Bioremediation of Commercial Polychlorinated Biphenyl Mixture … · 2018-11-13 · ii...

Bioremediation of Commercial

Polychlorinated Biphenyl Mixture

Aroclor 1260 by Naturally Occurring

Microorganisms

Pathiraja Mudiyanselage Gathanayana Pathiraja

B.Sc. (Honours) in Microbiology

M.Sc. in Environmental Science and Technology

Submitted in Fulfilment of the Requirements for the Award of the

Degree of Doctor of Philosophy

Science and Engineering Faculty

Queensland University of

Technology

2018

Bioremediation of Commercial Polychlorinated Biphenyl Mixture Aroclor 1260 by Naturally Occurring Microorganisms i

Keywords

Alternating anaerobic-aerobic treatment, Aroclor 1260, Biodegradation, Bioremediation,

Biosurfactants, Dechlorination, Exoproteome, Facultative anaerobic microorganisms,

Oxidation, Metaproteomics, Polychlorinated biphenyls, Secretome, Two stage

anaerobic-aerobic treatment.

ii Bioremediation of Commercial Polychlorinated Biphenyl Mixture Aroclor 1260 by Naturally Occurring Microorganisms

Abstract

Polychlorinated biphenyls (PCBs) are a group of synthetic organic chemical

compounds that consist of chlorinated aromatic hydrocarbons. PCBs had been

extensively used in numerous industrial applications. Commercial production of PCBs

ceased in 1993 with the wide recognition to their toxicity, bioaccumulation and

persistence characteristics. However, PCBs are still causing harmful effects due to

their presence in environmental systems such as in soil and sediment. Use of

microorganisms for remediation of PCB-contaminated soil has been widely

investigated due to the potential of microorganisms for breaking down complex

contaminants into less harmful by-products. However, the application of microbial

based bioremediation is not yet commonly practiced as the effectiveness is

influenced by a complexity of factors related to the microorganisms involved, the

nature of the PCB mixture and the contaminated environment.

This study was based primarily on the hypothesis that the complete degradation of

complex commercial PCB mixtures can be achieved through a combination of

anaerobic-aerobic treatment when appropriate microbial mixtures capable of

enhancing the aqueous solubility and degradation of PCBs under both anaerobic and

aerobic conditions were incorporated. To test this hypothesis, a detailed

experimental program was undertaken for isolating and identifying suitable PCB

degrading microorganisms from the natural environment, screening the identified

microorganisms for biosurfactant production, determining the PCB degradation

potential of facultative bacterial cultures under separate and combined anaerobic

and aerobic conditions, individually, and as a consortium. Lastly, the detection and

identification of the extracellular proteins released by microorganisms into the

external environment and analysis of their potential roles in PCB hydrolysis were

performed.

Through a selective enrichment process, two obligate aerobic and four facultative

anaerobic bacterial strains capable of utilizing a commercial PCB mixture, Aroclor

1260, as sole source of carbon were isolated. They were identified as

Bioremediation of Commercial Polychlorinated Biphenyl Mixture Aroclor 1260 by Naturally Occurring Microorganisms iii

Chryseobacterium sp. NP01, Delftia sp. NP02, Achromobacter sp. NP03,

Ochrobactrum sp. NP04, Lysinibacillus sp. NP05 and Pseudomonas sp. NP06 using full

length 16S rRNA gene sequence based molecular identification. Based on the results

and after an exhaustive review of the published literature, this study can be

considered as the first evidence of reporting the presence of Chryseobacterium and

Delftia species and their involvement in the remediation of soil contaminated with

PCBs.

The extreme hydrophobicity of PCBs makes them insoluble in aqueous medium and

is one of the main rate limiting factors in PCB bioremediation. Biosurfactants are

known to aid in solubility of hydrophobic chemicals, before certain microbes are able

to use them for energy. The selected bacterial cultures discovered here were further

tested for their ability to produce biosurfactants. Chryseobacterium sp. NP01 and

Lysinibacillus sp. NP05 demonstrated the highest biosurfactant production, PCB

solubility and degradation. The study created new knowledge on the ability of PCB

degrading bacteria to also produce biosurfactants that correlated well with increasing

PCB solubility and subsequently enhanced bioavailability and degradation.

PCB degradation efficiency of four facultative anaerobic bacterial strains

Achromobacter sp. NP03, Ochrobactrum sp. NP04, Lysinibacillus sp. NP05 and

Pseudomonas sp. NP06 were compared in parallel under aerobic, anaerobic and

combined anaerobic-aerobic conditions. This research can be considered as the first

comparative study assessing facultative anaerobic bacteria in degrading PCBs under

anaerobic, aerobic and two-stage anaerobic-aerobic cultivation conditions. The study

found that the four bacterial strains are capable of degrading commercial PCB

mixture as their sole source of carbon under all three tested conditions, with the

highest degradation under two stage anaerobic-aerobic conditions. Lysinibacillus sp.

NP05 performed the best with 9.16±0.8 mg/L maximum chloride yield under two stage

anaerobic-aerobic treatment, resulting in the removal of about one third of the total

chlorines present in the commercial PCB product Aroclor 1260. This positive finding

proves that in soil remediation applications, facultative microorganisms are better

candidates to survive and achieve higher PCB degradation rates under both anaerobic

and aerobic conditions.

iv Bioremediation of Commercial Polychlorinated Biphenyl Mixture Aroclor 1260 by Naturally Occurring Microorganisms

The study outcomes confirmed that a whilst a single bacterium may initially seem to

possess positive characteristics, it is highly probable that one microbe does not possess

the enzymatic capability to degrade all or even most of the PCB congeners present in the

contaminated environments. A consortium of carefully selected suitable microbial

species was expected to perform better than an individual microbe. Therefore, based on

the individual culture study results, three facultative anaerobic strains Achromobacter

sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05 were selected and used as

a consortium. A conventional two stage (TS) anaerobic-aerobic treatment was compared

to alternating (AN) anaerobic-aerobic treatment. The two stage process was set up to

provide an extended anaerobic phase of four weeks followed by a short aerobic phase

of two weeks. In contrast, weekly intervals of anaerobic and aerobic conditions were

applied in the alternating treatment. It was found that the alternating approach was

more efficient compared to the two stage treatment with yields of nearly 50% reduction

in total PCBs reached within the first two weeks compared to 24% reduction obtained in

two stage treatment. This finding is significant as one of the limiting factors in

bioremediation applications is the normal extended time span required and applied in

order to achieve satisfactory degradation of PCBs.

During this study, extracellular proteins released and/or secreted by the consortium

members under alternating and two stage anaerobic-aerobic-aerobic experimental

conditions were investigated. Proteins with diverse functional roles consisting of 319

non-secreted, 212 classically secreted and 87 non-classically secreted proteins were

identified. Out of 212 classically secreted proteins, 58% were identified as transport

related proteins. The identification of proteins involved in the intermediate steps of

PCB degradation pathways and in the detoxification of toxic chemicals provided key

evidences of the occurrence of PCB degradation, by the bacterial consortium. The

outcomes from the metaproteomics study will further advance the knowledge

background on the types and potential role of extracellular proteins and enzymes

detected in the culture supernatant, and their involvement in PCB degradation.

Bioremediation of Commercial Polychlorinated Biphenyl Mixture Aroclor 1260 by Naturally Occurring Microorganisms v

Table of Contents

Keywords .................................................................................................................. i

Abstract ................................................................................................................... ii

Table of Contents .....................................................................................................v

List of Figures .......................................................................................................... xii

List of Tables ......................................................................................................... xvii

List of Abbreviations ............................................................................................ xviii

Statement of Original Authorship .......................................................................... xx

Acknowledgements ............................................................................................... xxi

List of thesis associated publications ................................................................... xxii

Chapter 1: Introduction .................................................................................. 1

1.1 Background .................................................................................................. 1

1.2 Research Problem ........................................................................................ 2

1.3 Research Hypothesis ................................................................................... 3

1.4 Aims and Objectives .................................................................................... 4

1.5 Research Scope ............................................................................................ 4

1.6 Innovation and Contribution to Knowledge ................................................ 5

1.7 Research Design and Methodology ............................................................. 6

1.7.1 Critical review of research literature ...................................................... 9

1.7.2 Isolation, screening and identification of potential PCB degrading microorganisms .................................................................................... 9

1.7.3 Screening of bacterial isolates for their ability to produce biosurfactants to make hydrophobic PCBs soluble in aqueous media ...................... 10

1.7.4 Comparison of the individual facultative anaerobic bacterial isolates during PCB hydrolysis under aerobic, anaerobic and two stage anaerobic-aerobic conditions ............................................................. 11

1.7.5 Comparison of the bacterial consortium during PCB hydrolysis under two modes of combined anaerobic-aerobic treatments ................... 12

1.7.6 Analysis and characterization of proteins detected in the culture supernatants during PCB degradation. .............................................. 12

1.8 Thesis Outline ............................................................................................ 13

Chapter 2: Bioremediation of Polychlorinated biphenyls ............................... 15

2.1 Introduction ............................................................................................... 15

vi Bioremediation of Commercial Polychlorinated Biphenyl Mixture Aroclor 1260 by Naturally Occurring Microorganisms

2.2 Overview of polychlorinated biphenyls ..................................................... 16

2.2.1 Structure, properties and applications ................................................. 16

2.2.2 Influence of PCBs on human and ecosystem health ............................ 19

2.2.3 Regulations and monitoring .................................................................. 20

2.3 Removal of PCBs from contaminated soil ................................................. 21

2.3.1 PCBs as a soil contaminant ................................................................... 21

2.3.2 Remediation of PCB contaminated soil ................................................ 24

2.3.3 In-situ bioremediation .......................................................................... 24

2.3.3.1 Phytoremediation .................................................................... 24

2.3.3.2 Fungal bioremediation ............................................................. 28

2.3.3.3 Bacterial bioremediation ......................................................... 29

2.4 Biodegradation pathways .......................................................................... 29

2.4.1 Anaerobic reductive dechlorination ..................................................... 30

2.4.1.1 Anaerobic reductive dechlorination pathways ........................ 31

2.4.2 Aerobic oxidative degradation .............................................................. 33

2.4.2.1 The upper biphenyl degradation pathway .............................. 34

2.4.2.2 The lower biphenyl degradation pathway ............................... 37

2.4.3 Sequential anaerobic-aerobic degradation .......................................... 39

2.5 Factors affecting microbial remediation ................................................... 40

2.5.1 Soil properties ....................................................................................... 40

2.5.2 Environmental factors ........................................................................... 41

2.5.2.1 pH ............................................................................................. 41

2.5.2.2 Temperature ............................................................................ 42

2.5.2.3 Soil moisture ............................................................................ 42

2.5.3 PCB related properties .......................................................................... 42

2.5.4 Natural microbial diversity .................................................................... 43

2.6 Enhancement of bioremediation ............................................................... 46

2.6.1 Biostimulation ....................................................................................... 46

2.6.2 Bioaugmentation .................................................................................. 47

2.6.3 Mixed microbial consortia .................................................................... 48

2.6.4 Surfactants ............................................................................................ 48

2.6.5 Secondary carbon sources .................................................................... 50

2.6.6 Biocarriers ............................................................................................. 51

2.7 Monitoring of PCB degradation ................................................................. 51

2.8 Summary .................................................................................................... 55

Bioremediation of Commercial Polychlorinated Biphenyl Mixture Aroclor 1260 by Naturally Occurring Microorganisms vii

Chapter 3: Materials and Methods ................................................................ 57

3.1 Chemicals, media and reagents ................................................................. 57

3.1.1 PCB source ............................................................................................ 57

3.1.2 General Media and reagents ................................................................ 58

3.1.2.1 Media used for cultivating PCB degrading microorganisms .... 58

3.1.2.2 Media used in 16S rRNA based bacteria identification ........... 60

3.1.2.3 Media used in biosurfactant screening tests ........................... 61

3.1.2.4 Reducing sugar analysis in microbial growth studies .............. 62

3.1.2.5 Chemicals used in PCB extraction and analysis ....................... 63

3.1.2.6 Chemicals, buffers and materials used in protein extraction .. 64

3.2 Methods ..................................................................................................... 66

3.2.1 Selective enrichment of potential PCB utilizing bacteria ..................... 66

3.2.2 Genetic characterization based on 16S rRNA sequences ..................... 66

3.2.3 Bacterial growth profiles ...................................................................... 66

3.2.4 Bacteria cell density measurement ...................................................... 66

3.2.5 pH 68

3.2.6 Glucose concentration .......................................................................... 68

3.2.7 Chloride ion concentration ................................................................... 68

3.2.8 PCB extraction ....................................................................................... 69

3.2.9 PCB analysis .......................................................................................... 71

3.2.10 Screening tests for biosurfactant production ....................................... 73

3.2.11 Extracellular protein visualization, quantification, extraction and analysis ............................................................................................... 73

3.2.12 Storage of culture supernatant samples .............................................. 73

3.2.13 Bacterial culture maintenance .............................................................. 73

3.3 PCB degradation studies ............................................................................ 73

3.4 Cleaning of glassware used in PCB extraction ........................................... 74

3.5 Quality assurance ...................................................................................... 75

Chapter 4: Selective Enrichment and Identification of Potential PCB Degrading Microorganisms ........................................................................... 77

4.1 Background ................................................................................................ 77

4.2 Materials and Methods ............................................................................. 78

4.2.1 Soil and sediment sample collection and preparation ......................... 78

4.2.2 Selective enrichment of potential PCB utilizing bacteria ..................... 79

4.2.3 Characterization of potential PCB degrading bacteria ......................... 80

4.2.3.1 Morphological characteristics .................................................. 80

viii Bioremediation of Commercial Polychlorinated Biphenyl Mixture Aroclor 1260 by Naturally Occurring Microorganisms

4.2.3.2 Oxygen requirements............................................................... 81

4.2.3.3 Identification of bacterial isolates using 16S rRNA full length gene sequencing ...................................................................... 81

4.2.3.4 Confirmation of the ability to grow on PCBs as sole carbon source ....................................................................................... 84

4.2.3.5 Growth profiles of bacteria ...................................................... 85

4.3 Results and Discussion ............................................................................... 86

4.3.1 Screening and Identification of PCB utilizing culture members ........... 86

4.3.1.1 16S rRNA gene based identification ........................................ 86

4.3.1.2 Morphological characteristics of identified bacteria ............... 93

4.3.1.3 Screening of microorganisms based on tolerance to atmospheric oxygen ................................................................. 96

4.3.2 Basic growth profiles using glucose as the carbon source ................... 98

4.4 Conclusions .............................................................................................. 102

Chapter 5: Screening of bacterial isolates for biosurfactant production ....... 103

5.1 Background .............................................................................................. 103

5.2 Materials and Methods ........................................................................... 104

5.2.1 Experimental setup ............................................................................. 104

5.2.2 Evaluation of PCB solubility and degradation ..................................... 105

5.2.3 Screening for biosurfactant production .............................................. 105

5.2.3.1 Drop collapse test .................................................................. 105

5.2.3.2 Emulsification index (EI24) ...................................................... 106

5.2.3.3 Haemolysis ............................................................................. 106

5.3 Results and Discussion ............................................................................. 107

5.3.1 PCB solubility ....................................................................................... 107

5.3.2 Bacterial growth, chloride ion accumulation and pH ......................... 108

5.3.2.1 Bacterial growth ..................................................................... 108

5.3.2.2 Chloride ion concentration .................................................... 110

5.3.2.3 pH ........................................................................................... 111

5.3.3 Biosurfactant production .................................................................... 112

5.4 Conclusions .............................................................................................. 116

Chapter 6: PCB degradation potential of facultative anaerobic bacterial isolates .................................................................................................. 119

6.1 Background .............................................................................................. 119


6.2.1 Experimental setup ............................................................................. 120

Bioremediation of Commercial Polychlorinated Biphenyl Mixture Aroclor 1260 by Naturally Occurring Microorganisms ix

6.2.2 Bacteria seed culture preparation ...................................................... 121

6.2.3 Controls ............................................................................................... 122

6.2.4 Sample collection, analysis and preservation ..................................... 122

6.3 Results and discussion ............................................................................. 123

6.3.1 Growth profiles - aerobic vs anaerobic vs two stage anaerobic-aerobic 123

6.3.2 PCB degradation and solubilisation .................................................... 126

6.3.3 Chloride ion accumulation .................................................................. 129

6.4 Conclusions .............................................................................................. 133

Chapter 7: Effective degradation of polychlorinated biphenyls by three facultative anaerobic bacterial species Achromobacter, Ochrobactrum and Lysinibacillus as a consortium ........................ 135

7.1 Background .............................................................................................. 135


7.2.1 Laboratory microcosm batch experiments ......................................... 137

7.2.2 Frequency, collection and preservation of samples ........................... 137

7.2.3 Total PCB extraction and analysis ....................................................... 139

7.2.4 Carbon and nitrogen source utilization profiling of bacterial cultures 139

7.2.4.1 Procedure for Gram positive bacteria ................................... 140

7.2.4.2 Procedure for Gram negative bacteria .................................. 141


7.3.1 Test for competition ........................................................................... 142

7.3.2 PCB degradation by the bacterial consortium under AN and TS treatments ........................................................................................ 143

7.3.2.1 Total PCB degradation ........................................................... 143

7.3.2.2 PCB Homolog analysis ............................................................ 146

7.3.3 Chloride ion accumulation and pH variation ...................................... 151

7.3.3.1 Chloride ion analysis .............................................................. 151

7.3.3.2 pH analysis ............................................................................. 154

7.3.4 Carbon and nitrogen wide substrate utilization tests ........................ 156

7.4 Conclusions .............................................................................................. 160

Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05 ................................................................ 163

8.1 Background .............................................................................................. 163

x Bioremediation of Commercial Polychlorinated Biphenyl Mixture Aroclor 1260 by Naturally Occurring Microorganisms


8.2.1 Protein visualization ............................................................................ 166

8.2.1.1 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) ............................................................................. 166

8.2.1.2 Coomassie blue staining......................................................... 166

8.2.2 Protein quantification using Bicinchoninic acid assay (BCA) .............. 166

8.2.3 Trypsin digestion of extracellular proteins ......................................... 167

8.2.4 Desalting of samples prior to mass spectroscopy analysis ................. 168

8.2.5 Secretome analysis ............................................................................. 169

8.2.5.1 Liquid chromatography – mass spectroscopy ....................... 169

8.2.5.2 Protein identification using ProteinPilot v5 ........................... 171

8.2.5.3 Protein quantitation using PeakView .................................... 171

8.2.5.4 Bioinformatics analysis of peptide sequences ....................... 172


8.3.1 SDS-PAGE analysis: Extracellular protein detection and visualization 173

8.3.1.1 Individual cultures .................................................................. 173

8.3.1.2 Consortium study under AN and TS treatment conditions ... 176

8.3.2 Proteomics analysis ............................................................................. 178

8.3.2.1 Non-secretory proteins .......................................................... 179

8.3.2.2 Secretory proteins: Secretome .............................................. 182

8.4 Conclusions .............................................................................................. 193

Chapter 9: Conclusions, practical applications and recommendations for future research..................................................................................... 197

9.1 Conclusions .............................................................................................. 197

9.1.1 Isolation, screening and identification of potential PCB degrading microorganisms ................................................................................ 201

9.1.2 Screening of bacterial isolates for their ability to produce biosurfactants to make hydrophobic PCBs soluble in aqueous media .................... 201

9.1.3 Comparison of the individual facultative anaerobic bacterial isolates during PCB hydrolysis under aerobic, anaerobic and two stage anaerobic-aerobic conditions ........................................................... 202

9.1.4 Comparison of the bacterial consortium during PCB hydrolysis under two modes of combined anaerobic-aerobic treatments ................. 203

9.1.5 Analysing and characterization of proteins detected in the culture supernatants during PCB degradation ............................................. 204

9.2 Practical applications of research outcomes ........................................... 206

9.3 Recommendations for future research ................................................... 207

Bioremediation of Commercial Polychlorinated Biphenyl Mixture Aroclor 1260 by Naturally Occurring Microorganisms xi

References…….. ................................................................................................ 211

Appendices….. .................................................................................................. 245

Appendix A: PCB analysis ........................................................................... 246

Appendix B: PCB data related to bacterial consortium study ....................... 251

Appendix C: Extracellular protein analysis .................................................. 255

Supplimentary Material .................................................................................... 291

Supplementary Material 1: Abstracts of conference papers relevant to the thesis .......................................................................................... 293

Supplementary Material 2: Publications relevant to the thesis ................... 295

xii Bioremediation of Commercial Polychlorinated Biphenyl Mixture Aroclor 1260 by Naturally Occurring Microorganisms

List of Figures

Figure 1.1 Schematic representation of the research methodology ........................... 8

Figure 2.1 Structural form of PCB. Clx and Cly are number of chlorines attached to each benzene ring and x + y = 1 to 10. Adapted from Wiegel and Wu (2000). .......................................................................................................... 17

Figure 2.2 Concentrations of total PCBs in surface soils at global background sites (Li et al., 2010). .................................................................................... 22

Figure 2.3 Potential pathway for anaerobic dechlorination of highly chlorinated congeners. Adapted from Borja et al. (2005). ............................................. 31

Figure 2.4 The upper biphenyl degradation pathway. Modified from Field and Sierra-Alvarez (2008). ................................................................................... 35

Figure 2.5 The lower biphenyl degradation pathways (a) Mineralization of 2-hydroxypenta -2,4-dienoate. Modified from Field and Sierra-Alvarez (2008). (b) Mineralization of chlorobenzoic acid. Modified from ATSDR (2000). .......................................................................................................... 38

Figure 2.6 Overview of factors affecting microbial remediation ............................... 40

Figure 2.7 Variation of the bacterial community at genus level in the bulk, top and rhizosphere soils. The phylum of each genus is reported in brackets (Acid-Acidobacteria; Act-Actinobacteria; Alph-Alphaproteobacteria; Beta-Betaproteobacteria; Chlo-Chloroflexi; Firm-Firmicutes; Gamm-Gammaproteobacteria; Gemm-Gemmatimonatedes). Adapted from Stella et al. (2015). ........................... 45

Figure 2.8 The omics pyramid. Modified from NASEM (2016). ................................. 52

Figure 2.9 Organism based and community based “omic” approaches for assessing bioremediation approaches. Modified from Chovanec et al. (2011). .......................................................................................................... 54

Figure 3.1 Standard plate count technique ............................................................... 67

Figure 3.2 Recovery of Aroclor 1260 soluble in the aqueous minimal salt medium as a percentage of total PCBs added to the mixture, using diethyl ether (DEE) and hexane as the extraction solvents. Error bars represent the standard deviation of mean values (n = 3). .......................... 70

Figure 3.3 The Coy anaerobic chamber with the connecting airlock to the right, used in this research. ................................................................................... 74

Figure 4.1 Selective enrichment of potential PCB degrading bacteria, under aerobic and anaerobic conditions at 28 °C. ................................................. 80

Figure 4.2 Agarose gel electrophoresis of genomic DNA isolated from bacterial isolates using Isolate II genomic DNA kit (Bioline). A1 to A5 were from

Bioremediation of Commercial Polychlorinated Biphenyl Mixture Aroclor 1260 by Naturally Occurring Microorganisms xiii

aerobic selective enrichments and AN1 to AN6 were from anaerobic selective enrichments. ................................................................................. 87

Figure 4.3 Blue-white screening on LB/ampicillin/IPTG/X-Gal plate after the transformation into high efficiency E. coli JM109 competent cells. White colour colonies representing the positive transformations. ............ 88

Figure 4.4 Agarose gel electrophoresis of purified recombinant plasmids. 5 µL from each sample was loaded into the corresponding well. A1 to A5 were from aerobic selective enrichments and AN1 to AN6 were from anaerobic selective enrichments. Instead of plasmids, sterile MilliQ water was used in the negative control. ..................................................... 88

Figure 4.5 Pairwise alignment of forward and reverse DNA sequences of bacterial isolate AN2 using Emboss Needle software. Total length after the alignment was 1459 and number of similarities between two sequences were 738/1459 (50.6%). ............................................................ 89

Figure 4.6 Colony morphology of the bacterial cultures on nutrient agar after 48 h incubation at 28 °C. Culture A to F were under aerobic conditions and culture G was under anaerobic conditions. .......................................... 94

Figure 4.7 Gram stained bacterial cultures under the light microscope (100x magnification). ............................................................................................. 95

Figure 4.8 Bacterial colonies on minimal salt agar with 50 mg/L Aroclor 1260 as sole source of carbon after 48 hrs at 28 °C (A) under aerobic conditions, (B) facultative anaerobic cultures under anaerobic conditions, (C) negative controls under aerobic conditions (D) negative controls under anaerobic conditions. ........................................................................ 97

Figure 4.9 Growth of obligate anaerobic Novosphingobium sp. NP07 on 25 mg/L and 50 mg/L Aroclor 1260 containing minimal salt agar after 48 h incubation at 28 °C under anaerobic conditions. ........................................ 98

Figure 4.10 Basic bacterial growth profiles of the six bacterial isolates grown in minimal salt medium over a nine hour period at 28 °C and 150 rpm using glucose as the carbon source. Error bars represent the standard deviation of mean values (n = 3). ................................................................ 99

Figure 4.11 Variation of pH in the culture medium during the bacterial growth profile studies. Error bars represent the standard deviation of mean values (n = 3). ............................................................................................. 101

Figure 5.1 Variation of the total solubility of Aroclor 1260 in the aqueous minimal salt media inoculated with bacterial cultures. Error bars represent the standard deviation of mean values (n=3 for bacterial cultures and n=2 for abiotic controls). ...................................................... 107

Figure 5.2 Bacterial growth as optical density (OD600) in the batch mesocosms at 28 °C and 150 rpm. Error bars represent the standard deviation of mean values (n=3). ..................................................................................... 109

xiv Bioremediation of Commercial Polychlorinated Biphenyl Mixture Aroclor 1260 by Naturally Occurring Microorganisms

Figure 5.3 Chloride ion accumulation in the batch mesocosms after six weeks of incubation at 28 °C and 150 rpm. The background values from the controls of (1) minimal salt medium only and (2) seed cultures only were subtracted first. Error bars represent the standard deviation of mean values (n=3). ..................................................................................... 110

Figure 5.4 pH variation in the batch mesocosms at 28 °C and 150 rpm. Error bars represent the standard deviation of mean values (n=3). .................. 112

Figure 5.5 Drop collapse test. 1% (w/v) sodium dodecyl sulphate (SDS) solution was used as the positive control. The phosphate buffered saline (PBS) solution and abiotic control (minimal salt medium only) were used as negative controls. ....................................................................................... 114

Figure 5.6 Haemolysis of sheep blood in Tryptone soya agar after incubation at 28 °C for 48 hours (A) Chryseobacterium sp. NP01, (B) Delftia sp. NP02, (C) Achromobacter sp. NP03, (D) Ochrobactrum sp. NP04, (E) Lysinibacillus sp. NP05, (F) Pseudomonas sp. NP06, (G) 1% SDS as positive control, and (H) abiotic control. ................................................... 115

Figure 6.1 Growth of the four facultative anaerobic bacterial strains under (a) aerobic, (b) anaerobic, and (c) two stage anaerobic-aerobic conditions. Error bars represent the standard deviation of mean values (n = 3). ....... 125

Figure 6.2 PCB solubility under (a) aerobic, (b) anaerobic, and (c) two stage anaerobic-aerobic conditions. Prior to the addition of microbes, samples were removed and analysed for Initial soluble PCBs measurement and then after adding microbes, samples were removed immediately and represent week 0. Error bars represent the standard deviation of mean values from triplicates. ................................................ 128

Figure 6.3 Chloride ion accumulation in the culture media after six weeks. The background values from the controls of (1) minimal salt medium only and (2) seed cultures only were subtracted first. Error bars represent the standard deviation of mean values from triplicates. .......................... 130

Figure 6.4 Variation of pH and chloride ion concentrations after six weeks under aerobic, anaerobic and two stage anaerobic-aerobic conditions (Initial pH was adjusted to 7.0). Error bars represent the standard deviation of mean values from triplicates. ................................................................ 132

Figure 6.5 Growth profile, PCB hydrolysis and pH variation of Lysinibacillus sp. NP05 under two stage anaerobic-aerobic conditions. Error bars represent the standard deviation of mean values from triplicates. .......... 133

Figure 7.1 Inoculation of batch mesocosms with bacterial seed cultures inside the anaerobic chamber. ............................................................................. 137

Figure 7.2 Growth of Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05 on minimal salt-Aroclor 1260 agar at 28 °C after 72 h (a) under aerobic conditions, (b) under anaerobic conditions. ......... 143

Figure 7.3 Total PCB degradation as a percentage and bacterial growth as OD600 under (a) alternating and (b) two stage anaerobic-aerobic treatments

Bioremediation of Commercial Polychlorinated Biphenyl Mixture Aroclor 1260 by Naturally Occurring Microorganisms xv

by the bacterial consortium. Error bars represent the standard deviation of mean values (n = 3). .............................................................. 145

Figure 7.4 Variation of PCB homolog groups following AN treatment; (a) lower chlorinated congener groups (mono to tetra), and medium to highly chlorinated congener groups, (b) penta to hepta, (c) octa and nona. Error bars represent the standard deviation of mean values (n = 3). ....... 148

Figure 7.5 Variation of PCB homolog groups following TS conditions; (a) lower chlorinated congener groups (mono to tetra), and highly chlorinated congener groups, (b) penta to hepta, (c) octa and nona. Error bars represent the standard deviation of mean values (n = 3). ........................ 150

Figure 7.6 Chloride ion accumulation under alternating (AN) and two stage (TS) anaerobic-aerobic conditions. The background chloride values from the minimal salt medium were first subtracted from experimental values and media controls. Error bars represent the standard deviation of mean values (n=3 for experimental values and n=2 for media controls). .................................................................................................... 151

Figure 7.7 Measured chloride ion buildup in the culture medium and calculated chloride ion removal from the PCB mixture based on homolog group reductions under; (a) alternating (AN), and (b) two stage (TS) anaerobic-aerobic conditions. Error bars represent the standard deviation of mean values (n = 3). .............................................................. 153

Figure 7.8 pH trends relative to chloride ion concentration. (a) AN and (b) TS anaerobic-aerobic treatments. Error bars represent the standard deviation of mean values (n = 3). .............................................................. 155

Figure 8.1 The secretome and exoproteome of a Gram negative bacterial cell. (Armengaud et al., 2012) ........................................................................... 164

Figure 8.2 Role of extracellular enzymes in insoluble compound metabolism. ...... 165

Figure 8.3 SDS-PAGE analysis of Lysinibacillus sp. NP05 containing controls (minimal salt medium with no added PCBs) under anaerobic conditions at 28 °C. Lane 1, SeeBlue Protein standard as the protein molecular weight markers; lanes 2, time 0 immediately after addition of seed culture; lane 3 to 8, week 1 to week 6....................................................... 173

Figure 8.4 SDS-PAGE analysis of extracellular proteins of Achromobacter sp. NP03 under (A) aerobic and (B) anaerobic conditions at 28 °C. Lane 1, SeeBlue Protein standard as the protein molecular weight markers; lanes 2, time 0 immediately after addition of seed culture; lane 3 to 8, week 1 to week 6. ...................................................................................... 174

Figure 8.5 SDS-PAGE analysis of extracellular proteins of Ochrobactrum sp. NP04 under (A) aerobic and (B) anaerobic conditions at 28 °C. Lane 1, SeeBlue Protein standard as the protein molecular weight markers; lanes 2, time 0 immediately after addition of seed culture; lane 3 to 8, week 1 to week 6. ...................................................................................... 175

xvi Bioremediation of Commercial Polychlorinated Biphenyl Mixture Aroclor 1260 by Naturally Occurring Microorganisms

Figure 8.6 SDS-PAGE analysis of extracellular proteins of Lysinibacillus sp. NP05 under (A) aerobic and (B) anaerobic conditions at 28 °C. Lane 1, SeeBlue Protein standard as the protein molecular weight markers; lanes 2, time 0 immediately after addition of seed culture; lane 3 to 8, week 1 to week 6. ...................................................................................... 176

Figure 8.7 SDS-PAGE analysis of the extracellular proteins of the bacterial consortium consisting of Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05 under (A) AN, and (B) TS anaerobic-aerobic conditions at 28 °C. Lane 1, SeeBlue Protein standard as the protein molecular weight markers; lanes 2, time 0 immediately after addition of seed culture; lane 2 to 5, at fortnightly intervals up to week 6. ................................................................................................................. 177

Figure 8.8 Proportions of classically and non-classically secreted proteins in the culture supernatant of the bacterial consortium Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05. ..................... 183

Figure 8.9 Functional groupings of proteins identified as classically secreted proteins. ..................................................................................................... 184

Figure 8.10 The concentration of the sulfate transporter protein detected in the culture supernatant over time, under the alternating anaerobic-aerobic (AN) conditions. ............................................................................ 188

Figure 8.11 Venn diagram of the distribution of proteins identified as classically secreted proteins among (A) alternating (AN) anaerobic-aerobic, and (B) two stage (TS) anaerobic-aerobic conditions. ...................................... 190

Figure 8.12 Functional groupings of proteins identified as non-classically secreted proteins. ...................................................................................... 191

Figure 8.13 Variation of glutamine synthetase concentration and pH level in the culture supernatant under alternating anaerobic-aerobic conditions. ..... 192

Figure 8.14 Distribution of non-classically secreted proteins under (A) AN anaerobic-aerobic treatment, and (B) TS anaerobic-aerobic treatment. .................................................................................................................... 193

Figure 9.1 A schematic representation of the summary of major findings of the research study. ........................................................................................... 200

Bioremediation of Commercial Polychlorinated Biphenyl Mixture Aroclor 1260 by Naturally Occurring Microorganisms xvii

List of Tables

Table 2.1 PCB homologs and their chlorine substitutions ......................................... 18

Table 2.2 Comparison of PCB levels (µg/kg dry weight) in soils based on selected international studies. ................................................................................... 23

Table 2.3 Plant–microbial relationships associated with PCB degradation .............. 27

Table 2.4 Microbial dechlorination pathways (Wiegel & Wu, 2000). ....................... 32

Table 2.5 Types and microbial origin of biosurfactants. ............................................ 49

Table 3.1 Summary of physicochemical properties of Aroclor 1260 ........................ 57

Table 3.2 Average weight percent of PCB homolog groups and chlorines in Aroclor 1260. ................................................................................................ 72

Table 4.1 Comparison of closest relatives of isolated bacteria based on NCBI and RDP databases ...................................................................................... 91

Table 4.2 Final nomenclature and identification of the pure bacterial isolates ....... 92

Table 4.3 Specific growth rate of bacterial cultures during the growth profile studies using 2 g/L glucose as the carbon source. ..................................... 100

Table 5.1 Summary of biosurfactant screening tests .............................................. 113

Table 6.1 Bacteria cell count in overnight Luria–Bertani liquid medium at 28 °C..................................................................................................................... 123

Table 7.1 Ingredients for the nutrient additive solution, 12x (PM additive) ........... 140

Table 7.2 Preparation of final inoculation fluid to inoculate the Biolog plates....... 141

Table 7.3 Carbon source utilization by consortium members Achromobacter sp. NP03, Ochrobactrum sp. NP04, and Lysinibacillus sp. NP05. .................... 158

Table 7.4 Nitrogen source utilization by consortium members Achromobacter sp. NP03, Ochrobactrum sp. NP04, and Lysinibacillus sp. NP05. ............... 159

Table 8.1 Standard dilutions preparation for BCA assay ......................................... 167

Table 8.2 Analysis of extracellular proteins identified in culture supernatants of consortium under AN and TS conditions. .................................................. 178

Table 8.3 Functional classification of 319 identified proteins that were predicted to be non-secretory proteins by the SignalP 3.0 and SecretomeP 2.0 servers. ............................................................................ 179

Table 8.4 Heat map showing the relative abundance of ten highly secreted extracellular proteins detected in the culture supernatant of the bacterial consortium Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05. .............................................................. 186

xviiiBioremediation of Commercial Polychlorinated Biphenyl Mixture Aroclor 1260 by Naturally Occurring Microorganisms

List of Abbreviations

Abbreviations

AN Alternating anaerobic-aerobic treatment

CFU Colony forming units

DEE Di ethyl ether

DNS Dinitrosalicylic acid

GCMS Gas chromatography-mas spectroscopy

gDNA Genomic DNA

IPTG Isopropyl β-D-1-thiogalactopyranoside

LB Luria–Bertani medium

LCMS Liquid chromatography–mass spectrometry

NA Nutrient Agar

NCBI National Centre for Biotechnology Information

MSM Minimal salt medium

OD600 Optical density at 600 nm

PBS phosphate buffer saline

PCBs Poly chlorinated biphenyls

PCR Polymerase chain reaction

RDP Ribosomal database project

RSDV Relative standard deviation

SDS Sodium dodecyl sulphate

SOC Super optimal broth with catabolite repression

SRM Selective reaction monitoring

SWATH Sequential window acquisition of all theoretical mass spectra

TSA Tryptone soya agar

TS Two stage anaerobic-aerobic treatment

Bioremediation of Commercial Polychlorinated Biphenyl Mixture Aroclor 1260 by Naturally Occurring Microorganisms xix

Units

bp Base pair(s)

g Gram(s)

g Relative centrifugal force in units of gravity

h Hour(s)

kb Kilobase

kDa Kilodalton

min Minute(s)

mol Mole(s)

rpm Revolutions per minute

s Second(s)

v/v Volume per volume

w/v Weight per volume

xx Bioremediation of Commercial Polychlorinated Biphenyl Mixture Aroclor 1260 by Naturally Occurring Microorganisms

Statement of Original Authorship

The work contained in this thesis has not been previously submitted to meet

requirements for an award at this or any other higher education institution. To the

best of my knowledge and belief, the thesis contains no material previously published

or written by another person except where due reference is made.

Signature:

Date: November 2018

QUT Verified Signature

Bioremediation of Commercial Polychlorinated Biphenyl Mixture Aroclor 1260 by Naturally Occurring Microorganisms xxi

Acknowledgements

The successful completion of my thesis would not have been possible without the

guidance, support, encouragement and help from my supervisory committee,

technical staff, scholarship from the Australian Government, my family and friends.

I am extremely thankful to my principal supervisor, Associate Professor V. S. Junior

Te'o, for his invaluable guidance and support throughout my PhD research. I would

also like to express my sincere thanks to my associate supervisor, Professor Ashantha

Goonetilleke for his guidance, support and encouragement to complete my research

study successfully. My sincere thanks is extended to my associate supervisor, Dr.

Prasanna Egodawatta for giving me the opportunity to undertake my PhD study at

QUT and for his continuous support and guidance.

I sincerely acknowledge Queensland University of Technology (QUT) for providing me

financial support through a RTPSD scholarship to conduct this doctoral research

study. I would also wish to acknowledge the staff of Central Analytical Research

Facility (CARF) of QUT, especially Mr. Vincent Chand and Mr. Shane Russell for their

technical support, Ms. Silvia Gemme for supporting me with the GCMS analysis, Dr.

Pawel Sadowski for helping me with proteomics. I am very thankful to Mr. Tony Tuong

Ngo, Powerlink Queensland Oil testing services, for supplying me transformer oil

samples without any hesitation to initiate my experiments.

I would like to extend my thanks to my fellow HDR colleagues for their support,

friendship, and encouragement. I am very grateful to my husband Samanola and kids

Vinuri, Mindulie and Biman for their love, support, patience, and encouragement to

complete my PhD study. Finally, my parents are remembered with love and gratitude

for their guidance and blessings throughout my life.

xxii Bioremediation of Commercial Polychlorinated Biphenyl Mixture Aroclor 1260 by Naturally Occurring Microorganisms

List of thesis associated publications

Conference Papers

• Pathiraja P.M.G., Egodawatta P., Goonetilleke A., Te'o V.S. J. Degradation of

commercial polychlorinated biphenyl mixture by naturally occurring facultative

microorganisms.

Nineteenth International Conference on Environmental Biodegradation

Rates, Sydney, Australia, 2017.

Journal Papers (published)

• Pathiraja, G., Egodawatta, P., Goonetilleke, A., & Te'o, V. S. J. (2019). Solubilization

and degradation of polychlorinated biphenyls (PCBs) by naturally occurring

facultative anaerobic bacteria. Science of The Total Environment, 651, 2197-2207.

Journal Impact Factor: 4.9, SJR Rank Q1.

Journal papers (Under review)

• Effective degradation of polychlorinated biphenyls by three facultative anaerobic

bacterial species Achromobacter, Ochrobactrum and Lysinibacillus as a consortium.

Submitted to Science of the Total Environment, Journal Impact Factor: 4.9, SJR Rank

Q1.

Chapter 1: Introduction 1

Chapter 1: Introduction

1.1 Background

The diversity and magnitude of man-made toxic chemicals released into the

environment are creating long-term ecological impacts. Polychlorinated biphenyls

(PCBs) are one such toxic chemical group, consisting of 209 different chlorinated

organic compounds. Due to their unique chemical and physical properties, PCBs had

been used widely in many industrial applications, especially as insulating fluids in

transformers, capacitors, hydraulic systems, and as flame retardants. As a result,

since 1930, PCBs have been manufactured commercially in large scale as complex

mixtures.

The low reactivity, high chemical stability and complex nature of commercial PCB

mixtures have made them highly persistent and less environmentally desirable than

many other organic chemicals (Beyer & Biziuk, 2009). Their affinity to build up in living

organisms though bioaccumulation over time, biomagnification along the food chains

and resistance to biotransformation have led to numerous health implications in

humans and animals. As a result, PCBs were categorized as one of the original twelve

worldwide priority persistent organic pollutants (POPs) covered by the Stockholm

Convention (Bedard et al., 2007). Even though commercial production and use of

PCBs were banned or restricted decades ago, there is still a substantial amount of

PCBs present in the environment due to the continuing use and disposal of

equipment containing PCBs, recycling of PCB-contaminated products, emissions from

combustion of PCB contaminated waste, contaminated sites and disposal areas

(Breivik et al., 2007).

Treating environmental media such as soil contaminated with PCBs is of vital

importance to safeguard human and ecosystem health. In this regard, a range of

physical, chemical and biological treatment technologies have been investigated to

identify new remediation possibilities (Gomes et al., 2013). Bioremediation, the use

of microorganisms or microbial processes to degrade environmental contaminants,

2 Chapter 1: Introduction

is one of the methods intensively investigated for treating PCB-contaminated soils

(Boopathy, 2000).

1.2 Research Problem

Developing an environmentally sustainable and economically viable biological

treatment method as an alternative to existing physical and chemical treatment

methods for treating soil contaminated with PCBs is of critical importance. Over the

last decades, some aerobic and anaerobic microorganisms capable of degrading a

broad range of PCBs have been identified (Dercova et al., 2008; Sowers & May, 2013).

However, the application of microbial based bioremediation to treat PCB

contaminated soil is not yet widely practiced. The effectiveness of bioremediation is

determined by a number of factors, such as the lack of suitable microorganisms in

the contaminated environment, difficulties in maintaining the PCB degrading

bacterial communities in real-world environmental conditions, the nature of the PCB

mixture and the severity of contamination, low bioavailability of PCBs and the

characteristics of the contaminated environment.

As noted by Passatore et al. (2014), complete degradation of commercial PCB

mixtures that consist of compounds with varying degree of chlorination by

microorganisms can be achieved through a combination of anaerobic and aerobic

processes. It was recognized that the anaerobic dechlorination of more highly

chlorinated congeners followed by the aerobic degradation of those dechlorinated

products are the most likely degradation pathways occurring in the environment

(Payne et al., 2013). However, due to the complexity of commercial PCB mixtures

with the varying degree of chlorination, a single bacterium is not capable of degrading

all or even most of the PCB congeners present in contaminated environments (Pieper,

2005). Therefore, the search for appropriate microorganisms with the ability to

survive and degrade complex PCB mixtures under both anaerobic and aerobic

conditions would be a potential solution to achieve an efficient and effective process

for the biodegradation of PCBs.


Stella et al. (2015) noted that the extremely hydrophobic nature of PCBs makes them

poorly soluble in aqueous media, and this attribute can lead to PCBs being less

bioavailable for microbial degradation. Therefore, an increase in solubilisation would

enhance the bioavailability and subsequent biodegradation of PCBs (Ohtsubo et al.,

2004). However, the application of chemical and biological surfactants to increase

PCB solubility is limited due to various factors, such as chemical toxicity and high cost.

Therefore, the identification of suitable microorganisms that are capable of surviving

under a PCB contaminated environment, while producing biosurfactants would

accelerate PCB solubility and subsequent degradation.

Moreover, both anaerobic and aerobic PCB degradation pathways so far identified in

microorganisms were found to have occurred intracellularly (Wiegel & Wu, 2000;

Pieper, 2005; Agullo et al., 2017). However, the mechanism of transportation of PCB

molecules across the cytoplasmic membrane of microorganisms is not clear (Parales

& Ditty, 2017). The proteins or enzymes released and/or secreted by the

microorganisms to the extracellular environment may provide novel insights into the

mechanisms involved in modifying and transporting hydrophobic PCB molecules into

the cell (Basak & Dey, 2015). Therefore, identification of proteins released by

microorganisms into their surrounding extracellular environment and the functional

relationship with the PCB uptake into the bacterial cells would be important in order

to accelerate the degradation process.

Accordingly, this study aimed to discover microorganisms that can survive and

effectively degrade PCBs under both anaerobic and aerobic conditions, while

facilitating aqueous solubility and cellular uptake.

1.3 Research Hypothesis

The research study was based on the following hypotheses:

• Complete degradation of complex PCB mixtures can be achieved through a

combination of anaerobic-aerobic treatments.


• Microbial mixtures with varied capabilities for increasing the aqueous

solubility of PCBs, dechlorinating highly chlorinated congeners and complete

degradation of lower chlorinated congeners are required for comprehensive

degradation of PCBs.

• Extracellular proteins or enzymes released and/or secreted by the

microorganisms cultivated in minimal salts based medium with PCBs as a

carbon source, facilitate the uptake of PCB molecules into the bacterial cells

for degradation.

1.4 Aims and Objectives

The primary objective of this research was to identify suitable microorganisms from

the natural environment, which are capable of solubilizing and degrading complex

PCB mixtures under varying anaerobic and aerobic conditions in order to effectively

treat soil contaminated with PCBs.

To achieve this objective, the study aimed to:

• Isolate, screen, identify and characterize naturally occurring microorganisms

and select the best ones for further work, based on their ability to effectively

degrade PCBs under aerobic and anaerobic conditions.

• Screen the identified microorganisms for biosurfactant production in order to

increase the water solubility of PCBs.

• Determine the PCB degradation potential of facultative bacterial cultures

under anaerobic and aerobic conditions both individually and as a consortium.

• Analyse the extracellular proteins released by microorganisms into the

external environment.

1.5 Research Scope

The scope of this research study was as follows:


• The PCB source used in the present study was limited to Aroclor 1260, one of

the most commonly used and highly chlorinated commercial PCB mixtures

(USEPA, 2013). Its abundant usage has resulted in contamination of many

sites worldwide and has proven to be difficult to biodegrade due to its high

levels of chlorination (Bedard et al., 2007). Aroclor 1260 contains 60 to 90

different PCB congeners out of 209 possibilities (ATSDR, 2000; Breivik et al.,

2007). The outcomes of the study may not be applicable to PCB congeners

outside of the congeners available in Aroclor 1260.

• The research study was confined to treating soil contaminated with Aroclor

1260. However, the knowledge created in relation to the PCB solubility,

uptake and degradation by microorganisms is applicable to other

contaminated media such as sediments.

1.6 Innovation and Contribution to Knowledge

Investigating the performance of facultative anaerobic bacteria on PCB degradation

is expected to provide new insights to the knowledge base and research field. The

use of facultative anaerobic bacteria under anaerobic and aerobic conditions has led

to opportunities for further research based on their ability to degrade PCBs under

both aerobic and anaerobic conditions. Though a range of studies have so far

discussed PCB degradation potential of aerobic and anaerobic bacteria, their practical

use in the real-world environment is limited. Therefore, the innovative outcomes

from this study can be effectively utilized to treat contaminated soil under varying

anaerobic aerobic conditions in the field without losing the degradability and viability

potential.

The study also created new knowledge on the ability of PCB degrading bacteria to

produce biosurfactants. This finding is essential for designing an effective process to

enhance the bioavailability of PCBs in remediation applications, without addition of

toxic chemical surfactants or costly biological surfactants. Furthermore, the new

knowledge created relating to the identification of the alternating anaerobic-aerobic

treatment method as a rapid and efficient treatment option over the conventional


long-term two stage anaerobic-aerobic treatment can be successfully applied in

order to increase the effectiveness and reduce the cost of lengthy treatments. These

discoveries will contribute towards enhancing current microorganism based

bioremediation approaches to treat soils contaminated with PCBs and other similar

toxic chemical contaminants, in order to reduce and eventually eliminate their

harmful impacts on ecosystems and human health.

1.7 Research Design and Methodology

Effective biodegradation of PCBs is a complex process that involves a combination of

factors such as having suitable microbial cultures, complexity, concentration and

bioavailability of PCB congener mixtures, and environmental conditions. Therefore, the

research methodology was formulated in order to first, isolate, screen and identify

appropriate microorganisms with PCB degradation potential, second, react selected

microorganisms with PCBs, and third, analyse the results and characterize the key

contributing factors influencing the extent of PCB degradation by the microorganisms.

This section outlines the research methodology adopted.

The research methodology consisted of the following phases:

• Critical review of research literature.

• Isolation, screening and identification of potential PCB degrading

microorganisms.

• Screening of bacterial isolates for their ability to produce biosurfactants to make

hydrophobic PCBs soluble in aqueous media.

• Comparison of PCB degradation rates of facultative anaerobic bacterial isolates

individually under aerobic, anaerobic and two stage anaerobic-aerobic

conditions.

• Comparison of PCB degradation efficiency of selected bacterial isolates as a

consortium under two modes of combined anaerobic-aerobic treatments.

• Detection, identification and bioinformatics analysis of peptides produced from

proteins found in the culture supernatants during PCB hydrolysis indicative of

protein or enzyme secretion.


• Assessment of the types of proteins and a discussion of their possible roles in PCB

hydrolysis, and potential mechanisms of protein externalization by the

microorganisms.

The critical review of the research literature was first undertaken to identify the

knowledge gaps and research questions in PCB bioremediation, and thereby to

formulate the approaches to investigate the research problem. Figure 1.1 shows the

schematic of the research methodology describing the development of key study steps,

which are discussed in Chapters 4 to 8.


Figure 1.1 Schematic representation of the research methodology

Identification and bioinformatics analysis

Selective enrichment and identification of

potential PCB degrading microorganisms (Chapter 4)

Sediment and soil samples

Contamination with Aroclor 1260

Selective enrichment

Isolation of potential PCB degrading microorganisms

Identification using 16S rDNA and morphology

Long-term storage

PCB degradation potential of facultative anaerobic bacterial isolates under aerobic, anaerobic and two stage anaerobic– aerobic conditions (Chapter 6)

MSM medium + 50 mg/L Aroclor 1260

Analyze for PCB degradation

Develop microbial consortium

PCB degrading microorganism

s

Effective degradation PCBs by selected bacterial isolates as a consortium under combined anaerobic– aerobic conditions (Chapter 7)

Metaproteomics analysis of proteins detected in the culture supernatants (Chapter 8)

Analyse for PCB solubilisation, biosurfactants


PCB degrading microorganisms

Screening of bacterial isolates for biosurfactant production (Chapter 5)


Microbial consortium

Comparison of PCB degradation under AN and TS conditions

Visualization Quantification


1.7.1 Critical review of research literature

A critical literature review was conducted to identify the current status and

subsequent knowledge gaps in the area of environmental pollution on the use of

polychlorinated biphenyls. Of particular interest was the state of play on known types

of biodegradation pathways, factors affecting PCB degradation, and importantly, the

potential opportunities to improve PCB biodegradation processes using

microorganisms. The main areas of focus for the literature review were:

• Overview of polychlorinated biphenyls

• Different methods for removal of PCBs from contaminated soil

• Biodegradation pathways

• Factors affecting microbial remediation

• Enhancement of bioremediation

• Monitoring of PCB degradation

1.7.2 Isolation, screening and identification of potential PCB degrading

microorganisms

Due to the complexity of the study, it was essential to initially undertake the research

under laboratory conditions in order to limit the number of environmental variables.

This approach would then provide a robust platform to assess the applicability of the

outcomes under field conditions and to undertake appropriate modifications as

needed. The potential for field application of the laboratory study outcomes have

been further discussed in Section 9.3 of Chapter 9, Recommendations for future

research.

Accordingly, soil and sediment samples collected from different sites (locations

around the Brisbane City Botanical Gardens (27.4745° S, 153.0293° E), Brisbane River

(27.4745° S, 153.0293° E) and Coombabah Lake, Gold Coast (27.54° S, 153.22° E )

were artificially mixed with Aroclor 1260 and used to isolate PCB degrading

microorganisms. PCB concentration in soil varies widely depending on number of

factors such as distance to the point of contamination, source of contamination, and


soil depth. However, if the concentration of PCBs is ≥ 50 mg/kg in any soil type, then

such type of soil is regarded as PCB remediation waste and under the regulation of

the Toxic Substances Control Act. The PCB concentration used in this study was

limited to 50 mg/L, in order to limit the usage, minimize the waste generated and to

reduce the number of variables.

According to the current literature, soils contaminated with PCBs are most commonly

associated with transformer oil. Therefore, instead of direct contamination with the

commercial PCB mixture, Aroclor 1260, transformer oil samples contaminated with

Aroclor 1260 (supplied by the Powerlink oil testing laboratory) were used to

contaminate the soil and sediment samples. Although the focus of the study was to

remediate the contaminated soil, both soil and sediment samples were used during

the screening process with the aim of obtaining a combination of aerobic and

anaerobic bacteria as complete degradation of complex PCB mixtures favours the use

of both anaerobic degradation and aerobic oxidation.

For bacterial identification, the isolation and sequencing of good quality full length

16S rRNA gene DNAs (1.5kb) were performed through PCR and cloning using plasmids

and the bacterium Escherichia coli. The resulting full length 1.5kb 16S gene DNA

sequences on recombinant plasmids were submitted to the commonly used GenBank

National Center for Biotechnology Information (NCBI)

(https://www.ncbi.nlm.nih.gov/) as a non-curated database (Wang et al., 2007), and

also to the curated database Ribosomal Database Project (RDP)

(http://rdp.cme.msu.edu/). The details of methodologies used during the molecular

identification process are provided in Chapter 4.

1.7.3 Screening of bacterial isolates for their ability to produce biosurfactants to

make hydrophobic PCBs soluble in aqueous media

PCBs are inherently hydrophobic chemicals. As a consequence, insoluble PCBs are

considered one of the limiting factors in microbial bioremediation. Therefore, an

increase in the rate of solubilisation would potentially aid in the internalization

process of PCBs into the cells and subsequent degradation (Ohtsubo et al., 2004). The

http://rdp.cme.msu.edu/


identified microorganisms with PCB degradation potential were tested for

biosurfactant production. However, the extent of the study was limited to initial

screening for the potential biosurfactant production and PCB solubilisation using

emulsification index and drop collapse tests as quantitative tests and a haemolytic

assay as a qualitative test. The details of the experimental setup and analysis

methods used are discussed in Chapters 3 and 5, respectively.

1.7.4 Comparison of the individual facultative anaerobic bacterial isolates during

PCB hydrolysis under aerobic, anaerobic and two stage anaerobic-aerobic

conditions

Complete degradation of complex PCB mixtures needs a combination of anaerobic

dechlorination and aerobic oxidation bioconversion processes. Therefore, during this

study, the performance of four selected facultative anaerobic bacterial strains were

compared in parallel under anaerobic, aerobic and combined anaerobic-aerobic

conditions to evaluate their capability for PCB degradation. A total of 462 samples

were produced here as a result of replicates, number of strains, variables tested and

the low water solubility of Aroclor 1260. Therefore, it was not possible to maintain

and sacrifice a high number of whole flasks at weekly intervals to extract and measure

the total and congener specific PCB reductions.

Instead, removal of aliquots from each flask at weekly intervals and extraction of

PCBs were performed according to methods that measure the variation of total

soluble PCB levels in the aqueous culture medium (Adrian et al., 2009; Wang & He,

2013b). In addition, PCB hydrolysis was indirectly measured as cell growth and

chloride ion accumulation. All the experiments were conducted at 28 °C as the

average temperature due to the practical difficulties for maintaining the varied real

field conditions in the laboratory environment. The details of the calibration curve

preparation for PCB analysis are shown in the Appendix A while the details of testing

procedures and experimental setup are given in Chapters 3 and 6.


1.7.5 Comparison of the bacterial consortium during PCB hydrolysis under two

modes of combined anaerobic-aerobic treatments

Based on the results obtained for PCB solubility, cell growth and chloride

accumulation, the three facultative anaerobic bacteria, Achromobacter sp. NP03,

Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05 were chosen to form the

consortium. Two combined anaerobic-aerobic treatment modes namely alternating

anaerobic-aerobic treatment (AN) and two stage anaerobic-aerobic treatment (TS)

were selected to compare the rates and efficiencies of PCB degradation by the

consortium. Usually, anaerobic degradation is a long-term process and is one of the

limiting factors in the use of bioremediation as a treatment option.

Therefore, the aim of the two stage (TS) process was to mimic the conventional

combined anaerobic-aerobic treatment with an initial long term anaerobic phase

(four weeks) followed by a short term (two weeks) aerobic phase (Master et al., 2002;

Payne et al., 2013). In contrast, the aim of the alternating (AN) mode was to apply

equally short durations (weekly) of anaerobic and aerobic phases alternatively

throughout the six weeks period to allow the facultative anaerobic microorganisms

to convert the highly chlorinated congeners to lower chlorinated congeners under

anaerobic conditions and further break down the resulting lower chlorinated

congeners under aerobic conditions. However, the PCB degradation study was

limited to the total and homolog group based PCB analysis, as the purpose of the

research project was to study the overall PCB degradation potential. Therefore, the

congener specific degradation was not analysed. The data related to PCB analysis are

given in the Appendix B and the details of the total PCB extractions and the

experimental setups are given in Chapters 3 and 7, respectively.

1.7.6 Analysis and characterization of proteins detected in the culture supernatants

during PCB degradation.

PCB degradation by bacteria is believed to be an intracellular process. However, there

is paucity of knowledge available about the mechanism of how the hydrophobic PCB

molecule is transported into the cell across the cytoplasmic membrane. Therefore,


the extracellular proteins and/or enzymes either released or secreted by the

microorganisms into their surrounding environment were analysed to identify any

relationships between the rate of PCB degradation in the presence of extracellular

proteins and their concentrations and the potential mechanisms for the uptake of

PCBs. However, detailed analysis of extracellular proteins was limited to the bacterial

consortium as it is an exhaustive and time consuming process. Therefore, it was not

practical to focus on each individual bacterium under variable environmental

conditions. The data related to the relative abundance and categories of proteins and

enzymes is presented in Appendix C. The details of the analytical procedures used are

provided in Chapter 8.

1.8 Thesis Outline

This thesis consists of nine chapters. Chapter 1 introduces the research study providing

details of the research problem, hypotheses, aims and objective, scope, innovation and

contribution to knowledge, and research design and methodology.

The introduction to the research study is followed by the critical review of research

literature in Chapter 2. It provides a critical review of the research literature in the area

of environmental pollution due to polychlorinated biphenyls. It also identified the

knowledge gaps and provided research directions into advancing knowledge on

polychlorinated biphenyls, their biodegradation pathways, and factors affecting the

degradation and potential opportunities to improve the PCB biodegradation processes.

Chapter 3 outlines the general materials and methods used in the research study. It

further includes details on sample preservation and storage, bacterial culture

maintenance and quality assurance.

Chapters 4 to 8 are research chapters that describe the laboratory based experiments

and discuss the results.

Chapter 4 discusses the methodology used for the isolation, screening and identification

of potential PCB degrading microorganisms from soil and sediment samples artificially

contaminated with Aroclor 1260.


Chapter 5 presents the relationship between the biosurfactant production ability of

bacterial isolates with PCB solubility in aqueous media.

Chapter 6 compares PCB degradation rates of four facultative anaerobic bacterial isolates

individually under aerobic, anaerobic and two stage anaerobic-aerobic conditions.

Chapter 7 analyses and compares the PCB degradation efficiency of the selected bacterial

isolates as a consortium under alternating (AN) and two stage (TS) anaerobic-aerobic

treatment conditions.

Chapter 8 is the final research chapter and it analyses the extracellular proteins released

by the bacterial consortium members into the culture supernatant. It was important to

identify the types of proteins and establish any relationships between the proteins and

PCB degradation by the consortium members.

Chapter 9 is the final chapter. It provides the overall discussion, important conclusions

derived from the research and recommendations for future research.

Chapter 2: Bioremediation of Polychlorinated biphenyls 15

Chapter 2: Bioremediation of Polychlorinated biphenyls

2.1 Introduction

Contamination of the environment by hazardous and toxic synthetic chemicals is one

of the major problems facing the world today. Polychlorinated biphenyls (PCBs) are

one such synthetic chemical group, which consist of chlorinated aromatic

hydrocarbons. Due to their stable chemical and physical properties, PCBs were widely

used in many industrial applications since the 1930s. Low reactivity and high chemical

stability of PCBs have made them highly persistent (Beyer & Biziuk, 2009). High

lipophilicity makes PCBs soluble in fats and concentrates in body tissues. Resistance

to biodegradation results in increasingly higher concentrations as these compounds

move upwards through the food chain (ATSDR, 2000). Bioaccumulation,

bioconcentration and biomagnification along the food chain contribute to multiple

health effects in humans and animals. PCBs elicit a range of toxic responses including

acute lethality, carcinogenesis, dermal toxicity, genotoxicity, neurotoxicity and

immunosuppressive effects (ASTDR, 2014). As a result, the use of PCBs has been

restricted worldwide and they are categorised as a priority persistent organic

pollutant (POP) under the Stockholm Convention (Bedard et al., 2007; UNEP, 2009).

Among the approximate 1.3 million tons of cumulative global production until 1993,

about one third of PCBs are estimated to have been emitted to the environment prior

to restrictions being imposed. Emissions were due to releases from equipment still in

use and emissions from contaminated waste, contaminated sites and disposal areas

(Breivik et al., 2007; Li et al., 2010). This problem has led to extensive contamination

of all environmental media including soil, sediment and water through accidental

releases and inappropriate disposal.

Remediating soils contaminated with PCBs is a challenging task. The most widely used

method for treatment of PCB contaminated soil is high temperature incineration.

Additionally, various physical, chemical and biological treatment technologies have

16 Chapter 2: Bioremediation of Polychlorinated biphenyls

been investigated (Gomes et al., 2013). Microwave generated steam technology,

dechlorination using metals, base catalyzed decomposition, thermal desorption,

mechanochemical destruction and electrolysis are some of the novel physical and

chemical remediation technologies investigated (Liu et al., 2014; Qi et al., 2014;

Cagnetta et al., 2016). However, these technologies adopt harsh reaction conditions

such as high temperature, pressure, radiation and extreme pH, which have led to loss

of soil fertility (Sharma et al., 2014). PCBs in soils are relatively low in concentration,

being generally less than 1% of the contaminated mass, even at severely

contaminated sites (Passatore et al., 2014). While chemical and physical methods are

economically viable for the destruction of concentrated PCBs in oils and equipment,

the ability of these technologies to effectively treat large volumes of soil with low

concentrations of PCBs is questionable (Passatore et al., 2014).

Use of microorganisms for remediation of PCB-contaminated soil has been widely

investigated (Chuanmin et al., 2015). Microorganisms have the potential to break

down complex contaminants without transferring the pollution to another

environmental medium such as water, sediment or air. However, field scale

bioremediation approaches are not yet widely used as the effectiveness of using

microorganisms to treat PCB contaminated environments is determined by a number

of factors: (1) the nature of PCB mixture; (2) microorganisms involved; and (3) the

properties of the contaminated environment. Therefore, this critical review of

research literature specifically focuses on bringing together the current state of

knowledge on the properties of PCBs, known metabolic pathways, contributing

factors, their limitations as well as the enhancement opportunities in bioremediation

to identify knowledge gaps that hinder bioremediation.

2.2 Overview of polychlorinated biphenyls

2.2.1 Structure, properties and applications

Polychlorinated biphenyls comprise of two benzene rings joined by a carbon-carbon

bond, and on any or all of the remaining 10 carbon atoms, hydrogen atoms are

substituted with chlorine atoms (see Figure 2.1). Depending on the positions where


the substituted chlorine is located, chemical characteristics of biphenyls can be

altered. Hence, it is important to use a standard naming convention to distinguish

different chlorinated biphenyls. In the standard convention, the positions of carbons

are numbered 1 to 6 on one ring, and 1′ to 6′ on the other. 2, 2′, 6, and 6′ positions

are called as “ortho,” 3, 3′, 5 and 5′ positions as “meta” while 4 and 4′ positions are

called as “para” (WHO, 2016). Depending on the number of chlorine substituents and

their ortho, meta and/or para positioning in the biphenyl structure, 209 different

chlorinated compounds are possible as outlined in Table 2.1. These compounds are

commonly referred to as “congeners” (ATSDR, 2000). Based on the degree of

chlorination, PCBs with equal number of chlorines are called “homolog” (Robertson

& Hansen, 2015). Homologs that have different substitution patterns are called

“isomers” (Srirangam, 2007).

Figure 2.1 Structural form of PCB. Clx and Cly are number of chlorines attached to

each benzene ring and x + y = 1 to 10. Adapted from Wiegel and Wu (2000).

Physical and chemical characteristics such as low water solubility, low vapor pressure,

low electrical conductivity, non-explosive nature, high solubilisation in nonpolar

solvents, great thermal conductivity, high thermal and chemical stability and high

ignition temperature have made PCBs suitable for many industrial applications (Beyer

& Biziuk, 2009). As a result, from 1930 to 1993, PCBs were mass-produced worldwide

as complex mixtures comprising 60 to 90 different congeners (ATSDR, 2000; Breivik

et al., 2007). They were sold in different trade names, including Aroclor, Pyranol,

Pyroclor (USA), Clophen, Elaol (Germany), Fenchlor, Apirolio (Italy), Fenochlor


(Spain), Kanechlor, Santotherm (Japan) Phenoclor, Pyralene (France) and Sovol

(USSR) (IOMC, 2003). Among them, most commonly known and used PCB mixtures

were manufactured under the trade name Aroclor (USEPA, 2013). Approximately

48% of these commercial PCB mixtures were used as transformer oil, 21% for small

capacitors and 10% for other closed systems such as heat transfer systems and

hydraulic systems. Approximately 21% were used for open applications as

caulks/sealants, paints, plasticizers, anti-corrosion coatings, copy paper and flame

retardants (Breivik et al., 2007).

Table 2.1 PCB homologs and their chlorine substitutions

As there are 209 congeners with differing chemical structures, having a standard

nomenclature to identify and label PCBs is important. Currently, International Union

of Pure and Applied Chemistry (IUPAC) and Ballschmiter & Zell (1980) systems are in

use (IOMC, 2003). The IUPAC nomenclature is primarily based on identifying the

positioning of chlorine atoms in the aromatic carbon structure and lists the sequence

Chlorine

Substitutions

Homolog

group

Molecular

weight

Chlorine

(%)

Number of

Possible

Isomers

PCB

number

Mono C12H9Cl 154.1 19 3 1 - 3

Di C12H8Cl2 188 32 12 4 -15

Tri C12H7Cl3 222 41 24 16 -39

Tetra C12H6Cl4 256 49 42 40 -81

Penta C12H5Cl5 289.9 54 46 82 -127

Hexa C12H4Cl6 323.9 59 42 128 -169

Hepta C12H3Cl7 357.8 63 24 170 -193

Octa C12H2Cl8 391.8 66 12 194 -205

Nona C12HCl9 425.8 69 3 206 -208

Deca C12Cl10 459.7 71 1 209

Total 209


in name convention. For example, the PCB congener with chlorine atoms in 3, 4, 5,

and 3’, 4’ carbon positions is identified as 33’44’5 pentachlorobiphenyl (see Figure

2.1 for positioning).

In the Ballschmiter & Zell (1980) naming system, congeners are numbered from PCB

1 through PCB 209 depending on the structural arrangement of the PCB congeners in

an ascending order of the number of chlorine substitutions within each sequential

homologue. The commercial PCB mixture Aroclor is identified by a four digit number.

The first two digits represent the 12 carbon atoms in the biphenyl rings and the last

two digits represent the percentage of chlorines in the PCB mixture (Borja et al.,

2005). As an example, in the commercial PCB mixture Aroclor 1260, 12 indicates the

12 carbon atoms and 60 indicates the presence of 60% chlorine by weight.

2.2.2 Influence of PCBs on human and ecosystem health

Low solubility in water and high adsorption capability to soil make PCBs stable in the

environment (Aken et al., 2009). Once contaminated, it is extremely difficult to

remove PCBs from sediment and soil matrices. They are highly soluble in non-polar

organic solvents and biological lipids (US EPA, 1980). Para and meta saturated

congeners are the most non-polar and hence most soluble in lipids (IOMC, 2003). This

extreme solubility of PCBs in oils and fats leads them to bioaccumulate in animal cells

and pass through the food chains (Passatore et al., 2014). Because of this, PCBs are

considered as one of the most extensively distributed chlorinated chemicals in the

food chains (Borja et al., 2005). Among all, ortho chlorinated congeners have the

highest solubility in water, which is attributed to the hydrogen bonding associated

with the more polar character of the molecules (IOMC, 2003). Solubility of PCBs in

aqueous media decreases with increase in the degree of chlorination. Mono

chlorobiphenyl with one chlorine atom has solubility of 6 ppm while the octa

chlorobiphenyl with eight chlorine atoms is 0.007 ppm (Borja et al., 2005).

After entering the body, PCBs are reported to be transported through the blood

stream and accumulate in liver, muscles and adipose tissues (Borja et al., 2005). It

was revealed through various research studies that there is a possibility of having


multiple health impacts in exposed animals and humans including immune system

suppression, decrements in cognitive and neurobehavioral function, disruption of sex

steroid and thyroid function (Kjellerup et al., 2012; Yang et al., 2015). The extent of

accumulation and their health effects can vary based on the extent of exposure,

variances in the diet and metabolic processes, age, gender, size, season, physiological

condition and the area of the body where PCBs are concentrated (Brazova et al.,

2012). Worldwide monitoring programs have shown that PCBs are present in breast

milk, where illnesses and symptoms related to PCB poisoning can be shown from a

very young age (Lauby-Secretan et al., 2013). The International Agency for Research

on Cancer (IARC) has categorized PCBs as group I human carcinogens in 2013, based

on the evidence of carcinogenicity in humans and experimental animals (Chen et al.,

2015b).

2.2.3 Regulations and monitoring

In 2001, PCBs were listed as one of the twelve persistent organic pollutants by the

Stockholm Convention (UNEP, 2009). Parties to the Convention are obliged to

eliminate equipment and oils containing PCBs from use by the year 2025 and treat

them under environmentally sound waste management by 2028 (UNEP, 2009). In

Australia, material or waste containing PCBs at a concentration of more than 2 mg/kg

are subjected to regulation (VEPA, 2017). According to Canadian soil quality

guidelines for total PCBs, 0.5 mg/kg for agricultural soil, 1.3 mg/kg for residential soil

and 33 mg/kg for commercial and industrial soil are the recommended acceptable

background concentrations (CCME, 1999). PCBs are also listed as priority organic

pollutants by the United States Environmental Protection Agency (USEPA) (Sowers &

May, 2013). Clean up and disposal of soil and sediments contaminated with PCBs are

regulated under Toxic Substances Control Act (TSCA). The date of contamination, the

concentration in the source of PCB as well as the current PCB concentration in the

contaminated mass are taken into consideration during this process. If PCB level is ≥

50 mg/kg in any soil or sediments, they are regarded as PCB remediation waste and

regulated by the TSCA. Moreover, if the PCB level in soil or sediments is between 2

to 50 mg/kg from a spill after 1978 from a source that contains ≥ 50 mg/ kg or from


a source that is unauthorized for use, such contaminated soil or sediment are

regulated as PCB remediation waste (Davila et al., 1993).

For the determination of PCBs in soil, reliable sample collection, storage, extraction,

clean-up, detection and quality assurance techniques are needed (Muir & Sverko,

2006). Ultrasonic extraction and microwave-assisted extraction are superior to the

traditional Soxhlet extraction due to better recovery, and precision (Camel, 2000;

Arulpriya & Lalitha, 2013). For qualitative and quantitative analyses, gas

chromatographic methods based on single column and single electron capture

detector (ECD) were developed in early stages and later upgraded to double column

- double ECD and mass spectrometry detection capabilities (Muir & Sverko, 2006;

USEPA, 2007; Chuanmin et al., 2015).

2.3 Removal of PCBs from contaminated soil

2.3.1 PCBs as a soil contaminant

Soil is considered as a significant environmental sink for PCBs due to their strong

sorption potential with soil colloids and resistance to physicochemical degradation

and biodegradation. Sorption of PCBs to soil particles primarily depends on the

degree of chlorination of the PCB congeners and the properties of soil such as, type

of soil, organic matter content, soil moisture content and pH (CCME, 1999). These

factors are discussed in detail in section 2.5.

Atmospheric transport and deposition of PCBs is regarded as one of the major

sources of surface soil contamination (ATSDR, 2000). This was further confirmed by a

study based on 191 global background surface soils (up to 5 cm depth) collected from

sites, far away from human activities. (Meijer et al., 2003). The study further revealed

that the estimated total global PCB level only in background soils is 21,000 tons. As

summarized in Figure 2.2, total PCBs in background soil are highly variable (ranging

from 0.04 to 100 ng/g dry weight; mean, 4.9 ng/g dry weight) with highest

concentrations in Europe and North America (Li et al., 2010). Although PCB data

coverage is relatively good for Europe, East Asia, and the Great Lakes region of North

America, in many parts of the world such as Africa, Australasia, central and northern


Asia, South America, and the Caribbean region, data is relatively scarce (Li et al.,

2010). However, as given in the Table 2.2, the highest concentrations of PCBs in soils

are in the localized regions close to their production, application and disposal

(Vasilyeva & Strijakova, 2007).

Figure 2.2 Concentrations of total PCBs in surface soils at global background sites (Li

et al., 2010).

A recent study conducted by Desborough et al. (2016) found that the total PCB levels

in UK soils (average 4.7 ng/g, range 0.39 - 21 ng/g) have not significantly reduced

when compared to the levels reported in the mid-1980s. They suggests that the rate

of decline of concentrations of PCBs in soils are slower than expected due to the

continuing emissions from the remaining stocks in use in the built environment

(Desborough et al., 2016).


Table 2.2 Comparison of PCB levels (µg/kg dry weight) in soils based on selected

international studies.

Location PCBs, as mean value

and (range)

Number of

samples and

(depths)

Reference

Central Region, Ghana

7.99 ± 3.8 (1.32 -12.94)a

9.15±0.52

7.55±0.56

7.82±0.55

n=78

(0-10 cm)

(10-20 cm)

(20-30 cm)

(Bentum et al., 2016)

Dump site, Italy 920 x103 NM (Di Toro et al., 2006)

Ex-landfill site, Brighton, UK

(1.7 - 13.2) b n=48, (20 cm) (Zhou et al., 2014)

UK 4.7 (0.39-21) n=24, (5 cm) (Desborough et al., 2016)

Cambridgeshire, UK

5.03(0.27 - 80.5)c n=201, (NM) (Heywood et al., 2006)

England, Wales, Scotland, UK

31.8 (1.7 – 1199) n=100, (5 cm) (Creaser et al., 1989)

Dump site, Czech Republic

705.95±22.85 x103, d

375.81±19.45 x103, e

n=10, (2 m)

n-10, (10 cm)

(Stella et al., 2015)

Switzerland (0.86 – 12)f n=23, (10cm) (Schmid et al., 2005)

South Sweden (2.3 - 986) n=66, (5 cm) (Backe et al., 2004)

Dalian, Liaoning Province, China

2.8 (1.3 - 4.8)g n=14, (5 cm) (Wang et al., 2008)

KwaZulu-Natal, South Africa

109.64±116.07h

19.22±33.23i

n=15, (0.5 cm)

n=15, (1–2 cm)

(Batterman et al., 2009)

Notes:

n – number of samples, NM – Not mentioned a 31.89% hexa, 23.98% penta, 18.47% tri, 13.67% tetra & 11.99% hepta PCBs b All PCB congeners were present except PCB 18 c Sum of 33 PCB congeners with 9% PCB 153, 8.6% PCB18, 8.8% PCB28 & 7% PCB138. d 16.6% PCB 56, 9.8% PCB 52, 9.6% PCB 44 e 20.9% PCB 56 f Sum of 7 PCBs g Sum of 57 PCBs h, i As wet weight of 38 congeners, most prevalent PCBs - 41+ 71, 153+132, 138 +163


2.3.2 Remediation of PCB contaminated soil

Bioremediation is defined as a process that uses microorganisms, green plants or

their enzymes to treat the polluted sites for regaining their original condition (Glazer

and Nikaido, 1995). It is considered very promising as natural biological activities are

utilized either to partially degrade the contaminants to less harmful products or to

completely mineralize them (Tomei & Daugulis, 2013; Sharma et al., 2014). Generally,

biological treatments are long-term processes as they take a long time to show

satisfactory performance. However, in comparison to the other physical and chemical

treatment methods, it is less expensive and environmentally friendly while having

higher public acceptance (Busset et al., 2012; Tomei & Daugulis, 2013; Sharma et al.,

2014). In bioremediation approaches, in-situ (on site) bioremediation is preferred

over ex-situ (off site) as it involves the treatment of polluted material at the site

without removing the contaminated soil for ex-situ treatment.

2.3.3 In-situ bioremediation

In-situ bioremediation consists of the use of green plants (phytoremediation),

microorganisms (fungi and bacteria), or their enzymes to treat the polluted materials

at the site and is discussed in section 2.3.3.1 to 2.3.3.3.

2.3.3.1 Phytoremediation

In phytoremediation, plants and their associated microorganisms are used for the

treatment of contaminated soil (Aken et al., 2009). Extensive laboratory and

greenhouse studies have been undertaken to remediate PCB contaminated soils

through different phytoremediation approaches that are summarized below.

Phytoextraction is to grow suitable plant varieties capable of accumulating significant

amounts of the contaminant from the soil and storing it in the plant shoots. These

plants with high PCB levels can then be harvested and treated using suitable method

such as high temperature incineration (Aslund et al., 2007; Anyasi & Atagana, 2014;

Luo et al., 2015). This method cannot be regarded as a direct treatment option as it

use plants as a vector to physically transfer the contaminants from one place to other.


In phytotransformation, once the pollutants are absorbed by the plant through the

root system, enzymatic breakdown of pollutants takes place inside the plant tissues

(Aken et al., 2009). However, plants generally lack the enzymes that are necessary to

achieve complete degradation of recalcitrant organic compounds and therefore,

results in slow and incomplete degradation (Eapen et al., 2007).

In contrast, rhizoremediation is more effective as some secondary plant metabolites,

which are structurally similar to contaminants are released by plant roots. They

stimulate the growth of PCB degrading microbial community associated with the root

zone (Meggo et al., 2013; Liang et al., 2014; Jha et al., 2015; Liang et al., 2015).

Microorganisms utilize these secondary plant metabolites such as naringin, myricetin

and flavonone as their growth substrate while inducing PCB cometabolism (Leigh et

al., 2006; Musilova et al., 2016). In cometabolism, contaminants which cannot serve

as the primary substrate to provide energy for the microorganisms are degraded

cometabolically while utilizing some other substrate as their primary substrate

(Musilova et al., 2016). Fully developed roots and rhizospheres in switchgrass

(Panicum virgatum) and poplar (Populus deltoids x nigra) planted microcosms have

shown better biotransformation potential than the unplanted reactors (Meggo &

Schnoor, 2013). Table 2.3 summarises recent studies based on plant-microbe

combinations in PCB degradation. Plant growth-promoting rhizobacteria can survive

and multiply under extreme weather and climatic conditions while improving the

plant biota against stress imposed by contaminants and increase their

biomass/efficiency to take up contaminants (Asad, 2017).

Higher degradation rates in the mesocosms of switchgrass rhizospheres

bioaugmented with PCB degrading bacteria suggest that the use of phytoremediation

and bioaugmentation in combination could be an efficient and sustainable strategy

for the treatment of PCB contaminated soil (Liang et al., 2015). However, success in

remediation relies on several factors such as selection of suitable plant - microbial

combinations based on soil conditions, level of contamination and bioavailability of

PCBs as well as maintenance of appropriate conditions such as pH, moisture, and

other growth requirements. Most of the available information are based on small-

scale research studies conducted in confined environments such as hydroponics and


greenhouses. Performance under full scale is not yet known as full scale studies are

limited to a few trials and are not yet completed due to the long time span required

for plant based remediation approaches (Passatore et al., 2014). Furthermore, as a

contaminant, PCBs are frequently associated with other environmental contaminants

like dioxins and heavy metals. Therefore, an in-depth knowledge is needed to

understand the effects exerted by these co-contaminants on microorganisms and

plants associated with PCB remediation (Wang & He, 2013a).


Table 2.3 Plant–microbial relationships associated with PCB degradation

Microorganism/s Plant PCB reduction % Type of treatment Reference

Pseudomonas putida

Stenotrophomonas maltophilia

Mustard (B. juncea) 7.2–30.3% after 2

months

Greenhouse pots with

biochar as bio-carrier

(Pino et al., 2016)

Actinobacteria sp.

Chloroflexi sp.

Alfalfa 31.4% in 1 yr.

78.4% in 2 yrs.

Contaminated site (Tu et al., 2011)

Rhodococcus sp.

Burkholderia sp.

Austrian pine (P. nigra)

Goat Willow (S. caprea)

- Contaminated site (Leigh et al., 2006)

Geobacter sp. Switchgrass (Panicum

virgatum)

30–40 % after 6 months Soil microcosms (Liang et al., 2015)

Bioaugmentation with Burkholderia

xenovorans LB400

Switchgrass (P. virgatum) 47.3 ± 1.22% after 6

months

Soil microcosms (Liang et al., 2014)

Pseudomonas sp.

Agrobacterium sp.

Ochrobactrum sp.

Horseradish (A. rusticana)

Goat willow (S. caprea)

28.0 %

31.8 % after 6 months

Open air pots

(Ionescu et al., 2009)

Mixed microbial consortium Poplar > 90% after 14 months Contaminated site (Ancona et al., 2016)


2.3.3.2 Fungal bioremediation

Knowledge on the potential of applying fungi for PCB breakdown and their

occurrence in contaminated soils is limited. Penicillium chrysogenum, Penicillium

digitatum, Scedosporium apiospermum, and Fusarium solani have been found to

display rapid growth in mineral media containing PCB and glucose while degrading

PCBs (Tigini et al., 2009). However, none of the taxa was able to grow on PCBs as their

sole source of carbon. In microbial community analysis of soil historically

contaminated with high concentrations of PCBs, the microbial community was found

to consist of both bacteria and fungi although their individual contribution towards

PCB degradation was not known (Stella et al., 2015). Fate of some selected mono (2-

chlorobiphenyl and 4-chlorobiphenyl), tri (3,4,5-trichlorobiphenyl) and penta

(3,3`,4,4`,5-pentachlorobiphenyl) chloro biphenyls in a pure culture of Aspergillus

niger were determined. Only 2-chlorobiphenyl and 4-chlorobiphenyl were

transformed to hydrophilic metabolites with 38 and 52 % reductions, respectively,

while there was very low reduction of trichlorobiphenyl (2%) and no observable

reduction of pentachlorobiphenyl concentration (Kim et al., 2016). This result

suggests that the degradation ability of A. niger is limited to simple monochlorinated

PCB congeners.

Federici el al (2012) reported 33.8% overall PCB removal in soils contaminated with

Aroclor 1260 after bioaugmentation with maize stalk-immobilized Lentinus tigrinus.

However, the incubation control which only consisted of residential microflora also

demonstrated 28% overall PCB degradation (Federici et al., 2012). It is not clear that

the 5.8% difference between the L. tigrinus bioaugmented and control microcosms

was due to the degradation ability of L. tigrinus itself, its indirect contribution to

proliferate the residential soil microflora or direct absorption of PCBs into the fungal

mycelium. Other than the degradation, translocation of PCBs into fruiting bodies

were observed in white rot fungus Pleurotus ostreatus (oyster mushroom) growing

on straw spiked with a Delor 103 commercial PCB mixture (Moeder et al., 2005).

Pandey et al (2016) suggest that the metabolism of PCB compounds and their

metabolites by the ligninolytic enzymes secreted by white rot fungi is due to the


structural similarities between PCB molecules and lignin molecules (Pandey et al.,

2016).

It can be concluded that the fungal action takes place primarily in the extracellular

environment due to the secretion of enzymes to the external environment (Passatore

et al., 2014). Moreover, fungal based degradation seems to be most effective against

the lower chlorinated PCBs. Assessing the applicability of some fungal strains, which

have the ability to live in symbiosis with plant roots, for remediating PCB

contaminated soils will be beneficial, as they can survive under extreme

environmental conditions which would be an added advantage in remediation

applications.

2.3.3.3 Bacterial bioremediation

When compared to plants (Section 2.3.3.1) and fungi (Section 2.3.3.2), bacteria are

the most widely studied microorganisms in PCB bioremediation due to their

ubiquitous distribution in the environment and the ability to degrade lower and

highly chlorinated congeners by numerous bacterial strains through different

anaerobic metabolism, aerobic co-metabolism and aerobic metabolism pathways

(Passatore et al., 2014). Though the findings of many research studies are available,

bacteria based bioremediation of PCBs is not yet widely practiced as a field scale

treatment technique. Therefore, bacterial bioremediation of PCBs was chosen in the

current study as a way forward to minimize the gaps in available knowledge.

In the following sections, different types of biodegradation pathways, known types

of bacteria with PCB degradation potential, and their applications are discussed.

2.4 Biodegradation pathways

In PCB degradation, two distinctive microbial processes have been identified:

anaerobic reductive dechlorination; and aerobic oxidative degradation. The higher

chlorinated biphenyls are often subjected to anaerobic dechlorination while the

lower chlorinated biphenyls are often subjected to aerobic oxidation (Demirtepe et

al., 2015).


2.4.1 Anaerobic reductive dechlorination

The conversion of highly chlorinated congeners (congeners with five or more chlorine

atoms) to less chlorinated congeners (congeners with four or less chlorine atoms) by

replacing a single chlorine atom by a hydrogen atom is termed as dechlorination

(Hughes et al., 2009). This process reduces the potential toxicity and bioaccumulation

of highly chlorinated congeners. As illustrated in Equation 2.1, in the reductive

dechlorination process, PCBs are used as electron acceptors and the chlorine is

replaced with hydrogen (Borja et al., 2005).

R⎼Cl + 2e¯+ H+→ R⎼H + Cl¯ Equation 2.1

Anaerobic dechlorination of PCBs was detected in the sediments of the Hudson River,

Massachusetts (Furukawa & Fujihara, 2008). An escalation in the lower chlorinated

congeners and a reduction in the highly chlorinated congeners were observed when

analyzing the PCBs inanaerobic sediments (Borja et al., 2005). a similar trend was

exhibited during a laboratory study based on electronic waste contaminated soil

using dissimilatory Fe(III) reducing and arylhalorespiring bacteria (Song et al., 2015).

Specialized populations of microorganisms with distinct dehalogenating enzymes

have the potential to carry out reductive dechlorination. So far, several anaerobic

dechlorinating bacteria have been isolated. The microorganisms frequently belong to

the Dehalorespiring Chloroflexi group, such as Dehalococcoides spp. (Adrian et al.,

2009; Praveckova et al., 2015) and Dehalobium spp. (Payne et al., 2011). In addition,

Bacteroides and Betaproteobacterium, Pseudomonas (Bedard et al., 2006),

Desulfomonile, Desulfitobacterium, Dehalobacter, Dehalospirillum, Desulforomonas

(Borja et al., 2005) have also been identified as being able to undertake

dechlorination of PCBs. Furthermore, dissimilatory iron-reducing bacteria (DIRB)

belonging to Geobacteraceae are strictly anaerobic and capable of utilizing

chlorinated hydrocarbons directly as terminal electron acceptors (Song et al., 2015).


2.4.1.1 Anaerobic reductive dechlorination pathways

Studies conducted to investigate anaerobic dechlorination suggest that different

microbial populations are responsible for different dechlorination patterns based on

the number and position of chlorine substitutions in the biphenyl structure (Beyer &

Biziuk, 2009). These patterns confirm that dechlorination occurs mainly to meta and

para substituted chlorines producing less chlorinated, mostly ortho-substituted

products as illustrated in Figure 2.3. The rate of dechlorination of meta or para

positions increases when chlorine ions are available in the neighbouring positions.

When there are two chlorines in adjacent positions, it is called as doubly flanked

chlorine and when a single chlorine ion is present in one neighbouring position, it is

referred to as flanked chlorine. The descending order of preference for

dechlorination is reported in the form of doubly flanked chlorine > singly flanked

chlorine > unflanked chlorine (Wiegel & Wu, 2000).

Figure 2.3 Potential pathway for anaerobic dechlorination of highly chlorinated

congeners. Adapted from Borja et al. (2005).

However, the dechlorination order could vary depending on the types of the

microbial population present (Bedard et al., 2007). Dehalogenation is found to be

microbial species specific in terms of the substitution positions attacked and the

numbers of chlorine atoms removed. This conclusion is based on the observations of

different microbial populations generating different dehalogenation patterns under


different environmental conditions (Gomes et al., 2013; Song et al., 2015). Based on

congener loss and product accumulation, eight distinct reductive dechlorination

pathways have been recognized so far as M, Q, H’, H, P, N, LP and T (Wiegel & Wu,

2000) (see Table 2.4). It was observed that the removal of chlorines is highly selective

in each dechlorination pathway. As an example, in the pathway P, para chlorines

flanked by at least one meta chlorine were selectively removed from the biphenyl

molecule while the removal of meta chlorines flanked by at least one chlorine in

either the para or the ortho position happened in Pathway N (Bedard & May, 1995).

Table 2.4 Microbial dechlorination pathways (Wiegel & Wu, 2000).

Dechlorination Pathway Chlorines Removed

M Flanked and unflanked meta

Q Flanked and unflanked para, meta of 23-group

H’ Flanked para, meta of 23- and 234-groups

H Flanked para, doubly flanked meta

P Flanked para

N Flanked meta

LP Flanked and unflanked para

T Flanked meta of 2345-group, in hepta and octa

chlorobiphenyls

Imamoglu, et al., (2002) introduced an anaerobic dechlorination model (ADM). ADM

(Imamoglu et al., 2002) helps to identify the possible anaerobic dechlorination

patterns and to quantify the relevant dechlorination pathways in sediment

mesocosms. The model was further improved by Demirtepe et al. (2015), to identify

the possible anaerobic dechlorination patterns and to quantify the relevant

dechlorination pathways. In the modified ADM (Demirtepe et al., 2015),

dechlorination pathways are defined by the targeted chlorines (i.e. meta and para

positions, either as singly or doubly flanked) unlike the dechlorination pathways

based on congener loss and product accumulation (Wiegel & Wu, 2000).

Improvements to the previously modified ADM model have enabled a better


understanding of multiple dechlorination activities and co-elution of congeners with

increased accuracy, speed and flexibility by incorporating constraints and a new

goodness of fit evaluation, as reported by Karakas & Imamoglu (2017).

2.4.2 Aerobic oxidative degradation

Aerobic degradation typically occurs in topsoil and sediment layers. As noted by Field

& Sierra-Alvarez (2008), lower chlorinated congeners and the products of anaerobic

reductive dechlorination are subjected to aerobic degradation leading to complete

mineralization (Field & Sierra-Alvarez, 2008). Microbial oxidation of low (mono to

tetra) chlorinated biphenyls is catalysed by aerobic bacteria, which are capable of

degrading biphenyls as their sole source of carbon and energy (Pieper & Seeger,

2008). Various gram negative bacteria such as Acinetobacter, Alcaligenes,

Achromobacter, Burkholderia, Comamonas, Pseudomonas, Ralstonia, Sphingomonas

and gram positive bacteria such as Bacillus, Corynebacterium and Rhodococcus are

responsible for aerobic oxidative degradation of PCBs (Furukawa & Fujihara, 2008;

Hassan, 2014). Complete genomes of Burkholderia xenovorans LB400 and

Rhodococcus jostii RHA1, two of the most widely studied and characterised PCB-

metabolizing strains contributed to improve knowledge on their overall metabolism,

physiology, and evolution as well as their defence mechanisms against PCB toxicity

(Atago et al., 2016; Agullo et al., 2017).

Some intermediate products such as dihydrodiols and dihydroxybiphenyls that are

produced during the PCB degradation by diverse bacteria, including Burkholderia sp.

are referred to as dead-end intermediates as they are highly toxic to bacteria (Camara

et al., 2004). The reason for the toxicity is associated with the increased polarity of

these dihydroxylated metabolic intermediates as it make them more soluble in

aqueous medium (Seeger & Pieper, 2010). Only a few microorganisms have been

reported so far with the capacity to completely mineralize PCBs without

accumulating dead-end intermediates. A novel bacterium, Sphingobium fuliginis was

found to degrade biphenyl, lower chlorinated (mono, di and tri) PCBs and mono

chlorobenzoates without accumulation of dead-end intermediates (Hu et al., 2015).

More recently, Qui et al. (2016), demonstrated that after a 40% inoculation volume,


Comamonas testosteroni was capable of completely degrading 100–500 mg/L

decachlorobiphenyl PCB209 as the sole carbon source in 140 hr at low temperature

(10 °C ±0.5 °C) and pH 7–8. The strain was also effective in practical applications of

PCB209 biodegradation in contaminated soil (Qiu et al., 2016).

The aerobic oxidative degradation of PCBs occurs through two distinctive pathways.

Firstly, less chlorinated congeners are converted to the corresponding chlorinated

benzoic acid through the upper biphenyl pathway (Field & Sierra-Alvarez, 2008) as

illustrated in Figure 2.4. Chlorobenzoate is further mineralized to carbon dioxide and

inorganic chlorides through lower biphenyl pathways (ATSDR, 2000; Field & Sierra-

Alvarez, 2008) as illustrated in Figure 2.5 a and b. Utilization of biphenyl, an

unchlorinated analogue of PCBs and benzoate, an intermediate of biphenyl

degradation pathways are mainly performed by distinct bacterial groups with

different ecological growth strategies (Leewis et al., 2016). However, only a small

proportion of the microbial community have demonstrated their ability to assimilate

carbon from both biphenyls and benzoates. This may be due to the possessing of

enzymes that catalyses biochemical reactions in both upper and lower biphenyl

pathways.

2.4.2.1 The upper biphenyl degradation pathway

Four key enzymes are involved and play major roles in the upper biphenyl

degradation pathway (see Figure 2.4).


Figure 2.4 The upper biphenyl degradation pathway. Modified from Field and Sierra-Alvarez (2008).


(A) Biphenyl-2,3-Dioxygenases (BphA)

Biphenyl dioxygenases initiate the oxidation of polychlorinated biphenyls. The

enzyme belongs to the toluene / biphenyl branch of Rieske non-heme iron

oxygenases (Pieper & Seeger, 2008). It catalyses the formation of chlorinated dihydro

biphenyl-2,3-diol. It was revealed that there are considerable differences in various

biphenyl 2,3-dioxygenases for their congener selectivity patterns, as well as their

preference for the attacked ring structure (Seeger & Pieper, 2010). The order of

preference for the deoxygenation on the biphenyl pathway has been observed based

on Burkholderia xenovorans strain LB400 as; unsubstituted > 2-chloro > 2,5-dichloro

> 2,4-dichloro > 3-chloro > 4-chloro > 2,3-dichloro (Pieper & Seeger, 2008). The bphA1

gene encodes the large subunit of biphenyl 2, 3-dioxygenase enzyme. Major amino

acid differences are found in this subunit in different PCB degrading bacterial species.

This feature may explain the reason for the affinity of different bacteria for different

PCB congeners (Furukawa & Fujihara, 2008; Hoostal & Bouzat, 2016). The presence

of bphA1 gene has been used as a measure of genetic potential for PCB

biodegradation (Dercova et al., 2008).

(B) Cis-2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase (Cis-dihydrodiol

dehydrogenase) (BphB)

BphB is responsible for the dehydrogenation of cis-2,3-dihydro-2,3-

dihydroxychlorobiphenyls to 2,3 –dihydroxy chlorobiphenyl in the second step of the

upper biphenyl pathway (Furukawa & Fujihara, 2008). They are short chain alcohol

dehydrogenases and show common features such as an absolute requirement for the

coenzyme nicotinamide adenine dinucleotide (NAD+). This group of dehydrogenases

show broad substrate specificity and oxidized the cis-di-hydrodiols derived from

biphenyl and other polycyclic hyrocarbons to catecols through various aromatic

degradation pathways (Jouanneau & Meyer, 2006).

(C) 2,3-dihydroxybiphenyl-1,2-dioxygenase (BphC)

This is the critical step in upper biphenyl degradation pathway as 2, 3-

dihydroxybiphenyl-1,2-dioxygenase catalyses aromatic ring cleavage (Dai et al.,


2002). 2,3 –dihydroxy chlorobiphenyl is converted into a yellow meta-cleavage

product called chlorinated 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA)

by the enzyme 2,3-dihydroxybiphenyl-1,2-dioxygenase (Field & Sierra-Alvarez, 2008).

This enzyme belongs to type I extradiol dioxygenases (Pieper & Seeger, 2008). The

development of yellow coloration from the meta cleavage product was used to

screen the bacteria capable of producing dioxygenases as they are potential

candidates for PCB degradation (Dercova et al., 2008). Though they differ in substrate

specificity, BphC enzymes have the ability to transform various chloro substituted

derivatives into meta cleavage products (Dai et al., 2002). However, it was found that

the presence of 3,4-dihydroxybiphenyl and ortho chlorinated PCB metabolites

strongly inhibited the BphC enzymes responsible for aromatic ring cleavage by

promoting their suicide inactivation (Furukawa, 2006).

(D) 2-hydroxy-6-phenyl-6-oxohexa-2,4-dieneoate (HOPDA) hydrolase (BphD)

The enzyme catalysing the final conversion step for the upper biphenyl pathway is

BphD belonging to α / β hydrolase enzyme superfamily. It hydrolyses HOPDA down

to chlorinated 2-hydroxy-penta-2,4-dienoate and chlorobenzoate (Pieper & Seeger,

2008).

2.4.2.2 The lower biphenyl degradation pathway

Chlorobenzoates and 2-hydroxypenta-2,4-dienoates generated from the upper

biphenyl degradation pathway are further degraded through the lower biphenyl

pathway as shown in Figure 2.5 (a) and (b). Three enzymes called 2-hydroxypenta-

2,4-dienoate hydratase (BphH), 4-hydroxy-2-oxovalerate aldolase (BphI) and an

acylating acetaldehyde dehydrogenase (BphJ) are responsible for the conversion of

2-Hydroxypenta-2,4-dienoate to pyruvate and acetyl-CoA (Seeger & Pieper, 2010).

The resulting acetyl-CoA then enters the tricarboxylic acid (TCA) cycle, which is used

by all aerobic organisms to release their stored energy. Benzoate, the other end

product generated in the upper biphenyl degradation pathway is a growth substrate

for a broad range of bacteria. It can be mineralized to CO2 and H2O either via

chlorocatechol or 3-oxoadipate pathways under aerobic conditions (Pieper, 2005).

https://en.wikipedia.org/wiki/Aerobic_organism


Figure 2.5 The lower biphenyl degradation pathways (a) Mineralization of 2-hydroxypenta -2,4-dienoate. Modified from Field and Sierra-Alvarez

(2008). (b) Mineralization of chlorobenzoic acid. Modified from ATSDR (2000).


2.4.3 Sequential anaerobic-aerobic degradation

To achieve a complete degradation of PCBs, a combination of anaerobic reductive

dechlorination and aerobic oxidation is essential. Recent reviews and studies discuss the

possibility of promising results in biological removal of highly chlorinated PCB congeners

in sediments when anaerobic and aerobic processes are combined (Pieper, 2005; Bedard

et al., 2006; Wang & He, 2013b). The study by Master et al. (2002) observed no PCB

degradation without prior anaerobic treatment in soil slurry mesocosm containing

weathered Aroclor 1260 (Master et al., 2002). During their initial anaerobic treatment,

highly chlorinated congeners of Aroclor 1260 were completely or partially transformed

to less chlorinated congeners. Sequential anaerobic-aerobic composting of 2:3 soil to

waste ratio led to the mineralization of highly chlorinated PCBs in soil, with 25%

reduction of total PCBs and 61% reduction of PCBs containing two to five chlorine atoms

(Long et al., 2015). However, except for some soil–slurry based studies (Tartakovsky et

al., 2001; Master et al., 2002; Rodrigues et al., 2006), limited knowledge is available to-

date in relation to soil based sequential anaerobic-aerobic degradation of PCBs (Long et

al., 2015).

A sediment based study conducted in Guanica Bay, Puerto Rico, contaminated with

relatively high concentrations of PCBs, showed a high relative abundance of Chloroflexi

group, which included known anaerobic PCB-degrading bacteria in bottom sediments

and the presence of prominent aerobic PCB degraders in the intertidal sediments (Klaus

et al., 2016). This is a good example of the occurrence of combination of aerobic and

anaerobic biodegradation processes simultaneously in nature to decontaminate PCB

mixtures. Promising results have been obtained in concurrent anaerobic-aerobic

degradation of weathered Aroclor 1260 contaminated sediment and soil in laboratory

mesocosms (Kjellerup et al., 2012; Payne et al., 2013). Weathered Aroclor-contaminated

sediment mesocosms bioaugmented with anaerobic halorespiring Dehalobium

chlorocoercia and aerobic Burkholderia xenovorans showed 80% reduction in total PCBs

(by mass) after 120 days (Payne et al., 2013). Concurrent anaerobic dechlorination by D.

chlorocoercia and aerobic degradation by B. xenovorans was evident by no substantial

increase in lower chlorinated congeners. Therefore, concurrent degradation would be a

preferred alternative to the sequential anaerobic-aerobic degradation as it more closely


mimics natural attenuation processes. In two stage and concurrent degradation

processes, use of facultative bacteria with the ability of both anaerobic dechlorination

and aerobic degradation of PCBs would be a potential area for future research as they

have the ability to survive under aerobic and anaerobic conditions.

2.5 Factors affecting microbial remediation

The rate, extent, and route of PCB degradation in soil are determined by a variety of

biological, chemical and physical factors as they have a direct impact on the specific

bacterial communities involved in the degradation process and the bioavailability

fraction of PCBs (Camara et al., 2004; Vasilyeva & Strijakova, 2007; Chen et al.,

2015a). The major contributing factors are summarised in Figure 2.6 as presented

below.

Figure 2.6 Overview of factors affecting microbial remediation

2.5.1 Soil properties

Soil is an extremely complex system when compared to other environmental media.

Soil has variable physical, chemical and biological characteristics and such variation

influences the rate of PCB biodegradation in soil. As hydrophobic compounds, PCBs


are strongly sorbed to soil particles. The sorption capacity is determined primarily by

the quantity and quality of organic matter present in soil (Haluska et al., 1995).

Consequently, organic matter content is an important soil parameter that influences

the effectiveness of soil bioremediation as it has a direct impact on the bioavailability

of PCBs. Therefore, organic matter content is one of the rate limiting factors for the

persistence of inoculated microbial strains in soil and their ability to degrade PCBs

(Mrozik & Piotrowska-Seget, 2010). The presence of aromatic carbon in humic acids

also plays an important role in the survival and activity of inoculants (Long et al.,

2015). A recent study on sediments from the Hudson and Grasse river in the USA

revealed that the degree of dechlorination was mainly determined by the sediment

itself, not by the composition of PCBs (Xu et al., 2016). However, very little is known

about the activity of PCB degrading microorganisms in different soil types, though it

is an important requirement in in-situ bioremediation applications.

2.5.2 Environmental factors

Environmental factors such as pH, temperature and moisture levels play a major role

in PCB degradation. Their influences on PCB degradation are discussed below.

2.5.2.1 pH

Concentration of hydrogen cations (H+) plays an important role in the growth and

metabolism of microorganisms. Sediment samples contaminated with Aroclor

mixtures revealed that the optimal pH for the removal of chlorine under anaerobic

conditions was 7.0–7.5 (Borja et al., 2005). Degradation of PCBs in liquid cultures by

Pseudomonas sp. under aerobic conditions also indicated that near neutral pH

conditions were favourable, whereas a lower pH severely inhibited PCB

biodegradation (Chen et al., 2015a). In their study, the degradation efficiencies of

total PCBs were 4.2 %, 57 %, 63.6 %, and 44.3 % at pH 4, 6, 7, and 8 respectively.

Similarly, a 100 % degradation rate of PCB 209 was achieved with the cold resistant

Comamonas testosteroni strain between pH 7 to 8, whereas the degradation rates

were reduced to 80.6 % and 66.6 %, respectively, at pH 9 and 10 (Qiu et al., 2016).


2.5.2.2 Temperature

Temperature can significantly influence the bioavailability of PCBs in the environment

by creating an imbalance between the ratio of PCBs that are in dissolved form and

those adsorbed to organic matter (Asad, 2017). This imbalance can hinder the growth

of microorganisms by having an impact on physiological activities such as uptake and

enzymatic breakdown (Wiegel & Wu, 2000). Investigation of sediment contaminated

with Aroclor 1260 revealed that different optimal temperature ranges were

responsible for different dechlorination patterns (ATSDR, 2000; Borja et al., 2005).

However, it is difficult to draw conclusions solely based on laboratory experiments

where samples are incubated at constant temperature, whereas in nature, soil is

subjected to temperature fluctuations between day and night and seasonally.

Therefore, field scale studies and laboratory scale studies, which mimic real field

conditions, are important to assess the variation of the degradation efficiencies due

to diurnal effects and seasonal climatic changes.

2.5.2.3 Soil moisture

Soil moisture is critical, and a 43% degradation rate of highly chlorinated biphenyls

was obtained at 60% moisture content in anaerobic composting of PCB-contaminated

soil using pig manure (Zhang et al., 2013). Activity of bacteria is reduced when the

soil water content is low as it limits the supply of substrates through diffusion and

causes cell dehydration (Mrozik & Piotrowska-Seget, 2010).

2.5.3 PCB related properties

As a whole, PCBs are relatively insoluble in water, and the solubility decreases with

increased chlorination. As an example, solubility of Aroclor 1216 with 16% chlorines

is 0.42 mg/L in water while the solubility of Aroclor 1260 with 60% chlorine is 0.003

to 0.08 mg/L (ATSDR, 2000). The number and position (ortho, meta and para) of

chlorine atoms in a PCB molecule is directly related to the type, extent and pathway

for bioremediation. PCB congeners having chlorines in one biphenyl ring breakdown

more easily than those having chlorines in both rings, while the congeners with


double ortho substituted chlorines (chlorine at position 2, 6- or 2, 2′) are hard to

degrade (Furukawa & Fujihara, 2008). The preferred positions for overall

dechlorination were found to be, meta > para > ortho, while the ortho and double

flanked meta chlorines were primarily targeted followed by single and double flanked

para chlorines (Xu et al., 2016).

Effective rates for PCB dechlorination have been found to happen in the range of

100–1,000 mg/kg (wet weight) concentration in contaminated sediments (ATSDR,

2000). Concentrations above 1000 mg/kg may cause toxicity in bacterial communities

(Passatore et al., 2014). Conversely, at concentrations lower than 50 mg/kg, the

dechlorination process significantly reduced due to the insufficient induction of the

degradative enzymes and difficulty in sustaining the growth of competent organisms

(Passatore et al., 2014). However, it has been observed in some laboratory based

experiments that the reductive dechlorination of many PCB congeners in commercial

PCB mixtures take place even when their individual concentrations are less than 1

mg/kg (ATSDR, 2000). This finding is significant as it demonstrates the ability of PCB

dechlorinating microorganisms to remove the traces of PCBs from contaminated

environments.

2.5.4 Natural microbial diversity

The bioremediation of PCBs is possible due to the activities and interactions of

aerobic and/or anaerobic heterotrophic microorganisms present in soil. Natural

bioremediation of soils contaminated with PCBs is frequently affected by low

bioavailability and the inadequacy of PCB degrading autochthonous microorganisms

(Di Toro et al., 2006). Higher microbial density and diversity can be expected in

surface soil than in the sub surface soil due to the high organic matter content created

by plants (Boopathy, 2000). Organic matter serves as a reserve of carbon, energy and

macronutrients for microorganisms. With depth, microorganisms such as fungi and

actinomycetes decrease in numbers and bacteria become more dominant in the

microbial community (Boopathy, 2000). This is attributed to the ability of bacteria to

use alternative electron acceptors other than oxygen, under anaerobic conditions.


Microbial community composition analysis of bulk soil (depth of 2m), top soil (depth

up to 10 cm) and rhizosphere soil samples collected from a historically PCB

contaminated dump site in Czech Republic using pyrosequencing revealed that

proteobacteria were the most dominant group consisting nearly 25% of the total

population (see Figure 2.7) (Stella et al., 2015). Previously isolated bacteria with high

degradation potential to a wide range of PCBs and their halogenated metabolites

belonged to Sphingomonas, Burkholderia and Arthrobacter, which were among the

most abundant genera. Abundance of Burkholderia in the rhizosphere soil was

considerably high when compared to the other two soils, indicating their association

with plants.


Figure 2.7 Variation of the bacterial community at genus level in the bulk, top and

rhizosphere soils. The phylum of each genus is reported in brackets (Acid-

Acidobacteria; Act-Actinobacteria; Alph-Alphaproteobacteria; Beta-

Betaproteobacteria; Chlo-Chloroflexi; Firm-Firmicutes; Gamm-

Gammaproteobacteria; Gemm-Gemmatimonatedes). Adapted from Stella et al.

(2015).

One of the factors that hinders the complete degradation of PCB mixtures by diverse

bacteria is accumulation of different metabolic intermediates including

dihydroxylated derivatives (Camara et al., 2004). They act as dead-end metabolites


and the increased concentration of dihydroxylated intermediates causes cell lysis and

reduction of viable cell count.

2.6 Enhancement of bioremediation

Biological treatments offer environmentally friendly remediation options. However,

prolonged time frames before being able to observe results is one of the most limiting

factors in bioremediation of PCBs when compared to physical and chemical treatment

technologies. This section summarizes the various techniques proposed to overcome

the limitations due to factors such as low water solubility and complexity of PCBs,

inability of natural microbial flora to carry out PCB degradation and the lack of

additional carbon sources as discussed in section 2.5.

2.6.1 Biostimulation

Biostimulation of native PCB degrading microorganisms has been achieved by

introducing PCBs or other halogenated aromatic compounds to the contaminated soil

as alternate halogenated electron acceptors/co-substrates (haloprimers) (Krumins et

al., 2009). This approach can result in increased biomass of dehalogenating microbial

population, induce genes required for dechlorination and support either

dehalorespiration or cometabolism of PCB compounds (Sowers & May, 2013).

However, to introduce toxic compounds to soil as a treatment option is not an

environmentally acceptable approach as it may worsen the situation. Addition of

carvone to induce the biphenyl pathway (PetricHrsak et al., 2011) and the addition of

elemental ion to supply hydrogen as electron donor through the anaerobic corrosion

of iron (Varadhan et al., 2011) were observed to stimulate the indigenous population

of dechlorinators significantly in contaminated surface sediments. It was observed

that the oxidation of Fe stimulated microbial activity, while the resulting Fe2+

sequestered potentially inhibitory sulfides (Rysavy et al., 2005). However, it is

obvious that biostimulation will not be successful if the soil is deficient in viable

indigenous bacterial population with the PCB degradation potential.


2.6.2 Bioaugmentation

Bioaugmentation is the application of native or allochthonous wild type or genetically

modified microorganisms to sites contaminated with hazardous waste to speed up the

removal of undesirable compounds (Mrozik & Piotrowska-Seget, 2010). Successful

bioaugmentation of soil requires knowledge on the nature and level of contaminants and

the suitable microbial strains. Limited data is available on in-situ bioaugmentation as

most experiments are conducted under controlled laboratory environments (Adrian et

al., 2009; Krumins et al., 2009; Payne et al., 2011; Praveckova et al., 2015). However, 56%

reduction of higher chlorinated PCBs was observed after 120 days in Dehalobium

chlorocoercia DF1 bioaugmented sediment mesocosms contaminated with weathered

Aroclor 1260 (Payne et al., 2011). According to Winchell and Novak, (2008), addition of

iron failed to stimulate dechlorination of PCBs in sediment based microcosms without

bioaugmentation of suitable microbial culture (Winchell & Novak, 2008). Appearance of

congeners that were not products of Dehalobium chlorocoercia DF1, indicated the

possibility of stimulating of native PCB halorespiring populations after the

bioaugmentation (Payne et al., 2013).

Competition with indigenous microorganisms for limited resources, their antagonistic

interactions and predation play essential roles in bioaugmentation (Mrozik &

Piotrowska-Seget, 2010; Joutey et al., 2013). Repeated inoculation of the soil with an

active bacterial culture has been suggested to improve the efficiency of bioaugmentation

(Singer et al., 2000; Megharaj et al., 2011). A total of 57% PCB removal was observed

after repeated applications of a co-inoculation consisting of Arthrobacter sp. and

Ralstonia eutrophus, two PCB degrading bacteria with different congener specificities, 17

times during nine weeks duration to the Aroclor 1242 contaminated soil (Singer et al.,

2000). Pre-adaptation of catabolic bacteria to the target environment prior to inoculation

enhanced the rate of remediation (Megharaj et al., 2011), through improved survival,

persistence and degradative activities of microorganisms.


2.6.3 Mixed microbial consortia

Bioaugmentation with bacterial consortia having PCB degradation potential and the

ability to respond to different environmental stimuli are more desirable than single

strains (Bedard et al., 2006; Di Toro et al., 2006; Wang & He, 2013b; Sharma et al.,

2014). PCBs are present as complex congener mixtures in the environment and

different catabolic enzymes have been identified as bottlenecks for each congener

(Seeger & Pieper, 2010). Each microbial strain used in the consortium can exhibit a

particular activity range on the type and the extent of PCB metabolized, when

individually tested (Liz et al., 2009). PCB degradation potential of two bacterial

cultures individually and as a consortium were assessed for 15 days and the GC MS

analysis indicated that the 97% of PCB-44 (tetrachlorobiphenyl) reduction in

treatment containing consortium, whereas the individual organisms showed much

lower degradation activity yields at 67% and 82% (Sharma et al., 2014).

2.6.4 Surfactants

Lack of bioaccessibility due to the hydrophobic nature of PCBs is one of the most

limiting factors (Stella et al., 2015). This limitation can be overcome by the addition

of mobilizing agents such as chemical or biological surfactants to the polluted soil

(Banat et al., 2000; Singer et al., 2000; Fava & Di Gioia, 2001; Occulti et al., 2008).

However, use of chemical surfactants requires proper selection, planning and dosing.

Furthermore, prior information is needed about the type and fate of the surfactant

as well as PCBs to avoid possible leaching, ground and surface water pollution.

Biosurfactants are preferred over chemical surfactants due to their high interfacial

tension reduction activities, no or low toxicity and being readily biodegradable

(Mulligan, 2005). So far, various biosurfactants produced by microorganisms have

been identified (see Table 2.5).


Table 2.5 Types and microbial origin of biosurfactants.

Type of

biosurfactant

Microorganism Reference

Alasan Acinetobacter

radioresistens

(Navon-Venezia et al., 1995)

Arthrofactin Arthrobacter sp. (Morikawa et al., 2000)

Biosur PM Pseudomonas maltophilla (Phale et al., 1995)

Glycolipid Alcanivorax borkumensis

Serratia rubidea

Serratia marcescens

(Abraham et al., 1998)

(Matsuyama et al., 1990)

(Pruthi & Cameotra, 1997)

Lichenysin Bacillus licheniformis (Lin et al., 1994)

Particulate

surfactant

Pseudomonas marginalis (Burd & Ward, 1996)

Rhamnolipid Pseudomonas aeruginosa

Pseudoxanthomonas sp.

(Abdel-Mawgoud et al., 2010)

(Nayak et al., 2009)

Sophorose lipid Candida bombicola

Candida apicola

(Van Bogaert et al., 2007)

Streptofactin Streptomyces tendae (Richter et al., 1998)

Surfactin Bacillus pumilus

Bacillus subtilis

(Morikawa et al., 1992)

(Kakinuma et al., 1969)

Trehalose lipid Norcardia sp.

Rhodococcus sp.

(Kosaric & Choi, 1990)

(Singer et al., 1990)

Trehalose

tetraester

Arthrobacter sp.

Rhodococcus

wratislaviensis

(Passeri et al., 1991)

(Tuleva et al., 2008)

Viscosin Pseudomonas fluorescens (Laycock et al., 1991)

Application of microbial strains that are equally capable of biosurfactant production

and PCB degradation would be an attractive alternative to addition of surfactants.

Selection of an appropriate host strain/s for the expression of PCB-degrading

enzymes can include biosurfactant producing strains as they are ubiquitously

distributed in the environment (Bodour et al., 2003; Ohtsubo et al., 2004). Bodour et

al. (2003) screened soil samples collected from contaminated and uncontaminated

soils for biosurfactant-producing isolates. Out of the 20 soils tested, 13 including all

undisturbed soils contained a total of 45 putative biosurfactant producers. However,

the authors concluded that further research on the interactions among the possible

microbial candidates, biosurfactants and PCBs is needed as most research studies so


far conducted were based on the addition of either chemical or biological surfactants

alone, but not the direct application of surfactant producing microorganisms.

2.6.5 Secondary carbon sources

In anaerobic PCB dechlorination, PCBs are used as electron acceptors, but the rings are

not cleaved. Consequently, the PCB dechlorinating microorganisms require additional

sources of carbon and electrons for their growth. However, the presence of other carbon

sources could either stimulate the use of substances that hinder the PCB dechlorination

or inhibit PCB dechlorination by facilitating to out-compete the non-PCB dechlorinating

microorganisms or providing more preferred electron acceptors than PCBs to the

dechlorinating population (Wiegel & Wu, 2000; Yang et al., 2008; Parnell et al., 2010).

Indeed, the addition of glucose, tryptone, yeast extract (Hu et al., 2015), fumarate,

lactate (Song et al., 2015), malate (Bedard et al., 2006), fatty acids like acetate (Adrian et

al., 2009), propionate, butyrate, and hexanoic acid has been found to stimulate the

dechlorination of PCBs in carbon limited soils and sediments (ATSDR, 2000; Wu et al.,

2000; Sowers & May, 2013; Passatore et al., 2014; Wang et al., 2014).

In addition, enhanced dechlorination of low concentrations of weathered PCBs was

observed in Dehalococcoides ethenogenes bioaugmented sediment microcosms

amended with pentachloronitrobenzene (Krumins et al., 2009). Biphenyl addition was

also shown to enhance the mineralization of PCBs in bioaugmented and non-

bioaugmented treatments (Di Toro et al., 2006; Field & Sierra-Alvarez, 2008; Parnell et

al., 2010; Luo & Hu, 2013). However, the addition of harmful chemicals like

pentachloronitrobenzene and biphenyls to the environment as inducers may not be

appropriate (Ohtsubo et al., 2004) as it might lead to increased pollution levels. The

applicability of inexpensive and non toxic alternatives like agricultural residues, mulch

and compost as carbon sources is a preferred approach as these will also contribute to

upgrading the quality of contaminated soil through increasing water holding capacity,

porosity and microbial diversity.


2.6.6 Biocarriers

The introduction and ongoing maintenance of microbial communities in

contaminated sites are important to achieve and sustain successful bioremediation

efforts (Mrozik & Piotrowska-Seget, 2010). In most PCB-related degradation studies,

bacteria are directly introduced as liquid cultures to the contaminated soil though

PCB molecules are hydrophobic and poorly soluble in water (Di Toro et al., 2006; Liz

et al., 2009; Liang et al., 2014). Immobilizing microorganisms to suitable carrier

material helps to disperse the cells in the field while providing a protective niche for

the microorganisms against harsh environmental conditions and competitive

indigenous microflora (Power et al., 2011).

Clay particles have been reported to facilitate biofilm formation by acting as a

nutrient shuttle for the transfer of hydrophobic contaminants to biodegrading

microorganisms (Payne et al., 2013). In addition, bioaugmentation of PCB

contaminated soil by free, alginate and biochar immobilized microbial consortium

have shown that the survival rate is equally high in alginate and biochar than the free

microorganisms, while the highest PCB removal was noted in biochar immobilized

mesocosms (Pino et al., 2016). Furthermore, properly selected granulated activated

carbon (GAC) was tested and found to facilitate immobilization of microorganisms for

efficient PCB degradation by effectively sequestering PCBs from contaminated soil

and sediment (Mercier et al., 2014). The selection of carriers that can play a dual role,

as an absorbent for PCB molecules and an immobilizing agent for microorganisms

would be beneficial in order to concentrate low levels of PCBs while facilitating

microbial survival and PCB breakdown.

2.7 Monitoring of PCB degradation

The effectiveness of bioremediation efforts to remove the PCBs from the environment is

traditionally monitored through the assessment of loss of chlorines from the biphenyl

molecule, disappearance of PCB congeners of interest, total PCB reduction or by

analysing the intermediates generated and metabolic end products (Ganey & Boyd,

2005). However, for the successful development and implementation of bioremediation


strategies, understanding and monitoring of indigenous microbial communities, their

metabolic capabilities, and the way they control the major degradation pathways under

in-situ conditions are important.

Introduction of culture independent molecular approaches for the assessment of

diversity, structure, dynamics and functions of the microbial communities within

contaminated environments have revolutionized the concept of bioremediation (Sar &

Islam, 2012). Invention and advancement of molecular tools as well as a wide range of

“omics” technologies now permit analysis of microbial community composition and

activity, while preserving the fingerprints of biotic and abiotic factors, opening up new

perspectives in pollution control (Bell et al., 2014). Genomics, proteomics and

metabolomics are some of the important omics technologies useful in studying the

microorganisms involved in bioremediation by means of their genomes, the protein

products synthesized based on the genetic instructions, and the type of molecules they

metabolize, respectively (see Figure 2. 8). All the small molecules involved in the chemical

reactions in an organism is included in the metabolome. Therefore, metabolomics can

be used to detect the changes occur in the genomes and proteomes of organisms in

response to exposure of the chemical contaminants present in the environment.

Figure 2.8 The omics pyramid. Modified from NASEM (2016).


Genomics aims to understand the structure of the genome of single microorganism

through mapping genes and sequencing the DNA while metagenomics gives the

complete picture of microbial communities (microbiome) directly in their natural

environments without isolation and culturing of individual microorganisms (Thomas

et al., 2012). Similarly, proteomics can provide information on the composition of

proteins or proteome of the individual microbial species or the metaproteome of the

microbial community under specific environmental conditions (Figure 2.9) (Chovanec

et al., 2011).

In metagenomics, identification of microbial communities with PCB degradation

potential in contaminated environments is done using culture independent

molecular methods based on direct soil DNA extraction and analyses (PetricHrsak et

al., 2011). In oxidative PCB degradation, the degradation and transformation of PCBs

are generally taking place through the upper and lower biphenyl (bph) pathways as

described in detail under in Section 2.4.2. Two dioxygenases known as BphA and BphC

are the key enzymes responsible for catalyzing the initial steps in the upper biphenyl

pathway. Cell free polymerase chain reaction (PCR) based screening assay was

developed by Hoostal et al. (2002) to determine the PCB degradation potential of

resident microbial populations in PCB contaminated sediments through amplification

of bphA1 gene that encodes the biphenyl dioxygenase enzyme (Hoostal et al., 2002).

Abundance of the PCB degrading functional community in soil was successfully

assessed through the amplification of bphA and bphC sequences by quantitative PCR

assays (PetricHrsak et al., 2011).


Figure 2.9 Organism based, and community based “omic” approaches for assessing

bioremediation approaches. Modified from Chovanec et al. (2011).

Environmental proteomics applied to on-site microbial mineralization applications

can provide important information about the different proteins or enzymes and their

potential roles during bioremediation. Such information can be used to develop

biomarker proteins relevant to bioremediation and to build up a proteomic databank

to enable better understanding of the catalytic roles of microorganisms (Ahmad &

Ahmad, 2014). Recent advances in mass spectrometry, with increased resolution,

sensitivity, and throughput has led to an important development in analysing total

proteins (proteomics) (Chovanec et al., 2011). Development of more efficient, simple

and cost-effective methods for protein extraction from microbial communities and


improvement in bioinformatics tools to simplify the massive amount of data and

analyses would be helpful for reliable decision making in bioremediation applications.

Metabolomics is the scientific study of small molecules and biochemical

intermediates produced from cellular metabolism. It is a comprehensive extension of

traditional metabolite analysis. Further, the extremely diverse chemical structures of

metabolites can be complex, but there is progress with identification and

quantification being enabled using various technology platforms. In particular, GCMS

based metabolite profiling of microcosms and culture media is rapidly becoming one

of the cornerstones of functional metabolomics (Ahmad & Ahmad, 2014). These

advanced technologies enables a better understanding of the physiological state of

the microorganism and its response to different types of stimuli, including pollutants

(NASEM, 2016).

2.8 Summary

Although commercial production of PCBs was terminated in 1993, recent data have

revealed that there is no significant decline in historic levels of contamination

(Bentum et al., 2016; Desborough et al., 2016). Most of the commonly used physical

and chemical treatment methods to remediate PCB contaminated soil have serious

shortcomings of being unsustainable mainly due to the practical impossibility of

treating large quantities of soil containing low concentration of pollutants and

destruction of soil ecosystem by harsh treatment conditions. Thus, there is a vital

need to search for sustainable alternative approaches. Efficient bioremediation

techniques using sustainable microorganisms have the potential to fulfil this need.

This critical review of research literature has outlined the properties of

polychlorinated biphenyls, their impacts on human and ecosystem health while

critically evaluating the current state of knowledge on bioremediation of PCB

impacted environments with emphasis on potential areas for further improvement.

Factors that limit the efficacy of microbial remediation of PCBs are numerous. As soil

is a heterogeneous medium, the degradation rate is dependent on environmental

conditions as well as the other stressors faced by microorganisms. On the other hand,

PCBs are present in the environment as complex congener mixtures, and


bioremediation approaches generally need a longer time span due to the slow

degradation rates. Therefore, improvement and optimization of existing

bioremediation technologies are essential in order to produce viable treatment

options. Based on the promising results of some recent studies, potential areas for

further investigations are: (1). implementation of microbial based treatments which

mimic the real field situations; (2). introduction of microbial communities capable of

degrading multiple PCB congeners and their intermediates instead of using single

microbial strains; and (3). finding strains which are able to tolerate a wide range of

environmental variations, concomitant biosurfactant production and PCB

degradation.

Parallel to bioremediation studies, similar attention needs to be given to developing

reliable and standard monitoring technologies focused on microbial community

structure and their metabolic pathways. Application and development of biomarkers

or bio indicators using novel “omics” based approaches would be an enhanced

alternative to existing techniques that identify and quantify large numbers of

different congeners and their intermediate products. These new technologies require

highly sophisticated instruments, technical knowledge, upskilling, and bioinformatics

tools for data analyses, which are not yet freely available worldwide.

Chapter 3: Materials and Methods 57

Chapter 3: Materials and Methods

This chapter describes the general materials and methods used in the project.

Further details and specific materials and methods are provided in the Chapters 4 - 8

where appropriate.

3.1 Chemicals, media and reagents

All the chemical compounds, reagents and solvents used in the study were of high

purity analytical grade. The water was of MilliQ grade (Millipore), sterilized at 121 °C

for 30 min before use. Solvents used in PCB extraction were GCMS grade and the

chemicals and water used in protein extraction were of LCMS grade. During media

preparation, either HCl or NaOH was used to adjust the pH up to the required level.

3.1.1 PCB source

The PCB source, Aroclor 1260 was obtained as a gas chromatography/ flame

ionization detector (GC/FID) grade technical mixture from AccuStandard Inc. (New

Haven, CT, USA). The complex mixture consisted of about 75 penta to nona chloro

biphenyls with an average of 6.3 chlorines per biphenyl molecule (Bedard et al., 2007)

(Table 3.1).

Table 3.1 Summary of physicochemical properties of Aroclor 1260

(CAS No.110-968-25)

Property

Molecular weight (g/mole) 357.7

Colour Light yellow

Physical state Sticky resin

Boiling point, (°C) 385–420

Density, (g/cm3) at 25 °C 1.62

Water Solubility, (g/m3) 0.003- 0.08

58 Chapter 3: Materials and Methods

3.1.2 General Media and reagents

3.1.2.1 Media used for cultivating PCB degrading microorganisms

(A) Modified mineral salt medium (DSMZ medium 465a)

The mineral salt medium (DSMZ medium 465a) for the cultivation of hydroxybiphenyl

utilizing bacteria (Atlas, 2005) was used in all the culture enrichments and batch

experiments with the following modifications. Aroclor 1260 stock solution was

prepared in GCMS grade acetone (50 mg Aroclor 1260/mL acetone) instead of

ethanol, and used as the sole carbon and energy source to give 50 mg/L final PCB

concentration in the mineral salt medium.

Preparation of PCB stock solution:

50 mg of Aroclor 1260 was dissolved in 1 mL of analytical grade acetone in order to

make 50 mg/mL PCB stock solution. 1 µL of stock solution contained 0.05 mg of

Aroclor 1260. As the intention was to use PCBs as the sole source of carbon, when

dissolving PCBs, a small quantity of acetone (100 µL acetone for every 100 mL of

minimal salt medium) was used to prevent the solvent from being a significant supply

of substrate for dechlorinating microorganisms.

Composition of the mineral salt medium (MSM) per litre:

Na2HPO4·2H2O 3.5 g

KH2PO4 1.0 g

(NH4)2SO4 0.5 g

MgCl2·6H2O 0.1 g

Ca(NO3)2·4H2O 0.05 g

Trace elements solution SL-4 1.0 mL

pH 7.25 ± 0.2 at 25 °C

Components of the minimal salt medium were added and brought to 1.0 L volume

and pH was adjusted to 7. The medium was autoclaved for 30 min at 121 °C. Aroclor

1260 stock solution was aseptically added to the sterile MS medium to give 50 mg/L


concentration. To prepare mineral salt agar medium, bacteriological agar was added

to minimal salt medium before autoclaving to give a final concentration of 1.5% (w/v).

Typical working agar volumes were made up to 500 mL total.

Composition of the trace Elements Solution SL-4 (per 250 mL):

EDTA 125 mg

FeSO4·7H2O 50 mg

Trace elements solution SL-6 100 mL

Volume was brought to 250 mL using deionized water after all the components were

added and thoroughly mixed.

Composition of the trace Elements Solution SL-6 (per 250 mL):

H3BO3 75 mg

CoCl2·6H2O 50 mg

ZnSO4·7H2O 25 mg

MnCl2·4H2O 7.5 mg

Na2MoO4·H2O 7.5 mg

NiCl2·6H2O 5 mg

CuCl2·2H2O 2.5 mg

pH was adjusted to 3-4.

Volume was brought to 250 mL using deionized water after all the components were

added. Solution was mixed thoroughly and the pH was adjusted to 3-4.

(B) Nutrient agar (NA)

Nutrient agar was used in bacterial working culture preparation and in bacterial cell

counts. NA was prepared by suspending 28 g of nutrient agar (CM003, Oxoid) powder

in 1 L of distilled water and autoclaving for 30 min at 121 °C. When the mixture cooled

down to about 50 °C, 25-30 mL aliquots were poured into sterile petri dishes and

allowed to solidify.


(C) Luria- Bertani (LB) liquid medium

LB medium was used for bacterial seed culture preparations (Dercova et al., 2008).

Composition (per litre)

Bacto tryptone 10 g

BactoR-yeast extract 5 g

NaCl 5 g

Components were added and brought to 1.0 L volume and pH was adjusted to 7.0

with NaOH. The medium was sterilized by autoclaving for 30 min at 121 °C.

3.1.2.2 Media used in 16S rRNA based bacteria identification

(A) 100 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) stock solution

For 1.2 g IPTG (Promega), sterile deionised water was added to a final volume of 50

mL, vortexed until the powder was fully dissolved, filter-sterilized using sterile 0.2 µm

filter discs and stored at 4 °C until use.

(B) 50 mg/mL X-Gal solution (2 mL)

100 mg 5-bromo-4-chloro-3-indolyl-β-d-galactoside (Thermo Scientific) was dissolved

in 2 mL of N,N´-dimethyl-formamide (DMF, Ambion), covered with aluminium foil and

stored at –20 °C until use.

(C) LB plates with ampicillin/IPTG/X-Gal

15 g bacteriological agar (Oxoid) was added to 1 L LB medium and autoclaved. Once

the medium was cooled to 50 °C, filter sterile ampicillin (AppliChem) was added to

get the 100 μg/mL final concentration. 30–35 mL of medium was poured into 85 mm

petri dishes. When the agar was hardened, 100 μL of 100 mM IPTG and 20 μL of 50

mg/mL X-Gal were spread on the agar surface and allowed to absorb for 30 minutes

at 37 °C prior to use.


(D) Super optimal broth with catabolite repression (SOC) medium (per 100 mL)

Tryptone 2 g

Yeast extract 0.5g

1 M NaCl 1 mL

1 M KCl 0.25 mL

2 M Mg2+ stock, filter-sterilized 1mL

2 M glucose, filter-sterilized 1mL

Tryptone, Yeast extract, NaCl and KCl were added to 97 mL distilled water, stirred to

dissolve, and autoclaved and cooled to room temperature. 2M Mg2+ stock solution

and 2M glucose solutions were added to get a final concentration of 20 mM. The

volume was adjusted to 100 mL with sterile, distilled water. The final pH was adjusted

to 7.0. The final solution was sterilized by passing through a 0.2 µm filter. Stored at

4 °C until use.

(E) 2M Mg2+ stock (per 100 mL)

MgCl2.6H2O 20.33 g

MgSO4.7H2O 24.65 g

Powders were weighed and added into 50 mL of distilled water and the final volume

was brought to 100 mL using distilled water. The resulting solution was sterilized by

passing through a 0.2 µm filter.

3.1.2.3 Media used in biosurfactant screening tests

(A) Tryptone soya agar (TSA) + sheep blood (per litre)

Tryptone 15 g

Soya Peptone 5 g

Sodium Chloride 5 g

Bacteriological agar 15 g

Sheep Blood - Defibrinated 50 mL

pH 7.3 ± 0.2

https://en.wikipedia.org/wiki/Catabolite_repression


Readymade agar plates were purchased from Thermo Fisher Scientific and used in

the haemolytic assay test.

(B) Phosphate buffered saline (PBS) solution (per 250 mL)

NaCl 2 g

KCl 0.05 g

Na2HPO4 0.36 g

KH2PO4 0.06 g

Distilled water 200 mL

pH was adjusted to 7.4 with HCl.

All powders were weighed and added into 200 mL distilled water and the final volume

was adjusted to 250 mL with distilled water. PBS solution was filter sterilized by

passing through a 0.2 µm filter, stored at room temperature and used as the negative

control in the biosurfactant screening tests.

(C) 1 % Sodium dodecyl sulphate (SDS) solution

1 g SDS (Merk) was dissolved in 80 ml of distilled water and the volume was adjusted

up to 100 mL with distilled water. The SDS solution was filter sterilized by passing

through a 0.2 µm filter, stored at room temperature and used as the positive control

in biosurfactant screening tests.

3.1.2.4 Reducing sugar analysis in microbial growth studies

The following chemicals were made and used for the analysis of glucose utilization

during microbial cultivations grown on glucose as a carbon source, and was based on

the method by Miller (1959).


(A) Dinitrosalicylic acid (DNS) reagent, 1% (per litre)

10 g Dinitrosalicylic acid, 0.5 g sodium sulfite and 10 g sodium hydroxide were

dissolved in distilled water and the volume was brought to 1 L.

(B) Potassium sodium tartrate solution, 40%

40 g of Potassium sodium tartrate (Sigma Aldrich) was added into 80 mL of distilled

water and brought the volume up to 100 mL using distilled water.

3.1.2.5 Chemicals used in PCB extraction and analysis

(A) Sodium Sulfate, anhydrous granular reagent grade

200 g Sodium sulfate (Merk) was heated at 400 °C for 4 h, cooled in a desiccator, and

stored in a glass Schott bottle until use.

(B) Potassium Carbonate, anhydrous granular reagent grade

200 gm of Potassium carbonate (Merk) was heated at 400 °C for 4 h, cooled in a

desiccator, and stored in a glass Schott bottle until use.

(C) PCB Surrogate standard solutions

2,4,5,6-Tetrachloro-m-xylene (TCMX), 500 µg/mL stock solution in acetone was

purchased as GC/FID grade solution from AccuStandard Inc. (New Haven, CT,

USA). 10 µg/mL surrogate working solutions were prepared in 1 mL aliquots by

adding 980 µL of hexane to 20 µL of TCMX stock solution.

(D) PCB Internal standard stock solution

2,2',4,4',5,5'-Hexabromobiphenyl, 100 µg/mL in Hexane was purchased as GC/FID

grade solution from AccuStandard Inc. (New Haven, CT, USA). PCB internal standard

working solutions were prepared in 1 mL aliquots at a concentration of 10 µg/mL in

hexane.


3.1.2.6 Chemicals, buffers and materials used in protein extraction

(A) SDS-Tris lysis buffer (per 10 mL)

10% (w/v) SDS 4 mL

1.5 M Tris (pH 8.5) 667 µL

Dithiothreitol (Agilent) 0.154 g

Dithiothreitol was freshly added.

(B) Urea-Tris buffer (50 mL)

24 g urea was topped up to 50 mL with 100 mM Tris pH 8.5. Buffer was prepared

fresh.

(C) Dithiothreitol-Urea buffer (per 10 mL)

0.038 g Dithiothreitol was dissolved in 10 mL Urea-Tris buffer.

(D) Iodoacetamide-Urea buffer (per 10 mL)

0.092 g Iodoacetamide (Sigma) was dissolved in 10 mL Urea-Tris buffer. Buffer was

freshly prepared and light protected.

(E) 200 mM Ammonium bicarbonate

0.78 g Ammonium bicarbonate was dissolved in 50 mL LC-MS grade water.

(F) Preparation of Trypsin stocks

100 μg trypsin (Promega, catalogue number: V5280) reconstituted in 1 mL 50 mM

ammonium bicarbonate and aliquot 10 μL volumes into 98 tubes. The preparation

was done on ice in a keratin-free environment. The aliquots were stored at -80 °C

until use.


(G) iRT (indexed Retention Time) buffer

1 mL of 100 x concentrated iRT peptides (Biognosis) dissolved in 40% (v/v) acetonitrile

(ACN) was aliquot into 10 μL volumes. Aliquots were stored at -80 °C until use. Before

use, vials were transferred on to ice and each 10 µL aliquot was diluted 100 times by

mixing with 990 μL 2% (v/v) ACN/0.1% (v/v) formic acid.

(H) DW

0.2% (v/v) Trifluoro acetic acid (TFA) was prepared using LC-MS grade water.

(I) DE

5% (v/v) Ammonium hydroxide / 80% (v/v) ACN was prepared in a fume hood using

LC-MS grade water.

(J) Preparation of Stage Tips-SCX for desalting of protein samples

An SCX (Empore 3M) single membrane was placed in a clean petri dish. The blunt-end

needle was gently pressed into the membrane to cut out the disks. Needle was

attached to a 1 mL syringe and the membrane disk attached into the needle was

released into a regular 200 µL yellow (Eppendorf, catalogue number: 0030 014.464)

pipette tip using the plunger.


3.2 Methods

3.2.1 Selective enrichment of potential PCB utilizing bacteria

Refer to Chapter 4.

3.2.2 Genetic characterization based on 16S rRNA sequences

Refer to Chapter 4.

3.2.3 Bacterial growth profiles

Refer to Chapter 4.

3.2.4 Bacteria cell density measurement

Bacterial growth was measured as biomass concentration using two standard

techniques; optical density (OD600) measurement and standard plate count (SPC).

Although the standard plate count can detect viable cells, it is time consuming as it

requires preparation of serial dilutions, NA plates in duplicates for each dilution

incubation for 24 - 48 h for microbial growth as described in Section 3.3.1 B. On the

other hand, optical density measurement is simple and effective as the background

minimal medium used in all the growth studies was colorless. Therefore, optical

density was used as the main bacterial growth measurement method and SPC was

mainly used to countercheck the optical density measurements and to calculate the

number of colony forming units (CFU) used as the seed cultures.

(A) Optical density (OD600)

During the PCB degradation studies, the cell growth of microorganisms grown in

MSM spiked with Aroclor 1260 was measured as optical density at 600 nm using the

DU730 Beckman Coulter UV/VIS spectrophotometer. Cell free minimal salt medium

spiked with Aroclor 1260 was used as the media blank. Samples were diluted

appropriately to get the absorbance readings ≤ 0.3, to take into account the


spectrophotometer detection limit. The final optical density readings were then

produced by multiplying the OD600 readings by the dilution factor.

(B) Standard Plate count

Bacterial cell counts were determined for each bacterial strain. A serial dilution was

carried out using 0.85% (w/v) sterile saline solution as shown in Figure 3.1. Bacterial

cell counts in the original samples were calculated as colony forming units (CFU)/mL

using the following equation.

cfu/mL = (number of colonies x dilution factor) / volume plated on NA plate

Equation 3.1

Figure 3.1 Standard plate count technique


3.2.5 pH

Hanna HI 2221 pH meter was used to measure the pH according to the APHA method

4500-H (APHA, 2012). The pH meter was calibrated using pH 4.0, 7.0 and 10.0

calibration buffers each time before use.

3.2.6 Glucose concentration

The Dinitrosalicylic colorimetric method (Miller, 1959) was used to measure the

glucose concentration during the bacterial growth profile studies. The process

involves the oxidation of the aldehyde functional group in glucose. It reduces 3,5-

dinitrosalicylic acid (DNS) to 3- amino-5-nitrosalicylic acid which under alkaline

conditions is converted to a reddish brown coloured complex. DNS reagent and

potassium sodium tartrate solution were prepared as per the Section 3.1.2.4 A and

Section 3.1.2.4 B respectively. A Glucose standard curve was prepared using 0.2 g/L,

0.4 g/L, 0.6 g/L, 0.8 g/L and 1.0 g/L glucose solutions. 3 mL of DNS reagent was added

to 3 mL of glucose sample in a lightly capped test tube and the mixture was heated

in a water bath maintained at 90 °C for 5-15 min to develop the red-brown colour. 1

mL of 40% potassium sodium tartrate solution was added to stabilize the colour and

cooled to room temperature in a cold water bath. Absorbance was measured at 575

nm using the DU730 Beckman Coulter UV/VIS spectrophotometer, and glucose

concentration was calculated using the standard curve of glucose.

3.2.7 Chloride ion concentration

Chloride ion concentration in the culture medium was measured in order to

determine the amount of chlorines released from the PCB mixture, by the direct

action of the microbes. If bacteria were able to dechlorinate the PCB molecules, the

released chloride ions need to be accumulated in the culture medium. Therefore, the

chloride ions accumulated in the culture medium is directly proportional to the

degree of dechlorination. Samples were first centrifuged at 5000 x g for 10 min and

the resultant supernatant was filtered through 0.2 µm sterile filter discs to remove

the bacterial cells. Cell free culture supernatant was used to measure the chloride ion


concentration using the Dionex ICS-2100 ion chromatography according to USEPA

method 300.0 (USEPA, 1993). For the calibration curve preparation, 0.1 ppm, 1 ppm,

5 ppm, 10 ppm, 20 ppm and 100 ppm standard sodium chloride solutions were used.

The following controls were used in order to subtract the background chloride levels

coming from the minimal salt medium, lysis of bacterial cells and leaking of any

chloride occurred, and from any traces from the LB medium used to cultivate the

bacterial seed cultures.

(A) Abiotic controls: Minimal salt medium spiked with 50 mg/L Aroclor 1260.

(B) Media controls: Minimal salt medium inoculated with similar amount of

bacterial seed cultures used in the experiment. Chloride measurements were

done before and after the sonication to see the contribution from cell lysis.

(C) Minimal salt medium only.

3.2.8 PCB extraction

During the experiments, PCB concentrations were measured in two ways as; (1) total

soluble PCBs (used in Chapter 5 and Chapter 6), and (2) total PCBs (used in Chapter

7). PCBs are poorly soluble in water and the solubility of Aroclor 1260 is reported to

be in the range of 0.003-0.08 g/m3 (ATSDR, 2000). Therefore, before starting the PCB

extractions, the extraction efficiencies of two solvents (1), hexane and (2), diethyl

ether (DEE) were tested in order to determine the best solvent to use. 1 mL aliquots

of minimal salt media containing PCBs (50 mg/L, 25 mg/L and 10 mg/L in triplicates)

were transferred to duplicate 8 mL glass vials fitted with Teflon-lined screw caps

(THC15151083 and THC15091703, Thermo Fisher Scientific). PCBs that were soluble

in the aquous medium were extracted with 2.5 mL of GC grade n-hexane (USEPA,

2007) and GC grade DEE ((Bedard et al., 2006; Adrian et al., 2009) by vigorous

horizontal shaking on a platform shaker at 250 rpm for 4 h under room temperature,

followed by centrifugation at 5000 rpm for 10 min (Adrian et al., 2009).

The solvent phase was removed and used for subsequent analysis using the GCMS as

described under Section 3.3.4.2. 50 ppm/mL Aroclor 1260 in hexane was used to

compare the recovery efficiencies of PCBs dissolved in the aqueous medium as given

in Figure 3.2. All the DEE extracts were evaporated to dryness and redissolved in 1


mL hexane before analysis. Although recovery rates of PCBs were comparatively low

in hexane, the variation of the results in triplicate samples were minimal. Extraction

using DEE also needed additional step as once the PCBs are extracted, DEE need to

be removed by evaporation and resuspension of PCBs in hexane need to be done

before injecting samples to the gas chromatograph. Accordingly, hexane was chosen

as the extraction solvent.

Figure 3.2 Recovery of Aroclor 1260 soluble in the aqueous minimal salt medium as

a percentage of total PCBs added to the mixture, using diethyl ether (DEE) and hexane

as the extraction solvents. Error bars represent the standard deviation of mean values

(n = 3).

(A) Total soluble PCB extraction

Liquid aliquots (1 mL) withdrawn from batch culture flasks were transferred into 8

mL glass vials fitted with Teflon-lined screw caps. PCBs were extracted with 2.5 mL of

GC grade n-hexane (USEPA, 2007) by vigorous horizontal shaking at 250 rpm on a

platform shaker for 4 h at room temperature, followed by centrifugation at 5000 rpm

for 10 min (Adrian et al., 2009). The solvent phase was separated and used for

subsequent total soluble PCB analysis. Before extraction, 25 µL of 2, 4, 5, 6-


tetrachloro-m-xylene (10 µg/mL in hexane) was added to each sample as the

surrogate standard to determine the extraction efficiency (USEPA, 2007). The

surrogate recovery was 98.98% ± 19.11% (n=462).

(B) Total PCB extraction

Total PCB extraction was done by sacrificing the content of the whole flask based on

the method described by Murinova et al. (2014) with some modifications. 5 mL of n-

hexane was added to each flask and the flasks were sonicated in an ultrasonic bath

at medium speed for 10 min to disrupt the cells and release the bound PCBs. The

content of the flask was transferred into a 250 mL separatory funnel and vigorously

shaken for 2 min. Anhydrous K2CO3 (1 g) was added to the mixture to reduce the foam

generated from biomass and shaken for 15 s. The n-hexane layer was collected into

a flask containing anhydrous Na2SO4. The extraction was repeated twice with 5 mL of

n-hexane at each time. The content was centrifuged at 4500 rpm for 10 min to

separate the emulsion layer. The content was added to 25 mL volumetric flask and

the final volume was adjusted to a 25 mL with n-hexane and stored in amber colour

glass bottles at 4 °C until subsequent analysis by gas chromatography (GC). To

determine the extraction efficiency, 250 µL of 100 ppm of GC grade 2,4,5,6-

tetrachloro-m-xylene was used as the surrogate standard and added to flasks

immediately before starting the extraction.

3.2.9 PCB analysis

PCB extracts and standards were spiked with 2,2',4,4',5,5'-hexabromobiphenyl stock

solution as an internal standard before GCMS analysis (USEPA, 2007) to give 100 ppb

final concentration. PCB levels were determined as per the USEPA method 8082A

(USEPA, 2007). Thermo Scientific Trace 1310 (in splitless mode, at 260 °C inlet

temperature, 80 mL/min split flow and 1.2 min splitless time) equipped with SSL

injector with splitless liner with glass wool, Thermo TG-5SilMS analytical column (30

m × 0.25 mm ID×0.25 µm), TriPlus RSH auto sampler was used for PCB analysis.

Helium was used as the carrier gas at 1.2 mL/min constant flow. The column was kept

at 40 °C for 2 min, and then the temperature was raised to 300 °C at 15 °C/min and


kept for 5 min. The Thermo Scientific TraceFinder EFS software was used for the

screening and quantitation of total PCBs and PCB homolog groups using the Thermo

Scientific triple stage quadrupole Mass spectrometer (TSQ8000 EVO). Retention

times were taken from the existing literature (Walker & Feyerherm, 2013) and full

scan acquisition and timed selective reaction monitoring (SRM) modes were used to

distinguish the PCB homolog groups. 5 ppb, 10 ppb, 20 ppb, 50 ppb, 250 ppb, 500

ppb, 1000 ppb, 2000 ppb and 10 000 ppb Aroclor 1260 standard solutions were used

for calibration curve preparation. Average weight percent of PCB homolog groups

and chlorines in Aroclor 1260 used in the study were calculated based on the

calibration curve and given in Table 3.2. Relative amounts of PCB levels and homolog

groups in the samples were determined by nine-point calibration using Aroclor 1260

standard solutions and area integration. Details of calibration and surrogate standard

preparations were given in Appendix A.

Table 3.2 Average weight percent of PCB homolog groups and chlorines in Aroclor

1260.

Chlorine

Substitutions

Homolog group Homolog group (%)

(by weight)

Cl %

(by weight)

mono C12H9Cl 0.24 ± 0.03 0.05 ± 0.00

Di C12H8Cl2 0.82 ± 0.07 0.26 ± 0.02

Tri C12H7Cl3 1.97 ± 0.20 0.81 ± 0.08

Tetra C12H6Cl4 1.59 ± 0.06 0.78 ± 0.03

Penta C12H5Cl5 20.06 ± 0.33 10.83 ± 0.18

Hexa C12H4Cl6 50.69 ± 0.47 29.91 ± 0.28

Hepta C12H3Cl7 21.22 ± 0.61 13.37 ± 0.38

Octa C12H2Cl8 2.72 ± 0.23 1.80 ± 0.15

Nona C12HCl9 0.63 ± 0.03 0.43 ± 0.02

Deca C12Cl10 0.05 ± 0.00 0.04 ± 0.00

Total 100.00 ± 2.03 58.27 ± 1.15


3.2.10 Screening tests for biosurfactant production

Refer to Chapter 5.

3.2.11 Extracellular protein visualization, quantification, extraction and analysis

Refer to Chapter 8.

3.2.12 Storage of culture supernatant samples

Samples withdrawn during batch cultivations for protein extraction (1 mL aliquots),

chloride analysis (5 mL aliquots) and extraction of total soluble PCBs (1 mL aliquots)

were stored at -80 °C until analysis.

3.2.13 Bacterial culture maintenance

(A) Long term storage at -80 °C using glycerol stocks

For long term storage of bacterial cultures, 1 mL of samples containing bacterial cells

from 20 mL cultures grown overnight (at 28 °C and 150 rpm) in LB were removed, and

centrifuged at 5000 rpm for 10 min. For each bacterium, the supernatant was

discarded, and the cells were taken up in 1.5 mL sterile 50% glycerol solution and

aliquot to 3 x 500 µL samples, and stored at -80 °C.

(B) Bacteria working cultures

Bacterial cultures were streaked onto nutrient agar (NA) plates in duplicate and

stored at 4 °C. Sub culturing was performed at monthly intervals by streaking onto

fresh NA plates.

3.3 PCB degradation studies

All the PCB degradation studies were conducted at 28 °C. The experiments conducted

under aerobic conditions were done at 150 rpm in a platform shaker incubator. The

experiments conducted under anaerobic conditions were performed inside an

anaerobic chamber (COY laboratory products, Inc.) under strict anaerobic conditions.


Palladium catalysts located inside the chamber were used to scrub any residual

oxygen present in the chamber. Shake flasks and agar plates were transferred under

anaerobic conditions without changes to the internal atmosphere through a heavy

duty vacuum airlock compartment. The anaerobic environment inside the chamber

was maintained constant under 4.9% H2, 10.7% CO2 and 84.4% N2 (BOC Australia).

Once minimal salt medium containing flasks were prepared, they were kept

equilibrated for one week inside the anaerobic chamber with loosed caps before they

were inoculated with the bacterial cultures. Anaerobic experiments carried out at 28

°C were kept in a temperature controlled incubator inside the anaerobic chamber

(see Figure 3.3).

Figure 3.3 The Coy anaerobic chamber with the connecting airlock to the right, used

in this research.

3.4 Cleaning of glassware used in PCB extraction

All the glassware used in the PCB extractions were first soaked in detergent and

thoroughly washed with hot water. They were then rinsed with tap and distilled

water respectively followed by a thorough rinse with hexane. After drying at 50 °C,

all the cleaned glassware to be used in PCB extraction were sealed and stored in a

clean environment to prevent any accumulation of dust or other contaminants.


3.5 Quality assurance

To obtain accurate data is critical in scientific research and therefore, quality control

and quality assurance are very important. As such, the following measures were

taken to minimize errors. All the experiments were run in triplicate with identical

conditions to test the repeatability and the results were reported as means ±

standard deviations. In all the measurements and analyses, the Relative Standard

Deviation (RVSD) was calculated as defined by Equation 3.2.

RVSD (%)= (σr

μr

) ×100 Equation 3.2

Where:

µr – mean concentration of replicates

σr – standard deviation of the concentrations of replicates

With every set of experiments, controls (as described under each chapter) were run

and used in sample and data analysis. In PCB analysis, a series of blanks and standard

solutions were used as specified in the method 8082A (USEPA, 2007). This approach

included method blanks, calibration blanks, calibration standards, intermediate

checks, internal standards and surrogate standards. The surrogate standard 2,4,5,6-

Tetrachloro-m-xylene was added to all samples, controls, method blanks and

calibration standards as the surrogate standard, prior to extraction at the

concentrations described in the Sections 3.1.2.5 C. The recoveries of the surrogate

standards were calculated using the standard curve prepared for surrogate standard

in order to find out the extraction efficiencies of PCBs. Similarly, 5 µL of a 10 µg/mL

internal standard in hexane (see Section 3.1.2.5 D and Appendix A) was added to all

standard solutions used in the calibration curve, intermediate checks, and the

samples including the method blanks and controls before GCMS analysis. A nine point

calibration curve was prepared using a range of Aroclor 1260 concentrations from 5

ppb to 10,000 ppb and used in quantification of PCBs. In addition to that, method

blanks were run using similar volumes of deionized water only through the entire

analytical procedure for each batch of extractions. During PCB analysis, 5 ppm, 10


ppm, 20 ppm and 100 ppm Aroclor 1260 standard solutions were prepared and run

in between each of the 25 samples as intermediate checks.

In chloride analysis, six calibration standards 0.1 ppm, 1 ppm, 5 ppm, 10 ppm, 20 ppm

and 100 ppm sodium chloride solutions were used to prepare the calibration curve.

Additionally, in between each of the 20 samples, 5 ppm and 20 ppm sodium chloride

standards were run as intermediate checks.

Chapter 4: Selective Enrichment and Identification of Potential PCB Degrading Microorganisms 77

Chapter 4: Selective Enrichment and Identification of Potential PCB Degrading Microorganisms

4.1 Background

Bioremediation, the use of microorganisms capable of degrading toxic compounds to

decontaminate the polluted environments, has become an attractive alternative to

existing physical and chemical treatments. However, commercial application of

bioremediation to remediate PCB contaminated environments is not yet widely

practiced mainly due to long reaction times and lower degradation rates. Therefore,

further research is needed to improve and optimize the existing bioremediation

technologies in order to be cost effective as a viable treatment option for onsite

applications.

The aim of this chapter is to identify aerobic and anaerobic PCB degrading

microorganisms from the natural environment for use in subsequent PCB

degradation related studies discussed in Chapter 5 to Chapter 8. Soil and sediment

samples collected from the natural environment were contaminated with Aroclor

1260 as the PCB source and subjected to selective enrichment to isolate

microorganisms. During the selective enrichment, samples went through four serial

transfers using a fresh minimal salt PCB liquid medium at weekly intervals in order to

obtain soil and sediment free enrichment medium as a source of microorganisms that

are capable of utilizing PCBs as their sole source of carbon. Once the initial screening

was done, bacterial isolates were tested for their oxygen tolerance while searching

for any facultative anaerobic organisms, as the ability to survive under both anaerobic

and aerobic conditions would be an added advantage in field scale remediation

applications.

Full length 16S rRNA gene (1.5kb) isolation and sequencing was used for identification

of the bacterial isolates at the genus level as a minimum. The 16S rRNA gene is the

most commonly used genetic marker in bacterial identification due to a number of

78 Chapter 4: Selective Enrichment and Identification of Potential PCB Degrading Microorganisms

reasons, namely; (i) 16S rRNA is a component of the small subunit (30S) of ribosomal

RNA genes and is found in all bacteria (Janda & Abbott, 2007), and (ii) it is

comparatively short (~1.5 kb) and comprise a particular combination of conserved,

variable and hypervariable regions that have evolved at different rates (Srinivasan et

al., 2015). These characteristics allow the use of 16S rRNA to identify bacteria to the

genus or the species level by comparing against the existing databases that include

near full length sequences of a large number of bacterial strains.

4.2 Materials and Methods

4.2.1 Soil and sediment sample collection and preparation

In order to isolate possible aerobic PCB degrading bacteria, soil samples were

collected from locations around the Brisbane City Botanical Gardens and Queensland

University of Technology, Gardens Point Campus (27.4745° S, 153.0293° E). Samples

were collected into 500 mL sterile glass Schott bottles and transported in ice to the

laboratory. Samples were homogenised, and 50 g aliquots of the composite was

added to two 250 mL sterile Erlenmeyer flasks. Each sample was contaminated using

transformer oil mixed with Aroclor 1260 to obtain 50 mg/kg PCB concentration.

Flasks containing soil were mixed thoroughly and incubated stationary under aerobic

conditions at room temperature (23 °C ± 1 °C) for one month period to obtain PCB

tolerant microorganisms for selective enrichment screening.

In order to isolate potential anaerobic PCB degrading bacteria, three sites were

chosen for soil and sediment sampling. The sites were the Brisbane City Botanical

Gardens, Brisbane River (27.4745° S, 153.0293° E) and Coombabah Lake, Gold Coast

(27.54° S, 153.22° E). Samples were collected into 250 mL sterile glass Schott bottles

and transported under anaerobic conditions in an anaerobic jar. Similar to the

process adopted for aerobic soil samples, 50 g of composite samples were added to

two 50 mL polypropylene vials with screw caps under anaerobic conditions and

contaminated with Aroclor 1260 containing transformer oil to obtain 50 mg/kg PCB

concentration. The vials were sealed and incubated stationary inside the anaerobic

chamber (COY laboratory products) main compartment at room temperature (23 °C

± 1 °C) for one month. The atmosphere inside the anaerobic chamber was maintained


constant using a special mixed gas made up of 4.9% H2, 10.7% CO2 and 84.4 % N2

(BOC, Australia).

4.2.2 Selective enrichment of potential PCB utilizing bacteria

Isolation of microorganisms capable of utilising PCBs as carbon and energy source

under aerobic conditions was carried out through a series of selective enrichments

as illustrated in Figure 4.1a. Modified DSMZ medium 465a (see Section 3.1.2.1 A in

Chapter 3) was used as the base minimal salt medium with 50 mg/L Aroclor 1260 as

the sole source of carbon. During the study, two 250 mL Erlenmeyer flasks each

containing 100 mL of sterile minimal salt medium were prepared and inoculated with

10 g of composite soil previously contaminated with transformer oil containing

Aroclor 1260. In order to enrich the possible aerobic PCB degrading bacteria, flasks

were incubated aerobically in a platform shaker at 28 °C and 150 rpm for one week.

Three further serial transfers (10% of the enrichment medium) were carried out every

7 days into fresh sterile minimal salt medium. From the final flask, 1 mL of the

supernatant was serially diluted with sterile 0.85% saline water and plated in

duplicate on nutrient agar (CM003, Oxoid) and incubated for 24 to 48 h at 28 °C.

Morphologically different colonies obtained from the selective enrichment were

isolated and streaked on fresh nutrient agar plates.

The same procedure was followed for the enrichment of possible anaerobic PCB

degrading bacteria with the following modifications as illustrated in Figure 4.1b. After

inoculation with 10 g of composite soil and sediment samples previously

contaminated with Aroclor 1260, flasks were incubated anaerobically under static

conditions in an incubator kept inside the anaerobic chamber with the temperature

maintained at 28 °C (see Figure 3.3 in Chapter 3).


Figure 4.1 Selective enrichment of potential PCB degrading bacteria, under aerobic

and anaerobic conditions at 28 °C.

4.2.3 Characterization of potential PCB degrading bacteria

Bacterial isolates obtained from the selective enrichment were further characterized

based on their colony morphology on nutrient agar and cell morphology using Gram

staining. Genomic DNAs were first isolated from pure isolates, before full-length 16S

rRNA gene sequences were PCR amplified for sequencing, in order to ascertain their

identification.

4.2.3.1 Morphological characteristics

Smears of bacterial isolates were prepared on microscopic slides and stained using

the standard Gram staining technique to differentiate the Gram positive and negative

bacteria (Barthomolew & Mittwer, 1952). Bacterial cells were examined under oil

immersion (100x magnification) using Nikon Eclipse Ni light microscope.


4.2.3.2 Oxygen requirements

Bacterial isolates obtained from selective enrichment were inoculated in duplicate

on nutrient agar plates and incubated at 28 °C in parallel under anaerobic and aerobic

conditions to determine their growth tolerance in the presence and absence of

oxygen.

4.2.3.3 Identification of bacterial isolates using 16S rRNA full length gene

sequencing

The pair of 27F and 1492R universal primers were used for the amplification of the

full-length 1.5 kb 16S RNA gene sequences (Fredriksson et al., 2013). The resulting

1.5 kb polymerase chain reaction (PCR) products for the different bacterial isolates

were cloned into the pGEM-T vector plasmid (Promega) and the 1.5 kb RNA gene

inserts on the recombinant plasmids were sequenced using the dideoxy Sanger

sequencing method of the same plasmid in two separate reactions (Sanger &

Coulson, 1975). In a typical run, up to 900 bp of good quality readable nucleotide

sequences are obtained in both, forward and reverse directions.

(A) Genomic DNA (gDNA) extraction

Bacterial cultures initially isolated from selective enrichment were separately

inoculated into 5 mL of sterile Luria-Bertani broth and incubated overnight at 28 °C

and 150 rpm in a rotary incubator. Isolate II genomic DNA kit (Bioline) was used to

extract the bacterial DNA following the manufacturer’s protocol. The gDNA isolation

kit was successful in the isolation of good quality gDNAs from all the isolates except

two isolates. In order to extract the gDNA of these two isolates, the same procedure

was repeated, but with following modification. After addition of the Lysis buffer and

proteinase K solution, the tubes were incubated overnight at 37 °C to help with cell

wall Lysis, instead of the standard 3 hour incubation at 56 °C. The quality of the

extracted gDNA was determined by electrophoresis at 100 V for 1 h using 1% agarose

gel using 1KB plus hyper ladder (Bioline) as the marker.


(B) Polymerase chain reactions (PCR)

The extracted gDNA was diluted 1:10 with sterile ultra-pure water, and 1 µL aliquot

was used as a template for PCR amplification in a 50 µL reaction mixture. Universal

16S rRNA primers (Integrated DNA Technologies, Inc.), 27F (5'-

AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTA CCTTGTTACGACTT-3') were

used to amplify the 16S rRNA genes of isolated bacteria according to the MyTaq HS

Red DNA polymerase protocol (Bioline). One µL of ultra-pure water without DNA was

used as the negative control. The Eppendorf Master cycler machine was used for the

PCR reactions with the following PCR cycling conditions: initial denaturation at 95 °C

for 10 min, 30 cycles of 30 s at 95 °C, 30 s at 50 °C, and 2 min at 72 °C followed by

final extension at 72 °C for 7 min and a final hold at 4 °C. The quality of the PCR

products was checked by electrophoresis by running 5 µL on a 1% agarose gel at 100

V for one hour next to a 1KB plus hyper ladder as the marker.

The remainder of the PCR samples were run on a new 1% agarose gel and the DNA

fragments were excised and purified according to the Isolate II PCR and gel kit

protocol (Bioline). Following gel extractions, the concentrations of the DNA was

determined using the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific)

set at 260/280nm, expressed as ng/µL, as well as checking 5 µL on a 1% agarose gel

for 1 h at 100 V.

(C) Ligation, transformation and cloning in Escherichia coli (E. coli)

pGEM-T Easy Vector system (Promega) was used to clone the 16S rRNA gene PCR

product into E. coli. The gel purified ~1.5 kb 16S gene fragments were ligated into

the pGEM-T Easy Vector plasmids (Promega) according to the ligation protocol of

pGEM-T Easy Vector system (Promega) using 1:3 insert:vector ratio. The 16S rRNA

gene ligated vector plasmids were transformed into high efficiency E. coli JM 109

competent cells. The transformed cells were screened for blue and white colonies by

plating on Luria-Bertani (LB) agar plates containing ampicillin (100 µg/mL), Isopropyl

β-D-1-thiogalactopyranoside (IPTG, 0.5mM) and 5-bromo-4-chloro-3-indolyl-β-D-

galactopyranoside (X-Gal, 80 µg/mL). Three white colonies indicative of being

positive for recombinant clones were picked from plates representing each bacterial


isolate and patched onto fresh LB plates containing ampicillin, IPTG and X-Gal.

Potential recombinant bacterial clones were checked for the presence of 16S gene

inserts by colony PCR with M13 forward and reverse primers (Integrated DNA

Technologies, Inc.) that anneal to outside of the pGEM-T vector multiple cloning site

(Promega) as follows: PCR mixtures were prepared with no template using 5µL 5X

MyTaq reaction buffer, 1 µL of M13 forward and reverse primers, 0.25 µL MyTaq DNA

polymerase and 17.75 µL ultra-pure water to make 20 µL. Then a sterile inoculating

needle was used to gently touch a white colony and a few cells were transferred to

the reaction mixture. The following amplification program was used: 1 cycle at 94 °C

for 15 min, and then 35 cycles of 30 s at 94 °C, 30 s at 50 °C, and 2 min at 72 °C

followed by final hold at 4 °C. Resulting PCR products (5 µL) were checked on a 1%

agarose gel at 100 V for 1 h.

(D) Plasmid isolation and Sanger sequencing

After checking for the presence of 1.5 kb 16S gene fragments on the pGEM-T vector

by PCR, a single positive recombinant clone for each bacterial isolate was grown in 5

mL LB broth containing ampicillin (100 µg/mL) and incubated for 12 to 16 h at 37 °C

with vigorous shaking at 250 rpm. The plasmid isolation and purification was

performed using the QIAprep Spin Miniprep Kit (QIAGEN) according to the

manufacturer’s protocol. The purified plasmids were checked using gel

electrophoresis.

For Sanger sequencing, two separate sequence reactions using the M13F and M13R

primers were carried out for each recombinant plasmid, according to the standard

BigDye Terminator (BDT) v3.1 cycle sequencing kit protocol (Thermo Fisher

Scientific). The following cycling conditions were used: 1 cycle at 96 °C for 1 min, and

then 25 cycles of 10 s at 96 °C, 5 s at 50 °C, and 4 min at 60 °C followed by a final hold

at 4 °C.

Ethanol/EDTA precipitations were carried out to purify and precipitate the sequence

reactions. DNA sequencing was performed using 3500 Genetic Analyzer (Applied

Biosystems, Hitachi). FinchTV 1.4.0 (Geospiza Inc.) was used for editing raw

nucleotide DNA sequence data. The raw DNA sequences were further polished by


the removal of the plasmid vector sequences. Edited forward and reverse sequences

were aligned to obtain full length 1.5 kb contiguous sequences using the EMBOSS

Needle tool of the European Bioinformatics Institute (EMBL-EBI). The full length 16S

rRNA gene sequences were then submitted to determine the closest matching

sequences by comparing with the bacterial and archaeal 16S RNA databases using

the Sequence Match tool of the Ribosomal Database Project (RDP) and Basic Local

Alignment Search Tool (BLAST) of NCBI, USA. The searches were limited to curated

‘Type’ strains.

There is no universal definition existing for species level identification using 16S rRNA

gene sequencing and use of acceptable criteria for establishing a species match varies

widely in different studies (Janda & Abbott, 2007). However, a study based on 571

distinct pairs of strains, a threshold between 98.2 - 99.0% 16S rRNA gene sequence

identity has been suggested as a reasonable species boundary (Meier-Kolthoff et al.,

2013). Yarza et al. (2014) used a 98.7% 16S rRNA gene threshold in estimating global

species richness based on the 98.7 – 99% similarity threshold recommended by

Stackebrandt and Eber (2006). Therefore, if similarity was not 100%, identification

was limited up to the generic level as there is no guarantee of having 1% divergence

to obtain an accurate identification.

4.2.3.4 Confirmation of the ability to grow on PCBs as sole carbon source

All the bacterial strains identified in Section 4.2.3.2 found to be able to grow in the

presence of oxygen were cultivated in triplicate on minimal salt agar containing 50

mg/L of Aroclor 1260 as the sole source of carbon and incubated at 28 °C under

aerobic conditions for 24 to 72 h. This was to further confirm the ability of identified

bacteria strains to grow under aerobic conditions using PCBs as the sole source of

carbon. Similarly, all the facultative anaerobic cultures were streaked onto minimal

salt agar containing 50 mg/L of Aroclor 1260 as sole source of carbon and incubated

at 28 °C under anaerobic conditions for 24 to 72 h. Minimal salt agar with no added

Aroclor 1260, but an equal volume of acetone that was used to add Aroclor 1260 into

the minimal salt agar in the growth experiment, was used as the negative control to

determine whether there was any contribution from leftover acetone on bacterial


growth. In comparison, the minimal salt agar with 0.1% (w/v) glucose added and solid

rich nutrient agar medium alone were used as the positive controls.

4.2.3.5 Growth profiles of bacteria

To understand the growth of isolated bacteria under controlled conditions, bacterial

cultures were grown in a closed system as pure cultures and the rate of cell

population increase, rate of glucose consumption and the variation of pH over time

were measured over a nine hour period.

For each bacterial culture, triplicate 500 mL Erlenmeyer flasks containing 250 mL

sterile MSM and 2 g/L glucose were used to develop their growth curves. Flasks were

inoculated with 10% (v/v) overnight grown bacterial cultures in liquid LB broth as the

seed and incubated at 28 °C and 150 rpm in a shaker incubator. Cell density of the

bacterial cultures were calculated as colony forming units (CFU) using standards plate

count as described in Section 3.3.1.B to obtain the growth curves. The specific growth

rate (µ) of each bacteria were calculated for the exponential phase using Equation

4.1 (Maier, 2009).

𝑋 = Xₒeµᵗ Equation. 4.1

Where:

X – number of cells after time t (as CFU)

Xₒ- initial number of cells (as CFU)

µ- specific growth rate

Negative controls were also adopted and conducted parallel to each bacterial isolate.

In this regard, minimal salt medium containing 2 g/L glucose without inoculation with

bacterial seed culture, and minimal salt medium without glucose inoculated with

10% (v/v) seed culture were used as negative controls. Immediately after the

inoculation at time 0 and at hourly intervals up to nine hours, 6 mL aliquots were

withdrawn from each flask and analysed for optical density at 600 nm, pH and glucose


as reducing sugars using dinitrosalicylic colorimetric method (see Section 3.2.4 A,

Section 3.2.5 and Section 3.2.6 in Chapter 3 respectively for methods adopted).

4.3 Results and Discussion

4.3.1 Screening and Identification of PCB utilizing culture members

After the selective enrichment screening, five microbial isolates from aerobic

enrichments (labelled as A1 to A5) and six bacterial isolates from anaerobic

enrichments (labelled as AN1 to AN6) that grew on the minimal salt medium with

Aroclor 1260 as the sole carbon source were isolated, as pure cultures. The 11 isolates

were subjected to further analyses and their identities determined using 16S rRNA

gene sequencing and morphological characterization.

4.3.1.1 16S rRNA gene based identification

Full length (1.5kb) 16S rRNA gene isolation and sequencing was performed as

described in Section 4.2.3.3 in order to identify the eleven purified bacterial isolates.

Genomic DNAs were first isolated from overnight grown cells using Isolate II genomic

DNA kit (Bioline) and run on a 1% agarose gel as shown in Figure 4.2. Although equal

volume (5 mL) from each genomic DNA sample was loaded on to the gel, the intensity

of the DNA bands on the gel was different. The possible reasons for the differences

in extraction efficiency is attributed to the variations of the cell densities of each

bacterial culture and the differences of the bacterial strains, constitution of the cell

wall and the physiological state of the cells (Van Tongeren et al., 2011).


Figure 4.2 Agarose gel electrophoresis of genomic DNA isolated from bacterial

isolates using Isolate II genomic DNA kit (Bioline). A1 to A5 were from aerobic

selective enrichments and AN1 to AN6 were from anaerobic selective enrichments.

The genomic DNA of two bacterial isolates AN3 and AN4 (as shown in Figure 4.2) were

unable to be extracted using the standard Isolate II genomic DNA kit (Bioline)

protocol. The standard lysis protocol is not adequate to lyse some bacterial cell walls

and subsequent release of DNA (Van Tongeren et al., 2011). If they are Gram positive

bacteria, the thick peptidoglycan layer in the cell walls usually made them difficult to

break using the normal cell lysis method when compared to Gram negative bacteria

(Vingataramin & Frost, 2015).

Once the PCR amplifications of extracted genomic DNA was extracted from agarose

gel and purified, the resulting 1.5 kb DNA fragments were ligated into pGEMR-T Easy

Vector to obtain recombinant DNA. The ligated products were then transformed into

high efficiency E. coli JM109 competent cells to produce multiple copies of

recombinant DNA molecules as described in Section 4.2.3.3 C. After transformation,

positive white colonies were observed on the LB/ampicillin/IPTG/X-Gal agar plates

representing all the different bacterial isolates (Figure 4.3). Recombinant plasmid

DNAs were then isolated from E. coli, purified and the checked on a 1 % agarose gel

to assess the yield and quality (Figure 4.4), before subjecting them for Sanger

sequencing.


Figure 4.3 Blue-white screening on LB/ampicillin/IPTG/X-Gal plate after the

transformation into high efficiency E. coli JM109 competent cells. White colour

colonies representing the positive transformations.

Figure 4.4 Agarose gel electrophoresis of purified recombinant plasmids. 5 µL from

each sample was loaded into the corresponding well. A1 to A5 were from aerobic

selective enrichments and AN1 to AN6 were from anaerobic selective enrichments.

Instead of plasmids, sterile MilliQ water was used in the negative control.

Raw DNA sequences in the forward and reverse directions were first edited using

bioinformatics tool FinchTV 1.4.0 (Geospiza Inc.). The two sequences were merged

using EMBOSS Needle tool (EMBL-EBI) as described in 4.2.3.3 D and as shown in

Figure 4.5, before the 1.5kb 16S rRNA contiguous sequences were submitted to NCBI

and RDP databases for matches.


Figure 4.5 Pairwise alignment of forward and reverse DNA sequences of bacterial

isolate AN2 using Emboss Needle software. Total length after the alignment was 1459

and number of similarities between two sequences were 738/1459 (50.6%).


The closest matching sequences were obtained from NCBI and RDP databases and

are summarized in Table 4.1. The Ribosomal Database Project (RDP) is a curated

database as it provides the aligned and annotated rRNA gene sequence data (Cole et

al., 2014; Wang et al., 2007) while NCBI is not a curated database. There were no

differences between the two databases for culture number NP02, NP04, NP05 and

NP07 as they obtained the same closest relatives from both databases. However,

similarity of culture number NP01, NP03 and NP06 were limited to the generic level

between the two databases. None of the cultures showed 100% similarity to the

existing bacterial cultures in the databases. Therefore, during this study, the different

isolates were identified and labelled according to the genus name followed by strain

number (see Table 4.2). 16S rRNA sequences of the six viable cultures (NP01 to NP06)

were deposited in GenBank (National Centre for Biotechnology Information-NCBI)

database under the accession numbers KY427122, KY427123, KY711179, KY711180,

KY711181 and KY711182 respectively.


Table 4.1 Comparison of closest relatives of isolated bacteria based on NCBI and RDP databases

Notes:

a Similarity score - percent sequence identity over all pairwise comparable positions when ran with aligned myRDP sequences.

b S_ab score (Seqmatch score) - number of (unique) 7-base oligomers shared between the tested sequence and a given RDP sequence divided by the lowest

number of unique oligos in either of the two sequences (Wang et al., 2007)

Culture No.

RDP NCBI

Similarity score a

S_ab score b

Unique common oligomers

Name Maximum Score

Identities Gaps Name

NP 01 0.990 0.956 1344 Chryseobacterium rhizoplanae; JM-534; KP033261

2564 1422/1439(99%) 0 (0%) Chryseobacterium lactis KC1864

NP 02 0.995 0.947 1435 Delftia lacustris; 332; EU888308 2671 1479/1495(99%) 4 (0%) Delftia lacustris 332

NP 03 0.996 0.943 1364 Achromobacter insolitus; LMG 6003; AY170847

2660 1476/1493(99%) 5(0%) Achromobacter denitrificans DSM 30026

NP 04 0.994 0.979 1394 Ochrobactrum lupini; LUP21; AY457038

2625 1437/1445(99%) 0(0%) Ochrobactrum lupini LUP21

NP 05 0.997 0.967 1413 Lysinibacillus macroides; LMG 18474; AJ628749

2717 1486/1494(99%) 0(0%) Lysinibacillus macroides LMG 18474

NP 06 0.995 0.948 1416 Pseudomonas citronellolis; DSM 50332T; Z76659

2686 1486/1501(99%) 4(0%) Pseudomonas knackmussii B13

NP 07 0.985 0.926 1390 Novosphingobium naphthalenivorans; TUT562; AB177883

2564 1430/1451(99%) 0(0%) Novosphingobium naphthalenivorans TUT562


In summary, the eleven bacterial isolates initially identified were reduced to seven

distinct strains based on the outcomes of comparison with existing databases (Table

4.1) and labelled as NP01 to NP07 as shown in Table 4.2.

Table 4.2 Final nomenclature and identification of the pure bacterial isolates

Bacterial isolate New culture no. Proposed name

A1, A4 NP01 Chryseobacterium sp. NP01

A2 NP02 Delftia sp. NP02

A3, A5, AN5 NP03 Achromobacter sp. NP03

AN1 NP04 Ochrobactrum sp. NP04

AN3, AN4 NP05 Lysinibacillus sp. NP05

AN6 NP06 Pseudomonas sp. NP06

AN2 NP07 Novosphingobium sp. NP07

Various microorganisms associated with either anaerobic dechlorination (Adrian et

al., 2009; Payne et al., 2011; Praveckova et al., 2015) or aerobic oxidative

degradations (Borja et al., 2005; (Furukawa & Fujihara, 2008; Dudasova et al., 2014;

Hassan, 2014; Murinova & Dercova, 2014) of PCBs have previously been isolated from

different sediment and soil samples. However, there is no information available

about the bacterial species capable of utilizing PCBs as their sole carbon source

simultaneously under both, anaerobic and aerobic conditions.

Furthermore, a comprehensive search of published literature failed to identify any

studies reporting on the ability of Chryseobacterium sp. and Delftia sp. to grow on

PCBs. Therefore, the two obligate aerobic organisms identified as Chryseobacterium

sp. NP01 and Delftia sp. NP02 in the present study can be considered as new additions

to the existing list of PCB degrading bacteria.


4.3.1.2 Morphological characteristics of identified bacteria

The seven strains identified have shown different colony morphology on nutrient

agar as shown in Figure 4.6. Chryseobacterium sp. NP01 and Ochrobactrum sp. NP04

had mucilaginous colonies while Chryseobacterium sp. NP01 colonies were yellow

pigmented. Novosphingobium sp. NP07 colonies were orange pigmented on nutrient

agar. After the Gram staining, Lysinibacillus sp. NP05 was identified as a Gram

positive, rod shape, spore forming bacterium while all the other six bacterial strains

were identified as Gram negative, rod shape bacteria (see Figure 4.7 for cell

morphology).

When the Gram staining results were compared with the genomic DNA extraction

results as stated in Section 4.3.1.1, the two bacterial isolates AN3 and AN4 that was

not able to lyse using standard cell lysis protocol were identified as Gram positive,

spore forming Lysinibacillus sp. NP05. This further confirmed their resistance to cell

lysis due to the thick peptidoglycan layer (Vingataramin & Frost, 2015).


Figure 4.6 Colony morphology of the bacterial cultures on nutrient agar after 48 h incubation at 28 °C. Culture A to F were under aerobic

conditions and culture G was under anaerobic conditions.


Figure 4.7 Gram stained bacterial cultures under the light microscope (100x magnification).


4.3.1.3 Screening of microorganisms based on tolerance to atmospheric oxygen

The seven pure isolates were also tested for growth tolerance to atmospheric oxygen.

After growing them parallel under both, anaerobic and aerobic conditions, NP01 and

NP02 were identified as obligate aerobic bacteria as they have shown growth only

under aerobic conditions. NP03, NP04, NP05 and NP06 were able to grow under both,

aerobic and anaerobic conditions and therefore identified as facultative anaerobic

bacteria. NP07 was found to be obligate anaerobic as it was able to grow only under

anaerobic conditions.

Importantly, all the bacterial cultures confirmed their ability to grow and utilize PCBs

as their sole source of carbon by showing growth on minimal salt agar containing

Aroclor 1260 (Figure 4.8A and Figure 4.8B). The four facultative anaerobic bacterial

strains Achromobacter sp. NP03, Ochrobactrum sp. NP04, Lysinibacillus sp. NP05 and

Pseudomonas sp. NP06 were able to grow on minimal salt Aroclor 1260 agar under

both, aerobic (Figure 4.8A) and anaerobic (Figure 4.8B) conditions indicating their

ability to utilize PCBs as their sole source of carbon under both environmental

conditions. There was no visible growth in negative controls that contained solid

MSM with equal volume of acetone used to dissolved Aroclor 1260 (Figure 4.8C and

Figure 4.8D) This confirmed that the bacteria were not able to utilize acetone as their

carbon source even if there was residual acetone left in the agar medium after

evaporation. The characteristic yellow colour shown by Chryseobacterium sp. NP01

on nutrient agar (Figure 4.6A) was not present when growing on minimal salt agar as

shown in Figure 4.8A.


Figure 4.8 Bacterial colonies on minimal salt agar with 50 mg/L Aroclor 1260 as sole

source of carbon after 48 hrs at 28 °C (A) under aerobic conditions, (B) facultative

anaerobic cultures under anaerobic conditions, (C) negative controls under aerobic

conditions (D) negative controls under anaerobic conditions.

Out of the five bacterial cultures identified through anaerobic enrichment (NP03 to

NP07), only one culture, identified as Novosphingobium sp. NP07, was found to be

an obligate anaerobe. As shown in Figure 4.9, Novosphingobium sp. NP07

demonstrated the fastest growth on 25 mg/L and 50 mg/L Aroclor 1260 containing

MSA after 48 hrs. However, after few transfers to fresh minimal salt agar, the culture

lost its viability and was not able to recover. According to the literature, most PCB

dechlorinating obligate anaerobic cultures require the presence of sediment or


sediment substitutes to maintain their dechlorination ability. Therefore, it is difficult

to conduct subsequent bacterial enrichment and isolation processes involving such

bacteria (Wiegel & Wu, 2000). One of the main functions of added sediments to PCB

dechlorinating cultures is to enhance the bioavailability of PCB congener mixtures

(Adrian et al., 2009). There are only a few studies on maintaining sediment free

cultures by adding lactate (Wang & He, 2013b) and acetate (Bedard et al., 2006) as

carbon sources to the medium other than PCBs. This may explain why the viability of

obligate anaerobe Novosphingobium sp. NP07 was lost, and it was not included in

subsequent studies.

Figure 4.9 Growth of obligate anaerobic Novosphingobium sp. NP07 on 25 mg/L and

50 mg/L Aroclor 1260 containing minimal salt agar after 48 h incubation at 28 °C

under anaerobic conditions.

4.3.2 Basic growth profiles using glucose as the carbon source

Typical growth profiles of different and newly isolated microorganisms have generally

been performed using glucose as the carbon source, as it is readily used by most

microorganisms. During this study, cultures were set up as described in Section

4.2.3.5 and incubated over a nine hour period at 28 °C and 150 rpm in a shaker

incubator. Bacterial growth curves were developed based on the samples obtained

and analyzed for bacterial growth (optical density measurements) and glucose

consumption (Figure 4.10). The glucose test was based on the DNS reducing sugar

assay described in Section 3.2.6 in Chapter 3.


Figure 4.10 Basic bacterial growth profiles of the six bacterial isolates grown in minimal salt medium over a nine hour period at 28 °C and 150

rpm using glucose as the carbon source. Error bars represent the standard deviation of mean values (n = 3).

100 Selective Enrichment and Identification of Potential PCB Degrading Microorganisms

It was observed that only Chryseobacterium sp. NP01, Delftia sp. NP02,

Achromobacter sp. NP03 and Ochrobactrum sp. NP04 were able to utilize glucose as

their carbon source. The specific growth rate (µ) of these bacterial cultures were

calculated using the equation 4.1 as described in Section 4.2.3.5 and Table 4.3.

Samples removed over time demonstrated no significant change in the pH in all the

cultures throughout the study period (see Figure 4.11 for pH variation). However,

parallel to the glucose consumption, initial 7.2 pH in the Chryseobacterium sp. NP01,

Delftia sp. NP02 and Achromobacter sp. NP03 containing culture media indicated

slight reduction of pH over time until six to seven hours and started to increase again

once the glucose utilization was stopped. The possible reason for the pH reduction at

the beginning is attributed to the breakdown of glucose into acidic metabolites. Once

the glucose utilization was over, further combustion of acidic metabolites would have

again contributed to increase the pH.

Table 4.3 Specific growth rate of bacterial cultures during the growth profile studies

using 2 g/L glucose as the carbon source.

Bacterial culture Specific growth rate (µ) (h-1)

Chryseobacterium sp. NP01 0.47

Delftia sp. NP02 0.45

Achromobacter sp. NP03 0.34

Ochrobactrum sp. NP04 0.3

Lysinibacillus sp. NP05 Slight growth

Pseudomonas sp. NP06 No growth

There was no glucose reduction or biomass increase observed in the flasks containing

Pseudomonas sp. NP06 indicating the inability of this strain to consume glucose as its

carbon source. Interestingly, the glucose concentration measured as reducing sugars

in the Lysinibacillus sp. NP05 containing flasks started to increase slowly with time.

At the end of the nine hours, it increased up to ~ 2.27 g/L from the initially added

glucose level of 2 g/L. There was no noticeable increase in the glucose level in the

negative control as indicated in the Figure 4.10. This results suggested that under


nutrient limiting conditions, microbes such as Lysinibacillus sp. NP05 may have the

ability to synthesize some extracellular polymeric substances (EPS) such as

exopolysaccharides to protect the cells through cellular adhesion (Characklis &

Cooksey, 1983). The common sugars such as D-glucose, D-galactose and D-mannose

are frequently found in bacterial exopolysaccharides (Flemming et al., 2016). Some

Lysinibacillus species were previously identified with the ability to secrete some EPS

with high protein content (Francois et al., 2012). Parallel to the increase in glucose

level in the culture medium, Lysinibacillus sp. NP05 cell density as the OD600 also

indicated a gradual increase. The research literature suggests the ability of some

bacteria to utilize the secreted exopolysaccharides for protein production under

nutrient limiting conditions (Flemming et al., 2016).

Figure 4.11 Variation of pH in the culture medium during the bacterial growth profile

studies. Error bars represent the standard deviation of mean values (n = 3).

102 Selective Enrichment and Identification of Potential PCB Degrading Microorganisms

4.4 Conclusions

The overall aim of the selective enrichment screening in the present study was to

isolate, screen and identify potential PCB degrading microorganisms from the natural

environment for use in PCB degradation. Two obligate aerobic, four facultative

anaerobic and one obligate anaerobic bacterial strains were isolated by selective

enrichment screening. Based on the 16S rRNA sequence based molecular

identification, they were identified as Chryseobacterium sp. NP01, Delftia sp. NP02,

Achromobacter sp. NP03, Ochrobactrum sp. NP04, Lysinibacillus sp. NP05,

Pseudomonas sp. NP06 and Novosphingobium sp. NP07. Their survival on PCB was

confirmed by the growth on minimal salt agar containing commercial PCB mixture,

Aroclor 1260. However, the obligate anaerobe Sphingomonas sp. NP07 lost its

viability to grow and therefore, was not able to be used in further degradation

studies.

Importantly, this is also the first instance of reporting of the presence of

Chryseobacterium and Delftia species involved in PCB contaminated and hydrolysis

conditions. Furthermore, to date, information is not available on the isolation of

facultative anaerobic microorganisms capable of growing under both, anaerobic and

aerobic conditions while utilizing PCBs as their sole source of carbon. Therefore,

based on the results obtained in this study, the isolated facultative anaerobic strains


Pseudomonas sp. NP06 were chosen and individually compared for their PCB

degradation potential under parallel; aerobic, anaerobic and combined anaerobic-

aerobic conditions, as described in Chapter 6.

Chapter 5: Screening of bacterial isolates for biosurfactant production 103

Chapter 5: Screening of bacterial isolates for biosurfactant production

5.1 Background

PCBs are hydrophobic chemicals with poor water solubility. This feature and the low

abundance of suitable microbial communities in the contaminated environments can

prevent natural biodegradation occurring at faster rates (Edwards & Kjellerup, 2013;

Stella et al., 2015). Past research literature on biochemical pathways and intracellular

localization of enzymes responsible for PCB degradation suggest that PCBs have to

be solubilized for easier passage through the cell membrane and into the cytoplasm

prior to being metabolized. Therefore, an increase in the rate of solubilisation may

accelerate the entrance of PCBs into cells and their subsequent degradation (Ohtsubo

et al., 2004). One way of increasing the rate of solubilisation of hydrophobic PCBs is

the addition of mobilizing agents such as chemical and biological surfactants (Singer

et al., 2000; Fava & Di Gioia, 2001; Occulti et al., 2008). Chemical surfactants have the

advantage of being economical, but are often toxic to biological systems (Abraham

et al., 2002). In comparison, biosurfactants generally exhibit higher interfacial tension

reduction activities compared to chemical surfactants, and are less toxic and readily

biodegradable (Viisimaa et al., 2013). However, the main disadvantage of the use of

commercially available biosurfactants is the high cost (Aparna et al., 2012).

The use of suitable biosurfactant producing microbial strains, which are also capable

of degrading PCBs, would be an attractive alternative to the use of chemical and

biological surfactants. Indeed, the application of biosurfactant-producing and

pollutant-degrading microorganisms offers a dual advantage of a continuous supply

of biodegradable surfactant and the ability to degrade pollutants (Megharaj et al.,

2011). However, limited information is available on the fundamentals regarding the

regulation and mechanisms on why and how microbial cultures produce

biosurfactants, and the eventual hydrolysis PCBs. Most studies on PCB solubility have

been based on the addition of chemical or biological surfactants with no investigation

104 Chapter 5: Screening of bacterial isolates for biosurfactant production

into the actual application of microorganisms producing surfactants (Singer et al.,

2000; Fava & Di Gioia, 2001; Occulti et al., 2008; Viisimaa et al., 2013).

In this context, this chapter focuses on investigating the microorganisms with PCB

degradation potential described in Chapter 4, and whether they can also produce

biosurfactants during the hydrolysis of PCBs. The main outcome from this part of the

study was used in the selection of suitable bacterial strains to form the consortium

for further tests, as described in Chapter 7.


In this study, the six bacterial cultures isolated through selective enrichment

described in Chapter 4, namely, Chryseobacterium sp. NP01, Delftia sp. NP02,


Pseudomonas sp. NP06 were individually tested for their PCB solubilization and

biosurfactant production ability.

5.2.1 Experimental setup

Batch mesocosm experiments were conducted for each bacterial strain under aerobic

conditions for six weeks. Erlenmeyer flasks containing 75 mL of sterile minimal salt

medium spiked with 50 mg/L Aroclor 1260 were prepared as described in Section

3.1.2.1 A. Triplicate flasks were tested for each microorganism. Two flasks containing

75 mL of sterile minimal salt medium spiked with 50 mg/L Aroclor 1260 (without

adding bacteria) were used as abiotic controls. Additionally, media controls were

prepared by spiking 75 mL of sterile minimal salt medium with an equal volume of

acetone used to dissolve Aroclor 1260. Parallel to each bacterial batch culture

experiment, two media controls were conducted after inoculation with bacterial seed

culture in a similar way to the experiments.

Each bacterial culture was inoculated into 40 mL of sterile LB medium and incubated

overnight at 150 rpm and 28 °C in a platform shaker. Contents were transferred to 50

mL falcon tubes and centrifuged at 5000 x g for 10 minutes to separate the cell

pellets. Cell pellets were washed twice in minimal salt medium and re-suspended in


5 mL of fresh minimal salt medium. 1 mL aliquots of the seed culture suspension were

used to inoculate the batch mesocosms and media controls. All the mesocosms,

abiotic controls and media controls were incubated at 28 °C and 150 rpm in a rotary

shaker for six weeks.

5.2.2 Evaluation of PCB solubility and degradation

PCB solubilisation was measured in the form of total PCBs in solution as hydrophobic

portion of PCBs remained at the bottom of the flask and on the surface of the

aqueous medium. PCB degradation was measured indirectly using cell density, pH

variation and chloride ion accumulation in the batch mesocosms. Initially and at

weekly intervals, 1 mL aliquots of culture supernatants were removed, extracted (as

per Section 3.2.8 A) and analysed (as per Section 3.2.9) for total soluble PCBs. In order

to have homogeneous samples, aliquots were withdrawn from the middle part of the

liquid in the flasks. Parallel to the sampling for PCB analysis, 1 mL aliquots were

removed to measure the cell growth as optical density as per Section 3.2.4 A and 2

mL aliquots were removed to measure the pH as per Section 3.2.5. At the end of week

six, 10 mL from the remaining culture medium was removed, centrifuged at 5000 x g

for 10 min and passed through 0.2 µm filters to separate the bacterial cells and the

filtrate was analysed for chlorine ion accumulation as described in Section 3.2.7.

5.2.3 Screening for biosurfactant production

After six weeks of cultivation, 10 mL culture supernatant from each flask of the batch

experiment were centrifuged at 5000 x g for 10 min and filtered through 0.2 µm filters

to separate the bacterial cells. The cell free supernatants were used for the

subsequent biosurfactant screening tests.

5.2.3.1 Drop collapse test

The drop collapse test was used as a primary screening to determine the ability of

bacterial strains for their biosurfactant production capacity based on past literature

(Bodour et al., 2003; Alvarez et al., 2015; Panjiar et al., 2015; Joy et al., 2017). 20 μL


cell free culture supernatant was mixed with 5 μL of 0.1% methylene blue solution

and placed on a parafilm as a drop using a 100 μL micropipette (Panjiar et al., 2015).

The purpose of adding methylene blue was for easy visualization of the droplet.

Diameter of the drop was measured after one minute using 1 mm grit paper placed

underneath the parafilm paper. 1% (w/v) sodium dodecyl sulphate (SDS) solution was

used as the positive control while phosphate buffered saline (PBS) solution and

abiotic controls were used as negative controls. The results where the diameter of

the droplet was at least one millimetre larger than the one made by the negative

control were considered as positive for biosurfactant production.

5.2.3.2 Emulsification index (EI24)

3 mL of cell free culture supernatant and 3 mL of mineral oil were taken into a

graduated test tube and vigorously shaken for 2 min to form an emulsion. The

mixture was allowed to stand still for 24 h and the height of emulsion layer was

measured (Nayak et al., 2009). The emulsification index was calculated using

Equation 5.1 (Panjiar et al., 2015).

EI₂₄(%) = Height of emulsion formed after 24 hrs / Total height of solution x 100

Equation 5.1

Emulsions formed following the reactions with different culture supernatants were

compared to the controls. The positive control used was a 1% (w/v) solution of

synthetic surfactant sodium dodecyl sulphate in deionised water, whereas the abiotic

and medium only control samples were used as the negative controls.

5.2.3.3 Haemolysis

To detect the haemolytic activity indicative of biosurfactant production, 50 µL of the

cell free culture supernatant was spotted on the middle of Tryptone soya agar plates

containing 5% sheep blood (Thermo Fisher Scientific) and the plates were incubated

at 28 °C for 48 h (Alvarez et al., 2015). Plates were visually examined for haemolysis.

The level of clearance of red blood cells was considered proportional to the

concentration of biosurfactant. A yellow transparent zone indicated complete lysis of


red blood cells and was regarded as beta () or complete haemolysis. The appearance

of dark green zones beneath the place where the supernatant was spotted was

considered as alpha () or partial haemolysis of blood cells. Alpha and beta

haemolysis were considered as positive for biosurfactant production. No change in

the blood agar plates indicates gamma () or no haemolysis (Joy et al., 2017).


5.3.1 PCB solubility

At the beginning of the experiment, Aroclor 1260 was added to the minimal salt

medium to give 50 mg/L total PCB concentration. Due to the low water solubility of

this highly chlorinated PCB mixture, the initial soluble PCB concentration in the

aqueous minimal salt medium was very low. The results are shown in Figure 5.1.

Figure 5.1 Variation of the total solubility of Aroclor 1260 in the aqueous minimal salt

media inoculated with bacterial cultures. Error bars represent the standard deviation

of mean values (n=3 for bacterial cultures and n=2 for abiotic controls).

As shown in Figure 5.1, the results for the abiotic controls demonstrated that the

total soluble PCB concentration remained stable throughout the six weeks study


period and it was measured and found to be 0.15±0.03 mg/L (Figure 5.1). The results

from the obligate aerobic Chryseobacterium sp. NP01 and facultative anaerobic

Lysinibacillus sp. NP05 demonstrated increased concentrations of PCBs in solution

over time. In comparison, there was no significant increase in the PCB concentrations

in solution for the other four culture members (Figure 5.1).

For Lysinibacillus sp. NP05, the PCB concentration in the solution gradually increased

over time and reached a maximum solubility of 14.9±0.55 mg/L at the end of week 5.

In comparison, nearly half of the initially added 50 mg/L Aroclor 1260 became soluble

at the end of the week 6 for Chryseobacterium sp. NP01 and it was measured as

23.8±1.03 mg/L. However, increase in PCB concentration in solution does not

necessarily indicate the net solubility of PCBs. This is due to the potential of bacteria

for increasing solubility and breaking down of PCBs concomitantly under the provided

conditions. When the rate of degradation is similar to the rate of solubility, it would

not indicate any increase of PCB concentration in the solution.

5.3.2 Bacterial growth, chloride ion accumulation and pH

A holistic investigation into the performance of the bacterial strains was carried out

based on one experimental approach while obtaining results for; (1) PCB solubility,

(2) bacterial growth, (3) pH, and (4) chloride ion accumulation in the culture medium.

This is primarily due to the potential of the bacterial strains to increase PCB solubility,

while removing chlorines from biphenyl rings and breaking of the biphenyl rings

concomitantly when PCBs is the only carbon source available. Therefore, this

approach warranted a detailed assessment of bacterial growth, chloride ion build up

and pH variation, in addition to the testing for biosurfactant production during the

bacterial cultivations with PCBs, as an energy source.

5.3.2.1 Bacterial growth

During the six weeks experimental period, growth of all six cultures were very low

and optical density values were less than one (Figure 5.2). Chryseobacterium sp. NP01

reached its maximum growth within the first week of incubation with an OD600 value

of 0.65±0.03 despite the increasing PCB solubility over time (Figure 5.2 vs 5.1). In


comparison, the OD600 readings of Achromobacter sp. NP03, Ochrobactrum sp. NP04,

Lysinibacillus sp. NP05 and Pseudomonas sp. NP06 showed minor increase over time.

Figure 5.2 Bacterial growth as optical density (OD600) in the batch mesocosms at 28

°C and 150 rpm. Error bars represent the standard deviation of mean values (n=3).

When cell growth results (Figure 5.2) were compared to PCB solubility results (Figure

5.1), the total PCB solubility capacity of Chryseobacterium sp. NP01 and Lysinibacillus

sp. NP05 were significantly high (see Figure 5.1), but there was no significant increase

in growth. In particular, Chryseobacterium sp. NP01 reached high cell growth by the

end of week 1, followed by a steep decline between weeks 2 and 3, and then followed

a similar trend like most of the other bacteria in weeks, 4, 5 and 6 (Figure 5.2). At the

other extreme, Delftia sp. NP02 seemed to have little growth.

These results suggested that although Chryseobacterium sp. NP01 and Lysinibacillus

sp. NP05 were capable of transforming an extremely hydrophobic PCB mixture

soluble in aqueous medium, their PCB degradation ability under aerobic conditions

was limited to lower chlorinated congeners as suggested by Borja et al. (2005).


5.3.2.2 Chloride ion concentration

At the end of week six, the chloride ion concentrations were measured and are shown

in Figure 5.3. The contribution of chloride ion ions from controls (from minimal salt

medium and seed cultures) for all six cultures (NP01 to NP06) were 33.88±0.13,

34.78±0.08, 34.70±0.10, 35.33±0.03, 34.29±0.08 and 34.24±0.09 mg/L respectively.

The background chloride values were first subtracted from the experimental values

before the final values were plotted in order to clearly demonstrate the contribution

of chlorides due to the activity of each bacterial culture.

Figure 5.3 Chloride ion accumulation in the batch mesocosms after six weeks of

incubation at 28 °C and 150 rpm. The background values from the controls of (1)

minimal salt medium only and (2) seed cultures only were subtracted first. Error bars

represent the standard deviation of mean values (n=3).

As shown in Figure 5.3, the highest chloride ion concentrations were from

Chryseobacterium sp. NP01 and Achromobacter sp. NP03 both indicating similar

values of 2.4±0.3 mg/L and 2.4±0.5 mg/L respectively (Figure 5.3). These chloride

concentrations were followed by Lysinibacillus sp. NP05 (2.0±0.3 mg/L) and

Ochrobactrum sp. NP04 (1.5±0.3 mg/L). According to past research literature, under


aerobic conditions, bacteria are usually capable of degrading lower chlorinated

congeners through biphenyl ring cleavage (Borja et al., 2005; Field & Sierra-Alvarez,

2008). As previously analysed, the percentage of total chlorine in the Aroclor 1260

mixture used in this study was 58.27±1.15 (see Table 3.2 in Chapter 3). Based on the

homolog group composition in Table 3.2, out of 58.27±1.15% total chlorine, only

1.9±0.13% of chlorine was from the lower chlorinated congeners (congeners with 4

or less chlorines). Therefore, the theoretical maximum chloride concentration

expected to be released from a complete degradation of lower chlorinated congeners

in a 50 mg/L Aroclor 1260 solution is 0.95±0.06 mg/L. As shown in Figure 5.3, except

for Pseudomonas sp. NP06, the rest of the bacterial strains all released different, but

higher amounts of chlorides compared to the expected theoretical maximum yield.

These results suggested that under aerobic conditions, the six bacteria tested have

different mechanisms to attack PCBs, with the concomitant release of different

concentration of chloride ions, depending on their ability to degrade different lower

and moderately chlorinated homolog groups.

5.3.2.3 pH

There was no significant change in pH in all the batch mesocosms and they were in

the range of 7.02±0.01 to 7.78±0.02 as shown in Figure 5.4. However, throughout the

six weeks period, pH variation of flasks containing Chryseobacterium sp. NP01 and

Lysinibacillus sp. NP05 were similar and always slightly higher than the flasks

containing rest of the cultures. As PCB solubility levels were also high in the

mesocosms inoculated with Chryseobacterium sp. NP01 and Lysinibacillus sp. NP05,

any potential relationship between the pH of the medium and the agents responsible

for increased PCB solubility needs to be further investigated. One possible

explanation for all the pH to move towards the alkaline range is the potential build-

up of CO2 in the form of HCO3- in the medium, during cellular respiration under

aerobic cultivation with agitation set at 150 rpm.


Figure 5.4 pH variation in the batch mesocosms at 28 °C and 150 rpm. Error bars

represent the standard deviation of mean values (n=3).

5.3.3 Biosurfactant production

The bacterium Chryseobacterium sp. (formerly known as Flavobacterium) was

previously reported with the ability to produce biosurfactants named flavolipids. The

mixture was found to consist of at least 37 strong and stable different flavolipids even

at low concentrations (Bodour et al., 2004). When tested with the hydrocarbon

hexadecane, the apparent solubility of hexadecane increased by several orders of

magnitude. An extracellular bioemulsifier producing bacterium Lysinibacillus sp. was

also isolated from soil contaminated with petroleum oil and found to be positive with

different aliphatic and aromatic hydrocarbons like hexane, benzene, toluene, diesel

and kerosene for its biosurfactant production ability (Panjiar et al., 2015). In addition,

a bacterium isolated from refinery wastewater was identified as Ochrobactrum sp.

with the potential to produce exopolysaccharide bioemulsifier and was able to

degrade diesel oil (Ramasamy et al., 2014), n-octane, mineral light and heavy oils,

crude oil (Calvo et al., 2008). However, there is no research literature available that

confirms the ability of the same bacterial species to produce biosurfactants, which


makes extreme hydrophobic PCBs soluble in aqueous medium while concomitantly

degrading PCBs. Therefore, this study can be considered a first to report the potential

of Chryseobacterium, Lysinibacillus and Ochrobactrum species to produce

biosurfactants that ultimately increased the solubility of PCBs.

In this study, the drop collapse and emulsification index tests were used as

quantitative methods (Ramasamy et al., 2014), while the haemolytic assay was used

as the qualitative method (Thavasi et al., 2011) to test for biosurfactant production.

Results of these three tests are summarised in Table 5.1.

Table 5.1 Summary of biosurfactant screening tests

Notes:

* The zones of clearing were scored as follows: ‘+’ incomplete haemolysis; ‘++’ incomplete

haemolysis with semitransparent zones surrounded by green colour areas; ‘+++’ complete

haemolysis.

**1% (w/v) sodium dodecyl sulphate (SDS) solution

***Phosphate buffered saline (PBS) solution

ND - not detected.

Among the six bacterial cultures, four cultures showed positive results for the drop

collapse test. A maximum drop diameter of 6 mm was observed with the culture

Description Drop collapse test

(Diameter in mm)

Emulsification

index (%)

Haemolysis*

Positive Control** 5.3±0.3 50.0 +++

Negative Control*** 3.3±0.3 ND ND

Abiotic control 3.2±0.3 ND ND

Chryseobacterium sp. NP01 6.0±0.0 50.0 +++

Delftia sp. NP02 4.0±0.0 16.7 +

Achromobacter sp. NP03 5.3±0.3 33.3 ++

Ochrobactrum sp. NP04 5.0±0.0 16.7 ++

Lysinibacillus sp. NP05 5.0±0.0 50.0 +++

Pseudomonas sp. NP06 3.5±0.0 ND +


supernatant of Chryseobacterium sp. NP01. Achromobacter sp. NP03, Ochrobactrum

sp. NP04 and Lysinibacillus sp. NP05 were also demonstrated to have relatively high

diameters of 5.3±0.3 mm, 5.0±0.0 mm and 5.0±0.0 mm, respectively, when compared

to 3.3±0.3 mm diameter in the negative control (see Table 5.1 and Figure 5.5).

Figure 5.5 Drop collapse test. 1% (w/v) sodium dodecyl sulphate (SDS) solution was

used as the positive control. The phosphate buffered saline (PBS) solution and abiotic

control (minimal salt medium only) were used as negative controls.

As summarised in Table 5.1, the highest emulsification index of 50% was observed in

culture supernatants of both Chryseobacterium sp. NP01 and Lysinibacillus sp. NP05.

This high result was followed by Achromobacter sp. NP03 (33.3%), Ochrobactrum sp.

NP04 (16.7%) and Delftia sp. NP02 (16.7%). Emulsion formation was not observed in

the culture supernatant of Pseudomonas sp. NP06.

Similar to positive results from the emulsification index test, Chryseobacterium sp.

NP01 and Lysinibacillus sp. NP05 performed well during the haemolytic assay. Both

microbes showed strong yellow transparent zones in Tryptone soya agar containing

sheep blood after incubation at 28 °C for 48 hours indicative of complete haemolysis

of red blood cells (Figure 5.6). In contrast, Achromobacter sp. NP03 and

Ochrobactrum sp. NP04 showed partial haemolysis having semi-transparent zones

surrounded by dark green areas. Delftia sp. NP02 and Pseudomonas sp. NP06 showed


negative to minor haemolysis, when compared to other positive results and this also

coincides with the low chloride accumulation observed.

Figure 5.6 Haemolysis of sheep blood in Tryptone soya agar after incubation at 28 °C

for 48 hours (A) Chryseobacterium sp. NP01, (B) Delftia sp. NP02, (C) Achromobacter

sp. NP03, (D) Ochrobactrum sp. NP04, (E) Lysinibacillus sp. NP05, (F) Pseudomonas

sp. NP06, (G) 1% SDS as positive control, and (H) abiotic control.

When all three biosurfactant tests conducted were considered, the descending order

of preference for the potential of biosurfactant production is Chryseobacterium sp.

NP01 > Lysinibacillus sp. NP05 > Achromobacter sp. NP03 > Ochrobactrum sp. NP04

> Delftia sp. NP02 > Pseudomonas sp. NP06. According to Ohtsubo et al. (2004), the

following events need to take place for PCBs to be degraded by microorganisms: (1)

solubilisation of PCBs and their entry into cells; (2) expression of PCB degrading

enzymes in the cells; and (3) catalytic breakdown of the PCBs. When the results of

biosurfactant screening tests were compared with PCB solubility and chloride ion

accumulation, Chryseobacterium sp. NP01 and Lysinibacillus sp. NP05 that exhibited

the highest biosurfactant production potential also demonstrated the highest total

PCB solubility and chloride ion accumulation. These results provide a clear and

positive correlation between biosurfactant production and PCB solubilization and

subsequent degradation. Although the solubility of PCBs by the other four cultures


were comparatively low, they all displayed different levels of chloride ion

accumulation suggesting they had different mechanisms for attacking PCBs.

Not surprisingly, the two cultures, Delftia sp. NP02 and Pseudomonas sp. NP06 that

demonstrated the lowest biosurfactant production capability also had the lowest

chloride ion levels in the culture supernatant. When the growth profiles of bacterial

cultures were considered, the low growth rate would suggest it is due to the

availability of limited lower chlorinated congeners in the medium. Under aerobic

conditions, these bacteria are expected to only attack and consume lower

chlorinated congeners as their carbon source. Therefore, it is important to study the

performance of these culture members with secondary carbon sources, for cell

growth. The outcomes of such an experiment can potentially help enhance the

biosurfactant production and PCB degradation.

All these bacterial cultures were initially screened as discussed in Chapter 4 for their

ability to survive by utilizing PCBs as their sole source of carbon. According to the

results presented in this Chapter, it can be argued that the rate of microbial

degradation of PCBs is decided not only by their ability for breaking down the PCB

molecules, but also by their ability for making hydrophobic PCBs soluble in the

aqueous medium. Therefore, it is important to include suitable and different culture

members with the ability to produce biosurfactants in order to facilitate the

bioavailability of PCBs. Such a strategy may assist in producing effective on field and

scale up bioremediation applications.

5.4 Conclusions

Six bacterial isolates identified during an initial selective screening for PCB

degradation potential were also individually tested for their ability to produce

biosurfactants. Emulsification index and drop collapse tests were used as quantitative

tests and a haemolytic assay was used as a qualitative test to measure the

biosurfactant production ability of the selected microorganisms. Concomitantly, the

total PCB solubility, bacterial growth, and chloride ion accumulation were also

analysed to measure the PCB solubilisation and degradation potential.


Biosurfactants produced by the microorganisms were found to increase the solubility

of hydrophobic PCB mixture in the aqueous medium and consequently enhanced the

bioavailability and degradation of PCBs. The rate of PCB solubility was positively

correlated with the rate of biosurfactant production. Chryseobacterium sp. NP01 and

Lysinibacillus sp. NP05 demonstrated the highest biosurfactant production, PCB

solubilisation and chloride ion accumulation when compared to the other four

bacterial cultures, Delftia sp. NP02, Achromobacter sp. NP03, Ochrobactrum sp. NP04

and Pseudomonas sp. NP06.

In conclusion, the results suggested that microorganisms capable of degrading PCBs

also have a potential to produce some surface active substances to facilitate

hydrophobic PCBs soluble in aqueous media to make them available for utilization.

However, as this study was an initial screening to see the potential of isolated PCB

degrading microorganisms for biosurfactant production, further research is needed

to identify the types and physicochemical properties of the biosurfactants through

extraction, quantification and characterization and to assess their performance in

PCB contaminated soil remediation applications. This latter part was outside the

scope of this study.

Chapter 6: PCB degradation potential of facultative anaerobic bacterial isolates 119

Chapter 6: PCB degradation potential of facultative anaerobic bacterial isolates

6.1 Background

Biological conversion of highly and moderately chlorinated congeners into less

chlorinated congeners has been reported to take place under anaerobic conditions

(Praveckova et al., 2015; Agullo et al., 2017). In comparison to highly chlorinated

congeners, lower and moderately chlorinated congeners can be degraded by

oxidative bacteria under aerobic conditions through the upper and lower biphenyl

degradation pathways (Field & Sierra-Alvarez, 2008). This highlights the need of a

bioremediation strategy involving a combined approach of anaerobic dechlorination

and aerobic oxidation steps, in order to obtain the complete degradation of PCBs

(Passatore et al., 2014). Studies undertaken to understand PCB degradation by

combined anaerobic-aerobic conditions have used two separate groups of bacteria,

one capable of reductive dechlorination and the other capable of aerobic oxidation

(Evans et al., 1996; Master et al., 2002). However, there has been no comprehensive

study undertaken to understand PCB degradation using facultative anaerobic

microorganisms under a two-stage anaerobic-aerobic (dechlorination and oxidation)

bioprocess approach. Use of facultative anaerobic microorganisms can potentially

break down PCBs using the two stage anaerobic-aerobic processes while being able

to be used in field scale bioremediation applications as they have the potential to

survive and carry out degradation under varying environmental conditions.

The main aim of Chapter 6 is to investigate the capability of four facultative anaerobic

bacterial cultures Achromobacter sp. NP03, Ochrobactrum sp. NP04, Lysinibacillus sp.

NP05 and Pseudomonas sp. NP06 isolated through selective enrichment as described

in Chapter 4, for degrading Aroclor 1260. Experiments were conducted in parallel for

each facultative anaerobic culture member under (1) anaerobic, (2) aerobic and (3)

two stage (combined) anaerobic-aerobic conditions. The outcomes from this part of

120 Chapter 6: PCB degradation potential of facultative anaerobic bacterial isolates

the study are expected to contribute towards the development of more efficient and

effective bacterial mediated bioremediation treatment discussed in Chapter 7.


The batch mesocosm experiments were conducted for four facultative anaerobic

bacterial cultures Achromobacter sp. NP03, Ochrobactrum sp. NP04, Lysinibacillus sp.

NP05 and Pseudomonas sp. NP06 isolated and identified in Chapter 4 with the

potential to degrade PCBs under both aerobic and anaerobic conditions. For each

facultative anaerobic culture, three separate sets of experiments were conducted in

parallel under aerobic, anaerobic and two-stage anaerobic aerobic conditions,

respectively. All the experiments were conducted in triplicate for each

microorganism under each tested condition.

According to past research literature, the cleavage of the biphenyl ring usually does

not occur during PCB dechlorination, but uses PCBs as electron acceptors (Wiegel &

Wu, 2000). Therefore, it is expected that suitable secondary or additional carbon

sources would be needed and be accessible by the microorganisms for growth.

However, based on the results obtained during the initial selective screening

described in Chapter 4, all the facultative anaerobic microorganisms demonstrated

growth on minimal salt agar containing 50 mg/L of Aroclor 1260 as the only carbon

source available, under anaerobic conditions (see Figure 4.8B in Chapter 4).

Therefore, no additional carbon sources were added to the culture medium other

than PCBs, during the study.

6.2.1 Experimental setup

Erlenmeyer flasks containing 75 mL of sterile minimal salt medium (see Section

3.1.2.1A for mesocosm preparations) were prepared and Aroclor 1260 stock solution

in GCMS grade acetone was added to each flask as the sole source of carbon so that

the final Aroclor 1260 concentration was 50 mg/L. Contents of the flasks were

vigorously shaken for few minutes to evaporate acetone. In all anaerobic and two

stage experiments, flasks were prepared, inoculated and incubated in an anaerobic


chamber (COY laboratory products, Inc.) to maintain strict anaerobic conditions. The

flasks with minimal salt medium used for anaerobic and two stage experiments were

kept equilibrated for one week inside the anaerobic chamber main compartment

before they were inoculated with the seed cultures. The environment inside the

anaerobic chamber was maintained at a condition with 4.9 % H2, 10.7 % CO2 and 84.4

% N2 (BOC Australia).

Anaerobic dechlorination is a reductive process. PCBs act as electron acceptors and

the chlorine substituents are replaced with hydrogen (Borja et al., 2005). Therefore,

suitable electron donors are needed for the dechlorination process by

microorganisms, which may ultimately lead to the conversion of highly chlorinated

congeners into the lower chlorinated compounds. Hydrogen is assumed to act

directly or indirectly as the electron donor in the dechlorination process (Wiegel &

Wu, 2000). Therefore, the hydrogen gas present inside the anaerobic chamber and

controlled at 4.9% (mol/mol) concentration was the potential electron donor in the

present study.

In aerobic experiments, flasks were incubated at 28 °C and 150 rpm in a rotary shaker

for six weeks. Flasks used in the anaerobic experiments were retained at 28 °C in an

incubator kept inside the anaerobic chamber, with occasional gentle shaking by hand

over the 6 weeks period. The two-stage experiment started at 28 °C under anaerobic

conditions (with occasional shaking by hand) for the first four weeks, and then

removed from the anaerobic chamber and transferred to aerobic conditions at 28 °C

and 150 rpm for the last two weeks.

6.2.2 Bacteria seed culture preparation

Bacteria cultures were inoculated into sterile LB medium in 50 mL falcon tubes and

incubated overnight at 150 rpm and 28 °C in a platform shaker. 7.5 mL of culture

suspension was aseptically transferred to a sterile vial and centrifuged at 5000 x g for

10 min. The supernatant was discarded, and the resulting cell pellet was washed

twice in minimal salt medium. The cell pellet was collected again by centrifugation

and resuspended in 1 mL of minimal salt medium to be used as the inoculum.


Bacterial cell densities of the seed cultures were calculated as colony forming units

(CFU) using standard plate count as described in Section 3.2.4B.

6.2.3 Controls

The following controls were tested in parallel under each condition.

1. Abiotic controls: 75 mL of minimal salt medium spiked with 50 mg/L Aroclor

1260 without the addition of bacteria seed culture.

2. Media controls: 75 mL minimal salt medium spiked with an equal volume of

acetone used to dissolve 50 mg/L Aroclor 1260 and inoculated with an equal

amount of seed culture used in the experiment.

6.2.4 Sample collection, analysis and preservation

Samples were withdrawn from each flask at time zero and at weekly intervals for the

analyses of pH (2 mL), cell growth (1 mL), extracellular proteins (1 mL) and PCB

solubility (1 mL). In order to have representative samples, liquid aliquots were

removed from the middle of the culture medium while flasks were kept under

agitation. The cell growth was measured as the optical density as per Section 3.2.4A.

The pH and chloride ion concentration were measured as per Section 3.2.5 and

Section 3.2.7 respectively. The total soluble PCBs were extracted as per Section 3.2.8

A and analysed as per Section 3.2.9. Samples withdrawn for extracellular protein

analysis were stored in sterile low protein binding Eppendorf tubes at -80 °C until

subsequent analysis. Analytical processes and outcomes of extracellular protein

analysis are discussed in Chapter 8.


6.3 Results and discussion

Cell densities of the overnight grown four bacterial seed cultures are summarized in

Table 6.1.

Table 6.1 Bacteria cell count in overnight Luria–Bertani liquid medium at 28 °C.

Bacterial culture No. of colonies in 10-5 dilution

using 100 µL seed culture

No. of CFU/mL

seed culture

1 2 3

Achromobacter sp. NP03 296 288 282 2.9x108 ± 0.07x108

Ochrobactrum sp. NP04 151 144 155 1.5x108 ± 0.09x108

Lysinibacillus sp. NP05 162 143 152 1.5x108 ± 0.09x108

Pseudomonas sp. NP06 180 168 173 1.7x108 ± 0.12x108

6.3.1 Growth profiles - aerobic vs anaerobic vs two stage anaerobic-aerobic

As described in Section 6.2.1, the four facultative strains were tested for growth on

Aroclor 1260 as a carbon source in liquid batch cultures. All tests were monitored at

weekly intervals under three different conditions; aerobic, anaerobic and two stage

anaerobic-aerobic. The results are shown in Figure 6.1.

During aerobic conditions as shown in Figure 6.1a, all four strains reached saturation

by week one with optical density (OD600) readings of 0.51, 0.7, 0.62 and 0.46 for NP03,

NP04, NP05 and NP06 respectively. These optical densities are relatively similar and

demonstrated less variations over time. According to the literature, these results

suggest the possibility of NP03, NP04, NP05 and NP06 consuming lower chlorinated

PCB congeners within the first week and were unable to degrade highly chlorinated

congeners under aerobic conditions (Field & Sierra-Alvarez, 2008). This concept can

be supported by the fact that Aroclor 1260, the PCB source used in this study consists

of less than 10% (by weight) of lower chlorinated homolog groups (containing four or

fewer chlorines per biphenyl molecule) compared to the highly chlorinated homologs

(Mayes et al., 1998; ATSDR, 2000) (see Table 3.2 in Chapter 3). To provide further

support for these observations, Pieper and Seeger (2008) reported that aerobic


bacteria were capable of degrading biphenyl as the sole source of carbon and energy,

and usually involves the biodegradation of PCBs with less than four chlorine atoms.

Based on these results, under aerobic conditions, the four facultative anaerobic

strains NP03, NP04, NP05 and NP06 may have hydrolysed the lower chlorinated PCBs

homolog groups present in Aroclor 1260. The reactions appeared to occur within the

first one to two weeks of incubation (see Figure 6.1a).

In comparison, during anaerobic conditions, all four strains of NP03, NP04, NP05 and

NP06, showed higher cell densities with OD600 of 1.22, 1.31, 1.04 and 0.82,

respectively. Variations of cell densities indicate that the maximum growth was

reached by week 4 (see Figure 6.1b). Significantly high growth rates under anaerobic

conditions without the presence of carbon sources other than PCBs is an indication

of biphenyl ring cleavage in addition to dechlorination. According to the current

literature, anaerobic dechlorination is a reductive process that uses PCBs as electron

acceptors, but the carbon rings are usually not cleaved (Wiegel & Wu, 2000; Hughes

et al., 2009). In cases where bacteria are not capable of breaking down the carbon

ring structure, they will require additional carbon and electron sources in order to

maintain their growth during dechlorination of PCBs (Wu et al., 2000; Bedard et al.,

2006; Adrian et al., 2009; Wang & He, 2013b). In this case, the only carbon source

available is PCBs. During this study, 8.7x106, 5.0x106, 4.9x106 and 4.5x106 cells/mL

initial cell densities at week zero were increased by 200 fold to 2.0x108, 1.3x108,

1.3x108 and 1.0x108 cells/mL at week four for NP03, NP04, NP05 and NP06,

respectively. This fact confirms the capability of these microorganisms to utilize PCBs

as their carbon source under anaerobic conditions without the requirement for

additional carbon sources.


Figure 6.1 Growth of the four facultative anaerobic bacterial strains under (a) aerobic,

(b) anaerobic, and (c) two stage anaerobic-aerobic conditions. Error bars represent

the standard deviation of mean values (n = 3).

In the two-stage anaerobic-aerobic cultivations as shown in Figure 6.1c, all four

strains showed similar growth patterns to the anaerobic conditions (Figure 6.1b) up

to week four. However, after switching from anaerobic to aerobic conditions


(between weeks four to six), Lysinibacillus sp. NP05 started increasing in growth from

OD600 of 1.04±0.07 to 2.63±0.22, which is close to three times its original cell density.

Similarly, Ochrobactrum sp. NP03, Achromobacter sp. NP04 and Pseudomonas sp.

NP06 also showed a slight increase in optical densities of 1.39±0.08, 1.48±0.23 and

0.99±0.06, respectively, when conditions changed from anaerobic to aerobic as

indicated in Figure 6.1c. Based on these results, it can be postulated that all four

organisms are capable of performing dechlorination under anaerobic conditions in a

similar way at different rates and Lysinibacillus sp. NP05 subsequently hydrolyses the

carbon ring structure extensively under aerobic conditions compared to the other

three organisms. However, these earlier observations based on cell growth need

further confirmation through chemical analyses of PCB degradation and release of

chlorides into the culture supernatant.

6.3.2 PCB degradation and solubilisation

PCB concentrations were measured as total soluble PCBs in order to investigate the

change of solubility levels in the medium resulting from potential bacterial activity

under aerobic, anaerobic and two stage conditions. Solubility of PCBs in the medium

was an indirect measure of the microorganisms attacking the PCBs and converting

them from insoluble to soluble forms. The results are shown in Figure 6.2.

At the initial concentration of 50 mg/L, it was observed that the PCBs added to the

flasks appeared not completely soluble, but remained at the bottom as small clumps,

prior to the addition of the bacterial cultures. The total soluble PCB levels in the

abiotic controls with no bacteria added was measured and found to remain very low

throughout the cultivation period, with values found to be 0.15±0.02 mg/L, 0.57±0.07

mg/L and 0.5±0.23 mg/L under aerobic, anaerobic and two stage anaerobic-aerobic

conditions, respectively. According to Figure 6.2a and under aerobic conditions,

Lysinibacillus sp. NP05 showed the highest capacity to solubilize PCBs compared to

Ochrobactrum sp. NP03, Achromobacter sp. NP04 and Pseudomonas sp. NP06. This

increase of activity by Lysinibacillus sp. NP05 started after week one and reached

optimal activity by week five before it started to decrease. However, Lysinibacillus sp.

NP05 did not increase in cell growth, but showed similar cell densities to the rest of


the three strains as shown in Figure 6.1a. The increased solubility over the aerobic

incubation period with no corresponding increase in cell growth shown by

Lysinibacillus sp. NP05 could possibly be due to two reasons. First, Lysinibacillus sp.

NP05 is capable of creating some surface-active compounds or biosurfactants that

can ultimately lead to increased solubility of hydrophobic PCBs as discussed Chapter

5. Second, the bacteria is unable to carry out the dechorination of highly chlorinated

congeners under aerobic conditions even though it may have the capacity to

solubilize them as reported by Furukawa (2000). Camara et al. (2004) reported that

the accumulation of different metabolic intermediates of PCBs such as dihydrodiols

and dihydroxybiphenyls by aerobic oxidation are highly toxic to bacteria. Therefore,

while increased polarity of these dihydroxylated metabolites can increase their

aqueous solubility, toxic effects can ultimately lead to the inhibition of microbial cell

growth (Seeger & Pieper, 2010). Although the presence of these metabolites was not

analyzed, it was unlikely that cell growth was inhibited by such metabolites, indicated

by steady cell growth during the aerobic period.

In contrast to the aerobic conditions and as shown in Figure 6.2b, PCB solubility of all

four cultures increased significantly under anaerobic conditions. A similar trend was

also observed in the first four weeks during the anaerobic stage of the two stage

anaerobic-aerobic conditions (see Figure 6.2c). It was noted that soon after the

addition of bacterial cultures, the insoluble PCB pellets started to disappear in the

flasks, presumably as the PCBs became soluble due to the action of the

microorganisms. There are two possible explanations for the mode of action by the

microorganisms resulted in the increase in solubility of PCBs. First reason is that

dechlorination of PCBs could transform low water soluble highly chlorinated

congeners into more water soluble lower chlorinated congeners as reported by Yin

et al. (2011). If highly chlorinated congeners were dechlorinated into lower

chlorinated congeners, there should be more chlorides released to the medium.

Therefore, measurement of chloride ions accumulated in the medium is a direct

indication of dechlorination, which was discussed in Section 6.3.3. The second reason

is that the facilitation of the solubility of hydrophobic PCBs due to the production of


bioemulsifiers or surface-active molecules (biosurfactants) by microorganisms

(FedericiGiubileiCovino et al., 2012) as discussed in details in Chapter 5.

Figure 6.2 PCB solubility under (a) aerobic, (b) anaerobic, and (c) two stage anaerobic-

aerobic conditions. Prior to the addition of microbes, samples were removed and

analysed for Initial soluble PCBs measurement and then after adding microbes,

samples were removed immediately and represent week 0. Error bars represent the

standard deviation of mean values from triplicates.


Findings of the two stage experiments are shown in Figure 6.2c. As shown in Figure

6.2c, first four weeks were dedicated to anaerobic conditions before switching over

to aerobic conditions during the last two weeks. All four strains demonstrated similar

trends in the first four weeks under anaerobic conditions (Figure 6.2b vs Figure 6.2c).

However, in the second stage, when the conditions shifted from anaerobic to aerobic,

the solubility of PCBs reduced in Achromobacter sp NP03, Ochrobactrum sp. NP04

and Pseudomonas sp. NP06, whereas in Lysinibacillus sp. NP05, there was a

significant increase in the PCB solubility. Parallel to the increased PCB solubility,

growth of Lysinibacillus sp. NP05 also increased significantly during this period as

shown in Figure 6.1c aerobic phase.

6.3.3 Chloride ion accumulation

The release of chlorides and their concentration in the liquid medium were measured

as explained in Section 3.2.7. These measurements were taken as an indication of the

dechlorination process that took place following the addition of the four bacteria

strains. The chloride ion concentrations in the abiotic controls were also measured

and found to be relatively constant throughout the experiment and there was no

considerable difference between the initial and final chloride levels under aerobic,

anaerobic and two stage anaerobic-aerobic conditions. The chloride ion

concentrations in the controls were in the range of 37.0±0.9 mg/L. These background

levels were concluded to come from chloride containing compounds in the basal

minimal salt medium comprised of MgCl2·6H2O, CoCl2·6H2O, MnCl2·4H2O, NiCl2·6H2O

and CuCl2·2H2O. The background chloride values were first subtracted from the

experimental values obtained from samples of the medium containing cells. Similarly,

background values from the controls of seed cultures only were also subtracted from

the experimental values, before the final values were plotted as shown in Figure 6.3.


Figure 6.3 Chloride ion accumulation in the culture media after six weeks. The

background values from the controls of (1) minimal salt medium only and (2) seed

cultures only were subtracted first. Error bars represent the standard deviation of

mean values from triplicates.

According to Figure 6.3, accumulation of chloride ions in aerobic and anaerobic

conditions provided clear evidence that the four facultative microbes discovered

have the ability to dechlorinate the Aroclor 1260 mixture under both, aerobic and

anaerobic conditions. Significantly high chloride ion concentrations in the combined

anaerobic-aerobic treatments of all four cultures when compared to aerobic and

anaerobic treatments further confirms the effectiveness of combining anaerobic and

aerobic degradation rather than isolated aerobic or anaerobic applications

(Tartakovsky et al., 2001; Long et al., 2015). Increasing levels of chloride ions in the

culture medium were also reported as a direct indication of dechlorination of PCB

molecules by Yin et al. (2011). Under anaerobic and two stage conditions,

Lysinibacillus sp. NP05 demonstrated the highest chloride ion levels when compared

to the other three cultures and they were 9.16±0.8 mg/L and 5.2±0.7 mg/L,

respectively. Pseudomonas sp. NP06 has the lowest chloride levels under all three

conditions. As Aroclor 1260 theoretically contains 60% chlorine by weight, maximum

chlorine level expected in 50 mg/L Aroclor 1260 concentration is 30 mg/L. Therefore,


maximum chloride yield of 9.16±0.8 mg/L observed in Lysinibacillus sp. NP05 under

two stage anaerobic-aerobic treatment is an indication of the removal of one third of

total chlorine present in the Aroclor 1260 mixture.

The pH of the samples was also monitored, and the results from week 6 are shown

together with chloride ions accumulated in Figure 6.4. Before inoculation of the

cultures, flasks were maintained at pH 7. The pH values of the abiotic controls

remained relatively constant throughout the experiment (7.07±0.14) under

anaerobic, aerobic and two stage conditions. At the end of the aerobic and anaerobic

experiments, the pH did not significantly change and were 7.49±0.07 and 6.95±0.03,

respectively. However, significant pH reduction was observed at the end of all the

two stage anaerobic-aerobic experiments with values of 5.15±0.03, 4.98±0.06,

4.97±0.01 and 6.14±0.03 obtained for Achromobacter sp. NP03, Ochrobactrum sp.

NP04, Lysinibacillus sp. NP05, and Pseudomonas sp. NP06, respectively, as shown in

Figure 6.4. When variations of the pH values were compared with the accumulation

of the chloride ion levels in the medium, the negative correlation is clearly visible.

Based on these results, the lowering of pH under two stage anaerobic-aerobic

conditions appeared to have correlated well with the increase in chloride ions. The

high levels of chlorides under combined anaerobic-aerobic conditions is attributed to

the dechlorination of highly chlorinated congeners under anaerobic conditions first,

followed by oxidation of resulting lower chlorinated congeners into the

corresponding chlorinated intermediates through the upper biphenyl pathway as

reported by Field and Sierra-Alvarez (2008). Finally, it can be postulated that further

mineralization of the intermediates into carbon dioxide and inorganic chlorides

through the lower biphenyl pathways (ATSDR, 2000; FedericiGiubileiCovino et al.,

2012) has occurred after switching to aerobic conditions.


Figure 6.4 Variation of pH and chloride ion concentrations after six weeks under

aerobic, anaerobic and two stage anaerobic-aerobic conditions (Initial pH was

adjusted to 7.0). Error bars represent the standard deviation of mean values from

triplicates.

Overall, the data from the growth characteristics (Figure 6.1), PCB solubility (Figure

6.2), chloride accumulation levels (Figure 6.3), and pH (Figure 6.4) support the

identification of Lysinibacillus sp. NP05 as the best performer out of the four

facultative anaerobic strains tested followed by Achromobacter sp. NP03 and

Ochrobactrum sp. NP04 for PCB degradation. As shown in Figure 6.5, increase in

growth rate parallel to the increased PCB solubility under combined anaerobic-

aerobic treatment by Lysinibacillus sp. NP05 is an indication of the consumption of

Aroclor 1260 as their carbon and energy source other than solubilisation. Moreover,

decrease in pH over the two weeks of aerobic phase as per Figure 6.5 would coincide

with the occurrence of advanced PCB degradation steps to further breakdown the

chlorinated intermediates.


Figure 6.5 Growth profile, PCB hydrolysis and pH variation of Lysinibacillus sp. NP05

under two stage anaerobic-aerobic conditions. Error bars represent the standard

deviation of mean values from triplicates.

6.4 Conclusions

Based on an exhaustive review of past research literature, this can be considered as

the first comparative study assessing the capability and growth characteristics of

facultative anaerobic bacteria in degrading PCBs under anaerobic, aerobic and two-

stage anaerobic-aerobic cultivation conditions. The study found four bacterial strains

identified as Achromobacter sp. NP03, Ochrobactrum sp. NP04, Lysinibacillus sp.

NP05 and Pseudomonas sp. NP06, to have the capability for degrading the

commercial PCB mixture, Aroclor 1260 as the sole source of carbon under both,

anaerobic and aerobic conditions. Among the four strains tested, Lysinibacillus sp.

NP05 performed best based on the results from the comparative experiments on cell

growth, PCB solubility and chloride release and accumulation, under the three

process conditions tested.


All four strains performed better under anaerobic conditions compared to aerobic

conditions. However, the two stage anaerobic-aerobic conditions produced the best

overall results when assessed on cell growth, PCB solubility and chloride build up.

Importantly, it was found that these microorganisms can carry out these reactions

relatively rapidly within the first one to two weeks. This is despite the fact that a

number of research studies carried out so far have shown comparatively long

durations ranging from three to six months for the reactions to be completed (Master

et al., 2002; Chen et al., 2014).

The presence of chloride ions under all the three conditions tested found that the

highest chloride ion concentrations was achieved under two stage anaerobic-aerobic

conditions which suggest that the combined anaerobic-aerobic conditions could

further enhance the chlorine removal from the biphenyl structure. Therefore, in field

scale soil remediation applications, facultative microorganisms have the potential to

be better candidates as they can survive and degrade PCBs under both anaerobic and

aerobic conditions, while achieving relatively higher degradation rates.

The limitations of biological breakdown due to the characteristic hydrophobic

properties of PCBs can be overcome by the use of suitable bacterial strains, which

can simultaneously solubilize and breakdown PCBs. During this study, Lysinibacillus

sp. NP05 was found to have high potential as a candidate to effectively

decontaminate environmental pollutants such as highly chlorinated complex PCB

mixture, Aroclor 1260. Therefore, based on these results there is an opportunity to

produce and apply tailored-made consortia for future process designs and

applications resulting in shorter time frames, while effectively hydrolyzing PCBs.

Chapter 7: Effective degradation of polychlorinated biphenyls by three facultative anaerobic bacterial species Achromobacter, Ochrobactrum and Lysinibacillus as a consortium 135

Chapter 7: Effective degradation of polychlorinated biphenyls by three facultative anaerobic bacterial species Achromobacter, Ochrobactrum and Lysinibacillus as a consortium

7.1 Background

Reductive dechlorination and oxidative degradation are the two main processes

involved in the biodegradation of PCBs. Therefore, a combination of anaerobic

dechlorination and aerobic oxidation is essential to effectively degrade these

complex polychlorinated biphenyl mixtures to less toxic products. Due to the complex

metabolic network responsible for PCB degradation, a single bacterium does not

possess the enzymatic capability to degrade all or even most of the PCB congeners

present in polluted environments (Fritsche & Hofrichter, 2005; Pieper, 2005). Based

on the availability of genes encoding enzymes that degrade PCBs (Seeger & Pieper,

2010; Hassan, 2014; Wang et al., 2014), types of degradation products (Petric et al.,

2007; PetricBru et al., 2011) and information on how well individual microorganisms

degrade PCBs, a consortium of carefully selected microbial species is expected to

perform better than the application of individual microbes (Liz et al., 2009; Chen et

al., 2015a).

Currently, two modes of combined anaerobic dechlorination and aerobic oxidation

applications have been reported in PCB bioremediation studies, and have been

labelled as: (1) two stage (or sequential) anaerobic-aerobic degradation; and (2)

concurrent (or alternating) anaerobic-aerobic degradation (Master et al., 2002;

Payne et al., 2013; Chen et al., 2014; Long et al., 2015). Based on the results discussed

in Chapter 6, three individual strains Achromobacter sp. NP03, Ochrobactrum sp.

NP04 and Lysinibacillus sp. NP05 provided positive indicators as potential candidates

to undergo further tests as a consortium.

136 Chapter 7: Effective degradation of polychlorinated biphenyls by three facultative anaerobic bacterial species Achromobacter, Ochrobactrum and Lysinibacillus as a consortium

Accordingly, Chapter 7 primarily focuses on testing a consortium made up of

facultative anaerobic Achromobacter sp. NP03, Ochrobactrum sp. NP04 and

Lysinibacillus sp. NP05 based on their ability as individuals to degrade the PCB mixture

Aroclor 1260 under both, anaerobic and aerobic conditions. The rates and efficiencies

of PCB biodegradation by the selected three strains as a consortium were compared

under; (1) alternating (AN), and (2) two stage (TS) anaerobic-aerobic modes.


Three facultative anaerobic bacterial strains Achromobacter sp. NP03, Ochrobactrum

sp. NP04 and Lysinibacillus sp. NP05 capable of degrading PCBs under both, anaerobic

and aerobic conditions were used as a consortium in the present study. Initially they

were streaked on solid minimal salt medium containing 50 mg/L Aroclor 1260 and

duplicate plates were incubated anaerobically and aerobically at 28 °C to see whether

there was any visible competition among them for the growth substrate.

The two modes of treatments were tested at 28 °C for six weeks in order to compare

PCB degradation rates under; (1) two stage anaerobic-aerobic (TS), and (2)

alternating anaerobic-aerobic (AN) conditions. The TS experiment continued for four

weeks under anaerobic static conditions (as described in Chapter 6.2.1) and then

switched to aerobic mixing conditions at 150 rpm for the last two weeks. Under AN

conditions, the experiment was kept under anaerobic static conditions in the first

week and switched to aerobic mixing conditions in the second week. Switching from

one week of anaerobic to one week of aerobic incubations was continued throughout

the six-week study. In anaerobic stages, flasks were kept at 28 °C in an incubator kept

inside the anaerobic chamber with occasional gentle shaking by hand. Hydrogen gas

(H2) at 4.92% (BOC Australia) was provided as the electron donor during the

anaerobic phases.

Additionally, three culture members used in the consortium were checked for their

carbon and nitrogen source preferences. Understanding their preferences is

important so that cost, availability and applicability of carbon and nitrogen sources


during inoculation preparation, scale up and on-site remediation applications can be

determined.

7.2.1 Laboratory microcosm batch experiments

The liquid microcosms were prepared using 250 mL Erlenmeyer flasks containing 20

mL of sterile minimal salt medium (see Section 3.1.2.1A). 50 mg/mL Aroclor 1260

stock solution in GCMS grade acetone was added to each flask as sole source of

carbon to give 50 mg/L Aroclor 1260 final concentration.

For seed culture preparations, single bacterial colonies were picked and inoculated

into 250 mL of sterile Luria-Bertani (LB) broth and grown overnight at 28 °C, 150 rpm.

The seed cultures were collected by centrifuging at 5000 x g for 10 min, washed twice

in minimal salt medium, and the resulting cell pellets were resuspended in 1 mL of

sterile minimal salt medium. Previously prepared batch mesocosms were inoculated

with seed cultures under anaerobic conditions (Figure 7.1).

Figure 7.1 Inoculation of batch mesocosms with bacterial seed cultures inside the

anaerobic chamber.

7.2.2 Frequency, collection and preservation of samples

In order to obtain the total PCBs concentrations, the entire content in each flask was

extracted and tested. This was done as PCBs are poorly soluble in water and taking


aliquots may result in unrepresentative samples. Due to this, the experiment was set

up to sacrifice six whole flasks at each sampling from each TS and AN experiment.

These six flasks included one set of triplicate flasks for total PCB extraction and

another set of triplicate flasks for the analyses of bacterial growth, pH, chloride ion

concentration and extracellular proteins. Sampling was done initially (at time zero)

and at fortnightly intervals (at week 2, 4 and 6) for both, AN and TS treatments.

Two controls were also prepared to test parallel to the samples. First control was an

abiotic control without bacterial seed culture additions (i.e. Minimal salt medium

spiked with 50 mg/L Aroclor 1260) and the second control was a growth medium

control (i.e. minimal salt medium with bacterial seed culture addition, but no Aroclor

1260 was added). These controls were conducted in parallel to the experiments

under each TS and AN treatments, and duplicate flasks from each abiotic and growth

medium controls were sacrificed at each sampling time for analyses.

The following is the summary of the test set up of the mesocosms at each sampling

under AN and TS conditions:

• Three flasks for extractions for total PCBs and PCB homolog groups;

• Three flasks for bacterial growth, pH, chlorides and proteomic analyses;

• Two flasks as abiotic controls (minimal salt medium spiked with 50 mg/L

Aroclor 1260, but no seed culture added) ; and

• Two flasks as media controls (minimal salt medium spiked with an equal

volume of acetone used to dissolve Aroclor 1260 and inoculated with seed

culture, but no 50 mg/L Aroclor 1260 added).

Additionally, to see the difference when switching from one week of anaerobic to

one week of aerobic under AN treatment conditions, additional three flasks from AN

treatment and two flasks from each control were used for each consecutive week (at

week 1, 3 and 5) to take samples for cell growth, chloride, pH and extracellular

proteins.

Bacterial growth, pH and chloride ion concentrations were measured as per Section

3.2.4A, Section 3.2.5 and Section 3.2.7, respectively. Final chloride ion concentrations


were determined by subtracting the chloride concentrations of the controls. To see

whether there was any contribution of chloride from the cell lysis was also tested by

comparing the chloride levels of samples before and after being subjected to 10 min

sonication for cell disruption. Samples collected for protein analysis were stored at -

80 °C freezer until subsequent analysis. Analytical processes and outcomes of

extracellular protein analysis are discussed in Chapter 8.

7.2.3 Total PCB extraction and analysis

PCB extraction was performed according to the method described by Murinova et al.

(2014) using GC grade n-hexane as the extraction solvent. Further details of the

extraction method is as described in Section 3.2.8B. Recovery of the surrogate

standard was obtained at 77.0% ± 4.3% (n = 61) and found to be within the USEPA

recommended recovery limits of 70-130% (Eichelberger et al., 1995). A method blank

was also carried out in parallel to each batch of PCB extractions through the entire

procedure using sterile distilled water to evaluate any presence of contamination

from the complete preparation and analytical procedure.

PCB extracts were diluted four fold using n-hexane and analyzed using gas

chromatography (GC) as per the method described in Section 3.2.9 to determine the

total and homolog group specific PCB levels. PCB degradation rates were calculated

as a percentage using Equation 7.1.

PCB degradation (%)

=(Initial PCB concentration - PCB concentration in culture suspension)

Initial PCB concentrationx 100

Equation 7.1

7.2.4 Carbon and nitrogen source utilization profiling of bacterial cultures

Phenotype microarray plates (PM1 and PM3B, Biolog) containing 95 separate sole

carbon and nitrogen sources were used to identify carbon and nitrogen utilization

preferences of the three facultative bacterial consortium members. Depending on

the ability of bacteria to oxidize carbon and nitrogen sources in the wells, initially


colourless tetrazolium dye was reduced to form a purple colour (Miller & Rhoden,

1991). The colour intensity ranged from dark purple to colourless depending on the

intensity of bacterial growth. Biolog phenotype microarray (PM) procedures for Gram

negative and positive bacteria were followed during the cell suspension preparation

and inoculation.

7.2.4.1 Procedure for Gram positive bacteria

(A) Bacterial cell suspension preparation

Gram positive Lysinibacillus sp. NP05 was grown overnight at 30 °C on nutrient agar

to produce pure and isolated colonies. A cell suspension was prepared by transferring

bacterial colonies from the nutrient agar plate into 5 mL 1x inoculating fluid (Biolog

IF-0a GN/GP base) using a sterile cotton swab until the transmittance of 81% (0.0915

absorbance at 600 nm) was reached.

(B) Nutrient additive solution preparation

Nutrient solutions as indicated in Table 7.1 were prepared to use in the final

inoculation fluid, filter sterilized through 0.2 µm filters and stored at 4 °C until use.

Table 7.1 Ingredients for the nutrient additive solution, 12x (PM additive)

Ingredient Gm/50 mL PM1 (mL) PM3 (mL)

Sodium succinate 4.0515 - 3

MgCl2.6H2O

CaCl2.2H2O

2.44

0.88 1 1

L-arginine, HCl

L-glutamate, Na

0.0315

0.0505 1 -

L-cystine, pH 8.5 0.006 3 -

Yeast extract 0.3 1 1

Tween 80 0.3 1 1

Sterile water 3 4

Total 10 10


(C) Final inoculation fluid preparation

The recipe as indicated in Table 7.2 was used to prepare the final inoculating fluids.

Duplicate PM1 and PM3 Biolog plates were inoculated with respective final

inoculation fluid (100 µL/well). Plates were incubated at 30 °C and colour

development was monitored for 24 h in 30 min intervals and measured at 595 nm

wavelength using a plate reader (Synergy HTX). Equivalent volumes of bacterial cell

suspensions with no added carbon and nitrogen sources were used as negative

controls.

Table 7.2 Preparation of final inoculation fluid to inoculate the Biolog plates

Description mL/PM1 Biolog plate

(For carbon sources)

mL/PM3 Biolog plate

(For nitrogen sources)

1.2X stock inoculation fluid

(Biolog IF-0a GN/GP base)

10.0 10.0

Redox dye mix D, 100x (Biolog,

for Gram positive bacteria)

0.12 0.12

Nutrient additive solution, 12x

(PM additive)

1.0* 1.0**

Cell suspension (with 81% T) 0.88 0.88

Total 12.0 12.0

* From PM1 nutrient solution from Table 7.1.

**From PM3 nutrient solution from Table 7.1.

7.2.4.2 Procedure for Gram negative bacteria

(A) Bacterial cell suspension preparation

Gram negative bacteria, Achromobacter sp. NP03 and Ochrobactrum sp. NP04 were

separately grown overnight at 30 °C on nutrient agar to obtain pure colonies. Cell

suspensions were prepared by transferring the bacterial colonies from the nutrient

agar plates into 5 mL 1x inoculating fluid (Biolog IF-0a GN/GP base) using sterile

cotton swabs until the transmittance of 42% (0.3768 absorbance at 600 nm) was

reached.


(B) Final inoculation fluid preparation and inoculation of PM1 and PM3 plates

0.6 mL redox dye H (Biolog, for Gram negative bacteria) and 7.734 mL sterile water

were added to a sterile 50 mL falcon tube containing 41.666 mL of 1.2X stock

inoculation fluid (Biolog IF-0a GN/GP base) and thoroughly mixed to form a uniform

solution. To prepare the final inoculation fluid, 40 mL of the mixture was transferred

into a sterile 50 mL falcon tube and mixed gently with 8 mL of previously prepared

cell suspension having 42% Transmittance. 24 mL from the final inoculation fluid was

transferred to a sterile reservoir and PM1 plates were inoculated in duplicate for each

bacterial culture (100 µL/well). Then, 240 µL of the previously prepared 2M sodium

succinate solution was added to the remaining 24 mL of the final inoculation fluid.

The mixture was gently mixed to make a uniform suspension and used to inoculate

in duplicate PM3 Biolog plates (100 µL/well). Similar to the plates for the Gram

positive bacterium, plates for the two Gram negative bacteria were incubated at 30

°C and the colour development was monitored for 24 h in 30 min intervals and

measured as absorbance at 595 nm wavelength using a plate reader (Synergy HTX).

Bacterial cell suspensions with no added carbon or nitrogen sources were used as

negative controls.


7.3.1 Test for competition

At the start of the experiment, the three chosen bacterial cultures Achromobacter sp.

NP03, Ochrobactrum sp. NP 04 and Lysinibacillus sp. NP05 were streaked on to minimal

salt agar plates spiked with 50 mg/L of Aroclor 1260 as the sole source of carbon and

grown under both, aerobic and anaerobic conditions to test for growth inhibition. All

three strains grew well without any visible growth inhibition from the neighbouring

organism after 72 hours, and in particular, no obvious zones of inhibition were observed

at the areas where each strain merged with each other (Figure 7.2).


Figure 7.2 Growth of Achromobacter sp. NP03, Ochrobactrum sp. NP04 and

Lysinibacillus sp. NP05 on minimal salt-Aroclor 1260 agar at 28 °C after 72 h (a) under

aerobic conditions, (b) under anaerobic conditions.

7.3.2 PCB degradation by the bacterial consortium under AN and TS treatments

7.3.2.1 Total PCB degradation

Typically, anaerobic processes have been shown to be slow and require long

durations in order to achieve effective and higher degradation of PCBs. Adrian et al.

(2009) reported that 64% total PCB reduction was observed after four months of

incubation under anaerobic conditions using anaerobic Dehalococcoides sp. CBDB1,

whereas 36% reduction of total PCBs by a consortium of anaerobic microorganisms

after 10 months of anaerobic incubation was reported by Praveckova et al. (2015) .

Differences in rate of PCB degradation could be due to the differences in types of

microorganisms used and the application or treatment conditions. However, as

discussed in Chapter 6, when facultative anaerobic bacterial strains were tested

during this work as individuals for their PCB degradation potential under aerobic,

anaerobic and combined anaerobic-aerobic conditions, the highest results were

obtained under the combined anaerobic-aerobic conditions.

There are only limited research studies on conventional combined anaerobic-aerobic

treatments (Master et al., 2002; Chen et al., 2014; Long et al., 2015). Most of these


studies consist of lengthy (70 to 180 days) anaerobic phase followed by short aerobic

phase (28 to 60 days). Based on these earlier studies, a two stage (TS) treatment

process was designed for this study, which included a four week anaerobic stage

followed by a two week aerobic stage. In the alternating (AN) treatment mode,

cultures were placed in anaerobic conditions for one week and then switched to one

week long aerobic conditions. Alternation of anaerobic and aerobic conditions were

repeated throughout the six week testing period.

The results of PCB degradation in relation to cell growth under the AN vs TS

conditions are shown in Figure 7.3. According to Figure 7.3, the rate of PCB

degradation occurred faster under AN compared to TS conditions, with 49.2±2.5%

total PCB reduction reached during the first two weeks under AN (Figure 7.3a). In

comparison, only about 25% of PCB degradation was obtained in the first two weeks

of just anaerobic conditions under TS (Figure 7.3b). The results from the AN

conditions showed better degradation efficiency within a short period than that of

previous combined anaerobic-aerobic studies. In the study conducted by Master et

al. (2002), it took four months of anaerobic treatment followed by 28 days of aerobic

treatment to achieve 66% PCB reduction. Total PCB reduction reported by Long et al.

(2015) after 70 days of anaerobic treatment and 28 days of aerobic treatment was

only 25%. Therefore, the results of this study are superior in terms of treatment

efficiency compared to past studies.

According to Figure 7.3, though 49.2±2.5% total PCB reduction occurred after the first

two weeks under AN, only a 4.9% further reduction occurred during the last four

weeks reaching up to 54.1±0.49% degradation by week 6. However, despite the fact

that there was no considerable increase in PCB reduction after the second week,

bacterial growth continued to rise from week 2, demonstrated by the optical density

increase from 0.59±0.01 to 2.49±0.16 (Figure 7.3a). This suggests the possibility of a

consortium utilizing the intermediate products produced during the degradation of

Aroclor 1260 as their growth substrates. This hypothesis was confirmed by the fact

that there was no other carbon source added to the medium other than Aroclor 1260

and the increase in growth cannot be expected while there is no substantial PCB

reduction. However, the types of intermediate products produced and their fate


during the degradation process needed to be confirmed through further

investigation.

Figure 7.3 Total PCB degradation as a percentage and bacterial growth as OD600 under

(a) alternating and (b) two stage anaerobic-aerobic treatments by the bacterial

consortium. Error bars represent the standard deviation of mean values (n = 3).

The results from the two stage (TS) anaerobic-aerobic approach are shown in Figure

7.3b. Under TS conditions, the total PCB degradation rate after two weeks of

anaerobic treatment was 24.44±2.46% compared to 49.2±2.5% achieved during the

AN conditions (Figure 7.3b vs Figure 7.3a). From week 2 to 4 under continuing

anaerobic conditions, PCBs were further degraded up to 34.89±2.26%. Anaerobic PCB

dechlorination is a reductive process that uses PCBs as electron acceptors, but the

rings are not usually cleaved as reported by Borja et al. (2005). Therefore, PCB

dechlorinating only microorganisms require additional carbon and electrons sources

for their growth. During the first four weeks of anaerobic growth, the gradual

increase in the growth based on values of OD600 0.18±0.01 to 0.66±0.14,

corresponded well with total PCB concentration reduction (Figure 7.3b). These

results indicate that the consortium members were able to utilize PCBs as their

carbon source under anaerobic conditions. Furthermore, these results were

comparable to the findings from the comparative growth studies including the same


three microorganisms when tested individually under similar conditions as described

in Chapter 6.

Under TS conditions, when the cultures were switched from anaerobic to aerobic

conditions during the last two weeks, the PCB degradation rate increased from

34.89±2.26% (in week 4) to 47.99±1.55% (in week 6, Figure 7.3b). Meanwhile the

optical density value increased nearly three times from 0.66±0.14 in week 4 to

1.56±0.03 in week 6. Microbial growth thrived once it changed from anaerobic to

aerobic conditions. The increased PCB degradation and growth rates were highly

likely due to ring cleavage and hydrolysis of lower chlorinated PCB congeners

generated during the initial anaerobic phase.

7.3.2.2 PCB Homolog analysis

PCB congeners are categorized into ten homolog groups and labelled from

monochlorobiphenyls to decachlorobiphenyls based on the degree of chlorination in

the biphenyl molecule (ATSDR, 2000). However, decachlorobiphenyls were not

included in the analysis as they were present in trace quantities (Less than 0.05% of

the total PCB mixture). According to the existing literature, highly chlorinated

congeners (with 5 or more chlorines) are converted into lower chlorinated (four or

less) congeners under anaerobic conditions. Under aerobic oxidations, lower

chlorinated congeners breakdown into intermediate products such as

chlorobenzoates and benzoates based on upper biphenyl pathway (Borja et al.,

2005). Benzoate is a growth substrate for a broad range of bacteria and it can be

further mineralized to CO2 and H2O via lower biphenyl pathways such as the

chlorocatechol or 3-oxoadipate pathways (Pieper, 2005).

To determine the capability of the consortium to degrade the different PCB homolog

groups under alternating (AN) and two stage (TS) anaerobic-aerobic conditions,

samples were removed, extracted and analysed as described in Sections 7.2.2, 7.2.3

and 7.2.4, respectively. The results are shown in Figure 7.4 for the AN treatment and

in Figure 7.5 for the TS treatment. Since the range of homolog concentrations were

too broad to illustrate in a single graph, three graphs are plotted for each of the AN


and TS treatments in three different scales as: (a) homologs containing lower

chlorinated congeners (from mono to tetra); and (b) penta, hexa, hepta groups; and

(c) octa, nona groups of highly chlorinated congeners.

As shown in Figure 7.4, the concentrations of all the PCB homolog groups decrease

significantly during the first two weeks under AN conditions. Similar to total PCBs,

there was no considerable reduction in the concentrations of homolog groups after

the second week indicating the ability of the consortium to reach their optimum

degradation within the first two weeks under AN conditions. However, when

individual homolog groups were considered, the rate of degradation was negatively

correlated with the increasing number of chlorine atoms in the PCB molecules.

Congeners with single chlorine atoms (monochlorobiphenyl) displayed the highest

reduction rate reaching a 98.69±0.05% overall reduction at the end of week 6 (Figure

7.4a), whereas the nonachlorobiphenyls showed the lowest reduction rate of

47.12±1.64% at the end of week 6 (Figure 7.4c). Based on these results, it can be

concluded that the higher the number of chlorines attached to the biphenyl rings, the

more resistant the PCBs are to microbial biodegradation. A similar observation was

noted by Furukuwa et al (2006). When compared to other homolog groups,

hexachloribiphenyls represent 50.69±0.47% (by weight) of Aroclor 1260 mixture.

Therefore, reduction of initial 19.73±1.12 mg/L hexachlorobiphenyl level to

10.46±0.05 mg/L by week 2 is nearly 47% reduction of the original

hexachlorobiphenyl concentration and this is the main contribution to 49.2±2.5%

overall total PCB reduction (Figure 7.4b).


Figure 7.4 Variation of PCB homolog groups following AN treatment; (a) lower

chlorinated congener groups (mono to tetra), and medium to highly chlorinated

congener groups, (b) penta to hepta, (c) octa and nona. Error bars represent the

standard deviation of mean values (n = 3).

In the TS treatment, the first four weeks were kept under anaerobic conditions and

switched to aerobic conditions in the last two weeks. When lower chlorinated

congener groups were analyzed, the concentration of each group slightly increased

during the anaerobic phase with the highest increase observed in dichlorobiphenyl

from 0.21±0.02 mg/L in week 0 to 1.55±0.11 mg/L by the end of week 4 (Figure 7.5a).


However, once conditions changed from anaerobic to aerobic after week 4,

concentrations of all the lower chlorinated congener groups reduced far below the

initial concentrations (Figure 7.5a). These results indicate that the microorganisms

utilized the lower chlorinated PCBs as an energy source under the aerobic conditions.

The subsequent increase in cell density during the last two weeks under aerobic

conditions as demonstrated in Figure 7.3b would support this conclusion.

Furthermore, these findings are in agreement with the study outcomes by Borja et

al. (2005).

In contrast, the concentrations of highly chlorinated congeners gradually decreased

during the four week anaerobic phase as shown in Figure 7.5. The highest reductions

were observed for penta, hexa and octa homolog groups at the end of anaerobic

phase with 48.2±3.85%, 42.09±1.8% and 39.28±1.62% reductions, respectively (see

Figure 7.5b and Figure 7.5c). These reductions in the highly chlorinated congener

groups suggested the possibility of dechlorination during the anaerobic conditions. It

can also be pointed out that if these highly chlorinated congener groups were

dechlorinated into lower chlorinated congeners, increase in the lower chlorinated

groups need to be higher than what is shown in Figure 7.5a. As there is no significant

increase in lower chlorinated congeners during the anaerobic phase as expected, this

was due to the ability of consortium members to utilize the lower chlorinated

congeners as their carbon and energy source under anaerobic conditions. This can be

further confirmed by the increase in cell density during the anaerobic phase as shown

in Figure7.3b. After the conditions changed from anaerobic to aerobic, there was no

substantial reduction of any highly chlorinated congener groups. However, the

overall reduction in homolog groups was higher in the AN treatment with nearly 90%

of the total reductions achieved within the first two weeks, when compared to the

TS treatment.


Figure 7.5 Variation of PCB homolog groups following TS conditions; (a) lower

chlorinated congener groups (mono to tetra), and highly chlorinated congener

groups, (b) penta to hepta, (c) octa and nona. Error bars represent the standard

deviation of mean values (n = 3).


7.3.3 Chloride ion accumulation and pH variation

7.3.3.1 Chloride ion analysis

Monitoring of chloride ion accumulation in the culture medium was performed to

further confirm the removal of chlorines from the PCB mixture. It was expected that

the chloride ion build up in the culture medium would increase proportionately to

the decrease in PCB homolog groups over time. Accordingly, the presence and

buildup of chloride ion concentrations in both AN and TS treatments were measured

as described in Section 7.2.2, and the results are shown in Figure 7.6.

Figure 7.6 Chloride ion accumulation under alternating (AN) and two stage (TS)

anaerobic-aerobic conditions. The background chloride values from the minimal salt

medium were first subtracted from experimental values and media controls. Error

bars represent the standard deviation of mean values (n=3 for experimental values

and n=2 for media controls).


Primary contributors of chloride levels in the experimental setup are minimal salt

medium used for the experiments and dechlorination of PCBs. In order to eliminate

the contributions of chlorides from minimal salt medium, chloride concentrations of

controls were first examined. The chloride ions in the abiotic controls were expected

to come from chloride containing compounds in the basal minimal salt medium and

the average chloride level in the abiotic control was 37.36±0.47 mg/L. To remove the

contribution from the minimal salt medium, chloride values of the abiotic controls

were first subtracted from the experimental values and media controls before final

values were plotted as shown in Figure 7.6. There was no significant difference of the

chloride levels before and after sonication of the samples and the difference is less

than 1 mg/L. This is indicative of no significant contribution of chlorides to the final

chloride concentration from cell lysis.

As shown in Figure 7.6, increasing levels of chloride concentrations occurred over the

six weeks under both AN and TS treatments. However, higher yields of chlorides were

detected in AN compared to TS at the end of week 4. By the end of week 6, a yield of

17.63±0.91 mg/L chlorides were measured under AN conditions, compared to 11.79±

1.28 mg/L measured under TS conditions.

In addition to the measurement of chloride ion build up in the culture medium, the

theoretical chloride ion removal from Aroclor 1260 present in the medium was also

calculated based on the PCB homolog group reduction rates as discussed in Section

7.3.2.2. Calculated and measured chloride ion levels under each treatment condition

are shown in Figure 7.7.


Figure 7.7 Measured chloride ion buildup in the culture medium and calculated

chloride ion removal from the PCB mixture based on homolog group reductions

under; (a) alternating (AN), and (b) two stage (TS) anaerobic-aerobic conditions. Error

bars represent the standard deviation of mean values (n = 3).

It can be postulated that the release of chlorines is proportional to the homolog

group reduction rates and released chlorides are accumulating in the culture

medium. This leads to an ideal scenario where calculated chloride ions based on

homolog group reductions is equal to the measured chloride levels in the culture

medium. As shown in Figure 7.3a, Figure 7.4b and Figure 7.4c, nearly 50% reduction

in total PCBs and highly chlorinated penta to nona homolog groups were recorded in

week 2 under AN conditions and this would result in high chloride ion concentration

in the medium. Based on the PCB homolog group reduction rates, the calculated

chloride ions released at week 2 was 14.09±0.41 mg/L. However, measured chloride

ion concentration in the culture medium was only 3.9±0.8 mg/L in week 2 (Figure

7.7a) and it was significantly lower than that of week 4 and the calculated chloride

level. This suggests the possibility of transformation of PCBs into chlorinated

intermediate products such as chlorobenzoates and chlorocatechols rather than

releasing chlorine from biphenyl molecules as suggested by Petric et al. (2007).

However, by week 4, measured chloride level increased significantly up to 16.03±0.7

mg/L, a similar value in comparison to calculated chloride level of 14.19±0.65 mg/L.

This indicates the breakdown of intermediate products and release of bound

chlorides. This was further confirmed by the continued growth demonstrated by the


bacterial consortium from week 2 to week 4 with increased optical density from

0.59±0.01 at week 2 to 1.11±0.21 at week 4 as demonstrated in Figure 7.3a, even

when there was no significant increase in the PCB reduction after the second week.

When compared to AN treatment, there was no significant difference between the

calculated and measured chloride concentrations in TS treatment. As both values

were lower than that of AN treatment, it indicates that AN treatment is more

effective than TS treatment.

7.3.3.2 pH analysis

The pH of the culture medium was also measured as described in Section 7.2.2 and

the results are shown in Figure 7.8. As expected, the controls remained almost

constant throughout the study period (7.09±0.1) and were not included in Figure 7.8.

Overall, the pH in both AN and TS treatments under anaerobic conditions

demonstrated negative trends, while the pH trend was positive under aerobic

condition (Figure 7.8). In the AN treatment, the pH dropped from 7.0±0.04 to

6.67±0.09 at the end of each anaerobic phase. However, when switching from

anaerobic to aerobic, the pH appeared to have self-recovered and adjusted back to

its original neutral condition irrespective of the increasing chloride ion concentration

(Figure 7.8a). According to White (1986), some bacteria can undergo metabolic

processes that can lead to the secretion of compounds with the ability to alter the pH

of the medium . Under the aerobic conditions, it is possible that the consortium

members have metabolized the salts in the minimal salt medium such as ammonium

sulfate as their nitrogen source that resulted in the generation of ammonia (Park &

Lee, 1998). Moreover, under aerobic conditions, the flasks are shaken at 150 rpm, in

the presence of oxygen. It is common that microbes under respiration produce CO2,

which can be converted to HCO3 that will also contribute to the alkalinity of the

medium (Hem & Survey, 1989).


Figure 7.8 pH trends relative to chloride ion concentration. (a) AN and (b) TS

anaerobic-aerobic treatments. Error bars represent the standard deviation of mean

values (n = 3).

In the TS treatment, the initial pH dropped from 7.0±0.04 to about 5.51±0.27 by the

end of week 4 (Figure 7.8b). In contrast, the chloride ions from PCB degradation

increased linearly reaching 20.76±0.46 mg/L by the end of week 4. When compared

between the two conditions, it was of interest to see that during the first four weeks

under AN conditions, the buildup in chloride concentration was more gradual

compared to the linear and exponential increase under TS conditions (see Figure 7.8a

vs Figure 7.8b). However, once the conditions switched from anaerobic to aerobic

after week 4, pH level gradually recovered back to its original neutral value of 7.05.

Although, under TS conditions three consortium members individually were not able

to change the pH back to its original neutral position as shown in Figure 6.4 in Chapter

6. When used as a consortium, the ability to self-recover pH from acidic to neutral

condition is an added advantage in soil remediation applications as low pH severely

inhibit PCB biodegradation while.prolonged acidic pH can lead to leaching of acid

soluble toxic compounds present in the soil (Chen et al., 2015a).


7.3.4 Carbon and nitrogen wide substrate utilization tests

Availability of different carbon and nitrogen sources can be a major growth

requirement for microorganisms and play a significant role in the metabolism and

hydrolysis of environmental pollutants such as PCBs. Anaerobic dechlorination is a

reductive process and biphenyl rings are not cleaved unless the microorganisms are

capable of utilizing PCBs as a carbon source as reported by Wiegel and Wu (2000).

Therefore, supplementing a suitable additional carbon source would be essential for

the microorganisms for their survival while dechlorinating PCBs, under anaerobic

conditions. It was apparent that during the first four weeks, the growth rate under TS

conditions was much slower than the AN treatment (see Figure 7.3). Therefore,

providing an additional carbon source, which can be utilized by all the consortium

members, could enhance the PCB dechlorination rate. Addition of a desirable carbon

source was also shown to enhance the mineralization of PCBs under aerobic

conditions through co-metabolism of biphenyl as reported by Beyer and Biziuk

(2009). In the present study, (NH4)2SO4 in the minimal salt medium was provided as

the inorganic nitrogen source for the microorganisms. However, addition of an

economical organic nitrogen source which can be utilized by all the consortium

members would be important to maintain bacterial growth, either under laboratory

conditions, or in on-site applications.

Therefore, as part of further strain characterization and potential future applications,

the three strains Achromobacter sp. NP03, Ochrobactrum sp. NP04, and Lysinibacillus

sp. NP05 were subjected to a carbon and nitrogen-wide substrate utilization screen.

The Biolog system PM1 for carbon and PM3B for nitrogen substrate were used as

described in Section 7.2.4.

The results for different carbon substrate tests are shown in Table 7.3. Likewise,

results for the different nitrogen substrate tests are summarized in Table 7.4. If

bacteria were able to utilize particular carbon or nitrogen source, the initially

colourless tetrazolium dye is reduced to purple colour compound. The intensity of

colour change is proportional to the the degree of substrate utilization. Based on the

colour intensities, L-proline was identified as the best substrate that all three


consortium members, Achromobacter sp. NP03, Ochrobactrum sp. NP04, and

Lysinibacillus sp. NP05, could utilize as their sole source of carbon and nitrogen. L-

Proline is one of the twenty amino acids used by living organisms as the building

blocks of proteins (Al-Mailem et al., 2018). L-proline is produced at industrial scale

and some of the raw materials rich with L-proline are algae Chlorella (Leavitt, 1983)

and keratin separated from waste biomass (Sharma & Gupta, 2016). In addition to L-

proline, all three consortium culture members were able to utilize L-lactic acid and

methyl pyruvate as carbon sources (Table 7.3) and L-Glutamic acid, Ala-His, Ala-Leu

and Gly-Gln as nitrogen sources (Table 7.4) at high rates.

However, it should be further confirmed by additional research as the presence of

other carbon sources could either stimulate or inhibit PCB dechlorination by

facilitating out-competing the non-PCB dechlorinators or providing more preferred

electron acceptors other than PCBs to the dechlorinators in the natural environment

(Wiegel & Wu, 2000; Yang et al., 2008; Parnell et al., 2010). Additionally, potential for

the use of these chemical compounds as nutritional supplements to the soils in on-

site applications need to be carefully decided based on the associated cost and their

impact on the soil ecosystem.


Table 7.3 Carbon source utilization by consortium members Achromobacter sp. NP03, Ochrobactrum sp. NP04, and Lysinibacillus sp. NP05.

A - Achromobacter sp. NP03, O – Ochrobactrum sp. NP04, L – Lysinibacillus sp. NP05

High Moderate Low Negative

Carbon Source A O L Carbon Source A O L Carbon Source A O L Carbon Source A O L

L-Arabinose Citric Acid D,L-α-Glycerol- Phosphate Tricarballylic Acid

Adonitol D,L-Malic Acid D-Glucose-1-Phosphate L-Serine

D-saccharic Acid D-Ribose D-Fructose-6-Phosphate L-Threonine

Succinic Acid Tween 20 D-Glucose-6-Phosphate L-Alanine

D-galactose L-Rhamnose α-hydroxy glutaric acid-γ-lactone L-Alanyl-Glycine

L-aspartic Acid D-Fructose α-Hydroxy Butyric Acid Acetoacetic Acid

L-proline Acetic Acid β-Methyl-D-Glucoside N-Acetyl-β-D-Mannosamine

D-alanine α-D-Glucose N-acetyl-D-glucosamine Mono Methyl Succinate

D-Trehalose Maltose Maltotriose Methyl Pyruvate

D-Mannose D-Melibiose 2-Deoxy Adenosine D-Malic Acid

Dulcitol Thymidine Adenosine L-Malic Acid

D-Serine L-Asparagine Glycyl-L-Aspartic Acid Glycyl-L-Proline

D-Sorbitol D-Aspartic Acid D-galactonic acid-γ-lactone p-hydroxy phenyl acetic acid

Glycerol D-Glucosaminic Acid m-Inositol m-hydroxy phenyl acetic acid

L-Fucose 1,2-Propanediol D-Threonine Tyramine

D-Glucuronic Acid Tween 40 Fumaric Acid D-Psicose

D-Gluconic Acid α-Keto-Glutaric Acid Bromo Succinic Acid L-Lyxose

m-Tartaric Acid α-Keto-Butyric Acid Propionic Acid Glucuronamide

D-Xylose α-Methyl-D-Galactoside Mucic Acid Pyruvic Acid

L-Lactic Acid α-D-Lactose Glycolic Acid L-Galactonic Acid-γ-Lactone

Formic Acid Lactulose Glyoxylic Acid D-Galacturonic Acid

D-Mannitol Sucrose D-Cellobiose Phenylethyl-amine

L-Glutamic Acid Uridine Inosine 2-Aminoethanol

Tween 80 L-Glutamine Glycyl-L-Glutamic Acid


Table 7.4 Nitrogen source utilization by consortium members Achromobacter sp. NP03, Ochrobactrum sp. NP04, and Lysinibacillus sp. NP05.

Nitrogen Source A O L Nitrogen Source A O L Nitrogen Source A O L Nitrogen Source A O L

Ammonia L-Valine -Phenylethyl- amine Xanthosine

Nitrite D-Alanine Tyramine Uric Acid

Nitrate D-Asparagine Acetamide Alloxan

Urea D-Aspartic Acid Formamide Allantoin

Biuret D-Glutamic Acid Glucuronamide Parabanic Acid

L-Alanine D-Lysine D,L-Lactamide D,L--Amino-N-Butyric Acid

L-Arginine D-Serine D-Glucosamine -Amino-N-Butyric Acid

L-Asparagine D-Valine D-Galactosamine -Amino-N-Caproic Acid

L-Aspartic Acid L-Citrulline D-Mannosamine D,L--Amino- Caprylic Acid

L-Cysteine L-Homoserine N-Acetyl-D-Glucosamine -Amino-N-Valeric Acid

L-Glutamic Acid L-Ornithine N-Acetyl-D-Galactosamine -Amino-N-Valeric Acid

L-Glutamine N-Acetyl-L-Glutamic Acid N-Acetyl-D-Mannosamine Ala-Asp

Glycine N-Phthaloyl-L-Glutamic Acid Adenine Ala-Gln

L-Histidine L-Pyroglutamic Acid Adenosine Ala-Glu

L-Isoleucine Hydroxylamine Cytidine Ala-Gly

L-Leucine Methylamine Cytosine Ala-His

L-Lysine N-Amylamine Guanine Ala-Leu

L-Methionine N-Butylamine Guanosine Ala-Thr

L-Phenylalanine Ethylamine Thymine Gly-Asn

L-Proline Ethanolamine Thymidine Gly-Gln

L-Serine Ethylenediamine Uracil Gly-Glu

L-Threonine Putrescine Uridine Gly-Met

L-Tryptophan Agmatine Inosine Met-Ala

L-Tyrosine Histamine Xanthine

A - Achromobacter sp. NP03, O – Ochrobactrum sp. NP04, L – Lysinibacillus sp. NP05

High Moderate Low Negative


7.4 Conclusions

Based on an extensive search of published literature, this appears to be the first

comparative study assessing PCB degradation efficiency of three facultative

anaerobic bacteria as a consortium under two different combined anaerobic-aerobic

modes; alternating (AN) and two stage (TS). Based on the literature, microbial

degradation of PCBs under anaerobic conditions has been shown to be a long-term

process ranging from 70 to 180 days. Therefore, the two stage (TS) process was set

up to have an extended anaerobic phase of four weeks followed by a short aerobic

phase of two weeks. In contrast, weekly intervals of anaerobic and aerobic

conditions, and vice versa was applied in the alternating (AN) study, over a six week

period. The study found that the alternating approach was more efficient compared

to the two stage treatment conditions with nearly 50% reduction in total PCBs

achieved within the first two weeks compared to only 24% from the two stage

treatment. This is significant because one of the limiting factors in bioremediation

applications is the long time span required to achieve a satisfactory degradation rate.

During both treatments, the consortium exhibited a greater ability to degrade the

lower chlorinated PCB homolog groups under aerobic conditions compared to highly

chlorinated homolog groups. However, the total PCB reductions were always higher

under alternating conditions, suggesting the weekly switching between anaerobic

and aerobic conditions favored the consortium to change between dechlorination

and oxidation activities. Even after the PCB degradation appeared to have reached a

plateau at week two, the bacterial cell density and chloride ion concentration in the

culture medium under alternating conditions kept increasing, suggesting the ability

of the consortium to further break down the intermediate products as their sole

carbon source and release the bound chlorides.

L-proline was found to be the best substrate to be utilized by all three consortium

members as their carbon and nitrogen source. Additionally, all three bacterial strains

were able to utilize L-lactic acid and methyl pyruvate as their carbon source and L-

Glutamic acid, Ala-His, Ala-Leu and Gly-Gln as their nitrogen source. However,

applicability of the addition of such chemical compounds as nutritional supplements


to soil in bioremediation applications needs to be considered only after additional

research on their suitability for the soil ecosystem.

Finally, it can be concluded that the alternating (AN) anaerobic-aerobic treatment

would be a preferred approach compared to the two stage (TS) anaerobic-aerobic

process in PCB remediation applications when compared to degradation efficiencies

and time. Importantly, the use of facultative anaerobic bacteria such as

Achromobacter sp. NP03, Ochrobactrum sp. NP04, and Lysinibacillus sp. NP05 as a

consortium would be advantageous as they have the ability to survive and degrade

PCBs under both, aerobic and anaerobic conditions more efficiently than the

individual organisms.

Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05 163

Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05

8.1 Background

Recent research literature provide confirming evidences to suggest that the

intracellular degradation of PCBs occurs via a series of enzymes through anaerobic

dechlorination and aerobic oxidation (Wiegel & Wu, 2000; Pieper, 2005; Agullo et al.,

2017). However, there is no real knowledgeabout specific proteins involved in active

transport of hydrocarbons across the biological membranes (Parales & Ditty, 2017).

As PCBs are insoluble in aqueous media, it is a prerequisite to transform PCBs into

soluble and / or smaller molecules to diffuse more easily into the cells across the cell

membranes (Hearn et al., 2008). Extracellular enzymes released by microorganisms

into their surrounding environment may play a major role in transformation of

hydrophobic pollutants (Basak & Dey, 2015).

According to Desvaux et al. (2009), actively secreted and non-secreted proteins by

an organism are collectively called as exoproteome (see illustration in Figure 8.1).

However, the total proteins or enzymes exported from inside the cells to the external

environment by an organism is defined as the secretome. The secretome consists of

proteins that are actively transported to outside the cell through the cytoplasmic

membrane via classical or non-classical secretion mechanisms, proteins shedding

from the cell membrane, proteins released from the membrane vesicles, as well as

from the secretion machinery itself (Caccia et al., 2013).

164 Chapter 8: Metaproteomics analysis of extracellular proteins detected in the culture supernatant during PCB hydrolysis by the bacterial consortium: Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05

Figure 8.1 The secretome and exoproteome of a Gram negative bacterial cell.

(Armengaud et al., 2012)

As illustrated in Figure 8.2, oxidoreductases and hydrolases are some of the secreted

proteins from microorganisms with the ability to convert polymeric chemical

substances such as poly aromatic hydrocarbons to partially degraded or oxidized

products that can be easily absorbed by microorganisms (Gianfreda & Rao, 2004;

Basak & Dey, 2015). Hydrolases or hydrolytic enzymes facilitate the cleavage of major

chemical bonds such as C–C, C–O, C–N in toxic molecules and help to reduce their

toxicity (Karigar & Rao, 2011). However, there is limited knowledge about the role of

extracellular proteins or enzymes secreted by that can attacking and hydrolysing PCB

molecules to produce more easily diffusible intermediates. Proteins that facilitate

uptake of those intermediates into cells for further degradation, and subsequent

conversion to energy are also poorly known.


Figure 8.2 Role of extracellular enzymes in insoluble compound metabolism.

Chapter 8 focuses on extracellular proteins detected in the culture supernatants of

Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05 both

individually as described in Chapter 6 and as a consortium reacting with PCBs under

AN and TS conditions, as described in Chapter 7. This chapter also discusses; (1) mass

spectrometry based proteomics approaches for the identification of proteins present

in bacterial culture meda (2) bioinformatics analyses of these protein sequences, and

(3) their possible roles during the degradation of PCBs by the three facultative

anaerobic bacteria acting as a consortium.


Supernatant samples (1 mL) were collected from individual culture experiments of

facultative anaerobic members Achromobacter sp. NP03, Ochrobactrum sp. NP04

and Lysinibacillus sp. NP05 (as described in Chapter 6), and from the bacterial

consortium based experiments (as described in Chapter 7). These were used for the


analysis of extracellular proteins found in the culture supernatants. The samples were

centrifuged at 10,000 × g for 15 min at 4 °C to remove the bacterial cells, and the

resulting cell free culture supernatants containing proteins were used for protein

visualization, quantification and extraction prior to mass spectrometry analyses.

8.2.1 Protein visualization

8.2.1.1 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

SDS-PAGE was used to visualise the proteins in the culture supernatants. Protein

sample (32.5 µL) was mixed with 12.5 µL of 4x Laemmli loading buffer (Thermo Fisher

Scientific) and 5 µL of 10x Bolt reducing agent (Thermo Fisher Scientific). The mixture

was then denatured by boiling at 95 ̊ C for 5 min. Samples were resolved by SDS-PAGE

using Bolt 4-12% Bis-Tris Plus SDS-PAGE gels (Invitrogen, Thermo Fisher scientific).

SeeBlue Protein standard (Thermo Fisher scientific) was used as the protein

molecular weight (MW) marker. A 40 µL aliquot was loaded to each well and

electrophoresis was performed under constant voltage of 200 V for 30 min in mini

gel tanks (Life Technologies) using 1x Bolt MES SDS running buffer (Novex).

8.2.1.2 Coomassie blue staining

Following electrophoresis, gels were removed from tank and fixed by adding 50 mL

fixing solution (10% (v/v) acetic acid and 40% (v/v) ethanol) and agitating gently for

five minutes in a shallow tray. Gels were then rinsed with deionised water and stained

in QC Coomassie stain (Biorad) overnight at room temperature with gentle agitation.

The next day, gels were destained in deionized water for 1 to 3 hr with gentle

agitation at room temperature until the background cleared, and the protein bands

became clearly visible.

8.2.2 Protein quantification using Bicinchoninic acid assay (BCA)

The total protein concentrations in the culture supernatants were quantified using a

microplate bicinchoninic acid assay as per the Pierce BCA Protein Assay Kit protocol

(Catalog number 23227, Thermo Scientific). The protein concentrations were


determined relative to the triplicates of standard dilutions (0-2000 ng/μL) of bovine

serum albumin (BSA, stock solution of 2 mg/mL, Sigma-Aldrich (Table 8.1). 25 μL of

each sample and 200 μL working reagent (50 parts of BCA reagent A with 1 part of

BCA reagent B) was mixed to give 1:8 ratio in a 96-well microtiter plate and incubated

at 37 ˚C for 30 min. The plate was cooled to room temperature and the absorbance

was measured at 562 nm using a microplate reader (FLUOStar Optima, BMG Labtech).

Table 8.1 Standard dilutions preparation for BCA assay

Vial Volume of

Diluent* (μL)

Volume and Source of BSA

(μL)

Final BSA concentration

(μg/mL)

A 0 300 of Stock 2000

B 125 375 of Stock 1500

C 325 325 of Stock 1000

D 175 175 of vial B 750

E 325 325 of vial C 500

F 325 325 of vial E 250

G 325 325 of vial F 125

H 400 0 0 = Blank

*Sterile MilliQ water was used as the diluent.

8.2.3 Trypsin digestion of extracellular proteins

A modified in-filter digestion (FASP) protocol based on the work of Wisniewski et al.

(2009) was used to digest the proteins in the culture supernatant into peptides prior

to mass spectroscopy. Based on the protein concentration measurements in Section

8.2.2, supernatants containing 20 µg of proteins were used for the trypsin digestion.

Parallel to each batch of samples, 10 µg of BSA was also analyzed as a quality control

measure. Buffers and chemicals needed for the protein extractions were prepared as

per Section 3.1.2.6 of Chapter 3.

During sample preparation, protein samples were combined with 200 μL of

Dithiothreitol -Urea buffer and loaded onto 30 kDa Microcon YM-30 centrifugal filter


tubes (catalogue number MRCF0R030, Millipore) and incubated at room

temperature for 60 min in an agitator. Filters were centrifuged at 14,000 x g, 21 °C

for 15 min and the filtrate was discarded. Filters were washed with 200 μL Urea-Tris

buffer and centrifuged at 14,000 x g, 21 °C for 15 min, and the filtrate was discarded.

100 μL Iodoacetamide-Urea buffer was then added to each of the filters and

incubated at room temperature for 20 min in an agitator. Filters were centrifuged at

14,000 x g, 21 °C for 10 min and the filtrate was discarded. The filters were washed

twice with 100 μL of Urea-Tris buffer and centrifuged at 14,000 x g, 21 °C for 15 min

each, with the filtrate discarded after each time. The filters were then equilibrated

by washing twice in 100 μL of 100 mM ammonium bicarbonate buffer and

centrifuged at 14,000 x g, 21 °C for 10 min. The filtrate was removed after both

centrifugation steps.

Trypsin stock (10 µL of 1 µg/µL in 100 mM ammonium bicarbonate buffer) was

defrosted on ice and the working trypsin solution was prepared by adding 90 μL of

100 mM ammonium bicarbonate buffer to the trypsin stock. The working trypsin

solution was added to the tubes containing filters to achieve an enzyme : protein

ratio of 1:50 and the tubes were placed in a humidified chamber and incubated

overnight at 37 °C with gentle agitation.

Following protein tryptic digestions, the filters were transferred to clean 1.5 mL

Eppendorf tubes and the resultant peptides were eluted by centrifugation at 14,000

x g, 21 °C for 15 min. An additional elution step was performed with one wash of the

filters with 20 μL of 100 mM ammonium bicarbonate buffer and collected at 14,000

x g, 21 °C for 15 min. Samples containing tryptic peptides were vacuum dried using a

rotational vacuum concentrator (RVC 2-33IR, John Morris Scientific) for 30 min at 40

°C and reconstituted in 10 μL of 2% (v/v) Acetonitrile (ACN) / 0.1% (v/v) trifluoroacetic

acid (TFA) solution, before proceeding to desalting.

8.2.4 Desalting of samples prior to mass spectroscopy analysis

Following elution, the peptide samples were further purified using solid phase

extraction as described by Rappsilber et al (2003). In particular, 200 µL pipette tips


were filled with single SCX membrane disc (Empore 3M) as described in Section

3.1.2.6 J in Chapter 3 and activated by passing 30 μL of 100% ACN through using a

centrifugal force (2 min spin at 2,000 rpm). Next, 30 μL of 5% (v/v) ammonium

hydroxide/80% (v/v) ACN was added to the tips just before the last remainder of ACN

left the tip and collected for 2 min at 2,000 rpm. Finally, 30 μL of 0.2% TFA (v/v) was

added and collected for 2 min at 2,000 rpm. Peptide samples were then loaded onto

the tips and centrifuged for 2 min at 2,000 rpm. A 30 μL 0.2% TFA solution was added

and the tips were centrifuged for 2 min at 2,000 rpm and this step was repeated.

Peptide samples were eluted to clean 1.5 mL Eppendorf tubes by adding 30 μL of 5%

ammonium hydroxide/80% ACN to the tips followed by 2 min spin at 2,000 rpm. The

eluted peptide samples were vacuum dried as before using a rotational vacuum

concentrator and the pellets were reconstituted in 10 μL iRT buffer (see Section

3.1.2.6 G for preparation) ready for liquid chromatography - mass spectroscopy (LC-

MS) analyses.

8.2.5 Secretome analysis

8.2.5.1 Liquid chromatography – mass spectroscopy

(A) Chromatography

The peptide samples were analysed using the TripleTOF® 5600+ mass spectrometer

(SCIEX) in either DDA or DIA mode as described below. Approximately 400 ng – 1 µg

of peptide material was injected to the instrument. Peptides were separated by

performing reversed-phase chromatography using an Eksigent ekspert™ nanoLC 400

System directly interfaced to the MS/MS instrument. The LC platform was set up in a

trap and elute configuration with a 10 mm × 0.3 mm trap cartridge packed with

ChromXP C18CL 5 µm 120 Å material and a 150 mm × 75 µm analytical column packed

with ChromXP C18 3 µm 120 Å (Eksigent Technologies, Dublin, CA). The mobile phase

solvents were composed of mobile phase A: water/0.1% formic acid (FA); mobile

phase B: ACN/0.1% FA; and mobile phase C: water/2% ACN/0.1% FA. Trapping was

performed in mobile phase C for 5 min at 5 µL/min followed by an elution

configuration across either 9.5 min or 40 min gradient (depending whether 25 min or


65 min method was used) using mobile phases A and B at a conserved flowrate of

300 nL/min. The proportions of both solvents (A and B) were adjusted at specified

time-points of:

a) 0, 30, 35, 40, 49, 50 and 60 min corresponding to 98, 60, 35, 10, 10, 98 and 98 % of

solvent A in the case of the 65 min method.

b) 0, 5, 7, 9.5, 10.2 and 20 min corresponding to 95, 60, 10, 10, 95 and 95 % of solvent

A in the case of the 25 min method.

To minimise retention time drift, the analytical column was maintained at 40 °C.

(B) Data-dependent acquisition (DDA)

For spectral library generation, peptides were analysed by data-dependent

acquisition (DDA) using 25 min (for BSA QC samples) and 65 min method (for culture

supernatant samples). The DDA mode of the instrument was set to obtain high

resolution (resolving power: 30,000) TOF-MS scans over a range of 350-1350 m/z,

followed by up to 15 (in the case of 25 min method) or 40 (in the case of 65 min

method) high sensitivity MS/MS scans of the most abundant peptide ions per cycle

over the range of 100-2000 m/z. The selection criteria for the peptide ions included

intensity greater than 150 cps and charge state of 2-5. The dynamic exclusion

duration was set at either 3 s or 9 s to account for the difference in chromatographic

peak width between the two methods used. Each survey (TOF-MS) scan lasted 250

ms and the product ion (MS/MS) scan was acquired for 50 ms, resulting in a total

cycle time of either 1 s or 2.3 s depending on the method. The ions were fragmented

in the collision cell using rolling collision energy, and collision energy spread (CES) was

set to 5. The collected peptide ion fragmentation spectra were stored in two files per

sample with extensions format of .wiff and .wiff.scan (SCIEX).

(C) Data-independent acquisition (DIA)

For quantitation, eluted peptides were subjected to a cyclic data-independent

acquisition (DIA) using even isolation windows SWATH-MS™ acquisition 65 min

method (SCIEX) based on its earlier description (Gillet et al., 2012). In particular, a


survey scan data (MS) was acquired for 80 ms, followed by MS/MS on all precursors

within a particular isolation window in a cyclic manner using an accumulation time of

80 ms per individual SWATH-MS window. 36 overlapping windows, each 26 m/z units

wide, were used to cover the peptide ions in a range of 350 – 1250 m/z which resulted

in the cycle time of 3 s. Fragment ions were recorded in a high sensitivity mode and

in a range of 100 – 1800 m/z. Given the peptide chromatographic peaks were

approximately 18 s wide, the above parameters allowed the collection of at least 6

data points for each chromatographic peak to ensure accurate quantitation. The

collected data were stored in two files per sample with extensions format of .wiff and

.wiff.scan (SCIEX).

8.2.5.2 Protein identification using ProteinPilot v5

All DDA data files of individual culture samples of Achromobacter sp. NP03,

Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05 (as discussed in Chapter 6) were

submitted together for peak picking and database search through ProteinPilot (V5.0,

ABSciex) software using Paragon algorithm (Shilov et al, 2007). The database was

created using the existing protein sequences of the three microorganisms used in the

consortium study at genus level and protein sequences of the microorganisms

identified with PCB degradation potential in the present and previously published

studies downloaded (on 09.08.2017) from the NCBI protein database. The resulting

protein list was stored as a single .group file.

8.2.5.3 Protein quantitation using PeakView

The resulting .group file generated from ProteinPilot software via SWATH microapp

to create a spectral library with false discovery rate set to 1% for protein identification

and requirement of only unmodified peptides to be used. Next, SWATH™-MS data

files were imported into PeakView and SWATH microapp was used for targeted

extraction of fragment ion signals specific to peptides represented in the library from

each individual SWATH™-MS run. This peak detection step was conducted under

stringent criteria (99% confidence and 0% false discovery rate). SWATH™-MS runs

were performed in triplicate for each sample analyzed. At least four different


peptides were required per protein and their signals were averaged at a protein level

inside SWATH microapp. A protein was considered valid when it was detected in all

three replicates, and absent in the media controls. Protein level data were normalized

using the Most Likely Ratio (MLR) method using MarkerView software (V1.3.1, SCIEX)

which was also used to statistically identify the significantly changed proteins through

principal component analysis (PCA) before any further analysis using bioinformatics.

Finally, using Excel, relative abundance of significantly changed proteins were

determined as heat maps.

8.2.5.4 Bioinformatics analysis of peptide sequences

Once the identity of significantly changed proteins was established, the

corresponding peptide sequences were analysed whether that protein would fall

under a ‘secreted’ or ‘non-secreted’ protein using bioinformatics tools. The proteins

that would fall under classically secreted extracellular proteins were identified using

SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP) (Armengaud et al., 2012).

Default cut-off values were used during the search for both Gram positive and Gram

negative bacteria. In contrast, the proteins classified as non-classical secreted

proteins were predicted with SecretomeP 2.0

(http://www.cbs.dtu.dk/services/SecretomeP) (Vazquez-Gutierrez et al., 2017). A

protein without a signal peptide was considered positive as a non-classically secreted

protein when the SecP score was above 0.5. Analysis using SignalP first, followed by

SecretomeP, was considered as the preferential order when the same protein was

found positive under both prediction tools. All protein sequences were further

scrutinized through the redundancy check using Geneious (R10.2.3, Biomatters Ltd.).

Proteins that were grouped under secreted and non-secreted proteins were also

analysed according to their potential cellular and biological functions, following NCBI,

UniProtKB (UniProt Consortium, 2016), SwissProt (Bairoch & Apweiler, 2000)

databases and close homolog searches.

http://www.cbs.dtu.dk/services/SignalP

http://www.cbs.dtu.dk/services/SecretomeP



8.3.1 SDS-PAGE analysis: Extracellular protein detection and visualization

8.3.1.1 Individual cultures

As an initial screening step, 1 mL samples were collected at weekly intervals from the

supernatants of individual batch culture experiments of the three facultative

anaerobic culture members Achromobacter sp. NP03, Ochrobactrum sp. NP04 and

Lysinibacillus sp. NP05 as described in Chapter 6. Following separation of supernatant

from cells by centrifugation at 10,000 × g for 15 min at 4 °C, a total of 32.5 µL of clear

culture supernatants was checked from each sample, in order to visualize the

presence or absence of proteins in the supernatant over time (see SDS-PAGE protein

analysis in Section 8.2.1). Parallel to each set of samples, separate SDS-PAGE gels

were run for control samples collected at weekly intervals from flasks containing

minimal salt medium inoculated with bacterial seed cultures, containing no PCBs and

there were no visible proteins detected in any of the control SDS-PAGE gels (see

Figure 8.3 for Lysinibacillus sp. NP05 controls under anaerobic conditions).

Figure 8.3 SDS-PAGE analysis of Lysinibacillus sp. NP05 containing controls (minimal

salt medium with no added PCBs) under anaerobic conditions at 28 °C. Lane 1,

SeeBlue Protein standard as the protein molecular weight markers; lanes 2, time 0

immediately after addition of seed culture; lane 3 to 8, week 1 to week 6.


The SDS-PAGE results are shown in Figure 8.4 for Achromobacter sp. NP03, Figure 8.5

for Ochrobactrum sp. NP04 and Figure 8.6 for Lysinibacillus sp. NP05, respectively.

Notably, none of the three strains had any visible protein bands present in week 0,

directly after the addition of the seed cultures.

According to Figure 8.4, the main proteins detected in the supernatants of

Achromobacter sp. NP03 cultures are shown running at ~ 40 kDa that first appeared

in week 1 and were present up to week 6, during anaerobic conditions (Figure 8.4 B).

In contrast, lower yield proteins running at about 50 kDa appeared at weeks 1-6

under aerobic conditions. However, more proteins around the 90 and 40 kDa were

present during week 5 (Figure 8.4 A).

Figure 8.4 SDS-PAGE analysis of extracellular proteins of Achromobacter sp. NP03

under (A) aerobic and (B) anaerobic conditions at 28 °C. Lane 1, SeeBlue Protein

standard as the protein molecular weight markers; lanes 2, time 0 immediately after

addition of seed culture; lane 3 to 8, week 1 to week 6.

Similar to Achromobacter sp. NP03, low amounts of proteins were detected for

Ochrobactrum sp. NP04 under aerobic conditions during weeks 1 to 4 and 6, with

even low to no proteins appearing in week 5 (Figure 8.5 A). More proteins were

present under anaerobic conditions in weeks 2 to 6, with no obvious proteins

detected in week 1 (Figure 8.5 B).


Figure 8.5 SDS-PAGE analysis of extracellular proteins of Ochrobactrum sp. NP04

under (A) aerobic and (B) anaerobic conditions at 28 °C. Lane 1, SeeBlue Protein

standard as the protein molecular weight markers; lanes 2, time 0 immediately after

addition of seed culture; lane 3 to 8, week 1 to week 6.

The proteins detected in the Lysinibacillus sp. NP05 culture supernatants are shown

in Figure 8.6. Under aerobic conditions, a dominant high molecular weight proteins

running at about 100 kDa appeared during week 1 and was show continuing presence

from weeks 2 to 6 (Figure 8.6 A). In contrast, more proteins were detected under

anaerobic conditions, with the yield and pattern of proteins appearing similar in

weeks 1 and 3, and, weeks 4 and 6 (Figure 8.6 B). It was of interest that high amounts

of proteins running approximately at 100 kDa appeared in weeks 1 and 3, while lesser

amounts in weeks 2, 4 and 6. When compared to other weeks, relatively low amounts

of proteins were present in the supernatant in week 5.


Figure 8.6 SDS-PAGE analysis of extracellular proteins of Lysinibacillus sp. NP05 under

(A) aerobic and (B) anaerobic conditions at 28 °C. Lane 1, SeeBlue Protein standard

as the protein molecular weight markers; lanes 2, time 0 immediately after addition

of seed culture; lane 3 to 8, week 1 to week 6.

Despite the fact that the samples were normalized to the volume and not protein

amount, and therefore, change in the protein signal likely correlated with bacterial

density/growth as well as increased/decreased secretion. In summary, Lysinibacillus

sp. NP05 under anaerobic conditions showed the highest number of protein bands

present in the culture supernatants among all the samples (compare Figures 8.4, 8.5

and 8.6). In addition, protein levels in the extracellular environment under aerobic

conditions seemed lower than under anaerobic conditions. This may be due to the

low concentration of lower chlorinated PCB congeners present in the PCB mixture

(see Table 3.2 in Chapter 3) as the only available carbon source for the

microorganisms to utilize under aerobic conditions.

8.3.1.2 Consortium study under AN and TS treatment conditions

In the consortium experiments as described in Chapter 7, supernatant samples

collected at fortnightly intervals from the alternating (AN) and the two stage (TS)

anaerobic-aerobic experiments were also analysed using SDS-PAGE protein analysis.

The results are shown in Figure 8.7. Similar to the individual cultures (see Figures 8.4,


8.5 and 8.6), no obvious proteins can be seen in week 0, straight after the addition of

the seed comprising of Achromobacter sp. NP03, Ochrobactrum sp. NP04 and

Lysinibacillus sp. NP05, as a consortium. However, obvious proteins at around 38-39

kDa are present in week 2, and lesser amounts of the similar sized proteins in weeks

4 and 6 during the AN conditions (Figure 8.7 A). Another protein at about 29 kDa is

present in higher amounts in week 2, under the AN conditions compared to the week

2 results under the TS conditions (Figure 8.7 B). Incidentally, the high levels of

extracellular proteins in week 2 under the AN conditions coincides with the high PCB

degradation rate of 49.2±2.5% observed in week 2 under AN treatment conditions as

discussed in Section 7.3.2.1 in Chapter 7.

Figure 8.7 SDS-PAGE analysis of the extracellular proteins of the bacterial consortium

consisting of Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp.

NP05 under (A) AN, and (B) TS anaerobic-aerobic conditions at 28 °C. Lane 1, SeeBlue

Protein standard as the protein molecular weight markers; lanes 2, time 0

immediately after addition of seed culture; lane 2 to 5, at fortnightly intervals up to

week 6.

In comparison, similar sized proteins (~38-39 and 29 kDa) are also present in weeks

2 and 4 under anaerobic conditions in TS treatment (Fig. 8.7 B). However, it appeared

that the same proteins are not present in the culture medium, during the last 2 weeks

of cultivation when the condition was changed from anaerobic to aerobic in TS

conditions (Figure 8.7 B).


8.3.2 Proteomics analysis

The proteins in the culture supernatants from the consortium study were further

analysed using mass spectrometry in order to enable their identification. Proteins

isolated from the culture supernatants were digested into peptides, purified using

solid phase extraction and resultant peptides were submitted for mass spectrometric

analysis. After quantification using SWATH, a total of 618 proteins representing the

extracellular protein fractions in both AN and TS conditions were identified for

further analyses, following quality control measures as described in 8.2.3.4.

All the 618 total proteins were then subjected to bioinformatics analyses in order to

determine: (1) the presence of signal peptides characteristic of classically secreted

proteins using the SignalP 3.0 server (Petersen et al., 2011); and (2) proteins

characterized as non-classically secreted proteins using the SecretomeP 2.0 server, a

prediction method for identifying the proteins using the signal peptide independent

secretion pathways (Bendtsen et al., 2005). The remaining proteins were grouped as

non-secretory proteins. Analysis of 618 total proteins from AN and TS conditions over

the six weeks period is shown in Table 8.2. The highest number of total proteins (n-

542) detected in week 6 under AN conditions coincides with the maximum PCB

degradation rate (54.1±0.49%), optical density (2.49±0.16 (see Figure 7.3a in Chapter

7) and chloride ion accumulation (17.63±0.91 mg/L) (see Figure 7.6 in Chapter 7).

Table 8.2 Analysis of extracellular proteins identified in culture supernatants of

consortium under AN and TS conditions.

Category

Number of proteins

AN Treatment (weeks) TS Treatment (weeks)

2 4 6 2 4 6

Non secretory 265 220 273 261 249 263

Secretory 260 254 269 266 278 254

Total 525 474 542 527 527 517


8.3.2.1 Non-secretory proteins

Out of the 618 total proteins, 319 (52%) of the proteins found in the culture

supernatant were predicted to be non-secretory by the SignalP 3.0 and SecretomeP

2.0 servers. Both the highest (n=273) and lowest (n=220) number of non-secretory

proteins were detected in week 6 and week 4 under AN conditions as summarized in

Table 8.2. The predicted 319 non-secretory proteins were grouped according to their

potential functional activities (Table 8.3) and the detailed functional classification of

predicted non-secretory proteins in the culture supernatant is given in Table C.3 in

Annex C.

Table 8.3 Functional classification of 319 identified proteins that were predicted to

be non-secretory proteins by the SignalP 3.0 and SecretomeP 2.0 servers.

Function Number of proteins

Amino acid metabolism 43

Aromatics & Xenobiotic degradation 16

Carbohydrate metabolism 32

Carboxylic acid metabolism 15

Elongation factors and chaperones 11

Genetic Information Processing 41

Hypothetical 20

Lipid metabolism 22

Membrane related 6

Metabolism of cofactors and vitamins 6

Nucleotide metabolism 21

Signal transduction and chemotaxis 11

Stress and redox signalling 26

Transport and secretion 8

Other (uncategorised) 41

Total 319

The functional classification of these predicted non-secretory proteins revealed that

the large proportion matched to proteins involved in major metabolic processes

essential for the survival of cells such as amino acid metabolism (n=43), genetic

information processing (n=41) and carbohydrate metabolism (n=32) as shown in


Table 8.3. There were also 16 proteins related to aromatics and xenobiotic

degradation including 4-oxalocrotonate tautomerase, phenyl phosphate carboxylase,

3-oxoadipate CoA transferase, carboxymuconolactone decarboxylase, 2-

aminobenzoate-CoA ligase, and glutathione S-transferase.

4-Oxalocrotonate tautomerase enzyme is part of the bacterial plasmid-encoded

pathway, which allows the microorganisms harbouring the plasmid to use various

aromatic hydrocarbons as their sole sources of carbon and energy (Whitman, 2002).

Phenylphosphate carboxylase has been found to be responsible for catalysing the

para carboxylation of phenylphosphate to 4-hydroxybenzoate in anaerobic

metabolism of phenol (Schuhle & Fuchs, 2004). During aerobic degradation, aromatic

compounds are transformed to intermediates like catechol and protocatechuate

(Altenschmidt & Fuchs, 1992). However, there is a limited number of bacterial species

which are able to both, convert the chloroaromatics into catechol and

protocatechuate intermediates and further mineralize them, due to the lack of

chlorocatechol pathway enzymes (Pieper & Reineke, 2004). Both 3-oxoadipate CoA-

transferase and carboxymuconolactone decarboxylase detected here, are two such

enzymes involved in the catechol and protocatechuate branches of the 3-oxoadipate

pathway, which are important for the degradation of aromatic compounds (Eulberg

et al., 1998). 2-aminobenzoate-CoA ligase was found to be able to catalyse the first

step of aerobic metabolism of 2-aminobenzoate, an intermediate of PCB degradation

into the coenzyme A thioester of 2-aminobenzoate (Altenschmidt & Fuchs, 1992).

Bacterial glutathione transferases (GSTs) are one of the superfamily of enzymes that

play an important role in cellular detoxification against toxic xenobiotics via

conjugation of reduced glutathione and protection against chemical and oxidative

stresses (Nebert & Vasiliou, 2004; Allocati et al., 2009). Glutathione S-transferase is

also known as BphK and catalyzes the dechlorination of 3-chloro-2-hydroxy-6-oxo-6-

phenyl-2,4-dienoates (HOPDAs) compounds that are produced during the

degradation of PCBs in some bacterial biphenyl catabolic (bph) pathways. The

enzyme also catalyzed the dechlorination of 5 chloro HOPDA and 3,9,11-trichloro

HOPDA (Fortin et al., 2006). Additionally, GSTs are also involved in the reductive

dechlorination of pentachlorophenol (Van Agteren et al., 2013).


Oxidoreductases were present in culture supernatant throughout the study period

under both AN and TS conditions with the maximum concentration observed at week

2 under AN conditions. These enzymes are responsible for bacterial mediation of

detoxification of toxic organic compounds through oxidative coupling.

Microorganisms extract energy via energy-yielding biochemical reactions mediated

by oxidoreductases to cleave chemical bonds and to assist in the transfer of electrons

from a reduced organic substrate (donor) to another chemical compound (acceptor)

(Karigar & Rao, 2011). As a result of such oxidation-reduction reactions, the toxic

contaminants are oxidized to harmless compounds.

In addition to these results, the release of certain proteins without known peptide

secretion signals have also been reported in some comparative proteomics studies of

extracellular proteins of Escherichia coli. (Li et al., 2004; Xia et al., 2008). Li et al.

(2004) reported the presence of some enzymes that are involved in metabolic

processes such as nucleotide metabolism, glycolytic pathway, amino acid metabolism

as well as some outer membrane, periplasmic, and cytosolic proteins in the

extracellular proteome of some virulent Escherichia coli strains. Enzymes related to

metabolism and cellular processes such as isocitrate lyase, malate synthase without

signal peptide sequences were also detected in the extracellular proteome of some

other Escherichia coli (9BL21 and W3110) strains (Xia et al., 2008).

Importantly, the potential for cell lysis could not be excluded as a cause for the

release of these proteins, since increased concentrations of some major cytoplasmic

proteins such as chaperonin GroEL, DnaK suppressor protein and thioredoxin TrxA

were detected in the culture medium over time under both AN and TS treatments.

However, occurrence of cell lysis leading to cell death is less likely since there is a

strong trend in increasing cell density and subsequent PCB degradation rates in the

culture medium over time (see Figure 7.3 in Chapter 7).

Selective leakage of some cytoplasmic proteins by unidentified mechanisms was

previously reported (Xia et al., 2008). Therefore, alterations to the cell membrane

and cell wall structures is likely to occur rather than complete cell lysis due to various

stresses such as osmotic shock-like response (Kaakoush et al., 2010). Vazquez-Laslop

et al. (2001) reported that the increased permeability of the outer membrane due to


sudden drop in osmolarity in the medium leads to selective release of some small

cytoplasmic proteins. Thioredoxin and DnaK, which were present in high

concentrations in the exoproteome of the present study, were also among the highly

released proteins due to the osmolarity shock (Vazquez-Laslop et al., 2001).

Furthermore, Cl- ions build up during PCB dechlorination (see Figures 7.6 in Chapter

7) may have contributed to changing the osmolality of the culture medium and may

have some impact on the release of non-secretory proteins. However, this needs to

be further investigated.

Overall, the presence of enzymes that are responsible for the intermediate steps of

PCB degradation pathways and detoxification of toxic chemicals in the extracellular

environment by cell lysis or by selective straining provides strong support for the

occurrence of PCB degradation by the bacterial consortium.

8.3.2.2 Secretory proteins: Secretome

Out of 618 total proteins found in the exoproteome, 299 (48%) were

bioinformatically predicted to be secreted. Of the 299 total proteins identified to be

secreted in the culture supernatant, 71% (212 proteins) were predicted to have signal

peptides and grouped to be classically secreted proteins, while only 29% (87) proteins

were predicted to be secreted by non-classical secretion pathways (Figure 8.8).

Detailed information related to secretory proteins under AN and TS conditions are

given in Table C.1 in Appendix C.


Figure 8.8 Proportions of classically and non-classically secreted proteins in the

culture supernatant of the bacterial consortium Achromobacter sp. NP03,

Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05.

4.3.2.2.1 Classically secreted proteins

Classically secreted proteins were further categorized based on their functional

groups in Figure 8.9. Some proteins such as superoxide dismutase that were found to

have a specific function in the cytoplasm were also recognized to be actively involved

in some biological processes in the extracellular environment (Bendtsen et al., 2005;

Henderson & Martin, 2011). The functions they perform in the cytoplasmic and

extracellular environments are not always identical. Such proteins involved in

multiple functions are known as moonlighting proteins (Jeffery, 2003).


Figure 8.9 Functional groupings of proteins identified as classically secreted proteins.

As shown in Figure 8.9, most abundantly detected classically secreted proteins were

transporters and they represented 58% (122 proteins) of the total classically secreted

proteins. Transporter proteins are one of organism’s primary interfaces with the

environment. Bacterial transport proteins comprise a heterogeneous group

representative of their diverse biological functions (Giuliani et al., 2011). The ability

of bacteria to occupy specific environments mainly depends on their capacity to

obtain sufficient supplies of nutrients that are essential for their growth (Vazquez-

Gutierrez et al., 2017). Bacterial transport proteins play an important role in

facilitating the uptake of essential nutrients, regulate the cytoplasmic concentrations

of metabolites, export large molecules to the outer surface of the cell and prevent

toxic effects of toxins by catalyzing their active efflux (Yen et al., 2009). These proteins

comprise a heterogeneous group representative of their diverse functional and

cellular roles.

Among the ten extracellular proteins identified with the highest protein

concentrations, eight were also transport related classically secreted proteins (see

Table 8.4). The majority of the transporters such as sulfate transporter, leucine ABC

transporter subunit substrate-binding protein, glutamine ABC transporter substrate-

binding protein, amino acid ABC transporter substrate-binding protein, branched

chain amino acid ABC transporter substrate-binding protein, iron ABC transporter


substrate-binding protein, molybdate ABC transporter substrate-binding protein,

were present in the extracellular environment and are involved in the transport of

nutrients. The synthesis of a wide range such transporters in the cell is essential for

the microorganisms to uptake the nutrients from the environment (Christie-Oleza et

al., 2012).

Bacterial biodegradation of hydrocarbons, an important process for environmental

remediation, requires the passage of hydrophobic substrates across the cell

membrane. Membrane proteins are believed to be required to facilitate the

transportation of hydrophobic compounds across the outer membrane of Gram-

negative bacteria surrounded by a hydrophilic lipopolysaccharide layer (Hearn et al.,

2008). ATP-binding cassette (ABC) transporters couple ATP hydrolysis to aid in the

transport of various molecules across cellular membranes (Michalska et al., 2012).

The involvement of ABC transporters, via their associated substrate binding protein

specificity in the uptake and metabolism of benzoic acid and other aromatics was

revealed by Giuliani et al., (2011).


Table 8.4 Heat map showing the relative abundance of ten highly secreted extracellular proteins detected in the culture supernatant of the

bacterial consortium Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05.

No. Accession number

Name AN

(Weeks) TS

(Weeks)

Secreted*

2 4 6 2 4 6

1 OLU07503.1 Sulfate transporter subunit Yes (27-28) 2 EHK64178.1 Histone-like DNA-binding protein Yes# 3 WP_088138276.1 ABC transporter substrate-binding protein Yes (23-24) 4 WP_062683598.1 Amino acid ABC transporter substrate-binding protein Yes (22-23) 5 WP_062682236.1 Leucine ABC transporter subunit substrate-binding protein LivK Yes (25-26) 6 KOQ40547.1 Membrane protein Yes (22-23) 7 OLU06668.1 Iron ABC transporter substrate-binding protein Yes (28-29) 8 KGD98287.1 Glutamine ABC transporter substrate-binding protein Yes (26-27) 9 OLU08696.1 ABC transporter substrate-binding protein Yes (28-29)

10 WP_088595533.1 Glutamine ABC transporter substrate-binding protein GlnH Yes (26-27) Notes:

AN – Alternating anaerobic-aerobic treatment, TS – Two stage anaerobic-aerobic treatment

* Numbers given within brackets correspond to the signal peptide cleavage site location of classically secreted proteins.

# Non-classically secreted proteins.

Protein yield

Low <----------> high


There were three classically secreted proteins identified here that matched to

glutathione transporter proteins. They are; glutathione ABC transporter substrate-

binding protein, Glutathione-binding protein precursor and glutathione-binding

protein (Table C.1 in Appendix C). One of the main roles of glutathione is protection

of cells through the regulation of the concentrations of xenobiotics within the cell by

conjugating xenobiotics and exporting them from the cells (Cole & Deeley, 2006).

PCBs are highly hydrophobic and lipophilic molecules, and therefore they are

believed to be capable of unregulated entry into the cells through the cytoplasmic

membrane which can ultimately cause intracellular toxicity (Parales & Ditty, 2017).

Therefore, bacteria capable of utilizing PCBs as their growth substrate must have

some mechanism to control the intracellular PCB concentration at sufficient level to

permit cell growth while maintaining below the intracellular toxic levels. Therefore,

the presence of glutathione related transporter proteins in the culture supernatant

might have a role in maintaining the internal PCB concentration at sub-toxic levels.

The protein with the highest concentration found in the secretome was identified as

a sulfate transporter protein (Table 8.4). Sulfur plays several key roles in bacterial

cells as it is a part of some amino acids such as cysteine, methionine and some cellular

cofactors such as biotin, coenzyme A, S-adenosylmethionine, thiamine, glutathione,

and iron-sulfur clusters (Scott et al., 2007). Sulfate is the preferred sulphur source for

most of the organisms (Aguilar-Barajas et al., 2011). The concentration of the sulfate

transporter was analyzed at different time intervals and found that increased

amounts were detected during the aerobic stages compared to lower yields in the

anaerobic stages of both AN and TS conditions (see Table C.2 in Appendix C, and

Figure 8.10). However, role of sulphate transporters in PCB hydrolysis was not clear.


Figure 8.10 The concentration of the sulfate transporter protein detected in the

culture supernatant over time, under the alternating anaerobic-aerobic (AN)

conditions.

Lykidis et al. (2010) reported that the Cupriavidus necator, a Gram negative

bacterium that utilizes a variety of chloroaromatic compounds as its sole carbon and

energy source contains several ABC transporters that are able to transport aromatic

compounds. Aroclor 1260 is a synthetic chlorinated aromatic hydrocarbon mixture

used as the sole source of carbon, but there were no specific classically secreted

aromatic and xenobiotic degradation related proteins identified in the culture

supernatant. However, about 10% of uncategorized proteins were grouped together

and labelled as unclassified (Figure 8.9). In addition, about 10% of the classically

secreted proteins were grouped as ‘hypothetical’ proteins with unknown functions.

It is possible that these unclassified and unknown proteins identified here may form

new and novel secretion systems used by bacteria for growth depending on the

medium and substrates (Maffei et al., 2017).

About 10% of membrane type proteins were predicted as classically secreted

proteins (Table C.1 in Appendix C). However, it was not clear whether they are

actually secreted proteins or false positives, as they are usually not considered as

secreted proteins, but remain within the membranes of the bacterium. It has been


suggested that the SecretomeP server would identify them as secreted proteins due

to the presence of cleavable signal peptide sequences at the N-terminus (Song et al.,

2009). However, Maffei et al. (2017) recently revealed that the lipoproteins that are

fully or partially exposed to the surface occur either through known secretion

systems or by novel mechanisms. Additionally, some lipoproteins were identified as

surface-active amphiphilic metabolites or biosurfactants that play a major role in

bioremediation of hydrophobic chemical pollutants such as PCBs (Basak & Dey,

2015). The biosurfactants found during the initial biosufactant screening in Chapter

5 may have some link to the lipoproteins identified as part of the classically secreted

proteins.

The distribution of the 212 identified as classically secreted proteins at fortnightly

intervals were compared under AN and TS anaerobic-aerobic treatment conditions

(Figure 8.11). Out of 211 proteins identified in the AN conditions, 160 proteins were

found common in weeks 2, 4 and 6, with the highest number of proteins (193)

identified at Week 6 (see Figure 8.11A). 209 proteins were identified in the TS

conditions of which 161 proteins were found common in weeks 2, 4 and 6 (see Figure

8.11B). The highest number of proteins (199) with identifications were present at

week 4 under TS conditions. Even though most (140) of the proteins were found

common in all three fortnightly intervals under both AN and TS conditions, they were

present in different concentrations under each condition as indicated in the heat map

in Table C.1, Appendix C. Sulfate ABC transporter substrate-binding protein and

ethanolamine utilization protein were present only in week 6, under AN conditions.

Proteins involved in ethanolamine utilization (EUTs) have been studied and found to

be part of bacterial micro compartments (BMC) whereby BMC functions to control

metabolic reactions by confining volatile/toxic metabolite intermediates such as

alcohols and acids (Axen et al., 2014; Sturms et al., 2015). In the TS treatment,

ferrichrome ABC transporter substrate-binding protein was absent during the first

four weeks of anaerobic treatment and only present under aerobic condition at week

6. Sixteen out of twenty one common proteins found in week 2 and week 4

(anaerobic phase) of TS treatment were found to be transport related proteins.


Figure 8.11 Venn diagram of the distribution of proteins identified as classically

secreted proteins among (A) alternating (AN) anaerobic-aerobic, and (B) two stage

(TS) anaerobic-aerobic conditions.

8.3.2.2.2 Non-classically secreted proteins

Cytoplasmic proteins lacking typical signal peptides or secretion motifs, but found in

the extracellular environment of bacteria are called non-classically secreted proteins

(Zhao et al., 2017). A total of 87 proteins quantified using SWATH were predicted to

be non-classically secreted proteins by the SecretomeP server (Figure 8.8). However,

the mechanisms and cellular membrane locations and features responsible for non-

classical protein secretion are not well understood (Bendtsen et al., 2005).

Similar to the analysis undertaken for classically secreted proteins, non-classically

secreted proteins were further analyzed based on their potential functions and

outcomes are presented in Figure 8.12. Out of the 87 proteins, 17% (15 proteins)

were grouped as hypothetical proteins, indicative of novel or new proteins. Six non-

classically secreted proteins were identified to be involved in motility while five

proteins were related to transporter proteins.

Only one protein matched to an enzyme called Dienelactone hydrolase in relation to

aromatic and xenobiotic degradation. This enzyme plays a crucial role in microbial

degradation of chloroaromatics via the modified chlorocatechol ortho cleavage

pathway (Schlomann et al., 1993) by catalysing the hydrolysis of dienelactone to

maleylacetate (Park et al., 2010).


Figure 8.12 Functional groupings of proteins identified as non-classically secreted

proteins.

The first published study on non-classical protein secretion in bacteria reported the

secretion of an enzyme called glutamine synthetase (GlnA) (Bendtsen et al., 2005).

The enzymes glutamine synthetase and transglutaminase were detected in the

culture supernatants. These two enzymes may have had a direct impact on the

variation of pH in the culture medium. In the AN anaerobic-aerobic conditions, weeks

1, 3 and 5 were maintained under anaerobic conditions while weeks 2, 4 and 6 were

maintained under aerobic conditions. The starting pH of the medium was set at 7.0.

At each time, when the conditions changed to anaerobic, the pH slightly dropped

(Figure 8.13). However, once conditions were shifted from anaerobic to aerobic, the

pH was found to be closer to its original neutral value of 7, especially in weeks 2, 4

and 6 (Figure 8.13). Glutamine synthetase uses ammonia during the metabolism of

nitrogen by catalysing the condensation of glutamate and ammonia to form

glutamine (Takeo et al., 2013). At each anaerobic phase, the concentration of

glutamine synthetase was relatively high when compared to aerobic phase, indicating

more ammonium ion removal from the medium. The increased glutamine synthetase

concentrations may have coincided with the pH drop under anaerobic conditions as

shown in Figure 8.13. In contrast to glutamine synthetase, the enzyme

transglutaminase concentration was relatively high under each aerobic phase when


compared to anaerobic phases (see heat map in Table C.2, Appendix C). The enzyme

transglutaminase is responsible for catalysing the formation of a crosslink between a

free amine group and the γ-carboxamide group of a protein bound glutamine. The

reaction also produces ammonia (Zhang et al., 2009). Therefore, it can be assumed

that the variation of glutamine synthetase and transglutaminase concentrations

under aerobic and anaerobic conditions may have contributed to the variation of pH

levels in the medium (Figure 8.13).

Figure 8.13 Variation of glutamine synthetase concentration and pH level in the

culture supernatant under alternating anaerobic-aerobic conditions.

The distribution of the 87 non-classically secreted proteins at fortnightly intervals

were also compared under AN and TS anaerobic-aerobic treatment conditions (Figure

8.14). Similar to the classically secreted proteins, a high amount of the released

proteins under both AN and TS conditions were shared among all the fortnightly

collected samples (see Figure 8.14A and Figure 8.14B). However, and perhaps not

surprisingly, the concentrations of each protein in the culture supernatant varied at

different time intervals as shown in the Table C.1, Appendix C. When AN and TS

treatments were compared, the number of proteins shared during weeks 2, 4 and 6


was high in the culture supernatant under the TS treatment (63 proteins) compared

to the AN treatment (51 proteins). Under TS conditions, transglutaminase and

glutamine synthase were absent in anaerobic phase (in week 2 and week 4) and only

present in aerobic phase (week 6).

Figure 8.14 Distribution of non-classically secreted proteins under (A) AN anaerobic-

aerobic treatment, and (B) TS anaerobic-aerobic treatment.

Under both AN and TS conditions, a phospholipid-binding protein, dienelactone

hydrolase family protein, histone-like DNA-binding protein and flagella hook-

associated protein FlgK demonstrated relatively high concentrations throughout the

six weeks study period. An elongation factor protein concentration was relatively

high only during the initial two weeks in both, AN and TS conditions. In the TS

treatment, the only protein detected in week 2 was an efflux RND transporter

periplasmic adaptor subunit. Resistance nodulation division (RND) family

transporters belong to the bacterial efflux pumps and are widespread among Gram

negative bacteria (Nikaido & Takatsuka, 2009). They are known to catalyse and

regulate the active transport of substrates such as antibiotics and chemotherapeutic

agents and therefore may have a similar role during cell growth on PCBs.

8.4 Conclusions

So far, various studies have been conducted to identify the PCB degradation

pathways and the relevant enzymes responsible by analysing the bacterial


proteomes. However, no information is available on the types and potential role of

the enzymes secreted to the extracellular environment during microbial

bioremediation of pollutants such as PCBs. The proteomic approach to investigate

the exoproteome of the bacterial consortium grown on PCBs allowed a detailed study

on the composition and concentration of the proteins extracted from the culture

supernatant, under the AN and TS anaerobic-aerobic cultivation conditions.

During protein visualization using SDS-PAGE, it was revealed that proteins were

detected externally under both anaerobic and aerobic conditions, by the individual

facultative anaerobic bacterial culture members Achromobacter sp. NP03,

Ochrobactrum sp NP04 and Lysinibacillus sp. NP05 while growing on Aroclor 1260 as

the sole source of carbon and energy. These protein results suggested that these

bacteria were capable of secreting proteins or enzymes to the external environment

containing PCBs. In general, protein levels in the extracellular environment under

aerobic conditions were found to be lower than under anaerobic conditions.

Lysinibacillus sp. NP05 under anaerobic conditions demonstrated the highest number

of protein bands among all the samples, although the concentrations were different

for each week. In contrast and in the experiments using the consortium, the second

week of the alternating mode showed relatively high number of protein bands when

compared to the other samples, and this result correlated with the high PCB

degradation rate (49.2±2.5%) observed at week two under the alternating treatment

conditions (Figure 7.3 in Chapter 7).

Analysis of the exoproteome from the bacterial consortium reaction with Aroclor

1260 as the sole source of carbon resulted in the identification of 319 non-secreted,

212 classically secreted and 87 non-classically secreted proteins in the culture

supernatant. These proteins consisted of a heterogeneous group representative of

their diverse functional and cellular roles. Although the contribution of a high number

of cytoplasmic proteins in the extracellular environment is not clear, potentially

resulting from cell lysis or from selective straining, the presence of some proteins

involved in the intermediate steps of the PCB degradation pathways and

detoxification of toxic chemicals is a direct indication for the occurrence of PCB


degradation by the bacterial consortium. Transporters were the largest protein group

that represented 58% of the total classically secreted proteins. However, the

mechanisms for protein or enzyme release, adaptations for cell protection from toxic

pollutants and their specific functions in PCB degradation as an extracellular protein

or enzyme remains to be further investigated.

Chapter 9: Conclusions, practical applications and recommendations for future research 197

Chapter 9: Conclusions, practical applications and recommendations for future research

9.1 Conclusions

Polychlorinated biphenyls are regarded as legacy pollutants. Even though commercial

production and use ceased in 1993, extremely stable properties have made PCBs

persistent in the environment, leading to a range of problems with ecosystem and

human health. There is a vital need to search for sustainable treatment approaches

to remediate PCB contaminated soil due to the serious shortcomings of current

physical and chemical treatment methods. Development of sustainable and effective

bioremediation techniques using microorganisms have the potential to fulfil this

need.

The effectiveness of microbial remediation of PCB contaminated soil is determined

by number of factors. The degradation rate is mainly dependent on the

environmental conditions, soil characteristics, complexity and severity of PCB

contamination, ability of native or bioaugmented microorganisms to survive and to

degrade the PCB congeners and their metabolic intermediates. As PCBs are usually

manufactured and applied as complex congener mixtures, treatment of

contaminated environments is also a complex process. As a result, bioremediation

approaches generally need a longer time span due to the slow degradation rates.

Therefore, improvement and optimization of existing bioremediation technologies

are essential in order to produce viable and cost effective treatment approaches.

The present research study focused on addressing the knowledge gaps in PCB

bioremediation that have been identified in the research literature. The study was

based on the primary hypotheses that complete degradation of complex

commercially available PCB mixtures can be achieved through a combination of

anaerobic-aerobic treatment when appropriate microbial mixtures which are capable

198 Chapter 9: Conclusions, practical applications and recommendations for future research

of increasing the aqueous solubility of PCBs and degrading PCB congeners under both

anaerobic and aerobic conditions are incorporated.

Accordingly, potential PCB degrading microorganisms were isolated from the natural

environment and their PCB degradation potentials were determined as individuals

and as a consortium under different environmental conditions. The ability of bacteria

to make hydrophobic PCBs soluble in aqueous media and to facilitate the uptake of

PCBs into the cells by releasing extracellular proteins and/or secreted enzymes were

also tested. The findings presented in this thesis details the new knowledge created

to bridge a number of current knowledge gaps in the microbial based bioremediation

of PCBs.

The schematic representation of the summary of major research findings described

in Chapters 4-8, is given in Figure 9.1 and the main conclusions derived from the study

are discussed below in Sections 9.1.1 to 9.1.5.

According to Figure 9.1, PCBs were added into the minimal salts based bacterial

growth medium as the sole source of carbon. Biosurfactants that were released and

detected in the extracellular environment showed a direct impact on increasing the

solubility of hydrophobic PCBs in the aqueous medium by the PCB degrading

microorganisms, as discussed in detailed in Chapter 5. The major proteins that were

found to be related to PCB degradation and detoxification and discussed in Chapter

8 are only shown in the Figure 9.1. The lipoproteins detected in the culture

supernatant may have increased PCB solubilization together with the biosurfactants.

According to the existing literature (see chapters 8.1), PCB degradation is believed to

be an intracellular process and therefore the enzymes responsible for anaerobic

degradation and aerobic oxidation pathways shown inside the cell in the Figure 9.1

would not be expected in the extracellular environment. However, some of the

previously identified enzymes such as dienelactone hydrolase and 3-oxoadipate co A

transferase that are responsible for some of the intermediate steps in PCB

degradation pathways were found outside in the culture supernatant as highlighted

in Figure 9.1 (also see Sections 8.3.2.1 and 8.3.2.2). Relatively high levels of different

oxidoreductases were also found in the culture supernatant as both secretory and


non-secretory proteins, which are usually engaged in detoxification of toxic

hydrocarbons.

It is postulated that bacteria capable of utilizing PCBs as their growth substrate need

some mechanism to control the intracellular PCB concentration at sufficient level to

permit cell growth while maintaining and controlling the intracellular toxic levels.

Therefore, as shown in Figure 9.1, the presence of some classically secreted

glutathione related transporter proteins in the culture supernatant might have a key

role in maintaining the internal PCB concentration at sub-toxic levels as one of the

main functions of glutathione is protection of cells through the regulation of internal

xenobiotic concentrations by conjugating and exporting them from the cells. Nearly

half of the secretory proteins were related to cellular transport (see Figures 8.9 and

9.1) and they were found to be involved in various cellular functions such as uptake

of nutrients, regulation of cytoplasmic metabolites concentration, and prevention of

the effects of toxins by catalyzing their active efflux.


Figure 9.1 A schematic representation of the summary of major findings of the research study.


9.1.1 Isolation, screening and identification of potential PCB degrading

microorganisms

As discussed in Chapter 4, the selective enrichment screening was done to isolate and

identify the potential PCB degrading microorganisms from the natural environment.

The main conclusions derived are as follows:

• From 11 microorganisms initially tested, two obligate aerobic and four

facultative anaerobic bacterial strains that can utilize commercial PCB mixture,

Aroclor 1260 as their sole source of carbon were isolated from the natural

environment.

• They were identified as Chryseobacterium sp. NP01, Delftia sp. NP02,


Pseudomonas sp. NP06 using 16S rRNA gene sequence based molecular

identification.

• This finding is also the first evidence of reporting the PCB degradation ability of

Chryseobacterium and Delftia species.

9.1.2 Screening of bacterial isolates for their ability to produce biosurfactants to

make hydrophobic PCBs soluble in aqueous media

As discussed in Chapter 5, six bacterial cultures isolated during Chapter 4 were further

tested for their ability to produce biosurfactants as one of the limitations in biological

breakdown is poor bioavailability due to the extreme hydrophobic nature of PCBs. The

main conclusions from chapter 5 are as follows:

• Microorganisms capable of degrading PCBs discovered in this study, were also

found to have the potential to produce some surface active substances to facilitate

the enhancement of solubility of the hydrophobic PCBs.

• The PCB solubility results was positively correlated with the biosurfactant

production.


• Chryseobacterium sp. NP01 and Lysinibacillus sp. NP05 demonstrated the highest

biosurfactant production, PCB solubility and chloride ion accumulation when

compared to Delftia sp. NP02, Achromobacter sp. NP03, Ochrobactrum sp. NP04

and Pseudomonas sp. NP06

9.1.3 Comparison of the individual facultative anaerobic bacterial isolates during

PCB hydrolysis under aerobic, anaerobic and two stage anaerobic-aerobic

conditions

PCB degradation efficiency of four facultative anaerobic culture members, namely,


Pseudomonas sp. NP06 were compared under aerobic, anaerobic and combined

anaerobic-aerobic conditions for their PCB degradation potential as discussed in Chapter

6. The significant conclusions of the Chapter 6 are as follows:

• Based on the review of publicly available literature, this is the first comparative

study assessing the capability of facultative anaerobic bacteria for degrading PCBs

under anaerobic, aerobic and two-stage anaerobic-aerobic cultivation conditions.

• All four facultative anaerobic bacterial strains were found to be capable of

degrading the commercial PCB mixture, Aroclor 1260 as a sole source of carbon

under both, anaerobic and aerobic conditions at varying degrees.

• All four stains showed limitations in growth under aerobic conditions. This is

attributed to the low number of lower chlorinated congeners available in the PCB

mixture. In contrast, the growth increased under anaerobic conditions, suggesting

the microbes were capable in attacking the biphenyl ring and used PCBs as their

carbon source.

• The highest chloride ion accumulation from all the four facultative anaerobic

cultures was observed under two stage anaerobic-aerobic conditions, suggesting

the dechlorination of highly chlorinated congeners under anaerobic conditions

occurred first, followed by the degradation of the resulting lower chlorinated

congeners under aerobic conditions.


• A maximum chloride yield of 9.16±0.8 mg/L was observed for Lysinibacillus sp.

NP05 under the two stage anaerobic-aerobic treatment. This value corresponded

to one third of the total chlorine present in the Aroclor 1260 mixture indicating

33% total chlorine removal.

• In field scale soil remediation applications, facultative microorganisms have the

potential as better candidates as they can survive and degrade PCBs under both,

anaerobic and aerobic conditions while achieving high PCB degradation rates.

• Out of the four facultative anaerobic microorganisms, Lysinibacillus sp. NP05 was

found to have high potential as a single candidate to effectively dechlorinate the

highly chlorinated complex PCB mixture, Aroclor 1260.

9.1.4 Comparison of the bacterial consortium during PCB hydrolysis under two

modes of combined anaerobic-aerobic treatments

According to previous studies, when the two stage (or combined) anaerobic-aerobic

treatment was employed to treat the PCBs, a relatively long anaerobic phase (from 70 -

180 days) followed by a short aerobic phase (from 28-60 days) is the usual approach used

as anaerobic degradation is believed to be a slow process. In addition to that, anaerobic

PCB dechlorinating bacteria have been used under the anaerobic phase while only

aerobic PCB degrading bacteria were used under the aerobic phase. In Chapter 7, a

conventional two stage anaerobic-aerobic treatment (TS) with lengthy anaerobic phase

(of four weeks) followed by short aerobic phase (of two weeks) was compared to the

alternating anaerobic-aerobic-aerobic treatment (AN) having equally short anaerobic

and aerobic phases (one week each). Based on the results obtained in Chapters 5 and 6,

the bacterial consortium was developed consisting of Achromobacter sp. NP03,

Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05 and used in the comparison of TS and

AN anaerobic-aerobic treatments. The major conclusions derived from Chapter 7 are

summarized below:

• Based on the review of publicly available literature, this is the first comparative

study assessing the PCB degradation efficiency under two different combined

anaerobic-aerobic modes; alternating (AN) and two stage (TS), using a consortium


of three facultative anaerobic bacteria Achromobacter sp. NP03, Ochrobactrum sp.

NP04 and Lysinibacillus sp. NP05 that were found capable of degrading PCBs under

both, anaerobic and aerobic conditions.

• In both treatments, lower chlorinated PCB homolog groups exhibited greater

degradation capability under aerobic conditions when compared to highly

chlorinated homolog groups, although the degradation rates were always high

under alternating conditions.

• Even after the PCB degradation reached a plateau at week two, continuous

increases in bacterial cell density and chloride ion concentration in the culture

medium under alternating conditions highlighted the ability of the consortium

member/s to further breaking down the intermediate products.

• The alternating treatment (AN) was found to be the preferred method over the

two stage (TS) treatment with nearly 50% reduction in total PCBs within the first

two weeks when compared to 24% reduction achieved under the two stage

treatment.

• The switching between short anaerobic and aerobic phases at weekly intervals

under alterating conditions favoured the consortium to change in between

dechlorination and oxidation. This alternating switching between two phases

facilitated the overall PCB degradation rate due to increased PCB degradation

under aerobic phases through ring cleavage and hydrolysis of lower chlorinated

PCB congeners that were generated during the initial anaerobic phases.

9.1.5 Analysing and characterization of proteins detected in the culture

supernatants during PCB degradation

Various studies have been conducted to-date to identify the PCB degradation

pathways and the relevant enzymes responsible by analysing the bacterial

proteomes. However, knowledge is limited about the types and roles of proteins

and/or enzymes secreted into the extracellular environment during PCB degradation.

Therefore, research discussed in Chapter 8 aimed to investigate the composition,


concentration and characteristics of extracellular proteins released by three PCB

degrading facultative anaerobic bacteria as a consortium under alternating and two

stage anaerobic-aerobic-aerobic experimental conditions and to relate them to PCB

degradation data. The important conclusions derived from the extracellular protein

analysis are given below:

• During protein visualization, it was found that under both anaerobic and

aerobic conditions, three facultative anaerobic bacterial culture members

Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05

were individually capable of secreting proteins into the liquid medium at

varying levels while Lysinibacillus sp. NP05 under anaerobic conditions

demonstrated the highest number of protein bands.

• From a metaproteomics approach, a total of 618 proteins consisted of a

heterogeneous group, representative of their diverse functional and cellular

roles including 319 non-secreted, 212 classically secreted and 87 non-

classically secreted proteins were identified in the culture supernatant of the

bacterial consortium.

• Transport related proteins represented 58% of the total classically secreted

proteins. This is a significant finding because bacterial transport proteins play

an important role in facilitating the uptake of essential nutrients, regulate the

cytoplasmic concentrations of metabolites, export large molecules to the

outer surface of the cell and prevent toxic effects of toxins by catalyzing their

active efflux.

• The presence of large number of non-secretory proteins (n=319) in the

extracellular environment was not clear whether it was from cell lysis or from

any other cellular mechanism such as selective straining.

• The occurrence and identification of some proteins involved in the

intermediate steps of the known PCB degradation pathways, regulation of

internal xenobiotic concentration by conjugating and exporting excess

xenobiotic from the cells, detoxification of toxic chemicals and allows

microorganisms to use aromatic hydrocarbons as their sole source of carbon


and energy was a direct indication of the occurrence of PCB degradation by

the bacterial consortium.

9.2 Practical applications of research outcomes

The objective of the research study was to identify suitable microorganisms from the

natural environment that are capable of solubilizing and degrading complex PCB

mixtures under varying anaerobic and aerobic conditions to effectively treat the

contaminated soil. Although many studies have been conducted to assess the possibility

of using bioremediation as a biological treatment option to treat the soils contaminated

with PCBs, bioremediation is still not widely practiced as a field scale remediation option

due to various challenges.

In biological degradation of PCBs, two main processes are fundamentally involved,

namely, anaerobic dechlorination and aerobic oxidation. Highly chlorinated PCB

congeners first need to be dechlorinated into lower chlorinated congeners. Under

anaerobic conditions, dechlorinating bacteria are usually involved in this step. The next

step is partial or complete degradation of lower chlorinated congeners, which is an

oxidative process and aerobic PCB degrading bacteria are responsible for the conversion.

The studies undertaken in the past have mainly reported on the use of two separate

groups of microorganisms when both anaerobic dechlorination and aerobic oxidation

steps are involved in combination. These microorganisms and process conditions are

referred to as, anaerobic dechlorinating microorganisms under anaerobic conditions and

aerobic degradative microorganisms under aerobic conditions. However, both,

anaerobic and aerobic conditions are present in the natural soil environment and

changes in the environmental conditions can lead to death of PCB degrading

microorganisms introduced to the soil if they cannot survive under varying oxygen levels.

During the present study, it was found that there are some facultative anaerobic

microorganisms in the natural environment that can survive and carryout PCB

degradation successfully under both anaerobic and aerobic conditions simultaneously.

This novel finding can be incorporated into bioremediation based soil decontamination

applications.


The existing studies on the combined anaerobic and aerobic treatments usually use a

prolonged initial anaerobic phase followed by short aerobic phase assuming that the

anaerobic dechlorination is a slow process, and therefore requires a long period ranging

from 70 to 180 days to achieve the expected outcome. However, in the present study,

application of equally short alternating anaerobic and aerobic phases demonstrated

higher PCB degradation rates within two weeks compared to the conventional two stage

treatments having lengthy anaerobic phase typically in months, followed by a short

aerobic phase. This research outcome will create new opportunities to overcome one of

the major limitations in bioremediation by reducing the extended time required for

conventional bioremediation applications.

Extreme hydrophobic nature of PCB mixtures is another rate limiting factor that hinders

the effective biodegradation as it reduces the bioavailability of PCBs to the potential

degrading bacterial community. Research has been undertaken to improve the aqueous

solubility of PCBs by adding chemical surfactants and biosurfactants to the soil. However,

such applications were limited due to the toxicity of chemical surfactants to the PCB

degrading and other native soil microbial community and to the high cost associated with

commercially available biosurfactants. In the present study, some of the

microorganisms, which were identified as having PCB degradation potential, were also

found to be capable of making the hydrophobic PCBs soluble in aqueous medium by

producing biosurfactants. This is an added advantage in the field of bioremediation as

the appropriate microbial mixture having both degradation and solubilization capabilities

can replace the addition of toxic chemicals or biological surfactants externally.

In summary, the outcomes from this research study can be incorporated in future

bioremediation applications to effectively treat soils contaminated with PCBs and other

similar toxic xenobiotic compounds.

9.3 Recommendations for future research

The outcomes of this research study have contributed new knowledge on

bioremediation of contaminated environments by PCBs. In order to further consolidate

the outcomes of this study, the following additional research studies are recommended.


Based on the review of publicly available literature, the present study is the first to

identify the facultative anaerobic microorganisms with PCB degradation ability under

both anaerobic and aerobic conditions. However, total PCB degradation was

determined using PCB homolog group concentration and chloride ion accumulation.

Therefore, in future studies, incorporation of the analysis of PCB degradation at

individual congener level is recommended to understand the potential pathways

used by microorganisms during PCB degradation.

In the present study, a commercial PCB mixture, Aroclor 1260 was used as the sole source

of carbon for PCB degrading microorganisms. According to the literature, during the

anaerobic PCB dechlorination, the biphenyl ring is usually not cleaved unless the

microorganisms are capable of utilizing PCBs as their carbon source. Therefore, providing

a suitable carbon source is essential for the microorganisms for their survival while

carrying out PCB dechlorination. Even under aerobic conditions, addition of a desirable

carbon source was shown to enhance the mineralization of PCBs through co-metabolism.

Therefore, providing a suitable secondary carbon source, which can be utilized by all the

members in the bacterial consortium, could enhance the PCB degradation rate. During

the screening for potential secondary carbon sources, L-proline, L-lactic acid and methyl

pyruvate were identified as sources that can be utilized by all three consortium

members. In addition, L-proline was found to be utilized by all the bacterial strains used

in the consortium as their nitrogen source. Therefore, it needs to be further confirmed

by additional research to see whether the addition of secondary carbon sources will

either stimulate or inhibit PCB degradation and their capability in field scale remediation

applications.

The reason for the increased solubility of hydrophobic PCB mixture in the aqueous

medium over time was found to be due to the biosurfactants produced by the PCB

degrading bacterial strains. However, as this study was an initial screening to see the

potential of isolated PCB degrading microorganisms for biosurfactant production, further

research is needed to identify the types and physico-chemical properties of the

biosurfactants through extraction, quantification and characterization and to assess their

performance in PCB contaminated soil.


The study found that the alternating approach was more efficient compared to the two

stage treatment suggesting the switching between short anaerobic and aerobic phases

of weekly intervals favoured the consortium to changing between dechlorination and

oxidation. However, the sampling frequency in this study under both treatment

conditions was limited to fortnightly intervals and PCB degradation analysis were limited

to total PCB degradation based on the PCB homolog concentration. Therefore, it is

recommended to closely monitor the behaviour of PCB degradation and product build

up under each anaerobic and aerobic phase at daily intervals to get detailed information

to further confirm the scientific principles behind the alternating approach.

Generally, a project of this nature has to be undertaken in the laboratory first and

then transferred to the field. As the present study was limited to liquid based

experiments, it is highly recommended to study the performance of bacterial strains on

PCB degradation and variations in composition profile of the bacterial consortium in PCB

contaminated soil under alternating anaerobic-aerobic conditions.

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Appendices 245

Appendices

246 Appendix A: PCB analysis

Appendix A: PCB analysis

Appendix A: PCB analysis 247

Table A. 1 Selective reaction monitoring (SRM) transitions

Name Retention

Time Ion

Polarity Window

Pre-width

Post-width

Mass Product

Mass Collision Energy

mono 10 Positive 1 7 13 188 152 22

mono 10 Positive 1 7 13 190 152 22

di 10 Positive 1 7 13 222 152 22

di 10 Positive 1 7 13 224 152 22

tri 10 Positive 1 7 13 256 186 22

tri 10 Positive 1 7 13 258 186 22

tetra 10 Positive 1 7 13 289.9 219.9 22

tetra 10 Positive 1 7 13 291.9 222 22

penta 10 Positive 1 7 13 323.9 253.9 22

penta 10 Positive 1 7 13 325.9 255.9 22

hexa 10 Positive 1 7 13 357.9 287.9 22

hexa 10 Positive 1 7 13 359.8 289.9 22

hepta 10 Positive 1 7 13 391.9 321.9 22

hepta 10 Positive 1 7 13 393.8 323.9 22

octa 10 Positive 1 7 13 427.8 355.8 22

octa 10 Positive 1 7 13 429.8 357.8 22

nona 10 Positive 1 7 13 461.7 391.8 22

nona 10 Positive 1 7 13 463.8 393.8 22

deca 10 Positive 1 7 13 495.7 425.7 22

deca 10 Positive 1 7 13 497.7 427.7 22

2456 tetrachloro-m-xylene 12.53 Positive 2 1 1 242 207 15



224455-hexabromobiphenyl 20.74 Positive 2 1 1 468 306 30




Table A. 2 Retention times used for homolog groups and standards

Retention Time (min) Compound

11.50-12.70 Mono

12.53 2,4,5,6-tetrachloro-m-xylene

12.50-13.50 Di

13.40-14.95 Tri

14.60-15.81 Tetra

15.00-17.00 Penta

16.45-18.20 Hexa

17.20-18.75 Hepta

17.90-19.55 Octa

18.70-20.00 Nona

20.20 Deca

20.74 2,2′,4,4′,5,5′-hexabromo biphenyl

Appendix B: PCB data related to bacterial consortium study 249

Table A. 3 Aroclor 1260 calibration curve preparation from 50 mg/L stock solution

Notes: * Internal standard 2,2′,4,4′,5,5′-hexabromo biphenyl

**Surrogate – 2,4,5,6-tetrachloro-m-xylene

Aroclor 1260 V2 (µL) Final concentration of surrogate in

calibration levels (ppb)

Ratio M1/M2

M1 (ppb)

V1 (µL)

M2 (ppb) V2 (µL) Solvent

(Hexane)

Internal standard*

(10000 ppb)

Surrogate**

(1000 ppb)

1.0 50000 200 10000 500 395 5 250 250

0.5 10000 500 5000 500 245 5 0 125

0.25 5000 400 2000 500 295 5 0 50

0.125 2000 250 1000 500 245 5 0 25

0.05 2000 125 500 500 340 5 0 12.5

0.5 2000 62.5 250 500 432.5 5 0 6.25

0.2 2000 25 100 500 470 5 0 2.5

0.1 100 250 50 500 245 5 0 1.25

0.05 100 100 20 500 395 5 0 0.5

100 50 10 500 445 5 0 0.25


Table A. 4 Peak area values obtained for each homolog group at different Aroclor 1260 standard concentrations

Homolog

group

Peak area

5 ppb 10 ppb 20 ppb 50 ppb 250 ppb 500 ppb 1000 ppb 2000 ppb 10000 ppb

mono 1889.348 1707.92589 2468.0219 3465.0801 15574.9853 30401.777 76422.231 129119.086 750636.22

di 550.9803 1188.7858 5080.5965 8521.2593 56355.3414 117940.77 270306.73 498293.522 2950682.9

tri 4497.273 4884.74261 11812.728 24380.686 122937.635 251359.76 594197.13 1087982.18 6107066.8

tetra 2845.426 3695.28486 7324.9622 17024.727 124256.795 257673.05 627861.52 1121852.91 6265611.3

penta 30426.95 45411.7234 105299.96 240614.42 1319259.75 2751272.7 6568636.5 11776736.5 67231764

hexa 67817.83 109574.906 259315.13 595372.54 3350325.52 6928845.8 16565257 30207732.6 173350129

hepta 30819.58 49218.3871 115007.26 268515.69 1546399.88 3202036.2 7659288.8 13826669.3 80016143

octa 4640.051 8502.89597 15613.646 26168.11 176444.942 354261.01 852198.37 1538777.25 8835023.5

nona 730.4066 1319.00774 2175.074 5331.9759 41249.352 79538.784 192950.88 350151.504 2011603.3

deca 12.63421 385.909366 250.08227 437.05678 3016.55984 5866.5805 13860.713 28517.889 160508.93

Total 144230.5 225889.569 524347.46 1189831.6 6755820.76 13979196 33420980 60565832.8 347679170


Appendix B: PCB data related to bacterial

consortium study

252 Appendix B: PCB data related to bacterial consortium study

Table B. 1 Homolog and total PCB composition in the Two stage anaerobic-aerobic treatment

Concentration

(µg/L)

Uninoculated

control STDV Week 0 STDV Week 2 STDV Week 4 STDV Week 6 STDV

mono 25.75 1.74 22.09 1.27 83.04 5.83 354.37 30.29 0.90 0.50

di 217.32 19.06 209.73 20.69 314.97 7.99 1550.56 110.93 33.35 3.60

tri 593.56 26.00 591.56 12.17 596.27 38.47 942.59 83.67 164.46 16.30

tetra 790.20 30.73 728.15 103.91 725.93 25.73 831.86 42.64 236.22 86.16

penta 7974.02 238.11 8285.12 675.16 5621.28 406.15 4130.70 307.46 3658.28 384.09

hexa 19728.43 1125.69 18060.61 338.91 14691.42 313.85 11424.79 356.11 10576.86 910.62

hepta 7777.20 743.16 7283.13 292.32 6035.75 144.05 4958.36 157.50 4545.18 221.73

octa 926.88 86.36 798.70 69.01 689.67 10.16 562.77 15.06 536.66 48.25

nona 202.99 21.01 189.99 9.78 153.26 4.36 127.29 1.59 124.55 9.46

deca 18.51 1.78 15.91 0.75 13.05 0.83 10.57 0.75 9.29 0.74

Total PCB 38232.24 1934.50 36184.98 85.69 28924.63 944.11 24893.86 863.58 19885.74 1452.83


Table B. 2 Homolog and total PCB concentrations in the alternating anaerobic-aerobic treatment

Concentration

(µg/L)

uninoculated

control STDV Week 0 STDV Week 2 STDV Week 4 STDV Week 6 STDV

mono 25.755 1.735 22.085 1.268 1.380 0.718 0.725 0.066 0.336 0.013

di 217.322 19.063 209.733 20.693 15.404 1.676 10.060 0.299 9.542 1.197

tri 593.557 25.997 591.555 12.168 125.624 19.200 120.412 10.854 95.087 2.713

tetra 790.205 30.734 728.152 103.905 280.507 37.452 288.415 7.861 278.337 4.132

penta 7974.023 238.110 8285.116 675.162 3485.743 181.287 3653.442 50.885 3233.831 226.179

hexa 19728.433 1125.692 18060.611 338.912 10462.199 549.223 10309.500 358.778 9527.195 372.236

hepta 7777.203 743.160 7283.129 292.323 4450.881 218.022 4312.760 158.724 3875.390 24.000

octa 926.876 86.362 798.696 69.013 481.680 27.793 500.159 16.609 418.755 16.982

nona 202.990 21.005 189.989 9.779 108.509 5.285 109.904 0.671 107.336 3.326

deca 18.509 1.784 15.914 0.751 8.925 1.391 8.542 1.284 9.568 0.881

Total PCB 38232.240 1934.498 36184.980 85.689 19420.851 957.890 19313.918 564.092 17555.378 188.643

254 Appendix B: PCB data related to bacterial consortium study

Appendix C: Extracellular protein analysis 255

Appendix C: Extracellular protein analysis

256 Appendix C: Extracellular protein analysis

Table C.1 Relative abundance of extracellular proteins of bacterial consortium Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05 found in the culture supernatant under alternating (AN) and two stage (TS) anaerobic-aerobic conditions as a heat map.

No. Accession number Protein

AN (Weeks)

TS (Weeks) Secreted*

2 4 6 2 4 6

Transport related

1 WP_054452161.1 ABC transporter Yes (28-29)


3 WP_088148777.1 ABC transporter permease Yes (22-23)






9 KNY13962.1 ABC transporter permease Yes (20-21)

10 WP_088447256.1 ABC transporter substrate-binding protein Yes (28-29)




14 ASC66145.1 ABC transporter substrate-binding protein Yes (22-23)





19 OWT57704.1 ABC transporter substrate-binding protein Yes (35-36)


21 OAD17456.1 ABC transporter substrate-binding protein Yes (26-27)



AN

(Weeks)

TS

(Weeks) Secreted*

2 4 6 2 4 6


23 OCZ60996.1 ABC transporter substrate-binding protein Yes (26-27)

24 OAE68865.1 ABC transporter substrate-binding protein Yes (26-27)



27 KGD96204.1 ABC transporter substrate-binding protein Yes (25-26)



30 OLT99158.1 ABC transporter substrate-binding protein Yes (21-22)


32 OLU02054.1 ABC transporter substrate-binding protein Yes (24-25)




36 OWG18014.1 ABC transporter substrate-binding protein Yes (25-26)



39 KXO76717.1 ABC transporter substrate-binding protein Yes (27-28)

40 OOC59559.1 ABC transporter substrate-binding protein Yes (37-38)

41 KXO75178.1 amino acid ABC transporter Yes (21-22)

42 WP_088589881.1 amino acid ABC transporter substrate-binding protein Yes (22-23)



45 KGD90086.1 amino acid ABC transporter substrate-binding protein Yes (22-23)

46 KGE00355.1 amino acid ABC transporter substrate-binding protein Yes (26-27)



AN

(Weeks)

TS

(Weeks) Secreted*

2

4

6

2

4

6


48 KNE29344.1 amino acid ABC transporter substrate-binding protein Yes (31-32)

49 OLU07976.1 amino acid ABC transporter substrate-binding protein Yes (22-23)




53 WP_088146425.1 branched-chain amino acid ABC transporter substrate-binding protein Yes (23-24)


55 AKP90359.1 Branched-chain amino acid ABC transporter, amino acid-binding protein Yes (28-29)


57 KGY25861.1 C4-dicarboxylate ABC transporter Yes (24-25)

58 WP_056567163.1 C4-dicarboxylate ABC transporter substrate-binding protein Yes (23-24)



61 WP_088148288.1 carbohydrate ABC transporter substrate-binding protein Yes (22-23)

62 EEQ96006.1 extracellular solute-binding protein Yes (27-28)

63 EHK65400.1 extracellular solute-binding family protein Yes (22-23)

64 ABS16514.1 extracellular solute-binding protein family 3 Yes (23-24)

65 WP_048394203.1 ferrichrome ABC transporter substrate-binding protein Yes (27-28)

66 KGD98287.1 glutamine ABC transporter substrate-bindnig protein Yes (26-27)

67 OLU03919.1 glutamine ABC transporter substrate-binding protein Yes (25-26)

68 KNE26968.1 glutathione ABC transporter substrate-binding protein Yes (28-29)

69 CUJ34547.1 Glutathione-binding protein gsiB precursor Yes (28-29)

70 ADP14939.1 glutathione-binding protein GsiB Yes (28-29)



AN

(Weeks)

TS

(Weeks) Secreted*

2 4 6 2 4 6

71 WP_057284748.1 glycine/betaine ABC transporter substrate-binding protein Yes (27-28)

72 WP_063952087.1 hemin ABC transporter substrate-binding protein Yes (18-19)

73 WP_088146328.1 iron ABC transporter substrate-binding protein Yes (29-30)


75 KGD93619.1 iron ABC transporter substrate-binding protein Yes (24-25)

76 OLU06668.1 iron ABC transporter substrate-binding protein Yes (28-29)

77 KMN36448.1 iron ABC transporter substrate-binding protein Yes (33-34)

78 KXO74055.1 iron ABC transporter substrate-binding protein Yes (23-24)


80 OCX15298.1 iron ABC transporter substrate-binding protein Yes (22-23)

81 CUJ13421.1 leucine ABC transporter subunit substrate-binding protein Yes (24-25)

82 KGD96135.1 membrane protein Yes (22-23)

83 KOQ40547.1 membrane protein Yes (22-23)

84 WP_008160825.1 metal ABC transporter substrate-binding protein Yes (29-30)

85 WP_088153545.1 molybdate ABC transporter substrate-binding protein Yes (24-25)

86 WP_043212569.1 MULTISPECIES: ABC transporter permease Yes (24-25)

87 WP_062685045.1 MULTISPECIES: ABC transporter substrate-binding protein Yes (30-31)


89 WP_062683233.1 MULTISPECIES: amino acid ABC transporter substrate-binding protein Yes (23-24)




93 WP_056317574.1 MULTISPECIES: branched chain amino acid ABC transporter substrate-binding protein Yes (25-26)

94 WP_088595533.1 MULTISPECIES: glutamine ABC transporter substrate-binding protein GlnH Yes (26-27)

95 WP_062684476.1 MULTISPECIES: hemin ABC transporter substrate-binding protein Yes (19-20)



AN

(Weeks)

TS

(Weeks) Secreted*

2

4

6

2

4

6

96 WP_062682236.1 MULTISPECIES: leucine ABC transporter subunit substrate-binding protein LivK Yes (25-26)

97 WP_034397436.1 MULTISPECIES: outer membrane protein assembly factor BamE Yes (22-23)

98 WP_082601759.1 peptide ABC transporter Yes (31-32)

99 ASC64693.1 peptide ABC transporter substrate-binding protein Yes (28-29)

100 WP_056569658.1 peptide ABC transporter substrate-binding protein Yes (27-28)

101 KGD99111.1 peptide ABC transporter substrate-binding protein Yes (28-29)


103 WP_088145960.1 phosphate ABC transporter substrate-binding protein PstS Yes (24-25)

104 WP_054423127.1 phosphate/phosphite/phosphonate ABC transporter substrate-binding protein Yes (24-25)

105 WP_043544234.1 polyamine ABC transporter substrate-binding protein Yes (25-26)

106 WP_049054386.1 sugar ABC transporter substrate-binding protein Yes (30-31)

107 KMN40622.1 sugar ABC transporter substrate-binding protein Yes (28-29)

108 WP_061346078.1 sulfate ABC transporter substrate-binding protein Yes (21-22)

109 OLU07503.1 sulfate transporter subunit Yes (27-28)

110 WP_078339521.1 thiamine ABC transporter substrate binding subunit Yes (23-24)

111 WP_088147934.1 transporter Yes (21-22)

112 KGD94171.1 transporter Yes (24-25)

113 AOU92447.1 tripartite tricarboxylate transporter Yes (31-32)

114 WP_008160088.1 tripartite tricarboxylate transporter substrate binding protein Yes (29-30)








AN

(Weeks)

TS

(Weeks) Secreted*

2 4 6 2 4 6



122 OMG92355.1 urea ABC transporter substrate-binding protein Yes (27-28)

123 OLU07484.1 ABC transporter substrate-binding protein Yes#

124 WP_043547903.1 C4-dicarboxylate ABC transporter Yes#

125 WP_076815687.1 electron transporter Yes#

126 CUJ75845.1 Membrane protein involved in colicin uptake Yes#

127 WP_063959745.1 MULTISPECIES: efflux RND transporter periplasmic adaptor subunit Yes#

Membrane related

128 KGD89451.1 lipoprotein Yes (17-18)

129 WP_043542536.1 lipoprotein, YaeC family Yes (25-26)




133 WP_043542943.1 membrane protein Yes (20-21)


135 WP_062681332.1 MULTISPECIES: outer membrane protein assembly factor BamC Yes (18-19)

136 WP_062681256.1 MULTISPECIES: peptidoglycan-associated lipoprotein Yes (22-23)

137 ADP14169.1 NLPA lipoprotein family protein 1 Yes (25-26)

138 WP_088146536.1 peptidoglycan-binding protein Yes (24-25)

139 SIT23972.1 periplasmic chaperone for outer membrane proteins SurA Yes (29-30)

140 CUJ96470.1 Probable phospholipid-binding protein mlaC precursor Yes (28-29)

141 EZP50560.1 putative lipoprotein Yes(23-24)

142 WP_043547462.1 TonB-dependent siderophore receptor Yes (21-22)




AN

(Weeks)

TS

(Weeks) Secreted*

2

4

6

2

4

6

144 WP_088141626.1 TonB-dependent receptor Yes (22-23)



147 WP_043548035.1 carboxypeptidase regulatory-like domain-containing protein [ Yes (22-23)

148 WP_079242778.1 SusC/RagA family TonB-linked outer membrane protein Yes (22-23)


150 WP_054481236.1 MULTISPECIES: phospholipid-binding protein Yes#

151 OWT75564.1 phospholipid-binding protein Yes#

152 KGD93668.1 phospholipid-binding protein Yes#

153 WP_043544462.1 TonB-dependent receptor Yes#

154 WP_043547031.1 TonB-dependent siderophore receptor Yes#

Amino acids and protein

155 KGD95192.1 amino acid-binding protein Yes (24-25)

156 WP_085946870.1 argininosuccinate lyase Yes (16-17)

157 OLU10332.1 argininosuccinate lyase Yes (23-24)

158 CUI77092.1 Leucine-isoleucine-valine-threonine-alanine-binding protein precursor Yes (25-26)

159 KGD89270.1 peptidase Yes (21-22)

160 CUI97220.1 Peptidyl-prolyl cis-trans isomerase cyp18 Yes (21-22)

161 KGD95891.1 peptidylprolyl isomerase Yes (19-20)

162 KGD90035.1 porin Yes (32-33)

163 WP_009370175.1 protein sphX precursor Yes (26-27)

164 WP_088154790.1 serine peptidase Yes (34-35)

165 WP_057286328.1 transglutaminase Yes (26-27)

166 WP_043548331.1 twin-arginine translocation pathway signal protein Yes (34-35)



AN

(Weeks)

TS

(Weeks) Secreted*

2 4 6 2 4 6

167 WP_054453210.1 peptidase Yes (21-22)

168 OLU07391.1 S9 family peptidase Yes (23-24)

169 WP_043541316.1 thiol:disulfide interchange protein Yes (27-28)

170 EZP63473.1 Glutamine synthetase Yes#

171 KGD87014.1 glutamine synthetase Yes#

172 KGE00438.1 leucine--tRNA ligase Yes#

173 WP_043547732.1 peptidase M20 Yes#

174 WP_025139792.1 transglutaminase Yes#

175 WP_061346249.1 type I glutamate-ammonia ligase Yes#

176 OLU09649.1 serine peptidase Yes#

177 WP_062682562.1 MULTISPECIES: peptidase Yes#

178 KGD95842.1 sulfoxide reductase catalytic subunit YedY Yes#

179 WP_036567469.1 sarcosine oxidase subunit alpha family protein Yes#

Energy Metabolism

180 ASC63703.1 cytochrome Yes (22-23)

181 WP_068983280.1 cytochrome B6 Yes#

182 WP_088147109.1 cytochrome C biogenesis protein Yes#

183 WP_058665008.1 NAD(P)H-dependent oxidoreductase Yes#

184 WP_088139396.1 energy transducer TonB Yes#

185 WP_008164054.1 electron transfer flavoprotein subunit alpha/FixB family protein Yes#

186 WP_042792534.1 MULTISPECIES: electron transfer flavoprotein subunit alpha/FixB family protein Yes#

Carbohydrate metabolism

187 ADP18914.1 VI polysaccharide biosynthesis protein VipB/TviC Yes#

188 SEJ19686.1 glyceraldehyde-3-phosphate dehydrogenase (NAD+) Yes#

189 WP_078339602.1 alcohol dehydrogenase AdhP

Yes#



AN

(Weeks)

TS

(Weeks) Secreted*

2 4 6 2 4 6

190 OPD83684.1 aldehyde dehydrogenase, partial Yes#

Aromatics & Xenobiotic degradation

191 WP_043544412.1 dienelactone hydrolase family protein Yes#

Genetic Information Processing

192 WP_088146602.1 LacI family transcriptional regulator Yes (27-28)

193 OOC51395.1 LacI family transcriptional regulator yes (23-24)

194 WP_016992165.1 MULTISPECIES: ethanolamine utilization protein EutJ Yes (30-31)

195 SDY72801.1 nucleoside-binding protein Yes (24-25)

196 WP_088159385.1 transcription initiation protein Yes#

197 WP_043541446.1 MULTISPECIES: 30S ribosomal protein S16 Yes#

198 KGD95836.1 50S ribosomal protein L32 Yes#


200 OBY85802.1 50S ribosomal protein L6 Yes#

201 KEH14151.1 50S ribosomal protein L27 Yes#

202 WP_088587769.1 nucleotide exchange factor GrpE Yes#

203 KGD93761.1 translocation protein TolB Yes#

204 EHK64371.1 endoribonuclease L-PSP family protein Yes#

205 EHK64178.1 histone-like DNA-binding protein Yes#

206 OLU00781.1 guanylate kinase Yes#

Hypothetical

207 WP_013394864.1 hypothetical protein Yes (22-23)


209 OWT64198.1 hypothetical protein Yes (21-22)




AN

(Weeks)

TS

(Weeks) Secreted*

2 4 6 2 4 6

211 KXO76461.1 hypothetical protein Yes (19-20)


213 WP_054500944.1 hypothetical protein yes (23-24)









222 ASC65605.1 hypothetical protein Yes (50-51)

223 KGD93886.1 hypothetical protein Yes (25-26)

224 KMN36246.1 hypothetical protein Yes (23-24)





229 WP_088447542.1 hypothetical protein Yes#





234 KGE00062.1 hypothetical protein

Yes#



AN

(Weeks)

TS

(Weeks) Secreted*

2

4

6

2

4

6

235 SEJ51186.1 hypothetical protein Yes#




239 KOF53565.1 hypothetical protein Yes#



242 KGD93750.1 hypothetical protein Yes#


Mineral absorption

244 WP_061347015.1 iron uptake system protein EfeO Yes (25-26)

245 KGD96157.1 zinc protease Yes (27-28)

246 KGD88097.1 superoxide dismutase Yes (22-23)

247 WP_063643475.1 MULTISPECIES: copper chaperone PCu(A)C Yes (20-21) 248 WP_008167322.1 copper chaperone PCu(A)C Yes (26-27)

249 WP_043541183.1 copper chaperone PCu(A)C Yes (22-23)

250 WP_050446790.1 Co2+/Mg2+ efflux protein ApaG Yes#

251 WP_088598926.1 MULTISPECIES: iron-sulfur cluster insertion protein ErpA Yes#

252 WP_056559067.1 superoxide dismutase Yes#

253 ASC66955.1 superoxide dismutase [Fe] Yes#

Fatty acid metabolism

254 WP_056567753.1 beta-ketoacyl-ACP reductase Yes#

Motility

255 OWT67573.1 elongation factor P Yes#



AN

(Weeks)

TS

(Weeks) Secreted*

2 4 6 2 4 6

256 WP_057284408.1 flagellar biosynthesis protein FlgE Yes#

257 WP_076521318.1 flagellar hook protein FliD Yes#

258 OLT99557.1 flagellar hook protein FliD Yes#

259 ASC68125.1 flagellar hook-associated protein 3 Yes#

260 WP_043545126.1 flagellar hook-associated protein FlgK Yes#

Other

261 WP_043547752.1 dehydratase Yes (24-25)

262 WP_013391069.1 DUF3300 domain-containing protein Yes (24-25)




266 WP_062683029.1 MULTISPECIES: BON domain-containing protein Yes (24-25)

267 WP_010658359.1 MULTISPECIES: DUF1344 domain-containing protein Yes (21-22)


269 EJO30942.1 extra-cytoplasmic solute receptor family protein 174 Yes (23-24)

270 WP_065345553.1 Ig family protein Yes (20-21)

271 WP_061345471.1 immunogenic protein Yes (28-29)

272 WP_062681529.1 MULTISPECIES: immunogenic protein Yes (25-26)

273 WP_061347347.1 ligand-binding protein SH3 Yes (22-23)

274 WP_050447996.1 nitrate reductase Yes (24-25)

275 WP_047422444.1 OmpA family protein Yes (21-22)



AN

(Weeks)

TS

(Weeks) Secreted*

2 4 6 2 4 6

276 EHK66933.1 periplasmic protein Yes (20-21)

277 WP_088159604.1 peroxiredoxin Yes (21-22)

278 WP_052097643.1 polyisoprenoid-binding protein Yes (23-24)

279 WP_076408159.1 polyketide cyclase Yes (24-25)

280 WP_088148447.1 receptor Yes (28-29)

281 CUI89627.1 Uncharacterised protein Yes (22-23)

282 SFB23670.1 2-oxoglutarate dehydrogenase E2 component Yes#

283 WP_076519821.1 arsenate reductase Yes#

284 ADP18479.1 carboxymethylenebutenolidase 4 Yes#

285 WP_062676177.1 catalase/peroxidase HPI Yes#

286 WP_071633427.1 ferredoxin family protein Yes#

287 WP_061347851.1 hemolysin expression modulating protein Yes#

288 WP_088138258.1 histone Yes#

289 WP_076519581.1 MULTISPECIES: peroxidase Yes#


291 WP_043547559.1 peroxiredoxin OsmC Yes#

292 WP_088141124.1 OsmC family peroxiredoxin Yes#

293 KGD97163.1 peroxidase Yes#

294 WP_043543107.1 nitronate monooxygenase Yes#

295 KGY26538.1 nucleoid-associated protein Yes#

296 OLU09883.1 Phasin (PHA-granule associated protein) Yes#

297 WP_054550133.1 SRPBCC domain-containing protein Yes#



AN

(Weeks)

TS

(Weeks) Secreted*

2 4 6 2 4 6

298 OLU07233.1 TIGR01244 family protein Yes#

299 WP_088153633.1 DUF2950 domain-containing protein Yes#

Notes:

AN – Alternating anaerobic-aerobic treatment, TS – Two stage anaerobic-aerobic treatment

* Numbers given within brackets correspond to the cleavage site location within the amino acid sequence of classically secreted proteins.


Protein yield

Negative Low <----------> high


Table C.2 Relative abundance of extracellular proteins of bacterial consortium Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05

found in the culture supernatant under alternating (AN) anaerobic-aerobic conditions as a heat map. Classically secreted proteins were predicted using the

SignalP 3.0 Server and the non-classically secreted proteins were predicted using the SecretomeP 2.0 Server. Week 1, 3, 5 were kept under anaerobic and

week 2, 4, 6 were kept under aerobic conditions.

No. Accession number Protein Weeks

Secreted* 1 2 3 4 5 6

Transport related









9 KNY13962.1 ABC transporter permease Yes (20-21)












Secreted* 1 2 3 4 5 6

19 OWT57704.1 ABC transporter substrate-binding protein Yes (35-36)


21 OAD17456.1 ABC transporter substrate-binding protein Yes (26-27)


23 OCZ60996.1 ABC transporter substrate-binding protein Yes (26-27)

24 OAE68865.1 ABC transporter substrate-binding protein Yes (26-27)



27 KGD96204.1 ABC transporter substrate-binding protein Yes (25-26)



30 OLT99158.1 ABC transporter substrate-binding protein Yes (21-22)






36 OWG18014.1 ABC transporter substrate-binding protein Yes (25-26)



39 KXO76717.1 ABC transporter substrate-binding protein Yes (27-28)

40 OOC59559.1 ABC transporter substrate-binding protein Yes (37-38)

41 KXO75178.1 amino acid ABC transporter Yes (21-22)






Weeks

Secreted* 1

2

3

4

5

6


46 KGE00355.1 amino acid ABC transporter substrate-binding protein Yes (26-27)


48 KNE29344.1 amino acid ABC transporter substrate-binding protein Yes (31-32)

49 OLU07976.1 amino acid ABC transporter substrate-binding protein Yes (22-23)






55 AKP90359.1 Branched-chain amino acid ABC transporter, amino acid-binding protein Yes (28-29)


57 KGY25861.1 C4-dicarboxylate ABC transporter Yes (24-25)




61 WP_088148288.1 carbohydrate ABC transporter substrate-binding protein Yes (22-23)

62 EEQ96006.1 extracellular solute-binding protein Yes (27-28)

63 EHK65400.1 extracellular solute-binding family protein Yes (22-23)

64 ABS16514.1 extracellular solute-binding protein family 3 Yes (23-24)

65 WP_048394203.1 ferrichrome ABC transporter substrate-binding protein Yes (27-28)

66 KGD98287.1 glutamine ABC transporter substrate-bindnig protein Yes (26-27)

67 OLU03919.1 glutamine ABC transporter substrate-binding protein Yes (25-26)

68 KNE26968.1 glutathione ABC transporter substrate-binding protein Yes (28-29)



Secreted* 1 2 3 4 5 6

69 CUJ34547.1 Glutathione-binding protein gsiB precursor Yes (28-29)

70 ADP14939.1 glutathione-binding protein GsiB Yes (28-29)

71 WP_057284748.1 glycine/betaine ABC transporter substrate-binding protein Yes (27-28)

72 WP_063952087.1 hemin ABC transporter substrate-binding protein Yes (18-19)



75 KGD93619.1 iron ABC transporter substrate-binding protein Yes (24-25)

76 OLU06668.1 iron ABC transporter substrate-binding protein Yes (28-29)

77 KMN36448.1 iron ABC transporter substrate-binding protein Yes (33-34)



80 OCX15298.1 iron ABC transporter substrate-binding protein Yes (22-23)

81 CUJ13421.1 leucine ABC transporter subunit substrate-binding protein Yes (24-25)



84 WP_008160825.1 metal ABC transporter substrate-binding protein Yes (29-30)

85 WP_088153545.1 molybdate ABC transporter substrate-binding protein Yes (24-25)

86 WP_043212569.1 MULTISPECIES: ABC transporter permease Yes (24-25)







93 WP_056317574.1 MULTISPECIES: branched chain amino acid ABC transporter substrate-binding protein Yes (25-26)

94 WP_088595533.1 MULTISPECIES: glutamine ABC transporter substrate-binding protein GlnH Yes (26-27)


No.

Accession number

Protein

Weeks

Secreted*

1 2 3 4 5 6

95 WP_062684476.1 MULTISPECIES: hemin ABC transporter substrate-binding protein Yes (19-20)

96 WP_062682236.1 MULTISPECIES: leucine ABC transporter subunit substrate-binding protein LivK Yes (25-26)

97 WP_034397436.1 MULTISPECIES: outer membrane protein assembly factor BamE Yes (22-23)

98 WP_082601759.1 peptide ABC transporter Yes (31-32)

99 ASC64693.1 peptide ABC transporter substrate-binding protein Yes (28-29)


101 KGD99111.1 peptide ABC transporter substrate-binding protein Yes (28-29)


103 WP_088145960.1 phosphate ABC transporter substrate-binding protein PstS Yes (24-25)

104 WP_054423127.1 phosphate/phosphite/phosphonate ABC transporter substrate-binding protein Yes (24-25)

105 WP_043544234.1 polyamine ABC transporter substrate-binding protein Yes (25-26)

106 WP_049054386.1 sugar ABC transporter substrate-binding protein Yes (30-31)

107 KMN40622.1 sugar ABC transporter substrate-binding protein Yes (28-29)

108 WP_061346078.1 sulfate ABC transporter substrate-binding protein Yes (21-22)

109 OLU07503.1 sulfate transporter subunit Yes (27-28)

110 WP_078339521.1 thiamine ABC transporter substrate binding subunit Yes (23-24)

111 WP_088147934.1 transporter Yes (21-22)

112 KGD94171.1 transporter Yes (24-25)

113 AOU92447.1 tripartite tricarboxylate transporter Yes (31-32)








Secreted* 1 2 3 4 5 6




122 OMG92355.1 urea ABC transporter substrate-binding protein Yes (27-28)

123 OLU07484.1 ABC transporter substrate-binding protein Yes#

124 WP_043547903.1 C4-dicarboxylate ABC transporter Yes#

125 WP_076815687.1 electron transporter Yes#


127 WP_063959745.1 MULTISPECIES: efflux RND transporter periplasmic adaptor subunit Yes#

Membrane related

128 KGD89451.1 lipoprotein Yes (17-18)





133 WP_043542943.1 membrane protein Yes (20-21)


135 WP_062681332.1 MULTISPECIES: outer membrane protein assembly factor BamC Yes (18-19)

136 WP_062681256.1 MULTISPECIES: peptidoglycan-associated lipoprotein Yes (22-23)

137 ADP14169.1 NLPA lipoprotein family protein 1 Yes (25-26)

138 WP_088146536.1 peptidoglycan-binding protein Yes (24-25)

139 SIT23972.1 periplasmic chaperone for outer membrane proteins SurA Yes (29-30)

140 CUJ96470.1 Probable phospholipid-binding protein mlaC precursor Yes (28-29)

141 EZP50560.1 putative lipoprotein Yes(23-24)




Weeks

Secreted* 1

2

3

4

5

6

Amino acids and protein


144 WP_088141626.1 TonB-dependent receptor Yes (22-23)



147 WP_043548035.1 carboxypeptidase regulatory-like domain-containing protein [ Yes (22-23)

148 WP_079242778.1 SusC/RagA family TonB-linked outer membrane protein Yes (22-23)


150 WP_054481236.1 MULTISPECIES: phospholipid-binding protein Yes#

151 OWT75564.1 phospholipid-binding protein Yes#

152 KGD93668.1 phospholipid-binding protein Yes#

153 WP_043544462.1 TonB-dependent receptor Yes#

154 WP_043547031.1 TonB-dependent siderophore receptor Yes#

155 KGD95192.1 amino acid-binding protein Yes (24-25)

156 WP_085946870.1 argininosuccinate lyase Yes (16-17)

157 OLU10332.1 argininosuccinate lyase Yes (23-24)

158 CUI77092.1 Leucine-isoleucine-valine-threonine-alanine-binding protein precursor Yes (25-26)

159 KGD89270.1 peptidase Yes (21-22)

160 CUI97220.1 Peptidyl-prolyl cis-trans isomerase cyp18 Yes (21-22)

161 KGD95891.1 peptidylprolyl isomerase Yes (19-20)

162 KGD90035.1 porin Yes (32-33)

163 WP_009370175.1 protein sphX precursor Yes (26-27)

164 WP_088154790.1 serine peptidase Yes (34-35)

165 WP_057286328.1 transglutaminase Yes (26-27)

166 WP_043548331.1 twin-arginine translocation pathway signal protein Yes (34-35)



Secreted* 1 2 3 4 5 6

167 WP_054453210.1 peptidase Yes (21-22)

168 OLU07391.1 S9 family peptidase Yes (23-24)

169 WP_043541316.1 thiol:disulfide interchange protein Yes (27-28)

170 EZP63473.1 Glutamine synthetase Yes#

171 KGD87014.1 glutamine synthetase Yes#

172 KGE00438.1 leucine--tRNA ligase Yes#

173 WP_043547732.1 peptidase M20 Yes#

174 WP_025139792.1 transglutaminase Yes#

175 WP_061346249.1 type I glutamate-ammonia ligase Yes#

176 OLU09649.1 serine peptidase [Achromobacter xylosoxidans] Yes#

177 WP_062682562.1 MULTISPECIES: peptidase Yes#


179 WP_036567469.1 sarcosine oxidase subunit alpha family protein Yes#

Energy Metabolism

180 ASC63703.1 cytochrome Yes (22-23)

181 WP_068983280.1 cytochrome B6 Yes#

182 WP_088147109.1 cytochrome C biogenesis protein Yes#


184 WP_088139396.1 energy transducer TonB Yes#

185 WP_008164054.1 electron transfer flavoprotein subunit alpha/FixB family protein Yes#

186 WP_042792534.1 MULTISPECIES: electron transfer flavoprotein subunit alpha/FixB family protein Yes#


187 ADP18914.1 VI polysaccharide biosynthesis protein VipB/TviC Yes#

188 SEJ19686.1 glyceraldehyde-3-phosphate dehydrogenase (NAD+) Yes#

189 WP_078339602.1 alcohol dehydrogenase AdhP Yes#

190 OPD83684.1 aldehyde dehydrogenase, partial Yes#



Secreted* 1 2 3 4 5 6


191 WP_043544412.1 dienelactone hydrolase family protein Yes#


192 WP_088146602.1 LacI family transcriptional regulator Yes (27-28)

193 OOC51395.1 LacI family transcriptional regulator yes (23-24)

194 WP_016992165.1 MULTISPECIES: ethanolamine utilization protein EutJ Yes (30-31)

195 SDY72801.1 nucleoside-binding protein Yes (24-25)

196 WP_088159385.1 transcription initiation protein Yes#

197 WP_043541446.1 MULTISPECIES: 30S ribosomal protein S16 Yes#



200 OBY85802.1 50S ribosomal protein L6 Yes#

201 KEH14151.1 50S ribosomal protein L27 Yes#

202 WP_088587769.1 nucleotide exchange factor GrpE Yes#

203 KGD93761.1 translocation protein TolB Yes#

204 EHK64371.1 endoribonuclease L-PSP family protein Yes#

205 EHK64178.1 histone-like DNA-binding protein Yes#

206 OLU00781.1 guanylate kinase Yes#

Hypothetical



209 OWT64198.1 hypothetical protein Yes (21-22)




Secreted* 1 2 3 4 5 6



213 WP_054500944.1 hypothetical protein yes (23-24)









222 ASC65605.1 hypothetical protein Yes (50-51)

223 KGD93886.1 hypothetical protein Yes (25-26)

224 KMN36246.1 hypothetical protein Yes (23-24)








232 WP_048395293.1 hypothetical protein Yes# 233 WP_063584394.1 hypothetical protein Yes#

234 KGE00062.1 hypothetical protein Yes#

235 SEJ51186.1 hypothetical protein Yes#



Weeks

Secreted* 1

2

3

4

5

6




239 KOF53565.1 hypothetical protein Yes#



242 KGD93750.1 hypothetical protein Yes#


Mineral absorption

244 WP_061347015.1 iron uptake system protein EfeO Yes (25-26)

245 KGD96157.1 zinc protease Yes (27-28)

246 KGD88097.1 superoxide dismutase Yes (22-23)

247 WP_063643475.1 MULTISPECIES: copper chaperone PCu(A)C Yes (20-21)



250 WP_050446790.1 Co2+/Mg2+ efflux protein ApaG Yes#

251 WP_088598926.1 MULTISPECIES: iron-sulfur cluster insertion protein ErpA Yes#

252 WP_056559067.1 superoxide dismutase Yes#

253 ASC66955.1 superoxide dismutase [Fe] Yes#

Fatty acid metabolism

254 WP_056567753.1 beta-ketoacyl-ACP reductase Yes#

Motility

255 OWT67573.1 elongation factor P Yes#

256 WP_057284408.1 flagellar biosynthesis protein FlgE Yes#

257 WP_076521318.1 flagellar hook protein FliD Yes#



Secreted* 1 2 3 4 5 6

258 OLT99557.1 flagellar hook protein FliD Yes#

259 ASC68125.1 flagellar hook-associated protein 3 Yes#

260 WP_043545126.1 flagellar hook-associated protein FlgK Yes#

Others

261 WP_043547752.1 dehydratase Yes (24-25)





266 WP_062683029.1 MULTISPECIES: BON domain-containing protein Yes (24-25)



269 EJO30942.1 extra-cytoplasmic solute receptor family protein 174 Yes (23-24)

270 WP_065345553.1 Ig family protein Yes (20-21)

271 WP_061345471.1 immunogenic protein Yes (28-29)

272 WP_062681529.1 MULTISPECIES: immunogenic protein Yes (25-26)

273 WP_061347347.1 ligand-binding protein SH3 Yes (22-23)

274 WP_050447996.1 nitrate reductase Yes (24-25)

275 WP_047422444.1 OmpA family protein Yes (21-22)

276 EHK66933.1 periplasmic protein Yes (20-21)

277 WP_088159604.1 peroxiredoxin Yes (21-22)

278 WP_052097643.1 polyisoprenoid-binding protein Yes (23-24)

279 WP_076408159.1 polyketide cyclase Yes (24-25)

280 WP_088148447.1 receptor Yes (28-29)

281 CUI89627.1 Uncharacterised protein Yes (22-23)

282 WP_076519821.1 arsenate reductase Yes#


No. Accession number Protein Weeks Secreted* 1 2 3 4 5 6

283 ADP18479.1 carboxymethylenebutenolidase 4 Yes#

284 WP_062676177.1 catalase/peroxidase HPI Yes#

285 WP_071633427.1 ferredoxin family protein Yes#

286 WP_061347851.1 hemolysin expression modulating protein Yes#

287 WP_088138258.1 histone Yes#

288 WP_076519581.1 MULTISPECIES: peroxidase Yes#


290 WP_043547559.1 peroxiredoxin OsmC Yes#

291 WP_088141124.1 OsmC family peroxiredoxin Yes#


293 KGD97163.1 peroxidase Yes#

294 WP_043543107.1 nitronate monooxygenase Yes#

295 KGY26538.1 nucleoid-associated protein Yes#

296 OLU09883.1 Phasin (PHA-granule associated protein) Yes#

297 WP_054550133.1 SRPBCC domain-containing protein Yes#

298 OLU07233.1 TIGR01244 family protein Yes#

299 WP_088153633.1 DUF2950 domain-containing protein Yes#

Notes:

* Numbers given within brackets correspond to the cleavage site location within the amino acid sequence of classically secreted proteins.


Week 1, 3 and 5 were maintained under anaerobic conditions and week 2, 4 and 6 were maintained under aerobic conditions.

Protein yield

Negative Low <----------> high


Table C.3 Detailed functional classification of predicted non-secretory proteins in the culture

supernatant of the bacterial consortium Achromobacter sp. NP03, Ochrobactrum sp. NP04

and Lysinibacillus sp. NP05 grown in minimal salt medium using 50 mg/L Aroclor 1260 as the

sole source of carbon

Function Accession No. nnumbernumber

Protein


OWG17491.1 glutathione S-transferase

ASC68025.1 glutathione peroxidase

WP_088447182.1

carboxymuconolactone decarboxylase family protein WP_043547979.

1 carboxymuconolactone decarboxylase family protein WP_088164555.

1 glutathione-dependent disulfide-bond oxidoreductase WP_088146345.

1 glutathione S-transferase

KGD95298.1 carboxymuconolactone decarboxylase

WP_006391478.1

glutathione S-transferase

WP_043546720.1

glutathione S-transferase

KGD86957.1 glutathione S-transferase

KGD90634.1 3-oxoadipate CoA-transferase

KGD95901.1 carboxymuconolactone decarboxylase

WP_013391342.1

2-aminobenzoate-CoA ligase

WP_088139343.1

carboxymethylenebutenolidase

WP_088146045.1

phenylphosphate carboxylase subunit delta

OAD15100.1 4-oxalocrotonate tautomerase


WP_048395937.1

MULTISPECIES: malate synthase A

OLE09158.1 malate synthase G

KXO78488.1 glutamine--fructose-6-phosphate aminotransferase

CUI32503.1 L-lactate dehydrogenase

A9BQA5.1 Succinate--CoA ligase [ADP-forming] subunit beta

APE47142.1 succinate--CoA ligase subunit alpha

WP_088154574.1

malate synthase G

WP_043544785.1

carbohydrate kinase family protein

KGD89899.1 fructose-1,6-bisphosphate aldolase

WP_062684727.1

acyl-ACP--UDP-N-acetylglucosamine O-acyltransferase KIU67857.1 isocitrate lyase

KGE00280.1 isocitrate lyase

EHK65803.1 fructose-1,6-bisphosphate aldolase

AMG47497.1 fructose-bisphosphatase

WP_088148834.1

malate synthase G

OWT65266.1 class II fumarate hydratase

WP_004233046.1

MULTISPECIES: isocitrate lyase

KGD95789.1 succinyl-CoA:3-ketoacid-CoA transferase

WP_008158821.1

isocitrate dehydrogenase (NADP(+))

OAD13600.1 succinate--CoA ligase subunit beta

CUI44931.1 Malate dehydrogenase

WP_061344542.1

malate dehydrogenase

WP_043542435.1

2-dehydro-3-deoxy-phosphogluconate aldolase

WP_088138267.1

glutamate--tRNA ligase

OWT65337.1 inositol monophosphatase

WP_043542539.1

glycosyl hydrolase


Function Accession No. Protein

WP_061347956.1

NAD-glutamate dehydrogenase

ASC68281.1 N-formylglutamate deformylase

WP_046807028.1

triose-phosphate isomerase

KGD98528.1 3-isopropylmalate dehydrogenase

OJV49620.1 NAD-dependent epimerase

WP_006389696.1

NAD dependent epimerase/dehydratase

Elongation factors and chaperones

SDY63415.1 chaperonin GroEL

WP_081955120.1

Tir chaperone family protein

KZK25390.1 ATP-dependent chaperone ClpB

KGD90204.1 molecular chaperone GroEL

KRB12805.1 Fe-S protein assembly chaperone HscA

KRC86368.1 chaperonin

WP_054451905.1

chaperonin GroEL

OCZ53579.1 translation elongation factor G

KZK30311.1 elongation factor Tu

WP_088148204.1

elongation factor G

WP_088140169.1

elongation factor Ts


OLU08504.1 asparaginyl/glutamyl-tRNA amidotransferase subunit C WP_043543299.

1 ribonuclease III

WP_057283073.1

non-canonical purine NTP pyrophosphatase

KOQ46086.1 50S ribosomal protein L29

WP_088446340.1

nicotinate-nucleotide diphosphorylase (carboxylating) WP_062681662.

1 MULTISPECIES: GntR family transcriptional regulator

KFJ12144.1 ribosomal protein L19

KOF53552.1 ATP synthase F0F1 subunit epsilon

KOF53046.1 transcriptional regulator

KNE28388.1 glutamyl-tRNA amidotransferase

KEH14150.1 50S ribosomal protein L21

WP_008164520.1

ribose-5-phosphate isomerase RpiA

KOQ22336.1 DNA-directed RNA polymerase subunit omega

ADP15126.1 translation initiation factor IF-2

KGD94250.1 transcriptional regulator

WP_043211401.1

MULTISPECIES: transcription elongation factor GreB

WP_061344667.1

DNA starvation/stationary phase protection protein Dps APX77825.1 50S ribosomal protein L24

APE50426.1 translation elongation factor Ts

OLU10224.1 DNA polymerase III subunit beta

WP_043549030.1

transcription termination/antitermination protein NusA KGD85622.1 50S ribosomal protein L9

WP_062683079.1

MULTISPECIES: 50S ribosomal protein L25

CUJ77722.1 Transcription antitermination protein nusG

ASC68086.1 30S ribosomal protein S2

WP_043545730.1

MULTISPECIES: DNA-binding response regulator

ASC68083.1 ribosome-recycling factor

OWT65989.1 adenylate kinase

OAE68850.1 DNA starvation/stationary phase protection protein



Protein

EHK64962.1 acetaldehyde dehydrogenase

CUI94695.1 DnaK suppressor protein

WP_071633166.1

heat-inducible transcriptional repressor HrcA

ASC68256.1 exodeoxyribonuclease III

WP_088165862.1

phosphoribosylaminoimidazolesuccinocarboxamide synthase WP_062682787.

1 MULTISPECIES: transcriptional repressor LexA

WP_056568752.1

ferric iron uptake transcriptional regulator

OFL34502.1 DNA-directed RNA polymerase subunit alpha

BAP44485.1 transaldolase B

WP_088148869.1

molybdenum-dependent transcriptional regulator

WP_057285634.1

GTPase ObgE

WP_076519393.1

MULTISPECIES: GTPase Era

Hypothetical WP_028691874.1

MULTISPECIES: hypothetical protein

AIK41363.1 hypothetical protein DR92_4327

WP_062681152.1


KXO79421.1 hypothetical protein AYJ56_01890

WP_062681189.1


WP_043548951.1

hypothetical protein

WP_006391526.1


WP_043212744.1


WP_088141988.1


OCX62666.1 hypothetical protein BFM98_01295

EIK53096.1 hypothetical protein YO5_02683

ASC64511.1 hypothetical protein B9P52_09445

KGE01814.1 hypothetical protein JL37_00900

WP_054458252.1


WP_036077843.1


WP_043544852.1


WP_088138363.1


WP_082775659.1


WP_076521683.1


OLU09627.1 hypothetical protein BVK87_03785

Lipid metabolism

WP_010661441.1

acetyl-CoA C-acetyltransferase

OAE44373.1 3-hydroxyacyl-[acyl-carrier-protein] dehydratase FabZ OLU06419.1 acyl dehydratase

WP_016453937.1

acyl-CoA dehydrogenase

WP_062681131.1

MULTISPECIES: acyl carrier protein

ASC68058.1 enoyl-CoA hydratase

WP_076521293.1

3-hydroxyacyl-[acyl-carrier-protein] dehydratase FabZ WP_088587684.

1 enoyl-CoA hydratase

WP_061346094.1

acyl-CoA dehydrogenase

WP_050446775.1

enoyl-CoA hydratase

OWT77500.1 acyl carrier protein

WP_021586121.1

beta-ketoacyl-ACP reductase

WP_088147327.1

beta-ketoacyl-ACP reductase

WP_012203459.1

MULTISPECIES: enoyl-CoA hydratase

OKO45451.1 REVERSED long-chain-acyl-CoA synthetase

CUI26675.1 Uncharacterized acyl-CoA thioester hydrolase HI_0827



WP_034394989.1

MULTISPECIES: 3-hydroxyacyl-CoA dehydrogenase

AEF90758.1 Enoyl-(acyl-carrier-protein) reductase (NADH)

KGD95832.1 3-ketoacyl-ACP reductase

ASC66946.1 3-hydroxyacyl-CoA dehydrogenase

WP_043543263.1

propionate--CoA ligase

WP_061346536.1

3-hydroxyacyl-CoA dehydrogenase

Membrane related

WP_043547278.1

membrane protein

AKP92101.1 putative lipoprotein

WP_034397436.1

MULTISPECIES: outer membrane protein assembly factor BamE WP_043544857.

1 outer protein B

WP_057284478.1

outer membrane protein chaperone

WP_043544088.1

exopolyphosphatase

Nucleotide metabolism

WP_088153872.1

oxidoreductase

OWT72173.1 NADP-dependent oxidoreductase

KGD93926.1 cytochrome C550

WP_058665727.1

redox-regulated ATPase YchF

WP_076518982.1

fumarate hydratase, class II

WP_061071973.1

ribose-5-phosphate isomerase RpiA

WP_076815687.1

electron transporter RnfB

OLU00760.1 phosphoribosylamine--glycine ligase

KGD96735.1 ATP synthase F0F1 subunit delta

WP_006218219.1

electron transfer flavoprotein subunit beta

WP_048395540.1

MULTISPECIES: ATP-dependent Clp protease ATP-binding subunit WP_054452508.

1 ATP-dependent chaperone ClpB

WP_020924632.1

MULTISPECIES: dihydrolipoyl dehydrogenase

WP_062684019.1

MULTISPECIES: NAD(P)(+) transhydrogenase (Re/Si-specific) subunit alpha SEI90423.1 dihydrolipoamide dehydrogenase

WP_088419875.1

orotate phosphoribosyltransferase

WP_046804414.1

phosphoribosylaminoimidazolesuccinocarboxamide synthase WP_043210047.

1 MULTISPECIES: glycosyl transferase

KNE24372.1 inosine-5-monophosphate dehydrogenase

KGY26696.1 nucleotide-binding protein

KGD90752.1 phosphoglyceromutase

Protein and amino acid metabolism

WP_043547631.1

endopeptidase La

WP_068986654.1

type I methionyl aminopeptidase

WP_088147382.1

arginine--tRNA ligase

WP_062682748.1

MULTISPECIES: succinyl-diaminopimelate desuccinylase WP_006390545.

1 MULTISPECIES: isocitrate lyase

WP_012206888.1

MULTISPECIES: 4-hydroxy-tetrahydrodipicolinate synthase WP_062679845.

1 MULTISPECIES: branched-chain amino acid transaminase OCZ66373.1 cysteine synthase B

WP_061345481.1

urease subunit alpha

EFI67045.1 threonine synthase

WP_066036939.1

peptide chain release factor 1

WP_088146963.1

serine--tRNA ligase

KXO79104.1 cysteine synthase



Protein

SEI93094.1 cysteine synthase

KGE00121.1 indole-3-glycerol-phosphate synthase

WP_071632907.1

tryptophan--tRNA ligase

WP_062681333.1

MULTISPECIES: 4-hydroxy-tetrahydrodipicolinate synthase SIT05832.1 protease I

ASC68057.1 3-hydroxyisobutyrate dehydrogenase

KXO76179.1 UDP-N-acetylmuramoyl-L-alanyl-D-glutamate--2,6-diaminopimelate ligase WP_071632036.

1 methylcrotonoyl-CoA carboxylase

WP_081010950.1

MULTISPECIES: aminopeptidase P family protein

WP_062682745.1

MULTISPECIES: chorismate mutase

WP_043543277.1

leucyl aminopeptidase

ASC63154.1 cysteine synthase B

OAD13310.1 aromatic amino acid aminotransferase

ASC64081.1 serine hydroxymethyltransferase

WP_085943758.1

thioredoxin TrxA

EFI68384.1 cysteine synthase

WP_016995150.1

NADP-specific glutamate dehydrogenase

KGD86968.1 pyrroline-5-carboxylate reductase

ADP13864.1 thiazole biosynthesis protein ThiG family protein

WP_063327214.1

3-deoxy-7-phosphoheptulonate synthase

KGD97156.1 aspartate aminotransferase

KRC76684.1 ornithine carbamoyltransferase

WP_071630174.1

3-deoxy-7-phosphoheptulonate synthase class II

WP_025140033.1

acetylornithine transaminase

KGD90458.1 proline iminopeptidase

KGD87877.1 peptide chain release factor 2

OLU09675.1 acetylornithine deacetylase

WP_043547594.1

2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N-succinyltransferase KOF52692.1 acetolactate synthase

WP_043544334.1

threonine synthase [

Stress and redox signalling

WP_050448221.1

universal stress protein

WP_062683504.1

MULTISPECIES: universal stress protein

CUJ55705.1 General stress protein 17o

WP_049075673.1

MULTISPECIES: universal stress protein

ASC63586.1 universal stress protein

KGR86650.1 chemical-damaging agent resistance protein C

OLU07232.1 universal stress protein

WP_043544650.1

universal stress protein

WP_006393278.1

peroxide stress protein YaaA

ASC63654.1 peroxide stress protein YaaA

EZP63695.1 Peroxiredoxin Q/BCP

WP_076521074.1

MULTISPECIES: thioredoxin

WP_062684140.1

MULTISPECIES: peroxiredoxin

OXC90540.1 thioredoxin-disulfide reductase

WP_061072286.1

thioredoxin-disulfide reductase

OMG90645.1 oxidoreductase

OAD13580.1 alkyl hydroperoxide reductase

KRC70798.1 oxidoreductase



WP_063960063.1

MULTISPECIES: NAD(P)H-quinone oxidoreductase

OAE65212.1 alkyl hydroperoxide reductase

KGD92171.1 oxidoreductase

WP_075041485.1

gfo/Idh/MocA family oxidoreductase

SFB54940.1 Nucleotide-binding universal stress protein, UspA family WP_062683028.

1 CsbD family protein

WP_088148284.1

NADP-dependent oxidoreductase

WP_057286974.1

N-acetyl-gamma-glutamyl-phosphate reductase

Signal transduction and chemotaxis

WP_047377097.1

glucokinase

OLU00781.1 guanylate kinase

WP_054441174.1

MULTISPECIES: homoserine kinase

KGD99719.1 adenylate kinase

ASC66688.1 histidine kinase

KGY28956.1 phosphoglycerate kinase

SIT27796.1 nucleoside diphosphate kinase

WP_088446300.1

pyruvate kinase

SEK01422.1 purine-binding chemotaxis protein CheW

WP_047470594.1

methyl-accepting chemotaxis protein

WP_054427381.1

translocation protein TolB

Transport and secretion

WP_063952513.1

electron transporter RnfB

WP_088139428.1

phosphate transport system regulatory protein PhoU KRB12177.1 ABC transporter substrate-binding protein

OCR22505.1 transporter

WP_006218564.1

thiol:disulfide interchange protein DsbD

WP_062684286.1

MULTISPECIES: PTS sugar transporter subunit IIA

WP_088146814.1

diguanylate cyclase

WP_066037445.1

HPr family phosphocarrier protein

Carboxylic acid metabolism

OCA80479.1 aldehyde dehydrogenase

WP_054550108.1

acetate--CoA ligase

KGD97042.1 2-methylisocitrate lyase

WP_076411594.1

O-acetylhomoserine aminocarboxypropyltransferase ASC63275.1 propionate--CoA ligase

OLU08467.1 3-hydroxybutyrate dehydrogenase

WP_088446703.1

2-hydroxyhepta-2,4-diene-1,7-dioate isomerase

WP_062683435.1

MULTISPECIES: lactoylglutathione lyase

OXC92513.1 uroporphyrinogen decarboxylase

KFJ11781.1 acetyl-CoA carboxylase, biotin carboxyl carrier protein WP_013800519.

1 NAD(P)-dependent alcohol dehydrogenase

WP_029926123.1

MULTISPECIES: glyoxylate/hydroxypyruvate reductase A KNY09037.1 2,3,4,5-tetrahydropyridine-2,6-carboxylate N-succinyltransferase WP_054548608.

1 2-oxoglutarate dehydrogenase E1 component

WP_088139068.1

acetate--CoA ligase

Metabolism of cofactors and vitamins

WP_010658361.1

3,4-dihydroxy-2-butanone-4-phosphate synthase

WP_062683274.1

MULTISPECIES: molybdopterin adenylyltransferase

WP_062680922.1

MULTISPECIES: iron donor protein CyaY

WP_056560178.1

bacterioferritin



Protein

SIT19086.1 bacterioferritin

APE48441.1 3,4-dihydroxy-2-butanone-4-phosphate synthase

Others WP_043543107.1

nitronate monooxygenase

WP_043517811.1

nitronate monooxygenase

WP_047993075.1

DUF779 domain-containing protein

WP_088143023.1


CUJ39015.1 Uncharacterized protein conserved in bacteria

KGY24760.1 CBS domain-containing protein

OMG77958.1 gamma carbonic anhydrase family protein

WP_043544356.1

GNAT family N-acetyltransferase

WP_043541508.1

CBS domain-containing protein

WP_008165630.1

diaminopimelate epimerase

WP_043547280.1

threonylcarbamoyl-AMP synthase

WP_025135677.1


WP_088159720.1

ubiquinone-binding protein

WP_043545226.1

MULTISPECIES: nitrogen regulatory protein P-II 1

CUK06870.1 Arsenate reductase and related proteins glutaredoxin family WP_082400968.

1 HAD family hydrolase, partial

WP_063959978.1

MULTISPECIES: superoxide dismutase

WP_006218564.1

thiol:disulfide interchange protein DsbD

WP_049667999.1

response regulator

WP_062684908.1

MULTISPECIES: dUTPase

WP_062682845.1

MULTISPECIES: DUF2513 domain-containing protein

KXO75649.1 histidine--tRNA ligase

WP_063587851.1

cell division protein FtsA

WP_088138739.1

hydrolase

OWT64701.1 cupredoxin-domain containing protein

OAE50901.1 deoxycytidine triphosphate deaminase

WP_088148655.1

glycerophosphodiester phosphodiesterase

KMN39865.1 reductase

WP_088147916.1

hydroxyacid dehydrogenase

CUK07244.1 Uncharacterized protein conserved in bacteria

WP_061072417.1

amidohydrolase

WP_041653427.1

MULTISPECIES: nitroreductase

WP_071632572.1

RidA family protein

EEQ95822.1 Nitrogen regulatory protein P-II

WP_076516740.1

MULTISPECIES: MBL fold metallo-hydrolase

EFF77190.1 SWIB/MDM2 domain protein

WP_088148618.1

bifunctional ornithine acetyltransferase/N-acetylglutamate synthase KGE01752.1 acetylornithine aminotransferase

ASC62876.1 quinone oxidoreductase

CUJ75942.1 2-hydroxy-3-oxopropionate reductase

OLU06774.1 aconitate hydratase 1

Supplimentary Material 291

Supplimentary Material

292 Supplimentary Material

Supplementary Material 1: Abstracts of conference papers relevant to the thesis 293

Supplementary Material 1: Abstracts of

conference papers relevant to the thesis

294 Supplementary Material 1: Abstracts of conference papers relevant to the thesis

Supplementary Material 2: Publications relevant to the thesis 295

Supplementary Material 2: Publications

relevant to the thesis

Science of the Total Environment 651 (2019) 2197–2207

Contents lists available at ScienceDirect

Science of the Total Environment

j ourna l homepage: www.e lsev ie r .com/ locate /sc i totenv

Solubilization and degradation of polychlorinated biphenyls (PCBs) bynaturally occurring facultative anaerobic bacteria

Gathanayana Pathiraja a, Prasanna Egodawatta a, Ashantha Goonetilleke b, Valentino S. Junior Te'o a,⁎a School of Earth, Environmental and Biological Sciences, Queensland University of Technology (QUT), Brisbane 4001, Queensland, Australiab School of Civil Engineering and Built Environment, Queensland University of Technology (QUT), Brisbane 4001, Queensland, Australia

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• The best strains degraded PCBs underboth anaerobic and aerobic conditions.

• Natural bacterial isolates exhibitedconcomitant PCB solubilization anddegradation.

• PCB solubility positively correlated withpotential biosurfactant production.

• Highest chlorine removal was achievedunder two stage anaerobic-aerobicconditions.

• Lysinibacillus sp. demonstrated thehighest PCB solubility and chloridebuild up.

⁎ Corresponding author.E-mail addresses: [email protected] (G

[email protected] (P. Egodawatta), [email protected] (V.S.J. Te'o).

https://doi.org/10.1016/j.scitotenv.2018.10.1270048-9697/© 2018 Published by Elsevier B.V.

a b s t r a c t
a r t i c l e i n f o
Article history:Received 20 August 2018Received in revised form 9 October 2018Accepted 9 October 2018Available online 11 October 2018

Editor: Jay Gan

A combination of solubilization and degradation is essential for the bioremediation of environments contami-natedwith complex polychlorinated biphenyls (PCB)mixtures. However, the application of facultative anaerobicmicroorganisms that can both solubilize and breakdown hydrophobic PCBs in aqueousmedia under both anaer-obic and aerobic conditions, has not been reported widely. In this comprehensive study, four bacteria discoveredfrom soil and sediments and identified as Achromobacter sp. NP03,Ochrobactrum sp. NP04, Lysinibacillus sp. NP05and Pseudomonas sp. NP06, were investigated for their PCB degradation efficiencies. Aroclor 1260 (50 mg/L), acommercial and highly chlorinated PCB mixture was exposed to the different bacterial strains under aerobic,anaerobic and two stage anaerobic–aerobic conditions. The results confirmed that all four facultative anaerobicmicroorganisms were capable of degrading PCBs under both anaerobic and aerobic conditions. The highest chlo-rine removal (9.16± 0.8mg/L), PCB solubility (14.7± 0.93mg/L) and growth rates as OD600 (2.63 ± 0.22)wereobtained for Lysinibacillus sp. NP05 under two stage anaerobic-aerobic conditions. The presence of biosurfactantsin the culturemedium suggested their role in solubility of PCBs. Overall, the positive results obtained suggest thathigh PCB hydrolysis can be achieved using suitable facultative anaerobic microorganisms under two stageanaerobic-aerobic conditions. Such facultative microbial strains capable of solubilization as well as degradationof PCBs under both anaerobic and aerobic conditions provide an efficient and effective alternative to commonlyused bioaugmentation methods utilizing specific obligate aerobic and anaerobic microorganisms, separately.

© 2018 Published by Elsevier B.V.

Keywords:BioremediationBiosurfactantChloride build upTwo stage anaerobic-aerobic treatment

. Pathiraja),@qut.edu.au (A. Goonetilleke),

1. Introduction

The diversity and magnitude of synthetic toxic chemicals releasedinto the environment are creating long-term human and ecosystem

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health impacts. Polychlorinated biphenyls (PCBs) are one such toxicchemical group consisting of 209 different chlorinated organic com-pounds. PCBs are persistent in the environment due to low reactivity,high chemical stability and extreme hydrophobicity (Beyer and Biziuk,2009). Conversely, high lipophilicity makes PCBs soluble in fats.Such characteristics results in bioaccumulation, bioconcentration andbiomagnification along the food chains leading to numerous healthimplications in humans and animals (ATSDR, 2000). Due to theirpersistence in the environment, sites contaminated with PCBs such aselectricity distribution stations, service areas and dumpsites as well assediments in the nearby waterbodies are still posing significant threatsto human and ecosystem health.

Microorganisms play an important role in the removal of toxicchemical compounds from the environment. Biological conversion ofhighly and moderately chlorinated PCB congeners into less chlorinatedcongeners has been reported to take place through dechlorinationunder anaerobic conditions (Praveckova et al., 2015; Agullo et al.,2017). In comparison, lower and moderately chlorinated congenerscan be degraded by oxidative bacteria under aerobic conditions throughupper and lower biphenyl degradation pathways (Field and Sierra-Alvarez, 2008). Therefore, to achieve complete degradation, one of themost promising bioremediation strategies is to combine the anaerobicdechlorination and aerobic oxidation (Passatore et al., 2014). Althoughnumerous sediment and soil based studies have been conducted eitherunder anaerobic or aerobic conditions separately, using potentialPCB degrading bacteria (Adrian et al., 2009; Payne et al., 2011; Wangand He, 2013; Murinova et al., 2014), studies based on combinedanaerobic-aerobic conditions are limited (Evans et al., 1996; Masteret al., 2002; Long et al., 2015). Studies by Evans et al. (1996) andMaster et al. (2002) used two separate groups of bacteria capable ofreductive dechlorination and aerobic oxidation, respectively, to degradePCBs in contaminated soil slurry. No studies appear to have beenundertaken so far on PCB degradation using facultative anaerobic mi-croorganisms under two-stage anaerobic-aerobic conditions. However,there is a recent study based on facultative anaerobic bacteria mediatedin situ delignification and enhanced gas release under microaerophilicconditions in soil containing lignocellulose (Rashid et al., 2017).

Past research literature on biochemical pathways and intracellularlocalization of enzymes responsible for PCB degradation suggest thatPCBs have to be solubilized first for easier passage through the cellwall and into the cytoplasm prior to being metabolized. Therefore, anincrease in the rate of solubilization could accelerate the entrance ofPCBs into the cells and their subsequent degradation (Ohtsubo et al.,2004). Most of the research studies undertaken so far on PCB solubilityhave been based on the addition of chemical or biological surfactantswith limited investigation into the actual application ofmicroorganismsproducing surfactants (Singer et al., 2000; Fava and Di Gioia, 2001;Occulti et al., 2008; Viisimaa et al., 2013). Chemical surfactants havethe advantage of being economical, but are often toxic to biologicalsystems (Abraham et al., 2002). In comparison, biosurfactants generallyexhibit higher interfacial tension reduction activities compared tochemical surfactants, and are less toxic and readily biodegradable(Viisimaa et al., 2013). However, the main disadvantage in the use ofcommercially available biosurfactants is the high cost (Aparna et al.,2012).

Therefore, the use of suitable biosurfactant producing microbialstrains, which are also capable of degrading PCBs under both, anaerobicand aerobic conditions, would be an attractive alternative to the use ofeither chemical or biological surfactants or PCB degrading aerobicand anaerobic bacterial groups, separately. Indeed, the application ofbiosurfactant-producing and pollutant-degrading microorganismsoffers the dual advantage of a continuous supply of biodegradablesurfactants and the ability to degrade pollutants (Megharaj et al., 2011).

The aim of this study was to isolate potential naturally occurringfacultative anaerobic bacteria from soil and sediment environmentsand to investigate their capability for degrading Aroclor 1260, a complex

and widely used commercial PCB mixture, under comparative anaero-bic, aerobic and two stage anaerobic-aerobic conditions while solubiliz-ing a hydrophobic PCBmixture. The outcomes of the study are expectedto contribute to the development of more efficient and effective bacte-rial mediated bioremediation treatment of PCB contaminated soils andsediments.

2. Materials and methods

2.1. PCB source

Aroclor 1260 was selected as the commercial grade PCB source forthis study and obtained as a GC/FID grade technical mixture fromAccuStandard Inc. (New Haven, CT, USA). Aroclor 1260 represents acomplex PCB mixture consisting of about 75 different penta to nonachloro biphenylswith an average of 6.3 chlorines per biphenylmolecule(Bedard et al., 2007). Aroclor 1260was prepared as a 50mg/mL stock inGCMS grade acetone, before use.

2.2. Screening, enrichment and identification of possible PCB degradingmicroorganisms

2.2.1. Sampling of soil and sedimentsIn order to isolate possible facultative anaerobic PCB degrading bac-

teria, six soil samples around the Brisbane City area and six sedimentsamples from Brisbane River (27.4745° S, 153.0293° E) and CoombabahLake, Gold Coast (27.54° S, 153.22° E), Australia were collected intosterile glass bottles and transported on ice to the laboratory. Soil andsediment samples were separately homogenised and 50 g from eachcomposite soil and sediment mixtures were added to duplicate250mLErlenmeyer flasks. After contaminationwith Aroclor 1260 to ob-tain 50 mg/kg PCB concentration, flasks were incubated stationaryunder aerobic conditions at room temperature (23 °C ± 1 °C) for onemonth. Similarly, 50 g of each composite soil and sediment mixturewere added to duplicate 50 mL polypropylene vials with screw caps,contaminated with Aroclor 1260 to obtain 50 mg/L concentration andincubated stationary inside the anaerobic chamber (COY lab products)main compartment at room temperature (23 °C ± 1 °C) for onemonth. The atmosphere inside the anaerobic chamber was maintainedconstant at 4.9% H2, 10.7% CO2 and 84.4% N2.

2.2.2. Selective enrichment of possible PCB degrading bacteriaIsolation ofmicroorganisms capable of utilizing PCBswas carried out

through a series of selective enrichments using DSMZ medium 465a(Atlas, 2005) as the base minimal salt medium (MSM) with the follow-ing modifications. After autoclaving the medium, instead of 0.5 g/Lhydroxybiphenyl in ethanol, Aroclor 1260 in GCMS grade acetone wasadded as the sole carbon and energy source to give 50 mg/L final PCBconcentration. Four 250 mL Erlenmeyer flasks containing 100 mL ofsterile MSM medium were prepared. Two flasks were inoculated with10 g of composite soil, and the other two were inoculated with 10 gof sediment previously contaminated with Aroclor 1260. Two flasks(onewith soil and the other with sediment) were incubated aerobicallyin a platform shakermaintained at 150 rpmand 28 °C. The other two re-maining flasks were incubated anaerobically under static conditions inan incubator kept inside the anaerobic chamber with the temperaturekept at 28 °C. Four serial transfers (10% of the enrichment medium)were carried out from each flask at weekly intervals into fresh sterileMSM containing 50mg/LAroclor 1260. From the final flask, supernatantwas plated on nutrient agar (CM003, Oxoid) and incubated overnight at28 °C. Morphologically different colonies were isolated and streaked onfresh nutrient agar plates.

2.2.3. Characterization of potential PCB degrading bacteriaBacterial isolates obtained from selective enrichment were inocu-

lated in duplicate on minimal salt agar plates containing 50 mg/L of

2199G. Pathiraja et al. / Science of the Total Environment 651 (2019) 2197–2207

Aroclor 1260 as sole source of carbon and incubated at 28 °C in parallelunder anaerobic and aerobic conditions to determine their growth tol-erance in the presence/absence of atmospheric oxygen. Minimal saltagar with no added Aroclor 1260, but an equal volume of acetone thatwas used to add Aroclor 1260 into the minimal salt agar in the growthexperiment, was used as the negative control to determine whetherthere was any contribution from leftover acetone on bacterial growth.In comparison, nutrient agar medium alone was used as the positivecontrol. Isolates with the ability to grow on PCBs under both, anaerobicand aerobic conditions were selected for further characterization basedon colony morphology on nutrient agar plates and cell morphologyusing Gram staining. Additionally, the bacterial isolates with highPCB solubility and degradation potential were further characterizedbased on their ability to utilize different carbon and nitrogen sourcesusing Biolog PM1 and PM3B plates containing 95 separate sole carbonand nitrogen sources. Full-length 16S rRNA gene sequences were PCRamplified from purified genomic DNA (gDNA) isolated from puremicrobial isolates in order to ascertain their identification as shown inFig. 1.

2.2.3.1. Genomic DNA extraction and polymerase chain reactions (PCR).Bacterial gDNAs were extracted using Isolate II genomic DNA kit(Bioline) and different gDNAs were used as templates for PCRamplification of 16S rRNA genes. Full length universal 16S rRNAprimers, 27F and 1492R (Integrated DNA Technologies, Inc.), wereused and amplifications were performed according to the MyTaq HSRed DNA polymerase protocol (Bioline). The following PCR cyclingconditions were used: 1 × cycle: 95 °C/10 min, 30 × cycles: 95 °C/30 s,50 °C/30 s, 72 °C/2 min, 1 × cycle: 72 °C/7 min, and held at 4 °C. Thequality of the PCR products was checked by electrophoresis at 100 Vfor 1 h using a 1% agarose gel. The ~1.5 kb RNA gene PCR productswere extracted and purified using the Isolate II PCR and gel extractionkit protocol (Bioline).

2.2.3.2. Ligation, transformation and cloning of PCR products for sequencing.The gel purified ~1.5 kb 16S RNA gene fragments were ligated intothe pGEM-T Easy Vector (Promega) and transformed into high efficiencyE. coli JM 109 competent cells. The transformed cells were screened forblue andwhite colonies by plating on Luria-Bertani (LB) agar plates con-taining ampicillin (100 μg/mL), Isopropyl β-D-1-thiogalactopyranoside

Fig. 1. Process overview for full length (1.5 kb) 16S rRNA based genomic D

(IPTG, 0.5mM) and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside(X-Gal, 80 μg/mL). Recombinant colonies were selected and checked forthe presence of 16S gene inserts by colony PCR using M13 forward andreverse primers (Integrated DNA Technologies, Inc.) that anneal to out-side of the pGEM-T vector (Promega) multiple cloning site.

2.2.3.3. Recombinant plasmid isolation and sequencing. Recombinantclones of each bacterial culture were grown in liquid LB broth contain-ing ampicillin (100 μg/mL), before plasmid isolation and purificationswere performed using the QIAprep (QIAGEN) Spin Miniprep Kit. Twoseparate sequence reactions using M13F and M13R primers werecarried out for each recombinant plasmid, according to the standardBigDye Terminator (BDT) v3.1 cycle sequencing kit (Thermo FisherScientific) protocol. DNA sequencing was performed using 3500Genetic Analyzer (Applied Biosystems, Hitachi) and the programFinchTV 1.4.0 (Geospiza Inc.) was used for editing of raw nucleotideDNA sequence data. The DNA sequences were polished by the removalof the plasmid vector sequences and merging of the two overlappingsequences to obtain a full length 1.5 kb contiguous sequence wasdone using the Emboss Needle tool of the European BioinformaticsInstitute (EMBL-EBI). The resulting full length 16S rRNA genesequences were then submitted to determine the closest matchingsequences by comparing with the bacterial and archaeal 16S RNAdatabases using the Sequence Match tool of the Ribosomal DatabaseProject (RDP) (Cole et al., 2014) and Basic Local Alignment SearchTool (BLAST) of NCBI, USA. The searches were limited to ‘Type’ bacterialstrains.

2.3. PCB degradation by individual bacterial cultures under aerobic, anaer-obic and two-stage anaerobic-aerobic conditions

Erlenmeyer flasks containing 75 mL of sterile MSM (same mediumused for selective enrichment) were prepared and 50 mg/mL Aroclor1260 stock solution in GCMS grade acetone was added to each flask assole source of carbon to give 50 mg/L Aroclor 1260 concentration.Contents of theflaskswere vigorously shaken for 3min to allow acetoneto evaporate. For each bacterial culture, three separate sets of experi-ments were carried out in parallel under aerobic, anaerobic and two-stage anaerobic-aerobic conditions. In all anaerobic and two stageexperiments, flasks containing media were prepared and incubated in

NA isolation, cloning and sequencing based molecular identification.


an anaerobic chamber (COY laboratory products, Inc.) under strict an-aerobic conditions. The liquid minimal salt medium in the flasks werekept equilibrated for oneweek inside the anaerobic chambermain com-partment before theywere inoculatedwith the seed cultures. Palladiumcatalysts located inside the chamberwere used to scrub any residual ox-ygen present in the chamber and the samples were transferred underanaerobic conditions without changes to the internal atmosphere inthe chamber through a heavy duty vacuum airlock compartment. Theenvironment inside the anaerobic chamber was maintained constantunder 4.9% H2, 10.7% CO2 and 84.4% N2 (BOC Australia). Under these an-aerobic conditions, hydrogen gas present inside the anaerobic chamberand controlled at 4.9% (mol/mol) concentration as thepotential electrondonor in the present study.

Aliquots of bacterial seed cultures (7.5 mL) grown overnight in LBbroth were centrifuged at 5000 ×g for 10 min. The resulting cell pelletswere washed twice in MSM, resuspended in 1 mL of the samemedium,and then transferred into the anaerobic chamber and used as theinoculum for each flask. Before inoculation of bacterial cultures, flaskswere maintained at pH 7. Flasks used in anaerobic experiments werekept at 28 °C in an incubator kept inside the anaerobic chamber, withoccasional gentle shaking by hand over the six weeks period. In aerobicexperiments, after inoculationwith seed cultures,flaskswere incubatedat 28 °C and 150 rpm under aerobic conditions for six weeks. The two-stage experiment was started and continued at 28 °C under similaranaerobic conditions (with occasional shaking by hand) for the firstfour weeks, and the flasks were removed from the anaerobic chamberand transferred to aerobic conditions at 28 °C and 150 rpm during thelast two weeks. The experiments were conducted in triplicate whilethe controls were conducted in duplicate. Minimal salt media spikedwith 50 mg/L Aroclor 1260 with no added bacteria seed cultures wereused as abiotic controls. Minimal salt media spiked with equal volumesof acetone that were used to dissolve 50 mg/L Aroclor 1260 were inoc-ulated with bacterial seed cultures similar to the experiment and usedas media controls.

2.3.1. Sample collection and analysisSamples (4mL) were withdrawn from each flask at time zero and in

weekly intervals to determine pH, cell growth and PCB solubility. Inorder to have representative samples, liquid aliquots were removedfrom themiddle of the culturemediumwhile flaskswere kept under ag-itation. Bacterial cell densities were calculated as colony forming units(CFU) using standard plate count. The DU730 Beckman Coulter UV/VISspectrophotometer was used to measure the cell growth at 600 nmoptical density and calibrated Hanna HI 2221 pH meter was used tomeasure the pH according to the APHA method 4500-H (APHA, 2012).At the end of six weeks, chloride ion concentration in each flask wasmeasured as described below in order to determine the amount ofchlorines released from the PCB mixture, by the direct action of themicrobes. If bacteria were able to dechlorinate the PCB molecules, thereleased chloride ions were expected to accumulate in the culturemedium. To see whether there is any significant contribution to thechloride level in the culture medium due to the lysis of bacterialcells, the chloride measurements were performed before and afterthe sonication of the samples. Samples were first centrifuged at5000 ×g for 10 min and the resultant supernatant was filteredthrough 0.2 μm sterile filter discs to remove the bacterial cells. Thecell free culture supernatant was used to measure the chloride ionconcentration using the Dionex ICS-2100 ion chromatographyaccording to USEPA method 300.0 (USEPA, 1993). For the calibrationcurve preparation, 0.1 ppm, 1 ppm, 5 ppm, 10 ppm, 20 ppm and100 ppm standard sodium chloride solutions were used. The abioticcontrols andmedia controls (see Section 2.2) were used in parallel inorder to subtract the background chloride levels coming from theminimal salt medium, leaking of any chloride due to bacterial celllysis and any traces left from the LB medium used to cultivate thebacterial seed cultures.

2.3.2. PCB extraction and analysisLiquid aliquots (1mL) sampled in weekly intervals were transferred

to 8 mL glass vials fitted with Teflon-lined screw caps. PCBs were ex-tracted with 2.5 mL of GC grade n-hexane (USEPA, 2007) by vigoroushorizontal shaking on a platform shaker for 4 h at 250 rpm underroom temperature, followed by centrifugation at 5000 rpm for 10 min(Adrian et al., 2009). The solvent phase was used for subsequentPCB analysis. Before extraction, 25 μL of 2, 4, 5, 6-tetrachloro-m-xylene(10 μg/mL in hexane) was added to each sample as the surrogate stan-dard to determine the extraction efficiency (USEPA, 2007). Surrogaterecovery was 98.98% ±19.11% (n = 308).

PCB extracts and standards were spiked with 2,2′,4,4′,5,5′-hexabromobiphenyl as an internal standard (USEPA, 2007). Total solu-ble PCB levels were determined as per the USEPA method 8082A(USEPA, 2007). Thermo Scientific Trace 1310 (in splitless mode, at260 °C inlet temperature, 80 mL/min split flow and 1.2 min splitlesstime) equipped with SSL injector with splitless liner with glass wool,Thermo TG-5SilMS analytical column (30m× 0.25mm ID × 0.25 μm)and, TriPlus RSH auto sampler were used for PCB analysis. Heliumwas used as the carrier gas at 1.2 mL/min constant flowrate. Thecolumn was kept at 40 °C for 2 min, and then the temperature wasraised to 300 °C at 15 °C/min and kept for 5 min. The ThermoScientific Trace Finder EFS software was used for the screening andquantitation of total soluble PCBs and PCB homolog groups usingthe Thermo Scientific triple stage quadrupole Mass spectrometer(TSQ8000 EVO). Retention times for each PCB homolog group wastaken from the existing literature (Walker and Feyerherm, 2013)and full scan acquisition and timed selective reaction monitoring(SRM) modes were used to distinguish the PCB homolog groups.Relative amounts of dissolved PCB levels and homolog groups weredetermined by nine-point calibration using Aroclor 1260 standardsolutions and area integration.

2.4. Screening for biosurfactant production

At the end of the six week experiment, 10 mL culture supernatantfrom each flask of the aerobic batch experiment were centrifuged at5000 ×g for 10 min and filtered through 0.2 μm filters to separate thebacterial cells. The cell free supernatants were used for the subsequentbiosurfactant screening tests.

2.4.1. Drop collapse testThe drop collapse test was used as a primary screening to deter-

mine the ability of bacterial strains for their biosurfactant productioncapacity based on past literature (Bodour et al., 2003; Alvarez et al.,2015; Panjiar et al., 2015; Joy et al., 2017). 20 μL cell free culturesupernatant was mixed with 5 μL of 0.1% methylene blue solutionand placed on parafilm paper as a drop using a pipette. The purposeof adding methylene blue was for easy visualization of the droplet.Diameter of the drop was measured after 1 min using 1 mm gritpaper placed underneath the parafilm paper. 1% (w/v) sodium dode-cyl sulphate (SDS) solution was used as the positive control whilephosphate buffered saline (PBS) solution and abiotic controls wereused as negative controls. The results where the diameter of thedroplet was at least 1 mL larger than the one made by the negativecontrol were considered as positive for biosurfactant production.When there is no biosurfactant present, the droplet remains stableas the polar water molecules are repelled from the hydrophobicsurface. When the interfacial tension between the liquid droplet andthe hydrophobic surface is reduced, the drop spreads or collapses(Alvarez et al., 2015).

2.4.2. Emulsification index (EI24)3 mL of cell free culture supernatant and 3 mL of mineral oil were

taken into a graduated test tube and vigorously shaken for 2 min toform an emulsion. The mixture was allowed to stand still for 24 h and


the height of emulsion layer was measured (Nayak et al., 2009). Theemulsification index was calculated using Eq. (1) (Panjiar et al., 2015).

EI₂₄ %ð Þ ¼ Height of emulsion formed after 24 hrs=Total height of solution� 100

ð1Þ

Emulsions formed following the reactions with different culturesupernatants were compared to the controls. The positive control usedwas a 1% (w/v) solution of synthetic surfactant sodiumdodecyl sulphatein deionised water, whereas the abiotic and medium only controlsamples were used as the negative controls.

2.4.3. HaemolysisTo detect the haemolytic activity indicative of biosurfactant

production, 50 μL of the cell free culture supernatant was spotted onthe middle of Tryptone soya agar plates containing 5% sheep blood(Thermo Fisher Scientific) and the plates were incubated at 28 °C for48 h (Alvarez et al., 2015). Plates were visually examined forhaemolysis. The level of clearance of red blood cellswas considered pro-portional to the concentration of biosurfactant. A yellow transparentzone indicated complete lysis of red blood cells and was regarded asbeta (β) or complete haemolysis. The appearance of dark green zonesbeneath the place where the supernatant was spotted was consideredas alpha (α) or partial haemolysis of blood cells. Alpha and betahaemolysis were considered as positive for biosurfactant production.No change in the blood agar plates indicated gamma (γ) or nohaemolysis (Joy et al., 2017).

3. Results and discussion

3.1. Identification of PCB utilizing facultative anaerobic culture members

After selective enrichment screening, three Gram negative (NP 03,04 and 06) and one Gram-positive (NP05) rod-like shape bacterialstrains capable of utilizing PCBs as sole source of carbon under bothanaerobic and aerobic conditions were isolated. During 16S rRNA genesequencing based identification, the closest matching sequences wereobtained from National Center for Biotechnology Information (NCBI)and Ribosomal Database Project (RDP) databases and are summarisedin Table 1. The RDP database was used as a curated database as itprovides the aligned and annotated rRNA gene sequence data (Coleet al., 2014; Wang et al., 2007), while NCBI was used as a non-curateddatabase. There were no differences between the two databases forculture numbers NP04 and NP05 as they obtained the same closestrelatives from both databases. However, similarity of culture numbersNP03 and NP06 were limited to the generic level between the twodatabases. There is no universal definition existing for species levelidentification using 16S rRNA gene sequencing and use of acceptablecriteria for establishing a species match varies widely in differentstudies (Janda and Abbott, 2007). Therefore, if similarity was not

Table 1Comparison of closest relatives of isolated bacteria based on NCBI and RDP databases.

Culture no. RDP database NCBI d

Similarityscorea

Unique commonoligomers

Closest match Maximscore

NP 03 0.996 1364 Achromobacter insolitus LMG6003, AY170847

2660

NP 04 0.994 1394 Ochrobactrum lupini LUP21,AY457038

2625

NP 05 0.997 1413 Lysinibacillus macroides LMG18474, AJ628749

2717

NP 06 0.995 1416 Pseudomonas citronellolis DSM50332T, Z76659

2686

a Similarity score - percent sequence identity over all pairwise comparable positions when r

100%, then identification was limited up to the generic level as there isno guarantee of having 1% divergence to obtain accurate identification.As none of the cultures showed 100% similarity to the existing data-bases, the four bacterial cultures were named according to their genusfollowed by the strain number. 16S rRNA sequences of the four cultures(NP03, NP04, NP05 and NP06) were deposited in NCBI GenBank data-base under the accession numbers KY711179, KY711180, KY711181and KY711182, respectively.

There was no visible growth in the media controls that containedequal volume of acetone used to dissolve Aroclor 1260. This findingconfirmed that the bacteria were not able to utilize acetone as their car-bon source even if there was residual acetone left in the medium afterevaporation. Bacteria such as Achromobacter sp. and Ochrobactrum sp.have been found in PCB contaminated sediments, but only under aero-bic conditions (Dudasova et al., 2014; Murinova and Dercova, 2014).However, in this study, it was found that the four facultative anaerobicbacterial strains Achromobacter sp. NP03, Ochrobactrum sp. NP04,Lysinibacillus sp. NP05 and Pseudomonas sp. NP06 were able to growon minimal salt agar containing Aroclor 1260 under aerobic and anaer-obic conditions indicating their ability to utilize PCBs as their sole sourceof carbon under both environmental conditions.

The facultative anaerobic bacterial strains with high PCB solubilityand degradation potential were further characterized based on theirability to utilize different carbon and nitrogen sources using BiologPM1 and PM3B plates. Achromobacter sp. NP03, Ochrobactrum sp.NP04 and Lysinibacillus sp. NP05 were able to utilize L-Proline as solesource of carbon and nitrogen. In addition, L-lactic acid and methyl py-ruvate were utilized as sole source of carbon (Table S1, SupplementaryInformation) and L-Glutamic acid, Ala-His, Ala-Leu and Gly-Gln wereused as nitrogen sources (Table S2, Supplementary Information) athigh rates by Achromobacter sp. NP03, Ochrobactrum sp. NP04 andLysinibacillus sp. NP05.

3.2. PCB degradation under aerobic, anaerobic and two stage anaerobic-aerobic conditions

3.2.1. Growth profiles measured at OD600 while grown on Aroclor 1260 ascarbon source

Fig. 2 shows the growth profiles of four facultative anaerobic strainsunder aerobic, anaerobic and two stage anaerobic-aerobic conditions.As evident in Fig. 2a, all four strains reached saturation by week oneunder aerobic conditions (OD600 of 0.51, 0.7, 0.62 and 0.46 for NP03,04, 05 and 06, respectively) and did not improve much further withtime. According to the literature, these results suggest the possibilityof all four strains consuming lower chlorinated PCB congeners withinthe firstweek andwere unable to degrade highly chlorinated congenersunder aerobic conditions (Field and Sierra-Alvarez, 2008). This conceptcan be supported by the fact that Aroclor 1260, the PCB source used inthis study consists of less than 10% (by weight) of lower chlorinatedhomolog groups (containing four or fewer chlorines per biphenyl

atabase Proposed name

um Identities Gaps Closest match

1476/1493(99%)

5(0%) Achromobacter denitrificansDSM 30026

Achromobacter sp.NP03

1437/1445(99%)

0(0%) Ochrobactrum lupini LUP21 Ochrobactrum sp.NP04

1486/1494(99%)

0(0%) Lysinibacillus macroidesLMG 18474

Lysinibacillus sp.NP05

1486/1501(99%)

4(0%) Pseudomonas knackmussiiB13

Pseudomonas sp.NP06

an with aligned myRDP sequences.

Fig. 2. Growth of four facultative anaerobic bacterial strains under (a) aerobic,(b) anaerobic, and (c) two stage anaerobic-aerobic conditions. Error bars represent thestandard deviation of mean values (n = 3).


molecule) compared to the highly chlorinated homologs (Mayes et al.,1998; ATSDR, 2000). To provide further support for these observations,Pieper and Seeger (2008) reported that aerobic bacteria are capable ofdegrading biphenyls as the sole source of carbon and energy, andusually involves the biodegradation of PCBs with less than four chlorineatoms. Therefore, the inability of bacterial cultures to degrade highlychlorinated congeners and the limited availability of lower chlorinatedcongeners in the medium may have negatively contributed to the lim-ited growth rate under aerobic conditions.

In comparison, during anaerobic conditions, all four strains of NP03,NP04, NP05 and NP06, showed higher cell densities with OD600 of 1.22,1.31, 1.04 and 0.82, respectively, reaching maximum growth by week 4(Fig. 2b). Significantly high growth rates under anaerobic conditionswithout the presence of carbon sources other than PCBs is an indicationof biphenyl ring cleavage in addition to dechlorination. According toexisting literature, anaerobic dechlorination is a reductive process thatuses PCBs as electron acceptors, but the carbon rings are usually notcleaved (Wiegel andWu, 2000;Hughes et al., 2009). In caseswhere bac-teria are not capable of breaking down the carbon ring structure, they

will require additional carbon sources in order tomaintain their growthduring dechlorination of PCBs (Wu et al., 2000; Bedard et al., 2006;Adrian et al., 2009; Wang and He, 2013). During this study, 8.7 × 106,5.0 × 106, 4.9 × 106 and 4.5 × 106 cells/mL initial cell densitiesat week zero were increased by 200 folds to 2.0 × 108, 1.3 × 108,1.3 × 108 and 1.0 × 108 cells/mL at week four for NP03, NP04, NP05and NP06, respectively. This fact confirmed the capability of these mi-croorganisms to utilize PCBs as their carbon source other than electronacceptors under anaerobic conditions without the requirement foradditional carbon sources.

In the two-stage anaerobic-aerobic cultivations as shown in Fig. 2c,all four strains showed similar growth patterns to the anaerobic condi-tions (Fig. 2b) up to week four during anaerobic conditions. However,after switching from anaerobic to aerobic conditions between weekfour to six, Lysinibacillus sp. NP05 started increasing in growth fromOD600 of 1.04± 0.07 to 2.63± 0.22, which is nearly three times its orig-inal cell density. Similarly, Ochrobactrum sp. NP03, Achromobacter sp.NP04 and Pseudomonas sp. NP06 also indicated slight increase in celldensities (1.39 ± 0.08, 1.48 ± 0.23 and 0.99 ± 0.06, respectively)when changing conditions from anaerobic to aerobic as indicated inFig. 2c. Based on these results, it can be postulated that all fourorganisms are capable of performing dechlorination under anaerobicconditions in a similar way at different rates and Lysinibacillus sp.NP05 subsequently hydrolyses the carbon ring structure extensivelyunder aerobic conditions compared to the other three organisms.

3.2.2. Variation of PCB solubility in aqueous mediumPCB concentrations were measured as total soluble PCBs in order to

investigate the change of solubility levels in themedium resulting frompotential bacterial activity under aerobic, anaerobic and two stage con-ditions. Solubility of PCBs in themediumwas an indirectmeasure of themicroorganisms attacking the PCBs and converting them from insolubleto soluble forms. Theoutcomes of this experiment are shown in Fig. 3. Atthe initial concentration of 50mg/L, it was observed that the PCBs addedto the flasks appeared to be not completely soluble, but most remainedat the bottom as small clumps prior to the addition of the bacterialcultures. The total soluble PCB levels in the abiotic controls with no bac-teria addedwasmeasured and found to remain very low throughout thecultivation period, with values found to be 0.15 ± 0.02 mg/L, 0.57 ±0.07 mg/L and 0.5 ± 0.23 mg/L under aerobic, anaerobic and twostage anaerobic-aerobic conditions, respectively. Such solubility rangesare consistentwith previous reports (ATSDR, 2000; Bedard et al., 2007).

Under aerobic conditions, Lysinibacillus sp. NP05 showed the highestcapacity to solubilize PCBs compared to Ochrobactrum sp. NP03,Achromobacter sp. NP04 and Pseudomonas sp. NP06 (Fig. 3a). Thisincrease in activity by Lysinibacillus sp. NP05 started after week oneand reached optimal activity by week five before it started to decrease.However, Lysinibacillus sp. NP05 did not increase in cell growth, butshowed similar cell densities to the rest of the three strains as shownin Fig. 2a. The increased solubility over the aerobic incubation periodwith no corresponding increase in cell growth shown by Lysinibacillussp. NP05 could possibly be due to two reasons. Firstly, their ability tosecrete some surface-active compounds that ultimately lead to in-creased solubility of the hydrophobic PCB mixture (Singer et al., 2000;Cameotra and Bollag, 2003). Secondly, the bacterium was unable tocarry out the dechorination of highly chlorinated congeners under aer-obic conditions as reported by Furukawa (2000) even though it mayhave the capacity to solubilize them.

In contrast to the aerobic conditions and as shown in Fig. 3b, PCBsolubility of all four cultures increased significantly under anaerobicconditions. A similar trendwas also observed in thefirst fourweeks dur-ing the anaerobic stage of the two stage anaerobic-aerobic conditions(see Fig. 3c). It was noted that soon after the addition of bacterialcultures, the insoluble PCB pellets started to disappear in the flasks,presumably as the PCBs became soluble due to the action of the micro-organisms. There are two possible explanations for the mode of action

Fig. 3. PCB solubility under (a) aerobic, (b) anaerobic, and (c) two stage anaerobic-aerobicconditions. Error bars represent the standard deviation of mean values from triplicates.Initial is the total soluble PCBs prior to the addition of microbes and week 0 isimmediately after addition of microbes.


by themicroorganisms that resulted in the increase in solubility of PCBs.Firstly, the dechlorination of PCBs could transform low water solublehighly chlorinated congeners intomorewater soluble lower chlorinatedcongeners as reported by Yin et al. (2011). If highly chlorinated conge-ners were dechlorinated into lower chlorinated congeners, there shouldbe more chlorides released to the medium. Therefore, measurementof chloride ions accumulated in the medium is a direct indicationof dechlorination, which is discussed in Section 3.2.3. Secondly, theenhancement of the solubility of hydrophobic PCBs was due to the pro-duction of bioemulsifiers or surface-active molecules (biosurfactants)by the microorganisms (Federici et al., 2012).

Findings of the two stage experiments are shown in Fig. 3c. Duringthe two stage study, the first four weeks were dedicated to anaerobicconditions before switching over to aerobic conditions in the last twoweeks. All four strains demonstrated similar trends in the first fourweeks under anaerobic conditions (Fig. 3b vs Fig. 3c). However, in thesecond stage, when the conditions shifted from anaerobic to aerobic,

the solubility of PCBs declined in Achromobacter sp. NP03,Ochrobactrumsp. NP04 and Pseudomonas sp. NP06, whereas in Lysinibacillus sp. NP05,there was a significant increase in PCB solubility. Parallel to theincreased PCB solubility, the growth of Lysinibacillus sp. NP05 also in-creased significantly during this period as shown in Fig. 2c aerobicphase. This confirmed the ability of Lysinibacillus to make hydrophobicPCB mixture soluble in the aqueous medium under both, anaerobicand aerobic conditions. Furthermore, it also confirmed the ability to de-chlorinate the highly chlorinated congeners during the anaerobic phaseand to degrade the resulting lower chlorinated congeners during theaerobic phase. This is an added advantage in bioremediation applica-tions as the organism can survive and degrade the PCB compoundsunder varying anaerobic and aerobic conditions.

3.2.3. Chloride ion build up and variation of pH in the aqueous mediumThe release of chlorides and their concentrations in the liquid me-

dium were measured as explained in Section 2.2.1. These measure-ments were taken as an indication of the dechlorination process thatoccurred following the addition of the four bacteria strains. The chlorideion concentrations in the abiotic controlswere alsomeasured and foundto be relatively constant throughout the experiment and there was noconsiderable difference between the initial and final chloride levelsunder aerobic, anaerobic and two stage anaerobic-aerobic conditions.The chloride ion concentrations in the controls were in the range of37.0 ± 0.9 mg/L. These background levels were concluded to comefrom chloride containing compounds in the basal minimal salt mediumcomprising of MgCl2·6H2O, CoCl2·6H2O, MnCl2·4H2O, NiCl2·6H2O andCuCl2·2H2O. The background chloride values coming from the abioticcontrols and media controls were first subtracted from the experimen-tal values, before the final values were plotted as shown in Fig. 4.

According to Fig. 4, accumulation of chloride ions in aerobic andanaerobic conditions provided clear evidence that the four facultativemicrobes discovered have the ability to dechlorinate the Aroclor 1260mixture under both, aerobic and anaerobic conditions. Significantlyhigh chloride ion concentrations in the combined anaerobic-aerobictreatment of all four cultures when compared to separate aerobic andanaerobic treatments further confirmed the effectiveness of combininganaerobic and aerobic degradation rather than isolated aerobic oranaerobic applications (Tartakovsky et al., 2001; Long et al., 2015). In-creasing levels of chloride ions in the culture medium were reportedby Yin et al. (2011) as a direct indication of dechlorination of PCB mol-ecules. Under anaerobic and two stage conditions, Lysinibacillus sp.NP05 demonstrated the highest chloride ion levels when compared tothe other three cultures and they were 5.2 ± 0.7 mg/L and 9.16 ±0.8 mg/L, respectively. Pseudomonas sp. NP06 had the lowest chloridelevels under all three conditions. As Aroclor 1260 theoretically contains60% chlorine by weight, the maximum chlorine level expected in50 mg/L Aroclor 1260 concentration is 30 mg/L. Therefore, the chlorideyield of 9.16 ± 0.8 mg/L observed in Lysinibacillus sp. NP05 under twostage anaerobic-aerobic treatment is an indication of the removal ofone third of total chlorine present in the Aroclor 1260 mixture.

The pH of the culture supernatants were also monitored togetherwith chloride ion build up and are shown in Fig. 5. The pH values ofthe abiotic controls remained relatively constant throughout the exper-iment (7.07 ± 0.14) under anaerobic, aerobic and two stage conditions.At the end of the aerobic and anaerobic experiments, the pHdid not sig-nificantly change and were 7.49 ± 0.07 and 6.95 ± 0.03, respectively.However, significant pH reduction was observed at the end of all thetwo stage anaerobic-aerobic experiments with values of 5.15 ± 0.03,4.98 ± 0.06, 4.97 ± 0.01 and 6.14 ± 0.03 obtained for Achromobactersp. NP03,Ochrobactrum sp. NP04, Lysinibacillus sp. NP05, and Pseudomo-nas sp. NP06, respectively (Fig. 5). When variations of the pH valueswere compared with the accumulation of the chloride ion levels in themedium, the negative correlation is clearly visible. Based on these re-sults, the lowering of pH under two stage anaerobic-aerobic conditionsappeared to have correlated well with the increase in chloride ions. The

Fig. 4. Chloride ion accumulation in the culturemedia after six weeks. The values shown here are after subtracting the background chloride levels coming from abiotic controls andmediacontrols. Error bars represent the standard deviation of mean values from triplicates.


high levels of chlorides under combined anaerobic-aerobic conditions isattributed to the dechlorination of highly chlorinated congeners underanaerobic conditions first, followed by further degradation of resultinglower chlorinated congeners after switching to aerobic conditions.

3.3. Biosurfactant production

An extracellular biosurfactant producing Lysinibacillus sp. waspreviously reported with the ability to solubilize different aliphaticand aromatic hydrocarbons such as hexane, benzene, toluene, dieseland kerosene (Panjiar et al., 2015). In addition, a bacterium isolatedfrom refinery wastewater was identified as Ochrobactrum sp. with thepotential to produce exopolysaccharide bioemulsifier and was able todegrade diesel oil (Ramasamy et al., 2014), n-octane, mineral light andheavy oils, crude oil (Calvo et al., 2008). However, there is no research

Fig. 5. Variation of pH and chloride ion concentrations after six weeks under aerobic, anaerobicrepresent the standard deviation of mean values from triplicates.

literature available that confirms the ability of these bacterial speciesto produce biosurfactants, which makes extremely hydrophobic PCBssoluble in aqueous medium while concomitantly degrading PCBs.Therefore, this study can be considered a first to report the potentialof Lysinibacillus, Achromobacter and Ochrobactrum species to producebiosurfactants that ultimately increased the solubility of PCBs.

In this study, the drop collapse and emulsification index tests wereused as quantitative methods (Ramasamy et al., 2014), while thehaemolytic assay was used as the qualitative method (Thavasi et al.,2011) to test for biosurfactant production. Results of these three testsare summarised in Table 2.

Among the four bacterial cultures, three showed positive results forthe drop collapse test. The drop spreads or collapses proportional to theconcentration of biosurfactants in the supernatant, due to the reductionof interfacial tension between the liquid droplet and the hydrophobic

and two stage anaerobic-aerobic conditions the initial pH was adjusted to 7.0. Error bars

Table 2Summary of biosurfactant screening tests.

Description Drop collapse test(diameter in mm)

Emulsificationindex (%)

Haemolysisa

Positive controlb 5.3 ± 0.3 50.0 +++Negative controlc 3.3 ± 0.3 ND NDAbiotic control 3.2 ± 0.3 ND NDAchromobacter sp. NP03 5.3 ± 0.3 33.3 ++Ochrobactrum sp. NP04 5.0 16.7 ++Lysinibacillus sp. NP05 5.0 50.0 +++Pseudomonas sp. NP06 3.5 ND +

ND - not detected.a The zones of clearingwere scored as follows: ‘+’ incomplete haemolysis; ‘++’ partial

haemolysis with semitransparent zones surrounded by green colour areas; ‘+++’ com-plete haemolysis.

b 1% (w/v) sodium dodecyl sulphate (SDS) solution.c Phosphate buffered saline (PBS) solution.


surface (Alvarez et al., 2015). A maximum drop diameter of 5.3 ±0.3 mmwas observed for the culture supernatant of Achromobacter sp.NP03. Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05 also demon-strated to have relatively high diameters of 5.0 mm, when comparedto 3.3 ± 0.3mmdiameter in the negative control (see Table 2). The rel-atively high droplet diameters of the supernatants confirms the releaseof biosurfactants by these bacterial cultures into the culture medium.

As summarised in Table 2, the highest emulsification index of 50%was observed in culture supernatants of Lysinibacillus sp. NP05. Thishigh result was followed by Achromobacter sp. NP03 (33.3%) andOchrobactrum sp. NP04 (16.7%). Emulsion formation was not observedin the culture supernatant of Pseudomonas sp. NP06. The production ofbiosurfactants was evident by the formation of the emulsion layer byLysinibacillus, Achromobacter and Ochrobactrum strains. The ability ofbiosurfactant production by these potential PCB degradingmicroorgan-isms to increase the solubility of hydrophobic PCBs without addition ofchemical or biological surfactant is an added advantage in bioremedia-tion applications.

Similar to positive results from the emulsification index test,Lysinibacillus sp. NP05 performed well during the haemolytic assay. Itshowed strong yellow transparent zones in Tryptone soya agar contain-ing sheep blood after incubation at 28 °C for 48 h indicative of completehaemolysis of red blood cells. In contrast, Achromobacter sp. NP03and Ochrobactrum sp. NP04 showed partial haemolysis having semi-

Fig. 6. Growth profile, PCB hydrolysis and pH variation of Lysinibacillus sp. NP05 under two stagfrom triplicates.

transparent zones surrounded by dark green areas. Pseudomonas sp.NP06 showed negative to minor haemolysis and this also coincideswith the low chloride accumulation observed.

Based on the three biosurfactant tests, the descending order of pref-erence for the potential of biosurfactant production is Lysinibacillus sp.NP05 N Achromobacter sp. NP03 N Ochrobactrum sp. NP04 N Pseudomo-nas sp. NP06. When the results of biosurfactant screening testswere compared with PCB solubility and chloride ion accumulation,Lysinibacillus sp. NP05 that exhibited the highest biosurfactant produc-tion potential, also demonstrated the highest total PCB solubility andchloride ion accumulation. These results provide a clear and positivecorrelation between biosurfactant production and PCB solubilizationand subsequent degradation. Not surprisingly, Pseudomonas sp. NP06that demonstrated the lowest biosurfactant production capability alsohad the lowest chloride ion levels in the culture supernatant.

According to the results presented in this study, it can be argued thatthe rate of microbial degradation of PCBs is decided not only by theirability of break down the PCB molecules, but also bymaking the hydro-phobic PCBs soluble in the aqueous medium. Therefore, it is importantto include suitable and different culture members with the ability toproduce biosurfactants in order to facilitate the bioavailability of PCBs.Such a strategy will be highly effective in field and scale up bioremedi-ation applications.

Overall, the data from the growth characteristics (Fig. 2), PCBsolubility (Fig. 3), chloride build up (Fig. 4), pH variation (Fig. 5)and biosurfactant production (Table 2) support the identificationof Lysinibacillus sp. NP05 as the best performer out of four facultativeanaerobic strains tested followed by Achromobacter sp. NP03,Ochrobactrum sp. NP04 and Pseudomonas sp. NP06. As shown inFig. 6, increase in growth rate parallel to the increased PCB solubilityunder combined anaerobic-aerobic treatment by Lysinibacillus sp.NP05 is an indication of the consumption of Aroclor 1260 as its carbonand energy source other than solubilization. Moreover, decrease in pHover the two weeks of aerobic phase as per Fig. 6 would coincide withthe occurrence of advanced PCB degradation steps to further break-down the chlorinated intermediates.

4. Conclusions

Based on an exhaustive review of past research literature, this re-search can be considered as the first comparative study which assessed

e anaerobic-aerobic conditions. Error bars represent the standard deviation of mean values


the capability and growth characteristics of facultative anaerobicbacteria in degrading PCBs under anaerobic, aerobic and two-stageanaerobic-aerobic cultivation conditions. The study found four bacterialstrains identified as Achromobacter sp. NP03, Ochrobactrum sp. NP04,Lysinibacillus sp. NP05 and Pseudomonas sp. NP06, to have the capabilityfor degrading commercial PCB mixture, Aroclor 1260 as the sole sourceof carbon under both, anaerobic and aerobic conditions. Among the fourstrains tested, Lysinibacillus sp. NP05 performed the best based on theresults from the comparative experiments on cell growth, PCB solubil-ity, chloride build up and biosurfactant production.

The results suggested that microorganisms capable of degradingPCBs also have the potential to produce surface active substances tofacilitate hydrophobic PCBs which are soluble in aqueous media andconsequently enhanced the bioavailability and degradation of PCBs.The two stage anaerobic-aerobic conditions produced the best overallresults when assessed on cell growth, PCB solubility and chloride buildup. In field scale soil remediation applications, facultative microorgan-isms have the potential to be better candidates as they can surviveand degrade PCBs under both anaerobic and aerobic conditions, whileachieving relatively higher degradation rates. Furthermore, based onthese results there is an opportunity to produce and apply tailor-madebacterial consortia for future process designs and applications resultingin shorter time frames, while effectively hydrolyzing PCBs.

Acknowledgement

The authors would like to acknowledge the Queensland University ofTechnology (QUT) for the Australian Government Research TrainingProgram (RTP) scholarship provided to the first author for undertakingthis doctoral study. We also gratefully thank the QUT Central AnalyticalResearch Facility (CARF) operated by the Institute of Future Environments(IFE) where the analytical data reported in this paper were obtained.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.scitotenv.2018.10.127.

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https://doi.org/10.1016/j.ecoenv.2015.05.038

https://doi.org/10.1007/s12182-014-0359-9

https://doi.org/10.1007/s12182-014-0359-9







https://doi.org/10.1016/S0043-1354(01)00175-0

https://doi.org/10.1016/S0043-1354(01)00175-0

https://doi.org/10.4172/2157-7463.S1-001








https://doi.org/10.1371/journal.pone.0059178

https://doi.org/10.1371/journal.pone.0059178

https://doi.org/10.1128/AEM.00062-07






https://doi.org/10.1007/s11274-011-0728-0

Bioremediation of Commercial Polychlorinated Biphenyl Mixture … · 2018-11-13 · ii...

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