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BACTERIAL INOCULUM DEVELOPMENT FOR EFFLUENTS

TREATMENT OF SHRIMP FARMING INDUSTRY

Evayantie Wahyuni Binti Zamudin

A thesis submitted

In fulfillment of the requirements for the degree in Master of Science

Faculty of Resource Science and Technology UNIVERSITI MALAYSIA SARAWAK

2013

ii

DECLARATION

I hereby declare that no portion of the work referred to in this dissertation has been submitted

in support of an application for another degree of qualifications of this or any other university

or institution of higher learning.

________________________

Evayantie Wahyuni Zamudin

Department of Molecular Biology Faculty of Resource Science and Technology Universiti Malaysia Sarawak

iii

ACKNOWLEDGEMENTS

Writing the acknowledgements is a wonderful phase to express in so few word all the

gratitude and deepest appreciation to people who made this Msc. dissertation possible.

Thank you to Prof. Dr. Kasing Apun, my supervisor for her dedicated supervision, guidance

and comments. The quality of her supervision is the best. Thank you for guiding me all the

steps to achieve this goal. Thank you indeed. My sincere gratitude also goes to Assoc. Prof.

Dr. Awang Ahmad Sallehin Awang Husaini and Dr. Micky Vincent, my co-supervisors for all

their concern and support.

My appreciation also goes to Universiti Malaysia Sarawak (UNIMAS) and Ministry of

Science, Technology and Innovation (MOSTI) for the opportunity given to pursue my goals

and awarding me with Post-Graduate Research Fellowship.

I wish to dedicate my dissertation to my family who is always proud of me, believed in me

and knew that I would do well. Their encouragement during the hard and stressful times for

the completion of this intense project was very crucial. They gave real inspiration for me to

achieve my Master degree into reality. I believe that my achievements are a reflection of the

love, effort and pray that I have received from them. My success belongs to them. My special

thanks go to my friends; Mr Tan Sia Hong, Mdm Lim Mui Hua, Ms Wan Adenawani, Ms

Chong Yee Ling and Ms Hashimatul Fatma, for their outstanding support and service

throughout my master programme. My deepest thanks to Mr Aziz Ajim, Mr Amin Manggi,

Ms Limjatai, Mdm Sheila Ungau and Ms Siti Hawa for excellent technical support. May God

bless all of you.

Evayantie Wahyuni Zamudin, 2013.

iv

ABSTRACT

In shrimp-pond farming, effective water quality management is critical for shrimp growth and

survival as well as to reduce the impact of shrimp farming on the environment. Therefore, this

study was conducted with the objective of developing indigenous bacterial formulation, which

can effectively treat shrimp pond effluents thus improving the water quality in shrimp

farming. To ascertain possible bacterial species to be used in the formulation, water samples

were collected from two shrimp farms in Muara Tebas and Santubong, Kuching, Sarawak,

Malaysia. Eleven water samples were collected from these two shrimp farms. Microorganisms

in the water samples with the necessary characteristics such as active proteolytic, cellulolytic

and amylolytic were isolated. These eight species of microorganisms were then identified

based on biochemical test, API 20NE and API 20E kit, followed by Polymerase Chain

Reaction (PCR) technique. The main species that had been identified as potential degraders of

pond effluents belonged to the Bacillus sp.. From the primary screening on solid media, eight

active isolates were chosen based on the size of the diameter of the clear zone surrounding the

colonies in the plate-screening medium. A time course study was performed for 72 hours for

each of the potential isolates in liquid media to analyze their growth curve and enzymes

production with respect to time. These isolates were individually further grown in shaked

flask cultures to determine the pattern of enzyme production, protein content and bacterial

growth. All of the monocultures (eight isolates) were then combined in minimal media for 24

hours at 37oC to determine the pattern of growth and enzymes production when in

combination of 1 : 1, 1 : 2, 2 : 1, 1 : 3, 3 : 1, 3 : 2 and 2 : 3. From the results obtained from

biculture combination, the best combination of bacteria was then tested for their ability to

grow and respond in a triculture system. 1% of Bacillus megaterium (S120): Bacillus cereus

v

strain 3 (S65): Enterococcus casseliflavus (S11) was selected as the best triculture

combination because they produced the highest enzymes of cellulase, amylase and protease.

Therefore, the formulation consists of three bacterial species identified as Bacillus

megaterium, Bacillus cereus and Enterococcus casseliflavus. The bacterial formulation was

then tested on shrimp pond water from two different aquaculture farms and UNIMAS’s pond

water. Chemical and biological parameters in the water were analyzed for seven days except

for UNIMAS pond where the treatment was extended to fourteen days. There were no

significant differences (P > 0.05) in water quality parameters such as orthophosphate, total

phosphorus, bacterial count, temperature and pH for samples from the shrimp pond water.

However, ammonia-nitrogen, nitrite, nitrate, turbidity, suspended solid, chemical oxygen

demand (COD), sulfide and biochemical oxygen demand (BOD) differed significantly (P <

0.05). Meanwhile for water samples from UNIMAS pond, nitrate, bacterial count,

temperature and pH showed no significant differences (P > 0.05). Other parameters such as

ammonia-nitrogen, nitrite, orthophosphate, total phosphorus, turbidity, suspended solid,

chemical oxygen demand (COD), sulfide and biochemical oxygen demand (BOD) differed

significantly (P < 0.05) compared to control experiment. The ability to reduce more than 50%

of eight water quality parameters in this study clearly showed that the indigenous bacterial

inoculums were a good shrimp pond effluents degrader.

vi

PEMBANGUNAN INOKULUM BAKTERIA UNTUK RAWATAN EFLUEN INDUSTRI

PENTERNAKAN UDANG

ABSTRAK

Pengurusan kualiti air yang berkesan adalah amat kritikal bagi ladang penternakan udang

yang intensif. Kesan penternakan udang terhadap alam sekitar juga tidak boleh di pandang

ringan. Oleh itu, kajian ini dijalankan bagi membangunkan formulasi bakteria natif, yang

efektif dalam proses rawatan kumbahan daripada kolam udang. Ini sekaligus akan

memperbaiki mutu kualiti air kolam udang terbabit. Spesis-spesis ini telah dipencilkan

daripada dua ladang penternakan udang yang terdapat di Muara Tebas dan Santubong,

Kuching, Sarawak, Malaysia. Di ladang-ladang tersebut, sebelas sampel air daripada kolam-

kolam yang berlainan diperolehi. Mikoorganisma di dalam sampel air yang mempunyai ciri-

ciri penting seperti aktif proteolitik, selulolitik dan amilolitik diasingkan. Kesemua lapan

spesis bakteria yang berpotensi ini tadi, kemudiannya dikenalpasti berdasarkan penggunaan

kit API 20E dan API 20NE, ujian biokimia dan seterusnya menggunakan teknik tindakbalas

rantai polimerase. Spesis utama yang berjaya dikenalpasti berpotensi menguraikan bahan

kumbahan kolam udang tergolong dalam spesis Bacillus sp.. Melalui ujian awal pada media

pepejal, lapan pencilan yang aktif telah dipilih berdasarkan diameter zon lutsinar yang

mengelilingi koloni-koloni bakteria di dalam piring medium ujikaji. Satu kajian yang

berterusan selama 72 jam telah dijalankan di dalam media cecair bagi setiap pencilan yang

berpotensi supaya pola pertumbuhan dan pengeluaran enzim pencilan terbabit dapat

dianalisis. Pencilan-pencilan ini tadi kemudiannya dibiakkan secara individu di dalam

kelalang kultur yang bergetar bagi menentukan corak pengeluaran enzim, kandungan protein

dan pertumbuhan bakteria. Kesemua monokultur (lapan pencilan) ini seterusnya

digabungkan di dalam media yang minimum selama 24 jam pada suhu 37oC bagi

vii

menentukan corak pertumbuhan dan pengeluaran enzim apabila di dalam kombinasi 1 : 1, 1 :

2, 2 : 1, 1 : 3, 3 : 1, 3 : 2 dan 2 : 3. Kombinasi bikultur yang terbaik akan dipilih untuk

dibiakkan di dalam sistem trikultur bagi melihat kebolehan mereka bertindakbalas dan

membiak. 1% Bacillus megaterium (S120): Bacillus cereus strain 3 (S65) : Enterococcus

casseliflavus (S11) telah dipilih sebagai kombinasi trikultur yang terbaik di atas kebolehan

mereka menghasilkan enzim selulase, amilase dan protease yang tertinggi. Oleh itu,

formulasi ini terdiri daripada tiga spesis bakteria yang dikenalpasti sebagai Bacillus

megaterium, Bacillus cereus dan Enterococcus casseliflavus. Formulasi bakteria ini

kemudiannya diuji pada air kolam udang daripada dua ladang berbeza dan juga pada air

kolam yang terdapat di UNIMAS. Parameter biologi dan kimia di dalam air kolam kemudian

dianalisis selama tujuh hari kecuali bagi air kolam di UNIMAS, di mana proses rawatan

diteruskan selama empat belas hari. Hasil kajian mendapati, tiada perbezaan yang signifikan

(P > 0.05) bagi parameter kualiti air kolam udang seperti ortofosfat, jumlah fosforus,

bilangan bakteria, suhu dan pH. Bagaimanapun, bagi ammonia-nitrogen, nitrit, nitrat,

kekeruhan air, pepejal terlarut, keperluan oksigen kimia, sulfida dan keperluan oksigen

biokimia menunjukkan perbezaan yang signifikan (P < 0.05). Sementara bagi sampel air

daripada kolam UNIMAS, nilai nitrat, bilangan bakteria, suhu dan pH tidak menunjukkan

perbezaan signifikan (P > 0.05). Parameter lain seperti ammonia-nitrogen, nitrit, orthofosfat,

jumlah fosforus, kekeruhan air, pepejal terlarut, keperluan oksigen kimia, sulfide dan

keperluan oksigen biokimia menunjukkan perbezaan yang signifikan (P < 0.05) jika

dibandingkan dengan eksperimen kawalan. Kebolehan untuk mengurangkan lebih daripada

50% daripada lapan parameter kualiti air di dalam kajian ini, jelas menunjukkan

keberkesanan inokulum bakteria natif di dalam proses penguraian bahan kumbahan daripada

kolam udang.

viii

TABLE OF CONTENTS

DECLARATION ii

ACKNOWLEDGEMENT iii

ABSTRACT iv

TABLE OF CONTENTS viii

LIST OF TABLES xii

LIST OF FIGURES xiv

LIST OF ABBREVIATIONS xvii

LIST OF PUBLICATIONS xix

CHAPTER 1 INTRODUCTION

1.1 Introduction 1

1.2 Objectives 10

CHAPTER 2 LITERATURE REVIEW

2.1 The Use of Microorganisms in Aquaculture Industry 11

2.2 Shrimp Pond Effluents 15

2.2.1 Effect of Shrimp Pond Effluents 16

2.2.1.1 Effects on Shrimp Culture 16

2.2.1.2 Impact on Environment 17

2.3 Bioremediation In Shrimp Farming Industry 18

2.3.1 Bioremediation of Organic Detritus 19

2.3.2 Bioremediation of Hydrogen Sulphide 20

2.3.3 Bioremediation of Nitrogenous Compounds 21

2.4 Molecular Characterization of Microbial Species 22

ix

2.4.1 Polymerase Chain Reaction (PCR) 23

2.4.2 16S ribosomal RNA gene sequencing 25

2.5 Application of Enzymes in Breakdown of the Feed Wastes 25

2.6 Application of Beneficial Microbes in Aquaculture 28

CHAPTER 3 MATERIALS AND METHODS

3.1 Isolation of Potential Bacteria from Shrimp Farms 30

3.1.1 Bacterial Sampling 30

3.1.2 Bacterial Isolation 30

3.1.2.1 Isolation of Cellulose Degrading Bacteria 31

3.1.2.2 Isolation of Starch Degrading Bacteria 32

3.1.2.3 Isolation of Protein Degrading Bacteria 32

3.2 Identification of Potential Bacteria 33

3.2.1 Gram Staining and Spore Staining 33

3.2.2 Biochemical Characterization of Bacteria 33

3.3 Working and Stock Cultures of Isolates 34

3.4 Identification Using Molecular Approach 35

3.4.1 Isolation of DNA 35

3.4.2 PCR Analysis 36

3.4.3 Agarose Gel Electrophoresis and Gel Documentation 38

3.4.4 DNA Purification from Agarose Gel 38

3.4.5 Quantification of DNA Concentration 40

3.4.6 16S rRNA Analysis 40

3.4.6.1 Sequence Analysis 40

x

3.5 Enzyme Assay 41

3.5.1 Cellulase Assay 42

3.5.2 Amylase Assay 42

3.5.3 Protease Assay 43

3.5.4 Protein Determination 44

3.6 Inoculum Development 44

3.7 Performance of Indigenous Bacterial Formulation 47

CHAPTER 4 RESULTS

4.1 Bacterial identification 49

4.1.1 Morphology on Selective Media 49

4.1.2 Biochemical Tests and Test Kits 53

4.2 Enzyme Studies 56

4.2.1 Screening for the Potential Amylase, Protease and 56

Cellulase Producer

4.2.2 Growth and Enzyme Production of Selected Isolates 59

4.3 Inoculum Development 67

4.3.1 Biculture Combination 67

4.3.2 Triculture Combination 79

4.4 Molecular Approach Using 16S Ribosomal RNA Gene to 81

Confirm Identity of Active Isolate

4.4.1 DNA Isolation 81

4.4.2 The Use of 16s Ribosomal RNA Gene for PCR Amplification 82

xi

4.5 Performance of Indigenous Bacterial Formulation in Laboratory Trial 84

To Improve Water Quality

CHAPTER 5 DISCUSSION 89

CHAPTER 6 CONCLUSIONS 97

REFERENCES 99

APPENDIXES 130

xii

LIST OF TABLES

Table 3.1 Designation, sequence, size and amplication size of

Primer pA and Primer pH.

37

Table 3.2 Specific PCR reaction mixture of 25 µl volume

reaction.

37

Table 3.3 Specific PCR amplication parameter. 38

Table 3.4 Bicombination of individual pure cultures

(monoculture + monoculture) 1%, 1.5% and 2%.

46

Table 3.5 Tricombination of individual pure cultures

(monoculture + monoculture+ monoculture).

46

Table 4.1 The morphology, colour, size, Gram staining and

spore staining of the 73 isolates.

50

Table 4.2 Number of potential isolates from each sampling site

and samples.

53

Table 4.3 Biochemical test results of the 65 bacterial isolates. 54

Table 4.4 Identified species of the 73 bacterial isolates. 56

Table 4.5 Potential enzyme producer among the isolates as

represented by clearing zones on minimal plate agar.

57

Table 4.6 Isolates producing the biggest clearing zones in

diameter on solid media containing substrate of

interest.

58

Table 4.7 A combination of Bacillus megaterium (S120), an

amylase producer and Bacillus cereus strain 3 (S65),

a cellulase producer after 24 hours in minimal

medium.

67

Table 4.8 A combination of Neisseria mucosa (S39), an amylase

producer and Bacillus cereus strain 1 (MT14), a cellulase

producer after 24 hours in minimal medium.

69

xiii


producer and Bacillus cereus strain 3 (S65),a cellulase


71


producer and Enterococcus casseliflavus (S11), a protease


73

Table 4.11 A combination of Bacillus cereus strain 1 (MT14), a

cellulase producer and Enterococcus casseliflavus (S11),

a protease producer after 24 hours in minimal medium.

75

Table 4.12 A combination of Bacillus cereus strain 3 (S65), a

cellulase producer and Enterococcus casseliflavus (S11), a

protease producer after 24 hours in minimal medium.

77

Table 4.13 Bacterial count, cellulase production, amylase production

and protease production when three isolates were

combined in minimal medium after 24 hours of

incubation.

80

Table 4.14 DNA quality of the three isolates as determined by

spectrophotometer reading.

81

Table 4.15 Comparison of the identities of the 3 isolates from

sequencing results and biochemical identification.

84

Table 4.16 Water quality parameters from two shrimp culture

ponds after 7 days of treatment. Values are mean

concentrations ± standard deviations.

86

Table 4.17 Water quality parameters from UNIMAS pond after 7

and 14 days of treatment. Values are mean

concentrations ± standard deviations.

87

xiv

LIST OF FIGURES

Figure 1.1 Diagram shows how biostimulation react. 5

Figure 1.2 Diagram shows how bioaugmentation react. 6

Figure 2.1 Tentative classification of microbial treatments used in

aquaculture, according to current terminology. 13

Figure 3.1 Tank 1 (Control): Filled with 20L water from shrimp

farm 1. Aerated without inoculums. Treatment Tank:

Tank 2: Shrimp Pond 1 water. Aerated + inoculum;

Tank 1 (Control): Filled with 20L water from shrimp

farm 2. Aerated without inoculums. Treatment Tank:

Tank 2: Shrimp Pond 2 water. Aerated + inoculum;

Tank 1 (Control): Filled with 20L water from UNIMAS

pond. Aerated without inoculums. Treatment Tank:

Tank 2: UNIMAS Pond water. Aerated + inoculums.

47

Figure 4.1 Hydrolysis zones around the colonies indicated

that the isolates can degrade: starch (a); protein

(b) and cellulose (c).

58

Figure 4.2 Growth pattern, protein content and cellulase production

of the isolate Lactococcus casei (MT53). The growth,

protein content and cellulase production were

investigated in minimal media at 37o

61

C.

Figure 4.3 Growth pattern, protein content and cellulase

production by Bacillus cereus strain 1 (MT14).

0.5% (w/v) carboxymetylcellulose (CMC) was used

to induce the enzyme synthesis.

61


of the isolate Bacillus cereus strain 2 (S67). The growth,



C.

62

xv


of the isolate Bacillus cereus strain 3 (S65). The growth,



62

C.

Figure 4.6 Growth pattern, protein content and amylase production

of the isolate Neisseria sicca (S38). The growth, protein

content and amylase production were investigated in

minimal media at 37o

64

C.


of the isolate Bacillus megaterium (S120). The growth,

protein content and amylase production were


64

C.


of the isolate Neisseria mucosa (S39). The growth,

protein content and amylase production were


65

C.

Figure 4.9 Growth pattern, protein content and protease production

of the isolate Enterococcus casseliflavus (S11). The

growth, protein content and protease production were


66

C.

Figure 4.10 Growth, protein content, amylase and cellulase

production of Bacillus megaterium (S120) and Bacillus

cereus strain 3 (S65) after 24 hours of incubation.

68


production of Neisseria mucosa (S39) and Bacillus

cereus strain 1 (MT14) after 24 hours of incubation.

70


production of Neisseria mucosa (S39) and Bacillus

cereus strain 3 (S65).

72

Figure 4.13 Growth, amylase and protease production of Neisseria

mucosa (S39) and Enterococcus casseliflavus (S11)

after 24 hours of incubation.

74

Figure 4.14 Growth, cellulase and protease production of Bacillus

cereus strain 1 (MT14) and Enterococcus casseliflavus

(S11) after 24 hours of incubation.

76

xvi

Figure 4.15 Growth, cellulase and protease production of Bacillus

cereus strain 3(S65) and Enterococcus casseliflavus

(S11) after 24 hours of incubation.

78

Figure 4.16 1% agarose gel electrophoresis of the PCR product from

the three isolates using Primer PA and Primer PH.

Lanes: 1- M [GeneRuler ™ 1 kb DNA ladder

(Fermentas)], 2-S11, 3- S120 and 4- S65.

82

Figure 4.17 1% agarose gel electrophoresis of the purified

DNA. Lanes: 1- M [GeneRuler ™ 1 kb DNA

ladder (Fermentas)], 2-S11, 3- S120 and 4- S65.

83

Figure 4.18 Graph showing 7 days of the bacterial count for two

shrimp ponds water and UNIMAS’s pond water.

88

xvii

LIST OF ABBREVIATIONS

α Alpha

β Beta

µl Micro-liter

mM Milimolar

Bp Base pair

DNA Deoxyribonuclease acid

DNS Dinitrosalicylic acid

dNTPs Deoxynucleoside-5’-triphosphates

TAE Tris-acetate-EDTA

EDTA Ethylene diamine tetraacetic acid

Hr(s) Hour(s)

Kbp Kilo base pair

M Molar

mg/mL Miligram per milliliter

min(s) Minute(s)

sec(s) Second(s)

mL Mililiter

NCBI National Centre for Biotechnology Information

Cm Centimeter

o Degree Celcius (temperature) C

OD Optical density

PCR Polymerase chain reaction

xviii

RNA Ribonucleic acid

rRNA Ribosomal ribonucleic acid

U Unit(s)

CFU/ mL Colony forming unit per milliliter

U/ µl Units per microliter

ug/mL Micrograms per milliliter

uM/ mL Micromolar per milliliter

v/ v Volume per volume

% Percent

Rpm

V

Revolutions per minute

pmol/ µl

Volt

UV

Picomole per microliter

ANOVA

Ultraviolet

Analysis of Variance

xix

LIST OF PUBLICATIONS

• Evayantie Wahyuni Zamudin and Kasing Apun (2007). Performance of an Indigenous

Bacterial Formulation in Improving the Water Quality of Shrimp Farm. Proceedings

of the International Conference on Natural Resources and Environmental Management

and Environmental Safety and Health. p 233 – 237. ISBN No.: 978-983-9257-79-3.

• Evayantie Wahyuni Zamudin, Kasing Apun, Awang Ahmad Sallehin Awang Hussaini

and Micky Vincent (2007). Effects of Microorganisms Application on Shrimp Pond

Water Quality. Proceedings of the 9th Symposium of the Malaysian Society of Applied

Biology.P-72, 30th - 31st

May 2007.

• Evayantie Wahyuni Zamudin, Kasing Apun, Micky Vincent and Awang Ahmad

Sallehin Awang Hussaini (2006). Screening and Isolation of Active Microbial

Degraders from Shrimp Farms. Presented at the 31st Annual Conference of the

Malaysian Society for Biochemistry and Molecular Biology. p 62, 17th

August 2006.

1

CHAPTER 1

INTRODUCTION

1.1 Introduction

The Tiger shrimp, Penaeus monodon Fabricus, is the main species cultured in aquaculture

industry and it contributed 87.4% (2,818.04 tonnes) of the total brackish water pond

production in Malaysia. The estimated wholesale of tiger shrimp production alone in 2007

generated RM 664,510,000.00 which is RM 121,814,000.00 more than the previous year

(Anon, 2007).

Intensive one-species aquaculture such as intensive shrimp pond-farming is not stable and

risky. It requires large applications of organic feed and mechanical energy per unit water

volume. This application focus on productivity from much larger land and oceanic areas

within a smaller area of shrimp grow out ponds (Folke and Kautsky, 1992; Kautsky et al.,

2000). As a result, large amounts of feed, feces and metabolic wastes accumulate in pond

waters and pond soils. These wastes are degraded through nitrogen degraders and other

decomposition processes to produce among other things such as ammonia, nitrite, nitrate and

phosphate (Suppaditl et al., 2005).

An important issue that needs to be addressed in this industry is the problem arising from the

discharge of shrimp effluents. Although there is legislation on discharge of the effluents, the

2

effluents are commonly discharged into rivers, thus polluting the river system. This has

become a major concern among environmentalists and government agencies.

There are many approaches being employed in trying to minimize the impact of shrimp

farming on the environment which can be divided into two practices. The first practice

involves direct treatment on the hatcheries ponds and separate treatment on effluent

discharged from these water bodies. Such method involved the use of bio-reactors, specially-

designed micro-niche systems (mats), filters (bio-filters and sand filters), weed or algae and

selected bivalves. The second practice uses the latest technology by using beneficial microbes

and enzymes. This type of application uses certain types of microbes and enzymes that

consume sludge constantly to provide cleaner, healthier and reusable water for the ponds.

Thus, frequent water intake or discharge is less needed. This new application is known as

bioremediation.

Bioremediation is defined as the process whereby organic wastes are biologically degraded

under controlled conditions to an innocuous state, or to level below concentration limits

established by regulatory authorities (Mueller et al., 1996; Vidali, 2001). Bioremediation is a

process that uses naturally occurring bacteria to degrade the environmental contaminants into

less toxic forms. The microorganisms may be indigenous to a contaminated area or they may

be isolated from elsewhere and brought to the contaminated site. Contaminant compounds are

transformed through reactions that take place as a part of their metabolic processes.

In order for bioremediation to be effective, microorganisms must enzymatically attack the

pollutants and convert them to harmless product. As bioremediation can be effective only

3

during the environmental conditions permit microbial growth and activity, its application

often involves the manipulation of environmental parameters to allow microbial growth and

degradation to proceed at a faster rate.

Although relatively few contamination compounds have been successfully bioremediated to

date, research is continuing to identify organisms and methods capable of degrading a variety

of other pollutants, and bioremediation remains an innovative and attractive pollution

abatement technology (King et al., 1997; Vidali, 2001).

An alternative to the enhancement of bioremediation by indigenous microorganisms is the use

of an inoculum of an appropriate pure or mixed culture of degrading microorganisms to effect

the removal of the undesired compounds (Gibson and Sayler, 1992; Rhee et al., 2004).

Bioremediation embraces biodegradation, which is often defined as complete mineralization

of the organic substrates to inorganic product such as carbon dioxide, water, inorganic

compounds and cell protein. Biodegradation of organic constituents is accomplished by

enzymes produced by microbes which are specific to the substances (Kim et al., 2007). In

addition, biodegradation of a compound is a stepwise process which is fundamentally an

electron transfer process that catalyzed by microbial enzymes.

Bioremediation can occur under two main conditions. Conditions that require all viable

atmospheric oxygen is known as aerobic condition. Aerobic bioremediation is usually

preferred because it degrades pollutants 10 to 100 times faster than anaerobic bioremediation

and it is usually the most effective for a complete degradation of the majority of pollutants

(Allard, 1997). The presence of appropriate redox conditions will determine which redox

4

regime will occur, and in turn, which kind of substance will be degraded. When oxygen

amount is limited, degradation may even occur, because various microbial communities are

able to be active under anaerobic conditions and to use alternate acceptors. In the absence of

oxygen, nitrate, manganese and iron oxides, and sulphate may act as suitable electron

acceptors (Liliana et al., 2006).

The two main types of bioremediation are in situ bioremediation and ex situ bioremediation.

In situ bioremediation is when the contaminated site is cleaned up exactly where it occurred.

It is the most commonly used type of bioremediation because it is the cheapest and most

efficient, so it is generally better to use (Vidali, 2001). There are two main types of in situ

bioremediation which is intrinsic bioremediation and accelerated bioremediation. In

accelerated bioremediation, either substrate or nutrients are added to the environment to help

break down the toxic spill by making the microorganisms grow more rapidly. The process

usually involves stimulating the indigenous subsurface microflora to degrade the

contaminants; although in most recent cases where genetically engineered microbes with

specialized metabolite activity have also been used (Kaplan and Kitts, 2004).

Meanwhile, biostimulation is the process of providing bacterial communities with a favorable

environment in which they can effectively

degrade contaminants (Kaplan and Kitts, 2004).

This is employed by introducing nutrients including nitrogen and phosphorous sources, pH

adjustments and soil aerations thereby enhancing the biodegradation process as shown in

Figure 1.1.

5

Figure 1.1: Diagram shows how biostimulation react.

Source:

www.ecocycle.co.jp/e_bioremediation/e_bioremediation.html

When biostimulation of the indigenous microflora present at the contaminated site is not

effective, a “bioaugmentation” strategy may be adopted. Bioaugmentation frequently involves

the addition of of preselected microorganisms, indigenous or exogenous, to the contaminated

sites. This ensure the proper team of microorganisms are present in the environment in term

of organisms type, number and compatibility, to effectively and efficiently seize the waste

constituents and break them down into their most basic compounds as shown in Figure 1.2

(Edgehill, 1999).

Bioaugmentation may be more suitable than biostimulation in the following cases: treatment

of high recalcitrant compounds, treatment of contaminant presents in relatively high or very

low concentrations, treatment of sites contaminated by very recent spillages or treatment of

environments where a particular physicochemical characteristic inhibits the activity of

indigenous microbiota. Bioaugmentation however involves addition when natural

communities of degrading bacteria are at low levels or not present, the addition of

contaminant-degrading organism is believed to speed up the process (Kaplan and Kitts, 2004).

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