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Varo-Arguello, W. E.; Camacho-Prez, B.; Ros-Leal, E.; Vazquez-Landaverde, P. A.; Ponce-Noyola, M.T.; Barrera-
Corts, J.; Sastre-Conde, I.; Rindernknecht-Seijas, N.F.; Poggi-Varaldo, H.M.* Triphasic slurry bioreactors for the
bioremediation of lindane-impacted soil under aerobic and anaerobic conditions.Environmental Engineering and
Management Journal; 2012, 11 (10): 1811-1823.
TRIPHASIC SLURRY BIOREACTORS FOR THE BIOREMEDIATION OF
LINDANE-IMPACTED SOIL UNDER AEROBIC AND ANAEROBIC
CONDITIONS
Wendy E. Varo-Arguello1, Beni Camacho-Prez
1, Elvira Ros-Leal
2, Pedro A. Vazquez-
Landaverde3, Mara T. Ponce-Noyola
4, Josefina Barrera-Corts
5, Isabel Sastre-Conde
6,
Noem F. Rindernknecht-Seijas7, Hctor M. Poggi-Varaldo
1
1Environmental Biotechnology and Renewable Energies R&D Group, Department of Biotechnology
and Bioengineering, CINVESTAV del IPN, P.O. Box 14-740, 07000 Mxico D.F., Mxico; 2Central Analtica, Department of Biotechnology and Bioengineering, CINVESTAV del IPN
3CICATA-IPN, Qro., Mxico;
4Microbial Genetics Group, Department of Biotechnology and Bioengineering, CINVESTAV del
IPN;5Control inteligente de Procesos, Department of Biotechnology and Bioengineering, CINVESTAV
del IPN6IRFAP, Conselleria d Agricultura i Pesca, Palma de Mallorca, Islas Baleares, Spain;
7
ESIQIE del IPN, Mexico D.F., Mxico
Abstract
The objective of this study was two-fold: (i) to evaluate the effect of co-substrate supplementation
and possible synergistic effect of the indigenous population and a lindane-acclimated inoculum on
the removal of lindane in three-phase, aerobic slurry bioreactors (SB) , and (ii) to evaluate the
effect final electron acceptor (O2, CO2 and SO4-2, or A, M, and SR, respectively) and
supplementation with carbon source (sucrose, 1 and 0 g/L; C or NC, respectively) on the removal of
lindane in triphasic lab scale SB. In a first experiment lindane was significantly removed in the first
week of operation (55-70%); its reduction further continued at a lower rate. Both factors had a
moderately significant effect; on the one hand, sucrose supplementation enhanced the removal of
lindane (p < 0.08); on the other hand the indigenous microflora and lindane-acclimated inoculum
In memoriam Dr. Y. M. Cabidoche who made outstanding contributions to the remediation of soils polluted with chlordecone and other
pesticides
Author to whom all correspondence should be addressed: e-mail: [email protected], Tel.: (5255) 5747 3800 ext 4324
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exhibited some kind of antagonism (p < 0.07), since removals in SB with sterile soil were higher
than those with live soil. In a second experiment, there was a significant effect of factor electron
acceptors on removal of lindane (p < 0.0001): lindane removal followed the order A > SR > M.
Supplementation with sucrose had a significant positive effect (p < 0.004). Main metabolites from
lindane degradation were chlorobenzene (CB), 1,2-dichlorobenzene (1,2-DCB) 1,3-dichlorobenzene
(1,3-DCB) and 1,2,4-trichlorobenzene (1,2,4-TCB) in aerobic and sulfate reducing slurry
bioreactors, only CB and 1,2-DCB were found in methanogenic units. Metabolites were consistent
with those reported in aerobic and anaerobic degradation pathways of lindane.
Keywords: bioremediation, clayish soil, lindane, slurry bioreactors, solvent, triphasic
1. Introduction
The application of lindane and technical HCH during the last six decades has resulted in
environmental contamination of global dimensions and concern (Camacho-Prez et al., 2012; Li et
al., 2003; Li, 1999; Li and MacDonald, 2005; Vijgen et al., 2011) due to its widespread use in the
past, pronounced toxicity as well as persistence against biotic or abiotic degradation, and its trend to
bioconcentration. Production and agricultural use of lindane has been banned in the StockholmConvention on Persistent Organic Pollutants (POPs) held in 26th August 2009 (UNEP, 2009).
However, in some countries, lindane is still used in public health applications to control insect-borne
diseases, and there are some unofficial reports that point out to illegal agricultural use in several
developing countries. More importantly, past use of lindane has created an environmental burden
and contaminated sites that are still waiting for remediation initiatives (International HCH and
Pesticides Association, 2012; Ramos et al., 2011; Romero et al., 2004; Zhang et al., 2005).
Lindane is introduced into the environment mainly via diffuse sources (agricultural runoff),
but also from point sources like production sites and pesticide spills (Wauchope et al ., 2002). In a
large set of countries (Austria, Brazil, China, Czech Republic, France, Germany, Hungary, India,
Italy, Japan, Macedonia, Mexico, Nigeria, Poland, Romania, Slovakia, South Africa, Spain,
Switzerland, Turkey, The Netherlands, UK, USA, and former USSR) between 4 and 7 million
tonnes of wastes of toxic, persistent and bioaccumulative lindane residues have been produced and
discarded during 60 years of lindane production. The amounts of lindane wastes and number of
countries with lindane problems increase if wastes and contaminated sites from lindane application
are considered (Fuentes et al., 2010; Ramos et al., 2011; Vijgen et al., 2011; Zhang et al., 2005).
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Human exposure to lindane and the negative effects on human health have been reviewed by
Nolan et al. (2011). The most important route of human exposure to lindane is ingestion of food
contaminated with this pesticide as well as dermal exposure. In Mxico, lindane has been detected
and its levels have been measured in human blood, human breast milk and human adipose tissue,
demonstrating that humans still are being exposed to lindane (Lpez-Carrillo et al., 2002; Ramos et
al., 2011).
In soils and sediments, lindane is degraded primarily by biotransformation, although
volatilization may play a positive role. It ks known that lindane can be uptaken by natural microbial
populations owing to their extremely high heterogeneity and adaptability to unfavourable
environmental conditions (Fuentes et al., 2010; Phillips et al., 2004). Yet, the hydrophobic nature of
organic pesticides leads to a strong sorption with the organic matter and clay contents of the soil and
this, in turn, significantly slows down any remediation process (Cabidoche et al., 2006, 2009; Poggi-
Varaldo et al., 2002, Poggi-Varaldo and Rinderknecht-Seijas, 2003).
Bioremediation constitutes a feasible approach to clean up soils and water systems
contaminated by -HCH because of its advantages over other alternatives such as incineration,
storage, or soil washing (Lal et al., 2010; Phillips et al., 2005; Robles-Gonzalez et al., 2012). As
we mentioned above, one challenge faced by bioremediation is to overcome the strong, irreversible
sorption of hydrophobic contaminants onto solid matrices (Robles-Gonzlez et al., 2008 and 2006;Zanaroli et al., 2010). In this regard, it is known that solvents added to a variety of biological
treatments allows increased mass transfer of oxygen and enhanced desorption of hydrophobic
pollutants sorbed onto soils (Dumont et al., 2006 and 2011; Zilouei et al., 2008 ). Former work of
our Group has shown the advantages of using silicone oil as a solvent in triphasic slurry bioreactors
(Robles-Gonzalez et al., 2012)). Thus, the objective of this study was two-fold: (i) to evaluate the
effect of co-substrate supplementation and the synergistic effect of the indigenouspopulation and a
lindane- acclimated inoculum on the removal of lindane in three-phase, aerobic slurry bioreactors,
in lab scale units, and (ii) to evaluate the effect of final electron acceptor (O2, CO2, and SO4-2
) and
biostimulation with a degradable carbon source (sucrose, 1 and 0 g/L; C and NC, respectively) on
the removal of lindane in triphasic lab scale slurry bioreactors.
2. Materials and methods
2.1. Soil and pesticide
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The soil used in the experiment originated from San Miguel Tequixtepec, Oaxaca (Table 1).
Its pH was 7.2, and had high contents of organic matter (8.1%). It contained 35, 33, and 42% of
sand, silt, and clay, respectively (Table 1). Lindane, or -1,2,3,4,5,6-hexachloro-cyclohexane reagent
grade from Aldrich Chemical Company, Inc., with 97% purity was used. Lindane is a moderately
lipophilic, organo-chlorinated substance characterized by a high partition coefficient octanol-water
Kow 4*103
(Robles-Gonzlez et al., 2012), with a low solubility in water, approx. 7 mg/L at 20C,
and slight polarity due to the strong electronegative effects of chlorine atoms bound to the aliphatic
ring.
Table 1. Main physico-chemical characteristics of mineral agricultural soil
used in this work
Parameter Value
Source San Miguel Tequixtepec, Oaxaca
Soil type Cambisol
pH 7.2 0.1
Organic matter (%) 8.1 0.1
COD a (mg COD/ kg dry soil) 5100 436
BOD (mg BOD5/kg dry soil) 3725 350
Clay (%) 42.3 0.8
Sand (%) 36.5 2.7
Silt (%) 21.2 3.3Texture Clayish
Hydraulic conductivity LowNotes:
aCOD: Chemical Oxygen Demand,
bBOD: Biochemical Oxygen Demand
2.2. Preparation and contamination of soil
The soil was sieved through a 20 mesh screen. Afterwards, it was sterilized by tyndalization
(three times at 121C for 60 min, with a 24 h period of incubation between treatments) except for the
soil used with live indigenous microbial population (LS). All soil samples were contaminated with a
dose of 100 mg lindane/kg dry soil: the adequate amount of lindane was dissolved in 500 mL of
acetoneand thoroughly mixed. The polluted soil was vacuum-dried at 40C in order to evaporate the
acetone.
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2.3. Seed bioreactors
Lindane-acclimated inocula for the slurry bioreactors were drawn from continuous suspended
growth reactors (aerobic, methanogenic, and sulfate-reducing units). The aerobic seed bioreactor
consisted of an aerated Eckenfelder cell of 14 L capacity operated at ambient temperature. It was fed
an influent containing (per liter of tap water): lactose (1g), KH2PO4 (0.17 g), K2HPO4 (0.57g)
Na2HPO4 (0.668g), NaHCO3 (0.02g), NH4NO3 (0.1855g), MgSO4 (0.022g); CaCl2 (0.0275g) and
lindane (7 mg). The methanogenic seed reactor was a 4 L capacity glass bottle and working volume
3 L that operated at 35C. It received an influent containing (per liter of tap water): sucrose (5g),
NaHCO3 (1.5g), K2HPO4 (0.6g), Na2CO3 (3g), NH4Cl (0.6g), CH3COOHglacial (1.5mL) and lindane
(7mg). The sulfate-reducing seed reactor was a 4 L capacity glass bottle and working volume 3 L
that operated at 35C. Its influent contained (per liter of water) sucrose (5 g), NaHCO3 (1.5 g),
K2HPO4 (0.6 g), Na2CO3(3 g), NH4Cl (0.6 g), Na2SO4 (13g), sulfuric acid (0.75 mL) and lindane (7
mg). Influents to seed bioreactors were prepared in batches of 2 L that were kept under refrigeration
until use. 14 mg of lindane in powder (Aldrich Chemical Co., Inc., 97% purity) was weighed in an
electronic balance Sartorius brand, model Micro, capacity 1500 mg, with precision 0.001 mg. The
lindane was added to each batch and solubilized by energic magnetic mixing.
All the seed bioreactors were operated for 1 year or more with lindane in their feeds.
2.4. Experimental design
2.4.1. First experiment
A 22
factorial experiment with two replicates was carried out. The effect of (i) co-substrate
sucrose concentration at 0 to 1 g/L (abbreviations NC, C, respectively), and (ii) indigenous soil
microbial population (live soil, LS; and sterile or dead soil, DS) were evaluated. Also an abiotic
control was made (sterile both soil and inoculum) was implemented.
The experimental setup for evaluating the kinetics of lindane removal was carried-out in lab
scale slurry bioreactors, which were prepared as follows: 20 g of soil contaminated with lindane
(100 mg/kg), 30 mg of volatile solids suspended (VSS) of inoculum acclimatized to lindane, 60 mL
of mineral medium and 20 mL of silicone oil, either with or without sucrose. Bioreactors were
incubated under aerobic conditions and stirred at 120 rpm at 25 C for 4 weeks; samples were
taken every week by destructive sampling (0,1,2,3,4 weeks).
The medium for aerobic slurry bioreactors contained the following (per liter): 33 mL of
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NaH2PO4 (0.2 M), 67 mL Na2HPO4 (0.2M), 180 mg (NH4)3PO4, 31.5 mg CaSO4, 23 mg FeSO4 and
22.5 mg MgSO4 (Pesce and Wunderlin, 2004).The pH of the medium (before mixing with soil) was7.2.
2.4.2. Second experiment
The effect of 2 factors at 3 levels was evaluated: co-substrate sucrose (1 and 0 g/L; C and NC,
respectively) and final electron acceptor (O2, CH4 and SO-4; or aerobic A, methanogenic M, and
sulfate-reducing SR, respectively). Abiotic controls were run (sterilized soil and inoculum).
Bioreactors were implemented and operated as described elsewhere (Robles-Gonzalez et al., 2012).
Methanogenic mineral medium was prepared as follows (per liter of deionized water) (modified
from Bachmann et al., 1988): 60 mg of K2HPO4, 100 mg of (NH4)3PO4. Sulfate reducing mineral
medium was prepared as follows (per liter of desionized water): 100ml phosphate buffer; 100mg
(NH4)3PO4; 10ml of vitamin stock, with a ratio COD/SO4= 2.5.
Both anaerobic media were formulated with a ratio 16C:1N depending of co-substrate
supplementation and 10mL of resazurin at (1g/L) as redox indicator. Lab scale slurry bioreactors
were seeded with corresponding inocula sampled from either aerobic, methanogenic or sulfate
reducing seed bioreactors. Liquors from seed bioreactors were sedimented (under anoxic conditions
when required) and decanted, and the biomass of concentrated liquors was determined asconcentration of VSS in mg/L. Taking into account the target seed biomass per slurry bioreactor (30
mg VSS per bottle), and the concentration of biomass in the concentrated liquors, the volumes of
concentrated liquor to be transferred to slurry bioreactors could be easily found with the mass
balance
VSL = 30 mg/CSL (1)
where
VSL stands for the volume of sedimented liquor, in L; and
CSL is the concentration of biomass in the sedimented liquor, in mgVSS/L
The slurry bioreactors were also loaded with 20 g of soil polluted with 100 mg lindane/kg dry soil,
60 mL of medium, and 20 mL of silicone oil.
2.5. Analytical methods
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The concentration of lindane was measured by Headspace-Solid Phase Microextraction-Gas
Chromatography- Electron Capture Detector in a Perkin-Elmer chromatograph equipped with
electron capture detector, and a Supelco capillary column (30m x 0.25mm x 0.5m). Temperatures
of column, injector, and detector were 210, 250C and 350C respectively. Nitrogen at 8 mL/min
was the carrier gas. A sample of 10 mL slurry was placed in a vial and stirred and heated at 80C for
10 min. After that, a solid phase microextraction fiber was immersed into it for 10 min at 80C. The
fiber was then placed into the injector of a gas chromatograph for thermal desorption of adsorbed
compounds for 5 min. Limits of detection for quantification was of 0.001 mg lindane/L level
(Prosen et al., 2002); lindane metabolites were extracted and analized as described by Quintero et al.
(2006).
Microbial counts of aerobic lindane-clastic microorganisms were carried out by plating. Petri
dishes were prepared by triplicate for each dilution (from 1X101 to 1X105); they were incubated at
30C for 10 days in a static incubator. After incubation, colonies were counted and results were
expressed on the basis of colony-forming unit (CFU) per mL of sample.
The medium R2A agar described in APHA (1987) Method 9215 A section 6, p. 9-55 was
used (per L of distilled water): K2HPO4, 0.3g; MgSO47H2O, 0.05g; agar, 15g; lindane, 7mg.
The liquid mixture pH was adjusted to 7.2 with either 2N HCl or NaOH before adding agar.
Afterwards, the medium was heated to dissolve agar and further sterilized at 121C for 15min.While warm, lindane was added and quickly dissolved by mixing with a sterile magnetic mixer in a
laminar flow hood surrounded by four burners to keep sterile and warm conditions. While warm,
the medium was poured into Petri dishes.
The soil pH was determined in a slurry soil/deionized water 1:2 (w/w) (Robles-Gonzlez et
al., 2012), soil texture was measured by the hydrometer method, soluble biochemical oxygen
demand (BOD) and soluble chemical oxygen demand were s estimated according to the Standard
Methods (American Public Health Association, 1981) in a warm water soil extract 1:5. Total soil
organic matter content was estimated by the method of oxidation with K2Cr2O7 (Robles-Gonzlez
et al., 2006). In the seed bioreactors were determined pH, sulphate (for the sulphate-reducing unit),
organic matter content as COD and biomass according to the Standard Methods (methods 423,
426C, 508 and 209E respectively). Lindane and metabolites sorbed onto biomass of seed bioreactors
were determined intermittently. The alkalinity and ratio were determined according to Poggi-
Varaldo and Oleszkiewicz (1992).
2.6. Statistical analysis
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Results of the experiment were subjected to analysis of variance using the software
Design-Expert (Stat-Ease, Inc., Minneapolis, MN, USA). Duncan test of means was performed
according to procedures described in Montgomery (1997).
3. Results and discussion
3.1. Seed bioreactors
Table 2 shows a summary of the performance of the seed bioreactors. Sulfate-reducing
SBs displayed the highest lindane removal. This is consistent with reports of Elango et al. (2011)
who mentioned that there are many sulfate reducing bacteria such asDesulfovibrio gigas, D.
africanus and Desulfococcus multivorans, able to dechlorinate organic compounds. Concentration
of lindane and its metabolites sorbed onto biomass in the seed bioreactors was negligible (data not
shown). Thus, lindane removal in the seed bioreactors was essentially biologically-mediated. The
specific removal rates in seed bioreactors were 336, 155, and 120 mg lindaneremoved/(kgbiomassd) for
the A, M, and SR seed bioreactors, respectively. These values were estimated on the basis of
average mass of biomass, other average data from Table 2 for the seed bioreactors, and an inputconcentration of lindane of 7 mg/L, using the corresponding mass balances.
3.2. First experiment
3.2.1. Lindane removal
The general trend of lindane concentration in all treatments was a drastic removal of lindane in
the first week (55-70%); lindane reduction further continued at a lower rate (Fig. 1). In thediscussion below, treatments have received convenient abbreviations as follows: NC-DS stands for
without sucrose and sterile soil, C-DS means supplemented with sucrose and sterile soil; NC-LS
stands for without sucrose and live soil, and C-LS represents supplemented with sucrose and live
soil. At the end of the experiment, the highest lindane removal was observed in treatment C-DS
(90%). NC-DS, C-LS, and NC-LS treatments showed removals of 87, 87, and 83%, respectively
(Fig. 1). Both factors had a moderate significant effect; sucrose supplementation slightly enhanced
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Table 2. Average performance of suspended growth biomass seed reactors used for
inoculation of slurry bioreactors
Parameter Average Standard deviation
Aerobic Methanogenic Sulfate reducing
Lindane (%)a 76.3 3.4 70.1 4.5 80.7 6.2
pH (-) 7.95 0.25 7.69 0.31 8.03 0.27
COD (%)b 69.8 9.2 67.3 10.1 59.9 9.5
VSS (mg/L) 1318 251 1463 274 1893 295
Coefficient NA 0.20 0.09 0.15 0.07
sulfate (%)c NA NA 76.5 7.9
CH4 (%) NA 44.7 13.1 NA
Notes:aLindane removal efficiency,
bChemical oxygen demand removal efficiency,
csulfate removal efficiency, VSS:
Volatile suspended solids, NA: Not applicable. Standard deviations calculated with respect to time.
the removal of lindane (p < 0.08). Indigenous microflora and lindane-aclimated inoculum exhibited
some kind of antagonism (p < 0.07), since removals of DS treatments were higher than those of LS
units. In contrast, Di Toro et al. (2008) reported an improved degradation of initial 10 g diesel/kg
soil in polluted soils using slurry reactors bioaugmented and biostimulated with a commercial
preparation Enzyveba, after 4.5 months of treatment. They suggested the occurrence of some kind of
synergism between indigenous and exogenous microflora.
Our abiotic control showed a low-to-moderate removal of lindane (11%). This indicates that
biotic removal contributed up to 79% of the overall lindane reduction.
3.2.2. Lindaneclastic bacteria counts
Lindaneclastic bacteria counts in our slurry bioreactors in treatments with sterilized soil and
live acclimated inoculum were in the order of 5 log CFU (Fig. 2). In contrast, the counts in the units
with live soil (live indigenous microflora) and live acclimated inocula were in the order of 3.5 log
CFU, independently of sucrose or no sucrose supplementation. This points out to some kind of
antagonism that was more evident after the second week of operation of the slurry bioreactors.
3.2.3. Intermediate metabolites
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Fig. 3 shows a typical chromatogram of the aerobic, triphasic slurry bioreactor supplemented
0
0.2
0.4
0.6
0.8
1
1.2
0 1 2 3 4
Lindane(C/C0)
t (wk)
a) b)
Fig. 1. (a) Time course of remaining lindane concentration in polluted soils treated in aerobic,
triphasic slurry bioreactors; (b) average lindane removals after 30 days batch operation treated in
aerobic, triphasic slurry bioreactors (Keys to cadre (a): () Abiotic control (AC); () supplemented
with cosubstrate, live indigenous soil microflora plus acclimated inoculum (C-LS); () non
supplemented, live indigenous soil microflora plus acclimated inoculum (NC-LS); () non
supplemented, sterilized soil plus live acclimated inoculum (NC-DS); () supplemented with co-
substrate, sterilized soil plus live acclimated inoculum (C-DS);
Keys to cadre (b): the error bar represents the standard error of the experiment SEE = (MSE/r)1/2,
where MSE is the mean square error and r is the number of replicates. AC: abiotic control; C-LS:
supplemented with cosubstrate, live soil and live acclimated inoculum; NC-LS, non supplemented,
live soil and live acclimated inoculum; NC-DS, non-supplemented, sterile soil and live acclimated
inoculum; C-DS: supplemented with cosubstrate, sterile soil and live acclimated inoculum)
with sucrose and bioaugmented with lindane-acclimated inoculum. The soil was sterilized.
Chlorobenzene (CB), 1,2-dichlorobenzene (1,2-CB) and a couple of trichlorobenzenes were found.
These metabolites are consistent with those found in the last stages of lindane aerobic degradation
(Camacho-Prez et al., 2012).
Interestingly, the highly chlorine substituted metabolites characteristic of the early stages of
lindane transformation pentachlorocyclohexane (PCCH) and tetrachlorocyclohexane TCCH (Lal et
al., 2010) were not detected in our SBs (Table 3).
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0
2
4
6
0 1 2 3 4 5
LogCFU
Time (week)
Fig. 2. Time course of colony-forming units (CFU) of lindaneclastic microorganisms in aerobic
slurry bioreactors (Keys: () supplemented with co-substrate sucrose, sterilized soil plus live
acclimated inoculum (C-DS); () non supplemented, sterilized soil plus live acclimated inoculum
(NC-DS); () supplemented with cosubstrate, live indigenous soil microflora plus live acclimated
inoculum (C-LS); (), not supplemented, live indigenous soil microflora plus live acclimated
inoculum (NC-LS)
3.3. Second experiment
3.3.1. Lindane removal
Lindane removal followed the order A > SR > M (Fig. 4). The ANOVA indicated that there
was a significant effect of factor electron acceptors on removal of lindane (p < 0.0001, Fig. 5).
The order of lindane removal in our work was slightly different to that reported by Robles-Gonzlez
et al. (2012) who found lindane removals in the order SR > A >> M in experiments with lab scale
slurry bioreactors without silicone oil. The difference was possibly due to the positive effect of
silicone oil on oxygen transfer (Daugulis, 2001) in A-SB of our current work. Sucrose
supplementation had a significant effect on the removal of lindane (p < 0.004, Fig 5), particularly in
the anaerobic treatments. Interestingly the increase of lindane removal was lower than expected
probably due to the important amount of indigenous soluble and degradable organic matter in the
soil (3.6 g soluble BOD/kg, Table 1). Interestingly, soluble organic removal efficiencies in sucrose-
supplemented SBs were consistently higher than those of the corresponding non-supplemented units
(methanogenic SB with sucrose, 74%; methanogenic SB without sucrose, 60%; sulphate-reducing
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Fig. 3. GC-MS detection of intermediate metabolites of lindane degradation in aerobic triphasic SB
at 30 day of batch operation (Supplementation with 1 g/L sucrose; sterile soil, bioaugmented with
lindane-acclimated inoculum from seed bioreactors)
SB with sucrose, 76%; sulphate-reducing SB without sucrose, 66%; aerobic SB, with sucrose, 78%;
and the aerobic SB without sucrose, 68%.) This was somewhat expected because sucrose is more
easily degradable than natural soluble organic matter of the soil.
The average specific removal rates of lindane rL/S in our best slurry bioreactors were approx.
rL/S = 90 mg removed lindane/((kgsoil)(30 d)) = 3 mg removed lindane/(kgsoil.d) (2)
This value is in the middle to high side of the range of specific removal rates reported in the
literature dealing with bioremediation of soils polluted with lindane (Table 3).
Quintero et al. (2006) observed nearly 100% removal of lindane in anaerobic slurry
bioreactors; their high results could be ascribed to the use of a massive inoculum (8 g VSS/L), the
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Table 3. Treatment of contaminated soil with lindane in slurry bioreactors
Microorganism
Externalsources of
carbon and
energy/Electrondonors/
Electronacceptors
Initialconcentration
of-HCH
MatrixExperimental
conditions
Intermediate
metabolites
Removal(%)and
removal rateRef.
Anaerobic
Granular sludge
(8 g VSS/ L)
Starch (2g
COD/L)
100 mg/kg
soil
Soil slurry
(Sandyslime soil)
pH 7
Temp. 30C
350 RPM
V: 4000mL
PCCH
TCCH
1,2,3-TCB
1,3-DCB
CB
100 % in 3 days
33.33mg/kg*d
1
Anaerobic
granular sludge
--------- 4.1 mg/kg soil Soil slurry
(sandyclayloam)
and 2.3%organicmatter
Temp. 30C
V:30mL
ND 95% in 2 weeks
0.24 mg/kg*d
2
Lindaneacclimatedinocula
(500 mgVSS/L)
Sucrose/sulphate 100mg/kg Soil slurry(clayishsoil with
8%organicmatter)
pH 7
Vt: 100 mL
120 RPM
PCCH
1,2,4-TCB;
1,2,3-TCB;
CB,
B
88% in 30 days
2.93 mg/kg*d
3
Lindaneacclimatedinocula
(500 mg
VSS/L)
Sequential
M-SR
100 mg/kg Soil slurry
(clayishsoil with8%
organicmatter)
pH 7
Vt: 100 mL
120 RPM
PCCH,
1,2,4-TCB
98% in 30 days
3.26 mg/kg*d
4
Lindaneacclimated
inocula
(500 mgVSS/L)
Sucrose/CH4 100 mg/kg Soil slurry(clayish
soil with8%organicmatter)
pH 7
Vt: 100 mL
120 RPM
NR 47% in 30days
1.57 mg/kg*d
5
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Mixed nativemicrobial
populationaclimated
glucose plusacetate (1:1) at
of 630 mg/liter;
SO4 ; HNO3
-HCH
400mg/kg
SoilSlurry
dark at 30Cin rotary
shaker (8 rpm;amplitude, 20
cm)
(30% water;wt/wt),
CB 3,5-DCP,TCP
6% in 100 days;
0.24 mg/kg*d;nitrifying
insignificant
transformation
6
acclimatedinocula
Sucrose / SO24 -HCH
100mg/kg
Soil slurry Triphasicreactor: 20%
v/v silicone oil
1,2,4-TCB
1,2-DCB
1,3-DCB
CB
84% in 30 days;2.8mg/kg*d
Thiswork
acclimatedinocula
NC / SO24 -HCH
100mg/kg
Soil slurry
(clayish
soil with8%organicmatter)
Triphasicreactor: 20%
v/v silicone oil
1,2,4-TCB
1,2-DCB
1,3-DCB
CB
78% in 30 days;
2.6 mg/kg*d
Thiswork
Mixed native
microbialpopulationaclimated
glucose plus
acetate (1:1) atof 630 mg/liter
CH4
-HCH
400mg/kg
Soil
Slurry
dark at 30C
in rotaryshaker (8 rpm;amplitude, 20
cm)
(30% water;wt/wt),
CB 3,5-DCP,
TCP
85% in 100 days;
3.4 mg/kg*d.
6
Lindaneacclimatedinocula
Sucrose/
CH4
-HCH
100mg/kg
Soil slurry
(clayish
soil with8%organic
matter)
Triphasicreactor: 20%v/v silicone oil
1,2-DCB
CB
35 % in 30days;
1.16 mg/kg*d
Thiswork
Lindaneacclimatedinocula
NC/CH4 -HCH
100mg/kg
Soil slurry(clayishsoil with
8%organic
matter)
Triphasicreactor: 20%v/v silicone oil
1,2-DCB
CB
22% in 30 days;
0.73 mg/kg*d
Thiswork
Aerobic
Pseudomonas
aeruginosaITRC-5
Withoutadditional
source of carbon Isomers of
HCH
2000mg/kg
soilslurry
rotary shakerat 28C at
200rpm
-PCCH
1,2,4-TCB
CHQ
98% in 15 days
130.67 mg/kg*d 7
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Microbacterium
sp. ITCR 1Isomers ofHCH and the
intermediatesas its sole
source of
carbon andenergy
Soil slurry 2,5 DCP 96% in 28 days
6.85 mg/kg*d
8
Lindane
acclimatedinocula
O2 -HCH
100 mg/L
Soil slurry
(clayishsoil with
8%
organicmatter)
NR 86% in 30 days
2.86 mg/L*d
5
Lindane
acclimated
inocula
Sucrose/ O2 -HCH
100mg/kg
Soil slurry
(clayish
soil with
8%organic
matter))
Sequential M-
A with
silicone oil
1,4 DCB
CB
82% in 30 days
2.73 mg/kg*d
9
Lindane
acclimatedinocula
O2 -HCH
100mg/kg
Soil slurry
(clayishsoil with
8%
organicmatter)
Partially-
aerated slurrybioreactor.
Triphasic
reactor: 20%v/v silicone oil
NR 39% in 30 days
1.3 mg/kg*d
5
Mixed nativemicrobialpopulation
O2 -HCH400mg/kg
Soil slurry dark at 30Cin rotaryshaker (8 rpm;amplitude, 20
cm)
(30% water;wt/wt),
-PCCH 100% in 100days
4 mg/kg*d
6
Pandoraeaspecies
O2 -HCH, -
HCH
100 mg/ L
of eachisomer
Soil slurry(sandy
loam soil)
orbitalincubator (160
rpm) at 25, 30,and 35 C) at
7-9pH
NR -HCH 60% in
30 days;
2 mg/L*d
10
Pandoraeaspecies O2 150 mg/L Soil slurry(15%) (30C,160rpm) NR 59.6 and 53.3%-HCH and -
HCH in 9 weeks
11
Lindane
acclimated
inocula
Sucrose/ O2 -HCH
100mg/kg
Soil slurry
(clayish
soil with8%
organic
matter)
Triphasic
reactor: 20%
v/v silicone oil
1,2,4-TCB
1,2-DCB
1,3-DCB
CB
90% in 30 days
3 mg/kg*d
This
work
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Lindaneacclimated
inocula
O2 -HCH
100mg/kg
Soil slurry
(clayishsoil with
8%
organicmatter)
Triphasicreactor: 20%
v/v silicone oil
1,2,4-TCB
1,2-DCB
1,3-DCB
CB
88% in 30 days
2.9 mg/kg*d
Thiswork
Notes: -HCH: hexachlorocyclohexane; 1,2,3.TCB:1,2,3-trichorobenzene,1,2,4-TCB:1,2,4-trichlorobenzene; 1,2-
DCB:1,2-dichlorobenzene; 1,3-DCB:1,3-dichlorobenzene; CB: Clorobenzene; COD: chemical oxygen demand; M-SR:
methanogenic-sulfate reducing; ND: Not detected; NR: Not reported; PCCH: Pentachlorocyclohexene; UASB: upflow
anaerobic sludge blanket; VSS: Volatile Suspended Solids; TCCH: Tetrachlocyclohexene; THCH: technical grade
hexachlorocyclohexane; References:1Quintero et al.(2006);
2Baczynski et al. (2010);;
3Robles-Gonzalez et al. (2008);
4Camacho-Prez et al. (2010);
5Robles-Gonzalez et al. (2008);
6Bachman et al. (1988) ;
7Manickam et al. (2008) ;
8Manickam et al. (2006a) ;
9Camacho-Prez et al. (2010) ;
10Siddique et al., (2002);
11Okeke et al. (2002)
coarse texture of their soil (sandy) where lindane is more easily desorbed and available for further
degradation, as well as the semicontinuous supplementation with organic co-substrate (Table 3).
Bachmann et al. (1988) also reported high removals of-HCH (100%) although after a long lag time
(about 25 days) in methanogenic microcosms loaded with polluted soil (Table 3). In contrast,
Robles-Gonzalez et al. (2012) found that methanogenic slurry bioreactors exhibited a poor
performance with a low 20 to 30% of lindane removal. Our results and those of Robles-Gonzalez et
al. (2012) with M-SB disagree with several works where it was observed that -HCH could bemore easily degraded by anaerobic cultures than aerobic ones (Bachmann et al., 1988; Buser and
Muller, 1995). Okeke et al. (2002) carried out experiments with SB inoculated with the bacterium
Pandorea sp., with a presumably anaerobic operation of 9 weeks duration. Initial lindane
concentration was 100 mg/kg; they found removals of 59.6 and 53.3% for - and -HCH,
respectively (Table 3). Lindane removals in our A- and SR-SB (90 and 88%, respectively) were
significantly higher and 2.4 times faster than results reported by Okeke et al. (2002).
Quintero et al. (2005) treated a sandy soil polluted with a mixture of isomers , , y -HCH
(100 mg/kg each isomers) in anaerobic SBs (Table 3). Starch was supplemented at 2 g/L every 3
days. They observed high removals of nearly 100% for the isomers and of HCH after 60 day
treatment.
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a) b)
Fig. 4. Time course of lindane concentration in slurry bioreactors (a) supplemented with sucrose; (b)
without sucrose (Keys: (): abiotic control; ( ): methanogenic bioreactor; (): sulfate reducing
bioreactor; (): aerobic units)
Fig.5. Overall lindane removal efficiency in slurry bioreactors (Keys: black bars supplemented with
sucrose and dotted bars without sucrose. A: aerobic SB; M: methanogenic SB; SR: sulfate-reducing
SB. *Error bars represent the standard error of the experiment SEE= (MSE/r) 1/2)
3.3.2. Intermediate metabolites
Metabolites from lindane degradation found in A- and SR-SBs were chlorobenzene, 1,2
dichlorobenzene, 1,3 dichlorobenzene 1,2,4-trichlorobenzene (Fig. 6 and 7). In M slurry
bioreactors we found chlorobenzene and 1,2- dichlorobenzene (Fig. 8). There was no apparent
accumulation of these metabolites; they are present at trace levels so it is likely that the lindane
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molecule was significantly dechlorinated, although lindane removal did not reach 100%. Most
metabolites identified in the final stage of operation of the A-SB are consistent with lindane aerobic
degradation pathways reported by Camacho-Prez et al. (2012) (Table 3).
Several microorganisms such as Pseudomonas sp (Tu, 1975; Sahu et al. 1995) can transform
-HCH to -pentachlorocyclohexene (-PCCH) in aerobic culture.Furthermore, Nagata et al. (1999)
found that the -HCH-dehydrochlorinase (LinA) of Sphingomonas paucimobilis catalyzes the
bioconversion of -HCH to 1,2,4-trichlorobenzene (1,2,4- TCB) via -1,3,4,5,6-pentachloro-
cyclohexene. On the other hand, Quintero et al. (2006) in a study on the degradation of HCH
isomers in anaerobic slurry reactors, found traces of diverse intermediate metabolite, such as
pentachlorocyclohexane isomers, tetrachlorocyclohexene, 1,2,3-trichlorobenzene, 1,3-
dichlorobenzene and clorobenzene. They concluded that low concentrations of the metabolites
indicated that intermediate compounds were not accumulated and they proceed to their further
degradation to CB, the end product in the degradation mechanism.
Interestingly, we could not find PCCH and TCCH in our SBs; it is known that these highly-
substituted metabolites are characteristic of the first stages in aerobic and anoxic pathways of
lindane degradation (Camacho-Prez et al., 2012). This would be consistent with our hypothesis
mentioned above regarding that the lindane molecule was significantly dechlorinated in our SBs,
although lindane removal did not reach 100%. Camacho-Prez (2010; reported also in
Camacho-Prez et al., 2012) studied the effect of adding silicone oil (as a desorption-aid) on the
performance of slurry sequential bioreactors treating a heavy soil polluted with lindane (Table 3).
They found that the sequential methanogenic-sulfate-reducing slurry bioreactors without silicone oil
showed the highest lindane removal efficiency 98%. Unexpectedly, in their work the units added
with silicone oil showed a close removal (up to 93%), although slightly lower than that of SB
without silicone oil. The sulfate-reducing stage of operation contributed the most to lindane
reduction: 41% of lindane was removed in the 15-d methanogenic stage whereas the sulfate-
reducing stage was responsible for 57% of lindane elimination. After 15 d operation PCCH was
detected in the slurries, whereas traces of 1,2,4-trichlorobenzene was present after 30 d incubation.
In another work Robles-Gonzalez (2008) assessed the bioremediation of a heavy soil polluted
with 100 mg lindane/kg in full (dominant) sulfate-reducing slurry bioreactors with no silicone oil.
Lindane removal was 88% whereas the detected metabolites after 30 d operation were PCCH; 1,2,4-
TCB; 1,2,3-TCB; CB, and benzene.
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Fig. 6. GC-MS detection of intermediate metabolites of lindane degradation in aerobic -SB (a) at 15
days and (b) at 30 days of batch operation (Peaks after 55 min retention time are tetradecamethyl-
hexasiloxane, piperidine, 1-(5-trifluoromethyl-2-pyridyl)-4-(1H-pyrrol-1-yl), hexadecamethyl-
Heptasiloxane, 1,1,3,3,5,5,7,7,9,9,11,11,13,13,15,15-hexadecamethyl-octasiloxane, probably from
the chromatographic column phase or silicone oil)
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Fig. 7. GC-MS detection of intermediate metabolites of lindane degradation in sulfate-reducing SB
(a) 15 days and (b) 30 days of batch operation
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600000 -
500000 -
400000 -
300000
200000 -
100000
Abundance y
x
z
Fig. 8. GC-MS detection of intermediate metabolites of lindane degradation in methanogenic SB at
30 day of batch operation
4. Conclusions
From the first experiment, we can conclude that:
Sucrose supplementation had a slight positive effect on lindane removal from soil with highcontents of clay and organic matter in aerobic, triphasic slurry bioreactors.
Indigenous soil microflora and inocula acclimated to lindane seem to be somewhatantagonistic regarding lindane removal.
Overall, aerobic triphasic slurry bioreactors bioaugmented with a lindane-acclimated inoculumand supplemented with sucrose could achieve a fast bioremediation of an agricultural soil with
high contents of clay and organic matter polluted with lindane.
In the second experiment, we also found that
There was a significant effect of factor electron acceptors on removal of lindane (p< 0.0001):lindane removal followed the order A > SR > M (90%, 84% and 35% with sucrose
supplementation, and 88%, 78 and 21% without sucrose, respectively).
Supplementation with sucrose also had a significant positive effect, particularly in theanaerobic slurry bioreactors (M- and SR-SB). Interestingly the increase of lindane removal was
lower than expected probably due to the important amounts of soluble and degradable organic
matter in the soil (3.6 g soluble BOD/kg ).
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Main metabolites from lindane degradation were chlorobenzene (CB), 1,2-dichlorobenzene(1,2-DCB) 1,3-dichlorobenzene (1,3-DCB) and 1,2,4-trichlorobenzene (1,2,4-TCB) in aerobic
and sulfate slurry bioreactors; only CB and 1,2-DCB were found in methanogenic units.
Highly-Cl-substituted metabolites such as PCCH and TCCH were not detected in our SBRs. It is
likely that a high extent of dechlorination occurred for the removed lindane in our work.
Acknowledgements
The authors express their sincere recognition to the Editor and the anonymous Reviewers of the
Journal; their insightful comments and suggestions helped to considerably improve the article. The
authors also wish to thank financial support from CINVESTAV-IPN. The excellent technical help of
Mr. Rafael Hernndez-Vera, MSBiol (GBAER-EBRE, Dept. of Biotechnology and Bioengineering,
CINVESTAV del IPN) Mr. Cirino Rojas and Mr. Gustavo Medina-Mendoza, both BSChemEng
(Central Analtica, ibidem) is sincerely appreciated. Two of the authors (WEV-A and BC-P) wish to
acknowledge graduate scholarships from CONACYT, Mexico. Design-Ease Inc. kindly provided a
free license of its software Design-Expert v8 to HMP-V, which is warmly appreciated.
Nomenclature
1,4 DCB 1,4-dichlorobenzeneA Aerobic
AC Abiotic control
ANOVA Analysis of variance
BOD Biochemical oxygen demandC Supplemented with co-susbtrate
CB Chlorobenzene
C-DS Supplemented with co-substrate, sterile soil and live acclimated inoculum
CFU Colony-forming unit
C-LS Supplemented with co-susbtrate, live soil
COD Chemical oxygen demand
CSL Concentration of biomass in the sedimented liquorDS Sterile soilGC-ECD Gas Chromatography- Electron Capture Detector
HCH Hexachlorocyclonexane
HS-SPME Head-space with solid-phase microextraction
LS Live soil
M Methanogenic
MSE Mean of the sum of squares of the error
NC Non-supplemented with cosusbtrateNC-DS Non-supplemented sterile soil and live acclimated inoculum
NC-LS Non supplemented, live soil and live acclimated inoculumPCCH Pentachloro cyclohexene
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POPs Persistent organic pollutants
r Number of replicates
rL/S Average specific removal rate of lindane in slurry bioreactors
SR Sulfate reducing
TCCH Tetrachloro cyclohexeneVSL Volume of sedimented liquorVSS Volatile suspended solids
Greek characters
Alpha coefficient, ratio of alkalinities
lindane Lindane removal efficiency
sulfate Sulfate removal efficiency
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