International Journal of Environmental Research University of Tehran ISSN: 1735-6865 EISSN: 2008-2304 Vol. 4, Num. 3, 2010, pp. 471-478

International Journal of Environmental Research, Vol. 4, No. 3, July-September, 2010, pp. 471-478

Article

Degradation of technical grade hexachlorocyclohexane in soil slurry by a defined microbial consortium

Rajashekara Murthy, H . M . , Thakur, M . S. and Manonmani, H . K . *

Department of Fermentation Technology and Bioengineering, Central Food Technological Research Institute, Mysore-570020, India

Correspondence Address: *Department of Fermentation Technology and Bioengineering, Central Food Technological Research Institute, Mysore-570020, India
manonmani_99@yahoo.com

Date of Submission: 04-Oct-2009
Date of Decision: 12-Mar-2010
Date of Acceptance: 22-Mar-2010

Code Number: er10052

Abstract

Keywords: Pesticide, Bioremediation, Bacterial isolates, Aerobic cultivation, Food safety

Introduction

Hexachlorocyclohexane (HCH) is an organochlo-rine insecticide that has been used world wide in agri-culture and public health programmes (Breivik et al., 1999).During the production of technical grade HCH, 85% of the product containing other isomers mainly α-, β- and δ-HCH would be dumped as waste, as they are not insecticidal.This causes serious soil pollution (Braun et al., 1991).Bioremediation of soil polluted with HCH is a subject of basic research.Biodegradation of HCH in soil has been attempted (MacRae et al., 1967, Bachmann et al., 1988, Doelman et al., 1985).

The degradation of HCH in soil by microorgan-isms depends on adsorption rate to the soil and its restricted availability for biological action (Bollag et al., 1992; Harms and Bosma, 1997).One possibility to help the mobility of the pollutant is its transfer from the soil to the liquid phase.The slurry phase system is a bioremediation technique where the contaminated soil is combined with water and other additives in stirred tank bioreactor (Alexander, 1994). This system mini-mizes the formation of aggregates and allows a faster equilibrium between the solid and the liquid phases by means of the suspension and vigorous agitation of the soil in the liquid medium.Both effects have been shown to favor mass transfer and thus enhance biodegrada-tion rate of the pollutants (Rogers et al., 1993). Soil slurry bioreactors have been used in the degradation of many pollutants such as pesticides, explosives, polynuclear aromatic hydrocarbons, and chlorinated organic pollutants (Robles-Gonzalez et al., 2008) The degradation of HCH isomers in soil slurry in labora-tory scale has been reported (Bachmann et al., 1988; Van Eekert et al., 1998).There are hardly any reports on the studies on a higher scale of degradation.We have developed a microbial consortium capable of degrading technical grade HCH by long term enrich-ment under aerobic conditions. Studies on the optimi-zation of degradation conditions in laboratory level is has been done(Murthy and Manonmani 2007 ) It was intended to assess the degradation capability of the developed consortium in a bioreactor in soil slurry Different environmental conditions optimized under shake flask experiments were adopted in bioreactor studies.The slurry experiment was performed in an aerobic slurry reactor.In this bioreactor, the spiked soil was suspended in stirred reactor vessel and mixed with water. HCH alone was supplied as a source of carbon.

Materials and Methods

Technical grade hexachlorocyclohexane (tech-HCH) was obtained from Hindustan Insecticides Pvt. Ltd. Mumbai, India. This was found to have all four isomers of HCH. Pure grade α-, β-, γ- and δ-isomers of HCH were purchased from Sigma-Aldrich Chemical Company, MO, USA. The bioreactor used was a 3 L capacity tank with 24 cm x 11 cm x 24 cm dimensions.Sterile soil (500g) spiked with required quantity of tech-HCH was taken in the reactor and mineral salts medium (M4 medium) (Murthy and Manonmani, 2007) was added up to 1L level. Mixing was done by the passage of air through the slurry at required air volumes. Soil slurry in the bioreactor was inoculated with induced microbial consortium at 500 ΅g protein/ g soil. Slurry was mixed well by bubbling air.Sampling was done at regular intervals and ana-lyzed for residual HCH, and surviving microorganisms (CFU).

Effect of aeration on degradation studied by pass-ing (bubbling) air at 0.5, 1.0 and 2.0vvm levels.Other experimental protocol was same as given for degrada-tion studies. Residual HCH was extracted from soil slurry by extracting with twice the volume of dichloromethane.Extraction was done thrice.The sol-vent fractions were pooled, passed over a bed of an-hydrous sodium sulphate, concentrated and purified by passing through florisil column.The fraction con-taining residual substrate was evaporated to dryness, resuspended in a known volume of acetone and used for quantification by Thin Layer Chromatography (TLC) and Gas Chromatography (GC). TLC was done using silica gel-G TLC plates.Residual substrate samples dissolved in required quantity of acetone were spotted on TLC plates and these plates were devel-oped in cyclohexane.The residual tech-HCH spots were identified after spraying the air-dried developed plates with O-tolidine in acetone.The residual substrate spots were delineated by marking with a needle and the area was measured.The concentration of residual substrate was computed from a standard plot of log concentra-tions versus square root of the area prepared for stan-dard tech-HCH.The acetone layer containing residual substrate after appropriate dilution was injected into gas chromatograph (Fison's model) equipped with ⁶³ Ni detector and SS column (200 cm X 2 mm) packed with 1.5% OV 17 plus 1.95 QF1 on chromosorb W 80/100 mesh.The column, injector and detector were main-tained at 230 ⁰ C, 250 ⁰ C and 320 ⁰ C respectively with a flow rate of carrier gas nitrogen at 50 mL/min. Under these conditions, the retention time of HCH-isomers was:α-HCH, 3.34 min; γ-HCH, 3.98 min; β-HCH, 4.42 min; δ-HCH, 5.1 min.The recovery of HCH isomers ranged from 92 to 95% from mineral salts medium. All the data presented in this study are based on triplicate estimations.The survival of individual members of the HCH degrading microbial consortium was done by estimating the Colony Forming Units (CFU) (Sahu et al., 1995).

Results and Discussion

The study is meant to assess the efficiency of an aerobic reactor to degrade HCH isomers contained in soil slurry.The optimized conditions obtained in our previous study were adopted in this reactor study to find out the applicability of shake flask trials in small reactors. Only HCH concentration was varied during degradation period.The optimized parameters used were: inoculum level 500 ΅g proteins/ g soil, pH 7.5 and incubation temperature 30 ⁰ C.

The soil used was red soil with no history of HCH applications [Table - 1]. Soil had a particle size less than 0.5 mm. This size was chosen to provide high superfi-cial area for interaction between HCH and microorgan-isms. [Table - 2].

At 10 ppm of HCH concentration, all the four isomers were degraded during incubation period. γ- isomer was degraded faster by 72 h [Figure - 1] with a degradation rate of 0.0179 mg /L/ h [Table - 3]. Other isomers α-, β - and δ-HCH took 120 h for complete degradation with rates of 0.0186, 0.0136 and 0.0176 mg /L/ h respec-tively [Table - 3]. There was no initial lag. Degradation of all isomers started even at 3 h.At 25 ppm of tech-HCH level, time required for degradation was more. Among four isomers, degradation of -, β - and δ-HCH started immediately without any lag [Figure - 2]. But deg-radation of δ-isomer started only after 12 h of incuba-tion. γ-isomer was degraded completely by 96 h with a degradation rate of 0.0126 mg/ L/ h [Table - 3]. α-,and β -isomers were degraded completely by 168 h of incu-bation at rates of 0.0122 and 0.0144 mg/ L/ h respectively.δ-isomer was not completely degraded even by 168 h with still nearly 10% of the substrate remaining after degradation period.

The survivability of microbial cells in both 10 and 25 ppm were studied. At 10 ppm of tech-HCH level, all the members of the consortium survived till the end of degradation period [Table - 4]. However, isolate T ₇ was reduced in number, compared to other isolates.At 25 ppm of tech-HCH concentration, the survivability pattern was almost the same as that in 10 ppm. All the isolates survived till the end of degradation period.

At 0.5 vvm aeration rate, degradation was slow. Incubation time of 168 h was required for complete degradation of all isomers [Figure - 3]. The easily degrad-able γ-isomer and α- and β-, took 144 h for complete degradation. δ-isomer was degraded completely by 168 h. The degradation rate was 0.0092, 0.0137, 0.0147 and 0.0129 mg/ L/ h for α-, β-, γ- and δ-isomers respectively.At aeration rate of 1.00 vvm [Figure - 4]. the degradation of α-, β- and γ- isomers were complete by 168 h of incubation. δ- isomer was not degraded com-pletely with nearly 15% of the isomer remaining at 168 h. The rates of degradation were 0.0144, 0.0086, 0.0128 and 0.0100 mg/ L/ h respectively for α-, β-, γ- and δ--isomers [Table - 5].The degradation decreased with in-crease in aeration to 2.0 vvm [Figure - 5]. None of the isomers were degraded completely even by 144 h of incubation. Nearly 35% of α-, 54% of β-, 28% of γ-and 52% of δ-isomers of 10 ppm of tech-HCH were found to be remaining even after 144 h of incubation. The degradation rates were 0.0061, 0.0049, 0.009 and 0.0051 mg/ L/ h, for α-, β-, γ- and δ-isomers, respec-tively [Table - 5].

HCH has been used world wide as general broad spectrum insecticide for a variety of purposes includ-ing fumigation of the house hold and commercial stor-age areas, pest control on domestic animals, mosquito control and to eradicate soil-dwelling and plant-eating insects. Although only lindane has insecticidal prop-erty, HCHs as group are toxic and considered potential carcinogens (Walker et al., 1999) and listed as priority pollutants by the US EPA. Due to their persistence and recalcitrance, HCHs continue to pose a serious toxicological problem at industrial sites where post production of lindane along with unsound disposal practices has led to serious contamination. In addi-tion, many countries including India have permitted HCH production (lindane is permitted to be used) and use. This has become a global issue due to problems of volatility and transportation of HCH isomers by air to remote locality (Galiulin et al., 2002; Walker et al., 1999). Due to the toxicity and persistence of HCH, soils contaminated with HCHs have been targeted for remediation. Biodegradation of α-, β-, γ- and δ- iso-mers of HCH have been extensively studied in the labo-ratory at individual level.But information is insufficient on pilot or full-scale in situ field settings. The HCH-isomers have been shown to differ in their persistence in soil and in their properties like solubility and volatil-ity that determine their rates of biodegradation.Earlier studies suggested that degradation of HCH was faster under anoxic conditions and that microbial degrada-tion was primary route of HCH disappearance from soil (MacRae et al., 1967).Microbial degradation of all the HCH-isomers has since been observed under oxic conditions both in soil (Bachmann et al., 1988; Doelman et al., 1985; Sahu et al., 1993) and in pure cultures of microorganisms (Bhuyan et al., 1993; Thomas et al., 1996).We have isolated in our laboratory a microbial consortium consisting of ten bacterial isolates which have got the capacity to degrade HCH (Manonmani et al., 2000; Murthy and Manonmani, 2007) under oxic conditions. Translation of the laboratory scale trials to small reactors was studied in soil slurry. Soil slurry has been adopted for the microbial degradation of pes-ticides, explosives, polynuclear aromatic hydrocar-bons, and chlorinated organic pollutants (Robles-Gonzalez et al., 2008) . An anaerobic reactor was used to degrade HCH isomers contained in soil slurry cul-tures The influence of different environmental condi-tions was evaluated: the HCH concentration (25-100 mg HCH/ kg), the type of substrate (volatile fatty acids or starch), the sludge concentration (2-8 g VSS /L ) and the replacement of spiked soil to simulate a fed-batch operation (10-50%) were tried (Quintero, 2005, 2006).The slurry-phase system was adopted to mini-mize the formation of aggregates and to allow faster equilibrium between solid and liquid phases by means of the suspension and vigorous agitation of the soil in the liquid medium. Both these effects have favored mass transfer and thus enhance the biodegradation rate of pollutants (Rogers et al., 1993). To achieve these advantages the degradation of HCH in soil was stud-ied in a slurry system. The moisture content in soil has been shown to influence greatly HCH degrada-tion. Chessells et al. (1988) have reported a correlation between soil moisture content and removal rates of HCH isomers in field agricultural soils. Enhanced removal of HCH in soils with higher moisture contents has been reported. This has been made possible due to prevailing anoxic conditions during flooding. Thus anaerobic metabolism has been reported to exist in these soils.In our earlier studies, soil moisture content of 15 to 20% was found to give good biodegradation of HCH-isomers (unpublished data).As our microbial consortium consisted of aerobic microorganisms, air was passed through soil slurry to maintain oxic condi-tion. Similar degradation of α-HCH under oxic condi-tions in either moist soil or soil slurries has been re-ported (Doelman et al., 1990). Degradation of 23 mg/ kg/ day of α-HCH in soil under oxic conditions have been obtained (Bachmann et al., 1988). However, re-duction of 13 mg/ kg/ day was obtained under methanogenic conditions.Van Eekert et al. (1998) have reported the removal of α-HCH from a sandy soil con-taining low concentration of the isomer in slurries where lactate or sulfide had been added to reduce redox potential.Degradation of a-HCH in glass columns packed with contaminated sediments and held under methanogenic conditions has been reported (Middeldorp et al., 1996), although degrading popula-tion of microorganisms appeared not to be methanogens. Degradation of-HCH under oxic con-ditions has also been reported (Yule et al., 1967). 13-HCH isomer, an indisputably most recalcitrant isomer, does not undergo biodegradaion easily. The concen-tration did not decrease noticeably in field study un-der any treatment (moist soil and oxic soil slurries in small pots) (Doelman et al., 1985).

Conclusion

Although HCH removal has been observed under both oxic and anoxic bioremediation treatments, treat-ments under oxic condition have resulted in the almost complete removal of HCH via mineralization. These observations are on par with our results wherein un-der oxic conditions good degradation of HCH-isomers of technical mixture has been observed. Even though all the four isomers were present together in technical mixture, no adverse or inhibitory effects were observed by either parent compounds or their metabolites.We have tried to address the inadequately addressed topic of bioremediation of HCH contaminated soils in slurry. With the disadvantages of slurry bioreactors such as requirements for soil excavation, handling, condition-ing and bioreactor construction/operation that typi-cally increase treatment costs compared to most simple bioremediation techniques (Cookson 1995).The suc-cessful results obtained in small scale soil bioreactors need to be addressed during translation further to still larger scale.

Acknowledgements

Authors thank Director, Central Food Technologi-cal Research Institute, Mysore for his constant sup-port to this work.Financial assistance from CSIR, New Delhi in the form of SRF is gratefully acknowledged.^[26]

References

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