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Short communication

Degradation of hexachlorocyclohexane (HCH;

a

,

b

,

g

and

d

) by

Bacillus circulans

and

Bacillus brevis

isolated from soil

contaminated with HCH

Archna Gupta, C.P. Kaushik*, A. Kaushik

Department of Environmental Science and Engineering, Guru Jambheshwar University, Hisar-125 001, Haryana, India

Accepted 3 April 2000

Abstract

Hexachlorocyclohexane (HCH;a,b,gandd) degradation eciency of acclimatized Bacillus circulansandBacillus breviswas studied under aerobic conditions on nutrient agar. These bacteria were isolated from HCH contaminated soil and acclimatized to dierent concentrations of HCH for more than 2 years. Acclimatized bacteria degraded at a signi®cantly high rate not only theaandgisomers but also the thermodynamically stablebanddisomers at dierent concentrations, suggesting their potential for degradation of even the aged residues.72000 Elsevier Science Ltd. All rights reserved.

Keywords:Bioremediation; HCH degradation;Bacillus circulans;Bacillus brevis; Pesticide; Soil bacteria

Bacillus circulans and Bacillus brevis, isolated from

the soil contaminated with hexachlorocyclohexane (HCH), degraded the four isomers (a, b, g and d) of HCH, under aerobic conditions on nutrient medium. These bacteria were acclimatized to dierent concen-trations of HCH for more than 2 years. Degradation occurred at a signi®cantly high rate with acclimatized bacteria. These acclimatized bacteria were also capable of eciently degrading HCH isomers at a lower con-centration of 1 mg/ml. While the widespread use of insecticide HCH has been discontinued for a number of years in certain countries, the problem of residues of all isomers of HCH remains because of the high persistence and interconversion of these isomers in soil (Steinwandter and SchluÈter, 1978). Attempt has been made to accelerate the degradation of these residues by the use of micro-organisms. Our study was aimed at isolating the micro-organisms from soil which have had long exposure to HCH and acclimatizing them

under laboratory conditions to increase their HCH degradation eciency. Samples from HCH contami-nated soil were taken upto a depth of 5 cm, from four dierent sites in the surroundings of Hindustan Insecti-cides Ltd., New Delhi, India. Soil samples were pooled and 5 g of soil, after homogenisation, was added to 50 ml sterile water and stirred vigorously for 30 min. This suspension was serially diluted (10) and the 10ÿ6 di-lution was used as inoculum for isolating the micro-organisms required to degrade the isomers of HCH. Inoculum (1 ml) was added to an Erlenmeyer ¯ask containing 50 ml nutrient broth (Difco Manual, 1953) and 5 mg/ml HCH. It was incubated at 33228C for 3 days. The culture was serially diluted and plated on petridishes containing 25 ml of nutrient agar and 5mg/ ml HCH. Bacterial colonies appearing on the plates were streaked till the pure colonies were obtained. Pure micro-organisms were tested for their ability to degrade HCH isomers. The bacterial colonies showing HCH degradation capability were acclimatized by reg-ularly exposing them to HCH for more than 2 years and then used for further study.

The isolated bacteria were identi®ed to be B. circu-Soil Biology & Biochemistry 32 (2000) 1803±1805

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* Corresponding author. Tel.: 28459; fax: +91-1662-31240.

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lans. and B. brevis according to the criteria shown in Table 1.

One millilitre of 7-day-old bacterial inoculum was added to an Erlenmeyer ¯ask with 50 ml nutrient agar broth and either 1 or 5 mg/ml of the HCH isomer. Each of the four isomers was tested separately in tripli-cate. Uninoculated medium containing the same HCH concentration was kept as control. Immediately after adding the inoculum, 2 ml medium was withdrawn and extracted thrice with 10 ml hexane each time, and analysed for HCH concentration at zero time. Ana-lyses for HCH concentration in the medium were car-ried out at 2-day intervals for a, g and d isomers and at 7-day intervals for b HCH. HCH isomers obtained from EPA, USA were used as standards.

HCH isomers, extracted in hexane, were analysed on GC (Chemito 2865), having methyl silicone (1 m ®lm thickness) capillary column (25 m, 0.25 mm i.d.) and

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Ni electron capture detector. The temperatures of oven, column and detector were 175, 175 and 2408C, respectively. Nitrogen ¯ow was 10 ml/min. The results obtained were statistically analysed and are presented in Table 2.

The concentration of the a, g and d isomers of HCH as determined by GC decreased rapidly after in-oculating the medium with these two bacterial strains

B. brevis. and B. circulans, under aerobic conditions.

All the isomers, except b HCH, showed more than 80% degradation 8 days after inoculation, whereas decrease in uninoculated medium of the a, b, g and d

isomers was negligible. The percentage loss of HCH was signi®cant for both 1 and 5 mg/ml concentrations. Isomer b, the most recalcitrant isomer (Kaushik, 1989), was analysed for a total period of 28 days at 7-day intervals. Both strains were capable of degrading this isomer at both concentrations. Loss of 51.3 and 36.3% was observed at 1 mg/ml concentration for B.

circulans and B. brevis, respectively. Isomers a and g,

the fast degrading isomers (Kaushik, 1991), degraded from 81 to 95% and from 95 to 100%, respectively after 8 days. Both the strains were capable of degrad-ingdHCH at both the concentrations.

Degradation of HCH at two concentrations by these species was tested for signi®cance of dierence using t -test. Degradation of a, b and g isomers by B. brevis

did not vary signi®cantly for the two concentrations. However, degradation of various isomers of HCH by

B. circulans varied signi®cantly P<0:01 with the

in-itial HCH level, as shown in Table 2.

B. brevis showed signi®cantly greater degradation

P<0:01ofa andgisomers at 5 mg/ml and ofd

iso-mer at 1 mg/ml as compared to that by the other species. B. circulans, on the other hand, was signi®-cantly more eective than B. brevis in degrading the recalcitrant b isomer at 1 mg/ml concentration P<

0:01anddisomer at 5mg/mlP<0:05).

The metabolites of HCH seem to be more volatile than the parent molecule and possibly escape into the atmosphere as volatiles, immediately after formation as no metabolite was observed in the form of any extra peak in the chromatograms. These species aerobic in nature have not been reported earlier for

Table 1

Identi®cation of HCH degrading soil isolates

Test Bacillus circulans Bacillus brevis

Gram reaction + +

Degradation of HCH isomers byBacillus circulansandBacillus brevisa

HCH isomer Incubation period (day)

Bacillus circulans Bacillus brevis

0 Concentration = 1mg/ml Concentration = 5mg/ml Concentration = 1mg/ml Concentration = 5mg/ml

a 8 0.03520.001 0.96220.043 0.14620.028 0.25320.011

(96.520.26)b (80.721.1)b.q (85.424.45) (94.920.44)q b 28 0.48720.005 3.01420.043 0.63720.006 3.02920.035

(51.321.04)c.r (39.721.35)c (36.321.12)r (39.421.14) g 8 Not detectable 0.26820.004 Not detectable 0.079620.0003

(94.620.178)s (98.420.02)s

d 8 0.19420.0003 0.74620.008 0.11420.012 1.16620.031 (80.620.98)d.u (85.120.15)d.t (88.621.53)a.u (76.820.82)a.t

a

Values in parentheses represent percentage degradation of HCH (mean2S.E.). The means followed by the same letter are signi®cantly dier-ent from each other based ont-test (after arcsin transformation). The dierences are signi®cant atP<0:01, except d, whereP<0:05.

A. Gupta et al. / Soil Biology & Biochemistry 32 (2000) 1803±1805

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the degradation of HCH. Earlier reports favour the anaerobic degradation (MacRae et al., 1967; Castro and Yoshida, 1974), but Tu (1976) isolated several aerobic species of bacteria and fungi which were able to degrade g HCH isomer. Jagnow et al. (1977) iso-lated facultative anaerobes, that could dechlorinate the four isomers under aerobic conditions. Bachmann et al. (1988) also found that aerobic conditions are more feasible for HCH degradation.

Only one bacterium, Pseudomonas spp., has been reported to degrade the four isomers of HCH (Sahu et al., 1992). The species B. brevis and B. circulans, iso-lated in this study, are capable of degrading the four isomers of HCH. Once adapted to degrade HCH, species of Bacillus degraded the lower concentration of pesticide at a very similar rate and degraded not only the a and g isomers, but also the thermodynamically stablebanddisomers. These observations may be use-ful in developing technologies for bioremediation of soils contaminated with HCH.

Acknowledgements

One of the authors (Archna Gupta) was supported by a research fellowship from the University Grants Commission, New Delhi, India, which is thankfully acknowledged.

References

Bachmann, A., Walet, P., Wijnen, P., deBruin, W., Huntjens, J.L.M., Roelofsen, W., Zehnder, A.J.B., 1988. Biodegradation of a and b hexachlorocyclohexane in a soil slurry under dierent redox conditions. Applied and Environmental Microbiology 54, 143±149.

Castro, T.F., Yoshida, T., 1974. Eect of organic matter on the bio-degradation of some organochlorine insecticides in submerged soils. Soil Science and Plant Nutrition 20, 363±370.

Difco Manual, 1953. 9th ed. Difco Laboratories, Detroit, MI, USA. Jagnow, G., Haider, K., Ellwardt, P., 1977. Anaerobic

dechlorina-tion and degradadechlorina-tion of hexachlorocyclohexane isomers by an-aerobic and facultative anaerobic bacteria. Archives of Microbiology 115, 285±292.

Kaushik, C.P., 1989. Loss of HCH from surface soil layers under sub-tropical conditions. Environmental Pollution 59, 253±264. Kaushik, C.P., 1991. Persistence and metabolism of HCH and DDT

in soil under subtropical conditions. Soil Biology and Biochemistry 23 (2), 131±134.

MacRae, I.C., Raghu, K., Castro, T.F., 1967. Persistence and biode-gradation of four common isomers of benzene hexachloride in submerged soils. Agricultural and Food Chemistry 15, 911±914. Sahu, S.K., Patnaik, K.K., Sethunathan, N., 1992.

Dehydrochlorination of disomer of hexachlorocyclohexane by a soil bacterium, Pseudomonas spp. Bulletin of Environmental Contamination and Toxicology 48, 265±268.

Steinwandter, H., SchluÈter, H., 1978. Experiments on lindane metab-olism in plants. IV: A kinetic investigation. Bulletin of Environmental Contamination and Toxicology 20, 174±179. Tu, C.M., 1976. Utilization and degradation of lindane by soil

micro-organisms. Archives of Microbiology 108, 259±263.