Soil Biology & Biochemistry 32 (2000) 1803±1805
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, g and d) degradation eciency of acclimatized Bacillus circulans and Bacillus brevis was 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 the a and g isomers but also the thermodynamically stable b and d isomers at dierent concentrations, suggesting their potential for degradation of even the aged residues. 7 2000 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 concentrations 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 concentration 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 * Corresponding author. Tel.: +91-1662-28459; fax: +91-166231240. E-mail address: [email protected]
under laboratory conditions to increase their HCH degradation eciency. Samples from HCH contaminated soil were taken upto a depth of 5 cm, from four dierent sites in the surroundings of Hindustan Insecticides 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 dilution was used as inoculum for isolating the microorganisms 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 33 2 28C for 3 days. The culture was serially diluted and plated on petridishes containing 25 ml of nutrient agar and 5 mg/ 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 regularly 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-
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A. Gupta et al. / Soil Biology & Biochemistry 32 (2000) 1803±1805
Table 1 Identi®cation of HCH degrading soil isolates Test
Gram reaction Endospore Motility Fluorescence (UV) Starch hydrolysis Nitrate reduction Nitrite reduction Oxidase test Arabinose test Lactose test Mannose test Xylose test
+ + + ÿ + + + ÿ + + ÿ +
+ + + ÿ ÿ + ÿ ÿ ÿ ÿ + ÿ
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 triplicate. 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. Analyses for HCH concentration in the medium were carried 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 63 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 inoculating 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 7day 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 degrading d HCH at both the concentrations. Degradation of HCH at two concentrations by these species was tested for signi®cance of dierence using ttest. 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 initial HCH level, as shown in Table 2. B. brevis showed signi®cantly greater degradation
P < 0:01 of a and g isomers at 5 mg/ml and of d isomer 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 < 0:01 and d isomer at 5 mg/ml
P < 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 2 Degradation of HCH isomers by Bacillus circulans and Bacillus brevisa HCH isomer
Incubation period (day) 0
Bacillus circulans Concentration = 1 mg/ml
Concentration = 5 mg/ml
Concentration = 1 mg/ml
Concentration = 5 mg/ml
0.03520.001 (96.520.26)b 0.48720.005 (51.321.04)c.r Not detectable
0.14620.028 (85.424.45) 0.63720.006 (36.321.12)r Not detectable
0.96220.043 (80.721.1)b.q 3.01420.043 (39.721.35)c 0.26820.004 (94.620.178)s 0.74620.008 (85.120.15)d.t
0.25320.011 (94.920.44)q 3.02920.035 (39.421.14) 0.079620.0003 (98.420.02)s 1.16620.031 (76.820.82)a.t
0.11420.012 (88.6 21.53)a.u
Values in parentheses represent percentage degradation of HCH (mean2S.E.). The means followed by the same letter are signi®cantly dierent from each other based on t-test (after arcsin transformation). The dierences are signi®cant at P < 0:01, except d, where P < 0:05.
A. Gupta et al. / Soil Biology & Biochemistry 32 (2000) 1803±1805
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) isolated 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, isolated 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 stable b and d isomers. These observations may be useful 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.
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