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International Biodeterioration & Biodegradation 57 (2006) 107–113

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Degradation and detoxification of hexachlorocyclohexaneisomers by Pseudomonas aeruginosa ITRC-5

Pankaj Chaudhary, Manish Kumar, Bhom S. Khangarot, Ashwani Kumar�

Industrial Toxicology Research Centre, Post Box No 80, Mahatma Gandhi Marg, Lucknow 226 001, India

Abstract

Technical-hexachlorocyclohexane (t-HCH) consists of four major isomers i.e. a-, b-, g- and d-HCH. The insecticidal g-HCH is

separated from it by solvent extraction, and the remaining ‘muck’ is discarded. HCH-isomers from the ‘muck’ can potentially enter the

environment and impart toxicity. For its biological treatment, biodegradation of HCH-isomers by the isolated bacterium Pseudomonas

aeruginosa ITRC-5 was evaluated. In its presence, from 1.7mM ‘muck’, 498% a- and 480% b-, g- and d-HCH were degraded after 24

days of incubation. The degradation was optimal at 1.7mM input concentration of ‘muck’, pH 9.0, and temperature 20–30 1C. Under

these conditions, from 2 g ‘muck’, 490% S-HCH, i.e. the sum of a, b, g, and d-HCH, were degraded in a 1.0-liter batch-reactor after

incubation for 4 cycles of 5 weeks each. The degradation was accompanied with 90% reduction in the toxicity of ‘muck’ to the aquatic

test organism Daphnia magna. The isolated bacterium ITRC-5 can therefore be used for the degradation and detoxification of HCH-

wastes, prior to their disposal.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: Hexachlorocyclohexane; Pseudomonas aeruginosa; Degradation; Detoxification

1. Introduction

Technical-hexachlorocyclohexane (t-HCH) consists offour major isomers, a-, b-, g- and d-HCH, in anapproximate ratio of 73%, 6%, 13% and 7%, respectively.The insecticidal g-isomer is purified from it by solventextraction, and is used for the protection of crops andcontrol of vector borne diseases (Willett et al., 1998;ATSDR, 1999). The remaining ‘muck’, consisting predo-minantly of a-, b- and d-isomers, along with the residualg-HCH, is stored at secured dumpsites. These, besidesoccupying valuable space, represent a serious risk as theHCH-residues from it can enter the environment and causetoxicity (Willett et al., 1998; ATSDR, 1999).

Bioremediation, which includes the gainful utilization ofmicroorganisms for the biodegradation of target pollu-tants, is a potential technique for the biological treatmentof industrial wastes and contaminated soils (Crawford andCrawford, 1996; Alexander, 1999). For this reason, several

e front matter r 2006 Elsevier Ltd. All rights reserved.

iod.2005.12.003

ing author. Tel.: +91522 2620107; fax: +91 522 2628227.

ess: ashwani26@rediffmail.com (A. Kumar).

laboratories have isolated and characterized the micro-organisms that can effect the degradation of differentHCH-isomers under anaerobic (MacRae et al., 1969;Jagnow et al., 1977; van Eekert et al., 1998; van Doesburget al., 2005) and aerobic conditions (Senoo and Wada,1989; Sahu et al., 1990; Thomas et al., 1996; Gupta et al.,2000; Manonmani et al., 2000; Okeke et al., 2002; Boltneret al., 2005; Kumar et al., 2005). In almost all the reports,aerobic degradation of pure HCH-isomers was studied,and their rate of degradation was in the order of g-4a-4d-and 4b-HCH (Gupta et al., 2000; Sahu et al., 1990;Kumar et al., 2005). Recently, however, the degradation ofHCH-isomers was studied when these are present togetheras t-HCH, and the degradation of b- and d-isomers wasfound to be substantially faster, compared to when thesewere present individually (Kumar et al., 2005).The pathway for the degradation of g-HCH has been

comprehensively worked out in the bacterium Sphingomo-

nas paucimobilis UT26, and the genes for its differentenzymes have been characterized (Nagata et al., 1999).Briefly, it proceeds by two steps of dehydrochlorina-tion, two steps of hydrolytic dehalogenation, and one

ARTICLE IN PRESS

ToVacuum

Fig. 1. Schematic diagram of 1.0 liter ‘batch reactor’ for the degradation

of HCH-isomers by Pseudomonas aeruginosa ITRC-5. Arrows indicate the

direction of airflow.

P. Chaudhary et al. / International Biodeterioration & Biodegradation 57 (2006) 107–113108

dehydrogenation step, catalyzed by the gene products linA,linB and linC, respectively, to form 2,5-dichlohydroqui-none, which undergoes further degradation and is miner-alized (Nagata et al., 1999). Presence of similar genes hasbeen reported in many other bacteria as well (Thomaset al., 1996; Kumari et al., 2002; Boltner et al., 2005). Thedegradation of other HCH-isomers is less understood.Recent studies have demonstrated the metabolism ofb- and d-HCH to pentachlorocyclohexanol, suggestingthat their degradation might proceed via a distinct pathway(Kumar et al., 2005; Nagata et al., 2005).

Environmental factors viz. temperature, pH, etc. haveprofound influence on the microbial activity as well as thebioavailability of target chemicals (Kastner et al., 1998;Awasthi et al., 2000; Siddique et al., 2002). Theiroptimization, therefore, is necessary to obtain meaningfuldegradation of pollutants. In this report, we determined theoptimal conditions i.e. pH, temperature, and initial inputconcentration of ‘muck’, for the degradation of HCH-isomers. The influence of biodegradation on the reductionof their toxicity was also evaluated.

2. Material and methods

2.1. Chemicals

Technical HCH (67.4% a-, 6.8% b-, 17.3% g- and 7.4% d-HCH) and

‘HCH muck’ (77.5% a-, 10.5% b-, 3.8% g- and 3.9% d-HCH) were

obtained from India Pesticides Ltd, Lucknow, India, and Kanoria

Chemicals & Industries Limited, Renukoot, India, respectively. Mercuric

thiocyanate, Tween 20s and 2-phenoxy-ethanol were purchased from

Sigma Chemical Co, St. Louis, USA.

2.2. Inoculum

The bacterium Pseudomonas aeruginosa ITRC-5, isolated earlier by

‘selective enrichment’ from rhizosphere soil of an HCH-contaminated site

for the degradation of HCH-isomers (Kumar et al., 2005), was used. It

causes rapid degradation of a- and g-isomers with a concomitant release of

5.6mmol chloride ions and 4.1mmol CO2 mmol�1 HCH isomer. The

degradation of b- and d-HCH is slower, is accompanied with the release of

0.9mmol chloride ions mmol�1 HCH-isomer, and results in the accumula-

tion of a metabolite pentachlorocyclohexanol (Kumar et al., 2005). The

bacterium was grown for 1 week in the medium (KH2PO4, 170mg;

Na2HPO4, 980mg; (NH4)2SO4, 100mg; MgSO4 � 7H2O, 4.87mg; FeSO4 �

7H2O, 0.05mg; CaCO3, 0.20mg; ZnSO4 � 7H2O, 0.08mg; CuSO4 � 5H2O,

0.016mg; H3BO3, 0.006mg; yeast extract 10mg and glucose 10mg,

dissolved in 100ml distilled water, pH 7.4) containing 0.68mM t-HCH, at

28 1C with shaking at 180 revmin�1, and was used as inoculum for all the

experiments.

2.3. Biodegradation of HCH-isomers

Biodegradation of HCH-isomers was studied in 100ml Erlenmeyer

flasks that were containing 18ml medium and 1.7mM ‘muck’. Briefly, 24

flasks were divided equally into two sets. While flasks of set 1 received 2ml

inoculum (2.8� 108 colony forming unitsml�1), flasks of set 2 remained

un-inoculated and received 2ml medium only. All the flasks were

incubated at 28 1C with shaking at 180 revmin�1. Three flasks from each

set were removed after 0, 8, 16 and 24 days, respectively. One-milliliter

sample from each flask was aspirated for the estimation of released

chloride by a colorimetric method using AgNO3 and mercuric thiocyanate

reagent (Bergman and Sanik, 1957). The remaining medium was acidified

to pHo2.0 with HCl. Residual HCH-isomers as well as the formed

metabolites were extracted with hexane–acetone, and analyzed by a gas

chromatograph that was equipped with 63Ni-ECD, as described earlier

(Awasthi et al., 2003).

In the experiments where effect of initial concentration of ‘muck’ on the

degradation of HCH-isomers was to be studied, 30 flasks (Erlenmeyer;

250ml) were divided equally into five sets. Sets 1–5 received 0.68, 1.71,

3.42, 6.84 and 17.1mM ‘muck’, respectively. Fifty-milliliter medium was

added to all the flasks. While 3 flasks from each set received 5ml

inoculum, the other 3 remained un-inoculated. After incubation, 1ml

sample was removed from each flask after 0, 4, 8, 12, 16, 20, 24 and 28

days, and the released chloride was estimated as described above.

Similarly, 66 flasks (Erlenmeyer; 100ml) that were containing 1.7mM

‘muck’ were taken for evaluating the influence of pH on the degradation of

HCH-isomers. These were divided in 11 sets of six flasks each. Sets 1–11

received 18ml medium that was adjusted at pH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11

and 12, respectively. From each set, 3 flasks were inoculated with 2ml

inoculum, and 3 others were un-inoculated. After incubation for 2 weeks,

the residual HCH-isomers were extracted and analyzed as described

above.

To evaluate the effect of temperature on the biodegradation of HCH-

isomers, 36 flasks (Erlenmeyer; 100ml) that were containing 0.34mM

‘muck’ and 18ml medium were taken. These were divided equally in 6 sets.

While 3 flasks from each set were inoculated with 2ml inoculum, 3 others

remained un-inoculated. Sets 1–6 were incubated at 5, 10, 20, 30 40 and

50 1C, respectively, for 1 week without shaking. Residual HCH-isomers

were extracted and analyzed. Similarly, to evaluate the effect of Tween-

20s on the degradation of HCH isomers, the flasks containing 0.34mM

HCH-muck, and 0%, 0.05%, 0.1%, 0.5% or 1.0% (v/v) Tween-20s were

incubated at 28 1C with shaking at 180 revmin�1. Both un-inoculated and

inoculated flasks were removed after 0, 1, 2 and 3 days, and residual HCH-

isomers were analyzed.

For the ‘bench-scale’ batch reactor, two one-liter flasks, each contain-

ing 450ml medium, 1.7mM ‘muck’, and 50ml inoculum, were joined

(Fig. 1). These were incubated at 28 1C with aeration that was provided

daily for 30min by flushing the vacuum driven filtered air. Samples were

drawn weekly for the estimation of released chloride ions. After 5 weeks,

450ml sample was removed from each flask for the extraction of residual

HCH-isomers. To the remaining 50ml, fresh 450ml medium along with

1.7mM HCH was added and the reaction was continued as before. After

four cycles of such treatment, total released chloride and the residual

HCH-isomers were quantified, as above.

2.4. Aquatic toxicity of residual HCH isomers

Two sets of flasks, containing 0.34mM ‘muck’ in 18ml mineral

medium, were prepared. One set was inoculated with 2ml inoculum, and

the other, un-inoculated set, was run as control. After 4 days of incubation

at 28 1C with shaking at 180 revmin�1, addition of 2ml DMSO to all the

flasks terminated the reaction. Aquatic toxicity of the residual HCH-

isomers along with the formed metabolites was determined as described

(EPA, 1993). Briefly, 0, 1, 2, 3, 4 and 5ml aliquots were drawn from both

un-inoculated and inoculated sets and were made upto 5ml with 10%

ARTICLE IN PRESSP. Chaudhary et al. / International Biodeterioration & Biodegradation 57 (2006) 107–113 109

DMSO. These were added to 30mm Petri-plates, containing 45ml tap

water and 20 test-organisms Daphnia magna. Mortality of these was

scored after 48 h incubation at 28 1C, and was used as the index of the

toxicity of ‘muck’.

0.68

1.71

3.42

6.84

17.1

6.84 UI

Rel

ease

d ch

lori

de (

mM

)

Days0 4 8 12 16 20 24 28

0

10

20

30

Fig. 2. Release of chloride ions after the degradation of HCH-isomers by

Pseudomonas aeruginosa ITRC-5, when the input concentrations of ‘muck’

were 0.68–17.1mM. Chloride released from 6.84mM ‘muck’, under un-

inoculated conditions, is also shown, and was comparable at other

concentrations. Values given are mean7standard deviation (o5%) of the

three replicates.

3. Results and discussion

3.1. Biodegradation of HCH-isomers

From the input 1.7mM ‘muck’, consisting of 1.33, 0.17,0.06 and 0.06mM a-, b-, g- and d-HCH, respectively,o5%degradation of different HCH-isomers was observed after24 days of incubation under un-inoculated conditions(Table 1). This could be due to their hydrolysis, photo-decomposition, volatilization, or other abiotic transforma-tions (ATSDR, 1999). Addition of Pseudomonas aeruginosa

ITRC-5 enhanced their degradation, and 498% a-, and480% b-, g- and d-HCH, respectively, were degraded afterthe same period (Table 1). The degradation of HCH-isomers was extensive, as it was accompanied with therelease of 8.4mM chloride ions (Table 1), and noaccumulation of any metabolite was observed. Similarobservations were also made in earlier studies on thedegradation of a- and g-HCH (Manonmani et al., 2000;Okeke et al., 2002; Pesce and Wunderlin, 2004). Accumula-tion of the metabolite pentachlorocyclohexanol (PCCOL)reportedly formed during the degradation of b- andd-HCH (Sahu et al., 1995; Kumar et al., 2005; Nagataet al., 2005), was also not observed in this study. This couldbe due to its enhanced metabolism in the presence of a- andg-HCH, as shown earlier for b- and d-HCH (Kumar et al.,2005). The observed release of 4.9mM chloride ions mM�1

‘muck’ is lesser than the expected 6.0mM, based on 6molchloride from 1mol HCH-isomer, possibly because oflimited degradation of b- and d-HCH (Kumar et al., 2005).

3.2. Effect of input concentration of ‘muck’ on the

degradation HCH-isomers

Influence of different input concentration of ‘muck’ onthe degradation of HCH-isomers was evaluated byestimating the release of chloride ions. At the input 0.68,

Table 1

Degradation of HCH-isomers, and release of chloride ions, under un-inoculat

Incubation (days) mM HCH recovereda

a-HCH b-HCH g

UI I UI I U

0 1.28 (100)b 1.28 (100) 0.17 (100) 0.17 (100) 0

8 1.26 (98) 0.33 (26) 0.17 (100) 0.10 (59) 0

16 1.25 (97) 0.06 (4.7) 0.17 (100) 0.06 (35) 0

24 1.23 (96) 0.01 (0.8) 0.17 (100) 0.03 (17) 0

BDL; below detection limit (o0.03mM).aValues given are mean of triplicate experiments where the standard deviatibValues in parenthesis represent percent, taking the amount recovered at 0-

1.71, 3.42, 6.84 or 17.1mM ‘muck’, no detectable release ofchloride ions was observed under un-inoculated conditions(Fig. 2). Addition of inoculum initiated the degradation ofHCH-isomers, which was comparable at early time pointsfor all the concentrations, and �1.1mM chloride ionsday�1 were released (Fig. 2). Thus, at the input concentra-tion of 0.68 and 1.7mM ‘muck’, 3.36 and 8.4mM chloride,respectively, was released after 4 and 8 days of incubation.It represented the release of 4.9mM chloride ions mM�1

‘muck’, and did not increase after prolonged incubation(Fig. 2). At higher input of ‘muck’ i.e. 3.42mM or more,however, while the degradation of HCH-isomers at earlytime points was comparable till around 15mM chlorideions were released, it was inhibited progressively at latertime points and was nearly 100% after the release of22mM chloride ions. Thus, from the input 3.42, 6.84 or17.1mM ‘muck’, the maximum of 4.4, 3.2, and 1.3mMchloride ions mM�1 ‘muck’, respectively, were released(Fig. 2). This inhibition could be due to the toxicity ofmetabolites, which might have formed but not detectedin this study by the methods employed. Similar inhibitionof HCH-degradation has been reported when the initial

ed and inoculated conditions

mM Cl-released

-HCH d-HCH

I I UI I UI I

.06 (100) 0.06 (100) 0.06 (100) 0.06 (100) BDL BDL

.06 (100) 0.02 (33) 0.06 (100) 0.02 (33) BDL 5.9

.06 (100) 0.01 (17) 0.06 (100) 0.01 (16) BDL 8.2

.06 (100) 0.01 (17) 0.06 (100) 0.01 (16) BDL 8.4

on was less than 5%.

time as 100%.

ARTICLE IN PRESSP. Chaudhary et al. / International Biodeterioration & Biodegradation 57 (2006) 107–113110

content of a-HCH was 4900mg g�1 soil (Bachmannet al., 1988). Likewise, in another study, the degradationof g-HCH was inhibited when its initial concentration was41.03mM (Pesce and Wunderlin, 2004). The inhibition ofHCH- degradation in the present study does not appear tobe due to decrease in the pH of reaction medium to �6.7after incubation for 12 days, as its correction to 7.5 did notimprove the degradation rates (data not given).

3.3. Effect of pH on the degradation of HCH-isomers

The pH of medium plays an important role in thephysiology of microorganisms as well as the bioavailablib-ity of pollutants, and consequently influences theirdegradation (van Veen et al., 1997; Kastner et al., 1998).At pH 2.0 or 3.0, no degradation of HCH-isomers wasobserved (Fig. 3), which could be due to the diminishedmicrobial activity under these conditions. At pH 4.0,however, 10–12% of a-, g- and d-HCH were degraded after2 weeks of incubation, but no degradation of b-HCH wasobserved (Fig. 3). The degradation of HCH-isomersincreased progressively at higher pH and was optimal at

2 3 4 5 6 7 8 9 10 11 12

pH

1.4

0.7

0

0.18

0.09

0

0.06

0.03

0

0.06

0.03

0

Res

idua

l HC

H is

omer

s (m

M)

α

β

γ

δ

Fig. 3. Degradation of a-, b-, g- and d-HCH under un-inoculated

conditions (shaded bars), and in the presence of Pseudomonas aeruginosa

ITRC-5 (open bars), after incubation for 2 weeks at 28 1C at different pH.

Values given are mean7standard deviation (o5%) of the three replicates.

pH 9.0. At this pH, 82%, 45%, 90% and 72% of a-, b-, g-and d-HCH, respectively, were degraded after incubationfor 2 weeks (Fig. 3). The degradation was inhibited at pH10.0. At higher pH i.e. 11.0 and 12.0, HCH levels declinedin both un-inoculated and inoculated flasks (Fig. 3),possibly due to their abiotic destruction under theseconditions. The results are in agreement with earlier studies(Manonmani et al., 2000; Siddique et al., 2002), where theoptimal pH for the degradation of a- or g-HCH has beenreported to be around 8.0–9.0, and their destruction wasobserved at higher pH.

3.4. Effect of temperature on the degradation of

HCH-isomers

Temperature of the reaction medium influences themicrobial activity, as well as the bioavailability of thepollutants, and is therefore an important determinant forpollutant degradation (Alexander, 1999). Addition of ITRC-5did not initiate the degradation of HCH-isomers at 5 1C, butreasonable degradation was observed at 10–40 1C (Fig. 4).The degradation was optimal at 20–30 1C, when from theinput 0.34mM ‘muck’, 93%, 55%, 85% and 82% a, b-, g-,and d-HCH, respectively, were degraded after 1 week of

280

140

0

12

6

0

36

18

0

12

6

0

5 10 20 30 40 50

α

β

γ

δ

Res

idua

l HC

H is

omer

s (µ

M)

Temperature (°C)

Fig. 4. Degradation of a-, b-, g- and d- HCH under un-inoculated

conditions (shaded bars), and in the presence of Pseudomonas aeruginosa

ITRC-5 (open bars), after incubation at different temperatures for 1 week.

Values given are mean7standard deviation (o5%) of the three replicates.

ARTICLE IN PRESSP. Chaudhary et al. / International Biodeterioration & Biodegradation 57 (2006) 107–113 111

incubation (Fig. 4). At 50 1C, degradation of 70–75% g- andd-HCH, and 20% a-HCH, was observed under both un-inoculated and inoculated conditions, possibly due to theirvolatilization or abiotic destruction. b-HCH, however, wasnot affected at this temperature (Fig. 4). The results are inagreement with earlier reports where optimal degradation ofa- and g-HCH was observed at 20–30 1C (Bachmann et al.,1988; Siddique et al., 2002).

3.5. Effect of Tween-20s on the degradation of HCH-

isomers

Limited bioavailability of target pollutants is amongstthe major causes of their slow biodegradation. Addition ofsurfactants has, therefore, been demonstrated to be highly

Res

idua

l HC

H is

omer

s (µ

M)

280

140

0

α

36

18

0

β

12

6

0

γ

12

6

0

δ

Days

UI

I

I+ 0.05%

I+ 0.1%

I+ 0.5%

I+ 1.0%

0 1 2 3 0 1 2 3

Fig. 5. Degradation of a-, b-, g- and d- HCH under un-inoculated

conditions (closed squares), and in the presence of Pseudomonas

aeruginosa ITRC-5, both with and without the additional presence of

different concentrations of Tween-20s. Values given are mean7standard

deviation (o5%) of the three replicates.

Chl

orid

e (m

M)

Incubati

0 2 6 8 10

2

4

8

6

4

Fig. 6. Release of chloride ions after the degradation of HCH-isomers. Results

cycle, when 450ml of culture medium was removed, and fresh 450ml medium

useful in such cases (Aronstein et al., 1991; Tiehm, 1994;Mulligan, 2005). In this study, from 0.34mM ‘muck, 65%,40%, 75%, and 76% a-, b-, g- and d-HCH, respectively,were degraded upon incubation with ITRC-5 for 3 days(Fig. 5). Presence of additional 0.05% or 0.1% non-ionicdetergent Tween-20s did not improve the degradation(Fig. 5), suggesting that the solubility of HCH-isomersmight not be a limiting factor. The degradation of HCH-isomers was progressively inhibited in the presence ofhigher Tween-20s i.e. 0.5% and 1.0%, which could be dueto its cytotoxicity. Presence of higher concentration ofdetergents has been reported to be detrimental to thedegradation of target pollutants in other studies also (Lahaand Luthy, 1992; Liu et al., 1995; Volkering et al., 1995).

3.6. Biodegradation of HCH-isomers in a ‘bench-scale’

reactor

One liter ‘batch reactor’ was run for 4 cycles of 5 weekseach, at 28 1C and pH 9.0. In the first cycle, from the input1.7mM ‘HCH-muck’, 7.6mM chloride ions were releasedafter 5 weeks of incubation (Fig. 6). The released chloridedecreased progressively in subsequent cycles and 6.9, 6.4 and5.8mM chloride was released after 2nd, 3rd and 4th cycle,respectively (Fig. 6). The progressive decrease in the amountof released chloride might be due to the accumulation oftoxic metabolites and consequent decrease in the degrada-tion of HCH-isomers, as discussed in Section 3.2. After 4cycles, from the total 1.7mM ‘muck’ in 4 liters, 91%, 80%,97% and 96% a-, b-, g- and d-HCH, respectively, weredegraded, which was accompanied with the cumulativerelease of 6.62mM chloride ions (Table 2). Thus, from theinput 2.0 g ‘muck’, 490% of S-HCH i.e. the sum of all theisomers was degraded in 4 cycles of 5 weeks each.

3.7. Biodegradation mediated detoxification of the ‘muck’

Mortality of test organisms is a sensitive endpoint fordetermining the eco-toxicological risk of environmental

on (weeks)

0 12 14 16 18 20

of four cycles of 5 week each are presented. Arrows indicate the end of each

that contained 1.7mM ‘muck’ was added.

ARTICLE IN PRESS

Table 2

Degradation of HCH-isomers in 1-litre batch reactor, after 4-cycles of 35 days each, under un-inoculated and inoculated conditions

mM HCH recovereda

a-HCH b-HCH g-HCH d-HCH S-HCH Cl� released

Un-inoculated 1.223 (92)b 0.171 (95) 0.054 (91) 0.065 (93) 1.513 (92.2) BDL

Inoculated 0.118 (8.8) 0.037 (20.5) 0.002 (3.3) 0.004 (5.7) 0.161 (9.8) 6.62

BDL; below detection limit (o0.03mM).aValues given are mean of triplicate experiments where the standard deviation was less than 5%.bValues in parenthesis represent percent, taking the amount recovered at 0-time as 100%.

Table 3

Biodegradation mediated reduction in the toxicity of HCH-isomers

present in ‘muck’

HCH (mM) % Mortalitya

Un-inoculated sample Inoculated sample

34.0 100 5

27.2 100 0

20.4 50 0

13.6 40 0

6.8 10 0

3.4 5 0

0 0 0

aValues given are mean of triplicates where the standard deviation was

less than 5%.

P. Chaudhary et al. / International Biodeterioration & Biodegradation 57 (2006) 107–113112

pollutants and has, therefore, been taken as an index oftheir biological treatment (Jarvis et al., 1998; Awasthiet al., 2003; Frische, 2003; Gemini et al., 2005). In this study,biodegradation mediated decrease in the toxicity to the testorganisms D. magna was evaluated. Incubation of test-organisms for 48h with the un-inoculated sample that wascontaining 34.0mM ‘muck’, led to 100% mortality, whichwas progressively lesser at lower concentrations (Table 3).On the other hand, the samples that were originallycontaining the same amount of ‘muck’, but had undergonebiodegradation for 4 days, only 5% mortality of the testorganisms was observed. No mortality was observed with itsdiluted samples (Table 3). Thus, 90% detoxification ofHCH-isomers was achieved upon their degradation by theisolated bacterium P. aeruginosa ITRC-5.

In conclusion, incubation of ‘muck’ with the isolatedbacterium P. aeruginosa ITRC-5 under optimized condi-tions i.e. 1.7mM input concentration, pH 9.0, andtemperature 20–30 1C, causes substantial degradation ofHCH-isomers, which is accompanied with reduction oftheir toxicity. The bacterium, therefore, can be used for thebiological treatment and safe disposal of HCH-wastes.

Acknowledgements

This work was supported by a grant from Department ofBiotechnology, Ministry of Science and Technology,Government of India.

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