Enhanced Biodegradation of â- andδ-Hexachlorocyclohexane in thePresence of r- and γ-Isomers inContaminated SoilsM A N I S H K U M A R , P A N K A J C H A U D H A R Y ,M A N I S H D W I V E D I , R A N J A N K U M A R ,D E B A R A T I P A U L , † R A K E S H K . J A I N , †

S A T Y E N D R A K . G A R G , ‡ A N DA S H W A N I K U M A R *

Industrial Toxicology Research Centre, Post Box No 80,Mahatma Gandhi Marg, Lucknow- 226 001, India, Institute ofMicrobial Technology, Chandigarh, India, and Department ofMicrobiology, Dr. R. M. L. Avadh University,Faizabad- 224 001, India

The chlorinated insecticide hexachlorocyclohexane (HCH)has been used extensively in the past, and contaminatedsites are present throughout the world. Toward theirbioremediation, we isolated a bacterium Pseudomonasaeruginosa ITRC-5 that mediates the degradation of all thefour major isomers of HCH under aerobic conditions,both in liquid-culture and contaminated soils. In liquid-culture, the degradation of R- and γ-HCH is rapid and isaccompanied with the release of 5.6 µmole chloride ions and4.1 µmole CO2 µmole-1 HCH-isomer. The degradation ofâ- and δ-isomers is slow, accompanied with the release of0.9 µmole chloride ions µmole-1 HCH-isomer, and resultsin a transient metabolite 2,3,4,5,6-pentachlorocyclohexan-1-ol. The strain ITRC-5 also mediates the degradation ofR-, â-, γ-, and δ-isomers in contaminated soils, wheredegradation of otherwise persistent â- and δ-HCH is enhancedseveralfold in the presence of R- or γ-HCH. The degradationof soil-applied â- and δ-HCH under aerobic conditionshas not been reported earlier. The isolate ITRC-5 thereforedemonstrates potential for the bioremediation of HCH-wastes and contaminated soils.

IntroductionThe insecticidal formulation of technical-hexachlorocyclo-hexane (t-HCH) predominantly consists of four majorisomers, that is, R- (60-70%), â- (5-12%), γ- (10-18%), andδ-HCH (6-10%). It has been used extensively in the past forthe protection of crops and control of vector borne diseases(1, 2). Its residues persist in the environment, undergovolatilization in tropical conditions, migrate to long distanceswith air current, deposit in colder regions, and causewidespread contamination (1-4). The HCH-residues enterthe food chain and impart toxicity. Accordingly, the use oft-HCH has either stopped or is severely restricted in mostcountries, and only the insecticidal γ-isomer is permitted (1,2). Because of extensive use of t-HCH in the past, numerouscontaminated sites are present throughout the world (5). At

these sites, being most persistent, â-HCH is usually presentin disproportionately higher amounts (6-8).

Stimulation of autochthonous microorganisms and aug-mentation by the isolated microbes are among the attractiveoptions for enhanced bioremediation of contaminated soils(9). For this reason, microbial transformation of HCH-isomersboth under aerobic (10-15) and anaerobic (16-18) condi-tions has been extensively studied. â-HCH was least sus-ceptible to microbial degradation, possibly because of theall-equatorial arrangement of chlorine atoms on the cyclo-hexane ring (19). Under methanogenic conditions, microfloraenriched from river sediments (17), or granular sludge of asugar-beet refinery wastewater treatment reactor (18), hasbeen shown to degrade all the four major isomers of HCH.Chlorobenzene and benzene were the major metabolitesformed (17, 18). Under aerobic conditions, complete deg-radation of R- and γ-HCH (10-15) but limited degradationof â- and δ-HCH (10, 20) has been reported. The degradationof R- and γ-isomers has been demonstrated in contaminatedsoils also, but despite prolonged incubations no degradationof soil applied â- or δ-HCH was observed (10, 20).

In this report, we describe the characterization of abacterial strain ITRC-5 that under aerobic conditions medi-ates the degradation of all the four major HCH-isomers inliquid-culture as well as contaminated soils. In the con-taminated soils, the degradation of otherwise persistent â-and δ- isomers was enhanced in the presence of additionalR- or γ-HCH.

Experimental SectionChemicals. Technical HCH, consisting of R- (67.4%), â-(6.8%), γ- (17.3%), and δ-HCH (7.4%), was obtained fromIndia Pesticides Limited, Lucknow, India. Individual isomersof HCH were purchased from Riedel-deHaen, Germany.Mercuric thiocyanate, 2-phenoxy-ethanol, and 2,5-dichlo-rophenol were from Sigma Chemical Co, St. Louis, MO.

Soils. Two soils were used. Soil-A was from the garden ofIndustrial Toxicology Research Centre, Lucknow, India. Itcontained 43.6% clay, 20% silt, 36.4% sand, and 0.47% organiccarbon, and the pH was 7.9. Soil-B was from the surroundingsof an industry (India Pesticides Ltd, Lucknow, India), whichhad been manufacturing t-HCH in the past for several years.It contained 58% clay, 24% silt, 17.6% sand, and 0.15% organiccarbon, and the pH was 9.72. Soil-B was contaminated with51.54 µmole R- and 206.18 µmole â-HCH g-1 soil. Both thesoils were air-dried and sieved through a 2-mm mesh beforeuse.

Isolation and Characterization of HCH-Degrading Bac-terium. The bacterium was isolated from the HCH-contaminated rhizosphere soil-B by “selective enrichmentmethod”. Briefly, 5 g soil was added to the flasks that wereprecoated with 6.87 µmole t-HCH and that contained 20 mLmedium (KH2PO4, 170 mg; Na2HPO4, 980 mg; (NH4)2 SO4,100 mg; MgSO4, 4.87 mg; FeSO4, 0.05 mg; CaCO3, 0.20 mg;ZnSO4, 0.08 mg; CuSO4‚5H2O, 0.016 mg; H3BO3, 0.006 mg;yeast extract 10 mg and glucose 10 mg, dissolved in 100 mLdistilled water, pH 7.6). After incubation at 28 °C with shakingat 180 rpm for 1 week, 2 mL of the growing culture wastransferred to the fresh flasks and grown as above. After threemore cycles of this enrichment process, the cells were platedand grown on agar (1.5% w/v) medium, at 28 °C for 48 h. Thegrowing individual colonies were evaluated for their capabilitytoward the degradation of γ-HCH by release of chloride ions.One of the colonies that exhibited maximum degradationwas selected for further studies and designated as ITRC-5.Morphological and biochemical tests (21) and complete

* Corresponding author phone: +91-522-2620107; fax: +91-522-2628227; e-mail: ashwani26@rediffmail.com.

† Institute of Microbial Technology.‡ Dr. R. M. L. Avadh University.

Environ. Sci. Technol. 2005, 39, 4005-4011

10.1021/es048497q CCC: $30.25 2005 American Chemical Society VOL. 39, NO. 11, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 4005Published on Web 04/22/2005

sequencing of 16S ribosomal RNA gene were conducted forits identification Nucleotide sequencing was done on a cyclesequencer (ABI prism, model 310, version 3.4), using the bigdye terminator cycle sequencing ready reaction kit (PEBiosystems) according to the manufacturers instructions.The organism has been deposited at Microbial Type CultureCollection and Gene Bank, Chandigarh, India, as MTCC 4727.

Biodegradation of HCH-Isomers in Liquid-Culture. Theisolated bacterium was grown in 20 mL medium containing13.75 µmole t-HCH for 1 week at 28 °C with shaking at 180rpm. Two mL of this culture (2.8 × 108 cfu mL-1) was usedas inoculum for further experiments. Biodegradation/mineralization of R- or γ-HCH was studied in 250-mLErlenmeyer flasks that had a cylindrical well fused at thecenter of their base. Briefly, three sets of three flasks each,containing 45 mL of medium in their main body and 4.0 mLof 0.5 M KOH in the central well, were prepared. To themedium, 5 mL of inoculum, set one; 171.8 µmole R- or γ-HCH,set two; and both inoculum and R- or γ-HCH, set three, wereadded. After incubation at 28 °C with shaking at 180 rpm for0, 5, 10, 15, and 20 days, the KOH solution was aspirated andreplaced with the fresh solution. The aspirated KOH wastitrated against 0.1 N HCl. The difference in the amount ofHCl consumed by sets 1 and 3 was used for calculating theevolved CO2. No release of CO2 was observed in the flasks ofset 2, which had HCH isomers but were not inoculated. Inparallel sets, the reaction was terminated after 0-20 days byacidification to pH <2.0. Residual HCH (22) and releasedchloride were estimated (23).

Biodegradation of â- and δ-HCH was studied in 100-mLErlenmeyer flasks, which were precoated with 1.72 µmoleHCH-isomers. After addition of 18 mL medium and 2 mLinoculum, the flasks were incubated up to 8 days. ResidualHCH (22) and released chloride were estimated (23). A parallelset of uninoculated flasks was run as control.

Biodegradation of HCH Isomers in Contaminated Soil.One kg of soil-A was spiked with 3.43 mmol t-HCH in twosteps to prevent the loss of indigenous microflora duringspiking (24). Briefly, 3.43 mmol t-HCH was dissolved in 100mL hexane:acetone (9:1) and mixed with 250 g soil. Afterevaporation of the solvent, an additional 750 g soil-A wasmixed with it. Five grams of this spiked soil was added toeach of the 36 test tubes (flat bottom, 3.25 in. × 0.9 in.),which were divided into two sets. For set 1 (inoculated), 750µL of mineral medium containing 50 µL inoculum (2.8 × 108

cfu ml-1) was added to each tube, and for set 2 (uninoculated),only 750 µL mineral medium was added to each tube. Afterincubation at 28 °C, three tubes from each set were removedafter 0-25 days. Residual HCH-isomers were extracted andquantified by gas chromatography (22).

To study the degradation of soil-applied â- or δ-HCH, 1kg soil-A was spiked with 0.17 mmol in two steps as above,resulting in 0.17 µmole â- or δ-HCH g-1 test soil. Twentygrams of this soil was transferred to each of the six beakers.To beakers 3-6, an additional 0.31 mmole R- HCH, 0.31mmole γ- HCH, 0.5 mmol glucose, or 1.09 mmol sodiumacetate, respectively, was added. After evaporation of thesolvent, another 70 g of test soil was added to all the beakers.Beaker 1 received 13.5 mL medium and was run as unin-oculated control. To beakers 2-6, 13.5 mL medium, con-taining 1.2 mL inoculum, was added. From beakers 1-6, sets1-6 were prepared by apportioning the soils from each beakerto 18 tubes. After incubation at 28 °C, three tubes from eachset were removed after 0-25 days. Residual HCH-isomerswere extracted (22) and quantified by gas chromatography.

Extraction and Analysis of the Residual HCH-Isomersand the Formed Metabolites. The residual HCH-isomersand the formed metabolites were extracted from the reactionmedium with hexane-acetone and analyzed by a gas chro-matograph (GC, Netel Chromatograph; Michro 9100) fitted

with 63Ni-ECD, as described earlier (22). The retention timesfor R-, γ-, â-, and δ-HCH were 1:58, 2:31, 2:58, and 3:28 min,respectively. A Perkin-Elmer Autosystem GC, equipped withECD and a capillary column DB-5 MS, 30 m × 0.25 mm (ID)with a film thickness of 0.25 µm, was used for the analysisof 2,5-dichlorophenol (2,5-DCP). The oven temperatureprogramming was initially 150 °C, held for 1 min, then rampedto 200 °C at 5 °C min-1 and held for 2 min, and again rampedto 275 °C at 15 °C min-1 and held for 12 min. The injectorand detector temperatures were 250 °C and 300 °C, respec-tively. Nitrogen, at a pressure of 15 psi, was used as the carriergas. Under these conditions, the retention time for 2,5-DCPand γ-HCH was 9.7 and 22.0 min, respectively. Authenticstandards were used for the quantification of HCH isomersand formed 2,5-DCP.

Gas-chromatography-mass-spectroscopy analysis wasdone on a Perkin-Elmer Autosystem XL GC equipped witha PE Turbo Mass. The GC was fitted with a DB-5 low bleedMS capillary column (J&W Scientific, United States), 30 m ×0.25 mm (ID), 0.25-µm film thickness. The initial oventemperature was 60 °C for 2 min, ramped to 160 °C at 8 °Cmin-1 and held for 8 min, ramped again to 250 °C at 12 °Cmin-1 and held for 6 min, and then up to 280 °C at 15 °Cmin-1 and held for 6 min. Helium, at a pressure of 20 psi,was used as the carrier gas. The injector temperature was250 °C, and the ion source was held at 200 °C. Electronionization was conducted at 70 eV and quadrapole wasscanned from m/z 30 to 300.

NMR spectroscopy was done at Chemical Analysis Labo-ratory, Bulleen Victoria 3105, Australia. Briefly, the metaboliteM1, formed after the degradation of δ-HCH, was purified bysilica gel 60 (0.063-0.1 mm) column chromatography. It wasdissolved in 99.96% CDCl3 and analyzed by Bruker AvanceAV300 spectrometer, followed by COSY and HSQC sequencesanalysis.

Results and DiscussionBiodegradation of HCH-Isomers in Liquid-Culture. Theisolated bacterium ITRC-5 is rod-shaped, gram-negative,motile, and catalase- and oxidase-positive. It mediates thenitrate and nitrite reduction and was identified as Pseudo-monas aeruginosa. Nucleotide sequence of its 16S ribosomalRNA gene (NCBI Gene Bank accession no. AY 264292) was>99.8% homologous to the type strain that further confirmedthis identification. The bacterium mediated the biodegrada-tion of all four major isomers of HCH. The degradation ofγ-HCH was faster than R-HCH at early time points but was>95% for both the isomers after 20 days of incubation (Table1). The degradation of these isomers is accompanied withthe formation of 5.6 µmole chloride ions and 4.1 µmole CO2

µmole-1 HCH-isomer (Table 1). During the degradation ofγ-HCH, accumulation of a metabolite, 0.02 µmole µmole-1

γ-HCH, was also observed. The metabolite was identified as2,5-dichlorophenol, on the basis of its comigration with theauthentic compound on TLC (Retardation factor 0.34;developed in hexane:chloroform:acetone, 9:3:1, and visual-ized by Gibb’s reagent) and GC (Retention time 9.5). Theresult is in agreement with an earlier report (25) wherein2,5-DCP is formed as a dead-end metabolite by the non-enzymatic conversion of the intermediary metabolite 2,4,5-trichloro-2,5-cyclohexadiene-1-ol during the degradation ofγ-HCH by the strain Sphingomonas paucimobiliz UT26.Formation of 2,5-DCP and presence of linA, B, and C genes(data not given) suggest that the pathway for γ-HCHdegradation in ITRC-5 might be similar to that in the reportedstrains (25, 26).

The degradation of â- and δ-HCH by ITRC-5 wasconsiderably slower than R- or γ-HCH. From the input 1.72µmole, 34% of â- and >99% δ-HCH were degraded after 8days of incubation (Table 2). The degradation of â- and δ-HCH

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FIGURE 1. Degradation of â- and δ-HCH (squares) and formation of metabolite M1 (circles) under uninoculated (UI) and inoculated (I)conditions. Values given are the mean of three replicates, and the standard deviation was less than 10%. Gas chromatogram of the samples,after 6 (â-) and 2 (δ-) days of incubation, is also presented.

TABLE 1. Degradation of r- and γ-HCH, and the Release of Chloride and CO2, under Uninoculated and Inoculated Conditions

amount recovered (µmole)a

r-HCH γ-HCH

uninoculated inoculated uninoculated inoculated

days HCH HCH Cl CO2 HCH HCH Cl CO2

0 171.8 (100) 171.8 (100) 0 0 171.8 (100) 171.8 (100) 0 05 171.5 (99.8) 79.9 (46.5) 507.0 391.7 171.4 (99.7) 18.2 (10.6) 859.2 641.5

10 171.1 (99.5) 30.1 (17.5) 781.7 578.3 170.9 (99.4) 4.8 (2.8) 933.6 682.115 170.8 (99.4) 8.6 (5.0) 901.4 652.9 170.5 (99.2) 1.7 (1.0) 953.5 693.120 170.5 (99.2) 3.4 (1.9) 947.6 686.2 170.1 (99.0) 0.9 (0.5) 960.6 696.4

a Mean of four experiments. Reaction volume was 50 mL. The standard deviation was less than 5%. Values in parentheses are percent, takingthe recoveries at 0 time as 100%.

TABLE 2. Degradation of â- and δ-HCH, and Release of Chloride, under Uninoculated and Inoculated Conditions

amount recovered (µmole)a

â-HCH δ-HCH

uninoculated inoculated uninoculated inoculated

days HCH HCH Cl HCH HCH Cl

0 1.72 (100) 1.72 (100) 0 1.67 (100) 1.67 (100) 02 1.72 (100) 1.63 (94.7) BDL 1.62 (97.0) 0.75 (44.9) 1.104 1.70 (98.8) 1.50 (87.2) BDL 1.54 (92.2) 0.19 (11.3) 1.346 1.70 (98.8) 1.30 (75.5) 0.36 1.45 (86.8) 0.06 (3.6) 1.488 1.70 (98.8) 1.13 (65.7) 0.55 1.37 (82) 0.01 (0.9) 1.50

a The legend details are same as for Table 1 except reaction volume was 20 mL. BDL is below detection limit (0.3 µmole).

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was accompanied with the release of 0.9 µmole chloride ionsµmole-1 of these isomers (Table 2), suggesting that theirdegradation is limited. For this reason, release of CO2 aftertheir degradation was not measured. Concomitant to thedegradation of both â- and δ-HCH, a metabolite M1 wasformed, which undergoes further degradation (Figure 1). M1was tentatively identified as 2,3,4,5,6 pentachlorocyclohexan-1-ol (PCCOL) by mass spectroscopy (Figure 2), as its majorfragment ions m/z 237, 199, 157, 135, 125, 109, 99, and 85,matched well with those of PCCOL, reported earlier to beformed after the biodegradation of â-HCH by a Sphingomonaspaucimobiliz (27).

The metabolite M1 was further analyzed by NMR spec-troscopy (Figure 3). On the basis of the peak areas, proton-proton coupling constants, multiplet peak maximum “slopes”,and the COSY and HSQC (proton-detected proton-carboncorrelation) results, presence of an OH group and six protonswas detected and assigned (Table 3). The carbon-13 shiftswere determined by HSQC, and from the 2-D spectrum fourpeaks were deduced to be present (Table 4). It implied thepresence of a six-carbon ring with C2 symmetry about theC1-C4 axis. These assignments confirmed the identity ofM1 as PCCOL.

Formation of PCCOL during the degradation of δ-HCHby ITRC-5 is in contrast with earlier reports (26, 28) whereformation of δ-pentachlorocyclohexene, by the enzymedehydrochlorinase LinA that mediates the degradation of R-and γ-HCH in the strain UT 26, was observed (Figure 4).Formation of PCCOL during the degradation of both â- andδ-HCH by ITRC-5 suggests that their pathway might becommon in this strain.

The rate of degradation of different isomers by ITRC-5 isin the order of γ- > R- > δ- > â-HCH, which is distinct fromthat by a Sphingomonas paucimobiliz, R- > â- ) γ- > δ-HCH(29), and by a Pandoraea sp., δ- > â- ) R- > γ-HCH (15). Thereason for these differences is not clear at present.

Biodegradation of HCH Isomers in Contaminated Soils.In 5 g of soil-A, spiked with 17.15 µmole t-HCH (containing11.55, 1.17, 2.97, and 1.27 µmole of R-, â-, γ-, and δ-HCH,respectively), 8-12% decrease of all the HCH-isomers wasobserved after 25 days of incubation under uninoculatedconditions (Table 5). This could be due to the effect of variousabiotic factors, namely, volatilization, oxidation, photode-composition, and so forth, along with biotransformation bythe autochthonous microorganisms. Addition of ITRC-5 (2.8× 106 cfu g-1soil) enhanced the degradation of all the HCH-isomers. The degradation of R- and γ-HCH was rapid and>95% of these were degraded after 5 days of incubation (Table5). The degradation of â- and δ-HCH was slower, and 27%and 77% of these, respectively, were degraded after 15 days.Incubation for an additional 10 days led to only 2-6% increase

FIGURE 2. Mass spectrum of metabolite M1, formed during the biodegradation of â- (A) and δ-HCH (B).

TABLE 3. Assignment of the Peaks from 1H NMR Spectrum

peakno.

chemicalshift (ppm) multiplicity

relativearea assignment

1 2.80 broad doublet 1 -OH(J ) 2.5 Hz)

2 4.38 broad multiplet 1 H-1(overlapped)

3 4.39 doublet of doublets 2 H-2/H-6(J ) 2.2/J ) 11.5 Hz)

4 4.55 doublet of doublets 2 H-3/H-5(J ) 3/J ) 10.5 Hz)

5 4.72 triplet 1 H-4(J ) 2.5 Hz)

TABLE 4. Assignment of the Peaks from 13C NMR Spectrum

peak no. chemical shift (ppm) assignment

1 59.0 C-3/C-52 60.9 C-2/C-63 67.5 C-44 73.7 C-1

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in the degradation of â- and δ-HCH (Table 5). The degradationof HCH-isomers in the contaminated soil was accompaniedwith the release of 4.97 µmole chloride ions µmole-1 t-HCH(Table 5) suggesting that their degradation in soil-appliedconditions is similar to that in liquid-culture conditions.

While the degradation of soil-applied R- and γ-isomersby the isolate ITRC-5 is in agreement with the earlier studies(10, 20), to our knowledge this the first report wherein thedegradation of soil-applied â- and δ-isomers of HCH underaerobic conditions has been described.

Enhanced Degradation of â- and δ-HCH in the Soils inthe Presence of r- and γ-Isomers. The efficacy of the isolatedITRC-5 was evaluated toward the degradation of â- andδ-HCH, when these were the predominant contaminants insoils. Thus, in 5 g of soil-A that was contaminated with 0.85µmole â-HCH, <5% degradation of this isomer was observedafter 25 days of incubation under uninoculated conditions.Addition of ITRC-5 did not cause any enhancement in itsdegradation (Figure 5). The observation was surprising, asITRC-5 could affect the degradation of â-HCH in t-HCH

contaminated soils (Table 5). The degradation of â-HCH was,however, enhanced to 37% and 20% in the presence ofadditional 17 µmole R- or γ-HCH, respectively, after 10 daysof incubation. Similarly, from 5 g soil-A that was contaminatedwith 0.85 µmole δ-HCH, <10% was degraded after 10 daysof incubation under inoculated conditions. This was en-hanced to 92% and 70% in the presence of 17 µmole R- andγ-HCH, respectively (Figure 5). Further incubation up to 25days did not cause any significant increase in the degradationof soil-applied â- or δ-HCH (Figure 5).

The enhanced degradation of â- and δ-HCH is possiblydue to the proliferation of ITRC-5 in the presence of R- orγ-HCH as observed in liquid-culture (Table 1). No furtherdegradation of â- and δ-HCH was observed after 10 days ofincubation (Figure 5), possibly because the growth supportingR- or γ-isomers are depleted by this time. Other carbonsources, that is, glucose or sodium acetate, are also expectedto increase the growth of bacterium and consequently thedegradation of HCH-isomers. However, no increase in thedegradation of â- or δ-HCH in their presence was observed

FIGURE 3. 1H NMR spectra of metabolite M1.

TABLE 5. Biodegradation of HCH Isomers in Contaminated Soils

amount recovered (µmole)a

r-HCH â-HCH γ-HCH δ-HCH Cl

days UI I UI I UI I UI I UI I

0 11.58 (100) 11.58 (100) 1.17 (100) 1.17 (100) 2.97 (100) 2.97 (100) 1.27 (100) 1.27 (100) 0.0 0.05 11.44 (98.8) 0.41 (3.5) 1.16 (99.1) 1.02 (87.1) 2.89 (97.3) 0.14 (4.7) 1.25 (98.4) 0.53 (41.7) BDL 73.24

10 11.36 (98) 0.17 (1.4) 1.16 (99.1) 0.90 (76.9) 2.84 (95.6) 0.11 (3.7) 1.23 (96.8) 0.37 (29.1) BDL 79.4415 11.15 (96.2) 0.06 (0.5) 1.14 (97.4) 0.85 (72.6) 2.77 (93.2) 0.09 (3.0) 1.20 (94.4) 0.29 (22.8) BDL 80.5620 10.77 (93) 0.05 (0.4) 1.11 (94.8) 0.84 (71.7) 2.68 (90.2) 0.09 (3.0) 1.16 (91.3) 0.25 (19.6) BDL 82.5125 10.45 (90.2) 0.03 (0.2) 1.08 (2.3) 0.83 (70.9) 2.61 (87.8) 0.08 (2.7) 1.13 (89) 0.21 (16.5) BDL 84.51

a The details are same as for Table 1 except that the reaction was done in 5 g soil. UI, I, and BDL represent uninoculated, inoculated, and belowdetection limit (0.3 µmole), respectively.

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(Figure 5). This might be due to the repression of degradativegenes or their loss from the cells in the presence of favorablecarbon sources. Such repression/loss of degradative geneshas been reported earlier also in many cases (30, 31). Theobservations further explain that the degradation of soil-applied â-HCH was not observed in earlier studies (10, 20)because sufficient amount of additional R- or γ-isomer wasnot present.

In summary, the present study suggests that an effectiveremediation of HCH-contaminated sites, including those withthe otherwise persistent â- and δ-isomers of HCH, can beachieved by the addition of the isolated bacterium ITRC-5.Search for the gratuitous substances that can help inretaining/inducing the activity of ITRC-5 toward the deg-radation of â- or δ-HCH will be extremely useful.

AcknowledgmentsWe thank Dr. Tara Sutherland, CSIRO Entomology, Canberra,Australia for help in NMR analysis, and Dr. S.K. Tiwari,

National Botanical Research Institute, Lucknow, India, foranalysis of the soil samples. Financial assistance by Depart-ment of Biotechnology, India, is gratefully acknowledged.

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FIGURE 4. Biodegradation of δ-HCH to PCCH (26, 28) and PCCOL(this study).

FIGURE 5. Degradation of soil-applied â- and δ-HCH underuninoculated (UI) and inoculated (I) conditions. Degradation underinoculated conditions, in the presence of additional r-HCH (I + r),γ-HCH (I + γ), glucose (I + glu), or sodium acetate (I + ace), isalso shown. Values given are the mean of three replicates, and thestandard deviation was less than 10%.

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Received for review September 24, 2004. Revised manuscriptreceived March 22, 2005. Accepted March 24, 2005.

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