Environ. Sci. Technol. 2005, 39, 4005-4011
Enhanced Biodegradation of β- and δ-Hexachlorocyclohexane in the Presence of r- and γ-Isomers in Contaminated Soils MANISH KUMAR, PANKAJ CHAUDHARY, MANISH DWIVEDI, RANJAN KUMAR, DEBARATI PAUL,† RAKESH K. JAIN,† SATYENDRA K. GARG,‡ AND ASHWANI KUMAR* Industrial Toxicology Research Centre, Post Box No 80, Mahatma Gandhi Marg, Lucknow- 226 001, India, Institute of Microbial Technology, Chandigarh, India, and Department of Microbiology, Dr. R. M. L. Avadh University, Faizabad- 224 001, India
The chlorinated insecticide hexachlorocyclohexane (HCH) has been used extensively in the past, and contaminated sites are present throughout the world. Toward their bioremediation, we isolated a bacterium Pseudomonas aeruginosa ITRC-5 that mediates the degradation of all the four major isomers of HCH under aerobic conditions, both in liquid-culture and contaminated soils. In liquidculture, the degradation of R- and γ-HCH is rapid and is accompanied with the release of 5.6 µmole chloride ions and 4.1 µmole CO2 µmole-1 HCH-isomer. The degradation of β- and δ-isomers is slow, accompanied with the release of 0.9 µmole chloride ions µmole-1 HCH-isomer, and results in a transient metabolite 2,3,4,5,6-pentachlorocyclohexan-1ol. The strain ITRC-5 also mediates the degradation of R-, β-, γ-, and δ-isomers in contaminated soils, where degradation of otherwise persistent β- and δ-HCH is enhanced severalfold in the presence of R- or γ-HCH. The degradation of soil-applied β- and δ-HCH under aerobic conditions has not been reported earlier. The isolate ITRC-5 therefore demonstrates potential for the bioremediation of HCHwastes and contaminated soils.
Introduction The insecticidal formulation of technical-hexachlorocyclohexane (t-HCH) predominantly consists of four major isomers, that is, R- (60-70%), β- (5-12%), γ- (10-18%), and δ-HCH (6-10%). It has been used extensively in the past for the protection of crops and control of vector borne diseases (1, 2). Its residues persist in the environment, undergo volatilization in tropical conditions, migrate to long distances with air current, deposit in colder regions, and cause widespread contamination (1-4). The HCH-residues enter the food chain and impart toxicity. Accordingly, the use of t-HCH has either stopped or is severely restricted in most countries, and only the insecticidal γ-isomer is permitted (1, 2). Because of extensive use of t-HCH in the past, numerous contaminated sites are present throughout the world (5). At * Corresponding author phone: +91-522-2620107; fax: +91-5222628227; e-mail:
[email protected]. † Institute of Microbial Technology. ‡ Dr. R. M. L. Avadh University. 10.1021/es048497q CCC: $30.25 Published on Web 04/22/2005
2005 American Chemical Society
these sites, being most persistent, β-HCH is usually present in disproportionately higher amounts (6-8). Stimulation of autochthonous microorganisms and augmentation by the isolated microbes are among the attractive options for enhanced bioremediation of contaminated soils (9). For this reason, microbial transformation of HCH-isomers both under aerobic (10-15) and anaerobic (16-18) conditions has been extensively studied. β-HCH was least susceptible to microbial degradation, possibly because of the all-equatorial arrangement of chlorine atoms on the cyclohexane ring (19). Under methanogenic conditions, microflora enriched from river sediments (17), or granular sludge of a sugar-beet refinery wastewater treatment reactor (18), has been shown to degrade all the four major isomers of HCH. Chlorobenzene and benzene were the major metabolites formed (17, 18). Under aerobic conditions, complete degradation of R- and γ-HCH (10-15) but limited degradation of β- and δ-HCH (10, 20) has been reported. The degradation of R- and γ-isomers has been demonstrated in contaminated soils also, but despite prolonged incubations no degradation of soil applied β- or δ-HCH was observed (10, 20). In this report, we describe the characterization of a bacterial strain ITRC-5 that under aerobic conditions mediates the degradation of all the four major HCH-isomers in liquid-culture as well as contaminated soils. In the contaminated soils, the degradation of otherwise persistent βand δ- isomers was enhanced in the presence of additional R- or γ-HCH.
Experimental Section Chemicals. Technical HCH, consisting of R- (67.4%), β(6.8%), γ- (17.3%), and δ-HCH (7.4%), was obtained from India Pesticides Limited, Lucknow, India. Individual isomers of HCH were purchased from Riedel-deHae¨n, Germany. Mercuric thiocyanate, 2-phenoxy-ethanol, and 2,5-dichlorophenol were from Sigma Chemical Co, St. Louis, MO. Soils. Two soils were used. Soil-A was from the garden of Industrial Toxicology Research Centre, Lucknow, India. It contained 43.6% clay, 20% silt, 36.4% sand, and 0.47% organic carbon, and the pH was 7.9. Soil-B was from the surroundings of an industry (India Pesticides Ltd, Lucknow, India), which had been manufacturing t-HCH in the past for several years. It contained 58% clay, 24% silt, 17.6% sand, and 0.15% organic carbon, and the pH was 9.72. Soil-B was contaminated with 51.54 µmole R- and 206.18 µmole β-HCH g-1 soil. Both the soils were air-dried and sieved through a 2-mm mesh before use. Isolation and Characterization of HCH-Degrading Bacterium. The bacterium was isolated from the HCHcontaminated rhizosphere soil-B by “selective enrichment method”. Briefly, 5 g soil was added to the flasks that were precoated with 6.87 µmole t-HCH and that contained 20 mL medium (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 mL distilled water, pH 7.6). After incubation at 28 °C with shaking at 180 rpm for 1 week, 2 mL of the growing culture was transferred to the fresh flasks and grown as above. After three more cycles of this enrichment process, the cells were plated and grown on agar (1.5% w/v) medium, at 28 °C for 48 h. The growing individual colonies were evaluated for their capability toward the degradation of γ-HCH by release of chloride ions. One of the colonies that exhibited maximum degradation was selected for further studies and designated as ITRC-5. Morphological and biochemical tests (21) and complete VOL. 39, NO. 11, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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sequencing of 16S ribosomal RNA gene were conducted for its identification Nucleotide sequencing was done on a cycle sequencer (ABI prism, model 310, version 3.4), using the big dye terminator cycle sequencing ready reaction kit (PE Biosystems) according to the manufacturers instructions. The organism has been deposited at Microbial Type Culture Collection and Gene Bank, Chandigarh, India, as MTCC 4727. Biodegradation of HCH-Isomers in Liquid-Culture. The isolated bacterium was grown in 20 mL medium containing 13.75 µmole t-HCH for 1 week at 28 °C with shaking at 180 rpm. Two mL of this culture (2.8 × 108 cfu mL-1) was used as inoculum for further experiments. Biodegradation/ mineralization of R- or γ-HCH was studied in 250-mL Erlenmeyer flasks that had a cylindrical well fused at the center of their base. Briefly, three sets of three flasks each, containing 45 mL of medium in their main body and 4.0 mL of 0.5 M KOH in the central well, were prepared. To the medium, 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, were added. After incubation at 28 °C with shaking at 180 rpm for 0, 5, 10, 15, and 20 days, the KOH solution was aspirated and replaced with the fresh solution. The aspirated KOH was titrated against 0.1 N HCl. The difference in the amount of HCl consumed by sets 1 and 3 was used for calculating the evolved CO2. No release of CO2 was observed in the flasks of set 2, which had HCH isomers but were not inoculated. In parallel sets, the reaction was terminated after 0-20 days by acidification to pH 99.8% homologous to the type strain that further confirmed this identification. The bacterium mediated the biodegradation of all four major isomers of HCH. The degradation of γ-HCH was faster than R-HCH at early time points but was >95% for both the isomers after 20 days of incubation (Table 1). The degradation of these isomers is accompanied with the formation of 5.6 µmole chloride ions and 4.1 µmole CO2 µmole-1 HCH-isomer (Table 1). During the degradation of γ-HCH, accumulation of a metabolite, 0.02 µmole µmole-1 γ-HCH, was also observed. The metabolite was identified as 2,5-dichlorophenol, on the basis of its comigration with the authentic compound on TLC (Retardation factor 0.34; developed in hexane:chloroform:acetone, 9:3:1, and visualized by Gibb’s reagent) and GC (Retention time 9.5). The result is in agreement with an earlier report (25) wherein 2,5-DCP is formed as a dead-end metabolite by the nonenzymatic conversion of the intermediary metabolite 2,4,5trichloro-2,5-cyclohexadiene-1-ol during the degradation of γ-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-HCH degradation in ITRC-5 might be similar to that in the reported strains (25, 26). The degradation of β- and δ-HCH by ITRC-5 was considerably slower than R- or γ-HCH. From the input 1.72 µmole, 34% of β- and >99% δ-HCH were degraded after 8 days of incubation (Table 2). The degradation of β- and δ-HCH
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 uninoculated
γ-HCH inoculated
uninoculated
inoculated
days
HCH
HCH
Cl
CO2
HCH
HCH
Cl
CO2
0 5 10 15 20
171.8 (100) 171.5 (99.8) 171.1 (99.5) 170.8 (99.4) 170.5 (99.2)
171.8 (100) 79.9 (46.5) 30.1 (17.5) 8.6 (5.0) 3.4 (1.9)
0 507.0 781.7 901.4 947.6
0 391.7 578.3 652.9 686.2
171.8 (100) 171.4 (99.7) 170.9 (99.4) 170.5 (99.2) 170.1 (99.0)
171.8 (100) 18.2 (10.6) 4.8 (2.8) 1.7 (1.0) 0.9 (0.5)
0 859.2 933.6 953.5 960.6
0 641.5 682.1 693.1 696.4
a Mean of four experiments. Reaction volume was 50 mL. The standard deviation was less than 5%. Values in parentheses are percent, taking the recoveries at 0 time as 100%.
TABLE 2. Degradation of β- and δ-HCH, and Release of Chloride, under Uninoculated and Inoculated Conditions amount recovered (µmole)a β-HCH uninoculated
a
δ-HCH inoculated
uninoculated
inoculated
days
HCH
HCH
Cl
HCH
HCH
Cl
0 2 4 6 8
1.72 (100) 1.72 (100) 1.70 (98.8) 1.70 (98.8) 1.70 (98.8)
1.72 (100) 1.63 (94.7) 1.50 (87.2) 1.30 (75.5) 1.13 (65.7)
0 BDL BDL 0.36 0.55
1.67 (100) 1.62 (97.0) 1.54 (92.2) 1.45 (86.8) 1.37 (82)
1.67 (100) 0.75 (44.9) 0.19 (11.3) 0.06 (3.6) 0.01 (0.9)
0 1.10 1.34 1.48 1.50
The legend details are same as for Table 1 except reaction volume was 20 mL. BDL is below detection limit (0.3 µmole).
FIGURE 1. Degradation of β- 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 (β-) and 2 (δ-) days of incubation, is also presented. VOL. 39, NO. 11, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. Mass spectrum of metabolite M1, formed during the biodegradation of β- (A) and δ-HCH (B).
TABLE 3. Assignment of the Peaks from 1H NMR Spectrum peak no.
chemical shift (ppm)
1
2.80
2
4.38
3
4.39
4
4.55
5
4.72
multiplicity broad doublet (J ) 2.5 Hz) broad multiplet (overlapped) doublet of doublets (J ) 2.2/J ) 11.5 Hz) doublet of doublets (J ) 3/J ) 10.5 Hz) triplet (J ) 2.5 Hz)
relative area
assignment
1
-OH
1
H-1
2
H-2/H-6
2
H-3/H-5
1
H-4
was accompanied with the release of 0.9 µmole chloride ions µmole-1 of these isomers (Table 2), suggesting that their degradation is limited. For this reason, release of CO2 after their degradation was not measured. Concomitant to the degradation of both β- and δ-HCH, a metabolite M1 was formed, which undergoes further degradation (Figure 1). M1 was tentatively identified as 2,3,4,5,6 pentachlorocyclohexan1-ol (PCCOL) by mass spectroscopy (Figure 2), as its major fragment ions m/z 237, 199, 157, 135, 125, 109, 99, and 85, matched well with those of PCCOL, reported earlier to be formed after the biodegradation of β-HCH by a Sphingomonas paucimobiliz (27). The metabolite M1 was further analyzed by NMR spectroscopy (Figure 3). On the basis of the peak areas, protonproton coupling constants, multiplet peak maximum “slopes”, and the COSY and HSQC (proton-detected proton-carbon correlation) results, presence of an OH group and six protons was detected and assigned (Table 3). The carbon-13 shifts were determined by HSQC, and from the 2-D spectrum four peaks were deduced to be present (Table 4). It implied the presence of a six-carbon ring with C2 symmetry about the C1-C4 axis. These assignments confirmed the identity of M1 as PCCOL. 4008
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TABLE 4. Assignment of the Peaks from 13C NMR Spectrum peak no.
chemical shift (ppm)
assignment
1 2 3 4
59.0 60.9 67.5 73.7
C-3/C-5 C-2/C-6 C-4 C-1
Formation of PCCOL during the degradation of δ-HCH by ITRC-5 is in contrast with earlier reports (26, 28) where formation of δ-pentachlorocyclohexene, by the enzyme dehydrochlorinase LinA that mediates the degradation of Rand γ-HCH in the strain UT 26, was observed (Figure 4). Formation of PCCOL during the degradation of both β- and δ-HCH by ITRC-5 suggests that their pathway might be common in this strain. The rate of degradation of different isomers by ITRC-5 is in the order of γ- > R- > δ- > β-HCH, which is distinct from that by a Sphingomonas paucimobiliz, R- > β- ) γ- > δ-HCH (29), and by a Pandoraea sp., δ- > β- ) R- > γ-HCH (15). The reason 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 (containing 11.55, 1.17, 2.97, and 1.27 µmole of R-, β-, γ-, and δ-HCH, respectively), 8-12% decrease of all the HCH-isomers was observed after 25 days of incubation under uninoculated conditions (Table 5). This could be due to the effect of various abiotic factors, namely, volatilization, oxidation, photodecomposition, and so forth, along with biotransformation by the autochthonous microorganisms. Addition of ITRC-5 (2.8 × 106 cfu g-1soil) enhanced the degradation of all the HCHisomers. The degradation of R- and γ-HCH was rapid and >95% of these were degraded after 5 days of incubation (Table 5). The degradation of β- 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 3. 1H NMR spectra of metabolite M1.
TABLE 5. Biodegradation of HCH Isomers in Contaminated Soils amount recovered (µmole)a r-HCH
β-HCH
γ-HCH
Cl
δ-HCH
days
UI
I
UI
I
UI
I
UI
I
UI
I
0 5 10 15 20 25
11.58 (100) 11.44 (98.8) 11.36 (98) 11.15 (96.2) 10.77 (93) 10.45 (90.2)
11.58 (100) 0.41 (3.5) 0.17 (1.4) 0.06 (0.5) 0.05 (0.4) 0.03 (0.2)
1.17 (100) 1.16 (99.1) 1.16 (99.1) 1.14 (97.4) 1.11 (94.8) 1.08 (2.3)
1.17 (100) 1.02 (87.1) 0.90 (76.9) 0.85 (72.6) 0.84 (71.7) 0.83 (70.9)
2.97 (100) 2.89 (97.3) 2.84 (95.6) 2.77 (93.2) 2.68 (90.2) 2.61 (87.8)
2.97 (100) 0.14 (4.7) 0.11 (3.7) 0.09 (3.0) 0.09 (3.0) 0.08 (2.7)
1.27 (100) 1.25 (98.4) 1.23 (96.8) 1.20 (94.4) 1.16 (91.3) 1.13 (89)
1.27 (100) 0.53 (41.7) 0.37 (29.1) 0.29 (22.8) 0.25 (19.6) 0.21 (16.5)
0.0 BDL BDL BDL BDL BDL
0.0 73.24 79.44 80.56 82.51 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 below detection limit (0.3 µmole), respectively.
in the degradation of β- and δ-HCH (Table 5). The degradation of HCH-isomers in the contaminated soil was accompanied with the release of 4.97 µmole chloride ions µmole-1 t-HCH (Table 5) suggesting that their degradation in soil-applied conditions is similar to that in liquid-culture conditions. While the degradation of soil-applied R- and γ-isomers by the isolate ITRC-5 is in agreement with the earlier studies (10, 20), to our knowledge this the first report wherein the degradation of soil-applied β- and δ-isomers of HCH under aerobic conditions has been described. Enhanced Degradation of β- and δ-HCH in the Soils in the Presence of r- and γ-Isomers. The efficacy of the isolated ITRC-5 was evaluated toward the degradation of β- and δ-HCH, when these were the predominant contaminants in soils. Thus, in 5 g of soil-A that was contaminated with 0.85 µmole β-HCH,
