Title of Invention

A PROCESS FOR THE BIOREMEDIATION OF HEXACHLOROCYCLOHEXANE-CONTAMINATED SOILS

Abstract

The present invention relates to a process for the bioremediation of hexachlorocyclohexane contaminated soils. It relates to the degradation of hexachlorocyclohexane (HCH), a chlorinated pesticide consisting predominantly (>99%) of alpha-, gamma- and delta- isomers and marketed as technical Benzenehexachloride (BHC).In the prior art no one has described any process, wherein, all the four isomers of Hexachlorocyclohexane, present either alone or together (as in technical Benzenehexachloride) can be biodegraded from contaminated soils). Novelty of the present invention resides in the utilization of a novel strain of Pseudomonas sp. capable of biodegrading the major isomers of hexachlorocyclohexane resulting in the bioremedidiation of contaminated soils.

Full Text

The present invention relates to a process for the bioremediation of hexachlorocyclohexane contaminated soils. Particularly, it relates to the degradation of hexachlorocyclohexane (HCH), a chlorinated pesticide consisting predominantly (>99%) of, alpha-, beta-, gamma- and delta- isomers and marketed as technical Benzenehexachloride (BHC). Hexachlorocyclohexane (HCH) is used extensively, as an insecticide, for protection of crops and prevention of vector borne diseases. This chemical accumulates in the environment, enters the human body via food and causes toxicity. The soil and water at the sites where this chemical is manufactured, formulated, stored, spilled, dumped or intentionally used, become contaminated. Biodegradation ofjthis chemical by the process described herein offers significant potential for clean-up of contaminated sites, which could then be used for productive uses such as agriculture, aqua-culture etc. Bioremediation of HCH-contaminated soil will also help in the prevention of further pollution of water bodies through run-off of contaminated soil. The manufacturing industries can use it for treatment of effluents to meet the stipulated environmental standards and thereby proving the technology eco-friendly. Earlier, in literature, it has been shown that if agricultural soils, contaminated with hexachlorocyclohexane, are kept flooded for long periods (70-90 days), different isomers of hexachlorocyclohexane undergo biodegradation and eventually disappear (Mac Rae 1C, Raghu K and Castro T.F.(1967) Persistence and degradation of four common isomers of Benzenehexachloride. J. Agric.Food Chem. 15:911-914 ). This process is anaerobic and is extremely time consuming. Under aerobic conditions also, biodegradation of the isomers of hexachlorocyclohexane was studied (Bachmann A, DeBurien W, Jumelet J.C., Rijnaarts H.H.N and Zhender A.J.B.(1998) Aerobic mineralization of alpha-hexachlorocyclohexane in contaminated soils. Appl. Environ. Microbiol. 54: 548-554 ). In this study, alpha-isomer of hexachlorocyclohexane, after application at 300 mg/kg soil, was degraded in three days of incubation but the beta -isomer remained undegraded. Further, no attempt was made to study the degradation of gamma- and delta- isomers. In another study, from a contaminated soil, where multiple applications of gamma-hexachlorocyclohexane had been made, a rapid degradation of gamma-isomer has been demonstrated (Wada H., Senoo K. and Takai Y (1989) Rapid degradation of gamma- HCH in upland soil after multiple applications. Soil Sci. Plant Nutri. 35:71-77). A major drawback of this study is that the biodegradation of alpha-, beta-and delta- isomers was not studied. From this it is not possible to infer whether the degradation of other isomers can take place. Further more, the details of the microflora involved are not available. In yet another study, by the addition of an isolated organism, biodegradation of soil-applied isomers of hexachlorocyclohexane has been demonstrated (Sahu S.K., Patnaik K.K., Bhuyan S. and Sethunathan N. (1993) Degradation of soil applied isomers of hexachlorocyclohexane by Pseudomonas sp. Soil Biol. Biochem. 25:387-391). The limitation of this process was that the investigators demonstrated the degradation of alpha- and gamma- isomers of hexachlorocyclohexane, but not of the beta isomer. Further, the studies on the biodegradation of delta- isomer of hexachlorocyclohexane were not attempted. Thus, to-date, no one has described any process, wherein, all the four isomers of Hexachlorocyclohexane, present either alone or together (as in technical Benzenehexachloride) can be biodegraded from contaminated soils). The bacterial culture has been deposited at Institute of Microbial Technology MTCC/IDA,Chandigarh deposited on 22-2-1999.The number of deposit is MTCC B0017.the source of culture is Pesticide contaminated soil sediments from Lucknow,India. Novelty of the present invention resides in the utilization of a novel strain of Pseudomonas sp. Capable of biodegrading the major isomers of hexachlorocyclohexane resulting in the bioremedidiation of contaminated soils. These de-contaminated soils will now not contribute to environmental pollution and can be used fro productive purposes such as agriculture, aquaculture etc. The main object of the present invention is to provide "A process for the bioremediation of hexachlorocyclohexane contaminated soils" which obviates the drawbacks as detailed above. Accordingly, the present invention provides a process for the bioremediation of hexachlorocyclohexane contaminated soils which comprises; contacting hexachlorohexane (HCH-)- contaminated soil with a novel strain of Pseudomionas sp. capable of degrading isomers of HCH having characteristics as herein described at least for 48 hrs., optionally in presence of carbon sources and mineral ions to get HCH-decontaminated soil. In an embodiment of the present invention is the isolated bacterial strain, Pseudomonas sp. Designated as ITRC-1. This strain was isolated from a mixture of soils, obtained from four different contaminated sites in Lucknow, India, by "selective enrichment technique" wherein bacterial populations are selectively grown on target chemical (in this case, hexachlorocyclohexane) for 4-6 weeks. Growing cells were plated on an agar plate, made in nutrient media. Out of the several colonies that grew, one was selected for the degradation activity. This isolated strain mediates the biodegradation of all the four isomers of hexachlorocyclohexane, both in liquid culture and soil-bound conditions. The isolated bacterium is rod shaped gram negative, oxidase and catalase positive, motile and capable of growth at 42°C and in citrate medium. The intracellular enzymes responsible for this unique activity may be utilized for the biotransformation of other structurally related chemicals by fermentation. This novel strain has been deposited at Microbial Type Culture Collection at Institute of Microbial Technology, Chandigarh. In another embodiment of the present invention is that contacting may be effected up to four weeks. Still another embodiment of the present invention is that contacting of bacterium with HCH-contaminated soil may be effected by mixing or spraying the bacterium in slurry or powder form. In yet another embodiment of the present invention contacting may be effected at pH-5.0 to 10.0 "with optimal activity at neutral pH. Further to enhance the activity of bioremediation optionally carbon sources such as glucose, sodium succinate or sodium acetate and mineral ions such as calcium, magnesium, manganese may be added. The process of obtaining hexachlorocyclohexane-free-soil from the sites, contaminated with hexachlorocyclohexane, consists of addition of bacterial inoculums to the soils and their incubation for different time periods, depending upon the concentration of the pollutant. The degradation occurs, to varying extents, in wt (20% of the water holding capacity) to flooded soils, at pH 5-10 and also in presence of other carbon sources, such as glucose, sodium acetate & sodium succinate. The scientific explanation of this process is that, on inoculation of isolated microorganisms to the hexachlorocyclohexane contaminated soil, the isomers of hexachlorocyclohexane are degraded and utilized as source of carbon and energy by the organisms. The novelty of the process lies in the fact that unlike all the earlier reports in literature (discussed~earlier), this is the only process wherein all the four isomers of hexachlorocyclohexane, present in contaminated soil, are degraded. The following examples are given by way of illustration of the present invention and should not be construed to limit the scope of the present invention. Example 1: Degradation of Hexachlorocyclohexane isomers in contaminated soils. Ten grams of contaminated soil, obtained from an industrial site in Lucknow, was mixed with 240 gm of garden soil obtained from the campus of Industrial Toxicology Research Centre, Lucknow. Five gram portions of this soil were dispensed in sixteen containers (100 ml glass beakers), maintained as two groups of four sets each, in duplicate. To these containers 25 ml of mineral medium (KHjPC^O.l? g, Na2HPC>4 0.98 g; (NH4>2 SO4, 0.10 g; MgSO4, 4.87 mg; MgO, 0.10 mg; FeSO4 0.05 g; CaCO3, 0.20 mg, ZnSO4, 0.08 mg; CuSO4.5H2O, 0.16 mg; CoSO4, 0.015 mg; H3BO3, 0.006 mg; dissolved in 100 ml of distilled water) was added. Samples of group one were inoculated with 0.5 ml of bacterial suspension, prepared by inoculating the isolated bacterium, Pseudomonas sp. ITRC-1, in Luria-Bertoni (LB) medium, (5 g Bactotryptone, 2.5 g Yeast extract and 5.0 g of sodium chloride, adjusted to pH 7.4 and made up to 500 ml with distilled water), growth at 28° C overnight, centrifugation at 10,000 rpm for 10 min. and suspension in mineral medium to an optical density of 1.0 at 600 nm, and of group two remained uninoculated. Followed by incubation at 28° C, one set from each group was harvested after 0,2,3 and 4 weeks respectively. Isomers of Hexachlorocyclohexane were extracted by an organic solvent as described below. To each container 10 ml of acetone was added and the contents were stirred for 10 min. After addition of 25 ml of dichloromethane, the contents were stirred again for 10 min. The contents were allowed to settle and acetone/dichloromethane layer was recovered. To the remaining soil slurry, another 25 ml of dichloromethane was added. After stirring for 10 min, and leaving for another 20 min. for the contents to settle, the dichloromethane layer was recovered and added to the earlier acetone / dichloromethane fraction. This organic fraction was dried at room temperature and residual hexachlorocyclohexane isomers were quantified by Gas chromatography. The amounts of residual isomers of hexachlorocyclohexane (average of duplicate containers) were measured at different time periods and are presented below (Table 1). Table -1: Degradation of hexachlorocyclohexane isomers in contaminated soils (Table Removed)UI - Uninoculated, IN - Inoculated, values in parenthesis represent percent of HCH amounts present after different periods of incubation. The inference drawn from the results (Table 1) is, that in uninoculated samples, after four weeks of incubation, the residual alpha-, beta-, gamma- and delta-isomers of hexachlorocyclohexane were 54, 70, 58 and 85 percent respectively of the initial (100%) concentration. Addition of bacterial cells to these soil samples enhanced the degradation of hexachlorocyclohexane isomers, and the residual amounts of alpha-, beta-, gamma- and delta-isomers were 5, 20, 6 and 13 percent respectively of the initial (100%) concentration. Example 2: Biodegradation of hexachlorocyclohexane isomers, present individually, in liquid culture conditions by Pseudomonas sp. ITRC-1. To a group of ten sets consisting of two flasks (100 ml) each, 100 u,g of alpha- Hexachlorocyclohexane (HCH) and eight ml of mineral medium (KHjPO^ 0.17 g;Na2 HPO4, 0.098g; (NHt^ SO4, O.lOg; MgSO4, 4.87mg, MgO, 0.10 mg; FeSO4 O.OSmg; CaCO 3, 0.20mg; ZnSO4 0.08mg; CuSO4 ,5H2O, 0.016 mg; CoSO4, O.OlSmg; H3BO3, 0.006mg; dissolved in 100 ml of distilled water) was added. To sets 1-5 (uninoculated) 2 ml of mineral medium alone, and to sets 6-10 (inoculated) 2 ml of mineral medium containing 2x 106 cells was added. Likewise, separate groups often sets each were maintained for beta-, gamma- and delta-HCH. All the flasks were incubated at 28° C with shaking at 200 rpm for up to 5 days. A set of both uninoculated and inoculated flasks for each HCH-isomer was harvested daily for 5 days. The contents were extracted with hexane: acetone (2:1), evaporated and dissolved in 1 ml of hexane. The amounts of residual isomers (average of duplicate estimations) obtained are depicted in table 2. Table 2: Degradation of hexachlorocyclohexane (HCH) isomers in liquid culture conditions. (Table Removed)The inference drawn from the results of table 2 is that the bacterial addition causes the biodegradation of all the four isomers of HCH. The degradation of alpha-HCH is slow on day 1 but the rates of degradation of all the other HCH isomers are comparable. Example3: Growth, chloride release and degradation of different chlorinated hydrocarbons by isolated strain Pseudomonas sp. ITRC-1. To evaluate the efficacy of the isolated bacterium towards the degradation of other chlorinated compounds, ten conical flasks(50ml capacity) received 10 ml of mineral medium (KH2PO4, 0.17 g;Na2 HPO4, 0.098g; (NHOz SO4, O.lOg; MgSO4, 4.87mg, MgO, 0.10 mg; FeSO4 O.OSmg; CaCO 3, 0.20mg; ZnSO4 0.08mg; CuSO4 .5H2O, 0.016 mg; CoSO4, O.OlSmg; H3BO3, 0.006mg; dissolved in 100 ml of distilled water) containing either gamma-Hexachlorocyclohexane or Hexachlorobenzene or Pentachlorophenol, or 2,4-Dichlorophenol or 2,3,5- Trichlorophenol, or 2,4,5-Trichlorophenol or 1,2,3,4-Tetrachlorobenzene, or 1,2,4-Trichlorobenzene or 2,5-Dichlorophenol respectively at the concentration of 50 ppm. One flask (control) did not receive any carbon source. All the flasks were inoculated with 0.1 ml suspension of the isolated bacterium, Pseudomonas sp. ITRC-1, prepared by inoculation in LB medium, (5g Bactotryptone, 2.5g Yeast extract and 5g of Sodium chloride, adjusted to pH 7.4 and made up to 500 ml with distilled water) followed by incubation at 28°C overnight, separation of cells by centrifugation, and washed with mineral medium and suspended to final OD6oo=1.0. After incubation for 72 hrs the optical density of the medium, in all the flasks, was recorded. Two ml suspension from each flask was withdrawn and released chloride ions were measured by standard method (Bergmann J.G. and Sanik J.(1957)Determination of trace amounts of chloride in naptha. Anal. Chem. 29: 241-243 ). The results on the growth of cells (optical density) and released chloride ions are depicted in table 3. Table 3 : Growth and chloride release, in different chlorinated compounds, after bacterial inoculation. (Table Removed) • All measurements were done after 72 hrs of incubation. • At the start of experiment, ODeoo was 0.1. The inferences drawn from the above observations are that the isolated bacterium, can not-only grow on hexachlorocyclohexane, but can also grow on other chlorinated compounds like, Hexachlorobenzene, Pentachlorophenol, 2,4-Dichlorophenol and 2,4,5-Trichlorophenol. Growth on these chemicals is accompanied by release of chloride ions, suggesting an extensive mineralization of these chemicals. Isolated bacterial cells on other hand, cannot grow on 1,2,3,4-Tetrachlorobenzene, 1,2,4-Trichlorobenzene and 2,5-Dichlorophenol, and consequently chloride ions are not released. The main advantages of the present invention are: It provides a process to obtain Hexachlorocyclohexane-free-soil from the sites contaminated with the major isomers of hexachlorocyclohexane, a chlorinated pesticide. The decontaminated soil, thus obtained, can be useful for agriculture, aquaculture, human habitation and industrial activity. Bioremediation of a HCH-contaminated soil will help in prevention of further pollution of water bodies through run-off of contaminated soil. The manufacturing industries can use it for bio-treatment of effluents to meet the stipulated environmental standards and thereby proving the technology eco-friendly. We Claim: 1. A process for the bioremediation of hexachlorocyclohexane contaminated soils which comprises; contacting hexachlorohexane (HCH-)- contaminated soil with a novel strain of Pseudomionas sp. capable of degrading isomers of HCH having characteristics as herein described at least for 48 hrs., optionally in presence of carbon sources and mineral ions to get HCH-decontaminated soil. 2. A process as claimed in claim 1 wherein the contacting is effected up to four weeks. 3. A process as claimed in claims 1 and 2 wherein the contacting of bacterium with HCH- contaminated soil is effected by mixing or spraying the bacterium in slurry or powder form. 4. A process as claimed in claims 1 to 3 wherein the contacting is effected at pH-5.0 to 10.0 with optimal activity at neutral pH. 5. A process as claimed in claims 1 to 4 wherein the carbon sources such as glucose, sodium succinate or sodium acetate and mineral ions such as calcium, magnesium, manganese are optionally added fro-enhancing the activity of bioremediation. 6. A process for the bioremediation of hexachlorocyclohexane contaminated soils substantially as herein in described with reference to the examples accompanying this specification.

Documents:

275-del-2000-abstract.pdf

275-del-2000-claims.pdf

275-del-2000-correpondence-others.pdf

275-del-2000-correpondence-po.pdf

275-del-2000-description (complete).pdf

275-del-2000-form-1.pdf

275-del-2000-form-19.pdf

275-del-2000-form-2.pdf