Title of Invention	A PROCESS FOR DEGRATION OF DICHLORO DIPHENY LTRICHLORO ETHANE (DDT) USING AN IMPROVED STRAIN OF SERRATIA MARCESCENS
Abstract	The present invention provides a process for degradation of dichlorodiphenyltrichloroethane (DDT) using an improved bacterial strain of Serratia marcescens . A novel improved strain of Serratia marcescens having ability to degrade DDT was isolated by enrichment of DDT contaminated soil and culturing in medium containing 5ppm DDT as the sole source of carbon . The obtained strain was cultivated in a 0.5% basal glucose- minimal medium containing 10 ppm DDT and a co-substrate , at a temperature of 20-30°C for 2 h. The said culture acclimatized in shake culture for a period of 48 hours , the acclimatized culture degrade DDT within 72 h .

Title of Invention

A PROCESS FOR DEGRATION OF DICHLORO DIPHENY LTRICHLORO ETHANE (DDT) USING AN IMPROVED STRAIN OF SERRATIA MARCESCENS

Abstract

The present invention provides a process for degradation of dichlorodiphenyltrichloroethane (DDT) using an improved bacterial strain of Serratia marcescens . A novel improved strain of Serratia marcescens having ability to degrade DDT was isolated by enrichment of DDT contaminated soil and culturing in medium containing 5ppm DDT as the sole source of carbon . The obtained strain was cultivated in a 0.5% basal glucose- minimal medium containing 10 ppm DDT and a co-substrate , at a temperature of 20-30°C for 2 h. The said culture acclimatized in shake culture for a period of 48 hours , the acclimatized culture degrade DDT within 72 h .

Full Text	The present invention relates to a process for a degradation of An dichlorodiphenyltrichlproethane (DDT), mine an improved strain of Serratia marcesscens invention particularly relates to A process for the preparation of a biocatalyst formulation, which can be used for the removal of DDT residues from industrial effluents contaminated water bodies and other contaminated sites through degradation. 1,1,1-Trichloro-2,2-bis(4-chlorophenyl)ethane commonly called dichlorodipheny-trichloroethane (DDT) is one of the most persistent environmental pollutants introduced by man way back in 1940s. It has been extensively used world wide in both agriculture and public health programmes after World War II (Spindler ,M 1983, Residue Rev. 90, 1-34). Due to continued and descriminate use of this insecticide, DDT has emerged as one of the major pollutants in India and other third world countries. DDT appears to undergo slow degradation in the environment. Reference may be made to Addison. R.F.; Brodie P.F; Zinck M.E and Sergeant D.E, 1984, Environ. Sci. Technol. , 18, 935-937; Muir D.C.G, Yarechawski A.L; Corbet R.L; Webster G.R.B and Smith A.E 1985, J Agric. Food Chem. 33, 518-523; Wedemeyer G, Science, 1967, 152, 647; Wedemeyer G, 1967, Appl, Microbiol. , 15, 1494-1495. ; Focht D.D and Alexander M 1970, Appl. Microbiol. 20, 608-611; Focht D.D and Alexander M 1970, Science, 170, 91-92; Focht D.D. and Alexander M 1971 , J .Agric Food Chem. 19, 20-22; Pfaender F.K. and Alexander M 1972, J. Agric. Food Chem. 20, 842-846; Pfaender F.K. and Alexander.M 1973, J.Agric. Food Chem, 21, 397-399; Subba Rao R.V. and Alexander M ,1977, Appl. Environ. Microbiol. 33, 101-108; Subba Rao R.V and Alexander M, 1985, Appl. Environ. Microbiol, 49, 509-516, wherein biodegradation of DDT and DDT metabolites by microorganisms have been demonstrated. Two major pathways of DDT degradation have been reported: (1) Reductive dechlorination, favoured under anaerobic conditions and (2) dehydrochlorination favoured under aerobic conditions. The reports are mainly under anaerobic conditions. References may be made to Lai, R and Saxena D.M, 1982, Microbiol. Rev., 46, 95-127, wherein the involvement of microorganisms such as Enterobacter aerogenes, Pseudomonas fluorescens, Escherichia coli and Klebsiella pneumoniae have been shown. These organisms dechlorinate p,p'-DDT to 1,1-dichloro-2,2-bis(4-chlorophenyl) ethylene (DDE), 1,1-dichloro-2,2-bis(4-chlorophenyl)ethane (DDD), 1-chloro-2,2-bis(4-chlorophenyl)ethylene (DDMU), 1-chloro-2,2-bis(4-chlorophenyl)ethane (DDMS), unsym-bis(4-chlorophenyl)ethylene (DDNU) exclusively under anareobic conditions. Aerobic degradation of DDT by Alcaligenes eutrophus A5 has been described recently. Reference may be made to Nadeau, L,J, Menn, F,M, Breen, A and Sayler, G.S, 1994, Appl. Environ. Microbiol, 60,51-55. The initial aerobic step has been shown to be the oxidation of the phenyl ring at the adjacent ortho- and mete-positions to form hydroxy - DDTs, which form a dihydrodiol due to its instability. Dihydrodiol was further metabolised to 2,3-dihydroxy-DDT, which would be further catabolised to 4-chlorobenzoic acid. Among fungi, Phanerochaete chrysosporium , a white rot fungus, has been reported to mineralise DDT ( Bumpus, J. A. and Aust, S. D., 1985,Science, 228, 1434-1436) by first oxidising DDT to 2,2,2-trichloro-1,1-bis(4-chlorophenyl)ethanol (dicofol) and then dechlorinating to 2,2-dichloro-1,1-bis(4-chlorophenyl)ethanol followed by oxidative carbon-carbon bond cleavage to form 4,4'-dichlorobenzophenone (DBP). The drawback in these works is the degradation is at a very low concentrations and also incomplete. Reference may be made to a patent that has been issued in the U.S.A (compost decontamination of DDT- contaminated, .soil, Bernier. R. L. Gray, N.C.C. and Moser, L.E., US 5660612 dt. August 26th 1997) which deals with decontamination of soil containg.DDT.type contaminants by composting.. Reference may be made to incorporate Indian patent (pending) No. 256/ del / 2000 wherein the involvement of microorganism, Pseudomonas aeruginosa in the elimination of DDT under aerobic conditions in shake flasks and in soil has been demonstrated. The drawbacks in all the above works are that the concentration of DDT degraded by these bacterial and fungal isolates is very low and also the degradation is incomplete. Hence it is imperative to develop a process to degrade higher concentrations of DDT completely within a reasonably short period. The main object of the present invention is to provide a process for the preparation of a biocatalyst formulation useful for the degradation of dichlorodiphenyltrichloroethane (DDT) residues, which obviates the drawbacks as detailed above. Another object of the present invention is to prepare a biocatalyst for the treatment of DDT industry effluents for the elimination of DDT other contaminating residues. In the drawings accompanying this specification, Fig 1 represents degradation of 5ppm DDT by Serratia marcescens CFR 1020. Growth (OD 600) CI" released residual DDT Fig 2 represents degradation of 10 ppm of DDT by Serratia marcescens CFR 1020. All symbols are as given in Fig 1. Fig 3 represents degradation of 15 ppm of DDT by Serratia marcesscens CFR 1020. All symbols are as given in Fig 1. Fig 4 represents degradation of 20 ppm of DDT by Serratia marcesscens CFR 1020. All symbols are as given in fig. 1. All these experiments were done by incubating the flasks at 30°C on a rotary shaker at a speed of 150 rpm. Accordingly the present invention provides a process for the degradation of dichlorodiphenyltrichloroethane (DDT) which comprises preparing a novel improved strain of Serratia marcescens having characteristics as herein described by enrichment of DDT contaminated soil and culturing in medium containing 5ppm DDT as the sole source of carbon , culturing the obtained strain in a 0.5% basal glucose- minimal medium containing 15 ppm DDT and a co-substrate such as herein described , at a temperature of 20-30°C for 2 h followed by acclimatization for 48 h , getting degradation of DDT from mineral medium. In an embodiment of the present invention the degradation may be effective in presence of co-substrate selected from the group consisting of glycerol, peptone, yeast extract and tryptone soya broth at 10 ppm level of In an another embodiment of the present invention the degradation may be effected in the inoculum previously grown on various substrates selected from the group consisting of acetate, succinate, citrate, glucose, sucrose, glycerol, peptone, yeast extract and tryptone soya broth at 0.5 % level. In yet an another embodiment of the present invention the DDT used may be employed up to 15ppm. Serratia marcescens CFR1020 was cultivated on 0.5% glucose- mineral medium containing 10ppm DDT for 24 h. The cells were then subjected to acclimation by growing them in 10ppm DDT as the sole source of carbon in the mineral medium. This acclimated culture could degrade 15µg/ ml DDT in 120 h in shake flasks. In the presence of co-substrates, 10ppm DDT disappeared by 72h compared to flasks without co-substrates. This microbial culture can be effectively deployed for the treatment of DDT industry effluents, water bodies and other materials contaminated with DDT- residues. A novel process was deployed for elimination of DDT-residues from industrial effluents and other contaminated materials using a biocatalyst preparation of a bacterial culture Serratia marcescens CFR 1020. The microbial culture was developed by enrichment of DDT contaminated soil in shake flasks containing DDT as the sole source of carbon and energy. The microbial consortium that got established in the medium containing 5ppm of DDT was resolved into individual bacterial strains. Individual strains were tested for their ability to degrade DDT. Of the four bacterial strains, was obtained Serratia marcescens CFR 1020, that could degrade effectively 15µg/ ml, The culture characteristics of Serratia marcescens CFR 1020 are as follows: Orange to red colonies with ridges with curled margins on nutrient agar. During growth on pyocyanin agar the strain produces red coloured pigment, on fluorescene agar it produces red to orange pigment. The surface of the colony on nutrient agar is rough and umbonate with light buff coloured transluscent ring around. It grows from 8° C to 41°C very well. Older colonies become reddish brown and sticky in nature. Cells are cocco-bacilloid with a hollow ridge in the centre, measuring 0.57- 0.81(m in length and 0.32- 0.49(m in width. It is gram -ve, motile, aerobic, oxidase -ve, indole -ve, does not produce H2S. Subjecting the isolate to increasing concentrations of DDT as sole carbon source has developed the mutant of this isolate by conventional enrichment technique. Dichlorodiphenyltrichloroethane (DDT) used was 98% pure and was obtained from Sigma chemical company, Mo, USA. Other chemicals used in the media and the reagents were of analytical grade and were odtained, from standard Indian companies. The basal medium used for enrichment and for growth of the cultures contained (per liter of distilled water) 0.675g KH2 PO4 ; 5.455g Na2HPO4 ; 0.25g NH4N03 ; 0.2g MgS04 . 7H20; 0.1g Ca (NO3) 2 and 1.0 ml of trace mineral solution containing (mg/ ml)- FeSO4.7H2 O, 1.0; MnSO4 . H2 O, 1.0; CuCI2. 2 H2O, 0.25; Na2MoO4 . 2 H2O, 0.25; H3BO3, 0.1 and cone. H2SO4, 5.0 ml. The pH of the medium was 7.5. Required amount of DDT and / or co-substrate was added to the medium as described later. Nutrient agar plates were used for resolving the different bacterial strains from mixtures of acclimated isolates, as well as for the study of their taxanomy. Nutrient agar medium contained 3g beef extract, 5g peptone, 5g NaCI and 20g agar per liter of distilled water (pH 7). Cultivation in shake flasks was carried out in 50ml mineral medium containing required amount of growth substrate (DDT) taken in 250ml Erlenmeyer flasks and incubating at 30°C on a rotary shaker (150rpm). For maintaining the strain, mineral agar plates/ slopes containing DDT 25µg/ml) were used. The culture was incubated for 4-5 days at 30°C and then preserved at 4°C. The culture was maintained as liquid culture also, after growing it in mineral salts medium with 10µg/ml DDT for 48h and storing at 4°C. The growth of the culture was determined spectrophotometrically by measuring the turbidity at 600nm. Quantitative estimation of chloride was done by HNO3-AgNO3 method. To 1ml of the aliquot of culture supernatant, 1ml of 0.15N HNO2 was added followed by the addition of 0.1 N AgNO3. The contents of the tubes were mixed well and were allowed to stand at room temperature for 20 min. Then turbidity was measured at 600nm. The amount of chloride was computed from a standard curve prepared for NaCI. Quantitative determination of the substrate residues (DDT) was determined either by thin layer chromatography or gas chromatography with 63Ni electron capture detector. The culture broth was extracted thrice with equal volumes of diethyl ether. The solvent layers were pooled, evaporated and redissolved in convenient volume of acetone. Known volume of this was loaded on to a silica gel G- coated plate (0.3mm) and developed in cyclohexane. The plates were air-dried and DDT was detected by spraying the plate with 2% solution of o-tolidine in acetone and exposing to sunlight. The spots were delineated by marking with a needle and the area was measured. The concentration was computed from a standard plot of log concentrations versus square root of the area, prepared for reference standard of DDT. 63Ni electron capture detector was used. The conditions used were as follows: column, 2mm ID and 200mm length packed with 1.5% OV-17 plus 1.95QF1 on 80-100 mesh chromosorb W; carrier gas, nitrogen; flow at 50ml /min.; column temperature, 230 °C, injector temperature, 230 °C and detector temperature, 320 °C. The retention of DDT was 7.4 min under these conditions. This improved strain of Serratia marcescens CFR 1020 showed ability to degrade p,p'-DDT which took 96h to completely mineralise 10µg/ ml DDT. However in the presence of co-substrates at 0.5% level, 10µg/ ml DDT was degraded by 72h. The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention. EXAMPLE 1 The mineral medium contained 0.675g KHa PO4 ; 5.455g NaaHPO4 ; 0.25g NH4NO3 ; 0.2g MgS04 . 7H2O; 0.1g Ca (NO3) 2 and 1.0 ml of trace mineral solution per liter of distilled water. The substrate, DDT was added at 5µg/ ml level. The required amount of substrate was dissolved in 50µl of acetone and was added at the bottom of a dry, sterile 250ml conical flask, inside a laminar hood and kept open till the acetone evaporated completely. Then 25ml of mineral medium was added and inoculated with cells of Serratia marcescens CFR 1020. The inoculum added was cells equivalent to OD600 of 0.2 per ml after inoculation (0 hr). The flasks were incubated on a rotary shaker at 150rpm at 30° C. Samples were drawn at regular intervals and analysed for growth (OD600), inorganic chloride (CI) and residual substrate. The results are presented in Fig.1. (sheet No. 1 of the drawing accompanying the specification). Complete disappearance of the substrate and the release of 100% CI" was observed at 96 h of incubation. Biomass also increased during this period. These observations clearly indicate that DDT (5ppm) was completely mineralised. The microbial mass was separated by centrifugation at 8000rpm for 10 min. and lyophilised by freeze-drying. EXAMPLE 2 The mineral medium contained 0.675g KH2 PO4i 5.455g NaaHPO4 0.25g NH4NO3; 0.2g MgSO4. 7H2O; 0.1g Ca (N03) 2 and 1.0 ml of trace mineral solution per liter of distilled water. The substrate, DDT was added at 10ng/ml level. The required amount of substrate was dissolved in 50µl of acetone and was added at the bottom of a dry, sterile 250ml conical flask, inside a laminar hood and kept open till the acetone evaporated completely. Then 25ml of mineral medium was added and inoculated with cells of Serratia marcescens CFR 1020. The inoculum added was cells equivalent to OD600 of 0.2 per ml after inoculation (0 hr). The flasks were incubated on a rotary shaker at 150rpm at 30°C. Samples were drawn at regular intervals and analysed for growth (OD600), inorganic chloride (CI) and residual substrate. The results are presented in Fig.2. (sheet No.2 of the drawing accompanying the specification). Complete disappearance of the substrate and the release of 100% Cl was observed at 96h of incubation. The microbial mass was separated by centrifugation at 8000 pm for 10 min. and lyophilised by freeze-drying. EXAMPLE 3 The mineral medium contained 0.675g KH2 PO4; 5.455g Na2HPO4; 0.25g NH4NO3; 0.2g MgSO4. 7H2O; 0.1g Ca (NO3) 2 and 1.0 ml of trace mineral solution per liter of distilled water. The substrate, DDT was added at 15(g/ ml level. The required amount of substrate was dissolved in 50(l of acetone and was added at the bottom of a dry, sterile 250ml conical flask, inside a laminar hood and kept open till the acetone evaporated completely. Then 25ml of mineral medium was added and inoculated with cells of Serratia marcescens CFR 1020. The inoculum added was cells equivalent to OD6oo of 0.2 per ml after inoculation (0 hr). The flasks were incubated on a rotary shaker at 150rpm at 30°C. Samples were drawn at regular intervals and analysed for growth (OD600), inorganic chloride (Cl~) and residual substrate. The results are presented in Fig.3. (sheet No.3 of the drawing accompanying the specification). Complete disappearance of the substrate and the release of 100% CI" was observed at 120h of incubation. The microbial mass was separated by centrifugation at 8000rpm for 10 min. and lyophilised by freeze-drying. EXAMPLE 4 The mineral medium contained 0.675g KH2 PO4; 5.455g Na2HPO4; 0.25g NH4NO3; 0.2g MgSO4. 7H2O; 0.1g Ca (NO3) 2 and 1.0 ml of trace mineral solution per liter of distilled water. The substrate, DDT was added at 20µg/ ml level. The required amount of substrate was dissolved in 50(l of acetone and was added at the bottom of a dry, sterile 250ml conical flask, inside a laminar hood and kept open till the acetone evaporated completely. Then 25ml of mineral medium was added and inoculated with cells of Serratia marcescens CFR 1020. The inoculum added was cells equivalent to OD600 of 0.2 per ml after inoculation (0 hr). The flasks were incubated on a rotary shaker at 150rpm at 30°C. Samples were drawn at regular intervals and analysed for growth (OD600), inorganic chloride (Cl~) and residual substrate. The results are presented in Fig.4. (sheet No.4 of the drawing accompanying the specification). Complete disappearance of the substrate and the release of 90% CI" was observed at 144h of incubation. The microbial mass was separated by centrifugation at 8000rpm for 10 min. and lyophilised byfreeze-drying. EXAMPLE 5 Co-metabolism of 10(g/ ml DDT by Serratia marcescens CFR 1020 was studied in shake flasks. 25ml mineral medium was taken in 250 ml Erlenmeyer flasks containing 0.5 % of different substrates as listed in Table 1. The mineral medium contained 0.675g KH2 PO4; 5.455g Na2HPO4; 0.25g NH4NO3; 0.2g MgSO4. 7H2O; 0.1g Ca (NO3) 2 and 1.0 ml of trace mineral solution per liter of distilled water. The substrate, DDT was added at 10µg/ ml level. The required amount of substrate was dissolved in 50µl of acetone and was added at the bottom of a dry, sterile conical flask, inside a laminar hood and kept open till the acetone evaporated completely. Then 25ml of mineral medium containing 0.5 % of required co-substrate was added and inoculated with cells of Serratia marcescens CFR 1020. The inoculum added was cells equivalent to OD6oo of 0.2 per ml after inoculation (0 hr). The flasks were incubated on a rotary shaker at 150rpm at 30° C for 72 h. Samples were analysed for growth (OD600), inorganic chloride (Cl) and residual substrate. The inoculum was the colonies grown on nutrient agar plates, suspended in minimal medium, washed well and resuspended in minimal medium. The results are presented in Table 1. Complete disappearance of the substrate and the release of 100% Cl" was observed. Table: 1 (Table Removed) EXAMPLE 6 Effect of growth of Serratia marcescens CFR 1020 on different substrates and degradation of 10(g/ ml DDT was studied in shake flasks. 20 ml of mineral medium containing 0.5 % of different substrates (taken separately) was taken in 100 ml Erlenmeyer flasks. The mineral medium contained 0.675g KH2PO4; 5.455g Na2HPO4; 0.25g NH4NO3; 0.2g MgSO4. 7H2O; 0.1g Ca (NO3) 2 and 1.0 ml of trace mineral solution per liter of distilled water. Single colonies grown on nutrient agar were inoculated to these flasks and the flasks were incubated on a rotary shaker at 150rpm, 30°C for 24 h. The cells were harvested, washed well and used as inoculum. The substrate, DDT was added at lOµg/ ml level. The required amount of substrate was dissolved in 50µl of acetone and was added at the bottom of a dry, sterile 100ml conical flask, inside a laminar hood and kept open till the acetone evaporated completely. Then 20ml of mineral medium was added and inoculated with cells of Serratia marcescens CFR 1020 grown on different substrates. The inoculum added was cells equivalent to OD600 of 0.2 per ml after inoculation (0 hr). The flasks were incubated on a rotary shaker at 150rpm at 30°C for 72 h. Samples were analysed for growth (OD600), inorganic chloride (Cl~) and residual substrate. The results are presented in Table 2. Table: 2 (Table Removed) The main advantages of the present invention are: 1. The preparation can be in powder form. 2. The preparation can be applied to effluent treatment plants for elimination of DDT. 3. The preparation can eliminate DDT efficiently in presence of co- substrates. We Claim: 1. A process for the degradation of dichlorodiphenyltrichloroethane (DDT) using an improved strain of Serratia marcescens which comprises preparing a novel improved strain of Serratia marcescens having characteristics as herein described by enrichment of DDT contaminated soil and culturing in medium containing 5ppm DDT as the sole source of carbon , culturing the obtained strain in a 0.5% basal glucose- minimal medium containing 15 ppm DDT and a co-substrate such as herein described , at a temperature of 20-30°C for 2 h followed by acclimatization for 48 h , getting degradation of DDT from mineral medium. 2. A process as claimed in claim 1 wherein the co-substrate used is selected from the group consisting of glycerol, peptone, yeast extract and tryptone soya broth at 10ppm level of DDT. 3. A process as claimed in claims 1-2, wherein the degradation is effected in the inoculum previously grown on various substrates selected from the group consisting of acetate, succinate, citrate, glucose, sucrose, glycerol, peptone, yeast extract and tryptone soya broth at 0.5 % level. 4. A process for the degradation of dichlorodiphenyltrichloroethane (DDT) using an improved strain of Serratia marcescens substantially as herein described with reference to the examples and drawings accompanying this specification.

Full Text

The present invention relates to a process for a degradation of An dichlorodiphenyltrichlproethane (DDT), mine an improved strain of Serratia marcesscens invention particularly relates to A process for the preparation of a biocatalyst formulation, which can be used for the removal of DDT residues from industrial effluents contaminated water bodies and other contaminated sites through degradation.
1,1,1-Trichloro-2,2-bis(4-chlorophenyl)ethane commonly called dichlorodipheny-trichloroethane (DDT) is one of the most persistent environmental pollutants introduced by man way back in 1940s. It has been extensively used world wide in both agriculture and public health programmes after World War II (Spindler ,M 1983, Residue Rev. 90, 1-34). Due to continued and descriminate use of this insecticide, DDT has emerged as one of the major pollutants in India and other third world countries. DDT appears to undergo slow degradation in the environment.
Reference may be made to Addison. R.F.; Brodie P.F; Zinck M.E and Sergeant D.E, 1984, Environ. Sci. Technol. , 18, 935-937; Muir D.C.G, Yarechawski A.L; Corbet R.L; Webster G.R.B and Smith A.E 1985, J Agric. Food Chem. 33, 518-523; Wedemeyer G, Science, 1967, 152, 647; Wedemeyer G, 1967, Appl, Microbiol. , 15, 1494-1495. ; Focht D.D and Alexander M 1970, Appl. Microbiol. 20, 608-611; Focht D.D and Alexander M 1970, Science, 170, 91-92; Focht D.D. and Alexander M 1971 , J .Agric Food Chem. 19, 20-22; Pfaender F.K. and Alexander M 1972, J. Agric. Food Chem. 20, 842-846; Pfaender F.K. and Alexander.M 1973, J.Agric. Food Chem, 21, 397-399; Subba Rao R.V. and Alexander M ,1977, Appl. Environ.
Microbiol. 33, 101-108; Subba Rao R.V and Alexander M, 1985, Appl. Environ. Microbiol, 49, 509-516, wherein biodegradation of DDT and DDT metabolites by microorganisms have been demonstrated. Two major pathways of DDT degradation have been reported: (1) Reductive dechlorination, favoured under anaerobic conditions and (2) dehydrochlorination favoured under aerobic conditions. The reports are mainly under anaerobic conditions.
References may be made to Lai, R and Saxena D.M, 1982, Microbiol. Rev., 46, 95-127, wherein the involvement of microorganisms such as Enterobacter aerogenes, Pseudomonas fluorescens, Escherichia coli and Klebsiella pneumoniae have been shown. These organisms dechlorinate p,p'-DDT to 1,1-dichloro-2,2-bis(4-chlorophenyl) ethylene (DDE), 1,1-dichloro-2,2-bis(4-chlorophenyl)ethane (DDD), 1-chloro-2,2-bis(4-chlorophenyl)ethylene (DDMU), 1-chloro-2,2-bis(4-chlorophenyl)ethane (DDMS), unsym-bis(4-chlorophenyl)ethylene (DDNU) exclusively under anareobic conditions. Aerobic degradation of DDT by Alcaligenes eutrophus A5 has been described recently. Reference may be made to Nadeau, L,J, Menn, F,M, Breen, A and Sayler, G.S, 1994, Appl. Environ. Microbiol, 60,51-55. The initial aerobic step has been shown to be the oxidation of the phenyl ring at the adjacent ortho- and mete-positions to form hydroxy - DDTs, which form a dihydrodiol due to its instability. Dihydrodiol was further metabolised to 2,3-dihydroxy-DDT, which would be further catabolised to 4-chlorobenzoic acid. Among fungi, Phanerochaete chrysosporium , a white rot fungus, has been reported to mineralise DDT ( Bumpus, J. A. and Aust, S. D., 1985,Science, 228, 1434-1436) by first oxidising DDT to 2,2,2-trichloro-1,1-bis(4-chlorophenyl)ethanol (dicofol) and then
dechlorinating to 2,2-dichloro-1,1-bis(4-chlorophenyl)ethanol followed by oxidative carbon-carbon bond cleavage to form 4,4'-dichlorobenzophenone (DBP). The drawback in these works is the degradation is at a very low concentrations and also incomplete.
Reference may be made to a patent that has been issued in the U.S.A (compost decontamination of DDT- contaminated, .soil, Bernier. R. L. Gray, N.C.C. and Moser, L.E., US 5660612 dt. August 26th 1997) which deals with decontamination of soil containg.DDT.type contaminants by composting..
Reference may be made to incorporate Indian patent (pending) No. 256/ del / 2000 wherein the involvement of microorganism, Pseudomonas aeruginosa in the elimination of DDT under aerobic conditions in shake flasks and in soil has been demonstrated.
The drawbacks in all the above works are that the concentration of DDT degraded by these bacterial and fungal isolates is very low and also the degradation is incomplete. Hence it is imperative to develop a process to degrade higher concentrations of DDT completely within a reasonably short period.
The main object of the present invention is to provide a process for the preparation of a biocatalyst formulation useful for the degradation of dichlorodiphenyltrichloroethane (DDT) residues, which obviates the drawbacks as detailed above.
Another object of the present invention is to prepare a biocatalyst for the treatment of DDT industry effluents for the elimination of DDT other contaminating residues.
In the drawings accompanying this specification, Fig 1 represents degradation of 5ppm DDT by Serratia marcescens CFR 1020. Growth (OD 600) CI" released residual DDT
Fig 2 represents degradation of 10 ppm of DDT by Serratia marcescens CFR 1020. All symbols are as given in Fig 1.
Fig 3 represents degradation of 15 ppm of DDT by Serratia marcesscens CFR 1020. All symbols are as given in Fig 1.
Fig 4 represents degradation of 20 ppm of DDT by Serratia marcesscens CFR 1020. All symbols are as given in fig. 1.
All these experiments were done by incubating the flasks at 30°C on a rotary shaker at a speed of 150 rpm.
Accordingly the present invention provides a process for the degradation of dichlorodiphenyltrichloroethane (DDT) which comprises preparing a novel improved strain of Serratia marcescens having characteristics as herein described by enrichment of DDT contaminated soil and culturing in medium containing 5ppm DDT as the sole source of carbon , culturing the obtained strain in a 0.5% basal glucose- minimal medium containing 15 ppm DDT and a co-substrate such as herein described , at a temperature of 20-30°C for 2 h followed by acclimatization for 48 h , getting degradation of DDT from mineral medium.
In an embodiment of the present invention the degradation
may be effective in presence of co-substrate selected from the group consisting of glycerol, peptone, yeast extract and tryptone soya broth at 10 ppm level of In an another embodiment of the present invention the degradation may be effected in the inoculum previously grown on various substrates selected from the group consisting of acetate, succinate, citrate, glucose, sucrose, glycerol, peptone, yeast extract and tryptone soya broth at 0.5 % level.
In yet an another embodiment of the present invention the DDT used may be employed up to 15ppm.
Serratia marcescens CFR1020 was cultivated on 0.5% glucose- mineral medium containing 10ppm DDT for 24 h. The cells were then subjected to acclimation by growing them in 10ppm DDT as the sole source of carbon in the mineral medium. This acclimated culture could degrade 15µg/ ml DDT in 120 h in shake flasks. In the presence of co-substrates, 10ppm DDT disappeared by 72h compared to flasks without co-substrates. This microbial culture can be effectively deployed for the treatment of DDT industry effluents, water bodies and other materials contaminated with DDT- residues.
A novel process was deployed for elimination of DDT-residues from industrial effluents and other contaminated materials using a biocatalyst preparation of a bacterial culture Serratia marcescens CFR 1020. The microbial culture was developed by enrichment of DDT contaminated soil in shake flasks containing DDT as the sole source of carbon and energy. The microbial consortium that got established in the medium containing 5ppm of DDT was resolved into individual bacterial strains. Individual strains were tested for their ability to degrade DDT. Of the four bacterial strains, was obtained Serratia marcescens CFR 1020, that could degrade effectively 15µg/ ml,
The culture characteristics of Serratia marcescens CFR 1020 are as follows:
Orange to red colonies with ridges with curled margins on nutrient agar. During growth on pyocyanin agar the strain produces red coloured pigment, on fluorescene agar it produces red to orange pigment. The surface of the colony on nutrient agar is rough and umbonate with light buff coloured transluscent ring around. It grows from 8° C to 41°C very well. Older colonies become reddish brown and sticky in nature. Cells are cocco-bacilloid with a hollow ridge in the centre, measuring 0.57- 0.81(m in length and 0.32- 0.49(m in width. It is gram -ve, motile, aerobic, oxidase -ve, indole -ve, does not produce H2S. Subjecting the isolate to increasing concentrations of DDT as sole carbon source has developed the mutant of this isolate by conventional enrichment technique.
Dichlorodiphenyltrichloroethane (DDT) used was 98% pure and was obtained from Sigma chemical company, Mo, USA. Other chemicals used in the media and the reagents were of analytical grade and were odtained, from standard Indian companies.
The basal medium used for enrichment and for growth of the cultures contained (per liter of distilled water) 0.675g KH2 PO4 ; 5.455g Na2HPO4 ; 0.25g NH4N03 ; 0.2g MgS04 . 7H20; 0.1g Ca (NO3) 2 and 1.0 ml of trace mineral solution containing (mg/ ml)- FeSO4.7H2 O, 1.0; MnSO4 . H2 O, 1.0; CuCI2. 2 H2O, 0.25; Na2MoO4 . 2 H2O, 0.25; H3BO3, 0.1 and cone. H2SO4, 5.0 ml. The pH of the medium was 7.5. Required amount of DDT and / or co-substrate was added to the medium as described later.
Nutrient agar plates were used for resolving the different bacterial strains from mixtures of acclimated isolates, as well as for the study of their taxanomy.
Nutrient agar medium contained 3g beef extract, 5g peptone, 5g NaCI and 20g agar per liter of distilled water (pH 7).
Cultivation in shake flasks was carried out in 50ml mineral medium containing required amount of growth substrate (DDT) taken in 250ml Erlenmeyer flasks and incubating at 30°C on a rotary shaker (150rpm). For maintaining the strain, mineral agar plates/ slopes containing DDT 25µg/ml) were used. The culture was incubated for 4-5 days at 30°C and then preserved at 4°C. The culture was maintained as liquid culture also, after growing it in mineral salts medium with 10µg/ml DDT for 48h and storing at 4°C.
The growth of the culture was determined spectrophotometrically by measuring the turbidity at 600nm. Quantitative estimation of chloride was done by HNO3-AgNO3 method. To 1ml of the aliquot of culture supernatant, 1ml of 0.15N HNO2 was added followed by the addition of 0.1 N AgNO3. The contents of the tubes were mixed well and were allowed to stand at room temperature for 20 min. Then turbidity was measured at 600nm. The amount of chloride was computed from a standard curve prepared for NaCI.
Quantitative determination of the substrate residues (DDT) was determined either by thin layer chromatography or gas chromatography with 63Ni electron capture detector. The culture broth was extracted thrice with equal volumes of diethyl ether. The solvent layers were pooled, evaporated and redissolved in convenient volume of acetone. Known volume of this was loaded on to a silica gel G- coated plate (0.3mm) and developed in cyclohexane. The plates were air-dried and DDT was detected by spraying the plate with 2% solution of o-tolidine in acetone and exposing to sunlight. The spots were delineated by marking with a needle and the area was measured. The
concentration was computed from a standard plot of log concentrations versus square root of the area, prepared for reference standard of DDT. 63Ni electron capture detector was used. The conditions used were as follows: column, 2mm ID and 200mm length packed with 1.5% OV-17 plus 1.95QF1 on 80-100 mesh chromosorb W; carrier gas, nitrogen; flow at 50ml /min.; column temperature, 230 °C, injector temperature, 230 °C and detector temperature, 320 °C. The retention of DDT was 7.4 min under these conditions.
This improved strain of Serratia marcescens CFR 1020 showed ability to degrade p,p'-DDT which took 96h to completely mineralise 10µg/ ml DDT. However in the presence of co-substrates at 0.5% level, 10µg/ ml DDT was degraded by 72h.
The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention.
EXAMPLE 1
The mineral medium contained 0.675g KHa PO4 ; 5.455g NaaHPO4 ; 0.25g NH4NO3 ; 0.2g MgS04 . 7H2O; 0.1g Ca (NO3) 2 and 1.0 ml of trace mineral solution per liter of distilled water. The substrate, DDT was added at 5µg/ ml level. The required amount of substrate was dissolved in 50µl of acetone and was added at the bottom of a dry, sterile 250ml conical flask, inside a laminar hood and kept open till the acetone evaporated completely. Then 25ml of mineral medium was added and inoculated with cells of Serratia marcescens CFR 1020. The inoculum added was cells equivalent to OD600 of 0.2 per ml after inoculation (0 hr). The flasks were incubated on a rotary shaker
at 150rpm at 30° C. Samples were drawn at regular intervals and analysed for growth (OD600), inorganic chloride (CI) and residual substrate. The results are presented in Fig.1. (sheet No. 1 of the drawing accompanying the specification). Complete disappearance of the substrate and the release of 100% CI" was observed at 96 h of incubation. Biomass also increased during this period. These observations clearly indicate that DDT (5ppm) was completely mineralised.
The microbial mass was separated by centrifugation at 8000rpm for 10 min. and lyophilised by freeze-drying.
EXAMPLE 2
The mineral medium contained 0.675g KH2 PO4i 5.455g NaaHPO4 0.25g NH4NO3; 0.2g MgSO4. 7H2O; 0.1g Ca (N03) 2 and 1.0 ml of trace mineral solution per liter of distilled water. The substrate, DDT was added at 10ng/ml level. The required amount of substrate was dissolved in 50µl of acetone and was added at the bottom of a dry, sterile 250ml conical flask, inside a laminar hood and kept open till the acetone evaporated completely. Then 25ml of mineral medium was added and inoculated with cells of Serratia marcescens CFR 1020. The inoculum added was cells equivalent to OD600 of 0.2 per ml after inoculation (0 hr). The flasks were incubated on a rotary shaker at 150rpm at 30°C. Samples were drawn at regular intervals and analysed for growth (OD600), inorganic chloride (CI) and residual substrate. The results are presented in Fig.2. (sheet No.2 of the drawing accompanying the specification). Complete disappearance of the substrate and the release of 100% Cl was observed at 96h of incubation.
The microbial mass was separated by centrifugation at 8000 pm for 10 min. and lyophilised by freeze-drying.
EXAMPLE 3
The mineral medium contained 0.675g KH2 PO4; 5.455g Na2HPO4; 0.25g NH4NO3; 0.2g MgSO4. 7H2O; 0.1g Ca (NO3) 2 and 1.0 ml of trace mineral solution per liter of distilled water. The substrate, DDT was added at 15(g/ ml level. The required amount of substrate was dissolved in 50(l of acetone and was added at the bottom of a dry, sterile 250ml conical flask, inside a laminar hood and kept open till the acetone evaporated completely. Then 25ml of mineral medium was added and inoculated with cells of Serratia marcescens CFR 1020. The inoculum added was cells equivalent to OD6oo of 0.2 per ml after inoculation (0 hr). The flasks were incubated on a rotary shaker at 150rpm at 30°C. Samples were drawn at regular intervals and analysed for growth (OD600), inorganic chloride (Cl~) and residual substrate. The results are presented in Fig.3. (sheet No.3 of the drawing accompanying the specification). Complete disappearance of the substrate and the release of 100% CI" was observed at 120h of incubation.
The microbial mass was separated by centrifugation at 8000rpm for 10 min. and lyophilised by freeze-drying.
EXAMPLE 4
The mineral medium contained 0.675g KH2 PO4; 5.455g Na2HPO4; 0.25g NH4NO3; 0.2g MgSO4. 7H2O; 0.1g Ca (NO3) 2 and 1.0 ml of trace mineral solution per liter of distilled water. The substrate, DDT was added at 20µg/ ml level. The required amount of substrate was dissolved in 50(l of acetone and was added at the bottom of a dry, sterile 250ml conical flask, inside a laminar
hood and kept open till the acetone evaporated completely. Then 25ml of mineral medium was added and inoculated with cells of Serratia marcescens CFR 1020. The inoculum added was cells equivalent to OD600 of 0.2 per ml after inoculation (0 hr). The flasks were incubated on a rotary shaker at 150rpm at 30°C. Samples were drawn at regular intervals and analysed for growth (OD600), inorganic chloride (Cl~) and residual substrate. The results are presented in Fig.4. (sheet No.4 of the drawing accompanying the specification). Complete disappearance of the substrate and the release of 90% CI" was observed at 144h of incubation.
The microbial mass was separated by centrifugation at 8000rpm for 10 min. and lyophilised byfreeze-drying.
EXAMPLE 5
Co-metabolism of 10(g/ ml DDT by Serratia marcescens CFR 1020 was studied in shake flasks. 25ml mineral medium was taken in 250 ml Erlenmeyer flasks containing 0.5 % of different substrates as listed in Table 1.
The mineral medium contained 0.675g KH2 PO4; 5.455g Na2HPO4; 0.25g NH4NO3; 0.2g MgSO4. 7H2O; 0.1g Ca (NO3) 2 and 1.0 ml of trace mineral solution per liter of distilled water. The substrate, DDT was added at 10µg/ ml level. The required amount of substrate was dissolved in 50µl of acetone and was added at the bottom of a dry, sterile conical flask, inside a laminar hood and kept open till the acetone evaporated completely. Then 25ml of mineral medium containing 0.5 % of required co-substrate was added and inoculated with cells of Serratia marcescens CFR 1020. The inoculum added was cells equivalent to OD6oo of 0.2 per ml after inoculation (0 hr). The flasks were incubated on a rotary shaker at 150rpm at 30° C for 72 h. Samples were
analysed for growth (OD600), inorganic chloride (Cl) and residual substrate. The inoculum was the colonies grown on nutrient agar plates, suspended in minimal medium, washed well and resuspended in minimal medium. The results are presented in Table 1.
Complete disappearance of the substrate and the release of 100% Cl" was observed.
Table: 1

(Table Removed)
EXAMPLE 6
Effect of growth of Serratia marcescens CFR 1020 on different substrates and degradation of 10(g/ ml DDT was studied in shake flasks. 20 ml of mineral medium containing 0.5 % of different substrates (taken separately) was taken in 100 ml Erlenmeyer flasks.
The mineral medium contained 0.675g KH2PO4; 5.455g Na2HPO4; 0.25g NH4NO3; 0.2g MgSO4. 7H2O; 0.1g Ca (NO3) 2 and 1.0 ml of trace mineral
solution per liter of distilled water. Single colonies grown on nutrient agar were inoculated to these flasks and the flasks were incubated on a rotary shaker at 150rpm, 30°C for 24 h. The cells were harvested, washed well and used as inoculum. The substrate, DDT was added at lOµg/ ml level. The required amount of substrate was dissolved in 50µl of acetone and was added at the bottom of a dry, sterile 100ml conical flask, inside a laminar hood and kept open till the acetone evaporated completely. Then 20ml of mineral medium was added and inoculated with cells of Serratia marcescens CFR 1020 grown on different substrates. The inoculum added was cells equivalent to OD600 of 0.2 per ml after inoculation (0 hr). The flasks were incubated on a rotary shaker at 150rpm at 30°C for 72 h. Samples were analysed for growth (OD600), inorganic chloride (Cl~) and residual substrate. The results are presented in Table 2.
Table: 2

(Table Removed)
The main advantages of the present invention are:
1. The preparation can be in powder form.
2. The preparation can be applied to effluent treatment plants for
elimination of DDT.
3. The preparation can eliminate DDT efficiently in presence of co-
substrates.

We Claim:
1. A process for the degradation of dichlorodiphenyltrichloroethane (DDT) using
an improved strain of Serratia marcescens which comprises preparing a novel
improved strain of Serratia marcescens having characteristics as herein
described by enrichment of DDT contaminated soil and culturing in medium
containing 5ppm DDT as the sole source of carbon , culturing the obtained
strain in a 0.5% basal glucose- minimal medium containing 15 ppm DDT and a
co-substrate such as herein described , at a temperature of 20-30°C for 2 h
followed by acclimatization for 48 h , getting degradation of DDT from mineral
medium.
2. A process as claimed in claim 1 wherein the co-substrate used is selected from
the group consisting of glycerol, peptone, yeast extract and tryptone soya broth
at 10ppm level of DDT.
3. A process as claimed in claims 1-2, wherein the degradation is effected in the
inoculum previously grown on various substrates selected from the group
consisting of acetate, succinate, citrate, glucose, sucrose, glycerol, peptone,
yeast extract and tryptone soya broth at 0.5 % level.
4. A process for the degradation of dichlorodiphenyltrichloroethane (DDT) using an
improved strain of Serratia marcescens substantially as herein described with
reference to the examples and drawings accompanying this specification.

Documents:

226-del-2001-abstract.pdf

226-del-2001-claims.pdf

226-del-2001-correspondence-others.pdf

226-del-2001-correspondence-po.pdf

226-del-2001-description (complete).pdf

226-del-2001-drawings.pdf

226-del-2001-form-1.pdf

226-del-2001-form-19.pdf

226-del-2001-form-2.pdf

226-del-2001-form-3.pdf

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Patent Number

209454

Indian Patent Application Number

226/DEL/2001

PG Journal Number

38/2007

Publication Date

21-Sep-2007

Grant Date

30-Aug-2007

Date of Filing

28-Feb-2001

Name of Patentee

COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESARCH`

Applicant Address

RAFI MARG, NEW DELHI - 110 001, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	HARAVEY KSISHNANMANONMANI	CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE, MYSORE, INDIA.
2	RAJKUMAR BIDLAN	CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE, MYSORE, INDIA.
3	ANEBAGILU ABDULLAMOHAMMED KUNHI	CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE, MYSORE, INDIA.

PCT International Classification Number

C02C 5/10

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA