Title of Invention	A PROCESS FOR THE PREPARATION OF CONSORTIUM OF GENETICALLY MODIFIED BACTERIA USEFUL FOR REMEDIATION OF HAZARDOUS CHEMICAL WASTES
Abstract	A process for the preparation of consortium of genetically modified bacteria useful for remediation of hazardous chemical wastes by growing stress tolerant soil microorganisms like Pseudomonas sp. Rhizobium sp., Bacillus sp., Micrococcus sp., Flavobacterium sp., Arthrobacter sp., or Rhodococcus sp. in a conventional Luria broth medium comprising Tryptone 100 g/l, Yeast extract 5 g/l, Sodium chloride 10 g/l by adjusting pH at 7.0 with Sodium Hydroxide at least for a period of 19 hours under stirring in a known manner separating the cells by centrifugation so as to obtain a pellet, resuspending the said pellet in a conventional minimal salt medium (Mg) having been supplemented with clay or clay minerals so as to have moisture content of 60 percent and optionally lyophilizing or freeze drying to get the consortium.

Title of Invention

A PROCESS FOR THE PREPARATION OF CONSORTIUM OF GENETICALLY MODIFIED BACTERIA USEFUL FOR REMEDIATION OF HAZARDOUS CHEMICAL WASTES

Abstract

A process for the preparation of consortium of genetically modified bacteria useful for remediation of hazardous chemical wastes by growing stress tolerant soil microorganisms like Pseudomonas sp. Rhizobium sp., Bacillus sp., Micrococcus sp., Flavobacterium sp., Arthrobacter sp., or Rhodococcus sp. in a conventional Luria broth medium comprising Tryptone 100 g/l, Yeast extract 5 g/l, Sodium chloride 10 g/l by adjusting pH at 7.0 with Sodium Hydroxide at least for a period of 19 hours under stirring in a known manner separating the cells by centrifugation so as to obtain a pellet, resuspending the said pellet in a conventional minimal salt medium (Mg) having been supplemented with clay or clay minerals so as to have moisture content of 60 percent and optionally lyophilizing or freeze drying to get the consortium.

Full Text	The present invention relates to a process for the preparation of consortium of genetically modified bacteria useful for remediation of hazardous chemical wastes. The invention uses a procedure for binding natural or genetically engineered bacteria to clay or clay minerals so as to enhance the activity of the organism and the engineered function, if any, for application in bioremediation of hazardous industrial wastes, particularly halogenated and polynuclear aromatic hydrocarbons. The composition can be used for seeding containing bioreactors for transformation of polyhalogenated compounds like chlorinated hydrocarbons, insecticides like lindane. • In "pump and treat technologies" for removal of hazardous halogenated compounds from water. Pump and treat methods conventionally involve pumping contaminated water to the surface for treatment and may include a system for injection into groundwater as part of the remediation strategy. Biotechnological processes continue to play a central role in treatment of municipal, industrial and agricultural wastes. Commercial biotechnology has inevitably advanced into areas that depend on the introduction of both natural and genetically engineered microorganisms (GEMs) into the environment (Anon, (1982). The use of genetically engineered microorganisms in the environment. In genetic Technology : A new Frontier, Office of Technology Assessment, West View Press, Boulder, CO, USA pp.117-127). GEMs are being constructed for a variety of environmental applications including their use in agriculture as pesticides or for agronomic crop production, pollution control of toxic waste in land-fill sites, waste water treatment facilities or after accidental spillage, and in mining and the petrochemical industry for enhancing oil recovery or removal of sulphur. Presently, the release of GEMs is restricted (Freitas, J.R. D, Germida, J.J. (1991) Pseudomonas cepacia & Pseudomonas putida as winter wheat Inoculants for biocontrol of Rhizoctania solani. Can. J. Microbiol. 37 : 780-784 ; Howard, J. and Fox, S. (1994) Review of current research projects and innovations in bioremediation. Genetic Engnn. News 14 (17) : 8-9). Field trials have been carried out involving genetically engineered strain of Pseudomonas syringae, the first to be approved for release into the environment (USA) Pseudomonas fluorescens carrying a lac ZY marker gene (USA), Rhizobium meliloti carrying a Tn5 marker gene (UK), and a genetically engineered Baculovirus (UK) (De Leij, F.A.A.M., Sutton, E.J., Whipps, J.M., Fenton, J.S. and Lynch, J.M. (1995) Field Release of a genetically modified Pseudomonas fluorescens on wheat : Establishment, Survival and Dissemination. Bio/Technology 13: 1488-1492; Prosser, J.I, Killham, K., Glover, LA. and Rattray, E.A.S. (1996) Luminescence- based systems for detection of bacteria in the environment. Crit. Revs. Biotechnol. 16 : 157-183; Scott Angle, J.; Levin, M. A., Gagliardi, J.V.; Mclntosh, M.S. (1995). Validation of microcosms for examining the survival of Pseudomonas aureofaciens (lacZY) in soil. Appl. Environm. Microbiol. 61 : 2835-2839). These field trials have revealed the behaviour of Pseudomonas fluorescens lac ZY strains and Pseudomonas syringae can be closely predicted by the results of experiments done in contained systems before release into environment. This engenders certain level of confidence in the ability to define in laboratory cultures and in microcosms many of ecologically important characters that will be required in a GEM that is to be deliberately released. It cannot be disputed that in the last 20 years, recombinant DMA technology has contributed additional genetic diversity to laboratory microbial populations. However, it is on record that there have been no cases reported in which an organism has become a disease problem as a result of receiving recombinant DNA. On the other hand, the resistance to the use of genetically manipulated bacteria is not universal. A few US based companies like Polybac Corporation, Sybron Corporation and others have been selling for almost a decade bacteria that they claim outperform indigenous strains found in aerated lagoons used in biological treatment of industrial wastes containing dioxins, orthochlorophenol, grease and "nonbiodegradable" detergents. The introduction of industrial chemicals into sewage treatment as well as other waste treatment facilities has caused problems because many chemicals are not degraded in traditional waste treatment facilities and escape as pollutants into the environment. Hence, bioremediation envisages the most obvious use of living OEMs and is proposed as a lucrative market. Since many contaminated sites or hazardous industrial wastes contain more than one pollutant, it is often difficult for natural microorganisms to degrade efficiently and simultaneously, a mixture of pollutants. Indigenous microorganisms when exposed to a mixture of pollutants, may produce toxic intermediates that do not allow an overall reduction of toxicity of biotreated sample to any great extent. Further, mixed formulations will allow only preferential proliferation of bacteria that feed on the most abundant compound leading to survival of only one or few strains with ultimately no bacteria available to degrade certain chemicals, and therefore have limited usage. The need for designing genetically competent organisms is due to the non-availability of efficient enzyme systems to break down certain compounds like poiychlorinated hydrocarbons which have half-life of order of years (Shanker, R. and Atkins, W.M. (1996) Luciferase Dependent, Cytochrome P450 catalysed dehalogenation in genetically engineered Pseudomonas. Biotechnology Progress (ACS & AlChE) 12 (4): 474-479 ; Timmis, KM and Pieper, D.H. (1999) Bacteria designed for bioremediation. Trends in Biotechnology 17: 201-204 ; Trombly, J. (1995) Engineering enzymes for better bioremediation. Environm. Sci. Technol. 29:560-564A. ; Timmis, K.N., Steffan, R.J. and Unterman, R. (1994) Designing microorganisms for treatment of toxic wastes. Ann. Rev. Microbiol. 48 : 525-557; Warner, P.J. (1991) Prospects for use of genetically manipulated microorganisms in agriculture. Society for General Microbiology News (May issue) : 27-29.) Hence, genetically improved single cultures are considered more efficient than a mixer of cultures in degrading single or mixer of toxic chemicals. Therefore, genetically improved bacteria in combination with metabolically competent natural bacteria can be used to design stable composition with the desired traits. The prerequisite to use of microorganisms for detoxification of hazardous wastes is the preservation regime and shelf life of material. Hence, the preservation of both natural and genetically engineered microorganisms is becoming increasingly important due to their potential application in hazardous waste treatment in contained systems viz. soil slurry reactors as well as pump & treat systems. Lyophilization is the method most commonly used for the preservation of natural microorganisms. Although, survival after lyohilization and long term storage for 6-10 years have been reported for many genera of natural bacteria, yeasts and fungi, the methodology has not been explored for environmentally important organisms in terms of their metabolic competence after storage. The success of bacteria introduced into soil depends on their ability to survive in sufficiently high numbers and for longer periods of time. It has been shown that the addition of bentonite clay to fresh orfreeze-dried rhizobial inoculants significantly enhanced their survival in soil attributed to the creation of protective microhabitats due to the bentonite amendment. Similar results were obtained with a Tn5 mutant Pseudomonas fluorescens strain (Heijnin, C.E., Hok-A-Hin C.H. and Van Veen J.A. (1992) Improvements to the use of bentonite clay as a protective agent, increasing survival levels of bacteria introduced into soil. Soil Biol. Biochem. 24(6); 533-538). Thus, lyophilization of bacterial cultures in combination with the use of clay minerals like bentonite confers an advantage for their application in bioremediation. The main object of the present invention is to provide a process for the preparation of synergistic composition useful for remediation of hazardous chemical wastes. In the present invention, a genetically engineered soil bacterium Pseudomonas having Ace No. MTCC 102 has been used as a model organism to evaluate the technique developed for preparation of a synergistic composition. This can be applicable to other stress tolerant soil organisms such as herein defined. Accordingly, the present invention provides a process for the preparation of consortium of genetically modified bacteria useful for remediation of hazardous chemical wastes particularly halogenated and polynuclear aromatic hydrocarbon which comprises growing stress tolerant soil microorganisms like Pseudomonas sp. Rhizobium sp., Bacillus sp., Micrococcus sp., Flavobacterium sp., Arthrobacter sp., or Rhodococcus sp. in a conventional Luria broth medium comprising Tryptone 100 g/l, Yeast extract 5 g/l, Sodium chloride 10 g/l by adjusting pH at 7.0 with Sodium Hydroxide at least for a period of 19 hours under stirring in a known manner separating the cells by centrifugation so as to obtain a pellet, resuspending the said pellet in a conventional minimal salt medium (Mg) such as herein described, having been supplemented with clay or clay minerals so as to have moisture content of 60 percent and optionally lyophilizing or freeze drying to get the consortium. In an embodiment of the present invention, stress tolerant soil organisms used may be such as naturally occurring organism or genetically engineered organism such as Pseudomonas putida co-expressing chromosomally encoded Vibrio harveyi luciferase and plasmid encoded cytochrome P450 cam C monooxygenase, used as model organism having Ace No.MTCC 102. The genetically engineered bacterium Pseudomonas putida co-expressing cytochrome P450 cam and luciferase has been bound to clay or clay minerals to design a stable composition to impart longer shelf life and therefore viability as a commercial package. The preparation of composition involved the following steps : Inoculation of the bacteria in Luria broth pH 7.2 for growth at 30°C for 19-20 hours on an orbital shaker at 200 rpm. Harvesting of cells by centrifugation at 8000 x g for 10 minutes at 4°C. Pellet of cells was resuspended in M9 medium containing 0.1% magnesium sulfate, 0.003% sodium benzoate and 0.2% sodium citrate to which the clay-based binding matrix was added to provide moisture content of 60 percent and stored at 4°C. Alternatively, cells resuspended in M9 medium containing 0.1% magnesium sulfate were added to clay-based binding matrix and freeze dried. Lyophilization was performed at -25°C to -35°C at 0.60 to 1.032 mbar for 10-15 hours. Freeze-dried samples were stored desiccated at room temperature. The following examples are given by way of illustration of activity of the composition of the present inventon and therefore should not be construed to limit its scope: Example-1 The short term survival of clay-bound cells of genetically engineered P. putida co-expressing luciferase and cytochrome P450 cam on storage at 4°C was evaluated by measuring luminescence in 0.1 g clay paste suspended in Luria broth. Luminescence was measured for 30 seconds or 1 minute in a Tri- We claim: 1 . A process for the preparation of consortium of genetically modified bacteria useful for remediation of hazardous chemical wastes particularly halogenated and polynuclear aromatic hydrocarbon which comprises growing stress tolerant soil microorganisms like Pseudomonas sp. Rhizobium sp., Bacillus sp., Micrococcus sp., Flavobacterium sp., Arthrobacter sp., or Rhodococcus sp. in a conventional Luria broth medium comprising Tryptone 100 g/l, Yeast extract 5 g/l, Sodium chloride 10 g/l by adjusting pH at 7.0 with Sodium Hydroxide at least for a period of 19 hours under stirring in a known manner separating the cells by centrifugation so as to obtain a pellet, re suspending the said pellet in a conventional minimal salt medium (Mg) such as herein described, having been supplemented with clay or clay minerals so as to have moisture content of 60 percent and lyophilizing or freeze drying to get the consortium. 2. A process as claimed in claim 1 wherein stress tolerant soil organisms such as genetically engineered organisms used is Pseudomonas putida co- expressing chromosomally encoded Vibrio harveyi luciferase and plasmid encoded cytochrome P450 cam C monooxygenase. 3. A process for the preparation of consortium of genetically modified bacteria useful for remediation of hazardous chemical wastes substantially as herein described with reference to the examples and drawing accompanying this specification.

Full Text

The present invention relates to a process for the preparation of consortium of genetically modified bacteria useful for remediation of hazardous chemical wastes. The invention uses a procedure for binding natural or genetically engineered bacteria to clay or clay minerals so as to enhance the activity of the organism and the engineered function, if any, for application in bioremediation of hazardous industrial wastes, particularly halogenated and polynuclear aromatic hydrocarbons.
The composition can be used for seeding containing bioreactors for transformation of polyhalogenated compounds like chlorinated hydrocarbons, insecticides like lindane.
• In "pump and treat technologies" for removal of hazardous halogenated compounds from water. Pump and treat methods conventionally involve pumping contaminated water to the surface for treatment and may include a system for injection into groundwater as part of the remediation strategy.
Biotechnological processes continue to play a central role in treatment of
municipal, industrial and agricultural wastes. Commercial biotechnology has
inevitably advanced into areas that depend on the introduction of both natural and
genetically engineered microorganisms (GEMs) into the environment (Anon, (1982).
The use of genetically engineered microorganisms in the environment. In genetic
Technology : A new Frontier, Office of Technology Assessment, West
View Press, Boulder, CO, USA pp.117-127). GEMs are
being constructed for a variety of environmental applications including their use in agriculture as pesticides or for agronomic crop production, pollution control of toxic waste in land-fill sites, waste water treatment facilities or after accidental spillage, and in mining and the petrochemical industry for enhancing oil recovery or removal of sulphur. Presently, the release of GEMs is restricted (Freitas, J.R. D, Germida, J.J. (1991) Pseudomonas cepacia & Pseudomonas putida as winter wheat Inoculants for biocontrol of Rhizoctania solani. Can. J. Microbiol. 37 : 780-784 ; Howard, J. and Fox, S. (1994) Review of current research projects and innovations in bioremediation. Genetic Engnn. News 14 (17) : 8-9). Field trials have been carried out involving genetically engineered strain of Pseudomonas syringae, the first to be approved for release into the environment (USA) Pseudomonas fluorescens carrying a lac ZY marker gene (USA), Rhizobium meliloti carrying a Tn5 marker gene (UK), and a genetically engineered Baculovirus (UK) (De Leij, F.A.A.M., Sutton, E.J., Whipps, J.M., Fenton, J.S. and Lynch, J.M. (1995) Field Release of a genetically modified Pseudomonas fluorescens on wheat : Establishment, Survival and Dissemination. Bio/Technology 13: 1488-1492; Prosser, J.I, Killham, K., Glover, LA. and Rattray, E.A.S. (1996) Luminescence- based systems for detection of bacteria in the environment. Crit. Revs. Biotechnol. 16 : 157-183; Scott Angle, J.; Levin, M. A., Gagliardi, J.V.; Mclntosh, M.S. (1995). Validation of microcosms for examining the survival of Pseudomonas aureofaciens (lacZY) in soil. Appl. Environm. Microbiol. 61 : 2835-2839). These field trials have revealed the behaviour of Pseudomonas fluorescens lac ZY strains and Pseudomonas syringae can be closely predicted by the results of
experiments done in contained systems before release into environment. This engenders certain level of confidence in the ability to define in laboratory cultures and in microcosms many of ecologically important characters that will be required in a GEM that is to be deliberately released. It cannot be disputed that in the last 20 years, recombinant DMA technology has contributed additional genetic diversity to laboratory microbial populations. However, it is on record that there have been no cases reported in which an organism has become a disease problem as a result of receiving recombinant DNA.
On the other hand, the resistance to the use of genetically manipulated bacteria is not universal. A few US based companies like Polybac Corporation, Sybron Corporation and others have been selling for almost a decade bacteria that they claim outperform indigenous strains found in aerated lagoons used in biological treatment of industrial wastes containing dioxins, orthochlorophenol, grease and "nonbiodegradable" detergents.
The introduction of industrial chemicals into sewage treatment as well as other waste treatment facilities has caused problems because many chemicals are not degraded in traditional waste treatment facilities and escape as pollutants into the environment. Hence, bioremediation envisages the most obvious use of living OEMs and is proposed as a lucrative market. Since many contaminated sites or hazardous industrial wastes contain more than one pollutant, it is often difficult for natural microorganisms to degrade efficiently and simultaneously, a mixture of pollutants. Indigenous microorganisms when exposed to a mixture of pollutants, may produce toxic intermediates that do not

allow an overall reduction of toxicity of biotreated sample to any great extent. Further, mixed formulations will allow only preferential proliferation of bacteria that feed on the most abundant compound leading to survival of only one or few strains with ultimately no bacteria available to degrade certain chemicals, and therefore have limited usage. The need for designing genetically competent organisms is due to the non-availability of efficient enzyme systems to break down certain compounds like poiychlorinated hydrocarbons which have half-life of order of years (Shanker, R. and Atkins, W.M. (1996) Luciferase Dependent, Cytochrome P450 catalysed dehalogenation in genetically engineered Pseudomonas. Biotechnology Progress (ACS & AlChE) 12 (4): 474-479 ; Timmis, KM and Pieper, D.H. (1999) Bacteria designed for bioremediation. Trends in Biotechnology 17: 201-204 ; Trombly, J. (1995) Engineering enzymes for better bioremediation. Environm. Sci. Technol. 29:560-564A. ; Timmis, K.N., Steffan, R.J. and Unterman, R. (1994) Designing microorganisms for treatment of toxic wastes. Ann. Rev. Microbiol. 48 : 525-557; Warner, P.J. (1991) Prospects for use of genetically manipulated microorganisms in agriculture. Society for General Microbiology News (May issue) : 27-29.) Hence, genetically improved single cultures are considered more efficient than a mixer of cultures in degrading single or mixer of toxic chemicals. Therefore, genetically improved bacteria in combination with metabolically competent natural bacteria can be used to design stable composition with the desired traits.
The prerequisite to use of microorganisms for detoxification of hazardous wastes is the preservation regime and shelf life of material. Hence,

the preservation of both natural and genetically engineered microorganisms is becoming increasingly important due to their potential application in hazardous waste treatment in contained systems viz. soil slurry reactors as well as pump & treat systems. Lyophilization is the method most commonly used for the preservation of natural microorganisms. Although, survival after lyohilization and long term storage for 6-10 years have been reported for many genera of natural bacteria, yeasts and fungi, the methodology has not been explored for environmentally important organisms in terms of their metabolic competence after storage. The success of bacteria introduced into soil depends on their ability to survive in sufficiently high numbers and for longer periods of time. It has been shown that the addition of bentonite clay to fresh orfreeze-dried rhizobial inoculants significantly enhanced their survival in soil attributed to the creation of protective microhabitats due to the bentonite amendment. Similar results were obtained with a Tn5 mutant Pseudomonas fluorescens strain (Heijnin, C.E., Hok-A-Hin C.H. and Van Veen J.A. (1992) Improvements to the use of bentonite clay as a protective agent, increasing survival levels of bacteria introduced into soil. Soil Biol. Biochem. 24(6); 533-538). Thus, lyophilization of bacterial cultures in combination with the use of clay minerals like bentonite confers an advantage for their application in bioremediation.
The main object of the present invention is to provide a process for the
preparation of synergistic composition useful for remediation of hazardous chemical
wastes. In the present invention, a genetically engineered soil
bacterium Pseudomonas having Ace No. MTCC 102 has been
used as a model organism to evaluate the
technique developed for preparation of a synergistic composition. This can be applicable to other stress tolerant soil organisms such as herein defined.
Accordingly, the present invention provides a process for the preparation of consortium of genetically modified bacteria useful for remediation of hazardous chemical wastes particularly halogenated and polynuclear aromatic hydrocarbon which comprises growing stress tolerant soil microorganisms like Pseudomonas sp. Rhizobium sp., Bacillus sp., Micrococcus sp., Flavobacterium sp., Arthrobacter sp., or Rhodococcus sp. in a conventional Luria broth medium comprising Tryptone 100 g/l, Yeast extract 5 g/l, Sodium chloride 10 g/l by adjusting pH at 7.0 with Sodium Hydroxide at least for a period of 19 hours under stirring in a known manner separating the cells by centrifugation so as to obtain a pellet, resuspending the said pellet in a conventional minimal salt medium (Mg) such as herein described, having been supplemented with clay or clay minerals so as to have moisture content of 60 percent and optionally lyophilizing or freeze drying to get the consortium.
In an embodiment of the present invention, stress tolerant soil organisms used may be such as naturally occurring organism or genetically engineered organism such as Pseudomonas putida co-expressing chromosomally encoded Vibrio harveyi luciferase and plasmid encoded cytochrome P450 cam C monooxygenase, used as model organism having Ace No.MTCC 102.
The genetically engineered bacterium Pseudomonas putida co-expressing cytochrome P450 cam and luciferase has been bound to clay or clay minerals to design a stable composition to impart longer shelf life and
therefore viability as a commercial package. The preparation of composition
involved the following steps :
Inoculation of the bacteria in Luria broth pH 7.2 for growth at 30°C for 19-20 hours on an orbital shaker at 200 rpm.
Harvesting of cells by centrifugation at 8000 x g for 10 minutes at 4°C. Pellet of cells was resuspended in M9 medium containing 0.1% magnesium sulfate, 0.003% sodium benzoate and 0.2% sodium citrate to which the clay-based binding matrix was added to provide moisture content of 60 percent and stored at 4°C.
Alternatively, cells resuspended in M9 medium containing 0.1% magnesium sulfate were added to clay-based binding matrix and freeze dried. Lyophilization was performed at -25°C to -35°C at 0.60 to 1.032 mbar for 10-15 hours. Freeze-dried samples were stored desiccated at room temperature. The following examples are given by way of illustration of activity of the
composition of the present inventon and therefore should not be construed to limit
its scope:
Example-1
The short term survival of clay-bound cells of genetically engineered P.
putida co-expressing luciferase and cytochrome P450 cam on storage at 4°C was
evaluated by measuring luminescence in 0.1 g clay paste suspended in Luria broth.
Luminescence was measured for 30 seconds or 1 minute in a Tri-

We claim:
1 . A process for the preparation of consortium of genetically modified bacteria useful for remediation of hazardous chemical wastes particularly halogenated and polynuclear aromatic hydrocarbon which comprises growing stress tolerant soil microorganisms like Pseudomonas sp. Rhizobium sp., Bacillus sp., Micrococcus sp., Flavobacterium sp., Arthrobacter sp., or Rhodococcus sp. in a conventional Luria broth medium comprising Tryptone 100 g/l, Yeast extract 5 g/l, Sodium chloride 10 g/l by adjusting pH at 7.0 with Sodium Hydroxide at least for a period of 19 hours under stirring in a known manner separating the cells by centrifugation so as to obtain a pellet, re suspending the said pellet in a conventional minimal salt medium (Mg) such as herein described, having been supplemented with clay or clay minerals so as to have moisture content of 60 percent and lyophilizing or freeze drying to get the consortium.
2. A process as claimed in claim 1 wherein stress tolerant soil organisms such
as genetically engineered organisms used is Pseudomonas putida co-
expressing chromosomally encoded Vibrio harveyi luciferase and plasmid
encoded cytochrome P450 cam C monooxygenase.
3. A process for the preparation of consortium of genetically modified bacteria
useful for remediation of hazardous chemical wastes substantially as herein
described with reference to the examples and drawing accompanying this
specification.

Documents:

328-del-2000-abstract.pdf

328-del-2000-claims.pdf

328-del-2000-correspondence-others.pdf

328-del-2000-correspondence-po.pdf

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

328-del-2000-drawings.pdf

328-del-2000-form-1.pdf

328-del-2000-form-19.pdf

328-del-2000-form-2.pdf

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Patent Number

232420

Indian Patent Application Number

328/DEL/2000

PG Journal Number

13/2009

Publication Date

27-Mar-2009

Grant Date

17-Mar-2009

Date of Filing

28-Mar-2000

Name of Patentee

COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG NEW DELHI-110 001, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	RISHI SHANKER	NEERI, NEHRU MARG, NAGPUR-440 020 (M.S)INDIA
2	KAVITA RATTAN	NEERI,NEHRU MARG,NAGPUR-440 020 (M.S)INDIA
3	PURUSHOTTAM KHANNA	NEERI,NEHRU MARG,NAGPUR-440 020 ( M.S)INDIA

PCT International Classification Number

C12N 1/20

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA