Title of Invention	A MICRO EMULSIFIED OLEOPHILIC NUTRIENT COMPOSITION FOR OIL DEGRADING MICROORGANISMS AND A PROCESS FOR THE PREPARATION THEREOF
Abstract	This invention relates to a process for the preparation of micro emulsified oleophilic nutrient composition for oil degrading microorganisms comprising the steps of preparing phosphate solution by dissolving tri lauryl ethoxy phosphate, taken 10-25% by weight, in 2- butoxy ethanol, taken 5-15% by weight, at room temperature and heating the solution at 50-80°C in a water bath; adding lauryl alcohol, taken 5-7% by weight to the phosphate solution prepared in step (i) under continuous stirring, maintaining the temperature of the solution to 70°C for 5 minutes and adding oleic acid taken 25-30% by weight, preheated in a water bath, slowly under stirring; preparing solution of urea and 2-amino-2methyl-l-propanol, by taking urea 14-18% by weight, heating the solution to 50-80°C and adding 2-amino-2-methyl-l propanol, taken 0.5-1.0% by weight to it; adding the solution of urea prepared in step (iii) to the phosphate solution obtained at the end of step (ii) under continuous stirring, heating the entire solution to 70-85°C under intermittent stirring, and then allowing the solution to cool up to 60-40°C; adding 4-6% oleic acid to the solution obtained in step (iv) with stirring and allowing the solution to cool to room temperature to obtain a clear micro emulsion of the nutrient. Also this invention relates to a micro emulsified oleophilic nutrient composition for oil degrading microorganisms comprising of 10 to 25% by wt water, 6 to 9% by wt urea, 0-5 to 1% by wt of 2-amino-2-methyl-l-propanol, 10 to 25% by wt of alkyl ethoxy phosphate, 5 to 15% by wt butoxy ethanol, 5 to 7% by wt lauryl alcohol and 30 to 35% by wt oleic acid.

Title of Invention

A MICRO EMULSIFIED OLEOPHILIC NUTRIENT COMPOSITION FOR OIL DEGRADING MICROORGANISMS AND A PROCESS FOR THE PREPARATION THEREOF

Abstract

This invention relates to a process for the preparation of micro emulsified oleophilic nutrient composition for oil degrading microorganisms comprising the steps of preparing phosphate solution by dissolving tri lauryl ethoxy phosphate, taken 10-25% by weight, in 2- butoxy ethanol, taken 5-15% by weight, at room temperature and heating the solution at 50-80°C in a water bath; adding lauryl alcohol, taken 5-7% by weight to the phosphate solution prepared in step (i) under continuous stirring, maintaining the temperature of the solution to 70°C for 5 minutes and adding oleic acid taken 25-30% by weight, preheated in a water bath, slowly under stirring; preparing solution of urea and 2-amino-2methyl-l-propanol, by taking urea 14-18% by weight, heating the solution to 50-80°C and adding 2-amino-2-methyl-l propanol, taken 0.5-1.0% by weight to it; adding the solution of urea prepared in step (iii) to the phosphate solution obtained at the end of step (ii) under continuous stirring, heating the entire solution to 70-85°C under intermittent stirring, and then allowing the solution to cool up to 60-40°C; adding 4-6% oleic acid to the solution obtained in step (iv) with stirring and allowing the solution to cool to room temperature to obtain a clear micro emulsion of the nutrient. Also this invention relates to a micro emulsified oleophilic nutrient composition for oil degrading microorganisms comprising of 10 to 25% by wt water, 6 to 9% by wt urea, 0-5 to 1% by wt of 2-amino-2-methyl-l-propanol, 10 to 25% by wt of alkyl ethoxy phosphate, 5 to 15% by wt butoxy ethanol, 5 to 7% by wt lauryl alcohol and 30 to 35% by wt oleic acid.

Full Text	FIELD OF INVENTION: This invention relates to a micro emulsified oleophilic nutrient composition and the process of preparation thereof for culturing micro organisms used for combating oil pollution in water. PRIOR ART: Sea water gets polluted by accidental spillages of oil from oil tankers, offshore drilling operations etc. The spillage of oil results in the formation of a continuous film of slick of oil which tends to spread continuously. This oil film is undesirable because it constitutes a barrier to the transfer of air and light from the atmosphere to the sea water, which are indispensable to support the marine life. In costal water, the oily film also damages the beaches. One way of dealing with such pollution is to use oil dispersing compositions containing surface active compounds. When the dispersing compositions are sprayed on the oil slicks, they disintegrate the cohesive oil film into small droplets and disperse the droplets into the water column to a depth of several meters. The droplets, that are dispersed under the sea, are then biodegraded and consumed by microorganisms that are present in sea and are active in oil metabolism. This biological degradation is a slow process and is consequently unable to prevent the settling of un-degraded oil droplets and the formation of oil deposits on the floor of the sea. The biodegradation, as mentioned above, can be enhanced by increasing the growth of the microorganisms at the water oil interface. Controlling pollution caused by hydrocarbon compounds (oil) in water by bioremediation is acquiring increasing significance. The. principle of bioremediation is based on optimum promotion of the growth of the pollution consuming microorganisms population. Major advantage of this process is the inexpensive in situ process without producing any waste for further disposal.In this regard two important features are of paramount significance(i) presence of oil degrading microorganisms at the oil-water interface, and (ii) nutrients required for the growth of the oil degrading microorganisms. In the elimination of hydrocarbon slicks by biodegradation at sea, beaches or even in lakes, the biggest problem is the preliminary multiplication of the oil degrading microorganisms. As such, it becomes necessary to supply growth promoting elements of the microorganisms. Generally, these growth promoting elements are not present in sufficient concentration in the polluted area. These growth promoting elements are primarily inorganic or organic compounds of nitrogen and phosphorous and these are generally not available in sufficient concentration in the polluted area. These growth promoting elements are made available as nutrient concentrate for stimulation as well as growth aids for the accelerated growth of the microorganisms. The nutrient concentrate is made in the form of a micro emulsion, which is thermodynamically stable water in oil type. The internal phase of micro emulsion is aqueous solution of nutrient material and the external phase is a liquid immiscible with water. This helps nutrient substance to remain in the layer treated instead of being entrained by water. Mineral salts such as ammonium salts, phosphates etc. are commonly used as nutrients. However, these mineral salts are soluble in water and these have practically no affinity for the polluting oil where a strong growth of microorganism should occur. Further, nitrogen containing organic nutrients such as condensation products of urea or melamine with aldehydes is soluble in water. These readily go to aqueous phase when sprayed on oil slick. To eliminate this problem, various methods have been tried. The,US Patent No. 1959127 discloses a method, wherein a nutrient in the form of granules of nitrogenous compounds and solid phosphates coated with paraffin is made for the growth of the microorganisms. However, this method suffers from the disadvantage that it requires special equipment which increases the cost of the nutrient. Another disadvantage of this method, known in the art, is that it requires a separate surface-active agent for the treatment of the oil slicks. Still another disadvantage of this method, known in the prior art, is that the paraffin coating may get dissolved if the polluting oil contains aromatic hydrocarbons. US Patent No. 3883397 discloses a method wherein coating of fatty add salt instead of paraffin is made. However, this method suffers from the disadvantage that fatty acid coating material is difficult to degrade in the absence of external nitrogen and phosphorous, thereby making it difficult for the microorganisms to have the nutrient readily available. The disadvantages of the prior art, mentioned above, have been overcome by the oleophilic nutrient of the present invention for the growth of the microorganisms active in oil degradation and which are present in sea water. The nutrient of the present invention is in the form of a micro emulsion of water in oil type, the internal phase of which is an aqueous solution of nutrient material and the external phase is a liquid immiscible with water but miscible with the hydrophobic liquid to be biodegraded. The use of the nutrient helps in achieving rapid and complete biodegradation of the oil at low cost. The nutrient exhibits a low toxicity towards aquatic flora. OBJECTS OF THE INVENTION: The primary object of the invention is to provide a process for the preparation of a micro emulsifide oleiophilic nutrient composition for the growth of oil degrading microorganisms to combat oil pollution in water and in shore. Another object of the invention is to identify micro emulsifiers for the preparation of the micro emulsifide oleiophilic nutrient composition. Another object of the invention is to provide a process for the preparation of micro emulsifide oleiophilic nutrient composition, which does not require costly and highly purified chemicals but uses easily available chemicals.Yet another object of the invention is to provide a process for the preparation of the micro emulsified oleiphilic nutrient composition, which does not require any expensive equipment. Still further object of the invention is to provide a micro emulsified oleiphilic nutrient composition, which helps in the growth of oil degrading bacteria. Even further object of the present invention is to provide micro emulsified oleiphilic nutrient composition, which has a shelf life of more than one year. Still further object of the invention is to provide a micro emulsified oleiphilic nutrient composition, which has thermal stability over a temperature range 20°C to 85°C. STATEMENT OF INVENTION According to this invention is to provide a process for the preparation of micro emulsified oleophilic nutrient composition for oil degrading microorganisms comprising the steps of:- i) preparing phosphate solution by dissolving tri lauryl ethoxy phosphate, taken 10-25% by weight, in 2- butoxy ethanol, taken 5-15% by weight, at room temperature and heating the solution at 50-80°C in a water bath; ii) adding lauryl alcohol, taken 5-7% by weight to the phosphate solution prepared in step (i) under continuous stirring, maintaining the temperature of the solution to 70°C for 5 minutes and adding oleic acid taken 25-30% by weight, preheated in a water bath, slowly under stirring; iii) preparing solution of urea and 2-amino-2methyl-1-propanol, by taking urea 14-18% by weight, heating the solution to 50-80°C and adding 2-amino-2-methyl-l propanol, taken 0.5-1.0% by weight to it; iv) adding the solution of urea prepared in step (iii) to the phosphate solution obtained at the end of step (ii) under continuous stirring, heating the entire solution to 70-85°C under intermittent stirring, and then allowing the solution to cool up to 60-40°C; v) adding 4-6% oleic acid to the solution obtained in step (iv) with stirring and allowing the solution to cool to room temperature to obtain a clear micro emulsion of the nutrient. Further according to this invention, there is provided a micro emulsified oleophilic nutrient composition for oil degrading microorganisms comprising of 10 to 25% by wt water, 6 to 9% by wt urea, 0-5 to 1% by wt of 2-amino-2-methyl- 1-propanol, 10 to 25% by wt of alkyl ethoxy phosphate, 5 to 15% by wt butoxy ethanol, 5 to 7% by wt lauryl alcohol and 30 to 35% by wt oleic acid. DESCRIPTION OF INVENTION According to the present invention there is provided a micro emulsified oleiophilic nutrient composition and a process for the preparation thereof, which helps in accelerated growth of oil degrading microorganisms to combat oil pollution in sea water. The nutrient of the present invention is in the form of a micro emulsion of water in oil type, the internal phase of which is an aqueous solution of nutrient material and the external phase is a liquid immiscible with water but miscible with the hydrophobic liquid to be biodegraded. The use of the nutrient helps in achieving rapid and complete biodegradation of the oil at low cost. The nutrient exhibits a low toxicity towards aquatic flora. Further, it has a higher shelf life of one year and it is thermally stable over a temperature range of 20°C to 85°C. The process of the present invention involves in the micro emulsification of the nutrient with the help of micro emulsifying agents and optimizing the conditions for the preparation. The external phase of the micro emulsion is miscible with the hydrophobic liquid to be biodegraded. The nutrient composition of the present invention utilizes a unsaturated long chain fatty acid oleic acid as a source of carbon which is utilized by the oil degrading microorganisms and which is miscible with the external hydrophobic layer. This permits rapid development of the oil degrading microorganisms. Urea, which is a rich source of nitrogen and which is very much soluble in water is utilized as a source of nitrogen by the nutrient composition. The nutrient also utilizes lauryl ethoxy phosphate as a surfactant and as a source of phosphorous. Further, it utilizes 2-butoxy ethanol as the co-surfactant which helps in reducing the viscosity of the microemulsion. The nutrient composition also utilizes 2-amino-2-methyl-l-propanol which bind water at the interface and which helps in slow release of the nutrient. It also helps in maintaining the pH of the emulsion and works as a micro emulsifier. Slow release of the nutrient is required so that the nutrient is available to the oil degrading microorganisms for a longer duration. The micro emulsified oleophilic nutrient composition of the present invention helps in accelerated growth of the oil degrading microorganisms it utilizes a unsaturated long chain fatty acid oleic acid as a s source of carbon which is utilized by the oil degrading microorganisms and which is miscible with the external hydrophobic layer. This permits rapid development of the oil degrading microorganisms. Urea, which is a rich source of nitrogen and which is very much soluble in water is utilized as a source of nitrogen by the nutrient composition. The nutrient utilises lauryl ethoxy phosphate as a surfactant and as a source of phosphorous. It utilises 2-butoxy ethanol as the co-surfactant which helps in reducing the viscosity of the microemulsion. The nutrient composition also utilises 2-amino-2-methyl-1-propanol which bind water at the interface and which helps in slow release of the nutrient. It also helps in maintaining the Ph of the emulsion and works as a micro emulsifier. Slow release of the nutrient is required so that the nutrient is available to the oil degrading microorganisms for a longer duration. According to the process of the present invention, the process for the preparation of micro emulsified oleophilic nutrient composition comprises the steps of: (i) Preparing phosphate solution The phosphate solution is prepared by dissolving tri lauryl ethoxy phosphate [[C12H25OC2H4)3O]3PO]], taken 10-25% weight wise, in 2-butoxy ethanol (C4HgOCH2CH2OH), taken 5-15% weight wise, at room temperature in 1 liter glass beaker. The phosphate solution, thus prepared, is heated to about 50-80°C .preferably 70 °C. in a water bath. (ii) Adding lauryl alcohol & oelic acid to the pnosphate solution Lauryl alcohol (C12H25 OH), taken 5-7% weight wise, is added to the phosphate solution prepared in step (i) under continuous stirring and the temperature of the solution is maintained to 70°C for about 5 minutes. To this solution, oleic acid (H3C(CH2)7HC=CH(CH2)7COOH), taken 25-30%, preheated to 70°C in a water bath, is added slowly under stirring. (iii) Preparing solution of urea and 2-amino -2 methyl-1- propanol. A solution of urea is prepared by dissolving urea (H2N-CO-NH2), taken 14-18% weight wise in distilled water in a glass beaker. The solution is heated to about 50-80°C preferably 70°C in a water bath. To this, 2 amino-2 methyl-1-propanol (C4H11NO ), taken 0.5-1.0% weight wise, is added. (iv) Adding mixture obtained from step (iii) to the mixture obtained from step (ii) The solution of urea prepared in step (iii) is added to the phosphate solution obtained at the end of step (ii) under continuous stirring .and the entire solution is heated 70-85°C for about 15 minutes under intermittent stirring. Next, the solution is allowed to cool up to 60-40°C with intermittent stirring. (v) Adding oleic acid to the solution obtained from step (iv) Oleic acid, taken 4-6%, is added to the solution obtained in step (iv) with stirring and the solution is allowed to cool to room temperature Finally, a clear micro emulsion of nutrient is formed The invention shall now be illustrated with the following working example, which is intended to be a typical example to explain the technique of the present invention and which is not intended to be taken restrictively to imply any limitation to the scope of the present invention. WORKING EXAMPLE: For preparation of one liter of micro emulsified oleophilic nutrient, 240 gm of try lauryl ethoxy phosphate is dissolved in 110 ml of 2-butoxy ethanol .to prepare the phosphate solution. Next, 151 gm of urea is dissolved in 189 ml of water to prepare the urea solution. The phosphate solution and urea solution, thus obtained, and 280 gm of oleic acid are separately heated in water bath to 70°C. 60 gm of lauryl alcohol is added to the phosphate solution and temperature is maintained at 70°C for 5 minutes. Oleic acid is added to this with stirring. Next, 6 gm of 2-amino-2-methyl-1-propanol is added to the urea solution and then this entire solution is added to the phosphate solution under continuous stirring. Temperature of this mix is raised to 80-85°C and it is maintained for 15 minutes. The solution is allowed to cool slowly to 50°C with intermittent stirring. 50 gm of oleic acid is added to this under stirring and the solution is cooled to room temperature to finally obtain the micro emulsion of nutrient. The properties of nutrient, thus obtained, is provided in the following: Oleiophilic in nature pH 6.4-6.6 Viscosity at 20°C 260 cst Experiments were conducted to test the efficacy of the nutrient with respect to the growth of oil degrading bacteria. A mixed culture of oil degrading bacteria comprising of Pseudomonas alcaligenes, P stutzeri, Micrococcus Varians, M. roseus and Bacillus brevis was maintained in Basal salts solution having o.5% HSD as the source of carbon. For assessing the growth of bacteria in presence of oleophilic nutrient composition, the inoculum consisted of 5% (w/v) of 24- 28 hr old culture grown as above and transferred to conical flasks containing 100 ml sterile seawater having 1% HSD and 0.1 % nutrient. A control flask was also kept having bacterial culture and HSD but no nutrient. The culture flasks were then incubated at room temperature 28± 2°C on a rotary shaker at 160 rpm for a period of 5 days. The bacterial growth in terms of bacterial count was determined by the serial dilution technique and by growing the colonies on Zobell marine agar plates. The colony forming units / ml was then calculated. The results are provided in the following: Results: (Table Removed) SSW Sterile sea water, HSD High Speed Diesel, N Nutrient, BC Bacterial culture, CPU Colony Forming Units The present embodiment of the invention, which has been set forth above, was for the purpose of illustration and is not intended to limit the scope of the invention. It is to be understood that various changes, adaptations and modifications can be made in the invention described above by those skilled in the art without departing from the scope of the invention, which has been defined by following claims: I CLAIM: 1. A process for the preparation of micro emulsified oleophilic nutrient composition for oil degrading microorganisms comprising the steps of:- i) preparing phosphate solution by dissolving tri lauryl ethoxy phosphate, taken 10-25% by weight, in 2- butoxy ethanol, taken 5-15% by weight, at room temperature and heating the solution at 50-80°C in a water bath; ii) adding lauryl alcohol, taken 5-7% by weight to the phosphate solution prepared in step (i) under continuous stirring, maintaining the temperature of the solution to 70°C for 5 minutes and adding oleic acid taken 25-30% by weight, preheated in a water bath, slowly under stirring; iii) preparing solution of urea and 2-amino-2methyl-l-propanol, by taking urea 14-18% by weight, heating the solution to 50-80°C and adding 2-amino-2-methyl-l propanol, taken 0.5-1.0% by weight to it; iv) adding the solution of urea prepared in step (iii) to the phosphate solution obtained at the end of step (ii) under continuous stirring, heating the entire solution to 70-85°C under intermittent stirring, and then allowing the solution to cool up to 60-40°C; v) adding 4-6% oleic acid to the solution obtained in step (iv) with stirring and allowing the solution to cool to room temperature to obtain a clear micro emulsion of the nutrient. 2. A process for the preparation of micro emulsified oleophilic nutrient composition for oil deprading microorganisms prepared substantially from the preceding claims. 3. A micro emulsified oleophilic nutrient composition for oil degrading microorganisms comprising of 10 to 25% by wt water, 6 to 9% by wt urea, 0-5 to 1% by wt of 2-amino-2-methyl-l-propanol, 10 to 25% by wt of alkyl ethoxy phosphate, 5 to 15% by wt butoxy ethanol, 5 to 7% by wt lauryl alcohol and 30 to 35% by wt oleic acid. 4. A micro emulsified oleophilic nutrient composition for oil degrading microorganisms, as claimed in the preceeding claims, which is if water-in-oil type. 5. A micro emulsified oleophilic nutrient composition for oil degrading microorganisms and a process for the preparation thereof substantially as described and exemplified herein.

Full Text

FIELD OF INVENTION:
This invention relates to a micro emulsified oleophilic nutrient composition and the process of preparation thereof for culturing micro organisms used for combating oil pollution in water.
PRIOR ART:
Sea water gets polluted by accidental spillages of oil from oil tankers, offshore drilling operations etc. The spillage of oil results in the formation of a continuous film of slick of oil which tends to spread continuously. This oil film is undesirable because it constitutes a barrier to the transfer of air and light from the atmosphere to the sea water, which are indispensable to support the marine life. In costal water, the oily film also damages the beaches.
One way of dealing with such pollution is to use oil dispersing compositions containing surface active compounds. When the dispersing compositions are sprayed on the oil slicks, they disintegrate the cohesive oil film into small droplets and disperse the droplets into the water column to a depth of several meters. The droplets, that are dispersed under the sea, are then biodegraded and consumed by microorganisms that are present in sea and are active in oil metabolism. This biological degradation is a slow process and is consequently unable to prevent the settling of un-degraded oil droplets and the formation of oil deposits on the floor of the sea.
The biodegradation, as mentioned above, can be enhanced by increasing the growth of the microorganisms at the water oil interface. Controlling pollution caused by hydrocarbon compounds (oil) in water by bioremediation is acquiring increasing significance. The. principle of bioremediation is based on optimum promotion of the growth of the pollution consuming microorganisms population. Major advantage of this process is the inexpensive in situ process without producing any waste for further disposal.In this regard two important features are of paramount significance(i) presence of oil degrading microorganisms at the oil-water interface, and (ii) nutrients required for the growth of the oil degrading microorganisms. In the elimination of hydrocarbon slicks by biodegradation at sea, beaches or even in lakes, the biggest problem is the preliminary multiplication of the oil degrading microorganisms. As such, it becomes necessary to supply growth promoting elements of the microorganisms. Generally, these growth promoting elements are not present in sufficient concentration in the polluted area.
These growth promoting elements are primarily inorganic or organic compounds of nitrogen and phosphorous and these are generally not available in sufficient concentration in the polluted area. These growth promoting elements are made available as nutrient concentrate for stimulation as well as growth aids for the accelerated growth of the microorganisms. The nutrient concentrate is made in the form of a micro emulsion, which is thermodynamically stable water in oil type. The internal phase of micro emulsion is aqueous solution of nutrient material and the external phase is a liquid immiscible with water. This helps nutrient substance to remain in the layer treated instead of being entrained by water.
Mineral salts such as ammonium salts, phosphates etc. are commonly used as nutrients. However, these mineral salts are soluble in water and these have practically no affinity for the polluting oil where a strong growth of microorganism should occur. Further, nitrogen containing organic nutrients such as condensation products of urea or melamine with aldehydes is soluble in water. These readily go to aqueous phase when sprayed on oil slick.
To eliminate this problem, various methods have been tried. The,US Patent No. 1959127 discloses a method, wherein a nutrient in the form of granules of nitrogenous compounds and solid phosphates coated with paraffin is made for the growth of the microorganisms.
However, this method suffers from the disadvantage that it requires special equipment which increases the cost of the nutrient.
Another disadvantage of this method, known in the art, is that it requires a separate surface-active agent for the treatment of the oil slicks.
Still another disadvantage of this method, known in the prior art, is that the paraffin coating may get dissolved if the polluting oil contains aromatic hydrocarbons.
US Patent No. 3883397 discloses a method wherein coating of fatty add salt instead of paraffin is made. However, this method suffers from the disadvantage that fatty acid coating material is difficult to degrade in the absence of external nitrogen and phosphorous, thereby making it difficult for the microorganisms to have the nutrient readily available.
The disadvantages of the prior art, mentioned above, have been overcome by the oleophilic nutrient of the present invention for the growth of the microorganisms active in oil degradation and which are present in sea water. The nutrient of the present invention is in the form of a micro emulsion of water in oil type, the internal phase of which is an aqueous solution of nutrient material and the external phase is a liquid immiscible with water but miscible with the hydrophobic liquid to be biodegraded. The use of the nutrient helps in achieving rapid and complete biodegradation of the oil at low cost. The nutrient exhibits a low toxicity towards aquatic flora.
OBJECTS OF THE INVENTION:
The primary object of the invention is to provide a process for the preparation of a micro emulsifide oleiophilic nutrient composition for the growth of oil degrading microorganisms to combat oil pollution in water and in shore.
Another object of the invention is to identify micro emulsifiers for the preparation of the micro emulsifide oleiophilic nutrient composition.
Another object of the invention is to provide a process for the preparation of micro emulsifide oleiophilic nutrient composition, which does not require costly and highly purified chemicals but uses easily available chemicals.Yet another object of the invention is to provide a process for the preparation of the micro emulsified oleiphilic nutrient composition, which does not require any expensive equipment.
Still further object of the invention is to provide a micro emulsified oleiphilic nutrient composition, which helps in the growth of oil degrading bacteria.
Even further object of the present invention is to provide micro emulsified oleiphilic nutrient composition, which has a shelf life of more than one year.
Still further object of the invention is to provide a micro emulsified oleiphilic nutrient composition, which has thermal stability over a temperature range 20°C to 85°C.
STATEMENT OF INVENTION
According to this invention is to provide a process for the preparation of micro emulsified oleophilic nutrient composition for oil degrading microorganisms comprising the steps of:-
i) preparing phosphate solution by dissolving tri lauryl ethoxy
phosphate, taken 10-25% by weight, in 2- butoxy ethanol, taken 5-15% by weight, at room temperature and heating the solution at 50-80°C in a water bath;
ii) adding lauryl alcohol, taken 5-7% by weight to the phosphate solution prepared in step (i) under continuous stirring, maintaining the temperature of the solution to 70°C for 5 minutes and adding oleic acid taken 25-30% by weight, preheated in a water bath, slowly under stirring;
iii) preparing solution of urea and 2-amino-2methyl-1-propanol, by taking urea 14-18% by weight, heating the solution to 50-80°C and adding 2-amino-2-methyl-l propanol, taken 0.5-1.0% by weight to it;
iv) adding the solution of urea prepared in step (iii) to the phosphate solution obtained at the end of step (ii) under continuous stirring, heating the entire solution to 70-85°C under intermittent stirring, and then allowing the solution to cool up to 60-40°C;
v) adding 4-6% oleic acid to the solution obtained in step (iv) with
stirring and allowing the solution to cool to room temperature to obtain a clear micro emulsion of the nutrient.
Further according to this invention, there is provided a micro emulsified oleophilic nutrient composition for oil degrading microorganisms comprising of 10 to 25% by wt water, 6 to 9% by wt urea, 0-5 to 1% by wt of 2-amino-2-methyl- 1-propanol, 10 to 25% by wt of alkyl ethoxy phosphate, 5 to 15% by wt butoxy ethanol, 5 to 7% by wt lauryl alcohol and 30 to 35% by wt oleic acid.

DESCRIPTION OF INVENTION
According to the present invention there is provided a micro emulsified oleiophilic nutrient composition and a process for the preparation thereof, which helps in accelerated growth of oil degrading microorganisms to combat oil pollution in sea water. The nutrient of the present invention is in the form of a micro emulsion of water in oil type, the internal phase of which is an aqueous solution of nutrient material and the external phase is a liquid immiscible with water but miscible with the hydrophobic liquid to be biodegraded. The use of the nutrient helps in achieving rapid and complete biodegradation of the oil at low cost. The nutrient exhibits a low toxicity towards aquatic flora. Further, it has a higher shelf life of one year and it is thermally stable over a temperature range of 20°C to 85°C.
The process of the present invention involves in the micro emulsification of the nutrient with the help of micro emulsifying agents and optimizing the conditions for the preparation. The external phase of the micro emulsion is miscible with the hydrophobic liquid to be biodegraded. The nutrient composition of the present invention utilizes a unsaturated long chain fatty acid oleic acid as a source of carbon which is utilized by the oil degrading microorganisms and which is miscible with the external hydrophobic layer. This permits rapid development of the oil degrading microorganisms. Urea, which is a rich source of nitrogen and which is very much soluble in water is utilized as a source of nitrogen by the nutrient composition. The nutrient also utilizes lauryl ethoxy phosphate as a surfactant and as a source of phosphorous. Further, it utilizes 2-butoxy ethanol as the co-surfactant which helps in reducing the viscosity of the microemulsion. The nutrient composition also utilizes 2-amino-2-methyl-l-propanol which bind water at the interface and which helps in slow release of the nutrient. It also helps in maintaining the pH of the emulsion and works as a micro emulsifier. Slow release of the nutrient is required so that the nutrient is available to the oil degrading microorganisms for a longer duration.
The micro emulsified oleophilic nutrient composition of the present invention helps in accelerated growth of the oil degrading microorganisms it utilizes a unsaturated long chain fatty acid oleic acid as a s source of carbon which is utilized by the oil degrading microorganisms and which is miscible with the external hydrophobic layer. This permits rapid development of the oil degrading microorganisms. Urea, which is a rich source of nitrogen and which is very much soluble in water is utilized as a source of nitrogen by the nutrient composition. The nutrient utilises lauryl ethoxy phosphate as a surfactant and as a source of phosphorous. It utilises 2-butoxy ethanol as the co-surfactant which helps in reducing the viscosity of the microemulsion. The nutrient composition also utilises 2-amino-2-methyl-1-propanol which bind water at the interface and which helps in slow release of the nutrient. It also helps in maintaining the Ph of the emulsion and works as a micro emulsifier. Slow release of the nutrient is required so that the nutrient is available to the oil degrading microorganisms for a longer duration.
According to the process of the present invention, the process for the preparation of micro emulsified oleophilic nutrient composition comprises the steps of:
(i) Preparing phosphate solution
The phosphate solution is prepared by dissolving tri lauryl ethoxy phosphate [[C12H25OC2H4)3O]3PO]], taken 10-25% weight wise, in 2-butoxy ethanol (C4HgOCH2CH2OH), taken 5-15% weight wise, at room temperature in 1 liter glass beaker. The phosphate solution, thus prepared, is heated to about 50-80°C .preferably 70 °C. in a water bath.
(ii) Adding lauryl alcohol & oelic acid to the pnosphate solution
Lauryl alcohol (C12H25 OH), taken 5-7% weight wise, is added to the phosphate solution prepared in step (i) under continuous stirring and the temperature of the solution is maintained to 70°C for about 5 minutes. To this solution, oleic acid (H3C(CH2)7HC=CH(CH2)7COOH), taken 25-30%, preheated to 70°C in a water bath, is added slowly under stirring.
(iii) Preparing solution of urea and 2-amino -2 methyl-1- propanol.
A solution of urea is prepared by dissolving urea (H2N-CO-NH2), taken 14-18% weight wise in distilled water in a glass beaker. The solution is heated to about 50-80°C preferably 70°C in a water bath. To this, 2 amino-2 methyl-1-propanol (C4H11NO ), taken 0.5-1.0% weight wise, is added.
(iv) Adding mixture obtained from step (iii) to the mixture obtained from step (ii)
The solution of urea prepared in step (iii) is added to the phosphate solution obtained at the end of step (ii) under continuous stirring .and the entire solution is heated 70-85°C for about 15 minutes under intermittent stirring. Next, the solution is allowed to cool up to 60-40°C with intermittent stirring.
(v) Adding oleic acid to the solution obtained from step (iv)
Oleic acid, taken 4-6%, is added to the solution obtained in step (iv) with stirring and the solution is allowed to cool to room temperature Finally, a clear micro emulsion of nutrient is formed
The invention shall now be illustrated with the following working example, which is intended to be a typical example to explain the technique of the present invention and which is not intended to be taken restrictively to imply any limitation to the scope of the present invention.
WORKING EXAMPLE:
For preparation of one liter of micro emulsified oleophilic nutrient, 240 gm of try lauryl ethoxy phosphate is dissolved in 110 ml of 2-butoxy ethanol .to prepare the phosphate solution. Next, 151 gm of urea is dissolved in 189 ml of water to prepare the urea solution. The phosphate solution and urea solution, thus obtained, and 280 gm of oleic acid are separately heated in water bath to 70°C. 60 gm of lauryl alcohol is added to the phosphate solution and temperature is maintained at 70°C for 5 minutes. Oleic acid is added to this with stirring. Next, 6 gm of 2-amino-2-methyl-1-propanol is added to the urea solution and then this entire solution is added to the phosphate solution under continuous stirring. Temperature of this mix is raised to 80-85°C and it is maintained for 15 minutes. The solution is allowed to cool slowly to 50°C with intermittent stirring. 50 gm of oleic acid is added to this under stirring and the solution is cooled to room temperature to finally obtain the micro emulsion of nutrient. The properties of nutrient, thus obtained, is provided in the following:
Oleiophilic in nature
pH 6.4-6.6
Viscosity at 20°C 260 cst
Experiments were conducted to test the efficacy of the nutrient with respect to the growth of oil degrading bacteria.
A mixed culture of oil degrading bacteria comprising of Pseudomonas alcaligenes, P stutzeri, Micrococcus Varians, M. roseus and Bacillus brevis was maintained in Basal salts solution having o.5% HSD as the source of carbon. For assessing the growth of bacteria in presence of oleophilic nutrient composition, the inoculum consisted of 5% (w/v) of 24- 28 hr old culture grown as above and transferred to conical flasks containing 100 ml sterile seawater having 1% HSD and 0.1 % nutrient. A control flask was also kept having bacterial culture and HSD but no nutrient. The culture flasks were then incubated at room temperature 28± 2°C on a rotary shaker at 160 rpm for a period of 5 days. The bacterial growth in terms of bacterial count was determined by the serial dilution technique and by growing the colonies on Zobell marine agar plates. The colony forming units / ml was then calculated. The results are provided in the following:
Results:

(Table Removed)
SSW Sterile sea water, HSD High Speed Diesel,
N Nutrient, BC Bacterial culture,
CPU Colony Forming Units
The present embodiment of the invention, which has been set forth above, was for the purpose of illustration and is not intended to limit the scope of the invention. It is to be understood that various changes, adaptations and modifications can be made in the invention described above by those skilled in the art without departing from the scope of the invention, which has been defined by following claims:

I CLAIM:
1. A process for the preparation of micro emulsified oleophilic nutrient composition for oil degrading microorganisms comprising the steps of:-
i) preparing phosphate solution by dissolving tri lauryl
ethoxy phosphate, taken 10-25% by weight, in 2- butoxy ethanol, taken 5-15% by weight, at room temperature and heating the solution at 50-80°C in a water bath;
ii) adding lauryl alcohol, taken 5-7% by weight to the phosphate solution prepared in step (i) under continuous stirring, maintaining the temperature of the solution to 70°C for 5 minutes and adding oleic acid taken 25-30% by weight, preheated in a water bath, slowly under stirring;
iii) preparing solution of urea and 2-amino-2methyl-l-propanol, by taking urea 14-18% by weight, heating the solution to 50-80°C and adding 2-amino-2-methyl-l propanol, taken 0.5-1.0% by weight to it;
iv) adding the solution of urea prepared in step (iii) to the phosphate solution obtained at the end of step (ii) under continuous stirring, heating the entire solution to 70-85°C under intermittent stirring, and then allowing the solution to cool up to 60-40°C;
v) adding 4-6% oleic acid to the solution obtained in step (iv) with stirring and allowing the solution to cool to room temperature to obtain a clear micro emulsion of the nutrient.
2. A process for the preparation of micro emulsified oleophilic
nutrient composition for oil deprading microorganisms prepared
substantially from the preceding claims.
3. A micro emulsified oleophilic nutrient composition for oil degrading
microorganisms comprising of 10 to 25% by wt water, 6 to 9% by
wt urea, 0-5 to 1% by wt of 2-amino-2-methyl-l-propanol, 10 to
25% by wt of alkyl ethoxy phosphate, 5 to 15% by wt butoxy
ethanol, 5 to 7% by wt lauryl alcohol and 30 to 35% by wt oleic
acid.
4. A micro emulsified oleophilic nutrient composition for oil degrading
microorganisms, as claimed in the preceeding claims, which is if
water-in-oil type.
5. A micro emulsified oleophilic nutrient composition for oil degrading microorganisms and a process for the preparation thereof substantially as described and exemplified herein.

Documents:

770-DEL-2004-Abstract-(01-06-2011).pdf

770-del-2004-abstract.pdf

770-DEL-2004-Claims-(01-06-2011).pdf

770-del-2004-claims.pdf

770-DEL-2004-Correspondence Others-(01-06-2011).pdf

770-del-2004-correspondence-others.pdf

770-del-2004-correspondence-po.pdf

770-DEL-2004-Description (Complete)-(01-06-2011).pdf

770-del-2004-description (complete).pdf

770-del-2004-form-18.pdf

770-del-2004-form-2.pdf

770-DEL-2004-Form-5-(01-06-2011).pdf

770-del-2004-form1.pdf

770-del-2004-form26.pdf

770-DEL-2004-GPA-(01-06-2011).pdf

770-DEL-2004-Petition-137-(01-06-2011).pdf

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Patent Number

252627

Indian Patent Application Number

770/DEL/2004

PG Journal Number

22/2012

Publication Date

01-Jun-2012

Grant Date

24-May-2012

Date of Filing

23-Apr-2004

Name of Patentee

DIRECTOR GENERAL, DEFENCE RESEARCH AND DEVELOPMENT ORGANIZATION

Applicant Address

defence research & development organisation ministry of defence, Govt. of India dte of er & IPR/IPR group West Block 8, Wing 1, R K Puram New Delhi

Inventors:

#	Inventor's Name	Inventor's Address
1	PRAMIL CHANDRA DEB	Naval Materials Research Laboratory, Shil Badlapur Road, Midc Area, Additional Ambernath(E), Dist.Thane-421506
2	SAROJAM PARAMESWARAN	Naval Materials Research Laboratory, Shil Badlapur Road, Midc Area, Additional Ambernath(E), Dist.Thane-421506

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