Title of Invention	BIO-ASSISTED METHOD FOR DISPOSING THE EMULSIONS OF METAL WORKING FLUIDS
Abstract	Disclosed herein a bio-assisted method for disposal of emulsion generated from the metal working fluids used in manufacturing industries, the method comprises of isolating and selecting microbes to form consortia and employing the same for degradation of emulsion wherein said consortia comprising microbes selected from Pseudomonas aeruginosa, Rathayibacter rathayi or Achromobacter xylosoxidans in any combination. Further the method disclosed in the present invention provides about 90% or more degradation of emulsion.

Title of Invention

BIO-ASSISTED METHOD FOR DISPOSING THE EMULSIONS OF METAL WORKING FLUIDS

Abstract

Disclosed herein a bio-assisted method for disposal of emulsion generated from the metal working fluids used in manufacturing industries, the method comprises of isolating and selecting microbes to form consortia and employing the same for degradation of emulsion wherein said consortia comprising microbes selected from Pseudomonas aeruginosa, Rathayibacter rathayi or Achromobacter xylosoxidans in any combination. Further the method disclosed in the present invention provides about 90% or more degradation of emulsion.

Full Text	FORM 2 THE PATENT ACT 1970 (39 of 1970) & THE PATENTS (AMENDMENT) RULES, 2006 COMPLETE SPECIFICATION (See section 10 and rule 13) 1. TITLE OF THE INVENTION BIO-ASSISTED METHOD FOR DISPOSING THE EMULSIONS OF METAL WORKING FLUIDS 2. APPLICANT (a) NAME : Indian Oil Corporation Limited (b) NATIONALITY : INDIAN (c) ADDRESS : G-9, AH Yavar Jung Marg, Bandra (East), Mumbai - 400051, (India) 3. PREAMBLE TO THE DESCRIPTION COMPLETE The following specification particularly describes the invention and the manner in which it is to be performed. Field of the Invention This invention, in general, relates to the method for disposal of emulsions of metal working fluids (MVVFs). More particularly the invention provides a bio-assisted method for disposal of emulsions of MWFs employing selective microbial consortia. Background of the Invention Metal Working Fluids (MWFs) are extensively employed as coolant and lubricant in metal working and manufacturing/engineering industries in wide range of operations such as cutting (e.g. turning, milling, tapping, drilling, grinding, broaching etc.), rolling, drawing, rust prevention and stamping etc. Both neat and water-soluble type fluids are used in metal working operations. Out of these, water-soluble type fluids have complex chemical composition and include constituents like emulsifiers, corrosion inhibitors, biocides and other performance additives. After certain cycles of uses eventually the emulsion of MWFs reaches a point where the fluid is no longer capable of performing its function of cooling and lubricating. These used emulsions not only contain chemicals present in them but also other oils used in various machine parts and dirt etc. A large amount of used emulsion of metal working fluid is generated by user industries which are considered environmentally reactive as it contains oils, chemicals, microbial population etc. and disposal of such used and contaminated metal fluids becomes inevitable. The traditional options for disposal of such used emulsions containing hazardous compounds are either dumping in dumpsites or recycling of used and contaminated fluids or employing other physico-chemical methods for degradation of pollutants. However, all such methods are often ineffective, environmentally reactive, and economically unviable. These emulsions, if not disposed properly may cause adverse effect on health and environment by spillage or other means. Therefore, there is a need to develop an alternative sustainable, environmental friendly and cost effective route for disposal of emulsions, which can also meet the requirements of regulatory bodies. Description of the Prior Arts The disposal of emulsion from metal working fluid needs to be treated as a serious scientific challenge. The prior art discloses many conventional methods for disposing emulsions such as are dumping in dumpsites, recycling and physico-chemical methods Disposal by dumping in landfill as disclosed in many patents has the short coming as the cost involved in transportation, chemical analysis on each shipment, dumping fee, taxes and other miscellaneous expenses amount to the point that it is more expensive to dispose of a gallon of metal working fluid than it originally was to purchase it. Moreover, the dumping in dumpsites is not permanent solution of the problem and has been banned in most of the countries. Similarly, incineration is not eco-friendly and sustainable, besides being expensive. Further in various prior arts are described a physico-chemical method to dispose of a metal working fluid, such as US Patent No. 5,589,138 is related to method to regenerate and stabilize metalworking fluids by using additives. US Patent No. 5,445,945 uses specifically chelating agent as additive. US Patent No. 4,636,317 describes recycling of metalworking fluids employing mobile equipments. Similarly, US Patent No. 5,447,642 describes regeneration using pasteurization. US Patent No. 5,137, 654 and Nidal Hilal et.al (International Biodeteriotion & Biodegradation, 47, 4, 2001, 215-224) describes use of ultra filtration and nanoflltration membranes respectively. However, all such processes are expensive and are not effective for removal of smaller molecules e.g synthetics. US Patent No. 6,712,975 describes a method for treating metal-working fluid using heat treatment and filter treatment process in combination. The physicochemical methods like heat treatment often prove ineffective or economically unviable. As discussed above the traditional options for disposal of such used emulsions based on physicochemical methods, are often ineffective or costly. Moreover, the degradation of contaminants is not achieved to completion and generally the amount of decontamination achieved does not meet the tightened legislation rules. With recent advances, the bioremediation of persistent organic pollutants using microorganisms has become rapidly growing area of research for environmental protection. Bioremediation has distinct advantages over physico-chemical remediation methods, as it can be cost effective and the complete degradation of organic pollutants with-out collateral destruction of the site material or its indigenous flora and fauna The prior art discloses the treatment of waste MWFs exploiting microbes, as microorganisms can degrade numerous organic pollutants owing to their metabolic activity and their capacity to adapt to inhospitable environments. Thus, microorganisms are major players in site remediation. US Patent No. 5,401,400 discloses a membrane bioreactor system for treating synthetic metal-working fluids and oil-based products using undefined microorganisms. However, the bioreactor inoculated by undefined microbes has met with limited success and moreover these approaches are often not reproducible Further in some prior arts use of selected microbes for disposal of metal working fluids in bioreactor has also been disclosed such as_van der Gast et al 2002 (Journal of Industrial Microbiology and Biotechnology Volume 29, Number 1 Pages: 20 - 27) describes the effectiveness of a strategy for constructing microbial consortia comprising {Clavibacter michiganensis, Methylobacterium mesophilicum, and Rhodococcus erythropolis) for treating chemically mixed industrial effluent, which could utilize or tolerate all of the individual MWF components, including the biocide and the recalcitrant compound benzotriazole. The degradation achieved by this process although is reproducible; however, percent degradation is not complete. Christopher J van der Gast et al 2003 (Bioaugmentation strategies for remediating mixed chemical effluents, Biotechnol Prog. 2003 Jul-Aug;19(4):l 156-61) discloses the effectiveness of another defined bacterial consortium, constructed specifically for treating metal-working fluids (MWFs), and contrasted its performance to that of undefined inocula from activated sludge. Construction of the consortium was based on knowledge of the diversity of bacterial communities that naturally colonize MWF and determination of their catabolic abilities and tolerance to the chemical constituents. Chemical analysis of the inoculated MWF bioreactor revealed the defined inoculum reduced the pollution load by over 80% from an initial chemical oxygen demand of approximately 48 000 mg-L-1. The inocula performance was approximately 50% more effective than that of the undefined microbial community from the activated sludge. The percent degradation achieved with said consortium is not complete. Christopher J van der Gast et at 2004 (Environ Microbiol 2004 Mar; 6: 254-63) discloses the effectiveness of a bacterial consortium composed of four species (Clavibacter michiganensis, Methylobacterium mesophilicum, Rhodococcus erythropolis and Pseudomonas putida), selected on the basis of their apparent ubiquity in waste MWF, degradation ability and tolerance to fluctuating chemistry of the waste. In said process 30-40% more effective degradation than any other treatment (indigenous MWF community alone or activated sludge) was achieved. Furthermore, all the chemical constituents, including the biocide (a formaldehyde release agent) demonstrated > 60% reduction. Many chemical components of the MWF proved to be recalcitrant in the other treatments. The results of this study confirm that assemblage of an inoculum, based on a comprehensive knowledge of the indigenous microbial community, in the target habitat, is a highly effective way of selecting microbial populations for bioaugmentation of bioreactors. Christopher J Van der Gast and Thomson 2005 (Effects of pH amendment on metal working fluid wastewater biological treatment using a defined bacterial consortium. Biotechnol Bioeng. 2005 Feb 5;89(3):357-66) studied the effect of pH amendment of a highly alkaline metal working fluid (MWF) wastewater on biological treatment in a bioreactor system following introduction of a bacterial inoculum (comprised of the following strains: Agrobacterium radiobacter, Comamonas testosteroni, Methylobacterium mesophilicum, Microbacterium esteraromaticum, and Microbacterium saperdae). The pH values tested were 6, 7, 8, and 9. After 14 days, the final mean chemical oxygen demand (COD) reduction at pH 9 was 50 +/-1.4 %; at pH 8, 58 +/- 1.4 %; pH 7, 65 +/- 1.0 %; and pH 6, 75 +/- 2.7 % of the initial COD (approximately 10,000 mg L-1), respectively. The consortia required specific conditions to achieve effective degradation The bio assisted methods discussed above in the prior art provides a technique wherein the degradation achieved is not upto the levels of completion and require specific conditions to achieve the same. As the successful treatment of chemically mixed wastes by bioaugmentation requires the selection of suitable strains for exploitation, therefore, there is a need to develop an alternative environmental friendly and cost effective route for disposal of emulsions at ambient conditions. The use of other microbes with better metabolic activity and ability may provide an effective method for disposal of used emulsion. The present invention provides robust and effective route of disposal of emulsion and the desired degradation can be achieved. Further the method disclosed herein according to present invention is cost effective and capable of dealing with wide range of wastes including synthetic and semi-synthetic. Summary of the Invention It is a principal object of the present invention to devise a novel bio assisted method for disposal of emulsion generated from metal working fluids (MWFs) used in various operations related to manufacturing/engineering industries, wherein said method comprises of selecting and enriching a microbial consortia which is capable of achieving almost 84% or more degradation in an environmentally acceptable manner. It is another object of the present invention to provide a method for disposal of emulsion generated from the metal working fluids, wherein said method is carried out in in situ as well as ex situ conditions employing said selective microbial consortia. The above and other objects are attained in accordance with the present invention wherein there is provided following embodiments, however, the described embodiments hereinafter is in accordance with the best mode of practice and the invention is not restricted to the particular embodiments. In accordance with one preferred embodiment of the present invention, there is provided a method for disposal of emulsion generated from the metal working fluids, the method comprises of isolating selective microbes by enrichment for forming consortia, which is capable of degrading metal working fluids and its components from emulsion of MWFs, wherein said microbial consortia comprises Pseudomonas aeruginosa IOC1, Rathayibacter rathayi IOC2 or Achromobacter xylosoxida IOC3 in any effective combination and wherein each microbe in said combination can vary in a ratio ranging from 0.25 to 2. In accordance with one other embodiment of the present invention, there is provided a method for disposal of emulsion generated from the metal working fluids, wherein the method comprises preparing selected microbial consortia as inoculum and adding the same to the bioreactor operating under pre-defined controlled conditions. In accordance with another embodiment of the present invention, there is provided a method for disposal of emulsion generated from the metal working fluids, wherein the method comprises preparing selected microbial consortia as inoculum and adding the same to the soil system at ambient conditions in naturally occurring environment. In accordance with yet another embodiment of the present invention, there is provided a method for disposal of emulsion generated from the metal working fluids, wherein used microbes in the present invention have achieved degradation in fresh as well as the used emulsion in bioreactor and wherein the percent degradation achieved is at least 90% in case of fresh emulsion and at least 84% in case of used emulsion. In accordance with yet another embodiment of the present invention there is provided a method for disposal of emulsion generated from the metal working fluids, wherein used microbes in the present invention have achieved percent degradation of emulsion in soil farming is at least 89% in case of used emulsion and 91% in case of fresh emulsion. In accordance with yet another embodiment of the present invention there is provided a method for disposal of emulsion generated from the metal working fluids. The method comprises of isolating microbes by enrichment, using the same selected microbial consortia as inoculum for inoculating the bioreactor, optimizing the reactor condition to run the same to achieve desired result. In accordance with yet another embodiment of the present invention there is provided a method for disposal of emulsion generated from the metal working fluids. The method comprises of isolating microbes by enrichment, using the same selected microbial consortia as inoculum and binding it with carrier, adding the resultant inoculum to soil-off mixture, wherein said soii-oif mixture is optimized with nutrients, thereafter tilling and watering the same to enhance growth of microbes in soil system. Brief Description of the Drawings For the purpose of promoting an understanding of the principles of the invention, reference will be made to the drawings. Therefore the principles of the invention are illustrated herein contemplated as would normally occur to one skilled in the art to which the invention relates Fig 1: A graphical representation showing degradation of fresh emulsion by selected microbial consortia in a bioreactor Fig 2: A graphical representation showing degradation of used emulsion by selected microbial consortia in a bioreactor Fig 3: A graphical representation showing degradation of fresh emulsion by selected microbial consortia in a soil system Fig 4: A graphical representation showing degradation of used emulsion by selected microbial consortia in a soil system Detailed Description of the Invention While this specification concludes with claims particularly pointing out and distinctly claiming that, which is regarded as the invention, it is anticipated that the invention can be more readily understood through reading the following detailed description of the invention and study of the included examples. The subject invention is aimed to find suitable and effective bio-treatment process for disposal of metal working fluids waste which is achieved by selection of the appropriate microbial strains. The selection of strains is essentially based on their metabolic activity and tolerance for co-contaminants. The process of the present invention is equally efficient in in situ as well as ex situ conditions. The in situ bio-assisted method for disposal of metal working fluids as carried out in bioreactor in accordance with the present invention, comprises the steps of: isolation and selection of microbes capable of degrading typical components of a metal working fluids (i.e., base oil, metal paasivator, lubricity additive, corrosion inhibitor, emulsifier and biocides) alone and in mixture as carbon source, wherein the microbes capable of utilizing most of the individual components of the cutting oil as carbon source were selected. The bio-assisted method employs selected microbes, wherein said microbes are further used for inoculum preparation which is further utilized for inoculation of the bioreactor. The bio-assisted method carried out in the bioreactor comprises the steps of optimization of nutrient system in the bioreactor followed by inoculation of the bioreactor using said inoculum and, running the bioreactor at controlled conditions and thereafter carrying out the monitoring of the said process. The bio-assisted method in accordance with the present invention, as carried out at the sites wherein, the amount of used emulsion is less and the installation of bioreactor is not practically feasible; there is need for more effective method for treatment for bioremediation. Alternatively, in the present invention there has been disclosed equally efficient land farming approach using said selected microbes wherein, the oil part of the used emulsion can be disposed after breaking the emulsion by any method known in the prior art and subjecting the said oil part to the soil system. Land farming is controlled and repeated application of wastes to the soil surface, using microorganisms in the soil to naturally biodegrade hydrocarbon constituents, dilute and attenuate metals, and transform and assimilate waste constituents. Land farming can be a relatively low-cost waste management approach. In the prior art numerous patents have disclosed land faming approach for treating the waste, however, the prior art is silent on the methodology for disposal of emulsion of metal working fluids by land farming approach. The method for treatment in soil system in accordance with the present invention comprises the steps of: isolating and selecting of microbial consortia capable of degrading various components of the metal working fluids, breaking the used emulsion by any method known in prior art for the said purpose. The bio-assisted land farming method for disposal of emulsion in metal working fluids in soil system further comprises employing selected microbes for the preparation of inoculum and its subsequent binding with carrier. The bio assisted land farming method further comprises mixing of said oil with soil, followed by addition of said inoculum comprising microbial consortia of selected microbes of present invention to soil-oil mixture, and thereafter, tilling and watering said mixture at periodic intervals. The microbial cultures used in the bio-assisted method of the present invention are natural-occurring bacterial cultures, isolated from operationally exhausted emulsions. The operationally exhausted emulsion was used for more than a month containing around 3.5 % oil and 3 X 109 CFU/ml. The bacterial cultures are isolated by enrichment techniques and selected by their ability to grow on various components of metal working fluids as the predominant source of carbon in the basal medium. Typical enrichment techniques comprise adding a sample of used emulsion containing a large population of bacteria to an aqueous medium containing components of metal working fluid as suitable initial primary food source, acting as sole carbon source in a nutrient system. In the present study around 150 microbes capable of utilizing components of metal working fluids or oil part of used emulsion were isolated. The said isolated microbes were further screened for their ability to assimilate various components of cutting oil i.e., base oil, metal paasivator, lubricity additive, corrosion inhibitor, emulsifier and biocides as carbon source. The selected microbes were capable of utilizing most of the individual components of the MWFs. However, most of the strains could not grow in presence of biocide. The strains able to grow in presence of biocide were further adapted to biocide by growing in presence of increasing concentration of biocide, thereby enrichment of desired strains is achieved. The bacterial cultures used in the bio-assisted method of the present invention were further enriched and said, enrichment is achieved by a nutrient system which desirably includes a nitrogen source like ammonium chloride; phosphorus source like dipotassium phosphate; magnesium source, such as magnesium salt, and can optionally include other nutrients such as sodium, calcium and iron salts. A suitable nutrient system which effectively utilized during the enrichment process includes an ammonium salt and a phosphate compound, along with minor amounts of other conventional nutrients wherein, the molar ratio of elemental nitrogen to phosphorus is from about 5: 1 to about 15: 1, and more preferably from about 8: 1 to about 12: 1. A particularly preferred nutrient system for use during the enrichment process includes ammonium chloride, from about 5 to about 20 parts by weight of hydrated magnesium sulfate (MgSO4.7H2O) per 100 parts by weight of ammonium chloride, from about 5 to about 20 parts by weight of sodium chloride per 100 parts by weight of ammonium chloride, and from about 15 to about 50, and more preferably from about 20 to about 30 parts by weight of monobasic potassium phosphate (K.H2 PO4) per 100 parts by weight of ammonium chloride and traces of vitamins and trace elements. The foregoing nutrients are dissolved in a suitable amount of water to dissolve the nutrients and combined with appropriate quantities of a suitable initial primary food source and the mixed bacterial consortia. The microbes enriched by said enrichment process were isolated at the end of enrichment cycles. Selection of the pure desired isolates was done by streaking on minimal salt agar medium containing metal working fluids as carbon source. These microbes were further evaluated for their ability to utilize MWFs and its components. Three microbes were selected essentially based on their ability to degrade various components of MWFs effectively. The selected constituents microbes of the microbial consortia obtained were characterized using molecular biology tools like molecular typing with repetitive sequence (Rep) based PCR with primer sets ERIC (enterobacterial repetitive intergenic consensus), REP (repetitive extragenic pallindrome, and sequence of 16S rDNA gene, The microbes selected and employed in the method of the present invention were identified as Pseudomonas aeruginosa IOC1, Rathayibacter rathayi IOC2 or Achromobacter xylosoxida IOC3. The microbial consortia comprising said microbes was capable of degrading all the components of MWFs and MWFs itself. The microbial consortia used, in present invention achieved effective degradation of MWFs and said consortia is effective in bioreactor as well as in soil system. The selected microbes Pseudomonas aeruginosa IOC1, Rathayibacter rathayi IOC2 or Achromobacter xylosoxida IOC3 comprising the consortia employed in the method of the present invention are effective in any combination, wherein each microbe in the combination can vary in a ratio ranging from 0.25 to 2. The selected microbes employed in the method of the present invention were prepared as a high-density culture wherein, the said high density culture was used as an inoculum for degradation of emulsion. The said microbial consortia may be inoculated into an aqueous medium containing the Luria-Bertani broth as medium and the incubated in an aerated reactor or fermenter or other culture vessel. The said mixed culture is allowed to grow till the said culture exhibits successful growth; thereafter, said culture prepared is used as inoculum. The inoculum comprising selected microbes can be transferred to a suitable vessel containing emulsion, preferably 15 liter bioreactor. The preferred inoculum volume is 0.1-20% by volume of total volume of used emulsion, preferably 1-5% by volume. The reactor is operated at standardized predetermined conditions The emulsion is fed to the reactor as such can be taken about 70-90% of the volume of the bioreactor preferably 75% of the reactor volume wherein the MWFs provides the carbon source for growth The desired oxygen levels are obtained by aeration of the culture wherein typical aeration rate of the reactor is 0.1-1.0 volumes of air per volume of medium per minute. Further, the temperature of the bioreactor is set in the range 20-40.degree Celsius (°C) preferably at 30-35 °C, at a pH generally maintained in the range 6.5-8.0, preferably in the range 7-7.5. The reaction conditions in the reactor are further modified to achieve maximum degradation. This involves feeding the reactor with modified emulsion supplied with nutrients for the bacterial culture. A wide variety of nutrients for the bacterial culture may be used, as will be understood by persons skilled in the art. Such nutrients will include nitrogen, phosphorus and potassium compounds. In particular, the nutrients comprise bioavailable nitrogen and phosphorus compounds. In embodiments, the amount of nitrogen is in the range of 50-1000 ppm and preferably 400-700 ppm, and the amount of phosphate is in the range of 10-200 ppm and preferably 50-150 ppm. In addition to nitrogen and phosphorus compounds, the nutrient also contains optimized concentrations of compounds other than nitrogen, phosphorus, carbon, oxygen and sodium, required to support bacterial growth and therefore it is normally necessary to add to the reactor one or more of magnesium, manganese, inorganic or organic sulphur, calcium, iron, copper, cobalt, zinc, boron and molybdenum. As such a culture solution there is widely used a material having a meat juice, a yeast extract, a malt extract, bactopeptone, glucose, inorganic salts, mineral, etc. in admixture at a proper ratio is widely used. The bioreactor is run at optimized conditions for 7-30 days and the biodegradation was examined in 15 liter bioreactor. For monitoring the process, samples were collected and analyzed for reduction in total petroleum hydrocarbon (TPH). When the level of TPH in aqueous mixture is reduced to an acceptable level, the aqueous mixture is discharged from the reactor. The microbes employed in the present invention have shown better metabolic activity. The degradation was evaluated in fresh as well as the used emulsion in bioreactor. Table 1 indicates the degradation results of emulsion. The percent degradation achieved in 40 days is 90% for fresh emulsion which is more than as reported in the prior art (Figure 1). Similarly, the percent degradation of used emulsion is 84% (figure 2). Used oil takes little more time for degradation, which may be attributed to the presence of the oxidation products during machining operation Table 1 Days Fresh emulsion % degradation Used emulsion % degradation 10 49 22 20 77 35 30 88 81 40 90 84 In view of the advantages of land farming which include its simplicity and low capital cost, the ability to apply multiple waste loadings to the same parcel of land, and the potential to improve soil conditions, the biodegradation of the MWFs and emulsion of MWFs was also subjected to soil matrix using selected microbial consortia employed in the present invention. This novel land farming approach for degrading metal working fluids has not been disclosed in any prior art. In the present invention, the said bio-assisted method for soil treatment approach comprises the slep of first adsorbing the appropriately grown microbial consortia used in the present lirvemtion on suitable carrier. The said microorganisms capable of degrading hydrocarbon contaminants are dispersed in soil while being supported on, i. e., fixed in a carrier. The earner in accordance with the present invention required for supporting microorganisms may be used, is any known material, so far as it can be applied to soil with microorganisms supported thereon. The ideal carrier materials can firmly adsorb microorganisms to the surface :hereof, and be helpful in transport and dispersal of final bioremediation agent. The carrier employed in the present invention is made of the materials that can retain microorganisms thereon relatively mildly and thus allows easy release of microorganisms thus proliferated. The said carrier is inexpensive and can act as a nutrient source for the microorganisms thus applied, particularly a nutrient source, which can be gradually released to advantage. Further, as the preferred embodiment of the invention is the formation of a carrier by a biodegradable material is advantageous in that any problems arising from secondary contamination by residual carrier or the effect of applied microorganisms on the soil ecological system can be avoided. As such a biodegradable carrier is preferably used a material, which gradually decomposes and disappears after the remediation of soil by applied microorganisms. When said biodegradable carrier is used, the applied selected microorganisms are released into soil after the disappearance of the carrier, and are put in environments which are severe to growth. The selected microorganisms are then driven out of soil and gradually decrease in number to extinction. As a result, the ecological system in soil can be restored to the original state. The preferred carriers employed herein in the present invention are biodegradable carrier material which includes corn husk, sugar industry waste or any agricultural waste. The water content of the carrier is from 1% to 99% by weight, preferably from 5% to 90% by weight, more preferably from 10% to 85% by weight. When the water content of the carrier is too low, microorganisms find difficulty in survival. On the contrary, when the water content of the carrier is too high, the resulting carrier exhibits a deteriorated physical strength that makes itself difficult to handle, The carrier used in the bio-assisted land farming process in accordance with the present invention is adsorbed with microbial consortia comprising selected microbes of present invention, wherein it was tested for its efficacy in soil experiments. The parameters of soil studies may be volume of soil bed, sludge dosing and mixing, microbial consortia dosing, nutrient composition dosing, tilling and moisture content. The bio-assisted land faming method of the present invention, wherein the oil portion of the used emulsion may be collected by breaking the emulsion by any known method in prior art like heat treatment, acid treatment etc. In the soil, the oil portion of used emulsion was spread uniformly around 5-40%, preferably 15% (w/w) and thoroughly mixed with soil. The bio-assisted land faming method further comprises of mixing soil inoculation of mixed soil with microbial consortia around 0.1- 5% w/w based on emulsion concentration. Inoculum size was preferably 0. 2%. Nutrients were also applied at the rate of 100 mg to 1000 mg/100 kg of emulsion, after making its 5-25% solution in water. The bio-assisted land faming method in accordance with the present invention further comprises nutrient optimization wherein, nutrient optimization preferably comprise adding materials containing carbon, nitrogen and phosphorus. In this regard a culture solution suitable for the growth of microorganisms may be used. The culture solution as such comprises widely used material having a meat juice, a yeast extract, a malt extract, bactopeptone, glucose, inorganic salts, mineral, etc. in admixture at a proper ratio. The said components may be mixed at a proper ratio depending on the kind of microorganisms. The nutrients to be used in the invention may be any nutrients containing proper organic and inorganic nutrients besides, the aforesaid mentioned culture solution. In accordance the preferred embodiment, the preferable nutrient is mixture of yeast extract and potassium nitrate in 1: 1. In accordance with present invention, in order to enhance growth of applied microbes, tilling and watering of plot was carried out. The moisture content of the soil-system is 30% to 80% of the water retention capability of the soil- oil mix. To achieve further degradation by the bio-assisted method of the present invention, the important aspect of the remediation treatment is that mechanical mixing and tilling of soil sludge mix at starting of treatment and in between of treatment was also carried out. The said process helps in the aeration, which is essentially required by the microbes of present invention for growth and survival. Preferably the tiling was carried out in 15 days. Tilling of the soil creates better contact between organic compound contaminants, and promotes the microbes to faster reaction rates and increase the likelihood that the degradative reactions proceed to completion. Secondly, the thorough mixing enabled by mechanical agitation aids in controlling and dissipating the heat generated by the microbial growth. In accordance with the present invention, the amount of percent degradation achieved by land farming is high. In this regard Table 2 indicates that in case of used emulsion after two months, 62% degradation is achieved as compared to control wherein only 6.5 is achieved in case of control. Further, the percentage degradation achieved is significantly even higher in six months as 89% degradation is achieved as compared to control wherein only 23% is achieved (Figure 3). Similarly in case of fresh emulsion after two months, 67% degradation is achieved and after six months the degradation achieved is as high as 91% (Figure 4), Table 2 Control % Used Fresh Time degradation emulsion % emulsion % degradation degradation 2 6.5 62 67 months 6 23 89 91 months Following are examples, which illustrate procedures, including the best mode, for practicing the invention. These examples should not be construed as limiting. Example 1 Degradation of used emulsion in bioreactor Microbes capable of degrading emulsions of water-soluble metal working fluids were isolated from operationally exhausted metal working cutting fluids obtained from industrial workshop by enrichment technique. The isolated bacteria were evaluated for their ability to grow and assimilate various components of MWF as sole carbon source. Based on the growth ability, bacteria were combined in order to make suitable consortia/microbial blend. Biodegradation of used emulsion was examined in I5-Iit bioreactor (Applicon Bioconsole with controller ADI 1032) containing 8 lit of the operationally exhausted soluble cutting oil emulsion was obtained from industrial workshop (used for more than one month) and used with out dilution after amending with nutrients. The nutrient system contained yeast extract 0.2% (w.v), KNO3 ( 0.01%) and ammonium phosphate (.01%).The airflow within the bioreactor was maintained at 5 lit/min. Reactor temperature was maintained at 30°C using water heated jackets. At the timed interval, samples were withdrawn and solvent extractable total petroleum hydrocarbon (TPH) was determined, gravimetrically. Figure 1 depicts the degradation of used emulsion after one month Example 2 Degradation of used emulsion in soil The biodegradation of the oil obtained from the breaking of used emulsion by acid treatment, as per the art described in prior art for the purpose. The oil portion was subjected to soil matrix using microbial consortia along with optimized nutrient (KN03 and yeast extract in 1: 1 (w/w)). The samples were collected and analyzed for reduction in TPH .The microbial blend could degrade more than 89% of the used emulsion in 6 months in soil (Figure 4). While this invention has been described in detail with reference to certain preferred embodiments, it should be appreciated that the present invention is not limited to those precise embodiments rather, in view of the present disclosure, which describes the current best mode for practicing the invention, many modifications and variations, would present themselves to those skilled in the art without departing from the scope and spirit of this invention. We Claim: 1. A bio-assisted method for disposal of emulsion generated from the metalworking fluids, wherein the method comprises isolating and selecting effective strains of microbes from operationally exhausted emulsions by enrichment to form a microbial consortia and employing the same for degradation of emulsion, wherein said method is characterized in achieving 84-91% degradation of emulsion in in situ or ex situ conditions; wherein said isolation is carried out by employing enrichment technique; wherein said microbial consortia comprises any combination of the microbes selected from Pseudomonas aeruginosa IOC1, Rathayibacter rathayi IOC2 or Achromobacter xylosoxida IOC3, and each microbe in the combination can vary in a ratio ranging from 0.25 to 2. 2. The method as claimed in claim 1, wherein said degradation is performed in fresh emulsion or used emulsion. 3. The method as claimed in claim 2, wherein the percent degradation is 90-91% of fresh emulsion. 4. The method as claimed in claim 2, wherein the percent degradation is 84-89% of used emulsion. 5. The method as claimed in claim 1, wherein said method is carried out in a bioreactor or in a soil system. 6. The method as claimed in claim 1, the method comprising; isolating and selecting a group of microbes to form the microbial consortia, which is capable of degrading metal working fluids and its components from emulsion of metal working fluids, adding the resultant microbial consortia to a bioreactor having said metal working fluids, optimizing the conditions in the bioreactor, inoculating the bioreactor with said selective microbial consortia, and running the bioreactor to achieve desired degradation; wherein said isolation is carried out by employing enrichment technique, 7. The method as claimed in claim 6, wherein the emulsion in use is 70-90% of the volume of the bioreactor, preferably 75%. 8. The method as claimed in claim 6, wherein the optimized temperature of bioreactor is 20-40°C, preferably 30 -35°C. 9. The method as claimed in claim 1, the method comprising; isolating and selecting a group of microbes to form the microbial consortia, which is capable of degrading metal working fluids and its 'components from emulsion of metal working fluids, binding the resultant microbial consortia with biodegradable carrier. adding the resultant blend to soil-oil mixture, providing optimized nutrients in the soil, and periodic tilling and watering to enhance growth of said microbes to achieve desired degradation; wherein said isolation is carried out by employing enrichment technique. 10. The method as claimed in claim 9, wherein the soil-oil mixture comprises oil in the range of 5-40%, preferably 15% (w/w) and is thoroughly mixed with the soil. 11. The method as claimed in claim 9, wherein said carrier is selected from cornhusk, sugar industry waste or any agricultural waste. 12. The method as claimed in claim 9, wherein nutrients are in the range of 100 mg to 1000 mg/100 kg of emulsion. 13. The method as claimed in claim 9, wherein the nutrient is preferably a mixture of yeast extract and potassium nitrate in 1:1 ratio. 14. The method as claimed in any of the claims 1, 6 or 9, wherein the enrichment is performed by employing controlled nutrient system and in presence of biocide. 15. The method as claimed in claim 14, wherein the nutrients are selected from nitrogen source and phosphorus source. 16. The method as claimed in claim 15, wherein the molar ratio of nitrogen to phosphorus source is 5:1 to 15:1.

Full Text

FORM 2
THE PATENT ACT 1970
(39 of 1970)
&
THE PATENTS (AMENDMENT) RULES, 2006
COMPLETE SPECIFICATION
(See section 10 and rule 13)
1. TITLE OF THE INVENTION
BIO-ASSISTED METHOD FOR DISPOSING THE EMULSIONS OF METAL
WORKING FLUIDS
2. APPLICANT
(a) NAME : Indian Oil Corporation Limited
(b) NATIONALITY : INDIAN
(c) ADDRESS : G-9, AH Yavar Jung Marg, Bandra (East), Mumbai - 400051, (India)
3. PREAMBLE TO THE DESCRIPTION
COMPLETE
The following specification particularly describes the invention and the manner in which it is to be performed.

Field of the Invention
This invention, in general, relates to the method for disposal of emulsions of metal working fluids (MVVFs). More particularly the invention provides a bio-assisted method for disposal of emulsions of MWFs employing selective microbial consortia.
Background of the Invention
Metal Working Fluids (MWFs) are extensively employed as coolant and lubricant in metal working and manufacturing/engineering industries in wide range of operations such as cutting (e.g. turning, milling, tapping, drilling, grinding, broaching etc.), rolling, drawing, rust prevention and stamping etc. Both neat and water-soluble type fluids are used in metal working operations. Out of these, water-soluble type fluids have complex chemical composition and include constituents like emulsifiers, corrosion inhibitors, biocides and other performance additives. After certain cycles of uses eventually the emulsion of MWFs reaches a point where the fluid is no longer capable of performing its function of cooling and lubricating. These used emulsions not only contain chemicals present in them but also other oils used in various machine parts and dirt etc. A large amount of used emulsion of metal working fluid is generated by user industries which are considered environmentally reactive as it contains oils, chemicals, microbial population etc. and disposal of such used and contaminated metal fluids becomes inevitable.
The traditional options for disposal of such used emulsions containing hazardous compounds are either dumping in dumpsites or recycling of used and contaminated fluids or employing other physico-chemical methods for degradation of pollutants. However, all such methods are often ineffective, environmentally reactive, and economically unviable. These emulsions, if not disposed properly may cause adverse effect on health and environment by spillage or other means. Therefore, there is a need to develop an alternative sustainable, environmental

friendly and cost effective route for disposal of emulsions, which can also meet the requirements of regulatory bodies.
Description of the Prior Arts
The disposal of emulsion from metal working fluid needs to be treated as a serious scientific challenge. The prior art discloses many conventional methods for disposing emulsions such as are dumping in dumpsites, recycling and physico-chemical methods
Disposal by dumping in landfill as disclosed in many patents has the short coming as the cost involved in transportation, chemical analysis on each shipment, dumping fee, taxes and other miscellaneous expenses amount to the point that it is more expensive to dispose of a gallon of metal working fluid than it originally was to purchase it. Moreover, the dumping in dumpsites is not permanent solution of the problem and has been banned in most of the countries. Similarly, incineration is not eco-friendly and sustainable, besides being expensive.
Further in various prior arts are described a physico-chemical method to dispose of a metal working fluid, such as US Patent No. 5,589,138 is related to method to regenerate and stabilize metalworking fluids by using additives. US Patent No. 5,445,945 uses specifically chelating agent as additive. US Patent No. 4,636,317 describes recycling of metalworking fluids employing mobile equipments. Similarly, US Patent No. 5,447,642 describes regeneration using pasteurization. US Patent No. 5,137, 654 and Nidal Hilal et.al (International Biodeteriotion & Biodegradation, 47, 4, 2001, 215-224) describes use of ultra filtration and nanoflltration membranes respectively. However, all such processes are expensive and are not effective for removal of smaller molecules e.g synthetics.
US Patent No. 6,712,975 describes a method for treating metal-working fluid using heat treatment and filter treatment process in combination. The physicochemical methods like heat treatment often prove ineffective or economically unviable.
As discussed above the traditional options for disposal of such used emulsions based on physicochemical methods, are often ineffective or costly. Moreover, the degradation of

contaminants is not achieved to completion and generally the amount of decontamination achieved does not meet the tightened legislation rules.
With recent advances, the bioremediation of persistent organic pollutants using microorganisms has become rapidly growing area of research for environmental protection. Bioremediation has distinct advantages over physico-chemical remediation methods, as it can be cost effective and the complete degradation of organic pollutants with-out collateral destruction of the site material or its indigenous flora and fauna
The prior art discloses the treatment of waste MWFs exploiting microbes, as microorganisms can degrade numerous organic pollutants owing to their metabolic activity and their capacity to adapt to inhospitable environments. Thus, microorganisms are major players in site remediation.
US Patent No. 5,401,400 discloses a membrane bioreactor system for treating synthetic metal-working fluids and oil-based products using undefined microorganisms. However, the bioreactor inoculated by undefined microbes has met with limited success and moreover these approaches are often not reproducible
Further in some prior arts use of selected microbes for disposal of metal working fluids in bioreactor has also been disclosed such as_van der Gast et al 2002 (Journal of Industrial Microbiology and Biotechnology Volume 29, Number 1 Pages: 20 - 27) describes the effectiveness of a strategy for constructing microbial consortia comprising {Clavibacter michiganensis, Methylobacterium mesophilicum, and Rhodococcus erythropolis) for treating chemically mixed industrial effluent, which could utilize or tolerate all of the individual MWF components, including the biocide and the recalcitrant compound benzotriazole. The degradation achieved by this process although is reproducible; however, percent degradation is not complete.
Christopher J van der Gast et al 2003 (Bioaugmentation strategies for remediating mixed chemical effluents, Biotechnol Prog. 2003 Jul-Aug;19(4):l 156-61) discloses the effectiveness

of another defined bacterial consortium, constructed specifically for treating metal-working fluids (MWFs), and contrasted its performance to that of undefined inocula from activated sludge. Construction of the consortium was based on knowledge of the diversity of bacterial communities that naturally colonize MWF and determination of their catabolic abilities and tolerance to the chemical constituents. Chemical analysis of the inoculated MWF bioreactor revealed the defined inoculum reduced the pollution load by over 80% from an initial chemical oxygen demand of approximately 48 000 mg-L-1. The inocula performance was approximately 50% more effective than that of the undefined microbial community from the activated sludge. The percent degradation achieved with said consortium is not complete.
Christopher J van der Gast et at 2004 (Environ Microbiol 2004 Mar; 6: 254-63) discloses the effectiveness of a bacterial consortium composed of four species (Clavibacter michiganensis, Methylobacterium mesophilicum, Rhodococcus erythropolis and Pseudomonas putida), selected on the basis of their apparent ubiquity in waste MWF, degradation ability and tolerance to fluctuating chemistry of the waste. In said process 30-40% more effective degradation than any other treatment (indigenous MWF community alone or activated sludge) was achieved. Furthermore, all the chemical constituents, including the biocide (a formaldehyde release agent) demonstrated > 60% reduction. Many chemical components of the MWF proved to be recalcitrant in the other treatments. The results of this study confirm that assemblage of an inoculum, based on a comprehensive knowledge of the indigenous microbial community, in the target habitat, is a highly effective way of selecting microbial populations for bioaugmentation of bioreactors.
Christopher J Van der Gast and Thomson 2005 (Effects of pH amendment on metal working fluid wastewater biological treatment using a defined bacterial consortium. Biotechnol Bioeng. 2005 Feb 5;89(3):357-66) studied the effect of pH amendment of a highly alkaline metal working fluid (MWF) wastewater on biological treatment in a bioreactor system following introduction of a bacterial inoculum (comprised of the following strains: Agrobacterium radiobacter, Comamonas testosteroni, Methylobacterium mesophilicum, Microbacterium esteraromaticum, and Microbacterium saperdae). The pH values tested were 6, 7, 8, and 9. After 14 days, the final mean chemical oxygen demand (COD) reduction at pH 9 was 50 +/-1.4 %; at pH 8, 58 +/- 1.4 %; pH 7, 65 +/- 1.0 %; and pH 6, 75 +/- 2.7 % of the initial COD

(approximately 10,000 mg L-1), respectively. The consortia required specific conditions to achieve effective degradation
The bio assisted methods discussed above in the prior art provides a technique wherein the degradation achieved is not upto the levels of completion and require specific conditions to achieve the same. As the successful treatment of chemically mixed wastes by bioaugmentation requires the selection of suitable strains for exploitation, therefore, there is a need to develop an alternative environmental friendly and cost effective route for disposal of emulsions at ambient conditions. The use of other microbes with better metabolic activity and ability may provide an effective method for disposal of used emulsion.
The present invention provides robust and effective route of disposal of emulsion and the desired degradation can be achieved. Further the method disclosed herein according to present invention is cost effective and capable of dealing with wide range of wastes including synthetic and semi-synthetic.
Summary of the Invention
It is a principal object of the present invention to devise a novel bio assisted method for disposal of emulsion generated from metal working fluids (MWFs) used in various operations related to manufacturing/engineering industries, wherein said method comprises of selecting and enriching a microbial consortia which is capable of achieving almost 84% or more degradation in an environmentally acceptable manner.
It is another object of the present invention to provide a method for disposal of emulsion generated from the metal working fluids, wherein said method is carried out in in situ as well as ex situ conditions employing said selective microbial consortia.
The above and other objects are attained in accordance with the present invention wherein there is provided following embodiments, however, the described embodiments hereinafter is in accordance with the best mode of practice and the invention is not restricted to the particular embodiments.

In accordance with one preferred embodiment of the present invention, there is provided a method for disposal of emulsion generated from the metal working fluids, the method comprises of isolating selective microbes by enrichment for forming consortia, which is capable of degrading metal working fluids and its components from emulsion of MWFs, wherein said microbial consortia comprises Pseudomonas aeruginosa IOC1, Rathayibacter rathayi IOC2 or Achromobacter xylosoxida IOC3 in any effective combination and wherein each microbe in said combination can vary in a ratio ranging from 0.25 to 2.
In accordance with one other embodiment of the present invention, there is provided a method for disposal of emulsion generated from the metal working fluids, wherein the method comprises preparing selected microbial consortia as inoculum and adding the same to the bioreactor operating under pre-defined controlled conditions.
In accordance with another embodiment of the present invention, there is provided a method for disposal of emulsion generated from the metal working fluids, wherein the method comprises preparing selected microbial consortia as inoculum and adding the same to the soil system at ambient conditions in naturally occurring environment.
In accordance with yet another embodiment of the present invention, there is provided a method for disposal of emulsion generated from the metal working fluids, wherein used microbes in the present invention have achieved degradation in fresh as well as the used emulsion in bioreactor and wherein the percent degradation achieved is at least 90% in case of fresh emulsion and at least 84% in case of used emulsion.
In accordance with yet another embodiment of the present invention there is provided a method for disposal of emulsion generated from the metal working fluids, wherein used microbes in the present invention have achieved percent degradation of emulsion in soil farming is at least 89% in case of used emulsion and 91% in case of fresh emulsion.
In accordance with yet another embodiment of the present invention there is provided a method for disposal of emulsion generated from the metal working fluids. The method comprises of isolating microbes by enrichment, using the same selected microbial consortia as inoculum for

inoculating the bioreactor, optimizing the reactor condition to run the same to achieve desired result.
In accordance with yet another embodiment of the present invention there is provided a method for disposal of emulsion generated from the metal working fluids. The method comprises of isolating microbes by enrichment, using the same selected microbial consortia as inoculum and binding it with carrier, adding the resultant inoculum to soil-off mixture, wherein said soii-oif mixture is optimized with nutrients, thereafter tilling and watering the same to enhance growth of microbes in soil system.
Brief Description of the Drawings
For the purpose of promoting an understanding of the principles of the invention, reference will be made to the drawings. Therefore the principles of the invention are illustrated herein contemplated as would normally occur to one skilled in the art to which the invention relates
Fig 1: A graphical representation showing degradation of fresh emulsion by selected microbial
consortia in a bioreactor
Fig 2: A graphical representation showing degradation of used emulsion by selected microbial
consortia in a bioreactor
Fig 3: A graphical representation showing degradation of fresh emulsion by selected microbial
consortia in a soil system
Fig 4: A graphical representation showing degradation of used emulsion by selected microbial
consortia in a soil system
Detailed Description of the Invention
While this specification concludes with claims particularly pointing out and distinctly claiming that, which is regarded as the invention, it is anticipated that the invention can be more readily understood through reading the following detailed description of the invention and study of the included examples.

The subject invention is aimed to find suitable and effective bio-treatment process for disposal of metal working fluids waste which is achieved by selection of the appropriate microbial strains. The selection of strains is essentially based on their metabolic activity and tolerance for co-contaminants. The process of the present invention is equally efficient in in situ as well as ex situ conditions.
The in situ bio-assisted method for disposal of metal working fluids as carried out in bioreactor in accordance with the present invention, comprises the steps of: isolation and selection of microbes capable of degrading typical components of a metal working fluids (i.e., base oil, metal paasivator, lubricity additive, corrosion inhibitor, emulsifier and biocides) alone and in mixture as carbon source, wherein the microbes capable of utilizing most of the individual components of the cutting oil as carbon source were selected.
The bio-assisted method employs selected microbes, wherein said microbes are further used for inoculum preparation which is further utilized for inoculation of the bioreactor.
The bio-assisted method carried out in the bioreactor comprises the steps of optimization of nutrient system in the bioreactor followed by inoculation of the bioreactor using said inoculum and, running the bioreactor at controlled conditions and thereafter carrying out the monitoring of the said process.
The bio-assisted method in accordance with the present invention, as carried out at the sites wherein, the amount of used emulsion is less and the installation of bioreactor is not practically feasible; there is need for more effective method for treatment for bioremediation. Alternatively, in the present invention there has been disclosed equally efficient land farming approach using said selected microbes wherein, the oil part of the used emulsion can be disposed after breaking the emulsion by any method known in the prior art and subjecting the said oil part to the soil system.
Land farming is controlled and repeated application of wastes to the soil surface, using microorganisms in the soil to naturally biodegrade hydrocarbon constituents, dilute and

attenuate metals, and transform and assimilate waste constituents. Land farming can be a relatively low-cost waste management approach. In the prior art numerous patents have disclosed land faming approach for treating the waste, however, the prior art is silent on the methodology for disposal of emulsion of metal working fluids by land farming approach.
The method for treatment in soil system in accordance with the present invention comprises the steps of: isolating and selecting of microbial consortia capable of degrading various components of the metal working fluids, breaking the used emulsion by any method known in prior art for the said purpose.
The bio-assisted land farming method for disposal of emulsion in metal working fluids in soil system further comprises employing selected microbes for the preparation of inoculum and its subsequent binding with carrier.
The bio assisted land farming method further comprises mixing of said oil with soil, followed by addition of said inoculum comprising microbial consortia of selected microbes of present invention to soil-oil mixture, and thereafter, tilling and watering said mixture at periodic intervals.
The microbial cultures used in the bio-assisted method of the present invention are natural-occurring bacterial cultures, isolated from operationally exhausted emulsions. The operationally exhausted emulsion was used for more than a month containing around 3.5 % oil and 3 X 109 CFU/ml. The bacterial cultures are isolated by enrichment techniques and selected by their ability to grow on various components of metal working fluids as the predominant source of carbon in the basal medium. Typical enrichment techniques comprise adding a sample of used emulsion containing a large population of bacteria to an aqueous medium containing components of metal working fluid as suitable initial primary food source, acting as sole carbon source in a nutrient system. In the present study around 150 microbes capable of utilizing components of metal working fluids or oil part of used emulsion were isolated. The said isolated microbes were further screened for their ability to assimilate various components of cutting oil i.e., base oil, metal paasivator, lubricity additive, corrosion inhibitor, emulsifier

and biocides as carbon source. The selected microbes were capable of utilizing most of the individual components of the MWFs. However, most of the strains could not grow in presence of biocide. The strains able to grow in presence of biocide were further adapted to biocide by growing in presence of increasing concentration of biocide, thereby enrichment of desired strains is achieved.
The bacterial cultures used in the bio-assisted method of the present invention were further enriched and said, enrichment is achieved by a nutrient system which desirably includes a nitrogen source like ammonium chloride; phosphorus source like dipotassium phosphate; magnesium source, such as magnesium salt, and can optionally include other nutrients such as sodium, calcium and iron salts. A suitable nutrient system which effectively utilized during the enrichment process includes an ammonium salt and a phosphate compound, along with minor amounts of other conventional nutrients wherein, the molar ratio of elemental nitrogen to phosphorus is from about 5: 1 to about 15: 1, and more preferably from about 8: 1 to about 12: 1. A particularly preferred nutrient system for use during the enrichment process includes ammonium chloride, from about 5 to about 20 parts by weight of hydrated magnesium sulfate (MgSO4.7H2O) per 100 parts by weight of ammonium chloride, from about 5 to about 20 parts by weight of sodium chloride per 100 parts by weight of ammonium chloride, and from about 15 to about 50, and more preferably from about 20 to about 30 parts by weight of monobasic potassium phosphate (K.H2 PO4) per 100 parts by weight of ammonium chloride and traces of vitamins and trace elements. The foregoing nutrients are dissolved in a suitable amount of water to dissolve the nutrients and combined with appropriate quantities of a suitable initial primary food source and the mixed bacterial consortia.
The microbes enriched by said enrichment process were isolated at the end of enrichment cycles. Selection of the pure desired isolates was done by streaking on minimal salt agar medium containing metal working fluids as carbon source. These microbes were further evaluated for their ability to utilize MWFs and its components. Three microbes were selected essentially based on their ability to degrade various components of MWFs effectively.

The selected constituents microbes of the microbial consortia obtained were characterized using molecular biology tools like molecular typing with repetitive sequence (Rep) based PCR with primer sets ERIC (enterobacterial repetitive intergenic consensus), REP (repetitive extragenic pallindrome, and sequence of 16S rDNA gene,
The microbes selected and employed in the method of the present invention were identified as Pseudomonas aeruginosa IOC1, Rathayibacter rathayi IOC2 or Achromobacter xylosoxida IOC3. The microbial consortia comprising said microbes was capable of degrading all the components of MWFs and MWFs itself. The microbial consortia used, in present invention achieved effective degradation of MWFs and said consortia is effective in bioreactor as well as in soil system.
The selected microbes Pseudomonas aeruginosa IOC1, Rathayibacter rathayi IOC2 or Achromobacter xylosoxida IOC3 comprising the consortia employed in the method of the present invention are effective in any combination, wherein each microbe in the combination can vary in a ratio ranging from 0.25 to 2.
The selected microbes employed in the method of the present invention were prepared as a high-density culture wherein, the said high density culture was used as an inoculum for degradation of emulsion. The said microbial consortia may be inoculated into an aqueous medium containing the Luria-Bertani broth as medium and the incubated in an aerated reactor or fermenter or other culture vessel. The said mixed culture is allowed to grow till the said culture exhibits successful growth; thereafter, said culture prepared is used as inoculum.
The inoculum comprising selected microbes can be transferred to a suitable vessel containing emulsion, preferably 15 liter bioreactor. The preferred inoculum volume is 0.1-20% by volume of total volume of used emulsion, preferably 1-5% by volume.
The reactor is operated at standardized predetermined conditions The emulsion is fed to the reactor as such can be taken about 70-90% of the volume of the bioreactor preferably 75% of the reactor volume wherein the MWFs provides the carbon source for growth The desired

oxygen levels are obtained by aeration of the culture wherein typical aeration rate of the reactor is 0.1-1.0 volumes of air per volume of medium per minute. Further, the temperature of the bioreactor is set in the range 20-40.degree Celsius (°C) preferably at 30-35 °C, at a pH generally maintained in the range 6.5-8.0, preferably in the range 7-7.5.
The reaction conditions in the reactor are further modified to achieve maximum degradation. This involves feeding the reactor with modified emulsion supplied with nutrients for the bacterial culture. A wide variety of nutrients for the bacterial culture may be used, as will be understood by persons skilled in the art. Such nutrients will include nitrogen, phosphorus and potassium compounds. In particular, the nutrients comprise bioavailable nitrogen and phosphorus compounds. In embodiments, the amount of nitrogen is in the range of 50-1000 ppm and preferably 400-700 ppm, and the amount of phosphate is in the range of 10-200 ppm and preferably 50-150 ppm. In addition to nitrogen and phosphorus compounds, the nutrient also contains optimized concentrations of compounds other than nitrogen, phosphorus, carbon, oxygen and sodium, required to support bacterial growth and therefore it is normally necessary to add to the reactor one or more of magnesium, manganese, inorganic or organic sulphur, calcium, iron, copper, cobalt, zinc, boron and molybdenum. As such a culture solution there is widely used a material having a meat juice, a yeast extract, a malt extract, bactopeptone, glucose, inorganic salts, mineral, etc. in admixture at a proper ratio is widely used.
The bioreactor is run at optimized conditions for 7-30 days and the biodegradation was examined in 15 liter bioreactor. For monitoring the process, samples were collected and analyzed for reduction in total petroleum hydrocarbon (TPH). When the level of TPH in aqueous mixture is reduced to an acceptable level, the aqueous mixture is discharged from the reactor.
The microbes employed in the present invention have shown better metabolic activity. The degradation was evaluated in fresh as well as the used emulsion in bioreactor. Table 1 indicates the degradation results of emulsion. The percent degradation achieved in 40 days is 90% for fresh emulsion which is more than as reported in the prior art (Figure 1). Similarly, the percent

degradation of used emulsion is 84% (figure 2). Used oil takes little more time for degradation, which may be attributed to the presence of the oxidation products during machining operation

Table 1
Days Fresh emulsion % degradation Used emulsion % degradation
10 49 22
20 77 35
30 88 81
40 90 84
In view of the advantages of land farming which include its simplicity and low capital cost, the ability to apply multiple waste loadings to the same parcel of land, and the potential to improve soil conditions, the biodegradation of the MWFs and emulsion of MWFs was also subjected to soil matrix using selected microbial consortia employed in the present invention. This novel land farming approach for degrading metal working fluids has not been disclosed in any prior art.
In the present invention, the said bio-assisted method for soil treatment approach comprises the slep of first adsorbing the appropriately grown microbial consortia used in the present lirvemtion on suitable carrier. The said microorganisms capable of degrading hydrocarbon contaminants are dispersed in soil while being supported on, i. e., fixed in a carrier.
The earner in accordance with the present invention required for supporting microorganisms may be used, is any known material, so far as it can be applied to soil with microorganisms supported thereon. The ideal carrier materials can firmly adsorb microorganisms to the surface :hereof, and be helpful in transport and dispersal of final bioremediation agent.
The carrier employed in the present invention is made of the materials that can retain microorganisms thereon relatively mildly and thus allows easy release of microorganisms thus proliferated. The said carrier is inexpensive and can act as a nutrient source for the

microorganisms thus applied, particularly a nutrient source, which can be gradually released to advantage.
Further, as the preferred embodiment of the invention is the formation of a carrier by a biodegradable material is advantageous in that any problems arising from secondary contamination by residual carrier or the effect of applied microorganisms on the soil ecological system can be avoided. As such a biodegradable carrier is preferably used a material, which gradually decomposes and disappears after the remediation of soil by applied microorganisms.
When said biodegradable carrier is used, the applied selected microorganisms are released into soil after the disappearance of the carrier, and are put in environments which are severe to growth. The selected microorganisms are then driven out of soil and gradually decrease in number to extinction. As a result, the ecological system in soil can be restored to the original state.
The preferred carriers employed herein in the present invention are biodegradable carrier material which includes corn husk, sugar industry waste or any agricultural waste. The water content of the carrier is from 1% to 99% by weight, preferably from 5% to 90% by weight, more preferably from 10% to 85% by weight. When the water content of the carrier is too low, microorganisms find difficulty in survival. On the contrary, when the water content of the carrier is too high, the resulting carrier exhibits a deteriorated physical strength that makes itself difficult to handle,
The carrier used in the bio-assisted land farming process in accordance with the present invention is adsorbed with microbial consortia comprising selected microbes of present invention, wherein it was tested for its efficacy in soil experiments. The parameters of soil studies may be volume of soil bed, sludge dosing and mixing, microbial consortia dosing, nutrient composition dosing, tilling and moisture content.
The bio-assisted land faming method of the present invention, wherein the oil portion of the used emulsion may be collected by breaking the emulsion by any known method in prior art like heat treatment, acid treatment etc. In the soil, the oil portion of used emulsion was spread uniformly around 5-40%, preferably 15% (w/w) and thoroughly mixed with soil.

The bio-assisted land faming method further comprises of mixing soil inoculation of mixed soil with microbial consortia around 0.1- 5% w/w based on emulsion concentration. Inoculum size was preferably 0. 2%. Nutrients were also applied at the rate of 100 mg to 1000 mg/100 kg of emulsion, after making its 5-25% solution in water.
The bio-assisted land faming method in accordance with the present invention further comprises nutrient optimization wherein, nutrient optimization preferably comprise adding materials containing carbon, nitrogen and phosphorus. In this regard a culture solution suitable for the growth of microorganisms may be used. The culture solution as such comprises widely used material having a meat juice, a yeast extract, a malt extract, bactopeptone, glucose, inorganic salts, mineral, etc. in admixture at a proper ratio. The said components may be mixed at a proper ratio depending on the kind of microorganisms. The nutrients to be used in the invention may be any nutrients containing proper organic and inorganic nutrients besides, the aforesaid mentioned culture solution. In accordance the preferred embodiment, the preferable nutrient is mixture of yeast extract and potassium nitrate in 1: 1.
In accordance with present invention, in order to enhance growth of applied microbes, tilling and watering of plot was carried out. The moisture content of the soil-system is 30% to 80% of the water retention capability of the soil- oil mix.
To achieve further degradation by the bio-assisted method of the present invention, the important aspect of the remediation treatment is that mechanical mixing and tilling of soil sludge mix at starting of treatment and in between of treatment was also carried out. The said process helps in the aeration, which is essentially required by the microbes of present invention for growth and survival. Preferably the tiling was carried out in 15 days. Tilling of the soil creates better contact between organic compound contaminants, and promotes the microbes to faster reaction rates and increase the likelihood that the degradative reactions proceed to completion. Secondly, the thorough mixing enabled by mechanical agitation aids in controlling and dissipating the heat generated by the microbial growth.
In accordance with the present invention, the amount of percent degradation achieved by land farming is high. In this regard Table 2 indicates that in case of used emulsion after two months, 62% degradation is achieved as compared to control wherein only 6.5 is achieved in case of control. Further, the percentage degradation achieved is significantly even higher in six months

as 89% degradation is achieved as compared to control wherein only 23% is achieved (Figure 3). Similarly in case of fresh emulsion after two months, 67% degradation is achieved and after six months the degradation achieved is as high as 91% (Figure 4),

Table 2
Control % Used Fresh
Time degradation emulsion % emulsion %
degradation degradation
2 6.5 62 67
months
6 23 89 91
months
Following are examples, which illustrate procedures, including the best mode, for practicing the invention. These examples should not be construed as limiting.
Example 1
Degradation of used emulsion in bioreactor
Microbes capable of degrading emulsions of water-soluble metal working fluids were isolated from operationally exhausted metal working cutting fluids obtained from industrial workshop by enrichment technique. The isolated bacteria were evaluated for their ability to grow and assimilate various components of MWF as sole carbon source. Based on the growth ability, bacteria were combined in order to make suitable consortia/microbial blend. Biodegradation of used emulsion was examined in I5-Iit bioreactor (Applicon Bioconsole with controller ADI 1032) containing 8 lit of the operationally exhausted soluble cutting oil emulsion was obtained from industrial workshop (used for more than one month) and used with out dilution after amending with nutrients. The nutrient system contained yeast extract 0.2% (w.v), KNO3 ( 0.01%) and ammonium phosphate (.01%).The airflow within the bioreactor was maintained at 5 lit/min. Reactor temperature was maintained at 30°C using water heated jackets. At the timed interval, samples were withdrawn and solvent extractable total petroleum hydrocarbon (TPH)

was determined, gravimetrically. Figure 1 depicts the degradation of used emulsion after one month
Example 2
Degradation of used emulsion in soil
The biodegradation of the oil obtained from the breaking of used emulsion by acid treatment, as per the art described in prior art for the purpose. The oil portion was subjected to soil matrix using microbial consortia along with optimized nutrient (KN03 and yeast extract in 1: 1 (w/w)). The samples were collected and analyzed for reduction in TPH .The microbial blend could degrade more than 89% of the used emulsion in 6 months in soil (Figure 4).
While this invention has been described in detail with reference to certain preferred embodiments, it should be appreciated that the present invention is not limited to those precise embodiments rather, in view of the present disclosure, which describes the current best mode for practicing the invention, many modifications and variations, would present themselves to those skilled in the art without departing from the scope and spirit of this invention.

We Claim:
1. A bio-assisted method for disposal of emulsion generated from the
metalworking fluids, wherein the method comprises isolating and selecting effective strains of microbes from operationally exhausted emulsions by enrichment to form a microbial consortia and employing the same for degradation of emulsion, wherein said method is characterized in achieving 84-91% degradation of emulsion in in situ or ex situ conditions;
wherein said isolation is carried out by employing enrichment technique;
wherein said microbial consortia comprises any combination of the microbes selected from Pseudomonas aeruginosa IOC1, Rathayibacter rathayi IOC2 or Achromobacter xylosoxida IOC3, and each microbe in the combination can vary in a ratio ranging from 0.25 to 2.
2. The method as claimed in claim 1, wherein said degradation is performed in fresh emulsion or used emulsion.
3. The method as claimed in claim 2, wherein the percent degradation is 90-91% of fresh emulsion.
4. The method as claimed in claim 2, wherein the percent degradation is 84-89% of used emulsion.
5. The method as claimed in claim 1, wherein said method is carried out in a bioreactor or in a soil system.
6. The method as claimed in claim 1, the method comprising;
isolating and selecting a group of microbes to form the microbial consortia, which is capable of degrading metal working fluids and its components from emulsion of metal working fluids,
adding the resultant microbial consortia to a bioreactor having said metal working fluids,
optimizing the conditions in the bioreactor,
inoculating the bioreactor with said selective microbial consortia, and running the bioreactor to achieve desired degradation;

wherein said isolation is carried out by employing enrichment technique,
7. The method as claimed in claim 6, wherein the emulsion in use is 70-90% of the volume of the bioreactor, preferably 75%.
8. The method as claimed in claim 6, wherein the optimized temperature of bioreactor is 20-40°C, preferably 30 -35°C.
9. The method as claimed in claim 1, the method comprising;
isolating and selecting a group of microbes to form the microbial consortia, which is capable of degrading metal working fluids and its 'components from emulsion of metal working fluids,
binding the resultant microbial consortia with biodegradable carrier.
adding the resultant blend to soil-oil mixture,
providing optimized nutrients in the soil, and
periodic tilling and watering to enhance growth of said microbes to achieve
desired degradation;
wherein said isolation is carried out by employing enrichment technique.
10. The method as claimed in claim 9, wherein the soil-oil mixture comprises oil in
the range of 5-40%, preferably 15% (w/w) and is thoroughly mixed with the soil.
11. The method as claimed in claim 9, wherein said carrier is selected from cornhusk, sugar industry waste or any agricultural waste.
12. The method as claimed in claim 9, wherein nutrients are in the range of 100 mg to 1000 mg/100 kg of emulsion.
13. The method as claimed in claim 9, wherein the nutrient is preferably a mixture of yeast extract and potassium nitrate in 1:1 ratio.
14. The method as claimed in any of the claims 1, 6 or 9, wherein the enrichment is performed by employing controlled nutrient system and in presence of biocide.

15. The method as claimed in claim 14, wherein the nutrients are selected from nitrogen source and phosphorus source.
16. The method as claimed in claim 15, wherein the molar ratio of nitrogen to phosphorus source is 5:1 to 15:1.

Documents:

349-MUM-2006-ABSTRACT(24-8-2011).pdf

349-mum-2006-abstract(9-3-2007).pdf

349-mum-2006-claims(9-3-2007).pdf

349-MUM-2006-CLAIMS(AMENDED) (16-1-2012).pdf

349-MUM-2006-CLAIMS(AMENDED)-(24-8-2011).pdf

349-MUM-2006-CLAIMS(AMENDED)-(4-5-2012).pdf

349-MUM-2006-CLAIMS(MARKED COPY)-(4-5-2012).pdf

349-MUM-2006-CORRESPONDENCE(16-9-2009).pdf

349-MUM-2006-CORRESPONDENCE(4-5-2012).pdf

349-mum-2006-correspondence(9-3-2007).pdf

349-mum-2006-description (provisional).pdf

349-mum-2006-description(complete)-(9-3-2007).pdf

349-mum-2006-drawing(9-3-2007).pdf

349-mum-2006-drawings.pdf

349-mum-2006-form 1(28-3-2006).pdf

349-mum-2006-form 1(9-3-2007).pdf

349-MUM-2006-FORM 13(16-1-2012).pdf

349-MUM-2006-FORM 18(17-9-2009).pdf

349-mum-2006-form 2(complete)-(9-3-2007).pdf

349-mum-2006-form 2(title page)-(complete)-(9-3-2007).pdf

349-mum-2006-form 2(title page)-(provisional)-(10-3-2006).pdf

349-MUM-2006-FORM 26(4-5-2012).pdf

349-mum-2006-form 3(9-3-2007).pdf

349-mum-2006-form 5(9-3-2007).pdf

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349-mum-2006-form-5.pdf

349-MUM-2006-MARKED COPY(24-8-2011).pdf

349-MUM-2006-REPLY TO EXAMINATION REPORT(16-1-2012).pdf

349-MUM-2006-REPLY TO EXAMINATION REPORT(24-8-2011).pdf

349-MUM-2006-SPECIFICATION(AMENDED) (16-1-2012).pdf

349-MUM-2006-SPECIFICATION(AMENDED)-(24-8-2011).pdf

349-MUM-2006-SPECIFICATION(MARKED COPY) (16-1-2012).pdf

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Patent Number

253148

Indian Patent Application Number

349/MUM/2006

PG Journal Number

26/2012

Publication Date

29-Jun-2012

Grant Date

28-Jun-2012

Date of Filing

10-Mar-2006

Name of Patentee

INDIAN OIL CORPORATION LIMITED

Applicant Address

G-9, Ail Yavar Jung Marg, Bandra(E),Mumbai

Inventors:

#	Inventor's Name	Inventor's Address
1	UPRETI MANOJ KUMAR	Indian Oil Corporation Limited, Research and Develpoment Centre, Sector 13, Faridabad-121 007
2	SINGH M.P.	Indian Oil Corporation Limited, Research and Develpoment Centre, Sector 13, Faridabad-121 007
3	KAGDIYAL VIVEKANAND	Indian Oil Corporation Limited, Research and Develpoment Centre, Sector 13, Faridabad-121 007
4	KAUR HARINDER	Indian Oil Corporation Limited, Research and Develpoment Centre, Sector 13, Faridabad-121 007
5	SRIVASTAVA UMESH	Indian Oil Corporation Limited, Research and Develpoment Centre, Sector 13, Faridabad-121 007
6	BHATNAGER PANKAJ	Indian Oil Corporation Limited, Research and Develpoment Centre, Sector 13, Faridabad-121 007
7	MALHOTRA R.K.	Indian Oil Corporation Limited, Research and Develpoment Centre, Sector 13, Faridabad-121 007
8	VERMA R.P.	Indian Oil Corporation Limited, Research and Develpoment Centre, Sector 13, Faridabad-121 007

PCT International Classification Number

B01D35/10

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA