Title of Invention

"A BIOTECHNOLOGICAL METHOD FOR ENHANCING CRUDE OIL DEGRADATION"

Abstract

The present invention provides a biotechnological process for enhancing the crude oil degradation using a bacterial consortium, selected from a group of Pseudomonas strains and comprising of Pseudomonas strain NCC DSS8, Pseudomonas strain NCC DSS6, Pseudomonas strain NCC GSS3 and Pseudomonas putida comprising cultivating the individual member of the consortium in a mineral medium containing catechol as a sole carbon source followed by mixing the said bacterial culture grown individually in equal proportions with the environmental matrix of the kind such as herein described having crude oil or any other hydrocarbon to facilitate degradation of hydrocarbons from the said environment.

Full Text

This invention relates to a biotechnological method for enhancing crude oil degradation using a formulated bacterial consortium. More particularly, this invention relates to a method for using a bacterial consortium containing Pseudomonas strains for enhancing the crude oil degradation, which finds application in oil spills/hydrocarbon remediation. Microorganisms are known to attack crude oil which is a complex mixture of different hydrocarbons. Hence, the effective biodegradation of crude oil can only be achieved by the simultaneous action of the desired metabolically versatile microbes to accommodate the mix substrate characteristics of the crude oil. in oil spills, the microbial population in the environment goes to a process of adaptation, which is followed by the selection of metabolically active oil degrading organisms. The same can be achieved with the reduced lag phase, by the bio-augmentation of the required metabolic capacity. The necessity for seeding with the complementary hydrocarbon degrading bacteria arises from the rationale that indigenous microbial populations may not be capable of degrading a wide range of potential substrates in a complex mixture such as crude oil. In this invention, the ability of the designed bacterial consortium with wide hydrocarbonodastic capacity has been employed for degradation of various fractions of crude oil. The designed consortium has members with desired physiological capabilities. In this study, three isolates (NCC.DSS6, NCC.DSS8 and NCC.GSS3) were selected along with a strain of P. putida to design a consortium. These genetically different isolates were selected on the basis of their substrate utilization range. In designing the consortium, a key aspect taken into consideration was the solubility and accessibility of the compounds in crude oil to microorganisms. Since only 0.02% of crude oil is water soluble there is a need for emulsification. Hence, an organism NCC.DSS6, capable of producing a rhamnolipid by utilizing short chain aliphatic like dodecane, was included in the consortium as a member. The other two isolates NCC.DSSa and NCC.GSS3 were selected due to their specialized metabolic potential of utilizing long chain aliphatic and aromatics, respectively. Pseudomonas putida known for its capability to consume wide range of hydrocarbons including various downstream metabolites formed in the degradation was chosen as a member of the consortium. The lag period required after the inoculation of consortia in a degradative condition was removed considerably by adapting the culture on catechol. The studies were carried out in shake flask as well as with the contaminated soil and the efficacy of the designed consortia was evaluated. In the prior art, the use of pure culture for bio degradation of hydrocarbon has been attempted and reported by the various researchers. Oil degradation by pure culture is normally a time consuming and preceded by the initial significant lag phase. The pure culture were also reported such as Acinetobacter calcoaceticus RAG-1; shown similar kind of extended lag phase for utilization of hydrocarbons. The lage phase could be between 4 to 15 days period with no oil degradation. Involving pure culture approach, the total time taken for the oil degradation may range from 5 to 15 weeks. With the pure culture maximum 40-50%. degradation has been reported (Fought, J. M. et al, Applied and Environmental Microbiology, vol 55, p. 36-42, 1989). The other approach widely tested is the use of nutrient amendments at the contaminated site for the bioremediation. The approach considers that the balance of nutrients, will allow the natural population to use contaminant as a source of carbon such as crude oil. A field scale experiment was carried out for the oil spills at Exxon valdez where the nutrient amendment protocol was tried out (Lindstorm, J.E. et al, Applied and Environmental Microbiology, vol 57, p. 2514-2522,1991). It has been reported that the fertilizer as a source of nitrogen and phosphorus addition resulted in higher crude oil utilization than on untreated reference plots. The significant levels up to 60% hydrocarbons such as hexadecane and phenanthrene were removed from the contaminated site after 80 days of the holding period. Over the course of the study in both the treated and untreated reference plots, the microbial populations of hetrotrophs and oil degraders increased to two orders of magnitude. The hydrocarbon removal in sea sediments and sand on the beaches were in the range of 50% degradation. In another study, a bioremediation treatment for cleanup of various kinds of soils contaminated with hydrocarbons were evaluated with an induction in the indigenous microbial population. The study sown that there was 65% hydrocarbons were removed from the soil after 18 weeks of incubation. The levels of hydrocarbons used, were 140mg/g of soil (Song, H., et al, Applied and Environmental Microbiology, vol 56, p. 652-656,1990). The main objective of the present invention is to provide a biotechnological method for enhancing the crude oil degradation using a formulated bacterial consortium. Figures The explanations of the enclosed figures are as follows: Fig. 1 (a) depicts that the GC for the control experiment where the crude oil with out biotreatment was extracted in hexane and analyzed for the various constituents of the crude oil. Fig. 1 (b-d) depicts that the GC for the experiment where the crude oil was biodegraded with un-adapted cultures; and was extracted in hexane and analyzed for the various constituents of the crude oil. Fig.1 (e) depicts that the GC profiles for the experiment where the crude oil biodegradation by individual adapted cultures; and the residual crude after five days of incubation was extracted in hexane and analyzed for the various constituents of the crude oil. Fig.2 (a) depicts that the surface tension reduction by the strain NCC.DSS6 and Fig.2 (b) depicts the IR spectra of biosurfactant produced by the strain NCC.DSS6. Fig.3 depicts that the capillary-gas chromatographs of the biodegradation of Bombay High Crude oil by the formulated bacterial consortia; wherein the residual crude oil was extracted after 3 days of incubation. Fig.4 depicts that the various fractions of crude oil separated by column- chromatography; after the biodegradation of Bombay High Crude oil by the formulated bacterial consortia; wherein the residual crude oil was extracted after 3 days of incubation. Fig.5 (a-b) depicts that the gas chromatographs of the biodegradation of the designed model petroleum mix by the formulated bacterial consortia in back cotton soil spiked with the crude oil, wherein the residual crude oil was extracted after 8 days of incubation. Fig.6 (a-b) depicts that the gas chromatographs of the biodegradation of the designed model petroleum mix by the formulated bacterial consortia in back cotton soil spiked with the crude oil, wherein the residual crude oil was extracted after 8 days of incubation. Fig.7 (a-c) depicts that the capillary-gas chromatographs of the biodegradation of Gulf High Crude oil by the formulated bacterial consortia in the soil sample from the refinery premises contaminated with the crude oil; wherein the role of bioavailability has been demonstrated and was compared with synthetic detergent Tween-80, the residual crude oil was extracted after 8 days of incubation. Table 1 describes that the different contaminants identified in the refinery soil used in the experiments described in the Fig.7 (a-c). Table 2 describes the composition of model petroleum mix used in the experiments described in Fig.6 (a-b). Table 3 describes the screening approach and analysis for screening the thirty-five isolates. Table 3 a) describes the growth characteristics of the organisms. Table 3 b) describes the antibiotic sensitivity pattern and also the preferential utilization and survival of organisms in diesel, petrol and gulf high crude as sole source of carbon. Table 3 c describes, the utilization of selected compound which are representative in crude oil degradation by microorganisms; and set the criteria for designing of microbial consortia. Accordingly Nthe present invention jxpvides a biotechttolodical method for" Accordingly, the present invention provides a biotechnological process for enhancing the crude oil degradation using a bacterial consortium, selected from a group of Pseudomonas strains and comprising of Pseudomonas strain NCC DSS8, Pseudomonas strain NCC DSS6, Pseudomonas strain NCC GSS3 and Pseudomonas putida comprising cultivating the individual member of the consortium in a mineral medium containing catechol as a sole carbon source followed by mixing the said bacterial culture grown individually in equal proportions with the environmental matrix of the kind such as herein described having crude oil or any other hydrocarbon to facilitate degradation of hydrocarbons from the said environment. In an embodiment of the invention, a bacterial formulation used for the oil degradation contain a defined mix of at least four identified Pseudomonas strains. In another embodiment of the invention, the identified Pseudomonas strains are present in equal proportions. In yet another embodiment of the invention, degradation of hydrocarbons is achieved by the adaptation of the Pseudomonas strains by growing them in a medium with catechol as sole source of carbon. In a further embodiment in the invention, the bioavailability of hydrocarbon is enhanced by the use of Pseudomonas strains NCC DSS6 which provides the bioemulsifier in the medium containing soil, water and combination thereof contaminated with hydrocarbon.In further embodiment in the invention, the four Pseudomonas strains forming a bacterial consortium can tolerate 1% crude oil without loosing the degradative capacity. In yet another embodiment of the invention, the identified Pseudomonas strains from the bacterial formulation are adapted by growing the individual strain on 25-50ppm of catechol for enhancing the oil degrading capacity. In a further embodiment of the invention, the use of Pseudomonas strains NCC DSS6 helps in the subtained emulsification of hydrocarbon which helps in activating the growth of the other oil degrading Pseudomonas strains used in the consortia. In a another embodiment in invention, the matrix used for the oil degradation can be selected from soil, oil spills, refinery waste, effluents contaminated with hydrocarbons, waste generated in oil cleaning and drilling areas and combination there of. In a further embodiment, the enhancement of oil degrading capacity has reduced the time required for more than 60% hydrocarbon removal from 5 weeks to 3 days. In an another embodiment, the bacterial consortium used in the said method has degradative capacity for crude oil as a source of a carbon source irrespective of its origin viz. Bombay High crude or Gulf High crude; representing light and heavy crude respectively. The bacterial formulation was developed from the selected microorganisms from a library of oil degrading microorganisms. A semi continuous crude oil 5L batch fed reactor was used to enrich the crude oil degrading bacteria. The different soil and biological treatment plant samples from oil refinery premises were used as inoculum for the reactor. The reactor was fed once a day with 1% crude oil and 0.05% potassium phosphate. It stabilized with 60% efficiency for crude oil degradation in a period of 5 months. The activated sludge was used to isolate microorganisms and screening gave thirty-five genotypically different microorganisms. The organisms were characterized morphologically on the basis of gram staining, colony characteristics, incubation time, and optimum growth temperature. The antibiotic sensitivity patterns using four different antibiotics were developed, for differentiating the oil degrading bacteria, at extrachromosomal DMA levels. This screening is able to distinguish the selectively evolved strain even from the same population. The organisms were further characterized for their hydrocarbon utilization pattern. The crude oil degradation was done by designing a consortium having different bacterial isolates viz. NCC.DSS6, NCC.DSS8 and NCC.GSS3 and Pseudomonas putida. (The strains have been deposiedat NEERI Culture Collection Centre, Nagpur) Seed culture inoculum was prepared by adapting all the four members of consortium in Bushnell Haas minimal mineral medium ( BH medium ) containing (g/L): NH4NO3,. 1.0; CaCI2, 0.002; MgS04.7H20, 0.02; FeCI3.6H2O, 0.05; KH2P04, 1.0; pH 7.0±0.2, amended with catechol (200mg/L) as the sole source of carbon. After 48 hours the cells were pelleted, washed and inoculated in equal ratio into 100 ml of the BH medium with 1% crude oil i.e. Bombay High crude ( BHC ) oil to give the final optical density of 0.01 /ml at 620 nm. The culture flasks were incubated at 200 rpm on a rotary shaker at 30°C for 2 to 8 days. Biodegradation analysis was carried out by packed column gas chromatographic analysis or capillary column gas chromatographic analysts or fractionation of crude oil using silica gel columns. The biosurfactant production capacity for NCC.DSS6 was characterized by analysing the reduction in surface tension of the defined medium containing (g/L): glucose 10; NaN03, 1.4; MgSO4.7H2O, 0,220; KCI, 0.550; CaCl2.2H2O, 0.0275; FeCI3.6H2O 0.270; which was deficient in phosphates (45 mg/L KH2 PO4). The degradation capacity of the consortia and individual members of the consortia was assessed and exploited in shake flask cultures and soil contaminated with crude oil / hydrocarbons. This invention is described through the following examples, which are only illustrative in the nature and should not be construed to limit the scope of invention. Example 1 The selected members of the consortium in the beginning were tried individually in adapted and non-adapted conditions for the assessment of their degradative capacity. The culture were grown in nutrient rich medium and also adapted in BH medium for 48h with 100ppm of catechol. The culture grown from both the conditions were inoculated with 1.0 OD/ml at 620nm in BH medium containing crude oil as sole source of carbon. After 5 days of incubation the crude oil was extracted and analyzed using packed column gas chromatography as shown in Fig 1(a) - control, Ftg1(b), 1(c) and 1(d) the members of consortia were grown on only nutrient rich medium and inoculated for degradation studies, Figl(e). shows degradation pattern of catechol adapted cultures. The data supports that the degradative capacity can be enhanced by using the catechol adaptation in consortia members. Example 2 One of the isolates, NCC.DSS6 was found to produce biosurfactant as it lowered the surface tension of liquid media containing glucose as carbon source, to 37 dynes/cm (Fig.2(a)). The surfactant was extracted from cell free broth and subjected to IR analysis ( Fig.2(b)) which demonstrated close resemblance to the reported IR spectra of rhamnolipid produced by Pseudomonas aeruginosa. Example 3 On the basis of the degradative capacity described in the example 1, three isolates were selected for designing the consortium along with Pseudomonas putida. The isolate, NCC.DSS6 was selected on the basis of its capability to produce biosurfactant as described in example 2 and effective emulsification of crude oil thus making it bio-available to other members of consortium. The other two members NCC.DSS8 and NCC.GSS3 were found to be effective in consuming long chain aliphatic and aromatics, respectively. The designed consortium could degrade 65 to 70% of BHC in 72 hours as evident from gas chromatography data (Fig.3). The observations made in the present study suggest that the use of preconditioned bacterial consortium is a better approach over the use of pure culture as shown in example 1 for remediating oil spills as it offers effective degradation of various components of crude oil. Even the relatively recalcitrant aromatic fraction shows substantial reduction. The biosurfactant produced by a member of consortium plays a crucial rote in the clean up process as it makes the insoluble components of crude oil miscible in the water and available for metabolism. Such designed consortium not only can have applications in clean up of OH spills but also in the 'seeding1 of biotreatment processes such as petrochemical wastewaters. Example 4 In this report the ability of the designed bacterial consortium with wide hydrocarbonoclastic capacity has been employed for degradation of various fractions of crude oil. The desired physiological capabilities of the designed consortium was tested by monitoring the degradation of the different fractions of crude oil. After the desired incubation period, the samples were prepared by extracting the residual crude oil in hexane followed by concentrating on rotatory flash evaporators. The residual oil was suspended in 2 ml chloroform and adsorbed on 2 g of silica gel. The adsorbed oil was fractionated using a 30 x 15 mm glass column packed with activated silica gel. A layer of anhydrous sodium sulfate was placed over the sample to adsorb any water and minimize the interference of solvent with crude oil. The saturated, aromatic and asphaltene fractions of crude oil were eluted with 120 ml of hexane, benzene and chloroform: methanol (1:1, v/v), respectively. The remaining crude oil, after degradation was also subjected to column chromatography. Column chromatography, followed by gravimetric analysis indicated 81% and 65% degradation of the aliphatic and aromatic fractions after 72 hours ( Fig.4 ). However, no reduction was detected in the asphalt fraction of crude oil (Fig.4). Example 5 5 g. of black cotton soil sample were taken in glass tubes for experimentation, and autoclaved three times. Sterile soil was spiked with 100//I of Bombay high crude dissolved in 1ml chloroform which facilitated mixing of hydrocarbons in soil. After adding chloroform, the soil was mixed thoroughly. Solvent was allowed to evaporate prior to inoculation. Consortium members, individually grown on catechol, were inoculated to give the final optical density(A620) of 1.0 suspended in 100µl / g of soil. After inoculation, the culture tubes were incubated at 30°C in the dark. After 8 days, the oil was extracted with hexane (1:1 v/wt) and subjected to gas chromatography. The removal of hydrocarbons could only be attributed to the catabolic capabilities of designed consortium as bioaugmentation was carried out after sterilization. There was significant reduction in hydrocarbon peaks has been observed after chromatographic analysis of the samples as shown in Fig4. Example 6 The soil experiment with same setup as described in example 5 was repeated with designed model hydrocarbon mixture as shown in Table 1. After 8 days of incubation the soil sample was extracted and analyzed as shown in Fig 5. Pristane was used as internal standard. The data suggest as observed in example 5, similarly, there was utilization of all the constituents of the model hydrocarbon mixture as shown in Fig 5. It has been observed that with model hydrocarbon mix as carbon source dodecane (C12) and naphthalene were degraded more than 90% while pentadecane (C15) and hexadecane (C16) degradation was 60%. Phenanthrene (tricyclic), dibenzothiophene (S containing heterocyclic), eicosane (C20), tetracosane (C24) and octacosane (C28) were relatively less biodegradable (30-45%). Example 7 The role of culture NCC.DSS6 was further studied in facilitating the bio-avialability of the hydrocarbons. Soil collected from a refinery dump site was selected to study the efficacy of designed bacterial consortium in natural conditions. The soil contained various hydrocarbons of saturate and aromatic nature as well as organic compounds of plant origin. Some of the compounds have been identified by GC-MS and listed in Table 2. The microcosm experiments conducted with soil demonstrated that consortium effectively reduced contamination level (Fig.6 ).The biosurfactant producing organism, NCC.DSS6, played a key role in bioremediation of contaminated soil. Without NCC.DSS6, the consortium could not attack hydrocarbons even though a chemical detergent, Tween-80 was provided ( Fig.6c ). Tween-80 itself or its biodegradation products could have inhibited the growth of consortium members or the surfactant could have been used as preferential substrate or on the other hand the mobilization of high concentration of hydrocarbons could have proven toxic to the micro flora. The bioavailability mediated via NCC.DSS6 is at the cost of contaminating hydrocarbons ( Fig6b ). This results in sustained release of hydrocarbons from soil. Hence, at a given point of time, the hydrocarbon level would be well below the toxic level for consortium. This scenario provides a balanced availability of carbon source in favor of reduction of contamination levels in either of the conditions. In this invention, the ability of the formulated bacterial consortium with the desired physiological capacity has been employed for the degradation of various fractions of crude oil. In this study, three isolates (NCC.DSS6, NCC.DSS8 and NCC.GSS3) were selected along with a strain of P. putida to design a consortium. These genetically different isolates were used to formulate the consortia because of their wide hydrocarbon substrate utilization range. In designing the consortium, an important aspect taken into consideration was the solubility and accessibility of the compounds in crude oil to microorganisms. Since only 0.02% of crude oil is water soluble there is a need for em unification. Hence, an organism NCC.DSS6, capable of producing a rhamnolipid by utilizing short chain aliphatic like dodecane, was include in the consortium as a member. The other two isolates NC.DSS8 and NCC.GSS3 were selected due to their specialized metabolic potential of utilizing long chain aliphatic and aromatics, respectively. Pseudomonas putida known for its capability to consume wide range of hydrocarbons including various downstream metabolites formed in the degradation was chosen as a member of the consortium. The lag period required after the inoculation of consortia in a degradative condition was removed considerably by adapting the culture on catechol. The studies were carried out in shake flask as well as with the contaminated soil and the efficacy of the designed consortia was evaluated. ADVANTAGES: 1. The invention uses the defined mix of bacteria; hence, it is easy to monitor on release in the environment. 2. The emulsifying capacity of the consortia enhances the bioavailability of the hydrocarbons for the degradation. 3. The catechol adaptation reduces the lag phase in bioremediation of hydrocarbons in different types of matrix such as water, soil or combinations. 4. The mix culture can be used under bioaugmentation protocol in decontamination of hydrocarbons. 5. In the present invention, the enhancement of oil degrading capacity has reduced the time required for more than 60% hydrocarbon removal from 5 weeks to 3 days. Table-1: Composition of Model Petroleum* (Table Removed) * Pristane was used as internal standard (1.5 ml), the total volume of above standard mixture was 6.37 ml Table-2: GC Profile of Contaminants Present in Refinery Soil (Table Removed) 'Identified by GC-MS (Varians, Saturn-3) on DB-5 column Table 3 a: Primary Screening of the Isolates (Table Removed) Table 3 b: Secondary Screening of the Isolates (Table Removed) All isolates were found to be resistant to Amplcillin (10µg/disc) and Penicillin-G (10µg/disc) * a Table 3 c: Tertiary Screening of the Isolates (Table Removed) Growth/Utilization monitored in terms of absorbance at 620 nm +: O.D.0.05-0.20; ++: O.D. 0.21-0.40; +++ :O.D. > 0.40 We Claim: 1. A biotechnological process for enhancing the crude oil degradation using a bacterial consortium, selected from a group of Pseudomonas strains and comprising of Pseudomonas strain NCC DSS8, Pseudomonas strain NCC DSS6, Pseudomonas strain NCC GSS3 and Pseudomonas putida comprising cultivating the individual member of the consortium in a mineral medium containing catechol as a sole carbon source followed by mixing the said bacterial culture grown individually in equal proportions with the environmental matrix of the kind such as herein described having crude oil or any other hydrocarbon to facilitate degradation of hydrocarbons from the said environment. 2. A method as claimed in claim 1, wherein the environmental matrix used may be selected from soil, contaminated soil, waste water generated at refinery premises, oil spills leading to contamination in any environmental niches. 3. A method as claimed in claims 1 and 2, wherein the bio-availability is facilitated through culture of Pseudomonas strain NCC.DSS6 by production of surfactant in medium selected from soil or water or combinations thereof contaminated with hydrocarbons. 4. A method as claimed in claims 1 to 3, wherein the selected strains are adapted by growing the culture on catechol for obtaining increased degradation rates. 5. A method as claimed in claims 1 to 4, wherein oil spills are remediated from a contaminated medium by making hydrocarbons bio-available via culture of Pseudomonas strain NCC.DSS6 and affecting the remediation subsequently with other members of consortia by reducing the surface tension favorable for emulsification of hydrocarbon. 6. A method as claimed in claims 1 to 5, wherein the members of the bacterial consortium are isolated from different soil and biological treatment plant samples from oil refinery premises. 7. A biotechnological process for enhancing crude oil degradation substantially as hereinbefore described with reference to the foregoing examples and figures.

Documents:

2878-del-1998-abstract.pdf

2878-del-1998-claims.pdf

2878-del-1998-correspondence-others.pdf

2878-del-1998-correspondence-po.pdf

2878-del-1998-desciption (complete).pdf

2878-del-1998-drawings.pdf

2878-del-1998-form-1.pdf

2878-del-1998-form-19.pdf

2878-del-1998-form-2.pdf

2878-del-1998-form-3.pdf