Title of Invention

"A PROCESS FOR REMOVAL OF TAR BALL POLLUTANTS USING THRAUSTOCHYTRID FUNGI"

Abstract

A process for removal of tar ball pollutants using a marine thraustochytrid fungi by contacting tar ball-polluted sediments with the thraustochytrid fungus inoculum being grown in a sea-water medium in situ and seawater salinity being in the range of 25-35 parts per thousand, pH of 7,0-8.5, at a temperature ranging 25-35°C pH being 7.0-8.5 at a temperature ranging 25-35°C for a period ranging 20-30 days, separating the said grown fungus and sediments/soil free from tar ball pollutants by extracting the said sediment grown culture with hexane to remove the residual tar ball. The process is useful for removal of tar ball contaminants present in marine sediments.

Full Text

This invention relates to a process for removal of tar ball pollutants using a marine thraustochytrid fungi. The invention particularly relates to a process for removal of tar ball pollutants in marine sediments using thraustochytrid fungi. Thraustochytrids are heterotrophic microorganisms and are, therefore, capable of utilising organic compounds in their environment including hydrocarbons for their nutrition, and convert these to their cell biomass and to carbon dioxide through respiration. By this mechanism, they are also capable of degrading and removing tar ball contaminants present in the sediments. Hydrocarbons are one of the major pollutants in the marine environment. Sources of this pollutant are crude oil, as well as products of their distillation, such as gasolines, heating fuels, diesels and heavy fuel oils. Natural crude oils consist of hundreds of fractions, broadly classified into (a) the saturates or aliphatic fraction, including paraffins (n-alkanes and branched alkanes) and naphthanes (cycloalkanes) , (b) the aromatic fraction and the asphaltenes. Hydrocarbon pollution in certain areas such as harbours and boatyards may be chronic because low amounts of hydrocarbons are constantly released into these environments,. Occasionally, coastal waters and sediments may be exposed to acute, catastrophic oil spills such as when a tanker accident takes place. Once introduced into the water, hydrocarbons undergo a rapid process of weathering and biodegradation, whereby the volatile aromatics and small molecular aliphatics are removed. More complex components of the hydrocarbons, in the form of tar balls will persist for a longer time and continue to induce toxic effects. Such tar balls consist of long chain aliphatics, complex polyaromatics and asphaltenes (Bartha, R. & Atlas. 1977. Advances in Applied Microbiology; 22: 225-266; Atlas, R.M. 1981. Microbiological Review 45: 180 - 209). Hydrocarbon pollution, including that by tar balls, severely afects the productivity and health of the ecosystem by harming the resident plants and animals. Tar balls also negatively affect the aesthetics of beaches. Several physical, chemical and microbiological methods have been attempted to mitigate hydrocarbon pollution. Physical methods involve the containment, skimming or burning of oil. Chemical methods include the dispersal and emulsification of oil using various chemicals. These physical and chemical methods have limitations for use. Physical methods require immediate action after an oil spill and are expensive. Chemical methods introduce substances into the ecosystem, which themselves may be toxic. Microbiological methods are based on the fact that a wide variety of microorganisms, particularly bacteria, fungi and cyanobacteria are important hydrocarbon degraders in the sea by heterotrophic mechanisms, as mentioned above (Leahy, J.H. & R.R. Colwell, 1990, Microbiological Review 54: 305-315). The most important hydrocrbon-degrading bacteria belong to Achromobacter, Acinetobacter. Alcaligenes. Arthrobacter. Bacillus. Flavobacterium. Nocardia and Pseudomonas spp. and the coryneforms. Species of Aureobasidium. Candida. Rhodotorula and Sporobolomyces areA the most common hydrocarbon-degrading organisms among fungi. Since individual hydrocarbon-degrading microbes may be selective in utilising only a few of the numerous fractions of crude oil, a more complete degradation of oil in the marine environment has been attempted with mixed populations of microbes (Atlas, R.M. 1991, Journal of Chemical Technology and Biotechnology 52: 149-156; Venkateswaran, K. et al., 1991, FEMS Microbiology and Ecology 86: 113-122; Prince, R.G. 1993, Critical Reviews in Microbiology 217-242). Two differnt approaches have been tried using microorganisms to remove hydrocarbon pollution. (1) Instead of introducing known species of hydrocarbon-degrading species, the already existing natural hydrocarbon-degrading flora are stimulated by introducing fertilisers into the environment. Thus (i) Patent No. 538408; Title: Bioremediation of contaminated groundwater; Date of sanction: 24.1.1995. (ii) Patent No. 5436160; Title: Bioremediation of hydrocarbon contaminated soil; Date of sanction: 25.7.1995. and (iii) Patent No. 5443845; Title: Composition for enhanced bioremediation of petroleum; Date of sanction: 22.8.1995. These patents have claimed processes which have attempted to introduce nutrients to soils to promote in situ bacterial removal of pollutants. However, these methods to stimulate hydrocarbon removal may result in an upset of natural ecological balance by introducing extraneous nutrients. In addition, these patents do not address removal of tar ball pollutants, which are difficult to degrade by microbial processes. (2) In addition, microbiological methods have also considered using 'microbial inocula , either as single species or as a consortium of several species, wherein artificial assemblages of oil-degrading microbial isolates are formulated and applied in the field to- mitigate pollution as given below. Single species or strains capable of pollutant removal in the environment have been used. Thus: (i) Patent No. 5395535; Title: Removal of hazardous chemical substances floating on water; Date of issue: 7.3.1995. In this patent, a product and a process are disclosed for the purpose of biodegrading organic chemical spills on water or land in situ. The product is a dried, macerated plant or vegetable material having a small oil or wax content enabling it to preferentially absorb oil in the presence of water. The product, specifically cotton gin trash, carries a microbial inoculum consisting of indigenous microbes which biodegrade the organic chemical, specifically petroleum hydrocarbons. The process consists of applying the macerated cotton gin trash to the surface of the hydrocarbon floating on water or covering the land. Upon.contact of the product with water, a dormant inoculum of microorganisms is revived. They increase in numbers because of food present in the product, biodegrading the chemical spill in situ. However, this method does not address tar balls and it also introduces extraneous nutrients into the environment; (ii) Patent No> 5413933; Title: Microorganisms and methods for degrading plant cell walls and complex hydrocarbons; Date of issue: 9.5.1995. This patent describes a method of degrading a complex hydrocarbon comprising contacting the complex hydrocarbon with a biologically pure culture of a marine amoeba for a time and under conditions sufficient to degrade the complex hydrocarbon. The said amoeba is a mutant of a multinucleated marine amoeba ATCC 0319, retaining the ability of ATCC 40319 to degrade hydrocarbon and halogen-substituted hydrocarbon chains. However, this patent does not address tar balls in marine sediments, (iii) Patent No. 5420035; Title: Bacteria isolated from amoebae/bacteria consortium; Date of issue: 30.5.1995. This patent describes a biologically pure strain of a microbe comprising a heterotrophic bacterium isolated from an amoeba/bacteria consortium, consisting of ATCC 40908 or a mutant of the said consortium possessing all the characteristics of the said consortium. The said bacterium selected from the group consisting of ATCC 75527, ATCC 75529, ATCC 55638, ATCC 55639 and ATCC 55640. However, this patent does not address tar balls in marine sediments. (iv) Patent No. 5427944; Title: Bioremediation of polycyclic aromatic hydrocarbon-contaminated soil; Date of issue: 27.6.95. This patent describes a process for biodegrading polycyclic aromatic hydrocarbon contaminants, comprising the steps of: contacting a mixed bacteria culture comprising Achromobacter sp. and Mycobacterlum sp. with a material containing polycyclic aromatic hydrocarbon contaminants in the presence of nitrogen and phosphorous containing nutrients, and oxygen, hydrogen peroxide or both the ratio of Achromobacter sp. to Mycobacterium sp., on a biomass basis, being from about 10:1 to about 1:10, the ratio of elemental nitrogen to phosphorous in said nutrients being from about 5:1 to about 15:1. However, this patent does not address tar balls in marine sediments. (v) Patent No, 5459065; Title: Process for the degradation of coal tar and its constituents by Phanerochaete chrysosporium. Date of issue: 17.10.95. This patent describes a process for degrading degradation-resistant organic pollutants comprising the steps of: a. combining at least one hydrocarbon having three or more fused rings selected from the group consisting of coal tar, constituents of coal tar, distillation fractions of coal tar, and polycyclic aromatic organic compounds capable of being derived from coal tar or constituents or distillation fractions thereof, with the lignin-degrading fungal enzymes expressed by Phanerochaete chrysosporium placed in contact with the pollutant, in the presence of hydrogen peroxide; and (b) allowing .degradation reaction to proceed until the pollutant is converted to less toxic degradation products. However, this is a terrestrial fungus and cannot be used under marine situations, where seawater salinity inhibits the growth of terrestrial species. Thus: (1) Suitable microbial species, which can be used in formulations have still not been developed for combating tar ball pollution; (2) Tar balls are recalcitrant to microbial degradation and little information on microorganisms that degrade tar balls is available. Therefore, the application of the right microorganism, namely one that is capable of degrading tar balls may be successfully utilised in removal of these pollutants from marine sediments. The main object of the present invention is to provide a process for the removal of tar ball contamination in marine sediments by using a group/of marine fungi, the thraustochytrids, which have the capability to degrade crude oil and tar balls and which can be applied to soils contaminated with tar balls, thus resulting in a decrease of the said pollutant. The novel feature of this invention is that it removes tar balls, which are difficult to degrade, from marine sediments with the help of a group of heterotrophic microorganisms and without addition of supplementary nutrients. This invention relates to removal of tar balls in marine sediments by using a group of fungi, the thraustochytrids. Thraustochytrids are heterotrophic, colourless, unicellular, marine organisms and are ubiquitous in the sea (Moss, ST. (ed.), 1986, Biology of Marine Fungi. Cambridge University Press; Porter, D. 1989, In Handbook of Protoctista, pp. 388-398, L. Margulis et al. (Eds.), Jones and Bartlett Publishers, Boston; Raghukumar, S. 1996, In Advances in Zoosporic fungi. R. Dayal (ed.), M.D. Publications Pvt Ltd., New Delhi). Although far less abundant than bacteria, they have nevertheles been isolated from art equal variety of habitats, including seawater and sediments, macrophytic detritus, live algae, animal guts and live animals. Thraustochytrids were classified earlier under the Kingdom Fungi. However, it is now known that thraustochytrids belong to the Kingdom (Profista and their phylogenetic affinities are uncertain. Cells of thraustochytrids are 2-20 microns in size and produce a network of ectoplasmic net elements which are believed to produce degradative enzymes and absorb nutrients. Tar ball degrading thraustochytrids, which can be used in this process, can be cultured from coastal or oceanic waters and sediments and both from polluted and unpolluted areas. However, in order to obtain cultures/capable of substantial degradation of tar balls, it is preferable to isolate them from coastal waters subjected to oil pollution. The standard method of pine pollen baiting can be adopted for isolation of thraustochytrid fungi. Various amounts of water or sediment sample are added to sterile seawater. This is then baited by adding a small amount of oven-sterilised pine pollen. Thraustochytrids grow on the pollen in about 3 days. Thraustochytrid cells can be recognised as colourless, globose to subglobose cells of 2 - 20 microns diameter, growing on the surfaces of the pine pollen grains. Often, fine ectoplasmic net elements grow out from the cells into the surrounding medium. Frequently also, swimming zoospores possessing two flagella can be made out in the culture. These cultures are made bacteria-free by inoculating onto seawater/pine pollen containing antibiotics (streptomycin and penicillin). Bacteria-free cultures are streaked onto nutrient agar plates to verify absence of bacteria. Colonies of thraustochytrids that appear on the plates are then transferred to sterile seawater/pine pollen in tubes and maintained therein. The capability of thraustochytrids to degrade hydrocarbons can be judged by using the MATH (Microbial Adhesion to Hydrocarbons) assay (Rosenberg, M., 1991, Critical Reviews in Microbiology 18: 159), as shown in Fig. 1, accompanying this specification. This is an indirect indication that a given thraustochytrid culture is capable of hydrocarbon degradation. Thraustochytrid isolates that can be used in this process can be further screened and selected for tar ball degradation using the following method. A small quantity of tar balls (collected from beach soils and stored until further use for experiments) is dissolved in hexane. This is then added to a suitable liquid medium, for example, peptone broth, to yield a 0.5 % concentration of tar balls. Inocula of thraustochytrid cultures for the experiment are raised in a nutrient broth and the cells harvested by centrifugation and resuspended in a small amount of sterile seawater. Aliquots of this can be used to inoculate the tar balls in peptone broth. Growth of thraustochytrids can be analysed by estimating total proteins. Thraustochytrids grow better in the presence of tar balls than in their absence, as shown in Fig.2 accompanying this specification. Quantitative and qualitative degradation of tar balls in the broth can be estimated using standard gravimetric and gas chromatographic analyses (Fig. 3). Marine sediments containing tar balls can be treated by inoculating the most suitable thraustochytrid cultures as selected from the above methods. Tar ball contamination in marine sediments can be contained by the following manner in the present invention. A suitable strain of thraustochytrids can be grown in large quantities in any suitable nutrient medium. -Preferably, inocula for this treatment can be obtained by growing the thraustochytrids in seawater/peptone broth containing autoclaved crude oil. Autoclaved crude oil supports excellent growth of thraustochytrids, as compared to unautoclayed oil. Inocula can also be obtained by growing thraustochytrids in any standard nutrient broth containing seawater. Cells of these can be collected by centrifugation or by filtration methods and used directly as inocula. Alternatively, it might be possible to use cells which have been lyophilised. The inoculum can be applied directly to the polluted sediments. No supplementary addition of nutrients is necessary. The thraustochytrids grow in inoculated. sediments and would degrade the tar ball components. For purposes of experimental demonstration, tar balls can be dissolved in a small quantity of hexane and mixed with a marine sediment sample. After addition of thraustochytrids to such a sediment, degradation of tar balls can be monitored periodically using gas chromatography. Long chain aliphatics above 20 carbon length are generally less degradable than those below 20 carbon by microorganisms. Interestingly, with thraustochytrids, aliphatics above 20 C are better utilised. In addition, no nutrient supplement to marine sediments is required in the case of thraustochytrids. Accordingly, the present invention provides a process for removal of tar ball pollutants using a marine thraustochytrid fungi which comprises contacting tar ball-polluted sediments with the thraustochytrid fungus inoculum being grown in a sea-water medium in situ and seawater salinity being in the range of 25-35 parts per thousand, pH of 7.0-8.5, at a temperature ranging 25-35°C and pH being 7.0-8.5 for a period ranging from 20-30 days, separating the said grown fungus and sediments/soil free from tar ball pollutants by extracting the said sediment grown culture with hexane to remove the residual tar ball. A process for removal of tar ball pollutants in marine sediments using thraustochytrid fungi, which comprises contacting tar ball-polluted sediments with the thraustochytrid fungus grown in a seawater medium, in situ seawater salinity of 25-35 parts per thousand, pH of 7,0 8.5, at ambient temperature, without adding any nutrients, separating the said fungus and sediments / soil free from tar ball pollutants by conventional methods. A process for removal of tar ball pollutants in marine sediments using thraustochytrid fungi. The invention is further illustrated with the following Examples which should not, however, be construed to limit the scope of the invention. Example-1 Three strains of a thraustochytrid, NIO Cultue #7, #16 and #17 were obtained from surface sediments of Mormugao harbour, 15 m below water surface by using the seawater/pine pollen method. About 1 g of sediment was suspended in 100 ml sterile seawater. Four dilutions of the suspension, namely, 1, 0.5, 0.25 and O.I ml of the suspension,.each with 4 replicates were added to sterile, flat-bottomed, wide-mouthed, screw-capped tubes (total of 16 tubes). The final volume in each tube was adjusted to 20 ml using sterile seawater. A small amount of pine pollen sterilised at 90-100°C for 48 h was added as a bait to aid growth. All tubes were microscopically examined after 1 week for growth of thraustbchytrids on pine pollen. Axenic cultures were obtained from the tubes by inoculating a loopfulof the initial cultures onto 5 ml.of sterile seawater containing 0,05 g streptomycin and 1000 units of penicillin. After 5 days, the bacteria-free cultures were streaked onto plates containing a standard nutrient agar with seawater. Bacteria-free colonies from the plates were then transferred to seawater/pine pollen in tubes and maintained therein. All three strains of the thraustochytrid obtained in this manner correspond to ulkenie radiata Gaertner. The cells of this thraustochytrid are up to 15 microns in diameter, globose and hyaline. Fine ectoplasmic net elements are produced by the thraus tochytrid. Before formation of motile - zoospores, the cell wall of the mature zoosporangium disappears and the naked contents divide -radially to form zoospores. The zoospores, which number about 12, develop 2 flagella -each and swim away. The strains were subcultured and maintained every 20 days. Example 2 A thraustochytrid, NIO Culture # 12 was obtained from decaying mangrove leaves from Chorao mangroves, Goa by using the seawater/pine pollen method. Small fragments of the decomposing leaves were suspended in about 5 ml of sterile seawater in flat-bottomed screw cap tubes. A small amount of pine pollen sterilised at 90-100°C for 48 h was added as a bait to aid growth. All tubes were microscopically examined after 1 week for growth of thraustochytrids on pine pollen. Axenic cultures were obtained from the tubes by inoculating a loopful of the initial cultures onto 5 ml of sterile seawater containing 0.05 g streptomycin and 1000 units of penicillin. After 5 days, the bacteria-free cultures were streaked onto plates containing a standard nutrient agar with seawater. Bacteria-free colonies from the plates were then transferred to seawater/pine pollen in tubes and maintained therein. The thraustochytrid corresponds to Schizochytrium mangrovei Raghukumar. The cells of this thraustochytrid are 7- 10 microns in diameter, globose and hyaline. Fine ectoplasmic net elements are produced by the thraustochytrid. The mature cell divides repeatedly by binary divisions and forms about 12 cells, each of which is transformed into a zoospore. The zoospores develop 2 flagella and swim away. The strains were subcultured and maintained every 20 days. Example 3: A thraustochytrid, NIO Culture # 21 was obtained-from surface waters of Dona Paula Jetty, Goa by using the seawater/pine pollen method. Different quantities of the water sample, namely, 16, 8, 4 and 2 ml were added to flat- bottomed screw-capped tubes. The final volume was adjusted to 16 ml using sterilised seawater in such a manner that 4 different dilutions of the seawater sample were present. Four replicates were maintained for each dilution, with a total of 6 tubes. A small amount of pine pollen sterilised at 90 - 100°C for 48 h was added as a bait to aid growth. All tubes were microscopically examined after 1 week for growth of thraustochytrids onpine pollen. Axenic cultures were obtained from the tubes by inoculating a loopfulof the initial cultures onto 5 ml of sterile seawater containing 0.05 g streptomycin and 1000 units of penicillin. After 5 days, the bacteria-free cultures were streaked onto plates containing a standard nutrient agar with seawater. Bacteria-free colonies from the plates were then transferred to seawater/pine pollen in tubes and maintained therein. The thraustochytrid obtained in this manner corresponds to the genus Schizochytrium Goldstein and Belsky. This species is characterised by cells which are about 7.5 microns in diameter, globose and hyaline. Fine ectoplasmic net elements are produced by the thraustochytrid. Repeated binary divisions of the cell result in a large cluster of cells. Each cell of the cluster divides to produce zoospores. The zoospores, which number about 16 per cell, develop 2 flagella and swim away. The culture was subcultured and maintained every 20 days. Example 4: The thraustochytrids NIO Cultures #7, 16 and 17 as isolated above were tested for growth on tar balls in a nutrient medium. A 100 mg quantity of tar ball was dissolved in 2 ml of hexane, which was then added to 20 ml of a seawater/peptone broth containing 0.01 % peptone. These flasks were inoculated only after 3 days to allow evaporation of hexane. Inocula of thraustochytrids were raised in Modified Vishniac's broth containing 0.05 % gelatin hydrolysate, 0.001 % liver extract, 0.01 % yeast extract, 0.1 % peptone, 0.4 % dextrose and 100 ml seawater. Cells were harvested by centrifugation and resuspended in sterile seawater. About 8 ml of this was added to 12 ml of the peptone broth in seawater to yield a final concentration of 0.1 % peptone. Growth was estimated after about 7 days by centrifuging the cells, resuspending in distilled water and by estimating total proteins by a suitable method, such as Lowry's. Addition of tar balls to the peptone broth promoted growth of the thraustochytrids up to about 100 %, as compared to unamended peptone broth (Fig. 2). Example 5: The thraustochytrid NIO Culture #12 isolated as above was tested for growth on tar balls in a nutrient medium. For this purpose, the same procedures were used as given in Example 4. Addition of tar balls to the peptone broth promoted growth of the thraustochytrid up to about 100 %, as compared to unamended peptone broth (Fig. 2). Example 6: The thraustochytrid NIO Culture #21 isolated as above was tested for growth on tar balls in a nutrient medium. For this purpose, the same procedures were used as given in Example 4. Addition of tar balls to the peptone broth promoted growth of the thraustochytrid up to about 30 %, as compared-~to unamended peptone broth (Fig. 2). Example 7: The thraustochytrids, NIO Culture #7, #16 and #17 as described in Examples 1 and 4 were tested for removal of tar balls added to 0.1 % peptone broth. Tar balls were added to the peptone broth using the same methods described in Example 5. Following growth for a week, the amount of tar balls present in the culture medium was determined gravimetrically and by gas chromatography. The thraustochytrids removed up to 30 % of tar balls present in the medium during this period (Fig. 3). Example 8: The thraustochytrid, NIO Culture #12, as described in Examples 2 and 5 was tested for removal of tar balls added to peptone broth medium. For this purpose, the same method as given in Example 7 was used. The thraustochytrid removed up to 30 % of tar balls present in the nutrient medium during this period (Fig. 3). Example 9: The thraustochytrid, NIO Culture #21, as described in Examples 3 and 6 was tested for removal of tar balls added to peptone broth medium. For this purpose, the same method as given in Example 7 was used. The thraustochytrid removed up to 20 % of tar balls present in the nutrient medium during this period (Fig. 3). Example 10: Three isolates of thraustochytrids , #29, #66 and #67 were isolated from boatyards and jetties along Goa. Removal of tar balls added to sediments by these thraustochytrids was examined. For this purpose, a known weight of intertidal sediment (clay-sand) from Dona Paula beach, Goa was autoelaved in 5 cm acid-washed Petri dishes. A 2 ml of hexane containing 100 mg of tar balls was mixed with this. The dishes were left at 45°C overnight to allow the hexane to evaporate. The thraustochytrids were grown as described in Example 4. The plates containing sediments polluted with tar balls were inoculated with 1 ml cell suspension of the thraustochytrids in seawater, harvested from MV broth mentioned above, after harvesting and rinsing the cells in sterile seawater. After a period of 1 month, the sediment was placed in 100 ml Erlenmeyer flasks and the tar balls extracted thrice in 5 ml of double-distilled hexane, for 1 h each on a shaker. The extracts were pooled, a pinch of sodium sulphite added to remove moisture and filtered through a glassfibre filter. Fluorescence was read using an F-2000 fluorescence spectrophotometer with an excitation at 310 nm and emission at 360 nm. The values were read against standard curves prepared for various concentrations of Bombay High crude oil. The thraustochytrid removed up to 71 % of tar balls, extractive from sediments by using hexane during this period (Table 1). Organic nitrogen present in the sediment was simultaneously analysed. This showed that concomitant to growth of thraustochytrids in the sediments, as confirmed by microscopic examination, and removal of tar balls, the thraustochytrids removed and utilised the organic nitrogen present in the sediments for their growth (Table 1) Table 1. Degradation of tar balls in sediment after 1 month (Table Removed) Example-11 The aliphatic hydrocarbon fraction of the tar balls that is removed by the thraustochytrid NIO Culture #16 was examined using gas chromatography. The thraustochytrid culture was grown and inoculated onto a nutrient medium containing tar balls, as described in Examples 4. The thraustochytrid preferentially removed aliphatic fractions above 20°C chains in length (Fig. 4). There are several advantages in the present invention. Thraustochytrids can be easily mass-cultivated in fermentors. This makes it easy to obtain large amounts of cell material for bioremedial formulations and application. The cells can be used fresh or in a lyophilised form. The capability of thraustochytrids to degrade substantial amounts of tar balls is a distinqt advantage. Fractions of tar balls that are more refractory to bacterial degradation may be removed better by thraustochytrids. An added advantage is the fact that the sediments do not have to be treated with supplementary nutrients to aid the growth of thraustochytrids. We claim : 1. A process for removal of tar ball pollutants using a marine thraustochytrid fungi which comprises contacting tar ball-polluted sediments with the thraustochytrid fungus inoculum being grown in a sea-water medium in situ and seawater salinity being in the range of 25-35 parts per thousand, pH of 7.0-8.5, at a temperature ranging 25-35°C and pH being 7.0-8.5 for a period ranging from 20-30 days, separating the said grown fungus and sediments/soil free from tar ball pollutants by extracting the said sediment grown culture with hexane to remove the residual tar ball. 2. A process as claimed in claim 1 wherein the contacting is effected for 20- 30 days. 3. A process as claimed in claim 1 wherein tar ball pollutant used is native and not supplemented with any nutrients. 4. A process for removal of tar ball pollutants using a marine thraustochytrid fungi substantially as herein described with reference to the examples and accompanying the specification.

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