Title of Invention

"A METHOD FOR REMOVAL OF OIL POLLUTANTS FROM SEAWATER USING CYANOBACTERIA"

Abstract

A method for removal of oil pollutants from seawater using cyanobacteria by cultivating cyanobacteria such as Oscillatoria salina, Plectonema terebrans and Aphanocapsa sp. in a conventional seawater medium having salinity in the range of 25-35 parts per thousand, pH between 7.5 to 8.2 at room temperature upto 15 days, then contacting with oil pollutant in seashore oil polluted land or marine sediments *o get sediment or seashore land free of oil by known methods.

Full Text

This invention relates to a method for removal of oil pollutants from seawater using cyanobacteria. This invention particularly relates to a process for removal of crude oil pollutants in marina sediments of seashore land using cyanobacter ia. Cyanobacter ia are photosynthet ic microorganisms capable of growing in their native environment without addition of supplementary nutrients. T.hey only require light and the available micronutrients in their environment. Cyanobacter ia form an important component of the microbial community in marine sediments. They degrade crude oil as measures of detoxifications of their microenv ironment. By this method they are capable of removing oil and mitigate pollution. The seashore environment experiences pollution by crude oil through various sources. Natural crude oil consists of hundreds of fractions, broadly classified into (a) the saturates or aliphatic fraction, including paraffins (n-alkanes and branched alkanes) and naphthanes ( eye 1 oal kanes ) , (b) the aromatic fraction and (c) the asphaltenes. Hydrocarbon pollution becomes chronic in beaches in the vicinity of harbours and jetties and severely affects the plants and animals present in that ecosystem. Crude oil spill is relatively protected from wind and wave action in seashore sediments, unlike in open oceanic waters. Therefore, it is not dispersed easily and does not rapidly undergo natural weathering processes. Hydrocarbon pollution is combated using various physical, chemical and microbiological methods. Physical methods involve 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 is 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 yeasts are known to be important hydrocarbon degraders in the sea (Leah, J.J. and R.R. Colwell, 1990, Microbiol Rev. 54: 305-315). The most important hydrocarbon-degrading bacteria belong to "Achromobacter, Acinetobacter, Alcaligenes, Arthrobacter', "^Bacillus' , 'Flavobacterium' , "Nocaradia' and "Pseudomonas' spp. and the coryneforms. Species of Aureobasidium, Candida, Rhodotorula, and Sporobolomyces spp. are the most common hydrocarbon-degrading organisms among fungi. Hydrocarbon-degrading microbes are selective in utilising a few of the numerous fractions of crude oil. A more complete degradation of oil in the marine environment is possible with mixed populations of native microbes (Atlas, R.M.1991, J. Chem. Tech. Biotechnol. 52: 149-156. Venkateswaran K. et al.., 1991, FEMS Microbiology Ecology, 86: 113-122.). Two different approaches have been tried using microorganisms to remove hydrocarbon pollution. (1) Hydrocarbon-degrading activities of native mixed population of microorganisms are stimulated by introducing fertilisers into the environment. Thus: (i) the US Patent No. 5384048 "Bioremediation of contaminated groundwater" (issue date: 24.1.1995) describes method to encourage growth of natural aerobic microbes by introducing nutrient fluid. (ii) the US Patent No. 5436160 "Bioremediation of hydrocarbon contaminated soil" (issue date: 25.7.1995) describes injection of surfactants to promote growth of indigenous soil microorganisms, and (iii)the US Patent No. 5443845 /'Composition for enhanced bioremediation of petroleum" (issue date 22.8.1995) claims promoting growth of hydrocarbon degrading microorganisms by introducing external nutrient solution. However these methods to stimulate hydrocarbon removal may upset natural ecological balance by introducing extraneous nutrients. Besides, these do not address marine sediments for which microbes adapted to that habitat will be required. (2) In addition, microbiologists 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. Single species or strains capable of pollutant removal in the environment have been used. Thus: (i) U.S. Patent No. 5395535; Title: Removal of hazardous chemical subtances floating on water; Date of issue: 7.3.1995. In this patent, a product and a process is 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 hyrocarbons. 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, dormant inoculum of microorganisms are revived, They increase in numbers because of food present in the product, biodegrading the chemical spill in situ. However, this method also introduces extraneous nutrients into thr inviroment; (ii)U.S. Patent No 5413933;Title: Microorganisms and methods or degrading plant cell walls and complex hydrocarbons; Date of issue: 9.5.1995. This patent describes a method of degrading a 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 40319, retaining the ability of ACC 40319 to degrade hydrocarbon and halogen-substituted hydrocarbon chains. However, this patent does not address oil in marine sediments. (iii)U.S. 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 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, ATC 55639 and ATCC 55640. However, this patent uses heterotrophic bacteria in marine sediments. iv) U.S Patent No. 5427944; Title: Bioremediation of polycyclic aromatic hydrocarbon-contaminated soil; Date of issue: 27.6.1995. This patent describes a process for biodegradation of polycyclic aromatic hydrocarbon contaminants, comprising the steps of: contacting a mixed bacteria culture comprising of Achromobacter sp and Mycobacterium 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 describes enrichment of marine sediments with nutrients. v) U.S.Patent No. 5459065; Title: Process for the degradation of coal tar and its constituents by Phanerochaete chrysosporium. Date of issue: 17.10.95. The use of a single microorganism to remove tar balls has been addressed in this patent. 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 fraction thereof, with the lignin-degrading fungal enzymes expressed by Phanerchaete 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. The main objective of the present invention is to provide a process for the removal of crude oil pollutants using cyanobacte-ia using cyanobacteria. These can be applied to soils and waters contaminated with crude oil and result in breakdown of long chains of hydrocarbon in shorter less toxic compounds. This results in a decrease of the said pollutants in the intertidal marine sediments. The novel feature of this invention is that it avoids the use of organic fertilizers to stimulate growth of native oil degrading cyanobacteria or blue-green algae. It uses an alternative method of applying native photosynthetic organisms adapted to presence of oil and capable of hydrocarbon degradation, as described herein. These do not need any external source of organic carbon for growth, but at the same time will degrade hydrocarbons. Cyanobacteria are important members of the microbial community in the seashore ecosystem. They are therefore, naturally adapted to growing in marine conditions. The present invention described a method for the removal of oil containing aliphatic, aromatic and other fractions using pure cultures of marine filamentous cyanobacteria such as Oscillatoria salina, Plectonema terebrans and unicellular colonial cyanobacterial culture such as Aphanocapsa sp. In addition, this invention also describes efficient degradation of fractionated aliphatic fraction of crude oil by the above mentioned three species of cyanobacteria. Cyanobacteria are prokaryotic phototrophic organisms. Being phototrophic, they use light, water and atmospheric carbon dioxide to fix energy in the form of reduced carbon. They are commonly called blue-green algae. They possese the green pigment chlorophyll as in the green algae besides the blue pigment phycocyanin which actively take part in photosynthesis. They are prokaryotic and lack a true nucleus as with bacteria. For these reasons therefore , they are more aptly called cyanobacteria. They are ubiquitous in their distribution, occurring from ice fields in the Antactic to flooded rice fields in the tropics. The cyanobacterial cultures for this work were obtained from the National facilty for marine cyanobacteria at Bharathidasan university, Trichy, Tamil Nadu. They are Oscillatoria salina (strain 3 # BDU 20631), Plectonema terebrans (strain # BDU 91211) and Aphanocapsa sp (strain # BDU 50261). The filamentous cyanobacterium O.salina having long twining trichomes with l0um diameter are blue-green in color. This species can be recognised easily by the tapering ends of its trichomes oscillating when mounted in water on a microscope slide. The filamentous cyanobacterium P.terebrans has trichomes of around lOum diameter and the culture appears blackish red. The red coloration is due to the presence of the water soluble pigment phycoerythrin. The species of Aphanocapsa has bright blue-green ovoid cells which are scatttered throughout gelatinous matrix. Cyanobacteria can be isolated from marine sediments, calcareous shells and corals. They can be grown in artificial seawater medium. The medium can contain macronutrients like sodium chloride, magnesium chloride, potassium chloride and micronutrients like sodium nitrate, potassium phosphate, magnesium sulfate and the like. It is advisable to add a trace metal mix, EDTA and ferric ammonium citrate for better growth. The pH of the medium can be adjusted to 7.5. The cyanobacteria can be grown in the liquid medium for 10-15 days in natural light near window at room temperature (30 C) as stationary cultures. They can also be grown in artificial light under fluorescent tubes. Cultures grown as above can be tested for degradation of crude oil. An inoculum can be prepared by decanting the culture medium, washing the cultures once with sterile seawater and suspending in filter sterilised seawater. The filamentous forms can be broken down mechanically into a homogenous suspension. The inoculum can be added to 50ml of synthetic seawater medium or filter sterilised seawater as the case may be, in a flask. The salinity of the medium can be 25-35 parts per thousand and the pH can be 7.5-8.2, as in natural seawater. The culture can be o allowed to grow at 25-30 C. Crude oil can be added to this culture and incubated. Two types of control can be maintained: a) one containing oil in filter sterilised seawater without inoculum b) one containing oil and filter sterilised seawater and killed culture. Purity of cultures can be checked during the experiments by inoculating a loopful of culture medium in peptone glucose water. After incubation for 24h, turbid medium indicates bacterial contamination and such experiments are to be repeated with fresh bacteria-free cultures. For gravimetric analysis of degradation, the entire content of the culture flask can be extracted twice with analytical grade carbon tetrachloride (CCl ), the pooled extract can be taken in a preweighed glass vial/evaporated to dryness in a dessicator and weighed in a precision balance. The difference in weight between uninoculated control and inoculated flask can be calculated as a loss in weight due to degradation. The difference in weight between initial cyanobacterial inoculum and the final can be taken as the biomass of the experimental culture in the presence of oil. An aliquot of CCI , extract can be injected into capillary column for analysis of degradation of various carbon chains by gas chromatography. The extract from uninoculated control and killed cultures serve as controls. The invention describes removal of crude oil using three species of cyanobacteria. These are the filamentous forms Oscillatoria salina, Plectonema terebrans and unicellular colonial from Aphanocapsa sp. In addition, they also degrade aliphatic fraction of the crude oil and pure compound Hexadecan. They can be used for field trials as homogeneous suspension. In this invention, a process is described wherein marine sediments contaminated with crude oil can be treated in the following manner. Cyanobacteria can be cultivated in defined synthetic media in large quantities. The cells can be homogenised using any conventional homogeniser and inoculum can be prepared. This inoculum can be directly added to polluted marine sediments. Alternatively, it might be possible to lyophilise the inoculum and use it subsequently. No additional nutrients need to be added. The photosynthetic cyanobacteria, which are native to marine sediments grow autotrophically and simultaneously can degrade the crude oil components in the sediments, thereby resulting in degradation of 50-70% of oil in 10 days. This degradation can take place under in situ salinity of 25-35 parts o per thousand, pH of 7.5-8.2 and temperature of 25-30 C. For the purpose of experimental demonstration, crude oil can be added to marine sediments sample. After addition of heavy inoculum of cyanobacterial culture to such sediment, degradation of crude oil can be monitored periodically using gas chromotography. Long chain aliphatics above 20 carbon length are generally less degradable than those below 20 carbon by microorganisms. Interestingly with cyanobacteria, aliphatics above 20 carbon are better degraded. In addition, no nutrient supplement to marine sediments is required in the case of cyanobacteria. The cyanobacteria Agmenellum quadruplicatum strain PR-6 and Oscillatoria sp. Strain JCM are reported to oxidize naphthalene predominantly to 1-naphthol. Naphthalene and alkyl-substituted naphthalene are among the most toxic components in the water soluble fractions of crude and fuel oils (Carniglia, 1992, Bio-degradation, 3:351-368). The strain PR-6 of A. quadruplicatum also metabolized phenanthrene to trans-dihydro-phenanthrene and phenanthrols. Cyanobacteria oxidize hydrocarbons as detoxification reactions and thus differ from bacterial and fungal catabolic reactions which utilize hydrocarbons as a source of carbon and energy and thus mineralize it to carbon dioxide. Accordingly, the present invention provides a method for removal of oil pollutants from seawater using cyanobacteria which comprises cultivating cyanobacteria such as Oscillatoria salina, Plectonema terebrans and Aphanocapsa sp. in a conventional seawater medium having salinity in the range of 25-35 parts per thousand, pH between 7.5 to 8.2 at room temperature upto 15 days, then contacting with oil pollutant in seashore oil polluted land or marine sediments to get sediment or seashore land free of oil by known methods. Example-1 Synthetic marine medium containing NaCI 25g; MgCI2.6H2Og; KCI 0.5g; NaNO3 0.75g; K2HPO4 .3H2O 0.02g; MgSO4.7H2O 3.5g; CaCI2.2H2O 0.5g; Citric acid 0.003g; Ferric ammonium citrate 0.003 g; EDTA (Disodium salt 0.005g; Na2CO3 0.02 g; Trace metal mix 1 ml, was prepared using 1L distilled water. The stock trace metal mixture contained H PO 2.86g; MnCl .4H 0 1.81g; ZnSO . 7H O 0.222g; Na MoO .2H 0 0.339g; CuSO .5H 0 0.079g; Co(NO ) .6H 0 0.49g; CoCl ,6H 0 0.49g in 1L distilled water. This mixture was stored in the dark at 5 C in refrigerator. EDTA was added always before adding ferric salt. The medium was sterilised by passing through sterilised 0.22 um filter and never autoclaved. 50 ml of this medium was distributed in 250 ml Erlenmeyer flask and inoculated with 10 ml of homogenised culture of Oscillatoria salina. For stationary cultures, the inoculated flasks were o incubated at room temperature of 25-30 C in diffuse natural light. Shake cultures of the organisms were maintained by incubating the flasks on an orbital shaker at 200 rpm speed. The initial weight of the inoculum was determined gravimetrically and the final weight after 10 days was compared. (Table Removed) Figures in brackets represent growth in presence of the crude oil. n.t = not tested. The example given here shows that Oscillatoria salina grows better in static culture and further its growth in the presence of crude oil is comparatively good. EXAMPLE 2 The experimental procedure here were the same as given in Example 1, but the cyanobacterial culture used was that of lectonema terebrans (Table Removed) Figures in brackets represent growth in presence of the crude oil. n.t = not tested The example shows that Plectonema terebrans grows much better in static cultures. EXAMPLE 3 The experimental procedure here were the same as in examples 1 & 2, but the cyanobacterial culture used was that of Aphanocapsa sp. (Table Removed) Figures in brackets represent growth in presence of the crude oil. n.t = not tested. The example given here shows that Aphanocapsa sp grows better in shake culture and its growth is stimulated in the presence of crude oil. These three examples show that there is no inhibition of growth of these three cyanobacteria by the crude oil used. EXAMPLE 4 Initially, degradation of oil by cyanobacterial cultures was tested using synthetic seawater medium in stionary cultues at 25- 35 part per thousand salinity, pH of 7.5 - 8.2 and temperature of 25-30 C. Crude oil to the final concentration of 0.5% was added to the medium and the cyanobacterial culture, Oscillatoria salina was allowed to grow in this oil enriched medium for 10 days. At the end of the incubation period the cultures were extracted with CCl as described above and the percentage of oil degraded waB quantified gravimetrically and chromatographically using gas chromatograph. The cyanobacteria break down crude oil in order to detoxify the environment for itself. (Table Removed) salina The example shows that under the above described conditions, Oscillatoria salina is able to remove 50-60% of crude oil pollutant. EXAMPLE 5 The experimental procedure was the same as that in the Example 4, but the cyanobacterial culture Plectonema terebrans was used for degradation of the crude oil. (Table Removed) The above example shows that Plectonema terebrans under the above mentioned conditions is able to remove 50-60% crude oil. EXAMPLE 6 The experimental procedure was the same as that in the example 5, but the cyanobacterial culture, Aphanocapsa sp was used for oil degradation studies. (Table Removed) The above example proves that Aphanocapsa sp is capable of removing 40-60% of crude oil under the above mentioned conditions. EXAMPLE 7 In order to keep experimental conditions nearer to the natural conditions, filter sterilised seawater instead of synthetic seawater medium (As shown in the above examples 4-6) was used for carrying out oil degradation experiments. Uniform homogenised culture suspension of Oscillatoria salina was added to filter sterilised seawater, allowed to grow for 5 days before adding 1% of crude oil. These flasks were further incubated for 5 days and extracted with CCl , for gravimetric and G.C analysis. Figures 1 & 2 show the G.C profile of crude oil and its degraded products respectively. As seen from the Fig 2, this organism degraded higher fractions of crude oil, that is aliphatics above 20 carbon length much more efficiently. (Table Removed) The above example demonstrated that the above described experimental conditions, Oscillatoria salina can degrade ca. 60% of crude oil. EXAMPLE 8 The experimental procedure remained the same as in the example 7, but the cyanobacterial culture Plectonema terebrans was used for oil degradation studies. The gas chromatographic profile of the degraded product is shown in the Fig.3. (Table Removed) The above example proves that Plectonema terebrans can degrade ca. 50% of crude oil under the above mentioned experimental conditions. EXAMPLE 9 The experimental procedures and conditions were the same as in the example 7 & 8 but the cyanobacterial culture Aphanocapsa sp was used for oil degradation studies. The gas chromatographic profile of the degraded oil is shown in the Fig. 4. (Table Removed) The above results demonstrated that Aphanocapsa sp is able to degrade 50-70% of crude oil under the experimental conditions described in this example. EXAMPLE 10 Crude oil was fractionated into aliphatics, aromatics and polar fractions using silica gel and alumina column. Aliphatics, aromatics and polar contribute 50.2, 14 and 4.8% respectively and the remaining 30% forms waxes and the like. Degradation of this fractionated aliphatic portion of the crude oil was tested using the cyanobacterial culture of Oscillatoria salina. Aliphatic fraction was dissolved in petroleum ether and a final concentration of 1% was added to the flasks containing filter sterilised seawater. The solvent was allowed to evaporate from the flasks before adding the inoculum. The cultures were incubated for 5 days and extracted with CCl . Gas chromatographic analysis were carried out with these 4 extracts. The G.C profile of the aliphatic fraction and the degraded products are shown in the Figs. 5 & 6 respectively. Degradation of pure aliphatic, Hexadecane was used for comparison at 0.1% concentration. (Table Removed) The above results demonstrate that 0.salina can degrade 87% of aliphatic fraction of the crude oil. EXAMPLE 11 The experimental procedures and conditions were the same as in the example 10, the cyanobacterial culture used was that of Plectonema terebrans. The G.C. profile of the degradtion of aliphatic fraction of the crude oil is shown in the Fig.7. (Table Removed) The results demonstrate that P.terebrans is capable of degrading ca.45% of aliphatic fraction of the crude oil. EXAMPLE 12 The experimental procedures and conditions were the same as in example 10 & 11 except thaat the cyanobacterial culture used here was that of Aphanocapsa sp. The G.C. profile of the degradation of the aliphatic fraction of the crude oil is shown in the Fig. 8. (Table Removed) The example shows that Aphanocapsa sp is capable of degrading about 85% of aliphatic fraction of the crude oil under the above described experimental conditions. This invention has several advantages. Cyanobacteria are easy to mass culture in simple synthetic seawater medium. They are natural inhabitants of marine seashore environment and therefore can be successfully used. They can be sprayed on to oil polluted sites. As they grow attached to a substratum, they do not get washed away with waves. Thay can grow well and easily on seashore sediments, on calcareous shells and rocky substratum in photic conditions in the absence of any added fertilizers. As these cyanobacteria degrade aliphatic fractions much more efficiently, they have an added advantage in bioremediation of oil spill in seashore ecosystem. We claim : 1. A method for removal of oil pollutants from seawater using cyanobacteria which comprises cultivating cyanobacteria such as Oscillatoria salina, Plectonema terebrans and Aphanocapsa sp. in a conventional seawater medium having salinity in the range of 25-35 parts per thousand, pH between 7.5 to 8.2 at room temperature upto 15 days, then contacting with oil pollutant in seashore oil polluted land or marine sediments to get sediment or seashore land free of oil by known methods. 2. A method for removal of oil pollutants from seawater using cyanobacteria substantially as herein described.

Documents:

1873-del-1997-abstract.pdf

1873-del-1997-claims.pdf

1873-del-1997-correspondence-others.pdf

1873-del-1997-correspondence-po.pdf

1873-del-1997-description (complete).pdf

1873-del-1997-drawings.pdf

1873-del-1997-form-1.pdf

1873-del-1997-form-19.pdf

1873-del-1997-form-2.pdf