Title of Invention

"A PROCESS FOR RECLAMATION OF CRUDE OIL CONTAMINATED SOIL"

Abstract

ABSTRACT The present invention provides an eco-friendly process for the reclamation of crude oii contaminated soil, wherein the process incorporates a bio-formulation which is used for reclamation of crude oii contaminated soil. In the process a bio-formulation is prepared consisting of a mixture of three types of bacterial strains belonging to the genus Thiobacillus, Azotobacter and Arthobacter and the said bio-formulation is applied to the crude oii contaminated soil to be reclaimed. The process of the present invention for the reclamation of crude oii contaminated soil will find wide usage particularly in and around oii field areas and generally in areas contaminated due to crude oii spillage.

Full Text

This invention relates to a process for the reclamation of crude oii contaminated soil. This invention particularly relates to an eco-friendly process for the reclamation of crude oii contaminated soil by using a bio-formulation. The process of the present invention which incorporates a bio-formulation can be used for reclamation of crude oii contaminated soil. The process of the present invention for the reclamation of crude oii contaminated soil will find wide usage particularly in and around oii field areas and generally in areas contaminated due to crude oii spillage. The North Eastern region of India, in particular, has vast petroleum reserves. In oii fields, during drilling, gathering, transporting or in other operations, some quantity of crude oii is spilled out from drilling and operating sites. The spilled crude oii is an ecologica! threat, particularly to the flora and fauna. Beneficia! microbes such as nitrogen fixers, phosphate solublisers, sulphur oxidisers, cellulase degraders, are also affected because of crude oi! spillage. Moreover, some quantity of Chemicals present in the spilled crude oi! leaches out or percoiates to the rhizosphere of the soil. The soil gets contaminated so badly that no vegetation takes place in such affected area. It takes years to return to he normal condition. The methods for treating soil contaminated by petroleum and its by-products are previously known in the art. The search of patent data bases provided the following prior art references: Reference may be made to patent no. EP-1008396. The invention relates to environmental control and can be used for cleaning soils polluted with recent or aged crude oii or petroleum product spills. The invention essentially relates to a method for cleaning soils polluted with petroleum products, and comprising mixing of the soil with thermally-split graphite, adding of water thereto and separating the sorbent floated to the surface together with the adsorbed petroleum products. The soil and sorbent can be mixed in situ using agricultura! or road-building machines, as well as in mixing vessels. To accelerate the process, treatment of the aged petroleum products spills can include adding of an extractive agent such as kerosene, the application of cavitation action, or a combination thereof. The thermally-split graphite can be regenerated repeatedly by sublimating the sorbent-recovered petroleum products, by extracting or squeezing processes. Reference may be made to patent no. US-6036849, a method of removing hydrocarbons from soils contaminated with various hydrocarbons such as gasoline, diesel fuel, solvents, motor oii and crude oii. The process first screens the soil to remove oversized rocks and debris and to reduce the contaminated soil to uniformly sized particles. The soil particles are moved along a conveyor and first sprayed with an oxidizer diluted with ionized water and then sprayed with only ionized water. The washed particles are then vigorously mixed with their entrained oxidizer and ionized water in an auger mixer for several minutes to oxidize almost all of the remaining hydrocarbons. The washed and hydrocarbon-free soil is then moved by conveyor to a stockpile for storage, testing and drying. Reference may be made to patent no. US-6444204, wherein the invention relates to the field of bio-technologies and environmental protection, and refers the cleaning of water, soil and other media polluted with petroleum products. The cleaning method of the present invention is carried out with a biological preparation that comprises a pool of yeast of the Candida maltosa genus from the strains deposited under No. VKPM Y-2256 and No. VKPM Y-2257. These strains rnay be present in the pool in any ratio but preferably in the ratio of 1:1. The biological preparation may be used for removing pollution caused by a wide range of hydrocarbons contained in crude oii and petroleum products, for example fuel oii, oils, etc. The biological preparation affords recovery of oii pollution at temperatures up to +41 C. and pH 3.0-9.0, and may be used in saline ecosystems. The process for producing the biological preparation eofnprises simultaneously cultivating the above-mentioned yeast strains on a liquid medium followed by separation and removal of the biomass thus grown. To obtain a powdered preparation, the biomass is subjected to drying. When cleaning the above-cited media from oii pollution, an aqueous suspension of the biological preparation with a nitrogen-phosphate additive is used Reference may be made to patent no. WO 9531408, wherein the proposed biological preparation for removing crude oii and petroleum-product contaminants from water and soil consists of a mixture of the following hydrocarbon-oxidising strains of micro-organism: rhodococcus sp. BKM-AC-1500 D, rhodococcus maris BKM-AC-1501 D, rhodococcus erythropolis BKM-AC-1502 D, pseudomonas stutzeri BKM-B-1972 D, Candida sp. BKM-Y-2778 D. The relative proportions of these strains are (0-1):(0-1):(0-1):(0-1):(0-1). The prepar ation in question is a naturally occurring association of microorganisms or mixture of individual micro-organism strains. The proposed method of removing crude oii and petroleum-product contaminants from water and soil involves treating the water or soil with the biological preparation containing the strains rh. sp. BKM-AC-1500 D, rh. maris BKM-AC-1501 D, rh. erythropolis BKM-AC-1502 D, ps. stutzeri BKM-B-1972 D, and C. sp. BKM-Y-2778 D. The preparation used contains no fewer than 2 x 10 live microbial cells for every square metre of contaminated area. Treatment of the water and/or soil is carried out by sprinkling a suspension of the preparation in a solution of mineral salts. For the treatment of soil, trenches are cut in the soil. Water and/or soil with a salinity of up to 10 % is treated in this way. The preparation can be used for treating reservoirs and saline soil and brackish water ecosystems. Reference may be made to patent no. US-6153017, a method for removal of hydrocarbon materials from solid particulate soil contaminated with the hydrocarbon materials. The method comprises forming an aqueous slurry of a mixture of a hydrophobic adsorbent selected from foamed synthetic materials or natural materials which entrap gas, with the soil in the presence of water. The adsorbent has a density less than water. The slurry is mixed for a period of time, and a gravity separation of the adsorbent from the aqueous admixture thus obtained is effected. Reference may be made to patent no. US-6110372, bioremediation of petroleum pollutants with alkane-utilizing bacteria, wherein alkane-utilizing bacteria are used to degrade pollutants comprising petroleum compounds. In-situ or ex-situ techniques may be used to reduce or eliminate petroleum pollutants from liquid, gas and solid sources. In a preferred embodiment, petroleum concentrations in various environments are reduced by contacting the petroleum pollutants with butane-utilizing bacteria in the presence of oxygen to degrade the petroleum pollutants by cometabolism or direct metabolism. Suitable alkane-utilizing bacteria include Pseudomonas, Variovorax, Nocardia, Chryseobacterium, Comamonas, Acidovorax, Rhodococcus, Aureobacterium, Micrococcus, Aeromonas, Stenotrophomonas, Sphingobacterium, Shewanella, Phyllobacterium, Clavibacter, Alcaligenes, Gordona, Corynebacterium and Cytophaga. Reference may be made to patent no. US-5415777, process for the decontamination of soils contaminated by petroleum products, wherein a method of decontaminating soil contaminated by petroleum products on the site of the contamination is described. Such decontamination is accomplished by chemical breakdown, enzymatic action and biologica! microbial degradation. Significant byproducts of this degradation of the hydrocarbon material are: water, sodium salts, ammonium salts, carbon dioxide, free amino acids and heat. The invention relates to a process for decontaminating soils contaminated by petroleum products. Specifically, the invention relates to a method of treating contaminated soils by applying carbonate salts, ammonium hydroxide, and dilute organic acids to the soil at the site of the contamination. This can be followed by the addition of proteinase enzymes and bacterial inoculates. Reference may be made to patent no. US-5128262, microbial decontamination of soils contaminated with hydrocarbons, in particular mineral oils by microbial oxidation. The microbial decontamination of soils contaminated with hydrocarbons, in particular with mineral oils, by microbial oxidation is improved by a process in which in addition to supplying the oxygen necessary for degradation, by aeration, soluble nutrients, water and soluble or dispersed biosurfactants as such or mixed with chemical surfactants are metered into the contaminated soils directly or into the removed contaminated soil layers, in one or more batches at time intervals, in an amount such that the formation of toxic intermediates which slow down or block microbial degradation in prevented, the accessibility of mineral oils and their products for the microorganisms is simultaneously increased and, as a result, accelerated, virtually complete mineralization by respiration to CO.sub.2 and H.sub.2 O is effected. The methods for treating soil contaminated by petroleum and its by-products which are previously known in the art, have all hâd disadvantages relating to the fact that, in most cases, the contaminated soil must be excavated and transported to a decontamination site with a resulting increase in expense and time. Furthermore, most of the methods involve the addition of strong Chemicals to the contaminated soil which themselves have the potenţial of being environmentally hazardous. Additionally, many of the known methods are not able to completely degrade the contaminating substances from soil, but instead, succeed only in separating the most hazardous of the contaminants from the soil in a form in which they can be more easily disposed of. There is a need to provide a process for reclamation of such contaminated soil, particularly by biological means, which is an ideal and eco-friendly method to revive soil condition rapidly and to restore the environment. The main object of the present invention is to provide a process for the reclamation of crude oii contaminated soil, which will obviate the drawbacks of the hitherto known prior art. Another object of the present invention is to provide an eco-friendly process for the reclamation of crude oii contaminated soil by using a bio-formulation. Yet another object of the present invention is to provide a process for the reclamation of crude oii contaminated soil by using a bio-formulation consisting of bacterial strains belonging to the genus Thiobacillus, Azotobacter and Arthobacter. Still another object of the present invention is to reclaim the crude oii contaminated soil for vegetation revival and development. Still yet another object of the present invention is to bring about the bio-chemical changes of the crude oii contaminated soil through application of bio-formulation for sustaining plant growth. A further object of the present invention is to enrich the beneficial microbial population and their activity in such crude oii affected area. The present invention provides an eco-friendly process for the reclamation of crude oii contaminated soil, wherein the process incorporates a bio-formulation which is used for reclamation of crude oii contaminated soil. In the process a bio-formulation is prepared essentially consisting of a mixture of three types of bacterial strains belonging to the genus Thiobacillus, Azotobacter and Arthobacter and the said bio-formulation is applied to the crude oii contaminated soil to be reclaimed. Accordingly the present invention provides a process for the reclamation of crude oii contaminated soil, which comprises: (a) preparing separate sterilized media for bacteria! strains belonging to the genus Thiobacillus, Azotobacter and Arthobacte, respectively; (b) inoculating the said medium(s) with respective strain(s) and incubating for a period of about 48 hours; (c) centrifuging the cultures separately at 4000 rpm for about 10 minutes and filtering to obtain supernatents; (d) mixing the supernatants obtained in step (c) in a ratio of 1 : 5 : 3 of culture filtrates of Thiobacillus, Azotobacter and Mhobacter, respectively, to obtain a bio-formulation; (e) diluting the said bio-formulation in a ratio of 1:1 and applying directly to the crude oii contaminated soil; (f) repeating the treatment of the said contaminated soil, if required, after three months, with the bio-formulation concentration diluted to a ratio of 1:2. In an embodiment of the present invention, the sterilized medium for bacterial străin belonging to the genus Thiobacillus is a mineral medium having pH in the range of 6.7 to 7.0, containing sodium thiosulphate: 5g, K2HPO4: 0.1 g , NaHC03 : 0.2g , NH4CI: 0.1 g and Agar 20 g per litre. In another embodiment of the present invention, the sterilized medium for bacterial străin belonging to the genus Azotobacter is a succinate medium having pH of 6.9, containing succinic acid : 4 g , K2HPO4 : 1g, KH2PO4:1g, NH4(SO4)2 :1.5g , in 1 litre distilled water. In still another embodiment of the present invention, the sterilized medium for bacterial străin belonging to the genus Arthobacter is a nitrogen free medium consisting of a mixture of 100ml of (a) and 900ml of (b), wherein (a) is K2HPO4: 5g, MgSO4 :2g , CaS04 :1g , FeS04: 0.2 g , MnSO4: 0.2 g , MoO3 Kl: 0.1g, in 1 litre distilled water and (b) is sucrose : 10 g, CaCO3: 3g , in 900 ml distilled water, pH 7.0. In yet another embodiment of the present invention, the sterilization of the media is done in an autoclave for about 15 minutes at a pressure of about 15 Ibs. In still yet another embodiment of the present invention, the incubation temperature for Thiobacillus was 30° C± 2° C . In a further embodiment of the present invention, the incubation temperature for Azotobacter and Arthobacter was 24° C. In a still further embodiment of the present invention, the Thiobacillus sp. used in the bio-formulation is an autotrophic bacteria isolated from coal deposition area. In another embodiment of the present invention, the Azotobacter sp. used in the bio-formulation is a heterotrophic bacteria isolated from tea garden soil. In still another embodiment of the present invention, the Arthobacter sp. used in the bio-formulation, is a nitrogen fixing străin which produces 70 jj,g of nitrogen per ml., isolated from tea garden soil. The novelty of the process of the present invention is that it provides an eco-friendly process for the reclamation of crude oii contaminated soil. The process of the present invention enables vegetation revival, sustains plant growth, brings about bio-chemical changes in the crude oii contaminated soil and enriches the beneficia! microbial population and their activity in such crude oii affected area. The process of the present invention achieves the novelty by the non-obvious inventive step of providing a bio-formulation consisting of a mixture of three types of bacterial strains belonging to the genus Thiobacillus, Azotobacter and Arthobacter. The said bio-formulation is applied to the crude oii contaminated soil to be reclaimed for sustaining plant growth. The following example is given by way of illustration of the detailed process steps of the present invention in actual practice and should not be construed to limit the scope of the present invention. EXAMPLE-1 (A) Three types of bacterial strains belonging to the genus: Thiobacillus, Azotobacter and Arthobacter were isolated. (i) The străin Thiobacillus sp. belonging to the genus Thiobacillus was isolated from the coal deposition area located near the pilot plant at the Regional Research Laboratory, Jorhat, Assam. (ii) The străin Azotobacter sp. belonging to the genus Azotobacter was isolated from tea garden soil. The Azotobacter sp. is a heterotrophic bacteria and grows well at a pH 6.9. (iii) The străin Arthobacter sp. belonging to the genus Arthobacter was isolated from Sangsua Tea Garden, Jorhat. (B) Separate sterilized media for bacterial strains belonging to the genus Thiobacillus, Azotobacter and Arthobacte, respectively, was prepared. (a) A mineral medium for Thiobacillus străin was prepared containing sodium thiosulphate: 5g , k2HPO4:0.1 g , NaHCO3 : 0.2g , NH4CI: 0.1 g , Agar 20 g, in one litre distilled water. The pH was maintained at 6.8. The sterilization of the media was done in an autoclave for 15 minutes at a pressure of 15 Ibs. (b) A medium for bacterial străin belonging to the genus Azotobacter was prepared containing succinic acid : 4 g , K2HPO4 : 1g, Kh^PO^Ig, NH4(SO4)2 :1.5g , in 1 litre distilled water. The pH was maintained at 6.9. The sterilization of the media was done in an autoclave for 15 minutes at a pressure of 15 Ibs. (c) A nitrogen free medium for Arthobacter was prepared consisting of a mixture of 100ml of (a) and QOOml of (b), wherein (a) is K2HP04 : 5g, MgS04 :2g , CaS04:1g , FeS04 : 0.2 g , MnSO4 : 0.2 g , MoO3 Kl: 0.1g, in 1 litre distilled water and (b) is sucrose : 10 g, CaCO3 : 3g , in 900 ml distilled water, pH 7.0. The sterilization of the media was done in an autoclave for 15 minutes at a pressure of 15 Ibs. (C) The mediums were inoculated with respective strains and incubated for 48 hours. Thiobacillus was incubated at 30° C± 2° C whereas for Azotobacter and Arthobacter the temperature was maintained at 24° C. (D) The cultures were centrifuged separately at 4000 rpm for 10 minutes. (E) A bio-formulation was prepared by mixing the supernatants i.e., culture filtrates of Thiobacillus , Azotobacter and Mhobacter in the ratio of 1 : 5 : 3 respectively. (F) The bio-formulation was applied to the crude oii contaminated soil by diluting the mixed culture filtrate in a ratio of 1:1. (G) The treatment was repeated after three months. The bio-formulation was applied to the partially de-contaminated soil by diluting the mixed culture filtrate in a ratio of 1:2. As a result of the treatment, as detailed above, of the crude oii contaminated soil by the process of the present invention, it was observed that: 1. The number of soil beneficial microbes like nitrogen fixers, sulphur oxidisers, phosphate solublisers, cellulase degraders were enhanced by an order 4 to 5 times, post-treatment of the crude oii contaminated soil. 2. The enzymatic activity, urease, phosphatase, rhodanase, cellulase, of the beneficial microbes were enhanced by a factor of 3 to 6, after the treatment of crude oii contaminated soil. 3. The chemical nature, such as nitrogen, sulphur, phosphorous, potassium, iron, aluminium, zinc content of the contaminated soil improved by a factor of 6, after the treatment. The main advantage and distinguishing features of the present invention are as follows: 1. Reclamation of crude oii contaminated soil by biologica! means is an ideal, safe and eco-friendly method. 2. Number of soil beneficial microbes like nitrogen fixers, sulphur oxidisers, phosphate solublisers were enhanced. 3. Enzymatic activity of the beneficial microbes enhanced after the treatment of crude oii contaminated soil. 4. Chemical nature of the contaminated soil improved after the treatment. 5. Bacterial strains used are from local sources, isolated from soils of Assam. 6. The chemicals needed for bacteria! culture are easily available. 7. The process is economical. We Clairn: 1. A process for the reclamation of crude oii contaminated soil, which comprises: (a) preparing separate sterilized media for bacteria! strains belonging to the genus Thiobacillus, Azotobacter and Arthobacte, respectively; (b) inoculating the said medium(s) with respective strain(s) and incubating for a period of about 48 hours; (c) centrifuging the cultures separately at 4000 rpm for about 10 minutes and filtering to obtain supernatents; (d) mixing the supernatants obtained in step (c) in a ratio of 1 : 5 : 3 of culture filtrates of Thiobacillus, Azotobacter and Mhobacter, respectively, to obtain a bio-formulation; s (e)diluting the said bio-formulation in a ratio of 1:1 and applying directly to the crude oii contaminated soil; (f) repeating the treatment of the said contaminated soil, if required, after three months, with the bio-formulation concentration diluted to a ratio of 1:2. 2. A process as claimed in claim 1, wherein the sterilized medium for bacteria! străin belonging to the genus Thiobacillus is a mineral medium having pH in the range of 6.7 to 7.0, containing sodium thiosulphate: 5g, K2HPO4:0.1 g , NaHCOs : 0.2g , NH4CI: 0.1 g and Agar 20 g per litre. 3. A process as claimed in claim 1-2, wherein the sterilized medium for bacterial străin belonging to the genus Azotobacter is a succinate medium having pH of 6.9, containing succinic acid : 4 g , K2HPO4 : 1g, KH2PO4:1g, NH4(SO4)2 :1.5g , in 1 litre distilled water. 4. A process as claimed in claim 1-3, wherein the sterilized medium for bacterial străin belonging to the genus Arthobacter is a nitrogen free medium consisting of a mixture of 100ml of (a) and 900ml of (b), wherein (a) is K2HPO4: 5g, MgSO4 :2g ,CaS04:1g , FeS04:0.2g, MnSO4: 0.2 g , MoO3 Kl: 0.1g, in 1 litre distilled water and (b) is sucrose : 10 g, CaCO3: 3g , in 900 ml distilled water, pH 7.0. 5. A process as claimed in claim 1-4, wherein the sterilization of the media is done in an autoclave for about 15 minutes at a pressure of about 15 Ibs. 6. A process as claimed in claim 1-5, wherein the incubation temperature for Thiobacillus was 30° C± 2° C . 7. A process as claimed in claim 1-6, wherein the incubation temperature for Azotobacter and Arthobacter was 24° C. 8. A process as claimed in claim 1-7, wherein the Thiobacillus sp. used in the bio-formulation is an autotrophic bacteria isolated from coal deposition area. 9. A process as claimed in claim 1-8, wherein the Azotobacter sp. used in the bio-formulation is a heterotrophic bacteria isolated from tea garden soil. 10. A process as claimed in claim 1-9, wherein the Arthobacter sp. used in the bio-formulation, is a nitrogen fixing străin which produces 70 |j,g of nitrogen per ml., isolated from tea garden soil. 11. A process for the reclamation of crude oii contaminated soil, substantially as herein described with reference to the example.

Documents:

470-DEL-2003-Abstract (12-02-2008).pdf

470-del-2003-abstract.pdf

470-DEL-2003-Claims (12-02-2008).pdf

470-DEL-2003-Claims 19-08-2008.pdf

470-del-2003-claims.pdf

470-DEL-2003-Correspondence-Others (12-02-2008).pdf

470-DEL-2003-Correspondence-Others-(19-08-2008).pdf

470-del-2003-correspondence-others.pdf

470-del-2003-correspondence-po.pdf

470-DEL-2003-Description (Complete) (12-02-2008).pdf

470-del-2003-description (complete)-19-08-2008.pdf

470-del-2003-description (complete).pdf

470-del-2003-form-1.pdf

470-del-2003-form-18.pdf

470-del-2003-form-2.pdf

470-DEL-2003-Form-3 (12-02-2008).pdf

470-del-2003-form-3.pdf