DESC:PROCESS FOR CONFINED BIOREMEDIATION OF HYDROCARBON SLUDGE

FIELD OF THE INVENTION

The present invention relates to a method for treatment of sludge produced during petroleum production, refining, storage and transport and oil contaminated soil. More particularly, the present invention provides a reactor based biological disposal methodology for oily sludge and oil contaminated soil.
BACKGROUND OF THE INVENTION

The production, processing, storage, transportation of petroleum and petrochemical products results in the generation of a considerable volume of oily sludges. These sludges come from a variety of sources including storage tank bottoms including crude oil and product storage tank, oil-water separators, dissolved air floatation units, bio-sludges from effluent treatment plant. Oily sludge is classified as hazardous waste and their safe disposal is essential.

A variety of physical, chemical and biological approaches like storage in secure landfill, incineration, indirect thermal treatment, aeration, venting, air sparging and land farming based natural bioremediation using biostimulation and bio-augmentation have been in use to dispose petroleum sludge and oil contaminated soil. Among these methods biological methods are considered more environmental friendly and cost effective this involves use of indigenous or augmented microorganisms to metabolize organic substrates. Most of the biological approaches including landfarming, composting or biopiling involve use of for treatment of oily sludges and oil contaminated soil. In this process sludges are sprayed on land together with nutrients and/or specified microbes and the soil is tilled to promote the activity of microbial population for the degradation of petroleum hydrocarbons. Although land farming may be a low cost method if land is available, it requires very long treatment times due to lack of control on environmental factors such as seasonal variation in temperature, pH, moisture, and natural microbial activity, mixing and circulation. In climates with limited rainfall, the cost to maintain the proper moisture content in the soil could be prohibitive. Lack of uniformity causes high contaminant concentration/toxicity in localized pockets and inconsistent permeability of soil makes it difficult to apply treatment additives like nutrients and oxygen. There is still the danger of groundwater contamination unaddressed.

These limitations can be overcome by using a bioreactor based methodology for oily sludge and oil contaminated soil remediation. It eliminates the need to spread high concentrations of petroleum hydrocarbons on large areas of land. The bioreactor-based process is characterized by much higher rates and extents of degradation than are observed in land farming systems due to the minimization of mass-transfer limitation and increased desorption of contaminants by continuous mixing, controlled environmental and nutritional factors. The deposition of heavy metals in the soil, and the potential contamination of ground water supplies have resulted in a major reduction in the use of land farming for oily wastes.

Beside the above issues, with increasing reliance on opportunity crudes like high TAN crude, high paraffinic crude, heavy crude and extra heavy crude with higher recalcitrant/hard fractions/molecules like naphthenic acid, asphaltene, waxes etc. needs specific treatment methodologies to degrade them effectively and cost effective manner by overcoming the mass transfer and low bioavailability issue.

The present invention provides a confined bioremediation process which can be effectively used to dispose the oily sludge but not limited to produce from crude oils, opportunity crudes like high TAN crude, high paraffinic crude, heavy crude and extra heavy crude etc. by using thermophilc microbes at higher temperature and including step to deploymerise the high molecular weight molecules.

US 6,153,017 discloses 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 affected. Mixing is accomplished using reactors, tanks with air spargers, impellers, rakes, screw assemblies or stirrers, rotating drums e.g. similar to a cement mixer, tumblers, reactors on reciprocating or orbital shaking machines, vibration or sonication mixers and screw or other conveyor equipment. This method teaches away from using microorganisms for degradation of harmful hydrocarbons and discloses treatments using solid hydrophobic adsorbent materials like foamed synthetic materials or natural materials which entrap gas and soil for its functioning.

U.S 6,652,752 discloses method for the biodegradation of an oil-based sludge comprising a mixture of petroleum hydrocarbons. The method comprises forming an aqueous solution in a reactor of an oil-in-water emulsion of the oil-based sludge, bacterial culture and nutrients for the bacterial culture, the bacterial culture having the ability to grow on petroleum hydrocarbons as sole carbon source and having been isolated from a hydrocarbon contaminated soil or hydrocarbon-containing sludge or other environments rich in hydrocarbon degrading bacteria, maintaining the aqueous solution under aerobic conditions in the reactor at a temperature of at least 10° C for a period of time sufficient to reduce the amount of hydrocarbon by at least 25%, and discharging aqueous solution having a reduced amount of hydrocarbons from the reactor. The method disclosed in this invention works at pH within the range 6.4-7.6 and does not disclose the consortium of micro-organisms as in the prevent invention for the bioremediation of oily sludge and contaminated soil.

Petrozyme Process© is also a reactor-based biological process for the degradation of oily sludge wastes from petroleum activities. The technology involves high rate biodegradation of hydrocarbons in an aerated bio-reactor supplied with a proprietary nutrient blend and bacterial culture. Typical total petroleum hydrocarbon (TPH) concentrations input to the Petrozyme Process© are in the 10 - 30% range. A typical treatment time at 28° - 32° C is 10 days. Laboratory tests have shown that most of bacteria involved are known oil-degrading bacteria such as species of Pseudomonas, Acinetobacter, Rhodococcus and Alcaligenes etc. The major residue at the end of treatment is a water phase having very low hydrocarbon concentration (<1% TPH). The water may be recycled or processed in a waste water treatment plant. As the water contains high populations of hydrocarbon degrading bacteria, it may also be used to treat hydrocarbon-contaminated soil. Small quantities of solids that settle can be delisted from the hazardous waste list for safe "non-hazardous" disposal (http://www.petrozyme.com and Ajay Singh,Bill Mullin Owen Ward. Reactor-Based Process for the Biological Treatment of Petroleum Wastes. PN # 2000, Petrotech 2001 Conference , Baharain 29-30 October 2001). The method
disclosed in this invention works at temperature range 28° - 32° C and use a proprietary nutrient blend and bacterial culture different than disclosed in present invention.

EP 2051784 A2 discloses a bio-assisted method for treatment of hydrocarbon contaminated soil employing novel microbes which are capable of decontaminating hydrocarbon contaminated soil having free flowing water or in slurry form or having large amount of gravels. The method comprises adding hydrocarbon releasing microbes followed by adding the microbes capable of degrading the released hydrocarbon to decontaminate the soil, wherein said microbes are grown separately in suitable vessels containing a suitable nutrient medium. However, this invention does not recite the consortium of microorganisms as done is in the present invention.
US 7445927 B2 discloses a sequencing batch reactor tank for the on-site bio-degradation of oily sludge. In this invention, bacteria already present in and adapted to oily sludge degrade the hydrocarbons found in oily sludge within two weeks from 20,000 ppm to less than 100 ppm. A degradation cycle requires 5 days. After five days a recirculation pump and aeration system are turned off and solids are allowed to settle to the bottom of the tank. An ultrafiltration unit connected to the tank requires approximately 16 hours processing the contents of the reactor tank. It is noteworthy that sludge which immediately taken out from the tank do not contain high concentration of hydrocarbon degrading bacteria. Hence, this method cannot be used for treatment of the sludge which does not contain adapted hydrocarbon degrading bacteria.

EP 0609399 B1 discloses a method for improved slurry-phase bioremediation treatment of organic sludge and mixtures of organic sludge and organics-contaminated soils by dissolving the contaminants into an aqueous phase and microbially degrading same. This invention performs on the mechanism of co-metabolism and requires addition of additional co-substrates for hydrocarbon degradation. The degradation of co-substrates may pose further disposal problem. Moreover, it requires large numbers of reactors for functioning of the invention.

US 5633164 relates to the aerobic degradation of organic compounds in the fluid or solid phase using some specific microbes. The method disclosed in the present invention works at limited pH and temperature range and bacterial culture different than disclosed in present invention.

US2009/0325271 discloses a bio-assisted method for treatment of hydrocarbon contaminated soil employing novel microbes which are capable of decontaminating hydrocarbon contaminated soil having free flowing water or in slurry form or having large amount of gravels. This invention relates to addition of the bio surfactant-producing bacteria as a beneficial remedy and more practical than exogenously adding purified bio surfactant to contaminated soils. However, there is no disclosure of a bioreactor based methodology and overcoming the problem of using bio surfactants on large areas of lands.

WO2014/033638 discloses a bio-inoculant composition comprising consortia of microorganisms selected from group of Bacillus sp., Pseudomonas putida, Bacillus subtilis and Pseudomonas putida and its use for reduction of contaminants in effluents from processing industries. However, this document does not disclose the novel and specific combination of microbes used differently at three different stages in a bioreactor for breaking down the hydrocarbons in the oily sludge and contaminated soil to water containing less than 5 ppm oil and grease.

SUMMARY OF THE INVENTION

Disclosed herein is a bio-assisted remediation method for the treatment of oily sludge or oil contaminated soil, in three stages, wherein:
(a) Stage-1 comprises subjecting the sludge or oil contaminated soil to depolymerisation under microaerophilic conditions using a consortium of microbes MBDP consisting of equal ratios of Pseudomonas putida IOC5a1 (MTCC 5385), Pseudomonas aeruginosa IOCX (MTCC 5389), Bacillus substilis (MTCC 5386), Enterobacter aerogens IOC-EA-6 (MTCC 25016), Achromobacter xylosoxidan IOC - SC-4 (MTCC 25024) in a reactor and a nutrient formulation;
(b) Stage-2 comprises degrading the resultant of Stage-1 into carbon dioxide and water under aerobic conditions using a consortium of microbes MBHD consisting of equal ratios of Pseudomonas putida IOCR1 (MTCC 5387), Pseudomonas fulva IOC SAM1 (MTCC 5802), Alcaligenes sp. IOC-MA-2 (MTCC 25022), Pseudomonas putida IOC-MA-1 (MTCC 25021) in the same or different reactor; and
(c) Stage-3 comprises transferring the resultant of Stage-2 to another reactor to obtain a water phase with less than 5 ppm of oil content by using a consortium of microbes MBLC consisting of equal ratios of Bacillus sp. IOC-EP-01 (MTCC 5870), Bacillus subtilis IOC EP-03 (MTCC 5851), Lysinibacillus sp. GP-ETPBA5 (MTCC 5666), Pseudomonas aeruginosa IOC-EA-106 (MTCC 25019).
Another embodiment of the present invention provides a bio-assisted remediation method for the treatment of oily sludge or oil contaminated soil, in three stages, wherein:
(a) Stage-1 comprises:
(i) providing high solid slurry of the sludge or oil contaminated soil (5-70%) in a reactor optionally comprising at least one anode and at least one cathode;
(ii) adding a nutrient formulation comprising at least one nitrogen source, one phosphorus source, one ester and one vitamin source to the slurry;
(iii) subjecting said slurry to depolymerisation under microaerophilic conditions using a consortium of microbes MBDP consisting of Pseudomonas putida IOC5a1, Pseudomonas aeruginosa IOCX, Bacillus substilis, Enterobacter aerogens IOC-EA-6, Achromobacter xylosoxidan IOC - SC-4;
(iv) stirring the slurry at low speed (100-500 rpm) under microaerophilc conditions;
(v) allowing reacting said slurry for 1-5 days at temperature of 5-70ºC and pH of 4-10, salinity of 0-5%;
(b) Stage-2 comprises:
(i) transferring the resultant slurry of Stage 1 to another reactor optionally comprising at least one anode and at least one cathode;
(ii) inoculating said slurry under aerobic conditions with a consortium of microbes MBHD consisting of equal ratios of Pseudomonas putida IOCR1, Pseudomonas fulva IOC SAM1, Alcaligenes sp. IOC-MA-2, Pseudomonas putida IOC-MA-1;
(iii) allowing reacting said slurry for 2-10 days at temperature and 5-70ºC and pH of 4-12,
(iv) allowing the settling of slurry;
(v) removing the settled oil free solids;
(c) Stage-3 comprises:
(i) transferring the resultant of Stage-2 to another reactor having aeration facility;
(ii) innoculating the slurry with an immobilized consortium of microbes MBLC consisting of equal ratios of Bacillus sp. IOC-EP-01, Bacillus subtilis IOC EP-03, Lysinibacillus sp. GP-ETPBA5, Pseudomonas aeruginosa IOC-EA-106;
(iii) allowing reacting said slurry for 2-10 days at temperature: 5-70 degree C and pH: 4-10;
(iv) obtaining water with less than 5 ppm oil and grease.
In another embodiment of the present invention, the nutrient formulation used in Stage-1 comprises at least one nitrogen source, one phosphorus source, one ester and one vitamin source to the slurry, wherein the concentration of the nitrogen and phosphorus source is at least 0.001% of the sludge and the concentration of fatty acid, vitamins and amino acids is at least 0.0001% of the sludge to be treated.
In yet another embodiment of the present invention, the nutrient formulation used in Stage-1 comprises KH2PO4, K2HPO4, MgSO4, (NH4)2SO4, KNO3, peptone, yeast extract, trace element and multi vitamin solution.
In another embodiment of the present invention, the depolymerization in Stage-1 comprises depolymerisation of resins, asphaltenes, waxes and other high molecular weight hydrocarbons to lower molecular weight compounds.
In another embodiment of the present invention, the microbial consortium of Stage 3 is immobilized on a matrix.
In yet another embodiment of the present invention, the matrix/membrane is selected from synthetic plastics, surface-modified carbon nanotubes, poly (tetrafluoroethylene) (PTFE) fibrils, zeolite, mesoporous silica, porous glass, activated charcoal, ceramics, acrylamide, polyurethane foams, polyurethane foams with impregnated with carbon nano tubes or charcoal, polyvinyl, resins and natural polymer etc.
In another embodiment of the present invention, Stage 1 and Stage 2 are performed in same reactor.
In another embodiment of the present invention, Stage 1 and Stage 2 are performed in different reactors.
In another embodiment of the present invention, the bio-assisted remediation can be performed in continuous, batch or semi continuous mode.
In another embodiment of the present invention, the reactors used in Stage-1 and/or Stage-2 optionally comprise at least two electrodes connected with each other by a conductive wire like titanium wire and poised at 200 mv to 1500 mV through a source of current.
In yet another embodiment of the present invention, the reactors used in Stage-1 and/or Stage-2 comprise at least one anode and at least one cathode.
In another embodiment of the present invention, the sludge comprises oily sludge and/or hydrocarbon-contaminated soil.
In another embodiment of the present invention, the oily sludge comprises all types of oil containing sludges including sludge obtained from conventional crude as well from opportunity crude, high TAN crude and sludge from product thereof.
In another embodiment of the present invention, the microbes are thermophilic.
In another embodiment of the present invention, the reactor in the third stage comprises single or multiple sub-chambers.
In another embodiment of the present invention, the reactor in the third stage has aeration facility.
DESCRIPTION OF THE INVENTION

While the invention is susceptible to various modifications and/or alternative processes and/or compositions, specific embodiment thereof has been shown by way of example in the drawings and tables and will be described in detail below. It should be understood, however that it is not intended to limit the invention to the particular processes and/or compositions disclosed, but on the contrary, the invention is to cover all modifications, equivalents, and alternative falling within the spirit and the scope of the invention as defined by the appended claims.

The protocols have been represented where appropriate by conventional representations, showing only those specific details that are pertinent to understanding the embodiments of the present invention so as not to obscure the disclosure with details that will be readily apparent to those of ordinary skill in the art having benefit of the description herein.

The following description is of exemplary embodiments only and is not intended to limit the scope, applicability or configuration of the invention in any way. Rather, the following description provides a convenient illustration for implementing exemplary embodiments of the invention. Various changes to the described embodiments may be made in the function and arrangement of the elements described without departing from the scope of the invention.

Definitions:

The term “oily sludge” as used in the context of the present invention means solid/semisolid waste generated due to storage of crude oil /products, various petroleum hydrocarbons, water, heavy metals, and solid particles.

The term “high TAN crude” as used in the context of the present invention means acidic crude oils that contain substantial amounts of naphthenic acids or other acids.

The term “opportunity crude” as used in the context of the present invention means crude oil with high sulfur and asphaltene content, low gravity, high viscosity and/or high paraffinic content.

The term “conventional crude” as used in the context of the present invention means crude oils having low sulphur content and are easily flowable at ambient temperature.

The term “MBDP” as used in the context of the present invention means microbial blend/consortium containing Pseudomonas putida IOC5a1 (MTCC 5385), Pseudomonas aeruginosa IOCX (MTCC 5389), Bacillus substilis (MTCC 5386), Enterobacter aerogens IOC-EA-6 (MTCC 25016), Achromobacter xylosoxidan IOC - SC-4 (MTCC 25024) in equal ratio.

The term “MBHD” as used in the context of the present invention means the microbial consortium containing Pseudomonas putida IOCR1 (MTCC 5387), Pseudomonas fulva IOC SAM1 (MTCC 5802), Alcaligenes sp. IOC-MA-2 (MTCC 25022), Pseudomonas putida IOC-MA-1 (MTCC 25021) in equal ratio.

The term “MBLC” as used in the context of the present invention means the microbial blend/consortium containing Bacillus sp. IOC-EP-01 (MTCC 5870), Bacillus subtilis IOC EP-03 (MTCC 5851), Lysinibacillus sp. GP-ETPBA5 (MTCC 5666), Pseudomonas aeruginosa IOC-EA-106 (MTCC 25019) in equal ratio.

The present invention discloses a methodology for treating oily sludge (all types of sludge including sludge obtained from conventional crude as well from opportunity crude, high TAN crude and sludge from product thereof) and hydrocarbon-contaminated soil. In particular, the present invention discloses a three-stage bioassisted remediation of oil-containing sludge.

The source of the microbes used in the present invention is soil and water. The geographical origin of the microbes is ¬India. The aforesaid microbes have been deposited with the Microbial Type Culture Collection (MTCC), Chandigarh as required under the Budapest Treaty. The MTCC identified the microbes and assigned their respective Accession numbers as recited hereunder.

In accordance with the present invention, the oil containing sludge is subjected to bioassisted remediation in three stages;
a. In the first stage, the sludge or contaminated soil is treated under microaerophilic conditions using a consortium of selected microbes for 1-5 days. The microbial consortium consisting Pseudomonas putida IOC5a1 (MTCC 5385), Pseudomonas aeruginosa IOCX (MTCC 5389), Bacillus substilis (MTCC 5386), Enterobacter aerogens IOC-EA-6 (MTCC 25016), Achromobacter xylosoxidan IOC - SC-4 (MTCC 25024) in equal ratio. This step results in depolymerization of the resins, asphaltenes, waxes and other high molecular weight hydrocarbons to lower molecular weight compounds. These microbes also produce extracellular long chain hydrocarbon emulsifying and solubilizing agents, resulting in the decreased surface tension and lowered interfacial tension between oil/water phases;
b. In the second stage, the sludge treated in stage-1 is subjected to treatment using a specific set of the microbes which degrades the hydrocarbon to carbon dioxide and water under aerobic conditions. The microbial consortium consists Pseudomonas putida IOCR1(MTCC 5387), Pseudomonas fulva IOC SAM1 (MTCC 5802), Alcaligenes sp. IOC-MA-2 (MTCC 25022), Pseudomonas putida IOC-MA-1 (MTCC 25021) in equal ratio;

c. In the third stage, the liquid phase from stage 2 is treated in a reactor having a specific set of microbes immobilized on suitable matrix. The microbial consortium consists Bacillus sp. IOC-EP-01 (MTCC 5870), Bacillus subtilis IOC EP-03 (MTCC 5851), Lysinibacillus sp. GP-ETPBA5 (MTCC 5666), Pseudomonas aeruginosa IOC-EA-106 (MTCC 25019) in equal ratio;

In an aspect of the present invention, Stage 1 and stage 2 can be performed in same reactor or different reactor.

In another aspect of the present invention, the process disclosed herewith can be performed in continuous, batch or semi continuous mode.

In yet another aspect, the microbe can be used as whole cell, cell fractions, enzyme extracted purified or their improved mutants.

In another aspect of the present invention, the degradation rate may be further enhanced by inserting suitably at least two electrodes (i.e., at least one anode and at least one cathode, made up of the carbon or other conductive material like steel) in the reactor at both in stage -1 and stage-2 or in any one of them where electrodes are connected with each other by a conductive wire like titanium wire and poised at 200 mv to 1500 mV through some source of current like solar panel, electricity etc. The electrodes in a system will potentially serve as a non-exhaustible electron acceptor for in situ bioremediation of hydrocarbon present in the sludge or contaminated soil; hence, it will significantly shorten the remediation period. It will also help in removing the metal content from the sludge.

In another aspect of the present invention, the nutrient formulation comprises at least one nitrogen source selected from yeast extract, urea, corn steep liquor, ammonium nitrate, potassium nitrate etc., at least one phosphorus source selected from diammonuim phosphate, potassium phosphate etc. , at least one fatty acid selected from oleic acid,caprylic acid, elaidic acid, linoleic acid, palmitic acid etc. , at least one amino acid source selected from tyrosine, serine, glutamine, valine etc. and at least one vitamin source having folic acid, p-aminobenzoic acid, biotin, thaiamine etc. to the slurry, wherein the concentration of the nitrogen and phosphorus source is at least 0.001% of the sludge and the concentration of fatty acid, vitamins and amino acids is at least 0.0001% of the sludge to be treated.
In another aspect of the present invention, the reactor used in Stage 3 may be of single or multiple sub-chambers and have aeration facility.
In another aspect of the present invention, the reactor used in Stage 3 has aeration facility.
In another aspect of the present invention, the bacterial consortium MBLC used in Stage 3 can be immobilized on a matrix/membrane.
In another aspect of the present invention, the bacterial consortium in an immobilized form provides enhanced microbial cell stability, allows continuous process operation and prevents the requirement to separate the biomass-liquid separation requirement. The immobilization can be done as per the method known in prior art.
In another aspect of the present invention, the matrix/membrane can be selected from synthetic plastics, surface-modified carbon nanotubes, poly (tetrafluoroethylene) (PTFE) fibrils, zeolite, mesoporous silica, porous glass, activated charcoal, ceramics, acrylamide, polyurethane foams, polyurethane foams with impregnated with carbon nano tubes or charcoal, polyvinyl, resins and natural polymer etc.
In another aspect of the present invention, the pH and temperature needs to be controlled only beyond the specified ranges in each stage.
Another aspect of the present invention discloses the process of treatment of oil contaminated sludge or soil. The various steps and operation parameters (but not limited) of the treatment methodology in accordance with the present invention are as follows:
1. A high solid slurry of the sludge or oil contaminated soil (5-70% w/v) in a reactor.
2. Adding a nutrient formulation containing at least one nitrogen source selected from yeast extract, urea, corn steep liquor, ammonium nitrate, potassium nitrate etc., at least one phosphorus source selected from diammonuim phosphate, potassium phosphate etc. , at least one fatty acid selected from oleic acid,caprylic acid, elaidic acid, linoleic acid, palmitic acid etc. , at least one amino acid source selected from tyrosine, serine, glutamine, valine etc. and at least one vitamin source having folic acid, p-aminobenzoic acid, biotin, thaiamine etc. to the slurry, wherein the concentration of the nitrogen and phosphorus source is at least 0.001% of the sludge and the concentration of fatty acid, vitamins and amino acids is at least 0.0001% of the sludge to be treated.
3. Inoculating thus obtained slurry with selected microbial blend MBDP and stirring the slurry at low speed (100-500 rpm) under microaerophilc conditions.
4. Allowing reacting for 1-5 days at temperature: 5-70 degree C and pH: 4-10, salinity: 0-5%. pH, salinity and temperature need not to be controlled within above mentioned range.
5. Transferring the above slurry obtained at step 3 to the other reactor and inoculating with selected microbes. This step can be performed in the same reactor used at step-1 or other reactor. The reactor used should have devices for temperature, pH, agitation, aeration and stirring control and monitoring as well as provisions for inserting electrodes.
6. Allowing reacting for 2-10 days at temperature: 5-70 degree C and pH: 4-12. pH and temperature need not to be controlled within above mentioned range.
7. Obtaining reduction in hydrocarbon content of reactor in the range of 95-99% in comparison to control reactor run parallel without bacteria.
8. Allowing the settling of the slurry.
9. Removing the settled oil free solids.
10. Transferring the liquid phase to a reactor having immobilized microbes and aeration facility.
11. Allowing reacting for 2-10 days at temperature: 5-70 degree C and pH: 4-10 and temperature need not be controlled within above range.
12. Obtaining water with less than 5 ppm oil and grease.

Accordingly, the main embodiment of the present invention provides for a bio-assisted remediation method for the treatment of oily sludge or oil contaminated soil, in three stages, wherein:
(a) Stage-1 comprises subjecting the sludge or oil contaminated soil to depolymerisation under microaerophilic conditions using a consortium of microbes MBDP consisting of equal ratios of Pseudomonas putida IOC5a1 (MTCC 5385), Pseudomonas aeruginosa IOCX (MTCC 5389), Bacillus substilis (MTCC 5386), Enterobacter aerogens IOC-EA-6 (MTCC 25016), Achromobacter xylosoxidan IOC - SC-4 (MTCC 25024) in a reactor and a nutrient formulation;
(b) Stage-2 comprises degrading the resultant of Stage-1 into carbon dioxide and water under aerobic conditions using a consortium of microbes MBHD consisting of equal ratios of Pseudomonas putida IOCR1 (MTCC 5387), Pseudomonas fulva IOC SAM1 (MTCC 5802), Alcaligenes sp. IOC-MA-2 (MTCC 25022), Pseudomonas putida IOC-MA-1 (MTCC 25021) in the same or different reactor; and
(c) Stage-3 comprises transferring the resultant of Stage-2 to another reactor to obtain a water phase with less than 5 ppm of oil content by using a consortium of microbes MBLC consisting of equal ratios of Bacillus sp. IOC-EP-01 (MTCC 5870), Bacillus subtilis IOC EP-03 (MTCC 5851), Lysinibacillus sp. GP-ETPBA5 (MTCC 5666), Pseudomonas aeruginosa IOC-EA-106 (MTCC 25019).
Another embodiment of the present invention provides a bio-assisted remediation method for the treatment of oily sludge or oil contaminated soil, in three stages, wherein:
(a) Stage-1 comprises:
(i) providing high solid slurry of the sludge or oil contaminated soil (5-70%) in a reactor optionally comprising at least one anode and at least one cathode;
(ii) adding a nutrient formulation comprising at least one nitrogen source, one phosphorus source, one ester and one vitamin source to the slurry;
(iii) subjecting said slurry to depolymerisation under microaerophilic conditions using a consortium of microbes MBDP consisting of Pseudomonas putida IOC5a1, Pseudomonas aeruginosa IOCX, Bacillus substilis, Enterobacter aerogens IOC-EA-6, Achromobacter xylosoxidan IOC - SC-4;
(iv) stirring the slurry at low speed (100-500 rpm) under microaerophilc conditions;
(v) allowing reacting said slurry for 1-5 days at temperature of 5-70ºC and pH of 4-10, salinity of 0-5%;
(b) Stage-2 comprises:
(i) transferring the resultant slurry of Stage 1 to another reactor optionally comprising at least one anode and at least one cathode;
(ii) inoculating said slurry under aerobic conditions with a consortium of microbes MBHD consisting of equal ratios of Pseudomonas putida IOCR1, Pseudomonas fulva IOC SAM1, Alcaligenes sp. IOC-MA-2, Pseudomonas putida IOC-MA-1;
(iii) allowing reacting said slurry for 2-10 days at temperature and 5-70ºC and pH of 4-12,
(iv) allowing the settling of slurry;
(v) removing the settled oil free solids;
(c) Stage-3 comprises:
(i) transferring the resultant of Stage-2 to another reactor having aeration facility;
(ii) innoculating the slurry with an immobilized consortium of microbes MBLC consisting of equal ratios of Bacillus sp. IOC-EP-01, Bacillus subtilis IOC EP-03, Lysinibacillus sp. GP-ETPBA5, Pseudomonas aeruginosa IOC-EA-106;
(iii) allowing reacting said slurry for 2-10 days at temperature: 5-70 degree C and pH: 4-10;
(iv) obtaining water with less than 5 ppm oil and grease.
In another embodiment of the present invention, the nutrient formulation used in Stage-1 comprises at least one nitrogen source, one phosphorus source, one ester and one vitamin source to the slurry, wherein the concentration of the nitrogen and phosphorus source is at least 0.001% of the sludge and the concentration of fatty acid, vitamins and amino acids is at least 0.0001% of the sludge to be treated.
In yet another embodiment of the present invention, the nutrient formulation used in Stage-1 comprises KH2PO4, K2HPO4, MgSO4, (NH4)2SO4, KNO3, peptone, yeast extract, trace element and multi vitamin solution.
In another embodiment of the present invention, the depolymerization in Stage-1 comprises depolymerisation of resins, asphaltenes, waxes and other high molecular weight hydrocarbons to lower molecular weight compounds.
In another embodiment of the present invention, the microbial consortium of Stage 3 is immobilized on a matrix.
In yet another embodiment of the present invention, the matrix/membrane is selected from synthetic plastics, surface-modified carbon nanotubes, poly (tetrafluoroethylene) (PTFE) fibrils, zeolite, mesoporous silica, porous glass, activated charcoal, ceramics, acrylamide, polyurethane foams, polyurethane foams with impregnated with carbon nano tubes or charcoal, polyvinyl, resins and natural polymer etc.
In another embodiment of the present invention, Stage 1 and Stage 2 are performed in same reactor.
In another embodiment of the present invention, Stage 1 and Stage 2 are performed in different reactors.
In another embodiment of the present invention, the bio-assisted remediation can be performed in continuous, batch or semi continuous mode.
In another embodiment of the present invention, the reactors used in Stage-1 and/or Stage-2 optionally comprise at least two electrodes connected with each other by a conductive wire like titanium wire and poised at 200 mv to 1500 mV through a source of current.
In yet another embodiment of the present invention, the reactors used in Stage-1 and/or Stage-2 comprises at least one anode and at least one cathode.
In another embodiment of the present invention, the sludge comprises oily sludge and/or hydrocarbon-contaminated soil.
In another embodiment of the present invention, the oily sludge comprises all types of oil containing sludges including sludge obtained from conventional crude as well from opportunity crude, high TAN crude and sludge from product thereof.
In another embodiment of the present invention, the microbes are thermophilic.
In another embodiment of the present invention, the reactor in the third stage comprises single or multiple sub-chambers.
In another embodiment of the present invention, the reactor in the third stage has aeration facility.
Having described the basic aspects of the present invention, the following non-limiting examples illustrate specific embodiment thereof.

EXAMPLES

Example-1: Oily sludge degradation using microbial blends

A controlled stirring tank reactor having impellers was charged with 5 Kg of crude oil tank bottom sludge and 2 Liters of media containing (g per liter) KH2PO4 (1.0), KNO3 (00.75), peptone 1.0, yeast extract 5; oleic acid (0.001), caprylic acid (0.001), tyrosine (0.001), folic acid (0.001), p-aminobenzoic acid (0.001), thiamine (0.001). The tank was inoculated with MBDP microbial blend containing Pseudomonas putida IOC5a1 (MTCC 5385), Pseudomonas aeruginosa IOCX (MTCC 5389), Bacillus substilis (MTCC 5386), Enterobacter aerogens IOC-EA-6 (MTCC 25016), Achromobacter xylosoxidan IOC-SC-4 (MTCC 25024) in equal ratio to achieve final microbial count of 3.8 x 105. The temperature was 50 degree C and pH 9. Stirring was done at 200 rpm. After two days of incubation under above conditions, the slurry was transferred to another bioreactor and inoculated with another consortium MBHD consisting Pseudomonas putida IOCR1 (MTCC 5387), Pseudomonas fulva IOC SAM1 (MTCC 5802), Alcaligenes sp. IOC-MA-2 (MTCC 25022), Pseudomonas putida IOC-MA-1 (MTCC 25021) in equal ratio to achieve final microbial count of 3.1 x 105 cfu/ml. The temperature was 20 degree C and pH 6. Stirring was done at 500 rpm with aeration @ 10L/min. A control was run parallely without microbes. After ten days of incubation under above conditions the hydrocarbon degradation profile was as follows:

Day % Total Petroleum Hydrocarbon (TPH)
Reactor with microbes Reactor without microbes
1 38.9 52.6
4 17.2 46.2
6 9.3 45.6
8 0.56 42.5
10 0.01 41.3

The slurry was allowed to settle after 10 days and it produced the solid around 3% of the total sludge loaded which was free of any hydrocarbon.
The liquid phase was transferred to another reactor having bacterial blend MBLC consisting Bacillus sp. IOC-EP-01 (MTCC 5870), Bacillus subtilis IOC EP-03 (MTCC 5851), Lysinibacillus sp. GP-ETPBA5 (MTCC 5666), Pseudomonas aeruginosa IOC-EA-106 (MTCC 25019) in equal ratio which was immobilized on polyurethane foams with impregnated with charcoal. The reactor was supplied air at the rate 25ml/min. Water was recycled in this reactor for 18hrs at pH 8, temperature 60 degrees C and resulted in oil and grease content less than 5 ppm.
Example-2: Oily sludge degradation using microbial blends with electrodes
A controlled stirring tank reactor having impellers was charged with 4 Kg of crude oil tank bottom sludge and 2 Liters of media containing (g per liter) KH2PO4 (1.0), KNO3 (00.75), peptone 1.0, yeast extract 5; oleic acid (0.001), caprylic acid (0.001), tyrosine (0.001), folic acid (0.001), p-aminobenzoic acid (0.001), thiamine (0.001). . The tank was inoculated with MBDP microbial blend containing Pseudomonas putida IOC5a1 (MTCC 5385), Pseudomonas aeruginosa IOCX (MTCC 5389), Bacillus substilis (MTCC 5386), Enterobacter aerogens IOC-EA-6 (MTCC 25016), Achromobacter xylosoxidan IOC-SC-4 (MTCC 25024) in equal ratio to achieve final microbial count of 3.8 x 105. The temperature was 50 degree C and pH 9. Stirring was done at 200 rpm. After two days of incubation under above conditions, the slurry was transferred to another bioreactor having one cathode and one anode made up of activated carbon. The electrodes were connected using conductive wire and 1000mV potential was applied to the electrodes through a solar panel. The bioreactor was inoculated with another consortium MBHD consisting Pseudomonas putida IOCR1 (MTCC 5387), Pseudomonas fulva IOC SAM1 (MTCC 5802), Alcaligenes sp. IOC-MA-2 (MTCC 25022), Pseudomonas putida IOC-MA-1 (MTCC 25021) in equal ratio to achieve final microbial count of 3.1 x 105 cfu/ml. The temperature was 20 degree C and pH 6. Stirring was done at 500 rpm with aeration @ 10L/min. A control was run parallely without microbes. After ten days of incubation under above conditions the hydrocarbon degradation profile was as follows:

Day % Total Petroleum Hydrocarbon (TPH)
Reactor with microbes and electrodes Reactor without microbes and electrodes
1 21.31 52.62
4 7.42 46.25
6 1.01 45.67
8 0.007 42.51

The slurry was allowed to settle after 8 days and it produced the solid around 3.2% of the total sludge loaded which was free of any hydrocarbon. The liquid phase was transferred to another reactor having bacterial blend MBLC consisting Bacillus sp. IOC-EP-01 (MTCC 5870), Bacillus subtilis IOC EP-03 (MTCC 5851), Lysinibacillus sp. GP-ETPBA5 (MTCC 5666), Pseudomonas aeruginosa IOC-EA-106 (MTCC 25019) in equal ratio which was immobilized on polyurethane foams with impregnated with charcoal. The reactor was supplied air at the rate 25ml/min. The water was recycled in this reactor at temperature 45 degrees C and pH 6 for 18 hrs and resulted in oil and grease content less than 4 ppm.

Example-3: Effectiveness of the microbial blend to depolymerize the oily sludge

A controlled stirring tank reactor having impellers was charged with 4 Kg of crude oil tank bottom sludge and 2 Liters of media containing (g per liter) KH2PO4 (1.0), KNO3 (00.75), peptone 1.0, yeast extract 5; oleic acid (0.001), caprylic acid (0.001), tyrosine (0.001), folic acid (0.001), p-aminobenzoic acid (0.001), thiamine (0.001). The tank was inoculated with MBDP microbial blend containing Pseudomonas putida IOC5a1 (MTCC 5385), Pseudomonas aeruginosa IOCX (MTCC 5389), Bacillus substilis (MTCC 5386), Enterobacter aerogens IOC-EA-6 (MTCC 25016), Achromobacter xylosoxidan IOC - SC-4 (MTCC 25024) in equal ratio to achieve final microbial count of 3.8 x 105. The temperature was 50 degree C and pH 9. Stirring was done at 200 rpm. After 2 days of incubation under above conditions the composition of the sludge was analysed by TLC-FID technique to estimate their Saturated Aromatic Resin and Asphaltene (SARA) contents as per the method known in the prior art for the same. The results are given in table-1.

Table-1: Composition of the sludge after treatment with microbial blend.

Components Sludge treated with microbial blend (%) Sludge not treated with microbial blend (%)
Saturates 59.1 21.2
Aromatics 18.6 16.8
Resins 9.8 29.4
Ashphaltene 12.5 32.6

The table-1 showed that the microbial blend could depolymerize the higher molecular weight components like resins and asphaltenes.

The aqueous phase of the both the reactor was studied for its emulsification index and surface tension as per the method known in the prior art for the same. The results are given in Table 2 below.

Table-2: Emulsification index and surface tension of the aqueous phase after treatment with microbial blend

Components Sludge treated with microbial blend Sludge not treated with microbial blend
Emulsification index (%) 86.3% 29.7%
Surface tension mN /m 21 56

The results in Table-2 showed microbes also produce extracellular long chain hydrocarbon emulsifying and solubilizing agents, resulting in the decreased surface tension and lowered interfacial tension between oil/water phases.

Example-4: Effectiveness of the microbial blend to depolymerize the oily sludge at lower temperature and electrodes

A controlled stirring tank reactor having impellers was charged with 4 Kg of crude oil tank bottom sludge and and 2 Liters of media containing (g per liter) KH2PO4 (1.0), KNO3 (00.75), peptone 1.0, yeast extract 5; oleic acid (0.001), caprylic acid (0.001), tyrosine (0.001), folic acid (0.001), p-aminobenzoic acid (0.001), thiamine (0.001). The tank was inoculated with MBDP microbial blend containing Pseudomonas putida IOC5a1 (MTCC 5385), Pseudomonas aeruginosa IOCX (MTCC 5389), Bacillus substilis (MTCC 5386), Enterobacter aerogens IOC-EA-6 (MTCC 25016), Achromobacter xylosoxidan IOC - SC-4 (MTCC 25024) in equal ratio to achieve final microbial count of 3.8 x 105. The temperature was 5 degree C and pH 9. Stirring was done at 200 rpm. It was provided one cathode and one anode made up of activated carbon. The electrodes were connected using conductive wire and 800 mV potential was applied to the electrodes solar panel. After 30 hours of incubation under above conditions the composition of the sludge was analysed by TLC-FID technique to estimate their Saturated Aromatic Resin and Asphaltene (SARA) contents as per the method known in the prior art for the same. The results are given in table-3.

Table-3: Composition of the sludge after treatment with microbial blend.

Components Sludge treated with microbial blend(%) Sludge not treated with microbial blend(%)
Saturates 64.5 21.2
Aromatics 17.2 16.8
Resins 3.4 29.4
Ashphaltene 9.9 32.6

,CLAIMS:We Claim:

1. A bio-assisted remediation method for the treatment of oily sludge or oil contaminated soil, in three stages, wherein:
(a) Stage-1 comprises subjecting the sludge or oil contaminated soil to depolymerisation under microaerophilic conditions using a consortium of microbes MBDP consisting of equal ratios of Pseudomonas putida IOC5a1 (MTCC 5385), Pseudomonas aeruginosa IOCX (MTCC 5389), Bacillus substilis (MTCC 5386), Enterobacter aerogens IOC-EA-6 (MTCC 25016), Achromobacter xylosoxidan IOC - SC-4 (MTCC 25024) in a reactor and a nutrient formulation;
(b) Stage-2 comprises degrading the resultant of Stage-1 into carbon dioxide and water under aerobic conditions using a consortium of microbes MBHD consisting of equal ratios of Pseudomonas putida IOCR1 (MTCC 5387), Pseudomonas fulva IOC SAM1 (MTCC 5802), Alcaligenes sp. IOC-MA-2 (MTCC 25022), Pseudomonas putida IOC-MA-1 (MTCC 25021) in the same or different reactor; and
(c) Stage-3 comprises transferring the resultant of Stage-2 to another reactor to obtain a water phase with less than 5 ppm of oil content by using a consortium of microbesMBLC consisting of equal ratios of Bacillus sp. IOC-EP-01 (MTCC 5870), Bacillus subtilis IOC EP-03 (MTCC 5851), Lysinibacillus sp. GP-ETPBA5 (MTCC 5666), Pseudomonas aeruginosa IOC-EA-106 (MTCC 25019).
2. The bio-assisted remediation method as claimed in claim 1 for the treatment of oily sludge or oil contaminated soil, in three stages, wherein:
(a) Stage-1 comprises:
(i) providing high solid slurry of the sludge or oil contaminated soil (5-70%) in a reactor optionally comprising at least one anode and at least one cathode;
(ii) adding a nutrient formulation comprising at least one nitrogen source, one phosphorus source, one ester and one vitamin source to the slurry;
(iii) subjecting said slurry to depolymerisation under microaerophilic conditions using a consortium of microbes MBDP consisting of Pseudomonas putida IOC5a1, Pseudomonas aeruginosa IOCX, Bacillus substilis, Enterobacter aerogens IOC-EA-6, Achromobacter xylosoxidan IOC - SC-4;
(iv) stirring the slurry at low speed (100-500 rpm) under microaerophilc conditions;
(v) allowing reacting said slurry for 1-5 days at temperature of 5-70ºC and pH of 4-10, salinity of 0-5%;
(b) Stage-2 comprises:
(i) transferring the resultant slurry of Stage 1 to another reactor optionally comprising at least one anode and at least one cathode;
(ii) inoculating said slurry under aerobic conditions with a consortium of microbes MBHD consisting of equal ratios of Pseudomonas putida IOCR1, Pseudomonas fulva IOC SAM1, Alcaligenes sp. IOC-MA-2, Pseudomonas putida IOC-MA-1;
(iii) allowing reacting said slurry for 2-10 days at temperature and 5-70ºC and pH of 4-12,
(iv) allowing the settling of slurry;
(v) removing the settled oil free solids;
(c) Stage-3 comprises:
(i) transferring the resultant of Stage-2 to another reactor having aeration facility;
(ii) innoculating the slurry with an immobilized consortium of microbes MBLC consisting of equal ratios of Bacillus sp. IOC-EP-01, Bacillus subtilis IOC EP-03, Lysinibacillus sp. GP-ETPBA5, Pseudomonas aeruginosa IOC-EA-106;
(iii) allowing reacting said slurry for 2-10 days at temperature: 5-70 degree C and pH: 4-10;
(iv) obtaining water with less than 5 ppm oil and grease.
3. The bio-assisted remediation method as claimed in claims 1 or 2, wherein the nutrient formulation used in Stage-1 comprises at least one nitrogen source, one phosphorus source, one ester and one vitamin source to the slurry, wherein the concentration of the nitrogen and phosphorus source is at least 0.001% of the sludge and the concentration of fatty acid, vitamins and amino acids is at least 0.0001% of the sludge to be treated.
4. The bio-assisted remediation method as claimed in claims 1 or 2, wherein the nutrient formulation used in Stage-1 comprises KH2PO4, K2HPO4, MgSO4, (NH4)2SO4, KNO3, peptone, yeast extract, trace element and multi vitamin solution.
5. The bio-assisted remediation method as claimed in claims 1 or 2, wherein depolymerization in Stage-1 comprises depolymerisation of resins, asphaltenes, waxes and other high molecular weight hydrocarbons to lower molecular weight compounds.
6. The bio-assisted remediation method as claimed in claims 1 or 2, wherein the microbial consortium of Stage 3 is immobilized on a matrix.
7. The bio-assisted remediation method as claimed in claims 1 or 2, wherein the matrix is selected from synthetic plastics, surface-modified carbon nanotubes, poly (tetrafluoroethylene) (PTFE) fibrils, zeolite, mesoporous silica, porous glass, activated charcoal, ceramics, acrylamide, polyurethane foams, polyurethane foams with impregnated with carbon nano tubes or charcoal, polyvinyl, resins and natural polymer etc.
8. The bio-assisted remediation method as claimed in claims 1 or 2, wherein Stage 1 and Stage 2 are performed in same reactor.
9. The bio-assisted remediation method as claimed in claims 1 or 2, wherein Stage 1 and Stage 2 are performed in different reactors.
10. The bio-assisted remediation method as claimed in claims 1 or 2, wherein the bio-assisted remediation can be performed in continuous, batch or semi continuous mode.
11. The bio-assisted remediation method as claimed in claims 1 or 2, wherein the reactors used in Stage-1 and/or Stage-2 optionally comprise at least two electrodes connected with each other by a conductive wire like titanium wire and poised at 200 mv to 1500 mV through a source of current.
12. The bio-assisted remediation method as claimed in claim 10, wherein the reactors used in Stage-1 and/or Stage-2 comprise at least one anode and at least one cathode.
13. The bio-assisted remediation method as claimed in in claims 1 or 2, wherein the sludge comprises oily sludge and/or hydrocarbon-contaminated soil.
14. The bio-assisted remediation method as claimed in claim 13, wherein the oily sludge comprises all types of oil containing sludges including sludge obtained from conventional crude as well from opportunity crude, high TAN crude and sludge from product thereof.
15. The bio-assisted remediation method as claimed in any of the preceding claims, wherein the microbes are thermophilic.
16. The bio-assisted remediation method as claimed in in claims 1 or 2, wherein the reactor in the third stage comprises single or multiple sub-chambers.
17. The bio-assisted remediation method as claimed in in claims 1 or 2, wherein the reactor in the third stage has aeration facility.