Form 2
THE PATENTS
ACT 1970 (39 of 1970)
COMPLETE SPECIFICATION
(SECTION 10, rule 13)
"A Method of Remediation of Acidic Sludge**
Indian Oil Corporation Limited, an Indian company, ofG-9, Ali Yavar Jung Marg, Bandra (East), Mumbai - 400051, Maharashtra, India.
The following specification particularly describes and ascertains the nature of this invention and the manner in which it is to be performed.

ORIGINAL
385/MUM/2000

GRANTED
30-3-2004

Field of invention
The present invention relates to a method of disposal of sludge and more particularly to a method of disposal of acidic sludge utilizing microorganisms capable of degrading of hydrocarbon i.e., bioremediation at acidic pH.
Background of the invention
Over the past several decades worldwide production, processing, storage, transportation and utilization of synthetic and naturally occurring chemical substances has led to the introduction of significant quantities of hazardous materials into the environment. Unintentional spillage of petroleum distillates, industrial solvents and other chemical substances has been caused, for example, by weathering, chemical corrosion and accidental damage to pipes, storage vessels, processing equipment, transportation vehicles, etc. Deliberate acts and carelessness have also contributed to the release of hazardous substances into the environment. The spillage of such materials has resulted in large numbers of polluted sites and enormous volumetric quantities of soil and groundwater, which have been contaminated with hazardous substances. Soil contamination can cause extensive damage to the local ecosystem by accumulating in the tissue of animals and plants and by causing death thereto and/or mutation to the progeny thereof. Such contamination can also present a serious health threat to humans, and, in extreme cases, can render the contaminated area unsuitable for human habitation. In many cases, contaminated sites can pose a danger to adjacent property, such as by entrainment of hazardous substances by local groundwater flow, and local laws frequently mandate remediation prior to the sale or lease of property wherein the soil has been contaminated with hazardous materials.
Various methods have been utilized for the treatment, remediation or disposal of petroleum sludge. These methods generally include permanent removal of the contaminated soil to a secure landfill, incineration, indirect thermal treatment, aeration, venting, air sparging and bioremediation. Removal of contaminated soil to landfills is no longer an attractive alternative on account of the high excavation, transportation and disposal costs, and because of the potential for residual liability. Incineration and indirect thermal treatment can be achieved either on-site or off-site, but in either case involves excavation, handling and treatment of substantially all of the contaminated soil as well as significant amounts of soil adjacent to the contaminated soil,. The soil must then either be transported to the treatment facility or else the treatment apparatus must be installed on-site. In either case, these methods generally involve enormous transportation and handling costs, and require large amounts of energy to combust or volatilize the contaminants. Other elaborate and expensive techniques, which have been utilized, involve excavation and treatment of the contaminated soil using multistep unit operations for separating and recovering the soil from the contaminants. Removal, incineration, indirect thermal treatment and other methods of handling contaminated soil, which involve complete excavation of the contaminated soil, have the advantage that they can be accomplished in a relatively short amount of time. These methods are particularly attractive in those situations where there is a substantial risk that the contamination will rapidly spread to adjacent property. However, in most cases such methods are prohibitively expensive.

' It is know to exploit microorganisms to detoxify or degrade hydrocarbon contaminants.This treatment method is known as bioremediation. Bioremediation may be effected under aerobic and anaerobic conditions. Major requirements for effective bioremediation are: a biodegradable organic substrate, an appropriate and active microbial community (consortium), bioavailabilty of the polluting substrate to the microorganisms, and the creation of optimal conditions for microbial metabolism. Sometimes bioremediation requires further biostimulation with nutrients or some specific analogue substrate; it may also require bioaugumentation of the microbial community if the site does not have an appropriate indigenous biodegrading population. Bioremedaitin is a cost effective, less energy intensive and ecofriendly method for disposal of sludge.
U.S. Pat. No. 3,152,983 discloses the microbial disposal of oily wastes. This method is designed for large industrial waste separation and disposal, beginning with an oil water separator.
U.S. Pat. No. 3,462,275 discloses a process for treating biodegradable organic waste material using a thermophilic aerobic microorganism culture capable of digesting cellulose and produce cellular proteinaceous material.
U. S. Pat. No. 3,769,164 teaches a process for the microbial degradation of spilled petroleum by treating it with specially mutated species of microorganisms Canadida parapsilosis (ATCC 20246), Aspergillus sp. (ATCC 20253), Nocardia corallina (ATCC 21504), etc.
U.S. Pat. No. 3,838,198 is directed at conditioning raw waste input for digestion by thermophilic aerobic microorganisms. The process is geared towards animal waste.
U.S. Pat. No. 3,871,956 features a method for cleaning accidental oil spills on water or in soil. The method of this patent does not utilize temperature or oxygen controls and is not a self-perpetuating process.
U.S. Pat. No. 3,871,957 teaches methods of applying certain microorganisms for rapid dispersal of oil spills. The microorganisms so employed include a wide variety of bacteria yeasts, actinomyces and filamentous fungi. This reference also teaches use of certain clays such as kaolin or zeolites as carriers for such microorganisms.
The US patent 4,415,662 discloses the use of a particular fungus to degrade petroleum crude oil and products. The method comprises the application to the crude petroleum and petroleum products in an environment of an effective amount of the fungus Actinomucor elegans (Ediam) Benj. and Hasselt., Strain No. TCM05, its enzymatic active principle compound, a broth comprising it, or a carrier medium comprising the fungus or its enzymatic active principal compound.
U.S. Pat. No. 4,668,388 discloses a high rate reactor for the treatment of biosludge, wherein the reaction is conducted in an enclosure.
U.S. Pat. No. 4,913,586, describes a process and apparatus for safely handling and detoxifying contaminated soil substantially saturated with contaminants such as fuel and petroleum hydrocarbons. The contaminated soil is treated with an additive consisting of low grade humic acid and lime that is mixed with the soil in the approximate ratio of nine parts soil to one part of additive. The treatment involves breaking down the contaminated soil particles to a fine silt or

sand, mixing the additive into the soil in a tumbler and, finally, discharging the thoroughly additive-covered and encapsulated toxic soil particles into a previously dug trench.
U.S. Pat. No. 5,035,537, discloses a method for treatment of soil, porous rock and similar material contaminated by petroleum, hydrocarbon and volatile organic compounds and includes the steps of gathering the contaminated soil, disbursing it uniformly on an impervious horizontal surface to a depth of four to six inches, treating it with an emulsifying agent and allowing the emulsifying agent to seep through the soil and volatilize the hydrocarbon and organic compounds in the soil.
U.S. Pat. No. 5,039,415, relates to a method of treating hydrocarbon contaminated soil by excavating the soil, forming the soil into a flowing particulate stream; forming an aqueous liquid mixture of water and a microbe-containing solution that reacts with hydrocarbon to form CO.sub.2 and water; dispersing the liquid mixture into the particulate soil stream to wet the particulate; and allowing the substance to react with the wetted soil particulate to thereby form CO.sub.2 and water.
U.S. Pat. No. 5,055,196, discloses a process for treating soil or sludge to remove contaminants in contact with the soil or sludge. More particularly, this method relates to a process in which inorganic contaminants, such as metal or metal salts, or organic contaminants, such as PCBs, are removed from water-wet soil and sludge.
United States Patent 5,059,252 discloses a method for on-site bioremediation of soils contaminated with petroleum derived hazardous wastes.This method for enhancing bioremediation includes the step of applying a cationic ion exchange resin to the contaminated soil in an amount sufficient to promote growth of organisms capable of degrading the hazardous waste.
United States Patent 5,415,777 discloses a method of decontaminating soil contaminated by petroleum products on the site of the contamination which is accomplished by chemical breakdown, enzymatic action and biological microbial degradation. Significant byproducts of this degradation of the hydrocarbon material are: water, sodium salts, ammonium salts, carbon dioxide, free amino acids and heat.
United States Patent 5,427,944 teaches a process for biodegrading polycyclic aromatic hydrocarbon contaminants using a mixed bacteria culture of Achromobacter sp. and Mycobacterium sp and nutrient. The mixed bacteria culture was utilized for in situ or ex situ bioremediation of contaminated soil, or in any of various conventional bioreactors to treat contaminated liquids such as landfill leachates, groundwater or industrial effluents.
U.S. Pat. No. 5,453,133 relates to a process for removing contaminants, such as hydrocarbons, from soil. The process involves contacting the contaminated soil with a suitable solvent for the contaminant, in the presence of a bridging liquid which is immiscible with the solvent, while agitating. The amount of the bridging liquid and the degree of agitation are balanced to control the particle size of the substantially contaminant- and solvent-free soil agglomerates so formed.
U.S. Pat. No. 5,494,580 relates to a method for decontamination of a hydrocarbon-polluted environment by the use of certain bacterial compositions.

United States Patent 5,609,667 discloses a method of treating hydrocarbon contaminated soils. A powdered cellulose, containing essentially 3-8% of ammonium sulfate forms a biologically active media which preferentially adsorbs hydrocarbons in the presence of water and supports the growth of naturally occurring hydrocarbon reducing bacterial forms resulting in rapid decomposition of the adsorbed hydrocarbons into water, carbon dioxide and other benign waste products.
United States Patent 5,624,843 discloses method for improving the bioremediation of hydrocarbon contaminated water with indigenous microorganisms comprising adding to the hydrocarbon contaminated water a hydrocarbon solution of an additive selected from the group consisting of (a) a mixture of a sorbitan ester of a C.sub.7 to a C.sub.22 monocarboxylic acid and a polyaxyalkylene adduct of a sorbitan monoester of a C.sub.7 to C.sub.22 monocarboxylic acid, the adduct having from 6 to 50 polyoxyalkylene units, (b) an alkylglycoside wherein the alkyl group has from about 8 to 18 carbon atoms and the glycoside is a mono or a diglycoside, and (c) a mixture of (a) and (b), the solution being added in amounts sufficient to promote the growth of indigenous micro organisms.
United States Patent 5,807,724 teaches a method and means for enabling the remediation and removal of spilled oils or fats (lipids) from land, moving or standing fluids ,slurries, or semi¬solid, processed manufactures, or from food substances using three particular species of the yeasts (Candida albicans; Candida guilliermondii; and Candida yarrowia) which possess the capability to produce an enzyme, lipase, which is capable of breaking down hydrocarbon-based substances, e.g., crude oil and other petroleum distillates, either paraffin-based or possessing other peculiar chemical bases.
US patent 5811290 describes a method for enhancing bioremediation of hydrocarbon contaminated soils,water and/or sludge using urea-surfactant clathrate and U.S. Pat. No. 5,834,540 relates to a composition and process for the remediation of contaminated materials, and in particular for soil remediation. The reference relates to compositions and processes that cause the breakdown of unwanted contaminates, such as hydrocarbon wastes. The reference relates to the further use of radiation, preferably microwave radiation, to effect the degradation of contaminates. The reference is equally effective in treating materials that have been exposed to harmful or infectious biological contaminates.
United States Patent 6,057,147 discloses an apparatus and method for enhanced bioremediation of hydrocarbons removed from a contaminated object. The device and method promote continuous microbial bioremediation of hydrocarbon contaminants in a self-propagating manner while cleaning the solution and filtering sediment without generating an environmentally dangerous waste trail. The cleaning solution comprises microorganisms of the genus Achromobacter, Actinobacter, Alcaligenes, Arthrobacter, Bacillus, Nocardia, Flavobacterium, Pseudomonas and mixtures thereof.
United States Patent 6,503,746 pertains to isolated strains Paenibacillns validus that degrade polyaromatic hydrocarbons, a method of isolating the strains of the invention and a method of using the strains of the invention for bioremediation.
The United States Patent 20020187545 application discloses a process for Bioremediation of hydrocarbon contaminated waste using corn material The hydrocarbonaceous contaminant is

contacted with the corn material in the presence of nutrients and bacteria effective for bioremediation.
The 20030100098 US patent application discloses the application of bacterial strains to bioremediate ployaromatic hydrocarbon contaminated soils and groundwater.
Objectives of the invention
Old refineries have long lasting problem of safe disposal of spent acid tar (acidic sludge), which
is accumulated over several decades as a result of acid treatment of wax and lube base oils.
However, the acid treatment of lube oil and wax has been discontinued but there is huge
inventory of this acid tar in refineries. For example Digboi refinery in India have an inventory of
20,000- 50,000 KL which is stored in the open sludge ponds. In past several attempts to dispose
the accumulated spent tar has been made but met with only a limited success. Conventional
practices generally do not provide a practical, affordable technology for remediating acidic
sludge in ecofriendly and intensive manner Biodegradation of acidic sludge is a difficult task
because at low pH the normal sludge degrading microorganisms are unable to grow.
The above prior art is silent regarding bioremediation of acidic petroleum sludge. There is a need for a process that will rapidly decontaminate acidic hydrocarbon sludge in an efficient and environmentally acceptable manner. Accordingly, the objective of the invention is to provide a method for bioremediation of acidic sludge by acid tolerant hydrocarbon assimilating microbes.
Summary of invention
The instant invention teaches a method and means for enabling the remediation and removal of acidic oily sludge, spilled oils, and slurries.
Present invention relates to isolation of two particular species of the yeast which possess the capability of breaking down hydrocarbon substances, e.g., crude oil and other petroleum distillates, either paraffin-based or possessing other peculiar chemical bases in acidic as well as in neutral pH.
These isolates have been used to develop a process for remediation of acidic sludge which comprises, mixing the sludge with soil, providing microbial consortia capable of degrading hydrocarbon to soil sludge mixture, in the presence of appropriate quantity of nutrients.
Detailed description of the invention
Old refineries have long lasting problem of safe disposal of spent acid tar (acidic sludge), which is accumulated over several decades as a result of acid treatment of wax and lube base oils. However, after development of the state of art technologies the, acid treatment of lube oil and wax has been discontinued, but there is huge inventory of this acid tar in refineries. For example Digboi refinery in India have an inventory of 20,000- 50,000 KL which is stored in the open sludge ponds.
In past several attempts to dispose the accumulated spent tar has been made but met with only a limited success. These methods generally include secure landfill, incineration, indirect thermal

treatment, aeration, venting, air sparging and bioremediation. Sludge stored in storage pits was source for contamination the ground water. Conventional practices generally do not provide a practical, affordable technology for remediating acidic sludge in ecofriendly and intensive manner. One of the methods was neutralization with lime followed by drying and then use of the material in brick kilns as fuel. This method was not also successful because of combustion-related problems and emission issues.
The microorganisms can be employed to detoxify or degrade hydrocarbon contaminants. This treatment method is known as bioremediation. Bioremedaitin is a cost effective, less energy intensive and ecofriendly method for disposal of sludge. But bioremediation of acidic sludge is a difficult task because at low pH the normal sludge degrading microorganisms are unable to grow. However, the specialized sludge degrading microorganism which have special adaptations to survive in acidic environment, can degrade the sludge. Such microbes can be developed from indigenous microbial population of acidic sludge by the selective enrichment technique
Major requirements for effective bioremediation is a biodegradable organic substrate, an appropriate and active microbial community (consortium), bioavailabilty of the polluting substrate to the microorganisms, and the creation of optimal conditions for microbial growth. Sometimes bioremediation requires further biostimulation with nutrients, surfactants, or some specific analogue substrate; it may also require bioaugumentation of the microbial community if the site does not have an appropriate indigenous biodegrading population. In the bioaugumentation the naturally occurring but selected specific microbes are cultivated in laboratory condition and then supplemented in the field.
The process of development of bioremediation techniques includes steps namely, Isolation and characterization naturally occurring microorganisms with bioremediation potential by selective enrichment technique , optimization of large-scale cultivation of these microbes and its absorption on suitable carrier . Next step is evaluation of its ability to degrade petroleum sludge and its different fraction separately in liquid media as well as in lab scale soil experiments and optimization of conditions like nutrient ,moisture condition ,aeration for enhanced biodegradation in experimental conditions. Then the process is tested and optimized in field.
The hydrocarbon degrading microbe of present invention was isolated by selective enrichment culture technique from indigenous microbial population of acidic sludge taking acidic sludge as carbon source and inoculum.
Enrichment culture technique is based on basic principle of selection and allows growth of organism of interest, and as unfavorable as possible for competing organisms. In this technique the growth parameters, including cultural nutrient medium, conditions are controlled to favor the development of a specific organism or group of organisms.
The first cycle of enrichment was carried out by adding 5 gms of the soil in 100 ml of medium and flask were incubated at 30 0c on shaking condition .An enrichment cycle was repeat four time using above mentioned minimal salt media and acid tar as sole carbon source.
A suitable nutrient medium for use during the enrichment process should, at minimum include a nitrogen source, such as an ammonium salt, and a phosphorus source, such as an alkali metal phosphate compound. The nutrient system used during the process of acclimating the mixed bacteria culture to hydrocarbon compounds desirably includes a magnesium source, such as a

magnesium salt, and can optionally include other nutrients such as sodium, calcium and iron salts. A suitable nutrient system which can be effectively utilized during the enrichment process includes an ammonium salt and a phosphate compound, along with minor amounts of other conventional nutrients, wherein the molar ratio of elemental nitrogen to phosphorus is from about 5: 1 to about 15: 1, and more preferably from about 8: 1 to about 12: 1. A particularly preferred nutrient system for use during the enrichment process includes ammonium chloride, from about 5 to about 20 parts by weight of hydrated magnesium sulfate (Msub.a SO.sub.4.7H.sub.2 0) per 100 parts by weight of ammonium chloride, from about 5 to about 20 parts by weight of sodium chloride per 100 parts by weight of ammonium chloride, and from about 15 to about 50, and more preferably from about 20 to about 30 parts by weight of monobasic potassium phosphate (KH.sub.2 PO.sub.4) per 100 parts by weight of ammonium chloride and traces of vitamins and trace elements. The pH of the medium was kept acidic preferably 3. The foregoing nutrients are dissolved in a suitable amount of water to dissolve the nutrients and combined with appropriate quantities of a suitable initial primary food source and the mixed bacteria culture. A suitable initial primary food source is generally total acidic sludge or or its total petroleum hydrocarbon or its saturates,aromatic ,asphaltene fractions. In particular, it is recommended that the acclimation process be carried out by adding samples of the soil, which is to be remediated to the mixed microbial culture.
Isolation of the pure desired isolates was done by streaking on minimal salt agar medium containing .05-5% acidic sludge as carbon source and pH of medium was maintained 3.0.These plates were incubated at 300 c. Eight microbial strains were obtained at the end of the enrichment cycle from the acidic tar sludge.
The two acidic sludge degrading isolates showing highest growth and degradation potential have been identified as Candida digboigensis (dig,boi.en'sis) L. nom.Fem.Adj.digboigenesis referes to place Digboi in Assam state of India. These cultures are deposited in MTCC (Microbial Type Culture Collection and Gene Bank) at Institute of Microbial Technology, Chandigarh. , under number MTCC 4371 and 4372 respectively.The type strain of C. digboigenesis is strain MTCC 4371 and also deposited in collection of Yeast Division of the Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands. These two microbes were combined to make the microbial blend for bioremediation and were used in present invention.
The isolates were evaluated separately and in combination for their potential to degrade acid tar and its fractions at acidic pH.For that the individual isolate or mixture was inoculated in 5 ml of mineral salt medium .After 48 hr growth this 5 ml culture was transferred in 20 ml fresh medium.After 48 hrs growth 20 ml culture was transferred to minimal salt 80 ml which contained acidic sludge or its toluene or hexane soluble fraction (1% ) as carbon source. All experiments were carried out at pH 3 and 30 0c temperature in shaking condition. After incubation of 10 days the residual substrate was measured. In addition, the ability of pure strains to grow separately on various fraction of acidic sludge at pH 3 was also examined.
After successful isolation and efficacy test in laboratory scale the microbial consortia /blend was
grown in bioreactor. An inoculum for bioreactor, of the mixed culture of the present invention
was prepared by first growing the individual microbe on separate agar plates in a conventional
manner. After sufficient growth of the individual microbe is achieved, both microbes can be
transferred to a fresh agar plate for simultaneous growth of both organisms together in a mixed
culture. After the mixed culture exhibits successful growth, it can be transferred to a suitable
vessel containing a nutrient solution. The vessel preferably should contain control devices for

tempreture, pH, agitation, aeration and stirring .Particularly preferred nutrient system for growth includes (g per liter) KH2P04 0.5-1.0, K2HP04 0.5-1.0,Mg S04 0.5-1.0,(NH4)2SO4 0.25-0.75,KNO3 0.25-0.75,Trace element 5 ml to 20 ml of solution and Multi vitamin solution 0.5-5 ml, fermentable sugars as carbon source 1 -5%.
The composition of Trace element solution (gram per litre) is Nitrilotriacetic acid 1-1.5 ,FeS04 . 7H20 0.05-0.15,MnCl2.4.H20 0.005-0.015,CoCl2.6,H20 0.15-0.2,CaCl2 . 2H20 0 .05-0.15,ZnCl2 0.05-0.15, CuCl2.H20 0.01-0.03^3BO3 0.01-0.02,Na2MoO4 0.01-0.02,Na2SeO3 0.015-0.02,NiSO4 0.01-0.03,SnCl2 0.01-0.03.The composition of Trace element solution Multivitamin solution (g/1) is Biotin 0.001-0.003,Folic acid 0.001-0.003,Pyridoxine HC1 0.05-0.02,Thiamine HC10.002-0.008,Riboflavin 0.001-0.01,Nicotinic acid 0.002-0.01,Ca-Pentotheonate0.002-0.01,Lipoic acid 0.0025-0.0075,Vitamin B12 0.0005-0.0015,PABA 0.0025-0.0075.
The appropriately grown mixed microbial culture of the invention was adsorbed on suitable carrier. In the invention, microorganisms capable of degrading hydrocarbon contaminants are dispersed in soil while being supported on, i.e., fixed in a carrier.
As the carrier for supporting microorganisms there may be used any known material so far as it can be applied to contaminated soil with microorganisms supported thereon. From the standpoint of effective supporting of useful microorganisms, carrier materials which can firmly adsorb microorganisms to the surface thereof be helpful in transport and dispersal of final bioremediation agent.
The carrier made of the materials that can retain microorganisms thereon relatively mildly and thus allows easy release of microorganisms thus proliferated. The carrier is inexpensive and can act as a nutrient source for the microorganisms thus applied, particularly a nutrient source, which can be gradually released to advantage.
Further, the formation of a carrier by a biodegradable material is advantageous in that any problems arising from secondary contamination by residual carrier or the effect of applied microorganisms on the soil ecological system can be avoided. As such a biodegradable carrier there is preferably used a material which gradually decomposes and disappears after the remediation of soil by applied microorganisms. When such a carrier is used, applied microorganisms which have been released into soil after the disappearance of the carrier are then put in environments which are severe to growth such as competition with predominant native-born microorganisms in soil and predation by protozoan. The microorganisms are then driven out of soil and gradually decrease in number to extinction. As a result, the ecological system in soil can be restored to the original state.
Preferred examples of the biodegradable carrier material employable herein include corn husk,sugar industry waste or any agricultural waste.
The water content of the carrier is from 1% to 99% by weight, preferably from 5% to 90% by weight, more preferably from 10% to 85% by weight. When the water content of the carrier is too low, microorganisms find difficulty in survival. On the contrary, when the water content of the carrier is too high, the resulting carrier exhibits a deteriorated physical strength that makes itself difficult to handle.

The carrier adsorbed microbial blend was tested for its efficacy in liquid medium as well in lab scale soil experiments. After successful lab scale validation the process was optimized in field. The parameters of field studies are volume of soil bed, sludge dosing and mixing, microbial blend dosing, nutrient composition and dosing and tilling and moisture content. A plot was brick lined and cemented and impermeable plastic sheet was put in bottom .This area was demarcated with cemented boundary preferably of 45 cm height.In this pit a uniform soil layer preferably of 15 cm was preapred.
On the plot, acidic sludge was spread uniformly around 5 -40% , preferably 10% (w/w) and thoroughly mixed with soil bed.
After mixing bed was inoculated with microbial blends around 0.1-5% w/w based on sludge concentration. Inoculum size was preferably 0.2%.
Nutrients were also applied at the rate of 10 mg to 100 mg /100 kg of sludge after making its 5-25% solution in water.
As nutrients there are preferably used materials containing carbon, nitrogen and phosphorus. A culture solution suitable for the growth of microorganisms may be used. As such a culture solution there is widely used a material having a meat juice, a yeast extract, a malt extract, bactopeptone, glucose, inorganic salts, mineral, etc. in admixture at a proper ratio is widely used. These components may be mixed at a proper ratio depending on the kind of microorganisms. As the nutrients to be used in the invention there may be used any nutrients containing proper organic and inorganic nutrients besides the aforementioned culture solution. Preferable nutrient is mixture of yeast extract and potassium nitrate in 1:1.
The site was again tilled to mix the content i.e. acid tar and microbial blend along with nutrient, to a depth of around 7-10 cm. Tilling and watering of plot was carried out to enhance growth of applied microbes. The moisture content of the soil-sludge mix should be more preferably from 30% to 80% of the water retention capability of the soil -sludge mix . When the water content is too low, microorganisms find difficulty in survival. On the contrary, when the water content is too highjit stops aeration.
An important aspect of the remediation treatment is that mechanical mixing and tilling of soil sludge mix at starting of treatment and in between of treatment. This helps in the aeration, which is essentially required of microbe of present invention for growth and survival. Preferably the tiling should be carried out in 15 days interval and it preferably up to depth of plastic sheet put in the bottom. The another advantages of mechanical agitation to promote mixing are several. Tilling of the soil creates better contact between organic compound contaminants, and microbes to promote faster reaction rates and increase the likelihood that the degradative reactions will proceed to completion. Secondly, the thorough mixing enabled by mechanical agitation aids in controlling and dissipating then heat generated by the microbial growth.
The temperature at which soil remediation is effected needs to be suitable for the action of microorganisms, i.e., from 3.degree. C. to 50.degree. C, preferably from lO.degree. C. to 45.degree. C, more preferably from 18,degree. C. to 40.degree. C. In order to keep the microorganisms within this temperature range, heating such as spraying and injection of hot water may be effected depending on the situation. In cold districts, a heat conductor may be inserted into soil so that heat from a heat source can be transferred to soil. Alternatively, the heat

conductor there may be used any material, which can transmit heat such as metal and ceramics.
The present invention therefore provides a safe, effective and inexpensive means for eliminating acidic oily sludge from environment.
To further illustrate the present invention, reference is made to the following examples-
Examplel
Isolation and Characterization of acidic sludge -degrading microbe
The nutrient media (mineral salt media, MSM) used for enrichment process includes 1.0% KH2P04, 1.0 % K2HP04, 0.5% Mg S04, 0.75%, NH4Cl 2.0%, NaCl 0.2 % (V/V) of trace element solution and 0.01% (V/V) vitamin solution. The trace element contained 0.15% nitrilotriacetic acid, 0.001% FeS04.7H20, 0.005% MnCl2.4.H20, 0.01 %CaCl2.2H2O, 0.05-% 0.05% ZnCl2,0.001%CuCl.2.H2O.The composition of Multivitamin 0.01%Biotin, 0.03% Folic acid, 0.02%, Thiamine HC1, 0.01% Riboflavin, 0.02% Nicotinic acid, and 0.005%Vitamin B12.The foregoing nutrients are dissolved in a suitable amount of water to dissolve the nutrients. In this medium 5% acidic sludge was used as inoculum and total carbon source. The pH of media kept acidic preferably 3.The first cycle of enrichment was carried out by adding 5 gms of the soil in 100 ml of medium and flask were incubated at 30 0c on shaking condition. The cultures were monitored for the presence of microorganisms by gram staining and microscopy and subcultured into fresh MSM medium when growth was detected. After 3 to 4 subcultures, the mixed culture was plated onto MSM agar plates containing the same substrate as the enrichment.
The mixed culture obtained by enrichment was evaluated for its potential to degrade acid tar .For that the mixed culture was inoculated in 5 ml of MSM medium. After 48 hr growth this 5 ml culture was transferred in 20 ml medium. After 48 hrs growth 20 ml culture was transferred to minimal salt 80 ml, which contained toluene and hexane extract of acidic sludge (1%) as carbon source. All experiments were carried out at pH3. After incubation of 10 days the residual substrate was measured. Pure desired isolates from the mixed culture was isolated by streaking on minimal salt (MSM) agar medium containing .05-5% acidic sludge as carbon source and pH of medium was maintained 3.0.These plates were incubated at 30°C. Eight microbial strains were obtained at the end of the enrichment cycle from the acidic tar sludge.
In addition, the ability of pure strains to grow separately on various fraction of acidic sludge at pH 3 was also examined. The two acidic sludge degrading isolates showing highest growth and degradation have been identified by MTCC (Microbial Type Culture Collection and Gene Bank) at Institute of Microbial Technology, Chandigarh. These were identified as Candida digboigensis (dig, boi.en'sis) L. nom.Fem.Adj.digboigenesis referes to place Digboi in Assam state of India. These cultures are deposited in MTCC under number MTCC 4371 and 4372 respectively. The type strain of C digboigenesis is strain MTCC 4371 and also deposited in collection of Yeast Division of the Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands.

Example 2
Large-scale cultivation and mixing with carrier
An inoculum of the mixed culture of the present invention was prepared by first growing
the individual microbe on separate Luria agar plates in a conventional manner. After
sufficient growth of the individual microbe is achieved, both microbes can be transferred to
a fresh agar plate for simultaneous growth of both organisms together in a mixed_culture.
After the mixed culture exhibits successful growth, it can be transferred to a suitable vessel
containing a nutrient solution. The vessel preferably should contain control devices for
temperature, pH, agitation, aeration and stirring. The temperature was kept 30°C, ph 3,
stirring 200 2pm and air 5 L /min. Used nutrient system for growth includes (g per liter)
KH2P04 0.5-1.0, K2HP04 0.5-1.0, Mg S04 0.5-1.0, (NH4SO4 0.25-0.75, KNO3 0.25-
0.75, Trace element 5 ml to 20 ml of solution and Multi vitamin solution 0.5-5 ml, 10%
sucrose as carbon source. The composition of Trace element solution (gram per liter) is
Nitrilotriacetic acid 1-1.5, FeS04. 7H20 0.05-0.15,MnCl2.4.H20 0.005-0.015,
C0Cl2.6H2O 0.15-0.2, CaCl2.2H20 0.05-0.15, ZnCl20.05- 0.15, CuCl.2.H20 0.01- 0.03, H3BO3 0.01- 0.02, Na2Mo04 0.01-0.02,Na2SeO3 0.015-0.02, N1SO4 0.01-0.03, SnCl20.01-0.03. The composition of Trace element solution Multivitamin solution (g/1) is Biotin0.01 0.003,Folic acid 0.001-0.003,PyridoxineHCl 0.05-0.02, Thiamine HC1 0.002-0.008, Riboflavin 0.001-0.01, Nicotinic acid0.002-0.01,Ca-Pentotheonate 0.002-0.01, Lipoic acid 0.0025-0.0075,VitaminB120.0005-0.0015,PABA 0.0025-0.0075.
The appropriately grown mixed microbial culture of the invention was adsorbed on previously UV sterilized corn floor in ratio l:6(v/w). This material was packed in UV sterilized plastic bag keeping 80% space for air and it was sealed.
Example 3
Biodegradation in liquid media
The media used for biodegradation consisted of Minimal Salts Medium (MSM) supplemented with 0.25% yeast extract. Runs to determine the biodegradation of sludge with respect to incubation time were carried out in 250-ml Erlenmeyer flasks containing 50 ml of media and 5 g of sludge. The flasks were inoculated with Candida digbioensis. The flasks were incubated at 30°C and at 220 rpm. The residual total petroleum hydrocarbons.(TPH) content was determined by extraction with 1:1 mixture of hexane and toluene. The solvent layer was separated in separating funnel and transferred to pre -weighted vial. The solvent was evaporated in fume hood and residual oil was weighed to determine total petroleum hydrocarbons.

The results are as follows-
Tablel Biodegradation of acidic sludge in soil in lquid media

Incubation time (days) TPH degradation (%)
7 22
14 41
21 63
28 78
56 92
It was found that over a period of 56 days ,about 92 % degradation of TPH occurred. Example 4
Degradation of Sludge in soil
A plot of 200 square meters was marked and lined with cemented brick and impermeable plastic sheet was put in bottom over the brick layer .This area was demarcated with cemented boundary of 45 cm height.In this pit auniform soil ayer preferably of 15 cm was prepared. On the plot around 5 MT of acidic sludge was spread uniformly This was thoroughly mixed with
After mixing bed was inoculated with 80 kg microbial blends along with 3 kg of nutrient
(KN03 and yeast extract in 1:1 (w/w) dissolved in 100 lit of tap water The site was again rilled
to mix the content .Mechanical tilling of site was carried out on every 15 days .Watering was
carried out to maintain appropriate moisture level.
Residual total petroleum hydrocarbons (TPH) content was determined as follows-At timed intervals, 10 g of representative soil-sludge mix was refluxed with 50 ml of hexane . The residual material was then refluxed with 50 ml of toluene The solvent layers was separated in separating funnel and pooled and transferred to pre -weighted vial.The solvent was evaporated in fume hoods and residual oil was weighted to determine total petroleum hydrocarbons. The results are as follows-
Table2 : Biodegradation of acidic sludge in soil

days TPH degardation (%)
30 23
60 36
90 52

120 76
150 91
The pH of the samples was determined by taking 25 g of representative and homogenized sample of sludge /soil mix in a beaker containing 25 ml ultra pure water and allowed to mix in rotatory shaker at 180 rpm for four hours. The content were allowed to settle and filtered. The pH of the supernatant was measured by pH meter on zero days and after every 30 day of bioremediation.
The results are as folio ws-
Table3. pH profile of soil mixed with acidic sludge under treatment

Days PH
0 2.9
60 3.6
90 4.3
120 5.2
150 5.7

We claim:
1. A method of remediation of acidic sludge, which comprises: mixing the sludge with soil, adding microbial consortia capable of degrading hydrocarbon to soil-sludge mixture, wherein the microorganisms are supported on a carrier; and providing nutrient for the microorganisms to the soil-sludge mixture.
2. A method of remediation of acidic sludge as claimed in claim 1. wherein the microbial consortia capable of degrading hydrocarbon includes microorganisms capable to degrade aromatic, aliphatic, naphthenic fraction of sludge as herein described at acidic pH.
3. A method of remediation of acidic sludge as claimed in claim 1, wherein the microorganism is yeast.
4. A method of remediation of acidic sludge as claimed in claim 3. wherein the microbial consortia includes Candida digboigensis MTCC 4371 and Candida digboigensis MTCC 4372.
5. A method of remediation of acidic sludge as claimed in claim 1. wherein the carrier for support of microorganisms is biodegradable.
6. A method of remediation of acidic sludge as claimed in claim 1, wherein the carrier is made of materials that can retain microorganisms thereon relatively mildly and thus allows easy release of microorganisms thus proliferated.

7. A method of remediation of acidic sludge as claimed in claim 1, wherein the microorganisms are supported on the carrier of natural origin including comhusk, sugar industry waste or agricultural waste.
8. A method of remediation of acidic sludge as claimed in claim 1, wherein the water content of the carrier is from 1% to 99% by weight.
9. A method of remediation of acidic sludge as claimed in claim 1, wherein the water content of the carrier is preferably 10% to 85% by weight.
10. A method of remediation of acidic sludge as claimed in claim 1, wherein the nutrients are compounds that provide nitrogen source and phosphorus source to microorganisms in preferable molar ratio of elemental nitrogen to phosphorus 5:1-15:1.
11. A method of remediation of acidic sludge as claimed in claim 10, wherein the nutrient system optionally includes a magnesium source.
12. A method of remediation of acidic sludge as claimed in claim 10, wherein the nutrient system optionally includes sodium, calcium and iron salts.

13. A method of remediation of acidic sludge as claimed in claim 10, wherein the nitrogen source is an ammonium salts.
14. A method of remediation of acidic sludge as claimed in claim 10, wherein the phosphorus source is an alkali metal phosphate compound.
15. A method of remediation of acidic sludge as claimed in claim 10. wherein the preferable molar ratio of elemental nitrogen to phosphorus is 8:1-12.1.
16. A method of remediation of acidic sludge as claimed in claim 1, wherein the nutrient system has the following composition ammonium chloride, from 5 to 20 parts by weight of hydrated magnesium sulfate (M.sub.a SO.sub.4.7H.sub.2 0) per 100 parts by weight of ammonium chloride, from 5 to 20 parts by weight of sodium chloride per 100 parts by weight of ammonium chloride, and from 15 to 50 and more preferably from 20 to 30 parts by weight of monobasic potassium phosphate (KH.sub.2 PO.sub.4) per 100 parts by weight of ammonium chloride and traces of vitamins and trace elements.
17. A method of claim 1, optionally comprising the step of:
periodic tilling the soil-sludge mixes to loosen and to increase aeration for bacterial
growth;
maintaining the moisture content, which is at least 80% of the water retention
capability of the soil -sludge mix; and
testing the soil-sludge mix periodically to monitor reduction in amount of
hydrocarbon contaminants contained in the soil.
Dated this 29th day of March 2004
Archana Shanker
of Anand and Anand, Advocates