Biodegradation of Hydrocarbons from Crude Oil by Pseudomonas Putida

Bio adulteration of Hydrocarbons from Crude Oil by Pseudomonas putida A Project dvirtuoso down the stairs the advocate of Dr. K. Bharathi plane section of Biotechnology. Submitted to the faculty Of Department of Biotechnology National Institute Of Technology, Warangal (A. P) Submitted By Febin P. Nalpady, Anzal Rahman, Shruti Sharma, Sindhuja Nandiraju, Giraboina Kranthi Kumar find out INSTITUTE OF TECHNOLOGY WARANGAL (A. P) (DEEMED UNIVERSITY) 2010-2011 DEPARTMENT OF BIOTECHNOLOGYNATIONAL INSTITUTE OF TECHNOLOGY, WARANGAL (A. P) certification This is to certify that the project entitled . carried away by .. , bearing wheeling no. . ,, final year B. Tech, Biotechnology, during academic year 2010-2011, is a bonafide pee-pee submitted to the National Institute of Technology, Warangal in partial fulfillment of the requirements for the requirements for the award of the micro place setting stage of Bachelor of Technology. Guide Dr. K Bharathi Dept. of Biotechnology NIT Waran gal ACKNOWLEDGEMENTWith great sport and deep sense of gratitude, we take this opportunity to express our sense of responsibility to Dr. K Bharathi, our project guide for accepting us under her skilful self to carry out this project hold up, and providing us his invaluable guidance and constant encouragement at each and every pervert passim the progress of this project. To be sincere it was an inextinguishable treasure of pleasure for us to plow under her ex stallent guidance. I would similarly like to thank the faculty of our department,Dr. P Sreenivasa Rao, Mr. Onkara Perumal and Mr. K. Narasimhulu.They were a pillar of strength for us and advance us to do our best. Name Roll no Table of content 1. Abstract 2. grounding 2. 1 Bioremediation 2. 2 The conventional proficiencys of remediation 2. 3 Advantages of Bioremediation 2. 4 Microbes that ar useful for bioremediation 3. Review of literature 3. 1 microbic humiliation 3. 2 Bio humiliation of crude colour hydrocarbo ns 3. 3 Factors affecting Degradation 3. 4 Mechanism of rock crude crude Hydrocarbon Degradation 4. Brief outline of the project work 5. Materials and Methods 5. 1 Preparation Of nutritive blood 5. 2 Preparation of food Agar Slants from the bacterial Strain 5. Preparation of SubCultures of Bacterial Strain 5. 4 Centrifugation of Crude Oil 5. 5 Subculturing Of Petri Plates with inunct 5. 6 Bio adulteration Studies 5. 7 hydrometric abstract 6. Results 6. 1 ontogenesis Analysis of Pseudomonas Putida 6. 2 gravimetric Analysis 7. Discussion 8. References 1. ABSTRACT Oil cast offs adjudge become a serious problem with the ever-increasing resource exploitation, transportation, store, and accidental leakage of embrocate. Several techniques, including forcible, chemic, and biological orders, argon utilize to recover spilled embrocate from the surround.Bioremediation is a promising plectrum for remediation since it is effective and economic in removing oil with less grou ndless environmental damages. However, it is a relatively slow exploit and the degree of supremacy depends on a exit of factors. These factors include the existence of a microbic population roofitalable of degrading the pollutants, the availability of contaminants to the microbial population and the environment factors argon fibre of injury, temperature, pH, the presence of oxygen and nutritives. This project aims to study the degradation extent of the pseudomonas putida on oil.The microbial strain utilize is procured from NCL pune. 2. Introduction In quantitative terms, crude oil is hotshot of the most crucial organic pollutants in naval environment and it has been estimated that worldwide somewhere in the midst of 1. 7- 8. 8? 106 tons of oil hydrocarbons impact shipboard soldier water systems and estuaries annually. Reports absorb been appearing since last three decades on the biodegradability of crude oil by bacteria which tush use hydrocarbons as source of car bon and energy. A way to mitigate the effects of oil spills is bioremediation. 2. 1 BioremediationIt is a process by which chemical substances argon degraded by bacteria and early(a) microorganisms. The use of these microorganisms has been successfully applied for the treatment of waste and wastewater in controlled systems. Several research studies comport recently been performed to investigate the use of bioremediation for oil-spill cleaning in seawater, freshwater and terrestrial aras. The technique has been form to have a potential for broad applications in terrestrial and freshwater environments for treating domains and sediments bemire with oil and other substances, as strong as for coastal environments impacted by oil spills.Water is a more sensitive medium than soil and requires dissimilar remediation techniques. Spills to sur present water are easier to clean up than spills to groundwater, for obvious reasons. It is non only much harder to see the extent of the con tamination, except also to pull back the source of the contamination as, for example, a leaking underground storage tank. 2. 2. The conventional techniques of remediation . The conventional techniques used for remediation have been to intrude up contaminated soil and take it to a landfill, or to poll and contain the contaminated areas of a spot.The methods have some drawbacks. The first method simply moves the contamination elsewhere and whitethorn create signifi flockt risks in the excavation, handling, and transport of hazardous material. Additionally, it is very difficult and increasingly expensive to keep new landfill sites for the final disposal of the material. A better approach than these handed- fell methods is to contendly destroy the pollutants if workable, or at least to transform them to innocent(p) substances.Some technologies that have been used are senior high-temperature incineration and various types of chemical degeneracy (e. g. , base-catalyzed dechlo rination, UV oxidation). They can be very effective at trim back levels of a image of contaminants, but have several drawbacks, principally their technological Byzantineity, the cost for small-scale application, and the lack of public acceptance, especially for incineration that may harvest-feast the exposure to contaminants for two the workers at the site and nearby re inclinents. . 3 Advantages of Bioremediation Bioremediation is an option that offers the possibility to destroy or render harmless various contaminants apply natural biological activity. As such, it uses relatively low-cost, low-technology techniques and can often be carried out on site. It go forth not always be suitable, however, as the range of contaminants on which it is effective is limited, the timescales involved are relatively long, and the rest contaminant levels achievable may not always be appropriate.Although the methodologies utilise are not technically complex, considerable experience and ex pertise may be required to design and implement a successful bioremediation program, receivable to the need to thoroughly assess a site for suitability and to optimise conditions to achieve a satisfactory result. Because bioremediation seems to be a good alternate(a) to conventional clean-up technologies research in this field, especially in the United States, apace increasing. Bioremediation has been used at a number of sites worldwide, including Europe, with varying degrees of success.Techniques are modify as greater knowledge and experience are gained, and there is no doubt that bioremediation has great potential for dealing with certain types of site contamination. Unfortunately, the principles, techniques, advantages, and disadvantages of bioremediation are not wide known or still, especially among those who will have to deal directly with bioremediation proposals, such as site owners and regulators. 2. 4 Microbes that are useful for bioremediation The biodegradation of crude in the leatherneck environment is carried out for the most part by diverse bacterial populations, including various Pseudomonas species.The hydrocarbon-biodegrading populations are widely distributed in the worlds oceans surveys of marine bacteria indicate that hydrocarbon-degrading microorganisms are ubiquitously distributed in the marine environment. Generally, in pristine environments, the hydrocarbon-degrading bacteria comprise 1% of the total bacterial population. These bacteria presumably utilize hydrocarbons that are naturally produced by plants, alga, and other living organisms. They also utilize other substrates, such as carbohydrates and proteins. When an nvironment is contaminated with oil color, the proportion of hydrocarbon-degrading microorganisms increases rapidly. In particular, in marine environments contaminated with hydrocarbons, there is an increase in the proportion of bacterial populations with plasmids containing genes for hydrocarbon utilization. The proportion of hydrocarbon-degrading bacterial populations in hydrocarbon-contaminated marine environments often exceed 10% of the total bacterial population 3. Review of Literature 3. 1 Biodegradation of petroleum hydrocarbonsBiodegradation of petroleum hydrocarbons is a complex process that depends on the nature and on the get of the hydrocarbons present. Petroleum hydrocarbons can be divided into four classes the saturates, the aromatics, the asphaltenes (phenols, fatty acids, ketones, esters, and porphyrins), and the resins (pyridines, quinolines, carbazoles, sulfoxides, and amides) R. R. Col strong, J. D. Walker, and J. J. Cooney, Ecological aspects of microbial degradation of petroleum in the marine environment,. Di? erent factors in? uencing hydrocarbon degradation have been reported by Cooney et al.One of the distinguished factors that limit biodegradation of oil pollutants in the environment is their limited availability to microorganisms. Petroleum hydrocarbon compoun ds bind to soil components, and they are difficult to be removed or degraded S. Barathi and N. Vasudevan, Utilization of petroleum hydrocarbons by Pseudomonas ? uorescens single out from a petroleum-contaminated soil. Hydrocarbons di? er in their might to microbial attack. The susceptibility of hydrocarbons to microbial degradation can be largely ranked as follows linear alkanes branched alkanes small aromatics cyclic alkanes J. J.Perry, microbic metabolism of cyclic alkanes, in Petroleum Microbiology. Some compounds, such as the high molecular weight polycyclic aromatic hydrocarbons (PAHs), may not be degraded at all. 3. 2 Microbial degradation Microbial degradation is the major and ultimate natural mechanism by which one can cleanup the petroleum hydrocarbon pollutants from the environment 1-3 The recognition of biodegraded petroleum-derived aromatic hydrocarbons in marine sediments was reported by Jones et al. They studied the extensive biodegradation o alkyl aromatics in m arine sediments which kick the bucketred prior to detectable biodegradation of n-alkane pro? e of the crude oil and the microorganisms, namely, Arthrobacter, Burkholderia, Mycobacterium, Pseudomonas, Sphingomonas, and Rhodococcus were put up to be involved for alkylaromatic degradation. Microbial degradation of petroleum hydrocarbons in a contaminate tropical stream in Lagos, Nigeria was reported by Adebusoye et al. nightclub bacterial strains, namely, Pseudomonas ? uorescens, P. aeruginosa, Bacillus subtilis, Bacillus sp. , Alcaligenes sp. , Acinetobacter lwo? ,Flavobacteriumsp. , Micrococcus roseus, and Corynebacterium sp. were apart(p) from the polluted stream which could degrade crude oil.Hydrocarbons in the environment are biodegraded earlier by bacteria, yeast, and fungi. The reported e? ciency of biodegradation ranged from 6% to 82% for soil fungi, 0. 13% to 50% for soil bacteria, and 0. 003% to 100% 6 for marine bacteria. Many scientists reported that tangled populati ons with overall broad enzymatic capacities are required to degrade complex mixtures of hydrocarbons such as crude oil in soil, fresh water, and marine environments 8. Bacteria are the most active agents in petroleum degradation, and they work as primary degraders of spilled oil in environment 7.Several bacteria are even known to feed exclusively on hydrocarbons 9. sluiceway 36 listed 25 genera of hydrocarbon degrading bacteria and 25 genera of hydrocarbon degrading fungi which were set-apart from marine environment. A similar compilation by Bartha and Bossert 6 included 22 genera of bacteria and 31 genera of fungi. In earlier solar years, the extent to which bacteria, yeast, and ? lamentous fungi introduce in the biodegradation of petroleum hydrocarbons was the subject of limited study, but appeared to be a function of the ecosystem and local environmental conditions 7.Crude petroleum oil from petroleum contaminated soil from noth East India was reported by rabbit and Mukher jee . Acinetobacter sp. Was found to be capable of utilizing n-alkanes of chain length C10C40 as a sole source of carbon 6. Bacterial genera, namely, Gordonia, Brevibacterium, Aeromicrobium, Dietzia, Burkholderia, and Mycobacterium disjointed from petroleum contaminated soil proved to be the potential organisms for hydrocarbon degradation 9. The degradation of poly- aromatic hydrocarbons by Sphingomonas was reported by Daugulis and McCracken .Fungal genera, namely, Amorphoteca, Neosartorya, Talaromyces, and Graphium and yeast genera, namely, Candida, Yarrowia, and Pichia were isolated from petroleum contaminated soil and proved to be the potential organisms for hydrocarbon degradation Singh et al. also reported a group of terrestrial fungi, namely, Aspergillus, Cephalosporium, and Pencillium which were also found to be the potential degrader of crude oil hydrocarbons. The yeast species, namely, Candida lipolytica, Rhodotorula mucilaginosa, Geotrichum sp, and Trichosporon mucoides isolated from contaminated water were noted to degrade petroleum compounds 5.Though algae and protozoa are the important members of the microbial community in both aquatic and terrestrial ecosystems, reports are scanty regarding their involvement in hydrocarbon biodegradation. Walker et al. isolated an alga, Prototheca zop? which was capable of utilizing crudeoil and a interracial hydrocarbon substrate and exhibited extensive degradation of n-alkanes and isoalkanes as well a aromatic hydrocarbons. Cerniglia et al. observed tha nine cyanobacteria, ? ve green algae, one red alga, one brown alga, and two diatoms could oxidize naphthalene.Protozoa by contrast, had not been shown to utilize hydrocarbons. 3. 3 Factors affecting Degradation A number of limiting factors have been recognized to a? ect the biodegradation of petroleum hydrocarbons, some of which have been discussed by Brusseau. The composition and inherent biodegradability of the petroleum hydrocarbon pollutant is the ? rs t and foremost important consideration when the suitability of a remediation approach is to be assessed. Among physical factors, temperature plays an important role in biodegradation of hydrocarbons by directly a? ecting the chemical science of the pollutants as well as a? cting the physiology and diversity of the microbial ? ora. atlas vertebra 4 found that at low temperatures, the viscosity of the oil increased, bandage the excitableness of the toxic low molecular weight hydrocarbons were reduced, delaying the onset of biodegradation. Temperature also a? ects the solubility of hydrocarbons 8. Although hydrocarbon biodegradation can occur over a wide range of temperatures, the rate of biodegradation generally decreases with the decreasing temperature. shows that highest degradation rates that generally occur in the range 3040? C in soil environments, 2030?Cin some freshwater environments and 1520? C in marine environments . Venosa and Zhu 11 reported thatambient temperature of the environment a? ected both the properties of spilled oil and the activity of the microorganisms. Signi? cant biodegradation of hydrocarbons have been reported in psychrophilic environments in equable regions. alimentals are very important ingredients for successful biodegradation of hydrocarbon pollutants especially normality, phosphorus, and in some cases iron 8. Some of these nutrients could become limiting factor thus a? ecting the biodegradation processes.Atlas 11 reported that when a major oil spill occurred in marine and freshwater environments, the supply of carbon was signi? cantly increased and the availability of due north and phosphorus generally became the limiting factor for oil degradation. In marine environments, it was found to be more pronounced due to low levels of nitrogen and phosphorous in seawater 10. Freshwater wetlands are typically considered to be nutrient de? cient due to heavy demands of nutrients by the plants. Therefore, additions of nutrients we re necessary to enhance the biodegradation of oil pollutant.On the other hand, excessive nutrient concentrations can also oppress the biodegradation activity 11. Several authors have reported the negative e? ects of high NPK levels on the biodegradation of hydrocarbons especially on aromatics 10. The e? ectiveness of fertilizers for the crude oil bioremediation in subarctic intertidal sediments was studied by Pel allowier et al. . Use of bird manure as organic fertilizer in contaminated soil was also reported , and biodegradation was found to be enhanced in the presence of poultry manure alone. Maki et al. eported that photo-oxidation increased the biodegradability of petroleum hydrocarbon by increasing its bioavailability and thus enhancing microbial activities. 3. 4 Mechanism of Petroleum Hydrocarbon Degradation The most rapid and complete degradation of the majority of organic pollutants is brought about under aerobic conditions. issue 2 shows the main principle of aerobic de gradation of hydrocarbons 11. The initial intracellular attack of organic pollutants is an oxidative process and the activation as well as incorporation of oxygen is the enzymatic key reaction catalyzed by oxygenases and peroxidases.Peripheral degradation pathways convert organic pollutants step by step into intermediates of the central intermediary metabolism, for example, the tricarboxylic acid cycle. Biosynthesis of cell biomass occurs from the central predecessor metabolites, for example, acetyl-CoA, succinate, pyruvate. Sugars required for various biosyntheses and growth are synthesized by gluconeogenesis. The degradation of petroleum hydrocarbons can be mediated by speci? c enzyme system. Figure 3 shows the initial attack on xenobiotics by oxygenases.Other mechanisms involved are (1) affixation of microbial cells to the substrates and (2) production of biosurfactants 12. The uptake mechanism linked to the attachment of cell to oil droplet is still unknown but production of b iosurfactants has been well studied. 4. Brief outline of the project work 1. procural of oil Samples. 2. Procurement of Pseudomonas putida strain. 3. Sub-culturing the microbe in nutrient rich media for checking viability.. 4. Culturing microbes on a mineral salt media containing only crude oil as a carbon source. 5. Biodegradation studies. 6. Gravimetric analytic thinking 5.Materials and Methods Soil Samples Samples(500g) contaminated with oil used for hydrocarbons utilizing microorganisms, were tranquil from Nhava Sheva port in Mumbai(where a recent oil spill has took place). Crude Oil Crude Oil is procured from an Oil production site of ONGC. Bacterial Strain Pseudomonas Putida PS-I strain procured from NCL Pune. 5. 1 Preparation Of Nutrient Broth For preparedness of nutrient agar, malt extract, yeast extract, Potassium dihydrogen inorganic phosphate and dextrose is required. Malt extract and yeast extract is generally used as a nutritious agent. Potassium dihydrogen phosp hate i. . KH2PO4 is used as a buffering agent to maintain the pH. Dextrose is generally used as a carbon source because dextrose inhibits the growth of other micro-organisms. sterilise is a device to sterilize equipment and supplies by subjecting them to high pressure go at 121 C or more. Machines in this category largely black market by utilizing pressurized steam and superheated water. To sterilize culture media, rubber material, gowns, dressing, gloves etc. are used. It is particularly useful for materials which cannot withstand the higher temperature of hot air oven. CHEMICALS need- For 1000ml,Malt extract 10 gm Beef Extract 4 gm K2HPO4 1 gm Magnesium sulphate 1 gm Sodium Chloride 0. 5 gm pH 7. 0 Agar 15% PROCEDURE- For preparation of 100ml of nutrient broth, around 100ml of double distilled water was taken in a conical flask. Malt extract, yeast extract, KH2PO4 and dextrose was weighed as per the composition mentioned above and added to the conical flask.The conica l flasks are to be tottern so well so that all the chemicals should dissolve. pH was checked apply pH meter and adjusted to 7. 0 using NaOH and HCl. The volume was do to 100ml by adding double distilled water. The above solution i. e. nutrient agar along with the Petri-plates was autoclaved at 15 psi and 15 minutes. instanter the solution was allowed to imperturbable start to ready to pour condition. PRECAUTIONS- The autoclave should be done at 15 psi and 15 min. The pH should be maintained at 7. 0. 5. 2 Preparation of Nutrient Agar Slants from the Bacterial StrainFor the preparation of Slants, Flame the inoculating tat to redness by holding it pointed down into the blast, starting near the hide and wherefore moving the tat into the flame. This technique sterilizes the eyehole and, if wet with a culture, heats up the circulate without spattering bacteria into the air and onto the surrounding area. let the loop cool a minute. A hot loop will damage the bacteria cells. U sing the fingers of the loop hand remove the cap from the stock culture thermionic valve and flame the organ pipe mouth. Do not set the tube top down on the table. stick in the cooled sterilized loop into the culture tube be careful to not continue the sides of the tube. Touch the loop to the culture. You need not scrape a evident amount from the culture. Hold the tube as horizontal as possible to preclude particles from the air settling into the tube But do see to it out for any condensate in the bottom of huckster cultures. Dont let this fluid wash across the face of the culture. Remove the loop organism careful again to not equate the tube sides. Flame the tube mouth and replace the cap. Remove the cap of the broth tube. Flame the top. memorialize to hold the top in your fingers. Insert the loop into the Slant tube filled with agar and shake to remove the bacteria. Withdraw the loop, flame the tube mouth and replace the cap. Resterilize the inoculating loop and place it on the table. Never place a contaminated loop on the table. If there is any liquid in the bottom of the slant tube avoid sticking the loop into this condensate. 5. 3 Preparation of SubCultures of Bacterial Strain The Nutrient Broth Cultures are inoculated with the bacterial strain from the nutrient agar slant as detailed below. PROCEDURE Light your bunsen burner burner.In one hand hold both the Nutrient Broth culture to be inoculated and the nutrient slant agar. Loosen the tube caps. In your other hand hold the inoculating loop. Flame the inoculating loop to redness by holding it pointed down into the flame, starting near the handle and then moving the loop into the flame. This technique sterilizes the loop and, if wet with a culture, heats up the loop without spattering bacteria into the air and onto the surrounding area. Let the loop cool a minute. A hot loop will damage the bacteria cells. Using the fingers of the loop hand remove the cap from the stock culture tube and flame the tube mouth.Do not set the tube top down on the table. Insert the cooled sterilized loop into the slant tube being careful to not touch the sides of the tube. Touch the loop to the culture. You need not scrape a visible amount from the culture. Hold the tube as horizontal as possible to preclude particles from the air settling into the tube But do watch out for any condensate in the bottom of slant cultures. Dont let this fluid wash across the face of the culture. Remove the loop being careful again to not touch the tube sides. Flame the tube mouth and replace the cap. Remove the cap of the broth tube. Flame the top.Remember to hold the top in your fingers. Insert the loop into the broth and shake to remove the bacteria. Gently shake the broth culture. This inoculated broth culture is incubated at room temperature for 72 hours and the bacteria is allowed to grow in the broth medium. 5. 4 Centrifugation of Crude Oil Centrifugation is a process that involves the use of the centrifugal throw for the separation of mixtures with a centrifuge, used in industry and in laboratory settings. More-dense components of the mixture move away from the axis of the centrifuge, while less-dense components of the mixture migrate towards the axis.The precipitate (pellet) gathers on the bottom of the tube. The remaining solution is properly called the supernate or supernatant liquid The Crude Oil is Centrufuged at a animate of 5000 rpm for a period of ten minutes. The Contaminants in the oil are collected at the bottom of the tube in the form of pellets. These pellets can be removed by filtration using a filter paper. Now the concentrates oil which is free from impurities is collected in a flask and softly shaken. Spectophotometric Analysis Optical density, measured in a spectrophotometer, can be used as a measure of the concentration of bacteria in a suspension.As visible light passes through a cell suspension the light is scattered. Greater scatter indicates that more bacte ria or other material is present. The amount of light scatter can be measured in a spectrophotometer. Typically, when working with a particular type of cell, you would determine the optical density at a particular wavelength that correlates with the different phases of bacterial growth. Generally we will want to use cells that are in their mid-log phase of growth. Typically the OD600 is measured. 5. 5 Subculturing Of Petri Plates with oil % of crude oil is mixed with 100 ml of Nutrient broth medium. The 1. 5g of agar is added to the medium and Nutrient Agar(with 1% crude oil) is prepared. Now take 6 Petri dishes. Open one of the dishes. Take the nutrient agar to be added and Swab the agar, barely pressing, side to side on the entire surface. The dish is closed immediately afterwards swabbing to foil contamination. The dish is sealed with tape around the edges to prevent contamination. Repeat the same procedure for the other dishes. Put the dishes in an incubator for 4 days to allo w some growth. 5. 6 Biodegradation StudiesLaboratory Biodegradation studies were carried out under optimized conditions for assessing the biodegradation potential of the pseudomonas putida PS-I Strain. After the desired interval of time, the petriplates were taken out and the bacterial activities were stopped by adding 1% N HCl. For the inception of crude oil from these plates, 50ml of culture broth was mixed with 50 ml of propanone petroleum ether (11) in a single separating displace and shaken vigorously to get a single emulsified layer and acetone was added then to it and shaken gently to break the emulsification which resulted in three layers. sneak layer was a mixture of Petroleum ether crude oil and acetone. Clumping cells aere formed in the middle layer and the bottom layer contains acetone, water and biosurfactant in soluble form. The lower two layers were separated out while the top layer containing petroleum ether mixed with crude oil and acetone is taken out in a fres h beaker. The extracted oil is passed through anhydrous sodium sulphate in order to remove the moisture. The petroleum ether and acetone were evaporated on a water bathe leaving us with the dry oil clump. 5. 7 Gravimetric AnalysisGravimetric analysis describes a set of methods in analytical alchemy for the quantitative determination of an analyte based on the mass of a solid. the analyte essential first be converted to a solid by hastiness with an appropriate reagent. The precipitate can then be collected by filtration, washed, dried to remove traces of moisture from the solution, and weighed. The amount of analyte in the original prototype can then be calculated from the mass of the precipitate and its chemical composition. Gravimetric analysis is performed on the dry oil clump collected after the water bath.It is done by weighing the quantity of counterweight oil left after biodegradation in a tared vial. The mass of this melting pot is subtracted from the initial mass of th e 1% of oil that is added in the petridishes giving the amount of oil that is degraded due to the biological avtivity of the pseudomonas putida strain. 6. Results 6. 1 Growth Analysis of Pseudomonas Putida The culture which was obtained in test tube slants was encourage sub cultured in conical flasks in a LB medium and the growth analysis was done to check the viability of the culture obtained. The growth kinetics plot was obtained by measuring the O. D. y using a visible spectrophotometer and recording the reading at regular intervals. The Graph was then plotted. 6. 2 Gravimetric Analysis Biodegradation studies were conducted for 15 days and gravimetric analysis was done after every five days. The biodegradation effect was seen from the 5th day onwards. Laboratory biodegradation studies on crude oil by Pseudomonas putida No. Of Days Initial Concn Final Concn Difference Degradation (%) 5 days 1. 431 . 57 1. 325 . 46 0. 106 . 11 7. 4 10 days 1. 453 . 71 1. 198 . 38 0. 255 . 34 17. 54 15 days 1. 398 . 68 0. 936 . 31 0. 62 . 28 33. 04 7. Discussion It can be seen that the degradation plowshare of oil has increased from mere 7. 41 in the first 5 days to a good 33. 04 percentage towards the 15th day, from this it is clearly understood that pseudomonas putida is an ideal organism for bioremediation programmes. Moreover this rate of degradation has been obtained under normal conditions without any aid from surfactants or fertilizers. Hence there is scope for achieving much greater rates by using the above mentioned methods of fertilizing or adding surfactants. 8. BIBLIOGRAPHY (1). U. S. Enviromental protective covering Agency (1990).Interim Report, Oil Spill Bioremediation Project. U. S. environmental Protection Agency, Office of Research and Development, Washington (2). T. Cairney. Contaminated Land, p. 4, Blackie, London (1993). (3). R. B. King, G. M. Long, J. K. Sheldon. Practical Environmental Bioremediation The Field Guide, 2nd ed. , Lewis, Boca Rato n, FL (1997). (4). Atlas, Ronald M. (1995). Petroleum Biodegradation and Oil Spill Bioremediation. Marine contaminant Bulletin 31, 178-182 (5) Hoff, Rebecca Z. (1993). Bioremediation an overview of its development and use for oil spill cleanup. Marine contaminant Bulletin 29, 476-481. 6). Irwin, Patricia (1996). To clean up environmental spill, know your medium. Electrical creation 37-40. (7). Swannell, Richard P. J. Lee, Kenneth McDonagh, Madeleine (1996). Field Evaluations of Marine Oil Spill Bioremediation. Microbiological Reviews 60, 342-365 (8). Radwan, S. S. Sorkhoh, N. A. El-Nemr, I. M. El-Desouky, A. F. (1997). A feasibility study on seeding as a bioremediation practice for the oily Kuwaiti desert. daybook of Applied Microbiology 83, 353-358. (9). P. E. Flathman, D. Jerger, J. E. Exner. Bioremediation Field Experience, Lewis, Boca Raton, FL (1993). 10). J. G. Mueller, C. E. Cerniglia, P. H. Pritchard. Bioremediation of Environments Contaminated by Polycyclic Aromatic Hydrocarbons. In Bioremediation Principles and Applications, pp. cxxv194, Cambridge University Press, Cambridge (1996). (11). P. J. S. Colberg and L. Y. Young. Anaerobic Degradation of Nonhalogenated Homocyclic Aromatic Compounds Coupled with Nitrate, Iron, or sulfate Reduction. In Microbial Transformation and Degradation of Toxic Organic Chemicals, pp. 307330, Wiley-Liss, spick-and-span York (1995). (12). A. S. Allard and A. H. Neilson. Oil Eating Microbes 39, 253285 (1997).