Oil Degradation

Identification and characterization of oil degrading genes of Pseudomonas sps and Cloning of oil degrading genes in E.
coli
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INTRODUCTION
Identification and characterization of oil degrading genes of Pseudomonas sps and Cloning of oil degrading genes in E.coli
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1. INTRODUCTION
In the last years, a large number of ecosystems have been changed by the growing influence of human activity. As a result, many people have become aware of the need to protect ecosystems as well as to evaluate the damage caused by contamination. During the previous years, the frequency and risk of oil pollution has lead to extensive research. Most of the petroleum goes in the ecosystem via leak of coastal oil refineries. This fact increased the interest of scientists to investigate the oil distribution and its fate in the environment, especially the marine environment. Approximately five million tons of crude oil and refined oil enter the environment each year as a result of anthropogenic sources such as oil spills (Hinchee and Kitte, 1995). Past analysis of reported oil spills indicated that most of the oil comes from tankers, barges and other vessels as well from land pipeline spills. Extensive changes in marine, as well as terrestrial ecosystems resulting from the grounding of the Exxon Valdez (1989), the Nahodka oil spill, the Erica spill (1999) and the Prestige spill (2002), have recently increased the attention of Environmentalists, Chemists, Biotechnologists and Engineers (Braddock et al., 1995).Shipping accidents have a serious impact on the surrounding environment. The consequences include serious, widespread and long-term damage to marine ecosystems, terrestrial life, human health and natural resources. It is very important to characterize oil spills as already known sources. This can help environmentalists to predict the behavior of oil and estimate the long-term impact on the environment. It is necessary to select an appropriate clean-up method. The recovery of oil was studied extensively since the 1970s. Conventional remediation methods include physical removal of contaminated material. These methods also use chemicals, especially shoreline cleaners, which are often organic solvents with or without surfactants (Riser-Roberts 1992). The shoreline cleaners with surfactants emulsify the adsorbed oil, which entrain adjacent waters or is transported deeper into the shoreline
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soil. The oil solvent mixtures are collected using conventional skimming methods. Mechanical recovery of oil includes the use of the oil absorbents. Sorbents help to transform oil to a transportable form for short-term storage. However, most of the used sorbents end up in the landfills. Most of the physicochemical methods use chemical agents, as well as their emulsion with oil cause toxicity, to aquatic organisms. They produce another source of pollution and also increase the oil recovery cost. Additionally, abiotic losses due to evaporation of low molecular hydrocarbons, dispersion and photo oxidation (involves only aromatic compounds) play a major role in decontamination of the oil spill environments (Mills et al., 2003). There is an increased interest in promoting environmental methods in the process of cleaning oil-polluted sites. These methods are less expensive and do not introduce additional chemicals to the environment. Compared to physiochemical methods, bioremediation offers a very feasible alternative for an oil spill response. This technique is considered an effective technology for treatment of oil pollution. One reason is that the majority of the molecules in the crude oil and refined products are biodegradable. 1.1 HYDROCARBONS Petroleum products are used as fuels, solvents and feed stocks in the textile, pharmaceutical and plastic industries. Petroleum is a complex mixture of hydrocarbons and other organic compounds, including some organometalloconstituents. Petroleum constituents represent: saturates, aromatics, resins and asphaltenes (Figure 1.1). Saturates are defined as hydrocarbons containing no double bonds. They are categorized according to their chemical structures into alkanes (paraffins) and cycloalkanes. Saturates represents the highest percentage of crude oil constituents. Aromatic hydrocarbons with one or several aromatic rings are usually substituted with different alkyl groups. In comparison to the saturated and aromatic fractions, the resin and asphaltenes contain non3
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hydrocarbon polar compounds. Resins and asphaltenes have very complex and mostly unknown carbon structure with addition of many nitrogen, sulfur and oxygen atoms (Harayama, 2004). Petroleum recovered from different reservoirs varies widely in compositional and physical properties. The composition of particular petroleum product ranges from the very low molecular weight hydrocarbons to the very high. A hydrocarbons chemical structure affects its biodegradation in two ways. First, the molecule may contain groups or substituents that cannot react with available or inducible enzymes. Second, the structure may determine the compound to be in a physical state where microbial degradation does not easily occur. Usually, the larger and more complex the structure of a hydrocarbon, the more slowly it is oxidized. Also the degree of substitution affects the degradation. Compounds that contain amine, methoxy and sulfonate groups, ether linkages, halogens and branched carbon chains are generally persistent. Adding aliphatic side-chains increases the susceptibility of cyclic hydrocarbons to microbial attack (Riser-Roberts, 1992). Hydrocarbon composition affects their physicochemical properties. Hydrocarbons differ in their solubility, from polar compounds, such as methanol to very low solubility non-polar compounds, such as high molecular weight polynuclear aromatic hydrocarbons.The solubilization is not the only factor determining the degradation of hydrocarbons. Many microorganisms, such as Pseudomonas aeruginosa, Pseudomonas putida and Bacillus subtilis, Bacillus cereus, Bacillus licheniformis and Bacillus laterospor excrete emulsifiers that increase the surface area of the substrate. On the other hand, these microorganisms modify their cell surface to increase its affinity for hydrophobic substrates and, thus facilitate their absorption (Cybulski et al., 2003; Carvalho and Fonseca, 2004). Hydrocarbons can be very fluid or very viscous and very volatile or relative nonvolatile. Viscosity of polluting oils is an important property. It determines the spreading and dispersion of the hydrocarbon mixture and
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also the surface area available for microbial attack. The variability in the physicochemical character of hydrocarbons causes changes in the behavior of individual hydrocarbons as well as mixtures. The concentration of organic compounds in the environment also affects the level of tolerance. At low concentrations, all fractions are likely to be attacked. However, at high concentrations, only those fractions most susceptible to degradation will be broken down. Also the concentration of contaminants will affect the number of organisms present. It has been shown that the higher concentrations of gasoline in contaminated water were related to higher counts of microorganisms (Doong and Wu, 1995).
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Figure 1.1
Structures of crude oil constituents: (A) substituted cyclopentane,

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cyclohexane, bicyclic species, (B) substituted aromatics (xylene, naphthalene, perylene), (C) thiophene, elemental sulfur, nonyl mercaptan, (D) substituted pyridine, pyrrole, carbazole, (E) phenol, long chain alcohol, (F) asphaltene model molecule (Machin et al., 2005). 1.2 MICROORGANISMS In recent years, many microbial ecologists have identified various microbial species that are effective degraders of hydrocarbons in natural environments. Many of these microbial consortia have been isolated from heavily contaminated coastal areas. They were isolated on their ability to metabolize various carbon sources, such as aliphatic and aromatic compounds and their chlorinated derivates. The microorganisms were obtained originally by enrichment culture procedures, where maximum specific growth rate or maximum final cell concentration was used as the selection criterion. Petroleum hydrocarbons can be degraded by microorganisms such as bacteria, fungi, yeast and microalgae (Riser-Roberts, 1992; Bundy at el., 2004). However, bacteria play the central role in hydrocarbon degradation. The driving force for petroleum biodegradation is the ability of microorganisms to utilize hydrocarbons to satisfy their cell growth and energy needs. A large number of studies report that low molecular weight alkanes are degraded most rapidly. Mixed cultures carry out more extensive biodegradation of petroleum than pure cultures (Ghazali at el., 2004; Oteyza at el., 2005; Sun at el., 2004; Gerdes at el., 2004; Trindade et al., 2004). In many ecosystems there is already an adequate indigenous microbial community capable of extensive oil biodegradation, provided that environmental conditions are favorable for oil-degrading metabolic activity (Capelli at el.,
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2001; Richard and Vogel, 1999; Kim at el., 2004). There are several advantages relying on indigenous microorganisms rather than adding microorganisms to degrade hydrocarbons. First, natural populations have developed through many years. These microorganisms are adapted for survival and proliferation in that environment. Secondly, the ability to utilize hydrocarbons is distributed among a diverse microbial population. This population occurs in natural ecosystems and either independently or in combination metabolizes various hydrocarbons. Many times, when the amount of microorganisms is sufficient in the contaminated environment, microbial seeding is not required. Nutrient availability, especially of nitrogen and phosphorus, seems to be the most limiting factor. It was confirmed that these nutrients enhance growth of microorganisms, which leads to more rapid decomposition of contaminants (Chaineau et al., 2005; Coulon et al., 2004). Accepted values for a mixed microbial population in the soil are C: N, 10:1; and C: P, 100:1. Nitrogen and phosphorus can be supplied with common inorganic fertilizers with the N:P ratio at 16:1 when the optimum nitrogen fertilization for a sandy matrix is lower than 100 mg N kg dry soilimates (Ferguson at el., 2003; Kim at el., 2004). Adding yeast extract or domestic sewage as source of nitrogen did not prove beneficial. Urea formaldehyde was found to be the most satisfactory nitrogen source (Riser-Roberts, 1992). Microorganisms are equipped with metabolic machinery to use petroleum products as a carbon and energy source. The metabolic pathways that hydrocarbon- degrading heterotrophy use can be either aerobic (i.e. they utilize oxygen as the primary electron acceptor) or anaerobic (i.e. they utilize an alternative electron acceptor such as nitrate or sulfate). Aerobic degradation usually proceeds more rapidly and is considered to be more effective than anaerobic degradation. One reason is that aerobic reactions require less free energy for initiation and yield more energy per reaction.
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1.3 METABOLIC MACHINERY 1.3.1 AEROBIC DEGRADATION Aerobic biodegradation of hydrocarbons and crude oil is a long known and well- studied process. However, the ability of anaerobic microorganisms to oxidize and utilize crude oil as a complex organic substrate under aerobic conditions was discovered just recently. These microorganisms decompose most organic compounds into carbon dioxide, water and mineral matter, such as sulfate, nitrate and other inorganic compounds. They do not produce hydrogen sulfide or methane as reaction products. The aerobic pathway proceeds most rapidly and most efficiently, because aerobic reactions require less free energy for initiation and yield more energy per reaction. The hydrocarbons are broken down by a series of enzyme-mediated reactions. Oxygen serves as an external electron acceptor, while an organic component of the contaminating substance functions as the electron donor or energy source. The general degradation pathway for an alkane involves sequential formation of an alcohol, an aldehyde and a fatty acid. The fatty acid is cleaved, releasing carbon dioxide and forming a new fatty acid that is two carbon units shorter than the parent molecule in a process known as betaoxidation. The initial enzymatic attack involves a group of mono oxygenases. The general pathway for aromatic hydrocarbons involves cishydroxylation of the ring structure forming a diol (e.g. catechol) using dioxygenase. The ring is oxidatively cleaved by dioxygenases, forming a dicarboxylic acid (e.g. muconic acid). Oxidation of substituted aromatics generally proceeds by initial beta-oxidation of the sidechain, followed by cleavage of the ring structure. The degradative pathway for a highly branched
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compound, such as pristane or phytane, may proceed by omega oxidation forming a dicarboxylic acid, instead of only monocarboxylic acid (Hamme et al., 2003). Aerobic degradation in soil is associated with a variety of microorganisms, including bacteria, actinomycetes and fungi. Acinetobacter and Rhodococcus are two bacterial strains often associated with petroleum contaminated habitats. Intense interest has arisen on the study of alkane oxidation by these bacteria in the last decade. Acinetobacter utilizes an alkane mono oxygenase (terminal oxidation) to convert the hydrocarbon to a primary alcohol to allow for the subsequent breakdown and utilization of the hydrocarbon (Figure 1.2). The degradation of alkylcyclohexane by Acinetobacter sp. ODDK71 was investigated by Koma (Koma et al., 2003). Strain ODDK71 degraded alkylcyclohexanes (alkyl sidechain length of >12) by co-metabolism when hexadecane was used as a growth substrate. GC/MS analysis of co-metabolized products from dodecylcyclohexane suggests that strain ODDK71 degraded dodecylcyclohexane via a ring oxidation and an alkyl sidechain oxidation pathways. The ring oxidation pathway of dodecylcyclohexane is a novel pathway of microbial degradation of dodecylcyclohexane (Koma et al., 2003). Significant correlation between aliphatic, aromatic and asphaltic fractions of the crude oil and the Rhodococcus genera able to utilize such hydrocarbons was observed in several studies. After oil pollution of soil, representative strains of the Rhodococcus showed the ability to metabolize a broad spectrum of hydrocarbons (Peressutti et al., 2003). Rhodococcus posses an alkane monooxygenase as Acinetobacter, but in which the second carbon atom is oxidized (subterminal oxidation) leading to the production of a secondary alcohol and the subsequent ketone is further metabolized to a primary alcohol for further breakdown (Figure
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1.3). Both microorganisms, Acinetobacter as well Rhodococcus, utilize respiration to generate ATP where oxygen serves as the terminal electron acceptor in electron transport.
Figure 1.2 Degradation of alkanes by Acinetobacter sp. (Hamme et al., 2003)

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NAD/NADH= Nicotine Amide Adenine Dinucleotide S-CoA= acetyl coenzyme A
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Pseudomonas appears to be the most ubiquitous bacteria found in oil contaminated soils and soil in general. This bacterium is able to adapt to many different hydrocarbons. They are responsible for degrading most of the aromatics in gasoline, although the efficiency in degrading aromatics hydrocarbons can vary among strains. The most extensively characterized polycyclic aromatic hydrocarbons degradation pathway is encoded by the NAH7 plasmids from Pseudomonas putida. The first operon encodes the pathway for naphthalene conversion to salicylate. The second codes for the conversion of salicylate via catechol meta-cleavage to acetaldehyde and pyruvate. Molecular oxygen is introduced into the aromatic nucleus via naphthalene dioxygenase (Hamme et al., 2003). The catabolic pathways for three- and four-ring PAHs in
Pseudomonas putida were examined in several studies. For example, phenanthrene was degraded by Pseudomonas sp. strain PP2 via a dioxygenase-initiated pathway that converged with the naphthalene degradation pathway. Secretion of a surfactant into the medium and increased cell-surface hydrophobicity during growth were postulated to increase uptake of poorly soluble phenanthrene (Parales and Haddock, 2004).Specific attention has also been given to other dioxygenases associated with the biodegradation of polycyclic aromatic hydrocarbons (PAHs). These enzymes represent multicomponent systems that catalyze a specific regioselective dioxygenation. These reactions are required for degradation of dibenzofurans, dibenzo- p-dioxin and carbazole. Carbazole dioxygenase activity had been observed only in Pseudomonas strains. The archetype carbazole 1,9-dioxygenase, isolated from Pseudomonas resinovorans CA10, has been recently characterized in detail (Pieper et al., 2004).
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Figure 1.3 Degradation of alkanes by Rhodococcus sp.(Hamme et al., 2003) NAD/NADH= nicotine amide adenine Dinucleotide S-CoA= acetyl coenzyme A
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A PAH - and phenol-degrading microorganism, Pseudomonas containing mixtures with p-cresol and sodium salicylate. Both Pseudomonas
putida (ATCC p-cresol and
17484), was also used to study the substrate interactions during cell growth on carbazolesodium salicylate could be utilized by the bacteria as the sole carbon and energy sources (Gen-Yu and Loh, 2002). Some other sp. were found to grow on aromatic constituents of gasoline as a sole source of carbon. Strains of the Bijerinckia genus are also very active in aerobic hydrocarbon degradation. The presence of biphenyl dioxygenase enable these microorganisms to oxidize benzo(a)pyrene, benzo(a)anthracene and the aromatic N-heterocycle carbazole (Resnicek at el., 1993). 1.3.2 ANAEROBIC DEGRADATION In contrast to the fact that aerobic microbial hydrocarbon metabolism has been extensively investigated, the same is not true about anaerobic hydrocarbon metabolism. The roles of bacteria that participate in these processes under anoxic/anaerobic conditions during biodegradation are not fully understood. Oxygen is not available in all environments where hydrocarbons occur (e.g. in deep sediments, flooded soils, eutrophic lagoons, stagnant fresh and ocean waters and in oil reservoirs). Several studies have investigated the question, whether or not the biodegradation of hydrocarbons is possible under anoxic conditions. It was not until the late 1980s that new groups of microorganisms were found to degrade hydrocarbons under strictly anoxic conditions. Studies have confirmed that these microorganisms activate organic compounds by special biochemical mechanisms that differ completely from those employed in aerobic hydrocarbon metabolism (Riser-Roberts, 1992).N-alkanes, branched alkanes, cycloalkanes, and some alkenes have been shown to be degraded under anaerobic conditions. For example, unsubstituted, methyl-substituted, and ethyl-substituted cyclopentenes, cyclopentanes and cyclohexanes were consumed without a substantial lag in the presence of sulfate but rather less effectively under methanogenic conditions. Dimethyl-substituted cyclopentanes and cyclohexanes were biodegraded only in the
presence of sulfate (Widdel and Rabus, 2001). Several laboratory and field studies conducted on biodegradation had clearly demonstrated biodegradation of BTEX (benzene, toluene, ethylbenzene and xylene isomers) compounds. The biodegradation of these individual compounds depends on the terminal electron acceptor. Usually, toluene and m, p-xylenes degrade under anaerobic conditions. One typical example is benzene degradation. Benzene is cleaved aerobically. On the other hand, it is usually recalcitrant under nitrate-reducing conditions. Benzene cleavage is highly site-specific in the presence of Fe (III) and sulfate terminal electron acceptor. Ethyl benzene degradation was described under aerobic as well as nitrate-reducing conditions. Nevertheless, degradation of this compound is site-specific under Fe (III) and sulfate reducing as well as methanogenic conditions. Utilization of o-xylene is enhanced by aerobic conditions, and is site-specific under anaerobic conditions (Schreiber et al.,2004).Hydrocarbons such as alkylbenzenes including m-, o-, and pxylene, trimethylbenzenes, naphthalene and phenanthrene as well as n-alkanes and branched alkanes can also be metabolized under anaerobic conditions (Figure 1.4A). These reactions may take place under Fe (III)-reducing, denitrifying and sulfate reducing conditions, by anoxygenic photosynthetic bacteria, or in syntrophic consortia of protonreducing and methanogenic bacteria. Other terminal electron acceptors than O2 shown to be used during this metabolism include manganese oxides, soil humic acids and fumarate in a fermentative oxidation. These microorganisms that use nitrate, ferric iron or sulphate as electron acceptors grow in syntrophic cocultures with other anaerobes (Boopathy, 2004).Most recent studies showed that n-hexane was activated under anaerobic conditions at carbon-2 in connection with an addition to fumarate, yielding (1methylpentyl)succinate (Figure 1.4B). The biochemistry of the degradation of alkylsuccinates has not been elucidated. However, it is expected that these reactions lead to fatty acid metabolism.Toluene has been the most studied hydrocarbon with respect to enzymatic and genetic characterization in the denitrifying bacteria Azoarcus sp. In the proposed pathway,fumarate addition to toluene is mediated by benzylsuccinate synthase
to form benzylsuccinate. Series of -oxidation reactions convert benzylsuccinate to acetyl CoA and benzoyl-CoA which is a central intermediate in the anaerobic degradation of aromatic compounds. Benzoyl-CoA undergoes reductive dearomatization and ring cleavage followed by reactions that again resemble those in the -oxidation reactions of fatty acids (Widdel and Rabus, 2001). For ethylbenzene, oxidation under denitrifying conditions starts with dehydrogenation by ethylbenzene dehydrogenase to produce 1 phenylethanol (Figure 1.4C). This reaction is followed by oxidation to acetophenone. Acetophenone is carboxylated and after activation yields 3-oxo-3phenylpropionyl-CoA and thiolytic cleavage to acetylCoA and benzoyl-CoA. The metabolic pathway for sulfate-reducing bacteria is similar to that of toluene metabolism. In this case (1-phenylethyl) succinate was detected in enrichment cultures of Azoarcus sp. indicating ethylbenzene addition to fumarate (Figure 1.4C).
Figure 1.4 The initial reactions during anaerobic degradation of saturated and aromatic hydrocarbons (Townsend et al., 2004). There is evidence that anaerobic degradation of m-xylene proceeds, in analogy to that of toluene, via m-methyl benzyl succinate to methyl benzoyl-CoA (Widdel and Rabus, 2001; Riser-Roberts, 1992).PAH are also metabolized under anaerobic conditions. Naphtalene degradation proceeds via carboxylation to form 2-naphtoate (the central intermediate in a pathway analogous to the benzoyl-CoA pathway for monoaromatic
compounds) in sulfate-reducing as well as denitrifying bacteria (Figure 1.4D). The identification of other metabolites in a sulphate-reducing enrichment culture indicated the further metabolism of 2-naphthoate (presumably as activated acid) via subsequent reduction of the two rings to yield decalin-2-carboxylate. Alkylnaphthalenes appear to be activated by a mechanism similar to that of toluene (Hamme et al., 2003).Previous studies investigated a phenanthrene-adapted anaerobic group capable of degrading phenanthrene in river sediment. The current studies explore the effects of several factors such as sludge source, the presence of individual or mixed PAHs, pH and the addition of electron donors on PAH degradation rates. The results show that the order of degradation rates for PAHs in municipal sludge under anaerobic conditions is: phenanthrene > pyrene > anthracene > fluorene > acenaphthene. In petrochemical sludge the order is acenaphthene > fluorene > phenanthrene > anthracene > pyrene (Chang et al., 2001, 2003; Meckenstock et al., 2004)The mechanism by which anaerobic benzene degradation occurs is unclear. Anaerobic degradation of benzene has not yet been described in detail because except for two Dechloromonas strains (RBC and JJ), no pure cultures of bacteria have yet been isolated for study (Riser-Roberts, 1992). Some studies indicate that benzene can be transformed by methanogenic cultures acclimated to lignin-derived aromatic acids under anaerobic conditions with several intermediates, including cresols, phenol, demethylation products, aromatic alcohols, aldehydes and acids. Phenol and benzoate have been consistently detected as intermediates of anaerobic benzene degradation suggesting that the hydroxylation of benzene to phenol is one of the initial steps in anaerobic benzene degradation. The conversion of phenol to benzoate could then occur by the carboxylation of phenol to form 4-hydroxybenzoate followed by the reductive removal of the hydroxyl group to form benzoate.13C-Labeling studies suggest that the carboxyl carbon of benzoate is derived from one of the carbons of benzene. Latest studies show the possibility of benzene degradation under anaerobic conditions. However, microorganisms with this ability are not ubiquitous. It is obvious that the degradation of petroleum and refined products proceedsmuch
faster in the presence of oxygen than under anoxic conditions. Furthermore, aerobic microorganisms degrade a larger range of hydrocarbon compounds than anaerobic. Aerobic bioremediation processes are very effective in treating hydrocarbon contamination, however they are often expensive (e.g. hydrogen peroxide). For this reason anaerobic biodegradation provides cost-effective and advantageous in situ bioremediation technology that can be used for the decontamination of soil, sediment, and ground water contaminated with petroleum hydrocarbons. It was observed that benzene, toluene, ethyl benzene, and xylene (BTEX) are degradable without oxygen in contaminated groundwater (Johnson et al., 2003; Coates et al., 2002). Polyaromatic hydrocarbons (PAHs) have been identified as hazardous chemicals by different State and Central Pollution Control Boards, because of their toxic, carcinogenic and tetragenic effects on living body. At present, hydrocarbon fuels (mainly diesel) contain an excessive quantity of PAHs, causing abundant distribution of the same in the ecosphere. In order to protect environment from such PAH emission from diesel oil, a stringent EURO III standard has recently been enforced. This specifies that the maximum allowable concentration of PAH in diesel oil to be used as automobile fuel should be 11% by weight.Conventional hydro-treatment of diesel (using Co-Mo/Ni-Mo catalysts) to reduce the PAH content below this permissible limit failed. Even under high pressure (80 kPa) and temperature (633K) conversion of aromatics to naphthenes has so far been achieved only in order of 40%1. Investigation on the biodegradation on PAH is being carried out for a fairly long time and despite of the fact, as observed by some of the investigators, that these compounds may resist degradation by microbial enzymes2, many papers are appearing in literatures describing the success of biodegradation process of PAH3-12. This relatively new technology demands proper coordination between classical microbiological work and bioprocess engineering, followed by bioseparation for its successful use in industry.
In the present investigation a systematic and programmed bioprocess study of a mixed culture system capable of degrading PAH from a simulated mixture has been reported. In order to initiate the bioprocess study, isolation of mixed culture from soil of petrol pump of three major Indian cities, has been carried out. Using microbiological and biochemical tests, different strains were identified. These mixed cultures constituted the living biocatalysts for degradation of the substrate under study. Based on the performance of mixed cultures collected from different cities, it was observed that sample collected from Delhi city worked more efficiently compared to the samples of the other two cities. Thus, for the subsequent bioprocess study, the mixed culture obtained from the soil of Delhi city was exclusively used. Study on the removal of PAH by the strains so isolated has been carried out using simulated mixture of either anthracene or naphthalene in methanol solution as substrate. Since such biodegradation reaction follows a complex path, the intention of this investigation was to establish a systematic reaction program under controlled condition and to evaluate intrinsic kinetic parameters essential for bioreactor design. By judiciously performing simulation work and comparing the experimental data with available model equations, it is observed that the reaction engineering behavior of both naphthalene and anthracene degradation system can successfully be described by Monods substrate uninhibited model equation. In a subsequent attempt, the complete rate equation relating cell growth and substrate depletion has been computed by evaluating the intrinsic kinetic parameters, viz., max and KS, present in the Monod equation using differential analysis of the experimental data. It is needless to state that these kinetic parameters are of essential requirements for bioreactor design. Petroleum hydrocarbon continues to be used as the principle source of energy and hence an important global environmental pollutant (Rahman et al., 2003). Environmental pollution with petroleum and petrochemical products (complex mixtures of hydrocarbons) has been recognised as one of the most serious current problem, especially
when associated with accidental spills on the large scale (Plohland Leskovsek, 2002, Ghosh et al., 2006). Crude oil is composed of a wide range of hydrocarbons. Microbial utilization of these compounds as sole carbon sources is highly dependent on the chemical nature of the compounds within the petroleum mixture and on the environmental determinants (Atlas,1981; Bharathi and Vasudevan, 2001; Chukwu and Odunzeb, 2006). Under ideal conditions, the hydrocarbons are completely mineralized to carbon dioxide and water, with some biomass production. Biodegradation efficiency depends on microorganisms, capability of producing enzymes and that will degrade the target compounds. Factor such as temperature, pH and nutrient status are of importance as moderators (Alexander, 1994). Degradation of these hydrocarbon by microorganisms has been assessed by a variety of strategies including the seeding of the environment with mixed oil-utilizing bacteria (Dave et al., 1994). Numerous microorganisms, including bacteria, fungi, and yeasts are known for their ability to degrade hydrocarbons (Chaillan et al., 2006). Surfactants are amphipathic molecules with both hydrophilic and hydrophobic (generally hydrocarbon) moieties that partition preferentially at the interface between fluid phases with different degrees of polarity and hydrogen bonding such as oil/water or air/ water interfaces (Desai and Banat, 1997). Therefore chemical surfactants are widely used in industrial applications; biosurfactants are biological molecules with similar properties to their chemical counterparts. Probably the most important advantage of biosurfactants over chemical surfactants is their ecological acceptability. Moreover, biosurfactants are non-toxic, natural biodegradable products and thus, essentially compatible with the biogeochemical cycle (Haba et al., 2000). There have been recent many reports on using them in enhanced oil biodegradation (Mulligan et al., 2001; Rahman et al., 2002a; Urum et al., 2003). Biosurfactants can be produced with high yield by some microorganisms, especially Pseudomonas sp. These microorganisms can use the various renewal resources, especially agro industrial wastes, as the potential carbon sources (Maneerat, 2005). Among the best studied biosurfactants are rhamnolipids that belong to the glycolipid class (Tuleva et al., 2002). Rhamnolipids have been identified predominantly from Pseudomonas aeruginosa (Burger et al., 1963; Tuleva et al., 2002).
In the process of fulfilling the energy requirement for today's population, various natural resources have been exploited. But the principal source of energy continues to be petroleum hydrocarbon and hence a global pollutant. During accidental spills, action will be taken to remove or remediate the contaminant immediately, whereas in the gasoline and diesel stations the spills due to leakage may be small but continuous and prolonged. Because of its persistence, the chance for groundwater contamination is high. Toxicity of crude oil includes liver necrosis, congestion of the liver, fat degeneration, and dissociation of hepatocytes. Birds and animals in oil-contaminated area are found to have black emulsion in the digestive tract with a petroleum odor. This leads to decrease in the absorption of nutrients and finally leads to death of these birds and animals due to rupture of capillaries and hemorrhage, hepatocellular dissociation, hemosiderosis, renal tubular necrosis, and anemia. The aromatics in crude oils also have numerous adverse effects on the environment particularly to the local microbial flora. It was shown that a-pinene, limonene, camphene, and isobornyl acetate were inhibitory to the microorganisms. The phenolic and quinonic naphthalene derivatives inhibited the growth of the cells. Calder and Lader demonstrated that increasing amounts of naphthalene, 2-methylnaphthalene, pyrene, and others resulted in an increased lag phase and lowered the growth rates of two bacteria growing on these compounds. Uribe et al. reported the toxic effects of cyclohexane on the energy transduction in Saccharomyces cerevisiae. Cyclohexane inhibited oxygen uptake in intact cells and isolated mitochondria. Studies on isolated mitochondria showed that ATP synthesis was impaired whereas ATP hydrolysis was slightly increased. Uptake of potassium ions was impaired, and dissipation of the mitochondrial membrane potential was observed . These studies indicate that the permeability barrier of the inner mitochondrial membrane was disrupted by cyclohexane. Oil contamination with petroleum and petroleum-based hydrocarbons has caused critical environmental and health defects and increasing attention has been paid for developing and implementing innovative technology for cleaning up this contaminant. Bioremediation methods are currently receiving favorable publicity as promising environmental friendly treatment technologies for the remediation of
hydrocarbons. Moreover, biological methods can have an edge over the physico-chemical treatment regimes in removing spills as they offer cost effective in situ biodegradation of oil fractions by the microorganisms. Bioremediation can be described as the conversion of chemical compounds by living organisms, especially microorganisms, into energy, cell mass, and biological waste product . The rates of uptake and mineralization of many organic compounds by a microbial population depend on the concentration of the compound. Inhibition of biodegradation by nutrient or oxygen limitation or through toxic effects exerted by volatile hydrocarbons may occur due to high concentrations of undispersed hydrocarbons in water. Extreme pH and temperature conditions are expected to have a negative influence on the ability of microbial populations to degrade the hydrocarbons. Since the fate of hydrocarbon degradation is largely determined by the local environmental conditions, which influence the microbial growth and enzymatic activities, this research was carried out to explore the possibility of the use of selected bacterial cultures and a mixed bacterial consortium to degrade a crude oil at various pH, temperatures, and oil concentrations.
REVIEW OF LITERATURE
2. REVIEW OF LITERATURE:Bioremediation is emerging as a promising technology for the treatment of soil and groundwater contamination. The technology is very effective particularly in dealing with petroleum hydrocarbon contamination. However, bioremediation is a site-specific process and feasibility studies are required before full-scale remediation can be successfully applied. The type and scale of the feasibility studies that will be needed are specific to the bioremediation approach to be employed during full-scale clean-up operation. In all cases however, these studies have the same goals: to accurately determine if specific hydrocarbon contaminants are amenable to biological treatment and to determine the time and cost required to treat the contaminants of concern according to
the regulated clean-up criteria. This contribution provides background information on the chemistry and microbiology of hydrocarbon contamination, discusses the prospective of using biological methods for addressing this problem and describes several microbiological methods which can be used for the feasibility assessment of soil bioremediation. The focus of this project is to highlight the needs for the integration of laboratory data to full-scale bioremediation. Today, application of microorganisms for removing crude oil pollution from contaminated sites as bioremediation studies, was considered by scientists because other methods such as surfactant washing and incineration lead to production of more toxic compounds and they are non-economic. Decomposition of polyaromatic hydrocarbons was studied using mixed culture. Mixed strains were isolated from soil of petrol stations of different Indian cities and the best performing strains from these sources were used for subsequent bioprocess study using simulated mixture of anthracene and naphthalene as carbon source in methanol solution. The cell growth curve and substrate depletion time history curve obtained from batch fermentative process show that the reaction engineering behaviour of the systems under study can well be represented by classical substrate uninhibited Monods model. In a separate attempt the intrinsic kinetic parameters max and KS were evaluated following differential analysis of experimental data (Pinaki Bhattacharya et al.2007). In this study, the growth of sixty-one bacterial strains in crude oil were determined spectrophotometrically at 620 nm. Pseudomonas aeruginosa G1, Pseudomonas fluorescens G6, Pseudomonas stutzeri G11 and Pseudomonas putida G15 were chosen for the study based on the efficiency of crude oil utilisation. At 1% (v/v) crude oil concentration, P. stutzeri G11 strain degraded a maximum of 69%. The percentage of degradation by the P. stutzeri G11 strain decreased from 69% to 59% as the concentration of crude oil was increased from 1% (v/v) to 2.5% (v/v). Strain G11 was selected to determine the effects of surfactants (Tween- 80 and TritonX-100) on the biodegradation of crude oil. While strain G11 showed 76% degradation at mineral salts medium (MSM)
containing 1% (v/v) crude oil + 1% (v/v) TritonX-100, it showed 61% degradation at MSM containing 2.5% (v/v) crude oil + 2.5% (v/v)TritonX-100. Also, degradation rate of this strain was 96% in the presence of 1% (v/v) crude oil + 1% (v/v) Tween-80, while degradation rate was 48% in the presence of 2.5% (v/v) crude oil + 2.5 % (v/v) Tween80. Additionally, the rhamnolipid production of P. stutzeri G11 strain both in crude oil and in crude oil + two different surfactants (TritonX-100 and Tween-80, separately) were investigated. These results suggest that surfactants have improved both crude oil degradation and rhamnolipid production and the degradation rates have depended very much on the chemical structure of surfactants .( Gokcen Yuvali Celik*1 et al.2008) A preliminary study was undertaken to determine the optimal conditions for the biodegradation of a crude oil. Among 57 oil-degrading bacterial cultures isolated from oil-contaminated soil samples, Bacillus sp. IOS1-7, Corynebacterium sp. BPS2-6, Pseudomonas sp. HPS2-5, and Pseudomonas sp. BPS1-8 were selected for the study based on the efficiency of crude oil utilization. Along with the selected individual strains, a mixed bacterial consortium prepared using the above strains was also used for degradation studies. The mixed bacterial consortium showed more growth and degradation than did individual strains. At 1% crude oil concentration, the mixed bacterial consortium degraded a maximum of 77% of the crude oil. This was followed by 69% by Pseudomonas sp. BPS1-8, 64% by Bacillus sp. IOS1-7, 45% by Pseudomonas sp. HPS2-5, and 41% by Corynebacterium sp. BPS2-6. The percentage of degradation by the mixed bacterial consortium decreased from 77 to 45% as the concentration of crude oil was increased from 1 to 12%. Temperature of 35 C and pH 7 were found to be optimum for maximum degradation. Muthuswamy Sathishkumar1, et al,2008)
(
Many Enzymes produced by Pseudomonas aeruginosa had been used in commercial scales, lipase is an example. P. aeruginosa has been investigated intensively during past decays regarding its medical and industrial importance. It was establish in different wastes treatment processes such as oil waste bioremediation. In this study, two
strains of P. aeruginosa were used to produce lipase from Castor oil aiming to control the different castor oil wastes and technical lipase production. Both strains were able to grow on media consists of 20 g/l Castor oil and 3 g/l Yeast extract at 37C. The lipase enzyme activity at 37, 60 and 75C in presence of 0.4, 0.8, 2 and 2.8 mg/ml p-nitrophenyl palmitate were investigated. The V and K using of both Hanes and max m LineweaverBurk plots were determined. Lipase produced from the two strains revealed mesophilic and thermophilic activities. The maximum activity at 37C was 1121.00 and 470.82 Units/ml, while at 75C was 358.72 and 147.18 Unit/ml for strain 1 and strain 2, respectively. P.aeruginosa lipases for castor oil waste control and for the production of mesophilic and thermophilic lipases under mesophilic condition was recommended.( Amro
A. Amara and Soheir R. Salem,2009)
Laboratory scale batch studies were performed to test the diesel oil biodegradation ability of ES1 cultures isolated from Arabian Sea sediments obtained from the vicinity of an oil field. This culture could utilize diesel as the sole source of carbon and energy. Under aerobic conditions, 39% loss of diesel oil was observed over 8 days where 80% of the loss was due to aliphatic constituents. Under anoxic nitrate reducing conditions the rate and extent of degradation was significantly lower, i.e., 18% over 50 days. Salt acclimatized cultures could tolerate salinities up to 3.5% and demonstrated optimal performance at a salinity of 0.5%. The optimum N/P ratio for these cultures was found to be in the range of 2:15:1. Addition of two trace elemental substance formulations exhibited a significant inhibitory effect on culture growth. This culture has good potential for decontamination of oil-contaminated marine and subsurface environments. (Suparna Mukherji et al) The bacterial diversity in a tropical soil experimentally polluted with crude oil during a 57 days bioremediation was investigated in five 1 m2 plots using total culturable hydrocarbon utilizing bacteria, heterotrophic bacteria and gas chromatographic analyses. Four out of the five experimental plots received each 4 L of Bonny light crude oil while
three treatment plots received 3 kg of NPK, urea fertilizers or poultry droppings with periodic tilling. Two plots, oil-contaminated and pristine served as controls. Bacterial counts increased 200 fold and 2 fold in the NPK treated and poultry-dropping-treated plots respectively, by day 31 post-inoculation. Detectable hydrocarbons in the treatment plots decreased by 84 - 95% and 96 - 99%, 31 and 57 days post inoculation, respectively, compared with the petroleum contaminated control. Bacterial strains isolated included Rhodococcus sp., Nocardia sp., Arthrobacter sp., Gordonia sp., Mycobacterium sp., Corynebacterium sp., Bacillus sp., Micrococcus sp., Flavobacterium sp., Pseudomonas sp. and Alcaligenes sp. The overall data suggest an important contribution of Actinobacteria during bioremediation of crude oil-polluted soil. ( Chikere1 et al ,2009) The potential biodegradation of crude oil was assessed based on the development of a fermentative process with a strain of Pseudomonas aeruginosa which produced 15.4 g/L rhamnolipids when cultured in a basal mineral medium using glycerol as a sole carbon source. However, neither cell growth nor rhamnolipid production was observed in the comparative culture system using crude oil as the sole carbon source instead. As rhamnolipid, an effective biosurfactant, has been reported to stimulate the biodegradation of hydrocarbons, 1 g/L glycerol or 0.22 g/L rhamnolipid was initially added into the medium to facilitate the biodegradation of crude oil. In both situations, more than 58% of crude oil was degraded and further converted into accumulated cell biomass and rhamnolipids. These results suggest that Pseudomonas aeruginosa could degrade most of crude oil with direct or indirect addition of rhamnolipid. And this conclusion was further supported by another adsorption experiment, where the adsorption capacity of crude oil by killed cell biomass was negligible in comparison with the biologic activities of live cell biomass. (ZHANG Guo-liang ()1 et al,2005) Potential mineral oil degrading bacterial isolations (Pseudomonas putida, Rhodococcus erythroplolis and Bacillus thermoleovorans) were studied from the contaminated soils. The bacterial populations of these polluted soils were 5.25 x 105,
1.76 x 106 and 5.11 x 105 cell/ml with three different colony types of bacterial strains, respectively. The study was conducted at laboratory research center in Hannover, Germany. On the other hand, microbial population diversity studies were carried out by microbial enumeration, identification, and determination of growth responses of bacterial isolates in different concentrations of mineral oil sample. Phenotypic examination of the heterotrophic bacteria belonged mainly to the genus Pseudomonas, Rhodococcus and Bacillus. The mixed populations were capable of degrading mineral oil of up to 120 ppm. The biodegradation of mineral oil was fast by the mixed culture comparing to the biodegradation of each strain separately. It could be noticed from this study how to restore contaminated soils to a non toxic state. Healthy soils are essential, not only to sustain production of food and fiber for citizens of the world, but also, to provide for a good quality of life. (Abdel-Megeed et al)
AIM AND OBJECTIVES
3. AIMS AND OBJECTIVES
The primary objectives of this study were to:-
I. Isolation, identification and characterization of hydrocarbon-degrading microorganisms from the terrestrial sites by selective enrichment technique.
II.
Investigate the biodegradation potential of our interest Pseudomonas strain.
III.
Cloning of oil degrading gene into E.coli
MATERIALS AND METHODS
4. MATERIALS AND METHODS

4.1. SOIL SAMPLE: Soil samples were collected from three different sources such as a) Petrol pump b) Mechanic shop
c) Yamuna Bank
4.2. SERIAL DILUTION:
In this technique the sample was serially diluted in sterile (isotonic) saline solution (Appendix-I), and. The basis of obtaining isolated colonies with this technique is the reduction in the number of bacteria per unit volume in the inoculum by dilution.
4.2.1. PROCEDURE: 1gm of soil sample was transferred aseptically into 9ml of saline water which gave 100 dilution and marked as test tube A The test tube A was vortexed and 1 ml of sol. Of test tube A was transferred aseptically into 9ml of saline water in test tube B which gave 10-1 dilution The test tube B was vortexed and 1 ml of sol. Of test tube B was transferred aseptically into 9ml of saline water in test tube C which gave 10-2 dilution
The test tube C was vortexed and 1 ml of sol. Of test tube C was transferred aseptically into 9ml of saline water in test tube D which gave 10-3 dilution The test tube D was vortexed and 1 ml of sol. Of test tube D was transferred aseptically into 9ml of saline water in test tube E which gave 10-4 dilution 4.3. SPREAD PLATE: This method is, used for quantitative estimation of microbial cells in a known volume of original sample. Finally one ml aliquot of 10 -4 dilution was added to a sterile Petri dish to which was added 15 ml of sterile, cool, molten nutrient agar medium (Appendix-I) and. spreaded with a sterile bent glass rod . The dishes are incubated at 370c temperature. After 2 days of incubation colonies of each kind of microbes grow in the dish. The number of colonies of each kind was counted. This number was then multiplied by the dilution factor to find the total number of cells per ml of the original sample. 4. 4. ISOLATION OF PURE CULTURE: If the bacterial species being sought comprises a suitably high proportion of the mixed population it can be isolated into pure culture. This method is used for separation of individual bacteria from each other in a mixed culture.
4.4.1. PROCEDURE: Each isolated colony (blue, green and white) of three different serially diluted samples was streaked aseptically on a nutrient agar plate. The same process was repeated 5 times to obtain pure culture of individual strain. 4. 5. OIL DEGRADING ABILITIES:-
The ability of the isolated colony to degrade the oil can be viewed by culturing the isolated colony in synthetic media broth (Appendix-I) containing Castrol oil. If the isolated strain thrives in the media it clearly depicts that it has the ability to degrade the oil and utilize it as the carbon source. 4.5.1. PROCEDURE: One loopful of isolated strain was inoculated in to 5ml of synthetic media and 2.5l of Castrol oil in a test tube.It was incubated in Incubated shaker at 36 C, 64 RPM for two days. This process was repeated for each strain isolated to check their oil degrading ability. 4.6. STREAKING ON KINGS B MEDIA: Kings B media (Appendix-I) is a selective media which favour the growth of only Pseudomonas. If the growth appears on Kings B media it will reveal that our isolated strain is of Pseudomonas.
4.6.1. PROCEDURE: One loopful of isolated strain which has capacity to degrade the oil was streaked aseptically on Kings B media and incubated at 37C for overnight.
4.7. STREAKING ON CETRIMIDE AGAR: -
Cetrimide Agar (Appendix-I) is used in the isolation and identification of Pseudomonas aeruginosa. Pseudomonas aeruginosa produces a number of watersoluble pigments, including the yellow-green or yellow-brown fluorescent pigment pyoverdin (fluorescein). When pyoverdin combines with the blue water-soluble pigment pyocyanin, the bright green color characteristic of Pseudomonas aeruginosa is
created. Agar containing Cetrimide has been used successfully to isolate Pseudomononas aeruginosa.
4.7.1. PROCEDURE: One loop of isolated strain cultured on Kings B media was streaked aseptically on Cetrimide Agar plate and incubated at 37C for overnight. If after incubation the strain on the plate appears green it may be Pseudomanas aeruginosa. It may be confirmed by performing further biochemical test. If the strain does not appears green it may belongs to any other species of Pseudomonas which can be further clarify by performing biochemical test.
4.8. BIOCHEMICAL TEST SOIL ISOLATES: (APPENDIX-1 & APPENDIX-2) 4.8.1. GRAM STAINING: A loopful of sterile distilled water was placed onto a microscope slide. The isolated colony was smeared with inoculating loop, dried at room temperature. The smear was heat fixed. The slide was flooded with crystal violet and left for one minute. The slide was washed with cold water and flooded with Grams iodine, left for one minute. Again the slide was washed with water and decolorize until the solvent flows colorlessly from the slide. The slide was flooded with safranine and left for 30 seconds. Finally the slide was washed with water, dried and examined under the microscope for Gram reaction.
4.8.2. INDOLE PRODUCTION TEST: In 5ml of peptone water 1ml of the inoculum was inoculated and incubated at 37C for 24 hours.
After incubation 5 drops of Kovacs reagent was added to the surface.

4.8.3. METHYL RED TEST:-
In 5ml of Glucose Phosphate broth, 1ml of inoculum was inoculated and incubated at 37C for 48 hours. After incubation, 5drops of Methyl red indicator was added to the surface. The tube was rolled between the palms of hands to disperse methyl red.
4.8.4. VOGES PROSKAUER TEST:

In 5ml of Glucose-Phosphate broth, 1ml of the inoculum was inoculated and incubated at 37C for 48 hours. After incubation, 0.6ml of -napthal was added and shaked. Then 0.2ml of 40% potassium hydroxide was added, mixed gently and left 1015 minutes for color development.
4.8.5. CITRATE UTILIZATION: One loop of inoculum was streaked and incubated at 37C for 48 hours.
4.8.6. OXIDASE: A piece of filter paper was placed on a cleaned microscope slide. 2- 3drops of oxidase reagent was placed on to the filter paper. The isolated colony was smeared with a loop on the filter paper. The presence of a dark purple color was observed. The reaction is positive, if the smear turns purple within 10-30sec.
4.8.7. CATALASE: A drop of 3% hydrogen peroxide was placed on to a clean microscope slide. The isolated colony was smeared with an inoculating loop in to the drop of hydrogen peroxide. Then the slide was observed for the evolution of bubbles. The reaction is positive if oxygen bubbles form rapid.
4.8.8. UREA HYDROLYSIS: The inoculum was inoculated in the media with a loop and incubated at 37C for 24hours.
4.8.9. MOTILITY TEST: The inoculum was inoculated deep with a inoculating loop and incubated at 37C for 24-48 hours.
4.8.10. MANNITOL FERMENTATION: The culture was streaked on Mannitol Salt Agar plate and incubated at 37C for 2 days.
4.8.11. NITRATE REDUCTION TEST: In order to determine if the bacteria can reduce nitrate, the organism was inoculated into nitrate reduction broth, an undefined medium that contains large amounts of nitrate (KNO3). After incubation, alpha-napthylamine and sulfanilic acid were added.
4.8.12. LACTOSE FERMENTATION:-
The culture was streaked on MacConkey agar plate and incubated at 37C for 2 days.
4.8.13. GELATIN LIQUEFACTION TEST: A heavy inoculum from a pure culture of the organism was stabbed into the media. The gelatin media was incubated for at least 48 hours, and then placed into the refrigerator for approximately 30 minutes.
4.8.14. STARCH AGAR TEST: The isolated culture was streaked on the starch agar media. The starch agar media was incubated for 24 hours at 37C.
4.8.15. CARBOHYDRATE FERMENTATION TEST:-
The isolated culture was inoculated in sucrose broth.
The broth was incubated at 37C for 24 hours.
4.9. OIL DEGRADATION STUDIES ON DIFFERENT pH :The influence of pH on the growth and degradation of Castrol oil was studied. The green isolated colony was inoculated into three test tubes, each containing 5ml of sterile synthetic media and 2.5l of sterile Castrol oil. Each test tube contained sterile mineral
salts medium with Castrol oil and inocula at different pH from 5 to 7. The three different pH were prepared by using 1N HCL and NaOH. The tubes were then incubated in Incubator Shaker at 36C for 64 rpm. After 2 days incubation the bacterial growth in the medium with Castrol oil were determined spectrophotometrically. The studies were detected spectrophotometrically at 550nm. 4.9.1. DEGRADATION STUDIES CONCENTRATIONS:WITH VARYING CASTROL OIL
Biodegradation of Castrol oil with the isolated colony was performed with various concentration of oil (2.5l, 3l, 3.5l----------------2ml) at pH 5, 6, 7 respectively. The inoculated tube were incubated for 2 days and bacterial growth, Castrol oil degradation were estimated.
4.10. MOLECULAR CLONING:4.10.1. MATERIALS AND METHODS:4.10.1.1. Chemicals. :-
Castrol - 2T Supreme (2 Stroke Engine oil), Castrol India Limited, Mumbai 400093. 4.10.1.2. BACTERIAL STRAIN, VECTOR, REAGENTS, AND GROWTH CONDITIONS:The bacterial strain used was Pseudomonas aeruginosa. The vector used was pBR 322 (Promega) (APPENDIX -2). E. coli strains were grown at 37C on Luria-Bertani (LB) medium (APPENDIX -1) supplemented with 100 and 15 g/ml of ampicillin, tetracycline respectively. The bacterial strain was grown at 36 C in synthetic media with 2% crude oil. 4.10.1.3. ISOLATION OF GENOMIC DNA:Genomic DNA from the efficient crude oil degrading isolate was obtained in the following way:Method: The isolate was grown over night in Nutrient broth (5ml) and cells were separated by centrifugation (10,000rpm for 2min).
The pellet obtained was suspended in 567l TE buffer by repeated pipetting. SDS (30l, 20%), proteinase K (5l, 25mg/ml) and RNase (5l, 10mg/ml) and lysozyme (30l, 100mg/ml) were added to the suspension and incubated at 37 0C for 1 hour. (APPENDIX-1) NaCl (100l, 5M) was added to the above suspension and mixed thoroughly. CTAB (cetryltrimethylammonium bromide) / NaCl solution (80l) was added and incubated at 65 0C for 30minutes. To the above suspension, equal amounts of chloroform and isoamyl alcohol (24:1) was added, mixed well and centrifuged at 15,000rpm for 15 minutes at room temperature. Upper aqueous layer formed during the above step was transferred into fresh eppendorf tube and equal volumes of phenol, chloroform and isoamyl alcohol (24:24:1) was added, mixed by inversion and finally centrifuged at 15,000rpm for 15 minutes at room temperature. To the supernatant obtained, equal volume of isopropanol was added, mixed well, incubated at 0C for 15 minute and again centrifuged at 15,000 rpm for 15 minutes. Supernatant was discarded from the above step and the pellet obtained was washed with 200 l of 70% ethanol by centrifuging at 15,000rpm for 10 minutes. Supernatant was discarded and the pellet was air dried. 40 l of TE buffer was added to the pellet. Electrophoresis of this DNA sample was done. 4.10.1.4. ISOLATION OF PLASMID: The isolate was grown over night in Nutrient broth (5ml) and cells were separated by centrifugation (10,000rpm for 2min).
The pellet obtained was suspended in 100 l ice cold TE buffer. 5 l of RNase was added and incubated in ice for 5 minute. 200 l of Lysis buffer was added and mixed gently by inverting & rolling. It was incubated on ice for 5 minute. 150 l of ice cold 3M potassium acetate was added and vortexed. It was incubated in ice for 10 minute and centrifuged at 12,000 rpm for 10 minute. In the supernatant equal amount of phenol : chloroform : isoamyl alcohol mixture was added and centrifuged at 12,000 rpm for 10 minute. The top layer was taken and equal amount of ice cold isopropanol was added and incubated at 0C for 15 minute. It was centrifuged at 12,000 for 10 minute. Supernatant was discarded from the above step and the pellet obtained was washed with 200 l of 70% ethanol by centrifuging at 15,000 rpm for 10 minutes. Supernatant was discarded and the pellet was air dried. 40 l of TE buffer was added to the pellet. Electrophoresis of this Plasmid sample was done. 4.10.1.5) Agarose Gel Electrophoresis:Gel Running Buffer: TAE Buffer (APPENDIX-1) is preferred for faster electrophoretic migration of linear DNA. DNA Loading Dye:6X gel loading buffer 30% Glycerol (v/v), 0.25% Xylene cyanol (w/v), 0.25% bromophenol blue (w/v).
Electrophoresis:-
For genomic DNA, 0.5gm of agarose was dissolved in 50ml of 1X TAE (1% agarose gel) and heated to melt agarose.
Then it was cooled down to 60-65 C and 5l of Ethydium bromide was added.
Finally the gel was run at 70-100 V (voltage gradient) containing DNA sample in the well.
Agarose gel was viewed directly on a UV trans illuminator and the DNA
sample was proceeded for PCR.
4.11. POLYMERASE CHAIN REACTION (PCR):PCR allows the production of more than 10 million of copies of a target sequences from only a few molecules. 4.11.1. MATERIALS: Template DNA (genomic and plasmid of Pseudomonas aerugenosa) and OPY 7, Sequences-5- AGAGCCGTCA 3) BTK Biosciences, New Delhi. Taq Polymerase GeNei, Bangalore. Distilled water Taq Buffer with MgCl2, dNTPs ,
Oligonucleotide primer (IBRC-RP07, Sequences- 5- TTGGCACGGG 3
4.11.2. REACTION MIXTURE SET UP: All solutions after thawing are gently vortexed and centrifuged.
In a PCR tube all the components were added in the following manner.
TABLE-1 (Reaction Mixture for PCR) REAGENT Template DNA IBRC-RP07 (Primer) 2mM dNTPs mix Taq Buffer with MgCl2 Taq DNA Polymerase Sterile deionized water FINAL CONCENTRATION 1g 0.1-1M 0.2mM of each 1X 1.25 l /50 l QUANTITY, for 20 l of Reaction Mixture 2 l 2 l 2 l 2 l 0.5 l 11.5 l
The reaction mixture sample was gently vortexed and centrifuged to collect all drops from walls of tube. Then the samples in the PCR tube were placed in the Thermo cycler and PCR was started. 4.11.3. CYCLING CONDITIONS:Lid temperature - 110C Initial Denaturation step - 94C 5 minute Denaturation step - 94C 1 minute Primer Annealing step - 36C 40 second Extending step - 72C 1 minute No of cycles 33 cycles Final Extending step - 72C 5 minute Hold - 4C Along with genomic DNA and plasmid of Pseudomonas aeruginosa, E.coli
genomic DNA and plasmid were amplified.

The PCR samples were run in 1.5% agarose gel, checked for amplification along
with 1Kb DNA ladder (Invitrogen).
4.11.4. PCR Result: The 600-650 base pair of DNA fragment of plasmid of Pseudomonas was amplified and was not present in E.coli plasmid. This fragment was the gene of interest (insert DNA) which was separated from the gel for cloning. 4.12. RECOVERY OF DNA FROM LMP (LOW MELTING POINT) AGAROSE GEL: The DNA fragment was detected by irradiating the gel with long-wave ultraviolet light (wave length: 355 - 360 nm).
T he region of the gel containing desired DNA was cut out. The gel slice was transferred into a microfuge tube.
To the gel slice about 1-3 volumes of TE buffer was added. 0.3 volume of 3M sodium acetate was added (pH7).
The tube was heated at 65 C. Eventually the tube was tapped to mix the melted gel and TE buffer.
Centrifuged for 2 min at room temperature, then for 5 min at 4 C.
Equal volume of isopropanol was added and microfuge tube was incubated for 1 hour at 4C.
DNA was concentrated with ethanol precipitation.
The insert DNA was again amplified with PCR.
4.13. RESTRICTION DIGESTION OF INSERT DNA: The components were combined in the following order in a microfuge tube: 10l 10X Restriction Endonuclease Buffer 1 l Bovine Serum Albumin 2 l Insert DNA(amplified) 1 l E co.R1 Enzyme Incubated for 2 hours at 37C in Bacteriological Incubator.
4.14. DIGESTION OF VECTOR DNA: The components were combined in the following order in a microfuge tube: 10l 10X Restriction Endonuclease Buffer 1 l Bovine Serum Albumin
2 l Vector DNA(pBR-322) 1 l E co.R1 Enzyme Incubated for 2 hours at 37C in Bacteriological Incubator.
4.15. LIGATION OF DNA INSERT WITH VECTOR DNA: The components were combined in the following order in a microfuge tube: Insert DNA digested - 10 l Vector DNA digested -5 l T4 DNA Ligase Buffer- 1 l T4 DNA Ligase -5 l
Incubated at 16 C for overnight. After incubation it was heated at 65C for 5-10 minutes for inactivation of the enzyme.
4.16. PREPARATION OF CHEMICALLY COMPETENT E. coli CELLS:Competent cells are those that possess more easily altered cell walls that DNA can be passed through easily. These cells readily incorporate foreign DNA. On example of a competent cell is E. coli. 4.16.1 PROTOCOL: Overnight pure E.coli (DH5) culture was inoculated in 50ml of LB broth (APPENDIX-1) and kept in shaker for overnight at 37C. 1ml of overnight kept E.coli culture was inoculated in fresh 50ml LB broth and kept in shaker for 3-4 hrs. 1ml culture was taken in an eppendorf tube and centrifuged at 5000 rpm for 10 minute.
The supernatant was discarded and 1ml of TSS Solution (Transformation storage solution) (APPENDIX-1) was added to the pellet and stored at -20C.
4.17 TRANSFORMATION OF PLASMID DNA INTO CHEMICALLY COMPETENT E.coli CELLS:The uptake of exogenous DNA by cells that alters the phenotype or genetic trait of a cell is transformation. For cells to uptake exogenous DNA they must first be made permeable so the DNA can enter the cells. This state is referred to as competency. 4.17.1. PROTOCOL: 100l of competent cell with 10l of pBR-322(ligated) were taken in an eppendorf. Incubated at 0C for 15 min, kept at 42C for 45 sec and again incubated at 0 C for 15 min (HEAT SHOCK). 900l of LB broth was added to the eppendorf and incubated at 37C for 1 hr. Centrifuged for 5000 rpm for 5 min. 700l of supernatant was discarded and the pellet was resuspended in the remaining solution. Finally spreaded on agar plates. 100l of resulting culture was spreaded on LB plates containing ampicillin (1mg/ml) or tetracycline (1mg/ml).
RESULTS AND DISCUSSION
5. RESULT AND DISCUSSION:5.1. ISOLATION AND CHARACTERIZATION STUDIES: The pure soil isolates are isolated from the single colonies on Nutrient Agar, Kings B Media and Cetrimide agar. Characteristics of soil isolates are given below in following Table 2: Table 2: CHARACTERIZATION OF SOIL ISOLATES:No. of colonies Oil degrading Ability Growth On Kings B Media
Place
Morphology
i ). Blue, Regular, Matt 1. Petrol Pump 4 ii ). Green, Regular, Glassy iii).White, Regular, Matt. iv).White, Regular, Glassy. i ). Blue, Regular, Glassy 2. Mechanic shop 3 ii). White, Regular, Matt. iii).White, Regular, Glassy i) .White, Irregular, Matt. 3. Yamuna Bank 3 ii ). White, Irregular, Glassy iii). White, Regular, Matt
Negative Negative Positive Positive Negative Positive Positive Positive Negative Positive
Negative Negative Positive Positive Negative Positive Positive Positive Negative Positive
Table 3: GROWTH OF THE ISOLATES IN CETRIMIDE AGAR:No. of colonies on Kings B Media 2 Morphology i).White, Regular, Matt. ii).White, Regular, Glassy. i . White, Regular, Matt. ii).White, Regular, Glassy i ) .White, Irregular, Matt. ii). White, Regular, Matt Cetrimide Agar Test Positive (Green) Negative Positive (Green) Negative Positive (Green) Negative
Place 1. Petrol Pump
2. Mechanic shop
3. Yamuna Bank
Figure2: Colonies grown on Nutrient Agar
Figure 3: Isolated colonies of nutrient agar streaked on KingsB Media
Figure 4: Isolated Pseudomonas sps. Streaked on Cetrimide Agar
Green colony on Cetrimide Agar(Pseudomonas aeruginosa) 5.1.1. BIOCHEMICAL STUDIES:
The pure soil isolates of Kings B Media and Cetrimide Agar are subjected to various Biochemical test for characterization of organisms. Figure5: Biochemical test of isolates of KingsB media and Cetrimide Agar
1- Urease Test of Plates Of Cetrimide Agar 2, 3, 4- Urease Test of Plates of KingsB Media 5- Methyl Red Test of Colonies of Cetrimide Agar 6- Voges-Proskauer Test of Colonies of Cetrimide Agar 7- Peptone Test of Colonies of KingsB Media 8- Peptone Test of Colonies of Cetrimide Agar 9, 10- Simmons Citrate Agar Test of Colonies of KingsB Media 11- Simmons Citrate Agar Test of Colonies of Cetrimide Agar
TABLE:4 : MORPHOLOGICAL AND BIOCHEMICAL TESTS FOR SOIL ISOLATES FROM DIFFERENT SOIL SOURCES
S.NO 1 2 3 4 5 6
MOPHOLOGICAL TESTS Media used for inoculation Colony character on selective medium Colony character on nutrient agar Gram-stain character Motility BIOCHEMICAL TESTS* a) Oxidase reaction b) Nitrate reduction c).Catalase test d) Citrate e) Gelatin liquefaction tests f) Indole test g) Methyl Red test h) Voges- Proskauer Test i) Mac Conkey agar test j) Sugar tests ( Sucrose, Mannitol) k) Urease l) starch Agar
Green colony on Cetrimide agar Synthetic media with Castrol oil White, irregular, matty Fluorescent and diffusible pigment Gram -ve rods motile + + + + Non- lactose fermenting -
Note: + = Positive
- = Negative
From the above biochemical tests it can be concluded that the green colonies grown in Cetrimide agar was found to be pseudomonas aeroginosa (confirmed from website (www.microbeid.com.) 5.2 BIODEGRATION STUDIES: 5.2.1. Degradation studies with varying Castrol oil concentrations and different pH:Various concentrations of Biodegradation of Castrol oil at different pH was estimated by using spectrophotometrically.
Table 5: (Optical density at 550 nm after 2 days incubation)
Sl .No
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
pH- 5
0.204 0.297 0.35 0.445 0.425 0.518 0.642 0.598 0.696 0.748 0.798 0.802 0.876 0.767 0.658 0.866 0.939 0.967 0.762 0.622 0.579 0.436 0.336 0.216
pH- 6
0.453 0.552 0.712 0.932 1.132 1.303 1.12 1.293 1.307 1.462 1.492 1.071 1.117 1.399 1.437 1.506 1.484 1.216 1.174 0.887 0.706 0.695 0.609 0.495
pH- 7
1.08 1.208 1.264 1.299 1.348 1.468 1.38 1.441 1.497 1.578 1.596 1.695 1.793 1.835 1.712 1.756 1.792 1.688 1.526 1.594 1.177 1.007 0.891 0.808
Castrol oil concentration(l)

2.5l 3l 3.5l 4l 4.5l 5l 6l 7l 8l 9l 10l 15l 20l 25l 30l 35l 40l 45l 50l 55l 60l 1000l 1500l 2000l
Out of 10 colonies isolated from different soil sources such as petrol pump, mechanic shop and Yamuna bank only 6 pure cultures able to grow in mineral salts medium with Castrol oil as carbon source were identified through enrichment and isolation procedure. After streaking on Kings B media and Cetrimide agar the isolated pure cultures were identified to belong to the genus Pseudomonas. The normal heterotrophic bacteria flora present in the soil.
However, it is the dominant strain. Studies on the effect of the pH showed that pH 7 was favorable for the bacterial isolates. Population of the individual culture i.e. Pseudomonas aeruginosa increased with time at pH 7. FIGURE 6: pH GRAPH
FIGURE7: PH STUDIES ON DIFFERENT CONCENTRATION OF OIL (2.5,3L,..2mL)
5.3. MOLECULAR CLONING STUDIES:

Figure 8: Isolation of Genomic DNA and Plasmid of E.coli and Pseudomonas aeruginosa
Figure 9: Polymerase chain reaction of Pseudomonas aeruginosa and E.coli plasmid
Gene of Interest lies between 600 bp to 500 bp
Figure 10: Restriction Digestion of gene of interest (Insert DNA of Pseudomonas aeruginosa) and pBR-322 (Vector DNA)
Figure:11: Transformed E.coli cells(insert DNA + Vector) on Luria Bertani Medium supplemented with Ampicillin.
Transformed E.coli cells on LB plate supplemented with ampicillin and tetracycline and it was showing oil degradation ability when grown on synthetic media with oil.
DISCUSSION:The isolate Pseudomonas aeruginosa was identified as hydrocarbon degrading
Microorganisms. Crude oil contains many kinds of hydrocarbons such as saturated and aromatic hydrocarbons, resins and asphaltenes. A number of compounds slightly degradable by microorganisms are contained in the fraction of aromatic hydrocarbons. Metagenomic analyses by many researchers have showed that only a few enzymes which degrade aromatic hydrocarbons are produced in most microorganisms in natural environments. Mixed populations with overall broad enzymatic capacities are required to degrade complex mixtures of hydrocarbons such as crude oil or diesel fuel. Such mixed cultures display metabolic versatility and superiority to pure cultures. A microbial consortium containing a number of microorganisms which synthesize the degradative enzymes for different parts of the decomposition pathway is considered to be well suited to the degradation of aromatic hydrocarbons. Microorganisms not directly involved in the degradation process also probably play a role by producing micronutrients or surfaceactive agents for the solubilization of aromatic hydrocarbons. Biodegradation caused by mixed cultures was more effective than that caused by pure cultures mainly due to the complexity of oil products. Various organisms have the capability of degrading various forms of hydrocarbons and thus when a consortium of these microbes is applied to degrade various forms of hydrocarbons in a single source like crude oil, the total degradation is more effective. The isolated individual oil degrades showed optimal values of pH 7 and temperature 36C for maximum degradation. pH 7 as the optimal range for hydrocarbon degradation. Extremes in pH were shown to have a negative influence on the ability of microbial populations to degrade hydrocarbons. Temperature influences petroleum biodegradation by its effect on the physical nature and chemical composition of the oil, rate of hydrocarbon metabolism by microorganisms and composition of the microbial community . At low temperatures, the viscosity of the oil is increased, volatilization of alkanes reduced, and the water solubility decreased, delaying and decreasing the onset of biodegradation. Above that temperature, degradation of hydrocarbons decreased, which may be attributed to the membrane toxicity of
hydrocarbons at elevated temperatures. 30C - 36C was considered to be the optimum temperature for microbial growth and PAH degradation. It has been observed that 75 and 85% degradation of total crude oil by Pseudomonas aeruginosa strain at 20 and 30C, respectively. Since all isolates were mesophilic in nature, they all exhibited optimum activity at 35C. Crude oil degradation was inversely proportional to the concentration of the oil. Compounds such as saturates, aromatics, and polar compounds present in different crude oil samples were degraded to different degrees by the same organisms. The degradability was not solely determined by the chemical structure but other factors as well. The bioavailability of these compounds in different crude oil samples may differ. Saturated compounds with molecular weight larger than 500 may not be degraded by the organisms, because this size corresponds to the exclusion size for passage through the outer membrane of Gram-negative bacteria. Generally, it is believed that microbes preferably degrade/metabolize C8C15 nalkanes followed by C16C36 n-alkanes due to the simplicity of these hydrocarbons. Saturated, cyclic high-molecular weight compounds like hopanes are usually not attacked by the microbes due to their complexity. Most of the light aromatics like toluene, xylene, etc. are reported to be degraded either completely or partially . Although it is not possible to specifically emphasize the metabolic pathway of degradation by individual microbes and microbial consortium without complete characterization of the crude oil before and after degradation. Due to the highly complex nature of the crude oil, it is very difficult to understand the degradation mechanism especially for aromatics. The study revealed that the isolate achieved maximum crude oil degradation at pH 7 and 35C. Hence, it is suggested that the use of isolate i.e. Pseudomonas aeruginosa under optimized conditions will be an effective and ecofriendly technology for the degradation of hydrocarbons from crude oil.
To test whether the 200bp, 400bp, 600bp or 800bp genes of Pseudomonas aeruginosa confers any aromatic-ring-hydroxylating abilities to E. coli, the genes were cloned into DH5 E.coli to construct the expression vector pBR322. The E. coli strain transformed with pBR 322 with 600 bp gene seemed to actively results in the hydroxylation of aromatic substrates. Since biological treatment can efficiently destroy the hydrocarbons and does not allow the contaminant to accumulate, it is considered to be a superior technology.
BIBLIOGRAPHY
BIBLIOGRAPHY: 1. Abdel-Megeed, A. A. M.1- and Abou-El Seoud I. I. A.2.Biodegradation of mineral oil by bacterial strains isolated from contaminated soils. Dept. of Plant Protection 2- Dept. Of Soil and Agric. Chem.Faculty of Agricultural (Saba Basha), Alexandria University, Egypt
2. Alexander, M.: Biodegradation and Bioremediation. Ca.: Academic Press, San Diego (1994). 3. Atlas, Ronald M.: Microbial degradation of petroleum hydrocarbons, an environmental perspective. Microbiol. Rev., 45, 180-209 (1981) 4. Ausubel FM, Brent R, et al. (1992) Current protocols in Molecular Biology. John Wiley and Sons, New York, ISBN: 0-47125092-9. 5. Bharathi, S. and N. Vasudevan: Utilazation of petroleum hydrocarbons by Pseudomonas fluorescens isolated from petroleum contaminated soil. Environ. Int., 26, 413-416 (2001). 6. Braddock FJ, Lindstrom EJ, Reisinger JH, and Brown JE. Distribution of hydroWilliam Sound, Alaska, follow Pollution Bulletin 1995;30:125-132. 7. Burger, M.M., L. Glaser and R.M. Burton: The enzymatic synthesis of rhamnose-containing glycolipid by extracts of Pseudomonas aeruginosa. J. Biol. Chem., 238, 2595-2602 (1963). 8. Capelli SM, Busalmen PJ, and De Snchez RS.Hydrocarbon bioremediation of a mineral-base contaminated waste from crude oil extraction by indigenous bacteria. International Biodeterioration and Biodegradation 2001;47:233-238 9. Chaillan, F., C.H. Chaineau, V. Point, A. Saliot and J. Oudot: Factors inhibiting bioremediation of soil contaminated with weathered oils and drill cuttings. Environ. Pollut., 144, 255-265 (2006). 10. Chaineau CH, Rougeux G, Yepremian C, and Oudot J.Effect of nutrient concentration on the biodegradation of crude oil and associated microbial populations in the soil. Soil Biology & Biochemistry 2005;xx:1-8 11. Chang BV, Chang JS, and Yuan SY. Anaerobic degradation of phenanthrene in river sediment under denitrifying conditions. Bulletin of Environmental Contamination and Toxicology 2001 (in press).
12. Chang VB, Chang WS, and Yuan YS. Anaerobic degradation of polycyclic aromatic hydrocarbons in sludge.Advances in Environmental Research 2003;7: 623-628 13. Characterization and identification of Bacillus cereus GMHS: An efficient 2picoline degrading bacterium. D Madhusudan Reddy1, Debarati Paul2, Harish Kumar Reddy 1, Gopal Reddy 14. Chikere1*, G. C. Okpokwasili1 and B. O. Chikere2 Bacterial diversity in a tropical crude oil-polluted soil undergoing bioremediation African Journal of Biotechnology Vol. 8 (11), pp. 2535-2540, 3 June, 2009 ISSN 16845315 2009 Academic Journals 15. Choi, K.D., G.H. Jeohn, J.S. Rhee and O.J. Yoo, 1990. Cloning and nucleotide sequence of an esterase gene from Pseudomonas fluorescens and expression of the gene in Escherichia coli. Agric. Biol. Chem., 54: 2039-2045. 16. Chukwu, L.O. and C.C. Odunzeh: Relative toxicity of spent lubricant oil and detergent against benthic macro-invertebrates of a west african estuarine lagoon. J. Environ. Biol., 27, 479-484 (2006). 17. Coates DJ, Chakraborty R, and McInerney JM. Anaerobic benzene biodegradationa new era. Research in Microbiology 2002;153:621-628. 18. Cohen, S.N., C.Y.A. Chang and H.S.U. Leslie, 1972. Non-chromosomal antibiotic resistance in bacteria: Genetic transformation of Escherichia coli by Rfactor DNA. Proc. Nat. Acad. Sci. USA., 69: 2110-2114. 19. Coulon F, Pelletier E, Gourhant L, and Delille D. Effects of nutrient and temperature on degradation of petroleum hydrocarbons in contaminated subAntarctic soil. Chemosphere 2005;58:1439-1448. 20. Cybulski Z, Dziurla E, Kaczorek E, and Olszanowski A. The Influence of Emulsifiers on Hydrocarbon Biodegradation by Pseudomonadacea and Bacillacea Strains. Spill Science and Technology Bulletin 2003;8:503-507.
21. Dave, H., C. Ramakrishna, B.D. Bhatt and J.D. Desai: Biodegradation of slop from a petrochemical industry and bioreclamation of slop oil contaminated soil. World. J. Microbiol. Biotechnol., 10, 653-656 (1994). 22. Desai, J.D. and I.M. Banat: Microbial production of surfactants and their commercial potential. Microbiol. Mol. Biol. Rev., 61, 47-64 (1997). 23. Doong R, and Wu S. Substrate effects on the enhanced biotransformation er anaerobic condition. Chemosphere 1995;30:1499-1511.
24. Egland, P. G., and C. S. Harwood. 1999. BadR, a new MarR family member,
regulates anaerobic benzoate degradation by Rhodopseudomonas palustris in concert with AadR, an Fnr family member. J. Bacteriol. 181:2102-2109. 25. Gen-Yu Y, and Loh K. Inhibition of p-cresol on aerobic biodegradation of carbazole, and sodium salicylate by Pseudomonas putida. Water Research 2002;36:1794-1802 26. Gerdes B, Brinkmeyer R, Dieckmann G, and Helmke E. Influence of crude oil on changes of bacterial communities in Artic sea-ice. FEMS Microbiology Ecology 2005;53:129-139 27. Ghazali MF, Zaliha NR, Abdul RN, Salleh AB, and Basri M. Biodegradation of hydrocarbons in soil by microbial consortium. International Biodeterioration and Biodegradation 2004;54:61-67.
28. Ghosh, Apurba Ratan, Padmanabha Chakraborti and Sandipan Pal: Impact of diesel oil effluent in the mucosal surface of the alimentary canal of Oreochromis nilotica (Linnaeus): A scanning electron microscopic study. J. Environ. Biol., 27, 129-134 (2006) 29. Gram negative aerobic/micro-aerophilic rods and cocci (group 4), in Bergeys manual of determinative bacteriology, 9th edn, edited by J G Holt, N R Kreig, P M A Sneath, J T
30. Stanley & S T Williams (Williams & Wilkins Co., Baltimore) 1994, 72168. 31. Gokcen Yuvali Celik*1, Belma Aslim2 and Yavuz Beyatli2. Enhanced crude oil biodegradation and rhamnolipid production by Pseudomonas stutzeri strain G11 in the presence of Tween-80 and Triton X-100 J. Environ. Biol. ISSN : 02548704 29(6), 867-870 (2008) 32. Haba, E., M.J. Espuny, M. Busquets and A. Manresa: Screening and production of rhamnolipids by Pseudomonas aeruginosa 47T2 NCIB 40044 from waste frying oils. J. Appl. Microbiol., 88, 379-387 (2000). 33. Hamme DJ, Singh A, and Ward PO.Recent advances in Petroleum Microbiology. Microbiology and Molecular Biology Reviews 2003;67:503-548 34. Harayama S, Kasai Y, and Hara H. Microbial communities in oil-contaminated seawater. Current Opinion in Biotechnology 2004;15:20535. Hinchee ER, Kitte AJ, and HJ. Applied Bioremediation of Petroleum Hydrocarbons. Columbus (OH): Battelle Press; 1995. 36. 1Amro A. Amara and 2Soheir R. Salem Degradation of Castor Oil and Lipase Production by Pseudomonas aeruginosa . American-Eurasian J. Agric. & Environ. Sci., 5 (4): 556-563, 2009 ISSN 1818-6769 IDOSI Publications, 2009 37. J. A. Calder, J. H. Lader, Effect of Dissolved Aromatic Hydrocarbons on the Growth of Marine Bacteria in Batch Culture, Appl. Environ. Microbiol. 1976, 32, 95 101
38. Jenkins, J. R., and R. A. Cooper. 1988. Molecular cloning, expression, and
analysis of the genes of the homoprotocatechuate catabolic pathway of Escherichia coli C. J. Bacteriol. 170:5317-5324. 39. J. G. Holt, N. R. Kreig, P. H. A. Sneath, J. T. Stanely, S. T. Williams, Bergey's Manual of Determinative Bacteriology, Williams and Wilkins Publishers, Maryland 1994
40. Johnson JS, Woolhouse JK, Prommer H, Barry AD and Christofi N.Contribution of anaerobic microbial activity to natural attenuation of benzene in groundwater. Engineering Geology 2003;70:343-349. 41. J. Sikkema, J. A. M. deBont, B. Poolman, Mechanisms of Membrane Toxicity of Hydrocarbons, Microbiol. Rev. 1995, 59, 201 222.
42. Kanaly, R. A., and S. Harayama. 2000. Biodegradation of high-molecular-weight
polycyclic aromatic hydrocarbons by bacteria. J. Bacteriol. 182:2059-2067.

43. Kim, S. J., R. C. Jones, C. J. Cha, O. Kweon, R. D. Edmondson, and C. E.
Cerniglia. 2004. Identification of proteins induced by polycyclic aromatic hydrocarbon in Mycobacterium vanbaalenii PYR-1 using two-dimensional polyacrylamide gel electrophoresis and de novo sequencing methods. Proteomics 4:3899-3908 44. Kim SJ, Choi DH, Sim DS, and Oh YS. Evaluation of bioremediation effectiveness on crude oil-contaminated sand. Chemosphere 2005;59:845-852
45. Kim, Y. H., K. H. Engesser, and C. E. Cerniglia . 2003. Two polycyclic aromatic
hydrocarbon o-quinone reductases from a pyrene-degrading Mycobacterium. Arch. Biochem. Biophys. 416:209-217.
46. Khan, A. A., R. F. Wang, W. W. Cao, D. R. Doerge, D. Wennerstrom, and C. E.
Cerniglia. 2001. Molecular cloning, nucleotide sequence, and expression of genes encoding a polycyclic aromatic ring dioxygenase from Mycobacterium sp. strain PYR-1. Appl. Environ. Microbiol. 67:3577-3585.
47. Krivobok, S., S. Kuony, C. Meyer, M. Louwagie, J. C. Willison, and Y.
Jouanneau. 2003. Identification of pyrene-induced proteins in Mycobacterium sp. strain 6PY1: evidence for two ring-hydroxylating dioxygenases. J. Bacteriol. 185:3828-3841. 48. K. S. M. Rahman, J. T. Rahman, P. Lakshmanaperumalsamy, I. M. Banat, Towards Efficient Crude Oil Degradation by a Mixed Bacterial Consortium, Bioresour. Technol. 2002, 85, 257 261.
49. Koma D, Hasumi S, and Motoki K. Biodegradation of n-alkylcyclohexanes by co-oxidation via multiple pathways in Acinetobacter sp. ODDK71. Journal of Bioscience and Bioengineering 2003;95:641-644 50. MacFaddin J F, Differentiation of most frequently isolated Pseudomonas spp., in: Biochemical tests for identification of medical bacteria, 3rd edn (Williams & Wilkins Co.,Baltimore) 1999, 690. 51. Machn I, de Jess CC, Rivas G, Higuerey I, Crdova C, Pereira P, Ruette F, and Sierraalta 52. Maniatis, T., E.F. Fritsch and J. Sambrock, 1982. Molecular Cloning: A Laboratory Manual. 3rd Edn., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 53. Maneerat, Suppasil: Production of biosurfactants using substrates from renewable-resources. Songklanakarin. J. Sci. Technol., 27, 675-683 (2005) 54. Meckenstock UR, Safinowski M, and Griebler CH. Anaerobic degradation of polycyclic aromatic hydrocarbons.FEMS Microbiology Ecology2004;49:27-36 55. Mills AM, Bonner SJ, McDonald JT, Page AC, and Autenrieth LR. Intrinsic bioremediation of a petroleum-impacted wetland. Marine Pollution Bulletin 2003;46:887-899 56. Mulligan, C.N., R.N. Yong and B.F. Gibbs: Surfactant-enhanced remediation of contaminated soil: A review. Eng. Geol., 60, 371-380 (2001) 57. Muthuswamy Sathishkumar1, 2 Arthur Raj Binupriya1 Sang-Ho Baik3 Sei-Eok Yun2 Biodegradation of Crude Oil by Individual Bacterial Strains and a Mixed Bacterial Consortium Isolated from Hydrocarbon Contaminated Areas. Clean 2008, 36 (1), 92 96 58. N. L. Olivera, J. L. Esteves, M. G. Commendator, Alkane Biodegradation by a Microbial Community from Contaminated Sediments in Patagonia, Argentina, Int. Biodeter. Biodegr. 1997, 40, 75 79.
59. Oteyza de TG, Grimalt JO, Lliros M, and Esteve I. Microcosm experiments of oil degradation by microbial mats. Science of The Total Environment Article in press. 60. Parales EP, and Haddock DJ.Biocatalytic degradation of pollutants. Current Opinion in Biotechnology 2004;15:374-379 61. Peressutti RS, Alvarez MH, and Pucci HO. Dynamics of hydrocarbon-degrading bacteriocenosis of an experimental oil pollution in Patagonian soil. International Biodeterioration and Biodegradation 2003;52:21-30 62. Pieper HD, Dos Santos MV, and Golyshin NP. Genomic and mechanistic insights into the biodegradation of organic pollutants. Current Opinion in Biotechnology 2004;15:215-224. 63. Plohl, K. and H. Leskovsek: Biological degradation of motor oil in water. Acta Chim. Slov., 49, 279-289 (2002 64. R. A. Khan, P. Ryan, Long Term Effects of Crude Oil on Common Murres (Uria aalge) Following Rehabilitation, Bull. Environ. Contamin. Toxicol. 1991, 46, 216 222. 65. Rahman, K.S.M., I.M. Banat, J. Thahira-Rahman, T. Thayumanavan and Lakshmanaperumalsamy: Bioremidation of gasoline contaminated soil by a bacterial consortium amended with poultry litter, coir pith and rhamnolipid biosurfactant. Biores. Technol., 81, 25-32 (2002a). 66. Rahman, K.S.M., J. Thahira-Rahman, P. Lakshmanaperumalsamy and I.M. Banat: Towards efficient crude oil degradation by a mixed bacterial consortium. Biores. Technol., 85, 257-261 (2002b). 67. Rahman, K.S.M., J. Thahira-Rahman, Y. Kourkoutas, I. Petsas, R. Marchantand I.M. Banat: Enhanced bioremediation of n-alkane in petroleumsludge using bacterial consortium amended with rhamnolipid and micronutrients. Biores. Technol., 90, 159-168 (2003
68. Resnicek MS, Torok SD, and Gibson TD. Oxidation of carbazole to\hydroxycarbazole by naphthalene 1,2-dioxygenase and biphenyl 2,3dioxygenase. FEMS Microbiology Letters 1993;113:297-302. 69. Richard JY, and Vogel MT. Characterization of a soil bacterial consortium capable of degrading diesel fuel. International Biodeterioration & Biodegradation 1999;44:93-100 70. Riser-Roberts E. Bioremediation of Petroleum Contaminated Sites. Boca Raton (FL): CRC Press Inc; 1992 71. Rose, O.C., M.I. Brookes and J.L.B. Mallet, 1994. A quick and simple nonlethal method for extracting DNA from butterfly wings. Mol. Ecol., 3: 275-275. 72. R. S. Kennedy, W. R. Finnerty, K. Sudarsanan, R. A. Young, Microbial Assimilation of Hydrocarbons the Fine Structure of a Hydrocarbon Oxidizing Acinetobacter sp. Arch. Microbiol. 1975, 102, 75 83. 73. Ruma Roy, Raja Ray1, Ranjana Chowdhury, Pinaki Bhattacharya* Degradation of polyaromatic hydrocarbons by mixed culture isolated from oil contaminated soilA bioprocess engineering study . .Indian Journal of Biotechnology Vol 6, January 2007, pp 107-113
74. Sambrook, J., and D. W. Russell. 2001. Molecular cloning: a laboratory manual,
3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 75. Schreiber EM, RG, Feinstein TD, and Bahr MJ.Mechanisms of electron acceptor utilization: implications for simulating anaerobic biodegradation. Journal of Contaminant Hydrology 2004;73:99-127. 76. Suparna Mukherji *, Sheeja Jagadevan, Gita Mohapatra, Avinash Vijay Biodegradation of diesel oil by an Arabian Sea sediment culture isolated from the vicinity of an oil field. Centre for Environmental Science and Engineering (CESE), Indian Institute of Technology Bombay (IIT), Powai, Mumbai 400076, India 77. S. P. Colowick, N. O. Kaplan, Methods in Enzymology, Academic Press, New York 1995, pp. 142 155.
78. S. Uribe, P. Rangel, G. Espinola, G. Aguirre, Effects of Cyclohexane, an Industrial Solvent, on the Yeast Saccharomyces cerevisiae and on Isolated Yeast Mitochondria, Appl. Environ. Microbiol. 1990, 56, 2114 2119 79. Trindade PVO, Sobral LG, Rizzo ACL, Leite SGF, and Soriano AU. Bioremediation of a weathered and recently oil-contaminated soils from Brazil: a comparison study. Chemosphere 2005;58:515-522 80. Townsend GT, Prince CR, and Suflita MJ. Anaerobic biodegradation of alicyclic constituents of gasoline and natural gas condensate by bacteria from an anoxic aquifer.FEMS Microbiology Ecology 2004;49:129-135. 81. Tuleva, B.K., G.R. Ivanov and N.E. Christova: Biosurfactant production by a new Pseudomonas putida strain. Z Naturforsch. C., 57, 356-360 (2002). 82. Urum, K., T. Pekdemir and M. Gopur: Optimum conditions for washing of crude oil- contaminated soil with biosurfactant solutions. Process Saf. Environ., 81, 203-209 (2003).

Oil Degradation

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Oil Degradation

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Identification and characterization of oil degrading genes of Pseudomonas sps and Cloning of oil degrading genes in E.

Structures of crude oil constituents: (A) substituted cyclopentane,

Figure 1.2 Degradation of alkanes by Acinetobacter sp. (Hamme et al., 2003)

NAD/NADH= Nicotine Amide Adenine Dinucleotide S-CoA= acetyl coenzyme A

putida (ATCC p-cresol and

AIM AND OBJECTIVES

3. AIMS AND OBJECTIVES

The primary objectives of this study were to:-

Investigate the biodegradation potential of our interest Pseudomonas strain.

Cloning of oil degrading gene into E.coli

MATERIALS AND METHODS

4. MATERIALS AND METHODS

4.2. SERIAL DILUTION:

After incubation 5 drops of Kovacs reagent was added to the surface.

4.8.4. VOGES PROSKAUER TEST:

4.8.12. LACTOSE FERMENTATION:-

4.8.15. CARBOHYDRATE FERMENTATION TEST:-

The isolated culture was inoculated in sucrose broth.

The broth was incubated at 37C for 24 hours.

4.10. MOLECULAR CLONING:4.10.1. MATERIALS AND METHODS:4.10.1.1. Chemicals. :-

sample was proceeded for PCR.

Oligonucleotide primer (IBRC-RP07, Sequences- 5- TTGGCACGGG 3

genomic DNA and plasmid were amplified.

with 1Kb DNA ladder (Invitrogen).

Centrifuged for 2 min at room temperature, then for 5 min at 4 C.

DNA was concentrated with ethanol precipitation.

The insert DNA was again amplified with PCR.

RESULTS AND DISCUSSION

Place 1. Petrol Pump

Figure2: Colonies grown on Nutrient Agar

Figure 3: Isolated colonies of nutrient agar streaked on KingsB Media

Figure 4: Isolated Pseudomonas sps. Streaked on Cetrimide Agar

Green colony on Cetrimide Agar(Pseudomonas aeruginosa) 5.1.1. BIOCHEMICAL STUDIES:

Table 5: (Optical density at 550 nm after 2 days incubation)

Castrol oil concentration(l)

FIGURE7: PH STUDIES ON DIFFERENT CONCENTRATION OF OIL (2.5,3L,..2mL)

5.3. MOLECULAR CLONING STUDIES:

Figure 9: Polymerase chain reaction of Pseudomonas aeruginosa and E.coli plasmid

Gene of Interest lies between 600 bp to 500 bp

DISCUSSION:The isolate Pseudomonas aeruginosa was identified as hydrocarbon degrading

polycyclic aromatic hydrocarbons by bacteria. J. Bacteriol. 182:2059-2067.

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