Research Journal of Environmental Toxicology 6 (1): 1-26, 2011 ISSN 1819-3420 / DOK: 10.3923Mrjet.2011.1.26 © 2011 Academic Journals Inc. Bioremediation of Petroleum oil Contaminated Soil and Water 'P.K. Jain, 'V.K. Gupta, 'R.K. Gaur, *M. Lowry, *D.P. Jaroli and °U.K. Chauhan ‘Department of Biotechnology, MITS University, Lakshmengarh, Sikar-88231 1, Rajasthan, India “Department of Zoology, University of Rajasthan, Jaipur-302004, Rajasthan, India "Department of Biotechnology, A.P.S. University, Rewa.486 003, Madhya Pradesh, India Correspondling Author:Dr. V.K, Gupta, Deparimentof Biotechnology, MITS University, Lakshmangarh, Sikar-35281 1, Rajasthan, India Tel: 0091 935 221 5170 ABSTRACT Environmental pollution with petroleum and petrochemical products (complex mixture of hydrocarbon) has been recognized as one of the most important serious current problem. People ‘working in garage ete. are always exposed with oily sludge which are potent immunotoxicants and carcinogenic. Acridental leakages from petroleum carrying ships lead to oily layers over the water surface, possessing great threat to the existing flora and fauna, The currently used physical and chemical treatments are effective for the degradation of petroleum products but they lag behind in the desired properties, apart they frequently produce many hazardous compounds which are potent immunotoxicants and careinogenie for living beings. In contrast, bioremediation is effective treatment in terms of efficacy, safety on long terms use, cost and simplicity of administration. However, for the foreseeable future, long term tolerance studies are needed before being recommended for large seale use. Key words: Petroleum oil, bioremediation, hydrocarbon degrading bacteria, 16S rDNA INTRODUCTION ‘The world depends on oil. Vast amount is used, transported, processed and stored around the ‘world. In 2008, the total world consumption of petroleum was over 13.1 billion liters per day, The United States Energy Information Administration projects (as of 2006) world consumption of oil to inevease to 98.3 million barrels per day (15.63*10" m* day~) in 2015 and 118 million barrels day” (18.8x10" m® day™) in 2080 (EIA, 2006). With such a high consumption, oil spills are inevitable. The most notable oil spills at sea involve large tankers, such as Exxon Valdez, which spilled thousands of tones oil (Paine ef al, 1996; Albaiges et a, 2006). These oil spills can cause severe damage to sea and shoreline organisms (Whitfield, 2003). Most responsible for the contamination are service stations, garages, scrap yards, waste treatment plants, sawmills and wood impregnation plants, Thereafter, several studies have been examined the fate of petroleum in various ecosystem (Boehm et al., 1995; Whittaker et al., 1999), The development of petroloum industry into new frontiers, the apparent inevitable spillages that oceur during routine operations and records of acute aevidents during transportation has called for more studies into oil pollution problems (Timmis ef al., 1998), which has been recognized as the most significant contamination problem (Snape et al., 2001). Oil is a complex mixture of hydrocarbons and other organi¢ compounds, ineluding some organometallic constituents (Butler and Mason, 1997). It contain hundreds or thousands of 1Res, J, Environ. Toxicol, 5 (1): 1-26, 2011 aliphatic, branched and avomatie hydrocarbons (Prince, 1993; Wang et al., 1998), most of whieh are toxic to living organisms (ATSDR, 1995), This engine oil renders the environment unsightly and constitutes a potential threat to humans, animals and vegetation (ATSDR, 1997; Edewor et al, 2004). Fat soluble components may accumulate in the organs of animals and may be enriched in the food chain, even up to humans (Mackay and Fraser, 2000). Prolonged exposure and high oil coneentration may cause the development of liver or kiciney disease, possible damage to the bone marrow and an increased risk of cancer (Propset et al., 1999; Lloyd and Cackette, 2001; Mishra et al., 2001). To animals, even a slight staining by oil can be fatal (Wells, 2001). Oil stains can decrease the insulation of a bird's feathers causing the bind to freeze to death, In addition, oil diminishes the ability of birds to fly, dive and swim, which leads to starvation. Birds also swallow oil when cleaning their feathers, which causes intoxication (EPA, 1999). In the long term, toxic and carcinogenic compounds can cause intoxication, diseases, cell damage, developmental disorders and reproduction problems (ATSDR, 1995). In addition to toxic effects, oil products can affect plant and animals physically. A thick layer of oil inhibits the metabolism of plants and suffoeates them, Destruction of plants affects the whole food web and decreases the natural habitats of numerous species (HELCOM, 2003), Hydrocarbon compounds such as petroleum are essential for life. Since, they do not naturally occur in the forms most useful to humans, they can be hazardous. Fuel and lubricating oil spills have become a major environmental hazard to-date, The contamination of the environment. with petroleum hydrocarbons provides serious problems for many countries. Man has dealt with the clean up of petroleum products contamination since the first day oil was discovered. REMEDIATION OF HYDROCARBON-CONTAMINATED ENVIRONMENT. Remediation refers to removing, degrading or transfornting: contaminants to harmless or less harmful substances. Additionally, it include methods that reduce mobility and migration of the contaminants, preventing their spreading to uncontaminated areas; toxicity of the contaminants remains unaltered, but the risk they pose to the environment is reduced (US, DOD, 1994). For the treatment of contaminated soil physiochemical and biological treatment is used. The physicochemical treatments are ineineration, thermal desorption, coker, cement kiln, solvent extraction and land filling etc. Incineration is a very effective treatment method, but itis costly and after burning, the soil has lost most of its nutritional value and structure. Land filling does remove the contaminants but only relocates the problem (Lageman et al., 2005), Furthermore, in Europe, legislation requires reduction of the number of landfills. In 2004, the number of landfills untreated contaminated material in England and Wales was reduced from over 200 to only eleven (Environment Agency, 2008). In Finland, the number of landfills has decreased from 232 in 1999 to 80 in 2005 (FEI, 2006), As a result, the cost of dumping contaminated soils into landfills has risen considerably. Its therefore evident that new, innovative methods are needed to treat, contaminated soils. Claude E. ZoBell had as far back as 1948, recognized that many microorganisms have the ability to utilize hydrocarbons as the sole souree of carbon and energy. He further reeognized that the microbial utilization of hydrocarbons were highly dependent on the chemical nature of the components in the petroleum mixture and environmental determinants (Atlas, 1981), Microbial biodegradation of pollutants has intensified in recent years as mankind strives to find sustainable ways to cleanup contaminated environments (Diaz, 2008). Biodegradation of hydrocarbons by natural populations of microorganisms represent, one of the primary mechanism by whieh 2Res, J, Environ. Toxicol, 5 (1): 1-26, 2011 petroleum and other hydrocarbon pollutants are eliminated from the environment. The effects of environmental parameters on microbial degradation of hydrocarbons, the elucidation of metabolic pathways, genetic basis for hydrocarbon dissimilation by microorganisms and the effects of hydrocarbon contamination on microbial communities have been the areas of intense interest and the subjects of several reviews (Atlas, 1981, 1994) Mierobial remediation of a hydrocarbon-contaminated site is accomplished with the help of a diverse group of microorganisms, particularly the indigenous bacteria present in soil. These microorganisms can degrade a wide range of target constituents present in oily sludge (Eriksson et al., 1999; Barathi and Vasudevan, 2001; Mishra ef al., 2001). A large number of Peeudomonas strains capable of degrading PAHs have been isolated from soil and aquifers (Kiyohara et al., 1992: dohnson et ai., 1996; Pathak ef al., 2008). Other petroleum hydrocarbon degraders include Yokenella sp., Alealigenes sp., Roseomonas sp., Stenotrophomonas sp., Acinetobacter sp., Flavobacter sp., Corynebacterium sp., Streptococcus sp... Providencia sp., Sphingobacterium sp., Capnocy-tophaga sp., Moraxella sp., Bacillus sp. and Enterobacter sp., Rusansky et al., 1987; Antai, 1990; Bhattacharya et ai., 2002; Jain et al., 2010), Other organisms such as fungi are also capablo of degrading the hydrocarbons of engine oil to a cortain extent. However, they take longer periods of time to grow as compared to their bacterial counterparts (Prenafota-Boldu et ai., 2001) Hydrocarbon degrading bacteria and fungi are widely distributed in marine, freshwater and soil habitats, Similarly, hydrocarbon degrading eyancbecteria have been reported (Lliros et al, 2008; Chaillan et al., 2004), although, contrasting reports indicated that growth of mats built by eyancbaetoria in the Saudi coast led to preservation of oil residues Barth, 2008). Typical bacterial groups already known for their capacity to degrade hydrocarbons include Pseudomonas sp., Marinobacter sp., Aleanivorax sp., Microbulbifer sp. Sphingomonas sp., Mierococeus sp., Cellulomonas sp., Dietzia sp. and Gordonia sp. (Brito et al., 2006). Molds belonging to the genera Aspergillus sp., Penicillium sp., Fusarium sp., Amorphoteca sp., Neosartoryt sp., Paecilomyces sp., ‘Talaromyces sp., Graphium sp. and the yeasts Candida sp., Yarrowia sp. and Pichia sp. have been implicated in hydrocarbon degradation (Chaillan et al., 2004). Petroleum oil degradation by bacteria can occur under both aerobic and anaerobic conditions (Zengler et al., 1998). In general acrobic metabolism of hydrocarbons requires oxygenase enzymes which ineorporate molecular oxygen into the reduced substrate, Typically with aliphatic hydrocarbons aleohols are initially produced; these are oxidized sequentially via dehydrogenases to carboxylic acids, which then undergo B-oxidation, In the case of aromatic substrates as well as Polyeyelie Aromatic Hydrocarbons (PAHs) hydroxylation of a ring occurs via mono or dioxygenase enzymes in eukaryotes and prokaryotes (Cerniglia, 1984). After diol formation the ring is cleaved then further degraded, Bacteria have developed two general strategies for enhancing contact with water-insoluble hydrocarbons: specific adhesion mechanisms and production of extracellular emulsifying agents. Many hydrocarbon-degrading microorganisms produce extracellular emulsifying agents. In some eases, emulsifier production is induced by growth on hydrocarbons (Hisatsuka et al., 1971) Several reviews have been published on the microbial metabolism of and aromatic hydrocarbons, (Cerniglia, 1984; Perry, 1984). It has been established that the first step in the aerobic degradation of hydrocarbons by bacteria is usually the introduetion of molecular oxygen into the hydrocarbon, Prokaryotes convert aromatic hydrocarbons by an initial dioxygenase attack to trans-dihydrodiols that are further oxidised to dihydroxy products example, catechol in the case of benzene (Atlas and