Microbial remediation is a modern method which utilizes the action of microorganisms to degrade the complex organic molecule such as hydrocarbons (oil and gas), metals, pesticides and plastics and it is a promising approach to improve the environmental conditions . One of the major problems faced by the modern industrialized world is the contamination of soil and water with complex hydrocarbons (organic compounds containingonly hydrogen and carbon atoms) such as crude oil and its derivatives . These toxic chemicals are found to be carcinogenic and they have to be removed from the environment. Some micro-organisms have the capacity to degrade these complex organic compounds to non toxic products such as water, carbon dioxide and mineral salts . The genera of micro organisms normally used for the biodegradation of hydrocarbons are Nocardia, Pseudomonas, Acinetobacter, Bacillus, Pencillium, Aspergillus, Candida and sporobolomyces .
The microbial biodegradation of hydrocarbons which present in the soil is depends upon many environmental factors. This includes the nature of micro organism used, nutrients, temperature, moisture, soil PH, soil-waste ratio, and oxygen content . The hydrocarbons can be removed by in-situ or ex-situ methods. The In situ method of soil treatment is more efficient and economical than ex-situ methods which are much more costly even though it may be well established. The ex situ methods can be done in two ways
- Provide nutrients to the localized micro organism in the form of nitrogen, carbon and promote their population growth. These micro organisms will metabolize all the complex molecules into non toxic compounds (Biostimulation).
- Spread over a particular micro organism into the contaminated soil which can adapt the environment and metabolize the complex compound in to non toxic 
The bio mass present in the soil should have the capacity to survive in the soil and degrade the complex molecule under proper environmental conditions (bioaugmentation).
Bioremediation also occurs naturally called Natural attenuation, i.e. bioremediation occurs without human interference . There are so many methods to determine the hydrocarbon contents such as Gravimetric method, infrared screening method, gas chromatographic method etc. . Nations are spending millions of dollars each year to remove the Hydrocarbon contaminants from the soil and ground water. The modern science and technology of bioremediation provides alternative technologies for detoxifying petroleum hydrocarbons present in the soil. The cases of oil refinery in the southern Poland and the former Guadalupe oil field in the USA shows the assistance of modern microbial techniques for the remediation process of hydrocarbons from the soil.
WHAT IS PETROLEUM HYDROCARBONS......?
Total petroleum hydrocarbon (TPH) is a term used to denote a number of complex hydrocarbons which are mainly derived from crude oil. Crude oil is the raw material which is collected from the underground and used to produce petroleum, a product which is more useful for the industrialized world but causes contamination in the environment and more problems in the ecosystem. Petroleum hydrocarbons (PHC) are long chain-branched complex molecules of carbon and hydrogen connected by strong chemical bonds . They are hydrophobic in nature and low soluble in water and easily absorbed by the soil. The density of the petroleum hydrocarbons are always less than that of water, so that they will be insoluble in the ground water and form an intermediate layer between soil and air .since the number of carbon chains are more in petrol they are more volatile in nature. The microbial remediation of the petroleum hydrocarbons depends up on the nature of PHC from which they arrived, nature of soil and environmental conditions .
PRINCIPLE OF MICROBIAL REMEDIATION
Conversional methods of clean up technologies such as sparging are not much capable of restoring the environmental conditions . So the researches thought an alternative way of remediation of contaminants. The knowledge about the micro organisms which clean up the contaminants of earth for billions of years gave new thoughts in Bioremediation. Micro organisms contain different enzymes for the degradation of complex chemicals to release energy for their vital activities and build more cells . Micro organisms have the capacity to detoxify large number of compounds via oxidation and reduction .This is the basic principle of microbial bioremediation. Bacteria are the more efficient organism for the bioremediation . So that depends up on the contamination and environmental conditions microbial remediation uses different aspects.
EFFECTS OF PETROLEUM HYDROCARBONS
Petroleum hydrocarbons are the major source of origin of pollution. This will automatically cause problems to the public health and environment. Petroleum hydrocarbons are entered to the soil due to some accidents or release from the industry and private use. However the pollution caused by the hydrocarbons is real nuisance to public and environment. Hydrocarbon spills can also make direct dangers and risk. The toxic vapours from the spills cause health problems such as head ache, skin allergy etc. Buried installations in the underground such as pipes and cables maybe attacked by the hydrocarbons in the soil and causes chemical changes on them. Sometimes the vapours from the hydrocarbons may lead to a massive explosion in the underground engineering construction sites and car parking. When a product which contains hydrocarbon enters to the soil, gravitational force will act on it and draw the fluid down wards, so that some hydrocarbons may be absorbed by soil particle or they may be trapped in the soil pores and at last they will reach to the ground water. When they mixed in ground water, it cannot be use for drinking or other purposes. Presents of hydrocarbons in the ground water is limited to less than ten micrograms per litter. Generally the petroleum hydrocarbon contamination in soil can create major economic and environmental problems [2, 6 and 7].
CHEMICAL REACTION OF MICRO-ORGANISM ON HYDROCARBONS
Generally there are five chemical reactions are present in the soil system for the degradation of contaminants. They are oxidation, reduction, elimination, hydrolysis and substitution. In these five reactions only oxidation and reduction are capable of transfer the petroleum hydrocarbon . During microbial remediation, micro-organisms uses the complex molecules present in the soil as a source of energy and degrade them to carbon dioxide, water and mineral salts which are less toxic substances than parent molecule. The complex enzymes present in the micro organisms help in the conversion process. These reactions are takes place in the presence or absence of oxygen. The chemical changes are taking place via oxidation and reduction of the molecules. In the case of Hydrocarbons it is oxidize in to carbon dioxide and water by microbes. In the presence of oxygen hydrocarbons looses one electron and oxidized in to water molecule and carbon dioxide (aerobic respiration), but in the absence or limited supply of oxygen the respiration (oxidation) process decreases. At this time inorganic substances present in the reaction environment such as nitrate compounds sulfate compounds, carbon dioxide etc. act as terminal electron acceptors in the reaction process and help the biodegradation process easier . A general enzymatic reaction which occurs during the degradation process can be expressed as ,
O + E ↔OE → RP + E
O= organic chemical E= enzyme
OE= enzyme organic chemical complex P= product, a modified or decomposed chemical
FACTORS AFFECTING SOIL BIOREMEDIATION
Although micro organism present in the soil, the bioremediation process depends up on a number of other factors. It may be environmental factors or biological factors. Environmental factors are geologic and hydrologic, contaminant migration, bioavailability; soil metric potential, redox potential etc. biological factors are rate of contamination, extent of contamination, nutrient availability, temperature, moisture, and PH. Soil PH is important because most of the micro organism survive only in a particular PH. if the temperature during the bioremediation varies, it will affect the consumption of hydrocarbons by the microbes because the enzymes are highly influenced by the temperature difference. Many soil bacterium having the ability to consume the PHC at its optimum conditions. For mesophiles, a soil bacterium- requires an optimum temperature to degrade PHC is 25-45 0C. Some thermopile bacteria's which occur in the hot springs can be modifying to degrade hydrocarbons in normal conditions [8, 9, 10].
HOW HYDROCARBONS REMOVE FROM THE SOIL.....?
Different techniques were used to remove the hydrocarbon contaminants from the soil depending up on the nature of the soil and microbial population. Natural attenuation, biostimulation and bioaugmentation are the main methods of soil treatments. Natural attenuation is the ability of microbes in the contaminated soil to degrade the hydrocarbons. Soil contains different types of micro organism and some of them have the capacity to consume the hydrocarbons directly in its optimum conditions. Sometimes the micro organism in the soil cannot be able to degrade the contaminants may be due to difference in temperature, moisture or nutrients from its optimum conditions. In this condition the requirements should be supplied for the process development, this is known as biostimulation. In some extreme conditions micro organisms present in the soil cannot digest the hydrocarbons, in this situation genetically modified micro organism which can act appropriately in that particular soil condition will introduce in the contaminated soil. This is known as bioaugmentation .
Technologies in the bioremediation process can be broadly classified into two- in situ and ex situ. The in situ technologies involve the treatment of contamination in place and in ex situ process the contaminants are physically removed from the site for the treatment .
IN SITU TECHNOLOGIES
Treatment of contaminated soil 'in place' can be done using in situ methods such as bioventing, biosparging, in situ biodegradation and bioaugmentation. Thein situmethods are well known and cost effective. During in situ method bio mass should be established and its environmental conditions should be controlled. The most common method in the in situ technology is bioventing. This method is used for the remediation of deep soil contamination .It involves the stimulation of soil bacteria by supplying nutrients and oxygen through pipes to the contaminated sites. In bioventing the air flow will be controlled, so that it will supply only the required amount of oxygen for the bacteria. This will help to remove the volatile substances to the air. This method is more applicable in the case of petroleum hydrocarbons [3; 10; 12; 13]
In situ biodegradation is another method which utilizes the micro organism by supplying oxygen and nutrients in the medium. By enhancing the bacterial colonies in the soil, they can remove the contamination very easily. To increase the oxygen concentration in the deep soil biosparging method is used. This involves the injection of air into the soil under controlled pressure through small diameter pipes. Normally air will inject under the water table so that it will enhance the organic consumption of bacterial colonies in the soil-water inter mediate zone. Bioaugmentation is an in situ technology in which micro organisms are frequently added to degrade the contaminants. Biosurfactants are the surface active chemical substances produced by the micro organisms which have the capacity to enhance the emulsification of hydrocarbons. Biosurfactants can solubilise the hydrocarbons into non toxic and biodegradable product. Several micro organisms have the capacity to produce Biosurfactants, in that most of them are bacteria and yeast [3, 10, 11, 12, and 13].
Ex situ technologies involves the evacuation of soil from the contamination sites to the bioremediation treatment site. Since it uses more engineering technology for evacuation and removal of contamination, it will becostlier than in situ bioremediation. The main advantage of ex situ bioremediation is that it requires only short time than in situ method. In this method the soil will be uniformly mixed with micro organisms and nutrients, so that it promises additional degradation of contaminants. The ex situ technologies involves land farming, composting, bio piles and bioreactors .
Land farming is based on a simple technology. In this method the contaminated soil is dig out from thesite and it will be spared into a prepared bed. This soil should be tilled periodically to provide maximum aeration to the micro organism. If the aeration is more, the degradation process will increasegradually. This method is normally applied to remove the petroleum refinery waste. This method is applicable only to the surface soil up to 15-30 cm depth. Land farming is costly and very difficult to maintain. In composting method the contaminated soil will be treated with animal manure normally pig and horse. This will enrich nutrients in the soil, so that the micro organisms can grow rapidly and can consume more contaminants.
Bio-pile is combination of land farming and composting. It uses genetically modified microorganisms for the treatment. This method is mainly used for the biodegradation of petroleum hydrocarbons. Bio-piles provide a favourable environment to grow and utilize the hydrocarbons for the microbes. Bioreactors are the specially constructed vessels to treat the contaminated soils. The proper mixing and aeration in the bioreactor increase the rate of bioremediation [3; 10, 12, 13, 15].
CASE STUDIES /APPLICATIONS
The oil refinery located in the southern part of Poland was approximately hundred years old and it created a lot of hydrocarbon contamination in the surroundings. The refinery deposited several millions of heterogeneous waste material in its surroundings. The new refinery management team decided to remove the contaminants from the soil. The main contaminant in the soil was petroleum hydrocarbon. For the remediation process they selected the biopile and biostimulation technology. They started the processing in 1996 and completed in 2001. The bioremediation process using biopile and biostimulation technology was successful to remove the hydrocarbons from the soil .
The former Guadalupe oil field on the central cost of California created a huge environmental problem during the 1990-1994. The Guadalupe Nipomo dune complex is home to numerous endangered plants and animal species. This dune is a valuable environmental source. The oil production created enormous environmental problems. The hydrocarbons polluted the soil and water of that dune. Due to this the refinery was closed and remediation process started in late 1994. The excavation activates were completed in 2001 February. The remediation process includes the microbial treatments such as natural attenuation and biosparging .
The hydrocarbon contamination is increasing in each year and the significance of microbial remediation is getting higher. Microbial remediation is an eco-friendly, cost-effective remediation method which is more suitable for the remediation of petroleum hydrocarbons from the soil. Microbial remediation has so many disadvantages such as supply of nutrients and oxygen during the process. Researches are undergoing to reduce the limitations in the microbial remediation and make this technique as a major one for the remediation of soil hydrocarbon contamination.
 Bioremediation of soil heavily contaminated with crude oil and its products: composition of the microbial consortium; Jelena s. Milic, vladimir p. Beškoski, mila v. Ilic, samira a. M. Ali, Gordana d. Gojgic-cvijovic and miroslav m. Vrvic
 Bioremediation of soil contaminated by lindane utilizing the fungus Ganoderma austral via response surface methodology: K. Papadopoulou, V. Dritsa, and D. Doulia ; National technique university of Athens.
 Bio surfactants and their application for soil bioremediation; Naim Kosaric, university of western Ontario.
 Bioremediation of oil contaminated soil: microbial methods and for feasibility assessment and field valuation; M T Bala, N. Al-Awadhi, R Al- Daher, Kuwait institute for science research.
 A suitable model to describe the bioremediation of petroleum contaminated soil; Danielle M. Pala, Denize D. de Carvalho, Jose Carlos Pinto
 Edward J.Calabrese , Paul T. Kosetechi - Hydrocarbon contaminated soil- vol.1, Lewis publications
 ATSDR; Agency for Toxic Substances and Disease Registry
 A General Essay on Bioremediation of Contaminated Soil; Dana L. Donlon and J.W.Bauder, Professor, Montana State University-Bozeman SSE Graduate Student and Professor, respectively
 Fundamental principles of bioremediation; Environmental response division, April 1998
 Bioremediation- An overview; M Vidali; Pure appl. Chem.; vol.73 No.7
 Laboratory scale bioremediation experiments on hydrocarbon contaminated soil; J. Sabate, A.M Solanas
Application of soil slurry respirometry to optimize and subsequently monitor ex situ bioremediation of hydrocarbon contaminated soil; T.J Aspray, D.J.C. Carvalho, J.C. Philp
 Factors limiting bioremediation technology; R. Boopathy, Nicholls state university
 FFTR- remediation technologies screening matrix and reference guide, version 4.0
 Ex-situ bioremediation of oil contaminated soil; Ta- Chen Lin, Po-Tsen Pan, Sheng-Shung Chen
 The JCCES: http://www.jcces.fsu.edu/index.cfm
 Characterization of aerobic and anaerobic microbial activity in hydrocarbon contaminated soil; a master's thesis presented to San Luis Obispo, California polytechnic state university