hydrocarbon degradation

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HYDROCARBON DEGRADATION INTRODUCTION AND LITERATURE SURVEY Pollution is one of the major global problems facing mankind today due to rapid industrial growth, urbanization, wrong implementation of agricultural practices, etc. resulting in deterioration of environment. Industrial revolution has resulted in many materials as product intermediates or wastes of our industries and other activities which do not resembles natural ones. Nature’s built in mechanisms and self regulation ability can not tackle these novel pollutants as a result of which they accumulate in the environment and can be detrimental. They enter in to the natural environment and can not become a part of degradation cycle for the simple reason that most of microorganisms do not find any resemblance between those materials and their natural food habits. However, some organisms exhibit the property of degrading certain compounds especially hydrocarbons.

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Page 1: Hydrocarbon Degradation

HYDROCARBON DEGRADATION

INTRODUCTION AND LITERATURE SURVEY

Pollution is one of the major global problems facing mankind today due to rapid industrial growth, urbanization, wrong implementation of agricultural practices, etc. resulting in deterioration of environment. Industrial revolution has resulted in many materials as product intermediates or wastes of our industries and other activities which do not resembles natural ones.

Nature’s built in mechanisms and self regulation ability can not tackle these novel pollutants as a result of which they accumulate in the environment and can be detrimental. They enter in to the natural environment and can not become a part of degradation cycle for the simple reason that most of microorganisms do not find any resemblance between those materials and their natural food habits. However, some organisms exhibit the property of degrading certain compounds especially hydrocarbons.

Hydrocarbons are organic compound consisting entirely of carbon and hydrogen(1). Hydrocarbon from which one hydrogen atom has been removed are fuctional groups called Hydrocarbyls(2). Aromatic hydrocarbons, alkanes, alkenes,Cycloalkanes and Alkyne based compounds are different types of hydrocarbons.

The majority of hydrocarbons are found to be naturally occur in crude oil where decomposed organic matter provides an abundance of carbon and hydrogen which, when bonded, can catenate to form seemingly limitless chains(2,4).Otherproducts such as petrol, diesel, kerosene etc. are obtained from crude oil by fractional distillation [plate 1]

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Plate 1: Fractional distillation of petroleum

Plate 2: Generation of polluting waste

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Extracted hydrocarbons in liquid form are referred to as petroleum or mineral oil where as hydrocarbon in gaseous form is referred to as natural gas. oil reservoirs in sedimentary rocks are the source of hydrocarbons for the energy, transport and petrochemical industry.

The presence of oil has significant social and environmental impacts from accidents and routine activities such as seismic exploration, drilling and generation of polluting waste [plate2].

Oil extraction is costly and sometimes environmentally damaging although over 70% of the reserves of the world are associated with visible macro seepages and many oil fields are found to have natural leaks. Accidental or natural leakage of oil affects the birds, animals, organisms and human beings.

Treatment of oil spill can be done by three methods: Traditional( physical), Conventional( chemical), and

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Bioremediation( biological). Traditional methods employed are Use of skimmers, Solvents, Sorbents, Booms, Barriers/ Spill Berms, Incineration [plate 3.1]. Conventional methods employed are Dispersants [plate 3.2], Solidifiers, Surface Washing Agents. All these methods remove oils by producing toxic end products.

Bioremediation is the process that uses microorganisms, fungi, green plants to rapidly degrade hazardous contaminants to environmentally safe levels. Bioremediation[plate 3.3] involves establishing the conditions in contaminated environment so that appropriate microorganisms florish and carry out the metabolic activities to detoxify the contaminant.

The chemical composition of crude oil and oil products also influences their susceptibility to biodegradation. The so called light oil mainly moderate chain aliphatic hydrocarbons and low content of aromatic hydrocarbons is more quickly infected by microorganisms compared to high aromatic oils.

The kerosene usually includes C10- C16 aliphatic hydrocarbons and antifreeze additives, glycols and ethers which can possess also biostatic properties (Neihof and Bailey 1978; Gaylarde et al 1999). Some microorganisms are capable of degrading antifreeze agents added to kerosene ( Gaylarde 1999)

Various grades of diesel contain mainly C15- C22 aliphatic hydrocarbons. The decrease in the concentration of sulphur containing compounds as well as presence of various additives such as stabilizing, chelating agents and surfactants serving as source of nutrients activate microbial growth( Lopes Ferreira et al 2006).

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Solid and liquid lubricants made of petroleum hydrocarbons are readily affected by Fungi and Bacteria (Vishnyakova et al 1970).

Most prevalent Bacterial, Fungal and Yeast hydrocarbon degraders are identified. Bacterial hydrocarbon degraders [Table 1] include in the genera Pseudomonas, Arthrobacter and other Coryneforms, Vibrio, Bacillus, Micrococcus and Acinetobacter. Fungal hydrocartbon degraders include in the genera Penicillium, Cladosporium resinae and Yeast like Candida, Rhodotorula, Aureobasidium, Sporobotomyces [plate 4].

Plate 3.1: Incineration method Plate 3.2: Dispersants

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Plate 3.3: Bioremediation

Biosurfactants or surface active compounds are heterogenous group of surface active molecules produced by microorganisms, which either adhere to cell surface or are excreted extracellularly in the growth medium (Fietcher 1992, Zajic and Stiffens 1994; Makker and Cameotra 1998). Several types of biosurfactants have been isolated and characterized including glycolipids, phospholipids, lipopeptides, natural lipids, fatty acids, lipopolysaccharides and other fully characterized [Table 2].

Chemically synthesized surfactants have been used in oil industry to aid clean up of oil spills as well as to enhance oil recovery from oil reserves. These compounds are not biodegradable and can be toxic to environment. Biosurfactant have special advantage over their commercially manufactured counterparts because of their lower toxicity, biodegradable nature and effectiveness at extreme temperature, pH, salinity and ease of synthesis. They are potential candidate for much commercial applications in the pharmaceutical and food processing and oil

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recovery industries(Banat 1995; Desai et al.1997; Makker and Cameotra 1998).

Plate 4: Hydrocarbon degrading Microorganisms

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Table 1: Microorganisms with hydrocarbon degrading potential

Sr.No Organisms Toxic Chemicals 1. Pseudomonas spp. Benzene, Anthracene,Naphthalene,

pCB’s, 4- alkyle benzoates, p-xylene, cyclic aromatics, toluene,phenols

2. Alcaligenes spp. Halogenated hydrocarbons, linear alkyl benzene sulphonates, polycyclic aromatics, PCB’s.

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3. Arthrobacter spp. Benzene, polycyclic aromatics. 4. Bacillus spp. Aromatics, long chain

alkanes,phenol,cresol. 5. Corynebacterium

spp.Halogenated hydrocarbons.

6. Azotobacter spp. Aromatics 7. Flavobacterium spp. Aromatics 8. Rhodococcus spp. Naphthalene 9. Mycobacterium spp. Aromatics, branched hydrocarbons,

benzene, cycloparaffins 10. Nocardia Hydrocarbons, alkyl benzene,

naphthalene, polycyclic aromatics 11. Methanogens Aromatics 12. Xanthomonas spp. Hydrocarbons, polycyclic

hydrocarbons

Table 2: The biosurfactants produced by various microorganisms

Surfactant SourceTrehalose dimycolates

Mycobacterium spp, Nocardia spp, Rhodococcus spp.

Trehalose dicornemycolates

Arthrobacter spp, Corynebacterium spp.

Rhamnolipids Pseudomonas spp.Lipoproteins Bacillus spp, Streptomyces spp,

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Corynebacterium spp, Mycobacterium spp.Ornithine lipid Pseudomonas spp, Gluconobacter spp.Phospholipids Candida spp, Corynebacterium spp,

Micrococcus spp,Thiobacillus spp.Fatty acid/ Natural lipids

Acinetobacter spp, Pseudomonas spp, Micrococcus spp, Candida spp, Penicillium spp, Aspergillus spp.

REVIEW OF THE LITERATURE:

Environmental pollution is a major cause of concern affecting ecosystems globally. Oil spill, an offshoot of this environmental pollution caused by spillage from the tankers, release of the effluents and offshore drilling activities in adversely affecting the aquatic ecosystem ( Atlas 1981). Use of ecofriendly methods such as bioremediation using microbes in the recent concern for environmental pollution.

A wide range of studies have dealt with biotransformation, biodegradation and bioremediation of petroleum hydrocarbons and interest in exploiting crude oil degrading microorganisms for environmental clean up has become central to petroleum microbiology (A.Akhoran Sepahi;

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M.Enrani 2008). There are so many bacteria and fungi with its ability and these organisms are widely distribute in marine, fresh water and soil habitats ( head and Swannel,1999) but scientist reported that indeginous and adapted microorganisms are more efficient for biodegradation of oil pollutants.

Many species of bacteria, microscopic fungi and yeast can utilize hydrocarbons. Bacterial species of Pseudomonas, Acinetobacter, Mycococcus, Flavobacterium, Aeromonas, Micrococcus, Geobacillus isolated from condensates of aviation fuel storage systems or from hot water reservoirs are responsible deteriorating crude oil and its products both in natural conditions and in storage tanks( odier 1976; Ferrari et al 1998;Gaylarde et al. 1999; Bento and Gaylarde 2001; Normen et al. 2002; Rahman et al 2002a, b; Bonch- Osmolovskaya et al.2003; Olliver and Magot 2005). Representatives of isolated microfungi are Cladosporium, Aspergillus, Penicillium, Hormoconis resinae, Paecilomyces, while yeast Candida, Rhodotorula glutinis have been identified from condensates of oil products storage systems(Odier 1976; Haggett and Morchat 1992; Yang et al 1992; Lopes and Gaylarde 1996; Ferrari et al 1998; Gaylarde et al 1999; Bento and Gaylarde 2001; Olliver and Magot 2005). Main products of hydrocarbon microbial metabolism are carbondioxide, water and in smaller quantities fatty acids and surfactants participating in stabilization of water oil emulsion( Odier 1976; Chesneau 2000; Olliver and Magot 2005).

The isolated anaerobic microorganisms capable of causing oil and oil products deterioration outnumber the aerobic ones. These anaerobic species belong to various microbial genera and families including Themosipho petrotoga,

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Thermoanaerobacter, Froidobacterium, Acetobacterium, Desulfotomaculum, Desulfovibrio, Desulfobacula, Desulfobacterium, Desulfosarcina, Desulfococcus, Thiomicrospira, Azoarcres etc.( Harms et al. 1999; Gevertz et al. 2000; Takahata et al. 2000; Gardner and Stewart 2002; Watanabe et al. 2002; Bonch – Osmolovskaya et al.2003; Head et al.2003; Kodama and Watanabe 2003; Roling et al. 2003; Olliver and Magot 2005.)

Biodegradation of hydrocarbons by natural population of microorganisms represents a one of the primary mechanism by which petroleum and other hydrocarbon pollutants are eliminated from the environment. There are so many bacterial strain that can degrade or transform the components of crude oil products to the nontoxic, nonhazardous, biodegradable and environmental friendly compounds ( Korde et al.1996; Kasley et al 1999; Dal Arco and De Franca 2001; Barathi and Vasudevan 2001). Supplementing source of microorganisms capable of degrading particular pollutants ( Bioaugmentation) and the enhancement of desorption of pollutants from particulate using surfactants can increase hydrocarbon degradation (I.rshira et al 1998).

Hydrocarbon degrading microorganisms often bacteria have been developed different adaptations for utilizing poorly soluble substrates. Most of them produce biosurfactants amphiphilic molecules of diverse chemical nature and molecular size in effective surface active and biological properties (Desai and Benet 1997; Rosenberg and Ron, 1999). They help in disperse the hydrocarbons increase the surface area of hydrophobic water insoluble substrate and increase bioavaibilty, thereby stimulating the growth of bacteria and rate of bioremediation (Ron and Rosenberg 2002). These properties create micro emulsion in which micelle formation occur where hydrocarbons can solubilize in water or water in

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hydrocarbons(Banat1995). The majority of known biosurfactants are synthesized by microorganisms grown on water immisible hydrocarbond but some have been produced on such water soluble substrates asglucose, glycerol and ethanol(ABU-Ruwaida et al.1991).

Plasmid have been found to harbour genes encoding the transformation of environmental pollution known as catabolic plasmids. Some of the plasmid mediated bacterial utilization of various carbon compounds which could be found in complex mixture of crude oil (Chakrabarti 1976). Heterotrophic bacteria a higher incidence of plasmid DNA in hydrocarbon contaminated environments such as offshore fields riverine sediments polluted by coking plant discharges (Burton et al 1982; Day et al 1988), ground water contaminated by aromatic hydrocarbon (Ogunneitan et al 1987) and a dystrophic lake containing naturally high concentration of aromatic humic compounds (Schutt 1989).

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AIM:

The discriminate growth of cities increase in human population, vast industrialization and transportation has led to increase accumulation of waste materials in soil as well as leading to soil and marine pollution. However many microorganisms undergo physiological, genetic changes and develop various enzymes require to degrade this waste. It is only limited number of organisms that have the ability to compete and grow in to the terrestrial and aquatic environment and breakdown such complex recalcitrant contaminants which can further be recycled. Realizing the immense potential of hydrocarbons degrading organisms as cleanup tool.

The present work was undertaken to envisage the following:

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Isolation of hydrocarbon degrading microorganisms from terrestrial colonies by selective cultivation.

Determination of colonial and morphological characteristics of the microorganisms obtained.

Purification and maintenance of the potential isolates.

Cultivation and degradive studies of pure isolates.

MATERIALS AND METHODS:

4.1 Collection of samples

Terrestrial ecosystem are known to contain heterogenous microbial population. Two soil samples from two places that is Vijayawada and Goa were selected to isolate diverse groups of microorganisms. However, new techniques can be utilized for the isolation, characterization of novel groups of organisms from such ecosystems.

4.2 selection of hydrocarbons

Hydrocarbons are referred to as backbone or skeleton composed entirely of carbon and hydrogen and other bonded

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compounds and lack a functional group. Hydrocarbons are classified into aliphatic and aromatic hydrocarbons. For ex.petrol, crude oil, kerosene, diesel, salts of benzene sodium benzoate anthracene, naphthalene etc. METHOD:In the present study the hydrocarbons such as petrol and diesel oil used as sole source of carbon.

4.3 Use of medium

Use Bushnell Hass medium(BH medium):For the selection cultivation and isolation of hydrocarbon degrading organisms, Bushnell Hass medium can be used which act as source of nitrogen and other salts.

Use of Nutrient medium: It is general purpose medium used for selective cultivation and isolation of hydrocarbon degrading organisms.

4.4 Isolation of hydrocarbon degrading microorganisms:

Growth of desired microbes can be achieved by using enrichment medium or by selective cultivation. The selective medium provides the type of the physical environment and a medium in or on the which desired organisms thrives ideally for the exclusion of all others.

METHOD: Environmental samples are diluted by using serial

dilution technique.

0.1ml of each dilution are spread on the BH agar and supplied with hydrocarbon as a sole carbon as a energy source that is petrol and diesel by placing it in a vapour

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tube(cut off micropipette tips, sealed at one end with heat in the lid of plate) or alternatively the filter paper is immersed in the hydrocarbons (petrol and diesel oil) and placed in a lid of petriplate. The control plate with out substrate are also inoculated.

Seal all plates with parafilm and then incubated at 25 degrees for atleast for 2-3 weeks.

4.5 Colonial and morphological characteristics of isolates :

The identification of organism is a process of determining its species. Determination of cultural characteristics is one of preliminary methods of systematic study of pure cultures and includes the observation of colonial characteristics such as:

Size in mm Shape - circular, irregular, radiate Color - pigment formation which changes in color Surface - smooth, rough, wavy and granular Elevation - flat elevated, low convex, umbonate Edges - entire, undulate, crenated, lobate,ciliated Opacity - translucent, transparent or opaque Consistency - mucoid, firm, friable, membranous

Method: Prepare nutrient agar medium as per the

composition. Transfer 0.1ml of cure suspension on each medium

and spread plate. Incubate the plates at 37degrees for 72 hrs.

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Observe the growth after 24hrs and record colony characters on media.

4.6 Purification and maintainance of isolates :

For maintainance and preservation of culture, various techniques like stab culture technique, overlaying with mineral oil, use of parafilm and periodic transfer of cultures are used. This technique helps to maintain original morphology, biochemical and genetic characters of the organisms.

METHOD: Pick up the isolated colonies H-1 to H14 from plates. Streak the bacterial cultures on nutrient agar plates using

quadrant streaking method. Incubate the plates at respective temperature till growth

appears. Pick isolated colony and streak on nutrient agar in

duplicate preservation of culture. Apply Para film on the cotton plug and maintain these

slants as stock cultures. Use the other set of slants as working stocks.

4.7 Staining method adapted for studying the isolates:

Staining methods adapted for studying the isolates is gram staining.This technique is used to study the gram character of the organism.

Method: Using the sterile nichrome loop, take a loopful of each of

culture on a grease free slide and make a thin film with a marked area in a drop of saline.

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Air dry the smear, heat fix and stains with crystal violet for a minute.

Drain the excess stain and wash the slide under stream of water.

Flood the smear with grams iodine for 30sec. Drain off excess stain and decolorize with alcohol. Counter stain with saffranine for a minute. Wash the slide, dry and observe under oil immersion

objective of microscope.

4.8 Cultivation and degradive studies of pure isolates:

Growth of bacterial culture in liquid medium is indicated by increase in turbidity which implies the breakdown of complex substances in to simpler forms termed as DEGRADATION.Aromatic hydrocarbons can also be oxidized and degraded by microbes under aerobic conditions, ultimately resulting in production of four major diphenolic intermediates Catechol, Protocatechuate, homogentisate and gentisate.

Method: Measure 75µl of petrol and diesel and dispense in 10ml BH

medium tubes. Adjust the pH of the medium to 7.2 for bacterial cultures. Inoculate 1ml of starter culture of H-1 to H-14 in respective

tubes. Incubate all the tubes at 37 degrees at 100rpm for 3-4 weeks. Observe the results after every 24hrs until the disappearance

of oil film/ luxuriant growth of organisms appears.

4.9 Greasy spot method :

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Oil released in to the environment is a well recognized problem in today’s world which affect plants, animals and even human beings. Biological degradation of oil by bacteria is an effective and efficient methods of oil removal. The following experiment can be used to demonstrate that some types of bacteria can degrade oil and also the other variables can be selected and incorporated into the lesson plan to allow additional experimentation. This test is performed as a confirmation test for degradation.

Method: Cut a brown paper bag in to appropriate size . Using a pipette or dropper, draw a small quantity of

liquid from above experimental broth. Deposit 14 drops of liquid from H-1 to H-14 broth tubes. After a few hours, the water will evaporate, leaving a

greasy spot on a brown paper. Measure and record the diameter of each spot.

4.10 Production of biosurfactants by potential isolates :

Mechanism of degradation involves the attachment of microbial cells to oil droplets and production of biosurfactants.The isolates have degradive ability to produce anionic glycolipids on solid media.

Methods: Blue agar plates containing cetyltrimethyl ammonium

bromide (CTAB) (0.2mg/ml) and methylene blue (5µm/ml) in BH medium.

Inoculate a loopful of isolates which have degradable ability. Incubate the plates at 37 degree centigrade for 24-48 hrs.

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RESULTS AND DISCUSSION:

Hydrocarbons are compounds made up of carbon and hydrogen and other bonded groups. They are classified in to aromatic and aliphatic. The presence of hydrocarbons has significant and various impacts resulting in pollution. Soil and water contaminated with toxic components such as hydrocarbons pose a major environmental and human health problem that needs an effective and affordable technological solution.

Bioremediation which involves application of biological treatment for cleaning up of hazard chemicals may provide an economically viable solution for remediating some of these sites. This process involves detoxification using microorganisms where the waste material is converted in to inorganic compounds such as carbon dioxide, water and methane (Alexander 1999).

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To assess the microbial diversity of hydrocarbon contaminated ecosystems. Garage soil from terrestrial environment from two places that is Vijayawada and Goa were selected for isolation of hydrocarbon degrading microorganisms.

Selection of samples and hydrocarbons Garage soil was selected for the purpose as it is known to contain different types of hydrocarbons like petrol and diesel among the two samples used. Garage soil from Vijayawada was found to be reddish brown in color, granular. Garage soil from Goa was found to be brownish black in color, sticky.

When analyzed in laboratory petrol was acquired from petrol pump for the work and was found to be highly volatile. When the diesel analyzed it was found to Abe volatile but less volatile than petrol.

Isolation of hydrocarbon degrading organisms:

The accurate measurement of living bacterial cells in very essential to understand the microbial ecology of any environment. For this purpose spread plate technique was employed as this technique is commonly used for the isolation of discrete colonies. BH medium with hydrocarbon as a sole source of carbon was used for isolation of hydrocarbon degrading microorganisms. In the present study four bacterial colonies were isolated on BH medium containing hydrocarbon. The colonies obtained were H-1 to H-14 [Photos].

Colonial characteristics:

The size of colonies were found to be ranging from pinpoint to 5mm. a wide variation in colors such as white to yellow to orange were observed [Table 3].

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Purification and maintainance of isolates The isolates were purified and maintained to study the morphology and physiology of organisms. For maintainance and preservation of culture various techniques like agar slants, applying Para film on cotton plug and periodic transfer of culture s are used. These techniques help to maintain original morphology, biochemical and genetical characters of organisms.

Table 3: Colonial Characteristics of Isolates:

Isolates Colony CharactersGram Stain

Color Growth Form Margin Elevation Density

H-1 White Abundant Circular Lobate Convex Opaque

H-2 Orange Abundant Irregular Entire Flat Opaque

H-3 Yellow slight Circular Undulate Raised Transparent

H-4 yellow Moderate Circular Lobate Convex Opaque

H-5 Orange Moderate Circular Entire Raised Opaque

H-6 White Moderate Irregular Lobate Convex Opaque

H-7 Orange Slight Circular Entire Convex Transparent

H-8 Light orange

Slight Rhizoid Undulate Convex Transparent

H-9 Light Slight Circular Entire Convex Transparent

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orangeH-10 Yellow Moderate Circular Entire Convex Transparent

H-11 Light orange

Moderate Circular Entire Raised Opaque

H-12 White Slight Circular Entire Convex Opaque

H-13 White Slight Circular undulate Convex Opaque

H-14 Yellow Moderate Circular Entire Raised Opaque

Staining of isolates: For microbiological analysis to determine the biological characteristics of microorganisms staining method was adapted for bacteria such as gram staining to obtain the gram character and morphology of all isolates. With respect to hydrocarbons degrading microorganisms it was interesting to know that the gram character of organisms were found to be [photos]

Degradation studies of pure isolates: To study the degradation ability of pure isolates, 14 cultures ie. H-1 to H-14 were purified in laboratory and cultivated in liquid BH medium with petrol and diesel as a sole source of carbon. Interestingly was found to be potential degrader of petrol and diesel.[tubes Photos]

Greasy spot method: The greasy spot method was performed with all 14 isolates that is H-1 to H14. The less diameter spot was observed in

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H-3, H-5, H12, H-13 and H14. While all other showed spot with large diameter. Indicating that five isolates was able to use and degrade hydrocarbon.[photos]

Production of biosurfactants Biosurfactants are produced by wide variety of microorganisms. Most of biosurfactants are different types of glycolipids (Desai and Benat 1997). Secreted surface active compounds improve cell growth and bioavailability of hydrophobic compounds thus accelerating their degradation. Five isolates formed dark blue halos on agar plates indicating the anionic glycolipids. This method gave a conformation that the above five isolates are potential degraders.[photos]

The results presented here will be particularly useful in choosing strains for environmental applications involving the implantation of microorganisms into soil matrix (Bioaugmentation). As contaminated sites usually contain heterogenous hydrocarbons it is promising to use for bioaugmented clean up strains with broad abilities to grow on different hydrocarbons, for this purpose a model consortium including isolates H-3, H-5, H-12, H13, H14 with good potential degradation ability and production of glycolipids was proposed for hydrocarbon waste treatment of polluted soil environments (Ergeniavasileva-Tonkova and Victoria Geshera 2003).

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