Isolation and Characterization of Biosurfactant-producing Bacteria From Waste Oil Contaminated Soil in Abakaliki

Isolation and Characterization of Biosurfactant-producing Bacteria From Waste Oil Contaminated Soil in Abakaliki

Several human activities have led to the pollution of the environment with crude oil and other related substances. In view of the present concern with protection of the environment, there is a renewed interest in the use of biological agents to remove contaminants from the environment. Chemically-synthesized surfactants have been used in the oil industry to aid clean – up of oil spills, as well as to enhance oil recovery from oil reservoirs. These compounds are non-biodegradable and can be toxic to the environment (Tabatabaee et al., 2005). Biosurfactants are biologically synthesized surface-active agents produced by many microorganisms (Banat, 1995; Nitschke and Pastore, 2006). They are complex molecules covering a wide range of chemical types including peptides, fatty acids, phospholipids, glycolipids, antibiotics, and lipopeptides (Anandaraj and Thivakaran, 2010). Despite their molecular structure, biosrufactants display excellent surface activities since they originate from living organisms such as bacteria, fungi and yeasts (Anandaraj and Thivakaran, 2010). Mechanisms of biosurfactants have the ability to reduce surface tension and interfacial tension between two liquid phases (Cameotra and Makkar, 2004). Due to their unique nature, the properties of biosurfactants can be explored for a variety of industrial and bioremediation applications such as in remediation of organic and metal contaminated – sites, in enhanced oil recovery, as cosmetic additives and in biological control (Banat et al., 2000). When compared to synthetic surfactant, biosurfactants have several advantages including high bio-degradability, low toxicity, low irritancy, ecological acceptability, compatibility with human skin, ability to act at wide range of temperature, pH and salinity levels, and ability to be produced from renewable and cheaper substances (Deleu and Paquot, 2004). It also has emulsification and foaming properties (Banat et al., 2000; Dehgan-Noudeh et al., 2009; Ghayyomi-Jazeh et al., 2012). On the other hand, chemically synthesized surfactants are toxic, non-degradable and their accumulation in living tissues over time can lead to the development of cancer (Seghal et al., 2009; Lakshmipathy, 2010). Therefore, it is reasonable to expect diverse properties and physiological functions from biosurfactants such as increasing the surface area and bio-availability of hydrophobic water-insoluble substrates through solubilization/desorption, metal binding, bacterial pathogenesis, quorum sensing, biofilm formation (Priya and Usharani, 2009). They are also found to enhance nutrient transport across membranes and affect various host interactions. Unlike synthetic surfactants, microbial-produced compounds are easily degraded, eco-friendly and particularly suited for environmental applications such as bioremediation and dispersion of oil spills (Mohan et al., 2006). However, the use of biosurfactants in industry is limited due to lack of public acceptance of biosurfactant-producing microorganisms, and the high purity necessary for food, cosmetics and pharmaceutical applications.

For these reasons, they are mainly used for environmental treatments (Xu et al., 2011; Desai and Banat, 1997; Comeotra and Makkar, 1998). Although biosurfactants exhibit important advantages, they have not yet been employed extensively in industry because of relatively high cost of production. One possible strategy for reducing costs is the utilization of alternative substrates such as agro-industrial wastes (Mercade and Manresa, 1994), but the main problem related to use of alternative substrates as culture medium is finding a waste with the right balance of nutrients that permits cell growth and product accumulation (makkar and Cameotra, 1999). Examples of such substrates include molasses, (Maneera, 2005; Raza et al., 2007; Onbasli and Aslim, 2009), peat hydrolysate (Sheppart and Milligan, 1987), and potato processing effluents (Fox and Bala, 2000). It is therefore necessary to increase the yield and product accumulation of biosurfactants through the development of economical engineering processes and the use of cost effective substrates for the growth of biosurfactant-producing microorganisms. The choice of inexpensive raw materials is important to the economy of the process (Mercade and Manresa, 1994; Nitschke et al., 2004). For instance, the use of vegetable oils and their wastes, and waste edible oils such as waste frying oil, as source of biosurfactants is promising but requires more research to full realization (Makkar et al., 2011). Theses waste oils are mostly not utilized efficiently, and sometimes are recovered improperly and sold illegally as waste edible oils (WEOs) for consumption, even though such oils can cause a myriad of health problems (Guo and Chen, 2009; Yao and Min, 2010; Zhang et al., 2012). There are few reports which utilized the vast potential of these frying oils for biosurfactant production (Nitschke et al., 2004; Fleurackers, 2006; Shah et al., 2007; Zhu et al., 2007). The type and amount of the microbial surfactants produced depend primarily on the producer organism, factors like carbon and nitrogen, trace elements, temperature, pH, NaCl concentration and aeration can affect their production by the organisms (Chakrabati, 2012). Hydrophobic pollutants present in petroleum hydrocarbons, and soil and water environment require solubilization before being degraded by microbial cells. Surfactants can increase the surface area of hydrophobic materials, such as pesticides in soil and water environment, thereby increasing their water solubility. Hence, the presence of biosurfactants enhances microbial degradation of pollutants (Cameotra and Makkar, 2004). Some examples of these groiup of biosurfactants are; rhamnolipids produced by different Pseudomonas species, sophorolipids produced by Torulopsis species (Gautain and Tuagi, 2006), lipopolysacchrides or non ionic surfactants produced by Candida lipoytica and C. tropicalis (Poremba et al., 1991), non ionic trehalose corynomycolates and emulsan synthesized by Rhodococcus erythropolis, and man Mycobacterium and Arthrobacter species respectively (Arima et al., 1968); lipoproteins or lipopeptides such as surfactin and subtilism produced by Bacillus subtilis (Besson et al., 1976). Harnessing this technology of biosurfactants can go a long way in the bioremediation of contaminated soil.

Statement of Problem
The negative impact of environmental pollution by crude oil and its products cannot be over emphasized. Oil spill is no longer news in crude oil producing countires such as Nigeria. Many agricultural lands which were known to be very fertile for crop production have been claimed by crude oil and its products, hence making them unfit for food production. Most of the aquatic lives and the activities of the hydrosphere have been adversely affected. A set of measures have been employed to remediate the impact of crude oil but have not generated much success due to the hydrophobicity of the hydrocarbon. This work aims at identifying organisms than can successfully degrade the crude oil with the help of the surface tension-reducing substances (Biosurfactants) which they produce.

This research is aimed at extracting and characterizing biosurfactants produced by bacteria isolated from different waste oil contaminated soil.

The objectives of this study include:
– To isolate bacteria capable of producing biosurfactants from waste oil contaminated soil.
– To identify / characterize the bacteria isolated
– To screen the isolates for their biosurfactant-producing ability.
– To screen the isolates for their biosurfactant-producing ability.
– To extract the biosurfactants produced.
– To screen and characterize the biosurfactants produced
– To test the potency of the biosurfactants produced in a contaminated soil



Microbiology of Biosurfactants
Biosurfactants are amphiphilic molecules consisting of a hydrophilic and a hydrophobic moiety that interact with the phase boundary in heterogeneous systems. The non-polar (hydrophobic) “tail” is usually made of saturated, unsaturated and hydroxylated fatty acids or fatty alcohols whereas the polar (hydrophilic) “head” consists of mono-, oligo- or polysaccharides, peptides or proteins, and may also contain phosphates, carboxylic acids or alcohols (Lang, 2002).
Microorganisms utilize a variety of organic compounds as their source of carbon and energy for their growth. When the carbon source is an insoluble substrate like a hydrocarbon, microorganisms facilitate their diffusion into the cell by producing a variety of substances one of which is the biosurfactants. Some bacteria and yeasts excrete surfactants which emulsify the hydrocarbon substrate in the growth medium. Some examples of this group of biosurfactants are rhamnolipids which are produced by different Pseudomonas spp. (Healy et ah, 1996), or the soropholipids which are produced by several Torulopsis spp, some other microorganisms are capable of changing the structure of their cell wall, which they achieve by synthesizing lipopolysaccharides on monomic surfactants in their cell wall. Examples of these groups are Candida lipolytica, Clostridium tropicalis and Rhodococcus erythropolis, which produces cell wall bound lipopolysaccharides when growing on alkanes; Mycobacterium spp. and Arthrobacter sp. which synthesizes non comic trehalose corynomycolates (Desai and Banat, 1997). There are lipopolysaccharides such as emulsan synthesized by Acinetobactersp and lipoprotein or lipopeptides such as surfactins and subtilism produced by Bacillus subtilis (Cameotra and Makkar, 1998). Other effective biosurfactants are mycolates and corynomycolates which are produced by Rhodococcus sp, Corynebacterium sp, Mycobacterium sp, and Nocardia sp. and/ or nithinliplids, which are produced by Pseudomonas rubescens, Glucobacter cerinus and Thiobacillus ferrooxidans. These organisms can be utilized efficiently in the recovery of petroleum polluted sites.

Classification of Biosurfactants
Bioisurfactants can be classified according to the nature of the charge on individual polar moiety. They include:

Anionic surfactants: These are negatively charged usually due to a sulphonate or sulphur group.

Non-ionic surfactants: These lack ionic constituent and the majority of all non-ionics are polymerization products of 1, 2-epoxyethane.

Cationic surfactants: These are characterized by a quaternary ammonium group which is , positively charged.

Amphoteric surfactants: These have both positively and negatively charged moieties in the same molecule.
Biosurfactants can also be grouped into two categories based on molecular weight, namely:

Low-molecular-mass molecules with lower surface and interfacial tensions. They include glycolipids, phospholipids and lipopeptides.

High-molecular-mass polymers, which include amphipathic polysaccharides, proteins, lipopolysaccharides, lipoproteins or complex mixtures of these biopolymers.
Low-molecular-mass biosurfactants are efficient in lowering surface and interfacial tensions, whereas high-molecular-mass biosurfactants are more effective at stabilizing oil-in-water emulsions (Rosenberg and Ron, 1999).
The mobilization mechanism occurs at concentrations below the biosurfactant critical micelle concentration (CMC). At such concentrations, biosurfactants reduce the surface and interfacial tension between air/water and soil/water systems. Due to the reduction of interfacial force, contact of biosurfactants with soil/oil system increases the contact angle and reduces the capillary force holding oil and soil together. On the other hand, above the biosurfactant CMC, the solubilization process takes place. At these concentrations biosurfactant molecules associate to form micelles, which dramatically increase the solubility of oil (Urum and Pekdemir, 2004). The, hydrophobic ends of biosurfactant molecules connect together inside the micelle while the hydrophilic ends are exposed to the aqueous phase on the exterior. Consequently, the interior of a micelle creates an environment compatible for hydrophobic organic molecules. The process of incorporation of these molecules into a micelle is known as solubilization.
Various microorganisms are known to produce specific kinds of biosurfactants. This depends mainly on the molecular composition of the type of biosurfactant produced. For instance, Pseudomonas aeruginosa DS10-129 was used to produce rhamnolipid (Rhaman et al., 2003), Sophorose lipid was produced by Torulopsis bombicola, and Bacillus subtilis ATCC2132 was used to produce surfactin. Kosari (1992) classified biosurfactants based on their structure namely; hydroxylated and cross-linked fatty acids, polysaccharide-lipid complexes, glycolipids, lipoproteins-lipopeptides, phospholipids and complete cell surfaces.

Properties of Biosurfactants
Biosurfactants are surface-active substances synthesized by living cells. They have the properties of reducing surface tension, stabilizing emulsions, promoting foaming and are generally non-toxic and biodegradable. Interest in microbial surfactants has been steadily increasing in recent years due to their diversity, environmentally friendly nature, possibility of large-scale production, selectivity, performance under extreme conditions and potential applications in environmental protection (Karanth et al., 1999).
The two most desirable properties of biosurfactants are biodegradability and low toxicity. While molecules of biological origin are almost always biodegradable, the rate at which they are degraded can vary significantly as does the degree at which they can be considered toxic. Though many biosurfactants are easily biodegradable and exhibit low toxicity, it is inappropriate to assume that all bio surfactants possess these same characteristics under all conditions (Deleu and Paquot, 2004).
Furthermore, even though a biosurfactant may not be toxic itself, the producing microorganism could be pathogenic and hence the production of such biosurfactant may lead to environmental concerns. For instance, Pseudomonas sp, including the opportunistic pathogen, P. aeruginosa, can be used to produce the biosurfactant rhamnolipid. Similar concerns have been expressed over the widespread production of citric acid by the opportunistic pathogen,. Aspergilus niger but were limited to ensuring that the final product was sufficiently pure and that the production process was contained. In the case of rhamnolipids, there are many non¬pathogenic strains of Pseudomonas which can be used to produce the molecule. Sophorolipids are synthesized by various non-pathogenic yeasts such as Candida apicola, Rhodotorula bogoriensis, Wickerhamiella domercqiae, and Starmerella bombicolq (Chm et al., 2006). Besides biodegradability, low toxicity, and high production potential, biosurfactants also have a high surface and interfacial activity compared to many chemically synthesized surfactants. These properties help justify the higher costs of production and purification associated with large scale production. Generally, a surfactant is considered as a good surfactant if it can lower surface tension (ST) of water from 72 to 30-35 mN/m and the interfacial tension (IT) of water/hexadecane from 40 to lmN/m.
In addition to this, biosurfactants are reported to function under wide range of temperatures, pressures and ionic strengths; and they also possess a number of other useful biological activities including antimicrobial, virucidal, anticancer, immunomodulatory properties.

Surface and interface activity of biosurfactants
Surfactants help in reducing surface tension and interfacial tension. Surfactin produced by B. subtilis can reduce surface tension of water to 25mNm-1 and interfacial tension of water/hexadecane to less than 1mNm-1 (Cooper et al., 1981).
P. aeruginosa produces the surfactant-rhamnoliphis which usually decreases surface tension of water from 26 Nm m-1 and interfacial tension of water/hexadecane to less than 1mN m-1 (Syldatk et al., 1985). Thus, biologically produced surfactants are generally more effective and efficient with their critical micelle concentration (CMC) several times lower than chemical surfactants. In other words, for maximum decrease on surface tension, biosurfactant is more effective while surfactant is unnecessary (Desai and Banat, 1997).

Temperature and pH Tolerance
Extremophiles are known to produce biosurfactants and these have gained attention in the last decades because of their commercial interests. Most of these biosurfactants and their surface activity are resistant to environmental factore such as temperature and pH. According to Mclnerney et al. (1990), Lichenysin produced by B. licheniformis of about 50ºC, pH of between 4.5-9.0 and NaCl and Ca concentrations of up to 50 and 25gL-1 respectively.
Arthrobacter protophormiae produces a biosurfactant that is both thermostable (30-100ºC) and pH stable (2-12) (Singh and Cameotra, 2004). These organisms are important in the industrial since most industrial processes involve exposure to extreme temperature, pH and pressure.

Microbially derived compounds are suitable for environmental applications such as bioremediation/biosorption (Mulligan et al., 2001). They are also easily degraded when compared to synthetic surfactants (Mohan et al., 2006). Due to the ever increasing environmental concerns, there is need to search for alternative products (Biosurfactants) which is suitable for the environment (Cameotra and Makkar, 2004) synthetic chemical surfactants impose environmental problems, hence biodegradable biossurfactants from microorganisms isolated from oil polluted marine environments are concerns for biosorption of poorly soluble polycyclic aromatic hydrocarbons, and phenanthrene contaminated aquatic surfaces (Olivera et al., 2003). Lee et al. (2008) in their research discovered that blooms of marine algae, cochlodinium can be controlled using the biodegradable biosurfactant soropholipid with an efficiency of 90% in 30 minutes treatment.

Low Toxicity
Biosurfactants are generally considered to be low in toxicity or non-toxic products and therefore appropriate for pharmaceutical, cosmetic and food industries. Poremba et al. (1991) in their research demonstrated that the chemically derived surfactants (eg corexit) showed an LC 50 against photobacterium phosphoreum and was found to be 10 times lower than that of rhamnolipids.
In like manner, Flasz et al. (1998) compared the toxicity and mutagenicity profile of biosurfactants produced by Pseudomonas aeruginosa and chemical surfactants, it was observed that biosurfactants were non-toxic and non-mutagenic. The low toxicity profile of sophorolipids from Candida bombicola made them useful. Biosurfactants form Bacillus licheniformis was found to be resistant to high temperature of about 50ºC, pH of between 4.5 and 9.0 and 25gL-1 respectively. Another biosurfactant produced by Arthrobacter protphormiae was found to be both thermostable (30-100ºC) and pH stable (2-12) (Singh and Cameotra, 2004).

Emulsion Forming and Emulsion Breaking
Biosurfactantas may act as emulsifiers or de-emulsifiers.
An emulsion can be described as a heterogenous system which consists of one immiscible liquid dispersed in another, in the form of droplets.
Emulsions are generally of two types; oil-in-water (O/W) or water in oil (W/O) emulsions. Emulsions possess a minimal stability which can be enhanced by additives such as biosurfactants and can be maintained as stable emulsions for many months or years (Velikonja and Kosaric, 1993).

Anti-adhesive Agents
Biosurfactants can act as anti-adhesives which alters formation of biofilms by microorganisms. Biosurfactants alters the hydrophobicity of the surface which in turn affects the adhesion of microbes over a surface. For example, surfactin from Streptococcus thermophilus slows down the colonization of other thermophilic strains of Streptococcus over steel which are responsible for failing. Similarly, a biosurfactant from Pseudomonas fluorescens inhibited the attachment of Listeria monocytogenes onto steel surface (Chakrabarti, 2012).

Sources of Biosurfactants
Biosurfactants can be extracted from bacteria, fungi and even yeasts.

Bacterial Biosurfactants
Microorganisms utilize a wide range of organic compounds as carbon and energy source. When the carbon source is in an insoluble form like a hydrocarbon, microorganisms enhance their diffusion into the cell by producing a variety of substances such excrete ionic surfactants which emulsify the hydrocarbon in the growth medium. Examples of such biosurfactants are rhamnolipids that are produced by different Pseudomonas species (Guerra-Santo et al., 1986), or sophorolipids that are produced by several Torulopsis species (Cutler and Light, 1979). Some other microorganisms are capable of changing the structure of their cell wall which are achieved through production of non ionic or lipopolysaccharide surfactants in their cell wall. Examples of this group of organisms are: Rhodococcus erythropolis and various species of Mycobacterium and Arthrobacter species which produce non ionic trehalose corynomycolates (Ristau and Wagner, 1983). Acubetobacter species produce lipopolysaccharides such as emulsan (Kretschmer et al., 1982). While Baccillus subtilis produces surfatin and subtilisin (Cooper et al., 1981).

Fungal Biosurfactants
In contrast to bacterial, relatively fewer fungi are known to produce biosurfactants. Among the known fungal species that produce biosurfactants are Candida bombicola (Casas et al., 1997), Candida lipolytica (Sarubbo et al., 2008), Candida ishiwadae (Thanomsub et al., 2004), Candida batistae (Konishi et al., 2007), Aspergillus ustus (Alejandro et al., 2011) and Trjichosporon ashii (Chandran and Das, 2010). Most of these organisms are known to produce biosurfactants on low cost raw materials. The most common type of biosurfactants produced by these species are glycolipids. Some others, example Candida lipolytica produces cell wall bound lipopolysaccharide awhen growing on n-alkanes (Rufino et al., 2007).

Types of Biosurfactants
There exist many types of biosurfactants each produced by a specific microorganism. Below are some of the various types of biosurfactants:

These are the most common low molecular weight group of biosurfactants consisting of carbohydrate and lipid components. They consist of mono-, di-, tri-and tetrasaccharides which include glucose, mannose, galactose, glucuronic acid, rhamnose and galactosesulphate. The fatty acid component usually has a composition similar to that of phospholipids of the same microorganism (Chen et al., 2007). Among the glycolipids, the best known are the rhamnolipids, trehalolipids and sophorolipids (Desai and Banat, 1997), and the best studied glycolipid bioemulsifiers, rhamnolipids, trehalolipids and sophorolipids are disaccharides that are acylated with long chain fatty acids or hydroxyl fatty acids (Rosenberg and Ron, 1999).
Rhamnolipids: Bacteria of the genus Pseudomonas are known to produce glycolipid surfactant containing rhamnose and 3-hydroxy fatty acids. Rhamnolipids produced by Pseudomonas aeruginosa have been widely studied using virgin olive oil and reported as a mixture of homologous species (Healy et al., 1996). Disaccharide rhamnolipids are found by condensing two moles of rhamnose sugar and an acetyl group links the hydrophobic group. However, the lipid part of the molecule contains ester and carboxyl groups.
Rhamnolipids produced by Pseudomonas aeruginosa strains are among the most effective surfactants when applied for the removal of hydrophobic compounds from contaminated soils (Rhaman et al., 2003). They possess low average minimum surface tension of (30-32 mNm-1); high average emulsifying activity of (10.4-15.5 µL-1 filterate), low critical micelle concentration (CMC) of 5-65mgL-1 and high affinity for hydrophobic organic molecules (Karanth et al., 1999).
Sophorolipids: They are group of biosurfactants produced by Torulopsis specie. Sophorolipids consist of a dimeric sugar (Sophorose) and a hydroxyl fatty acid, linked by a (3-glycosidic bond. Sophorolipids are generally a mixture of at least six to nine different « hydrophobic sophorolipids. There are two types of sophorolipids namely, the acidic (non-lactonic) sophorolipids and the lactonic sophorolipids. The hydroxyl fatty acid moiety of the acidic sophorolipids has a free carboxylic acid functional group while that of the lactonic sophorolipids form a macrocyclic lactone ring with the 4-hydroxyl group of the sophorose by intramolecular esterification. Until recently, lactonic sophorolipids have been reported to have attracted more commercial and scientific attention than their acidic counterparts. They have measurable biocide activity, while the acetylated lactonic sophorolipids have been applied in cosmetics as antidandruff, bacteriostatic agents and deodorants (Karanth et al., 1999).
Trehalolipids: Another group of glycolipids are the trehalolipids; the serpentine group seen in many members of the genus Mycobacterium is due to the presence of trehalose esters on the cell surface. Disaccharide trehalose linked at C6 to mycolic acid is associated with most species of Mycobacterium, Nocardia and Corynebacterium. Mycolic acids are long-chain, branched-p-hydroxy fatty acids. Trehalolipids from different organisms differ in the size and structure of mycolic acid, the number of C atoms and the degree of unsaturation (Desai and Banat, 1997). Trehalose lipids from Rhodococcus erythropolis and Arthrobacter sp. were found to lower the surface and interfacial tensions in culture broth from 25-40 and 1-5 mNm”1, respectively (Li et ah, 1984).

Lipoproteins and Lipopeptides: These consist of a lipid attached to a polypeptide chain (Rosenberg and Ron, 1999).
Surfactin: These are produced by Bacillus sp. containing seven amino acids bonded to a carboxyl and hydroxyl groups of a 14-carbon acid. Surfactin just as any other biosurfactant reduces surface tension from 72-27 mNm”1 with concentrations as low as 0.005%, making surfactin one of the most powerful biosurfactants. Another important characteristic of surfactin is its ability to lyse mammalian erythrocyte and to form sphaeroplasts. This property is been used to detect surfactin production through haemolysis on blood agar.
Several biosurfactants have shown antimicrobial action against various bacteria, algae, fungi, and viruses. Singh and Cameotra (2004) reported the antifungal and antibacterial property of the lipopeptide, iturin which was produced by B. subtilis. Iturin from B. subtilis was found to be active even after autoclaving. pH5-ll and with a shelf life of 6 months at -18°C (Nitshke and Pastore, 2006).
Lichenysin: Bacillus licheniformis produces several biosurfactants which exhibit excellent stability under extreme temperature, pH and salt conditions which are similar to surfactin. Also, lichenysin from B. licheniformis are able to reduce the surface tension and interfacial tension of water to 27 and 0.36 mNm” respectively.

Fatty Acids
Fatty acids produced from alkanes as a result of microbial oxidations have been considered as surfactants. In addition to the straight chain acids, microorganisms produce complex fatty acids containing OH groups and alkyl branches. Examples of such complex acids include Corynomycolic acids that are also surfactants. The hydrophilic or lipophilic balance of fatty acids is clearly related to the length of the hydrocarbon chain. For lowering surface and interfacial tensions, the most active saturated fatty acids are in the range of C12-C14 (Rosenberg and Ron, 1999). Fatty acids are released into the environment by Rhodococcus sp.

Phospholipids are known to form major components of microbial membranes. When certain hydrocarbon degrading bacteria or yeasts are grown on alkane substrates, the level of phospholipid increases greatly. For instance, using hexadecane-grown Acinetobacter sp. H01-N, phospholipids (mainly phosphatidyl ethanolamine) rich vesicles were produced. Phospholipids have been quantitatively produced from Thiobacillus thiooxidans that are responsible for wetting elemental sulphur necessary for grow. Phosphatidyl ethanolamine produced by Rhodococcus erythropolis grown on n-alkane resulted in the lowering of interfacial tension between water and hexadecane to less than 1 mNm and CMC of 30mgL-1. Ron and Rosenberg (1999) also reported that the phospholipids are mainly synthesized by representatives of the Acinetobacter and Corynebacterium genera.

Polymeric Biosurfactants
The best studied polymeric biosurfactants include emulsan, liposan, mannoprotein and polysaccharide-protein complexes (Desai and Banat, 1997). Emulsan is an effective emulsifying agent for hydrocarbons in water, even at a concentration as low as 0.001-0.01%. It is noticed as one of the most powerful emulsion stabilizers known with the ability to resist inversion even at a water-to-oil ratio of 1:4. Liposan is an extracellular water-soluble emulsifier synthesized by Candida lipolytica and is composed of 83% carbohydrate and 17% protein. The application of such polymeric biosurfactant, liposan, as emulsifier in food and cosmetic industries were discussed by Chakrabarti (2012). Other polymeric biosurfactants such as biodispersan, alasan, food emulsifiers, protein complexes and insecticides emulsifiers, have been reported. Ron and Rosenberg (2001) reported that they are produced Xanthomonas campestris, Acinetobacter radioresistens, and Acinetobactei venetianus.

Particulate Biosurfactants: The particulate surfactants are found in the extracellular membrane vesicles partition where they form a microemulsion which plays an important role in alkane uptake by microbial cells. Vesicles of Acinetobacter spp. strain H01-N with a diameter of 20-50nm and a buoyant density of 1.158 cubic gem are composed of protein, phospholipids and lipopolysaccharide (Chakrabarti, 2012).

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