Biosurfactants: producers, properties and their practical usage
I.N. Gogotov, S.V. Belonozhkin, R.S.Khodakov, A.N. Shkidchenko
Institute of Basic Biological Problems RAS, Mordva State University named by N.P.Ogarev, Institute of biochemistry and physiology of microorganisms named by G.K. Skryabin
It has been analyzed literature data and the data obtained by us on producers of surfactants materials (biosurfactants), properties and their possible usage in oil-producing, chemical, pharmaceutical and food-processing industries, agriculture, as well as purification of the environment from carbohydrates, heavy metals and other pollutants
Biosurfactants, being surfactants (SAF) which are formed by a wide variety of microorganisms, have attracted recently considerable interest both in theoretical and practical aspects. This is conditioned first of all by wide possibilities of their usage in oil-producing and mineral resource industry, chemical, pharmaceutical and food-processing industry, and agriculture and for purification of the environment from carbohydrates, heavy metals and other pollutants. Biosurfactants, formed by the microorganisms, do not yield to the synthetic surfactants by their efficiency to emulsification. However, unlike the synthetic surfactants they have such advantages as biodegradability and the absence of toxicity, as well as the ability to be synthesized from renewable sources, that makes them promising for the development of new ecologically friendly technologies.
Surfactants are ampiphatic molecules with both functional moieties: polar with the main hydrophilic group and non-polar with lipophilic trait. The properties of surfactant are determined by the balance between its hydrophilic and lipophilic components. In solution the surfactant’s molecules have a tendency to aggregation or with each other with formation of micelles or different polarities between the phases like oil|water. Surfactants are defined by their capability of reducing the surface tension (ST), critical micelle concentration (CCM), excess surface of Gibbs, interfacial tension and hydrophilic-lipophilic balance (Cooper, Zajic, 1980).
There are two wide classes of surfactants: chemically synthesized surfactants and that ones synthesized by microorganisms (biologically surface- active materials, bio SAM). Chemically synthesized surfactants are usually classified according to the nature of their polar grouping (cation, anion and non-polar type).Although there are ion and non ion biosurfactants they are usually characterized by the chemical composition and/or producer. At present 5 categories of biosurfactants are known: glycolipids; lipopolysaccharides and polysaccharide-lipid complexes; lipopeptides; fat acids and neutral lipids (Desai, Banat, 1997). According to the data available the physiological role of microbial biosurfactant is in adhesion to the substrate and emulsifying of nutrient components, desorption from the surface, antibacterial and antifungal activities and receptors for bacteriophages.
Unlike chemically synthesized surfactants biosurfactants are categorized by their chemical composition and producers. The biosurfactants’ structure includes hydrophilic moiety comprising amino acids or peptide anions and cations; mono-, di- or polysaccharides and hydrophobic components consisting from saturated and unsaturated fatty acids. Thus, according to the chemical nature biosurfactants are grouped as follows ( Desai, Banat, 1997):
1) glycolipids ( rhamnolipids- Pseudomonas aeruginaosa; trehalolipids – Rhodococcus erythropolis, Nocardia rhodochrous, N. erythropolis, mycobacterium phlei; sophorozolipids – Torulopsis bombicola, T.ampicola, T. petrophilum);
2) lipoprotein and lipopeptides ( lichenisin – Bacillus licheniformis; subtilisin – B. subtilis; cyrculosynes - B. circularis; polimyxines – B. subtilis; viscosine – Pseudomonas fluorescents; emulsan - Phormidium sp.; liposan – Candida lypolytical);
3) lipopolysaccharides ( emulsanes – Arthrobacter sp., A. calcoaceticus; Phormidium sp.: xantane – Xanthomonas campestris);
4) fatty acids – Candida spp C. lepus.;
5) phospholipids – Thiobacillus thiooxidans; Corynebacterium sp.; Candida sp.).
Regulation of biosurfactants synthesis
Bacteria of Rhodococcus synthesize biosurfactatnts in response to the presence of n- alcanes in the medium ( Pirog. Et al. 2004; Kostina et al., 2005). Biosurfactatnts of Rhodococcus represent glycolipids including trehalozolipids ( trehalozomono- and trehalazodikorinomikolat). Physiological role of biosurfactants at the growth of Rhodococcus on carbohydrates is in solubilization of hydrophobic substrates in the cells. Surface tension of the supernatant in the cultural liquid obtained upon growing of R. erythropolis AP-25 on hexadecane, is 30,3 mH/m and approximately 50 mH/m - on glucose. Trehalozomonokorinomicolate is absent in biosurfactant synthesized by this bacterium at the growth on glucose, and the spectrum of lipids are greatly poor than that at the growth of the culture with hexadecane.
The quantity of biosurfactants synthesized by Rhodococcus on hydrophobic substrates vary from 0,5 up to 30 g/l and does not depend on the concentration of the carbon source, the carbon/ nitrogen ratio, nature of the nutrient source, pH value, concentration of the oxygen dissolved and the number of other factors ( Pirog et al., 2004). At periodic cultivation of Rhodococcus in flasks in the media contaiting 2% of hexadecane or kerosene (carbon oil), 1-3 g/l of biosurfactant is synthesized.
Biosurfactants production and their properties
Biosurfactants have the same physical-chemical properties as the synthetic surfactants. They are known from the values of CCM (critical micelle concentration), HLB 9 hydrophilic-lipophylic balance), surface and interfacial tension and others. The coefficient of cultural liquid dilution, cell suspension or purified extracts up to the CCM value ( Eliseev, Kucher, 2001)is frequently used to measure biosurfactants concentration . Biosurfactants for which the HLB value is less that 6, show the tendency to stabilization of the emulsion “water in the oil” type , and at the HLB value equaled to 10-18 they form the systems such as “oil in the water” type. Emulsions are formed in the case when one liquid phase is dispersed as microscopic drops in other liquid continuous phase. Biosurfactants can stabilize (emulsifiers) and destabilize ( deemulsifiers) the emulsions. Emulsifiers are characterized by the period of hydrocarbon suspension decomposition in biosurfactants water solution, and deemulsifiers are considered by the effect of biosurfactant on the standard emulsion obtained with the use of synthetic surfactant ( Eliseev, Kucher, 2001).
Heteropolisaccharide xantan in a rather pure form represent mild fluffy powder which is easily dissolved in small water quantities with gels formation. It is heat stable, resistant to the effect of electrolytes, maintaining the viscosity in salted solutions and not adsorbed by the solid samples. The properties mentioned are kept while using heterosaccharide at the very low concentrations (Gogotov, 2005). Sklerglukan is easily dissolved in the water forming solutions with high resistance in the wide range of the temperatures, pH values and salts concentrations.
The industrial interest to emulsan is caused by its capability of stabilizing the emulsion, “oil in the water “type, and forming the emulsion “water in the oil” under conditions of low energy expenses. Emulsan makes emulsions both of aliphatic and aromatic hydrocarbon. It shows, nevertheless, the capability of forming the film of 2 nm on the surface of oil drop with hydrophilic groups oriented to the water phase in such a way that prevents their mixture and stabilization of emulsion is observed. The affinity of this bioemulsifier is extremely high even at centrifugation, since the emulsion is not decomposed though it is separated to the upper layer with cream, and the lower one representing the water phase. The cream named as emulsanosol belong to “oil in the water “type emulsion with approximately 70% oil ( Eliseev, Kucher, 2001).Active emulsan in the presence of high salts’ concentrations is not absorbed on the lime stones and sandstones. Highly efficient emulsifiers with high specificity to the crude oil are emulsanes obtained in the medium with ethanole. Protein free apoemulsanes are high temperature resistant. In neutral or alkaline medium at 100oC they keep completely their emulsifying properties for two hours. One of the major properties specifying bioemulsifiers usage is their capability of emulsifying hydrocarbons at the certain pH values. Biosurfactants seized oil from the sand surface hydrophilizing this surface and hence forming the conditions for the oil to be “ slided” freely in space between the sand grains. It is not excluded that lowering of the viscosity, as well as the surface tension of the oil is occurred in this case.
Maximal activity of liposan was achieved at the 50/1 ratio of hexane to liposan. The ratio higher that 50:1 caused the drop of emulsifying activity. Maximum emulsifying activity of it was at pH from 2 to 5. Liposan is heat resistant for 1 hour at 70oC and it lost up to 60% of its activity after warming for 1 hour at 100oC. At 10mM K and Na ions stimulate the activity by 1.2 times, whereas Mg, Mn and Ca ions have weak effect on the activity. At ion concentration in 1M emulsifying activity decreases from 1,2 to 2,5 times. The ability of liposan to stabilize or emulsify different waterinsoluble compounds manifest due to the length of their chain. Lipopeptide biosurfactant iturin isolated from the culture B. amylologuefacies shows significant stability under laboratory conditions. It keeps the surface and antibiotic properties while keeping for 2 months at 20oC and incubation for 30 minutes at 100oC.However, at autoclaving ( for 20 minutes, 121oC) the activity of iturin is decreased by 40%.At sun or ultraviolet light its antibiotic and surface activities remain unchanged.
Practical usage of biosurfactants
Biosurfactants are a structurally diverse group of surface active molecules synthesized by microorganisms. These compounds reduce the surface and interfacial tensions in both water solutions and hydrocarbon mixtures, which makes them potential candidates for enhancing oil recovery (Banat, 1995) and deemulsification processes (Desai, Banat, 1997). Biosurfactants have several advantages over the chemical surfactants, such as lower toxicity, high biodegradability: better environment compatibility (Georgiou et al., 1990); high foaming, selectivity and specific activity at extreme temperatures, pH, and salts (Velikonja, Kosaric, 1993); and the ability to be synthesized from renewable feedstoks. Hence, one of the potential users of biosurfactants is in the oil industry, which can use the preparations with minimum purification including whole- cell broth suspension (Banat, 1995).Compared with chemical surfactants they are very selective, required in small quantities, efficient under broad ranges of oil and reservoir conditions and environmentally friendly. It has been shown a 30% increase in total oil recovery from underground sandstones by using trehalolipids Nocardia rhodochrus. Multi-biotech Co., a subsidiary of Geodyne technology, mastered industrial production of biosurfactants for enhanced oil recovery. The treatment of crude heavy Venezuelan oil with emulsan has been shown to reduce its viscosity from 200.000 to 100 cP. Thus, it was feasibleto pump the heavy oil up to 26.000 miles in a commercial pipeline that was impossible after conventional chemical surfactant treatment. In Kuwait it has been demonstrated the possibility to use biosurfactant for pumping crude oil into the oil storage. Petrogen Inc. (USA) achieved 90% recovery of the oil trapped into the sludge by using biosurfactant-producing strain (Benerjee, 1998). An important trend in application of microorganisms forming biosurfactatnts is the technologies of soil remediation polluted with hydrocarbons ( Banat, 1995).Thus, rhamnolipid Pseudomonas aeruginosa has removed substantial quantities of oil from contaminated Alaskan gravel from Exxon Valdez. It has been also demonstrated that this biosurfactant can remove up to 25%-70% and 40-80% carbohydrates from contaminated sandy-loam and silt-loam soils, respectively. In addition high efficiency of biosurfactants was observed in bioremediation of soils from heavy metals including uranium, cadmium and lead ( Miller, 1995), phenatren and polychloride biphenyl ( Van Dyke et al., 1993).
Biosurfactants as food additives also have promising applications in the food-processing industry. Lecitin and its derivatives, fatty acids ethers containing glycerol sorbit or ethylene glycol and ethyoxylated derivatives of monoglycerides are currently in use as emulsifiers. Biosurfactants are also used in medicine and cosmetic industry ( Klekner, Kosaric , 1993).. Kao Co. Ltd applies sophorolipid as moisturizer for skin and hair in cosmetic products “Sophin” and in lipstick. Some biosurfactants are promising in the use for medicine and veterinary medicine. The succinol-trehalose lipid of Rhodococcus erythropolis has been shown to suppress herpex simplex virus and influenza virus with a lethal dose of 10-30 mg/ml. Biosurfactant surfactin can be used in peat dehydration, in the paper, coal, textile and uranium ore-processing industries ( Desai, Banat, 1997). High cost of biosurfactants’ production, which is 3-10 fold higher their chemical analogues, is the only hindrance for wide application of them.
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