Shulyakovà M.A., Konon A.D., Shevchuk T.A., Pirog T.P.

National University of Food Technologies, Kiev, Ukraine

SYNTHESIS OF SURFACE-ACTIVE SUBSTANCES UNDER THE CONDITIONS OF GROWTH OF RHODOCOCCUS ERYTHROPOLIS IMV Ac-5017 AND ACINETOBACTER CALCOACETICUS IMV B-7241 ON THE MIXTURE OF GROWTH SUBSTRATES

In recent years the interest to glycerol as a substrate for microbial synthesis is growing due to the expansion of volumes of biodiesel production in the world that caused the move of this alcohol from the category of "target" technology products into the category of wastes. Search for new, more advanced methods of utilizing this waste will not only help to get rid of the problems of its accumulation, but also increase the efficiency of biodiesel production. One of the possible ways of utilization of glycerol is to use it as a substrate in biotechnological processes for receiving of practically valuable products, including for surface-active substances (SAS) [1, 2]. SAS of the microbial origin are the subject of intensive theoretical and applied researches because of the possibility of their application in various industries and environmental biotechnologies. The advantages of microbial surfactants in comparison with chemical analogues are biodegradability, reduced toxicity and stable activity in a wide range of pH, salinity and temperatures [3 - 5].

In previous studies it was shown the possibility of use of glycerol as a source of carbon and energy for the SAS synthesis by Rhodococcus erythropolis IMV Ac-5017 and Acinetobacter calcoaceticus IMV B-7241 but the level of the SAS formed remained lower than on traditional substrates (hexadecane, ethanol) [6, 7]. Thus, the aim of this work was to investigate the possibility of intensifying of the SAS synthesis of R. erythropolis IMV Ac-5017 and A. calcoaceticus IMV B-7241 during the growth on glycerol.

One of the ways of improving of the technology of microbial synthesis is the cultivation of producers on a mixture of growth substrates that allows to avoid unproductive wastes of carbon and energy that occur in the time of use of monosubstrates and increase the efficiency of transformation of carbon of substrates in secondary metabolites [8].

The established broad substrate specificity of N,N-dimethylnitrosamine (NDMA)-dependent alcohol dehydrogenases of A.calcoaceticus IMV B-7241 and R. erythropolis IMV Ac-5017 [9] allowed us to suggest the possibility of improving of SAS synthesis, using a mixture of energy unequivalent growth substrates, particularly energy excess hexadecane and energy deficient glycerol.

Because the parameters of growth and synthesis of target product on mixed substrates depend on the inoculum quality [8], firstly we investigated the influence of the nature of the carbon source in the medium for inoculum preparation. The maximum amount of SAS synthesized for both strains was obtained after using the inoculums, gained on monosubstrate hexadecane. Under such conditions of cultivation of A. calcoaceticus IMV B-7241 the rates of SAS surfactant concentration was 1,5 and 3,6 fold, and for R. erythropolis IMV Ac-5017 – 1,3 and 1,6 fold higher compared to the rates gained on monosubstrates hexadecane and glycerol respectively.

During the cultivation of microorganisms on mixed substrates to secure maximum conversion of carbon into the target product it is necessary to determine the optimal for its synthesis molar ratio of monosubstrates in the mixture, which in turn requires theoretical calculations of the energy needs for SAS and biomass synthesis. [8]. For making such calculations it is need to know ways of metabolism of corresponding monosubstrates. As a result of enzymatic analysis it was found that glycerol metabolism in A. calcoaceticus IMV B-7241 and R. erythropolis IMV Ac-5017 is carried out in two ways: by the glycerol-3-phosphate pathway - involving glycerolkinase and glycerol-3-phosphate dehydrogenase and by dihydroxyacetone pathway, where work NDMA-dependent alcohol dehydrogenase and pyrroloquinoline quinone (PQQ) – dependent glyceroldehydrogenase, performing oxidation of glycerol, and dihydroxyacetone kinase. Theoretically calculated for A. calcoaceticus IMV B-7241 optimal molar ratio in the mixture of hexadecane and glycerol was 1:6,9, and for R. erythropolis IMV Ac-5017 – 1:7,7. Theoretically calculated ratios were confirmed experimentally: the maximum levels of SAS synthesis of R. erythropolis IMV Ac-5017 were in 1,5-2,2 fold and for A. calcoaceticus IMV B-7241 – in 1,2-4 fold higher than those gained on monosubstrates.

Thus, the proposed approaches such as use of glycerol as a cheap substrate, using a mixture of growth substrates may be the basis for the development of economically beneficial industrial technologies of producing of SAS of A. calcoaceticus IMV B-7241 and R. erythropolis IMV Ac-5017 and help to solve the problem of disposal of waste of biodiesel production.

References

1. da Silva G., Mack M., Contiero J. Glycerol: A promising and abundant carbon source for industrial microbiology // Biotechnol. Adv. – 2009. – V.27, ¹ 1. – P. 30–39.

2. Yazdani S., Gonzalez R. Anaerobic fermentation of glycerol: a path to economic viability for the biofuels industry // Curr. Opin. Biotechnol. – 2007. – V.18, ¹3. – P. 213–219.

3. Kaur J., Cameotra S.S., Makkar R. S., Mehta S.K. Synthesis of biosurfactants and their advantages to microorganisms and mankind // Adv. Exp. Med. Biol., 2010. – V.67, ¹2. – P. 261 – 280.

4. Robles-González I.V., Fava F., Poggi-Varaldo M.H. A review on slurry bioreactors for bioremediation of soils and sediments // Microb. Cell Fact., 2008. - V.7, ¹5. – Ð. 1–16.

5. Banat I., Franzetti A., Gandolfi I., Bestetti G., Martinotti M., Fracchia L., Smyth T., Marchant R. Microbial biosurfactants production, applications and future potential // Appl. Microbiol. Biotechnol., 2010. – V. 87, ¹ 2. – P. 427−444.

6. Pirog T.P., Shevchuk T.A., Voloshina I.N., Karpenko E.V. Production of surfactants by Rhodococcus erythropolis strain EK-1, grown on hydrophilic and hydrophobic substrates // Appl. Biochem. Microbiol. – 2004. – V.40, ¹5. – P. 470−475.

7. Pirog T.P., Antonuk S.I., Karpenko Y.V., Shevchuk T.A. The influence of conditions of Acinetobacter calcoaceticus K-4 strain cultivation on surface-active substances synthesis // Appl. Biochem. Microbiol., 2009. – V.45, ¹3. – P. 272−278.

8. Pidhorskyy V.S., Iutynska G.A., Pirog T.P. Intensification of the technologies of microbial synthesis. – Kyiv, "Naukova Dumka", 2010. – 327 p.

9. Pirog T.P., Shevchuk T.A., Konon A.D., Shulyakova M.A., Iutynskaya G.A. Glycerol as a substrate for the synthesis of surface-active substances of Acinetobacter calcoaceticus IMV B-7241 and Rhodococcus erythropolis IMV Ac-5017 // Mikrobiol.journal., 2012. – 74, ¹ 1. – Ñ. 20−27.