Due to its physicochemical properties and functional characteristics, microbial exopolysaccharides (EPS) have been successfully used in various industries for decades. At the same time, only some of them (xanthan, dextran, pullulan, etc.) have achieved a real commercial success, while the vast majority of these biopolymers are still in the initial stages of fundamental research and are of exclusively scientific interest. This is mainly due to their high cost and low concentration of the target product.
Obviously, to solve these problems, it is necessary to search for cheap raw materials for the polysaccharides production and, in general, to carry out complex optimization of cultivation conditions. It is known from the literature that one of the promising methods for increasing the indicators of microbial metabolites synthesis is the use of a mixture of growth substrates.
The dissertation is devoted to the establishment of the peculiarities of intensification of polysaccharide ethapolan synthesis during growth of the strain Acinetobacter sp. IMV B-7005 on the mixture of refined sunflower oil and C2-C6–substrates (acetate, ethanol, molasses), as well as the investigation of the possibility of replacing refined oil in the mixed substrate with a waste one.
In the dissertation, for the first time, the possibility of increasing the synthesis rates of ethapolan on the mixture of energy-excessive substrates was demonstrated. At the molar ratio of concentrations of ethanol and refined oil in the mixture of 1:0.056, as close as possible to the theoretically calculated (1:0.076), the concentration of polysaccharide was 2.6-2.8 times higher compared to that on the corresponding monosubstrates. At the same time, based on theoretical calculations of the energy requirements of EPS and biomass of Acinetobacter sp. IMV B-7005 synthesis on the energy-deficient substrate (acetate) it was determined that the molar ratio of concentrations of sodium acetate and oil in the mixture, at which the maximum EPS synthesis was achieved, should be 1:0.13.
It was established that regardless of the type of C2-substrate in the mixture with oil during the cultivation of the producer, a deviation of the pH of the culture liquid from the optimal level for the ethapolan synthesis (7.0-8.0) was observed. In order to stabilize pH, an ammonium source of nitrogen, which transport into bacterial cells takes place by antiport with proton and is accompanied by acidification of the culture liquid, was replaced with a nitrate source, transported by symport with proton. An additional decrease in the initial concentration of oil and C2-substrates in the culture medium followed by fractional application during producer growth on their mixture was accompanied by an increase in polysaccharide synthesis by 1.5-6 times.
Due to neutralization of the pre-hydrolyzed molasses in the mixture with oil, an additional increase in the parameters of the ethapolan synthesis by 20-25% was achieved.
The possibility of replacing refined sunflower oil mixed with acetate, ethanol, or molasses with different types of waste oil (after frying potatoes, meat, vegetables, and mixed after frying various products) has been established. At the same time, it was demonstrated that an increase in the content of magnesium cations from 1.6 to 5.0 mM, which are an activator of acetyl-CoA synthetase − the key enzyme of C2-metabolism in Acinetobacter sp. IMV B-7005, in the medium with waste oil and C2-substrates, was accompanied by an increase in the ethapolan synthesis by 11-58%.
Technologies for the ethapolan production on the mixture of waste sunflower oil and C2-C6–substrates (acetate, ethanol, molasses) have been developed, which include: 1) growing the inoculum on a waste oil monosubstrate; 2) using potassium nitrate as a nitrogen source; 3) increasing the content of Mg2+ in the culture medium to 5 mM; 4) fractional application of substrates. The implementation of such approaches made it possible to increase the ethapolan synthesis to 16-18 g/l, which is higher than the indicators obtained on the corresponding oil-containing monosubstrates (12-14 g/l), or the mixture of glucose (molasses) and C2-C4–substrates (acetate, ethanol, fumarate) (10-14 g/l).
The obtained results testify to the possibility of developing a universal technology for ethapolan production on the mixture of waste oil and C2-C6–substrates, independent of the type and supplier of refried oil.
