Синтез мікробного полісахариду етаполану на олієвмісних промислових відходах

Abstract

Дисертаційна робота присвячена розробці технології синтезу мікробного екзополісахариду етаполану штамом Acinetobacter sp. ІМВ В-7005 на відпрацьованих (пересмажених) оліях з різним вмістом полі- та мононенасичених жирних кислот. Максимальна концентрація етаполану (14–15 г/дм3) досягалася за концентрації відпрацьованої соняшникової олії 5 % (об’ємна частка), нітрату амонію 0,6 г/дм3 і використання інокуляту, вирощеного на відповідній рафінованій олії. Дробне внесення субстрату в процесі культивування штаму ІМВ В-7005 дало змогу підвищити кількість екзополісахариду на 20 %. Завдяки високій емульгувальній активності розчину етаполану (Е24=48–52 %), одержаного згідно розробленої технології, екзополісахарид штаму ІМВ В-7005 може бути використаний як складник косметичних кремів для рук. Розроблено тимчасовий технологічний регламент на виробництво культуральної рідини Acinetobacter sp. IMB B-7005 на основі відпрацьованої соняшникової олії для вторинного видобутку нафти. Dissertational work is devoted to the development of technology for the microbial exopolysaccharide (EPS) ethapolan synthesis by Acinetobacter sp. IMV B-7005 on waste oils with different contents of poly- and monounsaturated fatty acids. Today, microbial EPS are the subject of intense theoretical and applied researches. One of the most promising EPS is ethapolan. The producer of ethapolan is auxotroph for calcium pantothenate, industrial production of which is absent in Ukraine. A market research of multivitamin drugs that could be a source of pantothenate was conducted. Among the considered drugs the most promising for ussin was multivitamin complex “Complevit”. The optimal concentration of "Сomplevit" (in recount on pantothenate), which provides maximum ethapolan synthesis in a medium with different concentrations of sunflower oil, is 0.00085–0.00095 %. As in further work is planning to replace the refined oil on the waste, at the next stage the synthesis of ethapolan was investigated in a medium with the maximum possible concentration of refined sunflower oil. Increasing of sunflower oil concentration in basic medium for Acinetobacter sp. IMV B-7005 cultivation to 4−5 % was accompanied by decrease of ethapolan synthesis compared with those in the medium containing lower (2−3 %) substrate concentration. Increasing ammonium nitrate content to 0.6 g/cm3 and/or pantothenate concentration to 0.00095 % in a medium with 5% sunflower oil allowed to increase the amount of ethapolan synthesized up to 12.5 g/dm3, that is in 1.3−1.4 times higher than in the basic medium with the same concentration of the substrate but lower NH4NO3 (0.4 g/dm3) and pantothenate (0.00085 %). One of the approaches to the intensification of microbial synthesis technologies is adding of exogenous precursors of biosynthesis. Adding of exogenous glucose (a component of ethapolan) and fumarate (precursor of gluconeogenesis) at a concentration of 0.05 % in the cultivation medium of the strain IMV B-7005 with refined sunflower oil made it possible to increase the amount of synthesized ethapolan in 1.1−1.4 times, as well as to increase the viscosity of its solutions in 2−9.9 times compared with the indicators without predecessors. The possibility to replace refined oil on waste after meat and potato frying and unrefined oil for biosynthesis of ethapolan was studied. It is established that using inoculum grown on refined oil is accompanied by the synthesis of 14.4–15.5 g/dm3 of ethapolan on the waste oil after frying meat and unrefined sunflower oils (5 %). The indices of ethapolan synthesis in the medium containing waste oil after frying potato were increased from 4.2 g/dm3 to 8.1 g/dm3 with using inoculum grown on the same substrate. Synthesis of exopolysaccharide ethapolan on waste oils of various qualities (with different ratios of mono- and polyunsaturated fatty acids) was investigated as the generation of one or another waste depends on the region where the production is concentrated and the type of manufactured oil. It was established that the highest ethapolan concentration (11–14 g/dm3) was observed under Acinetobacter sp. IMV B-7005 cultivation on waste after frying meat sunflower and corn oils (that characterized by higher content of polyunsaturated fatty acids) at concentration 5 %, with using inoculum grown on refined oils. Replacing these oils in the cultivation medium on olive and rapeseed (with higher content of monounsaturated fatty acids) accompanied by some decrease in EPS concentrations (to 9‒10 g/dm3), but the EPS-synthesizing ability was higher in several times (6.3–7.6 g EPS/ g biomass). Also we have shown that regardless of the oil type in the inoculum obtaining medium (olive or sunflower), the ethapolan synthesis indexes on the mixture of waste sunflower and olive oils (in the ratio of 1:4; 4:1; 1:1) were slightly lower than in conditions of the producer growth on refined sunflower oil, but at the same time increasing of the EPS-synthesizing ability on 14−41 % was observed. Using mixed after frying meat, potatoes, onions and cheese sunflower oil as a substrate for the ethapolan production accompanied by the synthesis of the same polysaccharide concentration, as well as on refined oil. Reduction of the initial quantity of mixed sunflower oil to 1.25−2 % with followed fractional adding in portions of 1.25−1.5 % in the cultivation process to the final amount of 5% was accompanied by increase of ethapolan concentration on 15−20 % compared to a one-time addition of 5 % substrate. Solutions of the synthesized under such conditions polysaccharide at concentration of 0.05 % emulsified hexadecane, gasoline, diesel fuel (emulsification index 48−52 %), and the formed emulsion was stable for 20 days. Temporary technological regulation for the Acinetobacter sp. IMB B-7005 culture fluid obtaining from waste oil for secondary oil production was developed. Theoretical calculations of the cost for the nutrient medium preparing for Acinetobacter sp. IMB B-7005 culture fluid obtaining needed to increase secondary oil production at “OkhtyrkaNaftogaz” (262 units) is 3535.6 UAH, while for the production of the well-known EPS, xanthan (in the amount that will provide an equivalent amount of oil) – 44928 UAH. Due to the high emulsifying activity of the ethapolan solution, obtained according to the developed technology, the strain IMV B-7005 EPS can be used as a component of hand cosmetic creams.