Hordienko O. Stages optimization of the biotechnological process of long term storage of Master Seed Bacteria E.coli

PhD thesis deals with the optimization of biotechnological process long-term storage of production strains E. coli. Have been worked up the comprehensive systematic approach to the identification and optimization of long-term storage of critical stages of the production strain of E. coli 055 in consequence of researches leading. Have been identified the critical stages which optimization requiring: - the typical characteristics of the strain E. coli 055 restoration by cell culturing in laboratory animals; - parameter optimization stage of final drying in freeze-drying; The optimum parameters of final drying stage: the temperature and duration of heating have been determined. They are t - 23 С, 12 hours and concentration cells - 1 mln/cm3, respectively. Is optimized quantitative and qualitative composition of the protective environment at E. coli 055 freeze-drying; gelatins - 1 %, sucrose - 10 %, aerosil A-300 - 3 %. A multifaceted approach is developed which bases on definition and optimization of the critical phases of long-term storage technology of collection strains E. coli O55 using mathematical modeling research plan of Complete Factorial Experiment (CFE). At the critical stage - final drying by freeze-drying were optimized: completion of drying temperature (23 С) and completion of drying duration (12 hours). The quantitative and qualitative composition of protective environment is optimized (gelatins - 1 %, sucrose - 10 %, aerosil A-300 - 3 %). The obtained results showed that the optimization of the developed approach to the long-term storage technology of the collection strain is achieved by increasing the output of viable cells of E. coli 055 to 88 %. The studies on the sequencing of two samples of E. coli 055 strain: one sample - kept for 5 years in the collection and lost some characteristic features, the second one - cell culturing recovery through the body of laboratory animals. The obtained nucleotide sequences of 16S rRNA gene were compared with each other and with the database USA GenBank.