SCALING OF BIODEGRADABLE POLYHYDROXYALKANOATES SYNTHESIS TECHNOLOGY IN PILOT PRODUCTION CONDITIONS
Implementation of a new technology in pilot production (PP) conditions is a necessary step for scaling and subsequent industrial production. Technology testing in PP conditions makes it possible to specify the physical and chemical parameters of fermentation process, to create a mass and energy balance, to develop a process procedure and to get batches needed to conduct a complex of required tests. The first domestic PP of polyhydroxyalkanoates (PHAs) was constructed and put into operation in January 2005 on the basis of Krasnoyarsk Scientific Center of the Siberian Branch of the RAS during the collaboration between the Institute of Biophysics SB RAS and the Biotechnology Department of the “BioKhimMash” JSC (Moscow) as part of the project of the International Science and Technical Center (ISTC) (Volova et al., 2006). The PP featured standard equipment of domestic production and non-standard equipment manufactured by those who implemented the project. The process was based on the technology using the bacterial strain Ralstonia eutrophus B5786 with the biomass and polymer yield up to 50 g/l and 80-85 %, respectively. The achieved production rates with regard to total biomass and polymer were 0.045 0.036 kg/hr (1). The studies carried out as part of the mega-project "Biotechnology of New Biomaterials" implemented under the supervision of the leading scientist, MIT professor Anthony John Sinskey (USA) (Resolution of RF government No 220 of April 9, 2010), as a result of which it was possible to significantly increase the efficiency of PHA biosynthesis due to the obtained strain of bacteria Cupriavidus eutrophus B-10646 able to synthesize PHAs with different chemical structures on various carbon sources, made it possible to proceed to the drafting of a new and more productive pilot production (PP).
Baseline data for the design of the pilot production of degradable polyhydroxyalkanoates (PHAs)
Based on the analysis of the obtained polymer synthesis results in different modes (autotrophic and heterotrophic), it was decided to choose for scaling the option implemented on more affordable glucose. As a result of the work conducted, it was possible to significantly increase the yield of polymers from 2 kg per cycle to 9.5 kg per cycle. More affordable glucose was used as a carbon substrate. The extraction time was reduced from 12 to 8 hours. The long process of settling was replaced by filtering, which reduced the time of oilcake separation from the extract to 8 hours. The pretreatment of biomass by a detergent solution made it possible to increase the degree of polymer extraction to 98.5%, as well as to avoid the formation of azeotropic mixtures, which made possible the regeneration and return of the solvents to the process. The obtained baseline data were the basis of the project of PHA pilot production and the choice of production equipment.
As a result of the complex research performed using the obtained strain C. eutrophus V10646 and optimization of PHA biosynthesis, certain indicators were achieved which were taken as a basis for drafting pilot production. In accordance with the developed project, the pilot production was equipped and commissioned including the “Bioengineering” fermentation line (Switzerland) and blocks for production of inoculum and growth media, isolation and purification of polymers. The pilot plant was put into operation. The achieved results of bioplastics biosynthesis at a final concentration of cells biomass in the culture C. eutrophus V10646 and polymer 110 ± 10 g/L and 85 ± 5% were, with regard to biomass and PHA, 1.7 and 1.4 g/l ∙ hr, respectively, i.e. up to 500 kg of polymer/year. The pilot plant was put into operation, certified and is now successfully operating. The scaling of technology made it possible to produce experimental batches of polymers with different chemical structures in the amounts necessary to obtain specialized high-tech products (films, membranes, ultra-thin fibers, nano- and micro-particles, 3D carriers of drugs and cells). It facilitated the organization of regulated research and standardization of specialized polymer products. The pilot production is an educational base for training of graduate and post-graduate students in "Biotechnology".
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3. Volova T.G., Kiselev E.G., Vinogradova O.N., Nikolaeva E.D., Chistyakov A.A., Sukovatyi A.G., Shishatskaya E.I. (2014). A glucose-utilizing strain, Сupriavidus eutrophus В-10646: growth kinetics, characterization and synthesis of multicomponent PHAs. PloS One. 9. e87551.