The Co-production Of Poly-β-hydroxybutyrate And Ectoine

Poly-β-hydroxybutyrate (PHB) is a polyester with great material property and biodegradability. The demand of PHB for market is large since its significant potential for applications in the fields of industrial packaging, agricultural plastic film mulching, etc. Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) is a compatible solute produced under high osmotic stress by some microorganism. Ectoine has high commercial value and used in the areas of cytoprotective agents, biological preparation stability and pharmaceutics. As so far, the scale of commercial production and use was limited by the production efficiency and cost. It has become the research hotspot that the co-producing of PHB Ectoine in one process (PHB/Ect co-production) among the research of improving production efficiency. PHB/Ect co-production was a unique technical strategy and method which can realize the economic balance through producing the high-valued Ectoine and promote the commercial production and application of PHB. So far, the research of PHB/Ect co-production was limited to strain screening and condition optimizing, the technical problems such as high NaCl concentration, low production efficiency and complex extraction and purification process had not been solved.The main limiting factors of improving PHB/Ect co-production efficiency were revealed by experiments in this paper as follows. PHB was inhibited by the high NaCl concentration corresponding high Ectoine threshold value. The syntheses of Ectoine and PHB were not synchronized, PHB synthesis was in induction phase, while Ectoine stoped synthesizing or degraded partly. The existing extraction methods of PHB would damage the structure of Ectoine. Ectoine-excreting strain was used in this paper, which has the advantages as follows. PHB/Ect co-production can be proceeded under low NaC1 concentration by Ectoine-excreting strains. The synthesis of Ectoine and PHB was synchronized. The excreted Ectoine can be avoided the degradation in equilibrium phase. The technical obstacle in PHB/Ect co-production can be solved by using Ectoine-excreting strains. According to this, the main research content and results are as follows:(1) PHB/Ect co-production was proceeded by Ectoine-excreting strains. PHB-producing strains was screened from Ectoine-excreting strains (2 strains screened from salt lakes and 2 strains was saved in our lab). Halomonas salina DSM 5928 was selected since the high production of PHB and Ectoine. PHB produced by H. salina DSM 5928 and the Ectoine produced and excreted partly was identified by Nuclear Magnetic Resonance. H. salina DSM 5928 was the Ectoine-excreting strain used for PHB/Ect co-production firstly. This kind of strain has provided the basis to solve the problem of high NaCl concentration, low production efficiency and complex extraction and purification process.(2) The PHB/Ect co-production conditions by H. salina DSM 5928 were optimized. The effects of the NaCl concentration, the initial C/N ratio and the phosphate concentration were investigated with respect to PHB/Ect co-production. The highest PHB/Ect co-production of 9.4 g/L was obtained under the optimized conditions of 30 g/L NaCl, an initial C/N ratio of 15 and initial phosphate concentration of 12 g/L. The NaCl concentration of 30 g/L was lower than that disclosed to date in PHB/Ect co-production (no less than 75 g/L), which was also the optimum NaCl concentration for the PHB production by this strain.(3) The research of metabolism regulation was proceeded on the basis of the integration of PHB and Ectoine synthesis pathway and the analysis of both the similarities and differences in metabolic regulation. By monitoring the acetic acid production and pH before and after adding sodium citrate, the carbon overflow in PHB/Ect co-production was comfirmed. The carbon overflow was restrained by addition of 4 g/L sodium citrate, the productions of PHB and Ectoine were increased by 65.4% and 23.1%, respectively. The Tricarboxylic Acid Cycle flow was reduced by the oxygen limit in the equilibrium phase of cell growth, the PHB production precursor of Acetyl-CoA was accumulated and PHB production was increased by 30.9%. The metabolic flux of Ectoine synthesis was strengthened with mixed carbon sources of glucose and monosodium glutamate and Ectoine production was increased by 1.3 times. The study showed that comprehensive metabolic control methods of inhibiting carbon overflow by sodium citrate, reducing the Tricarboxylic Acid Cycle flow and mixed carbon sources of glucose and monosodium glutamate can significantly improve the efficiency of PHB/Ect co-production.(4) The PHB/Ect co-production fermentation in 2.5 L fermentator was proceeded. The kinetics models of cell growth and product formation were established. A fed-batch fermentation of PHB/Ect co-production was proceeded on the basis of analysis of kinetics models. The established kinetics model of cell growth was that of product formation was The PHB/Ect co-production system comprised growing and non-growing cell phases and was developed with NaCl concentration of 30 g/L, the concentrations of PHB (45.9 g/L) and Ectoine (11.2 g/L) were the highest level of PHB/Ect co-production as so far. The synchronous synthesis of PHB and Ectoine with high efficiency was basically realized since 68.8% of Ectoine was produced in cell growth equilibrium phase. The 80.4% of Ectoine was excreted which can be effectively avoided the degradation in equilibrium phase.(5) The pure aqueous extraction method of PHB based on osmotic downshock was studied. The effects of osmotic pressure, extraction temperature and extraction time on the extraction rate of PHB were investigated. The extraction rate of PHB was 87.5 (purity≥90%) under the conditions of osmotic downshock of 60 g/L NaCl, extraction temperature at 60℃ and extraction for 4 h. The extraction rate of Ectoine was 84.2% (purity≥90%). The extraction methods of PHB/Ect co-products were established with the advantages of simple technology, safety and environment-friendly, high efficiency and low cost.The Ectoine-excreting strain H. salina DSM 5928 was used for PHB/Ect co-production in this paper. It had solved the existing problems in PHB/Ect co-production such as high NaCl concentration, low production efficiency and complex extraction and purification process. A new and effective technical strategy and method has been put forward in further improving the PHB/Ect co-production efficiency. It has significance in the commercial production and application of PHB and Ectoine, as well as reducing the white pollution caused by the plastic products.