The Research On Co-production Of PHB And Ectoine By Ectoine-excreting Strain

Petrochemical industry plastics is widely used in the life. Because of its non-degradation, the long-term residue of waste has caused serious environmental pollution. Poly-β-hydroxybutyrate (PHB) and its miscibility substances are similar to the petroleum plastic materials. It is considered to be a good substitute for the traditional plastic as its property of complete biodegradation and biocompatibility. However, the low production efficiency and high production cost of PHB are the main problems that preclude its large-scale industrial production. The co-production of PHB and high added value products will reduce the production cost of PHB. Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid, Ect) is one of the compatible solute. It can be widely used as cell protective agent, stabilizing agent of biological agents, and other fields. It has a high commercial additional value. The co-production of ectoine with high additional value by using co-production technology of microorganism fermentation to synthesized PHB and ectoine (PHB/Ect) shows great potential to reduce production costs of PHB.The moderately halophilic bacteria was the mostly species in the microorganism used in PHB/Ect co-production. Many species of Halomonas genus has been used in this research. However, the research results showed that the PHB/Ect was not effective in solving the problems of low efficiency, high production cost and high cost.Ectoine-excreting strain in Halomonas can partly excrete the compatible solute ectoine which was excreted intracellular to extracellular under low NaCl concentration in the medium. Ectoine can be produce and excrete efficiently in equilibrium phase. The intracellular ectoine concentration was lower than ectoine threshold value caused by ectoine excreting. Consequently, the secreting-strain of ectoine realized the excessive synthesis of ectoine. It has great potential to solve the problem in PHB/Ect co-synthesis of microorganisms. Revealing secretion mechanism of ectoine is important to metabolic regulation of ectoine synthesis of excreting-strain, increase in ectoine synthesis and thus the improvement of PHB/Ect co-production efficiency.The excreting mechanism was studied in this paper. Under the condition of higher synthesis of ectoine, PHB/Ect co-production conditions of ectoine-excreting strain were optimized. After optimizing fermentation strategy by establishing the dynamic model of fermentation, the ectoine-excreting strain with down shock-tolerance has the potential to establish a PHB/Ect circulation system to further reduce the PHB/Ect co-production cost and improve economic efficiency. The main contents and results were as follows:1. The study of mechanism of ectoine-excreting. The excreting mechanism of Halomonas salina DSM 5928 was revealed and the generalized Kunte model was established. In ectoine-excreting strains, the release ability was stronger than absorb ability of ectoine, so that the ectoine excreting was more than uptake. The teaA gene expression quantity was low. The combining efficiency of TeaA to ectoine was low, so that the absorption amount of ectoine which combined to TeaA was few. Consequently, ectoine excreted. Moreover, based on the mechanism of excreting of ectoine, kunte model of the osmoregulation of intracellular ectoine concentration.2. Selecting the ectoine-excreting strain that is applied to co-production PHB/Ect. The conditions of synthesis and excreting ectoine of H. venusta DSM4743 was optimized. The excreting product was identified by Nuclear Magnetic Resonance. The ectoine synthesis and secretion amount was 299.5 mg/g CDW and 224.6 mg/g CDW, respectively under the condition of 30 g/L NaCl,80 g/L monosodium glutamate as the carbon source. Secretion rate was reached up to 75%.3. The identification of PHB synthesized by H. venusta DSM 4743 and the optimization of synthesis condition. PHB can be produced by H. venusta DSM 4743 under the identification of 1H-NMR. PHB can be produced by renewable resources-beet molasses by H. venusta DSM 4743. The optimal conditions of PHB production were 30 g/L NaCl,160 g/L molasses, C/N ratio of 15,3 g/L KH2PO4 and 9 g/L K2HPO4 and initial pH of 7.2. Under the optimal conditions, the PHB production was 7.8 g/L. Oxygen limitation was beneficial to PHB production under the same conditions in equilibrium phase, PHB production was increased to 8.1 g/L.4. The PHB/Ect co-production conditions by H. venusta DSM 4743 were optimized and the kinetics models of cell growth and product formation were establish- ed. Under the condition of molasses and monosodium glutamate as carbon source with the concentration of 110 g/L and 25 g/L, respectively, NaCl concentration of 30 g/L, the addition amount of phosphate with 3 g/L KH2PO4 and 9 g/L K2HPO4, PHB and ectoine synthesis amounts were reached the highest. The highest production of ectoine (4.2 g/L) and PHB (14 g/L) were obtained under optimal conditions after 28 h in a fermentation. The kinetics models were established according to the batch fermentation curve. A fed-batch fermentation of PHB/Ect co-production was proceeded on the basis of analysis of kinetics models. The concentrations of PHB (32.1 g/L) and ectoine (8.6 g/L) were at a high level of PHB/Ect co-production as so far. The CDW was 41.3 g/L and yield was 0.31 g/g.5. The cyclic preparation system of PHB/Ect co-production based on osmotic downshock was optimized and established. PHB synthesized and released by H. venusta DSM 4743 was identified by’H-NMR. The average PHB production was 6.5 g/L/circulation and total amount was 25.8 g/L. The average PHB release rate was 45%. The average Ectoine production was 1.7 g/L/circulation and total amount was 6.8 g/L. The average Ectoine excreting rate was 75.8% and release rate was 93.7%. H. venusta DSM 4743 was fermented by fermenter. In one culture period (4 cycles), the synthesis amounts of PHB and ectoine were 90.2 g/L and 27.8 g/L, respectively. The efficiency of co-production was 0.95 g/L/h. The recovery rate of PHB and ectoine were 87.9% and 88.9%, respectively. The co-production efficiency and recovery rate of PHB and ectoine were significantly improved.The Ectoine-excreting strain H. venusta DSM 4743 was used for PHB/Ect co-production in this paper. A new and effective technical strategy has been established in further improving the efficiency of PHB/Ect co-production. It is the foundation of solving the problems of PHB/Ect co-production. It has significance in the industrial production and application of PHB and Ectoine in large scale.