Screening And Optimizing Fermentation Conditions Of A High Yield Strain For Poly-β-hydroxybutyrate(PHB)

Poly-β-hydroxybutyrate(PHB) is a kind of thermoplastic polyester stored in cells as carbon source and energy because of the lack of nitrogen, phosphorus, magnesium and other nutrients. The physical properties of PHB are similar to the traditional polypropylene plastic, and it can completely degrade without producing any toxic substances.As a new type of environmentally friendly materials PHB has got more and more attention,but because of high production costs, PHB products are not popular in people’s daily lives,the reasons come from the costs of raw materials, process design, as well as downstream processing. In order to reduce production costs,the research screened out a novel wild-type strain with high-yield PHB and short fermentation time as the starting strain,and then optimized its fermentation medium and fermentation parameters.Finally, examined the potential of this strain in industrial production by 5L bioreactor.1 Used the method of plate stained by nile blue combining with glass staining by Sudan black,screening 9 strains from the sludge which can produce PHB. Products were extracted with chloroform after ultrasonic wall-breaking treatment.Finally, determined that No.3 was the best strain:28h, PHB1.76g/L, 59.85% of CDW.It was identified as Pseudomonas koreensis by morphological observation and 16 SrDNA sequence analysis,named Pk3.2 By single factor test determined the optimum fermentation medium of Pseudomonas koreensis that carbon,nitrogen species were glucose,ammonium chloride;At the same time, with the changing concentration of each component in the culture medium,the test results showed that the three factors of glucose,ammonium chloride, dipotassium hydrogenphosphate were major on PHB yield.Based on the single factor test,using Design Expert8.0 to design Box-Behnken response response surface experimental with three factors and three levels,five central point in 17 groups,get fit regression equation.Finally,determined the optimum fermentation medium:glucose 16.64g/L,NH4 Cl 3.94g/L, K2HPO4 1.45g/L,PHB production reached 3.18g/L, 66.11% of CDW.Compared with before increased by 1.8 times.3 Based on the results on the media formulations, optimized the fermentation parameters to determine the optimum fermentation parameters by response surface methodology and get further yield of PHB. First of all,through the single factor experiments,determined the level range of rotation speed,initial pH,inoculation amount and temperature;Then, based on the results of single factor experiments,using Design Expert8.0 to design Box-Behnken response surface experimental with four factors and three levels,five central point in 29 groups,and get fit regression equation;Finally, determined the optimum fermentation parameters through software analysis,the results were:rotation speed 170.91r/min,initial pH 7.64,inoculation amount 6.21%,temperature 32.15℃,PHB production reached 4.42g/L.The effect was 2.51 times as before.4 In order to examine the potential of Pk3 in industrial production,the strain was cultrued in 5L bioreactor on the basic of fermentation condition optimization, the optimum fermentation time was from 28 h to 20 h in advanced,PHB 4.72g/L,CDW 9.47g/L, PHB content 49.87%. Compared with shaking flask culture, the yield of PHB increased slightly,from 4.42g/L to 4.72g/L;the dry cell weight increased greatly, from 6.60g/L to 9.47g/L;PHB content reduced from 66.87% to 49.87%.According to the whole results, Pk3 could adapt to 5L bioreactor fermentation and had the potential in industrial PHB production.