Optimization Of Fermentation Conditions For Synthesizing PHA From Glycerol By Pseudomonas Putida And Its Mixed Consortium

Due to the vigorous development of biodiesel,a large amount of glycerol was produced as a by-product.Meanwhile,the high production cost of PHA greatly limits its application.Therefore,it is of great significance to produce PHA with glycerol.In this paper,PHA was synthesized from glycerol as substrate by adding sodium octanate.By optimizing the culture medium and culture condition of Pseudomonas putida and its mixed bacteria,the optimal conditions for PHA synthesis were obtained.In order to select excellent PHA producing strains,the levels of PHA produced by Pseudomonas putida NBRC 14164 and Pseudomonas putida KT2440 were compared at different nutrient levels.The PHA yield of Pseudomonas putida KT2440 was significantly higher than that of Pseudomonas putida NBRC 14164.At the same time,the nutrient two medium is the most suitable medium.Therefore,Pseudomonas putida KT2440 was selected as a PHA-producing strain.In addition,the growth of glycerol in various laboratory common molds was compared.Escherichia coli BL21,Escherichia coli MG1655,and Saccharomyces cerevisiae W303 were found to have a better growth in glycerol.These strains were selected for mixed fermentation with Pseudomonas putida KT2440.Based on the nutrient two medium,the culture conditions of Pseudomonas putida KT2440 were optimized,and the optimal growth conditions were obtained by single factor and response surface optimization: sodium octanate was 12.23 g/L,the liquid loading was 44.39 mL/250 mL,the speed was 220 rpm,the concentration of ammonium sulfate was 1 g/L,and glycerol was 40 g/L.Using this condition for shake flask fermentation experiments,the average yield of PHA was 4.56 g/L,which was significantly higher than that of the same type with glycerol and sodium octanoate as sole carbon sources.Pseudomonas putida KT2440 was cultured in a 5 L fermentor and optimized for fermentation.The PHA yield was up to 8.47 g/L by adjusting the speed and ventilation.The phase-limited oxygen and feed operations inhibited the growth and PHA synthesis of Pseudomonas putida KT2440.Because the low dissolved oxygen is not conducive to PHA synthesis,and the high feed concentration inhibits the growth and synthesis of PHA in Pseudomonas putida KT2440.Subsequent feeding operations can be performed by constant-current feeding or by reducing the initial sodium octanoate concentration in the medium while reducing thesodium caprylate feed concentration.The optimization of the mixed bacteria was carried out with the culture conditions optimized by the Pseudomonas putida KT2440.The selected model organisms Escherichia coli BL21,Escherichia coli MG1655 and Saccharomyces cerevisiae W303 were respectively mixed with Pseudomonas putida KT2440,and then the optimal fermentation conditions were obtained by single-factor and orthogonal experiments for each mixed-bacteria system: At PH=6.5,the optimal process for PHA production from Pseudomonas putida KT2440 and Escherichia coli MG1655 was 40 mL carrier liquid,3 mL inoculum,and 1:10 inoculum ratio.The optimum process for PHA production from Pseudomonas putida KT2440 and Escherichia coli BL21 is 30 mL carrier liquid,2 mL inoculum,and 1:5 inoculation ratio.The optimum process for producing PHA from Pseudomonas putida KT2440 and Saccharomyces cerevisiae W303 was 20 mL carrier liquid,3 mL inoculum,and1:1 inoculation ratio.The actual values obtained from the above optimal conditions are: the PHA yield of Pseudomonas putida KT2440 and Escherichia coli MG1655 was 4.01 g/L,the PHA yield of Pseudomonas putida KT2440 and Escherichia coli BL21 was 4.81 g/L.The PHA yield of Pseudomonas putida KT2440 and Saccharomyces cerevisiae W303 was 4.57 g/L.After the optimization of the PHA yield of the last two groups of mixed bacteria system is slightly higher than the yield of single bacteria.Subsequently,metabolic substances were detected and analyzed for these two groups of mixtures.The results showed that Pseudomonas putida KT2440 and Escherichia coli BL21 may communicate mainly through ribose,adenine nucleotides,lactic acid and vitamin C.Pseudomonas putida KT2440 and Saccharomyces cerevisiae W303 may communicate mainly through ribose,adenine nucleotide,cysteine,ornithine and vitamin C.