Screening And Identification Of PHB Strain And Optimization Of Fermentation Technology

PHB is a kind of biodegradable polymer materials,which can be used in medicine,environmental protection,agriculture and other fields,it has wide applications and wide industrial application value.The strain was identified as Pseudomonas by the 16 S r DNA gene sequence analysis,Then,the bacterium was identified as Pseudomonas putida according to the physiological and biochemical characteristics test.The strain was named PP25.The results of preliminary fermentation test showed the yield of PHB was about 2.456 g/L when 2% glycerol was used as the main carbon source.In this paper,UV method and HPLC method were established to determine the content of PHB in Pseudomonas sp.In the establishment of UV spectrophotometry,the method of ultrasonic wall breaking was as the pretreatment of cells,the optimum conditions were determined by the factors such as the ultrasonic time,the evaporation time,the water bath time and the dosage of sulfuric acid.The result was a linear relationship within 1.0 ~ 3.5 g/m L with UV spectrophotometric method,the recovery rate was95.93%~104.79% and the relative standard deviation was 1.13% ~ 4.27%.HPLC method was used to analyze the intracellular PHB by Aminex HPX-87 C column.The final results were obtained after optimizing the chromatographic conditions,5 mmol/L sulfuric acid was as the mobile phase,the flow rate was 0.7 m L/min,column temperature was 50 o C,the recovery rate of the sample was 98.91% ~ 101.97%,and the relative standard deviation was 0.763% ~ 2.014%.In the paper,the factors affecting the growth of PP25 and the accumulation of PHB were studied,including temperature,shaking speed,initial p H,inoculation amount,liquid volume,carbon source,nitrogen source,inorganic salt,metal ion and so on.The factors were screened by the Placket-Burman experiment,which had significant influence on the PHB synthesis of PP25.The results showed that glycerol,ammonium sulfate and initial p H had significant effects on the synthesis of PHB of PP25 strain.The relationship between 3 significant factors and PHB accumulation of PP25 were studied by steepest ascent test and response surface methodology.The results showed that the optimum conditions were glycerol at 26.892 g/L,ammonium sulphate at 3.043 g/L,and initial p H at 7.004,and the maximum estimated value of poly(hydroxybutyrate)was about 8.062 g/L.The actual PHB synthesis was 7.881 g/Lafter verification of the model,which was similar to the theoretical yield,which increased 32.52% at optimized condition.The optimal conditions were determined that Glycerol was 26.892 g/L,citric acid was2 g/L,ammonium sulfate was 3.043 g/L,two sodium hydrogen phosphate was 15 g/L,sodium dihydrogen phosphate was 4 g/L,seven water ferrous sulfate was 0.4 g/L,magnesium Sulfate was1.2 g/L,and initial p H was 7.004.In this paper,the effects of aeration rate,temperature and p H on the accumulation PHB of PP25 in 5 L fermentor were investigated.The results showed that the optimal fermentation conditions were aeration rate at 1.2 vvm,temperature at 28�C,and p H at 6.5,the biomass was 18.04 g/L and the yield of PHB was about 10.617 g/L under these conditions,which was about respectively 46.19%?78.95% greater than those obtained with initial fermentation conditions.On this basis,the feeding process of different time periods was studied,feeding composition was glycerol or different ratio of carbon and nitrogen sources.The results showed that the compound carbon and nitrogen source(glycerol 60 g,ammonium sulfate 12 g)was added after the fermentation 24 h,the C/N ratio was 5:1,finally,the dry weight and yield of PHB were further increased by 71.40% and by 54.95%,respectively.In this paper,the effects of some factors on the extraction rate of intracellular PHB were studied,including the time of bead milling,the quality of bead grinding,the time of chloroform extraction,temperature,cell concentration,alcohol precipitation time and the ratio of ethanol to chloroform.The results showed that the milling time was 3 min,bead grinding capacity of 100 mg,chloroform extraction time was 78.3 min,the temperature was 54.5o C,cell concentration was 20.5 mg/m L,the ratio of chloroform and alcohol was 1:4,and the alcohol precipitation time was 70 min,the intracellular PHB extraction rate was 84.20% with these condition.After purification,the extraction rate of PHB was73.51%,and the purity of PHB was 98.97%.