Study On The Production Of Polyhydroxyalkanoates By The Thermophilic Mixed Strains

Polyhydroxyalkanoates(PHAs)are polyesters that can be produced by microorganisms from renewable resources(such as carbohydrate and fatty acid)as intracellular carbon storage compounds.This biopolyester is regarded as a substitute for oil and petrochemicals not only for its similar material properties to conventional plastics but also for its biodegradable properties.However,the cost of PHA production hinders its commercial exploitation.To promote the application of PHA,some tactics were conducted to reduce cost,including screening some predominant strains,improving fer mentation process or employing much more effective extraction and separation methods.Commercial production of PHA is currently based on pure culture processes employing solo strain.Pure culture processes use generally pure sterile substrates and axenic reactors,leading to high production costs and thus relatively expensive products,which have prevented the use of this material on a real industrial scale.In comparison,microbial mixed culture adopt several highly efficient strains for PHA synthesis.The advantages of the microbial mixed culture lie in:(1)improve the utilization of substrate,equipment and energy,et al;(2)obtain high-purity PHA;(3)reduce the cost of PHA synthesis.On the other hand,temperate is a key factor for PHA synthesis.Higher temperate could accelerate diffusion of nutrients and exclude ambient strains.Therefore,it is feasible to get more PHA via mesophilic fermentation with thermophiles strains.In spite of above advantages,few PHA-accumulating thermophiles with have yet been found.Thus,the optimum culture conditions of thermophiles still need to be researched.In our group,we succeeded in isolating a thermophilic PHA-accumulating bacteria(Bacillus licheniformis JH8)from the biotrickling filter system.To the best of our knowledge,this is the first report on the production of PHA by the genus of Bacillus licheniformis.This study investigated the cytoplasmic PHA inclusions in Bacillus licheniformis JH8 by staining the biopolymer with Nile red and observing the cells under a fluorescence microscope.The architectural features and material features as well as content of PHA produced by Bacillus licheniformis JH8 were determined by gas chromatography,fourier transform infrared(FTIR)and nuclear magnetic resonance(NMR).This study then characterizes the relationship between the growth and the PHA accumulation under different condition.After that,further investigation refer to the influence of carbon sources,temperature and initial pH value on PHA accumulation were conducted to obtain the optimal PHA amount by Bacillus licheniformis JH8.According to our study on Bacillus licheniformis JH8 and C.daeguensis TAD1,this study investigated the optimum culture conditions for microbial mixed culture on PHA synthesis by Bacillus licheniformis JH8 and C.daeguensis TAD1(JH8-TAD1).Based on the result of shake-flashing culture,spreading cultivation for PHA synthesis by JH8-TAD1 was conducted via a 5-l bioreactor.After that,kinetics model for the growth of JH8-TAD1 and PHA synthesis under microbial mixed culture were set.At the end,two-stage batch-fed fermentation was established to assess the effect of microbial mixed culture on PHA synthesis by JH8-TAD1.The major results are listed as follow:1?The results show that the biopolyester in Bacillus licheniformis JH8 is verified as poly-?-hydroxybutyrate(PHB).As PHB synthesis coming with the growth of Bacillus licheniformis JH8,Bacillus licheniformis JH8 would be defined as a growth-associated PHA-accumulating bacteria2?Bacillus licheniformis JH8 could synthesize PHB with the range of 30-60 Co,and the optimal PHB synthesis was recorded at 60 Co.This show that the PHB synthetase of Bacillus licheniformis JH8 possess thermostability.On the other hand,the growth of Bacillus licheniformis JH8 and PHB synthesis would be affected by the initial pH value of substrate.The optimal cell dry weight(CDW)and PHB content were recorded at pH 7.5.It indicate that slight alkali environment is favor of the growth of Bacillus licheniformis JH8 and PHB synthesis.3?The process of JH8-TAD1 adopting glucose to synthesize PHB is classified as coupling model.Under the optimal conditions(loading volume 5%,glucose,NH4 Cl and fermentation time 28 h),the CDW,PHB content and PHB relative amount were recorded as 3.34 g/L?2.38 g/L and 71.32%,respectively;4?According to the result of Plackett-Burman,the main suppressive factors are NH4Cl?glucose and MgSO4·7H2O,that influence the effect of JH8-TAD1 synthetizing PHA.According to response surface methodology,the quadratic regression model of NH4Cl?glucose and MgSO4·7H2O were set up,respectively.Based on the model's calcu-lations,the optimal CDW(3.34 g/L),PHB content(2.38 g/L)and PHB relative amount(71.32%)could be obtained under the condition of glucose 29.37 g/L,NH4 Cl 2.35 g/L,MgSO4·7H2O 0.32 g/L,Na2HPO4·12H2O 9.0 g/L,KH2PO4 1.5g/L,CaCl2·2H2O 0.02 g/L and trace element solution 1 ml;5?With the aim of building up theoretical model for large scale production of PHB,a 5 L bioreactor was applied to investigate the characterization of PHB production by JH8-TAD1 under different fermentation condition.According to the model of PHA synthesis by JH8-TAD1 for large scale production,two-stage fed-batch fermentation tactic was set up to obtain the optimal CDW(10.93 g/L),PHB content(7.53 g/L)and PHB relative amount(68.92%)after fermentation time 36 h.This work initially reveals the characterization of the PHA accumulation by JH8-TAD1,which will play the theoretical foundation for later strain modification,process optimization of large-scale fermentation.