Research Of High-density Fermentation Of Chlorella

Chlorella as an edible microalgae has a high-value nutritional quality.Among the commercially microalga,Chlorella has been widely used in many ways such as food and healthy products.At present,Chlorella is mainly cultivated by photoautotrophic cultivation that has some disadvantages such as low biomass,long culture cycle,difficult to harvest,and so on;which has restricted the sustainable development of Chlorella.Heterotrophic cultivation of Chlorella mediated microbial fermentation technique,not only eliminates the light-dependence of microalgae and reduces the occupation of available land,but also enhances the growth rate and cells density.These advantages of microbial fermentation technique promote the industrial upgrading of Chlorella.In this dissertation,on the basis of the optimization of the culture medium of Chlorella sp.by heterotrophic method,50L fermenter and 2000L fermenter were used to carry out the larger scale-up culture in the pilot scale.The main studies and results are as follows:1.Chlorella.MBFJNU-17,which was stored in our laboratory,was found to be 99%homologous to the Chlorella sp.LBA50 by Blast comparison in NCBI through ITS sequences.Then,potassium nitrate,yeast powder,peptone powder and urea were selected to screen the suitable nitrogen source for the growth of the Chlorella.Furthermore,Response Surface Methodology(RSM)was used for optimizing the culture medium.The results showed that:(1)Urea was the most suitable nitrogen source for the cultivation of Chlorella sp..The highest biomass and protein content were 10.85 gL-1 and 53%,respectively.(2)Using the Plackett-Burman and Box-Behnken methods,dry cell biomass was applied to evaluate the performance of growth.Under the optimum conditions,the maximum dry biomass could reach 15.53 gL-1.2.The optimal medium formulation was applied to 50 L fermenter for batch culture of Chlorella sp..Based on the Logistics equation,the growth kinetics was fitted by nonlinear fitting method using software Oringin8.5.The mathematical models concerning cell growth,matrix consumption,and protein biosynthesis were constructed.According to the model characteristics,the cell density of Chlorella sp.increased to 40.75 gL-1 by supplementing the carbon source.Results suggested that the nitrogen source was the second limiting substrate for high-density culture of Chlorella sp..Through supplying carbon source and nitrogen source,the cell dry weight,average growth rate and glucose yield were 106.65 gL-1,0.89 gL-1h-1 and 0.56 gg-1 respectively.3.Pilot culture of 2000 L fermenter was performed to cultivate Chlorella sp.using fermentation parameters of 50 L.The microalga dry weight of 81.6 gL-1and the glucose yield of 0.54 gg-1 were achieved in the sugar consumption of 150.5 gL-1 and nitrogen consumption of 10.5 gL-1 in 120 h of cultivation.Protein content and chlorophyll content of algae powder were 34.60%and 7.75 mg/g,respectively.4.Real-time quantitative PCR was performed to determine the gene expression levels of key metabolic enzymes of Chlorella sp.,including citrate synthase(CS),6-phosphate Glucose dehydrogenase(G6PDF),diaminopimelateisomerase(dapF).It was found that dapF,G6DPH and CS were strongly inhibited when glucose and urea were at lower concentration in the logarithmic growth phase.However,the supplementation of C and N sources promoted the expression of dapF,G6DPH and CS and resulted in the increase in growth and metabolism of cells.Even though C and N source were sufficient in stability phase of cells,the expression of dapF,G6DPH and CS decreased to 0.30,0.31 and 0.22 times in comparison to the control group at the end of cultured,respectively.