Study On Fermentation Condition And Fermentation Kinetics Of High-yield Heterotrophic Microalgae

Microalgae are prokaryotic or eukaryotic photosynthetic microorganisms that can grow rapidly and live in harsh conditions due to their unicellular or simple multicellular structure.And they are present in all existing earth ecosystems,not just aquatic but also terrestrial,representing a big variety of species living in a wide range of environmental conditions.Owing to their diverse chemical composition,they can be acted as a nutritional supplement or represent a source of protein and biodieseL In particular,as a feed additive,the market demand gradually increased.Domestic and foreign commercial algae proteinproducers have reached annual production of 100 tons,the output value of billions of dollars,and only seaweed feed production of Taiwan Province reached 120 tons.Nevertheless,there is still a large gap in the supply and demand of microalgae feed protein in China;this is due to the difficulty in producing and high cost of natural nutrition pattern of microalgae.So more research turns to screen and select the facultative or chemical heterotrophic strains,and the production capacity will be improved by optimizing of the culture conditions and the culturestrategy.According to the theory of metabolic control fermentation and fermentation kinetics,the dissertation focuses on the microscopic morphology,fermentation condition and its fermentation kinetics of protein producing strain Nannochloropsis gaditana.The main research contents and results are as follows:(1)Effect of different culture conditions on microscopic morphology and protein production of N.gaditana.Morphological studies have shown that N.gaditana has two different forms of life:glial vesicles and free single cells,and the glial vesicles gradually evolved to free single cells during the fermentation process:0-20 h is for the lag phase and exponential growth phase,N.gaditana mainly in the form of glial vesicles,cell size gradually increased;during 20 h-28 h,glial vesicles survived with free single cells during the deceleration phase;after 28 h,with the progress of fermentation,the single cell enjoyed absolute advantage during the stability and decay phase.In addition,the study of culture conditions such as temperature,pH and stirring speed showed that the culture conditions had a great influence on the intracellular protein content and the time required for the final cell morphology,and the stirring speed had a great effect on morphology of the cells.(2)Optimization of fermentation conditions for protein production of N.gaditana was achieved by response surface methodology.The response surface methodology to improve microalgae protein productivity during stable phase,were designed to optimize the fermentation conditions of batch cultivation of N.gaditana.Based on the data from single-factor trial,four main conditions including the temperature,pH,stirring speed and aeration rate were selected as influencing factors,and then the experiments with four factors and three levels were designed according to a Box-Benhnken central composite rule.The results showed that the optimal fermentation conditions were attained as follows:temperature 30?,pH 6.9,stirring speed 340 r/min and aeration rate 0.65 vvm.Finally,the confirmatory experiment was performed under these optimal conditions:the final protein concentration increased to 6.18 g/L by 9.18%than before,consistent with the model prediction;the final biomass concentration increased to 6.18 g/L,and 70.3%increase was achieved compared to the other literature.(3)Batch kinetics of N.gaditana retardant growth was established by dual-power functions.Based on the above optimum conditions,the batch kinetics of N.gaditana fermentation was studied in 2 L fermenter.A mathematical equation of yield coefficient and a new dual-power function about cell retardant growth were established respectively,according to the practical dynamic conversion from substrates to microalgae cells and considering the characteristic of cell retardant growth.The kinetic models and parameters describing the course of Microalgae fermentation were obtained with nonlinear curve fit by Matlab,and the estimation of model kinetic parameters showed that the new models achieved a satisfactory prediction of the microalgae cultivation.The parameter ? is close to 0,which indicate that microalgae protein production belongs to growth associated.Additionally,we also carried out 50 L scale-up fermentation experiment and the result demonstrated that the developed models can considerably represent the dynamic cultivating status of the microalgae cell.Summarily,the kinetic models about retardant growth and yield coefficient could have a promising application in the future process control and optimization of industrial.(4)Fed-batch fermentation kinetics ofN.gaditana in fermenter was studied.Through the way of constant-rate feeding,the study kinetic models of batch fermentation for cell growth,protein production and total sugar consumption was established.According to the known model parameters,the nonlinear fitting was carried out.The results predicted by the model were in good agreement with the experimental observations(The correlation coefficient was more than 0.95)and showed that the models developed here reasonably describe the process and dynamic mechanism of fed-batch fermentation for microalgae proteins.