Optimization And Scale Up Of Chlorella Photoautotrophic Cultivation For High Lipid Productivity

Due to the lack of fossil energy and the urgency of CO2emission reduction, the development for industrilization of biofuels from microalgae has been paid much more attention. Microalage cultivation is the key to biofuels from microalgae, and high lipid productivity is the central aim of microalgae cultivation. Therefore, optimizaition and scale up of microalgae photoautotrophic cultivation for high lipid productivity has been a research focus in recent years. However, many researches in the literature mainly focused on indoor cultivation; some results refered to outdoor culture but the scales of them were not large. Chlorella spp. are main species of fresh oleaginous algae, however, few research has done into the optimization and scale up of Chlorella photoautotrophic cultivation for high lipid productivity.Chlorella sp. has been used as the research object. The effects of trophic modes, temperature, nutrient concentrations and culture modes on the lipid productivity have been investigated; the novel model of "heterotrophic seed-photoautotrophic cultivaton" of Chlorella has been found; photoautotrophic medium of C. pyrenoidosa for high lipid productivity has been selected and optimized; and the C. pyrenoidosa indoor photoautotrophic cultivation for high lipid productivity has been established. Based on the indoor results, the Chlorella outdoor photoautotrophic culture has been established and optimized in0.35m2open basins. Moreover, for outdoor photoautotrophic mass culture, the seed preparation scale has been magnified from5L flasks to50L and500L fermenters, and the photoautotrophic cultivation scale has been enlarged from0.35m2open basins to80m2raceway ponds. The main results are as follows:(1) Establish a novel Chlorella culture model of "heterotrophic seed-photoautotrophic cultivation"("HS-PC", the photoautotrophic culture with heterotrophic cells as seed). For overcoming the long period of seed cultured photoautotrophically, difficulty for algal species control and inadequate cells supply for the inoculation of microalgae photoautotrophic cultivation, a novel model of "HS-PC" has been firstly established. For C. pyrenoidosa, the biomass productivity of heterotrophy was20.9-fold higher than that of photoautotrophy during the seed preparation process; the lipid productivity of "HS-PC" was1.59times higher than that of "PS-PC"(the photoautotrophic culture with photoautotrophic cells as seed); The fatty acids of "HS-PC" model were in medium length (16-18carbons) with2or3unsaturation degree, which were suitable for biofuels-making. Furthermore, the "HS-PC" model also can be applied to C. ellipsoidea and C. vulgaris; the lipid productivity of C. ellipsoidea and C. vulgaris in "HS-PC" model were1.52-fold and1.58-fold higher than those in "PS-PC" model, respectively.(2) Obtain the optimum C. pyrenoidosa photoautotrophic medium for both indoor and outdoor culture through the selection and optimization. The results showed that the lipid productivity of the cells cultured with the optimized medium were1.61-fold and1.67-fold higher than those with the original one in the3L panel bioreactor for indoor and outdoor culture; the lipid productivity with the optimized medium was1.33-fold higher than that with the original one in the outdoor0.35m2open basins.(3) Temperatures during the daytime and night have great effects on lipid productivity. For outdoor culture with light-dark cycle, the losing of biomass and lipid at night decreased the biomass and lipid productivity. Hence, studies on the effects of temperature on the variations of biomass concentration, lipid content and fatty acids composition under a light-dark cycle culture were absolutely important. The results showed that30℃was the optimal temperature for achieving high biomass and lipid productivity. The night biomass loss decreased with the increasing daytime temperature or the decreasing night temperature; the extent to the lipid content at night increased with the increasing daytime temperature or the decreasing night temperature. In addition, the night biomass loss decreased with the increasing biomass concentration at any temperature; among the six main fatty acids, C16:0was the most abundant fatty acid and apparently influenced by the temperature, which was increased with the increasing temperature.(4) Establish an indoor photoautotrophic cultivation strategy of semi-continuous culture with pH-regulation by CO2and nitrogen-limitation. In the batch culture, the lipid productivity can be enhanced with pH-regulation at7±0.2by CO2, which was2.64-fold higher than that without pH control. The lipid productivity of the cells can be increased under the nitrogen-limitation conditions; the lipid content of Chlorella with the initial NaNO3concentration at200mg/L was38.7%, which was1.25times higher than that at500mg/L. Additionally, under the same culture conditions, the lipid productivity of the cells in the semi-continuous culture was1.47-fold higher than that in the batch culture. Hence, the culture strategy of integrating both nitrogen-limitation and pH-regulation by CO2in a semi-continuous cultivation was investigated for enhancing the lipid productivity. The results showed that the lipid productivity of the above semi-continuous system with one-third replacement and24h’s interval was3.64times higher than that in the batch culture without pH control and nitrogen limitation.(5) Establish an outdoor photoautotrophic cultivation in0.35m2open basins. The results showed that the optimal pH was7±0.2and the fermenter exhaust-gas can take place of pure CO2as carbon source and for pH-regulation; the optimal ventilation volumn, broth depth and inoculation concentration were0.5vvm,14cm and0.3g/L as well as no mixing during the night. Additionally, the Chlorella outdoor photoautotrophic culture has been optimized by indoor culture results:when the natural temperature was low (maximium temperature<30℃), the lipid productivity can be enhanced by81%through controlling the daytime temperature between30℃and36℃; Under the same culture conditions, the lipid productivity of Chlorella in the semi-continuous culture with nitrogen-limitation was1.85-fold higher than that in the batch culture.(6) The seed preparation process has been enlarged from5L flasks to500L fermenters for adequate cells supply for the inoculation of the photoautotrophic culture. The "HS-PC" model was firstly carried out for outdoor mass culture:the photoautotrophic culture scale was magnified to80m2raceway ponds. The maximum lipid productivity of Chlorella cultured in80m2raceway ponds reached46.4mg/L/d (5.22g/m2/d), which was the biggest scale and highest lipid productivity in the literature.Our results can not only load a good foundation for industrialization of Chlorella cultivation with high lipid productivity, but also provide a valuable reference for other oleaginous microalgae cultivation.