Study On The Isolation Of Oleaginous Microalgae And Their Co-cultivation

The unsustainable fossil fuels which can induce environmental pollution hasbecome a social focus. Biodiesel, as an ideal substitute of petroleum fuels has beendeveloped rapidly in recent years. It should be a green renewable new energy whichused widely. Realization of large-scale biodiesel production was restricted byinsufficient feedstock and high-cost.Microalgae, can be used to provide sufficient and qualified feedstock forbiodiesel because many different kinds of them cultivated easily and grow fast. Tosolve feedstock high-cost, it was key having excellent microalgae strains to improvelipid productivities under optimum culture condition. It could be further reduce theproduction cost to simplify operation and lower electric power consumption throughauto-flocculation methods for harvesting microalgae cells.In this experiment, lipid productivity is used as a key characteristic forcomprehensive evaluation of microalgae lipids production capacity, instead of thepartial measures by microalgae biomass or lipid content. According to this evaluationstandard, we isolated an oil microalgal strain for biodiesel production fromChengjiang Fuxian Lake, Yunnan Province. This microalgae was identified as thegenera Chlorella and named as Chlorella sp. U4341through morphologicalexamination and18S rRNA gene phylogenetic analysis. We established oil extractionmethod and improved in situ transesterification method. The lipid productivities andfatty acid profiles of Chlorella sp. U4341through in situ transesterification usingcommon medium BG-11through photoautotrophic cultivation, heterotrophiccultivation and mixotrophic cultivation were carried out. The results indicated that isworthy to carry out further research to increase the lipid productivities and improvefatty acid profiles for Chlorella sp. U4341.The biomass and lipids productivities of Chlorella sp. U4341were increasedsignificantly through modified Bold’s Basal medium and culture conditions optimizedusing Response Surface Methodology (RSM)-Central Composite Design (CCD) approach. In optimal culture conditions, the lipid productivities of Chlorella sp.U4341through photoautotrophic cultivation was17.99mg L-1d-1, which elevated0.22times and0.10times compared with the common photoautotrophic cultivation(14.77mg L-1d-1) and cultured using original BG-11medium (16.63mg L-1d-1)respectively. The lipid productivities through heterotrophic cultivation was273.34mgL-1d-1, which elevated0.58times and1.64times compared with the commonheterotrophic cultivation (173.06mg L-1d-1) and cultured using original BG-11medium (103.72mg L-1d-1) respectively. The lipid productivities of mixotrophiccultivation was280.57mg L-1d-1, which elevated0.54times and1.45timescompared with the common heterotrophic cultivation (181.60mg L-1d-1) and culturedusing original BG-11medium (114.45mg L-1d-1) respectively. The proportion ofmonounsaturated fatty acids was improved and the proportion of polyunsaturatedfatty acids decreased significantly using optimized culture conditions. It make thecetane number value tenuous increased. However, the value of lipid productivities isfar from being satisfactory, and fatty acid profiles has not met the biodieselproduction standards. Therefore, we should take a new method to further improvedculture method.Lipid productivities were enhanced in co-cultured system for microalgae andbacteria or microalgae and yeast cultivation, the researchers have done a large amountof work for this, and has made the rich achievement. In this experiment, microalgaeChlorella sp. U4341and microalgae Monoraphidium sp. FXY-10were used inco-cultured system. It is due to the high growth rate of U4341and lipid accumulationability of FXY-10. Different concentrations proportion of microalgae cells inco-cultivation system directly affected the total biomass, total oil content and lipidproductivities. Therefore, adjusting the ratio among the various microalgae toinoculate is more convenient to control the proportion of different microalgae cellsconcentration under co-cultivation system. In order to obtain the highest lipidproductivities, we selected different inoculation concentration whose initialconcentration of U4341and FXY-10were3.41×106and4.26×106cells mL-1,1.83×106and3.50×106cells mL-1,1.83×106and1.98×106cells mL-1in photoautotrophic cultivation, heterotrophic cultivation and mixotrophic cultivationunder the co-cultivation system, respectively.The growth rate of Chlorella sp. U4341and Monoraphidium sp. FXY-10inco-cultured system were inhibited and the biomass of them were lower than solecultivation respectively. The nitrate consumption rate has significantly acceleratedand the nitrogen limitation arrived earlier than them in mono-cultivation. It pushedthe stable phase of microalgae in co-cultured system arrived earlier. Inphotoautotrophic cultivation, the time of co-cultivation was26days shortenedseparate culture to20days. In heterotrophic cultivation and mixotrophic cultivation,the time was shortened from the6days and10days to5days respectively. The oilproduction were improved by shorter culture time and higher lipid productivities.The lipid productivity were29.63mg L-1d-1,315.60mg L-1d-1and293.23mgL-1d-1in photoautotrophic cultivation, heterotrophic cultivation and mixotrophiccultivation, respectively. The lipid productivities were induced by complicated factors.The interaction between the two strains and nutritional deficiencies especiallynitrogen starvation conditions maybe the important factors to improve lipidproductivity under the co-cultivation system.The oil quality were promoted in co-cultured system because the concentrationof C18:1fatty acid were increased significantly. The cetane number value wereincreased to49.04and52.26while cold filter plugging point values (-2.89to-6.64)had preferably fluidity at low temperature in heterotrophic and mixotrophicco-cultivation system. This cetane number value has achieved our national biodieselstandard.The auto-flocculation sedimentation of microalgae cells could simplify process,help biomass harvesting and reduce production costs. The light could makesedimentation rate lower. In order to improve the sedimentation rate, we performsedimentation experiments in the darkness condition. The sedimentation rate inco-cultivation system had higher sedimentation speed than it in mono-cultivation.And it could be90%in8hours. The main cells aggregation were U4341inheterotrophic cultivation, FXY-10in photoautotrophic cultivation and the cells ofU4341and FXY-10were enlargement and become aggregation during sedimentation process. The auto-flocculation sedimentation of microalgae cells were induced bycomplicated factors. The sole pH alternation could not influence the cellssedimentation significantly. Increasing the concentration of extracellular polymericsubstances (EPS) were the main influence factors.