The Optimization Of CO₂ Sequestration Using Chlorella Vulgaris And Preparation Of Cell Growth Factors

With the rapid development of industrialization,increased CO₂ emission has led to the“greenhouse effect”,which has become a major challenge for human society.Microalgae grows fast and has high carbon fixation efficiency.The use of microalgae for carbon dioxide sequestration is considered to be a promising method for immobilizing CO₂.As the second largest economic microalgae in China,Chlorella has achieved large-scale outdoor cultivation,its grows fast and relatively mature culture technology,and has a good scale of carbon sequestration.However,the large-scale cultivation of Chlorella is mainly cultivated using an organic carbon source,such as acetic acid or glucose,and the CO₂ utilization efficiency is low or substantially not utilized.Therefore,how to improve the CO₂ utilization in large-scale cultivation of Chlorella is of great significance for CO₂ sequestration.To address this issue,the feasibility of CO₂ photoautrophy culture was first studied.As a result,when 5%CO₂was introduced into the medium（the flow rate was set to 300 m L/min/L）,dispersed by aerated stone aeration,C.vulgaris biomass production growth rate can reach to0.21 g/L/d,which was 1.34 times that of acetic acid culture.On this basis,we studied the effect of CO₂/acetic acid mixotrophy culture,that is,the C.vulgaris was cultured by introducing CO₂ during the daytime,and acetic acid was added at nighttime to carry out heterotrophic culture through the tricarboxylic acid cycle and the glyoxylate cycle.Our results shows that the mixotrophy culture can increase the growth rate of C.vulgaris to 0.24 g/L/d,which was higher than 0.15 g/L/d and 0.21 g/L/d of cultures with acetic acid or CO₂ culture only,respectively.After ten days’culture,the biomass yield of mixotrophy culture could reach2.37 g/L,and the CO₂ biofixation was 0.294 g/L/d,which significantly improved the biomass production and CO₂ utilization efficiency.Physiological and biochemical analysis showed that under mixotrophy culture the contents of biomass,chlorophyll and protein of C.vulgaris were the highest,2.37 g/L,61.1mg/L and 361.54 mg/L,respectively,which were 7.2 and 6.4 and 6.4 times of the control group;the content of unsaturated fatty acids was also increased significantly to 54.2 mg/L,which was 5.6 times that of the control group.The results of flow cytometry showed that the size and density of C.vulgaris cells increased under the mixotrophy culture,and the morphology of algae cells was also characterized,which was beneficial to the centrifugal harvesting of downstream algae cells.The enzyme activity assays revealed that the carbonic anhydrase activity was inhibited and the Rubisco enzyme activity was enhanced in the mixotrophy culture.Transcriptome sequencing analysis revealed that the metabolism-related genes of the Calvin cycle and the Citric acid cycle were significantly up-regulated under the conditions of mixotrophy culture,which may be contribute to the increase in biomass.In addition,the preparation methods,biological activities and active components of Chlorella growth factor（CGF）were also studied.The results showed that the highest yield of CGF extraction by high pressure hot water method was 24.5%.The CGF extracted method can significantly promote cell proliferation rate and OH^-free radical scavenging rate,and its proliferation-promoting rate and clearance rate were 40%and 62.3%,respectively.The results of CGF active ingredient analysis showed that the cell proliferation and OH^-free radical scavenging activity of the protein degradation or extraction the lipids were reduced by about22.2%,13.5%and 15.3%,9.7%,respectively.After nucleic acid components degraded,it had no significant effect on its biological activity,suggesting that the protein and lipid components may be the main active components of CGF.In conclusion,the mixotrophy method developed in this study can significantly improve the biomass and carbon sequestration efficiency,and can be applied to large-scale production and carbon sequestration.At the same time,the extraction and active fraction analysis of CGF will promoting the development of high value-added products.Our study will also lay a foundation for the comprehensive utilization of C.vulgaris.