Promotion Of Microalgal Carbon Uptake And Biomass Production By Exogenous Carbonic Anhydrase

Microalgae are able to efficiently fix CO₂through photosynthesis to synthesize organic matter,including polysaccharides,proteins,lipids and carotenoids,and are an important raw material resource for the food and chemical industries.Compared to terrestrial plants,microalgae are 10-50 times more efficient at photosynthesis and contribute more than 70%of the Earth's oxygen at a level of less than 1%of the total plant population.In addition,microalgae also have the advantages of strong environmental adaptability,can use brackish or semi-saline water culture,and do not occupy arable land with cash crops,etc.Carbon source is an important factor for the growth of microalgae,and the uptake of carbon source by microalgae is directly related to its photosynthetic efficiency and culture cost.Especially in the context of the double carbon economy of"Carbon neutral and Carbon peak",it is undoubtedly important to develop microalgae industry and reduce the cost of microalgae culture by improving the efficiency of carbon source utilization in the process of microalgae culture.Based on the above background,this study was conducted to explore the improvement of the supply capacity of carbon source and the conversion efficiency of algal cells to carbon source in microalgae culture system as the starting point.Based on the determination of the role of carbonic anhydrase in the process of CO₂ mass transfer and absorption,symbiotic bacteria capable of secreting extracellular carbonic anhydrase were screened from the environment,isolated and identified,and the construction conditions of the algal-bacterial symbiotic system and its effect on the utilization of microalgae were optimized before and after The main results were as follows.The main results were as follows.1.Screening and identification of exogenous carbonic anhydrase expressing bacteriaSix strains of microorganisms with extracellular carbonic anhydrase activity were isolated and screened from the algal environment(lake water and soil).The three strains,YD-1,YD-4 and YD-6,were identified by sequencing as Gram-positive bacteria,among which YD-1 was homologous to Bacillus safensis(99%)and YD-4 was similar to Frigoribacterium faeni(94%),belonging to Frigoribacterium.YD-6 is homologous to Bacillus licheniformis(99.86%)and also has high similarity to Bacillus paralicheniformis(99.86%),and both YD-6 and YD-1 belong to Bacillus sp.2.Construction of algal co-culture system and its effect on carbon source utilization by microalgaeResults of algal inoculation ratio:Three strains of bacteria with carbonic anhydrase(CA)activity were screened and cultured in combination according to the ratio of the number of algal cells 1:10,1:1 and 10:1,respectively.The results showed that the three bacterial strains had effects on the growth,lipid content and extracellular carbonic anhydrase activity of Chlorella,and the combined culture with the addition of bacterial cells had more significant promotion effect than the addition of supernatant containing the same number of bacteria.When the ratio of algae to bacteria was 1:10,the maximum algal cell density of Chlorella+YD-1 culture system was 6.55×10~7 cells m L^-1,which was 54.48%and 9.71%higher than that of Chlorella pure culture system(control)and Chlorella+YD-4 culture system,respectively,and similar to that of Chlorella+YD-6 The extracellular carbonic anhydrase activity of the Chlorella+YD-1 culture system increased by 55.19%compared to the control.It can be seen that the Chlorella+YD-1 culture system with an algae to bacteria ratio of 1:10 was effective in promoting the growth of Chlorella.Bacteria promoted carbon uptake in Chlorella:Transcriptomic analysis was used to study the mechanism of bacterial YD-1 to promote carbon uptake in Chlorella,and the results of differential gene expression and metabolic pathways of differential gene enrichment in Chlorella+YD-1 culture system and Chlorella pure culture system(control)were found.The expression of apolipoproteins and other genes increased,the photosynthetic electron transport chain was strengthened,and the synthesis of ATP rose,which enhanced the photosynthesis of Chlorella,indicating that the addition of bacterial YD-1 increased the extracellular carbonic anhydrase activity,promoted the uptake of CO₂ by Chlorella,and enhanced the photosynthesis of Chlorella.(3)Construction and validation of combined culture system of immobilized bacteria and ChlorellaThe most suitable cross-linking time of 12 h was obtained by measuring the physical indices and carbonic anhydrase activity of cross-linked 4 h,12 h and 24 h immobilized pellets using sodium alginate+calcium chloride embedding method.In the first culture cycle,the maximum algal cell density and extracellular carbonic anhydrase activity were5.62×10~7 cells m L^-1 and 4.32 U m L^-1,which were 40.80%and 42.11%higher than those of the control group,respectively.The maximum biomass and extracellular carbonic anhydrase activity were increased by 31.46%and 34.09%,respectively,when the immobilized bacterial pellets were recovered for the second culture of Chlorella.After 14days of incubation,the immobilized pellets still maintained 90%of carbonic anhydrase activity.It can be seen that the immobilized pellets of bacteria can be used more than once,providing an effective way to reduce the cost of carbon source for large-scale culture of microalgae.