Enhancement Of CO₂ Fixation And Lipid Accumulation In Coccomyxa Subellipsoidea By Diethanolamine

Microalgae have the advantages of short growth cycle,high environmental adaptability and high photosynthesis efficiency,and can convert CO₂ into organic substances such as sugars,lipid and proteins through photosynthesis,among which lipid is ideal raw materials for biodiesel refining,so using microalgae to efficiently fix CO₂ and accumulate lipid is an effective strategy for both carbon bioreduction and renewable energy production.However,it is difficult to apply this technology industrially,mainly because of the low CO₂ absorption and mass transfer efficiency in the microalgae culture system,which limits the efficient CO₂ fixation and lipid accumulation by microalgae.In this paper,C.subellipsoidea was used as a research object,we enhanced the carbon sequestration and lipid production efficiency of C.subellipsoidea by adding diethanolamine(DEA)to the culture system of C.subellipsoidea to improve the gas-liquid mass transfer coefficient and CO₂ mass transfer efficiency,and used transcriptomics to elucidate the regulatory mechanism of CO₂ fixation and lipid accumulation in C.subellipsoidea enhanced by DEA.The main findings are as follows:1)The effects of different DEA concentrations on the carbon sequestration and lipid production performance of C.subellipsoidea were investigated.The results showed that at a CO₂ supply concentration of 2%,the action of 40 mg·L^-1 DEA increased the gas-liquid mass transfer coefficient K_Lavalue to 0.073/min,and the biomass,CO₂ fixation efficiency,oil content and oil yield of C.subellipsoidea reached the maximum values of 97 mg·L^-1,225.98 mg·L^-1·d^-1,64.33%,59.9 mg·L^-1·d^-1 which was 1.64,1.64,1.15 and 1.27 times higher than that of DEA-free control;Under the action of DEA,the percentage of C16-C18 fatty acids in C.subellipsoidea cells was92%,and the percentage of MUFAs was higher than that of SFAs and PUFAs,which is an ideal raw material for refining algae-based biodiesel.2)Transcriptional analysis was performed with 2%CO₂ cultured C.subellipsoidea cells as control and 2%CO₂ and 40 mg·L^-1 DEA cultured C.subellipsoidea cells as experimental group,and the results showed that there were 1944 genes differentially expressed in C.subellipsoidea cells under the effect of DEA,of which 1145 were up-regulated and 799 were down-regulated;with the transcriptional differential gene65708,gene52173,gene65708,gene52173,gene15759 and gene27575 were verified by q RT-PCR,and their expression patterns were consistent with the transcriptome,which confirmed that the transcriptome screening differential gene data were more reliable.The 1944 differentially expressed genes were annotated with GO and KEGG.2406entries were enriched by GO,including 11.35%for cellular composition,32%for molecular function and 56.65%for biochemical processes;KEGG enriched 107pathways,among which 72 genes were significantly enriched in cellular process peroxisome pathway,345 genes were significantly enriched in genetic information processing,including ribosome,ribosome biology in eukaryotes,RNA transport and other pathways;1417 genes were significantly enriched in metabolism,significantly enriched in glycolysis,fatty acid biosynthesis,pyruvate metabolism,photosynthetic carbon fixation,TCA.It is confirmed that DEA can significantly regulate carbon metabolism and energy metabolism pathways in the cell center of C.subellipsoidea,providing energy and precursors for lipid synthesis and accumulation in C.subellipsoidea.3)Based on the analysis of the metabolic pathway of the differentially expressed gene KEGG in C.subellipsoidea under the action of DEA,the carbon sequestration and lipid accumulation network C.subellipsoidea was reconstructed.In the inorganic carbon transport process of C.subellipsoidea,carbonic anhydrase CA catalyzed the CO₂hydration reaction,and its activity increased with the increase of culture time,reaching the highest CA activity of 150.65 U·L^-1 and 138.93 U·L^-1 in the experimental and control groups,respectively,by the 7th day of culture,which integrated more external carbon sources into the intracellular carbon metabolic flow and provided sufficient carbon skeleton for rapid cell growth.carbon skeleton;In the Calvin cycle pathway,the expression of gene39868 and gene53355 encoding PGKase and GAPDHase were significantly up-regulated compared with the control group,and their enhanced activities accelerated carbon fixation in C.subellipsoidea,However,there was no significant change in the gene65708 encoding Rubis CO enzyme,and the experimental group after five days of culture,the Rubis CO activity was enhanced and reached a maximum of 602.63 U·L^-1 on 7days,presumably g65708 was significantly expressed after transcription;The key regulatory gene52357,gene53369,gene29458,gene57881 in the glycolysis and TCA cycle pathways were significantly upregulated,and the gene encoding PEPC enzyme gene61449 expression was down-regulated,and its enzyme activity was at a lower level during C.subellipsoidea culture.The enhancement of glycolysis and TCA cycle metabolic pathway provided more energy and intermediate metabolites to C.subellipsoidea cells;gene15759 was significantly expressed in pyruvate metabolic pathway,and PDH enzyme activity was significantly different compared with no DEA treatment,which accelerated the metabolism of pyruvate and synthesized a large amount of acetyl accase.In lipid metabolic pathway,there was no significant change in the gene27575 encoding ACCase enzyme.The ACCase activity was measured at different incubation periods of C.subellipsoidea and reached a maximum of 195.93 U·L^-1 at day 7,which was 1.24 times higher than the maximum ACCase activity in cells without DEA treatment.Moreover,gene37141,the gene encoding ACAT,was significantly down-regulated,indicating that the degradation of long-chain fatty acids was slowed down and more lipids were accumulated intracellularly in late stages of C.subellipsoidea.