Enhanced Bioconversion And Molecular Mechanism Investigation Of The Mixed Culture Of Microalga And Yeast In Liquid Digestate

China is a large country of biogas production.The treatment and utilization of liquid digestate has been a bottleneck restricting the development of biogas industry.The deep utilization of liquid digestate is considered to be the only way to turn waste into treasure and realize circular economy.Microalgae can effectively assimilate nitrogen and phosphorus from wastewater and realize the purification of wastewater and the production of microalgae biomass.This process can reduce the cost of biogas slurry liquid digestate treatment,and microalgae biomass can be used as feedstocks for bioenergy,feed and fertilizer.Mixed culture of microalgae and yeast has obvious advantages in pollutant removal and microbial biomass due to the synergistic effect.In this paper,the systematically comparision of the effects of mono microalga,mono yeast and mixed culture on pollutant removal and biomass production in liquid digestate were carried out.The intrinsic molecular mechanism of bioconversion enhancement of liquid digestate by the mixed culture of microalga and yeast was elucidated through transcriptome analysis.The main findings are as follows:（1）Mixed culture of microalgae-yeast with liquid digestate of yeast industry showed that liquid digestate of yeast industry could be used as a low-cost substrate for Yarrowia lipolytica culture,and mixed culture with Clorella vulgaris could enhance the production of microbial biomass.The removal of NH₃-N in the mixed culture was better than those of mono cultures and the removal rate of NH₃-N increased with the dilution ratio.The removal rate of NH₃-N and the initial inoculation concentration of microalga were positively correlated between mixed culture groups.The lipid yield of the mixed culture system was higher than that of the mono cultures,and the higher heat value（HHV）yield of the mixed culture system was obtained.（2）Mixed culture of microalgae-yeast with liquid digestate of dairy wastewater showed that the mixed culture could enhance biomass production.Mixed culture fixed more C and N from the culture environment and enhanced the yield of lipid,protein and HHV.The removal efficiency of total phosphorus in the mixed culture（up to 100%in 48 hours）was better than that in mono cultures.The transcription levels of NR and glnII,the key genes of nitrogen assimilation in C.vulgaris,were lower in the mixed culture system than that in the mono microalga culture system when ammonia nitrogen was sufficient.The depletion of ammonia nitrogen in mono microalga culture initiated NO₃^-assimilation in microalgae,accompanied by a sharp increase in NR transcription levels.（3）Mixed culture of microalgae and yeast in simulated wastewater showed that the specific growth rate of microalgae and yeast cells in the mixed culture group(0.30 d^-1 and0.99 d^-1)was significantly higher than that in the corresponding mono culture.The cell volume of yeast in the mixed culture was significantly reduced,while the relative chlorophyll content of microalgae was significantly increased.Mixed culture can obtain higher carbon and nitrogen utilization rate and make carbon mainly flow to carbohydrate.Higher yield of lipid（0.77 g/L）,carbohydrate（1.82 g/L）,protein（1.99 g/L）and HHV（114.64 kJ/L）were obtained in mixed culture.（4）Global metabolic regulation studies of Y.lipolytica in response to mixed culture with Chlorella pyrenoidosa（24 hours）showed that 167 differentially expressed genes were identified,with 100 up-regulated and 67 down-regulated.GO enrichment analysis of DEGs showed that DEGs was significantly enriched in 69 terms.Analysis of representative18 GO terms of DEGs showed that the mixed culture had a certain effect on the expression of genes related to aerobic respiration and ribosome,which was mainly down-regulated in the mixed culture.This indicated that aerobic respiration and protein synthesis ability may be inhibited.Global metabolic pathways and KEGG enrichment analysis showed that the effect of mixed culture on the metabolism of Y.lipolytica cells was not obvious;just 11 differentially expressed genes were significantly enriched in Ribosome and One carbon pool by folate.The results showed that mixed culture had only a significant effect on these two metabolic pathways.DEGs which enrichment into one carbon pool by folate metabolic pathway were down-regulated in the metabolic pathway.（5）Global metabolic regulation studies of C.pyrenoidosa in response to the mixed culture with Y.lipolytica showed that 5731 unigenes were significantly altered by mixed culture,with 3593 were significantly up-regulated and 2138 were significantly down-regulated.GO enrichment analysis showed that 3174 significant differentially unigenes were significantly enriched in 251 terms and extremely significantly enriched in 113 terms.Excepting for down-regulated unigenes involved in photosynthesis,protein repair and chloroplast matrix thylakoids,the number of up-regulated genes in the rest of GO terms was significantly higher than that of down-regulated genes.Global metabolic pathways and KEGG analysis showed that differentially expressed unigenes were significantly enriched in a series of central metabolic pathways,including carbon metabolism,nitrogen metabolism,tryptophan metabolism,glycolysis/glyconeogenesis,pyruvate metabolism,citric acid cycle,fatty acid metabolism,oxidative phosphorylation,pentose phosphate pathway,etc.The inhibition photosynthetic reaction of the photosynthetic system in the mixed culture system resulted in the decrease of CO₂ fixation in the dark reaction stage.Carbon metabolism analysis showed that TCA cycle,glycolysis/gluconeogenesis,and pentose phosphate pathways were enhanced although CO₂ fixation pathways were inhibited in the mixed culture system.Nitrogen metabolism pathway analysis showed that the key enzymes involved in nitrate assimilation were up-regulated in the mixed culture system.