High-efficient Nitrogen Removal By Mixotrophic Chlorella And Molecular Regulatory Mechanism Of Enhanced Nitrogen Metabolism Under Light Induction

Nitrogen-rich industrial wastewater treatment is the world-wide challenge in industrial,traditional methods of physical and chemical methods are facing the bottlenecks such as low treatment efficiency,high cost,no economic returns,and the easily caused secondary pollution.Microalgae-based wastewater treatment system can effective achieve the nutrients removal from wastewater with the coproduction of high-value algal biomass,which is expected to be a new method in wastewater treatment and resource recovery.In this study,green microalgae Chlorella pyrenoidosa was used as the candidate strain to analysis the nitrate removal from high nitrate industrial wastewater,while the transcriptiomic analysis was further used to clarify the molecular mechanism for nitrogen metabolism of mixotrophic and heterotrophic Chlorella.And then the pilot demonstration technology for high-efficient nitrate removal by mixotrophic Chlorella based on the 10 m³photo-fermenter platform was establied,which clarified the anti-bacterial contamination characteristics and effluent safety in photofermentation system.Subsequently,the effect of light quality on the nitrate removal and protein production of mixotrophic Chlorella was further investigated and optimized in shake flask and 50-L photo-fermenter,along with the transcriptomic analysis used to elucidate the molecular mechanism of nitrogen metabolism induced by light quality in mixotrophic Chlorella.Finally,the application of photo-fermentation Chlorella for the high ammonium-sality industrial wastewater treatment was further investigated.The main research results are as follows:（1）The nitrate removal and protein production of Chlorella under heterotrophic and mixotrophic mode was compared and analyzed in 50-L photo-fermenter.Results showed that the nitrate consumption rate(3,465 mg L^-1d^-1),protein content（39.35%DW）and pigmetns content(12.09 mg g^-1)of Chlorella under mixotrophic mode was increased by 80.55%,16.6%and 40%,but the starch content decreased by 29.4%compared with the hetertrophic mode,which inducated that mixotrophic mode significantly promote the nitrate assimilation,protein and pigments biosynthesis,but inhibited the starch biosynthesis.The results from transcriptomic revealed that the genes eccoding key enzymes involved in GDH pathway and GS/GOGAT pathway was up-regulated for the enhanced nitrate assimilation induced by warm white light of photofementation Chlorella in comparision with heterotrophic mode;the genes eccoding key enzymes of glucose phosphate translocation enzyme（PGM）and amylase（GLGB）was down-regulated for inhibiting starch biosynthesis to decrease the carbohydrate content.Meanwhile,the genes eccoding key enzymes of fatty acid biosynthesis involved in fatty acid synthase（Fsb D）,3-coenzyme-reductase（Fab G）,enoyl reductase（Fab I）,and protein biosynthesis involved in glutamate synthase,glutamine synthase,diaminopimelate decarboxylase and glycine-glyoxylate transaminase was up-regulated to increased the content of fatty acid and protein in algal cells.These results indicated that the warm white light in mixotrophic mode has the obvious advantage in nitrate removal and protein biosynthesis in comparison with heterotrophic mode.（2）The axenic batch culture of photo-fermentation Chlorella in 10 m³ photo-fermenter platform was performed for the high nitrate synthetic wastewater treatment.Results showed that the average NO₃^-removal rate of 5,770 mg L^-1 d^-1 and biomass productivity of 11.06 g L^-1d^-1 achieved in 10,000-L photo-fermenter.Meanwhile,the average NO₃^-removal rate of 6,080mg L^-1 d^-1 and biomass productivity of 18.14 g L^-1 d^-1 could also be achieved under the semi-continuous culture with the bacteria contamination in 10,000-L photofermenter,which is the highest level of current reported,indicating the strong characteristic of anti-pollution in the photofermentation system.16S r DNA sequencing was applied to illustrate the variation of microbial community diversity under under the semi-continuous culture with the bacteria contamination.Results elucidated that the strains Bacillus nealsonii,Elizabethkingia and Pseudomonas mosselii were the dominant strain under different culture phase,Bacillus nealsonii was the first report in the algae-bacteria system.Meanwhile,relative abundance of pathogenic bacteria Elizabethkingia and Pseudomonas mosselii as initial time point of 99.88%and 86.89%was decreased to the level of 2.12%and 9.37%,indicated that the abundance of pathogenic bacteria significantly decreased after the photo-fermentation Chlorella,along with the enhanced safelty in effulent.（3）The effect of light quality on the nitrate removal and protein biosynthesis by mixotrophic Chlorella was investigated based on the shake flask culture system.Results verified that the mixed LED of red and blue was more conducive to cell growth,blue LED was benefit to nutrient assimilation,protein and polyunsaturated fatty acid production;while red led was demonstrated to promote the biosynthesis of starch and monounsaturated fatty acid.Therefore,the blue LED was demonstrated to be the optimal light quality for protein production in mixotrohic Chlorella.Subsequently,the effect of enhanced blue LED induction on the nitrate removal and protein biosynthesis by photofermentation Chlorella was further explored in 50-L photo-fermenter.Results from the two batches of constant blue LED ratio induction verified that increased the blue ratio from 6.3%to 9.2%could obviously increase the protein content from 36.06%to 38.9%,but the decrease the biomass productivity from7.59 g L^-1 d^-1 to 6.59 g L^-1 d^-1.While phase-increased of the blue ratio from 6.3%-9.2%-21.5%can simultaneously enhance the protein content and shorten the growth lag phase,along with the maximum protein content and productivity of 53.18%and 3.59 g L^-1 d^-1 achieved under W+B（B:6.3%-9.2%-21.5%）,which were 37.3%and 41.3%higher than those under W+B（B:9.2%）.（4）The warn white light was used as control,results from the analysis of phase-increased blue light ratio in mixotrophic Chlorella showed that glucose(16.7 g L^-1 d^-1),phosphate(405mg L^-1 d^-1),and nitrate(4,185 mg L^-1 d^-1)consumption rate,as well as the content of protein（53.18%DW）,amino acid(457.2 mg g^-1)and pigment（1.59%DW）under W+B（B:6.3%-9.2%-21.5%）was increased by14.4%,20.78%,58.8%,35.08%,28%and 32.06%with the warm white light.The differentially expressed gene of photo-fermentation Chlorella at same time point under the two modes showed that the genes eccoding key enzymes involved in glycolysis of glucose-6-phosphate isomerase（GPI）,phosphofructokinase（PFK）,enolase（ENO）,glyceraldehyde 3-phosphate dehydrogenase（GAP）and TCA cysle of citrate synthetase（CS）,succinyl coenzyme A（SCLA）,succinodehydrogenase（SDH）were up-regulated under blue light for improving the central carbon metabolism with the enhcaned glucose consumption rate to meet the substrate and energy for the rapid cell growth and metabolic process in mixotrophic Chlorella.Meanwhile,the genes encoding key enzymes involved in the glutamine synthetase,glutamate synthetase and glutamate dehydrogenase were up-regulated by blue light to enhace the nitrate assimilation.The genes encoding key enzymes of sphinggolipid delta-deasturase,PLP-depent threonine dehydratese,acetohydroxy acid isomeroreductase,and branched-chain amino acid aminotransferase were also up-regalated to enhance the serine,valine,and isoleucine biosynthesis to increase the protein content.Genes encoding coproporphyrinogen oxidase for catalyzing the synthesis of protoporphyrinogen IX and proporphyrinogen oxidase for catalyzing the synthesis of portophyrin IX,as well as the?-hydroxylase and zeaxanthin cyclooxygenase for catalyzing the synthesis of?-cryptoxanthin and violaxanthin were up-regalated for 0.15～2.0 fold with the enhanced the content of protoporphyrin,lutein and violaxanthin.Meanwhile,the genes encoding photosystem I,photosystemⅡ,cytochrome b6/f complex,F-type ATP synthease and light-harvesting system were up-regalated for 0.11～2.6 fold to promote the photoreaction in mixotorphic Chlorella.These results revealed the molecular mechanism of high-eifficient nitrogen removal and protein biosynthesis and the light-bioeffect induced by blue light.（5）The effect of high-efficient ammonium removal from the high ammonium-high salinity wastewater by photo-fermentation Chlorella was further investigated.Results from the batch culture conducted in the 50-L photo-fermenter with three inner LED lights verified that the high-efficient ammonium removal under photo-fermentation mode were feasible with the average NH₄⁺removal rate of 750 mg L^-1 d^-1and biomass productivity of 11.81 g L^-1 d^-1.Two-phased acclimated culure in the 50-L photo-fermenter can simultaneously improved the ammonium tolerance and removal rate with the highest NH₄⁺tolerance concentration of 3,000mg L^-1,maximum NH₄⁺removal rate of 2,212 mg L^-1 d^-1 and biomass productivity of 20.21 g L^-1 d^-1 were achieved by photo-fermentation of Chlorella,indicated that the acclimated culture can enhance the NH₄⁺tolerance and removal rate,as well as the reduced dilution process,which provides innovative technology for deep treatment treatment and resource recovery of high-ammonium-high salinity industrial wastewater...