High-Efficient Removal Of Ammonium By Mixotrophic Galdieria Sulphuraria And Its Molecular Mechanism Of Adaptation To High-Ammonium Industrial Wastewater

The rapid development of industry in China is stressing and threatening environmental protection,advocating sustainable economic development is thus,becoming the national development strategy.In the past few years,the discharge of ammonium-containing industrial wastewater was declining.However,the application of traditional wastewater treatment systems or conventional algal-based wastewater treatment systems cannot meet the demand of high removal efficiency or efficient resource utilization when treating with high-strength ammonium-containing industrial wastewater.In this study,a thermoacidophilic Galdieria sulphuraria（termed as G.sulphuraria in the text）was selected as a bioremediation candidate to explore a high-efficient technology for high-ammonium industrial wastewater(NH₄⁺:3,000～7,000 mg L^-1)treatment.First of all,mixotrophic inoculum was inoculated to high-NH₄⁺industrial wastewater medium for evaluating its growth and NH₄⁺removal efficiency,at the same time,biosafety risk of discharge water was elucidated through evaluating the variation of microbial community diversity under non-sterile culture.Subsequently,scaled-up cultivation was developed in a 5 L photo-fermenter for intensifying bioconversion efficiency of NH₄⁺to algal protein by mixotrophic G.sulphuraria,and eventually enhanced NH₄⁺removal rate and protein production.At last,the underlying mechanism of high-efficient ammonium removal and its adaptation to high-ammonium industrial wastewater by mixotrophic G.sulphuraria was illustrated through untargeted metabolomic analysis,transcriptomic analysis,and the united analysis.The main results can be summarized as follows:（1）The growth and NH₄⁺removal capacity of mixotrophic G.sulphuraria with wild-strain inoculum and high-NH₄⁺acclimated inoculum in sterile and non-sterile high-NH₄⁺wastewater medium was evaluated using flask culture.Results indicated that the highest biomass concentration of 12.85 g L^-1,biomass productivity of 2.12 g L^-1 d^-1,NH₄⁺removal rate of 0.35g L^-1 d^-1,NH₄⁺removal efficiency of 47.83%and protein content of 66.80%DW were attained in high-NH₄⁺acclimated inoculum under sterile wastewater medium.（2）16S rDNA sequencing was applied to illustrate the variation of microbial community diversity under non-sterile photo-fermentation by mixotrophic high-NH₄⁺acclimated inoculum.Results elucidated that the relative abundance of three dominant phylum of Proteobacteria,Actinobacteria and Acidobacteria at initial time point of 39.58%,14.75%and 10.84%was decreased to the level of 21.86%,11.73%and 5.89%,respectively.Moreover,α-diversity analysis indicated that Chao,ACE and Shannon index were decreased after culture,it thus revealed that the rapid cell growth which accelerated NH₄⁺assimilation led to the sharply decrease of p H in wastewater medium,and eventually inhibited the growth of indigenous bacteria.It thus,resulted in a decrease to relative abundance and diversity of microbial community,and lowered the biosafety risk of discharge water.（3）Fed-batch sterile and non-sterile culture in 5 L photo-fermenter was applied to evaluate the capacity of algal growth,NH₄⁺removal and protein production in high-NH₄⁺acclimated inoculum of G.sulphuraria.Results showed that the highest cell density of 3.03×10⁹ cells m L^-1,biomass concentration of 71.68 g L^-1,NH₄⁺removal rate of 1.41 g L^-1 d^-1,NH₄⁺removal efficiency of 100%and protein productivity of 5.54 g L^-1 d^-1 obtained in sterile culture were significantly higher than that in non-sterile culture.It illustrated that photo-fermentation coupled with fed-batch feeding strategy for high-NH₄⁺acclimated inoculum could significantly enhance biomass productivity and NH₄⁺removal rate.At the same time,rich protein content up to 42.57%DW,amino acids content of 46.46～50.28%DW,and total lipids content of21.93～39.10%DW were attained from the harvested algal biomass.（4）Fed-batch sterile culture in a 5 L photo-fermenter was utilized to evaluate the cell growth and its NH₄⁺removal capacity by mixotrophic G.sulphuraria with wild-strain inoculum under high-ammonium industrial wastewater medium(NH₄⁺concentration:1,500 mg L^-1,6,315mg L^-1,and 7,475 mg L^-1).Results indicated that specific growth rate of cells in three batches were 0.49 d^-1,0.18 d^-1 and 0.22 d^-1,while NH₄⁺removal rate were 404.17 mg L^-1 d^-1,46.11 mg L^-1 d^-1 and 269.17 mg L^-1 d^-1;and NH₄⁺removal efficiency was 83.62%,6.56%and 43.21%,respectively.The growth trend of cells and NH₄⁺removal rate was increased at first then declined,it thus,illustrated that a metabolic remodulation of cells were occurred when responding to high-NH₄⁺signal at batch 2,and gradually recovered at batch 3 because of the new homeostasis was constructed after long-term adaptation to high-NH₄⁺environment.In order to reveal the molecular mechanism of adaptation to high-NH₄⁺environment by mixotrophic G.sulphhuraria,untargeted metabolomics was introduced.Changes in metabolites profile of G.sulphuraria in response to and adapting to high-NH₄⁺environment under sterile photo-fermentation were deeply studied.Results illustrated that abundance of lipids-related metabolites such as linolenic acid and octadecenoic acids which could enhance cell membrane fluidity were sharply up-regulated for 2.76～4.13-fold and 1～2-fold at batch 2 and 3,respectively;naringenin and melitric acid which belongs to phenylpropanes were up-regulated for 0.11～0.98-fold and 2.74～3.98-fold at both batches,it thus illustrating that the antioxidant system were activated.Conversely,the abundance of most amino acids,especially glutamate and glutamine that involved in GS/GOGAT cycle and participated in NH₄⁺assimilation directly was slightly down-regulated.As for TCA cycle,the abundance ofα-ketoglutaric acid was down-regulated at first and then up-regulated at batch 3;abundance of aconitic acid and malate was sharply down-regulated for 3.98-fold and 3.36-fold respectively at batch2,then reduced at batch 3,it indicated that the metabolisms of carbon and nitrogen were recovering.These results above demonstrated that metabolic mechanisms related to stress response and resistance were activated when cells were responding and adapting to high-NH₄⁺environment,however,the TCA cycle,pathways related to NH₄⁺assimilation and nitrogen metabolism were going down in this case.（5）The global metabolic regulatory network of G.sulphuraria in response to and adapting to high-NH₄⁺environment under sterile photo-fermentation was deeply analyzed utilizing transcriptomics analysis.Results indicated that encoding genes for the synthesis of serine/threonine protein kinase（STPK）and ANTH domain-containing protein（CALM）in glycerophospholipid synthesis were up-regulated for 0.70～3.99-fold at batch 2 and 3,indicating that the alga improved its resistance against high-NH₄⁺environment through remodeling membrane composition.However,the encoding genes for synthesis of photosynthetic system protein subunit（Psb,Psa）,cytochrome complex（Cytb6）and F-type ATPase in photosynthesis,and magnesium-protoporphyrin IX monomethyl ester cyclase（MPEC）,protochlorophyllide reductase Chl N and Chl B subunits（chl N,and chl B）in chlorophyll synthetic pathway were sharply down-regulated for 1.28～4.99-fold at batch 2 and 3,it thus revealed that a decreased activity of light-harvesting complex and a block of electron transport were occurred,and eventually resulted in the inhibition of light absorption,transfer and conversion in microalgae.As for genes involved in TCA cycle,encoding genes of citrate synthase（CS）were down-regulated for 0.89～1.49-fold at batch 2 and 3,but that of succinyl-Co A synthetase（LSC1）,and malate dehydrogenase（MDH1）were up-regulated for 0.10～2.66-fold during cultivation,they thus indicated that the normal metabolism of TCA cycle was gradually recovered through its adaptation to high-NH₄⁺environment.It was worth noted that encoding genes related to glutamine synthase（gln A）were down-regulated slightly at the initial time point of batch 2（0h-B2）,but up-regulated to 0.80～2.52-fold in the following culture;encoding genes for glutamate synthase（glt S）were down-regulated in the whole culture,the maximum down-regulation was occurred at batch 2 for 3.06-fold,but reduced at batch 3,it indicated that the capacity of NH₄⁺assimilation in cells were gradually recovered during its adaptation to high-NH₄⁺environment.（6）United omic analysis was applied to elucidate the correlation between gene expression and metabolites synthesis.Results indicated that the significant correlation between differentially expressed genes and differential metabolites were mainly involved in lipids（such as linolenic acid,squalene,and methyl jasmonate）,carbohydrates（such as inositol and maltose）,and secondary metabolites（such as nicotine and luteolin）metabolism,but not in amino acids or NH₄⁺assimilation pathway.It thus demonstrated that the molecular regulation corresponding to abiotic resistance was significantly activated,while that related to amino acids synthesis,and NH₄⁺metabolism was going down during the alga was responding and adapting to high-NH₄⁺environment.