Exploration Of Changes In EPS Secretion And Related Resistance Genes And Metabolism Levels Of Microorganisms Under Copper Ion Stress

With the development of domestic electroplating and other industries,heavy metal wastewater treatment had become an urgent environmental issue.The traditional microbial method for treating heavy metal wastewater has the advantages of green and low-cost,and its research prospect is still very broad.However,the common dilemma is that heavy metals have widespread toxicity to microorganisms.But it had been pointed out that microorganisms themselves have certain resistance capabilities,such as excessive secretion of extracellular polymeric substances（EPS）or generation of resistance genes and so on.Therefore,this topic is established in the context of biological treatment of heavy metal wastewater.Applying the microorganisms into the treatment of different concentrations of copper-containing wastewater,and the EPS were extracted to analyze the variations in EPS contents and functional groups.The response changes of EPS secretion under different concentrations of copper ions were revealed as such.Finally,transcriptome sequencing was used to identify the differentially expressed genes and further explore the related resistance genes and metabolic levels regulation under copper stress.As such,the response changes of EPS secretion and resistance genes with metabolic level of microorganisms（bacteria and fungi）under copper stress could be explored comprehensively,which could provide reliable technical guidance and theoretical support for microbial treatment of heavy metal wastewater.The main research conclusions of this paper are as follows:（1）Isolation and identification of the strain and the wastewater treatment performance: The fungus Aspergillus terreus 2021,WLL-ISO was registered at Gene Bank（OK577948）.The removal efficiency for 10 and 30 mg/L copper containing wastewater were 100±0.18%and 100±0.30%by the fungus Aspergillus terreus 2021,WLL-ISO,but they were only 38.5±1.14%and 34.04±1.46%by the strain Bacillus sp..It indicated that the fungus showed more efficient heavy metal removal performance.（2）Response changes of EPS secreted by fungus Aspergillus terreus 2021,WLL-ISO:The EPS secreted under 0,10 and 30 mg/L Cu²⁺were 0.64±0.15,0.67±0.08 and 0.76±0.01 g/L,and the PN/PS（ratio of protein（PN）to polysaccharide（PS））ratio of decreased from 0.32± 0.024 to 0.15±0.027.With the rising of Cu²⁺concentration,the PS contents showed the most increased,which enhanced the barrier effect of EPS.The XPS spectrum showed an increase for the peaks of C-C and-C-OH.The FTIR spectrum showed the most enhancement at the peak at 730 cm^-1,1240 cm^-1and 1725 cm^-1,which were identified as the amideⅠandⅡin PN and the C=C bond in PS.And the 3D-EEM indicated that the PN contents have not interacted with copper ions and quenching.It validated that the response of the fungi secreted EPS under copper stress was identified as more PS contents secretion,which could enhance the barrier effect of EPS.（3）Response changes of EPS secreted by strain Bacillus sp.:The EPS secreted under 0,10 and 30 mg/L Cu²⁺were 3.48±0.19,11.38±0.57 and 12.95±0.58 g/CDW.And the stimulation increase of PS content is much greater than that of PN,which indicated that PS was the main response contents for copper resistance.The PN/PS ratio increased from 0.18±0.002to 0.67±0.007 and 0.77±0.052,which indicated the enhanced surface hydrophobicity and promote the strain self-flocculation.The FTIR spectrum showed the most enhancement for the two glycosidic bonds at the peak near 1070 cm^-1and 540 cm^-1.The XPS spectrum showed an most increase for the peaks at around 286.3 e V（-C=O/-C-O-C）under copper stress,which indicated more PN contents in EPS.And the 3D-EEM showed the most weaken of the Peak B（Ex/Em 350/450）,which indicated the quenching of PN contents after reacting with copper.It validated that the response of the strain secreted EPS under copper stress was identified as more PS contents secretion and higher PN/PS ratio,which could enhance the barrier effect of EPS and the surface hydrophobicity to alleviate the copper toxicity to cells.（4）Changes in copper resistance genes:Under the stress of copper ion,26 genes were upregulated while 93 genes were downregulated in Bacillus sp.And it showed that three copper-ion resistance genes were downregulated while the other seven genes were upregulated after analyzing ten differentially expressed genes related to copper resistance.The three down regulated genes are responsible for the expression of three cytochromes of Bacillus sp..It indicated that copper toxicity inhibited the level of microbial oxidative phosphorylation and reduced the efficiency of electron transfer.The most significantly upregulated genes are the active genes of metabolic enzymes related to cell synthesis and pyrimidine biosynthesis,and the copper ions binding and transport genes have also upregulated.It indicated that copper toxicity stress significantly inhibited the cell activity,but still triggered the upregulation of copper resistance genes in microorganisms.（5）Metabolic levels analysis:KEGG and GO enrichment analysis showed that the most significant upregulation under copper stress was the biosynthesis of uridine 5’-monophosphate synthetases（UMPs）,followed by the synthesis of pyrimidine.Upregulation of these two pathways promoted EPS secretion.The KO metabolic pathway analysis showed thatβ- fructofuranan glycosidase is the key enzyme in promoting PS secretion.While cysteine sulfideγLyase is the key enzyme that promotes PN secretion in EPS,which served as a global transcription regulatory factor in the copper resistance reaction,which could promote the secretion of six histidine kinases and the convert of ATP into ADP.Meanwhile,upregulation of pyrimidine and purine metabolism,which could promote the electron transmission and nucleic acid metabolism capacity.And thereby promoted the EPS secretion metabolism levels.