Bacterial Diversity In Biological Tannery Wastewater Treatment Process And Bioaugmented Nitrogen Removal

Biological wastewater treatment technology refers to the adsorption or degradation of pollutants in wastewater by biological physiological characteristics and metabolism.Bacterial communities present in activated sludge or biofilm are responsible for most of the carbon and nutrient removal from sewage and thus represent the core component of biological wastewater treatment system.Therefore,study of bacterial community structure and function in sewage treatment system is conducive to improving treatment process and efficiency.However,previous studies most focused on the bacterial community in aerobic or anaerobic unit at DNA level,which provided inaccurate information on metabolism of bacterial community.Meanwhile,biological nitrogen removal is the main component of biological wastewater treatment technology,whereas traditional nitrogen removal performs with poor efficiency and high operation cost.Bacteria capable of simultaneous heterotrophic nitrification and aerobic denitrification at the same time and space,provide new insights in biological nitrogen removal.In this work,polymerase chain reaction-denaturing gradient gel electrophoresis?PCR-DGGE?and Illumina high-throughout sequencing were combined to detect the diversity,structure,function and metabolism of bacterial community among tannery wastewater treatment at both DNA and RNA levels.A total of 9 isolates were screened from activated sludge.Among them,the characteristic and mechanism of nitrogen removal by strain A2 was studied and bioaugmented nitrogen removal by A2 was conducted to evaluate its potential in practical application.The results were listed as follows:?1?Chemical oxygen demand?COD?in sewage decreased along with the treatment of fatty wastewater and the process could stably remove 96.22%and 99.64%of COD and ammonium,respectively.The quality of the effluent could meet the first-order discharge standard of China.Compared with fatty wastewater,due to the carbon lackness in subsequent anoxic/oxic/oxic process,only 88.23%of COD and 88.95%of ammonium were removed after biological treatment of synthetic wastewater.?2?PCR-DGGE and Illumina high-throughout sequencing results indicated that Proteobacteria dominated all units of the integrated wastewater treatment system,followed by Bacteroidetes,Firmicutes,Actinobacteria,Chloroflexi and[Thermi],while their relative abundances were different among them.Quantative PCR suggested that copies of bacterial 16S rRNA gene in all samples ranged from 4.22×10⁸ to 4.93×10¹¹ copies/mL,among which anoxic tank and oxic tank possessed the highest 16S rRNA gene copies,further demonstrating that prokaryotic microorganisms played pivotal roles in biological wastewater treatment,especially in core units.?3?DNA and RNA-based Illumina high-throughout sequencing results revealed that bacterial community diversity showed no significant differences between DNA community and RNA community?P>0.61?,while RNA-based bacterial community structure significantly differed from DNA-based structure?F=13.14,P<0.001?.The significant distinctions between DNA community and RNA community resulted from the relative abundance of Proteobacteria,Firmicutes and Chloroflexi.Within Proteobacteria,Nitrosomonas and Nitrosococcus were only detected in RNA-based community.Furthermore,function and metabolic activity analyses of bacterial community in activated sludge found that metabolism and membrane transport were the main function of bacterial community and the functions were significantly different at both DNA and RNA levels?F=3.14,P=0.0142?,indicating that DNA-based bacterial community was not always keeping metabolic avtivity.Most of the bacterial community kept dormant or low activity among the integrated wastewater treatment system,while Proteobacteria and Firmicutes kept potential metabolic activity,suggesting that those two phyla played critical roles in wastewater treatment.Moreover,quantative PCR indicated that bacterial community were more active in homogenize tank and oxidation ditch than in anoxic tank,oxic tank and secondary oxic tank.Correlation analysis revealed that Proteobacteria and Firmicutes were both positively correlated with COD and ammonium,which played important role in shaping bacterial community,further indicating those two phyla were responsible for COD and ammonium removal.?4?According to the high-throughout sequencing resluts,a total of 9 bacteria that could perform hetertrophic nitrification and aerobic denitrification were isolated from activated sludge and all of them were affiliated to genus Pseudomonas within phylum Proteobacteria after identification of 16S rDNA.Among them,Pseudomonas sp.A2 could remove 97.99%of ammonium and 100%nitrate under optimum conditions,that is,sodium succinate as carbon source,pH of 7,temperature of 30 ^oC,C/N of 10:1 and shaking speed of 160 rpm.No nitrite was accumulated during heterotrophic nitrification,whereas the accumulations of nitrate and nitrite were observed during nitrification and aerobic denitrification by Pseudomonas sp.A2,respectively.Ammonium was preferentially utilized to achieve simultaneous nitrification and denitrification and then successively utilized nitrite and nitrate for denitrification by Pseudomonas sp.A2 in the ammonium-nitrite-nitrate mixed N-source.The addition of nitrite could inhibit the nitrification,nevertheless,nitrate could promote the denitrification rate and eliminate the inhibiton of nitrite.Complete genome sequencing showed Pseudomonas sp.A2contained genes encoding ammonia monooxygenase,nitrite reductase and nitrate reductase.Therefore,two different ammonium metabolic pathways might exist in Pseudomonas sp.A2.One is heterotrophic nitrification by oxidizing ammonium to nitrite and nitrate.The other is oxidizing ammonium to nitrogenous gas directly via hydroxylamine.?5?Pseudomonas sp.A2 was immobilized with sodium alginate and polyacrylamide and then inoculated into sequencing batch reactor?SBR?for bioaugmented ammonium removal.Bioaugmented with Pseudomonas sp.A2 could promote the COD and ammonium removal efficiencies.Ammonium removal efficiency of bioaugmented group was stabilized after 20cycles,while the control fluctuated all the time.This indicated that bioaugmented with Pseudomonas sp.A2 could improve the stability of SBR.Meanwhile,ammonium removal efficiency of bioaugmented group was 10.87%higher than control and the COD and ammonium concentration in the effluent of bioaugmented group individually stabilized at 130 mg/L and 20mg/L after 30 cycles treatment,which could meet the second-order discharge standard of China,thus suggesting the good application foreground of Pseudomonas sp.A2 in bioaugrmented nitrogen removal...