| Simultaneous methanogenic and denitrification coupling process(SMD)is a new decarburization and nitrogen removal process,which developed on the basis of traditional anaerobic-anoxic-aerobic combination biological treatment.The expanded granular sludge bed(EGSB)reactor is widely used because of its low sludge production,high treatment efficiency,less occupation area and high operating load.However,the influencing factors of anaerobic biological treatment process conditions are more difficult to control,and the factors affecting SMD coupling process and its microbial community in a single EGSB reactor are not very clear at present.The SMD coupling process performance in a single EGSB reactor was investi gated from four conditions: inoculation sludge,carbon source,carbon-nitrogen ratio and nitrate-nitrogen loading,and its microbial community and functional gene distribution were analyzed by high-throughput sequencing and metagenomics.1)The effects of two different inoculate sludge on SMD coupling process start-up and corresponding microbial community structure and functional genes were studied in the EGSB reactor.The results showed that the removal rate of NO3--N was over 98.6% and that of organic matter was about 65%,but the accumulation of nitrite nitrogen and ammonia nitrogen in effluent is different.Combined with the effect of gas production,the inoculation effect of anaerobic digestion sludge was considered to be relatively good.And through high-throughput sequencing analysis,it can be known that the dominant bacteria were Proteobacteria,Firmicutes,Euryarchaeota,Spirochaetes,after the aerobic activated sludge inoculating reactor was started,the relative abundance of Bacteroides(denitrifying bacteria)was 5.04% and the relative abundance of methanobacterium(methanogenic bacteria)increased from 0.067% to 0.87%.Moreover,the EGSB reactor inoculated with anaerobic sludge was started,the relative abundance of Proteobacteria,Firmicutes,Cloacimonetes,Spirochaetes increased significantly at the phylum level,the relative abundance of Bacteroides(denitrifying bacteria)increased from 0.085% to 4.1% and the relative abundance of Methanobacterium(methanogenic bacteria)increased from 0.15% to 1.18% at the genus level.The metagenomics analysis found that nap A and nar G functional genes were most distributed genes in SMD coupling process before and after start-up of anaerobic digestion sludge inoculation reactor.2)The effects of glucose,lactose,sodium formate,sodium acetate and sodium propionate as carbon source on the SMD process of EGSB reactor were studied.The results showed that sodium acetate was the best carbon source,although the removal rates of COD and nitrate nitrogen removal rates of the five carbon sources were above 85% and 99.5%,the accumulation of nitrite nitrogen and ammonia nitrogen in the effluent was quite different.When sodium acetate was used as carbon source,the nitrite nitrogen accumulation was the least and the ammonia nitrogen concentration was the lowest.Furthermore,the maximum nitrite nitrogen concentration was 0.0212 mg/L and the ammonia nitrogen concentration was less than 10 mg/L.High throughput sequencing and metagenomic analysis showed that the dominant denitrifying bacteria were Pseudomonas,Thauera,unidentified?Spirochaetaceae,Bacteroides,AUTHM297,Petrimonas,vadin BC27?wastewater-sludge?group and the methane producing dominant bacteria was Methanobacterium,Methanosaeta,Methanoregula,when sodium acetate was the carbon source.3)The effects of three different carbon-nitrogen ratios on the SMD coupling process and microbial community structure in EGSB reactor were investigated.The results showed that the ratio of carbon to nitrogen had a great effect on the concentration of ammonia nitrogen in the effluent,at the maximum carbon-nitrogen ratio(23.85),the concentration of ammonia-nitrogen in the effluent was the lowest,the removal rate of COD and nitrate-nitrogen was about 84.57% and 97.90% respectively.High-throughput sequencing analysis showed the dominant bacteria were Proteobacteria(22.57%-46.95%),Bacteroidetes(11.57%-23.77%),Cloacimonetes(0.04%-18.15%)and Thermotogae(0.0003%-11.15%)in the sample of XH,XH3,XH4,XH5;And the relative abundance of Euryarchaeota was 0.7%,4.68%,3.58%,2.64% respectively,and the corresponding dominant bacteria were Sulfuritalea(2.73%),Enterobacter(15.86%),AUTHM297(11.10%),Dechloromonas(13.97%);While Methanobacterium played a major role on methanogenic.At the same time,Metagenomic analysis showed that the abundance distribution of nap A,nar G,nir K,nir S,nor B,nos Z in XH3 samples was 253.47 ppm,148.09 ppm,37.88 ppm,24.42 ppm,26.77 ppm,17.24 ppm,respectively;Moreover,mcr A,a methanogenic functional gene,had the lowest abundance in XH samples and the highest abundance in XH3 samples,reaching 8.34 ppm.4)The effect of nitrate nitrogen volume loading(0.93–3.5(kg/m3·d))on the SMD process and its microbial community in EGSB reactor were investigated.The results showed that the removal rate of COD decreased with the increase of nitrate nitrogen volume load,and the gas production decreased slightly.When the nitrate nitrogen load was 0.93-1.45 kg/m3·d,the best effect was obtained.High throughput sequencing analysis showed that the dominant phylum in each sample was mainly Proteobacteria,followed by Firmicutes,Bacteroidetes,Lgnavibacteriae,Actinobacteriae,Euryarchaeota,Synergistetes,Chloroflexi,Gemmatimonadetes and other phylum.The relative abundance of Proteobacteria decreased from 76.17% to 51.32%,and the relative abundance of Bacteroidtes showed a decreasing trend and the relative abundance of Firmicutes increased from 7.88% to 39.99% with the increase of the NO 3--N load of nitrate nitrogen from 0.93 kg / m3·d to 3.5kg/m 3·d.Furthermore,the dominant bacteria in the BY18 samples at the genus level were Pseudomonas,Halomonas,Thauera,Luteimonas,vadin BC27?wastewater-sludge?group,as well as Methanobacterium.While the result of Metagenomic analysis showed that denitrifying functional genes were most abundant in sample BY18,and the abundance distribution mcr A methanogenic functional genes increased gradually with the increase of nitrate nitrogen volume loading. |
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