Enrichment Culture And Population Characteristics Of Comammox

Comammox has attracted much attention since it was discovered in 2015.Its discovery broke the traditional two-step nitrification theory and expanded people’s understanding of nitrification.At the same time,the phenomenon that ammonia oxidizing bacteria(AOB)have very low abundance and nitrite oxidizing bacteria(NOB)still have good nitrification performance in some ecological environments is explained.With the in-depth study on the physiological characteristics of comammox,it is found that the bacteria is widely distributed in various habitats.Compared with common nitrifying bacteria,it has higher affinity for ammonia nitrogen,more energy can be generated from the nitrification reaction,and it is more suitable for growth in poor nutrient environment.However,as comammox grows slowly and is difficult to be separated from common nitrifying bacteria,the enrichment process of comammox is difficult.So far,there is only one pure bacterium.In this paper,the enrichment culture of comammox was carried out.Two different types of reactors,sequentially batch membrane bioreactor(SBBR,R1、R2)and sequentially batch reactor(SBR,R3、R4),were used to create a low DO and low ammonia environment,and KAN was added to inhibit the growth of conventional nitrifying bacteria.Ordinary aerobic activated sludge and anammox activated sludge were inoculated for comammox enrichment culture.Changes in the nitrification performance of the system were observed by detecting changes in the effluent trinitrogen concentration.Changes in sludge properties before and after the test were compared by detecting Zeta potential and sludge particle size.Finally,16 S r RNA high-throughput sequencing and q PCR were used to detect changes in the activated sludge microbial community and changes in the abundance and types of comammox.In order to provide theoretical support for the promotion and application of whole-process nitrifying bacteria in the field of sewage biological treatment.The main research results are as follows:(1)During the experimental period,the nitrification performance of the four nitrification systems gradually improved,as shown by a decrease in the concentration of nitrite in the effluent,a gradual increase in the oxidation rate of ammonia nitrogen and a gradual increase in the oxidation rate of ammonia nitrogen.During the same period,the effluent nitrite concentration and the peak concentration of nitrite produced during the reaction were lower in the experimental group with KAN than in the empty control group,indicating that KAN had an inhibitory effect on normal nitrifying bacteria.After system stabilization,the very low concentration of nitrite in effluent from nitrification system under low DO and anaerobic conditions indicated that the nitrification system was dominated by comammox.(2)The sludge Zeta potential decreased after the test,but the experimental group was slightly higher than the blank group due to the addition of KAN.The particle size of sludge decreased after R1 and R2 tests,and increased after R3 and R4 tests,because the two groups of sludge culture methods were different.(3)Compared with the original sludge,the microbial community structure of the four nitrification systems had great changes,and the experimental group had the least amount of OTUs with the original sludge.The microbial community structure of R1,R2,R3 and R4 systems also had great differences.The main bacteria causing the differences were Denitratisoma,Run-SP154 and Methylocaldum.The richness and diversity of the microbial community were also significantly changed from that of the original mud.The richness of the experimental group was lower than that of the original mud,while the richness of the blank group was higher than that of the original mud.The diversity of the R1 and R2 microbial community increased while that of R3 and R4 decreased.(5)The relative abundance of Helicobacter nitrification in the experimental group increased significantly,while that in the blank group decreased,and the relative abundance of AOB in the four nitrification systems was very low,indicating that the nitrification reaction was dominated by comammox.The KEGG database was used to annotate the function of microbial nitrogen metabolizing proteins in the two groups of activated sludge.The results showed that the nitrification reactive enzyme proteins in the experimental group showed an obvious upward trend while the blank group showed the opposite trend.(6)During the experiment,the abundance of R3 system comammox increased steadily,while that of R4 decreased first(0-30 cycles)and then increased.At the end of the experiment,the abundance of comammox in R3 system increased 558 times to 2.59E+05 copies/g,and R4 increased only 3.2 times,indicating that KAN could selectively enrich comammox.(7)The species of comammox changed greatly before and after the test.The main bacterial species of comammox in the raw mud was CMX amo A-OTU2,which accounted for98.94% of the total amount of comammox.The main strain of comammox in R3 is Candidatus Nitrospira inopinata(98.62%).The main strains of comammox in R4 were Candidatus Nitrospira inopinata(60.88%)and CMX(MG387165.1)(38.60%).