Study On Denitrification Effect,Population Structure And Characteristic Metabolic Enzymes Of Anammox Bacteria

The anaerobic ammonia oxidation(Anammox)bacteria group has the characteristics of extremely slow growth and high requirements for the growth environment.The cell division time is about 11 days,and it is extremely sensitive to the growth environment,so it severely restricts the rapid development of the anaerobic ammonia oxidation processes.Because the growth of Anammox bacteria is very characteristic,it has attracted more and more researchers to study it in various aspects.In this paper,Anammox bacteria were enriched and cultured in a single-stage autotrophic denitrification reactor.During the cultivation process,the stability of anaerobic ammonia oxidation operation was judged by measuring the nitrogen content of its water quality.When the operation reached the stable period,the anaerobic ammonia oxidizing bacteria in the single-stage autotrophic denitrification reactor were qualitatively and quantitatively studied,the bacteria in the reactor were randomly sequenced,and the strains were separated,purified and sequenced.Real-time fluorescence quantitative PCR was used to quantitatively study the anaerobic ammonia-oxidizing bacteria in the reactor and Erlenmeyer flask in different environments.Hydrazine-Oxidizing Enzyme(HZO),a metabolic enzyme characteristic of anaerobic ammonia oxidizing bacteria,was preliminarily purified and the effect of quinone compounds on the activity of hydrazine oxidase was explored.HZO gene was cloned and analyzed and predicted for secondary structure and tertiary structure.The main results of this study are as follows:(1)The anaerobic ammonia-oxidizing bacteria were dark red in color,and were placed in a single-stage autotrophic denitrification reactor.The culture environment was set to 300mg/L of ammonia nitrogen and 400 mg/L of nitrous nitrogen.The removal rate of ammonia nitrogen was 96.8%and the removal rate of nitrous nitrogen was 97.4%after running for about 60 days.According to the nitrogen balance,it is calculated that some NO₃^--N are produced in the removal of NH₄⁺-N and NO₂^--N at the same time,and the proportion of the three is NH₄⁺-N?NO₂^--N?NO₃^--N=1?1.32?0.26.At this time,the Rs(Rs=?NO₂^--N/?NH₄⁺-N)and Rp(Rp=?NO₃^—N/?NH₄⁺-N)of the water samples in the reactor were about1.38 and 0.27,respectively.At this time,it could be regarded as that the single-stage autotrophic denitrification reactor for anaerobic ammonia oxidizing bacteria was successfully started.The anaerobic ammonia oxidizing bacteria were operating well,and the anaerobic ammonia oxidation reaction normally occurred.Under this condition,the next experiment could be carried out.(2)The results of the qualitative study of anammox bacteria in the reactor:the 16s rDNA whole sequence obtained by sequencing was submitted to the NCBI for BLAST similarity retrieval for homology analysis.The species of YS1 and YS2 belonging to anammox bacteria can not be judged,which species of YS3 belongs to can not be judged.The comparison result of YS4 shows that it belongs to Planctomycetales.The results show that there are anaerobic ammonia oxidation bacteria in activated sludge of single stage autotrophic nitrogen removal reactor.After the screening and purification of the total colony in the reactor,the strains isolated and purified on the inorganic medium were sequenced,and the sequencing results were compared in the NCBI.The results of comparison:the aerobic black bacteria purified on inorganic medium HYH belongs to Pseudomonas anguilliseptica,anaerobes YYR and anaerobes brown bacteria YYZ belongs to Diaphorobacter sp.,anaerobes YYH belongs to Streptomyces.On the other hand,the YYB of anaerobic white bacteria purified on organic medium belongs to the genus Serratia plymuthica,and the HYB of aerobic white bacteria belongs to Bacillus velezensis.(3)The quantitative results of the bacteria in single-stage autotrophic nitrogen removal reactor and the quantitative results of the bacteria in conical flask oscillatory culture were studied by fluorescence quantitative PCR(qPCR)analysis of the anaerobic ammonia oxidation bacteria.The results showed that the content of floating bacteria in the bacteria in single-stage autotrophic nitrogen removal reactor was higher,Brocadia was the dominant strain.The single stage autotrophic nitrogen removal reactor was more suitable for enrichment and culture of anammox bacteria.(4)The results of preliminary purification and enzymatic activity of the characteristic metabolic enzyme-hydrazine oxidase(HZO)of anammox bacteria:the optimum temperature for determination of hydrazine oxidase activity at different reaction temperatures was 35?,and the optimum pH at different reaction times was 7.5.By extracting coenzyme Q with n-heptane,the activity of the crude enzyme decreased,and the addition of coenzyme Q had the best effect on improving the enzyme activity,2-hydroxy-1,4-naphthoquinone had a weaker effect on enzyme activity improvement,and anthraquinone-2-sodium sulfonate salt had little effect on enzyme activity.The preliminary purification of HZO was carried out.By DEAE anion exchange chromatography,it was known that the concentration of the hydrazine oxidase in the 0.2 mol/L NaCl eluent increased the enzyme activity by 6.1 times compared with the crude enzyme solution.Ultrafiltration results showed that the molecular weight of purified hydrazine oxidase protein was between 50 and 100 kDa.(5)The results of gene cloning of the characteristic metabolic enzyme-hydrazine oxidase(HZO)of anammox bacteria showed that the sequence of the cloned gene was compared on the NCBI database,and the gene similarity reached up to 99%,which proved that the cloning was successful.SOPMA results of on-line secondary structure analysis showed that enzyme protein contained four structures.Swiss Model results of the three-stage structure analysis showed that the three-dimensional structure model for the amino acid sequence of ammonia oxidase had not been successfully established.The prediction results of signal peptide sequence indicated that the possibility of signal peptide of hydrazine oxidase protein was very low.