Effects Of Temperature,pH And Free Nitrous Acid On Kinetics Of Nitrite Oxidizing Bacteria And Microbial Population Structure

In order to explore the influence of environmental factors on the growth characteristics of nitrite oxidizing bacteria?NOB?,based on the study of the effects of temperature and pH on NOB activity,the research of matrix inhibitors of NOB was further carried out.Using a sequencing batch reactor?SBR?,the gradient increases the concentration of nitrite nitrogen?NO-2-N?to accumulate NOB,and uses NOB-rich activated sludge as the research object,based on metagenomic sequencing,16Sr RNA high-throughput sequencing,real-time PCR and functional gene analysis were used to investigate the changes of the rate of nitrogen oxidation?SNiOR?in different temperatures,pH,free nitrous acid?FNA?and free ammonia?FA?.The nonlinearity fitting of the experimental data was carried out by Monod model,matrix inhibition kinetics model and non-matrix inhibition kinetics model.The effects of temperature,pH,FNA and FA on NOB activity kinetics were investigated,and the fitting results were statistically analyzed.It aims to provide theoretical support for optimizing biological nitrogen removal technology to achieve efficient and stable nitrification.The NOB enrichment test found that under the condition of controlling dissolved oxygen concentration?DO?4 mg·L-1?and temperature equal to 25?,sodium nitrite?NaNO₂?was the only energy source,and the concentration of NO₂^--N was increased by gradient.Fast enrichment of NOB can be achieved.The metagenomic species annotation and abundance analysis showed that Nitrobacter accounted for 38.0%of all bacteria in the NOB enrichment system.In addition,the results of gene sequencing showed that the proportion of the remaining bacteria was less than 5%,indicating that NOB in the sludge microorganisms of the SBR system was dominant after enrichment and culture.The protein sequence was compared with the KEGG nitrogen metabolic pathway database.The protein sequence was compared with the KEGG nitrogen metabolic pathway database.The system mainly carried out nitrite oxidation and denitrification reactions.The nitrite oxidoreductase related to the nitrite oxidation process accounted for 13.5%of the total nitrogen metabolism gene.The effects of different temperatures on NOB activity kinetics were investigated based on the Monod model and statistical analysis was performed.The results show that SNiOR reaches the maximum at 30?,which is 1.31 gN·?gVSS·d?^-1.Statistical variance analysis showed that the Monod equation can better describe the effect of NO₂^--N concentration on NOB activity under different temperature conditions.Based on the variation of the temperature coefficient???in different temperature intervals,it is known that when the system temperature is lower than 25?or higher than 30?,the NOB activity is more sensitive to changes with temperature.Based on 16Sr RNA high-throughput sequencing technology,the changes of bacterial population structure in activated sludge samples under three different temperatures showed that the order of species richness and diversity was:25?>35?>40?.By analyzing the structure of the genus bacterial community,40?is not conducive to the growth and reproduction of the Nitrospira.The microbial correlation network map shows that SNiOR is positively correlated with Nitrospira,the correlation coefficient is 0.95,and Nitrospira is the only influencing factor of SNiOR in NOB enrichment system.Through the analysis of microbial metabolic function by KEGG database,the system's carbohydrate metabolism and energy metabolism decreased during the process of increasing temperature from 35?to 40?.Using nxrA and nxrB gene sequences as primers,based on real-time PCR and functional gene sequencing techniques,the effects of different pH on NOB activity and kinetic parameters of Monod equation were investigated.When the pH is between 6.0 and 7.0,the NOB activity increases with the increase of pH.When the pH is between 7.0 and 8.5,the NOB activity decreases with the increase of pH.The Monod model under different pH conditions can well describe the effect of substrate substrate concentration on NOB activity.The fitting results showed that r_max reached the maximum at pH=7.0,which was 1.134gN·?gVSS·d?^-1,indicating that NOB activity was the best under this pH condition.The results of real-time PCR showed that the nxrA gene copy number was pH_7.5>pH_8.5>pH_6.0 under different pH,and the nxrB gene copy number was:pH_7.5>pH_6.0>pH_8.5,indicating that acidic conditions have a greater effect on the activity of nxrA gene,and alkaline conditions have a greater effect on the activity of nxrB gene.Based on the substrate and inhibition kinetics to determine whether FNA is a true substrate and inhibitor of NOB,it is optimal to use the Aiba model to fit NOB activity under different pH conditions.The fitting results showed that when the pH was equal to 6.5,NO₂^--N was higher than 33.7 mg·L^-1,and FNA was higher than 0.07 mg·L^-1,SNiOR was decreased and NOB was inhibited.When the pH is equal to 7.0,NO₂^--N is higher than 261.8 mg·L^-1,and FNA is higher than 0.21 mg·L^-1,SNiOR is decreased and NOB is inhibited.When the pH is equal to 8.0,NO₂^--N is higher than 995.7 mg·L^-1,and FNA is higher than 0.08 mg·L^-1,SNiOR is decreased and NOB is inhibited.Since the system temperature is constant at 25?,and after eliminating the effect of pH on NOB activity,it is found that the FNA concentration has a strong inhibitory effect on NOB compared with the effect of NO₂^--N concentration on NOB activity,indicating that FNA is the true inhibitor.PCR analysis showed that the nxrA and nxrB genes were present in the NOB-enriched system at low NO₂^--N concentration.Under the condition of high NO₂^--N concentration,the nxrA gene accumulated and the nxrB gene gradually decreased.In addition,based on the non-substrate inhibition kinetics,found that FA inhibited NOB activity inhibition type in line with non-competitive reversible inhibition kinetics.