Nitrogen Metabolism Mechanism Of Pseudomonas Stutzeri XL-2 And Its Application In SBAR

In this dissertation,a heterotrophic nitrification-aerobic denitrifying bacteria Pseudomonas stutzeri XL-2?GenBank accession number KY435925?has been studied in the laboratory to investigate the heterotrophic nitrification characteristics and aerobic denitrification characteristics of the bacteria,and further use moleculars.Biological and bioinformatics analysis of the P.stutzeri XL-2 denitrification key enzyme genes was performed to clarify the nitrogen metabolism mechanism of bacteria XL-2.Finally,the bacteria XL-2 was put into the sequencing batch airlift reactor?SBAR?and mixed with activated sludge to culture granular sludge.The effect of the bacteria in SBAR promoted particle formation and enhanced biological nitrogen removal.The main conclusions of the paper are as follows:?1?P.stutzeri XL-2 can convert NH₄⁺-N to NO₃^--N under different organic carbon concentrations and different organic carbon types.It is believed that P.stutzeri XL-2 has heterotrophic nitrification properties.The results of PCR amplification showed that there was an ammonia monooxygenase gene?amo?and a hydroxylamine oxidase gene?hao?in P.stutzeri XL-2.Therefore,the heterotrophic nitrification pathway of P.stutzeri XL-2 may be NH₄⁺-N?NH₂OH?NO₂^--N?NO₃^--N.In different oxygen environment experiments,P.stutzeri XL-2 can reduce NO3--N under aerobic and anoxic conditions.PCR amplification results showed that there are Nitrate reductase gene?nap?,nitrite reductase?nir?,nitric oxide reductase?nor?and nitrous oxide reductase genes?nos?in P.stutzeri XL-2,and these four denitrification key enzyme genes can be expressed in fluorescence quantification experiments.Therefore,the denitrification pathway of P.stutzeri XL-2 may be NO₃^--N?NO₂^--N?N₂O?N₂.?2?SBAR1 and SBAR2 reactors were set up and P.stutzeri XL-2 was added to the SBAR2 reactor.In the initial stage of the reactor start-up,the sludge has no fixed form and edge profile and the whole is loose.In the first stage?NH₄⁺-N concentration was 50mg/L?,the proportion of particles with a sludge particle size greater than 0.36 mm in both reactors was less than 50%,and the granulation was incomplete;the second stage?NH₄⁺-N concentration was 80 mg/L?The volume fraction of SBAR1 sludge particles larger than 0.36 mm accounted for 68.2%;the particle volume of SBAR2 sludge particles larger than 0.36 mm accounted for 73.8%and the granulation was complete.At the end of the second phase,the particles in the two reactors were spherical,with smooth surfaces,sharp outlines,and the main surface of the pellets were cocci.At this time,the MLSS of SBAR1 and SBAR2 sludge was 9.2 g/L and 10.2g/L,respectively,and the average SV30 was reduced to 31%and 27%,corresponding to an average SVI of 34 mL/g and 27 mL/g,respectively.From the overall results,the SBAR2 sludge volume is higher than SBAR1,and the sedimentation is also superior.?3?The average removal rate of NH₄⁺-N in the middle and late stages of SBAR1was 96.0%and the average TN removal rate was 86.5%.The average NH₄⁺-N removal rate of SBAR2 was 96.1%and the average TN removal rate was 94.6%.After increasing the concentration of influent NH₄⁺-N,the addition of P.stutzeri XL-2 had almost no effect on the NH₄⁺-N degradation performance of the particles,but it could increase the removal rate of TN.At this stage,the average NO₃^--N accumulation in the effluent of SBAR1 is 6.4 mg/L,while the average NO₃^--N accumulation in SBAR2effluent is only 0.4 mg/L.The addition of P.stutzeri XL-2 may be able to optimize particle denitrification performance in terms of improving particle denitrification.The NO3--N difference in the effluent of SBAR1 and SBAR2 in the second stage is relatively obvious from the first stage.Increasing the influent substrate load may be beneficial to the enrichment of P.stutzeri XL-2.?4?The proportion of Flavobacterium in SBAR1 and SBAR2 sludge increased from 0.8%to 7.1%and from 0.6%to 10.2%,respectively.Flavobacterium is one of the signs of particle maturation,so SBAR2 sludge is more granular.At the end of the second phase,in SBAR1 and SBAR2 sludge,the proportion of filamentous fungi in the genus Hydrogenophaga was 13.4%and 1.8%,respectively;the percentage of filamentous bacteria in the genus Filimonas was 1.8%and 0.7%,respectively.The two genus bacteria participate in the formation of granules in a way that constitutes a skeleton.The bacteria of Acinetobacter,Enterobacter,Shinella,Flavobacterium,Diaphorobacter and Pseudomonas all have reported on heterotrophic nitrification and aerobic denitrification.Comamonas,Hydrogenophaga bacteria can perform anaerobic denitrification.There are autotrophic nitrification bacteria in the two reactor communities,such as Nitrosomonas genus.Both heterotrophic nitrification,autotrophic nitrification,aerobic denitrification,and aerobic denitrification were present in both reactors.?5?The proportion of P.stutzeri XL-2 in SBAR1 cultured sludge was always 0;the proportion of P.stutzeri XL-2 in SBAR2 sludge increased from 0.4%at the end of the first phase to 4.9%at the end of the second phase.This is consistent with the fact that the particle size of SBAR2 granular sludge is generally larger than that of SBAR1sludge,and the concentration of NO₃^--N in the effluent of SBAR2 is relatively low and the total nitrogen degradation efficiency is relatively high.