Research On Nitrogen Metabolic Mechanism Of Aerobic Denitrifier Pseudomonas Stutzeri T13 And Regulation Of Nitrite Accumulation

The application of traditional denitrification is being limited by its strict requirement to dissolved oxygen(DO).The conflict between nitrification and denitrification on different environment requirements causes the unrealizable unification in the same time or space.Aerobic denitrification,as a novel biological nitrogen removal technique,provides a potential possiblity of combining the both processes depending on its high tolerance on DO.Over more than 30 years research on aerobic denitrification,there are still many issues contained in the black box which slow down the technique incubation forwards application,eg.lack undersdanding of molacular mechanism,limited sepecific regulation methods targeting complicate infuence on nitrogen removal performance,as well as the unclear nitrogen metabolism pathways basing on different nitrogen compounds,including ammonium,nitrate and nitrite.The early research on Pseudomonas stutzeri T13 shows that the metabolism pathways of nitrogen compounds(ammonium metabolism and nitrate metabolism)were not clear.And the nitrite accumulated seriously during aerobic denitrification process.These issues were deeply investigated in this thesis.The molecular mechanism of aerobic denitrification and the nitrogen metabolism pathways of the strain T13 were revealed.Basing on aerobic denitrification mechanism,the methods of controlling nitrite accumulation during aerobic denitrification by the strain T13 were proposed.The molacular mechanism of aerobic denitrication was investigated.From analyzing the whole genome of the strain T13,the genes which encode 10 enzymes relating to nitrate metabolism were founded,including nap AB which encodes periplasmic nitrate reductase,nir K and nir S which encode dissimilatory nitrite reductase,nor BC-1 and nor BC-2 which encode nitric oxide reductase,nos Z which encodes nitrous oxide reductase,nar GHI which encodes respiration nitrate reductase,nas AB which encodes assimilatory nitrare reductase,as well as nir BD-1 and nir BD-2 which encode assimilatory nitrite reductase.By analyzing with KEGG database,it's known that the strain T13 has the potential capacity of conducting complete aerobic denitrification,anoxic denitrification and DNRA pathways.The transcription of these 10 genes under different nitrogen metabolism conditions was determined by RT-qPCR,and the nap AB,nir K,nor BC-2 and nor Z were found as the main functional genes involving aerobic denitrication.Noticeably,nitrous oxide maight be the terminal product of aerobic denitrification instead of nitrogen gas because of the inefficient transcription of nos Z.DNRA would help the microbes to obtrain available nitrogen source by transforming nitrate to ammonium if nitrate exists as the sole nitroge source.By contrast,it wouldn't be conducted in the presence of ammonium.In addition,because of lacking the Amo and Hao genes,the strain is unable to conduct heterotrophic nitrification process.The nitrogen metabolism pathways of the strain T13 using different nitrogen source and electron acceptor(ammonium,nitrate,ammonium + nitrate)was analyzed by stoichiometry method.Mass balance calculation of nitrogen shown that the strain T13 utilized ammonium completely through assimilation process.Meanwhile,ammonium assimilation was considerably affected by initial ammonium concentration and C/N.99% of ammonium assimilation rate was realized with initial ammonium concentration of around 100 mg/L and C/N of 10.When nitrate was used as the sole nitrogen source,the microbes obtained nitrogen source through DNRA which could transform nitrate to ammonium,and got extra energy from aerobic denitrification on the basis of oxygen respiration.46.44% and 53.56% of nitrate was transformed through DNRA and aerobic denitrification respectively.In comparison,the strain no longer conducted DNRA process for nitrogen source in the presence of ammonium.All of the nitrate was devoted to aerobic denitrification in the result of 79.51% TN removal rate.The essential reason of nitrite accumulation during aerobic denitrification by the strain T13 was investigated.According to the principle of enhancing the quantity and activity of nitrite reductase and optimizing the dynamic reaction balance,the essential factors including DO,nitrogen concentration and biomass quantity were regulated for the purpose of equilibrating both of nitrate reduction and nitrite reduction processes,and improving the nitrite accumulation.High concentration of DO slightly affected nitrate reduction,however seriously inhibited nitrite reduction.DO regulation and immobilization technique could achieve the enhancement of nitrite reduction in time or space,which increased the TN removal to 80.6% and 44.4%,respectively.In specific condition,deceasing nitrate concentration to 100 mg/L reduced the maximum accumulation of produced nitrite,which indirectly impaired its biological toxicity and substrate inhibition as well.As a result,the nitrite accumulation rate was decreased to 29%,and the TN removal rate of 71.4% was realized.Increasing the supply of ammonium from 0 mg/L to 100 mg/L,which enhanced the ammonium assimilation efficiency,raised the nitrate distribution towards aerobic denitrification from 76.4% to 100%.In such condition,the accumulation rate of nitrite decreased from 67.1% to 35.8%,and TN removal through aerobic denitrification went up from 9.3% to 64.2%.