Inhibition Mechanism Research Of Denitrifying Phosphorus Accumulating Organisms Based On Nitrite As Electron Acceptor

NO₂^-,as the nitrogen removal intermediate product,could serve as the electron acceptor for Denitrifying polyphosphate accumulating organisms（DPAOs）to anoxic phosphrus uptake,but its concentration had adverse effects on the activity of the microorganisms when it reached a certain value.However,the inhibitory effect of NO₂^-,p H value and its protonated product-Free nitrous acid（FNA）,on microbial activity has not been established.Therefore,this project takes NO₂^-type DPAOs as the research object to explore the metabolic characteristics of DPAOs under the condition of gradient addition of NO₂^-as well as the inhibitory effect of NO₂^-on DPAOs and its detoxification mechanism under different p H values and temperatures.With the help of molecular biological means,The effects of NO₂^-,p H value and FNA on microbial activity were analyzed from a comprehensive and multi-angle,in order to provide theoretical explanations for the controversial issues of NO₂^-inhibition,and at the same time to provide solutions for the stable operation of wastewater treatment plants affected by NO₂^-.The main results are as follows:（1）Polyphosphate accumulating organisms（PAOs）and DPAOs were enriched by anaerobic-aerobic alternations and anaerobic-anoxic-aerobic alternations,respectively.After 67days of system operation,DPAOs was successfully enriched in the SBR reactor.In the process of DPAOs acclimation,the inhibition of NO₂^-on DPAOs activity could be reduced by extending anoxia duration and adjusting the dosing mode of NO₂^-to batch dosing,and the dephosphorization rate of DPAOs could be improved.The highest dephosphorization rate of R1 and R2 systems was 60.30%and 64.17%,respectively.High throughput sequencing results show that the bacteria with denitrification and phosphorus removal function in R1 and R2systems are Flavobacterium,Candidatus Accumulibacter,Pseudomonas and Dechloromonas.Flavobacterium was the highest DPAOs in the two systems with a relative abundance of 24.71%and 9.86%,respectively.Meanwhile,the Glycogen accumulating organisms（GAOs）with a high proportion in the R1 and R2 systems are Candidatus Competibacter,with relative abundance of 4.28%and 15.61%,respectively.（2）The effects of NO₂^--N concentration on the metabolic performance of DPAOs showed that the optimal dosage of NO₂^--N in R1 and R2 systems was 20 mg/L and 30 mg/L,respectively,and the R2 system was more tolerantce to NO₂^-.It may be related to the relatively high abundance of GAOs in R2 system,and some evolutionary clays with Denitrifying glycogen accumulating organisms（DGAOs）,NO₂^-/NO₃^-can be used as electron acceptor to carry out endogenous denitrification process and share NO₂^-with DPAOs to reduce the inhibition of DPAOs activity by reducing the concentration of NO₂^--N.Under high concentration of NO₂^--N stress,PHA degradation is mainly used for NO₂^-reduction to detoxify rather than phosphorus absorption.Meanwhile,Glycogen（Gly）level does not rise in hypoxia stage,but drops.It may be converted to Poly-β-hydroxyalkanoates（PHA）to counteract the adverse effects of high NO₂^--N concentrations on DPAOs.The results of gradient NO₂^--N concentration addition showed that when there was no electron acceptor addition or insufficient electron acceptor addition at the anoxic stage,the metabolism of phosphorus uptake was transferred to that of aerobic phosphorus uptake.The post-aeration stage became the main contribution stage of phosphorus uptake.There was also phosphorus uptake at the anoxic stage when there was no electron acceptor addition,which might be related to the metabolism of Flavobacterium.Some of its clades may have metabolic mechanisms similar to those of Tetrasphaera.With the increase of NO₂^--N concentration,the phosphorus removal rate of hypoxia also increased.However,when the concentration of NO₂^--N reached a certain value,the phosphorus absorption activity of DPAOs was inhibited.The phosphorus removal performance of Enhanced biological phosphorus removal（EBPR）system is decreased.（3）NO₂^-stress experiments under different p H values showed that when the initial p H value of hypoxia was about 7.5,DPAOs had the strongest tolerance to NO₂^-,and 20 mg/L NO₂^--N was added to obtain the highest dephosphorization rate of 87.07%.Under this condition,In the anoxic stage of the main reactor,30 mg/L NO₂^--N was added three times,and the dephosphorization rate could reach about 95%.When the p H value is below 7.0,the lower the p H value,the more beneficial the reduction of NO₂^-.When the metabolic activity of DPAOs is completely inhibited,DPAOs generates energy for the reduction of NO₂^-to detoxify through phosphorus release in the hypoxia stage,and the lower the p H value,the higher the phosphorus release amount of hypoxia under the condition of the same concentration of NO₂^--N.When FNA value is used to evaluate the influence of DPAOs activity,attention should be paid to the range of p H value.When p H value is less than 7.5,the smaller p H value is,the larger the calculated value of FNA is,and the more obvious the inhibitory effect on DPAOs is under the condition of the same concentration of NO₂^--N.However,when p H value is greater than 7.5,it is no longer suitable to use FNA value to evaluate the influence on DPAOs activity.Under different temperature conditions,when FNA value is 8.0×10^-4 mg HNO₂-N/L in the range of20℃～30℃,the effect of hypoxia and phosphorus removal is the best.Beyond this temperature range,temperature affects the effect of hypoxia and phosphorus removal by affecting the metabolic activity of DPAOs,and temperature becomes the real reason for limiting the effect of hypoxia and phosphorus absorption of DPAOs.（4）The results of molecular biological tests at different p H values and temperatures showed that Flavobacterium was the highest relative abundance of DPAOs in the two systems,and low p H and high temperature conditions were not suitable for its growth and proliferation.The IIC and IID branches of Accumulibacter are the most dominant clades of the two systems,the IIC branch is the most tolerant of NO₂^-,while the IID branch is suitable for dominant growth in a range of NO₂^--N concentration.In addition,both R1 and R2 systems have achieved good denitrification and phosphorus removal performance under NO₃^-.On the one hand,Flavobacterium may also use NO₂^-/NO₃^-as an electron acceptor for denitrification and phosphorus removal when absorbing phosphorus from hypoxia.On the other hand,there is a synergistic metabolic effect among bacteria.Thauera and Candidatus Competibacter,with denitrification ability,can reduce NO₃^-to NO₂^-and serve as electron acceptor for phosphorus absorption by hypoxia in DPAOs.In conclusion,when exploring the adverse effects of NO₂^-on metabolic activity of DPAOs,only by comprehensively considering the effects of NO₂^-,FNA,p H,temperature and synergistic metabolism among bacteria can we better explain the inhibition mechanism and detoxification mechanism of DPAOs by NO₂^-under different environmental conditions,and provide better ideas for the development of more efficient and energy-saving wastewater treatment.