Fast Start-up And Performance Maintenance Of Denitratation And Its Coupling With Mainstream Anammox

Energy saving and emission reduction of urban sewage treatment facilities is very important in the context of carbon neutralization.The partial denitratation-anammox（PD-AMX）process has great advantages in reducing aeration energy consumption,carbon source demand and sludge production.It is one of the most promising technologies in the upgrading and reconstruction of urban sewage treatment plants in the future.At present,the anoxic zone in the sewage treatment plants using A²O process could be upgraded to PD-AMX process to achieve shortcut biological nitrogen removal.However,how to fast start PD in the anoxic zone and maintain stable performance is the primary problem to be solved in engineering practice.After the establishment of denitratation,the second key problem to be solved is how to enrich and retain anammox bacteria to realize the efficient coupling with PD for nitrogen removal.For these two issues,this paper conducted four parts of research as the follows.Given the guidance role of the metabolic characteristics in the development of targeted process control methods,a highly stable denitratation community was enriched from activated sludge by controlling C/N and p H,and the apparent characteristics of carbon and nitrogen conversion in typical cycles were investigated.Then,metagenomics and metatranscriptomics were integrated to identify the functional strain of acetotrophic denitratation,and its metabolic model was constructed based on its draft genome.Finally,the molecular mechanism of carbon and nitrogen metabolism and potential physiological benefits were analyzed by genome-centric metatranscriptomics.The results showed that in the feast stage with sufficient external carbon source,acetate was rapidly consumed with 65%of acetate stored in microbial cells in the form of polyhydroxyalkanoates（PHA）,and the accumulation rate of nitrite was 87%;After the depletion of acetate,PHA began to degrade to provide electrons for nitrate reduction.Thauera aminoaromatica TJ127 was the main contributor responsible for the uptake of acetate,the synthesis of PHA and the reduction of nitrate in the community.Although this strain contains a full set of denitrifying enzyme coding genes（nar G,nap A,nir S,nor B,nos Z）,the expression levels of nitrite reductase coding gene nir S and its electron transporters（complex III and cytochrome c）were very low at the feast stage,which may be the main reason for the high accumulation of nitrite.Further studies showed that the activities of nitrite reductase and its electron transporter was regulated by nitrate.When nitrate decreased below 2.6±1.1 mg N L^-1,nitrite reduction was significantly triggered.The genome of the strain also contains five copies of PHA synthase coding gene（pha C）and it was highly expressed in the feast stage,which may be the main reason for the high accumulation of PHA.Since the synthesis of PHA requires the consumption of reducing power and Acetyl-Co A,the energy generated by oxidative phosphorylation in the feast stage was mainly（69%）used to uptake and activate acetate,and the reducing power was preferentially allocated to carbon storage rather than nitrite reduction.This unique reducing power and energy distribution characteristics make the denitratation species to obtain more carbon sources at the cost of fewer electron acceptors in the feast stage(27.4 g COD mol^-1-e),which may be the main reason for their competitive advantage in coping with the alternation of feast/famine under the condition of nutrient fluctuation.According to the metabolic characteristics of denitratation species obtained above,a method based on nitrogen metabolism regulation to accelerate the start-up of denitratation was proposed and its feasibility was verified.Firstly,the regulation of nitrification intermediate hydroxylamine（HA）on denitrifying nitrogen metabolism was explored.It was found that HA could combine with the periplasmic cytochrome c to restrict the transfer of electrons from complex III to cytochrome c,so that the decrease of electron consumption rate of complex III was much greater than that of complex I,resulting in higher inhibition of nitrite reductase activity and more electrons flowing to nitrate reductase.Thus,the nitrite accumulation efficiency of activated sludge was improved.Then,the long-term effects of three levels of HA(0,0.5 and 2.5mg N L^-1)on the start-up of denitratation reactor were further compared.The results showed that the continuous addition of 0.5 and 2.5 mg N L^-1 can shorten the start-up time by 1/2 and 2/3,respectively.Further,combined the high-resolution genotypic information of species obtained through metagenomic binning with the phenotypic analysis,it was found that the continuous addition of HA promoted the proliferation of the sequential phenotype Thauera aminoaromatica TJ127.Although this strain had similar anoxic yield coefficient and lower maximum growth rate compared with the denitrifers in activated sludge,it showed higher tolerance to HA exposure.Especially,its apparent growth rate will be higher than that of the concurrent phenotype denitrifiers in activated sludge when the HA concentration is higher than 1.7 mg N L^-1.Therefore,the long-term targeted regulation of electron distribution between NOx reductases can promote the enrichment of sequential phenotype denitrifiers with priority to nitrate reduction,thereby accelerating the start-up of denitratation.Considering the carbon storage and consumption characteristics of denitratation in the feast/famine stages,the effects of carbon source types and quality on carbon metabolism were further investigated,and a method based on carbon metabolism regulation to maintain the high-efficiency performance of denitratation was proposed.The results showed that when the carbon source was changed from acetate to methanol or glucose,the denitratation performance immediately declined,and the effective denitratation could not be achieved after long-term operation.When acetate was replaced by propionate or butyrate or their mixture,the stable nitrite accumulation can be maintained by reducing the anoxic feast/famine ratios.In addition to butyrate-added reactor,high-efficiency performance with nitrite accumulation rate and nitrate removal rate higher than 80%could be achieved by adjusting C/N ratios.The optimal C/N ratio of volatile fatty acids（VFAs）was inversely proportional to the number of carbon atoms,that is,butyrate>mixed acids>propionate>acetate.The PHA components synthesized by denitrifiers were related to the parity of carbon atoms of VFAs,that is,propionate was mainly converted into PHV;acetate or butyrate was mainly converted into PHB,while the mixed VFAs were converted into a mixture of PHB and PHV.The results of metagenomic analysis showed that the abundance of Thauera aminoaromatica in the propionate-added or butyrate-added reactor was decreased,while other denitrifying strains with the ability of PHA synthesis were enriched（such as Zoogloea caeni TJ49,Zoogloea sp.TJ39 and Azospira restricta TJ56）.In contrast,the abundance of Thauera aminoaromatica was increased in the reactor fed with mixed acids,which indicated that the presence of acetate may promote the utilization of propionate and butyrate by Thauera aminoaromatica.Therefore,selecting VFAs that are easy to synthesize PHA as carbon source,improving the proportion of acetate in VFAs and maintaining a low feast/famine ratio are crucial to maintain the high efficiency of denitratation.Based on the key mechanism of rapid start-up and stable operation of denitratation obtained above,a method to realize the rapid was proposed and its feasibility was verified.Firstly,by comparing with the performance of the directly started reactor,it is found that the reactor started with sidestream effluent can achieve better benefits,including shorter time to achieve efficient denitratation,more stable operation performance of PD-AMX,higher total nitrogen removal rate,and higher contribution rate of anammox to total nitrogen removal.In addition,sidestream enhancement is conducive to the enrichment and maintenance of denitratation bacteria（Thauera）and anammox bacteria（Ca.Kuenenia）,thereby improving specific denitratation activity and specific anammox activity of sludge.Finally,the reasons for enhancing the coupling of PD-AMX are revealed:high p H of sidestream effluent is conducive to improving nitrite accumulation efficiency,and the soluble microbial products（SMPs）also promote the anammox activity.Notably,the enhancement of anammox activity by SMPs was obviously depedent on the density of anammox bacteria.It was effective during the initial start-up stage when the density of anammox bacteria is less than 5×10⁸ copies L^-1.Thus,this role was similar to the effect of quorum sensing induced by the signal molecules in the sidestrem effluent.At the later stage of start-up process when the density of anammox bacteria is higher than5×10⁸ copies L^-1,the enhancement effect was mainly attributed to the continuous input of free anammox bacteria in the sidestream effluent,which could be classified as the quantitative enhancement.Therefore,it is a feasible strategy to enhance the efficient coupling of PD-AMX with sidestream effluent.Overall,the above results provided new insights for solving the bottle-neck problems in the application of mainstream PD-AMX process,which would provide theoretical support for the upgrading of biological nitrogen removal process in the context of carbon neutralization.