Study On The Effect Of AHLs On The Characteristics And Nitrogen Removal Performance Of ANAMMOX Granules And The Regulation Mechanism

Compared with the traditional biological nitrogen removal process,ANAMMOX process has the advantages of saving energy consumption,low cost and low sludge production.However,the high sensitivity to environmental changes of ANAMMOX culture were pointed out obstacles when applied to practical treatment.For example,ANAMMOX granules were easy to float in high-loaded reactors and adverse pH or low temperature resulted in the decrease in the activity of granules and granules disintegration.Therefore,this study investigated the effects of Acylated Homoserine Lactones(AHLs)on the characteristics and nitrogen removal performance of ANAMMOX granules and the regulation mechanism to improve the stability and activity of ANAMMOX granules in the adverse environments by using AHLs,and promote the wide application of ANAMMOX process.This paper explored the relationship between the process of disintegration and structure recovery of ANAMMOX granules under substrate shock and in the recovery period and the release of endogenous AHLs in granules,and clarified the close relationship between the distribution and content of specific types of AHLs and the stability and activity of granules.On this basis,long-term effects of exogenous AHLs on the characteristics of ANAMMOX granules in high-loaded reactors were studied and the strategy of controlling ANAMMOX granules floatation in high-loaded reactor by using exogenous AHLs was put forward.After that,the relationship between AHLs release and ANAMMOX granules characteristics under different pH conditions was investigated,and the regulation of endogenous AHLs on granules under neutral and weakly acidic conditions was proved to be closely related to the biomass density of granules.Moreover,the strategy of using exogenous AHLs to enhance the stability and activity of granules with low biomass density was proposed.In order to further reduce the cost of the technology of using exogenous AHLs to enhance granules stability and activity under adverse environmental conditions,this study developed AHLs analogues(denitrification sludge extracellular polymeric substances)and investigated the effect of enhanced denitrification sludge extracellular polymeric substances(DS-EPS_en)on granules performance under extreme pH shock.Finally,the influence of AHLs on granules properties at low temperature was studied,and the strategy of using exogenous AHLs to enhance the stability and activity of granules at low temperature was proposed.The main conclusions were displayed as follows:The transient high total nitrogen(TN)concentration shock for 24h led to severe deterioration in the nitrogen removal performance of the system and granules characteristics.Compared to the stable period,the TN removal efficiency decreased from 89%to 43%under substrate shock and plenty of granules disintegrated.When the substrate concentration returned to the normal level,the granules stability and nitrogen removal performance could also recovered to the normal level.But the recovery process took a long time.The above changes in granules characteristics resulting from transient high-concentration substrate shock were closely related to the regulation of AHLs-based quorum sensing(AHLs-QS).Substrate shock stimulated the release of abundant N-octanoyl-DL-homoserinelactone(C8-HSL),which resulted in the excessive loosely-bound EPS(LB-EPS)secretion and the significant increase of the ratio of protein to carbohydrate(PN/PS)value,leading to the deterioration of granules stability and the decrease in nitrogen removal efficiency.However,because abundant N-hexanoyl-DL-homoserine lactone(C6-HSL)existed in EPS phase,the content of C6-HSL increased obviously after shock,which was beneficial to maintain granules activity.Substrate shock promoted the release of C8-HSL and led to the excessive secretion of LB-EPS,which could protect the activity of ANAMMOX microorganism in adverse environment,while this selfdefense behavior also resulted in unstable reactor operation performance.Under the high nitrogen load condition,short-term addition of C8-HSL had long-term positive effects on the settleability of ANAMMOX granules and could effectively control granules floatation.C8-HSL controlled effectively the excessive secretion of bound EPS(B-EPS),decreased the PN/PS by 48%and increased the RH of granules by 28%,therefore C8-HSL improved the settleability of granules.On day 150,when the nitrogen loading rates of all reactors were 13.4 kg TN m^-3d^-1,the nitrogen removal rate and nitrogen removal efficiency of the reactor with C₈-HSL(R3)were up to 11.2 kg TN m^-3d^-1and 88%,respectively.C6-HSL increased the activity of the granules by 16%.The long-term effects of C8-HSL on granules settleability and long-term effects of C6-HSL on granules activity may be attributed to sustainable release of endogenous signals induced by exogenous signal.The regulation of AHLs-based quorum sensing(QS)on granules characteristics under adverse pH conditions depended on pH and biomass density.Neutral and weakly acidic pH could stimulate the release of C8-HSL and C6-HSL in high biomass density granules(H-granules).And,the increase in C8-HSL content promoted the EPS production and the increase in PN/PS,which help to maintaining the stable structure of the granules.The increase in C6-HSL content served to maintain the stability and activity of granules,so the H-granules could maintain high stability and activity under neutral and weakly acidic conditions.However,the release amount of AHLs,EPS content and PN/PS value of low biomass density granules(L-granules)decreased,and the stability and activity of granules deteriorated.Under the peracid(pH 5.5)and alkaline(pH 7.5-9.0)conditions,the release of AHLs and the secretion of EPS were independent of the biomass density.Under the neutral and weakly acidic pHs,the generation mechanism of AHLs in L-granules could be activated by short-term exogenous addition of C6-HSL and C8-HSL,causing excessive endogenous release of C6-HSL and C8-HSL,which was conducive to improving the stability and activity of L-granules under adverse weakly acidic pHs.Semi-starvation fluctuation of C/N ratio culture strategy effectively enhanced the characteristics of EPS from denitrifying sludge(DS-EPS),which had the potential to replace purified AHLs to improve the stability and activity of granules under adverse pH conditions.The contents of aromatic protein and polysaccharide in enhanced DS-EPS(DS-EPS_en)significantly increased,which changed the molecular weight(MW)distribution of DS-EPS,so the flocculation efficiency of DS-EPS_enwas significantly improved.The flocculation efficiency of DS-EPS_enwas higher at low pH,while quite low at high pH.So DS-EPS_enimprove significantly the stability of L-granules and H-granules under extremely acidic shock.But DS-EPS_enhad no obvious effect on the stability of granules under extremely alkaline shock.In addition,DS-EPS_encontained abundant available AHLs,which promoted the growth of Candidatus?Kuenenia and the expression of ANAMMOX active genes,therefore DS-EPS_enpromoted the activity of L-granules and H-granules under extremely acidic shock.However,AHLs degraded rapidly under prealkaline conditions,so adding DS-EPS_endid not improve the activity of L-granules under extremely alkaline shock.But,the AHLs in DS-EPS_eninduced the sustainable release of endogenous AHLs in H-granules in alkaline condition,contributing to the increase in the activity of H-granules.Under different operating temperatures,the addition of different concentrations of AHLs inhibitors reduced the content of C6-HSL and C8-HSL of ANAMMOX granules on different degrees,which led to further the deterioration of granules activity and stability at adverse temperatures,and the important role of endogenous C6-HSL and C8-HSL signals in regulating granules stability and activity was confirmed at low temperatures.At the same times,N-(3-oxooctanoyl)-L-homoserine lactone(3OC8-HSL)was released by ANAMMOX granules,which was driven by the low temperatures.The effect of exogenous C8-HSL on the stability of granules was closely related to the operating temperature.When the operating temperature was within the range of 11?16?,the stability of granules could be significantly improved by exogenous addition of C8-HSL.When the operating temperatures of the reactors were 11?23?,exogenous C6-HSL and 3OC8-HSL significantly increased the activity of ANAMMOX granules.This study proposed a new approach to improve the stability and activity of ANAMMOX granules at low temperatures from the perspective of quorum sensing.