Impacts And Mechanisms Of Sodium Chloride Salinity On Performance And AnAOB In ANAMMOX-EGSB Reactor

The anaerobic ammonium oxidation（ANAMMOX）process has a broad application prospect in the field of biological nitrogen removal in wastewater due to its advantages of high efficiency,low energy consumption,high nitrogen loading and low sludge yield.The process is suitable for the treatment of wastewater with a high nitrogen concentration.However,industrial wastewater often has high salinity,which is unfavourable for ANAMMOX.Few studies have reported the effects of salinity on Anammox process nitrogen removal performance,particularly the inhibition of bacterium-induced on anammox bacteria and the adaptation mechanism of anammox bacteria（AnAOB）to salinity.In this study,an anaerobic ammonium oxidation-expanded granular sludge blanket（ANAMMOX-EGSB）reactor was employed,and different sodium chloride salinities were set for reasearch.Salinity in the influent was increased from 0 g/L to 15g/L,then further increased to 30 g/L.The effects of salinity on nitrogen removal performance and on the characteristics and microbial community structure of granular sludge in the reactor under different salinity levels were investigated.The effects of increasing salinity on the types and contents of the intact ladderane lipid were studied and was related to the physiological response of AnAOB to salinity.The effects of salinity on the activities of crucial nitrogen-converting enzymes,such as NarGH,NirS and Hzo,were determined.Through a combination of SAA test and enzyme abundance analysis,the impact of salinity on AnAOB was revealed on the basis of biochemical enzymes.Predictive functional profiling based on the COGs and KEGG pathway databases were analysed,and the related proteins and pathways which were greatly affected by salinity were identified by significant difference analysis,which revealed the metabolic mechanisms of salinity on AnAOB.（1）Nitrogen removal performance decreased during the twice salinity increasing,and then recovered with the extension of time.The total nitrogen removal rate was finally restored to over 85%,indicating salinity inhibits the activities of AnAOB,and AnAOB adapts to salinity.Salinity mainly affected the ammonium removal and had little effect on nitrite.Under the saline conditions,the granular sludge of the reactor was ellipsoidal and the surface was uneven.The average particle size increased with running time,but the average particle size of the granular sludge at 30 g/L was lower than at 0 g/L.The total amount of granular sludge EPS under long-term saline conditions were decreased.Moreover,protein content decreased whereas polysaccharide content were increased.Salinity affected the aggregation of granular sludge,as indicated by the decrease in PN/PS ratio,increase in the contact angle of the granular sludge suspension,and increase in the absolute value of the zeta potential.（2）Microbial diversity analysis showed that microbial diversity in the granular sludge decreased,and the microbial species considerably varied under saline conditions.Planctomycetes,Proteobacteria and Chloroflexi were dominant microbial in the granular sludge,and the total abundances of them was above 80%,and various phyla exhibited different abundances under increasing salinity levels.As salinity increased,the abundance of Planctomycetes increased,whereas the abundance of Proteobacteria decreased.However,the abundance of Chloroflexi fluctuated,that is,it decreased at 15 g/L salinity but increased at 30 g/L salinity.Candidatus Kuenenia,Candidatus Jettenia and unclassified Brocadiaceae genera were detected in the granular sludge.These relative abundance increased with the extension of running time.The Candidatus Kuenenia was the dominant genus.Theassociated bacteria provided support for the function of AnAOB under the saline conditions.（3）The method for extracting and detecting intact ladderane lipid from reactor was optimised.No difference in the type of intact ladderane lipid of granular sludge was observed at increased salinity.The relative content of the ladderane lipid I decreased initially and then increased at increasing salinity,indicating that AnAOB had a process of growth and then death.During the process of salinity increasing,the ratio of ladderane lipid II and ladderane lipid III increased with salinity,indicating that the permeability of Anammoxosme membrane decreased.The decreased permeability could prevent water loss and maintain the cell functions.No significant difference in intact ladderane lipid content with two branch chains was observed at increased salinity,and thus indicating that there was no significant change in the compactness of Anammoxosme membrane.（4）Regarded the enzyme activities at the same salinity to the reactor was 100%。The test results of key enzyme activities of nitrogen transformation under different saline conditions showed that salinity inhibited the activity of NarGH enzyme,but the activity could be remitted after the salinity was reduced.Salinity condition had no significant effect on NirS enzyme.Increase in salinity considerably inhibited the activity of the Hzo enzyme.The results of the SAA test showed that the inhibition of SAA_NH4+_-N was higher than that of SAA_NO2-_-N.The SAA_NH4+_-N decreased significantly when salinity decreased,whereas SAA_NO2-_-N showed no significant change.The abundance of NarGH and NirS enzymes in AnAOB and the abundance of ammonium and nitrite transporters increased under long-term saline conditions,reflecting the response of AnAOB to long-term salinity stress.（5）Significant difference analysis was performed for predictive functional profiling based on the COGs database under different salinity levels,and the abundance of down-regulated and up-regulated functional proteins under saline conditions were determined.The down-regulated functional protein characterizes the inhibition of AnAOB by salinity,including intracellular repair and inhibition of energy production.The up-regulated functional protein characterizes the adaptation of AnAOB to salinity,indicating that AnAOB can adapt to salinity by enhancing metabolism,increasing cell membrane stability,and enhancing inorganic ion transport.The analysis of odd ratio was performed for the predictive functional profiling based on KEGG pathway database under different salinity levels,and the types of down-regulated and up-regulated in pathway abundance under different saline conditions were determined.The down-regulated pathway represented the inhibition of salinity on AnAOB.The inhibition of AnAOB was mainly manifested by the weakening of intracellular carbohydrate and lipid metabolism,the blockage of intracellular energy supply and the reduction of membrane transport capacity.Up-regulated pathways indicated the adaptation of AnAOB to salinity stress.AnAOB might adapt to salinity stress by strengthening wall/membrane synthesis,essential cofactors（porphyrins）and energy productivity,enhancing intracellular material transformation and gene repair and changing its structure and group behaviour.This thesis systematically studied the effect of salinity on ANAMMOX-EGSB reactor nitrogen removal performance,granular sludge characteristics,microbial community structure,ladderane lipid,crucial enzymes of nitrogen conversion,COGs functional proteins and KEGG pathways and revealed the effects of salinity on AnAOB and the mechanism by which AnAOB adapts to salinity on the basis of physiological and biochemical processes and metabolism.The results of this paper may provide a theoretical basis for the application of ANAMMOX in wastewater treatment.