Effect Of Low Concentration Nitrate On The Regulation Mechanism Of Aerobic Sludge Granulation Formation And Succession Of Community Structure

Aerobic granular sludge(AGS)is a promising technology for wastewater treatment.It has the advantages of strong resistance to shock loading,higher treatment efficient,less sludge production and low operation cost.However,AGS is also limited by the drawback of long forming time,and the granulation process is also affected by oxygen mass transfer,temperature,p H and other factors.Nitrate,as a widely existing conventional water pollutant,may influence the formation process of AGS,but the specific mechanism has not been reported.In this paper,low concentration nitrate was added in five SBR reactors to explore the regulation mechanism on AGS formation process and the influence on microbial community structure.By monitoring the changes of water quality,AGS particle size,surface morphology,sedimentation performance,biomass,denitrification rate and other physiological and biochemical indicators during AGS formation process,the effect of low concentration nitrate on AGS formation process was explored.Through the detection of EPS amounts and compositions,c-di-GMP concentration and element proportion of AGS,the mechanism of low-concentration nitrate affecting AGS formation process was explored.Finally,by further analyzing the microbial community structure of AGS,the mechanism of low concentration of nitrate on sludge granulation process was explained from the aspect of microbial species succession.The results of apparent monitoring on the granulation process of sludge showed that the addition of nitrate at low concentrations(0-20 mg/L)had two-sided effects on the granulation formation of sludge.On the one hand,the addition of nitrate can accelerate the accumulation of organic matter by sludge and quickly reach the conditions for the beginning of granulation formation,thus accelerating the granulation process of sludge.Moreover,the formed granular sludge had a good appearance.The particle surface was more smooth and regular,and the particles were dense.The AGS surface was mainly dominated by spherical bacteria.However,on the other hand,the continuous addition of nitrate would make the inorganic matter grow too fast,resulting in the high inorganic matter content in the formed AGS,particle instability,dramatic changes in particle morphology,and large cracks on the surface.The debris and body shell of granular sludge are left in the reactor,and the sedimentation performance deteriorates and the biomass is lost.In addition,the formation of particles cultured by pure ammonia nitrogen is slow,but the particle size distribution in the system is more concentrated,and AGS is more dense,which has good settlement performance.In terms of pollutant removal capacity,compared with the culture conditions with pure ammonia nitrogen as the nitrogen source,the addition of nitrate can rapidly strengthen the adaptability of inoculated sludge,and can improve the simultaneous nitrification and denitrification rate of the reaction system,thus helping to improve and stabilize the denitrification capacity of the whole system.Even after the particle disintegration phenomenon occurs,the total nitrogen removal efficiency of the reaction system is not affected.Through the analysis of the mechanism of low concentration of nitrate affecting the process of sludge granulation,it can be concluded that the addition of nitrate can stimulate the secretion of c-di-GMP in the early stage of sludge granulation to promote the synthesis and secretion of EPS,especially the extracellular protein in EPS.At the same time,through the analysis of protein secondary structure,it was found that the addition of nitrate at the initial stage of granulation could also promote the secretion and synthesis of aggregated protein structures such as α-helix,β-sheet and 3-turn helix,which enhanced the aggregation of sludge and accelerated the granulation process of sludge.However,the continuous addition of nitrate may also lead to excessive secretion of EPS,which is easy to block the material transmission channel on the surface of the particles,resulting in the lack of substances required for metabolism in the particles and causing bacterial death.From CLSM,it is also observed that there are more dead bacteria in the center of AGS,which causes hydrolysis and cavitation in the particles,resulting in the disintegration of the particles.In addition,the continuous addition of nitrate inhibited the synthesis and secretion of β-polysaccharides which were beneficial to the stability of AGS structure,and promoted the increase of non-coiled folding and anti-parallel β-sheet/polymer chain,which were not conducive to the aggregation of AGS.In addition,the denitrification effect was low without adding nitrate,and there was sufficient calcium ion in the reactor.Therefore,the crystal nucleus effect of the calcification was obvious.Although the granulation rate was slow,the particles formed were more stable.The community structure analysis of AGS samples showed that the microbial species richness and diversity in granular sludge decreased significantly during the granulation process.Low concentration of nitrate has a positive effect on the richness and diversity of microbial flora,but the difference of nitrate concentration has no significant effect on species diversity and richness.The addition of nitrate can obtain faster community structure succession rate in AGS system,which makes the strains beneficial to sludge granulation quickly become dominant strains,thus accelerating the process of sludge granulation.At the same time,nitrate has a good promoting effect on nir-type denitrifying bacteria,which can not only improve the denitrification capacity of the system,but also accelerate the process of sludge granulation by promoting the production of EPS.In addition,the addition of nitrate can promote the growth of f＿A4b and g＿Leptothrix filamentous bacteria.In the early stage of sludge granulation,these two strains may intertwined to form a relatively close crystal nucleus to promote granulation.In the later stage,excessive EPS on the surface of the particles will hinder the mass transfer function of AGS,so that some of them die and will be dissolved by f＿＿Haliangiaceae with bacterial dissolution function,which makes the particles loose and leads to the disintegration of the particles.After functional prediction of the system,it was found that nitrate played a positive role in promoting GDP-mannose dehydrogenase,glutamate dehydrogenase and other enzymes,which could regulate EPS synthesis.