Effect Of Temperature On The Operation Characteristics And Microbial Interaction In Anammox System

Since the advantages of high nitrogen removal efficiency and low energy consumption,anaerobic ammonia oxidation（Anammox）technology,as an alternative to traditional nitrification and denitrification process in the future,has attracted a large number of attentions.However,since the process is easily affected by the external environment,such as temperature,salinity,organic matter,etc.,its practical application still faces many challenges.Therefore,in this study,a systematic study was carried out in the temperature range of5-45°C.This research investigated the effects of temperature change on system performance and sludge characteristics,and comprehensively examined the influence of temperature change on microbial community diversity and community composition in Anammox system,and clarified the relationship between the assembly mechanism of Anammox microbial community and temperature.Besides,molecular ecological network analysis was conducted to reveal the connection relationship between species in Anammox system under gradient cooling and gradient heating processes,and the metabolism interaction in the process of Anammox was further verified by performing metagenomic analysis.The main conclusions are as follows:（1）Anammox systems with gradient cooling（30-5°C）,gradient heating（35-45°C）and constant temperature（35°C）had been established to clarify the system performance and sludge characteristics of different processes.The nitrogen removal rate（NRR）slightly decreased to 2.10 kg N/m³d when temperature dropped to 5℃and the system performance was well;while the NRR rapidly decreased to 0.25 kg N/m³d when temperature increased to 45℃and the Anammox system collapsed.Temperature could regulate the secretion of Extracellular Polymeric Substances（EPS）,and the EPS content of Anammox system fluctuated at 15.95-32.03 mg·g^-1-VSS and 7.22-33.57mg·g^-1-VSS under gradient cooling and heating processes,respectively,which was greater than the fluctuation range under constant temperature.Low temperature of 25-5℃and high temperature of 40℃promoted sludge aggregation;while high temperature of 45℃led to particle disintegration,and more rod-shaped bacteria appeared on the surface of the particles at 45℃;and the particles were in a dynamic process of continuous cracking and formation at constant temperature.（2）Based on 16S r RNA gene sequencing technology,the variations of community structure and community assembly mechanism under gradient cooling,gradient heating and constant temperature were revealed.Both gradient cooling and gradient heating processes enhanced the richness and diversity of the Anammox microbial community.While the relative abundance of Candidatus Kuenenia was suppressed to the minimum of 17.47%at 15℃and 9.13%at 45℃,both of which were lower than the lowest value of 23.31%in the constant temperature process.Denitratisoma was the main heterotroph in the gradient cooling process,and norank＿o＿SBR1031 was the main heterotroph in the gradient heating process,and Denitratisoma and norank＿o＿SBR1031 were in the similar abundance under constant temperature.Based on i CAMP（quantitatively Inferring Community Assembly Mechanisms by Phylogenetic bin-based null model）analysis,it was found that the importance of stochastic ecological processes of both gradient cooling and heating processes showed a downward trend as a whole,and therefore the gradient change of temperature would increase the importance of deterministic ecological processes.（3）Based on the random matrix theory,microbial ecological networks under different processes were constructed to explore the interaction relationship between microorganisms and identify the keystone species in the networks.A certain low temperature（25-15℃）and high temperature（40-45℃）could promote the connection between bacteria and improve the complexity of networks.The subnetworks of Planctomycetota and Patescibacteria became more complex under lower and higher temperature,which indicated that Planctomycetota and Patescibateria occupied an important niche under temperature stress in Anammox community.22 of the26 positively correlated connections in the subnetworks of Anaerobic Ammonia Oxidizing Bacteria（An AOB）under gradient cooling process were all heterotrophic bacteria,suggesting that 25-15℃promoted the cooperation between An AOB and heterotrophic bacteria,while 5℃would inhibit these kind of cooperation relationships.There were more competitive relationships between An AOB and heterotrophs under gradient heating process,which illustrated that An AOB maintained the system stability by inhibiting the role of heterotrophic bacteria in the network.While An AOB lost the competitive advantage at 45℃because of the low abundance,leading to the deterioration of system.In addition,77.19%（44 species）of the 57 keystone species identified in the networks of the three processes were clearly heterotrophic bacteria with extremely low abundance,indicating that heterotrophic bacteria as well as rare species could maintain the stability of network structure under temperature stress.（4）Based on the metagenome technology,the distribution of functional genes and metabolism interaction between species in Anammox system were clarified.Anammox-related genes were slightly inhibited under gradient cooling process,while genes hdh and hzs were severely inhibited under gradient heating process.And it was found that gene hao could replace nir to provide NO in Anammox metabolism to complete nitrogen removal.Both gradient cooling and gradient heating processes promoted the abundance of genes related to most carbohydrate metabolism and amino acid metabolism,and the promotion effect of heating on system metabolic activities was more obvious.Binning analysis was performed based on metagenome data of gradient cooling and gradient heating processes,respectively.By constructing ecological model of the microbial community,it was found that there were consistent gene defects in AMX1 and AMX2 during the gradient cooling and gradient heating processes,and thus various metabolic activities might need to be completed by cross-feeding with symbiotic bacteria,mainly including nitrogen metabolism,carbon metabolism,EPS metabolism,and secondary metabolite metabolism.It was also found that the species involved in the metabolic interaction during gradient cooling and gradient heating processes were different.This study revealed the potential interaction relationship in Anammox community under temperature stress by constructing microbial ecological networks in the processes of gradient cooling and gradient heating.And metagenome technology combined with binning analysis was used to reconstruct the metabolism interaction relationship between Anammox and symbiotic bacteria in the community.This study could provide novel ideas for building a stable and efficient nitrogen removal system,and furthermore provided an important theoretical basis and technical reference for expanding the practical application of Anammox technology.