Analysis Of The Assembly Mechanism Of Anaerobic Ammonia Oxidation Functional Biofilm On Different Fillers

Excessive nitrogen in the water will have adverse effects on ecological environment and human health.Anaerobic ammonia oxidation（Anammox）is more cost-effective and environmentally friendly when treating sewage with high ammonia concentration and low carbon to nitrogen ratio owing to its no requirement of external carbon source,low power consumption,and less residual sludge.The anaerobic ammonium-oxidizing bacteria（An AOB）involved in Anammox reaction has a long doubling time and harsh living condition,so the filler is used for its enrichment.Numerous studies have been focused on the effects of fillers with different materials and sizes on An AOB enrichment and Anammox reaction,however,the biofilm assembly mechanism is still unclear.In this paper,five fillers are selected,and biofilms attached to these fillers under six sampling times are used as the research objects.The differences in the enrichment of the anammox functional biofilms attached to the five fillers are analyzed from the perspective of the microbial community assembly mechanism.The main conclusions as follows:（1）The succession of microbial community structure revealed that the difference in filler material and size mainly affected the accumulation of microbial biomass.The best filler to enrich the anammox functional biofilm was the cubic modified sponge with a side length of 1 cm,followed by the cubic modified sponge with a side length of 1.5 cm,followed by fiber ball,and then non-woven fabric,while polyurethane sponge performed the worst in enriching the anammox functional microbial community.The average maximum biomass of An AOB on modified sponge A was 1.22×10¹²copies/cm³,which was 2.4,5.4,9.9 and 34.9 times of the corresponding values of modified sponge B,fiber ball,non-woven fabric and polyurethane sponge,respectively.（2）The null model test showed that when the environmental condition was stable and substrates were sufficient,the biofilm assembly was predominated by stochastic mechanisms.When the temperature dropped below the optimum temperature for the growth of An AOB,deterministic mechanisms were the dominant.The CCA correlation analysis between environmental factors and microbial communities indicated that temperature,concentrations of NO₂^--N and NO₃^--N had a greater impact on the biofilm succession than p H and NH₄⁺-N concentration.When facing the shock of the temperature decrease,the microbial community of the cubic modified sponge with a side length of 1 cm performed the best in maintaining the structure stability,followed by the cubic modified sponge with a side length of 1.5 cm,followed by fiber ball,and then non-woven fabric,while polyurethane sponge had the worst ability to resist the same shock.（3）The CCA correlation analyses between the filler functional group absorption strength and filler characteristics with microbial biomass indicated that NH and OH were the most important functional groups affecting the accumulation of biomass on polyester fillers（fiber ball and non-woven fabric）and polyurethane sponge fillers（polyurethane sponge and modified sponges）,respectively.The functional groups of the cubic modified sponge with a side length of 1 cm had the greatest impact on biomass,followed by the cubic modified sponge with a side length of 1.5 cm,followed by non-woven fabric,and then fiber ball,while polyurethane sponge exerts the weakest effect,however,these influences were not significantly correlated（P>0.05）.Contact angle was the main factor affecting the microbial biomass（P<0.01）during the biofilm assembly.While with the attachment of microorganisms,the influence of the specific surface area on biomass decreased,and the density became the main factor affecting the microbial biomass in the end.The attachment of Candidatus Brocadia was mainly affected by the contact angle and the density of the filler（P<0.01）,the attachment of Candidatus Kuenenia was mainly affected by the functional group NH and the density of the filler（P<0.01）,while the selective enrichment of fillers on Candidatus Jettenia was not significant（P>0.05）.（4）The molecular ecological networks of the microbial communities on fillers during the succession of the biofilm indicated that at the initial stage of the biofilm assembly,the microbial interspecies relationships exerting an important impact on the biofilm structure mainly occurred within OTU999（Methanobacterium）,OTU750（Truepera）,OTU577（Truepera）,OTU954（phylum Patescibacteria,class WWE3）and OTU4242（Nitrosomonas）,related to anaerobic digestion,denitrification and nitrification reactions,and the microorganisms with the most complex interactions OTU954,as well as OTU577 and OTU750 only appeared on fiber ball and polyurethane sponge fillers,respectively.Although the drop of temperature changed the microorganisms significantly affecting the microbial community structure,they were still related to anaerobic digestion and denitrification processes.When the biofilm was mature and stable,the interspecies interactions affecting the biofilm structure significantly mainly occurred within OTU406（Candidatus Brocadia）,OTU1316（Limnobacter）and OTU3851（Bacteroidetes＿bacterium＿OLB10）,carrying out Anammox and organic matter degradation reactions,and OTU1316 mainly attached to non-woven fabric.