Non-aerated Algal-bacterial Partial Nitrification Process Regulation And Microbial Interactions

Given global climate change and China’s“dual carbon goals”,synergy between pollution control and carbon reduction is a hot research topic in the field of wastewater treatment.Traditional nitrogen removal processes are unable to meet the current requirements of green and low-carbon sustainable wastewater treatment technologies owing to high energy consumption,high carbon requirements and high greenhouse gas emissions.In recent years,the coupled partial nitrification（PN）and anaerobic ammonium oxidation（Anammox）nitrogen removal process has been a promising application due to their low energy consumption and high removal efficiency.However,the challenges of achieving stable PN,the need for mechanical aeration and the residual nitrate（NO₃^--N）after the Anammox reaction have limited its further application in municipal wastewater treatment.It has been suggested that algal-bacterial PN may be an effective alternative to the PN-Anammox treatment,as it can provide the required reaction substrate for the Anammox process by limiting the growth of nitrifying bacteria through in situ oxygen production by microalgae without mechanical aeration,and further improve the efficiency of nitrogen removal through the assimilation of algae.However,the microbial composition of the algal-bacterial consortia is complex and influenced by various environmental and operational factors,and thus how to ensure the stable algal-bacterial PN,the operation strategy and the related microbial interactions remain to be unclear.Therefore,in this study,algae-bacterial PN systems were constructed to assess the effects of reactor types and operating modes,and operating parameters（solid retention time（SRT）,light intensity,and light source）on the PN performance and related mechanisms,and to investigate the underlying relationships and interactions between the algal and bacterial communities.By characterizing the extracellular polymeric substance（EPS）and morphology of algal-bacterial sludge,the formation process and spatiotemporal heterogeneity of granular sludge in algal-bacterial PN systems were revealed using high-throughput sequencing and multi-omics（i.e.,metagenomic and metabolomics）approaches.The main contents and results are as follows:（1）Algae-bacterial partial nitrification process optimization and system operation.The PN system was constructed by inoculating algae-bacterial mixed sludge solution from the inner wall of the anaerobic reactor effluent pipe in stable laboratory operation under light exposure.The results of the batch experiments showed that ammonia nitrogen（NH₄⁺-N）concentration of 50-75 mg/L,dissolved Oxygen concentration of less than 0.5 mg/L,red light:blue light=5:1 LED lamp as the light source,light intensity of 30μmol m^-2·s^-1 and inoculum concentration of 0.5 g-TSS/L were applied,the best ammonia nitrogen removal and highest NO₂^--N accumulation were achieved.Two different algal-bacterial PN systems（photo sequencing batch reactor（PSBR）and photo up-flow sludge blanket bioreactor（PUSB））accomplished excellent PN.And the nitrite accumulation rates（NAR）in the PSBR and PUSB were92.70±7.81%and 81.44±11.28%,respectively.Microbial activity assay results indicated that ammonia-oxidizing bacteria（AOB）were more active in both reactors compared to nitrite-oxidizing bacteria（NOB）.Scanning electron microscopy observations and microbial community analysis showed that the microbial morphology,diversity,and network in the PUSB and PSBR had different characteristics.Denser algal-bacterial granular sludge was observed in PSBR,whereas looser algal-bacterial flocs were observed in PUSB.Phormidium and Leptolyngbya（two filamentous cyanobacteria）were enriched in PSBR and PUSB,respectively.The higher abundance of Nitrosomonas（AOB）in PSBR and the complete inhibition of Nitrospira were the main reasons for their good PN performance.The microbial network of PUSB had denser interspecific interactions and more cooperative behavior.This research enhances the understanding of the mode of operation or reactor type for the PN in algae-bacterial systems and provides theoretical support for the design of for the design of energy-saving and efficient photobiological systems.（2）Solid retention time impacted algal-bacterial partial nitrification performance and microbial interactions.Results showed that the PSBRs were operated for 140 d at different SRTs（5,10,20,and 40 d）,with a 10 d SRT being the optimum condition for stable NO₂^--N accumulation and its NOB activity being stably suppressed.The higher activity and abundance of NOB under the longer SRTs（20 and40 d）resulted in higher NO₃^--N concentrations in their effluent.It was found that SRTs were closely related to microbial diversity,which was mainly driven by NAR and NO₃^--N concentrations.In addition,functional microorganisms at 10 d SRT were strongly associated with AOB activity and NO₂^--N accumulation,while those at 20 and 40 d were associated with NOB activity and NO₃^--N production.Based on microbial co-occurrence network and metagenomic analyses,stronger competitive microbial interactions,and higher network complexity associated with higher species richness and more connected pairs at the 10 d SRT promoted the formation of stable communities,thus ensuring more efficient partial nitrification.Overall,relatively short SRTs in algal-bacterial system could achieve more stable PN by inhibiting NOB activity,influencing community structure and microbial interactions,and thus establishing a relatively stable and balanced system.（3）Solid retention time impacted the formation of algal-bacterial aggregates for partial nitrification.The results showed that SRTs had a strong influence on the formation and morphological structure of algal-bacterial aggregates.Compared to the longer SRT,the average particle size of algal-bacterial aggregates in the 10 d SRT reached 1.54 mm,which was favorable for sludge settling.Scanning electron microscopy observations showed that the large number of filamentous cyanobacteria in the SRT of 10 d and the sticky EPS on the surface of filamentous cyanobacteria facilitated the attachment of prokaryotic microorganisms.In contrast,the 10 d SRT exhibited lower extracellular polymer substance（EPS）concentrations,but the highest protein-to-polysaccharide ratio（EPS-PN/PS）.High EPS-PN/PS ratio has the advantage of accelerating granule formation.EPS of SRT 10 d contained a high hydrophobic group associated with EPS-PN and a lowerα-helix/（β-sheet+random coil）ratio,which could also facilitate the formation and stabilization of algal-bacterial aggregates.In addition,algal-bacterial aggregates were highly dependent on the composition and succession of filamentous cyanobacteria（unclassified＿o＿＿Oscillatoriales and Phormidium accounted for 56.29%of the identified algae at 10 d SRT）.Metagenomic analysis further revealed that functional genes related to amino acid metabolism were highly expressed within 10d of SRT.This research demonstrates the important role of EPS composition and filamentous cyanobacteria on the formation of algal-bacterial aggregates and uncovered the microbiological mechanisms driving structural and functional changes in algal-bacterial aggregates.（4）Microbial community assembly and interactions in spatial-temporal scale for algal-bacterial aggregates.The results showed that the size of the algal-bacterial granules affected microbial nitrification and denitrification activities.AOB and nitrite reduction activities were higher in larger algal-bacterial aggregates,probably due to their ability to host more Nitrosomonas（AOB）and weaker photosynthesis as well as diverse ecological niches.16S rRNA high-throughput sequencing results indicated that larger algal-bacterial aggregates had higher species richness.Stochastic processes（dispersal limitation and ecological drift）dominated algal-bacterial aggregates community assembly at both temporal and spatial scales by neutral model analysis.Algal-bacterial aggregates act as stochastic collision carriers in the reactor and were formed mainly through stochastic processes.Larger algal-bacterial aggregates had higher stochastic processes,which could be due to larger aggregates providing more ecological niches.Metabonomics analyses suggested that Sphingomonadaceae,Weeksellaceae,and Rhizobiaceae could act as the pioneers of algal-bacterial aggregates,thus allowing further attachment of other microorganisms.Furthermore,these microorganisms such as Sphingomonadaceae,Weeksellaceae,and Burkholderiaceae promoted the formation of large-size algal-bacterial aggregates by influencing amino acid,polysaccharide,and lipid metabolism.This research comprehensively reveals the formation of algal-bacterial aggregates and their effects on PN performance from spatial and temporal scales,providing theoretical support for maintaining a stable and efficient photoautotrophic nitrogen removal wastewater treatment system.Overall,this study improves our understanding of microbial interactions mechanisms in algae-bacterial PN systems.By optimizing the PN performance of algae-bacterial system and revealing the formation of algae-bacterial aggregates and microbial reactions or interaction mechanisms,this study provides a large amount of basic data for the configuration/process and operation optimization of mainstream wastewater nitrogen removal by algae-bacterial ecosystem.