The Mechanisms Of Microbiome Succession And Adaption To Environmental Changes In Sand Biofilters For Manganese Removal

Excessive dissolved manganese concentration in groundwater existed in many areas at home and abroad.Biofilm microbiome plays a crucial role in the process of Mn(II)removal.Factors such as influent water quality,operating parameters and filter media affected Mn(II)removal by affecting the formation of biofilms.The technology has been developed well,but still faces some problems,such as the formation of biofilms usually takes a long time at the initial stage of biofilter startup.In addition,a sudden increase of influent Mn(II)might occur during the stable operation of the biofilter,which would affect the Mn(II)removal.Therefore,it is very important to explore the succession of biofilm microbiome in sand biofilters in oligotrophic environments,as well as how to maintain Mn(II)oxidation performance.In this dissertation,high-throughput sequencing and metagenomics methods,combined with ecological network analysis,revealed the adaptive mechanism of microbiome in sand biofilters in response to manganese load.The influence of filter types on the succession of biofilm microbiome was also analyzed.In addition,the key factors affecting microbial community diversity,functional diversity and interspecific interactions were deeply explored.The biological mechanism of co-removal of nitrogen and manganese was revealed.The specific research content and achievements are as follows:Firstly,the self-adaptive mechanism of the biofilm microbial community to the continuous increase of Mn(II)load during the stable operation period was analyzed.The Mn(II)removal efficiency in sand biofilters remained at 99.8%despite an increase in influent Mn(II)from 2 mg/L to 4 mg/L,but significantly deteriorated(50.1%-58.5%)upon increasing the filtration rate.A canonical correlation analysis of the microbial communities indicated that the local Mn(II)concentration and biofilter depth impacted community compositions of biofilms.The dominant species within the biofilms exhibited clear stratification,with simple associations in the upper layer of the sand biofilters and more complex interspecific interactions in the bottom layers.Putative manganese-oxidizing bacteria Hyphomicrobium and Pedomicrobium dominated the microbiomes in different layers of sand biofilters and changed relatively little in abundance when Mn(II)and filtration rater increased.The community networks showed that biofilm microbiomes in sand biofilters were resilient to the disturbance of Mn(II)load,primarily via regulating microbial interactions.High manganese loads negatively affected the functional modules for Mn(II)removal.Furthermore,the relatively rare species Candidatus Entotheonella palauensis was identified as a module hub,implying taxa with low abundance can have important roles in maintaining the stability of biofiltration system.In addition,it was found that nutrient condition was an important factor affecting the composition of functional microorganisms by the enrichment culture of manganese oxidizing bacteria from eutrophic conditions.Experiments on co-cultivation of microorganisms and fillers showed that microorganisms can promote the formation of manganese oxides,and the accumulation of manganese oxides enhanced the resistance of biofilters to Mn(II)load.Optimizing the adaptability of the co-cultivation of filter media and manganese-oxidizing microbes is an important strategy to promote Mn(II)removal in sand biofilters.The influence of filter medium type(magnetite and manganese sand)on the Mn(II)removal efficiency and the formation of biofilm microbial communities at the initial stage of reactor startup was further analyzed.In the initial stage,the Mn(II)removal rate of manganese sand biofiler accounted for 40%-91.2%because the manganese sand had a strong physichemical function.There was no Mn(II)removal in magnetite sand biofilter.Although the accumulation of manganese oxides in the manganese sand biofilter was much higher than that in the magnetite sand biofilter,Mn(II)removal rate of magnetite sand biofilter was higher than that of the manganese sand biofilter after32 days,because the functional microbial community in the magnetite sand biofilter was cultured.Magnetite sand biofilter and manganese sand biofilter had similar Mn(II)removal rates(94.13%and 99.16%)over stable operation for 80 days.Mn(II)removal rates at different depths in the manganese sand biofilter significantly changed with operation time,and the filter in the upper layer made the largest contribution to Mn(II)oxidation once operation had stabilized.As the biofilters run,the number of shared OTUs in two filters was increased.The microbial community structure gradually converged,and the community structure of the upper filter material was more similar.Manganese-oxidizing bacteria with high abundances were identified including:Pseudomonas,Hyphomicrobium,Pedomicrobium and Leptothrix.Hyphomicrobium and Pseudomonas were dominant Mn OB in manganese sand biofilter and magnetite sand biofilter,respectively.Species-species co-occurrence networks indicated that the microbiome of manganese sand biofilter had more complex correlations than that of magnetite sand biofilter,implying that biofilter medium substantially shaped the microbial community in the RSBF.A hybrid biofilter medium with magnetite sand and manganese sand may therefore be best in rapid sand filtration for Mn(II)oxidation.The effects of NH₄⁺concentration on the Mn(II)removal efficiency and the succession of functional microorganisms were explored.The mechanism of co-removal of Mn(II)and low concentration of NH₄⁺in sand biofilters was revealed based on metagenomics.The Mn(II)removal rate in magnetite sand biofilter varied between 38.18%to 88.08%.It accounted above 99.05%without ammonia in manganese sand biofilter and decreased to 57.38%after 3.5 mg/L influent NH₄⁺adding.The correlation analysis indicated effluent Mn(II)was positively related with influent NH₄⁺and negatively related with p H.Nitrification was the main way for ammonia nitrogen conversion in sand biofilters.The maximum nitrogen removal rate of magnetite sand and manganese sand biofilter reached 33.89%and 35.32%,respectively.The enrichment of anammox bacteria suggested that the anammox process was the main cause of nitrogen loss.In addition to nitrifying and anammox bacteria,the sand biofilters had high abundances of complete ammonia oxidizing bacteria(comammox),whose ammonia monooxygenase protein(amo A)all belonged to the clade A.Magnetite sand was facilitated to the enrichment of comammox microorganisms.Hyphomicrobium spp.,a putative manganese-oxidizing bacterium,was abundant in the biofilter.Its relative abundance decreased with the operation.Leptothrix was positively correlated with the operating time.The environmental pressure caused by the influent NH₄⁺might be the main reason for the enrichment of Leptothrix.Metagenomic analysis showed that there were abundant manganese-oxidizing proteins in the biofilters.Among the ten known manganese-oxidizing proteins annotated based on metagenomic,the abundances of Mox A,Cue O and Cot A proteases were relatively higher and evenly distributed in the biofilter,was not affected by ammonia concentration.The above research results indicated that the biofilm microbiomes in sand biofilters responded to environmental changes by changing the community diversity and the complex interspecies relationship.In addition,the different characteristics of filter media can be used to control the colonization of functional microbiome.