【作者】 赵鑫；

【导师】 王秀蘅； 邢德峰；

【作者基本信息】 哈尔滨工业大学 ， 环境科学与工程， 2020， 博士

【摘要】 国内外很多地区地下水存在锰浓度超标的问题,生物滤池除锰是地下水净化中的一项重要工艺。生物滤池生物膜微生物组在除锰过程中发挥重要作用,进水水质、运行参数以及滤料特征等因素会影响生物膜形成及锰去除效能。该工艺在实际运行中已经比较成熟,但依然面临一些问题待优化,例如生物滤池启动初期,生物膜形成所需时间较长;生物滤池稳定运行阶段可能出现进水浓度波动,会影响生物滤池的稳定性。因此探究寡营养环境中生物滤池生物膜微生物群落的演替过程及如何快速、长期维持锰氧化性能显得十分重要。本文利用高通量测序手段及宏基因组学方法,结合生态网络分析,探究了生物滤池对锰负荷的自适应机制,解析了滤料类型对生物膜微生物群落演替的影响,深入挖掘了影响微生物群落多样性、功能多样性及种群互作关系的关键因素,揭示了氮锰共去除的生物学机制。具体研究内容及取得成果如下:首先,解析了生物滤池稳定运行期的生物膜微生物群落对持续增加锰(Mn(II))负荷的自适应机制。当进水Mn(II)浓度从2 mg/L增加到4 mg/L,生物滤池的Mn(II)去除效率仍保持在99.8%,但随着滤速的升高,Mn(II)去除率显着降低(50.1–58.5%)。微生物群落的典范对应分析(CCA)表明,局部Mn(II)浓度和生物滤池深度会影响生物膜微生物群落组成。生物膜的优势物种表现出明显的分层,滤池下层的微生物种间关系比上层更为复杂。假定的锰氧化细菌Hypomicrobium和Pedomicrobium在不同滤层深度处均为优势物种,其相对丰度未因进水Mn(II)浓度或滤速升高而发生明显改变。微生物共现网络结果表明生物滤池生物膜微生物组主要通过调节微生物的相互作用来抵抗Mn(II)负荷产生的干扰,锰负荷升高导致与锰浓度显著相关的模块特征基因数量下降。稀有物种Candidatus Entotheonella palauensis被鉴定为模块中心,这意味着低丰度物种在维持生态系统稳定中起重要作用。此外,通过对锰氧化细菌的富集培养,发现营养条件是影响功能微生物组成的重要因素。与填料共培养的实验表明生物作用促进了锰氧化物的形成,而锰氧化物的积累可以增强生物滤池对锰负荷冲击的耐受力。因此,优化滤料和锰氧化微生物共培养的适配性是强化生物滤池除锰的一个重要策略。进一步分析了滤料类型(磁铁矿和锰砂)对反应器启动初期的除锰效能及生物膜微生物群落形成的影响。锰砂滤料具有较强的物理化学作用,初始阶段锰砂反应器的Mn(II)去除率在40%-91.2%之间,磁铁矿反应器没有Mn(II)去除。虽然锰氧化物在锰砂反应器中的积累量远高于磁铁矿反应器,但在反应器运行至32天,随着磁铁矿反应器内功能微生物富集,其对锰的去除率高于锰砂反应器。反应器稳定运行80天后,两者具有相似的Mn(II)去除率(94.13%和99.16%)。锰砂反应器不同滤层深度处的Mn(II)去除率随运行时间发生明显变化,上层滤料对Mn(II)氧化的贡献逐渐增加。随着反应器运行,两个反应器微生物群落中共有的OTU数量增加,微生物群落结构逐渐趋同,上层滤料的群落结构尤为相似。丰度较高的锰氧化微生物包括:Pseudomonas,Hyphomicrobium,Pedomicrobium及Leptothrix。其中,Hyphomicrobium和Pseudomonas分别是锰砂和磁铁矿反应器中的优势物种。共现网络分析表明锰砂反应器生物膜微生物种间互作关系比磁铁矿反应器更复杂,意味着滤料实质上影响了生物滤池生物膜微生物组形成。因此,磁铁矿和锰砂混合的生物滤池可能是快速启动除锰滤池的最佳方法。再次探究了氨氮浓度对生物滤池除锰效能及微生物组演替的影响,基于宏基因组结果解析了氮锰共去除机制。氨氮加入前后,磁铁矿反应器中Mn(II)去除率在38.18%-88.08%之间波动。未加入氨氮前,锰砂反应器锰去除率为99.05%,进水氨氮浓度为3.5 mg/L左右时,锰去除率降至57.38%,相关性分析表明出水锰浓度与进水氨氮正相关,与p H负相关。硝化作用是生物滤池中氨氮转化的主要途径。在氨氮转化的过程中存在氮损失,磁铁矿和锰砂反应器氮的最高去除率分别达到33.89%和35.32%。厌氧氨氧化细菌的富集暗示厌氧氨氧化过程是氮损失的主要原因。除硝化细菌和厌氧氨氧化细菌外,生物滤池中存在高丰度的完全氨氧化细菌属(comammox),其氨单加氧酶蛋白全部属于clade A分支,研究表明磁铁矿更利于comammox微生物的富集。锰砂滤池可以实现Mn(II)和低浓度氨氮同步去除,Hyphomicrobium spp.是生物滤池中丰度较高的假定锰氧化菌,其相对丰度随反应器运行时间延长而降低。锰氧化细菌Leptothrix与反应器运行时间成正相关,进水氨氮造成的环境压力可能是Leptothrix富集的主要因素。宏基因组分析表明生物滤池中存在丰富的锰氧化蛋白,在注释到的十种已知锰氧化蛋白中,Mox A、Cue O和Cot A蛋白酶的丰度相对较高,滤层深度处无明显差异,且未受氨氮浓度影响。以上研究结果表明生物滤池生物膜微生物组通过改变微生物群落多样性及复杂的种间关系来响应环境变化。此外可以利用滤料类型的特征差异对功能微生物组进行定殖调控。更多还原

【Abstract】 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.更多还原