【作者】 李勇；

【导师】 张建强；

【作者基本信息】 西南交通大学 ， 市政工程， 2020， 博士

【摘要】 湖泊富营养化已成为全球面临的严峻环境问题之一,严重破坏水生生态系统并危及人类饮用水安全。在外源污染物得到有效控制的条件下,湖泊沉积物中磷的释放是引起水体富营养化的重要因素。目前学者主要集中于浅水湖泊富营养化进程中微生物多样性及环境影响因素等方面研究,而在亚深水型湖泊微生物群落结构及其在磷循环过程中的作用研究较少。三岔湖位于长江水系沱江支流绛溪河上游,是成都天府新区重要饮用水源地,属于内源污染、富营养化的亚深水型湖泊。本文以三岔湖为模式湖泊,运用高通量测序、荧光定量PCR、纯培养和室内静态模拟等技术手段,结合生物统计学分析方法,系统研究了水体和沉积物中微生物群落的结构、多样性和丰度及其对主要环境因子的响应机制,分析其在沉积物-水体界面间磷素迁移转换过程中的功能,以期探讨功能微生物对水体富营养化的贡献,为同类型湖泊富营养化的修复和管理提供理论依据。取得的主要成果如下:（1）查明了三岔湖上覆水体和沉积物中磷素形态的时空变化特征。2015～2017年四季上覆水体中总磷（TP）含量的变化范围为0.034～0.227 mg·L^-1。其中,库尾和湖湾区水体TP含量较高,而主要来水区含量较低。季节分布上,春冬季水体TP含量高而夏秋季低。溶解性总磷（DTP）是TP的重要组成部分。溶解性正磷酸盐（SRP）是DTP的主要组成部分,它们的时空变化特征与TP类似。水体中SRP和TP含量均与叶绿素a（Chla）含量极显著正相关（p<0.01）,表明磷是三岔湖水体初级生产力的关键限制性营养元素。沉积物中磷素变化特征:TP含量变化范围为0.243～3.774 mg·g^-1,其含量处于同类型湖泊的上游。原养殖区沉积物TP含量较高,而主要来水区和湖尾区较低。季节分布上,冬秋季沉积物TP高而春夏季低。盐酸磷（HCl-P）是沉积物中磷素的主要成分,有机磷（OP）次之,而氢氧化钠磷（Na OH-P）含量最低。在不同时空采样点,沉积物中有效态磷类别及其对水体磷的贡献呈现不同的变化格局。沉积物磷素的释放是三岔湖水体富营养化的内源污染物。（2）基于16S r RNA基因的Miseq高通量测序技术研究了2017年三岔湖春、夏、秋和冬季沉积物中细菌群落的结构组成、多样性及分布特征,结果发现,三岔湖沉积物中细菌物种多样性丰富,获得9,325个OTUs,归属于细菌域的64个门,259个纲,1310个属。其中,Proteobacteria、Actinobacteria和Chloroflexi门为主要类群。Metastats检验表明,Actinobacteria和Chloroflexi 2个优势门以及hgc I＿clade、Pseudomonas、Sulfuricurvum、Syntrophus、Dechloromonas和Thiobacillus 6个优势属的相对丰度季节性变化显著（p<0.05）。非度量多维标度分析（NMDS）和聚类分析表明,春季和非春季的沉积物细菌群落形成两个明显的特征类群。其中,春季样本类群聚集,而非春季样本类群彼此混杂。Pearson相关性和冗余分析（RDA）表明,溶解氧（DO）、p H值、温度（T）、磷素和总有机碳（TOC）是影响三岔湖沉积物细菌群落结构的主要环境因子。Pearson相关性和线性回归分析表明,沉积物中的优势门（Actinobacteri和Cyanobacteria）、优势纲（Actinobacteria、Cyanobacteria、Flavobacteriia、Alphaproteobacteria和Chloroplast）和优势属（hgc I＿clade、Pseudomonas和Thiobacillus）的相对丰度与上覆水SRP含量极显著正相关（p<0.01）,而优势门Chloroflexi和优势纲Deltaproteobacteria的相对丰度与上覆水SRP含量极显著负相关（p<0.01）。因此,沉积物细菌类群对水体磷素具有良好的响应,其机制可能与微生物溶磷作用有关。（3）2017年三岔湖春、秋季上覆水浮游细菌群落的高通量测序分析显示,三岔湖上覆水体细菌物种多样性丰富,获得9,557个OTUs,归属于细菌域的55个门,64个纲,732个属。其中,Proteobacteria、Actinobacteria和Firmicutes门是优势类群。Metastats检验表明,两个季节上覆水具有共同的细菌类群,但部分细菌类群的相对丰度季节性差异显著（p<0.05或p<0.01）。Pseudomonas和Bacillus优势属在秋季的相对丰度极显著高于春季（p<0.01）,这可能是它们对秋季浮游植物茂盛生长引起的缺磷水环境的响应。主成分分析（PCA）和聚类分析表明,春、秋季上覆水浮游细菌群落形成两个明显的分支类群。Pearson相关性和RDA分析表明,三岔湖上覆水体浮游细菌群落结构主要受磷素、高锰酸盐指数(COD_Mn)、DO和p H值的影响。（4）采用高通量测序技术,以碱性磷酸酶（ALP）基因pho D和葡萄糖酸酶（GDH）基因gcd为分子标记,研究了三岔湖沉积物中有机磷溶磷细菌（OPB）和无机磷溶磷细菌（IPB）的多样性、丰度及其对环境因子的响应。结果发现,溶磷细菌的物种多样性丰富,其中Pseudomonas、Streptomyces和Phaeobacter是OPB的优势属,Rhizobiales、Burkholderiales和Pseudomonadales是IPB的优势目。OPB群落在春、秋季的分布差异显著（p<0.05）,但空间异质性不明显（p>0.05）。IPB群落在湖湾和主要来水区呈现显著季节性变化（p<0.05）。p H、DO和上覆水及沉积物的磷素是三岔湖沉积物OPB和IPB细菌群落的关键影响因子。荧光定量PCR分析显示,OPB pho D基因拷贝数在秋季显著高于春季（p<0.05）,且与上覆水SRP含量显著正相关（p<0.05）。IPB gcd基因拷贝数在春季高于秋季,两季的gcd基因的拷贝数与上覆水SRP含量显著正相关（p<0.05）。因此,含pho D和gcd基因溶磷细菌的群落结构和基因拷贝数的变化与上覆水SRP含量变化之间存在时空耦合关系。OPB和IPB可能在三岔湖沉积物-水体界面磷素迁移转化过程中发挥重要的作用。（5）采用选择性培养法,从三岔湖沉积物中分离得到具有稳定溶磷活性的细菌共82株,并对其分类学地位进行了初步鉴定。39株OPB分布于5个属,其中Acidovorax sp.SWWO1711作为OPB鲜见报道。43株IPB分布于9个属,其中Caryophanon tenue SWSI1715和Rhodococcus aetherivorans SWSI178作为IPB鲜见报道。菌株SWSO1719、SWWO1721、SWSI1728、SWSI1734和SWSI1719为潜在的新分类单元。多相分类研究表明,菌株SWWO1721为Spirosoma属的一个潜在的新种,将其命名为三岔湖螺状菌（Spirosoma lacussanchae sp.nov.）。（6）采用液体摇瓶实验和室内静态模拟实验方法研究了分离菌株的溶磷特性。43株IPB能溶解磷酸钙,释放出溶解性磷酸盐（DIP）并使培养液p H值降低。其释磷能力存在差异,释磷量为1.79～102.77 mg·L^-1。分离菌株溶磷特性受培养液DIP含量和p H值的影响。分离菌株的释磷量与p H值成极显著的负相关曲线关系（p<0.01）,释磷量符合指数函数的释放特征。大部分IPB菌株培养液投加后,显著促进沉积物中HCl-P的分解和释放（p<0.05）。39株OPB能溶解卵磷脂,释磷量为0.47～70.91 mg·L^-1。其溶磷特性受培养液DIP含量和ALP活性相互作用的影响。部分OPB菌株培养液投加后,显著促进沉积物有机磷向无机磷的转化（p<0.05）。三岔湖溶磷细菌在沉积物磷释放过程中发挥重要作用。综上,三岔湖水体富营养化与上覆水和沉积物中微生物在磷循环过程中展现的生态功能相关。三岔湖沉积物中的溶磷微生物资源丰富,其结构组成与环境因子密切相关,在沉积物-水体界面磷的迁移转化过程中发挥重要作用,对水体富营养化有重要贡献。更多还原

【Abstract】 Eutrophication of lakes,causing severe damages to aquatic ecosystems and threatening the safety of drinking water,is a serious issue of the environment in the world.Without external pollution source,the release of phosphorus in sediment is an important factor causing eutrophication of a lake.The current studies mainly focus on the microbial diversity and environmental factors in the process of shallow lake eutrophication.The compositions of microbial communities and their roles in the phosphorus cycles of sub-deep lakes have been rarely investigated.The Sancha Lake,studied in this paper,is located in the upper reaches of Jiangxi River,a tributary in Tuojiang River of the Yangtze River system.This lake serves as an important drinking water source for the Tianfu New Area of Chengdu City and is a sub-deep lake with internal pollution and eutrophication.With the Sancha Lake as the study object,the structure,diversity and abundance of microbial communities in water and sediment and their response mechanism to major environmental factors were systematically studied in this paper by high-throughput sequencing,fluorescence quantitative PCR,pure culture technique,indoor static simulation,bioinformatics,and so forth.The roles of microorganisms in the interfacial transfer and transformation processes of phosphorus between sediment and water were analyzed to explore the contribution of functional microorganisms to eutrophication of water and to shed light on the restoration and management of similar lakes undergoing eutrophication.The primary results are presented as follows:（1）The temporal and spatial variation characteristics of phosphorus compounds in the water and sediment of the Sancha Lake were verified.From the year of 2015 to 2017,the total phosphorus（TP）contents in the water body were in the range of 0.034 to 0.227 mg·L^-1.The TP contents in the reservoir tail and bay area were higher,and those in spring and winter were higher.Dissolved total phosphorus（DTP）is an important component of TP,and DTP mainly consisted of dissolved orthophosphate（SRP）.The temporal and spatial variation characteristics of DTP and SRP were similar to those of TP.The contents of SRP and TP in the water body were significantly positively correlated to the content of chlorophyll a（Chla）（p<0.01）,indicating that phosphorus was the crucial determining nutrient for the primary productivity in the Sancha Lake.On the other hand,the TP contents in the sediment were in the range of 0.243to 3.774 mg·g^-1,higher than those of similar lakes.The TP content in the original culture area was higher,and those in winter and autumn were higher.The phosphorus in the sediment mainly consisted of phosphorus hydrochloride（HCl-P）,followed by organic phosphorus（OP）,and the content of sodium-hydroxide-type phosphate（Na OH-P）was the lowest.The types of available phosphorus and their contribution to water phosphorus varied under the different temporal and spatial sampling conditions.The release of phosphorus from sediment was the internal pollution source in the Sancha Lake.（2）The structural composition,diversity and distribution characteristics of bacterial communities in the sediment of Sancha Lake during spring,summer,autumn and winter in the year of 2017 were probed by Miseq high-throughput sequencing technology based on the 16S r RNA gene.The results showed that the diversity of bacterial species in the sediment was high,and 9,325 OTUs acquired were acquired belonging to 64 phyla,259 classes and 1310 genera.Proteobacteria,Actinobacteria and Chloroflexi phyla were dominant.Metastats test results showed that the relative abundances of dominant phyla Actinobacteria and Chloroflexi and dominant genera hgc I＿clade,Pseudomonas,Sulfuricurvum,Syntrophus,Dechloromonas,and Thiobacillus changed significantly against seasons（p<0.05）.The results of non-metric multidimensional scale analysis（NMDS）and clustering analysis showed that the bacterial communities in sediment in spring and other seasons formed distinct characteristic groups.The groups of sediment sampled in spring aggregated,but those sampled in other seasons were mixed with each other.Pearson correlation and redundancy analysis（RDA）results showed that dissolved oxygen（DO）,p H,temperature（T）,phosphorus and total organic carbon（TOC）were the dominant environmental factors affecting the bacterial community structure in the sediment of the Sancha Lake.The linear regression analysis results showed that the relative abundances of dominant phyla（Actinobacteri and Cyanobacteria）,dominant classes（Actinobacteria,Cyanobacteria,Flavobacteriia,Alphaproteobacteria,and Chloroplast）and dominant genera（hgc I＿clade,Pseudomonas and Thiobacillus）were significantly positively correlated with the SRP content in the overlying water（p<0.05）.However,the relative abundances of dominant phylum Chloroflexi and dominant class Deltaproteobacteria,were significantly negatively correlated with the SRP content in the overlying water（p<0.05）.Therefore,the bacteria in sediment sensitively responded to phosphorus in the water,and the mechanism may be related to the phosphorus dissolving function of microorganisms.（3）The high-throughput sequencing analysis results showed that the diversity of the bacterial communities in the overlying water of the Sancha Lake during the spring and autumn of 2017 was high,and 9,557 OTUs were obtained,belonging to 55 phyla,64 classes and 732genera.Among them,Proteobacteria,Actinobacteria and Firmicutes were the dominant groups.Metastats test results showed that the same bacterial groups were observed in the overlying water during both seasons,but the difference was considerable（p<0.05）.The relative abundances of dominant genera Pseudomonas and Bacillus in autumn were significantly higher than those in spring（p<0.01）,respectively.This should be attributed to the response of the vigorously growing phytoplankton in autumn to the phosphorus deficient water environment.Principal component analysis（PCA）and clustering analysis results showed that two distinct cladistic groups of bacterial communities in the overlying water were formed during the spring and autumn.Pearson correlation and RDA analysis results showed that the distribution of bacterial communities in the overlying water of the Sancha Lake was mainly affected by phosphorus,permanganate index(COD_Mn),DO and p H.（4）The diversity and abundance of organophosphorus dissolving bacteria（OPB）and inorganophosphorus dissolving bacteria（IPB）in the sediment of Sancha Lake and their response to environmental factors were studied by high-throughput sequencing with the molecular markers of alkaline phosphatase（ALP）gene pho D and glucosidase（GDH）gene gcd.The results showed that the diversity of phosphorus dissolving bacteria was high.Pseudomonas,Streptomyces and Phaeobacter were the dominant genera of OPB.Rhizobiales,Burkholderiales and Pseudomonadales were the dominant orders of IPB.The distributions of OPB bacterial communities during spring and autumn were significantly different,but the spatial heterogeneity was not obvious.The IPB bacterial communities in the bay and inlet areas presented significant seasonal changes.The p H,DO and phosphorus in the overlying water and sediment were the critical factors influencing OPB and IPB bacterial communities in the sediment of the Sancha Lake.Fluorescent quantitative PCR analysis results showed that the copy number of OPB pho D gene in autumn was significantly greater than that in spring（p<0.05）,and the copy number was positively correlated with the SRP content in the overlying water（p<0.05）.On another hand,the copy number of IPB gcd gene in spring was greater than that in autumn,and the copy numbers in both seasons were significantly positively correlated with the SRP content in the overlying water（p<0.05）.Therefore,the changes in community composition and absolute abundance of phosphorusdissolving bacteria containing pho D and gcd genes were coupled with the changes of SRP content in the overlying water.OPB and IPB may play an important role in the transport and transformation process of phosphorus at the sediment-water interface of the Sancha Lake.（5）A total of 82 bacterial strains with stable organic and inorganic phosphorus dissolving activities were isolated from the sediment of Sancha Lake by selective culture,and these strains were categorized by taxonomy.39 OPBs,including rare Acidovorax sp.SWWO1711,were assigned to 5 genera.43 IPBs,including rare Caryophanon tenue SWSI1715 and Rhodococcus aetherivorans SWSI178,were classified into other 9 genera.Strains SWSO1719,SWWO1721,SWSI1728,SWSI1734 and SWSI1719 were potential new types.The results of polyphasic taxonomy showed that SWWO1721,denoted by Spirosoma lacussanchae sp.nov.in this paper,should be a new potential species of Spirosoma genus.（6）The phosphorus dissolving characteristics of the strains isolated were studied by experiment with flasks and indoor static simulation experiment.Calcium phosphate could be dissolved by 43 IPB strains to release dissolved inorganic phosphate（DIP）,and the p H of the culture medium was reduced.The phosphorus-releasing abilities of these strains were different,with phosphorus releasing amounts of 1.79 to 102.77 mg·L^-1.The phosphorus dissolving characteristics were affected by the DIP content and p H.The DIP content was significantly negatively correlated with p H,and the release process was consistent with exponential functions.Lecithin could be dissolved by 39 OPB strains,with phosphorus releasing amounts of 0.47 to 70.91 mg·L^-1,and their phosphorus dissolving characteristics were affected by the interaction between DIP content and ALP activity.The culture solution of most of the IPB strains could significantly promote the decomposition and release of HCL-P in the sediment（p<0.05）.In contrast,the dosage of the culture media of some IPB strains could significantly facilitaed the transformation of organic phosphorus in the sediment into inorganic phosphorus（p<0.05）.The phosphorus dissolving bacteria in the Sancha Lake played an important role in the release of phosphorus from sediment.In summary,the eutrophication in the Sancha Lake was related to the ecological functions of microorganisms in the overlying water and sediment during the phosphorus cycle.The sediment of the Sancha Lake was rich in phosphorus dissolving microorganisms,of which the structure and composition were closely related to environmental factors.These microorganisms played an important role in the transfer and transformation of phosphorus at the sediment-water interface,and contributed greatly to water eutrophication of the Sancha Lake.更多还原