海洋生态与环境科学重点实验室知识库

    自2011年以来，我国广西北部湾海域每年都发生大规模球形棕囊藻赤潮，对当地工业、水产养殖业以及核电冷源安全产生了巨大的负面影响。赤潮期间，包括营养物质可利用性在内的环境条件与非赤潮环境显著不同，影响了包括海洋微生物群落在内的局域海洋生态系统。另外，海洋微生物对浮游植物的生长具有反馈机制，在赤潮消长过程中发挥重要作用。到目前为止，关于微生物在球形棕囊藻赤潮消亡过程中的群落动态及生态功能变化研究甚少。因此，本论文聚焦于2017年3月—6月和2018年1月—2月两次北部湾球形棕囊藻赤潮事件，采用宏基因组技术和宏转录组技术相结合的方法，研究了该海域球形棕囊藻赤潮生消过程中微生物的生物地理分布模式和群落构建，分析了活性微生物群体的结构特征和转录表达，探索了微生物群落在赤潮消退过程中的响应机制。主要发现如下：

1. 通过16S扩增子技术对赤潮期间微生物群落构建的解析，发现不同区域（赤潮区与非赤潮区）、不同水深（表层和底层）、不同微生物组分（浮游和附着）以及不同时间段（暴发期、衰退期和消亡期）微生物群落结构都存在显著差异。赤潮区域的细菌丰度、微生物OTUs丰富度和多样性均显著高于非赤潮区域，且在赤潮阶段的衰退期（4月）达到最高。包括球形棕囊藻丰度、盐度、DO、SiO₃^2-和PO₄^3-在内的环境参数显著影响了微生物群落构建。微生物群落相似度随空间距离增加而减小，表明扩散相关过程驱动微生物生物地理分布。微生物群落构建受环境因子、空间距离和球形棕囊藻丰度的共同驱动，其中环境因子单独解释的微生物群落差异最大。
2. 赤潮和非赤潮区域中微生物群落构建的非随机的共发生网络拓扑结构具有一定的差异性，且赤潮区微生物共发生网络比非赤潮区微生物共发生网络的联系更为紧密，表明赤潮的发生使得微生物网络结构更为复杂。通过网络分析确定了与赤潮相关的5个关键类群（Keystone）：OM60 clade、Sulfitobacter、Oleibacter、Altererythrobacter和Psychrobacter。节点之间的相关性表明，赤潮微生物群落变化可能是由占主要地位的环境选择和少数物种之间的竞争作用所驱动。
3. 结合宏基因组和宏转录组技术探究赤潮发生期和衰退期微生物总群体和活性群体结构特征，发现微生物总群体和活性群体的丰富度和多样性随着赤潮衰退而增加。活性微生物群体结构组成在赤潮不同阶段存在显著差异，Erythrobacter和Candidatuspuniceispirillum等微生物类群在衰退期的转录活性显著增加。Vibrio是赤潮发生期微生物差异表达基因的主要贡献者，与大多数差异表达基因相关。棕囊藻细胞丰度、PO₄^3-含量和DO含量显著影响活性微生物群体结构，表明微生物在转录活性水平响应赤潮水体环境变化，其中Nitrospina、Litoreibacter和Candidatuspuniceispirillum等类群与棕囊藻细胞丰度和囊体直径显著负相关。
4. 宏转录组进一步测序分析赤潮消退过程中活性微生物的转录表达差异，发现赤潮发生期和衰退期的微生物群落显示出不同的代谢潜能，两个时期微生物转录水平显著增加的基因主要参与的代谢通路存在差异。在关键代谢通路（营养盐同化、细胞运动、群体感应和胞外分泌）中，微生物在发生期对L-氨基酸的摄取增加，而在衰退期对磷酸盐、磷酸酯、谷氨酰胺、单糖和多糖等有机物质的摄取和转运增加；运动性和群体感应在衰退期增强，而II型分泌系统的胞外毒素分泌则在发生期和衰退期均高表达。

    研究结果系统刻画了广西北部湾球形棕囊藻赤潮发生到消亡过程中，不同区域、不同水深、不同微生物组分和不同赤潮阶段的微生物的地理分布特征和群落构建；分析了微生物总群体（DNA水平）、活性群体（RNA水平）和功能代谢相关群体（差异表达基因相关微生物）在赤潮发生期和衰退期的结构组成差异；初步揭示了赤潮期间微生物吸收利用无机和有机营养物质的潜在机制以及微生物关键细胞过程随着赤潮的消退而发生的响应变化，为进一步揭示棕囊藻赤潮发生后藻源有机质降解及关键生源要素的循环过程，及微生物群落的环境适应机制和生态功能提供了科学依据。

  Since 2011, large-scale algal blooms caused by Phaeocystisglobosa have occurred annually in the Beibu Gulf, Guangxi Province, China, and have resulted in significant negative impacts to local industry, aquaculture and safety of nuclear power cold sources. During bloom, the environmental conditions including the availability of nutrients were significantly different with the non-bloom conditions, which affects the entire ecosystem, including marine microbes. And marine microbes form a feedback mechanism for the growth of phytoplankton, which play fundamental roles in the developmental processes of algal blooms. The detailed ecological functions of microbes during extinctionP.globosa bloom still need to be investigated comprehensively.Thus, in the present study, we focused on two P. globosa bloom events in the Beibu Gulf from March to June 2017 and January to February 2018. Coupled metagenomic and metatranscriptomic sequencing derived from environmental DNA and RNA were applied to elucidate the biogeographic distribution pattern and community construction of microbes, and analyze the structural characteristics and transcriptional expression of active microbial communities from the blooming period to the recession period of P. globosa bloom. The result revealed the ecological responses of the microbial community along with subsiding of the P. globosa bloom.The major findings were listed as following:

  First, with the assistance of 16S amplicon technique, the microbial structure showed significant variation among samples fromdifferent sites (bloom and non-bloom sites), depth (surface and bottom), fraction (free-living and particle-attached), and time (outbreak, recession, and extinction period).The bacterial abundance, microbial OTUs richness, and Shannon diversity in the bloom area were significantly higher than those in the non-bloom area, and increased significantly in April and then decreased in June. The microbial structure was significantly affected by the abundance ofP. globosa and environmental parameters such as salinity, DO, SiO₃^2-, and PO₄^3-. The similarities of microbial community decreased with spatial distance, indicating that dispersal-related processes drive the biogeographic distribution of microbes. The variation of microbial communities was mostly attributed to environmental selection, spatial distance, and the abundance of P. globosa successively.

  Second, the co-occurrence networks of microbial communities in bloom and non-bloom waters differed in terms of structure and composition, and the bloom network had more links and closer relationships between genera than the non-bloom network.The top five genera identified as keystone taxa in the bloom network were OM60 clade, Sulfitobacter, Oleibacter, Altererythrobacter, and Psychrobacter. Network analysis indicated that microbial communities had non-random pattern and driven by high environmental selection and low competitive effect among a few species.

  Third, during the blooming and recession periods of P. globosa, total and active microbial community composition were assessed by integrated metagenomic and metatranscriptomic approach. Our results found the abundance and diversity of the total and active communities increased along with the decline of the P. globosa bloom. Taxonomic analysis uncovered the active communities experienced distinctly different metabolic conditions across the bloom onset and collapse stages.The transcriptional activity of microbial groups, such as Erythrobacterand Candidatuspuniceispirillum increased significantly during the recession periods. The transcriptionally active taxa of Vibrio, which correlated with most functional genes were enriched in blooming period. The active microbial structure was significantly affected bythe abundance ofP. globosa, DOand PO₄^3- content, indicating that the microbes respond to changes in water environment of bloom at the level of transcriptional activity. Among them, the transcript abundance ofNitrospina,Litoreibacter, and Candidatuspuniceispirillumwere significantly negatively correlated withthe abundance ofP. globosa.

  Fourth, the transcriptional analyses indicated the blooming and recession periods of P. globosa had significantly different metabolic potentials. The number of differentially expressed genes mapped to central metabolic pathways varied across communities. In the nutrient assimilation pathways, the uptake of L-amino acid by microbes increased significantlyduring the bloom period, while the utilize and transport of inorganic phosphorus,organic substances, such as phosphate, glutamine, monosaccharides and polysaccharides, increased during the recession period. Motility and quorum sensing were enhanced during the recession period, while the secretion of extracellular protein virulence factors in the Type II secretory system was highly transcribed in the whole period.

  The results of the study systematically depicted the spatiotemporal dynamics of microbial community composition in both FL and PA assemblages from blooming to subsiding of P. globosa blooms. And compared thecomposition differences among total microbial community (DNA level), active community (RNA level), and functional metabolism-related community (differentially expressed gene-related microbes) the blooming and recession periods of P. globosa.The integrated field investigation and in-depth analysis of molecular data preliminary revealed the underlying mechanism of microbial absorption and utilization of inorganic and organic nutrients, and the changes of microbial key cell processes in response to blooming and recession of the P. globosa bloom. It provides a scientific basis for further revealing the organic degradation of algae source and the circulation process of key source elements after P. globosa bloom, as well as the environmental adaptation mechanism and ecological function of microbial community.

目录

第1章文献综述... 1

1.1棕囊藻及棕囊藻赤潮... 1

1.1.1  棕囊藻... 1

1.1.2  棕囊藻赤潮现状... 2

1.1.3  棕囊藻赤潮影响因素... 5

1.2  赤潮与海洋微生物... 9

1.2.1  海洋微生物在赤潮生消过程中的作用... 9

1.2.2  赤潮过程中微生物群落组装的影响因素... 10

1.2.3  棕囊藻赤潮与微生物研究现状... 12

1.3  环境微生物研究及分析方法... 13

1.3.1  宏基因组学... 13

1.3.2  宏转录组学... 16

1.3.3  共发生网络分析... 18

1.4  本论文的研究目标、内容及意义... 19

1.4.1  研究目标... 20

1.4.2  研究内容... 20

1.4.3  拟解决的主要科学问题... 21

第2章棕囊藻赤潮发生过程中微生物群落的时空动态... 23

2.1  前言... 23

2.2  材料与方法... 24

2.2.1  航次信息... 24

2.2.2  样品采集与测定... 24

2.2.3  16S扩增子测序分析... 26

2.2.4  数据处理与统计分析... 27

2.3  结果与分析... 29

2.3.1  棕囊藻与细菌丰度时空分布变化... 30

2.3.2  微生物多样性和群落组成... 35

2.3.3  微生物群落结构差异分析... 42

2.3.4  微生物群落分布的影响因素... 48

2.3.5  共发生网络分析... 55

2.4  讨论... 59

2.4.1  赤潮与非赤潮期微生物群落时空分布模式... 59

2.4.2  环境因子对微生物群落分布的影响... 60

2.4.3  赤潮与非赤潮区域海洋微生物共发生模式... 62

2.5  小结... 63

第3章棕囊藻赤潮消亡过程中微生物活性表达群体结构特征... 65

3.1  前言... 65

3.2  材料与方法... 66

3.2.1  航次信息... 66

3.2.2  样品采集与测定... 66

3.2.3  宏基因组与宏转录组测序分析... 67

3.2.4  数据处理与统计分析... 69

3.3  结果与分析... 70

3.3.1  数据生成和分析概述... 71

3.3.2  赤潮过程中总群体结构特征... 73

3.3.3  赤潮过程中活性群体结构特征... 77

3.3.4  功能相关微生物群体... 79

3.3.5  环境因子与微生物群落相关性分析... 83

3.4  讨论... 88

3.4.1  赤潮消亡过程中微生物总群体与活性群体差异... 88

3.4.2  环境因子对微生物群落分布的影响... 89

3.5  小结... 90

第4章棕囊藻赤潮消亡过程中微生物群落功能代谢变化... 91

4.1  前言... 91

4.2  材料与方法... 92

4.2.1  宏转录组测序分析... 92

4.2.2  数据处理与统计分析... 92

4.3  结果与分析... 93

4.3.1  赤潮消亡过程中微生物群落功能总体分布... 93

4.3.2  微生物功能代谢差异性表达... 95

4.3.3  微生物差异基因富集通路... 97

4.3.4  关键代谢通路差异性解析及其物种贡献... 98

4.4  讨论... 110

4.4.1  微生物对无机和有机营养盐的利用... 110

4.4.2  微生物运动性和胞外分泌功能表达... 112

4.5  小结... 113

第5章结论与展望... 115

5.1  结论... 115

5.2  创新点... 116

5.3  不足与展望... 116

参考文献... 119

附录... 145

致谢... 149

作者简介及攻读博士学位期间发表的论文... 151

缩略词... 153