珠江河网冬季浮游细菌群落结构及其影响因素
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摘要
为了解珠江河网冬季浮游细菌群落结构,探讨影响其群落结构的环境因素,于2017年11月和2018年1月在珠江河网(112.18°E～113.51°E,22.38°N～23.17°N)采集表层水样,借助IlluminaMiSeq高通量测序平台,运用16S rDNA扩增子技术研究珠江河网冬季浮游细菌群落结构,使用R(3.5.2)软件包以及SPSS进行统计分析及作图。结果表明,变形菌门(Proteobacteria)、放线菌门(Actinobacteria)、拟杆菌门(Bacteroidetes)、蓝细菌门(Cyanobacteria)是珠江河网丰度较高的门类,细菌群落优势种群依次为γ-变形菌纲(γ-Proteobacteria)、放线菌纲(Actinobacteria)和α-变形菌纲(α-Proteobacteria)。珠江河网冬季浮游细菌具有较高的菌群多样性(两次样品的Shannon指数分别为6.78±0.29、6.23±0.71), NMDS分析表明,浮游细菌群落结构之间不存在显著的地域差异。Pearson相关性分析表明,温度与γ-变形菌纲(P<0.05)、放线菌纲(P<0.05)以及α-变形菌纲(P<0.01)的丰度显著正相关,pH与放线菌纲(P<0.05)、α-变形菌纲(P<0.01)的丰度以及多样性指数(Shannon:P<0.01; Simpson:P<0.01)显著负相关,溶解氧与α-变形菌纲的丰度显著负相关(P<0.05)。冗余分析(RDA)结果显示,温度、pH是影响浮游细菌群落的主要因子。
Along with the development of economy and the living standard of residents, the discharge of industrial and agricultural wastewater and municipal domestic sewage has a serious impact on the water quality of the Pearl River Delta. Bacterial communities are important components in riverine ecosystem and play key roles in the degradation and transformation of various pollutions in river environment. Bacterioplanktonic community responds to changes in biotic and abiotic factors that are amplified during spring and summer these wet seasons,however, whether communities respond to environmental disturbance in dry seasons remains unknown. In this study, we collected surface water samples from the Pearl River Delta(112.18°E-113.51°E, 22.38°N-23.17°N) in November 2017 and January 2018, using high-throughput sequencing of 16 S rDNA gene amplicons on the Illumina Miseq platform, to investigate the bacterioplanktonic community's composition and their environmental impact factors of the Pearl River Delta in winter. Total DNA was extracted from water samples by using DNA extraction kit(Magen Hipure Spil DNA Kit), and DNA concentration was determined by Qubit? dsDNA HS Assay Kit. The targeted V3-V4 regions were amplified with the primers set(341 f-CCTACGRRBGCASCAGKVRVGAA;806 r-GGACTACNVGGGTWTCTAATCC). The purified PCR products were sequenced on Illumina MiSeq(Illumina, San Diego, CA, USA) platform, and raw reads were screened by QIIME(1.9.1), with the removal of chimeric sequences by UNCHIME. Operational taxonomic units(OTUs) were generated by Vsearch(1.9.6) with similarity at 97%, and aligned against reference database SILVA(http://www.arb-sliva.de). Αlpha diversity indices such as Shannon and Chao 1 index, and beta diversity based on Bray-Curtis difference coefficients were calculated in R(3.5.2) software(http://www.r-project.org) according to normalized OTU abundance. Non-metric multidimensional scaling(NMDS) was used to test communities dissimilarity, and analysis of similarities(ANOSIM) was used to test the similarity among different communities, by using vegan(2.5.2) package. Redundancy analysis(RDA) was used to analyze the relationship between bacterial community and environmental factors by using vegan(2.5.2) and ggplot2. Pearson correlation analysis was carried out with SPSS(19.0) statistical software(IBM Corporation, USA) to determine the relationship between environmental factors and the diversity of planktonic bacteria(Shannon index and Simpson index) and the abundance of specific bacteria. The results showed that Proteobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria were abundant phyla in Pearl River Delta. γ-Proteobacteria was the most abundant class among the communities, followed by Actinobacteria and α-Proteobacteria.The bacterioplanktonic community showed a relatively high diversity in the Pearl River Delta in winter, with no significant differences observed in bacterioplanktonic community's composition among all sampling sites. Pearson correlation analysis showed that the abundance of γ-Proteobacteria(P<0.05), Actinobacteria(P<0.05) andα-Proteobacteria(P<0.01) was positively correlated with temperature, while the diversity index was negatively correlated with PH(Shannon, P≤0.01; Simpson, P<0.007) and the abundance of α-Proteobacteria(P<0.05) was negatively correlated with dissolved oxygen. RDA showed that temperature, pH were the main driving factors affecting the structure of bacterioplanktonic community. Temperature was identified as the main environmental factor affecting planktonic bacteria community. Previous studies shown the composition of bacterial community was driven more by temperature and the average cell size of planktonic bacteria community decreased with the increase of temperature. In addition, temperature was related to the diversity of estuarine ecosystems. Seasonal temperature variation was also considered to be the main variable affecting the dynamics of sediment bacterial community in the Pearl River Estuary. The pH was another major environmental factor affecting the structure and diversity of bacterial communities. pH was generally considered as an indicator of Actinobacter, in the study,negative correlation was observed between pH and Actinobacter(R=?0.469, P=0.016), which was consistent with the results of other studies. These results might provide fundamental information on bacterioplanktonic community composition and environmental factors in winter Pearl River Delta.
Along with the development of economy and the living standard of residents, the discharge of industrial and agricultural wastewater and municipal domestic sewage has a serious impact on the water quality of the Pearl River Delta. Bacterial communities are important components in riverine ecosystem and play key roles in the degradation and transformation of various pollutions in river environment. Bacterioplanktonic community responds to changes in biotic and abiotic factors that are amplified during spring and summer these wet seasons,however, whether communities respond to environmental disturbance in dry seasons remains unknown. In this study, we collected surface water samples from the Pearl River Delta(112.18°E-113.51°E, 22.38°N-23.17°N) in November 2017 and January 2018, using high-throughput sequencing of 16 S rDNA gene amplicons on the Illumina Miseq platform, to investigate the bacterioplanktonic community's composition and their environmental impact factors of the Pearl River Delta in winter. Total DNA was extracted from water samples by using DNA extraction kit(Magen Hipure Spil DNA Kit), and DNA concentration was determined by Qubit? dsDNA HS Assay Kit. The targeted V3-V4 regions were amplified with the primers set(341 f-CCTACGRRBGCASCAGKVRVGAA;806 r-GGACTACNVGGGTWTCTAATCC). The purified PCR products were sequenced on Illumina MiSeq(Illumina, San Diego, CA, USA) platform, and raw reads were screened by QIIME(1.9.1), with the removal of chimeric sequences by UNCHIME. Operational taxonomic units(OTUs) were generated by Vsearch(1.9.6) with similarity at 97%, and aligned against reference database SILVA(http://www.arb-sliva.de). Αlpha diversity indices such as Shannon and Chao 1 index, and beta diversity based on Bray-Curtis difference coefficients were calculated in R(3.5.2) software(http://www.r-project.org) according to normalized OTU abundance. Non-metric multidimensional scaling(NMDS) was used to test communities dissimilarity, and analysis of similarities(ANOSIM) was used to test the similarity among different communities, by using vegan(2.5.2) package. Redundancy analysis(RDA) was used to analyze the relationship between bacterial community and environmental factors by using vegan(2.5.2) and ggplot2. Pearson correlation analysis was carried out with SPSS(19.0) statistical software(IBM Corporation, USA) to determine the relationship between environmental factors and the diversity of planktonic bacteria(Shannon index and Simpson index) and the abundance of specific bacteria. The results showed that Proteobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria were abundant phyla in Pearl River Delta. γ-Proteobacteria was the most abundant class among the communities, followed by Actinobacteria and α-Proteobacteria.The bacterioplanktonic community showed a relatively high diversity in the Pearl River Delta in winter, with no significant differences observed in bacterioplanktonic community's composition among all sampling sites. Pearson correlation analysis showed that the abundance of γ-Proteobacteria(P<0.05), Actinobacteria(P<0.05) andα-Proteobacteria(P<0.01) was positively correlated with temperature, while the diversity index was negatively correlated with PH(Shannon, P≤0.01; Simpson, P<0.007) and the abundance of α-Proteobacteria(P<0.05) was negatively correlated with dissolved oxygen. RDA showed that temperature, pH were the main driving factors affecting the structure of bacterioplanktonic community. Temperature was identified as the main environmental factor affecting planktonic bacteria community. Previous studies shown the composition of bacterial community was driven more by temperature and the average cell size of planktonic bacteria community decreased with the increase of temperature. In addition, temperature was related to the diversity of estuarine ecosystems. Seasonal temperature variation was also considered to be the main variable affecting the dynamics of sediment bacterial community in the Pearl River Estuary. The pH was another major environmental factor affecting the structure and diversity of bacterial communities. pH was generally considered as an indicator of Actinobacter, in the study,negative correlation was observed between pH and Actinobacter(R=?0.469, P=0.016), which was consistent with the results of other studies. These results might provide fundamental information on bacterioplanktonic community composition and environmental factors in winter Pearl River Delta.
引文
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[2]Giuliano L,De Domenico M,De Domenico E,et al.Identification of culturable oligotrophic bacteria within naturally occurring bacterioplankton communities of the Ligurian Sea by 16S rRNA sequencing and probing[J].Microbial Ecology,1999,37(2):77-85.
[3]Wang J,Peng J F,Song Y H,et al.Seasonal changes of microbial community distribution in sediments of Hun River[J].Research of Environmental Science,2016,29(2):202-210.[王佳,彭剑峰,宋永会,等.浑河底泥微生物群落的季节性变化特征[J].环境科学研究,2016,29(2):202-210.]
[4]Bowen J L,Morrison H G,Hobbie J E,et al.Salt marsh sediment diversity:a test of the variability of the rare biosphere among environmental replicates[J].The ISME Journal,2012,6(11):2014-2023.
[5]Guo L D.Progress of microbial species diversity research in China[J].Biodiversity Science,2012,20(5):572-580.[郭良栋.中国微生物物种多样性研究进展[J].生物多样性,2012,20(5):572-580.]
[6]Lu L,Jia Z J.Urease gene-containing archaea dominate autotrophic ammonia oxidation in two acid soils[J].Environmental Microbiology,2013,15(6):1795-1809.
[7]Kim S J,Park S J,Cha I T,et al.Metabolic versatility of toluene-degrding,iron-reducing bacteria in tidal flat sediment,characterized by stable isotope probing-based metagenomic analysis[J].Environmental Microbiology,2014,16(1):189-204.
[8]Yu L Y,Zhang W J,Liu L M,et al.Determining microeukaryotic plankton community around Xiamen Island,Southeast China,using Illumina Mi Seq and PCR-DGGE techniques[J].PLoS ONE,2015,10(5):e0127721.
[9]Wu X,Xi W Y,Ye W J,et al.Bacterial community composition of a shallow hypertrophic freshwater lake in China,revealed by 16S r RNA gene sequences[J].FEMS Microbiology Ecology,2010,61(1):85-96.
[10]Gao Y,Lai Z N,Zeng Y Y,et al.Community structure of copepods and the relationship with aquatic environmental factors in the Pearl River Delta[J].Journal of Fishery Sciences of China,2015,22(2):302-310.[高原,赖子尼,曾艳艺,等.珠江三角洲河网桡足类群落结构及其与水环境因子的关系[J].中国水产科学,2015,22(2):302-310.]
[11]Huang X D.Discussion on ecological status of water and countermeasures of ecological restoration of small and middle-sized rivers in the Pearl River Delta of Guangdong Province[J].Guangdong Water Resources and Hydropower,2016(5):16-19.[黄显东.广东省珠江三角洲地区中小河流水生态现状及修复对策初探[J].广东水利水电,2016(5):16-19.]
[12]Wang C,Li X H,Lai Z N,et al.Temporal and spatial pattern of the phytoplankton biomass in the Pearl River Delta[J].Acta Ecologica Sinica,2013,33(18):5835-5847.[王超,李新辉,赖子尼,等.珠三角河网浮游植物生物量的时空特征[J].生态学报,2013,33(18):5833-5847.]
[13]Walters W,Hyde E R,Berg-Lyons D,et al.Improved bacterial 16S rRNA gene(V4 and V4-5)and fungal internal transcribed spacer marker gene primers for microbial community surveys[J].mSystems,2016,1(1):e00009-15.
[14]Edgar R C,Haas B J,Clemente J C,et al.UCHIME improves sensitivity and speed of chimera detection[J].Bioinformatics,2011,27(16):2194-2200.
[15]Quast C,Pruesse E,Yilmaz P,et al.The SILVA ribosomal RNA gene database project:improved data processing and web-based tools[J].Nucleic Acids Research,2013,41:D590-D596.
[16]Wang Y,Zhang R,He Z L,et al.Functional gene diversity and metabolic potential of the microbial community in an estuary-shelf environment[J].Frontiers in Microbiology,2017,8:Article No.1153.
[17]Lundin D,Severin I,Logue J B,et al.Which sequencing depth is sufficient to describe patterns in bacterialα-andβ-diversity?[J].Environmental Microbiology Reports,2012,4(3):367-372.
[18]Wu X,Xi W Y,Ye W J,et al.Bacterial community composition of a shallow hypertrophic freshwater lake in China,revealed by 16S r RNA gene sequences[J].FEMS Microbiology Ecology,2007,61(1):85-96.
[19]Zwart G,Crump B C,Agterveld M,et al.Typical freshwater bacteria:an analysis of available 16S rRNA gene sequences from plankton of lakes and rivers[J].Aquatic Microbial Ecology,2002,28(2):141-155.
[20]Huang W,Jiang X.Profiling of sediment microbial community in Dongting Lake before and after impoundment of the Three Gorges Dam[J].International Journal of Environmental Research and Public Health,2016,13(6):Article No.617.
[21]Tang J,Xu X R,Shang C Y,et al.Association of bacterial diversty in city area of Nanming river with environmental factors[J].Acta Microbiologica Sinica,2015,55(8):1050-1059.[唐婧,徐小蓉,商传禹,等.南明河城区河段细菌多样性与环境因子的关系[J].微生物学报,2015,55(8):1050-1059.]
[22]Xu A L,Ren J,Song Z W,et al.Microbial community of municipal discharges in a sewage treatment plant[J].Environmental Science,2014,35(9):3473-3479.[徐爱玲,任杰,宋志文,等.污水处理厂尾水细菌群落结构分析[J].环境科学,2014,35(9):3473-3479.]
[23]Sun Z L,Xuan Y M,Zhang H,et al.Bacterial diversity in the Penaeus vannamei Boone intestine and aquaculture environment[J].Journal of Fishery Sciences of China,2016,23(3):594-605.[孙振丽,宣引明,张浩,等.南美白对虾养殖环境及其肠道细菌多样性分析[J].中国水产科学,2016,23(3):594-605.]
[24]Ghylin T W,Garcia S L,Moya F,et al.Comparative single-cell genomics reveals potential ecological niches for the freshwater acI Actinobacteria lineage[J].The ISME Journal,2014,8(12):2503-2516.
[25]Liu J W,Fu B B,Yang H M,et al.Phylogenetic shifts of bacterioplankton community composition along the Pearl Estuary:the potential impact of hypoxia and nutrients[J].Frontiers in Microbiology,2015,6:Article No.64.
[26]Zhang Y J,Li K,Zhu H R,et al.Community structure of microorganisms and its seasonal variation in Beihai Lake[J].Environmental Science,2017,38(8):3319-3329.[张雅洁,李珂,朱浩然,等.北海胡微生物群落结构随季节变化特征[J].环境科学,2017,38(8):3319-3329.]
[27]Lv X F,Ma B,Yu J B,et al.Bacterial community structure and function shift along a successional series of tidal flats in the Yellow River Delta[J].Scientific Reports,2016,6:Article No.36550.
[28]Li J Y,Jiang X,Jing Z Y,et al.Spatial and seasonal distributions of bacterioplankton in the Pearl River Estuary:The combined effects of riverine inputs,temperature,and phytoplankton[J].Marine Pollution Bulletin,2017,125:199-207.
[29]Lindstr?m E S,Kamst-Van Agterveld M P,Zwart G.Distribution of typical freshwater bacterial groups is associated with pH,temperature,and lake water retention time[J].Applied and Environmental Microbiology,2005,71(12):8201-8206.
[30]Adams H E,Crump B C,Kling G W.Temperature controls on aquatic bacterial production and community dynamics in arctic lakes and streams[J].Environmental Microbiology,2010,12(5):1319-1333.
[31]Sun Z,Li G,Wang C W,et al.Community dynamics of prokaryotic and eukaryotic microbes in an estuary reservoir[J].Scientific Reports,2014,4:Article No.6966.
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