Environ Sci Pollut Res DOI 10.1007/s11356-015-4534-3
RESEARCH ARTICLE
Bacterial communities of Beijing surface waters as revealed by 454 pyrosequencing of the 16S rRNA gene Yu-Mei Wei 1,2 & Jing-Qi Wang 1 & Ting-Ting Liu 1 & Wei-Wen Kong 1 & Nan Chen 1 & Xiao-Qing He 1 & Yi Jin 1
Received: 7 January 2015 / Accepted: 12 April 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract As a better understanding of Beijing surface water ecosystems can provide clues for environmental management and public health, here, we report a study of the bacterial communities of five Beijing surface waters conducted using 454 pyrosequencing of 16S ribosomal RNA (rRNA) genes. We expected to observe a core bacterial community among the surface waters and differences in bacterial community abundance over the different locations of sampling. In this study, we obtained a total of 60,810 trimmed reads from the five samples after the removal of unqualified reads. Bacterial sequences from the five samples were classified into taxonomic classes using the default settings of the mothur platform. Our results provided insight into the bacterial community composition of surface waters and revealed that there was a core microbial community in the microbial populations of surface samples at different geographic locations, with 13 phyla and Responsible editor: Robert Duran Yu-Mei Wei and Jing-Qi Wang contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s11356-015-4534-3) contains supplementary material, which is available to authorized users. * Xiao-Qing He [email protected] * Yi Jin [email protected] 1
College of Biological Sciences and Technology, Beijing Forestry University, P. O. Box 162, Qinghua East Rd 35, Haidian District, Beijing 100083, People’s Republic of China
2
State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P. O. Box 2871, Beijing 100085, People’s Republic of China
40 genera in common. Our findings also revealed the differences in bacterial communities among five surface water samples obtained at different locations. Keywords Pyrosequencing . Surface waters . Bacterial communities The treated effluents discharged from sewage treatment plants (STPs) into other water systems may have detrimental environmental effects due to their abundant nutrients, organic matter, and bacteria (Cébron et al. 2004). Some of the surface waters in Beijing, such as environmental waters or garden waters, immediately receive the effluents from municipal STPs after activated sludge treatment processes (He et al. 2012). This measure has greatly alleviated the pressure on the limited water supply of Beijing and ameliorated the urban ecosystem. However, with high levels of bacteria from STPs, these effluents may pose a potential threat to the health of surface water ecosystems. Increased bacterial loading can lead to alterations to water quality through bacterial biogeochemical transformations such as nitrification and denitrification (Wakelin et al. 2008). Furthermore, bacteria, pathogenic bacteria in particular, have an underlying adverse influence on public health. Therefore, a proper understanding of the bacterial communities in Beijing surface waters is a requisite for providing clues about the current ecosystem functions. Additionally, most previous studies have focused on microbial structure and diversity in activated sludge samples in STPs (Ibarbalz et al. 2013; Kim et al. 2013; Yu and Zhang 2012), while few available studies have examined the bacterial communities of the water systems receiving effluent from STPs. According to a recent study, a core microbial community was shared by activated sludge samples from 14 wastewater treatment plants at different geographic locations in China (Wang
Environ Sci Pollut Res
et al. 2012b). Nevertheless, the bacterial communities of surface waters that receive effluent from STPs in Beijing are rarely examined. Microbial communities from specific ecosystems can be investigated using various techniques. Traditionally, culture-dependent techniques were used to study bacterial diversities (Alegría et al. 2012; Ferrando et al. 2012; Vandecandelaere et al. 2012), but these methods could not provide accurate representations of the bacterial populations, as most bacteria in the environment are viable, but currently unculturable. Molecular approaches, such as 16S ribosomal RNA (rRNA) clone libraries (Eichler et al. 2006; Gihring et al. 2012; Zhang et al. 2013a) and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) (Garrido et al. 2014; González-Arenzana et al. 2013; Sun et al. 2013; Zhang et al. 2013b), have greatly increased our comprehension of microbial communities, although these methods poorly represent the rare bacterial groups. In recent years, 454 pyrosequencing has been introduced as a next-generation sequencing method for revealing relatively detailed profiles of microbial communities (Beloshapka et al. 2013; Jackson et al. 2012). The main advantages of 454 pyrosequencing are a much lower cost per base, the omission of a plasmid cloning step, a large amount of sequence data per run, and long read lengths with an average of up to 400 bps (Glenn 2011; Margulies et al. 2005). With the implementation of 454 pyrosequencing, the microbial diversity and abundance in various ecosystems have been revealed, such as soil (Eilers et al. 2012), marine water (Qian et al. 2011), drinking water (Hong et al. 2010), activated sludge samples (Sánchez et al. 2013), hot springs (Hou et al. 2013), and human feces (Wang et al. 2012a). In the present study, PCR-based 454 pyrosequencing was used to investigate the bacterial communities of surface water samples in Beijing. The objective was to address the following three questions: (1) What is the bacterial community composition of surface waters in Beijing? (2) What is the core bacterial community among the surface waters? (3) What are the differences in bacterial communities from surface water samples at different geographic locations?
Materials and methods Ethics statement All experimental procedures involving Beijing surface waters were conducted with the permission of the water resource administrative department of the Beijing Water Authority in China. There were no endangered or protected species involved in this study.
Sampling and processing A total of five surface water samples were collected in September 2012, as shown in Fig. 1. Some information about these samples is also provided in Supplementary Table S1, which was supported by the Beijing Water Authority (Beijing, China) in September 2012. Sampling sites 1, 2, 3, and 4 receive the effluent of STP no. 1, which is the largest STP in Beijing and treats wastewater from 2.4 million inhabitants. Sampling site 5 receives the effluent of STP no. 2 which has a processing wastewater capacity for 0.8 million residents. All of these sampling sites are representatives of reclaimed water for reuse. Of the five sample sites at different geographic locations, sampling sites 1, 3, and 5 are garden lakes for boating and swimming in summer while sampling sites 2 and 4 are city canals for beautifying the environment. All of the sampling sites are surrounded by lawns and trees. Sterile bottles were used to collect 3 L of water from each sample site, including triplicate samples (1 L each) at each site. All samples were then transferred to the laboratory, where they were immediately stored at 4 C. Each sample was filtered through a sandwich consisting of a 0.2-μm polycarbonate filter (Millipore, Cork, Ireland) with a precombusted glass-fiber filter (Millipore) on top. After filtration, the samples were frozen in liquid nitrogen and stored at −70 C until further analysis. DNA extraction The filter sandwiches were cut into small pieces and stored in 2-mL tubes. Microbial genomic DNA was extracted using a sodium dodecyl sulfate (SDS)-based method (Zhou et al. 1996), respectively. Purified DNA of three replicates from each site was pooled before PCR to minimize DNA extraction bias. DNA concentration and quality (ratio of A260/A280) were determined using a Qubit Fluorometer (Invitrogen Inc., New York, USA). PCR amplification, purification, and pyrosequencing The V1–V3 region of the 16S rRNA gene is ∼526 nt in length. This region was selected for construction of a community library through tag pyrosequencing. PCR amplification was performed using the primers B-27F (5 -CCT ATC CCC TGT GTG CCT TGG CAG TCT CAG AGA GTT TGATCC TGG CTC AG-3 ) and A-533R (5 -CCA TCT CAT CCC TGC GTG TCT CCG ACT CAG NNN NNN NNN NTT ACC GCG GCT GCT GGC AC-3 ), which incorporated the A and B sequencing adaptors and a sample-specific barcode sequence described by Wu et al. (Wu et al. 2012). The A and B adaptors are underlined and the eight N bases represent the sample specific barcodes that were used to sort multiple samples in a single 454 GS-FLX run. The PCR was performed in
Environ Sci Pollut Res
Fig. 1 Sampling sites for Beijing surface waters
triplicate with 10 ng of sample DNA, 1.25 U of Taq polymerase, 0.25 mM of dNTPs, 0.1 mM of each primer, and 2.5 μl of 10×PCR buffer in a total volume of 25 μL. A total of 25 cycles (30 s at 95 °C, 30 s at 55 °C, and 30 s at 72 °C) with an initial denaturation for 2 min at 95 °C were followed by a final extension for 10 min at 72 °C. PCR amplicons were purified using an Agarose Gel DNA Purification Kit (TIANGEN, Beijing, China), then pooled in equimolar ratios based on concentration. Emulsion PCR and pyrosequencing were performed according to the manufacturer’s recommendations (Margulies et al. 2005). Amplicon pyrosequencing was performed on the Roche 454 FLX Titanium platform (Roche, Nutley, NJ, USA) using a 454/Roche sequencing primer kit at Majorbio Bio-Pharm Technology Co. Ltd., Shanghai, China. Sequence data analyses In our study, pyrosequencing data was processed using mothur platform (Schloss et al. 2009), which was based on Schloss standard operation procedure. The raw sequences are available through NCBI Sequence Read Archive (SRA) database with accession number SRA246892. In brief, all sequences were denoised using the mothur-based reimplementation of PyroNoise with the recommended parameters (Schloss et al. 2011). Denoised sequences with one mismatched base in the barcode, more than two mismatched bases in the primers, any
ambiguous base, homologous bases longer than 8 bp, a length
RESEARCH ARTICLE
Bacterial communities of Beijing surface waters as revealed by 454 pyrosequencing of the 16S rRNA gene Yu-Mei Wei 1,2 & Jing-Qi Wang 1 & Ting-Ting Liu 1 & Wei-Wen Kong 1 & Nan Chen 1 & Xiao-Qing He 1 & Yi Jin 1
Received: 7 January 2015 / Accepted: 12 April 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract As a better understanding of Beijing surface water ecosystems can provide clues for environmental management and public health, here, we report a study of the bacterial communities of five Beijing surface waters conducted using 454 pyrosequencing of 16S ribosomal RNA (rRNA) genes. We expected to observe a core bacterial community among the surface waters and differences in bacterial community abundance over the different locations of sampling. In this study, we obtained a total of 60,810 trimmed reads from the five samples after the removal of unqualified reads. Bacterial sequences from the five samples were classified into taxonomic classes using the default settings of the mothur platform. Our results provided insight into the bacterial community composition of surface waters and revealed that there was a core microbial community in the microbial populations of surface samples at different geographic locations, with 13 phyla and Responsible editor: Robert Duran Yu-Mei Wei and Jing-Qi Wang contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s11356-015-4534-3) contains supplementary material, which is available to authorized users. * Xiao-Qing He [email protected] * Yi Jin [email protected] 1
College of Biological Sciences and Technology, Beijing Forestry University, P. O. Box 162, Qinghua East Rd 35, Haidian District, Beijing 100083, People’s Republic of China
2
State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P. O. Box 2871, Beijing 100085, People’s Republic of China
40 genera in common. Our findings also revealed the differences in bacterial communities among five surface water samples obtained at different locations. Keywords Pyrosequencing . Surface waters . Bacterial communities The treated effluents discharged from sewage treatment plants (STPs) into other water systems may have detrimental environmental effects due to their abundant nutrients, organic matter, and bacteria (Cébron et al. 2004). Some of the surface waters in Beijing, such as environmental waters or garden waters, immediately receive the effluents from municipal STPs after activated sludge treatment processes (He et al. 2012). This measure has greatly alleviated the pressure on the limited water supply of Beijing and ameliorated the urban ecosystem. However, with high levels of bacteria from STPs, these effluents may pose a potential threat to the health of surface water ecosystems. Increased bacterial loading can lead to alterations to water quality through bacterial biogeochemical transformations such as nitrification and denitrification (Wakelin et al. 2008). Furthermore, bacteria, pathogenic bacteria in particular, have an underlying adverse influence on public health. Therefore, a proper understanding of the bacterial communities in Beijing surface waters is a requisite for providing clues about the current ecosystem functions. Additionally, most previous studies have focused on microbial structure and diversity in activated sludge samples in STPs (Ibarbalz et al. 2013; Kim et al. 2013; Yu and Zhang 2012), while few available studies have examined the bacterial communities of the water systems receiving effluent from STPs. According to a recent study, a core microbial community was shared by activated sludge samples from 14 wastewater treatment plants at different geographic locations in China (Wang
Environ Sci Pollut Res
et al. 2012b). Nevertheless, the bacterial communities of surface waters that receive effluent from STPs in Beijing are rarely examined. Microbial communities from specific ecosystems can be investigated using various techniques. Traditionally, culture-dependent techniques were used to study bacterial diversities (Alegría et al. 2012; Ferrando et al. 2012; Vandecandelaere et al. 2012), but these methods could not provide accurate representations of the bacterial populations, as most bacteria in the environment are viable, but currently unculturable. Molecular approaches, such as 16S ribosomal RNA (rRNA) clone libraries (Eichler et al. 2006; Gihring et al. 2012; Zhang et al. 2013a) and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) (Garrido et al. 2014; González-Arenzana et al. 2013; Sun et al. 2013; Zhang et al. 2013b), have greatly increased our comprehension of microbial communities, although these methods poorly represent the rare bacterial groups. In recent years, 454 pyrosequencing has been introduced as a next-generation sequencing method for revealing relatively detailed profiles of microbial communities (Beloshapka et al. 2013; Jackson et al. 2012). The main advantages of 454 pyrosequencing are a much lower cost per base, the omission of a plasmid cloning step, a large amount of sequence data per run, and long read lengths with an average of up to 400 bps (Glenn 2011; Margulies et al. 2005). With the implementation of 454 pyrosequencing, the microbial diversity and abundance in various ecosystems have been revealed, such as soil (Eilers et al. 2012), marine water (Qian et al. 2011), drinking water (Hong et al. 2010), activated sludge samples (Sánchez et al. 2013), hot springs (Hou et al. 2013), and human feces (Wang et al. 2012a). In the present study, PCR-based 454 pyrosequencing was used to investigate the bacterial communities of surface water samples in Beijing. The objective was to address the following three questions: (1) What is the bacterial community composition of surface waters in Beijing? (2) What is the core bacterial community among the surface waters? (3) What are the differences in bacterial communities from surface water samples at different geographic locations?
Materials and methods Ethics statement All experimental procedures involving Beijing surface waters were conducted with the permission of the water resource administrative department of the Beijing Water Authority in China. There were no endangered or protected species involved in this study.
Sampling and processing A total of five surface water samples were collected in September 2012, as shown in Fig. 1. Some information about these samples is also provided in Supplementary Table S1, which was supported by the Beijing Water Authority (Beijing, China) in September 2012. Sampling sites 1, 2, 3, and 4 receive the effluent of STP no. 1, which is the largest STP in Beijing and treats wastewater from 2.4 million inhabitants. Sampling site 5 receives the effluent of STP no. 2 which has a processing wastewater capacity for 0.8 million residents. All of these sampling sites are representatives of reclaimed water for reuse. Of the five sample sites at different geographic locations, sampling sites 1, 3, and 5 are garden lakes for boating and swimming in summer while sampling sites 2 and 4 are city canals for beautifying the environment. All of the sampling sites are surrounded by lawns and trees. Sterile bottles were used to collect 3 L of water from each sample site, including triplicate samples (1 L each) at each site. All samples were then transferred to the laboratory, where they were immediately stored at 4 C. Each sample was filtered through a sandwich consisting of a 0.2-μm polycarbonate filter (Millipore, Cork, Ireland) with a precombusted glass-fiber filter (Millipore) on top. After filtration, the samples were frozen in liquid nitrogen and stored at −70 C until further analysis. DNA extraction The filter sandwiches were cut into small pieces and stored in 2-mL tubes. Microbial genomic DNA was extracted using a sodium dodecyl sulfate (SDS)-based method (Zhou et al. 1996), respectively. Purified DNA of three replicates from each site was pooled before PCR to minimize DNA extraction bias. DNA concentration and quality (ratio of A260/A280) were determined using a Qubit Fluorometer (Invitrogen Inc., New York, USA). PCR amplification, purification, and pyrosequencing The V1–V3 region of the 16S rRNA gene is ∼526 nt in length. This region was selected for construction of a community library through tag pyrosequencing. PCR amplification was performed using the primers B-27F (5 -CCT ATC CCC TGT GTG CCT TGG CAG TCT CAG AGA GTT TGATCC TGG CTC AG-3 ) and A-533R (5 -CCA TCT CAT CCC TGC GTG TCT CCG ACT CAG NNN NNN NNN NTT ACC GCG GCT GCT GGC AC-3 ), which incorporated the A and B sequencing adaptors and a sample-specific barcode sequence described by Wu et al. (Wu et al. 2012). The A and B adaptors are underlined and the eight N bases represent the sample specific barcodes that were used to sort multiple samples in a single 454 GS-FLX run. The PCR was performed in
Environ Sci Pollut Res
Fig. 1 Sampling sites for Beijing surface waters
triplicate with 10 ng of sample DNA, 1.25 U of Taq polymerase, 0.25 mM of dNTPs, 0.1 mM of each primer, and 2.5 μl of 10×PCR buffer in a total volume of 25 μL. A total of 25 cycles (30 s at 95 °C, 30 s at 55 °C, and 30 s at 72 °C) with an initial denaturation for 2 min at 95 °C were followed by a final extension for 10 min at 72 °C. PCR amplicons were purified using an Agarose Gel DNA Purification Kit (TIANGEN, Beijing, China), then pooled in equimolar ratios based on concentration. Emulsion PCR and pyrosequencing were performed according to the manufacturer’s recommendations (Margulies et al. 2005). Amplicon pyrosequencing was performed on the Roche 454 FLX Titanium platform (Roche, Nutley, NJ, USA) using a 454/Roche sequencing primer kit at Majorbio Bio-Pharm Technology Co. Ltd., Shanghai, China. Sequence data analyses In our study, pyrosequencing data was processed using mothur platform (Schloss et al. 2009), which was based on Schloss standard operation procedure. The raw sequences are available through NCBI Sequence Read Archive (SRA) database with accession number SRA246892. In brief, all sequences were denoised using the mothur-based reimplementation of PyroNoise with the recommended parameters (Schloss et al. 2011). Denoised sequences with one mismatched base in the barcode, more than two mismatched bases in the primers, any
ambiguous base, homologous bases longer than 8 bp, a length
