454 Pyrosequencing reveals bacterial diversity(ismej2011188a

ORIGINAL ARTICLE

454Pyrosequencing reveals bacterial diversity of activated sludge from 14sewage treatment plants Tong Zhang,Ming-Fei Shao and Lin Ye

Environmental Biotechnology Laboratory,Department of Civil Engineering,The University of Hong Kong,Hong Kong

Activated sludge (AS)contains highly complex microbial communities.In this study,PCR-based 454

pyrosequencing was applied to investigate the bacterial communities of AS samples from 14

sewage treatment plants of Asia (mainland China,Hong Kong,and Singapore),and North America

(Canada and the United States).A total of 259K effective sequences of 16S rRNA gene V4region

were obtained from these AS samples.These sequences revealed huge amount of operational

taxonomic units (OTUs)in AS,that is,1183–3567OTUs in a sludge sample,at 3%cutoff level and

sequencing depth of 16489sequences.Clear geographical differences among the AS samples from

Asia and North America were revealed by (1)cluster analyses based on abundances of OTUs or the

genus/family/order assigned by Ribosomal Database Project (RDP)and (2)the principal coordinate

analyses based on OTUs abundances,RDP taxa abundances and UniFrac of OTUs and their

distances.In addition to certain unique bacterial populations in each AS sample,some genera were

dominant,and core populations shared by multiple samples,including two commonly reported

genera of Zoogloea and Dechloromonas ,three genera not frequently reported (i.e.,Prosthecobacter ,

Caldilinea and Tricoccus )and three genera not well described so far (i.e.,Gp4and Gp6in

Acidobacteria and Subdivision3genera incertae sedis of Verrucomicrobia ).Pyrosequencing

analyses of multiple AS samples in this study also revealed the minority populations that are hard

to be explored by traditional molecular methods and showed that a large proportion of sequences

could not be assigned to taxonomic affiliations even at the phylum/class levels.

The ISME Journal (2012)6,1137–1147;doi:10.1038/ismej.2011.188;published online 15December 2011

Subject Category:microbial population and community ecology

Keywords:pyrosequencing;activated sludge;bacteria;core population;cluster analysis;PCoA

Introduction

Similar to soil and sediment,activated sludge (AS)is a highly complex system of eukaryotes (protozoa and fungi),bacteria,archaea and viruses,in which bacteria are dominant.Despite its importance in biological wastewater treatment,the microbial influence on the formation,development and function of AS remains largely unstudied (Wagner and Loy,2002).This is partly attributable to the lack of robust techniques needed to explore the complex microbial communities.The low sequencing depth of the traditional PCR-cloning approach when compared with the vast genetic diversity present in AS systems hindered a comprehensive characterization of the microbial community structure.In this aspect,the current community analyses typically represent a mere snapshot of the dominant members,with little information on taxa with medium to low abun-dances.High-throughput sequencing is a promising

method,as it provides enough sequencing depth to cover the complex microbial communities (Shendure and Ji,2008).So far,it has been applied to analyze microbial communities in marine water (Qian et al .,2010),soil (Roesch et al .,2007;Lauber et al .,2009),human hand surface (Fierer et al .,2008),human distal intestine (Claesson et al .,2009),etc.But few studies have been conducted using this method to investigate AS,though this approach has been used to study the microbial community in the raw sewage (McLellan et al .,2010)and the residual biosolids after digestion of AS (Bibby et al .,2010).Using computational ecology tools such as the UniFrac b -diversity metric (Lozupone and Knight,2005)and principal coordinates analysis (PCoA),researchers can compare differences in microbial communities between ecosystems and along envi-ronmental gradients.With the aid of high-throughput sequencing technology and the well-established b -diversity analytical tool,an attempt could be made to answer some fundamental questions related to AS microbial communities.In this study,AS samples were collec-ted from 14sewage treatment plants (STPs)in mainland China,Hong Kong,Singapore,the United States and Canada.Pyrosequencing using the Received 22May 2011;revised 9November 2011;accepted 9

November 2011;published online 15December 2011Correspondence:T Zhang,Environmental Biotechnology Labora-

tory,Department of Civil Engineering,The University of Hong

Kong,Pokfulam Road,Hong Kong.

E-mail:zhangt@hku.hk The ISME Journal (2012)6,1137–1147

&2012International Society for Microbial Ecology All rights reserved 1751-7362/12

67dcb6076c85ec3a87c2c5f2/ismej

16S rRNA gene as the biomarker was conducted to examine the bacterial diversity of these AS samples, to evaluate the similarity/difference of different samples,to compare the unique dominant bacterial populations,and to identify the core populations shared by different samples.To the best of our knowledge,this study is the first application of PCR-based454pyrosequencing to characterize and compare multiple AS samples.

Materials and methods

STPs and AS

As shown in Table1,AS samples were taken from the aeration tanks of14full-scale STPs that treat municipal wastewater in Asia(mainland China, Hong Kong and Singapore)and North America (Canada and the United States).These STPs treat mainly municipal wastewater,except that CN-QD-TD,CN-GZ-DT and CN-NJ-SJ have a significant industrial wastewater component.The three STPs from North America apply the conventional AS (CAS)or oxidation ditch processes.Those from Asia use A/O(anoxic/aerobic)or A/A/O(anaerobic/ anoxic/aerobic)processes,two of them are membrane bioreactors,and one of them t …… 此处隐藏：43393字，全部文档内容请下载后查看。喜欢就下载吧 ……