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Environmental Microbiology Reports (2014) 6(1), 80–89


Metagenomic analysis on seasonal microbial
variations of activated sludge from a full-scale
wastewater treatment plant over 4 years
Feng Ju, Feng Guo, Lin Ye, Yu Xia and Tong Zhang*
Environmental Biotechnology Lab, The University of
Hong Kong SAR, Hong Kong, China.
Metagenomic technique was employed to characterize the seasonal dynamics of activated sludge (AS)
communities in a municipal wastewater treatment
plant (WWTP) over 4 years. The results indicated
that contrary to Eukaryota (mainly Rotifera and
Nematoda), abundances of Bacteria and Archaea
(mainly Euryarchaeota) were significantly higher in
winter than summer. Two-way analysis of variance
and canonical correspondence analysis revealed that
many functionally important genera followed strong
seasonal variation patterns driven by temperature
and salinity gradients; among them, two nitrifying
bacteria, Nitrospira and Nitrosomonas, displayed
much higher abundances in summer, whereas
phosphate-removing genus Tetrasphaera, denitrifier
Paracoccus and potential human faecal bacteria, i.e.
Bifidobacterium, Dorea and Ruminococcus, showed
significantly higher abundances in winter. Particularly, occurrence of dual variation patterns beyond
explanation merely by seasonality indicated that
multivariables (e.g. dissolved oxygen, sludge retention time, nutrients) participated in shaping AS community structure. However, SEED subsystems
annotation showed that functional categories in AS
showed no significant difference between summer
and winter, indicating that compared with its microbial components, the functional profiles of AS were
much more stable. Taken together, our study provides
novel insights into the microbial community variations in AS and discloses their correlations with
influential factors in WWTPs.
Activated sludge (AS) harbours highly complex microbial
consortium, and heavily relies on them for the removal of
Received 25 October, 2012; accepted 15 September, 2013. *For
correspondence. E-mail; Tel. (+852) 2859
1968 (lab), (+852) 2857 8551 (office); Fax (+852) 2559 5337.

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd

organic pollutants and nutrients (such as nitrogen and
phosphorus) from sewage to protect our environment and
human health, thereby the performance of biological
wastewater treatment plants (WWTPs) using AS is closely
associated with the structure and functions of microbes.
For example, ammonia-oxidizing bacteria and nitriteoxidizing bacteria (NOB) are highly dedicated bacteria
groups for nitrogen removal, while the polyphosphateaccumulating organisms (PAOs) are significant for phosphorus condensation (Wagner and Loy, 2002; Venter
et al., 2004). Although they played key roles in pollutant
removal and biogeochemistry cycling, however, the
dynamics and behaviours of these functional bacteria
under various conditions are still not clear. Furthermore,
the occurrence of certain bacterial species not fully
studied in AS could be harmful to nutrients removal in
WWTPs by outcompeting related functional bacterial
groups (Bassin et al., 2012).
Study on seasonal variation is an alternative way
for correlating environmental factors with bacterial
community and function. There are a lot of studies on
the microbial dynamics in different ecosystems, like
ocean (Barberán et al., 2011), lake (Pajdak-Stos and
Fialkowska, 2012) and soil (Venter et al., 2004),
which are usually lacked of either sufficient long-term
monitoring data (both chemical and biological) or
powerful tools with adequate coverage for profiling the
whole complex microbial communities. Particularly, AS in
WWTPs is monitored routinely and kept cultured in a
nearly artificially controlled environment, which would
favour our study on the impact of interior environmental
and operational variables on those functionally significant microbes. Moreover, with the advent of highthroughput sequencing (HTS) technology, the diversity
and abundances of microbial communities within AS
could be comprehensively investigated.
Previous studies of AS using HTS mainly adopt 454
pyrosequencing of 16S rRNA gene amplicons to monitor
the bacterial communities in various bioreactors, including
AS (McLellan et al., 2010; Zhang et al., 2011), membrane
bioreactors (Guo and Zhang, 2012a) and oxidation
ditches (Ben-Amor et al., 2005). These results based on
sequencing 16S rRNA gene amplicons, however, may be
limited by efficiencies of primers, Polymerase chain

with abundances between 85.87 85. The abundance of Bacteria in summer was 87. microbial communities in AS were predominated by Bacteria.Multivariables shape activated sludge communities reaction (PCR) biases.13 5. Domain distribution of the sequences in the eight data sets derived from AS samples.021 0. 2007) at a maximum e-value cut-off of 1e-20. these studies merely explored the bacterial diversity without relevance to their functions and seasonal dynamics within the AS system. in which the seasonal dynamics of microbial communities greatly affects the performance and stability of pollutants removal. lake (Pajdak-Stos and Fialkowska.03 93. such as soil (Costello et al. Analyses of these data sets shed light on disclosing enormous microbial diversity and plentiful functional genes in these environments..04 85. Importantly.020 0.83 0.23 1.0%. 2013).55 89.73 6. b. 2010). A total of 204 581 817 paired-end (PE) metagenomic reads were generated with a length of 100 bps..92 87. 2011) have been reported. 2012b. Results and discussion The metagenomic data sets in this study A total of 10 metagenomes were sequenced with DNA extracted from eight AS samples of Sha Tin WWTP using 81 Illumina HTS.6% and 93. Similar Table 1.72 6.3 0. Microbial composition and seasonal dynamics at domain and phylum levels The seasonality of microbial communities in different ecosystems like ocean (Barberán et al.8 (± 2. 2011) and etc. more informative and more in-depth coverage sequencing data sets derived from various ecosystems. 2009). 80–89 .4 0. 2010). The results will help us understand the effects of these parameters on the structure and diversity of AS communities. In the present study..0 0.28 7. Steele et al. Paired t-test was conducted to compare differentials in the domain distribution of microbial communities between summary and winter samples. with an average length of 167 (± 3. followed by Eukaryota (0. Environmental Microbiology Reports. these studies only based on analysis of a single sample with no observation of seasonal dynamics of microbial communities. Shotgun metagenomics.19 4. The analysis of the three replicated data sets of sample AS08-7 showed that Illumina HTS has good reproducibility based on values of slope (approach 1. 2004) have been well documented. Nevertheless. 1990) search against SILVA SSUref database (Pruesse et al. Those hits with similarity > 95% and alignment length > 100 nt were annotated by the lowest common ancestor (LCA) algorithm in MEGAN using default parameters (Huson et al. Illumina HTS-based metagenomic approach was employed to characterize seasonal variations of microbial compositions and functions in the AS of a full-scale WWTP using eight AS samples collected in two seasons (winter and summer) over 4 years. 2007.6)%].12 0.3%) and Archaea (0. and averaged 24 645 626 (± 345 341) PE reads remained for each sample after de-replication (Table S2)..8 0. with little attention being paid to the AS system (Kim et al. which adopts direct sequencing of metagenomic DNA instead of 16S rRNA amplicons. To the best of our knowledge.11 6.11–0. as well as the impacts of the diversity of functionally important microorganisms on the stability of the process.7 0. taxonomic classification effectiveness of variable regions selected and pyrosequencing noises (Guo and Zhang. cow rumen (Hess et al.3 0.94) (Fig.33 2.05) difference in domain distribution between winter and summer samples.13 1. 2013).52 7.6 0.0 0.0 (± 1.29 88..40%).71 6. P-value refers to the one-tailed probability value of the paired t-test using summer and winter samples. 2012) and soil (Venter et al.331 a. results in much larger.40 2.. As shown in Table 1.73–7.8)%.18 90. suggesting that a sequencing depth of 5 G for each AS sample adopted in this study was deep enough to obtain analytical results with high reproducibility.02 7. ocean water (Freilich et al. Among the obtained tags.. human gut (Qin et al.92 6.17 7. which was lower than that in winter [91. 6..08 7. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.74 6.90 6. Paired t-test indicated that there was a significant (P-value < 0. Summer abundance (%a) Bacteria Archaea Eukaryota Unassigned Winter abundance (%a) AS07-7 AS08-7 AS09-7 AS10-7 Average AS08-1 AS09-1 AS10-1 AS11-1 Average P-valuesb 91. only very limited work on metagenomic analysis of the functions and compositions of microbial communities in AS (Horner-Devine et al. Fayle et al. Metagenomic profiling of seasonal dynamics of AS communities was linked to environmental or operational conditions in a full-scale WWTP by canonical correspondence analysis (CCA)..8 0. The number of tags obtained after reads overlapping were 18 098 869 (± 523 507). The number of PE reads in each sample was normalized to 25 426 000. S1).27 1.. seasonal dynamics of metabolic profiles in AS were evaluated.3) nt.86 91. 2011).0) and high linear coefficient (R2 > 0. 12 624–15 473 were identified as 16–18S rRNA genes tags using NCBI’s BLASTN (Altschul et al.045 0.73 91.. The abundance in percentage was based on the taxonomic results using the identified 16/18S rRNA gene tags. Meanwhile.. 2007).2 0..

Bacteroidetes (10. as shown in Fig.90)%].8%) (Fig. results were observed for Archaea. and 22 of them were identified as major phyla (top 15 in each sample). improving the performance of WWTPs (Yiannakopoulou et al. Seasonal variation patterns were observed at phylum level.3)%] was much higher than that in winter [1. 80–89 . Notably. a total of 40 phyla were found in all eight samples. The phylum names in black. like Rotifera and Nematoda. red and green represent phyla that were affiliated to Bacteria. microarray (Xia et al. 1). 6.. The relative abundances of major phyla in the eight Sha Tin AS samples. Similar to previous findings on AS using cloning (Snaidr et al. As most of the Eukaryota found in Sha Tin AS were affiliated with Rotifera and Nematoda.3%) and Nematoda (0.. respectively.9%).7%) and Firmicutes (4.6%). abundance of © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd. Eukaryota and Archaea. Differently. which could affect the performance of WWTPs. The number above each bar represents the abundance ratio of each phylum in summer to winter samples. most of the Eukaryotal populations belonged to Rotifera (1. Environmental Microbiology Reports. 16 were from Bacteria.0 ± (2. with winter abundance doubling that of summer.92 ± (0.7%). Chloroflexi (8. Table S3). 1. 1. averaging at 40. and their occurrence in the AS is beneficial to the reduction of the biomass production by predation of the microorganisms. 2010) and 454 pyrosequencing (Zhang et al.82 F. 1997). and the subdominant phyla were Actinobacteria (averaging at 21. the great variation of Bacteria and Eukaryota during summer and winter could be associated with significant change of quantities of predator and prey in the AS system. Fig. 5 from Eukaryota and 1 from Archaea (Fig. Ju et al. metagenomic data sets in this study found that Proteobacteria was the most abundant phylum (38–43%. 2009). 2011). the average abundance of Eukaryota in summer [5. On the one hand.. in AS could reduce biomass production via predation of the microorganisms.8%) in all 4-year AS samples. Among the major phyla. Previous studies have widely demonstrated that bacteria-eating Eukaryota.. At phylum level. 1.

Similar seasonal dynamics was also observed for two other bacterial phyla. Rhodobacter and Rhodobium (1. Amaricoccus. Caldithrix.5%). Lewinella (1. Group III: samples collected in summer of 2009 and 2010 (AS09-7 and AS10-7). Pirellula and Haliea (Fig.7 ± 0. CA of the eight AS samples also showed the same seasonal grouping patterns using benchmarks of 0.6 and 0.Multivariables shape activated sludge communities Actinobacteria (phylum) was usually higher in winter 26.1). Rhodobacter (2. Candidatus Microthrix (4. and three eukaryotic phyla. with abundances in summer 1.7%) and Tetrasphaera (Guo and Zhang.0%) includes another four widely reported denitrifying-related genera.0 ± 1. Gordonia (2. with abundance ratio of summer to winter (P1-ratio) of 0. and one well-known ammonia-oxidizing © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd. S2 and Table S4). Apart from the above seasonal patterns.7. considering the fact that samples collected in the last 2 years (Groups III and IV) tended to cluster closer to each other (than to samples in Group I or Group II) regardless of their opposite seasonal characteristics as summer and winter samples. Iami.6%). i. plus five not well-described genera. 80–89 .7%). Noteworthy. As shown in Fig.5%) and Paracoccus (1. followed by Planctomyces (5. Tetrasphaera (2. 2C and D).1 ± 0.and foaming-related genera. Judging from the averaged abundance of each genus in all samples. As demonstrated by the CA and PCoA. total Kjeldahl nitrogen (TKN-N) and total phosphate (TP) concentrations (Table S1.3 ± 0. the grouping patterns of AS samples displayed by CA and PCoA were quite similar to the mixed variation patterns of chemical oxygen (COD). Group I: samples collected in summer of 2007 and 2008 (AS07-7 and AS 08-7). Similarly.4%) and Nitrospira (6.4 ± 2. Group II: samples collected in winter of 2008 and 2009 (AS08-1 and AS09-1). 2012a). two eukaryotic phyla. Cyanobacteria. sludge samples collected in summer or winter were certainly similar to each other. occupying 79. Other less abundant genera (< 1. four bulking. namely Rotifera. PCoA plots (Fig. indicating the existence of considerable amount of rare species in AS.9)% (Table S3).1 ± 4. Acidobacteria and Spirochaetes (with P1-ratios of 3.e. Bifidobacterium (1. revealing the existence of dual grouping patterns that were beyond reasonable explanation merely 83 by seasonality (summer and winter). including Streptococcus (1. 2A and B. Nematoda and Streptophyta (with P1-ratios of 4. S2 and Table S4.4% of the classified sequences at genus level.4%).9 ± 2. Diplolaimella (2. Appendix S1).1%). that is Azoarcus.1 ± 0.5). As shown in Table S5.81 for class level and 0. 100 out of the total 643 assigned genera were shared by all eight AS samples.3.2%) were found as the two most abundant genera in AS of Sha Tin WWTP.6). 2.5%). The other abundant genera (> 1. possibly due to the seasonal temperature difference.2% of total assigned sequences. showed an opposite variation dynamics.7%). Cluster I was composed of four samples (Groups I and II) collected in the first 2 years. and Cluster II comprised four samples (Groups III and IV) collected in the last 2 years.0 ± 0.0). Thauera and Hyphomicrobium. while 202 rare genera that only appeared in one sample accounted for merely 1.e. On the other hand. one marine nematode.5%).5 and 3.5 ± 0.0–6.4. such as salinity and sludge retention time.5 and 0. as shown in Fig.1)% than in summer 17. the eight AS samples were divided into two clusters using a benchmark of 0. i. that is Nitrospirae. 7.73 for family level respectively (as indicated by the red dotted lines in Fig.6). based on Bray–Curtis distance. two denitrifying bacteria.3 ± 3.e. The major genera (top 20 in each sample) were selected (a total of 43 genera for all eight samples) and compared with their abundances in other samples. 2C and D).0%).3 ± 1. that is Verrucomicrobia and Thermotogae (with P1-ratios of 0. PCoA bases on abundances of classes (A) and families (B) revealed that the microbial communities in the eight AS samples could be clustered into four subgroups. one hydrolyser-related genus. We hypothesized that temperature exerts the most significant effect on the differentials in microbial structures between summer and winter samples. namely Arthropoda and Gastrotricha (with P1-ratios of 0.78 (as indicated by the blue dotted lines in Fig. Seasonal genus dynamics and genus– environment relationship Comparative analysis revealed the core and distinct genera harboured in the AS of Sha Tin WWTP over a period of 4 years. four fermentative or photo-fermentative bacteria that could utilize a variety of organic substrates. 6.2%) and Caldilinea (4. 2A and B) also supported the existence of another grouping pattern beyond summer and winter.0%) included one genus of phosphate-accumulating organisms (PAO).9.5 times higher than those in winter. Nocardioides (1.87 and 0. Environmental Microbiology Reports.4 ± 0. and one archaeal phylum Euryarchaeota (with P1-ratio of 0. although their abundances were relatively lower (Table S3).0 and 2. another four bacterial phyla. i.4 ± 2. Mycobacterium (8. 2.0 ± (3. as well as other environmental or operational parameters. possibly revealing an intrinsic correlation between the AS microbial structure and the available nutrients for microbial growth. Zoogloea. and Group IV: samples collected in winter of 2010 and 2011 (AS10-1 and AS11-1).0 ± 2.9 ± (6.3%). Grouping of the eight AS samples The similarity patterns of the eight AS samples were evaluated at class and family levels through two independent methods: principal coordinate analysis (PCoA) and cluster analysis (CA).

false discovery rate (FDR) < 0. The blue and red dotted lines show the similarity cut-off levels to cluster the eight Sha Tin AS samples. and the relationships between their abundances and the operational conditions/wastewater characteristics of the AS process were explored by CCA using 4-year monitoring data in Sha Tin WWTP (Table S1) and the PAST software (Hammer et al. 3A and B. 2004). those genera with significantly changed abundances across P1. As shown in Fig. A B C D Fig. 2. Fig. Environmental Microbiology Reports. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd. The variations of AS communities over the 4-year sampling period were examined based on two-way analysis of variance. 15 (names in purple) had significantly changed abundances from the first 2 years to the last 2 years (P2. 3B). PCoA (A and B) was conducted using the Bray–Curtis distance and a transformation exponent of 2 (as recommended).05. Nitrosomonas. CA (C and D) was performed using unweighted pair group mean averages as algorithm and the Bray–Curtis distance for similarity measurement. genus. To unravel the underlying influential factors responsible for the complex variation patterns observed in the AS communities. 3A and D). P2 and P1P2 were extracted (as shown in Fig. Fig. Ju et al.3] abundances over the 4-year sampling period. 6. 2001). 12 (names in blue) manifested significantly changed abundances with seasonal alternations from summer to winter (P1. 80–89 . Similar grouping patterns were adopted as used in the cluster analysis.. Principal coordinate analysis (PCoA) and cluster analysis (CA) of eight activated sludge (AS) samples at class and family levels.. 3A). and 6 (names in green) displayed significantly changed abundances across both P1 and P2. which plays particularly significant roles in oxidizing ammonia into nitrite during nitrification process in WWTPs (Raponi et al. 33 genera displayed significantly changed [P-value < 0.84 F. Among these genera.

Environmental Microbiology Reports.1% respectively. each genus is represented by a coloured point (blue for P1.Multivariables shape activated sludge communities A 85 B C Fig. P1: summer vs winter samples. purple and green represent those genera with significantly changed abundances across P1. 3. P2 and P1P2 (both P1 and P2) respectively. all 12 P1-affiliated genera (the blue points) were distributed either along or very close to temperature and salinity lines (or their extension lines). the genus names in bold blue. 3C. 80–89 . 6. Environmental variables are indicated by thick green lines with variable names (in red) at the end. These strong correlations of P1-affiliated genera with either temperature or salinity indicated that temperature and salinity were the major variables that led to the significantly different abundances of these populations from summer to winter. and black for both P1 and P2). or even both. AS shown in Fig. B) with significantly changed abundances across Pattern 1 (P1) and/or Pattern 2 (P2) based on two-way analysis of variance. purple for P2.2% and 32. This positive corre- lation of abundances of NOB like Nitrospira and hydrolysers like Lewinella with temperature partially explains the decreased nitrification and hydrolysis activities at lower temperature. with those abundant in summer being either positively correlated with temperature or negatively correlated with salinity. eigenvalues of horizontal and vertical axes equal to percentage variances of 49. For subfigure C. On the contrary. For subfigures A and B. leading to the incomplete nitrification and hydrolysis problems that commonly occur within full-scale biological treatment units during winter season. accompanied by the genus name. and genus-conditional triplot (C) displaying variations of these significantly changed genera with respect to the environmental variables. Boxplots showing all genera (A. P2: first-2-years vs last-2-years samples. and simultaneously negatively correlated with temperature. based on canonical correspondence analysis (CCA). those P1-affiliated genera located on negative direction of horizontal axis were positively correlated with salinity. with P1-ratio between © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

six genera (the black points in Fig.86 F. 6. namely ‘nitrogen metabolism’ and ‘phosphorus metabolism’. their abundances in the first 2 years were usually lower than the last 2 years. indicating the coexistence of multiple variables shaping the abundances of these genera in AS. 2007).g. merely accounted for 1. were shared by all the eight samples. and cluster (II) COD. Moreover. and/or (iii) influential factors exert discrepant effects on abundances of microorganisms carrying similar functional genes.8 ± 0. suggesting their significant roles in microbial communities of AS. although they are of particular importance for biological nitrogen and phosphorus removal from wastewater. Due to the difference in environmental and operational parameters (e... perhaps implicating the combined influences of multivariables that led to the significantly changed abundances across P2 in P2-affiliated genera. had significantly higher abundance in the first 2 years than the last 2 years. but they had quite different abundances in summer and winter seasons.e.2%). marine environment (Chaffron et al.. bulking and foaming bacteria. significantly different abundances of functional genes were expected to occur in summer and winter.4 and 5. those P2-affiliated genera located on positive direction of vertical axis. 2012a). another two categories. 2010). considering the fact that relative standard deviations of those functional categories in all AS samples were no more than 9.. Global gene functional profiles and seasonal dynamics in AS The overall functional profiles were predicted for the eight AS metagenomic data sets using the SEED subsystem (Overbeek et al.g. 2009).. Further comparisons at subsystems Level 2 and Level 4 (Fig. For each data set. SRT). temperature. salinity.38 ± 0. Moreover. It could also be found that P2-affiliated genera were independent from the influences of temperature and salinity..2%). Environmental Microbiology Reports.1– 51. 80–89 .. freshwater (Pandit et al. with ratios of averaged abundances from first 2 years to last 2 years (P2-ratio) between 1.. fermentative bacteria.7% (averaging at 3.. Particularly.07% and 0. such as Clostidium.). 2009) and enhanced biological phosphorus removal (Horner-Devine et al. However. other three genera.1%). Among them. 2010). DO. which mainly originated from the sea water used for toilet flushing. Meanwhile. Kosmotoga. TKN-N and TP. for those P2-affiliated genera located on the negative direction of vertical axis (e. may be associated with bulking and foaming problems that commonly occurred in the AS of many WWTPs (Guo and Zhang. two genera. denitrifying bacteria.4% of the 17 001 280–18 608 516 tags contained predicted proteins assigned to known functions at Level 1. Pirellula.90 ± 0. 0. Fig.03 and 0. considering the fact that these genera located somewhere around the vertical directions of lines of temperature and salinity.1%) (Table S6). 2008). 3C showed that all P2-affiliated genera (the purple points) were distributed between the lines (or their extension lines) of two clusters of influential factors: i. Flavobacterium. and amino acids and derivatives (8.6.2 and 0. carbohydrates (10. that is Tetrasphaera and Nocardioides. However. Comparison with previous studies on AS This is the first systematic metagenomic profiling of seasonal dynamics of AS communities by conducting Illumina HTS. including Nitrosomonas. especially those belonging to functionally important groups in AS (such as nitrifying bacteria. (ii) gradients of influential factors are not strong enough to make significant changes in abundances of functional genes. 41. 2009). Amaricoccus and Pseudorhodobacter. This implicated that compared with its microbial compositions. PAO. displayed dual variation patterns characterized by significantly changed abundances across both P1 and P2. it seemed that differences in the 28 Level 1 functional categories between summer and winter were not significant. 3C). the functional categories in AS were much more stable. namely Bifidobacterium. hydrolyser. cluster (I) dissolved oxygen (DO). Moreover.2 ± 0. This was comparable to the percentage of annotated sequences (40%) in a previous study on AS using pyrosequencing (Sanapareddy et al.4. with P2-ratios between 0. as shown in Table S6.6 ± 0. Tetrasphaera and etc. 4) showed that except for a small number of low-abundance functional categories at Level 4.6 ± 0. protein metabolism (8. functional © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd. Dorea and Ruminococcus. Haliea and Aeromonas). the majority of functional categories showed no significant difference (using twofold as the cut-off) in abundance between summer and winter. The most dominant functional categories were those involved with clustering-based subsystems (15. 2005) in MG-RAST at the e-value cut-off of 10−5 (Meyer et al.8 (Table S4). indicating high similarity of Level 1 functional categories among these ecosystems.2%). mixed liquor suspended solids. There are several possible explanations for this: (i) high proportion of functional genes related to fundamental metabolism are shared in summer and winter. Ju et al. etc. have been regarded as human faecal bacteria (Qin et al. mean cell retention time and sludge retention time (SRT). Flexibacter. These dominant functional categories evident in our AS metagenomes were also highly represented in metagenomic surveys in other environments like grassland soil (Costello et al. Our analysis demonstrates that some core genera.02% of all the predicted proteins that were assigned to known functional categories.

Finally. temperature and salinity exert almost contrary effects on AS microbes. judged from the CCA plots. most studies on the diversity of AS microbial communities mainly rely on analysis of a single AS sample using clone library analysis (Sasaki et al. probably laboratory-scale ones designed with much simpler and more controllable experimental conditions. etc. which targeted specific genes (such as 16S rRNA) for exploring the microbial diversity without observing their functions within AS. biases are also likely to be introduced during the DNA extraction. about 167 bps in average. are needed to clarify the effect and significance of each variable on the AS microbial community dynamics as well as stability of the process. The signal intensities were indicated by the number of sequences that were assigned into each category. although metagenomic sequencing is neither low throughput nor PCR-based. 4. However. microbial communities in AS were correlated with 11 environmental and operational parameters in a full-scale WWTP by metagenomic technique and CCA analysis. analysis based on SEED subsystems reveals that the functional categories in AS showed no significant difference between summer and winter.) also play significant roles in shaping the overall AS community structure. indicated by the two diagonal lines. 1994). for two variables with opposite effects on abundances of the same species. DO.Multivariables shape activated sludge communities B Log2(Winter signal) Log2(Winter signal) A 87 Log2(Summer signal) Log2(Summer signal) Fig. other variables (such as SRT. Further studies. 2010) or 454 pyrosequencing (Kwon et al. or more affected by the negative effect of the other.. The signal intensities (reads number) of functional categories from summer (X-axis) and winter (Y-axis) samples. although the optimal extraction kit (after comparison with several other kits) has been used. The differentially detected genes were identified as signal intensity difference of ≥ 2 folds. although the effect of temperature is more significant. 80–89 . Previously. Environmental Microbiology Reports.. which provides a comprehensive understanding of the influences of different variables on the microbial component and dynamics of functionally significant microorganisms. and 200 bp might constitute an optimal trade-off between the rate of underprediction and the production cost of such reads (Huson et al. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd. 2007). 6. and Illumina HTS has been proved to possess good repeatability.. the length of tags obtained in this study was short. in this work a sequencing depth of 5G is still not deep enough to accurately explore the rare species within AS owing to the limited number (12 624– 15 473 for each sample) of the identified 16/18S rRNA gene tags. 2010).. The results are based on subsystem annotation at Level 2 (A) and Level 4 (B). Each point in the figure represents one functional category at Level 2 or Level 4 determined by subsystem implemented in MG-RAST. Several technical limitations may affect our results. it is not easy to distinguish whether the species is more positively affected by one variable. Unfortunately. For the first time. For example. Our findings indicate that beside temperature and salinity. microarray (Xia et al. First. salinity. Moreover. although taxonomic classification of this length by LCA in MEGAN indicates that 100 bp is long enough to identify a species. it is hard to distinguish the effects of different variables on microbial community in a fullscale WWTP since a lot of uncontrollable or even undetectable influential factors are involved in such a pollutant-removing process.

I. Funayama. D.. Knittel.. Raes.. (2012) Effect of different salt adaptation strategies on the microbial diversity. J Microbiol Biotechnol 20: 1717–1723. Environ Microbiol 12: 378–392. J. Chokhawala...C.. Kwon. A.. van Loosdrecht. Costello. D. Meilijson. Harper. Smidt..J.N...A. T.. Arumugam. E.V. BMC Cancer 4: 56–68.L. Science 326: 1694–1697. Kurata. C. (1990) Basic local alignment search tool... Genome Res 17: 377–386. Guo.J...S. Butler. (2007) SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. H. and von Mering. E. G. M.. Hammer. Rehrauer.. M. Appl Environ Microbiol 71: 4679–4689. and Foster.M..-Y. H. S.W.. Muyzer.. M. T.. Pajdak-Stos. G. C. Burgdorf.. and Ruppin..... Egan. Turner. Hamady. Bassin. (2013) Ant mosaics occur in SE Asian oil palm plantation but not rain forest and are influenced by the presence of nest-sites and non-native species. J Biol Chem 269: 14730–14737.. (2007) A comparison of taxon co-occurrence patterns for macro-and microorganisms. I. A. S.. Feng Ju would like to thank HKU for the postgraduate studentship.. (2010) Diversity and population structure of sewage-derived microorganisms in wastewater treatment plant influent. N. Ecography 36: 1–7. H. A... Begley. Heilig. S. (2013) General and rare bacterial taxa demonstrating different temporal dynamic patterns in an activated sludge bioreactor. Manichanh. E. (2001) PAST: paleontological statistics software package for education and data analysis. Fuhrman. Huson. Technical support from Ms. R.. Appl Environ Microbiol 75: 1688– 1696. (2012a) Profiling bulking and foaming bacteria in activated sludge by high throughput sequencing.O.. F. BMC Bioinformatics 9: 386–394.. Hamp. Rosado. Jeong. A.... R. R. and Zhang. S. M.. A. and Cottenie. J. (2010) A global network of coexisting microbes from environmental and whole-genome sequence data. K.. Yu.. Nagata. S. F... Ø.. K. Fierer. et al. T. and Zhang. (2010) The large-scale organization of the bacterial network of ecological co-occurrence interactions.. Ludwig. G. Kim. Overbeek. et al..F.K.C.. Ben-Amor. Acknowledgements The authors would like to thank GRF HKU 7201/11E for financial support of this research.F. Kleerebezem. Kim. Quast. W. injured. and Lipman.. Nucleic Acids Res 38: 3857–3868. M. Olson.W. Hilger.. et al. Silver.. Schroth. Nucleic Acids Res 35: 7188–7196.. T. et al.. M..T. A. C.88 F. J. E. J.H.M. J. Colwell. Bates. R. Dr. and dead fecal bacteria assessed by fluorescenceactivated cell sorting and 16S rRNA gene analysis. (2009) Bacterial community variation in human body habitats across space and time.M. Lauber... D. D’Souza.E. Kubal.. N. and Glöckner. activity. Ludwig.. (1994) Expression cloning of a novel alpha 1. (2011) Metagenomic discovery of biomass-degrading genes and genomes from cow rumen. Environmental Microbiology Reports..A... S... U. H. and Park.. K. Wells.. T. Vicky Fung is greatly appreciated. (2012b) Biases during DNA extraction of activated sludge samples revealed by high throughput sequencing. K. Gordon. Mueller-Spitz. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.M.. J.. Chuang. Snaidr.H. Nature 464: 59–65.. W.D. W. (2010) A human gut microbial gene catalogue established by metagenomic sequencing. Freilich.. and Wang.. Bohannan.C. T. (2011) Using network analysis to explore co-occurrence patterns in soil microbial communities.. Hess.. Fuchs. (2005) The subsystems approach to genome annotation and its use in the project to annotate 1000 genomes..S.-Y. Guo Feng would like to thank HKU for postdoctoral fellowship. (2009) Contrasts between habitat generalists and specialists: an empirical extension to the basic metacommunity framework. M. Fayle. J. R.. and Sogin. T.J. E. A. (2008) The metagenomics RAST server – a public resource for the automatic phylogenetic and functional analysis of metagenomes. R. Appl Microbiol Biotechnol 97: 4607–4616. (1997) Phylogenetic analysis and in situ identification of bacteria in activated sludge. Kolasa. Glass. E. Sharan. Myers. S. S. Pandit. M.... Qin. Appl Microbiol Biotechnol 97: 1755–1765..C.. et al.C. Abee.....A. J. Andreishcheva. Palaeontol Electron 4: 1–9. and Schuster. Karp.. Gophna.A. R.. Cohoon. E. J. S. 6. W. M. F. and settling of nitrifying sludge in sequencing batch reactors. and Fialkowska. Science 331: 463–467.. D. J.. G. (2004) Microarray analysis reveals genetic pathways modulated by tipifarnib in acute myeloid leukemia. Ju et al. and Knight. Gonzalez. M. L. W. Huber.. E.. H. R. Peplies. T. M.A. McLellan.. Leibold.H.. ISME J 6: 343– 351. Water Environ Res 84: 619–625. Ecology 90: 2253–2262. Huse. Amann. F. Atkins. and Clinton. S. (2007) MEGAN analysis of metagenomic data. P... Guo. Belly. S.. and Ryan. and Fierer. Lancet.K. E. (2009) Molecular diversity of a North Carolina wastewater treatment plant as revealed by pyrosequencing. Vaughan. B. S. J. Paarmann. and de Vos. (2010) Bacterial community composition and diversity of a full-scale integrated fixed-film activated sludge system as investigated by pyrosequencing. Appl Environ Microbiol 63: 2884–2896.-D. J.. R. Barberán..M. N. Pernthaler. Kim.. Casamayor. Sasaki. E.-D. and Dezotti...M. Sczyrba... K.. C. Pruesse. Genome Res 20: 947–959. Raponi.A. Miller.T. E. Ohta.-S.M.I. J. et al. K. Appl Microbiol Biotechnol 93: 1281–1294. B. D. Chaffron. M. Auch. Fodor. Kreimer.. Meyer. and Schleifer.. K. 3-fucosyltransferase that is involved in biosynthesis of the sialyl Lewis x carbohydrate determinants in leukocytes.P..E. H. T. M. and Park. Choudhuri.F. H.-H. Sanapareddy. (2005) Genetic diversity of viable. (2012) The influence of temperature on the effectiveness of filamentous bacteria removal from activated sludge by rotifers. J.. Watanabe. R.. Ecology 88: 1345–1353. J.. Nucleic Acids Res 33: 5691– 5702. Y. R. Li. Jung. Water Res 46: 2772–2782.-S.O. References Altschul.E. H. J Mol Biol 215: 403–410.. Qi.. J. Gish. 80–89 . W. K. Horner-Devine..

(2010) Bacterial community structure in geographically distributed biological wastewater treatment reactors. Li.Multivariables shape activated sludge communities Steele. Kaimakamidou. S.A. M.Y. Illumina sequencing metadata of the eight activated sludge samples in this study. K.. and Karagiannidis. T. Song.A.. J. and Loy. Fig. Percentages of the shared genera and their corresponding sequences. Beman.L.. Wagner. Abundances of the top 20 genera in each AS sample. Rusch.. P. A. Engineering. Heidelberg. 80–89 .. Venter. Duan. J. Planning and Economics (CEMEPE). (2002) Bacterial community composition and function in sewage treatment systems.D. Skiathos island. Heat map of major genera (top 20 in each sample) of the eight AS samples. L. Table S5. Science 304: 66–74. Eisen. Table S2. Zhang. ISME J 6: 1137–1147.. S1. Countway.. L. Table S3. G.. J.D. Environ Sci Technol 44: 7391–7396. et al.. 146–157.. D. A. (2011) Marine bacterial.. Kungolos... ISME J 5: 1414–1425. Y. P. A.L. Remington. J.. Y. Andersen.F.. Variation of environmental and operational parameters.. M. Greece. D.. S2. et al... Fig. Characteristics of the environmental and operational parameters in Shatin WWTP from July 2007 to January 2011. Environmental Microbiology Reports. et al. Emmanouil. C. L. The percentage abundances (%) of the major phyla (top 15 in each sample) in the eight AS samples. 24–28 June 2007: Parlar Scientific Publications.. Curr Opin Biotechnol 13: 218–227.M. 6. Xia. J. pp... 89 Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Table S1. Table S6. Halpern. Piceno. J. Appendix S1.. Kim. In Proceedings of the First International Conference on Environmental Management. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd. Table S4.C.F. (2004) Environmental genome shotgun sequencing of the Sargasso Sea. A.. (2009) Testing the reliability of protozoa as indicators of wastewater treatment plant performance. Shao. Xia. archaeal and protistan association networks reveal ecological linkages. The relative abundances of the 28 functional categories in four ecosystems based on SEED subsystems implemented in MG-RAST. T. and Ye.M. V. Reproducibility of Illumina high-throughput sequencing at different sequencing depths. (2011) 454 Pyrosequencing reveals bacterial diversity of activated sludge from 14 sewage treatment plants. Vigil.. Yiannakopoulou.