454 pyrosequencing reveals bacterial diversity of activated sludge from 14 sewage treatment plants

Abstract

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 259 K effective sequences of 16S rRNA gene V4 region were obtained from these AS samples. These sequences revealed huge amount of operational taxonomic units (OTUs) in AS, that is, 1183-3567 OTUs in a sludge sample, at 3% cutoff level and sequencing depth of 16,489 sequences. 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., Gp4 and Gp6 in Acidobacteria and Subdivision3 genera 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.

Figure 1

Abundances of different phyla and classes in Proteobacteria in the 15 AS samples. The abundance is presented in terms of percentage in total effective bacterial sequences in a sample, classified using RDP Classifier at a confidence threshold of 50%. Taxa represented occurred at >1% abundance in at least one sample. Minor phyla refer to the taxa with their maximum abundance <1% in any sample.

Figure 2

CA based on Bray–Curtis distances of 15 AS samples. (a) At order level; (b) at 3% cutoff-OTU level. The dot lines show the similarity cutoff levels to cluster the 15 AS samples into five groups: Group I contains all AS from mainland China and that from Singapore; Group II contains the three samples from North America; Group III is sludge from Sha-Tin (Hong Kong), which treats saline sewage; Group IV is sludge from Shek-Wu-Hui (Hong Kong), which treats sewage containing slaughterhouse wastewater; Group V is sludge from Stanley (Hong Kong), which is located inside a cave. For 3% cutoff, 0.25 was selected, whereas 0.6 were selected at the order level, as the lower taxonomy level shows more differences.

Figure 3

Principal coordinate analysis of 15 AS samples by weighted UniFrac. For UniFrac, Greengenes coreset tree was selected as the reference tree, a sample mapping file showing the frequency of each reference taxon in different AS samples was generated through local BLAST and was uploaded to http://bmf2.colorado.edu/fastunifrac/ to conduct weighted UniFrac PCoA following the instructions (Hamady et al., 2010). PCoA at order level (Supplementary Figure S5a) and 3% cutoff-OTU level (Supplementary Figure S5b) were also conducted using PAST in a way similar to the above CA. For these three PCoA results, similar group patterns were obtained as CA shown in Figure 2. The color reproduction of this figure is available at the ISME Journal online.

Figure 4

Heat map of top 10 genera in each sample. The top 10 abundant genera in each sample were selected (a total of 58 genera for all 15 samples) and compared with their abundances (percentages) in other samples. The color intensity (log scale) in each panel shows the percentage of a genus in a sample, referring to color key at the right bottom. Those in bold font are the core genera in different AS samples.

Figure 5

Average abundances (percentages) of the top 100 genera in eight AS samples of Group I (mainland China and Singapore), and their average percentages in three AS samples of Group II (North America). The blue line shows the average percentages in Group I and the shadow area shows the variation ranges (average ±s.d.). The up-bars and down-bars show the average percentages of the corresponding genera in Group II. ^*Shows the genera, which had significantly different average abundances in Groups I and II. The color reproduction of this figure is available at the ISME Journal online.