Assessing the complex sponge microbiota: core,variable and species-specific bacterial communities in marine sponges

Marine sponges are well known for their associations with highly diverse, yet very specific and often highly similar microbiota. The aim of this study was to identify potential bacterial sub-populations in relation to sponge phylogeny and sampling sites and to define the core bacterial community. 16S ribosomal RNA gene amplicon pyrosequencing was applied to 32 sponge species from eight locations around the world''s oceans, thereby generating 2567 operational taxonomic units (OTUs at the 97% sequence similarity level) in total and up to 364 different OTUs per sponge species. The taxonomic richness detected in this study comprised 25 bacterial phyla with Proteobacteria, Chloroflexi and Poribacteria being most diverse in sponges. Among these phyla were nine candidate phyla, six of them found for the first time in sponges. Similarity comparison of bacterial communities revealed no correlation with host phylogeny but a tropical sub-population in that tropical sponges have more similar bacterial communities to each other than to subtropical sponges. A minimal core bacterial community consisting of very few OTUs (97%, 95% and 90%) was found. These microbes have a global distribution and are probably acquired via environmental transmission. In contrast, a large species-specific bacterial community was detected, which is represented by OTUs present in only a single sponge species. The species-specific bacterial community is probably mainly vertically transmitted. It is proposed that different sponges contain different bacterial species, however, these bacteria are still closely related to each other explaining the observed similarity of bacterial communities in sponges in this and previous studies. This global analysis represents the most comprehensive study of bacterial symbionts in sponges to date and provides novel insights into the complex structure of these unique associations.     

Toward a census of bacteria in soil

For more than a century, microbiologists have sought to determine the species richness of bacteria in soil, but the extreme complexity and unknown structure of soil microbial communities have obscured the answer. We developed a statistical model that makes the problem of estimating richness statistically accessible by evaluating the characteristics of samples drawn from simulated communities with parametric community distributions. We identified simulated communities with rank-abundance distributions that followed a truncated lognormal distribution whose samples resembled the structure of 16S rRNA gene sequence collections made using Alaskan and Minnesotan soils. The simulated communities constructed based on the distribution of 16S rRNA gene sequences sampled from the Alaskan and Minnesotan soils had a richness of 5,000 and 2,000 operational taxonomic units (OTUs), respectively, where an OTU represents a collection of sequences not more than 3% distant from each other. To sample each of these OTUs in the Alaskan 16S rRNA gene library at least twice, 480,000 sequences would be required; however, to estimate the richness of the simulated communities using nonparametric richness estimators would require only 18,000 sequences. Quantifying the richness of complex environments such as soil is an important step in building an ecological framework. We have shown that generating sufficient sequence data to do so requires less sequencing effort than completely sequencing a bacterial genome.     

Investigating Microbial Eukaryotic Diversity from a Global Census: Insights from a Comparison of Pyrotag and Full-Length Sequences of 18S rRNA Genes

Next-generation DNA sequencing (NGS) approaches are rapidly surpassing Sanger sequencing for characterizing the diversity of natural microbial communities. Despite this rapid transition, few comparisons exist between Sanger sequences and the generally much shorter reads of NGS. Operational taxonomic units (OTUs) derived from full-length (Sanger sequencing) and pyrotag (454 sequencing of the V9 hypervariable region) sequences of 18S rRNA genes from 10 global samples were analyzed in order to compare the resulting protistan community structures and species richness. Pyrotag OTUs called at 98% sequence similarity yielded numbers of OTUs that were similar overall to those for full-length sequences when the latter were called at 97% similarity. Singleton OTUs strongly influenced estimates of species richness but not the higher-level taxonomic composition of the community. The pyrotag and full-length sequence data sets had slightly different taxonomic compositions of rhizarians, stramenopiles, cryptophytes, and haptophytes, but the two data sets had similarly high compositions of alveolates. Pyrotag-based OTUs were often derived from sequences that mapped to multiple full-length OTUs at 100% similarity. Thus, pyrotags sequenced from a single hypervariable region might not be appropriate for establishing protistan species-level OTUs. However, nonmetric multidimensional scaling plots constructed with the two data sets yielded similar clusters, indicating that beta diversity analysis results were similar for the Sanger and NGS sequences. Short pyrotag sequences can provide holistic assessments of protistan communities, although care must be taken in interpreting the results. The longer reads (>500 bp) that are now becoming available through NGS should provide powerful tools for assessing the diversity of microbial eukaryotic assemblages.     

Low sequencing efforts bias analyses of shared taxa in microbial communities

The potential for comparing microbial community population structures has been greatly enhanced by developments in next generation sequencing methods that can generate hundreds of thousands to millions of reads in a single run. Conversely, many microbial community comparisons have been published with no more than 1,000 sequences per sample. These studies have presented data on levels of shared operational taxonomic units (OTUs) between communities. Due to lack of coverage, that approach might compromise the conclusions about microbial diversity and the degree of difference between environments. In this study, we present data from recent studies that highlight this problem. Also, we analyzed datasets of 16 rRNA sequences with small and high sequence coverage from different environments to demonstrate that the level of sequencing effort used for analyzing microbial communities biases the results. We randomly sampled pyrosequencing-generated 16S rRNA gene libraries with increasing sequence effort. Sequences were used to calculate Good's coverage, the percentage of shared OTUs, and phylogenetic distance measures. Our data showed that simple counts of presence/absence of taxonomic unities do not reflect the real similarity in membership and structure of the bacterial communities and that community comparisons based on phylogenetic tests provide a way to test statistically significant differences between two or more environments without need an exhaustive sampling effort.     

Reproducibility and quantitation of amplicon sequencing-based detection

To determine the reproducibility and quantitation of the amplicon sequencing-based detection approach for analyzing microbial community structure, a total of 24 microbial communities from a long-term global change experimental site were examined. Genomic DNA obtained from each community was used to amplify 16S rRNA genes with two or three barcode tags as technical replicates in the presence of a small quantity (0.1% wt/wt) of genomic DNA from Shewanella oneidensis MR-1 as the control. The technical reproducibility of the amplicon sequencing-based detection approach is quite low, with an average operational taxonomic unit (OTU) overlap of 17.2%±2.3% between two technical replicates, and 8.2%±2.3% among three technical replicates, which is most likely due to problems associated with random sampling processes. Such variations in technical replicates could have substantial effects on estimating β-diversity but less on α-diversity. A high variation was also observed in the control across different samples (for example, 66.7-fold for the forward primer), suggesting that the amplicon sequencing-based detection approach could not be quantitative. In addition, various strategies were examined to improve the comparability of amplicon sequencing data, such as increasing biological replicates, and removing singleton sequences and less-representative OTUs across biological replicates. Finally, as expected, various statistical analyses with preprocessed experimental data revealed clear differences in the composition and structure of microbial communities between warming and non-warming, or between clipping and non-clipping. Taken together, these results suggest that amplicon sequencing-based detection is useful in analyzing microbial community structure even though it is not reproducible and quantitative. However, great caution should be taken in experimental design and data interpretation when the amplicon sequencing-based detection approach is used for quantitative analysis of the β-diversity of microbial communities.     

Down under the tunic: bacterial biodiversity hotspots and widespread ammonia-oxidizing archaea in coral reef ascidians

Ascidians are ecologically important components of marine ecosystems yet the ascidian microbiota remains largely unexplored beyond a few model species. We used 16S rRNA gene tag pyrosequencing to provide a comprehensive characterization of microbial symbionts in the tunic of 42 Great Barrier Reef ascidian samples representing 25 species. Results revealed high bacterial biodiversity (3 217 unique operational taxonomic units (OTU_0.03) from 19 described and 14 candidate phyla) and the widespread occurrence of ammonia-oxidizing Thaumarchaeota in coral reef ascidians (24 of 25 host species). The ascidian microbiota was clearly differentiated from seawater microbial communities and included symbiont lineages shared with other invertebrate hosts as well as unique, ascidian-specific phylotypes. Several rare seawater microbes were markedly enriched (200–700 fold) in the ascidian tunic, suggesting that the rare biosphere of seawater may act as a conduit for horizontal symbiont transfer. However, most OTUs (71%) were rare and specific to single hosts and a significant correlation between host relatedness and symbiont community similarity was detected, indicating a high degree of host-specificity and potential role of vertical transmission in structuring these communities. We hypothesize that the complex ascidian microbiota revealed herein is maintained by the dynamic microenvironments within the ascidian tunic, offering optimal conditions for different metabolic pathways such as ample chemical substrate (ammonia-rich host waste) and physical habitat (high oxygen, low irradiance) for nitrification. Thus, ascidian hosts provide unique and fertile niches for diverse microorganisms and may represent an important and previously unrecognized habitat for nitrite/nitrate regeneration in coral reef ecosystems.     

Metagenomic and Small-Subunit rRNA Analyses Reveal the Genetic Diversity of Bacteria, Archaea, Fungi, and Viruses in Soil

Recent studies have highlighted the surprising richness of soil bacterial communities; however, bacteria are not the only microorganisms found in soil. To our knowledge, no study has compared the diversities of the four major microbial taxa, i.e., bacteria, archaea, fungi, and viruses, from an individual soil sample. We used metagenomic and small-subunit RNA-based sequence analysis techniques to compare the estimated richness and evenness of these groups in prairie, desert, and rainforest soils. By grouping sequences at the 97% sequence similarity level (an operational taxonomic unit [OTU]), we found that the archaeal and fungal communities were consistently less even than the bacterial communities. Although total richness levels are difficult to estimate with a high degree of certainty, the estimated number of unique archaeal or fungal OTUs appears to rival or exceed the number of unique bacterial OTUs in each of the collected soils. In this first study to comprehensively survey viral communities using a metagenomic approach, we found that soil viruses are taxonomically diverse and distinct from the communities of viruses found in other environments that have been surveyed using a similar approach. Within each of the four microbial groups, we observed minimal taxonomic overlap between sites, suggesting that soil archaea, bacteria, fungi, and viruses are globally as well as locally diverse.     

A novel method for determining microflora composition using dynamic phylogenetic analysis of 16S ribosomal RNA deep sequencing data

Deep sequencing of the 16S rRNA gene provides a comprehensive view of bacterial communities in a particular environment and has expanded our ability to study the impact of the microflora on human health and disease. Current analysis methods rely on comparisons of the sequences generated with an expanding but limited set of annotated 16S rRNA sequences or phylogenic clustering of sequences based on arbitrary similarity cutoffs. We describe a novel approach to characterize bacterial composition using deep sequencing of 16S rRNA gene. Our method defines operational taxonomic units based on phylogenetic tree reconstruction and dynamic clustering of sequences using solely sequencing data. These OTUs can be used to identify differences in bacteria abundance between environments. This approach can perform better than previous phylogenetic methods and will significantly improve our understanding of the microfloral role on human diseases by providing a comprehensive analysis of the microbial composition from various bacterial communities.     

BIPES,a cost-effective high-throughput method for assessing microbial diversity

Pyrosequencing of 16S rRNA (16S) variable tags has become the most popular method for assessing microbial diversity, but the method remains costly for the evaluation of large numbers of environmental samples with high sequencing depths. We developed a barcoded Illumina paired-end (PE) sequencing (BIPES) method that sequences each 16S V6 tag from both ends on the Illumina HiSeq 2000, and the PE reads are then overlapped to obtain the V6 tag. The average accuracy of Illumina single-end (SE) reads was only 97.9%, which decreased from ∼99.9% at the start of the read to less than 85% at the end of the read; nevertheless, overlapping of the PE reads significantly increased the sequencing accuracy to 99.65% by verifying the 3′ end of each SE in which the sequencing quality was degraded. After the removal of tags with two or more mismatches within the medial 40–70 bases of the reads and of tags with any primer errors, the overall base sequencing accuracy of the BIPES reads was further increased to 99.93%. The BIPES reads reflected the amounts of the various tags in the initial template, but long tags and high GC tags were underestimated. The BIPES method yields 20–50 times more 16S V6 tags than does pyrosequencing in a single-flow cell run, and each of the BIPES reads costs less than 1/40 of a pyrosequencing read. As a laborsaving and cost-effective method, BIPES can be routinely used to analyze the microbial ecology of both environmental and human microbiomes.     

Bacterial diversity and community composition from seasurface to subseafloor

We investigated compositional relationships between bacterial communities in the water column and those in deep-sea sediment at three environmentally distinct Pacific sites (two in the Equatorial Pacific and one in the North Pacific Gyre). Through pyrosequencing of the v4–v6 hypervariable regions of the 16S ribosomal RNA gene, we characterized 450 104 pyrotags representing 29 814 operational taxonomic units (OTUs, 97% similarity). Hierarchical clustering and non-metric multidimensional scaling partition the samples into four broad groups, regardless of geographic location: a photic-zone community, a subphotic community, a shallow sedimentary community and a subseafloor sedimentary community (⩾1.5 meters below seafloor). Abundance-weighted community compositions of water-column samples exhibit a similar trend with depth at all sites, with successive epipelagic, mesopelagic, bathypelagic and abyssopelagic communities. Taxonomic richness is generally highest in the water-column O₂ minimum zone and lowest in the subseafloor sediment. OTUs represented by abundant tags in the subseafloor sediment are often present but represented by few tags in the water column, and represented by moderately abundant tags in the shallow sediment. In contrast, OTUs represented by abundant tags in the water are generally absent from the subseafloor sediment. These results are consistent with (i) dispersal of marine sedimentary bacteria via the ocean, and (ii) selection of the subseafloor sedimentary community from within the community present in shallow sediment.     

Spatial scales of bacterial community diversity at cold seeps (Eastern Mediterranean Sea)

Cold seeps are highly productive, fragmented marine ecosystems that form at the seafloor around hydrocarbon emission pathways. The products of microbial utilization of methane and other hydrocarbons fuel rich chemosynthetic communities at these sites, with much higher respiration rates compared with the surrounding deep-sea floor. Yet little is known as to the richness, composition and spatial scaling of bacterial communities of cold seeps compared with non-seep communities. Here we assessed the bacterial diversity across nine different cold seeps in the Eastern Mediterranean deep-sea and surrounding seafloor areas. Community similarity analyses were carried out based on automated ribosomal intergenic spacer analysis (ARISA) fingerprinting and high-throughput 454 tag sequencing and were combined with in situ and ex situ geochemical analyses across spatial scales of a few tens of meters to hundreds of kilometers. Seep communities were dominated by Deltaproteobacteria, Epsilonproteobacteria and Gammaproteobacteria and shared, on average, 36% of bacterial types (ARISA OTUs (operational taxonomic units)) with communities from nearby non-seep deep-sea sediments. Bacterial communities of seeps were significantly different from those of non-seep sediments. Within cold seep regions on spatial scales of only tens to hundreds of meters, the bacterial communities differed considerably, sharing <50% of types at the ARISA OTU level. Their variations reflected differences in porewater sulfide concentrations from anaerobic degradation of hydrocarbons. This study shows that cold seep ecosystems contribute substantially to the microbial diversity of the deep-sea.     

Taxonomic Classification of Bacterial 16S rRNA Genes Using Short Sequencing Reads: Evaluation of Effective Study Designs

Massively parallel high throughput sequencing technologies allow us to interrogate the microbial composition of biological samples at unprecedented resolution. The typical approach is to perform high-throughout sequencing of 16S rRNA genes, which are then taxonomically classified based on similarity to known sequences in existing databases. Current technologies cause a predicament though, because although they enable deep coverage of samples, they are limited in the length of sequence they can produce. As a result, high-throughout studies of microbial communities often do not sequence the entire 16S rRNA gene. The challenge is to obtain reliable representation of bacterial communities through taxonomic classification of short 16S rRNA gene sequences. In this study we explored properties of different study designs and developed specific recommendations for effective use of short-read sequencing technologies for the purpose of interrogating bacterial communities, with a focus on classification using naïve Bayesian classifiers. To assess precision and coverage of each design, we used a collection of ∼8,500 manually curated 16S rRNA gene sequences from cultured bacteria and a set of over one million bacterial 16S rRNA gene sequences retrieved from environmental samples, respectively. We also tested different configurations of taxonomic classification approaches using short read sequencing data, and provide recommendations for optimal choice of the relevant parameters. We conclude that with a judicious selection of the sequenced region and the corresponding choice of a suitable training set for taxonomic classification, it is possible to explore bacterial communities at great depth using current technologies, with only a minimal loss of taxonomic resolution.     

Archaea Appear to Dominate the Microbiome of Inflatella pellicula Deep Sea Sponges

Microbes associated with marine sponges play significant roles in host physiology. Remarkable levels of microbial diversity have been observed in sponges worldwide through both culture-dependent and culture-independent studies. Most studies have focused on the structure of the bacterial communities in sponges and have involved sponges sampled from shallow waters. Here, we used pyrosequencing of 16S rRNA genes to compare the bacterial and archaeal communities associated with two individuals of the marine sponge Inflatella pellicula from the deep-sea, sampled from a depth of 2,900 m, a depth which far exceeds any previous sequence-based report of sponge-associated microbial communities. Sponge-microbial communities were also compared to the microbial community in the surrounding seawater. Sponge-associated microbial communities were dominated by archaeal sequencing reads with a single archaeal OTU, comprising ∼60% and ∼72% of sequences, being observed from Inflatella pellicula. Archaeal sequencing reads were less abundant in seawater (∼11% of sequences). Sponge-associated microbial communities were less diverse and less even than any other sponge-microbial community investigated to date with just 210 and 273 OTUs (97% sequence identity) identified in sponges, with 4 and 6 dominant OTUs comprising ∼88% and ∼89% of sequences, respectively. Members of the candidate phyla, SAR406, NC10 and ZB3 are reported here from sponges for the first time, increasing the number of bacterial phyla or candidate divisions associated with sponges to 43. A minor cohort from both sponge samples (∼0.2% and ∼0.3% of sequences) were not classified to phylum level. A single OTU, common to both sponge individuals, dominates these unclassified reads and shares sequence homology with a sponge associated clone which itself has no known close relative and may represent a novel taxon.     

Influences of Plant Species,Season and Location on Leaf Endophytic Bacterial Communities of Non-Cultivated Plants

Bacteria are known to be associated endophytically with plants. Research on endophytic bacteria has identified their importance in food safety, agricultural production and phytoremediation. However, the diversity of endophytic bacterial communities and the forces that shape their compositions in non-cultivated plants are largely uncharacterized. In this study, we explored the diversity, community structure, and dynamics of endophytic bacteria in different plant species in the Tallgrass Prairie Preserve of northern Oklahoma, USA. High throughput sequencing of amplified segments of bacterial rDNA from 81 samples collected at four sampling times from five plant species at four locations identified 335 distinct OTUs at 97% sequence similarity, representing 16 phyla. Proteobacteria was the dominant phylum in the communities, followed by the phyla Bacteriodetes and Actinobacteria. Bacteria from four classes of Proteobacteria were detected with Alphaproteobacteria as the dominant class. Analysis of molecular variance revealed that host plant species and collecting date had significant influences on the compositions of the leaf endophytic bacterial communities. The proportion of Alphaproteobacteria was much higher in the communities from Asclepias viridis than from other plant species and differed from month to month. The most dominant bacterial groups identified in LDA Effect Size analysis showed host-specific patterns, indicating mutual selection between host plants and endophytic bacteria and that leaf endophytic bacterial compositions were dynamic, varying with the host plant’s growing season in three distinct patterns. In summary, next generation sequencing has revealed variations in the taxonomic compositions of leaf endophytic bacterial communities dependent primarily on the nature of the plant host species.     

Comparative Phylogenetic Assignment of Environmental Sequences of Genes Encoding 16S rRNA and Numerically Abundant Culturable Bacteria from an Anoxic Rice Paddy Soil

We used both cultivation and direct recovery of bacterial 16S rRNA gene (rDNA) sequences to investigate the structure of the bacterial community in anoxic rice paddy soil. Isolation and phenotypic characterization of 19 saccharolytic and cellulolytic strains are described in the accompanying paper (K.-J. Chin, D. Hahn, U. Hengstmann, W. Liesack, and P. H. Janssen, Appl. Environ. Microbiol. 65:5042–5049, 1999). Here we describe the phylogenetic positions of these strains in relation to 57 environmental 16S rDNA clone sequences. Close matches between the two data sets were obtained for isolates from the culturable populations determined by the most-probable-number counting method to be large (3 × 10⁷ to 2.5 × 10⁸ cells per g [dry weight] of soil). This included matches with 16S rDNA similarity values greater than 98% within distinct lineages of the division Verrucomicrobia (strain PB90-1) and the Cytophaga-Flavobacterium-Bacteroides group (strains XB45 and PB90-2), as well as matches with similarity values greater than 95% within distinct lines of descent of clostridial cluster XIVa (strain XB90) and the family Bacillaceae (strain SB45). In addition, close matches with similarity values greater than 95% were obtained for cloned 16S rDNA sequences and bacteria (strains DR1/8 and RPec1) isolated from the same type of rice paddy soil during previous investigations. The correspondence between culture methods and direct recovery of environmental 16S rDNA suggests that the isolates obtained are representative geno- and phenotypes of predominant bacterial groups which account for 5 to 52% of the total cells in the anoxic rice paddy soil. Furthermore, our findings clearly indicate that a dual approach results in a more objective view of the structural and functional composition of a soil bacterial community than either cultivation or direct recovery of 16S rDNA sequences alone.     

Identification of Lactobacillus Isolates from the Gastrointestinal Tract, Silage, and Yoghurt by 16S-23S rRNA Gene Intergenic Spacer Region Sequence Comparisons

Lactobacillus isolates were identified by PCR amplification and sequencing of the region between the 16S and 23S rRNA genes (spacer region). The sequences obtained from the isolates were compared to those of reference strains held in GenBank. A similarity of 97.5% or greater was considered to provide identification. To check the reliability of the method, the V2-V3 region of the 16S rRNA gene was amplified and sequenced in the case of isolates whose spacer region sequences were less than 99% similar to that of a reference strain. Confirmation of identity was obtained in all instances. Spacer region sequencing provided rapid and accurate identification of Lactobacillus isolates obtained from gastrointestinal, yoghurt, and silage samples. It had an advantage over 16S V2-V3 sequence comparisons because it distinguished between isolates of Lactobacillus casei and Lactobacillus rhamnosus.     

Large variability of bathypelagic microbial eukaryotic communities across the world's oceans

In this work, we study the diversity of bathypelagic microbial eukaryotes (0.8–20 μm) in the global ocean. Seawater samples from 3000 to 4000 m depth from 27 stations in the Atlantic, Pacific and Indian Oceans were analyzed by pyrosequencing the V4 region of the 18S ribosomal DNA. The relative abundance of the most abundant operational taxonomic units agreed with the results of a parallel metagenomic analysis, suggesting limited PCR biases in the tag approach. Although rarefaction curves for single stations were seldom saturated, the global analysis of all sequences together suggested an adequate recovery of bathypelagic diversity. Community composition presented a large variability among samples, which was poorly explained by linear geographic distance. In fact, the similarity between communities was better explained by water mass composition (26% of the variability) and the ratio in cell abundance between prokaryotes and microbial eukaryotes (21%). Deep diversity appeared dominated by four taxonomic groups (Collodaria, Chrysophytes, Basidiomycota and MALV-II) appearing in different proportions in each sample. Novel diversity amounted to 1% of the pyrotags and was lower than expected. Our study represents an essential step in the investigation of bathypelagic microbial eukaryotes, indicating dominating taxonomic groups and suggesting idiosyncratic assemblages in distinct oceanic regions.     

Defining seasonal marine microbial community dynamics

Here we describe, the longest microbial time-series analyzed to date using high-resolution 16S rRNA tag pyrosequencing of samples taken monthly over 6 years at a temperate marine coastal site off Plymouth, UK. Data treatment effected the estimation of community richness over a 6-year period, whereby 8794 operational taxonomic units (OTUs) were identified using single-linkage preclustering and 21 130 OTUs were identified by denoising the data. The Alphaproteobacteria were the most abundant Class, and the most frequently recorded OTUs were members of the Rickettsiales (SAR 11) and Rhodobacteriales. This near-surface ocean bacterial community showed strong repeatable seasonal patterns, which were defined by winter peaks in diversity across all years. Environmental variables explained far more variation in seasonally predictable bacteria than did data on protists or metazoan biomass. Change in day length alone explains >65% of the variance in community diversity. The results suggested that seasonal changes in environmental variables are more important than trophic interactions. Interestingly, microbial association network analysis showed that correlations in abundance were stronger within bacterial taxa rather than between bacteria and eukaryotes, or between bacteria and environmental variables.     

The activated sludge ecosystem contains a core community of abundant organisms

Understanding the microbial ecology of a system requires that the observed population dynamics can be linked to their metabolic functions. However, functional characterization is laborious and the choice of organisms should be prioritized to those that are frequently abundant (core) or transiently abundant, which are therefore putatively make the greatest contribution to carbon turnover in the system. We analyzed the microbial communities in 13 Danish wastewater treatment plants with nutrient removal in consecutive years and a single plant periodically over 6 years, using Illumina sequencing of 16S ribosomal RNA amplicons of the V4 region. The plants contained a core community of 63 abundant genus-level operational taxonomic units (OTUs) that made up 68% of the total reads. A core community consisting of abundant OTUs was also observed within the incoming wastewater to three plants. The net growth rate for individual OTUs was quantified using mass balance, and it was found that 10% of the total reads in the activated sludge were from slow or non-growing OTUs, and that their measured abundance was primarily because of immigration with the wastewater. Transiently abundant organisms were also identified. Among them the genus Nitrotoga (class Betaproteobacteria) was the most abundant putative nitrite oxidizer in a number of activated sludge plants, which challenges previous assumptions that Nitrospira (phylum Nitrospirae) are the primary nitrite-oxidizers in activated sludge systems with nutrient removal.