Analysis of bacterioplankton genes in an impaired Great Lakes harbour reveals seasonal metabolic shifts and a previously undetected cyanobacterium.

Hamilton Harbour is an impaired embayment of Lake Ontario that experiences seasonal algal blooms despite decades of remedial efforts. To study the harbour's cyanobacterial and heterotrophic bacterial communities, we extracted and sequenced community DNA from surface water samples collected biweekly from different sites during summer and fall. Assembled contigs were annotated at the phylum level, and Cyanobacteria were further characterized at order and species levels. Actinobacteria were most abundant in early summer, while Cyanobacteria were dominant in mid-summer. Microcystis aeruginosa and Limnoraphis robusta were most abundant throughout the sampling period, expanding the documented diversity of Cyanobacteria in Hamilton Harbour. Functional annotations were performed using the MG-RAST pipeline and SEED database, revealing that genes for photosynthesis, nitrogen metabolism, and aromatic compound metabolism varied in relative abundances over the season, while phosphorus metabolism was consistent, suggesting that these genes remained essential despite fluctuating environmental conditions and community succession. We observed seasonal shifts from anoxygenic to oxygenic phototrophy, and from ammonia assimilation to nitrogen fixation, coupled with decreasing heterotrophic bacteria and increasing Cyanobacteria relative abundances. Our data contribute important insights into bacterial taxa and functional potentials in Hamilton Harbour, revealing seasonal and spatial dynamics that can be used to inform ongoing remediation efforts.

The influence of biofertilizers on leaf economics spectrum traits in a herbaceous crop.

Microbial inoculations or 'biofertilizers' represent novel contributions to sustainable agriculture. While belowground mechanisms surrounding how biofertilizers enhance crop production are well described, their role in aboveground trait expression remains less well explored. We quantified infraspecific variation in leaf economics spectrum (LES) traits in response to 10 biofertilizer treatments in basil (Ocimum basiclicum) cultivated under hydroponic conditions. Multiple physiological (i.e. maximum photosynthesis rates (A), dark respiration (R), and leaf-level light compensation points) and morphological (i.e. leaf mass per area (LMA) and leaf thickness) traits varied significantly across microbial treatments. Following treatments, basil plants differentiated from one another along an infraspecific LES, with certain plants expressing more resource-acquiring LES trait values (i.e. high A, R, leaf N, and low LMA), versus others that expressed the opposite suite of resource-conserving LES trait values. Infraspecific trait covariation largely matched LES patterns observed among plants globally. Bivariate and multivariate trait analyses further revealed that certain treatments-namely those including closely related Bacillus and Brevibacillus species strains-increased leaf resource capture traits such as A and leaf N. Biofertilizers influence plant performance through a role in moderating infraspecific leaf trait variation, thereby suggesting aboveground leaf traits may be used to diagnose optimal biofertilizer formulations in basil and other crops.

Metagenomic Analysis of Virus Diversity and Relative Abundance in a Eutrophic Freshwater Harbour.

Aquatic viruses have been extensively studied over the past decade, yet fundamental aspects of freshwater virus communities remain poorly described. Our goal was to characterize virus communities captured in the >0.22 µm size-fraction seasonally and spatially in a freshwater harbour. Community DNA was extracted from water samples and sequenced on an Illumina HiSeq platform. Assembled contigs were annotated as belonging to the virus groups (i.e., order or family) Caudovirales, Mimiviridae, Phycodnaviridae, and virophages (Lavidaviridae), or to other groups of undefined viruses. Virophages were often the most abundant group, and discrete virophage taxa were remarkably stable across sites and dates despite fluctuations in Mimiviridae community composition. Diverse Mimiviridae contigs were detected in the samples and the two sites contained distinct Mimiviridae communities, suggesting that Mimiviridae are important algal viruses in this system. Caudovirales and Phycodnaviridae were present at low abundances in most samples. Of the 18 environmental parameters tested, only chlorophyll a explained the variation in the data at the order or family level of classification. Overall, our findings provide insight into freshwater virus community assemblages by expanding the documented diversity of freshwater virus communities, highlighting the potential ecological importance of virophages, and revealing distinct communities over small spatial scales.

Endophytic Bacterial Community Structure and Function of Herbaceous Plants From Petroleum Hydrocarbon Contaminated and Non-contaminated Sites.

Bacterial endophytes (BEs) are non-pathogenic residents of healthy plant tissues that can confer benefits to plants. Many Bacterial endophytes have been shown to contribute to plant growth and health, alleviation of plant stress and to in-planta contaminant-degradation. This study examined the endophytic bacterial communities of plants growing abundantly in a heavily hydrocarbon contaminated site, and compared them to those found in the same species at a non-contaminated. We used culture- dependent and independent methods to characterize the community structure, hydrocarbon degrading capabilities, and plant growth promoting traits of cultivable endophytes isolated from Achillea millefolium, Solidago Canadensis, and Daucus carota plants from these two sites. Culture- dependent and independent analyses revealed class Gammaproteobacteria predominated in all the plants regardless of the presence of petroleum hydrocarbon, with Pantoea spp. as largely dominant. It was interesting to note a >50% taxonomic overlap (genus level) of 16s rRNA high throughput amplicon sequences with cultivable endophytes. PERMANOVA analysis of TRFLP fragments revealed significant structural differences between endophytic bacterial communities from hydrocarbon-contaminated and non-contaminated soils-however, there was no marked difference in their functional capabilities. Pantoea spp. demonstrated plant beneficial characteristics, such as P solubilization, indole-3-acetic acid production and presence of 1-aminocyclopropane-1-carboxylate deaminase. Our findings reveal that functional capabilities of bacterial isolates being examined were not influenced by the presence of contamination; and that the stem endosphere supports ubiquitous BEs that were consistent throughout plant hosts and sites.

Rapid degradation of Deepwater Horizon spilled oil by indigenous microbial communities in Louisiana saltmarsh sediments.

The Deepwater Horizon oil spill led to the severe contamination of coastal environments in the Gulf of Mexico. A previous study detailed coastal saltmarsh erosion and recovery in a number of oil-impacted and nonimpacted reference sites in Barataria Bay, Louisiana over the first 18 months after the spill. Concentrations of alkanes and polyaromatic hydrocarbons (PAHs) at oil-impacted sites significantly decreased over this time period. Here, a combination of DNA, lipid, and isotopic approaches confirm that microbial biodegradation was contributing to the observed petroleum mass loss. Natural abundance (14)C analysis of microbial phospholipid fatty acids (PLFA) reveals that petroleum-derived carbon was a primary carbon source for microbial communities at impacted sites several months following oil intrusion when the highest concentrations of oil were present. Also at this time, microbial community analysis suggests that community structure of all three domains has shifted with the intrusion of oil. These results suggest that Gulf of Mexico marsh sediments have considerable biodegradation potential and that natural attenuation is playing a role in impacted sites.

Probing the functional diversity of global pristine soil communities with 3-chlorobenzoate reveals that communities of generalists dominate catabolic transformation.

Understanding of functional diversity of microbial populations has lagged description of their molecular diversity. Differences in substrate specificity, kinetics, products, and regulation can dramatically influence phenotypic variation among closely related strains, features that are missed when the strains studied are the fastest-growing and most easily isolated from serial enrichments. To investigate the broader bacterial diversity underlying degradation of anthropogenic chemicals in nature, we studied the 3-chlorobenzoate (3-CBA) degradation rate in a collection of aerobic 3-CBA degraders previously isolated from undisturbed soils in two representative ecosystems: (i) Mediterranean sclerophyllous woodlands in California, Chile, South Africa, and Australia and (ii) boreal forests in Canada and Russia. The majority of isolates degraded 3-CBA slowly and did not completely mineralize 1.0 mM 3-CBA within 1 week. Those with intermediate degradation rates had incomplete degradation pathways and produced colored intermediates indicative of chlorocatechol, a product likely metabolized by other members of the community. About 10% of the isolates grew rapidly and mineralized greater than 90% of the 3-CBA, but because of population heterogeneity in soil, they are likely not large contributors to a soil's total transformation capacity. This suggests that xenobiotic degradation in nature is carried out by a community of cometabolic generalists and not by the efficient specialists that have been traditionally studied in the laboratory. A subset of 58 genotypically distinct strains able to degrade >80% of the 3-CBA was examined for their catabolic versatility using 45 different compounds: mono- and dichlorinated benzoates, phenols, anilines, toluenes, nitrobenzenes, chlorobenzenes, and 2,4-dichlorophenoxyacetic acid. The isolates degraded from 2 to more than 30 compounds with a median of 7, but there was no correlation to habitat of isolation or 3-CBA activity. However, these findings were indicative of finer-scale functional diversity.

Assessing microbial carbon sources and potential PAH degradation using natural abundance 14C analysis.

Natural abundance (14)C analysis was applied to PLFAs collected from an industrial site in southern Ontario in order to assess microbial carbon sources and potential PAH biodegradation in soils. Δ(14)C of microbial phospholipid fatty acids (PLFA) at the site ranged from +54‰ to -697‰. Comparison of these values to surrounding carbon sources found that microbial carbon sources were derived primarily from vegetation and/or natural organic matter present in the soils rather than PAHs. This study highlights that microbes are able to utilize almost all available pools of organic matter including older pools which are thought to contain recalcitrant compounds. Furthermore, it shows that even with the presence of an active microbial community, there may be little biodegradation of PAHs. This study illustrates challenges in assessing microbial activity in the environment and the advantage of using natural abundance (14)C analysis as a tool to elucidate microbial carbon sources.

Land-use change and soil type are drivers of fungal and archaeal communities in the Pampa biome.

The current study aimed to test the hypothesis that both land-use change and soil type are responsible for the major changes in the fungal and archaeal community structure and functioning of the soil microbial community in Brazilian Pampa biome. Soil samples were collected at sites with different land-uses (native grassland, native forest, Eucalyptus and Acacia plantation, soybean and watermelon field) and in a typical toposequence in Pampa biome formed by Paleudult, Albaqualf and alluvial soils. The structure of soil microbial community (archaeal and fungal) was evaluated by ribosomal intergenic spacer analysis and soil functional capabilities were measured by microbial biomass carbon and metabolic quotient. We detected different patterns in microbial community driven by land-use change and soil type, showing that both factors are significant drivers of fungal and archaeal community structure and biomass and microbial activity. Fungal community structure was more affected by land-use and archaeal community was more affected by soil type. Irrespective of the land-use or soil type, a large percentage of operational taxonomic unit were shared among the soils. We accepted the hypothesis that both land-use change and soil type are drivers of archaeal and fungal community structure and soil functional capabilities. Moreover, we also suggest the existence of a soil microbial core.

Changes in bacterial community structure after exposure to silver nanoparticles in natural waters.

Silver nanoparticles (AgNPs) are widely used in commercial products as antibacterial agents, but AgNPs might be hazardous to the environment and natural aquatic bacterial communities. Our recent research demonstrated that AgNPs rapidly but temporarily inhibited natural bacterioplankton production. The current study investigates the mechanism for the observed bacterial reaction to AgNPs by examining how AgNPs impact bacterial abundance, metabolic activity (5-cyano-2,3-ditolyl tetrazolium chloride (CTC+) cells), and 16S rRNA community composition. Natural bacterioplankton communities were dosed with carboxy-functionalized AgNPs at four concentrations (0.01-1 mg-Ag/L), incubated in triplicate, and monitored over 5 days. Ionic silver (AgNO(3)) and Milli-Q water treatments were used as a positive and negative control, respectively. Four general AgNP exposure responses, relative to the negative control, were observed: (1) intolerant, (2) impacted but recovering, (3) tolerant, and (4) stimulated phylotypes. Relationships between cell activity indicators and bacterial phylotypes, suggested that tolerant and recovering bacteria contributed the most to the community's productivity and rare bacteria phylotypes stimulated by AgNPs did not appear to contribute much to cell activity. Overall, natural bacterial communities tolerated single, low level AgNP doses and had similar activity levels to the negative control within five days of exposure, but bacterial community composition was different from that of the control.

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.

Bacterial community dynamics in the hyporheic zone of an intermittent stream.

The dynamics of in situ bacterial communities in the hyporheic zone of an intermittent stream were described in high spatiotemporal detail. We assessed community dynamics in stream sediments and interstitial pore water over a two-year period using terminal-restriction fragment length polymorphism. Here, we show that sediments remained saturated despite months of drought and limited hydrologic connectivity. The intermittency of stream surface water affected interstitial pore water communities more than hyporheic sediment communities. Seasonal changes in bacterial community composition was significantly associated with water intermittency, phosphate concentrations, temperature, nitrate and dissolved organic carbon (DOC) concentrations. During periods of low- to no-surface water, communities changed from being rich in operational taxonomic units (OTUs) in isolated surface pools, to a few OTUs overall, including an overall decline in both common and rare taxa. Individual OTUs were compared between porewater and sediments. A total of 19% of identified OTUs existed in both porewater and sediment samples, suggesting that bacteria use hyporheic sediments as a type of refuge from dessication, transported through hydrologically connected pore spaces. Stream intermittency impacted bacterial diversity on rapid timescales (that is, within days), below-ground and in the hyporheic zone. Owing to the coupling of intermittent streams to the surrounding watershed, we stress the importance of understanding connectivity at the pore scale, consequences for below-ground and above-ground biodiversity and nutrient processing, and across both short- and long-time periods (that is, days to months to years).

Comparison of commercial DNA extraction kits for isolation and purification of bacterial and eukaryotic DNA from PAH-contaminated soils.

Molecular characterization of the microbial populations of soils and sediments contaminated with polycyclic aromatic hydrocarbons (PAHs) is often a first step in assessing intrinsic biodegradation potential. However, soils are problematic for molecular analysis owing to the presence of organic matter, such as humic acids. Furthermore, the presence of contaminants, such as PAHs, can cause further challenges to DNA extraction, quantification, and amplification. The goal of our study was to compare the effectiveness of four commercial soil DNA extraction kits (UltraClean Soil DNA Isolation kit, PowerSoil DNA Isolation kit, PowerMax Soil DNA Isolation kit, and FastDNA SPIN kit) to extract pure, high-quality bacterial and eukaryotic DNA from PAH-contaminated soils. Six different contaminated soils were used to determine if there were any biases among the kits due to soil properties or level of contamination. Extracted DNA was used as a template for bacterial 16S rDNA and eukaryotic 18S rDNA amplifications, and PCR products were subsequently analyzed using denaturing gel gradient electrophoresis (DGGE). We found that the FastDNA SPIN kit provided significantly higher DNA yields for all soils; however, it also resulted in the highest levels of humic acid contamination. Soil texture and organic carbon content of the soil did not affect the DNA yield of any kit. Moreover, a liquid-liquid extraction of the DNA extracts found no residual PAHs, indicating that all kits were effective at removing contaminants in the extraction process. Although the PowerSoil DNA Isolation kit gave relatively low DNA yields, it provided the highest quality DNA based on successful amplification of both bacterial and eukaryotic DNA for all six soils. DGGE fingerprints among the kits were dramatically different for both bacterial and eukaryotic DNA. The PowerSoil DNA Isolation kit revealed multiple bands for each soil and provided the most consistent DGGE profiles among replicates for both bacterial and eukaryotic DNA.

Rethinking microbial diversity analysis in the high throughput sequencing era.

The analysis of amplified and sequenced 16S rRNA genes has become the most important single approach for microbial diversity studies. The new sequencing technologies allow for sequencing thousands of reads in a single run and a cost-effective option is split into a single run across many samples. However for this type of investigation the key question that needs to be answered is how many samples can be sequenced without biasing the results due to lack of sequence representativeness? In this work we demonstrated that the level of sequencing effort used for analyzing soil microbial communities biases the results and determines the most effective type of analysis for small and large datasets. Many simulations were performed with four independent pyrosequencing-generated 16S rRNA gene libraries from different environments. The analysis performed here illustrates the lack of resolution of OTU-based approaches for datasets with low sequence coverage. This analysis should be performed with at least 90% of sequence coverage. Diversity index values increase with sample size making normalization of the number of sequences in all samples crucial. An important finding of this study was the advantage of phylogenetic approaches for examining microbial communities with low sequence coverage. However, if the environments being compared were closely related, a deeper sequencing would be necessary to detect the variation in the microbial composition.

Composition of activated sludge settling and planktonic bacterial communities treating industrial effluent and their correlation to settling problems.

Problems with deflocculation and solids separation in biological wastewater treatment systems are linked to fluctuations in physicochemical conditions. This study examined the composition of activated sludge bacterial communities in lab-scale sequencing batch reactors treating bleached kraft mill effluent, under transient temperature conditions (30 to 45 °C) and their correlation to sludge settleability problems. The bacterial community composition of settled and planktonic biomass samples in the reactors was monitored via denaturing gradient gel electrophoresis of 16S ribosomal RNA gene fragments. Our analysis showed that settled biomass has a different community composition from the planktonic biomass (49 ± 7% difference based on Jaccard similarity coefficients; p < 0.01). During times of poor sludge compression, the settled and planktonic biomass became more similar. This observation supports the hypothesis that settling problems observed were due to deflocculation of normally settling flocs rather than the outgrowth of non-settling bacterial species.

Distantly sampled soils carry few species in common.

The bacterial phylogenetic structure of soils from four distinctly different sites in South and North America was analyzed. One hundred and thirty-nine thousand sequences of the V9 region of the small subunit of the bacterial ribosomal RNA gene generated for a previous study were used for this work. Whereas the previous work estimated levels of species richness, this study details the degree of bacterial community overlap between the four soils. Sequences from the four soils were classified and grouped into different phyla and then assigned to operational taxonomic units (OTUs) as defined by 97 or 100% sequence similarity. Pairwise Jaccard and theta similarity indices averaged over all phyla equalled 6 and 12% respectively at the 97% similarity level, and 15% for both at the 100% similarity level. At 100 and 97% sequence similarity, 1.5 and 4.1% of OTUs were found in all four soils respectively, and 87.9 and 74.4%, respectively were a unique particular soil. These analyses, based on the largest soil bacterial sequence retrieval to date, establish the high degree of community structure difference for randomly sampled dissimilar soils and support the idea that wide sampling is important for bioprospecting. The 10 most abundant cultured genera were determined in each soil. These 10 genera comprised a significant proportion of the reads obtained from each soil (31.3-37.4%). Chitinophaga was the most abundant or the second most abundant genus in all four soils with 7.5-13.8% of the total bacterial sequences in these soils. The striking result is that several culturable genera, whose roles in soil are virtually unknown, were found among these dominant sequences.

Pyrosequencing enumerates and contrasts soil microbial diversity.

Estimates of the number of species of bacteria per gram of soil vary between 2000 and 8.3 million (Gans et al., 2005; Schloss and Handelsman, 2006). The highest estimate suggests that the number may be so large as to be impractical to test by amplification and sequencing of the highly conserved 16S rRNA gene from soil DNA (Gans et al., 2005). Here we present the use of high throughput DNA pyrosequencing and statistical inference to assess bacterial diversity in four soils across a large transect of the western hemisphere. The number of bacterial 16S rRNA sequences obtained from each site varied from 26,140 to 53,533. The most abundant bacterial groups in all four soils were the Bacteroidetes, Betaproteobacteria and Alphaproteobacteria. Using three estimators of diversity, the maximum number of unique sequences (operational taxonomic units roughly corresponding to the species level) never exceeded 52,000 in these soils at the lowest level of dissimilarity. Furthermore, the bacterial diversity of the forest soil was phylum rich compared to the agricultural soils, which are species rich but phylum poor. The forest site also showed far less diversity of the Archaea with only 0.009% of all sequences from that site being from this group as opposed to 4%-12% of the sequences from the three agricultural sites. This work is the most comprehensive examination to date of bacterial diversity in soil and suggests that agricultural management of soil may significantly influence the diversity of bacteria and archaea.

Effects of transient temperature conditions on the divergence of activated sludge bacterial community structure and function.

The effect of temperature fluctuations on bacterial community structure and function in lab-scale sequencing batch reactors treating bleached kraft mill effluent was investigated. An increase in temperature from 30 to 45 degrees C caused shifts in both bacterial community structure and function. Triplicate reactors were highly similar for 40 days following startup. After the temperature shift, their community structure and function started to diverge from each other and from the control. A multi-response permutation procedure confirmed that the variability in community structure between transient and control reactors were greater than that among the triplicate transient reactors. The fact that these disturbances manifest themselves in different ways in apparently identical reactors suggests a high degree of variability between replicate systems.

High-temperature biotrickling filtration of hydrogen sulphide.

Biofiltration of malodorous reduced sulphur compounds such as hydrogen sulphide has been confined to emissions that are at temperatures below 40 degrees C despite the fact that there are many industrial emissions (e.g. in the pulp and paper industry) at temperatures well above 40 degrees C. This paper describes our study on the successful treatment of hydrogen sulphide gas at temperatures of 40, 50, 60 and 70 degrees C using a microbial community obtained from a hot spring. Three biotrickling filter (BTF) systems were set up in parallel for a continuous run of 9 months to operate at three different temperatures, one of which was always at 40 degrees C as a mesophilic control and the other two were for exploring high-temperature operation up to 70 degrees C. The continuous experiment and a series of batch experiments in glass bottles (250 ml) showed that addition of glucose and monosodium glutamate enhanced thermophilic biofiltration of hydrogen sulphide gas and a removal rate of 40 g m(-3) h(-1) was achieved at 70 degrees C. We suggest that the glucose is acting as a carbon source for the existing microbial community in the BTFs, whereas glutamate is acting as a compatible solute. The use of such organic compounds to enhance biodegradation of hydrogen sulphide, particularly at high temperatures, has not been demonstrated to our knowledge and, hence, has opened up a range of possibilities for applying biofiltration to hot gas effluent.

Two kinds of chlorocatechol 1,2-dioxygenase from 2,4-dichlorophenoxyacetate-degrading Sphingomonas sp. strain TFD44.

Two kinds of chlorocatechol 1,2-dioxygenase (CCD), TfdC and TfdC2 were detected in Sphingomonas sp. strain TFD44. These two CCDs could be simultaneously synthesized in TFD44 during its growth with 2,4-D as the sole carbon and energy sources. The apparent subunit molecular masses of TfdC and TfdC2 estimated by SDS-PAGE analysis were 33.8 and 33.1 kDa, respectively. The genes encoding the two CCDs were cloned and expressed in Escherichia coli. The two purified CCDs showed broad substrate specificities but had different specificity patterns. TfdC showed the highest specificity constant for 3-chlorocatechol and TfdC2 showed the highest specificity constant for 3,5-dichlorocatechol. The substrate specificity difference seemed to correlate with the alternation of amino acid supposed to be involved in the interaction with substrates. Whereas phylogenetic analysis indicated that the CCDs of Sphingomonas constitute a distinctive group among Gram-negative bacteria, TfdC and TfdC2 of TFD44 have divergently evolved in terms of their substrate specificity.

A survey of the composition and diversity of bacterial populations in bleached kraft pulp-mill wastewater secondary treatment systems.

Bacterial community compositions from 10 pulp- and paper-mill treatment systems were compared using both traditional and molecular techniques. 16S-RFLP (Random Fragment Length Polymorphisms) analysis was used to examine the genotypic profiles of the whole bacterial community of each treatment system. Although all the communities shared approximately 60% of their DNA band pattern, as determined by computer-assisted cluster analysis, each community displayed a unique profile that was stable over time under normal operating parameters. Reverse Sample Genome Probing (RSGP) and 16S-RFLP were used to compare the culturable bacterial communities of several geographically separated pulp-mill biotreatment system communities. There was little overlap in the composition of the culturable community between mills at the genus level. Furthermore, RSGP variation was almost as high within a mill as between mills. Partial sequences of the 16S rRNA genes from culturable isolates identified Bacillus spp., Pseudomonas spp., and Xanthobacter as some of the dominant species. Finally, several 16S rRNA genes from two whole community 16S RNA gene libraries were partially sequenced and identified as similar to unknown alpha-, beta-, and gamma-Proteobacteria, Ralstonia, Alcaligenes, Nitrospira, Firmicutes, and clones representing the new Holophaga/Acidobacterium phylum. These findings suggest that although these pulp- and paper-mill biotreatment communities perform similar functions, they are populated by unique mixtures of species.