Next‐Generation Sequencing Assessment of Eukaryotic Diversity in Oil Sands Tailings Ponds Sediments and Surface Water

Tailings ponds in the Athabasca oil sands (Canada) contain fluid wastes, generated by the extraction of bitumen from oil sands ores. Although the autochthonous prokaryotic communities have been relatively well characterized, almost nothing is known about microbial eukaryotes living in the anoxic soft sediments of tailings ponds or in the thin oxic layer of water that covers them. We carried out the first next‐generation sequencing study of microbial eukaryotic diversity in oil sands tailings ponds. In metagenomes prepared from tailings sediment and surface water, we detected very low numbers of sequences encoding eukaryotic small subunit ribosomal RNA representing seven major taxonomic groups of protists. We also produced and analysed three amplicon‐based 18S rRNA libraries prepared from sediment samples. These revealed a more diverse set of taxa, 169 different OTUs encompassing up to eleven higher order groups of eukaryotes, according to detailed classification using homology searching and phylogenetic methods. The 10 most abundant OTUs accounted for > 90% of the total of reads, vs. large numbers of rare OTUs (< 1% abundance). Despite the anoxic and hydrocarbon‐enriched nature of the environment, the tailings ponds harbour complex communities of microbial eukaryotes indicating that these organisms should be taken into account when studying the microbiology of the oil sands.     

Application of Ion Torrent Sequencing to the Assessment of the Effect of Alkali Ballast Water Treatment on Microbial Community Diversity

The impact of NaOH as a ballast water treatment (BWT) on microbial community diversity was assessed using the 16S rRNA gene based Ion Torrent sequencing with its new 400 base chemistry. Ballast water samples from a Great Lakes ship were collected from the intake and discharge of both control and NaOH (pH 12) treated tanks and were analyzed in duplicates. One set of duplicates was treated with the membrane-impermeable DNA cross-linking reagent propidium mono-azide (PMA) prior to PCR amplification to differentiate between live and dead microorganisms. Ion Torrent sequencing generated nearly 580,000 reads for 31 bar-coded samples and revealed alterations of the microbial community structure in ballast water that had been treated with NaOH. Rarefaction analysis of the Ion Torrent sequencing data showed that BWT using NaOH significantly decreased microbial community diversity relative to control discharge (p<0.001). UniFrac distance based principal coordinate analysis (PCoA) plots and UPGMA tree analysis revealed that NaOH-treated ballast water microbial communities differed from both intake communities and control discharge communities. After NaOH treatment, bacteria from the genus Alishewanella became dominant in the NaOH-treated samples, accounting for <0.5% of the total reads in intake samples but more than 50% of the reads in the treated discharge samples. The only apparent difference in microbial community structure between PMA-processed and non-PMA samples occurred in intake water samples, which exhibited a significantly higher amount of PMA-sensitive cyanobacteria/chloroplast 16S rRNA than their corresponding non-PMA total DNA samples. The community assembly obtained using Ion Torrent sequencing was comparable to that obtained from a subset of samples that were also subjected to 454 pyrosequencing. This study showed the efficacy of alkali ballast water treatment in reducing ballast water microbial diversity and demonstrated the application of new Ion Torrent sequencing techniques to microbial community studies.     

Bioreactor studies predict whole microbial population dynamics in oil sands tailings ponds

Microorganisms in oil sands fluid fine tailings (FFT) are critical to biogeochemical elemental cycling as well as to the degradation of residual hydrocarbon constituents and subsequent methane and CO₂ production. Microbial activity enhances particulate matter sedimentation rates and the dewatering of FFT materials, allowing water to be recycled back into bitumen extraction. A bulk of this evidence comes from bioreactor studies and has implications for engineering and environmental management of the FFT ponds. Yet, it is largely uncertain whether such laboratory populations are representative of whole field scale microbial communities. By using population ecology tools, we compared whole microbial communities present in FFT bioreactors to reference populations existing in Syncrude's West In Pit (WIP) tailings pond. Bacteria were found to be persistent in a sulfidic zone in both the oxic and anoxic bioreactors at all occasions tested. In contrast to the WIP, archaea only became predominant in bioreactors after 300 days, at which point analysis of similarity (global R statistic p?<?0.5) revealed no significant dissimilarities between the populations present in either system. A whole community succession pattern from bacterial dominated prevalence to a new assemblage predominated by archaea was suggested. These results have implications for the stepwise development of microbial model systems for predictive management of field scale FFT basins.     

Methanogenic potential of tailings samples from oil sands extraction plants

Approximately 20% of Canada's oil supply now comes from the extraction of bitumen from the oil sands deposits in northeastern Alberta. The oil sands are strip-mined, and the bitumen is typically separated from sand and clays by an alkaline hot water extraction process. The rapidly expanding oil sands industry has millions of cubic metres of tailings for disposal and large areas of land to reclaim. There are estimates that the consolidation of the mature fine tails (MFT) in the settling ponds will take about 150 years. Some of the settling ponds are now evolving microbially produced methane, a greenhouse gas. To hasten consolidation, gypsum (CaSO4 x 2H2O) is added to MFT, yielding materials called consolidated or composite tailings (CT). Sulfate from the gypsum has the potential to stimulate sulfate-reducing bacteria (SRB) to out-compete methanogens, thereby stopping methanogenesis. This investigation examined three MFT and four CT samples from three oil sands extractions companies. Each was found to contain methanogens and SRB. Serum bottle microcosm studies showed sulfate in the CT samples stopped methane production. However, if the microcosms were amended with readily utilizable electron donors, the sulfate was consumed, and when it reached approximately 20 mg/L, methane production began. Some unamended microcosms were incubated for 372 days, with no methane production detected. This work showed that each MFT and CT sample has the potential to become methanogenic, but in the absence of exogenous electron donors, the added sulfate can inhibit methanogenesis for a long time.     

Heterotrophic Potentials and Hydrocarbon Biodegradation Potentials of Sediment Microorganisms Within the Athabasca Oil Sands Deposit

Techniques for the enumeration and the determination of the potential activity of disturbed sediment mixed populations at control sites and sites within the Athabasca oil sands formation were applied to August and December samples. These techniques included the determination of general heterotrophic potential for the assimilation and respiration of glutamate, which indicated no oil sand-related changes in the sediments but which indicated a significant seasonal change. Enumeration by epifluorescence direct counts, oil sand hydrocarbon plate counts, and most-probable-number determinations of [¹⁴C]hexadecane and [¹⁴C]-naphthalene degraders indicated that only the plate count was sensitive to increased numbers of oil sand-related hydrocarbon-oxidizing microorganisms within the oil sands deposit. Unlike the most probable number determinations of [¹⁴C]hexadecane and [¹⁴C]naphthalene degraders, however, the biodegradation potential results of these substrates indicated a significant increase in activity at oil sands sites. These biodegradation potentials also showed a marked seasonal fluctuation. Although the biodegradation potentials and the endogenous hydrocarbon plate counts indicated an oil sand-adapted mixed sediment population, the results of these techniques did not correlate well with the concentrations of bituminous hydrocarbons in the sediments. The results suggest that a general capability for hydrocarbon oxidation exists in the Athabasca River system and that this capability is enhanced within the natural bounds of the Athabasca oil sands.     

Bacterial diversity and composition of an alkaline uranium mine tailings-water interface

The microbial diversity and biogeochemical potential associated with a northern Saskatchewan uranium mine water-tailings interface was examined using culture-dependent and -independent techniques. Morphologically-distinct colonies from uranium mine water-tailings and a reference lake (MC) obtained using selective and non-selective media were selected for 16S rRNA gene sequencing and identification, revealing that culturable organisms from the uranium tailings interface were dominated by Firmicutes and Betaproteobacteria; whereas, MC organisms mainly consisted of Bacteroidetes and Gammaproteobacteria. Ion Torrent (IT) 16S rRNA metagenomic analysis carried out on extracted DNA from tailings and MC interfaces demonstrated the dominance of Firmicutes in both of the systems. Overall, the tailings-water interface environment harbored a distinct bacterial community relative to the MC, reflective of the ambient conditions (i.e., total dissolved solids, pH, salinity, conductivity, heavy metals) dominating the uranium tailings system. Significant correlations among the physicochemical data and the major bacterial groups present in the tailings and MC were also observed. Presence of sulfate reducing bacteria demonstrated by culture-dependent analyses and the dominance of Desulfosporosinus spp. indicated by Ion Torrent analyses within the tailings-water interface suggests the existence of anaerobic microenvironments along with the potential for reductive metabolic processes.     

Has Alberta Oil Sands Development Altered Delivery of Polycyclic Aromatic Compounds to the Peace-Athabasca Delta?

Background

The extent to which Alberta oil sands mining and upgrading operations have enhanced delivery of bitumen-derived contaminants via the Athabasca River and atmosphere to the Peace-Athabasca Delta (200 km to the north) is a pivotal question that has generated national and international concern. Accounts of rare health disorders in residents of Fort Chipewyan and deformed fish in downstream ecosystems provided impetus for several recent expert-panel assessments regarding the societal and environmental consequences of this multi-billion-dollar industry. Deciphering relative contributions of natural versus industrial processes on downstream supply of polycyclic aromatic compounds (PACs) has been identified as a critical knowledge gap. But, this remains a formidable scientific challenge because loading from natural processes remains unknown. And, industrial activity occurs in the same locations as the natural bitumen deposits, which potentially confounds contemporary upstream-downstream comparisons of contaminant levels.

Methods/Principal Findings

Based on analyses of lake sediment cores, we provide evidence that the Athabasca Delta has been a natural repository of PACs carried by the Athabasca River for at least the past two centuries. We detect no measureable increase in the concentration and proportion of river-transported bitumen-associated indicator PACs in sediments deposited in a flood-prone lake since onset of oil sands development. Results also reveal no evidence that industrial activity has contributed measurably to sedimentary concentration of PACs supplied by atmospheric transport.

Conclusions/Significance

Findings suggest that natural erosion of exposed bitumen in banks of the Athabasca River and its tributaries is a major process delivering PACs to the Athabasca Delta, and the spring freshet is a key period for contaminant mobilization and transport. This baseline environmental information is essential for informed management of natural resources and human-health concerns by provincial and federal regulatory agencies and industry, and for designing effective long-term monitoring programs for the lower Athabasca River watershed.     

Evaluating the Metal Tolerance Capacity of Microbial Communities Isolated from Alberta Oil Sands Process Water

Anthropogenic activities have resulted in the intensified use of water resources. For example, open pit bitumen extraction by Canada’s oil sands operations uses an estimated volume of three barrels of water for every barrel of oil produced. The waste tailings–oil sands process water (OSPW)–are stored in holding ponds, and present an environmental concern as they are comprised of residual hydrocarbons and metals. Following the hypothesis that endogenous OSPW microbial communities have an enhanced tolerance to heavy metals, we tested the capacity of planktonic and biofilm populations from OSPW to withstand metal ion challenges, using Cupriavidus metallidurans, a known metal-resistant organism, for comparison. The toxicity of the metals toward biofilm and planktonic bacterial populations was determined by measuring the minimum biofilm inhibitory concentrations (MBICs) and planktonic minimum inhibitory concentrations (MICs) using the MBEC ™ assay. We observed that the OSPW community and C. metallidurans had similar tolerances to 22 different metals. While thiophillic elements (Te, Ag, Cd, Ni) were found to be most toxic, the OSPW consortia demonstrated higher tolerance to metals reported in tailings ponds (Al, Fe, Mo, Pb). Metal toxicity correlated with a number of physicochemical characteristics of the metals. Parameters reflecting metal-ligand affinities showed fewer and weaker correlations for the community compared to C. metallidurans, suggesting that the OSPW consortia may have developed tolerance mechanisms toward metals present in their environment.     

Massive dominance of Epsilonproteobacteria in formation waters from a Canadian oil sands reservoir containing severely biodegraded oil

The subsurface microbiology of an Athabasca oil sands reservoir in western Canada containing severely biodegraded oil was investigated by combining 16S rRNA gene- and polar lipid-based analyses of reservoir formation water with geochemical analyses of the crude oil and formation water. Biomass was filtered from formation water, DNA was extracted using two different methods, and 16S rRNA gene fragments were amplified with several different primer pairs prior to cloning and sequencing or community fingerprinting by denaturing gradient gel electrophoresis (DGGE). Similar results were obtained irrespective of the DNA extraction method or primers used. Archaeal libraries were dominated by Methanomicrobiales (410 of 414 total sequences formed a dominant phylotype affiliated with a Methanoregula sp.), consistent with the proposed dominant role of CO(2) -reducing methanogens in crude oil biodegradation. In two bacterial 16S rRNA clone libraries generated with different primer pairs, >?99% and 100% of the sequences were affiliated with Epsilonproteobacteria (n?=?382 and 72 total clones respectively). This massive dominance of Epsilonproteobacteria sequences was again obtained in a third library (99% of sequences; n?=?96 clones) using a third universal bacterial primer pair (inosine-341f and 1492r). Sequencing of bands from DGGE profiles and intact polar lipid analyses were in accordance with the bacterial clone library results. Epsilonproteobacterial OTUs were affiliated with Sulfuricurvum, Arcobacter and Sulfurospirillum spp. detected in other oil field habitats. The dominant organism revealed by the bacterial libraries (87% of all sequences) is a close relative of Sulfuricurvum kujiense - an organism capable of oxidizing reduced sulfur compounds in crude oil. Geochemical analysis of organic extracts from bitumen at different reservoir depths down to the oil water transition zone of these oil sands indicated active biodegradation of dibenzothiophenes, and stable sulfur isotope ratios for elemental sulfur and sulfate in formation waters were indicative of anaerobic oxidation of sulfur compounds. Microbial desulfurization of crude oil may be an important metabolism for Epsilonproteobacteria indigenous to oil reservoirs with elevated sulfur content and may explain their prevalence in formation waters from highly biodegraded petroleum systems.     

In Vitro Microbial Degradation of Bituminous Hydrocarbons and In Situ Colonization of Bitumen Surfaces Within the Athabasca Oil Sands Deposit

Bituminous hydrocarbons extracted from the Athabasca oil sands of north-eastern Alberta were adsorbed onto filter supports and placed at sites in the Athabasca River and its tributaries where these rivers come in contact with the oil sands formation. Colonization of the hydrocarbon surfaces at summer and winter ambient temperatures was examined by scanning and transmission electron microscopy as well as by epifluorescence microscopy of acridine orange-stained cross sections. Ruthenium red and alkaline bismuth stains visualized an association of bacteria with the hydrocarbon surface which was mediated by bacterial polysaccharides. Bacteria apparently lacking a glycocalyx were also found closely associated with the surface of the hydrophobic substrate and in channels within the substrate. A solvent precipitation and column chromatographic fractionation of the bitumen was followed by cross-tests for growth on the fractions by various isolated sediment microorganisms, as determined by epifluorescence count. All fractions except the asphaltenes supported the growth of at least two of the isolates, although fractionation of degraded bitumen revealed that the saturate, aromatic, and first polar fractions were preferentially degraded.     

Bioprocessing-Based Approach for Bitumen/Water/Fines Separation and Hydrocarbon Recovery from Oil Sands Tailings

Oil sands are a major source of oil, but their industrial processing generates tailings ponds that are an environmental hazard. The main concerns are mature fine tailings (MFT) composed of residual hydrocarbons, water, and fine clay. Tailings ponds include toxic contaminants such as heavy metals, and toxic organics including naphthenics. Naphthenic acids and polyaromatic hydrocarbons (PAHs) degrade very slowly and pose a long-term threat to surface and groundwater, as they can be transported in the MFT. Research into improved technologies that would enable densification and settling of the suspended particles is ongoing. In batch tests, BioTiger?, a microbial consortium that can metabolize PAHs, demonstrated improved oil sands tailings settling from a Canadian tailings pond. Results also showed, depending on the timing of the measurements, lower suspended solids and turbidity. Elevated total organic carbon was observed in the first 48 hours in the BioTiger?-treated columns and then decreased in overlying water. Oil sands tailings mixed with BioTiger? showed a two-fold reduction in suspended solids within 24 hours as compared to abiotic controls. The tailings treated with BioTiger? increased in microbial densities three orders of magnitude from 8.5 × 105 CFU/mL to 1.2 × 108 CFU/mL without any other carbon or energy source added, indicating metabolism of hydrocarbons and other available nutrients. Results demonstrated that bioaugmentation of BioTiger? increased separation of organic carbon from particles in oil sands and enhanced settling with tailings with improved water quality. Journal style is for Abstract to be less than 200 words, and contain no citations to other sources; please edit as needed     

Microbial community structure of surface sediments from a tropical estuarine environment using next generation sequencing

Microbial community structure was analyzed from tropical monsoon influenced Mandovi-Zuari (Ma-Zu) estuarine sediment by means of Next Gen Sequencing (NGS) approach using Ion Torrent PGM™. The sequencing generated 80,282 raw sequence reads. Barcoding with Ion Tags allowed multiplex analysis of microbial community and helped in identifying shifts in microbial community structure. Analysis of sequence data revealed that sediment at both the stations in the Mandovi estuary was dominated by Archaeal group, Euryarchaeota (53.1% and 64.01%). Among Euryarchaeota, Methanomicrobia was dominant. Methanococci was present only at the mouth and Methanopyri was detected at the mid-estuarine station. Whereas, both the stations of Zuari estuary were dominated by Bacteria, Proteobacteria, mainly Gammaproteobacteria (97.67% and 54.41%). A clear influence of mangrove ecosystem on the bacterial diversity was evident in the Zuari estuary. These results suggest that the two estuaries have a very distinct microbial community structure. Characterization of microbial communities in this study area using NGS for the first time points out that even within geographically close habitats, the microbial population structure is significantly influenced by localized interactions. The signatures obtained from sediments can thus be used to reconstruct habitat characteristics and serve as biomarkers. Future studies should focus on the functional gene profiling of different microbial communities and the influence of seasons and tide in such monsoon influenced estuaries.     

Performance comparison of benchtop high-throughput sequencing platforms

Three benchtop high-throughput sequencing instruments are now available. The 454 GS Junior (Roche), MiSeq (Illumina) and Ion Torrent PGM (Life Technologies) are laser-printer sized and offer modest set-up and running costs. Each instrument can generate data required for a draft bacterial genome sequence in days, making them attractive for identifying and characterizing pathogens in the clinical setting. We compared the performance of these instruments by sequencing an isolate of Escherichia coli O104:H4, which caused an outbreak of food poisoning in Germany in 2011. The MiSeq had the highest throughput per run (1.6 Gb/run, 60 Mb/h) and lowest error rates. The 454 GS Junior generated the longest reads (up to 600 bases) and most contiguous assemblies but had the lowest throughput (70 Mb/run, 9 Mb/h). Run in 100-bp mode, the Ion Torrent PGM had the highest throughput (80–100 Mb/h). Unlike the MiSeq, the Ion Torrent PGM and 454 GS Junior both produced homopolymer-associated indel errors (1.5 and 0.38 errors per 100 bases, respectively).     

Anaerobic microbial communities and their potential for bioenergy production in heavily biodegraded petroleum reservoirs

Most of the oil in low temperature, non-uplifted reservoirs is biodegraded due to millions of years of microbial activity, including via methanogenesis from crude oil. To evaluate stimulating additional methanogenesis in already heavily biodegraded oil reservoirs, oil sands samples were amended with nutrients and electron acceptors, but oil sands bitumen was the only organic substrate. Methane production was monitored for over 3000 days. Methanogenesis was observed in duplicate microcosms that were unamended, amended with sulfate or that were initially oxic, however methanogenesis was not observed in nitrate-amended controls. The highest rate of methane production was 0.15 μmol CH₄ g⁻¹ oil d⁻¹, orders of magnitude lower than other reports of methanogenesis from lighter crude oils. Methanogenic Archaea and several potential syntrophic bacterial partners were detected following the incubations. GC–MS and FTICR–MS revealed no significant bitumen alteration for any specific compound or compound class, suggesting that the very slow methanogenesis observed was coupled to bitumen biodegradation in an unspecific manner. After 3000 days, methanogenic communities were amended with benzoate resulting in methanogenesis rates that were 110-fold greater. This suggests that oil-to-methane conversion is limited by the recalcitrant nature of oil sands bitumen, not the microbial communities resident in heavy oil reservoirs.     

Comparative analysis of metagenomes from three methanogenic hydrocarbon-degrading enrichment cultures with 41 environmental samples

Methanogenic hydrocarbon metabolism is a key process in subsurface oil reservoirs and hydrocarbon-contaminated environments and thus warrants greater understanding to improve current technologies for fossil fuel extraction and bioremediation. In this study, three hydrocarbon-degrading methanogenic cultures established from two geographically distinct environments and incubated with different hydrocarbon substrates (added as single hydrocarbons or as mixtures) were subjected to metagenomic and 16S rRNA gene pyrosequencing to test whether these differences affect the genetic potential and composition of the communities. Enrichment of different putative hydrocarbon-degrading bacteria in each culture appeared to be substrate dependent, though all cultures contained both acetate- and H₂-utilizing methanogens. Despite differing hydrocarbon substrates and inoculum sources, all three cultures harbored genes for hydrocarbon activation by fumarate addition (bssA, assA, nmsA) and carboxylation (abcA, ancA), along with those for associated downstream pathways (bbs, bcr, bam), though the cultures incubated with hydrocarbon mixtures contained a broader diversity of fumarate addition genes. A comparative metagenomic analysis of the three cultures showed that they were functionally redundant despite their enrichment backgrounds, sharing multiple features associated with syntrophic hydrocarbon conversion to methane. In addition, a comparative analysis of the culture metagenomes with those of 41 environmental samples (containing varying proportions of methanogens) showed that the three cultures were functionally most similar to each other but distinct from other environments, including hydrocarbon-impacted environments (for example, oil sands tailings ponds and oil-affected marine sediments). This study provides a basis for understanding key functions and environmental selection in methanogenic hydrocarbon-associated communities.     

Performance Comparison of Illumina and Ion Torrent Next-Generation Sequencing Platforms for 16S rRNA-Based Bacterial Community Profiling

High-throughput sequencing of the taxonomically informative 16S rRNA gene provides a powerful approach for exploring microbial diversity. Here we compare the performances of two common “benchtop” sequencing platforms, Illumina MiSeq and Ion Torrent Personal Genome Machine (PGM), for bacterial community profiling by 16S rRNA (V1-V2) amplicon sequencing. We benchmarked performance by using a 20-organism mock bacterial community and a collection of primary human specimens. We observed comparatively higher error rates with the Ion Torrent platform and report a pattern of premature sequence truncation specific to semiconductor sequencing. Read truncation was dependent on both the directionality of sequencing and the target species, resulting in organism-specific biases in community profiles. We found that these sequencing artifacts could be minimized by using bidirectional amplicon sequencing and an optimized flow order on the Ion Torrent platform. Results of bacterial community profiling performed on the mock community and a collection of 18 human-derived microbiological specimens were generally in good agreement for both platforms; however, in some cases, results differed significantly. Disparities could be attributed to the failure to generate full-length reads for particular organisms on the Ion Torrent platform, organism-dependent differences in sequence error rates affecting classification of certain species, or some combination of these factors. This study demonstrates the potential for differential bias in bacterial community profiles resulting from the choice of sequencing platform alone.     

Direct Chloroplast Sequencing: Comparison of Sequencing Platforms and Analysis Tools for Whole Chloroplast Barcoding

Direct sequencing of total plant DNA using next generation sequencing technologies generates a whole chloroplast genome sequence that has the potential to provide a barcode for use in plant and food identification. Advances in DNA sequencing platforms may make this an attractive approach for routine plant identification. The HiSeq (Illumina) and Ion Torrent (Life Technology) sequencing platforms were used to sequence total DNA from rice to identify polymorphisms in the whole chloroplast genome sequence of a wild rice plant relative to cultivated rice (cv. Nipponbare). Consensus chloroplast sequences were produced by mapping sequence reads to the reference rice chloroplast genome or by de novo assembly and mapping of the resulting contigs to the reference sequence. A total of 122 polymorphisms (SNPs and indels) between the wild and cultivated rice chloroplasts were predicted by these different sequencing and analysis methods. Of these, a total of 102 polymorphisms including 90 SNPs were predicted by both platforms. Indels were more variable with different sequencing methods, with almost all discrepancies found in homopolymers. The Ion Torrent platform gave no apparent false SNP but was less reliable for indels. The methods should be suitable for routine barcoding using appropriate combinations of sequencing platform and data analysis.     

Investigation of the bacterial retting community of kenaf (Hibiscus cannabinus) under different conditions using next-generation semiconductor sequencing

The microbial communities associated with kenaf (Hibiscus cannabinus) plant fibers during retting were determined in an effort to identify possible means of accelerating this process for industrial scale-up. Microbial communities were identified by semiconductor sequencing of 16S rRNA gene amplicons from DNA harvested from plant-surface associated samples and analyzed using an Ion Torrent PGM. The communities were sampled after 96 h from each of three different conditions, including amendments with pond water, sterilized pond water, or with a mixture of pectinolytic bacterial isolates. Additionally, plants from two different sources and having different pretreatment conditions were compared. We report that the best retting communities are dominated by members of the order Clostridiales. These bacteria appear to be naturally associated with the plant material, although slight variations between source materials were found. Additionally, heavy inoculations of pectinolytic bacteria established themselves and in addition their presence facilitated the rapid dominance of the original plant-associated Clostridiales. These data suggest that members of the order Clostridiales dominate the community and are most closely associated with efficient and effective retting. The results further suggest that establishment of the community structure is first driven by the switch to anaerobic conditions, and subsequently by possible competition for nitrogen. These findings reveal important bacterial groups involved in fiber retting, and suggest mechanisms for the manipulation of the community and retting efficiency by modifying nutrient availability.