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USGS2020

Life at the frozen limit: Microbial carbon metabolism across a Late Pleistocene permafrost chronosequence

Permafrost is an extreme habitat yet it hosts microbial populations that remain active over millennia. Using permafrost collected from a Pleistocene chronosequence (19 to 33 ka), we hypothesized that the functional genetic potential of microbial communities in permafrost would reflect microbial strategies to metabolize permafrost soluble organic matter (OM) in situ over geologic time. We also hypothesized that changes in the metagenome across the chronosequence would correlate with shifts in carbon chemistry, permafrost age, and paleoclimate at the time of permafrost formation. We combined high-resolution characterization of water-soluble OM by Fourier-transform ion-cyclotron-resonance mass spectrometry (FT-ICR MS), quantification of organic anions in permafrost water extracts, and metagenomic sequencing to better understand the relationships between the molecular-level composition of potentially bioavailable OM, the microbial community, and permafrost age. Both age and paleoclimate had marked effects on both the molecular composition of dissolved OM and the microbial community. The relative abundance of genes associated with hydrogenotrophic methanogenesis, carbohydrate active enzyme families, nominal oxidation state of carbon (NOSC), and number of identifiable molecular formulae significantly decreased with increasing age. In contrast, genes associated with fermentation of short chain fatty acids (SCFAs), the concentration of SCFAs and ammonium all significantly increased with age. We present a conceptual model of microbial metabolism in permafrost based on fermentation of OM and the buildup of organic acids that helps to explain the unique chemistry of ancient permafrost soils. These findings imply long-term in situ microbial turnover of ancient permafrost OM and that this pooled biolabile OM could prime ancient permafrost soils for a larger and more rapid microbial response to thaw compared to younger permafrost soils.

Alaska↗

USGS2012

Epidemiology of a Salmonella enterica subsp. Enterica serovar Typhimurium strain associated with a songbird outbreak.

Salmonella enterica subsp. enterica serovar Typhimurium is responsible for the majority of salmonellosis cases worldwide. This Salmonella serovar is also responsible for die-offs in songbird populations. In 2009, there was an S. Typhimurium epizootic reported in pine siskins in the eastern United States. At the time, there was also a human outbreak with this serovar that was associated with contaminated peanuts. As peanuts are also used in wild-bird food, it was hypothesized that the pine siskin epizootic was related to this human outbreak. A comparison of songbird and human S. Typhimurium pulsed-field gel electrophoresis (PFGE) patterns revealed that the epizootic was attributed not to the peanut-associated strain but, rather, to a songbird strain first characterized from an American goldfinch in 1998. This same S. Typhimurium strain (PFGE type A3) was also identified in the PulseNet USA database, accounting for 137 of 77,941 total S. Typhimurium PFGE entries. A second molecular typing method, multiple-locus variable-number tandem-repeat analysis (MLVA), confirmed that the same strain was responsible for the pine siskin epizootic in the eastern United States but was distinct from a genetically related strain isolated from pine siskins in Minnesota. The pine siskin A3 strain was first encountered in May 2008 in an American goldfinch and later in a northern cardinal at the start of the pine siskin epizootic. MLVA also confirmed the clonal nature of S. Typhimurium in songbirds and established that the pine siskin epizootic strain was unique to the finch family. For 2009, the distribution of PFGE type A3 in passerines and humans mirrored the highest population density of pine siskins for the East Coast.

Applied and Environmental Microbiology↗

USGS1992

Molecular cloning and sequence analysis of the gene coding for the 57kDa soluble antigen of the salmonid fish pathogen Renibacterium salmoninarum

The complete sequence coding for the 57-kDa major soluble antigen of the salmonid fish pathogen, Renibacterium salmoninarum , was determined. The gene contained an opening reading frame of 1671 nucleotides coding for a protein of 557 amino acids with a calculated M r value of 57190. The first 26 amino acids constituted a signal peptide. The deduced sequence for amino acid residues 27–61 was in agreement with the 35 N-terminal amino acid residues determined by microsequencing, suggesting the protein in synthesized as a 557-amino acid precursor and processed to produce a mature protein of M r 54505. Two regions of the protein contained imperfect direct repeats. The first region contained two copies of an 81-residue repeat, the second contained five copies of an unrelated 25-residue repeat. Also, a perfect inverted repeat (including three in-frame UAA stop codons) was observed at the carboxyl-terminus of the gene.

FEMS Microbiology Letters↗

USGS2006

Quantity-activity relationship of denitrifying bacteria and environmental scaling in streams of a forested watershed

The spatial variability of subreach denitrification rates in streams was evaluated with respect to controlling environmental conditions, molecular examination of denitrifying bacteria, and dimensional analysis. Denitrification activities ranged from 0 and 800 ng-N gsed-1 d-1 with large variations observed within short distances (<50 m) along stream reaches. A log-normal probability distribution described the range in denitrification activities and was used to define low (16% of the probability distributibn), medium (68%), and high (16%) denitrification potential groups. Denitrifying bacteria were quantified using a competitive polymerase chain reaction (cPCR) technique that amplified the nirK gene that encodes for nitrite reductase. Results showed a range of nirK quantities from 103 to 107 gene-copy-number gsed.-1 A nonparametric statistical test showed no significant difference in nirK quantifies among stream reaches, but revealed that samples with a high denitrification potential had significantly higher nirK quantities. Denitrification activity was positively correlated with nirK quantities with scatter in the data that can be attributed to varying environmental conditions along stream reaches. Dimensional analysis was used to evaluate denitrification activities according to environmental variables that describe fluid-flow properties, nitrate and organic material quantities, and dissolved oxygen flux. Buckingham's pi theorem was used to generate dimensionless groupings and field data were used to determine scaling parameters. The resulting expressions between dimensionless NO3- flux and dimensionless groupings of environmental variables showed consistent scaling, which indicates that the subreach variability in denitrification rates can be predicted by the controlling physical, chemical, and microbiological conditions. Copyright 2006 by the American Geophysical Union.

Journal of Geophysical Research G: Biogeosciences↗

USGS2009

Culture-independent characterization of bacterial communities associated with the cold-water coral Lophelia pertusa in the northeastern Gulf of Mexico

Bacteria are recognized as an important part of the total biology of shallow-water corals. Studies of shallow-water corals suggest that associated bacteria may benefit the corals by cycling carbon, fixing nitrogen, chelating iron, and producing antibiotics that protect the coral from other microbes. Cold-water or deep-sea corals have a fundamentally different ecology due to their adaptation to cold, dark, high-pressure environments and as such have novel microbiota. The goal of this study was to characterize the microbial associates of Lophelia pertusa in the northeastern Gulf of Mexico. This is the first study to collect the coral samples in individual insulated containers and to preserve coral samples at depth in an effort to minimize thermal shock and evaluate the effects of environmental gradients on the microbial diversity of samples. Molecular analysis of bacterial diversity showed a marked difference between the two study sites, Viosca Knoll 906/862 (VK906/862) and Viosca Knoll 826 (VK826). The bacterial communities from VK826 were dominated by a variety of unknown mycoplasmal members of the Tenericutes and Bacteroidetes , whereas the libraries from VK906/862 were dominated by members of the Proteobacteria . In addition to novel sequences, the 16S rRNA gene clone libraries revealed many bacterial sequences in common between Gulf of Mexico Lophelia corals and Norwegian fjord Lophelia corals, as well as shallow-water corals. Two Lophelia -specific bacterial groups were identified: a cluster of gammaproteobacteria related to sulfide-oxidizing gill symbionts of seep clams and a group of Mycoplasma spp. The presence of these groups in both Gulf and Norwegian Lophelia corals indicates that in spite of the geographic heterogeneity observed in Lophelia -associated bacterial communities, there are Lophelia -specific microbes.

Gulf of Mexico↗

USGS2015

A real-time, quantitative PCR protocol for assessing the relative parasitemia of Leucocytozoon in waterfowl

Microscopic examination of blood smears can be effective at diagnosing and quantifying hematozoa infections. However, this method requires highly trained observers, is time consuming, and may be inaccurate for detection of infections at low levels of parasitemia. To develop a molecular methodology for identifying and quantifying Leucocytozoon parasite infection in wild waterfowl (Anseriformes), we designed a real-time, quantitative PCR protocol to amplify Leucocytozoon mitochondrial DNA using TaqMan fluorogenic probes and validated our methodology using blood samples collected from waterfowl in interior Alaska during late summer and autumn (n = 105). By comparing our qPCR results to those derived from a widely used nested PCR protocol, we determined that our assay showed high levels of sensitivity (91%) and specificity (100%) in detecting Leucocytozoon DNA from host blood samples. Additionally, results of a linear regression revealed significant correlation between the raw measure of parasitemia produced by our qPCR assay (Ct values) and numbers of parasites observed on blood smears (R2 = 0.694, P = 0.003), indicating that our assay can reliably determine the relative parasitemia levels among samples. This methodology provides a powerful new tool for studies assessing effects of haemosporidian infection in wild avian species.

Journal of Microbiological Methods↗

USGS2013

Microbial community composition and endolith colonization at an Arctic thermal spring are driven by calcite precipitation

Environmental conditions shape community composition. Arctic thermal springs provide an opportunity to study how environmental gradients can impose strong selective pressures on microbial communities and provide a continuum of niche opportunities. We use microscopic and molecular methods to conduct a survey of microbial community composition at Troll Springs on Svalbard, Norway, in the high Arctic. Microorganisms there exist under a wide range of environmental conditions: in warm water as periphyton, in moist granular materials, and in cold, dry rock as endoliths. Troll Springs has two distinct ecosystems, aquatic and terrestrial, together in close proximity, with different underlying environmental factors shaping each microbial community. Periphyton are entrapped during precipitation of calcium carbonate from the spring's waters, providing microbial populations that serve as precursors for the development of endolithic communities. This process differs from most endolith colonization, in which the rock predates the communities that colonize it. Community composition is modulated as environmental conditions change within the springs. At Troll, the aquatic environments show a small number of dominant operational taxonomic units (OTUs) that are specific to each sample. The terrestrial environments show a more even distribution of OTUs common to multiple samples.

Environmental Microbiology Reports↗

USGS2017

Sulfolobus islandicus meta-populations in Yellowstone National Park hot springs

Abiotic and biotic forces shape the structure and evolution of microbial populations. We investigated forces that shape the spatial and temporal population structure of Sulfolobus islandicus by comparing geochemical and molecular analysis from seven hot springs in five regions sampled over 3 years in Yellowstone National Park. Through deep amplicon sequencing, we uncovered 148 unique alleles at two loci whose relative frequency provides clear evidence for independent populations in different hot springs. Although geography controls regional geochemical composition and population differentiation, temporal changes in population were not explained by corresponding variation in geochemistry. The data suggest that the influence of extinction, bottleneck events and/or selective sweeps within a spring and low migration between springs shape these populations. We suggest that hydrologic events such as storm events and surface snowmelt runoff destabilize smaller hot spring environments with smaller populations and result in high variation in the S. islandicus population over time. Therefore, physical abiotic features such as hot spring size and position in the landscape are important factors shaping the stability and diversity of the S. islandicus meta-population within Yellowstone National Park.

Wyoming↗

USGS2018

Chronic physical disturbance substantially alters the response of biological soil crusts to a wetting pulse, as characterized by metatranscriptomic sequencing

Biological soil crusts (biocrusts) are microbial communities that are a feature of arid surface soils worldwide. In drylands where precipitation is pulsed and ephemeral, the ability of biocrust microbiota to rapidly initiate metabolic activity is critical to their survival. Community gene expression was compared after a short duration (1 hour) wetting pulse in both intact and soils disturbed by chronic foot trampling. Across the metatranscriptomes the majority of transcripts were cyanobacterial in origin, suggesting that cyanobacteria accounted for the bulk of the transcriptionally active cells. Chronic trampling substantially altered the functional profile of the metatranscriptomes, specifically resulting in a significant decrease in transcripts for nitrogen fixation. Soil depth (biocrust and below crust) was a relatively small factor in differentiating the metatranscriptomes, suggesting that the metabolically active bacteria were similar between shallow soil horizons. The dry samples were consistently enriched for hydrogenase genes, indicating that molecular hydrogen may serve as an energy source for the desiccated soil communities. The water pulse was associated with a restructuring of the metatranscriptome, particularly for the biocrusts. Biocrusts increased transcripts for photosynthesis and carbon fixation, suggesting a rapid resuscitation upon wetting. In contrast, the trampled surface soils showed a much smaller response to wetting, indicating that trampling altered the metabolic response of the community. Finally, several biogeochemical cycling genes in carbon and nitrogen cycling were assessed for their change in abundance due to wetting in the biocrusts. Different transcripts encoding the same gene product did not show a consensus response, with some more abundant in dry or wet biocrusts, highlighting the challenges in relating transcript abundance to biogeochemical cycling rates. These observations demonstrate that metatranscriptome sequencing was able to distinguish alterations in the function of arid soil microbial communities at two varying temporal scales, a long-term ecosystems disturbance through foot trampling, and a short term wetting pulse. Thus, community metatranscriptomes have the potential to inform studies on the response and resilience of biocrusts to various environmental perturbations.

Frontiers in Microbiology↗

USGS2012

Comparison of DNA preservation methods for environmental bacterial community samples

Field collections of environmental samples, for example corals, for molecular microbial analyses present distinct challenges. The lack of laboratory facilities in remote locations is common, and preservation of microbial community DNA for later study is critical. A particular challenge is keeping samples frozen in transit. Five nucleic acid preservation methods that do not require cold storage were compared for effectiveness over time and ease of use. Mixed microbial communities of known composition were created and preserved by DNAgard™, RNAlater ® , DMSO–EDTA–salt (DESS), FTA ® cards, and FTA Elute ® cards. Automated ribosomal intergenic spacer analysis and clone libraries were used to detect specific changes in the faux communities over weeks and months of storage. A previously known bias in FTA ® cards that results in lower recovery of pure cultures of Gram-positive bacteria was also detected in mixed community samples. There appears to be a uniform bias across all five preservation methods against microorganisms with high G + C DNA. Overall, the liquid-based preservatives (DNAgard™, RNAlater ® , and DESS) outperformed the card-based methods. No single liquid method clearly outperformed the others, leaving method choice to be based on experimental design, field facilities, shipping constraints, and allowable cost.

FEMS Microbiology Ecology↗

USGS1994

Use of molecular techniques to evaluate the survival of a microorganism injected into an aquifer

A PCR primer set and an internal probe that are specific for Pseudomonas sp. strain B13, a 3-chlorobenzoate-metabolizing strain, were developed. Using this primer set and probe, we were able to detect Pseudomonas sp. strain B13 DNA sequences in DNA extracted from aquifer samples 14.5 months after Pseudomonas sp. strain B13 had been injected into a sand and gravel aquifer. This primer set and probe were also used to analyze isolates from 3-chlorobenzoate enrichments of the aquifer samples by Southern blot analysis. Hybridization of Southern blots with the Pseudomonas sp. strain B13-specific probe and a catabolic probe in conjunction with restriction fragment length polymorphism (RFLP) analysis of ribosome genes was used to determine that viable Pseudomonas sp. strain B13 persisted in this environment. We isolated a new 3-chlorobenzoate-degrading strain from one of these enrichment cultures. The B13-specific probe does not hybridize to DNA from this isolate. The new strain could be the result of gene exchange between Pseudomonas sp. strain B13 and an indigenous bacterium. This speculation is based on an RFLP pattern of ribosome genes that differs from that of Pseudomonas sp. strain B13, the fact that identically sized restriction fragments hybridized to the catabolic gene probe, and the absence of any enrichable 3-chlorobenzoate-degrading strains in the aquifer prior to inoculation.

Massachusetts↗

USGS2002

A single Alal 39-to-Glu substitution in the Renibacterium salmoninarum virulence-associated protein p57 results in antigenic variation and is associated with enhanced p57 binding to Chinook salmon leukocytes

The gram-positive bacterium Renibacterium salmoninarum produces relatively large amounts of a 57-kDa protein (p57) implicated in the pathogenesis of salmonid bacterial kidney disease. Antigenic variation in p57 was identified by using monoclonal antibody 4C11, which exhibited severely decreased binding to R. salmoninarum strain 684 p57 and bound robustly to the p57 proteins of seven other R. salmoninarum strains. This difference in binding was not due to alterations in p57 synthesis, secretion, or bacterial cell association. The molecular basis of the 4C11 epitope loss was determined by amplifying and sequencing the two identical genes encoding p57, msa1 and msa2 . The 5′ and coding sequences of the 684 msa1 and msa2 genes were identical to those of the ATCC 33209 msa1 and msa2 genes except for a single C-to-A nucleotide mutation. This mutation was identified in both the msa1 and msa2 genes of strain 684 and resulted in an Ala 139 -to-Glu substitution in the amino-terminal region of p57. We examined whether this mutation in p57 altered salmonid leukocyte and rabbit erythrocyte binding activities. R. salmoninarum strain 684 extracellular protein exhibited a twofold increase in agglutinating activity for chinook salmon leukocytes and rabbit erythrocytes compared to the activity of the ATCC 33209 extracellular protein. A specific and quantitative p57 binding assay confirmed the increased binding activity of 684 p57. Monoclonal antibody 4C11 blocked the agglutinating activity of the ATCC 33209 extracellular protein but not the agglutinating activity of the 684 extracellular protein. These results indicate that the Ala 139 -to-Glu substitution altered immune recognition and was associated with enhanced biological activity of R. salmoninarum 684 p57.

Applied and Environmental Microbiology↗

USGS2012

Prevalence, environmental loading, and molecular characterization of Cryptosporidium and Giardia</i isolates from domestic and wild animals along the Central California Coast

The risk of disease transmission from waterborne protozoa is often dependent on the origin (e.g., domestic animals versus wildlife), overall parasite load in contaminated waterways, and parasite genotype, with infections being linked to runoff or direct deposition of domestic animal and wildlife feces. Fecal samples collected from domestic animals and wildlife along the central California coast were screened to (i) compare the prevalence and associated risk factors for fecal shedding of Cryptosporidium and Giardia species parasites, (ii) evaluate the relative importance of animal host groups that contribute to pathogen loading in coastal ecosystems, and (iii) characterize zoonotic and host-specific genotypes. Overall, 6% of fecal samples tested during 2007 to 2010 were positive for Cryptosporidium oocysts and 15% were positive for Giardia cysts. Animal host group and age class were significantly associated with detection of Cryptosporidium and Giardia parasites in animal feces. Fecal loading analysis revealed that infected beef cattle potentially contribute the greatest parasite load relative to other host groups, followed by wild canids. Beef cattle, however, shed host-specific, minimally zoonotic Cryptosporidium and Giardia duodenalis genotypes, whereas wild canids shed potentially zoonotic genotypes, including G. duodenalis assemblages A and B. Given that the parasite genotypes detected in cattle were not zoonotic, the public health risk posed by protozoan parasite shedding in cattle feces may be lower than that posed by other animals, such as wild canids, that routinely shed zoonotic genotypes.

California↗

USGS2021

Evaluation of a modified rapid viability-polymerase chain reaction method for Bacillus atrophaeus spores in water matrices

A rapid method that provides information on the viability of organisms is needed to protect public health and ensure that remediation efforts following a release of a biological agent are effective. The rapid viability-polymerase chain reaction (RV-PCR) method combines broth culture and molecular methods to provide results on whether viable organisms are present in less than 15 h. In this study, a modified RV-PCR (mRV-PCR) method was compared to a membrane-filtration culture method for the detection of viable Bacillus spores in water matrices. Samples included small and large volumes of chlorine and non‑chlorine treated tap water. Large volume water samples (up to 100 L), were processed by ultrafiltration using a semi-automated waterborne pathogen concentrator, followed by centrifugation as a secondary concentration technique. The concentrated samples were analyzed by mRV-PCR and culture methods. The overall agreement between the mRV-PCR and culture methods when seed concentrations were greater than 10 spores per sample volume analyzed was 96%. The total time from the start of sample processing to the final sample result for the mRV-PCR method was decreased by approximately 2 h, in comparison to the previously published RV-PCR method because of the incorporation of shorter, more efficient primary and secondary concentration steps and a shorter DNA extraction technique. Overall, this study confirmed that RV-PCR is a promising approach for identifying viable Bacillus spores in small- and large-volume water samples and for producing results in less time than traditional culture methods.

Journal of Microbiological Methods↗

USGS2010

Microclimate and limits to photosynthesis in a diverse community of hypolithic cyanobacteria in northern Australia

Hypolithic microbes, primarily cyanobacteria, inhabit the highly specialized microhabitats under translucent rocks in extreme environments. Here we report findings from hypolithic cyanobacteria found under three types of translucent rocks (quartz, prehnite, agate) in a semiarid region of tropical Australia. We investigated the photosynthetic responses of the cyanobacterial communities to light, temperature and moisture in the laboratory, and we measured the microclimatic variables of temperature and soil moisture under rocks in the field over an annual cycle. We also used molecular techniques to explore the diversity of hypolithic cyanobacteria in this community and their phylogenetic relationships within the context of hypolithic cyanobacteria from other continents. Based on the laboratory experiments, photosynthetic activity required a minimum soil moisture of 15% (by mass). Peak photosynthetic activity occurred between approximately 8°C and 42°C, though some photosynthesis occurred between −1°C and 51°C. Maximum photosynthesis rates also occurred at light levels of approximately 150–550 μmol m −2 s −1 . We used the field microclimatic data in conjunction with these measurements of photosynthetic efficiency to estimate the amount of time the hypolithic cyanobacteria could be photosynthetically active in the field. Based on these data, we estimated that conditions were appropriate for photosynthetic activity for approximately 942 h (∼75 days) during the year. The hypolithic cyanobacteria community under quartz, prehnite and agate rocks was quite diverse both within and between rock types. We identified 115 operational taxonomic units (OTUs), with each rock hosting 8–24 OTUs. A third of the cyanobacteria OTUs from northern Australia grouped with Chroococcidiopsis , a genus that has been identified from hypolithic and endolithic communities from the Gobi, Mojave, Atacama and Antarctic deserts. Several OTUs identified from northern Australia have not been reported to be associated with hypolithic communities previously.

Environmental Microbiology↗

USGS2020

Utilization of multiple microbial tools to evaluate efficacy of restoration strategies to improve recreational water quality at a Lake Michigan Beach (Racine, WI)

Hydro-meteorological conditions facilitate transport of fecal indicator bacteria (FIB) to the nearshore environment, affecting recreational water quality. North Beach (Racine, Wisconsin, United States), is an exemplar public beach site along Lake Michigan, where precipitation-mediated surface runoff, wave encroachment, stormwater and tributary outflow were demonstrated to contribute to beach advisories. Multiple restoration actions, including installation of a stormwater retention wetland, were successfully deployed to improve recreational water quality. Implementation of molecular methods (e.g. human microbial source tracking markers and Escherichia coli ( E. coli ) qPCR) assisted in identifying potential pollution sources and improving public health response time. However, periodic water quality failures still occur. As local beach managers reassess restoration measures in response to climatic changes, use of expanded microbial methods (including bacterial community profiling) may contribute to a better understanding of these dynamic environments. In this 2-year study (2015 and 2019), nearshore/offshore Lake Michigan, stormwater, and tributary samples were collected to determine if, 1) the constructed wetland (~50 m from the shoreline) continued to provide stormwater separation/retention and 2) mixing between onshore sources, Root River and Lake Michigan, was increasing due to rising precipitation/lake levels. Monthly rainfall totals were 1.5× higher in 2019 than 2015, coinciding with a 0.63 m lake-level rise. The prevalence of more intense, onshore winds also increased, facilitating interaction between potential reservoirs of FIB with nearshore water through wind driven waves and lake intrusion, e.g. beach sands and the adjacent Root River. While a strong relationship existed between wet weather wetland and North Beach nearshore E. coli concentrations (all sites), bacterial communities were strikingly different. Conversely, bacterial community overlap existed between the Root River mouth and nearshore/offshore sites. These results suggest the constructed wetland can accommodate the climate-related changes observed in this study. Future restoration activities could be directed towards upstream tributary sources in order to minimize microbial contaminants entering Lake Michigan.

Wisconsin↗

USGS1998

Microbial diversity in a hydrocarbon- and chlorinated-solvent- contaminated aquifer undergoing intrinsic bioremediation

A culture-independent molecular phylogenetic approach was used to survey constituents of microbial communities associated with an aquifer contaminated with hydrocarbons (mainly jet fuel) and chlorinated solvents undergoing intrinsic bioremediation. Samples were obtained from three redox zones: methanogenic, methanogenic-sulfate reducing, and iron or sulfate reducing. Small-subunit rRNA genes were amplified directly from aquifer material DNA by PCR with universally conserved or Bacteria - or Archaea -specific primers and were cloned. A total of 812 clones were screened by restriction fragment length polymorphisms (RFLP), approximately 50% of which were unique. All RFLP types that occurred more than once in the libraries, as well as many of the unique types, were sequenced. A total of 104 (94 bacterial and 10 archaeal) sequence types were determined. Of the 94 bacterial sequence types, 10 have no phylogenetic association with known taxonomic divisions and are phylogenetically grouped in six novel division level groups (candidate divisions WS1 to WS6); 21 belong to four recently described candidate divisions with no cultivated representatives (OP5, OP8, OP10, and OP11); and 63 are phylogenetically associated with 10 well-recognized divisions. The physiology of two particularly abundant sequence types obtained from the methanogenic zone could be inferred from their phylogenetic association with groups of microorganisms with a consistent phenotype. One of these sequence types is associated with the genus Syntrophus ; Syntrophus spp. produce energy from the anaerobic oxidation of organic acids, with the production of acetate and hydrogen. The organism represented by the other sequence type is closely related to Methanosaeta spp., which are known to be capable of energy generation only through aceticlastic methanogenesis. We hypothesize, therefore, that the terminal step of hydrocarbon degradation in the methanogenic zone of the aquifer is aceticlastic methanogenesis and that the microorganisms represented by these two sequence types occur in syntrophic association.

Applied and Environmental Microbiology↗