Distribution and stability of sulfate-reducing prokaryotic and hydrogenotrophic methanogenic assemblages in nutrient-impacted regions of the Florida Everglades

Although the influence of phosphorus loading on the Everglades ecosystem has received a great deal of attention, most research has targeted macro indicators, such as those based on vegetation or fauna, or chemical and physical parameters involved in biogeochemical cycles. Fewer studies have addressed the role of microorganisms, and these have mainly targeted gross informative parameters such as microbial biomass, enzymatic activities, and microbial enumerations. The objectives of this study were to characterize the dynamics of sulfate-reducing and methanogenic assemblages using terminal restriction fragment length polymorphism (T-RFLP) targeting the dissimilatory sulfite reductase (dsrA) and methyl coenzyme M reductase (mcrA) genes, respectively, and assess the impact of nutrient enrichment on microbial assemblages in the northern Everglades. T-RFLP combined with principal component analysis was a powerful technique to discriminate between soils from sites with eutrophic, transitional, and oligotrophic nutrient concentrations. dsrA T-RFLP provided a higher level of discrimination between the three sites. mcrA was a relatively weaker system to distinguish between sites, since it could not categorically discriminate between eutrophic and transition soil samples, but may be useful as an early indicator of phosphorus loading which is altering hydrogenotrophic methanogenic community in the transition zones, making them more similar to eutrophic zones. Clearly, targeting a combination of different microbial communities provides greater insight into the functioning of this ecosystem and provides useful information for understanding the relationship between eutrophication effects and microbial assemblages.     

Structure and Function of Methanogens along a Short-Term Restoration Chronosequence in the Florida Everglades

The removal of plants and soil to bedrock to eradicate exotic invasive plants within the Hole-in-the-Donut (HID) region, part of the Everglades National Park (Florida), presented a unique opportunity to study the redevelopment of soil and the associated microbial communities in the context of short-term primary succession and ecosystem restoration. The goal of this study was to identify relationships between soil redevelopment and activity and composition of methanogenic assemblages in HID soils. Methane production potentials indicated a general decline in methanogenic activity with restoration age. Microcosm incubations strongly suggested hydrogenotrophic methanogenesis as the most favorable pathway for methane formation in HID soils from all sites. Culture-independent techniques targeting methyl coenzyme M reductase genes (mcrA) were used to assess the dynamics of methanogenic assemblages. Clone libraries were dominated by sequences related to hydrogenotrophic methanogens of the orders Methanobacteriales and Methanococcales and suggested a general decline in the relative abundance of Methanobacteriales mcrA with time since restoration. Terminal restriction fragment length polymorphism analysis indicated methanogenic assemblages remain relatively stable between wet and dry seasons. Interestingly, analysis of soils across the restoration chronosequence indicated a shift in Methanobacteriales populations with restoration age, suggesting genotypic shifts due to site-specific factors.     

Phylogenetic Characterization of Methanogenic Assemblages in Eutrophic and Oligotrophic Areas of the Florida Everglades

Agricultural activities have produced well-documented changes in the Florida Everglades, including establishment of a gradient in phosphorus concentrations in Water Conservation Area 2A (WCA-2A) of the northern Everglades. An effect of increased phosphorus concentrations is increased methanogenesis in the eutrophic regions compared to the oligotrophic regions of WCA-2A. The goal of this study was to identify relationships between eutrophication and composition and activity of methanogenic assemblages in WCA-2A soils. Distributions of two genes associated with methanogens were characterized in soils taken from WCA-2A: the archaeal 16S rRNA gene and the methyl coenzyme M reductase gene. The richness of methanogen phylotypes was greater in eutrophic than in oligotrophic sites, and sequences related to previously cultivated and uncultivated methanogens were found. A preferential selection for the order Methanomicrobiales was observed in mcrA clone libraries, suggesting primer bias for this group. A greater diversity within the Methanomicrobiales was observed in mcrA clone libraries than in 16S rRNA gene libraries. 16S rRNA phylogenetic analyses revealed a dominance of clones related to Methanosaeta spp., an acetoclastic methanogen dominant in environments with low acetate concentrations. A significant number of clones were related to Methanomicrobiales, an order characterized by species utilizing hydrogen and formate as methanogenic substrates. No representatives of the orders Methanobacteriales and Methanococcales were found in any 16S rRNA clone library, although some Methanobacteriales were found in mcrA libraries. Hydrogenotrophs are the dominant methanogens in WCA-2A, and acetoclastic methanogen genotypes that proliferate in low acetate concentrations outnumber those that typically dominate in higher acetate concentrations.     

Structure and function of methanogens along a short-term restoration chronosequence in the Florida Everglades

The removal of plants and soil to bedrock to eradicate exotic invasive plants within the Hole-in-the-Donut (HID) region, part of the Everglades National Park (Florida), presented a unique opportunity to study the redevelopment of soil and the associated microbial communities in the context of short-term primary succession and ecosystem restoration. The goal of this study was to identify relationships between soil redevelopment and activity and composition of methanogenic assemblages in HID soils. Methane production potentials indicated a general decline in methanogenic activity with restoration age. Microcosm incubations strongly suggested hydrogenotrophic methanogenesis as the most favorable pathway for methane formation in HID soils from all sites. Culture-independent techniques targeting methyl coenzyme M reductase genes (mcrA) were used to assess the dynamics of methanogenic assemblages. Clone libraries were dominated by sequences related to hydrogenotrophic methanogens of the orders Methanobacteriales and Methanococcales and suggested a general decline in the relative abundance of Methanobacteriales mcrA with time since restoration. Terminal restriction fragment length polymorphism analysis indicated methanogenic assemblages remain relatively stable between wet and dry seasons. Interestingly, analysis of soils across the restoration chronosequence indicated a shift in Methanobacteriales populations with restoration age, suggesting genotypic shifts due to site-specific factors.     

Syntrophic-Methanogenic Associations along a Nutrient Gradient in the Florida Everglades

Nutrient runoff from the Everglades Agricultural Area resulted in a well-documented gradient of phosphorus concentrations in soil and water, with concomitant ecosystem-level changes, in the northern Florida Everglades. It was recently reported that sulfate-reducing prokaryote assemblage composition, numbers, and activities are dependent on position along the gradient (H. Castro, K. R. Reddy, and A. Ogram, Appl. Environ. Microbiol. 68:6129-6137, 2002). The present study utilized a combination of culture- and non-culture-based approaches to study differences in composition of assemblages of syntrophic and methanogenic microbial communities in eutrophic, transition, and oligotrophic areas along the phosphorus gradient. Methanogenesis rates were much higher in eutrophic and transition regions, and sequence analysis of 16S rRNA gene clone libraries constructed from samples taken from these regions revealed differences in composition and activities of syntroph-methanogen consortia. Methanogens from eutrophic and transition regions were almost exclusively composed of hydrogenotrophic methanogens, with approximately 10,000-fold-greater most probable numbers of hydrogenotrophs than of acetotrophs. Most cultivable strains from eutrophic and transition regions clustered within novel lineages. In non-culture-based studies to enrich syntrophs, most bacterial and archaeal clones were either members of novel lineages or closely related to uncultivated environmental clones. Novel cultivable Methanosaeta sp. and fatty acid-oxidizing bacteria related to the genera Syntrophomonas and Syntrophobacter were observed in microcosms containing soil from eutrophic regions, and different lines of evidence indicated the existence of novel syntrophic association in eutrophic regions.     

Identification of Methyl Coenzyme M Reductase A (mcrA) Genes Associated with Methane-Oxidizing Archaea

Phylogenetic and stable-isotope analyses implicated two methanogen-like archaeal groups, ANME-1 and ANME-2, as key participants in the process of anaerobic methane oxidation. Although nothing is known about anaerobic methane oxidation at the molecular level, the evolutionary relationship between methane-oxidizing archaea (MOA) and methanogenic archaea raises the possibility that MOA have co-opted key elements of the methanogenic pathway, reversing many of its steps to oxidize methane anaerobically. In order to explore this hypothesis, the existence and genomic conservation of methyl coenzyme M reductase (MCR), the enzyme catalyzing the terminal step in methanogenesis, was studied in ANME-1 and ANME-2 archaea isolated from various marine environments. Clone libraries targeting a conserved region of the alpha subunit of MCR (mcrA) were generated and compared from environmental samples, laboratory-incubated microcosms, and fosmid libraries. Four out of five novel mcrA types identified from these sources were associated with ANME-1 or ANME-2 group members. Assignment of mcrA types to specific phylogenetic groups was based on environmental clone recoveries, selective enrichment of specific MOA and mcrA types in a microcosm, phylogenetic congruence between mcrA and small-subunit rRNA tree topologies, and genomic context derived from fosmid sequences. Analysis of the ANME-1 and ANME-2 mcrA sequences suggested the potential for catalytic activity based on conservation of active-site amino acids. These results provide a basis for identifying methanotrophic archaea with mcrA sequences and define a functional genomic link between methanogenic and methanotrophic archaea.     

Evaluation of PCR Amplification Bias by Terminal Restriction Fragment Length Polymorphism Analysis of Small-Subunit rRNA and mcrA Genes by Using Defined Template Mixtures of Methanogenic Pure Cultures and Soil DNA Extracts

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a widely used method for profiling microbial community structure in different habitats by targeting small-subunit (SSU) rRNA and also functional marker genes. It is not known, however, whether relative gene frequencies of individual community members are adequately represented in post-PCR amplicon frequencies as shown by T-RFLP. In this study, precisely defined artificial template mixtures containing genomic DNA of four different methanogens in various ratios were prepared for subsequent T-RFLP analysis. PCR amplicons were generated from defined mixtures targeting not only the SSU rRNA but also the methyl-coenzyme M reductase (mcrA/mrtA) genes of methanogens. Relative amplicon frequencies of microorganisms were quantified by comparing fluorescence intensities of characteristic terminal restriction fragments. SSU ribosomal DNA (rDNA) template ratios in defined template mixtures of the four-membered community were recovered absolutely by PCR-T-RFLP analysis, which demonstrates that the T-RFLP analysis evaluated can give a quantitative view of the template pool. SSU rDNA-targeted T-RFLP analysis of a natural community was found to be highly reproducible, independent of PCR annealing temperature, and unaffected by increasing PCR cycle numbers. Ratios of mcrA-targeted T-RFLP analysis were biased, most likely by PCR selection due to the degeneracy of the primers used. Consequently, for microbial community analyses, each primer system used should be evaluated carefully for possible PCR bias. In fact, such bias can be detected by using T-RFLP analysis as a tool for the precise quantification of the PCR product pool.     

Phylogenetic characterization of methanogenic assemblages in eutrophic and oligotrophic areas of the Florida Everglades

Agricultural activities have produced well-documented changes in the Florida Everglades, including establishment of a gradient in phosphorus concentrations in Water Conservation Area 2A (WCA-2A) of the northern Everglades. An effect of increased phosphorus concentrations is increased methanogenesis in the eutrophic regions compared to the oligotrophic regions of WCA-2A. The goal of this study was to identify relationships between eutrophication and composition and activity of methanogenic assemblages in WCA-2A soils. Distributions of two genes associated with methanogens were characterized in soils taken from WCA-2A: the archaeal 16S rRNA gene and the methyl coenzyme M reductase gene. The richness of methanogen phylotypes was greater in eutrophic than in oligotrophic sites, and sequences related to previously cultivated and uncultivated methanogens were found. A preferential selection for the order Methanomicrobiales was observed in mcrA clone libraries, suggesting primer bias for this group. A greater diversity within the Methanomicrobiales was observed in mcrA clone libraries than in 16S rRNA gene libraries. 16S rRNA phylogenetic analyses revealed a dominance of clones related to Methanosaeta spp., an acetoclastic methanogen dominant in environments with low acetate concentrations. A significant number of clones were related to Methanomicrobiales, an order characterized by species utilizing hydrogen and formate as methanogenic substrates. No representatives of the orders Methanobacteriales and Methanococcales were found in any 16S rRNA clone library, although some Methanobacteriales were found in mcrA libraries. Hydrogenotrophs are the dominant methanogens in WCA-2A, and acetoclastic methanogen genotypes that proliferate in low acetate concentrations outnumber those that typically dominate in higher acetate concentrations.     

Composition and function of sulfate-reducing prokaryotes in eutrophic and pristine areas of the Florida Everglades

As a result of agricultural activities in regions adjacent to the northern boundary of the Florida Everglades, a nutrient gradient developed that resulted in physicochemical and ecological changes from the original system. Sulfate input from agricultural runoff and groundwater is present in soils of the Northern Everglades, and sulfate-reducing prokaryotes (SRP) may play an important role in biogeochemical processes such as carbon cycling. The goal of this project was to utilize culture-based and non-culture-based approaches to study differences between the composition of assemblages of SRP in eutrophic and pristine areas of the Everglades. Sulfate reduction rates and most-probable-number enumerations revealed SRP populations and activities to be greater in eutrophic zones than in more pristine soils. In eutrophic regions, methanogenesis rates were higher, the addition of acetate stimulated methanogenesis, and SRP able to utilize acetate competed to a limited degree with acetoclastic methanogens. A surprising amount of diversity within clone libraries of PCR-amplified dissimilatory sulfite reductase (DSR) genes was observed, and the majority of DSR sequences were associated with gram-positive spore-forming Desulfotomaculum and uncultured microorganisms. Sequences associated with Desulfotomaculum fall into two categories: in the eutrophic regions, 94.7% of the sequences related to Desulfotomaculum were associated with those able to completely oxidize substrates, and in samples from pristine regions, all Desulfotomaculum-like sequences were related to incomplete oxidizers. This metabolic selection may be linked to the types of substrates that Desulfotomaculum spp. utilize; it may be that complete oxidizers are more versatile and likelier to proliferate in nutrient-rich zones of the Everglades. Desulfotomaculum incomplete oxidizers may outcompete complete oxidizers for substrates such as hydrogen in pristine zones where diverse carbon sources are less available.     

Geochemistry and Microbial Populations in Sediments of the Northern Baffin Bay,Arctic

The Northern Baffin Bay between Greenland and Canada is a remote Arctic area restricted in primary production by seasonal ice cover, with presumably low sedimentation rates, carbon content and microbial activities in its sediments. Our aim was to study the so far unknown subseafloor geochemistry and microbial populations driving seafloor ecosystems. Shelf sediments had the highest organic carbon content, numbers of Bacteria and Archaea, and microcosms inoculated from Shelf sediments showed highest sulfate reduction and methane production rates. Sediments in the central deep area and on the southern slope contained less organic carbon and overall lower microbial numbers. Similar 16S rRNA gene copy numbers of Archaea and Bacteria were found for the majority of the sites investigated. Sulfate in pore water correlated with dsrA copy numbers of sulfate-reducing prokaryotes and differed between sites. No methane was found as free gas in the sediments, and mcrA copy numbers of methanogenic Archaea were low. Methanogenic and sulfate-reducing cultures were enriched on a variety of substrates including hydrocarbons. In summary, the Greenlandic shelf sediments contain vital microbial communities adapted to their specific environmental conditions.     

Identification of Methanogenic archaea in the Hyporheic Sediment of Sitka Stream

Methanogenic archaea produce methane as a metabolic product under anoxic conditions and they play a crucial role in the global methane cycle. In this study molecular diversity of methanogenic archaea in the hyporheic sediment of the lowland stream Sitka (Olomouc, Czech Republic) was analyzed by PCR amplification, cloning and sequencing analysis of the methyl coenzyme M reductase alpha subunit (mcrA) gene. Sequencing analysis of 60 clones revealed 24 different mcrA phylotypes from hyporheic sedimentary layers to a depth of 50 cm. Phylotypes were affiliated with Methanomicrobiales, Methanosarcinales and Methanobacteriales orders. Only one phylotype remains unclassified. The majority of the phylotypes showed higher affiliation with uncultured methanogens than with known methanogenic species. The presence of relatively rich assemblage of methanogenic archaea confirmed that methanogens may be an important component of hyporheic microbial communities and may affect CH₄ cycling in rivers.     

Syntrophic-methanogenic associations along a nutrient gradient in the Florida Everglades

Nutrient runoff from the Everglades Agricultural Area resulted in a well-documented gradient of phosphorus concentrations in soil and water, with concomitant ecosystem-level changes, in the northern Florida Everglades. It was recently reported that sulfate-reducing prokaryote assemblage composition, numbers, and activities are dependent on position along the gradient (H. Castro, K. R. Reddy, and A. Ogram, Appl. Environ. Microbiol. 68:6129-6137, 2002). The present study utilized a combination of culture- and non-culture-based approaches to study differences in composition of assemblages of syntrophic and methanogenic microbial communities in eutrophic, transition, and oligotrophic areas along the phosphorus gradient. Methanogenesis rates were much higher in eutrophic and transition regions, and sequence analysis of 16S rRNA gene clone libraries constructed from samples taken from these regions revealed differences in composition and activities of syntroph-methanogen consortia. Methanogens from eutrophic and transition regions were almost exclusively composed of hydrogenotrophic methanogens, with approximately 10,000-fold-greater most probable numbers of hydrogenotrophs than of acetotrophs. Most cultivable strains from eutrophic and transition regions clustered within novel lineages. In non-culture-based studies to enrich syntrophs, most bacterial and archaeal clones were either members of novel lineages or closely related to uncultivated environmental clones. Novel cultivable Methanosaeta sp. and fatty acid-oxidizing bacteria related to the genera Syntrophomonas and Syntrophobacter were observed in microcosms containing soil from eutrophic regions, and different lines of evidence indicated the existence of novel syntrophic association in eutrophic regions.     

Distance-Decay and Taxa-Area Relationships for Bacteria,Archaea and Methanogenic Archaea in a Tropical Lake Sediment

The study of of the distribution of microorganisms through space (and time) allows evaluation of biogeographic patterns, like the species-area index (z). Due to their high dispersal ability, high reproduction rates and low rates of extinction microorganisms tend to be widely distributed, and they are thought to be virtually cosmopolitan and selected primarily by environmental factors. Recent studies have shown that, despite these characteristics, microorganisms may behave like larger organisms and exhibit geographical distribution. In this study, we searched patterns of spatial diversity distribution of bacteria and archaea in a contiguous environment. We collected 26 samples of a lake sediment, distributed in a nested grid, with distances between samples ranging from 0.01 m to 1000 m. The samples were analyzed using T-RFLP (Terminal restriction fragment length polymorphism) targeting mcrA (coding for a subunit of methyl-coenzyme M reductase) and the genes of Archaeal and Bacterial 16S rRNA. From the qualitative and quantitative results (relative abundance of operational taxonomic units) we calculated the similarity index for each pair to evaluate the taxa-area and distance decay relationship slopes by linear regression. All results were significant, with mcrA genes showing the highest slope, followed by Archaeal and Bacterial 16S rRNA genes. We showed that the microorganisms of a methanogenic community, that is active in a contiguous environment, display spatial distribution and a taxa-area relationship.     

Spatial and seasonal patterns of periphyton biomass and productivity in the northern Everglades,Florida, U.S.A.

We sampled periphyton in dominant habitats at oligotrophic and eutrophic sites in the northern Everglades during the wet and the dryseasons to determine the effects of nutrient enrichment on periphytonbiomass, taxonomic composition, productivity, and phosphorus storage. Arealbiomass was high (100–1600 g ash-free dry mass [AFDM]m⁻²) in oligotrophic sloughs and in stands of the emergentmacrophyte Eleocharis cellulosa, but was low in adjacent stands of sawgrass,Cladium jamaicense (7–52 g AFDM m⁻²). Epipelon biomasswas high throughout the year at oligotrophic sites whereas epiphyton andmetaphyton biomass varied seasonally and peaked during the wet season.Periphyton biomass was low (3–68 g AFDM m⁻²) and limitedto epiphyton and metaphyton in open-water habitats at eutrophic sites andwas undetectable in cattail stands (Typha domingensis) that covered morethan 90% of the marsh in these areas. Oligotrophic periphytonassemblages exhibited strong seasonal shifts in species composition and weredominated by cyanobacteria (e.g., Chroococcus turgidus, Scytonema hofmannii)during the wet season and diatoms (e.g. Amphora lineolata, Mastogloiasmithii) during the dry season. Eutrophic assemblages were dominated byCyanobacteria (e.g., Oscillatoria princeps) and green algae (e.g., Spirogyraspp.) and exhibited comparatively little seasonality. Biomass-specific grossprimary productivity (GPP) of periphyton assemblages in eutrophic openwaters was higher than for comparable slough assemblages, but areal GPP wassimilar in these eutrophic (0.9–9.1 g C m⁻²d⁻¹) and oligotrophic (1.75–11.49 g C m⁻²d⁻¹) habitats. On a habitat-weighted basis, areal periphytonGPP was 6- to 30-fold lower in eutrophic areas of the marsh due to extensiveTypha stands that were devoid of periphyton. Periphyton at eutrophic siteshad higher P content and uptake rates than the oligotrophic assemblage, butstored only 5% as much P because of the lower areal biomass.Eutrophication in the Everglades has resulted in a decrease in periphytonbiomass and its contribution to marsh primary productivity. These changesmay have important implications for efforts to manage this wetland in asustainable manner. This revised version was published online in July 2006 with corrections to the Cover Date.     

Enrichment of lignocellulose-degrading microbial communities from natural and engineered methanogenic environments

The aim of this study was to develop an effective bioaugmentation concept for anaerobic digesters treating lignocellulosic biomass such as straw. For that purpose, lignocellulose-degrading methanogenic communities were enriched on wheat straw from cow and goat rumen fluid as well as from a biogas reactor acclimated to lignocellulosic biomass (sorghum as mono-substrate). The bacterial communities of the enriched cultures and the different inocula were examined by 454 amplicon sequencing of 16S rRNA genes while the methanogenic archaeal communities were analyzed by terminal restriction fragment length polymorphism (T-RFLP) fingerprinting of the mcrA gene. Bacteroidetes was the most abundant phylum in all samples. Within the Bacteroidetes phylum, Bacteroidaceae was the most abundant family in the rumen-derived enrichment cultures, whereas Porphyromonadaceae was the predominant one in the reactor-derived culture. Additionally, the enrichment procedure increased the relative abundance of Ruminococcaceae (phylum: Firmicutes) in all cultures. T-RFLP profiles of the mcrA gene amplicons highlighted that the ruminal methanogenic communities were composed of hydrogenotrophic methanogens dominated by the order Methanobacteriales regardless of the host species. The methanogenic communities changed significantly during the enrichment procedure, but still the strict hydrogenotrophic Methanobacteriales and Methanomicrobiales were the predominant orders in the enrichment cultures. The bioaugmentation potential of the enriched methanogenic cultures will be evaluated in further studies.

     

Aquatic faunal responses to an induced regime shift in the phosphorus‐impacted Everglades

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Characterization of the methanogen community in a household anaerobic digester fed with swine manure in China

Household anaerobic digesters have been installed across rural China for biogas production, but information on methanogen community structure in these small biogas units is sparsely available. By creating clone libraries for 16S rRNA and methyl coenzyme M reductase alpha subunit (mcrA) genes, we investigated the methanogenic consortia in a household biogas digester treating swine manure. Operational taxonomic units (OTUs) were defined by comparative sequence analysis, seven OTUs were identified in the 16S rRNA gene library, and ten OTUs were identified in the mcrA gene library. Both libraries were dominated by clones highly related to the type strain Methanocorpusculum labreanum Z, 64.0 % for 16S rRNA gene clones and 64.3 % for mcrA gene clones. Additionally, gas chromatography assays showed that formic acid was 84.54 % of the total volatile fatty acids and methane was 57.20 % of the biogas composition. Our results may help further isolation and characterization of methanogenic starter strains for industrial biogas production.     

Diversity and Distribution of Sulfate-Reducing Bacteria in Permanently Frozen Lake Fryxell, McMurdo Dry Valleys, Antarctica

The permanently frozen freshwater Lake Fryxell, located in the Dry Valleys of Antarctica, exhibits an ideal geochemistry for microbial sulfate reduction. To investigate the population of sulfate-reducing bacteria in Lake Fryxell, both 16S rRNA gene and metabolic primer sets targeting the dsrA gene for the dissimilatory sulfite reductase alpha subunit were employed to analyze environmental DNA obtained from the water column and sediments of Lake Fryxell. In addition, enrichment cultures of sulfate-reducing bacteria established at 4°C from Lake Fryxell water were also screened using the dsrA primer set. The sequence information obtained showed that a diverse group of sulfate-reducing prokaryotes of the domain Bacteria inhabit Lake Fryxell. With one exception, the enrichment culture sequences were not represented within the environmental sequences. Sequence data were compared with the geochemical profile of Lake Fryxell to identify possible connections between the diversity of sulfate-reducing bacteria and limnological conditions. Several clone groups were highly localized with respect to lake depth and, therefore, experienced specific physiochemical conditions. However, all sulfate-reducing bacteria inhabiting Lake Fryxell must function under the constantly cold conditions characteristic of this extreme environment.