The effects of sediment slurrying on microbial processes,and the role of amino acids as substrates for sulfate reduction in anoxic marine sediments

In sediment slurry experiments with anoxic marine sediments collected in Cape Lookout Bight, NC, and a site in mid-Chesapeake Bay, the rates of sulfate reduction and ammonium production decrease with increasing dilution of sediment with oxygen-free sea-water. The effect of sediment dilution on the rates of these processes can be described by a simple mathematical relationship, and when these rates are corrected for sediment dilution they yield values which agree well with direct measurements of these processes.In sediment slurry studies of amino acid utilization in Cape Lookout Bight sediments, the fermentative decarboxylation of glutamic acid (to -aminobutyric acid) or aspartic acid (to alanine or -alanine) did not occur when either of these amino acids were added to Cape Lookout Bight slurries. The addition of glutamic acid did however lead to a small (1) transient build-up of -aminoglutaric acid. Measured rates of glutamic acid uptake in these slurries also decreased with increasing sediment dilution.Molybdate inhibition experiments demonstrated that dissolved free amino acids represent 1–3% of the carbon sources/electron donors used for sulfate reduction in Cape Lookout Bight sediments. The direct oxidation of amino acids by sulfate reducing bacteria also accounts for 13–20% of the total ammonium produced. Glutamic acid, alanine, -aminoglutaric acid, aspartic acid and asparagine are the major amino acids oxidized by sulfate reducing bacteria in Cape Lookout Bight sediments.     

Temperature regulation of anaerobic degradation of organic matter

Anaerobic degradation of organic matter follows similar pathways in digesters and anaerobic freshwater sediments. The responsible microorganisms are linked in a complex food web, where short chain fatty acids and H₂ are important intermediates. Degradation of short-chain fatty acids is endothermic under standard conditions and is only possible at low H₂ partial pressures maintained by exothermic methanogenesis. The coupling between these endothermic and exothermic processes is delicate, and hence sensitive to environmental changes such as temperature variations. The effect of temperature on thermodynamics and on kinetics of these and other anaerobic degradation processes with emphasis on freshwater ecosystems is discussed.The author is with the Department of General Microbiology, Institute of Molecular Biology, University of Copenhagen, Sølvgade 83 H, DK-1307 Copenhagen K, Denmark     

Hydrogen Profiles and Localization of Methanogenic Activities in the Highly Compartmentalized Hindgut of Soil-Feeding Higher Termites (Cubitermes spp.)

It has been shown that the coexistence of methanogenesis and reductive acetogenesis in the hindgut of the wood-feeding termite Reticulitermes flavipes is based largely on the radial distribution of the respective microbial populations and relatively high hydrogen partial pressures in the gut lumen. Using Clark-type microelectrodes, we showed that the situation in Cubitermes orthognathus and other soil-feeding members of the subfamily Termitinae is different and much more complex. All major compartments of agarose-embedded hindguts were anoxic at the gut center, and high H₂ partial pressures (1 to 10 kPa) in the alkaline anterior region rendered the mixed segment and the third proctodeal segment (P3) significant sources of H₂. Posterior to the P3 segment, however, H₂ concentrations were generally below the detection limit (<100 Pa). All hindgut compartments turned into efficient hydrogen sinks when external H₂ was supplied, but methane was formed mainly in the P3/4a and P4b compartments, and in the latter only when H₂ or formate was added. Addition of H₂ to the gas headspace stimulated CH₄ emission of living termites, indicating that endogenous H₂ production limits methanogenesis also in vivo. At the low H₂ partial pressures in the posterior hindgut, methanogens would most likely outcompete homoacetogens for this electron donor. This might explain the apparent predominance of methanogenesis over reductive acetogenesis in the hindgut of soil-feeding termites, although the presence of homoacetogens in the anterior, highly alkaline region cannot yet be excluded. In addition, the direct contact of anterior and posterior hindgut compartments in situ permits a cross-epithelial transfer of H₂ or formate, which would not only fuel methanogenesis in these compartments, but would also create favorable microniches for reductive acetogenesis. In situ rates and spatial distribution of H₂-dependent acetogenic activities are addressed in a companion paper (A. Tholen and A. Brune, Appl. Environ. Microbiol. 65:4497–4505, 1999).     

Microbial Fuel Cells and Microbial Ecology: Applications in Ruminant Health and Production Research

Microbial fuel cell (MFC) systems employ the catalytic activity of microbes to produce electricity from the oxidation of organic, and in some cases inorganic, substrates. MFC systems have been primarily explored for their use in bioremediation and bioenergy applications; however, these systems also offer a unique strategy for the cultivation of synergistic microbial communities. It has been hypothesized that the mechanism(s) of microbial electron transfer that enable electricity production in MFCs may be a cooperative strategy within mixed microbial consortia that is associated with, or is an alternative to, interspecies hydrogen (H₂) transfer. Microbial fermentation processes and methanogenesis in ruminant animals are highly dependent on the consumption and production of H₂in the rumen. Given the crucial role that H₂ plays in ruminant digestion, it is desirable to understand the microbial relationships that control H₂ partial pressures within the rumen; MFCs may serve as unique tools for studying this complex ecological system. Further, MFC systems offer a novel approach to studying biofilms that form under different redox conditions and may be applied to achieve a greater understanding of how microbial biofilms impact animal health. Here, we present a brief summary of the efforts made towards understanding rumen microbial ecology, microbial biofilms related to animal health, and how MFCs may be further applied in ruminant research.     

Epilimnetic sulfate reduction and its relationship to lake acidification

Sulfate reduction occurred from 0–3 cm below the surface of the epilimnetic sediments of three northwestern Ontario lakes, including L.223, which has been experimentally acidified by additions of sulfuric acid. Shallow water sites were conducive to SO₄ ^2– reduction because decomposition in these predominantly sandy sediments caused oxygen concentrations to decrease rapidly within mm below the interface. The occurrence of methanogenesis just below the depth of minimum SO₄ ^2- concentration demonstrated that availability of organic carbon was not a limiting factor for sulphate reduction.Laboratory studies showed that SO₄ ^2- reduction rates in mixed sediments were lower at pH 4 than at pH 6. However, sulfate gradients in sediments indicated that there was no effect of acidification on sulfate reduction in situ. This was probably because microbial H⁺ consumption in the epilimnetic sediments maintained steep pH gradients below the sediment-water interface. The pH increased from = 5.0 to 6.5 or higher by a depth of 3.0 cm into the sediments.     

Thermodynamic and kinetic analysis of the H2 threshold for Methanobacterium bryantii M.o.H

H₂ thresholds, concentrations below which H₂ consumption by a microbial group stops, have been associated with microbial respiratory processes such as dechlorination, denitrification, sulfate reduction, and methanogenesis. Researchers have proposed that observed H₂ thresholds occur when the available Gibbs free energy is minimal (ΔG ≈ 0) for a specific respiratory reaction. Others suggest that microbial kinetics also may play a role in controlling the thresholds. Here, we comprehensively evaluate H₂ thresholds in light of microbial thermodynamic and kinetic principles. We show that a thermodynamic H₂ threshold for Methanobacterium bryantii M.o.H. is not controlled by ΔG for methane production from H₂ + HCO₃⁻. We repeatedly attain a H₂ threshold near 0.4 nM, with a range of 0.2–1 nM, and ΔG for methanogenesis from H₂ + HCO₃⁻ is positive, +5 to +7 kJ/mol-H_2, at the threshold in most cases. We postulate that the H₂ threshold is controlled by a separate reaction other than methane production. The electrons from H₂ oxidation are transferred to an electron sink that is a solid-phase component of the cells. We also show that a kinetic threshold (S _min) occurs at a theoretically computed H₂ concentration of about 2400 nM at which biomass growth shifts from positive to negative.     

Carbon Monoxide, Hydrogen, and Formate Metabolism during Methanogenesis from Acetate by Thermophilic Cultures of Methanosarcina and Methanothrix Strains

CO and H₂ have been implicated in methanogenesis from acetate, but it is unclear whether they are directly involved in methanogenesis or electron transfer in acetotrophic methanogens. We compared metabolism of H₂, CO, and formate by cultures of the thermophilic acetotrophic methanogens Methanosarcina thermophila TM-1 and Methanothrix sp. strain CALS-1. M. thermophila accumulated H₂ to partial pressures of 40 to 70 Pa (1 Pa = 0.987 × 10^-5 atm), as has been previously reported for this and other Methanosarcina cultures. In contrast, Methanothrix sp. strain CALS-1 accumulated H₂ to maximum partial pressures near 1 Pa. Growing cultures of Methanothrix sp. strain CALS-1 initially accumulated CO, which reached partial pressures near 0.6 Pa (some CO came from the rubber stopper) during the middle of methanogenesis; this was followed by a decrease in CO partial pressures to less than 0.01 Pa by the end of methanogenesis. Accumulation or consumption of CO by cultures of M. thermophila growing on acetate was not detected. Late-exponential-phase cultures of Methanothrix sp. strain CALS-1, in which the CO partial pressure was decreased by flushing with N₂-CO₂, accumulated CO to 0.16 Pa, whereas cultures to which ca. 0.5 Pa of CO was added consumed CO until it reached this partial pressure. Cyanide (1 mM) blocked CO consumption but not production. High partial pressures of H₂ (40 kPa) inhibited methanogenesis from acetate by M. thermophila but not by Methanothrix sp. strain CALS-1, and 2 kPa of CO was not inhibitory to M. thermophila but was inhibitory to Methanothrix sp. strain CALS-1. Levels of CO dehydrogenase, hydrogenase, and formate dehydrogenase in Methanothrix sp. strain CALS-1 were 9.1, 0.045, and 5.8 μmol of viologen reduced min^-1 mg of protein^-1. These results suggest that CO plays a role in Methanothrix sp. strain CALS-1 similar to that of H₂ in M. thermophila and are consistent with the conclusion that CO is an intermediate in a catabolic or anabolic pathway in Methanothrix sp. strain CALS-1; however, they could also be explained by passive equilibration of CO with a metabolic intermediate.     

Hydrogen metabolism and sulfate-dependent inhibition of methanogenesis in a eutrophic lake sediment (Lake Mendota)

Hydrogen metabolism was studied in anoxic sediments of the stratified Lake Mendota; using a method which allowed the measurement of in situ H₂ concentrations and the headspace-free analysis of turnover of dissolved H₂. Addition of sulfate resulted in partial but immediate inhibition of H₂-dependent methanogenesis. Sulfate addition did not result in an immediate decrease in the steady state concentration of dissolved H₂, nor did it significantly stimulate the rate constant of H₂ turnover. Sulfate-induced decrease in dissolved H₂ was only observed after prolonged incubation or when endogenous H₂ production was stimulated by added glucose. The turnover of the in situ H₂ accounted for only 14% of the H₂-dependent methanogenesis from bicarbonate. While rates of methanogenesis increased during the season, rates of H₂ turnover decreased, accounting for only 2% of the H₂-dependent methanogenesis at the end of summer stratification. These observations indicate that increasing proportions of CH₄ were formed from H₂ being directly transferred in syntrophic methanogenic associations. The rapid inhibition of H₂-dependent methanogenesis by exogenous sulfate may be explained at least partially by assuming methanogenic associations in which syntrophic sulfate reducers change their metabolism from fermentative H₂ production to sulfate reduction.     

Microbial community structure in a biofilm anode fed with a fermentable substrate: The significance of hydrogen scavengers

We compared the microbial community structures that developed in the biofilm anode of two microbial electrolysis cells fed with ethanol, a fermentable substrate—one where methanogenesis was allowed and another in which it was completely inhibited with 2‐bromoethane sulfonate. We observed a three‐way syntrophy among ethanol fermenters, acetate‐oxidizing anode‐respiring bacteria (ARB), and a H₂ scavenger. When methanogenesis was allowed, H₂‐oxidizing methanogens were the H₂ scavengers, but when methanogenesis was inhibited, homo‐acetogens became a channel for electron flow from H₂ to current through acetate. We established the presence of homo‐acetogens by two independent molecular techniques: 16S rRNA gene based pyrosequencing and a clone library from a highly conserved region in the functional gene encoding formyltetrahydrofolate synthetase in homo‐acetogens. Both methods documented the presence of the homo‐acetogenic genus, Acetobacterium, only with methanogenic inhibition. Pyrosequencing also showed a predominance of ethanol‐fermenting bacteria, primarily represented by the genus Pelobacter. The next most abundant group was a diverse community of ARB, and they were followed by H₂‐scavenging syntrophic partners that were either H₂‐oxidizing methanogens or homo‐acetogens when methanogenesis was suppressed. Thus, the community structure in the biofilm anode and suspension reflected the electron‐flow distribution and H₂‐scavenging mechanism. Biotechnol. Bioeng. 2010;105: 69–78. © 2009 Wiley Periodicals, Inc.     

Trophic links between fermenters and methanogens in a moderately acidic fen soil

Trophic links between fermentation and methanogenesis of soil derived from a methane‐emitting, moderately acidic temperate fen (pH 4.5) were investigated. Initial CO₂:CH₄ production ratios in anoxic microcosms indicated that methanogenesis was concomitant to other terminal anaerobic processes. Methane production in anoxic microcosms at in situ pH was stimulated by supplemental H₂–CO₂, formate or methanol; supplemental acetate did not stimulate methanogenesis. Supplemental H₂–CO₂, formate or methanol also stimulated the formation of acetate, indicating that the fen harbours moderately acid‐tolerant acetogens. Supplemental monosaccharides (glucose, N‐acetylglucosamine and xylose) stimulated the production of CO₂, H₂, acetate and other fermentation products when methanogenesis was inhibited with 2‐bromoethane sulfonate 20 mM. Glucose stimulated methanogenesis in the absence of BES. Upper soil depths yielded higher anaerobic activities and also higher numbers of cells. Detected archaeal 16S rRNA genes were indicative of H₂–CO₂‐ and formate‐consuming methanogens (Methanomicrobiaceae), obligate acetoclastic methanogens (Methanosaetaceae) and crenarchaeotes (groups I.1a, I.1c and I.3). Molecular analyses of partial sequences of 16S rRNA genes revealed the presence of Acidobacteria, Nitrospirales, Clamydiales, Clostridiales, Alpha‐, Gamma‐, Deltaproteobacteria and Cyanobacteria. These collective results suggest that this moderately acidic fen harbours phylogenetically diverse, moderately acid tolerant fermenters (both facultative aerobes and obligate anaerobes) that are trophically linked to methanogenesis.     

Methane oxidation potential in the water column of two diverse coastal marine sites

Methane oxidation in the water column was investigated at two nearshore marine environments with relatively high concentrations of dissolved methane. In the northern Gulf of Mexico, high methane oxidation rates were observed at the pycnocline, with the highest oxidation rate corresponding to the most negative bacterial ¹³C values. These low isotopic values occurred during the winter when overall bacterial productivity was low, suggesting that at this time of the year, methanotrophs in the Gulf could make up a significant portion of the overall bacterial assemblage. Although methane oxidation also occurred during more productive times (i.e., summer), the isotopic signal of methane oxidation was not observed in the bacterial biomass because of the higher overall bacterial productivity. The other site, Cape Lookout Bight, NC, is a small marine embayment where methane is produced in the organic-rich sediments. No measurable rates of methane oxidation in the water column occurred, and no anomalously low ¹³C values of the bacterioplankton were measured. In both environments, methane production and oxidation appear to be spatially coupled, occurring at/near the pycnocline in the northern Gulf of Mexico and at the sediment-water interface at Cape Lookout Bight, NC.     

No acetogen is equal: Strongly different H2 thresholds reflect diverse bioenergetics in acetogenic bacteria

Acetogens share the capacity to convert H₂ and CO₂ into acetate for energy conservation (ATP synthesis). This reaction is attractive for applications, such as gas fermentation and microbial electrosynthesis. Different H₂ partial pressures prevail in these distinctive applications (low concentrations during microbial electrosynthesis [<40 Pa] vs. high concentrations with gas fermentation [>9%]). Strain selection thus requires understanding of how different acetogens perform under different H₂ partial pressures. Here, we determined the H₂ threshold (H₂ partial pressure at which acetogenesis halts) for eight different acetogenic strains under comparable conditions. We found a three orders of magnitude difference between the lowest and highest H₂ threshold (6 ± 2 Pa for Sporomusa ovata vs. 1990 ± 67 Pa for Clostridium autoethanogenum), while Acetobacterium strains had intermediate H₂ thresholds. We used these H₂ thresholds to estimate ATP gains, which ranged from 0.16 to 1.01 mol ATP per mol acetate (S. ovata vs. C. autoethanogenum). The experimental H₂ thresholds thus suggest strong differences in the bioenergetics of acetogenic strains and possibly also in their growth yields and kinetics. We conclude that no acetogen is equal and that a good understanding of their differences is essential to select the most optimal strain for different biotechnological applications.     

Pyruvate oxidation by Methanococcus spp.

In the absence of H₂, Methanococcus spp. utilized pyruvate as an electron donor for methanogenesis. For Methanococcus voltae A3, Methanococcus maripaludis JJ1, and Methanococcus vannielii, typical rates of pyruvate-dependent methanogenesis were 3.4, 2.8, and 3.9 nmol min^-1 mg^-1 cell dry wt, respectively. These rates were 1–4% of the rates of H₂-dependent methanogenesis. For M. voltae, the concentration of pyruvate required for one-half the maximum rate of methanogenesis was 7 mM, and pyruvate-dependent methanogenesis was linear for 3 days. Radiolabeled acetate was formed from [3-¹⁴C]pyruvate, and the stoichiometry of pyruvate consumed per acetate produced was 1.12±0.27. The stoichiometry of pyruvate consumed per CH₄ produced was 3.64±0.34. These values are close to the expected values of 1 acetate and 4 CH₄. Although 10–30% of total cell carbon could be obtained from exogenous pyruvate during growth with H₂, pyruvate did not replace the nutritional requirement for acetate in Methanococcus voltae A3 or two acetate auxotrophs of Methanococcus maripaludis, JJ6 and JJ7. These results suggest that pyruvate was not oxidized in the presence of H₂. The inability to oxidize pyruvate during H₂-dependent methanogenesis would prevent a futile cycle of pyruvate oxidation and biosynthesis during autotrophic growth.     

Determination of isotope fractionation factors and quantification of carbon flow by stable carbon isotope signatures in a methanogenic rice root model system

Methanogenic processes can be quantified by stable carbon isotopes, if necessary modeling parameters, especially fractionation factors, are known. Anoxically incubated rice roots are a model system with a dynamic microbial community and thus suitable to investigate principal geochemical processes in anoxic natural systems. Here we applied an inhibitor of acetoclastic methanogenesis (methyl fluoride), calculated the thermodynamics of the involved processes, and analyzed the carbon stable isotope signatures of CO₂, CH₄, propionate, acetate and the methyl carbon of acetate to characterize the carbon flow during anaerobic degradation of rice roots to the final products CO₂ and CH₄. Methyl fluoride inhibited acetoclastic methanogenesis and thus allowed to quantify the fractionation factor of CH₄ production from H₂/CO₂. Since our model system was not affected by H₂ gradients, the fractionation factor could alternatively be determined from the Gibbs free energies of hydrogenotrophic methanogenesis. The fractionation factor of acetoclastic methanogenesis was also experimentally determined. The data were used for successfully modeling the carbon flow. The model results were in agreement with the measured process data, but were sensitive to even small changes in the fractionation factor of hydrogenotrophic methanogenesis. Our study demonstrates that stable carbon isotope signatures are a proper tool to quantify carbon flow, if fractionation factors are determined precisely.     

Gas Metabolism Evidence in Support of the Juxtaposition of Hydrogen-Producing and Methanogenic Bacteria in Sewage Sludge and Lake Sediments

We developed new techniques to measure dissolved H₂ and H₂ consumption kinetics in anoxic ecosystems that were not dependent on headspace measurements or gas transfer-limited experimentation. These H₂ metabolism parameters were then compared with measured methane production rates, and estimates of H₂ production and interspecies H₂ transfer were made. The H₂ pool sizes were 205 and 31 nM in sewage sludge from an anaerobic digestor and in sediments (24 m) from Lake Mendota, respectively. The H₂ turnover rate constants, as determined by using in situ pool sizes and temperatures, were 103 and 31 h⁻¹ for sludge and sediment, respectively. The observed H₂ turnover rate accounted for only 5 to 6% of the expected H₂-CO₂-dependent methanogenesis in these ecosystems. Our results are in general agreement with the results reported previously and are used to support the conclusion that most of the H₂-dependent methanogenesis in these ecosystems occurs as a consequence of direct interspecies H₂ transfer between juxtapositioned microbial associations within flocs or consortia.     

Partitioning Effects during Terminal Carbon and Electron Flow in Sediments of a Low-Salinity Meltwater Pond near Bratina Island, McMurdo Ice Shelf, Antarctica

A study of anaerobic sediments below cyanobacterial mats of a low-salinity meltwater pond called Orange Pond on the McMurdo Ice Shelf at temperatures simulating those in the summer season (<5°C) revealed that both sulfate reduction and methane production were important terminal anaerobic processes. Addition of [2-¹⁴C]acetate to sediment samples resulted in the passage of label mainly to CO₂. Acetate addition (0 to 27 mM) had little effect on methanogenesis (a 1.1-fold increase), and while the rate of acetate dissimilation was greater than the rate of methane production (6.4 nmol cm⁻³ h⁻¹ compared to 2.5 to 6 nmol cm⁻³ h⁻¹), the portion of methane production attributed to acetate cleavage was <2%. Substantial increases in the methane production rate were observed with H₂ (2.4-fold), and H₂ uptake was totally accounted for by methane production under physiological conditions. Formate also stimulated methane production (twofold), presumably through H₂ release mediated through hydrogen lyase. Addition of sulfate up to 50-fold the natural levels in the sediment (interstitial concentration, ~0.3 mM) did not substantially inhibit methanogenesis, but the process was inhibited by 50-fold chloride (36 mM). No net rate of methane oxidation was observed when sediments were incubated anaerobically, and denitrification rates were substantially lower than rates for sulfate reduction and methanogenesis. The results indicate that carbon flow from acetate is coupled mainly to sulfate reduction and that methane is largely generated from H₂ and CO₂ where chloride, but not sulfate, has a modulating role. Rates of methanogenesis at in situ temperatures were four- to fivefold less than maximal rates found at 20°C.     

Coupling of Methanothermobacter thermautotrophicus Methane Formation and Growth in Fed-Batch and Continuous Cultures under Different H2 Gassing Regimens

In nature, H₂- and CO₂-utilizing methanogenic archaea have to couple the processes of methanogenesis and autotrophic growth under highly variable conditions with respect to the supply and concentration of their energy source, hydrogen. To study the hydrogen-dependent coupling between methanogenesis and growth, Methanothermobacter thermautotrophicus was cultured in a fed-batch fermentor and in a chemostat under different 80% H₂-20% CO₂ gassing regimens while we continuously monitored the dissolved hydrogen partial pressures (p_H2). In the fed-batch system, in which the conditions continuously changed the uptake rates by the growing biomass, the organism displayed a complex and yet defined growth behavior, comprising the consecutive lag, exponential, and linear growth phases. It was found that the in situ hydrogen concentration affected the coupling between methanogenesis and growth in at least two respects. (i) The microorganism could adopt two distinct theoretical maximal growth yields (Y_{CH4 max}), notably approximately 3 and 7 g (dry weight) of methane formed mol⁻¹, for growth under low (p_H2 < 12 kPa)- and high-hydrogen conditions, respectively. The distinct values can be understood from a theoretical analysis of the process of methanogenesis presented in the supplemental material associated with this study. (ii) The in situ hydrogen concentration affected the “specific maintenance” requirements or, more likely, the degree of proton leakage and proton slippage processes. At low p_H2 values, the “specific maintenance” diminished and the specific growth yields approached Y_{CH4 max}, indicating that growth and methanogenesis became fully coupled.     

Microbial Methanogenesis and Acetate Metabolism in a Meromictic Lake

Methanogenesis and the anaerobic metabolism of acetate were examined in the sediment and water column of Knaack Lake, a small biogenic meromictic lake located in central Wisconsin. The lake was sharply stratified during the summer and was anaerobic below a depth of 3 m. Large concentrations (4,000 μmol/liter) of dissolved methane were detected in the bottom waters. A methane concentration maximum occurred at 4 m above the sediment. The production of ¹⁴CH₄ from ¹⁴C-labeled HCOOH, HCO₃⁻, and CH₃OH and [2-¹⁴C]acetate demonstrated microbial methanogenesis in the water column of the lake. The maximum rate of methanogenesis calculated from reduction of H¹⁴CO₃⁻ by endogenous electron donors in the surface sediment (depth, 22 m) was 7.6 nmol/h per 10 ml and in the water column (depth, 21 m) was 0.6 nmol/h per 10 ml. The methyl group of acetate was simultaneously metabolized to CH₄ and CO₂ in the anaerobic portions of the lake. Acetate oxidation was greatest in surface waters and decreased with water depth. Acetate was metabolized primarily to methane in the sediments and water immediately above the sediment. Sulfide inhibition studies and temperature activity profiles demonstrated that acetate metabolism was performed by several microbial populations. Sulfide additions (less than 5 μg/ml) to water from 21.5 m stimulated methanogenesis from acetate, but inhibited CO₂ production. Sulfate addition (1 mM) had no significant effect on acetate metabolism in water from 21.5 m, whereas nitrate additions (10 to 14,000 μg/liter) completely inhibited methanogenesis and stimulated CO₂ formation.     

Microbial analysis of a phototrophic sludge producing hydrogen from acidified wastewater

Based on DNA-cloning analysis, the microbial community of a phototrophic sludge producing H₂ from an acidified wastewater was composed of 81% of a species resembling Rhodobacter capsulatus (with 99.2% similarity) and two unidentified species of the Bacillus/Clostridium group. The sludge produced a biogas comprising 82 ± 2% H₂, 13 ± 2% CO₂, 4.5 ± 0.5% N₂, and 0.5 ± 0.2% H₂S.     

Formate production and utilization by methanogens and by sewage sludge consortia — interference with the concept of interspecies formate transfer

Pure cultures of H₂/CO₂- and formate-utilizing methanogens or mixed consortia of sewage sludge generated some formate from H₂/CO₂ at H₂ partial pressure in the gas phase above 200 kPa. At decreasing H₂ partial pressure the formate was taken up again and converted to methane. If methanogenesis was inhibited by bromoethanesulphonic acid (BESA) or a high redox potential (–180 to –200 mV), formate-utilizing methanogens produced high amounts of formate from H₂/CO₂. No formate was excreted by the species, which could only utilize H₂/CO₂ for methanogenesis. In contrast, H₂ formation from formate was observed in cultures of Methanobacterium thermoformicicum and M. formicicum. Measurable amounts were, however, only formed if its immediate utilization for methane production was inhibited by BESA. In the light of the data on formate formation from H₂/CO₂ and its re-utilization by all formate-utilizing methanogens, the concept of interspecies formate transfer of Thiele and Zeikus should be reconsidered. In pure cultures of methanogens or complex ecosystems with excess H₂, formate formation seemed to serve more as a means of disposal of surplus reducing power than for H₂ transfer. Correspondence to: J. Winter