Kinetic Parameters of the Conversion of Methane Precursors to Methane in a Hypereutrophic Lake Sediment

The kinetic parameters K_m, V_max, T_t (turnover time), and v (natural velocity) were determined for H₂ and acetate conversion to methane by Wintergreen Lake sediment, using short-term (a few hours) methods and incubation temperatures of 10 to 14°C. Estimates of the Michaelis-Menten constant, K_m, for both the consumption of hydrogen and the conversion of hydrogen to methane by sediment microflora averaged about 0.024 μmol g⁻¹ of dry sediment. The maximal velocity, V_max, averaged 4.8 μmol of H₂ g⁻¹ h⁻¹ for hydrogen consumption and 0.64 μmol of CH₄ g⁻¹ h⁻¹ for the conversion of hydrogen to methane during the winter. Estimated natural rates of hydrogen consumption and hydrogen conversion to methane could be calculated from the Michaelis-Menten equation and estimates of K_m, V_max, and the in situ dissolved-hydrogen concentration. These results indicate that methane may not be the only fate of hydrogen in the sediment. Among several potential hydrogen donors tested, only formate stimulated the rate of sediment methanogenesis. Formate conversion to methane was so rapid that an accurate estimate of kinetic parameters was not possible. Kinetic experiments using [2-¹⁴C]acetate and sediments collected in the summer indicated that acetate was being converted to methane at or near the maximal rate. A minimum natural rate of acetate conversion to methane was estimated to be about 110 nmol of CH₄ g⁻¹ h⁻¹, which was 66% of the V_max (163 nmol of CH₄ g⁻¹ h⁻¹). A 15-min preincubation of sediment with 5.0 × 10⁻³ atm of hydrogen had a pronounced effect on the kinetic parameters for the conversion of acetate to methane. The acetate pool size, expressed as the term K_m + S_n (S_n is in situ substrate concentration), decreased by 37% and T_t decreased by 43%. The V_max remained relatively constant. A preincubation with hydrogen also caused a 37% decrease in the amount of labeled carbon dioxide produced from the metabolism of [U-¹⁴C]valine by sediment heterotrophs.     

Uncoupling of Methanogenesis from Growth of Methanosarcina barkeri by Phosphate Limitation

Production of methane by Methanosarcina barkeri from H₂-CO₂ was studied in fed-batch culture under phosphate-limiting conditions. A transition in the kinetics of methanogenesis from an exponentially increasing rate to a constant rate was due to depletion of phosphate from the medium. The period of exponentially increasing rate of methanogenesis was extended by increasing the initial concentration of phosphate in the medium. Addition of phosphate during the constant period changed the kinetics to an exponentially increasing rate of methanogenesis, indicating the reversibility of phosphate depletion. The relation between methanogenesis and growth of M. barkeri was investigated by measuring the incorporation of phosphorus, supplied as KH₂³²PO₄, in the medium. At a low (1 μM) initial concentration of phosphate in the medium and during the constant period of methanogenesis, there was no net cell growth. At a higher (10 μM) initial concentration of phosphate, cell growth proceeded linearly with time after phosphate had been removed from the medium by uptake into cells.     

Hydrogenotrophic Methanogenesis by Moderately Acid-Tolerant Methanogens of a Methane-Emitting Acidic Peat

The emission of methane (1.3 mmol of CH₄ m⁻² day⁻¹), precursors of methanogenesis, and the methanogenic microorganisms of acidic bog peat (pH 4.4) from a moderately reduced forest site were investigated by in situ measurements, microcosm incubations, and cultivation methods, respectively. Bog peat produced CH₄ (0.4 to 1.7 μmol g [dry wt] of soil⁻¹ day⁻¹) under anoxic conditions. At in situ pH, supplemental H₂-CO₂, ethanol, and 1-propanol all increased CH₄ production rates while formate, acetate, propionate, and butyrate inhibited the production of CH₄; methanol had no effect. H₂-dependent acetogenesis occurred in H₂-CO₂-supplemented bog peat only after extended incubation periods. Nonsupplemented bog peat initially produced small amounts of H₂ that were subsequently consumed. The accumulation of H₂ was stimulated by ethanol and 1-propanol or by inhibiting methanogenesis with bromoethanesulfonate, and the consumption of ethanol was inhibited by large amounts of H₂; these results collectively indicated that ethanol- or 1-propanol-utilizing bacteria were trophically associated with H₂-utilizing methanogens. A total of 10⁹ anaerobes and 10⁷ hydrogenotrophic methanogens per g (dry weight) of bog peat were enumerated by cultivation techniques. A stable methanogenic enrichment was obtained with an acidic, H₂-CO₂-supplemented, fatty acid-enriched defined medium. CH₄ production rates by the enrichment were similar at pH 4.5 and 6.5, and acetate inhibited methanogenesis at pH 4.5 but not at pH 6.5. A total of 27 different archaeal 16S rRNA gene sequences indicative of Methanobacteriaceae, Methanomicrobiales, and Methanosarcinaceae were retrieved from the highest CH₄-positive serial dilutions of bog peat and methanogenic enrichments. A total of 10 bacterial 16S rRNA gene sequences were also retrieved from the same dilutions and enrichments and were indicative of bacteria that might be responsible for the production of H₂ that could be used by hydrogenotrophic methanogens. These results indicated that in this acidic bog peat, (i) H₂ is an important substrate for acid-tolerant methanogens, (ii) interspecies hydrogen transfer is involved in the degradation of organic carbon, (iii) the accumulation of protonated volatile fatty acids inhibits methanogenesis, and (iv) methanogenesis might be due to the activities of methanogens that are phylogenetic members of the Methanobacteriaceae, Methanomicrobiales, and Methanosarcinaceae.     

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).     

Effect of Temperature on Anaerobic Ethanol Oxidation and Methanogenesis in Acidic Peat from a Northern Wetland

The effects of temperature on rates and pathways of CH₄ production and on the abundance and structure of the archaeal community were investigated in acidic peat from a mire in northern Scandinavia (68°N). We monitored the production of CH₄ and CO₂ over time and measured the turnover of Fe(II), ethanol, and organic acids. All experiments were performed with and without specific inhibitors (2-bromoethanesulfonate [BES] for methanogenesis and CH₃F for acetoclastic methanogenesis). The optimum temperature for methanogenesis was 25°C (2.3 μmol CH₄ · g [dry weight]⁻¹ · day⁻¹), but the activity was relatively high even at 4°C (0.25 μmol CH₄ · g [dry weight]⁻¹ · day⁻¹). The theoretical lower limit for methanogenesis was calculated to be at −5°C. The optimum temperature for growth as revealed by real-time PCR was 25°C for both archaea and bacteria. The population structure of archaea was studied by terminal restriction fragment length polymorphism analysis and remained constant over a wide temperature range. Hydrogenotrophic methanogenesis accounted for about 80% of the total methanogenesis. Most 16S rRNA gene sequences that were affiliated with methanogens and all McrA sequences clustered with the exclusively hydrogenotrophic order Methanobacteriales, correlating with the prevalence of hydrogenotrophic methanogenesis. Fe reduction occurred parallel to methanogenesis and was inhibited by BES, suggesting that methanogens were involved in Fe reduction. Based upon the observed balance of substrates and thermodynamic calculations, we concluded that the ethanol pool was oxidized to acetate by the following two processes: syntrophic oxidation with methanogenesis (i) as an H₂ sink and (ii) as a reductant for Fe(III). Acetate accumulated, but a considerable fraction was converted to butyrate, making volatile fatty acids important end products of anaerobic metabolism.     

Physiological Characterization of an Anaerobic Ammonium-Oxidizing Bacterium Belonging to the “Candidatus Scalindua” Group

The phylogenetic affiliation and physiological characteristics (e.g., K_s and maximum specific growth rate [μ_max]) of an anaerobic ammonium oxidation (anammox) bacterium, “Candidatus Scalindua sp.,” enriched from the marine sediment of Hiroshima Bay, Japan, were investigated. “Candidatus Scalindua sp.” exhibits higher affinity for nitrite and a lower growth rate and yield than the known anammox species.     

Cross-Epithelial Hydrogen Transfer from the Midgut Compartment Drives Methanogenesis in the Hindgut of Cockroaches

In the intestinal tracts of animals, methanogenesis from CO₂ and other C₁ compounds strictly depends on the supply of electron donors by fermenting bacteria, but sources and sinks of reducing equivalents may be spatially separated. Microsensor measurements in the intestinal tract of the omnivorous cockroach Blaberus sp. showed that molecular hydrogen strongly accumulated in the midgut (H₂ partial pressures of 3 to 26 kPa), whereas it was not detectable (<0.1 kPa) in the posterior hindgut. Moreover, living cockroaches emitted large quantities of CH₄ [105 ± 49 nmol (g of cockroach)⁻¹ h⁻¹] but only traces of H₂. In vitro incubation of isolated gut compartments, however, revealed that the midguts produced considerable amounts of H₂, whereas hindguts emitted only CH₄ [106 ± 58 and 71 ± 50 nmol (g of cockroach)⁻¹ h⁻¹, respectively]. When ligated midgut and hindgut segments were incubated in the same vials, methane emission increased by 28% over that of isolated hindguts, whereas only traces of H₂ accumulated in the headspace. Radial hydrogen profiles obtained under air enriched with H₂ (20 kPa) identified the hindgut as an efficient sink for externally supplied H₂. A cross-epithelial transfer of hydrogen from the midgut to the hindgut compartment was clearly evidenced by the steep H₂ concentration gradients which developed when ligated fragments of midgut and hindgut were placed on top of each other—a configuration that simulates the situation in vivo. These findings emphasize that it is essential to analyze the compartmentalization of the gut and the spatial organization of its microbiota in order to understand the functional interactions among different microbial populations during digestion.     

Kinetics of Formate Metabolism in Methanobacterium formicicum and Methanospirillum hungatei

The kinetics of formate metabolism in Methanobacterium formicicum and Methanospirillum hungatei were studied with log-phase formate-grown cultures. The progress of formate degradation was followed by the formyltetrahydrofolate synthetase assay for formate and fitted to the integrated form of the Michaelis-Menten equation. The K_m and V_max values for Methanobacterium formicicum were 0.58 mM formate and 0.037 mol of formate h⁻¹ g⁻¹ (dry weight), respectively. The lowest concentration of formate metabolized by Methanobacterium formicicum was 26 μM. The K_m and V_max values for Methanospirillum hungatei were 0.22 mM and 0.044 mol of formate h⁻¹ g⁻¹ (dry weight), respectively. The lowest concentration of formate metabolized by Methanospirillum hungatei was 15 μM. The apparent K_m for formate by formate dehydrogenase in cell-free extracts of Methanospirillum hungatei was 0.11 mM. The K_m for H₂ uptake by cultures of Methanobacterium formicicum was 6 μM dissolved H₂. Formate and H₂ were equivalent electron donors for methanogenesis when both substrates were above saturation; however, H₂ uptake was severely depressed when formate was above saturation and the dissolved H₂ was below 6 μM. Formate-grown cultures of Methanobacterium formicicum that were substrate limited for 57 h showed an immediate increase in growth and methanogenesis when formate was added to above saturation.     

Localization and In Situ Activities of Homoacetogenic Bacteria in the Highly Compartmentalized Hindgut of Soil-Feeding Higher Termites (Cubitermes spp.)

Methanogenesis and homoacetogenesis occur simultaneously in the hindguts of almost all termites, but the reasons for the apparent predominance of methanogenesis over homoacetogenesis in the hindgut of the humivorous species is not known. We found that in gut homogenates of soil-feeding Cubitermes spp., methanogens outcompete homoacetogens for endogenous reductant. The rates of methanogenesis were always significantly higher than those of reductive acetogenesis, whereas the stimulation of acetogenesis by the addition of exogenous H₂ or formate was more pronounced than that of methanogenesis. In a companion paper, we reported that the anterior gut regions of Cubitermes spp. accumulated hydrogen to high partial pressures, whereas H₂ was always below the detection limit (<100 Pa) in the posterior hindgut, and that all hindgut compartments turned into efficient H₂ sinks when external H₂ was provided (D. Schmitt-Wagner and A. Brune, Appl. Environ. Microbiol. 65:4490–4496, 1999). Using a microinjection technique, we found that only the posterior gut sections P3/4a and P4b, which harbored methanogenic activities, formed labeled acetate from H¹⁴CO₃⁻. Enumeration of methanogenic and homoacetogenic populations in the different gut sections confirmed the coexistence of both metabolic groups in the same compartments. However, the in situ rates of acetogenesis were strongly hydrogen limited; in the P4b section, no activity was detected unless external H₂ was added. Endogenous rates of reductive acetogenesis in isolated guts were about 10-fold lower than the in vivo rates of methanogenesis, but were almost equal when exogenous H₂ was supplied. We conclude that the homoacetogenic populations in the posterior hindgut are supported by either substrates other than H₂ or by a cross-epithelial H₂ transfer from the anterior gut regions, which may create microniches favorable for H₂-dependent acetogenesis.     

Phosphate Inhibits Acetotrophic Methanogenesis on Rice Roots

The contribution of acetate- and H₂/CO₂-dependent methanogenesis to total CH₄ production was determined in excised washed rice roots by radiolabeling, methyl fluoride inhibition, and stable carbon isotope fractionation. Addition of ≥20 mM phosphate inhibited methanogenesis, which then was exclusively from H₂/CO₂. Otherwise, acetate contributed about 50 to 60% of the total methanogenesis, demonstrating that phosphate specifically inhibited acetotrophic methanogens on rice roots.     

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.     

Effective Suppression of Methane Emission by 2-Bromoethanesulfonate during Rice Cultivation

2-bromoethanesulfonate (BES) is a structural analogue of coenzyme M (Co-M) and potent inhibitor of methanogenesis. Several studies confirmed, BES can inhibit CH₄ prodcution in rice soil, but the suppressing effectiveness of BES application on CH₄ emission under rice cultivation has not been studied. In this pot experiment, different levels of BES (0, 20, 40 and 80 mg kg^-1) were applied to study its effect on CH₄ emission and plant growth during rice cultivation. Application of BES effectively suppressed CH₄ emission when compared with control soil during rice cultivation. The CH₄ emission rates were significantly (P<0.001) decreased by BES application possibly due to significant (P<0.001) reduction of methnaogenic biomarkers like Co-M concentration and mcrA gene copy number (i.e. methanogenic abunadance). BES significantly (P<0.001) reduced methanogen activity, while it did not affect soil dehydrogenase activity during rice cultivation. A rice plant growth and yield parameters were not affected by BES application. The maximum CH₄ reduction (49% reduction over control) was found at 80 mg kg^-1 BES application during rice cultivation. It is, therefore, concluded that BES could be a suitable soil amendment for reducing CH₄ emission without affecting rice plant growth and productivity during rice cultivation.     

Effect of Temperature on Carbon and Electron Flow and on the Archaeal Community in Methanogenic Rice Field Soil

Temperature is an important factor controlling CH₄ production in anoxic rice soils. Soil slurries, prepared from Italian rice field soil, were incubated anaerobically in the dark at six temperatures of between 10 to 37°C or in a temperature gradient block covering the same temperature range at intervals of 1°C. Methane production reached quasi-steady state after 60 to 90 days. Steady-state CH₄ production rates increased with temperature, with an apparent activation energy of 61 kJ mol⁻¹. Steady-state partial pressures of the methanogenic precursor H₂ also increased with increasing temperature from <0.5 to 3.5 Pa, so that the Gibbs free energy change of H₂ plus CO₂-dependent methanogenesis was kept at −20 to −25 kJ mol of CH₄⁻¹ over the whole temperature range. Steady-state concentrations of the methanogenic precursor acetate, on the other hand, increased with decreasing temperature from <5 to 50 μM. Simultaneously, the relative contribution of H₂ as methanogenic precursor decreased, as determined by the conversion of radioactive bicarbonate to ¹⁴CH₄, so that the carbon and electron flow to CH₄ was increasingly dominated by acetate, indicating that psychrotolerant homoacetogenesis was important. The relative composition of the archaeal community was determined by terminal restriction fragment length polymorphism (T-RFLP) analysis of the 16S rRNA genes (16S rDNA). T-RFLP analysis differentiated the archaeal Methanobacteriaceae, Methanomicrobiaceae, Methanosaetaceae, Methanosarcinaceae, and Rice clusters I, III, IV, V, and VI, which were all present in the rice field soil incubated at different temperatures. The 16S rRNA genes of Rice cluster I and Methanosaetaceae were the most frequent methanogenic groups. The relative abundance of Rice cluster I decreased with temperature. The substrates used by this microbial cluster, and thus its function in the microbial community, are unknown. The relative abundance of acetoclastic methanogens, on the other hand, was consistent with their physiology and the acetate concentrations observed at the different temperatures, i.e., the high-acetate-requiring Methanosarcinaceae decreased and the more modest Methanosaetaceae increased with increasing temperature. Our results demonstrate that temperature not only affected the activity but also changed the structure and the function (carbon and electron flow) of a complex methanogenic system.     

Two Different Bacterial Community Types Are Linked with the Low-Methane Emission Trait in Sheep

The potent greenhouse gas methane (CH₄) is produced in the rumens of ruminant animals from hydrogen produced during microbial degradation of ingested feed. The natural animal-to-animal variation in the amount of CH₄ emitted and the heritability of this trait offer a means for reducing CH₄ emissions by selecting low-CH₄ emitting animals for breeding. We demonstrate that differences in rumen microbial community structure are linked to high and low CH₄ emissions in sheep. Bacterial community structures in 236 rumen samples from 118 high- and low-CH₄ emitting sheep formed gradual transitions between three ruminotypes. Two of these (Q and S) were linked to significantly lower CH₄ yields (14.4 and 13.6 g CH₄/kg dry matter intake [DMI], respectively) than the third type (H; 15.9 g CH₄/kg DMI; p<0.001). Low-CH₄ ruminotype Q was associated with a significantly lower ruminal acetate to propionate ratio (3.7±0.4) than S (4.4±0.7; p<0.001) and H (4.3±0.5; p<0.001), and harbored high relative abundances of the propionate-producing Quinella ovalis. Low-CH₄ ruminotype S was characterized by lactate- and succinate-producing Fibrobacter spp., Kandleria vitulina, Olsenella spp., Prevotella bryantii, and Sharpea azabuensis. High-CH₄ ruminotype H had higher relative abundances of species belonging to Ruminococcus, other Ruminococcaceae, Lachnospiraceae, Catabacteriaceae, Coprococcus, other Clostridiales, Prevotella, other Bacteroidales, and Alphaproteobacteria, many of which are known to form significant amounts of hydrogen. We hypothesize that lower CH₄ yields are the result of bacterial communities that ferment ingested feed to relatively less hydrogen, which results in less CH₄ being formed.     

Development of a Microbial Model for the Combined Effect of Temperature and pH on Spoilage of Ground Meat, and Validation of the Model under Dynamic Temperature Conditions

The changes in microbial flora and sensory characteristics of fresh ground meat (beef and pork) with pH values ranging from 5.34 to 6.13 were monitored at different isothermal storage temperatures (0 to 20°C) under aerobic conditions. At all conditions tested, pseudomonads were the predominant bacteria, followed by Brochothrix thermosphacta, while the other members of the microbial association (e.g., lactic acid bacteria and Enterobacteriaceae) remained at lower levels. The results from microbiological and sensory analysis showed that changes in pseudomonad populations followed closely sensory changes during storage and could be used as a good index for spoilage of aerobically stored ground meat. The kinetic parameters (maximum specific growth rate [μ_max] and the duration of lag phase [λ]) of the spoilage bacteria were modeled by using a modified Arrhenius equation for the combined effect of temperature and pH. Meat pH affected growth of all spoilage bacteria except that of lactic acid bacteria. The “adaptation work,” characterized by the product of μ_max and λ(μ_max × λ) was found to be unaffected by temperature for all tested bacteria but was affected by pH for pseudomonads and B. thermosphacta. For the latter bacteria, a negative linear correlation between ln(μ_max × λ) and meat pH was observed. The developed models were further validated under dynamic temperature conditions using different fluctuating temperatures. Graphical comparison between predicted and observed growth and the examination of the relative errors of predictions showed that the model predicted satisfactorily growth under dynamic conditions. Predicted shelf life based on pseudomonads growth was slightly shorter than shelf life observed by sensory analysis with a mean difference of 13.1%. The present study provides a “ready-to-use,” well-validated model for predicting spoilage of aerobically stored ground meat. The use of the model by the meat industry can lead to effective management systems for the optimization of meat quality.     

Methanogenic Pathway and Archaeal Community Structure in the Sediment of Eutrophic Lake Dagow: Effect of Temperature

Methanogenic degradation of organic matter is an important microbial process in lake sediments. Temperature may affect not only the rate but also the pathway of CH₄ production by changing the activity and the abundance of individual microorganisms. Therefore, we studied the function and structure of a methanogenic community in anoxic sediment of Lake Dagow, a eutrophic lake in north-eastern Germany. Incubation of sediment samples (in situ 7.5°C) at increasing temperatures (4, 10, 15, 25, 30°C) resulted in increasing production rates of CH₄ and CO₂ and in increasing steady-state concentrations of H₂. Thermodynamic conditions for H₂/CO₂ -dependent methanogenesis were only exergonic at 25 and 30°C. Inhibition of methanogenesis with chloroform resulted in the accumulation of methanogenic precursors, i.e., acetate, propionate, and isobutyrate. Mass balance calculations indicated that less CH₄ was formed via H₂ at 4°C than at 30°C. Conversion of ¹⁴CO₂ to ¹⁴CH₄ also showed that H₂/CO₂ -dependent methanogenesis contributed less to total CH₄ production at 4°C than at 30°C. [2–¹⁴ C]Acetate turnover rates at 4°C accounted for a higher percentage of total CH₄ production than at 30°C. Collectively, these results showed a higher contribution of H₂-dependent methanogenesis and a lower contribution of acetate-dependent methanogenesis at high versus low temperature. The archaeal community was characterized by cloning, sequencing, and phylogenetic analysis of the 16S rRNA genes retrieved from the sediment. Sequences were affiliated with Methanosaetaceae, Methanomicrobiaceae, and three deeply branching euryarchaeotal clusters, i.e., group III, Rice cluster V, and a novel euryarchaeotal cluster, the LDS cluster. Terminal restriction fragment length polymorphism (T-RFLP) analysis showed that 16S rRNA genes affiliated to Methanosaetaceae (20–30%), Methanomicrobiaceae (35–55%), and group III (10–25%) contributed most to the archaeal community. Incubation of the sediment at different temperatures (4–30°C) did not result in a systematic change of the archaeal community composition, indicating that change of temperature primarily affected the activity rather than the structure of the methanogenic community.     

Mass spectrometric control of the thermophilic anaerobic digestion process based on levels of dissolved hydrogen

Summary The continuous and simultaneous monitoring of dissolved CH₄ and H₂ in samples from a laboratory scale thermophilic anaerobic digester contents by use of a silicone rubber-covered probe has enabled control of methanogenesis: regulation of the hydrogen signal in a closed feedback loop was by controlled addition of the carbon source. Dissolved hydrogen became apparent in this system at a lower loading rate than was obtained for a mesophilic anaerobic digestion system (Whitmoreet al., 1986). Controlling the supply of glucose (25 mM) at a dilution rate of 0.02 h^–1 and at progressively lower preset hydrogen levels allowed methanogenesis to be significantly prolonged before inhibition of the process occurred.     

Anaerobic Degradation of Lactate by Syntrophic Associations of Methanosarcina barkeri and Desulfovibrio Species and Effect of H2 on Acetate Degradation

When grown in the absence of added sulfate, cocultures of Desulfovibrio desulfuricans or Desulfovibrio vulgaris with Methanobrevibacter smithii (Methanobacterium ruminantium), which uses H₂ and CO₂ for methanogenesis, degraded lactate, with the production of acetate and CH₄. When D. desulfuricans or D. vulgaris was grown in the absence of added sulfate in coculture with Methanosarcina barkeri (type strain), which uses both H₂-CO₂ and acetate for methanogenesis, lactate was stoichiometrically degraded to CH₄ and presumably to CO₂. During the first 12 days of incubation of the D. desulfuricans-M. barkeri coculture, lactate was completely degraded, with almost stoichiometric production of acetate and CH₄. Later, acetate was degraded to CH₄ and presumably to CO₂. In experiments in which 20 mM acetate and 0 to 20 mM lactate were added to D. desulfuricans-M. barkeri cocultures, no detectable degradation of acetate occurred until the lactate was catabolized. The ultimate rate of acetate utilization for methanogenesis was greater for those cocultures receiving the highest levels of lactate. A small amount of H₂ was detected in cocultures which contained D. desulfuricans and M. barkeri until after all lactate was degraded. The addition of H₂, but not of lactate, to the growth medium inhibited acetate degradation by pure cultures of M. barkeri. Pure cultures of M. barkeri produced CH₄ from acetate at a rate equivalent to that observed for cocultures containing M. barkeri. Inocula of M. barkeri grown with H₂-CO₂ as the methanogenic substrate produced CH₄ from acetate at a rate equivalent to that observed for acetate-grown inocula when grown in a rumen fluid-vitamin-based medium but not when grown in a yeast extract-based medium. The results suggest that H₂ produced by the Desulfovibrio species during growth with lactate inhibited acetate degradation by M. barkeri.     

The Effect of Ionic Strength and Specific Anions on Substrate Binding and Hydrolytic Activities of Na,K-ATPase

The physiological ligands for Na,K-ATPase (the Na,K-pump) are ions, and electrostatic forces, that could be revealed by their ionic strength dependence, are therefore expected to be important for their reaction with the enzyme. We found that the affinities for ADP³⁻, eosin²⁻, p-nitrophenylphosphate, and V_max for Na,K-ATPase and K⁺-activated p-nitrophenylphosphatase activity, were all decreased by increasing salt concentration and by specific anions. Equilibrium binding of ADP was measured at 0–0.5 M of NaCl, Na₂SO₄, and NaNO₃ and in 0.1 M Na-acetate, NaSCN, and NaClO₄. The apparent affinity for ADP decreased up to 30 times. At equal ionic strength, I, the ranking of the salt effect was NaCl ≈ Na₂SO₄ ≈ Na-acetate < NaNO₃ < NaSCN < NaClO₄. We treated the influence of NaCl and Na₂SO₄ on K _diss for E·ADP as a “pure” ionic strength effect. It is quantitatively simulated by a model where the binding site and ADP are point charges, and where their activity coefficients are related to I by the limiting law of Debye and Hückel. The estimated net charge at the binding site of the enzyme was about +1. Eosin binding followed the same model. The NO₃ ⁻ effect was compatible with competitive binding of NO₃ ⁻ and ADP in addition to the general I-effect. K _diss for E·NO₃ was ∼32 mM. Analysis of V_max/K _m for Na,K-ATPase and K⁺-p-nitrophenylphosphatase activity shows that electrostatic forces are important for the binding of p-nitrophenylphosphate but not for the catalytic effect of ATP on the low affinity site. The net charge at the p-nitrophenylphosphate-binding site was also about +1. The results reported here indicate that the reversible interactions between ions and Na,K-ATPase can be grouped according to either simple Debye-Hückel behavior or to specific anion or cation interactions with the enzyme.