Effect of Nitrogen Source on Growth and Trichloroethylene Degradation by Methane-Oxidizing Bacteria

The effect of nitrogen source on methane-oxidizing bacteria with respect to cellular growth and trichloroethylene (TCE) degradation ability were examined. One mixed chemostat culture and two pure type II methane-oxidizing strains, Methylosinus trichosporium OB3b and strain CAC-2, which was isolated from the chemostat culture, were used in this study. All cultures were able to grow with each of three different nitrogen sources: ammonia, nitrate, and molecular nitrogen. Both M. trichosporium OB3b and strain CAC-2 showed slightly lower net cellular growth rates and cell yields but exhibited higher methane uptake rates, levels of poly-β-hydroxybutyrate (PHB) production, and naphthalene oxidation rates when grown under nitrogen-fixing conditions. The TCE-degrading ability of each culture was measured in terms of initial TCE oxidation rates and TCE transformation capacities (mass of TCE degraded/biomass inactivated), measured both with and without external energy sources. Higher initial TCE oxidation rates and TCE transformation capacities were observed in nitrogen-fixing mixed, M. trichosporium OB3b, and CAC-2 cultures than in nitrate- or ammonia-supplied cells. TCE transformation capacities were found to correlate with cellular PHB content in all three cultures. The results of this study suggest that the nitrogen-fixing capabilities of methane-oxidizing bacteria can be used to select for high-activity TCE degraders for the enhancement of bioremediation in fixed-nitrogen-limited environments.     

Changes in Activity and Community Structure of Methane-Oxidizing Bacteria over the Growth Period of Rice

The activity and community structure of methanotrophs in compartmented microcosms were investigated over the growth period of rice plants. In situ methane oxidation was important only during the vegetative growth phase of the plants and later became negligible. The in situ activity was not directly correlated with methanotrophic cell counts, which increased even after the decrease in in situ activity, possibly due to the presence of both vegetative cells and resting stages. By dividing the microcosms into two soil and two root compartments it was possible to locate methanotrophic growth and activity, which was greatest in the rhizoplane of the rice plants. Molecular analysis by denaturing gradient gel electrophoresis and fluorescent in situ hybridization (FISH) with family-specific probes revealed the presence of both families of methanotrophs in soil and root compartments over the whole season. Changes in community structure were detected only for members of the Methylococcaceae and could be associated only with changes in the genus Methylobacter and not with changes in the dominance of different genera in the family Methylococcaceae. For the family Methylocystaceae stable communities in all compartments for the whole season were observed. FISH analysis revealed evidence of in situ dominance of the Methylocystaceae in all compartments. The numbers of Methylococcaceae cells were relatively high only in the rhizoplane, demonstrating the importance of rice roots for growth and maintenance of methanotrophic diversity in the soil.     

在静态和动态培养条件下杂交瘤细胞的生长和代谢

通过对WuT3杂交瘤细胞在静态和动态培养条件下的比较,发现细胞生长和代谢有很大不同。在静态培养条件下,细胞培养周期较长,细胞密度较高;但在动态培养条件下,细胞代谢更加旺盛,葡萄糖、氨基酸等营养物质的消耗加快,乳酸、氨、丙氨酸等代谢产物的比生成速率较大。     

Growth of Anaerobic Methane-Oxidizing Archaea and Sulfate-Reducing Bacteria in a High-Pressure Membrane Capsule Bioreactor

Communities of anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria (SRB) grow slowly, which limits the ability to perform physiological studies. High methane partial pressure was previously successfully applied to stimulate growth, but it is not clear how different ANME subtypes and associated SRB are affected by it. Here, we report on the growth of ANME-SRB in a membrane capsule bioreactor inoculated with Eckernförde Bay sediment that combines high-pressure incubation (10.1 MPa methane) and thorough mixing (100 rpm) with complete cell retention by a 0.2-μm-pore-size membrane. The results were compared to previously obtained data from an ambient-pressure (0.101 MPa methane) bioreactor inoculated with the same sediment. The rates of oxidation of labeled methane were not higher at 10.1 MPa, likely because measurements were done at ambient pressure. The subtype ANME-2a/b was abundant in both reactors, but subtype ANME-2c was enriched only at 10.1 MPa. SRB at 10.1 MPa mainly belonged to the SEEP-SRB2 and Eel-1 groups and the Desulfuromonadales and not to the typically found SEEP-SRB1 group. The increase of ANME-2a/b occurred in parallel with the increase of SEEP-SRB2, which was previously found to be associated only with ANME-2c. Our results imply that the syntrophic association is flexible and that methane pressure and sulfide concentration influence the growth of different ANME-SRB consortia. We also studied the effect of elevated methane pressure on methane production and oxidation by a mixture of methanogenic and sulfate-reducing sludge. Here, methane oxidation rates decreased and were not coupled to sulfide production, indicating trace methane oxidation during net methanogenesis and not anaerobic methane oxidation, even at a high methane partial pressure.     

Growth and Population Dynamics of Anaerobic Methane-Oxidizing Archaea and Sulfate-Reducing Bacteria in a Continuous-Flow Bioreactor

The consumption of methane in anoxic marine sediments is a biogeochemical phenomenon mediated by two archaeal groups (ANME-1 and ANME-2) that exist syntrophically with sulfate-reducing bacteria. These anaerobic methanotrophs have yet to be recovered in pure culture, and key aspects of their ecology and physiology remain poorly understood. To characterize the growth and physiology of these anaerobic methanotrophs and the syntrophic sulfate-reducing bacteria, we incubated marine sediments using an anoxic, continuous-flow bioreactor during two experiments at different advective porewater flow rates. We examined the growth kinetics of anaerobic methanotrophs and Desulfosarcina-like sulfate-reducing bacteria using quantitative PCR as a proxy for cell counts, and measured methane oxidation rates using membrane-inlet mass spectrometry. Our data show that the specific growth rates of ANME-1 and ANME-2 archaea differed in response to porewater flow rates. ANME-2 methanotrophs had the highest rates in lower-flow regimes (μ_ANME-2 = 0.167 · week⁻¹), whereas ANME-1 methanotrophs had the highest rates in higher-flow regimes (μ_ANME-1 = 0.218 · week⁻¹). In both incubations, Desulfosarcina-like sulfate-reducing bacterial growth rates were approximately 0.3 · week⁻¹, and their growth dynamics suggested that sulfate-reducing bacterial growth might be facilitated by, but not dependent upon, an established anaerobic methanotrophic population. ANME-1 growth rates corroborate field observations that ANME-1 archaea flourish in higher-flow regimes. Our growth and methane oxidation rates jointly demonstrate that anaerobic methanotrophs are capable of attaining substantial growth over a range of environmental conditions used in these experiments, including relatively low methane partial pressures.     

Effect of Nitrogen Source on Growth and Trichloroethylene Degradation by Methane-Oxidizing Bacteria

The effect of nitrogen source on methane-oxidizing bacteria with respect to cellular growth and trichloroethylene (TCE) degradation ability were examined. One mixed chemostat culture and two pure type II methane-oxidizing strains, Methylosinus trichosporium OB3b and strain CAC-2, which was isolated from the chemostat culture, were used in this study. All cultures were able to grow with each of three different nitrogen sources: ammonia, nitrate, and molecular nitrogen. Both M. trichosporium OB3b and strain CAC-2 showed slightly lower net cellular growth rates and cell yields but exhibited higher methane uptake rates, levels of poly-β-hydroxybutyrate (PHB) production, and naphthalene oxidation rates when grown under nitrogen-fixing conditions. The TCE-degrading ability of each culture was measured in terms of initial TCE oxidation rates and TCE transformation capacities (mass of TCE degraded/biomass inactivated), measured both with and without external energy sources. Higher initial TCE oxidation rates and TCE transformation capacities were observed in nitrogen-fixing mixed, M. trichosporium OB3b, and CAC-2 cultures than in nitrate- or ammonia-supplied cells. TCE transformation capacities were found to correlate with cellular PHB content in all three cultures. The results of this study suggest that the nitrogen-fixing capabilities of methane-oxidizing bacteria can be used to select for high-activity TCE degraders for the enhancement of bioremediation in fixed-nitrogen-limited environments.

Optimal bioremediation conditions within contaminated aquifers are often found to be limited by the availability of nutrients, including nitrogen. Consequently, microorganisms that are capable of degrading contaminants as well as fixing molecular nitrogen as their sole nitrogen source could have a growth advantage in fixed-nitrogen-deficient environments that would be favorable for promoting in situ bioremediation.Trichloroethylene (TCE) is a major groundwater contaminant of concern in the United States due to its suspected carcinogenity and persistence in subsurface environments (31). However, a number of laboratory (1, 4, 13, 16, 18, 19, 22, 23, 26–28, 34) and field studies (3, 15, 24, 25) have shown that TCE can be cometabolically transformed into nontoxic end products (CO₂ and Cl⁻) by methane-oxidizing bacteria at the expense of reducing energy in the form of NADH. Many studies have also reported that some methane-oxidizing cultures (type II) are able to utilize different sources of nitrogen (N) for cellular growth (32, 33), including molecular nitrogen at reduced oxygen partial pressures (11, 12, 20, 33). The types of methanotrophs that are capable of nitrogen fixation also produce a type of oxygenase (i.e., soluble methane monooxygenase [sMMO]) which exhibits high activity with respect to the oxidation of TCE.Poly-β-hydroxybutyrate (PHB) is an internal reducing-energy storage polymer that can be used as an alternative reducing-energy source by a number of methane-oxidizing cultures under starvation conditions (9). Recently, a number of studies observed a correlation between TCE transformation capacities (T_c; mass of TCE transformed per mass of cells inactivated) and microbial PHB content (7, 16, 17), suggesting that PHB might be used as an alternative NADH source for TCE oxidation by methane-oxidizing bacteria in the absence of growth substrate. It has also been shown that the synthesis of PHB is stimulated in cells grown under nutrient-limited conditions, including nitrogen-fixing conditions (2, 9, 10, 21). As a result of the characteristics of methane-oxidizing microorganisms described above, it may be possible to select for nitrogen-fixing methane oxidizers in fixed-nitrogen-limited subsurface environments such that the burden of nutrient addition to the subsurface for the sustained growth of these contaminant degraders is diminished while contaminant degradation is enhanced during in situ bioremediation.A recent study conducted by us (7) explored the feasibility of using the nitrogen-fixing capabilities of methane oxidizers for the enhancement of bioremediation. Our results suggested that nitrogen-fixing mixed cultures were able to degrade TCE as effectively as nitrate-supplied cultures. Further, higher T_c and higher cellular PHB contents were observed in nitrogen-fixing cultures. Of particular interest were observations of lower TCE product toxicity, measured in terms of methane uptake rates following TCE exposure, for nitrogen-fixing cultures than for nitrate- or ammonia-supplied cultures. Since that study was conducted with mixed cultures, it was difficult to elucidate the reasons for the enhanced degradation performance of the nitrogen-fixing methane oxidizers. An understanding of the effects of nitrogen source on cell growth and TCE degradation ability will be particularly beneficial for designing, operating, and implementing in situ- or ex situ-engineered bioremediation systems. This study evaluates nitrogen source effects on methane-oxidizing bacteria, using two pure strains and one mixed chemostat culture. Nitrogen source effects are examined with regard to cellular growth, specific methane uptake rates, specific naphthalene oxidation rates, and TCE degradation ability.     

Characterization of Growth and Metabolism of Commercial Strains of Propionic Acid Bacteria by Pressure Measurement

Dairy propionibacteria are essential starters for Emmental cheese manufacture. The behavior of three commercial strains of Propionibacterium freudenreichii subsp. shermanii (P.f. 1, P.f. 2 and P.f 3) were studied in a liquid medium under air and N₂ atmosphere using an on‐line pressure measurement technique. Growth kinetics and metabolite production were characterized under conditions usually reported as “optimal conditions” (pH 6.5, NaCl 0 %, temperature 30 °C) and also evaluated under “stressful conditions” (pH 5.2, NaCl 2 %, temperature 20 °C) simulating the cheese ripening conditions. In both cases, the effects of oxygen on growth were strain‐dependent. Under “stressful conditions”, two of the three strains were inhibited by oxygen under conditions of air atmosphere, while all three strains grew under conditions of N₂ atmosphere. In the latter case, the duration of the lag phase and the maximum rate of pressure variation were significantly different, however, no significant differences were found between the strains with regard to the total fermentation time. Under “optimal conditions” metabolite production was strain‐dependent. In an air atmosphere, all strains produced more acetate and CO₂ and less propionate than in a nitrogen atmosphere.     

Identification of Aerobic Methane-Oxidizing Bacteria in Coastal Sediments of the Crimean Peninsula

Microbiology - Methane oxidation rates and diversity of methane-oxidizing microorganisms in the upper sediment layers of the Crimean Peninsula coastal regions (Black Sea) were investigated....     

Comparative Analysis of Methane-Oxidizing Archaea and Sulfate-Reducing Bacteria in Anoxic Marine Sediments

The oxidation of methane in anoxic marine sediments is thought to be mediated by a consortium of methane-consuming archaea and sulfate-reducing bacteria. In this study, we compared results of rRNA gene (rDNA) surveys and lipid analyses of archaea and bacteria associated with methane seep sediments from several different sites on the Californian continental margin. Two distinct archaeal lineages (ANME-1 and ANME-2), peripherally related to the order Methanosarcinales, were consistently associated with methane seep marine sediments. The same sediments contained abundant ¹³C-depleted archaeal lipids, indicating that one or both of these archaeal groups are members of anaerobic methane-oxidizing consortia. ¹³C-depleted lipids and the signature 16S rDNAs for these archaeal groups were absent in nearby control sediments. Concurrent surveys of bacterial rDNAs revealed a predominance of δ-proteobacteria, in particular, close relatives of Desulfosarcina variabilis. Biomarker analyses of the same sediments showed bacterial fatty acids with strong ¹³C depletion that are likely products of these sulfate-reducing bacteria. Consistent with these observations, whole-cell fluorescent in situ hybridization revealed aggregations of ANME-2 archaea and sulfate-reducing Desulfosarcina and Desulfococcus species. Additionally, the presence of abundant ¹³C-depleted ether lipids, presumed to be of bacterial origin but unrelated to ether lipids of members of the order Desulfosarcinales, suggests the participation of additional bacterial groups in the methane-oxidizing process. Although the Desulfosarcinales and ANME-2 consortia appear to participate in the anaerobic oxidation of methane in marine sediments, our data suggest that other bacteria and archaea are also involved in methane oxidation in these environments.     

Abundance, Activity, and Community Structure of Pelagic Methane-Oxidizing Bacteria in Temperate Lakes

The abundance and activity of methane-oxidizing bacteria (MOB) in the water column were investigated in three lakes with different contents of nutrients and humic substances. The abundance of MOB was determined by analysis of group-specific phospholipid fatty acids from type I and type II MOB, and in situ activity was measured with a ¹⁴CH₄ transformation method. The fatty acid analyses indicated that type I MOB most similar to species of Methylomonas, Methylomicrobium, and Methylosarcina made a substantial contribution (up to 41%) to the total bacterial biomass, whereas fatty acids from type II MOB generally had very low concentrations. The MOB biomass and oxidation activity were positively correlated and were highest in the hypo- and metalimnion during summer stratification, whereas under ice during winter, maxima occurred close to the sediments. The methanotroph biomass-specific oxidation rate (V) ranged from 0.001 to 2.77 mg CH₄-C mg⁻¹ C day⁻¹ and was positively correlated with methane concentration, suggesting that methane supply largely determined the activity and biomass distribution of MOB. Our results demonstrate that type I MOB often are a large component of pelagic bacterial communities in temperate lakes. They represent a potentially important pathway for reentry of carbon and energy into pelagic food webs that would otherwise be lost as evasion of CH₄.     

Toxicity of 1,1,1-Trichloroethane and Trichloroethene on a Mixed Culture of Methane-Oxidizing Bacteria

The influence of trichloroethene (TCE; 0 to 65 mg/liter) and 1,1,1-trichloroethane (1,1,1-TCA; 0 to 103 mg/liter) on methane consumption of a mixed culture of methane-oxidizing bacteria was studied in laboratory batch experiments. Increasing concentrations of TCE or 1,1,1-TCA resulted in decreasing methane consumption. Methane consumption was totally inhibited at a concentration of 13 mg of TCE per liter, while methane consumption was still observed at the upper studied concentration of 103 mg of 1,1,1-TCA per liter. The inhibition of methane consumption by TCE depended on the initial concentration of methane. A model accounting for competitive inhibition between methane and TCE or 1,1,1-TCA was used to simulate methane consumption at various concentrations of TCE or 1,1,1-TCA. The simulations indicated that competitive inhibition may be the mechanism causing the inhibitory effect of TCE on methane consumption, while this does not seem to be the case for 1,1,1-TCA.     

含无机盐沉淀菌悬液中微生物生长量的快速测定

目的:消除菌悬液中无机盐沉淀对比浊法测定生长量的干扰。方法:以嗜有机甲基杆菌ME25为材料,采用比浊法,研究了EDTA对菌悬液中无机沉淀物的清除效应以及对菌体生物量测定的影响。结果:在室温、pH 4~11,1.25×10^-2 mol/L EDTA与菌悬液样品作用1min,即可去除样品中无机盐沉淀,样品稳定,在1h内不影响样品中菌体吸光度的测定;实际样品和理论样品测定,相对误差小于3.0%,回收率为98%~100%,RSD均小于0.5%。结论:采用螯合剂EDTA可快速去除菌悬液中的无机盐沉淀,有效地消除沉淀物的干扰,明显提高了比浊法测定生长量的准确度,简便易行,具有较高的实用价值。     

Methane-Oxidizing Bacteria in a Finnish Raised Mire Complex: Effects of Site Fertility and Drainage

Methane-oxidizing bacteria (MOB) are the only biological sinks for methane (CH₄). Drainage of peatlands is known to decrease overall CH₄ emission, but the effect on MOB is unknown. The objective of this work was to characterize the MOB community and activity in two ecohydrologically different pristine peatland ecosystems, a fen and a bog, and their counterparts that were drained in 1961. Oligotrophic fens are groundwater-fed peatlands, but ombrotrophic bogs receive additional water and nutrients only from rainwater. The sites were sampled in August 2003 down to 10 cm below the water table (WT), and cores were divided into 10-cm subsamples. CH₄ oxidation was measured by gas chromatography (GC) to characterize MOB activity. The MOB community structure was characterized by polymerase chain reaction–denaturing gradient gel electrophoresis (DGGE) and sequencing methods using partial pmoA and mmoX genes. The highest CH₄ oxidation rates were measured from the subsamples 20–30 and 30–40 cm above WT at the pristine oligotrophic fen (12.7 and 10.5 μmol CH₄ dm⁻³ h⁻¹, respectively), but the rates decreased to almost zero in the vicinity of WT. In the pristine ombrotrophic bog, the highest oxidation rate at 0–10 cm was lower than in the fen (8.10 μmol CH₄ dm⁻³ h⁻¹), but in contrast to the fen, oxidation rates of 4.5 μmol CH₄ dm⁻³ h⁻¹ were observed at WT and 10 cm below WT. Drainage reduced the CH₄ oxidation rates to maximum values of 1.67 and 5.77 μmol CH₄ dm⁻³ h⁻¹ at 30–40 and 20–30 cm of the fen and bog site, respectively. From the total of 13 pmoA-derived DGGE bands found in the study, 11, 3, 6, and 2 were observed in the pristine fen and bog and their drained counterparts, respectively. According to the nonmetric multidimensional scaling of the DGGE banding pattern, the MOB community of the pristine fen differed from the other sites. The majority of partial pmoA sequences belonged to type I MOB, whereas the partial mmoX bands that were observed only in the bog sites formed a distinct group relating more to type II MOB. This study indicates that fen and bog ecosystems differ in MOB activity and community structure, and both these factors are affected by drainage.     

一株寡营养细菌胞外多糖的摇瓶发酵研究

从新疆的寡营养环境——古尔班通古特沙漠中分离到一株寡营养细菌Azotobacter sp．(1～15mg碳/L培养基),通过进行Azotobacter sp．菌的单因子优化培养基的试验、摇瓶培养工艺条件的优化试验(培养温度、培养时间、初始pH值、溶氧量),确定了菌种生长与营养需求等主要因子与胞外多糖产量、粘度的关系,结果表明,摇瓶发酵的最适宜条件为:以蔗糖为碳源,碳酸钙含量为2g/L,初始pH值为7左右,种龄72～84h,磷酸二氢钾、硫酸镁的含量分别为0．3g/L、0．1g/L,接种体积分数15%,于37℃摇瓶培养72h,250mL摇瓶装液量为50mL,在适宜条件下粘多糖的产量最大可达到1145．94μg/mL,粘性可达9200 mPa·s。     

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.     

Activity and Distribution of Methane-Oxidizing Bacteria in Flooded Rice Soil Microcosms and in Rice Plants (Oryza sativa)

The activity and distribution of CH(inf4)-oxidizing bacteria (MOB) in flooded rice (Oryza sativa) soil microcosms was investigated. CH(inf4) oxidation was shown to occur in undisturbed microcosms by using (sup14)CH(inf4), and model calculations indicated that almost 90% of the oxidation measured had taken place at a depth where only roots could provide the O(inf2) necessary. Slurry from soil planted with rice had an apparent K(infm) for CH(inf4) of 4 (mu)M and a V(infmax) of 0.1 (mu)mol g (dry weight)(sup-1) h(sup-1). At a depth of 1 to 2 cm, there was no significant difference (P > 0.05) in numbers of MOB between soil from planted and nonplanted microcosms (mean, 7.7 x 10(sup5) g [fresh weight](sup-1)). Thus, the densely rooted soil at 1 to 2 cm deep did not represent rhizospheric soil with respect to the number of MOB. A significantly increased number of MOB was found only in soil immediately around the roots (1.2 x 10(sup6) g [fresh weight](sup-1)), corresponding to a layer of 0.1 to 0.2 mm. Plant-associated CH(inf4) oxidation was shown in a double chamber with carefully washed intact rice plants. Up to 90% of the CH(inf4) supplied to the root compartment was oxidized in the plants. CH(inf4) oxidation on isolated roots was higher and had a larger variability than that in soil slurries. Roots had an apparent K(infm) for CH(inf4) of 6 (mu)M and a V(infmax) of 5 (mu)mol g (dry weight)(sup-1) h(sup-1). The average number of MOB in homogenized roots was larger than on the rhizoplane and increased with plant age. MOB also were found in surface-sterilized roots and basal culms, indicating the ability of these bacteria to colonize the interior of roots and culms.     

Existence of Novel Phylotypes of Nitrite-Dependent Anaerobic Methane-Oxidizing Bacteria in Surface and Subsurface Sediments of the South China Sea

Nitrite-dependent anaerobic methane oxidation (n-damo) is a recently discovered new microbial process performed by the Candidatus Methylomirabilis oxyfera with an unusual intra-aerobic pathway, but there is no report about n-damo bacteria in marine environments. M. oxyfera-like sequences were successfully retrieved for the first time from both surface and subsurface ocean sediments of the South China Sea (SCS) using both 16S rRNA and pmoA genes as biomarkers and PCR amplification in this study. The majority of M. oxyfera-like 16S rRNA gene-based PCR amplified sequences from the SCS sediments formed a new group distinctively different from those detected in freshwater habitats and the information is consistent phylogenetically with those obtained from the pmoA gene. This study showed the existence of n-damo in ocean sediments and suggests that marine sediments harbor n-damo phylotypes different from those in the freshwater. This finding here expands our understanding on the distribution of n-damo bacteria to marine ecosystem and implies their potential contribution to the marine C and N cycling.     

Minimum Threshold for Hydrogen Metabolism in Methanogenic Bacteria

Methanogenic isolates did not consume hydrogen below partial pressures of 6.5 Pa. Thus, in contrast to a previous report, results from pure-culture studies do not invalidate the threshold model for methane production from hydrogen in sediments.