Detection of luciferase gene sequences in nonluminescent bacteria from the Chesapeake Bay

Washed excised roots of rice (Oryza sativa) produced H(2), CH(4) and fatty acids (millimolar concentrations of acetate, propionate, butyrate; micromolar concentrations of isovalerate, valerate) when incubated under anoxic conditions. Surface sterilization of the root material resulted in the inactivation of the production of CH(4), a strong reduction of the production of fatty acids and a transient (75 h) but complete inhibition of the production of H(2). Radioactive bicarbonate was incorporated into CH(4), acetate, propionate and butyrate. About 20-40% of the fatty acid carbon originated from CO(2) reduction. In the presence of phosphate, CH(4) was exclusively produced from H(2)/CO(2), since phosphate selectively inhibited acetoclastic methanogenesis. Acetoclastic methanogenesis was also selectively inhibited by methyl fluoride, while chloroform or 2-bromoethane sulfonate inhibited CH(4) production completely. Production of CH(4), acetate, propionate and butyrate from H(2)/CO(2) was always exergonic with Gibbs free energies <-20 kJ mol(-1) product. Chloroform inhibited the production of acetate and the incorporation of radioactive CO(2) into acetate. Simultaneously, H(2) was no longer consumed and accumulated, indicating that acetate was produced from H(2)/CO(2). Chloroform also resulted in increased production of propionate and butyrate whose formation from CO(2) became more exergonic upon addition of chloroform. Nevertheless, the incorporation of radioactive CO(2) into propionate and butyrate was inhibited by chloroform. The accumulation of propionate and butyrate in the presence of chloroform probably occurred by fermentation of organic matter, rather than by reduction of acetate and CO(2). [U-(14)C]Glucose was indeed converted to acetate, propionate, butyrate, CO(2) and CH(4). Radioactive acetate, CO(2) and CH(4) were also products of the degradation of [U-(14)C]cellulose and [U-(14)C]xylose. Addition of chloroform and methyl fluoride did not affect the product spectrum of [U-(14)C]glucose degradation. The application of combinations of selective inhibitors may be useful to elucidate anaerobic metabolic pathways in mixed microbial cultures and natural microbial communities.     

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.     

Methanogenic archaea and CO2-dependent methanogenesis on washed rice roots

Washed excised roots of rice (Oryza sativa) immediately started to produce CH4 when they were incubated in phosphate buffer under anoxic conditions (N2 atmosphere), with initial rates varying between 2 and 70nmolh(-1)g(-1) dry weight of root material (mean +/- SE: 20.3 +/- 5.9 nmol h(-1) g(-1) dry weight; n = 18). Production of CH4 continued for at least 500 h, with rates usually decreasing slowly. CH4 production was not significantly affected by methyl fluoride, an inhibitor of acetoclastic methanogenesis. Less than 0.5% of added [2-14C]-acetate was converted to 14CH4, and conversion of 14CO2 to 14CH4 indicated that CH4 was almost exclusively produced from CO2. Occasionally, however, especially when the roots were incubated without additional buffer, CH4 production started to accelerate after about 200h reaching rates of > 100 nmol h(-1) g(-1) dry weight. Methyl fluoride inhibited methanogenesis by more than 20% only in these cases, and the conversion of 14CO2 to 14CH4 decreased. These results indicate that CO2-dependent rather than acetoclastic methanogenesis was primarily responsible for CH4 production in anoxically incubated rice roots. Determination of most probable numbers of methanogens on washed roots showed highest numbers (10(6)g(-1) dry roots) on H2 and ethanol, i.e. substrates that support CH4 production from CO2. Numbers on acetate (10(5) g(-1) dry roots) and methanol (10(4)g(-1) dry roots) were lower. Methanogenic consortia enriched on H2 and ethanol were characterized phylogenetically by comparative sequence analysis of archaeal small-subunit (SSU) ribosomal RNA-encoding genes (rDNA). These sequences showed a high similarity to SSU rDNA clones that had been obtained previously by direct extraction of total DNA from washed rice roots. The SSU rDNA sequences recovered from the H2/CO2-using consortium either belonged to a novel lineage of methanogens that grouped within the phylogenetic radiation of the Methanosarcinales and Methanomicrobiales or were affiliated with Methanobacterium bryantii. SSU rDNA sequences retrieved from the ethanol-using consortium either grouped within the genus Methanosarcina or belonged to another novel lineage within the phylogenetic radiation of the Methanosarcinales and Methanomicrobiales. Cultured organisms belonging to either of the two novel lineages have not been reported yet.     

Activity, structure and dynamics of the methanogenic archaeal community in a flooded Italian rice field

Methane production was studied in an Italian rice field over two consecutive years (1998, 1999) by measuring the rates of total and acetate-dependent methanogenesis in soil and root samples. Population dynamics of methanogens were followed by terminal restriction fragment length polymorphism and real-time PCR targeting archaeal SSU rRNA genes. Rates of total and acetate-dependent methanogenesis in soil increased during the season, reached a maximum at about 70-80 days after flooding and then decreased again. In contrast, the size of the archaeal community remained relatively constant. Therefore, the seasonal changes in the methanogenic processes were probably not caused by changes in the size of the methanogenic community but in its activity. During the 1998/1999 winter period, a slight decrease in archaeal cell numbers was found. In both years, the dominant groups were methanogens affiliated with Rice cluster I, Methanosaetaceae, Methanosarcinaceae and Methanobacteriaceae. Correspondence analysis showed, however, that the archaeal community structure was different in 1998 and 1999. Methanogens with potential acetoclastic activity made up a larger fraction of the total archaeal community in 1999 (32-53%) than in 1998 (20-32%). Furthermore, the frequency of Methanosaetaceae relative to Methanosarcinaceae was significantly higher in 1999 than in 1998. This difference could be explained by the much lower soil acetate concentrations in 1999, to which Methanosaetaceae are physiologically better adapted than Methanosarcinaceae. Over the season, however, the composition of the archaeal community remained relatively constant and thus did not reflect the observed seasonal change in CH(4) production activity. The analysis of rice root samples in 1999 showed that the archaeal community structure on the roots was similar to that in soil but with acetoclastic methanogens being relatively less common. This observation is in agreement with domination of CH(4) production by H(2)/CO(2)-dependent methanogenesis on roots. Our study provided a link between size, structure and function of the methanogenic community in an Italian rice field.     

Electron donor effect on nitrate reduction pathway and kinetics in a mixed methanogenic culture

The effect of different electron donors on the pathway and kinetics of nitrate reduction in a sulfide-acclimated mixed, mesophilic (35 degrees C) methanogenic culture was investigated. A mixture of dextrin and peptone, glucose, propionate, acetate, and H(2)/CO(2) were used as substrates at an initial chemical oxygen demand of 1,500 mg/L and the initial nitrate concentration ranged between 0 and 300 mg N/L. The fastest nitrate reduction was observed in the H(2)/CO(2) and acetate-fed cultures. In the case of propionate, nitrate reduction was the slowest followed by partial recovery of methanogenesis and accumulation of volatile fatty acids due to inhibition as a result of accumulation of denitrification intermediates. Similarly, accumulation of nitrite and nitric oxide and partial or complete inhibition of methanogenesis was observed in the H(2)/CO(2)-fed cultures. Methanogenesis completely recovered in the dextrin/peptone-, glucose-, and acetate-fed cultures at all nitrate levels. Denitrification was the dominant pathway of nitrate reduction in the propionate-, acetate-, and H(2)/CO(2)-fed cultures regardless of the COD/N value. However, both denitrification and dissimilatory nitrate reduction to ammonia (DNRA) were observed in the dextrin/peptone- and glucose-fed cultures and the degree of predominance of either of the two pathways was a function of the COD/N value. Therefore, the type of electron donor used affected both the nitrate reduction pathway and kinetics, as well as the recovery of fermentation and/or methanogenesis in the mixed methanogenic culture.     

Effect of inhibition of acetoclastic methanogenesis on growth of archaeal populations in an anoxic model environment

Methyl fluoride is frequently used to specifically inhibit acetoclastic methanogenesis, thus allowing determination of the relative contribution of acetate versus H2/CO2 to total CH4 production in natural environments. However, the effect of the inhibitor on growth of the target archaeal population has not yet been studied. Therefore, we incubated rice roots as an environmental model system under anoxic conditions in the presence and absence of CH3F, measured the activity and Gibbs free energy (DeltaG) of CH4 production, and determined the abundance of individual archaeal populations by using a combination of quantitative (real-time) PCR and analysis of terminal restriction fragment length polymorphism targeting the 16S rRNA gene. It was shown that CH3F specifically inhibited not only acetoclastic methanogenic activity but also the proliferation of Methanosarcina spp, which were the prevalent acetoclastic methanogens in our environmental model system. Therefore, inhibition experiments with CH3F seem to be a suitable method for quantifying acetoclastic CH4 production. It is furthermore shown that the growth and final population size of methanogens were consistent with energetic conditions that at least covered the maintenance requirements of the population.     

Influence of temperature and high acetate concentrations on methanogenesis in lake sediment slurries

Methanogenesis from main methane precursors H(2)/CO(2) and acetate was investigated in a temperature range of 2-70 degrees C using sediments from Lake Baldegg, Switzerland. Psychrophilic, psychrotrophic, mesophilic, and thermophilic methanogenic microbial communities were enriched by incubations for 1-3 months of nonamended sediment slurries at 5, 15, 30, and 50 degrees C. Isotope experiments with slurries amended with (14)C-labeled bicarbonate and (14)C-2-acetate showed that in the psychrophilic community (enriched at 5 degrees C), about 95% of methane originated from acetate, in contrast to the thermophilic community (50 degrees C) where up to 98% of methane was formed from bicarbonate. In the mesophilic community (30 degrees C), acetate was the precursor of about 80% of the methane produced. When the hydrogen-carbon dioxide mixture (H(2)/CO(2)) was used as a substrate, it was directly converted to methane under thermophilic conditions (70 and 50 degrees C). Under mesophilic conditions (30 degrees C), both pathways, hydrogenotrophic and acetoclastic, were observed. At low temperatures (5 and 15 degrees C), H(2)/CO(2) was converted into methane by a two-step process; first acetate was formed, followed by methane production from acetate. When slurries were incubated at high partial pressures of H(2)/CO(2), the high concentrations of acetate produced of more than 20 mM inhibited acetoclastic methanogenesis at a temperature below 15 degrees C. However, slow adaptation of the psychrophilic microbial community to high acetate concentrations was observed.     

Proton-motive-force-driven formation of CO from CO2 and H2 in methanogenic bacteria

Cell suspensions of methanogenic bacteria (Methanosarcina barkeri, Methanospirillum hungatei, Methano-brevibacter arboriphilus, and Methanobacterium thermoautotrophicum) were found to form CO from CO2 and H2 according to the reaction: CO2 + H2----CO + H2O; delta G0 = +20 kJ/mol. Up to 15,000 ppm CO in the gas phase were reached which is significantly higher than the equilibrium concentration calculated from delta G0 (95 ppm under the experimental conditions). This indicated that CO2 reduction with H2 to CO is energy-driven and indeed the cells only generated CO when forming CH4. The coupling of the two reactions was studied in more detail with acetate-grown cells of M. barkeri using methanogenic substrates. The effects of the protonophore tetrachlorosalicylanilide (TCS) and of the proton-translocating ATPase inhibitor N,N'-dicyclohexylcarbodiimide (cHxN)2C were determined. TCS completely inhibited CO formation from CO2 and H2 without affecting methanogenesis from CH3OH and H2. In the presence of the protonophore the proton motive force delta p and the intracellular ATP concentration were very low. (cHxN)2C, which partially inhibited methanogenesis from CH3OH and H2, had no effect on CO2 reduction to CO. In the presence of (cHxN)2C delta p was high and the intracellular ATP content was low. These findings suggest that the endergonic formation of CO from CO2 and H2 is coupled to the exergonic formation of CH4 from CH3OH and H2 via the proton motive force and not via ATP. CO formation was not stimulated by the addition of sodium ions.     

Methanogenic activity and diversity in the centre of the Amsterdam Mud Volcano, Eastern Mediterranean Sea

Marine mud volcanoes are geological structures emitting large amounts of methane from their active centres. The Amsterdam mud volcano (AMV), located in the Anaximander Mountains south of Turkey, is characterized by intense active methane seepage produced in part by methanogens. To date, information about the diversity or the metabolic pathways used by the methanogens in active centres of marine mud volcanoes is limited. (14)C-radiotracer measurements showed that methylamines/methanol, H(2)/CO(2) and acetate were used for methanogenesis in the AMV. Methylotrophic methanogenesis was measured all along the sediment core, Methanosarcinales affiliated sequences were detected using archaeal 16S PCR-DGGE and mcrA gene libraries, and enrichments of methanogens showed the presence of Methanococcoides in the shallow sediment layers. Overall acetoclastic methanogenesis was higher than hydrogenotrophic methanogenesis, which is unusual for cold seep sediments. Interestingly, acetate porewater concentrations were extremely high in the AMV sediments. This might be the result of organic matter cracking in deeper hotter sediment layers. Methane was also produced from hexadecanes. For the most part, the methanogenic community diversity was in accordance with the depth distribution of the H(2)/CO(2) and acetate methanogenesis. These results demonstrate the importance of methanogenic communities in the centres of marine mud volcanoes.     

Thermophilic sulfate reduction and methanogenesis with methanol in a high rate anaerobic reactor

Sulfate reduction outcompeted methanogenesis at 65 degrees C and pH 7.5 in methanol and sulfate-fed expanded granular sludge bed reactors operated at hydraulic retention times (HRT) of 14 and 3.5 h, both under methanol-limiting and methanol-overloading conditions. After 100 and 50 days for the reactors operated at 14 and 3.5 h, respectively, sulfide production accounted for 80% of the methanol-COD consumed by the sludge. The specific methanogenic activity on methanol of the sludge from a reactor operated at HRTs of down to 3.5 h for a period of 4 months gradually decreased from 0. 83 gCOD. gVSS(-1). day(-1) at the start to a value of less than 0.05 gCOD. gVSS(-1). day(-1), showing that the relative number of methanogens decreased and eventually became very low. By contrast, the increase of the specific sulfidogenic activity of sludge from 0. 22 gCOD. gVSS(-1). day(-1) to a final value of 1.05 gCOD. gVSS(-1). day(-1) showed that sulfate reducing bacteria were enriched. Methanol degradation by a methanogenic culture obtained from a reactor by serial dilution of the sludge was inhibited in the presence of vancomycin, indicating that methanogenesis directly from methanol was not important. H(2)/CO(2) and formate, but not acetate, were degraded to methane in the presence of vancomycin. These results indicated that methanol degradation to methane occurs via the intermediates H(2)/CO(2) and formate. The high and low specific methanogenic activity of sludge on H(2)/CO(2) and formate, respectively, indicated that the former substrate probably acts as the main electron donor for the methanogens during methanol degradation. As sulfate reduction in the sludge was also strongly supported by hydrogen, competition between sulfate reducing bacteria and methanogens in the sludge seemed to be mainly for this substrate. Sulfate elimination rates of up to 15 gSO(4)(2-)/L per day were achieved in the reactors. Biomass retention limited the sulfate elimination rate.     

Production of aromatic compounds during methanogenic degradation of straw in rice field soil

Production of CH(4) in anoxic rice field soil is stimulated by the addition of rice straw. Previous experiments showed that acetate and propionate are the most important intermediates of the carbon flow to CH(4), and accumulate if CH(4) production is inhibited by 2-bromoethanesulfonate (BES). However, some unidentified compounds were found to accumulate in addition. We now identified them as benzoate, phenylpropionate, and phenylacetate by comparison of the retention times in HPLC chromatograms with authentic standards and by mass spectrometry. These aromatic compounds accumulated only to concentrations <100 microM, especially in soil amended with rice straw (stem, sheath or blade straw). Phenylpropionate and benzoate were the most abundant aromatic intermediates contributing up to 4% to total CH(4) production. Phenylacetate, on the other hand, contributed very little (<0.3%). Gibbs free energies (DeltaG) were calculated for different anaerobic degradation pathways of the aromatic compounds at the actual incubation conditions. Conversion of benzoate to acetate, CO(2) and H(2) was strongly exergonic (DeltaG = -86 kJ mol(-1)) under methanogenic conditions, but became less exergonic (DeltaG = -30 kJ mol(-1)) when CH(4) production was inhibited. The primary oxidation of phenylpropionate was only exergonic for alpha-oxidation (i.e. phenylacetate as product) but not for beta-oxidation (i.e. benzoate as product). However, the DeltaG values for the complete degradation of phenylpropionate to acetate, CO(2) and H(2) were similar for both pathways and were also similar to those of benzoate degradation. Collectively, the results suggest that aromatic compounds are minor intermediates of anaerobic degradation of organic matter in rice field soil, and are syntrophically degraded by coupling to methanogenesis.     

Relationship of formate to growth and methanogenesis by Methanococcus thermolithotrophicus

Methanococcus thermolithotrophicus is a methanogenic archaebacterium that can use either H2 or formate as its source of electrons for reduction of CO2 to methane. Growth and suspended-whole-cell experiments show that H2 plus CO2 methanogenesis was constitutive, while formate methanogenesis required adaptation time; selenium was necessary for formate utilization. Cells grown on formate had 20 to 100 times higher methanogenesis rates on formate than cells grown on H2-CO2 and transferred into formate medium. Enzyme assays with crude extracts and with F420 or methyl viologen as the electron acceptor revealed that hydrogenase was constitutive, while formate dehydrogenase was regulated. Cells grown on formate had 10 to 70 times higher formate dehydrogenase activity than cells grown on H2-CO2 with Se present in the medium; when no Se was added to H2-CO2 cultures, even lower activities were observed. Adaptation to and growth on formate were pH dependent, with an optimal pH for both about one pH unit above that optimal for H2-CO2 (pH 5.8 to 6.5). When cells were grown on H2-CO2 in the presence of formate, formate (greater than or equal to 50 mM) inhibited both growth and methanogenesis at pH 5.8 to 6.2, but not at pH greater than 6.6. Both acetate and propionate produced similar inhibition. Formate inhibition was also observed in Methanospirillum hungatei.     

Relationship of formate to growth and methanogenesis by Methanococcus thermolithotrophicus.

Methanococcus thermolithotrophicus is a methanogenic archaebacterium that can use either H2 or formate as its source of electrons for reduction of CO2 to methane. Growth and suspended-whole-cell experiments show that H2 plus CO2 methanogenesis was constitutive, while formate methanogenesis required adaptation time; selenium was necessary for formate utilization. Cells grown on formate had 20 to 100 times higher methanogenesis rates on formate than cells grown on H2-CO2 and transferred into formate medium. Enzyme assays with crude extracts and with F420 or methyl viologen as the electron acceptor revealed that hydrogenase was constitutive, while formate dehydrogenase was regulated. Cells grown on formate had 10 to 70 times higher formate dehydrogenase activity than cells grown on H2-CO2 with Se present in the medium; when no Se was added to H2-CO2 cultures, even lower activities were observed. Adaptation to and growth on formate were pH dependent, with an optimal pH for both about one pH unit above that optimal for H2-CO2 (pH 5.8 to 6.5). When cells were grown on H2-CO2 in the presence of formate, formate (greater than or equal to 50 mM) inhibited both growth and methanogenesis at pH 5.8 to 6.2, but not at pH greater than 6.6. Both acetate and propionate produced similar inhibition. Formate inhibition was also observed in Methanospirillum hungatei.     

Endogenous methanogenesis stimulates oxidation of atmospheric CH(4) in alpine tundra soil

Experiments were done to test the hypothesis that atmospheric CH(4) oxidizers in a well-drained alpine tundra soil are supported by CH(4) production from anaerobic microsites in the soil. Soil was subjected to 22 days of anaerobic conditions with elevated H(2) and CO(2) in order to stimulate methanogenesis. This treatment stimulated subsequent atmospheric CH(4) consumption, probably by increasing soil methanogenesis. After removal from anaerobic conditions, soils emitted CH(4) for up to 6 h, then oxidized atmospheric CH(4) at 111 (+/- 5.7) pmol (g dry weight)(-1) h(-1), which was more than 3 times the rate of control soils. Further supporting our hypothesis, additions of lumazine, a highly specific inhibitor of methanogenesis, prevented the stimulation of atmospheric CH(4) oxidation by the anaerobic treatment. The method used to create anaerobic conditions with elevated H(2) and CO(2) also elevated headspace CH(4) concentrations. However, elevated CH(4) concentrations under aerobic conditions did not stimulate CH(4) oxidation as much as preexposure to H(2) and CO(2) under anaerobic conditions. Anaerobic conditions created by N(2) flushing did not stimulate atmospheric CH4 oxidation, probably because N2 flushing inhibited methanogenesis by removing necessary precursors for methane production. We conclude that anaerobic conditions with elevated H(2) and CO(2) stimulate atmospheric CH(4) oxidation in this dry alpine tundra soil by increasing endogenous CH(4) production. This effect was prevented by inhibiting methanogenesis, indicating the importance of endogenous CH(4) production in a CH(4-) consuming soil.     

Detecting active methanogenic populations on rice roots using stable isotope probing

Methane is formed on rice roots mainly by CO2 reduction. The present study aimed to identify the active methanogenic populations responsible for this process. Soil-free rice roots were incubated anaerobically under an atmosphere of H2/(13CO2) or N2/(13CO2) with phosphate or carbonate (marble) as buffer medium. Nucleic acids were extracted and fractionated by caesium trifluoroacetate equilibrium density gradient centrifugation after 16-day incubation. Community analyses were performed for gradient fractions using terminal restriction fragment polymorphism analysis (T-RFLP) and sequencing of the 16S rRNA genes. In addition, rRNA was extracted and analysed at different time points to trace the community change during the 16-day incubation. The Methanosarcinaceae and the yet-uncultured archaeal lineage Rice Cluster-I (RC-I) were predominant in the root incubations when carbonate buffer and N2 headspace were used. The analysis of [13C]DNA showed that the relative 16S rRNA gene abundance of RC-I increased whereas that of the Methanosarcinaceae decreased with increasing DNA buoyant density, indicating that members of RC-I were more active than the Methanosarcinaceae. However, an unexpected finding was that RC-I was suppressed in the presence of high H2 concentrations (80%, v/v), which during the early incubation period caused a lower CH4 production compared with that with N2 in the headspace. Eventually, however, CH4 production increased, probably because of the activity of Methanosarcinaceae, which became prevalent. Phosphate buffer appeared to inhibit the activity of the Methanosarcinaceae, resulting in lower CH4 production as compared with carbonate buffer. Under these conditions, Methanobacteriaceae were the prevalent methanogens. Our study suggests that the active methanogenic populations on rice roots change in correspondence to the presence of H2 (80%, v/v) and the type of buffer used in the system.     

Genetic analysis of mch mutants in two Methanosarcina species demonstrates multiple roles for the methanopterin-dependent C-1 oxidation/reduction pathway and differences in H(2) metabolism between closely related species

A mutation in the mch gene, encoding the enzyme 5,10-methenyl tetrahydromethanopterin (H(4)MPT) cyclohydrolase, was constructed in vitro and recombined onto the chromosome of the methanogenic archaeon Methanosarcina barkeri. The resulting mutant does not grow in media using H(2)/CO(2), methanol, or acetate as carbon and energy sources, but does grow in media with methanol/H(2)/CO(2), demonstrating its ability to utilize H(2) as a source of electrons for reduction of methyl groups. Cell suspension experiments showed that methanogenesis from methanol or from H(2)/CO(2) is blocked in the mutant, explaining the lack of growth on these substrates. The corresponding mutation in Methanosarcina acetivorans C2A, which cannot grow on H(2)/CO(2), could not be made in wild-type strains, but could be made in strains carrying a second copy of mch, suggesting that M. acetivorans is incapable of methyl group reduction using H(2). M. acetivorans mch mutants could also be constructed in strains carrying the M. barkeri ech hydrogenase operon, suggesting that the block in the methyl reduction pathway is at the level of H(2) oxidation. Interestingly, the ech-dependent methyl reduction pathway of M. acetivorans involves an electron transport chain distinct from that used by M. barkeri, because M. barkeri ech mutants remain capable of H(2)-dependent methyl reduction.     

The transmembrane electrochemical gradient of Na+ as driving force for methanol oxidation in Methanosarcina barkeri

A sodium ion gradient (inside low) across the cytoplasmic membrane of Methanosarcina barkeri was required for methanogenesis from methanol. This could be concluded from the following results. (a) Inhibition of the Na+/H+ antiporter by K+ or amiloride led to an inhibition of methanogenesis from methanol. (b) Upon addition of the sodium ionophore monensin the Na+ gradient was abolished and at the same time methanogenesis from methanol was inhibited. (c) Methanogenesis was impaired when the Na+ gradient had the opposite orientation (inside high). All these inhibitory effects were not observed when H2 was present in addition to methanol indicating that the oxidation of methanol to CO2 was driven by a sodium-motive force. In accordance with this, a methanol-dependent influx of Na+ and a corresponding decrease of the membrane potential could be observed, when the Na+/H+ antiporter was inhibited by amiloride. This influx was indicative of the presence of a Na+ transport system which was functional when the oxidation of methanol had to be driven, but was not functional when H2 was present for reduction of methanol to methane.     

Expression of ascorbate peroxidase and glutathione reductase in roots of rice seedlings in response to NaCl and H2O2

The accumulation of H2O2 by NaCl was observed in the roots of rice seedlings. Treatment with NaCl caused an increase in the activities of ascorbate peroxidase (APX) and glutathione reductase (GR) and the expression of OsAPX and OsGR in rice roots. Exogenously applied H2O2 also enhanced the activities of APX and GR and the expression of OsAPX and OsGR in rice roots. The accumulation of H2O2 in rice roots in response to NaCl was inhibited by the NADPH oxidase inhibitors, diphenyleneiodonium chloride (DPI) and imidazole (IMD). However, DPI, IMD, and dimethylthiourea, a H2O2 trap, did not reduce NaCl-enhanced activities of APX and GR and expression of OsAPX and OsGR. It appears that H2O2 is not involved in the regulation of NaCl-induced APX and GR activities and OsAPX and OsGR expression in rice roots.     

Anaerobic Degradation of Lactate by Syntrophic Associations of Methanosarcina barkeri and Desulfovibrio Species and Effect of H(2) 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 H(2) and CO(2) for methanogenesis, degraded lactate, with the production of acetate and CH(4). When D. desulfuricans or D. vulgaris was grown in the absence of added sulfate in coculture with Methanosarcina barkeri (type strain), which uses both H(2)-CO(2) and acetate for methanogenesis, lactate was stoichiometrically degraded to CH(4) and presumably to CO(2). 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 CH(4). Later, acetate was degraded to CH(4) and presumably to CO(2). 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 H(2) was detected in cocultures which contained D. desulfuricans and M. barkeri until after all lactate was degraded. The addition of H(2), but not of lactate, to the growth medium inhibited acetate degradation by pure cultures of M. barkeri. Pure cultures of M. barkeri produced CH(4) from acetate at a rate equivalent to that observed for cocultures containing M. barkeri. Inocula of M. barkeri grown with H(2)-CO(2) as the methanogenic substrate produced CH(4) 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 H(2) produced by the Desulfovibrio species during growth with lactate inhibited acetate degradation by M. barkeri.     

Biogeochemistry and biodiversity of methane cycling in subsurface marine sediments (Skagerrak, Denmark)

This biogeochemical, molecular genetic and lipid biomarker study of sediments ( approximately 4 m cores) from the Skagerrak (Denmark) investigated methane cycling in a sediment with a clear sulfate-methane-transition zone (SMTZ) and where CH(4) supply was by diffusion, rather than by advection, as in more commonly studied seep sites. Sulfate reduction removed sulfate by 0.7 m and CH(4) accumulated below. (14)C-radiotracer measurements demonstrated active H(2)/CO(2) and acetate methanogenesis and anaerobic oxidation of CH(4) (AOM). Maximum AOM rates occurred near the SMTZ ( approximately 3 nmol cm(-3) day(-1) at 0.75 m) but also continued deeper, overall, at much lower rates. Maximum rates of H(2)/CO(2) and acetate methanogenesis occurred below the SMTZ but H(2)/CO(2) methanogenesis rates were x 10 those of acetate methanogenesis, and this was consistent with initial values of (13)C-depleted CH(4) (delta(13)C c.-80 per thousand). Areal AOM and methanogenic rates were similar ( approximately 1.7 mmol m(-2) day(-1)), hence, CH(4) flux is finely balanced. A 16S rRNA gene library from 1.39 m combined with methanogen (T-RFLP), bacterial (16S rRNA DGGE) and lipid biomarker depth profiles showed the presence of populations similar to some seep sites: ANME-2a (dominant), ANME-3, Methanomicrobiales, Methanosaeta Archaea, with abundance changes with depth corresponding to changes in activities and sulfate-reducing bacteria (SRB). Below the SMTZ to approximately 1.7 m CH(4) became progressively more (13)C depleted (delta(13)C -82 per thousand) indicating a zone of CH(4) recycling which was consistent with the presence of (13)C-depleted archaeol (delta(13)C -55 per thousand). Pore water acetate concentrations decreased in this zone (to approximately 5 microM), suggesting that H(2), not acetate, was an important CH(4) cycling intermediate. The potential biomarkers for AOM-associated SRB, non-isoprenoidal ether lipids, increased below the SMTZ but this distribution reflected 16S rRNA gene sequences for JS1 and OP8 bacteria rather than those of SRB. At this site peak rates of methane production and consumption are spatially separated and seem to be conducted by different archaeal groups. Also AOM is predominantly coupled to sulfate reduction, unlike recent reports from some seep and gassy sediment sites.