Substrate-dependent incorporation of carbon and hydrogen for lipid biosynthesis by Methanosarcina barkeri.

Dual stable isotope probing has been used to infer rates of microbial biomass production and modes of carbon fixation. In order to validate this approach for assessing archaeal production, the methanogenic archaeon Methanosarcina barkeri was grown either with H2 , acetate or methanol with D2 O and 13 C-dissolved inorganic carbon (DIC). Our results revealed unexpectedly low D incorporation into lipids, with the net fraction of water-derived hydrogen amounting to 0.357 ± 0.042, 0.226 ± 0.003 and 0.393 ± 0.029 for growth on H2 /CO2 , acetate and methanol respectively. The variability in net water H assimilation into lipids during the growth of M. barkeri on different substrates is possibly attributed to different Gibbs free energy yields, such that higher energy yield promoted the exchange of hydrogen between medium water and lipids. Because NADPH likely serves as the portal for H transfer, increased NADPH production and/or turnover associated with high energy yield may explain the apparent differences in net water H assimilation into lipids. The variable DIC and water H incorporation into M. barkeri lipids imply systematic, metabolic patterns of isotope incorporation and suggest that the ratio of 13 C-DIC versus D2 O assimilation in environmental samples may serve as a proxy for microbial energetics in addition to microbial production and carbon assimilation pathways.

Energy Conservation via Hydrogen Cycling in the Methanogenic Archaeon Methanosarcina barkeri.

Energy conservation via hydrogen cycling, which generates proton motive force by intracellular H2 production coupled to extracellular consumption, has been controversial since it was first proposed in 1981. It was hypothesized that the methanogenic archaeon Methanosarcina barkeri is capable of energy conservation via H2 cycling, based on genetic data that suggest that H2 is a preferred, but nonessential, intermediate in the electron transport chain of this organism. Here, we characterize a series of hydrogenase mutants to provide direct evidence of H2 cycling. M. barkeri produces H2 during growth on methanol, a phenotype that is lost upon mutation of the cytoplasmic hydrogenase encoded by frhADGB, although low levels of H2, attributable to the Ech hydrogenase, accumulate during stationary phase. In contrast, mutations that conditionally inactivate the extracellular Vht hydrogenase are lethal when expression of the vhtGACD operon is repressed. Under these conditions, H2 accumulates, with concomitant cessation of methane production and subsequent cell lysis, suggesting that the inability to recapture extracellular H2 is responsible for the lethal phenotype. Consistent with this interpretation, double mutants that lack both Vht and Frh are viable. Thus, when intracellular hydrogen production is abrogated, loss of extracellular H2 consumption is no longer lethal. The common occurrence of both intracellular and extracellular hydrogenases in anaerobic microorganisms suggests that this unusual mechanism of energy conservation may be widespread in nature.IMPORTANCE ATP is required by all living organisms to facilitate essential endergonic reactions required for growth and maintenance. Although synthesis of ATP by substrate-level phosphorylation is widespread and significant, most ATP is made via the enzyme ATP synthase, which is energized by transmembrane chemiosmotic gradients. Therefore, establishing this gradient across the membrane is of central importance to sustaining life. Experimental validation of H2 cycling adds to a short list of mechanisms for generating a transmembrane electrochemical gradient that is likely to be widespread, especially among anaerobic microorganisms.

Co-cultivation of the strictly anaerobic methanogen Methanosarcina barkeri with aerobic methanotrophs in an oxygen-limited membrane bioreactor.

Wetlands contribute to 30% of global methane emissions due to an imbalance between microbial methane production and consumption. Methanogenesis and methanotrophy have mainly been studied separately, and little is known about their potential interactions in aquatic environments. To mimic the interaction between methane producers and oxidizers in the environment, we co-cultivated the methanogenic archaeon Methanosarcina barkeri with aerobic Methylocystaceae methanotrophs in an oxygen-limited bioreactor using acetate as methanogenic substrate. Methane, acetate, dissolved oxygen, available nitrogen, pH, temperature, and cell density were monitored to follow system stability and activity. Stable reactor operation was achieved for two consecutive periods of 2 months. Fluorescence in situ hybridization micrographs indicated close association between both groups of microorganisms. This association suggests that the methanotrophs profit from direct access to the methane that is produced from acetate, while methanogens are protected by the concomitant oxygen consumption of the methanotrophs. This proof of principle study can be used to set up systems to study their responses to environmental changes.

Effect of Nickel Levels on Hydrogen Partial Pressure and Methane Production in Methanogens.

Hydrogen (H2) consumption and methane (CH4) production in pure cultures of three different methanogens were investigated during cultivation with 0, 0.2 and 4.21 µM added nickel (Ni). The results showed that the level of dissolved Ni in the anaerobic growth medium did not notably affect CH4 production in the cytochrome-free methanogenic species Methanobacterium bryantii and Methanoculleus bourgensis MAB1, but affected CH4 formation rate in the cytochrome-containing Methanosarcina barkeri grown on H2 and CO2. Methanosarcina barkeri also had the highest amounts of Ni in its cells, indicating that more Ni is needed by cytochrome-containing than by cytochrome-free methanogenic species. The concentration of Ni affected threshold values of H2 partial pressure (pH2) for all three methanogen species studied, with M. bourgensis MAB1 reaching pH2 values as low as 0.1 Pa when Ni was available in amounts used in normal anaerobic growth medium. To our knowledge, this is the lowest pH2 threshold recorded to date in pure methanogen culture, which suggests that M.bourgensis MAB1 have a competitive advantage over other species through its ability to grow at low H2 concentrations. Our study has implications for research on the H2-driven deep subsurface biosphere and biogas reactor performance.

Physicochemical analysis of initial adhesion and biofilm formation of Methanosarcina barkeri on polymer support material.

The retention of selective biofilms of Methanosarcina species within anaerobic digesters could reduce start-up times and enhance the efficiency of the process in treating high-strength domestic sewage. The objective of the study was to examine the effect of the surface characteristics of six common polymer support materials on the initial adhesion of the model methanogen, Methanosarcina barkeri, and to assess the potential of these support materials as selective biofilm carriers. Results from both the initial adhesion tests and extended DLVO (xDLVO) model correlated with each other, with PVC (12% surface coverage/mm(2)), PTFE (6% surface coverage/mm(2)), and PP (6% surface coverage/mm(2)), shown to be the better performing support materials for initial adhesion, as well as subsequent biofilm formation by M. barkeri after 72h. Experimental results of these three support materials showed that the type of material strongly influenced the extent of adhesion from M. barkeri (p<0.0001), and the xDLVO model was able to explain the results in these environmental conditions. Therefore, DLVO physicochemical forces were found to be influential on the initial adhesion of M. barkeri. Scanning electron microscopy suggested that production of extracellular polymeric substances (EPS) from M. barkeri could facilitate further biofilm development. This study highlights the potential of using the xDLVO model to rapidly identify suitable materials for the selective adhesion of M. barkeri, which could be beneficial in both the start-up and long-term phases of anaerobic digestion.

Single-cell analysis reveals gene-expression heterogeneity in syntrophic dual-culture of Desulfovibrio vulgaris with Methanosarcina barkeri.

Microbial syntrophic metabolism has been well accepted as the heart of how methanogenic and other anaerobic microbial communities function. In this work, we applied a single-cell RT-qPCR approach to reveal gene-expression heterogeneity in a model syntrophic system of Desulfovibrio vulgaris and Methanosarcina barkeri, as compared with the D. vulgaris monoculture. Using the optimized primers and single-cell analytical protocol, we quantitatively determine gene-expression levels of 6 selected target genes in each of the 120 single cells of D. vulgaris isolated from its monoculture and dual-culture with M. barkeri. The results demonstrated very significant cell-to-cell gene-expression heterogeneity for the selected D. vulgaris genes in both the monoculture and the syntrophic dual-culture. Interestingly, no obvious increase in gene-expression heterogeneity for the selected genes was observed for the syntrophic dual-culture when compared with its monoculture, although the community structure and cell-cell interactions have become more complicated in the syntrophic dual-culture. In addition, the single-cell RT-qPCR analysis also provided further evidence that the gene cluster (DVU0148-DVU0150) may be involved syntrophic metabolism between D. vulgaris and M. barkeri. Finally, the study validated that single-cell RT-qPCR analysis could be a valuable tool in deciphering gene functions and metabolism in mixed-cultured microbial communities.

Promoting interspecies electron transfer with biochar.

Biochar, a charcoal-like product of the incomplete combustion of organic materials, is an increasingly popular soil amendment designed to improve soil fertility. We investigated the possibility that biochar could promote direct interspecies electron transfer (DIET) in a manner similar to that previously reported for granular activated carbon (GAC). Although the biochars investigated were 1000 times less conductive than GAC, they stimulated DIET in co-cultures of Geobacter metallireducens with Geobacter sulfurreducens or Methanosarcina barkeri in which ethanol was the electron donor. Cells were attached to the biochar, yet not in close contact, suggesting that electrons were likely conducted through the biochar, rather than biological electrical connections. The finding that biochar can stimulate DIET may be an important consideration when amending soils with biochar and can help explain why biochar may enhance methane production from organic wastes under anaerobic conditions.

A functional approach to uncover the low-temperature adaptation strategies of the archaeon Methanosarcina barkeri.

Low-temperature anaerobic digestion (LTAD) technology is underpinned by a diverse microbial community. The methanogenic archaea represent a key functional group in these consortia, undertaking CO2 reduction as well as acetate and methylated C1 metabolism with subsequent biogas (40 to 60% CH4 and 30 to 50% CO2) formation. However, the cold adaptation strategies, which allow methanogens to function efficiently in LTAD, remain unclear. Here, a pure-culture proteomic approach was employed to study the functional characteristics of Methanosarcina barkeri (optimum growth temperature, 37°C), which has been detected in LTAD bioreactors. Two experimental approaches were undertaken. The first approach aimed to characterize a low-temperature shock response (LTSR) of M. barkeri DSMZ 800(T) grown at 37°C with a temperature drop to 15°C, while the second experimental approach aimed to examine the low-temperature adaptation strategies (LTAS) of the same strain when it was grown at 15°C. The latter experiment employed cell viability and growth measurements (optical density at 600 nm [OD600]), which directly compared M. barkeri cells grown at 15°C with those grown at 37°C. During the LTSR experiment, a total of 127 proteins were detected in 37°C and 15°C samples, with 20 proteins differentially expressed with respect to temperature, while in the LTAS experiment 39% of proteins identified were differentially expressed between phases of growth. Functional categories included methanogenesis, cellular information processing, and chaperones. By applying a polyphasic approach (proteomics and growth studies), insights into the low-temperature adaptation capacity of this mesophilically characterized methanogen were obtained which suggest that the metabolically diverse Methanosarcinaceae could be functionally relevant for LTAD systems.

Genomically and biochemically accurate metabolic reconstruction of Methanosarcina barkeri Fusaro, iMG746.

Methanosarcina barkeri is an Archaeon that produces methane anaerobically as the primary byproduct of its metabolism. M. barkeri can utilize several substrates for ATP and biomass production including methanol, acetate, methyl amines, and a combination of hydrogen and carbon dioxide. In 2006, a metabolic reconstruction of M. barkeri, iAF692, was generated based on a draft genome annotation. The iAF692 reconstruction enabled the first genome-Scale simulations for Archaea. Since the publication of the first metabolic reconstruction of M. barkeri, additional genomic, biochemical, and phenotypic data have clarified several metabolic pathways. We have used this newly available data to improve the M. barkeri metabolic reconstruction. Modeling simulations using the updated model, iMG746, have led to increased accuracy in predicting gene knockout phenotypes and simulations of batch growth behavior. We used the model to examine knockout lethality data and make predictions about metabolic regulation under different growth conditions. Thus, the updated metabolic reconstruction of M. barkeri metabolism is a useful tool for predicting cellular behavior, studying the methanogenic lifestyle, guiding experimental studies, and making predictions relevant to metabolic engineering applications.

Desiccation as a long-term survival mechanism for the archaeon Methanosarcina barkeri.

Viable methanogens have been detected in dry, aerobic environments such as dry reservoir sediment, dry rice paddies and aerobic desert soils, which suggests that methanogens have mechanisms for long-term survival in a desiccated state. In this study, we quantified the survival rates of the methanogenic archaeon Methanosarcina barkeri after desiccation under conditions equivalent to the driest environments on Earth and subsequent exposure to different stress factors. There was no significant loss of viability after desiccation for 28 days for cells grown with either hydrogen or the methylotrophic substrates, but recovery was affected by growth phase, with cells desiccated during the stationary phase of growth having a higher rate of recovery after desiccation. Synthesis of methanosarcinal extracellular polysaccharide (EPS) significantly increased the viability of desiccated cells under both anaerobic and aerobic conditions compared with that of non-EPS-synthesizing cells. Desiccated M. barkeri exposed to air at room temperature did not lose significant viability after 28 days, and exposure of M. barkeri to air after desiccation appeared to improve the recovery of viable cells compared with that of desiccated cells that were never exposed to air. Desiccated M. barkeri was more resistant to higher temperatures, and although resistance to oxidative conditions such as ozone and ionizing radiation was not as robust as in other desiccation-resistant microorganisms, the protection mechanisms are likely adequate to maintain cell viability during periodic exposure events. The results of this study demonstrate that after desiccation M. barkeri has the innate capability to survive extended periods of exposure to air and lethal temperatures.

A vacuum-vortex technique for preparation of anoxic solutions or liquid culture media in small volumes for cultivating methanogens or other strict anaerobes.

A highly efficient method is described for producing at room temperature anoxic solutions of 50 ml or less in test tubes or serum vials by combining negative pressure with strong vortexing so that the liquid-surface, gas exchange area is increased by orders of magnitude. Liquid media suitable for the cultivation of methanogens may be rendered anoxic after three short vacuum-vortex steps.

Hydrogen is a preferred intermediate in the energy-conserving electron transport chain of Methanosarcina barkeri.

Methanogens use an unusual energy-conserving electron transport chain that involves reduction of a limited number of electron acceptors to methane gas. Previous biochemical studies suggested that the proton-pumping F(420)H(2) dehydrogenase (Fpo) plays a crucial role in this process during growth on methanol. However, Methanosarcina barkeri Delta fpo mutants constructed in this study display no measurable phenotype on this substrate, indicating that Fpo plays a minor role, if any. In contrast, Delta frh mutants lacking the cytoplasmic F(420)-reducing hydrogenase (Frh) are severely affected in their ability to grow and make methane from methanol, and double Delta fpo/Delta frh mutants are completely unable to use this substrate. These data suggest that the preferred electron transport chain involves production of hydrogen gas in the cytoplasm, which then diffuses out of the cell, where it is reoxidized with transfer of electrons into the energy-conserving electron transport chain. This hydrogen-cycling metabolism leads directly to production of a proton motive force that can be used by the cell for ATP synthesis. Nevertheless, M. barkeri does have the flexibility to use the Fpo-dependent electron transport chain when needed, as shown by the poor growth of the Delta frh mutant. Our data suggest that the rapid enzymatic turnover of hydrogenases may allow a competitive advantage via faster growth rates in this freshwater organism. The mutant analysis also confirms the proposed role of Frh in growth on hydrogen/carbon dioxide and suggests that either Frh or Fpo is needed for aceticlastic growth of M. barkeri.

Methane production by methanogens following an aerobic washing procedure: simplifying methods for manipulation.

The recent discovery of methane in the martian atmosphere is arguably one of the most important discoveries in the field of astrobiology. One possible source of this methane could be a microorganism analogous to those on Earth in the domain Archaea known as methanogens. Methanogens are described as obligately anaerobic, and methods developed to work with methanogens typically include anaerobic media and buffers, gassing manifolds, and possibly anaerobic chambers. To determine if the time, effort, and supplies required to maintain anaerobic conditions are necessary to maintain viability, we compared anaerobically washed cells with cells that were washed in the presence of atmospheric oxygen. Anaerobic tubes were opened, and cultures were poured into plastic centrifuge tubes, centrifuged, and suspended in fresh buffer, all in the presence of atmospheric oxygen. Washed cells from both aerobic and anaerobic procedures were inoculated into methanogenic growth media under anaerobic conditions and incubated at temperatures conducive to growth for each methanogenic strain tested. Methane production was measured at time intervals using a gas chromatograph. In three strains, significant differences were not seen between aerobically and anaerobically washed cells. In one strain, there was significantly less methane production observed following aerobic washing at some time points; however, substantial methane production occurred following both procedures. Thus, it appears that aerobic manipulations for relatively short periods of time with at least a few species of methanogens may not lead to loss of viability. With the discovery of methane in the martian atmosphere, it is likely that there will be an increase in astrobiology-related methanogen research. The research reported here should simplify the methodology.

Heme biosynthesis in Methanosarcina barkeri via a pathway involving two methylation reactions.

The methanogenic archaeon Methanosarcina barkeri synthesizes protoheme via precorrin-2, which is formed from uroporphyrinogen III in two consecutive methylation reactions utilizing S-adenosyl-L-methionine. The existence of this pathway, previously exclusively found in the sulfate-reducing delta-proteobacterium Desulfovibrio vulgaris, was demonstrated for M. barkeri via the incorporation of two methyl groups from methionine into protoheme.

Survival of methanogens during desiccation: implications for life on Mars.

The relatively recent discoveries that liquid water likely existed on the surface of past Mars and that methane currently exists in the martian atmosphere have fueled the possibility of extant or extinct life on Mars. One possible explanation for the existence of the methane would be the presence of methanogens in the subsurface. Methanogens are microorganisms in the domain Archaea that can metabolize molecular hydrogen as an energy source and carbon dioxide as a carbon source and produce methane. One factor of importance is the arid nature of Mars, at least at the surface. If one is to assume that life exists below the surface, then based on the only example of life that we know, liquid water must be present. Realistically, however, that liquid water may be seasonal just as it is at some locations on our home planet. Here we report on research designed to determine how long certain species of methanogens can survive desiccation on a Mars soil simulant, JSC Mars-1. Methanogenic cells were grown on JSC Mars-1, transferred to a desiccator within a Coy anaerobic environmental chamber, and maintained there for varying time periods. Following removal from the desiccator and rehydration, gas chromatographic measurements of methane indicated survival for varying time periods. Methanosarcina barkeri survived desiccation for 10 days, while Methanobacterium formicicum and Methanothermobacter wolfeii were able to survive for 25 days.

Washing methanogenic cells with the liquid fraction from a Mars soil simulant and water mixture.

Certain methanogens have been shown to grow on a Mars soil simulant following a washing procedure using a carbonate buffer. In experiments where liquid fractions from the soil simulant and water mixtures were used in place of the buffer, two out of three of the species demonstrated significantly greater methane production compared to the buffer.

The catalase and superoxide dismutase genes are transcriptionally up-regulated upon oxidative stress in the strictly anaerobic archaeon Methanosarcina barkeri.

Methanosarcina barkeri is a strictly anaerobic methanogenic archaeon, which can survive oxidative stress. The oxidative stress agent paraquat (PQ) suppressed growth of M. barkeri at concentrations of 50-200 microM. Hydrogen peroxide (H2O2) inhibited growth at concentrations of 0.4-1.6 mM. Catalase activity in cell-free extracts of M. barkeri increased about threefold during H2O2 stress (1.3 mM H2O2, 2-4 h exposure) and nearly twofold during superoxide stress (160 microM PQ, 2 h exposure). PQ (160 microM, 2-4 h exposure) and H2O2 (1.3 mM, 2 h exposure) also influenced superoxide dismutase activity in cell-free extracts of M. barkeri. Dot-blot analysis was performed on total RNA isolated from H2O2- and PQ-exposed cultures, using labelled internal DNA fragments of the sod and kat genes. It was shown that H2O2 but not PQ strongly induced up-regulation of the kat gene. PQ and to a lesser degree H2O2 induced the expression of superoxide dismutase. The results indicate the regulation of the adaptive response of M. barkeri to different oxidative stresses.

Formaldehyde activating enzyme (Fae) and hexulose-6-phosphate synthase (Hps) in Methanosarcina barkeri: a possible function in ribose-5-phosphate biosynthesis.

Formaldehyde activating enzyme (Fae) was first discovered in methylotrophic bacteria, where it is involved in the oxidation of methanol to CO2 and in formaldehyde detoxification. The 18 kDa protein catalyzes the condensation of formaldehyde with tetrahydromethanopterin (H4MPT) to methylene-H4MPT. We describe here that Fae is also present and functional in the methanogenic archaeon Methanosarcina barkeri. The faeA homologue in the genome of M. barkeri was heterologously expressed in Escherichia coli and the overproduced purified protein shown to actively catalyze the condensation reaction: apparent Vmax = 13 U/mg protein (1 U = micromol/min); apparent Km for H4MPT = 30 microM; apparent Km for formaldehyde = 0.1 mM. By Western blot analysis the concentration of Fae in cell extracts of M. barkeri was determined to be in the order of 0.1% of the soluble cell proteins. Besides the faeA gene the genome of M. barkeri harbors a second gene, faeB-hpsB, which is shown to code for a 42 kDa protein with both Fae activity (3.6 U/mg) and hexulose-6-phosphate synthase (Hps) activity (4.4 U/mg). The results support the recent proposal that in methanogenic archaea Fae and Hps could have a function in ribose phosphate synthesis.

Loss of the mtr operon in Methanosarcina blocks growth on methanol, but not methanogenesis, and reveals an unknown methanogenic pathway.

In the methanogenic archaeon Methanosarcina barkeri Fusaro, the N5-methyl-tetrahydrosarcinapterin (CH3-H4SPT):coenzyme M (CoM) methyltransferase, encoded by the mtr operon, catalyzes the energy-conserving (sodium-pumping) methyl transfer from CH3-H4SPT to CoM during growth on H2/CO2 or acetate. However, in the disproportionation of C-1 compounds, such as methanol, to methane and carbon dioxide, it catalyzes the reverse, endergonic transfer from methyl-CoM to H4SPT, which is driven by sodium uptake. It has been proposed that a bypass for this energy-consuming reaction may occur via a direct methyl transfer from methanol to H4SPT. To test this, an mtr deletion mutant was constructed and characterized in M. barkeri Fusaro. The mutant is unable to grow on methanol, acetate or H2/CO2, but can grow on methanol with H2/CO2 and, surprisingly, methanol with acetate. 13C labeling experiments show that growth on acetate with methanol involves a previously unknown methanogenic pathway, in which oxidation of acetate to a mixture of CO2 and formic acid is coupled to methanol reduction. Interestingly, although the mutant is unable to grow on methanol alone, it remains capable of producing methane from this substrate. Thus, the proposed Mtr bypass does exist, but is unable to support growth of the organism.