N 5, N10-Methylenetetrahydromethanopterin reductase from Methanosarcina barkeri

N ⁵ N ¹⁰-Methylenetetrahydromethanopterin reductase was purified 13-fold to apparent homogeneity from methanol grown Methanosarcina barkeri . The colourless enzyme was found to be composed of four identical subunits of apparent molecular mass 36 kDa. It catalysed the reduction of methylenetetrahydromethanopterin ( K _m=15 μM) to methyltetrahydromethanopterin with reduced coenzyme F₄₂₀ ( K _m=12 μM) at a specific rate ( V _max) of 2200 μmol min⁻¹· mg protein⁻¹ ( K _cat=1320 s⁻¹). With respect to coenzyme specificity, molecular properties and catalytic mechanism the enzyme was found to be similar to CH₂=H₄MPT reductase of Methanobacterium thermoautotrophicum which phylogenetically is only distantly related to M. barkeri .     

Methanogenesis from methanol in Methanosarcina barkeri studied using membrane inlet mass spectrometry

Abstract A mass spectrometer with membrane inlet was used to study methanol metabolism by Methanosarcina barkeri strain MS. The addition of methanol to methanol grown culture samples in the mass spectrometer vessel stimulated methanogenesis and hydrogen production. The apparent K _s for methanol was determined as 0.5 mM and the V _max as 8.14 mmol g (dry weight) h⁻¹. The V _max for methane production was fairly constant during growth of the culture on methanol implying that growth is tightly coupled to methanogenesis. The addition of methanol to culture samples in the mass spectrometer vessel stimulated methanogenesis with no lag which indicated that methanogenesis can be uncoupled from growth. Exposure of the culture sample in the mass spectrometer vessel to an atmosphere of 2 kPa oxygen for 80 min resulted in a decrease in the rate of methanogenesis from methanol but on returning the atmosphere to nitrogen the addition of further methanol stimulated methanogenesis. The effect of other inhibitors of methanogenesis (2-bromoethane sulphonate and monensin); K _j values 21.5 μM and 0.3 mM, respectively) were also studied.     

H2 and CO2 production from methanol or formaldehyde by the methanogenic bacterium strain Gö1 treated with 2-bromoethanesulfonic acid

Abstract In cell suspensions of the methanogenic bacterium strain Gö1 or Methanosarcina barkeri H₂ formation from methanol in the presence of 2-bromoethanesulfonic acid (BES) was strictly dependent on sodium ions; apparent K _S for Na⁺, 1.3±0.3 mM.H₂ formation was inhibited by the uncoupler tetrachlorosalicylanilide (TCS), but this inhibition could be temporarily overcome, when a sodium pulse (100 mM) was given to the cell suspension. On the other hand, H₂ formation from formaldehyde in the presence of BES (rate: 300 nmol H₂/h·mg protein as compared to 25 nmol H₂/h·mg protein from methanol) was not sodium-dependent, not TCS-sensitive and not inhibited by addition of monensin. H₂ formation was accompanied by CO₂ formation in stoichiometric amounts, 3 H₂:1 CO₂ for methanol and 2 H₂:1 CO₂ for formaldehyde oxidation.     

The effect of detergent on methanogenesis by Methanosarcina barkeri

Abstract both growth and methanogenesis of Methanosarcina barkeri are completely inhibited by sodium dodecylbenzene sulphonate at between 15 and 20 mg·1⁻¹. At lower concentrations growth of cultures was delayed, but no uncoupling of methanogenesis from growth was observed. Higher concentrations of detergent (50 mg·1⁻¹) produced marked alterations in the surface structures of organisms observed in scanning electron micrographs. Thus levels of a detergent common in anaerobic sewage treatment plants can inhibit methanogenesis, the terminal stage in the anaerobic digestion process.     

Effect of trimethylamine on acetate utilization by Methanosarcina barkeri

During growth of Methanosarcina barkeri strain Fusaro on a mixture of trimethylamine and acetate, methane production and acetate consumption were biphasic. In the first phase trimethylamine (33 mmol x l^-1) was depleted and some acetate (11–14 from 50 mmol x l^-1) was metabolized simultaneously. In the second phase the remaining acetate was cleaved stoichiometrically into CH₄ and CO₂. Kinetic experiments with (2-¹⁴C)acetate revealed that only 2.5% of the methane produced in the first phase originated from acetate: 18% of the acetate metabolized was cleaved into CH₄ and CO₂, 23% of the acetate was oxidized, and 55% was assimilated. Methane produced from CD₃–COOH in the first phase consisted of CD₂H₂ and CD₃H in a ratio of 1:1.     

Acetate catabolism by Methanosarcina barkeri: Hydrogen-dependent methane production from acetate by a soluble cell protein fraction

Abstract Cell extracts prepared from Methanosarcina barkeri converted acetate into methane and carbon dioxide under a hydrogen atmosphere. Methanogenesis by cell extracts required acetate and ATP and, the in vitro rate was 5 to 10% of the rate of methanogenesis observed during exponential growth of cells on acetate. Methane and carbon dioxide produced by cell extracts originated predominantly from the methyl and carboxyl groups of acetate, respectively, in a manner consistent with that observed in whole cells. Acetate degradation activity was detected in the soluble (150000 × g supernatant) fraction and not in the membrane fraction. These results are discussed in relation to a proposed model for ATP generation from acetate that involves both membrane-bound and soluble enzymatic components such as CO dehydrogenase.     

Methyltetrahydromethanopterin as an intermediate in methanogenesis from acetate in Methanosarcina barkeri

Cell extracts (100,000×g) of acetate grown Methanosarcina barkeri (strain MS) catalyzed CH₄ and CO₂ formation from acetyl-CoA with specific activities of 50 nmol·min^-1·mg protein^-1. CH₄ formation was found to be dependent on tetrahydromethanopterin (H₄MPT) (apparent K _M=4 μM), coenzyme M (H-S-CoM), and 7-mercaptoheptanoylthreonine phosphate (H-S-HTP=component B) rather than on methanofuran (MFR) and coenzyme F₄₂₀ (F₄₂₀). Methyl-H₄MPT was identified as an intermediate. This compound accumulated when H-S-CoM and H-S-HTP were omitted from the assays. These and previous results indicate that methanogenesis from acetate proceeds via acetyl phosphate, acetyl-CoA, methyl-H₄MPT, and CH₃-S-CoM as intermediates. The disproportionation of formaldehyde to CO₂ and CH₄ was also studied. This reaction was shown to be dependent on H₄MPT, MFR, F₄₂₀, H-S-CoM, and H-S-HTP.     

Sodium dependent acetate formation from CO2 in Peptostreptococcus productus (strain Marburg)

Abstract Washed cells of Peptostreptococcus productus (strain Marburg), which were incubated in the presence of CO/CO₂/N₂ (50%/ 17%/ 33%; 200 kPa) catalyzed the synthesis of acetate from carbon monoxide. The rate of acetate formation from CO was stimulated more than threefold by the addition of sodium (10 mM); potassium did not effect acetate synthesis. The degree of stimulation was dependent on the sodium concentration; the dependence followed simple Michaelis-Menten kinetics. The apparent K _m for sodium was determined to be about 2 mmol/1. Sodium also stimulated acetate synthesis from H₂ plus CO₂. In the absence of added sodium the formation of formate as an intermediate in methyl group synthesis was stimulated. It is suggested that the sodium dependent reaction(s) is one (or more) of the reactions involved in methyl group synthesis from CO₂.     

A role for mitochondrial carbonic anhydrase in limiting CO2 leakage from low CO2-grown cells of Chlamydomonas reinhardtii

A model is presented which quantifies a possible role for the carbonic anhydrase in the mitochondrial matrix of Chlamydomonas reinhardtii which incorporates the observation that the expression of this enzyme is increased under growth conditions in which the expression of the carbon dioxide-concentrating mechanism is increased. It is assumed that the inorganic carbon enters the cytosol from the medium, and leaves the cytosol to the plastids, as HCO₃⁻ and that there is negligible carbonic anhydrase activity in the cytosol. The role of the mitochondrial carbonic anhydrase is suggested to be the conversion to HCO₃^– of the CO₂ produced in the mitochondria in the light from tricarboxylic acid cycle activity and from decarboxylation of glycine in any photorespiratory carbon oxidation cycle activity which is not suppressed by the carbon concentrating mechanism. If there is a HCO₃⁻ channel in the inner mitochondrial membrane then almost all of the inorganic carbon leaves the mitochondria as HCO₃⁻, thus limiting the potential for CO₂ leakage through the plasmalemma. This mechanism could increase inorganic C supply to ribulose bisphosphate carboxylase-oxygenase by some 10% at the energetic expense of less than 1% of the total ATP generation by plastids plus mitochondria.     

In vitro methanol production from methyl coenzyme M using the Methanosarcina barkeri MtaABC protein complex

Methanol:coenzyme M methyltransferase is an enzyme complex composed of three subunits, MtaA, MtaB, and MtaC, found in methanogenic archaea and is needed for their growth on methanol ultimately producing methane. MtaABC catalyzes the energetically favorable methyl transfer from methanol to coenzyme M to form methyl coenzyme M. Here we demonstrate that this important reaction for possible production of methanol from the anaerobic oxidation of methane can be reversed in vitro. To this effect, we have expressed and purified the Methanosarcina barkeri MtaABC enzyme, and developed an in vitro functional assay that demonstrates MtaABC can catalyze the energetically unfavorable (ΔG° = 27 kJ/mol) reverse reaction starting from methyl coenzyme M and generating methanol as a product. Demonstration of an in vitro ability of MtaABC to produce methanol may ultimately enable the anaerobic oxidation of methane to produce methanol and from methanol alternative fuel or fuel‐precursor molecules. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1243–1249, 2017     

Oceanic sinks for atmospheric CO2

There is approximately 50 times more inorganic carbon in the global ocean than in the atmosphere. On time scales of decades to millions of years, the interaction between these two geophysical fluids determines atmospheric CO₂ levels. During glacial periods, for example, the ocean serves as the major sink for atmospheric CO₂, while during glacial–interglacial transitions, it is a source of CO₂ to the atmosphere. The mechanisms responsible for determining the sign of the net exchange of CO₂ between the ocean and the atmosphere remain unresolved. There is evidence that during glacial periods, phytoplankton primary productivity increased, leading to an enhanced sedimentation of particulate organic carbon into the ocean interior. The stimulation of primary production in glacial episodes can be correlated with increased inputs of nutrients limiting productivity, especially aeolian iron. Iron directly enhances primary production in high nutrient (nitrate and phosphate) regions of the ocean, of which the Southern Ocean is the most important. This trace element can also enhance nitrogen fixation, and thereby indirectly stimulate primary production throughout the low nutrient regions of the central ocean basins. While the export flux of organic carbon to the ocean interior was enhanced during glacial periods, this process does not fully account for the sequestration of atmospheric CO₂. Heterotrophic oxidation of the newly formed organic carbon, forming weak acids, would have hydrolyzed CaCO₃ in the sediments, increasing thereby oceanic alkalinity which, in turn, would have promoted the drawdown of atmospheric CO₂. This latter mechanism is consistent with the stable carbon isotope pattern derived from air trapped in ice cores. The oceans have also played a major role as a sink for up to 30% of the anthropogenic CO₂ produced during the industrial revolution. In large part this is due to CO₂ solution in the surface ocean; however, some, poorly quantified fraction is a result of increased new production due to anthropogenic inputs of combined N, P and Fe. Based on ‘circulation as usual’, models predict that future anthropogenic CO₂ inputs to the atmosphere will, in part, continue to be sequestered in the ocean. Human intervention (large-scale Fe fertilization; direct CO₂ burial in the deep ocean) could increase carbon sequestration in the oceans, but could also result in unpredicted environmental perturbations. Changes in the oceanic thermohaline circulation as a result of global climate change would greatly alter the predictions of C sequestration that are possible on a ‘circulation as usual’ basis.     

Effects of raised CO2 on potential CH4 production and oxidation in, and CH4 emission from, a boreal mire

1 In a glasshouse experiment we studied the effect of raised CO₂ concentration (720 p.p.m.) on CH₄ emission at natural boreal peat temperatures using intact cores of boreal peat with living vascular plants and Sphagnum mosses. After the end of the growing season half of the cores were kept unnaturally warm (17–20 °C). The potential for CH₄ production and oxidation was measured at the end of the emission experiment.
2 The vascular cores ('Sedge') consisted of a moss layer with sedges, and the moss cores (' Sphagnum ') of Sphagnum mosses (some sedge seedlings were removed by cutting). Methane efflux was 6–12 times higher from the Sedge cores than from the Sphagnum cores. The release of CH ₄ from Sedge cores increased with increasing temperature of the peat and decreased with decreasing temperature. Methane efflux from Sphagnum cores was quite stable independent of the peat temperatures.
3 In both Sedge and Sphagnum samples, CO₂ treatment doubled the potential CH₄ production but had no effect on the potential CH₄ oxidation. A raised concentration of CO₂ increased CH₄ efflux weakly and only at the highest peat temperatures (17–20 °C).
4 The results suggest that in cool regions, such as boreal wetlands, temperature would restrict decomposition of the extra substrates probably derived from enhanced primary production of mire vegetation under raised CO₂ concentrations, and would thus retard any consequent increase in CH₄ emission.     

Uptake of CO2 by aquatic vegetation

Abstract Photosynthesis by aquatic plants based on the supply of CO₂ from air-equilibrated solutions may be limited by the low diffusion coefficient of CO₂ in water. For plants in which the transport of CO₂ from the bulk medium is by diffusion, and the initial carboxylation uses RUBISCO, CO₂ supply can be increased by growth in habitats with fast water flow over the surface (reducing unstirred layer thickness), or with heterotrophically-augmented CO₂ levels, including the direct use of sediment CO₂. Many aquatic plants using RUBISCO as their initial carboxylase counter the limitations on CO₂ supply via the operation of biophysical CO₂ concentrating mechanisms which are based on active transport of HCO^?₃, CO₂ or H⁺ at the plasmalemma, and use bulk-phase HCO^?₃ or CO₂ as the C source. A final group of aquatic plants use biochemical CO₂ concentrating mechanisms based on auxiliary carboxylation by PEPc: C₄-like and Crassulacean Acid Metabolism–like processes are involved. These various mechanisms for increasing CO₂ supply to RUBISCO also help to offset the low specific reaction rate of aquatic plant RUBISCOs at low [CO₂] and low [CO₂]: [CO₂]. In addition to overcoming restrictions on CO₂ supply, the various methods of increasing inorganic C availability may also be important in alleviating shortages of nitrogen or photons.     

Sensing of atmospheric CO2 by plants

Abstract. Despite recent interest in the effects of high CO₂ on plant growth and physiology, very little is known about the mechanisms by which plants sense changes in the concentration of this gas. Because atmospheric CO₂ concentration is relatively constant and because the conductance of the cuticle to CO₂ is low, sensory mechanisms are likely to exist only for intercellular CO₂ concentration. Therefore, responses of plants to changes in atmospheric CO₂ will depend on the effect of these changes on intercellular CO₂ concentration. Although a variety of plant responses to atmospheric CO₂ concentration have been reported, most of these can be attributed to the effects of intercellular CO₂ on photosynthesis or stomatal conductance. Short-term and long-term effects of CO₂ on photosynthesis and stomatal conductance are discussed as sensory mechanisms for responses of plants to atmospheric CO₂. Available data suggest that plants do not fully realize the potential increases in productivity associated with increased atmospheric CO₂. This may be because of genetic and environmental limitations to productivity or because plant responses to CO₂ have evolved to cope with variations in intercellular CO₂ caused by factors other than changes in atmospheric CO₂.     

CO2 and intracellular pH

Abstract The experimental determination of cytoplasmic and vacuolar pH values is discussed. Despite variation in these values evidence indicates that intracellular pH values are normally regulated within narrow limits. The regulatory mechanisms proposed involve the metabolic consumption of OH^& and the active efflux of H ⁺. The evidence for intracellular pH modification in response to CO₂ hydration and the production of HCO^?₃ and H⁺ is examined. Theoretical calculations and experimental data indicate that CO₂ concentrations as high as 5% will lower intracellular pH. Conversely, variation in CO₂ levels around atmospheric concentrations is unlikely to perturb intracellular pH. High CO₂ levels are found in bulky tissues, and flooded root systems. Evidence is presented that the slow diffusion of dissolved CO₂ compared to gaseous CO₂ results in its accumulation. It is proposed that the accumulation of respiratory CO₂ may reduce intracellular pH values when plant tissues, cells or protoplasts are maintained in a liquid culture medium. Finally, the possible role of dark CO₂ fixation and organic acid synthesis in the regulation of intracellular pH is examined.