Characterization of a new mesophilic,secondary alcohol-utilizing methanogen,Methanobacterium palustre spec. nov. from a peat bog

The isolation and characterization of a new methanogen from a peat bog, Methanobacterium palustre spec. nov., strain F, is described. Strain F grew on H₂/CO₂ and formate in complex medium. It also grew autotrophically on H₂/CO₂. Furthermore, growth on 2-propanol/CO₂ was observed. Methane was formed from CO₂ by oxidation of 2-propanol to acetone or 2-butanol to 2-butanone, but growth on 2-butanol plus CO₂ apparently was too little to be measurable. Similarly, Methanobacterium bryantii M. o. H. and M. o. H. G formed acetone and 2-butanone from 2-propanol and 2-butanol, but no growth was measurable.On the basis of morphological and biochemical features strain F could be excluded from the genus Methanobrevibacter. Due to its cell morphology, lipid composition and polyamine pattern it belonged to the genus Methanobacterium. From known members of this genus strain F could be distinguished either by a different G+C content of the DNA, low DNA-DNA homology with reference strains, lacking serological reactions with anti-S probes and differences in the substrate spectrum.An alcohol dehydrogenase activity, specific for secondary alcohols and its substrate specificity was determined in crude extracts of strain F. NADP⁺ was the only electron carrier that was utilized. No reaction was found with NAD⁺, F₄₂₀, FMN and FAD.Abbreviations NAD⁺ nicotinamide adenine dinucleotide - NADH₂ reduced form of NAD⁺ - NADP⁺ nicotinamide adenine dinucleotide phosphate - NADPH₂ reduced form of NADP⁺ - FMN flavin adenine mononucleotide - FAD flavin adenine dinucleotide - ADH alcohol dehydrogenase - F₄₂₀ 8-hydroxy-7,8-didemethyl-5-deazaflavin - SSC standard saline citrate (0.15 M NaCl, 0.015 M trisodium citrate, pH 7.5)     

Growth of Methanogenic Bacteria in Pure Culture with 2-Propanol and Other Alcohols as Hydrogen Donors

Two types of mesophilic, methanogenic bacteria were isolated in pure culture from anaerobic freshwater and marine mud with 2-propanol as the hydrogen donor. The freshwater strain (SK) was a Methanospirillum species, the marine, salt-requiring strain (CV), which had irregular coccoid cells, resembled Methanogenium sp. Stoichiometric measurements revealed formation of 1 mol of CH₄ by CO₂ reduction, with 4 mol of 2-propanol being converted to acetone. In addition to 2-propanol, the isolates used 2-butanol, H₂, or formate but not methanol or polyols. Acetate did not serve as an energy substrate but was necessary as a carbon source. Strain CV also oxidized ethanol or 1-propanol to acetate or propionate, respectively; growth on the latter alcohols was slower, but final cell densities were about threefold higher than on 2-propanol. Both strains grew well in defined, bicarbonate-buffered, sulfide-reduced media. For cultivation of strain CV, additions of biotin, vitamin B₁₂, and tungstate were necessary. The newly isolated strains are the first methanogens that were shown to grow in pure culture with alcohols other than methanol. Bioenergetic aspects of secondary and primary alcohol utilization by methanogens are discussed.     

Classification of secondary alcohol-utilizing methanogens including a new thermophilic isolate

A thermophilic coccoid methanogenic bacterium, strain TCI, that grew optimally around 55° C was isolated with 2-propanol as hydrogen donor for methanogenesis from CO₂. H₂, formate or 2-butanol were used in addition. Each secondary alcohol was oxidized to its ketone. Growth occurred in defined freshwater as well as salt (2% NaCl, w/v) medium. Acetate was required as carbon source, and 4-aminobenzoate and biotin as growth factors. A need for molybdate or alternatively tungstate was shown.Strain TCI was further characterized together with two formerly isolated mesophilic secondary alcohol-utilizing methanogens, the coccoid strain CV and the spirilloid strain SK. The guanine plus cytosine content of the DNA of the three strains was 55,47, and 39 mol%, respectively. Determination of the molecular weights of the methylreductase subunits and sequencing of ribosomal 16S RNA of strains TCI and CV revealed close relationships to the genus Methanogenium. The new isolate TCI is classified as a strain of the existing species, Methanogenium thermophilum (thermophilicum). For strain CV, that uses ethanol or 1-propanol in addition, a classification as new species, Methanogenium organophilum, is proposed. Strain SK is affiliated with the existing species, Methanospirillum hungatei. The ability to use secondary alcohols was also tested with described species of methanogens. Growth with secondary alcohols was observed with Methanogenium marisnigri, Methanospirillum hungatei strain GP1 and Methanobacterium bryantii, but not with Methanospirillum strains JF1 and M1h, Methanosarcina barkeri, Methanococcus species or thermophilic strains or species other than the new isolate TCI.     

Anaerobic Degradation of Propionate by a Mesophilic Acetogenic Bacterium in Coculture and Triculture with Different Methanogens

A mesophilic acetogenic bacterium (MPOB) oxidized propionate to acetate and CO₂ in cocultures with the formate- and hydrogen-utilizing methanogens Methanospirillum hungatei and Methanobacterium formicicum. Propionate oxidation did not occur in cocultures with two Methanobrevibacter strains, which grew only with hydrogen. Tricultures consisting of MPOB, one of the Methanobrevibacter strains, and organisms which are able to convert formate into H₂ plus CO₂ (Desulfovibrio strain G11 or the homoacetogenic bacterium EE121) also degraded propionate. The MPOB, in the absence of methanogens, was able to couple propionate conversion to fumarate reduction. This propionate conversion was inhibited by hydrogen and by formate. Formate and hydrogen blocked the energetically unfavorable succinate oxidation to fumarate involved in propionate catabolism. Low formate and hydrogen concentrations are required for the syntrophic degradation of propionate by MPOB. In triculture with Methanospirillum hungatei and the aceticlastic Methanothrix soehngenii, propionate was degraded faster than in biculture with Methanospirillum hungatei, indicating that low acetate concentrations are favorable for propionate oxidation as well.     

Methane Production from Formate by Syntrophic Association of Methanobacterium bryantii and Desulfovibrio vulgaris JJ

Coculture of a sulfate-reducing bacterium, when grown in the absence of added sulfate, with Methanobacterium bryantii, which uses only H₂ and CO₂ for methanogenesis, degraded formate to CH₄. A pure culture of Desulfovibrio vulgaris JJ was able to produce small amounts of H₂. Such a syntrophic relationship might provide an additional way to avoid formate accumulation in anaerobic environments.     

Interspecific hydrogen transfer during methanol degradation by Sporomusa acidovorans and hydrogenophilic anaerobes

In the presence of active hydrogenophilic sulfate-reducing bacteria, the homoacetogenic bacterium Sporomusa acidovorans did not produce acetate during methanol degradation. H₂S and presumably CO₂ were the only end products. Since the sulfate-reducer did not degrade methnol or acetate, the sulfidogenesis from methanol was related to a complete interspecific hydrogen transfer between both species.In coculture with hydrogenophilic methanogenic bacteria (Methanobacterium formicicum, Methanospirillum hungatei), the interspecific hydrogen transfer with S. acidovorans was incomplete. Beside CH₄ and presumably CO₂, acetate was produced. The results suggested that H₂-production and H₂-consumption were involved during anaerobic methanol degradation by S. acidovorans and the hydrogenophilic anaerobes play an important role during methanol degradation by homoacetogenic bacteria in anoxic environments.     

Transient Production of Formate During Chemolithotrophic Growth of Anaerobic Microorganisms on Hydrogen

The homoacetogenic bacteria Acetobacterium woodii, A. carbinolicum, Sporomusa ovata, and Eubacterium limosum, the methanogenic archaeon Methanobacterium formicicum, and the sulfate-reducing bacterium Desulfotomaculum orientis all produced formate as an intermediate when they were growing chemolithoautotrophically with H₂ and CO₂ as sources of energy, electrons, and carbon. The sulfate-reducing bacterium Desulfovibrio vulgaris grew chemolithoheterotrophically with H₂ and CO₂ using acetate as carbon source, but also produced formate when growth was limited by sulfate. All these bacteria were also able to grow on formate as energy source. Formate accumulated transiently while H₂ was consumed. The maximum formate concentrations measured in cultures of A. woodii and A. carbinolicum were proportional to the initial H₂ partial pressure, giving a ratio of about 0.5 mM formate per 10 kPa H₂. The methanogen Methanobacterium bryantii, on the other hand, was unable to grow on formate and did not produce formate during chemolithoautotrophic growth on H₂. The results indicate that the ability to utilize formate, that is, to possess a formate dehydrogenase, was the precondition for the production of formate during chemolithotrophic growth on H₂. Received: 24 November 1998 / Accepted: 30 December 1998     

Growth of Methanobacterium thermoautotrophicum on H2CO2: High CH4 productivities in continuous culture

The thermophilic methanogenic bacterium, Methanobacterium thermoautotrophicum, was grown on H₂CO₂. In continuous culture, high CH₄ productivities were obtained (288 litres litre⁻¹ day⁻¹) with 96% CH₄ in the effluent gas, i.e. the productivity was twice as high as that obtained previously by other authors, with pure or mixed cultures; the biomass was 3·6 g dry wt litre⁻¹.     

Alcohol conversion by Desulfobulbus propionicus Lindhorst in the presence and absence of sulfate and hydrogen

Ethanol was rapidly degraded to mainly acetate in anaerobic freshwater sediment slurries. Propionate was produced in small amounts. Desulfovibrio species were the dominant bacteria among the ethanol-degrading organisms. The propionate-producing Desulfobulbus propionicus came to the fore under iron-limited conditions in an ethanol-limited chemostat with excess sulfate inoculated with anaerobic intertidal freshwater sediment. In the absence of sulfate, ethanol was fermented by D. propionicus Lindhorst to propionate and acetate in a molar ratio of 2.0.l-Propanol was intermediately produced during the fermentation of ethanol. In the presence of H₂ and CO₂, ethanol was quantitatively converted to propionate. H₂-plus sulfate-grown cells of D. propionicus Lindhorst were able to oxidize l-propanol and l-butanol to propionate and butyrate respectively with the concomitant reduction of acetate plus CO₂ to propionate. Growth was also observed on acetate alone in the presence of H₂ and CO₂ D. propionicus was able to grow mixotrophically on H₂ plus an organic compound. Finally, a brief discussion has been given of the ecological niche of D. propionicus in anaerobic freshwater sediments.     

Gas Exchange in the Filamentous Cyanobacterium Nostoc punctiforme Strain ATCC 29133 and Its Hydrogenase-Deficient Mutant Strain NHM5

Nostoc punctiforme ATCC 29133 is a nitrogen-fixing, heterocystous cyanobacterium of symbiotic origin. During nitrogen fixation, it produces molecular hydrogen (H₂), which is recaptured by an uptake hydrogenase. Gas exchange in cultures of N. punctiforme ATCC 29133 and its hydrogenase-free mutant strain NHM5 was studied. Exchange of O₂, CO₂, N₂, and H₂ was followed simultaneously with a mass spectrometer in cultures grown under nitrogen-fixing conditions. Isotopic tracing was used to separate evolution and uptake of CO₂ and O₂. The amount of H₂ produced per molecule of N₂ fixed was found to vary with light conditions, high light giving a greater increase in H₂ production than N₂ fixation. The ratio under low light and high light was approximately 1.4 and 6.1 molecules of H₂ produced per molecule of N₂ fixed, respectively. Incubation under high light for a longer time, until the culture was depleted of CO₂, caused a decrease in the nitrogen fixation rate. At the same time, hydrogen production in the hydrogenase-deficient strain was increased from an initial rate of approximately 6 μmol (mg of chlorophyll a)⁻¹ h⁻¹ to 9 μmol (mg of chlorophyll a)⁻¹ h⁻¹ after about 50 min. A light-stimulated hydrogen-deuterium exchange activity stemming from the nitrogenase was observed in the two strains. The present findings are important for understanding this nitrogenase-based system, aiming at photobiological hydrogen production, as we have identified the conditions under which the energy flow through the nitrogenase can be directed towards hydrogen production rather than nitrogen fixation.     

Isolation and Characterization of a Thermophilic Bacterium Which Oxidizes Acetate in Syntrophic Association with a Methanogen and Which Grows Acetogenically on H2-CO2

We previously described a thermophilic (60°C), syntrophic, two-membered culture which converted acetate to methane via a two-step mechanism in which acetate was oxidized to H₂ and CO₂. While the hydrogenotrophic methanogen Methanobacterium sp. strain THF in the biculture was readily isolated, we were unable to find a substrate that was suitable for isolation of the acetate-oxidizing member of the biculture. In this study, we found that the biculture grew on ethylene glycol, and an acetate-oxidizing, rod-shaped bacterium (AOR) was isolated from the biculture by dilution into medium containing ethylene glycol as the growth substrate. When the axenic culture of the AOR was recombined with a pure culture of Methanobacterium sp. strain THF, the reconstituted biculture grew on acetate and converted it to CH₄. The AOR used ethylene glycol, 1,2-propanediol, formate, pyruvate, glycine-betaine, and H₂-CO₂ as growth substrates. Acetate was the major fermentation product detected from these substrates, except for 1,2-propanediol, which was converted to 1-propanol and propionate. N,N-Dimethylglycine was also formed from glycine-betaine. Acetate was formed in stoichiometric amounts during growth on H₂-CO₂, demonstrating that the AOR is an acetogen. This reaction, which was carried out by the pure culture of the AOR in the presence of high partial pressures of H₂, was the reverse of the acetate oxidation reaction carried out by the AOR when hydrogen partial pressures were kept low by coculturing it with Methanobacterium sp. strain THF. The DNA base composition of the AOR was 47 mol% guanine plus cytosine, and no cytochromes were detected.     

Hydrogenase mediated nitrite reduction in chlorella

The assay of the hydrogenase of glucose-grown cells of Chlorella pyrenoidosa, strain 7-11-05 by means of nitrite reduction with molecular hydrogen is described. The hydrogenase of Chlorella shows maximum activity immediately after equilibration in the hydrogen atmosphere. The hydrogenase mediated reduction of nitrite to ammonia requires the presence of CO₂. However, at pH 6.4. when the reaction proceeds optimally, there is apparently sufficient retention of metabolic CO₂ to support the reaction, which goes to completion, at near maximum rates.

Reduction of nitrite in the hydrogenase system when CO₂ is present results in the uptake of 3 moles of H₂ per mole of nitrite and ammonia is the product. When CO₂ is absent or limiting, ammonia is also formed from nitrite but with the uptake of less than the stoichiometric amount of H₂. It is concluded that CO₂ is essential for the uptake of H₂, and that in the absence of CO₂ internal hydrogen donors support nitrite reduction.

The possibility that CO₂ exerts a catalytic effect in all reductions mediated by hydrogenase in algae is considered, and a further hypothesis, that hydrogenase arises from that portion of the photosynthetic machinery which also shows a catalytic requirement for CO₂, is proposed.

     

Selection of sulfur source for methane production by Methanobacterium thermoautotrophicum

Sulfur sources capable of replacing sulfide were surveyed for biomethanation from H₂ and CO₂ by thermoautotrophic methanogen, Methanobacterium thermoautotrophicum. Among sulfur containing compounds tested, l-cysteine, thiosulfate and coenzyme M gave poor growth when added as sulfur sources, whereas simultaneous addition of two sulfur sources, l-cysteine+thiosulfate, l-cysteine+l-methionine or l-cysteine+coenzyme M stimulated the growth.In a pressure-controlled fermentor system developed to obtain stoichiometry between input and output gases, the ratio of H₂ and CO₂ consumption to CH₄ production was almost stoichiometric, and when l-cysteine and thiosulfate or l-methionine were used in place of sulfide (control) similar growth patterns were observed. In a culture with continuous supply of substrates gases (1.3 vvm) and sulfur sources of 1 mM l-cysteine+2 mM thiosulfate, specific growth rate and specific methane production rate were 0.35 h⁻ and 3.24 l g⁻¹h⁻¹, respectively, compared to 0.22 h⁻¹ and 5.76 l g⁻h⁻¹ with Na₂ S.     

Photochemical disproportionation of sulfur into sulfide and sulfate byChlorobium limicola formathiosulfatophilum

The anaerobic bacteriumChlorobium assimilates carbon dioxide in the light with various sulfur compounds as electron donors. The well-known metabolic pathway proceeds from the oxidation of sulfide via sulfur to sulfate. In the dark the reaction is partially reversed when sulfur is reduced to hydrogen sulfide. The fermenting cells thereby release an excess of reductant. We have now found a hydrogen sulfide production from sulfur, which is light-dependent. It is more than ten times faster than the dark reaction. This appears in experiments where the cell suspension is illuminated in absence of CO₂ and flushed continuously with H₂ or Ar. The H₂S is trapped with ZnCl₂ and the S^2- titrated with iodine. The total amount of H₂S evolved in the light increases proportionally with the amount of sulfur added, and about one-half of the added sulfur is converted to H₂S. Another part of the metabolized sulfur appears at the same time as sulfate, but all the sulfur oxidized to sulfate does not account for the larger amount of sulfur reduced to hydrogen sulfide. Very likely other unanalyzed oxidized sulfur compounds must also have been produced. Use of H₂ instead of Ar as the anaerobic gas phase does not increase the amount of H₂S produced, nor does the addition of thiosulfate; sulfur itself is the preferred electron donor for the sulfur reduction. Up to a light intensity of 10000 ergs cm^-2sec^-1 CO₂ does not affect H₂S production. Without CO₂, saturation of the light-dependent evolution of H₂S is reached at about 40000 ergs cm^-2sec^-1. In contrast, presence of CO₂ at this light intensity makes the sulfide production disappear completely. On application of mass spectrometry to the gas exchange upon illumination, at high light intensity a H₂S gush is found during the first 3 min. This is followed by CO₂ fixation, while simultaneously the reductant H₂S is now taken up. WithRhodospirillum rubrum, the addition of sulfur leads to a moderate evolution of H₂S. In contrast toChlorobium this reaction inR. rubrum is not light-sensitive, nor does it produce detectable amounts of sulfate. After addition of malate the rate of H₂S evolution does increase in the light, since the cells use malate as an electron donor during their photochemical metabolism.     

Carbon monoxide-dependent evolution of hydrogen by the homoacetate-fermenting bacteriumClostridium thermoaceticum

Clostridium thermoaceticum was found to form H₂ when cultivated heterotrophically on dextrose under a carbon monoxide (CO) gas phase. In contrast, when cultivated under CO₂, only minimal levels of hydrogen were detected. Resting cells from the CO-grown cultures also formed H₂ when incubated under CO with dextrose, while a comparative study with resting cells from CO₂-grown cultures demonstrated that the CO₂-grown cells were not competent in H₂ formation when incubated under CO. When dextrose was deleted, CO-cultivated resting cells did not form H₂ when incubated under CO.     

Extracts of interferon-treated cells can inhibit reticulocyte lysate protein synthesis.

Rumen bacteria retained methanogenic activity when stored at ?60° under H₂. This activity, which resides in $\text{Methanobacterium ruminantium}$ and $\text{Methanobacterium mobilis}$, is not lost when the cells are broken, as has been suggested. Unlike in $\text{Methanosarcina barkerii}$ and $\text{Methanobacterium M.o.H.}$, in rumen bacteria methanogenic enzymes are not soluble but readily precipitated at 15,000 g. Methane was synthesized from tetrahydrofolate derivatives but at slower rates than from CO₂. From the data, it was not possible to determine if methyl- and methylene tetrahydrofolate were oxidized to CO₂ prior to reduction to CH₄. In room light, CH₃-B₁₂ was reduced to CH₄ non-enzymatically in the presence of protein. When the reactions were carried out in the dark, very little CH₄ was formed from CH₃-B₁₂ by rumen bacterial enzymes. The cell-free particulate fraction did not require added ATP for methanogenesis but showed an absolute requirement for H₂.     

Formation of D-1-amino-2-propanol from L-threonine by enzymes from Escherichia coli K-12

Escherichia coli K-12 cells contain two dehydrogenases which in sequence catalyze the net conversion of L-threonine to the D-isomer of 1-amino-2-propanol. These two enzymes are L-threonine dehydrogenase (L-threonine + NAD⁺ → aminoacetone + CO₂ + NADH + H⁺) and D-1-amino-2-propanol dehydrogenase (aminoacetone + NADH + H⁺D-1-amino-2-propanol + NAD⁺). Each enzyme has been obtained in purified form free of the other; the nature of the reaction catalyzed by the latter dehydrogenase alone and in a coupled system with the former enzyme has been studied. The results provide an explanation on the enzymological level for the utilization of L-threonine by cell suspensions of certain microorganisms for the biosynthesis of the D-1-amino-2-propanol moiety of Vitamin B₁₂.     

Role of Carbon Dioxide in Catabolism of Propane by “Nocardia paraffinicum” (Rhodococcus rhodochrous)

The catabolism of propane by “Nocardia paraffinicum” (Rhodococcus rhodochrous) has been shown to involve CO₂ fixation after its oxidation to propionic acid. “N. paraffinicum” failed to grow on either propane or 1-propanol in the absence of CO₂. The rate of propane utilization was directly related to the initial CO₂ concentration, and Warburg respirometry suggested that CO₂ was required for the catabolism of 1-propanol, propionaldehyde, and propionate but not for 2-propanol. These data also suggested that the predominant pathway for the utilization of propane by “N. paraffinicum” was through 1-propanol. The use of [2-¹⁴C]propane and ¹⁴CO₂ confirmed the catabolism of propane and the fixation of CO₂. Through the use of these isotopes and the pyruvate carboxylase inhibitor sodium arsenite, the labeled 2,4-dinitrophenylhydrazine derivative of pyruvate was trapped and isolated via thin-layer chromatography. The trapping of [¹⁴C]pyruvate in this manner was considered to be indicative of the presence of the methylmalonyl coenzyme A pathway for CO₂ fixation.     

Purine and pyrimidine biosynthesis in methanogenic bacteria

Following long-term labeling with [1-¹³C]acetate, [2-¹³C]acetate, ¹³CO₂, H¹³COOH, or ¹³CH₃OH, NMR spectroscopy was used to determine the labeling patterns of the purified ribonucleosides of Methanospirillum hungatei, Methanococcus voltae, Methanobrevibacter smithii, Methanosphaera stadtmanae, Methanosarcina barkeri and Methanobacterium bryantii. Major differences were observed among the methanogens studied, specifically at carbon positions 2 and 8 of the purines, positions at which one-carbon carriers are involved during synthesis. In Methanospirillum hungatei and Methanosarcina barkeri, the labcl at both positions came from carbon atom C-2 of acetate, as predicted from known eubacterial pathways, whereas in Methanococcus voltae and Methanobacterium bryantii both originated from CO₂. In Methanosphaera stadtmanae grown in the presence of formate, the C-2 of purines originated exclusively from formate and the C-8 was labeled by the C-2 of acetate. When grown in media devoid of formate, the C-2 of the purine ring originated mainly from the C-2 of acetate and in part from CH₃OH. In Methanobrevibacter smithii grown in the presence of formate, C-2 and C-8 of purines were derived from CO₂ and/or formate. The labeling patterns obtained for pyrimidines are consistent with the biosynthetic pathways common to eubacteria and eucaryotes.Abbreviations CODH Carbon monoxide dehydrogenase - FH₄ tetrahydrofolate - H₄MPT tetrahydromethanopterin Issued as NRCC Publication No. 37383     

Kinetics of Formate Metabolism in Methanobacterium formicicum and Methanospirillum hungatei

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