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The biochemical diversity of life reaches an extreme in a group of highly specialized archaebacteria known as the methanogens where novel coenzymes are involved in the biochemistry of methanogenesis.
We describe a K+ transport system in Methanospirillum hungatei cells depleted of cytoplasmic K+ via an ammonia/K+ exchange reaction (Sprott, G. D., Shaw, K. M., and Jarrell, K. F. (1984) J. Biol. Chem. 259, 12602-12608). Ammonia-treated cells contained low concentrations of ATP and were unable to make CH4 or to transport 86Rb+. All of these properties were restored by CaCl2, MgCl2, or MnCl2, and not by CoCl2 or NiCl2. The Rb+ transport system had a Km of 0.42 and Vmax of 29 nmol/min X mg; K+ inhibited competitively. Both H2 and CO2 were required for appreciable transport, whereas air, valinomycin, or nigericin were potent inhibitors. The influx of Rb+ was electrogenic and associated with proton efflux, producing a delta pH (alkaline inside) in acidic media. In the absence of K+ (or Rb+), the activation of CH4 synthesis by Mg2+ produced little change in the cytoplasmic pH, showing that methanogenesis did not elicit a net efflux of protons. The pH optimum for transport was in the range 6.0-7.3 where the transmembrane pH gradient would contribute minimally to the proton motive force. Protonophores at pH 6.3 caused a partial decline in CH4 synthesis and the ATP content and dramatically collapsed Rb+ transport. These and other inhibitor experiments, coupled with the fact that the Rb+ gradient was too large to be in equilibrium with the proton motive force alone, suggest a role for both ATP and the proton motive force in Rb+ transport. Also, a role for K+ in osmoregulation is indicated.
Evidence obtained by 13C NMR spectroscopy indicates that tetrahydromethanopterin (H4MPT) serves as a carbon carrier for C1 units at the methine, methylene, and methyl levels of oxidation. All three derivatives of H4MPT served as substrates for methanogenesis by cell extracts under a hydrogen atmosphere; in each instance, methane evolved at a rate comparable to that obtained when 2-(methylthio)ethanesulfonic acid was used as the substrate. Each C1 derivative of H4MPT stimulated the reduction of CO2 as efficiently as 2-(methylthio)ethanesulfonic acid. High resolution fast atom bombardment mass spectrometry indicated that the product of the spontaneous reaction of formaldehyde with H4MPT was methylene-H4MPT, with the molecular formula C31H45N6O16P. 13C NMR spectroscopy of hexamethylenetetramine, a model compound, suggested that the methylene group in methylene-H4MPT was bound to two nitrogen atoms. Molecular formulas of C31H44N6O16P and C31H47N6O16P were assigned to methenyl-H4MPT+, and methyl-H4MPT, by high resolution fast atom bombardment mass spectrometry. 1H NMR spectroscopy of methyl-H4MPT indicated that the methyl group was bound to a nitrogen atom. Sensitivity of each derivative to oxygen was noted. Apparent extinction coefficients of H4MPT and its derivatives were recorded. Evidence for the enzymatic synthesis of methylene-H4MPT from methenyl-H4MPT+ is presented.
The basic aspects of organic matter breakdown through methanogenesis are dealt with. The process is referred to as a method of obtaining energy-- biogas --and a way to solve a number of problems associated with the protection of the environment from waste products of the agricultural industry. Both traditional and modern technical means and basic parameters of the technology of biogas production are presented. Stated are also the important practical aspects of the biochemistry and kinetics of the process.
The presence of significant numbers of intestinal methanogens among vertebrates does neither depend on elaborated morphological structures nor on predominantly plant-based diets. Phylogenetic rather than dietary restrictions limit the occurrence of methanogenic bacteria also in the hindguts of primates. The Old-World monkeys are methanogenic--with the only exceptions of Cheirogaleid lemurs and bush-babies. In contrast, among New World monkeys the lack of intestinal methanogens is observed frequently in capuchins and marmosets. Since the absence of methanogens does not parallel distinct morphological, physiological, or ethological characteristics, it is likely that methanogenesis depends on a hereditary predisposition. In humans, methane-producers account for approximately 50% of the European populations. In this study, 56 individuals belonging to 5 families were studied for the occurrence of methane in the breath. The results of this screen are compatible with the assumption that the trait "methane emission" segregates as an autosomal, dominant character. Our findings suggest a high specificity of the symbiosis between primates and methanogenic bacteria. Therefore, the persistence of significant numbers of methanogens in the hindgut might be facilitated by a specific receptor for methanogenic bacteria.
The formation of a sulfuranyl radical intermediate followed by methyl transfer to the nickel(I) center of coenzyme F430 and generation of the disulfide has been proposed as a possible mechanism for the formation of methane catalyzed by methyl coenzyme M reductase in methanogenic archaea. In order to test this hypothesis, a sterically shielded, bifunctional model substrate that contained a methyl thioether and a sulfhydryl functional group, which could form a five-membered cyclic sulfuranyl radical according to the postulated mechanism, was synthesized. The corresponding thiolate reacted with Ni(II) salts to give a diamagnetic, square-planar Ni(II) dithiolate complex, which was characterized by X-ray diffraction. Upon irradiation of this complex with light of lambda > 300 nm, methane and the cyclic disulfide were formed, whereas irradiation of the thiolate in the absence of nickel gave only traces of methane and no cyclic disulfide. The observed products are consistent with the postulated mechanism via a sulfuranyl radical, and the role of light is interpreted as the formation of a Ni(I)/thiyl radical pair upon excitation of a charge-transfer band of the Ni(II) dithiolate. In the presence of a large excess of thiolate, the diamagnetic complex was transformed into a paramagnetic, five- or six-coordinate complex that proved to be more active in the generation of both methane and the cyclic disulfide, than the square-planar diamagnetic dithiolate.
Coenzyme F430 is the prosthetic group of methyl-coenzyme-M reductase, which catalyses the final step of methane formation in methanogenic bacteria. The coenzyme is a nickel-containing macrocyclic tetrapyrrole of unique structure. We describe the biosynthesis of this nickel porphinoid from L-glutamate via 5-aminolaevulinic acid, uroporphyrinogen III and dihydrosirohydrochlorin, the binding of the coenzyme to methyl-coenzyme-M reductase and the regulation of coenzyme F430 biosynthesis. We end with some evolutionary considerations on the biosynthesis of macrocyclic tetrapyrroles and remarks on the degradation of these compounds under anaerobic conditions.
To answer the intriguing question whether or not endothermic microbial growth exists, and in particular, to verify Heijnen and van Dijken's prediction (1992), acetotrophic methanogen, Methanosarcina barkeri, has been cultivated in a highly sensitive bench-scale calorimeter (an improved Bio-RC1 reaction calorimeter) in a pH auxostat fashion. A growth yield of 0.043 C-mol C-mol(-1) has been obtained and a cell density as high as 3 g L(-1) was attained. Heat uptake during growth has indeed been quantitatively measured with calorimetry, resulting in a heat yield of +145 kJ C-mol(-1). Thermodynamics of the growth of acetotrophic methanogens was analyzed in detail. The changes in Gibbs energy, enthalpy, and entropy during growth of M. barkeri were compared with some typical aerobic and anaerobic growth processes of different microorganisms on various substrates. In the growth of M. barkeri on acetate, the retarding effect of the positive enthalpy change on the driving force of growth is overcompensated by the large positive entropy change, resulting from converting one organic molecule (acetic acid) to two gaseous products, CH(4) and CO(2). Both the enthalpy and the entropy increases are due partially to the transition of these two products into the gaseous phase. The thermodynamic role of this phase transition for the growth process is analyzed. Microbial growth characterized by enthalpy increase and correspondingly by a large increase in entropy may be called enthalpy-retarded growth.
Batch anaerobic codigestion of municipal household solid waste (MHSW) and digested manure in mesophilic conditions was carried out. The different waste-to-biomass ratios and intensity of mixing were studied theoretically and experimentally. The experiments showed that when organic loading was high, intensive mixing resulted in acidification and failure of the process, while low mixing intensity was crucial for successful digestion. However, when loading was low, mixing intensity had no significant effect on the process. We hypothesized that mixing was preventing establishment of methanogenic zones in the reactor space. The methanogenic zones are important to withstand inhibition due to development of acids formed during acidogenesis. The 2D distributed models of symmetrical cylinder reactor are presented based on the hypothesis of the necessity of a minimum size of methanogenic zones that can propagate and establish a good methanogenic environment. The model showed that at high organic loading rate spatial separation of the initial methanogenic centers from active acidogenic areas is the key factor for efficient conversion of solids to methane. The initial level of methanogenic biomass in the initiation centers is a critical factor for the survival of these centers. At low mixing, most of the initiation methanogenic centers survive and expand over the reactor volume. However, at vigorous mixing the initial methanogenic centers are reduced in size, averaged over the reactor volume, and finally dissipate. Using fluorescence in situ hybridization, large irregular cocci of microorganisms were observed in the case with minimal mixing, while in the case with high stirring mainly dead cells were found.
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A methanogenic bacterial consortium was obtained after inoculation of benzoate medium under N2/CO2 atmosphere with intertidal sediment. A hydrogen donating organotroph and Methanococcus mazei were isolated from this enrichment. H2-utilising sulphate reducing bacteria were isolated under H2/CO2 in the absence of organic electron donors. The Methanococcus was able to produce methane in yeast extract medium under N2/CO2 if the H2 donating organism was present, and sulphate reduction occurred if the hydrogen utilising sulphate reducing bacteria were grown with the H2 donating organism. The ability of the H2 utilising sulphate reducing bacteria to inhibit Methanococcus competitively was shown in clutures containing both of these H2 utilising bacteria.
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The objective of this study was to evaluate the effects of bioaugmenting anaerobic biosolids digestion with a commercial product containing selected strains of bacteria from genera Bacillus, Pseudomonas, and Actinomycetes, along with ancillary organic compounds containing various micronutrients. Specifically, the effects of the bioaugment in terms of volatile solids destruction and generation and fate of odor-causing compounds during anaerobic digestion and during storage of the digested biosolids were studied. Two bench-scale anaerobic digesters receiving primary and secondary clarifier biosolids from various full-scale biological wastewater treatment plants were operated. One of the digesters received the bioaugment developed by Organica Biotech, while the other was operated as control. The bioaugmented digester generated 29% more net CH(4) during the 8 weeks of operation. In addition, the average residual propionic acid concentration in the bioaugmented digester was 54% of that in the control. The monitoring of two organic sulfide compounds, methyl mercaptan (CH(3)SH) and dimethyl sulfide (CH(3)SCH(3)), clearly demonstrated the beneficial effects of the bioaugmentation in terms of odor control. The biosolids digested in the bioaugmented digester generated a negligible amount of CH(3)SH during 10 days of post-digestion storage, while CH(3)SH concentration in the control reached nearly 300 ppm(v) during the same period. Similarly, peak CH(3)SCH(3) generated by stored biosolids from the bioaugmented digester was only 37% of that from the control.
The effects of numerous ionophores and inhibitors were tested on methane synthesis, intracellular ATP and potassium concentrations, and the proton motive force of the methanogenic archaebacterium Methanobacterium bryantii. M. bryantii had an internal pH near 6.8 (and hence little delta pH during growth) with an electrical potential of --127 mV in growth medium and --105 mV in a pH 6.5 buffer. The study has identified agents which, in M. bryantii, can effectively cause a decline of intracellular ATP (gramicidin, acetylene) and potassium concentrations (gramicidin, nigericin), inhibit methane synthesis (acetylene, gramicidin, nigericin, triphenylmethylphosphonium bromide), eliminate the electrical potential (high extracellular potassium ion concentrations), and dissipate artificially imposed, inside alkaline, pH gradients (monensin, nigericin, carbonyl cyanide m-chlorophenylhydrazone). Carbonyl cyanide m-chlorophenylhydrazone was generally ineffective in media or buffers reduced with cysteine-sulfide but could be effective in cysteine-free solutions reduced with hydrogen sulfide.
Factors F430 are nickel tetrapyrroles from methanogenic bacteria. Two methods are described to extract these compounds from cells of Methanobacterium thermoautotrophicum, namely, by boiling with 40% ethanol and by treatment of disrupted cells with HClO4 at pH 2 and 0 degrees C. The subsequent purification procedures involving column chromatography are outlined. Ethanol extraction yielded one yellow compound which will be denoted coenzyme MF430 (CoMF430). Extraction with HCIO4 yielded a yellow derivative, called Factor F430II, and a red component (F560). In addition, a number of derivatives were prepared by preparative thin-layer chromatography, acid hydrolysis, and methanolysis of acid hydrolyzates. On the basis of ultraviolet-visible light absorption and mass spectral data, it was concluded that the methylated chromophores obtained by treatment of acid hydrolyzates are derivatives of Ni(II)sirohydrochlorin and its pi-cation radical. Reduction studies and ultraviolet-visible light, 1H-NMR, and mass spectroscopy indicate that the chromophoric derivatives of CoMF430 differ from the native compound with respect to the reduction level of the tetrapyrrole and the structural elements that are attached to the chromophore.