Methanol biosynthesis by covalently immobilized cells of Methylosinus trichosporium: batch and continuous studies

The DEAE-cellulose linked cells of Methylosinus trichosporium displaying high specific methane mono-oxygenase activity (66 mumol methane oxidized/h mg cells) were used for methanol biosynthesis from biogas derived methane in a batch and a continuous cell reactor. The optimum cell-to-carrier ratio was determined to be 0.5 g cells/g dry weight cellulose. Batch experiments indicated that 100 mM phosphate ion concentration was necessary to inhibit further oxidation of methanol; excess oxygen supply favored methanol accumulation with an increase in methane conversion efficiency to 27%. A pulse of 40 mM sodium formate at the end of 6 h resulted in restoration of methanol accumulation by regenerating NADH(2) required for the sustained activity of methane mono-oxygenase. Maximum methanol level of 50 mumol/mg cells was obtained in the batch reactor. In a standard 50-mL ultrafiltration continuous reactor, the covalently linked cells produced methanol at a continuous rate of 100 mumol/h for the first 10 h, after which the methanol accumulation rate fell low due to the depletion of NADH(2). The methanol accumulation could be stimulated by supplying sodium formate (40 mM) in either 20 or 100 mM phosphate buffer. Maximum methanol accumulation rate of 267 mumol/h was obtained when 20 mM formate was supplied in the feed stream containing 100 mM phosphate ions, and this level of biosynthesis was maintained for over 72 h. The stoichiometric balance made at various points of formate addition indicated that the molar amount of methanol generated at steady state is dependent on the equimolar addition of sodium formate to the feed. The half-life t(1/2) and thermal denaturation rate constant K(d) were computed to be 108 h and 6.42 x 10(-3) h(-1), respectively.     

Oxidation of C1 Compounds by Particulate fractions from Methylococcus capsulatus: distribution and properties of methane-dependent reduced nicotinamide adenine dinucleotide oxidase (methane hydroxylase).

Cell-free particulate fractions of extracts from the obligate methylotroph Methylococcus capsulatus catalyze the reduced nicotinamide adenine dinucleotide (NADH) and O2-dependent oxidation of methane (methane hydroxylase). The only oxidation product detected was formate. These preparations also catalyze the oxidation of methanol and formaldehyde to formate in the presence or absence of phenazine methosulphate with oxygen as the terminal electron acceptor. Methane hydroxylase activity cannot be reproducibly obtained from disintegrated cell suspensions even though the whole cells actively respired when methane was presented as a substrate. Varying the disintegration method or extraction medium had no significant effect on the activities obtained. When active particles were obtained, hydroxylase activity was stable at 0 C for days. Methane hydroxylase assays were made by measuring the methane-dependent oxidation of NADH by O2. In separate experiments, methane consumption and the accumulation of formate were also demonstrated. Formate is not oxidized by these particulate fractions. The effects of particle concentration, temperature, pH, and phosphate concentration on enzymic activity are described. Ethane is utilized in the presence of NADH and O2. The stoichiometric relationships of the reaction(s) with methane as substrate were not established since (i) the presumed initial product, methanol, is also oxidized to formate, and (ii) the contribution that NADH oxidase activity makes to the observed consumption of reactants could not be assessed in the presence of methane. Studies with known inhibitors of electron transport systems indicate that the path of electron flow from NADH to oxygen is different for the NADH oxidase, methane hydroxylase, and methanol oxidase activities.     

Mechanistic Analysis of Ammonium Inhibition of Atmospheric Methane Consumption in Forest Soils

Methane consumption by forest soil was studied in situ and in vitro with respect to responses to nitrogen additions at atmospheric and elevated methane concentrations. Methane concentrations in intact soil decreased continuously from atmospheric levels at the surface to 0.5 ppm at a depth of 14 cm. The consumption rate of atmospheric methane in soils, however, was highest in the 4- to 8-cm depth interval (2.9 nmol per g of dry soil per day), with much lower activities below and above this zone. In contrast, extractable ammonium and nitrate concentrations were highest in the surface layer (0 to 2 cm; 22 and 1.6 μmol per g of dry soil, respectively), as was potential ammonium-oxidizing activity (19 nmol per g of dry soil per day). The difference in zonation between ammonium oxidation and methane consumption suggested that ammonia-oxidizing bacteria did not contribute significantly to atmospheric methane consumption. Exogenous ammonium inhibited methane consumption in situ and in vitro, but the pattern of inhibition did not conform to expectations based on simple competition between ammonia and methane for methane monooxygenase. The extent of ammonium inhibition increased with increasing methane concentration. Inhibition by a single ammonium addition remained constant over a period of 39 days. In addition, nitrite, the end product of methanotrophic ammonia oxidation, was a more effective inhibitor of methane consumption than ammonium. Factors that stimulated ammonium oxidation in soil, e.g., elevated methane concentrations and the availability of cosubstrates such as formate, methanol, or β-hydroxybutyrate, enhanced ammonium inhibition of methane oxidation, probably as a result of enhanced nitrite production.     

水稻土中CH4氧化的研究

在实验室条件下研究了水稻土中CH₄氧化的特性.结果表明,在早稻种植前采集的水稻土不能氧化大气中的CH₄,但当所供给的CH₄浓度>10μl·L^-1时,能迅速氧化CH₄,所供给的CH₄浓度越高,氧化CH₄的速度越大.经高浓度(>1000μl·L^-1)的CH₄预培养10d,可使本来不具有氧化大气CH₄能力的土壤氧化大气CH_4.大田CH₄排放通量高的水稻土,氧化CH₄的能力较大.     

Reduction of technetium by Desulfovibrio desulfuricans: biocatalyst characterization and use in a flowthrough bioreactor.

Resting cells of Desulfovibrio desulfuricans coupled the oxidation of a range of electron donors to Tc(VII) reduction. The reduced technetium was precipitated as an insoluble low-valence oxide. The optimum electron donor for the biotransformation was hydrogen, although rapid rates of reduction were also supported when formate or pyruvate was supplied to the cells. Technetium reduction was less efficient when the growth substrates lactate and ethanol were supplied as electron donors, while glycerol, succinate, acetate, and methanol supported negligible reduction. Enzyme activity was stable for several weeks and was insensitive to oxygen. Transmission electron microscopy showed that the radionuclide was precipitated at the periphery of the cell. Cells poisoned with Cu(II), which is selective for periplasmic but not cytoplasmic hydrogenases, were unable to reduce Tc(VII), a result consistent with the involvement of a periplasmic hydrogenase in Tc(VII) reduction. Resting cells, immobilized in a flowthrough membrane bioreactor and supplied with Tc(VII)-supplemented solution, accumulated substantial quantities of the radionuclide when formate was supplied as the electron donor, indicating the potential of this organism as a biocatalyst to treat Tc-contaminated wastewaters.     

Reduction of Technetium by Desulfovibrio desulfuricans: Biocatalyst Characterization and Use in a Flowthrough Bioreactor

Resting cells of Desulfovibrio desulfuricans coupled the oxidation of a range of electron donors to Tc(VII) reduction. The reduced technetium was precipitated as an insoluble low-valence oxide. The optimum electron donor for the biotransformation was hydrogen, although rapid rates of reduction were also supported when formate or pyruvate was supplied to the cells. Technetium reduction was less efficient when the growth substrates lactate and ethanol were supplied as electron donors, while glycerol, succinate, acetate, and methanol supported negligible reduction. Enzyme activity was stable for several weeks and was insensitive to oxygen. Transmission electron microscopy showed that the radionuclide was precipitated at the periphery of the cell. Cells poisoned with Cu(II), which is selective for periplasmic but not cytoplasmic hydrogenases, were unable to reduce Tc(VII), a result consistent with the involvement of a periplasmic hydrogenase in Tc(VII) reduction. Resting cells, immobilized in a flowthrough membrane bioreactor and supplied with Tc(VII)-supplemented solution, accumulated substantial quantities of the radionuclide when formate was supplied as the electron donor, indicating the potential of this organism as a biocatalyst to treat Tc-contaminated wastewaters.     

Production of methanol from methane by methanotrophic bacteria

Methanotrophs can oxidize methane to carbon dioxide through sequential reactions catalyzed by a series of enzymes including methane monooxygenase, methanol dehydrogenase, formaldehyde dehydrogenase, and formate dehydrogenase. When suspensions of methanotrophic bacteria of Methylosinus trichosporium IMV 3011 were incubated at 32°C with methane and oxygen, there was an extracellular accumulation of methanol from methane oxidation in response to carbon dioxide addition. Maximal accumulation of methanol was achieved with 40% carbon dioxide in the mixed reaction gases. A continuous experiment was performed in a continuous ultrafiltration reactor. The optimum gas mixture containing 20% (v v^?1) methane, 20% oxygen, 20% nitrogen and 40% carbon dioxide was used to provide substrates and to maintain the transmembrane pressure. The product (methanol) was removed in the eluate buffer. The initial methanol concentration in the eluate buffer was 8.22 μmol L^?1. The bioreactor was operated continuously for 198 h without obvious loss of productivity.     

Production of methanol from methane by methanotrophic bacteria

Methanotrophs can oxidize methane to carbon dioxide through sequential reactions catalyzed by a series of enzymes including methane monooxygenase, methanol dehydrogenase, formaldehyde dehydrogenase, and formate dehydrogenase. When suspensions of methanotrophic bacteria of Methylosinus trichosporium IMV 3011 were incubated at 32°C with methane and oxygen, there was an extracellular accumulation of methanol from methane oxidation in response to carbon dioxide addition. Maximal accumulation of methanol was achieved with 40% carbon dioxide in the mixed reaction gases. A continuous experiment was performed in a continuous ultrafiltration reactor. The optimum gas mixture containing 20% (v v^-1) methane, 20% oxygen, 20% nitrogen and 40% carbon dioxide was used to provide substrates and to maintain the transmembrane pressure. The product (methanol) was removed in the eluate buffer. The initial methanol concentration in the eluate buffer was 8.22 μmol L^-1. The bioreactor was operated continuously for 198 h without obvious loss of productivity.     

Physiological Studies of Methane and Methanol-Oxidizing Bacteria: Oxidation of C-1 Compounds by Methylococcus capsulatus

Methylococcus capsulatus grows only on methane or methanol as its sole source of carbon and energy. Some amino acids serve as nitrogen sources and are converted to keto acids which accumulate in the culture medium. Cell suspensions oxidize methane, methanol, formaldehyde, and formate to carbon dioxide. Other primary alcohols are oxidized only to the corresponding aldehydes. Oxidation of formate by cell suspensions is more sensitive to inhibition by cyanide than is the oxidation of other one carbon compounds. This is due to the cyanide sensitivity of a soluble nicotinamide adenine dinucleotide-specific formate dehydrogenase. Oxidation of formaldehyde and methanol is catalyzed by a nonspecific primary alcohol dehydrogenase which is activated by ammonium ions and is independent of pyridine nucleotides. Some comparisons are made with a strain of Pseudomonas methanica.     

Methanol Promotes Atmospheric Methane Oxidation by Methanotrophic Cultures and Soils

Two methanotrophic bacteria, Methylobacter albus BG8 and Methylosinus trichosporium OB3b, oxidized atmospheric methane during batch growth on methanol. Methane consumption was rapidly and substantially diminished (95% over 9 days) when washed cell suspensions were incubated without methanol in the presence of atmospheric methane (1.7 ppm). Methanotrophic activity was stimulated after methanol (10 mM) but not methane (1,000 ppm) addition. M. albus BG8 grown in continuous culture for 80 days with methanol retained the ability to oxidize atmospheric methane and oxidized methane in a chemostat air supply. Methane oxidation during growth on methanol was not affected by methane deprivation. Differences in the kinetics of methane uptake (apparent K_m and V_max) were observed between batch- and chemostat-grown cultures. The V_max and apparent K_m values (means ± standard errors) for methanol-limited chemostat cultures were 133 ± 46 nmol of methane 10⁸ cells⁻¹ h⁻¹ and 916 ± 235 ppm of methane (1.2 μM), respectively. These values were significantly lower than those determined with batch-grown cultures (V_max of 648 ± 195 nmol of methane 10⁸ cells⁻¹ h⁻¹ and apparent K_m of 5,025 ± 1,234 ppm of methane [6.3 μM]). Methane consumption by soils was stimulated by the addition of methanol. These results suggest that methanol or other nonmethane substrates may promote atmospheric methane oxidation in situ.     

Nitrous oxide production and methane oxidation by different ammonia-oxidizing bacteria.

Ammonia-oxidizing bacteria (AOB) are thought to contribute significantly to N2O production and methane oxidation in soils. Most of our knowledge derives from experiments with Nitrosomonas europaea, which appears to be of minor importance in most soils compared to Nitrosospira spp. We have conducted a comparative study of levels of aerobic N2O production in six phylogenetically different Nitrosospira strains newly isolated from soils and in two N. europaea and Nitrosospira multiformis type strains. The fraction of oxidized ammonium released as N2O during aerobic growth was remarkably constant (0.07 to 0.1%) for all the Nitrosospira strains, irrespective of the substrate supply (urea versus ammonium), the pH, or substrate limitation. N. europaea and Nitrosospira multiformis released similar fractions of N2O when they were supplied with ample amounts of substrates, but the fractions rose sharply (to 1 to 5%) when they were restricted by a low pH or substrate limitation. Phosphate buffer (versus HEPES) doubled the N2O release for all types of AOB. No detectable oxidation of atmospheric methane was detected. Calculations based on detection limits as well as data in the literature on CH4 oxidation by AOB bacteria prove that none of the tested strains contribute significantly to the oxidation of atmospheric CH4 in soils.     

Anaerobic catabolism of formate to acetate and CO2 by Butyribacterium methylotrophicum.

The catabolism of sodium formate to acetate and carbon dioxide by the anaerobic acetogen Butyribacterium methylotrophicum was analyzed by fermentation time course and 13C nuclear magnetic resonance studies. Significant hydrogen production and consumption fluxes were observed during formate catabolism but not during the catabolism of formate plus CO. In the latter case, formate and CO were simultaneously consumed and label distribution studies with mixtures of 13C-labeled CO and formate demonstrated their preferential incorporation into the acetate carboxyl and methyl groups, respectively. Hydrogen consumption was inhibited by CO when both were present, whereas hydrogen and formate were simultaneously consumed when CO2 was supplied. Carbon dioxide was required for the conversion of CO to acetate, but a similar need was not observed when methanol plus CO or formate plus CO was present. These analyses indicate a bifurcated single-carbon catabolic pathway in which CO2 is the sole single-carbon compound that directly supplies the carbonyl and methyl group synthesis pathways leading to the formation of acetyl coenzyme A, the primary reduced product. We discuss causes for the reported inability of B. methylotrophicum to use formate as a sole substrate.     

Optimization of methanol biosynthesis from methane using Methylosinus trichosporium OB3b

Methylosinus trichosporium OB3b oxidized methane to methanol in the presence of a high concentration of Cu2+. Further oxidation of methanol to formaldehyde was prevented by adding 200 mM NaCl which acted as a methanol dehydrogenase H inhibitor. The bacterium, 0.6 mg dry cell ml(-1), in methane/air (1:4, v/v) at 25 degrees C in 12.9 mM phosphate buffer (pH 7) containing 20 mM sodium formate and 200 mM NaCl accumulated 7.7 mM methanol over 36 h.     

Inhibition of trichloroethylene oxidation by the transformation intermediate carbon monoxide

Inhibition of trichloroethylene (TCE) oxidation by the transformation intermediate carbon monoxide (CO) was evaluated with the aquifer methanotroph Methylomonas sp. strain MM2. CO was a TCE transformation intermediate. During TCE oxidation, approximately 9 mol% of the TCE was transformed to CO. CO was oxidized by Methylomonas sp. strain MM2, and when formate was provided as an electron donor, the CO oxidation rate doubled. The rate of CO oxidation without formate was 4.6 liter mg (dry weight)-1 day-1, and the rate with formate was 10.2 liter mg (dry weight)-1 day-1. CO inhibited TCE oxidation, both by exerting a demand for reductant and through competitive inhibition. The Ki for CO inhibition of TCE oxidation, 4.2 microM, was much less than the Ki for methane inhibition of TCE oxidation, 116 microM. CO also inhibited methane oxidation, and the degree of inhibition increased with increasing CO concentration. When CO was present, formate amendment was necessary for methane oxidation to occur and both substrates were simultaneously oxidized. CO at a concentration greater than that used in the inhibition studies was not toxic to Methylomonas sp. strain MM2.     

Inhibition of trichloroethylene oxidation by the transformation intermediate carbon monoxide.

Inhibition of trichloroethylene (TCE) oxidation by the transformation intermediate carbon monoxide (CO) was evaluated with the aquifer methanotroph Methylomonas sp. strain MM2. CO was a TCE transformation intermediate. During TCE oxidation, approximately 9 mol% of the TCE was transformed to CO. CO was oxidized by Methylomonas sp. strain MM2, and when formate was provided as an electron donor, the CO oxidation rate doubled. The rate of CO oxidation without formate was 4.6 liter mg (dry weight)-1 day-1, and the rate with formate was 10.2 liter mg (dry weight)-1 day-1. CO inhibited TCE oxidation, both by exerting a demand for reductant and through competitive inhibition. The Ki for CO inhibition of TCE oxidation, 4.2 microM, was much less than the Ki for methane inhibition of TCE oxidation, 116 microM. CO also inhibited methane oxidation, and the degree of inhibition increased with increasing CO concentration. When CO was present, formate amendment was necessary for methane oxidation to occur and both substrates were simultaneously oxidized. CO at a concentration greater than that used in the inhibition studies was not toxic to Methylomonas sp. strain MM2.     

Microbial Oxidation of Methane and Methanol: Isolation of Methane-Utilizing Bacteria and Characterization of a Facultative Methane-Utilizing Isolate

A methane-utilizing organism capable of growth both on methane and on more complex organic substrates as a sole source of carbon and energy, has been isolated and studied in detail. Suspensions of methane-grown cells of this organism oxidized C-1 compounds (methane, methanol, formaldehyde, formate); hydrocarbons (ethane, propane); primary alcohols (ethanol, propanol); primary aldehydes (acetaldehyde, propionaldehyde); alkenes (ethylene, propylene); dimethylether; and organic acids (acetate, malate, succinate, isocitrate). Suspensions of methanol-or succinate-grown cells did not oxidize methane, ethane, propane, ethylene, propylene, or dimethylether, suggesting that the enzymatic systems required for oxidation of these substrates are induced only during growth on methane. Extracts of methane-grown cells contained a particulate reduced nicotinamide adenine dinucleotide-dependent methane monooxygenase activity. Oxidation of methanol, formaldehyde, and primary alcohols was catalyzed by a phenazine methosulfate-linked, ammonium ion-requiring methanol dehydrogenase. Oxidation of primary aldehydes was catalyzed by a phenazine methosulfate-linked, ammonium ion-independent aldehyde dehydrogenase. Formate was oxidized by a nicotinamide adenine dinucleotide-specific formate dehydrogenase. Extracts of methane-grown, but not succinate-grown, cells contained the key enzymes of the serine pathway, hydroxypyruvate reductase and malate lyase, indicating that the enzymes of C-1 assimilation are induced only during growth on C-1 compounds. Glucose-6-phosphate dehydrogenase was induced during growth on glucose. Extracts of methane-grown cells contained low levels of enzymes of the tricarboxylic acid cycle, including alpha-keto glutarate dehydrogenase, relative to the levels found during growth on succinate.     

Enzymology of one-carbon metabolism in methanogenic pathways

Methanoarchaea, the largest and most phylogenetically diverse group in the Archaea domain, have evolved energy-yielding pathways marked by one-carbon biochemistry featuring novel cofactors and enzymes. All of the pathways have in common the two-electron reduction of methyl-coenzyme M to methane catalyzed by methyl-coenzyme M reductase but deviate in the source of the methyl group transferred to coenzyme M. Most of the methane produced in nature derives from acetate in a pathway where the activated substrate is cleaved by CO dehydrogenase/acetyl-CoA synthase and the methyl group is transferred to coenzyme M via methyltetrahydromethanopterin or methyltetrahydrosarcinapterin. Electrons for reductive demethylation of the methyl-coenzyme M originate from oxidation of the carbonyl group of acetate to carbon dioxide by the synthase. In the other major pathway, formate or H2 is oxidized to provide electrons for reduction of carbon dioxide to the methyl level and reduction of methyl-coenzyme to methane. Methane is also produced from the methyl groups of methanol and methylamines. In these pathways specialized methyltransferases transfer the methyl groups to coenzyme M. Electrons for reduction of the methyl-coenzyme M are supplied by oxidation of the methyl groups to carbon dioxide by a reversal of the carbon dioxide reduction pathway. Recent progress on the enzymology of one-carbon reactions in these pathways has raised the level of understanding with regard to the physiology and molecular biology of methanogenesis. These advances have also provided a foundation for future studies on the structure/function of these novel enzymes and exploitation of the recently completed sequences for the genomes from the methanoarchaea Methanobacterium thermoautotrophicum and Methanococcus jannaschii.     

Dichloromethane and trichloroethylene inhibition of methane oxidation by the membrane-associated methane monooxygenase of Methylosinus trichosporium OB3b

Whole-cell assays were used to measure the effect of dichloromethane and trichloroethylene on methane oxidation by Methylosinus trichosporium OB3b synthesizing the membrane-associated or particulate methane monooxygenase (pMMO). For M. trichosporium OB3b grown with 20 μM copper, no inhibition of methane oxidation was observed in the presence of either dichloromethane or trichloroethylene. If 20 mM formate was added to the reaction vials, however, methane oxidation rates increased and inhibition of methane oxidation was observed in the presence of dichloromethane and trichloroethylene. In the presence of formate, dichloromethane acted as a competitive inhibitor, while trichloroethylene acted as a noncompetitive inhibitor. The finding of noncompetitive inhibition by trichloroethylene was further examined by measuring the inhibition constants K _iE and K _iES. These constants suggest that trichloroethylene competes with methane at some sites, although it can bind to others if methane is already bound. Whole-cell oxygen uptake experiments for active and acetylene-treated cells also showed that provision of formate could stimulate both methane and trichloroethylene oxidation and that trichloroethylene did not affect formate dehydrogenase activity. The finding that different chlorinated hydrocarbons caused different inhibition patterns can be explained by either multiple substrate binding sites existing in pMMO or multiple forms of pMMO with different activities. The whole-cell analysis performed here cannot distinguish between these models, and further work should be done on obtaining active preparations of the purified pMMO. Received: 3 November 1998 / Accepted: 1 March 1999     

The effect of oxygen on methanol oxidation by an obligate methanotrophic bacterium studied by in vivo 13C nuclear magnetic resonance spectroscopy

¹³C NMR was used to study the effect of oxygen on methanol oxidation by a type II methanotrophic bacterium isolated from a bioreactor in which methane was used as electron donor for denitrification. Under high (35–25%) oxygen conditions the first step of methanol oxidation to formaldehyde was much faster than the following conversions to formate and carbon dioxide. Due to this the accumulation of formaldehyde led to a poisoning of the cells. A more balanced conversion of ¹³C-labelled methanol to carbon dioxide was observed at low (1–5%) oxygen concentrations. In this case, formaldehyde was slowly converted to formate and carbon dioxide. Formaldehyde did not accumulate to inhibitory levels. The oxygen-dependent formation of formaldehyde and formate from methanol is discussed kinetically and thermodynamically. Journal of Industrial Microbiology & Biotechnology (2001) 26, 9–14. Received 04 March 2000/ Accepted in revised form 07 November 2000     

Isolation of Methanobrevibacter smithii from human feces.

Fecal specimens from nine adults were examined for the presence of methanogenic bacteria. Enrichment cultures of five specimens produced methane in 5 days. Of these five specimens, three were tested and produced methane during a short-term incubation. Four specimens did not produce methane in either short-term incubation or in enrichment culture. Each methanogenic culture contained methanogens similar in morphology to organisms of the genus Methanobrevibacter and showed factor-420 fluorescence by fluorescence microscopy. Pure cultures were obtained from four of the five methanogenic enrichment cultures. Each isolate grew and formed methane from either H2-CO2 or formate, but growth obtained with formate was poor. None of the isolates used acetate, methanol, or trimethylamine. All isolates grew in the presence of bile salts. In immunological studies, each isolate was closely related to the type strain of Methanobrevibacter smithii, a finding consistent with the physiological and morphological similarities between the isolates and the type strain.