Fermentation of Cellulose to Methane and Carbon Dioxide by a Rumen Anaerobic Fungus in a Triculture with Methanobrevibacter sp. Strain RA1 and Methanosarcina barkeri

The fermentation of cellulose by a rumen anaerobic fungus in the presence of Methanobrevibacter sp. strain RA1 and Methanosarcina barkeri strain 227 resulted in the formation of 2 mol each of methane and carbon dioxide per mol of hexose fermented. Coculture of the fungus with either Methanobrevibacter sp. or M. barkeri produced 0.6 and 1.3 mol of methane per mol of hexose, respectively. Acetate, formate, ethanol, hydrogen, and lactate, which are major end products of cellulose fermentation by the fungus alone, were either absent or present in very low quantities at the end of the triculture fermentation (≤0.08 mol per mol of hexose fermented). During the time course of cellulose fermentation by the triculture, hydrogen was not detected (<1 × 10⁻⁵ atm; <0.001 kPa) and only acetate exhibited transitory accumulation; the maximum was equivalent to 1.4 mol per mol of hexose at 6 days which was higher than the total acetate yield of 0.73 in the fungus monoculture. The effect of methanogens is interpreted as a shift in the flow of electrons away from the formation of electron sink products lactate and ethanol to methane via hydrogen, favoring an increase in acetate, which is in turn converted to methane and carbon dioxide by M. barkeri. The maximum rate of cellulose degradation in the triculture (3 mg/ml per day) was faster than previously reported for bacterial cocultures and within 16 days degradation was complete. The triculture was used successfully also in the production of methane from cellulose in the plant fibrous materials, sisal (fiber from leaves of Agave sisalona L.) and barley straw leaf.     

Methanogenesis from Choline by a Coculture of Desulfovibrio sp. and Methanosarcina barkeri

A sulfate-reducing vibrio was isolated from a methanogenic enrichment with choline as the sole added organic substrate. This organism was identified as a member of the genus Desulfovibrio and was designated Desulfovibrio strain G1. In a defined medium devoid of sulfate, a pure culture of Desulfovibrio strain G1 fermented choline to trimethylamine, acetate, and ethanol. In the presence of sulfate, more acetate and less ethanol were formed from choline than in the absence of sulfate. When grown in a medium containing sulfate, a coculture of Desulfovibrio strain G1 and Methanosarcina barkeri strain Fusaro degraded choline almost completely to methane, ammonia, and hydrogen sulfide and presumably to carbon dioxide. Methanogenesis occurred in two distinct phases separated by a lag of about 6 days. During the first phase of methanogenesis choline was completely converted to trimethylamine, acetate, hydrogen sulfide, and traces of ethanol by the desulfovibrio. M. barkeri fermented trimethylamine to methane, ammonia, and presumably carbon dioxide via dimethyl- and methylamine as intermediates. Simultaneously, about 60% of the acetate expected was metabolized. In the second phase of methanogenesis, the residual acetate was almost completely catabolized.     

Factors Involved in Hydrolysis of Microcrystalline Cellulose by Acetivibrio cellulolyticus

Acetivibrio cellulolyticus cellulase obtained by the water elution of residual cellulose from the growth medium was compared with the cellulase activity present in culture supernatants. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that water elution released most of the protein bands which adhered to undigested cellulose from the culture medium. The enzyme in the culture supernatant and that eluted from residual cellulose had specific activities for Avicel hydrolysis that were 20- to 40-fold greater than that of Trichoderma reesei cellulase. However, Ca²⁺ and a reducing agent such as dithiothreitol were required for maximum Avicel hydrolysis rates by these A. cellulolyticus enzyme preparations. The effect of these agents on p-nitrophenyl lactopyranoside hydrolysis suggested that they were required by an exoglucanase component. Supernatant enzyme preparations contained large amounts of carbohydrate which was separated from most of the cellulase protein by phenyl-Sepharose chromatography. Removal of this carbohydrate, which interfered with protein fractionations, allowed for an activity stain analysis of the supernatant enzyme.     

Bioconversion of Gelatin to Methane by a Coculture of Clostridium collagenovorans and Methanosarcina barkeri

A simple, stable, and transferable coculture of Clostridium collagenovorans and Methanosarcina barkeri that readily degraded gelatin into methane and carbon dioxide was developed. In monoculture, C. collagenovorans fermented all of the amino acids in gelatin except proline into acetate and carbon dioxide as the main products, with hydrogen, isovalerate, and isobutyrate detected in trace amounts (<1 mM). In coculture with M. barkeri, gelatin was transformed into methane and carbon dioxide, with varying levels of intermediary acetate formed as a function of incubation time. Various complex proteinaceous polymers could be readily transformed into methane and carbon dioxide at 30 to 40°C by a stable coculture which did not require exogenous growth factor additions. In addition, the coculture was readily transferable and preserved in the viable state for long periods, and methanogenesis could be initiated rapidly without the need for exogenous pH control.     

Stable Carbon Isotope Fractionation by Methanosarcina barkeri during Methanogenesis from Acetate, Methanol, or Carbon Dioxide-Hydrogen

Methanosarcina barkeri was cultured on methanol, H₂-CO₂, and acetate, and the ¹³C/¹²C ratios of the substrates and the methane produced from them were determined. The discrimination against ¹³C in methane relative to substrate decreased in the order methanol > CO₂ > acetate. The isotopic fractionation for methane derived from acetate was only one-third of that observed with methanol as the substrate. The data presented indicate that the last enzyme of methanogenesis, methylreductase, is not the primary site of isotopic discrimination during methanogenesis from methanol or CO₂. These results also support biogeochemical interpretations that gas produced in environments in which acetate is the primary methane precursor will have higher ¹³C/¹²C ratios than those from environments where other substrates predominate.     

Metabolism of Acetivibrio cellulolyticus during optimized growth on glucose,cellobiose and cellulose

Summary The growth of Acetivibrio cellulolyticus in 2.5 l batch cultures was optimized by controlling the growth pH at 6.7, the dissolved inorganic sulphide concentration at 0.4–0.6 mM, and by constant removal of hydrogen from the cultures by sparging with N₂/CO₂ or N₂ gas. An initial ethanol concentration of 0.15% (w/v) in cellobiose media resulted in specific growth rates which were reduced by about 75% compared to growth rates of 0.17 h^–1 in control cultures. Acetivibrio cellulolyticus had to be adapted for growth on glucose and ¹⁴C-radiotracer studies indicated that glucose was metabolized by the Embden-Meyerhof pathway. The specific growth rate (=0.03h^–1) and molar growth yield (Y_glucose=21.5) were considerably lower than those obtained (=0.17 h^–1, Y_cellobiose=68.9) in cellobiose media. A Y_ATP of 12.8 was obtained during growth on cellobiose. The mol product formed per mol Avicel cellulose fermented (on anhydroglucose equivalent basis) were 3.70 H₂, 2.64 CO₂, 0.73 acetate, 0.39 ethanol and 0.03 total soluble sugars on glucose basis. Maximum cellulase activity was observed in cellulose-grown cultures.National Research council of Canada No. 20826     

Mass-spectrometric studies of the interrelations among hydrogenase, carbon monoxide dehydrogenase, and methane-forming activities in pure and mixed cultures of Desulfovibrio vulgaris, Desulfovibrio desulfuricans, and Methanosarcina barkeri

The activities of pure and mixed cultures of Desulfovibrio vulgaris and Methanosarcina barkeri in the exponential growth phase were monitored by measuring changes in dissolved-gas concentration by membrane-inlet mass spectrometry. M. barkeri grown under H2-CO2 or methanol produced limited amounts of methane and practically no hydrogen from either substrate. The addition of CO resulted in a transient H2 production concomitant with CO consumption. Hydrogen was then taken up, and CH4 production increased. All these events were suppressed by KCN, which inhibited carbon monoxide dehydrogenase activity. Therefore, with both substrates, H2 appeared to be an intermediate in CO reduction to CH4. The cells grown on H2-CO2 consumed 4 mol of CO and produced 1 mol of CH4. Methanol-grown cells reduced CH3OH with H2 resulting from carbon monoxide dehydrogenase activity, and the ratio was then 1 mol of CH4 to 1 mol of CO. Only 12CH4 and no 13CH4 was obtained from 13CO, indicating that CO could not be the direct precursor of CH4. In mixed cultures of D. vulgaris and M. barkeri on lactate, an initial burst of H2 was observed, followed by a lower level of production, whereas methane synthesis was linear with time. Addition of CO to the mixed culture also resulted in transient extra H2 production but had no inhibitory effect upon CH4 formation, even when the sulfate reducer was D. vulgaris Hildenborough, whose periplasmic iron hydrogenase is very sensitive to CO. The hydrogen transfer is therefore probably mediated by a less CO-sensitive nickel-iron hydrogenase from either of both species.     

Anaerobic Degradation of Lactate by Syntrophic Associations of Methanosarcina barkeri and Desulfovibrio Species and Effect of H2 on Acetate Degradation

When grown in the absence of added sulfate, cocultures of Desulfovibrio desulfuricans or Desulfovibrio vulgaris with Methanobrevibacter smithii (Methanobacterium ruminantium), which uses H₂ and CO₂ for methanogenesis, degraded lactate, with the production of acetate and CH₄. When D. desulfuricans or D. vulgaris was grown in the absence of added sulfate in coculture with Methanosarcina barkeri (type strain), which uses both H₂-CO₂ and acetate for methanogenesis, lactate was stoichiometrically degraded to CH₄ and presumably to CO₂. During the first 12 days of incubation of the D. desulfuricans-M. barkeri coculture, lactate was completely degraded, with almost stoichiometric production of acetate and CH₄. Later, acetate was degraded to CH₄ and presumably to CO₂. In experiments in which 20 mM acetate and 0 to 20 mM lactate were added to D. desulfuricans-M. barkeri cocultures, no detectable degradation of acetate occurred until the lactate was catabolized. The ultimate rate of acetate utilization for methanogenesis was greater for those cocultures receiving the highest levels of lactate. A small amount of H₂ was detected in cocultures which contained D. desulfuricans and M. barkeri until after all lactate was degraded. The addition of H₂, but not of lactate, to the growth medium inhibited acetate degradation by pure cultures of M. barkeri. Pure cultures of M. barkeri produced CH₄ from acetate at a rate equivalent to that observed for cocultures containing M. barkeri. Inocula of M. barkeri grown with H₂-CO₂ as the methanogenic substrate produced CH₄ from acetate at a rate equivalent to that observed for acetate-grown inocula when grown in a rumen fluid-vitamin-based medium but not when grown in a yeast extract-based medium. The results suggest that H₂ produced by the Desulfovibrio species during growth with lactate inhibited acetate degradation by M. barkeri.     

Sulfate-Dependent Interspecies H2 Transfer between Methanosarcina barkeri and Desulfovibrio vulgaris during Coculture Metabolism of Acetate or Methanol

We compared the metabolism of methanol and acetate when Methanosarcina barkeri was grown in the presence and absence of Desulfovibrio vulgaris. The sulfate reducer was not able to utilize methanol or acetate as the electron donor for energy metabolism in pure culture, but was able to grow in coculture. Pure cultures of M. barkeri produced up to 10 μmol of H₂ per liter in the culture headspace during growth on acetate or methanol. In coculture with D. vulgaris, the gaseous H₂ concentration was ≤2 μmol/liter. The fractions of ¹⁴CO₂ produced from [¹⁴C]methanol and 2-[¹⁴C]acetate increased from 0.26 and 0.16, respectively, in pure culture to 0.59 and 0.33, respectively, in coculture. Under these conditions, approximately 42% of the available electron equivalents derived from methanol or acetate were transferred and were utilized by D. vulgaris to reduce approximately 33 μmol of sulfate per 100 μmol of substrate consumed. As a direct consequence, methane formation in cocultures was two-thirds that observed in pure cultures. The addition of 5.0 mM sodium molybdate or exogenous H₂ decreased the effects of D. vulgaris on the metabolism of M. barkeri. An analysis of growth and carbon and electron flow patterns demonstrated that sulfate-dependent interspecies H₂ transfer from M. barkeri to D. vulgaris resulted in less methane production, increased CO₂ formation, and sulfide formation from substrates not directly utilized by the sulfate reducer as electron donors for energy metabolism and growth.     

Mass-spectrometric studies of the interrelations among hydrogenase, carbon monoxide dehydrogenase, and methane-forming activities in pure and mixed cultures of Desulfovibrio vulgaris, Desulfovibrio desulfuricans, and Methanosarcina barkeri.

The activities of pure and mixed cultures of Desulfovibrio vulgaris and Methanosarcina barkeri in the exponential growth phase were monitored by measuring changes in dissolved-gas concentration by membrane-inlet mass spectrometry. M. barkeri grown under H2-CO2 or methanol produced limited amounts of methane and practically no hydrogen from either substrate. The addition of CO resulted in a transient H2 production concomitant with CO consumption. Hydrogen was then taken up, and CH4 production increased. All these events were suppressed by KCN, which inhibited carbon monoxide dehydrogenase activity. Therefore, with both substrates, H2 appeared to be an intermediate in CO reduction to CH4. The cells grown on H2-CO2 consumed 4 mol of CO and produced 1 mol of CH4. Methanol-grown cells reduced CH3OH with H2 resulting from carbon monoxide dehydrogenase activity, and the ratio was then 1 mol of CH4 to 1 mol of CO. Only 12CH4 and no 13CH4 was obtained from 13CO, indicating that CO could not be the direct precursor of CH4. In mixed cultures of D. vulgaris and M. barkeri on lactate, an initial burst of H2 was observed, followed by a lower level of production, whereas methane synthesis was linear with time. Addition of CO to the mixed culture also resulted in transient extra H2 production but had no inhibitory effect upon CH4 formation, even when the sulfate reducer was D. vulgaris Hildenborough, whose periplasmic iron hydrogenase is very sensitive to CO. The hydrogen transfer is therefore probably mediated by a less CO-sensitive nickel-iron hydrogenase from either of both species.     

Anaerobic bioconversion of cellulose by Ruminococcus albus, Methanobrevibacter smithii, and Methanosarcina barkeri

A system is described that combines the fermentation of cellulose to acetate, CH₄, and CO₂ by Ruminococcus albus and Methanobrevibacter smithii with the fermentation of acetate to CH₄ and CO₂ by Methanosarcina barkeri to convert cellulose to CH₄ and CO₂. A cellulose-containing medium was pumped into a co-culture of the cellulolytic R. albus and the H₂-using methanogen, Mb. smithii. The effluent was fed into a holding reservoir, adjusted to pH 4.5, and then pumped into a culture of Ms. barkeri maintained at constant volume by pumping out culture contents. Fermentation of 1% cellulose to CH₄ and CO₂ was accomplished during 132 days of operation with retention times (RTs) of the Ms. barkeri culture of 7.5–3.8 days. Rates of acetate utilization were 9.5–17.3 mmol l⁻¹ day⁻¹ and increased with decreasing RT. The K _s for acetate utilization was 6–8 mM. The two-stage system can be used as a model system for studying biological and physical parameters that influence the bioconversion process. Our results suggest that manipulating the different phases of cellulose fermentation separately can effectively balance the pH and ionic requirements of the acid-producing phase with the acid-using phase of the overall fermentation. Received: 7 December 1999 / Received revision: 28 April 2000 / Accepted: 19 May 2000     

Carbon monoxide dehydrogenase from Methanosarcina barkeri. Disaggregation, purification, and physicochemical properties of the enzyme

Carbon monoxide dehydrogenase from acetate-grown cells of Methanosarcina barkeri exists in a high molecular weight form (approximately 3 X 10(6)) under conditions of high ionic strength but is converted to a much smaller form by dialysis. The enzyme was purified by a procedure which exploits isolation of the aggregated form by gel filtration and subsequent dissociation. Following this, the enzyme was purified to within 92% of homogeneity by chromatography on phenyl-Sepharose and finally on hydroxylapatite. Due to the extreme oxygen lability of the enzyme, the entire procedure was carried out within the anaerobic laboratory at the National Institutes of Health. The enzyme has an alpha 2 beta 2 oligomeric structure composed of subunits with molecular weights of 19,700 and 84,500. The amino acid compositions of the individual subunits were determined. Analysis of the metal content by plasma emission spectroscopy indicated 1.3 +/- 0.3 (n = 4) nickel and 15.6 +/- 5.6 (n = 5) iron per mol of alpha 2 beta 2. The enzyme did not contain significant amounts of cobalt or molybdenum. Ferredoxin, FAD, FMN, 2,3,5-triphenyltetrazolium chloride, methyl viologen, and phenazine methosulfate served as electron acceptors; however, the enzyme failed to reduce NAD+, NADP+, or the 8-hydroxy-5-deazaflavin factor F420. The optimum pH was between 7 and 9. The apparent Km for methyl viologen was 7.1 mM, whereas the value for 2,3,5-triphenyltetrazolium chloride was below 0.5 mM. Strong inhibition was observed by oxygen and cyanide. Inactivation by glyoxaldehyde required enzymatic turnover which suggested that a reactive group was formed, or exposed, on an enzyme intermediate in catalysis. A high degree of thermostability was noted. Carbon monoxide, however, rendered the enzyme more susceptible to temperature inactivation.     

Evolution of the syntrophic interaction between Desulfovibrio vulgaris and Methanosarcina barkeri: Involvement of an ancient horizontal gene transfer

The sulfate reducing bacteria Desulfovibrio vulgaris and the methanogenic archaea Methanosarcina barkeri can grow syntrophically on lactate. In this study, a set of three closely located genes, DVU2103, DVU2104, and DVU2108 of D. vulgaris, was found to be up-regulated 2- to 4-fold following the lifestyle shift from syntroph to sulfate reducer; moreover, none of the genes in this gene set were differentially regulated when comparing gene expression from various D. vulgaris pure culture experiments. Although exact function of this gene set is unknown, the results suggest that it may play roles related to the lifestyle change of D. vulgaris from syntroph to sulfate reducer. This hypothesis is further supported by phylogenomic analyses showing that homologies of this gene set were only narrowly present in several groups of bacteria, most of which are restricted to a syntrophic lifestyle, such as Pelobacter carbinolicus, Syntrophobacter fumaroxidans, Syntrophomonas wolfei, and Syntrophus aciditrophicus. Phylogenetic analysis showed that all three individual genes in the gene set tended to be clustered with their homologies from archaeal genera, and they were rooted on archaeal species in the phylogenetic trees, suggesting that they were horizontally transferred from archaeal methanogens. In addition, no significant bias in codon and amino acid usages was detected between these genes and the rest of the D. vulgaris genome, suggesting the gene transfer may have occurred early in the evolutionary history so that sufficient time has elapsed to allow an adaptation to the codon and amino acid usages of D. vulgaris. This report provides novel insights into the origin and evolution of bacterial genes linked to the lifestyle change of D. vulgaris from a syntrophic to a sulfate-reducing lifestyle.     

Anaerobic Degradation of Lactate by Syntrophic Associations of Methanosarcina barkeri and Desulfovibrio Species and Effect of H(2) on Acetate Degradation

When grown in the absence of added sulfate, cocultures of Desulfovibrio desulfuricans or Desulfovibrio vulgaris with Methanobrevibacter smithii (Methanobacterium ruminantium), which uses H(2) and CO(2) for methanogenesis, degraded lactate, with the production of acetate and CH(4). When D. desulfuricans or D. vulgaris was grown in the absence of added sulfate in coculture with Methanosarcina barkeri (type strain), which uses both H(2)-CO(2) and acetate for methanogenesis, lactate was stoichiometrically degraded to CH(4) and presumably to CO(2). During the first 12 days of incubation of the D. desulfuricans-M. barkeri coculture, lactate was completely degraded, with almost stoichiometric production of acetate and CH(4). Later, acetate was degraded to CH(4) and presumably to CO(2). In experiments in which 20 mM acetate and 0 to 20 mM lactate were added to D. desulfuricans-M. barkeri cocultures, no detectable degradation of acetate occurred until the lactate was catabolized. The ultimate rate of acetate utilization for methanogenesis was greater for those cocultures receiving the highest levels of lactate. A small amount of H(2) was detected in cocultures which contained D. desulfuricans and M. barkeri until after all lactate was degraded. The addition of H(2), but not of lactate, to the growth medium inhibited acetate degradation by pure cultures of M. barkeri. Pure cultures of M. barkeri produced CH(4) from acetate at a rate equivalent to that observed for cocultures containing M. barkeri. Inocula of M. barkeri grown with H(2)-CO(2) as the methanogenic substrate produced CH(4) from acetate at a rate equivalent to that observed for acetate-grown inocula when grown in a rumen fluid-vitamin-based medium but not when grown in a yeast extract-based medium. The results suggest that H(2) produced by the Desulfovibrio species during growth with lactate inhibited acetate degradation by M. barkeri.     

Sulfate-Dependent Interspecies H(2) Transfer between Methanosarcina barkeri and Desulfovibrio vulgaris during Coculture Metabolism of Acetate or Methanol

We compared the metabolism of methanol and acetate when Methanosarcina barkeri was grown in the presence and absence of Desulfovibrio vulgaris. The sulfate reducer was not able to utilize methanol or acetate as the electron donor for energy metabolism in pure culture, but was able to grow in coculture. Pure cultures of M. barkeri produced up to 10 mumol of H(2) per liter in the culture headspace during growth on acetate or methanol. In coculture with D. vulgaris, the gaseous H(2) concentration was 相似文献   

Temperature Compensation in Methanosarcina barkeri by Modulation of Hydrogen and Acetate Affinity

The affinity of Methanosarcina barkeri 227 for acetate and hydrogen at different incubation temperatures was investigated. Increasing the temperature from 20 to 37°C resulted in a 4.5-fold increase in K_m for acetate and a 4.8-fold increase for hydrogen. The corresponding increase in V_max for acetate was 8.3-fold (5.4-fold for hydrogen). This response implied a decrease in the temperature coefficient (Q₁₀) and hence a decrease in the temperature dependency as a function of decreasing substrate concentration.     

Utilization of Methanol plus Hydrogen by Methanosarcina barkeri for Methanogenesis and Growth

Methanosarcina barkeri grew on methanol plus H₂. Both substrates were consumed in equimolar amounts. Growth was strictly dependent on the presence of acetate, which was required for the biosynthesis of cellular constituents. Only about 0.4% of the methane produced originated from acetate. By using deuterated methanol, it was demonstrated that methanogenesis from this compound under H₂ did not occur via oxidation of methanol to CO₂ and subsequent reduction but by direct reduction with H₂. Growth yields with methanol plus H₂ and with methanol alone were not significantly different: 2.8 g of cells per mol of methanol in mineral medium and 4.6 g of cells per mol of methanol in complex medium, respectively. Growth of M. barkeri on methanol plus H₂ depended strictly on the presence of sodium ions in the medium. In the presence of 50 mM K⁺ the K_s for Na⁺ was 5 mM.     

Purification, characterization, and primary structure of a monofunctional catalase from Methanosarcina barkeri

Methanosarcina barkeri is a strictly anaerobic, cytochrome-containing, methane-forming archaeon. We report here that the microorganism contains a catalase, which was purified and characterized. The enzyme with an apparent molecular mass of 190 kDa was shown to be composed of four identical subunits of apparent molecular mass of 54 kDa. The heme-containing enzyme did not exhibit peroxidase activity, which indicates that it is a monofunctional catalase. This is substantiated by the primary structure, which is related to that of other monofunctional catalases rather than to that of bifunctional catalase-peroxidases. The enzyme showed an [S]_0.5V for H₂O₂ of 25 mM and an apparent V _max of 200,000 U/mg; it was inhibited by azide ([I]_0.5V = 1 μM) and cyanide ([I]_0.5V = 5 μM) and inactivated by 1,2,4-aminotriazole. The activity was almost independent of the pH (between pH 4 and 10) and the temperature (between 15 °C and 55 °C). Comparison of the primary structure of monofunctional catalases revealed that the enzyme from M. barkeri is most closely related to the monofunctional catalase of Dictyostelium discoideum. Received: 29 December 1998 / Accepted: 1 March 1999