Interactions in syntrophic associations of endospore-forming, butyrate-degrading bacteria and h(2)-consuming bacteria

Butyrate is an important intermediate in the anaerobic degradation of organic matter. In sulfate-depleted environments butyrate is oxidized to acetate and hydrogen by obligate proton reducers, in syntrophic association with hydrogen-consuming methanogens. This paper describes two enrichments of endospore-forming bacteria degrading butyrate in consortia with methanogens. The isolates are readily established in coculture with H(2)-consuming, sulfate-reducing bacteria by pasteurizing the culture. The two original enrichments differed in that one grew to an optically dense culture while the second grew in clumps. Examination by scanning electron microscopy showed that clumping resulted from the production of large amounts of extracellular polymer. Several H(2)-consuming methanogens were identified in the enrichments. Some of them grew closely associated to the butyrate degraders. This attachment to the hydrogen producer may permit some methanogens to compete for the growth substrate against other bacteria having higher substrate affinity.     

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.     

End Products of Anaerobic Chitin Degradation by Salt Marsh Bacteria as Substrates for Dissimilatory Sulfate Reduction and Methanogenesis

The anaerobic pathway of chitin decomposition by chitinoclastic bacteria was examined with an emphasis on end product coupling to other salt marsh bacteria. Actively growing chitinoclastic bacterial isolates produced primarily acetate, H₂, and CO₂ in broth culture. No sulfate-reducing or methanogenic isolates grew on chitin as sole carbon source or produced any measurable degradation products. Mixed cultures of chitin degraders with sulfate reducers resulted in positive sulfide production. Mixed cultures of chitin-degrading isolates with methanogens resulted in the production of CH₄ with reductions in headspace CO₂ and H₂. The combination of all three metabolic types resulted in the simultaneous production of methane and sulfide, with more methane being produced in mixed cultures containing CO₂-reducing methanogens and acetoclastic sulfate reducers because of less interspecific H₂ competition.     

Hydrogenotrophic Methanogenesis by Moderately Acid-Tolerant Methanogens of a Methane-Emitting Acidic Peat

The emission of methane (1.3 mmol of CH₄ m⁻² day⁻¹), precursors of methanogenesis, and the methanogenic microorganisms of acidic bog peat (pH 4.4) from a moderately reduced forest site were investigated by in situ measurements, microcosm incubations, and cultivation methods, respectively. Bog peat produced CH₄ (0.4 to 1.7 μmol g [dry wt] of soil⁻¹ day⁻¹) under anoxic conditions. At in situ pH, supplemental H₂-CO₂, ethanol, and 1-propanol all increased CH₄ production rates while formate, acetate, propionate, and butyrate inhibited the production of CH₄; methanol had no effect. H₂-dependent acetogenesis occurred in H₂-CO₂-supplemented bog peat only after extended incubation periods. Nonsupplemented bog peat initially produced small amounts of H₂ that were subsequently consumed. The accumulation of H₂ was stimulated by ethanol and 1-propanol or by inhibiting methanogenesis with bromoethanesulfonate, and the consumption of ethanol was inhibited by large amounts of H₂; these results collectively indicated that ethanol- or 1-propanol-utilizing bacteria were trophically associated with H₂-utilizing methanogens. A total of 10⁹ anaerobes and 10⁷ hydrogenotrophic methanogens per g (dry weight) of bog peat were enumerated by cultivation techniques. A stable methanogenic enrichment was obtained with an acidic, H₂-CO₂-supplemented, fatty acid-enriched defined medium. CH₄ production rates by the enrichment were similar at pH 4.5 and 6.5, and acetate inhibited methanogenesis at pH 4.5 but not at pH 6.5. A total of 27 different archaeal 16S rRNA gene sequences indicative of Methanobacteriaceae, Methanomicrobiales, and Methanosarcinaceae were retrieved from the highest CH₄-positive serial dilutions of bog peat and methanogenic enrichments. A total of 10 bacterial 16S rRNA gene sequences were also retrieved from the same dilutions and enrichments and were indicative of bacteria that might be responsible for the production of H₂ that could be used by hydrogenotrophic methanogens. These results indicated that in this acidic bog peat, (i) H₂ is an important substrate for acid-tolerant methanogens, (ii) interspecies hydrogen transfer is involved in the degradation of organic carbon, (iii) the accumulation of protonated volatile fatty acids inhibits methanogenesis, and (iv) methanogenesis might be due to the activities of methanogens that are phylogenetic members of the Methanobacteriaceae, Methanomicrobiales, and Methanosarcinaceae.     

Methanogenesis at low temperatures by microflora of tundra wetland soil

Active methanogenesis from organic matter contained in soil samples from tundra wetland occurred even at 6 °C. Methane was the only end product in balanced microbial community with H₂/CO₂ as a substrate, besides acetate was produced as an intermediate at temperatures below 10°C. The activity of different microbial groups of methanogenic community in the temperature range of 6–28 °C was investigated using 5% of tundra soil as inoculum. Anaerobic microflora of tundra wetland fermented different organic compounds with formation of hydrogen, volatile fatty acids (VFA) and alcohols. Methane was produced at the second step. Homoacetogenic and methanogenic bacteria competed for such substrates as hydrogen, formate, carbon monoxide and methanol. Acetogens out competed methanogens in an excess of substrate and low density of microbial population. Kinetic analysis of the results confirmed the prevalence of hydrogen acetogenesis on methanogenesis. Pure culture of acetogenic bacteria was isolated at 6 °C. Dilution of tundra soil and supply with the excess of substrate disbalanced the methanoigenic microbial community. It resulted in accumulation of acetate and other VFA. In balanced microbial community obviously autotrophic methanogens keep hydrogen concentration below a threshold for syntrophic degradation of VFA. Accumulation of acetate- and H₂/CO₂-utilising methanogens should be very important in methanogenic microbial community operating at low temperatures.     

Populations of Methanogenic Bacteria in a Georgia Salt Marsh

Methanogens represented about 0.5% of the total bacteria in sediments from a Georgia salt marsh in which Spartina alterniflora is the predominant vegetation. The population of methanogens was composed of at least two groups of nearly equal size. One group was represented by cocci which were able to utilize trimethylamine and were unable to use H₂ or acetate. The second group was composed of two subgroups which were able to utilize H₂ but were unable to use trimethylamine or acetate. The more common subgroup included rod- or plate-shaped methanogens which could utilize isopropanol in addition to H₂ and formate. The second subgroup included Methanococcus maripaludis, which utilized only H₂ and formate. Other groups of methanogens were also present, including Methanosarcina sp. which utilized acetate, H₂, and methylamines. In addition to the overall variability in the types of methanogens, the numbers of methanogens in sediments also exhibited significant spatial variability both within and between tall- and short-Spartina zones.     

Growth of Desulfovibrio in Lactate or Ethanol Media Low in Sulfate in Association with H2-Utilizing Methanogenic Bacteria

In the analysis of an ethanol-CO₂ enrichment of bacteria from an anaerobic sewage digestor, a strain tentatively identified as Desulfovibrio vulgaris and an H₂-utilizing methanogen resembling Methanobacterium formicicum were isolated, and they were shown to represent a synergistic association of two bacterial species similar to that previously found between S organism and Methanobacterium strain MOH isolated from Methanobacillus omelianskii. In lowsulfate media, the desulfovibrio produced acetate and H₂ from ethanol and acetate, H₂, and, presumably, CO₂ from lactate; but growth was slight and little of the energy source was catabolized unless the organism was combined with an H₂-utilizing methanogenic bacterium. The type strains of D. vulgaris and Desulfovibrio desulfuricans carried out the same type of synergistic growth with methanogens. In mixtures of desulfovibrio and strain MOH growing on ethanol, lactate, or pyruvate, diminution of methane produced was stoichiometric with the moles of sulfate added, and the desulfovibrios grew better with sulfate addition. The energetics of the synergistic associations and of the competition between the methanogenic system and sulfate-reducing system as sinks for electrons generated in the oxidation of organic materials such as ethanol, lactate, and acetate are discussed. It is suggested that lack of availability of H₂ for growth of methanogens is a major factor in suppression of methanogenesis by sulfate in natural ecosystems. The results with these known mixtures of bacteria suggest that hydrogenase-forming, sulfate-reducing bacteria could be active in some methanogenic ecosystems that are low in sulfate.     

Cultivation of Methanogens under Low-Hydrogen Conditions by Using the Coculture Method

We previously reported the isolation of novel methanogens by using a new cultivation method, referred to as the coculture method. Here, we extended our coculture method to various anaerobic environmental samples. As a result, we successfully cultivated some uncharacterized methanogens in coculture enrichments and eventually isolated a new methanogen, within the order Methanomicrobiales.So far, almost all cases of the cultivation and isolation of H₂-utilizing methanogenic Archaea (methanogens) have been performed under high-H₂ concentrations (e.g., around 100 kPa), even though the concentrations in their natural habitat are far lower (10 to 100 Pa) than in laboratory cultures. This difference between in vitro and in situ physicochemical conditions very likely means that fast-growing methanogens that may prefer high concentrations of H₂ will have to be specifically selected; thus, laboratory cultures under such high-H₂ conditions result in the growth of a very limited range of species. To avoid this situation, we have proposed a new cultivation method, which we named the coculture method, for cultivating H₂-utilizing methanogens (14).Under anaerobic conditions, methanogens often partner with heterotrophic H₂-producing bacteria, which catalyze the oxidation of a variety of substrates (fatty acids, alcohols, and aromatic compounds). The methanogens use the H₂ produced by these heterotrophic bacteria, and in return, the bacteria benefit from the removal of the H₂ that would otherwise inhibit their growth. This lifestyle is commonly referred to as interspecies H₂ transfer, and the heterotrophic H₂-producing bacteria relying on H₂-utilizing methanogens are called syntrophs (11). In our previous studies, cultivation was performed with propionate as an indirect precursor substrate that is converted to H₂ by syntrophs, with the expectation that methanogens would grow as a result of interspecies H₂ transfer. Based on this strategy, two novel methanogens representing new genera, Methanocella paludicola strain SANAE and Methanolinea tarda strain NOBI-1, were successfully isolated (7, 13, 14).In this study, we extended the method to various types of environmental samples to cultivate and isolate uncharacterized methanogens. Moreover, we also extended the H₂-supplying substrates to include ethanol and butyrate in addition to propionate, because these substances are also known to be decomposed by a syntrophic association of substrate-oxidizing H₂-producing bacteria and H₂-utilizing methanogens (15).Nine anaerobic environmental samples (marine coastal sediment from Kashiwazaki, Niigata, Japan [KO]; freshwater lake sediment from Lake Suwa, Nagano, Japan [SL]; freshwater pond sediment from Shouzuma Pond, Nagano, Japan [SP]; river sediment of the Azusa River, Nagano, Japan [AR]; sediment from a lotus field located in Nagaoka, Niigata, Japan [LF]; rice field soil from Matsumoto, Nagano, Japan [NR]; rice field soil from Nagaoka, Niigata, Japan [SRP]; rice field soil from Tainan, Taiwan [TNR]; and methanogenic granular sludge obtained from a lab-scale upflow anaerobic sludge blanket reactor treating wastewater from the manufacture of palm oil in our laboratory [MP]) were anaerobically incubated with ethanol (10 mM), butyrate (20 mM), or propionate (20 mM) as the sole carbon and energy source. Additionally, we prepared propionate enrichment cultures with the addition of a pure culture of anaerobic syntrophic propionate-oxidizing bacterium Syntrophobacter fumaroxidans strain MPOB (DSMZ 10017) cells (inoculum size, 5% [vol/vol]) to obtain stable cultures (8, 18), except for enrichments from the marine sediment and granular sludge samples because the NaCl resistance of S. fumaroxidans was unknown (6) and the granular sludge was expected to contain a large amount of indigenous syntrophic bacteria (5, 16). Moreover, as control experiments, the same environmental samples were used in enrichments by the canonical cultivation method in the presence of high concentrations of H₂ (ca. 150 kPa in headspace) or formate (40 mM). All cultivations were performed anaerobically at 37°C without shaking. In total, 52 primary enrichment cultures were prepared for this study (see Table S1 in the supplemental material).When primary enrichment was made using high concentrations of H₂ and formate, the growth of methanogen-like microbes was confirmed within 3 to 5 days of incubation (as examples, photomicrographs of the enrichment cultures from TNR are shown in Fig. S1A to D in the supplemental material). After three consecutive transfers, 16S rRNA gene-based clone analysis was performed using an archaeal universal primer pair, Ar109f/1490R (14). Twenty-nine phylotypes were detected and were closely related to previously isolated methanogens (Fig. ​(Fig.11 and 2A and B; see Table S1 in the supplemental material). Among them, 26 phylotypes were classified into the genus Methanobacterium, two phylotypes in the genus Methanospirillum, and one in the genus Methanogenium. Moreover, 20 phylotypes showed high similarities (>97%) with the 16S rRNA genes of previously isolated methanogens, whereas the remaining nine phylotypes showed 95 to 96% similarities with the 16S rRNA genes of previously characterized methanogens (see Table S1 in the supplemental material). The methanogens possessing these sequences may be taxonomically novel at least at the species level (17), but they were all affiliated with the well-studied genera Methanobacterium and Methanospirillum.Open in a separate windowFIG. 1.Phylogenetic tree showing the placement of 16S rRNA gene sequences/clones obtained in this study. The colored phylotypes were obtained in this study. The difference in color among phylotypes indicates the different substrates used for the enrichment cultures (blue, hydrogen; green, formate; orange, ethanol; pink, butyrate; red, propionate and propionate plus S. fumaroxidans strain MPOB). The name of each phylotype is composed of the sample name, an abbreviation of the substrate for cultivation (H2, hydrogen; For, formate; Eth, ethanol; Buty, butyrate; Pro, propionate; ProM, propionate plus S. fumaroxidans), and the phylotype (for example, SRP-Pro-A is phylotype A recovered from the propionate enrichment culture cultivated from the environmental sample SRP). The number in parentheses indicates the number of identical clones obtained per number of clones analyzed for each phylotype. The accession numbers are also shown after each phylotype name. The phylotypes indicated by the same accession numbers have the same sequences (e.g., SP-For-A and SRP-Buty-C, AR-ProM-A and NR-ProM-A). All of the clonal sequences were greater than 1,000 nucleotides in length, with the exception of Methanospirillum sp. TM20-1 (GenBank acc. no. AB062404; 789 bp). Therefore, the initial tree was constructed with sequences greater than 1,000 nucleotides using the neighbor-joining method. Subsequently, the Methanospirillum sp. TM20-1 sequence was inserted into the tree by using the parsimony insertion tool of the ARB program. The scale bar indicates the estimated number of base changes per nucleotide sequence position. The symbols at the branch nodes indicate bootstrap values.Open in a separate windowFIG. 2.Phylogenetic affiliation of the identified phylotypes based on their cultivation substrates. The panels indicate the results of enrichment cultures with the following substrates: H₂ (A), formate (B), ethanol (C), butyrate (D), propionate (E), and propionate with the addition of the pure culture of S. fumaroxidans (F). The identified phylotypes were classified into their respective genera according to their 16S rRNA gene similarity with previously characterized methanogens. Phylotypes possessing sequence similarity greater than 92% were treated as the same genus. The number of phylotypes for each group is indicated in parentheses.In the coculture enrichments, substrate degradation concomitant with methane formation was confirmed after 1 week and more than 1 to 3 months of incubation in ethanol enrichment cultures and butyrate and propionate enrichment cultures, respectively. In particular, the growth of microbes in the propionate enrichments without the addition of S. fumaroxidans cells, except for the enrichments constructed from the RF and SRP samples, was very slow and unstable; the growth and methane production stopped unexpectedly and often made successive passages to fresh medium difficult. Additionally, two propionate enrichments in the absence of S. fumaroxidans inoculated from the KO and NR samples did not show methane production after a year of incubation. On the other hand, all of the propionate enrichments in the presence of S. fumaroxidans cells showed stable growth. During the incubation of the coculture enrichments, the H₂ partial pressures in the cultures were kept at <100 Pa in the ethanol enrichments and at <30 Pa in the butyrate and propionate cultures. Methane, H₂, short-chain fatty acids, and ethanol were measured as described previously (14). Microscopic observation after three to four transfers showed that those enrichments were comprised mainly of F₄₂₀-autofluorescent methanogen-like cells and oval- or rod-shaped bacterial cells, possibly syntrophs (see Fig. S1E to L in the supplemental material). These observations suggested that ethanol, butyrate, and propionate degradation were carried out by syntrophic association between syntrophic substrate-oxidizing H₂-producing bacteria and H₂-utilizing methanogens. To identify the methanogens present in those enrichments, archaeal 16S rRNA gene-based clone analyses were performed. A total of 52 phylotypes were obtained (Fig. ​(Fig.11 and 2C to F; see Table S1 in the supplemental material). Of these, 23 phylotypes were classified into the genera Methanobacterium (19 phylotypes) and Methanospirillum (4 phylotypes), which were very similar to those obtained from the H₂ and formate enrichments. On the other hand, the remaining 29 phylotypes were comprised of the orders Methanomicrobiales (20 phylotypes), Methanocellales (5 phylotypes), and Methanosarcinales (4 phylotypes, all belonging to the genus Methanosaeta). Within the order Methanomicrobiales, some phylotypes were affiliated with the genera Methanoculleus (nine phylotypes), Methanofollis (two phylotypes), Methanocalculus (one phylotype), and Methanoplanus (one phylotype). Additionally, sequences very closely related to the recently isolated methanogens Methanolinea tarda (six phylotypes) (7, 14) and “Candidatus Methanoregula boonei” (one phylotype) (1) were also obtained. Both M. tarda and “Ca. Methanoregula boonei” represent a family-level clade, which had long been recognized as an uncultured archaeal lineage called the group E1/E2 (1) (Fig. ​(Fig.1).1). Regarding the five phylotypes within the order Methanocellales, all were obtained from SRP and SP enrichments. Though the order Methanocellales had been recognized as the clone cluster rice cluster I, one strain has been isolated very recently (13, 14) and the rice cluster I methanogens are now being unveiled.Of the 52 phylotypes obtained from the coculture enrichments, 38 phylotypes (73% of the total phylotypes) were >97% similar to the 16S rRNA genes of the previously characterized (cultivated) methanogens. In contrast, 14 phylotypes (27%) had <96% sequence similarity with those of known methanogens. The organisms represented by these phylotypes were considered to be taxonomically novel at the species or even the genus level. Most of these phylotypes were affiliated with the orders Methanomicrobiales and Methanocellales with 92 to 96% sequence similarity (see Table S1 in the supplemental material). According to the 16S rRNA gene-based clone analysis, taxonomically novel methanogens were found in abundance in one ethanol, two butyrate, and eight propionate enrichments (from the KO, SP, SL, TNR, LF, and SRP samples). Especially, the ethanol and six propionate enrichments (from the SP, TNR, LF, and SRP samples) contained novel methanogens belonging to the group E1/E2 and/or the order Methanocellales (formerly known as rice cluster I), both of which contain only a few cultivated representatives so far. Therefore, we attempted to isolate these methanogens from the enrichments. After several attempts were performed over a year, a novel methanogen, designated strain TNR, was successfully isolated from the propionate enrichment culture (TNR) by serial dilution in liquid medium with H₂ (ca. 150 kPa) as the substrate.Strain TNR was a nonmotile, rod-shaped methanogen, which utilized H₂/CO₂ and formate for growth and methane production (see Fig. S2 in the supplemental material). The doubling time was 1.2 days at 37°C and pH 7. The most closely related methanogen cultivated so far was Methanolinea tarda that we have recently isolated (7), but the similarity of the 16S rRNA genes between the two was only 95% (Fig. ​(Fig.1).1). On the other hand, the isolation of methanogens from the other enrichments was not successful, i.e., when the coculture enrichments were inoculated into the serial dilution cultures with high concentrations of H₂ or formate, nontargeted methanogens, almost all of which had >97% sequence similarities to the 16S rRNA genes of known Methanobacterium and Methanoculleus species, outgrew in the cultures. The conventional method for final purification (i.e., using high concentrations of H₂ or formate as a direct substrate) has, therefore, a clear limitation, and new methods to overcome this will be needed.By using the coculture method, we successfully enriched methanogens that were absent in previous cultivation attempts and were only detected as environmental clones. In addition, we were able to isolate a methanogen belonging to the group E1/E2 of the order Methanomicrobiales. Our study clearly demonstrated that the coculture method is an effective way to cultivate hitherto uncharacterized methanogens. Interestingly, the taxonomic compositions of the phylotypes were clearly different depending on the substrates used in the coculture method (Fig. ​(Fig.11 and ​and2).2). When conventional cultivation was employed using high concentrations of H₂ and formate, only very limited phylotypes were obtained, namely, Methanobacterium- and Methanospirillum-related phylotypes in the H₂ cultures and Methanobacterium- and Methanogenium-related phylotypes in the formate cultures. When using the coculture method with ethanol or butyrate, Methanobacterium-related phylotypes were also dominant, accounting for 64.3% of the total phylotypes, whereas more diverse methanogen phylotypes than those in the H₂ and formate cultures were retrieved. Contrary to these results, propionate (with and without S. fumaroxidans) enrichments allowed quite a different pattern of methanogen phylotypes to become established. The most abundant phylotypes obtained from the propionate enrichments belonged to the orders Methanocellales and Methanomicrobiales, accounting for 72.8 and 84.7% of the clones examined. The addition of S. fumaroxidans cells into the propionate enrichments seemed to have no significant effect on the methanogenic community compositions that emerged, but it helped the stability of the whole community and the capability of the propionate degradation. The theoretical ranges of H₂ partial pressure that allow the anaerobic oxidation of ethanol, butyrate, and propionate to occur are 0.5 to 27,000 Pa; 0.5 to 60 Pa; and 0.5 to 28 Pa, respectively. These values were calculated based on the review on energy conservation by Thauer et al. (19), in which the concentrations of products and reactants were 0.35 atm , 0.65 atm , and 20 mM substances at 37°C and pH 7. For the calculation, a temperature correction was made using the van''t Hoff equation. Theoretically, the H₂ partial pressures in the various cultures differ depending on the substrates used, becoming lower in the order of substrates: ethanol > butyrate > propionate. Actually, the H₂ partial pressures measured during substrate degradation in the coculture enrichments remained within these theoretical ranges (data not shown). Given the above theoretical values, the apparent H₂ partial pressure that could be generated from a particular substrate would be the crucial factor affecting the change in the compositions of H₂-utilizing methanogens in the community. In fact, the relative abundance of members of the genera Methanobacterium and Methanospirillum increased as the given H₂ partial pressure became higher (propionate → butyrate → ethanol → H₂), and conversely, the relative abundance of members of the orders Methanomicrobiales (except for the genus Methanospirillum) and Methanocellales increased as the H₂ partial pressure became lower (Fig. ​(Fig.11 and ​and2).2). We assume that Methanocellales spp. and Methanomicrobiales spp. (except for Methanospirillum spp.) have higher affinities for H₂ than Methanobacterium spp. and Methanospirillum spp. Several previous studies also support this prediction. Lu et al. reported that Methanocellales methanogens incorporated ¹³C when rice roots were incubated in a low-H₂ atmosphere in the presence of ¹³CO₂, while Methanobacteriales and Methanosarcinales methanogens preferentially incorporated ¹³C in a high-H₂ atmosphere (10). Also, Methanocellales phylotypes were detected from methanogenic environments, usually with a low concentration of H₂, such as rice fields, fens, and peat bogs (e.g., see references 3, 4, and 9). In addition to the Methanocellales methanogens, members of the order Methanomicrobiales were frequently found in abundance in low-H₂-concentration methanogenic environments, such as peat bogs, fens, lake sediments, and rice fields (e.g., see references 2, 12, and 20). Detailed substrate affinity information will provide insight into the relevance between the population structures of methanogens and the H₂ concentrations of their habitats.     

Isolation of Key Methanogens for Global Methane Emission from Rice Paddy Fields: a Novel Isolate Affiliated with the Clone Cluster Rice Cluster I

Despite the fact that rice paddy fields (RPFs) are contributing 10 to 25% of global methane emissions, the organisms responsible for methane production in RPFs have remained uncultivated and thus uncharacterized. Here we report the isolation of a methanogen (strain SANAE) belonging to an abundant and ubiquitous group of methanogens called rice cluster I (RC-I) previously identified as an ecologically important microbial component via culture-independent analyses. To enrich the RC-I methanogens from rice paddy samples, we attempted to mimic the in situ conditions of RC-I on the basis of the idea that methanogens in such ecosystems should thrive by receiving low concentrations of substrate (H₂) continuously provided by heterotrophic H₂-producing bacteria. For this purpose, we developed a coculture method using an indirect substrate (propionate) in defined medium and a propionate-oxidizing, H₂-producing syntroph, Syntrophobacter fumaroxidans, as the H₂ supplier. By doing so, we significantly enriched the RC-I methanogens and eventually obtained a methanogen within the RC-I group in pure culture. This is the first report on the isolation of a methanogen within RC-I.     

The Microbial Logic behind the Prevalence of Incomplete Oxidation of Organic Compounds by Acetogenic Bacteria in Methanogenic Environments

Microbial degradation of organic material in methanogenic ecosystems is a multistep process in which subsequent groups use the products of the first groups of organisms in the chain as substrates. The acetogenic bacteria in these systems produce both H₂ and acetate. In the present minireview a thermodynamic approach is taken to evaluate the logic behind this duality. The evaluation shows that at the H₂ partial pressures that usually occur in methanogenic ecosystems the acetogenic oxidation of known acetogenic substrates such as propionate, butyrate, and benzoate yields more energy than their complete oxidation to H₂/CO₂. Also, H₂ partial pressures needed to achieve complete hydrogenogenic oxidation of these acetogenic substrates would have to be so low that H₂ would be virtually unavailable to the hydrogenotrophic bacteria, in casu the methanogens.     

Degradation of phenol via carboxylation to benzoate by a defined,obligate syntrophic consortium of anaerobic bacteria

Summary An obligate syntrophic culture was selected in mineral medium with phenol as the only carbon and energy source. The consortium consisted of a short and a long rod-shaped bacterium and of low numbers of Desulfovibrio cells, and grew only in syntrophy with methanogens, e. g. Methanospirillum hungatei. Under N₂/CO₂, phenol was degraded via benzoate to acetate, CH₄ and CO₂, while in the presence of H₂/CO₂ benzoate was formed, but not further degraded. When 4-hydroxybenzoate was fed to the mixed culture, it was decarboxylated to phenol prior to benzoate formation and subsequent ring cleavage. Isolation of pure cultures of the two rod-shaped bacteria failed. Microscopic observations during feeding of either 4-hydroxybenzoate, phenol or benzoate implied an obligate syntrophic interdependence of the two different rod-shaped bacteria and of the methanogen. The non-motile rods formed phenol from 4-hydroxybenzoate and benzoate from phenol, requiring an as yet unknown co-substrate or co-factor, probably cross-fed by the short, motile rod. The short, motile rodshaped bacterium grew only in syntrophy with methanogens and degraded benzoate to acetate, CO₂ and methane. Desulfovibrio sp., present in low numbers, apparently could not contribute to the degradation of phenol or 4-hydroxybenzoate.     

Growth of Syntrophic Propionate-Oxidizing Bacteria with Fumarate in the Absence of Methanogenic Bacteria

Oxidation of succinate to fumarate is an energetically difficult step in the biochemical pathway of propionate oxidation by syntrophic methanogenic cultures. Therefore, the effect of fumarate on propionate oxidation by two different propionate-oxidizing cultures was investigated. When the methanogens in a newly enriched propionate-oxidizing methanogenic culture were inhibited by bromoethanesulfonate, fumarate could act as an apparent terminal electron acceptor in propionate oxidation. ¹³C-nuclear magnetic resonance experiments showed that propionate was carboxylated to succinate while fumarate was partly oxidized to acetate and partly reduced to succinate. Fumarate alone was fermented to succinate and CO₂. Bacteria growing on fumarate were enriched and obtained free of methanogens. Propionate was metabolized by these bacteria when either fumarate or Methanospirillum hungatii was added. In cocultures with Syntrophobacter wolinii, such effects were not observed upon addition of fumarate. Possible slow growth of S. wolinii on fumarate could not be demonstrated because of the presence of a Desulfovibrio strain which grew rapidly on fumarate in both the absence and presence of sulfate.     

Regulation of hydrogen metabolism in Butyribacterium methylotrophicum by substrate and pH

 Exogenous H₂/CO₂ and glucose were consumed simultaneously by Butyribacterium methylotrophicum when grown under glucose-limited conditions. CO₂ reduction to acetate was coupled to H₂ consumption. The addition of either H₂ or CO₂ to glucose batch fermentation resulted in an increase in cell density, hydrogenase (H₂-consuming and -producing) activities and fatty acid production by B. methylotrophicum as compared to when N₂ was the feed gas. Hydrogenase activities appeared to be tightly regulated and were produced at higher rates during the exponential phase when CO₂ was the feed gas as compared to H₂ or N₂. The increase in H₂-consuming activity and decrease in H₂-producing activity was correlated with an increase in butyrate synthesis. H₂-consuming and ferredoxin (Fd)–NAD reductase activities increased while H₂-producing and NADH–Fd reductase activities decreased in cells grown at pH 5.5 compared to those at pH 7.0. The molar ratio of butyrate/acetate was shifted from 0.35 at pH 7.0 to 1.22 at pH 5.5. The addition of exogenous H₂ did not decrease the butyrate/acetate ratio at pH 7.0 nor at pH 5.5. The results indicated that growth pH values regulated both hydrogenase and Fd–NAD oxidoreductase activities such that, at acid pH, more intermediary electron flow was directed towards butyrate synthesis than H₂ production. Received: 22 August 1995/Received revision: 18 December 1995/Accepted: 22 January 1996     

Effects of Hydrogen Pressure during Growth and Effects of Pregrowth with Hydrogen on Acetate Degradation by Methanosarcina Species

Methanosarcina barkeri 227 and Methanosarcina mazei S-6 grew with acetate as the substrate; we found little effect of H₂ on the rate of aceticlastic growth in the presence of various H₂ pressures between 2 and 810 Pa. We used physical (H₂ addition or flushing the headspace to remove H₂) and biological (H₂-producing or -utilizing bacteria in cocultures) methods for controlling H₂ pressure in Methanosarcina cultures growing on acetate. Added H₂ (ca. 100 Pa) was removed rapidly (a few hours) by M. barkeri and slowly (within a day) by M. mazei. When the H₂ produced by the aceticlastic methanogens was removed by coculturing with an H₂-using Desulfovibrio sp., the H₂ pressure was about 2.2 Pa. Under these conditions the stoichiometry of aceticlastic methanogenesis did not change. H₂-grown inocula of M. barkeri grew with acetate as the sole catabolic substrate if the inoculum culture was transferred during logarithmic growth to acetate-containing medium or if the transfer was accomplished within 1 or 2 days after exhaustion of H₂. H₂-grown cultures incubated for 4 or more days after exhaustion of H₂ were able to grow with H₂ but not with acetate as the sole catabolic substrate. Addition of small quantities of H₂ to acetate-containing medium permitted these cultures to initiate growth on acetate.     

Butyrate oxidation by Syntrophospora bryantii in co-culture with different methanogens and in pure culture with pentenoate as electron acceptor

Syntrophospora bryantii degraded butyrate in co-culture with methanogens that can use both H₂ and formate for growth, but not in co-culture with methanogens that metabolize only H₂, suggesting that in suspended cultures formate may be a more important electron carrier in the syntrophic degradation of butyrate than H₂. Syntrophic butyrate oxidation was inhibited by the addition of 20 mm formate or the presence of 130 kPa H₂. In the absence of methanogens, S. bryantii is able to couple the oxidation of butyrate to acetate with the reduction of pentenoate to valerate. Under these conditions, up to 300 Pa H₂ was measured in the gas phase and up to 0.3 mm formate in the liquid phase. S. bryantii was unable to grow syntrophically with the aceticlastic methanogen Methanothrix soehngenii. However in triculture with Methanospirillum hungatei and Methanothrix soehngenii, S. bryantii degraded butyrate faster than in a biculture with only M. hungatei. Hydrogenase and formate dehydrogenase activities were demonstrated in cell-free extracts of S. bryantii.     

Establishment and Development of Ruminal Hydrogenotrophs in Methanogen-Free Lambs

The aim of this work was to determine whether reductive acetogenesis can provide an alternative to methanogenesis in the rumen. Gnotobiotic lambs were inoculated with a functional rumen microbiota lacking methanogens and reared to maturity on a fibrous diet. Lambs with a methanogen-free rumen grew well, and the feed intake and ruminal volatile fatty acid concentrations for lambs lacking ruminal methanogens were lower but not markedly dissimilar from those for conventional lambs reared on the same diet. A high population density (10⁷ to 10⁸ cells g⁻¹) of ruminal acetogens slowly developed in methanogen-free lambs. Sulfate- and fumarate-reducing bacteria were present, but their population densities were highly variable. In methanogen-free lambs, the hydrogen capture from fermentation was low (28 to 46%) in comparison with that in lambs containing ruminal methanogens (>90%). Reductive acetogenesis was not a significant part of ruminal fermentation in conventional lambs but contributed 21 to 25% to the fermentation in methanogen-free meroxenic animals. Ruminal H₂ utilization was lower in lambs lacking ruminal methanogens, but when a methanogen-free lamb was inoculated with a methanogen, the ruminal H₂ utilization was similar to that in conventional lambs. H₂ utilization in lambs containing a normal ruminal microflora was age dependent and increased with the animal age. The animal age effect was less marked in lambs lacking ruminal methanogens. Addition of fumarate to rumen contents from methanogen-free lambs increased H₂ utilization. These findings provide the first evidence from animal studies that reductive acetogens can sustain a functional rumen and replace methanogens as a sink for H₂ in the rumen.     

Methanogenic food web in the gut contents of methane-emitting earthworm Eudrilus eugeniae from Brazil

The anoxic saccharide-rich conditions of the earthworm gut provide an ideal transient habitat for ingested microbes capable of anaerobiosis. It was recently discovered that the earthworm Eudrilus eugeniae from Brazil can emit methane (CH₄) and that ingested methanogens might be associated with this emission. The objective of this study was to resolve trophic interactions of bacteria and methanogens in the methanogenic food web in the gut contents of E. eugeniae. RNA-based stable isotope probing of bacterial 16S rRNA as well as mcrA and mrtA (the alpha subunit of methyl-CoM reductase and its isoenzyme, respectively) of methanogens was performed with [¹³C]-glucose as a model saccharide in the gut contents. Concomitant fermentations were augmented by the rapid consumption of glucose, yielding numerous products, including molecular hydrogen (H₂), carbon dioxide (CO₂), formate, acetate, ethanol, lactate, succinate and propionate. Aeromonadaceae-affiliated facultative aerobes, and obligate anaerobes affiliated to Lachnospiraceae, Veillonellaceae and Ruminococcaceae were associated with the diverse fermentations. Methanogenesis was ongoing during incubations, and ¹³C-labeling of CH₄ verified that supplemental [¹³C]-glucose derived carbon was dissimilated to CH₄. Hydrogenotrophic methanogens affiliated with Methanobacteriaceae and Methanoregulaceae were linked to methanogenesis, and acetogens related to Peptostreptoccocaceae were likewise found to be participants in the methanogenic food web. H₂ rather than acetate stimulated methanogenesis in the methanogenic gut content enrichments, and acetogens appeared to dissimilate supplemental H₂ to acetate in methanogenic enrichments. These findings provide insight on the processes and associated taxa potentially linked to methanogenesis and the turnover of organic carbon in the alimentary canal of methane-emitting E. eugeniae.     

Diversity of H2/CO2-Utilizing Acetogenic Bacteria from Fecesof Non-Methane-Producing Humans

The purpose of this work was to study H₂/CO₂-utilizing acetogenic population in the colons of non-methane-producing individuals harboring low numbers of methanogenic archaea. Among the 50 H₂-consuming acetogenic strains isolated from four fecal samples and an in vitro semi-continuous culture enrichment, with H₂/CO₂ as sole energy source, 20 were chosen for further studies. All isolates were Gram-positive strict anaerobes. Different morphological types were identified, providing evidence of generic diversity. All acetogenic strains characterized used H₂/CO₂ to form acetate as the sole metabolite, following the stoichiometric equation of reductive acetogenesis. These bacteria were also able to use a variety of organic compounds for growth. The major end product of glucose fermentation was acetate, except for strains of cocci that mainly produced lactate. Yeast extract was not necessary, but was stimulatory for growth and acetogenesis from H₂/CO₂. Received: 28 December 1995 / Accepted: 30 January 1996     

Isolation and physiological characterization of Syntrophus buswellii strain GA from a syntrophic benzoate-degrading,strictly anaerobic coculture

A stable, syntrophic benzoate-degrading bacterial consortium was enriched from sewage sludge. It oxidized benzoate or 3-phenylpropionate to acetate, H₂ and CO₂. As hydrogen scavengers Methanospirillum hungatei and Desulfovibrio sp. were present. The benzoate-degrading bacteria of this syntrophic culture and of Syntrophus buswelli were able to grow with benzoate/crotonate or crotonate alone in the absence of a hydrogen-utilizing partner organism. If crotonate was the only substrate, acetate and butyrate were produced, while during growth on benzoate or 3-phenylpropionate crotonate served as a reducible co-substrate and was exclusively converted to butyrate. In the presence of crotonate interspecies hydrogen transfer was not necessary as a hydrogen sink. The benzoate degrader was isolated as a pure culture with crotonate as the only carbon source. The pure culture could also grow with benzoate/crotonate or 3-phenylpropionate/crotonate. The effect of high concentrations of crotonate and of acetate or butyrate on growth of the benzoate degrader was investigated. The benzoate degrader was compared with S. buswellii for its morphology, physiology and DNA base composition. Except for the fact that S. buswellii was also able to grow on cinnamate, no differences between the two organisms were detected. The isolate is named S. buswelli, strain GA.     

Syntrophic interactions among anode respiring bacteria (ARB) and Non‐ARB in a biofilm anode: electron balances

We demonstrate that the coulombic efficiency (CE) of a microbial electrolytic cell (MEC) fueled with a fermentable substrate, ethanol, depended on the interactions among anode respiring bacteria (ARB) and other groups of micro‐organisms, particularly fermenters and methanogens. When we allowed methanogenesis, we obtained a CE of 60%, and 26% of the electrons were lost as methane. The only methanogenic genus detected by quantitative real‐time PCR was the hydrogenotrophic genus, Methanobacteriales, which presumably consumed all the hydrogen produced during ethanol fermentation (～30% of total electrons). We did not detect acetoclastic methanogenic genera, indicating that acetate‐oxidizing ARB out‐competed acetoclastic methanogens. Current production and methane formation increased in parallel, suggesting a syntrophic interaction between methanogens and acetate‐consuming ARB. When we inhibited methanogenesis with 50 mM 2‐bromoethane sulfonic acid (BES), the CE increased to 84%, and methane was not produced. With no methanogenesis, the electrons from hydrogen were converted to electrical current, either directly by the ARB or channeled to acetate through homo‐acetogenesis. This illustrates the key role of competition among the various H₂ scavengers and that, when the hydrogen‐consuming methanogens were present, they out‐competed the other groups. These findings also demonstrate the importance of a three‐way syntrophic relationship among fermenters, acetate‐consuming ARB, and a H₂ consumer during the utilization of a fermentable substrate. To obtain high coulombic efficiencies with fermentable substrates in a mixed population, methanogens must be suppressed to promote new interactions at the anode that ultimately channel the electrons from hydrogen to current. Biotechnol. Bioeng. 2009;103: 513–523. © 2009 Wiley Periodicals, Inc.