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.     

Methanogen Colonisation Does Not Significantly Alter Acetogen Diversity in Lambs Isolated 17?h After Birth and Raised Aseptically

Reductive acetogenesis is not competitive with methanogenesis in adult ruminants, whereas acetogenic bacteria are the dominant hydrogenotrophs in the early rumen microbiota. The ecology of hydrogenotrophs in the developing rumen was investigated using young lambs, raised in sterile isolators, and conventional adult sheep. Two lambs were born naturally, left with their dams for 17?h and then placed into a sterile isolator and reared aseptically. They were inoculated with cellulolytic bacteria and later with Methanobrevibacter sp. 87.7 to investigate the effect of methanogen establishment on the rumen acetogen population since they lacked cultivable representatives of methanogens. Putative acetogens were investigated by acetyl-CoA synthase and formyltetrahydrofolate synthetase gene analysis and methanogens by methyl coenzyme reductase A gene analysis. Unexpectedly, a low abundant but diverse population of methanogens (predominantly Methanobrevibacter spp.) was identified in isolated lambs pre-inoculation with Mbb. sp 87.7, which was similar to the community structure in conventional sheep. In contrast, potential acetogen diversity in isolated lambs and conventional sheep was different. Potential acetogens affiliated between the Lachnospiraceae and Clostridiaceae in conventional sheep and with the Blautia genus and the Lachnospiraceae in isolated lambs. The establishment of Mbb. sp. 87.7 (1,000-fold increase in methanogens) did not substantially affect acetogen diversity.     

Influence of the composition of the cellulolytic flora on the development of hydrogenotrophic microorganisms, hydrogen utilization, and methane production in the rumens of gnotobiotically reared lambs

We investigated the influence of the composition of the fibrolytic microbial community on the development and activities of hydrogen-utilizing microorganisms in the rumens of gnotobiotically reared lambs. Two groups of lambs were reared. The first group was inoculated with Fibrobacter succinogenes, a non-H(2)-producing species, as the main cellulolytic organism, and the second group was inoculated with Ruminococcus albus, Ruminococcus flavefaciens, and anaerobic fungi that produce hydrogen. The development of hydrogenotrophic bacterial communities, i.e., acetogens, fumarate and sulfate reducers, was monitored in the absence of methanogens and after inoculation of methanogens. Hydrogen production and utilization and methane production were measured in rumen content samples incubated in vitro in the presence of exogenous hydrogen (supplemented with fumarate or not supplemented with fumarate) or in the presence of ground alfalfa hay as a degradable substrate. Our results show that methane production was clearly reduced when the dominant fibrolytic species was a non-H(2)-producing species, such as Fibrobacter succinogenes, without significantly impairing fiber degradation and fermentations in the rumen. The addition of fumarate to the rumen contents stimulated H(2) utilization only by the ruminal microbiota inoculated with F. succinogenes, suggesting that these communities could play an important role in fumarate reduction in vivo.     

The immune response to Lactococcus lactis: Implications for its use as a vaccine delivery vehicle

Abstract The development of hydrogenotrophic bacteria in the rumen of lambs was investigated by culture and labeling experiments. ¹⁴CO₂ and ¹³CO₂ incorporation by the rumen microflora of a 24-h-old lamb showed that while there was no labeled methane, double-labeled acetate was formed indicating the presence of hydrogen-dependent acetogenesis. In vitro counts from rumen fluid of 20-h-old lambs confirmed an extensive colonization of acetogenic bacteria while methanogens were absent. Methanogens appeared in the rumen of 30-h-old lambs, and as they developed there was a proportional decrease in the numbers of acetogens, indicating a competition for hydrogen between these two groups. Hydrogen-utilizing sulfate-reducing bacteria, which were established by the 3rd day after birth, did not seem to be affected by this competition.     

Diversity of the Formyltetrahydrofolate Synthetase Gene (fhs), a Key Enzyme for Reductive Acetogenesis,in the Bovine Rumen

A clone library of the partial formyltetrahydrofolate synthetase gene (fhs), a key enzyme in reductive acetogenesis, was constructed from the DNA of bovine rumen contents. Diverse sequences were recovered, the majority of which were clustered with the fhs of authentic acetogens. Low similarity values to known fhs were observed in all sequences, suggesting the presence of unknown acetogens.     

Assessment of Reductive Acetogenesis with Indigenous Ruminal Bacterium Populations and Acetitomaculum ruminis

The objective of this study was to evaluate the role of reductive acetogenesis as an alternative H₂ disposal mechanism in the rumen. H₂/CO₂-supported acetogenic ruminal bacteria were enumerated by using a selective inhibitor of methanogenesis, 2-bromoethanesulfonic acid (BES). Acetogenic bacteria ranged in density from 2.5 × 10⁵ cells/ml in beef cows fed a high-forage diet to 75 cells/ml in finishing steers fed a high-grain diet. Negligible endogenous acetogenic activity was demonstrated in incubations containing ruminal contents, NaH¹³CO₃, and 100% H₂ gas phase since [U-¹³C]acetate, as measured by mass spectroscopy, did not accumulate. Enhancement of acetogenesis was observed in these incubations when methanogenesis was inhibited by BES and/or by the addition of an axenic culture of the rumen acetogen Acetitomaculum ruminis 190A4 (10⁷ CFU/ml). To assess the relative importance of population density and/or H₂ concentration for reductive acetogenesis in ruminal contents, incubations as described above were performed under a 100% N₂ gas phase. Both selective inhibition of methanogenesis and A. ruminis 190A4 fortification (>10⁵ CFU/ml) were necessary for the detection of reductive acetogenesis under H₂-limiting conditions. Under these conditions, H₂ accumulated to 4,800 ppm. In contrast, H₂ accumulated to 400 ppm in incubations with active methanogenesis (without BES). These H₂ concentrations correlated well with the pure culture H₂ threshold concentrations determined for A. ruminis 190A4 (3,830 ppm) and the ruminal methanogen 10-16B (126 ppm). The data demonstrate that ruminal methanogenic bacteria limited reductive acetogenesis by lowering the H₂ partial pressure below the level necessary for H₂ utilization by A. ruminis 190A4.     

瘤胃甲烷菌及甲烷生成的调控

甲烷菌属于古细菌 ,参与有机物的厌氧降解 ,生成甲烷。反刍动物瘤胃内甲烷的生成损耗 2 %～ 12 %的饲料能量 ,并且通过嗳气排入大气。甲烷不仅是温室气体之一 ,而且还会破坏大气臭氧层。每年全球反刍动物排放大量的甲烷 ,减少瘤胃内甲烷的生成对提高饲料能量利用率和改善环境具有重要意义。近年来 ,有关瘤胃甲烷菌及甲烷生成调控的报道日益增多。概述甲烷菌的特性以及瘤胃内甲烷生成的途径 ,综述甲烷生成的调控手段 ,主要包括去原虫、日粮配合、添加电子受体、增加乙酸生成菌等方法     

Functional gene analysis suggests different acetogen populations in the bovine rumen and tammar wallaby forestomach

Reductive acetogenesis via the acetyl coenzyme A (acetyl-CoA) pathway is an alternative hydrogen sink to methanogenesis in the rumen. Functional gene-based analysis is the ideal approach for investigating organisms capable of this metabolism (acetogens). However, existing tools targeting the formyltetrahydrofolate synthetase gene (fhs) are compromised by lack of specificity due to the involvement of formyltetrahydrofolate synthetase (FTHFS) in other pathways. Acetyl-CoA synthase (ACS) is unique to the acetyl-CoA pathway and, in the present study, acetyl-CoA synthase genes (acsB) were recovered from a range of acetogens to facilitate the design of acsB-specific PCR primers. fhs and acsB libraries were used to examine acetogen diversity in the bovine rumen and forestomach of the tammar wallaby (Macropus eugenii), a native Australian marsupial demonstrating foregut fermentation analogous to rumen fermentation but resulting in lower methane emissions. Novel, deduced amino acid sequences of acsB and fhs affiliated with the Lachnospiraceae in both ecosystems and the Ruminococcaeae/Blautia group in the rumen. FTHFS sequences that probably originated from nonacetogens were identified by low "homoacetogen similarity" scores based on analysis of FTHFS residues, and comprised a large proportion of FTHFS sequences from the tammar wallaby forestomach. A diversity of FTHFS and ACS sequences in both ecosystems clustered between the Lachnospiraceae and Clostridiaceae acetogens but without close sequences from cultured isolates. These sequences probably originated from novel acetogens. The community structures of the acsB and fhs libraries from the rumen and the tammar wallaby forestomach were different (LIBSHUFF, P < 0.001), and these differences may have significance for overall hydrogenotrophy in both ecosystems.     

16S ribosomal DNA-directed PCR primers for ruminal methanogens and identification of methanogens colonising young lambs

The population densities and identities of methanogens colonising new-born lambs in a grazing flock were determined from rumen samples collected at regular intervals after birth. Methanogen colonisation was found at the first sampling (1-3 days after birth) and population densities reached around 10(4) methanogens per gram at 1 week of age. Population densities increased in an exponential manner to a maximum of 10(8)-10(9) per gram at 3 weeks of age. To identify methanogens, PCR primers specific for each of the Archaea; a grouping of the orders Methanomicrobiales, Methanosarcinales and Methanococcales; the order Methanobacteriales; the order Methanococcales; the order Methanosarcinales; the genus Methanobacterium; and the genus Methanobrevibacter were designed. Primer-pair specificities were confirmed in tests with target and non-target micro-organisms. PCR analysis of DNA extracts revealed that all the detectable ruminal methanogens belonged to the order Methanobacteriales, with no methanogens belonging to the Methanomicrobiales, the Methanosarcinales, or the Methanococcales being detected. In 3 lambs, the initial colonising methanogens were Methanobrevibacter spp. and in 2 lambs were a mixture of Methanobrevibacter and Methanobacterium spp. In the latter case, the initial colonising Methanobacterium spp. subsequently disappeared and were not detectable 12-19 days after birth. Seven weeks after birth, lambs contained only Methanobrevibacter spp. This study, the first to provide information on the identities of methanogens colonising pre-ruminants, suggests that the predominant methanogens found in the mature rumen establish very soon after birth and well before a functioning rumen develops.     

Effect of 2-bromoethanesulfonic acid and Peptostreptococcus productus ATCC 35244 addition on stimulation of reductive acetogenesis in the ruminal ecosystem by selective inhibition of methanogenesis.

Evidence is provided that reductive acetogenesis can be stimulated in ruminal samples during short-term (24-h) incubations when methanogenesis is inhibited selectively. While addition of the reductive acetogen Peptostreptococcus productus ATCC 35244 alone had no significant influence on CH4 and volatile fatty acid (VFA) production in ruminal samples, the addition of this strain together with 2-bromoethanesulfonic acid (BES) (final concentration, 0.01 or 0.03 mM) resulted in stimulation of acetic acid production and H2 consumption. Since acetate production exceeded amounts that could be attributed to reductive acetogenesis, as measured by H2 consumption, it was found that P. productus also fermented C6 units (glucose and fructose) heterotrophically to mainly acetate (> 99% of the total VFA). Using 14CH3COOH, we concluded that addition of BES and BES plus P. productus did not alter the consumption of acetate in ruminal samples. The addition of P. productus to BES-treated ruminal samples caused supplemental inhibition of CH4 production and stimulation of VFA production, representing a possible energy gain of about 13 to 15%.     

Development of the cellulolytic microflora in the rumen of lambs transferred into sterile isolators a few days after birth

Seven lambs, separated from their dam 24 h after birth, were kept in a conventional environment until transferred to sterile isolators between 1 and 9 days of age: two on day 1 (IA and IB), two on day 4 (IVA and IVB), one on day 8 (VIIIA), and two on day 9 (IXA and IXB). The lambs were reared in these isolators until 120 days of age. Lambs IA, IB, IXA, and IXB were free of cellulolytic bacteria when they were placed in the isolators. They were then inoculated with Bacteroides succinogenes S85 which became established in the four lambs. Until the age of 2 months, the population of this strain fluctuated and then stabilized at a high level (10(8)-10(9) cells/mL). Cellulolytic bacteria were present in the rumen of lambs IVA, IVB, and VIIIA when they were transferred to the isolators. In IVA, and IVB, the cellulolytic population slowly increased with the animal age. In contrast, in VIIIA, the cellulolytic bacteria disappeared within a few days. Bacteroides succinogenes S85 inoculated thereafter became established rapidly and reached a level comparable to that observed in lambs IA and IB. The total number of viable rumen bacteria in the isolated lambs was similar to that observed in conventionally raised animals, but differences were observed in the selective enumeration of bacteria utilizing specific energy sources.     

Effect of Methanobrevibacter sp MF1 inoculation on glycoside hydrolase and polysaccharide depolymerase activities, wheat straw degradation and volatile fatty acid concentrations in the rumen of gnotobiotically-reared lambs

Four naturally born lambs were placed in sterile isolators 24 h after birth before the natural establishment of cellulolytic microorganisms and archaea methanogens. At the age of 6 weeks they were inoculated with pure cultures of the strains FD1 and 007 of Ruminococcus flavefaciens and at the age of 4 months with a pure culture of Methanobrevibacter sp. MF1. Following the establishment of MF1, the population of R. flavefaciens slightly increased in the rumen of the four lambs, there was also an increase in straw degradation, in the activity of some glycoside and polysaccharide hydrolases of the adherent microbial populations and in the concentration of acetate in ruminal contents.     

Methanogens and Methanogenesis in the Rumens and Ceca of Lambs Fed Two Different High-Grain-Content Diets

The amount and nature of dietary starch are known to influence the extent and site of feed digestion in ruminants. However, how starch degradability may affect methanogenesis and methanogens along the ruminant''s digestive tract is poorly understood. This study examined the diversity and metabolic activity of methanogens in the rumen and cecum of lambs receiving wheat or corn high-grain-content diets. Methane production in vivo and ex situ was also monitored. In vivo daily methane emissions (CH₄ g/day) were 36% (P < 0.05) lower in corn-fed lambs than in wheat-fed lambs. Ex situ methane production (μmol/h) was 4-fold higher for ruminal contents than for cecal contents (P < 0.01), while methanogens were 10-fold higher in the rumen than in the cecum (mcrA copy numbers; P < 0.01). Clone library analysis indicated that Methanobrevibacter was the dominant genus in both sites. Diet induced changes at the species level, as the Methanobrevibacter millerae-M. gottschalkii-M. smithii clade represented 78% of the sequences from the rumen of wheat-fed lambs and just about 52% of the sequences from the rumen of the corn-fed lambs. Diet did not affect mcrA expression in the rumen. In the cecum, however, expression was 4-fold and 2-fold lower than in the rumen for wheat- and corn-fed lambs, respectively. Though we had no direct evidence for compensation of reduced rumen methane production with higher cecum methanogenesis, the ecology of methanogens in the cecum should be better considered.     

Effect ofMethanobrevibactersp MF1 Inoculation on Glycoside Hydrolase and Polysaccharide Depolymerase Activities, Wheat Straw Degradation and Volatile Fatty Acid Concentrations in the Rumen of Gnotobiotically-reared Lambs

Four naturally born lambs were placed in sterile isolators 24 h after birth before the natural establishment of cellulolytic microorganisms and archaea methanogens. At the age of 6 weeks they were inoculated with pure cultures of the strains FD1 and 007 ofRuminococcus flavefaciensand at the age of 4 months with a pure culture ofMethanobrevibactersp. MF1. Following the establishment of MF1, the population ofR. flavefaciensslightly increased in the rumen of the four lambs, there was also an increase in straw degradation, in the activity of some glycoside and polysaccharide hydrolases of the adherent microbial populations and in the concentration of acetate in ruminal contents.     

Acetogenesis from H2 and CO2 by methane- and non-methane-producing human colonic bacterial communities

Abstract: The purpose of the study was to define the potential for reductive acetogenesis of colonic microflora from six non-methane- and four methane-excreting human subjects in relation to numbers of the different H₂-utilizing microorganisms. Faecal bacterial suspensions were incubated in the presence of NaH¹³CO₃ and under a gas phase composed of either 100% N₂ (control) or 80% H₂–20% N₂. The effects of a specific methanogenesis inhibitor or of sulfate supplementation were also determined. Quantitative nuclear magnetic resonance showed the presence of both single- and double-labelled acetate in all incubations under hydrogen. H₂/CO₂-acetogenesis appears to be a quantitatively important activity only in the presence of very low numbers of methanogens. Inhibition of methanogenesis induced a large increase in ¹³CO₂ incorporation into acetate in CH₄-producing samples. These results showed that methanogens can efficiently outcompete acetogens in human colonic contents. In contrast, no clear-cut competition for H₂ between acetogenesis and dissimilatory sulfate-reduction could be demonstrated. A slight reduction of the acetogenic activity was only observed at the highest sulfate addition (100 mM).     

Influence of ciliate protozoa on biochemical changes and hydrolytic enzyme profile in the rumen ecosystem

AIMS: To assess the effect of presence or absence of rumen protozoa on fermentation characteristics and enzyme profile in growing lambs. METHODS AND RESULTS: Weaner lambs (G1, G2, G3, G4, G5 and G6 groups) were defaunated by oral administration of sodium laurel sulphate (at 8 g 100 kg(-1) body weight). The lambs of G4, G5 and G6 groups were refaunated. The roughage and concentrate ratio in the diet of G1 and G4, G2 and G5, and G3 and G6 were 50:50 (R1), 65:35 (R2) and 80:20 (R3), respectively. Daily dry matter intake was similar in defaunated and faunated lambs. However, digestibility of organic matter (OM), cellulose and gross energy were lower in defaunated lambs while crude protein (CP) digestibility was similar in both defaunated and faunated lambs. The rumen pH and NH3-N were lower (P < 0.01) while TVFA, total-N and TCA-ppt-N were higher (P < 0.01), in defaunated lambs. Ruminal activity of carboxymethyl cellulase was lower (P < 0.01) in defaunated lambs and amylase, xylanase, protease and urease were similar in faunated and defaunated lambs. Nutrient utilization, rumen metabolites and ciliate protozoal count were higher, whereas digestibility of fibre fractions was lower in high rather than low concentrate fed lambs. The rumen protozoa present before defaunation were B-type and the protozoa which re-established on refaunation were also B-type. CONCLUSIONS: Absence of ciliate protozoa decreased nutrient digestibility and increased ruminal TVFA and total-N with lower NH3-N concentration, indicating better energy and protein utilization in defaunated lambs. SIGNIFICANCE AND IMPACT OF THE STUDY: Defaunation improved energy and protein utilization in lambs.     

H2/CO2 metabolism in acetogenic bacteria isolated from the human colon

The present work reports on autotrophic metabolism in four H2/CO2-utilizing acetogenic bacteria isolated from the human colon (two Clostridium species, one Streptococcus species, and Ruminococcus hydrogenotrophicus). H2/CO2-utilization by these human acetogenic strains occurred during both exponential and stationary phases of growth. Acetate was the major metabolite produced by all isolates following the stoichiometric equation of reductive acetogenesis. Furthermore, the ability of these acetogenic bacteria to incorporate 13CO2 into acetate in the presence of H2 in the gas phase demonstrated the utilization of the reductive pathway of acetate formation from a one-carbon compound. Energy conservation during the autotrophic metabolism in colonic acetogens might involve sodium- or proton-chemiosmotic mechanisms. A sodium-dependent ATP generation was only demonstrated in one Clostridium species, whereas sodium could be replaced by potassium in other strains. The minimal thresholds of hydrogen uptake were determined and varied from 1100 to 3680 ppm depending on the acetogenic strain. These values appeared higher than those measured for the colonic methanogen,Methanobrevibacter smithii.     

Spatial succession and metabolic properties of functional microbial communities in an anaerobic baffled reactor

The spatial successions of bacterial and archaeal communities in anaerobic digestion were investigated in a glucose-degrading five-compartment anaerobic baffled reactor (ABR). The distributions of H₂-producing acetogens, H₂-utilizing acetogens and methanogens in different anaerobic-digestion stages were quantitatively analyzed using functional probes. The results show that the acidogenesis stage and acetogenesis stage were located in the first two compartments, while the methanogenesis were located in the last two compartments. In acidogenesis/acetogenesis stage of anaerobic digestion, H₂-producing acetogens (19.7%) and H₂-utilizing acetogens (8.3%) were the dominant bacterial community. While in methanogenesis stage, methanogens became the dominant (40.2%) with H₂-producing acetogens and H₂-utilizing acetogens only accounting for 6.6% and 4.8%, respectively. With the bacterial population decreasing from 7.2 ± 0.5 × 10¹² cells mL⁻¹ to 0.6 ± 0.3 × 10¹² cells mL⁻¹ along water flowing direction, their diversity increased from 2.79 to 299. The acidogenic bacteria, such as Lactococcus sp., Uncultured Firmicutes bacterium, and Uncultured Clostridium sp., etc., dominated in the acidogenesis/acetogenesis stage, while Uncultured Desulfobacterales bacterium became dominant in the methanogenesis stage. A two-stage anaerobic process may be suitable for easily degradable organic matters removal.     

Effect of tea saponin on methanogenesis,microbial community structure and expression of mcrA gene,in cultures of rumen micro‐organisms

Aims: To determine the in‐vitro effect and mode of action of tea saponin on the rumen microbial community and methane production. Methods and Results: Saponin extracted from tea seeds was added to (1) an in‐vitro fermentation inoculated with rumen fluid and (2) a pure culture of Methanobrevibacter ruminantium. Methane production and expression of the methyl coenzyme‐M reductase subunit A (mcrA) were monitored in both cultures. Abundance of methanogens, protozoa, rumen fungi and cellulolytic bacteria were quantified using real‐time PCR, and bacterial diversity was observed using denaturing gradient gel electrophoresis. Addition of tea saponin significantly reduced methane production and mcrA gene expression in the ruminal fermentation but not with the pure culture of M. ruminantium. The abundance of protozoa and fungi were significantly decreased 50% and 79% respectively but methanogen numbers were not affected, and Fibrobacter succinogenes increased by 41%. Bacterial diversity was similar in cultures with or without tea saponin. Conclusions: Tea saponin appeared to reduce methane production by inhibiting protozoa and presumably lowering methanogenic activity of protozoal‐associated methanogens. Significance and Impact of the Study: Tea saponin may be useful as a supplement to indirectly inhibit methane production in ruminants without a deleterious effect on rumen function.     

Localization and in situ activities of homoacetogenic bacteria in the highly compartmentalized hindgut of soil-feeding higher termites (Cubitermes spp.).

Methanogenesis and homoacetogenesis occur simultaneously in the hindguts of almost all termites, but the reasons for the apparent predominance of methanogenesis over homoacetogenesis in the hindgut of the humivorous species is not known. We found that in gut homogenates of soil-feeding Cubitermes spp., methanogens outcompete homoacetogens for endogenous reductant. The rates of methanogenesis were always significantly higher than those of reductive acetogenesis, whereas the stimulation of acetogenesis by the addition of exogenous H(2) or formate was more pronounced than that of methanogenesis. In a companion paper, we reported that the anterior gut regions of Cubitermes spp. accumulated hydrogen to high partial pressures, whereas H(2) was always below the detection limit (<100 Pa) in the posterior hindgut, and that all hindgut compartments turned into efficient H(2) sinks when external H(2) was provided (D. Schmitt-Wagner and A. Brune, Appl. Environ. Microbiol. 65:4490-4496, 1999). Using a microinjection technique, we found that only the posterior gut sections P3/4a and P4b, which harbored methanogenic activities, formed labeled acetate from H(14)CO(3)(-). Enumeration of methanogenic and homoacetogenic populations in the different gut sections confirmed the coexistence of both metabolic groups in the same compartments. However, the in situ rates of acetogenesis were strongly hydrogen limited; in the P4b section, no activity was detected unless external H(2) was added. Endogenous rates of reductive acetogenesis in isolated guts were about 10-fold lower than the in vivo rates of methanogenesis, but were almost equal when exogenous H(2) was supplied. We conclude that the homoacetogenic populations in the posterior hindgut are supported by either substrates other than H(2) or by a cross-epithelial H(2) transfer from the anterior gut regions, which may create microniches favorable for H(2)-dependent acetogenesis.