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.     

Hydrogen Profiles and Localization of Methanogenic Activities in the Highly Compartmentalized Hindgut of Soil-Feeding Higher Termites (Cubitermes spp.)

It has been shown that the coexistence of methanogenesis and reductive acetogenesis in the hindgut of the wood-feeding termite Reticulitermes flavipes is based largely on the radial distribution of the respective microbial populations and relatively high hydrogen partial pressures in the gut lumen. Using Clark-type microelectrodes, we showed that the situation in Cubitermes orthognathus and other soil-feeding members of the subfamily Termitinae is different and much more complex. All major compartments of agarose-embedded hindguts were anoxic at the gut center, and high H₂ partial pressures (1 to 10 kPa) in the alkaline anterior region rendered the mixed segment and the third proctodeal segment (P3) significant sources of H₂. Posterior to the P3 segment, however, H₂ concentrations were generally below the detection limit (<100 Pa). All hindgut compartments turned into efficient hydrogen sinks when external H₂ was supplied, but methane was formed mainly in the P3/4a and P4b compartments, and in the latter only when H₂ or formate was added. Addition of H₂ to the gas headspace stimulated CH₄ emission of living termites, indicating that endogenous H₂ production limits methanogenesis also in vivo. At the low H₂ partial pressures in the posterior hindgut, methanogens would most likely outcompete homoacetogens for this electron donor. This might explain the apparent predominance of methanogenesis over reductive acetogenesis in the hindgut of soil-feeding termites, although the presence of homoacetogens in the anterior, highly alkaline region cannot yet be excluded. In addition, the direct contact of anterior and posterior hindgut compartments in situ permits a cross-epithelial transfer of H₂ or formate, which would not only fuel methanogenesis in these compartments, but would also create favorable microniches for reductive acetogenesis. In situ rates and spatial distribution of H₂-dependent acetogenic activities are addressed in a companion paper (A. Tholen and A. Brune, Appl. Environ. Microbiol. 65:4497–4505, 1999).     

Hydrogen profiles and localization of methanogenic activities in the highly compartmentalized hindgut of soil-feeding higher termites (Cubitermes spp.).

It has been shown that the coexistence of methanogenesis and reductive acetogenesis in the hindgut of the wood-feeding termite Reticulitermes flavipes is based largely on the radial distribution of the respective microbial populations and relatively high hydrogen partial pressures in the gut lumen. Using Clark-type microelectrodes, we showed that the situation in Cubitermes orthognathus and other soil-feeding members of the subfamily Termitinae is different and much more complex. All major compartments of agarose-embedded hindguts were anoxic at the gut center, and high H(2) partial pressures (1 to 10 kPa) in the alkaline anterior region rendered the mixed segment and the third proctodeal segment (P3) significant sources of H(2). Posterior to the P3 segment, however, H(2) concentrations were generally below the detection limit (<100 Pa). All hindgut compartments turned into efficient hydrogen sinks when external H(2) was supplied, but methane was formed mainly in the P3/4a and P4b compartments, and in the latter only when H(2) or formate was added. Addition of H(2) to the gas headspace stimulated CH(4) emission of living termites, indicating that endogenous H(2) production limits methanogenesis also in vivo. At the low H(2) partial pressures in the posterior hindgut, methanogens would most likely outcompete homoacetogens for this electron donor. This might explain the apparent predominance of methanogenesis over reductive acetogenesis in the hindgut of soil-feeding termites, although the presence of homoacetogens in the anterior, highly alkaline region cannot yet be excluded. In addition, the direct contact of anterior and posterior hindgut compartments in situ permits a cross-epithelial transfer of H(2) or formate, which would not only fuel methanogenesis in these compartments, but would also create favorable microniches for reductive acetogenesis. In situ rates and spatial distribution of H(2)-dependent acetogenic activities are addressed in a companion paper (A. Tholen and A. Brune, Appl. Environ. Microbiol. 65:4497-4505, 1999).     

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.     

Phylogenetic Diversity, Abundance, and Axial Distribution of Bacteria in the Intestinal Tract of Two Soil-Feeding Termites (Cubitermes spp.)

The hindgut of soil-feeding termites is highly compartmentalized and characterized by pronounced axial dynamics of the intestinal pH and microbial processes such as hydrogen production, methanogenesis, and reductive acetogenesis. Nothing is known about the bacterial diversity and the abundance or axial distribution of the major phylogenetic groups in the different gut compartments. In this study, we showed that the variety of physicochemical conditions is reflected in the diversity of the microbial communities in the different gut compartments of two Cubitermes species (Termitidae: Termitinae). 16S rRNA gene clones from the highly alkaline first proctodeal segment (P1) of Cubitermes orthognathus represented almost exclusively gram-positive bacteria with low G+C content (LGC bacteria). In the posterior gut segments, their proportion decreased progressively, and the clone libraries comprised a variety of phyla, including the Cytophaga-Flexibacter-Bacteroides group, various subgroups of Proteobacteria, and the spirochetes. Phylogenetic analysis revealed that many of the clones clustered with sequences from the guts of other termites, and some even formed clusters containing only clones from C. orthognathus. The abundance and axial distribution of major phylogenetic groups in the gut of Cubitermes ugandensis were determined by fluorescence in situ hybridization with group-specific oligonucleotide probes. While the results were generally in good agreement with those of the clonal analysis, direct counts with probes specific for the Planctomycetales revealed a severe underestimation of representatives of this phylum in the clone libraries. Results obtained with newly designed FISH probes directed against two clusters of LGC clones from C. orthognathus indicated that the clones were restricted to specific gut regions. A molecular fingerprinting analysis published in a companion paper (D. Schmitt-Wagner, M. W. Friedrich, B. Wagner, and A. Brune, Appl. Environ. Microbiol. 69:6018-6024, 2003) corroborated the presence of compartment-specific bacterial communities in the gut of different Cubitermes species.     

Coupling glucose fermentation and homoacetogenesis for elevated acetate production: Experimental and mathematical approaches

Homoacetogenesis is an important potential hydrogen sink in acetogenesis, in which hydrogen is used to reduce carbon dioxide to acetate. So far the acetate production from homoacetogenesis, especially its kinetics, has not been given sufficient attention. In this work, enhanced production of acetate from anaerobic conversion of glucose through coupling glucose fermentation and homoacetogenesis is investigated with both experimental and mathematical approaches. Experiments are conducted to explore elevated acetate production in a coupled anaerobic system. Acetate production could be achieved by homoacetogenesis with a relative high acetate yield under mixed fermentation conditions. With the experimental observations, a kinetic model is formulated to describe such a homoacetogenic process. The maximum homoacetogenic rate (k_m,homo) is estimated to be 28.5 ± 1.7 kg COD kg⁻¹ COD day⁻¹ with an uptake affinity constant of 3.7 × 10⁻⁵ ± 3.1 × 10⁻⁶ kg COD m⁻³. The improved calculation of homoacetogenic kinetics by our approach could correct the underestimation of homoacetogenesis in anaerobic fermentation processes, as it often occurs in these systems supported by literature analysis. The model predictions match the experimental results in different cases well and provide insights into the dynamics of anaerobic glucose conversion and acetate production. Furthermore, acetate production via homoacetogenesis increases by about 40% through utilizing the fed‐batch coupling system, attributed to a balance between the hydrogen production in the acetogenesis phase and the hydrogen consumption in the homoacetogenesis phase. This work provides an effective way for increased anaerobic acetate production, and gives us a better understanding about the homoacetogenic kinetics in the anaerobic fermentation process. Biotechnol. Bioeng. 2011;108: 345–353. © 2010 Wiley Periodicals, Inc.     

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.     

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.     

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.     

Cross-Epithelial Hydrogen Transfer from the Midgut Compartment Drives Methanogenesis in the Hindgut of Cockroaches

In the intestinal tracts of animals, methanogenesis from CO₂ and other C₁ compounds strictly depends on the supply of electron donors by fermenting bacteria, but sources and sinks of reducing equivalents may be spatially separated. Microsensor measurements in the intestinal tract of the omnivorous cockroach Blaberus sp. showed that molecular hydrogen strongly accumulated in the midgut (H₂ partial pressures of 3 to 26 kPa), whereas it was not detectable (<0.1 kPa) in the posterior hindgut. Moreover, living cockroaches emitted large quantities of CH₄ [105 ± 49 nmol (g of cockroach)⁻¹ h⁻¹] but only traces of H₂. In vitro incubation of isolated gut compartments, however, revealed that the midguts produced considerable amounts of H₂, whereas hindguts emitted only CH₄ [106 ± 58 and 71 ± 50 nmol (g of cockroach)⁻¹ h⁻¹, respectively]. When ligated midgut and hindgut segments were incubated in the same vials, methane emission increased by 28% over that of isolated hindguts, whereas only traces of H₂ accumulated in the headspace. Radial hydrogen profiles obtained under air enriched with H₂ (20 kPa) identified the hindgut as an efficient sink for externally supplied H₂. A cross-epithelial transfer of hydrogen from the midgut to the hindgut compartment was clearly evidenced by the steep H₂ concentration gradients which developed when ligated fragments of midgut and hindgut were placed on top of each other—a configuration that simulates the situation in vivo. These findings emphasize that it is essential to analyze the compartmentalization of the gut and the spatial organization of its microbiota in order to understand the functional interactions among different microbial populations during digestion.     

Impact of oxygen on metabolic fluxes and in situ rates of reductive acetogenesis in the hindgut of the wood-feeding termite Reticulitermes flavipes

The symbiotic digestion of lignocellulose in the hindgut of the wood-feeding termite Reticulitermes flavipes is characterized by two major metabolic pathways: (i) the oxidation of polysaccharides to acetate by anaerobic hydrogen-producing protozoa; and (ii) the reduction of CO2 by hydrogenotrophic acetogenic bacteria. Both reactions together would render the hindgut largely homoacetogenic. However, the results of this study show that the situation is more complex. By microinjection of radiolabelled metabolites into intact agarose-embedded hindguts, we showed that the in situ rates of reductive acetogenesis (3.3 nmol termite(-1) h(-1)) represent only 10% of the total carbon flux in the living termite, whereas 30% of the carbon flux proceeds via lactate. The rapid turnover of the lactate pool (7.2 nmol termite(-1) h(-1)) consolidates the previously reported presence of lactic acid bacteria in the R. flavipes hindgut and the low lactate concentrations in the hindgut fluid. However, the immediate precursor of lactate remains unknown; the low turnover rates of injected glucose (< 0.5 nmol termite(-1) h(-1)) indicate that free glucose is not an important intermediate under in situ conditions. The influence of the incubation atmosphere on the turnover rate and the product pattern of glucose and lactate confirmed that the influx of oxygen via the gut epithelium and its reduction in the hindgut periphery have a significant impact on carbon and electron flow within the hindgut microbial community. The in situ rates of reductive acetogenesis were not significantly affected by the presence of oxygen or exogenous H2, which is in agreement with a localization of homoacetogens in the anoxic gut lumen rather than in the oxic periphery. This adds strong support to the hypothesis that the co-existence of methanogens and homoacetogens in this termite is based on the spatial arrangement of the different populations of the gut microbiota. A refined model of metabolic fluxes in the hindgut of R. flavipes is presented.     

Acetate Synthesis from H2 plus CO2 by Termite Gut Microbes

Gut microbiota from Reticulitermes flavipes termites catalyzed an H₂-dependent total synthesis of acetate from CO₂. Rates of H₂-CO₂ acetogenesis in vitro were 1.11 ± 0.37 μmol of acetate g (fresh weight)⁻¹ h⁻¹ (equivalent to 4.44 ± 1.47 nmol termite⁻¹ h⁻¹) and could account for approximately 1/3 of all the acetate produced during the hindgut fermentation. Formate was also produced from H₂ + CO₂, as were small amounts of propionate, butyrate, and lactate-succinate. However, H₂-CO₂ formicogenesis seemed largely unrelated to acetogenesis and was believed not to be a significant reaction in situ. Little or no CH₄ was formed from H₂ + CO₂ or from acetate. H₂-CO₂ acetogenesis was inhibited by O₂, KCN, CHCl₃, and iodopropane and could be abolished by prefeeding R. flavipes with antibacterial drugs. By contrast, prefeeding R. flavipes with starch resulted in almost complete defaunation but had little effect on H₂-CO₂ acetogenesis, suggesting that bacteria were the acetogenic agents in the gut. H₂-CO₂ acetogenesis was also observed with gut microbiota from Prorhinotermes simplex, Zootermopsis angusticollis, Nasutitermes costalis, and N. nigriceps; from the wood-eating cockroach Cryptocercus punctulatus; and from the American cockroach Periplaneta americana. Pure cultures of H₂-CO₂-acetogenic bacteria were isolated from N. nigriceps, and a preliminary account of their morphological and physiological properties is presented. Results indicate that in termites, CO₂ reduction to acetate, rather than to CH₄, represents the main electron sink reaction of the hindgut fermentation and can provide the insects with a significant fraction (ca. 1/3) of their principal oxidizable energy source, acetate.     

Trophic link between syntrophic acetogens and homoacetogens during the anaerobic acidogenic fermentation of sewage sludge

Acetate production during anaerobic sludge treatment has significant economic and environmental benefits. In this study, trophic links between syntrophic acetogens and homoacetogens in the anaerobic acidogenic fermentation of sewage sludge were investigated using methanogenic inhibitor 2-bromoethanesulfonate (BES) to block the methanogenesis pathway and butyrate to enhance syntrophic acetogenesis. The Gibbs free energies (ΔG) of the butyrate-degrading and homoacetogenic processes were close to the thermodynamic threshold of the reaction activity (−15 kJ/mol). In addition, microbial quantification analysis revealed that the growth of syntrophic acetogenic bacteria and homoacetogens in the treatment incubations was higher than that of the control. The results indicated that hydrogen-producing butyrate degraders are stimulated with homoacetogens when methanogenesis was specifically inhibited.     

Hydrogen-Dependent Oxygen Reduction by Homoacetogenic Bacteria Isolated from Termite Guts

Although homoacetogenic bacteria are generally considered to be obligate anaerobes, they colonize the intestinal tracts of termites and other environments that are not entirely anoxic in space or time. In this study, we investigated how homoacetogenic bacteria isolated from the hindguts of various termites respond to the presence of molecular oxygen. All strains investigated formed growth bands in oxygen gradient agar tubes under a headspace of H₂-CO₂. The position of the bands coincided with the oxic-anoxic interface and depended on the O₂ partial pressure in the headspace; the position of the bands relative to the meniscus remained stable for more than 1 month. Experiments with dense cell suspensions, performed with Clark-type O₂ and H₂ electrodes, revealed a large capacity for H₂-dependent oxygen reduction in Sporomusa termitida and Sporomusa sp. strain TmAO3 (149 and 826 nmol min⁻¹ mg of protein⁻¹, respectively). Both strains also reduced O₂ with endogenous reductants, albeit at lower rates. Only in Acetonema longum did the basal rates exceed the H₂-dependent rates considerably (181 versus 28 nmol min⁻¹ mg of protein)⁻¹). Addition of organic substrates did not stimulate O₂ consumption in any of the strains. Nevertheless, reductive acetogenesis by cell suspensions of strain TmAO3 was inhibited even at the lowest O₂ fluxes, and growth in nonreduced medium occurred only after the bacteria had rendered the medium anoxic. Similar results were obtained with Acetobacterium woodii, suggesting that the results are not unique to the strains isolated from termites. We concluded that because of their tolerance to temporary exposure to O₂ at low partial pressures (up to 1.5 kPa in the case of strain TmAO3) and because of their large capacity for O₂ reduction, homoacetogens can reestablish conditions favorable for growth by actively removing oxygen from their environment.     

Physicochemical Conditions and Microbial Activities in the Highly Alkaline Gut of the Humus-Feeding Larva of Pachnoda ephippiata (Coleoptera: Scarabaeidae)

The soil macrofauna plays an important role in the carbon and nitrogen cycle of terrestrial ecosystems. In order to gain more insight into the role of the intestinal microbiota in transformation and mineralization of organic matter during gut passage, we characterized the physicochemical conditions, microbial activities, and community structure in the gut of our model organism, the humus-feeding larva of the cetoniid beetle Pachnoda ephippiata. Microsensor measurements revealed an extreme alkalinity in the midgut, with highest values (pH > 10) between the second and third crown of midgut ceca. Both midgut and hindgut were largely anoxic, but despite the high pH, the redox potential of the midgut content was surprisingly high even in the largest instar. However, reducing conditions prevailed in the hindgut paunch of all instars (E_h ~ −100 mV). Both gut compartments possessed a pronounced gut microbiota, with highest numbers in the hindgut, and microbial fermentation products were present in high concentrations. The stimulation of hindgut methanogenesis by exogenous electron donors, such as H₂, formate, and methanol, together with considerable concentrations of formate in midgut and hemolymph, suggests that midgut fermentations are coupled to methanogenesis in the hindgut by an intercompartmental transfer of reducing equivalents via the hemolymph. The results of a cultivation-based enumeration of the major metabolic groups in midgut and hindgut, which yielded high titers of lactogenic, propionigenic, and acetogenic bacteria, are in good agreement not only with the accumulation of microbial fermentation products in the respective compartments but also with the results of a cultivation-independent characterization of the bacterial communities reported in the companion paper (M. Egert, B. Wagner, T. Lemke, A. Brune, and M. W. Friedrich, Appl. Environ. Microbiol. 69:6659-6668, 2003).     

Structure and Topology of Microbial Communities in the Major Gut Compartments of Melolontha melolontha Larvae (Coleoptera: Scarabaeidae)

Physicochemical gut conditions and the composition and topology of the intestinal microbiota in the major gut compartments of the root-feeding larva of the European cockchafer (Melolontha melolontha) were studied. Axial and radial profiles of pH, O₂, H₂, and redox potential were measured with microsensors. Terminal restriction fragment length polymorphism (T-RFLP) analysis of bacterial 16S rRNA genes in midgut samples of individual larvae revealed a simple but variable and probably nonspecific community structure. In contrast, the T-RFLP profiles of the hindgut samples were more diverse but highly similar, especially in the wall fraction, indicating the presence of a gut-specific community involved in digestion. While high acetate concentrations in the midgut and hindgut (34 and 15 mM) corroborated the presence of microbial fermentation in both compartments, methanogenesis was confined to the hindgut. Methanobrevibacter spp. were the only methanogens detected and were restricted to this compartment. Bacterial 16S rRNA gene clone libraries of the hindgut were dominated by clones related to the Clostridiales. Clones related to the Actinobacteria, Bacillales, Lactobacillales, and γ-Proteobacteria were restricted to the lumen, whereas clones related to the β- and δ-Proteobacteria were found only on the hindgut wall. Results of PCR-based analyses and fluorescence in situ hybridization of whole cells with group-specific oligonucleotide probes documented that Desulfovibrio-related bacteria comprise 10 to 15% of the bacterial community at the hindgut wall. The restriction of the sulfate-reducer-specific adenosine-5′-phosphosulfate reductase gene apsA to DNA extracts of the hindgut wall in larvae from four other populations in Europe suggested that sulfate reducers generally colonize this habitat.     

Folate Cross-Feeding Supports Symbiotic Homoacetogenic Spirochetes

Treponema primitia, an H₂-consuming CO₂-reducing homoacetogenic spirochete in termite hindguts, requires an exogenous source of folate for growth. Tetrahydrofolate (THF) acts as a C₁ carrier in CO₂-reductive acetogenesis, a microbially mediated process important to the carbon and energy requirements of termites. To examine the hypothesis that other termite gut microbes probably supply some form of folate to T. primitia in situ, we used a bioassay to screen for and isolate folate-secreting bacteria from hindguts of Zootermopsis angusticollis, which is the host of T. primitia. Based on morphology, physiology, and 16S rRNA gene sequences, the major folate secretors were identified as strains of Lactococcus lactis and Serratia grimesii. During growth, these isolates secreted 5-formyl-THF at levels up to 146 ng/ml, and their cell-free culture fluids satisfied the folate requirement of T. primitia strains in vitro. Analysis of Z. angusticollis hindgut fluid revealed that 5-formyl-THF was the only detectable folate compound and occurred at an in situ concentration (1.3 μg/ml) which was more than sufficient to support the growth of T. primitia. These results imply that cross-feeding of 5-formyl-THF by other community members is important for growth of symbiotic hindgut spirochetes and thus termite nutrition and survival.     

Physiology and Nutrition of Treponema primitia, an H2/ CO2-Acetogenic Spirochete from Termite Hindguts

Treponema primitia strains ZAS-1 and ZAS-2, the first spirochetes to be isolated from termite hindguts (J. R. Leadbetter, T. M. Schmidt, J. R. Graber, and J. A. Breznak, Science 283:686-689, 1999), were examined for nutritional, physiological, and biochemical properties relevant to growth and survival in their natural habitat. In addition to using H₂ plus CO₂ as substrates, these strains were capable of homoacetogenic growth on mono- and disaccharides and (in the case of ZAS-2) methoxylated benzenoids. Cells were also capable of mixotrophic growth (i.e., simultaneous utilization of H₂ and organic substrates). Cell extracts of T. primitia possessed enzyme activities of the Wood/Ljungdahl (acetyl coenzyme A) pathway of acetogenesis, including tetrahydrofolate-dependent enzymes of the methyl group-forming branch. However, a folate compound was required in the medium for growth. ZAS-1 and ZAS-2 growing on H₂ plus CO₂ displayed H₂ thresholds of 650 and 490 ppmv, respectively. Anoxic cultures of ZAS-1 and ZAS-2 maintained growth after the addition of as much as 0.5% (vol/vol) O₂ to the headspace atmosphere. Cell extracts exhibited NADH and NADPH peroxidase and NADH oxidase activities but neither catalase nor superoxide dismutase activity. Results indicate that (i) T. primitia is able to exploit a variety of substrates derived from the food of its termite hosts and in so doing contributes to termite nutrition via acetogenesis, (ii) in situ growth of T. primitia is likely dependent on secretion of a folate compound(s) by other members of the gut microbiota, and (iii) cells possess enzymatic adaptations to oxidative stress, which is likely to be encountered in peripheral regions of the termite hindgut.     

Volatile Fatty Acid Production by the Hindgut Microbiota of Xylophagous Termites

Acetate dominated the extracellular pool of volatile fatty acids (VFAs) in the hindgut fluid of Reticulitermes flavipes, Zootermopsis angusticollis, and Incisitermes schwarzi, where it occurred at concentrations of 57.9 to 80.6 mM and accounted for 94 to 98 mol% of all VFAs. Small amounts of C₃ to C₅ VFAs were also observed. Acetate was also the major VFA in hindgut homogenates of Schedorhinotermes lamanianus, Prorhinotermes simplex, Coptotermes formosanus, and Nasutitermes corniger. Estimates of in situ acetogenesis by the hindgut microbiota of R. flavipes (20.2 to 43.3 nmol · termite⁻¹ · h⁻¹) revealed that this activity could support 77 to 100% of the respiratory requirements of the termite (51.6 to 63.6 nmol of O₂ · termite⁻¹ · h⁻¹). This conclusion was buttressed by the demonstration of acetate in R. flavipes hemolymph (at 9.0 to 11.6 mM), but not in feces, and by the ability of termite tissues to readily oxidize acetate to CO₂. About 85% of the acetate produced by the hindgut microbiota was derived from cellulose C; the remainder was derived from hemicellulose C. Selective removal of major groups of microbes from the hindgut of R. flavipes indicated that protozoa were primarily responsible for acetogenesis but that bacteria also functioned in this capacity. H₂ and CH₄ were evolved by R. flavipes (usually about 0.4 nmol · termite⁻¹ · h⁻¹), but these compounds represented a minor fate of electrons derived from wood dissimilation within R. flavipes. A working model is proposed for symbiotic wood polysaccharide degradation in R. flavipes, and the possible roles of individual gut microbes, including CO₂-reducing acetogenic bacteria, are discussed.     

Effect of Temperature on Carbon and Electron Flow and on the Archaeal Community in Methanogenic Rice Field Soil

Temperature is an important factor controlling CH₄ production in anoxic rice soils. Soil slurries, prepared from Italian rice field soil, were incubated anaerobically in the dark at six temperatures of between 10 to 37°C or in a temperature gradient block covering the same temperature range at intervals of 1°C. Methane production reached quasi-steady state after 60 to 90 days. Steady-state CH₄ production rates increased with temperature, with an apparent activation energy of 61 kJ mol⁻¹. Steady-state partial pressures of the methanogenic precursor H₂ also increased with increasing temperature from <0.5 to 3.5 Pa, so that the Gibbs free energy change of H₂ plus CO₂-dependent methanogenesis was kept at −20 to −25 kJ mol of CH₄⁻¹ over the whole temperature range. Steady-state concentrations of the methanogenic precursor acetate, on the other hand, increased with decreasing temperature from <5 to 50 μM. Simultaneously, the relative contribution of H₂ as methanogenic precursor decreased, as determined by the conversion of radioactive bicarbonate to ¹⁴CH₄, so that the carbon and electron flow to CH₄ was increasingly dominated by acetate, indicating that psychrotolerant homoacetogenesis was important. The relative composition of the archaeal community was determined by terminal restriction fragment length polymorphism (T-RFLP) analysis of the 16S rRNA genes (16S rDNA). T-RFLP analysis differentiated the archaeal Methanobacteriaceae, Methanomicrobiaceae, Methanosaetaceae, Methanosarcinaceae, and Rice clusters I, III, IV, V, and VI, which were all present in the rice field soil incubated at different temperatures. The 16S rRNA genes of Rice cluster I and Methanosaetaceae were the most frequent methanogenic groups. The relative abundance of Rice cluster I decreased with temperature. The substrates used by this microbial cluster, and thus its function in the microbial community, are unknown. The relative abundance of acetoclastic methanogens, on the other hand, was consistent with their physiology and the acetate concentrations observed at the different temperatures, i.e., the high-acetate-requiring Methanosarcinaceae decreased and the more modest Methanosaetaceae increased with increasing temperature. Our results demonstrate that temperature not only affected the activity but also changed the structure and the function (carbon and electron flow) of a complex methanogenic system.