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).     

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.     

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 H₂ 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 H₂ was always below the detection limit (<100 Pa) in the posterior hindgut, and that all hindgut compartments turned into efficient H₂ sinks when external H₂ 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 H¹⁴CO₃⁻. 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 H₂ 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 H₂ was supplied. We conclude that the homoacetogenic populations in the posterior hindgut are supported by either substrates other than H₂ or by a cross-epithelial H₂ transfer from the anterior gut regions, which may create microniches favorable for H₂-dependent acetogenesis.     

Nitrate reduction, nitrous oxide formation, and anaerobic ammonia oxidation to nitrite in the gut of soil-feeding termites (Cubitermes and Ophiotermes spp.)

Soil-feeding termites play important roles in the dynamics of carbon and nitrogen in tropical soils. Through the mineralization of nitrogenous humus components, their intestinal tracts accumulate enormous amounts of ammonia, and nitrate and nitrite concentrations are several orders of magnitude above those in the ingested soil. Here, we studied the metabolism of nitrate in the different gut compartments of two Cubitermes and one Ophiotermes species using (15)N isotope tracer analysis. Living termites emitted N(2) at rates ranging from 3.8 to 6.8 nmol h(-1) (g fresh wt.)(-1). However, in homogenates of individual gut sections, denitrification was restricted to the posterior hindgut, whereas nitrate ammonification occurred in all gut compartments and was the prevailing process in the anterior gut. Potential rates of nitrate ammonification for the entire intestinal tract were tenfold higher than those of denitrification, implying that ammonification is the major sink for ingested nitrate in the intestinal tract of soil-feeding termites. Because nitrate is efficiently reduced already in the anterior gut, reductive processes in the posterior gut compartments must be fuelled by an endogenous source of oxidized nitrogen species. Quite unexpectedly, we observed an anaerobic oxidation of (15)N-labelled ammonia to nitrite, especially in the P4 section, which is presumably driven by ferric iron; nitrification and anammox activities were not detected. Two of the termite species also emitted substantial amounts of N(2) O, ranging from 0.4 to 3.9 nmol h(-1) (g fresh wt.)(-1), providing direct evidence that soil-feeding termites are a hitherto unrecognized source of this greenhouse gas in tropical soils.     

High-resolution analysis of gut environment and bacterial microbiota reveals functional compartmentation of the gut in wood-feeding higher termites (Nasutitermes spp.)

Higher termites are characterized by a purely prokaryotic gut microbiota and an increased compartmentation of their intestinal tract. In soil-feeding species, each gut compartment has different physicochemical conditions and is colonized by a specific microbial community. Although considerable information has accumulated also for wood-feeding species of the genus Nasutitermes, including cellulase activities and metagenomic data, a comprehensive study linking physicochemical gut conditions with the structure of the microbial communities in the different gut compartments is lacking. In this study, we measured high-resolution profiles of H(2), O(2), pH, and redox potential in the gut of Nasutitermes corniger termites, determined the fermentation products accumulating in the individual gut compartments, and analyzed the bacterial communities in detail by pyrotag sequencing of the V3-V4 region of the 16S rRNA genes. The dilated hindgut paunch (P3 compartment) was the only anoxic gut region, showed the highest density of bacteria, and accumulated H(2) to high partial pressures (up to 12 kPa). Molecular hydrogen is apparently produced by a dense community of Spirochaetes and Fibrobacteres, which also dominate the gut of other Nasutitermes species. All other compartments, such as the alkaline P1 compartment (average pH, 10.0), showed high redox potentials and comprised small but distinct populations characteristic for each gut region. In the crop and the posterior hindgut compartments, the community was even more diverse than in the paunch. Similarities in the communities of the posterior hindgut and crop suggested that proctodeal trophallaxis or coprophagy also occurs in higher termites. The large sampling depths of pyrotag sequencing in combination with the determination of important physicochemical parameters allow cautious conclusions concerning the functions of particular bacterial lineages in the respective gut sections to be drawn.     

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.     

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.     

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.     

Expression profiles of fhs (FTHFS) genes support the hypothesis that spirochaetes dominate reductive acetogenesis in the hindgut of lower termites

Reductive acetogenesis is an important metabolic process in the hindgut of wood-feeding termites. We analysed diversity and expression profiles of the bacterial fhs gene, a marker gene encoding a key enzyme of reductive acetogenesis, formyl tetrahydrofolate synthetase (FTHFS), to identify the active homoacetogenic populations in representatives of three different termite families. Clone libraries of polymerase chain reaction-amplified fhs genes from hindgut contents of Reticulitermes santonensis (Rhinotermitidae) and Cryptotermes secundus (Kalotermitidae) were compared with previously published fhs gene sequences obtained from Zootermopsis nevadensis (Termopsidae). Most of the clones clustered among the 'Termite Treponemes', which comprise also the fhs genes of the two strains of the homoacetogenic spirochaete Treponema primitia. The high abundance of treponemal fhs genes in all clone libraries was in agreement with the results of DNA-based terminal-restriction fragment length polymorphism (T-RFLP) analysis. Moreover, in mRNA-based T-RFLP profiles of the three termites, only expression of fhs genes of 'Termite Treponemes' was detected, albeit at different levels. In C. secundus, only one of the dominating phylotypes was transcribed, while in R. santonensis, the apparently less abundant fhs genes were the most actively expressed. Our results strongly support the hypothesis that spirochaetes are responsible for reductive acetogenesis in the hindgut of lower, wood-feeding termites.     

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 H(2)-CO(2). The position of the bands coincided with the oxic-anoxic interface and depended on the O(2) 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 O(2) and H(2) electrodes, revealed a large capacity for H(2)-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 O(2) with endogenous reductants, albeit at lower rates. Only in Acetonema longum did the basal rates exceed the H(2)-dependent rates considerably (181 versus 28 nmol min(-1) mg of protein)(-1)). Addition of organic substrates did not stimulate O(2) consumption in any of the strains. Nevertheless, reductive acetogenesis by cell suspensions of strain TmAO3 was inhibited even at the lowest O(2) 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 O(2) at low partial pressures (up to 1.5 kPa in the case of strain TmAO3) and because of their large capacity for O(2) reduction, homoacetogens can reestablish conditions favorable for growth by actively removing oxygen from their environment.     

Competition between reductive acetogenesis and methanogenesis in the pig large-intestinal flora

Washed bacterial suspensions obtained from the pig hindgut were incubated under ¹³CO₂ in a buffer containing NaH¹³CO₃ and carbohydrates. Incorporation of ¹³C into short chain fatty acids was assayed by quantitative nuclear magnetic resonance. The effects of different levels of H₂ added to the gas phase (0, 20 and 80% v/v) and of the specific methanogenesis inhibitor 2-bromoethane-sulphonic acid (BES) were determined. In control incubations increasing the concentration of H₂ markedly increased methane production. Single- and double-labelled acetate and butyrate were formed in all incubations. In the absence of BES, increasing H₂ significantly increased the incorporation of ¹³CO² into butyrate and the proportion of double-labelled acetate in total labelled acetate. The addition of BES proved to be very successful as a methane inhibitor and greatly enhanced the amount of mono- and double-labelled acetate, especially at the highest H₂ partial pressure. The results suggest that methanogenesis inhibited both routes of reductive acetogenesis, i.e. the homoacetate fermentation of hexose (represented for the most part by single labelling) and the synthesis of acetate from external CO₂ and H₂ (represented mostly by double labelling). A highly significant interaction between BES and H₂ concentration was observed. At the highest pH2 BES increased the proportion of labelled acetate in total acetate from 17.1% for the control to 50.9%. It was concluded that although acetogenesis and methanogenesis can occur simultaneously in the pig hindgut, reductive acetogenesis may become a significant pathway of acetate formation in the absence of methanogenesis.     

Axial differences in community structure of Crenarchaeota and Euryarchaeota in the highly compartmentalized gut of the soil-feeding termite Cubitermes orthognathus

Methanogenesis represents an important electron sink reaction in the hindgut of soil-feeding termites. This is the first comprehensive analysis of the archaeal community structure within the highly compartmentalized intestinal tract of a humivorous insect, combining clonal analysis and terminal restriction fragment (T-RF) length polymorphism (T-RFLP) fingerprinting of the archaeal communities in the different gut compartments of Cubitermes orthognathus. We found that the morphological and physicochemical heterogeneity of the gut is reflected in a large phylogenetic diversity and pronounced axial differences in the composition of the archaeal gut microbiota, notably among those clones or ribotypes that could be assigned to methanogenic taxa. Comparative analysis of the relative frequencies of different archaeal lineages among the small-subunit rRNA gene (SSU rDNA) clones and their corresponding T-RF indicated that the archaeal community in the anterior, extremely alkaline hindgut compartment (P1) consists mainly of members of the Methanosarcinaceae, whereas Methanobacteriaceae and Methanomicrobiales predominate in the subsequent, more posterior compartments (P3/4a and P4b). The relative abundance of Thermoplasmales increased towards the rectum (P5). SSU rDNA sequences representing Crenarchaeota, which have not yet been reported to occur in the intestinal tracts of arthropods, were detected in all gut sections. We discuss how the spatial distribution of methanogenic populations may be linked to axial heterogeneity in the physicochemical gut conditions and to functional adaptations to their respective ecological niches.     

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.     

Acetate Synthesis from H(2) plus CO(2) by Termite Gut Microbes

Gut microbiota from Reticulitermes flavipes termites catalyzed an H(2)-dependent total synthesis of acetate from CO(2). Rates of H(2)-CO(2) acetogenesis in vitro were 1.11 +/- 0.37 mumol of acetate g (fresh weight) h (equivalent to 4.44 +/- 1.47 nmol termite h) and could account for approximately 1/3 of all the acetate produced during the hindgut fermentation. Formate was also produced from H(2) + CO(2), as were small amounts of propionate, butyrate, and lactate-succinate. However, H(2)-CO(2) formicogenesis seemed largely unrelated to acetogenesis and was believed not to be a significant reaction in situ. Little or no CH(4) was formed from H(2) + CO(2) or from acetate. H(2)-CO(2) acetogenesis was inhibited by O(2), KCN, CHCl(3), 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 H(2)-CO(2) acetogenesis, suggesting that bacteria were the acetogenic agents in the gut. H(2)-CO(2) 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 H(2)-CO(2)-acetogenic bacteria were isolated from N. nigriceps, and a preliminary account of their morphological and physiological properties is presented. Results indicate that in termites, CO(2) reduction to acetate, rather than to CH(4), 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.     

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%.     

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) approximately -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 H(2), 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).     

Axial Differences in Community Structure of Crenarchaeota and Euryarchaeota in the Highly Compartmentalized Gut of the Soil-Feeding Termite Cubitermes orthognathus

Methanogenesis represents an important electron sink reaction in the hindgut of soil-feeding termites. This is the first comprehensive analysis of the archaeal community structure within the highly compartmentalized intestinal tract of a humivorous insect, combining clonal analysis and terminal restriction fragment (T-RF) length polymorphism (T-RFLP) fingerprinting of the archaeal communities in the different gut compartments of Cubitermes orthognathus. We found that the morphological and physicochemical heterogeneity of the gut is reflected in a large phylogenetic diversity and pronounced axial differences in the composition of the archaeal gut microbiota, notably among those clones or ribotypes that could be assigned to methanogenic taxa. Comparative analysis of the relative frequencies of different archaeal lineages among the small-subunit rRNA gene (SSU rDNA) clones and their corresponding T-RF indicated that the archaeal community in the anterior, extremely alkaline hindgut compartment (P1) consists mainly of members of the Methanosarcinaceae, whereas Methanobacteriaceae and Methanomicrobiales predominate in the subsequent, more posterior compartments (P3/4a and P4b). The relative abundance of Thermoplasmales increased towards the rectum (P5). SSU rDNA sequences representing Crenarchaeota, which have not yet been reported to occur in the intestinal tracts of arthropods, were detected in all gut sections. We discuss how the spatial distribution of methanogenic populations may be linked to axial heterogeneity in the physicochemical gut conditions and to functional adaptations to their respective ecological niches.     

Managing methanogens and homoacetogens to promote reductive dechlorination of trichloroethene with direct delivery of H2 in a membrane biofilm reactor

A study with H(2)-based membrane biofilm reactors (MBfRs) was undertaken to examine the effectiveness of direct H(2) delivery in ex-situ reductive dechlorination of chlorinated ethenes. Trichloroethene (TCE) could be reductively dechlorinated to ethene with up to 95% efficiency as long as the pH-increase effects of methanogens and homoacetogens were managed and dechlorinators were selected for during start-up by creating H(2) limitation. Based on quantitative PCR, the dominant bacterial groups in the biofilm at the end of reactor operation were Dehalococcoides, Geobacter, and homoacetogens. Pyrosequencing confirmed the dominance of the dechlorinators and identified Acetobacterium as the key homoacetogen. Homoacetogens outcompeted methanogens for bicarbonate, based on the effluent concentration of acetate, by suppressing methanogens during batch start-up. This was corroborated by the methanogenesis functional gene mcrA, which was 1-2 orders of magnitude lower than the FTHFS functional gene for homoacetogens. Imaging of the MBfR fibers using scanning electron microscopy showed a distinct Dehalococcoides-like morphology in the fiber biofilm. These results support that direct addition of H(2) can allow for efficient and complete reductive dechlorination, and they shed light into how H(2)-fed biofilms, when operated to manage methanogenic and homoacetogenic activity, can be used for ex-situ bioremediation of chlorinated ethenes.     

Characterization and phylogenomic analysis of Breznakiella homolactica gen. nov. sp. nov. indicate that termite gut treponemes evolved from non-acetogenic spirochetes in cockroaches

Spirochetes of the genus Treponema are surprisingly abundant in termite guts, where they play an important role in reductive acetogenesis. Although they occur in all termites investigated, their evolutionary origin is obscure. Here, we isolated the first representative of ‘termite gut treponemes’ from cockroaches, the closest relatives of termites. Phylogenomic analysis revealed that Breznakiella homolactica gen. nov. sp. nov. represents the most basal lineage of the highly diverse ‘termite cluster I', a deep-branching sister group of Treponemataceae (fam. ‘Termitinemataceae’) that was present already in the cockroach ancestor of termites and subsequently coevolved with its host. Breznakiella homolactica is obligately anaerobic and catalyses the homolactic fermentation of both hexoses and pentoses. Resting cells produced acetate in the presence of oxygen. Genome analysis revealed the presence of pyruvate oxidase and catalase, and a cryptic potential for the formation of acetate, ethanol, formate, CO₂ and H₂ - the fermentation products of termite gut isolates. Genes encoding key enzymes of reductive acetogenesis, however, are absent, confirming the hypothesis that the ancestral metabolism of the cluster was fermentative, and that the capacity for acetogenesis from H₂ plus CO₂ - the most intriguing property among termite gut treponemes - was acquired by lateral gene transfer.     

Structure, microbial associations and function of the so-called "mixed segment" of the gut in two soil-feeding termites, Procubitermes aburiensis and Cubitermes severus (Termitidae, Termitinae)

The morphology, histology and ultrastructure of the mesenteric and proctodaeal components of the mixed segment are described in detail, together with the disposition of the associated gut musculature, the patterns of peristalsis and the streaming movements of soil particles in the gut lumen. The mesenteric epithelium is characterized by very extensive basal infoldings which are associated with a large population of mitochondria, but evidence of significant protein synthesis and secretion is lacking. It is proposed that this tissue is a transporting epithelium whose major function is the secretion of a copious, K⁺ -rich fluid into the intestinal lumen to irrigate the hindgut (proctodaeum) and that alkaline hydrolysis of humic fractions by this fluid is an essential component of digestion.
Malpighian tubules are present, but do not participate in fluid excretion. Since the hind-guts of soil-feeding termites are exceptionally voluminous and elongated and the major symbiotic micro-organisms occupy fixed positions by virtue of their attachment to the lining cuticle, it is argued that flushing is necessary to sustain microbial activity and for the recovery of end-products by the host. A survey of the protodaeal epithelium suggests that the anterior colon (P4a) is the most likely site of fluid reabsorption and that the wall of the hindgut anterior to this site is impermeable. The greater degree of ultrastructural differentiation of the mixed segment of Cubitermes severus and its more complete morphological separation from the midgut suggests that this species is a more advanced soil-feeder than Procubitermes aburiensis.