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.     

Culturable populations of Sporomusa spp. and Desulfovibrio spp. in the anoxic bulk soil of flooded rice microcosms.

Most-probable-number (MPN) counts were made of homoacetogenic and other bacteria present in the anoxic flooded bulk soil of laboratory microcosms containing 90- to 95-day-old rice plants. MPN counts with substrates known to be useful for the selective enrichment or the cultivation of homoacetogenic bacteria (betaine, ethylene glycol, 2, 3-butanediol, and 3,4,5-trimethoxybenzoate) gave counts of 2.3 x 10(3) to 2.8 x 10(5) cells per g of dry soil. Homoacetogens isolated from the terminal positive steps of these dilution cultures belonged to the genus Sporomusa. Counts with succinate, ethanol, and lactate gave much higher MPNs of 5.9 x 10(5) to 3.4 x 10(7) cells per g of dry soil and led to the isolation of Desulfovibrio spp. Counting experiments on lactate and ethanol which included Methanospirillum hungatei in the medium gave MPNs of 2.3 x 10(6) to 7.5 x 10(8) cells per g of dry soil and led to the isolation of Sporomusa spp. The latter strains could grow with betaine, ethylene glycol, 2, 3-butanediol, and/or 3,4,5-trimethoxybenzoate, but apparently most cells of Sporomusa spp. did not initiate growth in counting experiments with those substrates. Spores apparently accounted for 2. 2% or less of the culturable bacteria. It appears that culturable Desulfovibrio spp. and Sporomusa spp. were present in approximately equal numbers in the bulk soil. Multiple, phylogenetically-distinct, phenotypically-different, strains of each genus were found in the same soil system.     

Simultaneous methanogenesis and oxygen reduction by Methanobrevibacter cuticularis at low oxygen fluxes

Methanogenic Archaea are often encountered in habitats that are not entirely anoxic in space or time. Recent biochemical and genomic studies have revealed the capacity of methanogens to reduce molecular oxygen. O(2) reduction by Methanobrevibacter species was investigated. Cell suspensions incubated in agar tubes under a headspace of H(2)-CO(2) and increasing concentrations of O(2) formed a distinct growth band, which coincided with the oxic-anoxic interface and indicated that the influx of O(2) into the band was balanced by its consumption. However, in batch cultures methanogenesis ceased as soon as traces of O(2) were added. Focusing on Methanobrevibacter cuticularis, a species colonizing the microoxic gut epithelium of termites, a diffusion-limited setup was used that allowed the exposure of dense cell suspensions to controlled O(2) fluxes. Here, Methanobrevibacter cuticularis was capable of simultaneous CH(4) production and O(2) consumption. Low O(2) fluxes (10% of the CH(4) production rate) had virtually no influence on methanogenesis [4.5 micromol CH(4) (mg dry wt)(-1) h(-1)], whereas higher O(2) fluxes (up to 30% of the initial CH(4) production rate) caused a reversible decrease in methanogenesis, which was accompanied by a reversible, partial conversion of coenzyme F(420) to factor F(390). The maximum O(2) reduction rate [4.8 micromol O(2) (mg dry wt)(-1) h(-1)] that could be maintained over extended time periods (>30 min) was similar to the CH(4) production rate under anoxic conditions.     

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.     

Characterization of abundance and diversity of lactic acid bacteria in the hindgut of wood- and soil-feeding termites by molecular and culture-dependent techniques

Lactic acid bacteria have been identified as typical and numerically significant members of the gut microbiota of Reticulitermes flavipes and other wood-feeding lower termites. We found that also in the guts of the higher termites Nasutitermes arborum (wood-feeding), Thoracotermes macrothorax, and Anoplotermes pacificus (both soil-feeding), lactic acid bacteria represent the largest group of culturable carbohydrate-utilizing bacteria (3.6-5.2x10(4) bacteria per gut; 43%-54% of all colonies). All isolates were coccoid and phenotypically difficult to distinguish, but their enterobacterial repetitive intergenic consensus sequence (ERIC) fingerprint patterns showed a significant genetic diversity. Six different genotypes each were identified among the isolates from R. flavipes and T. macrothorax, and representative strains were selected for further characterization. By 16S rRNA gene sequence analysis, strain RfL6 from R. flavipes was classified as a close relative of Enterococcus faecalis, whereas strain RfLs4 from R. flavipes and strain TmLO5 from T. macrothorax were closely related to Lactococcus lactis. All strains consumed oxygen during growth on glucose and cellobiose; oxygen consumption of these and other isolates from both termite species was due to NADH and pyruvate oxidase activities, but did not result in H2O2 formation. In order to assess the significance of the isolates in the hindgut, denaturing gradient gel electrophoresis was used to compare the fingerprints of 16S rRNA genes in the bacterial community of R. flavipes with those of representative isolates. The major DNA band from the hindgut bacterial community was further separated by bisbenzimide-polyethylene glycol electrophoresis, and the two resulting bands were sequenced. Whereas one sequence belonged to a spirochete, the second sequence was closely related to the sequences of the Lactococcus strains RfLs4 and TmLO5. Apparently, those isolates represent strains of a new Lactococcus species which forms a significant fraction of the complex hindgut community of the lower termite R. flavipes and possibly also of other termites.     

Methane oxidation in termite hindguts: absence of evidence and evidence of absence

A steep oxygen gradient and the presence of methane render the hindgut internal periphery of termites a potential habitat for aerobic methane-oxidizing bacteria. However, methane emissions of various termites increased, if at all, only slightly when termites were exposed to an anoxic (nitrogen) atmosphere, and (14)CH(4) added to the air headspace over live termites was not converted to (14)CO(2). Evidence for the absence of methane oxidation in living termites was corroborated by the failure to detect pmoA, the marker gene for particulate methane monooxygenase, in hindgut DNA extracts of all termites investigated. This adds robustness to our concept of the degradation network in the termite hindgut and eliminates the gut itself as a potential sink of this important greenhouse gas.     

Identification of phenolyl cobamide from the homoacetogenic bacterium Sporomusa ovata

Phenolyl cobamide was isolated from cyanide extractions of the anaerobic eubacterium Sporomusa ovata. The proposed corrinoid structure [Co alpha,Co beta-(monocyano,monoaquo)-phenolyl cobamide] has been deduced from 1H NMR, fast-atom-bombardment mass spectroscopy and ultraviolet/visible spectroscopy data. The complete corrinoid resembled p-cresolyl cobamide [Co alpha,Co beta-(monocyano,monoaquo)-p-cresolyl cobamide], which recently has been obtained from cyanide extractions of the same bacterium. The structures and chemical properties of both cobamides with uncoordinated nucleotides differed significantly from those of vitamin B12 [Co alpha-[alpha-(5,6-dimethylbenzimidazolyl)]-Co beta-cyanocobamide]. Sporomusa synthesized coenzymes of phenolyl cobamide and p-cresolyl cobamide in considerable amounts of 400 nmol/g and 1700 nmol/g dry cells, respectively. More than 90% of the complete corrinoid pool of the homoacetogenic bacterium consisted of these two corrinoids, indicating that they are physiologically important coenzymes of the bacterial metabolism.     

Aerotaxis in Desulfovibrio

Aerotaxis of two sulphate-reducing bacteria, the freshwater strain Desulfovibrio desulfuricans CSN (DSM 9104) and the marine strain Desulfovibrio oxyclinae N13 (DSM 11498), was studied using capillary microslides, microscopy and oxygen microsensors. The bacteria formed ring-shaped bands in oxygen diffusion gradients surrounding O₂ bubbles, which were placed into anoxic sulphate-free cell suspensions in capillary microslides. The radial expansion of the oxic volume by diffusion was stopped by aerobic respiration. Bands were formed by cells avoiding high O₂ levels near the O₂ bubble, as well as by cells entering from the surrounding anoxic zone. At the inner edge of the bands, O₂ levels of up to 20% air saturation (50 μM O₂) were found, while the outer edge always coincided with the oxic–anoxic interface. Ring diameters and O₂ concentrations at the inner edge of the band depended on the cell density and the strain used in the suspension. Band formation did not occur in the absence of an electron donor (5 mM lactate) or when N₂ gas bubbles were used. Both strains were highly motile with velocities of ≈ 32 μm s⁻¹ during forward runs, and 7 μm s⁻¹ during backward runs respectively. Within the bands, cells moved in circles of about 20 μm diameter, while cells outside the band exhibited straighter or only slightly bent traces. It is concluded that the capacity of respiration at high rates and the positive and negative aerotactical responses of Desulfovibrio provide an efficient strategy for removing O₂ from the habitat in situations where sufficient electron donors and high cell densities are present.     

Transformation of tetrachloroethylene to trichloroethylene by homoacetogenic bacteria

Abstract Eight homoacetogenic strains of the genera Acetobacterium, Clostridium and Sporomusa were tested for their ability to dechlorinate tetrachloroethylene (perchloroethene, PCE). Of the organisms tested only Sporomusa ovata was able to reductively dechlorinate PCE with methanol as an electron donor. Resting cells of S. ovata reductively dechlorinated PCE at a rate of 9.8 nmol h⁻¹ (mg protein)⁻¹ to trichloroethylene (TCE) as the sole product. The dechlorination activity depended on concomitant acetogenesis from methanol and CO₂. Cell-free extracts of S. ovata, Clostridium formicoaceticum, Acetobacterium woodii , and the methanogenic bacterium Methanolobus tindarius transformed PCE to TCE with Ti(III) or carbon monoxide as electron donors. Corrinoids were shown in S. ovata to be involved in the dechlorination reaction of PCE to TCE as evident from the reversible inhibition with propyl iodide. Rates of dechlorination followed a pseudo-first-order kinetic.     

Interspecific hydrogen transfer during methanol degradation by Sporomusa acidovorans and hydrogenophilic anaerobes

In the presence of active hydrogenophilic sulfate-reducing bacteria, the homoacetogenic bacterium Sporomusa acidovorans did not produce acetate during methanol degradation. H₂S and presumably CO₂ were the only end products. Since the sulfate-reducer did not degrade methnol or acetate, the sulfidogenesis from methanol was related to a complete interspecific hydrogen transfer between both species.In coculture with hydrogenophilic methanogenic bacteria (Methanobacterium formicicum, Methanospirillum hungatei), the interspecific hydrogen transfer with S. acidovorans was incomplete. Beside CH₄ and presumably CO₂, acetate was produced. The results suggested that H₂-production and H₂-consumption were involved during anaerobic methanol degradation by S. acidovorans and the hydrogenophilic anaerobes play an important role during methanol degradation by homoacetogenic bacteria in anoxic environments.     

Hydrogen Concentration Profiles at the Oxic-Anoxic Interface: a Microsensor Study of the Hindgut of the Wood-Feeding Lower Termite Reticulitermes flavipes (Kollar)

Molecular hydrogen is a key intermediate in lignocellulose degradation by the microbial community of termite hindguts. With polarographic, Clark-type H(inf2) microelectrodes, we determined H(inf2) concentrations at microscale resolution in the gut of the wood-feeding lower termite Reticulitermes flavipes (Kollar). Axial H(inf2) concentration profiles obtained from isolated intestinal tracts embedded in agarose Ringer solution clearly identified the voluminous hindgut paunch as the site of H(inf2) production. The latter was strictly coupled with both a low redox potential (E(infh) = -200 mV) and the absence of oxygen, in agreement with the growth requirements of the cellulolytic, H(inf2)-producing flagellates located in the hindgut paunch. Luminal H(inf2) partial pressures were much higher than expected (ca. 5 kPa) and increased more than threefold when the guts were incubated under a N(inf2) headspace. Radial H(inf2) concentration gradients showed a steep decrease from the gut center towards the periphery, indicating the presence of H(inf2)-consuming activities both within the lumen and at the gut epithelium. Measurements under controlled gas headspace showed that the gut wall was also a sink for externally supplied H(inf2), both under oxic and anoxic conditions. With O(inf2) microelectrodes, we confirmed that the H(inf2) sink below the gut epithelium is located within the microoxic gut periphery, but the H(inf2)-consuming activity itself, at least a substantial part of it, was clearly due to an anaerobic process. These results are in accordance with the recently reported presence of methanogens attached in large numbers to the luminal side of the hindgut epithelium of R. flavipes. If the oxygen partial pressure was increased, O(inf2) penetrated deeper and H(inf2) production was suppressed; it ceased completely as soon as the gut was fully oxic. In experiments with living termites, externally supplied H(inf2) (20 kPa) stimulated methane formation five- to sixfold to 0.93 (mu)mol (g of termite)(sup-1) h(sup-1), indicating that the methanogenic activity in R. flavipes hindguts is not saturated for hydrogen under in situ conditions. This rate was in good agreement with the H(inf2) uptake rates exhibited by isolated hindguts, which would account for more than half of the CH(inf4) formed by living termites under comparable conditions.     

Methane Oxidation in Termite Hindguts: Absence of Evidence and Evidence of Absence

A steep oxygen gradient and the presence of methane render the hindgut internal periphery of termites a potential habitat for aerobic methane-oxidizing bacteria. However, methane emissions of various termites increased, if at all, only slightly when termites were exposed to an anoxic (nitrogen) atmosphere, and ¹⁴CH₄ added to the air headspace over live termites was not converted to ¹⁴CO₂. Evidence for the absence of methane oxidation in living termites was corroborated by the failure to detect pmoA, the marker gene for particulate methane monooxygenase, in hindgut DNA extracts of all termites investigated. This adds robustness to our concept of the degradation network in the termite hindgut and eliminates the gut itself as a potential sink of this important greenhouse gas.     

Non-growth-associated demethylation of dimethylsulfoniopropionate by (homo)acetogenic bacteria

The demethylation of the algal osmolyte dimethylsulfoniopropionate (DMSP) to methylthiopropionate (MTPA) by (homo)acetogenic bacteria was studied. Five Eubacterium limosum strains (including the type strain), Sporomusa ovata DSM 2662(T), Sporomusa sphaeroides DSM 2875(T), and Acetobacterium woodii DSM 1030(T) were shown to demethylate DMSP stoichiometrically to MTPA. The (homo)acetogenic fermentation based on this demethylation did not result in any significant increase in biomass. The analogous demethylation of glycine betaine to dimethylglycine does support growth of acetogens. In batch cultures of E. limosum PM31 DMSP and glycine betaine were demethylated simultaneously. In mixed substrates experiments with fructose-DMSP or methanol-DMSP, DMSP was used rapidly but only after exhaustion of the fructose or the methanol. In steady-state fructose-limited chemostat cultures (at a dilution rate of 0.03 h(-1)) with DMSP as a second reservoir substrate, DMSP was biotransformed to MTPA but this did not result in higher biomass values than in cultures without DMSP; cells from such cultures demethylated DMSP at rates of approximately 50 nmol min(-1) mg of protein(-1), both after growth in the presence of DMSP and after growth in its absence. In cell extracts of glycine betaine-grown strain PM31, DMSP demethylation activities of 21 to 24 nmol min(-1) mg of protein(-1) were detected with tetrahydrofolate as a methyl acceptor; the activities seen with glycine betaine were approximately 10-fold lower. A speculative explanation for the demethylation of DMSP without an obvious benefit for the organism is that the DMSP-demethylating activity is catalyzed by the glycine betaine-demethylating enzyme and that a transport-related factor, in particular a higher energy demand for DMSP transport across the cytoplasmic membrane than for glycine betaine transport, may reduce the overall ATP yield of the fermentation to virtually zero.     

Microbial production and consumption of dimethyl sulfide (DMS) in a sea grass (Zostera noltii)-dominated marine intertidal sediment ecosystem (Bassin d'Arcachon, France)

The relation between net dimethyl sulfide (DMS) production and changes in near surface (0-5 mm) oxygen concentrations in a sea grass (Zostera noltii Hornem)-covered intertidal sediment ecosystem was examined during a diel cycle. Sediment covered with Zostera was found to be more oxygenated than uncovered sediment during the period of photosynthesis. This phenomenon was probably caused by radial oxygen loss of the Zostera root-rhizome system. The population sizes of the three functional groups of microbes mainly responsible for the concentration of DMS, the dimethylsulfoniopropionate (DMSP)-demethylating, DMSP-cleaving and DMS-oxidizing bacteria, were quantified by most probable number (MPN) methodologies. Sediments with Zostera supported substantially higher populations of both aerobic (149x10(6) cm(-3) DMSP-utilizing and 0.4x10(6) cm(-3) DMS-oxidizing) and anaerobic (43x10(6) cm(-3) DMSP-utilizing and 0.4x10(6) cm(-3) DMS-oxidizing) microorganisms than sediments without Zostera (DMSP-utilizing aerobes and anaerobes both 2x10(6) cm(-3) and DMS-oxidizing aerobes and anaerobes both 0.2x10(6) cm(-3)). Experiments conducted with sediment cores and sediment slurries suggested that the net production of DMS in these sediments was significantly lower during oxic periods than during anoxic periods. Intact sediment cores with and without Zostera produced DMS when incubated under anoxic/dark conditions (97.0 and 53.6 nmol DMS m(-2) h(-1), respectively), while oxic/light-incubated cores did not produce detectable amounts of DMS. In addition, kinetic parameter values (V(max) and K(m)) for DMSP degradation in cell suspensions of isolated DMSP-demethylating and DMSP-cleaving bacteria were measured and compared to documented values for other strains. Both V(max) and K(m) values for DMSP-demethylating organisms were found to be relatively low (14.4-20.1 nmol DMSP mg protein(-1) min(-1) and 4.1-15.5 μM, respectively) while these parameter values varied widely in the group of the DMSP-cleaving organisms (6.7-1000 nmol DMSP mg protein(-1) min(-1) and 2-2000 μM, respectively). It was hypothesized that a diel rhythm in DMS emission occurred, with a relatively low net production during the day and a high net production during the night. Environmental changes which result in increased anoxic conditions in coastal sediments, such as an increase in eutrophication, may therefore result in increased atmospheric DMS emission rates.     

Influence of CO2 and low concentrations of O2 on fermentative metabolism of the ruminal ciliate Polyplastron multivesiculatum.

The effects of ruminal concentrations of CO2 and oxygen on the end products of endogenous metabolism and fermentation of D-glucose by the ruminal entodiniomorphid ciliate Polyplastron multivesiculatum were investigated. The principal metabolic products were butyric, acetic, and lactic acids, H2, and CO2. 13C nuclear magnetic resonance spectroscopy identified glycerol as a previously unknown major product of D-[1-13C]glucose fermentation by this protozoan. Metabolite formation rates were clearly influenced by the headspace gas composition. In the presence of 1 to 3 microM O2, acetate, H2, and CO2 formation was partially depressed. A gas headspace with a high CO2 content (66 kPa) was found to suppress hydrogenosomal pathways and to favor butyrate accumulation. Cytochromes were not detected (less than 2 pmol/mg of protein) in P. multivesiculatum; protozoal suspensions, however, consumed O2 for up to 3 h at 1 kPa of O2. Under gas phases of greater than 2.6 kPa of O2, the organisms rapidly became vacuolate and the cilia became inactive. The results suggest that fermentative pathways in P. multivesiculatum are influenced by the O2 and CO2 concentrations that prevail in situ in the rumen.     

Sulfate-reducing bacteria in rice field soil and on rice roots.

Rice plants that were grown in flooded rice soil microcosms were examined for their ability to exhibit sulfate reducing activity. Washed excised rice roots showed sulfate reduction potential when incubated in anaerobic medium indicating the presence of sulfate-reducing bacteria. Rice plants, that were incubated in a double-chamber (phylloshpere and rhizosphere separated), showed potential sulfate reduction rates in the anoxic rhizosphere compartment. These rates decreased when oxygen was allowed to penetrate through the aerenchyma system of the plants into the anoxic root compartment, indicating that sulfate reducers on the roots were partially inhibited by oxygen or that sulfate was regenerated by oxidation of reduced S-compounds. The potential activity of sulfate reducers on rice roots was consistent with MPN enumerations showing that H2-utilizing sulfate-reducing bacteria were present in high numbers on the rhizoplane (4.1 x 10(7) g-1 root fresh weight) and in the adjacent rhizosperic soil (2.5 x 10(7) g-1 soil dry weight). Acetate-oxidizing sulfate reducers, on the other hand, showed highest numbers in the unplanted bulk soil (1.9 x 10(6) g-1 soil dry weight). Two sulfate reducing bacteria were isolated from the highest dilutions of the MPN series and were characterized physiologically and phylogenetically. Strain F1-7b which was isolated from the rhizoplane with H2 as electron donor was related to subgroup II of the family Desulfovibrionaceae. Strain EZ-2C2, isolated from the rhizoplane on acetate, grouped together with Desulforhabdus sp. and Syntrophobacter wolinii. Other strains of sulfate-reducing bacteria originated from bulk soil of rice soil microcosms and were isolated using different electron donors. From these isolates, strains R-AcA1, R-IbutA1, R-PimA1 and R-AcetonA170 were Gram-positive bacteria which were affiliated with the genus Desulfotomaculum. The other isolates were members of subgroup II of the Desulfovibrionaceae (R-SucA1 and R-LacA1), were related to Desulforhabdus sp. (strain BKA11), Desulfobulbus (R-PropA1), or culstered between Desulfobotulus sapovorans and Desulfosarcina variabilis (R-ButA1 and R-CaprA1).     

Epimerization of chenodeoxycholic acid to ursodeoxycholic acid by Clostridium baratii isolated from human feces

Several H2-producing fermentative anaerobic bacteria including Clostridium, Klebsiella and Fusobacteria degraded octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) (36 microM) to formaldehyde (HCHO) and nitrous oxide (N2O) with rates ranging from 5 to 190 nmol h(-1)g [dry weight] of cells(-1). Among these strains, C. bifermentans strain HAW-1 grew and transformed HMX rapidly with the detection of the two key intermediates the mononitroso product and methylenedinitramine. Its cellular extract alone did not seem to degrade HMX appreciably, but degraded much faster in the presence of H2, NADH or NADPH. The disappearance of HMX was concurrent with the release of nitrite without the formation of the nitroso derivative(s). Results suggest that two types of enzymes were involved in HMX metabolism: one for denitration and the second for reduction to the nitroso derivative(s).     

Isolation and characterization of two new homoacetogenic hydrogen-utilizing bacteria from the human intestinal tract that are closely related to Clostridium coccoides.

Two gram-positive, strictly anoxic, coccoid- to rod-shaped strains of bacteria, Clostridium coccoides 1410 and C. coccoides 3110, were isolated from human feces on the typical homoacetogenic substrates formate plus H2 plus CO2 (strain 1410) and vanillate plus H2 plus CO2 (strain 3110) in the presence of 2-bromoethanesulfonate to inhibit methanogenesis. On the basis of 16S rRNA sequencing, DNA-DNA hybridization, and physiological and morphological parameters, both isolates are closely related to C. coccoides DSM 935T. The G+C contents of the DNA were 46.1 and 46.2 mol% for C. coccoides 1410 and C. coccoides 3110, respectively. Cytochromes could not be detected. Formate was degraded exclusively to acetate, whereas vanillate was O-demethylated, resulting in acetate and 3,4-dihydroxybenzoate, the latter being further decarboxylated to catechol. In the presence of organic substrates, H2 was cometabolized to acetate, but both strains failed to grow autotrophically. Lactose, lactulose, sorbitol, glucose, and various other carbohydrates supported growth as well. Untypical of homoacetogens, glucose and sorbitol were fermented not exclusively to acetate; instead, considerable amounts of succinate and D-lactate were produced. H2 was evolved from carbohydrates only in negligible traces. Acetogenesis from formate plus H2 plus CO2 or vanillate plus H2 plus CO2 was constitutive, whereas utilization of carbohydrates was inducible. Hydrogenase, CO dehydrogenase, formate dehydrogenase, and all of the tetrahydrofolic acid-dependent, C1 compound-converting enzymes of the acetyl-coenzyme A pathway of homoacetogenesis were present in cell extracts.     

Influence of CO2 and low concentrations of O2 on fermentative metabolism of the ruminal ciliate Polyplastron multivesiculatum

The effects of ruminal concentrations of CO2 and oxygen on the end products of endogenous metabolism and fermentation of D-glucose by the ruminal entodiniomorphid ciliate Polyplastron multivesiculatum were investigated. The principal metabolic products were butyric, acetic, and lactic acids, H2, and CO2. 13C nuclear magnetic resonance spectroscopy identified glycerol as a previously unknown major product of D-[1-13C]glucose fermentation by this protozoan. Metabolite formation rates were clearly influenced by the headspace gas composition. In the presence of 1 to 3 microM O2, acetate, H2, and CO2 formation was partially depressed. A gas headspace with a high CO2 content (66 kPa) was found to suppress hydrogenosomal pathways and to favor butyrate accumulation. Cytochromes were not detected (less than 2 pmol/mg of protein) in P. multivesiculatum; protozoal suspensions, however, consumed O2 for up to 3 h at 1 kPa of O2. Under gas phases of greater than 2.6 kPa of O2, the organisms rapidly became vacuolate and the cilia became inactive. The results suggest that fermentative pathways in P. multivesiculatum are influenced by the O2 and CO2 concentrations that prevail in situ in the rumen.