Determination of the G + C content of twoSyntrophus buswellii strains by ultracentrifugation techniques

An anaerobic syntrophic bacterial culture degrading benzoate was isolated from a river sediment. The syntrophic organism was grown in coculture in the presence of a hydrogenotrophic strain,Desulfovibrio fructosovorans orMethanospirillum hungatei. The G+C content of the syntrophic benzoate degrader determined by density gradient ultracentrifugation was similar to that ofSyntrophus buswellii (54.3%). A method ensuring the G+C% determination of syntrophic bacteria is presented.     

Biochemical evidence for formate transfer in syntrophic propionate-oxidizing cocultures of Syntrophobacter fumaroxidans and Methanospirillum hungatei

The hydrogenase and formate dehydrogenase levels in Syntrophobacter fumaroxidans and Methanospirillum hungatei were studied in syntrophic propionate-oxidizing cultures and compared to the levels in axenic cultures of both organisms. Cells grown syntrophically were separated from each other by Percoll gradient centrifugation. In S. fumaroxidans both formate dehydrogenase and hydrogenase levels were highest in cells which were grown syntrophically, while the formate-H(2) lyase activities were comparable under the conditions tested. In M. hungatei the formate dehydrogenase and formate-H(2) lyase levels were highest in cells grown syntrophically, while the hydrogenase levels in syntrophically grown cells were comparable to those in cells grown on formate. Reconstituted syntrophic cultures from axenic cultures immediately resumed syntrophic growth, and the calculated growth rates of these cultures were highest for cells which were inoculated from the axenic S. fumaroxidans cultures that exhibited the highest formate dehydrogenase activities. The results suggest that formate is the preferred electron carrier in syntrophic propionate-oxidizing cocultures of S. fumaroxidans and M. hungatei.     

Anaerobic degradation of isovalerate by a defined methanogenic coculture

Isovalerate-oxidizing strictly aneerobic bacteria were isolated from marine sediment and sewage sludge in coculture with Desulfovibrio sp. Cells stained Gram positive and behaved Gram positive also in Gram classification with KOH. Isovalerate degradation depended on interspecies hydrogen transfer to syntrophic hydrogen-oxidizing sulfate reducers or methanogens. Isovalerate was the only substrate utilized and was fermented to 3 mol acetate and 1 mol hydrogen per mol substrate. The degradation pathway was studied by enzyme assays in crude cell extracts, and included acetyl-CoA dependent activation of isovalerate, oxidation to methylcrotonyl-CoA and carboxylation to methylgluta-conyl-CoA which is hydrated and cleaved to acetoacetate and acetyl-CoA. Studies with inhibitors and ionophores suggest that energy conservation with this organism depends on either acetate efflux-driven proton symport or on an ion-gradient driven carboxylation mechanism.     

Acetogenesis from Dichloromethane by a Two-Component Mixed Culture Comprising a Novel Bacterium

A strictly anaerobic two-component culture able to grow exponentially with a doubling time of 20 h on a medium containing dichloromethane as the carbon and energy source was characterized. On a medium without sulfate, we observed (per mol of dichloromethane) a mass balance of 2 mol of chloride, 0.26 mol of acetate, 0.05 mol of formate, and 0.25 mol of carbon in biomass. One component of the culture, strain DMB, was identified by a 16S ribosomal DNA analysis as a Desulfovibrio sp. The other component, the gram-positive organism strain DMC, could not be isolated. It was possible, however, to associate strain DMC on a medium containing dichloromethane in a coculture with Acetobacterium woodii or Methanospirillum hungatei. Coculture of strain DMC with the Archaeon M. hungatei allowed us to specifically amplify by PCR the 16S rRNA gene of strain DMC. A phylogenetic analysis of the 16S ribosomal DNA sequence revealed that this organism groups within the radiation of the Clostridium-Bacillus subphylum and exhibits the highest levels of sequence similarity (89%) with Desulfotomaculum orientis and Desulfitobacterium dehalogenans. Since the novel organism strain DMC was able to grow acetogenically with dichloromethane when it was associated with one of three metabolically different partners and since, in contrast to strain DMB, strain DMC contained carbon monoxide dehydrogenase activity, this bacterium is responsible for both the dehalogenation of dichloromethane and the acetogenesis observed in the original two-component culture. The obligatory dependence of strain DMC on a partner during growth with dichloromethane is thought to stem from the need for a growth factor produced by the associated organism.     

Energetics of syntrophic propionate oxidation in defined batch and chemostat cocultures

Propionate consumption was studied in syntrophic batch and chemostat cocultures of Syntrophobacter fumaroxidans and Methanospirillum hungatei. The Gibbs free energy available for the H(2)-consuming methanogens was <-20 kJ mol of CH(4)(-1) and thus allowed the synthesis of 1/3 mol of ATP per reaction. The Gibbs free energy available for the propionate oxidizer, on the other hand, was usually >-10 kJ mol of propionate(-1). Nevertheless, the syntrophic coculture grew in the chemostat at steady-state rates of 0.04 to 0. 07 day(-1) and produced maximum biomass yields of 2.6 g mol of propionate(-1) and 7.6 g mol of CH(4)(-1) for S. fumaroxidans and M. hungatei, respectively. The energy efficiency for syntrophic growth of S. fumaroxidans, i.e., the biomass produced per unit of available Gibbs free energy was comparable to a theoretical growth yield of 5 to 12 g mol of ATP(-1). However, a lower growth efficiency was observed when sulfate served as an additional electron acceptor, suggesting inefficient energy conservation in the presence of sulfate. The maintenance Gibbs free energy determined from the maintenance coefficient of syntrophically grown S. fumaroxidans was surprisingly low (0.14 kJ h(-1) mol of biomass C(-1)) compared to the theoretical value. On the other hand, the Gibbs free-energy dissipation per mole of biomass C produced was much higher than expected. We conclude that the small Gibbs free energy available in many methanogenic environments is sufficient for syntrophic propionate oxidizers to survive on a Gibbs free energy that is much lower than that theoretically predicted.     

Thermodynamics of hydrogen metabolism in methanogenic cocultures degrading ethanol or lactate

Pure cultures of Desulfovibrio vulgaris or Pelobacter acetylenicus do not grow with lactate or ethanol, respectively, under obligately proton- reducing conditions. However, a small part of these substrates was oxidized and molecular hydrogen was produced up to 4.2 and 3.2 kPa, respectively. During growth in syntrophic methanogenic cocultures with Methanospirillum hungatei as partner, maximum hydrogen partial pressures were significantly lower (0.7 to 2.5 kPa) than in the corresponding pure cultures. Calculation of Gibbs free energies for the prevailing culture conditions showed that H₂ partial pressures were kept in a range at which both, H₂-producing and H₂-consuming reactions, were thermodynamically permissive in pure as well as in syntrophic mixed cultures.     

"Candidatus contubernalis alkalaceticum," an obligately syntrophic alkaliphilic bacterium capable of anaerobic acetate oxidation in a coculture with Desulfonatronum cooperativum

From the silty sediments of the Khadyn soda lake (Tuva), a binary sulfidogenic bacterial association capable of syntrophic acetate oxidation at pH 10.0 was isolated. An obligately syntrophic, gram-positive, spore-forming alkaliphilic rod-shaped bacterium performs acetate oxidation in a syntrophic association with a hydrogenotrophic, alkaliphilic sulfate-reducing bacterium; the latter organism was previously isolated and characterized as the new species Desulfonatronum cooperativum. Other sulfate-reducing bacteria of the genera Desulfonatronum and Desulfonatronovibrio can also act as the hydrogenotrophic partner. Apart from acetate, the syntrophic culture can oxidize ethanol, propanol, isopropanol, serine, fructose, and isobutyric acid. Selective amplification of 16S rRNA gene fragments of the acetate-utilizing syntrophic component of the binary culture was performed; it was found to cluster with clones of uncultured gram-positive bacteria within the family Syntrophomonadaceae. The acetate-oxidizing bacterium is thus the first representative of this cluster obtained in a laboratory culture. Based on its phylogenetic position, the new acetate-oxidizing syntrophic bacterium is proposed to be assigned, in a Candidate status, to a new genus and species: "Candidatus Contubernalis alkalaceticum."     

Comparison of diffusion and reaction rates in anaerobic microbial aggregates

The ability of hydrogen diffusion to account for the rates of methane production in microbial aggregates was studied in a defined coculture consisting of a sulfate reducer grown as a syntrophic hydrogen producer in the absence of sulfate and a methanogen. The hydrogen uptake kinetics of the methanogen were determined using the infinite dilution technique. The maximum hydrogen uptake velocity was 7.1 nmol/min/μg protein and the half saturation constant for hydrogen uptake was 386 nmol/liter. A threshold of 28 nmol/liter below which no further hydrogen consumption occurred was observed. The reconstituted co-culture was shown to produce methane at rates similar to mixed culture enrichments grown on lactate. The diffusion model demonstrated that for the particular system studied, the rates of hydrogen diffusion could account for the overall rate of methane production.     

<Emphasis Type="Italic">Sporotomaculum syntrophicum</Emphasis> sp. nov., a novel anaerobic,syntrophic benzoate-degrading bacterium isolated from methanogenic sludge treating wastewater from terephthalate manufacturing

An anaerobic, mesophilic, syntrophic benzoate-degrading bacterium, designated strain FB(T), was isolated from methanogenic sludge which had been used to treat wastewater from the manufacture of terephthalic acid. Cells were non-motile gram-positive rods that formed spores. The optimum temperature for growth was 35-40 degrees C, and the optimum pH was 7.0-7.2. A co-culture with the hydrogenotrophic methanogen Methanospirillum hungatei converted benzoate to acetate, carbon dioxide, and methane. Butyrate transiently accumulated at a high concentration of 2.5 mM during degradation. Besides benzoate, no other compound tested supported growth of the co-culture. Crotonate supported growth of strain FB(T) in pure culture. Furthermore, the strain degraded benzoate in pure culture with crotonate as co-substrate to produce acetate and butyrate. The strain was not able to utilize sulfate, sulfite, thiosulfate, nitrate, fumarate, or Fe(III) as electron acceptor. The G+C content of the DNA was 46.8 mol%. Strain FB(T) contained MK-7 as the major quinone and C(16:1) as the major fatty acid. 16S rDNA sequence analysis revealed that the strain was a member of the genus Sporotomaculum, even though it exhibited significant differences, such as the capacity for syntrophic growth, to the known member of the genus. Hence, we propose the name Sporotomaculum syntrophicum sp. nov. for strain FB(T). The type strain is strain FB(T) (DSM 14795, JCM 11475).     

Coexistence of a sulphate-reducing Desulfovibrio species and the dehalorespiring Desulfitobacterium frappieri TCE1 in defined chemostat cultures grown with various combinations of sulfate and tetrachloroethene

A two-member co-culture consisting of the dehalorespiring Desulfitobacterium frappieri TCE1 and the sulphate-reducing Desulfovibrio sp. strain SULF1 was obtained via anaerobic enrichment from soil contaminated with tetrachloroethene (PCE). In this co-culture, PCE dechlorination to cis -dichloroethene was due to the activity of the dehalorespiring bacterium only. Chemostat experiments with lactate as the primary electron donor for both strains along with varying sulphate and PCE concentrations showed that the sulphate-reducing strain outnumbered the dehalogenating strain at relatively high ratios of sulphate/PCE. Stable co-cultures with both organisms present at similar cell densities were observed when both electron acceptors were supplied in the reservoir medium in nearly equimolar amounts. In the presence of low sulphate/PCE ratios, the Desulfitobacterium sp. became the numerically dominant strain within the chemostat co-culture. Surprisingly, in the absence of sulphate, strain SULF1 did not disappear completely from the co-culture despite the fact that there was no electron acceptor provided with the medium to be used by this sulphate reducer. Therefore, we propose a syntrophic association between the sulphate-reducing and the dehalorespiring bacteria via interspecies hydrogen transfer. The sulphate reducer was able to sustain growth in the chemostat co-culture by fermenting lactate and using the dehalogenating bacterium as a 'biological electron acceptor'. This is the first report describing growth of a sulphate-reducing bacterium in a defined two-member continuous culture by syntrophically coupling the electron and hydrogen transfer to a dehalorespiring bacterium.     

Pure-culture growth of fermentative bacteria, facilitated by H2 removal: bioenergetics and H2 production

We used an H2-purging culture vessel to replace an H2-consuming syntrophic partner, allowing the growth of pure cultures of Syntrophothermus lipocalidus on butyrate and Aminobacterium colombiense on alanine. By decoupling the syntrophic association, it was possible to manipulate and monitor the single organism's growth environment and determine the change in Gibbs free energy yield (DeltaG) in response to changes in the concentrations of reactants and products, the purging rate, and the temperature. In each of these situations, H2 production changed such that DeltaG remained nearly constant for each organism (-11.1 +/- 1.4 kJ mol butyrate(-1) for S. lipocalidus and -58.2 +/- 1.0 kJ mol alanine(-1) for A. colombiense). The cellular maintenance energy, determined from the DeltaG value and the hydrogen production rate at the point where the cell number was constant, was 4.6 x 10(-13) kJ cell(-1) day(-1) for S. lipocalidus at 55 degrees C and 6.2 x 10(-13) kJ cell(-1) day(-1) for A. colombiense at 37 degrees C. S. lipocalidus, in particular, seems adapted to thrive under conditions of low energy availability.     

Identification and cultivation of anaerobic, syntrophic long-chain fatty acid-degrading microbes from mesophilic and thermophilic methanogenic sludges

We investigated long-chain fatty acid (LCFA)-degrading anaerobic microbes by enrichment, isolation, and RNA-based stable isotope probing (SIP). Primary enrichment cultures were made with each of four LCFA substrates (palmitate, stearate, oleate, or linoleate, as the sole energy source) at 55 degrees C or 37 degrees C with two sources of anaerobic granular sludge as the inoculum. After several transfers, we obtained seven stable enrichment cultures in which LCFAs were converted to methane. The bacterial populations in these cultures were then subjected to 16S rRNA gene-based cloning, in situ hybridization, and RNA-SIP. In five of seven enrichment cultures, the predominant bacteria were affiliated with the family Syntrophomonadaceae. The other two enrichment cultures contained different bacterial populations in which the majority of members belonged to the phylum Firmicutes and the class Deltaproteobacteria. After several attempts to isolate these dominant bacteria, strain MPA, belonging to the family Syntrophomonadaceae, and strain TOL, affiliated with the phylum Firmicutes, were successfully isolated. Strain MPA converts palmitate to acetate and methane in syntrophic association with Methanospirillum hungatei. Even though strain TOL assimilated [(13)C]palmitate in the original enrichment culture, strain TOL has not shown the ability to degrade LCFAs after isolation. These results suggest that microbes involved in the degradation of LCFAs under methanogenic conditions might not belong only to the family Syntrophomonadaceae, as most anaerobic LCFA-degrading microbes do, but may also be found in phylogenetically diverse bacterial groups.     

“Candidatus Contubernalis alkalaceticum,” an Obligately Syntrophic Alkaliphilic Bacterium Capable of Anaerobic Acetate Oxidation in a Coculture with Desulfonatronum cooperativum

From the silty sediments of the Khadyn soda lake (Tuva), a binary sulfidogenic bacterial association capable of syntrophic acetate oxidation at pH 10.0 was isolated. An obligately syntrophic, gram-positive, spore-forming alkaliphilic rod-shaped bacterium performs acetate oxidation in a syntrophic association with a hydrogenotrophic, alkaliphilic sulfate-reducing bacterium; the latter organism was previously isolated and characterized as the new species Desulfonatronum cooperativum. Other sulfate-reducing bacteria of the genera Desulfonatronum and Desulfonatronovibrio can also act as the hydrogenotrophic partner. Apart from acetate, the syntrophic culture can oxidize ethanol, propanol, isopropanol, serine, fructose, and isobutyric acid. Selective amplification of 16S rRNA gene fragments of the acetate-utilizing syntrophic component of the binary culture was performed; it was found to cluster with clones of uncultured gram-positive bacteria within the family Syntrophomonadaceae. The acetate-oxidizing bacterium is thus the first representative of this cluster obtained in a laboratory culture. Based on its phylogenetic position, the new acetate-oxidizing syntrophic bacterium is proposed in the Candidatus status for a new genus and species: “Candidatus Contubernalis alkalaceticum.”     

Enrichment of thermophilic syntrophic anaerobic glutamate-degrading consortia using a dialysis membrane reactor

A dialysis cultivation system was used to enrich slow-growing moderately thermophilic anaerobic bacteria at high cell densities. Bicarbonate buffered mineral salts medium with 5 mM glutamate as the sole carbon and energy source was used and the incubation temperature was 55 degrees C. The reactor inoculum originated from anaerobic methanogenic granular sludge bed reactors. The microbial population was monitored over a period of 2 years using the most probable number (MPN) technique. In the reactor glutamate was readily degraded to ammonium, methane, and carbon dioxide. Cell numbers of glutamate-degrading organisms increased 400-fold over the first year. In medium supplemented with bromoethane sulfonic acid (BES, an inhibitor of methanogenesis), tenfold lower cell numbers were counted, indicating the syntrophic nature of glutamate degradation. After 2 years of reactor operation the predominant organisms were isolated and characterized. Methanobacterium thermoautotrophicum (strain R43) and a Methanosaeta thermophila strain (strain A) were the predominant hydrogenotrophic and acetoclastic methanogens, respectively. The numbers in which the organisms were present in the reactor after 24 months of incubation were 8.6 x 10(9) and 3.8 x 10(7) mL(-1) sludge, respectively. The most predominant glutamate-degrading organism (8.6 x 10(7) mL(-1) sludge), strain Z, was identified as a new species, Caloramator coolhaasii. It converted glutamate to hydrogen, acetate, some propionate, ammonium, and carbon dioxide. Growth of this syntrophic organism on glutamate was strongly enhanced by the presence of methanogens.     

Variability in fermentation patterns of sugar-utilizing bacteria isolated from anaerobic,intertidal sediments

Three dominant types of sugar-fermenting bacteria were isolated from the anaerobic, intertidal sediments of the Eems-Dollard estuary by applying techniques involving anaerobic agar shake tubes. One of the isolated types was tentatively identified as aStreptococcus species, the other two asBacteroides species. All types were versatile with respect to the utilization of sugars. The fermentation patterns of two types were dependent upon conditions of cultivation. In glucose-limited cultures ofStreptococcus strain NS.G52, the production of lactate was suppressed in favor of formate, acetate, and ethanol. In glucose-limited syntrophic cultures withMethanospirillum hungatei, Bacteroides strain NS.G42 was forced to produce acetate and hydrogen at the expense of ethanol. The fermentation pattern ofBacteroides strain NS.S42, which consisted of acetate, propionate, and succinate, was not affected by conditions of cultivation.     

Sulfate reduction in methanogenic bioreactors

Abstract: In the anaerobic treatment of sulfate-containing wastewater, sulfate reduction interferes with methanogenesis. Both mutualistic and competitive interactions between sulfate-reducing bacteria and methanogenic bacteria have been observed. Sulfate reducers will compete with methanogens for the common substrates hydrogen, formate and acetate. In general, sulfate reducers have better growth kinetic properties than methanogens, but additional factors which may be of importance in the competition are adherence properties, mixed substrate utilization, affinity for sulfate of sulfate reducers, relative numbers of bacteria, and reactor conditions such as pH, temperature and sulfide concentration. Sulfate reducers also compete with syntrophic methanogenic consortia involved in the degradation of substrates like propionate and butyrate. In the absence of sulfate these methanogenic consortia are very important, but in the presence of sulfate they are thought to be easily outcompeted by sulfate reducers. However, at relatively low sulfate concentrations, syntrophic degradation of propionate and butyrate coupled to HZ removal via sulfate reduction rather than via methanogenesis may become important. A remarkable feature of some sulfate reducers is their ability to grow fermentatively or to grow in syntrophic association with methanogens in the absence of sulfate.     

Degradation of unsaturated hydrocarbons by methanogenic enrichment cultures

Abstract The biodegradability of hydrocarbons under anaerobic conditions was studied in enrichment cultures using mineral media inoculated with sewage sludge or sediment samples of limnic and marine origin. No indication of methanogenic degradation was obtained with either n -hexane, n -hexadecane, n -heptadecane, 1-hexene, cis -2-hexene, trans -2-hexene, isoprene, 1-hexine, benzene, toluene, xylene, cyclohexene, cycloheptatriene, cyclopentadiene, styrene, naphthalene, azulene, or β-carotene. Squalene was incompletely converted to methane and carbon dioxide. Complete degradation was observed with 1-hexadecene. Methanogenic subcultures were maintained on 1-hexadecene and squalene. Both enrichments contained after several transfers Methanospirillum hungatei and Methanothrix soehngenii as prevalent methanogenic bacteria. Acetate (≤80 μ M) was the only intermediary product detected indicating that degradation proceeded via hydrogen-dependent syntrophic β-oxidations. Short rods on hexadecene and cocci on squalene were found to be associated with substrate degradation. The results indicate that terminal double bonds can be sufficient to allow methanogenic degradation of hydrocarbons whereas branching and terminal ring closures may significantly contribute to hydrocarbon stability in anoxic environments.     

Growth of a facultative anaerobe under oxygen-limiting conditions in pure culture and in co-culture with a sulfate-reducing bacterium

Abstract The occurrence and properties were studied of glucose-metabolizing bacteria present in the anaerobic sediment 5–10 cm below the surface of an estuarine tidal mud-flat. Of all these bacteria (10⁴– 10⁵ per g wet sediment) 80–90% were facultatively anaerobic species. Chemostat enrichments on glucose under aerobic, oxygen-limited and alternately aerobic-anaerobic conditions also yielded cultures dominated by facultative anaerobes. One of the dominant species, tentatively identified as a Vibrio sp., was studied in more detail under oxygen-limiting conditions. Fermentative and respiratory metabolisms were found to operate simultaneously, and the ratio between the two was regulated by the extent of oxygen limitation. A small fraction of the acetate formed under such growth conditions was shown to be subsequently respired. A co-culture was established of the Vibrio sp. and a sulfate-reducing bacterium ( Desulfovibrio HL21 ) in an aerated chemostat. The importance of these observations is discussed in relation to the role of facultative anaerobes in anaerobic habitats.     

Sulfate reduction by a syntrophic propionate-oxidizing bacterium

The syntrophic propionate-oxidizing bacterium MPOB was able to grow in the absence of methanogens by coupling the oxidation of propionate to the reduction of sulfate. Growth on propionate plus sulfate was very slow (=0.024 day^–1). An average growth yield was found of 1.5 g (dry weight) per mol of propionate. MPOB grew even slower than other sulfate-reducing syntrophic propionate-oxidizing bacteria. The growth rates and yields of strict sulfate-reducing bacteria (Desulfobulbus sp.) grown on propionate plus sulfate are considerably higher.