Interspecies distances between propionic acid degraders and methanogens in syntrophic consortia for optimal hydrogen transfer

A mixed culture from an anaerobic biowaste digester was enriched on propionate and used to investigate interspecies hydrogen transfer in dependence of spatial distances between propionate degraders and methanogens. From 20.3 mM propionate, 20.8 mM acetate and 15.5 mM methane were formed. Maximum specific propionate oxidation and methane formation rates were 49 and 23 mmol?mg^?1?day^?1, respectively. Propionate oxidation was inhibited by only 20 mM acetate by about 50 %. Intermediate formate formation during inhibited methanogensis was observed. The spatial distribution and the biovolume fraction of propionate degraders and of methanogens in relation to the total population during aggregate formation were determined. Measurements of interbacterial distances were conducted with fluorescence in situ hybridization by application of group-specific 16S rRNA-targeted probes and 3D image analyses. With increasing incubation time, floc formation and growth up to 54 μm were observed. Propionate degraders and methanogens were distributed randomly in the flocs. The methanogenic biovolume fraction was high at the beginning and remained constant over 42 days, whereas the fraction of propionate degraders increased with time during propionate feeding. Interbacterial distances between propionate degraders and methanogens decreased with time from 5.30 to 0.29 μm, causing an increase of the maximum possible hydrogen flux from 1.1 to 10.3 nmol?ml^?1?min^?1. The maximum possible hydrogen flux was always higher than the hydrogen formation and consumption rate, indicating that reducing the interspecies distance by aggregation is advantageous in complex ecosystems.     

Effects of sulfate on lactate and C2-, C3- volatile fatty acid anaerobic degradation by a mixed microbial culture

The effects of sulfate on the anaerobic degradation of lactate, propionate, and acetate by a mixed bacterial culture from an anaerobic fermenter fed with wine distillery waste water were investigated. Without sulfate and with both sulfate and molybdate, lactate was rapidly consumed, and propionate and acetate were produced; whereas with sulfate alone, only acetate accumulated. Propionate oxidation was strongly accelerated by the presence of sulfate, but sulfate had no effect on acetate consumption even when methanogenesis was inhibited by chloroform. The methane production was not affected by the presence of sulfate. Counts of lactate- and propionate-oxidizing sulfate-reducing bacteria in the mixed culture gave 4.5×10⁸ and 1.5×10⁶ viable cells per ml, respectively. The number of lactate-oxidizing fermentative bacteria was 2.2×10⁷ viable cells per ml, showing that sulfate-reducing bacteria outcompete fermentative bacteria for lactate in the ecosystem studied. The number of acetoclastic methanogens was 3.5×10⁸ viable cells per ml, but only 2.5×10⁴ sulfate reducers were counted on acetate, showing that acetotrophic methanogens completely predominated over acetate-oxidizing sulfate-reducing bacteria. The contribution of acetate as electron donor for sulfate reduction in the ecosystem studied was found to be minor.     

Growth of Syntrophic Propionate-Oxidizing Bacteria with Fumarate in the Absence of Methanogenic Bacteria

Oxidation of succinate to fumarate is an energetically difficult step in the biochemical pathway of propionate oxidation by syntrophic methanogenic cultures. Therefore, the effect of fumarate on propionate oxidation by two different propionate-oxidizing cultures was investigated. When the methanogens in a newly enriched propionate-oxidizing methanogenic culture were inhibited by bromoethanesulfonate, fumarate could act as an apparent terminal electron acceptor in propionate oxidation. ¹³C-nuclear magnetic resonance experiments showed that propionate was carboxylated to succinate while fumarate was partly oxidized to acetate and partly reduced to succinate. Fumarate alone was fermented to succinate and CO₂. Bacteria growing on fumarate were enriched and obtained free of methanogens. Propionate was metabolized by these bacteria when either fumarate or Methanospirillum hungatii was added. In cocultures with Syntrophobacter wolinii, such effects were not observed upon addition of fumarate. Possible slow growth of S. wolinii on fumarate could not be demonstrated because of the presence of a Desulfovibrio strain which grew rapidly on fumarate in both the absence and presence of sulfate.     

Cultivation and In Situ Detection of a Thermophilic Bacterium Capable of Oxidizing Propionate in Syntrophic Association with Hydrogenotrophic Methanogens in a Thermophilic Methanogenic Granular Sludge

The thermophilic, anaerobic, propionate-oxidizing bacterial populations present in the methanogenic granular sludge in a thermophilic (55°C) upflow anaerobic sludge blanket reactor were studied by cultivation and in situ hybridization analysis. For isolation of propionate-degrading microbes, primary enrichment was made with propionate as the sole energy source at 55°C. After several attempts to purify the microbes, a thermophilic, syntrophic, propionate-oxidizing bacterium, designated strain SI, was isolated in both pure culture and coculture with Methanobacterium thermoautotrophicum. Under thermophilic (55°C) conditions, strain SI oxidized propionate, ethanol, and lactate in coculture with M. thermoautotrophicum. In pure culture, the isolate was found to ferment pyruvate. 16S ribosomal DNA sequence analysis revealed that the strain was relatively close to members of the genus Desulfotomaculum, but it was only distantly related to any known species. To elucidate the abundance and spatial distribution of organisms of the strain SI type within the sludge granules, a 16S rRNA-targeted oligonucleotide probe specific for strain SI was developed and applied to thin sections of the granules. Fluorescence in situ hybridization combined with confocal laser scanning microscopy revealed that a number of rod-shaped cells were present in the middle and inner layers of the thermophilic granule sections and that they formed close associations with hydrogenotrophic methanogens. They accounted for approximately 1.1% of the total cells in the sludge. These results demonstrated that strain SI was one of the significant populations in the granular sludge and that it was responsible for propionate oxidation in the methanogenic granular sludge in the reactor.     

The effect of pharmaceuticals on the kinetics of methanogenesis and acetogenesis

In this study, the widely used anaerobic digestion model (ADM1) was used in order to simulate the inhibition of three pharmaceuticals, propranolol hydrochloride, ofloxacin and diclofenac sodium, on two groups of microorganisms, acetogens and acetoclastic methanogens, the most sensitive microorganisms groups involved in the anaerobic digestion process. The specific maximum consumption rate and saturation constant of acetate and propionate degraders were estimated through fitting the model to experimental data taken from continuous and batch experiments. A modified non-competitive inhibition function was used, and the inhibition constants were estimated using data from Batch experiments conducted at various concentrations of pharmaceuticals using enriched cultures with propionate and acetate degraders. It was found that propranolol hydrochloride was the most inhibitory pharmaceutical to both microorganisms groups.     

Biomass Measurement of Methanogens in the Sediments of Tokyo Bay Using Archaeol Lipids

An archaeal ether-linked lipid, archaeol, was determined to be a biomass indicator for methanogens both in the laboratory enriched culture and in marine sediments. The archaeol measurement method described by Ohtsubo et al. in 1993 was modified and applied to marine sediments. We compared the amount of archaeol with the cell number of methanogens or methane concentration in laboratory enriched culture of methanogens from marine sediment. Good correlations were obtained as follows: (Methane, mmol) = 11.2 × (Archaeol, mg): r= .996 or (Cell number) = 1.13 × 10¹¹× (Archaeol, mg): r= .995, respectively. In the sediments of Tokyo Bay, archaeol was measured from approximately 46 to 561 ng/dry g sediment at the entrance to 267 to 4160 ng/dry g sediment at the innermost area. Using the coefficient from the laboratory experiment, these data corresponded to cell numbers of 5.2 × 10⁶ to 4.7 × 10⁸/dry g sediment. These values were 1 or 2 orders of magnitude higher than those obtained by culture methods in previous studies. Although dead or decomposed cells might be detected, archaeol measurement is useful for estimating the biomass of methanogens because of the good correlation between methane concentration and archaeol content in marine environments. In this study, we found a correlation of (Methane, mmol) = 0.012 × (Archaeol, mg): r= .932, n= 17 in marine sediments. Received December 21, 1998; accepted June 16, 1999     

Enrichment of syngas‐converting communities from a multi‐orifice baffled bioreactor

The substitution of natural gas by renewable biomethane is an interesting option to reduce global carbon footprint. Syngas fermentation has potential in this context, as a diverse range of low‐biodegradable materials that can be used. In this study, anaerobic sludge acclimatized to syngas in a multi‐orifice baffled bioreactor (MOBB) was used to start enrichments with CO. The main goals were to identify the key players in CO conversion and evaluate potential interspecies metabolic interactions conferring robustness to the process. Anaerobic sludge incubated with 0.7 × 10⁵ Pa CO produced methane and acetate. When the antibiotics vancomycin and/or erythromycin were added, no methane was produced, indicating that direct methanogenesis from CO did not occur. Acetobacterium and Sporomusa were the predominant bacterial species in CO‐converting enrichments, together with methanogens from the genera Methanobacterium and Methanospirillum. Subsequently, a highly enriched culture mainly composed of a Sporomusa sp. was obtained that could convert up to 1.7 × 10⁵ Pa CO to hydrogen and acetate. These results attest the role of Sporomusa species in the enrichment as primary CO utilizers and show their importance for methane production as conveyers of hydrogen to methanogens present in the culture.     

Molecular analysis of the biomass of a fluidized bed reactor treating synthetic vinasse at anaerobic and micro-aerobic conditions

The microbial communities (Bacteria and Archaea) established in an anaerobic fluidized bed reactor used to treat synthetic vinasse (betaine, glucose, acetate, propionate, and butyrate) were characterized by denaturing gradient gel electrophoresis (DGGE) and phylogenetic analysis. This study was focused on the competitive and syntrophic interactions between the different microbial groups at varying influent substrate to sulfate ratios of 8, 4, and 2 and anaerobic or micro-aerobic conditions. Acetogens detected along the anaerobic phases at substrate to sulfate ratios of 8 and 4 seemed to be mainly involved in the fermentation of glucose and betaine, but they were substituted by other sugar or betaine degraders after oxygen application. Typical fatty acid degraders that grow in syntrophy with methanogens were not detected during the entire reactor run. Likely, sugar and betaine degraders outnumbered them in the DGGE analysis. The detected sulfate-reducing bacteria (SRB) belonged to the hydrogen-utilizing Desulfovibrio. The introduction of oxygen led to the formation of elemental sulfur (S⁰) and probably other sulfur compounds by sulfide-oxidizing bacteria (γ-Proteobacteria). It is likely that the sulfur intermediates produced from sulfide oxidation were used by SRB and other microorganisms as electron acceptors, as was supported by the detection of the sulfur respiring Wolinella succinogenes. Within the Archaea population, members of Methanomethylovorans and Methanosaeta were detected throughout the entire reactor operation. Hydrogenotrophic methanogens mainly belonging to the genus Methanobacterium were detected at the highest substrate to sulfate ratio but rapidly disappeared by increasing the sulfate concentration.     

Alcohol conversion by Desulfobulbus propionicus Lindhorst in the presence and absence of sulfate and hydrogen

Ethanol was rapidly degraded to mainly acetate in anaerobic freshwater sediment slurries. Propionate was produced in small amounts. Desulfovibrio species were the dominant bacteria among the ethanol-degrading organisms. The propionate-producing Desulfobulbus propionicus came to the fore under iron-limited conditions in an ethanol-limited chemostat with excess sulfate inoculated with anaerobic intertidal freshwater sediment. In the absence of sulfate, ethanol was fermented by D. propionicus Lindhorst to propionate and acetate in a molar ratio of 2.0.l-Propanol was intermediately produced during the fermentation of ethanol. In the presence of H₂ and CO₂, ethanol was quantitatively converted to propionate. H₂-plus sulfate-grown cells of D. propionicus Lindhorst were able to oxidize l-propanol and l-butanol to propionate and butyrate respectively with the concomitant reduction of acetate plus CO₂ to propionate. Growth was also observed on acetate alone in the presence of H₂ and CO₂ D. propionicus was able to grow mixotrophically on H₂ plus an organic compound. Finally, a brief discussion has been given of the ecological niche of D. propionicus in anaerobic freshwater sediments.     

Start-up and operation of a propionate-degrading fluidized-bed reactor

Summary A laboratory-scale fluidized-bed reactor was inoculated with a syntrophically propionate-degrading co-culture. After 24 days of batch operation with propionate and acetate in the medium, the reactor was operated for 8 months with propionate as the sole substrate. The loading rate was 8.5 kg chemical oxygen demand/m³ ·day, yielding a maximal gas production of 4.5 l/l·day at a removal efficiency of 94–99%. The degradation was not inhibited by up to 85mm propionate in pulse experiments, but short-time changes in redox potential above — 300 mV led to a drop in the propionate degradation rate. While Methanocorpusculum sp. and Methanospirillum sp. adhered to the sand in the early phase of the start-up, the consortium in the mature biofilm consisted of Desulfobulbus sp., Methanothrix soehngenii and species of at least three different genera of hydrogenotrophic methanogens. A syntrophic relationship between the sulphate-reducing Desulfobulbus sp. and hydrogenotrophic and acetotrophic methanogens is discussed.Offprint requests to: G. Zellner     

Cultivation and in situ detection of a thermophilic bacterium capable of oxidizing propionate in syntrophic association with hydrogenotrophic methanogens in a thermophilic methanogenic granular sludge

The thermophilic, anaerobic, propionate-oxidizing bacterial populations present in the methanogenic granular sludge in a thermophilic (55 degrees C) upflow anaerobic sludge blanket reactor were studied by cultivation and in situ hybridization analysis. For isolation of propionate-degrading microbes, primary enrichment was made with propionate as the sole energy source at 55 degrees C. After several attempts to purify the microbes, a thermophilic, syntrophic, propionate-oxidizing bacterium, designated strain SI, was isolated in both pure culture and coculture with Methanobacterium thermoautotrophicum. Under thermophilic (55 degrees C) conditions, strain SI oxidized propionate, ethanol, and lactate in coculture with M. thermoautotrophicum. In pure culture, the isolate was found to ferment pyruvate. 16S ribosomal DNA sequence analysis revealed that the strain was relatively close to members of the genus Desulfotomaculum, but it was only distantly related to any known species. To elucidate the abundance and spatial distribution of organisms of the strain SI type within the sludge granules, a 16S rRNA-targeted oligonucleotide probe specific for strain SI was developed and applied to thin sections of the granules. Fluorescence in situ hybridization combined with confocal laser scanning microscopy revealed that a number of rod-shaped cells were present in the middle and inner layers of the thermophilic granule sections and that they formed close associations with hydrogenotrophic methanogens. They accounted for approximately 1.1% of the total cells in the sludge. These results demonstrated that strain SI was one of the significant populations in the granular sludge and that it was responsible for propionate oxidation in the methanogenic granular sludge in the reactor.     

Growth of Methanogenic Bacteria in Pure Culture with 2-Propanol and Other Alcohols as Hydrogen Donors

Two types of mesophilic, methanogenic bacteria were isolated in pure culture from anaerobic freshwater and marine mud with 2-propanol as the hydrogen donor. The freshwater strain (SK) was a Methanospirillum species, the marine, salt-requiring strain (CV), which had irregular coccoid cells, resembled Methanogenium sp. Stoichiometric measurements revealed formation of 1 mol of CH₄ by CO₂ reduction, with 4 mol of 2-propanol being converted to acetone. In addition to 2-propanol, the isolates used 2-butanol, H₂, or formate but not methanol or polyols. Acetate did not serve as an energy substrate but was necessary as a carbon source. Strain CV also oxidized ethanol or 1-propanol to acetate or propionate, respectively; growth on the latter alcohols was slower, but final cell densities were about threefold higher than on 2-propanol. Both strains grew well in defined, bicarbonate-buffered, sulfide-reduced media. For cultivation of strain CV, additions of biotin, vitamin B₁₂, and tungstate were necessary. The newly isolated strains are the first methanogens that were shown to grow in pure culture with alcohols other than methanol. Bioenergetic aspects of secondary and primary alcohol utilization by methanogens are discussed.     

Characteristic microbial community of a dry thermophilic methanogenic digester: its long-term stability and change with feeding

Thermophilic dry anaerobic digestion of sludge for cellulose methanization was acclimated at 53 °C for nearly 5 years using a waste paper-based medium. The stability of the microbial community structure and the microbial community responsible for the cellulose methanization were studied by 16S rRNA gene-based clone library analysis. The microbial community structure remained stable during the long-term acclimation period. Hydrogenotrophic methanogens dominated in methanogens and Methanothermobacter, Methanobacterium, Methanoculleus, and Methanosarcina were responsible for the methane production. Bacteria showed relatively high diversity and distributed mainly in the phyla Firmicutes, Bacteroidetes, and Synergistetes. Ninety percent of operational taxonomic units (OTUs) were affiliated with the phylum Firmicutes, indicating the crucial roles of this phylum in the digestion. Relatives of Clostridium stercorarium, Clostridium thermocellum, and Halocella cellulosilytica were dominant cellulose degraders. The acclimated stable sludge was used to treat garbage stillage discharged from a fuel ethanol production process, and the shift of microbial communities with the change of feed was analyzed. Both archaeal and bacterial communities had obviously changed: Methanoculleus spp. and Methanothermobacter spp. and the protein- and fatty acid-degrading bacteria became dominant. Accumulation of ammonia as well as volatile fatty acids led to the inhibition of microbial activity and finally resulted in the deterioration of methane fermentation of the garbage stillage.     

典型煤层水微生物产甲烷潜力及其群落结构研究

内蒙古自治区二连盆地、海拉尔盆地是我国重要的煤层气产区,其中生物成因煤层气是煤层气的重要来源,但复杂物质转化产甲烷相关微生物群落结构及功能尚不清楚。【目的】研究煤层水中的微生物代谢挥发性脂肪酸产甲烷的生理特征及群落特征。【方法】以内蒙古自治区二连盆地和海拉尔盆地的四口煤层气井水作为接种物,分别添加乙酸钠、丙酸钠和丁酸钠厌氧培养;定期监测挥发性脂肪酸降解过程中甲烷和底物的变化趋势,应用高通量测序技术,分析原始煤层气井水及稳定期产甲烷菌液的微生物群落结构。【结果】除海拉尔盆地H303煤层气井微生物不能代谢丙酸外,其他样品均具备代谢乙酸、丙酸和丁酸产生甲烷的能力,其生理生态参数存在显著差异,产甲烷延滞期依次是乙酸丁酸丙酸;最大比产甲烷速率和底物转化效率依次是丙酸乙酸丁酸。富集培养后,古菌群落结构与煤层气井水的来源显著相关,二连盆地优势古菌为氢营养型产甲烷古菌Methanocalculus (相对丰度13.5%–63.4%)和复合营养型产甲烷古菌Methanosarcina (7.9%–51.3%),海拉尔盆地的优势古菌为氢营养型产甲烷古菌Methanobacterium(24.3%–57.4%)和复合营养型产甲烷古菌Methanosarcina(29.6%–66.5%);细菌群落则与底物类型显著相关,硫酸盐还原菌Desulfovibrio(12.0%–41.0%)、互营丙酸氧化菌Syntrophobacter(39.6%–75.5%)和互营丁酸菌Syntrophomonas(8.5%–21.9%)分别在乙酸钠、丙酸钠和丁酸钠处理组显著富集。【结论】煤层气井水微生物可降解挥发性脂肪酸(乙酸、丙酸和丁酸)并具有产甲烷潜力;乙酸可能被古菌直接代谢产甲烷,而丙酸和丁酸通过互营细菌和产甲烷古菌代谢产甲烷。Desulfovibrio、Syntrophobacter和Syntrophomonas分别在乙酸、丙酸和丁酸代谢过程中发挥了重要作用。这些结果为煤层气生物强化开采提供了一定的微生物资源基础。     

Clostridium neopropionicum sp. nov., a strict anaerobic bacterium fermenting ethanol to propionate through acrylate pathway

Strain X4 was isolated several years ago from an anaerobic mesophilic plant treating vegetable cannery waste waters. It was the first example of propionic fermentation from ethanol. Morphologic and physiologic characterizations of the strain are presented here. This strain is described as type strain of a new species, Clostridium neopropionicum sp. nov. Whole cells of strain X4 ferment [1-¹³C]ethanol and CO₂ to [2-¹³C]propionate, [1-¹³C]acetate and [2-¹³C]propanol, suggesting the absence of a randomizing pathway during the propionate formation. Enzymes involved in this fermentation were assayed in cell-free extracts of cells grown with ethanol as sole substrate. Alcohol dehydrogenase, aldehyde dehydrogenase, phosphate acetyl transferase, acetate kinase, pyruvate synthase, lactate dehydrogenases, and the enzymes of the acrylate pathway were detected at activities sufficient to be involved in ethanol fermentation. The same pathway may be used for the degradation of lactate or acrylate to acetate.     

Methanosarcina as the dominant aceticlastic methanogens during mesophilic anaerobic digestion of putrescible waste

Taking into account isotope ¹³C value a mathematical model was developed to describe the dynamics of methanogenic population during mesophilic anaerobic digestion of putrescible solid waste and waste imitating Chinese municipal solid waste. Three groups of methanogens were considered in the model including unified hydrogenotrophic methanogens and two aceticlastic methanogens Methanosaeta sp. and Methanosarcina sp. It was assumed that Methanosaeta sp. and Methanosarcina sp. are inhibited by high volatile fatty acids concentration. The total organic and inorganic carbon concentrations, methane production, methane and carbon dioxide partial pressures as well as the isotope ¹³C incorporation in PSW and CMSW were used for the model calibration and validation. The model showed that in spite of the high initial biomass concentration of Methanosaeta sp. Methanosarcina sp. became the dominant aceticlastic methanogens in the system. This prediction was confirmed by FISH. It is concluded that Methanosarcina sp. forming multicellular aggregates may resist to inhibition by volatile fatty acids (VFAs) because a slow diffusion rate of the acids limits the VFA concentrations inside the Methanosarcina sp. aggregates.     

Cellulose Fermentation by a Rumen Anaerobic Fungus in Both the Absence and the Presence of Rumen Methanogens

The fermentation of cellulose by an ovine rumen anaerobic fungus in the absence and presence of rumen methanogens is described. In the monoculture, moles of product as a percentage of the moles of hexose fermented were: acetate, 72.7; carbon dioxide, 37.6; formate, 83.1; ethanol, 37.4; lactate, 67.0; and hydrogen, 35.3. In the coculture, acetate was the major product (134.7%), and carbon dioxide increased (88.7%). Lactate and ethanol production decreased to 2.9 and 19%, respectively, little formate was detected (1%), and hydrogen did not accumulate. Substantial amounts of methane were produced in the coculture (58.7%). Studies with [2-¹⁴C]acetate indicated that acetate was not a precursor of methane. The demonstration of cellulose fermentation by a fungus extends the range of known rumen organisms capable of participating in cellulose digestion and provides further support for a role of anaerobic fungi in rumen fiber digestion. The effect of the methanogens on the pattern of fermentation is interpreted as a shift in flow of electrons away from electron sink products to methane via hydrogen. The study provides a new example of intermicrobial hydrogen transfer and the first demonstration of hydrogen formation by a fungus.     

Bacteria and archaea involved in anaerobic digestion of distillers grains with solubles

Cereal distillers grains, a by-product from bioethanol industry, proved to be a suitable feedstock for biogas production in laboratory scale anaerobic digesters. Five continuously stirred tank reactors were run under constant conditions and monitored for biogas production and composition along with other process parameters. Iron additives for sulfide precipitation significantly improved the process stability and efficiency, whereas aerobic pretreatment of the grains had no effect. The microbial communities in the reactors were investigated for their phylogenetic composition by terminal restriction fragment length polymorphism analysis and sequencing of 16S rRNA genes. The bacterial subcommunities were highly diverse, and their composition did not show any correlation with reactor performance. The dominant phylotypes were affiliated to the Bacteroidetes. The archaeal subcommunities were less diverse and correlated with the reactor performance. The well-performing reactors operated at lower organic loading rates and amended with iron chloride were dominated by aceticlastic methanogens of the genus Methanosaeta. The well-performing reactor operated at a high organic loading rate and supplemented with iron hydroxide was dominated by Methanosarcina ssp. The reactor without iron additives was characterized by propionate and acetate accumulation and high hydrogen sulfide content and was dominated by hydrogenotrophic methanogens of the genus Methanoculleus.     

Distribution of culturable microorganisms in Fennoscandian Shield groundwater

Microbial populations in 16 groundwater samples from six Fennoscandian Shield sites in Finland and Sweden were investigated. The average total cell number was 3.7×10⁵ cells ml⁻¹, and there was no change in the mean of the total cell numbers to a depth of 1390 m. Culture media were designed based on the chemical composition of each groundwater sample and used successfully to culture anaerobic microorganisms from all samples between 65 and 1350 m depth. Between 0.0084 and 14.8% of total cells were cultured from groundwater samples. Sulfate-reducing bacteria, iron-reducing bacteria and heterotrophic acetogenic bacteria were cultured from groundwater sampled at 65–686 m depth in geographically distant sites. Different microbial populations were cultured from deeper, older and more saline groundwater from 863 to 1350 m depth. Principal component analysis of groundwater chemistry data showed that sulfate- and iron-reducing bacteria were not detected in the most saline groundwater. Iron-reducing bacteria and acetogens were cultured from deep groundwater that contained 0.35–3.5 mM sulfate, while methanogens and acetogens were cultured from deep sulfate-depleted groundwater. In one borehole from which autotrophic methanogens were cultured, dissolved inorganic carbon was enriched in ¹³C compared to other Fennoscandian Shield groundwater samples, suggesting that autotrophs were active. It can be concluded that a diverse microbial community is present from the surface to over 1300 m depth in the Fennoscandian Shield.     

Enrichment of Thermophilic Propionate-Oxidizing Bacteria in Syntrophy with Methanobacterium thermoautotrophicum or Methanobacterium thermoformicicum

Thermophilic propionate-oxidizing, proton-reducing bacteria were enriched from the granular methanogenic sludge of a bench-scale upflow anaerobic sludge bed reactor operated at 55°C with a mixture of volatile fatty acids as feed. Thermophilic hydrogenotrophic methanogens had a high decay rate. Therefore, stable, thermophilic propionate-oxidizing cultures could not be obtained by using the usual enrichment procedures. Stable and reproducible cultivation was possible by enrichment in hydrogen-pregrown cultures of Methanobacterium thermoautotrophicum ΔH which were embedded in precipitates of FeS, achieved by addition of FeCl₂ to the media. The propionate-oxidizing bacteria formed spores which resisted pasteurization for 30 min at 90°C or 10 min at 100°C. Highly purified cultures were obtained with either M. thermoautotrophicum ΔH or Methanobacterium thermoformicicum Z245 as the syntrophic partner organism. The optimum temperature for the two cultures was 55°C. Maximum specific growth rates of cultures with M. thermoautotrophicum ΔH were somewhat lower than those of cultures with M. thermoformicicum Z245 (0.15 and 0.19 day^-1, respectively). Growth rates were even higher (0.32 day^-1) when aceticlastic methanogens were present as well. M. thermoautotrophicum ΔH is an obligately hydrogen-utilizing methanogen, showing that interspecies hydrogen transfer is the mechanism by which reducing equivalents are channelled from the acetogens to this methanogen. Boundaries of hydrogen partial pressures at which propionate oxidation occurred were between 6 and 34 Pa. Formate had a strong inhibitory effect on propionate oxidation in cultures with M. thermoautotrophicum. Inhibition by formate was neutralized by addition of the formate-utilizing methanogen or by addition of fumarate. Results indicate that formate inhibited succinate oxidation to fumarate, an intermediate step in the biochemical pathway of propionate oxidation.