Rapid Methane Oxidation in a Landfill Cover Soil

Methane oxidation rates observed in a topsoil covering a retired landfill are the highest reported (45 g m⁻² day⁻¹) for any environment. This microbial community had the capacity to rapidly oxidize CH₄ at concentrations ranging from <1 ppm (microliters per liter) (first-order rate constant [k] = −0.54 h⁻¹) to >10⁴ ppm (k = −2.37 h⁻¹). The physiological characteristics of a methanotroph isolated from the soil (characteristics determined in aqueous medium) and the natural population, however, were similar to those of other natural populations and cultures: the Q₁₀ and optimum temperature were 1.9 and 31°C, respectively, the apparent half-saturation constant was 2.5 to 9.3 μM, and 19 to 69% of oxidized CH₄ was assimilated into biomass. The CH₄ oxidation rate of this soil under waterlogged (41% [wt/vol] H₂O) conditions, 6.1 mg liter⁻¹ day⁻¹, was near rates reported for lake sediment and much lower than the rate of 116 mg liter⁻¹ day⁻¹ in the same soil under moist (11% H₂O) conditions. Since there are no large physiological differences between this microbial community and other CH₄ oxidizers, we attribute the high CH₄ oxidation rate in moist soil to enhanced CH₄ transport to the microorganisms; gas-phase molecular diffusion is 10⁴-fold faster than aqueous diffusion. These high CH₄ oxidation rates in moist soil have implications that are important in global climate change. Soil CH₄ oxidation could become a negative feedback to atmospheric CH₄ increases (and warming) in areas that are presently waterlogged but are projected to undergo a reduction in summer soil moisture.     

Degradation of Methanethiol by Methylotrophic Methanogenic Archaea in a Lab-Scale Upflow Anaerobic Sludge Blanket Reactor

In a lab-scale upflow anaerobic sludge blanket reactor inoculated with granular sludge from a full-scale wastewater treatment plant treating paper mill wastewater, methanethiol (MT) was degraded at 30°C to H₂S, CO₂, and CH₄. At a hydraulic retention time of 9 h, a maximum influent concentration of 6 mM MT was applied, corresponding to a volumetric loading rate of 16.5 mmol liter⁻¹ day⁻¹. The archaeal community within the reactor was characterized by anaerobic culturing and denaturing gradient gel electrophoresis analysis, cloning, and sequencing of 16S rRNA genes and quantitative PCR. Initially, MT-fermenting methanogenic archaea related to members of the genus Methanolobus were enriched in the reactor. Later, they were outcompeted by Methanomethylovorans hollandica, which was detected in aggregates but not inside the granules that originated from the inoculum, the microbial composition of which remained fairly unchanged. Possibly other species within the Methanosarcinacaea also contributed to the fermentation of MT, but they were not enriched by serial dilution in liquid media. The archaeal community within the granules, which was dominated by Methanobacterium beijingense, did not change substantially during the reactor operation. Some of the species related to Methanomethylovorans hollandica were enriched by serial dilutions, but their growth rates were very low. Interestingly, the enrichments could be sustained only in the presence of MT and did not utilize any of the other typical substrates for methylotrophic methanogens, such as methanol, methyl amine, or dimethylsulfide.     

Phylogenetic Identification and Substrate Uptake Patterns of Sulfate-Reducing Bacteria Inhabiting an Oxic-Anoxic Sewer Biofilm Determined by Combining Microautoradiography and Fluorescent In Situ Hybridization

We simultaneously determined the phylogenetic identification and substrate uptake patterns of sulfate-reducing bacteria (SRB) inhabiting a sewer biofilm with oxygen, nitrate, or sulfate as an electron acceptor by combining microautoradiography and fluorescent in situ hybridization (MAR-FISH) with family- and genus-specific 16S rRNA probes. The MAR-FISH analysis revealed that Desulfobulbus hybridized with probe 660 was a dominant SRB subgroup in this sewer biofilm, accounting for 23% of the total SRB. Approximately 9 and 27% of Desulfobulbus cells detected with probe 660 could take up [¹⁴C]propionate with oxygen and nitrate, respectively, as an electron acceptor, which might explain the high abundance of this species in various oxic environments. Furthermore, more than 40% of Desulfobulbus cells incorporated acetate under anoxic conditions. SRB were also numerically important members of H₂-utilizing and ¹⁴CO₂-fixing microbial populations in this sewer biofilm, accounting for roughly 42% of total H₂-utilizing bacteria hybridized with probe EUB338. A comparative 16S ribosomal DNA analysis revealed that two SRB populations, related to the Desulfomicrobium hypogeium and the Desulfovibrio desulfuricans MB lineages, were found to be important H₂ utilizers in this biofilm. The substrate uptake characteristics of different phylogenetic SRB subgroups were compared with the characteristics described to date. These results provide further insight into the correlation between the 16S rRNA phylogenetic diversity and the physiological diversity of SRB populations inhabiting sewer biofilms.     

Successional Development of Sulfate-Reducing Bacterial Populations and Their Activities in a Wastewater Biofilm Growing under Microaerophilic Conditions

A combination of fluorescence in situ hybridization, microprofiles, denaturing gradient gel electrophoresis of PCR-amplified 16S ribosomal DNA fragments, and 16S rRNA gene cloning analysis was applied to investigate successional development of sulfate-reducing bacteria (SRB) community structure and in situ sulfide production activity within a biofilm growing under microaerophilic conditions (dissolved oxygen concentration in the bulk liquid was in the range of 0 to 100 μM) and in the presence of nitrate. Microelectrode measurements showed that oxygen penetrated 200 μm from the surface during all stages of biofilm development. The first sulfide production of 0.32 μmol of H₂S m⁻² s⁻¹ was detected below ca. 500 μm in the 3rd week and then gradually increased to 0.70 μmol H₂S m⁻² s⁻¹ in the 8th week. The most active sulfide production zone moved upward to the oxic-anoxic interface and intensified with time. This result coincided with an increase in SRB populations in the surface layer of the biofilm. The numbers of the probe SRB385- and 660-hybridized SRB populations significantly increased to 7.9 × 10⁹ cells cm⁻³ and 3.6 × 10⁹ cells cm⁻³, respectively, in the surface 400 μm during an 8-week cultivation, while those populations were relatively unchanged in the deeper part of the biofilm, probably due to substrate transport limitation. Based on 16S rRNA gene cloning analysis data, clone sequences that related to Desulfomicrobium hypogeium (99% sequence similarity) and Desulfobulbus elongatus (95% sequence similarity) were most frequently found. Different molecular analyses confirmed that Desulfobulbus, Desulfovibrio, and Desulfomicrobium were found to be the numerically important members of SRB in this wastewater biofilm.     

Mixed-Culture Fermentor for Simulating Methanogenic Digestors

Propionate degradation in an anaerobic digestor degrading animal waste (10-day retention time, 5.75 g liter⁻¹ day⁻¹ volatile solids loading rate, 40°C) was 0.304 mM h⁻¹, measured with [2-¹⁴C]propionate; this value indicated that CH₄ produced from propionate accounted for 14.8% of the CH₄ produced in the digestor (34.5%, including acetate produced from propionate). The mean propionate concentration was 0.67 mM, giving a propionate turnover rate of 0.46 h⁻¹. A continuous-, mixed-culture fermentor was developed to mimic the digestor. When degradation rates of methanogenic precursors (H₂, CO₂, and acetate) equalled those measured in the digestor, propionate degradation was inhibited. When the H₂ turnover rate was lowered by decreasing addition of H₂-generating substrates or by allowing a portion of the H₂ degradation to occur in an isolated compartment, propionate degradation in the fermentor resumed. The possibility is discussed that in digestors, much of the H₂ is produced and degraded within microenvironments associated with particles. Thus, the gross turnover rate of H₂ measured in digestors is an average, and specific microenvironments within the digestor may have different rates of turnover.     

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

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

Hydrogenotrophic Methanogenesis by Moderately Acid-Tolerant Methanogens of a Methane-Emitting Acidic Peat

The emission of methane (1.3 mmol of CH₄ m⁻² day⁻¹), precursors of methanogenesis, and the methanogenic microorganisms of acidic bog peat (pH 4.4) from a moderately reduced forest site were investigated by in situ measurements, microcosm incubations, and cultivation methods, respectively. Bog peat produced CH₄ (0.4 to 1.7 μmol g [dry wt] of soil⁻¹ day⁻¹) under anoxic conditions. At in situ pH, supplemental H₂-CO₂, ethanol, and 1-propanol all increased CH₄ production rates while formate, acetate, propionate, and butyrate inhibited the production of CH₄; methanol had no effect. H₂-dependent acetogenesis occurred in H₂-CO₂-supplemented bog peat only after extended incubation periods. Nonsupplemented bog peat initially produced small amounts of H₂ that were subsequently consumed. The accumulation of H₂ was stimulated by ethanol and 1-propanol or by inhibiting methanogenesis with bromoethanesulfonate, and the consumption of ethanol was inhibited by large amounts of H₂; these results collectively indicated that ethanol- or 1-propanol-utilizing bacteria were trophically associated with H₂-utilizing methanogens. A total of 10⁹ anaerobes and 10⁷ hydrogenotrophic methanogens per g (dry weight) of bog peat were enumerated by cultivation techniques. A stable methanogenic enrichment was obtained with an acidic, H₂-CO₂-supplemented, fatty acid-enriched defined medium. CH₄ production rates by the enrichment were similar at pH 4.5 and 6.5, and acetate inhibited methanogenesis at pH 4.5 but not at pH 6.5. A total of 27 different archaeal 16S rRNA gene sequences indicative of Methanobacteriaceae, Methanomicrobiales, and Methanosarcinaceae were retrieved from the highest CH₄-positive serial dilutions of bog peat and methanogenic enrichments. A total of 10 bacterial 16S rRNA gene sequences were also retrieved from the same dilutions and enrichments and were indicative of bacteria that might be responsible for the production of H₂ that could be used by hydrogenotrophic methanogens. These results indicated that in this acidic bog peat, (i) H₂ is an important substrate for acid-tolerant methanogens, (ii) interspecies hydrogen transfer is involved in the degradation of organic carbon, (iii) the accumulation of protonated volatile fatty acids inhibits methanogenesis, and (iv) methanogenesis might be due to the activities of methanogens that are phylogenetic members of the Methanobacteriaceae, Methanomicrobiales, and Methanosarcinaceae.     

Distribution of Sulfate-Reducing and Methanogenic Bacteria in Anaerobic Aggregates Determined by Microsensor and Molecular Analyses

Using molecular techniques and microsensors for H₂S and CH₄, we studied the population structure of and the activity distribution in anaerobic aggregates. The aggregates originated from three different types of reactors: a methanogenic reactor, a methanogenic-sulfidogenic reactor, and a sulfidogenic reactor. Microsensor measurements in methanogenic-sulfidogenic aggregates revealed that the activity of sulfate-reducing bacteria (2 to 3 mmol of S²⁻ m⁻³ s⁻¹ or 2 × 10⁻⁹ mmol s⁻¹ per aggregate) was located in a surface layer of 50 to 100 μm thick. The sulfidogenic aggregates contained a wider sulfate-reducing zone (the first 200 to 300 μm from the aggregate surface) with a higher activity (1 to 6 mmol of S²⁻ m⁻³ s⁻¹ or 7 × 10⁻⁹ mol s⁻¹ per aggregate). The methanogenic aggregates did not show significant sulfate-reducing activity. Methanogenic activity in the methanogenic-sulfidogenic aggregates (1 to 2 mmol of CH₄ m⁻³ s⁻¹ or 10⁻⁹ mmol s⁻¹ per aggregate) and the methanogenic aggregates (2 to 4 mmol of CH₄ m⁻³ s⁻¹ or 5 × 10⁻⁹ mmol s⁻¹ per aggregate) was located more inward, starting at ca. 100 μm from the aggregate surface. The methanogenic activity was not affected by 10 mM sulfate during a 1-day incubation. The sulfidogenic and methanogenic activities were independent of the type of electron donor (acetate, propionate, ethanol, or H₂), but the substrates were metabolized in different zones. The localization of the populations corresponded to the microsensor data. A distinct layered structure was found in the methanogenic-sulfidogenic aggregates, with sulfate-reducing bacteria in the outer 50 to 100 μm, methanogens in the inner part, and Eubacteria spp. (partly syntrophic bacteria) filling the gap between sulfate-reducing and methanogenic bacteria. In methanogenic aggregates, few sulfate-reducing bacteria were detected, while methanogens were found in the core. In the sulfidogenic aggregates, sulfate-reducing bacteria were present in the outer 300 μm, and methanogens were distributed over the inner part in clusters with syntrophic bacteria.     

Transition from Anoxygenic to Oxygenic Photosynthesis in a Microcoleus chthonoplastes Cyanobacterial Mat

Benthic cyanobacterial mats with the filamentous Microcoleus chthonoplastes as the dominant phototroph grow in oxic hypersaline environments such as Solar Lake, Sinai. The cyanobacteria are in situ exposed to chemical variations between 200 μmol of sulfide liter⁻¹ at night and 1 atm pO₂ during the day. During experimental H₂S to O₂ transitions the microbial community was shown to shift from anoxygenic photosynthesis, with H₂S as the electron donor, to oxygenic photosynthesis. Microcoleus filaments could carry out both types of photosynthesis concurrently. Anoxygenic photosynthesis dominated at high sulfide levels, 500 μmol liter⁻¹, while the oxygenic reaction became dominant when the sulfide level was reduced below 100 to 300 μmol liter⁻¹ (25 to 75 μmol of H₂S liter⁻¹). An increasing inhibition of the oxygenic photosynthesis was observed upon transition to oxic conditions from increasing sulfide concentrations. Oxygen built up within the Microcoleus layer of the mat even under 5 mmol of sulfide liter⁻¹ (500 μmol of H₂S liter⁻¹) in the overlying water. The implications of such a localized O₂ production in a highly reducing environment are discussed in relation to the evolution of oxygenic photosynthesis during the Proterozoic era.     

Tin and Tin-Resistant Microorganisms in Chesapeake Bay

Sediment and water samples from nine stations in Chesapeake Bay were examined for tin content and for microbial populations resistant to inorganic tin (75 mg of Sn liter⁻¹ as SnCl₄·5H₂O) or to the organotin compound dimethyltin chloride [15 mg of Sn liter⁻¹ as (CH₃)₂SnCl₂]. Tin concentrations in sediments were higher (3.0 to 7.9 mg kg⁻¹) at sites impacted by human activity than at open water sites (0.8 to 0.9 mg kg⁻¹), and they were very high (239.6 mg kg⁻¹) in Baltimore Harbor, which is impacted by both shipping and heavy industry. Inorganic tin (75 mg Sn liter⁻¹) in agar medium significantly decreased viable counts, but its toxicity was markedly reduced in liquid medium; it was not toxic in medium solidified with silica gel. Addition of SnCl₄·5H₂O to these media produced a tin precipitate which was not involved in the metal's toxicity. The data suggest that a soluble tin-agar complex which is toxic to cells is formed in agar medium. Thus, the toxicity of tin depends more on the chemical species than on the metal concentration in the medium. All sites in Chesapeake Bay contained organisms resistant to tin. The microbial flora was more sensitive to (CH₃)₂SnCl₂ than to SnCl₄·5H₂O. The elevated level of tin-resistant microorganisms in some aeas not containing unusually high tin concentrations suggests that factors other than tin may participate in the selection for a tin-tolerant microbial flora.     

Effect of Temperature on Anaerobic Ethanol Oxidation and Methanogenesis in Acidic Peat from a Northern Wetland

The effects of temperature on rates and pathways of CH₄ production and on the abundance and structure of the archaeal community were investigated in acidic peat from a mire in northern Scandinavia (68°N). We monitored the production of CH₄ and CO₂ over time and measured the turnover of Fe(II), ethanol, and organic acids. All experiments were performed with and without specific inhibitors (2-bromoethanesulfonate [BES] for methanogenesis and CH₃F for acetoclastic methanogenesis). The optimum temperature for methanogenesis was 25°C (2.3 μmol CH₄ · g [dry weight]⁻¹ · day⁻¹), but the activity was relatively high even at 4°C (0.25 μmol CH₄ · g [dry weight]⁻¹ · day⁻¹). The theoretical lower limit for methanogenesis was calculated to be at −5°C. The optimum temperature for growth as revealed by real-time PCR was 25°C for both archaea and bacteria. The population structure of archaea was studied by terminal restriction fragment length polymorphism analysis and remained constant over a wide temperature range. Hydrogenotrophic methanogenesis accounted for about 80% of the total methanogenesis. Most 16S rRNA gene sequences that were affiliated with methanogens and all McrA sequences clustered with the exclusively hydrogenotrophic order Methanobacteriales, correlating with the prevalence of hydrogenotrophic methanogenesis. Fe reduction occurred parallel to methanogenesis and was inhibited by BES, suggesting that methanogens were involved in Fe reduction. Based upon the observed balance of substrates and thermodynamic calculations, we concluded that the ethanol pool was oxidized to acetate by the following two processes: syntrophic oxidation with methanogenesis (i) as an H₂ sink and (ii) as a reductant for Fe(III). Acetate accumulated, but a considerable fraction was converted to butyrate, making volatile fatty acids important end products of anaerobic metabolism.     

Activity and Diversity of Methanogens in a Petroleum Hydrocarbon-Contaminated Aquifer

Methanogenic activity was investigated in a petroleum hydrocarbon-contaminated aquifer by using a series of four push-pull tests with acetate, formate, H₂ plus CO₂, or methanol to target different groups of methanogenic Archaea. Furthermore, the community composition of methanogens in water and aquifer material was explored by molecular analyses, i.e., fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes amplified with the Archaea-specific primer set ARCH915 and UNI-b-rev, and sequencing of DNA from dominant DGGE bands. Molecular analyses were subsequently compared with push-pull test data. Methane was produced in all tests except for a separate test where 2-bromoethanesulfonate, a specific inhibitor of methanogens, was added. Substrate consumption rates were 0.11 mM day⁻¹ for methanol, 0.38 mM day⁻¹ for acetate, 0.90 mM day⁻¹ for H₂, and 1.85 mM day⁻¹ for formate. Substrate consumption and CH₄ production during all tests suggested that at least three different physiologic types of methanogens were present: H₂ plus CO₂ or formate, acetate, and methanol utilizers. The presence of 15 to 20 bands in DGGE profiles indicated a diverse archaeal population. High H₂ and formate consumption rates agreed with a high diversity of methanogenic Archaea consuming these substrates (16S rRNA gene sequences related to several members of the Methanomicrobiaceae) and the detection of Methanomicrobiaceae by using FISH (1.4% of total DAPI [4′,6-diamidino-2-phenylindole]-stained microorganisms in one water sample; probe MG1200). Considerable acetate consumption agreed with the presence of sequences related to the obligate acetate degrader Methanosaeata concilii and the detection of this species by FISH (5 to 22% of total microorganisms; probe Rotcl1). The results suggest that both aceticlastic and CO₂-type substrate-consuming methanogens are likely involved in the terminal step of hydrocarbon degradation, while methanogenesis from methanol plays a minor role. DGGE profiles further indicate similar archaeal community compositions in water and aquifer material. The combination of hydrogeological and molecular methods employed in this study provide improved information on the community and the potential activity of methanogens in a petroleum hydrocarbon-contaminated aquifer.     

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 (ΔG) 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 ΔG 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 ΔG value and the hydrogen production rate at the point where the cell number was constant, was 4.6 × 10⁻¹³ kJ cell⁻¹ day⁻¹ for S. lipocalidus at 55°C and 6.2 × 10⁻¹³ kJ cell⁻¹ day⁻¹ for A. colombiense at 37°C. S. lipocalidus, in particular, seems adapted to thrive under conditions of low energy availability.     

Assessing Phytoplankton and Bacterioplankton Production During Early Spring in Lake Erken, Sweden

The spring development of both phytoplankton and bacterioplankton was investigated between 18 April and 7 May 1983 in mesotrophic Lake Erken, Sweden. By using the lake as a batch culture, our aim was to estimate, via different methods, the production of phytoplankton and bacterioplankton in the lake and to compare these production estimates with the actual increase in phytoplankton and bacterioplankton biomass. The average water temperature was 3.5°C. Of the phytoplankton biomass, >90% was the diatom Stephanodiscus hantzchii var. pusillus, by the peak of the bloom. The ¹⁴C and O₂ methods of estimating primary production gave equivalent results (r = 0.999) with a photosynthetic quotient of 1.63. The theoretical photosynthetic quotient predicted from the C/NO₃ N assimilation ratio was 1.57. The total integrated incorporation of [¹⁴C]bicarbonate into particulate material (>1 μm) was similar to the increase in phytoplankton carbon determined from cell counts. Bacterioplankton increased from 0.5 × 10⁹ to 1.52 × 10⁹ cells liter⁻¹ (~0.5 μg of C liter⁻¹ day⁻¹). Estimates of bacterioplankton production from rates of [³H]thymidine incorporation were ca. 1.2 to 1.7 μg of C liter⁻¹ day⁻¹. Bacterial respiration, measured by a high-precision Winkler technique, was estimated as 4.8 μg of C liter⁻¹ day⁻¹, indicating a bacterial growth yield of 25%. The bulk of the bacterioplankton production was accounted for by algal extracellular products. Gross bacterioplankton production (production plus respiration) was 20% of gross primary production, per square meter of surface area. We found no indication that bacterioplankton production was underestimated by the [³H]thymidine incorporation method.     

Streptomyces thermoautotrophicus sp. nov., a Thermophilic CO- and H2-Oxidizing Obligate Chemolithoautotroph

The novel thermophilic CO- and H₂-oxidizing bacterium UBT1 has been isolated from the covering soil of a burning charcoal pile. The isolate is gram positive and obligately chemolithoautotrophic and has been named Streptomyces thermoautotrophicus on the basis of G+C content (70.6 ± 0.19 mol%), a phospholipid pattern of type II, MK-9(H₄) as the major quinone, and other chemotaxonomic and morphological properties. S. thermoautotrophicus could grow with CO (t_d = 8 h), H₂ plus CO₂ (t_d = 6 h), car exhaust, or gas produced by the incomplete combustion of wood. Complex media or heterotrophic substrates such as sugars, organic acids, amino acids, and alcohols did not support growth. Molybdenum was required for CO-autotrophic growth. For growth with H₂, nickel was not necessary. The optimum growth temperature was 65°C; no growth was observed below 40°C. However, CO-grown cells were able to oxidize CO at temperatures of 10 to 70°C. Temperature profiles of burning charcoal piles revealed that, up to a depth of about 10 to 25 cm, the entire covering soil provides a suitable habitat for S. thermoautotrophicus. The K_m was 88 μl of CO liter⁻¹ and V_max was 20.2 μl of CO h⁻¹ mg of protein⁻¹. The threshold value of S. thermoautotrophicus of 0.2 μl of CO liter⁻¹ was similar to those of various soils. The specific CO-oxidizing activity in extracts with phenazinemethosulfate plus 2,6-dichlorophenolindophenol as electron acceptors was 246 μmol min⁻¹ mg of protein⁻¹. In exception to other carboxydotrophic bacteria, S. thermoautotrophicus CO dehydrogenase was able to reduce low potential electron acceptors such as methyl and benzyl viologens.     

Characterization of an Autotrophic Nitrogen-Removing Biofilm from a Highly Loaded Lab-Scale Rotating Biological Contactor

In this study, a lab-scale rotating biological contactor (RBC) treating a synthetic NH₄⁺ wastewater devoid of organic carbon and showing high N losses was examined for several important physiological and microbial characteristics. The RBC biofilm removed 89% ± 5% of the influent N at the highest surface load of approximately 8.3 g of N m⁻² day⁻¹, with N₂ as the main end product. In batch tests, the RBC biomass showed good aerobic and anoxic ammonium oxidation (147.8 ± 7.6 and 76.5 ± 6.4 mg of NH₄⁺-N g of volatile suspended solids [VSS]⁻¹ day⁻¹, respectively) and almost no nitrite oxidation (< 1 mg of N g of VSS⁻¹ day⁻¹). The diversity of aerobic ammonia-oxidizing bacteria (AAOB) and planctomycetes in the biofilm was characterized by cloning and sequencing of PCR-amplified partial 16S rRNA genes. Phylogenetic analysis of the clones revealed that the AAOB community was fairly homogeneous and was dominated by Nitrosomonas-like species. Close relatives of the known anaerobic ammonia-oxidizing bacterium (AnAOB) Kuenenia stuttgartiensis dominated the planctomycete community and were most probably responsible for anoxic ammonium oxidation in the RBC. Use of a less specific planctomycete primer set, not amplifying the AnAOB, showed a high diversity among other planctomycetes, with representatives of all known groups present in the biofilm. The spatial organization of the biofilm was characterized using fluorescence in situ hybridization (FISH) with confocal scanning laser microscopy (CSLM). The latter showed that AAOB occurred side by side with putative AnAOB (cells hybridizing with probe PLA46 and AMX820/KST1275) throughout the biofilm, while other planctomycetes hybridizing with probe PLA886 (not detecting the known AnAOB) were present as very conspicuous spherical structures. This study reveals that long-term operation of a lab-scale RBC on a synthetic NH₄⁺ wastewater devoid of organic carbon yields a stable biofilm in which two bacterial groups, thought to be jointly responsible for the high autotrophic N removal, occur side by side throughout the biofilm.     

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 H₂-consuming methanogens was <−20 kJ mol of CH₄⁻¹ 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 CH₄⁻¹ 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.     

Improved Most-Probable-Number Method To Detect Sulfate-Reducing Bacteria with Natural Media and a Radiotracer

A greatly improved most-probable-number (MPN) method for selective enumeration of sulfate-reducing bacteria (SRB) is described. The method is based on the use of natural media and radiolabeled sulfate (³⁵SO₄²⁻). The natural media used consisted of anaerobically prepared sterilized sludge or sediment slurries obtained from sampling sites. The densities of SRB in sediment samples from Kysing Fjord (Denmark) and activated sludge were determined by using a normal MPN (N-MPN) method with synthetic cultivation media and a tracer MPN (T-MPN) method with natural media. The T-MPN method with natural media always yielded significantly higher (100- to 1,000-fold-higher) MPN values than the N-MPN method with synthetic media. The recovery of SRB from environmental samples was investigated by simultaneously measuring sulfate reduction rates (by a ³⁵S-radiotracer method) and bacterial counts by using the T-MPN and N-MPN methods, respectively. When bacterial numbers estimated by the T-MPN method with natural media were used, specific sulfate reduction rates (qSO₄²⁻) of 10⁻¹⁴ to 10⁻¹³ mol of SO₄²⁻ cell⁻¹ day⁻¹ were calculated, which is within the range of qSO₄²⁻ values previously reported for pure cultures of SRB (10⁻¹⁵ to 10⁻¹⁴ mol of SO₄²⁻ cell⁻¹ day⁻¹). qSO₄²⁻ values calculated from N-MPN values obtained with synthetic media were several orders of magnitude higher (2 × 10⁻¹⁰ to 7 × 10⁻¹⁰ mol of SO₄²⁻ cell⁻¹ day⁻¹), showing that viable counts of SRB were seriously underestimated when standard enumeration media were used. Our results demonstrate that the use of natural media results in significant improvements in estimates of the true numbers of SRB in environmental samples.     

An Outbreak of Nonflocculating Catabolic Populations Caused the Breakdown of a Phenol-Digesting Activated-Sludge Process

Activated sludge was fed phenol as the sole carbon source, and the phenol-loading rate was increased stepwise from 0.5 to 1.0 g liter⁻¹ day⁻¹ and then to 1.5 g liter⁻¹ day⁻¹. After the loading rate was increased to 1.5 g liter⁻¹ day⁻¹, nonflocculating bacteria outgrew the sludge, and the activated-sludge process broke down within 1 week. The bacterial population structure of the activated sludge was analyzed by temperature gradient gel electrophoresis (TGGE) of PCR-amplified 16S ribosomal DNA (rDNA) fragments. We found that the population diversity decreased as the phenol-loading rate increased and that two populations (designated populations R6 and R10) predominated in the sludge during the last several days before breakdown. The R6 population was present under the low-phenol-loading-rate conditions, while the R10 population was present only after the loading rate was increased to 1.5 g liter⁻¹ day⁻¹. A total of 41 bacterial strains with different repetitive extragenic palindromic sequence PCR patterns were isolated from the activated sludge under different phenol-loading conditions, and the 16S rDNA and gyrB fragments of these strains were PCR amplified and sequenced. Some bacterial isolates could be associated with major TGGE bands by comparing the 16S rDNA sequences. All of the bacterial strains affiliated with the R6 population had almost identical 16S rDNA sequences, while the gyrB phylogenetic analysis divided these strains into two physiologically divergent groups; both of these groups of strains could grow on phenol, while one group (designated the R6F group) flocculated in laboratory media and the other group (the R6T group) did not. A competitive PCR analysis in which specific gyrB sequences were used as the primers showed that a population shift from R6F to R6T occurred following the increase in the phenol-loading rate to 1.5 g liter⁻¹ day⁻¹. The R10 population corresponded to nonflocculating phenol-degrading bacteria. Our results suggest that an outbreak of nonflocculating catabolic populations caused the breakdown of the activated-sludge process. This study also demonstrated the usefulness of gyrB-targeted fine population analyses in microbial ecology.     

Impact of Different In Vitro Electron Donor/Acceptor Conditions on Potential Chemolithoautotrophic Communities from Marine Pelagic Redoxclines

Anaerobic or microaerophilic chemolithoautotrophic bacteria have been considered to be responsible for CO₂ dark fixation in different pelagic redoxclines worldwide, but their involvement in redox processes is still not fully resolved. We investigated the impact of 17 different electron donor/acceptor combinations in water of pelagic redoxclines from the central Baltic Sea on the stimulation of bacterial CO₂ dark fixation as well as on the development of chemolithoautotrophic populations. In situ, the highest CO₂ dark fixation rates, ranging from 0.7 to 1.4 μmol liter⁻¹ day⁻¹, were measured directly below the redoxcline. In enrichment experiments, chemolithoautotrophic CO₂ dark fixation was maximally stimulated by the addition of thiosulfate, reaching values of up to 9.7 μmol liter⁻¹ CO₂ day⁻¹. Chemolithoautotrophic nitrate reduction proved to be an important process, with rates of up to 33.5 μmol liter⁻¹ NO₃⁻ day⁻¹. Reduction of Fe(III) or Mn(IV) was not detected; nevertheless, the presence of these potential electron acceptors influenced the development of stimulated microbial assemblages. Potential chemolithoautotrophic bacteria in the enrichment experiments were displayed on 16S ribosomal complementary DNA single-strand-conformation polymorphism fingerprints and identified by sequencing of excised bands. Sequences were closely related to chemolithoautotrophic Thiomicrospira psychrophila and Maorithyas hadalis gill symbiont (both Gammaproteobacteria) and to an uncultured nitrate-reducing Helicobacteraceae bacterium (Epsilonproteobacteria). Our data indicate that this Helicobacteraceae bacterium could be of general importance or even a key organism for autotrophic nitrate reduction in pelagic redoxclines.