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.     

产甲烷菌研究进展

产甲烷菌是重要的环境微生物,在自然界的碳素循环中起重要作用。迄今已有5种产甲烷菌基因组测序完成。基因组信息使人们对产甲烷茵的细胞结构、进化、代谢及环境适应性有了更深的理解。目前已知的甲烷生物合成途径有3种,它们以乙酸、甲基化合物、氢/二氧化碳为起始,通过不同的反应途径都形成了甲基辅酶M,在甲基辅酶M还原酶的催化下最终形成甲烷。     

Molecular Ecological Analysis of Methanogens and Methanotrophs in Blanket Bog Peat

Abstract Methane production and methane oxidation potential were measured in a 30 cm peat core from the Moorhouse Nature Reserve, UK. The distribution of known groups of methanogens and methane oxidizing bacteria throughout this peat core was assessed. Using 16S rRNA gene retrieval and functional gene probing with genes encoding key proteins in methane oxidation and methanogenesis, several major groups of microorganisms were detected. Methane production and oxidation was detected in all depths of the peat core. PCR amplification and oligonucleotide probing experiments using DNA isolated from all sections of the peat core detected methanotrophs from the groups Methylosinus and Methylococcus and methanogens from the groups Methanosarcinaceae, Methanococcaceae, and Methanobacteriaceae. 16S rDNA sequences amplified with the Methylosinus-specific primer were shown to have a high degree of identity with 16S rDNA sequences previously detected in acidic environments. However, no methanogen sequences were detected by the probes available in this study in the sections of the peat core (above 7 cm) where the majority of methanogenesis occurred, either because of low methanogen numbers or because of the presence of novel methanogen sequences. Received: 9 March 1999; Accepted: 21 June 1999     

Methanotrophic Bacteria of Acidic Sphagnum Peat Bogs

Acidic Sphagnum peat bogs cover a considerable part of the territory of Russia and are an important natural source of biogenic methane, which is formed in their anaerobic layers. A considerable portion of this methane is consumed in the aerobic part of the bog profile by acidophilic methanotrophic bacteria, which comprise the methane filter of Sphagnum peat bogs and decrease CH₄ emission to the atmosphere. For a long time, these bacteria escaped isolation, which became possible only after the elucidation of the optimal conditions of their functioning in situ: pH 4.5–5.5; temperature, from 15 to 20°C; and low salt concentration in the solution. Imitation of these conditions and rejection of earlier used media with a high content of biogenic elements allowed methanotrophic bacteria of two new genera and species—Methylocella palustris and Methylocapsa acidiphila—to be isolated from the peat of Sphagnum peat bogs of European northern Russia and western Siberia. These bacteria are well adapted to the conditions in cold, acidic, oligotrophic Sphagnum peat bogs. They grow in a pH range of 4.2–7.5 with an optimum at 5.0–5.5, prefer moderate temperatures (15–25°C) and media with a low content of mineral salts (200–500 mg/l), and are capable of active dinitrogen fixation. Design of fluorescently labeled 16S rRNA–targeted oligonucleotide probes for the detection of Methylocella palustris and Methylocapsa acidiphila and their application to the analysis of sphagnum peat samples showed that these bacteria represent dominant populations of methanotrophs with a density of 10⁵–10⁶ cells/g peat. In addition to Methylocella and Methylocapsa populations, one more abundant population of methanotrophs was revealed (10⁶ cells/g peat), which were phylogenetically close to the genus Methylocystis.     

Methanogens and Methanogenesis in the Rumens and Ceca of Lambs Fed Two Different High-Grain-Content Diets

The amount and nature of dietary starch are known to influence the extent and site of feed digestion in ruminants. However, how starch degradability may affect methanogenesis and methanogens along the ruminant''s digestive tract is poorly understood. This study examined the diversity and metabolic activity of methanogens in the rumen and cecum of lambs receiving wheat or corn high-grain-content diets. Methane production in vivo and ex situ was also monitored. In vivo daily methane emissions (CH₄ g/day) were 36% (P < 0.05) lower in corn-fed lambs than in wheat-fed lambs. Ex situ methane production (μmol/h) was 4-fold higher for ruminal contents than for cecal contents (P < 0.01), while methanogens were 10-fold higher in the rumen than in the cecum (mcrA copy numbers; P < 0.01). Clone library analysis indicated that Methanobrevibacter was the dominant genus in both sites. Diet induced changes at the species level, as the Methanobrevibacter millerae-M. gottschalkii-M. smithii clade represented 78% of the sequences from the rumen of wheat-fed lambs and just about 52% of the sequences from the rumen of the corn-fed lambs. Diet did not affect mcrA expression in the rumen. In the cecum, however, expression was 4-fold and 2-fold lower than in the rumen for wheat- and corn-fed lambs, respectively. Though we had no direct evidence for compensation of reduced rumen methane production with higher cecum methanogenesis, the ecology of methanogens in the cecum should be better considered.     

Hydrogenotrophic methanogenesis by moderately acid-tolerant methanogens of a methane-emitting acidic peat

The emission of methane (1.3 mmol of CH(4) 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 CH(4) (0.4 to 1.7 micro mol g [dry wt] of soil(-1) day(-1)) under anoxic conditions. At in situ pH, supplemental H(2)-CO(2), ethanol, and 1-propanol all increased CH(4) production rates while formate, acetate, propionate, and butyrate inhibited the production of CH(4); methanol had no effect. H(2)-dependent acetogenesis occurred in H(2)-CO(2)-supplemented bog peat only after extended incubation periods. Nonsupplemented bog peat initially produced small amounts of H(2) that were subsequently consumed. The accumulation of H(2) was stimulated by ethanol and 1-propanol or by inhibiting methanogenesis with bromoethanesulfonate, and the consumption of ethanol was inhibited by large amounts of H(2); these results collectively indicated that ethanol- or 1-propanol-utilizing bacteria were trophically associated with H(2)-utilizing methanogens. A total of 10(9) anaerobes and 10(7) hydrogenotrophic methanogens per g (dry weight) of bog peat were enumerated by cultivation techniques. A stable methanogenic enrichment was obtained with an acidic, H(2)-CO(2)-supplemented, fatty acid-enriched defined medium. CH(4) 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 CH(4)-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 H(2) that could be used by hydrogenotrophic methanogens. These results indicated that in this acidic bog peat, (i) H(2) 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.     

Methanogenesis in McLean Bog,an Acidic Peat Bog in Upstate New York: Stimulation by H2/CO2 in the Presence of Rifampicin,or by Low Concentrations of Acetate

Acidic peat bog soils produce CH₄ and although molecular biological studies have demonstrated the presence of diverse methano-genic populations in them, few studies have sustained methanogenesis by adding the CH₄ precursors H₂/CO₂ or acetate, and few indigenous methanogens have been cultured. McLean Bog is a small (ca. 70 m across), acidic (pH 3.4–4.3) Sphagnum -dominated bog in upstate New York. Although addition of H₂/CO₂ or 10 mM acetate stimulated methanogenesis in soils from a nearby circumneutral-pH fen, neither of these substrates led to sustained methanogenesis in McLean Bog soil slurries. After a brief period of stimulation by H₂/CO₂, methanogenesis in McLean Bog soil declined, which could be attributed to buildup of large amounts of acetic acid produced from the H₂/CO₂ by acetogens. Addition of the antibiotic rifampicin inhibited acetogenesis (carried out by Bacteria) and allowed methanogenesis (carried out by Archaea) to continue. Using rifampicin, we were able to study effects of temperature, pH, and salts on methanogenesis from H₂/CO₂ in McLean Bog soil samples. The enriched H₂/CO₂-utilizing methanogens showed an optimum for activity near pH 5, and a temperature optimum near 35°C. Methanogenesis was not stimulated by addition of 10 mM acetate, but it was stimulated by 1 mM acetate, and multiple additions were consumed at increasing rates and nearly stoichiometrically converted to CH₄. In conclusion, we have found that both hydrogentrophic and aceticlastic methanogens are present in McLean Bog soils, and that methanogenic activity can be stimulated using H₂/CO₂ in the presence of rifampicin, or using low concentrations of acetate.     

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.     

Association of Novel and Highly Diverse Acid-Tolerant Denitrifiers with N2O Fluxes of an Acidic Fen

Wetlands are sources of denitrification-derived nitrous oxide (N₂O). Thus, the denitrifier community of an N₂O-emitting fen (pH 4.7 to 5.2) was investigated. N₂O was produced and consumed to subatmospheric concentrations in unsupplemented anoxic soil microcosms. Total cell counts and most probable numbers of denitrifiers approximated 10¹¹ cells·g_DW⁻¹ (where DW is dry weight) and 10⁸ cells·g_DW⁻¹, respectively, in both 0- to 10-cm and 30- to 40-cm depths. Despite this uniformity, depth-related maximum reaction rate (v_max) values for denitrification in anoxic microcosms ranged from 1 to 24 and −19 to −105 nmol N₂O h⁻¹· g_DW⁻¹, with maximal values occurring in the upper soil layers. Denitrification was enhanced by substrates that might be formed via fermentation in anoxic microzones of soil. N₂O approximated 40% of total nitrogenous gases produced at in situ pH, which was likewise the optimal pH for denitrification. Gene libraries of narG and nosZ (encoding nitrate reductase and nitrous oxide reductase, respectively) from fen soil DNA yielded 15 and 18 species-level operational taxonomic units, respectively, many of which displayed phylogenetic novelty and were not closely related to cultured organisms. Although statistical analyses of narG and nosZ sequences indicated that the upper 20 cm of soil contained the highest denitrifier diversity and species richness, terminal restriction fragment length polymorphism analyses of narG and nosZ revealed only minor differences in denitrifier community composition from a soil depth of 0 to 40 cm. The collective data indicate that the regional fen harbors novel, highly diverse, acid-tolerant denitrifier communities capable of complete denitrification and consumption of atmospheric N₂O at in situ pH.Nitrous oxide (N₂O) is a potent greenhouse gas with a global warming potential that is 300-fold higher than that of CO₂, and its concentration increased from 270 ppb in 1750 to 319 ppb in 2005 (17). N₂O can be produced in soils during denitrification, nitrification, the dissimilatory reduction of nitrate to nitrite and/or ammonium (hereafter referred to as dissimilatory nitrate reduction), or the chemical transformation of nitrite or hydroxylamine (5, 7, 49). The percentage of N₂O produced in any of these processes is variable, depending mainly on the redox potential, pH, and C/N ratio (49). In anoxic ecosystems such as waterlogged soils, most of the N₂O is considered to be denitrification derived (7, 9). Complete denitrification is the sequential reduction of nitrate to dinitrogen (N₂) via nitrite, nitric oxide (NO), and N₂O (75). The main product of denitrification varies with the organism and in situ conditions and is usually either N₂O or N₂ (68). N₂O can occur as a by-product during dissimilatory nitrate reduction when accumulated nitrite interacts with nitrate reductase to form N₂O (59). The production of N₂O by dissimilatory nitrate reducers is favored in environments with large amounts of readily available organic carbon (65). Thus, their contribution to nitrate-dependent production of N₂O in soils is likely insignificant compared to that of denitrifiers.The oxidoreductases involved in denitrification are termed dissimilatory nitrate reductase (Nar, encoded by narGHJI, or Nap, encoded by napEDABC), nitrite reductase (Nir, encoded by nirK and nirS), NO reductase (cNor and qNor, encoded by norBC and norB, respectively), and N₂O reductase (Nos, encoded by nosZ) (75). Nitrate reductase is also found in dissimilatory nitrate reducers (60). narG can therefore be used as a molecular marker to assess both denitrifiers and dissimilatory nitrate reducers, whereas nosZ is specific for the assessment of denitrifiers (25, 43, 48).Denitrification in soils is regulated by temperature, pH, substrate (i.e., carbon) availability, and water content (10, 24, 66). Although denitrification increases with increasing temperature, it can still occur at temperatures below 0°C (10, 24). Low temperatures appear to limit the activity of N₂O reductase more severely than other enzymes involved in denitrification and thus yield higher relative amounts of denitrification-derived N₂O (24). Although denitrification activity usually decreases under acidic conditions, the relative percentage of N₂O to total denitrification-derived nitrogenous gases increases with increasing acidity, a result attributed to the sensitivity of N₂O reductase to low pH (27, 70). However, denitrifier communities can be adapted to the in situ pH of the system (40, 58, 73).Wetlands are ecosystems in which denitrification is likely a dominant source of emitted N₂O (7, 44, 45). The identification and analysis of main drivers for N₂O production (i.e., the microbiota catalyzing N₂O production and consumption) is thus of major concern in such environments. Fens are specialized wetlands characterized by soil acidity (67). However, information on acid-tolerant denitrifier communities of such wetlands is scarce. It is hypothesized that fens harbor a diverse, hitherto unknown, denitrifier community that is adapted to in situ conditions and associated with N₂O fluxes (i.e., fen denitrifiers are acid tolerant and have a high affinity for nitrate and N₂O). Thus, the main objectives of the present study were to evaluate the capacities of denitrifier communities of an N₂O-emitting fen (20) to produce or consume N₂O and to determine if a novel and diverse denitrifier community was associated with these capacities.     

Hitherto Unknown [Fe-Fe]-Hydrogenase Gene Diversity in Anaerobes and Anoxic Enrichments from a Moderately Acidic Fen

Newly designed primers for [Fe-Fe]-hydrogenases indicated that (i) fermenters, acetogens, and undefined species in a fen harbor hitherto unknown hydrogenases and (ii) Clostridium- and Thermosinus-related primary fermenters, as well as secondary fermenters related to sulfate or iron reducers might be responsible for hydrogen production in the fen. Comparative analysis of [Fe-Fe]-hydrogenase and 16S rRNA gene-based phylogenies indicated the presence of homologous multiple hydrogenases per organism and inconsistencies between 16S rRNA gene- and [Fe-Fe]-hydrogenase-based phylogenies, necessitating appropriate qualification of [Fe-Fe]-hydrogenase gene data for diversity analyses.Molecular hydrogen (H₂) is important in intermediary ecosystem metabolism (i.e., processes that link input to output) in wetlands (7, 11, 12, 33) and other anoxic habitats like sewage sludges (34) and the intestinal tracts of animals (9, 37). H₂-producing fermenters have been postulated to form trophic links to H₂-consuming methanogens, acetogens (i.e., organisms capable of using the acetyl-coenzyme A [CoA] pathway for acetate synthesis) (7), Fe(III) reducers (17), and sulfate reducers in a well-studied moderately acidic fen in Germany (11, 12, 16, 18, 22, 33). 16S rRNA gene analysis revealed the presence of Clostridium spp. and Syntrophobacter spp., which represent possible primary and secondary fermenters, as well as H₂ producers in this fen (11, 18, 33). However, H₂-producing bacteria are polyphyletic (30, 31, 29). Thus, a structural marker gene is required to target this functional group by molecular methods. [Fe-Fe]-hydrogenases catalyze H₂ production in fermenters (19, 25, 29, 30, 31), and genes encoding [Fe-Fe]-hydrogenases represent such a marker gene. The objectives of this study were to (i) develop primers specific for highly diverse [Fe-Fe]-hydrogenase genes, (ii) analyze [Fe-Fe]-hydrogenase genes in pure cultures of fermenters, acetogens, and a sulfate reducer, (iii) assess [Fe-Fe]-hydrogenase gene diversity in H₂-producing fen soil enrichments, and (iv) evaluate the limitations of the amplified [Fe-Fe]-hydrogenase fragment as a phylogenetic marker.     

Protease Formation by a Moderately Halophilic Bacillus Strain

A moderately halophilic strain of Bacillus, isolated from unrefined solar salt, was capable of growth in the presence of 4 M NaCl. Maximal growth was obtained in a medium containing 1 to 2 M NaCl. The organism produced protease when cultivated aerobically in media containing 0 to 3 M NaCl or 0 to 2 M KCl. The protease activity was optimal at 0.5 M NaCl and 0.75 M KCl.     

Inhibition of Rumen Methanogenesis by Methane Analogues

Extremely low concentrations of chloroform and carbon tetrachloride and somewhat larger concentrations of methylene chloride inhibited the formation of methane by the rumen microbiota in the presence or absence of added substrate. The accumulation of hydrogen at these low concentrations indicates a selective inhibition of methanogenesis. Presumably, these inhibitors affect one or more of the reactions by which methane is formed from hydrogen and carbon dioxide.     

Molecular and Physiological Analysis of Al3+ and H+ Rhizotoxicities at Moderately Acidic Conditions

Al³⁺ and H⁺ toxicities predicted to occur at moderately acidic conditions (pH [water] = 5–5.5) in low-Ca soils were characterized by the combined approaches of computational modeling of electrostatic interactions of ions at the root plasma membrane (PM) surface and molecular/physiological analyses in Arabidopsis (Arabidopsis thaliana). Root growth inhibition in known hypersensitive mutants was correlated with computed {Al³⁺} at the PM surface ({Al3+}_PM); inhibition was alleviated by increased Ca, which also reduced {Al3+}_PM and correlated with cellular Al responses based on expression analysis of genes that are markers for Al stress. The Al-inducible Al tolerance genes ALUMINUM-ACTIVATED MALATE TRANSPORTER1 and ALUMINUM SENSITIVE3 were induced by levels of {Al3+}_PM too low to inhibit root growth in tolerant genotypes, indicating that protective responses are triggered when {Al3+}_PM was below levels that can initiate injury. Modeling of the H⁺ sensitivity of the SENSITIVE TO PROTON RHIZOTOXICITY1 knockout mutant identified a Ca alleviation mechanism of H⁺ rhizotoxicity, possibly involving stabilization of the cell wall. The phosphatidate phosphohydrolase1 (pah1) pah2 double mutant showed enhanced Al susceptibility under low-P conditions, where greater levels of negatively charged phospholipids in the PM occur, which increases {Al3+}_PM through increased PM surface negativity compared with wild-type plants. Finally, we found that the nonalkalinizing Ca fertilizer gypsum improved the tolerance of the sensitive genotypes in moderately acidic soils. These findings fit our modeling predictions that root toxicity to Al³⁺ and H⁺ in moderately acidic soils involves interactions between both toxic ions in relation to Ca alleviation.Aluminum (Al), principally in the form of Al³⁺ released from soil clay minerals, is one of the most important rhizotoxic ions in acidic soils and is abundant in soil solutions at pH (water) of less than 5.0. Many forest and grass land species naturally adapted to acid soils are very tolerant of H⁺ and Al³⁺ and thrive in soils where the pH is less than 4.0. However, most crop plants used for agriculture show inhibitory growth effects, even when the soil pH is neutralized by liming to moderately acidic pH values in the range of pH 5 to 5.5. For example, crops sensitive to Al³⁺ and H⁺ such as turnip (Brassica rapa; Kinraide and Parker, 1990) and alfalfa (Medicago sativa; Yokota and Ojima, 1995) show growth inhibition at these soil pH values. Field research and soil experiments have shown that inhibitory effects of moderately acidic soils (pH > 5) can be ameliorated by the application of Ca fertilizers, even if they are nonalkalinizing, such as gypsum, and this leads to improvement in crop productivity (Carvalho and Van Raij, 1997; Mora et al., 1999). This indicates that the soil Ca status is an important factor in determining crop yield at moderately low soil pH values with regard to Al³⁺ and H⁺ rhizotoxicity occurring in these soils. An understanding of the complex situation of acid soil stress in soil pH in the range of pH (water) 5 to 5.5 is important for designing efficient soil acidity management and breeding programs for resistant crop use in low-input agricultural systems.The complex rhizotoxicities at moderately acidic conditions that can be alleviated by Ca have been predicted by modeling studies in wheat (Triticum aestivum; Kinraide, 2003). The model first computes the activity of the rhizotoxicants and alleviants at the plasma membrane (PM) surface, for example {Al3+}_PM and {H+}_PM, and {Ca2+}_PM, using a speciation-based Gouy-Chapman-Stern electrostatic (SGCS) model (Kinraide and Wang, 2010). The mechanisms of toxicity and alleviation are then modeled by regression analyses for root growth inhibition fitted to the nonlinear equations (Kinraide, 2003; Kinraide et al., 2004). The modeling studies well describe the complicated events in the Al-toxic solutions near the PM surface under moderately acidic conditions. For example, the elevation of pH from 4.5 to between 5 and 5.5 decreases the activity of the most rhizotoxic Al species, Al³⁺ in the solution ({Al3+}_bulk), while it increases the negativity of the PM surface because of dissociation of H⁺ from potentially negative ligands such as phospholipids. As a result, {Al3+}_PM remains at moderately high levels at the PM surface at pH greater than 5 due to the attraction to negative charges on the PM surface, but it can be alleviated by coexisting cations such as Ca²⁺ and even by another rhizotoxic cation, H⁺. These modeling studies have proposed different mechanisms of Ca alleviation in this complex situation (Kinraide, 1998; Kinraide et al., 2004). Mechanisms I (the electrostatic displacement of toxicant at the PM surface) and II (the restoration at the PM surface of Ca²⁺ electrostatically displaced by the toxicant) are events at the PM surface, but mechanism III, which explains the remaining portion of the Ca alleviation, may involve other physiological responses, including unknown mechanisms. These predictions, derived from the modeling study, likely explain the complex nature of moderately acidic soils but may require further validation because they were developed using root growth as the sole criterion for rhizotoxicity.Although these types of modeling approaches have not been performed using Arabidopsis (Arabidopsis thaliana) plants, clear symptoms of Al³⁺ and H⁺ rhizotoxicity at moderately acidic conditions (pH ≥ 5) has been identified in Arabidopsis (Kobayashi and Koyama, 2002; Iuchi et al., 2007). A quantitative trait locus study of Al tolerance at moderately acidic conditions (4 μm Al, pH 5; Kobayashi and Koyama, 2002) identified a very similar genetic architecture of Al tolerance to that derived from a study that employed a lower pH value but with a greater level of Al (50 μm Al, pH 4.2; Hoekenga et al., 2003). The former conditions employed a lower Ca concentration (200 μm) than the latter (3 mm), which accounted for the predictions of {Al3+}_PM in relation to {Ca2+}_PM by electrostatics. On the other hand, several Al³⁺- and H⁺-sensitive transfer DNA insertion knockout (KO) mutant genotypes have been identified using the lower ionic-strength moderately acidic media (Sawaki et al., 2009). These lines exhibit different degrees of hypersensitivity to moderately acidic conditions because of the dysfunction of different tolerance genes, suggesting the involvement of different mechanisms. In Arabidopsis, ALUMINUM-ACTIVATED MALATE TRANSPORTER1 (AtALMT1) regulates Al-activated root malate excretion that protects the root tip from acute Al toxicity by Al exclusion (Hoekenga et al., 2006), and ALUMINUM SENSITIVE3 (ALS3) regulates internal Al sequestration involved in long term Al tolerance (Larsen et al., 1997, 2005). The KO mutants for these genes display Al hypersensitivity. In addition, SENSITIVE TO PROTON RHIZOTOXICITY1 (STOP1)-KO, a suppressor of multiple genes for Al and H⁺ tolerance, shows sensitivity to Al³⁺ and H⁺ (Iuchi et al., 2007; Sawaki et al., 2009). These sensitive genotypes are useful tools for evaluating Al³⁺ and H⁺ toxicity in the pH range 5 to 5.5. On the other hand, several cellular responses, such as the induction of gene expression, have been identified in Arabidopsis that could be useful in the estimation of the attraction of {Al³⁺} to the PM, which is computed by our electrostatic-based model. Therefore, Arabidopsis appears to be a useful model system for the validation of modeling based on the SGCS model and to further our understanding of Al³⁺ and H⁺ rhizotoxicities at moderately acidic conditions in relation to Ca²⁺ alleviation.Computation of {Al3+}_PM requires accurate speciation of Al and other solutes in the bulk solution. The original SGCS program is suitable for relatively simple solutions (Kinraide and Wang, 2010). However, the rooting medium used for the Arabidopsis assays exceeds the number of solutes that can be accurately assessed by the SGCS program (Kobayashi et al., 2007). Consequently, we updated the modeling methodology using the speciation program GEOCHEM-EZ, which is suitable for complex media (Famoso et al., 2010; Shaff et al., 2010). This improved model, used in conjunction with molecular biological assays such as biomarker analysis of Al-inducible gene expression, has allowed us in this study to validate the predicted {Al3+}_PM rhizotoxicity in relation to {Ca2+}_PM alleviation from the wheat modeling studies. The updated modeling of Ca alleviation in mutants uncovered one of the mechanisms of Ca alleviation in the H⁺-sensitive mutant and identified an Al-sensitive double mutant genotype, phosphatidate phosphohydrolase1 (pah1) and pah2 (Nakamura et al., 2009), that fitted previous predictions. Finally, we demonstrate the ability of gypsum to ameliorate the sensitive phenotype of selected genotypes, when they were grown in moderately acidic soil culture. Taken together, we present here experimental validation of the SGCS-based modeling, and its combination with molecular physiology provides a deeper understanding of plant Al³⁺ and H⁺ toxicity in relation to Ca²⁺ alleviation at pΗ of at least 5.0.     

Immunization against Rumen Methanogenesis by Vaccination with a New Recombinant Protein

Vaccination through recombinant proteins against rumen methanogenesis provides a mitigation approach to reduce enteric methane (CH₄) emissions in ruminants. The objective of present study was to evaluate the in vivo efficacy of a new vaccine candidate protein (EhaF) on methanogenesis and microbial population in the rumen of goats. We amplified the gene mru 1407 encoding protein EhaF using fresh rumen fluid samples of mature goats and successfully expressed recombinant protein (EhaF) in Escherichia coli Rosetta. This product was evaluated using 12 mature goats with half for control and other half injected with 400ug/goat the purified recombinant protein in day 1 and two subsequent booster immunizations in day 35 and 49. All measurements were undertaken from 63 to 68 days after the initial vaccination, with CH₄ emissions determined using respiration calorimeter chambers. The results showed that the vaccination caused intensive immune responses in serum and saliva, although it had no significant effect on total enteric CH₄ emissions and methanogen population in the rumen, when compared with the control goats. However, the vaccination altered the composition of rumen bacteria, especially the abundance of main phylum Firmicutes and genus Prevotella. The results indicate that protein EhaF might not be an effective vaccine to reduce enteric CH4 emissions but our vaccine have potential to influence the rumen ecosystem of goats.     

Shift from Acetoclastic to H2-Dependent Methanogenesis in a West Siberian Peat Bog at Low pH Values and Isolation of an Acidophilic Methanobacterium Strain

Methane production and archaeal community composition were studied in samples from an acidic peat bog incubated at different temperatures and pH values. H₂-dependent methanogenesis increased strongly at the lowest pH, 3.8, and Methanobacteriaceae became important except for Methanomicrobiaceae and Methanosarcinaceae. An acidophilic and psychrotolerant Methanobacterium sp. was isolated using H₂-plus-CO₂-supplemented medium at pH 4.5.     

Phylogenetic Analysis and In Situ Identification of Bacteria Community Composition in an Acidic Sphagnum Peat Bog

The Bacteria community composition in an acidic Sphagnum peat bog (pH 3.9 to 4.5) was characterized by a combination of 16S rRNA gene clone library analysis, rRNA-targeted fluorescence in situ hybridization (FISH), and cultivation. Among 84 environmental 16S rRNA gene clones, a set of only 16 cloned sequences was closely related (≥95% similarity) to taxonomically described organisms. Main groups of clones were affiliated with the Acidobacteria (24 clones), Alphaproteobacteria (20), Verrucomicrobia (13), Actinobacteria (8), Deltaproteobacteria (4), Chloroflexi (3), and Planctomycetes (3). The proportion of cells that hybridized with oligonucleotide probes specific for members of the domains Bacteria (EUB338-mix) and Archaea (ARCH915 and ARC344) accounted for only 12 to 22% of the total cell counts. Up to 24% of the EUB338-positive cells could be assigned by FISH to specific bacterial phyla. Alphaproteobacteria and Planctomycetes were the most numerous bacterial groups (up to 1.3 × 10⁷ and 1.1 × 10⁷ cells g⁻¹ peat, respectively). In contrast to conventional plating techniques, a novel biofilm-mediated enrichment approach allowed us to isolate some representatives of predominant Bacteria groups, such as Acidobacteria and Planctomycetes. This novel strategy has great potential to enable the isolation of a significant proportion of the peat bog bacterial diversity.     

Methanogenesis from lactate by a co-culture of Clostridium formicoaceticum and Methanosarcina mazei

Summary A co-culture of Clostridium formicoaceticum and Methanosarcina mazei converted lactate to methane and carbon dioxide at mesophilic temperatures and pH values near 7.0. Lactate was first converted to acetate by the homoacetogen, and then to CH₄ and CO₂ by the methanogen, with the second reaction as the rate-limiting step. The methane yield was about 1.45 mol/mol lactate. These two organisms formed a mutualistic association and may be useful together with the homolactic bacterium Stretococcus lactis to convert lactose to methane. Offprint requests to: S. T. Yang     

Inhibition of Butyrate Oxidation by Formate during Methanogenesis

A mixed methanogenic culture fed with glucose was perturbed with butyrate and formate to investigate the role of formate in the acetogenesis of butyrate. A free energy analysis suggests that formate rather than H₂ was the interspecies electron carrier for butyrate conversion into acetate for the culture studied.     

Growth of Methanogens on a Mars Soil Simulant

Currently, the surface of Mars is probably too cold, too dry, and too oxidizing for life, as we know it, to exist. But the subsurface is another matter. Life forms that might exist below the surface could not obtain their energy from photosynthesis, but rather they would have to utilize chemical energy. Methanogens are one type of microorganism that might be able to survive below the surface of Mars. A potential habitat for existence of methanogens on Mars might be a geothermal source of hydrogen, possibly due to volcanic or hydrothermal activity, or the reaction of basalt and anaerobic water, carbon dioxide, which is abundant in the martian atmosphere, and of course, subsurface liquid water. We report here that certain methanogens can grow on a Mars soil simulant when supplied with carbon dioxide, molecular hydrogen, and varying amounts of water.     

Propionic Acid Production by a Propionic Acid-Tolerant Strain of Propionibacterium acidipropionici in Batch and Semicontinuous Fermentation

A propionic acid-tolerant derivative of Propionibacterium acidipropionici P9 was obtained by serially transferring strain P9 through broth that contained increasing amounts of propionic acid. After 1 year of repeated transfers, a strain (designed P200910) capable of growth at higher propionic acid concentrations than P9 was obtained. An increase in the proportion of cellular straight-chain fatty acids and uncoupling of propionic acid production from growth were observed for strain P200910. Growth rate, sugar utilization, and acid production were monitored during batch and semicontinuous fermentations of semidefined medium and during batch fermentation of whey permeate for both strain P200910 and strain P9. The highest propionic acid concentration (47 g/liter) was produced by P200910 in a semicontinuous fermentation. Strain P200910 produced a higher ratio of propionic acid to acetic acid, utilized sugar more efficiently, and produced more propionic acid per gram of biomass than did its parent in all fermentations.