Influence of Methanogenic Populations in Holocene Lacustrine Sediments Revealed by Clone Libraries and Fatty Acid Biogeochemistry

Methanogenic populations were investigated in subsaline Laguna Potrok Aike sediments, southern Argentina. Microbial density and activity were assessed via cell count and in situ ATP detection for the last ～11K years. Methanogen phylogenetics highlighted species stratification throughout depth, whereas CO₂ reduction was the major pathway leading to methane production. Organic substrates, characterized using pore water analysis, bulk organic fractions and saturated fatty acids, showed a clear link between sediment colonization and initial organic sources. Concentrations and δ¹³C compositions of methane and fatty acids provided final evidence of a microbial imprint on Holocene organic proxies in the most colonized intervals.     

Purification and properties of a F420-nonreactive,membrane-bound hydrogenase from Methanosarcina strain Gö1

The distribution of the F₄₂₀-reactive and F₄₂₀-nonreactive hydrogenases from the methylotrophic Methanosarcina strain Gö1 indicated a membrane association of the F₄₂₀-nonreactive enzyme. The membrane-bound F₄₂₀-nonreactive hydrogenase was purified 42-fold to electrophoretic homogeneity with a yield of 26.7%. The enzyme had a specific activity of 359 mol H₂ oxidized · min^-1 · mg protein^-1. The purification procedure involved dispersion of the membrane fraction with the detergent Chaps followed by anion exchange, hydrophobic and hydroxylapatite chromatography. The aerobically prepared enzyme had to be reactivated anaerobically. Maximal activity was observed at 80°C. The molecular mass as determined by native gel electrophoresis and gel filtration was 77000 and 79000, respectively. SDS gel electrophoresis revealed two polypeptides with molecular masses of 60000 and 40000 indicating a 1:1 stoichiometry. The purified enzyme contained 13.3 mol S^2-, 15.1 mol Fe and 0.8 mol Ni/mol enzyme. Flavins were not detected. The amino acid sequence of the N-termini of the subunits showed a higher degree of homology to cubacterial uptake-hydrogenases than to F₄₂₀-dependent hydrogenases from other methanogenic bacteria. The physiological function of the F₄₂₀-nonreactive hydrogenase from Methanosarcina strain Gö1 is discussed.Abbreviations transmembrane electrochemical gradient of H^- - CoM-SH 2-mercaptoethanesulfonate - F₄₂₀ (N-l-lactyl--l-glutamyl)-l-glutamic acid phospodiester of 7,8-didemethyl-8-hydroxy-5-deazariboflavin-5-phosphate - F₄₂₀H₂ reduced F₄₂₀ - HTP-SH 7-mercaptoheptanoylthreonine phosphate - Mb. Methanobacterium - PMSF phenylmethyl-sulfonylfluoride - Cl₃AcOH trichloroacetic acid     

Interconversion of F430 derivatives of methanogenic bacteria

F₄₃₀ is the prosthetic group of the methylcoenzyme M reductase of methanogenic bacteria. The compound isolated from Methanosarcina barkeri appears to be identical to the one obtained from the only distinctly related Methanobacterium thermoautotrophicum. F₄₃₀ is thermolabile and in the presence of acetonitrile or C10 _in4 ^sup- two epimerization products are obtained upon heating; in the absence of these compounds F₄₃₀ is oxidized to 12, 13-didehydro-F₄₃₀. The latter is stereoselectively reduced under H₂ atmosphere to F₄₃₀ by cell-free extracts of M. barkeri or M. thermoautotrophicum. H₂ may be replaced by the reduced methanogenic electron carrier coenzyme F₄₂₀.Abbreviations CH₃S-CoM methylcoenzyme M, 2-methylthioethanesulfonic acid - HS-CoM coenzyme M, 2-mercaptoethanesulfonic acid - F₄₃₀ Ni(II) tetrahydro-(12, 13)-corphin with a uroporphinoid (III) ligand skeleton - 13-epi-F₄₃₀ and 12,13-di-epi-F₄₃₀ the 12, 13- and 12, 13-derivatives of F₄₃₀ - 12, 13-didehydro-F₄₃₀ F₄₃₀ oxidized at C-12 and C-13 - coenzyme F₄₂₀ 7,8-didemethyl-8-hydroxy-5-deazaflavin derivative - coenzyme F₄₂₀H₂ reduced coenzyme F₄₂₀ - MV⁺ methylviologen semiquinone - HPLC high-performance liquid chromatography     

Efficient separation of subfossil Chironomidae from lake sediments

Electron transport system (ETS) activity, CO₂ evolution, O₂ consumption, N₂-fixation (C₂H₂ reduction) and methanogenesis were appropriately measured in aerobic and anaerobically incubated sediment at 4, 10 and 20 ° C to better characterize these activities under different incubation conditions. ETS activity was always higher in the aerobically incubated sediment at all three incubation temperatures, whereas (C₂H₂ reduction was always greater in the anaerobic sediment. Carbon dioxide evolution was detected only in the aerobic sediment at 10 and 20 ° C but not at 4 ° C. Methane evolution in anaerobic sediment increased gradually with an increase in the incubation temperature.     

Methanogenesis and microbial lipid synthesis in anoxic salt marsh sediments

In anoxic salt marsh sediments of Sapelo Island, GA, USA, the vertical distribution of CH₄ production was measured in the upper 20 cm of surface sediments in ten locations. In one section of high marsh sediments, the concentration and oxidation of acetate in sediment porewaters and the rate and amount of¹⁴C acetate and¹⁴CO₂ incorporation into cellular lipids of the microbial population were investigated. CH₄ production rates ranged from <1 to 493 nM CH₄ gram sediment⁻¹ day⁻¹ from intact subcores incubated under nitrogen. Replacement with H₂ stimulated the rate of methane release up to nine fold relative to N₂ incubations. Rates of lipid synthesis from CO₂ averaged 39.2 ×10⁻²nanomoles lipid carbon cm³ sediment⁻¹ hr⁻¹, suggesting that CO₂ may be an important carbon precursor for microbial membrane synthesis in marsh sediments under anoxic conditions. Qualitative measurements of lipid synthesis rates from acetate were found to average 8.7 × 10⁻² nanomoles. Phospholipids were the dominant lipids synthesized by both substrates in sediment cores, accounting for an average of 76.6% of all lipid radioactivity. Small amounts of ether lipids indicative of methanogenic bacteria were observed in cores incubated for 7 days, with similar rates of synthesis for both CO₂ and acetate. The low rate of ether lipid synthesis suggests that either methanogen lipid biosynthesis is very slow or that methanogens represent a small component of total microbial lipid synthesis in anoxic sediments. present address: The University of Maryland,, Chesapeake Biological Laboratory, Box 38, Solomons, MD 20688, USA     

微生物互营产甲烷过程中的种间电子传递

甲烷作为全球第二大温室气体,是典型的可再生清洁能源,也是碳循环中的重要物质组成。大气中约74%的甲烷由产甲烷古菌和其他微生物的互营产生,种间电子传递(interspecies electron transfer, IET)是微生物菌群降低热力学能垒、实现互营产甲烷的核心过程。IET可分为间接种间电子传递(mediated interspecies electron transfer,MIET)和直接种间电子传递(direct interspecies electron transfer, DIET)两种类型,其中MIET依赖氢气、甲酸等载体完成电子的远距离传输,而DIET则依赖导电菌毛、细胞色素c等膜蛋白,通过微生物的直接接触实现电子传递。本文将从IET的研究历程出发,从电子传递机制、微生物种类、生态多样性等方面对微生物互营产甲烷过程中的两种IET类型进行比较,最后对未来待探索的方向进行展望。本综述有助于加深对微生物互营产甲烷过程中IET的理解,为解决由甲烷引发的全球气候变暖等生态问题提供理论支撑。     

Enrichment of homoacetogens converting H2/CO2 into acids and ethanol and simultaneous methane production

An anaerobic granular sludge was enriched to utilize H₂/CO₂ in a continuous gas-fed up-flow anaerobic sludge reactor by applying operating conditions expected to produce acetic acid, butyric acid, and ethanol. Three stages of fermentation were found: Stage I with acetic acid accumulation with the highest concentration of 35 mM along with a pH decrease from initial 6 to 4.5. In Stage II, H₂/CO₂ was replaced by 100% H₂ to induce solventogenesis, whereas butyric acid was produced with the highest concentration of 2.5 mM. At stage III with 10 µM tungsten (W) addition, iso-valeric acid, valeric acid, and caproic acid were produced at pH 4.5–5.0. In the batch tests inoculated with the enriched sludge taken from the bioreactor (day 70), however, methane production occurred at pH 6. Exogenous 15 mM acetate addition enhanced both the H₂ and CO₂ consumption rate compared to exogenous 10, 30, and 45 mM acetate by the enriched sludge. Exogenous acetate was failed to be converted to ethanol using H₂ as electron donor by the enriched acetogens.     

Anaerobic bioprocessing of organic wastes

Anaerobic digestion of dissolved, suspended and solid organics has rapidly evolved in the last decades but nevertheless still faces several scientific unknowns. In this review, some fundamentals of bacterial conversions and adhesion are addressed initially. It is argued in the light of G-values of reactions, and in view of the minimum energy quantum per mol, that anaerobic syntrophs must have special survival strategies in order to support their existence: redistributing the available energy between the partners, reduced end-product fermentation reactions and special cell-to-cell physiological interactions. In terms of kinetics, it appears that both reaction rates and residual substrate thresholds are strongly related to minimum G-values. These new fundamental insights open perspectives for efficient design and operation of anaerobic bioprocesses. Subsequently, an overview is given of the current anaerobic biotechnology. For treating wastewaters, a novel and high performance new system has been introduced during the last decade; the upflow anaerobic sludge blanket system (UASB). This reactor concept requires anaerobic consortia to grow in a dense and eco-physiologically well-organized way. The microbial principles of such granular sludge growth are presented. Using a thermodynamic approach, the formation of different types of aggregates is explained. The application of this bioprocess in worldwide wastewater treatment is indicated. Due to the long retention times of the active biomass, the UASB is also suitable for the development of bacterial consortia capable of degrading xenobiotics. Operating granular sludge reactors at high upflow velocities (5–6 m/h) in expanded granular sludge bed (EGSB) systems enlarges the application field to very low strength wastewaters (chemical oxygen demand < 1 g/l) and psychrophilic temperatures (10°C). For the treatment of organic suspensions, there is currently a tendency to evolve from the conventional mesophilic continuously stirred tank system to the thermophilic configuration, as the latter permits higher conversion rates and easier sanitation. Integration of ultrafiltration in anaerobic slurry digestion facilitates operation at higher volumetric loading rates and at shorter residence times. With respect to organic solids, the recent trend in society towards source separated collection of biowaste has opened a broad range of new application areas for solid state anaerobic fermentation.W. Verstraete and D. de Beer are with the Center for Environmental Sanitation, University of Gent, Coupure L 653, B-9000 Gent, Belgium; D. de Beer is also with the Max Plank Institut für Marine Mikrobiologie-Microzensor Group, Fahrenstrasse 1, 28359 Bremen, Germany. M. Pena is with the Groupo de Biotechnologia Ambiental, Departamento de Ingenieria Quimica, Universidad de Valladolid, Prado de la Magdalena, 47005 Valladolid, Spain. G. Lettinga is with the Department of Environmental Technology, Wageningen Agricultural University, Bomenweg 2, 6703 HD Wageningen, The Netherlands. P. Lens is with the Environmental Research Unit. Department of Microbiology, University College Galway, Galway, Ireland.     

Methanogenesis in thermophilic biogas reactors

Methanogenesis in thermophilic biogas reactors fed with different wastes is examined. The specific methanogenic activity with acetate or hydrogen as substrate reflected the organic loading of the specific reactor examined. Increasing the loading of thermophilic reactors stabilized the process as indicated by a lower concentration of volatile fatty acids in the effluent from the reactors. The specific methanogenic activity in a thermophilic pilot-plant biogas reactor fed with a mixture of cow and pig manure reflected the stability of the reactor. The numbers of methanogens counted by the most probable number (MPN) technique with acetate or hydrogen as substrate were further found to vary depending on the loading rate and the stability of the reactor. The numbers of methanogens counted with antibody probes in one of the reactor samples was 10 times lower for the hydrogen-utilizing methanogens compared to the counts using the MPN technique, indicating that other non-reacting methanogens were present. Methanogens that reacted with the probe againstMethanobacterium thermoautotrophicum were the most numerous in this reactor. For the acetate-utilizing methanogens, the numbers counted with the antibody probes were more than a factor of 10 higher than the numbers found by MPN. The majority of acetate utilizing methanogens in the reactor wereMethanosarcina spp. single cells, which is a difficult form of the organism to cultivatein vitro. No reactions were observed with antibody probes raised againstMethanothrix soehngenii orMethanothrix CALS-1 in any of the thermophilic biogas reactors examined. Studies using 2-¹⁴C-labeled acetate showed that at high concentrations (more than approx. 1 mM) acetate was metabolized via the aceticlastic pathway, transforming the methyl-group of acetate into methane. When the concentration of acetate was less than approx. 1 mM, most of the acetate was oxidized via a two-step mechanism (syntrophic acetate oxidation) involving one organism oxidizing acetate into hydrogen and carbon dioxide and a hydrogen-utilizing methanogen forming the products of the first microorganism into methane. In thermophilic biogas reactors, acetate oxidizing cultures occupied the niche ofMethanothrix species, aceticlastic methanogens which dominate at low acetate concentrations in mesophilic systems. Normally, thermophilic biogas reactors are operated at temperatures from 52 to 56° C. Experiments using biogas reactors fed with cow manure showed that the same biogas yield found at 55° C could be obtained at 61° C after a long adaptation period. However, propionate degradation was inhibited by increasing the temperature.     

The effect of sulfate and nitrate on methane formation in a freshwater sediment

A freshwater sediment from a ditch of a peat grassland near Zegveld (Province of Utrecht, The Netherlands) was investigated for its potential methanogenic and syntrophic activity and the influence of sulfate and nitrate on these potential activities. Methanogenesis started after a 10 days lagphase. After 35–40 days aceticlastic methanogens were sufficiently enriched to cause a net decrease of acetate. In the presence of sulfate methane formation was only slightly affected. The addition of nitrate led to an outcompetion of aceticlastic methanogens by nitrate reducers. When inorganic electron acceptors were absent, substrates like propionate and butyrate were converted by syntrophic methanogenic consortia. Addition of inorganic electron acceptors resulted in an outcompetition of the syntrophic propionate and butyrate degrading consortia by the sulfate and nitrate reducers.