Showing papers on "Methanogen published in 1984"

Non-aceticlastic methanogenesis from acetate: acetate oxidation by a thermophilic syntrophic coculture

Abstract: Methanogenesis from acetate by a rod-shaped enrichment culture grown at 60° C was found to require the presence of two organisms rather than a single aceticlastic methanogen. A thermophilic Methanobacterium which grew on H2/CO2 or formate was isolated from the enrichment. Lawns of this methanogen were used to co-isolate an “acetate oxidizer” in roll tubes containing acetate agar. The rod-shaped acetate oxidizer was morphologically distinct from the methanogen and did not show F420 autofluorescence. The coculture completely degraded 40 μmol/ml acetate, and produced nearly equal quantities of methane, and methanogenesis was coupled with growth. The doubling time for the coculture at 60°C was 30–40 h and the yield was 2.7±0.3 g dry wt/mol CH4. Studies with 14C-labelled substrates showed that the methyl group and the carboxyl group of acetate were both converted primarily to CO2 by the coculture and that CO2 was concurrently reduced to CH4. During growth, there was significant isotopic exchange between CO2 and acetate, especially with thecarboxyl position of acetate. These results support a mechanism for methanogenesis from acetate by the coculture in which acetate was oxidized to CO2 and H2 by one organism, while H2 was subsequently used by a second organism to reduce CO2 to CH4. Since the H2 partial pressure must be maintained below 10-4 atm by the methanogen for acetate oxidation to be thermodynamically feasible, this is an example of obligate interspecies hydrogen transfer. This mechanism was originally proposed for a single organism by Barker in 1936.

Methanogenesis in a Thermophilic (58°C) Anaerobic Digestor: Methanothrix sp. as an Important Aceticlastic Methanogen

Abstract: Aceticlastic methanogens and other microbial groups were enumerated in a 58°C laboratory-scale (3 liter) anaerobic digestor which was fed air-classified municipal refuse, a lignocellulosic waste (loading rate = 1.8 to 2.7 g of volatile solids per liter per day; retention time = 10 days). Two weeks after start-up, Methanosarcina sp. was present in high numbers (105 to 106 CFU/ml) and autofluorescent Methanosarcina-like clumps were abundant in sludge examined by using epifluorescence microscopy. After about 4 months of digestor operation, numbers of Methanosarcina sp. dropped 2 to 3 orders of magnitude and large numbers (most probable number = 106 to 107/ml) of a thermophilic aceticlastic methanogen morphologically resembing Methanothrix sp. were found. Methanothrix sp. had apparently displaced Methanosarcina sp. as the dominant aceticlastic methanogen in the digestor. During the period when Methanothrix sp. was apparently dominant, acetate concentrations varied between 0.3 and 1.5 μmol/ml during the daily feeding cycle, and acetate was the precursor of 63 to 66% of the methane produced during peak digestor methanogenesis. The apparent Km value obtained for methanogenesis from acetate, 0.3 μmol/ml, indicated that the aceticlastic methanogens were nearly saturated for substrate during most of the digestor cycle. CO2-reducing methanogens were capable of methanogenesis at rates more than 12 times greater than those usually found in the digestor. Added propionate (4.5 μmol/ml) was metabolized slowly by the digestor populations and slightly inhibited methanogenesis. Added n-butyrate, isobutyrate, or n-valerate (4.5 μmol/ml each) were broken down within 24 h. Isobutyrate was oxidized to acetate, a novel reaction possibly involving isomerization to n-butyrate. The rapid growth rate and versatile metabolism of Methanosarcina sp. make it a likely organism to be involved in start-up, whereas the low Km value of Methanothrix sp. for acetate may cause it to be favored in stable digestors operated with long retention times. Images

Characterization and nutritional properties of Methanothrix concilii sp. nov., a mesophilic, aceticlastic methanogen

Abstract: A nonmotile, rod-shaped, nonspore-forming, mesophilic methanogenic bacterium was isolated from sewage sludge The cells stained Gram negative In the presence of CO2, the isolate was able to grow and produce significant amounts of methane from acetic acid No growth or methane formation was observed when H2, methanol, pyruvate, propionate, butyrate, formate, or trimethylamine were provided as substrates in the presence of CO2 About 095 mol of CH4 was produced per mole of acetic acid consumed The optimum pH and temperature for growth were 71–75 and 35–40 °C, respectively The mass doubling time was about 24 h under optimum growth conditions The almost complete inhibition of methane formation by 10 μM 2-bromoethanesulfonic acid (2-BES) was reversed in the presence of 50 μM 2-mercaptoethanesulfonic acid (HS-CoM) D-Cycloserine at 01 mg/mL concentration caused complete inhibition of growth Sludge fluid enhanced the rate of methane formation, whereas 05% (w/v) yeast extract was inhibitory The optimum

Methanogenesis from Sucrose by Defined Immobilized Consortia

Abstract: A bacterial consortium capable of sucrose degradation primarily to CH4 and CO2 was constructed, with acetate as the key methanogenic precursor. In addition, the effect of agar immobilization on the activity of the consortium was determined. The primary fermentative organism, Escherichia coli, produced acetate, formate, H2, and CO2 (known substrates for methanogens), as well as ethanol and lactate, compounds that are not substrates for methanogens. Oxidation of the nonmethanogenic substrates, lactate and ethanol, to acetate was mediated by the addition of Acetobacterium woodii and Desulfovibrio vulgaris. The methanogenic stage was accomplished by the addition of the acetophilic methanogen Methanosarcina barkeri and the hydrogenophilic methanogen Methanobacterium formicicum. Results of studies with low substrate concentrations (0.05 to 0.2% [wt/vol]), a growth-limiting medium, and the five-component consortium indicated efficient conversion (40%) of sucrose carbon to CH4. Significant decreases in yields of CH4 and rates of CH4 production were observed if any component of the consortium was omitted. Approximately 70% of the CH4 generated occurred via acetate. Agar-immobilized cells of the consortium exhibited yields of CH4 and rates of CH4 production from sucrose similar to those of nonimmobilized cells. The rate of CH4 production decreased by 25% when cysteine was omitted from reaction conditions and by 40% when the immobilized consortium was stored for 1 week at 4°C.

Isolation and characterization of a novel thermophilic, freshwater methanogen.

Abstract: A novel thermophilic, coccoid methanogen isolated from nonthermal freshwater sediments is described. Hydrogen plus carbon dioxide and formate were substrates for methanogenesis, and methane production was stimulated by yeast extract, Casamino Acids, and tryptose. Growth also occurred autotrophically. Elevated levels of sodium chloride were not required for maximum growth and were inhibitory above 2%. The minimum doubling time occurred at 57 degrees C, and the upper and lower limits for methane production were 62 and 26 degrees C, respectively. The optimum pH for growth was between 7.0 and 7.5. Inhibitory antibiotics included metronidazole, anisomycin, chloramphenicol, and lasalocid. Colonies were circular, dark yellow, shiny, and convex with entire edges. Cells were 1 to 2.5 mum in diameter, nonmotile, occurring singly or in pairs, and fimbriated. Cells were lysed by pronase or trypsin digestion, glass-distilled water, and 1% sodium dodecyl sulfate. Electron micrographs of thin sections showed a monolayered cell wall ca. 20 nm thick. The DNA base ratio was 49.2 mol% guanine plus cytosine. The whole cell protein pattern differed from that of other named coccoid methanogens.

Hydrogen-using bacteria in a methanogenic acetate enrichment culture

Abstract: In a study of the anaerobic utilization of acetate, an enrichment culture of sewage sludge organisms was initiated with calcium acetate as the sole carbon and energy source. A mixed bacterial population became established from which 14 anaerobic species were isolated. Two of the isolates were methanogenic bacteria but only one of these, Methanosarcina barkeri, utilised acetate as an energy source in axenic culture. The other methanogenic isolate, a Methanobacterium sp., utilised H2/CO2 but not acetate. A third methanogen, which was morphologically identical to Methanothrix soehngenii, was detected in the enrichment but was not obtained in monoculture. 2-Bromoethanesulphonate, a specific inhibitor of methanogenesis, completely inhibited the enrichment at a concentration of 10 mumol/l. Addition of H2 formate or methanol to the enrichment did not affect the rate of methanogenesis. An H2-utilizing Desulfovibrio sp. was also isolated from the enrichment.

Methanogenesis fromSucrose byDefined Immobilized Consortia

Abstract: A bacterial consortium capable ofsucrosedegradation primarily toCH4andCO,was constructed, with acetate asthekeymethanogenic precursor.Inaddition, theeffect ofagar immobilization on theactivity of theconsortium was determined. Theprimary fermentative organism, Escherichia (0li, produced acetate, formate, H2,andCO2(knownsubstrates formethanogens), aswell asethanol andlactate, compoundsthat arenotsubstrates formethanogens. Oxidation ofthenonmethanogenic substrates, lactate andethanol, to acetate was mediated bytheaddition ofAcetobacterium wvoodii andDesulfoviibrio i'ulgairis. Themethanogenic stagewas accomplished bytheaddition oftheacetophilic methanogen Methanosar(Ina batrkeri and thehydrogenophilic methanogen Methanobachterium formicicum. Results ofstudies withlowsubstrate concentrations (0.05 to0.2%[wt/vol]), a growth-limiting medium,andthefive-component consortium indicated efficient conversion (40%)ofsucrosecarbon toCH4.Significant decreases inyields ofCH4and ratesofCH4production were observed ifany componentoftheconsortium was omitted. Approximately 70%oftheCH4generated occurred viaacetate. Agar-immobilized cells oftheconsortium exhibited yields ofCH4andratesofCH4production fromsucrosesimilar tothose ofnonimmobilized cells. TherateofCH4 production decreased by25%whencysteine was omitted fromreaction conditions andby40%whenthe immobilized consortium was stored for1weekat4°C. Thedegradation ofcarbohydrates tomethane bydefined mixedcultures hasbeenstudied byseveral investigators (8, 21,26-28). WinterandWolfe(27)studied thecomplete conversion offructose toCH4andCO,withcocultures of Acetobacterium wioodii andMethanosarcuina barkeri. These authors alsostudied methane formation fromglucose and fructose withdifferent mixtures ofA.wvoodii andvarious methanogenic bacteria (28). Winter(26)studied glucose degradation toCH4andCO,withvarious associations of Bifidobacteriium bifidius orEscherichia coli asprimary fermentative bacteria inassociation withDesulfoi'ibrio desiilfiuricans andM.barkeri. Additional studies involving cellulosedegradation bymethanogenic mixedcultures havebeen reported (16, 18,19,25). Theimmobilization ofbacteria provides anattractive meansofproduct separation fromcellular material with retention ofbiocatalysts. However, fewstudies areavailable dealing withimmobilized defined mixedcultures. Karube et al.(15) studied methane production byusingagar-immobilized bacterial enrichments fromdigested sewagesludge, andScherer etal.(22)studied methanol utilization by alginate-immobilized M.bar-keri. Asamodelsystem, wehaveconstructed amixedbacterial culture capable ofsucrose degradation primarily toCH4and CO2,withacetate asthekeydirect methanogenic precursor. Inaddition, wehavestudied theeffect ofimmobilization on theperformance ofthedefined consortium. Thistypeof methanogenic consortium resembles mixedculture processesfoundinanaerobic environments suchassediments or anaerobic digestors (14, 23). Ourexperiments weredesigned specifically totest theability ofaconsortium tocarry outthe biogenesis ofmethane inanutrient-limited medium.