Simultaneous butyrate oxidation by Syntrophomonas wolfei and catalytic olefin reduction in absence of interspecies hydrogen transfer

Methanogenesis by a Syntrophomonas wolfei/ Methanospirillum hungatei coculture was inhibited in presence of ethylene and the hydrogenation catalyst Pd-BaSO₄. However, butyrate oxidation by S. wolfei continued and ethylene was reduced to ethane. Per mol of butyrate oxidized, 2.4 mol acetate was produced and 0.8 mol ethylene was reduced. Acetylene, propylene and butene were less effective as H₂ acceptors than ethylene, and addition of bromoethanesulfonic acid was necessary to inhibit methanogenesis in the presence of the two longer-chain olefins. Other hydrogenation catalysts were less effective in the order Pd-charcoal < PE-asbestos < Pd-PEI beads < Pt-Al₂O₃, Pd-CaCO₃. Optimal ethylene hydrogenation was achieved with still incubation in presence of 7.2 mg Pd-BaSO₄ and 0.7 g sand per ml medium. The higher catabolic rate of S. wolfei in presence of the methanogen indicated that the biological H₂ removal mechanism was more efficient than the catalytic olefin reduction.Abbreviations BES bromoethane sulfonic acid - VFA volatile fatty acid     

Methanogenesis from Choline by a Coculture of Desulfovibrio sp. and Methanosarcina barkeri

A sulfate-reducing vibrio was isolated from a methanogenic enrichment with choline as the sole added organic substrate. This organism was identified as a member of the genus Desulfovibrio and was designated Desulfovibrio strain G1. In a defined medium devoid of sulfate, a pure culture of Desulfovibrio strain G1 fermented choline to trimethylamine, acetate, and ethanol. In the presence of sulfate, more acetate and less ethanol were formed from choline than in the absence of sulfate. When grown in a medium containing sulfate, a coculture of Desulfovibrio strain G1 and Methanosarcina barkeri strain Fusaro degraded choline almost completely to methane, ammonia, and hydrogen sulfide and presumably to carbon dioxide. Methanogenesis occurred in two distinct phases separated by a lag of about 6 days. During the first phase of methanogenesis choline was completely converted to trimethylamine, acetate, hydrogen sulfide, and traces of ethanol by the desulfovibrio. M. barkeri fermented trimethylamine to methane, ammonia, and presumably carbon dioxide via dimethyl- and methylamine as intermediates. Simultaneously, about 60% of the acetate expected was metabolized. In the second phase of methanogenesis, the residual acetate was almost completely catabolized.     

Degradation of benzoate by an anaerobic consortium and some properties of a hydrogenotrophic methanogen and sulfate-reducing bacterium in the consortium

A highly simplified anaerobic consortium which was able to degrade benzoate under mesophilic conditions was obtained from digested sludge acclimatized with benzoate. It converted 5 mM benzoate to methane quantitatively within 3 weeks in the absence of any organic nutrients under an N₂/CO₂ atmosphere. Degradation of benzoate was strictly inhibited by hydrogen. The consortium consisted of at least three microorganisms including an autofluorescent irregular coccus which was identified as Methanogenium sp., a short rod which did not autofluoresce and was considered to be a benzoate degrader, and a filamentous bacterium apparently classified as Methanothrix (= “Methanosaeta”. When sulfate was added to the medium, the methanogens were readily replaced by a sulfate-reducing bacterium, probably belonging to the genus Desulfovibrio, which had still remained in very low number in the consortium in the absence of sulfate, and benzoate was stoichiometrically converted to acetate without methanogenesis. Of various compounds which were expected to be intermediates in the benzoate degradation, only crotonate was degraded by concentrated cells of the consortium.     

Uncoupling of Methanogenesis from Growth of Methanosarcina barkeri by Phosphate Limitation

Production of methane by Methanosarcina barkeri from H₂-CO₂ was studied in fed-batch culture under phosphate-limiting conditions. A transition in the kinetics of methanogenesis from an exponentially increasing rate to a constant rate was due to depletion of phosphate from the medium. The period of exponentially increasing rate of methanogenesis was extended by increasing the initial concentration of phosphate in the medium. Addition of phosphate during the constant period changed the kinetics to an exponentially increasing rate of methanogenesis, indicating the reversibility of phosphate depletion. The relation between methanogenesis and growth of M. barkeri was investigated by measuring the incorporation of phosphorus, supplied as KH₂³²PO₄, in the medium. At a low (1 μM) initial concentration of phosphate in the medium and during the constant period of methanogenesis, there was no net cell growth. At a higher (10 μM) initial concentration of phosphate, cell growth proceeded linearly with time after phosphate had been removed from the medium by uptake into cells.     

Mass spectrometric control of the thermophilic anaerobic digestion process based on levels of dissolved hydrogen

Summary The continuous and simultaneous monitoring of dissolved CH₄ and H₂ in samples from a laboratory scale thermophilic anaerobic digester contents by use of a silicone rubber-covered probe has enabled control of methanogenesis: regulation of the hydrogen signal in a closed feedback loop was by controlled addition of the carbon source. Dissolved hydrogen became apparent in this system at a lower loading rate than was obtained for a mesophilic anaerobic digestion system (Whitmoreet al., 1986). Controlling the supply of glucose (25 mM) at a dilution rate of 0.02 h^–1 and at progressively lower preset hydrogen levels allowed methanogenesis to be significantly prolonged before inhibition of the process occurred.     

Production of oligosaccharides and cellobionic acid by <Emphasis Type="Italic">Fibrobacter succinogenes</Emphasis> S85 growing on sugars,cellulose and wheat straw

Extracellular culture fluid of Fibrobacter succinogenes S85 grown on glucose, cellobiose, cellulose or wheat straw was analysed by 2D-NMR spectroscopy. Cellodextrins did not accumulate in the culture medium of cells grown on cellulose or straw. Maltodextrins and maltodextrin-1P were identified in the culture medium of glucose, cellobiose and cellulose grown cells. New glucose derivatives were identified in the culture fluid under all the substrate conditions. In particular, a compound identified as cellobionic acid accumulated at high levels in the medium of F. succinogenes S85 cultures. The production of cellobionic acid (and cellobionolactone also identified) was very surprising in an anaerobic bacterium. The results suggest metabolic shifts when cells were growing on solid substrate cellulose or straw compared to soluble sugars.     

Isolation and Characterization of an Enterobacter cloacae Strain That Reduces Hexavalent Chromium under Anaerobic Conditions

An Enterobacter cloacae strain (HO1) capable of reducing hexavalent chromium (chromate) was isolated from activated sludge. This bacterium was resistant to chromate under both aerobic and anaerobic conditions. Only the anaerobic culture of the E. cloacae isolate showed chromate reduction. In the anaerobic culture, yellow turned white with chromate and the turbidity increased as the reduction proceeded, suggesting that insoluble chromium hydroxide was formed. E. cloacae is likely to utilize toxic chromate as an electron acceptor anaerobically because (i) the anaerobic growth of E. cloacae HO1 accompanied the decrease of toxic chromate in culture medium, (ii) the chromate-reducing activity was rapidly inhibited by oxygen, and (iii) the reduction occurred more rapidly in glycerol- or acetate-grown cells than in glucose-grown cells. The chromate reduction in E. cloacae HO1 was observed at pH 6.0 to 8.5 (optimum pH, 7.0) and at 10 to 40°C (optimum, 30°C).     

Nutritional Requirements of Methanosarcina sp. Strain TM-1

Methanosarcina sp. strain TM-1, an acetotrophic, thermophilic methanogen isolated from an anaerobic sludge digestor, was originally reported to require an anaerobic sludge supernatant for growth. It was found that the sludge supernatant could be replaced with yeast extract (1 g/liter), 6 mM bicarbonate-30% CO₂, and trace metals, with a doubling time on methanol of 14 h. For growth on either methanol or acetate, yeast extract could be replaced with CaCl₂ · 2H₂O (13.6 μM minimum) and the vitamin p-aminobenzoic acid (PABA, ca. 3 nM minimum), with a doubling time on methanol of 8 to 9 h. Filter-sterilized folic acid at 0.3 μM could not replace PABA. The antimetabolite sulfanilamide (20 mM) inhibited growth of and methanogenesis by Methanosarcina sp. strain TM-1, and this inhibition was reversed by the addition of 0.3 μM PABA. When a defined medium buffered with 20 mM N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid was used, it was shown that Methanosarcina sp. strain TM-1 required 6 mM bicarbonate-30% CO₂ for optimal growth and methanogenesis from methanol. Cells growing on acetate were less dependent on bicarbonate-CO₂. When we used a defined medium in which the only organic compounds present were methanol or acetate, nitrilotriacetic acid (0.2 mM), and PABA, it was possible to limit batch cultures of Methanosarcina sp. strain TM-1 for nitrogen at NH₄⁺ concentrations at or below 2.0 mM, in marked contrast with Methanosarcina barkeri 227, which fixes dinitrogen when grown under NH₄⁺ limitation.     

Metaproteomics of cellulose methanisation under thermophilic conditions reveals a surprisingly high proteolytic activity

Cellulose is the most abundant biopolymer on Earth. Optimising energy recovery from this renewable but recalcitrant material is a key issue. The metaproteome expressed by thermophilic communities during cellulose anaerobic digestion was investigated in microcosms. By multiplying the analytical replicates (65 protein fractions analysed by MS/MS) and relying solely on public protein databases, more than 500 non-redundant protein functions were identified. The taxonomic community structure as inferred from the metaproteomic data set was in good overall agreement with 16S rRNA gene tag pyrosequencing and fluorescent in situ hybridisation analyses. Numerous functions related to cellulose and hemicellulose hydrolysis and fermentation catalysed by bacteria related to Caldicellulosiruptor spp. and Clostridium thermocellum were retrieved, indicating their key role in the cellulose-degradation process and also suggesting their complementary action. Despite the abundance of acetate as a major fermentation product, key methanogenesis enzymes from the acetoclastic pathway were not detected. In contrast, enzymes from the hydrogenotrophic pathway affiliated to Methanothermobacter were almost exclusively identified for methanogenesis, suggesting a syntrophic acetate oxidation process coupled to hydrogenotrophic methanogenesis. Isotopic analyses confirmed the high dominance of the hydrogenotrophic methanogenesis. Very surprising was the identification of an abundant proteolytic activity from Coprothermobacter proteolyticus strains, probably acting as scavenger and/or predator performing proteolysis and fermentation. Metaproteomics thus appeared as an efficient tool to unravel and characterise metabolic networks as well as ecological interactions during methanisation bioprocesses. More generally, metaproteomics provides direct functional insights at a limited cost, and its attractiveness should increase in the future as sequence databases are growing exponentially.     

Ab initio molecular orbital calculations on the conformations of some unsaturated fragments of lipid molecules: 1-Butene, cis-2-pentene and 1,4-pentadiene

Conformational energies have been calculated for the title compounds which are basic fragments of physiologically active polyunsaturated fatty acids. For 1-butene extremely good accordance has been obtained with experimental data. In cis-2-pentene a strong influence of the cis methyl group on the potential energies for internal rotation has been found. For 1,4-pentadiene no great influence has been found from a possible coupling of the two rotors on the relative stabilities of the two stable conformations. Reasonable agreement has been observed with similar calculations published recently.     

Ethanol-to-methane activity of Geobacter-deprived anaerobic granules enhanced by conductive microparticles

Direct interspecies electron transfer (DIET) has been typically proposed as mechanism of electron transfer among methanogenic populations in granules during anaerobic digestion where Geobacter species play a key role. Using anaerobic granules where Geobacteraceae members were not prevalent − representing only 0.3% of total bacteria −, tests incubated with two co-substrates showed that the rate of methanogenesis from formate and hydrogen diminished in the presence of a non-methanogenic co-substrate such as ethanol. This could indicate that biological DIET occurs and competes with hydrogen and formate during methanogenesis. Moreover, the addition of conductive microparticles, such as stainless steel and granular activated carbon, was found to increase methanogenic activity in disintegrated granules by 190 ± 18% and 175 ± 22% respectively as compared to disintegrated granules devoid of microparticles. The addition of non-conductive microparticles such as porcelain however decreased methanogenic activity by 65 ± 3% of the disrupted granules without microparticle activity. These results indicate that syntrophic bacteria from anaerobic sludge excluding Geobacter species can also carry out conductive mineral mediated DIET.     

Complete Genome Sequence of Desulfurococcus fermentans,a Hyperthermophilic Cellulolytic Crenarchaeon Isolated from a Freshwater Hot Spring in Kamchatka,Russia

Desulfurococcus fermentans is the first known cellulolytic archaeon. This hyperthermophilic and strictly anaerobic crenarchaeon produces hydrogen from fermentation of various carbohydrates and peptides without inhibition by accumulating hydrogen. The complete genome sequence reported here suggested that D. fermentans employs membrane-bound hydrogenases and novel glycohydrolases for hydrogen production from cellulose.     

Sulfide enhances methanogenesis in nitrate-containing methanogenic cultures

The effects of sulfide on nitrate reduction and methanogenesis using butyrate as a carbon source were investigated in a mixed mesophilic, methanogenic culture. In the sulfide-free medium, 25-75 mg l⁻¹ nitrate markedly inhibited the efficiencies of acetogenesis and methanogenesis processes. Adding 25 mg-S l⁻¹ increased methane production in nitrate-amended medium. Low sulfide levels shifted the nitrate reduction pathway from denitrification to dissimilatory nitrate reduction to ammonia (DNRA), thereby reducing the amounts of toxic nitric oxide and nitrous oxide produced that inhibit methanogenesis. The dose of 25 mg l⁻¹ sulfide was oxidized completely, during which heterotrophic DNRA predominated. The oxidized forms of sulfide reformed, limiting induction of the heterotrophic denitrification pathway. The actions of heterotrophic and autotrophic DNRA bacteria, denitrifiers, sulfate-reducing bacteria and methanogens mitigate nitrate toxicity during methanogenesis in an anaerobic process.     

Co-digestion of manure and whey for in situ biogas upgrading by the addition of H2: process performance and microbial insights

In situ biogas upgrading was conducted by introducing H₂ directly to the anaerobic reactor. As H₂ addition is associated with consumption of the CO₂ in the biogas reactor, pH increased to higher than 8.0 when manure alone was used as substrate. By co-digestion of manure with acidic whey, the pH in the anaerobic reactor with the addition of hydrogen could be maintained below 8.0, which did not have inhibition to the anaerobic process. The H₂ distribution systems (diffusers with different pore sizes) and liquid mixing intensities were demonstrated to affect the gas-liquid mass transfer of H₂ and the biogas composition. The best biogas composition (75:6.6:18.4) was obtained at stirring speed 150 rpm and using ceramic diffuser, while the biogas in the control reactor consisted of CH₄ and CO₂ at a ratio of 55:45. The consumed hydrogen was almost completely converted to CH₄, and there was no significant accumulation of VFA in the effluent. The study showed that addition of hydrogen had positive effect on the methanogenesis, but had no obvious effect on the acetogenesis. Both hydrogenotrophic methanogenic activity and the concentration of coenzyme F₄₂₀ involved in methanogenesis were increased. The archaeal community was also altered with the addition of hydrogen, and a Methanothermobacter thermautotrophicus related band appeared in a denaturing gradient gel electrophoresis gel from the sample of the reactor with hydrogen addition. Though the addition of hydrogen increased the dissolved hydrogen concentration, the degradation of propionate was still thermodynamically feasible at the reactor conditions.     

Characteristics of anaerobic oxalate-degrading enrichment cultures from the rumen.

Enrichment cultures of rumen bacteria degraded oxalate within 3 to 7 days in a medium containing 10% rumen fluid and an initial level of 45 mM sodium oxalate. This capability was maintained in serially transferred cultures. One mole of methane was produced per 3.8 mol of oxalate degraded. Molecular hydrogen and formate inhibited oxalate degradation but not methanogenesis; benzyl viologen and chloroform inhibited both oxalate degradation and methanogenesis. Attempts to isolate oxalate-degrading bacteria from these cultures were not successful. Oxalate degradation was uncoupled from methane production when enrichments were grown in continuous culture at dilution rates greater than or equal to 0.078 h-1. Growth of the uncoupled population (lacking methanogens) in batch culture was accompanied by degradation of 45 mM oxalate within 24 h and production of 0.93 mol of formate per mol of oxalate degraded. Oxalate degradation by the uncoupled population was not inhibited by molecular hydrogen or formate. Cell yields (grams [dry weight]) per mole of oxalate degraded by the primary enrichment and the uncoupled populations were 1.7 and 1.0, respectively.     

Bacterial Cellulose-Binding Domain Modulates in Vitro Elongation of Different Plant Cells

Recombinant cellulose-binding domain (CBD) derived from the cellulolytic bacterium Clostridium cellulovorans was found to modulate the elongation of different plant cells in vitro. In peach (Prunus persica L.) pollen tubes, maximum elongation was observed at 50 μg mL⁻¹ CBD. Pollen tube staining with calcofluor showed a loss of crystallinity in the tip zone of CBD-treated pollen tubes. At low concentrations CBD enhanced elongation of Arabidopsis roots. At high concentrations CBD dramatically inhibited root elongation in a dose-responsive manner. Maximum effect on root hair elongation was at 100 μg mL⁻¹, whereas root elongation was inhibited at that concentration. CBD was found to compete with xyloglucan for binding to cellulose when CBD was added first to the cellulose, before the addition of xyloglucan. When Acetobacter xylinum L. was used as a model system, CBD was found to increase the rate of cellulose synthase in a dose-responsive manner, up to 5-fold compared with the control. Electron microscopy examination of the cellulose ribbons produced by A. xylinum showed that CBD treatment resulted in a splayed ribbon composed of separate fibrillar subunits, compared with a thin, uniform ribbon in the control.     

Influence of redox potential on methanation of methanol by Methanosarcina barkeri in Eh-stat batch cultures

The effect of Eh on the methanogenesis of methanol by Methanosarcina barkeri strain Fusaro was studied in pH-controlled anaerobic batch cultures at 37°C, in which the Eh of the culture medium was controlled by the addition of Ti(III)-citrate at values ranging from −340 to −520 mV. The changes in Eh revealed that the specific growth rate, μ, specific methane production rate, Q_CH4 and growth yield, Y_X/S were optimum under an Eh between −430 and −520 mV, while they decreased at the higher Eh of −340 mV. The maximum values of Q_CH4 and μ under the optimum Eh condition were 210 ml CH₄/g dry cell weight·h⁻¹ and 0.11 h⁻¹, respectively.     

Identification, Detection, and Spatial Resolution of Clostridium Populations Responsible for Cellulose Degradation in a Methanogenic Landfill Leachate Bioreactor

An anaerobic landfill leachate bioreactor was operated with crystalline cellulose and sterile landfill leachate until a steady state was reached. Cellulose hydrolysis, acidogenesis, and methanogenesis were measured. Microorganisms attached to the cellulose surfaces were hypothesized to be the cellulose hydrolyzers. 16S rRNA gene clone libraries were prepared from this attached fraction and also from the mixed fraction (biomass associated with cellulose particles and in the planktonic phase). Both clone libraries were dominated by Firmicutes phylum sequences (100% of the attached library and 90% of the mixed library), and the majority fell into one of five lineages of the clostridia. Clone group 1 (most closely related to Clostridium stercorarium), clone group 2 (most closely related to Clostridium thermocellum), and clone group 5 (most closely related to Bacteroides cellulosolvens) comprised sequences in Clostridium group III. Clone group 3 sequences were in Clostridium group XIVa (most closely related to Clostridium sp. strain XB90). Clone group 4 sequences were affiliated with a deeply branching clostridial lineage peripherally associated with Clostridium group VI. This monophyletic group comprises a new Clostridium cluster, designated cluster VIa. Specific fluorescence in situ hybridization (FISH) probes for the five groups were designed and synthesized, and it was demonstrated in FISH experiments that bacteria targeted by the probes for clone groups 1, 2, 4, and 5 were very abundant on the surfaces of the cellulose particles and likely the key cellulolytic microorganisms in the landfill bioreactor. The FISH probe for clone group 3 targeted cells in the planktonic phase, and these organisms were hypothesized to be glucose fermenters.     

Attack on Lignified Grass Cell Walls by a Facultatively Anaerobic Bacterium

A filamentous, facultatively anaerobic microorganism that attacked lignified tissue in forage grasses was isolated from rumen fluid with a Bermuda grass-containing anaerobic medium in roll tubes. The microbe, designated 7-1, demonstrated various colony and cellular morphologies under different growth conditions. Scanning electron microscopy revealed that 7-1 attacked lignified cell walls in aerobic and anaerobic culture. 7-1 predominately degraded tissues reacting positively for lignin with the chlorine-sulfite stain (i.e., sclerenchyma in leaf blades and parenchyma in stems) rather than the more resistant acid phloroglucinol-positive tissues (i.e., lignified vascular tissue and sclerenchyma ring in stems), although the latter tissues were occasionally attacked. Turbidimetric tests showed that 7-1 in anaerobic culture grew optimally at 39°C at a pH of 7.4 to 8.0. Tests for growth on plant cell wall carbohydrates showed that 7-1 grew on xylan and pectin slowly in aerobic cultures but not with pectin and only slightly with xylan in anaerobic culture. 7-1 was noncellulolytic as shown by filter paper tests. The microbe used the phenolic acids sinapic, ferulic, and p-coumaric acids as substrates for growth; the more highly methoxylated acids were used more effectively.     

Influence of selected carbohydrates on rhizogenesis of shoots saucer magnolia in vitro

Nodal segements were taken from juvenile shoots of mature 100 year-old trees of saucer magnolia (Magnolia x soulangiana Soul.-Bod.) and cultured on Standardi and Catalano medium supplemented with 1.33 μmol·dm⁻³ BA, 0.54 μmol·dm⁻³ NAA, 58 μmol·dm⁻³ sucrose and 6.0 g·l⁻¹ agar-agar. After 8 weeks, separated shoots were transferred to rooting medium with half-strength macronutrients (basal medium) supplemented with 0.3% activated charcoal and one of carbohydrates: arabinose, cellulose, fructose, galactose, glucose, lactose, mannose, rhamnose, ribose, sorbose, sucrose or xylose at 20 g·dm⁻³ and 7.0 g·dm⁻³ agar-agar. After 13 weeks of culture, shoot number, fresh and dry weight of shoots and roots, total root length and number of roots/per shoot were recorded. Percentages of rooted shoots were calculated. Fructose, mannose and xylose were the most effective carbon source on shoot proliferation followed by sucrose. The rooting response was induced by cellulose and xylose. Arabinose, rhamnose and sorbose inhibited root formation. The number of adventitious roots produced per shoot was stimulated by cellulose and xylose. Total biomass (shoot plus roots) of the plantlets was the highest at fructose and cellulose.