Conversion of lactose to methane by defined bacterial cocultures

The conversion of lactose — the main constituent of whey — to methane and carbon dioxide was studied using different defined constructed cultures, imploying strains of Methanosarcina barkeri, Methanobacterium bryantii, Escherichia coli, Acetobacterium woodii, Lactobacillus casei, and Lactobacillus plantarum. The following combinations of strains (food chains) were studied with respect to efficiency and yield of lactose conversion (methane yield in parentheses): E. coli and M. barkeri (4.5–7.6%), E. coli and M. bryantii (13.3%),E. coli, M. barkeri and M. bryantii (54%), L. casei, A. woodii and M. barkeri (93.3%). These conversions were carried out in pH controlled batch fermentations. A very efficient coculture was a combination of L. plantarum with A. woodii and M. barkeri: in chemostat cultures lactose was converted to methane and carbon dioxide with a yield of about 90%, at dilution rates of 0.27 d^-1to 0.37 d^-1.     

Methane formation from fructose by syntrophic associations of Acetobacterium woodii and different strains of methanogens

When Acetobacterium woodii was co-cultured in continuous or in stationary culture with Methanobacterium strain AZ, fructose instead of being converted to 3 mol of acetate was converted to 2 mol of acetate and 1 mol each of carbon dioxide and methane, showing that interspecies hydrogen transfer occurred. In continous culture the organisms formed a close physical association in clumps; the doubling time for each organism was 6h at 33°C. Methane mainly was derived from carbon positions 3 and 4 of the sugar, but other carbons also yielded methane; this was shown to be due to carbon dioxide-acetate exchange reactions by A. woodii in a manner similar to that carried out by Clostridium thermoaceticum. Four other methanogens, Methanobacterium M.o.H. and M.o.H. G, Methanobacterium formicicum, and Methanosarcina barkeri (not acetate-adapted) also produced similar results, when co-cultured with A. woodii.     

Acetate synthesis from 2 CO2 in acetogenic bacteria: is carbon monoxide an intermediate?

Cultures of Acetobacterium woodii and Clostridium thermoaceticum growing on fructose or glucose, respectively, were found to produce small, but significant amounts of carbon monoxide. In the gas phase of the cultures up to 53 ppm CO were determined. The carbon monoxide production was completely inhibited by 1 mM cyanide. Cultures and cell suspensions of both acetogens incorporated ¹⁴CO specifically into the carboxyl group of acetate. This CO fixation into C₁ of acetate was unaffected by cyanide (1 mM). The findings are taken to indicate that CO (in a bound form) is the physiological precursor of the C₁ of acetate in acetate synthesis from CO₂. The cyanide inhibition experiments support the hypothesis that the cyanide-sensitive carbon monoxide dehydrogenase may serve to reduce CO₂ to CO rather than to incorporate the carbonyl into C₁ of acetate.     

Growth rate influences reductive biodegradation of the organophosphorus pesticide demeton by Corynebacterium glutamicum

The organophosphorous pesticide, demeton-S-methyl was transformed byCorynebacterium glutamicum in co-metabolism with more readilydegradable substrates. Glucose, acetate and fructose were tested as growth substrates, and the highest demeton-S-methyl biotransformation average rate (0.78 mg l^-1 h^-1) and maximum instantaneous rate (1.4 mg l^-1 h^-1) were achieved on fructose. This higher efficiency seems to be linked to the atypical behavior of C. glutamicum grown on fructose, characterized by a prolonged period of accelerating growth instead of a constant growth rate observed on glucose or acetate. More precisely, for growth rates in the 0.1–0.4 h^-1 range, a direct coupling between the specific demeton-S-methyl consumption rate and the growth rate was demonstrated on fructose during batch –, steady state continuous – or continuous cultures with a controlled transient growth rate (accelerostat technology). The demeton-S-methyl biotransformation was more favoured during an acceleration phase of the growth rate.     

Complete degradation of carbohydrate to carbon dioxide and methane by syntrophic cultures of Acetobacterium woodii and Methanosarcina barkeri

Methanosarcina barkeri (strain MS) grew and converted acetate to CO₂ and methane after an adaption period of 20 days. Growth and metabolism were rapid with gas production being comparable to that of cells grown on H₂ and CO₂. After an intermediary growth cycle under a H₂ and CO₂ atmosphere acetateadapted cells were capable of growth on acetate with formation of methane and CO₂. When acetate-adapted Methanosarcina barkeri was co-cultered with Acetobacterium woodii on fructose or glucose as substrate, a complete conversion of the carbohydrate to gases (CO₂ and CH₄) was observed.Abbreviation CMC carboxymethyl cellulose     

Efficient mediator-independent hydrogenation of carbon-carbon double bonds,using the obligate anaerobe,Acetobacterium woodii,with fructose as an electron donor

Fructose and H₂ were compared as electron donors for hydrogenation of carbon-carbon double bonds using Acetobacterium woodii. Caffeate was used as a model substrate. An electron donor was required and both fructose and H₂ were suitable. With fructose as the donor, the K _s for caffeate was 0.5 mM and the V _max was 678 mmol kg_dry weight ⁻¹ h⁻¹.␣Fructose oxidation was coupled very efficiently to caffeate reduction by an alteration in the fructose fermentation so that acetate was no longer produced. Received: 24 June 1996 / Accepted: 1 July 1996     

Improvement of the long-term survival of Acetobacterium woodii by limiting acetate production

Summary The viable count in fructose-grown cultures of Acetobacterium woodii fell to undetectable levels 186 h after the stationary phase was reached. The loss of viability was due to accumulation of undissociated acetic acid, since loss of viability could be induced artificially in cultures grown at pH 6.8 by adjusting the pH to 5.0 or by adding high concentrations of acetate. Survival was greatly improved when cultures were grown at low initial fructose concentrations ( 2 g/l) to limit acetate production, since viable cells could still be detected after 73d.     

Differential effects of sodium ions on motility in the homoacetogenic bacteriaAcetobacterium woodii andSporomusa sphaeroides

The strictly anaerobic homoacetogenic bacteria Acetobacterium woodii and Sporomusa sphaeroides differ with respect to their energy metabolism. Since growth as well as acetate and ATP formation of A. woodii is strictly dependent on Na⁺, but that of S. sphaeroides is not, the question arose whether these organisms also use different coupling ions for mechanical work, i.e. flagellar rotation. During growth on fructose in the presence of Na⁺ (50 mM), cells of A. woodii were vigorously motile, as judged by light microscopy. At low Na⁺ concentrations (0.3 mM), the growth rate decreased by only 15%, but the cells were completely non-motile. Addition of Na⁺ to such cultures restored motility instantaneously. Motility, as determined in swarm agar tubes, was strictly dependent on Na⁺; Li⁺, but not K⁺ partly substituted for Na⁺. Of the amilorides tested, phenamil proved to be a specific inhibitor of the flagellar motor of A. woodii. Growth and motility of S. sphaeroides was neither dependent on Na⁺ nor inhibited by amiloride derivatives. These results indicate that flagellar rotation is driven by Δμ_Na ⁺ in A. woodii, but by Δμ_H ⁺ in S. sphaeroides. Received: 30 May 1995 / Accepted: 31 August 1995     

Reaction engineering analysis of hydrogenotrophic production of acetic acid by Acetobacterium woodii

Great interest has emerged in biological CO₂‐fixing processes in the context of current climate change discussions. One example for such a process is the hydrogenotrophic production of acetic acid by anaerobic microorganisms. Acetogenic microorganisms make use of carbon dioxide in the presence of hydrogen to produce acetic acid and biomass. In order to establish a process for the hydrogenotrophic production of acetic acid, the formation of acetate by Acetobacterium woodii was studied in a batch‐operated stirred‐tank bioreactor at different hydrogen partial pressures (pH₂) in the gas phase. The volumetric productivity of the batch processes increased with increasing hydrogen partial pressure. A maximum of the volumetric productivity of 7.4 g_acetate L⁻¹ day⁻¹ was measured at a pH₂ of 1,700 mbar. At this pH₂ a final acetate concentration of 44 g L⁻¹ was measured after a process time of 11 days, if the pH was controlled at pH 7.0 (average cell density of 1.1 g L⁻¹ cell dry weight). The maximum cell specific actetate productivity was 6.9 g_acetate g day⁻¹ under hydrogenotrophic conditions. Biotechnol. Bioeng. 2011;108: 470–474. © 2010 Wiley Periodicals, Inc.     

Selection of sulphur sources for the growth of Butyribacterium methylotrophicum and Acetobacterium woodii

Sulphide and cysteine inhibited growth of batch cultures of Butyribacterium methylotrophicum at moderate concentrations (above 0.5 mM) during growth on glucose (10 mM). The ability of several sulphur sources to replace sulphide was tested in cultures of B. methylotrophicum or Acetobacterium woodii. With sulphite (1 mM), thiosulphate (0.5 mM), elemental sulphur, and dithionite (1 mM), but not sulphate (1 mM), cultures of both organisms grew and produced some sulphide. With elemental sulphur as the sulphur source, toxic levels of sulphide accumulated. Optimal levels for the cultivation of B. methylotrophicum with sulphite were 0.5–2.0 mM, but at higher concentrations the growth rate decreased rapidly, while with dithionite up to 4.0 mM the growth rate was relatively unaffected. In chemostat cultures of B. methylotrophicum with dithionite (1 mM) as the sulphur source and glucose as the limiting substrate, dilution rates up to 0.40 h^–1 were obtained. Thiosulphate could only be used in batch cultures in combination with the reductant titanium(III)nitriloacetate, but in continuous cultures the addition of the reductant to the reservoir was not necessary, because once growth had started enough sulphide was produced to keep the fermentor reduced. The maximum growth rate of B. methylotrophicum with thiosulphate in batch and continuous culture was 0.26 h^–1. Both thiosulphate and dithionite are more convenient sulphur sources than sulphide, but dithionite is more versatile because of its reductive properties and the faster growth it allows.Offprint requests to: T. A. Hansen     

The effect of carbon dioxide on the growth kinetics of fructose-limited chemostat cultures of Acetobacterium woodii DSM 1030

The growth of the anaerobic acetogenic bacterium Acetobacterium woodii DSM 1030 was investigated in fructose-limited chemostat cultures. A defined medium was developed which contained fructose, mineral salts, cysteine · HCl and Ca pantothenate (1 mg · 1^–1) supplied in a vitamin supplement. Growth at high dilution rates was dependent on the presence of CO₂ in the gas phase. The ^max was found to be 0.16 h^–1 and the fructose maintenance requirement was 0.1 to 0.13 mmol fructose · (g dry wt)^–1 · h^–1. A growth yield of 61 g dry wt · (mol fructose)^–1, corrected for the cell maintenance requirement and for incorporation of fructose carbon into cell biomass, was determined from the fructose consumption. A corresponding growth yield of 69 g dry wt · (mol fructose)^–1 was calculated from the acetate production assuming that fructose fermentation was homoacetogenic. A Y_ATP of 12.2 to 13.8 g dry wt · (mol ATP)^–1 was calculated from these growth yields using a value of 5 mol ATP · (mol fructose)^–1 as an estimate of the amount of ATP synthesised from fructose fermentation. The addition of yeast extract (0.5 g · 1^–1) to the medium did not influence the ^max or cell yield. After prolonged growth under fructose-limited conditions the requirement of the culture for CO₂ in the gas phase was reduced.Abbreviations YE yeast extract - IC inorganic carbon - D fermenter dilution rate : h^–1 - M_X maintenance requirement for X: mmol X · (g dry wt)^–1 · h^–1 - X may be fructose (Fruct), fructose consumed in energy metabolism (Fruct [E]), acetate (Ac) - ATP CO₂, NH _inf4 ^sup+ or Pi - qX specific rate of utilisation or consumption of X: mmol X · (g dry wt)^–1 · h^–1 - V fermenter volume: litre - rC · Cell, fermenter cell carbon production: mmol C · h^–1 - Y_X yield of cells on X: g dry wt · (mol X)^–1 - Y _infx ^supmax the yield corrected for cell maintenance: g dry wt · (mol X)^–1 - S_ATP stoichiometry of ATP synthesis from fructose: mol ATP · (mol frucose)^–1 - x cell concentration: g dry wt · 1^–1 - specific growth rate : h^–1 - ^max maximum specific growth rate: h^–1     

Fermentation of triacetin and glycerol by Acetobacterium sp. No energy is conserved by acetate excretion

Two strains of homoacetogenic bacteria similar to Acetobacterium carbinolicum were enriched and isolated from freshwater and marine sediment samples with triacetin (glycerol triacetylester) as sole carbon and energy source. Also the type strains of A. carbinolicum and A. woodii were found to be able to grow with triacetin, and to convert it nearly exclusively to acetate. The triacetin-hydrolyzing enzyme was inducible, and was localized in the cytoplasmic fraction of both species at an activity of 0.21–0.26 U mg protein^-1. During fermentation of glycerol, varying amounts of 1,3-propranediol were produced which could be kept at a minimum in a glycerol-limited chemostat. Growth yields in batch and continuous culture experiments varied between 9.2 and 10.9 g mol glycerol^-1 and 6.5 and 7.6 g mol triacetin^-1 with five strains of homoacetogenic bacteria tested. These results indicate that excretion of acetate across the cytoplasmic membrane does not contribute to the energy conservation budget of these homoacetogenic bacteria.Dedicated to Prof. Dr. Holger W. Jannasch on occasion of his 60th birthday     

Growth yield increase linked to caffeate reduction in Acetobacterium woodii

Growth yields were determined with Acetobacterium woodii strain NZva 16 on hydrogen and CO₂, formate, methanol, vanillate, ferulate and fructose in mineral medium in the absence and presence of 0.05% yeast extract. Yeast extract was not essential for growth but enhanced growth yields by 25–100% depending on the substrate fermented. Comparison of yields on formate or methanol allowed calculation of an energy yield in the range of 1.5–2 mol ATP per mol acetate formed during homoacetate fermentation of A. woodii. In the presence of 6 mM caffeate, growth yields were determined with the substrates formate or methanol. Caffeate was reduced to hydrocaffeate and increased growth yields were obtained. An ATP yield of about 1 mol per mol of caffeate reduced was calculated. Cytochromes were not detectable in cell free extracts or membrane preparations.     

2,3-Butanediol Metabolism in the Acetogen Acetobacterium woodii

The acetogenic bacterium Acetobacterium woodii is able to reduce CO₂ to acetate via the Wood-Ljungdahl pathway. Only recently we demonstrated that degradation of 1,2-propanediol by A. woodii was not dependent on acetogenesis, but that it is disproportionated to propanol and propionate. Here, we analyzed the metabolism of A. woodii on another diol, 2,3-butanediol. Experiments with growing and resting cells, metabolite analysis and enzymatic measurements revealed that 2,3-butanediol is oxidized in an NAD⁺-dependent manner to acetate via the intermediates acetoin, acetaldehyde, and acetyl coenzyme A. Ethanol was not detected as an end product, either in growing cultures or in cell suspensions. Apparently, all reducing equivalents originating from the oxidation of 2,3-butanediol were funneled into the Wood-Ljungdahl pathway to reduce CO₂ to another acetate. Thus, the metabolism of 2,3-butanediol requires the Wood-Ljungdahl pathway.     

Non-Growth-Associated Demethylation of Dimethylsulfoniopropionate by (Homo)acetogenic Bacteria

The demethylation of the algal osmolyte dimethylsulfoniopropionate (DMSP) to methylthiopropionate (MTPA) by (homo)acetogenic bacteria was studied. Five Eubacterium limosum strains (including the type strain), Sporomusa ovata DSM 2662^T, Sporomusa sphaeroides DSM 2875^T, and Acetobacterium woodii DSM 1030^T were shown to demethylate DMSP stoichiometrically to MTPA. The (homo)acetogenic fermentation based on this demethylation did not result in any significant increase in biomass. The analogous demethylation of glycine betaine to dimethylglycine does support growth of acetogens. In batch cultures of E. limosum PM31 DMSP and glycine betaine were demethylated simultaneously. In mixed substrates experiments with fructose-DMSP or methanol-DMSP, DMSP was used rapidly but only after exhaustion of the fructose or the methanol. In steady-state fructose-limited chemostat cultures (at a dilution rate of 0.03 h⁻¹) with DMSP as a second reservoir substrate, DMSP was biotransformed to MTPA but this did not result in higher biomass values than in cultures without DMSP; cells from such cultures demethylated DMSP at rates of approximately 50 nmol min⁻¹ mg of protein⁻¹, both after growth in the presence of DMSP and after growth in its absence. In cell extracts of glycine betaine-grown strain PM31, DMSP demethylation activities of 21 to 24 nmol min⁻¹ mg of protein⁻¹ were detected with tetrahydrofolate as a methyl acceptor; the activities seen with glycine betaine were approximately 10-fold lower. A speculative explanation for the demethylation of DMSP without an obvious benefit for the organism is that the DMSP-demethylating activity is catalyzed by the glycine betaine-demethylating enzyme and that a transport-related factor, in particular a higher energy demand for DMSP transport across the cytoplasmic membrane than for glycine betaine transport, may reduce the overall ATP yield of the fermentation to virtually zero.     

Microbial acetate conversion to methane: kinetics,yields and pathways in a two-step digestion process

Summary Organic waste is converted in a two-stage process to methane and carbon dioxide by mixed cultures of microorganisms. Acetate, a product of acidogenic and acetogenic bacteria and the main substrate for methanogenic bacteria, is an important intermediate of the anaerobic degradation process, which results in the generation of methane. It was shown by labelling experiments using (U-¹⁴C) acetate that as much as 65%–96% of the total methane produced came from the acetate. The first order utilization rate for acetate in the methanogenic stages of a two-stage digestion process was between 0.17 h^-1 and 0.5 h^-1. The kinetics as well as the mass flow and yields of acetate and the methyl group of acetate were determined by pulse-labelling experiments with (U-¹⁴C) acetate and (2-¹⁴C) acetate without a significant rise of the total concentrations. Up to 58% of the acetate carbon was transformed to methane, and about 30% to carbon dioxide; only 4%–15% was incorporated into the biomass. There are at least two parallel degradation mechanisms in the metabolic transformation of acetate to methane: acetate is cleaved either to form methane and carbon dioxide or to form hydrogen and carbon dioxide, which can be transformed by an additional reaction to methane. Labelling experiments with (2-¹⁴C) acetate show that both mechanisms took place at similar order.     

Effect of molecular hydrogen and carbon dioxide on chemo-organotrophic growth of Acetobacterium woodii and Clostridium aceticum

During growth of Acetobacterium woodii on fructose, glucose or lactate in a medium containing less than 0.04% bicarbonate, molecular hydrogen was evolved up to 0.1 mol per mol of substrate. Under an H₂-atmosphere growth of A. woodii with organic substrates was completely inhibited whereas under an H₂/CO₂-atmosphere rapid growth occurred. Under these conditions H₂+CO₂ and the organic substrate were utilized simultaneously indicating that A. woodii was able to grow mixotrophically. Clostridium aceticum differed from A. woodii in that H₂ was only evolved in the stationary phase, that the inhibition by H₂ was observed at pH 8.5 but not at pH 7.5, anf that in the presence of fructose and H₂+CO₂ only fructose was utilized.The hydrogenase activity of fructose-grown cells of C. aceticum amounted to only 12% of that of H₂+CO₂-grown cells. With A. woodii a corresponding decrease of the activity of this enzyme was not observed.     

Kinetic analysis of ethanol production by an acetate-resistant strain of recombinant Zymomonas mobilis

Zymomonas mobilis ZM4/Ac^R (pZB5), a mutant recombinant strain with increased acetate resistance, has been isolated following electroporation of Z. mobilis ZM4/Ac^R. This mutant strain showed enhanced kinetic characteristics in the presence of 12 g sodium acetate l^–1 at pH 5 in batch culture on 40 g glucose, 40 g xylose l^–1 medium when compared to ZM4 (pZB5). In continuous culture, there was evidence of increased maintenance energy requirements/uncoupling of metabolism for ZM4/Ac^R (pZB5) in the presence of sodium acetate; a result confirmed by analysis of the effect of acetate on other strains of Z. mobilis. Nomenclature m Cell maintenance energy coefficient (g g^–1 h^–1)Maximum overall specific growth rate (1 h^–1)Maximum specific ethanol production rate (g g^–1 h^–1)Maximum specific total sugar utilization rate (g g^–1 h^–1)Biomass yield per mole of ATP (g mole^–1 Ethanol yield on total sugars (g g^–1)Biomass yield on total sugars (g g^–1)True biomass yield on total sugars (g g^–1)