Methanogenesis from Sucrose by Defined Immobilized Consortia

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.     

Formate utilization by members of the genus Methanobacterium

Washed cell suspensions of Methanobacterium formicicum MF, Methanobacterium bryantii M.o.H.G. and Methanobacterium strain FR-2 but not Methanobacterium bryantii M.o.H., were shown to produce hydrogen and methane from formate. Levels of dissolved gases (H₂ and CH₄) were continuously and simultaneously monitored within a closed reaction vessel using membrane inlet mass spectrometry. Growth on formate (0–50mM), measured by methane production and increase in absorbance, was observed for both M. formicicum MF and M. bryantii M.o.H.G. but not with Methanobacterium strain FR-2 or M. bryantii M.o.H.     

Production of methane and hydrogen by anaerobic ciliates containing symbiotic methanogens

Rates of methane production by three anaerobic ciliates containing symbiotic methanogens (the marine Metopus contortus and Plagiopyla frontata, and the limnic Metopus palaeformis) were quantified. Hydrogen production by normal (containing active symbionts), aposymbiotic and BES-treated cells was also measured in the case of the marine species. Methanogenesis was closely coupled to host metabolism and growth; at maximum ciliate growth rates (20°C) each methanogen produced about 1 fmol CH₄ per hour corresponding to about 7, 4 and 0.35 pmol per ciliate per hour for M. contortus, P. frontata and M. palaeformis, respectively. Normal cells produced traces of H₂. Hydrogen production by BES-treated or aposymbiotic cells accounted for 75 and 45% of the methane production of normal M. contortus and P. frontata cells, respectively. However, it is possible that hydrogen production was partly inhibited in the absence of methanogens. Theoretical considerations suggest that hydrogen transfer is significant to the metabolism of larger anaerobic ciliates. Ciliates with methanogens produced CH₄ under microaerobic conditions due to their ability to maintain an anoxic intracellular environment at low external oxygen tensions. Methanogenesis was still detectable at a pO₂ of 0.63 kPa (3 %atm sat).     

Anaerobic bioconversion of cellulose by Ruminococcus albus, Methanobrevibacter smithii, and Methanosarcina barkeri

A system is described that combines the fermentation of cellulose to acetate, CH₄, and CO₂ by Ruminococcus albus and Methanobrevibacter smithii with the fermentation of acetate to CH₄ and CO₂ by Methanosarcina barkeri to convert cellulose to CH₄ and CO₂. A cellulose-containing medium was pumped into a co-culture of the cellulolytic R. albus and the H₂-using methanogen, Mb. smithii. The effluent was fed into a holding reservoir, adjusted to pH 4.5, and then pumped into a culture of Ms. barkeri maintained at constant volume by pumping out culture contents. Fermentation of 1% cellulose to CH₄ and CO₂ was accomplished during 132 days of operation with retention times (RTs) of the Ms. barkeri culture of 7.5–3.8 days. Rates of acetate utilization were 9.5–17.3 mmol l⁻¹ day⁻¹ and increased with decreasing RT. The K _s for acetate utilization was 6–8 mM. The two-stage system can be used as a model system for studying biological and physical parameters that influence the bioconversion process. Our results suggest that manipulating the different phases of cellulose fermentation separately can effectively balance the pH and ionic requirements of the acid-producing phase with the acid-using phase of the overall fermentation. Received: 7 December 1999 / Received revision: 28 April 2000 / Accepted: 19 May 2000     

Degradation of Cinnamate via β-Oxidation to Benzoate by a Defined,Syntrophic Consortium of Anaerobic Bacteria

A syntrophic consortium was enriched in a basal medium containing cinnamate as the carbon and energy source. It was found to consist of three morphologically distinct microbes, viz., a short, rod-shaped, non-motile bacterium with distinctly pointed ends, Papillibacter cinnamivorans; a rod-shaped, motile bacterium with rounded ends, Syntrophus sp.; and a methanoarchaeon, Methanobacterium sp. This methanogen was then replaced by a collection strain of Methanobacterium formicicum. A syntrophic interdependency of the three partners of the consortium was observed during growth on cinnamate. In the presence of bromoethanesulfonic acid (BESA), cinnamate was transformed to benzoate, whereas under methanogenic conditions without BESA, cinnamate was first transformed to benzoate via β-oxidation and subsequently completely degraded into acetate, CH₄, and CO₂. Papillibacter cinnamivorans was responsible for benzoate production from cinnamate, whereas a syntrophic association between Syntrophus sp. and the methanogen degraded benzoate to acetate, CH₄, and CO₂. A new anaerobic degradation pathway of cinnamate into benzoate via β-oxidation by a pure culture of P. cinnamivorans is proposed. Received: 27 December 2001 / Accepted: 28 March 2002     

Pyruvate oxidation by Methanococcus spp.

In the absence of H₂, Methanococcus spp. utilized pyruvate as an electron donor for methanogenesis. For Methanococcus voltae A3, Methanococcus maripaludis JJ1, and Methanococcus vannielii, typical rates of pyruvate-dependent methanogenesis were 3.4, 2.8, and 3.9 nmol min^-1 mg^-1 cell dry wt, respectively. These rates were 1–4% of the rates of H₂-dependent methanogenesis. For M. voltae, the concentration of pyruvate required for one-half the maximum rate of methanogenesis was 7 mM, and pyruvate-dependent methanogenesis was linear for 3 days. Radiolabeled acetate was formed from [3-¹⁴C]pyruvate, and the stoichiometry of pyruvate consumed per acetate produced was 1.12±0.27. The stoichiometry of pyruvate consumed per CH₄ produced was 3.64±0.34. These values are close to the expected values of 1 acetate and 4 CH₄. Although 10–30% of total cell carbon could be obtained from exogenous pyruvate during growth with H₂, pyruvate did not replace the nutritional requirement for acetate in Methanococcus voltae A3 or two acetate auxotrophs of Methanococcus maripaludis, JJ6 and JJ7. These results suggest that pyruvate was not oxidized in the presence of H₂. The inability to oxidize pyruvate during H₂-dependent methanogenesis would prevent a futile cycle of pyruvate oxidation and biosynthesis during autotrophic growth.     

A semi-continuous culture system for production of cellulolytic and xylanolytic enzymes by the anaerobic fungus Piromyces sp. strain E2

Summary A system was developed for the semi-continuous cultivation of an anaerobic fungus, Piromyces sp. strain E2 (isolated from an Indian elephant), on Avicel (microcrystalline cellulose). The fungus was grown in a semi-continuous culture system: solids and fungal biomass was retained by means of a simple filter construction whereas the culture fluid was removed continuously. The production of fermentation products (acetate, ethanol, formate, lactate, hydrogen or methane), cellulolytic and xylanolytic enzymes, and protein by the fungus in monoculture or co-culture with Methanobacterium formicicum during growth on Avicel was monitored up to 45 days. These productions stabilized after an adaptation period of 24 and 30 days in the semi-continuous co-culture and monoculture, respectively. After this period the average (±SD) avicelase, -glucosidase, endoglucanase, and xylanase production in the semi-continuous monoculture were 27±6, 140±16, 1057±120 and 5012±583 IU.l^–1.dya^–1, respectively. Co-culture with the methanogen caused a shift in fermentation products to more acetate, and less ethanol and lactate. Furthermore, the production of all cellulolytic enzymes increased (40%) and xylanolytic enzyme production decreased (35%).Correspondence to: H. J. M. Op den Camp     

Cellulose fermentation by continuous cultures of Ruminococcus albus and Methanobrevibacter smithii

Summary The hydrolysis and fermentation of cellulose (Avicel) by continuous cultures of Ruminococcus albus strain 7 and Methanobrevibacter smithii strain PS were studied. Cellulose destruction ranged from ca. 22% to 71% for 0.25 to 2.27 days solids retention time, respectively. The cellulose hydrolysis rate constant (k) was 1.3 days^–1. Concentrations of soluble reducing sugars were low, showing that cellulose hydrolysis was the rate-limiting step of cellulose fermentation. The estimated methane-based molar growth yield for M. smithii was 2.8 g mol^–1. Its maximum specific growth rate was ca. 4 days^–1. The dissolved H₂ half-saturation constant (K _s ) for methanogenesis was ca. 1 M. The final products of the co-culture were primarily acetate, CH₄ and CO₂ and low levels of ethanol and H₂. The co-culture produced more H₂ (used for reduction of CO₂ to CH₄) and acetate than a monoculture of R. albus. These differences coulb be accounted for by the lower production of ethanol, confirming to the theory of interspecies H₂ transfer. Offprint requests to: M. J. Wolin     

Biomethanation of a cellulose-based substrate in the presence and absence of a cellulolytic bacterium

The effect of co-culturing a methanogen isolated from a paper mill waste (PMW) with cellulolytic bacteria isolated from the intestinal fluids of the silver cricket (Lepisma saccharina) on the biomethanation of filter paper strips was examined. The autoclaved filter paper strips were subjected to biomethanation in AC 21 medium inoculated with methanogen PMW in the presence and in absence of a co-culture of cellulolytic bacteria. In spite of poor initial response, methane production in the presence of the cellulolytic co-culture were found to increase gradually upto 25 days, after which a reduction in methane production was observed. Analysis of the results in terms of increased cellulose degradation in the presence of cellulolytic bacteria has been made. This revised version was published online in July 2006 with corrections to the Cover Date.     

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     

Effect of coculture of anaerobic fungi isolated from ruminants and non-ruminants with methanogenic bacteria on cellulolytic and xylanolytic enzyme activities

Neocallimastix strain N1, an isolate from a ruminant (sheep), was cocultured with three Methanobacterium formicicum strains, Methanosarcina barkeri, and Methanobrevibacter smithii. The coculture with Methanobacterium formicicum strains resulted in the highest production of cellulolytic and xylanolytic enzymes. Subsequently four anaerobic fungi, two Neocallimastix strains (N1 and N2) from a ruminant and two Piromyces species from non-ruminants (E2 and R1), were grown in coculture with Methanobacterium formicicum DSM 3637 on filter paper cellulose and monitored over a 7-day period for substrate utilisation, fermentation products, and secretion of cellulolytic and xylanolytic enzymes. Methanogens caused a shift in fermentation products to more acetate and less ethanol, lactate and succinate. Furthermore the cellulose digestion rate increased by coculture. For cocultures of Neoallimastix strains with Methanobacterium formicicum strains the cellulolytic and xylanolytic enzyme production increased. Avicelase, CMCase and xylanase were almost completely secreted into the medium, while 40–60% of the -glucosidase was found to be cell bound. Coculture had no significant effect on the location of cellulolytic and xylanolytic enzymes.     

Ethanol and acetate production by <Emphasis Type="Italic">Clostridium ljungdahlii</Emphasis> and <Emphasis Type="Italic">Clostridium autoethanogenum</Emphasis> using resting cells

Combined gasification and fermentation technologies can potentially produce biofuels from renewable biomass. Gasification generates synthesis gas consisting primarily of CO, CO₂, H₂, N₂, with smaller amounts of CH₄, NO_x, O₂, C₂ compounds, ash and tars. Several anaerobic bacteria species can ferment bottled mixtures of pure synthesis gas constituents. However, there are challenges to maintaining culture viability of synthesis gas exposed cells. This study was designed to enhance culture stability and improve ethanol-to-acetate ratios using resting (non-growing) cells in synthesis gas fermentation. Resting cell states were induced in autotrophic Clostridium ljungdahlii cultures with minimal ethanol and acetate production due to low metabolic activity compared to growing cell production levels of 5.2 and 40.1 mM of ethanol and acetate. Clostridium autoethanogenum cultures were not induced into true resting states but did show improvement in total ethanol production (from 5.1 mM in growing cultures to 9.4 in one nitrogen-limited medium) as well as increased shifts in ethanol-to-acetate production ratios.     

Long-term adaptation of <Emphasis Type="Italic">Escherichia coli</Emphasis> to methanogenic co-culture enhanced succinate production from crude glycerol

Escherichia coli can hardly grow anaerobically on glycerol without exogenous electron acceptor. The formate-consuming methanogen Methanobacterium formicicum plays a role as a living electron acceptor in glycerol fermentation of E. coli. Wild-type and mutant E. coli strains were screened for succinate production using glycerol in a co-culture with M. formicicum. Subsequently, E. coli was adapted to glycerol fermentation over 39 rounds (273 days) by successive co-culture with M. formicicum. The adapted E. coli (19.9 mM) produced twice as much succinate as non-adapted E. coli (9.7 mM) and 62% more methane. This study demonstrated improved succinate production from waste glycerol using an adapted wild-type strain of E. coli with wild-type M. formicicum, which is more useful than genetically modified strains. Crude glycerol, an economical feedstock, was used for the cultivation. Furthermore, the increase in methane production by M. formicicum during co-culture with adapted E. coli illustrated the possibility of energy-saving effects for the fermentation process.     

Activity and Viability of Methanogens in Anaerobic Digestion of Unsaturated and Saturated Long-Chain Fatty Acids

Lipids can be anaerobically digested to methane, but methanogens are often considered to be highly sensitive to the long-chain fatty acids (LCFA) deriving from lipids hydrolysis. In this study, the effect of unsaturated (oleate [C_18:1]) and saturated (stearate [C_18:0] and palmitate [C_16:0]) LCFA toward methanogenic archaea was studied in batch enrichments and in pure cultures. Overall, oleate had a more stringent effect on methanogens than saturated LCFA, and the degree of tolerance to LCFA was different among distinct species of methanogens. Methanobacterium formicicum was able to grow in both oleate- and palmitate-degrading enrichments (OM and PM cultures, respectively), whereas Methanospirillum hungatei only survived in a PM culture. The two acetoclastic methanogens tested, Methanosarcina mazei and Methanosaeta concilii, could be detected in both enrichment cultures, with better survival in PM cultures than in OM cultures. Viability tests using live/dead staining further confirmed that exponential growth-phase cultures of M. hungatei are more sensitive to oleate than are M. formicicum cultures; exposure to 0.5 mM oleate damaged 99% ± 1% of the cell membranes of M. hungatei and 53% ± 10% of the cell membranes of M. formicicum. In terms of methanogenic activity, M. hungatei was inhibited for 50% by 0.3, 0.4, and 1 mM oleate, stearate, and palmitate, respectively. M. formicicum was more resilient, since 1 mM oleate and >4 mM stearate or palmitate was needed to cause 50% inhibition on methanogenic activity.     

Isolation of propionate degrading bacterium in co-culture with a methanogen from a cattle dung biogas plant

Various anaerobic hydrolytic and methanogenic bacteria active in cattle dung biogas plants are reported in the literature. Anaerobic bacteria with ability to use volatile fatty acids constitute a vital bridge between hydrolytic bacteria and methanogenic bacteria. The present paper describes the isolation ofSyntrophobacter wolinii a propionate degrading bacterium in co-culture with a hydrogen utilizing methanogenviz.,Methanobacterium formicicum from the fermenting slurry of cattle dung biogas plant. Earlier studies on propionate and butyrate degradation indicatedMethanospirillum hungatei as the hydrogen utilizing partner of the co-culture whereas in the present studies this was not the case. Temperature 35° C, pH 7.5 and 20 mM of propionate were found optimal for growth and activity of co-culture.     

Biomethanation of H2 and CO2 by Methanobacterium thermoautotrophicum in membrane and ceramic bioreactors

Direct conversion of gaseous H₂ and CO₂ to CH₄ was achieved with Methanobacterium thermoautotrophicum ΔH (DSM 1053) cells fixed either on a cellulose acetate membrane or inside a porous silica-alumina ceramic support.In a membrane bioreactor with cellulose acetate (5 μmø), methane production rate increased in proportion to the contact area between the gases and the methanogen cells, giving a methane production rate of 0.75 ml CH₄/cm² contact area/h. The initial fixed-cell mass of 0.2 mg dry cell/cm² of contact area increased to 1 mg/cm² after 12 h of cultivation (steady state).In the ceramic bioreactor (cylindrical, 30 mmø × 70; av. pore size 100 μ, and porosity 79.7%), the methane production rate at steady state was 6 l CH₄/l ceramic/l. The methanogen cells grew homogeneously inside the ceramic up to 7 cm depth, and the cell density ranged from 20 to 30 mg dry cell/cm³ ceramic.     

Carbonic anhydrase activity in acetate grown Methanosarcina barkeri

Cell extracts (27000xg supernatant) of acetate grown Methanosarcina barkeri were found to have carbonic anhydrase activity (0.41 U/mg protein), which was lost upon heating or incubation with proteinase K. The activity was inhibited by Diamox (apparent K _i=0.5 mM), by azide (apparent K _i=1 mM), and by cyanide (apparent K _i=0.02 mM). These and other properties indicate that the archaebacterium contains the enzyme carbonic anhydrase (EC 4.2.1.1). Evidence is presented that the protein is probably located in the cytoplasm. Methanol or H₂/CO₂ grown cells of M. barkeri showed no or only very little carbonic anhydrase activity. After transfer of these cells to acetate medium the activity was induced suggesting a function of this enzyme in acetate fermentation to CO₂ and CH₄. Interestingly, Desulfobacter postgatei and Desulfotomaculum acetoxidans, which oxidize acetate to 2 CO₂ with sulfate as electron acceptor, were also found to exhibit carbonic anhydrase activity (0.2 U/mg protein).     

Anaerobic Degradation of Lactate by Syntrophic Associations of Methanosarcina barkeri and Desulfovibrio Species and Effect of H2 on Acetate Degradation

When grown in the absence of added sulfate, cocultures of Desulfovibrio desulfuricans or Desulfovibrio vulgaris with Methanobrevibacter smithii (Methanobacterium ruminantium), which uses H₂ and CO₂ for methanogenesis, degraded lactate, with the production of acetate and CH₄. When D. desulfuricans or D. vulgaris was grown in the absence of added sulfate in coculture with Methanosarcina barkeri (type strain), which uses both H₂-CO₂ and acetate for methanogenesis, lactate was stoichiometrically degraded to CH₄ and presumably to CO₂. During the first 12 days of incubation of the D. desulfuricans-M. barkeri coculture, lactate was completely degraded, with almost stoichiometric production of acetate and CH₄. Later, acetate was degraded to CH₄ and presumably to CO₂. In experiments in which 20 mM acetate and 0 to 20 mM lactate were added to D. desulfuricans-M. barkeri cocultures, no detectable degradation of acetate occurred until the lactate was catabolized. The ultimate rate of acetate utilization for methanogenesis was greater for those cocultures receiving the highest levels of lactate. A small amount of H₂ was detected in cocultures which contained D. desulfuricans and M. barkeri until after all lactate was degraded. The addition of H₂, but not of lactate, to the growth medium inhibited acetate degradation by pure cultures of M. barkeri. Pure cultures of M. barkeri produced CH₄ from acetate at a rate equivalent to that observed for cocultures containing M. barkeri. Inocula of M. barkeri grown with H₂-CO₂ as the methanogenic substrate produced CH₄ from acetate at a rate equivalent to that observed for acetate-grown inocula when grown in a rumen fluid-vitamin-based medium but not when grown in a yeast extract-based medium. The results suggest that H₂ produced by the Desulfovibrio species during growth with lactate inhibited acetate degradation by M. barkeri.     

Effect of trimethylamine on acetate utilization by Methanosarcina barkeri

During growth of Methanosarcina barkeri strain Fusaro on a mixture of trimethylamine and acetate, methane production and acetate consumption were biphasic. In the first phase trimethylamine (33 mmol x l^-1) was depleted and some acetate (11–14 from 50 mmol x l^-1) was metabolized simultaneously. In the second phase the remaining acetate was cleaved stoichiometrically into CH₄ and CO₂. Kinetic experiments with (2-¹⁴C)acetate revealed that only 2.5% of the methane produced in the first phase originated from acetate: 18% of the acetate metabolized was cleaved into CH₄ and CO₂, 23% of the acetate was oxidized, and 55% was assimilated. Methane produced from CD₃–COOH in the first phase consisted of CD₂H₂ and CD₃H in a ratio of 1:1.     

Transient Production of Formate During Chemolithotrophic Growth of Anaerobic Microorganisms on Hydrogen

The homoacetogenic bacteria Acetobacterium woodii, A. carbinolicum, Sporomusa ovata, and Eubacterium limosum, the methanogenic archaeon Methanobacterium formicicum, and the sulfate-reducing bacterium Desulfotomaculum orientis all produced formate as an intermediate when they were growing chemolithoautotrophically with H₂ and CO₂ as sources of energy, electrons, and carbon. The sulfate-reducing bacterium Desulfovibrio vulgaris grew chemolithoheterotrophically with H₂ and CO₂ using acetate as carbon source, but also produced formate when growth was limited by sulfate. All these bacteria were also able to grow on formate as energy source. Formate accumulated transiently while H₂ was consumed. The maximum formate concentrations measured in cultures of A. woodii and A. carbinolicum were proportional to the initial H₂ partial pressure, giving a ratio of about 0.5 mM formate per 10 kPa H₂. The methanogen Methanobacterium bryantii, on the other hand, was unable to grow on formate and did not produce formate during chemolithoautotrophic growth on H₂. The results indicate that the ability to utilize formate, that is, to possess a formate dehydrogenase, was the precondition for the production of formate during chemolithotrophic growth on H₂. Received: 24 November 1998 / Accepted: 30 December 1998