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     

Purine and pyrimidine biosynthesis in methanogenic bacteria

Following long-term labeling with [1-¹³C]acetate, [2-¹³C]acetate, ¹³CO₂, H¹³COOH, or ¹³CH₃OH, NMR spectroscopy was used to determine the labeling patterns of the purified ribonucleosides of Methanospirillum hungatei, Methanococcus voltae, Methanobrevibacter smithii, Methanosphaera stadtmanae, Methanosarcina barkeri and Methanobacterium bryantii. Major differences were observed among the methanogens studied, specifically at carbon positions 2 and 8 of the purines, positions at which one-carbon carriers are involved during synthesis. In Methanospirillum hungatei and Methanosarcina barkeri, the labcl at both positions came from carbon atom C-2 of acetate, as predicted from known eubacterial pathways, whereas in Methanococcus voltae and Methanobacterium bryantii both originated from CO₂. In Methanosphaera stadtmanae grown in the presence of formate, the C-2 of purines originated exclusively from formate and the C-8 was labeled by the C-2 of acetate. When grown in media devoid of formate, the C-2 of the purine ring originated mainly from the C-2 of acetate and in part from CH₃OH. In Methanobrevibacter smithii grown in the presence of formate, C-2 and C-8 of purines were derived from CO₂ and/or formate. The labeling patterns obtained for pyrimidines are consistent with the biosynthetic pathways common to eubacteria and eucaryotes.Abbreviations CODH Carbon monoxide dehydrogenase - FH₄ tetrahydrofolate - H₄MPT tetrahydromethanopterin Issued as NRCC Publication No. 37383     

Conversion of Cellulose to Methane and Carbon Dioxide by Triculture of Acetivibrio cellulolyticus, Desulfovibrio sp., and Methanosarcina barkeri

The fermentation of cellulose by monocultures of Acetivibrio cellulolyticus and cocultures of A. cellulolyticus-Methanosarcina barkeri, A. cellulolyticus-Desulfovibrio sp., and A. cellulolyticus-M. barkeri-Desulfovibrio sp. was studied. The monoculture produced ethanol, acetate, H₂, and CO₂. More acetate and less ethanol was formed by the cocultures than by the monoculture. Acetate was utilized by M. barkeri in coculture with A. cellulolyticus after a lag period, whereas ethanol was metabolized by the sulfate reducer only under conditions of low H₂ partial pressure, i.e., when cocultured with A. celluloyticus-M. barkeri or when grown together with the methanogen. Only the three-component culture carried out the rapid conversion of cellulose to CO₂ and methane. Furthermore, this culture hydrolyzed the most cellulose—85% of that initially present. This amount was increased to 90% by increasing the population of M. barkeri in the triculture. Methane production was also increased, and a quicker fermentation rate was achieved.     

Photosynthetic pathway and ontogeny affect water relations and the impact of CO2 on Bouteloua gracilis (C4) and Pascopyrum smithii (C3)

The eastern Colorado shortgrass steppe is dominated by the C₄ grass, Bouteloua gracilis, but contains a mixture of C₃ grasses as well, including Pascopyrum smithii. Although the ecology of this region has been extensively studied, there is little information on how increasing atmospheric CO₂ will affect it. This growth chamber study investigated gas exchange, water relations, growth, and biomass and carbohydrate partitioning in B. gracilis and P. smithii grown under present ambient and elevated CO₂ concentrations of 350 μl l⁻¹and 700 μl l⁻¹, respectively, and two deficit irrigation regimes. The experiment was conducted in soil-packed columns planted to either species over a 2-month period under summer-like conditions and with no fertilizer additions. Our objective was to better understand how these species and the functional groups they represent will respond in future CO₂-enriched environments. Leaf CO₂ assimilation (A _n), transpiration use efficiency (TUE, or A _n/transpiration), plant growth, and whole-plant water use efficiency (WUE, or plant biomass production/water evapotranspired) of both species were greater at elevated CO₂, although responses were more pronounced for P. smithii. Elevated CO₂ enhanced photosynthesis, TUE, and growth in both species through higher soil water content (SWC) and leaf water potentials (Ψ) and stimulation of photosynthesis. Consumptive water use was greater and TUE less for P. smithii than B. gracilis during early growth when soil water was more available. Declining SWC with time was associated with a steadily increased sequestering of total non-structural carbohydrates (TNCs), storage carbohydrates (primarily fructans for P. smithii) and biomass in belowground organs of P. smithii, but not B. gracilis. The root:shoot ratio of P. smithii also increased at elevated CO₂, while the root:shoot ratio of B. gracilis was unresponsive to CO₂. These partitioning responses may be the consequence of different ontogenetic strategies of a cool-season and warm-season grass entering a warm, dry summer period; the cool-season P. smithii responds by sequestering TNCs belowground in preparation for summer dormancy, while resource partitioning of the warm-season B. gracilis remains unaltered. One consequence of greater partitioning of resources into P. smithii belowground organs in the present study was maintenance of higher Ψ and A _n rates. This, along with differences in photosynthetic pathway, may have accounted for the greater responsiveness of P. smithii to CO₂ enrichment compared to B. gracilis. Received: 21 July 1997 / Accepted: 16 December 1997     

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     

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.     

Interactions of culture vessels, media volume, culture density, and carbon dioxide levels on lettuce and spearmint shoot growth in vitro

 The influence of culture chamber capacity, medium volume and culture density on the growth yields of lettuce (Lactuca sativa L.) and spearmint (Mentha spicata L.) shoots were determined in an environment containing either 350 or 10,000 μmol mol^–1 CO₂ after 8 weeks of incubation. High positive correlations occurred between the culture vessel capacity and spearmint fresh weight, leaf number, root number, and shoot number. Similarly, high positive correlations occurred between culture vessel capacity and lettuce fresh weight, leaf number, and root number. Higher fresh weights, leaf numbers, and root numbers were obtained from lettuce and spearmint shoots when cultured in 1-quart Mason jars containing 100- or 150-ml aliquots of medium compared to jars containing 25- or 50-ml aliquots of medium within an environment containing either 350 or 10,000 μmol mol^–1 CO₂. High culture density decreased growth yields, and this phenomenon could only be slightly off-set by the employment of an elevated CO₂ environment or larger culture vessels. Received: 22 December 1998 / Revision received: 2 July 1999 / Accepted: 12 July 1999     

Outdoor culture of a cyanobacterium with a vertical flat-plate photobioreactor: effects on productivity of the reactor orientation, distance setting between the plates, and culture temperature

The ability of a photobioreactor to fix CO₂ was evaluated with the thermophilic cyanobacterium, Synechocystis aquatilis SI-2. The reactor consisted of three to five flat plates of transparent acrylic plastic standing upright and in parallel and giving a 0.015-m light path. The reactor was 0.8 m high and 1 m long with 9 l working volume. The effects of the orientation of the vertical bioreactor, distance between the plates, and culture temperature on the productivity of biomass were investigated during the summer of 1998 in Kamaishi (39°N, 142°E), Japan. When the illuminated surface reactor was placed in an east–west-facing orientation, the biomass productivity was roughly 1.4-fold higher than that obtained in a north–south-facing orientation, because the former received more solar energy than the latter. The productivity based on the overall land area was the same for plates set either 0.25 m or 0.5 m apart. However, the volumetric productivity of the reactor in which the plates were set 0.25 m apart was lower than that when the plates were set 0.5 m apart, since the former plates received relatively lower solar irradiation because of severe mutual shading. When the culture temperature was maintained in its optimal range (37–43 °C), the productivity was 50% greater than that obtained in a culture at ambient temperature (20–44 °C). The biomass productivity and CO₂ fixation rate were investigated under various experimental conditions. The maximum rate of 53 g CO₂ m⁻² day⁻¹ was achieved in the temperature-regulated culture with the reactor set in an east–west-facing orientation, the distance between plates being 0.25 m. Received: 6 may 1999 / Received revision: 14 June 1999 / Accepted 5 July 1999     

Ultrahigh-cell-density culture of a marine green alga Chlorococcum littorale in a flat-plate photobioreactor

To test the feasibility of CO₂ remediation by microalgal photosynthesis, a modified type of flat-plate photobioreactor [Hu et al. (1996) Biotechnol Bioeng 51:51–60] has been designed for cultivation of a high-CO₂-tolerant unicellular green alga Chlorococcum littorale. The modified reactor has a narrow light path in which intensive turbulent flow is provided by streaming compressed air through perforated tubing into the culture suspension. The length of the reactor light path was optimized for the productivity of biomass. The interrelationship between cell density and productivity, as affected by incident light intensity, was quantitatively assessed. Cellular ultrastructural and biochemical changes in response to ultrahigh cell density were investigated. The potential of biomass production under extremely high CO₂ concentrations was also evaluated. By growing C. littorale cells in this reactor, a CO₂ fixation rate of 16.7 g CO₂ l⁻¹ 24 h⁻¹ (or 200.4 g CO₂ m⁻² 24 h⁻¹) could readily be sustained at a light intensity of 2000 μmol m⁻² s⁻¹ at 25 °C, and an ultrahigh cell density of well over 80 g l⁻¹ could be maintained by daily replacing the culture medium. Received: 20 October 1997 / Received revision: 19 December 1997 / Accepted: 24 January 1998     

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.     

Effects of increased CO2 and N on CH4 efflux from a boreal mire: a growth chamber experiment

Increases in the supply of atmospheric CO₂ and N are expected to alter the carbon cycle, including CH₄ emissions, in boreal peatlands. These effects were studied in a glasshouse experiment with peat monoliths cored from an oligotrophic pine fen. The cores with living plants were kept in 720 ppm_v and 360 ppm_v CO₂ atmospheres for about 6 months under imitated natural temperature cycle. Fertilisation with NH₄NO₃ (3 g m⁻² for 25 weeks) was applied to 18 of the 36 monoliths. The rate of CH₄ flux was non-linearly dependent on the number of Eriophorum vaginatum shoots growing in the monoliths, probably due to the gas transport properties of the aerenchyma. The average CH₄ efflux rate, standardised by the number of shoots, was increased by a maximum of 10–20% in response to the raised CO₂ level. In the raised-NH₄NO₃ treatment, the increase in CH₄ release was lower. The effect of combined CO₂+NH₄NO₃ on CH₄ release was negligible and even lower than in the single treatments. Both potential CH₄ production and oxidation rates at 5, 15 and 25°C were higher near the surface than at the bottom of the core. As expected, the rates clearly depended on the incubation temperature, but the different treatments did not cause any consistent differences in either CH₄ production or oxidation. The determination of potential CH₄ production and oxidation in the laboratory is evidently too crude a method of differentiating substrate-induced differences in CH₄ production and oxidation in vivo. These results indicate that an increase in atmospheric CO₂ or N supply alone, at least in the short term, slightly enhances CH₄ effluxes from boreal peatlands; but together their effect may even be restrictive. Received: 18 June 1998 / Accepted: 25 January 1999     

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     

The geochemistry of methane in Lake Fryxell,an amictic,permanently ice-covered,antarctic lake

The abundance and distribution of dissolved CH₄ were determined from 1987–1990 in Lake Fryxell, Antarctica, an amictic, permanently ice-covered lake in which solute movement is controlled by diffusion. CH₄ concentrations were < 1 υM in the upper oxic waters, but increased below the oxycline to 936 μM at 18 m. Sediment CH₄ was 1100 μmol (1 sed)⁻¹ in the 0–5 cm zone. Upward flux from the sediment was the source of the CH₄, NH₄ ⁺, and DOC in the water column; CH₄ was 27% of the DOC+CH₄ carbon at 18 m. Incubations with surficial sediments indicated that H¹⁴CO₃ ⁻ reduction was 0.4 μmol (1 sed)⁻¹ day⁻¹ or 4× the rate of acetate fermentation to CH₄. There was no measurable CH₄ production in the water column. However, depth profiles of CH₄, NH₄, and DIC normalized to bottom water concentrations demonstrated that a significant CH₄ sink was evident in the anoxic, sulfate-containing zone of the water column (10–18 m). The δ¹³CH₄ in this zone decreased from −72 % at 18 m to −76% at 12 m, indicating that the consumption mechanism did not result in an isotopic enrichment of ¹³CH₄. In contrast, δ¹³CH₄ increased to −55 % at 9 m due to aerobic oxidation, though this was a minor aspect of the CH₄ cycle. The water column CH₄ profile was modeled by coupling diffusive flux with a first order consumption term; the best-fit rate constant for anaerobic CH₄ consumption was 0.012 yr⁻¹. On a total carbon basis, CH₄ consumption in the anoxic water column exerted a major effect on the flux of carbonaceous material from the underlying sediments and serves to exemplify the importance of CH₄ to carbon cycling in Lake Fryxell.     

Activities of formylmethanofuran dehydrogenase,methylenetetrahydromethanopterin dehydrogenase,methylenetetrahydromethanopterin reductase,and heterodisulfide reductase in methanogenic bacteria

The activities of formylmethanofuran dehydrogenase, methylenetetrahydromethanopterin dehydrogenase, methylenetetrahydromethanopterin reductase, and heterodisulfide reductase were tested in cell extracts of 10 different methanogenic bacteria grown on H₂/CO₂ or on other methanogenic substrates. The four activities were found in all the organisms investigated: Methanobacterium thermoautotrophicum,M. wolfei, Methanobrevibacter arboriphilus, Methanosphaera stadtmanae, Methanosarcina barkeri (strains Fusaro and MS), Methanothrix soehngenii, Methanospirillum hungatei, Methanogenium organophilum, and Methanococcus voltae. Cell extracts of H₂/CO₂ grown M. barkeri and of methanol grown M. barkeri showed the same specific activities suggesting that the four enzymes are of equal importance in CO₂ reduction to methane and in methanol disproportionation to CO₂ and CH₄. In contrast, cell extracts of acetate grown M. barkeri exhibited much lower activities of formylmethanofuran dehydrogenase and methylenetetrahydromethanopterin dehydrogenase suggesting that these two enzymes are not involved in methanogenesis from acetate. In M. stadtmanae, which grows on H₂ and methanol, only heterodisulfide reductase was detected in activities sufficient to account for the in vivo methane formation rate. This finding is consistent with the view that the three other oxidoreductases are not required for methanol reduction to methane with H₂.     

Hydrogen formation by an arsenate-reducing Pseudomonas putida, isolated from arsenic-contaminated groundwater in West Bengal, India

Anaerobic growth of a newly isolated Pseudomonas putida strain WB from an arsenic-contaminated soil in West Bengal, India on glucose, l-lactate, and acetate required the presence of arsenate, which was reduced to arsenite. During aerobic growth in the presence of arsenite arsenate was formed. Anaerobic growth of P. putida WB on glucose was made possible presumably by the non-energy-conserving arsenate reductase ArsC with energy derived only from substrate level phosphorylation. Two moles of acetate were generated intermediarily and the reducing equivalents of glycolysis and pyruvate decarboxylation served for arsenate reduction or were released as H₂. Anaerobic growth on acetate and lactate was apparently made possible by arsenate reductase ArrA coupled to respiratory electron chain energy conservation. In the presence of arsenate, both substrates were totally oxidized to CO₂ and H₂ with part of the H₂ serving for respiratory arsenate reduction to deliver energy for growth. The growth yield for anaerobic glucose degradation to acetate was Y _Glucose = 20 g/mol, leading to an energy coefficient of Y _ATP = 10 g/mol adenosine-5'-triphosphate (ATP), if the Emden–Meyerhof–Parnas pathway with generation of 2 mol ATP/mol glucose was used. During growth on lactate and acetate no substrate chain phosphorylation was possible. The energy gain by reduction of arsenate was Y _Arsenate = 6.9 g/mol, which would be little less than one ATP/mol of arsenate.     

Pyruvate — a novel substrate for growth and methane formation in Methanosarcina barkeri

Methanosarcina barkeri strain Fusaro was found to grow on pyruvate as sole carbon and energy source after an incubation period of 10–12 weeks in the presence of high pyruvate concentrations (100 mM). Growth studies, cell suspension experiments and enzymatic investigations were performed with pyruvate-utilizing M. barkeri. For comparison acetate-adapted cells of M. barkeri were analyzed.

1. Pyruvate-utilizing M. barkeri grew on pyruvate (100 mM) with an initial doubling time of about 25 h (37 °C, pH 6.5) up to cell densities of about 0.8 g cell dry weight/l. The specific growth rate was linearily dependent on the pyruvate concentration up to 100 mM indicating that pyruvate was taken up by passive diffusion. Only CO₂ and CH₄ were detected as fermentation products. As calculated from fermentation balances pyruvate was converted to CH₄ and CO₂ according to following equation: Pyruvate^-+H⁺+0.5 H₂O » 1.25 CH₄+1.75 CO₂. The molar growth yield (Ych ₄) was about 14 g dry weight cells/mol CH₄. In contrast the growth yield (Ych ₄) of M. barkeri during growth on acctate (Acetate^-+H⁺ » CH₄+CO₂) was about 3 g/mol CH₄.
2. Cell suspensions of pyruvate-grown M. barkeri catalyzed the conversion of pyruvate to CH₄, CO₂ and H₂ (5–15 nmol pyruvate consumed/min x mg protein). At low cell concentrations (0.5 mg protein/ml) 1 mol pyruvate was converted to 1 mol CH₄, 2 mol CO₂ and 1 mol H₂. At higher cell concentration less H₂ and CO₂ and more CH₄ were formed due to CH₄ formation from H₂/CO₂. The rate of pyruvate conversion was linearily dependent on the pyruvate concentration up to about 30 mM. Cell suspensions of acetate-grown M. barkeri also catalyzed the conversion of 1 mol pyruvate to 1 mol CH₄, 2 mol CO₂ and 1 mol H₂ at similar rates and with similar affinity for pyruvate as pyruvate-grown cells.
3. Cell extracts of both pyruvate-grown and acetate-grown M. barkeri contained pyruvate: ferredoxin oxidoreductase. The specific activity in pyruvate-grown cells (0.8 U/mg) was 8-fold higher than in acetate-grown cells (0.1 U/mg). Coenzyme F₄₂₀ was excluded as primary electron acceptor of pyruvate oxidoreductase. Cell extracts of pyruvate-grown M. barkeri contained carbon monoxide dehydrogenase activity and hydrogenase activity catalyzing the reduction by carbon monoxide and hydrogen of both methylviologen and ferredoxin (from Clostridium).

This is the first report on growth of a methanogen on pyruvate as sole carbon and energy source, i.e. on a substrate more complex than acetate.     

The effect of low temperature (5–29 °C) and adaptation on the methanogenic activity of biomass

The influence of low temperature (5–29 °C) on the methanogenic activity of non-adapted digested sewage sludge and on temperature/leachate-adapted biomass was assayed by using municipal landfill leachate, intermediates of anaerobic degradation (propionate) and methane precursors (acetate, H₂/CO₂) as substrates. The temperature dependence of methanogenic activity could be described by Arrhenius-derived models. However, both substrate and adaptation affected the temperature dependence. The adaptation of biomass in a leachate-fed upflow anaerobic sludge-blanket reactor at approximately 20 °C for 4 months resulted in a sevenfold and fivefold increase of methanogenic activity at 11 °C and 22 °C respectively. Both acetate and H₂/CO₂ were methanized even at 5 °C. At 22 °C, methanogenic activities (acetate 4.8–84 mM) were 1.6–5.2 times higher than those at 11 °C. The half-velocity constant (K _s) of acetate utilization at 11 °C was one-third of that at 22 °C while a similar K _i was obtained at both temperatures. With propionate (1.1–5.5 mM) as substrate, meth‐anogenic activities at 11 °C were half those at 22 °C. Furthermore, the residual concentration of the substrates was not dependent on temperature. The results suggest that the adaptation of biomass enables the achievement of a high treatment capacity in the anaerobic process even under psychrophilic conditions. Received: 23 December 1996 / Received last revision: 18 June 1997 / Accepted: 23 June 1997     

Quantitative evaluation of ruminal methane and carbon dioxide formation from formate through C-13 stable isotope analysis in a batch culture system

Methane produced from formate is one of the important methanogensis pathways in the rumen. However, quantitative information of CH₄ production from formate has been rarely reported. The aim of this study was to characterize the conversion rate (CR) of formic acid into CH₄ and CO₂ by rumen microorganisms. Ground lucerne hay was incubated with buffered ruminal fluid for 6, 12, 24 and 48 h. Before the incubation, ¹³C-labeled H¹³COOH was also supplied into the incubation bottle at a dose of 0, 1.5, 2.2 or 2.9 mg/g of DM substrate. There were no interactions (P>0.05) between dose and incubation time for all variables evaluated. When expressed as an absolute amount (ml in gas sample) or a relative CR (%), both ¹³CH₄ and ¹³CO₂ production quadratically increased (P<0.01) with the addition of H¹³COOH. The total ¹³C (¹³CH₄ and ¹³CO₂) CR was also quadratically increased (P<0.01) when H¹³COOH was added. Moreover, formate addition linearly decreased (P<0.031) the concentrations of NH₃-N, total and individual volatile fatty acids (acetate, propionate and butyrate), and quadratically decreased (P<0.014) the populations of protozoa, total methanogens, Methanosphaera stadtmanae, Methanobrevibacter ruminantium M1, Methanobrevibacter smithii and Methanosarcina barkeri. In summary, formate affects ruminal fermentation and methanogenesis, as well as the rumen microbiome, in particular microorganisms which are directly or indirectly involved in ruminal methanogenesis. This study provides quantitative verification for the rapid dissimilation of formate into CH₄ and CO₂ by rumen microorganisms.     

Rhythmic components of photoperiodic time measurement in the pitcher-plant mosquito, Wyeomyia smithii

Photoperiodic time measurement regulating larval diapause in the pitcher-plant mosquito, Wyeomyia smithii, varies in a close relationship with latitude. The critical photoperiod mediating the maintenance and termination of diapause is positively correlated with latitude (r ² = 0.977) among six populations from southern (30–31° N), intermediate (40° N), and northern (46–49° N) latitudes in North America. The developmental response to unnaturally short and to unnaturally long photoperiods declines with increasing latitude, so that longer critical photoperiods are associated with a downward rather than a lateral shift in the photoperiodic response curve. Exotic light and dark cycles of varying period (T) with a short (10 h) photophase and a scotophase ranging from 14 (T = 24) to 62 (T = 72) h, reveal two geographic patterns: a decline in perturbability of the photoperiodic clock with increasing latitude, and no change with latitude in the 21-h period of rising and falling development with increasing T. These results show (1) that there is a rhythmic component to photoperiodic time measurement in W. smithii, (2) that the period of this rhythm is about 21 h in all populations, and (3) that more northern populations show decreasing responsiveness to photoperiod and increasing stability against perturbation by exotic period lengths (T > 24). Previous studies on W.␣smithii indicate that this single temperate species of a tropical and subtropical genus has evolved from south to north. We therefore conclude that the evolution of increasing critical photoperiod in W. smithii during its adaptive radiation into North America has more likely involved the amplitude and not the period of the underlying circadian pacemaker. Received: 22 July 1996 / Accepted: 30 September 1996     

Methyltetrahydromethanopterin as an intermediate in methanogenesis from acetate in Methanosarcina barkeri

Cell extracts (100,000×g) of acetate grown Methanosarcina barkeri (strain MS) catalyzed CH₄ and CO₂ formation from acetyl-CoA with specific activities of 50 nmol·min^-1·mg protein^-1. CH₄ formation was found to be dependent on tetrahydromethanopterin (H₄MPT) (apparent K _M=4 μM), coenzyme M (H-S-CoM), and 7-mercaptoheptanoylthreonine phosphate (H-S-HTP=component B) rather than on methanofuran (MFR) and coenzyme F₄₂₀ (F₄₂₀). Methyl-H₄MPT was identified as an intermediate. This compound accumulated when H-S-CoM and H-S-HTP were omitted from the assays. These and previous results indicate that methanogenesis from acetate proceeds via acetyl phosphate, acetyl-CoA, methyl-H₄MPT, and CH₃-S-CoM as intermediates. The disproportionation of formaldehyde to CO₂ and CH₄ was also studied. This reaction was shown to be dependent on H₄MPT, MFR, F₄₂₀, H-S-CoM, and H-S-HTP.