Isolation and Characterization of an H2-Oxidizing Thermophilic Methanogen

A thermophilic methanogen was isolated from enrichment cultures originally inoculated with sludge from an anaerobic kelp digester (55°C). This isolate exhibited a temperature optimum of 55 to 60°C and a maximum near 70°C. Growth occurred throughout the pH range of 5.5 to 9.0, with optimal growth near pH 7.2. Although 4% salt was present in the isolation medium, salt was not required for optimal growth. The thermophile utilized formate or H₂-CO₂ but not acetate, methanol, or methylamines for growth and methanogenesis. Growth in complex medium was very rapid, and a minimum doubling time of 1.8 h was recorded in media supplemented with rumen fluid. Growth in defined media required the addition of acetate and an unknown factor(s) from digester supernatant, rumen fluid, or Trypticase. Cells in liquid culture were oval to coccoid, 0.7 to 1.8 μm in diameter, often occurring in pairs. The cells were easily lysed upon exposure to oxygen or 0.08 mg of sodium dodecyl sulfate per ml. The isolate was sensitive to tetracycline and chloramphenicol but not penicillin G or cycloserine. The DNA base composition was 59.69 mol% guanine plus cytosine.     

Isolation and Characterization of Methanomicrobium paynteri sp. nov., a Mesophilic Methanogen Isolated from Marine Sediments

A new mesophilic methanogenic bacterial species isolated from marine sediments collected in the Cayman Islands is described. Cells are small rods occuring singly without filaments, are not motile, and do not possess flagella. Colonies are semitransparent and off-white in color. After 2 weeks of incubation at 37°C colonies are 1 to 2 mm in size, circular, and have entire edges. Only hydrogen-carbon dioxide is a substrate for growth and methane formation. Cells can tolerate a variety of organic secondary buffers (bicarbonate-CO₂ being the primary buffer). Cells do not require yeast extract or Trypticase, but do require acetate, for growth. The optimum growth temperature is 40°C. The optimum sodium concentration is 0.15 M. The optimum pH for growth is 7.0. The minimum generation time is 4.8 h. The DNA base composition is 44.9 mol% guanine plus cytosine. The name Methanomicrobium paynteri is proposed in honor of M. J. B. Paynter. The type strain is G-2000 (=ATCC 33997, =DSM 2545).     

Unexpected Errors in Gas Chromatographic Analysis of Methane Production by Thermophilic Bacteria

Unexpected errors in methane measurement by gas chromatography occurred when samples at thermophilic temperatures were analyzed. With a standard curve prepared at room temperature (25°C), stoppered bottles incubated and sampled at 37 to 85°C showed more methane upon analysis than bottles incubated at 25°C: values at 50, 63, and 85°C were 109, 126, and 125%, respectively, of the 25°C value. All variation between 4 and 50°C can be explained by the temperature difference between culture bottle and sampling syringe, and the variation of methane concentration can be predicted by the gas law. Between 50 and 63°C, there was a more dramatic rise than predicted by theory. These variations are important to consider if thermophilic methane production is to be measured accurately. Methods to avoid errors are discussed.     

Pressure and Temperature Effects on Growth and Methane Production of the Extreme Thermophile Methanococcus jannaschii

The marine archaebacterium Methanococcus jannaschii was studied at high temperatures and hyperbaric pressures of helium to investigate the effect of pressure on the behavior of a deep-sea thermophile. Methanogenesis and growth (as measured by protein production) at both 86 and 90°C were accelerated by pressure up to 750 atm (1 atm = 101.29kPa), but growth was not observed above 90°C at either 7.8 or 250 atm. However, growth and methanogenesis were uncoupled above 90°C, and the high-temperature limit for methanogenesis was increased by pressure. Substantial methane formation was evident at 98°C and 250 atm, whereas no methane formation was observed at 94°C and 7.8 atm. In contrast, when argon was substituted for helium as the pressurizing gas at 250 atm, no methane was produced at 86°C. Methanogenesis was also suppressed at 86°C and 250 atm when the culture was pressurized with a 4:1 mix of H₂ and CO₂, although limited methanogenesis did occur when the culture was pressurized with H₂.     

Methanosarcina acetivorans sp. nov., an Acetotrophic Methane-Producing Bacterium Isolated from Marine Sediments

A new acetotrophic marine methane-producing bacterium that was isolated from the methane-evolving sediments of a marine canyon is described. Exponential phase cultures grown with sodium acetate contained irregularly shaped cocci that aggregated in the early stationary phase and finally differentiated into communal cysts that released individual cocci when ruptured or transferred to fresh medium. The irregularly shaped cocci (1.9 ± 0.2 mm in diameter) were gram negative and occurred singly or in pairs. Cells were nonmotile, but possessed a single fimbria-like structure. Micrographs of thin sections showed a monolayered cell wall approximately 10 nm thick that consisted of protein subunits. The cells in aggregates were separated by visible septation. The communal cysts contained several single cocci encased in a common envelope. An amorphous form of the communal cyst that had incomplete septation and internal membrane-like vesicles was also present in late exponential phase cultures. Sodium acetate, methanol, methylamine, dimethylamine, and trimethylamine were substrates for growth and methanogenesis; H₂-CO₂ (80:20) and sodium formate were not. The optimal growth temperature was 35 to 40°C. The optimal pH range was 6.5 to 7.0. Both NaCl and Mg²⁺ were required for growth, with maximum growth rates at 0.2 M NaCl and 0.05 M MgSO₄. The DNA base composition was 41 ± 1% guanine plus cytosine. Methanosarcina acetivorans is the proposed species. C2A is the type strain (DSM 2834, ATCC 35395).     

Effect of Temperature and Retention Time on Methane Production from Beef Cattle Waste

The effect of temperature and retention time on the rate of methane production from waste of beef cattle fed a finishing diet was investigated by using continuously mixed 3-liter working volume anaerobic fermentors. The temperatures ranged from 30 to 65°C with 5°C increments between fermentors. The fermentors were fed once per day with 6% volatile solids (organic matter). Retention time for each temperature was varied from 18 to 2.5 days. After 3-volume turnovers, samples were obtained on 4 consecutive days. The highest methane production rate (liters/liter of fermentor per day) and methane yield at that rate (liters/gram of volatile solids) were 1.27 and 0.19 at 9 days and 30°C, 1.60 and 0.16 at 6 days and 35°C, 2.28 and 0.23 at 6 days and 40°C, 2.42 and 0.24 at 6 days and 45°C, 2.83 and 0.14 at 3 days and 50°C, 2.75 and 0.14 at 3 days and 55°C, 3.18 and 0.14 at 2.5 days and 60°C, and 1.69 and 0.17 at 6 days and 65°C. Volatile solids degradation at these retention times and temperatures was between 46 and 54%. The concentrations of volatile acids in the 30 to 55°C fermentors were generally below 2,000 mg/liter, with the exception of the 3-day retention time. The 60 and 65°C fermentors were usually above this level for all retention times. These studies indicate potential rates of methane production from the fermentation of untreated waste of beef cattle fed high-grain finishing diets. This information should serve as preliminary guidelines for various kinetic analyses and aid in economic evaluations of the potential feasibility of fermenting beef cattle waste to methane.     

Isolation and Characterization of a Methylotrophic Marine Methanogen, Methanococcoides methylutens gen. nov., sp. nov

A new genus of marine methanogenic bacteria is described that utilizes trimethylamine, diethylamine, monomethylamine, and methanol as substrates for growth and methanogenesis. Methane was not produced from H₂-CO₂, sodium formate, or sodium acetate. Growth on trimethylamine was stimulated by yeast extract, Trypticase (BBL Microbiology Systems, Cockeysville, Md.), rumen fluid, or B vitamins. The optimal growth temperature was 30 to 35°C. The maximum growth rate was between pH 7.0 and 7.5. Na⁺ (0.4 M) and MgSO₄ (0.05 M) were required for maximum growth. Colonies of the type strain, TMA-10, were yellow, circular, and convex with entire edges. Cells were nonmotile, nonsporeforming, irregular cocci 1 μm in diameter which stained gram negative and occurred singly or in pairs. Micrographs of thin sections revealed a monolayered cell wall approximately 10-nm thick which consisted of protein. Cells were lysed in 0.01% sodium dodecyl sulfate or 0.001% Triton X-100. The DNA base composition was 42 mol% guanine plus cytosine. Methanococcoides is the proposed genus and Methanococcoides methylutens is the type species. TMA-10 is the type strain (ATCC 33938).     

Effects of Hyperbaric Pressure on a Deep-Sea Archaebacterium in Stainless Steel and Glass-Lined Vessels

The effects of hyperbaric helium pressures on the growth and metabolism of the deep-sea isolate ES4 were investigated. In a stainless steel reactor, cell growth was completely inhibited but metabolic gas production was observed. From 85 to 100°C, CO₂ production proceeded two to three times faster at 500 atm (1 atm = 101.29 kPa) than at 8 atm. At 105°C, no CO₂ was produced until the pressure was increased to 500 atm. Hydrogen and H₂S were also produced biotically but were not quantifiable at pressures above 8 atm because of the high concentration of helium. In a glass-lined vessel, growth occurred but the growth rate was not accelerated by pressure. In most cases at temperatures below 100°C, the growth rate was lower at elevated pressures; at 100°C, the growth rates at 8, 250, and 500 atm were nearly identical. Unlike in the stainless steel vessel, CO₂ production was exponential during growth and continued for only a short time after growth. In addition, relatively little H₂ was produced in the glass-lined vessel, and there was no growth or gas production at 105°C at any pressure. The behavior of ES4 as a function of temperature and pressure was thus very sensitive to the experimental conditions.     

Biophysics of the inhibition of the growth of maize roots by lowered temperature

Roots of hydroponically grown maize (Zea mays cv LG11) have a greatly reduced growth rate at 5°C (0.02 millimeters per hour) compared with those at 20°C (1.2 millimeters per hour). Various physical parameters of roots growing at each temperature were compared. Turgor pressure of cells in the elongation zone increased from 0.59 ± 0.05 megapascal at 20°C to 0.82 ± 0.04 megapascal after 70 hours at 5°C; thus, growth rate was not limited by decreased pressure. On cooling, tissue plasticity (measured by Instron/tensiometer) decreased slowly over 70 hours. On rewarming to 20°C from 5°C, growth rate, turgor pressure, and tissue plasticity all returned concertedly to their original values over a period of days. However, immediately following cooling growth rate dropped rapidly from 1.8 to 0.12 millimeters per hour in 30 minutes but turgor pressure and tissue Instron plasticity remained unchanged. A plot of turgor pressure against growth rate indicated that, following cooling from 30 to 15°C, the in vivo wall extensibility of the tissue was reduced by 75%. Yield threshold was unchanged. Cells whose expansion was arrested in the long-term cold treatment do not resume growth. Root growth recovers by the expansion of cells newly produced by the meristem. Cessation of extension growth is an effect on the individual expanding cell. Growth recovery is not a reverse of this effect but requires the generation of fresh cells.     

Metabolism of Formate in Methanobacterium formicicum

Methanobacterium formicicum strain JF-1 was cultured with formate as the sole energy source in a pH-stat fermentor. Growth was exponential, and both methane production and formate consumption were linear functions of the growth rate. Hydrogen was produced in only trace amounts, and the dissolved H₂ concentration of the culture medium was below 1 μM. The effect of temperature or pH on the rate of methane formation was studied with a single fermentor culture in mid-log phase that was grown with formate under standard conditions at 37°C and pH 7.6. Methane formation from formate occurred over the pH range from 6.5 to 8.6, with a maximum at pH 8.0. The maximum temperature of methanogenesis was 56°C. H₂ production increased at higher temperatures. Hydrogen and formate were consumed throughout growth when both were present in saturating concentrations. The molar growth yields were 1.2 ± 0.06 g (dry weight) per mol of formate and 4.8 ± 0.24 g (dry weight) per mol of methane. Characteristics were compared for cultures grown with either formate or H₂-CO₂ as the sole energy source at 37°C and pH 7.6; the molar growth yield for methane of formate cultures was 4.8 g (dry weight) per mol, and that of H₂-CO₂ cultures was 3.5 g (dry weight) per mol. Both formate and H₂-CO₂ cultures had low efficiencies of electron transport phosphorylation; formate-cultured cells had greater specific activities of coenzyme F₄₂₀ than did H₂-CO₂-grown cultures. Hydrogenase, formate dehydrogenase, chromophoric factor F₃₄₂, and low levels of formyltetrahydrofolate synthetase were present in cells cultured with either substrate. Methyl viologen-dependent formate dehydrogenase was found in the soluble fraction from broken cells.     

Evidence for the Existence of Psychrophilic Methanogenic Communities in Anoxic Sediments of Deep Lakes

In order to obtain evidence for the existence of psychrophilic methanogenic communities in sediments of deep lakes that are low-temperature environments (4 to 5°C), slurries were first incubated at temperatures between 4 and 60°C for several weeks, at which time they were amended, or not, with an additional substrate, such as cellulose, butyrate, propionate, acetate, or hydrogen, and further incubated at 6°C. Initial methane production rates were highest in slurries preincubated at temperatures between 4 and 15°C, with maximal rates in slurries kept at 6°C. Hydrogen-amended cultures were the only exceptions, with the highest methane production rates at 6°C after preincubation at 30°C.     

Effect of Low Temperatures on Growth, Structure, and Metabolism of Campylobacter coli SP10

The effect of low temperatures on the survival, structure, and metabolism of Campylobacter coli SP10, a virulent strain, was investigated. C. coli became nonculturable rapidly at 20 and 10°C and slightly later at 4°C. Incubation in a microaerobic atmosphere improved survival, but after day 8, campylobacters were detectable by direct-count procedures only. The increase in the number of coccoid cells was most pronounced at 37°C but also was noticeable at 20 and 10°C. Two forms of coccoid cells were seen electron microscopically, but only one (20 and 10°C) seemed to be a degenerative form. The flagella were shorter at 20 and 10°C, a result which correlates well with the observed slight changes in the 62-kDa protein band. The fatty acid composition of bacterial cells was influenced significantly by low temperatures. An increase in the short-chain and unsaturated acids was noted; above all, a drastic increase in C_19:0 cyc at 20°C with a concomitant decrease in C_18:1 trans9,cis11 was seen. The concentrations of excreted metabolites were analyzed to obtain information on metabolic activity. Depending on the magnitude of the temperature downshift, the production of organic acids decreased, but it was always observable after a temperature-specific lag phase and regardless of ability to be cultured. Under optimal conditions, succinate, lactate, and acetate were the main metabolites, other acids being of less importance. The pattern changed significantly at lower temperatures. Succinate was never detected at 20°C and was only occasionally detected at 10 and 4°C. At the same time, fumarate concentrations, which are normally not detectable at 37°C, were highest at 20°C and reduced at 10 and 4°C. Inactivation of fumarate reductase was considered to be a possible explanation.     

Nitrate Transport and Not Photoinhibition Limits Growth of the Freshwater Cyanobacterium Synechococcus Species PCC 6301 at Low Temperature

The effect of low temperature on cell growth, photosynthesis, photoinhibition, and nitrate assimilation was examined in the cyanobacterium Synechococcus sp. PCC 6301 to determine the factor that limits growth. Synechococcus sp. PCC 6301 grew exponentially between 20°C and 38°C, the growth rate decreased with decreasing temperature, and growth ceased at 15°C. The rate of photosynthetic oxygen evolution decreased more slowly with temperature than the growth rate, and more than 20% of the activity at 38°C remained at 15°C. Oxygen evolution was rapidly inactivated at high light intensity (3 mE m⁻² s⁻¹) at 15°C. Little or no loss of oxygen evolution was observed under the normal light intensity (250 μE m⁻² s⁻¹) for growth at 15°C. The decrease in the rate of nitrate consumption by cells as a function of temperature was similar to the decrease in the growth rate. Cells could not actively take up nitrate or nitrite at 15°C, although nitrate reductase and nitrite reductase were still active. These data demonstrate that growth at low temperature is not limited by a decrease in the rate of photosynthetic electron transport or by photoinhibition, but that inactivation of the nitrate/nitrite transporter limits growth at low temperature.     

Methanosarcina mazei LYC, a New Methanogenic Isolate Which Produces a Disaggregating Enzyme

A methanogenic coccoid organism, Methanosarcina mazei LYC, was isolated from alkaline sediment obtained from an oil exploration drilling site. The isolate resembled M. mazei S-6 by exhibiting different morphophases during its normal growth cycle. It differed from M. mazei S-6 by undergoint a spontaneous shift from large, irregular aggregates of cells to small, individual, irregular, coccoid units. In batch cultures at pH 7.0, M. mazei LYC grew as aggregates during the early growth stage. As the batch culture began exponential growth, the cell aggregates spontaneously dispersed: the culture liquid became turbid, and myriads of tiny (diameter, 1 to 3 μm) coccoid units were observed under phase-contrast microscopy. Disaggregation apparently was accomplished by the production of an enzyme which hydrolyzed the heteropolysaccharide component of the cell wall; the enzyme was active on other Methanosarcina strains as well. Although the enzyme was active when tested at pH 6.0, it apparently was not produced at that pH: when strain LYC was grown at pH 6.0, only cell aggregates were present throughout batch growth. Individual coccoid cells of M. mazei LYC were sensitive to sodium dodecyl sulfate, but the large aggregates of cells were not. Strain LYC rapidly used H₂-CO₂, in addition to methanol, and mono-, di-, and trimethylamine as methanogenic substrates; acetate was used very slowly. Its optimum growth temperature was 40°C, and its optimum pH was 7.2.     

Acetate Degradation at 70°C in Upflow Anaerobic Sludge Blanket Reactors and Temperature Response of Granules Grown at 70°C

Anaerobic acetate degradation at 70°C and at 55°C (as a reference) was studied by running laboratory upflow anaerobic sludge blanket (UASB) reactors inoculated with mesophilic granular sludge. In UASB reactors fed with acetate-containing media (3 g of chemical oxygen demand [COD] per liter, corresponding to 47 mM acetate) approximately 50 days was needed at 70°C and less than 15 days was needed at 55°C to achieve an effluent COD of 500 to 700 mg/liter. In the UASB reactors at both 70 and 55°C up to 90% of the COD was removed. Batch assays showed that sludges from two 70°C UASB reactors, one run at a low effluent acetate concentration and the other run at a high effluent acetate concentration, exhibited slightly different responses to temperatures in the range from 37 to 70°C. Both 70°C sludges, as well as the 55°C sludge, produced methane at temperatures of 37 to 73°C. The 55°C sludge exhibited shorter lag phases than the 70°C sludges and higher specific methane production rates between 37 and 65°C.     

Effects of Temperature and Relative Humidity on the Growth of and Enzyme Production by Actinomucor taiwanensis during Sufu Pehtze Preparation

The growth of and production of protease, α-amylase, α-galactosidase, and lipase by Actinomucor taiwanensis in relation to temperature and relative humidity during the preparation of sufu (Chinese cheese) pehtze were investigated. The incubation temperature, humidity, and cultivation time greatly affected the growth of and enzyme production by A. taiwanensis on tofu. It grew best at 97% humidity and 30°C. The highest yields of protease (112 U/g of dry tofu) and lipase (1,448 U/g of dry tofu) were found after 60 h of incubation at 97% humidity and 25°C. On the other hand, the highest yield of α-amylase (1,949 U/g of dry tofu) was observed after 48 h of incubation at 96 to 97% humidity and 30°C, and the highest amount of α-galactosidase (387 U/g of dry tofu) was observed at 35°C and 96% humidity after 60 h of growth. The results suggest that the temperature and humidity should be controlled at 25 to 30°C and around 97%, respectively, during the commercial preparation of sufu pehtze for better growth of and production of enzymes by A. taiwanensis.     

Additive Effects of Alcohols, Their Acidic By-Products, and Temperature on the Yeast Pachysolen tannophilus

The effects of alcohols on the growth and fermentation of the yeast Pachysolen tannophilus were investigated at both 30 and 35°C. Addition of alcohols to the culture medium decreased both the growth rate and the final cell yield in a dose-dependent manner, and this decrease was more severe at 35°C. The concentration for 50% growth rate inhibition decreased as the chain length of the alcohol increased. In fermentations using a high initial cell density, production of acids was always observed when the medium was supplemented with alcohols. Supplementation of the culture medium with a short-chain alcohol plus the corresponding acid was shown to exert an additive deleterious effect on fermentation, and this effect increased with temperature. Production of acids was associated with the presence of alcohol dehydrogenase activity in cell extracts.     

Effect of Chilling Temperatures upon Cell Cultures of Tomato

The effect of chilling temperatures upon cell cultures of tomato (Lycopersicon esculentum Mill cv `VF36,' and cv `VFNT Cherry,' and L. hirsutum Humb. & Bonpl.) was tested. Doubling times for L. esculentum were 2 to 3 days at 28°C, and 3 to 8 days at 12°C. No growth was observed at 8°C, indicating an abrupt limit to growth between 8 and 12°C. Fluorescein diacetate staining indicated that 80 to 90% of the cells were alive when cells were maintained at 8°C for up to 2 weeks. When cultures kept at 8°C for up to 30 days were transferred to 28°C, growth resumed quickly, and at a rate virtually identical to that for unchilled cells. Similar results were found for cells maintained at 0°C, and for cells of `VFNT Cherry' and of L. hirsutum. Under certain conditions, cultures slowly doubled in fresh weight and cell volume at 8 or 9°C but additional growth at 8°C did not occur, nor could growth be maintained by subculture at 8 or 9°C. The results are contrary to reports that cell cultures of tomato die when exposed to temperatures below 10°C for 1 or 2 weeks. Our observations indicate that chilling temperatures quickly inhibit growth of tomato cells, but do not kill them.     

Thermostable Amylolytic Enzymes from a New Clostridium Isolate

A new Clostridium strain was isolated on starch at 60°C. Starch, pullulan, maltotriose, and maltose induced the synthesis of α-amylase and pullulanase, while glucose, ribose, fructose, and lactose did not. The formation of the amylolytic enzymes was dependent on growth and occurred predominantly in the exponential phase. The enzymes were largely cell bound during growth of the organism with 0.5% starch, but an increase of the starch concentration in the growth medium was accompanied by the excretion of α-amylase and pullulanase into the culture broth; but also by a decrease of total activity. α-Amylase, pullulanase, and α-glucosidase were active in a broad temperature range (40 to 85°C) and displayed temperature optima for activity at 60 to 70°C. During incubation with starch under aerobic conditions at 75°C for 2 h, the activity of both enzymes decreased to only 90 or 80%. The apparent K_m values of α-amylase, pullulanase, and α-glucosidase for their corresponding substrates, starch, pullulan, and maltose were 0.35 mg/ml, 0.63 mg/ml, and 25 mM, respectively.     

Temperature Adaptations in the Terminal Processes of Anaerobic Decomposition of Yellowstone National Park and Icelandic Hot Spring Microbial Mats

The optimum temperatures for methanogenesis in microbial mats of four neutral to alkaline, low-sulfate hot springs in Yellowstone National Park were between 50 and 60°C, which was 13 to 23°C lower than the upper temperature for mat development. Significant methanogenesis at 65°C was only observed in one of the springs. Methane production in samples collected at a 51 or 62°C site in Octopus Spring was increased by incubation at higher temperatures and was maximal at 70°C. Strains of Methanobacterium thermoautotrophicum were isolated from 50, 55, 60, and 65°C sites in Octopus Spring at the temperatures of the collection sites. The optimum temperature for growth and methanogenesis of each isolate was 65°C. Similar results were found for the potential rate of sulfate reduction in an Icelandic hot spring microbial mat in which sulfate reduction dominated methane production as a terminal process in anaerobic decomposition. The potential rate of sulfate reduction along the thermal gradient of the mat was greatest at 50°C, but incubation at 60°C of the samples obtained at 50°C increased the rate. Adaptation to different mat temperatures, common among various microorganisms and processes in the mats, did not appear to occur in the processes and microorganisms which terminate the anaerobic food chain. Other factors must explain why the maximal rates of these processes are restricted to moderate temperatures of the mat ecosystem.