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 degrees C), slurries were first incubated at temperatures between 4 and 60 degrees 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 degrees C. Initial methane production rates were highest in slurries preincubated at temperatures between 4 and 15 degrees C, with maximal rates in slurries kept at 6 degrees C. Hydrogen-amended cultures were the only exceptions, with the highest methane production rates at 6 degrees C after preincubation at 30 degrees C.     

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.     

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.     

Microbial Formation of Ethane in Anoxic Estuarine Sediments

Estuarine sediment slurries produced methane and traces of ethane when incubated under hydrogen. Formation of methane occurred over a broad temperature range with an optimum above 65°C. Ethane formation had a temperature optimum at 40°C. Formation of these two gases was inhibited by air, autoclaving, incubation at 4 and 80°C, and by the methanogenic inhibitor, 2-bromoethanesulfonic acid. Ethane production was stimulated by addition of ethylthioethanesulfonic acid, and production from ethylthioethanesulfonic acid was blocked by 2-bromoethanesulfonic acid. A highly purified enrichment culture of a methanogenic bacterium obtained from sediments produced traces of ethane from ethylthioethanesulfonic acid. These results indicate that the small quantities of ethane found in anaerobic sediments can be formed by certain methanogenic bacteria.     

Effects of Temperature on Methanogenesis in a Thermophilic (58°C) Anaerobic Digestor

The short-term effects of temperature on methanogenesis from acetate or CO₂ in a thermophilic (58°C) anaerobic digestor were studied by incubating digestor sludge at different temperatures with ¹⁴C-labeled methane precursors (¹⁴CH₃COO⁻ or ¹⁴CO₂). During a period when Methanosarcina sp. was numerous in the sludge, methanogenesis from acetate was optimal at 55 to 60°C and was completely inhibited at 65°C. A Methanosarcina culture isolated from the digestor grew optimally on acetate at 55 to 58°C and did not grow or produce methane at 65°C. An accidental shift of digestor temperature from 58 to 64°C during this period caused a sharp decrease in gas production and a large increase in acetate concentration within 24 h, indicating that the aceticlastic methanogens in the digestor were the population most susceptible to this temperature increase. During a later period when Methanothrix sp. was numerous in the digestor, methanogenesis from ¹⁴CH₃COO⁻ was optimal at 65°C and completely inhibited at 75°C. A partially purified Methanothrix enrichment culture derived from the digestor had a maximum growth temperature near 70°C. Methanogenesis from ¹⁴CO₂ in the sludge was optimal at 65°C and still proceeded at 75°C. A CO₂-reducing Methanobacterium sp. isolated from the digestor was capable of methanogenesis at 75°C. During the period when Methanothix sp. was apparently dominant, sludge incubated for 24 h at 65°C produced more methane than sludge incubated at 60°C, and no acetate accumulated at 65°C. Methanogenesis was severely inhibited in sludge incubated at 70°C, but since neither acetate nor H₂ accumulated, production of these methanogenic substrates by fermentative bacteria was probably the most temperature-sensitive process. Thus, there was a correlation between digestor performance at different temperatures and responses to temperature by cultures of methanogens believed to play important roles in the digestor.     

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.     

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.     

Adaptation of Phytoplankton-Degrading Microbial Communities to Thermal Reactor Effluent in a New Cooling Reservoir

In water column and sediment inocula from a nuclear reactor cooling reservoir, natural phytoplankton substrate labeled with ¹⁴C was used to determine aerobic and anaerobic mineralization rates for a range of temperatures (25, 40, 55, and 70°C) expected during reactor operation. For experiments that were begun during reactor shutdown, aerobic decomposition occurred at temperatures of <55°C. After 2 months of reactor operation, aerobic rates increased substantially at 55 and 70°C, although maximum rates were observed at temperatures of ≤40°C. The temperature range for which maximum anaerobic mineralization (i.e., the sum of CH₄ and CO₂) was observed was 25 to 40°C when the reactor was off, expanding to 25 to 55°C during reactor operation. Increased rates at 55°C, but not 70°C, correlated with an increase in the ratio of cumulative methane to carbon dioxide produced over 21 days. When reduced reactor power lowered the maximum temperature of the reservoir to 42°C, aerobic decomposition at 70°C was negligible, but remained substantial at 55°C. Selection for thermophilic decomposers occurred rapidly in this system in both aerobic and anaerobic communities and did not require prolonged exposure to elevated temperatures.     

Respiration and Alternative Oxidase in Corn Seedling Tissues during Germination at Different Temperatures

Respiration rates of Zea mays L. seedling tissues grown at 30 and 14°C were measured at 25°C at different stages of seedling growth. Accumulation of heat units was used to define the developmental stages to compare respiration between the two temperatures. At both temperatures, respiration rates of most tissues were highest at the youngest stages, then declined with age. Respiration rates of mesocotyl tissue were the most responsive to temperature, being nearly twofold higher when grown at 14 compared to 30°C. Alternative pathway respiration increased concomitantly with respiration and was higher in mesocotyls grown in the cold. When seedlings were started at 30 then transferred to 14°C, the increase in alternative pathway respiration due to cold was not observed unless the seedlings were transferred before 2 days of growth. Seedlings transferred to 14°C after growth at 30°C for 2 days had the same alternative oxidase capacity as seedlings grown at 30°C. Seedlings grown at 14°C for 10 to 12 days, then transferred to 30°C, lost alternative pathway respiratory capacity over a period of 2 to 3 days. Western blots of mitochondrial proteins indicated that this loss of capacity was due to a loss of the alternative oxidase protein. Some in vitro characteristics of mitochondria were determined. The temperature optimum for measurement of alternative oxidase capacity was 15 to 20°C. At 41°C, very little alternative oxidase was measured, i.e., the mitochondrial oxygen uptake was almost completely sensitive to cyanide. This inactivation at 41°C was reversible. After incubation at 41°C, the alternative oxidase capacity measured at 25°C was the similar to when it was measured at that temperature directly. Isolated mitochondria lost alternative oxidase capacity at the same rate when incubated at 41°C as they did when incubated at 25°C. Increasing the supply of electrons to isolated mitochondria increased the degree of engagement of the alternative pathway, whereas lower temperature decreased the degree of engagement. Lower temperatures did not increase the degree of engagement of the pathway in intact tissues. We interpret these observations to indicate that the greater capacity of alternative oxidase in cold-grown seedlings is a consequence of development at these low temperatures which results in elevated respiration rates. Low temperature itself does not cause greater capacity or engagement of the alternative oxidase in mitochondria that have developed under warm temperatures. Our hypothesis would be that the low growth temperatures require the seedlings to have a higher respiration rate for some reason, e.g., to prevent the accumulation of a toxic metabolite, and that the alternative pathway functions in that respiration.     

Partitioning Effects during Terminal Carbon and Electron Flow in Sediments of a Low-Salinity Meltwater Pond near Bratina Island, McMurdo Ice Shelf, Antarctica

A study of anaerobic sediments below cyanobacterial mats of a low-salinity meltwater pond called Orange Pond on the McMurdo Ice Shelf at temperatures simulating those in the summer season (<5°C) revealed that both sulfate reduction and methane production were important terminal anaerobic processes. Addition of [2-¹⁴C]acetate to sediment samples resulted in the passage of label mainly to CO₂. Acetate addition (0 to 27 mM) had little effect on methanogenesis (a 1.1-fold increase), and while the rate of acetate dissimilation was greater than the rate of methane production (6.4 nmol cm⁻³ h⁻¹ compared to 2.5 to 6 nmol cm⁻³ h⁻¹), the portion of methane production attributed to acetate cleavage was <2%. Substantial increases in the methane production rate were observed with H₂ (2.4-fold), and H₂ uptake was totally accounted for by methane production under physiological conditions. Formate also stimulated methane production (twofold), presumably through H₂ release mediated through hydrogen lyase. Addition of sulfate up to 50-fold the natural levels in the sediment (interstitial concentration, ~0.3 mM) did not substantially inhibit methanogenesis, but the process was inhibited by 50-fold chloride (36 mM). No net rate of methane oxidation was observed when sediments were incubated anaerobically, and denitrification rates were substantially lower than rates for sulfate reduction and methanogenesis. The results indicate that carbon flow from acetate is coupled mainly to sulfate reduction and that methane is largely generated from H₂ and CO₂ where chloride, but not sulfate, has a modulating role. Rates of methanogenesis at in situ temperatures were four- to fivefold less than maximal rates found at 20°C.     

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.     

Methanogenesis from Methylated Amines in a Hypersaline Algal Mat

Methane ebullition and high rates of methane production were observed in sediments of a hypersaline pond (180‰) which contained sulfate in excess of 100 mM. The highest rates of methane production were observed in surface sediments associated with an algal mat dominated by a Spirulina sp. The mat contained a methylated amine, glycine betaine (GBT), at levels which accounted for up to 20% of the total mat nitrogen. GBT was apparently the source of trimethylamine (TMA), which was also present in the sediment at relatively high concentrations. Patterns of substrate metabolism by the methanogenic populations in sediment slurries suggested that TMA was a major methane precursor. Neither exogenous hydrogen nor acetate stimulated methanogenesis, while addition of a variety of amines including TMA, trimethylamine oxide, GBT, and choline resulted in substantial increases with yields of >70%. The temperature optimum for methanogenesis in this system was 45 to 55°C, which coincided with the observed sediment temperature. Patterns and rates of methane production in this and other hypersaline algal mats may be determined by a complex interaction between salinity, the use of methylated amines for osmoregulation by algae, and the formation of TMA by fermentation.     

Influence of Temperature on Multiplication and Egg Hatching of Longidorus africanus

Longidorus africanus multiplication on tomato was highest at 29 °C. Few nematodes were recovered after 6 weeks at soil temperatures of 35 °C or below 23 °C. The time to egg hatching was shortest and the percentage of eggs hatching was highest at 29 °C. The minimum temperature and the heat sum above this temperature required for egg development were calculated to be 14.3 °C and 94.08 degree-days, respectively. The thermal times required for egg development by L. africanus and L. elongatus were nearly identical. For both species the product of the base temperature and the heat sum was near constant, and at a temperature of 22.3 °C the rates of egg development were equal.     

Methane Production in Minnesota Peatlands

Rates of methane production in Minnesota peats were studied. Surface (10- to 25-cm) peats produced an average of 228 nmol of CH₄ per g (dry weight) per h at 25°C and ambient pH. Methanogenesis rates generally decreased with depth in ombrotrophic peats, but on occasion were observed to rise within deeper layers of certain fen peats. Methane production was temperature dependent, increasing with increasing temperature (4 to 30°C), except in peats from deeper layers. Maximal methanogenesis from these deeper regions occurred at 12°C. Methane production rates were also pH dependent. Two peats with pHs of 3.8 and 4.3 had an optimum rate of methane production at pH 6.0. The addition to peat of glucose and H₂-CO₂ stimulated methanogenesis, whereas the addition of acetate inhibited methanogenesis. Cysteine-sulfide, nitrogen-phosphorus-trace metals, and vitamins-yeast extract affected methane production very little. Various gases were found to be trapped or dissolved (or both) within peatland waters. Dissolved methane increased linearly to a depth of 210 cm. The accumulation of metabolic end products produced within peat bogs appears to be an important mechanism limiting carbon turnover in peatland environments.     

TIME-TEMPERATURE RELATIONS IN THE INCUBATION OF THE WHITEFISH,COREGONUS CLUPEAFORMIS (MITCHILL)

1. Whitefish eggs incubated in aerated lake water at controlled tempera tures of 0°, 0.5°, 2°, 4°, 6°, 8°, 10°, and 12°C., failed to hatch at either 0° or 12°C. 0.6 per cent hatched alive at 10°C., 72.67 per cent hatched alive at 0.5°C., and an intermediate proportion hatched at intermediate temperatures. 2. The percentage of abnormal embryos which developed to the hatching stage varied directly with temperature between 4° and 12°, all embryos being abnormal at 12°C.; but none were abnormal at either 0.5°, or 2°C. Normal development predominated from 0.5 to 6°C. The highest proportion of embryos to hatch alive was 72.67 per cent at 0.5°C., which is, hence, the optimum temperature. 3. Total incubation time ranged from 29.6 days at 10°C. to 141 days at 0.5°C. 4. The time (T) required to attain any given stage of development is expressed in equations See PDF for Equation where temperature, t, is a negative exponent of the constant, A, whose value differs above or below 6°C., a critical temperature. Values of A above 6° fluctuate about 1.13; those of A below 6° fluctuate about 1.19 as a mean. 5. Applying Arrhenius'' equation µ values for the total incubation period are 27,500 below 6° and 27,100 above it. 6. The relative magnitude of A values of the exponential equation and µ values of Arrhenius'' equation show corresponding changes from one developmental period to another. 7. When plotted, thermal increments show cyclic variations, with maxima during periods of cleavage and of organogenesis. These may indicate the interaction of two separate sets of embryonic processes, which give a maximal response to temperature differences during these two separate periods. 8. Above 6°, µ values during the hatching process are distinct from those of developmental stages and are regarded as being due to the action of hatching enzymes.     

Seasonal Rates of Methane Oxidation in Anoxic Marine Sediments

Methane concentrations and rates of methane oxidation were measured in intact sediment cores from an inshore marine sediment at Jutland, Denmark. The rates of methane oxidation, determined by the appearance of ¹⁴CO₂ from injected ¹⁴CH₄, varied with sediment depth and season. Most methane oxidation was anoxic, but oxygen may have contributed to methane oxidation at the sediment surface. Cumulative rates (0- to 12-cm depth) for methane oxidation at Kysing Fjord were 3.34, 3.48, 8.60, and 17.04 μmol m⁻² day⁻¹ for April (4°C), May (13°C), July (17°C), and August (21°C), respectively. If all of the methane was oxidized by sulfate, it would account for only 0.01 to 0.06% of the sulfate reduction. The data indicate that methane was produced, in addition to being oxidized, in the 0- to 18-cm sediment stratum.     

Inactivation of Ascaris Eggs in Source-Separated Urine and Feces by Ammonia at Ambient Temperatures

Sustainable management of toilet waste must prevent disease transmission but allow reuse of plant nutrients. Inactivation of uterus-derived Ascaris suum eggs was studied in relation to ammonia in source-separated urine without additives and in human feces to which urea had been added, in order to evaluate ammonia-based sanitation for production of safe fertilizers from human excreta. Urine was used concentrated or diluted 1:1 and 1:3 with tap water at 4, 14, 24, and 34°C. Fecal material, with and without ash, was treated with 1% or 2% (wt/wt) urea at 24 and 34°C. At 34°C eggs were inactivated in less than 10 days in urine and in amended feces. At 24°C only feces with 2% (wt/wt) urea or 1% (wt/wt) urea at high pH (10) inactivated all eggs within 1 month, and no inactivation was observed after 75 days in urine diluted 1:3 (18 ± 11 mM NH₃). At temperatures of ≥24°C, NH₃ proved to be an efficient sanitizing agent in urine and feces at concentrations of ≥60 mM. Treating fecal material at 34°C can give a 6-log₁₀ egg inactivation within 1 month, whereas at 24°C 6 months of treatment is necessary for the same level of egg inactivation. At temperatures of 14°C and below, inactivation rates were low, with viable eggs after 6 months even in concentrated urine.     

Temperature-Driven Adaptation of the Bacterial Community in Peat Measured by Using Thymidine and Leucine Incorporation

The temperature-driven adaptation of the bacterial community in peat was studied, by altering temperature to simulate self-heating and a subsequent return to mesophilic conditions. The technique used consisted of extracting the bacterial community from peat using homogenization-centrifugation and measuring the rates of thymidine (TdR) or leucine (Leu) incorporation by the extracted bacterial community at different temperatures. Increasing the peat incubation temperature from 25°C to 35, 45, or 55°C resulted in a selection of bacterial communities whose optimum temperatures for activity correlated to the peat incubation temperatures. Although TdR and Leu incorporations were significantly correlated, the Leu/TdR incorporation ratios were affected by temperature. Higher Leu/TdR incorporation ratios were found at higher temperatures of incubation of the extracted bacterial community. Higher Leu/TdR incorporation ratios were also found for bacteria in peat samples incubated at higher temperatures. The reappearance of the mesophilic community and disappearance of the thermophilic community when the incubation temperature of the peat was shifted down were monitored by measuring TdR incorporation at 55°C (thermophilic activity) and 25°C (mesophilic activity). Shifting the peat incubation temperature from 55 to 25°C resulted in a recovery of the mesophilic activity, with a subsequent disappearance of the thermophilic activity. The availability of substrate for bacterial growth varied over time and among different peat samples. To avoid confounding effects of substrate availability, a temperature adaptation index was calculated. This index consisted of the log₁₀ ratio of TdR incorporation at 55 and 25°C. The temperature index decreased linearly with time, indicating that no thermophilic activity would be detected by the TdR technique 1 month after the temperature downshift. There were no differences between the slopes of the temperature adaptation indices over time for peat samples incubated at 55°C 3 or 11 days before incubation at 25°C. Thus, different levels of bacterial activity did not affect the temperature-driven adaptation of the bacterial community.     

Photoinduced Seed Germination of Oenothera biennis L: III. Analysis of the Postinduction Period by Means of Temperature

The postinduction period of Oenothera biennis L. seed germination was examined by temperature treatments. For all experiments, seeds received a standard 24 hour/24°C preinduction period and 12 hour/32°C photoinduction period. Germination is inhibited by postinduction temperatures above 32°C. When seeds are briefly incubated at 44°C and then transferred to 28°C, they germinate at a much lower percentage than 28°C controls. When thermally inhibited seeds are placed in the dark at 28°C for 20 hours, they can be promoted to germinate by a single pulse of red light. Seeds incubated at 12°C or below immediately after photoinduction enter a lag period in which they germinate slowly or not at all for a long time and then resume germination. The length of the lag period is exponentially related to the postinduction temperature. When seeds are incubated at a low temperature and then transferred to a warm temperature, they germinate much more rapidly than seeds not incubated at a low temperature. A model is proposed which is consistent with these and additional results. In the model, a germination promoter is irreversibly formed from a precursor and the synthesis of the precursor is favored at low temperatures and its degradation is favored at high temperatures.     

Methane Production by Fermentor Cultures Acclimated to Waste from Cattle Fed Monensin, Lasalocid, Salinomycin, or Avoparcin

The ability of microorganisms to ferment waste from cattle fed monensin, lasalocid, or salinomycin to methane was determined. Continuously mixed anaerobic fermentors with 3-liter working volumes at 55°C were used; fermentors were fed once per day. Initially, all fermentors were fed waste without antibiotics at 6% volatile solids (VSs, organic matter) and a 20-day retention time (RT) for 60 days. Waste from animals fed monensin, lasalocid, or salinomycin at 29, 20, and 16.5 mg per kg of feed, respectively, was added to duplicate fermentors at the above VSs, and RT. Avoparcin (5 to 45 mg/liter) was not fed to animals but was added directly to duplicate fermentors. Lasalocid and salinomycin had minimal effects on the rate of methane production at RTs of 20 days and later at 6.5 days. Avoparcin caused an increase in organic acids from 599 to 1,672 mg/liter (as acetate) after 4 weeks, but by 6 weeks, acid concentrations declined and the rate of methane production was similar to controls at a 6.5-day RT. The monensin fermentors stopped producing methane 3 weeks after antibiotic addition. However, after a 6-month acclimation period, the microorganisms apparently adapted, and methane production rates of 1.65 and 2.51 liters per liter of fermentor volume per day were obtained with 6% VSs, and RTs of 10 and 6.5 days, respectively. This compares with 1.78 and 2.62 liters/liter per day for controls (P > 0.05). All fermentors that were fed waste containing antibiotics had lower pH values and ammonia and alkalinity concentrations, suggesting less buffering capacity and protein catabolism than in controls. Acclimation results obtained with fermentors at 35°C were similar to those for fermentors at 55°C. These studies indicate that waste from cattle fed these selected growth-promoting antibiotics can be thermophilically fermented to methane at RTs of 6.5 days or longer and VS concentrations of 6%, at rates comparable to waste without antibiotics.