Adaptation of mesophilic anaerobic sewage fermentor populations to thermophilic temperatures.

Thermophilic (50 degrees C) and obligately thermophilic (60 degrees C) anaerobic carbohydrate- and protein-digesting and methanogenic bacterial populations were enumerated in a mesophilic (35 degrees C) fermentor anaerobically digesting municipal primary sludge. Of the total bacterial population in the mesophilic fementor, 9% were thermophiles (36 x 10(6)/ml) and 1% were obligate thermophiles (4.5 x 10(6)/ml). Of these 10%, the percentages of bacteria (thermophiles and obligate thermophiles, respectively) able to use specific substrates were further enumerated as follows: bacteria able to digest albumin, casein, starch, and mono- and disaccharides, 30 and 10%; pectin degraders, 10 and 0.2%; cellulose degraders, 2 and 0.06%; methanogens that grow with H2 and CO2, methanol, and dimethylamine, 9 and 1%; methanogens that grow with formate, 8 and 5%; and methanogens that grow with acetate, 25 and less than 0.8%. Shortly after the temperature was elevated from 35 to 50 or 60 degrees C, the digestion of albumin, casein, starch, and mono- and disaccharides was detected, and methane was produced from H2 and CO2. Methane produced from acetate was not delayed at 50 degrees C, but was delayed by 29 days at 60 degrees C. Methane produced from formate was delayed by 3 days, from methanol by 7 days, and from dimethylamine by 5 days at 50 and 60 degrees C. A 10- and 20-day acclimation period was required for hydrolysis of pectin and cellulose, respectively, at 50 degrees C. Digestion of pectin required 20 days and cellulose longer than 85 days when the temperature was elevated abruptly from 35 to 60 degrees C. The acclimation period for the digestion of pectin and cellulose at 60 degrees C was shortened to 3 and 15 days, respectively, by seeding with a small amount of a culture acclimated to 50 degrees C. The data suggest that enrichment of cellulolytic, pectinolytic, and acetate-utilizing bacteria is crucial for the digestion of sewage sludge at 60 degrees C.     

Adaptation of mesophilic anaerobic sewage fermentor populations to thermophilic temperatures

Thermophilic (50 degrees C) and obligately thermophilic (60 degrees C) anaerobic carbohydrate- and protein-digesting and methanogenic bacterial populations were enumerated in a mesophilic (35 degrees C) fermentor anaerobically digesting municipal primary sludge. Of the total bacterial population in the mesophilic fementor, 9% were thermophiles (36 x 10(6)/ml) and 1% were obligate thermophiles (4.5 x 10(6)/ml). Of these 10%, the percentages of bacteria (thermophiles and obligate thermophiles, respectively) able to use specific substrates were further enumerated as follows: bacteria able to digest albumin, casein, starch, and mono- and disaccharides, 30 and 10%; pectin degraders, 10 and 0.2%; cellulose degraders, 2 and 0.06%; methanogens that grow with H2 and CO2, methanol, and dimethylamine, 9 and 1%; methanogens that grow with formate, 8 and 5%; and methanogens that grow with acetate, 25 and less than 0.8%. Shortly after the temperature was elevated from 35 to 50 or 60 degrees C, the digestion of albumin, casein, starch, and mono- and disaccharides was detected, and methane was produced from H2 and CO2. Methane produced from acetate was not delayed at 50 degrees C, but was delayed by 29 days at 60 degrees C. Methane produced from formate was delayed by 3 days, from methanol by 7 days, and from dimethylamine by 5 days at 50 and 60 degrees C. A 10- and 20-day acclimation period was required for hydrolysis of pectin and cellulose, respectively, at 50 degrees C. Digestion of pectin required 20 days and cellulose longer than 85 days when the temperature was elevated abruptly from 35 to 60 degrees C. The acclimation period for the digestion of pectin and cellulose at 60 degrees C was shortened to 3 and 15 days, respectively, by seeding with a small amount of a culture acclimated to 50 degrees C. The data suggest that enrichment of cellulolytic, pectinolytic, and acetate-utilizing bacteria is crucial for the digestion of sewage sludge at 60 degrees C.     

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.     

Methanosarcinaceae and Acetate-Oxidizing Pathways Dominate in High-Rate Thermophilic Anaerobic Digestion of Waste-Activated Sludge

This study investigated the process of high-rate, high-temperature methanogenesis to enable very-high-volume loading during anaerobic digestion of waste-activated sludge. Reducing the hydraulic retention time (HRT) from 15 to 20 days in mesophilic digestion down to 3 days was achievable at a thermophilic temperature (55°C) with stable digester performance and methanogenic activity. A volatile solids (VS) destruction efficiency of 33 to 35% was achieved on waste-activated sludge, comparable to that obtained via mesophilic processes with low organic acid levels (<200 mg/liter chemical oxygen demand [COD]). Methane yield (VS basis) was 150 to 180 liters of CH₄/kg of VS_added. According to 16S rRNA pyrotag sequencing and fluorescence in situ hybridization (FISH), the methanogenic community was dominated by members of the Methanosarcinaceae, which have a high level of metabolic capability, including acetoclastic and hydrogenotrophic methanogenesis. Loss of function at an HRT of 2 days was accompanied by a loss of the methanogens, according to pyrotag sequencing. The two acetate conversion pathways, namely, acetoclastic methanogenesis and syntrophic acetate oxidation, were quantified by stable carbon isotope ratio mass spectrometry. The results showed that the majority of methane was generated by nonacetoclastic pathways, both in the reactors and in off-line batch tests, confirming that syntrophic acetate oxidation is a key pathway at elevated temperatures. The proportion of methane due to acetate cleavage increased later in the batch, and it is likely that stable oxidation in the continuous reactor was maintained by application of the consistently low retention time.     

Thermoanaerobacteriaceae oxidize acetate in methanogenic rice field soil at 50°C

Rice field soils contain a thermophilic microbial community. Incubation of Italian rice field soil at 50°C resulted in transient accumulation of acetate, but the microorganisms responsible for methane production from acetate are unknown. Without addition of exogenous acetate, the δ(13)C of CH(4) and CO(2) indicated that CH(4) was exclusively produced by hydrogenotrophic methanogenesis. When exogenous acetate was added, acetoclastic methanogenesis apparently also operated. Nevertheless, addition of [2-(13)C]acetate (99% (13)C) resulted in the production not only of (13)C-labelled CH(4) but also of CO(2), which contained up to 27% (13)C, demonstrating that the methyl group of acetate was also oxidized. Part of the (13)C-labelled acetate was also converted to propionate which contained up to 14% (13)C. The microorganisms capable of assimilating acetate at 50°C were targeted by stable isotope probing (SIP) of ribosomal RNA and rRNA genes using [U-(13)C] acetate. Using quantitative PCR, (13)C-labelled bacterial ribosomal RNA and DNA was detected after 21 and 32 days of incubation with [U-(13)C]acetate respectively. In the heavy fractions of the (13)C treatment, terminal restriction fragments (T-RFs) of 140, 120 and 171 bp length predominated. Cloning and sequencing of 16S rRNA showed that these T-RFs were affiliated with the bacterial genera Thermacetogenium and Symbiobacterium and with members of the Thermoanaerobacteriaceae. Similar experiments targeting archaeal RNA and DNA showed that Methanocellales were the dominant methanogens being consistent with the operation of syntrophic bacterial acetate oxidation coupled to hydrogenotrophic methanogenesis. After 17 days, however, Methanosarcinacea increasingly contributed to the synthesis of rRNA from [U-(13)C]acetate indicating that acetoclastic methanogens were also active in methanogenic Italian rice field soil under thermal conditions.     

Effects of Environmental Parameters on the Formation and Turnover of Acetate by Forest Soils

The capacity to form acetate from endogenous matter was a common property of diverse forest soils when incubated under anaerobic conditions. At 15 to 20(deg)C, acetate synthesis occurred without appreciable delay when forest soils were incubated as buffered suspensions or in microcosms at various percentages of their maximum water holding capacity. Rates for acetate formation with soil suspensions ranged from 35 to 220 (mu)g of acetate per g (dry weight) of soil per 24 h, and maximal acetate concentrations obtained in soil suspensions were two- to threefold greater than those obtained with soil microcosms at the average water holding capacity of the soil. Cellobiose degradation in soil suspensions yielded H(inf2) as a transient product. Under anaerobic conditions, supplemental H(inf2) and CO(inf2) were directed towards the acetogenic synthesis of acetate, and enrichments yielded a syringate-H(inf2)-consuming acetogenic consortium. At in situ temperatures, acetate was a relatively stable anaerobic end product; however, extended incubation periods induced acetoclastic methanogenesis and sulfate reduction. Higher mesophilic and thermophilic temperatures greatly enhanced the capacity of soils to form methane. Although methanogenic and sulfate-reducing activities under in situ-relevant conditions were negligible, these findings nonetheless demonstrated the occurrence of methanogens and sulfate-reducing bacteria in these aerated terrestrial soils. In contrast to the protracted stability of acetate under anaerobic conditions at 15 to 20(deg)C with unsupplemented soils, acetate formed by forest soils was rapidly consumed in the presence of oxygen and nitrate, and substrate-product stoichiometries indicated that acetate turnover was coupled to oxygen-dependent respiration and denitrification. The collective results suggest that acetate formed under anaerobic conditions might constitute a trophic link between anaerobic and aerobic processes in forest soils.     

Acetogens and acetoclastic methanosarcinales govern methane formation in abandoned coal mines

In abandoned coal mines, methanogenic archaea are responsible for the production of substantial amounts of methane. The present study aimed to directly unravel the active methanogens mediating methane release as well as active bacteria potentially involved in the trophic network. Therefore, the stable-isotope-labeled precursors of methane, [(13)C]acetate and H(2)-(13)CO(2), were fed to liquid cultures from hard coal and mine timber from a coal mine in Germany. Guided by methane production rates, samples for DNA stable-isotope probing (SIP) with subsequent quantitative PCR and denaturing gradient gel electrophoretic (DGGE) analyses were taken over 6 months. Surprisingly, the formation of [(13)C]methane was linked to acetoclastic methanogenesis in both the [(13)C]acetate- and the H(2)-(13)CO(2)-amended cultures of coal and timber. H(2)-(13)CO(2) was used mainly by acetogens related to Pelobacter acetylenicus and Clostridium species. Active methanogens, closely affiliated with Methanosarcina barkeri, utilized the readily available acetate rather than the thermodynamically more favorable hydrogen. Thus, the methanogenic microbial community appears to be highly adapted to the low-H(2) conditions found in coal mines.     

Methanogenic activity and diversity in the centre of the Amsterdam Mud Volcano, Eastern Mediterranean Sea

Marine mud volcanoes are geological structures emitting large amounts of methane from their active centres. The Amsterdam mud volcano (AMV), located in the Anaximander Mountains south of Turkey, is characterized by intense active methane seepage produced in part by methanogens. To date, information about the diversity or the metabolic pathways used by the methanogens in active centres of marine mud volcanoes is limited. (14)C-radiotracer measurements showed that methylamines/methanol, H(2)/CO(2) and acetate were used for methanogenesis in the AMV. Methylotrophic methanogenesis was measured all along the sediment core, Methanosarcinales affiliated sequences were detected using archaeal 16S PCR-DGGE and mcrA gene libraries, and enrichments of methanogens showed the presence of Methanococcoides in the shallow sediment layers. Overall acetoclastic methanogenesis was higher than hydrogenotrophic methanogenesis, which is unusual for cold seep sediments. Interestingly, acetate porewater concentrations were extremely high in the AMV sediments. This might be the result of organic matter cracking in deeper hotter sediment layers. Methane was also produced from hexadecanes. For the most part, the methanogenic community diversity was in accordance with the depth distribution of the H(2)/CO(2) and acetate methanogenesis. These results demonstrate the importance of methanogenic communities in the centres of marine mud volcanoes.     

Thermophilic sulfate-reducing bacteria in cold marine sediment

Abstract Sulfate reduction was measured with the ³⁵SO₄²⁻ -tracer technique in slurries of sediment from Aarhus Bay, Denmark, where seasonal temperatures range from 0° to 15°C. The incubations were made at temperatures from 0°C to 80°C in temperature increments of 2°C to search for presence of psychrophilic, mesophilic and thermophilic sulfate-reducing bacteria. Detectable activity was initially only in the mesophilic range, but after a lag phase sulfate reduction by thermophilic sulfate-reducing bacteria were observed. No distinct activity of psychrophilic sulfate-reducing bacteria was detected. Time course experiments showed constant sulfate reduction rates at 4°C and 30°C, whereas the activity at 60°C increased exponentially after a lag period of one day. Thermophilic, endospore-forming sulfate-reducing bacteria, designated strain P60, were isolated and characterized as D esulfotomaculum kuznetsovii . The temperature response of growth and respiration of strain P60 agreed well with the measured sulfate reduction at 50°–70°C. Bacteria similar to strain P60 could thus be responsible for the measured thermophilic activity. The viable population of thermophilic sulfate-reducing bacteria and the density of their spores was determined in most probable number (MPN) dilutions. The density was 2.8·10⁴ cells·.g⁻¹ fresh sediment, and the enumerations suggested that they were all present as spores. This result agrees well with the observed lag period in sulfate reduction above 50°C. No environment with temperatures supporting the growth of these thermophiles is known in the region around Aarhus Bay.     

Acetoclastic and hydrogenotrophic methane production and methanogenic populations in an acidic West-Siberian peat bog

Sites in the West Siberian peat bog 'Bakchar' were acidic (pH 4.2-4.8), low in nutrients, and emitted CH4 at rates of 0.2-1.5 mmol m(-2) h(-1). The vertical profile of delta13CH4 and delta13CO2 dissolved in the porewater indicated increasing isotope fractionation and thus increasing contribution of H2/CO2-dependent methanogenesis with depth. The anaerobic microbial community at 30-50 cm below the water table produced CH4 with optimum activity at 20-25 degrees C and pH 5.0-5.5 respectively. Inhibition of methanogenesis with 2-bromo-ethane sulphonate showed that acetate, phenyl acetate, phenyl propionate and caproate were important intermediates in the degradation pathway of organic matter to CH4. Further degradation of these intermediates indicated that 62-72% of the CH4 was ultimately derived from acetate, the remainder from H2/CO2. Turnover times of [2-14C]acetate were on the order of 2 days (15, 25 degrees C) and accounted for 60-65% of total CH4 production. Conversion of 14CO2 to 14CH4 accounted for 35-43% of total CH4 production. These results showed that acetoclastic and hydrogenotrophic methanogenesis operated closely at a ratio of approximately 2 : 1 irrespective of the incubation temperature (4, 15 and 25 degrees C). The composition of the archaeal community was determined in the peat samples by terminal restriction fragment length polymorphism (T-RFLP) analysis and sequencing of amplified SSU rRNA gene fragments, and showed that members of Methanomicrobiaceae, Methanosarcinaceae and Rice cluster II (RC-II) were present. Other, presumably non-methanogenic archaeal clusters (group III, RC-IV, RC-V, RC-VI) were also detected. Fluorescent in situ hybridization (FISH) showed that the number of Bacteria decreased (from 24 x 10(7) to 4 x 10(7) cells per gram peat) with depth (from 5 to 55 cm below the water table), whereas the numbers of Archaea slightly increased (from 1 x 10(7) to 2 x 10(7) cells per gram peat). Methanosarcina spp. accounted for about half of the archaeal cells. Our results show that both hydrogenotrophic and acetoclastic methanogenesis are an integral part of the CH4-producing pathway in acidic peat and were represented by appropriate methanogenic populations.     

Detection of luciferase gene sequences in nonluminescent bacteria from the Chesapeake Bay

Washed excised roots of rice (Oryza sativa) produced H(2), CH(4) and fatty acids (millimolar concentrations of acetate, propionate, butyrate; micromolar concentrations of isovalerate, valerate) when incubated under anoxic conditions. Surface sterilization of the root material resulted in the inactivation of the production of CH(4), a strong reduction of the production of fatty acids and a transient (75 h) but complete inhibition of the production of H(2). Radioactive bicarbonate was incorporated into CH(4), acetate, propionate and butyrate. About 20-40% of the fatty acid carbon originated from CO(2) reduction. In the presence of phosphate, CH(4) was exclusively produced from H(2)/CO(2), since phosphate selectively inhibited acetoclastic methanogenesis. Acetoclastic methanogenesis was also selectively inhibited by methyl fluoride, while chloroform or 2-bromoethane sulfonate inhibited CH(4) production completely. Production of CH(4), acetate, propionate and butyrate from H(2)/CO(2) was always exergonic with Gibbs free energies <-20 kJ mol(-1) product. Chloroform inhibited the production of acetate and the incorporation of radioactive CO(2) into acetate. Simultaneously, H(2) was no longer consumed and accumulated, indicating that acetate was produced from H(2)/CO(2). Chloroform also resulted in increased production of propionate and butyrate whose formation from CO(2) became more exergonic upon addition of chloroform. Nevertheless, the incorporation of radioactive CO(2) into propionate and butyrate was inhibited by chloroform. The accumulation of propionate and butyrate in the presence of chloroform probably occurred by fermentation of organic matter, rather than by reduction of acetate and CO(2). [U-(14)C]Glucose was indeed converted to acetate, propionate, butyrate, CO(2) and CH(4). Radioactive acetate, CO(2) and CH(4) were also products of the degradation of [U-(14)C]cellulose and [U-(14)C]xylose. Addition of chloroform and methyl fluoride did not affect the product spectrum of [U-(14)C]glucose degradation. The application of combinations of selective inhibitors may be useful to elucidate anaerobic metabolic pathways in mixed microbial cultures and natural microbial communities.     

Methanogenic population structure in a variety of anaerobic bioreactors

The methanogenic community structures of six anaerobic sludges were examined using culture-independent techniques. The sludges were obtained from full-scale and laboratory-scale bioreactors, treating a variety of low- and high-strength, simple and complex wastewaters at psychrophilic (10-14 degrees C), mesophilic (37 degrees C) and thermophilic (55 degrees C) temperatures. Amplified rDNA restriction analysis identified 18 methanogenic operational taxonomic units in the six samples. 16S rRNA gene sequencing and phylogenetic reconstruction demonstrated that five separate groups of methanogens were represented with Methanosaeta-like species dominant in all sludges, but particularly in samples from a psychrophilic bioreactor treating low-strength synthetic sewage (75% of all clones detected).     

Effect of temperature on carbon and electron flow and on the archaeal community in methanogenic rice field soil

Temperature is an important factor controlling CH(4) production in anoxic rice soils. Soil slurries, prepared from Italian rice field soil, were incubated anaerobically in the dark at six temperatures of between 10 to 37 degrees C or in a temperature gradient block covering the same temperature range at intervals of 1 degrees C. Methane production reached quasi-steady state after 60 to 90 days. Steady-state CH(4) production rates increased with temperature, with an apparent activation energy of 61 kJ mol(-1). Steady-state partial pressures of the methanogenic precursor H(2) also increased with increasing temperature from <0.5 to 3.5 Pa, so that the Gibbs free energy change of H(2) plus CO(2)-dependent methanogenesis was kept at -20 to -25 kJ mol of CH(4)(-1) over the whole temperature range. Steady-state concentrations of the methanogenic precursor acetate, on the other hand, increased with decreasing temperature from <5 to 50 microM. Simultaneously, the relative contribution of H(2) as methanogenic precursor decreased, as determined by the conversion of radioactive bicarbonate to (14)CH(4), so that the carbon and electron flow to CH(4) was increasingly dominated by acetate, indicating that psychrotolerant homoacetogenesis was important. The relative composition of the archaeal community was determined by terminal restriction fragment length polymorphism (T-RFLP) analysis of the 16S rRNA genes (16S rDNA). T-RFLP analysis differentiated the archaeal Methanobacteriaceae, Methanomicrobiaceae, Methanosaetaceae, Methanosarcinaceae, and Rice clusters I, III, IV, V, and VI, which were all present in the rice field soil incubated at different temperatures. The 16S rRNA genes of Rice cluster I and Methanosaetaceae were the most frequent methanogenic groups. The relative abundance of Rice cluster I decreased with temperature. The substrates used by this microbial cluster, and thus its function in the microbial community, are unknown. The relative abundance of acetoclastic methanogens, on the other hand, was consistent with their physiology and the acetate concentrations observed at the different temperatures, i.e., the high-acetate-requiring Methanosarcinaceae decreased and the more modest Methanosaetaceae increased with increasing temperature. Our results demonstrate that temperature not only affected the activity but also changed the structure and the function (carbon and electron flow) of a complex methanogenic system.     

Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria, XI. Engineering thermostability and activity of lactate dehydrogenases from bacilli

An extensive comparative structural analysis of lactate dehydrogenase (LDH) sequences from thermophilic, mesophilic and psychrophilic bacilli revealed characteristic primary structural differences. These specific amino-acid substitutions were found in the entire LDH molecule. However, in certain regions of the LDH an accumulation of these exchanges could be detected. These regions seem to be particularly important for the temperature adaptation of the enzyme. The influence of one of such regions at the N-terminus on stability and activity of LDHs was analysed by the construction of hybrid mutants between LDH sequences from thermophilic, mesophilic and psychrophilic bacilli and also by site-directed mutagenesis experiments at five different positions. The substitutions of Thr-29 or Ser-39 to Ala residues in the LDH from the mesophilic B. megaterium increased the thermostability of the enzyme drastically (15 degrees C). An increase of 20 degrees C could be observed when both amino-acid substitutions were introduced. These amino-acid substitutions resulted in an increase of Km for pyruvate and led to a three-fold reduction of the activity (kcat/Km) at 40 degrees C compared with the wild type enzyme. The influence of these amino-acid substitutions was also investigated in the LDHs from thermophilic and psychrophilic bacilli. The high heat resistance of the LDH from the thermophilic B. stearothermophilus was not altered by the Ala to Thr and Ser substitutions at positions 29 and 39, respectively. This indicates a cooperatively stabilized conformation of this LDH. However, in this mutant of the B. stearothermophilus LDH the activity (kcat/Km) was increased two-fold.     

Directed evolution study of temperature adaptation in a psychrophilic enzyme

We have used laboratory evolution methods to enhance the thermostability and activity of the psychrophilic protease subtilisin S41, with the goal of investigating the mechanisms by which this enzyme can adapt to different selection pressures. A combined strategy of random mutagenesis, saturation mutagenesis and in vitro recombination (DNA shuffling) was used to generate mutant libraries, which were screened to identify enzymes that acquired greater thermostability without sacrificing low-temperature activity. The half-life of seven-amino acid substitution variant 3-2G7 at 60 degrees C is approximately 500 times that of wild-type and far surpasses those of homologous mesophilic subtilisins. The dependence of half-life on calcium concentration indicates that enhanced calcium binding is largely responsible for the increased stability. The temperature optimum of the activity of 3-2G7 is shifted upward by approximately 10 degrees C. Unlike natural thermophilic enzymes, however, the activity of 3-2G7 at low temperatures was not compromised. The catalytic efficiency, k(cat)/K(M), was enhanced approximately threefold over a wide temperature range (10 to 60 degrees C). The activation energy for catalysis, determined by the temperature dependence of k(cat)/K(M) in the range 15 to 35 degrees C, is nearly identical to wild-type and close to half that of its highly similar mesophilic homolog, subtilisin SSII, indicating that the evolved S41 enzyme retained its psychrophilic character in spite of its dramatically increased thermostability. These results demonstrate that it is possible to increase activity at low temperatures and stability at high temperatures simultaneously. The fact that enzymes displaying both properties are not found in nature most likely reflects the effects of evolution, rather than any intrinsic physical-chemical limitations on proteins.     

Metabolic Activity of Fatty Acid-Oxidizing Bacteria and the Contribution of Acetate, Propionate, Butyrate, and CO(2) to Methanogenesis in Cattle Waste at 40 and 60 degrees C

The quantitative contribution of fatty acids and CO(2) to methanogenesis was studied by using stirred, 3-liter bench-top digestors fed on a semicontinuous basis with cattle waste. The fermentations were carried out at 40 and 60 degrees C under identical loading conditions (6 g of volatile solids per liter of reactor volume per day, 10-day retention time). In the thermophilic digestor, acetate turnover increased from a prefeeding level of 16 muM/min to a peak (49 muM/min) 1 h after feeding and then gradually decreased. Acetate turnover in the mesophilic digestor increased from 15 to 40 muM/min. Propionate turnover ranged from 2 to 5.2 and 1.5 to 4.5 muM/min in the thermophilic and mesophilic digestors, respectively. Butyrate turnover (0.7 to 1.2 muM/min) was similar in both digestors. The proportion of CH(4) produced via the methyl group of acetate varied with time after feeding and ranged from 72 to 75% in the mesophilic digestor and 75 to 86% in the thermophilic digestor. The contribution from CO(2) reduction was 24 to 29% and 19 to 27%, respectively. Propionate and butyrate turnover accounted for 20% of the total CH(4) produced. Acetate synthesis from CO(2) was greatest shortly after feeding and was higher in the thermophilic digestor (0.5 to 2.4 muM/min) than the mesophilic digestor (0.3 to 0.5 muM/min). Counts of fatty acid-degrading bacteria were related to their turnover activity.     

Methanogenic population dynamics during start-up of anaerobic digesters treating municipal solid waste and biosolids

An aggressive start-up strategy was used to initiate codigestion in two anaerobic, continuously mixed bench-top reactors at mesophilic (37 degrees C) and thermophilic (55 degrees C) conditions. The digesters were inoculated with mesophilic anaerobic sewage sludge and cattle manure and were fed a mixture of simulated municipal solid waste and biosolids in proportions that reflect U.S. production rates. The design organic loading rate was 3.1 kg volatile solids/m3/day and the retention time was 20 days. Ribosomal RNA-targeted oligonucleotide probes were used to determine the methanogenic community structure in the inocula and the digesters. Chemical analyses were performed to evaluate digester performance. The aggressive start-up strategy was successful for the thermophilic reactor, despite the use of a mesophilic inoculum. After a short start-up period (20 days), stable performance was observed with high gas production rates (1.52 m3/m3/day), high levels of methane in the biogas (59%), and substantial volatile solids (54%) and cellulose (58%) removals. In contrast, the mesophilic digester did not respond favorably to the start-up method. The concentrations of volatile fatty acids increased dramatically and pH control was difficult. After several weeks of operation, the mesophilic digester became more stable, but propionate levels remained very high. Methanogenic population dynamics correlated well with performance measures. Large fluctuations were observed in methanogenic population levels during the start-up period as volatile fatty acids accumulated and were subsequently consumed. Methanosaeta species were the most abundant methanogens in the inoculum, but their levels decreased rapidly as acetate built up. The increase in acetate levels was paralleled by an increase in Methanosarcina species abundance (up to 11.6 and 4.8% of total ribosomal RNA consisted of Methanosarcina species ribosomal RNA in mesophilic and thermophilic digesters, respectively). Methanobacteriaceae were the most abundant hydrogenotrophic methanogens in both digesters, but their levels were higher in the thermophilic digester.     

Detection of novel syntrophic acetate‐oxidizing bacteria from biogas processes by continuous acetate enrichment approaches

To enrich syntrophic acetate‐oxidizing bacteria (SAOB), duplicate chemostats were inoculated with sludge from syntrophic acetate oxidation (SAO)‐dominated systems and continuously supplied with acetate (0.4 or 7.5 g l^?1) at high‐ammonia levels. The chemostats were operated under mesophilic (37°C) or thermophilic (52°C) temperature for about six hydraulic retention times (HRT 28 days) and were sampled over time. Irrespective of temperature, a methane content of 64–69% and effluent acetate level of 0.4–1.0 g l^?1 were recorded in chemostats fed high acetate. Low methane production in the low‐acetate chemostats indicated that the substrate supply was below the threshold for methanization of acetate via SAO. Novel representatives within the family Clostridiales and genus Syntrophaceticus (class Clostridia) were identified to represent putative SAOB candidates in mesophilic and thermophilic conditions respectively. Known SAOB persisted at low relative abundance in all chemostats. The hydrogenotrophic methanogens Methanoculleus bourgensis (mesophilic) and Methanothermobacter thermautotrophicus (thermophilic) dominated archaeal communities in the high‐acetate chemostats. In line with the restricted methane production in the low‐acetate chemostats, methanogens persisted at considerably lower abundance in these chemostats. These findings strongly indicate involvement in SAO and tolerance to high ammonia levels of the species identified here, and have implications for understanding community function in stressed anaerobic processes.     

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

The short-term effects of temperature on methanogenesis from acetate or CO(2) in a thermophilic (58 degrees C) anaerobic digestor were studied by incubating digestor sludge at different temperatures with C-labeled methane precursors (CH(3)COO or CO(2)). During a period when Methanosarcina sp. was numerous in the sludge, methanogenesis from acetate was optimal at 55 to 60 degrees C and was completely inhibited at 65 degrees C. A Methanosarcina culture isolated from the digestor grew optimally on acetate at 55 to 58 degrees C and did not grow or produce methane at 65 degrees C. An accidental shift of digestor temperature from 58 to 64 degrees 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 CH(3)COO was optimal at 65 degrees C and completely inhibited at 75 degrees C. A partially purified Methanothrix enrichment culture derived from the digestor had a maximum growth temperature near 70 degrees C. Methanogenesis from CO(2) in the sludge was optimal at 65 degrees C and still proceeded at 75 degrees C. A CO(2)-reducing Methanobacterium sp. isolated from the digestor was capable of methanogenesis at 75 degrees C. During the period when Methanothix sp. was apparently dominant, sludge incubated for 24 h at 65 degrees C produced more methane than sludge incubated at 60 degrees C, and no acetate accumulated at 65 degrees C. Methanogenesis was severely inhibited in sludge incubated at 70 degrees C, but since neither acetate nor H(2) 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.     

Trichloroethylene biodegradation by mesophilic and psychrophilic ammonia oxidizers and methanotrophs in groundwater microcosms.

This study investigated the efficiency of methane and ammonium for stimulating trichloroethylene (TCE) biodegradation in groundwater microcosms (flasks and batch exchange columns) at a psychrophilic temperature (12 degrees C) typical of shallow aquifers in the northern United States or a mesophilic temperature (24 degrees C) representative of most laboratory experiments. After 140 days, TCE biodegradation rates by ammonia oxidizers and methanotrophs in mesophilic flask microcosms were similar (8 to 10 nmol day-1), but [14C]TCE mineralization (biodegradation to 14CO2) by ammonia oxidizers was significantly greater than that by methanotrophs (63 versus 53%). Under psychrophilic conditions, [14C]TCE mineralization in flask systems by ammonia oxidizers and methanotrophs was reduced to 12 and 5%, respectively. In mesophilic batch exchange columns, average TCE biodegradation rates for methanotrophs (900 nmol liter-1 day-1) were not significantly different from those of ammonia oxidizers (775 nmol liter-1 day-1). Psychrophilic TCE biodegradation rates in the columns were similar with both biostimulants and averaged 145 nmol liter-1 day-1. Methanotroph biostimulation was most adversely affected by low temperatures. At 12 degrees C, the biodegradation efficiencies (TCE degradation normalized to microbial activity) of methanotrophs and ammonia oxidizers decreased by factors of 2.6 and 1.6, respectively, relative to their biodegradation efficiencies at 24 degrees C. Collectively, these experiments demonstrated that in situ bioremediation of TCE is feasible at the psychrophilic temperatures common in surficial aquifers in the northern United States and that for such applications biostimulation of ammonia oxidizers could be more effective than has been previously reported.