Effect of propionate toxicity on methanogen-enriched sludge, Methanobrevibacter smithii, and Methanospirillum hungatii at different pH values.

The effect of propionate toxicity at different pH values (6.5, 7.0, and 8.0) on methanogen-enriched sludge. Methanobrevibacter smithii, and Methanospirillum hungatii was studied. Organisms were grown in Balch medium 3 in Hungate tubes, and toxicity was characterized by a decrease in production of methane and in bacterial numbers. Propionate inhibited bacterial growth and cumulative methane production at concentrations as low as 20 mM. In the absence of propionate, the methanogen-enriched sludge and M. smithii showed better cumulative methane production at pH 6.5 and 7.0 than at pH 8.0. However, in the presence of propionate, these organisms showed better cumulative methane production at pH 8.0. M. hungatii differed in its behavior; the best values of cumulative methane production for this organism occurred at pH 7.0. Bacterial numbers reflected the microbial response to the presence of propionate. The highest counts of methanogenic bacteria were observed at pH 6.5 and 8.0. The numbers of methanogens were affected by the presence of propionate even at concentrations as low as 20 or 30 mM; at propionate concentrations above 80 mM, the methanogen count was affected by at least 2 orders of magnitude. Upon comparison of the responses of the pure cultures and the methanogen-enriched sludge to increasing propionate concentrations, it was found that the sensitivity of the pure cultures was similar to that of the methanogens in the sludge.     

Effect of propionate toxicity on methanogenesis of night soil at phychrophilic temperature

The effect of propionate concentrations on biodegradation of human waste (night soil) was studied at 10 degrees C. Propionate was toxic for the biomethanation at all the pH tested (6.0, 7.0 and 8.0). The maximum reduction in biogas production in presence of 200 mM propionate was observed at pH 7.0 followed by 8.0. The methane content in biogas also followed a similar trend and at pH 7.0 an 11.5% decrease was observed. Propionate caused the reduction of methanogenic count by an approximately 2log value. Total volatile fatty acids increased with the increase in propionate concentration and particularly accumulation of propionate was observed. The results were also compared with the 30 degrees C fermentation.     

Effects of o-cresol co-disposal and pH on the methanogenic degradation of refuse

SULISTI, I.A. WATSON-CRAIK AND E. SENIOR. 1996. Both maximum o -cresol degradation and activity of sulphate-reducing bacteria (SRB) were observed at refuse pH values between 7.0 and 8.0. Optimum pH values for methane release were between 6.5 and 7.5. Partial inhibition of methane production was recorded at pH 5.7, 6.0 and 8.0, whilst sulphate reduction was inhibited partially at pH values 5.7–6.5. Both sulphate reduction and methanogenesis were completely inhibited in refuse with initial pH 4.0. The catabolism of acetate occurred under similar conditions to methane production, and was promoted at pH 6.5–7.5. It appeared that propionate oxidation depended upon the activities of SRB. Optimum conditions for the metabolism of propionate and other volatile fatty acids were between pH 7.0 and 8.0.     

Inhibition of methanogenesis in pure cultures by ammonia, fatty acids, and heavy metals, and protection against heavy metal toxicity by sewage sludge

The effect of ammonium chloride, sodium butyrate, sodium propionate, and the heavy metals nickel, zinc, and copper on methanogenesis by pure cultures of Methanospirillum hungatei, Methanosarcina barkeri, Methanobacterium thermoautotrophicum, and Methanobacterium formicicum at pH 6.5 was studied. The latter three strains were resistant to greater than 60 g/L of the volatile fatty acids and to greater than 10 g/L of NH3 N. Methanospirillum hungatei was somewhat more sensitive with 50% inhibition of methanogenesis occurring at 4.2 g/L NH3 N, 27 g/L butyrate, and 41 g/L propionate. All strains were very sensitive to both copper (1-5 mg/L) and zinc (1-10 mg/L), but much more resistant to nickel. Zinc and copper concentrations 30 to 270 times higher were required to cause inhibition of Msp. hungatei incubated in sewage sludge compared with buffer, indicating a strong protective environment was afforded the methanogens against heavy metal toxicity in the sludge.     

Acetate threshold values and acetate activating enzymes in methanogenic bacteria

Abstract The minimum threshold concentrations of acetate utilization and the enzymes responsible for acetate activation of several methanogenic bacteria were investigated and compared with literature data. The minimum acetate concentrations reached by hydrogenotrophic methane bacteria, which require acetate as carbon source, were between 0.4 and 0.6 mM. The acetoclastic Methanosarcina achieves acetate concentrations between 0.2 and 1.2 mM and Methanothrix between 7 and 70 μM. For the activation of acetate most of the hydrogenotrophic methane bacteria investigated use an acetyl-CoA synthetase with a relatively low K _m (40–90 μM) for acetate. although the affinity for acetate was high, the hydrogenotrophic methane bacteria were not able to remove acetate to lower concentrations than the acetoclastic methane bacteria, neither in pure cultures nor in anaerobic granular sludge samples. Based on these observations, it is not likely that hydrogenotrophic methanogens compete strongly for acetate with the acetoclastic methane bacteria.     

Characterization of a strain of Methanospirillum hungatti.

The results of morphological, base ratio, nutritional, temperature, and pH studies on a strain of Methanospirillum hungatii, isolated from an anaerobic pear waste digester, are described. The isolate, designated as strain GP 1, was compared with some of the characteristics of type-strain M. hungatii JF 1. Strain GP 1 is Gram-negative, weakly motile, and a strict anaerobe with a guanine plus cytosine (G +C) content of 46.5 mol%. The preferred substrates for methane production are hydrogen, carbon dioxide, and formate. Acetate is used under certain conditions but its specific contribution to cell carbon and (or) methane formation was not established. The optimum temperature for both growth and methane production is 35 degrees C, but growth and methane production occur over the range 25-45 degrees C. Methane production is optimal at pH 7.0.     

Evaluation of complementary effects of 9,10-anthraquinone and fumaric acid on methanogenesis and ruminal fermentation in vitro

The objective of the present study was to investigate the hypothesis that 9,10-anthraquinone (AQ) in combination with fumaric acid (FMA) may provide complementary effects to inhibit methanogens and enhance rumen's capacity for better utilisation of FMA towards propionate production. Three levels of AQ and four levels of FMA were tested in a 3 x 4 factorial design using in vitro gas production technique. AQ reduced the total gas and methane production significantly. The combination of 4 ppm AQ with FMA had additive effect on concentration of propionate. Supplementation of AQ alone resulted in hydrogen accumulation (p < 0.001), whereas presence of FMA (up to 6.5 mM) along with AQ declined hydrogen concentration (p < 0.001). The level of 4 ppm AQ did not affect in vitro digestibility, however, a reduction of organic matter digestibility was caused by 8 ppm AQ (p < 0.001), which was partially compensated by the addition of FMA (p = 0.06). The optimum FMA level depended on the AQ concentration. At 4 ppm AQ, a FMA level of 3.5 mM had best possible effect on partitioning factor and microbial biomass production (p < 0.001), though, at 8 ppm AQ the higher level of FMA (6.5 mM) responded better. Overall, FMA in combination with AQ provided an alternative hydrogen sink and might be introduced as a novel strategy for mitigation of enteric methane emission. Nevertheless, the result should be proved by in vivo experiments.     

pH and peptide supply can radically alter bacterial populations and short-chain fatty acid ratios within microbial communities from the human colon

The effects of changes in the gut environment upon the human colonic microbiota are poorly understood. The response of human fecal microbial communities from two donors to alterations in pH (5.5 or 6.5) and peptides (0.6 or 0.1%) was studied here in anaerobic continuous cultures supplied with a mixed carbohydrate source. Final butyrate concentrations were markedly higher at pH 5.5 (0.6% peptide mean, 24.9 mM; 0.1% peptide mean, 13.8 mM) than at pH 6.5 (0.6% peptide mean, 5.3 mM; 0.1% peptide mean, 7.6 mM). At pH 5.5 and 0.6% peptide input, a high butyrate production coincided with decreasing acetate concentrations. The highest propionate concentrations (mean, 20.6 mM) occurred at pH 6.5 and 0.6% peptide input. In parallel, major bacterial groups were monitored by using fluorescence in situ hybridization with a panel of specific 16S rRNA probes. Bacteroides levels increased from ca. 20 to 75% of total eubacteria after a shift from pH 5.5 to 6.5, at 0.6% peptide, coinciding with high propionate formation. Conversely, populations of the butyrate-producing Roseburia group were highest (11 to 19%) at pH 5.5 but fell at pH 6.5, a finding that correlates with butyrate formation. When tested in batch culture, three Bacteroides species grew well at pH 6.7 but poorly at pH 5.5, which is consistent with the behavior observed for the mixed community. Two Roseburia isolates grew equally well at pH 6.7 and 5.5. These findings suggest that a lowering of pH resulting from substrate fermentation in the colon may boost butyrate production and populations of butyrate-producing bacteria, while at the same time curtailing the growth of Bacteroides spp.     

Carbon nanotubes accelerate methane production in pure cultures of methanogens and in a syntrophic coculture

Carbon materials have been reported to facilitate direct interspecies electron transfer (DIET) between bacteria and methanogens improving methane production in anaerobic processes. In this work, the effect of increasing concentrations of carbon nanotubes (CNT) on the activity of pure cultures of methanogens and on typical fatty acid‐degrading syntrophic methanogenic coculture was evaluated. CNT affected methane production by methanogenic cultures, although acceleration was higher for hydrogenotrophic methanogens than for acetoclastic methanogens or syntrophic coculture. Interestingly, the initial methane production rate (IMPR) by Methanobacterium formicicum cultures increased 17 times with 5 g·L^?1 CNT. Butyrate conversion to methane by Syntrophomonas wolfei and Methanospirillum hungatei was enhanced (～1.5 times) in the presence of CNT (5 g·L^?1), but indications of DIET were not obtained. Increasing CNT concentrations resulted in more negative redox potentials in the anaerobic microcosms. Remarkably, without a reducing agent but in the presence of CNT, the IMPR was higher than in incubations with reducing agent. No growth was observed without reducing agent and without CNT. This finding is important to re‐frame discussions and re‐interpret data on the role of conductive materials as mediators of DIET in anaerobic communities. It also opens new challenges to improve methane production in engineered methanogenic processes.     

Effects of pH shifts, bile salts, and glucose on sporulation of Clostridium perfringens NCTC 8798

The sporulation of Clostridium perfringens NCTC 8798 was studied after exposing vegetative cells to: pH values of 1.5 to 8.0 in fluid thioglycolate broth (for 2h) and then transferring them to Duncan-Strong (DS) sporulation medium; sodium cholate or sodium deoxycholate (0.3 to 6.5 mM) in DS medium; or Rhia-Solberg medium with 0.4% (wt/wt) starch, glucose, or both added at 0 to 55 mM. At pH 1.5, no culturable heat-resistant spores were formed. For cells exposed to pH 3.0, 4.0, 5.0, or 6.0, increases in heat-resistant spores were not seen until after a lag of 12 to 13 h, whereas the lag was only 2 to 3 h for cells exposed to pH 7.0 or 8.0. Maximal spore crops were produced after only 6 to 8 h for cells exposed to pH 7 or 8, but 16 to 18 h was required for production of maximal spore crops by cells exposed to the lower-pH media. The addition of sodium cholate (3.5 to 6.5 mM) to DS medium only slightly reduced the culturable heat-resistant spore count from 1.9 X 10(7) to 3 X 10(6)/ml. The addition of 1.8 mM or more sodium deoxycholate reduced the culturable heat-resistant spore count to less than 10/ ml. When either starch or glucose alone was added to Rhia-Solberg medium there was no production of culturable heat-resistant spores, but a combination of 0.4% (wt/wt) starch and 4.4 mM glucose yielded 6 X 10(5) spores/ml. The spore production remained at this level for glucose concentrations of 6 to 22 mM, but then declined to about 3 X 10(3) spores per ml at higher concentrations.     

Effects of pH shifts, bile salts, and glucose on sporulation of Clostridium perfringens NCTC 8798.

The sporulation of Clostridium perfringens NCTC 8798 was studied after exposing vegetative cells to: pH values of 1.5 to 8.0 in fluid thioglycolate broth (for 2h) and then transferring them to Duncan-Strong (DS) sporulation medium; sodium cholate or sodium deoxycholate (0.3 to 6.5 mM) in DS medium; or Rhia-Solberg medium with 0.4% (wt/wt) starch, glucose, or both added at 0 to 55 mM. At pH 1.5, no culturable heat-resistant spores were formed. For cells exposed to pH 3.0, 4.0, 5.0, or 6.0, increases in heat-resistant spores were not seen until after a lag of 12 to 13 h, whereas the lag was only 2 to 3 h for cells exposed to pH 7.0 or 8.0. Maximal spore crops were produced after only 6 to 8 h for cells exposed to pH 7 or 8, but 16 to 18 h was required for production of maximal spore crops by cells exposed to the lower-pH media. The addition of sodium cholate (3.5 to 6.5 mM) to DS medium only slightly reduced the culturable heat-resistant spore count from 1.9 X 10(7) to 3 X 10(6)/ml. The addition of 1.8 mM or more sodium deoxycholate reduced the culturable heat-resistant spore count to less than 10/ ml. When either starch or glucose alone was added to Rhia-Solberg medium there was no production of culturable heat-resistant spores, but a combination of 0.4% (wt/wt) starch and 4.4 mM glucose yielded 6 X 10(5) spores/ml. The spore production remained at this level for glucose concentrations of 6 to 22 mM, but then declined to about 3 X 10(3) spores per ml at higher concentrations.     

pH and Peptide Supply Can Radically Alter Bacterial Populations and Short-Chain Fatty Acid Ratios within Microbial Communities from the Human Colon

The effects of changes in the gut environment upon the human colonic microbiota are poorly understood. The response of human fecal microbial communities from two donors to alterations in pH (5.5 or 6.5) and peptides (0.6 or 0.1%) was studied here in anaerobic continuous cultures supplied with a mixed carbohydrate source. Final butyrate concentrations were markedly higher at pH 5.5 (0.6% peptide mean, 24.9 mM; 0.1% peptide mean, 13.8 mM) than at pH 6.5 (0.6% peptide mean, 5.3 mM; 0.1% peptide mean, 7.6 mM). At pH 5.5 and 0.6% peptide input, a high butyrate production coincided with decreasing acetate concentrations. The highest propionate concentrations (mean, 20.6 mM) occurred at pH 6.5 and 0.6% peptide input. In parallel, major bacterial groups were monitored by using fluorescence in situ hybridization with a panel of specific 16S rRNA probes. Bacteroides levels increased from ca. 20 to 75% of total eubacteria after a shift from pH 5.5 to 6.5, at 0.6% peptide, coinciding with high propionate formation. Conversely, populations of the butyrate-producing Roseburia group were highest (11 to 19%) at pH 5.5 but fell at pH 6.5, a finding that correlates with butyrate formation. When tested in batch culture, three Bacteroides species grew well at pH 6.7 but poorly at pH 5.5, which is consistent with the behavior observed for the mixed community. Two Roseburia isolates grew equally well at pH 6.7 and 5.5. These findings suggest that a lowering of pH resulting from substrate fermentation in the colon may boost butyrate production and populations of butyrate-producing bacteria, while at the same time curtailing the growth of Bacteroides spp.     

Characterization of metabolic performance of methanogenic granules treating brewery wastewater: role of sulfate-reducing bacteria.

Granules from an upflow anaerobic sludge blanket system treating a brewery wastewater that contained mainly ethanol, propionate, and acetate as carbon sources and sulfate (0.6 to 1.0 mM) were characterized for their physical and chemical properties, metabolic performance on various substrates, and microbial composition. Transmission electron microscopic examination showed that at least three types of microcolonies existed inside the granules. One type consisted of Methanothrix-like rods with low levels of Methanobacterium-like rods; two other types appeared to be associations between syntrophic-like acetogens and Methanobacterium-like organisms. The granules were observed to be have numerous vents or channels on the surface that extended into the interior portions of the granules that may be involved in release of gas formed within the granules. The maximum substrate conversion rates (millimoles per gram of volatile suspended solids per day) at 35 degrees C in the absence of sulfate were 45.1, 8.04, 4.14, and 5.75 for ethanol, acetate, propionate, and glucose, respectively. The maximum methane production rates (millimoles per gram of volatile suspended solids per day) from H2-CO2 and formate were essentially equal for intact granules (13.7 and 13.5) and for physically disrupted granules (42 and 37). During syntrophic ethanol conversion, both hydrogen and formate were formed by the granules. The concentrations of these two intermediates were maintained at a thermodynamic equilibrium, indicating that both are intermediate metabolites in degradation. Formate accumulated and was then consumed during methanogenesis from H2-CO2. Higher concentrations of formate accumulated in the absence of sulfate than in the presence of sulfate. The addition of sulfate (8 to 9 mM) increased the maximum substrate degradation rates for propionate and ethanol by 27 and 12%, respectively. In the presence of this level of sulfate, sulfate-reducing bacteria did not play a significant role in the metabolism of H2, formate, and acetate, but ethanol and propionate were converted via sulfate reduction by approximately 28 and 60%, respectively. In the presence of 2.0 mM molybdate, syntrophic propionate and ethanol conversion by the granules was inhibited by 97 and 29%, respectively. The data show that in this granular microbial consortium, methanogens and sulfate-reducing bacteria did not compete for common substrates. Syntrophic propionate and ethanol conversion was likely performed primarily by sulfate-reducing bacteria, while H2, formate, and acetate were consumed primarily by methanogens.     

Characterization of metabolic performance of methanogenic granules treating brewery wastewater: role of sulfate-reducing bacteria.

Granules from an upflow anaerobic sludge blanket system treating a brewery wastewater that contained mainly ethanol, propionate, and acetate as carbon sources and sulfate (0.6 to 1.0 mM) were characterized for their physical and chemical properties, metabolic performance on various substrates, and microbial composition. Transmission electron microscopic examination showed that at least three types of microcolonies existed inside the granules. One type consisted of Methanothrix-like rods with low levels of Methanobacterium-like rods; two other types appeared to be associations between syntrophic-like acetogens and Methanobacterium-like organisms. The granules were observed to be have numerous vents or channels on the surface that extended into the interior portions of the granules that may be involved in release of gas formed within the granules. The maximum substrate conversion rates (millimoles per gram of volatile suspended solids per day) at 35 degrees C in the absence of sulfate were 45.1, 8.04, 4.14, and 5.75 for ethanol, acetate, propionate, and glucose, respectively. The maximum methane production rates (millimoles per gram of volatile suspended solids per day) from H2-CO2 and formate were essentially equal for intact granules (13.7 and 13.5) and for physically disrupted granules (42 and 37). During syntrophic ethanol conversion, both hydrogen and formate were formed by the granules. The concentrations of these two intermediates were maintained at a thermodynamic equilibrium, indicating that both are intermediate metabolites in degradation. Formate accumulated and was then consumed during methanogenesis from H2-CO2. Higher concentrations of formate accumulated in the absence of sulfate than in the presence of sulfate. The addition of sulfate (8 to 9 mM) increased the maximum substrate degradation rates for propionate and ethanol by 27 and 12%, respectively. In the presence of this level of sulfate, sulfate-reducing bacteria did not play a significant role in the metabolism of H2, formate, and acetate, but ethanol and propionate were converted via sulfate reduction by approximately 28 and 60%, respectively. In the presence of 2.0 mM molybdate, syntrophic propionate and ethanol conversion by the granules was inhibited by 97 and 29%, respectively. The data show that in this granular microbial consortium, methanogens and sulfate-reducing bacteria did not compete for common substrates. Syntrophic propionate and ethanol conversion was likely performed primarily by sulfate-reducing bacteria, while H2, formate, and acetate were consumed primarily by methanogens.     

Enrichment of thermophilic syntrophic anaerobic glutamate-degrading consortia using a dialysis membrane reactor

A dialysis cultivation system was used to enrich slow-growing moderately thermophilic anaerobic bacteria at high cell densities. Bicarbonate buffered mineral salts medium with 5 mM glutamate as the sole carbon and energy source was used and the incubation temperature was 55 degrees C. The reactor inoculum originated from anaerobic methanogenic granular sludge bed reactors. The microbial population was monitored over a period of 2 years using the most probable number (MPN) technique. In the reactor glutamate was readily degraded to ammonium, methane, and carbon dioxide. Cell numbers of glutamate-degrading organisms increased 400-fold over the first year. In medium supplemented with bromoethane sulfonic acid (BES, an inhibitor of methanogenesis), tenfold lower cell numbers were counted, indicating the syntrophic nature of glutamate degradation. After 2 years of reactor operation the predominant organisms were isolated and characterized. Methanobacterium thermoautotrophicum (strain R43) and a Methanosaeta thermophila strain (strain A) were the predominant hydrogenotrophic and acetoclastic methanogens, respectively. The numbers in which the organisms were present in the reactor after 24 months of incubation were 8.6 x 10(9) and 3.8 x 10(7) mL(-1) sludge, respectively. The most predominant glutamate-degrading organism (8.6 x 10(7) mL(-1) sludge), strain Z, was identified as a new species, Caloramator coolhaasii. It converted glutamate to hydrogen, acetate, some propionate, ammonium, and carbon dioxide. Growth of this syntrophic organism on glutamate was strongly enhanced by the presence of methanogens.     

Contribution of anaerobic protozoa and methanogens to hindgut metabolic activities of the American cockroach, Periplaneta americana.

The ciliate Nyctotherus ovalis occurs in high numbers in the hindgut of the American cockroach (Periplaneta americana) and harbors methanogenic bacteria as endosymbionts. The contribution of these hindgut microorganisms to metabolic and developmental processes of P. americana was studied by comparing cultures of cockroaches in which the composition of the hindgut microbial population was altered in various ways. Rearing the insects protozoan free resulted in increased insect generation time, decreased adult body weight, and absence of methane production. After feeding of protozoan-free adult cockroaches with a hindgut suspension containing N. ovalis and methanogens, methane increased to normal values and insect body weight was restored during the development of the second generation of insects. Feeding the protozoan-free cockroaches a hindgut suspension which was made free of N. ovalis resulted in an increase in methane production to only about 20% of the normal methane production level. This suggests that the methanogenic endosymbionts of N. ovalis are the major source of methane production in the hindgut. Inhibition of methanogens by addition of bromoethanesulfonic acid to the drinking water of a normal cockroach culture resulted in a reduction of methane production to about 2% of the normal level. No effects on insect body weight or the number of N. ovalis organisms were observed, but the fermentation pattern in the hindgut was shifted towards a relative increase in propionate levels. Similar results were obtained for in vitro cultures of hindgut microorganisms treated with bromoethanesulfonic acid. The results suggest a major role for hindgut protozoa in cockroach metabolic activities, especially during the insect growth period. The relatively large amounts of methane produced by cockroaches and by other methane-producing xylophagous insects suggest a major contribution by insects to global methane production.     

Methane production in an UASB reactor operated under periodic mesophilic-thermophilic conditions

Methane production was studied in a laboratory-scale 10 L anaerobic upflow sludge bed (UASB) reactor with periodic variations of the reactor temperature. On a daily basis the temperature was varied between 35 and 45 degrees C or 35 and 55 degrees C with a heating period of 6 h. Each temperature increase was accompanied by an increase in methane production and a decrease in the concentration of soluble organic matter in the effluent. In comparison to a reactor operated at 35 degrees C, a net increase in methane production of up to 22% was observed. Batch activity tests demonstrated a tolerance of mesophilic methanogenic populations to short-term, 2-6 h, temperature increases, although activity of acetoclastic methanogens decreased after 6 h exposure to a temperature of 55 degrees C. 16S sequencing of DGGE bands revealed proliferation of temperature-tolerant Methanospirillum hungatii sp. in the reactor.     

Methanogenesis in thermophilic biogas reactors

Methanogenesis in thermophilic biogas reactors fed with different wastes is examined. The specific methanogenic activity with acetate or hydrogen as substrate reflected the organic loading of the specific reactor examined. Increasing the loading of thermophilic reactors stabilized the process as indicated by a lower concentration of volatile fatty acids in the effluent from the reactors. The specific methanogenic activity in a thermophilic pilot-plant biogas reactor fed with a mixture of cow and pig manure reflected the stability of the reactor. The numbers of methanogens counted by the most probable number (MPN) technique with acetate or hydrogen as substrate were further found to vary depending on the loading rate and the stability of the reactor. The numbers of methanogens counted with antibody probes in one of the reactor samples was 10 times lower for the hydrogen-utilizing methanogens compared to the counts using the MPN technique, indicating that other non-reacting methanogens were present. Methanogens that reacted with the probe againstMethanobacterium thermoautotrophicum were the most numerous in this reactor. For the acetate-utilizing methanogens, the numbers counted with the antibody probes were more than a factor of 10 higher than the numbers found by MPN. The majority of acetate utilizing methanogens in the reactor wereMethanosarcina spp. single cells, which is a difficult form of the organism to cultivatein vitro. No reactions were observed with antibody probes raised againstMethanothrix soehngenii orMethanothrix CALS-1 in any of the thermophilic biogas reactors examined. Studies using 2-¹⁴C-labeled acetate showed that at high concentrations (more than approx. 1 mM) acetate was metabolized via the aceticlastic pathway, transforming the methyl-group of acetate into methane. When the concentration of acetate was less than approx. 1 mM, most of the acetate was oxidized via a two-step mechanism (syntrophic acetate oxidation) involving one organism oxidizing acetate into hydrogen and carbon dioxide and a hydrogen-utilizing methanogen forming the products of the first microorganism into methane. In thermophilic biogas reactors, acetate oxidizing cultures occupied the niche ofMethanothrix species, aceticlastic methanogens which dominate at low acetate concentrations in mesophilic systems. Normally, thermophilic biogas reactors are operated at temperatures from 52 to 56° C. Experiments using biogas reactors fed with cow manure showed that the same biogas yield found at 55° C could be obtained at 61° C after a long adaptation period. However, propionate degradation was inhibited by increasing the temperature.     

Effects of pH and hydraulic retention time on hydrogen production versus methanogenesis during anaerobic fermentation of organic household solid waste under extreme-thermophilic temperature (70 degrees C)

Two continuously stirred tank reactors were operated with household solid waste at 70 degrees C, for hydrogen and methane production. The individual effect of hydraulic retention time (HRT as 1, 2, 3, 4, and 6 days) at pH 7 or pH (5, 5.5, 6, 6.5, 7) at 3-day HRT was investigated on the hydrogen production versus methanogenesis. It was found that at pH 7, the maximum hydrogen yield was 107 mL-H(2)/g VS(added) (volatile solid added) but no stable hydrogen production was obtained as after some time methanogenesis was initiated at all tested HRTs. This demonstrated that sludge retention time alone was not enough for washing out the methanogens at pH 7 under extreme-thermophilic conditions. Oppositely, we showed that keeping the pH level at 5.5 was enough to inhibit methane and produce hydrogen stably at 3-day HRT. However, the maximum stable hydrogen yield was low at 21 mL-H(2)/g VS(added).     

Effects of acetate, propionate, and butyrate on the thermophilic anaerobic degradation of propionate by methanogenic sludge and defined cultures.

The effects of acetate, propionate, and butyrate on the anaerobic thermophilic conversion of propionate by methanogenic sludge and by enriched propionate-oxidizing bacteria in syntrophy with Methanobacterium thermoautotrophicum delta H were studied. The methanogenic sludge was cultivated in an upflow anaerobic sludge bed (UASB) reactor fed with propionate (35 mM) as the sole substrate for a period of 80 days. Propionate degradation was shown to be severely inhibited by the addition of 50 mM acetate to the influent of the UASB reactor. The inhibitory effect remained even when the acetate concentration in the effluent was below the level of detection. Recovery of propionate oxidation occurred only when acetate was omitted from the influent medium. Propionate degradation by the methanogenic sludge in the UASB reactor was not affected by the addition of an equimolar concentration (35 mM) of butyrate to the influent. However, butyrate had a strong inhibitory effect on the growth of the propionate-oxidizing enrichment culture. In that case, the conversion of propionate was almost completely inhibited at a butyrate concentration of 10 mM. However, addition of a butyrate-oxidizing enrichment culture abolished the inhibitory effect, and propionate oxidation was even stimulated. All experiments were conducted at pH 7.0 to 7.7. The thermophilic syntrophic culture showed a sensitivity to acetate and propionate similar to that of mesophilic cultures described in the literature. Additions of butyrate or acetate to the propionate medium had no effect on the hydrogen partial pressure in the biogas of an UASB reactor, nor was the hydrogen partial pressure in propionate-degrading cultures affected by the two acids.(ABSTRACT TRUNCATED AT 250 WORDS)