Effects of hydrogen and formate on the degradation of propionate and butyrate in thermophilic granules from an upflow anaerobic sludge blanket reactor.

Degradation of propionate and butyrate in whole and disintegrated granules from a thermophilic (55 degrees C) upflow anaerobic sludge blanket reactor fed with acetate, propionate, and butyrate as substrates was examined. The propionate and butyrate degradation rates in whole granules were 1.16 and 4.0 mumol/min/g of volatile solids, respectively, and the rates decreased 35 and 25%, respectively, after disintegration of the granules. The effect of adding different hydrogen-oxidizing bacteria (both sulfate reducers and methanogens), some of which used formate in addition to hydrogen, to disintegrated granules was tested. Addition of either Methanobacterium thermoautotrophicum delta H, a hydrogen-utilizing methanogen that does not use formate, or Methanobacterium sp. strain CB12, a hydrogen- and formate-utilizing methanogen, to disintegrated granules increased the degradation rate of both propionate and butyrate. Furthermore, addition of a thermophilic sulfate-reducing bacterium (a Desulfotomaculum sp. isolated in our laboratory) to disintegrated granules improved the degradation of both substrates even more than the addition of methanogens. By monitoring the hydrogen partial pressure in the cultures, a correlation between the hydrogen partial pressure and the degradation rate of propionate and butyrate was observed, showing a decrease in the degradation rate with increased hydrogen partial pressure. No significant differences in the stimulation of the degradation rates were observed when the disintegrated granules were supplied with methanogens that utilized hydrogen only or hydrogen and formate. This indicated that interspecies formate transfer was not important for stimulation of propionate and butyrate degradation.     

Role of formate and hydrogen in the degradation of propionate and butyrate by defined suspended cocultures of acetogenic and methanogenic bacteria

The butyrate-degradingSyntrophospora bryantii degrades butyrate and a propionate-degrading strain (MPOB) degrades propionate in coculture with the hydrogen- and formate-utilizingMethanospirillum hungatii orMethanobacterium formicicum. However, the substrates are not degraded in constructed cocultures with twoMethanobrevibacter arboriphilus strains which are only able to consume hydrogen. Pure cultures of the acetogenic bacteria form both hydrogen and formate during butyrate oxidation with pentenoate as electron acceptor and during propionate oxidation with fumarate as electron acceptor. Using the highest hydrogen and formate levels which can be reached by the acetogens and the lowest hydrogen and formate levels which can be maintained by the methanogens it appeared that the calculated formate diffusion rates are about 100 times higher than the calculated hydrogen diffusion rates.     

Methanogenesis from acetate and propionate by thermophilic down-flow anaerobic packed-bed reactor

The maximum propionate removal rate was 13.7 g/L-reactor/day at the organic loading rate of 66.4 kg-CODcr/m3-reactor/day (HRT, 4.75 h); however, the removal efficiency was very low. Clone library analysis and quantification by real-time PCR using 16S rRNA gene revealed that the population of methanogenic archaea in the biofilm fraction that developed on the packed bed was higher than that in the liquid fraction. The clone, which is related to Methanosarcina, was detected only in the biofilm fraction. The clones closely related to Pelotomaculum, which is capable of degrading propionate, and the hydrogenotrophic methanogen Methanothermobactor were also detected only in the biofilm fraction in the acetate and propionate-fed reactor. The experimental results indicate that the packed-bed design can maintain a sufficiently high density of methanogenic microorganisms within the system even at reduced HRTs as well as facilitate an efficient degradation of propionate and acetate, possibly through syntrophic reactions.     

Kinetics of lactate,acetate and propionate in unadapted and lactate-adapted thermophilic,anaerobic sewage sludge: the influence of sludge adaptation for start-up of thermophilic UASB-reactors

Summary A thermophilic anaerobic sludge digestor was adapted to lactate metabolism. The adapted sludge showed an improved capacity for lactate degradation when tested by a batch activity test, compared to the performance of unadapted sludge. Acetate was the major intermediate produced during the degradation. When adapted sludge was used as the inoculum for a lactate-fed, upflow anaerobic sludge blanket (UASB) reactor, the chemical oxygen demand reduction rate was higher than with unadapted sludge. After 39 days, however, the difference vanished due to an extensive wash-out of sludge from the reactor inoculated with adapted sludge.Offprint requests to: B. K. Ahring     

Inhibition of propionate degradation by acetate in methanogenic fluidized bed reactors

Summary The degradation of acetate, propionate and butyrate was monitored during start-up of five lab-scale methanogenic fluidized bed reactors on an artificially prepared waste water. The acetate concentration in the reactor content was found to influence the degradation of propionate but not of butyrate. In general, at acetate levels over 200 mg/l the degradation of propionate was below 60%, whereas the degradation was complete at acetate levels under 100 mg/l. The rationale of the inhibition of propionate degradation by acetate is discussed.     

Toxicity of nitrite toward mesophilic and thermophilic sulphate-reducing, methanogenic and syntrophic populations in anaerobic sludge

The various problems associated with treating sulphate-containing wastewaters stem inherently from successful competitive interactions between sulphate reducing bacteria (SRB) and other bacteria involved in the process, resulting in the formation of H₂S. Prevention of in-reactor sulphide generation by use of specific SRB inhibitors presents a potential solution. Nitrite has been reported to be a specific inhibitor of SRB but its possible toxicity to syntrophic and methanogenic members of the anaerobic consortium has not been investigated. In batch activity and toxicity tests, under both mesophilic and thermophilic conditions, nitrite, at concentrations of up to 150 mg L^–1, was found to be ineffective as a specific inhibitor of SRB, and was also shown to have an inhibitory effect on the activity of syntrophic and methane-producing bacteria in mesophilic and thermophilic digester sludge samples.     

Thermophilic anaerobic degradation of butyrate by a butyrate-utilizing bacterium in coculture and triculture with methanogenic bacteria

We studied syntrophic butyrate degradation in thermophilic mixed cultures containing a butyrate-degrading bacterium isolated in coculture with Methanobacterium thermoautotrophicum or in triculture with M. thermoautotrophicum and the TAM organism, a thermophilic acetate-utilizing methanogenic bacterium. Butyrate was beta-oxidized to acetate with protons as the electron acceptors. Acetate was used concurrently with its production in the triculture. We found a higher butyrate degradation rate in the triculture, in which both hydrogen and acetate were utilized, than in the coculture, in which acetate accumulated. Yeast extract, rumen fluid, and clarified digestor fluid stimulated butyrate degradation, while the effect of Trypticase was less pronounced. Penicillin G, d-cycloserine, and vancomycin caused complete inhibition of butyrate utilization by the cultures. No growth or degradation of butyrate occurred when 2-bromoethanesulfonic acid or chloroform, specific inhibitors of methanogenic bacteria, was added to the cultures and common electron acceptors such as sulfate, nitrate, and fumarate were not used with butyrate as the electron donor. Addition of hydrogen or oxygen to the gas phase immediately stopped growth and butyrate degradation by the cultures. Butyrate was, however, metabolized at approximately the same rate when hydrogen was removed from the cultures and was metabolized at a reduced rate in the cultures previously exposed to hydrogen.     

Kinetics of acetate, propionate and butyrate removal in the treatment of a semi-synthetic landfill leachate on anaerobic filter

The kinetics of acetate, propionate, and butyrate removal was studied in conditions of leachate treatment in a plug flow anaerobic fixed-film reactor made of a sequence of seven perfectly mixed compartments. An original experimental procedure was followed under sequential feeding conditions so as to maintain the Bacteriol biomass in a quasi-steady state all along the study. With an appropriate computer program based on the least squares method, the apparent kinetic parameters of VFA removal were calculated within concentration ranges below the levels of salt inhibition. The models proposed are based on simple theoretical considerations. For acetate and n-butyrate removal, the best fits were given by the Michaelis-Menten equation with respectively: V(m) (spec) = 0.49 +/- 0.06 g CH(3) COOH g(-1) biomass h(-1)and 0.18 +/- 0.02 g n-CH(3)CH(2)CH(2)COOH g(-1) biomass h(-1) and: K(s) = 21.2 +/- 0.9 g CH(3)COOH L(-1) liquid phase and 8.2 +/- 0.9 g n-CH(3)CH(2)CH(2)COOH L(-1) liquid phase, Iso-butyrate was produced during n-butyrate catabolism and the apparent removal rate of (n + iso)-butyrate considered as a whole was also described by the Michaelis-Menten equation with V(m) (spec) = 0.14 +/- 0.02 g(n + iso)-butyrate g(-1) biomass h(-1) and K(s) = 9.0 +/- 1.2 g (n + iso) butyrate L(-1) liquid phase. On the other hand in the case of propionate, the best fit was obtained with a first-order equation with K(spec) = (0.88 +/- 0.05) 10(-2) L liquid phase g(-1) biomass h(-1). These constants were subsequently used to predict the removal of mixtures of the three major VFAs under study, at various feed concentrations. Three sets of concentrations were tested, and the experimental data were compared to the simulations. This study, together with other experimental observations previously reported, tends to show that under sequential feeding conditions the classical assumption of butyrate beta-oxidation should be rejected. Butyrate seems to be anaerobically decarboxylated, but propionate thus formed inside the biofilm is degraded as soon as its formation proceeds. It was therefore considered that butyrate degradation produces, through propionate intermediate, 1 mole acetate per mole butyrate removed. When propionate or butyrate concentrations were high, the same phenomenon was noted, to a much lower extent, for the degradation of acetate formed inside the biofilm.     

Inorganic composition and microbial characteristics of methanogenic granular sludge grown in a thermophilic upflow anaerobic sludge blanket reactor

An upflow anaerobic sludge blanket reactor was operated under thermophilic conditions (55° C) for 160 days by feeding a wastewater containing sucrose as the major carbon source. The reactor exhibited a satisfactory performance due to the formation of well-settling granulated sludge, achieving a total organic carbon (TOC) removal of above 80% at an organic loading rate of 30 kg total organic C m^–3 day^–1. Structural and microbial properties of the methanogenic granular sludge were examined using scanning electron microscope X-ray analyses and serum vial activity tests. All the thermophilic granules developed showed a double-layered structure, comprised of a black core portion and a yellowish exterior portion. The interior cope portion contained abundant crystalline precipitates of calcium carbonate. Calcium-bound phosphorus was also present more prominently in the core portion than in the exterior portion. Methanogenic activities of the thermophilic granules both from acetate and from H₂ increased with increasing vial-test temperature in the range of 55–65° C [from 1.43 to 2.36 kg CH₄ chemical oxygen demand (COD) kg volatile suspended solids (VSS)^–1 day^–1 for acetate and from 0.85 to 1.11 kg CH₄ COD kg VSS^–1 day^–1 for H₂]. On the other hand, propionate-utilizing methanogenic activity was independent of vial-test temperature, and was much lower (0.1–0.12 kg CH₄ COD kg VSS^–1 day^–1) than that from either acetate or H₂. Acetate consumption during vial tests was considerably inhibited by the presence of H₂ in the headspace, indicating that a syntrophic association between acetate oxidizers and H₂-utilizing methane-producing bacteria was responsible for some portion of the overall acetate elimination by the theromophilically grown sludge.     

Interactions of acetate, propionate and butyrate in sheep liver mitochondria

1. Interactions in the rates of consumption of acetate, propionate and butyrate in sheep liver mitochondria were examined in the presence and absence of l-malate and alpha-oxoglutarate. 2. Acetate was not consumed in absence of ancillary substrate but utilization of acetate (7.2nmol/min per mg of protein) occurred in the presence of alpha-oxoglutarate. This consumption was abolished by propionate or butyrate but the presence of acetate did not affect consumption of propionate or butyrate. 3. Propionate consumption (10.1nmol/min per mg of protein) was unaffected by malate but was stimulated by 63% by butyrate or by 180% by alpha-oxoglutarate. 4. Butyrate consumption (3.3nmol/min per mg of protein) was stimulated by 117% by malate, by 151% by propionate and by 310% by alpha-oxoglutarate. 5. In the absence of ancillary substrates the maximum rate of total volatile fatty acid utilization (24.7nmol/min per mg of protein) occurred with a mixture of propionate and butyrate. When both propionate and butyrate were present total consumption was not affected by malate but was stimulated by 24% by alpha-oxoglutarate. With alpha-oxoglutarate present, propionate and butyrate each decreased the other's consumption by about 26%, but the total utilization was the greatest observed. 6. The inhibition of acetate consumption by propionate or butyrate is unexplained, but the remaining effects are consistent with an interaction of propionate and butyrate through oxaloacetate together with a general limitation imposed by a need for GTP to rephosphorylate AMP formed during activation of the volatile fatty acids.     

Monitoring of specific methanogenic activity of granular sludge by confocal laser scanning microscopy during start-up of thermophilic upflow anaerobic sludge blanket reactor

Confocal, laser-scanning microscopy was applied to acquire coenzyme F₄₂₀-based autofluorescence images of middle sections of sludge granules during start-up of a thermophilic reactor that were seeded with mesophilically-grown microorganisms of granular sludge. Digital images were analyzed to calculate weighted averages of autofluorescence. The values were related (r ²=0.97) to specific methanogenic activities of granular sludge as the granules developed to steady state.     

Anaerobic degradation of isovalerate by a defined methanogenic coculture

Isovalerate-oxidizing strictly aneerobic bacteria were isolated from marine sediment and sewage sludge in coculture with Desulfovibrio sp. Cells stained Gram positive and behaved Gram positive also in Gram classification with KOH. Isovalerate degradation depended on interspecies hydrogen transfer to syntrophic hydrogen-oxidizing sulfate reducers or methanogens. Isovalerate was the only substrate utilized and was fermented to 3 mol acetate and 1 mol hydrogen per mol substrate. The degradation pathway was studied by enzyme assays in crude cell extracts, and included acetyl-CoA dependent activation of isovalerate, oxidation to methylcrotonyl-CoA and carboxylation to methylgluta-conyl-CoA which is hydrated and cleaved to acetoacetate and acetyl-CoA. Studies with inhibitors and ionophores suggest that energy conservation with this organism depends on either acetate efflux-driven proton symport or on an ion-gradient driven carboxylation mechanism.     

Limited degradation of chlorophenols by anaerobic sludge granules.

To better understand the fate of chlorophenols treated in upflow anaerobic sludge bed reactors, we examined the ability of sludge granules from such bioreactors to degrade two trichlorophenols and one dichlorophenol in batch incubations under controlled conditions. Biodegradation was primarily limited to two distinct activities, reductive dehalogenation of ortho- and of meta-chlorine substituents. Both 3- and 4-monochlorophenol were persistent degradation products, while 2-monochlorophenol was further degraded. We also examined factors potentially affecting the rate and extent of 2,3,6-trichlorophenol degradation. An initial concentration of up to 1.75 mM (346 mg/liter) was dehalogenated. At that concentration, dehalogenation was partially inhibited but methanogenesis from formate was not. The initial concentration affected both the extent of dehalogenation and which products were detected. The maximum dechlorination rate observed was 1.4 mumol of Cl- h-1 g of volatile suspended solids-1. Dechlorination had a temperature optimum of 50 degrees C, was inhibited by added electron acceptors, and was not appreciably affected by added electron donors. The availability of electron acceptors and electron donors did not affect the extent of chlorophenol degradation. These particular sludge granules do not appear to be capable of mineralizing phenols with meta- or para-chlorine substituents.     

Limited degradation of chlorophenols by anaerobic sludge granules.

To better understand the fate of chlorophenols treated in upflow anaerobic sludge bed reactors, we examined the ability of sludge granules from such bioreactors to degrade two trichlorophenols and one dichlorophenol in batch incubations under controlled conditions. Biodegradation was primarily limited to two distinct activities, reductive dehalogenation of ortho- and of meta-chlorine substituents. Both 3- and 4-monochlorophenol were persistent degradation products, while 2-monochlorophenol was further degraded. We also examined factors potentially affecting the rate and extent of 2,3,6-trichlorophenol degradation. An initial concentration of up to 1.75 mM (346 mg/liter) was dehalogenated. At that concentration, dehalogenation was partially inhibited but methanogenesis from formate was not. The initial concentration affected both the extent of dehalogenation and which products were detected. The maximum dechlorination rate observed was 1.4 mumol of Cl- h-1 g of volatile suspended solids-1. Dechlorination had a temperature optimum of 50 degrees C, was inhibited by added electron acceptors, and was not appreciably affected by added electron donors. The availability of electron acceptors and electron donors did not affect the extent of chlorophenol degradation. These particular sludge granules do not appear to be capable of mineralizing phenols with meta- or para-chlorine substituents.     

Inhibition of the anaerobic acetate degradation by formate

Summary Granular sludge from an UASB reactor fed with VFA showed a very low affinity for formate which provide little support to the theory of interspecies formate transfer. It is shown that formate can inhibit acetate degradation by anaerobic sludge.     

Effects of propionate and acetate additions on solvent production in batch cultures of Clostridium acetobutylicum.

Addition of acetate or propionate to uncontrolled-pH batch cultures does not affect the initiation of solventogenesis but does enhance final solvent concentrations compared with those of unchallenged cultures. This observation can be explained in terms of the increased buffering capacity of the medium brought about by the added acids, resulting in protection against premature growth inhibition due to low culture pH values at the end of the fermentation. The uptake of propionic acid from the medium does not proceed solely via the coenzyme A-transferase pathway, since less acetone than propanol is formed. Therefore, at least 50% of the propionic acid is taken up through the reversed kinase-phosphotransbutyrylase reaction pathway.     

Methanogenesis from acetate: Physiology of a thermophilic, acetate-utilizing methanogenic bacterium

Abstract The biosynthesis of K88, K99 and F41 fibrillae by enterotoxigenic Escherichia coli strains was shown to be dependent on the growth phase of the cultures. An increase in adhesin production was observed, during exponential growth reaching its maximum at the end of this phase; thereafter adhesin production was arrested.
A simple and rapid purification procedure was developed for adhesins isolated from exponentially growing cells.     

Effect of sulfur-containing compounds on anaerobic degradation of cellulose to methane by mixed cultures obtained from sewage sludge.

Tests were made to determine the effects of inorganic and organic sulfur sources on the degradation of cellulose to methane in a chemically defined medium with sulfur-poor inoculum prepared from sewage sludge. The results show that a sulfur source of about a 0.85 mM concentration is essential for the degradation of cellulose to CH4. However, the production of CH4 from CO2 and H2 provided in the headspace occurred with 0.1 mM sulfate or sulfide. At a 9 mM concentration, all inorganic sulfur compounds other than sulfate inhibited both cellulose degradation and methane formation, and this inhibition increased in the order thiosulfate less than sulfite less than sulfide less than H2S. It appears that the degradation of cellulose to CH4 in a sulfate-free medium by inoculum maintained in a low-sulfur medium is inhibited because of the lack of availability of sulfur for growth of bacteria and synthesis of cell materials and sulfur-containing cofactors involved in cellulose degradation and methanogenesis. The reduction of methanogenesis by higher levels of sulfate probably occurs as a result of stimulation of reactions converting acetate and H2 to end products other than CH4.     

Cultivation and in situ detection of a thermophilic bacterium capable of oxidizing propionate in syntrophic association with hydrogenotrophic methanogens in a thermophilic methanogenic granular sludge

The thermophilic, anaerobic, propionate-oxidizing bacterial populations present in the methanogenic granular sludge in a thermophilic (55 degrees C) upflow anaerobic sludge blanket reactor were studied by cultivation and in situ hybridization analysis. For isolation of propionate-degrading microbes, primary enrichment was made with propionate as the sole energy source at 55 degrees C. After several attempts to purify the microbes, a thermophilic, syntrophic, propionate-oxidizing bacterium, designated strain SI, was isolated in both pure culture and coculture with Methanobacterium thermoautotrophicum. Under thermophilic (55 degrees C) conditions, strain SI oxidized propionate, ethanol, and lactate in coculture with M. thermoautotrophicum. In pure culture, the isolate was found to ferment pyruvate. 16S ribosomal DNA sequence analysis revealed that the strain was relatively close to members of the genus Desulfotomaculum, but it was only distantly related to any known species. To elucidate the abundance and spatial distribution of organisms of the strain SI type within the sludge granules, a 16S rRNA-targeted oligonucleotide probe specific for strain SI was developed and applied to thin sections of the granules. Fluorescence in situ hybridization combined with confocal laser scanning microscopy revealed that a number of rod-shaped cells were present in the middle and inner layers of the thermophilic granule sections and that they formed close associations with hydrogenotrophic methanogens. They accounted for approximately 1.1% of the total cells in the sludge. These results demonstrated that strain SI was one of the significant populations in the granular sludge and that it was responsible for propionate oxidation in the methanogenic granular sludge in the reactor.     

Anaerobic degradation of long-chain dicarboxylic acids by methanogenic enrichment cultures

Abstract Methanogenic enrichment cultures fermented the long-chain dicarboxylates adipate, pimelate, suberate, azelate, and sebacate (C₆-C₁₀) stoichiometrically to acetate and methane. After several transfers, the cultures contained cells of only a few morphologically distinguishable types. During anaerobic degradation of dicarboxylic acids with even-numbered carbon atoms, propionate accumulated intermediately, and butyrate was the intermediate product of degradation of those with an odd number of carbon atoms. Degradation of the long-chain dicarboxylates depended strictly on the presence of hydrogenotrophic methanogens. The primary attack in these processes was β-oxidation rather than decarboxylation. A general scheme of anaerobic degradation of long-chain dicarboxylic acids has been deduced from these results.