Relative importance of trophic group concentrations during anaerobic degradation of volatile fatty acids

Although obligate syntrophic reactions cannot proceed without hydrogenotrophs, it has been unclear from the literature whether potential improvements are achievable with higher concentrations of hydrogenotrophs. In this study, the relative importance of formate-/H(2)-utilizing and acetate-utilizing trophic groups in the anaerobic degradation of butyrate and propionate was assessed by adding various proportions of these enriched cultures to a mixed anaerobic seed inoculum. The improvement resulting from the additional acetate-utilizing cultures was much greater than with formate/H(2) utilizers. Furthermore, formate/H(2) utilizers did not improve propionate utilization significantly, suggesting the importance of optimum utilization of hydrogenotrophic capacity. During most of the volatile fatty acid (VFA) degradation period, the system responded with characteristic hydrogen levels to maintain the Gibbs free energy of oxidation approximately constant for both butyrate (-6 kJ) and propionate (-14 kJ). These free-energy values were independent of methanogenic activity, as well as the volume of the seed inoculum and the VFA concentrations present. By comparing the experimental results with kinetic and mass transfer models, it was postulated that the diffusional transfer of reducing equivalents was the major limiting factor for efficient VFA degradation. Therefore, for optimum utilization of the hydrogenotrophs, low acetate concentrations are vital to enable the system to respond with higher formate/H(2) levels, thus leading to improved transfer of reducing equivalents. Due to the small number of propionate utilizers (and hence their limited surface area) and low bulk liquid concentrations, the additional formate/H(2) utilizers were of minimal use for improving the degradation rate further. The butyrate degradation rates strongly correlated with the cumulative activity of hydrogenotrophs and acetotrophs over the experimental range studied, indicating the need to model obligate syntrophic reactions as a dependent function of methanogenic activity.     

Relative Importance of Trophic Group Concentrations during Anaerobic Degradation of Volatile Fatty Acids

Although obligate syntrophic reactions cannot proceed without hydrogenotrophs, it has been unclear from the literature whether potential improvements are achievable with higher concentrations of hydrogenotrophs. In this study, the relative importance of formate-/H₂-utilizing and acetate-utilizing trophic groups in the anaerobic degradation of butyrate and propionate was assessed by adding various proportions of these enriched cultures to a mixed anaerobic seed inoculum. The improvement resulting from the additional acetate-utilizing cultures was much greater than with formate/H₂ utilizers. Furthermore, formate/H₂ utilizers did not improve propionate utilization significantly, suggesting the importance of optimum utilization of hydrogenotrophic capacity. During most of the volatile fatty acid (VFA) degradation period, the system responded with characteristic hydrogen levels to maintain the Gibbs free energy of oxidation approximately constant for both butyrate (−6 kJ) and propionate (−14 kJ). These free-energy values were independent of methanogenic activity, as well as the volume of the seed inoculum and the VFA concentrations present. By comparing the experimental results with kinetic and mass transfer models, it was postulated that the diffusional transfer of reducing equivalents was the major limiting factor for efficient VFA degradation. Therefore, for optimum utilization of the hydrogenotrophs, low acetate concentrations are vital to enable the system to respond with higher formate/H₂ levels, thus leading to improved transfer of reducing equivalents. Due to the small number of propionate utilizers (and hence their limited surface area) and low bulk liquid concentrations, the additional formate/H₂ utilizers were of minimal use for improving the degradation rate further. The butyrate degradation rates strongly correlated with the cumulative activity of hydrogenotrophs and acetotrophs over the experimental range studied, indicating the need to model obligate syntrophic reactions as a dependent function of methanogenic activity.     

Quantitative analysis of the trophic groups with a fluorescence in situ hybridization (FISH) and the competitive kinetics of a propionate enriched anaerobic culture

An anaerobic culture had been enriched with propionate, which was a critical compound inhibiting the total anaerobic process when significantly accumulated. In addition, the distribution of the trophic groups was investigated using the tool of fluorescence in situ hybridization with 16S rRNA targeted oligonucleotide probes to understand the bacterial communication. Then the kinetic constants in the competitive inhibition models were obtained. The ratio of the methanogens was increased as the culture was enriched, whereas that of the acidogens was decreased. The sum of the acetoclastic and hydrogenotrophic methanogens was 34% and the methanogens was 50% at the 68^th day. Approximately 16% of the cells detected by DAPI staining were unknown. The degradation of ethanol is thermodynamically more favorable. However, the substrate affinity to propionate was presumably higher than to ethanol since the culture had been acclimated to the propionate-rich environment. Ethanol and acetate did not show any inhibition effect on the methane fermentation of propionate.     

Parameter and process significance in mechanistic modeling of cellulose hydrolysis

A process-based model relevant to landfill and anaerobic digesters was developed, which included a novel approach to biomass transfer between a cellulose-bound biofilm and biomass in the bulk liquid. Model results highlighted the significance of the bacterial colonization of cellulose particles by attachment through contact in solution. Simulations revealed that both enhanced colonization and cellulose degradation are associated with reduced cellulose particle size, increased biomass populations in solution and increased cellulose-binding ability of the biomass. This suggests that transportation of biomass into the system from elsewhere and/or bacterial inoculation of such systems could enhance degradation significantly. A sensitivity analysis of the system parameters revealed the biological rate and yield properties of the hydrolyzing bacteria are most significant with regard to cellulose degradation in the system.     

Some Experiments and Conclusions about the Influence of Pure H2 Gason the Anaerobic Degradation of Propionate by Mixed Cultures

Reaction enthalpy for propionate degradationΔG₀ is only negative when the partial pressure ofhydrogen pH₂ is less than 10^—4 bar. This means that for pH₂ more than 10^—4 bar, a total anaerobic degradation of propionate is impossible for thermodynamic reasons. Therefore, with increasing pH₂, the anaerobic degradation rate of propionate via acetate is inhibited. There are two ways to investigate the inhibitory effect of pH₂: to keep the concentration of hydrogen consuming bacteria low or to increase the mass transfer by feeding the hydrogen at higher flow rates. The author used an extended fixed bed reactor filled with polyurethane particles as a carrier for the bacteria, aerated with pure H₂ gas. The results, compared with the literature by using model equations in order to standardize the data, correspond well: The addition of pure H₂ gas has no observable effects on propionate degradation.In the fixed bed reactor with immobilized bacteria, it was not possible to reach an inhibitory concentration of H₂ and high process stability could be maintained.     

Thermodynamic evidence of trophic microniches in methanogenic granular sludge-bed reactors

Summary The Gibbs free energy changes in methanogenic granular biomass from sludge-bed reactors were evaluated using the in situ concentrations and partial pressures of metabolites during the metabolism of acetate, hydrogen, formate and propionate. Based on mass balance calculations it appeared that the degradation of propionate into acetate, hydrogen and bicarbonate was endergonic, even if propionate was effectively degraded. On the other hand, the methane-producing reactions, both from acetate and from hydrogen plus bicarbonate, were found to be exergonic and the free energy change was sufficient for the formation of ATP. Formate was detected in only one of the two reactors. When formate, instead of hydrogen, was considered as the electron carrier between propionate-degrading and methanogenic bacteria, similar thermodynamic results were obtained. The existence of trophic microniches in the granular biomass is suggested to explain propionate degradation even though the Gibbs free energy change in the liquid surrounding the granules was positive. Hence, to make propionate degradation exergonic the dissolved hydrogen concentration surrounding the propionate-degrading bacteria would have to be about 30 times lower than in the free liquid. Offprint requests to: S. Guiot     

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.     

Characteristics of granular methanogenic sludge grown on glucose in a UASB reactor

The formation of granules grown on glucose in an upflow anaerobic sludge blanket (UASB) reactor was investigated. Total granular sludge concentration retained in the UASB reactor was 34.5 g MLSS/l (30.0 g MLVSS/l) during 240 d operation on glucose minimum medium with the supplementation of 1.07 g NaHCO₃ per 1 g glucose. This realized a high-rate methanogenic fermentation of glucose of 17.6 g COD/l-reactor-d at 3.4 d⁻¹ of space velocity. The granules formed were relatively small, ranging mainly from 0.4 to 0.5 mm, had a relatively low cell density of 0.0542–0.0560 g MLVSS/ml, and had low specific gravity (0.97–1.19) due to very low ash content (11–13%). Electron microscopic analysis showed that Methanothrix spp. appeared dominant over the granules. The specific metabolic activities of bacterial trophic groups were the highest for H₂ followed by glucose, acetate, and propionate.     

Contribution of 13C-NMR spectroscopy to the elucidation of pathways of propionate formation and degradation in methanogenic environments

Propionate is an important intermediate in the anaerobic degradation of complex organic matter to methane and carbon dioxide. The metabolism of propionate-forming and propionate-degrading bacteria is reviewed here. Propionate is formed during fermentation of polysaccharides, proteins and fats. The study of the fate of 13C-labelled compounds by nuclear magnetic resonance (NMR) spectroscopy has contributed together with other techniques to the present knowledge of the metabolic routes which lead to propionate formation from these substrates. Since propionate oxidation under methanogenic conditions is thermodynamically difficult, propionate often accumulates when the rates of its formation and degradation are unbalanced. Bacteria which are able to degrade propionate to the methanogenic substrates acetate and hydrogen can only perform this reaction when the methanogens consume acetate and hydrogen efficiently. As a consequence, propionate can only be degraded by obligatory syntrophic consortia of microorganisms. NMR techniques were used to study the degradation of propionate by defined and less defined cultures of these syntrophic consortia. Different types of side-reactions were reported, like the reductive carboxylation to butyrate and the reductive acetylation to higher fatty acids.     

Energetics of syntrophic fatty acid oxidation

Abstract: Fatty acids are key intermediates in methanogenic degradation of organic matter in sediments as well as in anaerobic reactors. Conversion of butyrate or propionate to acetate, (CO₂), and hydrogen is endergonic under standard conditions, and becomes possible only at low hydrogen concentrations (10^-4-10^-5 bar). A model of energy sharing between fermenting and methanogenic bacteria attributes a maximum amount of about 20 kJ per mol reaction to each partner in this syntrophic cooperation system. This amount corresponds to synthesis of only a fraction (one-third) of an ATP to be synthesized per reaction. Recent studies on the biochemistry of syntrophic fatty acid-oxidizing bacteria have revealed that hydrogen release from butyrate by these bacteria is inhibited by a protonophore or the ATPase inhibitor DCCD ( N , N '-dicyclohexyl carbodiimide), indicating that a reversed electron transport step is involved in butyrate or propionate oxidation. Hydrogenase, butyryl-CoA dehydrogenase, and succinate dehydrogenase acitivities were found to be partially associated with the cytoplasmic membrane fraction. Also glycolic acid is degraded to methane and CO₂ by a defined syntrophic coculture. Here the most difficult step for hydrogen release is the glycolate dehydrogenase reaction ( E '₀=−92 mV). Glycolate dehydrogenase, hydrogenase, and ATPase were found to be membrane-bound enzymes. Membrane vesicles produced hydrogen from glycolate only in the presence of ATP; protonophores and DCCD inhibited this hydrogen release. This system provides a suitable model to study reversed electron transport in interspecies hydrogen transfer between fermenting and methanogenic bacteria in methanogenic biomass degradation.     

Modeling dynamic experiments on the anaerobic degradation of molasses wastewater

The kinetics of anaerobic degradation of a molasses wastewater were measured under constant pH conditions in a laboratory scale packed bed reactor. In continuous and batch experiments the formation and degradation rates of the organic acids (butyric, propionic and acetic) have been followed. The influence of hydrogen gas on the acid degradation rates has been measured and, contrary to the literature and the thermo-dynamic calculations, no inhibition was detected, biofilm diffusional effects may be the reason. Two dynamic simulation models were tested, a heterogeneous model, which considered the biofilm diffusion-reaction phenomena and a quasihomogeneous model with the same kinetics. Except for hydrogen, the diffusion effects were found to be negligible. Otherwise both models gave essentially the same results and the time profiles of acids, hydrogen, carbon dioxide and methane agreed relatively well with dynamic startup experiments. Batch experiments showed the acid concentrations to be highly sensitive to the initial molasses concentration. This aspect was not included in the model but is being investigated further.     

A two-dimensional mass transfer model for an annular bioreactor using immobilized photosynthetic bacteria for hydrogen production

A two-dimensional model for substrate transfer and biodegradation in a novel, annular fiber-illuminating bioreactor (AFIBR) is proposed in which photosynthetic bacteria are immobilized on the surface of a side-glowing optical fiber to form a stable biofilm. When excited by light, the desired intensity and uniform light distribution can be obtained within the biofilm zone in bioreactor and then realize continuous hydrogen production. Substrate transfer and biodegradation within the biofilm zone, as well as substrate diffusion and convection within bulk fluid regions are considered simultaneously in this model. The validity of the model is verified experimentally. Based on the model analysis, influences of flow rate and light intensity on the substrate consumption rate and substrate degradation efficiency were investigated. The simulation results show that the optimum operational conditions for the substrate degradation within the AFIBR are: flow rate 100 ml h^?1 and light intensity 14.6 μmol photons m^?2 s^?1.     

Kinetic parameters and relative turnovers of some important catabolic reactions in digesting sludge.

The kinetics of propionate degradation, acetate splitting, and hydrogen consumption in digesting sludge were investigated in a lab-scale digester. At natural steady-state conditions, the acetate-splitting systems in well-digested sludge were about half saturated. Propionate-degrading systems were saturated to only 10 to 15%, and hydrogen removal was less than 1% of the maximum possible rate. It was concluded that acetate splitting rather than "methanogenesis from fatty acids" is the rate-limiting reaction in the anaerobic degradation of dissolved organic matter and that a methoanogenic anaerobic ecosystem is stabilized by its large unused capacity of hydrogen consumption which is "buffering" the partial pressure of dissolved hydrogen in the system at sufficiently low values to permit rapid fatty acid oxidation. A tentative scheme of the substrate flow in sludge digestion is presented. It suggests that acid formation coupled with hydrogen formation via pyridine dinucleotide oxidation yields the immediate substrates, namely acetate and hydrogen, for about 54% of the total methanogenesis.     

Interspecies Electron Transfer during Propionate and Butyrate Degradation in Mesophilic, Granular Sludge

Granules from a mesophilic upflow anaerobic sludge blanket reactor were disintegrated, and bacteria utilizing only hydrogen or formate or both hydrogen and formate were added to investigate the role of interspecies electron transfer during degradation of propionate and butyrate. The data indicate that the major electron transfer occurred via interspecies hydrogen transfer, while interspecies formate transfer may not be essential for interspecies electron transfer in this system during degradation of propionate and butyrate.     

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)     

Hydrogen production and anaerobic decolorization of wastewater containing Reactive Blue 4 by a bacterial consortium of Salmonella subterranea and Paenibacillus polymyxa

Anaerobic biodegradability of wastewater (3,000 mg CODcr/l) containing 300 mg/l Reactive Blue 4, with different co-substrates, glucose, butyrate and propionate by a bacterial consortium of Salmonella subterranea and Paenibacillus polymyxa, concomitantly with hydrogen production was investigated at 35°C. The accumulative hydrogen production at 3,067 mg CODcr/l was obtained after 7 days of incubation with glucose, sludge, the bacterial consortium. The volatile fatty acids, residual glucose and the total organic carbon were correlated to hydrogen obtained. Interestingly, the bacterial consortium possess decolorization ability showing approximately 24% dye removal after 24 h incubation using glucose as a co-substrate, which was about two and eight times those of butyrate (10%), propionate (12%) and control (3%), respectively. RB4 decolorization occurred through acidogenesis, as high volatile fatty acids but low methane was detected. The bacterial consortium will be the bacterial strains of interest for further decolorization and hydrogen production of industrial waste water.     

Impact of liquid-to-gas hydrogen mass transfer on substrate conversion efficiency of an upflow anaerobic sludge bed and filter reactor

Efficient anaerobic degradation may be completed only under low levels of dissolved hydrogen in the liquid surrounding the microorganisms. This restraint can be intensified by the limitations of liquid-to-gas H₂ mass transfer, which results in H₂ accumulation in the bulk liquid of the reactor. Dissolved hydrogen proved to be an interesting parameter for reactor monitoring by showing a good correlation with short-chain volatile fatty acid concentration, namely propionate, which was not the case for the H₂ partial pressure. Biogas recycle was performed in a upflow anaerobic sludge bed and filter reactor. The effects of varying the ratio of recycled-to-produced gas from 2:1 (9 l/l reactor per day) to 8:1 (85 l/l reactor per day) were studied. By increasing the liquid—gas interface with biogas recycling, the dissolved hydrogen concentration could be lowered from 1.1 to 0.4 μ . Accordingly, the H₂ sursaturation factor was also reduced, leading to an important improvement of the H₂ mass transfer rate, which reached 20.86 h⁻¹ (±9.79) at a 8:1 gas recycling ratio, compared to 0.72 h⁻¹ (±0.24) for the control experiment. Gas recycling also lowered the propionate concentration from 655 to 288 mg l⁻¹ and improved the soluble chemical oxygen demand removal by 10–15%. The main problem encountered was the shorter solid retention time, which could lead to undesirable biomass washout at high gas recycling ratio. This could be circumvented by improving the reactor design to reduce the turbulence within the biomass bed.     

Evaluation of analytical methods for determining the distribution of biofilm and active bacteria in a commercial heating system

Danish district heating systems have good water quality, but continue to suffer from biofouling and biocorrosion. Localisation analyses of bacteria using microautoradiography were performed for one system in order to obtain detailed information for solving these problems. A mass balance showed that 77% of the bacteria were located at surfaces, with 23% in the bulk water, and 9% of the total carbon originated from biomass, while 91% was dissolved in the bulk water. The presence of active bacteria was determined with microautoradiography which showed that biofilms contained 99% and 1% were in the bulk water. A high bacterial functional diversity was observed, with active mesophilic and thermophilic bacteria under aerobic and anaerobic conditions and with potentially corrosive biofilm bacteria present. The study reveals that by applying the activity based approach, the ratio of living and dead bacteria in the biofilm and bulk water in this type of system could be accurately determined. Also, the results emphasise that to minimise biofilm growth and biocorrosion, monitoring should be established focusing on the surfaces, since bulk water parameters do not reflect bacterial activity.     

Inhibition of a methanogenic population by thioglycollate

Thioglycollate added at 3.2 mM inhibited methane (CH₄) production from propionate by a methanogenic bacterial population obtained from an anaerobic digester. Gas production from acetate was not inhibited at this concentration. Glucose added in a semi-synthetic medium was degraded in the presence of 3.2 mM thioglycollate but propionate accumulated in the medium and the methanol yield was reduced. The severity of inhibition of propionate degradation increased with further incubation.     

Alcohol and acid formation during the anaerobic decomposition of propylene glycol under methanogenic conditions

Intermediates formed during the anaerobic decomposition of propylene glycol under methanogenic conditions were studied using a serum bottle technique. The pathway is similar to the anaerobic decomposition of ethylene glycol as previously reported. For both compounds, the decomposition is believed to proceed via an initial disproportionation of the glycol to form equal molar amounts of the volatile fatty acid and normal alcohol of the same chain length. In the case of ethylene glycol, disproportionation results in the formation of acetate and ethanol, while disproportionation of propylene glycol produces propionate and n-propanol. Following disproportionation, the alcohols produced from glycol fermentation are oxidized to their corresponding volatile fatty acid with the reduction of protons to form hydrogen. Ethanol and propionate oxidation to acetate proceeds via a well-established syntrophic pathway that is favorable only under low hydrogen partial pressures. Subsequent degradation of acetate proceeds via acetoclastic methanogenesis with the production of carbon dioxide and methane. Despite the production of hydrogen in the initial steps of glycol degradation, both compounds are completely degradable under the methanogenic conditions tested in this study.