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.     

Decreased activity of a propionate degrading community in a UASB reactor fed with synthetic medium without molybdenum,tungsten and selenium

The composition and dynamics of the propionate degrading community in a propionate-fed upflow anaerobic sludge bed (UASB) reactor with sludge originating from an alcohol distillery wastewater treating UASB reactor was studied. The rather stable propionate degrading microbial community comprised relatives of propionate degrading Syntrophobacter spp., the hydrogen and formate consuming Methanospirillum hungatei and the acetate consuming Methanosaeta concilii. The effect of the long-term absence of molybdenum, tungsten and selenium from the feed to the UASB reactor on microbial community dynamics and activity was examined. Measurements for metal concentrations of the sludge and specific methanogenic activity tests with supplied molybdenum, tungsten and selenium were found to be unsuitable to detect the potential limitation of the microbial activity of the UASB sludge by these trace metals. During a long-term absence of molybdenum, tungsten and selenium from the feed to the UASB reactor, the methanogenic activity decreased while relatives of Smithella propionica and Pelotomaculum spp. competed with Syntrophobacter spp. for propionate consumption.     

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.     

Start-up and operation of a propionate-degrading fluidized-bed reactor

Summary A laboratory-scale fluidized-bed reactor was inoculated with a syntrophically propionate-degrading co-culture. After 24 days of batch operation with propionate and acetate in the medium, the reactor was operated for 8 months with propionate as the sole substrate. The loading rate was 8.5 kg chemical oxygen demand/m³ ·day, yielding a maximal gas production of 4.5 l/l·day at a removal efficiency of 94–99%. The degradation was not inhibited by up to 85mm propionate in pulse experiments, but short-time changes in redox potential above — 300 mV led to a drop in the propionate degradation rate. While Methanocorpusculum sp. and Methanospirillum sp. adhered to the sand in the early phase of the start-up, the consortium in the mature biofilm consisted of Desulfobulbus sp., Methanothrix soehngenii and species of at least three different genera of hydrogenotrophic methanogens. A syntrophic relationship between the sulphate-reducing Desulfobulbus sp. and hydrogenotrophic and acetotrophic methanogens is discussed.Offprint requests to: G. Zellner     

Influence of waste water composition on biofilm development in laboratory methanogenic fluidized bed reactors

Summary The influence of the volatile fatty acid composition of waste waters on biofilm development and on the time course of reactor start-up was investigated in laboratory scale fluidized bed reactors. It was found that biofilm development proceeded in a similar way with either acetate, butyrate, propionate or a mixture of these compounds as carbon source in the waste water. Startup was retarded, however, with propionate as sole carbon source. Scanning electron microscopic examination revealed that immobilization of bacteria on the sand used as adhesive support initially occurred in crevices and that thereupon the surface of the sand particles became colonized. The composition of the newly developed biomass was determined when reactors reached steady state. Significant differences in the relative substrate spectra and in the amounts of hydrogenotrophic and acetotrophic methanogenic bacteria were measured. The differences reflected the differences in the composition of the waste waters. The results obtained emphasize the role of the structure of the carrier surface in start-up of methanogenic fluidized bed reactors.Abbreviations used Aw ash weight - COD chemical oxygen demand - EB fluidized bed - hbi vitamin B₁₂-HBI - spt sarcinapterin - UASB upflow anaerobic sludge blanket - VFA volatile fatty acid - VSS volatile suspended solids - Ww wet weight     

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.     

Enrichment of acetate-, propionate-, and butyrate-degrading co-cultures from the biofilm of an anaerobic fluidized bed reactor

Summary Scanning electron microphotographs from the biofilm of a pilot scale anaerobic fluid-ized-bed reactor fed with acetate, propionate, and butyrate as carbon sources showed a predominance of filamentous organisms resembling Methanothrix sp. which could be isolated as an al-most pure culture as well as a Methanosarcina strain. Three syntrophic cultures, enriched in the medium of Boone and Xun, contained four or five microscopically distinguishable microorganisms, among them Methanospirillum sp., Methanothrix sp., Methanosarcina sp., and rods of acetogenic bacteria degrading propionate or butyrate effectively.     

Performance of a down-flow fluidized bed reactor under sulfate reduction conditions using volatile fatty acids as electron donors

The use of a down-flow fluidized bed (DFFB) reactor for the treatment of a sulfate-rich synthetic wastewater was investigated to obtain insight into the outcome of sulfate reduction in a biofilm attached to a plastic support under a down-flow regime. Fine low-density polyethylene particles were used as support for developing a biofilm within the reactor. The reactor treated a volatile fatty acids mixture of acetate or lactate, propionate, and butyrate at different chemical oxygen demand (COD) to sulfate ratios ranging from 1.67 to 0.67 (g/g). Organic loading rate changed from 2.5 to 5 g COD/L x day and sulfate loading rate increased from 1.5 to 7.3 g SO(4) (2-)/L x day. At the beginning of continuous operation, methanogenesis was the predominant process; however, after 187 days, sulfate reduction became the main ongoing biological process. After 369 days, a COD removal of 93% and a sulfate removal of 75% were reached. Total sulfide concentrations in the reactor ranged from 105, when the reactor was mainly methanogenic, to around 1,215 mg/L at the end of the experiment. The high sulfide concentrations did not affect the performance of the reactor. Results demonstrated that the configuration of the DFFB reactor was suitable for the anaerobic treatment of sulfate-rich wastewater.     

Scanning electron microscopy of biofilm development in anaerobic fixed-bed reactors: Influence of the inoculum

Summary Scanning electron microscopy was applied to evaluate the influence of inoculum on efficiency of initial biofilm formation and reactor performance. Five anaerobic fixed-bed reactors were inoculated with anaerobic sludges from different sources and operated in parallel under identical conditions with defined wastewater and acetate, propionate and butyrate as constituents In all sludges Methanothrix sp. was the predominant acetotroph. The reactors inoculated with anaerobic sludge adapted to the wastewater achieved the highest space loading with 21.0 g COD/l·d after 58 days. The inoculation with granular sludge from an upflow anaerobic sludge blanket (UASB) reactor resulted in significantly less reactor efficiency. Time course of biofilm formation and biofilm thickness (ranging from 20–200 m) depended on the type of inoculum.     

Consortium diversity of a sulfate‐reducing biofilm developed at acidic pH influent conditions in a down‐flow fluidized bed reactor

Sulfate reduction is an appropriate approach for the treatment of effluents with sulfate and dissolved metals. In sulfate‐reducing reactors, acetate may largely contribute to the residual organic matter, because not all sulfate reducers are able to couple the oxidation of acetate to the reduction of sulfate, limiting the treatment efficiency. In this study, we investigated the diversity of a bacterial community in the biofilm of a laboratory scale down‐flow fluidized bed reactor, which was developed under sulfidogenic conditions at an influent pH between 4 and 6. The sequence analysis of the microbial community showed that the 16S rRNA gene sequence of almost 50% of the clones had a high similarity with Anaerolineaceae. At second place, 33% of the 16S rRNA phylotypes were affiliated with the sulfate‐reducing bacteria Desulfobacca acetoxidans and Desulfatirhabdium butyrativorans, suggesting that acetotrophic sulfate reduction was occurring in the system. The remaining bacterial phylotypes were related to fermenting bacteria found at the advanced stage of reactor operation. The results indicate that the acetotrophic sulfate‐reducing bacteria were able to remain within the biofilm, which is a significant result because few natural consortia harbor complete oxidizing sulfate‐reducers, improving the acetate removal via sulfate reduction in the reactor.     

Influence of the acetate concentration on the recovery time of a perturbed anaerobic digester

Summary Imbalance in a fluidized bed reactor was simulated by injecting pure or mixed acetate and propionate salts into the digester. For acetate levels higher than 13–15 mM, propionate degradation was inhibited. In these cases, the time required by the system to reduce the propionate concentration up to steady-state values was approximately 60–70 % higher than the recovery time found in the cases in which this inhibition was not present.     

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)     

Characterization of sulfate-reducing bacteria dominated surface communities during start-up of a down-flow fluidized bed reactor

An anaerobic down-flow fluidized bed reactor was inoculated with granular sludge and started-up with sulfate containing synthetic wastewater to promote the formation of a biofilm enriched in sulfate-reducing bacteria (SRB), to produce biogenic sulfide. The start-up was done in two stages operating the reactor in batch for 45 days followed by 85 days of continuous operation. Low-density polyethylene was used as support. The biofilm formation was followed up by biochemical and electron microscopy analyses and the composition of the community was examined by 16S rDNA sequence analysis. Maximum immobilized volatile solids (1.2 g IVS/L_support) were obtained after 14 days in batch regime. During the 85 days of continuous operation, the reactor removed up to 80% of chemical oxygen demand (COD), up to 28% of the supplied sulfate and acetate was present in the effluent. Sulfate-reducing activity determined in the biofilm with ethanol or lactate as substrate was 11.7 and 15.3 g COD/g IVS per day, respectively. These results suggested the immobilization of sulfate reducers that incompletely oxidize the substrate to acetate; the phylogenetic analysis of the cloned 16S rDNA gene sequences showed high identity to the genus Desulfovibrio that oxidizes the substrates incompletely. In contrast, in the granular sludge used as inoculum a considerable number of clones showed homology to Methanobacterium and just few clones were close to SRB. The starting-up approach allowed the enrichment of SRB within the diverse community developed over the polyethylene support.     

Sulfate-reducing bacterial community structure and their contribution to carbon mineralization in a wastewater biofilm growing under microaerophilic conditions

The community structure of sulfate-reducing bacteria (SRB) and the contribution of SRB to carbon mineralization in a wastewater biofilm growing under microaerophilic conditions were investigated by combining molecular techniques, molybdate inhibition batch experiments, and microelectrode measurements. A 16S rDNA clone library of bacteria populations was constructed from the biofilm sample. The 102 clones analyzed were grouped into 53 operational taxonomic units (OTUs), where the clone distribution was as follows: Cytophaga-Flexibacter-Bacteroides (41%), Proteobacteria (41%), low-G+C Gram-positive bacteria (18%), and other phyla (3%). Three additional bacterial clone libraries were also constructed from SRB enrichment cultures with propionate, acetate, and H₂ as electron donors to further investigate the differences in SRB community structure due to amendments of different carbon sources. These libraries revealed that SRB clones were phylogenetically diverse and affiliated with six major SRB genera in the delta-subclass of the Proteobacteria. Fluorescent in situ hybridization (FISH) analysis revealed that Desulfobulbus and Desulfonema were the most abundant SRB species in this biofilm, and this higher abundance (ca. 2–4×10⁹ cells cm^–3 and 5×10⁷ filaments cm^–3, respectively) was detected in the surface of the biofilm. Microelectrode measurements showed that a high sulfate-reducing activity was localized in a narrow zone located just below the oxic/anoxic interface when the biofilm was cultured in a synthetic medium with acetate as the sole carbon source. In contrast, a broad sulfate-reducing zone was found in the entire anoxic strata when the biofilm was cultured in the supernatant of the primary settling tank effluent. This is probably because organic carbon sources diffused into the biofilm from the bulk water and an unknown amount of volatile fatty acids was produced in the biofilm. A combined approach of molecular techniques and batch experiments with a specific inhibitor (molybdate) clearly demonstrated that Desulfobulbus is a numerically important member of SRB populations and the main contributor to the oxidation of propionate to acetate in this biofilm. However, acetate was preferentially utilized by nitrate-reducing bacteria but not by acetate-utilizing SRB.     

An experimental study of mass diffusion and reaction rate in an anaerobic biofilm

An experimental reactor consisting of two chambers, separated by a porous ceramic immobilization matrix, was constructed to measure the effective diffusivity of different compounds and the consumption rates of acetate in developing biofilms. In initial experiments, effective diffusivities for acetate, propionate, isopropanol, and lithium salt through the ceramic immobilization matrix in the absence of biofilm were determined to be 40% to 50% less than in water at infinite dilution. The effective diffusivity of the lithium salt was similar to that of acetate. The effective diffusivity of the lithium salt through biofilms of thickness in the range of 200 to 1200 mum was essentially constant with a value of approximately 7% of that in water at infinite dilution. Acetate consumption in the biofilm was linearly proportional to biofilm thickness up to a biofilm depth of 800 mum. Deviation from linearity appeared in biofilm thicknesses greater than 800 mum. Results of these experiments support previous reports that immobilized cell reactors have significantly higher bioconversion rates than suspended cell systems.     

High-Rate Anaerobic Treatment of Wastewater at Low Temperatures

Anaerobic treatment of a volatile fatty acid (VFA) mixture was investigated under psychrophilic (3 to 8°C) conditions in two laboratory-scale expanded granular sludge bed reactor stages in series. The reactor system was seeded with mesophilic methanogenic granular sludge and fed with a mixture of VFAs. Good removal of fatty acids was achieved in the two-stage system. Relative high levels of propionate were present in the effluent of the first stage, but propionate was efficiently removed in the second stage, where a low hydrogen partial pressure and a low acetate concentration were advantageous for propionate oxidation. The specific VFA-degrading activities of the sludge in each of the modules doubled during system operation for 150 days, indicating a good enrichment of methanogens and proton-reducing acetogenic bacteria at such low temperatures. The specific degradation rates of butyrate, propionate, and the VFA mixture amounted to 0.139, 0.110, and 0.214 g of chemical oxygen demand g of volatile suspended solids⁻¹ day⁻¹, respectively. The biomass which was obtained after 1.5 years still had a temperature optimum of between 30 and 40°C.     

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.     

High-rate anaerobic treatment of wastewater at low temperatures

Anaerobic treatment of a volatile fatty acid (VFA) mixture was investigated under psychrophilic (3 to 8 degrees C) conditions in two laboratory-scale expanded granular sludge bed reactor stages in series. The reactor system was seeded with mesophilic methanogenic granular sludge and fed with a mixture of VFAs. Good removal of fatty acids was achieved in the two-stage system. Relative high levels of propionate were present in the effluent of the first stage, but propionate was efficiently removed in the second stage, where a low hydrogen partial pressure and a low acetate concentration were advantageous for propionate oxidation. The specific VFA-degrading activities of the sludge in each of the modules doubled during system operation for 150 days, indicating a good enrichment of methanogens and proton-reducing acetogenic bacteria at such low temperatures. The specific degradation rates of butyrate, propionate, and the VFA mixture amounted to 0.139, 0.110, and 0.214 g of chemical oxygen demand g of volatile suspended solids-1 day-1, respectively. The biomass which was obtained after 1.5 years still had a temperature optimum of between 30 and 40 degrees C.     

Modeling and analysis of layered stationary anaerobic granular biofilms

A model that portrays substrate profiles in a steady-state multispecies granular biofilm is developed and coupled with a biofilm detachment model. The model accounts for glucose, propionate, hydrogen, and acetate transformations performed by three bacterial trophic groups: acidogens, syntrophic bacterial consortia, and methanogens. This model adequately describes the phenomenon of propionate degradation under thermodynamically unfavorable bulk hydrogen concentrations. Also suggested is the superiority of the layered biofilm structure over homogeneous distribution of the trophic groups for anaerobic degradation of organic compounds. Furthermore, model analysis suggests that with increasing bulk glucose concentration biofilm thickness reaches a maximum that is then followed by biofilm disintegration. These results may have an important impact on the design and control of upflow anaerobic sludge bed reactors. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 122-130, 1997.     

Determining specific biomass activity in anaerobic wastewater treatment processes

An experimental method for the measurement of specific gas production rate was developed and tested with biomass samples taken from anaerobic fluidized bed reactors, operating with a variety of carriers with molasses, condensate from cellulose production and brewery wastewater as feeds. The method is based on reactor sampling and offline gas volume measurement during a known time interval. Important factors are biomass and liquid sampling under oxygen-free conditions, using the liquid from the reactor as substrate, providing sufficient mixing and maintaining the physical integrity of the biomass. The method was developed in such a way that small samples (20 ml) were taken under anaerobic conditions (poising agent) for short-term (2–3 min.) gas rate measurements in a small fluidized bed (25 ml) batch reactor with U-tube. Biomass content was measured by an instrumental nitrogen method (Dumas), followed by weight determination of the carrier. The gas rates measured with the test system, and their dependence on substrate concentration, were in good agreement with those directly measured from the continuous fluidized bed reactor. Additions of molasses and acetate to the sample proved that the influence of concentration on the biomass activity can be obtained only by operating the continuous reactor at the concentration levels of interest. Comparison between the reactors showed large differences in the specific activity and the total reactor activity. It was found when comparing two reactors, that the values of the specific and the total activities permitted the calculation of the relative biomass quantities. In this way the influence of the carrier-type could be evaluated.