Influence of Environmental Conditions on Methanogenic Compositions in Anaerobic Biogas Reactors

The influence of environmental parameters on the diversity of methanogenic communities in 15 full-scale biogas plants operating under different conditions with either manure or sludge as feedstock was studied. Fluorescence in situ hybridization was used to identify dominant methanogenic members of the Archaea in the reactor samples; enriched and pure cultures were used to support the in situ identification. Dominance could be identified by a positive response by more than 90% of the total members of the Archaea to a specific group- or order-level probe. There was a clear dichotomy between the manure digesters and the sludge digesters. The manure digesters contained high levels of ammonia and of volatile fatty acids (VFA) and were dominated by members of the Methanosarcinaceae, while the sludge digesters contained low levels of ammonia and of VFA and were dominated by members of the Methanosaetaceae. The methanogenic diversity was greater in reactors operating under mesophilic temperatures. The impact of the original inoculum used for the reactor start-up was also investigated by assessment of the present population in the reactor. The inoculum population appeared to have no influence on the eventual population.     

Influence of environmental conditions on methanogenic compositions in anaerobic biogas reactors

The influence of environmental parameters on the diversity of methanogenic communities in 15 full-scale biogas plants operating under different conditions with either manure or sludge as feedstock was studied. Fluorescence in situ hybridization was used to identify dominant methanogenic members of the Archaea in the reactor samples; enriched and pure cultures were used to support the in situ identification. Dominance could be identified by a positive response by more than 90% of the total members of the Archaea to a specific group- or order-level probe. There was a clear dichotomy between the manure digesters and the sludge digesters. The manure digesters contained high levels of ammonia and of volatile fatty acids (VFA) and were dominated by members of the Methanosarcinaceae, while the sludge digesters contained low levels of ammonia and of VFA and were dominated by members of the Methanosaetaceae. The methanogenic diversity was greater in reactors operating under mesophilic temperatures. The impact of the original inoculum used for the reactor start-up was also investigated by assessment of the present population in the reactor. The inoculum population appeared to have no influence on the eventual population.     

Magnetite accelerates syntrophic acetate oxidation in methanogenic systems with high ammonia concentrations

Ammonia accumulation is a major inhibitory substance causing anaerobic digestion upset and failure in CH₄ production. At high ammonia levels, CH₄ production through syntrophic acetate oxidization (SAO) pathways is more tolerant to ammonia toxicity than the acetoclastic methanogenesis pathway, but the low CH₄ production rate through SAO constitutes the main reason for the low efficiency of energy recovery in anaerobic digesters treating ammonia‐rich substrates. In this study, we showed that acetate fermentation to CH₄ and CO₂ occurred through SAO pathway in the anaerobic reactors containing a high ammonia concentration (5.0 g l^?1 NH₄⁺–N), and the magnetite nanoparticles supplementation increased the CH₄ production rates from acetate by 36–58%, compared with the anaerobic reactors without magnetite under the same ammonia level. The mechanism of facilitated methanogenesis was proposed to be the establishment of direct interspecies electron transfer (DIET) for SAO, in which magnetite facilitated DIET between syntrophic acetate oxidizing bacteria and methanogens. High‐throughput 16S rRNA gene sequencing analysis revealed that the bacterial Geobacteraceae and the archaeal Methanosarcinaceae and Methanobacteriaceae might be involved in magnetite‐mediated DIET for SAO and CH₄ production. This study demonstrated that magnetite supplementation might provide an effective approach to accelerate CH₄ production rates in the anaerobic reactors treating wastewater containing high ammonia.     

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.     

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

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

Enrichment and description of novel bacteria performing syntrophic propionate oxidation at high ammonia level

Inefficient syntrophic propionate degradation causes severe operating disturbances and reduces biogas productivity in many high-ammonia anaerobic digesters, but propionate-degrading microorganisms in these systems remain unknown. Here, we identified candidate ammonia-tolerant syntrophic propionate-oxidising bacteria using propionate enrichment at high ammonia levels (0.7–0.8 g NH₃ L⁻¹) in continuously-fed reactors. We reconstructed 30 high-quality metagenome-assembled genomes (MAGs) from the propionate-fed reactors, which revealed two novel species from the families Peptococcaceae and Desulfobulbaceae as syntrophic propionate-oxidising candidates. Both MAGs possess genomic potential for the propionate oxidation and electron transfer required for syntrophic energy conservation and, similar to ammonia-tolerant acetate degrading syntrophs, both MAGs contain genes predicted to link to ammonia and pH tolerance. Based on relative abundance, a Peptococcaceae sp. appeared to be the main propionate degrader and has been given the provisional name “Candidatus Syntrophopropionicum ammoniitolerans”. This bacterium was also found in high-ammonia biogas digesters, using quantitative PCR. Acetate was degraded by syntrophic acetate-oxidising bacteria and the hydrogenotrophic methanogenic community consisted of Methanoculleus bourgensis and a yet to be characterised Methanoculleus sp. This work provides knowledge of cooperating syntrophic species in high-ammonia systems and reveals that ammonia-tolerant syntrophic propionate-degrading populations share common features, but diverge genomically and taxonomically from known species.     

Short-term effect of acetate and ethanol on methane formation in biogas sludge

Biochemical processes in biogas plants are still not fully understood. Especially, the identification of possible bottlenecks in the complex fermentation processes during biogas production might provide potential to increase the performance of biogas plants. To shed light on the question which group of organism constitutes the limiting factor in the anaerobic breakdown of organic material, biogas sludge from different mesophilic biogas plants was examined under various conditions. Therefore, biogas sludge was incubated and analyzed in anaerobic serum flasks under an atmosphere of N₂/CO₂. The batch reactors mirrored the conditions and the performance of the full-scale biogas plants and were suitable test systems for a period of 24 h. Methane production rates were compared after supplementation with substrates for syntrophic bacteria, such as butyrate, propionate, or ethanol, as well as with acetate and H₂+CO₂ as substrates for methanogenic archaea. Methane formation rates increased significantly by 35 to 126 % when sludge from different biogas plants was supplemented with acetate or ethanol. The stability of important process parameters such as concentration of volatile fatty acids and pH indicate that ethanol and acetate increase biogas formation without affecting normally occurring fermentation processes. In contrast to ethanol or acetate, other fermentation products such as propionate, butyrate, or H₂ did not result in increased methane formation rates. These results provide evidence that aceticlastic methanogenesis and ethanol-oxidizing syntrophic bacteria are not the limiting factor during biogas formation, respectively, and that biogas plant optimization is possible with special focus on methanogenesis from acetate.     

Microbial community structure and dynamics during anaerobic digestion of various agricultural waste materials

The influence of the feedstock type on the microbial communities involved in anaerobic digestion was investigated in laboratory-scale biogas reactors fed with different agricultural waste materials. Community composition and dynamics over 2 months of reactors’ operation were investigated by amplicon sequencing and profiling terminal restriction fragment length polymorphisms of 16S rRNA genes. Major bacterial taxa belonged to the Clostridia and Bacteroidetes, whereas the archaeal community was dominated by methanogenic archaea of the orders Methanomicrobiales and Methanosarcinales. Correlation analysis revealed that the community composition was mainly influenced by the feedstock type with the exception of a temperature shift from 38 to 55 °C which caused the most pronounced community shifts. Bacterial communities involved in the anaerobic digestion of conventional substrates such as maize silage combined with cattle manure were relatively stable and similar to each other. In contrast, special waste materials such as chicken manure or Jatropha press cake were digested by very distinct and less diverse communities, indicating partial ammonia inhibition or the influence of other inhibiting factors. Anaerobic digestion of chicken manure relied on syntrophic acetate oxidation as the dominant acetate-consuming process due to the inhibition of aceticlastic methanogenesis. Jatropha as substrate led to the enrichment of fiber-degrading specialists belonging to the genera Actinomyces and Fibrobacter.     

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

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

Response surface methodology analysis of anaerobic syntrophic degradation of volatile fatty acids in an upflow anaerobic sludge bed reactor inoculated with enriched cultures

Anaerobic oxidation of volatile fatty acids (VFAs) as the key intermediates is restricted thermodynamically. Presently, enriched acetogenic and methanogenic cultures were used for syntrophic anaerobic digestion of VFAs in an upflow anaerobic sludge bed reactor fed with acetic, propionic, and butyric acids at maximum concentrations of 5.0, 3.0, and 4.0 g/L, respectively. Interactive effects of propionate, butyrate and acetate were analyzed. Hydraulic retention time (HRT) and acetate oxidizing syntrophs and methanogen (hydrogenotrophs) to syntrophic bacteria (propionate- and butyrate-oxidizing bacteria) population ratio (M/A) were investigated as key microbiological and operating variables of VFA anaerobic degradations. M/A did not affect the size distribution and had little effect on extracellular polymer contents of the granules. Granular sludge with close spatial microbial proximity enhanced syntrophic degradation of VFAs compared to other cultures, such as suspended cultures. Optimum conditions were found to be propionate = 1.93 g/L, butyrate = 2.15 g/L, acetate = 2.50 g/L, HRT = 22 h, and M/A = 2.5 corresponding to maximum VFA removal and biogas production rate. Results of verification experiments and predicted values from fitted correlations were in close agreement at the 95% confidence interval. Granules seemed to be smaller particles and less stable in construction with an irregular fractured surface compared to the original granules.     

Methanogenesis at low temperatures by microflora of tundra wetland soil

Active methanogenesis from organic matter contained in soil samples from tundra wetland occurred even at 6 °C. Methane was the only end product in balanced microbial community with H₂/CO₂ as a substrate, besides acetate was produced as an intermediate at temperatures below 10°C. The activity of different microbial groups of methanogenic community in the temperature range of 6–28 °C was investigated using 5% of tundra soil as inoculum. Anaerobic microflora of tundra wetland fermented different organic compounds with formation of hydrogen, volatile fatty acids (VFA) and alcohols. Methane was produced at the second step. Homoacetogenic and methanogenic bacteria competed for such substrates as hydrogen, formate, carbon monoxide and methanol. Acetogens out competed methanogens in an excess of substrate and low density of microbial population. Kinetic analysis of the results confirmed the prevalence of hydrogen acetogenesis on methanogenesis. Pure culture of acetogenic bacteria was isolated at 6 °C. Dilution of tundra soil and supply with the excess of substrate disbalanced the methanoigenic microbial community. It resulted in accumulation of acetate and other VFA. In balanced microbial community obviously autotrophic methanogens keep hydrogen concentration below a threshold for syntrophic degradation of VFA. Accumulation of acetate- and H₂/CO₂-utilising methanogens should be very important in methanogenic microbial community operating at low temperatures.     

Inhibition of mesophilic solid-substrate anaerobic digestion by ammonia nitrogen

This work focused on determining the effects of ammonia-nitrogen supplementation on the mesophilic solid-substrate anaerobic digestion of municipal wastes and waste activated sludge (biosolids). Bench-scale, semi-continuous, mesophilic reactors were operated with a 21-day mass-retention time and dosed with NH₄Cl, such that the corresponding chemical O₂ demand (COD)/N ratios in their feeds were 90, 80, 65 and 50 (reactors R1 or control, R2, R3 and R4 respectively). Reactor performance was evaluated in terms of the efficiency of volatile solid removal (efficiency for short), biogas productivity, methane content in the biogas, pH and volatile organic acid contents, among other monitoring and analytical parameters. The feedstock was a mixture of urban wastes with biosolids. It was found that the process performance deteriorated at increasing dosages of ammonia N, the process practically ceasing at COD/N = 50 (R4). Inhibition was characterized by efficiency and biogas productivity decreases and a more sudden drop of methane content in biogas and pH. A significant rise of propionic, butyric and valeric acid was found in reactors receiving the highest doses of ammonia N (R3 and R4). This suggested that inhibition of the syntrophic bacteria present in the anaerobic consortia also occurred. Luong and Pearson inhibition models were fitted to the data. Both models represented very well the acute effects of N supplementation on solid-substrate anaerobic digestion. However, the Luong model could also represent the process ceasing at a critical ammonia N concentration of 2800 mg/kg mixed solids. Received: 12 April 1996 / Received revision: 23 July 1996 / Accepted: 5 August 1996     

Polyphasic Analyses of Methanogenic Archaeal Communities in Agricultural Biogas Plants

Knowledge of the microbial consortia participating in the generation of biogas, especially in methane formation, is still limited. To overcome this limitation, the methanogenic archaeal communities in six full-scale biogas plants supplied with different liquid manures and renewable raw materials as substrates were analyzed by a polyphasic approach. Fluorescence in situ hybridization (FISH) was carried out to quantify the methanogenic Archaea in the reactor samples. In addition, quantitative real-time PCR (Q-PCR) was used to support and complete the FISH analysis. Five of the six biogas reactors were dominated by hydrogenotrophic Methanomicrobiales. The average values were between 60 to 63% of archaeal cell counts (FISH) and 61 to 99% of archaeal 16S rRNA gene copies (Q-PCR). Within this order, Methanoculleus was found to be the predominant genus as determined by amplified rRNA gene restriction analysis. The aceticlastic family Methanosaetaceae was determined to be the dominant methanogenic group in only one biogas reactor, with average values for Q-PCR and FISH between 64% and 72%. Additionally, in three biogas reactors hitherto uncharacterized but potentially methanogenic species were detected. They showed closest accordance with nucleotide sequences of the hitherto unclassified CA-11 (85%) and ARC-I (98%) clusters. These results point to hydrogenotrophic methanogenesis as a predominant pathway for methane synthesis in five of the six analyzed biogas plants. In addition, a correlation between the absence of Methanosaetaceae in the biogas reactors and high concentrations of total ammonia (sum of NH₃ and NH₄⁺) was observed.During the last decade the production of biogas from organic materials and residues has increased continuously in order to reduce the greenhouse gas emission resulting from the use of fossil energy sources. The energy-bearing substance of biogas is methane, which is produced as an end product of microbial anaerobic degradation of organic substrates, such as energy crops like maize, grains, grasses, or beets. Research for optimization of biogas production from renewable materials was initially focused on the evaluation of substrate eligibility and on the development and optimization of technical systems. However, biogas formation primarily depends on the structure and activity of the microbial community (28).The key microorganisms in the biogas formation process are the methane-generating microorganisms (methanogens). The capacity for methanogenesis is limited to members of the domain Archaea and, within this domain, on the phylum Euryarchaeota. With respect to the main metabolic precursors used, methanogens are usually divided into two groups: the aceticlastic methanogens that strictly metabolize acetate and the hydrogenotrophic methanogens that use H₂ or formate as an electron donor and CO₂ as a carbon source for their metabolism. Besides these major groups, certain methanogens are also able to convert methyl groups, methylamines, or methanol to methane (23, 40). The substrates for the methanogens are provided by several physiological groups of bacteria which degrade organic matter, sometimes in close syntrophic interaction with the methanogens (1).Several studies on the microbial diversity present in lab-scale biogas reactors supplied with renewable raw material (7, 57) have been recently published. However, analyses under laboratory conditions do not necessarily reflect conditions in full-scale reactors (35). Therefore, further research on the methanogenic community in full-scale biogas reactors is crucial.Generally, studies regarding the microbial community structure in full-scale biogas reactors have focused on different systems for wastewater treatment or classical biogas plants based on manure digestion (32, 38, 43). In most systems, approximately 70% of the carbon fixed in methane was derived from acetate. Only minor amounts, up to approximately 30%, were deduced from CO₂ (1, 42). Together with the presence of huge assemblages of Methanosarcina sp., it was assumed by some authors that aceticlastic methanogenesis was the predominant pathway for methane formation. Moreover, as shown by other studies, the relative contribution of H₂/CO₂ versus acetate as metabolic precursors for methanogens can be quite different in other anaerobic environments (10, 33, 37). However, the methanogenic microfloras in full-scale biogas reactors supplied with energy crops as a primary or sole substrate have rarely been studied (35, 37, 45).The aim of this study was to gain insight into the diversity of methane-producing Archaea in six full-scale biogas plants supplied with renewable raw material and different types of liquid manure as substrates. Therefore, a polyphasic approach with three different culture-independent techniques (fluorescence in situ hybridization [FISH], quantitative PCR [Q-PCR], and 16S rRNA gene analysis) to analyze methanogen diversity was carried out to overcome the known limitations of each single approach (15, 46). To analyze potential effects of different process parameters on the methanogenic archaeal community, the reactor performances were correlated with the apparent archaeal diversity.     

Changing Feeding Regimes To Demonstrate Flexible Biogas Production: Effects on Process Performance,Microbial Community Structure,and Methanogenesis Pathways

Flexible biogas production that adapts biogas output to energy demand can be regulated by changing feeding regimes. In this study, the effect of changes in feeding intervals on process performance, microbial community structure, and the methanogenesis pathway was investigated. Three different feeding regimes (once daily, every second day, and every 2 h) at the same organic loading rate were studied in continuously stirred tank reactors treating distiller''s dried grains with solubles. A larger amount of biogas was produced after feeding in the reactors fed less frequently (once per day and every second day), whereas the amount remained constant in the reactor fed more frequently (every 2 h), indicating the suitability of the former for the flexible production of biogas. Compared to the conventional more frequent feeding regimes, a methane yield that was up to 14% higher and an improved stability of the process against organic overloading were achieved by employing less frequent feeding regimes. The community structures of bacteria and methanogenic archaea were monitored by terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA and mcrA genes, respectively. The results showed that the composition of the bacterial community varied under the different feeding regimes, and the observed T-RFLP patterns were best explained by the differences in the total ammonia nitrogen concentrations, H₂ levels, and pH values. However, the methanogenic community remained stable under all feeding regimes, with the dominance of the Methanosarcina genus followed by that of the Methanobacterium genus. Stable isotope analysis showed that the average amount of methane produced during each feeding event by acetoclastic and hydrogenotrophic methanogenesis was not influenced by the three different feeding regimes.     

Methanosarcina Play an Important Role in Anaerobic Co-Digestion of the Seaweed Ulva lactuca: Taxonomy and Predicted Metabolism of Functional Microbial Communities

Macro-algae represent an ideal resource of third generation biofuels, but their use necessitates a refinement of commonly used anaerobic digestion processes. In a previous study, contrasting mixes of dairy slurry and the macro-alga Ulva lactuca were anaerobically digested in mesophilic continuously stirred tank reactors for 40 weeks. Higher proportions of U. lactuca in the feedstock led to inhibited digestion and rapid accumulation of volatile fatty acids, requiring a reduced organic loading rate. In this study, 16S pyrosequencing was employed to characterise the microbial communities of both the weakest (R1) and strongest (R6) performing reactors from the previous work as they developed over a 39 and 27-week period respectively. Comparing the reactor communities revealed clear differences in taxonomy, predicted metabolic orientation and mechanisms of inhibition, while constrained canonical analysis (CCA) showed ammonia and biogas yield to be the strongest factors differentiating the two reactor communities. Significant biomarker taxa and predicted metabolic activities were identified for viable and failing anaerobic digestion of U. lactuca. Acetoclastic methanogens were inhibited early in R1 operation, followed by a gradual decline of hydrogenotrophic methanogens. Near-total loss of methanogens led to an accumulation of acetic acid that reduced performance of R1, while a slow decline in biogas yield in R6 could be attributed to inhibition of acetogenic rather than methanogenic activity. The improved performance of R6 is likely to have been as a result of the large Methanosarcina population, which enabled rapid removal of acetic acid, providing favourable conditions for substrate degradation.     

Acetate Oxidation Is the Dominant Methanogenic Pathway from Acetate in the Absence of Methanosaetaceae

The oxidation of acetate to hydrogen, and the subsequent conversion of hydrogen and carbon dioxide to methane, has been regarded largely as a niche mechanism occurring at high temperatures or under inhibitory conditions. In this study, 13 anaerobic reactors and sediment from a temperate anaerobic lake were surveyed for their dominant methanogenic population by using fluorescent in situ hybridization and for the degree of acetate oxidation relative to aceticlastic conversion by using radiolabeled [2-¹⁴C]acetate in batch incubations. When Methanosaetaceae were not present, acetate oxidation was the dominant methanogenic pathway. Aceticlastic conversion was observed only in the presence of Methanosaetaceae.     

Anaerobic degradation of volatile fatty acids at different sulphate concentrations

The effect of sulfate on the anaerobic breakdown of mixtures of acetate, propionate and butyrate at three different sulfate to fatty acid ratios was studied in upflow anaerobic sludge blanket reactors. Sludge characteristics were followed with time by means of sludge activity tests and by enumeration of the different physiological bacterial groups. At each sulfate concentration acetate was completely converted into methane and CO₂, and acetotrophic sulfate-reducing bacteria were not detected. Hydrogenotrophic methanogenic bacteria and hydrogenotrophic sulfate-reducing bacteria were present in high numbers in the sludge of all reactors. However, a complete conversion of H₂ by sulfate reducers was found in the reactor operated with excess sulfate. At higher sulfate concentrations, oxidation of propionate by sulfate-reducing bacteria became more important. Only under sulfate-limiting conditions did syntrophic propionate oxidizers out-compete propionate-degrading sulfate reducers. Remarkably, syntrophic butyrate oxidizers were well able to compete with sulfate reducers for the available butyrate, even with an excess of sulfate. Correspondence to: A. Visser     

Multiple syntrophic interactions in a terephthalate-degrading methanogenic consortium

Terephthalate (TA) is one of the top 50 chemicals produced worldwide. Its production results in a TA-containing wastewater that is treated by anaerobic processes through a poorly understood methanogenic syntrophy. Using metagenomics, we characterized the methanogenic consortium inside a hyper-mesophilic (that is, between mesophilic and thermophilic), TA-degrading bioreactor. We identified genes belonging to dominant Pelotomaculum species presumably involved in TA degradation through decarboxylation, dearomatization, and modified β-oxidation to H₂/CO₂ and acetate. These intermediates are converted to CH₄/CO₂ by three novel hyper-mesophilic methanogens. Additional secondary syntrophic interactions were predicted in Thermotogae, Syntrophus and candidate phyla OP5 and WWE1 populations. The OP5 encodes genes capable of anaerobic autotrophic butyrate production and Thermotogae, Syntrophus and WWE1 have the genetic potential to oxidize butyrate to CO₂/H₂ and acetate. These observations suggest that the TA-degrading consortium consists of additional syntrophic interactions beyond the standard H₂-producing syntroph–methanogen partnership that may serve to improve community stability.     

Microbial Community Dynamics and Stability during an Ammonia-Induced Shift to Syntrophic Acetate Oxidation

Anaerobic digesters rely on the diversity and distribution of parallel metabolic pathways mediated by complex syntrophic microbial communities to maintain robust and optimal performance. Using mesophilic swine waste digesters, we experimented with increased ammonia loading to induce a shift from aceticlastic methanogenesis to an alternative acetate-consuming pathway of syntrophic acetate oxidation. In comparison with control digesters, we observed shifts in bacterial 16S rRNA gene content and in functional gene repertoires over the course of the digesters'' 3-year operating period. During the first year, under identical startup conditions, all bioreactors mirrored each other closely in terms of bacterial phylotype content, phylogenetic structure, and evenness. When we perturbed the digesters by increasing the ammonia concentration or temperature, the distribution of bacterial phylotypes became more uneven, followed by a return to more even communities once syntrophic acetate oxidation had allowed the experimental bioreactors to regain stable operation. The emergence of syntrophic acetate oxidation coincided with a partial shift from aceticlastic to hydrogenotrophic methanogens. Our 16S rRNA gene analysis also revealed that acetate-fed enrichment experiments resulted in communities that did not represent the bioreactor community. Analysis of shotgun sequencing of community DNA suggests that syntrophic acetate oxidation was carried out by a heterogeneous community rather than by a specific keystone population with representatives of enriched cultures with this metabolic capacity.