Syntrophic Propionate Oxidation Research Articles

Operational mode and powdered activated carbon promoting syntrophic propionate oxidation during anaerobic digestion of complex organic substances

Operational mode and powdered activated carbon (PAC) are key factors facilitating microbial syntrophy and interspecies electron transfer during anaerobic digestion, consequently benefiting process stability and efficient methanogenesis. In this study, continuous-flow reactor (CFR) and sequencing batch reactor (SBR), with and without the addition of PAC, respectively, were operated to examine their effects on system performance and methanogenic activity. Based on the cycle-test result, the PAC-amended CFR (CFRPAC) recorded both the highest methane yield (690.1 mL/L) and the maximum CH4 production rate (28.8 mL/(L·h)), while SBRs exhibited slow methanogenic rates. However, activity assays indicated that SBRs were beneficial for organics removal in batch experiments fed with peptone. Taxonomic and functional analysis confirmed that CFRs were optimal for proliferating oligotrophs (e.g., Geobacter) and SBRs were more suitable for copiotrophs (e.g., Desulfobulbus). Metagenomic analysis revealed that CFRs had efficient acetate metabolic pathways from propionate and ethanol, whereas SBRs did not, resulting in the buildup of propionate. Furthermore, Methanobacterium and Methanothrix were acclimated to the different operational conditions, while acetoclastic Methanosarcina and hydrogenotrophic Methanolinea were acclimated in SBRs (5.1–13.4%) and CFRs (0.3–1.7%), respectively. This study confirmed the enhancement of microbial syntrophy by the addition of PAC as well as the acclimation of electroactive bacteria (e.g., Geobacter) with complex organic substances.

Potential of acetic acid to restore methane production in anaerobic reactors critically intoxicated by ammonia as evidenced by metabolic and microbial monitoring

BackgroundBiogas and biomethane production from the on-farm anaerobic digestion (AD) of animal manure and agri-food wastes could play a key role in transforming Europe’s energy system by mitigating its dependence on fossil fuels and tackling the climate crisis. Although ammonia is essential for microbial growth, it inhibits the AD process if present in high concentrations, especially under its free form, thus leading to economic losses. In this study, which includes both metabolic and microbial monitoring, we tested a strategy to restore substrate conversion to methane in AD reactors facing critical free ammonia intoxication.ResultsThe AD process of three mesophilic semi-continuous 100L reactors critically intoxicated by free ammonia (> 3.5 g_N L−1; inhibited hydrolysis and heterotrophic acetogenesis; interrupted methanogenesis) was restored by applying a strategy that included reducing pH using acetic acid, washing out total ammonia with water, re-inoculation with active microbial flora and progressively re-introducing sugar beet pulp as a feed substrate. After 5 weeks, two reactors restarted to hydrolyse the pulp and produced CH4 from the methylotrophic methanogenesis pathway. The acetoclastic pathway remained inhibited due to the transient dominance of a strictly methylotrophic methanogen (Candidatus Methanoplasma genus) to the detriment of Methanosarcina. Concomitantly, the third reactor, in which Methanosarcina remained dominant, produced CH4 from the acetoclastic pathway but faced hydrolysis inhibition. After 11 weeks, the hydrolysis, the acetoclastic pathway and possibly the hydrogenotrophic pathway were functional in all reactors. The methylotrophic pathway was no longer favoured. Although syntrophic propionate oxidation remained suboptimal, the final pulp to CH4 conversion ratio (0.41 ± 0.10 LN_CH4 g_VS−1) was analogous to the pulp biochemical methane potential (0.38 ± 0.03 LN_CH4 g_VS−1).ConclusionsDespite an extreme free ammonia intoxication, the proposed process recovery strategy allowed CH4 production to be restored in three intoxicated reactors within 8 weeks, a period during which re-inoculation appeared to be crucial to sustain the process. Introducing acetic acid allowed substantial CH4 production during the recovery period. Furthermore, the initial pH reduction promoted ammonium capture in the slurry, which could allow the field application of the effluents produced by full-scale digesters recovering from ammonia intoxication.

Thermodynamic restrictions determine ammonia tolerance of functional floras during anaerobic digestion

Ammonia inhibition is a major challenge in anaerobic digestion processes, affecting the activity and performance of functional floras, including syntrophic butyrate oxidation (FSBO), syntrophic propionate oxidation (FSPO), acetoclastic methanogenesis (FAMs), and hydrogenotrophic methanogenesis (FHMs). FHMs was more tolerant to ammonia, with a half maximal inhibitory concentration (IC50) of 18.80 g/L, followed by FSBO (IC50 = 14.26 g/L) and FSPO (IC50 = 10.47 g/L), and FAMs was the most sensitive to ammonia with the lowest IC50 (1.74 g/L). The order of ammonia tolerance (IC50) was found to be correlated with the Gibbs free energy of metabolic reactions in each functional flora. The results suggested the functional flora with higher energy availability (exergonic capacity) exhibited enhanced ammonia resistance ability. These findings provide insights into the thermodynamic restrictions and ammonia tolerance mechanisms of functional floras, which can guide the optimization and operation of anaerobic digestion systems for efficient methane production.

Syntrophic Propionate Oxidation: One of the Rate-Limiting Steps of Organic Matter Decomposition in Anoxic Environments.

Syntrophic propionate oxidation is one of the rate-limiting steps during anaerobic decomposition of organic matter in anoxic environments. Syntrophic propionate-oxidizing bacteria (SPOB) are members of the "rare biosphere" living at the edge of the thermodynamic limit in most natural habitats. Hitherto, only 10 bacterial species capable of syntrophic propionate oxidization have been identified. SPOB employ different metabolisms for propionate oxidation (e.g., methylmalonyl-CoA pathway and C6 dismutation pathway) and show diverse life strategies (e.g., obligately and facultatively syntrophic lifestyle). The flavin-based electron bifurcation/confurcation (FBEB/C) systems have been proposed to help solve the thermodynamic dilemma during the formation of the low-potential products H2 and formate. Molecular ecological approaches, such as DNA stable isotope probing (DNA-SIP) and metagenomics, have been used to detect SPOB in natural environments. Furthermore, the biogeographical pattern of SPOB has been recently described in paddy soils. A comprehensive understanding of SPOB is essential for better predicting and managing organic matter decomposition and carbon cycling in anoxic environments. In this review, we described the critical role of syntrophic propionate oxidation in anaerobic decomposition of organic matter, phylogenetic and metabolic diversity, life strategies and ecophysiology, composition of syntrophic partners, and pattern of biogeographic distribution of SPOB in natural environments. We ended up with a few perspectives for future research.

Conductive materials enhance microbial salt-tolerance in anaerobic digestion of food waste: Microbial response and metagenomics analysis

Previous studies have shown that high salinity environments can inhibit anaerobic digestion (AD) of food waste (FW). Finding ways to alleviate salt inhibition is important for the disposal of the growing amount of FW. We selected three common conductive materials (powdered activated carbon, magnetite, and graphite) to understand their performance and individual mechanisms that relieve salinity inhibition. Digester performances and related enzyme parameters were compared. Our data revealed that under normal and low salinity stress conditions, the anaerobic digester ran steady without significant inhibitions. Further, the presence of conductive materials promoted conversion rate of methanogenesis. This promotion effect was highest from magnetite > powdered activated carbon (PAC) > graphite. At 1.5% salinity, PAC and magnetite are beneficial in maintaining high methane production efficiency while control and the graphite added digester acidified and failed rapidly. Additionally, metagenomics and binning were used to analyze the metabolic capacity of the microorganisms. Some species enriched by PAC and magnetite possessed higher cation transport capacities and were to accumulate compatible solutes. PAC and magnetite promoted direct interspecies electron transference (DIET) and syntrophic oxidation of butyrate and propionate. Also, the microorganisms had more energy available to cope with salt inhibition in the PAC and magnetite added digesters. Our data imply that the promotion of Na+/H+ antiporter, K+ uptake, and osmoprotectant synthesis or transport by conductive materials may be crucial for their proliferation in highly stressful environments. These findings will help to understand the mechanisms of alleviate salt inhibition by conductive materials and help to recover methane from high-salinity FW.

Key players in syntrophic propionate oxidation revealed by metagenome-assembled genomes from anaerobic digesters bioaugmented with propionic acid enriched microbial consortia.

Propionic acid (HPr) is frequently accumulated in anaerobic digesters due to its thermodynamically unfavorable degradation reaction. Here, we identify key players in HPr oxidation and organic overloading recovery from metagenome-assembled genomes (MAGs) recovered from anaerobic digesters inoculated with HPr-enriched microbial consortia before initiating organic overloading. Two independent HPr-enrichment cultures commonly selected two uncultured microorganisms represented with high relative abundance: Methanoculleus sp002497965 and JABUEY01 sp013314815 (a member of the Syntrophobacteraceae family). The relative abundance of JABUEY01 sp013314815 was 60 times higher in bioaugmented bioreactors compared to their unaugmented counterparts after recovery from organic overloading. Genomic analysis of JABUEY01 sp013314815 revealed its metabolic potential for syntrophic propionate degradation when partnered with hydrogenotrophic methanogens (e.g., Methanoculleus sp002497965) via the methylmalonyl-CoA pathway. Our results identified at least two key species that are responsible for efficient propionate removal and demonstrate their potential applications as microbial cocktails for stable AD operation.

Thermodynamics of volatile fatty acid degradation during anaerobic digestion under organic overload stress: The potential to better identify process stability

Anaerobic digestion (AD) operating under organic overload stress usually increases the potential for process instability, leading to significant economic and ecological consequences. Volatile fatty acids (VFAs) accumulation is regularly considered a major factor during AD and their degradation is subject to thermodynamic constraints. To date, no study has systematically investigated the mechanisms of VFA degradation on process stability from the perspective of thermodynamics. Hence, increased substrate-to-inoculum ratio was applied in this study to simulate organic overload stress using batch tests with Hybrid Pennisetum. As a result, VFAs accumulation increased, accompanied by decreased methane yield, slower methane production kinetics and even severe process instability. Metagenomic analysis demonstrated that the accumulated propionate and butyrate were degraded by methyl-malonyl-CoA and the β-oxidation pathway while syntrophic acetate oxidation was preferred during acetate degradation. The deviation of stability parameters to varying degrees from the recommended threshold values was observed. However, a subsequent thermodynamic analysis revealed that moderate organic overload stress merely retarded the syntrophic oxidation of propionate, butyrate, and acetate. As a result, the methanogenic activity decreased, and the lag phase of AD was extended, but no adverse thermodynamic effects actually occurred. Changes in the Gibbs free energy for syntrophic propionate and acetate oxidation have the potential to better identify process stability. This study provided novel insights into the underlying thermodynamic mechanisms of VFA degradation and may have important implications for improving the current diagnostic mode for AD process stability.

Integrated multi-omics analyses reveal the key microbial phylotypes affecting anaerobic digestion performance under ammonia stress

Ammonia inhibition is one of the most common causes of instability during the operation of commercial biogas plants. Here, the sensitivity of different functional bacteria to ammonia stress, the ability of functional bacteria to adapt to ammonia stress, and the key phylotypes affecting anaerobic digestion (AD) performance were studied by evaluating the process performance, active microbiome, and protein expression patterns during endogenous ammonia accumulation using integrated metagenomics and metaproteomics analyses. Acetate metabolism was most sensitive to ammonia stress, and the expression activity of methyl-CoM reductase of Methanothrix was inhibited at relatively low ammonia concentrations, which resulted in the accumulation of acetate and other short-chain volatile fatty acids (VFAs) through feedback effects. As the AD process progressed, the abundance of active Methanosarcina with high ammonia tolerance increased, and the activity of their enzymes related to acetoclastic methanation was significantly up-regulated, which resulted in the complete restoration of acetate metabolism and AD performance. Thus, microbial communities can cope with acetate metabolic repression through self-optimization. In contrast, when the total ammonia nitrogen (TAN) and free ammonia nitrogen (FAN) increased to 4846.95 mg N/L and 337.46 mg N/L, respectively, propionate (and no other VFAs) accumulated in the digester, which was accompanied by a decrease in methane yield by more than 65%. At this time, the abundance of active syntrophic propionate-oxidizing bacteria (SPOB) decreased by 52%, and the expression of key enzymes related to propionate degradation was significantly down-regulated. The proportion of down-regulated differentially expressed proteins in the dominant Pelotomaculum was as high as 94%, indicating the severe suppression of the growth of these functional bacteria as well as their inability to easily acclimate to ammonia stress. Thus, SPOB appeared to be the key microbial phylotypes affecting AD performance under ammonia stress. Ammonia inhibited the methylmalonyl-CoA pathway of Pelotomaculum by inhibiting the expression of succinyl-CoA synthase, which resulted in the suppression of syntrophic propionate oxidation. The results of this study provide new insights into the microbial mechanism of ammonia inhibition and identify the key phylotypes affecting AD performance under ammonia stress. Our findings also shed light on the microbial regulatory targets of nitrogen-rich waste anaerobic digesters.

Directional Selection of Microbial Community Reduces Propionate Accumulation in Glycerol and Glucose Anaerobic Bioconversion Under Elevated pCO2.

Volatile fatty acid accumulation is a sign of digester perturbation. Previous work showed the thermodynamic limitations of hydrogen and CO2 in syntrophic propionate oxidation under elevated partial pressure of CO2 (pCO2). Here we study the effect of directional selection under increasing substrate load as a strategy to restructure the microbial community and induce cross-protection mechanisms to improve glucose and glycerol conversion performance under elevated pCO2. After an adaptive laboratory evolution (ALE) process, viable cell density increased and predominant microbial groups were modified: an increase in Methanosaeta and syntrophic propionate oxidizing bacteria (SPOB) associated with the Smithella genus was found with glycerol as the substrate. A modest increase in SPOB along with a shift in the predominance of Methanobacterium toward Methanosaeta was observed with glucose as the substrate. The evolved inoculum showed affected diversity within archaeal spp. under 5 bar initial pCO2; however, higher CH4 yield resulted from enhanced propionate conversion linked to the community shifts and biomass adaptation during the ALE process. Moreover, the evolved inoculum attained increased cell viability with glucose and a marginal decrease with glycerol as the substrate. Results showed differences in terms of carbon flux distribution using the evolved inoculum under elevated pCO2: glucose conversion resulted in a higher cell density and viability, whereas glycerol conversion led to higher propionate production whose enabled conversion reflected in increased CH4 yield. Our results highlight that limited propionate conversion at elevated pCO2 resulted from decreased cell viability and low abundance of syntrophic partners. This limitation can be mitigated by promoting alternative and more resilient SPOB and building up biomass adaptation to environmental conditions via directional selection of microbial community.

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.

Blackwater biomethane recovery using a thermophilic upflow anaerobic sludge blanket reactor: Impacts of effluent recirculation on reactor performance

Thermophilic anaerobic digestion is a promising process for high-solid blackwater (BW) treatment due to improved hydrolysis rates, high methanogenesis efficiency, and pathogen removal, when compared with mesophilic treatment. In the present work, the effects of effluent recirculation (i.e., mixing) on thermophilic blackwater treatment were studied. A laboratory-scale thermophilic upflow anaerobic sludge blanket reactor was operated with and without effluent recirculation. The methanogenesis efficiency of the BW treatment increased from 45.0 ± 2.9% when effluent recirculation was applied to 56.7 ± 5.5% without effluent recirculation. Without effluent recirculation, the COD accumulation in the bioreactor was reduced from 17.2 to 3.8% and the effluent volatile fatty acids (VFA) concentration was reduced from 0.64 ± 0.18 to 0.15 ± 0.10 g/L. Further, both acetoclastic and hydrogenotrophic methanogenic activity increased from 101.3 ± 10.8 and 93.9 ± 6.1 to 120.4 ± 9.4 and 118.2 ± 13.2 mg CH4-COD/(gVSS⋅d), respectively, after effluent recirculation was discontinued. The predominant methanogens changed from Methanothermobacter (67%) with effluent recirculation to Methanosarcina (62%) without effluent recirculation. As compared to the effluent recirculation conditions, the enhanced biomethane recovery and treatment performance without effluent recirculation can be attributed to the close proximity of bacteria and archaea groups and the reduced VFA accumulation. Predicted functional gene comparison showed higher prevalence of function for intermediate metabolite transportation (transporters, ATP-binding cassette (ABC) transporters, and two-component system) after discontinuing effluent circulation, which contributed to improved syntrophic propionate oxidation and syntrophic acetate oxidization and enhanced hydrogenotrophic methanogenesis.

Biochar and magnetite promote methanogenesis during anaerobic decomposition of rice straw

Conductive materials like magnetite nanoparticles (nanoFe3O4) are known to stimulate methanogenesis from syntrophic oxidation of butyrate and propionate. Whether conductive materials increase the decomposition of complex organic substrates like plant residues is unknown. Here, we investigated the effects of biochar and nanoFe3O4 on anaerobic decomposition of rice straw and methanogenesis in a rice paddy soil. The straw decomposition and CH4 production were greatly stimulated by both treatments. Major bacterial populations were Bacteroidetes, Clostridia, Deltaproteobacteria, Actinobacteria and Anaerolineae, while methanogens were predominately Methanosarcina, Methanosaeta and Methanobacterium. Bacteroides were relatively enriched in the biochar treatment, while the Deltaproteobacteria and Anaerolineae were favored by nanoFe3O4. The relative abundance of typical syntrophs including Syntrophomonas, Syntrophus and Smithella were positively correlated with the degradation of intermediate short-chain fatty acids. Notably, the relative abundance of Geobacter, which increased during the incubation, was approximately tenfold greater than all other syntrophs. The co-occurrence of Geobacter together with Methanosarcina and Methanosaeta suggests that the enhanced decomposition of rice straw and methanogenesis by biochar and magnetite may be related to direct interspecies electron transfer involved in the syntrophic oxidation of intermediate products.

Candidatus Syntrophosphaera thermopropionivorans: a novel player in syntrophic propionate oxidation during anaerobic digestion.

Propionate is an important intermediate in the anaerobic mineralization of organic matter. In methanogenic environments, its degradation relies on syntrophic associations between syntrophic propionate-oxidizing bacteria (SPOB) and Archaea. However, only 10 isolated species have been identified as SPOB so far. We report syntrophic propionate oxidation in thermophilic enrichments of Candidatus Syntrophosphaera thermopropionivorans, a novel representative of the candidate phylum Cloacimonetes. In enrichment culture, methane was produced from propionate, while Ca. S. thermopropionivorans contributed 63% to total bacterial cells. The draft genome of Ca. S. thermopropionivorans encodes genes for propionate oxidation via methymalonyl-CoA. Phylogenetically, Ca. S. thermopropionivorans affiliates with the uncultured Cloacimonadaceae W5 and is more distantly related (86.4% 16S rRNA gene identity) to Ca. Cloacimonas acidaminovorans. Although Ca. S. thermopropionivorans was enriched from a thermophilic biogas reactor, Ca. Syntrophosphaera was in particular associated with mesophilic anaerobic digestion systems. 16S rRNA gene amplicon sequencng and a novel genus-specific quantitative PCR assay consistently identified Ca. Syntrophosphaera/Cloacimonadaceae W5 in 9 of 12 tested full-scale biogas reactors thereby outnumbering other SPOB such as Pelotomaculum, Smithella and Syntrophobacter. Taken together the ubiquity and abundance of Ca. Syntrophosphaera, those SPOB might be key players for syntrophic propionate metabolism that have been overlooked before.

Stimulation of Smithella-dominating propionate oxidation in a sediment enrichment by magnetite and carbon nanotubes.

Recent studies have shown that application of conductive materials including magnetite and carbon nanotubes (CNTs) can promote the methanogenic decomposition of short-chain fatty acids and even more complex organic matter in anaerobic digesters and natural habitats. The linkage to microbial identity and the mechanisms, however, remain poorly understood. Here, we evaluate the effects of nanoscale magnetite (nanoFe3 O4 ) and multiwalled CNTs on the syntrophic oxidation of propionate in an enrichment obtained from lake sediment. The microbial populations were composed mainly of Smithella, Syntrophomonas, Methanosaeta, Methanosarcina and Methanoregula. In addition to acetate, butyrate was transiently accumulated indicating that propionate was oxidized by Smithella via the dismutation pathway and part of the leaked butyrate was oxidized by Syntrophomonas. Propionate oxidation and CH4 production were significantly accelerated in the presence of nanoFe3 O4 and CNTs. While propionate oxidation was suppressed upon H2 application and suspended completely upon formate application in the control, this suppressive effect was substantially compromised in the presence of nanoFe3 O4 and CNTs. The tests on hydrogenotrophic methanogenesis of a pure culture methanogen and of the enrichment culture without propionate showed negative effect by both materials. The positive effect of nanoFe3 O4 disappeared when it was insulated by surface-coating with silica. Observations made with fluorescence in situ hybridization and scanning electron microscope indicated the extensive formation of microbial cell-conductive material mixture aggregates. Our results suggest that direct interspecies electron transfer is likely activated by the conductive materials and operates in concert with H2 /formate-dependent electron transfer for syntrophic propionate oxidation in the sediment enrichment.

Formate and Hydrogen as Electron Shuttles in Terminal Fermentations in an Oligotrophic Freshwater Lake Sediment.

The energetic situation of terminal fermentations in methanogenesis was analyzed by pool size determinations in sediment cores taken in the oligotrophic Lake Constance, Germany. Distribution profiles of fermentation intermediates and products were measured at three different water depths (2, 10, and 80 m). Methane concentrations were constant below 10 cm of sediment depth. Within the methanogenic zone, concentrations of formate, acetate, propionate, and butyrate varied between 1 and 40 μM, and hydrogen was between 0.5 and 5 Pa. From the distribution profiles of the fermentation intermediates, Gibbs free energy changes for their interconversion were calculated. Pool sizes of formate and hydrogen were energetically nearly equivalent, with -5 ± 5 kJ per mol difference of free energy change (ΔG) for a hypothetical conversion of formate to hydrogen plus CO2 The ΔG values for conversion of fatty acids to methanogenic substrates and their further conversion to methane and CO2 were calculated with hydrogen and with formate as intermediates. Syntrophic propionate oxidation reached energetic equilibrium with formate as the sole electron carrier but was sufficiently exergonic if at least some of the electrons were transferred via hydrogen. The energetic consequences of formate versus hydrogen transfer in secondary and methanogenic fermentations indicate that both carrier systems are probably used simultaneously to optimize the energy yields for the partners involved.IMPORTANCE In the terminal steps of methane formation in freshwater lake sediments, fermenting bacteria cooperate syntrophically with methanogens and homoacetogens at minimum energy increments via interspecies electron transfer. The energy yields of the partner organisms in these cooperations have so far been calculated based mainly on in situ hydrogen partial pressures. In the present study, we also analyzed pools of formate as an alternative electron carrier in sediment cores of an oligotrophic lake. The formate and hydrogen pools appeared to be energetically nearly equivalent and are likely to be used simultaneously for interspecies electron transfer. Calculations of reaction energies of the partners involved suggest that propionate degradation may also proceed through the Smithella pathway, which converts propionate via butyrate and acetate to three acetate residues, thus circumventing one energetically difficult fatty acid oxidation step.

Inhibitory effects of ammonia on syntrophic propionate oxidation in anaerobic digester sludge

Syntrophic propionate oxidation (SPO) coupled with methanogenesis is often inhibited under high ammonium concentrations in anaerobic digesters. However, the inhibitory mechanism remains poorly understood. We conducted two independent laboratory experiments with a swine manure digester sludge. In experiment I, RNA-based stable isotope probing (SIP) was applied to determine the active players of both bacteria and methanogens involved in SPO under different ammonium concentrations (0, 3 and 7 g NH4+N L−1). In experiment II, the dynamics of the bacterial community under ammonia stress was monitored using the 16S rRNA pyrosequencing and quantitative PCR under similar conditions as in experiment I but without the addition of external propionate. An additional higher ammonium treatment (10 g NH4+N L−1) was applied in experiment II to maximize the ammonia stress. We identified that the Smithella bacteria and the Methanosaetaceae and Methanospirillaceae archaea were the most active players involved in SPO and methanogenesis. We revealed that Smithella, Methanosaetaceae and Methanospirillaceae were moderately and severely inhibited at 3 and 7–10 g NH4+N L−1, respectively. However, the fermentative bacteria appeared to be more tolerant to ammonia stress. The microbial responses were corroborated with the accumulation of VFAs and the repression of methanogenesis under high ammonium conditions.

Metatranscriptomics reveals a differential temperature effect on the structural and functional organization of the anaerobic food web in rice field soil

BackgroundThe expected increase in global surface temperature due to climate change may have a tremendous effect on the structure and function of the anaerobic food web in flooded rice field soil. Here, we used the metatranscriptomic analysis of total RNA to gain a system-level understanding of this temperature effect on the methanogenic food web.ResultsMesophilic (30 °C) and thermophilic (45 °C) food web communities had a modular structure. Family-specific rRNA dynamics indicated that each network module represents a particular function within the food webs. Temperature had a differential effect on all the functional activities, including polymer hydrolysis, syntrophic oxidation of key intermediates, and methanogenesis. This was further evidenced by the temporal expression patterns of total bacterial and archaeal mRNA and of transcripts encoding carbohydrate-active enzymes (CAZymes). At 30 °C, various bacterial phyla contributed to polymer hydrolysis, with Firmicutes decreasing and non-Firmicutes (e.g., Bacteroidetes, Ignavibacteriae) increasing with incubation time. At 45 °C, CAZyme expression was solely dominated by the Firmicutes but, depending on polymer and incubation time, varied on family level. The structural and functional community dynamics corresponded well to process measurements (acetate, propionate, methane). At both temperatures, a major change in food web functionality was linked to the transition from the early to late stage. The mesophilic food web was characterized by gradual polymer breakdown that governed acetoclastic methanogenesis (Methanosarcinaceae) and, with polymer hydrolysis becoming the rate-limiting step, syntrophic propionate oxidation (Christensenellaceae, Peptococcaceae). The thermophilic food web had two activity stages characterized first by polymer hydrolysis and followed by syntrophic oxidation of acetate (Thermoanaerobacteraceae, Heliobacteriaceae, clade OPB54). Hydrogenotrophic Methanocellaceae were the syntrophic methanogen partner, but their population structure differed between the temperatures. Thermophilic temperature promoted proliferation of a new Methanocella ecotype.ConclusionsTemperature had a differential effect on the structural and functional continuum in which the methanogenic food web operates. This temperature-induced change in food web functionality may not only be a near-future scenario for rice paddies but also for natural wetlands in the tropics and subtropics.

Response of methane production via propionate oxidation to carboxylated multiwalled carbon nanotubes in paddy soil enrichments.

Carboxylated multiwalled carbon nanotubes (MWCNTs-COOH) have become a growing concern in terms of their fate and toxicity in aqueous environments. Methane (CH4) is a major product of organic matter degradation in waterlogged environments. In this study, we determined the effect of MWCNTs-COOH on the production of CH4 from propionate oxidation in paddy soil enrichments. The results showed that the methanogenesis from propionate degradation was accelerated in the presence of MWCNTs-COOH. In addition, the rates of CH4 production and propionate degradation increased with increasing concentrations of MWCNTs-COOH. Scanning electron microscopy (SEM) observations showed that the cells were intact and maintained their structure in the presence of MWCNTs-COOH. In addition, SEM and fluorescence in situ hybridization (FISH) images revealed that the cells were in direct contact with the MWCNTs and formed cell-MWCNTs aggregates that contained both bacteria and archaea. On the other hand, nontoxic magnetite nanoparticles (Fe3O4) had similar effects on the CH4 production and cell integrity as the MWCNTs-COOH. Compared with no nanomaterial addition, the relative abundances of Geobacter and Methanosarcina species increased in the presence of MWCNTs-COOH. This study suggests that MWCNTs-COOH exerted positive rather than cytotoxic effects on the syntrophic oxidation of propionate in paddy soil enrichments and affected the bacterial and archaeal community structure at the test concentrations. These findings provide novel insight into the consequences of nanomaterial release into anoxic natural environments.

The Establishment and Characteristics of Dominant Syntrophic Propionate Oxidation Bacteria and Sulfate-Reducing Bacteria in a Mixed Culture

The anaerobic bio-treatment of lower COD/SO42- ratio wastewater constitutes a bottle-aqneck due to the limited carbon source. In this type of environment, interactions between acidogenic fermentation bacteria and methane-producing bacteria (MPB) are aided by syntrophic fatty acid oxidation bacteria present in the functional niche; MPB and sulfate-reducing bacteria (SRB) compete with each other due to the availability of sulfate. Therefore, introduction of syntrophic fatty acid oxidation bacteria into an SRB dominated culture can achieve maximum utilization of the substrate, weakening the competition of MPB. In this study, the mixed culture of dominant syntrophic propionate oxidation bacteria (SPOB) and SRB was established and characterized at 35°C. The dominant SPOB and SRB were enriched in a continuous flow stirred-tank reactor and in batch experiments, respectively. Subsequently, an equal proportion of the enriched sludge was mixed and incubated in conical flasks with propionate as the sole substrate. Six parallel experiments (SP1–SP6) were conducted and two of them (SP2 and SP4) were chosen. The results revealed that the sulfate removal efficiencies of SP2 and SP4 were 91.83% and 93.03%, respectively. Furthermore, the outcome of the microbial analysis showed that the dominant microbial communities included Uncultured Smithella spp., Uncultured Desulfobacteraceae bacterium and sequences related to members of the family Desulfovibrio. Excess propionate was mainly syntrophically decomposed via sulfate reduction. Findings from this study could provide valuable information for the establishment of new processes and the regulation of parameters in the treatment of lower COD/SO42- ratio wastewater.

Peat: home to novel syntrophic species that feed acetate- and hydrogen-scavenging methanogens.

Syntrophic bacteria drive the anaerobic degradation of certain fermentation products (e.g., butyrate, ethanol, propionate) to intermediary substrates (e.g., H2, formate, acetate) that yield methane at the ecosystem level. However, little is known about the in situ activities and identities of these syntrophs in peatlands, ecosystems that produce significant quantities of methane. The consumption of butyrate, ethanol or propionate by anoxic peat slurries at 5 and 15 °C yielded methane and CO2 as the sole accumulating products, indicating that the intermediates H2, formate and acetate were scavenged effectively by syntrophic methanogenic consortia. 16S rRNA stable isotope probing identified novel species/strains of Pelobacter and Syntrophomonas that syntrophically oxidized ethanol and butyrate, respectively. Propionate was syntrophically oxidized by novel species of Syntrophobacter and Smithella, genera that use different propionate-oxidizing pathways. Taxa not known for a syntrophic metabolism may have been involved in the oxidation of butyrate (Telmatospirillum-related) and propionate (unclassified Bacteroidetes and unclassified Fibrobacteres). Gibbs free energies (ΔGs) for syntrophic oxidations of ethanol and butyrate were more favorable than ΔGs for syntrophic oxidation of propionate. As a result of the thermodynamic constraints, acetate transiently accumulated in ethanol and butyrate treatments but not in propionate treatments. Aceticlastic methanogens (Methanosarcina, Methanosaeta) appeared to outnumber hydrogenotrophic methanogens (Methanocella, Methanoregula), reinforcing the likely importance of aceticlastic methanogenesis to the overall production of methane. ΔGs for acetogenesis from H2 to CO2 approximated to -20 kJ mol(-1) when acetate concentrations were low, indicating that acetogens may have contributed to the flow of carbon and reductant towards methane.