Syntrophic Butyrate Oxidation Research Articles

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.

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.

Phase separation enhances bioenergy recovery in sugarcane vinasse biodigestion: Absolute or relative truth?

Phase separation has been indicated as the best approach to maximize energy production from sugarcane vinasse via biodigestion. However, process specificities, such as non-removing sulfate during fermentation, may negatively impact methanogenesis. This study assessed methane evolution in batch systems fed with different vinasse types, i.e., raw (RVin), sulfate-rich fermented (FVin+SO4) and sulfate-poor fermented (FVin-SO4) vinasses, detailing the metabolic shifts required to supply methanogens with acetate in each case. The incomplete oxidation of glycerol and lactate by sulfate reducers provided acetate in a shortcut-like pathway, promptly boosting methane evolution from RVin and FVin+SO4. However, the stimulus to methanogenesis takes its toll by impairing the final methane yield and biogas quality (up to 5.6% of sulfide). Acetate provision exclusively by the syntrophic oxidation of butyrate and phenol-derived intermediates slowed down methanogenesis. Controlling vinasse fermentation towards enhanced sulfate removal and acetate production will be imperative for maximizing bioenergy recovery in methanogenic systems.

Ecogenomics reveals community interactions in a long-term methanogenic bioreactor and a rapid switch to sulfate-reducing conditions.

The anaerobic digestion of wastes is globally important in the production of methane (CH4) as a biofuel. When sulfate is present, sulfate-reducing bacteria (SRB) are stimulated, competing with methanogens for common substrates, which decreases CH4 production and results in the formation of corrosive, odorous hydrogen sulfide gas (H2S). Here, we show that a population of SRB within a methanogenic bioreactor fed only butyrate for years immediately (within hours) responded to sulfate availability and shifted the microbial community dynamics within the bioreactor. By mapping shotgun metatranscriptomes to metagenome-assembled genomes, we shed light on the transcriptomic responses of key community members in response to increased sulfate provision. We link these short-term transcriptional responses to long-term niche partitioning using comparative metagenomic analyses. Our results suggest that sulfate provision supports a syntrophic butyrate oxidation community that disfavors poly-β-hydroxyalkanoate storage and that hydrogenotrophic SRB populations effectively exclude obligately hydrogenotrophic, but not aceticlastic, methanogens when sulfate is readily available. These findings elucidate key ecological dynamics between SRB, methanogens and syntrophic butyrate-oxidizing bacteria, which can be applied to a variety of engineered and natural systems.

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.

Stimulation of carbon nanomaterials on syntrophic oxidation of butyrate in sediment enrichments and a defined coculture

It remains elusive if direct interspecies electron transfer (DIET) occurs in canonical syntrophy involving short-chain fatty acids oxidation. In the present study, we determined the effects of carbon nanomaterials on syntrophic oxidation of butyrate in two lake sediment enrichments and a defined coculture comprising Syntrophomonas wolfei and Methanococcus Maripaludis. After four continuous transfers of enrichment cultivation, Syntrophomonas dominated the bacterial populations in enrichments, and the dominated methanogens comprised Methanosarcina and Methanospirillum in one enrichment (from Weiming Lake) and Methanoregula and Methanospirillum in another (from Erhai Lake). Butyrate oxidation and CH4 production was significantly accelerated by carbon nanotubes (CNTs) in both enrichments. Replacement of CNTs by magnetite caused similar stimulating effect. For the defined coculture, two carbon nanomaterials, CNTs and reduced graphene oxide (rGO), were tested, both showed consistently stimulating effects on butyrate oxidation. Addition of kaolinite, an electric nonconductive clay mineral, however, revealed no effect. The test on M. maripaludis in pure culture showed no effect by rGO and a negative effect by CNTs (especially at a high concentration). Fluorescence in situ hybridization (FISH) and scanning electron microscopy (SEM) revealed that microbial cells were interwoven by CNTs forming cell-CNT mixture aggregates, and in case of rGO, cells were attached to surface or wrapped-up by rGO thin sheets. Collectively, our data suggest that the presence of conductive nanomaterials likely induces DIET in syntrophic butyrate oxidation.

Reduction of Gibbs free energy and enhancement of Methanosaeta by bicarbonate to promote anaerobic syntrophic butyrate oxidation

Bicarbonate (HCO3−) has been extensively researched as a buffer in anaerobic digestion. The effect of HCO3− concentration on syntrophic butyrate oxidation process was evaluated by batch culturing of anaerobic activated sludge, and the mechanism was further revealed by the changes of Gibbs free energy (ΔG) and the interspecies transfers of electron and proton. The results showed that butyrate degradation rate was enhanced by 32.07% when the supplement of HCO3− increased from 0 to 0.20 mol/L. However, methane production and acetate degradation were strongly inhibited by HCO3− more than 0.10 mol/L. More function of HCO3− was found as 1) decreasing the ΔG of syntrophic methanogenesis of butyrate while increasing the ΔG of methanogenesis of acetate, 2) enriching M. harundinacea and M. concilii, 3) increasing the diffusion rate of protons between the syntrophic consortia. This work would increase the anaerobic digestion efficiency by enhancing the interaction of the syntrophic consortia.

Stimulatory Effect of Magnetite Nanoparticles on a Highly Enriched Butyrate-Oxidizing Consortium.

Syntrophic oxidation of butyrate is catabolized by a few bacteria specialists in the presence of methanogens. In the present study, a highly enriched butyrate-oxidizing consortium was obtained from a wetland sediment in Tibetan Plateau. During continuous transfers of the enrichment, the addition of magnetite nanoparticles (nanoFe3O4) consistently enhanced butyrate oxidation and CH4 production. Molecular analysis revealed that all bacterial sequences from the consortium belonged to Syntrophomonas with the closest relative of Syntrophomonas wolfei and 96% of the archaeal sequences were related to Methanobacteria with the remaining sequences to Methanocella. Addition of graphite and carbon nanotubes for a replacement of nanoFe3O4 caused the similar stimulatory effect. Silica coating of nanoFe3O4 surface, however, completely eliminated the stimulatory effect. The control experiment with axenic cultivation of a Syntrophomonas strain and two methanogen strains showed no effect by nanoFe3O4. Together, the results in the present study support that syntrophic oxidation of butyrate is likely facilitated by direct interspecies electron transfer in the presence of conductive nanomaterials.

Biochar assisted thermophilic co-digestion of food waste and waste activated sludge under high feedstock to seed sludge ratio in batch experiment

Batch experiments were conducted using biochar (BC) to promote stable and efficient methane production from thermophilic co-digestion of food waste (FW) and waste activated sludge (WAS) at feedstock/seed sludge (F/S) ratios of 0.25, 0.75, 1.5, 2.25, and 3. The results showed that the presence of BC dramatically shortened the lag time of methane production and increased the methane production rate with increased organic loading. The higher buffer capacity and large specific surface area of BC promoted microorganism growth and adaption to VFAs accumulation. Additionally, the electron exchange in syntrophic oxidation of butyrate and acetate as intermediate products was significantly facilitated by BC possibly due to the selective succession of bacteria and methanogens which may have participated in direct interspecies electron transfer, in contrast with the control group with low-efficient electron ferried between syntrophic oxidizers and methanogens using hydrogen as the electron carrier.

Syntrophomonas wolfei Uses an NADH-Dependent, Ferredoxin-Independent [FeFe]-Hydrogenase To Reoxidize NADH.

ABSTRACTSyntrophomonas wolfei syntrophically oxidizes short-chain fatty acids (four to eight carbons in length) when grown in coculture with a hydrogen- and/or formate-using methanogen. The oxidation of 3-hydroxybutyryl-coenzyme A (CoA), formed during butyrate metabolism, results in the production of NADH. The enzyme systems involved in NADH reoxidation in S. wolfei are not well understood. The genome of S. wolfei contains a multimeric [FeFe]-hydrogenase that may be a mechanism for NADH reoxidation. The S. wolfei genes for the multimeric [FeFe]-hydrogenase (hyd1ABC; SWOL_RS05165, SWOL_RS05170, SWOL_RS05175) and [FeFe]-hydrogenase maturation proteins (SWOL_RS05180, SWOL_RS05190, SWOL_RS01625) were coexpressed in Escherichia coli, and the recombinant Hyd1ABC was purified and characterized. The purified recombinant Hyd1ABC was a heterotrimer with an αβγ configuration and a molecular mass of 115 kDa. Hyd1ABC contained 29.2 ± 1.49 mol of Fe and 0.7 mol of flavin mononucleotide (FMN) per mole enzyme. The purified, recombinant Hyd1ABC reduced NAD+ and oxidized NADH without the presence of ferredoxin. The HydB subunit of the S. wolfei multimeric [FeFe]-hydrogenase lacks two iron-sulfur centers that are present in known confurcating NADH- and ferredoxin-dependent [FeFe]-hydrogenases. Hyd1ABC is a NADH-dependent hydrogenase that produces hydrogen from NADH without the need of reduced ferredoxin, which differs from confurcating [FeFe]-hydrogenases. Hyd1ABC provides a mechanism by which S. wolfei can reoxidize NADH produced during syntrophic butyrate oxidation when low hydrogen partial pressures are maintained by a hydrogen-consuming microorganism.IMPORTANCE Our work provides mechanistic understanding of the obligate metabolic coupling that occurs between hydrogen-producing fatty and aromatic acid-degrading microorganisms and their hydrogen-consuming partners in the process called syntrophy (feeding together). The multimeric [FeFe]-hydrogenase used NADH without the involvement of reduced ferredoxin. The multimeric [FeFe]-hydrogenase would produce hydrogen from NADH only when hydrogen concentrations were low. Hydrogen production from NADH by Syntrophomonas wolfei would likely cease before any detectable amount of cell growth occurred. Thus, continual hydrogen production requires the presence of a hydrogen-consuming partner to keep hydrogen concentrations low and explains, in part, the obligate requirement that S. wolfei has for a hydrogen-consuming partner organism during growth on butyrate. We have successfully expressed genes encoding a multimeric [FeFe]-hydrogenase in E. coli, demonstrating that such an approach can be advantageous to characterize complex redox proteins from difficult-to-culture microorganisms.

Conductive Fe3O4 Nanoparticles Accelerate Syntrophic Methane Production from Butyrate Oxidation in Two Different Lake Sediments

Syntrophic methanogenesis is an essential link in the global carbon cycle and a key bioprocess for the disposal of organic waste and production of biogas. Recent studies suggest direct interspecies electron transfer (DIET) is involved in electron exchange in methanogenesis occurring in paddy soils, anaerobic digesters, and specific co-cultures with Geobacter. In this study, we evaluate the possible involvement of DIET in the syntrophic oxidation of butyrate in the enrichments from two lake sediments (an urban lake and a natural lake). The results showed that the production of CH4 was significantly accelerated in the presence of conductive nanoscale Fe3O4 or carbon nanotubes in the sediment enrichments. Observations made with fluorescence in situ hybridization and scanning electron microscope indicated that microbial aggregates were formed in the enrichments. It appeared that the average cell-to-cell distance in aggregates in nanomaterial-amended enrichments was larger than that in aggregates in the non-amended control. These results suggested that DIET-mediated syntrophic methanogenesis could occur in the lake sediments in the presence of conductive materials. Microbial community analysis of the enrichments revealed that the genera of Syntrophomonas, Sulfurospirillum, Methanosarcina, and Methanoregula were responsible for syntrophic oxidation of butyrate in lake sediment samples. The mechanism for the conductive-material-facilitated DIET in butyrate syntrophy deserves further investigation.

Direct interspecies electron transfer accelerates syntrophic oxidation of butyrate in paddy soil enrichments.

Syntrophic interaction occurs during anaerobic fermentation of organic substances forming methane as the final product. H2 and formate are known to serve as the electron carriers in this process. Recently, it has been shown that direct interspecies electron transfer (DIET) occurs for syntrophic CH4 production from ethanol and acetate. Here, we constructed paddy soil enrichments to determine the involvement of DIET in syntrophic butyrate oxidation and CH4 production. The results showed that CH4 production was significantly accelerated in the presence of nanoFe3 O4 in all continuous transfers. This acceleration increased with the increase of nanoFe3 O4 concentration but was dismissed when Fe3 O4 was coated with silica that insulated the mineral from electrical conduction. NanoFe3 O4 particles were found closely attached to the cell surfaces of different morphology, thus bridging cell connections. Molecular approaches, including DNA-based stable isotope probing, revealed that the bacterial Syntrophomonadaceae and Geobacteraceae, and the archaeal Methanosarcinaceae, Methanocellales and Methanobacteriales, were involved in the syntrophic butyrate oxidation and CH4 production. Among them, the growth of Geobacteraceae strictly relied on the presence of nanoFe3 O4 and its electrical conductivity in particular. Other organisms, except Methanobacteriales, were present in enrichments regardless of nanoFe3 O4 amendment. Collectively, our study demonstrated that the nanoFe3 O4 -facilitated DIET occurred in syntrophic CH4 production from butyrate, and Geobacter species played the key role in this process in the paddy soil enrichments.

A proteomic view at the biochemistry of syntrophic butyrate oxidation in Syntrophomonas wolfei.

In syntrophic conversion of butyrate to methane and CO2, butyrate is oxidized to acetate by secondary fermenting bacteria such as Syntrophomonas wolfei in close cooperation with methanogenic partner organisms, e.g., Methanospirillum hungatei. This process involves an energetically unfavourable shift of electrons from the level of butyryl-CoA oxidation to the substantially lower redox potential of proton and/or CO2 reduction, in order to transfer these electrons to the methanogenic partner via hydrogen and/or formate.In the present study, all prominent membrane-bound and soluble proteins expressed in S. wolfei specifically during syntrophic growth with butyrate, in comparison to pure-culture growth with crotonate, were examined by one- and two-dimensional gel electrophoresis, and identified by peptide fingerprinting-mass spectrometry. A membrane-bound, externally oriented, quinone-linked formate dehydrogenase complex was expressed at high level specifically during syntrophic butyrate oxidation, comprising a selenocystein-linked catalytic subunit with a membrane-translocation pathway signal (TAT), a membrane-bound iron-sulfur subunit, and a membrane-bound cytochrome. Soluble hydrogenases were expressed at high levels specifically during growth with crotonate. The results were confirmed by native protein gel electrophoresis, by formate dehydrogenase and hydrogenase-activity staining, and by analysis of formate dehydrogenase and hydrogenase activities in intact cells and cell extracts. Furthermore, constitutive expression of a membrane-bound, internally oriented iron-sulfur oxidoreductase (DUF224) was confirmed, together with expression of soluble electron-transfer flavoproteins (EtfAB) and two previously identified butyryl-CoA dehydrogenases.Our findings allow to depict an electron flow scheme for syntrophic butyrate oxidation in S. wolfei. Electrons derived from butyryl-CoA are transferred through a membrane-bound EtfAB:quinone oxidoreductase (DUF224) to a menaquinone cycle and further via a b-type cytochrome to an externally oriented formate dehydrogenase. Hence, an ATP hydrolysis-driven proton-motive force across the cytoplasmatic membrane would provide the energy input for the electron potential shift necessary for formate formation.

Syntrophomonadaceae-affiliated species as active butyrate-utilizing syntrophs in paddy field soil.

DNA-based stable-isotope probing was applied to identify the active microorganisms involved in syntrophic butyrate oxidation in paddy field soil. After 14 and 21 days of incubation with [U-(13)C]butyrate, the bacterial Syntrophomonadaceae and the archaeal Methanosarcinaceae and Methanocellales incorporated substantial amounts of (13)C label into their nucleic acids. Unexpectedly, members of the Planctomycetes and Chloroflexi were also labeled with (13)C by yet-unclear mechanisms.

Involvement of NADH:acceptor oxidoreductase and butyryl coenzyme A dehydrogenase in reversed electron transport during syntrophic butyrate oxidation by Syntrophomonas wolfei.

Methanogenic oxidation of butyrate to acetate requires a tight cooperation between the syntrophically fermenting Syntrophomonas wolfei and the methanogen Methanospirillum hungatei, and a reversed electron transport system in S. wolfei was postulated to shift electrons from butyryl coenzyme A (butyryl-CoA) oxidation to the redox potential of NADH for H(2) generation. The metabolic activity of butyrate-oxidizing S. wolfei cells was measured via production of formazan and acetate from butyrate, with 2,3,5-triphenyltetrazolium chloride as electron acceptor. This activity was inhibited by trifluoperazine (TPZ), an antitubercular agent known to inhibit NADH:menaquinone oxidoreductase. In cell extracts of S. wolfei, the oxidation of NADH could be measured with quinones, viologens, and tetrazolium dyes as electron acceptors, and also this activity was inhibited by TPZ. The TPZ-sensitive NADH:acceptor oxidoreductase activity appeared to be membrane associated but could be dissociated from the membrane as a soluble protein and was semipurified by anion-exchange chromatography. Recovered proteins were identified by peptide mass fingerprinting, which indicated the presence of an NADH:acceptor oxidoreductase as part of a three-component [FeFe] hydrogenase complex and a selenocysteine-containing formate dehydrogenase. Furthermore, purification of butyryl-CoA dehydrogenase (Bcd) activity and peptide mass fingerprinting revealed two Bcd proteins different from the Bcd subunit of the Bcd/electron-transfer flavoprotein complex (Bcd/EtfAB) predicted from the genome sequence of S. wolfei. The results suggest that syntrophic oxidation of butyrate in S. wolfei involves a membrane-associated TPZ-sensitive NADH:acceptor oxidoreductase as part of a hydrogenase complex similar to the recently discovered "bifurcating" hydrogenase in Thermotoga maritima and butyryl-CoA dehydrogenases that are different from Bcd of the Bcd/EtfAB complex.

Localization of the enzymes involved in H2 and formate metabolism in Syntrophospora bryantii.

Cell-free extracts of crotonate-grown cells of the syntrophic butyrate-oxidizing bacterium Syntrophospora bryantii contained high hydrogenase activities (8.5-75.8 mumol.min-1mg-1 protein) and relatively low formate dehydrogenase activities (0.04-0.07 mumol.min-1 mg-1 protein). The KM value and threshold value of the hydrogenase for H2 were 0.21 mM and 18 microM, respectively, whereas the KM value and threshold value of the formate dehydrogenase for formate were 0.22 mM and 10 microM, respectively. Hydrogenase, butyryl-CoA dehydrogenase and 3-OH-butyryl-CoA dehydrogenase were detected in the cytoplasmic fraction. Formate dehydrogenase and CO2 reductase were membrane-bound, likely located at the outer aspect of the cytoplasmic membrane. Results suggest that during syntrophic butyrate oxidation H2 is formed intracellularly while formate is formed at the outside of the cell.

Butyrate oxidation by Syntrophospora bryantii in co-culture with different methanogens and in pure culture with pentenoate as electron acceptor

Syntrophospora bryantii degraded butyrate in co-culture with methanogens that can use both H2 and formate for growth, but not in co-culture with methanogens that metabolize only H2, suggesting that in suspended cultures formate may be a more important electron carrier in the syntrophic degradation of butyrate than H2. Syntrophic butyrate oxidation was inhibited by the addition of 20 mm formate or the presence of 130 kPa H2. In the absence of methanogens, S. bryantii is able to couple the oxidation of butyrate to acetate with the reduction of pentenoate to valerate. Under these conditions, up to 300 Pa H2 was measured in the gas phase and up to 0.3 mm formate in the liquid phase. S. bryantii was unable to grow syntrophically with the aceticlastic methanogen Methanothrix soehngenii. However in triculture with Methanospirillum hungatei and Methanothrix soehngenii, S. bryantii degraded butyrate faster than in a biculture with only M. hungatei. Hydrogenase and formate dehydrogenase activities were demonstrated in cell-free extracts of S. bryantii.