Syntrophic Propionate-oxidizing Bacteria Research Articles

Metagenomic insights into the enhanced methane production by hydrochar at varied propionate concentrations

Propionate is a crucial intermediate product in anaerobic digestion (AD), but its degradation is thermodynamically unfavorable. Its accumulation in the AD system can significantly reduce methane production efficiency and may lead to severe acidification. The present study investigated the methane production at various propionate concentrations (15, 30 and 60 mM) with and without hydrochar and also explored the microbial shifts by metagenomic analysis. The findings revealed that higher propionate concentrations reduced methane production and propionate degradation efficiency, leading to acetate accumulation. The addition of hydrochar mitigated the inhibitory effects of increased propionate concentrations, notably reduced the lag phase by 20.13 % and enhanced the methane production rate by 138.43 % under 30 mM propionate. This also resulted in faster degradation of propionate and acetate. Hydrochar impacted the microbial communities’ functional potentials, enhancing methane metabolic potential in all conditions. Genomic-centric analysis revealed that hydrochar selectively enriched syntrophic propionate-oxidizing bacteria (SPOB) candidate MAG79 Syntrophopropionicum sp., which could participate in DIET, across different concentrations of propionate, with the most pronounced effect at 30 mM propionate. This was distinct from the dominant species in the control groups, MAG63 Syntrophosphaera sp. and MAG69 Syntrophobacteraceae sp.. Hydrochar further supported the growth of another DIET candidate MAG4 Methanosarcina mazei and increased the abundance of proposed syntrophic acetate-oxidizing bacterium (SAOBs), across all propionate concentrations. These effects collectively led to more efficient propionate degradation and 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.

Membrane Potential-requiring Succinate Dehydrogenase Constitutes the Key to Propionate Oxidation and Is Unique to Syntrophic Propionate-oxidizing Bacteria

Propionate oxidation in Pelotomaculum thermopropionicum is performed under a thermodynamic limit. The most energetically unfavorable reaction in the propionate oxidation pathway is succinate oxidation. Based on previous genomic and transcriptomic ana-lyses, succinate oxidation in P. thermopropionicum under propionate-oxidizing conditions is conducted by the membrane-bound forms of two succinate dehydrogenases (SDHs). We herein examined the activity of SDH, the mechanisms underlying the succinate oxidation reaction in P. thermopropionicum, and the importance of the protein sequences of related genes. SDH activity was highly localized to the membrane fraction. An ana-lysis of the soluble fraction revealed that fumarate reductase received electrons from NADH, suggesting the involvement of membrane-bound SDH in propionate oxidation. We utilized an uncoupler and inhibitors of adenosine triphosphate (ATP) synthase and membrane-bound SDH to investigate whether the membrane potential of P. thermopropionicum supports propionate oxidation alongside hydrogen production. These chemicals inhibited hydrogen production, indicating that membrane-bound SDH requires a membrane potential for succinate oxidation, and this membrane potential is maintained by ATP synthase. In addition, the phylogenetic distribution of the flavin adenine dinucleotide-binding subunit and conserved amino acid sequences of the cytochrome b subunit of SDHs in propionate-oxidizing bacteria suggests that membrane-bound SDHs possess specific conserved amino acid residues that are strongly associated with efficient succinate oxidation in syntrophic propionate-oxidizing bacteria.

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.

Bioaugmentation with a propionate-degrading methanogenic culture to improve methane production from chicken manure

Volatile fatty acid (VFA) accumulation caused by high ammonia concentrations is often encountered during the anaerobic digestion (AD) of ammonia-rich substrates. In this study, propionate-degrading methanogenic cultures were introduced to augment the semi-continuous AD of chicken manure under high ammonia levels. Introduction of a methanogenic culture enhanced the methane yield in the bioaugmented digester by 17–26% when the organic loading rate (OLR) was 2–4 g L-1d-1 compared to that in the control. When the OLR was further increased from 4.0 L-1d-1 to 5.0 g L-1d-1, and bioaugmentation ceased, methane yield improved by 15–18% under a high total ammonia nitrogen level of 5.0–8.4 g NH4+-N/L. Moreover, bioaugmentation reconstructed the methanogenic community in the digester, promoting the dominance of hydrogenotrophic Methanobacterium and slightly increasing the abundance of aceticlastic Methanothrix and the syntrophic propionate-oxidizing bacteria Syntrophobacter, which were the key contributors to the improved AD under high ammonia concentrations.

Syntrophic propionate-oxidizing bacteria in methanogenic systems.

ABSTRACTThe mutual nutritional cooperation underpinning syntrophic propionate degradation provides a scant amount of energy for the microorganisms involved, so propionate degradation often acts as a bottleneck in methanogenic systems. Understanding the ecology, physiology and metabolic capacities of syntrophic propionate-oxidizing bacteria (SPOB) is of interest in both engineered and natural ecosystems, as it offers prospects to guide further development of technologies for biogas production and biomass-derived chemicals, and is important in forecasting contributions by biogenic methane emissions to climate change. SPOB are distributed across different phyla. They can exhibit broad metabolic capabilities in addition to syntrophy (e.g. fermentative, sulfidogenic and acetogenic metabolism) and demonstrate variations in interplay with cooperating partners, indicating nuances in their syntrophic lifestyle. In this review, we discuss distinctions in gene repertoire and organization for the methylmalonyl-CoA pathway, hydrogenases and formate dehydrogenases, and emerging facets of (formate/hydrogen/direct) electron transfer mechanisms. We also use information from cultivations, thermodynamic calculations and omic analyses as the basis for identifying environmental conditions governing propionate oxidation in various ecosystems. Overall, this review improves basic and applied understanding of SPOB and highlights knowledge gaps, hopefully encouraging future research and engineering on propionate metabolism in biotechnological processes.

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.

The effect of propionic acid accumulation on methane production in dry mesophilic anaerobic fermentation

The dry anaerobic digestion performances were investigated at the temperature of 35±1°Cwith addition of propionic acid at different concentrations in five lab-scale reactors (R1-R5) to investigate the effect of propionic acid accumulation on methane production in dry mesophilic anaerobic digestion. And the volatile fatty acids, biogas and microbial community were detected. The active of microbial community and the methane generation would be inhibited when the propionic acid exceeded 30 gL1, which affected not only syntrophic propionate-oxidizing bacteria but also acetate-utilizing methanogens. Eubacteria decreased sharply accompany with the higher concentration of propionic in R4 and R5. The relative abundance of acetoclastic methanogens was directly correlated with volatile solids removals and methane generation in dry anaerobic reactors. Otherwise, the quantity of syntrophic propionate-oxidizing bacteria was positively linear correlated with propionic acid when the concentration was below 30 gL1. The methanogenic bacteria activity in R3 was the best with the methane yield of 27.01 mLCH4 g-1 VS d-1. The results suggested that appropriate propionate could enhance methane production in the dry anaerobic fermentation.

Application of Polyacrylamide Flocculant for Stabilization of Anaerobic Digestion under Conditions of Excessive Accumulation of Volatile Fatty Acids

Excessive accumulation of volatile fatty acids (VFA) is one of the major factors destabilizing methanogenic digestion of organic wastes in anaerobic bioreactors. Existing methods of stabilization of this process are mostly expensive and labor-intensive, often requiring removal of a considerable portion of acidified biomass from the bioreactor. We propose a method for methanogenesis restoration in such soured reactors by the addition of a cationic polyacrylamide flocculant (PAM) at 20 mg/g total solids. After flocculant addition, mixing should be minimized to prolong the existence of the floccules formed in the presence of the flocculant. While partial microbial degradation of the polyacrylamide flocculant was observed during the thermophilic anaerobic process, complete PAM mineralization did not occur. Significant inhibition of anaerobic processes, primarily in the activity of syntrophic propionate-oxidizing bacteria, was observed at PAM concentrations above 40 mg/g total solids.

Response of syntrophic bacterial and methanogenic archaeal communities in paddy soil to soil type and phenological period of rice growth

Methanogenesis and syntrophy are the most important microbial metabolic processes for the anaerobic decomposition of organic matters in paddy soils. However, the diversity and abundance of syntrophic communities and their ecological determinants remain largely unexplored. In this study, we aimed to unravel the impacts of soil type and rice phenology on both syntrophic and methanogenic communities. The relative abundances of syntrophic bacteria and methanogenic archaea were significantly affected by soil type and rice growth stages, with 0.39–1.66% and 1.68–3.95% for syntrophic bacteria and 0.49–6.04% and 9.29–13.69% for methanogenic archaea, respectively. Besides, the populations of short-chain fatty acid-degrading syntrophs (SFAS) varied across soil types (range: 8.21 × 106–7.83 × 107 16S rRNA gene copies g−1 dry soil) and rice growth stages (range: 2.27 × 107–3.88 × 107 16S rRNA gene copies g−1 dry soil). Moreover, the populations of syntrophic propionate-oxidizing bacteria were 1.2–6.2 times higher than those of butyrate-oxidizing bacteria in the 10 paddy soils during the off-rice season, indicating that propionate is the main intermediate product during anaerobic decomposition of organic matter. However, Syntrophomonas was the most dominant throughout the rice growing season, implying that syntrophic pathways may vary between off-rice and growing seasons. Interestingly, the populations of SFAS and methanogens showed a significantly positive correlation (P < 0.05). This suggests that syntrophic bacteria, in cooperation with methanogenic archaea, can affect methane production in paddy soils. Furthermore, C/N ratio, soil moisture, soil pH, oxalate, citrate, and their interaction accounted for 65.20% of the changes in syntrophic communities. Altogether, our findings indicate that soil type, rice phenological stages and soil properties can shape the distribution of syntrophic communities in rice paddy fields.

Impacts of conductive materials on microbial community during syntrophic propionate oxidization for biomethane recovery.

Propionate is one of the most important intermediates in anaerobic digestion, and its degradation requires a syntrophic partnership between propionate-oxidizing bacteria and hydrogenotrophic methanogens. Anaerobic digestion efficiency can be improved by direct interspecies electron transfer (DIET) through conductive materials. This study aimed to investigate the effects of DIET on syntrophic propionate oxidization under room temperature (20°C) and reveal the syntrophic partners. Firstly, conventional anaerobic consortium and conductive material-enriched consortium were tested for DIET under high H2 partial pressure. The latter supplemented with granular activated carbon (GAC) can mitigate H2 inhibition through DIET. Secondly, a DIET consortium was enriched for testing GAC and magnetite, both showed DIET facilitation. Microbial communities in GAC- and magnetite-supplemented reactors were similar. Syntrophic propionate-oxidizing bacteria, for example, Smithella (3.9%-9.9%) and a genus from the family Syntrophaceae (1.9%-3.6%) and methanogens Methanobacterium (30.3%-75.2%), Methanolinea (8.5%-25.2%), Methanosaeta (11.4%-36.7%), and Candidatus Methanofastidiosum (3.6%-6.6%), were predominant. Functional genes for cell mobility and membrane transport (3.3% and 9.5% in control reactor) increased with GAC (3.7% and 11.1%, respectively) and magnetite (3.7% and 10.9%, respectively) addition. Syntrophic propionate-oxidizing bacteria and methanogenesis partners were revealed by co-occurrence network, for example, Methanobacterium with Smithella, Syntrophobacter, Dechloromonas, and Trichococcus, signifying the importance of the syntrophic partnership in DIET environment. PRACTITIONER POINTS: DIET improved syntrophic propionate oxidization under room temperature condition (20°C). Microbial communities were similar for GAC- and magnetite-supplemented reactors, different with control reactor. Syntrophic propionate-oxidizing bacteria and methanogenesis partners were revealed by co-occurrence network. Methanobacterium and Smithella, Syntrophobacter, Dechloromonas, and Trichococcus were correlated.

Methanogenic Activity and Microbial Community Structure in Response to Different Mineralization Pathways of Ferrihydrite in Paddy Soil

In soils and sediments, microbial reduction of iron (hydr)oxides and consequent formation of secondary iron minerals are important factors influencing many biogeochemical cycles and processes that including microbial methanogenesis. Here, we investigated methanogenic activity and microbial community of a paddy soil enrichment in response to different biomineralization pathways of ferrihydrite, which was reduced and transformed to magnetite and vivianite in the absence and presence of phosphate, respectively. For methanogenic degradation of both acetate and propionate, CH4 production rates in the magnetite-cultures were significantly enhanced compared with the vivianite-cultures. Characterization of 16S rRNA genes from methanogenic soils microbial community indicated that: (i) Methanocellaceae and Methanosarcinaceae were the dominant methanogens in all soil enrichments, (ii) syntrophic acetate-oxidizing bacteria of Clostridiaceae and syntrophic propionate-oxidizing bacteria of Syntrophobacteraceae were highly enriched in the magnetite-cultures, and (iii) the abundance of Anaerolineaceae was increased in the magnetite-cultures for both acetate- and propionate-fed incubations. The facilitated CH4 production in the magnetite-cultures might be related to the dynamic cycle of Fe(III)−Fe(II)−Fe(III) or magnetite-stimulated direct interspecies electron transfer between syntrophic acetate/propionate oxidizers and methanogens, which need further investigation. These results lend insight into the biogeochemical cycling of Fe, C and P in anaerobic soils and sediments, as well as the development of mitigation strategies of CH4 production.

Using DNA-based stable isotope probing to reveal novel propionate- and acetate-oxidizing bacteria in propionate-fed mesophilic anaerobic chemostats

Propionate is one of the most important intermediates of anaerobic fermentation. Its oxidation performed by syntrophic propionate-oxidizing bacteria coupled with hydrogenotrophic methanogens is considered to be a rate-limiting step for methane production. However, the current understanding of SPOB is limited due to the difficulty of pure culture isolation. In the present study, two anaerobic chemostats fed with propionate as the sole carbon source were operated at different dilution rates (0.05 d−1 and 0.15 d−1). The propionate- and acetate-oxidizing bacteria in the two methanogenic chemostats were investigated combining DNA-stable isotope probing (DNA-SIP) and 16S rRNA gene high-throughput sequencing. The results of DNA-SIP with 13C-propionate/acetate suggested that, Smithella, Syntrophobacter, Cryptanaerobacter, and unclassified Rhodospirillaceae may be putative propionate-oxidizing bacteria; unclassified Spirochaetaceae, unclassified Synergistaceae, unclassified Elusimicrobia, Mesotoga, and Gracilibacter may contribute to acetate oxidation; unclassified Syntrophaceae and Syntrophomonas may be butyrate oxidizers. By DNA-SIP, unclassified OTUs with 16S rRNA gene abundance higher than 62% of total Bacteria in the PL chemostat and 38% in the PH chemostat were revealed to be related to the degradation of propionate. These results suggest that a variety of uncultured bacteria contribute to propionate degradation during anaerobic digestion. The functions and metabolic characteristics of these bacteria require further investigation.

Different inhibitory mechanisms of chlortetracycline and enrofloxacin on mesophilic anaerobic degradation of propionate.

In anaerobic digestion, propionate is a key intermediate whose degradation is thermodynamically challenging and accumulation is detrimental to the process. Many wastewater streams contain antibiotics due to its globally increasing use, and these compounds can inhibit methane production. However, the effect of antibiotics on propionate degradation in anaerobic digestion remains unclear. In this study, the influence of two antibiotics (chlortetracycline [CTC] and enrofloxacin [EFX]) on biogas production and mesophilic propionate-degrading microbial community was investigated. CTC strongly repressed propionate oxidation, acetate utilization, and methane production, while EFX only inhibited propionate oxidation and methane production to a lesser extent. Microbial community analyses showed that syntrophic propionate-oxidizing bacteria (SPOB) Syntrophobacter had strong tolerance to both CTC and EFX. CTC inhibition mainly acted on the activity of acetate-oxidizing bacteria (Mesotoga, Geovibrio, Tepidanaerobacter, unclassified Bacteroidetes, and unclassified Clostridia) and acetoclastic methanogen, while EFX inhibition applied to the SPOB Smithella and acetoclastic methanogen. Network analysis further indicated that more complicated correlation among bacterial genera occurred in CTC treatments. These results suggested that CTC and EFX inhibited propionate degradation via different mechanisms, which was the result of joint action by antibiotics and microbial interactions.

Identification of novel potential acetate-oxidizing bacteria in thermophilic methanogenic chemostats by DNA stable isotope probing.

Syntrophic oxidization of acetate and propionate are both critical steps of methanogenesis during thermophilic anaerobic digestion. However, knowledge on syntrophic acetate-oxidizing bacteria (SAOB) and syntrophic propionate-oxidizing bacteria (SPOB) is limited because of the difficulty in pure culture isolation due to symbiotic relationship. In this study, two thermophilic acetate-fed anaerobic chemostats, ATL (dilution rate of 0.025 day-1) and ATH (0.05 day-1) and one thermophilic propionate-fed anaerobic chemostat PTL (0.025 day-1) were constructed, AOB and POB in these chemostats were studied via microbial community analysis and DNA stable-isotope probing (SIP). The results showed that, in addition to Tepidanaerobacter, a known SAOB, species of Thauera, Thermodesulfovibrio, Anaerobaculum, Ruminiclostridium, Comamonas, and uncultured bacteria belonging to Lentimicrobiaceae, o_MBA03, Thermoanaerobacteraceae, Anaerolineaceae, Clostridiales, and Ruminococcaceae were determined to be potential AOB in chemostats. Pelotomaculum was the key SPOB detected in the propionate-fed chemostat. Based onthe intense fluorescence of coenzyme F420, majority of Methanosarcina cells in acetate-fed chemostats were involved in hydrogenotrophic methanogenesis, suggesting the existence of highly active SAOB among the detected AOB. In the propionate-fed chemostat, most of the species detected as AOB were similar to those detected in the acetate-fed chemostats, suggesting the contribution of the syntrophic acetate oxidization pathway for methane generation. These results revealed the existence of previously unknown AOB with high diversity in thermophilic chemostats and suggested that methanogenesis from acetate via the syntrophic oxidization pathway is relevant for thermophilic anaerobic digestion.

Regulation of endogenous acyl homoserine lactones by microbial density to enhance syntrophism of acetogens and methanogens in anaerobic digestion

To understand the syntrophic mechanism of acetogens and methanogens in anaerobic digestion (AD), functional floras of syntrophic propionate-oxidizing bacteria (SPOB), syntrophic butyrate-oxidizing bacteria (SBOB), aceticlastic methanogens (AM) and hydrogenotrophic methanogens (HM) were separately tested by microbial density to reveal their performance in metabolic activity, extracellular polymeric substances (EPS) production and quorum sensing (QS) signal molecules (acyl homoserine lactones, AHLs) secretion, as well as the regulation of endogenous AHLs in balancing the social behaviors of syntrophic microorganism. The results showed that methanogenesis, EPS production and AHLs secretion of the functional floras were all observably affected by microbial density. The metabolic activity of SBOB, SPOB, AM and HM decreased with a microbial density more than 344, 295, 229 and 237 mgMLVSS/L, respectively. The intraspecific regulation of SPOB and HM were achieved with the mediation of OXOC6-HSL and C10-HSL, respectively. With the mediation of enjoyed signal molecules, metabolic coordination between SPOB and AM was of bidirectional regulation, but it was a unidirectional regulation from HM to SPOB. Syntrophic acetogens and methanogens in AD could bidirectionally regulate their EPS production via QS system to construct syntrophic floras that make the methane fermentation of propionate and butyrate more efficient. The present research work provides a practical strategy to regulate endogenous AHLs by microbial density to enhance the syntrophism of acetogens and methanogens in AD.

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.

Metatranscriptomic evidence for classical and RuBisCO-mediated CO2 reduction to methane facilitated by direct interspecies electron transfer in a methanogenic system

In a staged anaerobic fluidized-bed ceramic membrane bioreactor, metagenomic and metatranscriptomic analyses were performed to decipher the microbial interactions on the granular activated carbon. Metagenome bins, representing the predominating microbes in the bioreactor: syntrophic propionate-oxidizing bacteria (SPOB), acetoclastic Methanothrix concilii, and exoelectrogenic Geobacter lovleyi, were successfully recovered for the reconstruction and analysis of metabolic pathways involved in the transformation of fatty acids to methane. In particular, SPOB degraded propionate into acetate, which was further converted into methane and CO2 by M. concilii via the acetoclastic methanogenesis. Concurrently, G. lovleyi oxidized acetate into CO2, releasing electrons into the extracellular environment. By accepting these electrons through direct interspecies electron transfer (DIET), M. concilii was capable of performing CO2 reduction for further methane formation. Most notably, an alternative RuBisCO-mediated CO2 reduction (the reductive hexulose-phosphate (RHP) pathway) is transcriptionally-active in M. concilii. This RHP pathway enables M. concilii dominance and energy gain by carbon fixation and methanogenesis, respectively via a methyl-H4MPT intermediate, constituting the third methanogenesis route. The complete acetate reduction (2 mole methane formation/1 mole acetate consumption), coupling of acetoclastic methanogenesis and two CO2 reduction pathways, are thermodynamically favorable even under very low substrate condition (down to to 10−5 M level). Such tight interactions via both mediated and direct interspecies electron transfer (MIET and DIET), induced by the conductive GAC promote the overall efficiency of bioenergy processes.

Specific quorum sensing signal molecules inducing the social behaviors of microbial populations in anaerobic digestion

A series of dominant functional floras involved in anaerobic digestion was obtained by subculturing anaerobic sludge with specific substrates, respectively, and their specific quorum sensing signals (AHLs) were identified. It was found that most of the AHLs were secreted in starvation of the functional floras. One or two AHLs were found to have a significant positive correlation with the behavior of hydrolytic-fermentative bacteria, homoacetogens, syntrophic propionate-oxidizing bacteria, syntrophic butyrate-oxidizing bacteria and aceticlastic methanogens, respectively. Hydrogenotrophic methanogens had a various self-organization means and broader relations with the other populations, which was induced by six AHLs at least. Performance of an upflow anaerobic sludge blanket in the startup process revealed that the dominant populations involved in anaerobic digestion would secret specific AHLs to induce K-strategy in resource-limited situation for reproduction and regulating their cooperation. This work presents a novel perspective on the social behaviors of microbial populations in anaerobic digestion.

Re-activation and succession of functional microbial communities during long-term storing sludge granulation

The reactivation and granulation processes of damaged anaerobic microbial communities were studied. The changes of population structure of several typical functional microbes like E. limosum (E-HOMA), Methanomicrobiales (M-methanogens), Syntrophics fatty acid-β-oxidizing syntrophic bacteria and Syntrophic propionate-oxidizing bacteria (FP-HPA) were investigated. The results showed that the maximum abundance of bacteria and archaea was observed only through 33 days continuous incubation, much less than the time needed for sludge granular size increase (>180 days) and for achieving good COD removal (about 50 days). In comparison with FP-HPA and E-HOMA, the M-methanogens had stronger resistance for poor living conditions and enriched more quickly with environment improvement. During sludge granulation, the growth rate of microbial species was ranked in the order of M-methanogens > FP-HPA > E-HOMA. Compared with the initial microbial abundance of seed sludge, the initial microbial diversity may play more important role during sludge granulation. The reactivation of damaged microbial communities during sludge granulation was composed by three distinct periods, namely adaptation period, growth period and stable period.