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Propionate is one of the major intermediates in anaerobic digestion of organic waste to CO2 and CH4. In methanogenic environments, propionate is degraded through a mutualistic interaction between symbiotic propionate oxidizers and methanogens. Although temperature heavily influences the microbial ecology and performance of methanogenic processes, its effect on syntrophic interaction during propionate degradation remains poorly understood. In this study, metagenomics and metatranscriptomics were employed to compare mesophilic and thermophilic propionate degradation communities. Mesophilic propionate degradation involved multiple syntrophic organisms (Syntrophobacter, Smithella, and Syntrophomonas), pathways, interactions, and preference toward formate-based electron transfer to methanogenic partners (i.e., Methanoculleus). In thermophilic propionate degradation, one syntrophic organism predominated (Pelotomaculum), interspecies H2 transfer played a major role, and phylogenetically and metabolically diverse H2-oxidizing methanogens were present (i.e., Methanoculleus, Methanothermobacter, and Methanomassiliicoccus). This study showed that microbial interactions, metabolic pathways, and niche diversity are distinct between mesophilic and thermophilic microbial communities responsible for syntrophic propionate degradation.

Soil biogeochemical processes are not only gauged by the dominant taxa in the microbiome but also depend on the critical functions of its 'rare biosphere' members. Here, we evaluated the biogeographical pattern of 'rare biosphere' propionate-oxidizing syntrophs in 113 paddy soil samples collected across China. The relative abundance, activity and growth potential of propionate-oxidizing syntrophs were analysed to provide a panoramic view of syntroph biogeographical distribution at the continental scale. The relative abundances of four syntroph genera, Syntrophobacter, Pelotomaculum, Smithella and Syntrophomonas were significantly greater at the warm low latitudes than at the cool high latitudes. Correspondingly, propionate degradation was faster in the low latitude soils compared with the high latitude soils. The low rate of propionate degradation in the high latitude soils resulted in a greater increase of the total syntroph relative abundance, probably due to their initial low relative abundances and the longer incubation time for propionate consumption. The mean annual temperature (MAT) is the most important factor shaping the biogeographical pattern of propionate-oxidizing syntrophs, with the next factor being the soil's total sulfur content (TS). We suggest that the effect of MAT is related to the thermodynamic conditions, in which the endergonic constraint of propionate oxidation is leveraged with the increase of MAT. The TS effect is likely due to the ability of some propionate syntrophs to facultatively perform sulfate respiration.

Propionate is a crucial intermediate during methane fermentation. Investigating the effects of different kinds of inhibitors on the propionate-degrading microbial community is necessary to develop countermeasures for improving process stability. In the present study, under inhibitory conditions (acetate, propionate, sulfide, and ammonium addition), the dynamic changes of the propionate-degrading microbial community from a mesophilic chemostat fed with propionate as the sole carbon source were investigated using high-throughput sequencing of 16S rRNA. Sulfide and/or ammonia inhibited specific species in the microbial community. Compared with Syntrophobacter, Smithella was more resistant to inhibition by sulfide and/or ammonia. However, Syntrophobacter demonstrated greater tolerance than Smithella under acid inhibition conditions. Some genera that had close phylogenetic relationships and similar functions showed similar responses to different inhibitors.

Methanogenesis is central to anaerobic digestion processes. The conversion of propionate as a key intermediate for methanogenesis requires syntrophic interactions between bacterial and archaeal partners. In this study, a series of methanogenic enrichments with propionate as the sole substrate were developed to identify microbial populations specifically involved in syntrophic propionate conversion. These rigorously controlled propionate enrichments exhibited functional stability with consistent propionate conversion and methane production; yet, the methanogenic microbial communities experienced substantial temporal dynamics, which has important implications on the understanding of mechanisms involved in microbial community assembly in anaerobic digestion. Syntrophobacter was identified as the most abundant and consistent bacterial partner in syntrophic propionate conversion regardless of the origin of the source culture, the concentration of propionate, or the temporal dynamics of the culture. In contrast, the methanogen partners involved in syntrophic propionate conversion lacked consistency, as the dominant methanogens varied as a function of process condition and temporal dynamics. Methanoculleus populations were specifically enriched as the syntrophic partner at inhibitory levels of propionate, likely due to the ability to function under unfavorable environmental conditions. Syntrophic propionate conversion was carried out exclusively via transformation of propionate into acetate and hydrogen in enrichments established in this study. Microbial populations highly tolerant of elevated propionate, represented by Syntrophobacter and Methanoculleus, are of great significance in understanding methanogenic activities during process perturbations when propionate accumulation is frequently encountered. Key points • Syntrophobacter was the most consistent bacterial partner in propionate metabolism. • Diverse hydrogenotrophic methanogen populations could serve as syntrophic partners. • Methanoculleus emerged as a methanogen partner tolerant of elevated propionate.

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.

Gene and genome-centric approaches were applied to investigate the microbial taxonomic and metabolic diversity profiles from a lab-scale anaerobic reactor applied to laundry wastewater treatment with increasing concentrations of linear alkylbenzene sulfonate (LAS). Therefore, metagenomic analysis was carried out with reactor samples collected under three conditions, (i) Stage I, the reactor was fed with synthetic medium, (ii) Stage II, synthetic medium was replaced by diluted laundry wastewater with specific LAS loading rates (SLLRs) of 1.0 ± 0.3 mgLASgTVS−1d−1 and (iii) Stage III, SLLRs increased to 2.7 ± 0.7 mgLASgTVS−1d−1. Shotgun metagenomic sequencing was performed on an Illumina HiSeq 2 × 150 bp platform. Gene-centric analysis of each step of the metabolic pathway (fumarate addition, β-oxidation, ring cleavage and desulfonation) for anaerobic aromatic degradation showed eight over-representative genera (Achromobacter, Pelodictyon, Pseudomonas, Psychrobacter, Rhodococcus, Stenotrophomonas, Sulfurovum and Syntrophobacter), suggesting a microbial core with an important role in LAS biodegradation. Some of these genera were also recovered through a differential binning method, representing fifteen bacterial and one archaeal metagenome-assembled genomes (MAGs). Biodegradation pathway reconstruction of LAS using six MAGs unveiled the syntrophism for complete degradation of the LAS molecule. Only the MAGs with taxonomic annotation for Syntrophobacter showed genetic potential for fumarate addition, whereas in the ring cleavage, there was a predominance of genes in MAGs with taxonomic annotation for Pseudomonas fragi and Rhodococcus. This work represents the first report of genome-centric approach to study biological reactors applied in anionic surfactant degradation, contributing with detailed metabolic information of the key microbial actors in LAS degradation and opening perspectives for future biotechnological strategies aiming at bioaugmentation and/or biostimulation of indigenous microbial populations.

Methanogenic bioreactors have been applied to treat purified terephthalic acid (PTA) wastewater containing complex aromatic compounds, such as terephthalic acid, para-toluic acid and benzoic acid. This study characterized the interaction of microbial populations in 42 samples obtained from 10 PTA-degrading methanogenic bioreactors. Approximately, 54 dominant populations (11 methanogens, 8 syntrophs and 35 functionally unknown clades) that represented 73.9% of total 16S rRNA gene iTag sequence reads were identified. Co-occurrence analysis based on the abundance of dominant OTUs showed two non-overlapping networks centred around aromatic compound- (group AR: Syntrophorhabdaceae, Syntrophus and Pelotomaculum) and fatty acid- (group FA: Smithella and Syntrophobacter) degrading syntrophs. Group AR syntrophs have no direct correlation with hydrogenotrophic methanogens, while those from group FA do. As degradation of aromatic compounds has a wider thermodynamic window than fatty acids, Group AR syntrophs may be less influenced by fluctuations in hydrogenotrophic methanogen abundance or may non-specifically interact with diverse methanogens. In both groups, network analysis reveals full-scale- and lab-scale-specific uncultivated taxa that may mediate interactions between syntrophs and methanogens, suggesting that those uncultivated taxa may support the degradation of aromatic compounds through uncharted ecophysiological traits. These observations suggest that organisms from multiple niches orchestrate their metabolic capacity in multiple interaction networks to effectively degrade PTA wastewater.

The acid-tolerant methanogenic propionate degradation culture was acclimated in a propionate-fed semi-continuous bioreactor by daily adjusting the digestate pH. The performance of propionate fermentation, the respond of microbial community structure to the acidic environment, and the microbial network for propionate degradation in the acid-tolerant culture was investigated. The results demonstrated that after long term of acclimation to low pH, the digester could produce methane from propionate at pH 4.8–5.5 with 0.3–0.4 L g−1 propionic acid (HPr) d−1 of the volatile solids (VS) methane production. The predominant methanogens shifted from acetoclastic methanogens (∼87%) to hydrogenotrophic methanogens (∼67%) in the bioreactor with the dropping pH, indicating that hydrogenotrophic methanogens were more acid-tolerant than acetoclastic methanogens. Smithella (∼11%), Syntrophobacter (∼7%) and Pelotomaculum (∼3%) were the main propionate oxidizers in the acid-tolerant propionate-utilizing culture. Methanothrix dominant acetoclastic methanogens, while Methanolinea and Methanospirillum were the major H2 scavengers to support Syntrophobacter and Pelotomaculum syntrophic propionate degradation.

The upflow anaerobic sludge blanket (UASB) process is an anaerobic biological system that is widely used for the treatment of a variety of wastewater owing to its benefits such as low energy requirements and energy recovery as methane gas; however, unstable reactor performance is often observed. To solve this problem, several inert inorganic carriers and polymer materials have been studied for stabilization of the sludge microbial community and promotion of methane production. In this study, we demonstrate the effects of green tuff (GT) as a novel natural inorganic carrier on methane-producing activity (MPA) in combination with syntroph/methanogen-utilizable substrates and on the microbial community assembly of methanogenic batch cultures fed with granular sludge collected from UASB reactors treating milk- (MMW) or soy sauce- (SSW) manufacturing wastewater. The MPAs of MMW cultures with GT were 4.56- (propionate), 2.01- (n-butyrate), 1.37- (n-valerate), and 1.22- (benzoate) fold higher than those without GT. Likewise, SSW cultures with GT showed 1.34- (acetate) and 1.42- (propionate) fold higher MPA than those without GT. These results indicate that GT has potential to enhance MPAs during methanogenic degradation of these substrates. According to 16S rRNA transcripts-based community profiling, populations of syntrophic bacteria (Syntrophobacter and Smithella) and hydrogenotrophic methanogens (Methanobacterium and Methanolinea) in the cultures with GT were higher than in those without GT. Therefore, GT can be a potential solution for the activation of syntrophic partnerships and improvement/stabilization of reactor performance.

The methanogenic propionate degradation consortia were enriched in a propionate-fed semi-continuous bioreactor. The microbial community shift with depth, the microbial network and its correlation with metabolic pathway were also investigated. The results demonstrated that the maximum organic loading rate (OLR) of the reactor was 2.5 g propionic acid (HPr) L−1 d−1 with approximately 1.20 LL−1 d−1 of volumetric methane production (VMP). The organisms in the enrichment were spanning 36 bacterial phyla and 7 archaeal orders. Syntrophobacter, the main Hpr oxidizer in the digester, dominated bacteria with relative abundance changing from 63% to 37% with depth. The predominant methanogens shift from hydrogenotrophic Methanoculleus (∼60%) at the upper liquid layer to acetoclastic Methanothrix (∼51%) at the lower sediment layer in the bioreactor. These methanogens syntrophically support Syntrophobacter by degrading HPr catabolism by-products (H2 and acetate). Other bacteria could scavenge anabolic products (carbohydrate and protein) presumably derived from detrital biomass produced by the HPr-degrading community.

Direct interspecies electron transfer (DIET) among the cometabolism microbes plays a key role in the anaerobic degradation of persistent organic pollutants and stability of anaerobic bioreactor. In this study, the COD removal efficiency increased to 99.0% during the start-up stage in the combined bioelectrode-UASB system (R1) with magnetite nanoparticles addition, which was higher than those in the coupled bioelectrode-UASB (R2; 83.2%) and regular UASB (R3; 71.0%). During the stable stage, the increase of 2,4-dichloronitrobenzene (2,4-DClNB) concentration from 25 mg L−1 to 200 mg L−1 did not affect the COD removal efficiencies in R1 and R2, whereas the performance of R3 was deteriorated obviously. Further intermediates analysis indicated that magnetite nanoparticles enhanced the reductive dechlorination of 2,4-DClNB. High-throughput sequencing results showed that the functional microbes like Syntrophobacter and Syntrophomonas which have been reported to favor the DIET, were predominant on the cathode surface of R1 reactor. It is speculated that the addition of magnetite nanoparticles favors the cooperative metabolism of dechlorinating microbes and electricigens during 2,4-DClNB degradation process in the combined bioelectrode-UASB reactor. This study may provide a new strategy to improve the performance of microbial electrolysis cells and enhance the pollutant removal efficiency.

BACKGROUND Propionate accumulation may lead to digester failure. This study aimed to investigate the effect of ammonia, a metabolic product of protein, on propionate degradation. The shift of microbial community was also investigated. RESULTS Propionate accumulated over the experimental period in the reactor with a total ammonia nitrogen (TAN) concentration of 2.5 g N L−1, as a result the digester failed after 4 hydraulic retention times (HRT) at an organic load rate (OLR) of 0.8 g propionic acid (HPr) L−1 d−1. The average HPr degradation rate was below 54% during the fourth HRT, while >97% of the degraded HPr was converted to methane. The reactor without ammonia stress did not experience HPr accumulation and OLR was increased stepwise to 1.2 g L−1 d−1 at the 8th HRT. The average HPr degradation rate and methane recovery rate of this reactor in the last HRT was 99% and 74%, respectively. According to the shifts of microbial community, acetoclastic methanogen was more vulnerable to ammonia than hydrogenotrophic methanogen CONCLUSION TAN concentration of 2.5 g N L−1 inhibited propionate degradation more severely than methanogenesis. The loss of abundance of Clostridiaceae and Syntrophobacter might be the main reason for the poor performance under ammonia stress. © 2017 Society of Chemical Industry

Summary Methane is an important greenhouse gas and propionate is next to acetate the main intermediate (average 23%) of the carbon flow to CH4 in paddy fields. Sulfate (e.g., gypsum) application can reduce CH4 emissions up to 70%. However, the effect of gypsum application on propionate degradation and the microbial communities involved are not well understood. Therefore, we studied propionate-dependent sulfate reduction in anoxic microcosms of paddy soils from Italy and the Philippines, combining 16S rRNA and dissimilatory sulfite reductase (dsrB) gene profiling and co-occurrence network analysis. Sulfate was stoichiometrically reduced in treatments with propionate addition, while CH4 production was partially suppressed. Methane production but not sulfate reduction were suppressed and acetate accumulated after addition of methyl fluoride or fluoroacetate. With methyl fluoride in the presence of sulfate, the accumulated acetate was consumed after the depletion of propionate. Simultaneously, the relative abundances of Syntrophobacteraceae and Desulfovibrionaceae were significantly enhanced, while fluoroacetate repressed Desulfobulbaceae in both soils. Syntrophobacter 16S rRNA and dsrB gene copy numbers were also remarkably increased with gypsum amendment. Network analysis of both 16S rRNA and dsrB genes illustrated a strong co-occurrence of operational taxonomic units belonging to Syntrophobacteraceae, Desulfovibrionaceae and Desulfobulbaceae. In summary, Syntrophobacteraceae affiliated species were identified as the major propionate-dependent sulfate reducers in paddy soil. They (together with Desulfobulbaceae) oxidized propionate directly to acetate and CO2, or coupled the oxidation syntrophically to H2/formate-utilizing Desulfovibrionaceae. The transiently accumulating acetate was preferentially consumed by acetoclastic Methanosarcinaceae.

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.

In this study, the removal of anionic surfactant Linear Alkylbenzene Sulfonate (LAS) from laundry wastewater was evaluated in co-digestion with domestic sewage, using a pilot-scale Expanded Granular Sludge Bed reactor. Surfactant influent concentration was enhanced from 5 ± 3 mg LAS L−1 (stage I) to 19 ± 10 mg LAS L−1 (stage II) and 36 ± 19 mg LAS L−1 (stage III) throughout reactor operation. Sulfide levels higher than 20 mg L−1 influenced LAS removal efficiency, which decreased from 71% to 55% and 32% in stage I, II and III, respectively. Acclimation of microbial population was verified and higher relative abundance of the genera similar to Cytophaga, Bacteroides, Syntrophus and Syntrophobacter in the early stages (adaptation and stage I) was replaced by higher relative abundance of the genera Anaerophaga, Nitrosovibrio, Sulfurovum and Desulfovibrio in the last stages (stage II and III).

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.

Methanogenesis can be inhibited by volatile fatty acids (VFAs) accumulation and sulfate during anaerobic wastewater treatment. In this study, effects of ferroferric oxide (Fe3O4) on VFAs degradation and methanogenesis in sulfate-containing environment were investigated. Methanogenesis in reactors with or without sulfate were both favored through the addition of Fe3O4. In reactors without sulfate, the dosage of Fe3O4 increased the maximum methane production rate by 21.7% accompanied with faster acetate and propionate degradation. Metagenomic analysis showed that Fe3O4 mainly promoted electron exchange between Mesotoga, Syntrophobacter, Smithella and Methanosaeta without altering the syntrophic patterns. However, in the sulfate-containing reactor with low methanogenic efficiency, syntrophic ethanol users and Methanosaeta were replaced by sulfate-reducing bacteria and Methanosarcina, respectively. The supplement of Fe3O4 re-enriched the syntrophic partners inhibited by sulfate and rebuilt a new syntrophic interaction with high efficiency similar to that in sulfate-free environment, leading to better methanogenic performance in sulfate-containing environment.

Syngas based co-digestion is not only more economically attractive than separate syngas methanation but also able to upgrade biogas and increase overall CH4 amount simultaneously. However, high H2 concentration in the syngas could inhibit syntrophic degradation of propionate, resulting in propionate accumulation and even failure of the co-digestion system. In an attempt to reduce propionate accumulation via enhancing both H2 interspecies transfer (HIT) and direct interspecies electron transfer (DIET) pathways, layered granule-based biofilms induced by conductive carbon felt particles (CCFP) was employed. The results showed that propionate accumulation was effectively reduced with influent COD load up to 7gL(-1)d(-1). Two types of granule-based biofilms, namely biofilm adhered to CCFP (B-CCFP) and granules formed by self-immobilization (B-SI) were formed in the reactor. Clostridium, Syntrophobacter, Methanospirillum were possibly involved in HIT and Clostridium, Geobacter, Anaerolineaceae, Methanosaeta in DIET, both of which might be responsible for the high-rate propionate degradation.

Aiming at relieving acid accumulation in anaerobic digestion (AD), syntrophic associations of syntrophic fatty acid-oxidation bacteria and H2/formate-scavenging methanogens were enriched by feeding propionate, butyrate and formate in an up-flow anaerobic sludge blanket (UASB) reactor. Results showed that methane yield increased by 50% with increasing formate concentration (0–2000 mg COD/L). In addition, the abundance and quantity of SFOB (Syntrophobacter, Smithella and Syntrophomonas) and H2/formate-scavenging methanogens (Methanobacteriales and Methanomicrobiales) were increased after microbial acclimation. The enriched syntrophic associations showed higher propionate and butyrate removal efficiencies of 98.48 ± 1.14% and 99.71 ± 0.71%, respectively. Furthermore, encoding genes of formate dehydrogenase and hydrogenases presented higher abundances after microbial enrichment, which suggested that the enhancements of interspecies formate transfer and interspecies hydrogen transfer between syntrophic associations benefited volatile fatty acids (VFAs) conversion. This research provided an effective strategy to relieve acid accumulation.

Direct interspecies electron transfer (DIET) is a new electron-transfer strategy for enhanced propionate degradation. Ethanol can enrich the DIET species of Geobacter and conductive ferroferric oxide (Fe3O4) can promote DIET. Therefore, coupled effects of ethanol and Fe3O4 on propionate degradation were investigated. The maximum CH4 production rate was increased by 81.4% by adding Fe3O4 when simultaneously fed with ethanol and propionate, while the improvement could not be observed without ethanol. The sludge conductivity and the electron transfer system activity by adding Fe3O4 were increased by 2.66 and 2.73 times, respectively. Besides, the relative abundance of functional microbes such as Geobacter, Syntrophobacter, Smithella, and Methanosaeta, and their functional genes were increased by the supplement of Fe3O4. The improvement of propionate degradation by adding Fe3O4 was largely attributed to the co-existence of ethanol degradation. The DIET between Geobacter and Methanosaeta might provide more energies or rapidly consume the oxidation products to promote the propionate degradation.

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.

This study investigated the effects and fate of CIP on anaerobic sludge over a wide range of concentrations (0.05–50 mg/L), and 0.5–50 mg/L significantly inhibited organic removal and methanogenic activity, increased volatile fatty acids accumulation and low molecular weight soluble microbial products (SMPs), including p-cresol and nitrogen-containing compounds. Although microbial communities exposed to CIP did not differ significantly from the control in species diversity indices, Syntrophobacter and Methanothrix associated with acetogenesis and acetoclastic methanogenesis, respectively, were underrepresented in the CIP-exposed communities. Our study advances understanding of how environmentally relevant concentrations of CIP disrupts anaerobic digestion, which has important implications for anaerobic engineered systems treating CIP-bearing waste streams.

Anode potential has been shown to be a critical factor in the rate of acetate removal in microbial electrolysis cells (MECs), but studies with fermentable substrates and set potentials are lacking. Here, we examined the impact of three different set anode potentials (SAPs; −0.25, 0, and 0.25 V vs. standard hydrogen electrode) on the electrochemical performance, electron flux to various sinks, and anodic microbial community structure in two-chambered MECs fed with propionate. Electrical current (49–71%) and CH4 (22.9–41%) were the largest electron sinks regardless of the potentials tested. Among the three SAPs tested, 0 V showed the highest electron flux to electrical current (71 ± 5%) and the lowest flux to CH4 (22.9 ± 1.2%). In contrast, the SAP of −0.25 V had the lowest electron flux to current (49 ± 6%) and the highest flux to CH4 (41.1 ± 2%). The most dominant genera detected on the anode of all three SAPs based on 16S rRNA gene sequencing were Geobacter, Smithella and Syntrophobacter, but their relative abundance varied among the tested SAPs. Microbial community analysis implies that complete degradation of propionate in all the tested SAPs was facilitated by syntrophic interactions between fermenters and Geobacter at the anode and ferementers and hydrogenotrophic methanogens in suspension.

Inocula play an important role in anaerobic reactor startup by balancing the populations of Syntrophobacter and methanogens. Such balances make syntrophic metabolism thermodynamically feasible in anaerobic digestion. In this study, the effect of inocula on the performance of dairy manure digestion was investigated by analyzing the change in volatile fatty acids (VFA), total solids (TS), volatile solids (VS), specific biogas production (SGPR), and specific methane production (SPMP) as well as scanning and transmission electron micrographs. The study was performed at four treatments. Treatment one was granular sludge (GM); treatment two was non-granular sludge (SM); treatment three was mixed culture from an anaerobic lagoon (LM); while the fourth treatment (the control denoted MM) did not receive any exogenous inocula. In addition, stirred and unstirred conditions were maintained in the reactors to determine their effect on reactor startup. Performance ranking based on the SGPR and SPMP of treatments (in descending order) was: GM, SM, LM and MM under stirred conditions. Under unstirred conditions, performance ranking (also in descending order) was: SM, GM, LM, and MM. Results of the examination of microcolonies in the granular, non-granular sludge, and dairy manure suggest that syntrophic juxtaposition of methanogens and Syntrophobacter in granular inoculum was common while it was less visible in non-granular sludge, and completely absent in dairy manure.

Formate dehydrogenases and hydrogenases contain molybdenum or tungsten and/or selenium. These enzymes are crucial for interspecies formate and hydrogen transfer between propionate degrading Syntrophobacter spp. and methanogenic Methanospirillum spp. Here we used reverse transcription of total RNA followed by quantitative PCR (RT-qPCR) with specific primers to get insight into interspecies formate and hydrogen transfer. Transcriptional regulation of formate dehydrogenases and hydrogenases in Syntrophobacter and Methanospirillum spp. in a propionate-fed up-flow anaerobic sludge bed (UASB) reactor was examined. In both microorganisms formate dehydrogenase and hydrogenase coding genes (fdh and hyd respectively) were transcribed simultaneously. During 249 days in which molybdenum, tungsten and selenium were not supplied to the reactor feed, the microbial activity and transcription of fdh and hyd in Syntrophobacter spp. decreased. Transcription of fdh and hyd in Methanospirillum spp. did not decrease, but transcription of fdh increased when after 249 days molybdenum, tungsten and selenium were supplied to the reactor feed. The developed RT-qPCR is a technique that can give rapid information about active processes in methanogenic granular sludge and may contribute to predict metal limitation and failure in UASB reactors.

The effects of two typical methanogenic inhibitors [2-bromoethanesulfonate (BES) and chloroform (CHCl(3))] on the bacterial populations were investigated using molecular ecological techniques. Terminal restriction fragment length polymorphism analyses (T-RFLP) in combination with clone library showed that both the toxicants not only inhibited methanogenic activity but also considerably altered the bacterial community structure. Species of low % G + C Gram-positive bacteria (Clostridiales), high % G + C Actinomycetes, and uncultured Chloroflexi showed relatively greater tolerance of CHCl(3), whereas the BES T-RFLP patterns were characterized by prevalence of Geobacter hydrogenophilus and homoacetogenic Moorella sp. In addition, due to indirect thermodynamic inhibition caused by high hydrogen partial pressures, the growth of obligately syntrophic acetogenic Syntrophomonas and Syntrophobacter was also affected by selective inhibition of methanogenesis. Interestingly, by comparing the fermentative intermediates detected in BES- and CHCl(3)-treated experiments, it was furthermore found that when methanogenesis is specifically inhibited, the syntrophic interaction between hydrogen-producing fatty acid degraders and hydrogen-utilizating homoacetogens seemed to be strengthened.

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

In methanogenic habitats, volatile fatty acids (VFA), such as propionate and butyrate, are major intermediates in organic matter degradation. VFA are further metabolized to H(2), acetate and CO(2) by syntrophic fatty acid-degrading bacteria (SFAB) in association with methanogenic archaea. Despite their indispensable role in VFA degradation, little is known about SFAB abundance and their environmental distribution. To facilitate ecological studies, we developed four novel genus-specific quantitative PCR (qPCR) assays, with primer sets targeting known SFAB: Syntrophobacter, Smithella, Pelotomaculum and Syntrophomonas. Primer set specificity was confirmed using in silico and experimental (target controls, clone libraries and melt-curve analysis) approaches. These qPCR assays were applied to quantify SFAB in a variety of mesophilic methanogenic habitats, including a laboratory propionate enrichment culture, pilot- and full-scale anaerobic reactors, cow rumen, horse faeces, an experimental rice paddy soil, a bog stream and swamp sediments. The highest SFAB 16S rRNA gene copy numbers were found in the propionate enrichment culture and anaerobic reactors, followed by the bog stream and swamp sediment samples. In addition, it was observed that SFAB and methanogen abundance varied with reactor configuration and substrate identity. To our knowledge, this research represents the first comprehensive study to quantify SFAB in methanogenic habitats using qPCR-based methods. These molecular tools will help investigators better understand syntrophic microbial communities in engineered and natural environments.

Biological technology used to treat flue gas is useful to replace conventional treatment, but there is sulfide inhibition. However, no sulfide toxicity effect was observed in haloalkaliphilic bioreactors. The performance of the ethanol-fed bioreactor was better than that of lactate-, glucose-, and formate-fed bioreactor, respectively. To support this result strongly, Illumina MiSeq paired-end sequencing of 16S rRNA gene was applied to investigate the bacterial communities. A total of 389,971 effective sequences were obtained and all of them were assigned to 10,220 operational taxonomic units (OTUs) at a 97% similarity. Bacterial communities in the glucose-fed bioreactor showed the greatest richness and evenness. The highest relative abundance of sulfate-reducing bacteria (SRB) was found in the ethanol-fed bioreactor, which can explain why the performance of the ethanol-fed bioreactor was the best. Different types of SRB, sulfur-oxidizing bacteria, and sulfur-reducing bacteria were detected, indicating that sulfur may be cycled among these microorganisms. Because high-throughput 16S rRNA gene paired-end sequencing has improved resolution of bacterial community analysis, many rare microorganisms were detected, such as Halanaerobium, Halothiobacillus, Desulfonatronum, Syntrophobacter, and Fusibacter. 16S rRNA gene sequencing of these bacteria would provide more functional and phylogenetic information about the bacterial communities. (C) 2015 Elsevier B.V. All rights reserved.

The community composition of microbial cultures degrading tetrachloroethene (PCE), trichloroethene (TCE), cis-1,2-dichloroethene (cDCE) and vinyl chloride (VC) to ethene was studied. A combination of PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and 16S rRNA gene sequence analysis revealed that all cultures contained Dehalococcoides populations, but that the populations of other organisms varied widely. Based on the sequences of cloned 16S rRNA genes, real-time PCR methods were developed for several of these phylotypes affiliated with the putative dechlorinators Sulfurospirillum and Geobacter, the putative methanogens Methanomethylovorans, Methanomicrobiales, Methanosaeta and Methanosarcina, the putative acetogens Acetobacterium, Spirochaetes, and Sporomusa, and the putative fermenters Bacteroidetes, Syntrophus, and Syntrophobacter. These novel quantitative PCR methods were then used to estimate relative abundances of each phylotype in several individual cultures maintained on each chlorinated ethene. Dehalococcoides populations were the dominant phylotypes assayed in most KB-1 cultures, agreeing with the DGGE and cloning results. A Geobacter phylotype was also strongly represented in most PCE and TCE cultures, but not in cDCE or VC cultures, suggesting a possible role for this organism as a PCE-to-cDCE dechlorinator. The Sulfurospirillum phylotype was estimated to comprise a minor fraction of 16S rRNA gene copies and did not appear to have an important role in dechlorination.

Hydrogen sulphide (H2S) contributes a lot to odours at landfills, which is a threat to the environment and the health of the staff therein. To mitigate its emission, the bioconversion within landfill cover soils (LCSs) was introduced. H2S emission and concentration both in the field air above the landfill and in microcosm testing were surveyed. Results indicated that H2S emission and concentration in the landfill varied with landfill seasons and sites. There existed relationship between H2S concentration and fluxes spatially and temporally. To characterize and assess the spatial and temporal diversity of sulphur-oxidizing bacteria (SOB) and sulphate-reducing bacteria (SRB) in the LCSs, the terminal-restriction fragment length polymorphism technique was employed. Using the functional genes of dsrB and soxB, SOB, including Halothiobacillus, Rhodothalassium, Paracocccus, Allochromatium, and Thiobacillus, and SRB, including Desulfovibrio, Syntrophobacter, Desulfomonile and Desulfobacca, were identical and exhibited the dominant role in the LCSs. By employing an alternative available corona reactor, more than 90% removal efficiencies of sulphides were demonstrated, suggesting that the LCSs for eliminating odours in a lower concentration would be feasible.

Volatile fatty acid (VFA) metabolism and population changes during re-start of a wet anaerobic digester for treatment of biowaste suspensions were investigated. Initially acetate and propionate accumulated. However, VFA degradation rates improved within 2 weeks, reaching a balanced metabolism with constantly low VFA concentrations. The total microbial population consisted of 60% acidogenic + acetogenic and 40% methanogenic bacteria. Maximally 5.1% of all bacteria in the digester were propionate-oxidizing bacteria. Pelotomaculum species were dominant, followed by much lower numbers of Smithella species and Syntrophobacter species. Pelotomaculum and Smithella spec. decreased during acetate and propionate accumulation and recovered later on, whereas Syntrophobacter spec. was steadily increasing during start-up. A nearly constant proportion of Methanosaeta spec. was present all time, while Methanosarcina spec. decreased and Methanomicrobiales increased during accumulation and degradation of acetate and propionate. During steady state proportions as in the inoculum were obtained.

This paper reports on propionic acid (HPr) degradation in a laboratory scale two-phase anaerobic system, where HPr was accumulated in the acidogenic reactor and degraded in the methanogenic reactor. Batch tests using biomass from the two-phase anaerobic system showed HPr degradation was rarely detectable in the acidogenic reactor when HPr concentration ranged from 639 to 4531 mg HPr L−1 and at pH 4.50 to 6.50. Biomass from the methanogenic reactor could, however, successfully degrade HPr at its initial concentration of up to 4585 mg HPr L−1 at pH 6.40–7.30. ATP results showed that differences in the degradation ability of HPr by the acidogenic and methanogenic biomass may be related with their respective different biomass activities. Results from pyrosequencing showed that the predominant propionic acid oxidizing bacteria (POB) in the methanogenic reactor were Smithella (2.68%) and Syntrophobacter (0.35%); while poor degradation of HPr in the acidogenic reactor may be associated with the low abundance of POB (0.02% Desulfacinum and 0.08% Desulfobulbus). This might have been induced by the long-term unfavorable environment for POB growth in the acidogenic reactor.

Propionate is an important intermediate in the conversion of organic matter to methane and carbon dioxide. In methanogenic environments, the degradation of propionate to acetate and CO2 may account for 6 to 35 mol% of the total methanogenesis (17). Propionate oxidation itself is energetically very unfavorable under standard thermodynamic conditions (see Table ​Table1).1). Under methanogenic conditions, proton-reducing acetogenic bacteria are only able to gain energy from this reaction when the concentration of products is kept low. Thus, the degradation of propionate is only accomplished in obligate syntrophic consortia of proton-reducing acetogenic bacteria and methanogenic archaea (27, 28, 33). So far, three syntrophic propionate-oxidizing bacteria and some highly purified enrichment cultures have been described (3, 11, 20, 21, 22, 32, 43, 44, 49). TABLE 1 Reactions involved in the degradation of propionate and their standard Gibbs free-energy changes, corrected for a pH of 6.9 and a temperature of 37°Ca Studies have shown that most of the known syntrophic propionate-oxidizing bacteria degrade propionate via the methylmalonyl-coenzyme A (CoA) pathway (14, 25, 43). During the oxidation of propionate in the methylmalonyl-CoA pathway, electrons are released in three reactions, namely, the oxidation of succinate to fumarate, malate to oxaloacetate, and pyruvate to acetyl-CoA (25). In methanogenic environments, the H2 partial pressure is low enough to allow the direct reduction of protons with the electrons released during the oxidation of pyruvate and malate. However, the H2 partial pressure is not sufficient to allow this reduction during the oxidation of succinate to fumarate. It was hypothesized that the electrons released during the oxidation of succinate are shifted to a lower redox potential via reversed electron transport. This transport would be driven by the hydrolysis of 2/3 mol of ATP (27, 28, 37). Some evidence has been obtained for the presence of a reversed electron transport system in syntrophic propionate-degrading bacteria (41). However, the methylmalonyl-CoA pathway yields only 1 mol of ATP via substrate level phosphorylation. Therefore, if such a reversed electron transport is occurring, only 1/3 mol of ATP per mol of propionate is left for growth. Under physiological conditions, the Gibbs free-energy change needed for ATP synthesis must amount to a minimum of 70 kJ mol of ATP−1. Thus, the minimum Gibbs free-energy quantum that can generate 1/3 mol of ATP would amount to approximately −23 kJ mol of propionate−1. It has been suggested that this amount of Gibbs free-energy change corresponds to the minimum energy quantum required to sustain microbial life (27, 28). In several methanogenic environments, the apparent Gibbs free-energy change for propionate oxidation was on the order of −3 to −15 kJ mol of propionate−1 and thus was rather small (5, 19, 26, 46). This amount of free-energy change is less than the minimum energy quantum needed to sustain microbial life. It is not clear why such small free-energy changes are observed during the degradation of propionate. Most syntrophic propionate-oxidizing bacteria are not obligate syntrophs but are also able to grow on other substrates, such as fumarate, malate, and pyruvate, in the absence of a partner microorganism. A remarkable feature of the propionate-oxidizing Syntrophobacter species is their ability to couple the oxidation of propionate not only to an H2-consuming syntrophic partner but also to the reduction of sulfate. In fact, phylogenetic analysis of Syntrophobacter fumaroxidans has revealed that this bacterium is indeed related to sulfate-reducing bacteria (12). Perhaps the ability to reduce sulfate is of importance to explain the energetics of syntrophic propionate-oxidizing bacteria. Besides propionate, many alcohols, fatty acids, amino acids, and aromatic compounds are anaerobically degraded by syntrophy. In each case, the available free energy is relatively low and has to be shared between the two syntrophic partners (27, 28). The energetics of syntrophic interspecies H2 transfer has been studied in defined cocultures of benzoate-, lactate-, ethanol-, propionate-, and butyrate-oxidizing fermenting bacteria with H2-consuming methanogens (1, 8, 9, 30, 31, 28, 39, 45). Propionate oxidation, however, has not yet been investigated in continuous-culture experiments. Therefore, we studied the energetics of propionate consumption in syntrophic cocultures of S. fumaroxidans and Methanospirillum hungatei in the absence and presence of sulfate by determining the Gibbs free energy available for both the propionate oxidizers and the H2-consuming methanogens under steady-state conditions in batch and chemostat cultures. The growth yields and maintenance coefficients of the syntrophic propionate oxidizer were also determined.

Anaerobic digestion (AD) is an attractive microbiological technology for both waste treatment and energy production. Syntrophic acetogenic bacteria are an important guild because they are essential for maintaining efficient and stable AD operation. However, this guild is poorly understood due to difficulties to culture them. In this study, we developed specific PCR assays targeting the propionate-CoA transferase genes (pct) to investigate their diversity and distribution in several mesophilic anaerobic digesters and a bench-scale temperature-phased AD (TPAD) system. Phylogenetic analysis of sequenced pct amplicons revealed the occurrence of Syntrophobacter fumaroxidans and six other clusters of putative pct genes. Principal coordinate analysis (PCoA) showed that pct diversity and abundance were largely correlated to the feedstocks of the digesters, while little difference was seen between the granular and the liquid fractions of each digester or between the two digesters of the TPAD system. Cluster-specific qPCR analysis revealed major impact of feedstocks and fractions on the abundance of pct genes. Readily fermentable substrates such as sugar- or starch-rich feedstocks selected for pct genes (Cluster I) related to Syntrophobacter, while manure feedstock selected for pct clusters related to pct of Clostridium spp. These results suggest that propionate metabolism can be affected by feedstocks and partition differently between solid and liquid phases in digesters. The PCR assays developed in this study may serve as a tool to investigate propionate-oxidizing bacteria in anaerobic digesters and other anaerobic environments.

Propionate is one of the major intermediary products in the anaerobic decomposition of organic matter in wetlands and paddy fields. Under methanogenic conditions, propionate is decomposed through syntrophic interaction between proton-reducing and propionate-oxidizing bacteria and H 2 -consuming methanogens. Temperature is an important environmental regulator; yet its effect on syntrophic propionate oxidation has been poorly understood. In the present study, we investigated the syntrophic oxidation of propionate in a rice field soil at 15°C and 30°C. [U- 13 C]propionate (99 atom%) was applied to anoxic soil slurries, and the bacteria and archaea assimilating 13 C were traced by DNA-based stable isotope probing. Syntrophobacter spp., Pelotomaculum spp., and Smithella spp. were found significantly incorporating 13 C into their nucleic acids after [ 13 C]propionate incubation at 30°C. The activity of Smithella spp. increased in the later stage, and concurrently that of Syntrophomonas spp. increased. Aceticlastic Methanosaetaceae and hydrogenotrophic Methanomicrobiales and Methanocellales acted as methanogenic partners at 30°C. Syntrophic oxidation of propionate also occurred actively at 15°C. Syntrophobacter spp. were significantly labeled with 13 C, whereas Pelotomaculum spp. were less active at this temperature. In addition, Methanomicrobiales , Methanocellales , and Methanosarcinaceae dominated the methanogenic community, while Methanosaetaceae decreased. Collectively, temperature markedly influenced the activity and community structure of syntrophic guilds degrading propionate in the rice field soil. Interestingly, Geobacter spp. and some other anaerobic organisms like Rhodocyclaceae , Acidobacteria , Actinobacteria , and Thermomicrobia probably also assimilated propionate-derived 13 C. The mechanisms for the involvement of these organisms remain unclear.

The spatial distribution of microorganisms and their in situ activities in anaerobic granules were investigated by fluorescence in situ hybridization (FISH), beta imaging and microsensors. FISH results revealed a layered structure of microorganisms in the granule, where Chloroflexi was present in the outermost layer, Smithella spp. and Syntrophobacter spp. were found in a depth of ca. 100 μm, and Archaea was restricted to the inner layer (below ca. 300 μm from the surface). Substrate uptake patterns elucidated by beta imaging demonstrated that glucose uptake was highest at 50 μm depth, whereas propionate uptake had a peak at 200 μm depth. In addition, microsensor measurements revealed that acid was produced mainly at 100 μm depth and H 2 production was detected at a depth from 100 to 200 μm. H 2 consumption and corresponding CH 4 production were found below 200 μm from the surface. Direct comparison of these results implied sequential degradation of complex organic compounds in anaerobic granules; Chloroflexi contributed to fermentation of organic compounds and acid production in the outermost layer, volatile fatty acids were oxidized and H 2 was produced mainly by Smithella spp. and Syntrophobacter spp. at a depth from 100 to 200 μm, and Archaea produced CH 4 below ca. 300 μm from the surface.

We investigated the major phylogenetic groups and population size of glucose-, propionate-, and acetate-degrading bacteria in the glucose-degrading anaerobic digester sludge by stable-isotope probing analysis of 16S rRNA (RNA-SIP) with [(13)C(6)]glucose followed by time course analysis of microautoradiography combined with fluorescent in situ hybridization (MAR-FISH) with [U-(14)C]glucose. The results indicated that glucose was predominately degraded to CH(4) and CO(2) by glucose-degrading Propionibacterium and Olsenella that are belonging to the phylum Actinobacteria, propionate-degrading Smithella and Syntrophobacter, and acetate-degrading Methanosaeta and Synergistes group 4 in this anaerobic sludge. The population size of propionate degraders was the smallest among three trophic groups and the specific degradation rate of propionate was also low. The specific degradation rate of acetate was low even though their population size was comparable to the glucose degraders. These results could explain why the degradation of propionate and acetate was the rate-limiting step in methanogenic glucose degradation.

Quantitative monitoring method of two important trophic groups of bacteria in methanogenic communities was established and applied to six different anaerobic processes. The method we employed was based upon our previous sequence-specific rRNA cleavage method that allows quantification of rRNA of target groups so that the populations reflecting in situ activity could be determined. We constructed a set of scissor probes targeting the Chloroflexi group known as ‘semi-syntrophic’ heterotrophic bacteria and fatty acid-oxidizing syntrophs to determine their relative abundance in the processes. By using the method, we found that several reactors harbored a large amount of organisms belonging to the phylum Chloroflexi accounting for up to 20% of the total prokaryotic populations. Propionate-oxidizing syntrophs, Syntrophobacter, Smithella and Pelotomaculum were also found to be significant comprising up to 3.9% of the total populations, but their distribution is highly dependent on the process examined. This is the first clear, non-PCR based quantitative evidence that those organisms play active roles under in situ methanogenic conditions.

The dissimilatory reduction of sulfate contributes to the retention of sulfur in acidic mineratrophic peatlands. Novel sulfate-reducing prokaryotes (SRPs) colonize these low-sulfate fens. This study assessed the community structures of SRPs in a depth gradient (0-50 cm) in a fen, located in the Fichtelgebirge (Spruce Mountains), Germany. Detection of SRPs with multiplex (terminal-) restriction fragment length polymorphism analysis of amplified dissimilatory (bi)sulfite reductase genes (dsrAB) separated three subgroups derived from (i) the upper 5 and 10 cm, (ii) 15-25 cm, and (iii) 30-50 cm depth. Biogeochemical parameters measured in the soil solution from July 2001 to July 2004 documented that the upper 5-10 cm were exposed to drying and oxygenation prior to sampling. Periodic oxygenation reached a maximum depth of 25 cm in the water-saturated fen and was concomitant with relative high concentrations of nitrate (120 microM) and sulfate (up to 310 microM). The fen soil was permanently anoxic below 30 cm depth with average concentrations of sulfate below 40 microM and maximum concentrations of methane. Cloning of dsrAB PCR products from 5, 20 and 40 cm depth yielded a total of 84 unique dsrAB restriction patterns. Partial sequencing of 61 distinct clones resulted in 59 unique partial protein sequences that mainly clustered with DsrA sequences of uncultivated sulfate reducers. Syntrophobacter fumaroxidans- and Syntrophobacter wolinii-related bacteria appeared to be present only in 40 cm depth. Differences in the SRP community structures suggested that SRPs present in the upper fen soil have to tolerate O(2) and even drying, whereas SRPs present in deep anoxic zones may act as syntrophic fermentors in cooperation with H(2)-utilizing methanogens.

The phylogenetic and functional diversity of syntrophic propionate-oxidizing bacteria (POB) present in an anaerobic digester was investigated by microautoradiography combined with fluorescent in situ hybridization (MAR-FISH) that can directly link 16S rRNA phylogeny with in situ metabolic function. The syntrophic POB community in the anaerobic digester sludge consisted of at least four phylogenetic groups: Syntrophobacter, uncultured short rod Smithella (Smithella sp. SR), uncultured long rod Smithella (Smithella sp. LR), and an unidentified group. The activities of these POB groups were dependent on the propionate concentrations. The uncultured Smithella sp. SR accounted for 52-62% of the total active POB under all the propionate concentrations tested (0.5-15 mM). In contrast, uncultured Smithella sp. LR was active only at lower propionate concentrations and became a dominant active POB at 0.5 mM of propionate. Syntrophobacter accounted for 16-31% of the total active POB above 2.5 mM propionate, whereas the active Syntrophobacter population became low (ca. 6%) at 0.5 mM of propionate. The anaerobic digester was operated in a fill and draw mode, resulting in periodical changes in propionate concentration ranging from 0 to 10 mM. These phylogenetically and functionally diverse, to some extent functionally redundant, active POB communities were dynamically responding to the periodical changes in propionate concentration.

Propionate is an important intermediate of the degradation of organic matter in many anoxic environments. In methanogenic environments, due to thermodynamic constraints, the oxidation of propionate requires syntrophic cooperation of propionate-fermenting proton-reducing bacteria and H 2 -consuming methanogens. We have identified here microorganisms that were active in syntrophic propionate oxidation in anoxic paddy soil by rRNA-based stable-isotope probing (SIP). After 7 weeks of incubation with [ 13 C]propionate (13 C-labeled substrate per g dry weight of soil, we found that archaeal nucleic acids were 13 C labeled to a larger extent than those of the bacterial partners. Nevertheless, both terminal restriction fragment length polymorphism and cloning analyses revealed Syntrophobacter spp., Smithella spp., and the novel Pelotomaculum spp. to predominate in “heavy” 13 C-labeled bacterial rRNA, clearly showing that these were active in situ in syntrophic propionate oxidation. Among the Archaea , mostly Methanobacterium and Methanosarcina spp. and also members of the yet-uncultured “rice cluster I” lineage had incorporated substantial amounts of 13 C label, suggesting that these methanogens were directly involved in syntrophic associations and/or thriving on the [ 13 C]acetate released by the syntrophs. With this first application of SIP in an anoxic soil environment, we were able to clearly demonstrate that even guilds of microorganisms growing under thermodynamic constraints, as well as phylogenetically diverse syntrophic associations, can be identified by using SIP. This approach holds great promise for determining the structure and function relationships of further syntrophic or other nutritional associations in natural environments and for defining metabolic functions of yet-uncultivated microorganisms.

Nutrient runoff from the Everglades Agricultural Area resulted in a well-documented gradient of phosphorus concentrations in soil and water, with concomitant ecosystem-level changes, in the northern Florida Everglades. It was recently reported that sulfate-reducing prokaryote assemblage composition, numbers, and activities are dependent on position along the gradient (H. Castro, K. R. Reddy, and A. Ogram, Appl. Environ. Microbiol. 68: 6129-6137, 2002). The present study utilized a combination of culture- and non-culture-based approaches to study differences in composition of assemblages of syntrophic and methanogenic microbial communities in eutrophic, transition, and oligotrophic areas along the phosphorus gradient. Methanogenesis rates were much higher in eutrophic and transition regions, and sequence analysis of 16S rRNA gene clone libraries constructed from samples taken from these regions revealed differences in composition and activities of syntroph-methanogen consortia. Methanogens from eutrophic and transition regions were almost exclusively composed of hydrogenotrophic methanogens, with approximately 10,000-fold-greater most probable numbers of hydrogenotrophs than of acetotrophs. Most cultivable strains from eutrophic and transition regions clustered within novel lineages. In non-culture-based studies to enrich syntrophs, most bacterial and archaeal clones were either members of novel lineages or closely related to uncultivated environmental clones. Novel cultivable Methanosaeta sp. and fatty acid-oxidizing bacteria related to the genera Syntrophomonas and Syntrophobacter were observed in microcosms containing soil from eutrophic regions, and different lines of evidence indicated the existence of novel syntrophic association in eutrophic regions.

An anaerobic, thermophilic, syntrophic propionate-oxidizing bacterium, strain SI(T), isolated previously from granular sludge in a thermophilic upflow anaerobic sludge blanket (UASB) reactor, was characterized. The strain could grow fermentatively on pyruvate and fumarate in pure culture. The strain grew on propionate, ethanol, lactate, 1-butanol, 1-pentanol, 1,3-propanediol, 1-propanol and ethylene glycol in co-culture with the hydrogenotrophic methanogen Methanothermobacter thermautotrophicus strain deltaH(T). The optimum temperature for growth was 55 degrees C and the pH optimum was 7.0. The G+C content of the DNA was 52.8 mol %. Strain SI(T) contained MK-7 and MK-7(H4) as the major quinones and contained iso-C15:0 as the major fatty acid. Based on 16S rDNA sequence analysis, strain SI(T) formed a novel lineage within the gram-positive, spore-forming, sulphate-reducing bacterial group Desulfotomaculum. However, the strain lacked the ability to conduct dissimilatory sulphate reduction. Instead, it could reduce fumarate to succinate with concomitant growth on several organic substances as electron donor. These phenotypic and genetic properties support the formation of a novel species of a new genus, for which the name Pelotomaculum thermopropionicum gen. nov., sp. nov. is proposed. The type strain is strain SI(T) (= DSM 13744T = JCM 10971T).