Metabolism of novel potential syntrophic acetate-oxidizing bacteria in thermophilic methanogenic chemostats.

Acetate is a major intermediate in the anaerobic digestion of organic waste to produce CH4. In methanogenic systems, acetate degradation is carried out by either acetoclastic methanogenesis or syntrophic degradation by acetate oxidizers and hydrogenotrophic methanogens. Due to challenges in the isolation of syntrophic acetate-oxidizing bacteria (SAOB), the diversity and metabolism of SAOB and the mechanisms of their interactions with methanogenic partners are not fully characterized. In this study, the in situ activity and metabolic characteristics of potential SAOB and their interactions with methanogens were elucidated through metagenomics and metatranscriptomics. In addition to the reported SAOB classified in the genera Tepidanaerobacter, Desulfotomaculum, and Thermodesulfovibrio, we identified a number of potential SAOB that are affiliated with Clostridia, Thermoanaerobacteraceae, Anaerolineae, and Gemmatimonadetes. The potential SAOB possessing the glycine-mediated acetate oxidation pathway dominates SAOB communities. Moreover, formate appeared to be the main product of the acetate degradation by the most active potential SAOB. We identified the methanogen partner of these potential SAOB in the acetate-fed chemostat as Methanosarcina thermophila. The dominated potential SAOB in each chemostat had similar metabolic characteristics, even though they were in different fatty-acid-fed chemostats. These novel syntrophic lineages are prevalent and may play critical roles in thermophilic methanogenic reactors. This study expands our understanding of the phylogenetic diversity and in situ biological functions of uncultured syntrophic acetate degraders and presents novel insights into how they interact with methanogens.IMPORTANCECombining reactor operation with omics provides insights into novel uncultured syntrophic acetate degraders and how they perform in thermophilic anaerobic digesters. This improves our understanding of syntrophic acetate degradation and contributes to the background knowledge necessary to better control and optimize anaerobic digestion processes.

Unique H2-utilizing lithotrophy in serpentinite-hosted systems.

Serpentinization of ultramafic rocks provides molecular hydrogen (H2) that can support lithotrophic metabolism of microorganisms, but also poses extremely challenging conditions, including hyperalkalinity and limited electron acceptor availability. Investigation of two serpentinization-active systems reveals that conventional H2-/CO2-dependent homoacetogenesis is thermodynamically unfavorable in situ due to picomolar CO2 levels. Through metagenomics and thermodynamics, we discover unique taxa capable of metabolism adapted to the habitat. This included a novel deep-branching phylum, "Ca. Lithacetigenota", that exclusively inhabits serpentinite-hosted systems and harbors genes encoding alternative modes of H2-utilizing lithotrophy. Rather than CO2, these putative metabolisms utilize reduced carbon compounds detected in situ presumably serpentinization-derived: formate and glycine. The former employs a partial homoacetogenesis pathway and the latter a distinct pathway mediated by a rare selenoprotein-the glycine reductase. A survey of microbiomes shows that glycine reductases are diverse and nearly ubiquitous in serpentinite-hosted environments. "Ca. Lithacetigenota" glycine reductases represent a basal lineage, suggesting that catabolic glycine reduction is an ancient bacterial innovation by Terrabacteria for gaining energy from geogenic H2 even under hyperalkaline, CO2-poor conditions. Unique non-CO2-reducing metabolisms presented here shed light on potential strategies that extremophiles may employ for overcoming a crucial obstacle in serpentinization-associated environments, features potentially relevant to primordial lithotrophy in early Earth.

Novel Syntrophic Isovalerate-Degrading Bacteria and Their Energetic Cooperation with Methanogens in Methanogenic Chemostats.

Isovalerate is an important intermediate in anaerobic degradation of proteins/amino acids. Little is known about how this compound is degraded due to challenges in cultivation and characterization of isovalerate-degrading bacteria, which are thought to symbiotically depend on methanogenic archaea. In this study, we successfully enriched novel syntrophic isovalerate degraders (uncultivated Clostridiales and Syntrophaceae members) through operation of mesophilic and thermophilic isovalerate-fed anaerobic reactors. Metagenomics- and metatranscriptomics-based metabolic reconstruction of novel putative syntrophic isovalerate metabolizers uncovered the catabolic pathway and byproducts (i.e., acetate, H2, and formate) of isovalerate degradation, mechanisms for electron transduction from isovalerate degradation to H2 and formate generation (via electron transfer flavoprotein; ETF), and biosynthetic metabolism. The identified organisms tended to prefer formate-based interspecies electron transfer with methanogenic partners. The byproduct acetate was further converted to CH4 and CO2 by either Methanothrix (mesophilic) and Methanosarcina (thermophilic), which employed different approaches for acetate degradation. This study presents insights into novel mesophilic and thermophilic isovalerate degraders and their interactions with methanogens.

Deep Sequencing Uncovers Caste-Associated Diversity of Symbionts in the Social Ant Camponotus japonicus.

Symbiotic microorganisms can have a profound impact on the host physiology and behavior, and novel relationships between symbionts and their hosts are continually discovered. A colony of social ants consists of various castes that exhibit distinct lifestyles and is, thus, a unique model for investigating how symbionts may be involved in host eusociality. Yet our knowledge of social ant-symbiont dynamics has remained rudimentary. Through 16S rRNA gene deep sequencing of the carpenter ant Camponotus japonicus symbiont community across various castes, we here report caste-dependent diversity of commensal gut microbiota and lineage divergence of "Candidatus Blochmannia," an obligate endosymbiont. While most prevalent gut-associated bacterial populations are found across all castes (Alphaproteobacteria, Gammaproteobacteria, Bacteroidetes, and Cyanobacteria), we also discovered uncultured populations that are found only in males (belonging to Corynebacteriales, Alkanindiges, and Burkholderia). Most of those populations are not detected in laboratory-maintained queens and workers, suggesting that they are facultative gut symbionts introduced via environmental acquisition. Further inspection of "Ca. Blochmannia" endosymbionts reveals that two populations are dominant in all individuals across all castes but that males preferentially contain two different sublineages that are diversified from others. Clearly, each caste has distinct symbiont communities, suggesting an overlooked biological aspect of host-symbiont interaction in social insects.IMPORTANCE Social animals, such as primates and some insects, have been shown to exchange symbiotic microbes among individuals through sharing diet or habitats, resulting in increased consistency of microbiota among social partners. The ant is a representative of social insects exhibiting various castes within a colony; queens, males, and nonreproductive females (so-called workers) show distinct morphologies, physiologies, and behaviors but tightly interact with each other in the nest. However, how this social context affects their gut microbiota has remained unclear. In this study, we deeply sequenced the gut symbiont community across various castes of the carpenter ant Camponotus japonicus We report caste-dependent diversity of commensal gut microbial community and lineage divergence of the mutualistic endosymbiont "Candidatus Blochmannia." This report sheds light on the hidden diversity in microbial populations and community structure associated with guts of males in social ants.

Different Interspecies Electron Transfer Patterns during Mesophilic and Thermophilic Syntrophic Propionate Degradation in Chemostats.

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.

Response of Isovalerate-Degrading Methanogenic Microbial Community to Inhibitors.

Isovalerate is one of the key intermediates during anaerobic digestion treating protein-containing waste/wastewater. Investigating the effect of different kinds of inhibitors on isovalerate-degrading microbial community is necessary to develop measures for improving the effectiveness of the treatment plants. In the present study, dynamic changes in the isovalerate-degrading microbial community in presence of inhibitors (ammonium, sulfide, mixed ammonium and sulfide, and chlortetracycline (CTC)) were investigated using high-throughput sequencing of 16S rRNA gene. Our observations showed that the isovalerate-degrading microbial community responded differently to different inhibitors and that the isovalerate degradation and gas production were strongly repressed by each inhibitor. We found that sulfide inhibited both isovalerate oxidation followed by methanogenesis, while ammonium, mixed ammonium and sulfide, and CTC mainly inhibited isovalerate oxidation. Genera classified into Proteobacteria and Chloroflexi were less sensitive to inhibitors. The two dominant genera, which are potential syntrophic isovalerate oxidizers, exhibited different responses to inhibitors that the unclassified_Peptococcaceae_3 was more sensitive to inhibitors than the unclassified_Syntrophaceae. Upon comparison to acetoclastic methanogen Methanosaeta, hydrogenotrophic methanogens Methanoculleus and Methanobacterium were less sensitive to inhibitors.

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 on the 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.

Effects of the Wastewater Flow Rate on Interactions between the Genus Nitrosomonas and Diverse Populations in an Activated Sludge Microbiome.

The present study characterized the interactions of microbial populations in activated sludge systems during the operational period after an increase in the wastewater flow rate and consequential ammonia accumulation using a 16S rRNA gene sequencing-based network analysis. Two hundred microbial populations accounting for 81.8% of the total microbiome were identified. Based on a co-occurrence analysis, Nitrosomonas-type ammonia oxidizers had one of the largest number of interactions with diverse bacteria, including a bulking-associated Thiothrix organism. These results suggest that an increased flow rate has an impact on constituents by changing ammonia concentrations and also that Nitrosomonas- and Thiothrix-centric responses are critical for ammonia removal and microbial community recovery.

Thermodynamically diverse syntrophic aromatic compound catabolism.

Specialized organotrophic Bacteria 'syntrophs' and methanogenic Archaea 'methanogens' form a unique metabolic interaction to accomplish cooperative mineralization of organic compounds to CH4 and CO2 . Due to challenges in cultivation of syntrophs, mechanisms for how their organotrophic catabolism circumvents thermodynamic restrictions remain unclear. In this study, we investigate two communities hosting diverse syntrophic aromatic compound metabolizers (Syntrophus, Syntrophorhabdus, Pelotomaculum and an uncultivated Syntrophorhabdacaeae member) to uncover their catabolic diversity and flexibility. Although syntrophs have been generally presumed to metabolize aromatic compounds to acetate, CO2 , H2 and formate, combined metagenomics and metatranscriptomics show that uncultured syntrophs utilize unconventional alternative metabolic pathways in situ producing butyrate, cyclohexanecarboxylate and benzoate as catabolic byproducts. In addition, we also find parallel utilization of diverse H2 and formate generating pathways to facilitate interactions with partner methanogens. Based on thermodynamic calculations, these pathways may enable syntrophs to combat thermodynamic restrictions. In addition, when fed with specific substrates (i.e., benzoate, terephthalate or trimellitate), each syntroph population expresses different pathways, suggesting ecological diversification among syntrophs. These findings suggest we may be drastically underestimating the biochemical capabilities, strategies and diversity of syntrophic bacteria thriving at the thermodynamic limit.

Impacts of biostimulation and bioaugmentation on the performance and microbial ecology in methanogenic reactors treating purified terephthalic acid wastewater.

Up-flow anaerobic sludge blanket (UASB) processes treating purified terephthalic acid (PTA) wastewater often face challenges associated with biomass loss. As excessive biomass loss could lead to deterioration of PTA removal, biostimulation and bioaugmentation were often practiced without understanding the microbial impact in UASB. Three laboratory-scale UASB reactors were operated with synthetic PTA wastewater as the feed, with two added with co-substrate (glucose or molasses) on Day 170 for 90 days, and one with external granules on Day 118. Throughout the operation, treatment performance was measured together with the analysis of microbial communities of biomass samples using 16S rRNA-based gene Illumina sequencing. Glucose amendment destabilized both terephthalic acid and para-toluic acid removal, while molasses amendment improved para-toluic acid removal. Both substrate addition generally led to decreases in the abundances of syntrophs and methanogens and increases in carbohydrate-fermenting bacteria in the granular sludge. Regarding bioaugmentation, paper mill granule addition led to a temporary crash of terephthalic acid removal for 42 days, and deterioration of para-toluic acid removal throughout the operation. Syntrophs and methanogens were observed to colonize on the paper mill granules after three months, meanwhile growth of methanogens were stimulated on the PTA granules added initially. Overall, proper level of molasses amendment and external granule inoculation could be promising strategies to make up for biomass loss during the operation of PTA-degrading UASB.

Chasing the elusive Euryarchaeota class WSA2: genomes reveal a uniquely fastidious methyl-reducing methanogen.

The ecophysiology of one candidate methanogen class WSA2 (or Arc I) remains largely uncharacterized, despite the long history of research on Euryarchaeota methanogenesis. To expand our understanding of methanogen diversity and evolution, we metagenomically recover eight draft genomes for four WSA2 populations. Taxonomic analyses indicate that WSA2 is a distinct class from other Euryarchaeota. None of genomes harbor pathways for CO2-reducing and aceticlastic methanogenesis, but all possess H2 and CO oxidation and energy conservation through H2-oxidizing electron confurcation and internal H2 cycling. As the only discernible methanogenic outlet, they consistently encode a methylated thiol coenzyme M methyltransferase. Although incomplete, all draft genomes point to the proposition that WSA2 is the first discovered methanogen restricted to methanogenesis through methylated thiol reduction. In addition, the genomes lack pathways for carbon fixation, nitrogen fixation and biosynthesis of many amino acids. Acetate, malonate and propionate may serve as carbon sources. Using methylated thiol reduction, WSA2 may not only bridge the carbon and sulfur cycles in eutrophic methanogenic environments, but also potentially compete with CO2-reducing methanogens and even sulfate reducers. These findings reveal a remarkably unique methanogen 'Candidatus Methanofastidiosum methylthiophilus' as the first insight into the sixth class of methanogens 'Candidatus Methanofastidiosa'.