Anaerobic Degradation of Alkanes by Marine Archaea.

Alkanes are saturated apolar hydrocarbons that range from their simplest form, methane, to high-molecular-weight compounds. Although alkanes were once considered biologically recalcitrant under anaerobic conditions, microbiological investigations have now identified several microbial taxa that can anaerobically degrade alkanes. Here we review recent discoveries in the anaerobic oxidation of alkanes with a specific focus on archaea that use specific methyl coenzyme M reductases to activate their substrates. Our understanding of the diversity of uncultured alkane-oxidizing archaea has expanded through the use of environmental metagenomics and enrichment cultures of syntrophic methane-, ethane-, propane-, and butane-oxidizing marine archaea with sulfate-reducing bacteria. A recently cultured group of archaea directly couples long-chain alkane degradation with methane formation, expanding the range of substrates used for methanogenesis. This article summarizes the rapidly growing knowledge of the diversity, physiology, and habitat distribution of alkane-degrading archaea.

Bacterial interactions on nutrient-rich surfaces in the gut lumen.

The intestinal lumen is a turbulent, semi-fluid landscape where microbial cells and nutrient-rich particles are distributed with high heterogeneity. Major questions regarding the basic physical structure of this dynamic microbial ecosystem remain unanswered. Most gut microbes are non-motile, and it is unclear how they achieve optimum localization relative to concentrated aggregations of dietary glycans that serve as their primary source of energy. In addition, a random spatial arrangement of cells in this environment is predicted to limit sustained interactions that drive co-evolution of microbial genomes. The ecological consequences of random versus organized microbial localization have the potential to control both the metabolic outputs of the microbiota and the propensity for enteric pathogens to participate in proximity-dependent microbial interactions. Here, we review evidence suggesting that several bacterial species adopt organized spatial arrangements in the gut via adhesion. We highlight examples where localization could contribute to antagonism or metabolic interdependency in nutrient degradation, and we discuss imaging- and sequencing-based technologies that have been used to assess the spatial positions of cells within complex microbial communities.

Promethearchaeum syntrophicum gen. nov., sp. nov., an anaerobic, obligately syntrophic archaeon, the first isolate of the lineage 'Asgard' archaea, and proposal of the new archaeal phylum Promethearchaeota phyl. nov. and kingdom Promethearchaeati regn. nov.

An anaerobic, mesophilic, syntrophic, archaeon strain MK-D1T, was isolated as a pure co-culture with Methanogenium sp. strain MK-MG from deep-sea methane seep sediment. This organism is, to our knowledge, the first cultured representative of 'Asgard' archaea, an archaeal group closely related to eukaryotes. Here, we describe the detailed physiology and phylogeny of MK-D1T and propose Promethearchaeum syntrophicum gen. nov., sp. nov. to accommodate this strain. Cells were non-motile, small cocci, approximately 300-750 nm in diameter and produced membrane vesicles, chains of blebs and membrane-based protrusions. MK-D1T grew at 4-30 °C with optimum growth at 20 °C. The strain grew chemoorganotrophically with amino acids, peptides and yeast extract with obligate dependence on syntrophy with H2-/formate-utilizing organisms. MK-D1T showed the fastest growth and highest maximum cell yield when grown with yeast extract as the substrate: approximately 3 months to full growth, reaching up to 6.7×106 16S rRNA gene copies ml-1. MK-D1T had a circular 4.32 Mb chromosome with a DNA G+C content of 31.1 mol%. The results of phylogenetic analyses of the 16S rRNA gene and conserved marker proteins indicated that the strain is affiliated with 'Asgard' archaea and more specifically DHVC1/DSAG/MBG-B and 'Lokiarchaeota'/'Lokiarchaeia'. On the basis of the results of 16S rRNA gene sequence analysis, the most closely related isolated relatives were Infirmifilum lucidum 3507LTT (76.09 %) and Methanothermobacter tenebrarum RMAST (77.45 %) and the closest relative in enrichment culture was Candidatus 'Lokiarchaeum ossiferum' (95.39 %). The type strain of the type species is MK-D1T (JCM 39240T and JAMSTEC no. 115508). We propose the associated family, order, class, phylum, and kingdom as Promethearchaeaceae fam. nov., Promethearchaeales ord. nov., Promethearchaeia class. nov., Promethearchaeota phyl. nov., and Promethearchaeati regn. nov., respectively. These are in accordance with ICNP Rules 8 and 22 for nomenclature, Rule 30(3)(b) for validation and maintenance of the type strain, and Rule 31a for description as a member of an unambiguous syntrophic association.

Impact of additives on syntrophic propionate and acetate enrichments under high-ammonia conditions.

High ammonia concentrations in anaerobic degradation systems cause volatile fatty acid accumulation and reduced methane yield, which often derive from restricted activity of syntrophic acid-oxidising bacteria and hydrogenotrophic methanogens. Inclusion of additives that facilitate the electron transfer or increase cell proximity of syntrophic species by flocculation can be a suitable strategy to counteract these problems, but its actual impact on syntrophic interactions has yet to be determined. In this study, microbial cultivation and molecular and microscopic analysis were performed to evaluate the impact of conductive (graphene, iron oxide) and non-conductive (zeolite) additives on the degradation rate of acetate and propionate to methane by highly enriched ammonia-tolerant syntrophic cultures derived from a biogas process. All additives had a low impact on the lag phase but resulted in a higher rate of acetate (except graphene) and propionate degradation. The syntrophic bacteria 'Candidatus Syntrophopropionicum ammoniitolerans', Syntrophaceticus schinkii and a novel hydrogenotrophic methanogen were found in higher relative abundance and higher gene copy numbers in flocculating communities than in planktonic communities in the cultures, indicating benefits to syntrophs of living in close proximity to their cooperating partner. Microscopy and element analysis showed precipitation of phosphates and biofilm formation in all batches except on the graphene batches, possibly enhancing the rate of acetate and propionate degradation. Overall, the concordance of responses observed in both acetate- and propionate-fed cultures highlight the suitability of the addition of iron oxide or zeolites to enhance acid conversion to methane in high-ammonia biogas processes. KEY POINTS: • All additives promoted acetate (except graphene) and propionate degradation. • A preference for floc formation by ammonia-tolerant syntrophs was revealed. • Microbes colonised the surfaces of iron oxide and zeolite, but not graphene.

Effect of micro-aeration on syntrophic and methanogenic activity in anaerobic sludge.

Micro-aeration was shown to improve anaerobic digestion (AD) processes, although oxygen is known to inhibit obligate anaerobes, such as syntrophic communities of bacteria and methanogens. The effect of micro-aeration on the activity and microbial interaction in syntrophic communities, as well as on the potential establishment of synergetic relationships with facultative anaerobic bacteria (FAB) or aerobic bacteria (AB), was investigated. Anaerobic sludge was incubated with ethanol and increasing oxygen concentrations (0-5% in the headspace). Assays with acetate or H2/CO2 (direct substrates for methanogens) were also performed. When compared with the controls (0% O2), oxygen significantly decreased substrate consumption and initial methane production rate (MPR) from acetate or H2/CO2. At 0.5% O2, MPR from these substrates was inhibited 30-40%, and close to 100% at 5% O2. With ethanol, significant inhibition (>36%) was only observed for oxygen concentrations higher than 2.5%. Oxygen was consumed in the assays, pointing to the stimulation of AB/FAB by ethanol, which helped to protect the syntrophic consortia under micro-aerobic conditions. This highlights the importance of AB/FAB in maintaining functional and resilient syntrophic communities, which is relevant for real AD systems (in which vestigial O2 amounts are frequently present), as well as for AD systems using micro-aeration as a process strategy. KEY POINTS: •Micro-aeration impacts syntrophic communities of bacteria and methanogens. •Oxygen stimulates AB/FAB, maintaining functional and resilient consortia. •Micro-aeration studies are critical for systems using micro-aeration as a process strategy.

Mechanisms of microbial co-aggregation in mixed anaerobic cultures.

Co-aggregation of anaerobic microorganisms into suspended microbial biofilms (aggregates) serves ecological and biotechnological functions. Tightly packed aggregates of metabolically interdependent bacteria and archaea play key roles in cycling of carbon and nitrogen. Additionally, in biotechnological applications, such as wastewater treatment, microbial aggregates provide a complete metabolic network to convert complex organic material. Currently, experimental data explaining the mechanisms behind microbial co-aggregation in anoxic environments is scarce and scattered across the literature. To what extent does this process resemble co-aggregation in aerobic environments? Does the limited availability of terminal electron acceptors drive mutualistic microbial relationships, contrary to the commensal relationships observed in oxygen-rich environments? And do co-aggregating bacteria and archaea, which depend on each other to harvest the bare minimum Gibbs energy from energy-poor substrates, use similar cellular mechanisms as those used by pathogenic bacteria that form biofilms? Here, we provide an overview of the current understanding of why and how mixed anaerobic microbial communities co-aggregate and discuss potential future scientific advancements that could improve the study of anaerobic suspended aggregates. KEY POINTS: • Metabolic dependency promotes aggregation of anaerobic bacteria and archaea • Flagella, pili, and adhesins play a role in the formation of anaerobic aggregates • Cyclic di-GMP/AMP signaling may trigger the polysaccharides production in anaerobes.

The missing enzymatic link in syntrophic methane formation from fatty acids.

The microbial production of methane from organic matter is an essential process in the global carbon cycle and an important source of renewable energy. It involves the syntrophic interaction between methanogenic archaea and bacteria that convert primary fermentation products such as fatty acids to the methanogenic substrates acetate, H2, CO2, or formate. While the concept of syntrophic methane formation was developed half a century ago, the highly endergonic reduction of CO2 to methane by electrons derived from ß-oxidation of saturated fatty acids has remained hypothetical. Here, we studied a previously noncharacterized membrane-bound oxidoreductase (EMO) from Syntrophus aciditrophicus containing two heme b cofactors and 8-methylmenaquinone as key redox components of the redox loop-driven reduction of CO2 by acyl-coenzyme A (CoA). Using solubilized EMO and proteoliposomes, we reconstituted the entire electron transfer chain from acyl-CoA to CO2 and identified the transfer from a high- to a low-potential heme b with perfectly adjusted midpoint potentials as key steps in syntrophic fatty acid oxidation. The results close our gap of knowledge in the conversion of biomass into methane and identify EMOs as key players of ß-oxidation in (methyl)menaquinone-containing organisms.

Unnatural Direct Interspecies Electron Transfer Enabled by Living Cell-Cell Click Chemistry.

Direct interspecies electron transfer (DIET) is essential for maintaining the function and stability of anaerobic microbial consortia. However, only limited natural DIET modes have been identified and DIET engineering remains highly challenging. In this study, an unnatural DIET between Shewanella oneidensis MR-1 (SO, electron donating partner) and Rhodopseudomonas palustris (RP, electron accepting partner) was artificially established by a facile living cell-cell click chemistry strategy. By introducing alkyne- or azide-modified monosaccharides onto the cell outer surface of the target species, precise covalent connections between different species in high proximity were realized through a fast click chemistry reaction. Remarkably, upon covalent connection, outer cell surface C-type cytochromes mediated DIET between SO and RP was achieved and identified, although this was never realized naturally. Moreover, this connection directly shifted the natural H2 mediated interspecies electron transfer (MIET) to DIET between SO and RP, which delivered superior interspecies electron exchange efficiency. Therefore, this work demonstrated a naturally unachievable DIET and an unprecedented MIET shift to DIET accomplished by cell-cell distance engineering, offering an efficient and versatile solution for DIET engineering, which extends our understanding of DIET and opens up new avenues for DIET exploration and applications.

Heterotrophic denitrification: An overlooked factor that contributes to nitrogen removal in n-DAMO mixed culture.

Nitrate/nitrite-dependent anaerobic methane oxidation (n-DAMO) has been recognized as a sustainable process for simultaneous removal of nitrogen and methane. The metabolisms of denitrifying anaerobic methanotrophs, including Candidatus Methanoperedens and Candidatus Methylomirabilis, have been well studied. However, potential roles of heterotrophs co-existing with these anaerobic methanotrophs are generally overlooked. In this study, we pulse-fed methane and nitrate into an anaerobic laboratory sequencing batch bioreactor and enriched a mixed culture with stable nitrate removal rate (NRR) of ∼28 mg NO3--N L-1 d-1. Microbial community analysis indicates abundant heterotrophs, e.g., Arenimonas (5.3%-18.9%) and Fimbriimonadales ATM1 (6.4%), were enriched together with denitrifying anaerobic methanotrophs Ca. Methanoperedens (10.8%-13.2%) and Ca. Methylomirabilis (27.4%-34.3%). The results of metagenomics and batch tests suggested that the denitrifying anaerobic methanotrophs were capable of generating methane-derived intermediates (i.e., formate and acetate), which were employed by non-methanotrophic heterotrophs for denitrification and biomass growth. These findings offer new insights into the roles of heterotrophs in n-DAMO mixed culture, which may help to optimize n-DAMO process for nitrogen removal from wastewater.

Interspecies Formate Exchange Drives Syntrophic Growth of Syntrophotalea carbinolica and Methanococcus maripaludis.

The complete remineralization of organic matter in anoxic environments relies on communities of microorganisms that ferment organic acids and alcohols to CH4. This is accomplished through syntrophic association of H2 or formate producing bacteria and methanogenic archaea, where exchange of these intermediates enables growth of both organisms. While these communities are essential to Earth's carbon cycle, our understanding of the dynamics of H2 or formate exchanged is limited. Here, we establish a model partnership between Syntrophotalea carbinolica and Methanococcus maripaludis. Through sequencing a transposon mutant library of M. maripaludis grown with ethanol oxidizing S. carbinolica, we found that genes encoding the F420-dependent formate dehydrogenase (Fdh) and F420-dependent methylene-tetrahydromethanopterin dehydrogenase (Mtd) are important for growth. Competitive growth of M. maripaludis mutants defective in either H2 or formate metabolism verified that, across multiple substrates, interspecies formate exchange was dominant in these communities. Agitation of these cultures to facilitate diffusive loss of H2 to the culture headspace resulted in an even greater competitive advantage for M. maripaludis strains capable of oxidizing formate. Finally, we verified that an M. maripaludis Δmtd mutant had a defect during syntrophic growth. Together, these results highlight the importance of formate exchange for the growth of methanogens under syntrophic conditions. IMPORTANCE In the environment, methane is typically generated by fermentative bacteria and methanogenic archaea working together in a process called syntrophy. Efficient exchange of small molecules like H2 or formate is essential for growth of both organisms. However, difficulties in determining the relative contribution of these intermediates to methanogenesis often hamper efforts to understand syntrophic interactions. Here, we establish a model syntrophic coculture composed of S. carbinolica and the genetically tractable methanogen M. maripaludis. Using mutant strains of M. maripaludis that are defective for either H2 or formate metabolism, we determined that interspecies formate exchange drives syntrophic growth of these organisms. Together, these results advance our understanding of the degradation of organic matter in anoxic environments.

Comparative Analysis of Brucepastera parasyntrophica gen. nov., sp. nov. and Teretinema zuelzerae gen. nov., comb. nov. (Treponemataceae) Reveals the Importance of Interspecies Hydrogen Transfer in the Energy Metabolism of Spirochetes.

Most members of the family Treponemataceae (Spirochaetales) are associated with vertebrate hosts. However, a diverse clade of uncultured, putatively free-living treponemes comprising several genus-level lineages is present in other anoxic environments. The only cultivated representative to date is Treponema zuelzerae, isolated from freshwater mud. Here, we describe the isolation of strain RmG11 from the intestinal tract of cockroaches. The strain represents a novel genus-level lineage of Treponemataceae and is metabolically distinct from T. zuelzerae. While T. zuelzerae grows well on various sugars, forming acetate and H2 as major fermentation products, strain RmG11 grew poorly on glucose, maltose, and starch, forming mainly ethanol and only small amounts of acetate and H2. In contrast to the growth of T. zuelzerae, that of strain RmG11 was strongly inhibited at high H2 partial pressures but improved considerably when H2 was removed from the headspace. Cocultures of strain RmG11 with the H2-consuming Methanospirillum hungatei produced acetate and methane but no ethanol. Comparative genomic analysis revealed that strain RmG11 possesses only a single, electron-confurcating hydrogenase that forms H2 from NADH and reduced ferredoxin, whereas T. zuelzerae also possesses a second, ferredoxin-dependent hydrogenase that allows the thermodynamically more favorable formation of H2 from ferredoxin via the Rnf complex. In addition, we found that T. zuelzerae utilizes xylan and possesses the genomic potential to degrade other plant polysaccharides. Based on phenotypic and phylogenomic evidence, we describe strain RmG11 as Brucepastera parasyntrophica gen. nov., sp. nov. and Treponema zuelzerae as Teretinema zuelzerae gen. nov., comb. nov. IMPORTANCE Spirochetes are widely distributed in various anoxic environments and commonly form molecular hydrogen as a major fermentation product. Here, we show that two closely related members of the family Treponemataceae differ strongly in their sensitivity to high hydrogen partial pressure, and we explain the metabolic mechanisms that cause these differences by comparative genome analysis. We demonstrate a strong boost in the growth of the hydrogen-sensitive strain and a shift in its fermentation products to acetate during cocultivation with a H2-utilizing methanogen. Our results add a hitherto unrecognized facet to the fermentative metabolism of spirochetes and also underscore the importance of interspecies hydrogen transfer in not-obligately-syntrophic interactions among fermentative and hydrogenotrophic guilds in anoxic environments.

Evolutionary biology of lichen symbioses.

Lichens are the symbiotic outcomes of open, interspecies relationships, central to which are a fungus and a phototroph, typically an alga and/or cyanobacterium. The evolutionary processes that led to the global success of lichens are poorly understood. In this review, we explore the goods and services exchange between fungus and phototroph and how this propelled the success of both symbiont and symbiosis. Lichen fungal symbionts count among the only filamentous fungi that expose most of their mycelium to an aerial environment. Phototrophs export carbohydrates to the fungus, which converts them to specific polyols. Experimental evidence suggests that polyols are not only growth and respiratory substrates but also play a role in anhydrobiosis, the capacity to survive desiccation. We propose that this dual functionality is pivotal to the evolution of fungal symbionts, enabling persistence in environments otherwise hostile to fungi while simultaneously imposing costs on growth. Phototrophs, in turn, benefit from fungal protection from herbivory and light stress, while appearing to exert leverage over fungal sex and morphogenesis. Combined with the recently recognized habit of symbionts to occur in multiple symbioses, this creates the conditions for a multiplayer marketplace of rewards and penalties that could drive symbiont selection and lichen diversification.

Ecological co-occurrence and soil physicochemical factors drive the archaeal community in Amazonian soils.

We evaluated the co-occurrence of archaeal taxonomic groups and soil physicochemical characteristics in relation to the structuring of the archaeal community in Amazonian soil under different land use systems. Soil samples were collected in primary forest (PF), secondary forest (SF), agricultural systems (AG) and cattle pastures (PA). Archaeal community composition was revealed based on high-throughput amplicon sequencing of the 16S rRNA gene. The results revealed co-occurrence of archaeal classes, with two groups formed: Thaumarchaeota classes, including South Africa Gold Mine-Group 1 (SAGMG-1), Crenarchaeotic group (SCG) and Crenarchaeota candidate division YNPFFA, with predominance in PF and SF; and Bathyarchaeota_unclassified, Methanomicrobia and Methanobacteria (Euryarchaeota) with the FHMa11 terrestrial group, with predominance in PA. The number of co-occurrences between groups was lower in SF, AG and PA (approximately 30%) than in PF. The qPCR analysis revealed that PF also had the largest number of archaeal representatives. Soil texture may be a limiting factor of interactions between groups since the most representative groups, SAGMG-1 and the SCG (over 20% in all sites), were positively associated with coarse sand, the soil factor most correlated with the groups (33% of the total). These results suggest that interactions between archaeal classes belonging to different phyla may be dependent on the number of individuals in the soil environment. In this context, differences in soil physical structure among the land use systems can reduce the representatives of key groups and consequently the co-occurrence of Archaea, which could compromise the natural dynamics of this complex environment.

Downstream resource leakage a necessary condition for the stress-gradient hypothesis in processing chain commensalisms.

The stress-gradient hypothesis (SGH) in ecology predicts that the strength and frequency of positive interspecific interactions, including processing chain commensalisms (PCCs), increase with environmental stress. Although observed in some empirical PCC studies, a recent theoretical study of PCCs using a consumer-resource-type model found that, given the model's assumptions, the SGH never occurs. To investigate if this is a true reflection of PCCs or merely an artefact of the model, in this study, we modified this earlier model formulation by incorporating generalized, monotonically increasing resource uptake functions in place of linear functions, and added a term to represent the spontaneous leakage of the downstream resource to the environment. Mathematical analyses of the model revealed two key insights: 1) the specific algebraic forms of the functional responses of the species in a PCC do not affect the long-term behaviour of the system; 2) the SGH can occur in a facilitative interaction only if the consumer-independent leakage rate of the downstream resource exceeds the consumer-independent input rate. The first insight shows that the outcomes of consumer-resource interactions are robust to details of the functional responses when the functional responses are monotonically increasing, while the second insight shows that the SGH is not a universal feature of positive interactions but instead holds only under a well-defined set of conditions which may vary between PCC interactions and the environmental contexts in which they take place.

Bacteria and Methanogens in the Human Microbiome: a Review of Syntrophic Interactions.

Methanogens are microorganisms belonging to the Archaea domain and represent the primary source of biotic methane. Methanogens encode a series of enzymes which can convert secondary substrates into methane following three major methanogenesis pathways. Initially recognized as environmental microorganisms, methanogens have more recently been acknowledged as host-associated microorganisms after their detection and initial isolation in ruminants in the 1950s. Methanogens have also been co-detected with bacteria in various pathological situations, bringing their role as pathogens into question. Here, we review reported associations between methanogens and bacteria in physiological and pathological situations in order to understand the metabolic interactions explaining these associations. To do so, we describe the origin of the metabolites used for methanogenesis and highlight the central role of methanogens in the syntrophic process during carbon cycling. We then focus on the metabolic abilities of co-detected bacterial species described in the literature and infer from their genomes the probable mechanisms of their association with methanogens. The syntrophic interactions between bacteria and methanogens are paramount to gut homeostasis. Therefore, any dysbiosis affecting methanogens might impact human health. Thus, the monitoring of methanogens may be used as a bio-indicator of dysbiosis. Moreover, new therapeutic approaches can be developed based on their administration as probiotics. We thus insist on the importance of investigating methanogens in clinical microbiology.

Pyrite formation from FeS and H2S is mediated through microbial redox activity.

The exergonic reaction of FeS with H2S to form FeS2 (pyrite) and H2 was postulated to have operated as an early form of energy metabolism on primordial Earth. Since the Archean, sedimentary pyrite formation has played a major role in the global iron and sulfur cycles, with direct impact on the redox chemistry of the atmosphere. However, the mechanism of sedimentary pyrite formation is still being debated. We present microbial enrichment cultures which grew with FeS, H2S, and CO2 as their sole substrates to produce FeS2 and CH4 Cultures grew over periods of 3 to 8 mo to cell densities of up to 2 to 9 × 106 cells per mL-1 Transformation of FeS with H2S to FeS2 was followed by 57Fe Mössbauer spectroscopy and showed a clear biological temperature profile with maximum activity at 28 °C and decreasing activities toward 4 °C and 60 °C. CH4 was formed concomitantly with FeS2 and exhibited the same temperature dependence. Addition of either penicillin or 2-bromoethanesulfonate inhibited both FeS2 and CH4 production, indicating a coupling of overall pyrite formation to methanogenesis. This hypothesis was supported by a 16S rRNA gene-based phylogenetic analysis, which identified at least one archaeal and five bacterial species. The archaeon was closely related to the hydrogenotrophic methanogen Methanospirillum stamsii, while the bacteria were most closely related to sulfate-reducing Deltaproteobacteria, as well as uncultured Firmicutes and Actinobacteria. Our results show that pyrite formation can be mediated at ambient temperature through a microbially catalyzed redox process, which may serve as a model for a postulated primordial iron-sulfur world.

Symposium review: Understanding the role of the rumen microbiome in enteric methane mitigation and productivity in dairy cows.

Ruminants are one of the largest sources of global CH4 emissions. This enteric CH4 is exclusively produced by methanogenic archaea as a natural product during microbial fermentation in the reticulorumen. As CH4 formation leads to a gross energy loss for the ruminant host and is also an environmental issue, several CH4 mitigation approaches have been investigated, but results have been inconsistent, which may be partially attributed to a lack of understanding of the mechanistic basis of methanogenesis and the effect of inhibitors on individual methanogenic lineages and other fermenting microbes in the rumen. Methanogenic archaea are obligatory anaerobes that can reduce CO2, methanol, or methylamines or cleave acetate to form CH4. Although methanogens work toward a common goal of generating energy through the formation of CH4, individual methanogenic lineages differ in their physiological and metabolic capabilities, which can differentially affect H2 transactions and CH4 formation. Using advanced omic approaches, recent research has revealed that less abundant methanol-utilizing Methanosphaera and methylamine- and methanol-utilizing Methanomassiliicoccales lineages are positively correlated with CH4 emissions and may have a greater share in overall CH4 production compared with more abundant CO2-reducing methanogens than previously thought. These data imply that the diversity as well as the abundance of methanogens is important in CH4 formation, and that this diversity is influenced by H2 availability and interactions within and between H2-producing microbes in the rumen. These complex interactions between microbes and H2 are further influenced by variations in dietary, host, and environmental conditions. This review discusses critical knowledge gaps underlying methanogen diversity and its link to CH4 formation, formation of specific bacteria-archaeal cohorts, and how H2 production and utilization are regulated between these cohorts during normal and inhibited methanogenesis. Addressing these knowledge gaps has the potential to lead to the development of novel strategies or to complement existing strategies to effectively reduce CH4 formation while also improving productivity in dairy cows.

Mapping the biological activities of filamentous oxygenic photogranules.

Oxygenic photogranules have been suggested as alternatives to activated sludge in wastewater treatment. Challenging for modeling photogranule-based processes is the heterogeneity of photogranule morphologies, resulting in different activities by photogranule type. The measurement of microscale-activities of filamentous photogranules is particularly difficult because of their labile interfaces. We present here an experimental and modeling approach to quantify phototrophic O2 production, heterotrophic O2 consumption, and O2 diffusion in filamentous photogranules. We used planar optodes for the acquisition of spatio-temporal oxygen distributions combined with two-dimensional mathematical modeling. Light penetration into the photogranule was the factor controlling photogranule activities. The spatial distribution of heterotrophs and phototrophs had less impact. The photosynthetic response of filaments to light was detectable within seconds, emphasizing the need to analyze dynamics of light exposure of individual photogranules in photobioreactors. Studying other recurring photogranule morphologies will eventually enable the description of photogranule-based processes as the interplay of interacting photogranule populations.

In Situ Spectroelectrochemical Characterization Reveals Cytochrome-Mediated Electric Syntrophy in Geobacter Coculture.

Direct interspecies electron transfer (DIET) between microbial species prevails in some key microbial consortia. However, the electron transfer mechanism(s) in these consortia is controversial due to lack of efficient characterization methods. Here, we provide an in situ anaerobic spectroelectrochemical coculture cell (in situ ASCC) to induce the formation of DIET coculture biofilm on the interdigitated microelectrode arrays and characterize the electron transfer directly. Two typical Geobacter DIET cocultures, Geobacter metallireducens and wild-type Geobacter sulfurreducens (G.m&G.s) and G. metallireducens and a G. sulfurreducens strain deficient in citrate synthase (G.m&G.s-ΔgltA), were selected. In situ Raman and electrochemical Fourier transform infrared (FTIR) spectroscopy indicated that cytochromes are abundant in the electric syntrophic coculture. Cyclic voltammetry and potential step experiment revealed a diffusion-controlled electron transfer process and the electrochemical gating measurements further demonstrated a cytochrome-mediated electron transfer in the DIET coculture. Furthermore, the G.m&G.s-ΔgltA coculture displayed a higher redox conductivity than the G.m&G.s coculture, consistent with the existence of an intimate and efficient electrical connection between these two species. Our findings provide the first report of a redox-gradient-driven electron transport facilitated by c-type cytochromes in DIET coculture, supporting the model that DIET is mediated by cytochromes and suggest a platform to explore the other DIET consortia.

Patterns of syntrophic interactions in methanogenic conversion of propionate.

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.