CO2 supply is a powerful tool to control homoacetogenesis, chain elongation and solventogenesis in ethanol and carboxylate fed reactor microbiomes.

Anaerobic fermentation technology enables the production of medium chain carboxylates and alcohols through microbial chain elongation. This involves steering reactor microbiomes to yield desired products, with CO2 supply playing a crucial role in controlling ethanol-based chain elongation and facilitating various bioprocesses simultaneously. In the absence of CO2 supply (Phase I), chain elongation predominantly led to n-caproate with a high selectivity of 96 Cmol%, albeit leaving approximately 80% of ethanol unconverted. During this phase, C. kluyveri and Proteiniphilum-related species dominated the reactors. In Phase II, with low CO2 input (2.0 NmL L-1 min-1), formation of n-butyrate, butanol, and hexanol was stimulated. Increasing CO2 doses in Phase III (6 NmL L-1 min-1) led to CO2 utilization via homoacetogenesis, coinciding with the enrichment of Clostridium luticellarii, a bacterium that can use CO2 as an electron acceptor. Lowering CO2 dose to 0.5 NmL L-1 min-1 led to a shift in microbiome composition, diminishing the dominance of C. luticellarii while increasing C. kluyveri abundance. Additionally, other Clostridia, Proteiniphilum, and Lactobacillus sakei-related species became prevalent. This decrease in CO2 load from 6 to 0.5 NmL L-1 min-1 minimized excessive ethanol oxidation from 30%-50% to 0%-3%, restoring a microbiome favoring net n-butyrate consumption and n-caproate production. The decreased ethanol oxidation coincided with the resurgence of hydrogen formation at partial pressures above 1%. High concentrations of butyrate, caproate, and ethanol in the reactor, along with low acetate concentration, promoted the formation of butanol and hexanol. It is evident that CO2 supply is indispensable for controlling chain elongation in an open culture and it can be harnessed to stimulate higher alcohol formation or induce CO2 utilization as an electron acceptor.

Understanding the Role of Free-Living Bacteria in the Gut of the Lower Termite Coptotermes gestroi Based on Metagenomic DNA Analysis.

Termites' digestive systems, particularly in lower termites with the presence of protozoa, are unique ecological niches that shelter a diverse microbiota with a variety of functions for the host and the environment. In 2012, the metagenomic DNA (5.4 Gb) of the prokaryotes that freely live in the gut of the lower termite Coptotermes gestroi were sequenced. A total of 125,431 genes were predicted and analyzed in order to mine lignocellulolytic genes. however, the overall picture of the structure, diversity, and function of the prokaryotic gut microbiota was not investigated. In the present study, these 125,431 genes were taxonomically classified by MEGAN and functionally annotated by the Kyoto Encyclopedia of Genes and Genomes (KEGG) and by the Carbohydrate-Active enZYmes (CAZy) and HMMER databases. As a result, 95,751 bacterial genes were classified into 35 phyla. The structure of the bacteria, typified by a high ratio of Firmicutes to Bacterioidetes, was distinct from the structure of the entirety of the bacteria in the lower or higher termites' guts. The archaea (533 genes) were distributed into 4 phyla, 10 classes, 15 orders, 21 families, 47 genera, and 61 species. Although freely living in the guts, the prokaryotic community was formed, developed, and adapted to exhibit unique interactions in order to perform mutual roles of benefit to their hosts. Methanobacteriales, accounting for 61% of the archaea symbionts, seem to play an important role in methanogenesis. Concomitantly, bacterial methanotrophs in the gut utilize methane and combine with other bacterial groups, including potential lignocellulolytic degraders, acetogens, sulfur bacteria, and nitrogen-recycling bacteria, to efficiently convert wood with little nitrogen into acetates via certain pathway modules specified by prokaryotes that freely live in the gut. This forms an important energy source for the termites. Furthermore, bacteria carry 2223 genes involved in the biosynthesis of 17 antibiotic groups. The gut bacteria also possess genes for the degradation of 18 toxic aromatic compounds, of which four are commercial pesticides against termites commonly used for the preservation of wooden constructions. Eight of the eighteen pathways were the first to be reported from the termite gut. Overall, this study sheds light on the roles of the freely living bacteria and archaea in the C. gestroi gut, providing evidence that the gut microbiome acts as the second host genome, contributing both nutrients and immunity to support the host's existence, growth, and development.

Sequential bioconversion of C1-gases (CO, CO2, syngas) into lipids, through the carboxylic acid platform, with Clostridium aceticum and Rhodosporidium toruloides.

Syngas (CO, CO2, H2) was effectively bioconverted into lipids in a two-stage process. In the first stage, C1-gases were bioconverted into acetic acid by the acetogenic species Clostridium aceticum through the Wood-Ljungdahl metabolic pathway in a stirred tank bioreactor, reaching a maximum acetic acid concentration of 11.5 g/L, with a production rate of 0.05 g/L·h. Throughout this experiment, samples were extracted at different periods, i.e., different concentrations, to be used in the second stage, aiming at the production of lipids from acetic acid. The yeast Rhodosporidium toruloides, inoculated in the acetogenic medium, was able to efficiently accumulate lipids from acetic acid generated in the first stage. The best results, in terms of lipid content, dry biomass, biomass yield (Y(X/S)) and lipid yield (Y(L/S)) were 39.5% g/g dry cell weight, 3 g/L, 0.35 and 0.107, respectively. In terms of abundance, the lipid profile followed the order: C18:1 > C16:0 > C18:2 > C18:0 > Others. Experiments were also performed to determine the toxicity exerted by high concentrations of acetic acid on R. toruloides, resulting in inhibition at initial acid concentrations around 18 g/L leading to a higher lag phase and being lethal to the yeast at initial acetic acid concentrations around 22 g/L and above. This research paves the way for a novel method of growing oleaginous yeasts to produce sustainable biofuels from syngas or C1-pollutant gases.

Carbon dioxide reduction to high-value chemicals in microbial electrosynthesis system: Biological conversion and regulation strategies.

Large emissions of atmospheric carbon dioxide (CO2) are causing climatic and environmental problems. It is crucial to capture and utilize the excess CO2 through diverse methods, among which the microbial electrosynthesis (MES) system has become an attractive and promising technology to mitigate greenhouse effects while reducing CO2 to high-value chemicals. However, the biological conversion and metabolic pathways through microbial catalysis have not been clearly elucidated. This review first introduces the main acetogenic bacteria for CO2 reduction and extracellular electron transfer mechanisms in MES. It then intensively analyzes the CO2 bioconversion pathways and carbon chain elongation processes in MES, together with energy supply and utilization. The factors affecting MES performance, including physical, chemical, and biological aspects, are summarized, and the strategies to promote and regulate bioconversion in MES are explored. Finally, challenges and perspectives concerning microbial electrochemical carbon sequestration are proposed, and suggestions for future research are also provided. This review provides theoretical foundation and technical support for further development and industrial application of MES for CO2 reduction.

Isolation and characterization of reductive acetogens from rumen fluid samples of Murrah buffaloes.

In the present study, attempts have been made to isolate reductive acetogens from the rumen fluid samples of Murrah buffaloes (Bubalus bubalis). Out of 32 rumen samples 51 isolates were isolated, and based on autotrophic growth for production of acetate and presence of formyltetrahydrofolate synthetase gene (FTHFS) 12 isolates were confirmed as reductive acetogens. Microscopic observations showed that ten isolates as Gram-positive rods (ACB28, ACB29, ACB66, ACB73, ACB81, ACB91, ACB133, ACB229, ACB52, ACB95) and two isolates as Gram-positive cocci (ACB19, ACB89). All isolates tested negative for catalase, oxidase, and gelatin liquefaction, whereas the production of H2S was detected for two (ACB52 and ACB95) of the above isolates. All these isolates showed autotrophic growth from H2 and CO2, and heterotrophic growth with different fermentable sugars, viz., d-glucose, D-fructose, and D-trehalose but failed to grow on salicin, raffinose, and l-rhamnose. Out of the isolates, two showed amylase activity (ACB28 and ACB95), five showed CMCase activity (ACB19, ACB28, ACB29, ACB73 and ACB91), three showed pectinase activity (ACB29, ACB52 and ACB89), whereas none of the isolates was found positive for avicellase and xylanase activity. Based on 16S rDNA gene sequence analysis, the isolates showed their phylogenetic relationship with maximum similarity up to 99% to different strains of earlier reported known acetogens of clostridia group including Clostridium sp. (6), Eubacterium limosum (1), Ruminococcus sp. (1) and Acetobacterium woodii (1) except one, i.e., Vagococcus fluvialis. The results indicate that reductive acetogens isolated from the rumen fluid samples of Murrah buffalos are both autotrophic and heterotrophic in nature and further investigations are required to exploit and explore their potential as an alternate hydrogen sink.

Acetogen and acetogenesis for biological syngas valorization.

The bioconversion of syngas using (homo)acetogens as biocatalysts shows promise as a viable option due to its higher selectivity and milder reaction conditions compared to thermochemical conversion. The current bioconversion process operates primarily to produce C2 chemicals (e.g., acetate and ethanol) with sufficient technology readiness levels (TRLs) in process engineering (as midstream) and product purification (as downstream). However, the economic feasibility of this process could be improved with greater biocatalytic options in the upstream phase. This review focuses on the Wood-Ljungdahl pathway (WLP) which is a biological syngas-utilization pathway, redox balance and ATP generation, suggesting that the use of a specific biocatalysts including Eubacterium limosum could be advantageous in syngas valorization. A pertinent strategy to mainly produce chemicals with a high degree of reduction is also provided with examples of flux control, mixed cultivation and mixotrophy. Finally, this article presents future direction of industrial utilization of syngas fermentation.

Deep Subseafloor Biogeochemical Processes and Microbial Populations Potentially Associated with the 2011 Tohoku-oki Earthquake at the Japan Trench Accretionary Wedge (IODP Expedition 343).

Post-mega-earthquake geochemical and microbiological properties in subseafloor sediments of the Japan Trench accretionary wedge were investigated using core samples from Hole C0019E, which was drilled down to 851| |m below seafloor (mbsf) at a water depth of 6,890 m. Methane was abundant throughout accretionary prism sediments; however, its concentration decreased close to the plate boundary decollement. Methane isotope systematics indicated a biogenic origin. The content of mole-cular hydrogen (H2) was low throughout core samples, but markedly increased at specific depths that were close to potential faults predicted by logging-while-drilling ana-lyses. Based on isotopic systematics, H2 appeared to have been abundantly produced via a low-temperature interaction between pore water and the fresh surface of crushed rock induced by earthquakes. Subseafloor microbial cell density remained constant at approximately 105| |cells| |mL-1. Amplicon sequences revealed that predominant members at the phylum level were common throughout the units tested, which also included members frequently found in anoxic subseafloor sediments. Metabolic potential assays using radioactive isotopes as tracers revealed homoacetogenic activity in H2-enriched core samples collected near the fault. Furthermore, homoacetogenic bacteria, including Acetobacterium carbinolicum, were isolated from similar samples. Therefore, post-earthquake subseafloor microbial communities in the Japan Trench accretionary prism appear to be episodically dominated by homoacetogenic populations and potentially function due to the earthquake-induced low-temperature generation of H2. These post-earthquake microbial communities may eventually return to the steady-state communities dominated by oligotrophic heterotrophs and hydrogenotrophic and methylotrophic methanogens that are dependent on refractory organic matter in the sediment.

Obligate autotrophy at the thermodynamic limit of life in a new acetogenic bacterium.

One of the important current issues of bioenergetics is the establishment of the thermodynamic limits of life. There is still no final understanding of what is the minimum value of the energy yield of a reaction that is sufficient to be used by an organism (the so-called "biological quantum of energy"). A reasonable model for determination of the minimal energy yield would be microorganisms capable of living on low-energy substrates, such as acetogenic prokaryotes. The most prominent metabolic feature of acetogens is autotrophic growth with molecular hydrogen and carbon dioxide as the substrates, which is hardly competitive in environments. Most probably, that is why only facultative autotrophic acetogens have been known so far. Here, we describe the first obligately autotrophic acetogenic bacterium Aceticella autotrophica gen. nov., sp. nov., strain 3443-3AcT. Phylogenetically, the new genus falls into a monophyletic group of heterotrophic bacteria of the genera Thermoanaerobacterium, Thermoanaerobacter, and Caldanaerobacter (hereinafter referred to as TTC group), where the sole acetogenic representative has so far been the facultatively autotrophic Thermoanaerobacter kivui. A. autotrophica and T. kivui both are acetogens employing energy-converting hydrogenase (Ech-acetogens) that are likely to have inherited the acetogenesis capacity vertically from common ancestor. However, their acetogenic machineries have undergone different adjustments by gene replacements due to horizontal gene transfers from different donors. Obligate autotrophy of A. autotrophica is associated with the lack of many sugar transport systems and carbohydrate catabolism enzymes that are present in other TTC group representatives, including T. kivui.

Anaerobic degradation of organic carbon supports uncultured microbial populations in estuarine sediments.

BACKGROUND: A large proportion of prokaryotic microbes in marine sediments remains uncultured, hindering our understanding of their ecological functions and metabolic features. Recent environmental metagenomic studies suggested that many of these uncultured microbes contribute to the degradation of organic matter, accompanied by acetogenesis, but the supporting experimental evidence is limited. RESULTS: Estuarine sediments were incubated with different types of organic matters under anaerobic conditions, and the increase of uncultured bacterial populations was monitored. We found that (1) lignin stimulated the increase of uncultured bacteria within the class Dehalococcoidia. Their ability to metabolize lignin was further supported by the presence of genes associated with a nearly complete degradation pathway of phenolic monomers in the Dehalococcoidia metagenome-assembled genomes (MAGs). (2) The addition of cellulose stimulated the increase of bacteria in the phylum Ca. Fermentibacterota and family Fibrobacterales, a high copy number of genes encoding extracellular endoglucanase or/and 1,4-beta-cellobiosidase for cellulose decomposition and multiple sugar transporters were present in their MAGs. (3) Uncultured lineages in the order Bacteroidales and the family Leptospiraceae were enriched by the addition of casein and oleic acid, respectively, a high copy number of genes encoding extracellular peptidases, and the complete ß-oxidation pathway were found in those MAGs of Bacteroidales and Leptospiraceae, respectively. (4) The growth of unclassified bacteria of the order Clostridiales was found after the addition of both casein and cellulose. Their MAGs contained multiple copies of genes for extracellular peptidases and endoglucanase. Additionally, 13C-labeled acetate was produced in the incubations when 13C-labeled dissolved inorganic carbon was provided. CONCLUSIONS: Our results provide new insights into the roles of microorganisms during organic carbon degradation in anaerobic estuarine sediments and suggest that these macro and single molecular organic carbons support the persistence and increase of uncultivated bacteria. Acetogenesis is an additional important microbial process alongside organic carbon degradation. Video Abstract.

Novel quantitative method for individual isotopomer of organic acids from 13C tracer experiments determines carbon flow in acetogenesis.

Anaerobic microbial acetogenesis is ubiquitous on Earth, and thus plays an important role in the global carbon cycle. The mechanism of carbon fixation in acetogens has attracted great interest from various studies for combatting climate change, and even for studying ancient metabolic pathways. Here, we developed a new, simple method for investigating carbon flows in the metabolic reaction of acetogen by conveniently and accurately determining the relative abundance of individual acetate- and/or formate-isotopomers formed in 13C labeling experiments. We measured the underivatized analyte by gas chromatography-mass spectrometry (GC-MS) coupled with a direct aqueous sample injection technique. The individual abundance of analyte isotopomers was calculated by the mass spectrum analysis using the least-squares approach. The validity of the method was demonstrated by determining known mixtures of unlabeled and 13C-labeled analytes. The developed method was applied to study the carbon fixation mechanism of the well-known acetogen Acetobacterium woodii grown on methanol and bicarbonate. We provided a quantitative reaction model for methanol metabolism of A. woodii, which indicated that methanol was not the sole carbon precursor of the acetate methyl group and that 20-22% of the methyl group was formed from CO2. In contrast, the carboxyl group of acetate appeared to form exclusively by CO2 fixation. Thus, our simple method, without laborious analytical procedures, has broad utility for the study of biochemical and chemical processes related to acetogenesis on Earth.

Enrichment of homoacetogens converting H2/CO2 into acids and ethanol and simultaneous methane production.

An anaerobic granular sludge was enriched to utilize H2/CO2 in a continuous gas-fed up-flow anaerobic sludge reactor by applying operating conditions expected to produce acetic acid, butyric acid, and ethanol. Three stages of fermentation were found: Stage I with acetic acid accumulation with the highest concentration of 35 mM along with a pH decrease from initial 6 to 4.5. In Stage II, H2/CO2 was replaced by 100% H2 to induce solventogenesis, whereas butyric acid was produced with the highest concentration of 2.5 mM. At stage III with 10 µM tungsten (W) addition, iso-valeric acid, valeric acid, and caproic acid were produced at pH 4.5-5.0. In the batch tests inoculated with the enriched sludge taken from the bioreactor (day 70), however, methane production occurred at pH 6. Exogenous 15 mM acetate addition enhanced both the H2 and CO2 consumption rate compared to exogenous 10, 30, and 45 mM acetate by the enriched sludge. Exogenous acetate was failed to be converted to ethanol using H2 as electron donor by the enriched acetogens.

Mechanism of action of single and mixed antibiotics during anaerobic digestion of swine wastewater: Microbial functional diversity and gene expression analysis.

The mechanism by which antibiotics in swine wastewater affect anaerobic digestion (AD) remains unclear. Herein, we investigated how single and mixed antibiotics affect AD in swine wastewater. Both single and mixed antibiotics stimulated methane production at actual concentrations of 0.5-2 mg/L. Low-dose antibiotics (0.5 mg/L) exerted the most significant stimulatory effect on methane production, which increased by 211.63% (single) and 60.93% (mixed), respectively. However, an increased dose decreased the stimulatory effect on methane production. Overall, single antibiotics were more beneficial for methane production than mixed antibiotics since single antibiotics could promote the conversion of propionic and butyric acid, while mixed antibiotics inhibited the process. Microbial community analysis showed that single and mixed antibiotics could also lead to large changes in functional acidogens, ultimately leading to changes in methanogenic pathways.

Anaerobic digestion of fruit and vegetable waste: a critical review of associated challenges.

The depletion of fossil fuels coupled with stringent environmental laws has encouraged us to develop sustainable renewable energy. Due to its numerous benefits, anaerobic digestion (AD) has emerged as an environment-friendly technology. Biogas generated during AD is primarily a mixture of CH4 (65-70%) and CO2 (20-25%) and a potent energy source that can combat the energy crisis in today's world. Here, an attempt has been made to provide a broad understanding of AD and delineate the effect of various operational parameters influencing AD. The characteristics of fruit and vegetable waste (FVW) and its feasibility as a potent substrate for AD have been studied. This review also covers traditional challenges in managing FVW via AD, the implementation of various bioreactor systems to manage large amounts of organic waste and their operational boundaries, microbial consortia involved in each phase of digestion, and various strategies to increase biogas production.

Microbial electrosynthesis of acetate from CO2 under hypersaline conditions.

Microbial electrosynthesis (MES) enables the bioproduction of multicarbon compounds from CO2 using electricity as the driver. Although high salinity can improve the energetic performance of bioelectrochemical systems, acetogenic processes under elevated salinity are poorly known. Here MES under 35-60 g L-1 salinity was evaluated. Acetate production in two-chamber MES systems at 35 g L-1 salinity (seawater composition) gradually decreased within 60 days, both under -1.2 V cathode potential (vs. Ag/AgCl) and -1.56 A m-2 reductive current. Carbonate precipitation on cathodes (mostly CaCO3) likely declined the production through inhibiting CO2 supply, the direct electrode contact for acetogens and H2 production. Upon decreasing Ca2+ and Mg2+ levels in three-chamber reactors, acetate was stably produced over 137 days along with a low cathode apparent resistance at 1.9 ± 0.6 mΩ m2 and an average production rate at 3.80 ± 0.21 g m-2 d-1. Increasing the salinity step-wise from 35 to 60 g L-1 gave the most efficient acetate production at 40 g L-1 salinity with average rates of acetate production and CO2 consumption at 4.56 ± 3.09 and 7.02 ± 4.75 g m-2 d-1, respectively. The instantaneous coulombic efficiency for VFA averaged 55.1 ± 31.4%. Acetate production dropped at higher salinity likely due to the inhibited CO2 dissolution and acetogenic metabolism. Acetobacterium up to 78% was enriched on cathodes as the main acetogen at 35 g L-1. Under high-salinity selection, 96.5% Acetobacterium dominated on the cathode along with 34.0% Sphaerochaeta in catholyte. This research provides a first proof of concept that MES starting from CO2 reduction can be achieved at elevated salinity.

Biohythane: a Potential Biofuel of the Future.

Today, the world is becoming more dependent on fossil fuels. The major drawbacks of these non-renewable energy resources include an extreme environmental pollution and an extinction threat. Several technologies including microalgal biodiesel production, biomass gasification, and bioethanol production have been explored for the generation of renewable energy especially, biofuels. One such promising research has been carried out in the generation of biohythane which has the potential to become an alternative fuel to the existing non-renewable ones. It has been reported that biohydrogen can be produced from organic wastes or agricultural feedstocks with the help of acidogens. Dark fermentation can be carried out by acidogens to produce biohydrogen under anaerobic conditions by utilizing lignocellulosic biomass or sugarcane feedstocks in the absence of light. The spent medium contains volatile short-chain fatty acids like acetate, butyrate, and propionate that can serve as substrates for acetogenesis followed by methane biosynthesis by methanogens. Therefore, the sequential two-stage anaerobic digestion (AD) involves a production of biohydrogen followed by the biosynthesis of methane. This combined process is termed as a single eponym "Biohythane" (hydrogen + methane). Several studies have demonstrated about the effectiveness of biofuel, and it is believed to have a greater energy recovery, environmental friendliness, and shorter fermentation time. Biohythane can serve as an alternative future green biofuel and solve the present energy crisis in India as well as the entire world.

Deep-branching acetogens in serpentinized subsurface fluids of Oman.

Little is known of acetogens in contemporary serpentinizing systems, despite widely supported theories that serpentinite-hosted environments supported the first life on Earth via acetogenesis. To address this knowledge gap, genome-resolved metagenomics was applied to subsurface fracture water communities from an area of active serpentinization in the Samail Ophiolite, Sultanate of Oman. Two deeply branching putative bacterial acetogen types were identified in the communities belonging to the Acetothermia (hereafter, types I and II) that exhibited distinct distributions among waters with lower and higher water-rock reaction (i.e., serpentinization influence), respectively. Metabolic reconstructions revealed contrasting core metabolic pathways of type I and II Acetothermia, including in acetogenic pathway components (e.g., bacterial- vs. archaeal-like carbon monoxide dehydrogenases [CODH], respectively), hydrogen use to drive acetogenesis, and chemiosmotic potential generation via respiratory (type I) or canonical acetogen ferredoxin-based complexes (type II). Notably, type II Acetothermia metabolic pathways allow for use of serpentinization-derived substrates and implicate them as key primary producers in contemporary hyperalkaline serpentinite environments. Phylogenomic analyses indicate that 1) archaeal-like CODH of the type II genomes and those of other serpentinite-associated Bacteria derive from a deeply rooted horizontal transfer or origin among archaeal methanogens and 2) Acetothermia are among the earliest evolving bacterial lineages. The discovery of dominant and early-branching acetogens in subsurface waters of the largest near-surface serpentinite formation provides insight into the physiological traits that likely facilitated rock-supported life to flourish on a primitive Earth and possibly on other rocky planets undergoing serpentinization.

The root zone of graminoids: A niche for H2-consuming acetogens in a minerotrophic peatland.

The importance of acetogens for H2 turnover and overall anaerobic degradation in peatlands remains elusive. In the well-studied minerotrophic peatland fen Schlöppnerbrunnen, H2-consuming acetogens are conceptualized to be largely outcompeted by iron reducers, sulfate reducers, and hydrogenotrophic methanogens in bulk peat soil. However, in root zones of graminoids, fermenters thriving on rhizodeposits and root litter might temporarily provide sufficient H2 for acetogens. In the present study, root-free peat soils from around the roots of Molinia caerulea and Carex rostrata (i.e., two graminoids common in fen Schlöpnnerbrunnen) were anoxically incubated with or without supplemental H2 to simulate conditions of high and low H2 availability in the fen. In unsupplemented soil treatments, H2 concentrations were largely below the detection limit (∼10 ppmV) and possibly too low for acetogens and methanogens, an assumption supported by the finding that neither acetate nor methane substantially accumulated. In the presence of supplemental H2, acetate accumulation exceeded CH4 accumulation in Molinia soil whereas acetate and methane accumulated equally in Carex soil. However, reductant recoveries indicated that initially, additional unknown processes were involved either in H2 consumption or the consumption of acetate produced by H2-consuming acetogens. 16S rRNA and 16S rRNA gene analyses revealed that potential acetogens (Clostridium, Holophagaceae), methanogens (Methanocellales, Methanobacterium), iron reducers (Geobacter), and physiologically uncharacterized phylotypes (Acidobacteria, Actinobacteria, Bacteroidetes) were stimulated by supplemental H2 in soil treatments. Phylotypes closely related to clostridial acetogens were also active in soil-free Molinia and Carex root treatments with or without supplemental H2. Due to pronounced fermentation activities, H2 consumption was less obvious in root treatments, and acetogens likely thrived on root organic carbon and fermentation products (e.g., ethanol) in addition to H2. Collectively, the data highlighted that in fen Schlöppnerbrunnen, acetogens are associated to graminoid roots and inhabit the peat soil around the roots, where they have to compete for H2 with methanogens and iron reducers. Furthermore, the study underscored that the metabolically flexible acetogens do not rely on H2, potentially a key advantage over other H2 consumers under the highly dynamic conditions characteristic for the root-zones of graminoids in peatlands.

Metabolic Strategies Shared by Basement Residents of the Lost City Hydrothermal Field.

Alkaline fluids venting from chimneys of the Lost City hydrothermal field flow from a potentially vast microbial habitat within the seafloor where energy and organic molecules are released by chemical reactions within rocks uplifted from Earth's mantle. In this study, we investigated hydrothermal fluids venting from Lost City chimneys as windows into subseafloor environments where the products of geochemical reactions, such as molecular hydrogen (H2), formate, and methane, may be the only available sources of energy for biological activity. Our deep sequencing of metagenomes and metatranscriptomes from these hydrothermal fluids revealed a few key species of archaea and bacteria that are likely to play critical roles in the subseafloor microbial ecosystem. We identified a population of Thermodesulfovibrionales (belonging to phylum Nitrospirota) as a prevalent sulfate-reducing bacterium that may be responsible for much of the consumption of H2 and sulfate in Lost City fluids. Metagenome-assembled genomes (MAGs) classified as Methanosarcinaceae and Candidatus Bipolaricaulota were also recovered from venting fluids and represent potential methanogenic and acetogenic members of the subseafloor ecosystem. These genomes share novel hydrogenases and formate dehydrogenase-like sequences that may be unique to hydrothermal environments where H2 and formate are much more abundant than carbon dioxide. The results of this study include multiple examples of metabolic strategies that appear to be advantageous in hydrothermal and subsurface alkaline environments where energy and carbon are provided by geochemical reactions. IMPORTANCE The Lost City hydrothermal field is an iconic example of a microbial ecosystem fueled by energy and carbon from Earth's mantle. Uplift of mantle rocks into the seafloor can trigger a process known as serpentinization that releases molecular hydrogen (H2) and creates unusual environmental conditions where simple organic carbon molecules are more stable than dissolved inorganic carbon. This study provides an initial glimpse into the kinds of microbes that live deep within the seafloor where serpentinization takes place, by sampling hydrothermal fluids exiting from the Lost City chimneys. The metabolic strategies that these microbes appear to be using are also shared by microbes that inhabit other sites of serpentinization, including continental subsurface environments and natural springs. Therefore, the results of this study contribute to a broader, interdisciplinary effort to understand the general principles and mechanisms by which serpentinization-associated processes can support life on Earth and perhaps other worlds.

Biosynthesis of butyrate from methanol and carbon monoxide by recombinant Acetobacterium woodii / Biosíntesis de butirato a partir de metanol y monóxido de carbono por Acetobacterium woodii recombinante

Methanol is one of the most widely produced organic substrates from syngas and can serve as a bio-feedstock to cultivate acetogenic bacteria which allows a major contribution to reducing greenhouse gas. Acetobacterium woodii is one of the very few acetogens that can utilize methanol to produce acetate as sole product. Since A. woodii is genetically tractable, it is an interesting candidate to introduce recombinant pathways for production of bio-commodities from methanol. In this study, we introduced the butyrate production operon from a related acetogen, Eubacterium callanderi KIST612, into A. woodii and show a stable production of butyrate from methanol. This study also reveals how butyrate production by recombinant A. woodii strains can be enhanced with addition of electrons in the form of carbon monoxide. Our results not only show a stable expression system of non-native enzymes in A. woodii but also increase in the product spectrum of A. woodii to compounds with higher economic value.(AU)

Proposed protocol for rate-limiting step determination during anaerobic digestion of complex substrates.

Anaerobic digestion is a complex process, involving various microorganism groups and, consequently, several reactions. An easy-to-use protocol for the rate-limiting step determination of the process is proposed. The hydrogen production, acetate production, and acetate consumption rates can be calculated, according to a structured algorithm. During the rate limiting step determination, several compounds (biopolymer and monomer representatives, as well as sodium acetate) were used, combined or not with the substrate, to draw the corresponding conclusions. Three substrates were tested, characterized by specific organic compound groups (carbohydrates, proteins, and fats). All three substrates followed the acetate-consuming pathway for the organic matter conversion to methane. In this study, the rate-limiting step for the pathway of acetate consumption was acetate production. Determining the rate-limiting step through the proposed protocol can point to the appropriate actions needed to boost methane production, like substrate pretreatment, using an acidogenic reactor, or checking for the presence of inhibitors.