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Nitrous oxide (N2O), a potent greenhouse gas, can be generated by multiple biological and abiotic processes in diverse contexts. Accurately tracking the dominant sources of N2O has the potential to improve our understanding of N2O fluxes from soils as well as inform the diagnosis of human infections. Isotopic Site Preference (SP) values have been used toward this end, as bacterial and fungal nitric oxide reductases (NORs) produce N2O with different isotopic fingerprints, spanning a large range. Here, we show that flavohemoglobin (Fhp), a hitherto biogeochemically neglected yet widely distributed detoxifying bacterial NO reductase, imparts a distinct SP value onto N2O under anoxic conditions (~+10‰) that correlates with typical environmental N2O SP measurements. Using Pseudomonas aeruginosa as a model organism, we generated strains that only contained Fhp or the dissimilatory NOR, finding that in vivo N2O SP values imparted by these enzymes differ by over 10‰. Depending on the cellular physiological state, the ratio of Fhp:NOR varies significantly in wild-type cells and controls the net N2O SP biosignature: When cells grow anaerobically under denitrifying conditions, NOR dominates; when cells experience rapid, increased nitric oxide concentrations under anoxic conditions but are not growing, Fhp dominates. Other bacteria that only make Fhp generate similar N2O SP biosignatures to those measured from our P. aeruginosa Fhp-only strain. Fhp homologs in sequenced bacterial genomes currently exceed NOR homologs by nearly a factor of four. Accordingly, we suggest a different framework to guide the attribution of N2O biological sources in nature and disease.

Marine macroalgae produce abundant and diverse polysaccharides, which contribute substantially to the organic matter exported to the deep ocean. Microbial degradation of these polysaccharides plays an important role in the turnover of macroalgal biomass. Various members of the Planctomycetes-Verrucomicrobia-Chlamydia (PVC) superphylum are degraders of polysaccharides in widespread anoxic environments. In this study, we isolated a novel anaerobic bacterial strain NLcol2T from microbial mats on the surface of marine sediments offshore Santa Barbara, CA, USA. Based on 16S ribosomal RNA (rRNA) gene and phylogenomic analyses, strain NLcol2T represents a novel species within the Pontiella genus in the Kiritimatiellota phylum (within the PVC superphylum). Strain NLcol2T is able to utilize various monosaccharides, disaccharides, and macroalgal polysaccharides such as agar and ɩ-carrageenan. A near-complete genome also revealed an extensive metabolic capacity for anaerobic degradation of sulfated polysaccharides, as evidenced by 202 carbohydrate-active enzymes (CAZymes) and 165 sulfatases. Additionally, its ability of nitrogen fixation was confirmed by nitrogenase activity detected during growth on nitrogen-free medium, and the presence of nitrogenases (nifDKH) encoded in the genome. Based on the physiological and genomic analyses, this strain represents a new species of bacteria that may play an important role in the degradation of macroalgal polysaccharides and with relevance to the biogeochemical cycling of carbon, sulfur, and nitrogen in marine environments. Strain NLcol2T (= DSM 113125T = MCCC 1K08672T) is proposed to be the type strain of a novel species in the Pontiella genus, and the name Pontiella agarivorans sp. nov. is proposed.IMPORTANCEGrowth and intentional burial of marine macroalgae is being considered as a carbon dioxide reduction strategy but elicits concerns as to the fate and impacts of this macroalgal carbon in the ocean. Diverse heterotrophic microbial communities in the ocean specialize in these complex polymers such as carrageenan and fucoidan, for example, members of the Kiritimatiellota phylum. However, only four type strains within the phylum have been cultivated and characterized to date, and there is limited knowledge about the metabolic capabilities and functional roles of related organisms in the environment. The new isolate strain NLcol2T expands the known substrate range of this phylum and further reveals the ability to fix nitrogen during anaerobic growth on macroalgal polysaccharides, thereby informing the issue of macroalgal carbon disposal.

Background: Dark anaerobiosis promotes the acidification of the thylakoid lumen and a reduction in the plastoquinone (PQ) pool. The relationship between the reduction in the PQ pool in the dark and the induction of the xanthophyll cycle under high light stress was investigated in Chlamydomonas reinhardtii. Methods: To achieve a comprehensive oxidative/reductive (aerobic/anaerobic conditions) state of the PQ pool, cultures were bubbled with air or nitrogen for 4 h. To induce the xanthophyll cycle, the cultures were then irradiated with 1200 µmolphotons m−2 s−1 white light for 1 h. Results: The anaerobic cultures exhibited a stronger induction of the xanthophyll cycle with a 3.4-fold higher de-epoxidation state than the aerobic cultures. Chlorophyll fluorescence measurements showed that this response was influenced by the previous redox state of the PQ pool, and that dark anaerobiosis triggers physiological responses, such as exposure to high light. Thus, the photosynthetic apparatus in anaerobic cultures was already alerted, at the moment of high light exposure, to give an appropriate response to the stress with a stronger induction of the xanthophyll cycle than in aerobic cultures. Conclusions: Our results provide new information on the importance of the redox signaling pathway and highlight the importance of the reductive conditions of the PQ pool in regulating the physiological responses of photosynthetic organisms to stress. [ABSTRACT FROM AUTHOR]

The waste generated by the mezcal and seafood restaurant industries has significant negative environmental impacts on soils, water, and air. However, these waste materials offer the potential for methane production through anaerobic digestion. This research focuses on evaluating a methodological strategy that involves using an enriched inoculum, co-digesting waste materials, and applying specific pretreatment (chemical, thermal, and adjusting the volumetric ratio of residues) to produce biogas with a high methane content, as assessed through the Biochemical Methane Potential (BMP) test. The results demonstrate that the enriched inoculum used in the test exhibited a pH of 8.18, an Alkalinity of 31 g CaCO3/L, and an alpha (α) ratio of 0.75. The BMP test was conducted over 29 days. In the most successful test, which involved pretreatment at a temperature of 50 °C, a residue ratio of 1 VSW (Volume of Seafood Waste)/2 VMV (Volume of Mezcal Vinasses), and a pH of 6.5, we achieved a methane production of 1420.51 ± 0.038 N mL CH4, with a biogas containing 81.5% methane. This process also yielded 175.37 N mL CH4 per gram of Volatile Solids (VS) and a degradation kinetic constant of 0.14 1/d. Through the methodological strategy used with the combination of an enriched and stabilized inoculum, the co-digestion of the waste for nutrient complementation, and the application of pretreatments, it was achieved that the different stages that constitute the anaerobic process allowed for the establishment of optimal conditions for the generation of biogas with a high methane content. [ABSTRACT FROM AUTHOR]

ImportanceThis work has broad relevance due to the ubiquity of dyes containing azo bonds in food and drugs. We report that azo dyes can be degraded by human gut bacteria through both enzymatic and nonenzymatic mechanisms, even from a single gut bacterial species. Furthermore, we revealed that environmental factors, oxygen, and L-Cysteine control the ability of E. coli to degrade azo dyes due to their impacts on bacterial transcription and metabolism. These results open up new opportunities to manipulate the azoreductase activity of the gut microbiome through the manipulation of host diet, suggest that azoreductase potential may be altered in patients suffering from gastrointestinal disease, and highlight the importance of studying bacterial enzymes for drug metabolism in their natural cellular and ecological context.

Anaerobic fungi (Neocallimastigomycetes) found in the guts of herbivores are biomass deconstruction specialists with a remarkable ability to extract sugars from recalcitrant plant material. Anaerobic fungi, as well as many species of anaerobic bacteria, deploy multi-enzyme complexes called cellulosomes, which modularly tether together hydrolytic enzymes, to accelerate biomass hydrolysis. While the majority of genomically encoded cellulosomal genes in Neocallimastigomycetes are biomass degrading enzymes, the second largest family of cellulosomal genes encode spore coat CotH domains, whose contribution to fungal cellulosome and/or cellular function is unknown. Structural bioinformatics of CotH proteins from the anaerobic fungus Piromyces finnis shows anaerobic fungal CotH domains conserve key ATP and Mg2+ binding motifs from bacterial Bacillus CotH proteins known to act as protein kinases. Experimental characterization further demonstrates ATP hydrolysis activity in the presence and absence of substrate from two cellulosomal P. finnis CotH proteins when recombinantly produced in E. coli. These results present foundational evidence for CotH activity in anaerobic fungi and provide a path towards elucidating the functional contribution of this protein family to fungal cellulosome assembly and activity.

Anaerobic fungi found in the guts of large herbivores are prolific biomass degraders whose genomes harbor a wealth of carbohydrate-active enzymes (CAZymes), of which only a handful are structurally or biochemically characterized. Here, we report the structure and kinetic rate parameters for a glycoside hydrolase (GH) family 5 subfamily 4 enzyme (CelD) from Piromyces finnis, a modular, cellulosome-incorporated endoglucanase that possesses three GH5 domains followed by two C-terminal fungal dockerin domains (double dockerin). We present the crystal structures of an apo wild-type CelD GH5 catalytic domain and its inactive E154A mutant in complex with cellotriose at 2.5 and 1.8 Å resolution, respectively, finding the CelD GH5 catalytic domain adopts the (β/α)8-barrel fold common to many GH5 enzymes. Structural superimposition of the apo wild-type structure with the E154A mutant-cellotriose complex supports a catalytic mechanism in which the E154 carboxylate side chain acts as an acid/base and E278 acts as a complementary nucleophile. Further analysis of the cellotriose binding pocket highlights a binding groove lined with conserved aromatic amino acids that when docked with larger cellulose oligomers is capable of binding seven glucose units and accommodating branched glucan substrates. Activity analyses confirm P. finnis CelD can hydrolyze mixed linkage glucan and xyloglucan, as well as carboxymethylcellulose (CMC). Measured kinetic parameters show the P. finnis CelD GH5 catalytic domain has CMC endoglucanase activity comparable to other fungal endoglucanases with kcat = 6.0 ± 0.6 s-1 and Km = 7.6 ± 2.1 g/L CMC. Enzyme kinetics were unperturbed by the addition or removal of the native C-terminal dockerin domains as well as the addition of a non-native N-terminal dockerin, suggesting strict modularity among the domains of CelD. KEY POINTS: • Anaerobic fungi host a wealth of industrially useful enzymes but are understudied. • P. finnis CelD has endoglucanase activity and structure common to GH5_4 enzymes. • CelDs kinetics do not change with domain fusion, exhibiting high modularity.

Nanaerobes are a newly described class of microorganisms that use a unique cytochrome bd oxidase to achieve nanaerobic respiration at

Low carbon fuel and waste management policies at the federal and state levels have catalyzed the construction of Californias wet anaerobic digestion (AD) facilities. Wet ADs can digest food waste and dairy manure to produce compressed natural gas (CNG) for natural gas vehicles or electricity for electric vehicles (EVs). Carbon capture and sequestration (CCS) of CO2 generated from AD reduces the fuel carbon intensity by carbon removal in addition to avoided methane emissions. Using a combined lifecycle and techno-economic analysis, we determine the most cost-effective design under current and forthcoming federal and state low carbon fuel policies. Under many scenarios, designs that convert biogas to electricity for EVs (Biogas to EV) are favored; however, CCS is only cost-effective in these systems with policy incentives that exceed $200/tonne of CO2 captured. Adding CCS to CNG-producing systems (Biogas to CNG) only requires a single unit operation to prepare the CO2 for sequestration, with a sequestration cost of $34/tonne. When maximizing negative emissions is the goal, incentives are needed to either (1) fund CCS with Biogas to EV designs or (2) favor CNG over electricity production from wet AD facilities.

Complex interactions exist among microorganisms in a community to carry out ecological processes and adapt to changing environments. Here, we constructed a quad-culture consisting of a cellulolytic bacterium (Ruminiclostridium cellulolyticum), a hydrogenotrophic methanogen (Methanospirillum hungatei), an acetoclastic methanogen (Methanosaeta concilii), and a sulfate-reducing bacterium (Desulfovibrio vulgaris). The four microorganisms in the quad-culture cooperated via cross-feeding to produce methane using cellulose as the only carbon source and electron donor. The community metabolism of the quad-culture was compared with those of the R. cellulolyticum-containing tri-cultures, bi-cultures, and mono-culture. Methane production was higher in the quad-culture than the sum of the increases in the tri-cultures, which was attributed to a positive synergy of four species. In contrast, cellulose degradation by the quad-culture was lower than the additive effects of the tri-cultures which represented a negative synergy. The community metabolism of the quad-culture was compared between a control condition and a treatment condition with sulfate addition using metaproteomics and metabolic profiling. Sulfate addition enhanced sulfate reduction and decreased methane and CO2 productions. The cross-feeding fluxes in the quad-culture in the two conditions were modeled using a community stoichiometric model. Sulfate addition strengthened metabolic handoffs from R. cellulolyticum to M. concilii and D. vulgaris and intensified substrate competition between M. hungatei and D. vulgaris. Overall, this study uncovered emergent properties of higher-order microbial interactions using a four-species synthetic community. IMPORTANCE A synthetic community was designed using four microbial species that together performed distinct key metabolic processes in the anaerobic degradation of cellulose to methane and CO2. The microorganisms exhibited expected interactions, such as cross-feeding of acetate from a cellulolytic bacterium to an acetoclastic methanogen and competition of H2 between a sulfate reducing bacterium and a hydrogenotrophic methanogen. This validated our rational design of the interactions between microorganisms based on their metabolic roles. More interestingly, we also found positive and negative synergies as emergent properties of high-order microbial interactions among three or more microorganisms in cocultures. These microbial interactions can be quantitatively measured by adding and removing specific members. A community stoichiometric model was constructed to represent the fluxes in the community metabolic network. This study paved the way toward a more predictive understanding of the impact of environmental perturbations on microbial interactions sustaining geochemically significant processes in natural systems.

Lignocellulose forms plant cell walls, and its three constituent polymers, cellulose, hemicellulose and lignin, represent the largest renewable organic carbon pool in the terrestrial biosphere. Insights into biological lignocellulose deconstruction inform understandings of global carbon sequestration dynamics and provide inspiration for biotechnologies seeking to address the current climate crisis by producing renewable chemicals from plant biomass. Organisms in diverse environments disassemble lignocellulose, and carbohydrate degradation processes are well defined, but biological lignin deconstruction is described only in aerobic systems. It is currently unclear whether anaerobic lignin deconstruction is impossible because of biochemical constraints or, alternatively, has not yet been measured. We applied whole cell-wall nuclear magnetic resonance, gel-permeation chromatography and transcriptome sequencing to interrogate the apparent paradox that anaerobic fungi (Neocallimastigomycetes), well-documented lignocellulose degradation specialists, are unable to modify lignin. We find that Neocallimastigomycetes anaerobically break chemical bonds in grass and hardwood lignins, and we further associate upregulated gene products with the observed lignocellulose deconstruction. These findings alter perceptions of lignin deconstruction by anaerobes and provide opportunities to advance decarbonization biotechnologies that depend on depolymerizing lignocellulose.

A system for co-cultivation of anaerobic fungi with anaerobic bacteria was established based on lactate cross-feeding to produce butyrate and butanol from plant biomass. Several co-culture formulations were assembled that consisted of anaerobic fungi (Anaeromyces robustus, Neocallimastix californiae, or Caecomyces churrovis) with the bacterium Clostridium acetobutylicum. Co-cultures were grown simultaneously (e.g., 'one pot'), and compared to cultures where bacteria were cultured in fungal hydrolysate sequentially. Fungal hydrolysis of lignocellulose resulted in 7-11 mM amounts of glucose and xylose, as well as acetate, formate, ethanol, and lactate to support clostridial growth. Under these conditions, one-stage simultaneous co-culture of anaerobic fungi with C. acetobutylicum promoted the production of butyrate up to 30 mM. Alternatively, two-stage growth slightly promoted solventogenesis and elevated butanol levels (∼4-9 mM). Transcriptional regulation in the two-stage growth condition indicated that this cultivation method may decrease the time required to reach solventogenesis and induce the expression of cellulose-degrading genes in C. acetobutylicum due to relieved carbon-catabolite repression. Overall, this study demonstrates a proof of concept for biobutanol and bio-butyrate production from lignocellulose using an anaerobic fungal-bacterial co-culture system.

Sulfate-coupled anaerobic oxidation of methane (AOM) is performed by multicellular consortia of anaerobic methanotrophic archaea (ANME) in obligate syntrophic partnership with sulfate-reducing bacteria (SRB). Diverse ANME and SRB clades co-associate but the physiological basis for their adaptation and diversification is not well understood. In this work, we used comparative metagenomics and phylogenetics to investigate the metabolic adaptation among the 4 main syntrophic SRB clades (HotSeep-1, Seep-SRB2, Seep-SRB1a, and Seep-SRB1g) and identified features associated with their syntrophic lifestyle that distinguish them from their non-syntrophic evolutionary neighbors in the phylum Desulfobacterota. We show that the protein complexes involved in direct interspecies electron transfer (DIET) from ANME to the SRB outer membrane are conserved between the syntrophic lineages. In contrast, the proteins involved in electron transfer within the SRB inner membrane differ between clades, indicative of convergent evolution in the adaptation to a syntrophic lifestyle. Our analysis suggests that in most cases, this adaptation likely occurred after the acquisition of the DIET complexes in an ancestral clade and involve horizontal gene transfers within pathways for electron transfer (CbcBA) and biofilm formation (Pel). We also provide evidence for unique adaptations within syntrophic SRB clades, which vary depending on the archaeal partner. Among the most widespread syntrophic SRB, Seep-SRB1a, subclades that specifically partner ANME-2a are missing the cobalamin synthesis pathway, suggestive of nutritional dependency on its partner, while closely related Seep-SRB1a partners of ANME-2c lack nutritional auxotrophies. Our work provides insight into the features associated with DIET-based syntrophy and the adaptation of SRB towards it.

Some Alphaproteobacteria contain intracytoplasmic membranes (ICMs) and proteins homologous to those responsible for the mitochondrial cristae, an observation which has given rise to the hypothesis that the Alphaproteobacteria endosymbiont had already evolved cristae-like structures and functions. However, our knowledge of microbial fine structure is still limited, leaving open the possibility of structurally homologous ICMs outside the Alphaproteobacteria. Here, we report on the detailed characterization of lamellar cristae-like ICMs in environmental sulfate-reducing Desulfobacterota that form syntrophic partnerships with anaerobic methane-oxidizing (ANME) archaea. These structures are junction-bound to the cytoplasmic membrane and resemble the form seen in the lamellar cristae of opisthokont mitochondria. Extending these observations, we also characterized similar structures in Desulfovibrio carbinolicus, a close relative of the magnetotactic D. magneticus, which does not contain magnetosomes. Despite a remarkable structural similarity, the key proteins involved in cristae formation have not yet been identified in Desulfobacterota, suggesting that an analogous, but not a homologous, protein organization system developed during the evolution of some members of Desulfobacterota. IMPORTANCE Working with anaerobic consortia of methane oxidizing ANME archaea and their sulfate-reducing bacterial partners recovered from deep sea sediments and with the related sulfate-reducing bacterial isolate D. carbinolicus, we discovered that their intracytoplasmic membranes (ICMs) appear remarkably similar to lamellar cristae. Three-dimensional electron microscopy allowed for the novel analysis of the nanoscale attachment of ICMs to the cytoplasmic membrane, and these ICMs are structurally nearly identical to the crista junction architecture seen in metazoan mitochondria. However, the core junction-forming proteins must be different. The outer membrane vesicles were observed to bud from syntrophic Desulfobacterota, and darkly stained granules were prominent in both Desulfobacterota and D. carbinolicus. These findings expand the taxonomic breadth of ICM-producing microorganisms and add to our understanding of three-dimensional microbial fine structure in environmental microorganisms.

Increasingly stringent limits on nutrient discharges are motivating water resource recovery facilities (WRRFs) to consider the implementation of sidestream nutrient removal or recovery technologies. To further increase biogas production and reduce landfilled waste, WRRFs with excess anaerobic digestion capacity can accept other high-strength organic waste (HSOW) streams. The goal of this study was to characterize and evaluate the life-cycle global warming potential (GWP), eutrophication potential, and economic costs and benefits of sidestream nutrient management and biosolid management strategies following digestion of sewage sludge augmented by HSOW. Five sidestream nutrient management strategies were analyzed using environmental life-cycle assessment (LCA) and life-cycle cost analysis (LCCA) for codigestion of municipal sewage sludge with and without HSOW. As expected, thermal stripping and ammonia stripping were characterized by a much lower eutrophication potential than no sidestream treatment; significantly higher fertilizer prices would be needed for this revenue stream to cover the capital and chemical costs. Composting all biosolids dramatically reduced the GWP relative to the baseline biosolid option but had slightly higher eutrophication potential. These complex environmental and economic tradeoffs require utilities to consider their social, environmental, and economic values in addition to present or upcoming nutrient discharge limits prior to making decisions in sidestream and biosolids management.

Anaerobic methane oxidation exerts a key control on greenhouse gas emissions1, yet factors that modulate the activity of microorganisms performing this function remain poorly understood. Here we discovered extraordinarily large, diverse DNA sequences that primarily encode hypothetical proteins through studying groundwater, sediments and wetland soil where methane production and oxidation occur. Four curated, complete genomes are linear, up to approximately 1 Mb in length and share genome organization, including replichore structure, long inverted terminal repeats and genome-wide unique perfect tandem direct repeats that are intergenic or generate amino acid repeats. We infer that these are highly divergent archaeal extrachromosomal elements with a distinct evolutionary origin. Gene sequence similarity, phylogeny and local divergence of sequence composition indicate that many of their genes were assimilated from methane-oxidizing Methanoperedens archaea. We refer to these elements as 'Borgs'. We identified at least 19 different Borg types coexisting with Methanoperedens spp. in four distinct ecosystems. Borgs provide methane-oxidizing Methanoperedens archaea access to genes encoding proteins involved in redox reactions and energy conservation (for example, clusters of multihaem cytochromes and methyl coenzyme M reductase). These data suggest that Borgs might have previously unrecognized roles in the metabolism of this group of archaea, which are known to modulate greenhouse gas emissions, but further studies are now needed to establish their functional relevance.

All biological hydroxylation reactions are thought to derive the oxygen atom from one of three inorganic oxygen donors, O2, H2O2 or H2O. Here, we have identified the organic compound prephenate as the oxygen donor for the three hydroxylation steps of the O2-independent biosynthetic pathway of ubiquinone, a widely distributed lipid coenzyme. Prephenate is an intermediate in the aromatic amino acid pathway and genetic experiments showed that it is essential for ubiquinone biosynthesis in Escherichia coli under anaerobic conditions. Metabolic labeling experiments with 18O-shikimate, a precursor of prephenate, demonstrated the incorporation of 18O atoms into ubiquinone. The role of specific iron--sulfur enzymes belonging to the widespread U32 protein family is discussed. Prephenate-dependent hydroxylation reactions represent a unique biochemical strategy for adaptation to anaerobic environments. [ABSTRACT FROM AUTHOR]

Novel bacterial isolates with the capabilities of lignin depolymerization, catabolism, or both, could be pertinent to lignocellulosic biofuel applications. In this study, we aimed to identify anaerobic bacteria that could address the economic challenges faced with microbial-mediated biotechnologies, such as the need for aeration and mixing. Using a consortium seeded from temperate forest soil and enriched under anoxic conditions with organosolv lignin as the sole carbon source, we successfully isolated a novel bacterium, designated 159R. Based on the 16S rRNA gene, the isolate belongs to the genus Sodalis in the family Bruguierivoracaceae. Whole-genome sequencing revealed a genome size of 6.38 Mbp and a GC content of 55 mol%. To resolve the phylogenetic position of 159R, its phylogeny was reconstructed using (i) 16S rRNA genes of its closest relatives, (ii) multilocus sequence analysis (MLSA) of 100 genes, (iii) 49 clusters of orthologous groups (COG) domains, and (iv) 400 conserved proteins. Isolate 159R was closely related to the deadwood associated Sodalis guild rather than the tsetse fly and other insect endosymbiont guilds. Estimated genome-sequence-based digital DNA-DNA hybridization (dDDH), genome percentage of conserved proteins (POCP), and an alignment analysis between 159R and the Sodalis clade species further supported that isolate 159R was part of the Sodalis genus and a strain of Sodalis ligni. We proposed the name Sodalis ligni str. 159R (=DSM 110549 = ATCC TSD-177). IMPORTANCE Currently, in the paper industry, paper mill pulping relies on unsustainable and costly processes to remove lignin from lignocellulosic material. A greener approach is biopulping, which uses microbes and their enzymes to break down lignin. However, there are limitations to biopulping that prevent it from outcompeting other pulping processes, such as requiring constant aeration and mixing. Anaerobic bacteria are a promising alternative source for consolidated depolymerization of lignin and its conversion to valuable by-products. We presented Sodalis ligni str. 159R and its characteristics as another example of potential mechanisms that can be developed for lignocellulosic applications.

Syntrophic consortia of anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB) consume large amounts of methane and serve as the foundational microorganisms in marine methane seeps. Despite their importance in the carbon cycle, research on the physiology of ANME-SRB consortia has been hampered by the slow growth and complex physicochemical environment the consortia inhabit. Here, we report successful sediment-free enrichment of ANME-SRB consortia from deep-sea methane seep sediments in the Santa Monica Basin, California. Anoxic Percoll density gradients and size-selective filtration were used to separate ANME-SRB consortia from sediment particles and single cells to accelerate the cultivation process. Over a 3-year period, a subset of the sediment-associated ANME and SRB lineages, predominantly comprised of ANME-2a/2b ("Candidatus Methanocomedenaceae") and their syntrophic bacterial partners, SEEP-SRB1/2, adapted and grew under defined laboratory conditions. Metagenome-assembled genomes from several enrichments revealed that ANME-2a, SEEP-SRB1, and Methanococcoides in different enrichments from the same inoculum represented distinct species, whereas other coenriched microorganisms were closely related at the species level. This suggests that ANME, SRB, and Methanococcoides are more genetically diverse than other members in methane seeps. Flow cytometry sorting and sequencing of cell aggregates revealed that Methanococcoides, Anaerolineales, and SEEP-SRB1 were overrepresented in multiple ANME-2a cell aggregates relative to the bulk metagenomes, suggesting they were physically associated and possibly interacting. Overall, this study represents a successful case of selective cultivation of anaerobic slow-growing microorganisms from sediments based on their physical characteristics, introducing new opportunities for detailed genomic, physiological, biochemical, and ecological analyses. IMPORTANCE Biological anaerobic oxidation of methane (AOM) coupled with sulfate reduction represents a large methane sink in global ocean sediments. Methane consumption is carried out by syntrophic archaeal-bacterial consortia and fuels a unique ecosystem, yet the interactions in these slow-growing syntrophic consortia and with other associated community members remain poorly understood. The significance of this study is the establishment of sediment-free enrichment cultures of anaerobic methanotrophic archaea and sulfate-reducing bacteria performing AOM with sulfate using selective cultivation approaches based on size, density, and metabolism. By reconstructing microbial genomes and analyzing community composition of the enrichment cultures and cell aggregates, we shed light on the diversity of microorganisms physically associated with AOM consortia beyond the core syntrophic partners. These enrichment cultures offer simplified model systems to extend our understanding of the diversity of microbial interactions within marine methane seeps.

Fluorescent proteins, such as green fluorescent proteins, are invaluable tools for detecting and quantifying gene expression in high-throughput reporter gene assays. However, they introduce significant inaccuracies in studies involving microaerobiosis or anaerobiosis, as oxygen is required for the maturation of these proteins' chromophores. In this study, the authors highlight the errors incurred by using fluorescent proteins under limited oxygenation by comparing standard fluorescence-based reporter gene assays to quantitative real-time PCR data in the study of a complex oxygen-regulated gene network. Furthermore, a solution to perform quantification of anaerobic and microaerobic gene expression with fluorescent reporter proteins using a microplate reader with an oxygen control system and applying pulses of full oxygenation before fluorescence measurements is provided.

The recently discovered microbial reductive defluorination of two C6 branched and unsaturated fluorinated carboxylic acids (FCAs) provided valuable insights into the environmental fate of per- and polyfluoroalkyl substances (PFASs) and potential bioremediation strategies. However, a systematic investigation is needed to further demonstrate the role of C═C double bonds in the biodegradability of unsaturated PFASs. Here, we examined the structure-biodegradability relationships of 13 FCAs, including nine commercially available unsaturated FCAs and four structurally similar saturated ones, in an anaerobic defluorinating enrichment and an activated sludge community. The anaerobic and aerobic transformation/defluorination pathways were elucidated. The results showed that under anaerobic conditions, the α,β-unsaturation is crucial for FCA biotransformation via reductive defluorination and/or hydrogenation pathways. With sp2 C-F bonds being substituted by C-H bonds, the reductive defluorination became less favorable than hydrogenation. Moreover, for the first time, we reported enhanced degradability and defluorination capability of specific unsaturated FCA structures with trifluoromethyl (-CF3) branches at the α/β-carbon. Such FCA structures can undergo anaerobic abiotic defluorination in the presence of reducing agents and significant aerobic microbial defluorination. Given the diverse applications and emerging concerns of fluorochemicals, this work not only advances the fundamental understanding of the fate of unsaturated PFASs in natural and engineered environments but also may provide insights into the design of readily degradable fluorinated alternatives to existing PFAS compounds.

Background: Dark anaerobiosis promotes the acidification of the thylakoid lumen and a reduction in the plastoquinone (PQ) pool. The relationship between the reduction in the PQ pool in the dark and the induction of the xanthophyll cycle under high light stress was investigated in Chlamydomonas reinhardtii. Methods: To achieve a comprehensive oxidative/reductive (aerobic/anaerobic conditions) state of the PQ pool, cultures were bubbled with air or nitrogen for 4 h. To induce the xanthophyll cycle, the cultures were then irradiated with 1200 µmolphotons m−2 s−1 white light for 1 h. Results: The anaerobic cultures exhibited a stronger induction of the xanthophyll cycle with a 3.4-fold higher de-epoxidation state than the aerobic cultures. Chlorophyll fluorescence measurements showed that this response was influenced by the previous redox state of the PQ pool, and that dark anaerobiosis triggers physiological responses, such as exposure to high light. Thus, the photosynthetic apparatus in anaerobic cultures was already alerted, at the moment of high light exposure, to give an appropriate response to the stress with a stronger induction of the xanthophyll cycle than in aerobic cultures. Conclusions: Our results provide new information on the importance of the redox signaling pathway and highlight the importance of the reductive conditions of the PQ pool in regulating the physiological responses of photosynthetic organisms to stress.

Anaerobic methanotrophic (ANME) archaea obtain energy from the breakdown of methane, yet their extrachromosomal genetic elements are little understood. Here we describe large plasmids associated with ANME archaea of the Methanoperedens genus in enrichment cultures and other natural anoxic environments. By manual curation we show that two of the plasmids are large (155,605 bp and 191,912 bp), circular, and may replicate bidirectionally. The plasmids occur in the same copy number as the main chromosome, and plasmid genes are actively transcribed. One of the plasmids encodes three tRNAs, ribosomal protein uL16 and elongation factor eEF2; these genes appear to be missing in the host Methanoperedens genome, suggesting an obligate interdependence between plasmid and host. Our work opens the way for the development of genetic vectors to shed light on the physiology and biochemistry of Methanoperedens, and potentially genetically edit them to enhance growth and accelerate methane oxidation rates.

The anaerobic oxidation of methane coupled to sulfate reduction is a microbially mediated process requiring a syntrophic partnership between anaerobic methanotrophic (ANME) archaea and sulfate-reducing bacteria (SRB). Based on genome taxonomy, ANME lineages are polyphyletic within the phylum Halobacterota, none of which have been isolated in pure culture. Here, we reconstruct 28 ANME genomes from environmental metagenomes and flow sorted syntrophic consortia. Together with a reanalysis of previously published datasets, these genomes enable a comparative analysis of all marine ANME clades. We review the genomic features that separate ANME from their methanogenic relatives and identify what differentiates ANME clades. Large multiheme cytochromes and bioenergetic complexes predicted to be involved in novel electron bifurcation reactions are well distributed and conserved in the ANME archaea, while significant variations in the anabolic C1 pathways exists between clades. Our analysis raises the possibility that methylotrophic methanogenesis may have evolved from a methanotrophic ancestor.

Fossil benthic foraminifera are used to trace past methane release linked to climate change. However, it is still debated whether isotopic signatures of living foraminifera from methane-charged sediments reflect incorporation of methane-derived carbon. A deeper understanding of isotopic signatures of living benthic foraminifera from methane-rich environments will help to improve reconstructions of methane release in the past and better predict the impact of future climate warming on methane seepage. Here, we present isotopic signatures (δ13C and δ18O) of foraminiferal calcite together with biogeochemical data from Arctic seep environments from c. 1200 m water depth, Vestnesa Ridge, 79° N, Fram Strait. Lowest δ13C values were recorded in shells of Melonis barleeanus, - 5.2‰ in live specimens and - 6.5‰ in empty shells, from sediments dominated by aerobic (MOx) and anaerobic oxidation of methane (AOM), respectively. Our data indicate that foraminifera actively incorporate methane-derived carbon when living in sediments with moderate seepage activity, while in sediments with high seepage activity the poisonous sulfidic environment leads to death of the foraminifera and an overgrowth of their empty shells by methane-derived authigenic carbonates. We propose that the incorporation of methane-derived carbon in living foraminifera occurs via feeding on methanotrophic bacteria and/or incorporation of ambient dissolved inorganic carbon.

BackgroundQuantification of individual species in microbial co-cultures and consortia is critical to understanding and designing communities with prescribed functions. However, it is difficult to physically separate species or measure species-specific attributes in most multi-species systems. Anaerobic gut fungi (AGF) (Neocallimastigomycetes) are native to the rumen of large herbivores, where they exist as minority members among a wealth of prokaryotes. AGF have significant biotechnological potential owing to their diverse repertoire of potent lignocellulose-degrading carbohydrate-active enzymes (CAZymes), which indirectly bolsters activity of other rumen microbes through metabolic exchange. While decades of literature suggest that polysaccharide degradation and AGF growth are accelerated in co-culture with prokaryotes, particularly methanogens, methods have not been available to measure concentrations of individual species in co-culture. New methods to disentangle the contributions of AGF and rumen prokaryotes are sorely needed to calculate AGF growth rates and metabolic fluxes to prove this hypothesis and understand its causality for predictable co-culture design.ResultsWe present a simple, microplate-based method to measure AGF and methanogen concentrations in co-culture based on fluorescence and absorbance spectroscopies. Using samples of < 2% of the co-culture volume, we demonstrate significant increases in AGF growth rate and xylan and glucose degradation rates in co-culture with methanogens relative to mono-culture. Further, we calculate significant differences in AGF metabolic fluxes in co-culture relative to mono-culture, namely increased flux through the energy-generating hydrogenosome organelle. While calculated fluxes highlight uncertainties in AGF primary metabolism that preclude definitive explanations for this shift, our method will enable steady-state fluxomic experiments to probe AGF metabolism in greater detail.ConclusionsThe method we present to measure AGF and methanogen concentrations enables direct growth measurements and calculation of metabolic fluxes in co-culture. These metrics are critical to develop a quantitative understanding of interwoven rumen metabolism, as well as the impact of co-culture on polysaccharide degradation and metabolite production. The framework presented here can inspire new methods to probe systems beyond AGF and methanogens. Simple modifications to the method will likely extend its utility to co-cultures with more than two organisms or those grown on solid substrates to facilitate the design and deployment of microbial communities for bioproduction and beyond.

Abstract Background The endoplasmic reticulum (ER)-mitochondria membrane contact sites (MCS) are extensively studied in aerobic eukaryotes; however, little is known about MCS in anaerobes with reduced forms of mitochondria named hydrogenosomes. In several eukaryotic lineages, the direct physical tether between ER and the outer mitochondrial membrane is formed by ER-mitochondria encounter structure (ERMES). The complex consists of four core proteins (Mmm1, Mmm2, Mdm12, and Mdm10) which are involved in phospholipid trafficking. Here we investigated ERMES distribution in organisms bearing hydrogenosomes and employed Trichomonas vaginalis as a model to estimate ERMES cellular localization, structure, and function. Results Homology searches revealed that Parabasalia-Anaeramoebae, anaerobic jakobids, and anaerobic fungi are lineages with hydrogenosomes that retain ERMES, while ERMES components were gradually lost in Fornicata, and are absent in Preaxostyla and Archamoebae. In T. vaginalis and other parabasalids, three ERMES components were found with the expansion of Mmm1. Immunofluorescence microscopy confirmed that Mmm1 localized in ER, while Mdm12 and Mmm2 were partially localized in hydrogenosomes. Pull-down assays and mass spectrometry of the ERMES components identified a parabasalid-specific Porin2 as a substitute for the Mdm10. ERMES modeling predicted a formation of a continuous hydrophobic tunnel of TvMmm1-TvMdm12-TvMmm2 that is anchored via Porin2 to the hydrogenosomal outer membrane. Phospholipid-ERMES docking and Mdm12-phospholipid dot-blot indicated that ERMES is involved in the transport of phosphatidylinositol phosphates. The absence of enzymes involved in hydrogenosomal phospholipid metabolism implies that ERMES is not involved in the exchange of substrates between ER and hydrogenosomes but in the unidirectional import of phospholipids into hydrogenosomal membranes. Conclusions Our investigation demonstrated that ERMES mediates ER-hydrogenosome interactions in parabasalid T. vaginalis, while the complex was lost in several other lineages with hydrogenosomes.

Introduction: Arterial lactate, mixed venous O2 saturation, venous minus arterial CO2 partial pressure (Pv-aCO2) and the ratio between this gradient and the arterial minus venous oxygen content (Pv-aCO2/Ca-vO2) were proposed as markers of tissue hypoperfusion and oxygenation. The main goals were to characterize the determinants of Pv-aCO2 and Pv-aCO2/Ca-vO2, and the interchangeability of the variables calculated from mixed and central venous samples. Methods: 35 cardiac surgery patients were included. Variables were measured or calculated: after anesthesia induction (T1), end of surgery (T2), and at 6...8.ßhours intervals after ICU admission (T3 and T4). Results: Macrohemodynamics was characterized by increased cardiac index and low systemic vascular resistances after surgery (p.ß

Photogeneration of hydrogen in microalgae is thought to be among the mechanisms increasing their resilience to stresses including those caused by nutrient deprivation by re-routing the flow of electrons and reducing power in the cell. Metabolism of phosphorus (P), an essential nutrient, and its reserve forms such as polyphosphate (PolyP), is affected by and plays a role in the responses to diverse stresses, too. However, the potential interplay of the capability of photogeneration of hydrogen and turnover of phosphorus-rich inclusions in stressed microalgae cells so far escaped the attention of researchers. Here, we present a quantitative ultrastructural view of the turnover of P-rich inclusions in the model microalga Chlamydomonas reinhardtii strains, the parent strain CC-425 and its hydEF-1 mutant lacking hydrogenase activity as a function of sulfur and oxygen availability in the medium. In addition to the electron microscopy cell image analysis of the studied strains, we followed the elemental composition of the inclusions in different (sub) compartments of the cells obtained with energy dispersive X-ray spectroscopy. The stress caused by sulfur deprivation and subsequent transition of the microalgae culture to anaerobic conditions declined the size of phosphorus-containing inclusions but increased their number in the parent strain. Overall, the accumulation of the phosphorus-rich inclusions in hydEF-1 mutant was much lower than in the fully functional parent strain regardless of the cultivation conditions. We believe that impaired hydrogenase activity and correspondingly reduced sink of electrons and reducing power in the mutant strain indirectly affects the turnover of P and its reserves in the cell. These effects were manifested by the changes in the abundance, morphology, and elemental composition of the P-containing inclusions. We hypothesized that the sulfur-deprivation stress increased the initiation of the biosynthesis of PolyP chains, but their elongation and hence the formation of large PolyP-containing inclusions was hindered by anaerobiosis. [ABSTRACT FROM AUTHOR]

Anaerobic gut fungi (AGF, Neocallimastigomycota) inhabit the alimentary tract of herbivores. Although strict anaerobes, studies have suggested their capacity to retain viability after various durations of air exposure. It is currently unclear whether AGF can actively grow, and not merely survive, in redox potentials (Eh) higher than those encountered in the herbivorous gut. We evaluated the growth of two AGF strains (Orpinomyces joyonii and Testudinimyces gracilis) at various Eh levels, achieved by manipulating the concentrations of reductant (cysteine hydrochloride) in culture media. Both strains exhibited robust and sustainable growth at negative Eh (−50 mV or below). However, growth in the absence of cysteine hydrochloride (Eh value around +50 mV) was possible only for O. joyonii and only for one subcultivation. The capacity to grow at +50 mV was further confirmed in four additional taxa (Pecoramyces ruminatium, Anaeromyces mucronatus, Aklioshbmyces papillarum , and Piromyces communis), while two (Aestipascuomyces dupliciliberans and Capellomyces foraminis) failed to grow under these conditions. Our results establish the ability of AGF to grow at redox potential values higher than those encountered in their natural habitats. Such capability could contribute to efficient AGF dispersal and horizontal transmission between hosts, and could have important implications for industrial applications of AGF. [ABSTRACT FROM AUTHOR]

Anaeramoebae is a recently described phylum of anaerobic, marine amoebae, and amoeboflagellates belonging to the Metamonada supergroup. So far, six species have been described based on light microscopic morphology and sequences of the SSU rRNA gene. Here we present three new strains of Anaeramoeba with a description of their morphology, ultrastructure, and phylogenetic position based on the analysis of SSU rRNA gene sequences. Two of the strains represent a new species, Anaeramoeba pumila sp. nov., that has the smallest cells of all known Anaeramoeba species, and one that represents a species from the newly recognized Anaeramoeba flamelloides complex. Anaeramoebae are known to have a syntrophic relationship with prokaryotes. Our strains display two novel, remarkable types of symbioses, previously unknown from Anaeramoebae. [ABSTRACT FROM AUTHOR]

Molasses are a source of fermentable sugars, the substrate utilized for this study, as it is a subproduct from sugarcane processing for sucrose recovery. These sugars can be metabolized for bioH 2 production by bacterial strains, particularly strict-anaerobe Clostridium acetobutylicum (Clac) and its media preparation requires chemical of O 2 removal and N 2 flushing to promote anaerobiosis. Bacillus subtilis (Bsub) can deplete O 2 from culture media and promotes anaerobiosis, which is an aspect that would benefit from further research. Clac was grown in an anaerobic medium and produced 269 mL of hydrogen at 72 h. By co-culturing Clac and Bsub , 502 mL of bioH 2 were produced in a cysteine/N 2 flushed medium, while 203 mL of bioH 2 at 72 h were produced by aerobic co-culture. Additionally, in an untreated/aerobic conventional growth culture for Clac , no production of H 2 was detected, reinforcing the utility of Bsub for anaerobiosis generation. • Clostridium acetobutylicum consumes substrate approximately 6x faster than Bacillus subtilis. • Clostridium acetobutylicum produced 202 mL of bioH 2 in a culture with anaerobiosis assisted by Bacillus subtilis. • Bacillus subtilis CBB5555 can act as a biological O 2 depleter for anaerobic bioprocesses. [ABSTRACT FROM AUTHOR]

Green hydrogen, produced during microalgal photosynthesis, is regarded as one of the most promising sustainable energy sources. It utilizes sunlight and water, which are essentially unlimited, and its combustion results in only water as a waste product. In microalgal hydrogen energy production systems, the sensitivity of hydrogenase to O2 poses a significant challenge, limiting sustained photosynthetic H2 production in microalgae. Additionally, efficient photosynthetic H2 production in anaerobic microalgal cells is hindered by impaired electron source (photosystem II) and electron loss through the Calvin‐Benson cycle, cyclic electron transfer around photosystem I, and O2 photoreduction, which are identified as the other key challenges. Over the past eight decades, considerable progress has been made in addressing these challenges and regulating electron transfer to achieve sustainable and efficient photosynthetic H2 production in microalgae. In this review, we discuss a range of regulatory methods for achieving sustainable and efficient photosynthetic H2 production in microalgae. Emphasizing the significant progress made over the past eight decades, we also address current challenges and propose potential future solutions. [ABSTRACT FROM AUTHOR]

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Copyright of Investigación Agraria is the property of Investigacion Agraria and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Metal(loid) salts were used to treat infectious diseases in the past due to their exceptional biocidal properties at low concentrations. However, the mechanism of their toxicity has yet to be fully elucidated. The production of reactive oxygen species (ROS) has been linked to the toxicity of soft metal(loid)s such as Ag(I), Au(III), As(III), Cd(II), Hg(II), and Te(IV). Nevertheless, few reports have described the direct, or ROS-independent, effects of some of these soft-metal(loid)s on bacteria, including the dismantling of iron-sulfur clusters [4Fe-4S] and the accumulation of porphyrin IX. Here, we used genome-wide genetic, proteomic, and biochemical approaches under anaerobic conditions to evaluate the direct mechanisms of toxicity of these metal(loid)s in Escherichia coli. We found that certain soft-metal(loid)s promote protein aggregation in a ROS-independent manner. This aggregation occurs during translation in the presence of Ag(I), Au(III), Hg(II), or Te(IV) and post-translationally in cells exposed to Cd(II) or As(III). We determined that aggregated proteins were involved in several essential biological processes that could lead to cell death. For instance, several enzymes involved in amino acid biosynthesis were aggregated after soft-metal(loid) exposure, disrupting intracellular amino acid concentration. We also propose a possible mechanism to explain how soft-metal(loid)s act as proteotoxic agents. [ABSTRACT FROM AUTHOR]

Phage therapy is a promising alternative in treating bacterial infections; however, understanding how bacteria and phages interact according to the environment is crucial. Although most phage therapy applications are intended for anaerobic environments (such as intestine or wounds), research on the effects of oxygen absence on the phage infection process is scarce . Here, we studied the effect of anoxic conditions in a Salmonella sp. – phage model, given the prevalence of this bacterial pathogen in anaerobic environments, and the potential of phage therapy for its control. Our results show that oxygen-deprived conditions affect the life cycle length of the phage and its capacity to control the bacterial population. Changes in host cell size associated with the FtsZ protein were observed, along with a modified emergence of resistance against the phage under different oxygen conditions. Interestingly, transcriptomic analyses revealed that phage infection in aerobic conditions triggers the expression of genes associated with nitrate reduction, an alternative pathway for anaerobic respiration. Meanwhile, uninfected anaerobic cultures displayed the upregulation of genes involved in a different anaerobic metabolic route comprising the transport of thiosulfate and sulfate. Altogether, our results provide insights into the importance of considering oxygen availability as an important factor for phage therapy when working with facultative anaerobic bacteria. IMPORTANCE Many parameters affect phage-bacteria interaction. Some of these parameters depend on the environment in which the bacteria are present. Anaerobiosis effect on phage infection in facultative anaerobic bacteria has not yet been studied. The absence of oxygen triggers metabolic changes in facultative bacteria and this affects phage infection and viral life cycle. Understanding how an anaerobic environment can alter the behavior of phages during infection is relevant for the phage therapy success. [ABSTRACT FROM AUTHOR]

Anaerobic gut fungi (Neocallimastigomycetes) live in the digestive tract of large herbivores, where they are vastly outnumbered by bacteria. It has been suggested that anaerobic fungi challenge growth of bacteria owing to the wealth of biosynthetic genes in fungal genomes, although this relationship has not been experimentally tested. Here, we cocultivated the rumen bacteria Fibrobacter succinogenes strain UWB7 with the anaerobic gut fungi Anaeromyces robustus or Caecomyces churrovis on a range of carbon substrates and quantified the bacterial and fungal transcriptomic response. Synthetic cocultures were established for at least 24 h, as verified by active fungal and bacterial transcription. A. robustus upregulated components of its secondary metabolism in the presence of Fibrobacter succinogenes strain UWB7, including six nonribosomal peptide synthetases, one polyketide synthase-like enzyme, and five polyketide synthesis O-type methyltransferases. Both A. robustus and C. churrovis cocultures upregulated S-adenosyl-l-methionine (SAM)-dependent methyltransferases, histone methyltransferases, and an acetyltransferase. Fungal histone 3 lysine 27 trimethylation marks were more abundant in coculture, and heterochromatin protein-1 was downregulated. Together, these findings suggest that fungal chromatin remodeling occurs when bacteria are present. F. succinogenes strain UWB7 upregulated four genes in coculture encoding drug efflux pumps, which likely protect the cell against toxins. Furthermore, untargeted nonpolar metabolomics data revealed at least one novel fungal metabolite enriched in coculture, which may be a defense compound. Taken together, these data suggest that A. robustus and C. churrovis produce antimicrobials when exposed to rumen bacteria and, more broadly, that anaerobic gut fungi are a source of novel antibiotics. IMPORTANCE Anaerobic fungi are outnumbered by bacteria by 4 orders of magnitude in the herbivore rumen. Despite their numerical disadvantage, they are resilient members of the rumen microbiome. Previous studies mining the genomes of anaerobic fungi identified genes encoding enzymes to produce natural products, which are small molecules that are often antimicrobials. In this work, we cocultured the anaerobic fungus Anaeromyces robustus or Caecomyes churrovis with rumen bacteria Fibrobacter succinogenes strain UWB7 and sequenced fungal and bacterial active genes via transcriptome sequencing (RNA-seq). Consistent with production of a fungal defense compound, bacteria upregulated genes encoding drug efflux pumps, which often export toxic molecules, and fungi upregulated genes encoding biosynthetic enzymes of natural products. Furthermore, tandem mass spectrometry detected an unknown fungal metabolite enriched in the coculture. Together, these findings point to an antagonistic relationship between anaerobic fungi and rumen bacteria resulting in the production of a fungal compound with potential antimicrobial activity.

INTRODUCTION: In the multisystem inflammatory disorder systemic sclerosis (SSc), gastrointestinal tract (GIT) affliction is highly prevalent. There are no known disease modifying therapies and the negative impact is substantial. Aiming for a new therapeutic principle, and inspired by recent work showing associations between gut microbiota changes and GIT symptoms in SSc, we performed a pilot study on faecal microbiota transplantation (FMT) with the single-donor bacterial culture Anaerobic Cultivated Human Intestinal Microbiome (ACHIM). Motivated by positive pilot study signals, we designed the ReSScue trial as a phase II multicentre, placebo-controlled, randomised 20-week trial to evaluate safety and efficacy on lower GIT symptoms of FMT by ACHIM in SSc. METHODS AND ANALYSES: We aim to include 70 SSc participants with moderate to severe lower GIT symptoms, defined by the validated patient-reported University of California Los Angeles Scleroderma Clinical Trial Consortium GIT 2.0 2.0 questionnaire. The trial includes three parts. In part A1 (induction phase) lasting from week 0 to week 12, participants will be randomised 1:1 to repeat infusions of 30 mL ACHIM or placebo at week 0 and 2 by gastroduodenoscopy. In part A2, which is an 8-week subsequent maintenance phase, all study participants will receive 30 mL ACHIM at week 12 and followed until week 20 on continued blind. In part B, which will last until the last participant completes part A2, the participants will be followed through a maximum 16-week extended monitoring period, for longer-term data on safety and intervention effects. Primary endpoint is change from baseline to week 12 in UCLA GIT subscale scores of diarrhoea or bloating, depending on the worst symptom at baseline evaluated separately for each patient. Secondary endpoints are safety measures and changes in UCLA GIT scores (total, diarrhoea and bloating). ETHICS AND DISSEMINATION: This protocol was approved by the Northern Norwegian Committee for Medical Ethics. Study findings will be published. TRIAL REGISTRATION NUMBER: NCT04300426; Pre-results. PROTOCOL VERSION: V.3.1.

Diverting organic waste from landfills provides significant emissions benefits in addition to preserving landfill capacity and creating value-added energy and compost products. Dry anaerobic digestion (AD) is particularly attractive for managing the organic fraction of municipal solid waste because of its high-solids composition and minimal water requirements. This study utilizes empirical data from operational facilities in California in order to explore the key drivers of dry AD facility profitability, impacts of market forces, and the efficacy of policy incentives. The study finds that dry AD facilities can achieve meaningful economies of scale with organic waste intake amounts larger than 75,000 tonnes per year. Materials handling costs, including the disposal of inorganic residuals from contaminated waste streams and post-digester mass (digestate) management, are both the largest and the most uncertain facility costs. Facilities that utilize the biogas for vehicle fueling and earn associated fuel credits collect revenues that are 4-6x greater than those of facilities generating and selling electricity and 10-12x greater than facilities selling natural gas at market prices. The results suggest important facility design elements and enabling policies to support an increased scale of organic waste handling infrastructure.

Anaerobic fungi (class Neocallimastigomycetes) thrive as low-abundance members of the herbivore digestive tract. The genomes of anaerobic gut fungi are poorly characterized and have not been extensively mined for the biosynthetic enzymes of natural products such as antibiotics. Here, we investigate the potential of anaerobic gut fungi to synthesize natural products that could regulate membership within the gut microbiome. Complementary 'omics' approaches were combined to catalog the natural products of anaerobic gut fungi from four different representative species: Anaeromyces robustus (Arobustus), Caecomyces churrovis (Cchurrovis), Neocallimastix californiae (Ncaliforniae), and Piromyces finnis (Pfinnis). In total, 146 genes were identified that encode biosynthetic enzymes for diverse types of natural products, including nonribosomal peptide synthetases and polyketide synthases. In addition, N. californiae and C. churrovis genomes encoded seven putative bacteriocins, a class of antimicrobial peptides typically produced by bacteria. During standard laboratory growth on plant biomass or soluble substrates, 26% of total core biosynthetic genes in all four strains were transcribed. Across all four fungal strains, 30% of total biosynthetic gene products were detected via proteomics when grown on cellobiose. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) characterization of fungal supernatants detected 72 likely natural products from A. robustus alone. A compound produced by all four strains of anaerobic fungi was putatively identified as the polyketide-related styrylpyrone baumin. Molecular networking quantified similarities between tandem mass spectrometry (MS/MS) spectra among these fungi, enabling three groups of natural products to be identified that are unique to anaerobic fungi. Overall, these results support the finding that anaerobic gut fungi synthesize natural products, which could be harnessed as a source of antimicrobials, therapeutics, and other bioactive compounds.

Microorganisms play vital roles in modulating organic matter decomposition and nutrient cycling in soil ecosystems. The enzyme latch paradigm posits microbial degradation of polyphenols is hindered in anoxic peat leading to polyphenol accumulation, and consequently diminished microbial activity. This model assumes that polyphenols are microbially unavailable under anoxia, a supposition that has not been thoroughly investigated in any soil type. Here, we use anoxic soil reactors amended with and without a chemically defined polyphenol to test this hypothesis, employing metabolomics and genome-resolved metaproteomics to interrogate soil microbial polyphenol metabolism. Challenging the idea that polyphenols are not bioavailable under anoxia, we provide metabolite evidence that polyphenols are depolymerized, resulting in monomer accumulation, followed by the generation of small phenolic degradation products. Further, we show that soil microbiome function is maintained, and possibly enhanced, with polyphenol addition. In summary, this study provides chemical and enzymatic evidence that some soil microbiota can degrade polyphenols under anoxia and subvert the assumed polyphenol lock on soil microbial metabolism.

Coral reef health depends on an intricate relationship among the coral animal, photosynthetic algae, and a complex microbial community. The holobiont can impact the nutrient balance of their hosts amid an otherwise oligotrophic environment, including by cycling physiologically important nitrogen compounds. Here we use 15N-tracer experiments to produce the first simultaneous measurements of ammonium oxidation, nitrate reduction, and nitrous oxide (N2O) production among five iconic species of reef-building corals (Acropora palmata, Diploria labyrinthiformis, Orbicella faveolata, Porites astreoides, and Porites porites) in the highly protected Jardines de la Reina reefs of Cuba. Nitrate reduction is present in most species, but ammonium oxidation is low potentially due to photoinhibition and assimilatory competition. Coral-associated rates of N2O production indicate a widespread potential for denitrification, especially among D. labyrinthiformis, at rates of ~1 nmol cm-2 d-1. In contrast, A. palmata displays minimal active nitrogen metabolism. Enhanced rates of nitrate reduction and N2O production are observed coincident with dark net respiration periods. Genomes of bacterial cultures isolated from multiple coral species confirm that microorganisms with the ability to respire nitrate anaerobically to either dinitrogen gas or ammonium exist within the holobiont. This confirmation of anaerobic nitrogen metabolisms by coral-associated microorganisms sheds new light on coral and reef productivity.

The herbivore digestive tract is home to a complex community of anaerobic microbes that work together to break down lignocellulose. These microbiota are an untapped resource of strains, pathways and enzymes that could be applied to convert plant waste into sugar substrates for green biotechnology. We carried out more than 400 parallel enrichment experiments from goat faeces to determine how substrate and antibiotic selection influence membership, activity, stability and chemical productivity of herbivore gut communities. We assembled 719 high-quality metagenome-assembled genomes (MAGs) that are unique at the species level. More than 90% of these MAGs are from previously unidentified herbivore gut microorganisms. Microbial consortia dominated by anaerobic fungi outperformed bacterially dominated consortia in terms of both methane production and extent of cellulose degradation, which indicates that fungi have an important role in methane release. Metabolic pathway reconstructions from MAGs of 737 bacteria, archaea and fungi suggest that cross-domain partnerships between fungi and methanogens enabled production of acetate, formate and methane, whereas bacterially dominated consortia mainly produced short-chain fatty acids, including propionate and butyrate. Analyses of carbohydrate-active enzyme domains present in each anaerobic consortium suggest that anaerobic bacteria and fungi employ mostly complementary hydrolytic strategies. The division of labour among herbivore anaerobes to degrade plant biomass could be harnessed for industrial bioprocessing.

Phosphite is the most energetically favorable chemotrophic electron donor known, with a half-cell potential (Eo') of -650 mV for the PO43-/PO33- couple. Since the discovery of microbial dissimilatory phosphite oxidation (DPO) in 2000, the environmental distribution, evolution, and diversity of DPO microorganisms (DPOMs) have remained enigmatic, as only two species have been identified. Here, metagenomic sequencing of phosphite-enriched microbial communities enabled the genome reconstruction and metabolic characterization of 21 additional DPOMs. These DPOMs spanned six classes of bacteria, including the Negativicutes, Desulfotomaculia, Synergistia, Syntrophia, Desulfobacteria, and Desulfomonilia_A Comparing the DPO genes from the genomes of enriched organisms with over 17,000 publicly available metagenomes revealed the global existence of this metabolism in diverse anoxic environments, including wastewaters, sediments, and subsurface aquifers. Despite their newfound environmental and taxonomic diversity, metagenomic analyses suggested that the typical DPOM is a chemolithoautotroph that occupies low-oxygen environments and specializes in phosphite oxidation coupled to CO2 reduction. Phylogenetic analyses indicated that the DPO genes form a highly conserved cluster that likely has ancient origins predating the split of monoderm and diderm bacteria. By coupling microbial cultivation strategies with metagenomics, these studies highlighted the unsampled metabolic versatility latent in microbial communities. We have uncovered the unexpected prevalence, diversity, biochemical specialization, and ancient origins of a unique metabolism central to the redox cycling of phosphorus, a primary nutrient on Earth.

The genus Aspergillus encompasses human pathogens such as Aspergillus fumigatus and industrial powerhouses such as Aspergillus niger In both cases, Aspergillus biofilms have consequences for infection outcomes and yields of economically important products. However, the molecular components influencing filamentous fungal biofilm development, structure, and function remain ill defined. Macroscopic colony morphology is an indicator of underlying biofilm architecture and fungal physiology. A hypoxia-locked colony morphotype of A. fumigatus has abundant colony furrows that coincide with a reduction in vertically oriented hyphae within biofilms and increased low oxygen growth and virulence. Investigation of this morphotype has led to the identification of the causative gene, biofilm architecture factor A (bafA), a small cryptic open reading frame within a subtelomeric gene cluster. BafA is sufficient to induce the hypoxia-locked colony morphology and biofilm architecture in A. fumigatus Analysis across a large population of A. fumigatus isolates identified a larger family of baf genes, all of which have the capacity to modulate hyphal architecture, biofilm development, and hypoxic growth. Furthermore, introduction of A. fumigatusbafA into A. niger is sufficient to generate the hypoxia-locked colony morphology, biofilm architecture, and increased hypoxic growth. Together, these data indicate the potential broad impacts of this previously uncharacterized family of small genes to modulate biofilm architecture and function in clinical and industrial settings.IMPORTANCE The manipulation of microbial biofilms in industrial and clinical applications remains a difficult task. The problem is particularly acute with regard to filamentous fungal biofilms for which molecular mechanisms of biofilm formation, maintenance, and function are only just being elucidated. Here, we describe a family of small genes heterogeneously expressed across Aspergillus fumigatus strains that are capable of modifying colony biofilm morphology and microscopic hyphal architecture. Specifically, these genes are implicated in the formation of a hypoxia-locked colony morphotype that is associated with increased virulence of A. fumigatus Synthetic introduction of these gene family members, here referred to as biofilm architecture factors, in both A. fumigatus and A. niger additionally modulates low oxygen growth and surface adherence. Thus, these genes are candidates for genetic manipulation of biofilm development in aspergilli.

The rumen harbors a complex microbial mixture of archaea, bacteria, protozoa, and fungi that efficiently breakdown plant biomass and its complex dietary carbohydrates into soluble sugars that can be fermented and subsequently converted into metabolites and nutrients utilized by the host animal. While rumen bacterial populations have been well documented, only a fraction of the rumen eukarya are taxonomically and functionally characterized, despite the recognition that they contribute to the cellulolytic phenotype of the rumen microbiota. To investigate how anaerobic fungi actively engage in digestion of recalcitrant fiber that is resistant to degradation, we resolved genome-centric metaproteome and metatranscriptome datasets generated from switchgrass samples incubated for 48 h in nylon bags within the rumen of cannulated dairy cows. Across a gene catalog covering anaerobic rumen bacteria, fungi and viruses, a significant portion of the detected proteins originated from fungal populations. Intriguingly, the carbohydrate-active enzyme (CAZyme) profile suggested a domain-specific functional specialization, with bacterial populations primarily engaged in the degradation of hemicelluloses, whereas fungi were inferred to target recalcitrant cellulose structures via the detection of a number of endo- and exo-acting enzymes belonging to the glycoside hydrolase (GH) family 5, 6, 8, and 48. Notably, members of the GH48 family were amongst the highest abundant CAZymes and detected representatives from this family also included dockerin domains that are associated with fungal cellulosomes. A eukaryote-selected metatranscriptome further reinforced the contribution of uncultured fungi in the ruminal degradation of recalcitrant fibers. These findings elucidate the intricate networks of in situ recalcitrant fiber deconstruction, and importantly, suggest that the anaerobic rumen fungi contribute a specific set of CAZymes that complement the enzyme repertoire provided by the specialized plant cell wall degrading rumen bacteria.

Mechanisms controlling CO2 and CH4 production in wetlands are central to understanding carbon cycling and greenhouse gas exchange. However, the volatility of these respiration products complicates quantifying their rates of production in the field. Attempts to circumvent the challenges through closed system incubations, from which gases cannot escape, have been used to investigate bulk in situ geochemistry. Efforts towards mapping mechanistic linkages between geochemistry and microbiology have raised concern regarding sampling and incubation-induced perturbations. Microorganisms are impacted by oxygen exposure, increased temperatures and accumulation of metabolic products during handling, storage, and incubation. We probed the extent of these perturbations, and their influence on incubation results, using high-resolution geochemical and microbial gene-based community profiling of anaerobically incubated material from three wetland habitats across a permafrost peatland. We compared the original field samples to the material anaerobically incubated over 50 days. Bulk geochemistry and phylum-level microbiota in incubations largely reflected field observations, but divergence between field and incubations occurred in both geochemistry and lineage-level microbial composition when examined at closer resolution. Despite the changes in representative lineages over time, inferred metabolic function with regards to carbon cycling largely reproduced field results suggesting functional consistency. Habitat differences among the source materials remained the largest driver of variation in geochemical and microbial differences among the samples in both incubations and field results. While incubations may have limited usefulness for identifying specific mechanisms, they remain a viable tool for probing bulk-scale questions related to anaerobic C cycling, including CO2 and CH4 dynamics.

Antibiotic resistance genes (ARGs) are emerging contaminants causing serious global health concern. Interventions to address this concern include improving our understanding of methods for treating waste material of human and animal origin that are known to harbor ARGs. Anaerobic digestion is a commonly used process for treating dairy manure, and although effective in reducing ARGs, its mechanism of action is not clear. In this study, we used three ARGs to conducted a longitudinal bench scale anaerobic digestion experiment with various temperatures (28, 36, 44, and 52°C) in triplicate using fresh dairy manure for 30 days to evaluate the reduction of gene abundance. Three ARGs and two mobile genetic elements (MGEs) were studied: sulfonamide resistance gene (sulII), tetracycline resistance genes (tetW), macrolide-lincosamide-streptogramin B (MLSB) superfamily resistance genes (ermF), class 1 integrase gene (intI1), and transposase gene (tnpA). Genes were quantified by real-time quantitative PCR. Results show that the thermophilic anaerobic digestion (52°C) significantly reduced (p < 0.05) the absolute abundance of sulII (95%), intI1 (95%), tnpA (77%) and 16S rRNA gene (76%) after 30 days of digestion. A modified Collins-Selleck model was used to fit the decay curve, and results suggest that the gene reduction during the startup phase of anaerobic digestion (first 5 days) was faster than the later stage, and reductions in the first five days were more than 50% for most genes.

Background: The endoplasmic reticulum (ER)-mitochondria membrane contact sites (MCS) are extensively studied in aerobic eukaryotes; however, little is known about MCS in anaerobes with reduced forms of mitochondria named hydrogenosomes. In several eukaryotic lineages, the direct physical tether between ER and the outer mitochondrial membrane is formed by ER-mitochondria encounter structure (ERMES). The complex consists of four core proteins (Mmm1, Mmm2, Mdm12, and Mdm10) which are involved in phospholipid trafficking. Here we investigated ERMES distribution in organisms bearing hydrogenosomes and employed Trichomonas vaginalis as a model to estimate ERMES cellular localization, structure, and function. Results: Homology searches revealed that Parabasalia-Anaeramoebae, anaerobic jakobids, and anaerobic fungi are lineages with hydrogenosomes that retain ERMES, while ERMES components were gradually lost in Fornicata, and are absent in Preaxostyla and Archamoebae. In T. vaginalis and other parabasalids, three ERMES components were found with the expansion of Mmm1. Immunofluorescence microscopy confirmed that Mmm1 localized in ER, while Mdm12 and Mmm2 were partially localized in hydrogenosomes. Pull-down assays and mass spectrometry of the ERMES components identified a parabasalid-specific Porin2 as a substitute for the Mdm10. ERMES modeling predicted a formation of a continuous hydrophobic tunnel of TvMmm1-TvMdm12-TvMmm2 that is anchored via Porin2 to the hydrogenosomal outer membrane. Phospholipid-ERMES docking and Mdm12-phospholipid dot-blot indicated that ERMES is involved in the transport of phosphatidylinositol phosphates. The absence of enzymes involved in hydrogenosomal phospholipid metabolism implies that ERMES is not involved in the exchange of substrates between ER and hydrogenosomes but in the unidirectional import of phospholipids into hydrogenosomal membranes. Conclusions: Our investigation demonstrated that ERMES mediates ER-hydrogenosome interactions in parabasalid T. vaginalis, while the complex was lost in several other lineages with hydrogenosomes. [ABSTRACT FROM AUTHOR]