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BD1-5, OP11, and OD1 bacteria have been widely detected in anaerobic environments, but their metabolisms remain unclear owing to lack of cultivated representatives and minimal genomic sampling. We uncovered metabolic characteristics for members of these phyla, and a new lineage, PER, via cultivation-independent recovery of 49 partial to near-complete genomes from an acetate-amended aquifer. All organisms were nonrespiring anaerobes predicted to ferment. Three augment fermentation with archaeal-like hybrid type ll/lll ribulose-l, 5-bisphosphate carboxylase-oxygenase (RuBisCO) that couples adenosine monophosphate salvage with CO₂ fixation, a pathway not previously described in Bacteria. Members of OD1 reduce sulfur and may pump protons using archaeal-type hydrogenases. For six organisms, the UGA stop codon is translated as tryptophan. All bacteria studied here may play previously unrecognized roles in hydrogen production, sulfur cycling, and fermentation of refractory sedimentary carbon.

Oxygen availability is a major factor and evolutionary force determining the metabolic strategy of bacteria colonizing an environmental niche. In the soil, conditions can switch rapidly between oxia and anoxia, forcing soil bacteria to remodel their energy metabolism accordingly. Mycobacterium is a dominant genus in the soil, and all its species are obligate aerobes. Here we show that an obligate aerobe, the soil actinomycete Mycobacterium smegmatis , adopts an anaerobe-type strategy by activating fermentative hydrogen production to adapt to hypoxia. This process is controlled by the two-component system DosR-DosS/DosT, an oxygen and redox sensor that is well conserved in mycobacteria. We show that DosR tightly regulates the two [NiFe]-hydrogenases: Hyd3 (MSMEG_3931-3928) and Hyd2 (MSMEG_2719-2718). Using genetic manipulation and high-sensitivity GC, we demonstrate that Hyd3 facilitates the evolution of H ₂ when oxygen is depleted. Combined activity of Hyd2 and Hyd3 was necessary to maintain an optimal NAD ⁺/NADH ratio and enhanced adaptation to and survival of hypoxia. We demonstrate that fermentatively-produced hydrogen can be recycled when fumarate or oxygen become available, suggesting Mycobacterium smegmatis can switch between fermentation, anaerobic respiration, and aerobic respiration. Hydrogen metabolism enables this obligate aerobe to rapidly meet its energetic needs when switching between microoxic and anoxic conditions and provides a competitive advantage in low oxygen environments.

Key Points Plant polysaccharides of dietary origin are a major energy source for the dense anaerobic microbial communities that inhabit the mammalian large intestine and rumen. Microbial fermentation products, in turn, provide energy and nutrients to the host that would otherwise be unavailable. Only a few species of gut microorganism have the ability to act as primary degraders of insoluble substrates, such as plant cell walls, but many others benefit from cross-feeding interactions. The organization and spectrum of enzymes, substrate-binding modules and transport systems largely defines the types of substrate that can be exploited by a given gut bacterium, but only two gut anaerobes have so far been the subject of detailed functional studies. In the rumen cellulolytic bacterium Ruminococcus flavefaciens (phylum Firmicutes) enzymes that are involved in the degradation of insoluble plant cell walls are organized into a cellulosome complex that is bound to the cell surface. By contrast, the sequestration and breakdown of soluble starch molecules is achieved mainly in the periplasm of the Gram-negative human colonic species Bacteroides thetaiotaomicron . More information from microbial genomics is needed to reveal the diversity of the systems that are involved in polysaccharide utilization, and thus the extent of the variation in these two paradigms among gut bacteria. Gut bacteria are largely untapped sources of enzymes and binding domains that have potential for biotechnological application. Dietary plant polysaccharides are a major energy source for the anaerobic microbiota that inhabit the mammalian large intestine and rumen. Flint and colleagues discuss polysaccharide utilization by gut anaerobes, focusing on two examples, the use of insoluble structural polysaccharides by Ruminococcus flavefaciens and the use of starch by Bacteroides thetaiotaomicron . The microbiota of the mammalian intestine depend largely on dietary polysaccharides as energy sources. Most of these polymers are not degradable by the host, but herbivores can derive 70% of their energy intake from microbial breakdown — a classic example of mutualism. Moreover, dietary polysaccharides that reach the human large intestine have a major impact on gut microbial ecology and health. Insight into the molecular mechanisms by which different gut bacteria use polysaccharides is, therefore, of fundamental importance. Genomic analyses of the gut microbiota could revolutionize our understanding of these mechanisms and provide new biotechnological tools for the conversion of polysaccharides, including lignocellulosic biomass, into monosaccharides.

The rise of agro-industrial activities over recent decades has exponentially increased lignocellulose biomasses (LCB) production. LCB serves as a cost-effective source for fermentable sugars and other renewable chemicals. This study explores the use of microbial consortia, particularly thermophilic consortia, for LCB deconstruction. Thermophiles produce stable enzymes that retain activity under industrial conditions, presenting a promising approach for LCB conversion. This research focused on two microbial consortia (i.e., microbiomes) that were analyzed for enzyme production using a cheap medium, i.e., a mixture of spent mushroom substrate (SMS) and digestate. The secreted xylanolytic enzymes were characterized in terms of temperature and pH optima, thermal stability, and hydrolysis products from LCB-derived polysaccharides. These enzymes showed optimal activity aligning with common biorefinery conditions and outperformed a formulated enzyme mixture in thermostability tests in the digestate. Phylogenetic and genomic analyses highlighted the genetic diversity and metabolic potential of these microbiomes. Bacillus licheniformis was identified as a key species, with two distinct strains contributing to enzyme production. The presence of specific glycoside hydrolases involved in the cellulose and hemicellulose degradation underscores these consortia’s capacity for efficient LCB conversion. These findings highlight the potential of thermophilic microbiomes, isolated from an industrial environment, as a robust source of robust enzymes, paving the way for more sustainable and cost-effective bioconversion processes in biofuel and biochemical production and other biotechnological applications.

Biological (or reductive) soil disinfestation (BSD or RSD) is a bioremediation process to control soil-borne plant pathogens using activities of indigenous bacteria in the soil. Three obligate anaerobic bacterial strains (TW1, TW10, and TB10), which were isolated from anoxic soil subjected to BSD treatments, were examined for their abilities to produce anti-fungal enzymes. All strains were affiliated with the different lineages of the genus Clostridium. The three strains decomposed β-1,3-glucans (curdlan and laminarin), and β-1,3-glucanase activities were detected from their culture supernatants with these glucans. The three strains also produced the enzyme with wheat bran as a growth substrate and killed the Fusarium pathogen (Fusarium oxysporum f. sp. spinaciae) in the anaerobic co-incubation conditions. Observation by fluorescence microscopy of the pathogen cells showed that the three strains had degraded the fungal cells in different manners upon co-incubation with wheat bran. When the three strains were cultivated with the dead cells or the cell wall samples prepared from the Fusarium pathogen, strain TW1 utilized these materials as easily decomposable substrates by releasing β-1,3-glucanase. When observed by fluorescence microscopy, it appeared that strain TW1 degraded the mycelial cell wall nearly thoroughly, with the septa remaining as undecomposed luminous rings. In contrast, the other two strains decomposed neither the dead cells nor the cell wall samples directly. The results indicate that the various anaerobic bacteria proliferated in the soil under the BSD treatments should play key roles as an organized bacterial community to eliminate fungal pathogens, namely by release of anti-fungal enzymes with different properties.Key points•Three clostridial strains isolated from BSD-treated soils produced β-1,3-glucanase.•All strains killed the Fusarium pathogen in the anaerobic co-incubation conditions.•One of the strains produced β-1,3-glucanase with the fungal cell wall as a substrate.•The strain degraded the cell wall almost completely, except for the mycelial septa.

Production of biofuels and other value-added products from lignocellulose breakdown requires the coordinated metabolic activity of varied microorganisms. The increasing global demand for biofuels encourages the development and optimization of production strategies. Optimization in turn requires a thorough understanding of the microbial mechanisms and metabolic pathways behind the formation of each product of interest. Hydrolysis of lignocellulosic biomass is a bottleneck in its industrial use and often affects yield efficiency. The accessibility of the biomass to the microorganisms is the key to the release of sugars that are then taken up as substrates and subsequently transformed into the desired products. While the effects of different metabolic intermediates in the overall production of biofuel and other relevant products have been studied, the role of proteins and their activity under anaerobic conditions has not been widely explored. Shifts in enzyme production may inform the state of the microorganisms involved; thus, acquiring insights into the protein production and enzyme activity could be an effective resource to optimize production strategies. The application of proteomic analysis is currently a promising strategy in this area. This review deals on the aspects of enzymes and proteomics of bioprocesses of biofuels production using lignocellulosic biomass as substrate.

Recently, a new oxygenic pathway has been proposed based on the disproportionation of NO with putative NO dismutase (Nod). In addition to a new process in nitrogen cycling, this process provides ecological advantages for the degradation of substrates in anaerobic conditions, which is of great significance for wastewater treatment. However, the Nod distribution in aquatic environments is rarely investigated. In this study, we obtained the nod genes with an abundance of 2.38 ± 0.96 × 105 copies per gram of dry soil from the Zoige wetland and aligned the molecular characteristics in the corresponding Nod sequences. These Nod sequences were not only found existing in NC10 bacteria, but were also found forming some other clusters with Nod sequences from a WWTP reactor or contaminated aquifers. Moreover, a new subcluster in the aquifer-similar cluster was even dominant in the Zoige wetland and was named the Z-aquifer subcluster. Additionally, soils from the Zoige wetland showed a high potential rate (10.97 ± 1.42 nmol of CO2 per gram of dry soil per day) for nitrite-dependent anaerobic methane oxidation (N-DAMO) with low abundance of NC10 bacteria, which may suggest a potential activity of Nod in other clusters when considering the dominance of the Z-aquifer subcluster Nod. In conclusion, we verified the occurrence of Nod in an alpine wetland for the first time and found a new subcluster to be dominant in the Zoige wetland. Moreover, this new subcluster of Nod may even be active in the N-DAMO process in this alpine wetland, which needs further study to confirm.

The properties and microbial turnover of exopolymeric substances (EPS) were measured in a hypersaline nonlithifying microbial mat (Eleuthera, Bahamas) to investigate their potential role in calcium carbonate (CaCO₃) precipitation. Depth profiles of EPS abundance and enzyme activities indicated that c. 80% of the EPS were turned over in the upper 15-20 mm. Oxic and anoxic mat homogenates amended with low-molecular-weight (LMW) organic carbon, sugar monomers, and different types of EPS revealed rapid consumption of all substrates. When comparing the consumption of EPS with that of other substrates, only marginally longer lag times and lower rates were observed. EPS (5-8%) were readily consumed during the conversion of labile to refractory EPS. This coincided with a decrease in glucosidase activity and a decrease in the number of acidic functional groups on the EPS. Approximately half of the calcium bound to the EPS remained after 10 dialyses steps. This tightly bound calcium was readily available to precipitate as CaCO₃. We present a conceptual model in which LMW organic carbon complexed with the tightly bound calcium is released upon enzyme activity. This increases alkalinity and creates binding sites for carbonate and allows CaCO₃ to precipitate. Therefore, this model explains interactions between EPS and CaCO₃ precipitation, and underscores the critical role of aerobic and anaerobic microorganisms in early diagenesis and lithification processes.

The recalcitrance of plant biomass is the most important barrier to its economic conversion by microbes to products of interest. Thermophiles have special advantages for biomass conversion and members of the genus Caldicellulosiruptor are the most thermophilic cellulolytic microbes known. In this study, we report the construction of a replicating shuttle vector for Caldicellulosiruptor species based on pBAS2, the smaller of two native C. bescii plasmids. The entire plasmid was cloned into an E. coli cloning vector containing a pSC101 origin of replication and an apramycin resistance cassette for selection in E. coli. The wild-type C. bescii pyrF locus was cloned under the transcriptional control of the regulatory region of the ribosomal protein S30EA (Cbes2105), and the resulting vector was transformed into a new spontaneous deletion mutant in the pyrFA locus of C. bescii that allowed complementation with the pyrF gene alone. Plasmid DNA was methylated in vitro with a recently described cognate methyltransferase, M.CbeI, and transformants were selected for uracil prototrophy. The plasmid was stably maintained in low copy with selection but rapidly lost without selection. There was no evidence of DNA rearrangement during transformation and replication in C. bescii. A similar approach was used to screen for transformability of other members of this genus using M.CbeI to overcome restriction as a barrier and was successful for transformation of C. hydrothermalis, an attractive species for many applications. Plasmids containing a carbohydrate binding domain (CBM) and linker region from the C. bescii celA gene were maintained with selection and were structurally stable through transformation and replication in C. bescii and E. coli.

▪ Abstract  The natural production and anthropogenic release of halogenated hydrocarbons into the environment has been the likely driving force for the evolution of an unexpectedly high microbial capacity to dehalogenate different classes of xenobiotic haloorganics. This contribution provides an update on the current knowledge on metabolic and phylogenetic diversity of anaerobic microorganisms that are capable of dehalogenating—or completely mineralizing—halogenated hydrocarbons by fermentative, oxidative, or reductive pathways. In particular, research of the past decade has focused on halorespiring anaerobes, which couple the dehalogenation by dedicated enzyme systems to the generation of energy by electron transport–driven phosphorylation. Significant advances in the biochemistry and molecular genetics of degradation pathways have revealed mechanistic and structural similarities between dehalogenating enzymes from phylogenetically distinct anaerobes. The availability of two almost complete genome sequences of halorespiring isolates recently enabled comparative and functional genomics approaches, setting the stage for the further exploitation of halorespiring and other anaerobic dehalogenating microbes as dedicated degraders in biological remediation processes.