Nitrogen and sulfur for phosphorus: Lipidome adaptation of anaerobic sulfate-reducing bacteria in phosphorus-deprived conditions.

Understanding how microbial lipidomes adapt to environmental and nutrient stress is crucial for comprehending microbial survival and functionality. Certain anaerobic bacteria can synthesize glycerolipids with ether/ester bonds, yet the complexities of their lipidome remodeling under varying physicochemical and nutritional conditions remain largely unexplored. In this study, we thoroughly examined the lipidome adaptations of Desulfatibacillum alkenivorans strain PF2803T, a mesophilic anaerobic sulfate-reducing bacterium known for its high proportions of alkylglycerol ether lipids in its membrane, under various cultivation conditions including temperature, pH, salinity, and ammonium and phosphorous concentrations. Employing an extensive analytical and computational lipidomic methodology, we identified an assemblage of nearly 400 distinct lipids, including a range of glycerol ether/ester lipids with various polar head groups. Information theory-based analysis revealed that temperature fluctuations and phosphate scarcity profoundly influenced the lipidome's composition, leading to an enhanced diversity and specificity of novel lipids. Notably, phosphorous limitation led to the biosynthesis of novel glucuronosylglycerols and sulfur-containing aminolipids, termed butyramide cysteine glycerols, featuring various ether/ester bonds. This suggests a novel adaptive strategy for anaerobic heterotrophs to thrive under phosphorus-depleted conditions, characterized by a diverse array of nitrogen- and sulfur-containing polar head groups, moving beyond a reliance on conventional nonphospholipid types.

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

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

Pontiella agarivorans sp. nov., a novel marine anaerobic bacterium capable of degrading macroalgal polysaccharides and fixing nitrogen.

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 É©-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.

Tartrate fermentation with H2 production by a new member of Sporomusaceae enriched from rice paddy soil.

In rice paddies, soil and plant-derived organic matter are degraded anaerobically to methane (CH4), a powerful greenhouse gas. The highest rate of methane emission occurs during the reproductive stage of the plant when mostly dicarboxylic acids are exudated by the roots. The emission of methane at this stage depends largely on the cooperative interaction between dicarboxylic acid-fermenting bacteria and methanogenic archaea in the rhizosphere. The fermentation of tartrate, one of the major acids exudated, has been scarcely explored in rice paddy soils. In this work, we characterized an anaerobic consortium from rice paddy soil composed of four bacterial strains, whose principal member (LT8) can ferment tartrate, producing H2 and acetate. Tartrate fermentation was accelerated by co-inoculation with a hydrogenotrophic methanogen. The assembled genome of LT8 possesses a Na+-dependent oxaloacetate decarboxylase and shows that this bacterium likely invests part of the H2 produced to reduce NAD(P)+ to assimilate C from tartrate. The phylogenetic analysis of the 16S rRNA gene, the genome-based classification as well as the average amino acid identity (AAI) indicated that LT8 belongs to a new genus within the Sporomusaceae family. LT8 shares a few common features with its closest relatives, for which tartrate degradation has not been described. LT8 is limited to a few environments but is more common in rice paddy soils, where it might contribute to methane emissions from root exudates.IMPORTANCEThis is the first report of the metabolic characterization of a new anaerobic bacterium able to degrade tartrate, a compound frequently associated with plants, but rare as a microbial metabolite. Tartrate fermentation by this bacterium can be coupled to methanogenesis in the rice rhizosphere where tartrate is mainly produced at the reproductive stage of the plant, when the maximum methane rate emission occurs. The interaction between secondary fermentative bacteria, such as LT8, and methanogens could represent a fundamental step in exploring mitigation strategies for methane emissions from rice fields. Possible strategies could include controlling the activity of these secondary fermentative bacteria or selecting plants whose exudates are more difficult to ferment.

Genomic insights into clostridia in bioenergy production: Comparison of metabolic capabilities and evolutionary relationships.

Bacteria from diverse genera, including Acetivibrio, Bacillus, Cellulosilyticum, Clostridium, Desulfotomaculum, Lachnoclostridium, Moorella, Ruminiclostridium, and Thermoanaerobacterium, have attracted significant attention due to their versatile metabolic capabilities encompassing acetogenic, cellulolytic, and C1-metabolic properties, and acetone-butanol-ethanol fermentation. Despite their biotechnological significance, a comprehensive understanding of clostridial physiology and evolution has remained elusive. This study reports an extensive comparative genomic analysis of 48 fully sequenced bacterial genomes from these genera. Our investigation, encompassing pan-genomic analysis, central carbon metabolism comparison, exploration of general genome features, and in-depth scrutiny of Cluster of Orthologous Groups genes, has established a holistic whole-genome-based phylogenetic framework. We have classified these strains into acetogenic, butanol-producing, cellulolytic, CO2-fixating, chemo(litho/organo)trophic, and heterotrophic categories, often exhibiting overlaps. Key outcomes include the identification of misclassified species and the revelation of insights into metabolic features, energy conservation, substrate utilization, stress responses, and regulatory mechanisms. These findings can provide guidance for the development of efficient microbial systems for sustainable bioenergy production. Furthermore, by addressing fundamental questions regarding genetic relationships, conserved genomic features, pivotal enzymes, and essential genes, this study has also contributed to our comprehension of clostridial biology, evolution, and their shared metabolic potential.

Mechanisms of microbial co-aggregation in mixed anaerobic cultures.

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

Oxygen tolerance in anaerobes as a virulence factor and a health-beneficial property.

Oxygen tolerance of anaerobes is a virulence factor, but can also be a beneficial property. Many species have evolved to tolerate or take advantage of the presence of low, especially nanaerobic (≤0.14 %) oxygen concentrations. Oxygen tolerance is genus-, species- and strain-dependent according to their protective mechanisms. It was better expressed in some pathogenic species such as Bacteroides fragilis, Clostridioides difficile, and Clostridium perfringens, as well as in Akkermansia muciniphila than in other potential probiotics such as Alistipes, Blautia and Roseburia spp. Different degrees of oxygen sensitivity were found between the strains of Anaerostipes, Faecalibacterium, and Bifidobacterium spp. Importantly, clostridial spores and anaerobes in biofilms are protected from oxidation. Rubrerythrins and flavodiiron proteins and two regulators (sigma factor B and PerR) contribute to C. difficile protection from reactive oxygen species (ROS). The frequent pathogen, B. fragilis, has numerous protective factors such as enzymes (catalase, superoxide dismutase, alkyl hydroperoxidase, thioredoxin peroxidase, and aerobic-type NrdAB ribonucleotide reductase), and nanaerobic respiration. Seven proteins confer strain-specific oxygen adaptation of Faecalibacterium prausnitzii. Oxygen tolerance protects anaerobes from ROS, shields their DNA and modulates gene expression. Furthermore, oxygen can induce mutations leading to antibiotic resistance as shown in Prevotella melaninogenica. Some Faecalibacterium, Anaerostipes, Bifidobacterium, and Akkermansia strains from the intestinal microbiota exhibiting oxygen tolerance may become next-generation probiotic candidates. Further studies are needed to reveal oxygen effects on more anaerobic species and strains, and the influence of oxygen on antibiotic resistance. More studies on oxygen-tolerant probiotic strains can be useful to optimize biotechnological methods.

Enhanced biomethane production from waste activated sludge anaerobic digestion by ceramsite and amended Fe2O3 ceramsite.

Wastes recycling and reutilization technique could simultaneously fulfill waste control and energy recovery sustainably, which has attracted increasing attention. This work proposed a novel waste reuse technology utilizing ceramsite and amended Fe2O3-ceramsite made from waste activated sludge (WAS) as additives to promote the yield of methane from WAS anaerobic digestion (AD). Experimental results demonstrated that compared to the control (85.05 ± 0.2 mL CH4/g-VS), the cumulative methane yield was effectively enhanced by 14% and 40% when ceramsite and Fe2O3-ceramsite were added. Further investigation revealed that ceramsite, especially the Fe2O3-ceramsite, enriched the populations of key anaerobes involved in hydrolysis, acidification, and methanogenesis. Meanwhile, potential syntrophic metabolisms between syntrophic bacteria and methanogens were confirmed in the Fe2O3-ceramsite AD system. Mechanisms studies exhibited that ceramsite and Fe2O3-ceramsite reinforced intermediate processes for methane production. The favorable pore structure, enhanced Fe (III) reduction capacity and conductivity also contributed a lot to the AD process.

Achieving sustainable trichloroethylene removal from nitrate-containing groundwater: Effects of particle size and dosage of microscale zero-valent iron on its synergistic action with anaerobic bacteria.

The coupling of microscale zero-valent iron (mZVI) and anaerobic bacteria (AB) has gained increasing attention due to its ability to enhance dechlorination efficiency by combining the advantages of chemical and microbial reduction. However, the implementation of these coupling technologies at the field scale is challenging in terms of sustainability goals due to the coexistence of various natural electron acceptors in groundwater, which leads to limited electron selectivity and increased secondary risk. Therefore, this study used trichloroethylene (TCE) as a probe contaminant and nitrate (NO3-) as a typical co-occurring natural electron acceptor to optimize the overall sustainable remediation performance of an mZVI/AB coupled system by adjusting the mZVI particle size and dosage. Results revealed that mZVI particles of different sizes exhibit different microorganism activation capabilities. In contrast to its 2 µm and 7 µm counterparts, the 30 µm mZVI/AB system demonstrated a strong dosage-dependency in TCE removal and its product selectivity. Finally, multi-criteria analysis (MCA) methods were established to comprehensively rank the alternatives, and 30 µm mZVI (15 g/L dosage) was determined to be the best remediation strategy with the highest total sustainability score under all studied hydro-chemical conditions when equal weights were applied to technical, environmental, and economic indicators. Our work provides a paradigm for comprehensively assessing the sustainable remediation performance of chlorinated aliphatic hydrocarbons polluted groundwater in practical applications.

Enhancement of electron transfer via magnetite in nitrite-dependent anaerobic methane oxidation system.

Nitrite-dependent anaerobic methane oxidation (n-DAMO) is a novel denitrification process that simultaneously further removes and utilizes methane from anaerobic effluent from wastewater treatment plants. However, the metabolic activity of n-DAMO bacteria is relative low for practical application. In this study, conductive magnetite was added into lab-scale sequencing batch reactor inoculated with n-DAMO bacteria to study the influence on n-DAMO process. With magnetite amendment, the nitrogen removal rate could reach 34.9 mg N·L-1d-1, nearly 2.5 times more than that of control group. Magnetite significantly facilitated the interspecies electron transfer and built electrically connected community with high capacitance. Enzymatic activities of electron transport chain were significantly elevated. Functional gene expression and enzyme activities associated with nitrogen and methane metabolism had been highly up-regulated. These results not only propose a useful strategy in n-DAMO application but also provide insights into the stimulating mechanism of magnetite in n-DAMO process.

Isolation, Genomic Sequence and Physiological Characterization of Parageobacillus sp. G301, an Isolate Capable of Both Hydrogenogenic and Aerobic Carbon Monoxide Oxidation.

Prokaryotes that can oxidize carbon monoxide (CO oxidizers) can use this gas as a source of carbon or energy. They oxidize carbon monoxide with carbon monoxide dehydrogenases (CODHs): these are divided into nickel-containing CODH (Ni-CODH), which are sensitive to O2, and molybdenum-containing CODH (Mo-CODH), which can function aerobically. The oxygen conditions required for CO oxidizers to oxidize CO may be limited, as those which have been isolated and characterized so far contain either Ni- or Mo-CODH. Here, we report a novel CO oxidizer, Parageobacillus sp. G301, which is capable of CO oxidation using both types of CODH based on genomic and physiological characterization. This thermophilic, facultatively anaerobic Bacillota bacterium was isolated from the sediments of a freshwater lake. Genomic analyses revealed that strain G301 possessed both Ni-CODH and Mo-CODH. Genome-based reconstruction of its respiratory machinery and physiological investigations indicated that CO oxidation by Ni-CODH was coupled with H2 production (proton reduction), whereas CO oxidation by Mo-CODH was coupled with O2 reduction under aerobic conditions and nitrate reduction under anaerobic conditions. G301 would thus be able to thrive via CO oxidation under a wide range of conditions, from aerobic environments to anaerobic environments, even with no terminal electron acceptors other than protons. Comparative genome analyses revealed no significant differences in genome structures and encoded cellular functions, except for CO oxidation between CO oxidizers and non-CO oxidizers in the genus Parageobacillus; CO oxidation genes are retained exclusively for CO metabolism and related respiration. IMPORTANCE Microbial CO oxidation has received much attention because it contributes to global carbon cycling in addition to functioning as a remover of CO, which is toxic to many organisms. Some microbial CO oxidizers, including both bacteria and archaea, exhibit sister relationships with non-CO oxidizers even in genus-level monophyletic groups. In this study, we demonstrated that a new isolate, Parageobacillus sp. G301, is capable of both anaerobic (hydrogenogenic) and aerobic CO oxidation, which has not been previously reported. The discovery of this new isolate, which is versatile in CO metabolism, will accelerate research on CO oxidizers with diverse CO metabolisms, expanding our understanding of microbial diversity. Through comparative genomic analyses, we propose that CO oxidation genes are not essential genetic elements in the genus Parageobacillus, providing insights into the factors which shape the punctate distribution of CO oxidizers in the prokaryote tree, even in genus-level monophyletic groups.

A (S)-3-Hydroxybutyrate Dehydrogenase Belonging to the 3-Hydroxyacyl-CoA Dehydrogenase Family Facilitates Hydroxyacid Degradation in Anaerobic Bacteria.

Ketone bodies, including acetoacetate, 3-hydroxybutyrate, and acetone, are produced in the liver of animals during glucose starvation. Enzymes for the metabolism of (R)-3-hydroxybutyrate have been extensively studied, but little is known about the metabolism of its enantiomer (S)-3-hydroxybutyrate. Here, we report the characterization of a novel pathway for the degradation of (S)-3-hydroxybutyrate in anaerobic bacteria. We identify and characterize a stereospecific (S)-3-hydroxylbutyrate dehydrogenase (3SHBDH) from Desulfotomaculum ruminis, which catalyzes the reversible NAD(P)H-dependent reduction of acetoacetate to form (S)-3-hydroxybutyrate. 3SHBDH also catalyzes oxidation of d-threonine (2R, 3S) and l-allo-threonine (2S, 3S), consistent with its specificity for ß-(3S)-hydroxy acids. Isothermal calorimetry experiments support a sequential mechanism involving binding of NADH prior to (S)-3-hydroxybutyrate. Homologs of 3SHBDH are present in anaerobic fermenting and sulfite-reducing bacteria, and experiments with Clostridium pasteurianum showed that 3SHBDH, acetate CoA-transferase (YdiF), and (S)-3-hydroxybutyryl-CoA dehydrogenase (Hbd) are involved together in the degradation of (S)-3-hydroxybutyrate as a carbon and energy source for growth. (S)-3-hydroxybutyrate is a human metabolic marker and a chiral precursor for chemical synthesis, suggesting potential applications of 3SHBDH in diagnostics or the chemicals industry. IMPORTANCE (R)-3-hydroxybutyrate is well studied as a component of ketone bodies produced by the liver and of bacterial polyesters. However, the biochemistry of its enantiomer (S)-3-hydroxybutyrate is poorly understood. This study describes the identification and characterization of a stereospecific (S)-3-hydroxylbutyrate dehydrogenase and its function in a metabolic pathway for the degradation of (S)-3-hydroxybutyrate as a carbon and energy source in anaerobic bacteria. (S)-3-hydroxybutyrate is a mammalian metabolic marker and a precursor for chemical synthesis and bioplastics, suggesting potential applications of these enzymes in diagnostics and biotechnology.

Hydrogen online monitoring based on thermal conductivity for anaerobic microorganisms.

H2 -producing microorganisms are a promising source of sustainable biohydrogen. However, most H2 -producing microorganisms are anaerobes, which are difficult to cultivate and characterize. While several methods for measuring H2 exist, common H2 sensors often require oxygen, making them unsuitable for anaerobic processes. Other sensors can often not be operated at high gas humidity. Thus, we applied thermal conductivity (TC) sensors and developed a parallelized, online H2 monitoring for time-efficient characterization of H2 production by anaerobes. Since TC sensors are nonspecific for H2 , the cross-sensitivity of the sensors was evaluated regarding temperature, gas humidity, and CO2 concentrations. The systems' measurement range was validated with two anaerobes: a high H2 -producer (Clostridium pasteurianum) and a low H2 -producer (Phocaeicola vulgatus). Online monitoring of H2 production in shake flask cultivations was demonstrated, and H2 transfer rates were derived. Combined with online CO2 and pressure measurements, molar gas balances of the cultivations were closed, and an anaerobic respiration quotient was calculated. Thus, insight into the effect of medium components and inhibitory cultivation conditions on H2 production with the model anaerobes was gained. The presented online H2 monitoring method can accelerate the characterization of anaerobes for biohydrogen production and reveal metabolic changes without expensive equipment and offline analysis.

Deciphering different effects of ZVI and NaOH on metabolic characteristics in the process of methanogenesis recovery from VFA suppression.

Dosing zero valent iron (ZVI) or sodium hydroxide (NaOH) is the common method of addressing acidification in anaerobic digestion (AD) systems; however, few studies have discussed and compared their effects on microbial metabolism. In the present study, microbial syntrophy and metabolic pathways under ZVI and NaOH regulation are comparatively analyzed through microbial network analysis and metagenomic/metaproteomic analyses. CH4 yield in the ZVI reactor was 414 mL/gVS, an increase of 23% when compared with that in the reactor with NaOH dosing (336 mL/gVS). The methanogenesis recovery period in the ZVI reactor (37 days) was shorter than that in the NaOH reactor (48 days). Co-occurrence networks indicated that ZVI promoted Methanoculleus and Methanosarcina to establish a complex syntrophic association with SAO bacteria (Syntrophaceticus and Aminobacterium) and syntrophic acetogens (Syntrophomonas), strengthening SAO-hydrogenotrophic methanogenesis (HM) and acetoclastic methanogenesis (AM) pathways simultaneously. Metagenomic analysis showed that the relative abundance of mcrA and fwdB in the ZVI reactor was higher 27% than that in the NaOH reactor. Furthermore, through metaproteomics analysis, much more enzymes related to glucose degradation, bioconversion of butyric acid and pyruvate, conversion of formate and acetate to CO2, and production of CH4 from acetate and CO2 were significantly upregulated under ZVI regulation than under NaOH regulation (fold change relative to control [FC] > 1.5, p < 0.05). The results of the present study enhance our understanding of methanogenic mechanisms under the regulation of ZVI, providing a theoretical basis for its practical application in AD systems experiencing VFA suppression.

Anaerobic bacterial response to nitric oxide stress: Widespread misconceptions and physiologically relevant responses.

How anaerobic bacteria protect themselves against nitric oxide-induced stress is controversial, not least because far higher levels of stress were used in the experiments on which most of the literature is based than bacteria experience in their natural environments. This results in chemical damage to enzymes that inactivates their physiological function. This review illustrates how transcription control mechanisms reveal physiological roles of the encoded gene products. Evidence that the hybrid cluster protein, Hcp, is a major high affinity NO reductase in anaerobic bacteria is reviewed: if so, its trans-nitrosation activity is a nonspecific secondary consequence of chemical inactivation. Whether the flavorubredoxin, NorV, is equally effective at such low [NO] is unknown. YtfE is proposed to be an enzyme rather than a source of iron for the repair of iron-sulfur proteins damaged by nitrosative stress. Any reaction catalyzed by YtfE needs to be revealed. The concentration of NO that accumulates in the cytoplasm of anaerobic bacteria is unknown, but indirect evidence indicates that it is in the pM to low nM range. Also unknown are the functions of the NO-inducible cytoplasmic proteins YgbA, YeaR, or YoaG. Experiments to resolve some of these questions are proposed.

Metagenomic evidence of a novel family of anammox bacteria in a subsea environment.

Bacteria in the order 'Candidatus Brocadiales' within the phylum Planctomycetes (Planctomycetota) have the remarkable ability to perform anaerobic ammonium oxidation (anammox). Two families of anammox bacteria with different biogeographical distributions have been reported, marine Ca. Scalinduaceae and freshwater Ca. Brocadiaceae. Here we report evidence of three new species within a novel genus and family of anammox bacteria, which were discovered in biofilms of a subsea road tunnel under a fjord in Norway. In this particular ecosystem, the nitrogen cycle is likely fuelled by ammonia from organic matter degradation in the fjord sediments and the rock mass above the tunnel, resulting in the growth of biofilms where anammox bacteria can thrive under oxygen limitation. We resolved several metagenome-assembled genomes (MAGs) of anammox bacteria, including three Ca. Brocadiales MAGs that could not be classified at the family level. MAGs of this novel family had all the diagnostic genes for a full anaerobic ammonium oxidation pathway in which nitrite was probably reduced by a NirK-like reductase. A survey of published molecular data indicated that this new family of anammox bacteria occurs in many marine sediments, where its members presumably would contribute to nitrogen loss.

The aerobic electron flux is deficient in fumarate respiration of a strict anaerobe Bacteroides thetaiotaomicron.

Why oxygen ceases the growth of strictly anaerobic bacteria is a longstanding question, yet the answer remains unclear. Studies have confirmed that the dehydratase-fumarase containing an iron-sulfur cluster ([4Fe-4S]) is inactivated upon exposure to oxygen in the intestinal obligate anaerobe, Bacteroides thetaiotaomicron (B. thetaiotaomicron); this blocks fumarate respiration, which is the essential energy-producing pathway in anaerobes. Here, we substituted the [4Fe-4S]-dependent fumarase in B. thetaiotaomicron with an iron-free isozyme from E. coli (Ec-FumC). Results show that Ec-FumC successfully performed the catalytic function of fumarase in B. thetaiotaomicron, as the fum-mutant strain that expressed Ec-FumC exhibited succinate-producing ability under anaerobic growth conditions. Ec-FumC is oxygen-resistant and remains active to produce fumarate upon aeration; however, B. thetaiotaomicron mutant that expressed Ec-FumC did not convert fumarate to succinate during air exposure. Biochemical assays of inverted membrane vesicles from wild-type B. thetaiotaomicron confirmed that the electron flux from NADH to fumarate was less efficient in the presence of air as compared to that without oxygen. Our findings suggest that the anaerobic fumarate respiration might be paralyzed due to electron dissipations upon aeration of the obligate anaerobe.

A Type III Polyketide Synthase Specific for Sporulating Negativicutes is Responsible for Alkylpyrone Biosynthesis.

Genomic analyses indicate that anaerobic bacteria represent a neglected source of natural products. Whereas a limited number of polyketides have been reported from anaerobes, products of type III polyketide synthases (PKSs) have remained unknown. We found a highly conserved biosynthetic gene cluster (BGC) comprising genes putatively encoding a type III PKS and a methyltransferase in genomes of the Negativicutes, strictly anaerobic, diderm bacteria. By in vivo and in vitro expression of a type III PKS gene, dquA from the oak-associated Dendrosporobacter quercicolus in E. coli we show production of long-chain alkylpyrones. Intriguingly, this BGC is specific for sporulating Sporomusaceae but absent in related Negativicutes that do not sporulate, thus suggesting a physiological role.

Novel Long-Chain Fatty Acid (LCFA)-Degrading Bacteria and Pathways in Anaerobic Digestion Promoted by Hydrochar as Revealed by Genome-Centric Metatranscriptomics Analysis.

A large amount of long-chain fatty acids (LCFA) are generated after lipids hydrolysis in anaerobic digestion (AD), and LCFA are difficult to be biodegraded. This study showed that hydrochar (HC), which was produced during the hydrothermal liquefaction of organic wastes, significantly increased the methane production rate (by 56.9%) of oleate, a typical refractory model LCFA. Genomic-centric metatranscriptomics analysis revealed that three novel microbes (Bin138 Spirochaetota sp., Bin35 Smithellaceae sp., and Bin54 Desulfomonilia sp.) that were capable of degrading LCFA were enriched by HC, which played an important role in the degradation of oleate. LCFA was degraded to acetate through the well-known LCFA ß-oxidation pathway and the combined ß-oxidation and butyrate oxidation pathway. In addition, it was found that HC promoted the direct interspecies electron transfer (DIET) between Methanothrix sp. and Bin54 Desulfomonilia sp. The enriched new types of LCFA-degrading bacteria and the promotion of DIET contributed to the improved methane production rate of oleate by HC. IMPORTANCE Long-chain fatty acids (LCFA) are difficult to be degraded in anaerobic digestion (AD), and the known LCFA degrading bacteria are only limited to the families Syntrophomonadaceae and Syntrophaceae. Here, we found that hydrochar effectively promoted AD of LCFA, and the new LCFA-degrading bacteria and a new metabolic pathway were also revealed based on genomic-centric metatranscriptomic analysis. This study provided a new method for enhancing the AD of organic wastes with high content of LCFA and increased the understanding of the microbes and their metabolic pathways involved in AD of LCFA.

Hormesis-Like Effects of Tetrabromobisphenol A on Anaerobic Digestion: Responses of Metabolic Activity and Microbial Community.

Tetrabromobisphenol A (TBBPA) has extensive applications in various fields; its release into ecosystems and the potential toxic effects on organisms are becoming major concerns. Here, we investigated the effects of TBBPA on anaerobic digestion, whose process is closely related to the carbon cycles under anaerobic conditions. The results revealed that TBBPA exhibited dose-dependent hormesis-like effects on methane production from glucose, i.e., the presence of 0.1 mg/L TBBPA increased the methane production rate by 8.79%, but 1.0-4.0 mg/L TBBPA caused 3.45-28.98% of decrement. We found that TBBPA was bound by the tyrosine-like proteins of the extracellular polymeric substances of anaerobes and induced the increase of reactive oxygen species, whose slight accumulation stimulated the metabolism activities but high accumulation increased the apoptosis of anaerobes. Owing to the differences between individual anaerobes in tolerance, TBBPA at 0.1 mg/L stimulated the acidogenesis and hydrogenotrophic methanogenesis, whereas higher levels (i.e., 1.0-4.0 mg/L) severely restrained all of the processes of acidogenesis, acetogenesis, and methanogenesis. Along with the accumulation of bisphenol A (BPA) produced from TBBPA by Longilinea sp. and Pseudomonas sp., the methanogenic pathway was partly shifted from acetate-dependent to hydrogen-dependent direction, and the activities of carbon monoxide dehydrogenase and acetyl-CoA decarbonylase/synthase were inhibited, while acetate kinase and F420 were hormetically affected. These findings elucidated the mechanism of anaerobic syntrophic consortium responses to TBBPA, supplementing the potential environmental risks of brominated flame retardants.