Long-range electron proton coupling in respiratory complex I - insights from molecular simulations of the quinone chamber and antiporter-like subunits.

Respiratory complex I is a redox-driven proton pump. Several high-resolution structures of complex I have been determined providing important information about the putative proton transfer paths and conformational transitions that may occur during catalysis. However, how redox energy is coupled to the pumping of protons remains unclear. In this article, we review biochemical, structural and molecular simulation data on complex I and discuss several coupling models, including the key unresolved mechanistic questions. Focusing both on the quinone-reductase domain as well as the proton-pumping membrane-bound domain of complex I, we discuss a molecular mechanism of proton pumping that satisfies most experimental and theoretical constraints. We suggest that protonation reactions play an important role not only in catalysis, but also in the physiologically-relevant active/deactive transition of complex I.

Redox profile of silver catfish challenged with Aeromonas hydrophila and treated with hexane extract of Hesperozygis ringens (Benth.) Epling through immersion bath.

The growing increase in the fish farming sector has favored the establishment of bacterial outbreaks caused by Aeromonas hydrophila in several species. The hexane extract of Hesperozygis ringens (HEHR) (Lamiaceae) leaves increased the survival rate of silver catfish (Rhamdia quelen) experimentally infected by A. hydrophila. However, it is noteworthy that no reports have been found on the possible mechanisms of action of this extract in infected fish. This study aimed to evaluate the effect of the HEHR, administered through single immersion bath, on lipid peroxidation and antioxidant defenses in muscle and liver tissue of silver catfish challenged with A. hydrophila. The results showed that the oxidative status of silver catfish was altered, although oxidative stress was not triggered during the experiment. HEHR at 30 mg/L (HEHR30) was not characterized as a pro-oxidant agent in the presence of infection, unlike florfenicol and HEHR at 15 mg/L treatments in some cases. In short, HEHR30 provided an important increase in hepatic catalase activity, characterizing one of the possible mechanisms involved in the greater survival of fish experimentally infected by A. hydrophila. Additionally, HEHR30 did not induce lipid peroxidation, nor reduced antioxidant defenses of silver catfish infected or not by A. hydrophila.

Evolution of the redox-altered, two-tiered Muralha Flow in the Fronteira Oeste Rift, southern Paraná Volcanic Province.

The thorough redox alteration of a lava flow is an undescribed feature in intraplate basaltic provinces. The Early Cretaceous (134.5 Ma) Paraná Province displays that alteration in the major Muralha Flow. This oxidized and reduced flow from the southern part of the province was studied with satellite images, field surveying, petrography, and published whole rock geochemistry. The 100 x 100 km flow from the Cuesta de Haedo presents two hydrothermal tiers - lower Tier 1 is gray to white, upper Tier 2 is red. Iron oxyhydroxides characterize Tier 2. Tier 1 contains clay minerals, zeolites, pyrite and calcite, and agate (possibly amethyst) geodes. In a first event, the upper Tier 2 was oxidized by hot water from the underlying Guarani Paleoaquifer. The high water/rock ratio decreased due to porosity clogging by precipitation of secondary minerals, and the fluid became reducing. Lowering of Eh and pH was caused by reaction of water with reducing particles (calcite, organic molecules) present in the paleoerg sandstones and with fresh rock surfaces. A lower Tier 1 was then formed during slow, hot water percolation. Reduction was interrupted below 30 °C (calcite formation). Large scale, similar alteration occurred in all studied oceanic ridges and only rarely in continental environments.

FRBM Mini REVIEW: Chemogenetic approaches to probe redox dysregulation in heart failure.

Chemogenetics refers to experimental methods that use novel recombinant proteins that can be dynamically and uniquely regulated by specific biochemicals. Chemogenetic approaches allow the precise manipulation of cellular signaling to delineate the molecular pathways involved in both physiological and pathological disease states. Approaches utilizing yeast d-amino acid oxidase (DAAO) enable manipulation of intracellular redox metabolism through generation of hydrogen peroxide in the presence of d-amino acids and have led to the development of new and informative animal models to characterize the impact of oxidative stress in heart failure and neurodegeneration. These chemogenetic models, in which DAAO expression is regulated by different tissue-specific promoters, have led to a range of cardiac phenotypes. This review discusses chemogenetic approaches to manipulate oxidative stress in models of heart failure. These approaches provide new insights into the relationships between redox metabolism and normal and pathologic states in the heart, as well as in other diseases characterized by oxidative stress.

Mo than meets the eye: genomic insights into molybdoenzyme diversity of Seleniivibrio woodruffii strain S4T.

Seleniivibrio woodruffii strain S4T is an obligate anaerobe belonging to the phylum Deferribacterota. It was isolated for its ability to respire selenate and was also found to respire arsenate. The high-quality draft genome of this bacterium is 2.9 Mbp, has a G+C content of 48%, 2762 predicted genes of which 2709 are protein-coding, and 53 RNA genes. An analysis of the genome focusing on the genes encoding for molybdenum-containing enzymes (molybdoenzymes) uncovered a remarkable number of genes encoding for members of the dimethylsulfoxide reductase family of proteins (DMSOR), including putative reductases for selenate and arsenate respiration, as well as genes for nitrogen fixation. Respiratory molybdoenzymes catalyze redox reactions that transfer electrons to a variety of substrates that can act as terminal electron acceptors for energy generation. Seleniivibrio woodruffii strain S4T also has essential genes for molybdate transporters and the biosynthesis of the molybdopterin guanine dinucleotide cofactors characteristic of the active centers of DMSORs. Phylogenetic analysis revealed candidate respiratory DMSORs spanning nine subfamilies encoded within the genome. Our analysis revealed the untapped potential of this interesting microorganism and expanded our knowledge of molybdoenzyme co-occurrence.

A new strategy for improving the energy efficiency of electro-Fenton: Using N-doped activated carbon cathode with strong Fe(III) adsorption capacity to promote Fe(II) regeneration.

Fe(II) regeneration plays a crucial role in the electro-Fenton process, significantly influencing the rate of ·OH formation. In this study, a method is proposed to improve Fe(II) regeneration through N-doping aimed at enhancing the adsorption capacity of the activated carbon cathode for Fe(III). N-doping not only enriched the pore structure on the surface of activated carbon, providing numerous adsorption sites, but also significantly increased the adsorption energy for Fe(III). Among the types of nitrogen introduced, pyridine-N exhibited the most substantial enhancement effect, followed by pyrrole-N, while graphite-N showed a certain degree of inhibition. Furthermore, N-doping facilitated the adsorption of all forms of Fe(III) by activated carbon. The adsorption and electrosorption rates of the NAC-900 electrode for Fe(III) were 30.33% and 42.36%, respectively. Such modification markedly enhanced the Fe3+/Fe2+ cycle within the electro-Fenton system. The NAC-900 system demonstrated an impressive phenol degradation efficiency of 93.67%, alongside the lowest electricity consumption attributed to the effective "adsorption-reduction" synergy for Fe(III) on the NAC-900 electrode. Compared to the AC cathode electro-Fenton system, the degradation efficiency of the NAC-900 cathode electro-Fenton system at pH = levels ranging from 3 to 5 exceeded 90%; thus, extending the pH applicability of the electro-Fenton process. The degradation efficiency of phenol using the NAC-900 cathode electro-Fenton system in various water matrices approached 90%, indicating robust performance in real wastewater treatment scenarios. This research elucidates the impact of cathodic Fe(III) adsorption on Fe(II) regeneration within the electro-Fenton system, and clarifies the influence of different N- doping types on the cathodic adsorption of Fe(III).

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.

[Changes in protein requirements of elderly people over 5 years assessed using the indicator amino acid oxidation method].

OBJECTIVE: To evaluate the changes in protein requirements of the elderly during the past five years. METHODS: Based on the previous study of protein requirements of 14 elderly in 2017, 4 of these elderly(70-80 y) were included as study participants and protein requirements were re-evaluated using the indicator amino acid oxidation method. There were seven protein levels: 0.1, 0.3, 0.6, 0.9, 1.2, 1.5 and 1.8 g/(kg·d). Maintenance diets were given for the first two days of each protein level. A stable isotope study was conducted on the day 3, using L-~(13)C-phenylalanine as an indicator on the basis of an amino acid rationed diet, which was orally ingested into the body along with the amino acid rationed diet, and breath and urine samples were collected when the metabolism of L-~(13)C-phenylalanine reached steady state in the body. By measuring the kinetic parameters of labeled amino acids in the samples, a nonlinear mixed-effects model was constructed for the protein intake to be tested and the oxidation rate of labeled amino acids. The mean protein requirement of the study population was determined by the protein intake corresponding to the inflection point of the curve. RESULTS: Based on the production rate of ~(13)CO_2 in exhaled breath of four elderly people at different protein levels, the mean protein requirement was 1.05(95%CI 0.51-1.60) g/(kg·d). The protein recommended nutrient intake was 1.31(95%CI 0.64-2.00) g/(kg·d) was estimated by applying the coefficient of variation of the mean protein requirement to derive the recommended nutrient intake. CONCLUSION: Protein requirements in the elderly have increased over a five-year period and sarcopenia may be the main cause of increased protein requirements.

Potential Role of the Anammoxosome in the Adaptation of Anammox Bacteria to Salinity Stress.

The underlying adaptative mechanisms of anammox bacteria to salt stress are still unclear. The potential role of the anammoxosome in modulating material and energy metabolism in response to salinity stress was investigated in this study. The results showed that anammox bacteria increased membrane fluidity and decreased mechanical properties by shortening the ladderane fatty acid chain length of anammoxosome in response to salinity shock, which led to the breakdown of the proton motive force driving ATP synthesis and retarded energy metabolism activity. Afterward, the fatty acid chain length and membrane properties were recovered to enhance the energy metabolic activity. The relative transmission electron microscopy (TEM) area proportion of anammoxosome decreased from 55.9 to 38.9% under salinity stress. The 3D imaging of the anammox bacteria based on Synchrotron soft X-ray tomography showed that the reduction in the relative volume proportion of the anammoxosome and the concave surfaces was induced by salinity stress, which led to the lower energy expenditure of the material transportation and provided more binding sites for enzymes. Therefore, anammox bacteria can modulate nitrogen and energy metabolism by changing the membrane properties and morphology of the anammoxosome in response to salinity stress. This study broadens the response mechanism of anammox bacteria to salinity stress.

Enhancement Effect Induced by the Second Metal to Promote Ozone Catalytic Oxidation of VOCs.

It is a promising research direction to develop catalysts with high stability and ozone utilization for low-temperature ozone catalytic oxidation of VOCs. While bimetallic catalysts exhibit excellent catalytic activity compared with conventional single noble metal catalysts, limited success has been achieved in the influence of the bimetallic effect on the stability and ozone utilization of metal catalysts. Herein, it is necessary to systematically study the enhancement effect in the ozone catalytic reaction induced by the second metal. With a simple continuous impregnation method, a platinum-cerium bimetallic catalyst is prepared. Also highlighted are studies from several aspects of the contribution of the second metal (Ce) to the stability and ozone utilization of the catalysts, including the "electronic effect" and "geometric effect". The synergistic removal rate of toluene and ozone is nearly 100% at 30 °C, and it still shows positive stability after high humidity and a long reaction time. More importantly, the instructive significance, which is the in-depth knowledge of enhanced catalytic mechanism of bimetallic catalysts resulting from a second metal, is provided by this work.

Magnetic MoS2/Fe3O4 composite as an effective activator of persulfate for the degradation of tetracycline: performance, activation mechanisms and degradation pathways.

The activated persulfate (PS) process could produce sulfate radical (SO4·-) and rapidly degrade organic pollutants. The application of Fe3O4 as a promising PS activator was limited due to the rapid conversion of Fe2+ to Fe3+ on its surface. Mo4+ on MoS2 surface could be used as a reducing site to convert Fe3+ to Fe2+, but the separation and recovery of MoS2 was complex. In this study, MoS2/Fe3O4 was prepared to accelerate the Fe3+/Fe2+ cycle on Fe3O4 surface and achieved efficient separation of MoS2. The results showed that MoS2/Fe3O4 was more effective for PS activation compared to Fe3O4 or MoS2, with a removal efficiency of 91.8% for 20 mg·L-1 tetracycline (TC) solution under the optimal conditions. Fe2+ and Mo4+ on MoS2/Fe3O4 surface acted as active sites for PS activation with the generation of SO4•-, •OH, •O2-, and 1O2. Mo4+ acted as an electron donor to promote the Fe3+/Fe2+ cycling and thus improved the PS activation capability of MoS2/Fe3O4. The degradation pathways of TC were inferred as hydroxylation, ketylation of dimethylamino group and C-N bond breaking. This study provided a promising activated persulfate-based advanced oxidation process for the efficient degradation of TC by employing MoS2/Fe3O4 as an effective activator.

Bioenergetics of iron snow fueling life on Europa.

The main sources of redox gradients supporting high-productivity life in the Europan and other icy ocean world oceans were proposed to be photolytically derived oxidants, such as reactive oxygen species (ROS) from the icy shell, and reductants (Fe(II), S(-II), CH4, H2) from bottom waters reacting with a (ultra)mafic seafloor. Important roadblocks to maintaining life, however, are that the degree of ocean mixing to combine redox species is unknown, and ROS damage biomolecules. Here, we envisage a unique solution using an acid mine drainage (AMD)-filled pit lakes analog system for the Europan ocean, which previous models predicted to be acidic. We hypothesize that surface-generated ROS oxidize dissolved Fe(II) resulting in Fe(III) (hydr)oxide precipitates, that settle to the seafloor as "iron snow." The iron snow provides a respiratory substrate for anaerobic microorganisms ("breathing iron"), and limits harmful ROS exposure since they are now neutralized at the ice-water interface. Based on this scenario, we calculated Gibbs energies and maximal biomass productivities of various anaerobic metabolisms for a range of pH, temperatures, and H2 fluxes. Productivity by iron reducers was greater for most environmental conditions considered, whereas sulfate reducers and methanogens were more favored at high pH. Participation of Fe in the metabolic redox processes is largely neglected in most models of Europan biogeochemistry. Our model overcomes important conceptual roadblocks to life in icy ocean worlds and broadens the potential metabolic diversity, thus increasing total primary productivity, the diversity and volume of habitable environmental niches and, ultimately, the probability of biosignature detection.

Formation of Supplementary Metal-Binding Centers in Proteins under Stress Conditions.

In many proteins, supplementary metal-binding centers appear under stress conditions. They are known as aberrant or atypical sites. Physico-chemical properties of proteins are significantly changed after such metal binding, and very stable protein aggregates are formed, in which metals act as "cross-linking" agents. Supplementary metal-binding centers in proteins often arise as a result of posttranslational modifications caused by reactive oxygen and nitrogen species and reactive carbonyl compounds. New chemical groups formed as a result of these modifications can act as ligands for binding metal ions. Special attention is paid to the role of cysteine SH-groups in the formation of supplementary metal-binding centers, since these groups are the main target for the action of reactive species. Supplementary metal binding centers may also appear due to unmasking of amino acid residues when protein conformation changing. Appearance of such centers is usually considered as a pathological process. Such unilateral approach does not allow to obtain an integral view of the phenomenon, ignoring cases when formation of metal complexes with altered proteins is a way to adjust protein properties, activity, and stability under the changed redox conditions. The role of metals in protein aggregation is being studied actively, since it leads to formation of non-membranous organelles, liquid condensates, and solid conglomerates. Some proteins found in such aggregates are typical for various diseases, such as Alzheimer's and Huntington's diseases, amyotrophic lateral sclerosis, and some types of cancer.

Fenton Reaction in vivo and in vitro. Possibilities and Limitations.

The review considers the problem of hydrogen peroxide decomposition and hydroxyl radical formation in the presence of iron in vivo and in vitro. Analysis of the literature data allows us to conclude that, under physiological conditions, transport of iron, carried out with the help of carrier proteins, minimizes the possibility of appearance of free iron ions in cytoplasm of the cell. Under pathological conditions, when the process of transferring an iron ion from a donor protein to an acceptor protein can be disrupted due to modifications of the carrier proteins, iron ions can enter cytosol. However, at pH values close to neutral, which is typical for cytosol, iron ions are converted into water-insoluble hydroxides. This makes it impossible to decompose hydrogen peroxide according to the mechanism of the classical Fenton reaction. A similar situation is observed in vitro, since buffers with pH close to neutral are used to simulate free radical oxidation. At the same time, iron hydroxides are able to catalyze decomposition of hydrogen peroxide with formation of a hydroxyl radical. Decomposition of hydrogen peroxide with iron hydroxides is called Fenton-like reaction. Studying the features of Fenton-like reaction in biological systems is the subject of future research.

Free Radical Lipid Peroxidation Induced by Reactive Halogen Species.

The review is devoted to the mechanisms of free radical lipid peroxidation (LPO) initiated by reactive halogen species (RHS) produced in mammals, including humans, by heme peroxidase enzymes, primarily myeloperoxidase (MPO). It has been shown that RHS can participate in LPO both in the initiation and branching steps of the LPO chain reactions. The initiation step of RHS-induced LPO mainly involves formation of free radicals in the reactions of RHS with nitrite and/or with amino groups of phosphatidylethanolamine or Lys. The branching step of the oxidative chain is the reaction of RHS with lipid hydroperoxides, in which peroxyl and alkoxyl radicals are formed. The role of RHS-induced LPO in the development of human inflammatory diseases (cardiovascular and neurodegenerative diseases, cancer, diabetes, rheumatoid arthritis) is discussed in detail.

A Redox-Regulated, Heterodimeric NADH:cinnamate Reductase in Vibrio ruber.

Genes of putative reductases of α,ß-unsaturated carboxylic acids are abundant among anaerobic and facultatively anaerobic microorganisms, yet substrate specificity has been experimentally verified for few encoded proteins. Here, we co-produced in Escherichia coli a heterodimeric protein of the facultatively anaerobic marine bacterium Vibrio ruber (GenBank SJN56019 and SJN56021; annotated as NADPH azoreductase and urocanate reductase, respectively) with Vibrio cholerae flavin transferase. The isolated protein (named Crd) consists of the sjn56021-encoded subunit CrdB (NADH:flavin, FAD binding 2, and FMN bind domains) and an additional subunit CrdA (SJN56019, a single NADH:flavin domain) that interact via their NADH:flavin domains (Alphafold2 prediction). Each domain contains a flavin group (three FMNs and one FAD in total), one of the FMN groups being linked covalently by the flavin transferase. Crd readily reduces cinnamate, p-coumarate, caffeate, and ferulate under anaerobic conditions with NADH or methyl viologen as the electron donor, is moderately active against acrylate and practically inactive against urocanate and fumarate. Cinnamates induced Crd synthesis in V. ruber cells grown aerobically or anaerobically. The Crd-catalyzed reduction started by NADH demonstrated a time lag of several minutes, suggesting a redox regulation of the enzyme activity. The oxidized enzyme is inactive, which apparently prevents production of reactive oxygen species under aerobic conditions. Our findings identify Crd as a regulated NADH-dependent cinnamate reductase, apparently protecting V. ruber from (hydroxy)cinnamate poisoning.

Soil redox status governs within-field spatial variation in microbial arsenic methylation and rice straighthead disease.

Microbial arsenic (As) methylation in paddy soil produces mainly dimethylarsenate (DMA), which can cause physiological straighthead disease in rice. The disease is often highly patchy in the field, but the reasons remain unknown. We investigated within-field spatial variations in straighthead disease severity, As species in rice husks and in soil porewater, microbial composition and abundance of arsM gene encoding arsenite S-adenosylmethionine methyltransferase in two paddy fields. The spatial pattern of disease severity matched those of soil redox potential, arsM gene abundance, porewater DMA concentration, and husk DMA concentration in both fields. Structural equation modelling identified soil redox potential as the key factor affecting arsM gene abundance, consequently impacting porewater DMA and husk DMA concentrations. Core amplicon variants that correlated positively with husk DMA concentration belonged mainly to the phyla of Chloroflexi, Bacillota, Acidobacteriota, Actinobacteriota, and Myxococcota. Meta-omics analyses of soil samples from the disease and non-disease patches identified 5129 arsM gene sequences, with 71% being transcribed. The arsM-carrying hosts were diverse and dominated by anaerobic bacteria. Between 96 and 115 arsM sequences were significantly more expressed in the soil samples from the disease than from the non-disease patch, which were distributed across 18 phyla, especially Acidobacteriota, Bacteroidota, Verrucomicrobiota, Chloroflexota, Pseudomonadota, and Actinomycetota. This study demonstrates that even a small variation in soil redox potential within the anoxic range can cause a large variation in the abundance of As-methylating microorganisms, thus resulting in within-field variation in rice straighthead disease. Raising soil redox potential could be an effective way to prevent straighthead disease.

Bioinformatics-Guided Reconstitution of Biosynthetic Machineries of Fungal Eremophilane Sesquiterpenes.

Eremophilanes exhibit diverse biological activities and chemical structures. This study reports the bioinformatics-guided reconstitution of the biosynthetic machinery of fungal eremophilanes, eremofortin C and sporogen-AO1, to elucidate their biosynthetic pathways. Their biosyntheses include P450-catalyzed multistep oxidation and enzyme-catalyzed isomerization by the DUF3237 family protein. Successful characterization of six P450s enabled us to discuss the functions of eremophilane P450s in putative eremophilane biosynthetic gene clusters, providing opportunities to understand the oxidative modification pathways of fungal eremophilanes.

Comparing methanol and glycerol as carbon sources for mainstream partial denitrification/anammox in an IFAS process.

This study explored the implementation of mainstream partial denitrification with anammox (PdNA) in the second anoxic zone of a wastewater treatment process in an integrated fixed film activated sludge (IFAS) configuration. A pilot study was conducted to compare the use of methanol and glycerol as external carbon sources for an IFAS PdNA startup, with a goal to optimize nitrogen removal while minimizing carbon usage. The study also investigated the establishment of anammox bacteria on virgin carriers in IFAS reactors without the use of seeding, and it is the first IFAS PdNA startup to use methanol as an external carbon source. The establishment of anammox bacteria was confirmed in both reactors 102 days after startup. Although the glycerol-fed reactor achieved a higher steady-state maximum ammonia removal rate because of anammox bacteria (1.6 ± 0.3 g/m2/day) in comparison with the methanol-fed reactor (1.2 ± 0.2 g/m2/day), both the glycerol- and methanol-fed reactors achieved similar average in situ ammonia removal rates of 0.39 ± 0.2 g/m2/day and 0.40 ± 0.2 g/m2/day, respectively. Additionally, when the upstream ammonia versus NOx (AvN) control system maintained an ideal ratio of 0.40-0.50 g/g, the methanol-fed reactor attained a lower average effluent TIN concentration (3.50 ± 1.2 mg/L) than the glycerol-fed reactor (4.43 ± 1.6 mg/L), which was prone to elevated nitrite concentrations in the effluent. Overall, this research highlights the potential for PdNA in IFAS configurations as an efficient and cost-saving method for wastewater treatment, with methanol as a viable carbon source for the establishment of anammox bacteria. PRACTITIONER POINTS: Methanol is an effective external carbon source for an anammox startup that avoids the need for costly alternative carbon sources. The methanol-fed reactor demonstrated higher TIN removal compared with the glycerol-fed reactor because of less overproduction of nitrite. Anammox bacteria was established in an IFAS reactor without seeding and used internally stored carbon to reduce external carbon addition. Controlling the influent ammonia versus NOx (AvN) ratio between 0.40 and 0.50 g/g allowed for low and stable TIN effluent conditions.

Physiological versatility of ANME-1 and Bathyarchaeotoa-8 archaea evidenced by inverse stable isotope labeling.

BACKGROUND: The trophic strategy is one key principle to categorize microbial lifestyles, by broadly classifying microorganisms based on the combination of their preferred carbon sources, electron sources, and electron sinks. Recently, a novel trophic strategy, i.e., chemoorganoautotrophy-the utilization of organic carbon as energy source but inorganic carbon as sole carbon source-has been specifically proposed for anaerobic methane oxidizing archaea (ANME-1) and Bathyarchaeota subgroup 8 (Bathy-8). RESULTS: To further explore chemoorganoautotrophy, we employed stable isotope probing (SIP) of nucleic acids (rRNA or DNA) using unlabeled organic carbon and 13C-labeled dissolved inorganic carbon (DIC), i.e., inverse stable isotope labeling, in combination with metagenomics. We found that ANME-1 archaea actively incorporated 13C-DIC into RNA in the presence of methane and lepidocrocite when sulfate was absent, but assimilated organic carbon when cellulose was added to incubations without methane additions. Bathy-8 archaea assimilated 13C-DIC when lignin was amended; however, their DNA was derived from both inorganic and organic carbon sources rather than from inorganic carbon alone. Based on SIP results and supported by metagenomics, carbon transfer between catabolic and anabolic branches of metabolism is possible in these archaeal groups, indicating their anabolic versatility. CONCLUSION: We provide evidence for the incorporation of the mixed organic and inorganic carbon by ANME-1 and Bathy-8 archaea in the environment. Video Abstract.