Biology subjects

Rotaru, A.-E.

Publications and source records attributed to Rotaru, A.-E..

16 recordsLinked to original sources

bioRxiv2026

The metallome of Methanosarcina barkeri during electron uptake from acathode

Electromethanogenesis, the cathode-dependent reduction of CO2 to CH4 by methanogens, offers a sustainable route to methane fuel. Methanosarcina barkeri lacks surface-exposed multiheme cytochromes for extracellular electron transfer (EET). Instead, we recently showed that surface-bound G-quadruplex ribonucleic acids (G4-RNA) are required for EET, yet how electrons traverse this extracellular matrix remains unresolved. Here, we quantified metal accumulation during cathodic growth by inductively coupled plasma mass spectrometry in cells grown on cathodes poised at -430 mV versus the standard hydrogen electrode, using acetate-grown cells, open-circuit controls and abiotic cathodes for comparison. Cathode-grown M. barkeri showed CH4 buildup attributable to cathodic electrons (2.1 {+/-} 0.8% CH4), whereas open-circuit controls showed negligible increase (0.26 {+/-} 0.15% CH4). Cathode-bound cells exhibited [~]55-fold enrichment in Co, Ni and Mo, and 5- to 21-fold enrichment in Cu, Zn, and Fe relative to acetate-grown cells; neither acetate-grown cells nor abiotic cathodes accumulated metals. To resolve where metals reside, we mapped the elemental distribution in acetate-grown cells by scanning transmission electron microscopy-energy dispersive X-ray spectroscopy and high-resolution nano X-ray fluorescence. Fe co-localized with phosphorus in intracellular storage bodies, whereas Co and Zn localized within the extracellular capsule. Together, these data indicate selective metal sequestration during electromethanogenesis and raise the possibility that certain metals associate with G4-RNA and/or the methanochondroitin matrix to support charge transfer at the cell surface. This metalomic fingerprint provides a new proxy for dissecting archaeal EET strategies and may inform the design of more efficient bioelectrochemical systems.

microbiology↗

bioRxiv2026

Wastewater-impacted Skagerrak Sea microbiomes anaerobically demethylate micropollutants

Methylated micropollutants such as naproxen and caffeine persist in wastewater effluents and accumulate in coastal sediments, including Hakefjorden, Skagerrak Sea, yet their anaerobic fate and role in methane emissions remain unresolved. Here we traced the fate and microbiome responses to {superscript 1}3C-labeled naproxen and caffeine in sediment microcosms. We show that naproxen underwent rapid O-demethylation to desmethylnaproxen, with 90% {+/-} 15.5% removed within 25 days, producing primarily {superscript 1}3CO2 and some {superscript 1}3CH. Naproxen enriched methylotrophic and hydrogenotrophic Methanomicrobia, alongside Lokiarchaeia, Bathyarchaeia, and bacterial taxa like Eubacterium and Syntrophomonadaceae. Metagenomics revealed O-demethylation genes in enriched bacterial MAGs affiliated with uncultured Thermoanaerobaculia, indicating a bacterial demethylation potential. In contrast, caffeine was largely recalcitrant to degradation ([~]85% {+/-} 5% remaining), yet its 13C-labeled N-methyl groups fueled {superscript 1}3CH production, coinciding with enrichment of Bathyarchaeia and Methanosarcina. These results show that methylated micropollutants can activate both bacterial and archaeal demethylation pathways in coastal sediment microbiomes.

microbiology↗

bioRxiv2025

Genome-centric metagenomics reveals novel electroactive syntrophs in a conductive particle-dependent consortium from coastal sediments

Conductive particles are abundant in coastal sediments, yet the organisms and pathways that use them for methane production remain unclear. We applied long-read, genome-resolved metagenomics to a sediment-derived consortium that remained dependent on granular activated carbon (GAC) for a decade. We identified a particle-obligate food web of electrogenic syntrophic acetate oxidizers (SAO), an electrotrophic methanogen, and necromass recyclers. The dominant SAO electrogen was a new genus, Candidatus Geosyntrophus acetoxidans (<70% ANI/AAI to described taxa), encoding a streamlined extracellular electron-transfer system: one porin-cytochrome conduit (PCC), 47 multiheme cytochromes, conductive pili, and acetate uptake/utilization genes. A second SAO electrogen, Lentimicrobium sp., carried two giant PCC-like clusters, suggesting an alternative acetate-oxidation route. Electrons flowed via GAC to a Methanosarcina (<89% ANI/AAI to described taxa) equipped with the multiheme cytochrome MmcA and a Rnf/Fpo/HdrDE circuit for EET-driven CO2-reducing methanogenesis. Particle-free lines lost both partners and methanogenic activity, establishing particles as the determinant of persistence. This first genomic blueprint of a natural CIET-SAO consortium identifies potential genomic markers (distinct PCCs, MmcA) for in-situ detection and reveals a particle-bound route from acetate to methane likely operating as a fundamental electron-transfer unit in geoconductor-rich anoxic sediments.

microbiology↗

bioRxiv2025

Extracellular RNA drives Electromethanogenesis in a Methanogenic Archaeon

Methanogenic archaea account for two-thirds of global methane emissions. Some species, including Methanosarcina barkeri, reduce CO2 by directly acquiring electrons from solid substrates. However, the mechanism of electron acquisition in M. barkeri has remained unclear because this archaeon lacks the multiheme c-type cytochromes that drive extracellular electron transfer in many other microbes. Here we show that M. barkeri releases abundant extracellular nucleic acids during early growth, primarily short RNAs (78%). These extracellular nucleic acids assemble into G-quadruplexes (G4s) and B-DNA architectures that decorate cell surfaces and link aggregates. Surface-associated G4s are folded in vivo in a conformation compatible with cofactor binding and redox chemistry. Enzymatic degradation of extracellular nucleic acids abolished electron uptake and electromethanogenesis, whereas addition of synthetic G4-RNAs doubled methane yields and lowered cell-electrode interfacial resistance. These effects were not observed when cells were grown on soluble substrates. Together, these findings identify eRNA as a previously unrecognized electron conduit in methanogens, raising the possibility that RNA-based electron transfer may predate more elaborate protein-based electron conduits, with implications for models of early earth metabolism and for the design of next-generation bioenergy systems.

microbiology↗

bioRxiv2025

Cell Surface Differences within the Genus Methanosarcina Shape Interactions with the Extracellular Environment

Methanosarcina are metabolically versatile methanogenic archaea that can perform extracellular electron transfer (EET), with important ecological and biotechnological implications. These archaea are broadly classified into two types (Type I and Type II) based on their energy metabolism and are also differ in their aggregation-disaggregation behavior, cell surface properties, and electron transfer strategies. Type I Methanosarcina typically form large multicellular aggregates within a methanochondroitin extracellular matrix, thrive in organic-rich environments, play a key role in anaerobic digestion during wastewater treatment and can perform EET. However, their mechanism of EET remains unresolved. In contrast, Type II Methanosarcina rely on multiheme c-type cytochromes for EET and are better adapted to low-organic, mineral-rich environments such as deep-sea sediments and aquifers, where they contribute to methane emissions. Despite their significance, the molecular mechanisms behind EET in Methanosarcina-- particularly for Type I--remain poorly understood. This review highlights what is known and what is unknown regarding the surface biology of Methanosarcina, their EET strategies, and biogeochemical and industrial roles, emphasizing the need for further research to unlock their full potential in sustainable methane management.

microbiology↗

bioRxiv2024

Aerotolerant methanogens use seaweed and seagrass metabolites to drive marine methane emissions

Methanogenesis is classically thought to be limited to strictly anoxic environments. While oxygenated oceans are a known methane source, it is argued that methanogenesis is driven by methylphosphonate-degrading bacteria or potentially is associated to zooplankton gut microbiomes rather than by methanogenic archaea. Here we show through in situ monitoring and ex situ manipulations that methane is rapidly produced by archaea in frequently oxygenated sandy sediments. By combining biogeochemical, metagenomic, and culture-based experiments, we show this activity is driven by aerotolerant methylotrophic methanogens (Methanococcoides spp.) broadly distributed in surface layers of sandy sediments, providing evidence of a hidden process contributing to marine methane emissions. Moreover, we show that methane emissions are driven by methylated seaweed and seagrass metabolites, revealing an unexpected feedback loop between eutrophication-driven algal blooms and greenhouse gas emissions.

microbiology↗

bioRxiv2024

Adaptation of a methanogen to the constructed environment

Due to unique genomic adaptations, Methanococcus maripaludis Mic1c10 exhibits severe corrosive behavior when in direct contact with Fe0. These adaptations are linked to attachment and effective growth on constructed surfaces. One such adaptation is that of a specific [NiFe]-hydrogenase that may anchor on the cell surface via glycosyl-glycosyl interactions to receive Fe0-electrons directly. Such an evolutionary response to constructed environments requires us to rethink methane cycling in human-altered ecosystems.

microbiology↗

bioRxiv2022

The Fate of DIET Consortium exposed to continuous and intermittent electrochemical stimulation

Electromethanogenesis is the electrochemical conversion of carbon dioxide (CO2) to methane (CH4) with microorganisms as catalysts, and it is a promising approach for biogas upgrading. Many studies have shown increased methane production by electrochemical stimulation, allegedly due to the enhancement of direct interspecies electron transfer (DIET) between electrogenic bacteria and methanogens. However, these studies were done with mixed cultures where the interspecies interactions were undefined, so the impact of an electrochemical stimulus on these interactions remains unresolved. Here we report that an electrochemical stimulus diminished the survival and activity of a defined DIET consortium. We investigated the impact of electrochemical stimulation on a co-culture of Geobacter metallireducens and Methanosarcina barkeri, where the two partners interact syntrophically via DIET. The electrochemical stimulus was a cathode poised at - 700 mV (versus the standard hydrogen electrode, SHE). Electrochemical stimulation was provided either continuously or intermittently, and so was the food substrate. Compared to the untreated DIET co-culture, all treatment strategies relying on cathodic current additions resulted in acetate accumulation, lower methane production and lower cell numbers for both partners, indicating a detrimental impact of electrochemical stimulation to a DIET consortium. These findings suggest that future bioelectrochemical technologies must guarantee the survival of the syntrophic partners during current additions.

microbiology↗

bioRxiv2019

Extracellular electron uptake in Methanosarcinales is independent of multiheme c-type cytochromes

The co-occurrence of Geobacter and Methanosarcinales is often used as a proxy for the manifestation of direct interspecies electron transfer (DIET) in man-made and natural aquatic environments. We previously reported that not all Geobacter are capable of DIET with Methanosarcina. Here we tested 15 new artificial co-culture combinations with methanogens and electrogenic bacteria, including an electrogen outside of the Geobacter clade - Rhodoferax ferrireducens.\n\nConsistently, highly effective electrogenic bacteria (G. metallireducens, G. hydrogenophilus and R. ferrireducens) formed successful associations with Methanosarcinales. Highly effective electrogens could not sustain the growth of H2-utilizing methanogens of the genera Methanococcus, Methanobacterium, Methanospirillum, Methanolacinia or Methanoculleus.\n\nMethanosarcinales, including strict non-hydrogenotrophic methanogens of the genus Methanothrix (Mtx. harundinacea and Mtx. shoeghenii) and Methanosarcina horonobensis, conserved their ability to interact with electrogens. Methanosarcinales were classified as the only methanogens containing c-type cytochromes, unlike strict hydrogenotrophic methanogens. It was then hypothesized that multiheme c-type cytochromes give Methanosarcinales their ability to retrieve extracellular electrons. However, multiheme c-type cytochromes are neither unique to this group of methanogens nor universal. Only two of the seven Methanosarcinales tested had multiheme c-type cytochromes (MCH). In one of these two species - M. mazei a deletion mutant for its MCH was readily available. Here we tested if the absence of this MHC impacts extracellular electron uptake. Deletion of the MHC in M. mazei did not impact the ability of this methanogens to retrieve extracellular electrons from G. metallireducens or a poised cathode. Since Methanosarcina did not require multiheme c-type cytochromes for direct electron uptake we proposed an alternative strategy for extracellular electron uptake.

microbiology↗

bioRxiv2019

Lifeless Clostridia stimulate methanogenesis on Fe0 in an urban lake corrosive community

Microbial-induced corrosion has been extensively studied in pure cultures. However, Fe0 corrosion by complex environmental communities, and especially the interplay between microbial physiological groups, is still poorly understood. In this study, we combined experimental physiology and metagenomics to explore Fe0-dependent microbial interactions between physiological groups enriched from anoxic climate lake sediments. Then, we investigated how each physiological group interacts with Fe0. We offer evidence for a new interspecies interaction during Fe0 corrosion. We showed that acetogens enhanced methanogenesis but were negatively impacted by methanogens (opportunistic microbial interaction). Methanogens were positively impacted by acetogens. In the metagenome of the corrosive community, the acetogens were mostly represented by Clostridium and Eubacterium, the methanogens by Methanosarcinales, Methanothermobacter and Methanobrevibacter. Within the corrosive community, acetogens and methanogens produced acetate and methane concurrently, however at rates that cannot be explained by abiotic H2-buildup at the Fe0 surface. Thus, microbial-induced corrosion might have occurred via a direct or enzymatically mediated electron uptake from Fe0. The shotgun metagenome of Clostridium within the corrosive community contained several H2-releasing enzymes including [FeFe]-hydrogenases, which could boost Fe0-dependent H2-formation as previously shown for pure culture acetogens. Outside the cell, acetogenic hydrogenases could become a common good for any H2/CO2-consuming member in the microbial community including methanogens that rely on Fe0 as a sole energy source. However, the exact electron uptake mechanism from Fe0 remains to be unraveled.

microbiology↗

bioRxiv2019

Baltic Methanosarcina and Clostridium compete for electrons from metallic iron

Microbial induced corrosion of steel structures, used for transport or storage of fuels, chemical weapons or waste radionuclides, is an environmental and economic threat. In non-sulfidic environments, the exact role of methanogens in steel corrosion is poorly understood. From the non-sulfidic, methanogenic sediments of the Baltic Sea corrosive communities were enriched using exclusively Fe0 as electron donor and CO2 as electron acceptor. Methane and acetate production were persistent for three years of successive transfers. Methanosarcina and Clostridium were attached to the Fe0, and dominated metagenome libraries. Since prior reports indicated Methanosarcina were merely commensals, consuming the acetate produced by acetogens, we investigated whether these methanogens were capable of Fe0 corrosion without bacterial partners (inhibited by an antibiotic cocktail). Unassisted, methanogens corroded Fe0 to Fe2+ at similar rates to the mixed community. Surprisingly, in the absence of competitive bacteria, Baltic-Methanosarcina produced six times more methane than they did in the mixed community. This signifies that Baltic-Methanosarcina achieved better corrosion alone, exclusive of an operative bacterial partner. Our results also show that together with acetogens, Methanosarcina interact competitively to retrieve electrons from Fe0 rather than as commensals as previously assumed.

microbiology↗

bioRxiv2018

Syntrophus Conductive Pili Demonstrate that Common Hydrogen-Donating Syntrophs can have a Direct Electron Transfer Option

Syntrophic interspecies electron exchange is essential for the stable functioning of diverse anaerobic microbial communities. Hydrogen/formate interspecies electron transfer (HFIT), in which H2 and/or formate function as diffusible electron carriers, has been considered to be the primary mechanism for electron sharing because most common syntrophs were thought to lack biochemical components, such as electrically conductive pili (e-pili), necessary for direct interspecies electron transfer (DIET). Here we report that Syntrophus aciditrophicus, one of the most intensively studied microbial models for HFIT, produces e-pili and can grow via DIET. Pilin genes likely to yield e-pili were found in other genera of hydrogen/formate-producing syntrophs. The finding that DIET is a likely option for diverse syntrophs that are abundant in many anaerobic environments necessitates a reexamination of the paradigm that HFIT is the predominant mechanism for syntrophic electron exchange within anaerobic microbial communities of biogeochemical and practical significance.

microbiology↗

bioRxiv2018

Extracellular electron uptake by two Methanosarcina species

Direct electron uptake by prokaryotes is a recently described mechanism with a potential application for energy and CO2 storage into value added chemicals. Members of Methanosarcinales, an environmentally and biotechnologically relevant group of methanogens, were previously shown to retrieve electrons from an extracellular electrogenic partner performing Direct Interspecies Electron Transfer (DIET) and were therefore proposed to be electroactive. However, their intrinsic electroactivity has never been examined. In this study, we tested two methanogens belonging to the genus Methanosarcina, M. barkeri and M. horonobensis, regarding their ability to accept electrons directly from insoluble electron donors like other cells, conductive particles and electrodes. Both methanogens were able to retrieve electrons from Geobacter metallireducens via DIET. Furthermore, DIET was also stimulated upon addition of electrically conductive granular activated carbon (GAC) when each was co-cultured with G. metallireducens. However, when provided with a cathode poised at -400 mV (vs. SHE), only M. barkeri could perform electromethanogenesis. In contrast, the strict hydrogenotrophic methanogen, Methanobacterium formicicum, did not produce methane regardless of the type of insoluble electron donor provided (Geobacter cells, GAC or electrodes). A comparison of functional gene categories between the two Methanosarcina showed differences regarding energy metabolism, which could explain dissimilarities concerning electromethanogenesis at fixed potentials. We suggest that these dissimilarities are minimized in the presence of an electrogenic DIET partner (e.g. Geobacter), which can modulate its surface redox potentials by adjusting the expression of electroactive surface proteins.

microbiology↗

bioRxiv2018

Interspecies interactions mediated by conductive minerals in the sediments of the ferruginous Lake La Cruz, Spain

Lake La Cruz is considered a biogeochemical analogue to early Earth marine environments because its water column is depleted in sulfate, but rich in methane and iron, similar to conditions envisaged for much of the Precambrian. In this early Earth analogue environment, we show that conductive particles establish a tight metabolic coupling between electroactive microbial clades. We propose that mineral-based syntrophy is of potential relevance for the evolution of Earths earliest complex life forms. We show that the anoxic sediment of Lake La Cruz, which is rich in biogeochemically reactive iron minerals, harbors known electroactive species such as Geobacter and Methanothrix, in addition to other groups which have not been previously associated with an electroactive lifestyle. Slurry incubations on various substrates in the presence of conductive particles showed significant methanogenic activity, whereas incubations with non-conductive glass beads resulted in low methanogenic rates similar to slurries without added particles. In the absence of conductive particles, all tested substrates were metabolized to acetate, which accumulated to [~]10 mM. Similar to a previous study on iron-rich Baltic Sea sediments, we observed that conductive mineral additions to La Cruz slurries enabled acetate oxidation, thus preventing acetate accumulation. Acetate oxidation coupled to high methanogenic activity was only maintained in successive mud-free enrichments when these were amended with conductive minerals. In serial mud-free transfers, conductive particles conserved a consortium of Youngiibacter-Methanothrix, whereas Youngiibacter spp. died off in the absence of conductive particles. In contrast, mud-free enrichments without conductive particles ceased any metabolic activity during the second transfers. Syntrophic consortia from this early Earth analogue environment only survived in the presence of conductive particles. Mineral-mediated syntrophy could be one of the earliest evolutionary interspecies associations. Conductive minerals might have fueled metabolic exchange between cells via intercellular electron transfer prompting tight cell-to-cell associations and possibly eukaryogenesis.

microbiology↗

bioRxiv2017

Conductive particles enable syntrophic acetate oxidation between Geobacter and Methanosarcina from coastal sediments

Coastal sediments are rich in conductive minerals, which could impact microbial processes for which acetate is a central intermediate. In the methanogenic zone, acetate is consumed by methanogens and/or syntrophic acetate oxidizing (SAO) consortia. SAO consortia live under extreme thermodynamic pressure and their survival depends on successful partnership. Here we demonstrate that conductive minerals facilitate a SAO partnership between Geobacter and Methanosarcina from the coastal sediments of the Bothnian Bay, Baltic Sea. Bothnian methanogenic sediments showed a high apparent isotopic fractionation (c 1.07) characteristic of CO2-reductive methanogenesis. The native community was represented by electrogens such as Geobacter and methanogens like Methanosarcina. Upon the addition of conductive particles (activated carbon and magnetite) methanogenesis from acetate increased four fold. Geobacter (96% related to G. psychrophilus) and Methanosarcina (99% related to M. subterranea) dominated the conductive particle-spiked SAO communities. Using NanoSIMS we demonstrated that during SAO, Geobacter incorporated 82% of the labeled acetate as compared to only 18% by Methanosarcina. At the same time Geobacter converted 27% of the 13C-acetate to 13CO2 as detected by IRMS. Indigenous soluble shuttles were not involved in SAO, since spiking fresh cultures with spent-media filtrate had no effect on methanogenic rates. Our results demonstrate that Geobacter oxidizes acetate to CO2 while transferring electrons extracellularly via conductive particles to Methanosarcina, which utilizes them for CO2 reduction to methane. In natural environments, mediation of SAO by conductive particles between electrogens and methanogens could impact the iron and methane cycles.\n\nSignificanceAcetate oxidizing bacteria are known to thrive in mutualistic consortia in which H2 or formate is shuttled to a methane-producing Archaea partner. Here we discovered that they could instead transfer electrons via conductive minerals. Mineral-SAO (syntrophic acetate oxidation) could be a vital pathway for CO2-reductive methanogenesis in the environment, especially in sediments rich in conductive minerals. Mineral-SAO is therefore of potential importance for both iron and methane cycles in sediments and soils. Additionally, our observations imply that agricultural runoff or amendments with conductive chars could trigger a significant increase in methane emissions.

microbiology↗

bioRxiv2017

Potential for Methanosarcina to contribute to uranium reduction during acetate-promoted groundwater bioremediation

Previous studies of in situ bioremediation of uranium-contaminated groundwater with acetate injections have focused on the role of Geobacter species in U(VI) reduction because of a lack of other abundant known U(VI)-reducing microorganisms. Monitoring the levels of methyl CoM reductase subunit A (mcrA) transcripts during an acetate-injection field experiment demonstrated that acetoclastic methanogens from the genus Methanosarcina were enriched after 40 days of acetate amendment. The increased abundance of Methanosarcina corresponded with an accumulation of methane in the groundwater. An enrichment culture dominated by a Methanosarcina species with the same Methanosarcina mcrA sequence that predominated in the field experiment could effectively convert acetate to methane. In order to determine whether Methanosarcina species could be participating in U(VI) reduction in the subsurface, cell suspensions of M. barkeri were incubated in the presence of U(VI) with acetate provided as the electron donor. U(VI) was reduced by metabolically active M. barkeri cells, however, no U(VI) reduction was observed in inactive controls. These results demonstrate that Methanosarcina species could play an important role in the long-term bioremediation of uranium-contaminated aquifers after depletion of Fe(III) oxides limits the growth of Geobacter species. The results also suggest that Methanosarcina have the potential to influence uranium geochemistry in a diversity of anaerobic sedimentary environments.

microbiology↗