Environmental Microbiology Reports

Organohalide-respiring bacteria (OHRB), such as Dehalobacter, play key roles in the bioremediation of anoxic environments contaminated with chlorinated aromatic compounds. These obligate anaerobes rely on syntrophic interactions to obtain essential resources--hydrogen, acetate, and corrinoid cofactors--from acetogens and fermenters. However, the metabolic interactions enabling complete reductive dehalogenation of compounds like 1,2,4-trichlorobenzene (1,2,4-TCB) to benzene remain incompletely understood. In this study, we asked: (1) What are the key microbial taxa and their functional roles within a Dehalobacter-containing anaerobic microbial community detoxifying chlorinated benzenes? (2) How do syntrophic interactions enable complete dehalogenation of 1,2,4-TCB to benzene under anaerobic conditions? (3) Can genome-resolved metagenomics and genome-scale metabolic modeling elucidate the metabolic dependencies supporting organohalide respiration in complex consortia? To address these questions, we cultivated microbial communities in batch reactors using methanol as electron donor and either 1,2,4-TCB or monochlorobenzene (MCB) as electron acceptor. In active MCB-fed cultures, benzene increased from 0 to 62.3{micro}mol per bottle while MCB decreased from 88.3 to 22.0{micro}mol per bottle over 120 days, with this pattern repeating across multiple substrate additions. Using genome-resolved metagenomics to identify dominant taxa and select 12 high-quality metagenome-assembled genomes (MAGs) for modeling, we reconstructed genome-scale metabolic models (GEMs) to identify candidate metabolic interactions and predict syntrophic dependencies that may support organohalide respiration in these consortia. Community flux sampling predicted that methanol, H2, acetate, and CO2 formed the dominant exchange backbone of the modeled community, while also indicating competition for shared electron donors between the two Dehalobacter populations. Model-guided minimal-community analysis further identified a narrow dechlorinating core in which all feasible minimal consortia retained a Dehalobacter member together with Methanothrix. These results provide a modeling-informed framework for hypothesis generation and future experimental validation of anaerobic consortia relevant to bioremediation.

Plant growth promoting microbes enhance developmental progression of the host by influencing its nutrient availability or by deploying secondary metabolites responsible for manipulating the hormonal crosstalk. Microbacterium bengalense sp. nov. GB16_1_BI (Accession number: SRX9280401), a newly identified ammonium releasing Actinomycetota, could enhance plant growth by manipulating rhizosphere bacteria. Amplicon sequencing of the 16S rRNA V3-V4 region from the rhizosphere of the black rice (Chakhao Poireiton) showed that GB16_1_BI could inhibit most bacteria. However, GB16_1_BI inoculation encouraged the growth of rare bacteria specific to waterlogged rice rhizosphere. Analysis of the OTUs using PICRUSt2 (Phylogenetic investigation of communities by reconstruction of unobserved states) showed increased abundance in the marker genes for nitrogen cycling (nifH, nrfA and nrt) but not for nifD or nifK which was also reflected in the ANOSIM analysis in the OTUs of the N-fixing bacteria. Marker genes for methane metabolism (comA, comB, cofG and cofH) were also more abundant in the inoculated plants than the control; however, ANOSIM studies did not support this observation in the OTUs of methane cycling bacteria. Both Methylosinus and Methylocystis, the two most abundant methanotrophic OTUs, are also known to be nitrogen fixers. Hence, GB16_1_BI could influence plant growth predominantly by manipulating nitrogen cycling microbes. The genome sequence as well as untargeted metabolome analyses of GB16_1_BI showed abundance of secondary metabolites with probable antimicrobial activity. GB16_1_BI could utilize varied carbohydrates and amino acid as energy source and form persister-like cells may help it to survive in the soil in absence of the host plant.

Methane is a powerful greenhouse gas. Typically, a large fraction of the methane formed in coastal sediments is removed via anaerobic methane oxidation (AOM). Here, we demonstrate the potential for a range of AOM pathways in brackish coastal sediments by ANME-2a archaea. At our study site, geochemical profiles indicate that AOM is primarily restricted to a shallow, metal-oxide-rich sulfate-methane transition zone (SMTZ). ANME-2a were the sole methanotrophs detected, and metatranscriptomics showed the highest expression levels of the ANME-2a genes in the SMTZ. AOM activity was observed in sediment incubations with various electron acceptors, including sulfate, metal oxides, and the organic matter analogue graphene oxide. Highest potential rates were observed in sediments from below the SMTZ, pointing towards fast stimulation of the deeper methanotrophic community when alleviating the electron acceptor limitation. The variety of AOM pathways and persistence of methanotrophs below the SMTZ likely contribute to the resilience of the microbial methane filter in brackish coastal sediments.

Brown rot wood-degrading fungi release carbon (C) from deadwood but leave behind a large fraction of C sequestered in lignin residues or as fungal metabolites. The strength of sequestration in these C residuals remains unclear, but proteobacteria-dominated bacterial communities have been implicated in metabolizing C from decay residues, possibly erasing the C sequestration potential assumed for brown rot. Here, we paired a model brown rot fungus (Rhodonia placenta) with a model Alphaproteobacterium (Rhodopseudomonas palustris) to track fungal release and bacterial utilization of C derived from decaying wood. We found that fungal decay products generated by R. placenta could be used by R. palustris for growth, and later decay stages contained more usable substrates than early stages. High performance liquid chromatography with mass spectrometry identified a range of aromatic and non-aromatic compounds in the fungal-decayed wood, but after 95 days of bacterial growth, R. palustris preferentially consumed non-aromatic acids over aromatic lignin monomers. Genes involved with aromatic compound degradation were unimportant for bacterial growth, and RNA sequencing revealed that aromatic compound degradation genes were repressed on decayed wood extract. Randomly barcoded transposon sequencing failed to identify a solitary catabolic pathway used by R. palustris, suggestive of substrate co-utilization, and surprisingly, showed that genes involved with copper toxicity were essential. Finally, we found that genes involved with biosynthesis of certain cofactors and amino acids were no longer essential on decayed wood extract, suggesting these nutrients were readily accessible. This study helps lay the foundation to understand potential bacterial-fungal interactions in decayed wood. Graphical abstractTo explore how brown rot fungi support and compete with bacterial partners in the wood decay environment, the model brown rot fungus Rhodonia placenta was used to degrade aspen wafers which were then infused into bacterial growth medium. By leveraging the range of molecular biology tools available for the model Alphaproteobacterium Rhodopseudomonas palustris, we discovered that R. palustris preferentially consumes short organic acids instead of aromatic lignin monomers which it would otherwise consume if provided in isolation. Additionally, R. palustris scavenged certain amino acids (AAs) and enzyme cofactors including methionine, biotin, and PLP from the decayed wood extract, highlighting these as key shared resources for bacterial-fungal partnerships. We found that R. placenta increased the concentration of certain metals (Cu and Al) inducing a metal stress response in R. palustris, indicating that metal toxicity could be an important mode of competition between fungi and bacteria in the wood decay environment. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/723453v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@16f31fcorg.highwire.dtl.DTLVardef@13a9b34org.highwire.dtl.DTLVardef@a37dcforg.highwire.dtl.DTLVardef@198bf1c_HPS_FORMAT_FIGEXP M_FIG C_FIG

1Consortia of archaea and partner bacteria couple the anaerobic oxidation of alkanes to sulfate reduction. While catabolic pathways in anaerobic alkane-oxidizing archaea (ANKA) are increasingly understood, their anabolic capacities remain poorly characterized. Here, we examined nine enrichment cultures dominated by ANKA and their partner bacteria for small-molecular compounds using solvent extraction and gas chromatographic analysis of derivatized extracts. All hydrocarbon-degrading cultures contained substantial amounts of disaccharides in their metabolite pools. Cold-adapted methane-oxidizing cultures dominated by ANME-2c and Seep-SRB2 contained up to 1.5 mg of trehalose per mg soluble protein. Trehalose was also abundant in ethane-oxidizing cultures of Candidatus Ethanoperedens and its distinct partner SRBs, accounting for up to 75 % of the extracted metabolites. In contrast, thermophilic ANKA cultures dominated by ANME-1 or Ca. Syntropharchaeum and Ca. Desulfofervidus contained an abundant as-yet-unidentified glucose-containing disaccharide. Metagenomic analysis revealed widespread trehalose metabolism genes among partner Desulfobacterota and in ANME-2c and Ca. Ethanoperedens, but a lower potential in ANME-1 and Syntropharchaeum, consistent with metabolite profiles. If exogenous trehalose was added to the Ethane50 culture, we observed rapid metabolization by heterotrophic microorganisms, but poor assimilation by the Ca. Ethanoperedens/ Ca. Desulfofervidus core community, indicating that ANKA/SRB consortia do not consume externally supplied trehalose. Instead, Ca. Ethanoperedens/ Ca. Desulfofervidus, as well as other ANKA/SRB consortia, may use the disaccharides as energy-storage molecules, osmolytes, or components of the extracellular matrix. Notably, the disaccharides produced by the consortia also sustain ancillary heterotrophs, thereby linking alkane oxidation to broader sedimentary carbon cycling.

Numerous studies have shown that the abundance of antibiotic-resistant bacteria (ARBs) or antibiotic-resistance genes (ARGs) in soil increases after irrigation with wastewater. However, it is unclear whether this increase is due to the selection effects of pharmaceutical residues in the irrigation water or the continuous introduction of ARBs and ARGs with the wastewater. Further, it is unclear how the binding of antibiotics to natural colloids (1-1000 nm) affects their biological effects compared to truly dissolved substances (< 1 nm). We conducted competition experiments with resistant and susceptible Acinetobacter baylyi BD413 strains in wastewater, as well as in colloidal and truly dissolved extracts of soils irrigated with wastewater. Although the concentrations of our six target antibiotics were far below the measured minimum selective concentrations of the tested strains, we demonstrate that the resistant strain was favored in the wastewater and the colloidal extracts. In contrast, the truly dissolved fractions exhibited weaker and more variable selective effects. A non-targeted analysis revealed the presence of 82 additional substances in our extracts, including further antibiotics, pesticides, and different non-antibiotic drugs that may influence the selection of our resistant A. baylyi BD413 strain. Our findings suggest that antibiotic resistance is selected for in wastewater and wastewater-irrigated soils. This cannot be explained by antibiotic concentrations alone, but may also arise from the effects of complex mixtures of co-occurring contaminants, particularly those associated with colloidal particles.

BackgroundAdult vestimentiferan tubeworms inhabiting hydrothermal vents and cold seeps lack a mouth and anus and rely entirely on organic matter produced by sulfur -oxidizing autotrophic bacterial symbionts in their trophosomes. These symbionts, which predominantly belong to the genus Proteobacteria, are acquired horizontally from the environment. However, the effects of rearing conditions that differ from natural habitats on the microbiome composition or abundance of these bacteria remain unclear. MethodsWe conducted a metagenomic analysis of Lamellibrachia satsuma reared in an aquarium under sulfide-supplemented and sulfide-free conditions. ResultsImmediately after collection, the microbiome was dominated by known symbionts within {gamma}-Proteobacteria, exhibiting low species diversity. After 6 months of rearing, the abundance of these symbionts significantly decreased under both conditions, whereas overall bacterial diversity increased. In particular, -Proteobacteria became more abundant under sulfide-supplemented conditions, while {delta}-Proteobacteria predominated in the absence of sulfide. Despite these changes, symbionts were not entirely lost, and the hosts survived for 6 months, likely due to their low metabolic rate. These findings suggest that the microbiome of L. satsuma can respond flexibly to changes in the rearing environment. They also indicate that the hosts metabolism can be maintained even with a smaller quantity of symbiotic bacteria.

Microbes play a vital role in plant development, health, and resilience, yet relatively little is known about the specific metabolic mechanisms driving interactions in these host-associated communities. Systems biology models enable a computational approach to understanding metabolic interactions, which can be difficult to pinpoint experimentally; however, these methods cannot yet accommodate the large number of species in natural communities. Synthetic communities (SynComs) provide a more tractable alternative to explore targeted interactions. Here, we investigated metabolite exchange in a seven-member maize root-associated SynCom, specifically accounting for plant host context by designing a customized exudate medium. We constructed metabolic models for each bacterial species and curated them with in vitro phenotyping data to reflect experimentally based carbon uptake potential. Flux balance analysis of individual species demonstrated that integrating phenotype data and changing medium type had substantial impacts on predicted growth rates, which in turn shaped potential interspecies interactions. In silico community growth optimization of the seven-member community model showed that the exudate medium supported a more diverse community composition compared to minimal medium, with predictions of community member abundance closely aligned to literature-derived experimental results. Predicted metabolite exchange in the root exudate environment showed Enterobacter ludwigii as a community hub, and cross-feeding of indole suggested a potential effect of bacterial community interactions on the plant host. Our in silico findings indicate the host plays an important role in structuring microbial interactions and cross-feeding at the metabolic level, underscoring the importance of considering environmental context from both theoretical and experimental perspectives. IMPORTANCETrue understanding of a system is marked by the ability to predict its behavior. The complexity of natural host-microbe systems represents a frontier of knowledge that scientists are working to understand, and elucidating principles of interactions within multi-partite microbial communities remains a challenge in microbial ecology. Synthetic communities provide a tractable starting point for investigating interaction mechanisms, and computational approaches complement laboratory experiments by systematically evaluating multiple possibilities for metabolic pathway processing, thereby allowing us to comprehensively study the interconnected metabolic networks of host-associated microbiota. The model we developed for the seven-member maize root-associated bacterial community presents a step toward predicting plant-microbe behavior, providing hypotheses for future experimental testing and serving as a template for expanding model complexity to more members and other systems.

The maintenance of endosymbiosis in cnidarians depends on the tight regulation of host immunity, cell cycle, and nutrient exchange, yet how these processes are impacted by interacting environmental stressors remains largely unknown. To address this, we employed physiological metrics, gene expression analysis, microbiome characterization, imaging (NF-{kappa}B localization, endoplasmic reticulum ultrastructure, EdU labeling), and stable isotope tracing in the model sea anemone Exaiptasia diaphana to examine the effects of heat and nitrate on these regulatory processes, individually and in combination. Heat treatment led to NF-{kappa}B activation, proteostatic stress, suppression of nutrient exchange, decreased cell-cycle progression, and microbiome restructuring, with all effects more pronounced in symbiotic than aposymbiotic anemones. In symbiotic anemones, nitrate partially offset these heat-induced responses through sustained carbon translocation, suggesting that the presence of symbionts, in conjunction with elevated nitrate, can temporarily buffer host thermal stress. However, prolonged combined exposure resulted in holobiont failure. These findings reveal that while nitrate enrichment can transiently delay the onset of bleaching, it does not preserve the regulatory networks required for symbiotic stability -- underscoring the vulnerability of cnidarian holobionts to the compounding effects of warming and nitrate pollution.

Corals harbor a diverse bacterial community that facilitates adaptation and sustains their health. In coral holobiont research, culture-independent approaches have transformed the existing paradigm. Molecular techniques, such as metabarcoding, revealed a high diversity of previously unrecognized bacterial symbionts. Coral microbiota characterization has relied on these techniques over the last decade, but relying solely on them does not provide a detailed understanding of the dynamics of the coral holobiont complex. Returning to classic microbiological methods and in vitro experimentation can yield novel insights into symbiont roles, physiology, and interactions within the holobiont. Under this premise, we aimed to isolate and culture bacteria from four Mediterranean corals. The recovery of 84 pure bacterial isolates and their initial classification based on the 16S rRNA gene revealed substantial diversity among symbionts amenable to culture. Several isolates represent novel species within relevant genera, such as Vibrio, underscoring the value of culture-based studies. All cultures were cryopreserved to guarantee long-term accessibility for future projects. This represents a key step towards describing the roles of bacteria within the coral holobiont, as cultures enable in-depth morphological and physiological characterization of the symbionts and experimental ecology studies.

Northern peatlands store large carbon stocks but are sensitive to disturbance. Hydrology, vegetation, herbivory and snow conditions may affect the soil microorganisms driving methane (CH) and nitrous oxide (N2O) cycling. We investigated how reindeer exclusion and snow depth (increased and reduced relative to ambient) manipulations (ongoing for three seasons) influenced archaeal and bacterial communities in a boreal rich fen. Metagenomic (MG) and metatranscriptomic (MT) sequencing were combined with pore-water chemistry and CH flux measurements to link the microbiome to ecosystem processes. Microbial communities differed between outside and inside the exclosure. However, these patterns primarily reflected underlying hydrological variation. Slightly wetter inside plots showed higher expression of denitrification genes (norB, nosZ) and lower (nirS+nirK)/nosZ ratios, indicating greater potential for complete denitrification to N2 instead of N2O. Methane dynamics were mainly associated with vegetation: plots associated with Carex rostrata exhibited lower pmoA/mcrA ratios and elevated CH fluxes. Snow manipulations had subtle effects: reduced snow depth decreased the expression of taxa dependent on microbial interactions, while the effect to the investigated metabolic marker genes was small. Overall hydrology, leading to variations in redox conditions and nutrient availability, together with vegetation appeared as the primary drivers on microbial greenhouse gas processes in this peatland.

Many environmental bacteria do not readily grow under laboratory conditions and population establishment often occurs stochastically. Although the scout hypothesis has been proposed to explain stochastic population establishment in environmental bacteria, how stochastic population establishment is shaped by individual cell growth behaviors in environmental isolates remains unclear. In the present study, we focused on the ammonia-oxidizing bacterium Nitrosomonas sp. PY1 and showed that environmentally responsive individual cell growth behavior, incorporating time-dependent stochastic growth initiation, shapes both deterministic and stochastic population establishment dynamics. Using single-cell observation, we revealed that PY1 altered cell growth behavior in response to surrounding biomass production ({Delta}Vt). These {Delta}Vt-dependent changes in growth behavior were suppressed by the addition of its own cell-free supernatant (CFS), indicating the presence of a growth regulation mechanism via cell-cell communication. Replicate cultures under the same conditions showed that the population establishment of PY1 was stochastic, whereas the model strain Nitrosomonas europaea exhibited synchronized population establishment, consistent with previous reports. This stochasticity in PY1 was also eliminated by the addition of CFS. Finally, a simulation model based on {Delta}Vt-dependent cell growth behavior of PY1 successfully reproduced synchronized population establishment in the presence of CFS. By contrast, the stochastic population establishment observed in the absence of CFS was successfully reproduced by a model incorporating {Delta}Vt-independent growth initiation following a Weibull distribution. Such environmentally responsive changes in population establishment dynamics may contribute to the low isolation success of environmental bacteria and sudden blooms of the rare biosphere.

Microbial ammonia oxidation, the first and rate-limiting step of nitrification, plays a central role in soil nitrogen cycling. It is most relevant in agricultural soils as nitrifiers compete with crops for ammonia-based fertilizers. Therefore, synthetic nitrification inhibitors are widely used alongside fertilizers to reduce the activities of dominant drivers of this process, i.e. ammonia-oxidizing archaea (AOA) and bacteria (AOB). However, the physiological responses of ammonia oxidizers remain poorly resolved. Here the response of the AOA Nitrososphaera viennensis to the nitrification inhibitors 3,4-dimethylpyrazole phosphate (DMPP) and allylthiourea (ATU) were investigated using a combination of functional genomics, physiological assays, and relief experiments. The results overturn earlier assumptions that DMPP and ATU act by chelating free copper. Both compounds affected ammonia oxidation and triggered broader shifts in energy metabolism and stress-response pathways, which diverged markedly between the two inhibitors. We propose a competitive inhibition of the ammonia monooxygenase complex with DMPP as it can be alleviated by additional ammonia and elicits activation of urea acquisition, while ATU acted as a non-competitive inhibitor generally inducing quiescence. Both modes of inhibition were associated with clear transcriptomic and proteomic signals that will be advantageous for the identification of mechanisms of other nitrification inhibitors in the future. Key word: Ammonia-oxidizing archaea, nitrification, nitrification inhibitors, archaea, nitrogen cycle

As key drivers of nitrification, ammonia-oxidizing archaea (AOA) play a central role in the global nitrogen cycle and contribute significantly to the emissions of the potent greenhouse gas nitrous oxide (N2O). However, the ecological implications of AOA growth as biofilms, remain poorly understood. Since nitrite production can be used to follow cellular activities directly we were able to compare biofilms with planktonic cells of the terrestrial model AOA Nitrososphaera viennensis at ecologically and agriculturally relevant conditions. Biofilms were more resistant across nearly all tested conditions and remained active at lower temperatures, acidic pH, and high ammonium concentrations. Collectively, activities in biofilm help reconcile discrepancies between earlier laboratory and environmental observations of soil AOA. Additionally, biofilms showed a high general resilience and lowered sensitivities to nitrification inhibitors. Although in situ biofilms grown in microrespiratory chambers exhibited activity and ammonia affinity similar to planktonic cells, biofilm cultures produced only half as much N2O. The enhanced fitness of biofilms across all tested conditions vastly expands the potential ecophysiological niche of AOA and supports the hypothesis that biofilm growth represents the in situ phenotype of AOA in soil environments.

Microbial communities are central to the biogeochemical cycling of nutrients, critically shaping ecosystem functioning and influencing climate change mitigation. Mangrove ecosystems are among the most important global carbon sinks that enable large amounts of carbon to be sequestered and stored. However, gaps persist in understanding the fundamental aspects of microbial-driven carbon cycling in these environments. This research explores the microbial taxonomic and functional diversity related to carbon cycling in selected tropical mangrove sediments across various locations and depths. Sequencing data analyses based on the 16S rRNA gene revealed distinct microbial community composition but conserved predicted functions across the different mangrove locations. Depth was a strong influence on the functional composition, with carbon-related pathways and metabolic strategies differing between top and bottom sediments. Putative functional gene abundance analyses revealed that carbon fixation processes were among the top carbon-related pathways, suggesting the key role of mangrove microbial communities in sustaining long-term carbon storage. Within these communities, Desulfobacterota appeared as a primary contributor to carbon fixation, while Chloroflexota played a significant role in carbon metabolism and methane cycling. Co-occurrence network analyses also revealed that these microbial groups were among the keystone taxa in mangrove sediments. Our study adds on to the body of knowledge on the mangrove microbiome and their carbon metabolic processes, which helps to improve strategies for managing and leveraging these vital carbon sinks.

Ectomycorrhizal (EcM) fungi are well-recognized symbionts impacting tree health and ecosystem functioning globally, yet understanding of their timing of proliferation in soils across seasons and years remains limited. We analyzed monthly patterns of EcM fungal abundance and community structure over two years in five temperate monodominant forest plots via quantitative PCR and Illumina sequencing. We found that the phenological dynamics of EcM fungi differed significantly by host tree leaf habit, fungal exploration type, fungal genus, and soil moisture. Overall, total EcM fungal abundances based on qPCR consistently peaked in autumn, and were more dynamic in evergreen than deciduous plots, supporting ideas of surplus carbon and asymmetric above-belowground dynamics. Longer-distance exploration types peaked earlier and were more stable than shorter-distance types, suggesting an independent and supportive role in releasing spring nutrients. About half of 20 focal taxa consistently peaked in either autumn, summer, or spring, while others were either host- and/or year-dependent. Our findings highlight that phenology is a key EcM fungal trait best explained by both host and fungal contributions, and future studies across biomes should consider seasonal shifts and sampling to elucidate phenological traits. Summary- The timing of belowground production and seasonal community dynamics remain poorly understood for ectomycorrhizal (EcM) fungi. - We collected soils monthly for two years from five temperate monodominant forest plots. - Fungal production peaked in autumn, shorter-distance and evergreen-associated spanned wider ranges, and half of focal fungal genera showed seasonal preference, emphasizing autumn surplus carbon and spring nutrients from long-distance types. - Future studies should consider seasonal shifts when sampling EcM fungal communities, and forest carbon models should include asymmetric above-belowground phenology. Translated Summary (Spanish)- La fenologia de la produccion y composicion de comunidades de hongos ectomicorrizicos (EcM) es poco estudiada. - Recolectamos suelos mensualmente por dos anos de cinco parcelas mono-dominantes templados. - Produccion maxima de hongos ocurrio en otono, hongos asociados con arboles siempreverdes y de exploracion de corta-distancia observaron rangos mas amplios, y la mitad de generos de hongos focales observaron preferencia estacional, enfatizando extra carbono en otono y nutrientes en primavera de tipos larga-distancia. - Estudios deben considerar cambios estacionales para el muestreo de hongos EcM, y modelos de carbono deben incluir fenologia asimetrica entre hojas y hongos. Plain language summaryEctomycorrhizal fungi are critical for the global carbon cycle, but their seasonal and inter-annual growth patterns remain unclear. We sample soil DNA monthly over two years across five different monodominant temperate forest stands. We find an overall belowground peak in autumn, with significantly later growth under wetter conditions, more dynamism with evergreen trees, and distinct spring growth by longer-distance fungi.

Microbial colonization is a major cause of deterioration in paintings, leading to discoloration, pigment degradation, and loss of structural integrity. While biodeterioration of artworks has been studied in temperate climates, tropical environments remain underexplored despite their high humidity and temperature, which promote microbial growth. This study assessed the microbiological deterioration of two eighteenth-century oil paintings, La Muerte de San Jose and Virgen de Guadalupe, located in Orosis Colonial Church and Religious Art Museum, Costa Rica. Microorganisms were isolated and identified using VITEK(R) 2, microscopy, and MALDI-ToF analysis, and their biofilm-forming capacity was evaluated. Additionally, the antimicrobial activity of six essential oil components was tested using direct and indirect contact assays. Twenty-three bacterial species and fifteen fungal genera were identified, with Bacillus, Staphylococcus, Cladosporium, and Aspergillus among the most common. Notably, La Virgen de Guadalupe displayed the highest microbial diversity, reflected in a high Shannon index, indicative of a more complex microbial community. Several isolates displayed strong biofilm formation, particularly Bacillus subtilis/amyloliquefaciens/vallismortis and Staphylococcus saprophyticus. Linalool exhibited the strongest inhibitory activity, achieving complete bacterial growth inhibition in non-contact assays. Environmental monitoring revealed persistently elevated relative humidity and CO2 levels during the study period. Together, these results reveal the complex microbial ecology of tropical heritage paintings and demonstrate that volatile essential oil components can serve as candidates for low-impact antimicrobial strategies in preventive conservation. ImportanceUnderstanding the microbiological deterioration of cultural heritage in tropical environments is crucial for designing sustainable conservation strategies. While microbial colonization of artworks has been widely studied in temperate regions, data from tropical climates remain limited despite inherently favorable conditions for microbial proliferation. This study integrates microbiological, environmental, and physicochemical analyses to characterize microbial communities colonizing eighteenth-century oil paintings in Orosi, Costa Rica. By combining microbial identification, biofilm quantification, and essential oil biocide testing, it bridges applied microbiology and cultural heritage conservation. The finding that volatile components such as linalool inhibit biofilm-forming bacteria without direct contact highlights their potential as eco-friendly, noninvasive antimicrobial alternatives to conventional biocides. These results expand the understanding of biodeterioration dynamics under tropical conditions and offer a practical framework for developing sustainable, evidence-based conservation protocols that protect both heritage materials and the environment. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=171 SRC="FIGDIR/small/723565v1_ufig1.gif" ALT="Figure 1"> View larger version (98K): org.highwire.dtl.DTLVardef@16cd608org.highwire.dtl.DTLVardef@57aa00org.highwire.dtl.DTLVardef@159fcbeorg.highwire.dtl.DTLVardef@e0363b_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 0.C_FLOATNO Artistic visualization of the geographical context of the studied artworks and the multidisciplinary analytical approaches applied, highlighting the diversity of microorganisms identified (illustration by Keylin Urena-Alvarado). C_FIG

A challenge in using plant biomass is its highly recalcitrant nature, which makes it economically infeasible to utilize. In natural environments, various microbes, including bacteria and fungi, are reported to decompose plant cell wall materials such as cellulose and hemicellulose, and there may be undescribed microbes that contribute to the degradation of plant biomass. We focused on isolating novel plant biomass-degrading bacteria and screened more than 100 isolates from the Tomakomai experimental forest in Hokkaido, Japan. Among them, one novel Bacillus species was chosen for whole-genome sequencing. Comparative genomics and a carbon source utilization assay indicated that the isolate belongs to a subspecies of Bacillus subtilis, which we named B. sp. TTS1. Glucose, cellobiose, xylose, xylan, mannose, or mannan was used as the sole carbon source in the minimum medium, and the growth of this bacterium was determined. Furthermore, a proteomic analysis of B. sp. TTS1 was performed using culture supernatants from various polysaccharide-containing media. In the present study, several key enzymes involved in plant biomass degradation were identified, namely {beta}-1,4-mannanase and xylanase, and they were highly enriched in all tested polysaccharides.

Hydrogenophilus thermoluteolus TH-1 is a thermophilic hydrogen-oxidizing bacterium capable of producing poly(3-hydroxybutyrate) (PHB) from CO2. To redirect carbon flux for producing other useful biomaterials, we disrupted the acetoacetyl-CoA reductase genes (phaB1 and phaB2), which are central to the primary PHB synthesis pathway. Unexpectedly, the resulting {Delta}phaB1B2 mutant still accumulated PHB under autotrophic conditions, reaching approximately 25-35 % of the wild-type level. Furthermore, PHB accumulation in the mutant was significantly restored when fatty acids (butyrate and oleate) were used as carbon sources, whereas acetate and malate resulted in reduced accumulation. These results suggest the existence of a PhaB-independent PHB synthesis pathway. We propose that intermediates from the {beta}-oxidation of fatty acids are converted to (R)-3-hydroxybutyryl-CoA, bypassing the disrupted PhaB enzymes. Additionally, the basal PHB production from non-fatty acid sources implies the involvement of a reverse {beta}-oxidation pathway. This study highlights the metabolic versatility of strain TH-1 for future metabolic engineering.

Quantifying the lipid biosynthesis rate of archaea in hot spring sediments is necessary to interpret the abundance, isotopic patterns, and environmental significance of archaeal lipid biosignatures, with implications for modern biogeochemical cycling and astrobiology. Here, we performed lipid hydrogen stable isotope probing (LH-SIP) experiments on whole sediments collected from two high-temperature, suboxic, circumneutral hot springs in Yellowstone National Park (USA) and El Tatio Geyserfield (Chile). We determined the incorporation of 2H2O into intact polar lipids (IPLs) which provides a taxon- and metabolism-agnostic quantification of biosynthesis under near-natural conditions. We targeted isoprenoid glycerol dialkyl glycerol tetraether lipids (IPL iGDGTs) and recovered structures with 0 to 7 cyclopentyl rings from both springs. We observed minor 2H-uptake into archaeal IPLs in spring sediments in Yellowstone, corresponding to decadal-scale apparent generation times (16 {+/-} 7 years), and no uptake in El Tatio sediments (consistent with minimum generation times of 35 {+/-} 5 years). We infer that net production of sedimentary IPL-iGDGTs is very slow, consistent with a combination of slow archaeal growth, persistence of older IPLs, lipid recycling, and/or contributions from recently sedimented planktonic biomass. These are the first direct, ex situ estimates of archaeal lipid production rates in terrestrial hydrothermal systems using LH-SIP incubations and provide critical constraints for interpreting archaeal lipids in ancient hot spring deposits. This research establishes a framework for assessing activity by slow-growing extremophilic archaea in hydrothermal environments and provides support for targeting hydrothermal deposits on Mars for biosignature detection efforts. Plain Language SummaryHot springs on Earth are important natural laboratories for understanding how signs of life might form and be preserved in hydrothermal environments on early Earth or Mars. In this study, we examine the rate of archaeal lipid biosignature production in sediments from two hot springs in Yellowstone National Park and the El Tatio Geyserfield in Chile. We used a method that measures new microbial production by tracing heavy hydrogen from labeled water as microbes incorporate that hydrogen into newly made lipids in their cell membranes. We found that archaeal lipids in hot spring sediments are produced very slowly, on timescales of decades. This result, along with the chemical stability of lipids and the rapid mineralization rate in hot springs, may allow these molecular biosignatures to be entombed and preserved in hot spring mineral deposits. These results help us better interpret ancient hydrothermal deposits on Earth and support the idea that slowly growing microbial communities could still leave detectable molecular traces in similar environments on Mars and other rocky planets. Key PointsO_LILipid hydrogen stable isotope probing is applied to high temperature hot spring sediments for the first time C_LIO_LIIn hot spring sediments, archaeal lipid production occurs on decadal timescales comparable to some marine sediments, but are much faster than the century- to millennia-scale rates observed in the deep subsurface C_LIO_LIConfirmation of archaeal lipid synthesis in hot spring sediments adds additional support for targeting Martian hydrothermal deposits for biosignature detection efforts C_LI