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At least 37 records · Page 2

Age over sex: evaluating gut microbiota differences in healthy Chinese populations

Age and gender have been recognized as two pivotal covariates affecting the composition of the gut microbiota. However, their mediated variations in microbiota seem to be inconsistent across different countries and races. In this study, 613 individuals, whom we referred to as the “healthy” population, were selected from 1,018 volunteers through rigorous selection using 16S rRNA sequencing. Three enterotypes were identified, namely, Escherichia–Shigella , mixture ( Bacteroides and Faecalibacterium ), and Prevotella . Moreover, 11 covariates that explain the differences in microbiota were determined, with age being the predominant factor. Furthermore, age-related differences in alpha diversity, beta diversity, and core genera were observed in our cohort. Remarkably, after adjusting for 10 covariates other than age, abundant genera that differed between age groups were demonstrated. In contrast, minimal differences in alpha diversity, beta diversity, and differentially abundant genera were observed between male and female individuals. Furthermore, we also demonstrated the age trajectories of several well-known beneficial genera, lipopolysaccharide (LPS)-producing genera, and short-chain fatty acids (SCFAs)-producing genera. Overall, our study further elucidated the effects mediated by age and gender on microbiota differences, which are of significant importance for a comprehensive understanding of the gut microbiome spectrum in healthy individuals.

Wu, Jiacheng↗

Identification of shared viral sequences in peat moss metagenomes reveals elements of a possible Sphagnum core virome

Viruses are an understudied component of plant microbiomes. Identifying viruses that are shared between individual plants, or members of the “core virome”, could reveal stable viral populations with the potential to modulate the composition and function of the microbiome. Here, we examined the virome associated with Sphagnum mosses, a keystone species that has direct influence over the fate of peatland carbon stores. We analyzed bulk metagenomes and metatranscriptomes generated from Sphagnum field samples collected over a ten-month period to identify virus-like sequences shared among plants. Individual Sphagnum samples harbored distinct DNA and RNA viromes where only a small percentage (< 1%) of the total number of identified viral contigs were shared among all samples. Based on taxonomic classification, the shared viral contigs represent bacterial viruses, or phage (Caudoviricetes), as well as viruses of eukaryotes, namely nucleocytoplasmic large DNA viruses (Nucleocytoviricota) and RNA viruses (Riboviria). We linked the shared phage-like contigs to viral regions within sequenced genomes of bacterial taxa that are members of the Sphagnum core microbiome, suggesting that these contigs represent temperate phage or degraded prophage. The putative nucleocytoplasmic large DNA viruses and RNA viruses were phylogenetically diverse and showed sequence similarity to viruses associated with a broad range of hosts and environmental sources. The identification of shared viral contigs suggested that, despite the compositional heterogeneity between samples, Sphagnum mosses may harbor a core virome. Future work validating the presence of the core virome is warranted as it may aid in understanding how persistent viruses impact microbiome ecology and symbiont evolution within this climatically relevant keystone species.

Metagenomics↗

Randomized control trial of moderate dose vitamin D alters microbiota stability and metabolite networks in healthy adults

ABSTRACT Evidence indicates that both vitamin D and the gut microbiome are involved in the process of colon carcinogenesis. However, it is unclear what effects supplemental vitamin D 3 has on the gut microbiome and its metabolites in healthy adults. We conducted a double-blind, randomized, placebo-controlled trial to identify the acute and long-term microbiota structural and metabolite changes that occur in response to a moderate dose (4,000 IU) of vitamin D 3 for 12 weeks in healthy adults. Our results demonstrated a significant increase in serum 25-hydroxy-vitamin D (25(OH)D) in the treatment group compared to placebo ( P < 0.0001). Vitamin D 3 significantly increased compositional similarity ( P < 0.0001) in the treatment group, and enriched members of the Bifidobacteriaceae family. We also identified a significant inverse relationship between the percent change in serum 25(OH)D and microbial stability in the treatment group ( R = −0.52, P < 0.019). Furthermore, vitamin D 3 supplementation resulted in notable metabolic shifts, in addition to resulting in a drastic rewiring of key gut microbial-metabolic associations. In conclusion, we show that a moderate dose of vitamin D 3 among healthy adults has unique acute and persistent effects on the fecal microbiota, and suggest novel mechanisms by which vitamin D may affect the host-microbiota relationship. IMPORTANCE Preventative measures to reduce the rise in early-onset colorectal cancer are of critical need. Both vitamin D, dietary and serum levels, and the gut microbiome are implicated in the etiology of colorectal cancer. By understanding the intimate relationship between vitamin D, the gut microbiome, and its metabolites, we may be able to identify key mechanisms that can be targeted for intervention, including inflammation and metabolic dysfunction. Furthermore, the similarity of vitamin D to cholesterol, which is metabolized by the gut microbiome, gives precedence to its ability to produce metabolites that can be further studied and leveraged for controlling colorectal cancer incidence and mortality.

Wyatt, Madhur↗

Aboveground and Belowground Responses to Cyanobacterial Biofertilizer Supplement in a Semi-Arid, Perennial Bioenergy Cropping System

The need for sustainable agricultural practices to meet the food, feed, and fuel demands of a growing global population while reducing detrimental environmental impacts has driven research in multi-faceted approaches to agricultural sustainability. Perennial cropping systems and microbial biofertilizer supplements are two emerging strategies to increase agricultural sustainability that are studied in tandem for the first time in this study. During the establishment phase of a perennial switchgrass stand in SW Montana, USA, we supplemented synthetic fertilization with a nitrogen-fixing cyanobacterial biofertilizer (CBF) and were able to maintain aboveground crop productivity in comparison to a synthetic only (urea) fertilizer treatment. Soil chemical analysis conducted at the end of the growing season revealed that late-season nitrogen availability in CBF-supplemented field plots increased relative to urea-only plots. High-throughput sequencing of bacterial/archaeal and fungal communities suggested fine-scale responses of the microbial community and sensitivity to fertilization among arbuscular mycorrhizal fungi, Planctomycetes, Proteobacteria, and Actinobacteria. Given their critical role in plant productivity and soil nutrient cycling, soil microbiome monitoring is vital to understand the impacts of implementation of alternative agricultural practices on soil health.

biofertilizer↗

Culture-Independent Fungal Profiling for the International Space Station using Nanopore Sequencing: Method Development

Microbial monitoring of the International Space Station (ISS) environment is a crew health requirement that encompasses both bacterial and fungal identification. To achieve this currently, culture-based methods are used for sample collection, and these samples must be returned to the laboratory for analysis. The use of culture and the need for sample return to Earth results in a bias toward culturable organisms and causes a significant delay between sample collection and delivery of final data (weeks to months), respectively. Recently, advancements in molecular technology have aided a broad range of applications, including medical, industrial, and basic sciences. Additionally, increases in portability and ease-of-use of molecular platforms have provided point-of-use capabilities demonstrated by the miniPCR thermal cycler (miniPCR bio) and the MinION sequencer (Oxford Nanopore Technologies). Together, these devices have been applied to, and validated for, the identification of bacteria onboard the ISS. Building on this work, we have developed a spaceflight-compatible fungal workflow. Molecular-based fungal analysis is complicated by low biomass, difficult-to-lyse spores, debate regarding the region for taxonomic assignment, and the lack of bioinformatic pipelines and reference databases. To overcome these difficulties, primers yielding an ~ 2 Kb amplicon were validated against a wide range of ISS fungal isolates. The current spaceflight library preparation was substantially optimized, a bioinformatic pipeline was created, and refinements to the UNITE database were implemented. To compare this optimized method to the current culture-based standard, 30 sample sets (60 total swabs, two swabs held in tandem) were evaluated. Parallel fungal profiles were obtained between the two methods, with the culture-independent method revealing increased diversity. The addition of this method to the already established bacterial process fulfills the crew health identification requirement. Moreover, the implementation of this method onboard ISS will enhance our understanding of its unique fungal microbiome.

Hang N. Nguyen↗

Alkaloids are associated with increased microbial diversity and metabolic function in poison frogs

Shifts in host-associated microbiomes can impact both host and microbes. It is of interest to understand how perturbations, like the introduction of exogenous chemicals, impact microbiomes. In poison frogs (family Dendrobatidae), the skin microbiome is exposed to alkaloids that the frogs sequester for defense. These alkaloids are antimicrobial; however, their effect on the frogs’ skin microbiome is unknown. To test this, we characterized microbial communities from field-collected dendrobatid frogs. Then, we conducted a laboratory experiment to monitor the effect of the alkaloid decahydroquinoline (DHQ) on the microbiome of two frog species with contrasting alkaloid loads in nature. In both datasets, we found that alkaloid-exposed microbiomes were more phylogenetically diverse, with an increase in diversity among rare taxa. Further, to better understand the isolate-specific response to alkaloids, we cultured microbial isolates from poison frog skin and found that many isolates exhibited enhanced growth or were not impacted by the addition of DHQ. To further explore the microbial response to alkaloids, we sequenced the metagenomes from high- and low-alkaloid frogs and observed a greater diversity of genes associated with nitrogen and carbon metabolism in high-alkaloid frogs. From these data, we hypothesized that some strains may metabolize the alkaloids. We used stable isotope tracing coupled to nanoSIMS (nanoscale secondary ion mass spectrometry), which supported the idea that some of these isolates are able to metabolize DHQ. Together, these data suggest that poison frog alkaloids open new niches for skin-associated microbes with specific adaptations, such as alkaloid metabolism, that enable survival in this environment.

59 BASIC BIOLOGICAL SCIENCES↗

Synthetic microbial communities: Bridging research and application in second-generation bioenergy feedstock microbiomes

The sustainable production of purpose-grown bioenergy feedstocks is essential in transitioning away from fossil fuels. Synthetic communities (SynComs) are consortia of microorganisms that can be used as biological interventions to support objectives like plant growth and stress tolerance. This review examines the state of knowledge regarding microbiomes and SynComs of second-generation bioenergy feedstocks, focusing on the rhizosphere. We first provide an overview of second-generation feedstocks, including switchgrass (Panicum virgatum), miscanthus (Miscanthus × giganteus), sorghum (Sorghum spp.), sugarcane (Saccharum spp.), and poplar (Populus spp.), and summarize our current understanding of their plant-soil-microbiome ecology. We next discuss considerations in the objectives, design, and evaluation of SynComs to enhance feedstock production, and then critically review the literature around their use. Our literature analysis revealed that SynCom performance varied substantially between controlled pilot experiments and field trials, possibly due to system complexity that could not be fully considered in their design and pilot evaluation. We identified a gap in the use of SynComs to support the unique sustainability objectives of biofuel feedstock agriculture, presenting an opportunity to leverage these additional microbial traits in SynCom designs. Finally, we emphasize the importance of targeted research to identify the ecological principles that govern the assembly, activation, and persistence of microbes in the feedstock rhizosphere, thereby enhancing our capacity to manage microbiomes under diverse environmental conditions and ensure their functionality. Beyond biofuels, SynComs are a promising microbiome management strategy for crop production; however, an ecologically informed design and evaluation of SynComs are advised.

SynCom↗

Gut microbiota carbon and sulfur metabolisms support Salmonella infections

Abstract Salmonella enterica serovar Typhimurium is a pervasive enteric pathogen and ongoing global threat to public health. Ecological studies in the Salmonella impacted gut remain underrepresented in the literature, discounting microbiome mediated interactions that may inform Salmonella physiology during colonization and infection. To understand the microbial ecology of Salmonella remodeling of the gut microbiome, we performed multi-omics on fecal microbial communities from untreated and Salmonella-infected mice. Reconstructed genomes recruited metatranscriptomic and metabolomic data providing a strain-resolved view of the expressed metabolisms of the microbiome during Salmonella infection. These data informed possible Salmonella interactions with members of the gut microbiome that were previously uncharacterized. Salmonella-induced inflammation significantly reduced the diversity of genomes that recruited transcripts in the gut microbiome, yet increased transcript mapping was observed for seven members, among which Luxibacter and Ligilactobacillus transcript read recruitment was most prevalent. Metatranscriptomic insights from Salmonella and other persistent taxa in the inflamed microbiome further expounded the necessity for oxidative tolerance mechanisms to endure the host inflammatory responses to infection. In the inflamed gut lactate was a key metabolite, with microbiota production and consumption reported amongst members with detected transcript recruitment. We also showed that organic sulfur sources could be converted by gut microbiota to yield inorganic sulfur pools that become oxidized in the inflamed gut, resulting in thiosulfate and tetrathionate that support Salmonella respiration. This research advances physiological microbiome insights beyond prior amplicon-based approaches, with the transcriptionally active organismal and metabolic pathways outlined here offering intriguing intervention targets in the Salmonella-infected intestine.

59 BASIC BIOLOGICAL SCIENCES↗

Understanding the Impact of The Deep Space and Lunar Environment on Crop Production and the Associated Microbiome

As the Artemis mission advances to the Moon, women and men will be venturing into dangerous environments and facing unique hazards. In addition to living in closed environments, the deep space environment adds high levels of radiation, the absence of Earth’s magnetic field, and altered gravity. These elements present new challenges to long duration space flight that need to be overcome. To meet these challenges we have to understand:

C. D. Quincy↗

A stable 15-member bacterial SynCom promotes Brachypodium growth under drought stress

Introduction: Rhizosphere microbiomes are known to drive soil nutrient cycling and influence plant fitness during adverse environmental conditions. Field-derived robust Synthetic Communities (SynComs) of microbes mimicking the diversity of rhizosphere microbiomes can greatly advance a deeper understanding of such processes. However, assembling stable, genetically tractable, reproducible, and scalable SynComs remains challenging. Methods: Here, we present a systematic approach using a combination of network analysis and cultivation-guided methods to construct a 15-member SynCom from the rhizobiome of Brachypodium distachyon. This SynCom incorporates diverse strains from five bacterial phyla. Genomic analysis of the individual strains was performed to reveal encoded plant growth-promoting traits, including genes for the synthesis of osmoprotectants (trehalose and betaine) and Na+/K+ transporters, and some predicted traits were validated by laboratory phenotypic assays. Results: The SynCom demonstrates strong stability both in vitro and in planta. Most strains encoded multiple plant growth-promoting functions, and several of these were confirmed experimentally. The presence of osmoprotectant and ion transporter genes likely contributed to the observed resilience of Brachypodium to drought stress, where plants amended with the SynCom recovered better than those without. We further observed preferential colonization of SynCom strains around root tips under stress, likely due to active interactions between plant root metabolites and bacteria. Discussion: Our results demonstrate that trait-informed construction of synthetic communities can yield stable, functionally diverse consortia that enhance plant resilience under drought. Preferential colonization near root tips points to active, localized plant-microbe signaling as a component of stress-responsive recruitment. This stable SynCom provides a scalable platform for probing mechanisms of plant-microbe interaction and for developing microbiome-based strategies to improve soil and crop performance in variable environments.

Yadav, Archana↗

Data for Genetic Variation in Zea mays Influences Microbial Nitrification and DeNitrification in Conventional Agroecosystems

Nitrogenous fertilizers provide a short-lived benefit to crops in agroecosystems, but stimulate nitrification and denitrification, processes that result in nitrate pollution, N2O production, and reduced soil fertility. Recent advances in plant microbiome science suggest that genetic variation in plants can modulate the composition and activity of rhizosphere N-cycling microorganisms. Here we attempted to determine whether genetic variation exists in Zea mays for the ability to influence the rhizosphere nitrifier and denitrifier microbiome under “real-world” conventional agricultural conditions. To capture an extensive amount of genetic diversity within maize we grew and sampled the rhizosphere microbiome of a diversity panel of germplasm that included ex-PVP inbreds ( Z. mays ssp. mays ), ex-PVP hybrids ( Z. mays ssp. may s), and teosinte ( Z. mays ssp. mexicana and Z. mays ssp. parviglumis ). From these samples, we characterized the microbiome, a suite of microbial genes involved in nitrification and denitrification and carried out N-cycling potential assays. Here we are showing that populations/genotypes of a single species can vary in their ecological interaction with denitrifers and nitrifers. Some hybrid and teosinte genotypes supported microbial communities with lower potential nitrification and potential denitrification activity in the rhizosphere, while inbred genotypes stimulated/did not inhibit these N-cycling activities. These potential differences translated to functional differences in N2O fluxes, with teosinte plots producing less GHG than maize plots. Taken together, these results suggest that Zea genetic variation can lead to changes in N-cycling processes that result in N leaching and N2O production, and thereby are selectable targets for crop improvement. Understanding the underlying genetic variation contributing to belowground microbiome N-cycling into our conventional agricultural system could be useful for sustainability.

Nitrogen↗

Engineering Microbial Communities: Frontier Science for the Bioeconomy Workshop Series

In nature, biological systems are shaped by complex interactions of diverse microorganisms such as bacteria, archaea, fungi, and viruses living within communities called microbiomes (Berg et al. 2020; Prescott 2017). These collective interactions result in emergent community properties that can be leveraged for beneficial purposes such as bioenergy and biomolecule production. Given this potential and the immensity of microbial genomic diversity, the U.S. Department of Energy’s (DOE) Biological and Environmental Research (BER) program has long invested in research to better understand the biology of environmental microbes and microbiomes.

59 BASIC BIOLOGICAL SCIENCES↗

Breaking the reproducibility barrier with standardized protocols for plant–microbiome research

Inter-laboratory replicability is crucial yet challenging in microbiome research. Leveraging microbiomes to promote soil health and plant growth requires understanding underlying molecular mechanisms using reproducible experimental systems. In a global collaborative effort involving five laboratories, we aimed to help advance reproducibility in microbiome studies by testing our ability to replicate synthetic community assembly experiments. Our study compared fabricated ecosystems constructed using two different synthetic bacterial communities, the model grass Brachypodium distachyon, and sterile EcoFAB 2.0 devices. All participating laboratories observed consistent inoculum-dependent changes in plant phenotype, root exudate composition, and final bacterial community structure, where Paraburkholderia sp. OAS925 could dramatically shift microbiome composition. Comparative genomics and exudate utilization linked the pH-dependent colonization ability of Paraburkholderia, which was further confirmed with motility assays. The study provides detailed protocols, benchmarking datasets, and best practices to help advance replicable science and inform future multi-laboratory reproducibility studies.

Novak, Vlastimil↗

Pre-Bleaching Coral Microbiome Is Enriched in Beneficial Taxa and Functions

Coral reef health is tightly connected to the coral holobiont, which is the association between the coral animal and a diverse microbiome functioning as a unit. The coral holobiont depends on key services such as nitrogen and sulfur cycling mediated by the associated bacteria. However, these microbial services may be impaired in response to environmental changes, such as thermal stress. A perturbed microbiome may lead to coral bleaching and disease outbreaks, which have caused an unprecedented loss in coral cover worldwide, particularly correlated to a warming ocean. The response mechanisms of the coral holobiont under high temperatures are not completely understood, but the associated microbial community is a potential source of acquired heat-tolerance. Here we investigate the effects of increased temperature on the taxonomic and functional profiles of coral surface mucous layer (SML) microbiomes in relationship to coral–algal physiology. We used shotgun metagenomics in an experimental setting to understand the dynamics of microbial taxa and genes in the SML microbiome of the coral Pseudodiploria strigosa under heat treatment. The metagenomes of corals exposed to heat showed high similarity at the level of bacterial genera and functional genes related to nitrogen and sulfur metabolism and stress response. The coral SML microbiome responded to heat with an increase in the relative abundance of taxa with probiotic potential, and functional genes for nitrogen and sulfur acquisition. Coral–algal physiology significantly explained the variation in the microbiome at taxonomic and functional levels. These consistent and specific microbial taxa and gene functions that significantly increased in proportional abundance in corals exposed to heat are potentially beneficial to coral health and thermal resistance.

59 BASIC BIOLOGICAL SCIENCES↗

A roadmap to understanding and anticipating microbial gene transfer in soil communities

Engineered microbes are being programmed using synthetic DNA for applications in soil to overcome global challenges related to climate change, energy, food security, and pollution. However, we cannot yet predict gene transfer processes in soil to assess the frequency of unintentional transfer of engineered DNA to environmental microbes when applying synthetic biology technologies at scale. This challenge exists because of the complex and heterogeneous characteristics of soils, which contribute to the fitness and transport of cells and the exchange of genetic material within communities. Here, we describe knowledge gaps about gene transfer across soil microbiomes. Here, we propose strategies to improve our understanding of gene transfer across soil communities, highlight the need to benchmark the performance of biocontainment measures in situ, and discuss responsibly engaging community stakeholders. We highlight opportunities to address knowledge gaps, such as creating a set of soil standards for studying gene transfer across diverse soil types and measuring gene transfer host range across microbiomes using emerging technologies. By comparing gene transfer rates, host range, and persistence of engineered microbes across different soils, we posit that community-scale, environment-specific models can be built that anticipate biotechnology risks. Such studies will enable the design of safer biotechnologies that allow us to realize the benefits of synthetic biology and mitigate risks associated with the release of such technologies.

bioccontainment↗

Genetic variation in Zea mays influences microbial nitrification and denitrification in conventional agroecosystems

Nitrogenous fertilizers provide a short-lived benefit to crops in agroecosystems, but stimulate nitrification and denitrification, processes that result in nitrate pollution, N 2 O production, and reduced soil fertility. Recent advances in plant microbiome science suggest that genetic variation in plants can modulate the composition and activity of rhizosphere N-cycling microorganisms. Here we attempted to determine whether genetic variation exists in Zea mays for the ability to influence the rhizosphere nitrifier and denitrifier microbiome under “real-world” conventional agricultural conditions. To capture an extensive amount of genetic diversity within maize we grew and sampled the rhizosphere microbiome of a diversity panel of germplasm that included ex-PVP inbreds (Z. mays ssp. mays), ex-PVP hybrids (Z. mays ssp. mays), and teosinte (Z. mays ssp. mexicana and Z. mays ssp. parviglumis). From these samples, we characterized the microbiome, a suite of microbial genes involved in nitrification and denitrification and carried out N-cycling potential assays. Here we are showing that populations/genotypes of a single species can vary in their ecological interaction with denitrifers and nitrifers. Some hybrid and teosinte genotypes supported microbial communities with lower potential nitrification and potential denitrification activity in the rhizosphere, while inbred genotypes stimulated/did not inhibit these N-cycling activities. These potential differences translated to functional differences in N 2 O fluxes, with teosinte plots producing less GHG than maize plots. Taken together, these results suggest that Zea genetic variation can lead to changes in N-cycling processes that result in N leaching and N 2 O production, and thereby are selectable targets for crop improvement. Understanding the underlying genetic variation contributing to belowground microbiome N-cycling into our conventional agricultural system could be useful for sustainability.

59 BASIC BIOLOGICAL SCIENCES↗

Microbiome Comparison and Pathogen Identification for Three Migrating Passerines Captured During Spring Season in Jordan Using 16S rRNA Sequencing

Jordan is located on an important spot along the Mediterranean and Black Sea Flyway. Hundreds of migratory bird species have been identified stopping over in Jordan during spring and autumn migratory seasons. Compared to mammals and economically important birds, the microbiomes of wild bird species are severely understudied. Gut microbial composition is a valuable source of information that reflects food preferences, foraging behavior, and the risk of pathogen transmission to humans and other animals. In this study, we assessed the microbiome composition of three species of migrating passerines (willow warblers, lesser whitethroats, and common reed warblers) captured during the spring migration stopover in Jordan in 2023. A total of 59 fecal samples were selected evenly from the three species and subjected to 16S sequencing and microbiome analysis. Our objectives were to determine the diversity of bacteria in these three species, assess the amount of intra- and inter-specific variation, and detect pathogenic genera and species that could pose health risks to humans, domestic animals, and wildlife. Bacteria mainly belonged to the phyla Proteobacteria (62%), Actinobacteriota (18%), Firmicutes (13%), Cyanobacteria (5%), and Bacteroidota (1%). The results reveal that lesser whitethroats had the greatest variation in bacterial genus richness, Shannon diversity, and microbial composition compared to willow warblers and common reed warblers. The three bird species harbored several pathogenic genera and species, including Campylobacter, Enterococcus, Escherichia-Shigella, Mycoplasma, Rickettsia, Clostridium perfringens, and Vibrio cholerae. We suggest further investigation to understand the relationship between migratory behavior and their gut microbiome. We advocate for the use of advanced molecular techniques to characterize the pathogens found in migratory birds that might have public and environmental health impacts in addition to economic loss.

59 BASIC BIOLOGICAL SCIENCES↗

Response of soil nutrient pools and microbiomes to recurrent wildfire disturbance and varying burn severities in a mixed conifer forest

Wildfire is a pervasive disturbance in mixed-conifer forests, yet the relative influence of fire recurrence versus burn severity on soil biogeochemistry and microbial communities remains poorly quantified. We examined a natural gradient of fire history (0–3 prior fires) and burn severity (low–high) spanning 50 yr in a mixed-conifer ecosystem to assess how repeated fire shapes soil carbon (C) and nitrogen (N) pools, their isotopic signatures, mineral and particulate fractions, microbial community composition, carbon-use, CO₂ fluxes, and vegetation cover. Successive fires produced progressively higher bare-ground percentages and lower tree cover, which were tightly linked to declines in microbial diversity and reductions bulk %C, and %N. δ 13 C increased with fire frequency, indicating preferential loss of labile C through combustion or enhanced microbial oxidation, thereby explaining the observed net soil-C decline. Conversely, δ 15 N decreased and pH increased as tree density declined, reflecting altered N cycling and reduced acidification in post-fire soils. Fire recurrence, more than severity, corresponded with a marked shift in the bacterial community: for example, Xanthobacteraceae—key N-fixers and C-cyclers—diminished, while N-fixing Bacillaceae increased, underscoring the tightly coupled nature of soil nutrient dynamics and microbiome composition after repeated burns. Our results demonstrate that fire recurrence appears to be a stronger driver of post-fire soil ecosystem responses in this mixed-conifer forest, influencing both abiotic nutrient pools and the functional potential of the soil microbiome. These findings provide a more enhanced assessment and understanding to date of the biogeochemical consequences of repeated wildfire disturbance that can be used to inform management strategies aimed at preserving soil health in fire-prone landscapes.

54 ENVIRONMENTAL SCIENCES↗