Soil microbiota are key players of terrestrial ecosystem functioning, including decomposition, soil organic matter formation, and nutrient cycling, and interact strongly with plants in the rhizosphere. Several studies have demonstrated the potential of plants to alter soil microbiome assembly and functioning (i.e., through manipulation of soil organic matter pools via root exudation), which can be critical for sustaining soil ecosystem functioning. Using soil from a long-term biodiversity experiment in Germany, we investigated how soil microbial communities responded to variations in plant species richness (1–16 species), functional group richness (1–4 groups), and plant identity (grasses, legumes, small herbs, and tall herbs) using 16S rRNA gene and ITS amplicon sequencing. We examined bacterial and fungal community structure, metabolic potential, and microbial network architecture to better understand the role of the soil microbiome and its net positive relationship between biodiversity and ecosystem functioning. Plant diversity induced gradual shifts in microbial community composition, while increasing soil organic carbon and nitrogen stocks. Microbial networks exhibited increased connectivity, particularly between bacteria and fungi. Meanwhile, mutualistic and antagonistic functional guild representation increased, that is the sum total of plant-beneficial (i.e., endophytes) and plant- or fungi-detrimental (i.e., pathogens and parasites) fungal guilds, respectively. Key nodes shifted from generalist taxa at low plant diversity to more specialized communities at high plant diversity. Notably, fungi responded more strongly than bacteria, and their functional potential was driven by plant functional identity rather than species richness. At low plant diversity, generalist taxa likely exploit less complex and diverse organic carbon inputs, allowing them to dominate available niches. In contrast, higher plant diversity promotes a broader array of specialist taxa that likely benefit from the greater diversity of organic carbon compounds, and thus greater niche availability. As network complexity grows, ecosystem functions are being distributed across more taxa, leading to greater microbiome stability, and ultimately more efficient soil carbon and nutrient cycling. Our findings suggest that higher plant diversity strengthens microbial functioning and enhances microbiome resilience, that is the capacity of the microbial community to maintain soil functioning despite environmental disturbances.

Nonindigenous marine species are a growing threat to coastal ecosystems, but invasive dynamics are understudied in marine environments. The microbiome of invasive macroalgal species has been recognised as contributing to their invasive success, but research to date has largely focussed on the prokaryotic fraction. This research compared the epiphytic fungal communities between a native (Fucus serratus) and an invasive (Sargassum muticum) macroalga, across a relatively small spatial scale (< 800 m), in North Wales. DNA extracts from surfaces of macroalga were PCR-amplified with primers ITS1F and ITS2 and sequenced using Illumina MiSeq. Difference in fungal community composition was observed between all sample groups with the exception of S. muticum and F. serratus samples taken from within a few meters of each other, suggesting environmental drivers of the macroalgal fungal community are more important than host specificity. It also raises the question of whether S. muticum exerts a direct influence on the microbiome of neighbouring algal species, although further research is required to explore this potential interaction. No differences were detected in fungal biodiversity across any of the samples, supporting prior findings of a natural peak in fungal diversity at local scales.

Soil pH is a predominant factor in structuring microbial communities; however, its role in shaping microbial life-history traits across large spatial scales remains underexplored. Here, we hypothesised that bacterial ubiquity, or niche breadth, across a diverse collection of soils is linked to genomic traits. We leveraged a national-scale survey of UK soils (the Countryside Survey) and 16S rRNA gene sequencing data with trait annotations (estimated genome size, coding density, and rRNA operon copy number) to examine trait-environment-niche breadth relationships. Our analyses revealed that soil pH was the dominant environmental driver of niche classification and bacterial community traits along the niche range. Low pH soils (pH <5.5) hosted ubiquitous taxa with larger genome sizes, lower coding densities and lower rRNA copy numbers, implying slower growing taxa with higher genetic facilities. Mildly acidic soils (pH 5.5 to 7) favour higher rRNA copy numbers, intermediate genome sizes and moderate coding densities. Alkaline soils (pH >7) feature communities with the smallest niche range, smallest genomes and highest coding densities. Here, specialisation occurs through streamlining with simpler, smaller genomes favoured. We found that generalist taxa were widespread across the pH range, becoming dominant under acidic conditions, while taxa adapted to higher pH were comparatively scarce in their distribution. These findings identify soil pH as a key physiological filter that aligns microbial genomic traits and ecological strategies across landscapes. By extending prior site-specific results to a broad-scale context, our study highlights how trait-based metrics can predict microbial responses to soil conditions, with implications for understanding ecosystem carbon cycling and informing land management practices aimed at sustaining soil health in the future. ### Competing Interest Statement The authors have declared no competing interest. UK Research and Innovation, BBX011089/1, NE/S005137/1

Abstract Restoring agriculturally degraded habitats to species-rich grasslands is a vital conservation objective. During restoration, how the soil resistome matures alongside microbial community composition and function remains unclear. Here, we tested two competing hypotheses: whether the soil resistome co-occurs through a microbial structural maturation, in which soil restoration is associated with higher-order biotic interactions, or whether antimicrobial resistance (AMR) is instead associated with the competitive pressures and high bacterial taxonomic richness found in disturbed, eutrophic arable land. Using a unique land-use chronosequence on Salisbury Plain, UK, we investigated the trajectory of ecosystem reassembly following the cessation of agricultural activity. Our results demonstrate that AMR abundance increases significantly with restoration age, reaching a maximum in >143-year-old soils. The strongest predictor of this rise in AMR abundance was an increasing microbial eukaryotic signature rather than increasing microbial density, suggesting that resistome expansion is not associated with generalised spatial competition, but rather, co-occurs with structural maturation of the microbiome. We observed an order of magnitude increase in antibiotic biosynthetic potential, dominated by the emergence of streptomycin clusters. Microbial reorientation during soil maturation mirrors the expansion of a core resistome comprised of ancient, intrinsic mechanisms, such as MFS efflux pumps and RbpA target protection, in older soils. We demonstrate that endogenous AMR is a hallmark of healthy, restored soil ecosystems rather than a marker of anthropogenic soil degradation.

The restoration of species-rich calcareous grasslands is a critical conservation objective, yet the recovery of the invisible below-ground microbiome remains poorly quantified compared to above-ground vegetation. Using a unique 143-year land-use chronosequence on Salisbury Plain, UK, we investigated the trajectory of ecosystem reassembly across arable, regenerating (23 and 67 years), and ancient grasslands. By integrating vegetation surveys with soil physiochemistry, microbial profiling, and shotgun metagenomics, we identified a decoupling between floral and edaphic recovery. While the diversity of vegetation recovered relatively rapidly, approaching ancient grassland levels within 23-67 years, soil properties exhibited persistent legacy effects and slow convergence. Bacterial richness decreased with restoration age; this taxonomic contraction was conversely matched by an expansion in inferred metagenomic functional potential. This was reflected in increased functional gene richness and shifts in the relative abundance of specific SEED-annotated functions towards metabolic pathways associated with complex carbon cycling and stress tolerance. These shifts were congruent with the emergence of specific, unnamed genera belonging to Pseudomonadota and Actinomycetota, and the Bacillota species Pristimantibacillus. The soil ecosystem remained distinct from the 143-year stage even after 67 years of recovery, characterized by persistent legacy phosphorus and a slow accumulation of soil organic matter. These findings suggest that passive regeneration alone may be insufficient for full soil functional recovery, and that strategies targeting microbial assembly and long-term carbon dynamics warrant further evaluation.

Human degradation of peatlands worldwide has turned them into net carbon sources. In upland blanket peatlands, erosion disrupts new plant-derived carbon input and exposes deep peat, putting old carbon at risk of oxidation. The efficacy of restoration in preventing carbon loss and recovering ecosystem function depends on microbial responses to both water table manipulation and renewed litter input. Yet it is unclear how these factors alter the microbial communities that ultimately control carbon storage and emissions. We show that microbial community composition in the eroded peatland of Waun Fignen Felen, South Wales, was primarily governed by the bioavailability of organic matter rather than water-table position. Long-term erosion leaves behind a legacy of highly degraded organic matter, unaltered by re-wetting. Where plant litter accumulation is renewed on formerly eroded peat surfaces, the influx of bioavailable organic input supports a distinct microbial community with greater biomass, and evidence of elevated respiration.

Dissolved organic carbon (DOC) concentrations have increased in soil solutions and surface waters over the past 30 years in acid sensitive areas of Europe and Northern America. This has been linked to recovery from acidification of soils with decreasing levels of atmospheric pollution. Whilst previous research has found pH related DOC solubility is a likely driver of increased DOC concentrations in surface waters, it is unclear whether increased DOC production from organic matter decomposition, due to more favourable pH conditions for biological activity, has also contributed to the rising trends in surface waters. We investigated how acidity impacts microbial communities, the decomposition of litter types common to acid-sensitive ecosystems, and the impact on DOC production. A decomposition study using litter bags and the Tea Bag Index was incorporated into an established acidity manipulation field experiment across two sites with contrasting acid deposition histories, and two characteristic soil types (peats and peaty podzols). We found higher rates of litter decomposition in podzol soil than peat leading to higher DOC production and aromaticity, as indicated by absorbance properties. Acidity manipulations did not influence decomposition rates for most litter types, DOC production for any litter type, Tea Bag Index parameters (stabilisation factor and decomposition rate) or microbial diversity, for any site or soil type. Our study suggests that decomposition of aboveground-collected litter does not mediate acidity effects on DOC production, whilst soil type, and physiochemical soil responses to acidity, are more influential on DOC release from organic soils.

Understanding the relationships between bacteria, their ecological and genomic traits, and their environment is important to elucidate microbial community dynamics and their roles in ecosystem functioning. Here, we examined the relationships between soil properties and bacterial traits within highly managed agricultural soil systems subjected to arable crop rotations or management as permanent grass. We assessed the bacterial communities using metabarcoding and assigned each amplicon trait scores for rRNA copy number, genome size, and guanine-cytosine (GC) content, which are classically associated with potential growth rates and specialization. We also calculated the niche breadth trait of each amplicon as a measure of social ubiquity within the examined samples. Within this soil system, we demonstrated that pH was the primary driver of bacterial traits. The weighted mean trait scores of the samples revealed that bacterial communities associated with soils at lower pH (<7) tended to have larger genomes (potential plasticity), have more rRNA (higher growth rate potential), and are more ubiquitous (have less niche specialization) than the bacterial communities from higher pH soils. Our findings highlight not only the association between pH and bacterial community composition but also the importance of pH in driving community functionality by directly influencing genomic and niche traits.

As a promising strategy for improving soil health and mitigating climate change, biochar has received increasing attention in recent years. However, findings regarding its microbial effects are often context-dependent, and the complex impacts of biochar on soil microbial communities remain inadequately understood. Here, we integrated 843 samples of 16 S rRNA sequencing data from 24 independent studies, utilizing machine learning and co-occurrence network analysis to evaluate general patterns and mechanisms by which biochar alters bacteria community composition and survival strategies. Overall, biochar significantly increased soil organic C, total N, pH, and reduced bulk density, which in turn affected microbial communities. While biochar addition did not notably alter bacterial α-diversity (Shannon index or species richness), it significantly changed β-diversity, indicating that biochar addition altered microbial community composition. Biochar changed community composition and survival strategies by reducing the oligotroph/copiotroph ratio, favouring copiotrophs groups (e.g., Bacteroidota). K-strategists (e.g., Planctomycetota) were reduced by biochar, potentially due to increased nutrient availability and increased competition. Model selection analysis identified that soil physicochemical properties were key drivers of α-diversity, while climate variables were the main factors influencing β-diversity. Biochar properties represented key factors influencing changes in microbial survival strategy. Machine learning identified 138 biomarker genera, with biochar-treated soils showing an increase in Pseudomonadota. Co-occurrence network analysis indicated that biochar increased microbial stability, but reduced network complexity, suggesting a trade-off between resilience and functional redundancy following biochar addition. These findings provide new insights into how biochar influences the composition and ecological functions of soil bacterial communities and offer theoretical support for its scientific application in sustainable soil management.

Biodiversity surveys are critical for detecting environmental change; however, undertaking them at scale and capturing all available diversity through observation is challenging and costly. This study evaluated the potential of soil-extracted eDNA to describe plant communities and compared these findings to traditional, observation-based field surveys. We analyzed 789 soil samples using high-throughput amplicon sequencing and compared DNA-based diversity metrics, indicator taxa, predicted vegetation class, and plant cover in a comparison with co-located field survey data. The results indicated that taxonomically aggregated (genus) eDNA-derived data, while showing slightly reduced Shannon's diversity scores, yielded remarkably similar overall richness and composition estimates. However, the DNA indicator taxa and predictive power for vegetation community classification were also lower overall than those recorded by the field survey. In many cases, plant cover could be inferred from amplicon abundance data with some accuracy despite widely differing scales of sampling-0.25 g crumb of soil versus a 1 m2 quadrat. Overall, results from eDNA demonstrated lower sensitivity but were broadly in accordance with traditional surveys, with our findings revealing comparable taxonomic resolution at the genus level. We demonstrate the potential and limitations of a simple molecular method to inform landscape-scale plant biodiversity surveys, a vital tool in the monitoring of land use and environmental change.

Lignocellulose represents a primary input of organic carbon (C) into soils, yet the identity of specific microorganisms and genes which drive lignocellulose turnover in soils remains poorly understood. To address this knowledge gap, we used a 10-year grassland plant-exclusion experiment to investigate how reduced plant C inputs affect microbial communities and their lignocellulolytic potential using a combination of metagenomic sequencing and untargeted metabolomics. We specifically tested the hypothesis that microbial community function in bare fallow plots would transition towards microbiota with genes for recalcitrant biomass degradation (i.e., lignocellulose), when compared to grassland plots with high labile C inputs. Long-term plant exclusion lowered soil C and nitrogen (N) and reduced cellulose content, whilst hemicellulose and lignin were unchanged. Similarly soil microbiomes were highly distinct in long-term bare soils, along with soil extracellular enzyme profiles, though short-term plant-removal effects were less apparent. Plant exclusion resulted in a general enrichment of Firmicutes, Thaumarchaeota, Acidobacteria, Fusobacteria, and Ascomycota, with a general reduction in Actinobacteria. However, changes in bare soil lignocellulose degradation genes were more associated with discrete taxa from diverse lineages, particularly the Proteobacteria. Grouping of lignocellulose-degrading genes into broad substrate classes (cellulases, hemicellulases and lignases) revealed a possible increase in lignin degradation genes under plant exclusion confirming our hypothesis, although all other changes were at the level of the carbohydrate-active enzyme (CAZy) family. Intriguingly, untargeted metabolome profiles were highly responsive to plant exclusion, even after only one year. Bare soils were depleted in oligosaccharides and enriched in monosaccharides, fatty and carboxylic acids, supporting emerging evidence of long-term persistent C being within simple compounds. Together our data show that extracellular lignin degrading enzymes increase under long-term plant exclusion. There is now a need for increased focus on the microbial metabolic mechanisms which regulate the processing and persistence of enzymatically released compounds, particularly in energy limited soils.

The local adaption of soil microbial communities to native litter inputs, the so-called home field effect (HFE), is well established, though this phenomenon has yet to be demonstrated for agriculturally relevant inorganic nutrient sources. Using compound-specific N-15-stable isotope probing of proteinaceous amino acids (AAs), we investigated if continuous long-term grassland fertilisation with either ammonium or nitrate resulted in preferential assimilation by the soil microbial community of the 'home' N fertiliser. Relative ammonium uptake was maximal in historic ammonium treated soils and previously unfertilised soil, confirming a general microbial preference for ammonium likely due to biochemical transformation efficiencies. Assimilation of nitrate and ammonium into AAs was comparable for the historic nitrate fertilisation, indicating that microbial adaptive processes governed by historical land use can dictate the immobilisation efficiency of different fertilisers. This is the first observation of the HFE in long-term fertilised grassland soils, with further work required to investigate abiotic or biotic mechanisms underpinning this phenomena.

Soil organic carbon (SOC) is a soil health indicator and understanding dynamics changing SOC stocks will help achieving net zero goals. Here we present four datasets featuring 11,750 data points covering co-located aboveground and below-ground metrics for exploring ecosystem SOC dynamics. Five sites across England with an established land use contrast, grassland and woodland next to each other, were rigorously sampled for aboveground (n = 109), surface (n = 33 soil water release curves), topsoil, and subsoil metrics. Commonly measured soil metrics were analysed in five soil increments for 0–1 metre (n = 4550). Less commonly measured soil metrics which were assumed to change across the soil profile were measured on a subset of samples only (n = 3762). Additionally, we developed a simple method for soil organic matter fractionation using density fractionation which is part of the less common metrics. Finally, soil metrics which may impact SOC dynamics, but with less confidence as to their importance across the soil profile were only measured on topsoil (~5–15 cm = mineral soil) and subsoil (below 50 cm) samples (n = 2567).

The soil microbiome determines the fate of plant-fixed carbon. The shifts in soil properties caused by land use change leads to modifications in microbiome function, resulting in either loss or gain of soil organic carbon (SOC). Soil pH is the primary factor regulating microbiome characteristics leading to distinct pathways of microbial carbon cycling, but the underlying mechanisms remain understudied. Here, the taxa-trait relationships behind the variable fate of SOC were investigated using metaproteomics, metabarcoding, and a C-13-labeled litter decomposition experiment across two temperate sites with differing soil pH each with a paired land use intensity contrast. C-13 incorporation into microbial biomass increased with land use intensification in low-pH soil but decreased in high-pH soil, with potential impact on carbon use efficiency in opposing directions. Reduction in biosynthesis traits was due to increased abundance of proteins linked to resource acquisition and stress tolerance. These trait trade-offs were underpinned by land use intensification-induced changes in dominant taxa with distinct traits. We observed divergent pH-controlled pathways of SOC cycling. In low-pH soil, land use intensification alleviates microbial abiotic stress resulting in increased biomass production but promotes decomposition and SOC loss. In contrast, in high-pH soil, land use intensification increases microbial physiological constraints and decreases biomass production, leading to reduced necromass build-up and SOC stabilization. We demonstrate how microbial biomass production and respiration dynamics and therefore carbon use efficiency can be decoupled from SOC highlighting the need for its careful consideration in managing SOC storage for soil health and climate change mitigation.

Current understanding of soil carbon dynamics suggests that plant litter quality and soil mineralogy control the formation of mineral-associated soil organic carbon (SOC). Due to more efficient microbial anabolism, high-quality litter may produce more microbial residues for stabilisation on mineral surfaces. To test these fundamental concepts, we manipulate soil mineralogy using pristine minerals, characterise microbial communities and use stable isotopes to measure decomposition of low- and high-quality litter and mineral stabilisation of litter-C. We find that high-quality litter leads to less (not more) efficient formation of mineral-associated SOC due to soil microbial community shifts which lower carbon use efficiency. Low-quality litter enhances loss of pre-existing SOC resulting in no effect of litter quality on total mineral-associated SOC. However, mineral-associated SOC formation is primarily controlled by soil mineralogy. These findings refute the hypothesis that high-quality plant litters form mineral-associated SOC most efficiently and advance our understanding of how mineralogy and litter-microbial interactions regulate SOC formation. This study challenges the hypothesis that high-quality plant litters form stable, mineral-associated soil organic carbon most efficiently, providing evidence that litter-microbial interactions and soil mineralogy regulate soil organic carbon formation.

The genus Tetracladium has historically been regarded as an aquatic hyphomycete. However, sequencing of terrestrial ecosystems has shown that Tetracladium species might also be terrestrial soil and plant-inhabiting fungi. The diversity of Tetracladium species, their distribution across ecosystems, and the factors that shape community composition remain largely unknown. Using internal transcribed spacer (ITS) amplicon sequencing, we investigated the spatial distribution of Tetracladium in 970 soil samples representing the major ecosystems found across the British landscape. Species of the genus were found in 57% of the samples and across all vegetation types. The Tetracladium sequences we recovered included species common in aquatic ecosystems. However, we found five additional clades that clustered with environmental sequences previously found in terrestrial environments. The community composition of the Tetracladium OTUs was mainly related to vegetation type and soil pH. Strikingly, both taxon richness and overall abundance were highest in arable soils and showed positive relationships with soil pH. T. maxilliforme and a taxon of environmental sequences, Tetracladium group 1, was the biggest group, had the most relative abundance across ecosystems and was found in all vegetation types. Overall, this study provides insights into the community composition patterns of Tetracladium in terrestrial ecosystems and highlights the importance of vegetation characteristics in shaping Tetracladium communities.

The seasonal coupling of plant and soil microbial nutrient demands is crucial for efficient ecosystem nutrient cycling and plant production, especially in strongly seasonal alpine ecosystems. Yet, how these seasonal nutrient cycling processes are modified by climate change and what the consequences are for nutrient loss and retention in alpine ecosystems remain unclear. Here, we explored how two pervasive climate change factors, reduced snow cover and shrub expansion, interactively modify the seasonal coupling of plant and soil microbial nitrogen (N) cycling in alpine grasslands, which are warming at double the rate of the global average. We found that the combination of reduced snow cover and shrub expansion disrupted the seasonal coupling of plant and soil N-cycling, with pronounced effects in spring (shortly after snow melt) and autumn (at the onset of plant senescence). In combination, both climate change factors decreased plant organic N-uptake by 70% and 82%, soil microbial biomass N by 19% and 38% and increased soil denitrifier abundances by 253% and 136% in spring and autumn, respectively. Shrub expansion also individually modified the seasonality of soil microbial community composition and stoichiometry towards more N-limited conditions and slower nutrient cycling in spring and autumn. In winter, snow removal markedly reduced the fungal:bacterial biomass ratio, soil N pools and shifted bacterial community composition. Taken together, our findings suggest that interactions between climate change factors can disrupt the temporal coupling of plant and soil microbial N-cycling processes in alpine grasslands. This could diminish the capacity of these globally widespread alpine ecosystems to retain N and support plant productivity under future climate change.

Rainforests provide vital ecosystem services that are underpinned by plant-soil interactions. The forests of Borneo are globally important reservoirs of biodiversity and carbon, but a significant proportion of the forest that remains after large-scale agricultural conversion has been extensively modified due to timber harvest. We have limited understanding of how selective logging affects ecosystem functions including biogeochemical cycles driven by soil microbes. In this study, we sampled soil from logging gaps and co-located intact lowland dipterocarp rainforest in Borneo. We characterised soil bacterial and fungal communities and physicochemical properties and determined soil functioning in terms of enzyme activity, nutrient supply rates, and microbial heterotrophic respiration. Soil microbial biomass, alpha diversity, and most soil properties and functions were resistant to logging. However, we found logging significantly shifted soil bacterial and fungal community composition, reduced the abundance of ectomycorrhizal fungi, increased the abundance of arbuscular mycorrhizal fungi, and reduced soil inorganic phosphorous concentration and nitrate supply rate, suggesting some downregulation of nutrient cycling. Within gaps, canopy openness was negatively related to ectomycorrhizal abundance and phosphomonoesterase activity and positively related to ammonium supply rate, suggesting control on soil phosphorus and nitrogen cycles via functional shifts in fungal communities. We found some evidence for reduced soil heterotrophic respiration with greater logging disturbance. Overall, our results demonstrate that while many soil microbial community attributes, soil properties, and functions may be resistant to selective logging, logging can significantly impact the composition and abundance of key soil microbial groups linked to the regulation of vital nutrient and carbon cycles in tropical forests.

Globally, major efforts are being made to restore peatlands to maximise their resilience to anthropogenic climate change, which puts continuous pressure on peatland ecosystems and modifies the geography of the environmental envelope that underpins peatland functioning. A probable effect of climate change is reduction in the waterlogged conditions that are key to peatland formation and continued accumulation of carbon (C) in peat. C sequestration in peatlands arises from a delicate imbalance between primary production and decomposition, and microbial processes are potentially pivotal in regulating feedbacks between environmental change and the peatland C cycle. Increased soil temperature, caused by climate warming or disturbance of the natural vegetation cover and drainage, may result in reductions of long-term C storage via changes in microbial community composition and metabolic rates. Moreover, changes in water table depth alter the redox state and hence have broad consequences for microbial functions, including effects on fungal and bacterial communities especially methanogens and methanotrophs. This article is a perspective review of the effects of climate change and ecosystem restoration on peatland microbial communities and the implications for C sequestration and climate regulation. It is authored by peatland scientists, microbial ecologists, land managers and non-governmental organisations who were attendees at a series of three workshops held at The University of Manchester (UK) in 2019–2020. Our review suggests that the increase in methane flux sometimes observed when water tables are restored is predicated on the availability of labile carbon from vegetation and the absence of alternative terminal electron acceptors. Peatland microbial communities respond relatively rapidly to shifts in vegetation induced by climate change and subsequent changes in the quantity and quality of below-ground C substrate inputs. Other consequences of climate change that affect peatland microbial communities and C cycling include alterations in snow cover and permafrost thaw. In the face of rapid climate change, restoration of a resilient microbiome is essential to sustaining the climate regulation functions of peatland systems. Technological developments enabling faster characterisation of microbial communities and functions support progress towards this goal, which will require a strongly interdisciplinary approach.

Biological nitrogen fixation (BNF) performed by diazotrophs is vital to our understanding of ecosystem functions, as plant nitrogen (N) is commonly a limiting nutrient for primary productivity. However, significant limitations have remained in our knowledge of the controls and rates of this process, due to technical difficulties in directly quantifying nitrogen (N2) fixation rates. To address this, we developed a novel compound-specific 15N-stable isotope probing method involving analysis of acid hydrolysable soil amino acids (AAs) by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) for the quantification of BNF in soils. By analysing 15N-enriched AAs (as N-acetyl, O-isopropyl derivatives), this new approach aimed to provide greater specificity compared to existing methods, and to contribute previously unobtainable quantitative information on the capture and flow of N2 fixed in soils. Laboratory incubations using 15N2 gas were carried out on surface peat over 15 days to obtain quantitative measures of N2 fixation by the microbial community. Longer incubations with the addition of a glucose energy source significantly increased the level of 15N enrichment, i.e. N fixed. The enhanced detection limits of 15N-AAs by GC-C-IRMS, compared to bulk soil δ15N value determinations, was key to assessments of N2 fixation. Valuable insights into the assimilation pathway of the applied 15N2-substrate were revealed; for peat soils, 15N incorporation into glutamate dominated over other AAs.