Dr. Aline Frossard

Knowledge on microbial community shifts during ecosystem succession from bare surfaces resulting from massive landscape stripping is extremely limited. Here we took advantage of an artificially created experimental catchment (6 ha) to assess structural and functional changes of microbial communities in ephemeral stream sediments and adjacent soils between 3 and 13 years after catchment construction. The catchment has since developed in undisturbed conditions, with major transformations in its morphology, hydrology and vegetation reflected by changes in microbial community structure and function. Initially dominated by cyanobacteria (42% of 16S rRNA reads in 2008 and 0.3% in 2018), the bacterial community shifted to an essentially heterotrophic composition within 10 years, when Alphaproteobacteria (12 vs 21%) and Planctomycetes (3 vs 16%), in particular, gained in importance. Similarly, Sordariomycetes (5% of ITS reads in 2008 and 27% in 2018) replaced Dothideomycetes (53 vs 14%) as the prevailing fungal class. Microbial respiration rates increased tenfold, from an average of 0.5–4.4 μg CO₂ g⁻¹ DM h⁻¹, accompanied by an increase in potential enzyme activities. Seasonal patterns of microbial community functions were accentuated over a decade of catchment development, whereas structural community changes were less pronounced. Spatial variation of community composition also increased, with differences between soils and sediments intensifying over time. However, a striking disconnect between microbial community structure and function in 2008 had vanished by 2018. Thus, a decade of ecosystem succession resulted in fundamental shifts in microbial community structure and function, highlighting the intricate interplay between changing environmental conditions and microbial responses.

We present a comprehensive, customizable workflow for inferring prokaryotic phenotypic traits from marker gene sequences and modelling the relationships between these traits and environmental factors, thus overcoming the limited ecological interpretability of marker gene sequencing data. We created the trait sequence database ampliconTraits, constructed by cross-mapping species from a phenotypic trait database to the SILVA sequence database and formatted to enable seamless classification of environmental sequences using the SINAPS algorithm. The R package MicEnvMod enables modelling of trait – environment relationships, combining the strengths of different model types and integrating an approach to evaluate the models' predictive performance in a single framework. Traits could be accurately predicted even for sequences with low sequence identity (80 %) with the reference sequences, indicating that our approach is suitable to classify a wide range of environmental sequences. Validating our approach in a large trans-continental soil dataset, we showed that trait distributions were robust to classification settings such as the bootstrap cutoff for classification and the number of discrete intervals for continuous traits. Using functions from MicEnvMod, we revealed precipitation seasonality and land cover as the most important predictors of genome size. We found Pearson correlation coefficients between observed and predicted values up to 0.70 using repeated split sampling cross validation, corroborating the predictive ability of our models beyond the training data. Predicting genome size across the Iberian Peninsula, we found the largest genomes in the northern part. Potential limitations of our trait inference approach include dependence on the phylogenetic conservation of traits and limited database coverage of environmental prokaryotes. Overall, our approach enables robust inference of ecologically interpretable traits combined with environmental modelling allowing to harness traits as bioindicators of soil ecosystem functioning.

Mountain ecosystems, contributing substantially to the global carbon (C) and nitrogen (N) biogeochemical cycles, are heavily impacted by global changes. Although soil respiration and microbial activities have been extensively studied at different elevation, little is known on the relationships between environmental drivers, microbial functions, and greenhouse gas fluxes (GHGs; carbon dioxide [CO₂], methane [CH₄] and nitrous oxide [N₂O]) in soils of different elevation. Here, we measured how in situ GHG fluxes were linked to soil properties, soil organic matter (SOM) quantity and composition (the proportion of humic-like vs. protein-like OM), microbial biomass, enzyme activities and functional gene abundances in natural soils spanning an elevational gradient of ∼2400 m in Switzerland. Soil CO₂ fluxes did not significantly vary from low (lowland zone) to higher (montane and subalpine zones) elevation forests, but decreased significantly (P<0.001) from the treeline to the mountain summit. Multivariate analyses revealed that CO₂ fluxes were controlled by C-acquiring enzymatic activities which were mainly controlled by air mean annual temperature (MAT) and SOM quantity and composition. CH₄ fluxes were characterized by uptake of atmospheric CH₄, but no trend was observed along the elevation. N₂O fluxes were also dominated by uptake of atmospheric N₂O. The flux rates remained stable with increasing elevation below the treeline, but decreased significantly (P<0.001) from the treeline to the summit. N₂O fluxes were driven by specific nitrifying and denitrifying microbial genes (ammonia-oxidizing amoA and N₂O-producing norB), which were again controlled by SOM quantity and composition. Our study indicates the treeline as a demarcation point changing the patterns of CO₂ and N₂O fluxes along the elevation, highlighting the importance of SOM quantity and composition in controlling microbial enzyme activities and GHG fluxes.

Over recent decades, there has been a noticeable change in vegetation diversity accompanied by increased plant productivity in tundra systems of the High-Arctic, leading to elevated carbon and nutrient inputs into the soil. This shift can alter microbial community composition and activity in these ecosystems. In this study, we aimed to identify genes transcribed by active microorganisms and compare their expression in unamended and amended soils with labile carbon and/or nitrogen compounds. We also assessed gene expression differences in tundra soils with varying edaphic characteristics (upslope vs. downslope sites). We amended soils with either glycine or cellulose or left them unamended (i.e., control) for 7 days of incubation, and we isolated and sequenced RNA using Illumina technology. Whereas we observed only a weak transcriptional response after cellulose addition, the glycine addition significantly influenced transcriptional patterns, with upregulation of carbon- and nitrogen-cycling genes. Notably, microbial taxa from the Pseudomonadaceae and Micrococcaceae families showed the most pronounced response to glycine, indicating a shift of the communities towards copiotrophic organisms. This response was consistent across the two soil types, suggesting a common impact on microbial community activity. These findings suggest that an increase in carbon and nitrogen inputs could substantially affect microbial functioning in High-Arctic tundra soils, with potential implications for ecosystem dynamics under global warming.

Soil microbial traits and functions play a central role in soil organic carbon (SOC) dynamics. However, at the macroscale (regional to global) it is still unresolved whether (i) specific environmental attributes (e.g., climate, geology, soil types) or (ii) microbial community composition drive key microbial traits and functions directly. To address this knowledge gap, we used 33 grassland topsoils (0–10 cm) from a geoclimatic gradient in Chile. First, we incubated the soils for 1 week in favorable standardized conditions and quantified a wide range of soil microbial traits and functions such as microbial biomass carbon (MBC), enzyme kinetics, microbial respiration, growth rates as well as carbon use efficiency (CUE). Second, we characterized climatic and physicochemical properties as well as bacterial and fungal community composition of the soils. We then applied regression analysis to investigate how strongly the measured microbial traits and functions were linked with the environmental setting versus microbial community composition. We show that environmental attributes (predominantly the amount of soil organic matter) determined patterns of MBC along the gradient, which in turn explained microbial respiration and growth rates. However, respiration and growth normalized for MBC (i.e., specific respiration and growth) were more linked to microbial community composition than environmental attributes. Notably, both specific respiration and growth followed distinct trends and were related to different parts of the microbial community, which in turn resulted in strong effects on microbial CUE. We conclude that even at the macroscale, CUE is the result of physiologically decoupled aspects of microbial metabolism, which in turn is partially determined by microbial community composition. The environmental setting and microbial community composition affect different microbial traits and functions, and therefore both factors need to be considered in the context of macroscale SOC dynamics.

Nematodes are numerous in soils and play a crucial role in soil food-webs. DNA metabarcoding offers a time-effective alternative to morphology-based assessments of nematode diversity. However, it is unclear how different DNA extraction methods prior to metabarcoding could affect community analysis. We used soils with woody vegetation from a European latitudinal gradient (29 sites, 39 to 79°N, ∼4500 km, covering six biomes) to systematically evaluate the effect of two sources of nematode DNA either directly extracted from soils vs. extracted from nematodes previously isolated from soils hypothesizing that the DNA source material may produce different diversities, community structures and abundances of feeding types. Nematode-sample DNA exhibited a higher richness, while no difference in Shannon diversity was found between the approaches. The DNA sources also created significantly different community structures, with greater differences observed across soil-extracted DNA than nematode-sample DNA. The most overrepresented species in nematode-sample DNA were Heterocephalobus elongatus, Eucephalobus striatus and Hexatylus sp., whereas Phasmarhabditis sp. and Eumonhystera filiformis were overrepresented in soil-extracted DNA. Read abundances of feeding types significantly differed between the DNA sources and across sites, with a significant effect of biome on both ecto- and endoparasitic herbivores in soil-extracted DNA and for ectoparasitic herbivores only in nematode-sample DNA. Collectively, our data suggest that choice of the DNA source material may lead to different patterns of nematode community composition across space and environmental conditions. Improving the sensitivity of the soil-extracted DNA method by developing protocols using larger amounts of soil and designing nematode-specific primers will make this approach an efficient screening tool to analyse nematode diversity and community structure complementing the labour-intensive isolation of intact nematodes from soils (nematode-sample DNA).

Real-time quantitative PCR (qPCR) has been widely used to quantify gene copy numbers in microbial ecology. Despite its simplicity and straightforwardness, establishing qPCR assays is often impeded by the tedious process of producing qPCR standards by cloning the target DNA into plasmids. Here, we designed double-stranded synthetic DNA fragments from consensus sequences as qPCR standards by aligning microbial gene sequences (10–20 sequences per gene). Efficiency of standards from synthetic DNA was compared with plasmid standards by qPCR assays for different phylogenetic marker and functional genes involved in carbon (C) and nitrogen (N) cycling, tested with DNA extracted from a broad range of soils. Results showed that qPCR standard curves using synthetic DNA performed equally well to those from plasmids for all the genes tested. Furthermore, gene copy numbers from DNA extracted from soils obtained by using synthetic standards or plasmid standards were comparable. Our approach therefore demonstrates that a synthetic DNA fragment as qPCR standard provides comparable sensitivity and reliability to a traditional plasmid standard, while being more time- and cost-efficient.

Climate change and erosion from agricultural areas cause increased drying periods and bedform migration of riverbeds, respectively, worldwide. Both sediment drying and bedform migration can independently stress the microbial community residing in the riverbed. Here, we investigated the microbial response after exposure to these stressors with a focus on long-term recovery. We conducted an in situ experiment to investigate the long-term (8 months) functional and structural recovery of benthic microbial communities from either sediment drying (episodic severe stressor) or bedform migration (frequent moderate stressor). Stressed sediment associated communities were rewetted (dried sediments) and immobilized (migrated sediments) and exposed in the River Spree (north-eastern Germany) to initiate the recovery process. We then evaluated the microbial function (community respiration, net community production and extracellular enzymatic activities) as well as the bacterial, fungal and diatom community structure (16S rRNA gene and ITS region metabarcoding, and microscopic diatom morphotype classification). We observed different recovery times for community respiration (less than 7 days) and gross primary production (more than 5 months), implying a shift toward net heterotrophy in the first few months after stress exposure. Similarly, we observed a strong autotrophic community response (particularly associated with the diatoms Navicula and Fragilaria), especially in migrated sediments. The bacterial and fungal community response to sediment drying was stronger than to bedform migration (particularly associated with the bacterium Flavobacterium and the fungi Alternaria sp. and Aureobasidium pullulans). Our results show that sediment drying and bedform migration had a significant impact on the microbial community function and structure, which persisted for several months after the stress. Due to the surprising long period of recovery, successive stress events combined with seasonal effects will likely hamper the ongoing recovery process with severe alterations to the microbial function and structure. These findings extend the concept of ecosystem resilience and stability on the dimensions of timescale and seasonal environmental variations. Legacy effects are expected to play a key role when facing future stress.

Take-Home Message Climate warming is transforming the Arctic at an unprecedented rate with previously barren and sparsely vegetated landscapes undergoing “greening”. We postulate that the observed vegetation changes throughout the Arctic are not only tied to warming, but to changes in soil properties and their impacts on plants and soil microbial communities. A key to understanding extent and patterns of greening of formerly sparsely vegetated Arctic environments will be to unravel the interactions between the biosphere and the role of soil genesis.

The analysis of organic mercury (Hg) species in polluted soils is a necessary tool to assess the environmental risk(s) of mercury in contaminated legacy sites. The artificial formation of monomethylmercury (MeHg) during soil extraction and/or analysis is a well-known limitation and is especially relevant in highly polluted areas where MeHg/Hg ratios are notoriously low. Although this has been known for almost 30 years, the thorough characterisation of artificial formation rates is rarely a part of the method development in scientific literature. Here we present the application of two separate procedures (inorganic Hg (iHg) spiking and double-spike isotope dilution analyses (DSIDA)) to determine and correct for artificial Hg methylation in MeHg-selective acid-leaching/organic solvent extraction procedure. Subsequently, we combined corrected MeHg and ethylmercury (EtHg) measurements with PCR amplification of hgcA genes to distinguish between naturally formed MeHg from primary deposited MeHg in soils from a legacy site in a Swiss mountain valley. We found the DSIDA procedure incompatible with the organomercury selective extraction method due to the quantitative removal of iHg. Methylation factors from iHg spiking were in the range of (0.0075 ± 0.0001%) and were consistent across soils and sediment matrices. Further, we suggest that MeHg was deposited and not formed in-situ in two out of three studied locations. Our line of evidence consists of 1) the concomitant detection of EtHg, 2) the elevated MeHg concentrations (up to 4.84 μg kg^-1), and 3) the absence of hgcA genes at these locations. The combination of Hg speciation and methylation gene (hgcA) abundance analyses are tools suited to assess Hg pollution pathways at Hg legacy sites.

Soil invertebrates (i.e., soil fauna) are important drivers of many key processes in soils including soil aggregate formation, water retention, and soil organic matter transformation. Many soil fauna groups directly or indirectly participate in litter consumption. However, the quantity of litter consumed by major faunal groups across biomes remains unknown. To estimate this quantity, we reviewed > 1000 observations from 70 studies that determined the biomass of soil fauna across various biomes and 200 observations from 44 studies on litter consumption by soil fauna. To compare litter consumption with annual litterfall, we analyzed 692 observations from 24 litterfall studies and 183 observations from 28 litter stock studies. The biomass of faunal groups was highest in temperate grasslands and then decreased in the following order: boreal forest > temperate forest > tropical grassland > tundra > tropical forest > Mediterranean ecosystems > desert and semidesert. Tropical grasslands, desert biomes, and Mediterranean ecosystems were dominated by termites. Temperate grasslands were dominated by omnivores, while temperate forests were dominated by earthworms. On average, estimated litter consumption (relative to total litter input) ranged from a low of 14.9% in deserts to a high of 100.4% in temperate grassland. Litter consumption by soil fauna was greater in grasslands than in forests. This is the first study to estimate the effect of different soil fauna groups on litter consumption and related processes at global scale.

In the high-Arctic, increased temperature results in permafrost thawing and increased primary production. This fresh plant-derived material is predicted to prime microbial consortia for degradation of the organic matter stored in tundra soils. However, the effects of warming and plant input on the microbial community structure is hardly known. We assessed the use of glycine, a readily available C and N source, and cellulose, a long C-biopolymer, by prokaryotic and fungal communities using DNA-SIP in tundra soils incubated at 8 °C or 16 °C. Glycine addition contributed mainly to instantaneous microbial carbon use and priming of soil organic matter decomposition, particularly under elevated temperature. By contrast, cellulose was linked to the dominant and active microbial communities, with potential carbon stabilization in soils. Our findings stress the importance of the type of plant-derived material in relation to microbial metabolism in high-Arctic soils and their consequences for the carbon cycle in response to global warming.

The warming-induced thawing of permafrost promotes microbial activity, often resulting in enhanced greenhouse gas emissions. The ability of permafrost microorganisms to survive the in situ sub-zero temperatures, their energetic strategies and their metabolic versatility in using soil organic materials determine their growth and functionality upon thawing. Hence, functional characterization of the permafrost microbiome, particularly in the underexplored mid-latitudinal alpine regions, is a crucial first step in predicting its responses to the changing climate, and the consequences for soil-climate feedbacks. In this study, for the first time, the functional potential and metabolic capabilities of a temperate mountain permafrost microbiome from central Europe has been analysed using shotgun metagenomics. Permafrost and active layers from the summit of Muot da Barba Peider (MBP) [Swiss Alps, 2979 m above sea level (a.s.l.)] revealed a strikingly high functional diversity in the permafrost (north-facing soils at a depth of 160 cm). Permafrost metagenomes were enriched in stress-response genes (e.g. cold-shock genes, chaperones), as well as in genes involved in cell defence and competition (e.g. antiviral proteins, antibiotics, motility, nutrient-uptake ABC transporters), compared with active-layer metagenomes. Permafrost also showed a higher potential for the synthesis of carbohydrate-active enzymes, and an overrepresentation of genes involved in fermentation, carbon fixation, denitrification and nitrogen reduction reactions. Collectively, these findings demonstrate the potential capabilities of permafrost microorganisms to thrive in cold and oligotrophic conditions, and highlight their metabolic versatility in carbon and nitrogen cycling. Our study provides a first insight into the high functional gene diversity of the central European mountain permafrost microbiome. Our findings extend our understanding of the microbial ecology of permafrost and represent a baseline for future investigations comparing the functional profiles of permafrost microbial communities at different latitudes.

Combined effects of climate change and increasing anthropogenic water demand have increased and extended dry period occurrences in rivers worldwide. Riverbed drying can significantly affect sediment microorganisms, crucial drivers of biogeochemical processes in lotic systems. In this study, we evaluated how sediment bacterial and fungal community structure and composition (based on 16S rRNA gene and ITS metabarcoding) and microbial functions (community respiration and extracellular enzymatic activities) respond to different riverbed drying intensities over 90 days. Riverbed sediment collected in a flowing reach of the Spree river in northeastern Germany was dried under different rates in outdoor mesocosms during the summer months of 2018. Our results demonstrate that drying attributes (duration and intensity) and sediment organic matter (OM) content play a crucial role in sediment microbial community assembly and functioning throughout drying. Milder drying surprisingly triggered a more rapid and drastic change in the microbial community composition and diversity. After 90 days of drying, Bacilli (Firmicutes) became the dominant bacterial class in most treatments, except in sediments with low OM content under the most severe drying treatment. Fungal amplicon sequence variants (ASVs) from Dothideomycetes (Ascomycota) had by far the highest relative abundance in all our treatments at the end of the drying experiment, making up 65.1% to 94.0% of the fungal reads. CO₂ fluxes, a proxy for sediment community respiration, were rapidly and strongly affected by drying in all treatments. Our results imply that even short riverbed drying periods are likely to have significant consequences for the biogeochemical dynamics in recently formed non-perennial temperate rivers.

Mercury (Hg) is one of the most toxic heavy metals and is known for its persistence in the environment and potential to accumulate along the food chain. In many terrestrial polluted sites, earthworms are in direct contact with Hg contamination by ingesting large quantities of soil. However, little is known about the impact of Hg soil pollution on earthworms' gut microbiome. In this study, two incubation experiments involving earthworms in soils from a long-term Hg-polluted site were conducted to assess: (1) the effect of soil Hg contamination on the diversity and structure of microbial communities in earthworm, cast and soil samples; and (2) how the gut microbiome of different digestive track parts of the earthworm responds to soil Hg contamination. The large accumulation of total Hg and methyl-Hg within the earthworm tissues clearly impacted the bacterial and fungal gut community structures, drastically decreasing the relative abundance of the dominating gut bacterial class Mollicutes. Hg-tolerant taxa were found to be taxonomically widespread but consistent along the different parts of the earthworm digestive tract. This study revealed that although Hg might not directly affect the health of macro-organisms in the food-web such as earthworms, their metabolism and legacy in the soil might be impacted through changes in their gut microbiome.

The relatively poor simulation of the below-ground processes is a severe drawback for many ecosystem models, especially when predicting responses to climate change and management. For a meaningful estimation of ecosystem production and the cycling of water, energy, nutrients and carbon, the integration of soil processes and the exchanges at the surface is crucial. It is increasingly recognized that soil biota play an important role in soil organic carbon and nutrient cycling, shaping soil structure and hydrological properties through their activity, and in water and nutrient uptake by plants through mycorrhizal processes. In this article, we review the main soil biological actors (microbiota, fauna and roots) and their effects on soil functioning. We review to what extent they have been included in soil models and propose which of them could be included in ecosystem models. We show that the model representation of the soil food web, the impact of soil ecosystem engineers on soil structure and the related effects on hydrology and soil organic matter (SOM) stabilization are key issues in improving ecosystem-scale soil representation in models. Finally, we describe a new core model concept (KEYLINK) that integrates insights from SOM models, structural models and food web models to simulate the living soil at an ecosystem scale.

Ongoing climate change involves increasing snow scarcity, which results in more frequent freeze-thaw cycles (FTCs) in polar and alpine soils. Although repeated FTCs have been shown to alter the structure and functions of soil microbial communities, a thorough understanding on the influence of FTCs frequency on polar and especially alpine soil microbiomes is still elusive. Here, we investigated the impact of repeated weekly vs. daily FTC frequencies on the structure and functions of prokaryotic and fungal communities from north- and south-exposed soils from two mountain ridges, one in the Arctic and one in the High-Alps. FTCs affected prokaryotic communities more strongly than fungal communities, where mainly cold-tolerant and opportunistic fungi (e.g., Mrakia, Mortierella) were responsive. Prokaryotic communities were more affected by weekly FTCs than by daily FTCs. Daily FTCs favored fast-growing bacteria (e.g., Arthrobacter), while oligotrophic and largely uncultured taxa (e.g., Verrucomicrobia) benefited from weekly FTCs. FTCs negatively affected microbial respiration but had minor impacts on C-, N- and P-acquiring enzymatic activities. Plausible pre-adaptation of the microbial communities to naturally occurring frequent FTCs at their site of origin did not show a clear influence on the microbial responses to the tested FTCs. Altogether, our study provides an integrative overview on potential structural and functional changes of soil microbial communities in polar and alpine regions in response to the projected increase in FTCs; therefore advancing our understanding on the impact of climate change in these rapidly changing ecosystems.

The soil active layer in the High Arctic is a reservoir for nutrients and microbial communities, but their behavior and interactions remain unclear. Therefore, the objective of this study was to characterize soil organic matter (SOM) composition and potential enzyme activities in soil active layer of Svalbard. Samples from two different sites and two different soil layers (biocrust: 0-1 cm; mineral soil: 5-10 cm depth) were collected along a moisture gradient. The molecular SOM composition was assessed using pyrolysis-field ionization mass spectrometry (Py-FIMS), and the potential activities of seven extracellular enzymes characteristic for different soil microbial processes were measured. In general, SOM in both sites was dominated by lipids/sterols, alkylaromatics and phenols/lignin monomers. Dehydroergosterol prevailed in a majority of the samples. Biocrusts samples in both sites had higher total ion intensity and volatile matter, but lower lipid and alkylaromatics contents. The potential enzyme activities varied significantly across the sites, depths and moisture gradients and, in general, were higher in the biocrusts than in underlying soils. Besides, majority of measured enzyme activities were lower in the wetter plots. Taken together, our results expand the knowledge of SOM and enzyme activity in High Arctic topsoil, which is important for better understanding the dynamic of soil biotic and abiotic processes in polar environments, particularly in the context of climate change.

1. Carrion is long recognized as important to scavengers. How carrion may affect soil microbial biodiversity and ecosystem processes in natural systems is comparatively unknown, but is important for the intersection of vertebrate food webs, below-ground processes and ecological heterogeneity.
2. We assessed in situ soil and plant responses to wolf-killed mammal carrion in Yellowstone National Park, USA.
3. Bison and elk carcasses increased soil respiration and vegetation nutrient concentration and the carcasses contain altered bacterial and fungal communities compared to control plots. The ‘fingerprints’ of soil microbial taxa associated with bison compared to elk carcasses differed considerably and taxa are found dependent upon abiotic gradients and soil properties.
4. We found evidence that soil microbial community changes associated with carcasses may not be as generalizable as previously thought, which is important for a mechanistic understanding of the links between carrion and soil heterogeneity and potentially for applications in forensic science.
5. This work demonstrates the importance of carrion studies in natural systems. Our findings show that carrion creates distinct ecological patterns that contribute to both above- and below-ground biological heterogeneity, linking carrion distribution dynamics with soil microbial biodiversity and ecosystem functions.