Publications

Microbial necromass comprises a large fraction of soil organic matter (SOM) due to the accumulation and stabilization of microbial residues from dead archaea, bacteria and fungi. Amino sugars, neutral sugars and uronic acids have been used as microbial necromass biomarkers to trace the origin and composition of microbial residues in the SOM pool. Due to the structural complexity of sugars, derivatization reactions and high-throughput analytical methods are required to separate and quantify these sugar-related compounds. Our aim was to develop a rapid and sensitive assay to measure amino sugar, neutral sugar and uronic acid compounds using pre-column 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization. PMP-derivatives were separated and quantified via reversed phase (RP) ultra-high-performance liquid chromatography (UPLC) coupled to high-resolution Orbitrap mass spectrometry (MS). The method was validated and applied on hydrolyzed peptidoglycans and the biomass of archaeal, bacterial, fungal and plant species, as well as with soils. This developed PMP method allowed the separation and quantification of 18 sugar-related compounds, including four amino sugars, three N-acetyl amino sugars, eight neutral sugars, and three uronic acids within 20 min. This PMP method showed a precision for isotope enrichment detection of 0.03–0.05 atom % 13C for D-glucose and D-glucosamine. This is the first time talosaminuronic acid (deriving from archaeal pseudopeptidoglycan) was identified and quantified using PMP derivatization. The application of this novel PMP method on pure hydrolyzed biomass and soils, showed the successful chromatographic and mass spectrometric separation and quantification of amino sugar, neutral sugar and uronic acid compounds. A multivariate analysis using these sugar-related PMP derivatives showed a clustering of the species according to their respective taxonomic group (archaea, gram-positive bacteria, gram-negative bacteria, fungi and plants). The modified PMP method can be applied to identify and quantify soil microbial necromass biomarkers, as well as their contribution to SOM. The sensitive isotope tracer detection allows tracing isotopically labeled materials into necromass biomarkers in SOM pools.

Climate change increases the frequency and intensity of drought events, affecting soil functions including carbon sequestration and nutrient cycling, which are driven by growing microorganisms. Yet we know little about microbial responses to drought due to methodological limitations. Here, we estimate microbial growth rates in montane grassland soils exposed to ambient conditions, drought, and potential future climate conditions (i.e., soils exposed to 6 years of elevated temperatures and elevated CO2 levels). For this purpose, we combined 18O-water vapor equilibration with quantitative stable isotope probing (termed ‘vapor-qSIP’) to measure taxon-specific microbial growth in dry soils. In our experiments, drought caused >90% of bacterial and archaeal taxa to stop dividing and reduced the growth rates of persisting ones. Under drought, growing taxa accounted for only 4% of the total community as compared to 35% in the controls. Drought-tolerant communities were dominated by specialized members of the Actinobacteriota, particularly the genus Streptomyces. Six years of pre-exposure to future climate conditions (3 °C warming and + 300 ppm atmospheric CO2) alleviated drought effects on microbial growth, through more drought-tolerant taxa across major phyla, accounting for 9% of the total community. Our results provide insights into the response of active microbes to drought today and in a future climate, and highlight the importance of studying drought in combination with future climate conditions to capture interactive effects and improve predictions of future soil-climate feedbacks.

Plant roots release a variety of low-molecular weight compounds, such as sugars, amino acids or organic acids into the soil, impacting microbial activities and physico-chemical soil processes in their surroundings. These compounds are a source of easily available Carbon (C) and energy for soil microbes, potentially accelerating microbial decomposition of soil organic matter in the immediate vicinity of roots. However, knowledge about processes in root exudation hotspots remains limited due to experimental difficulties in investigating such hotspots in soil.

Microdialysis, a passive sampling technique based on diffusion, has been successfully used to collect soil solutes at small spatial scales. Reverse microdialysis, also termed retrodialysis, can be used to introduce solutes into the soil, mimicking passive root exudation. However, little is known about the dynamics of substances released by passive diffusion into intact soil, a crucial prerequisite for applying reverse microdialysis to study root exudation hotspots in undisturbed soils.

Here, we used reverse microdialysis to investigate the spatial and temporal dynamics of thirteen different organic compounds passively introduced into two different intact soils. Diffusion of compounds into soils was substantially lower than into water, and was not – as in water – determined by molecular size. Interestingly, butyrate, oxalate and propionate showed the highest diffusive fluxes into soil combined with the lowest rate of back retrieval after input, indicating that they were quickly removed from the soil solution by biotic or abiotic processes. In contrast, glucose and fructose unexpectedly accumulated around the membrane after input without removal. Furthermore, diffusive fluxes of compounds into soils showed a fluctuating temporal pattern, which may be explained by an observed 2-h delay of microbial respiration of added 13C-labelled compounds. During the course of 12 days, approximately one third of 13C-labelled compounds introduced into soil was respired while 8% ended up in microbial biomass.

Our results demonstrate that introducing compounds into intact soil triggers complex biotic and abiotic responses at the time scale of hours. Reverse microdialysis proved to be an excellent tool to investigate such responses as well as the dynamics and metabolic consequences of passively released compounds into intact soil, and – in combination with 13C labelled substrate and respiration measurements - to shed light on potential priming effects that may be triggered by them.

Fatty acid biomarkers have emerged as a useful tool to quantify biomass of various microbial groups. Here we focus on the frequent use of the fatty acid 16:1ω5 as a biomarker for arbuscular mycorrhizal (AM) fungi in soils. We highlight some issues with current applications of this method and use several examples from the literature to show that the phospholipid fatty acid (PLFA) 16:1ω5 can occur in high concentrations in soils where actively growing AM fungi are absent. Unless the study includes a control where the contribution of other microbes can be estimated, we advocate for the use of the neutral lipid fatty acid (NLFA) 16:1ω5. This biomarker has higher specificity, is more responsive to shifts in AM fungal biomass, and quantification can be conducted along with PLFA analysis without doubling analytical efforts. We conclude by contrasting various methods used to measure AM fungal biomass in soil and highlight future research needs to optimize fatty acid analyses.

Depolymerization of high-molecular weight organic nitrogen (N) represents the major bottleneck of soil N cycling and yet is poorly understood compared to the subsequent inorganic N processes. Given the importance of organic N cycling and the rise of global change, we investigated the responses of soil protein depolymerization and microbial amino acid consumption to increased temperature, elevated atmospheric CO2, and drought. The study was conducted in a global change facility in a managed montane grassland in Austria, where elevated CO2 (eCO2) and elevated temperature (eT) were stimulated for 4 years, and were combined with a drought event. Gross protein depolymerization and microbial amino acid consumption rates (alongside with gross organic N mineralization and nitrification) were measured using 15N isotope pool dilution techniques. Whereas eCO2 showed no individual effect, eT had distinct effects which were modulated by season, with a negative effect of eT on soil organic N process rates in spring, neutral effects in summer, and positive effects in fall. We attribute this to a combination of changes in substrate availability and seasonal temperature changes. Drought led to a doubling of organic N process rates, which returned to rates found under ambient conditions within 3 months after rewetting. Notably, we observed a shift in the control of soil protein depolymerization, from plant substrate controls under continuous environmental change drivers (eT and eCO2) to controls via microbial turnover and soil organic N availability under the pulse disturbance (drought). To the best of our knowledge, this is the first study which analyzed the individual versus combined effects of multiple global change factors and of seasonality on soil organic N processes and thereby strongly contributes to our understanding of terrestrial N cycling in a future world.

Ectomycorrhizal fungi live in close association with their host plants and form complex interactions with bacterial/archaeal communities in soil. We investigated whether abundant or rare ectomycorrhizal fungi on root-tips of young beech trees (Fagus sylvatica) shape bacterial/archaeal communities. We sequenced 16S rRNA genes and fungal internal transcribed spacer regions of individual root-tips and used ecological networks to detect the tendency of certain assemblies of fungal and bacterial/archaeal taxa to inhabit the same root-tip (i.e. modularity). Individual ectomycorrhizal root-tips hosted distinct fungal communities associated with unique bacterial/archaeal communities. The structure of the fungal-bacterial/archaeal association was determined by both, dominant and rare fungi. Integrating our data in a conceptual framework suggests that the effect of rare fungi on the bacterial/archaeal communities of ectomycorrhizal root-tips contributes to assemblages of bacteria/archaea on root-tips. This highlights the potential impact of complex fine-scale interactions between root-tip associated fungi and other soil microorganisms for the ectomycorrhizal symbiosis.

Network analysis has been used for many years in ecological research to analyze organismal associations, for example in food webs, plant-plant or plant-animal interactions. Although network analysis is widely applied in microbial ecology, only recently has it entered the realms of soil microbial ecology, shown by a rapid rise in studies applying co-occurrence analysis to soil microbial communities. While this application offers great potential for deeper insights into the ecological structure of soil microbial ecosystems, it also brings new challenges related to the specific characteristics of soil datasets and the type of ecological questions that can be addressed. In this Perspectives Paper we assess the challenges of applying network analysis to soil microbial ecology due to the small-scale heterogeneity of the soil environment and the nature of soil microbial datasets. We review the different approaches of network construction that are commonly applied to soil microbial datasets and discuss their features and limitations. Using a test dataset of microbial communities from two depths of a forest soil, we demonstrate how different experimental designs and network constructing algorithms affect the structure of the resulting networks, and how this in turn may influence ecological conclusions. We will also reveal how assumptions of the construction method, methods of preparing the dataset, and definitions of thresholds affect the network structure. Finally, we discuss the particular questions in soil microbial ecology that can be approached by analyzing and interpreting specific network properties. Targeting these network properties in a meaningful way will allow applying this technique not in merely descriptive, but in hypothesis-driven research. Analysing microbial networks in soils opens a window to a better understanding of the complexity of microbial communities. However, this approach is unfortunately often used to draw conclusions which are far beyond the scientific evidence it can provide, which has damaged its reputation for soil microbial analysis. In this Perspectives Paper, we would like to sharpen the view for the real potential of microbial co-occurrence analysis in soils, and at the same time raise awareness regarding its limitations and the many ways how it can be misused or misinterpreted.

Microbial community analysis via marker gene amplicon sequencing has become a routine method in the field of soil research. In this perspective, we discuss technical challenges and limitations of amplicon sequencing and present statistical and experimental approaches that can help addressing the spatio-temporal complexity of soil and the high diversity of organisms therein. We illustrate the impact of compositionality on the interpretation of relative abundance data and discuss effects of sample replication on the statistical power in soil community analysis. Additionally, we argue for the need of increased study reproducibility and data availability, as well as complementary techniques for generating deeper ecological insights into microbial roles and our understanding thereof in soil ecosystems. At this stage, we call upon researchers and specialized soil journals to consider the current state of data analysis, interpretation, and availability to improve the rigor of future studies.

Ectomycorrhizal plants trade plant-assimilated carbon for soil nutrients with their fungal partners. The underlying mechanisms, however, are not fully understood. Here we investigate the exchange of carbon for nitrogen in the ectomycorrhizal symbiosis of Fagus sylvatica across different spatial scales from the root system to the cellular level. We provided 15 N-labelled nitrogen to mycorrhizal hyphae associated with one half of the root system of young beech trees, while exposing plants to a 13 CO2 atmosphere. We analysed the short-term distribution of 13 C and 15 N in the root system with isotope-ratio mass spectrometry, and at the cellular scale within a mycorrhizal root tip with nanoscale secondary ion mass spectrometry (NanoSIMS). At the root system scale, plants did not allocate more 13 C to root parts that received more 15 N. Nanoscale secondary ion mass spectrometry imaging, however, revealed a highly heterogenous, and spatially significantly correlated distribution of 13 C and 15 N at the cellular scale. Our results indicate that, on a coarse scale, plants do not allocate a larger proportion of photoassimilated C to root parts associated with N-delivering ectomycorrhizal fungi. Within the ectomycorrhizal tissue, however, recently plant-assimilated C and fungus-delivered N were spatially strongly coupled. Here, NanoSIMS visualisation provides an initial insight into the regulation of ectomycorrhizal C and N exchange at the microscale.

Soil organic carbon management has the potential to aid climate change mitigation through drawdown of atmospheric carbon dioxide. To be effective, such management must account for processes influencing carbon storage and re-emission at different space and time scales. Achieving this requires a conceptual advance in our understanding to link carbon dynamics from the scales at which processes occur to the scales at which decisions are made. Here, we propose that soil carbon persistence can be understood through the lens of decomposers as a result of functional complexity derived from the interplay between spatial and temporal variation of molecular diversity and composition. For example, co-location alone can determine whether a molecule is decomposed, with rapid changes in moisture leading to transport of organic matter and constraining the fitness of the microbial community, while greater molecular diversity may increase the metabolic demand of, and thus potentially limit, decomposition. This conceptual shift accounts for emergent behaviour of the microbial community and would enable soil carbon changes to be predicted without invoking recalcitrant carbon forms that have not been observed experimentally. Functional complexity as a driver of soil carbon persistence suggests soil management should be based on constant care rather than one-time action to lock away carbon in soils.

Elevated CO2 (eCO2) experiments provide critical information
to quantify the effects of rising CO2 on vegetation1–6.
Many eCO2 experiments suggest that nutrient limitations
modulate the local magnitude of the eCO2 effect on plant
biomass1,3,5, but the global extent of these limitations has
not been empirically quantified, complicating projections of
the capacity of plants to take up CO2
7,8. Here, we present a
data-driven global quantification of the eCO2 effect on biomass
based on 138 eCO2 experiments. The strength of CO2
fertilization is primarily driven by nitrogen (N) in ~65% of
global vegetation and by phosphorus (P) in ~25% of global
vegetation, with N- or P-limitation modulated by mycorrhizal
association. Our approach suggests that CO2 levels expected
by 2100 can potentially enhance plant biomass by 12 ± 3%
above current values, equivalent to 59 ± 13 PgC. The globalscale
response to eCO2 we derive from experiments is similar
to past changes in greenness9 and biomass10 with rising CO2,
suggesting that CO2 will continue to stimulate plant biomass
in the future despite the constraining effect of soil nutrients.
Our research reconciles conflicting evidence on CO2 fertilization
across scales and provides an empirical estimate of
the biomass sensitivity to eCO2 that may help to constrain
climate projections.

Root exudation is an important process determining plant interactions with the soil environment. Many studies have linked this process to soil nutrient mobilization. Yet, it remains unresolved how exudation is controlled and how exactly and under what circumstances plants benefit from exudation. The majority of root exudates include primary metabolites (sugars, amino acids and organic acids) believed to be passively lost from the root and used by rhizosphere-dwelling microbes. In this review, we synthetize recent advances in ecology and plant biology to explain and propose mechanisms by which root exudation of primary metabolites is controlled, and what role their exudation plays in plant nutrient acquisition strategies. Specifically, we propose a novel conceptual framework for root exudates. This framework is built upon two main concepts: (i) root exudation of primary metabolites is driven by diffusion, with plants and microbes both modulating concentration gradients and therefore diffusion rates to soil depending on their nutritional status; (ii) exuded metabolite concentrations can be sensed at the root tip and signals are translated to modify root architecture. The flux of primary metabolites through root exudation is mostly located at the root tip, where the lack of cell differentiation favors diffusion of metabolites to the soil. We show examples of how the root tip senses concentration changes of exuded metabolites and translate that into signals to modify root growth. Plants can modify the concentration of metabolites either by controlling source/sink processes or by expressing and regulating efflux carriers, therefore challenging the idea of root exudation as a purely unregulated passive process. Through root exudate flux, plants can locally enhance concentrations of many common metabolites which can serve as sensors and integrators of the plant nutritional status and of the nutrient availability in the surrounding environment. Plant-associated micro-organisms also constitute a strong sink for plant carbon thereby increasing concentration gradients of metabolites and affecting root exudation. Understanding the mechanisms of, and the effects that, environmental stimuli have on the magnitude and type of root exudation will ultimately improve our knowledge of processes determining soil CO2 emissions, ecosystem functioning and how to improve the sustainability of agricultural production.

Plant roots release recent photosynthates into the rhizosphere, accelerating decomposition of organic matter by saprotrophic soil microbes (’rhizosphere priming effect’) which consequently increases nutrient availability for plants. However, about 90% of all higher plant species are mycorrhizal, transferring a significant fraction of their photosynthates directly to their fungal partners. Whether mycorrhizal fungi pass on plant-derived carbon (C) to bacteria in root-distant soil areas, i.e. incite a ‘hyphosphere priming effect’, is not known. Experimental evidence for C transfer from mycorrhizal hyphae to soil bacteria is limited, especially for ectomycorrhizal systems. As ectomycorrhizal fungi possess enzymatic capabilities to degrade organic matter themselves, it remains unclear whether they cooperate with soil bacteria by providing photosynthates, or compete for available nutrients.

To investigate a possible C transfer from ectomycorrhizal hyphae to soil bacteria, and its response to changing nutrient availability, we planted young beech trees (Fagus sylvatica) into ‘split-root’ boxes, dividing their root systems into two disconnected soil compartments. Each of these compartments was separated from a litter compartment by a mesh penetrable for fungal hyphae, but not for roots. Plants were exposed to a 13C-CO2–labeled atmosphere, while 15N-labeled ammonium and amino acids were added to one side of the split-root system.

We found a rapid transfer of recent photosynthates via ectomycorrhizal hyphae to bacteria in root-distant soil areas. Fungal and bacterial phospholipid fatty acid (PLFA) biomarkers were significantly enriched in hyphae-exclusive compartments 24 h after 13C-CO2–labeling. Isotope imaging with nanometer-scale secondary ion mass spectrometry (NanoSIMS) allowed for the first time in situ visualization of plant-derived C and N taken up by extraradical fungal hyphae, and in microbial cells thriving on hyphal surfaces. When N was added to the litter compartments, bacterial biomass and the amount of incorporated 13C strongly declined. Interestingly, this effect was also observed in adjacent soil compartments where added N was only available for bacteria through hyphal transport, indicating that ectomycorrhizal fungi were acting on soil bacteria. Together, our results demonstrate that (i) ectomycorrhizal hyphae rapidly transfer plant-derived C to bacterial communities in root-distant areas, and (ii) this transfer promptly responds to changing soil nutrient conditions.