Hydroxyl radicals (HO•), produced through reactions between H2O2 and iron oxides, drive biogeochemical transformations, mediate organism toxicity, and facilitate advanced oxidation processes. The effectiveness of these processes depends on the spatial proximity between HO• generation and target substrates. Consequently, the oxidation mechanisms should be governed by interfacial interactions among H2O2, substrates, and iron oxide surfaces. Substrate oxidation by iron oxide/H2O2 systems were studied using two probes simultaneously: terephthalate (TPA), forming outer-sphere surface complexes, and coumarin, exhibiting no surface interactions. The reactions were followed as a function of pH, time and H2O2 concentration. Complementary experiments were performed with oxalate inner-sphere surface complexes. Both ferrihydrite and goethite were studied, representing differences in reduction potential. Solution analyses were combined with in-situ infrared spectroscopy probing the interfacial reactions. Both probes were oxidized by ferrihydrite/H2O2. Between pH 5.5-6.5, substantial amounts of TPA outer-sphere surface complexes were oxidized, while coumarin was mainly oxidized at pH ≤ 4.5, coinciding with a decrease in TPA oxidation. At all investigated pH values, H2O2 reduced ferrihydrite, and the partitioning of Fe(II) controlled the location of HO• generation. At low pH, Fe(II) diffused into solution triggering homogeneous Fenton reactions, while adsorption and re-oxidation at higher pH confined radical generation to the near-surface region. Oxalate inner-sphere complexes resisted oxidation. Oxidation by the goethite/H2O2 system was low compared to ferrihydrite, consistent with the lower reduction potential of goethite. This work demonstrates that H2O2-promoted reduction of iron oxides is a key reaction leading to HO• oxidation of organic outer-sphere surface complexes.

Fungal necromass is increasingly recognized as a major component of soil organic matter, and identifying the factors that govern its formation is critical for understanding and predicting the global carbon cycle. Among these factors, the biochemical composition of mycelial residues at senescence, particularly melanin content, has been consistently identified as a key determinant of the fraction of fungal necromass that persists in soils. However, even non-melanized mycelial residues exhibit a recalcitrant fraction that resists microbial decomposition, and the reasons for this persistence are not well understood. To address this gap, we asked whether the growth stage at which a single non-melanized fungal species dies governs the decay of its necromass in soil. Using Neurospora crassa, we produced seven necromass types that ranged from early exponential growth to prolonged starvation and decomposed them in forest soil. Necromass derived from biomass experiencing net growth at the time of harvest decomposed up to ten times faster than necromass from starved cultures, which were undergoing biomass loss. By the end of decomposition, only about 10 % of necromass from early-growth-stage biomass remained, while nearly 65 % of necromass from starved biomass persisted. Differences in mycelial biochemical traits, particularly C:N ratio and the degree of branching of glucans, which varied with fungal growth stage at death, explained variation in both decay rates and the size of the persistent fractions. Our findings suggest that the growth stage of fungi at death is a key factor driving fungal necromass decay profiles, with potentially large consequences for the contribution of fungal necromass to soil organic matter stocks.

Soil organic matter represents the largest active reservoir of organic carbon in terrestrial ecosystems, playing a critical role in atmospheric carbon capture and climate change mitigation. Recent studies have demonstrated that mycelial residues, also known as fungal necromass, contribute significantly to fungal necromass stocks in soils. While the magnitude and distribution of fungal necromass stocks are increasingly well documented, the processes driving their formation remain poorly understood. Specifically, the transformation of recently senesced mycelial residues into stabilized soil organic matter during the early stages of decomposition is not fully elucidated. These residues form an ephemeral resource patch of energy and nutrients for soil microbial decomposers, with the unique aspect that the microorganisms responsible for producing them also serve as their primary decomposers, contrasting with the decay of plant residues. Thus, new concepts, theories, and approaches are needed to understand fungal necromass decomposition. Here, we assess the intrinsic drivers of necromass decay by evaluating how the physiological status of fungi at the time of death influences decomposition processes, and explore extrinsic drivers by characterizing the biodiversity and functional traits of microbial decomposer communities—including fungi and protists. Our goal is to develop a refined conceptual and research framework for microbial residue decomposition and promote the integration of these processes into soil biogeochemical models.

The keystone species concept holds that certain members of an ecological community, despite their low abundance, exert disproportionately large effects on species diversity and composition. In microbial ecology, experimental validation of this concept has been limited because targeted removal of individual species remains technically challenging. Here, we developed a procedure to test the keystone species concept within a soil microbial food web by selectively suppressing a protist predator at the microscale via ultraviolet-induced phototoxicity in a microfluidic soil chip system. We targeted a hypotrich ciliate (subclass Hypotrichia), and combined microscopy with high-throughput amplicon sequencing of microbial taxonomic markers to assess, across multiple trophic levels, how its suppression affected microbial community abundance, diversity, and composition. Over the 20-day incubation, the chip system supported complex communities of bacteria, fungi, and protists. Following Hypotrichia suppression, two distinct ecological responses were observed: first, an increase in the relative abundance of flagellates, consistent with mesopredator release, accompanied by a significant rise in overall protist diversity; second, a convergence in protist community composition, indicative of biotic homogenization. Bacterial community abundance, richness, and composition remained unchanged, likely due to compensatory predation from a relative increase in bacterivorous flagellates. In contrast, fungal diversity decreased, presumably because the altered protist community favored facultative fungal consumers. Collectively, these findings provide direct experimental evidence that low-abundance microbial predators can function as keystone species, modulating predator community composition and diversity, and exerting cascading effects on lower trophic levels within microbial brown food webs.

Dissolved organic matter (DOM) is a major carbon pool and considered the most bioavailable and most mobile fraction of organic matter. DOM is generally defined as the organic matter passing a filter pore size of 0.2 or 0.45 µm, and this size cut off means that DOM not only contains dissolved molecules but also colloidal objects and aggregates up to a few hundred nanometres. The properties of this colloidal DOM fraction, such as for example size, shape, and surface charge, will affect its actual bioavailability and mobility in the environment. Although previously not well studied, there has recently been a growing interest in this colloidal fraction of DOM. We have studied DOM extracted by water from a boreal spruce forest soil, filtered through a 0.2 µm pore size. By using a combination of spectroscopy techniques, such as NMR, and light (SLS, DLS), X-ray (SAXS) and neutron (SANS) scattering techniques, we can access chemical and physical information on both the molecular and colloidal fractions of DOM. Our results show that the colloidal DOM has a homogenous chemical composition, and that carbohydrates is the dominating chemical component in both the colloidal and molecular DOM. The colloids have a mass fractal structure which does not change upon dilution and they are electrostatically stabilised against aggregation. In a lab scale study, we investigated the bacterial decomposition of this DOM during a two-month incubation. The molecular fraction of DOM was quickly decomposed. However, no change was observed for the colloidal DOM, constituting ca. 50% of the carbon, indicating that it persisted bacterial decomposition. Our results suggest that colloidal properties could be an important but hitherto overlooked aspect to the central question of what dictates organic matter reactivity and persistency in different environments and across different time scales. Our current work extends from soil solution to aquatic ecosystems, to assess the ubiquity of the colloidal fraction of DOM.

Mushroom-forming wood-decay fungi are broadly categorized into white and brown rot. White-rot fungi decompose recalcitrant crystalline cellulose using a large array of hydrolytic and oxidative enzymes. Brown-rot fungi lack many of these enzymes but decompose cellulose via Fenton-generated hydroxyl radicals. To better understand these mechanisms, we developed a Raman spectroscopy-based method to study cellulose decomposition by two white-rot fungi ( Bjerkandera adusta and Trametes versicolor ), a brown-rot fungus ( Fomitopsis pinicola ), and a Stereaceae species of uncertain decay type. Raman spectra of fungi-decomposed cellulose were highly complex, reflecting physical and chemical cellulose modifications and fungal compounds like pigments. To extract signals only from decomposed cellulose and reduce data dimensionality, reference libraries were generated using chemicals that reduce crystallinity (NaOH) or oxidize cellulose (TEMPO). Chemical libraries reduced data complexity and facilitated extraction of cellulose decomposition signals, distinguishing white-rot from brown-rot effects. Wavenumbers related to oxidation better contributed to the separation of the two decomposition types. The reduced datasets also matched the decomposition characteristics of the uncertain decay fungus to those of the brown-rot fungus. The methodology developed here could be used to further characterize plant cell wall biopolymer decomposition in single fungus-single substrate setups and complex soil samples. Importance The degrading activity of saprotrophic fungi plays a crucial role in organic matter decomposition in terrestrial ecosystems, influencing nutrient cycling from plant material. Recently, there has been increasing interest in utilizing spectroscopic techniques for studying organic matter decomposition, as these methods are non-destructive. While Raman spectroscopy has been employed to identify and differentiate chemical compounds, its application to biological samples has been limited due to the complexity of spectral signals, which are challenging to interpret. In this study, we introduce a novel approach to reducing Raman spectral data to elucidate the mechanisms underlying fungal degradation of cellulose. This is achieved by utilizing reduced datasets derived from the spectral analysis of chemically modified cellulose. The dataset can be further expanded to include additional chemical treatments and fungal species, potentially revealing differences in cellulose degradation among various saprotrophs. Moreover, this approach can be adapted for use with other substrates or chemical processes and could be enhanced by integrating omics techniques.

Soil protists are increasingly recognized as key players in organic matter turnover, yet their role as direct decomposers (i.e., saprotrophs) remains underexplored compared to that of bacteria and fungi. Here, we synthesize ecological, physiological, and genomic evidence to highlight the potential of protists to actively decompose organic matter and influence soil carbon cycling. We distinguish two saprotrophic strategies within protists—lysotrophic (extracellular) and phagotrophic (intracellular)—with the latter being unique to protists among microbial decomposers. By directly ingesting particulate or dissolved organic matter, phagotrophic saprotrophic protists may bypass constraints associated with extracellular decomposition, potentially providing an advantage in breaking down recalcitrant substrates. In contrast, lysotrophic saprotrophy in protists involves the secretion of enzymes, similar to bacterial and fungal decomposers. We propose that integrating protist saprotrophy into conceptual and quantitative models of soil organic matter decomposition could address critical knowledge gaps. This integration involves employing functional genomics and functional ecology methodologies to determine, in vitro, the capacity of protists to function as saprotrophs, elucidate the genetic pathways underpinning saprotrophic activities, and assess, in situ, their direct contributions to organic matter decomposition processes. Ultimately, a clearer view of the organic matter decomposition capacities of soil protists will refine our understanding of microbially driven carbon fluxes.

Ectomycorrhizal fungi are critical mediators of nitrogen acquisition in forest ecosystems, exhibiting variation in both host association and metabolic traits that mediate differential responses to forest nitrogen availability. However, how nitrogen acquisition strategies vary among closely related fungal species, how these patterns manifest in conifer-associated ECM fungi, and whether they persist over changing nitrogen regimes, remains poorly understood. Using an integrative approach combining in silico genomic analysis, in vitro growth assays, and isotopic analysis of in situ specimens spanning six decades, we provide the first comprehensive examination of nitrogen assimilation in congeneric conifer-associated ectomycorrhizal fungi using six Suillus species. We found highly conserved genes for inorganic nitrogen assimilation across species, but striking interspecific variation in the genetic capacity for organic nitrogen metabolism. Interspecific differences were also observed in fungal growth on varying nitrogen substrates in the growth assays, as well as in the isotopic signatures of historical specimens. For the latter, carbon isotopic patterns showed divergent temporal trends among Suillus species, suggestive of differential N use over time. Collectively, these genomic, physiological, and isotopic findings support the presence of notable interspecific diversity in ectomycorrhizal fungal nitrogen acquisition and suggest that coniferous forests and their fungal symbionts exhibit distinct responses to shifts in nitrogen availability compared to broadleaf forests. The ability of even closely related ectomycorrhizal fungi to employ diverse nitrogen acquisition strategies has important implications for forest ecosystem resilience, as different species may provide complementary services to host trees under varying environmental conditions, potentially reducing competition, and influencing forest responses to altered nutrient availability.

We have investigated the bacterial decomposition of dissolved organic matter (DOM) extracted from the organic layer of a boreal forest soil and filtered at a pore size of 0.2 µm. This DOM source has previously been extensively characterized and contains approximately equal amounts by carbon of a colloidal fraction, mainly composed of carbohydrates, and a fraction of molecularly dissolved DOM. Here, extracts were inoculated with soil bacteria and the decomposition of DOM was followed over a period of 2 months, during which it was analyzed with scattering methods and 1H NMR, and by measuring the concentration of total organic carbon. A comparison was also made with dialyzed extract. Results showed that while the bacteria fully decomposed the molecular fraction within approximately two weeks, the colloidal fraction was stable with no visible decomposition within the 2 months. The results indicate the importance of distinguishing small molecules from colloidal aggregates in decomposition studies, and demonstrate the usefulness of combining scattering methods with 1H NMR for this purpose.

ABSTRACT In microbiological studies, a common goal is to link environmental factors to microbial activities. Both environmental factors and microbial activities are typically derived from bulk samples. It is becoming increasingly clear that such bulk environmental parameters poorly represent the microscale environments microorganisms experience. Using infrared (IR) microspectroscopy, the spatial distribution of chemical compound classes can be visualized, making it a useful tool for studying the interactions between microbial cells and their microenvironments. The spatial resolution of conventional IR microspectroscopy has been limited by the diffraction limit of IR light. The recent development of optical photothermal infrared (O-PTIR) microspectroscopy has pushed the spatial resolution of IR microspectroscopy beyond this diffraction limit, allowing the distribution of chemical compound classes to be visualized at sub-micrometer spatial scales. To examine the potential and limitations of O-PTIR microspectroscopy to probe the interactions between fungal cells and their immediate environments, we imaged the decomposition of cellulose films by cells of the ectomycorrhizal fungus Paxillus involutus and compared O-PTIR results using conventional IR microspectroscopy. Whereas the data collected with conventional IR microspectroscopy indicated that P. involutus has only a very limited ability to decompose cellulose films, O-PTIR data suggested that the ability of P. involutus to decompose cellulose was substantial. Moreover, the O-PTIR method enabled the identification of a zone located outside the fungal hyphae where the cellulose was decomposed by oxidation. We conclude that O-PTIR can provide valuable new insights into the abilities and mechanisms by which microorganisms interact with their surrounding environments. IMPORTANCE Infrared (IR) microspectroscopy allows the spatial distribution of chemical compound classes to be visualized. The use of conventional IR microspectroscopy in microbiological studies has been restricted by limited spatial resolution. Recent developments in laser technology have enabled a new class of IR microspectroscopy instruments to be developed, pushing the spatial resolution beyond the diffraction limit of IR light to approximately 500 nm. This improved spatial resolution now allows microscopic observations of changes in chemical compounds to be made, making IR microspectroscopy a useful tool to investigate microscale changes in chemistry that are caused by microbial activity. We show these new possibilities using optical photothermal infrared microspectroscopy to visualize the changes in cellulose substrates caused by oxidation by the ectomycorrhizal fungus Paxillus involutus at the interface between individual fungal hyphae and cellulose substrates.

Dissolved organic matter (DOM) plays a central role in soil carbon (C) dynamics, serving as both a substrate for microbial decomposers and a source of material stabilised via physical protection in molecular aggregates and associations with mineral particles. It is well established that soil microorganisms play a key role in mineral-associated C aggregates; however, their impacts on molecular aggregates is not clearly understood. Here, we examined the ability of an ectomycorrhizal fungus (Paxillus involutus) and a saprotrophic fungus (a strain of Gloeophyllum), two major functional groups of fungal decomposers in forest ecosystems, to decompose and process the molecular and colloidal size fractions of DOM. DOM was extracted by water from boreal forest soil, and the chemical composition and colloidal properties were followed over 11 days using nuclear magnetic resonance (NMR) spectroscopy and small-angle light and X-ray scattering techniques. Both fungi decompose various organic compounds into their molecular fractions in the presence of an energy source (i.e. glucose). The decomposition rate was significantly higher for Gloeophyllum than for P. involutus. When glucose was depleted, Gloeophyllum continued to decompose more complex carbohydrates, whereas the decomposition activity of P. involutus almost stopped. A large proportion of the C in the DOM was found in organic colloids. At later stages, Gloeophyllum but not P. involutus, significantly affected the colloids by promoting the formation of larger aggregates. Thus, saprotrophic fungi activity can significantly influence the colloidal properties of DOM. Our results support the view that ectomycorrhizal fungi decompose some of the soil organic C however, their overall capacity for DOM decomposition and transformation is significantly lower than that of saprotrophic fungi.

Organic colloids are an important part of dissolved organic matter (DOM) yet many of their properties remain elusive. The main aims of this study were to assess how the colloidal properties of DOM extracted with water from an organic boreal soil horizon varied with the extraction protocol, and thereby provide insight into the nature of the DOM colloids and develop a mechanistic understanding of how the colloids were generated from the parent soil aggregates. This was accomplished by systematic variations of extraction temperature (4 degrees C-100 degrees C), time, mechanical agitation, and pH, together with a combination of chemical analyses, and light and X-ray scattering. Our results agreed with the previous identification of two main colloidal DOM species, one fractal cluster and a second, smaller colloidal DOM species described as chains or coils. Fractal clusters completely dominated the colloidal DOM in extracts from our soil at room temperature and below. Colloidal coils only existed in DOM extracted above room temperature, and their amount increased significantly between 50 degrees C-100 degrees C. Moreover, the temperature variation indicated that the fractal clusters partly dissolved into colloidal coils at elevated temperatures. Mechanical agitation at 4 degrees C significantly increased the amount of DOM extracted, increasing the concentrations of both fractal clusters and low-molecular weight organic compounds. While the clusters were extracted from agitated and non-agitated soil suspensions, the low molecular weight organics were mainly released by agitation. Based on the experimental observations, we propose a conceptual model where parent soil aggregates contain the fractal clusters in mobile and occluded forms, and that the occluded clusters co-exist with occluded low molecular weight organics. These occluded forms may be released by mechanical forces, increasing pH and temperature. At higher temperatures, the soil aggregates and the fractal clusters start to break up, and subsequently individual colloidal coils, presumably carbohydrates, disperse in the water phase. The model explains the origin and properties of the fractal clusters that completely dominate the colloidal DOM extracted from our soil at room temperature and below.

Soil derived dissolved organic matter (DOM) is an important component of the carbon cycle and influences numerous biogeochemical processes, including the formation of mineral-organic associations. DOM ranges in size from small organic molecules to macromolecules and colloidal aggregates. In this study we have used small angle neutron (SANS) and X-ray (SAXS) scattering to characterize the colloidal DOM fraction from the organic layer of a boreal forest soil, and its interactions with hematite (α-Fe2O3) mineral nanoparticles. Comparison between SAXS and contrast variation SANS patterns revealed that the scattering form factor of the colloidal DOM aggregates was essentially independent of the scattering contrast, implying that the colloidal aggregates have an essentially homogeneous chemical composition, down to the nanometre length scale. Variation of the D2O/H2O ratio of the solvent yielded a SANS intensity minimum at ca. 40 ​vol % D2O, which was consistent with colloids composed of mainly polysaccharides. At pH 5.5 the pure hematite nanoparticles were colloidally stable in water and characterized by a ζ-potential of +25 ​mV and a hydrodynamic radius of ca. 70 ​nm. In the presence of DOM, the hematite nanoparticles lost the colloidal stability and aggregated into larger clusters, displaying a negative ζ-potential of ca. −25 ​mV. The charge reversal suggested that negatively charged polyanions of DOM adsorbed onto the hematite particles, possibly leading to bridging flocculation. Our results suggested that mainly low molecular weight components induced hematite aggregation because no or very limited interactions between DOM colloids and hematite were detected.

A major fraction of nitrogen (N) in boreal forest soils is found in organic forms associated with soil organic matter (SOM) and mineral particles. The capacity of ectomycorrhizal (ECM) fungal symbionts to access this N is debated, considering that these fungi have lost many of the genes for decomposing organic matter that were present in their saprotrophic ancestors. To gain a molecular-level understanding of the N-mining processes in ECM fungi, we developed an experimental approach where the processes of decomposition were studied in parallel with the changes in the structure and properties of the organic matter. We showed that ECM fungi have significant capacities to assimilate organic N associated with SOM and mineral surfaces. The decomposition mechanisms differ between species, reflecting the lignocellulose decomposition mechanisms found in their saprotrophic ancestors. During N-mining, the ECM fungi processed the SOM to a material with increased adsorptive properties to iron oxide mineral particles. Two pathways contributed to these changes: Extracellular modifications of the SOM and secretion of mineral surface reactive metabolites. Some of these metabolites have iron(III)-reducing activities and can participate in extracellular Fenton reactions and redox reactions at iron oxide mineral surfaces. We conclude that the traditional framework for understanding organic N acquisition by ECM fungi from recalcitrant SOM must be extended to a framework that includes how those decomposition activities affect the stabilization and reactivity of mineral-associated SOM. The activity through these complex networks of reactions is decisive for the overall effect of ECM fungal decomposition on nutrients and C-cycling in forest ecosystems.

Cellulose degradation by fungi plays a fundamental role in terrestrial carbon cycling, but the mechanisms by which fungi cope with the crystallinity of cellulose are not fully understood. We used X-ray scattering to analyze how fungi, a commercial enzyme mix, and a Fenton reaction-generated radical alter the crystalline structure of cellulose.

Recent studies have shown that dissolved organic matter (DOM) decomposed by ectomycorrhizal (ECM) fungi increases adsorptive properties of organic matter towards soil mineral surfaces. Concomitantly, ECM fungi secrete secondary metabolites with iron reducing capacity that are thought to participate in non-enzymatic Fenton-based decomposition of DOM. The aim of this study was to investigate if the iron reduction induced by the ECM fungus Paxillus involutus during organic matter decomposition was conserved in the decomposed DOM. We explored how the modified DOM reductively dissolved ferrihydrite and goethite nanoparticles and how these processes affected the reactions with H 2 O 2 and the Fenton-based oxidation of mineral-associated organic matter. Culture filtrates were obtained from incubation of the ECM fungus on DOM from forest litter of a spruce forest. This modified DOM was separated by extraction into an ethyl acetate and a water fraction. These fractions were reacted with ferrihydrite and goethite in absence and presence of H 2 O 2 . Dissolved Fe 2+ and HO • were measured and the reactions at the iron oxide-water interfaces were monitored in real-time with in-situ IR spectroscopy. Experiments showed that decomposition of DOM by P. involutus generated a modified DOM that displayed an increased and persistent reductive capacity. Most of the reductants were isolated in the aromatic- and carboxyl-dominated ethyl acetate fraction but some reduction capacity was also captured in the water fraction mainly containing carbohydrates. Reductive dissolution was more extensive for ferrihydrite than goethite, and this process generated significant oxidation of the DOM-ferrihydrite associations. Oxidation of adsorbed DOM was triggered by H 2 O 2 via heterogenous and homogeneous Fenton reactions. These oxidation processes were favored by ferrihydrite because of a high reduction potential and a high efficiency of heterogeneous Fenton as compared to goethite. An optimal timing between the heterogeneous and homogeneous Fenton processes triggered extensive radical oxidation of the DOM-ferrihydrite associations generating a high concentration of surface-associated oxalate. Overall, the results show that organic matter associated with ferrihydrite may be more susceptible to radical oxidation than on goethite, and that fungal decomposition of DOM in general may have consequences for other important soil processes such as mineral dissolution, adsorption and initiation of radical reactions.

Components of dissolved organic matter (DOM) span from sub-nm molecules to colloidal aggregates of several hundred nm. The colloidal fraction is important for the transport of organic matter and associated elements in the environment, and for the stability of DOM constituents with respect to microbial decomposition. This study focuses on the colloidal properties of DOM extracted from spruce forest soils of a chronosequence. The DOM samples were obtained by common water extraction procedures at 21 and 100°C, respectively. We applied an experimental approach combining chemical analysis with light and X-ray scattering techniques that informed on the colloidal size, charge, and structure of DOM. Results showed that two main types of colloids were present: semi-flexible cylinders and fractal aggregates. The cylinders consisted of carbohydrates, presumably hemicelluloses, while the aggregates were a composite material containing a large fraction of carbohydrates together with aliphatics and clay particles. These fractal aggregates dominated the cold-water extracts whereas the strong increase in total organic carbon by hot-water extraction caused a concomitantly strong increase of semi-flexible cylinders, which became the predominant species. Comparison between the chronosequence soils showed that with increasing forest age, the amount of carbon extracted per gram of soil declined and the concentration of the semi-flexible cylinders decreased. Thus, the distribution between the fractal aggregates and cylinders in the forest soil DOM samples depends on the composition of the soil organic matter and the leaching temperature. Changes in this distribution may have important implications for the reactivity and stability of DOM colloids.

A large fraction of nitrogen (N) in forest soils is present in mineral-associated proteinaceous compounds. The strong association between proteins and minerals limits microbial accessibility to this source, which is a relatively stable reservoir of soil N. We have shown that the ectomycorrhizal (ECM) fungus Paxillus involutus can acquire N from iron oxide-associated proteins. Using tightly controlled isotopic, spectroscopic and chromatographic experiments, we demonstrated that the capacity to access N from iron oxide-associated bovine serum albumin (BSA) is shared with the ECM fungi Hebeloma cylindrosporum and Piloderma olivaceum. Despite differences in evolutionary history, growth rates, exploration types and the decomposition mechanisms of organic matter, their N acquisition mechanisms were similar to those described for P. involutus. The fungi released N from mineral-associated BSA by direct action of extracellular aspartic proteases on the mineral-associated BSA, without initial desorption of the protein. Hydrolysis was suppressed by the adsorption of proteases to minerals, but this adverse effect was counteracted by the secretion of compounds that conditioned the mineral surface. These data suggest that the enzymatic exudate-driven mechanism to access N from mineral-associated proteins is found in ECM fungi of multiple lineages and exploration types.

Ectomycorrhizal fungi use both extracellular enzymes and hydroxyl radicals to decompose soil organic matter (SOM) in a way that is similar to that of their saprotrophic wood decomposing ancestors. Although it are ultimately the individual hyphae that decompose SOM, it has remained unclear if it is also the local environmental conditions experienced by individual hyphae that control the decomposition activity of these hyphae or if it is the overall physiological status of the mycelium these hyphae are connected to that drives decomposition activity of hyphae. We set up an experimental system in which the decomposition activity of individual hyphae could be imaged using infrared (IR) microspectroscopy. Colonies of the ectomycorrhizal fungus Paxillus involutus were grown on solid, sterile lignin films which were amended with ferrihydrite minerals or not. The decomposition activity of individual hyphae was subsequently related to the local environmental conditions experienced by subsets of hyphae (presence or absence of ferrihydrite in lignin substrates) of a mycelial colony and the overall physiological status of the mycelium (difference in hydroxyl radical producing capacity of the mycelium and organic versus inorganic nitrogen nutrition). Using this experimental set-up, we have shown that the local conditions experienced by individual hyphae plays a key role in determining the decomposition activity of these hyphae, but the overall decomposition activity of the mycelium these hyphae were connected to also played a clear role. We also showed that hyphae which more actively oxidized the lignin substrate, also secreted more extracellular matrix materials, suggesting a functional involvement of fungal extracellular matrices in this decomposition process. We conclude that phenotypic heterogeneity occurring between genetically identical hyphal tips may be an important strategy for filamentous fungi to cope with heterogeneous and constantly changing soil environments.

Filamentous fungi play a key role in the terrestrial carbon cycle as they are the primary decomposers of lignocellulose in soil organic matter (SOM). Fungi secrete a wide range of oxidative and hydrolytic enzymes, and generate radicals through extracellular secondary metabolites to decompose SOM. To study fungal decomposition of SOM, the activities of isolated enzymes are typically studied as proxies for the decomposition activity of fungi. However, extracellular enzymes involved in lignocellulose decomposition are often bound to fungal extracellular polymeric substance (EPS) matrices. This association between extracellular enzymes and EPS matrices affects the activities of the enzymes. Moreover, extracellular enzymes and fungal cells are prone to attack by radicals and proteolytic enzymes themselves. Hence, these seemingly incompatible decomposition mechanisms must be regulated in some way in the fungal extracellular space to allow efficient decomposition of SOM, while preventing damage to secreted extracellular enzymes or the fungal cells themselves. We here review studies investigating the associations between fungal extracellular enzymes and EPS matrices and how these associations affect hydrolytic and oxidative reactions involved in SOM decomposition. Based on the knowledge compiled in the current review, we propose that fungal EPS matrices should be viewed as highly dynamic and functional parts of the fungal extracellular decomposition machinery. We also build a conceptual illustration that describes how the molecular composition and structure of fungal EPS matrices ensure that extracellular decomposition reactions only proceed at the right time and in the right place.