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Fungal secretomes contain a wide range of hydrolytic and oxidative enzymes, including cellulases, hemicellulases, pectinases, and lignin-degrading accessory enzymes, that synergistically drive litter decomposition in the environment. While secretome studies of model organisms such as Phanerochaete chrysosporium and Aspergillus species have greatly expanded our knowledge of these enzymes, few have extended secretome characterization to environmental isolates or conducted side-by-side comparisons of diverse species. Thus, the mechanisms of carbon degradation by many ubiquitous soil fungi remain poorly understood. Here we use a combination of LC-MS/MS, genomic, and bioinformatic analyses to characterize and compare the protein composition of the secretomes of four recently isolated, cosmopolitan, Mn(II)-oxidizing Ascomycetes (Alternaria alternata SRC1lrK2f, Stagonospora sp. SRC1lsM3a, Pyrenochaeta sp. DS3sAY3a, and Paraconiothyrium sporulosum AP3s5-JAC2a). We demonstrate that the organisms produce a rich yet functionally similar suite of extracellular enzymes, with species-specific differences in secretome composition arising from unique amino acid sequences rather than overall protein function. Furthermore, we identify not only a wide range of carbohydrate-active enzymes that can directly oxidize recalcitrant carbon, but also an impressive suite of redox-active accessory enzymes that suggests a role for Fenton-based hydroxyl radical formation in indirect, non-specific lignocellulose attack. Our findings highlight the diverse oxidative capacity of these environmental isolates and enhance our understanding of the role of filamentous Ascomycetes in carbon turnover in the environment.

Despite the ubiquity of Mn oxides in natural environments, there are only a few observations of biological Mn(II) oxidation at pH < 6. The lack of low pH Mn-oxidizing bacteria (MOB) isolates limits our understanding of how pH influences biological Mn(II) oxidation in extreme environments. Here, we report that a novel MOB isolate, Mesorhizobium australicum strain T-G1, isolated from an acidic and metalliferous uranium mining area, can oxidize Mn(II) at both acidic and neutral pH using different enzymatic pathways. X-ray diffraction (XRD), Raman spectroscopy, and scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDS) revealed that T-G1 initiated bixbyite-like Mn oxide formation at pH 5.5 which coincided with multi-copper oxidase (MCO) expression from early exponential phase to late stationary phase. In contrast, reactive oxygen species (ROS), particularly superoxide, appeared to be more important for T-G1 mediated Mn(II) oxidation at neutral pH. ROS was produced in parallel with the occurrence of Mn(II) oxidation at pH 7.2 from early stationary phase. Solid phase Mn oxides did not precipitate, which is consistent with the presence of a high amount of H2O2 and lower activity of catalase in the liquid culture at pH 7.2. Our results show that M. australicum T-G1, an acid tolerant MOB, can initiate Mn(II) oxidation by varying its oxidation mechanisms depending on the pH and may play an important role in low pH manganese biogeochemical cycling.

Mercury (Hg) is a toxic heavy metal that poses significant human and environmental health risks. Soils and sediments, where Hg can exist as the Hg sulfide mineral metacinnabar (β-HgS), represent major Hg reservoirs in aquatic environments. Metacinnabar has historically been considered a sink for Hg in all but severely acidic environments, and thus disregarded as a potential source of Hg back to aqueous or gaseous pools. Here, we conducted a combination of field and laboratory incubations to identify the potential for metacinnabar as a source of dissolved Hg within near neutral pH environments and the underpinning (a)biotic mechanisms at play. We show that the abundant and widespread sulfur-oxidizing bacterium Thiobacillus extensively colonized metacinnabar chips incubated within aerobic, near neutral pH creek sediments. Laboratory incubations of axenic Thiobacillus cultures lead to the release of metacinnabar-hosted Hg(II) and subsequent volatilization to Hg(0). This dissolution and volatilization was greatly enhanced in the presence of the sulfur intermediate, thiosulfate, which served a dual role by enhancing HgS dissolution and providing an additional metabolic substrate for Thiobacillus. These findings reveal a new coupled abiotic-biotic pathway for the transformation of metacinnabar-bound Hg(II) to Hg(0), while expanding the sulfide substrates available for neutrophilic chemosynthetic bacteria to Hg-laden sulfides. They also point to mineral-hosted Hg as an underappreciated source of gaseous elemental Hg to the environment.

Research into the deep biosphere requires an understanding of both the microbial community at a given site and the geochemical and hydrological factors that support that microbial community. To highlight the interplay between geochemistry and microbiology in these deep environments, we characterized the hydrogeologic and geochemical systems of a 2.7 Ga banded iron formation within the Canadian Shield in the Soudan Underground Mine State Park in Minnesota, United States, a site known to host a lithotrophic microbial community. Calcium-sodium-chloride brines, characteristic of deep groundwaters throughout the Canadian Shield, were found in the site with total dissolved constituents (

Manganese (Mn) oxides are among the strongest oxidants and sorbents in the environment, and Mn(II) oxidation to Mn(III/IV) (hydr)oxides includes both abiotic and microbially-mediated processes. While white-rot Basidiomycete fungi oxidize Mn(II) using laccases and manganese peroxidases in association with lignocellulose degradation, the mechanisms by which filamentous Ascomycete fungi oxidize Mn(II) and a physiological role for Mn(II) oxidation in these organisms remain poorly understood. Here we use a combination of chemical and in-gel assays and bulk mass spectrometry to demonstrate secretome-based Mn(II) oxidation in three phylogenetically diverse Ascomycetes that is mechanistically distinct from hyphal-associated Mn(II) oxidation on solid substrates. We show that Mn(II) oxidative capacity of these fungi is dictated by species-specific secreted enzymes and varies with secretome age, and we reveal the presence of both Cu-based and FAD-based Mn(II) oxidation mechanisms in all 3 species, demonstrating mechanistic redundancy. Specifically, we identify candidate Mn(II)-oxidizing enzymes as tyrosinase and glyoxal oxidase in Stagonospora sp. SRC1lsM3a, bilirubin oxidase in Stagonospora sp. and Paraconiothyrium sporulosum AP3s5-JAC2a, and GMC oxidoreductase in all 3 species, including Pyrenochaeta sp. DS3sAY3a. The diversity of the candidate Mn(II)-oxidizing enzymes identified in this study suggests that the ability of fungal secretomes to oxidize Mn(II) may be more widespread than previously thought.

Selenium (Se) is an essential element for most organisms yet can cause severe negative biological consequences at elevated levels. The oxidized forms of Se, selenate [Se(VI)] and selenite [Se(IV)], are more mobile, toxic, and bioavailable than the reduced forms of Se such as volatile or solid phases. Thus, selenate and selenite pose a greater threat to ecosystems and human health. As current Se remediation technologies have varying efficiencies and costs, novel strategies to remove elevated Se levels from environments impacted by anthropogenic activities are desirable. Some common soil fungi quickly remove Se (IV and VI) from solution by aerobic reduction to solid or volatile forms. Here, we perform bench-scale culture experiments of two Se-reducing Ascomycota to determine their Se removal capacity in growth media conditions containing either Se(IV) or Se(VI) as well as in Se-containing municipal (∼25 μg/L Se) and industrial (∼2000 μg/L Se) wastewaters. Dissolved Se was measured throughout the experiments to assess Se concentration and removal rates. Additionally, solid-associated Se was quantified at the end of each experiment to determine the amount of Se removed to solid phases (e.g., Se(0) nanoparticles, biomass-adsorbed Se, or internal organic selenoproteins). Results show that under optimal conditions, fungi more efficiently remove Se(IV) from solution compared to Se(VI). Additionally, both fungi remove a higher percentage of Se from the filtered municipal wastewater compared to the industrial wastewater, though cultures in industrial wastewater retained a greater amount of solid-associated Se. Additional wastewater experiments were conducted with supplemental carbohydrate- or glycerin-based carbon products and additional nitrogen- and phosphorous-containing nutrients in some cases to enhance fungal growth. Relative to unamended wastewater experiments, supplemental carbohydrates promote Se removal from municipal wastewater but minimally impact industrial wastewater removal. This demonstrates that carbon availability and source impacts fungal Se reduction and removal from solution. Calculations to assess the leaching potential of solid-associated Se from fungal biomass show that wastewater Se release will not exceed regulatory limits. This study highlights the considerable potential for the mycoremediation of Se-contaminated wastewaters.

This article presents visual image data and detailed methodology for the use of a new method for quantifying the exudation of siderophores during fungal growth. The data include images showing time series for calibration, fungal exudation, and negative controls, as well as replication accuracy information. In addition, we provide detailed protocols for making CAS assay layer plates, the digital analysis protocol for determining area of color change, and discuss growth media that do and do not work with the layer plate method. The results of these data, their interpretation, and further discussion can be found in Andrews et al., 2016 [1].