Isolation and genomic analysis of "Metallumcola ferriviriculae" MK1, a Gram-positive, Fe(III)-reducing bacterium from the Soudan Underground Mine, an iron-rich Martian analog site.

The Soudan Underground Mine State Park, found in the Vermilion Iron Range in northern Minnesota, provides access to a ~ 2.7 billion-year-old banded iron formation. Exploratory boreholes drilled between 1958 and 1962 on the 27th level (713 m underground) of the mine intersect calcium and iron-rich brines that have recently been subject to metagenomic analysis and microbial enrichments. Using concentrated brine samples pumped from a borehole depth of up to 55 m, a novel Gram-positive bacterium was enriched under anaerobic, acetate-oxidizing, and Fe(III) citrate-reducing conditions. The isolated bacterium, designated strain MK1, is non-motile, rod-shaped, spore-forming, anaerobic, and mesophilic, with a growth range between 24°C and 30°C. The complete circular MK1 genome was found to be 3,720,236 bp and encodes 25 putative multiheme cytochromes, including homologs to inner membrane cytochromes in the Gram-negative bacterium Geobacter sulfurreducens and cytoplasmic membrane and periplasmic cytochromes in the Gram-positive bacterium Thermincola potens. However, MK1 does not encode homologs of the peptidoglycan (CwcA) and cell surface-associated (OcwA) multiheme cytochromes proposed to be required by T. potens to perform extracellular electron transfer. The 16S rRNA gene sequence of MK1 indicates that its closest related isolate is Desulfitibacter alkalitolerans strain sk.kt5 (91% sequence identity), which places MK1 in a novel genus within the Desulfitibacteraceae family and Moorellales order. Within the Moorellales order, only Calderihabitans maritimus strain KKC1 has been reported to reduce Fe(III), and only D. alkalitolerans can also grow in temperatures below 40°C. Thus, MK1 represents a novel species within a novel genus, for which we propose the name "Metallumcola ferriviriculae" strain MK1, and provides a unique opportunity to study a cytochrome-rich, mesophilic, Gram-positive, spore-forming Fe(III)-reducing bacterium.IMPORTANCEThe Soudan Underground Mine State Park gives access to understudied regions of the deep terrestrial subsurface that potentially predate the Great Oxidation Event. Studying organisms that have been relatively unperturbed by surface conditions for as long as 2.7 billion years may give us a window into ancient life before oxygen dominated the planet. Additionally, studying microbes from anoxic and iron-rich environments can help us better understand the requirements of life in analogous environments, such as on Mars. The isolation and characterization of "Metallumcola ferriviriculae" strain MK1 give us insights into a novel genus and species that is distinct both from its closest related isolates and from iron reducers characterized to date. "M. ferriviriculae" strain MK1 may also act as a model organism to study how the processes of sporulation and germination are affected by insoluble extracellular acceptors, as well as the impact of spores in the deep terrestrial biosphere.

Time-Resolved Examination of Fungal Selenium Redox Transformations.

Selenium (Se) is both a micronutrient required for most life and an element of environmental concern due to its toxicity at high concentrations, and both bioavailability and toxicity are largely influenced by the Se oxidation state. Environmentally relevant fungi have been shown to aerobically reduce Se(IV) and Se(VI), the generally more toxic and bioavailable Se forms. The goal of this study was to shed light on fungal Se(IV) reduction pathways and biotransformation products over time and fungal growth stages. Two Ascomycete fungi were grown with moderate (0.1 mM) and high (0.5 mM) Se(IV) concentrations in batch culture over 1 month. Fungal growth was measured throughout the experiments, and aqueous and biomass-associated Se was quantified and speciated using analytical geochemistry, transmission electron microscopy (TEM), and synchrotron-based X-ray absorption spectroscopy (XAS) approaches. The results show that Se transformation products were largely Se(0) nanoparticles, with a smaller proportion of volatile, methylated Se compounds and Se-containing amino acids. Interestingly, the relative proportions of these products were consistent throughout all fungal growth stages, and the products appeared stable over time even as growth and Se(IV) concentration declined. This time-series experiment showing different biotransformation products throughout the different growth phases suggests that multiple mechanisms are responsible for Se detoxification, but some of these mechanisms might be independent of Se presence and serve other cellular functions. Knowing and predicting fungal Se transformation products has important implications for environmental and biological health as well as for biotechnology applications such as bioremediation, nanobiosensors, and chemotherapeutic agents.

Aqueous Co removal by mycogenic Mn oxides from simulated mining wastewaters.

Naturally occurring manganese (Mn) oxide minerals often form by microbial Mn(II) oxidation, resulting in nanocrystalline Mn(III/IV) oxide phases with high reactivity that can influence the uptake and release of many metals (e.g., Ni, Cu, Co, and Zn). During formation, the structure and composition of biogenic Mn oxides can be altered in the presence of other metals, which in turn affects the minerals' ability to bind these metals. These processes are further influenced by the chemistry of the aqueous environment and the type and physiology of microorganisms involved. Conditions extending to environments that typify mining and industrial wastewaters (e.g., increased salt content, low nutrient, and high metal concentrations) have not been well investigated thus limiting the understanding of metal interactions with biogenic Mn oxides. By integrating geochemistry, microscopic, and spectroscopic techniques, we examined the capacity of Mn oxides produced by the Mn(II)-oxidizing Ascomycete fungus Periconia sp. SMF1 isolated from the Minnesota Soudan Mine to remove the metal co-contaminant Co(II) from synthetic waters that are representative of mining wastewaters currently undergoing remediation efforts. We compared two different applied remediation strategies under the same conditions: coprecipitation of Co with mycogenic Mn oxides versus adsorption of Co with pre-formed fungal Mn oxides. Co(II) was effectively removed from solution by fungal Mn oxides through two different mechanisms: incorporation into, and adsorption onto, Mn oxides. These mechanisms were similar for both remediation strategies, indicating the general effectiveness of Co(II) removal by these oxides. The mycogenic Mn oxides were primarily a nanoparticulate, poorly-crystalline birnessite-like phases with slight differences depending on the chemical conditions during formation. The relatively fast and complete removal of aqueous Co(II) during biomineralization as well as the subsequent structural incorporation of Co into the Mn oxide structure illustrated a sustainable cycle capable of continuously remediating Co(II) from metal-polluted environments.

Hydrobiogechemical interactions in the hyporheic zone of a sulfate-impacted, freshwater stream and riparian wetland ecosystem.

Coupled abiotic and biotic processes in the hyporheic zone, where surface water and groundwater mix, play a critical role in the biogeochemical cycling of carbon, nutrients, and trace elements in streams and wetlands. Dynamic hydrologic conditions and anthropogenic pollution can impact redox gradients and biogeochemical response, although few studies examine the resulting hydrobiogeochemical interactions generated within the hyporheic zone. This study examines the effect of hyporheic flux dynamics and anthropogenic sulfate loading on the biogeochemistry of a riparian wetland and stream system. The hydrologic gradient as well as sediment, surface water, and porewater geochemistry chemistry was characterized at multiple points throughout the 2017 spring-summer-fall season at a sulfate-impacted stream flanked by wetlands in northern Minnesota. Results show that organic-rich sediments largely buffer the geochemical responses to brief or low magnitude changes in hydrologic gradient, but sustained or higher magnitude fluxes may variably alter the redox regime and, ultimately, the environmental geochemistry. This has implications for a changing climate that is expected to dramatically alter the hydrological cycle. Further, increased sulfate loading and dissolved or adsorbed ferric iron complexes in the hyporheic zone may induce a cryptic sulfur cycle linked to iron and carbon cycling, as indicated by the abundance of intermediate valence sulfur compounds (e.g., polysulfide, elemental sulfur, thiosulfate) throughout the anoxic wetland and stream-channel sediment column. The observed deviation from a classical redox tower coupled with potential changes in hydraulic gradient in these organic-rich wetland and stream hyporheic zones has implications for nutrient, trace element, and greenhouse gas fluxes into surface water and groundwater, ultimately influencing water quality and global climate.

Bioaugmentation with As-transforming bacteria improves arsenic availability and uptake by the hyperaccumulator plant Pteris vittata (L).

Inorganic arsenic (As) is a toxic and carcinogenic pollutant that has long-term impacts on environmental quality and human health. Pteris vittata plants hyperaccumulate As from soils. Soil bacteria are critical for As-uptake by P. vittata. We examined the use of taxonomically diverse soil bacteria to modulate As speciation in soil and their effect on As-uptake by P. vittata. Aqueous media inoculated with Pseudomonas putida MK800041, P. monteilii MK344656, P. plecoglossicida MK345459, Ochrobactrum intermedium MK346993 or Agrobacterium tumefaciens MK346997 resulted in the oxidation of 5-30% As(III) and a 49-79% reduction of As(V). Soil inoculated with P. monteilii increased extractable As(III) and As(V) from 0.5 and 0.09 in controls to 0.9 and 0.39 mg As kg-1 soil dry weight, respectively. Moreover, and P. vittata plants inoculated with P. monteilii, P. plecoglossicida, O. intermedium strains, and A. tumefaciens strains MK344655, MK346994, MK346997, significantly increased As-uptake by 43, 32, 12, 18, 16, and 14%, respectively, compared to controls. The greatest As-accumulation (1.9 ± 0.04 g kg-1 frond Dwt) and bioconcentration factor (16.3 ± 0.35) was achieved in plants inoculated with P. monteilii. Our findings indicate that the tested bacterial strains can increase As-availability in soils, thus enhancing As-accumulation by P. vittata. Novelty statement Pteris vittata, a well-known As-hyperaccumulator, has the remarkable ability to accumulate higher levels of As in their above-ground biomass. The As-tolerant bacteria-plant interactions play a significant role in bioremediation by mediating As-redox and controlling As-availability and uptake by P. vittata. Our studies indicated that most of the tested bacterial strains isolated from As-impacted soil significantly enhanced As-uptake by P. vittata. P. monteilii oxidized 20% of As(III) and reduced 50% of As(V), increased As-extraction from soils, and increased As-uptake by 43% greater compared with control. Therefore, these strains associated with P. vittata can be used in large-scale field applications to remediate As-contaminated soil.

Mechanisms of Manganese(II) Oxidation by Filamentous Ascomycete Fungi Vary With Species and Time as a Function of Secretome Composition.

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.

Fungal Bioremediation of Selenium-Contaminated Industrial and Municipal Wastewaters.

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.

A Fungal-Mediated Cryptic Selenium Cycle Linked to Manganese Biogeochemistry.

Selenium (Se) redox chemistry is a determining factor for its environmental toxicity and mobility. Currently, millions of people are impacted by Se deficiency or toxicity, and in geologic history, several mass extinctions have been linked to extreme Se deficiency. Importantly, microbial activity and interactions with other biogeochemically active elements can drastically alter Se oxidation state and form, impacting its bioavailability. Here, we use wet geochemistry, spectroscopy, and electron microscopy to identify a cryptic, or hidden, Se cycle involving the reoxidation of biogenic volatile Se compounds in the presence of biogenic manganese [Mn(III, IV)] oxides and oxyhydroxides (hereafter referred to as "Mn oxides"). Using two common environmental Ascomycete fungi, Paraconiothyrium sporulosum and Stagonospora sp., we observed that aerobic Se(IV and VI) bioreduction to Se(0) and Se(-II) occurs simultaneously alongside the opposite redox biomineralization process of mycogenic Mn(II) oxidation to Mn oxides. Selenium bioreduction produced stable Se(0) nanoparticles and organoselenium compounds. However, mycogenic Mn oxides rapidly oxidized volatile Se products, recycling these compounds back to soluble forms. Given their abundance in natural systems, biogenic Mn oxides likely play an important role mediating Se biogeochemistry. Elucidating this cryptic Se cycle is essential for understanding and predicting Se behavior in diverse environmental systems.

Mn oxide formation by phototrophs: Spatial and temporal patterns, with evidence of an enzymatic superoxide-mediated pathway.

Manganese (Mn) oxide minerals influence the availability of organic carbon, nutrients and metals in the environment. Oxidation of Mn(II) to Mn(III/IV) oxides is largely promoted by the direct and indirect activity of microorganisms. Studies of biogenic Mn(II) oxidation have focused on bacteria and fungi, with phototrophic organisms (phototrophs) being generally overlooked. Here, we isolated phototrophs from Mn removal beds in Pennsylvania, USA, including fourteen Chlorophyta (green algae), three Bacillariophyta (diatoms) and one cyanobacterium, all of which consistently formed Mn(III/IV) oxides. Isolates produced cell-specific oxides (coating some cells but not others), diffuse biofilm oxides, and internal diatom-specific Mn-rich nodules. Phototrophic Mn(II) oxidation had been previously attributed to abiotic oxidation mediated by photosynthesis-driven pH increases, but we found a decoupling of Mn oxide formation and pH alteration in several cases. Furthermore, cell-free filtrates of some isolates produced Mn oxides at specific time points, but this activity was not induced by Mn(II). Manganese oxide formation in cell-free filtrates occurred via reaction with the oxygen radical superoxide produced by soluble extracellular proteins. Given the known widespread ability of phototrophs to produce superoxide, the contribution of phototrophs to Mn(II) oxidation in the environment may be greater and more nuanced than previously thought.

Persistent Bacterial and Fungal Community Shifts Exhibited in Selenium-Contaminated Reclaimed Mine Soils.

Mining and other industrial activities worldwide have resulted in Se-enriched surface soils, which pose risks to human and environmental health. Although not well studied, microbial activity can alter Se bioavailability and distribution, even in oxic environments. We used high-throughput sequencing to profile bacterial and fungal communities inhabiting mine soils in southeastern Idaho, comparing mined and unmined locations within two reclaimed phosphate mine areas containing various Se concentrations. The goal was to determine whether microbial communities differed in (i) different mines, (ii) mined areas compared to unmined areas, and (iii) various soil Se concentrations. Though reclamation occurred 20 to 30 years ago, microbial community structures in mined soils were significantly altered compared to unmined soils, suggesting persistent mining-related impacts on soil processes. Additionally, operational taxonomic unit with a 97% sequence similarity cutoff (OTU0.03) richness and diversity were significantly diminished with increasing Se, though not with other geochemical parameters, suggesting that Se contamination shapes communities in favor of Se-tolerant microorganisms. Two bacterial phyla, Actinobacteria and Gemmatimonadetes, were enriched in high-Se soils, while for fungi, Ascomycota dominated all soils regardless of Se concentration. Combining diversity analyses and taxonomic patterns enables us to move toward connecting physiological function of microbial groups to Se biogeochemical cycling in oxic soil environments.IMPORTANCE Selenium contamination in natural environments is of great concern globally, and microbial processes are known to mediate Se transformations. Such transformations alter Se mobility, bioavailability, and toxicity, which can amplify or mitigate Se pollution. To date, nearly all studies investigating Se-microbe interactions have used culture-based approaches with anaerobic bacteria despite growing knowledge that (i) aerobic Se transformations can occur, (ii) such transformations can be mediated by microorganisms other than bacteria, and (iii) microbial community dynamics, rather than individual organismal activities, are important for metal(loid) cycling in natural environments. We examined bacterial and fungal communities in Se-contaminated reclaimed mine soils and found significant declines in diversity at high Se concentrations. Additionally, we identified specific taxonomic groups that tolerate excess Se and may be useful for bioremediation purposes. These patterns were similar across mines of different ages, suggesting that microbial community impacts may persist long after physicochemical parameters indicate complete site recovery.

Quantitative iTRAQ-based secretome analysis reveals species-specific and temporal shifts in carbon utilization strategies among manganese(II)-oxidizing Ascomycete fungi.

Fungi generate a wide range of extracellular hydrolytic and oxidative enzymes and reactive metabolites, collectively known as the secretome, that synergistically drive plant litter decomposition in the environment. While secretome studies of model organisms have greatly expanded our knowledge of these enzymes, few have extended secretome characterization to environmental isolates, particularly filamentous Ascomycetes, or directly compared temporal patterns of enzyme utilization among diverse species. Thus, the mechanisms of carbon (C) degradation by many ubiquitous soil fungi remain poorly understood. Here we use a combination of iTRAQ proteomics and extracellular enzyme activity assays to compare the protein composition of the secretomes of four manganese(II)-oxidizing Ascomycete fungi over a three-week time course. We demonstrate that the fungi exhibit striking differences in the regulation of extracellular lignocellulose-degrading enzymes among species and over time, revealing species-specific and temporal shifts in C utilization strategies as they degrade the same substrate. Specifically, our findings suggest that Alternaria alternata SRC1lrK2f and Paraconiothyrium sporulosum AP3s5-JAC2a employ sequential enzyme secretion patterns concomitant with decreasing resource availability. Stagonospora sp. SRC1lsM3a preferentially degrades proteinaceous substrate before switching to carbohydrates, and Pyrenochaeta sp. DS3sAY3a utilizes primarily peptidases to aggressively attack carbon sources in a concentrated burst. This work highlights the diversity of operative metabolic strategies among understudied yet ubiquitous cellulose-degrading Ascomycetes, enhancing our understanding of their contribution to C turnover in the environment.

Fourier-transform infrared spectroscopy (FTIR) analysis of triclinic and hexagonal birnessites.

The characterization of birnessite structures is particularly challenging for poorly crystalline materials of biogenic origin, and a determination of the relative concentrations of triclinic and hexagonal birnessite in a mixed assemblage has typically required synchrotron-based spectroscopy and diffraction approaches. In this study, Fourier-transform infrared spectroscopy (FTIR) is demonstrated to be capable of differentiating synthetic triclinic Na-birnessite and synthetic hexagonal H-birnessite. Furthermore, IR spectral deconvolution of peaks resulting from MnO lattice vibrations between 400 and 750cm-1 yield results comparable to those obtained by linear combination fitting of synchrotron X-ray absorption fine structure (EXAFS) data when applied to known mixtures of triclinic and hexagonal birnessites. Density functional theory (DFT) calculations suggest that an infrared absorbance peak at ~1628cm-1 may be related to OH vibrations near vacancy sites. The integrated intensity of this peak may show sensitivity to vacancy concentrations in the Mn octahedral sheet for different birnessites.

Comparative Analysis of Secretome Profiles of Manganese(II)-Oxidizing Ascomycete Fungi.

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.

Iron Transformation Pathways and Redox Micro-Environments in Seafloor Sulfide-Mineral Deposits: Spatially Resolved Fe XAS and δ(57/54)Fe Observations.

Hydrothermal sulfide chimneys located along the global system of oceanic spreading centers are habitats for microbial life during active venting. Hydrothermally extinct, or inactive, sulfide deposits also host microbial communities at globally distributed sites. The main goal of this study is to describe Fe transformation pathways, through precipitation and oxidation-reduction (redox) reactions, and examine transformation products for signatures of biological activity using Fe mineralogy and stable isotope approaches. The study includes active and inactive sulfides from the East Pacific Rise 9°50'N vent field. First, the mineralogy of Fe(III)-bearing precipitates is investigated using microprobe X-ray absorption spectroscopy (µXAS) and X-ray diffraction (µXRD). Second, laser-ablation (LA) and micro-drilling (MD) are used to obtain spatially-resolved Fe stable isotope analysis by multicollector-inductively coupled plasma-mass spectrometry (MC-ICP-MS). Eight Fe-bearing minerals representing three mineralogical classes are present in the samples: oxyhydroxides, secondary phyllosilicates, and sulfides. For Fe oxyhydroxides within chimney walls and layers of Si-rich material, enrichments in both heavy and light Fe isotopes relative to pyrite are observed, yielding a range of δ(57)Fe values up to 6‰. Overall, several pathways for Fe transformation are observed. Pathway 1 is characterized by precipitation of primary sulfide minerals from Fe(II)aq-rich fluids in zones of mixing between vent fluids and seawater. Pathway 2 is also consistent with zones of mixing but involves precipitation of sulfide minerals from Fe(II)aq generated by Fe(III) reduction. Pathway 3 is direct oxidation of Fe(II) aq from hydrothermal fluids to form Fe(III) precipitates. Finally, Pathway 4 involves oxidative alteration of pre-existing sulfide minerals to form Fe(III). The Fe mineralogy and isotope data do not support or refute a unique biological role in sulfide alteration. The findings reveal a dynamic range of Fe transformation pathways consistent with a continuum of micro-environments having variable redox conditions. These micro-environments likely support redox cycling of Fe and S and are consistent with culture-dependent and -independent assessments of microbial physiology and genetic diversity of hydrothermal sulfide deposits.

Digital image quantification of siderophores on agar plates.

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].

Layer plate CAS assay for the quantitation of siderophore production and determination of exudation patterns for fungi.

The chrome azurol S (CAS) assay measures the chelating activity of siderophores, but its application (especially to fungi) is limited by toxicity issues. In this note, we describe a modified version of the CAS assay that is suitable for quantifying siderophore exudation for microorganisms, including fungi.

Characterization of pH dependent Mn(II) oxidation strategies and formation of a bixbyite-like phase by Mesorhizobium australicum T-G1.

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, Raman spectroscopy, and scanning electron microscopy with energy dispersive X-ray spectroscopy revealed that T-G1 initiated bixbyite-like Mn oxide formation at pH 5.5 which coincided with multi-copper oxidase 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.

Microbial- and thiosulfate-mediated dissolution of mercury sulfide minerals and transformation to gaseous mercury.

Mercury (Hg) is a toxic heavy metal that poses significant environmental and human health risks. Soils and sediments, where Hg can exist as the Hg sulfide mineral metacinnabar (ß-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 bacteria of the genus Thiobacillus extensively colonized metacinnabar chips incubated within aerobic, near neutral pH creek sediments. Laboratory incubations of axenic Thiobacillus thioparus cultures led 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 thiosulfate, which served a dual role by enhancing HgS dissolution through Hg complexation 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.

Nutrient input influences fungal community composition and size and can stimulate manganese (II) oxidation in caves.

Little is known about the fungal role in biogeochemical cycling in oligotrophic ecosystems. This study compared fungal communities and assessed the role of exogenous carbon on microbial community structure and function in two southern Appalachian caves: an anthropogenically impacted cave and a near-pristine cave. Due to carbon input from shallow soils, the anthropogenically impacted cave had an order of magnitude greater fungal and bacterial quantitative-polymerase chain reaction (qPCR) gene copy numbers, had significantly greater community diversity, and was dominated by ascomycotal phylotypes common in early phase, labile organic matter decomposition. Fungal assemblages in the near-pristine cave samples were dominated by Basidiomycota typically found in deeper soils (and/or in late phase, recalcitrant organic matter decomposition), suggesting more oligotrophic conditions. In situ carbon and manganese (II) [Mn(II)] addition over 10 weeks resulted in growth of fungal mycelia followed by increased Mn(II) oxidation. A before/after comparison of the fungal communities indicated that this enrichment increased the quantity of fungal and bacterial cells, yet decreased overall fungal diversity. Anthropogenic carbon sources can therefore dramatically influence the diversity and quantity of fungi, impact microbial community function, and stimulate Mn(II) oxidation, resulting in a cascade of changes that can strongly influence nutrient and trace element biogeochemical cycles in karst aquifers.