Biogenic manganese oxides as reservoirs of organic carbon and proteins in terrestrial and marine environments

Manganese (Mn) oxides participate in a range of interactions with organic carbon (OC) that can lead to either carbon degradation or preservation. Here, we examine the abundance and composition of OC associated with biogenic and environmental Mn oxides to elucidate the role of Mn oxides as a reservoir for carbon and their potential for selective partitioning of particular carbon species. Mn oxides precipitated in natural brackish waters and by Mn(II)‐oxidizing marine bacteria and terrestrial fungi harbor considerable levels of organic carbon (4.1–17.0 mol OC per kg mineral) compared to ferromanganese cave deposits which contain 1–2 orders of magnitude lower OC. Spectroscopic analyses indicate that the chemical composition of Mn oxide‐associated OC from microbial cultures is homogeneous with bacterial Mn oxides hosting primarily proteinaceous carbon and fungal Mn oxides containing both protein‐ and lipopolysaccharide‐like carbon. The bacterial Mn oxide‐hosted proteins are involved in both Mn(II) oxidation and metal binding by these bacterial species and could be involved in the mineral nucleation process as well. By comparison, the composition of OC associated with Mn oxides formed in natural settings (brackish waters and particularly in cave ferromanganese rock coatings) is more spatially and chemically heterogeneous. Cave Mn oxide‐associated organic material is enriched in aliphatic C, which together with the lower carbon concentrations, points to more extensive microbial or mineral processing of carbon in this system relative to the other systems examined in this study, and as would be expected in oligotrophic cave environments. This study highlights Mn oxides as a reservoir for carbon in varied environments. The presence and in some cases dominance of proteinaceous carbon within the biogenic and natural Mn oxides may contribute to preferential preservation of proteins in sediments and dominance of protein‐dependent metabolisms in the subsurface biosphere.     

A Fungal-Prokaryotic Consortium at the Basalt-Zeolite Interface in Subseafloor Igneous Crust

We have after half a century of coordinated scientific drilling gained insight into Earth´s largest microbial habitat, the subseafloor igneous crust, but still lack substantial understanding regarding its abundance, diversity and ecology. Here we describe a fossilized microbial consortium of prokaryotes and fungi at the basalt-zeolite interface of fractured subseafloor basalts from a depth of 240 m below seafloor (mbsf). The microbial consortium and its relationship with the surrounding physical environment are revealed by synchrotron-based X-ray tomographic microscopy (SRXTM), environmental scanning electron microscopy (ESEM), and Raman spectroscopy. The base of the consortium is represented by microstromatolites—remains of bacterial communities that oxidized reduced iron directly from the basalt. The microstromatolites and the surrounding basalt were overlaid by fungal cells and hyphae. The consortium was overgrown by hydrothermally formed zeolites but remained alive and active during this event. After its formation, fungal hyphae bored in the zeolite, producing millimetre-long tunnels through the mineral substrate. The dissolution could either serve to extract metals like Ca, Na and K essential for fungal growth and metabolism, or be a response to environmental stress owing to the mineral overgrowth. Our results show how microbial life may be maintained in a nutrient-poor and extreme environment by close ecological interplay and reveal an effective strategy for nutrient extraction from minerals. The prokaryotic portion of the consortium served as a carbon source for the eukaryotic portion. Such an approach may be a prerequisite for prokaryotic-eukaryotic colonisation of, and persistence in, subseafloor igneous crust.     

Carbon assimilating fungi from surface ocean to subseafloor revealed by coupled phylogenetic and stable isotope analysis

Fungi are ubiquitous in the ocean and hypothesized to be important members of marine ecosystems, but their roles in the marine carbon cycle are poorly understood. Here, we use ¹³C DNA stable isotope probing coupled with phylogenetic analyses to investigate carbon assimilation within diverse communities of planktonic and benthic fungi in the Benguela Upwelling System (Namibia). Across the redox stratified water column and in the underlying sediments, assimilation of ¹³C-labeled carbon from diatom extracellular polymeric substances (¹³C-dEPS) by fungi correlated with the expression of fungal genes encoding carbohydrate-active enzymes. Phylogenetic analysis of genes from ¹³C-labeled metagenomes revealed saprotrophic lineages related to the facultative yeast Malassezia were the main fungal foragers of pelagic dEPS. In contrast, fungi living in the underlying sulfidic sediments assimilated more ¹³C-labeled carbon from chemosynthetic bacteria compared to dEPS. This coincided with a unique seafloor fungal community and dissolved organic matter composition compared to the water column, and a 100-fold increased fungal abundance within the subseafloor sulfide-nitrate transition zone. The subseafloor fungi feeding on ¹³C-labeled chemolithoautotrophs under anoxic conditions were affiliated with Chytridiomycota and Mucoromycota that encode cellulolytic and proteolytic enzymes, revealing polysaccharide and protein-degrading fungi that can anaerobically decompose chemosynthetic necromass. These subseafloor fungi, therefore, appear to be specialized in organic matter that is produced in the sediments. Our findings reveal that the phylogenetic diversity of fungi across redox stratified marine ecosystems translates into functionally relevant mechanisms helping to structure carbon flow from primary producers in marine microbiomes from the surface ocean to the subseafloor.Subject terms: Microbial ecology, Fungal ecology, Microbiome, Biogeochemistry     

Fungal oxidative dissolution of the Mn(II)‐bearing mineral rhodochrosite and the role of metabolites in manganese oxide formation

Microbially mediated oxidation of Mn(II) to Mn(III/IV) oxides influences the cycling of metals and remineralization of carbon. Despite the prevalence of Mn(II)‐bearing minerals in nature, little is known regarding the ability of microbes to oxidize mineral‐hosted Mn(II). Here, we explored oxidation of the Mn(II)‐bearing mineral rhodochrosite (MnCO₃) and characteristics of ensuing Mn oxides by six Mn(II)‐oxidizing Ascomycete fungi. All fungal species substantially enhanced rhodochrosite dissolution and surface modification. Mineral‐hosted Mn(II) was oxidized resulting in formation of Mn(III/IV) oxides that were all similar to δ‐MnO₂ but varied in morphology and distribution in relation to cellular structures and the MnCO₃ surface. For four fungi, Mn(II) oxidation occurred along hyphae, likely mediated by cell wall‐associated proteins. For two species, Mn(II) oxidation occurred via reaction with fungal‐derived superoxide produced at hyphal tips. This pathway ultimately resulted in structurally unique Mn oxide clusters formed at substantial distances from any cellular structure. Taken together, findings for these two fungi strongly point to a role for fungal‐derived organic molecules in Mn(III) complexation and Mn oxide templation. Overall, this study illustrates the importance of fungi in rhodochrosite dissolution, extends the relevance of biogenic superoxide‐based Mn(II) oxidation and highlights the potential role of mycogenic exudates in directing mineral precipitation.     

Bacterial dominance in subseafloor sediments characterized by methane hydrates

The degradation of organic carbon in subseafloor sediments on continental margins contributes to the largest reservoir of methane on Earth. Sediments in the Andaman Sea are composed of ~ 1% marine-derived organic carbon and biogenic methane is present. Our objective was to determine microbial abundance and diversity in sediments that transition the gas hydrate occurrence zone (GHOZ) in the Andaman Sea. Microscopic cell enumeration revealed that most sediment layers harbored relatively low microbial abundance (10(3)-10(5) cells cm(-3)). Archaea were never detected despite the use of both DNA- and lipid-based methods. Statistical analysis of terminal restriction fragment length polymorphisms revealed distinct microbial communities from above, within, and below the GHOZ, and GHOZ samples were correlated with a decrease in organic carbon. Primer-tagged pyrosequences of bacterial 16S rRNA genes showed that members of the phylum Firmicutes are predominant in all zones. Compared with other seafloor settings that contain biogenic methane, this deep subseafloor habitat has a unique microbial community and the low cell abundance detected can help to refine global subseafloor microbial abundance.     

The igneous oceanic crust – Earth's largest fungal habitat?

In recent years the igneous oceanic crust has been recognized as a substantial microbial habitat and a scientific frontier within Geology, Biology, and Oceanography. A few successful metagenomic investigations have indicated the presence of Archaea and Bacteria, but also fungi in the subseafloor igneous crust. A comprehensive fossil record supports the presence of fungi in these deep environments and provides means of investigating the fungal presence that complements metagenomic methods. Considering the vast volume of the oceanic crust and that it is the largest aquifer on Earth, we put forward that it is the largest fungal habitat on the planet. This review aims to introduce a yet unexplored fungal habitat in an environment considered extreme from a biological perspective. We present the current knowledge of fungal abundance and diversity and discuss the ecological role of fungi in the igneous oceanic crust.     

Zinc Sorption During Bio-oxidation and Precipitation of Manganese Modifies the Layer Stacking of Biogenic Birnessite

The formation and structural evolution of fungal mediate biogenic birnessite are dynamic processes. Although the associations of Zn with the pre-formed biogenic Mn oxides are relatively well understood, the reactivity of the intermediate precipitate at the initial stage of Mn bio-oxidation appears to differ from the final precipitate. In the present work, Zn sorption during precipitation of biogenic Mn oxides was investigated contrasting Zn sorption to pre-formed biogenic Mn oxides, using the Mn-oxidizing fungus Paraconiothyrium sp. WL-2. A substantially higher Zn uptake was found during precipitation of biogenic Mn oxides compared to Zn sorption to pre-formed biogenic Mn oxides. The presence of Zn during Mn oxidation resulted in a biogenic Mn oxide with reduced ordering in the c-axis. The precipitate was identified by X-ray diffraction (XRD) as a layer-type Mn oxide with structural properties similar to hexagonal birnessite. Extended X-ray absorption fine structure (EXAFS) spectroscopy showed that Zn forms triple-corner-sharing tetrahedral coordination (^IVTCS-Zn) complexes on the surface of birnessite, which may inhibited layer stacking of birnessite in the final products. This study emphasizes the importance of the intermediate precipitates on Zn sorption, and provides insight regarding the dynamic interaction between Zn and Mn oxide in the process of microbiological oxidation. Supplemental materials are available for this article. Go to the publisher's online edition of Geomicrobiology Journal to view the supplemental file.     

Fossilized Life in Subseafloor Ultramafic Rocks

Ultramafic rocks are hypothesized to support a subseafloor hydrogen-driven biosphere because of extensive production of bioavailable energy sources like H₂ or CH₄ from fluid-rock interactions. Hence, the apparent lack of microbial remains in subseafloor ultramafic rocks, in contrast to their frequent observation in subseafloor basalts, is somewhat of a paradox. Here we report fossilized microbial remains in aragonite veins in ultramafic rocks from the 15°20′N Fracture Zone area on the Mid-Atlantic Ridge (MAR), collected during Ocean Drilling Program (ODP) Leg 209. The microbial remains consist of filamentous structures associated with biofilms. The young age (<1 Myr) and absence of diagenesis result in fossilized microbial communities with a pristine composition characterized by carbonaceous matter (CM) and the enrichment in trace elements such as Ni, Co, Mo and Mn. Our study confirms the presence of the hypothesized deep subseafloor biosphere hosted in ultramafic rocks. We further show that host rock composition may influence the microbial elemental composition, which is recorded during the fossilization.     

Microbial Community Stratification Controlled by the Subseafloor Fluid Flow and Geothermal Gradient at the Iheya North Hydrothermal Field in the Mid-Okinawa Trough (Integrated Ocean Drilling Program Expedition 331)

The impacts of lithologic structure and geothermal gradient on subseafloor microbial communities were investigated at a marginal site of the Iheya North hydrothermal field in the Mid-Okinawa Trough. Subsurface marine sediments composed of hemipelagic muds and volcaniclastic deposits were recovered through a depth of 151 m below the seafloor at site C0017 during Integrated Ocean Drilling Program Expedition 331. Microbial communities inferred from 16S rRNA gene clone sequencing in low-temperature hemipelagic sediments were mainly composed of members of the Chloroflexi and deep-sea archaeal group. In contrast, 16S rRNA gene sequences of marine group I Thaumarchaeota dominated the microbial phylotype communities in the coarse-grained pumiceous gravels interbedded between the hemipelagic sediments. Based on the physical properties of sediments such as temperature and permeability, the porewater chemistry, and the microbial phylotype compositions, the shift in the physical properties of the sediments is suggested to induce a potential subseafloor recharging flow of oxygenated seawater in the permeable zone, leading to the generation of variable chemical environments and microbial communities in the subseafloor habitats. In addition, the deepest section of sediments under high-temperature conditions (∼90°C) harbored the sequences of an uncultivated archaeal lineage of hot water crenarchaeotic group IV that may be associated with the high-temperature hydrothermal fluid flow. These results indicate that the subseafloor microbial community compositions and functions at the marginal site of the hydrothermal field are highly affected by the complex fluid flow structure, such as recharging seawater and underlying hydrothermal fluids, coupled with the lithologic transition of sediments.     

Production of Biogenic Manganese Oxides by Anamorphic Ascomycete Fungi Isolated from Streambed Pebbles

We characterized the production of biogenic Mn oxides by four anamorphic ascomycete fungi isolated from streambed pebbles with Mn oxide coatings. Based on the 18S rRNA gene sequences, one strain was related to members of the order Xylariales and the other three were within distinct lineages of the Pleosporales. These strains oxidized Mn(II) to deposit Mn oxides when their growth approached the stationary phase. The fungal Mn oxides showed X-ray diffraction patterns typical of poorly crystalline vernadite (δ -MnO₂), and X-ray absorption near-edge structure spectroscopy confirmed that the Mn phases consisted predominantly of Mn(IV). Mn(II) oxidation in the four strains proceeded enzymatically. The Mn(II)-oxidizing proteins were inhibited by azide and o-phenanthroline, and the proteins also oxidized typical laccase substrates including 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), showing the role of laccase or a laccase-like metalloenzyme. The mineralogical traits of the biogenic Mn oxides, and the participation of laccase-like enzymes, are in accordance with our previous results obtained with one Hypocreales ascomycete. In conclusion, phylogenetically diverse ascomycetes may use this common enzymatic system to produce solid Mn phases similar to δ -MnO₂.     

Fungal manganese oxidation in a reduced soil

Manganese chemistry in soils is a function of complex, competing biotic and abiotic reactions. The role of soil-borne fungi in mediating these reactions is poorly understood. The objective of this article is to document direct observation of fungal Mn oxidation in soil under near in situ conditions, and to isolate, describe and confirm the role of fungi in the observed Mn oxidation, and present a model to explain our observations. We incubated soil under different moisture contents in sample cells designed to allow us to use synchrotron microspectroscopic techniques to analyse areas as small as 38x40 microm2. Mn was redistributed and accumulated in distinct small circular shapes or in dendritic patterns near the air-soil interface when water-saturated soil was incubated for >or=7 days. Mn oxidation did not occur at 3 or 52 degrees C indicating that oxidation was caused by microbial activity. Mn-oxidizing fungi were isolated from the sample cells and cultured on agar. Reinoculation of sterile soil with the Mn-oxidizing isolates resulted in the formation of Mn oxides around fungal hyphae. A model to describe the distinct zonal distribution of Mn oxides in the sample cells is presented. We believe that our data are the first direct observation of Mn oxidation by soil-inhabiting fungi under in situ conditions. Mn-oxidizing fungi may play an underappreciated role in the cycling of Mn in soils.     

Identification of Mn(II)-Oxidizing Bacteria from a Low-pH Contaminated Former Uranium Mine

Biological Mn oxidation is responsible for producing highly reactive and abundant Mn oxide phases in the environment that can mitigate metal contamination. However, little is known about Mn oxidation in low-pH environments, where metal contamination often is a problem as the result of mining activities. We isolated two Mn(II)-oxidizing bacteria (MOB) at pH 5.5 (Duganella isolate AB_14 and Albidiferax isolate TB-2) and nine strains at pH 7 from a former uranium mining site. Isolate TB-2 may contribute to Mn oxidation in the acidic Mn-rich subsoil, as a closely related clone represented 16% of the total community. All isolates oxidized Mn over a small pH range, and isolates from low-pH samples only oxidized Mn below pH 6. Two strains with different pH optima differed in their Fe requirements for Mn oxidation, suggesting that Mn oxidation by the strain found at neutral pH was linked to Fe oxidation. Isolates tolerated Ni, Cu, and Cd and produced Mn oxides with similarities to todorokite and birnessite, with the latter being present in subsurface layers where metal enrichment was associated with Mn oxides. This demonstrates that MOB can be involved in the formation of biogenic Mn oxides in both moderately acidic and neutral pH environments.     

Biogenic Manganese Oxides Facilitate Iodide Oxidation at pH ≤ 5

Radioactive ¹²⁹I, a byproduct of nuclear power generation, can pose risks to human health if released into the environment, where its mobility is highly dependent on speciation. Based on thermodynamic principles, ¹²⁹I should exist primarily as iodide (I^?) in most terrestrial environments; however, organo-¹²⁹I and ¹²⁹iodate are also commonly detected in contaminated soils and groundwater. To investigate the capability of biogenic manganese oxides to influence iodide speciation, 17 manganese-oxidizing bacterial strains, representing six genera, were isolated from soils of the Savannah River Site, South Carolina. The isolates produced between 2.6 and 67.1 nmole Mn oxides (ml^?1 media after 25 days, pH 6.5). Results from inhibitor assays targeting extracellular enzymes and reactive oxygen species indicated that both play a role in microbe-induced Mn(II) oxidation among the strains examined. Iodide oxidation was not observed in cultures of the most active Mn-oxidizing bacteria, Chryseobacterium sp. strain SRS1 and Chromobacterium sp. strain SRS8, or the fungus, Acremonium strictum strain KR21–2. While substantial amounts of Mn(III/IV) oxides were only generated in cultures at ≥pH 6, iodide oxidation was only observed in the presence of Mn(III/IV) oxides when the pH was ≤5. Iodide oxidation was promoted to a greater extent by synthetic Mn(IV)O₂ than biogenic Mn(III/IV) oxides under these low pH conditions (≤pH 5). These results indicate that the influence of biogenic manganese oxides on iodide oxidation and immobilization is primarily limited to low pH environments.     

Geomicrobiology in oceanography: microbe-mineral interactions at and below the seafloor

Oceanography is inherently interdisciplinary and, since its inception, has included the study of microbe-mineral interactions. From early studies of manganese nodules, to the discovery of hydrothermal vents, it has been recognized that microorganisms are involved at various levels in the transformation of rocks and minerals at and below the seafloor. Recent studies include mineral weathering at low temperatures and microbe-mineral interactions in the subseafloor "deep biosphere". A common characteristic of seafloor and subseafloor geomicrobiological processes that distinguishes them from terrestrial or near-surface processes is that they occur in the dark, one or more steps removed from the sunlight that fuels the near-surface biosphere on Earth. This review focuses on geomicrobiological studies and energy flow in dark, deep-ocean and subseafloor rock habitats.     

Fungal diversity from communities to genes

Fungi are hyperdiverse organisms and assemble in complex communities, characterized by high levels of species richness, turnover, and endemism. However, the origins and maintenance of such high diversity and the role environments play in fungal adaptation are still elusive. Traditionally, efforts to understand fungal diversity in their environment have been divided between studies at the species level and below species level, with separate disciplines such as community ecology and population genetics working independently and with little communication. Here I argue that linking these different approaches is required to fully document the diversity of fungi in nature. Understanding the patterns and mechanisms of fungal diversity and composition requires not only the study of species assemblies and ranges, but also relies on comprehending fungal intra-specific variation, dispersal and establishment, including identifying key traits influencing fitness. This implies better integration and cross-fertilization between disciplines addressing fungi at a multitude of biological levels, ranging from genes to whole communities. Such approach will yield direct links between variation, adaptation and environments and provide a much more comprehensive understanding of fungal diversity.     

Exploration of cultivable fungal communities in deep coal‐bearing sediments from ∼1.3 to 2.5 km below the ocean floor

Although subseafloor sediments are known to harbour a vast number of microbial cells, the distribution, diversity, and origins of fungal populations remain largely unexplored. In this study, we cultivated fungi from 34 of 47 deep coal‐associated sediment samples collected at depths ranging from 1289 to 2457 m below the seafloor (mbsf) off the Shimokita Peninsula, Japan (1118 m water depth). We obtained a total of 69 fungal isolates under strict contamination controls, representing 61 Ascomycota (14 genera, 23 species) and 8 Basidiomycota (4 genera, 4 species). Penicillium and Aspergillus relatives were the most dominant genera within the Ascomycetes, followed by the members of genera Cladosporium, Hamigera, Chaetomium, Eutypella, Acremonium, Aureobasidium, Candida, Eurotium, Exophiala, Nigrospora, Bionectria and Pseudocercosporella. Four Basidiomycota species were identified as genera Schizophyllum, Irpex, Bjerkandera and Termitomyces. Among these isolates, Cladosporium sphaerospermum and Aspergillus sydowii relatives were isolated from a thin lignite coal‐sandstone formation at 2457 mbsf. Our results indicate that these cultivable fungal populations are indigenous, originating from past terrigenous environments, which have persisted, possibly as spores, through ～20 million years of depositional history.     

Phylogenetic and enzymatic diversity of deep subseafloor aerobic microorganisms in organics- and methane-rich sediments off Shimokita Peninsula

"A meta-enzyme approach" is proposed as an ecological enzymatic method to explore the potential functions of microbial communities in extreme environments such as the deep marine subsurface. We evaluated a variety of extra-cellular enzyme activities of sediment slurries and isolates from a deep subseafloor sediment core. Using the new deep-sea drilling vessel "Chikyu", we obtained 365 m of core sediments that contained approximately 2% organic matter and considerable amounts of methane from offshore the Shimokita Peninsula in Japan at a water depth of 1,180 m. In the extra-sediment fraction of the slurry samples, phosphatase, esterase, and catalase activities were detected consistently throughout the core sediments down to the deepest slurry sample from 342.5 m below seafloor (mbsf). Detectable enzyme activities predicted the existence of a sizable population of viable aerobic microorganisms even in deep subseafloor habitats. The subsequent quantitative cultivation using solid media represented remarkably high numbers of aerobic, heterotrophic microbial populations (e.g., maximally 4.4 x 10(7) cells cm(-3) at 342.5 mbsf). Analysis of 16S rRNA gene sequences revealed that the predominant cultivated microbial components were affiliated with the genera Bacillus, Shewanella, Pseudoalteromonas, Halomonas, Pseudomonas, Paracoccus, Rhodococcus, Microbacterium, and Flexibacteracea. Many of the predominant and scarce isolates produced a variety of extra-cellular enzymes such as proteases, amylases, lipases, chitinases, phosphatases, and deoxyribonucleases. Our results indicate that microbes in the deep subseafloor environment off Shimokita are metabolically active and that the cultivable populations may have a great potential in biotechnology.     

Geomicrobiology of manganese(II) oxidation

Mn(II)-oxidizing microbes have an integral role in the biogeochemical cycling of manganese, iron, nitrogen, carbon, sulfur, and several nutrients and trace metals. There is great interest in mechanistically understanding these cycles and defining the importance of Mn(II)-oxidizing bacteria in modern and ancient geochemical environments. Linking Mn(II) oxidation to cellular function, although still enigmatic, continues to drive efforts to characterize manganese biomineralization. Recently, complexed-Mn(III) has been shown to be a transient intermediate in Mn(II) oxidation to Mn(IV), suggesting that the reaction might involve a unique multicopper oxidase system capable of a two-electron oxidation of the substrate. In biogenic and abiotic synthesis experiments, the application of synchrotron-based X-ray scattering and spectroscopic techniques has significantly increased our understanding of the oxidation state and relatively amorphous structure (i.e. delta-MnO(2)-like) of biogenic oxides, providing a new blueprint for the structural signature of biogenic Mn oxides.