Mineralogical and Genomic Constraints on the Origin of Microbial Mn Oxide Formation in Complexed Microbial Community at the Terrestrial Hot Spring

Abstract

Manganese (Mn) oxides are widespread on the surface environments of the modern Earth. The role of microbial activities in the formation of Mn oxides has been discussed for several decades. However, the mechanisms of microbial Mn oxidation, and its role in complex microbial communities in natural environments, remain uncertain. Here, we report the geochemical, mineralogical, and metagenomic evidence for biogenic Mn oxides, found in Japanese hot spring sinters. The low crystallinity of Mn oxides, and their spatial associations with organic matter, support the biogenic origin of Mn oxides. Specific multicopper oxidases (MCOs), which are considered Mn-oxidizing enzymes, were identified using metagenomic analyses. Nanoscale nuggets of copper sulfides were, also, discovered in the organic matter in Mn-rich sinters. A part of these copper sulfides most likely represents traces of MCOs, and this is the first report of traces of Mn-oxidizing enzyme in geological samples. Metagenomic analyses, surprisingly, indicated a close association of Mn oxides, not only in aerobic but also in anaerobic microbial communities. These new findings offer the unique and unified positions of Mn oxides, with roles that have not been ignored, to sustain anaerobic microbial communities in hot spring environments.

Keywords: anaerobes; biogenic Mn oxides; hot spring; metagenome; multicopper oxidase.

Figure 1

(A) Location of Hachikuro hot spring in Japan. (B) Relative locations of the sampling points in the hot spring stream. (C) Fe-(hydro)oxide sinters near the venting site. (D) Mn-oxide layers, beneath the cyanobacterial mat and aragonite layers at Site 1. (E) View of the mid-stream area (Site 2). (F) Mn-rich sample (HKm) at Site 2. Primary carbonate layers (orange part) are brecciated, with development of Mn oxides (black part).

Figure 2

SEM image of Mn oxides and elemental distribution. (A) Well-faceted crystals represent aragonite and submicron spheres represent Mn oxides, in this image. (B) Back-scattered image of the cross section of a Mn-oxide sphere. Mn-oxide spheres show concentric inside structures, made of chemically distinct layers. (C–F) The distribution of Mn, C, O, and Ca, respectively. Bright layers in the back-scattered image (B) corresponds to MnO₂ layers (C,E). Dark layers are made of organic matter, which was suggested because Ca (F) and O (E) were not detected.

Figure 3

HR-TEM images of Mn oxides. (A) TEM image, (B) lattice image, and (C) diffraction pattern (HAADF) of Mn oxides in HKs-Mn. Based on HAADF, this crystal is identified as δ-MnO₂ (vernadite). (D) TEM image, (E) lattice image, and (F) diffraction pattern (HAADF) of Mn oxides in HKm. Based on HAADF, this crystal is identified as hausmannaite (Mn²⁺Mn³⁺₂O₄). (A,D) do not show clear crystal structures. A part of lattices of Mn oxides are captured in (B,E), but most parts do not show clear lattices. HAADF images of both samples are ringed, suggesting X-ray diffraction from multiple weakly crystalline crystals.

Figure 4

Community structure based on 16S rRNA of (A) HKs-Mn, (B) HKs-Fe, (C) HKm, and (D) HKd. Red color indicates anaerobes. The outer pie chart represents phylum level and the inner pie chart represents class level.

Figure 5

Cu-S nano-nuggets in MnO₂/Organic complex. (A) FE-SEM image showing distribution of nanoscale Cu-S nuggets in MnO₂/Organic complex structure (HKm). Yellow arrows indicate Cu- and S-bearing nuggets in organic matter. (B) TEM image showing Cu-S nuggets in organic matter and (C–F) distribution of S, Cu, C, and Ca, respectively. The spots of Cu are corresponded to those of S (yellow arrows in (D,E), but dark image in (F)).