Custom-made medium approach for effective enrichment and isolation of chemolithotrophic iron-oxidizing bacteria

Abstract

Chemolithotrophic neutrophilic iron (Fe)-oxidizing bacteria, which mainly belong to the family Gallionellaceae, universally prevail in terrestrial environments changing Fe cycling. However, they are typically recognized as difficult-to-culture microbes. Despite efforts, there are few Fe(II)-oxidizing lithotroph isolates; hence, their physiological and ecological knowledge remains limited. This limitation is largely owing to difficulties in their cultivation, and we hypothesize that the difficulty exists because substrate and mineral concentrations in the cultivation medium are not tuned to a specific environmental condition under which these organisms live. To address this hypothesis, this study proposes a novel custom-made medium approach for chemolithotrophic Fe(II)-oxidizing bacteria; a method that manipulates medium components through diligent analysis of field environment. A new custom-made medium simulating energy substrates and nutrients under the field condition was prepared by modifying both chemical composition and physical setup in the glass-tube medium. In particular, the modification of the physical setup in the tube had a significant effect on adjusting dissolved Fe(II) and O2 concentrations to the field environment. Using the medium, Gallionellaceae members were successfully enriched and a new Gallionellaceae species was isolated from a natural hot spring site. Compared with conventional medium, the custom-made medium has significantly higher ability in enriching Gallionellaceae members.

Keywords: Gallionellaceae; custom-made medium; enrichment; iron-oxidizing bacteria; isolation.

Figure 1.

(A) Sampling location in the Sambe hot spring site and (B) Fe precipitates with a soft structure at the spot 2.

Figure 2.

(A) Schematic figure showing medium Types 1 and 2. (B) Schematic figure of medium Type 3. (C) Photograph of the Types 1, 2, and 3 after an abiotic 7 days incubation. A white arrow in (C) indicates a distinct Fe(III) band typically observed in the conventional medium Type 1.

Figure 3.

(A) Pie chart showing the bacterial community at family level in the Fe precipitate collected at the Sambe spot 2 based on 16S rRNA (V3–V4 region) amplicon sequencing. (B) Pie chart at genus level in Gallionellaceae of (A). (C) Phylogenetic tree of Gallionellaceae ASVs (dominance in the whole bacterial community > 0.1%) found in the Sambe spot 2 and strains UT4 and UT5 based on Neighbor-Joining method. Leptothrix mobilis was chosen as the out group. In (C), the percentage value for each ASV represents the percentage in the bacterial community (not in the Gallionellaceae community).

Figure 4.

Depth profiles of dissolved O₂ and Fe(II) in the abiotic incubation with Type 1 (A), Type 2 (B) and Type 3 (C). Gray areas in figures indicate the on-site O₂ and Fe(II) levels. Brown box in the figure indicates the Fe(II)-oxidizer's habitat (Fe(III) deposit) layer expected in each medium type. In the figures, error bars represent 1 standard deviation from the mean.

Figure 5.

Gallionellaceae/Bacteria ratios after the first enrichment (14 days incubation) with medium Types 1–3 estimated by the qPCR with Gallionellaceae and Bacteria specific primers. The 10⁻⁴ to 10⁻⁶ diluted Fe precipitates were inoculated to the media.

Figure 6.

Growth curves (A) and total Fe(III) curves (B) for strains UT4 and UT5 cultivated in medium Type 3 with FeS plug at 28°C. Abiotic control (no-inoculation) was shown in (B). In (A) and (B), error bars represent 1 standard deviation from the mean. (C) Fluorescence images of UT4 and UT5 cells (left side) and phase contrast images (right side) of the same field of fluorescence images.

Figure 7.

Phylogenomic tree of the family Gallionellaceae. The genus-level clades were indicated based on the GTDB taxonomic classification, except for the genus Ferrigenium. The strains UT4 and UT5 were indicated in bold. Values of 1000 replicates for SH-aLRT (left, %) and ultrafast bootstrap (right, %) were indicated at the nodes (<50% are not shown). The sequences of Nitrosomonas mobilis Ms1 (GCF_900103035.1), Nitrosomonas cryotolerans ATCC 49181 (GCF_900143275.1), and Nitrosospira multiformis ATCC 25196 (GCF_000196355.1) were used as the outgroup (not shown).