Population structure of manganese-oxidizing bacteria in stratified soils and properties of manganese oxide aggregates under manganese-complex medium enrichment

Abstract

Manganese-oxidizing bacteria in the aquatic environment have been comprehensively investigated. However, little information is available about the distribution and biogeochemical significance of these bacteria in terrestrial soil environments. In this study, stratified soils were initially examined to investigate the community structure and diversity of manganese-oxidizing bacteria. Total 344 culturable bacterial isolates from all substrata exhibited Mn(II)-oxidizing activities at the range of 1 µM to 240 µM of the equivalent MnO2. The high Mn(II)-oxidizing isolates (>50 mM MnO2) were identified as the species of phyla Actinobacteria, Firmicutes and Proteobacteria. Seven novel Mn(II)-oxidizing bacterial genera (species), namely, Escherichia, Agromyces, Cellulomonas, Cupriavidus, Microbacterium, Ralstonia, and Variovorax, were revealed via comparative phylogenetic analysis. Moreover, an increase in the diversity of soil bacterial community was observed after the combined enrichment of Mn(II) and carbon-rich complex. The phylogenetic classification of the enriched bacteria represented by predominant denaturing gradient gel electrophoresis bands, was apparently similar to culturable Mn(II)-oxidizing bacteria. The experiments were further undertaken to investigate the properties of the Mn oxide aggregates formed by the bacterial isolates with high Mn(II)-oxidizing activity. Results showed that these bacteria were closely encrusted with their Mn oxides and formed regular microspherical aggregates under prolonged Mn(II) and carbon-rich medium enrichment for three weeks. The biotic oxidation of Mn(II) to Mn(III/IV) by these isolates was confirmed by kinetic examinations. X-ray diffraction assays showed the characteristic peaks of several Mn oxides and rhodochrosite from these aggregates. Leucoberbelin blue tests also verified the Mn(II)-oxidizing activity of these aggregates. These results demonstrated that Mn oxides were formed at certain amounts under the enrichment conditions, along with the formation of rhodochrosite in such aggregates. Therefore, this study provides insights into the structure and diversity of soil-borne bacterial communities in Mn(II)-oxidizing habitats and supports the contribution of soil-borne Mn(II)-oxidizing bacteria to Mn oxide mineralization in soils.

Figure 1. Phylogenetic relationship between the 16S rRNA gene sequences from the soil isolates with high Mn(II)-oxidizing activities (labeled with “□”) and their closest GenBank sequences with 16S rRNA gene from the known Mn(II)-oxidizing bacterial strains (labeled with “▪”) reported previously.

The GenBank accession numbers of these sequences are shown in brackets. Bootstrap values ≥50% with 1,000 replicates are indicated at the branch points.

Figure 2. DGGE analysis of bacterial 16S rRNA V3 genes amplified from the total community DNA of the untreated soil (represented by A, B, and C labeled in lanes) and Mn (II)/carbon-rich complex medium-enriched soil (represented by 0, 1, and 10 mM labeled in lanes; from different depths; 0, 1, and 10 mM are the Mn(II) concentration).

A: DGGE profile of the original soil from A-, B-, and C-horizon soils; B, C, and D: DGGE profile of Mn(II)-enriched soil from A-, B-, and C-horizon soils, respectively. Denaturant gradients of 50% to 70%, 45% to 65%, 45% to 55%, and 40% to 50% were used for the optimal separation of the products for A, B, C, and D, respectively. The numbers on the gels are the bands that were excised and sequenced corresponded to the list in Table S1.

Figure 3. Time course of the Mn(II)-oxidizing activities and the cell growth of several isolates from A-, B-, and C-layer soils.

The cells were grown in liquor K medium for 144(OD₆₀₀) and the concentration of Mn oxides were determined according to the procedures described in the “Materials and Methods” section. (○) Optical density of the cells (at 600 nm); (▪) Concentration of Mn oxides; (□) pH. A: Non-Mn(II)-oxidizing E. coli JM109 (as the negative control). B: A86. C: A101. D: B84. E: C19. F: C13.

Figure 4. SEM images of the mixture of bacteria and Mn oxides as well as EDX spectra of the selected areas.

In SEM, A101, B84, and C92 were the isolates from the A-, B-, and C-layer substrata, respectively, illustrating the formation of Mn oxide aggregates; C13 represents an SEM image of an isolate having no capability to produce Mn oxide aggregates. Two scanning areas for EDX analysis in the SEM images of A101, B84, and C92 were indicated by a, b, and arrows, respectively. A single scan was indicated for C13.

Figure 5. XRD patterns (A, B, and C) and LBB tests (D) of Mn oxides from different depths of soils and Mn oxides from different Mn(II)-oxidizing bacteria.

The experiments were performed using dried powdered Mn oxide samples. In A, B, and C, the red dashed lines indicate the overlapping peaks; In C, a HEPES buffer and a synthesized rhodochrosite sample were used as the negative controls.