Biofilm Formation and the Establishment of Anaerobic Environment on Excavation Damaged Zone Fractures in Deep Neogene Sedimentary Rock

17 Feb 2025

To dispose of radioactive waste in the deep subsurface, subsurface tunnels need to be excavated, which introduces O2 from the air into the deep geological environment. As the anoxic environment is favorable for disposing radioactive waste, microbial activity is expected to promote oxygen consumption after closing the subsurface tunnels by backfilling. Furthermore, microbial activity is associated with the corrosion of metal canisters and gas production by the decomposition of organic matter. In many countries, such microbial influences are therefore described in the Safety Case Report published by implementers (e.g., [1, 2]).

Microbial biofilms at excavation-damaged zone (EDZ) fractures were investigated by drilling from a 350-m-deep gallery and subsequent borehole logging at the Horonobe Underground Research Laboratory (URL). Horonobe URL is located in Horonobe town, Hokkaido, Japan, to enhance the reliability of technologies for radioactive waste disposal in Neogene sedimentary rocks. The previous study indicates that a reducing environment had been maintained in the EDZ around the Horonobe URL tunnels [3].

Using microscopic and spectroscopic techniques, the dense colonization of microbial cells was demonstrated at the surfaces of the EDZ fractures with high hydraulic conductivity. 16S rRNA gene sequence analysis revealed the dominance of gammaproteobacterial lineages, whose cultivated members are aerobic methanotrophs. Our results suggest that an aerobic environment is established in the EDZ fracture. This aerobic environment is hypothesized to be formed by microbial production of dark O2 [4, 5]. If dark O2 is present as dissolved species in groundwater, redox-sensitive radionuclides (e.g., U and Np) could be oxidation and mobilized. Consequently, it may be necessary for implementers to conduct a safety assessment on the production of dark O2 by subsurface microbes. The analysis results are presented below; for further details, refer to a published paper [4]. This study has been conducted as part of a joint research program between the Secretariat of the Nuclear Regulation Authority, the University of Toyo, and the Japan Atomic Energy Agency.

Biofilm Formation at EDZ Fractures

We characterized the frozen core samples in which a light brownish film extensively covered one of the EDZ fractures at ~36 cm (EDZ1 in Fig. 1a and 1b). Greenish signals from the film stained by SYBR-Green I was observed using a fluorescence stereo microscope (Fig. 1c and 1d). To exclude the possibility that greenish signals resulted from the introduction of microbial contamination during drilling and cutting with the diamond band saw, we observed the other sides of the core block after DNA staining (Fig. 1e). In contrast, greenish signals were not evident on the surfaces caused by drilling and cutting. This result is important for clarifying that the greenish signals were not derived from contamination. After the dispersion of the film detached from the fracture into the solution, the suspension was stained by SYBR-Green I and observed by a fluorescence microscope. The aggregation of coccoid cells was associated with the minor presence of rod-shaped cells (Fig. 1f and 1g).

$Figure 1 Result of observation on EDZ1 surface in the core sample [4]. Photographs of a half-split core sequence from ~0.3 to ~0.5 mabh (a), the fracture surface at EDZ1 on the lower block (b), and the fracture surface at EDZ1 created on the lower block by cutting with a band saw (c). Fluorescence stereo microscopic images of the fracture surface at EDZ1 from the top (d) and the side (e). The yellow arrow and yellow dotted line indicate the fracture at EDZ1 (a, e). Green and red arrows indicate the surfaces created by cutting with a band saw and drilling, respectively (e). Fluorescence microscopic images of the material detached from the fracture surface (f, g).$

Figure 1 Result of observation on EDZ1 surface in the core sample [4]. Photographs of a half-split core sequence from ~0.3 to ~0.5 mabh (a), the fracture surface at EDZ1 on the lower block (b), and the fracture surface at EDZ1 created on the lower block by cutting with a band saw (c). Fluorescence stereo microscopic images of the fracture surface at EDZ1 from the top (d) and the side (e). The yellow arrow and yellow dotted line indicate the fracture at EDZ1 (a, e). Green and red arrows indicate the surfaces created by cutting with a band saw and drilling, respectively (e). Fluorescence microscopic images of the material detached from the fracture surface (f, g).
(© This figure is licensed under CC BY 4.0.)

To confirm whether the greenish signals originated from microbial cells rather than materials strongly bound to SYBR-Green I and/or autofluorescence, the film attached to the fracture was directly analyzed by in situ infrared (IR) spectroscopy (Fig. 2). IR spectra characterized by two amide peaks at ~1530 and ~1640 cm−1 were highly similar to those obtained from microbial cultures. The amide I peak at ~1640 cm−1 is shifted in peak position due to the difference in chemical form, which is why the peaks are slightly shifted in the spectra from the fracture (Fig. 2).

$Figure 2 Submicron-scale spectroscopic analysis of an EDZ fracture (EDZ1) [4]. A photograph of the fracture surface (a), a counter map of the fracture surface highlighted with a yellow square based on the peak at 1530 cm−1 in optical photothermal infrared (O-PTIR) spectra (b). O-PTIR spectra of points 1 and 2 and cultured cells of Nanobdella aerobiophila strain MJ1T (=JCM33616T) and Metallosphaera sedula strain MJ1HA (=JCM33617) for an archaeal reference and Shewanella oneidensis strain MR-1 T (=ATCC 700550.T) for a bacterial reference (c). The peak assignment was based on Ellerbrock et al. and Movasaghi et al. [8, 9]. A blank spectrum was obtained from the surface created by cutting with a diamond band saw, especially where the cut surface intersected the EDZ fracture (indicated by the yellow arrow in (a)).$

Figure 2 Submicron-scale spectroscopic analysis of an EDZ fracture (EDZ1) [4]. A photograph of the fracture surface (a), a counter map of the fracture surface highlighted with a yellow square based on the peak at 1530 cm−1 in optical photothermal infrared (O-PTIR) spectra (b). O-PTIR spectra of points 1 and 2 and cultured cells of Nanobdella aerobiophila strain MJ1T (=JCM33616T) and Metallosphaera sedula strain MJ1HA (=JCM33617) for an archaeal reference and Shewanella oneidensis strain MR-1 T (=ATCC 700550.T) for a bacterial reference (c). The peak assignment was based on Ellerbrock et al. and Movasaghi et al. [8, 9]. A blank spectrum was obtained from the surface created by cutting with a diamond band saw, especially where the cut surface intersected the EDZ fracture (indicated by the yellow arrow in (a)).
(© This figure is licensed under CC BY 4.0.)

A light brownish film was also visible at an EDZ fracture at~39 cm next to EDZ1 (EDZ2). Greenish signals were observed at EDZ2 after SYBR-Green Ⅰ staining. The absence of greenish signals at the surfaces generated by cutting with the band saw and splitting the core into half excluded the possibility of microbial contamination during core processing. The in-situ IR spectroscopy also confirmed the spectra diagnostic of microbial cells at the EDZ2. These results clarified the biofilm formation at the EDZ fractures.

Taxonomic Profiling of Biofilm Communities at EDZ Fractures

16S rRNA gene amplicon analysis was performed for materials aseptically collected from EDZ1 and EDZ2 (Fig. 3), yielding 3119 and 2939 sequences, respectively. Both samples were dominated by members of the families Methylophilaceae and Methylomonadaceae, which include aerobic methanotrophs [6, 7]. Our results indicate that the presence of aerobic methanotrophs confirms the establishment of an aerobic environment in the EDZ fracture.

$Figure 3 Microbial community structures from biofilms on fractures named EDZ1 and EDZ2[4]. The phylum and class were classified by 16S rRNA gene sequences in the nomenclature based on the SILVA 138 database using QIIME2 software.$

Figure 3 Microbial community structures from biofilms on fractures named EDZ1 and EDZ2[4]. The phylum and class were classified by 16S rRNA gene sequences in the nomenclature based on the SILVA 138 database using QIIME2 software.
(© This figure is licensed under CC BY 4.0.)

References

[1] NUMO (2021) The NUMO Pre-siting SDM-based safety case. NUMO-TR-21–01

[2] Posiva SKB (2017) Safety functions, performance targets and technical design requirements for a KBS-3V repository. Posiva SKB Report 01.

[3] Mochizuki A, Ishii E, Miyakawa K, Sasamoto H (2020) Mudstone redox conditions at the Horonobe Underground Research Laboratory, Hokkaido, Japan: Effects of drift excavation. Engineering Geology. 267 (20): 105496_1–105496_11.

[4] Hirota, A., Kouduka, M., Fukuda, A. et al. Biofilm Formation on Excavation Damaged Zone Fractures in Deep Neogene Sedimentary Rock. Microb Ecol 87, 132 (2024). https://doi.org/10.1007/s00248-024-02451-7

[5] Ruff SE, Humez P, de Angelis IS et al (2023) Hydrogen and dark oxygen drive microbial productivity in diverse groundwater ecosystems. Nat Commun 14:3194.

[6] Chistoserdova L, Kalyuzhnaya MG, Lidstrom ME (2009) The expanding world of methylotrophic metabolism. Annu Rev Microbiol 63:477–499.

[7] Kalyuzhnaya MG, Gomez OA, Murrell JC (2019) The methane-oxidizing bacteria (methanotrophs). In: McGenity T (ed) Taxonomy, Genomics and Ecophysiology of Hydrocarbon-Degrading Microbes. Handbook of Hydrocarbon and Lipid Microbiology, Springer, Cham, pp 1–34.

[8] Ellerbrock R, Stein M, Schaller J (2022) Comparing amorphous silica, short-range-ordered silicates and silicic acid species by FTIR. Sci Rep 12(1):11708.

[9] Movasaghi Z, Rehman S, Rehman UI (2008) Fourier transform infrared (FTIR) spectroscopy of biological tissues. Appl Spectrosc Rev 43(2):134–179.

Contact

Akinari Hirota
NRA
hirota_akinari_ni7@nra.go.jp