273 projects under construction. It is generally recognized that one of the difficulties involved in the initiation of CCS projects is to find an appropriate geological layer with high permeability overlain by an impermeable geological layer that does not intersect with major faults and/or fractured shear zones, which could lead to the leakage of injected CO2. Hence, it is of paramount importance in ascertaining such geological layers and monitoring injected CO2, but this can lead to a delay and/or an increase in the cost. In this regard, abandoned deep underground mines have a great potential to store CO2, as an alternative to such ordinary geological layers used for CCS, when the host rock is composed of ultramafic rocks. Recent studies have revealed that carbonate minerals are precipitated when such mafic rocks have a contact with carbonic water, due to the chemical reaction between carbonate ions and those released from the rock (Cao et al., 2024). However, such experiments are predominantly performed under high temperature ranges more than 100℃. Under low temperature ranges, the chemical interaction proceeds at a slow pace. This can be a possible obstacle to the implementation of CCS in abandoned deep hard rock mines. Hence, the present study aims to enhance the CO2 mineralization efficiency with microbial enzyme, considering its application to ultramafic rocks in deep hard rock mines. 2. Method Basalt and peridotite are used for the experiment. The basalt and peridotite rocks were collected from quarries located in Nagasaki prefecture and Hokkaido, Japan, respectively. According to XRF analyses, the basalt contains 2.48% of Mg, 4.73% of Ca, 13.85% of Fe, whilst the peridotite contains 24.09% of Mg, 0.28% of Ca, and 6.23% of Fe. The rocks were first fragmented with a hammer, which were then powdered with a mortar. The experimental strains, Bacillus subtilis 168 (JCM 10629) was obtained from the Japan Collection of Microorganisms (RIKEN BRC, Tsukuba, Japan). Lysogeny Broth (LB) medium, consisting of 1% (m/v) tryptone, 0.5% (m/v) yeast extract, and 1% (m/v) NaCl, was used as the culture medium, adjusted to a pH of 7.0–7.3. Cultivation was performed at 37°C. Thereafter, microbial solution containing carbonic anhydrase (CA) was obtained by centrifuging the microbial suspension at 8000 rpm for 10 minutes. CA is a microbial enzyme that catalyzes the generation of bicarbonate ions when CO2 is dissolved in the water. Sealing tests were performed for the following three conditions: Case-1) the rock powders and water are put in a plastic container, which is then placed in a high-pressure vessel. After creating an anaerobic condition and injecting CO2 under 9 MPa, the vessel is sealed and kept at room temperature for one week, Case-2) instead of water, the microbial solution containing CA is used, Case-3) First, the mixed solution is put into the vessel in the same way as Case-1, and sealed. Then, after the elapse of one week, the microbial solution is added to the solution under an aerobic condition, followed by one-week sealing. Table 1 – Experimental conditions Case Rock mass (g) Water (g) Microbial solution Duration Temp 1 5 5 N/A 1 Room 2 1 N/A 9 2 37℃
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