The aragonite nanoneedles are immobilized on stable TiO2 ceramic20. For comparison, we also evaluated the bactericidal action of unmodified TiO2 ceramic and calcite nanocrystals immobilized on TiO2 ceramic against E. coli K-12. The bactericidal evaluations were based on two experimental procedures. In the first procedure, water loaded with E. coli K-12 was poured into a petri dish containing aragonite-modified (or calcite-modified) TiO2 ceramic and maintained for a maximum of 16 h under static and agitated conditions. The second experiment involved a closed circulation system, used to simulate actual conditions, featuring a TiO2 ceramic monolith–packed Pyrex glass tube (300 mm × 10 mm (internal diameter)). In this system, we observed a decrease in the number of E. coli K-12 cells in water as a function of circulation time. The E. coli K-12 cells on the surface of aragonite-modified (or calcite-modified) TiO2 ceramic were observed by confocal laser scanning microscopy.
Calcium carbonate accumulates on the TiO2 ceramic surface, as shown in Fig. 1, during the long-term circulation of mineral water containing calcium bicarbonate under UV irradiation. Mineral water containing only calcium bicarbonate produces a precipitate characterized by hexagonal crystals, while mineral water (such as Evian® and Contrex®) containing magnesium and strontium ions along with calcium bicarbonate produces a precipitate characterized by acicular crystals (Fig. 2). The X-ray diffraction (XRD) and laser-Raman spectra in Fig. 3, respectively, reveal that the crystal structures of the hexagonal and acicular calcium carbonate are calcite and aragonite, respectively.
Acicular aragonite crystals do not form on the TiO2 ceramic surface when water is circulated over the photocatalyst in the absence of UV irradiation. Whereas, under UV irradiation, bicarbonate ions are converted to carbonate ions by photocatalysis to form insoluble acicular calcium carbonate, i.e., aragonite, on the TiO2 ceramic surface. The lengths of the acicular crystals range from 10 nm to a few micrometers, depending on the duration of circulation and the concentration of calcium bicarbonate in the mineral water. It is expected that the large (micrometer) acicular crystals will capture the bacteria in a water-flow system, while the small (sub-micrometer) acicular crystals exert a mechano-bactericidal effect.
The changes in the number of E. coli K-12 cells in an aqueous phase over time are shown in Fig. 4. The number of E. coli K-12 cells does not change significantly in a saline water system. In a system containing unmodified TiO2 ceramics, the number of E. coli K-12 cells decreases slightly. Finally, in a system containing aragonite-modified TiO2, the number of E. coli K-12 cells decreases remarkably. The decrease in the number of bacterial cells under static conditions indicates that E. coli K-12 is captured by acicular aragonite owing to its own motor function, and the improvement in the antibacterial action achieved by agitation is less significant than the antibacterial action associated with the motor function of the bacteria.
Figure 5 display SEM images of the surfaces of unmodified, calcite-modified, and aragonite-modified TiO2 ceramics that were immersed in saline water containing E. coli K-12 for 16 h. The distinctive shapes of E. coli K-12 cells on the unmodified (Fig. 5 (1)) and calcite-modified TiO2 ceramic surfaces (Fig. 5 (2) and (2’)) do not change after 16 h; in contrast, the distinct shapes of E. coli K-12 cells are absent on the aragonite-modified TiO2 ceramic surfaces. In Figs. 5(3) and 5(3'), the cell membrane was observed to be stretched and leathery. In Fig. 5(3"), a situation was observed in which a substance that appeared to be protoplasm was ejected from the stab wounds of E. coli.
Figure 6 show images of live/dead E. coli K-12 cells on naked TiO2 ceramic (a) and aragonite-modified TiO2 ceramics (b) obtained by confocal microscopy after 16 h in an E. coli K-12 solution under static condition (5 × 103 CFU/mL). The green color indicates viable cells, and the red color indicates dead cells. The number of cells (dead and alive) on the aragonite-modified TiO2 ceramic surface was much smaller than the number of cells on the unmodified TiO2 ceramic surface. However, the small number of dead cells observed suggests that the nucleic acid, which is stained with a fluorescent dye, eluted into the aqueous phase through the punctured cell wall (impaled by acicular aragonite: as shown in Fig. 5(3”)); this loss of nucleic acid (and fluorescent marker) accounts for the low incidence of cells (dead) observed by confocal microscopy on the aragonite-modified TiO2 ceramic surface.
The fate of bacteria captured by nanoneedle structures has already been reported. Hazel et al. and Jenkins et al. found that nanoneedles penetrate the cell membranes of bacteria11, 25, resulting in sterilization. Wu et al. considered the relationship between the length of the nanoneedle that penetrates the bacterial-cell wall and the inter-needle distance9. They reported that the stretching of the cell membrane increases with the increasing density of the nanoneedles. Their experiment investigated the bacteria at the gas–solid interface; however, we expect that the mechano-bactericidal mechanism at the gas–solid interface should be similar to that at the liquid–solid interface in this study because we observed bacteria impaled on the acicular aragonite.
Therefore, we investigated the mechano-bactericidal performance of acicular aragonite with different nanoneedle sizes. As shown in Fig. 7, the mechano-bactericidal performance of acicular aragonite was dependent on the size of the nanoneedles. That is, the mechano-bactericidal action of acicular aragonite characterized by small needles with a length and diameter of 1–2 and 0.05–0.1 µm, respectively, is high; while that of acicular aragonite characterized by larger needles with a length and diameter of 2–6 and 0.2–0.6 µm, respectively, is low. This needle-size dependence of mechano-bactericidal performance is consistent with the results of earlier studies for the gas-sold phase7, 9, 11, 17, 21.
Figure 8 reveals the mechano-bactericidal action in circulation systems. Unmodified, calcite-modified, and aragonite-modified TiO2 ceramics were packed into Pyrex glass tubes (300 mm × 10 mm (internal diameter)) and water containing E. coli K-12 (5 × 103 CFU/mL) were circulated through these tubes at a rate of 50 mL/min. Figures 3a, 3b, and 3c show images of the live/dead E. coli K-12 cells on the surfaces of the unmodified, calcite-modified, and aragonite-modified TiO2 ceramics, respectively, after circulating the aqueous phase for 3 h. A significant amount of viable E. coli K-12 cells is observed on the surface of the unmodified TiO2 ceramics (Fig. 8 (a)). The total number of bacterial cells on the surface of the calcite-modified TiO2 ceramics (Fig. 8 (b)) is less than that on the surface of the unmodified TiO2 ceramics; however, both viable and dead bacteria are observed. As shown in Fig. 8 (c), very few dead bacterial cells are observed on the surface of the aragonite-modified TiO2 ceramics, despite the presence of viable bacteria; this result is consistent with the result obtained under static conditions (Fig. 6).
The most significant finding of this research is the aqueous mechano-bactericidal action of aragonite consisting of calcium carbonate. Almost all of the reported nanoneedle, nanopillar, and whisker-shaped materials exhibit mechano-bactericidal action. However, it is known that asbestos, potassium titanate, carbon nanotubes, and metal nanowires demonstrate lung toxicity due to oxidative stress induced by their shape22–24. Acicular aragonite consists of nanoneedle-shaped crystals as well and its associated toxicity is still subject to further study. However, acicular aragonite does not show toxicity toward lung tissue, unlike the aforementioned nanoneedle, nanopillar, and whisker-shaped materials18. Calcium carbonate, which is the main component of aragonite, easily dissolves in the human body and, as a result, morphology-induced toxicity does not manifest. In the course of water treatment through the mechano-bactericidal action of aragonite-modified TiO2 ceramics, we must assume the defluxion of nanoneedles into the water as a result of fracturing. Unlike body-soluble acicular aragonite, nanoneedle, nanopillar, and whisker-shaped nanomaterials are unsuitable for drinking water treatment because of the risk of fragment effluence.
We have already mentioned the possibility of damage to acicular aragonite during water treatment; however, it is also expected that the acicular aragonite will recover/self-repair in natural water flow. More specifically, the self-replication ability of the system, and its mechano-bactericidal action, is anticipated. In fact, we found that acicular aragonite nucleates from dead E. coli K-12 cells during long-term circulation (Fig. 9).
Photocatalytic environmental purification can only be performed during the daytime and, for optimum performance, under sunny conditions26–28. However, ideal photocatalytic drinking-water purification systems for developing countries should achieve absolute performance under all weather conditions, not only sunny but also cloudy or rainy; possibly by combining photocatalytic activity and mechano-bactericidal action. Figure 10 shows the change in number of E. coli K-12 cells in circulation systems featuring tubes containing unmodified and aragonite-modified TiO2 ceramics under dark and UV light conditions. The number of E. coli K-12 cells in the unmodified TiO2 ceramic system decreases significantly more under UV-A irradiation than dark conditions due to photocatalysis29, 30. However, the number of E. coli K-12 cells in the aragonite-modified TiO2 ceramic system under dark conditions is lower than that in the unmodified TiO2 ceramic system under UV-A irradiation. Significantly, the absolute rate of E. coli K-12 cell reduction in the aragonite-modified TiO2 ceramic system under UV-A irradiation (Sterilization rate const. = 1.22 h− 1) was ~ 2.0 times of under dark (Reduction rate const. = 0.809 h− 1), and ~ 3.0 times of the UV light conditions in the unmodified TiO2 ceramic system (Photocatalytic rate const. = 0.618 h− 1).
In a previous study, we revealed why the photocatalytic activity of TiO2 does not decrease with increasing aragonite accumulation on its surface19. The photocatalytic reaction mainly involves the formation and migration of active species, such as •OH, by UV irradiation of the TiO2 photocatalyst. The lifetime of •OH, of around 2.7 µs38, is considered sufficient to allow it to migrate into the aqueous phase through the layer of aragonite. The densification of the aragonite layer is expected to impede the migration of active species. In reality, the aragonite layer on the TiO2 surface is very porous and a few micrometers thick. This porosity facilitates the migration of the active species, generated by photocatalysis, to the surface of the aragonite layer. This is one of the reasons why the photocatalytic sterilization performance of the aragonite-modified TiO2 ceramic photocatalyst exceeds that of the unmodified TiO2 ceramic photocatalyst. Moreover, the mineralization of bacteria on the aragonite-modified TiO2 ceramic photocatalyst is promoted by photocatalysis; as a result, the surface of this material will maintain a clean condition unlike that of an aragonite system without photocatalytic materials.
As described above, we demonstrated the mechano-bactericidal treatment of water by acicular aragonite. However, several aspects require elucidation, such as the relationship between the optimum needle size of aragonite or flow speed and bactericidal performance, and the mechano-bactericidal action of acicular aragonite against other bacterial species. As previously reported, the TiO2 ceramic photocatalyst, which is used as a substrate for acicular aragonite growth, is extremely strong and does not deteriorate during long-term use. Therefore, it is expected that access to safe water can be achieved in many developing countries with systems that combine mechano-bactericidal action and photocatalysis, such as the proposed acicular aragonite-modified TiO2 ceramic system, that obviate the use of disinfectants and concomitant chemical risks and high running costs.