*3.4. Spherulite Pyrite Crystals*

Pyrite spherules were observed in sediments of all hot springs (Figure 10). The size of pyrite spherules is usually around 500 nm to 1 μm, aggregated either loosely (Figure 10a) or tightly (Figure 10b). Their assemblages are different from framboidal pyrite, which is made of more euhedral pyrite nanocrystals arranged in a particular order [20]. Framboidal pyrites dominate the crystal forms in black shales (a hydrothermal environment without biologically induced pyrite mineralization, e.g., [76–78]). However, no pyrite framboid was observed in any of the studied hot springs. All of these pyrite spherules are covered by a relatively thick crust of clay rich in organic matter, as indicated by EDS measurements (Figure 10c–f).

**Figure 10.** SEM images and the EDS result of spherulitic pyrite crystals that are characterized by biofilm covering materials (**a**–**e**), aggregated either loosely (**a**) or tightly (**b**). (**f**) is the EDS profile of the pyrite crystal in (**e**).

In summary, the diversity of pyrite crystal habits described in this study was found to be much higher than that of authigenic pyrites in deep-sea hydrothermal vents [41,79]. This diversity has been preserved in those small hot springs for >40,000 years [52–54]. The record of the complex crystallographic features over such a long time reflects secular interactions between the continuous supply of energy and nutritional elements by the active hot springs and the metabolisms of the microbial communities.

We can consider a complex assemblage of pyrite forms as the reflection of the interactions between the microbial communities with their geochemical environments, even though there is no direct record of a biologically mediated mineralization. The complex pyrite crystal habits is coincident with the thriving of microbial communities in the Kamchatka volcanic hot springs through time. Further evaluation of the complexity may develop links between the microbial physiology (e.g., energy metabolism, metal respiration, and nutritional cycles) and the evolution of iron sulfide mineralogy.

This complex set of pyrite crystals may also be a useful reference for the identification of biogenic iron sulfides on Mars. Potential Martian biosignatures using crystal morphology and traits include the deposition of the digenetic apatite 'flowers' as the result of the biological cycle of phosphorus [48], single-domain magnetite formed at low temperatures [80–82], etched pits on the surface of crystals that have cell characteristics [83], abnormally tiny crystal sizes (~2–10 nm) [6,81], unusual crystal lengths in one or more dimensions [84,85], and mineral casts or encrustations preserving biological characteristics [6,85,86]. The formation of a jarosite-goethite-gypsum assemblage was considered to be the result of oxidation of pyrite [87]. The Chemistry and Mineralogy (CheMin) x-ray diffraction (XRD) analyses confirmed the existence of pyrite in mudstone at John Klein by Curiosity rover [88]. However, detailed observations of pyrite crystal on Mars have been limited by in situ measurement methods so far. Some of researchers have tried to look for clues in Marian meteorites. Euhedral pyrite crystals have not often been observed in many meteorites [89,90]. Pyrites in Northwest Africa (NWA) 7533 are cubic or octahedral crystals with average grains sizes of 30–40 μm [91]. Euhedral octahedral pyrite crystals (~50 μm) were observed in Marian meteorite NWA 7475 [92]. These assemblages of pyrite are not as complex as those we observed in the Kamchatka hot springs and their possible hydrothermal genesis [91] may have made the difference. Putative microbial activity in the hot spring environments on Mars in the distant past [93,94] might also have formed similar complex pyrite deposits. Due to the lack of dynamic geological activity and the freezing temperatures on Mars, the microstructures of any complex pyrite deposits in the near-subsurface sediments could be well-preserved for multiple billions of years. However, to fully understand the validity of a well-preserved pyrite complex on Mars, further experiments will be required. Such experiments will need to concentrate on the stability of complex pyrite deposits under various environmental conditions, such as burial, desiccation, heating, and other processes that are likely to have occurred throughout Martian history. Therefore, this presents a new avenue in the search for signs of ancient biota on Mars, and these pyrite complexes should be added to the list of potential signatures of Martian life.
