**3. Results and Discussion**

The Mössbauer spectra of Oil Pool and Jen's Vent 1 hot spring sediments are shown in Figure 2 and the fitting results are listed in Table 2. Samples from Jen's Vent 1 showed two fitted doublets with chemical isomer shifts (IS) of 0.30 mm/s and 1.06 mm/s, and quadrupole splitting (QS) of 0.60 mm/s and 1.99 mm/s, respectively. The doublet with small IS and QS values was assigned to low-spin Fe2<sup>+</sup> in pyrite [66], while that with large IS and QS values was assigned to high-spin Fe2<sup>+</sup> on the lattice of clay minerals. Although Fe2<sup>+</sup> in pyrite has similar hyperfine parameters to those of Fe3<sup>+</sup> on the lattice of ferric iron oxides, detailed SEM observations and previous geochemical measurements (Table 1; [41,55]) confirmed the existence of pyrite rather than ferric iron oxides. The parameters in agreement with those of pyrite and Fe2<sup>+</sup> in silicates [67,68] suggest that iron in Jen's Vent1 mainly existed as Fe2<sup>+</sup> in pyrite and Fe2<sup>+</sup>-bearing silicates. For the Oil Pool, two doublets with similar parameters were also observed, but showed the high level of Fe2<sup>+</sup> in pyrite (96.65%).

**Figure 2.** Room temperature 57Fe Mössbauer spectra of low-spin Fe2<sup>+</sup> in pyrite and high-spin Fe2<sup>+</sup> on the lattice of silicates (Jen's Vent 1 and Oil Pool).


**Table 2.** Mössbauer spectroscopic parameters of hot spring sediments.

a. IS = isomer shift. b. QS = quadrupole splitting.

Eh-pH diagrams were plotted based on geochemical data of Jen's Vent 1 hot springs (83 ◦C, activity [Fe2+] = 10<sup>−</sup>6.7, [SO4 2-] = 10<sup>−</sup>2.8, according to [65]) which show the thermodynamic stabilities of sulfur species with the current geochemical conditions of these hot springs (Figure 3). It can be seen that the influences of the temperature and ion concentrations on thermodynamic equilibrium in the hot springs studied were insignificant. The electrochemical potentials of Kamchatka hot springs (Table 1) favor the stability of Fe(II) in silicates and pyrite, which is consistent with the Mössbauer spectroscopic results. If only ideal thermodynamic equilibrium is taken into account (Figure 3b), elemental sulfur seems unable to exist in the current springs. However, elemental sulfur was commonly observed in the sediments. The SEM observations showed that some of the elemental sulfur crystals were irregular (Figure 4a) and needed confirmation by EDS analysis (Figure 4b), while others could only be detected based on EDS microanalysis (Figure 4c,d). We also observed a small number of monoclinic sulfur crystals with well-developed crystal faces (Figure 4e) that were chemically confirmed by EDS analysis (Figure 4f). They imply that conditions in favor of sulfur deposition have existed previously.

**Figure 3.** Eh-pH diagrams calculated using Geochemist's Workbench illustrating [69]: (**a**) Eh, pH, temperature and estimated ion concentrations in Kamchatka hot springs Jen's Vent 1, Vent 2, Zavarzin and Burlyashii (83 ◦C, Activities: Fe2<sup>+</sup> = 10<sup>−</sup>6.715, SO4 <sup>2</sup><sup>−</sup> = 10<sup>−</sup>2.796); (**b**) The thermodynamic stability of elemental sulfur under the current geochemical conditions (83 ◦C, Activity: SO4 <sup>2</sup><sup>−</sup> = 10<sup>−</sup>2).

**Figure 4.** Scanning electron microscope (SEM) images (**a**,**c**,**e**) and energy-dispersive X-ray spectroscopy (EDS)-measured chemical composition of element sulfur (**b**,**d**,**f**) in different morphologies observed in Kamchatka hot springs. The signal of Pd in the spectrum of (**b**) should be ignored because it was from the instrumental background.

Anaerobic chemoorganoheterotrophic and chemolithoautotrophic bacteria and archaea have been identified and isolated from the Kamchatka hot springs [43,56,70]. Thermophilic sulfate-reducing bacteria (e.g., *Thermoanaerobacterium aciditolerans*), sulfur-reducing bacteria (e.g., *Thermanaerovibrio velox*), sulfur-reducing archaea (e.g., *Thermoproteus uzoniensis, Thermoplasmatales*), along with the other thermophilic microorganisms, build up a biological system that interplays with the geochemical system in those hot springs [43,61,71]. Besides those bacterial sulfur redox processes, *Thermoanaerobacter* *ethanolicus* and *Carboxydocella manganica* sp. nov. isolated from the hot springs in Kamchatka can reduce Fe(III) for respiration [70,72].

The coexistence of pyrite crystals with a variety of sizes and morphologies were observed and characterized in all the studied hot spring sediments. The size of single euhedral crystals is in a range from ~100 nm to ~40 μm, with various crystal habits including cubic {100}, pyritohedral {210}, octahedral {111}, pyritohedral {310} forms, and their combinations. Irregular or spherical aggregates of pyrite crystals appearing loosely or tightly were also common in the sediments. Most pyrite crystals showed smooth surfaces, while rough surfaces were observed on pyrite covered by clay minerals, organic matter, or the even finer pyrite nanocrystals. However, the framboidal structure of pyrite that is very common in sedimentary rocks and modern marine or lake sediments [19,29,73] was absent in the hot spring sediments. Below are some detailed descriptions of crystal habits.
