Abstract:Sulfur Cave (Puturosu Mountain, Romania) is an extreme environment, unique for displaying life in a gas chemocline. The lower part of the cave is filled with CO 2 , CH 4 , and H 2 S of mofettic origin, while the upper part contains air that floats above the heavier volcanic gasses. S° and H 2 SO 4 (from sulfur-oxidation) cover the cave wall at and below the CO 2 -H 2 S:O 2 gas/gas interface. On the cave wall, near the interface the pH is <1 and unusual microbial biofilms occur on the rock's surface. We provide context information on the geology, mineralogy, chemistry and biology to better understand this unique environment. We have used X-ray diffraction, optical microscopy, scanning electron microscopy with EDAX capabilities, stable isotope analysis and 16S and 18S rDNA amplicon sequencing. The most common taxa in the microbial biofilms are Mycobacteria, Acidithiobacillus and Ferroplasmaceae. Liquid water in this system originates solely from condensation of water vapor onto the cave walls making inflow of organic carbon from outside unlikely. The most likely primary source of energy for this microbial community is sulfur oxidation with H 2 S and S° as main reductants and atmospheric O 2 as the main oxidant. Ferric iron from the rock surface is another potential oxidant. In Sulfur Cave, gaseous CO 2 (from mofettic emission) maintains the stability of the gas chemocline. Sulfur Cave biofilms can help the search for extreme life in the subsurface, near volcanic systems on Earth and Mars. The Sulfur Cave example shows that a habitable environment can be established underground in gas chemoclines near CO 2 -dominated gas discharge zones, where it can have a steady supply of water and energy.

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