| Summary: | Methane and carbon dioxide are one of the most important greenhouse gases and significant components of the carbon cycle. Biogeochemical methane transformation may occur even in the extreme conditions of deep subsurface ecosystems. This study presents methane-related biological processes in saline sediments of the Miocene Wieliczka Formation, Poland. Rock samples (W2, W3, and W4) differed in lithology (clayey salt with veins of fibrous salt and lenses of gypsum and anhydrite; siltstone and sandstone; siltstone with veins of fibrous salt and lenses of anhydrite) and the accompanying salt type (spiza salts or green salt). Microbial communities present in the Miocene strata were studied using activity measurements and high throughput sequencing. Biological activity (i.e., carbon dioxide and methane production or methane oxidation) occurred in all of the studied clayey salt and siltstone samples but mainly under water-saturated conditions. Microcosm studies performed at elevated moisture created more convenient conditions for the activity of both methanogenic and methanotrophic microorganisms than the intact sediments. This points to the fact that water activity is an important factor regulating microbial activity in saline subsurface sediments. Generally, respiration was higher in anaerobic conditions and ranged from 36 ± 2 (W2<sub>200%t.w.c</sub>) to 48 ± 4 (W3<sub>200%t.w.c</sub>) nmol CO<sub>2</sub> gdw<sup>−1</sup> day<sup>−1</sup>. Methanogenic activity was the highest in siltstone and sandstone (W3, 0.025 ± 0.018 nmol CH<sub>4</sub> gdw<sup>−1</sup> day<sup>−1</sup>), while aerobic methanotrophic activity was the highest in siltstone with salt and anhydrite (W4, 220 ± 66 nmol CH<sub>4</sub> gdw<sup>−1</sup> day<sup>−1</sup>). The relative abundance of CH<sub>4</sub>-utilizing microorganisms (<i>Methylomicrobium</i>, <i>Methylomonas</i>, <i>Methylocystis</i>) constituted 0.7–3.6% of all taxa. Methanogens were represented by <i>Methanobacterium</i> (0.01–0.5%). The methane-related microbes were accompanied by a significant number of unclassified microorganisms (3–64%) and those of the <i>Bacillus</i> genus (4.5–91%). The stable isotope composition of the CO<sub>2</sub> and CH<sub>4</sub> trapped in the sediments suggests that methane oxidation could have influenced δ<sup>13</sup>C<sub>CH4</sub>, especially in W3 and W4.
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