Organic-rich diatomaceous ooze was reacted with seawater and a Na-Ca-K-Cl fluid of seawater chlorinity at 325–400°C, 400–500 bars, and fluid/sediment mass ratios of 1.56–2.35 to constrain factors regulating the abundance and stable isotope composition of C and S species during hydrothermal alteration of sediment from Guaymas Basin, Gulf of California. Alteration of inorganic and organic sedimentary components resulted in extensive exchange reactions, the release of abundant H₂S, CO₂, CH₄, and C_organic, to solution, and recrystallization of the sediment to an assemblage containing albitic plagioclase, quartz, pyrrhotite, and calcite.

The δ³⁴S_cdt values of dissolved H₂S varied from −10.9 to +4.3‰ during seawater-sediment interaction at 325 and 400°C and from −16.5 to −9.0‰ during Na-Ca-K-Cl fluid-sediment interaction at 325 and 375°C. In the absence of seawater SO₄, H₂S is derived from both the transformation of pyrite to pyrrhotite and S released during the degradation of organic matter. In the presence of seawater SO₄, reduction of SO₄ contributes directly to H₂S production. Sedimentary organic matter acts as the reducing agent during pyrite and SO₄ reduction. Requisite acidity for the reduction of SO₄ is provided by Mg fixation during early-stage sediment alteration and by albite and calcite formation in Mg-free solutions.

Organically derived CH₄ was characterized by δ13Cpdb values ranging between −20.8 and −23.1‰, whereas δ¹³C_pdb values for dissolved C_organic ranged between −14.8 and −17.7%. Mass balance calculations indicate that δ¹³C values for organically derived CO₂ were ≥ − 14.8%. Residual solid sedimentary organic C showed small (≤ 0.7‰) depletions in ¹³C relative to the starting sediment.

The experimental results are consistent with the isotopic and chemical composition of natural hydrothermal fluids and minerals at Guaymas Basin and permit us to better constrain sources and sinks for C and S species in subseafloor hydrothermal systems at sediment-covered spreading centers. Our data show that the sulfur isotope composition of hydrothermal Sulfide minerals in Guaymas Basin can be explained by derivation of S from diagenetic sulfide and seawater sulfate. Basaltic S may also contribute to hydrothermal sulfide precipitates but is not required to explain their isotopic composition. Estimates of seawater/ sediment mass ratios based on sulfur isotopic composition of sulfide minerals and the abundance of dissolved NH₃ in vent fluids range from 3–29 during hydrothermal circulation. Sources of C in Guaymas Basin hydrothermal fluids include thermal degradation of organic matter, bacteriogenic methane production, and dissolution of diagenetic carbonate.