Carbon fixation assays.

Chemosynthetic and photosynthetic carbon fixation was measured for the Australian soil microcosm samples by tracing incorporation of ¹⁴C-labeled CO₂. Gaseous ¹⁴CO₂ (1% [vol/vol]) was generated by mixing 15 μl of radiolabeled sodium bicarbonate solution (NaH¹⁴CO₃, 56.6 mCi nmol⁻¹; Perkin Elmer) with equal volumes of 10% HCl solution in a sealed 5-ml glass vial and incubating it for 2 h at room temperature. Samples (0.5 g) of either live soil or heat-killed controls (autoclaved at 121°C and 15 lb/in² for 60 min) were collected from each microcosm at each sampling point (T1 to T4 and C1 to C4). They were transferred to sterile 5-ml glass vials sealed with rubber septa. Each vial was injected with ¹⁴CO₂ (1% [vol/vol]) gas to achieve a final initial headspace concentration of 400 ppmv, which otherwise comprised ambient air. The samples were incubated for 24 h at 28°C in technical quadruplicate under four conditions: (i) light plus ¹⁴CO₂, (ii) light plus ¹⁴CO₂ + 100 ppmv of H₂, (iii) dark plus ¹⁴CO₂, and (iv) dark plus ¹⁴CO₂ plus 100 ppmv of H₂. Samples incubated under dark conditions were covered with aluminum foil prior to incubation, while those incubated under light conditions were incubated under a light box. Unfixed ¹⁴CO₂ was removed by transferring soils to 10-ml scintillation vials and suspending them in 2 ml of 10% acetic acid in ethanol. Samples were left to dry in a chemical fume hood at ambient temperature for 48 h. Approximately 10 ml of scintillation cocktail (EcoLume) was added and ¹⁴C counts were measured with a liquid scintillation counter (Tri-Carb 2810 TR; Perkin Elmer) operating at 93% efficiency. Background luminescence and chemiluminescence were corrected through internal calibration standards and by including an additional background vial. Background scintillation counts were subtracted from sample counts prior to calculations. The amount of ¹⁴CO₂ fixed per sample was quantified as the mass of ¹⁴CO₂ per mass of soil per time unit (picomoles per gram per day).