Isotopic labelling tests

For each of the duplicate tests, a subsample of bioreactor biomass (250 mL) was transferred anaerobically to a 330 mL glass vessel. The biomass was flushed with a mixed gas (90% CH₄, 5% CO₂, and 5% N₂) for 10 min. Approiximately 10 mL ¹³C-labelled methane (Sigma-Aldrich, 99 atom % ¹³C, USA) and ~0.03 g ⁵⁷Fe-labelled ferrihydrite were added to each vessel on Day 34. Each vessel was pressurised to 1.2 atm through injecting helium. Each test lasted for ~110 days.

During the tests, Fe(III) and Fe(II) in liquid and solid phases were sampled every 3–7 days and measured for their ⁵⁷Fe(III)/⁵⁶Fe(III), ⁵⁴Fe(III)/⁵⁶Fe(III), ⁵⁷Fe(II)/⁵⁶Fe(II) and ⁵⁴Fe(II)/⁵⁶Fe(II) ratios. After the ⁵⁷Fe(III)/⁵⁶Fe(III) ratio reached a steady state (Supplementary Figure 2), methane in gas phase, CO₂ in gas, liquid and solid phases, Fe(III)/Fe(II) in liquid and solid phases were sampled every week and measured for their concentrations and the respective isotopic fractions.

Total methane, Fe(III) and Fe(II) and their consumption/production rates were calculated as described in section “mass- and electron balance tests”. Total CO₂ was quantified by considering the amounts in gas, liquid and solid phases, and the profile of CO₂ was used to determine its production rate (rCO₂) via linear regression. The amounts of methane, CO₂, Fe(III) and Fe(II) and their respective isotopic fractions were used to calculate the amounts of ¹³CH₄, ¹³CO₂, ⁵⁷Fe(III), and ⁵⁷Fe(II). The profiles of ¹³CH₄, ¹³CO₂, ⁵⁷Fe(III) and ⁵⁷Fe(II) were used to determine the consumption rates of ¹³CH₄ (r¹³CH₄) and ⁵⁷Fe(III) (r⁵⁷Fe(III)), and the production rates of ¹³CO₂ (r¹³CO₂) and ⁵⁷Fe(II) (r⁵⁷Fe(II)) via linear regression.