Fig. 5

Thermodynamically feasible envelopes for the production of a acetate, b pyruvate, and c formate as a function of consumed Fe³⁺ as the external electron acceptor. Horizontal lines denote the measured amount of acetate and formate, respectively. Both lines intersect with the thermodynamically feasible envelope suggested by the model. Acetyl-CoA competes with iron for electrons, thus resulting in a reduction of maximum acetate production with increasing Fe³⁺ consumption. At lower Fe³⁺ consumption, the electron transport chain is not sufficient to meet the required ATP demands, thus making acetate production essential. Increasing Fe³⁺ uptake increases the flux through the methylotrophic, and consequently, the electron transport chain. At a Fe³⁺ uptake of 0.28 mol/mol methane, thermodynamic limitations restrict pyruvate production, thereby resulting in a reduced sensitivity of minimum acetate to Fe³⁺ uptake. A Fe³⁺ uptake of 0.6 mol/mol methane is sufficient to meet the ATP demands using the electron transport chain alone, thus decreasing the minimum required acetate production to zero. Since CO₂ was not observed as a secreted product, the end product of the methylotrophic pathway was hypothesized to be formate produced by abiotic deformylation of formylmethanofuran, which competes with acetate and pyruvate for carbons causing the maximum pyruvate production to decline when Fe³⁺ uptake surpasses 0.6 mol/mol methane. However, the minimum formate production still remains zero due to the fact that formate can be oxidized to produce CO₂ for acetate and pyruvate production. Beyond a Fe³⁺ uptake of 2.3 mol/mol methane, the thermodynamics of the system do not favor the production of acetate, thus making formate and pyruvate production mandatory