In silico Analysis of Clostrdidium Acetobutylicum ATCC 824 Metabolic Response to an External Electron Supply

The main problems of the production of butanol using Clostridium spp. are the low yield due to the generation of several by-products and the toxic effects that this compound exerts on the producing microorganisms, which ultimately result in a low butanol volumetric productivity and titer.

Among the multiple variables that affect the production of butanol by solventogenic clostridia, the oxido-reduction potential (ORP) has been recognized as of major importance. Therefore, most of the strategies focussed on optimizing this process are directly or indirectly related with this variable, being the ultimate goal to redirect the carbon and electron flow from the production of acids and hydrogen to the production of butanol.

The ORP can be controlled by adding a chemical oxidant, and a reductant, to the culture medium. Also, electron carriers have shown to affect the intracellular ORP by deviating the electron flow. The process in which electrons from a cathode are utilized as co-reducing equivalents in addition to carbon source-derived reducing equivalents has been called electrofermentation, and offers an interesting approach to optimize the production of butanol by Clostridium spp.. It has been reported that an external electron supply, using or not a mediator,  increases the NADH/NAD⁺ ratio in solventogenic clostridia and the production of reduced compounds such as butanol.

This work makes use of the iCAC490 model of Clostridium acetobutylicum ATCC 824 and the Constrained Based Reconstruction Analysis (COBRA) toolbox to carry out a systematic in silico evaluation of the metabolic capabilities of C. acetobutylicum ATCC 824 and its response to intracellular redox changes, as a result of an electron uptake flux. Several analyses were conducted, which included robustness, production envelope and phenotypic phase planes. The optknock function was used to identify possible butanol-overproducing phenotypes. Overall, the results obtained qualitatively coincide with experimental results reported in literature. The model showed that the use of an external electron supply, which acts as co-reducing agent along with glucose-derived reducing power, results in an increase in the butanol specific productivity. However, a proportional increase in the butyrate uptake flux is required. Besides, the uptake of external butyrate leads to the coupling of butanol production and growth. Phenotypic phase planes of C. acetobutylicum ATCC 824 showed that the reducing capacity is a growth limiting resource when butanol is produced, which becomes more limiting at high butyrate uptake fluxes. An electron uptake flux allows the metabolism to reach the growth optimallity line until a butyrate uptake of 20 mmol gDW^-1 h^-1. Although the maximum butanol flux does not coincide with the growth optimallity line, a butyrate uptake combined with an electron uptake flux would result in an increased butanol volumetric productivity, which would be maximal within a butyrate uptake flux range between 20 and 30 mmol gDW^-1 h^-1. Based on the results obtained, the supplementation of butyrate to the culture medium arises as a potential strategy to optimize the production of butanol by C. acetobutylicum ATCC 824 under an electron flux supply.