Figure 1: [A] traditional aerobic fermentation process, where glucose is partially oxidized to CO2 to provide metabolic energy and the remainder is used as carbon source for biosynthesis. [B] alternative ‘closed carbon loop’ process where co-fed, CO2-derived formic acid serves as energy source and glucose uniquely serves as carbon source.
To illustrate these process benefits, we postulate the following typical microbial stoichiometry for aerobic conversion of glucose into biomass:
\begin{equation} C_{6}H_{12}O_{6}+2.85\ O_{2}+0.6\ \text{NH}_{3}\ \ \rightarrow\ 3\ CH_{1.8}O_{0.5}N_{0.2}+\ 3\ \text{CO}_{2}+\ 4.2\ H_{2}\text{O\ \ \ \ \ }\left[eq.3\right]\nonumber \\ \end{equation}
which gives a yield of biomass on O2(Yxo) of 1.05 C-molx/molo2, a yield of biomass on sugar (Yxs) of 3 C-molx/mols, and 1 C-mol of biomass formed per mol of CO2 released (Yxc). Under the assumptions of no energetic costs in cross membrane metabolite transport, and a P/O ratio of 1.0 for respiration of NAD(P)H the catabolic subreaction:
\begin{equation} 0.475\ C_{6}H_{12}O_{6}+2.85\ O_{2}\ \rightarrow\ 2.85\ \text{CO}_{2}+\ 2.85\ H_{2}O\ (+\ 7.6\ ATP)\ \ \ \ \ \left[eq.4\right]\nonumber \\ \end{equation}
can be completely replaced by dissimilation of formic acid:
\begin{equation} 7.6\ \text{CH}_{2}O_{2}+\ 3.8\ O_{2}\rightarrow\ 7.6\ \text{CO}_{2}+\ 7.6\ H_{2}O\ (+\ 7.6\ ATP)\ \ \ \ \ \left[eq.5\right]\nonumber \\ \end{equation}
resulting in the overall stoichiometry:
\begin{equation} {0.\ 525\ C}_{6}H_{12}O_{6}+7.6\ \text{CH}_{2}O_{2}+\ 3.8\ O_{2}+0.6\ \text{NH}_{3}\rightarrow\ 3\ CH_{1.8}O_{0.5}N_{0.2}+\ 7.75\ \text{CO}_{2}+\ 8.95\ H_{2}\text{O\ \ \ \ \ }\left[eq.6\right]\ \nonumber \\ \end{equation}
which gives a Yxo of 0.79 C-molx/molo2, a Yxs of 5.71 C-molx/mols, and a Yxc of 0.39 C-molx/molco2. Clearly, the Yxs is higher, but Yxo and Yxc are lower, which is undesired. However, combining the electrocatalytical reaction of [eq.1] with [eq.6] shows the synergy of the two processes:
\begin{equation} {0.\ 525\ C}_{6}H_{12}O_{6}+0.6\ \text{NH}_{3}\text{\ \ }\ 3\ CH_{1.8}O_{0.5}N_{0.2}+\ 0.15\ \text{CO}_{2}+\ 1.35\ H_{2}\text{O\ \ \ \ \ }\left[eq.7\right]\ \nonumber \\ \end{equation}
This overall stoichiometry gives an infinitely high Yxo, a Yxs of 5.71 C-molx/mols, and a Yxcof 20 C-molx/molco2. All three yields are improved relative to [eq.3]. Note that the O2produced in [eq.1] will be produced in a separate unit operation from the fermentation process where O2 is consumed so even though the overall process does not consume O2, aeration of the fermentation is still required. The O2produced in the reduction of CO2 can be used to intensify the fermentation process by injecting pure O2or enriching the fermentation air (Groen et al. , 2005).
Applying this theoretical concept to Yarrowia lipolytica as a model strain, two factors that impact the overall yield are the mechanisms for formic acid transport (passive vs. active) and the overall stoichiometry of NADH dissimilation by the respiratory chain (P/O ratio). Both passive diffusion of formic acid, as well as anion/proton-symport have been described in earlier research in the yeast S. cerevisiae (Overkamp et al. , 2002; Geertmanet al. , 2006), and neither of these mechanisms results in a net expenditure of ATP in transport of formic acid. Moreover, metabolic modelling studies in cultures with Penicillium chrysogenum , grown on mixtures of formic acid and glucose, also indicated no ATP expenditure in formic acid transport (Harris et al. , 2007). In light of these observations and since no data is reported on formic acid uptake in Y. lipolytica , no ATP expenditure for formic acid uptake was expected in this organism.
In Y. lipolytica the mitochondria contain a branched respiratory chain, constituted by the classic internal, proton pumping Complex I and an alternative, external NADH dehydrogenase (Kerscher et al., 1999), combined with the other classical mitochondrial complexes (III and IV) involved in electron transport from NADH. Complex I and the alternative NADH dehydrogenase provide two entry points for NADH-derived electrons into the respiratory chain. Since proton pumping by Complex I contributes to the proton gradient across the mitochondrial membrane whereas the alternative NADH dehydrogenase does not, the overall stoichiometry (P/O ratio and the equivalent ATP/NADH yield) differs depending on the entry point used. The physiological contribution of Complex I and alternative NADH dehydrogenase(s) remains enigmatic (Juergens et al. , 2020 & 2021), which impedes accurate theoretical prediction of the ATP yield of aerobic substrate dissimilation. Therefore, the optimal molar ratio between glucose and formic acid in the feed, which is the ratio where formic acid is exactly sufficient to replace glucose dissimilation, must be determined experimentally.
In addition to the physiology of Y. lipolytica , practical success of the proposed approach requires (1) a technologically and economically feasible process to capture CO2 and reduce it to formic acid, and (2) an industrially relevant fermentation process design in which the formic acid does not accumulate to a level that affects cell metabolism. The former requirement, capture and conversion of CO2 to formic acid, has been addressed elsewhere (see e.g. Claassens et al. , 2019; Malkhandi et al. , 2019; Pérez-Gallent et al. , 2021) and is out of scope of this work. This study covers the latter requirement for the industrially important yeast species Yarrowia lipolytica for which formic acid consumption has been previously demonstrated (Nsoe et al. , 2018; Vartiainen et al. , 2019).