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).