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Energy & Environmental Science DOI: 10.1039/C6EE01108J

Energy & Environmental Science Accepted Manuscript

ARTICLE

Ethanol production in syngas-fermenting Clostridium ljungdahlii is

controlled by thermodynamics rather than by enzyme expression
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Received 00th January 20xx, a,† a, † a b a,‡ a,‡

Accepted 00th January 20xx H. Richter , B. Molitor , H. Wei , W. Chen , L. Aristilde , L. T. Angenent
DOI: 10.1039/x0xx00000x Acetogenic bacteria are attracting interest as biocatalysts in the biotechnology industry, since they are able to ferment
www.rsc.org/ carbon monoxide (CO)-rich gases. Wild-type strains produce mainly acetate and ethanol, but genetic modifications have
already broadened the product portfolio. To enhance the production of intrinsic or heterologous biochemicals, knowledge
on the microbial physiology is necessary. This physiology includes two different phases: acidogenesis (growth/acetate
production) and solventogenesis (starvation/ethanol production). We operated two sequential, continuous bioreactors
with a pure culture of Clostridium ljungdahlii to achieve steady-state conditions in an acetate- and an ethanol-producing
stage to spatially separate acidogenesis and solventogenesis. Here, nearly 2000 proteins and their differential abundances
between acidogenesis and solventogenesis were identified. In addition, we measured important metabolites. The results
showed that nutrient-limited conditions triggered a transition to solventogenesis without altering the differential
abundances of enzymes in the central energy metabolism. Our proteomics results revealed that the enzymes for ethanol
production (AOR/ADH) were consistently abundant, even during acidogenesis. Based on this work, we developed an
overflow model with thermodynamic rather than genetic regulation. The model identifies acetic acid and reduced
cofactors as the saturation reactants. When the intracellular concentration of undissociated acetic acid reaches a
thermodynamic threshold, C. ljungdahlii will be able to shunt surplus reducing equivalents toward ethanol immediately.
This is important during retarded growth, when reducing equivalents can no longer be shunted toward biomass
production, while the supply of CO-rich gas is still high. Nutrient availability and pH can be manipulated to achieve the
desirable level of solventogenesis during bioprocessing.

Broader Context

Syngas fermentation is a quickly emerging research field with biotechnological importance. First industrial scale operations are being
pursued to produce ethanol from gaseous waste streams with acetogenic bacteria. The development of molecular biology tools is just
about to allow for metabolic engineering to optimize the production of intrinsic and heterologous products. Understanding the microbial
physiology of acidogenesis (acetate production and growth phase) and solventogenesis (ethanol production and starvation phase) in
acetogenic bacteria is of great interest to generate intrinsic or heterologous products from syngas. We performed a proteome and
metabolome analysis of Clostridium ljungdahlii, which was grown in two sequential bioreactors to spatially separate acidogenesis from
solventogenesis. Our results showed that the metabolic shift between these two phases involved redirecting reducing equivalents based on
thermodynamics rather than regulating the abundance of central metabolic enzymes. With an overflow model, we are able to explain
ethanol production during syngas fermentation. In addition, we provide information on details of the utilized pathway for ethanol
production in C. ljungdahlii. All together, these findings are important for the syngas fermentation community to design future strategies to
improve the production of intrinsic or heterologous products with acetogenic bacteria.

Introduction convert a gas mixture consisting of carbon monoxide (CO),
Syngas fermentation is a biotechnology production platform to hydrogen (H2), and carbon dioxide (CO2) into valuable products
such as acetate, ethanol, and 2,3-butanediol. This platform
utilizes acetogenic bacteria, including Clostridium ljungdahlii
1,2
and Clostridium autoethanogenum, as biocatalysts.
C. ljungdahlii has become a popular microbe for chemical and
fuel production and the genome sequence has been published
3
in 2010. Syngas-fermenting bacteria utilize the Wood
4
Ljungdahl pathway (WLP) for carbon fixation. This pathway
reduces CO2 to acetyl-CoA in a step-wise manner, consuming

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one mole of ATP. While one mole of acetyl-CoA is converted the proteomes from a pure culture of C. ljungdahlii that Online
View Article was
into one mole of acetate, one mole of ATP is recovered (Figure grown in two sequential bioreactors, which were separating
DOI: 10.1039/C6EE01108J
1). Considering only substrate-level phosphorylation as a steady-state acidogenesis and solventogenesis phases.
source of ATP, the WLP by itself does not conserve energy. The Surprising to us, we did not find substantial differential

Energy & Environmental Science Accepted Manuscript

+
function of the reduced ferredoxin:NAD oxidoreductase (Rnf) abundances of central metabolic enzymes. Therefore, factors
complex and the ATPase – an energy-producing machinery – is other than regulating the differential abundance of enzymes
5,6
essential for growth of C. ljungdahlii on syngas. had to be responsible for a metabolic shift from acidogenesis
For syngas-fermenting bacteria, alcohol production occurs to solventogenesis. In addition, with the proteome analysis we
preferably when growth is limited due to nutrient limitations found indications of considerable nutrient limitations during
7-10
or a low pH. This combination of solvent production and solventogenesis and stress conditions during both phases. To
low growth is known from the well-studied energy metabolism further shed light on the metabolic differences in C. ljungdahlii
of acetone/butanol/ethanol (ABE)-fermenting clostridia, during acidogenesis and solventogenesis, we performed a
Published on 20 May 2016. Downloaded by UNIVERSITY OF NEBRASKA on 22/05/2016 06:46:29.

including Clostridium acetobutylicum and Clostridium metabolome analysis. Based on our results and the literature,
beijerinckii. For these bacteria the metabolism is clearly we developed a simplified overflow model that explains the
separated into two phases: 1) acidogenesis; and 2) redirection of reducing equivalents away from biomass
solventogenesis. Acidogenesis has been associated with production toward ethanol production without a genetic
growth during which acetate and n-butyrate are produced regulatory system.
(from sugars), while solventogenesis has been associated with
a stationary phase and sporulation during which ethanol, n- Experimental
butanol, and acetone are produced (from sugars and by re- Materials and methods
11-13
assimilation of acetate and n-butyrate). ABE-fermenting
Clostridium ljungdahlii strain PETC (ATCC55383) was obtained
clostridia will preferably perform acidogenesis because of a
from ATCC, Manassas, VA. We performed proteomics and
higher ATP yield of 3 mole of ATP/mole of glucose and 2 mole
metabolomics analyses from two independent bioreactor runs
of ATP/mole of glucose during acidogenesis and
of a two-stage fermentation system under similar conditions.
solventogenesis, respectively. When undissociated short-chain
Quantification in the proteomics experiment was conducted
carboxylic acids accumulate and become toxic, the clostridia
using mass spectrometry-based iTRAQ technology.
will undergo a phase transition from acidogenesis to
Intracellular metabolites were measured using liquid
solventogenesis to continue their metabolism without
chromatography-mass spectrometry following extraction from
acidifying the fermentation broth any further. In fact, the
cell-rich samples, and blank experiments were performed to
short-chain carboxylates, which are produced during
account for accumulation of metabolites in the cell-free
acidogenesis, are re-assimilated into their corresponding
bioreactor broth. Detailed description of the bioreactor runs
alcohols, leading to an increase in the pH of the fermentation
11 and sampling and analysis of proteomics and metabolomics
broth. Here, we refer to carboxylates as both the total of
data can be found in ESI Materials and Methods.
undissociated and dissociated species.

With proteome analyses researchers have observed a Results and Discussion
differential abundance of enzymes in the central metabolic
The metabolic shift from acidogenesis to solventogenesis did not
pathways for phase transition with ABE-fermenting clostridia.
The difference in the central metabolic pathways included involve changes in relative abundances of central metabolic
increased abundances of enzymes for solvent production and enzymes
decreased abundances of enzymes for acid production during C. ljungdahlii was grown in a two-stage, continuous syngas-
14,15
solventogenesis compared to acidogenesis. Others have fermentation system with an excess gaseous stream consisting
14,16 9,10
used transcriptome analysis to study ABE fermentation , of 60% CO, 35% H2, and 5% CO2. As a continuous system,
but results between studies were not straightforward because steady-state conditions were reached in both bioreactor
transcriptome data do not always correlate well with protein stages before samples for the proteome analysis were taken.
abundances due to post-transcriptional or translational The culture in Stage A (1-L bioreactor optimized for
17,18
regulation and proteolysis. Further, the proteome is more acidogenesis) produced 83.97 mM acetate and 12.10 mM
directly related to cellular functions and metabolism than the ethanol at an OD600 of 2.12. Meanwhile, the culture in Stage B
14
transcriptome. Therefore, a proteome analysis is the (4-L bioreactor optimized for solventogenesis) produced 33.09
preferred method, especially since current MS-based methods mM acetate and 188.2 mM ethanol at an OD600 of 7.25 (Figure
are quantitative: 1) within a single proteome; and 2) between S2, Table S2). The volumetric acetate production rate for the
samples with the same microbe to compare protein culture in Stage B was considerably lower than in Stage A,
19-21
abundances under different environmental conditions. while the volumetric ethanol production rate remained similar
Here, we had hypothesized that for the metabolic shift from for both stages (Table S2), resulting in a net acetate
acidogenesis to solventogenesis for syngas-fermenting consumption in Stage B with acetate being fed from Stage A.
bacteria, changes in enzyme abundances in central metabolic Consequently, the ethanol-to-acetate ratios were 0.15 and 5.7
pathways would be involved (similar to ABE-fermenting for Stage A and Stage B, respectively.
clostridia). To test this hypothesis, we analyzed and compared

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Recently, Mock et al. published a metabolic scheme with solventogenesis is known as the stationary phase. All relevant
View Article Online
stoichiometries of ATP and reducing equivalents (ferredoxin, enzymes in the central anabolic pathways could
DOI: be identified
10.1039/C6EE01108J
NAD(P)H) for fermentation of H2/CO2 based on experiments in our study, but only citrate synthase (#12, not highly
with C. autoethanogenum. We verified and further built on abundant) and glyceraldehyde-3-phosphate dehydrogenase

Energy & Environmental Science Accepted Manuscript

this scheme by including fermentation of CO and fructose (#18) were down-regulated during solventogenesis compared
(Figure S1; ESI Results and Discussion – metabolic scheme to acidogenesis (Figure 2, Table S6). Thus, the metabolic shift
S1.1). In summary, the net ATP yield for acetate and ethanol from acidogenesis to solventogenesis did not involve changes
production was always positive, regardless of the substrate. In in differential abundance of central metabolic enzymes, but
addition, the indirect route of ethanol production via acetate rather had to be caused on a thermodynamic level.
resulted in a higher net ATP yield compared to the direct
route, despite that reduced ferredoxin is used in the indirect Proteome analysis indicated severe nutrient limitations
route (Figure S1, Table S1, ESI Results and Discussion – during solventogenesis, and general stress conditions in both
Published on 20 May 2016. Downloaded by UNIVERSITY OF NEBRASKA on 22/05/2016 06:46:29.

proteome S2.1). bioreactor stages

With our proteome analysis, we identified 1988 proteins in the Within the bioreactor study, we had found that the ethanol
samples (acidogenesis and solventogenesis) from which 1743 production rate in Stage B was not limited by the gas/liquid
10
proteins met the criteria to obtain comparative quantitative mass-transfer rate of CO and H2. Therefore, one or several
data. The criteria included that these proteins had to be unknown medium components, such as macronutrients, trace
present in both samples with at least two unique peptides (ESI elements, or vitamins, must have been limiting the metabolic
Material and Methods). These 1743 proteins were not equally activity of C. ljungdahlii in the Stage B of our two-stage
10
abundant, though. In fact, only 61 proteins were highly bioreactor system. With the proteome analysis we were able
abundant (≥ 2500 μmol/mol), exceeding 60% of the total to observe which medium components were likely limiting.
proteome based on moles (Table S3). From the 245 proteins Among the proteins most up-regulated during
that did not meet our criteria, none of them was highly solventogenesis, 18 have functions either related to sulfur-
abundant in the total proteome based on the threshold of ≥ containing amino acid metabolism or transport of sulfuric
2500 μmol/mol. We observed 46% and 53% of the 1743 compounds (Table S4), and 3 of these 18 proteins are highly
proteins that met our criteria in the acidogenesis and abundant (Table S3). This indicates a severe limitation in
solventogenesis proteome from Stage A and Stage B, sulfur. Based on this proteome analysis, we revisited the
respectively. Our analysis included two technical replicates for sulfur-assimilation pathway in C. ljungdahlii and found that the
23
each bioreactor stage (4 proteome samples), with almost genes for sulfate reduction found in Clostridium kluyveri are
identical values between the replicates (ESI Results and absent in C. ljungdahlii. We learned that the sulfur added as
24
discussion – proteome S2.2). MgSO4 in the standard 1754 PETC- (ATCC) or P7- medium for
We did not find a difference in abundances for enzymes of the syngas-fermenting bacteria can, therefore, not be utilized by
central metabolic pathways for both catabolism and anabolism C. ljungdahlii. The only available sulfur-source is the cysteine
in our proteome analysis between solventogenic and added at a final concentration of 1 mM in our modified P7
acidogenic conditions (Figures 1 and 2, Table S6). For medium as a reducing agent (SI Material and Methods). Up-
catabolism, phosphotransacetylase (Pta) and acetate kinase regulation of the cysteine synthases CLJU_c24370 (2031
(AckA) for acetate synthesis, and aldehyde:ferredoxin μmol/mol, 21.8-fold) and CLJU_c39310 (12324 μmol/mol,
oxidoreductase (AOR) and alcohol dehydrogenase (ADH) for 26.7-fold), which catalyze the assimilation of sulfide into
ethanol synthesis were highly abundant under both conditions cysteine, indicate the attempt of the cells to incorporate
(Figure 1). Neither aldehyde dehydrogenase (ALDH) nor additional sulfur as sulfide, while this was absent from the
bifunctional aldehyde/alcohol dehydrogenase (AdhE) were medium. Hence, all future studies with syngas-fermentation
detected in substantial amounts under both conditions (Figure should replace sulfate by either sulfide or cysteine as the sulfur
1). With our proteome analysis we, therefore, found that source.
ethanol production is exclusively achieved through reduction Indications for limitations of other nutrients besides sulfur can
of undissociated acetic acid via AOR during fermentation of be found in the proteome, albeit never as defined as for sulfur.
syngas (SI Results and Discussion – proteome S2.1). All Several proteins involved in nutrient transport, vitamin or
enzymes of the WLP were detected, but not found to be cofactor biosynthesis (e.g., thiamine), amino acid metabolism-
differentially abundant during acidogenesis and related proteins, and nitrogen-limitation/-fixation were found
solventogenesis (Figure 1). Enzymes that are involved in to be up-regulated during solventogenesis (Table S4), but
energy conservation were not found to be differentially there was no clear pattern. The down-regulated proteins
abundant, which includes the hydrogenases, the NADH- during solventogenesis (i.e., up-regulated proteins during
+
dependent reduced ferredoxin:NADP oxidoreductase (Nfn) acidogenesis), which identify nutrient limitations, were never
complex, the membrane-associated Rnf complex, and the highly abundant in the total proteome, and thus not very
ATPase (ESI Results and Discussion – proteome S2.3 and S2.4). important (Table S5).
For anabolism, we also did not observe considerable changes The proteome analysis also found that 7 stress-related
in protein abundance between conditions, even though proteins were highly abundant (≥ 2500 μmol/mol; out of 61)
acidogenesis is known as the growth phase, while during both acidogenesis and solventogenesis (Table S3). We

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ARTICLE Journal Name

observed a slightly elevated stress response during μmol/mol, 2.6-fold) (Table S4). This is logical View because the
Article Online
solventogenesis, but only 1 of the up-regulated proteins was cellular strategy to overcome nitrogen-limitation in microbes
DOI: 10.1039/C6EE01108J
highly abundant (Tables S3 and S4; ESI Results and Discussion that are capable of fixing molecular nitrogen (N2), including
– proteome S2.5). Therefore, the shift toward solventogenesis C. ljungdahlii, would be to up-regulate nitrogenase-related

Energy & Environmental Science Accepted Manuscript

25
could not be explained by a general stress response. Since we components to enable nitrogen-fixation. However, we did
had not observed any considerable differences in enzyme not expect that C. ljungdahlii would be able to overcome
abundances in the central metabolic pathways (Figures 1 and nitrogen-limitation through this strategy due to the absence of
2), we decided to perform a metabolome analysis of N2 in our gas mixture.
C. ljungdahlii during acidogenesis and solventogenesis during a Third, we found a significant accumulation of alanine (5-fold)
subsequent experimental run (ESI Results and Discussion – during solventogenesis compared to acidogenesis (Figure 2,
metabolome S3.1). Our two objectives of this approach were Table S7). Because we anticipated that all amino acids to
to: 1) identify factors important for the metabolic shift from synthesize new proteins would get depleted during nutrient
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acidogenesis to solventogenesis; and 2) investigate the limitation, accumulation of any amino acid is counter-intuitive
redirection of reducing equivalents toward ethanol rather than under the nutrient- (nitrogen-) limitation indicated by the
biomass after the cells had stopped growing due to nutrient proteome and metabolome analyses. Alanine biosynthesis
limitations. involves transamination reactions of pyruvate (Pyr) with other
26,27
amino acids, such as glutamate or valine. The
The metabolome analysis verified the proteome analysis in transamination enzyme activities would result in depletion of
regards to nutrient limitations glutamate and valine, respectively, to synthesize alanine.
We measured a total of 27 intracellular metabolites with the Indeed, we found depletion in the glutamate level (-4-fold) and
metabolome analysis (Figures 2 and 3; Tables S7 and S8), and the valine level (-4-fold, although not significant) during
discuss here three amino acids that are significantly different solventogenesis compared to acidogenesis (Figure 2,
between acidogenesis and solventogenesis and that are Table S7). We hypothesize that the elevated levels of the
related to the observed nutrient limitations (Table S7). First, intracellular alanine serve as a quick and reversible means of
we found a significant depletion in the levels of the sulfur- storing carbon and nitrogen during a nutrient-limiting
containing amino acid methionine (-2-fold) under condition. Alanine can be easily invested back into the central
solventogenic conditions compared to acidogenic conditions metabolism as Pyr or other amino acids by transamination
26,27
(Figure 2, Table S7). This is a verification of the proteome reactions. This hypothesis, however, would need further
analysis for which we already observed the effect of sulfur investigation.
limitation. Based on this result, we further analyzed the
proteome data and found that enzymes in methionine Establishing solventogenic conditions involved redirections of
biosynthesis were up-regulated significantly during reducing equivalents and carbon due to nutrient limitations
solventogenesis, including: O-acetylhomoserine sulfydrylase It is critical for bacteria to balance catabolic NAD(P)H
28
(CLJU_c06640, 7657 μmol/mol, 37.6-fold); homoserine O- formation with anabolic consumption. Because of the growth
succinyltransferase (CLJU_c24330, 1238 μmol/mol, 7.1-fold); limitation in our Stage B bioreactor, reducing equivalents can
and cystathionine gamma-synthase (CLJU_c24380, 1042 no longer be re-oxidized by producing new biomass. At the
μmol/mol, 26.4-fold) (Table S4). One explanation is to same time, oxidation of CO and H2 continues to supply
overcome depletion of methionine by increasing the levels of reducing equivalents, energy, and carbon, and therefore CO
enzymes for biosynthesis of this amino acid; however, this and/or CO2 are fixed into acetyl-CoA through the WLP.
would need to be further investigated. Together with high concentrations of acetate in Stage B (Table
Second, we also found a significant depletion of the N-rich S2) and enzymes for solventogenesis always present (no
amino acid glutamate (-4-fold; and glutamine, -1.5-fold, different abundance found with proteomics, Figure 1), surplus
although not significant) in the metabolome analysis (Figure 2, reducing equivalents are re-oxidized by producing ethanol via
Table S7). Since glutamate and glutamine are the primary sites solventogenesis. This is at the expense of a lower ATP-yield per
for ammonia (nitrogen) assimilation, this was a confirmation of substrate molecule from ethanol production compared to
the nitrogen-limitation for which we had found indications in acetate production (Figure S1, Table S1). However, during
the proteome. Ammonia was the only nitrogen source in our nutrient-limited conditions this may be the only possibility for
bioreactor system. Based on this finding, we again analyzed C. ljungdahlii to keep up its energy metabolism, and in
the proteome data. We found nitrogenase-related proteins addition, provide a means to avoid further acidification.
(for N-fixation) to be up-regulated in our proteome analysis, For ABE-fermenting clostridia, the solventogenesis phase is
+
including: nitrogenase molybdenum-iron protein, alpha chain, characteristic for recovering oxidized cofactors (NAD and
+
NifD2 (CLJU_c23520, 423 μmol/mol, 19.5-fold); nitrogenase NADP ) by re-assimilation of short-chain carboxylic acids,
molybdenum-iron protein, beta chain, NifK2 (CLJU_c23510, which were produced during sugar oxidation, and reducing
11,15
495 μmol/mol, 14.8-fold); nitrogenase iron protein, NifH them into their corresponding alcohols. Indeed, a less
(CLJU_c23530, 884 μmol/mol, 8.8-fold); nitrogen fixation reduced redox state (i.e., a low ratio of reduced to oxidized
+ +
protein, NifS (CLJU_c12110, 2778 μmol/mol, 3.1-fold); and cofactors [NADH/NAD and NADPH/NADP ]) has been found
nitrogen fixation protein, NifU, N-terminal (CLJU_c12120, 6357 during solventogenesis compared to acidogenesis in

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29,30
solventogenic clostridia. This, because of the high activity fermentation of sugars in syngas-fermenting bacteria. The
View Article Online
of the solvent-producing pathways that utilize reduced higher ATP yields during sugar fermentation may enable a
DOI: 10.1039/C6EE01108J
30
cofactors, while acidogenesis is switched off. more sophisticated mechanism of metabolic regulation in
Based on these considerations, we analyzed the levels of C. ljungdahlii and other syngas-fermenting bacteria.

Energy & Environmental Science Accepted Manuscript

reducing equivalents obtained in our metabolome analysis to
gain insights into the differences in the redox state of A model is proposed to explain biomass, acetate, and ethanol
C. ljungdahlii during solventogenesis and acidogenesis (Figure production for C. ljungdahlii
3). Contrarily to ABE-fermentation, we found an accumulation Building on our results and the literature, we here propose a
of NADH in the total bioreactor environment (including the simplified overflow model to describe the fluxes of reducing
blank; an in-depth discussion is given in ESI Results and equivalents and carbon during syngas fermentation in
+
Discussion – metabolome S3.2), while the intracellular NAD C. ljungdahlii (Figure 4). In our model, CODH and hydrogenase
level was decreased during solventogenesis compared to are constantly supplying reducing equivalents from the
Published on 20 May 2016. Downloaded by UNIVERSITY OF NEBRASKA on 22/05/2016 06:46:29.

acidogenesis (Figure 3). This result suggests to us a more oxidation of CO or H2 (excess gas supply), and the WLP
reduced redox state during solventogenesis compared to provides fixed carbon in the form of acetyl-CoA from syngas
acidogenesis for the total bioreactor environment. Specifically (Figure 4, A). Biomass is produced and consumes reducing
for the intracellular redox state, however, no reliable equivalents and carbon (Figure 4, B). A large fraction of the
statement can be made. Regardless, the redox state in our carbon, however, is directed toward acetate production, to
study was clearly not more oxidized during solventogenesis conserve cellular energy (Figure 4, C). Acetate is transported
compared to acidogenesis, which is the case for ABE- out of the cell by active transport processes. In Stage B of our
30 +
fermenting clostridia. In addition, our NADPH/NADP ratios two-stage bioreactor system, additional carbon in the form of
were not different between solventogenesis and acidogenesis, acetate is provided from Stage A. Depending on the total
again implying that solventogenesis could not create an acetate concentration and the extracellular pH, a varying
oxidized redox state. This may be a logical result because of concentration of undissociated acetic acid is present, which
the high syngas supply into Stage B resulting in continuous can freely diffuse between the intracellular and the
generation of reducing equivalents by oxidation of CO and H2, extracellular milieu (further discussion on the effect of the
while anabolic consumption of reducing equivalents was extracellular pH and total acetate concentration are in ESI
halted due to growth stagnation. Results and Discussion – overflow model S4.1). The enzymes
In summary, our results suggest that nutrient limitations and for the production of ethanol (AOR, ADH) are always abundant
the resulting growth limitation redirected reducing equivalents (Figure 4, D). Thus, ethanol can be immediately produced as
toward ethanol production (solventogenesis in Stage B) an overflow product, as soon as the concentration of overflow
without altering enzyme levels in central metabolic pathways. reactants (i.e., undissociated acetic acid and reducing
The imbalance of oxidative and reductive pathways during equivalents) reach a critical concentration when an excess of
growth limitation likely resulted in an accumulation of the acetyl-CoA and reducing equivalents can no longer be directed
reduced cofactor NADH and its utilization for solventogenesis. toward biomass (Figure 4, E). AOR has been shown to catalyze
The question that remains to be answered is why ABE- the reduction of only undissociated acetic acid, and therefore
32,33
fermenting clostridia evolved with genetic regulation for the overflow reactant is acetic acid and not acetate.
acidogenesis and solventogenesis and syngas-fermenting Importantly, the direct reduction of undissociated acetic acid
bacteria evolved without such regulation? Both detoxify the to acetaldehyde with reduced ferredoxin, which is catalyzed by
majority of the short-chain acids that are generated during AOR, is only thermodynamically feasible when the substrate
acidogenesis by reducing them into their corresponding concentrations (acetic acid and reduced ferredoxin) are high
alcohols during solventogenesis, which is a process that occurs and the product concentrations (acetaldehyde and oxidized
22
at the expense of a lower ATP-yield. ferredoxin) are low. The reduction of acetaldehyde to
On one hand, the re-oxidation of redox cofactors during ethanol (Eº’ = -200 mV) with NADH (Eº’ = -320 mV) is exergonic
solventogenesis seems more efficient in ABE-fermenting and will maintain a low product concentration in the AOR
clostridia for which enzymes involved in alcohol production are reaction (i.e., acetaldehyde). Therefore, it is of utmost
up-regulated, while enzymes involved in acid production are importance to reach an intracellular thermodynamic threshold
down-regulated. Therefore, ABE-fermenting clostridia avoid concentration of undissociated acetic acid to render the
producing costly enzymes that are not required. On the other reduction to acetaldehyde thermodynamically feasible (Figure
hand, without any regulation and the permanent presence of 4, F). The biomass concentration of the syngas-fermenting
all these enzymes, C. ljungdahlii may be more flexible with the bacteria will further influence the production of ethanol as an
advantageous amenity to quickly redirect reducing equivalents overflow product, because critical concentrations of
coming from the oxidation of CO and H2 toward either biomass undissociated acetic acid and reducing equivalents must first
or ethanol. This would facilitate the difficult ATP production be reached. With this simplified overflow model, we can
from syngas, which is at the thermodynamic limits of bacterial explain not only our results but also the results of another
31 22
metabolism. To answer the evolution question, it might be study with a single stage bioreactor in which ethanol
interesting to investigate whether abundances of enzymes are production occurred without nutrient limitation (ESI Results
regulated between acidogenesis and solventogenesis during and Discussion – overflow model SI 4.2). Following our

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simplified overflow model, addition of external acetic acid to Acknowledgements View Article Online
the bioreactor would trigger solventogenesis when threshold DOI: 10.1039/C6EE01108J
Funding was provided by Jossie Hollander and the Foundation
concentrations are reached for: 1) pH-dependent intracellular
des Fondateurs. B.M. was funded through a postdoctoral
undissociated acetic acid; and 2) reducing equivalents. Mock

Energy & Environmental Science Accepted Manuscript

22 research fellowship from the German Research Foundation
et. al added ~40 mM ammonium acetate as a nitrogen
(DFG, MO2933/1-1). Metabolomics analysis was supported by
source to their medium, resulting in external acetate addition.
a research starter grant from the U.S. National Science
This may explain their high ethanol production rates with C.
Foundation (Division of Molecular and Cellular Biosciences,
autoethanogenum. However, to validate our simplified
MCB 133292), which was awarded to LA. The proteomics
overflow model, further research is needed.
experiments were conducted in the Proteomics Core Facility at

the Institute for Biotechnology, Cornell University.
Future perspective – molecular biology and metabolic
engineering of syngas-fermenting bacteria
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Molecular biological tools for syngas fermenting bacteria are

Notes and references
emerging and recent developments will allow for metabolic
engineering of syngas-fermenting bacteria in the future.
34,35
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DOI: 10.1039/C6EE01108J

Energy & Environmental Science Accepted Manuscript

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Figure 1. Schematic of the central energy metabolism in C. ljungdahlii during syngas fermentation. Reactions are shown with substrates, products, and involved
redox mediators, without representing stoichiometric balances. The involved enzymes are given in different boxes with the corresponding accession numbers
(CLJU_c#) in the first column, relative abundance of the protein in our proteome analysis (μmol/mol) in the second column, and the fold-change (solventogenesis
vs. acidogenesis) in the third column. Horizontal lines in the boxes separate individual enzymes (homologs) or enzyme complexes. Tables are shaded regarding to
their function: light brown, Wood-Ljungdahl pathway; light grey, hydrogenases; light red, acetate production; blue, ethanol production; and green, energy
conservation. The CODH from the ACS/CODH complex is linked to the reversible oxidation of CO to CO2 , which is shown here by a purple-dotted box and line to
connect with the reaction (ESI Results and Discussion – proteome S2.2). FDH and the Hyt H2 ase can form a complex to directly reduce CO2 to formate with H2,
which is depicted by orange-dotted boxes that are connected by orange-dotted lines with each other and with the reaction. ACS, acetyl-CoA synthase; AcsE,
methyltransferase; CODH, CO dehydrogenase; CoFeS-P, corrinoid iron-sulfur protein; FchA/FolD, bifunctional methylene-THF dehydrogenase/ formyl-THF
cyclohydrolase; Fdh, formate dehydrogenase; Fhs, formyl-THF synthetase; MetFV, methylene-THF reductase; THF, tetrahydrofolate.

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Figure 2. Schematic of the central anabolism to provide metabolite precursors. Intracellular metabolite levels and blank measurements (ESI Materials and Methods)
obtained in the two different growth conditions (acidogenesis: sample, red; blank, light red; solventogenesis: sample, blue; blank, light blue) are shown in boxes close to the
position of the metabolite in the reaction scheme; Ac-P shown in the dashed box is part of the reaction scheme in Figure 1 (error bars give standard deviations of three
independent biological replicates with three technical replicates each [n = 9]; Tables S7 and S8 for the measured values and statistics). Reactions catalyzed by bi-directional
enzymes are shown with lines with arrows on both sides; otherwise, separate lines are used to indicate different enzymes involved. Dashed black lines link metabolite
precursors to biomass components listed on the right. Three reaction paths involving enzyme #24 (Transaldolase) and #25 (Transketolase) with always two substrates and
two products for each pathway are shown as different colors. Each reaction path is bi-directional and shown here as a combination of color-coded solid lines and dashed
lines. Color code: orange, GAP + F-6-P <-> Xyl-5-P + Ery-4-P; blue, Xyl-5-P + Ribose-5-P <-> GAP + Sedo-7-P; red, GAP + Sedo-7-P <-> F-6-P + Ery-4-P. Solid lines with arrows
point between the substrates and the enzyme. Dashed lines with arrows point between the enzyme and the products. Enzyme abundances and fold-changes
(solventogenesis vs. acidogenesis) are in Table S6. Ac-P, acetyl-phosphate; Cit, citrate; Isocit, isocitrate; 2-KG, 2-ketoglutarate; Pyr, pyruvate; PEP, phosphoenolpyruvate; OA,
oxaloacetate; Mal, malate; Fum, fumarate; Suc, succinate; 2-P-G, 2-phosphoglycerate; 3-P-G, 3-phosphoglycerate; 1,3-BPG, 1,3-bisphosphoglycerate; GAP, glyceraldehyde 3-
phosphate; DHAP, dihydroxyacetone phosphate; F-1,6-BP, fructose-1,6-bisphosphate; Sedo-7-P, sedoheptulose-7-phosphate; Ery-4-P, erythrose-4-phosphate; F-6-P,
fructose-6-phosphate; G-6-P, glucose-6-phosphate; Xyl-5-P, xyloulose-5-phosphate; R-5-P, ribulose-5-phosphate.

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Figure 3. Redox mediators in C. ljungdahlii. Intracellular distribution and blank
+ +
measurements of redox mediators (NAD , NADH, NADP , NADPH) in C. ljungdahlii
during acidogenesis (red, sample; light red, blank) and solventogenesis (blue,
sample; light blue, blank). Error bars give standard deviations of three
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independent biological replicates with three technical replicates each (n = 9).

Tables S7 and S8 for measured values and statistics.

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Figure 4. Overflow model for fluxes of reducing equivalents and carbon

in C. ljungdahlii during syngas fermentation. A. CO is oxidized to CO2 to
feed this electron acceptor into the Wood-Ljungdahl pathway (WLP) and
to generate reducing equivalents ([H]). The level of reducing equivalents
can accumulate under certain conditions, which is indicated by the brown
color-gradient. The WLP produces acetyl-CoA with the methyl branch
(left) and the carbonyl branch (right). B. Acetyl-CoA is utilized to produce
biomass, which consumes reducing equivalents and carbon. C. The
acetyl-CoA is also directed toward acetate production, which generates
cellular energy (ATP) to balance the ATP consumption by acetyl-CoA
production. Acetate can be transported out of the cell by active transport
processes (dashed line), and ~5% of the total acetate will be in the
undissociated acetic acid form at an assumed intracellular pH of 6. The
total intracellular acetate concentration will be low (*). D. The enzymes
for ethanol production are always abundant and reducing undissociated
acetic acid via the intermediate acetaldehyde, which consumes reducing
equivalents, can immediately generate ethanol. E. When growth is
limited and biomass can no longer be produced, the total intracellular
acetate concentration will increase (**). F. At a thermodynamic threshold
concentration of undissociated acetic acid with a surplus of reducing
equivalents, ethanol production will occur as an overflow mechanism
(blue color gradient). The extracellular pH and total acetate
concentration have an influence on the intracellular total acetate
concentration (pH 5.5 and pH 4.5 [* vs. **]), since undissociated acetic
acid can freely diffuse over the cell membrane (light red and dark red
color gradient).

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