Microorganisms 11 01610

microorganisms
Article
Exploring the Influence of pH on the Dynamics of
Acetone–Butanol–Ethanol Fermentation
Manish Kumar 1, * , Supreet Saini 2 and Kalyan Gayen 3
1 Department of Chemical Engineering, Indian Institute of Technology Gandhinagar, Palaj,

Gandhinagar 382055, Gujarat, India
2 Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai,
Mumbai 400076, Maharashtra, India; [email protected]
3 Department of Chemical Engineering, National Institute of Technology Agartala,
Agartala 799053, Tripura, India; [email protected]
* Correspondence: [email protected]
Abstract: Clostridium acetobutylicum is an anaerobic bacterium that is extensively studied for its
ability to produce butanol. Over the past two decades, various genetic and metabolic engineering
approaches have been used to investigate the physiology and regulation system of the biphasic
metabolic pathway in this organism. However, there has been a relatively limited amount of research
focused on the fermentation dynamics of C. acetobutylicum. In this study, we developed a pH-based
phenomenological model to predict the fermentative production of butanol from glucose using
C. acetobutylicum in a batch system. The model describes the relationship between the dynamics
of growth and the production of desired metabolites and the extracellular pH of the media. Our
model was found to be successful in predicting the fermentation dynamics of C. acetobutylicum, and
the simulations were validated using experimental fermentation data. Furthermore, the proposed
model has the potential to be extended to represent the dynamics of butanol production in other
fermentation systems, such as fed-batch or continuous fermentation using single and multi-sugars.
Keywords: ABE fermentation; Clostridium acetobutylicum; phenomenological model; butanol;

acidogenesis; solventogenesis; pH
Citation: Kumar, M.; Saini, S.; Gayen,
K. Exploring the Influence of pH on
the Dynamics of
Acetone–Butanol–Ethanol
1. Introduction
Fermentation. Microorganisms 2023,
11, 1610. https://doi.org/10.3390/ Clostridium acetobutylicum is a versatile microorganism capable of fermenting both
microorganisms11061610 hexose and pentose sugars into biofuels such as butanol and ethanol, as well as other
valuable solvents such as acetone, and acids including acetic and butyric acid, and even
Academic Editors: Thomas Brück
hydrogen [1]. As a result of this unique ability, researchers have focused on enhancing
and Dania Awad
its potential for producing target chemicals on a global scale [2–4]. One of the most re-
Received: 23 April 2023 cent areas of investigation has been to identify the functional modules of genes involved
Revised: 4 June 2023 in the production of acids and solvents, granulose formation, sporulation, sugar uptake
Accepted: 14 June 2023 systems, and phase transitions, while uncovering their regulatory mechanisms [5]. How-
Published: 18 June 2023 ever, despite significant progress, several challenges remain to be addressed in order to
bridge the information gap at both the system and reactor-levels and improve the yield of
butanol [6–8].
On the basis of available physiological and genomic-level information, research efforts
Copyright: © 2023 by the authors.
must be made towards developing the system and reactor-level understanding of acetone–
Licensee MDPI, Basel, Switzerland.
This article is an open access article
butanol–ethanol (ABE) fermentation with the help of mathematical models and their
distributed under the terms and
validation with experimental data. Precise system-level modeling and simulation can
conditions of the Creative Commons incorporate a large volume of experimental data, and also lead to design of physiologically
Attribution (CC BY) license (https:// significant experiments [9]. In this direction, limited data are available to capture the
creativecommons.org/licenses/by/ dynamics of fermentation in C. acetobutylicum [10].
4.0/).
Microorganisms 2023, 11, 1610. https://doi.org/10.3390/microorganisms11061610 https://www.mdpi.com/journal/microorganisms

Microorganisms 2023, 11, 1610 2 of 23
To advance our understanding of acetone–butanol–ethanol (ABE) fermentation, re-

search efforts should focus on developing mathematical models based on physiological and
genomic-level data and validating these models with experimental results. With precise
system-level modeling, we can integrate a large volume of experimental data, leading to the
design of more physiologically meaningful experiments [9]. By combining mathematical
models with empirical data, we can gain a deeper understanding of the ABE fermentation
process at both the system and reactor levels. This can help us optimize fermentation condi-
tions and improve product yields. Ultimately, the use of rigorous modeling techniques can
enhance our ability to harness the usefulness of ABE fermentation for a range of industrial
applications [9,10].
In particular, kinetic studies of ABE fermentation have shown promise in optimizing
operational parameters and improving the design of bioreactors for achieving high-yield
fermentation processes [11–14]. To this end, several kinetic models have been constructed
for this fermentation process, capturing sugar and production profiles for batch [15–18],
continuous [19,20], and integrated systems with recovery processes [21–23]. Some recent
efforts have also incorporated detailed biochemical reactions in the metabolic network of
the bacterium to enhance modeling accuracy [24–26].
ABE fermentation is a complex process influenced by various factors that significantly
impact its efficiency and yield. One crucial factor is the composition of the fermentation
medium, including the types and concentrations of substrates and nutrients available for
the microorganisms. The pH level of the medium also plays a critical role, as it affects
the metabolic activities of the microorganisms and the production of desired products.
Additionally, the temperature and oxygen availability can significantly influence ABE
fermentation, as they influence the growth and metabolic rates of the microorganisms.
Other factors such as inoculum cocentration fermentation time, and agitation rate also
contribute to the overall fermentation performance [27]. Understanding and optimizing
these factors is essential for achieving high ABE production and improving the efficiency
of this fermentation process. In this study, we focused on effect of pH level of the medium
on C. acetobutylicum’s growth and production profiles.
Although media pH is known to be a crucial factor for regulating biomass growth
and the transition from acidogenesis to solventogenesis in Clostridium cultures [16,28,29],
only a limited number of reports on ABE fermentation have investigated this variable.
Incorporating pH into a physiological model can provide valuable insights for designing a
fermentation process. Some studies have included pH dependence to develop mathematical
models that were validated using experimental results from a continuous system [30,31].
However, these studies employed controlled pH profiles that were specific to each phase of
fermentation (e.g., 5.7 for acidogenesis and 4.4 for solventogenesis).
In this study, we propose a phenomenological model to gain insights into the dynamics
of ABE fermentation process across different pH profiles. Our model specifically describes
the dynamics of biomass growth and synthesis of desired products in a batch process. By
accounting for the biphasic metabolic network and variation in pH profiles, our model
successfully captures the complexity of the fermentation process. We validate our model
through simulations that closely match experimental fermentation data. Moreover, our
presented model has the potential to be extended for the analysis of ABE fermentation
on consumption of multiple pentose and hexose sugars in other fermentation processes,
including fed-batch and continuous systems, both at controlled and uncontrolled pH. The
versatility of our model opens up avenues for better understanding the dynamics of fermen-
tation processes, which could lead to more efficient and sustainable industrial processes.
2. Materials and Methods

2.1. Model Development
Assumptions for phenomenological modeling of ABE fermentation in C. acetobutylicum.
It was assumed that in the absence of any external stress, there is no production of
acids in solventogenesis phase and no production of solvents in acidogenesis [32]. By
external stress, we mean any external addition of acid/base to control pH.
Acidogenesis shifts to solventogenesis when bacterial growth enters in stationary
phase [33].
pH variation in culture depends on capacity of buffer (KH2 PO4 and K2 HPO4 ) and
assimilation of produced acids (acetic and butyric acid) during fermentation.
Effect of buffer (KH2 PO4 and K2 HPO4 ) was taken in account for maintaining the
pH value until phosphate of buffering agents was not consumed by the organism [34,35].
Once the effect of the buffer was exhausted, pH was altered according to the variation in
hydrogen ion concentration dissociated from acetic and butyric acid.
High concentration of substrates inhibits synthesis of biomass and butyric acid [25,36,37].
Substrate inhibition was not incorporated in the case of acetic acid as rapid production
of acid was observed in the early hours of fermentation experiments while substrate
concentration was high.
Various studies report product inhibition in ABE fermentation by acids (acetic and
butyric acid) and solvents (acetone, butanol, and ethanol) [37,38]. Therefore, product
inhibition was taken in account for modeling production profiles of both metabolic phases
in C. acetobutylicum.
Bacterial culture of C. acetobutylicum became metabolically inactive once pH fell below
3.0. Metabolically inactivation is meant complete cessation of growth and synthesis of
metabolites and substrate utilization. This fact was observed from batch fermentation
experiments at uncontrolled pH levels during this study.
2.2. pH
It was observed from fermentation experiments that pH of culture remained relatively
unchanged for approximately the first 10 h of incubation as assimilation of acids in media
starts thereafter. Therefore, pH was unchanged in the starting hours of fermentation
because of the buffering effect of buffer agents (KH2 PO4 and K2 HPO4 ) present in media [35].
Once accumulation of H+ ions form produced acids which crossed the buffering zone due
to simultaneous production of acetic and butyric acid; pH decreased rapidly. In our
model, pH of buffering zone in early hours of fermentation was determined by Henderson–
Hasselbalch’s equation (Equation (1)), while out of the buffering zone it was calculated on
the basis of H+ ions dissociated from acids produced (Equation (2)).
Salt − H +

pH = pKa + log10 (1)
Acid + H +
pH = −log10 H + (2)
+
where, H + = H A + HB+ . H A+
and HB+ indicate H+ ions from acetic and butyric acid,
respectively. In this biological system, KH2 PO4 and K2 HPO4 act as an acid and a salt,
+
respectively, to generate a buffering zone in media. H A and HB+ were calculated as follows:
+ +
−
Hint + HA (A )
KaA = (3)
Amol
+
+ HB+ ( B− )

Hint
KaB = (4)
Bmol
where, KaA and KaB denote dissociation constants for acetic and butyric acid, respectively.
+
Hint indicates the initial hydrogen ions in the media. Concentrations of acetic acid (Amol )
with corresponding anion (A− ) and butyric acid (Bmol ) with related anion (B− ) played a
major role for variation in pH profiles. In our simulations, we considered the initial pH as
an input parameter. Subsequently, changes in pH were modeled to account for the presence
of buffer agents and acid production during the fermentation process. This approach
allowed us to capture the dynamic nature of pH variations throughout the fermentation.
By incorporating the influence of buffer agents and acid production, we aimed to provide a
more realistic representation of the pH dynamics during ABE fermentation.
2.3. pH Effect on Growth and Production Profiles

Activity of enzymes depend on hydrogen ions present in solution and hence, each
enzyme acts optimally at a specific value of pH [39].
Enzyme.H + S K ⇔ Enzyme.H.S ⇒ Enzyme.H + P
Enzyme.H + H + δ pH1 ⇔ Enzyme.H2+

Enzyme.H δ pH2 ⇔ Enzyme− + H +
where, S and P indicate substrate and product.
The fact was incorporated in the proposed model and following expression of substrate
saturation constant was found successful to confine pH-dependency in all rates involved
in the model:
i.pH2
 
H + δ
i.pH j
Kj = K ij 1 + i.pH1 +  (5)
δ H+
j
i.pH
where, K ij andKj are substrate saturation constant and pH-dependent substrate satu-
ration constant, respectively, for all rates involved in model. i denotes substrate utilized
for the production of metabolite j. H + indicates hydrogen ion concentration present in the
culture, which was calculated according to the method described in the previous section.
i.pH1 i.pH2
δj and δj denote equilibrium constants for key enzyme(s) when its active sites act
as a base and an acid.
2.4. Growth
C. acetobutylicum contains a biphasic metabolic pathway [40]. The first phase of
metabolism (acidogenesis) incorporates the exponential growth of the biomass associated
with the production of acetic and butyric acid consuming glucose as the sole carbon source.
Production of acetone, ethanol, and butanol occurs along with the consumption of glucose,
acetic acid, and butyric acid while cells reach the stationary phase. This phase of organism’s
metabolism is called solventogenesis [33] (Figure 1).
Biomass growth kinetics can be described in the following equations:
dX
= α1 ( µ − K d ) X (6)
dt
µmax G

X
µ= 1− (7)
G.pH G2 X max
K Biomass +G+ X
K IG
where, X indicates dry biomass (g·L−1 ), and Kd is endogenous metabolism coefficient
(g·L−1 ), which incorporates the consumption of cell substances (The term Kd X is called
as cell death rate.). α1 is represented as a control coefficient to regulate the first phase of
metabolic pathway (acidogenesis). µ and µmax are specific growth rate (h−1 ) and maximum
specific growth rate (h−1 ), respectively. G indicates glucose
concentration
(g·L−1 ). X max
represents maximum dry biomass (g·L−1 ) and term 1 − XX max describes the decrease in
growth due to synthesis of toxic chemicals and depletion of nutrients in the media with the
G.pH
Microorganisms 2023, 11, x FOR PEERcourse fermentation time [11,17]. K Biomass and K X
REVIEWof
IG are the pH-dependent glucose satura-
5 of 23
tion constant (g·L−1 ) and the substrate inhibition constant for glucose (g·L−1 ), respectively.
Figure 1.
Figure 1. Schematic
Schematic representation
representation of involvement of
of involvement of substrates
substrates and products in
and products in both
both metabolic
metabolic
phases (acidogenesis and solventogenesis) of fermentation in C. acetobutylicum. (A) Acidogenesis
phases (acidogenesis and solventogenesis) of fermentation in C. acetobutylicum. (A) Acidogene-
contains one substrate (glucose) and three products (biomass, acetic acid, and butyric acid). (B) Sol-
sis contains one substrate (glucose) and three products (biomass, acetic acid, and butyric acid).
ventogenesis phase incorporates three substrates (glucose, acetic acid, and butyric acid) and three
(B) Solventogenesis
products phase incorporates
(acetone, butanol, and ethanol).three substrates
Apart (glucose,
from biomass and acetic acid, and
metabolites, butyric
gases, acid) and
viz. hydrogen
three products (acetone, butanol, and ethanol). Apart from biomass and metabolites,
and carbon dioxide are also evolved throughout the fermentation (not shown in figure). During gases, viz.
hydrogen and carbon dioxide are also evolved throughout the fermentation (not
model development, all intracellular steps of metabolic pathway were assumed in steady-state and shown in figure).
only extracellular
During substratesall
model development, and products were
intracellular steps taken into account.
of metabolic pathway The figure
were also shows
assumed rates of
in steady-state
and only extracellular
production regulated by substrates and products
control coefficients forwere taken into
acidogenesis (𝛼account. The figure also(𝛼shows
) and solventogenesis ). Torates
sim-
plify
of the complex
production metabolism
regulated of C. acetobutylicum,
by control coefficients forwe have created
acidogenesis (α1a) schematic representation
and solventogenesis (α2 ).that
To
illustratesthe
simplify thecomplex
two metabolic phases.
metabolism Inacetobutylicum,
of C. order to enhancewe clarity and readability,
have created a schematicwerepresentation
have divided
this representation
that illustrates the twointometabolic
two separate figures.
phases. In order to enhance clarity and readability, we have divided
this representation into two separate figures.
Biomass growth kinetics can be described in the following equations:
2.5. Acidogenesis
𝑑𝑋
= 𝛼 (𝜇 − 𝐾 in)𝑋C. acetobutylicum, which, on con-
Acidogenesis is the first phase of fermentation (6)
𝑑𝑡
sumption of carbon source in the culture media, incorporates rapid biomass growth and
synthesis of acids (acetic and butyric acid). Rates of production of acids are described
µ 𝐺 𝑋
as follows: µ= r A G𝐺
max 1 −
𝑋
(7)
r A =𝐾 . G.pH + 𝐺 + (8)
(K A + G ) 1/ 𝐾1 − K A
IA
where, 𝑋 indicates dry biomass (g·L−1), and 𝐾 is endogenous metabolism coefficient

r max
B G (The term 𝐾 𝑋 is called(9)
(g·L−1), which incorporates = consumption
r B the G.pH of 2 cell substances
as
cell death rate.). 𝛼 is represented (K Bas a + G + KGcoefficient
control B ) 1/ 1 − to
B
Kregulate
IB the first phase of met-
IG
abolic pathway (acidogenesis). µ and µ are specific growth rate (h−1)−and maximum
where, r A and r B are rate of synthesis for acetic (A) and butyric (B) acid (h 1 ),−1respectively.
specific growth rate (h ), respectively. 𝐺 indicates glucose concentration (g·L−).
−1 𝑋 rep-
r max
A and r B
max are maximum rates of synthesis for acetic and butyric acid (h 1 ) and their
resents maximum dry biomass (g·L−1)−and term 1 − describes
G.pH the
G.pH decrease in
pH-dependent saturation constants (g·L 1 ) are indicated as K A and K B , respectively.
growth due
Previous to synthesis
reports postulated of toxic chemicals andofdepletion
that accumulation acids is oneof nutrients in the
of the critical mediawhich
factors with
.
the course
create of fermentation
cellular stress resultingtime [11,17]. 𝐾transition
in metabolic andto𝐾solventogenesis
are the pH-dependent glucose
[41]. Additionally,
saturation constant (g·L −1) and the substrate inhibition constant for glucose (g·L−1), respec-
acids control their own production via negative feedback loops [32]. To account for this
tively.
fact in rates of acid production, two product inhibition constants, (g·L−1 ) as K I A and K IB ,
are introduced for acetic and butyric acid, respectively. Previous results from fermentation
2.5. Acidogenesis
experiments illustrated that butyric acid was produced at a lower rate than acetic acid and
Acidogenesis
its expression only is theplace
took first after
phasea oflagfermentation
in the early hoursin C. ofacetobutylicum,
fermentation.which, on con-
For capturing
sumption
these of carbon
features of the source in the culture
fermentation dynamics,media, incorporates
substrate inhibition rapid
wasbiomass
taken ingrowth
accountand
in
synthesis of acids (acetic and butyric acid). Rates of production of acids are described as
follows:
the rate of butyric acid production (unlike in the rate of acetic acid formation). K BIG denotes
glucose inhibition constant (g·L−1 ) for butyric acid production.
2.6. Solventogenesis
In the solventogenesis phase, the organism consumes the produced acids in the
acidogenesis phase along with the carbon source in the media and produces solvents
(acetone, butanol, and ethanol) (Figure 1). It was observed from fermentation experiment
that consumption of glucose and both acids occurred simultaneously. The fact was included
in following rate equation of solvent production:
riG.max riA.max riB.max GAB

ri = G.pH

A.pH

B.pH
(10)
i
G + Ki A + Ki B + Ki 1/ 1 − K Ii
where, i stands for acetone, butanol, and ethanol. riG.max , riA.max , and riB.max denote maxi-
mum rate of solvent production(h−1 ) for glucose, acetic acid, and butyric acid, respectively.
G.pH A.pH B.pH
Ki , Ki , and Ki are pH-dependent glucose, acetic acid, and butyric acid saturation
constants for solvents production (g·L−1 ). K Ii symbolizes product inhibition constant for
solvents. Equation (10) was found successful to incorporate the simultaneous consumption
of three substrates [42].
Apart from acids and solvents, hydrogen and carbon dioxide are also the evolved in
both phases of ABE fermentation. Hydrogenase catalyzes oxidation of reduced ferredoxin
for hydrogen production. Carbon dioxide is evolved through two conversions, pyruvate to
acetyl-CoA and acetoacetate to acetone catalyzed by pyruvate-ferredoxinoxidoreductase
and acetoacetate decarboxylase, respectively [43]. Consumption of glucose in acidogenesis
and simultaneous consumption of glucose, acetic acid, and butyric acid in solventogenesis
phase yield carbon dioxide. In addition, hydrogen is also released throughout the fermen-
tation with lower rate than carbon dioxide [41]. Rates of hydrogen and carbon dioxide
evolution in both phases are described as follows:
r G.max
H2 G
rG
H2 = G.pH
(11)
K H2 +G
G.max G
rCO
G 2
rCO2
= G.pH
(12)
KCO2 + G
A.max r B.max AB
rCO
AB 2 CO2
rCO 2
= A.pH

B.pH
(13)
A + KCO2 B + KCO2
where, rate and maximum rate of hydrogen evolution are denoted as r G G.max ,
H2 and r H2
G.pH G
respectively, and K H2 is glucose saturation constant for hydrogen evolution. rCO 2
and
AB − 1
rCO2 represent the rate of carbon dioxide evolution (h ) from glucose alone and from
G.max , r A.max , and r B.max
simultaneous utilization of acetic acid + butyric acid, respectively. rCO 2 CO2 CO2
− 1
denote maximum carbon dioxide evolution rate (h ) from glucose, acetic acid, and butyric
G.pH A.pH B.pH
acid, respectively. KCO2 , KCO2 , and KCO2 are pH-dependent glucose, acetic acid, and
butyric acid saturation constants (g·L−1 ) for carbon dioxide evolution.
2.7. Ordinary Differential Equations Encompassing Mass Balance of Fermentation

Following equations were constructed to represent the growth and production profiles
of fermentation:
2.7.1. Consumption of Glucose

Glucose was used as a main carbon source in this fermentation for yielding biomass
and other metabolites. More precisely, glucose is the sole carbon source in acidogenesis,
while glucose is utilized simultaneously with acetic and butyric acid for synthesizing
solvents in solventogenesis. Therefore, the consumption rate of glucose for production of
biomass, acids, solvents, and carbon dioxide is represented as follows:
− dG
dt = −Y X
G α1 µ X − Y G α1 r A X − Y G α1 r B X − Y G α2 r Act X − Y G α2 r Et X
A B Act Et
G (14)
−Y G α2 r But X − Y G rCO 2
X
But CO2
where, Y G , Y G , Y G , Y G , YG , Y G , and Y G denote the yield coefficients (g·g−1 ) of glucose

X A B Act Et But CO2
consumption with respect to biomass, acetic acid, butyric acid, acetone, ethanol, butanol
formation, and carbon dioxide evolution, respectively. α1 and α2 indicate control coefficients
for acidogenesis and solventogenesis, respectively.
2.7.2. Synthesis and Consumption of Acetic and Butyric Acid

C. acetobutylicum produces acetic and butyric acid utilizing glucose as the sole carbon
source in the acidogenesis phase of metabolism, and thereafter consumes the produced
acids simultaneously with glucose in second phase for solvent production. Mole balances
of acetic and butyric acid are represented as follows in ordinary differential equations:
dA AB
= α1 r A X − Y A α2 r Act X − Y A α2 r Et X − Y A α2 r But X − Y A α2 rCO X (15)
dt Act Et But CO2 2
dB AB
= α1 r B X − Y B α2 r Act X − Y B α2 r Et X − Y B α2 r But X − Y B α2 rCO X (16)
dt Act Et But CO2 2
where, Y A , YA , Y A , and Y A indicate the yield coefficients (g·g −1 ) of acetic acid con-
Act Et But CO2
sumption with respect to acetone, ethanol, butanol formation, and carbon dioxide evolution.
Similarly, Y B , Y B , Y B , and Y B indicate the yield coefficients (g·g −1) of butyric acid con-
Act Et But CO2
sumption with respect to acetone, ethanol, butanol formation, and carbon dioxide evolution.
2.7.3. Synthesis of Acetone, Ethanol, Butanol

The bacterium grows rapidly in acidogenesis and produces acids. Accumulation of
acids drives a significant decrease in pH in media leading causes toxic effect for cells. To
overcome the toxic effect from produced acids, cells shift their metabolism to solventoge-
nesis, which leads to consumption of acids and synthesis of solvents. The production of
solvents can be written as the following equations:
d( Act)
= α2 r Act X (17)
dt
d( Et)
= α2 r Et X (18)
dt
d( But)
= α2 r But X (19)
dt
2.7.4. Hydrogen Evolution and Carbon Dioxide Evolution

C. acetobutylicum releases hydrogen and carbon dioxide in both phases of metabolism.
It has been reported that hydrogen evolution rate is higher in acidogenesis than solvento-
genesis and carbon dioxide evolution rate is approximately similar in both phases [41,43].
Following equations represent the evolution of hydrogen and carbon dioxide:
d( H2 )
= rG
H2 X (20)
dt
d(CO2 )
G AB

= rCO + α 2 r CO X (21)
dt 2 2
2.7.5. Consumption of Phosphate

Previous studies reported that KH2 PO4 and K2 HPO4 act as buffer agents in bacterial
media [35]. However, KH2 PO4 and K2 HPO4 are also the source of phosphate during
metabolic activities of organism [34]. Therefore, we took phosphate consumption and its
effect on buffer strength of solution into account for modeling the pH profile of fermentation.
Equations (22) and (23) represent consumption of KH2 PO4 and K2 HPO4 , respectively.
d(KH2 PO4 )
= −γKH2 PO4 µX − mKH2 PO4 X (22)
dt
d(K2 HPO4 )
= −γK2 HPO4 µX − mK2 HPO4 X (23)
dt
where, γKH2 PO4 and γK2 HPO4 are the control coefficients for consumption of KH2 PO4 and
K2 HPO4 , respectively. mKH2 PO4 and mK2 HPO4 are maintenance coefficients of KH2 PO4 and
K2 HPO4 for biomass, respectively.
2.8. Experimental Data

The experimental data used in this study for parameter estimation and model valida-
tion were obtained from our previously published paper [32]. The reference paper provides
detailed descriptions of all experimental procedures. In this study [32], all fermentation
experiments were conducted at a temperature of 37 ◦ C in triplicate. To measure the biomass,
optical density (A600 ) was utilized as a proxy, which was then converted to dry cell weight
(g/L) using a correlation curve established between the absorbance measured at 600 nm and
the corresponding dry cell weight. Specifically, Gholizadeh el al. [44] determined that one
unit of OD600 roughly corresponded to 0.79 g/L of dry cell weight for C. acetobutylicum cells.
2.9. Validation of Model

Model parameters were estimated by fitting the data set of Experiment 1, and data
of Experiment 2 and Experiment 3 were used to validate the model prediction. Ordinary
differential equations (Equations (1)–(25)) were solved by using ODE 15 s solver available
in MATLAB (Mathworks, Natick, MA, USA) for simulating experimental results. Figures 2
and 3 show a comparative fit of model predictions to experimental data.
acetobutylicum cells.
termined that one unit of OD600 roughly corresponded to 0.79 g/L of dry cell weight for C.
acetobutylicum cells.
2.9. Validation of Model
Model parameters
2.9. Validation of Model were estimated by fitting the data set of Experiment 1, and data of
Experiment 2 and Experiment 3 were used to validate the model prediction. Ordinary
Model parameters were estimated by fitting the data set of Experiment 1, and data of
Microorganisms 2023, 11, 1610 differential equations (Equations (1)–(25)) were solved by using ODE 15 s solver available 9 of 23
Experiment 2 and Experiment 3 were used to validate the model prediction. Ordinary
in MATLAB (Mathworks, Natick, MA, USA) for simulating experimental results. Figures
differential equations (Equations (1)–(25)) were solved by using ODE 15 s solver available
2 and 3 show a comparative fit of model predictions to experimental data.
in MATLAB (Mathworks, Natick, MA, USA) for simulating experimental results. Figures
2 and 3 show a comparative fit of model predictions to experimental data.
Figure Figure 2. Depiction

2. Depiction of of a dynamics of
a dynamics of 90
90hhlong
longbatch
batch fermentation system
fermentation using C.
system acetobutylicum
using at
C. acetobutylicum at
starting pH of 6.4 (“Experiment 1”). Figure shows comparative scenario of experimental (dots) and
starting pH2.of
Figure
simulation 6.4 (“Experiment
Depiction
(solid of a dynamics
lines) 1”). Figure
of 90 h longshows
data of fermentation. batch
This comparative
setfermentation scenario
system
of experimental was of
datausing C. experimental
acetobutylicum
used at(dots) and
to estimate the
starting
simulation pH of
(solid
parameters of6.4 (“Experiment
lines) data
proposed 1”). Figure shows
of fermentation.
model. comparative
This scenario of experimental
set of experimental data was used (dots)toand
estimate the
simulation (solid lines) data of fermentation. This set of experimental data was used to estimate the
parameters of proposed model.
parameters of proposed model.
(A)
(A)
Microorganisms 2023, 11, x FOR PEER REVIEW 10 of 23
(B)
Figure 3. Demonstration of comparative profiles of experimental (dots) and simulation (solid lines)
data of ABE fermentation in a batch process at starting of (A) pH 5.7 (“Experiment 2”) and (B) pH 4.4
(“Experiment 3”). These sets
Figure 3. Demonstration of experimental
of comparative profilesdata were used(dots)
of experimental to validate the predictions
and simulation of model.
(solid lines)
data of ABE fermentation in a batch process at starting of (A) pH 5.7 (“Experiment 2”) and (B) pH
4.4 (“Experiment 3”). These sets of experimental data were used to validate the predictions of model.
3. Results
Numerous reports have suggested that the pH level of the fermentation medium
plays a crucial role in regulating the physiological behavior of Clostridium acetobutylicum
3. Results
Numerous reports have suggested that the pH level of the fermentation medium plays
a crucial role in regulating the physiological behavior of Clostridium acetobutylicum during
acetone–butanol–ethanol (ABE) fermentation [31,45]. It has been observed that the optimal
pH range for acidogenesis is between 5.0–6.0, while for solventogenesis, it lies in the range
of 4.0–5.0 [45]. In light of these findings, we have developed a physiological model that
accounts for the pH profile in conjunction with other extracellular metabolites during ABE
fermentation. The model, as illustrated in Figure 1, incorporates two control strategies:
(a) metabolic transition from acidogenesis to solventogenesis is regulated through biomass
growth, with acidogenesis occurring during the exponential phase and solventogenesis
during the stationary phase, as supported by experimental data; and (b) the rates of
metabolite formation are controlled by the organism’s modulation of its metabolism for
optimum growth and synthesis of metabolites in both phases, according to different pH
levels in the medium. We have employed three sets of fermentation experimental data,
starting with initial pH values of 6.4, 5.7, and 4.4, which are referred to as “Experiment 1,”
“Experiment 2,” and “Experiment 3,” respectively, throughout the text of the paper [32].
The pH of a fermentation media was determined by calculating the hydrogen ion
concentration dissociated from two acids, acetic acid (Ka = 1.7378 × 10−5 ) and butyric acid
(Ka = 1.5136 × 10−5 ). During the initial phase of the fermentation (up to about 10 h), the pH
remained stable due to the buffer capacity provided by KH2 PO4 and K2 HPO4 (0.75 g·L−1 )
present in the media. Experimental observations indicated that during this period, a small
amount of acetic acid (1.4–1.7 g·L−1 ) and butyric acid (0.08–0.7 g·L−1 ) were produced, and
the H+ ions produced from the acids were balanced by the buffer agents to maintain a
constant pH. However, after this initial phase, the pH of the media rapidly decreased due
to the production of acids, and the buffer was no longer effective in controlling the pH
(Figures 2 and 3).
Furthermore, variations in culture performance due to different starting pH values
were incorporated into a model using an expression that related the substrate saturation
constant and the hydrogen ion concentration present in the culture (Equation (5)). The
i.pH1 i.pH2
values of equilibrium constants (δj and δj ) used in the model were determined by
optimizing the production rates of individual products at a specific pH (Table 1). The
suitability of this expression for accounting for the effect of pH variation in the rates of
products was demonstrated through comparative observations between simulation and
experimental data (Figure 4). The following sections of the study include the estimation of
model
Microorganisms 2023, 11, x FOR PEER REVIEWparameters, details of the control strategy, and outcomes of model simulations,12andof 23
their comparison with experimental data.
Figure 4. Rates of biomass accounting the variation in starting pH level.

Figure 4. Rates of biomass accounting the variation in starting pH level.
3.1. Estimation of Model Parameters

The model parameters were estimated by fitting the fermentation data of Experiment
1, which had an initial pH of 6.4 (Table 1). Our approach involved an iterative process,
where we adjusted the parameter values until the simulation data aligned well with the
experimental data. Subsequently, the model, incorporating these estimated parameter val-
ues, was utilized to simulate the growth, consumption, and production profiles under dif-
ferent pH conditions. Preliminary attempts were made, and the parameters were im-
proved using a dynamic optimization algorithm called “fmincon” in MATLAB (Math-
works, Natick, MA, USA). The estimated model parameters can be found in Table 1. The
Table 1. Estimated parameter values used in proposed model.
Parameter Unit Value Parameter Unit Value Parameter Unit Value

µmax h−1 0.78 KG Act g ·L−1 3.2 YG g·gg·g−1 1.07
Act
S.pH1
KX IG g ·L−1 170 δAct - 5.00 × 10−5 YG g ·g−1 0.93
Et
S.pH2
G
K Biomass g ·L−1 3 δAct - 8.1 × 10−12 YG g ·g−1 1.1
But
G.pH1
δBiomass - 4.00 × 10−5 A
K Act g ·L−1 3 Y G g ·g−1 2.2
CO2
G.pH2
δBiomass - 9.00 × 10−7 K BAct g ·L−1 3 Kd h−1 0.000001
r max
A h−1 0.9 K I Act g ·L−1 24 YA g ·g−1 0.057
Act
KG A g ·L−1 0.1 G.max
r But h−1 0.87 YA g ·g−1 0.028
Et
G.pH1
δA - 5.00 × 10−7 A.max
r But h−1 0.79 Y A g ·g−1 0.3
But
G.pH2
δA - 5.00 × 10−7 B.max
r But h−1 0.83 Y A g ·g−1 0.4
CO2
KI A g ·L−1 2.8 G
K But g ·L−1 3.5 Y B g ·g−1 0.28
Act
S.pH1
r max
B h−1 1.8 δBut - 7.00 × 10−5 YB g ·g−1 0.15
Et
S.pH2
K BIG g ·L−1 80 δBut - 8.1 ×10−12 Y B g ·g−1 0.33
But
K BG g ·L−1 30.2 A
K But g ·L−1 3.1 Y B g ·g−1 0.4
CO2
G.pH1
δB - 4.00 × 10−5 B
K But g ·L−1 3.3 γKH2 PO4 - 1 × 10−10
G.pH2
δB - 1.00 × 10−8 K IBut g ·L−1 35 mKH2 PO4 h−1 1 × 10−8
K IB g ·L−1 8 G.max
rCO 2
h−1 0.35 γK2 HPO4 - 1.7 × 10−1
G.max
r Et h−1 0.8 G
KCO 2
g ·L−1 12.5 mK2 HPO4 h−1 9.9 × 10−3
S.pH1
A.max
r Et h−1 0.54 δgas - 0.002 pKa - 6.11
S.pH2
B.max
r Et h−1 0.57 δgas - 8.1e-12 KaA - 1.7378 × 10−5
KEtG g ·L−1 4.1 A.max
rCO 2
h−1 0.1 KaB 1.5136 × 10−5
S.pH1
δEt - 3.00 × 10−5 B.max
rCO 2
h−1 0.1
S.pH2
δEt - 8.1 × 10−12 A
KCO 2
g ·L−1 5
KEtA g ·L−1 3.5 B
KCO 2
g ·L−1 5
KEtB g ·L−1 3.8 r G.max
H2 h−1 0.19
K IEt g ·L−1 20 KGH2 g ·L−1 2
r G.max
Act h−1 0.8 YG g ·g−1 0.42
X
A.max
r Act h−1 0.6 YG g ·g−1 0.33
A
r B.max
Act h−1 0.75 YG g ·g−1 0.33
B
3.1. Estimation of Model Parameters

The model parameters were estimated by fitting the fermentation data of Experiment 1,
which had an initial pH of 6.4 (Table 1). Our approach involved an iterative process, where
we adjusted the parameter values until the simulation data aligned well with the exper-
imental data. Subsequently, the model, incorporating these estimated parameter values,
was utilized to simulate the growth, consumption, and production profiles under different
pH conditions. Preliminary attempts were made, and the parameters were improved using
a dynamic optimization algorithm called “fmincon” in MATLAB (Mathworks, Natick,
MA, USA). The estimated model parameters can be found in Table 1. The initial values of
solvents (acetone, butanol, and ethanol) and gases (hydrogen and carbon dioxide) were
set to zero, as it was assumed that inoculums in the late exponential phase were used
during the experiments before entering the solventogenesis phase. The initial values of all
variables are reported in Table 2. The initial values of substrates (glucose, KH2 PO4 , and
K2 HPO4 ) were taken from experimental data, while the initial values of all other variables
were estimated based on the fitting of experimental data from Experiment 1.
Table 2. Table represents initial values employed for parameters estimation using experimental data
(starting pH 6.4). Table also contains validation of initial values during the simulation to predict the
production profiles of other two batch fermentation experiments (starting pH 5.7 and 4.4).
Substrate/Product Initial Values (g·L−1 )

Starting pH 6.4 Starting pH 5.7 Starting pH 4.4
Glucose 43.981 42.226 45.391
Biomass 0.02 0.02 0.02
Acetic acid 0.002 0.002 0.002
Butyric acid 0.004 0.004 0.004
Ethanol 0 0 0
Acetone 0 0 0
Butanol 0 0 0
Hydrogen 0 0 0
Carbon dioxide 0 0 0
KH2 PO4 0.75 0.75 0.75
K2 HPO4 0.75 0.75 0.75
3.2. Control Mechanism for Metabolic Switch from Acidogenesis to Solventogenesis

Various fermentations were carried out at different uncontrolled pH, and it was found
that acid production only occurred during the exponential phase of biomass growth, while
solvent production began when the growth entered the stationary phase. Therefore, the
transition of biomass growth from the exponential to the stationary phase was used as an
indicator for the metabolic shift in the development of the current model. To this end, two
control coefficients (α1 and α2 ) were included in the model for the two metabolic phases.
α1 was used to control acid production, while α2 was used to control solvent production.
The coefficients were assigned values of 0 and 1 to represent the on and off states for
maintaining only one active phase at a given time. Additionally, it was observed that
the time for metabolic switching varied from approximately 18 to 30 h in different sets
of experimental data and depended on the growth conditions. The following inequality
expressions were used in model to confine the metabolic shift:
For acidogenesis
X X
α1 = 1; i f 0 ≤ ≤ R A and α2 = 0; i f > RS (24)
X max X max
For solventogenesis
X X
α2 = 1; i f 0 ≤ ≤ RS and α1 = 0; i f max > R A (25)
X max X
where, R A and RS represent constant ratios of biomass concentration and maximum
biomass concentration for acidogenesis and solventogenesis respectively. Their values
(R A = 0.99 and RS = 0.82) were estimated on the basis on experimental data (from
Experiment 1). The inequalities were such that value of both control coefficients was
1 when XX max lies between R A and R S . This means that both metabolic phases were active
in this interval, consistent with experimental observations. Individually, acidogenesis and
solventogenesis were active before (α1 = 1 and α2 = 0) and after (α1 = 0 and α2 = 1) this
interval respectively (Figure 5).
1). The inequalities were such that value of both control coefficients was 1 when lies
between 𝑅 and 𝑅 . This means that both metabolic phases were active in this interval,
consistent with experimental observations. Individually, acidogenesis and solventogene-
sis were active before (𝛼 = 1 and 𝛼 = 0) and after (𝛼 = 0 and 𝛼 = 1) this interval respec-
Microorganisms 2023, 11, 1610 tively (Figure 5). 13 of 23
Figure 5. Representation of control strategy used in model to confine the metabolic transition from
Figure 5. Representation of control strategy used in model to confine the metabolic transition from
acidogenesis to solventogenesis. Figure shows profiles of control coefficients for acidogenesis (α ,
acidogenesis to solventogenesis. Figure shows profiles of control coefficients for acidogenesis (𝛼 ,1
solid lines) and solventogenesis (α , dotted lines) over the time course of fermentation. The values of
solid lines) and solventogenesis (𝛼 2, dotted lines) over the time course of fermentation. The values
of0 0and
and 1 of control
1 of coefficients
control coefficients (α1(𝛼
andand
α2 ) 𝛼embrace switch-off
) embrace and and
switch-off -on of
-onproduction of each
of production of metabolite
each me-
from specific substrate(s) consistent with the decision-making mechanism of the organism.
tabolite from specific substrate(s) consistent with the decision-making mechanism of the organism.
3.3. Acidogenesis Incorporates Rapid Biomass Growth and Production of Acids over the
3.3. Acidogenesis
Consumption Incorporates Rapid Biomass Growth and Production of Acids over the
of Glucose
Consumption of Glucose
The production kinetics of biomass was modeled using Equations (1) and (2). Ex-
The production
perimental kineticsindicated
measurements of biomass
thatwas modeledbiomass
maximum using Equations (1) and
concentrations (2). Exper-
ranging from
imental measurements
− 1 indicated that maximum biomass concentrations
1.8–2.0 g·L were achieved under different initial pH conditions. The close proximity ranging from
1.8–2.0 g·L−1 were achieved
of the maximum under differentover
biomass concentrations initial pH conditions.
a significant changeThein close
mediaproximity of
pH (4.5–6.8)
the maximum
suggests biomassisconcentrations
that growth over a significant
relatively insensitive to initial pHchange in media
variations in thispH (4.5–6.8)
range. sug-
However,
gests that growth
low biomass growthis relatively insensitive
was observed during to
theinitial pH variations
fermentation process,inwhich
this range.
can beHowever,
attributed
low biomass growth was observed
to the presence of toxic chemicals in during
the the
media fermentation
and substrate process, which
inhibition. To can be at-
account for
tributed to the presence of toxic chemicals Xin the media and substrate inhibition. To ac-

the slow biomass growth, a term of 1 − X max and substrate inhibition were incorporated
count forgrowth
into the the slow biomass
kinetics growth,
model. Theasubstrate 1−
term of inhibition and substrate
constant inhibition were
was determined to be
170 g·L−1 . Acid production rates were simulated using Equations (3) and (4). Experimental
data indicated that the rate of acetic acid formation was higher than butyric acid in the
early stages of acidogenesis [32]. To account for the delay in butyric acid synthesis, sub-
strate inhibition was included in the model. Additionally, the well-known phenomenon of
product inhibition was successfully incorporated into the rate expressions [18].
Figure 2 presents the simulation results in comparison with the fermentation data
obtained from Experiment 1. During this experiment, the initial pH of 6.4 was maintained,
but due to the production of a considerable amount of acetic acid (2.7 g·L−1 ) and butyric
acid (3.9 g·L−1 ) in the first metabolic phase, the pH level dropped to 3.0. The metabolic
activity of cells ceased once the pH level went below 3.0. During this phase, the maximum
concentration of acids recorded during this phase was 1.8 g·L−1 , while approximately
10 g·L−1 of glucose was consumed. Acid production was halted after 18 h of fermentation
time, and at the same time, the biomass entered the stationary phase.
Subsequently, the model was simulated using the inputs obtained from Experiment 2.
The comparison between the predictions generated by the model and the measurements can
be observed in Figure 3A. The pH was initially set to a value of 5.7 during the simulations
and was not controlled thereafter, mimicking the experimental conditions. The production
of acid led to a further reduction in pH, reaching a final value of 4.4. It is noteworthy that
the cellular metabolism remained active throughout the remaining fermentation duration
despite the acidic conditions. The acidogenesis phase resulted in the detection of total
concentrations of acetic and butyric acid, amounting to 2.2 and 4.0 g·L−1 , respectively, while
approximately 10 g·L−1 of glucose was consumed. Notably, acid production persisted for a
longer duration of 27 h compared to Experiment 1.
To mimic the growth conditions described in Experiment 3, we conducted simulations

using an initial pH of 4.4 and a glucose concentration of 45.39 g·L−1 , as detailed in Table 2.
The resulting model outcomes were compared to experimental measurements (Figure 3B).
During the experiment, the pH decreased from 4.4 to 3.0, negatively impacting cellular
metabolic activities. This pH profile facilitated the display of dynamic metabolite profiles
by the cells, similar to those observed in Experiment 1. Acid production occurred for up to
30 h, resulting in final concentrations of 2.3 g·L−1 1 and 2.5 g·L−1 for acetic acid and butyric
acid, respectively. Although acid production continued for the full 30 h, it was lower than
in the other two pH profiles, possibly due to the cells already being under stress from the
low starting pH. This led to a decreased metabolic rate.
Upon reaching its peak, the biomass elicited a shift in metabolism from acidogenesis
to solventogenesis. In terms of modeling, during the second phase of metabolism (sol-
ventogenesis), the values of phase control coefficients, α1 became 0 and α2 was set to 1.
In the brief interim period between acidogenesis and solventogenesis, both phase control
coefficients were set to 1, indicating the simultaneous occurrence of active acidogenesis
and solventogenesis. This approach proved advantageous in delineating the variance in
switch-time between different growth conditions, as depicted in Figure 4.
3.4. Production of Solvents Is Arisen in Solventogenesis Phase over the Consumption of Glucose
and Acids Produced in Acidogenesis Phase
During the solventogenesis phase, cells consume the acids produced in the acidogene-
sis phase along with the glucose present in the growth medium. In our proposed model,
the formation rate of solvents is described by Equation (5), which comprises three distinct
maximum rates and three substrate saturation constants for each solvent derived from all
three substrates. Based on experimental observations, we determined that glucose, acetic
acid, and butyric acid are all utilized simultaneously for solvent production, as illustrated
in Figure 3A, and this was incorporated into our kinetic model. Additionally, our rate
expression for solvent production takes into account product inhibition.
In Experiment 2, which had an initial pH of 5.7, a greater amount of solvents (ace-
tone, butanol, and ethanol) were produced compared to Experiments 1 and 3. The final
concentrations of these solvents were recorded as 3.8 g·L−1 , 2.3 g·L−1 , and 7.7 g·L−1 , re-
spectively. This pH profile allowed for the cells to remain metabolically active throughout
the fermentation process and to consume glucose, acetic acid, and butyric acid until they
were almost completely exhausted. As a result of this acid consumption, the pH of the
medium slightly increased from its lowest value of 4.4 to 4.6 (Figure 3A). In contrast,
Experiments 1 (initial pH of 6.4) and 3 (initial pH of 4.4) exhibited very low production
of solvents. In Experiment 1, the concentrations of acetone, butanol, and ethanol were
0.96 g·L−1 , 1.2 g·L−1 , and 0.25 g·L−1 , respectively. In Experiment 3, their concentrations
were recorded as 0.91 g·L−1 , 1.23 g·L−1 , and 0.28 g·L−1 , respectively. The low solvent
production in these fermentations was due to a significant decrease in pH to a level of 3.0,
which was unfavorable for metabolic activity in the cells. Collectively, during Experiments
1 and 3, the cells were unable to maintain a favorable pH level for solvent production,
whereas in Experiment 2, the cells performed well at an initial pH of 5.7. Therefore, an
uncontrolled initial pH of 5.7 was found to be favorable for higher solvent production and
for maintaining the cells in an active state for a longer period in a batch system.
3.5. Hydrogen and Carbon Dioxide Are Evolved in Both Metabolic Phases
In this study, we also aimed to predict the dynamics of hydrogen and carbon dioxide
evolution across various pH profiles during fermentation experiments. The results are
presented in Figure 6, which illustrates the evolution and rate profiles for gases during the
experiments. The findings suggest that the evolution of gases stopped in Experiment 1 and
3 when the culture reached a pH of 3.0. The concentration of gases in both experiments re-
mained below 15 g·L−1 , which was released during acidogenesis and short solventogenesis
stages before the metabolic activities ceased due to stressed growth conditions. However,
the exhaustion of available carbon sources.
Experiment 2 displayed higher concentrations of hydrogen (26 g·L−1 ) and carbon dioxide
(30 g·L−1 ) during both phases of metabolism, as cells were functional up to the exhaustion
of available carbon sources.
(A)
Hydrogen/Cabon dioxide Hydrogen/Cabon dioxide

Concentration (g/L) 30
Rate (g/L.h)
2
20
10 1
Microorganisms 2023, 11, x FOR PEER REVIEW 16 of 23
0 0
0 20 40 60 80 0 20 40 60 80
Time (h) Time (h)
(B)

Concentration (g/L)
30
Rate (g/L.h)
2
20
10 1
0 0
0 20 40 60 80 0 20 40 60 80
Time (h) Time (h)
(C)

Concentration (g/L)
30
Rate (g/L.h)
2
20
10 1
0 0
0 20 40 60 80 0 20 40 60 80
Time (h) Time (h)
Figure 6. The figure shows predicted data of rates and production profiles of hydrogen (solid
lines) and carbon dioxide (dotted lines) evolution at starting pH of (A) 6.4 (“Experiment 1”)
Figure 6. The figure shows predicted data of rates and production profiles of hydrogen (solid lines)
(B)
and5.7 (“Experiment
carbon 2”) and lines)
dioxide (dotted (C) 4.4evolution
(“Experiment 3”). pH of (A) 6.4 (“Experiment 1”) (B) 5.7 (“Ex-
at starting
periment 2”) and (C) 4.4 (“Experiment 3”).
Furthermore, we conducted an analysis of glucose consumption rate, as well as
the productivity and yield of fermentation products under various growth conditions
Furthermore, we conducted an analysis of glucose consumption rate, as well as the
(Figures 7 and 8A,B). During acidogenesis, the consumption rate of glucose (g·L−1 ·h−1 )
productivity and yield of fermentation products under various growth conditions (Fig-
was found to be higher in Experiment 1 and 3 compared to Experiment 2 (up to−1approxi-
ures 7 and 8A,B). During acidogenesis, the consumption rate of glucose (g·L .h−1) was
mately 30 h) (Figure 7). Specifically, the starting pH of 6.4 enabled cells to consume glucose
found to be higher in Experiment 1 and 3 compared to Experiment 2 (up to approximately
rapidly in the early stages of fermentation, as cells exhibited a higher growth rate and acid
30 h) (Figure 7). Specifically, the starting pH of 6.4 enabled cells to consume glucose rap-
production at this pH level. However, after 30 h (during solventogenesis), the consumption
idly in the early stages of fermentation, as cells exhibited a higher growth rate and acid
rate of glucose was almost negligible at starting pH levels of 6.4 and 4.4 due to the unfa-
production at this pH level. However, after 30 h (during solventogenesis), the consump-
tion rate of glucose was almost negligible at starting pH levels of 6.4 and 4.4 due to the
unfavorable pH level (pH 3.0) reached by the culture. At a starting pH of 5.7, cells con-
sumed a greater amount of glucose during solventogenesis than in acidogenesis, as carbon
flow favored solvent production. The highest consumption rate was observed between 40
vorable pH level (pH 3.0) reached by the culture. At a starting pH of 5.7, cells consumed
a greater amount of glucose during solventogenesis than in acidogenesis, as carbon flow
favored solvent production. The highest consumption rate was observed between 40 and
Microorganisms 2023, 11, x FOR 50 h of fermentation time, after which the rates decreased due to the accumulation
17 ofof
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products and a reduced amount of remaining glucose in the batch fermenter (Figure 7).
Figure 7. Variation in consumption rate of glucose at different starting pH levels of 6.4, 5.7 and 4.4.
Figure 7. Variation in consumption rate of glucose at different starting pH levels of 6.4, 5.7 and 4.4.
Figure 7. Variation in consumption rate of glucose at different starting pH levels of 6.4, 5.7 and 4.4.
(A)
(A)
(B)
(B)
Figure 8. Figure shows (A) productivity and (B) yield of batch fermentation runs at different initial
pH using C. acetobutylicum.
Figure 8. Additionally,
Figure
Figure shows our
8. Figure evaluation
(A)shows of and
productivity yield(B)
(A) productivity(g·g−1) and
yield
and productivity
ofyield
(B) batch (g·L−1.h
offermentation
−1) revealed
runs
batch fermentation atruns at that,
different initialinitial
different
in Experiments
pH using C. 1 and
acetobutylicum. 3, cells
pH using C. acetobutylicum. were more involved in acidogenesis than solventogenesis,
while the opposite was observed in Experiment 2 (Figure 8A,B). At a starting pH of 5.7,
both yieldAdditionally,
and productivity values were
our evaluation approximately
of yield (g·g−1) and two-fold higher
productivity (g·Lthan those
−1.h−1) revealed that,
in Experiments 1 and 3, cells were more involved in acidogenesis than solventogenesis,
while the opposite was observed in Experiment 2 (Figure 8A,B). At a starting pH of 5.7,
both yield and productivity values were approximately two-fold higher than those
Additionally, our evaluation of yield (g·g−1 ) and productivity (g·L−1 ·h−1 ) revealed
that, in Experiments 1 and 3, cells were more involved in acidogenesis than solventogenesis,
while the opposite was observed in Experiment 2 (Figure 8A,B). At a starting pH of 5.7, both
yield and productivity values were approximately two-fold higher than those observed
at starting pH levels of 6.4 and 4.4. Thus, our modeling and experimental approaches
suggested that a high starting pH level (6.4) may promote a high rate of biomass and acid
production to shorten the metabolic shift time. However, to maintain cells in an active
condition and produce a greater amount of solvents in the second phase, an intermediate
pH level should be maintained above a certain threshold (pH 4.4).
4. Discussion
The model was successful in predicting the profiles of glucose consumption, biomass
formation, acids’ and solvents’ production, pH, and gases (hydrogen and carbon dioxide)
evolution in a batch fermentation system of C. acetobutylicum. The model was used to
predict fermentation profiles at starting pH levels of 5.7 and 4.4, and these predictions were
validated using fermentation data at both pH levels. Our study emphasizes the crucial
role of pH in achieving optimal biomass yield of C. acetobutylicum and maximizing solvent
production. Additionally, the data predicted by our model suggests, that while a high
biomass yield does not necessarily result in high solvent concentrations, rapid growth
during the initial hours of a batch fermentation promotes increased acid production (Fig.
8B). However, if the objective is to enhance acetic and butyric acid yield instead of solvent
production, it is favorable to prioritize high growth during the early stages of fermentation
to yield higher acid yields.
The results showed that a starting pH of 5.7 was more suitable than pH levels of
6.4 and 4.4 for higher production of solvents. In the proposed model, it was found that
when the starting pH was higher than the optimum pH (pH 5.7), the cells grew faster and
produced acids rapidly, which led to shock and caused the cells to enter sporulation instead
of solventogenesis. Similarly, when the cells grew at a lower pH level than the optimum
level, a small amount of acids lowered the pH level significantly and forced the cells to
enter sporulation. Therefore, due to the initialization of sporulation, cells produced lower
concentrations of solvents at higher and lower starting levels of pH than pH 5.7.
It has been observed that sporulation and solventogenesis are regulated by one activa-
tor, Spo0A [46]. However, the reasons for Spo0A activating sporulation or solventogenesis
under different growth conditions are unknown [47]. At a starting pH level of 5.7, the
cells were able to hold the pH at a certain favorable level (pH 4.4) for growth through the
consumption of acids. Therefore, in this case, the cells performed metabolically well and
consumed glucose and acids up to almost exhaustion for producing high concentrations
of solvents. Our study revealed a significant impact of pH level on the utilization rate of
glucose (g/L·h) during fermentation. Specifically, we observed that maintaining a pH level
of 5.7 facilitated continuous glucose consumption throughout the process, resulting in a
higher yield of solvents (Figures 7 and 8B).
The maximum concentration of biomass achieved in the batch fermentation was not
found to be very sensitive to the starting pH level as the biomass achieved a similar maxi-
mum concentration for all three uncontrolled growth conditions. Additionally, biomass
growth showed a decline in the case of a starting pH of 6.4, which might be due to the rapid
biomass and acids production rate compared to the other pH levels. However, this decline
in biomass growth was not predicted by the proposed model. Along with the profiles of
other products, the model was also capable of predicting the concentration of hydrogen
and carbon dioxide in both phases of metabolism. Furthermore, it was observed from the
predicted profiles that under the starting pH of 5.99, solventogenesis was active for 65 h,
which is around five-fold higher than the period for the starting pH of 6.8 and 4.5. While
the activation time of acidogenesis was similar in all three different pH environments,
it indicates that the activation period of the solventogenesis phase is dependent on the
starting pH level.
5. Conclusions
We developed an uncontrolled pH-based kinetic model to capture the fermentation
dynamics of C. acetobutylicum in a batch system. The model accurately described the
consumption of glucose, formation of biomass, production of acids and solvents, pH
changes, and evolution of hydrogen and carbon dioxide gases under various experimental
conditions. Our study revealed that maintaining a pH above 4.4 is crucial for keeping
the cells in a metabolically active state, resulting in higher solvent production during the
second phase of fermentation. This model can be extended to represent the dynamics of
other types of fermentation processes, such as fed-batch and continuous systems, both with
controlled and uncontrolled pH. Furthermore, the proposed model can aid in developing
the kinetics of ABE fermentation using other sugars, such as arabinose and xylose, either
individually or in combination with glucose. While significant efforts are still needed, our
study provides a crucial foundation for identifying optimal parameters in the production
of biobutanol at an industrial scale. By elucidating the relationship between medium pH
and the production of biobutanol, our findings contribute to the development of efficient
and scalable processes. Although additional research and optimization are necessary, our
study serves as an important steppingstone towards realizing the industrial production
of biobutanol.
Author Contributions: M.K., S.S. and K.G. conceived and designed the study. M.K. developed the
modeling framework and executed all simulations. M.K., S.S. and K.G. discussed the results. M.K.
wrote the first draft of the manuscript. M.K., S.S. and K.G. reviewed and prepare the final version of
the manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: The authors would like to express their gratitude for the generous financial support
provided by the Department of Science and Technology (DST) of the Government of India (grant
number SR/FTP/ETA-0006/2010).
Data Availability Statement: The data from this research is available upon request to the correspond-
ing author.
Acknowledgments: We express our gratitude to the Indian Institute of Technology Gandhinagar
(IITGN) and the National Institute of Technology Agartala (NITA) for their invaluable support in
facilitating and enabling the successful execution of this research.
Conflicts of Interest: The authors declare no conflict of interest.
Nomenclature
A
KCO2
Acetic acid saturation constant for CO2 evolution (g·L−1 )
A.pH
KCO2 pH-dependent acetic acid saturation constant for CO2 evolution (g·L−1 )
KCOB
2
Butyric acid saturation constant for CO2 evolution (g·L−1 )
B.pH
KCO2 pH-dependent butyric acid saturation constant for CO2 evolution (g·L−1 )
KCOG
2
Glucose saturation constant for CO2 evolution (g·L−1 )
G.pH
KCO2 pH-dependent glucose saturation constant for CO2 evolution (g·L−1 )
KG H2 Glucose saturation constant for H2 evolution (g·L−1 )
G.pH
K H2 pH-dependent glucose saturation constant for H2 evolution (g·L−1 )
KG A Glucose saturation constant for acetic acid formation (g·L−1 )
G.pH
KA pH-dependent glucose saturation constant for acetic acid formation (g·L−1 )
A
K Act Acetic acid saturation constant for acetone formation (g·L−1 )
A.pH
K Act pH-dependent acetic acid saturation constant for acetone formation (g·L−1 )
K BAct Butyric acid saturation constant for acetone formation (g·L−1 )
B.pH
K Act pH-dependent butyric acid saturation constant for acetone formation (g·L−1 )
KG Act Glucose saturation constant for acetone formation (g·L−1 )
G.pH
K Act pH-dependent glucose saturation constant for acetone formation (g·L−1 )
K BG Glucose saturation constant for butyric acid formation (g·L−1 )
G.pH
KB pH-dependent glucose saturation constant for butyric acid formation (g·L−1 )
G
K Biomass Glucose saturation constant for biomass synthesis (g·L−1 )
G.pH pH-dependent glucose saturation constant for biomass
K Biomass
synthesis (g·L−1 )
A
K But Acetic acid saturation constant for butanol formation (g·L−1 )
A.pH pH-dependent acetic acid saturation constant for butanol
K But
formation (g·L−1 )
B
K But Butyric acid saturation constant for butanol formation (g·L−1 )
B.pH pH-dependent butyric acid saturation constant for butanol
K But
G
K But Glucose saturation constant for butanol formation (g·L−1 )
G.pH pH-dependent glucose saturation constant for butanol
K But
A
KEt Acetic acid saturation constant for ethanol formation (g·L−1 )
A.pH pH-dependent acetic acid saturation constant for ethanol
KEt
B
KEt Butyric acid saturation constant for ethanol formation (g·L−1 )
B.pH pH-dependent butyric acid saturation constant for ethanol
KEt
G
KEt Glucose saturation constant for ethanol formation (g·L−1 )
G.pH pH-dependent glucose saturation constant for ethanol
KEt
KI A Product inhibition constant for acetic acid formation (g·L−1 )
K I Act Product inhibition constant for acetone formation (g·L−1 )
K IB Product inhibition constant for butyric acid formation (g·L−1 )
K IBut Product inhibition constant for butanol formation (g·L−1 )
K IEt Product inhibition constant for ethanol formation (g·L−1 )
K BIG Substrate inhibition constant for butyric acid formation (g·L−1 )
KX IG Glucose inhibition constant for biomass synthesis (g·L−1 )
KaA Dissociation constant for acetic acid
KaB Dissociation constant for butyric acid
Kd Endogenous metabolism constant for cell growth (h−1 )
X max Maximum dry cell weight (g·L−1 )
Acetic acid consumption yield coefficient with respect to CO2
Y A
CO2 evolution (g·g−1 )
Acetic acid consumption yield coefficient with respect to acetone
Y A
Act formation (g·g−1 )
Acetic acid consumption yield coefficient with respect to butanol
Y A
But formation (g·g−1 )
Acetic acid consumption yield coefficient with respect to ethanol
YA
Et formation (g·g−1 )
Butyric acid consumption yield coefficient with respect to CO2
Y B
Butyric acid consumption yield coefficient with respect to acetone
Y B
Butyric acid consumption yield coefficient with respect to butanol
Y B
Butyric acid consumption yield coefficient with respect to ethanol
YB
Glucose consumption yield coefficient with respect to CO2
Y G
Glucose consumption yield coefficient with respect to acetic acid
YG
A formation (g·g−1 )
Glucose consumption yield coefficient with respect to acetone
Y G
Glucose consumption yield coefficient with respect to butyric acid

YG
B formation (g·g−1 )
Glucose consumption yield coefficient with respect to butanol
Y G
Glucose consumption yield coefficient with respect to ethanol
YG
Glucose consumption yield coefficient with respect to biomass
YG
X growth (g·g−1 )
mK2 HPO4 maintenance coefficients of K2 HPO4 for biomass (h−1 )
mKH2 PO4 maintenance coefficients of KH2 PO4 for biomass (h−1 )
A.max
rCO 2
Maximum rate of CO2 evolution from acetic acid (h−1 )
B.max
rCO2 Maximum rate of CO2 evolution from butyric acid (h−1 )
G.max
rCO 2
Maximum rate of CO2 evolution from glucose (h−1 )
G.max
r H2 Maximum rate of H2 evolution from glucose (h−1 )
A.max
r Act Maximum rate of acetone formation for acetic acid (h−1 )
B.max
r Act Maximum rate of acetone formation for butyric acid (h−1 )
r G.max
Act Maximum rate of acetone formation for glucose (h−1 )
r max
A Maximum rate of acetic acid formation (h−1 )
r max
B Maximum rate of butyric acid formation (h−1 )
A.max
r But Maximum rate of butanol formation for acetic acid (h−1 )
B.max
r But Maximum rate of butanol formation for butyric acid (h−1 )
G.max
r But Maximum rate of butanol formation for glucose (h−1 )
A.max
r Et Maximum rate of ethanol formation for acetic acid (h−1 )
B.max
r Et Maximum rate of ethanol formation for butyric acid (h−1 )
G.max
r Et Maximum rate of ethanol formation for glucose (h−1 )
A Acetic acid concentration (g·L−1 )
Act Acetone concentration (g·L−1 )
B Butyric acid concentration (g·L−1 )
But Butanol concentration (g·L−1 )
Et Ethanol concentration (g·L−1 )
G Glucose concentration (g·L−1 )
X Dry cell weight (g·L−1 )
pKa pKa value for buffer system in media
Greek letters
µmax Maximum specific growth rate (h−1 )
γK2 HPO4 Control coefficients for consumption of K2 HPO4
γKH2 PO4 Control coefficients for consumption of KH2 PO4
α1 Control coefficient for phase 1 (acidogenesis)
α2 Control coefficient for phase 2 (solventogenesis)
S.pH1 Equilibrium constants for key enzyme(s) involved in gases (CO2 and
δgas
H2 ) formation from substrate when its active sites act as a base
S.pH2 Equilibrium constants for key enzyme(s) involved in gases (CO2 and
δgas
H2 ) formation from acetic acid when its active sites act as an acid
G.pH1 Equilibrium constants for key enzyme(s) involved in acetic acid
δA
formation from glucose when its active sites act as a base
G.pH2 Equilibrium constants for key enzyme(s) involved in acetic acid
δA
formation from glucose when its active sites act as an acid
S.pH1 Equilibrium constants for key enzyme(s) involved in acetone formation
δAct
from substrate when its active sites act as a base
S.pH2 Equilibrium constants for key enzyme(s) involved acetone formation
δAct
from substrate when its active sites act as an acid
G.pH1 Equilibrium constants for key enzyme(s) involved in butyric acid

δB
formation from glucose when its active sites act as a base
G.pH2 Equilibrium constants for key enzyme(s) involved in butyric acid
δB
formation from glucose when its active sites act as an acid
G.pH1 Equilibrium constants for key enzyme(s) involved in biomass formation
δBiomass
from glucose when its active sites act as a base
G.pH2 Equilibrium constants for key enzyme(s) involved in biomass formation
δBiomass
from glucose when its active sites act as an acid
S.pH1 Equilibrium constants for key enzyme(s) involved in butanol formation
δBut
S.pH2 Equilibrium constants for key enzyme(s) involved in butanol formation
δBut
S.pH1 Equilibrium constants for key enzyme(s) involved in ethanol formation
δEt
S.pH2 Equilibrium constants for key enzyme(s) involved in ethanol formation
δEt
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microorganisms

1 Department of Chemical Engineering, Indian Institute of Technology Gandhinagar, Palaj,

Keywords: ABE fermentation; Clostridium acetobutylicum; phenomenological model; butanol;

Microorganisms 2023, 11, 1610. https://doi.org/10.3390/microorganisms11061610 https://www.mdpi.com/journal/microorganisms

To advance our understanding of acetone–butanol–ethanol (ABE) fermentation, re-

2. Materials and Methods

2.3. pH Effect on Growth and Production Profiles

Enzyme.H + S K ⇔ Enzyme.H.S ⇒ Enzyme.H + P

Enzyme.H + H + δ pH1 ⇔ Enzyme.H2+

where, 𝑋 indicates dry biomass (g·L−1), and 𝐾 is endogenous metabolism coeﬃcient

riG.max riA.max riB.max GAB

2.7. Ordinary Differential Equations Encompassing Mass Balance of Fermentation

2.7.1. Consumption of Glucose

where, Y G , Y G , Y G , Y G , YG , Y G , and Y G denote the yield coefficients (g·g−1 ) of glucose

2.7.2. Synthesis and Consumption of Acetic and Butyric Acid

2.7.3. Synthesis of Acetone, Ethanol, Butanol

2.7.4. Hydrogen Evolution and Carbon Dioxide Evolution

2.7.5. Consumption of Phosphate

2.8. Experimental Data

2.9. Validation of Model

Figure Figure 2. Depiction

Microorganisms 2023, 11, x FOR PEER REVIEW 10 of 23

Figure 4. Rates of biomass accounting the variation in starting pH level.

3.1. Estimation of Model Parameters

Table 1. Estimated parameter values used in proposed model.

Parameter Unit Value Parameter Unit Value Parameter Unit Value

3.1. Estimation of Model Parameters

Substrate/Product Initial Values (g·L−1 )

3.2. Control Mechanism for Metabolic Switch from Acidogenesis to Solventogenesis

To mimic the growth conditions described in Experiment 3, we conducted simulations

Microorganisms 2023, 11, 1610 15 of 23

Hydrogen/Cabon dioxide Hydrogen/Cabon dioxide

Hydrogen/Cabon dioxide Hydrogen/Cabon dioxide

Hydrogen/Cabon dioxide Hydrogen/Cabon dioxide

Glucose consumption yield coefficient with respect to butyric acid

G.pH1 Equilibrium constants for key enzyme(s) involved in butyric acid

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