Biochemical Engineering Journal 99 (2015) 156–166

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Biochemical Engineering Journal

journal homepage: www.elsevier.com/locate/bej

Kinetic study of butanol production from various sugars by

Clostridium acetobutylicum using a dynamic model
Francesca Raganati a , Alessandra Procentese a , Giuseppe Olivieri a,b,∗ , Peter Götz c ,
Piero Salatino a , Antonio Marzocchella a
a
Dipartimento di Ingegneria Chimica, Dei Materiali e Della Produzione Industriale – Università Degli Studi di Napoli Federico II, P.le V. Tecchio 80, 80125
Napoli Italy
b
Bioprocess Engineering, Wageningen University, AlgaePARC, P.O. Box 16, 6700 AA Wageningen, The Netherlands
c
Department of Life Sciences and Technology/Bioprocess Engineering, Beuth University of Applied Sciences Berlin, Seestrasse 64, 13347 Berlin Germany

a r t i c l e i n f o a b s t r a c t

Article history: This paper presents a kinetic dynamic model of acetone–butanol–ethanol production by Clostridium
Received 27 July 2014 acetobutylicum DSM 792 developed with the biochemical networks simulator COPASI. This model is
Received in revised form 25 February 2015 an evolution of previous models described in the literature, updated by including various mono-, di-,
Accepted 1 March 2015
hexose and pentose sugars: glucose, mannose, fructose, sucrose, lactose, xylose and arabinose. The kinetic
Available online 28 March 2015
relationships of uptake of substrate, butanol production, cell growth and cell death are also included.
The batch fermentation tests were carried out at an initial sugar concentration ranging from 5 to 100 g/L.
Keywords:
The data from the batch tests were used to assess the kinetic parameters of the model. This model gave
Substrate inhibition
Product inhibition
satisfactory results for each sugar, both in terms of simulation of fermentation – the square correlation
Clostridium acetobutylicum coefficient of metabolite concentrations, calculated by comparing experiments and simulations, ranged
Dynamic modelling between 0.87 and 0.925 – and of comparison with the models reported in the literature.
Biokinetics The effects of mono-, di-, hexose and pentose sugars on the growth and production of metabolites,
COPASI including acids and solvents, were reviewed according to the proposed model. The low fermentation
performance measured for xylose and lactose were interpreted taking into account the sugar uptake, the
acid production and the hydrolysis path.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction
Butanol is an energy carrier with remarkable features –
hydrophobicity, high energy density, possibility to be used in the
current internal combustion engines without any upgrade, dis-
tribution by the current infrastructures – and that has already
been proposed as a substitute/supplement of gasoline [1–3].
Acetone–butanol–ethanol (ABE)-producing Clostridia produce sol-
vents by fermenting several biomasses, such as palm oil waste [4],
agro-industrial waste [5], and agricultural crops [6,7]. Clostridium
saccharoperbutylacetonicum, Clostridium acetobutylicum, Clostrid-
ium beijerinckii, and Clostridium aurantibutyricum can metabolize a
great variety of substrates: pentoses, hexoses, mono-, di- and poly-
saccharides [8]. Under batch conditions the fermentation process
of solvent-producing Clostridium strains proceeds with the produc-
∗ Corresponding author at: Bioprocess Engineering, Wageningen University,
AlgaePARC, P.O. Box 16, 6700 AA Wageningen, The Netherlands.
Tel.: +31 6 19304246.
E-mail addresses: giolivie@unina.it, giuseppe.olivieri@wur.nl (G. Olivieri).
tion of cells, hydrogen, carbon dioxide, acetic acid and butyric acid
during the initial growth phase (acidogenesis) [8]. As the acid con-
centration increases (pH decreases), the cell metabolism shifts to
solvent production (solventogenesis). The acidogenic cells – able to
reproduce themselves – enter the solventogenesis state undergoing
a morphological change [8]. During solventogenesis, the active cells
become endospores unable to reproduce themselves. Therefore,
two physiological states must be taken into account for Clostridia:
one for the acidogenic phase, and one for the solventogenic phase.
Considerable research has been carried out on the ABE fermentation
systems to enhance butanol production [9–11]. Yet several ques-
tions are still open as to how to optimise the industrial processes
to produce butanol by fermentation.
The number of models reported in the literature is limited [12],
which confirms the complexity of the metabolic pathway involved
in ABE production. Papoutsakis [13] developed a stoichiometric
model: it may be used to calculate or estimate the rates of the reac-
tions occurring in the pathway in several ABE-producing Clostridia.
Votruba et al. [14] developed a mathematical model of batch cul-
tures of C. acetobutylicum based on the biochemistry and physiology

http://dx.doi.org/10.1016/j.bej.2015.03.001
1369-703X/© 2015 Elsevier B.V. All rights reserved.
 F. Raganati et al. / Biochemical Engineering Journal 99 (2015) 156–166 157

described in the literature are generally focused on individual sug-

Nomenclature ars (glucose and xylose). No effort has been deployed to develop
models able to simulate the fermentation of the wide spectrum of
[AACoA] Acetoacetyl-CoA concentration (mM) sugars that can be used as carbon source [12].
ABE Acetone–butanol–ethanol This study proposes a kinetic dynamic model to investigate
[Acet] Acetate concentration (mM) the effects of the carbon source on the ABE production by C. ace-
[A] Acetone concentration (mM) tobutylicum DSM 792. The model was applied to several sugars
[ACoA] Acetyl-CoA concentration (mM) – glucose, mannose, fructose, sucrose, lactose, xylose, and arabi-
Ar Arabinose nose – using the specific metabolic pathway of each sugar: the
[BCoA] Butyrl-CoA concentration (mM) Embden–Meyerhof–Parnas (EMP) pathway equations for hexose
[Biomass] Biomass concentration (mM) and disaccharide sugars; and the pentose phosphate (PP) pathway
[B] Butanol concentration (mM) equations for pentose sugars. Two main issues were addressed: (1)
[Butyr] Butyrate concentration (mM) the dynamic behaviour of the metabolites involved in ABE produc-
[E] Ethanol concentration (mM) tion, and (2) the inhibitory and activatory mechanisms. The model
F Switching factor of on-off mechanism was aimed to reproduce the main features of batch fermentations,
[F6P] Fructose 6-phosphate concentration (mM) characterized by a transient behaviour dominated by the accumu-
Fr Fructose lation of the inhibiting metabolites. The model may also support
G Glucose the simulation of biofilm behaviour where non-homogenous con-
[G3P] Glyceraldehyde 3-phosphate concentration (mM) centration of metabolites is expected throughout the biofilm.
j Number of the corresponding reaction in Fig. 1A-B
[Pyr] Pyruvate concentration (mM)
Kaj Activation constant for activator (mM) 2. Materials and methods
Kiij Inhibition constant for inhibitor (mM)
Kisj Inhibition constant for substrate (mM) 2.1. Microorganism and media
Kmj Concentration of metabolite where the rate is equal
to half the value ov Vmax (mM) C. acetobutylicum DSM 792 was supplied by DSMZ (Braun-
Kmsj Specific activation constant (mM) schweig, Germany). Stock cultures were reactivated according
L Lactose to the DSMZ procedure. The reactivated cultures were stored at
M Mannose −80 ◦ C. The thawed cells were inoculated into 12 mL synthetic
rj Rate equation of metabolic reaction medium containing glucose (30 g/L) and yeast extract (5 g/L) in
Vmaxj Maximum reaction rate (h−1 ) 15 mL Hungate tubes (pre-cultures). The cells were grown under
S Sucrose anaerobic conditions for 48 h at 37 ◦ C. Then they were transferred
[Sugar] Sugar concentration (mM) into fermentation bottles. Each test was carried out in duplicate
YE Yeast extract and the mean values are reported as results. The error was typically
X Xylose within 5%.
The fermentation medium consisted of 5 g/L yeast extract, 2 g/L
ammonium chloride (N-source) and 5 g/L CaCO3 supplemented
to a P2 stock solution: buffer) 0.25 g/L KH2 PO4 , 0.25 g/L K2 HPO4 ;
of metabolite growth and synthesis. Desai et al. [15] analysed the
mineral) 0.2 g/L MgSO4 ·7H2 O, 0.01 g/L MnSO4 ·7H2 O, 0.01 g/L
contribution of acid formation pathways to the metabolism of C.
FeSO4 ·7H2 O [22]. The medium was sterilized in an autoclave before
acetobutylicum ATCC824T according to the metabolic flux analysis
the carbon source addition. Chemicals (CaCO3 , KH2 PO4 , K2 HPO4 ,
(MFA). Shinto et al. [16,17] reported a kinetic simulation model to
MgSO4 ·7H2 O, MnSO4 ·7H2 O, and FeSO4 ·7H2 O) and yeast extract
describe the dynamic behaviour of metabolites in ABE production
were from Sigma–Aldrich.
by C. saccharoperbutylacetonicum N1-4 ATCC13564 using glucose or
The sugars investigated were: glucose, mannose, fructose,
xylose as carbon source. Kim et al. [18] developed a kinetic model
sucrose, lactose, arabinose and xylose (Sigma–Aldrich). The
describing the metabolism in acetone–butanol–ethanol (ABE)-
concentrated sugar solutions were sterilized by filtration and
fermentation by C. acetobutylicum ATCC 824; in particular they
supplemented to the autoclaved medium. The stock solution con-
used an optimization algorithm combining a genetic algorithm
centration of each investigated sugar was 300 g/L.
and the Levenberg–Marquardt algorithm in order to estimate the
kinetic parameters of the model. Millat et al. [19] presented a
method for linking the pH shift, the clostridial growth and the 2.2. Analytical methods
acetone–butanol–ethanol fermentation metabolic network sys-
tematically into a model that combines the dynamics of the external The pH was measured off-line using a pH-meter (Hanna Instru-
pH and optical density with a metabolic model. Mayank et al. [12] ments). The samples taken from the cultures were analysed to
pointed out the necessity to develop dynamic models to simu- measure the liquid phase concentration of biomass, sugars and
late steady state fermentations as well as transient behaviours to metabolites. The cell density was measured as optical absorbance
support the development and optimization of butanol production at 600 nm (OD600 ) using a spectrophotometer (Cary 50 Varian). The
processes. Moreover, Mayank et al. [12] concluded that the effi- calibration tests for C. acetobutylicum dried mass indicated that 1
cient design of large-scale fermenters requires the combination OD600 = 0.4 gDM /L [10].
of reliable dynamic models and the description of the bioreactor The concentration of soluble species was measured in the liq-
hydrodynamics, in particular when immobilized cells are used. uid supernatant after cell separation by centrifugation. The sugar
The sugars present in the most promising feedstock (raw and concentration was measured by high performance liquid chro-
treated) for the ABE fermentation are widely assorted (glucose, matography (HPLC) using an Agilent 1100 system (Palo Alto, CA).
arabinose, mannose, xylose, fructose, sucrose, lactose, etc.). There The sugars were separated on a 8 ␮m Hi-Plex H, 30 cm × 7.7 mm
are several experimental studies on the effects of these sugars on at room temperature and detected with a refractive index
fermentation performance [20–22], but the theoretical investiga- detector. Deionized water was used as mobile phase at a flow
tions are quite limited. To the authors’ knowledge, the models rate of 0.6 mL/min. Acetone, butanol, ethanol, acetic and butyric
158 F. Raganati et al. / Biochemical Engineering Journal 99 (2015) 156–166
acid concentrations were measured by means of a GC apparatus
equipped with a FID, and outfitted with a capillary column poraplot
Q (25 m × 0.32 mm). An internal standard (hexanoic acid) was used
to assess acids and alcohols and their concentrations.
2.3. Operating conditions and procedures
Screw-cap Pyrex bottles (100 mL) containing 75 mL medium
were used as fermenters. All the cultivations were carried out at
37 ◦ C and without pH control. The cultures were agitated by means
of a rotary shaker at 110 rpm. The medium was inoculated with a
6.25% (v/v) suspension of actively growing pre-cultures. The initial
cell concentration was set at 150 mg/L. Three millilitres of cultures
were sampled periodically for cell/metabolites characterization.
The initial cell concentration corresponded to 1.5 mM when the
cell molecular weight was assumed equal to 101 g/mol [23,24]. The
initial concentration of the investigated sugar in each batch test
ranged between 5 and 100 g/L.
3. Theoretical framework
3.1. Model
A kinetic simulation model was developed using the biochemi-
cal networks simulator software COPASI [25]. COPASI supports the
development and the analyses of a reaction network and includes
approximate velocity functions of enzyme kinetics.
The proposed model includes the substrate utilization rate, the
production rate and the cell growth rate and is based on the
metabolic pathways of C. acetobutylicum [16,17]. The pathways
reported in Fig. 1A (ModelHex/Disacc ) were used when glucose, man-
nose, fructose, sucrose, and lactose were the carbon source. The
pathways reported in Fig. 1B (ModelPent ) were used when xylose
and arabinose were the carbon source. Glucose, fructose, mannose,
Fig. 1. (A) Metabolic pathways of C. acetobutylicum. Carbon source: glucose, mannose, fructose, sucrose, and lactose. Enzymes (bold style): PTA, phosphotransacetylase; AK,
acetate kinase; CoAT, CoA transferase; PTB, phosphotransbutyrylase; BK, butyrate kinase; BADH, butyraldehyde dehydrogenase; BDH, butanol dehydrogenase. (B) Metabolic
PP pathways of C. acetobutylicum. Carbone source: xylose and arabinose. Enzymes (bold style): TA, transaldolase; TK, transketolase.
sucrose and lactose are metabolized via the EMP pathway, and
xylose and arabinose are metabolized via the PP pathway [16,17].
The models are detailed hereinafter.
ModelHex/Disacc – the single step of the metabolic pathway
described in Fig. 1A – hexose and disaccharide sugars – is reported
in Table 1 as Reaction r1 through r2.
ModelPent – the first steps of xylose and arabinose metabolism
are reported in Table 1 as Reaction r20 through r25 . Reaction r13
through r16 also hold for the conversion of xylose and arabinose
provided that G3P is produced.
The main assumptions made in the model by Shinto et al. [16,17]
are as follows:
• A Michaelis–Menten type kinetics characterized by butanol non-
competitive inhibition is assumed to exist for the rate equation
∗ .
of cell growth r15
• According to the Eq. (1.16), the death rate of the cell – r16 – is
assumed to be a first order kinetics in the biomass concentration.
• The kinetic rates of Eqs. (1.1) and (1.20) – r ∗ and r ∗ , respec-
1 20
tively – are obtained by combining the substrate-sugar inhibition
and the butanol-uncompetitive inhibition behaviour, according
to the known effects of inhibition by substrate and solvent on the
substrate conversion rate [8].
• The kinetic rate of Eq. (1.10) – r ∗ – is obtained by combining
10
the butyrate activation and the butanol-uncompetitive inhibition
behaviour, according to the enhancement effects of butyrate on
the butanol production rate reported by Shinto et al. [16]
The assumptions made in the kinetic model proposed (herein,
referred to as proposed model) are summarised in the following.
The kinetic relationships are reported without the superscript “*”.
• The kinetic expressions proposed by Shinto et al. [16,17] for sugar
uptake (r1∗ and r20
∗ ), butanol production (r ∗ ) and cell growth
10
 F. Raganati et al. / Biochemical Engineering Journal 99 (2015) 156–166 159

Table 1
Reaction steps of the EMP and PP metabolic pathways and associated kinetics.
Eqs.
Name Reactions Kinetics Refs.
r1 G/M/F → F6P or r1 =  nB1 a
(1.1)
 [S] 2
Vmax1 [S]
1− [B]
BMAX1
F
S → 2 F6P or Km1 +[S]+Km1
Kis1
b

L → F6P + 2G3P
Vmax1 [S]
r1∗ = F a
Km1 +Km1 ([S]/Kis1 )2 +[S](1+[B]/Kii1 )

Vmax2 [F6P]
r2 F6P → 2 G3P r2 = Km2 +[F6P]
F a
(1.2)
Vmax3 [G3P]
r3 G3P → Pyr r3 = Km3 +[G3P]
F a
(1.3)
Vmax4 [Pyr]
r4 Pyr → ACoA r4 = Km4 +[Pyr]
F a
(1.4)
Vmax5 [ACoA]
r5 ACoA → Acetate r5 = Km5 +[ACoA]
F a
(1.5)
Vmax6 [Acet]
r6 Acetate → ACoA r6 = Km6 +[Acet]
F a
(1.6)
Vmax7 [ACoA]
r7 ACoA → E r7 = Km7 +[ACoA]
F a
(1.7)
Vmax8 [ACoA]
r8 ACoA → ½ AACoA r8 = Km8 +[ACoA]
a
(1.8)
Vmax9 [AACoA]
r9 AACoA → BCoA r9 = Km9 +[AACoA]
F a
(1.9)

Vmax10 [BCoA]
 [B]
nB10
r10 BCoA → B r10 = Km10 (1+Ka10 /[Butyr])+S
1− BMAX10
F b
(1.10)

∗ Vmax10 [BCoA]
r10 = Km10 (1+Ka10 /[Butyr])+[BCoA](1+[B]/Kii10 )
F a

 1
 1

r11 Acet + AACoA → A + ACoA r11 = Vmax11 1+Km11A /[Acet] 1+Km11B /[AACoA]
a
(1.11)
 1
 1

r12 Butyr + AACoA → A + BCoA r12 = Vmax12 1+Km12A /[Butyr] 1+Km12B /[AACoA]
a
(1.12)

Vmax13 [BCoA]
r13 BCoA → Butyr r13 = Km13 +[BCoA]
F a
(1.13)
Vmax14 [Butyr]
r14 Butyr → BCoA r14 = Km14 +[Butyr]
F a
(1.14)

Vmax15 [ACoA]
 [Acet]
nAcetate  [Butyr]
nButyrate  [A]
nA  [E]
nE  [B]
nB15
r15 ACoA → Biomass r15 = Km15 +[ACoA]
1− AcetMAX
1− ButyrMAX
1− AMAX
1− EMAX
1− BMAX15
b
(1.15)

∗ Vmax15 [ACoA]
r15 = a
Km15 (1+[B]/Kii15 )+[ACoA](1+[B]/Kii15 )

Vmax16 [Biomass][B]
r16 Biomass → Inactive Cells r16 = Kms16 ×Ka16 +(Kms16 +[Biomass])[B]
b
(1.16)

∗
r16 = Vmax16 [Biomass] a

 nB20
r20 X/Ar → X5P r20 =
Vmax20 [S]
 [S]
2 1− [B]
BMAX20
F b
(1.17)
Km20 +[S]+Km20
Kis20

∗ Vmax20 [S]
r20 = F a
Km20 +Km20 ([S]/Kis20 )2 +[S](1+[B]/Kii20 )

Vmax21 [X5P]
r21 X5P → R5P r21 = Km21 +[X5P]
a
(1.18)
Vmax22 [R5P]
r22 R5P → X5P r22 = Km22 +[R5P]
a
(1.19)
 1
 1

r23 R5P + X5P→G3P + S7P r23 = Vmax23 1+Km23A /[R5P] 1+Km23B /[X5P]
a
(1.20)
 1
 1

r24 G3P + S7P → E4P + F6P r24 = Vmax24 1+Km24A /[S7P] 1+Km24B /[G3P]
a
(1.21)
 1
 1

r25 E4P + X5P → F6P + G3P r25 = Vmax25 1+Km25A /[X5P] 1+Km25B /[E4P]
a
(1.22)

a
[16,17].
b
[27].

∗ ) tend to zero as butanol concentration approaches infi-

(r15
nite. However, this behaviour does not fit the metabolism of C.
acetobutylicum that is characterized by full inhibition as the con-
centration of inhibitor metabolites approaches a critical value
[23,24,26]. Moreover, the rate of the cell death (1.16) is acti-
vated by butanol [27]. In the proposed model no reactivation
of death cells (spores) was taken in consideration and a modi-
fied set of reaction rates was used to substitute the uptake rates
r1∗ and r20
∗ , the butanol production rate r ∗ , the growth rate r ∗ ,
10 15
the cell death rate r16∗ . Table 1 reports the proposed reaction
rate as: r1 and r20 , complete inhibition as butanol concentra-
tion approaches the critical value BMAX was included; r15 , an
interactive multiproduct-inhibited model was used for the cell
growth kinetics; r16 , a specific butanol activation expression was
included.
• Acetoacetyl-CoA transferase (CoAT) has a broad carboxylic acid
specificity and can catalyse the transfer of CoA to either acetate
and butyrate [16,28]. According to this observation, a specific
expression of the reaction rate of CoAT was proposed for each
substrate in agreement with the random bi bi model [27]: r11
and r12 .
• The reaction rate equations of TK and TA consisted of random
bi–bi mechanisms (Eqs. (1.20)–(1.22)).
• Several metabolic reactions of ABE fermentation require ATP or
NADH (Fig. 1A). These reactions are not expected to happen if
energy source depletion occurs – e.g. sugar exhaustion – and
160 F. Raganati et al. / Biochemical Engineering Journal 99 (2015) 156–166

an on–off mechanism was used. A switch-factor F was intro-

nButyr

0.13
duced and its value may be 1 or 0 depending on the substrate

a
concentration in the broth. According to the observation by
Okamoto et al. [29], the threshold value of substrate concentra-
tion at which F switches from 1 to 0 is larger than zero: F is set to

nAcet

0.15
1 for substrate concentrations larger than 1.00 mM. The switch-

a
factor was used for the rate of reactions that include ATP, ADP,

nE

1
a
NADH, and NAD+ amongst the substrates: r1 –r7 , r9 , r10 , r13 , r14

0.71

0.25

0.22
and r20 .

nBj

a
nA

1
Although there is experimental evidence that the sugar concen-

a
tration is not the key to cause and effect in regulation, this approach

ButyrMAX
is well suited to represent batch fermentations, where sugar deple-

(mM)

177
tion and regulatory onset of solventogenesis are coupled.

a
AcetMAX
3.2. Assessment of the model parameters

(mM)

124.5
a
The main assumptions made to assess the model parameters are
reported hereinafter.

(mM)
EMAX

712
• According to the kinetics of enzymatic reactions, the maximum

a
(mM)
reaction rate is proportional to the effective concentration of the

BMAXj

193
220

180
enzymes. In turn, this effective concentration of the enzymes

a
depends on the sugar type. Therefore, it is reasonable to assume

(mM)
AMAX

968
that the maximum reaction rate depends on the sugar type;
• The “affinity” constants – Kaj , Kiij , Kisj , Kms16 and Kmj (except for

a
Kinetic parameters assessed by processing experimental data according to the presented and Shinto et al.’s model. Carbon source: glucose.
j = 1 and 20) – do not depend on the sugar because they depend on

299
250
the enzyme responsible for the reaction step but not on its con-

b
centration. The values of the “affinity” constants were assumed
(mM)
KmjB

0.001 0.0002276
197
to be constant for all the investigated sugars and were assessed
a

for glucose;
• The parameters of the sugar uptake Reactions (r1 and r20 ) were

217
assessed for each sugar. Indeed, Servinsky et al. [30] reported
b

that C. acetobutylicum has sugar-specific mechanisms for the

(mM)
KmjA

241
transcriptional regulation of transport and metabolism genes. In
a

particular, C. acetobutylicum utilizes: (a) symporters and ATP-

(mM)

binding cassette (ABC) transporters for the uptake of pentose

10.5
Kmsj

sugars; (b) phosphotransferase system (PTS) transporters and a

a

gluconate – H+ (GntP) transporter – for the uptake of disaccha-

rides and hexoses. Moreover, Servinsky et al. [30] reported that
12.5

the transcription of some transporter genes is induced by specific

b

sugars. Sugar-specific transport roles are suggested for various

(mM)

7.41

4.37
Kaj

transporters of the PTS and of the ABC superfamily [30].

a
(mM)

299

287

299

The assessment of the model parameters was carried out accord-

Kiij

ing to the following procedure.

135
b

i.) The proposed model was applied to the data measured dur-
(mM)

7.75

ing the glucose fermentation tests. The values of the kinetic

Kisj

parameters were estimated by fitting the experimental data

measured during the batch cultures of C. acetobutylicum in
0.39

0.59
0.29

3.24
0.37

7.32
0.65
0.07

glucose-based medium with glucose initial concentration rang-

27

273
145
239

204

ing between 5 and 555 mM;

b

0.0004

ii.) The kinetic parameters for the hexoses (except glucose) and
0.11

0.14

9.56
0.42

4.13
0.07

7.20

0.09

0.05
(mM)

disaccharides (sucrose and lactose) were assessed. Except for

212
286

162
Kmj

Kaj , Kiij , Kisj , Kmj , Kms16 (i and j ranging between 2 and 16), the
a

maximum reaction rate Vmaxj of each reaction step and the

9.44
0.59
0.46

7.49

2.61
6.80

0.05

0.04

value of BMAXj , AcetMAX , ButyrMAX , AMAX , EMAX , nBJ , nAcet , nButyr ,

114
265
254

297
292
79

709
198

Shinto-parameter set.
b

nA , and nE were assessed. The parameters of the reaction rate

Presented Model.
6.79

5.23
0.48

6.67
0.34

2.43
1.89
8.20

r1 were also estimated for each sugar.

(h−1 )
Vmaxj

95
305
276

310
248
92

722
207

iii.) The kinetic parameters of the xylose pathway (r20 –r25 ) were
a

assessed. Except for Kaj , Kiij , Kisj , Kmj , Kms16 (i and j ranging
Reactions

between 2 and 16), the maximum reaction rate Vmaxj of each

Table 2

reaction step and the value of BMAXj , AcetMAX , ButyrMAX , AMAX ,

r10
r11
r12
r13
r14
r15
r16
r1
r2
r3
r4
r5
r6
r7
r8
r9

EMAX , nBJ , nAcet , nButyr , nA , and nE were assessed.

a

b
 F. Raganati et al. / Biochemical Engineering Journal 99 (2015) 156–166 161

Fig. 2. Experimental time-course data and simulation results of target metabolites for glucose, mannose, fructose, sucrose, lactose, arabinose and xylose using the proposed
model. Initial sugar concentration 60 g/L.

Table 3
Kinetic parameters assessed by processing experimental data according to the presented and Shinto’s model. Carbon source: xylose.

Reactions Vmax Kmi Kis Kii KmA KmB BMAX nB

(h−1 ) (mM) (mM) (mM) (mM) (mM) (mM)
a b a b a b b a b a b a a

r20 1.50 4.32 54 0.40 293 9.3 14.8 172 1.79

r21 296 299 27 186
r22 218 129 43 210
r23 216 149 0.08 69 3.74 25.6
r24 205 61 5 212 22 0.36
r25 298 113 0.21 127 2.98 1.14

(a) Presented Model.

(b) Shinto-parameter set.

iv.) The kinetic parameters for the arabinose pathway were
assessed: the parameters of the reaction rate r20 , Vmaxj of each
reaction step, and BMAXj , AcetMAX , ButyrMAX , AMAX , EMAX , nBJ ,
nAcet , nButyr , nA , and nE . All the other parameters were set to
the values measured for the xylose pathway.
The simulated annealing (SA) method – an optimization algo-
rithm of COPASI – was used for the parameter fitting.
The study also included the assessment of the kinetic parame-
ters of the model by Shinto et al. [16,17] based on the experimental
data collected in the present investigation (hereinafter, referred to
162 F. Raganati et al. / Biochemical Engineering Journal 99 (2015) 156–166

Fig. 3. (A) Time-resolved concentration of glucose, biomass and metabolites. Experimental data vs. simulation results. (B) Time-resolved concentration of xylose, biomass
and metabolites. Experimental data vs. simulation results. Initial sugar concentration 60 g/L.

Table 4
Average squared correlation coefficients (r2 ) between simulation results and experimental data.

r2 Glucose Mannose Fructose Sucrose Lactose Arabinose Xylose

a
Shinto et al.’s simulation 0.855 0.812 0.820 0.800 0.904 0.848 0.830
Presented Model 0.894 0.887 0.870 0.880 0.925 0.904 0.890
a
[16,17].

as Shinto-parameter set). The Shinto-parameter set was assessed
for C. acetobutylicum and for all the seven sugars investigated.
The soundness of the model was tested according to the follow-
ing two procedures:
• The assessment of the average squared correlation coefficients
(r2 ) between the simulation results and the experimental data.
• The comparison of the results of the proposed model with those
of the model by Shinto et al. [16,17] using the Shinto-parameter
set.
4. Results and discussion
4.1. Batch cultures
The batch cultures were carried out at initial sugar concentra-
tions ranging between 5 and 555 mM for hexose sugars (glucose,
mannose, and fructose), 14 and 278 mM for disaccharide sugars
(sucrose and lactose), 33 and 667 mM for pentose sugars (xylose
and arabinose). The time resolved concentration of sugar and
metabolites was measured. As expected, the pH (data not reported)
decreased with the time since the beginning of the fermentation
and stabilized at about 4.7 under the solventogenic phase.
The experimental data reported in Fig. 2 refer to the batch fer-
mentation tests carried out at an initial sugar concentration of
335, 167 and 400 mM for hexose, disaccharide and pentose sugars,
respectively.
The data reported in Fig. 2 highlight that solvent production
is sugar specific: the glucose fermentation was characterized by
the highest butanol concentration (169 mM); the performance
of mannose, fructose, and sucrose fermentations was slightly
lower (butanol concentration 140, 134, and 135 mM, respectively);
the performance of arabinose and xylose fermentations was the
lowest (116, and 112 mM, respectively); the lactose fermenta-
tion was characterized by the lowest final butanol concentration
(19 mM).
 F. Raganati et al. / Biochemical Engineering Journal 99 (2015) 156–166 163

Table 5
Sugar uptake (r1 /r20 ), butanol production (r10 ) and cell growth (r15 ) kinetic parameters for mannose, fructose, sucrose, lactose, arabinose and xylose.

Reaction Parameters Mannose Fructose Sucrose Lactose Arabinose Xylose

r1 Vmax (h−1 ) 4.71 4.64 3.4 1.11 2.7 1.5
Km (mM) 0.1 0.04 0.49 40.8 46 54
Kis (mM) 9 6 20 100 200 210
BMAX (mM) 199 199 199 153 186 171
nB 0.94 0.95 1.13 1.85 1.79 1.79
r10 BMAX (mM) 180 181 172 135 163 158
nB 0.46 0.47 0.58 1.62 0.62 0.64
r15 AcetMAX (mM) 116 124 114 119 100 82
ButyrMAX (mM) 179 150 141 141 137 127
AMAX (mM) 991 920 1096 972 875 810
BMAX (mM) 151 148 140 110 136 137
EMAX (mM) 722 760 765 725 689 661
nAcet 0.72 0.79 0.93 1.39 0.91 1.21
nButyr 0.26 0.28 0.37 0.87 0.72 0.87
nA 1 1 1 1 1 1
nB 0.76 0.73 0.83 1.93 1.18 1.85
nE 1 1 1 1 1 1

4.2. Effect of inhibition and activation terms
The data reported in Fig. 2 were processed according to the
proposed model to assess the kinetic parameters of the metabolic
pathways of C. acetobutylicum for hexoses/disaccharide (Fig. 1A)
and pentose sugars (Fig. 1B). The procedure is reported in Section
3.2.
The kinetic parameters assessed by processing the experimen-
tal data measured during the glucose fermentation are reported
in Table 2. Fig. 3A reports the experimental data and the results
of the plots from both the proposed model and the Shinto et al.’s
model [16,17]. The Shinto-parameter set was used for the simula-
tions made by means of the Shinto et al.’s model. The agreement
of the proposed model with the experimental data is very satis-

Fig. 4. Deviation of Vmax of Reaction r1 through r16 assessed for hexoses/disaccharides/pentoses with respect to the value assessed for glucose as a function of the sugar.
164 F. Raganati et al. / Biochemical Engineering Journal 99 (2015) 156–166
Fig. 5. Deviation of Vmax of Reaction r20 through r25 assessed for arabinose with
respect to the value assessed for xylose as a function of the sugar.
factory. The average correlation coefficient (r2 ) reported in Table 4
highlights that the proposed model is more likely than (r2 = 0.894)
than the Shinto et al.’s simulation (0.855).
Table 3 reports the kinetic parameters of the PP pathway (r20
through r25 ) assessed by evaluating data measured during xylose
fermentation. Fig. 3B reports the experimental data and the results
of the plots from both the proposed model and the Shinto et al.’s
model [16,17]. The Shinto-parameter set was used for the simula-
tions made by means of the Shinto et al.’s model. The agreement
of the proposed model with the experimental data is very satis-
factory. The average correlation coefficient (r2 ) reported in Table 4
highlights that the proposed model is more likely (r2 = 0.89) than
the Shinto et al.’s simulation (0.83).
Tables 2 and 3 also report the Shinto-parameter set assessed
for the Shinto et al.’s model of the C. acetobutylicum (b-data group
in both tables). The direct comparison of the parameters of the
same reaction in the two models cannot be carried out without
taking into account the overall structure of the models. The main
parameter changes were recorded for the reactions characterized
by different relationships: Reactions r1 , r10 , r15 and r16 . The Vmax
assessed for the reactions does not change regardless of the model
used (present vs. Shinto’s). The Vmax of Reaction r16 (cell death rate)
does depend on the model because the Shinto’s version does not
take into account the expected activation by butanol.
The kinetic parameters of the proposed model as applied to
mannose, fructose, sucrose, lactose, and arabinose were assessed
according to the procedure reported in Section 3.2. The simulations
of the proposed model are plotted in Fig. 2 for all the investigated
sugars: the experimental dynamic behaviour of target metabolites
is also reported. Table 5 reports the kinetic parameters of the sugar
uptake (r1 and r20 ), the butanol production (r10 ) and the cell growth
(r15 ) assessed for the fermentation of mannose, fructose, sucrose,
lactose, arabinose, and xylose. The average r2 for each sugar with
reference to the proposed model and Shinto et al.’s simulation
[16,17] were calculated and are reported in Table 4. The analysis
of the table suggests that in the proposed model r2 increases as
compared to Shinto et al.’s simulation [16,17], whatever the sugar
tested. The results confirm that the structure of the proposed model
improves the simulation results. The results of this model are more
likely than Shinto et al.’s model, which is particularly useful when
the kinetic model is included in an overall model of the conversion
process (e.g. biofilm reactors). The main updates with respect to
Shinto et al.’s model [16,17] are: full inhibition term for butanol
in the uptake kinetics (r1 and r20 ) and in the butanol production
kinetics (r10 ); an interactive multiproduct-inhibited model for the
cell growth kinetics (r15 ); a specific butanol activation expression
for the cell death kinetics (r16 ).
The results of the proposed simulations were also analysed to
investigate the sensitivity of each reaction step to every sugar. Fig. 4
reports the variation of the maximum reaction rate (Vmax ) of the
reaction steps with respect to the value assessed for glucose fer-
mentation, for the hexose/disaccharide tested. Fig. 5 reports the
variation of Vmax of the reaction steps of the arabinose PP path-
way with respect to the value assessed for xylose fermentation.
The main observations are reported hereinafter.
(a) The sugar uptake Reactions – r1 and r20 – strongly
depend on the sugar type. The values of the parameters
suggest that the C. acetobutylicum preference scale is: glu-
cose > mannose/fructose > sucrose > arabinose > xylose > lactose.
This scale agrees with the literature on sugar conversion
[20–22].
(b) Except for the cell death Reaction (r16 ), the Vmax of each reac-
tion step of hexose/disaccharides is smaller than that of the
homologous step of glucose fermentation. The Vmax16 assessed
for glucose fermentation is smaller than that measured for the
hexose/disaccharides investigated.
(c) The reaction steps r2 , r3 , r5 , r6 , r8 , r9 , r13 , r15 for mannose and
fructose fermentations are just barely affected by the sugar
type: the parameters differ by less than 20% with respect to the
homologous parameters measured for glucose fermentation.
(d) For sucrose fermentation the reaction steps r3 , r5 , r6 , r8 , r9 are
just barely affected by the sugar type: the parameters differ
by less than 20% with respect to the homologous parameters
measured for glucose fermentation.
(e) Except for r3 , when lactose, arabinose, and xylose are used
as carbon source the reaction steps of the C. acetobutylicum
fermentation are strongly affected by the sugar type: the param-
eters differ by more than 20% with respect to the homologous
parameters measured for glucose fermentation.
(f) Except for the sugar uptake reaction r20 , the reactions of the
PP pathway are not affected by the sugar. The kinetics of the
reaction r20 strongly depends on the sugar: the arabinose uptake
is significantly faster than xylose.
Based on these observations it is possible to discuss the effects
of the sugars on the proposed model.
Observation (a) would confirm the first assumption reported in
Section 3.2. The concentration of the enzymes responsible for the
investigated reaction steps is expected to be high when glucose is
used as carbon source.
The fermentation of the hexoses does not strongly depend on
the sugar type. Although the glucose fermentation is characterized
by faster reaction rates, the difference of the rate assessed for the
other hexoses is within 20%.
As regards the fermentation of the di-saccharide sucrose and
lactose, the results may be interpreted in terms of the different
hydrolysis path of the two sugars. Both sugars are transported into
the cells according to the same PTS mechanism, and hydrolysed
into simple sugars. The sucrose is hydrolysed into fructose-
6-P and glucose-6-P: both sugars can be metabolized via the
Embden–Meyerhof pathway [31]. The lactose is hydrolysed into
glucose and galactose-6-P: “the glucose may be phosphorylated
and incorporated into the glycolytic pathway whereas galactose-
6-P is generally metabolized by the tagatose 6-P pathway” [32].
Therefore, the lower performance of the lactose and sucrose with
respect to the glucose may be due to the sugar transport across the
cell membrane. Moreover, the lower performances of the lactose
with respect to the sucrose may be due to the bottleneck of the
galactose-6-P pathway.
The acidogenic phase length is not affected by the sugar type
except for lactose and xylose. These two sugars are characterized
by a longer acidogenic phase (Fig. 2). This observation agrees with
the Vmax value assessed for acid production (Reactions r5 and r13 )
that is smaller than that assessed for glucose (see Fig. 4e and m). The
 F. Raganati et al. / Biochemical Engineering Journal 99 (2015) 156–166
observed behaviour of lactose is in agreement with the lower sugar
uptake discussed in the previous paragraph. A peculiar behaviour
has been observed when comparing arabinose and xylose, two
sugars characterized by slow fermentation. Both pentoses are char-
acterized by a sugar uptake rate lower than that of glucose but that
of xylose is even lower. Moreover, the acid production for xylose is
lower than that assessed for arabinose. The combination of these
two steps produces an acidogenic phase longer than that of arabi-
nose.
The proposed model may be used as an update of the dynamic
model proposed by Shinto et al. [16,17] to support the design and
optimization of fermenters dedicated to butanol production. The
model reproduces the main features of batch fermentations that
are generally characterized by a transient behaviour dominated
by the accumulation of the inhibiting metabolites. Furthermore,
this model is expected to support the simulation of continuous
fermenters that are aimed at producing butanol at the maximum
possible concentration. The model may also support the simula-
tion of biofilm behaviour where non-homogenous concentration of
metabolites is expected and in particular when butanol concentra-
tion is expected to reach maximum/inhibiting values in the inner
part [12]. Of course, the development of a biofilm reactor model
would benefit from a more detailed dynamic model [33,34] but
such a detailed model would be so complex that the simulations
would hardly be feasible.
5. Conclusions
A kinetic dynamic model of acetone–butanol–ethanol (ABE)
production by C. acetobutylicum DSM 792 developed with the bio-
chemical networks simulator COPASI is illustrated in this paper.
It is an updated version of the model by Shinto et al. [16,17]. The
kinetic relationships included in this model are more representa-
tive of the fermentation behaviour. Full inhibition by metabolites is
included in the kinetics of sugar uptake, of butanol production, and
cell growth rate. The butanol activation is used for the cell death
kinetics. The proposed kinetics have a key role when it is included
in a model of butanol continuous production in a structured fer-
menter such as a biofilm reactor. The knowledge of this kinetics is
fundamental for the reliability of the models aimed at supporting
the operation of the fermenters and at investigating the dynamics
of the conversion process. The operation of this reactor typology is
usually aimed at producing high butanol concentration stream and
the inhibition role of this solvent is crucial for the success of the
process.
The effects of the substrate were studied by applying the model
to several sugars: glucose, mannose, fructose, sucrose, lactose,
xylose, and arabinose. The parameters assessed for the glucose
were used for the simulations with the other sugars except for the
value of the sugar uptake Reactions (r1 and r20 ) and the maximum
reaction rates of all reaction steps.
The model gave satisfactory results: the squared correlation
coefficient (r2 ) between experimental and calculated time-course
of metabolites ranges between 0.87 and 0.925.
Acknowledgements
The Università Degli Studi di Napoli Federico II is acknowl-
edged for the grant of F. Raganati to be at the University of Berlin.
The authors thank the Ministero dello Sviluppo Economico (Italy)
for the financial support to the project EuroTransBio ETB-2012-16
OPTISOLV (Development, optimization and scale-up of biological
solvent production) (C01/0878/01-03/X21) as well as the BMBF
(Germany) for supporting the projects COSMIC2 (FKZ: 0315782B)
and OPTISOLV (FKZ: 031A231C).
165
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