Biochemical Engineering Journal 99 (2015) 156166

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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 Gtz c ,
Piero Salatino a , Antonio Marzocchella a
a

Dipartimento di Ingegneria Chimica, Dei Materiali e Della Produzione Industriale Universit 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

Article history:
Received 27 July 2014
Received in revised form 25 February 2015
Accepted 1 March 2015
Available online 28 March 2015
Keywords:
Substrate inhibition
Product inhibition
Clostridium acetobutylicum
Dynamic modelling
Biokinetics
COPASI

a b s t r a c t
This paper presents a kinetic dynamic model of acetonebutanolethanol production by Clostridium
acetobutylicum DSM 792 developed with the biochemical networks simulator COPASI. This model is
an evolution of previous models described in the literature, updated by including various mono-, di-,
hexose and pentose sugars: glucose, mannose, fructose, sucrose, lactose, xylose and arabinose. The kinetic
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.
The data from the batch tests were used to assess the kinetic parameters of the model. This model gave
satisfactory results for each sugar, both in terms of simulation of fermentation the square correlation
coefcient of metabolite concentrations, calculated by comparing experiments and simulations, ranged
between 0.87 and 0.925 and of comparison with the models reported in the literature.
The effects of mono-, di-, hexose and pentose sugars on the growth and production of metabolites,
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, distribution by the current infrastructures and that has already
been proposed as a substitute/supplement of gasoline [13].
Acetonebutanolethanol (ABE)-producing Clostridia produce solvents by fermenting several biomasses, such as palm oil waste [4],
agro-industrial waste [5], and agricultural crops [6,7]. Clostridium
saccharoperbutylacetonicum, Clostridium acetobutylicum, Clostridium beijerinckii, and Clostridium aurantibutyricum can metabolize a
great variety of substrates: pentoses, hexoses, mono-, di- and polysaccharides [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).
http://dx.doi.org/10.1016/j.bej.2015.03.001
1369-703X/ 2015 Elsevier B.V. All rights reserved.

tion of cells, hydrogen, carbon dioxide, acetic acid and butyric acid
during the initial growth phase (acidogenesis) [8]. As the acid concentration 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 [911]. Yet several questions 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 conrms 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 reactions occurring in the pathway in several ABE-producing Clostridia.
Votruba et al. [14] developed a mathematical model of batch cultures of C. acetobutylicum based on the biochemistry and physiology

F. Raganati et al. / Biochemical Engineering Journal 99 (2015) 156166

Nomenclature
[AACoA] Acetoacetyl-CoA concentration (mM)
ABE
Acetonebutanolethanol
[Acet]
Acetate concentration (mM)
[A]
Acetone concentration (mM)
[ACoA] Acetyl-CoA concentration (mM)
Ar
Arabinose
[BCoA] Butyrl-CoA concentration (mM)
[Biomass] Biomass concentration (mM)
[B]
Butanol concentration (mM)
[Butyr] Butyrate concentration (mM)
[E]
Ethanol concentration (mM)
F
Switching factor of on-off mechanism
[F6P]
Fructose 6-phosphate concentration (mM)
Fr
Fructose
G
Glucose
[G3P]
Glyceraldehyde 3-phosphate concentration (mM)
j
Number of the corresponding reaction in Fig. 1A-B
[Pyr]
Pyruvate concentration (mM)
Kaj
Activation constant for activator (mM)
Kiij
Inhibition constant for inhibitor (mM)
Kisj
Inhibition constant for substrate (mM)
Concentration of metabolite where the rate is equal
Kmj
to half the value ov Vmax (mM)
Kmsj
Specic activation constant (mM)
L
Lactose
M
Mannose
rj
Rate equation of metabolic reaction
Vmaxj
Maximum reaction rate (h1 )
S
Sucrose
[Sugar] Sugar concentration (mM)
YE
Yeast extract
X
Xylose

of metabolite growth and synthesis. Desai et al. [15] analysed the

contribution of acid formation pathways to the metabolism of C.
acetobutylicum ATCC824T according to the metabolic ux analysis
(MFA). Shinto et al. [16,17] reported a kinetic simulation model to
describe the dynamic behaviour of metabolites in ABE production
by C. saccharoperbutylacetonicum N1-4 ATCC13564 using glucose or
xylose as carbon source. Kim et al. [18] developed a kinetic model
describing the metabolism in acetonebutanolethanol (ABE)fermentation by C. acetobutylicum ATCC 824; in particular they
used an optimization algorithm combining a genetic algorithm
and the LevenbergMarquardt 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
acetonebutanolethanol fermentation metabolic network systematically into a model that combines the dynamics of the external
pH and optical density with a metabolic model. Mayank et al. [12]
pointed out the necessity to develop dynamic models to simulate steady state fermentations as well as transient behaviours to
support the development and optimization of butanol production
processes. Moreover, Mayank et al. [12] concluded that the efcient design of large-scale fermenters requires the combination
of reliable dynamic models and the description of the bioreactor
hydrodynamics, in particular when immobilized cells are used.
The sugars present in the most promising feedstock (raw and
treated) for the ABE fermentation are widely assorted (glucose,
arabinose, mannose, xylose, fructose, sucrose, lactose, etc.). There
are several experimental studies on the effects of these sugars on
fermentation performance [2022], but the theoretical investigations are quite limited. To the authors knowledge, the models

157

described in the literature are generally focused on individual sugars (glucose and xylose). No effort has been deployed to develop
models able to simulate the fermentation of the wide spectrum of
sugars that can be used as carbon source [12].
This study proposes a kinetic dynamic model to investigate
the effects of the carbon source on the ABE production by C. acetobutylicum DSM 792. The model was applied to several sugars
glucose, mannose, fructose, sucrose, lactose, xylose, and arabinose using the specic metabolic pathway of each sugar: the
EmbdenMeyerhofParnas (EMP) pathway equations for hexose
and disaccharide sugars; and the pentose phosphate (PP) pathway
equations for pentose sugars. Two main issues were addressed: (1)
the dynamic behaviour of the metabolites involved in ABE production, and (2) the inhibitory and activatory mechanisms. The model
was aimed to reproduce the main features of batch fermentations,
characterized by a transient behaviour dominated by the accumulation of the inhibiting metabolites. The model may also support
the simulation of biolm behaviour where non-homogenous concentration of metabolites is expected throughout the biolm.
2. Materials and methods
2.1. Microorganism and media
C. acetobutylicum DSM 792 was supplied by DSMZ (Braunschweig, Germany). Stock cultures were reactivated according
to the DSMZ procedure. The reactivated cultures were stored at
80 C. The thawed cells were inoculated into 12 mL synthetic
medium containing glucose (30 g/L) and yeast extract (5 g/L) in
15 mL Hungate tubes (pre-cultures). The cells were grown under
anaerobic conditions for 48 h at 37 C. Then they were transferred
into fermentation bottles. Each test was carried out in duplicate
and the mean values are reported as results. The error was typically
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 ;
mineral) 0.2 g/L MgSO4 7H2 O, 0.01 g/L MnSO4 7H2 O, 0.01 g/L
FeSO4 7H2 O [22]. The medium was sterilized in an autoclave before
the carbon source addition. Chemicals (CaCO3 , KH2 PO4 , K2 HPO4 ,
MgSO4 7H2 O, MnSO4 7H2 O, and FeSO4 7H2 O) and yeast extract
were from SigmaAldrich.
The sugars investigated were: glucose, mannose, fructose,
sucrose, lactose, arabinose and xylose (SigmaAldrich). The
concentrated sugar solutions were sterilized by ltration and
supplemented to the autoclaved medium. The stock solution concentration of each investigated sugar was 300 g/L.
2.2. Analytical methods
The pH was measured off-line using a pH-meter (Hanna Instruments). The samples taken from the cultures were analysed to
measure the liquid phase concentration of biomass, sugars and
metabolites. The cell density was measured as optical absorbance
at 600 nm (OD600 ) using a spectrophotometer (Cary 50 Varian). The
calibration tests for C. acetobutylicum dried mass indicated that 1
OD600 = 0.4 gDM /L [10].
The concentration of soluble species was measured in the liquid supernatant after cell separation by centrifugation. The sugar
concentration was measured by high performance liquid chromatography (HPLC) using an Agilent 1100 system (Palo Alto, CA).
The sugars were separated on a 8 m Hi-Plex H, 30 cm 7.7 mm
at room temperature and detected with a refractive index
detector. Deionized water was used as mobile phase at a ow
rate of 0.6 mL/min. Acetone, butanol, ethanol, acetic and butyric

158

F. Raganati et al. / Biochemical Engineering Journal 99 (2015) 156166

acid concentrations were measured by means of a GC apparatus

equipped with a FID, and outtted 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 biochemical 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, mannose, 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,

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 rst 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 MichaelisMenten type kinetics characterized by butanol noncompetitive 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 rst order kinetics in the biomass concentration.
The kinetic rates of Eqs. (1.1) and (1.20) r and r , respec1
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
), butanol production (r ) and cell growth
uptake (r1 and r20
10

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.

F. Raganati et al. / Biochemical Engineering Journal 99 (2015) 156166

159

Table 1
Reaction steps of the EMP and PP metabolic pathways and associated kinetics.
Name

Reactions

Kinetics

r1

G/M/F F6P or

r1 =

S 2 F6P or
L F6P + 2G3P

Km1 +[S]+Km1

r1 =
F6P 2 G3P

r2

G3P Pyr

r3

Pyr ACoA

r4

ACoA Acetate

r5

Acetate ACoA

r6

ACoA E

r7

ACoA AACoA

r8

AACoA BCoA

r9
r10

BCoA B

 [S] 2

Vmax1 [S]

(1.2)

r3 =

Vmax3 [G3P]
F
Km3 +[G3P]

(1.3)

r4 =

Vmax4 [Pyr]
F
Km4 +[Pyr]

(1.4)

r5 =

Vmax5 [ACoA]
F
Km5 +[ACoA]

(1.5)

r6 =

Vmax6 [Acet]
F
Km6 +[Acet]

(1.6)

r7 =

Vmax7 [ACoA]
F
Km7 +[ACoA]

(1.7)

r8 =

Vmax8 [ACoA]
Km8 +[ACoA]

(1.8)

r9 =

Vmax9 [AACoA]
F
Km9 +[AACoA]

(1.9)

(1.10)

r10 =

=
r10

Vmax10 [BCoA]
F
Km10 (1+Ka10 /[Butyr])+[BCoA](1+[B]/Kii10 )

r13

BCoA Butyr

r13 =



1
1+Km11A /[Acet]

Vmax13 [BCoA]
F
Km13 +[BCoA]

(1.13)

r14 =

Vmax14 [Butyr]
F
Km14 +[Butyr]

(1.14)

r15 =

Vmax15 [ACoA]
Km15 +[ACoA]

(1.15)

r16 =

[Acet]
AcetMAX

Vmax20 [S]

r20 =

[Butyr]
ButyrMAX

nButyrate 

[A]
AMAX

nA 

[E]
EMAX

nE 

[B]
BMAX15

nB15

[S]
Kis20

(1.16)

2

[B]
BMAX20

Vmax20 [S]
Km20 +Km20 ([S]/Kis20 )2 +[S](1+[B]/Kii20 )

nB20

(1.17)

r21 =

Vmax21 [X5P]
Km21 +[X5P]

(1.18)

Vmax22 [R5P]
Km22 +[R5P]

(1.19)

(1.20)

(1.21)

(1.22)

r23

R5P + X5PG3P + S7P

r23 = Vmax23

r24

G3P + S7P E4P + F6P

r24 = Vmax24

nAcetate 

Vmax16 [Biomass][B]
Kms16 Ka16 +(Kms16 +[Biomass])[B]

r22 =

E4P + X5P F6P + G3P

1
1+Km12B /[AACoA]

Km15 (1+[B]/Kii15 )+[ACoA](1+[B]/Kii15 )

R5P X5P



Vmax15 [ACoA]

r22

r25

1
1+Km11B /[AACoA]

(1.12)

=
r20

X5P R5P

1
1+Km12A /[Butyr]

Km20 +[S]+Km20

r21

(1.11)

= Vmax16 [Biomass]
r16

X/Ar X5P

[B]
BMAX10

r15

r20

nB10

Vmax10 [BCoA]
Km10 (1+Ka10 /[Butyr])+S

r12 = Vmax12

Biomass Inactive Cells

Butyr + AACoA A + BCoA

r16

Vmax2 [F6P]
F
Km2 +[F6P]

r12

ACoA Biomass

(1.1)

r2 =

r11 = Vmax11

r15

Eqs.

Kis1

Vmax1 [S]

Acet + AACoA A + ACoA

Butyr BCoA

nB1

[B]
BMAX1

Km1 +Km1 ([S]/Kis1 )2 +[S](1+[B]/Kii1 )

r11

r14

Refs.
a

r25 = Vmax25





1
1+Km23A /[R5P]
1
1+Km24A /[S7P]
1

1+Km25A /[X5P]





1
1+Km23B /[X5P]
1
1+Km24B /[G3P]
1

1+Km25B /[E4P]





[16,17].
[27].

) tend to zero as butanol concentration approaches in(r15

nite. However, this behaviour does not t the metabolism of C.
acetobutylicum that is characterized by full inhibition as the concentration of inhibitor metabolites approaches a critical value
[23,24,26]. Moreover, the rate of the cell death (1.16) is activated by butanol [27]. In the proposed model no reactivation
of death cells (spores) was taken in consideration and a modied set of reaction rates was used to substitute the uptake rates
, the butanol production rate r , the growth rate r ,
r1 and r20
10
15
. Table 1 reports the proposed reaction
the cell death rate r16
rate as: r1 and r20 , complete inhibition as butanol concentration approaches the critical value BMAX was included; r15 , an
interactive multiproduct-inhibited model was used for the cell

growth kinetics; r16 , a specic butanol activation expression was

included.
Acetoacetyl-CoA transferase (CoAT) has a broad carboxylic acid
specicity and can catalyse the transfer of CoA to either acetate
and butyrate [16,28]. According to this observation, a specic
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
bibi 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

0.15

0.22
1

0.25

0.71

177
124.5
712
180

193
287

968

220

299
250
0.001 0.0002276
241
217
197

b
a
b

10.5

12.5

299

135

Presented Model.
Shinto-parameter set.

0.05
4.13
0.0004

0.07
7.32
0.65

7.75
27
0.39
273
145
239
0.59
0.29
204
3.24
0.37
0.07
0.11
212
286
7.20
0.14
0.09
162
9.56
0.42

6.80
114
265
254
9.44
0.59
0.46
297
292
79
7.49
0.05
709
198
2.61
0.04
6.79
95
305
276
8.20
5.23
0.48
310
248
92
6.67
0.34
722
207
2.43
1.89

b
a
b
a

4.37

7.41

b
b

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

a
a
a
a
a

299

BMAXj
(mM)
AMAX
(mM)
KmjB
(mM)
KmjA
(mM)
Kmsj
(mM)
Kaj
(mM)
Kiij
(mM)
Kisj
(mM)
Kmj
(mM)

i.) The proposed model was applied to the data measured during the glucose fermentation tests. The values of the kinetic
parameters were estimated by tting the experimental data
measured during the batch cultures of C. acetobutylicum in
glucose-based medium with glucose initial concentration ranging between 5 and 555 mM;
ii.) The kinetic parameters for the hexoses (except glucose) and
disaccharides (sucrose and lactose) were assessed. Except for
Kaj , Kiij , Kisj , Kmj , Kms16 (i and j ranging between 2 and 16), the
maximum reaction rate Vmaxj of each reaction step and the
value of BMAXj , AcetMAX , ButyrMAX , AMAX , EMAX , nBJ , nAcet , nButyr ,
nA , and nE were assessed. The parameters of the reaction rate
r1 were also estimated for each sugar.
iii.) The kinetic parameters of the xylose pathway (r20 r25 ) were
assessed. Except for Kaj , Kiij , Kisj , Kmj , Kms16 (i and j ranging
between 2 and 16), the maximum reaction rate Vmaxj of each
reaction step and the value of BMAXj , AcetMAX , ButyrMAX , AMAX ,
EMAX , nBJ , nAcet , nButyr , nA , and nE were assessed.

Vmaxj
(h1 )

The assessment of the model parameters was carried out according to the following procedure.

Table 2
Kinetic parameters assessed by processing experimental data according to the presented and Shinto et al.s model. Carbon source: glucose.

According to the kinetics of enzymatic reactions, the maximum

reaction rate is proportional to the effective concentration of the
enzymes. In turn, this effective concentration of the enzymes
depends on the sugar type. Therefore, it is reasonable to assume
that the maximum reaction rate depends on the sugar type;
The afnity constants Kaj , Kiij , Kisj , Kms16 and Kmj (except for
j = 1 and 20) do not depend on the sugar because they depend on
the enzyme responsible for the reaction step but not on its concentration. The values of the afnity constants were assumed
to be constant for all the investigated sugars and were assessed
for glucose;
The parameters of the sugar uptake Reactions (r1 and r20 ) were
assessed for each sugar. Indeed, Servinsky et al. [30] reported
that C. acetobutylicum has sugar-specic mechanisms for the
transcriptional regulation of transport and metabolism genes. In
particular, C. acetobutylicum utilizes: (a) symporters and ATPbinding cassette (ABC) transporters for the uptake of pentose
sugars; (b) phosphotransferase system (PTS) transporters and a
gluconate H+ (GntP) transporter for the uptake of disaccharides and hexoses. Moreover, Servinsky et al. [30] reported that
the transcription of some transporter genes is induced by specic
sugars. Sugar-specic transport roles are suggested for various
transporters of the PTS and of the ABC superfamily [30].

EMAX
(mM)

The main assumptions made to assess the model parameters are

reported hereinafter.

3.2. Assessment of the model parameters

AcetMAX
(mM)

ButyrMAX
(mM)

Although there is experimental evidence that the sugar concentration is not the key to cause and effect in regulation, this approach
is well suited to represent batch fermentations, where sugar depletion and regulatory onset of solventogenesis are coupled.

nA

nBj

nE

nAcet

nButyr

an onoff mechanism was used. A switch-factor F was introduced and its value may be 1 or 0 depending on the substrate
concentration in the broth. According to the observation by
Okamoto et al. [29], the threshold value of substrate concentration at which F switches from 1 to 0 is larger than zero: F is set to
1 for substrate concentrations larger than 1.00 mM. The switchfactor was used for the rate of reactions that include ATP, ADP,
NADH, and NAD+ amongst the substrates: r1 r7 , r9 , r10 , r13 , r14
and r20 .

0.13

F. Raganati et al. / Biochemical Engineering Journal 99 (2015) 156166

Reactions

160

F. Raganati et al. / Biochemical Engineering Journal 99 (2015) 156166

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 Shintos model. Carbon source: xylose.
Reactions

r20
r21
r22
r23
r24
r25

Vmax
(h1 )

Kmi
(mM)

Kis
(mM)

Kii
(mM)

KmA
(mM)
a

1.50
296
218
216
205
298

4.32
299
129
149
61
113

54
27
43

0.40
186
210

293

9.3

14.8

0.08
5
0.21

KmB
(mM)
b

69
212
127

3.74
22
2.98

BMAX
(mM)

nB

172

1.79

25.6
0.36
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 algorithm of COPASI was used for the parameter tting.
The study also included the assessment of the kinetic parameters of the model by Shinto et al. [16,17] based on the experimental
data collected in the present investigation (hereinafter, referred to

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F. Raganati et al. / Biochemical Engineering Journal 99 (2015) 156166

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 coefcients (r2 ) between simulation results and experimental data.
r2

Glucose

Shinto et al.s simulation

Presented Model
a

0.855
0.894

Mannose

Fructose

Sucrose

Lactose

Arabinose

Xylose

0.812
0.887

0.820
0.870

0.800
0.880

0.904
0.925

0.848
0.904

0.830
0.890

[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 following two procedures:
The assessment of the average squared correlation coefcients
(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 concentrations 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 fermentation 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 specic: 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 fermentation was characterized by the lowest nal butanol concentration
(19 mM).

F. Raganati et al. / Biochemical Engineering Journal 99 (2015) 156166

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

Arabinose

Xylose

r1

Vmax (h1 )
Km (mM)
Kis (mM)
BMAX (mM)
nB

4.71
0.1
9
199
0.94

4.64
0.04
6
199
0.95

3.4
0.49
20
199
1.13

1.11
40.8
100
153
1.85

2.7
46
200
186
1.79

1.5
54
210
171
1.79

r10

BMAX (mM)
nB

180
0.46

181
0.47

172
0.58

135
1.62

163
0.62

158
0.64

r15

AcetMAX (mM)
ButyrMAX (mM)
AMAX (mM)
BMAX (mM)
EMAX (mM)
nAcet
nButyr
nA
nB
nE

116
179
991
151
722
0.72
0.26
1
0.76
1

124
150
920
148
760
0.79
0.28
1
0.73
1

114
141
1096
140
765
0.93
0.37
1
0.83
1

119
141
972
110
725
1.39
0.87
1
1.93
1

100
137
875
136
689
0.91
0.72
1
1.18
1

82
127
810
137
661
1.21
0.87
1
1.85
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.

Lactose

The kinetic parameters assessed by processing the experimental 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 simulations 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.

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F. Raganati et al. / Biochemical Engineering Journal 99 (2015) 156166

reports the variation of the maximum reaction rate (Vmax ) of the

reaction steps with respect to the value assessed for glucose fermentation, for the hexose/disaccharide tested. Fig. 5 reports the
variation of Vmax of the reaction steps of the arabinose PP pathway with respect to the value assessed for xylose fermentation.
The main observations are reported hereinafter.

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 coefcient (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 simulations made by means of the Shinto et al.s model. The agreement
of the proposed model with the experimental data is very satisfactory. The average correlation coefcient (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. Shintos). The Vmax of Reaction r16 (cell death rate)
does depend on the model because the Shintos 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 conrm 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. biolm 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 specic 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

(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: glucose > mannose/fructose > sucrose > arabinose > xylose > lactose.
This scale agrees with the literature on sugar conversion
[2022].
(b) Except for the cell death Reaction (r16 ), the Vmax of each reaction 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 parameters 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 signicantly faster than xylose.
Based on these observations it is possible to discuss the effects
of the sugars on the proposed model.
Observation (a) would conrm the rst 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 fructose6-P and glucose-6-P: both sugars can be metabolized via the
EmbdenMeyerhof pathway [31]. The lactose is hydrolysed into
glucose and galactose-6-P: the glucose may be phosphorylated
and incorporated into the glycolytic pathway whereas galactose6-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) 156166

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 characterized 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 arabinose.
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 simulation of biolm behaviour where non-homogenous concentration of
metabolites is expected and in particular when butanol concentration is expected to reach maximum/inhibiting values in the inner
part [12]. Of course, the development of a biolm reactor model
would benet 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 acetonebutanolethanol (ABE)
production by C. acetobutylicum DSM 792 developed with the biochemical 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 representative 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 fermenter such as a biolm 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
coefcient (r2 ) between experimental and calculated time-course
of metabolites ranges between 0.87 and 0.925.
Acknowledgements
The Universit Degli Studi di Napoli Federico II is acknowledged for the grant of F. Raganati to be at the University of Berlin.
The authors thank the Ministero dello Sviluppo Economico (Italy)
for the nancial 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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