Continuous lactose fermentation by Clostridium acetobutylicum Assessment

of acidogenesis kinetics
Fabio Napoli, Giuseppe Olivieri, Maria Elena Russo, Antonio Marzocchella

, Piero Salatino
Chemical Engineering Department, Universit degli Studi di Napoli Federico II, P.le V. Tecchio n. 80, 80125 Napoli, Italy
a r t i c l e i n f o
Article history:
Received 4 June 2010
Received in revised form 1 September 2010
Accepted 2 September 2010
Available online 7 September 2010
Keywords:
Clostridium acetobutylicum
Acidogenesis
Product inhibition
Growth model
pH effects
a b s t r a c t
An assessment of the growth kinetics of acidogenic cells of Clostridium acetobutylicum DSM 792 is
reported in the paper. Tests were carried out in a continuous stirred tank reactor under controlled con-
ditions adopting a complex medium supplemented with lactose as carbon source to mimic cheese whey.
The effects of acids (acetic and butyric), solvents (acetone, ethanol and butanol) and pH on the growth
rate of acidogenic cells were assessed. The conversion process was characterized under steady-state con-
ditions in terms of concentration of lactose, cells, acids, total organic carbon and pH. The growth kinetics
was expressed by means of a multiple product inhibition and interacting model including a novel formu-
lation to account for the role of pH. The model has the potential to predict microorganism growth rate
under a broad interval of operating conditions, even those typical of solvents production.
2010 Elsevier Ltd. All rights reserved.
1. Introduction
Acetonebutanolethanol (ABE) production by Clostridium acet-
obutylicum used to be a major bioprocess until the mid-20th cen-
tury when the petrochemical route to butanol made it cost-
ineffective (Jones and Woods, 1986). The recent advances in geno-
mic and metabolic engineering associated to the new economic
scenario of the last twenty years have contributed to renew the
interest in ABE process (Cascone, 2008). To increase the economic
competitiveness of the bioprocess, the attention has been focused
on three main issues: (i) improvement of the metabolic capability
of industrial solventogenic clostridia by mutation or other types of
genetic manipulation (Papoutsakis, 2008); (ii) process intensica-
tion through the adoption of innovative reactor congurations
(Qureshi et al., 2005; Nielsen et al., 2010); (iii) use of inexpensive
renewable resources as substrates for fermentations (Gutierrez
et al., 1998; Ezeji and Blaschek, 2008; Qureshi et al., 2008).
As reviewed by Jones and Woods (1986), two pathways have
been identied in the metabolism of clostridia strains: acidogene-
sis, characterized by substrate conversion into acids (acetic and bu-
tyric acids) by high motile cells (acidogenic cells); solventogenesis,
characterized by substrate and acid conversion into solvents (ABE)
by clostridial-form cells unable to grow (solventogenic cells). The
shift of the two pathways strongly depends on pH and concentra-
tion of metabolites. Studies on continuous production of solvents
by means of clostridia strains conned in reactors by immobiliza-
tion or cell-recycling report the simultaneous production of acids
and solvents suggesting that acidogenic and solventogenic cells
coexist in the cultures (Mutschlechner et al., 2000; Huang et al.,
2004; Qureshi and Blaschek, 2005; Tashiro et al., 2005; Ezeji
et al., 2007; Napoli et al., 2009). The simultaneous production of
acids and solvents coupled with the simultaneous presence of both
acidogenic and solventogenic cells make the ABE production pro-
cess a very complex system to be investigated.
The ABE production by clostridia has been characterized by
adopting several techniques/methodologies. The metabolic ux
analysis and the metabolic control analysis have proven to be pow-
erful tools in promoting genetic engineering applications to modify
the regulation network in order to improve cell performances (Vil-
ladsen, 2007). The analyses are based on a detailed model of the
cell metabolic network and are complicated by the strong inter-
connections between substrate/metabolite uptake/production.
But benets in bioreactor design, scale-up and optimization may
also derive from unstructured models, simple relationships of
key variables. Moreover, the characterization of both bifurcational
patterns and dynamical behavior of bioreactors is made possible by
nimble unstructured models (Russo et al., 2008).
Typically, the models proposed in the literature are character-
ized using fermentation tests carried out under batch conditions
(Jones and Woods, 1986; Yang and Tsao, 1994). But the peculiar
behavior of batch fermentations may introduce transient effects
on cells growth and metabolite production/uptake that are not
0960-8524/$ - see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2010.09.004

Corresponding author. Address: Chemical Engineering Department, School of

Biotechnological Sciences, P.le V. Tecchio 80, 80125 Napoli, Italy. Tel.: +39 081
7682541; fax: +39 081 5936936.
E-mail address: antonio.marzocchella@unina.it (A. Marzocchella).
Bioresource Technology 102 (2011) 16081614
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j our nal homepage: www. el sevi er . com/ l ocat e/ bi or t ech
representative of steady-state continuous cultures, typically
adopted to enhance bioreactor productivity. Therefore, the model
parameters assessed by batch cultures may differ from parameters
assessed by steady-state cultures (Li and Humphrey, 1989), the
difference being remarkable when metabolic processes are charac-
terized by substrate/product inhibition (Schrder et al., 1996).
There is a great deal of literature on ABE production by clostridia
adopting glucose as substrate. The ability of clostridia strains to
metabolize lactose is also reported in the literature (Qureshi
et al., 2005; Napoli et al., 2009, 2010). The interest in latter hexose
has to do with its massive presence in the whey permeate and its
removal being a critical issue for dairy industry.
The present study moves one step further toward the character-
ization of butanol production by C. acetobutylicum. In consideration
of the complexity of the metabolic pathways and with a view to
obtain an unstructured kinetic model, in the present study the
choice has been made to investigate the behavior of acidogenic
cells as a rst step of the whole process, acidogenic cells being still
active during solvents production. The kinetics of acidogenic cell
growth in a CSTR operated under controlled pH is the focus of
the present paper. The study aims at providing kinetic models
and parameters for butanol production adopting a lactose solution
to mimic cheese whey. The inuence of acids (acetic and butyric),
solvents (acetone, ethanol and butanol) and pH on the growth rate
of C. acetobutylicum cells under acidogenesis phase conditions has
been addressed. Process conditions characterized by high ABE con-
centrations have been included in the analysis with the aim to ex-
tend the model to describe cell growth rate also under
solventogenesis conditions. A specic procedure has been pro-
posed to assess model parameters minimizing the number of tests.
2. Materials and procedure
2.1. Microorganism and culture media
C. acetobutylicum DSM 792 was supplied by DSMZ. Stock cul-
tures were reactivated according to the method suggested by the
supplier. Semi-synthetic culture medium (Napoli et al., 2009)
was supplied with 5 g/L Yeast Extract (YE) and D-Lactose as carbon
source. The medium was sterilized in autoclave (121 C, 20 min).
2.2. Apparatus
The reactor was a magnetically stirred 1 Liter vessel (Pyrex

)
equipped with temperature and pH controller. Temperature was
controlled by means of a water jacket connected to a thermostatic
water bath. Both inlet and outlet liquid streams were handled by
peristaltic pumps (Gilson Minipuls 3). The volume of the liquid
phase in the reactor was set at 0.6 L.
The pH was adjusted at the desired value by a controller (Appli-
kon Bio Controller ADI 1030) equipped with 1 M NaOH solution
tank. Nitrogen was sparged (10 nL/h) at the bottom of the reactor
to preserve the anaerobic condition.
2.3. Analytical methods
The analysis of culture samples withdrawn from the chemostat
provided biomass concentration, lactose and metabolite concen-
tration and total organic carbon (TOC) in the liquid phase. Cell den-
sity was measured as optical absorbance at 600 nm (OD
600
) using a
spectrophotometer (Cary-50 Varian). Calibration tests for C. acet-
obutylicum dried mass indicated that 1 OD
600
= 0.4 g
DM
/L. The ele-
mental analysis of dry biomass was performed by means of a C/
H/N 2000 LECO

analyzer.
The concentration of soluble species was measured in the liquid
phase after cell harvesting by centrifugation. Lactose concentration
was measured by means of an enzymatic kit (Biopharm). Metabolite
concentrations were measured by means of a gas-chromatograph
(Agilent 7890AGC) equipped witha FIDand with a capillary column
poraplot Q(25 m 0.32 mm), adopting external standards. The TOC
was measured by a Shimadzu TOC V-CSH analyzer.
2.4. Operating conditions and procedures
Pre-cultures were prepared by inoculating 2 mL of the reacti-
vated cells into 50 mL of lactose supplemented semi-synthetic
medium and incubated for two days.
The pre-cultures were inoculated into the reactor containing
0.6 L of semi-synthetic medium consisting of YE and lactose at
xed concentration. Typically, after 6 h of batch culture the lac-
tose-bearing stream was fed to the reactor at the desired dilution
rate (D). The culture was periodically sampled to measure biomass
and metabolite concentrations until steady-state conditions were
reached. Under each set of operating conditions, the steady state
was characterized in terms of concentration of lactose, cells and
metabolites, averaged over a culture-time longer than 56 times
the liquid spacetime.
Tests were carried out at 35 C under anaerobic conditions. The
lactose concentration in the inlet stream was set between 4 and
50 g/L. The set-point for the pH controller was xed at values rang-
ing between 4.0 and 7.0. The dilution rate was changed between
0.05 and 0.77 h
1
.
The reactor vessel and the medium were sterilized in autoclave.
The gas stream was sterilized by ltration (cut-off 0.2 lm,
Millipore).
3. Design of the experiments and analysis of data
3.1. Assessment of material balances
The reliability of the data measured during the tests was
checked by assessing the closure on carbon mass balance. The
Nomenclature
AA, Ac, B, BA, Et, L concentration of acetic acid, acetone, butanol,
butyric acid, ethanol, (mg/L); lactose (g/L)
D dilution rate, 1/h
H
+
hydrogen ion/hydroxylic concentration, M
R
i
inhibition ratio referred to the species i,
TOC total organic carbon, g/L
X cells concentration, g
DM
/L
Greek letters
d error dened by Eq. (2),
l specic growth rate, h
-1
r
c
carbon fraction of cells, g/g
DM
Subscripts
0 feeding medium
AA, Ac, B, BA, Et, L acetic acid, acetone, butanol, butyric acid, eth-
anol, lactose
F. Napoli et al. / Bioresource Technology 102 (2011) 16081614 1609
following assumptions were made: (i) steady-state operation; (ii)
only the acidogenic pathway was active; (iii) carbon conversion
into CO
2
followed the EmbdenMeyerhof pathway (Jones and
Woods, 1986); (iv) sterile feedings. Accordingly the mass balance
on carbon reads:
TOC
0
TOC X r
C
4
MW
C
MW
L
L
0
L 0 1
where r
c
is the carbon fractional mass of C. acetobutylicum, MW
C
and MW
L
are the molecular weight of carbon and lactose, respec-
tively, X, L and TOC the concentrations in the liquid phase of bio-
mass, lactose and total organic carbon, respectively. Subscript 0
refers to the feeding. The accuracy of the measurements is ex-
pressed by d dened as:
TOC
0
TOC X r
C
4
MW
C
MW
L
L
0
L
TOC
0
d 2
It is noteworthy that the assumption (ii) was supported by tests,
as reported in the results section.
Results of the continuous tests were worked out considering the
mass balance on cells extended to the continuous reactor. Under
steady-state conditions the biomass balance reads:
D l 3
where l is the specic growth rate, and D the dilution rate.
3.2. Growth rate model
The growth kinetics of C. acetobutylicum is characterized by
products inhibition. With reference to single-product inhibited
growth, the models available in the literature typically conform
to one of the following expressions (Han and Levenspiel, 1988):
Ierusalimsky-type l l
max
S
S K
S
1
1 P=K
P
4
Aiba-type l l
max
S
S K
S
e
P=K
P
5
Luong-type l l
max
S
S K
S
1
P
P
max

n
6
Expressions (4) and (5) imply that cells may have a nite,
though small, growth rate even at very large product concentra-
tions (P ?1). However, the cell growth of C. acetobutylicum is
characterized by full inhibition as the concentration of inhibitor
metabolites approaches a critical value (P
max
). Model (6) better de-
scribes this kind of behavior, and was selected to correlate the data.
In agreement with Zeng et al. (1994) the growth kinetics of C.
acetobutylicum under acidogenesis conditions at a xed pH can
be described by the interactive multiproduct-inhibited model:
l l
max

L
L K
L
1
AA
AA
max

n
AA
1
BA
BA
max

n
BA
1
Ac
Ac
max

n
Ac
1
Et
Et
max

n
Et
1
B
B
max

n
B
7
where l
max
is the maximum specic growth rate, K
L
the Monod
coefcient for lactose, L, AA, BA, Ac, Et and B the concentration of lac-
tose, acetic acid, butyric acid, acetone, ethanol and butanol,
respectively.
The inhibitory effects of solvents are included in Eq. (7), even
though these metabolites are not present during acidogenesis.
The interest in solvent effects is justied by the need to describe
the kinetics of acidogenic cells even during solvent production.
Due to the complexity of the model expressed by Eq. (7) and the
number of parameters to be determined (l
max
, K
L
, AA
max
, BA
max
,
Ac
max
, Et
max
, B
max
, n
AA
, n
BA
, n
Ac
, n
Et
and n
B
), a multistep parameter
inference procedure based on an experimental matrix designed
ad hoc was required to infer the kinetic parameters.
Step (1) Preliminary estimate of l
max
and K
L
: Once the pH is
xed, rst-step estimates of l
max
and K
L
(l
I
max
and K
I
L
) were ob-
tained by a best-t procedure applied to data obtained during tests
carried out at dilution rates D approaching wash-out, with lactose
concentrations ranging over a broad interval. Under these condi-
tions, metabolite concentration was negligible and Eq. (7) reduces
to the classical Monod model.
Step (2) Preliminary estimate of AA
max
and BA
max
: At the pre-
set value of pH and using values of l
I
max
and K
I
L
determined in the
previous stage, preliminary estimates (AA
I
max
and BA
I
max
) of the crit-
ical concentration of acids AA
max
, BA
max
were assessed by working
out data obtained in experiments carried out at nearly wash-out
conditions with controlled addition of acids in the feeding. Under
these conditions, due to the very low consumption of the substrate,
acid production is limited, and the concentration of acids to be con-
sidered in Eq. (7) is dictated by the amount of acids purposely sup-
plemented to the feeding. Assuming n
AA
= n
BA
= 1, data regression
provided the rst-step estimate of AA
I
max
and BA
I
max
.
Step (3) Exact determination of l
max
, K
L
, AA
max
, BA
max
, n
AA
and
n
BA
at the pre-set pH: The complete set of these parameters was -
nally determined by a parametric inference procedure applied to
Eq. (7) matched against data measured under a wide interval of
operating conditions. The preliminary estimates obtained in the
previous steps were used as the starting point of the multivariate
best-t convergence procedure.
Step (4) Estimation of Ac
max
, Et
max
, B
max
, n
Ac
, n
Et
and n
B
: The
assessment of these parameters was accomplished at the pre-xed
pH by working out data measured in tests in which solvents were
purposely added to the feedstock.
3.3. Modeling the effect of pH
The role of pH in the acidogenesis kinetics can be twofold. It can
affect both the maximum specic growth rate and the critical con-
centration of acids. The latter could depend on the concentration of
undissociated acids.
The relationship between the parameter l
max
of a microorgan-
ism and pH is typically described by the model proposed by Tang
et al. (1989):
l
max

l
max
1
H

K
H

K
OH
H

8
where H
+
is the concentration of hydrogen ions in the medium and
l
max
, K
H
and K
OH
are model parameters. The pH (pH
OPT
) at which
the cell growth rate is maximum is related to the parameters K
H
and K
OH
according to:
log

K
H
K
OH
p
pH
OPT
9
Huang et al. (1985) investigated the growth of C. acetobutylicum
ATCC 824 on glucose pointing out the relevance of intracellular pH.
In particular, they reported that the transmembrane pH (DpH)
difference of pH between the medium and the intracellular value
decreased with the medium pH and increased up to 1.5 when
the pH of the medium was at its minimum value. Considering
these ndings, a modied version of Eq. (8) is put forward in the
present study:
l
max

l
max
1
H

K
H

K
OH
H

1
10
pH
OPT
pH
10
DpH
!
10
where DpH is set to 1.5.
The effects of pH on the maximum specic growth rate and on
the critical concentrations of metabolites were assessed by
1610 F. Napoli et al. / Bioresource Technology 102 (2011) 16081614
working out the data measured during tests carried out at pH
ranging between 4 and 7. It should be noted that the dilution rate
interval at pH smaller than 5.0 is drastically reduced. Actually low
D promotes solvent production, while large values of D bring about
reactor wash-out.
4. Results and discussion
Table 1 reports some selected results of a representative set of
runs carried out at pH = 5.0, at dilution rates approaching the
wash-out conditions. The elemental analysis of the dry biomass
sampled at the end of each run yielded r
C
= 44%
W
. Table 1 also re-
ports the error associated with material balance on carbon, d, com-
puted according to Eq. (2). It resulted smaller than 10%, whatever
the operating conditions tested.
Solvents were absent in the reactor whatever the operating con-
ditions adopted, but when purposely supplemented in the feeding.
Accordingly, the assumption (ii) reported in the Section 3.1 held.
Data reported in Table 1 were worked out for a rough estimate
of l
max
and K
L
at pH = 5, in agreement with step (1) of 3.2.
Accordingly the inhibition terms of Eq. (7) were neglected and data
regression yielded l
I
max
= 0.80 h
1
and K
I
L
=2.0 g/L.
4.1. Inhibition by acids
Table 2 reports data resulting from tests carried out with the
aim of determining the critical concentrations AA
I
max
and BA
I
max
.
According to step (2) of 3.2, the values of n
AA
and n
BA
were both
set equal to 1 at this stage. The rst four tests in Table 2 refer to
operations characterized by limited lactose consumption and feed-
ing supplemented with either acetic or butyric acid. Assuming that
under the operating conditions selected the inhibition effects of
the acid not supplemented may be neglected, Eq. (7) yields:
l l
I
max

L
L K
I
L
1
AA
AA
I
max
11
l l
I
max

L
L K
I
L
1
BA
BA
I
max
12
for tests carried out with addition of acetic acid and butyric acid to
the feeding, respectively. The inhibitory effect of the generic acid
A may be assessed by analyzing the ratio:
R
A

l
l
I
max
L
L K
I
L
1
A
A
max
13
as a function of the acid concentration. Working out the data of
the rst four rows of Table 2 tests carried out supplementing ace-
tic or butyric acid to the feeding in agreement with Eq. (13), a
rst estimate of the critical concentration of acids has been as-
sessed, AA
I
max
= 1.66 g/L and BA
I
max
= 3.34 g/L.
According to the procedure described in step (3) of 3.2, the ex-
act estimate of the parameters in Eq. (7) (l
max
, K
L
, AA
I
max,
BA
I
max
) at
pH = 5.0 was accomplished by regression of data in Tables 1 and 2,
characterized by broad intervals of dilution rate, lactose and
metabolites concentration. The nal results of the complete multi-
variate best-t procedure were:
l
max
0:95 h
1
K
L
1:34 g=L
AA
max
1:56 g=L n
AA
0:98
BA
max
3:00 g=L n
BA
0:96
14
It is noteworthy that the critical concentration of butyric acid is
larger than that of acetic acid.
It is interesting to compare the critical concentrations deter-
mined in the present study with data previously reported in the lit-
erature. The critical concentration of butyric acid (3.00 g/L) is about
twice that of the acetic acid (1.56 g/L), consistently with the larger
selectivity towards butyric acid observed during runs carried out
without any supplemented acid. Yang and Tsao (1994) determined
a toxic level of acetic acid and butyric acid concentrations of about
12 and 11 g/L, respectively, based on a batch culture of C. acetobu-
tylicum ATCC824 on complex medium supplemented with glucose.
For the industrial strain C. acetobutylicum P262 the growth rate
vanishes when the concentration of acetic and butyric acids ap-
proach the limiting values of 4.8 and 10.6 g/L, respectively (Ennis
and Maddox, 1987). The analysis of the critical concentrations sug-
gests that the toxic concentration of acids depends on the strain as
well as on the medium composition.
4.2. Inhibition by solvents
The inhibition effects of solvents on the cell growth rate un-
der acidogenic conditions were investigated by supplementing
acetone, butanol and ethanol to the reference medium. The lac-
tose concentration in the feeding was set in these tests at about
ten times the value of the Monod constant K
L
. Selected data
from tests carried out by supplementing individual solvents to
the medium are reported in Table 3. S being the concentration
of the generic solvent, an inhibition ratio R
s
could be dened
as the ratio between the measured growth rate l and the
growth rate calculated adopting the exact values of the param-
eters (14):
R
S

l
l
max
1
AA
AAmax

n
AA
1
BA
BAmax

n
BA
1
S
S
max

ns
15
Table 1
Results of selected tests carried out at pH = 5.
Feeding D = l
(h
1
)
Concentration in the efuent stream d (Eq. (2))
%
L
0
(g/L)
Acetic acid
(mg/L)
Butyric acid
(mg/L)
Lactose
(g/L)
4.6 0.22 420 810 1.1 4
4.6 0.44 320 590 2.6 2
4.6 0.49 280 560 2.5 3
13.6 0.71 140 250 12.7 5
13.6 0.71 140 220 12.9 3
25.0 0.71 170 430 23.8 1
36.0 0.74 160 290 34.0 7
24.9 0.76 250 530 22.4 4
13.9 0.77 90 170 13.8 8
Table 2
Results of tests carried out with medium supplemented with acetic or butyric acid.
Supplemented
product
D = l
(h
1
)
Concentration in the efuent d (Eq. (2))%
Acetic acid
(mg/L)
Butyric acid
(mg/L)
Lactose
(g/L)
Acetic acid 0.32 820 780 11.5 3
0.24 1050 250 7.1 4
Butyric acid 0.38 270 1410 12.7 6
0.15 260 2250 5.2 2
None 0.05 1090 2260 6.2 3
0.12 950 1990 8.8 4
0.15 760 1880 44.0 10
0.18 770 1620 8.4 3
0.33 440 810 12.0 2
0.50 290 760 40.8 5
0.56 270 670 22.2 2
0.63 180 390 12.2 1
F. Napoli et al. / Bioresource Technology 102 (2011) 16081614 1611
Tests carried out by supplementing butanol to the feeding at a
concentration larger than 16.5 g/L (data not reported) were charac-
terized by complete growth inhibition, then by reactor wash-out.
The analysis of data reported in Table 3 suggests that the depen-
dence of l on butanol concentration is non-linear. Working out
the data reported in the Table 3 regarding the tests carried out
by supplementing butanol to the feeding in agreement with Eqs.
(7) and (15) yields B
max
= 16.5 g/L and n
B
= 0.47. The critical con-
centration assessed for butanol is in agreement with values re-
ported in the literature (Yang and Tsao, 1994).
The analysis of data reported in Table 3 regarding the tests car-
ried out by supplementing either ethanol or acetone to the feeding
considers that the solvent concentration were quite larger than
those usually achieved during the ABE fermentation (Jones and
Woods, 1986; Napoli et al., 2009). Despite the high solvent concen-
tration tested, the R
S
assessed in agreement with Eq. (15) decreases
linearly with the solvent concentration. Accordingly, the exponent
n for both solvent was set at 1 and the critical concentration as-
sessed by linear extrapolation of data at R
s
= 0. It resulted
Ac
max
= 64.5 g/L and Et
max
= 35.0 g/L.
The comparison between the critical concentration of acids and
solvents indicates that the most oxidized form of metabolites is
typically more toxic than the solvents. The analysis of the critical
concentration of the three solvents suggests that the inhibitory ef-
fect of acetone and ethanol is typically negligible with respect to
that of butanol, the main product of ABE fermentation. Therefore,
strategies aiming at the optimization of the process should focus
on butanol inhibition.
4.3. The role of pH
Table 4 reports the results obtained in tests carried out with
variable values of pH, ranging between 4.0 and 7.0. Data were
worked out according to Eq. (7) under two alternative hypotheses:
the concentration of either the undissociated or the total acids was
considered in the kinetic model Eq. (7). Regression of data based on
the concentration of undissociated acids yielded poor correlation.
On the contrary, the regression of data assuming the total concen-
tration of acids successfully correlated data at different pH. In par-
ticular it was shown that: (i) the value of pH affects the specic
growth rate; (ii) the critical concentration of acids (AA
max
, BA
max
)
does not change with pH.
Fig. 1 reports the specic growth rate vs. the pH of the medium.
The best-t procedure yielded the following values of parameters
in Eq. (8):
Table 4
Results of tests carried with different values of pH of the medium.
pH D = l
(h
1
)
Concentration in the efuent d (Eq. (2))
%
Acetic acid
(mg/L)
Butyric acid
(mg/L)
Lactose
(g/L)
4.0 0.24 380 800 12.5 2
0.32 120 260 14.0 4
4.2 0.12 900 1940 8.7 2
0.18 840 2060 9.0 3
0.35 540 1220 10.8 3
0.52 210 500 13.1 4
4.5 0.09 710 2280 8.1 4
0.27 640 1400 10.8 3
0.51 350 770 12.8 4
6.0 0.06 920 2230 4.9 4
0.30 490 870 10.7 3
0.55 210 450 13.1 3
0.60 70 110 6.4 2
7.0 0.10 80 290 7.7 2
Fig. 1. Maximum specic growth rate of C. acetobutylicum as a function of pH.
Table 3
Results of tests carried out with the medium supplemented with solvents.
Supplemented product D = l (h
1
) Concentration in the efuent d (Eq. (2)) %
Acetic acid
(mg/L)
Butyric acid
(mg/L)
Butanol
(mg/L)
Ethanol
(mg/L)
Acetone
(mg/L)
Lactose
(g/L)
Butanol 0.51 280 550 3070 11.7 3
0.52 200 460 6330 12.2 5
0.24 560 1200 5840 9.0 5
0.26 440 1020 8960 10.9 1
0.27 360 890 12080 10.7 2
0.19 290 680 14640 10.3 2
0.47 320 500 5360 6.7 5
0.32 240 430 11900 11.8 3
Acetone 0.36 460 940 5080 11.4 2
0.20 660 1390 14900 9.5 3
0.20 570 1220 25200 9.4 2
Ethanol 0.49 300 560 490 13.1 3
0.45 270 530 970 11.3 2
0.47 270 560 1850 14.0 1
0.47 300 630 5800 14.8 1
0.25 490 1020 11200 12.6 1
0.23 360 730 20700 12.2 1
1612 F. Napoli et al. / Bioresource Technology 102 (2011) 16081614
K
H
3:33 10
4
M K
OH
5:04 10
7
M l
max
1:06 h
1
pH
OPT
4:9
The dashed line in Fig. 1 results from computations based on Eq.
(8) with the parameters reported above. The agreement with data
points is fair for pH larger than 5.0, but poor at low pH. The sym-
metry of Eq. (8) does not reproduce the experimental data, which
are instead characterized by a steep decrease of the specic growth
rate at pH < 5. Alternatively, regression analysis of data was
accomplished according to Eq. (10) assuming pH
OPT
= 4.9 and
DpH = 1.5. Computations corresponding to Eq. (10) are reported
in Fig. 1 as solid line, and showthat the agreement with data points
is satisfactory over the entire range of pH tested.
The pH appears to have a twofold effect on the specic growth
rate of acidogenic cells of C. acetobutylicum. The rst effect is com-
mon for most bacterial strains and is related to the active fraction
of enzymes, as detailed by Tang et al. (1989). The model proposed
by Tang et al. (1989) is adequate to reproduce data points for pH
larger than the optimal pH value. The second effect is related to
the ability of the cells to survive far from optimal environmental
conditions: the cells counteract the reduction of the medium pH
below the optimal value by extruding protons, so as to keep the
intracellular pH close to the optimum (Huang et al., 1985). The va-
lue of pH
OPT
depends on the strain. Several C. acetobutylicum DSM
strains show maximum growth rate at pH 5 and C. acetobutyli-
cum ATCC 824 at 6 (Jones and Woods, 1986).
4.4. Overall assessment of the kinetic model
Fig. 2 reports a parity plot comparing experimental values of the
specic growth rate with those predicted according to Eqs (7) and
(10), using model parameters reported in Table 5. Data points are
those obtained in tests performed at pH > 4.2.
Inspection of the parity plot indicates that the departure of
model predictions from experimental data points is fairly small,
typically within 10%. This nding suggests that the model based
on application of Eqs (7) and (10) is able to correctly reproduce
the growth kinetics of C. acetobutylicum DSM 792 on lactose over
a broad range of operating conditions, even at low product
concentrations.
5. Conclusions
The kinetics of C. acetobutylicum DSM 792 growth on lactose
was investigated by means of a chemostat. The inuence of etha-
nol, acetone and butanol on the growth kinetics was characterized
to adopt the model for acidogenic cells during solvent production.
The experimental results were successfully correlated by a mul-
tiple product-inhibited and interacted growth model, embodying a
term related to the pH effect. An optimized experimental matrix
and a multi-step parametric inference method were proposed.
The kinetic model is relevant to design and to optimize a continu-
ous bioreactor to produce ABE, and to characterize both the bifur-
cational patterns and the dynamic behavior of bioreactors.
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Table 5
Model parameters of Eqs (7) and (10).
lmax = 1.06 h
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K
L
= 1.34 g/L
AA
max
= 1.56 g/L n
AA
= 0.98
BA
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= 3.00 g/L n
BA
= 0.96
B
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= 16.50 g/L n
B
= 0.47
Ac
max
= 64.5 g/L n
Ac
= 1
Et
max
= 35.0 g/L n
Et
= 1
K
H
= 3.33 10
4
M K
OH
= 5.04 10
7
M
pH
OPT
= 4.9 DpH = 1.5
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