Biochemical Engineering Journal 19 (2004) 189197

Optimization of dissimilation of glycerol to 1,3-propanediol by Klebsiella

pneumoniae in one- and two-stage anaerobic cultures
Zhi-Long Xiu a, , Bing-Hui Song b , Zong-Tao Wang b , Li-Hua Sun b ,
En-Min Feng b , An-Ping Zeng c
a

Biotechnology Department, Dalian University of Technology, Linggong Road 2, Dalian 116024, PR China
b Applied Mathematics Department, Dalian University of Technology, Dalian 116024, PR China
Biochemical Engineering Division, GBF-German Research Center for Biotechnology, D-38106 Braunschweig, Germany
Received 31 May 2003; received in revised form 20 November 2003; accepted 9 December 2003

Abstract
In this study, the optimal conditions of batch and continuous glycerol fermentations by Klebsiella pneumoniae were investigated using
the volumetric productivity of 1,3-propanediol (1,3-PD) as an optimization target based on a mathematical model that considers the growth
kinetics of multiple inhibitions and the metabolic overflow of substrate consumption and product formation. For batch culture with a
given inoculation of 0.1 g biomass l1 , the optimal initial glycerol concentration was found to be 960 mmol l1 , which lead to the highest
volumetric productivity (52.6 mmol l1 h1 ) of 1,3-propanediol. For continuous fermentations, the optimal dilution rate and initial glycerol
concentration in feed were 0.29 h1 and 731 mmol l1 , respectively. The corresponding productivity was 114 mmol l1 h1 that was more
than twice the productivity of an optimal batch culture. A comparison between experimental and computational results of the concentration,
yield and productivity of 1,3-propanediol showed that most of the continuous fermentation data were lower than or approached to the
calculated values. Stability analysis of continuous cultivations indicated that two regions of multiple states occurred at relatively high
concentrations of initial glycerol in feed. One of them approached to the wash-out line. A two-stage continuous process was proposed,
in which the first stage was operated at the optimal conditions and the second one was used to consume the residual glycerol in the
first one. Model analysis showed that the dilution rate should be much higher in the second stage than in the first one. In terms of the
concentration, yield and productivity of 1,3-propanediol, a two-step bioprocess of two bioreactors in series appeared to be more favorable
for 1,3-propanediol production than a single bioreactor system with the same volume.
2004 Elsevier B.V. All rights reserved.
Keywords: 1,3-Propanediol; Glycerol; Bioconversion; Klebsiella pneumoniae; Process optimization; Two-stage bioprocess

1. Introduction
Because 1,3-propanediol (1,3-PD) possesses potential
applications on a large commercial scale, especially as a
monomer of polyesters, polyethers or polyurethanes, its microbial production has recently paid much attention [1]. It is
considered to be one of the bulk chemicals, which is likely
to be produced by bioprocesses on large scales [2]. Both
bioconversions of glucose and of glycerol to 1,3-PD by a
single microorganism are two main kinds of microbial production of 1,3-PD. The former utilizes a recombinant strain
combining both glycerol and 1,3-PD producing genes [3].
The latter can be naturally conducted by bacteria belonging
Corresponding author. Tel.: +86-411-4706369;
fax: +86-411-4706369.
E-mail address: zlxiu@mail.dlptt.ln.cn (Z.-L. Xiu).

1369-703X/$ see front matter 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.bej.2003.12.005

to the genera of Klebsiella, Citrobacters, Enterobacter and

Clostridia and so on [1], in which Klebsiella pneumoniae
has been widely investigated since 1980s due to its high
productivity. The experimental investigations showed that
the fermentation of glycerol by K. pneumoniae is a complex bioprocess, since the microbial growth is subjected to
multiple inhibitions of substrate and products, e.g. glycerol,
1,3-PD, acetate and ethanol [4]. In addition, the kinetics
of substrate consumption and product formation behave
metabolic overflow at substrate-excess conditions [5,6].
The metabolic overflow of product and its inhibition on cell
growth lead to nonlinear phenomena, such as multiplicity,
hysteresis and oscillation [79]. Nevertheless, the metabolic
overflow can also lead to a high concentration and high productivity of 1,3-PD [10]. To achieve this, the concentration
of residual unconsumed glycerol in the bioreactor must be
high, but this raises considerable problems in the separation

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Z.-L. Xiu et al. / Biochemical Engineering Journal 19 (2004) 189197

Nomenclature
ai (i = 14) coefficients of characteristic equation
aij
elements of Jacobian matrix
b1 , b2
parameters for determination of
yield of ethanol on glycerol
(mmol l1 h1 )
c1 , c2
parameters for determination of yield of
ethanol on glycerol (mmol l1 h1 )
CP
product concentration (mmol l1 )

CP
maximal product concentration
(mmol l1 )
CS , CS0
substrate concentration in reactor and
feed medium, respectively (mmol l1 )

CS
maximum residual substrate
concentration (mmol l1 )
D
dilution rate (h1 )
KS
Monod saturation constant (mmol l1 )
KS , KP
saturation constants for substrate and
product in kinetic equations with excess
terms (mmol l1 )
J
Jacobian stability matrix
ms , mP
maintenance term of substrate
consumption and product formation
under substrate-limited conditions
(mmol g1 h1 )
qs , qP
specific rates of substrate uptake and
product formation (mmol g1 h1 )
m
m
qS , qP
maximum increment of substrate
consumption rate and product formation
rate under substrate-sufficient conditions
(mmol g1 h1 )
Q
volumetric productivity of 1,3-propanediol
(mmol l1 h1 )
t
time (h)
X
biomass concentration (g l1 )
m
m
YS , YP
maximum growth yield (g mmol1 ) and
product yield (mmol g1 )
Y
yield of 1,3-propanediol on glycerol
(mol mol1 )
Greek letters

eigenvalues
, m
specific and maximum specific
growth rate (h1 )
Subscripts
EtOH
HAc
PD
s
S
t
X

ethanol
acetic acid
1,3-propanediol
single bioreactor system
glycerol
total bioprocess of a two-stage system
biomass

of target product from the fermentation broth due to similar

physical and chemical properties of glycerol and 1,3-PD.
It is thus desirable to convert the high residual glycerol to
1,3-propanediol in a subsequent bioreactor. The previous
theoretical analysis of an ideal microbial system with a single substrate and a single product indicated that two-stage
fermentations present a better performance compared with
the one-stage cultures in terms of concentration, productivity and the yield of product [11]. However, no model analysis and simulations have been done to determine the optimal
operation conditions in a complex bioprocess involving
metabolic overflow and multiplicity such as the microbial
production of 1,3-PD, especially for a two-stage process.
In this study, a model-guided analysis and optimization
of glycerol fermentation are conducted. The volumetric productivity of 1,3-PD is used as an objective function of optimization to determine the optimal dilution rate and initial
glycerol concentration in the feed in a one-stage continuous anaerobic cultivation. The concentration of 1,3-PD and
its yield on glycerol are investigated and compared with the
experimental results. In addition, a two-stage bioprocess is
designed and analyzed theoretically so as to efficiently convert the high residual glycerol in the first bioreactor.

2. Kinetic models
K. pneumoniae DSM2026 was used. The medium composition, cultivation conditions, determination of biomass,
substrate and metabolites, and experimental data have been
previously reported [46,10,12].
Mass balances of biomass, substrate and products in continuous microbial cultures are written as follows:
dX
= ( D)X
dt

(1)

dCS
= D(CS0 CS ) qS X
dt

(2)

dCPi
= qPi X DCPi
dt

(3)

(Pi = PD, HAc, EtOH)

where the specific growth rate of cells (), specific consumption rate of substrate (qS ) and specific formation rate
of product (qPi ) are expressed by Eqs. (4)(6), respectively,
on the basis of previous work [46].





CS
CS
CPD
1
1
KS + C S
CS
CPD





CHAc
CEtOH
1
1
CHAc
CEtOH

= m

qS = mS +

CS
+ qSm
m
YS
CS + KS

(4)

(5)

Z.-L. Xiu et al. / Biochemical Engineering Journal 19 (2004) 189197

Table 1
Parameters in the kinetic models (5) and (6)
Substrate/products

Ym

qm

Glycerol
Propanediol
Acetate

2.20
2.69
0.97

0.0082
67.69
33.07

28.58
26.59
5.74

11.43
15.50
85.71

m
m
qPi = mPi + YPi
+ qPi

CS

CS + KPi

(Pi = PD, HAc)

(6)

The specific formation rate of ethanol can be described by

the following Eq. (7):
m
qEtOH = qS YEtOH/S

(7)

in which the yield of ethanol on glycerol is a function of

product of dilution rate or specific growth rate and glycerol
concentration in bioreactor [12].
b1
b2
m
YEtOH/S
=
+
(8)
(c1 + DCS ) (c2 + DCS )
For K. pneumoniae cultivated under anaerobic conditions
at 37 C and pH 7.0, the maximum specific growth rate
(m ) and the saturation constant for glycerol (KS ) in Eq. (4)
present the values of 0.67 h1 and 0.28 mmol l1 , respectively [4]. The critical concentrations of glycerol, 1,3-PD,
acetate and ethanol for cell growth are 2039, 939.5, 1026
and 360.9 mmol l1 [10], respectively. In addition, the parameters b1 , b2 , c1 , c2 in Eq. (8) are 0.025, 5.18, 0.06, and
50.45 mmol l1 h1 , respectively, while the ones for Eqs. (5)
and (6) are listed in Table 1 as previously described [12].
The governing equations for a two-stage fermentation process as depicted in Fig. 1 can be described by a group of
Eq. (9).

dX1

= X1 (1 D1 )

dt

dCS1

= D1 (CS0 CS1 ) X1 qS1

dt

dCPD1

= D1 CPD1 + X1 qPD1

dt

dCHAc1

= D1 CHAc1 + X1 qHAc1

dt

dCEtOH1

= D1 CEtOH1 + X1 qEtOH1

dt
(9)

dX2 = X2 (2 D2 ) + D2 X1

dt

dC

S2

= D2 (CS1 CS2 ) X2 qS2

dt

dCPD2

= D2 (CPD1 CPD2 ) + X2 qPD2

dt

dCHAc2

= D2 (CHAc1 CHAc2 ) + X2 qHAc2

dt

dCEtOH2 = D2 (CEtOH1 CEtOH2 ) + X2 qEtOH2

dt
The specific growth rates of cells in bioreactors I and II
are expressed by Eqs. (10) and (11), respectively.

191




CS1
CS1
CPD1
1
1
1 = m
CS1 + KS
C
C



S
 PD
CHAc1
CEtOH1
1
1
CHAc
CEtOH





CS2
CS2
CPD2
2 = m
1
1
CS2 + KS
C
C



S
 PD
CHAc2
CEtOH2
1
1
CHAc
CEtOH

(10)

(11)

The specific consumption rate of glycerol and specific formation rates of 1,3-PD, acetate, ethanol in bioreactors I and
II can be described by equations similar to (5)(8) replacing
CS , with CS1 , 1 and CS2 , 2 , respectively. The parameters are the same as those in Eqs. (5)(8).

3. Methods of optimization and criteria for

multiplicity
For batch cultures, the dilution rate (D) in Eqs. (1)(3)
is zero and is replaced by the specific growth rate () in
Eq. (8). The initial conditions are as follows:
t = 0,
CPi = 0

CS = CS0 ,

X = X0 ,

(Pi = PD, HAc, EtOH)

The Eqs. (1)(3) are resolved at a given initial biomass concentration (X0 ) and different initial glycerol concentration
(CS0 ). An optimal initial glycerol concentration can be determined at a maximum volumetric productivity of 1,3-PD.
The volumetric productivity of 1,3-PD, CPD /t, is chosen as
an objective function of optimization for batch culture at the
given initial biomass concentration.
For continuous cultures, a volumetric productivity of
1,3-PD (DCPD ) as an objective function of optimization is
obtained by letting left hands of Eqs. (1)(3) be zero and
resolving a steady-state solution at a given dilution rate (D)
and initial glycerol concentration in feed (CS0 ). The optimal
operation condition can be got at the maximum volumetric
productivity.
The Hurwitz criteria is used to analyze the stability of
a nonlinear system composed of Eqs. (1)(8) or equation
group (9), which is similar to the previous work [8,11]. The
stability of the second bioreactor depends on the first one
in a two-stage bioprocess. The Jacobian matrix of the first
bioreactor in Eq. (9) can be written as follows:

a11 a12 a13 a14 a15

a21 a22 a23 a24 a25

J =
a31 a32 a33 a34 a35
a41 a42 a43 a44 a45
a51 a52 a53 a54 a55
where aij (i = 1 5, j = 1 5) is the partial derivative of the former five equations in Eq. (9) versus
X1 , CS1 , CPD1 , CHAc1 , CEtOH1 , respectively.

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Z.-L. Xiu et al. / Biochemical Engineering Journal 19 (2004) 189197

Fig. 1. Flow chart of continuous operation of two reactors in series.

of biomass (0.05 and 0.1 g l1 ) in batch cultures. A maximum productivity (52.6 mmol l1 h1 ) could be obtained
at CS0 = 960 mmol l1 and X0 = 0.1 g l1 . With the initial
biomass concentration decreases, the volumetric productivity also decreases, but the optimal initial glycerol concentration does not change significantly. For instance, an optimal
CS0 is determined to be 1000 mmol l1 at X0 = 0.05 g l1
and the maximum productivity of 45.1 mmol l1 h1 . The
experimental data of batch cultures [13] at an inoculation
amount of 0.1 g biomass l1 are less than the predicted upper limit of productivity of 1,3-PD, especially in the range
of optimum initial glycerol concentration. The experimental concentrations of biomass and 1,3-PD are also below
their theoretical limits (Fig. 3). The upper limit of 1,3-PD
concentration increases with initial glycerol concentration
increases. A comparison of computational results between
Figs. 2 and 3 shows that the optimal initial glycerol concentration for biomass (1180 mmol l1 ) is higher than that
for the productivity of 1,3-PD (960 mmol l1 ) at an inoculation of 0.1 g biomass l1 . However, no optimal X0 can be

The characteristic equation can get from the above Jacobian matrix:
5 + a 1 4 + a 2 3 + a 3 2 + a 4 + a 5 = 0

(12)

in which a1 , ..., a5 are determined by the steady-state solutions of Eqs. (1)(8) or (9). The roots of Eq. (12) are the
eigenvalues of the matrix J, which can be used to analyze
the stability of the corresponding steady-state solution.

4. Results and discussion

4.1. One-stage fermentation

Productivity of 1,3-PD (mmol/L.h)

4.1.1. Batch cultures

Two operational conditions need to be determined for
batch cultures: the initial inoculation amount of biomass
(X0 ) and the initial glycerol concentration (CS0 ). Fig. 2
depicts the volumetric productivities of 1,3-PD at different
initial concentrations of glycerol and constant inoculation

60

X 0 =0.1 g/L
50

X 0 =0.05g/L

40
30
20
10
0
0

200

400

600

800

1000

1200

1400

1600

1800

Initial concentration of glycerol (m m ol/L)

Fig. 2. Model calculation of effects of initial glycerol concentration on the productivity of 1,3-propanediol at a constant amount of inoculation in batch
culture of glycerol fermentation by K. pneumoniae (points represent the experimental data and the curves are the upper limits of productivity).

Z.-L. Xiu et al. / Biochemical Engineering Journal 19 (2004) 189197

193

Fig. 3. Comparison of biomass and 1,3-PD between experimental data and calculated results for batch cultures (circle points are the experimental
concentrations of 1,3-PD; up triangle points are the experimental biomass concentrations; curves represent the upper limits of biomass and 1,3-PD
concentrations).

CS(mmol/L)

X (g/L)

6
4
2
0
2000
1500
1000
500
0

CPD(mmol/L)

4.1.2. Continuous cultures

Fig. 4 shows the experimental and computational results
of a continuous culture by K. pneumoniae at a dilution rate
of 0.1 h1 and different initial glycerol concentrations. The
computation shows that there are two hysteresis loops at
given operational conditions, i.e. initial glycerol concentration and dilution rate. The first hysteresis loop, BCEDB, as
shown in Fig. 4 had been discussed in detail in the previous
studies [8,12]. The second one, FGJHF, occurs at high initial glycerol concentrations. The states on the line HJ are the
so-called wash-out steady states. Stability analysis shows
that the states on the lines CD and GH are unstable. The
other ones are stable. In this work, the stable steady state
with a high concentration of 1,3-PD is chosen to calculate
the volumetric productivity of 1,3-PD.
According to the occurrence of multiplicity, the operational regime can be divided into three regions as depicted
in Fig. 5: Regions I and II with multiple states, region III
with single steady states. The first hysteresis loop occurs
in the region II and the second one in region I. The region I approaches to the wash-out line ABC. The dash line

HG represents the optimum steady states for continuous

cultures.
Fig. 6 shows comparisons between experimental and computational results of volumetric productivities. The computational values represent the upper limits of productivity of

800
600
400
200
0

CEtOH(mmol/L) CHAc(mmol/L)

determined for batch culture because the volumetric productivity is a monotonously increasing function of X0 at a
constant initial glycerol concentration.
It needs to be mentioned that Eqs. (1)(8) are limited to
describe the exponential phase of a batch culture [12]. It
is also difficult to describe the long lag phase or stationary
phase of batch cultivation with such an unstructured kinetic
model. Therefore, the above optimal result is only a theoretical value for special cases, e.g. a batch culture with a short
or no lag phase and the maximum productivity obtained at
exponential phase.

B
D

C
E

F
H

G
J

160
120
80
40
0

300
225
150
75
0
0

300

600

900 1200 1500 1800 2100

Initial glycerol concentration in feed (mmol/L)

Fig. 4. Comparison of biomass, substrate and product concentrations
between experimental and simulated results at a dilution rate of 0.1 h1
in continuous cultures (data from [10]).

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Z.-L. Xiu et al. / Biochemical Engineering Journal 19 (2004) 189197

Fig. 5. Division of the operating regime of continuous cultures according

to the occurrence of multiplicity. Regions I (BCDB) and II (EFGE): two
stable steady states and one unstable steady state; region III (below the
line ABD except region II): single steady state; line HG: steady states
with optimum productivity; point P: an optimal operational condition with
a maximum productivity.

1,3-PD. The corresponding operational line is the dash line

HG in Fig. 5. It lies mostly in the stable region III and partly
in the two-state region II. A theoretically optimal initial glycerol concentration and dilution rate can be inferred from
Fig. 6A and B, respectively, i.e. 731 mmol l1 and 0.29 h1 .
It is depicted by the point P on the dash line HG in Fig. 5
and is a single stable steady state. Its corresponding concentration, volumetric productivity, and molar yield of 1,3-PD
are 400 mmol l1 , 114 mmol l1 h1 , and 0.63 mol mol1 ,
respectively. It should be noted that the maximum theoretical 1,3-PD volumetric productivity obtained is close to the
highest ones achieved in the literature, for single-stage continuous cultures of K. pneumoniae [10] or C. butyricum [14],
growing on pure or industrial glycerol.
Based on the mass, ATP and reducing equivalent balances,
a pathway analysis showed that a maximum yield of 1,3-PD

on glycerol could be obtained for a special case of no formation of ethanol and hydrogen [15]. Under these conditions, the concentration of acetic acid is stoichiometrically
to be 0.31 times of 1,3-PD concentration. An optimal dilution rate of 0.3 h1 could be calculated from the cell growth
kinetics at a maximum volumetric productivity [10]. This
agrees well with the calculated results in this work. However, no optimal initial glycerol concentration was reported
in previous work.
Moreover, it is noted that most of the experimental volumetric productivities of 1,3-PD present lower values compared with the upper limits besides few cases (Fig. 6). The
same remark may be done considering experimental and theoretical concentrations of 1,3-PD (data not presented). Furthermore, the theoretical maximum concentration of 1,3-PD
decreases as dilution rate but increases as initial glycerol
concentration in feed, while the yield of 1,3-PD on glycerol declines as dilution rate increases but increases as initial
glycerol concentration increases, as shown in Fig. 7. This
fact should be attributed to additional generation of NADH2 ,
occurring under low dilution rates and glycerol-excess conditions [1]. Similarly, most of the experimental data are
below the theoretical limits except few data. Few are beyond the theoretical values even over the maximum yield,
0.72 mol mol1 [15], especially at dilution rates of more than
0.45 h1 . The reason might be a measurable deviation for
1,3-PD or glycerol. On the other hand, the maximum yield
of 1,3-PD was previously estimated to be less than that of
recent analysis, being 0.85 mol mol1 [16].
4.2. Two-stage fermentation
The concentrations of biomass, glycerol, 1,3-PD, acetate,
and ethanol in the first stage fermentor are 2.89 g l1 , 98.16,
400.07, 116.57, and 42.31 mmol l1 , respectively, at the
maximum volumetric productivity of 1,3-PD, i.e. at a dilution rate of 0.29 h1 and an initial glycerol concentration of
730.80 mmol l1 .

(A)

140

160

(B)

140

120

120

100

100

80

80

60

60
40

40

20

20

0
0

200

400

600

800

1000

1200

1400

0.0

Initial concentration of glycerol in feed (mmol/L)

0.1

0.2

0.3

0.4

0.5

Productivity (mmol/L.h)

Volumetric productivity (mmol/L.h)

160

0.6

-1

Dilution rate (h )

Fig. 6. Effects of the initial glycerol concentration in feed (A) and the dilution rate (B) on the volumetric productivity of 1,3-propanediol in continuous
cultures. The curves are the predicted optimum values and points represent the steady-state experimental results from [47,9,10].

195

1.0
0.9

1.0

(B)

(A)

0.9

0.8

0.8

0.7

0.7

0.6

0.6

0.5

0.5

0.4

0.4

0.3

0.3

0.0

0.1

0.2

0.3

0.4

0.5

0.6

300

600

900

1200

1500

Yield of propanediol on glycerol (mol/mol)

Yield of propanediol to glycerol (mol/mol)

Z.-L. Xiu et al. / Biochemical Engineering Journal 19 (2004) 189197

-1

Dilution rate (h )

Initial concentration of glycerol in feed (mmol/L)

Fig. 7. Comparison of theoretical optimum yields of propanediol to glycerol (curves) with experimental results (points) at different dilution rates (A) and
initial glycerol concentrations in feed (B) in continuous cultures (dash line in A represents the maximum yield according to ref. [15]).

CS2 (mmol/L)

There is no doubt that the residual glycerol in bioreactor needs to be further converted for a complete use of
the substrate. Fig. 8 depicts the calculated residual glycerol concentration and 1,3-PD concentration in the second
bioreactor and the total volumetric productivity and molar yield of 1,3-PD on glycerol in a bioprocess with two
bioreactors in series at different dilution rates of the second bioreactor when the first bioreactor is operated at the
60
40
20

L1

L2

CPD2 (mmol/L)

450
400
350
300

Yt (mol/mol)

Qt (mmol/L.h)

120
80
40
0

0.6
0.5
0.4
0

-1

D2 (h )
Fig. 8. Model simulations of substrate and product concentrations in
the second bioreactor and total productivity and yield of a two-stage
fermentation.

optimal conditions. As the dilution rate of the second

bioreactor increases, the residual glycerol concentration
in the second bioreactor also increases, e.g. from 1.90
to 15.09 mmol l1 when a dilution rate varies in a range
between 1.25 and 2.0 h1 . In contrast, the final concentration of 1,3-PD firstly increases and then slowly decreases. The maximum final concentration of 1,3-PD is
447.36 mmol l1 at a dilution rate of 1.75 h1 . The corresponding residual glycerol concentration, total volumetric
productivity and molar yield of 1,3-PD are 9.23 mmol l1 ,
109.87 mmol l1 h1 , and 0.62 mol mol1 , respectively, as
shown in Table 2. In fact, the total volumetric productivity
and molar yield of 1,3-PD behave as saturated curves shown
in Fig. 8. Table 2 also indicates that both objective parameters will continue to increase if D2 is larger than 1.75 h1 .
However, the residual glycerol concentration dramatically
increases, while the concentration of 1,3-PD decreases very
little. From an integrated view of residual glycerol concentration, final 1,3-PD concentration, volumetric productivity
and molar yield, hence, the dilution rate of the second
bioreactor should be in a range between 1.0 and 1.75 h1 .
Even if the minimum dilution rate in Table 2 (0.5 h1 )
is adopted, these results are not expected. Normally, the
dilution rate of the second bioreactor is considered to be
less than that of the first one in order to further consume
the residual glycerol and increase the final concentration
of target product. The dilution rate of the first fermentor
is usually less than 0.5 h1 , otherwise wash-out is easy to
occur. Therefore, D2 > 0.5 h1 is beyond belief.
So far, no experimental work has been reported for
two-stage continuous 1,3-propanediolic fermentation by K.
pneumoniae. Two-stage continuous fermentation of glycerol has only been studied with Clostridium butyricum [14]
and Citrobacter freundii [17]. In the both fermentations, the
dilution rate was chosen to be lower in the second stage
than in the first. It has also been reported that in these
cases biomass concentration was always less in the second
fermentor than in the first one, and this was attributed to

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Z.-L. Xiu et al. / Biochemical Engineering Journal 19 (2004) 189197

Table 2
Computational results of two-stage fermentation at different dilution rates (operational conditions of the first bioreactor: D1 =0.29 h1 , CS0 = 730.80 mmol)
D2 (h1 )

X2 (g l1 )

CS2
(mmol l1 )

CPD2
(mmol l1 )

CHAc2
(mmol l1 )

CEtOH2
(mmol l1 )

Yt
(mol mol1 )

Qt
(mmol l1 h1 )

0.5
0.75
1.0
1.25
1.5
1.75
2.0

3.54
3.57
3.57
3.53
3.45
3.38
3.32

0.16
0.35
0.76
1.91
4.66
9.23
15.09

427.15
436.30
441.09
444.27
446.54
447.36
446.48

131.39
134.67
135.84
135.48
133.83
132.06
130.62

70.04
59.96
55.12
52.73
51.08
49.37
47.72

0.585
0.597
0.604
0.610
0.615
0.620
0.624

77.66
90.26
98.02
103.32
107.16
109.87
111.61

bacterial autolysis occurred in the second stage of the culture due to either microbial propagation under permanently
unfavorable conditions (increased contact time between the
cells and the metabolic products) or excretion of autolytic
enzymes [14]. It should be mentioned that the computational results of Fig. 8 and Table 2 do not agree with the
experimental observations, the key question, hence, that is
posed, is whether the cell growth kinetics of the second
stage culture is the same as the first stage. An experimental
study in this respect is needed.
In a two-stage fermentation, the theoretical biomass and
1,3-PD concentrations are higher in the second step than the
first. In contrast, the total yield and productivity of 1,3-PD
decrease in comparison with that of the first stage culture.
On the other hand, the total yield and productivity of 1,3-PD
of a two-stage culture are much higher than that of a single bioreactor with the same volume of double bioreactors
in two-step fermentation. Thus a two-stage fermentation is
theoretically favorable for 1,3-PD production.

5. Conclusion
The optimal conditions of batch and continuous anaerobic glycerol fermentations by K. pneumoniae are obtained
by using the volumetric productivity of 1,3-propanediol as
an optimization target. When an initial glycerol concentration was 960 mmol l1 , the highest volumetric productivity
of 1,3-PD of a batch culture could reach 52.6 mmol l1 h1
at a given inoculation of 0.1 g biomass l1 . The optimal
conditions of a continuous fermentation are a dilution rate
of 0.29 h1 and an initial glycerol concentration in feed
of 731 mmol l1 . The corresponding highest productivity
is 114 mmol l1 h1 , which is more than twice of a batch
culture. The experimental results agree very well with the
computational results in term of the concentration, yield
and productivity of 1,3-PD in continuous fermentations by
K. pneumoniae. The stability analysis of continuous cultivations indicates that two regions of multiple states occur at
relatively high concentrations of initial glycerol in the feed.
One of them approaches to the wash-out line. The theoretical
analysis of two-stage continuous processes shows that the
dilution rate of the second stage should be much higher than
the first one operated at the optimal conditions. A two-step

bioprocess is also favorable for 1,3-PD production in comparison with a single bioreactor system with the same volume of double bioreactors in series of a two-stage process.

Acknowledgements
This work was supported by the National Natural Science
Foundation of China (grant no. 20176005) and the tenth 5
years projects of Science and Technology Administration
of China (grant no. 2001BA708B01 04).
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