Biotechnology Letters 23: 1087–1093, 2001.

© 2001 Kluwer Academic Publishers. Printed in the Netherlands.

1087

Mathematical modelling of ethanol production from glucose/xylose

mixtures by recombinant Zymomonas mobilis

Noppol Leksawasdi, Eva L. Joachimsthal & Peter L. Rogers∗

Department of Biotechnology, University of New South Wales, Sydney, NSW 2052, Australia
∗ Author for correspondence (Fax: +61 2 9313-6710; E-mail: p.rogers@unsw.edu.au)

Received 16 March 2001; Revisions requested 10 April 2001; Revisions received 9 May 2001; Accepted 9 May 2001

Key words: ethanol, modelling, recombinant Zymomonas mobilis

Abstract
A model has been developed for the fermentation of mixtures of glucose and xylose by recombinant Zymomonas
mobilis strain ZM4(pZB5), containing additional genes for xylose assimilation and metabolism. A two-substrate
model based on substrate limitation, substrate inhibition, and product (ethanol) inhibition was evaluated, and
experimental data was compared with model simulations using a Microsoft EXCEL based program and methods
of statistical analysis for error minimization. From the results it was established that the model provides good
predictions of experimental batch culture data for 25/25, 50/50, and 65/65 g l−1 glucose/xylose media.

Nomenclature: 1 – glucose; 2 – xylose; x – biomass concentration (g l−1 ); s – substrate concentration (g l−1 );

p – ethanol concentration (g l−1 ); µmax – maximum overall specific growth rate (1 h−1 ); qs,max – overall max-
imum specific substrate utilization rate (g g−1 h−1 ); qp,max – overall maximum specific ethanol production rate
(g g−1 h−1 ); α – weighting factor for glucose consumption; Pm – maximum ethanol concentration (g l−1 ); Pi
– threshold ethanol concentration (g l−1 ); Ks – substrate limitation constant (g l−1 ); Ki – substrate inhibition
constant (g l−1 ); R 2 – correlation coefficient; RSS – residual sum of squares; RRS – relative residual summation;
RRStotal – sum of all RRS values.

Introduction Rogers 2000, Lawford & Rousseau 2000) has con-

Ethanol, produced from renewable resources such as
lignocellulosic residues, has the potential to be a cost-
effective and environmentally sustainable liquid fuel
(Zhang et al. 1995). However the significant con-
centrations of glucose and xylose, which are present
in lignocellulosic hydrolysates, must be fully fer-
mentable for an economically viable process. The aim
in recent years has been to develop recombinant strains
which can utilize these sugars with reasonable yields
and rates (Laplace et al. 1995, Olsson et al. 1995,
Zhang et al. 1995, Ho et al. 1996). Genetic engi-
neering with the ethanologenic Zymomonas mobilis
has achieved a recombinant strain able to simultane-
ously ferment glucose and xylose (Zhang et al. 1995)
and subsequent kinetic analysis (Joachimsthal et al.
1999, Lawford & Rousseau 1999, Joachimsthal &
firmed that relatively high ethanol concentrations and
productivities can be achieved with high yields.
Optimization of ethanol fermentations is based on
the development of realistic growth and fermentation
models. Previous kinetic models for Z. mobilis have
been proposed in the literature (Lee & Rogers 1983,
Nipkow et al. 1986, Veermallu & Agrawal 1990,
Garro et al. 1995). These have generally been mod-
ifications of the Monod equation (Monod 1941) and
have included substrate inhibition, product inhibition,
as well as substrate limitation effects. Unstructured
models such as these can also provide a general under-
standing of the metabolic processes involved as well as
the basis for process optimization.
The unstructured mathematical model presented in
this study is concerned with simultaneous fermenta-
tion of two sugars, glucose and xylose, by the recom-
1088
binant Zymomonas mobilis ZM4(pZB5). There have
been previous reports of models for Saccharomyces
cerevisiae on mixtures of glucose/maltose (Lee et al.
1995) and glucose/galactose (Gadgil et al. 1996), as
well as for Z. mobilis on glucose/fructose (Lee &
Huang 2000). Finding a model which simulates the
fermentation characteristics of recombinant Z. mobilis
ZM4(pZB5) was initiated by modelling growth and
fermentation on single sugars (glucose and xylose).
These models for individual sugars were then com-
bined to form a model to simulate the kinetics of
glucose/xylose fermentation in batch systems.
Materials and methods
Microorganism
Recombinant Zymomonas mobilis ZM4(pZB5) has the
pZB5 plasmid transformed into the host strain and
was kindly made available by Dr Min Zhang (NREL,
Golden, CO). The Escherichia coli genes for produc-
tion of xylose isomerase, xylulokinase, transketolase,
and transaldolase have been introduced into the pZB5
plasmid. These confer xylose assimilation and fermen-
tation capability. The strain was maintained as frozen
stock culture in growth media (see below) supple-
mented with 10 µg ml−1 tetracycline and 10% (v/v)
glycerol at – 70 ◦ C.
Media composition and preparation
Growth media for Z. mobilis: carbon source(s); yeast
extract (10 g l−1 inoculum, 5 g l−1 fermentation me-
dia); KH2 PO4 (2 g l−1 ); mgSO4 · 7H2 O (1 g l−1 );
(NH4 )2 SO4 (2 g l−1 ). Media were sterilized by au-
toclaving at 121 ◦ C for 10 min. Selective pressure for
recombinants was applied using a medium supplement
of 10 µg ml−1 tetracycline.
Fermentation studies
All fermentation inocula were prepared in stationary
cultivations incubated at 30 ◦ C. Fermentations were
initiated with a 10% (v/v) inoculum. Experiments
were conducted in a 2-l controlled fermenter using
a working volume of 1 l with an agitation rate of
200 rpm, at 30 ◦ C, and a pH of 5. Control of pH
was provided by the automatic addition of 3 M NaOH.
Samples were removed at various times and stored at
−20 ◦ C until sample analysis.
Analytical methods
Biomass concentration was determined turbidometri-
cally (at 660 nm). Absorbance measurements were
made using whole broth samples that were diluted into
the linear range of the instrument. A correlation fac-
tor (0.23) previously determined for the strain, was
used to convert the absorbance values into biomass
concentrations.
Sample supernatants were analyzed to determine
the concentrations of glucose, xylose, and ethanol.
Analysis was performed using HPLC with an Aminex
HPX-87H column (Bio-Rad) with 5 mm H2 SO4 (at
65 ◦ C, 0.6 ml min−1 ) as the mobile phase. Standards
containing mixed components were periodically run to
verify calibration accuracy.
The batch kinetic data for 25/25, 50/50, and
65/65 g l−1 glucose/xylose media using the above
analytical methods have been reported previously
(Joachimsthal & Rogers 2000).
The program for modelling and simulation
The design of VBA (Visual Basic for Applications)
program codes in Microsoft EXCEL for modelling
was based on the well established Gauss–Newton
method for non-linear regression with step size halv-
ing (Draper & Smith 1981, Bates & Watts 1988, My-
ers 1990, Ratkowsky 1990). The simulation program
was designed to achieve the minimal total Residual
Sum of Squares (RSS) and acceptable curve fitting of
experimental values.
RSStotal = RSSx + RSSs1 + RSSs2 + RSSp , (1)
where: x = biomass concentration (g l−1 ); s1 = glu-
cose concentration (g l−1 ); s2 = xylose concentration
(g l−1 ); p = ethanol concentration (g l−1 ).
Development of a double substrate model
Microbial growth
For formulation of the double substrate model, the
microbial growth on each sugar is represented by
the specific growth rates of recombinant Z. mobilis
ZM4(pZB5) on glucose and xylose as single carbon
sources. The basis for these equations was taken from
a previous model development for Z. mobilis ZM4 for
growth and fermentation of glucose (Lee & Rogers
1983). The equations assume Monod kinetics for sub-
strate limitation and ethanol inhibition, with both a
 1089

threshold level and a maximum inhibitory concentra- dx

= [arx,1 + (1 − α)rx,2 ]x. (6)
tion, as well as a typical substrate inhibition term. dt
These relationships are represented by Equations (2)
and (3). Glucose and xylose uptake
For glucose:

 For sugar uptake, glucose and xylose are considered
s1
rx,1 = µmax,1 × in separate rate equations. The same constraint is
Ksx,1 + s1


 placed upon these proportioning factors to indicate
p − Pix,1 Kix,1 an unchanged activity and constant total sugar uptake
1− . (2)
Pmx,1 − Pix,1 Kix,1 + s1 rate via the Glf diffusion transport protein for glu-
cose/xylose. The glucose and xylose uptakes can be
For xylose:

 represented by Equations (7) and (8), respectively,
s2

rx,2 = µmax,2 × ds1 s1
Ksx,2 + s2 = −αqs,max,1 ×


 dt Kss,1 + s1
p − Pix,2 Kix,2


1− . (3) p − Pis,1 Kis,1
Pmx,2 − Pix,2 Kix,2 + s2 1− x, (7)
Pms,1 − Pis,1 Kis,1 + s1
The terms used are defined fully in the Nomencla-

ture section, with subscript 1 referring to glucose and ds2 s2
= −(1 − α)qs,max,2 ×
subscript 2 to xylose. dt Kss,2 + s2
As growth occurs simultaneously on both glucose


p − Pis,2 Kis,2
and xylose, and competition for uptake occurs be- 1− x. (8)
tween the two sugars, the contribution of glucose and Pms,2 − Pis,2 Kis,2 + s2
xylose to biomass formation is assumed to be:
dx Ethanol production
= j1 rx,1 x + j2 rx,2 x, (4)
dt
where the weighting factor j is dependent on the For ethanol production, the rate is given by Equation
relative consumption rates of the two sugars. The (9). The rate of ethanol production can be related to
weighting factors for glucose and xylose uptakes are the rates of glucose and xylose uptake as shown in
specified as j1 and j2 , respectively. An important as- Equations (10) and (11).
sumption of the model is that the sum of the weighting dp
= [αrp,1 + (1 − α)rp,2 ]x (9)
factors for glucose and xylose uptake is unity. This dt
is based on the assumption that both glucose and xy- For glucose:
lose compete for uptake via a common and unchanged

s1
sugar transport system in Z. mobilis. This system has rp,1 = qp,max,1 ×
been reported previously as the glucose facilitated Ksp,1 + s1
transport system (mediated by the Glf gene) (DiMarco


p − Pip,1 Kip,1
& Romano 1985, Parker et al. 1995, Weisser et al. 1− . (10)
Pmp,1 − Pip,1 Kip,1 + s1
1995, 1996). Other authors have reported simulta-
neous glucose and xylose uptake by recombinant Z. For xylose:
mobilis (Zhang et al. 1995, Krishnan et al. 2000, Law-

s2
ford & Rousseau 1999, 2000, Lawford et al. 2000). It rp,2 = qp,max,2 ×
Ksp,2 + s2
is evident from our earlier kinetic data also that both



glucose and xylose can be taken up simultaneously, p − Pip,2 Kip,2
with xylose at a considerably slower rate. 1− . (11)
Pmp,2 − Pip,2 Kip,2 + s2
As a result of this assumption, we can write:
j1 + j2 = 1 ⇒ j2 = 1 − j1 . (5) Parameter values which resulted in minimization of
the differences between the simulation and experimen-
For simplification, j1 is designated as α. The modifica- tal data were calculated using computational strategies
tion of Equation (4) to include both glucose and xylose outlined in the Materials and methods section.
is shown in Equation (6):
1090

Fig. 1. Simulation of the mixed sugar system and experimental data Fig. 2. Simulation of the mixed sugar system and experimental data
for ZM4(pZB5) on 25 g l−1 glucose and 25 g l−1 xylose medium. for ZM4(pZB5) on 50 g l−1 glucose and 50 g l−1 xylose medium.

, Glucose; , xylose; , ethanol; •, biomass.
, Glucose; , xylose; , ethanol; •, biomass.

Glucose/xylose fermentation simulation

Simplification of the derived model and determination

of optimal parameter values

Conditions were imposed upon the parameters to re-

late to their microbial/biochemical relevance. These
conditions are listed in Table 1. Initial parameter
values used to define ‘local search region’ were deter-
mined from previously published values (Joachimsthal
& Rogers 2000).

Simulation of the batch glucose/xylose fermentations Fig. 3. Simulation of the mixed sugar system and experimental data
for ZM4(pZB5) on 65 g l−1 glucose and 65 g l−1 xylose medium.
The initial concentration values of each component
, Glucose; , xylose; , ethanol; •, biomass.
(Table 2a) and the values of the kinetic parameters (Ta-
ble 2b) which resulted in the minimization process of individual profile errors contributing disproportion-
RSStotal value were determined and the fit of the model ately to the RSStotal . For this reason, an RRStotal was
to the experimental data calculated. The RSStotal and evaluated.
correlation coefficient (R 2 ) values were used to assess The RRStotal was determined using the following
the fit of the model to the experimental data. equation.
As shown in Figures 1–3, the glucose/xylose
model demonstrates excellent simulation of the exper- RRStotal = RRSx + RRSs1 RRSs2 RRSp (12)
imental data for glucose and xylose media containing where
25/25, 50/50, and 65/65 g l−1 of each sugar. N  
  ipredicted 
RRSm = 
Sensitivity analysis 1 − i ,
exp
n=1

The model for the fermentation of glucose/xylose in
a batch system was examined for its sensitivity to
changes in the value of α with all other initial and
parameter values being allowed to float within previ-
ously defined limits. RSStotal was not considered to
be the best indicator for sensitivity analysis because
it measures fitting only in ‘absolute terms’, with the
ipredicted = predicted value; iexp = experimental value;
m = x, s1 , s2 , or p (designated variable in each
data set); N = total number of data points in each
experiment; n = 1 to N.
The lowest RRStotal (the sum of all RRS values),
and hence the best fit, was determined to be for α =
0.65 (Table 3).
 1091
Table 1. Conditions used for initial model parameter simplification based on microbial/biochemical
relevance.

Condition Assumption

1 qp,max,1 < 0.51qs,max,1 Based on theoretical yields of ethanol from glucose and xylose
qp,max,2 < 0.53qs,max,2

2 Kss,1 = Ksp,1 When sugar uptake is affected by low sugar concentrations the
Kss,2 = Ksp,2 ethanol production rate is affected in the same way

3 Pms,1 = Pmp,1 When sugar uptake is inhibited completely by ethanol no further

Pms,2 = Pmp,2 ethanol can be produced

4 Pis,1 = Pip,1 The ethanol concentration that begins to inhibit sugar uptake also
Pis,2 = Pip,2 begins to inhibit ethanol production

5 Kis,1 = Kip,1 When sugar uptake is limited by the concentration of sugar,

Kis,2 = Kip,2 ethanol production is limited in the same way

Discussion this parameter (designated α) showed that the specific

A model representing fermentation of glucose and xy-
lose by the recombinant Z. mobilis ZM4(pZB5) has
been successfully developed. As seen from Figures 1–
3, the predicted results are in good agreement with the
experimental batch culture data. The parameters esti-
mated in the simulation can be related to sensible mi-
crobial/biochemical values. The model describes the
high ethanol production rate during the initial phase
of primary glucose/xylose fermentation, followed by
the slower production rate from xylose after glucose is
consumed. The model also accommodates the experi-
mentally observed simultaneous utilization of glucose
and xylose in batch culture.
From an evaluation of the parameter values deter-
mined using the minimization routine, some conclu-
sions may be drawn. In the present model for example,
the substrate limitation (Monod or saturation) constant
for biomass production by xylose (Ksx,2) was approx-
imately 3 times that of glucose (Ksx,1) indicating a
higher affinity for glucose than xylose at substrate
limiting levels. Other reports comparing the glucose
and xylose saturation constants for organisms such as
Saccharomyces cerevisiae (Krishnan et al. 1999) and
Klebsiella oxytoca (Turner et al. 1988) relate a similar
result with the Ksx value for xylose being 3–6 times
that of glucose.
This study incorporated weighting factors to repre-
sent the relative preference for sugar utilization and the
production of ethanol and biomass from either glucose
or xylose. Assuming direct competition between glu-
cose and xylose for the transport system in Z. mobilis,
rate of glucose uptake was 65% of its maximum value,
while that of xylose was 35% of its maximum value
when the two sugars were present initially in a 1:1
ratio. These values were found to give the best fit, as
was determined by sensitivity analysis.
From the model parameter values, ethanol would
become inhibitory to growth from glucose and xy-
lose above a threshold level of approximately 27–29
g l−1 (Pix,1 , Pix,2 ). Threshold ethanol concentrations
of 42.6 g l−1 (Pip,1 ) and 53.1 g l−1 (Pip,2 ) were deter-
mined for glucose and xylose uptake respectively. In
general, growth would be subject to complete ethanol
inhibition at a lower ethanol concentration than for
sugar uptake. The maximum inhibitory ethanol con-
centration for growth on glucose (Pmx,1 = 57.2 g l−1 )
was similar to that for growth on xylose (Pmx,2 =
56.3 g l−1 ). The maximum inhibitory ethanol concen-
tration for glucose uptake (Pms,1 = 75.4 g l−1 ) was
slightly lower than that for xylose uptake (Pms,2 =
81.2 g l−1 ). The model parameter values indicate that
biomass production from glucose or xylose was more
sensitive to ethanol inhibition, than sugar uptake and
subsequent ethanol production.
The model parameter values indicate that signif-
icant glucose inhibition of the rate of growth would
occur at concentrations above 200 g l−1 (Kix,1 ), which
is lower than the concentration above which xylose
inhibits biomass production (Kix,2 = 600 g l−1 ). Glu-
cose would become significantly inhibitory to ethanol
production at concentrations above 186 g l−1 (Kip,1 ),
whereas xylose inhibition effects on ethanol produc-
tion are insignificant at xylose levels used in the
1092
Table 2. a. Initial values for the batch fermentation of ZM4(pZB5) Table 3. Sensitivity of the mixed sugar model to
on various glucose/xylose media with α = 0.65. weighting factor for glucose consumption (α).

Glucose/xylose 25/25 50/50 65/65 α RRStotal

(g l−1 )
0.40 27.8
x0 0.028 0.017 0.003 0.45 26.2
s01 25.08 51.08 59.30 0.50 25.7
s02 27.65 51.00 63.24 0.53 23.7
p0 1.41 2.84 3.83 0.57 20.4
0.60 19.1
b. Optimal kinetic parameters for all data sets with α = 0.65. 0.63 18.8
0.65 18.4
Glucose Xylose 0.67 19.5
Biomass production model 0.70 20.3
0.75 25.2
µmax,1 0.31 µmax,2 0.1
Ksx,1 1.45 Ksx,2 4.91
Pmx,1 57.2 Pmx,2 56.3
Kix,1 200 Kix,2 600
Pix,1 28.9 Pix,2 26.6
Acknowledgements

Glucose and xylose consumption model The financial support of NREL (National Renewable
qs,max,1 10.9 qs,max,2 3.27
Energy Laboratory, USA) and the Thai Government
Kss,1 6.32 Kss,2 0.03 (N. Leksawasdi) is gratefully acknowledged. The
Pms,1 75.4 Pms,2 81.2 project is funded partially under the US Department
Kis,1 186 Kis,2 600 of Energy Sub Contract XDH-9-29055-01.
Pis,1 42.6 Pis,2 53.1

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