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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-


firmed that relatively high ethanol concentrations and
Ethanol, produced from renewable resources such as productivities can be achieved with high yields.
lignocellulosic residues, has the potential to be a cost- Optimization of ethanol fermentations is based on
effective and environmentally sustainable liquid fuel the development of realistic growth and fermentation
(Zhang et al. 1995). However the significant con- models. Previous kinetic models for Z. mobilis have
centrations of glucose and xylose, which are present been proposed in the literature (Lee & Rogers 1983,
in lignocellulosic hydrolysates, must be fully fer- Nipkow et al. 1986, Veermallu & Agrawal 1990,
mentable for an economically viable process. The aim Garro et al. 1995). These have generally been mod-
in recent years has been to develop recombinant strains ifications of the Monod equation (Monod 1941) and
which can utilize these sugars with reasonable yields have included substrate inhibition, product inhibition,
and rates (Laplace et al. 1995, Olsson et al. 1995, as well as substrate limitation effects. Unstructured
Zhang et al. 1995, Ho et al. 1996). Genetic engi- models such as these can also provide a general under-
neering with the ethanologenic Zymomonas mobilis standing of the metabolic processes involved as well as
has achieved a recombinant strain able to simultane- the basis for process optimization.
ously ferment glucose and xylose (Zhang et al. 1995) The unstructured mathematical model presented in
and subsequent kinetic analysis (Joachimsthal et al. this study is concerned with simultaneous fermenta-
1999, Lawford & Rousseau 1999, Joachimsthal & tion of two sugars, glucose and xylose, by the recom-
1088

binant Zymomonas mobilis ZM4(pZB5). There have Analytical methods


been previous reports of models for Saccharomyces
cerevisiae on mixtures of glucose/maltose (Lee et al. Biomass concentration was determined turbidometri-
1995) and glucose/galactose (Gadgil et al. 1996), as cally (at 660 nm). Absorbance measurements were
well as for Z. mobilis on glucose/fructose (Lee & made using whole broth samples that were diluted into
Huang 2000). Finding a model which simulates the the linear range of the instrument. A correlation fac-
fermentation characteristics of recombinant Z. mobilis tor (0.23) previously determined for the strain, was
ZM4(pZB5) was initiated by modelling growth and used to convert the absorbance values into biomass
fermentation on single sugars (glucose and xylose). concentrations.
These models for individual sugars were then com- Sample supernatants were analyzed to determine
bined to form a model to simulate the kinetics of the concentrations of glucose, xylose, and ethanol.
glucose/xylose fermentation in batch systems. 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
Materials and methods containing mixed components were periodically run to
verify calibration accuracy.
Microorganism The batch kinetic data for 25/25, 50/50, and
65/65 g l−1 glucose/xylose media using the above
Recombinant Zymomonas mobilis ZM4(pZB5) has the analytical methods have been reported previously
pZB5 plasmid transformed into the host strain and (Joachimsthal & Rogers 2000).
was kindly made available by Dr Min Zhang (NREL,
Golden, CO). The Escherichia coli genes for produc- The program for modelling and simulation
tion of xylose isomerase, xylulokinase, transketolase,
and transaldolase have been introduced into the pZB5 The design of VBA (Visual Basic for Applications)
plasmid. These confer xylose assimilation and fermen- program codes in Microsoft EXCEL for modelling
tation capability. The strain was maintained as frozen was based on the well established Gauss–Newton
stock culture in growth media (see below) supple- method for non-linear regression with step size halv-
mented with 10 µg ml−1 tetracycline and 10% (v/v) ing (Draper & Smith 1981, Bates & Watts 1988, My-
glycerol at – 70 ◦ C. ers 1990, Ratkowsky 1990). The simulation program
was designed to achieve the minimal total Residual
Media composition and preparation Sum of Squares (RSS) and acceptable curve fitting of
experimental values.
Growth media for Z. mobilis: carbon source(s); yeast RSStotal = RSSx + RSSs1 + RSSs2 + RSSp , (1)
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 ); where: x = biomass concentration (g l−1 ); s1 = glu-
(NH4 )2 SO4 (2 g l−1 ). Media were sterilized by au- cose concentration (g l−1 ); s2 = xylose concentration
toclaving at 121 ◦ C for 10 min. Selective pressure for (g l−1 ); p = ethanol concentration (g l−1 ).
recombinants was applied using a medium supplement
of 10 µg ml−1 tetracycline.
Development of a double substrate model
Fermentation studies
Microbial growth
All fermentation inocula were prepared in stationary
cultivations incubated at 30 ◦ C. Fermentations were For formulation of the double substrate model, the
initiated with a 10% (v/v) inoculum. Experiments microbial growth on each sugar is represented by
were conducted in a 2-l controlled fermenter using the specific growth rates of recombinant Z. mobilis
a working volume of 1 l with an agitation rate of ZM4(pZB5) on glucose and xylose as single carbon
200 rpm, at 30 ◦ C, and a pH of 5. Control of pH sources. The basis for these equations was taken from
was provided by the automatic addition of 3 M NaOH. a previous model development for Z. mobilis ZM4 for
Samples were removed at various times and stored at growth and fermentation of glucose (Lee & Rogers
−20 ◦ C until sample analysis. 1983). The equations assume Monod kinetics for sub-
strate limitation and ethanol inhibition, with both a

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