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Biocatalysis, 1988, Vol. 1, pp.

217-229 0 1988 Harwood Academic Publishers GmbH

Reprints available directly from the publisher Printed in the United Kingdom
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S. L. PATERSON, A. G. FANE,* and C. J. D. FELL
School of Chemical Engineering and Industrial Chemistry, University of New
South Wales, Kensington, NSW, 2033, Australia


Department of Biotechnology, University of New South Wales
(Received September 21, 1987)

This study describes the results of a hollow fibre membrane reactor with immobilized treated cells of
Zymomonas mobilis which produced sorbitol and gluconic acid continuously from fructose and
glucose respectively. A productivity of 10-20 g sorbitol . L-' * h-' and 10-20 gluconate .L-' . h-'
(based on total bioreactor volume) from a feed of 100 g . L-' each of glucose and fructose was
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possible at high dilution rates. Kinetic parameters describing the reaction rate of treated cells in batch
reactors were used to analyse the performance of the hollow fibre membrane reactor employing
significant convective mass transfer. No significant mass transfer limitation was apparent.

KEY WORDS Membrane reactor, sorbitol and gluconate production, starling flow


Dilution rate based on a volume of 500 ml, h-'.

HFR-CSTR feed rate, L h-'. -
Fructose concentration, g * L-'.
Glucose concentration, g - L-'.
Combined saturation constant, g L-l.
Saturation constant for fructose, g L-l.
Saturation constant for glucose, g . L-'.
Saturation constant for inhibition by sorbitol, g . L-'.
Sorbitol concentration, g * L-'.
Hollow fibre inlet pressure, kPa.
Hollow fibre outlet pressure, kPa.
e.-Po, kPa.
Glucose and or fructose concentration, g L-l.
Concentration at HFR CSTR stage inlet, g - L-l.

* To whom correspondence should be addressed.

218 S . L. PATERSON ET A L .

[$lo Concentration at HFR CSTR stage outlet, g . L-'.

V Rate of uptake of glucose and or fructose and rate of production of
sorbitol and or gluconate, g - g-I . h-'.
V* Reaction rate corrected to eliminate the effects of sorbitol inhibition,
g . g-' . h-l.
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V,, Maximum rate of reaction, g.g-'. h-I.

X Biomass loading to reactor, g DW.


The bacterium Zymomonas mobilis has been shown to be a very fast, efficient
producer of ethanol from sugars (Rogers, Lee and Tribe, 1979). However,
Viikari (1984) and Barrow et al. (1984) demonstrated that quite high levels of the
industrially important sugar alcohol, sorbitol, accumulated when either sucrose or
a mixture of glucose and fructose was used as the carbon source during growth.
Sorbitol was shown to be derived only from fructose by NMR and HPLC (Barrow
et al., 1984). Further work by Leigh, Scopes and Rogers (1984) identified an
enzyme complex capable of converting glucose to gluconic acid in association with
reduction of fructose to sorbitol. The enzyme responsible for glucose oxidation
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and fructose reduction has been described as a glucose-fructose oxidoreductase,

which, in combination with gluconolactonase (EC, is capable of
converting totally equimolar fructose and glucose solutions of over 500 g . L-'
(total sugar) to sorbitol and gluconic acid when titrated with a suitable base at a

pH close to 6.2 (Zachariou and Scopes, 1986). Thus,
gluconate + H+

where enzyme A is glucose-fructose oxidoreductase and enzyme B is gluconolacto-

Both the enzymes taking part in the reaction described above are present at
significant levels in 2. mobilk, especially when the organism is grown on glucose
as a carbon source (Zachariou and Scopes, 1984). Scopes, Rogers and Leigh
(1985) showed that harvested cells of Z . mobilis treated with toluene in a buffer
at pH 7 became permeable. Soluble co-factors and high energy compounds
necessary for the complete conversion of glucose to ethanol could be washed out
of the cells. The oxidoreductase and gluconolactonase remained active at
significant levels within the cells and are assumed to be membrane bound.
This study addresses some of the reaction engineering questions relating to the
use of the Z. mobilis enzyme complex as part of a feasibility study being
undertaken in our laboratories for the continuous production of sorbitol and
gluconic acid. In particular, the hollow fibre membrane reactor (HFR) is
examined as a potential immobilization system for toluene-treated cells of Z.
Results are presented showing the efficacy of the immobilized biocatalyst under
different operating conditions of dilution rate and mass transfer regime in the
HFR. Stability of the enzyme complex is also reported.

The HFR as an Immobilization System

Because toluene-treated cells are non-viable, immobilization is a logical path to
follow for the development of a highly productive continuous process for sorbitol
and gluconate production. However, the conversion of glucose to sorbitol
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described earlier occurs at a relatively high rate (Zachariou and Scopes, 1986)
and consequently a large amount of base must be supplied for pH control at the
optimum of 6.2. In such an immobilized cell system, it is possible that the need
for pH control may place an upper limit on the productivity of the process.
Unfortunately, the majority of immobilization systems rely on diffusive mass
transfer for the supply of reactants and removal of products. However, HFR
processes can be augmented by convective mass transfer facilitated by operating
parameters such as pressure gradients and the hydraulic permeabilities of the cell
and membrane layers. The role of convective mass transfer in HFRs is supported
by both theoretical analyses (Libicky, 1985) and experimental evidence (Park and
Kim, 1985); Paterson et al., 1987). Significant convective mass transfer may help
also with pH control within the immobilized biocatalyst, and this would be
The majority of studies dealing with HFR enzyme processes have made use of
ultrafiltration (UF) membranes, which pose a significant resistance to convective
mass transfer in comparison with microporous membranes (Paterson et al. , 1987).
Enzymes, due to their relatively small size, may wash through microporous
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membranes, unless they are chemically bonded to the membrane with an agent
such as glutaraldehyde (Gekas, 1986). However, in the present study the
enzymes are held within the treated Z. mobilis cells, which can be retained by a
microporous membrane without the need for chemical bonding, thereby simplify-
ing the process.
Convective supply of reactants and removal of products in HFRs can be
achieved by a number of modes of operation (Tharakan and Chau, 1986), all of
which rely on a pressure differential across the membrane to drive the process
(Figure 1). In the present work the closed shell mode of operation was used to
promote convective mass transfer as “Starling flow” because of the compactness
of the bioreactor and the design of the system to give a high feed recirculation
rate at a moderate pressure drop over the the HFR, leading to good pH control.
Starling flow, which is discussed in detail elsewhere (Libicky, 1985; Paterson et
al., 1987), was preferred to the cross flow ultrafiltration (CFUF) mode that
Tharakan and Chau (1986) suggest is the superior HFR operating mode. CFUF
would have relatively poor pH control because low flux across the cell and
membrane layers would result in a slow recirculation rate. The open shell UF

c c t t
(A) Open shell UF (El Closed shell “Starling flow” (C) Cross flow UF

Figure 1 The three modes of convective mass transfer in HFRs. The closed shell mode (B) was used
in this study.
220 S . L. PATERSON ET A L .

mode by Park and Kim (1985) was also not appropriate in this case because the
non-viable Z . mobilis cells would quickly wash out of the shell unless they were
chemically bonded to the membrane surface.
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Kinetic Analysis of Batch Results

A kinetic analysis of batch reactor results obtained from Masters (1986) and
Rogers and Chun (1987) was performed as part of this work in order to develop a
rate expression for sorbitol production in the absence of mass transfer limitation.
Numerical values generated from this expression were compared with reaction
rate data from HFR experiments to identify any mass transfer limitations in the
HFR. Some variation in the predicted kinetic parameters from various batch
reactor experiments was expected due to slightly different toluene treatments.
The predicted maximum reaction rate, V,,,, for toluene-treated cells was
compared with the predicted V,,, for exponentially growing cells, using ex-
perimental data for Z . mobilis growth (Barrow et al., 1984) and kinetic data for
the free enzyme (Zachariou and Scopes, 1986). From this comparison it was
possible to determine the extent of enzyme deactivation due to complete toluene
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Determination of Kinetic Parameters

The reaction rate of the glucose-fructose oxidoreductase enzyme can be assumed
to be rate limiting for sorbitol and gluconate formation and can be described by
ping-pong kinetics (Zachariou and Scopes, 1986) thus,

Ping-pong kinetics describe a reaction in which two substrates must combine at an

enzyme site to release a product or products. Zachariou and Scopes (1986)
established the values of Kmfand K,, as 252 and 5.4 g - L-' respectively for the
oxidoreductase enzyme with excess gluconolactonase present. However, both the
activity of the enzyme complex in Z . mobilis cells and the effect of toluene
treatment of the activity of the enzyme complex is not known. A kinetic analysis
of data using treated cells is therefore necessary. For batch and HFR processes
the concentrations of glucose and fructose will be equal at any time, assuming
they were equal initially and that no unidentified side reactions occur. In this
case equation (1) can be simplified to the familiar Michaelis-Menten form and
Kmr and Kmg can be combined as a single saturation parameter, K,,,, with the
main contribution being from Kmf. Thus,

where K,,, = K m j + K,,,, and, [g] = [f] = [s].

Zachariou and Scopes (1986) also showed that at a sorbitol concentration of
0.8M (144g * L-I) the oxidoreductase enzyme was inhibited by 27%. In

analysing the experimental batch data of Masters (1986) and Rogers and Chun
(1987) this must be taken into account, as the concentration of sorbitol was often
greater than 0.8 M in the reaction mixture. A general expression for end product
inhibition can be combined with equation (2) to give;
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In order to write equation (3) in the convenient Lineweaver-Burk double

reciprocal form for graphical prediction of V,,, and K,,,, a corrected reaction rate
V * must be introduced. V * is defined as the instantaneous reaction rate that
would have occurred had there been no sorbitol inhibition. V * can be expressed
as a function of V and the inhibition term in equation (3) as;

Writing equation (4) in terms of V and substituting into equation (3), a double
reciprocal form of equation (3) can be written for analysis of batch data.
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Assuming 27% inhibition at a sorbitol concentration of 144 g . L-', Kp can then

be estimated from the inhibition expression in equation (3) as 383g 'L-'. In
principle the rate expression could also contain a term for substrate inhibition.
However, there was no evidence for this, even at concentrations of 300 g - L-' of
both glucose and fructose in the results of Rogers and Chun (1987) or in our
work. Consequently substrate inhibition was not included in the rate expression.
Following standard procedures described by Segel (1975) for the analysis of
enzyme kinetic data, the experimental batch data of Masters (1986) and Rogers
and Chun (1987), for the change in glucose and sorbitol concentration with time,
have been analysed using equation ( 5 ) to yield V,, and K,,,values.

Preparation of Toluene-Treated Cells

The bacterial strain 2. mobilis ZM4 (ATCC 31821) was stored at -20°C in
glycerol prior to inoculum preparation. Both the inoculum medium and the
medium for cell growth contained; glucose (100 g - L-'), yeast extract (5 g - L-I),
(NH4)*S04 (1 g . L-') and MgS04 7H20 (1 g - L-I), autoclaved for 20 min at
100 kPa. No phosphate was included in the medium as it would be difficult to
wash completely from the treated cells, thus promoting some ethanol and CO,
formation during the HFR run. For cell production, well-stirred fermenters (4 L
capacity) were inoculated at 5% (v/v) seed level. The pH of the fermentation
broth was maintained at 5.0 and the temperature at 30°C. Cells were harvested in
the late exponential phase after 20 h growth by centrifugation (5700 g) prior to
treatment with toluene (1% (v/v) in 0.1 N citrate buffer at pH6.8) for lOmin, as
suggested by Masters (1986) for complete treatment without excessive deactiva-
tion of the enzyme complex. Treated cells were then washed with buffer prior to
charging to the HFR shell. The yield of cells from the fermenter was
approximately 2.5 g dry weight (DW) - L-'.

HFR Design
HFR units were made in-house with the assistance of Memtec (Australia) Ltd.
Each unit consisted of loo0 polypropylene, isotropic, hydrophobic, hollow fibres
(I.D. = 270 pm, O.D. = 515 pm, 0.2 pm nominal pore size), potted with poly-
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urethane rubber in a glass tube (3cm I.D. x 20cm long). With these
specifications, fibres had packing densities similar to commercial hollow fibre
membrane units.
The HFRs were run continuously incorporating a recycle loop of product stream
(Figure 2) so as to give an appreciable pressure drop per pass and thereby induce
Starling flow. Recycle also enabled the pH to be controlled at 6.2 by addition of
6NNaOH to the recycle vessel. The hollow fibre unit had a volume of 140ml
and the recycle vessel, including all lines, a volume of 360 ml. Dilution rates were
calculated using the total volume of 500 ml unless otherwise specified.

Analysis Methods
Samples were taken from the HFR experiments and immediately frozen.
Glucose was assayed on a daily basis using the UV/hexokinase method
(Boehringer Mannheim). Analysis of glucose, fructose and sorbitol was
performed using a Waters model 660HPLC with a BioRad Animex HPX-87C
column. Gluconic acid was assayed by the UV/glucose kinase NADPH method
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(Boehringer Mannheim). Ethanol was assayed using a Technicon Auto Analyser.

HRF Experimental Protocol

HFRs were sterilized with 5% (v/v) formaldehyde for 24 h prior to washing with
sterile distilled water (20 L). Treated cells (20 g DW) were charged to the shell
space and any remaining air was bled from the shell after “start-up” to allow
Starling flow to take place. After each experiment, the membranes were washed
with sodium dodecyl sulphate (1% in 5NNaOH) for 24h at 40°C. This was
sufficient for complete restoration of water flux.

Figure 2 The HFR experimental set-up showing; 1-HFR unit of 140ml. 2-recycle vessel of 360m1,
%feed vessel, k u t l e t vessel, %feed peristaltic pump, 6-6 N NaOH addition peristaltic pump,
7-micro pump. 8-pressure gauges, %water bath at 3YC, l h a m p l e line, 11-pH control loop.

The characteristics of the HFR process examined were mass transfer effects,
conversion vs dilution rate profiles, and long term stability of the catalyst. The
pH indicator bromocresol purple was added to the sugar feed at 0.01 g . L-' so
that the pH in the shell of the HFR and the cell layers could be observed
qualitatively, thereby indicating the extent of mass transfer limitation.
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In the present study we report the results from three HFR experiments (Rl,
R2, and R3), all of which had a pH of 6.2, the same cell loadings (20 g DW), cell
treatments and feed concentrations of 100g * L-' of both glucose and fructose.
The only difference between experiments was the recirculation rate leading to
different pressure drops over the hollow fibre unit. Pressure drops for R1, R2,
and R3 were 30, 50 and 70 kPa respectively at the start of the experiments.

Kinetics of Treated Cells Charged to Batch Reactors
Figure 3 shows the production of sorbitol with time for three batch experiments
taken from Masters (1986) and Rogers and Chun (1987). Gluconate (not shown
in Figure 3) exhibited similar trends to the sorbitol profiles. Glucose and fructose
profiles (also not shown) exhibited the inverse trends of sorbitol and gluconate.
Figure 4 shows a sample plot of 1/V* vs l/[s], according to equation (5) of data
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from Rogers and Chun (1987) (shown in Figure 3). The pH was maintained at
6.2 by addition of 2NNaOH and the temperature was controlled at 39°C.
Ethanol was produced to 5.5 g L-', indicating incomplete toluene treatment.
The predicted V,,, was 3.4 g sorbitol- g cell-' * h-' and K , was estimated as
25Og.L-'. Results for V, and K , corresponding to the three batch

TIME (h)

Figure 3 Sorbitol profiles with time for three batch experiments. Symbols are; C f o r 20% (w/v)
initial sugars from Masters (1986), +for 40% (w/v)initial sugars from Rogers and Chun (1987),
A-for 60% (w/v)initial sugars from Rogers and Chun (1987).
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5 10 15
I 1is] (L/g)x I 03

Figure 4 1/V* vsl/[s] for a batch experiment with an initial sugar level of 40% (w/v) and cell
loading of 3 4 . 9 g D W . L-I. V,,, equals 3 . 4 g . g-' h-' (l/vertical axis intercept) and K,,, equals
250g. L-' ( - l/horizontal axis intercept).
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experiments (Figure 3) are given in Table 1. There was some variation in V,,
probably due to slight variations in toluene treatment.
From the batch fermentation data of growing 2. mobilis (Barrow et al., 1984),
the maximum specific rate of sorbitol production is approximately 2 g g-' h-'.
Under these conditions the fermentation broth contained approximately
25g-L-I of glucose and 4Og.L-' of fructose. Leigh (1987) also observed a
maximum sorbitol production rate of 2 g . g-' . h-' during continuous culture
studies of 2. mobilis. Substituting the values of Kmr and Kmg(Zachariou and
Scopes, 1986) and other relevant data reported above (ie; [ g ] , [f],and V) into
equation [l],the maximum potential reaction rate of untreated 2. mobilis cells is
calculated to approximately 15 g . g-I - h-'. This is considerably higher than the
V,, of toluene-treated cells reported in Table 1 (in the range of 2.8 to 4.0).

Table 1 Kinetic constants V,, and K , derived from batch experiments

Reference Initial Cell Treatment Ethanol V, Km

concen- loading (% Toluene formed (g .g .h - I ) (g .L -')
tration of (g . L - I ) x mins) (g ' L -9
(g L -9
1 100 20 1 x 10 0.0 2.8 250
2 200 34.9 1 x 10 5.5 3.4 250
2 300 39.7 1 x 10 5.0 4.0 250

I Masters (1986).
Rogers and Chun (1981).

HFR Experiments
Time profiles of sorbitol and fructose concentrations for HFR R1 at a dilution
rate of 0.056h-' (based on the total volume of 500ml) are shown in Figure 5.
Very little enzyme deactivation occurred over 250 h and the yield of sorbitol from
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fructose was close to 100% (mole basis). Gluconate and glucose showed similar
profiles to sorbitol and fructose respectively and are not reproduced here.
Figure 6 shows conversion vs HFR pressure drop for different dilution rates for
R1, R2, and R3. At a dilution rate of 0.056h-' the system showed no
improvement in conversion from 50 kPa (R2) to 70 kPa (R3), indicating reaction
limitation under these conditions. However at higher dilution rates improve-
ments in conversion and therefore reaction rate were apparent with increasing
pressure drop.
Figure 7 shows the specific reaction rate of HFR R3 at different dilution rates
compared with predicted reaction rates (using equation (3)) for a batch reactor
with free treated cells having a V,, of 2.8 g a 8 - l - h-', K,,,of 250 g L-' and K p
of 383 g . L-'. The treated cells charged to both HFR R3 and those exhibiting
the kinetic constants above showed no ethanol or gas formation. Very good
agreement is observed between the HFR and batch results, indicating that, under
the experimental operating conditions of HFR R3, no significant mass transfer
limitation occurred.
Studies with the pH indicator bromocresol purple in the feed indicated that at
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high dilution rates, and therefore faster overall reactions, some low pH regions
were present in the shell and around the cell layer. HFR R3 exhibited a smaller
pH drop in the shell and around the cell layers than HFRs R2 and R1.

Development of a Reactor for a High Yield Conversion (>98%)

Because the K , value for the oxidoreductase enzyme is quite high (250 g L-') a
conversion approaching 100% would be difficult to obtain from a continuous

Figure 5 Sorbitol (W) and fructose (0)profiles with time for HFR R l at a dilution rate of 0.056 h-'
(0.2 h-' based on the hollow fibre unit volume).
226 S. L. PATERSON E T A L .


- 75 -
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5 50- v/

25 -

0 I I 1 I I
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O6 t

0 02-

I I I 1
0 20 LO 60
Figore 7 Specific reaction rate (g .g-' . h-') vs glucose or fructose substrate concentrations for
HFR R3 (H) compared with the predicted reaction rate for free cells in a batch reactor (0)
[V,,, = 2.8 g . g-' . hK', K,,, = 250 g . L-', K,, = 383 g . L-' and initial sugars of 100 g . L-' both
glucose and fructose]. HFR R3 o/c conversion is also shown on the horizontal axis.

stirred tank reactor (CSTR) process, unless a long residence time was employed.
The HFR process was run as a CSTR in this work to obtain pH control and can
be referred to as an HFR-CSTR. In the manufacture of sorbitol and gluconate
using the Z . mobilk enzyme complex a high degree of conversion is desirable,
because glucose and fructose would be very difficult to separate from sorbitol on
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an industrial scale. This requirement can be satisfied by placing HFR-CSTRs in

series and thereby attaining some characteristics of a plug flow reactor (Wang et
al., 1979). Assuming no mass transfer limitations occur, a rate equation for HFR
R3 can be derived from equation [3].
V=2.8[ ][
383 ]
250 + [ s ] 383 [ p ]+
Since [ p ]= [sIi - [s],, a mass balance on glucose around each HFR-CSTR stage
at steady state produces the following expression;

Equation (7) was used to derive [sI0 for each stage of the HFR-CSTR loops
assuming 20 g DW treated cells, a feed glucose and fructose concentration each of
100 g - L-l and a dilution rate of 0.2 h-l. This was compared with a plug flow
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process by solving equation (7) for many very small HFR-CSTRs in series with
the same cell loading per unit volume and feed rate but much shorter residence

0 5 10 15 20
F p r e 8 Predicted conversion (%) vs residence time (h) for four HFR CSTRs in series (shown as
blocks) with the same operating characteristics as HFR R3 at D = 0.2 h-' compared with a plug flow
reactor (dashed line).
228 S. L. PATERSON E T A L .

times. Figure 8 shows the conversion vs residence time profile for four
HFR-CSTRs in series compared with a plug flow profile. It is apparent that four
stages could achieve a conversion greater than 98%, with the majority of the
reaction occurring in the first stage. As expected, the plug flow process would be
significantly better than the HFR-CSTR process in series. Increased cell loading
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per unit reactor volume would improve the process, assuming no mass transfer
limitation occurred.


The kinetic constants V,,, and K,,, for treated cells charged to batch reactors
show only small variations between experiments. In a few cases ethanol (and
presumably carbon dioxide) was produced, corresponding to those experiments in
which higher V,, values were calculated (Table 1). For the HFR process, cells
must be treated sufficiently with toluene so that significant metabolism and gas
evolution will not occur. Gas formation will increase the shell pressure in the
HFR, displacing the sugar solution and disrupting Starling flow. Extended
toluene treatment appeared to deactivate the enzyme complex when compared to
the predicted activity of the oxidoreductase enzyme system in untreated Z.
mobilis cells. However a treatment of 1% (v/v) toluene for 10 minutes provided
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a good balance between complete permeablisation and excessive deactivation.

Results from HFR R3 suggest that, with a feed concentration of 100 g * L-' of
both glucose and fructose, no appreciable mass transfer limitations occurred,
even at reaction rates of 0.45 g - g-' h-' (Figure 7). For all HFRs reported in
this work the cell loading based on the HFR unit volume of 140ml was
140 g DW - L-', which can be considered reasonably high. The HFR unit volume
was small when compared to the volume of the recycle loop in the experimental
set-up used (1:2.4). However, a system designed for industrial use may well
function with a HFR unit volume to recycle loop volume ratio of 10 to 1 as the
recycle loop is only required for pH control. Productivities of 10-20 g sorbitol or
gluconate - L-' . h-' were found for dilution rates D = 0.2-0.4 h-' based on the
total volume of the hollow fibre reactor and the recycle loop. For a larger pilot
scale or industrial unit, the relative volume of the recycle loop to the reactor is
likely to be much smaller, thereby giving an appreciable increase in productivity.
The current industrial chemical process for sorbitol manufacture involves high
temperature (140°C) and pressure (12.7 MPa) hydrogenation of dextrose syrup
over a Raney nickel catalyst ((Benson, 1978). In a batch reactor containing
treated cells of 2. mobilis and initial glucose and fructose concentrations of
300 g L-' (Rogers and Chun, 1987) the time of conversion compares favorably
with the chemical process described by Phillips (1963). However, it occurs at
39°C and 1 atmosphere without the requirement for H2. A possible feedstock for
the Z. mobilis enzyme process would be high fructose corn syrup. In the USA
this is significantly cheaper than the dextrose (Hebeda, 1978) used for commercial
production of sorbitol (Benson, 1978) and gluconate (Rehm and Reed, 1984).
The HFR-CSTR process is easy to operate and straight forward to scale-up. A
series configuration should be able to convert greater than 98% of sugars (Figure
8) whilst maintaining a reasonable productivity.
Further work on this process will involve higher feed concentrations of glucose

and fructose (up to 300 g/L each). From the experimental and modelling results
presented in this work the HFR process should be able to reach considerably
higher productivities, especially with better Starling flow and therefore enhanced
pH control. The HFR process for sorbitol and gluconate production has
considerable commercial potential.
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The authors would like to thank Memtec (Australia) Ltd. for financial support
and supply of the membrane unit materials and the Australian Government for
support in the form of a Commonwealth Scholarship (SLP). The technical
assistance of S. Masters is gratefully acknowledged.

Barrow, K. D., Leigh, D. A., Rogers, P. L. and Warr, R. G. (1984) Sorbitol production by
Zymomonas mobilis. Appl. Microbiol Biotechnol., 20, 225-232
Benson, F. R. (1978) Alcohols, polyhydric.
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(3rd. ed.), 1, 754-778
Gekas. V. C. (1986) Artificial membranes as carriers for the immobilization of biocatalvsts. Enzvme
Miciob. Technol.’8, 450-460
Hebeda, R. E. (1978) Syrups. Kirk Othmer Encyclopedia of Chemical Technology. (3rd. ed.), 22,
For personal use only.

Leigh, D. A., Scopes, R. K. and Rogers, P. L. (1984) A proposed pathway for sorbitol production by
Zymomonas mobilis. App. Microbiol. Biotechnol., 20, 413-415
Leigh, D. A. (1987) Some studies on substrate catabolism in Zymomonas mobilis, strain ZM4. PhD
Thesis, University of New South Wales.
Libicky, S . B. (1985) Transport of gases and liquids through dense microbial cell aggregates cultured
within hollow fibre membrane bioreactors. PhD Thesis, Stanford University.
Masters, S . (1986) Sorbitol production by Zymomonas mobilis. BSc (Hons) Thesis, University of
Park, T. H. and Kim, I. H. (1985) Hollow fibre fermentor using ultrafiltration. Appl. Microbiol.
Biotechnol., 22, 190-194
Paterson, S. L., Fane, A. G., Fell, C. J. D. and Rogers, P. L. (1987) Influence of convective mass
transfer via Starling flow in a hollow fibre reactor employing E. Coli to produce phenylalanine. in
Phillips, M. A. (1963). Catalytic hydrogenation of glucose to sorbitol using a highly active catalyst.
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