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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
INTRODUCTION
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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fructose
--
(A)
gluconolactone
sorbitol
-
pH close to 6.2 (Zachariou and Scopes, 1986). Thus,
glucose
(B)
gluconate + H+
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
desirable.
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
I
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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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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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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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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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.
SORBITOL AND GLUCONATE PRODUCTION 223
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.
RESULTS
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).
224 S. L. PATERSON ET AL.
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I I L
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).
I Masters (1986).
Rogers and Chun (1981).
SORBITOL AND GLUCONATE PRODUCTION 225
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.
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 .
100
-m
- 75 -
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5
z
0
v,
LL
W
>
5 50- v/
a
L L
I
25 -
0 I I 1 I I
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c
CI
O6 t
I
m
\
m
v
2
W
OL-
K
z
0
k
0
a
W
K
0 02-
Y
0
W
a
v)
I I I 1
0 20 LO 60
SUGAR CONCENTRATION <g/L)
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.
SORBITOL AND GLUCONATE PRODUCTION 227
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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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
RESIDENCE T I M E Ih)
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.
DISCUSSION
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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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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Acknowledgements
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.
References
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
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Miciob. Technol.’8, 450-460
Hebeda, R. E. (1978) Syrups. Kirk Othmer Encyclopedia of Chemical Technology. (3rd. ed.), 22,
499-522
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Microbiol. Biotechnol., 19, 252-255
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(1979) Fermentation and enzyme technology., Chapter 14, Wiley Interscience, NY.
Zachariou, M. and Scopes, R. K. (1986) Glucose-fructose oxidoreductase, a new enzyme isolated
from Zymomonas mobilis that is responsible for sorbitol production. J. Bacteriol., 167, 863-869