Biotech. Adv. Vol. 4, pp. 207-218, 1986 0734-9750/86 $0.00 + .

50
Printed in Great Britain. All rights reserved. Pergamon Journals Ltd

FED-BATCH CULTURE TECHNOLOGY

B. J. M I N I H A N E a n d D. E. B R O W N

Biotechnology Centre, Crenfield Institute of Technology, Crenfield,

Bedfordshire, MK43 OAL, UK

ABSTRACT

The use of fed-batch procedures offers distinct advantages over other modes of
operation of btoreactors, and is a widely researched technique. These advantages
are discussed; some uses of fed-batch procedures and the associated methods of
modelling and control are reviewed.

KEYMORDS

Fed-batch, glucose effect, catabolite repression, bakers' yeast, cellulase,

penicillin, structured model, unstructured model, feed-back control,
feed-forward control.

I ffrROOOCTIOM

There are three modes of bioreactor operation identified, namely pure batch,
continuous, and fed or seml-batch. In a batch fermentation process all the
required nutrients, with the exception of pH correction and oxygen if aerobic,
are in the culture medium prior to innoculation. Continuous fermentation
processes have both an inflow and outflow of nutrient medium such that the medium
volume In the bloreactor remains constant and all the nutrients in the medium are
present at a constant concentration. In the case of a fed-batch process, one or
more nutrients are supplied to the bioreactor but with no outflow during the run.
This procedure means that the concentration of one or more of the nutrients in
the medium can be externally manipulated by altering the feed rate during the run
according to the feed-back of control parameters such as dissolved o~gen (DO)

207
208 B.J. MINIHANE and D.E. BROWN

(41), pH, or respiratory quotient (RQ) (55). Alternatively, feed-forward

strategies can be employed using pre-programmedfeed profiles. The advantages
that these techniques give to the operator for improved process technology has
resulted in their widespreadindustrial application. Yamaneand Shimizu (59)
i n i t i a l l y classify fed-batch operations into those with feed-back control and
those without, and then sub-divide according to the feeding strategy adopted.
These include constant and exponential feed rates, and optimised procedures.
Early work tended to use intermltent feeding or constant feed rates, but recent
advances in measurementand control techniques make i t possible to use more
sophisticated programmes. The corresponding advance in computer systems has
resulted in their extensive use for control and optimisation of fed-batch
operations. Several reviews of such applications have recently been published
(18, 39, 43).

The major advantage that the fed-batch technique offers over p u r e batch
cultivation is the a b i l i t y to control or a l t e r the concentration of one or more
nutrients within the culture medium. I t is thus ideal for processes in which
either cell growth or product formation is sensitive to the concentration of the
limiting substrate. I t may also allow the replacement of complex sources of
nutrients with more definable components so that high levels of reproducibility
and p r e d i c t a b i l i t y becomepossible.

SUBSTRATE INHIBITION

In many processes the nutrient utilised for growth by the organism, such as
ethanol or aromatic compounds,w i l l i n h i b i t the growth process except at low
concentrations. This inhibition can be overcomeby using a fed-batch procedure.
Renard et al (41) prevented the toxic effect of methanol on PseudomonasAMI by
using methanol additions in response to the measured dissolved oxygen tension.
This avoided growth limitation either due to methanol t o x i c i t y or lack of
methanol substrate. Similarly Silman (46) found glucose inhibition of Zymomonas
mobills at concentrations above 8% w/v, but overcamethis inhibition by
feeding the glucose and was thus able to maintain ethanol productivities greater
than 4.5 g ethanol/L/h.

To obtain high cell concentrations in a batch culture i t is necessary to supply

high i n i t i a l concentrations of nutrients. Such high values may not be possible,
either because of substrate inhibition, not seen at lower concentrations, or
because of low s o l u b i l i t i e s of the substrates; however some processes are only
economically attractive i f high cell concentrations are possible. Suzuki et al
(49) produced yeast cell concentrations greater than 150 g/L by using a feed of
 FED-BATCH CULTURE TECHNOLOGY 209

a l l nutrients. They controlled the substrate feed (ethanol) using a porous Teflon
tubing sensor and a pH control for their nitrogen source. A mass balancing
,principle was used to prepare and feed a combined salts solution at a rate linked
to either the ethanol or ammoniafeed. Monitoring of ethanol concentation was
u t i l i s e d by Huang and Chu (21) to feed ethanol for Candida u t i l i s , obtaining
cell concentrations of 64 g / l . A problem of substrate i n s o l u b i l i t y was overcome
by Rodriguez and Enriquez (42) to obtain yields of 19.9 g/l of Cellulomonas on
sugarcane bagasse pith.

CATABOLITE REPRESSION

I f an organism is u t i l i s i n g a rapidly metabolizable carbon energy source such as

glucose, the resultant decrease in i n t r a c e l l u l a r cyclic AMP concentration causes
enzyme biosynthesis to cease. This phenomenonis .known as catabolite repression.
Fed-batch processes, by l i m i t i n g the growth rate due to the slow feeding of the
carbon source, derepress the formation of the desired products. Where the
direct measurementof specific growth rates or substrate concentrations is
d i f f i c u l t , indirect correlations have to be used for control purposes. The
comnonest exan~}le of this is in the production of antibiotics such as p e n i c i l l i n .
To create a control strategy for sugar addition in the production of p e n i c i l l i n ,
both the cell concentration and the growth rate BlaSt be known. However continuous
measurement of glucose in the mediumis d i f f i c u l t and so a technique of carbon
balancing under carbon limited conditions has been developed by Mou and Cooney
(30,31). I n i t i a l l y , the carbon dioxide evolution rate (CER) was used to calculate
the growth rate and total carbon dioxide evolved was found to give an accurate
estimation of the quantity of ~celium present. Their adaptive control strategy
allowed for the addition of carbon to control the growth rate until the desired
cell concentration was obtained. At that point in time, the production phase was
i n i t i a t e d by slow feeding, controlled by an estimate of the specific growth rate
and a value of the cell concentration obtained by overall and instantaneous
carbon balances. Nestaas and Wang (33, 34, 35, 36) used an on-llne f i l t r a t i o n
probe to characterise the build-up and degradation of ~ c e l i a l blomass
quantitatively and were able to correlate k~vphal density with the rate of
p e n i c i l l i n synthesis (35). This was then conVDinedwith a pre-determined feed
p r o f i l e to optimise the p e n i c i l l i n synthesis (35, 36). The probe provided a
feed-back control system to manipulate the glucose feed p r o f i l e that was
calculated from a mathematical model. Nelligan and Calum ( 3 7 ) used an
anticipatory control system measuring the carbon dioxide output rather than f u l l
carbon balancing, and were able to obtain a good correlation with their predicted
behaviour using a micro-coR~uter to control to optimumconditions. Earlier work
on p e n i c i l l i n production is tabulated by Yamaneand Shimizu in their review of
210 B.J. MINIHANE and D.E. BROWN

fed-batch techniques (5g).

Another area of recent interest in which catabollte repression ts overcome is in

the production of cellulase using fed-batch fermentation (1, 16, 17, 19, 20, 26).
This technique has been shown to produce higher concentrations of enzyme than
previous work in batch or continuous culture. McLeanand Podruzny (26) quote
productlvlties up to 160 U.FPA /L/h using intermitent feeding of Solkafloc, and
also report the advantageof easier agitation and aeration due to the lower
medium densities than in batch growth. Hendy et al (19, 20) used a continuous
rather than intermitent feed of Solkafloc and quoted a maximumproductivity of
247 U./L/h at an addition rate of 2.5 g cellulose/h.

Work has recently been done on the production of tylosin by Streptonlyces

fradlae, In which Vu-Trong and Gray ( 5 1 ) found enhancedyields over those they
prevlously obtained using a constant feed rate (52, 53) by feeding both glucose
and monosodium glutamate in a cyclic fashion. The production of L-lysine by
CoKynebacterlum ~luta~cum, using a glucose based co~lex medium, has also
been examined ( 4 4 ) and the successful production of Cephalosporin C by
Cephalosporiumacremonium, using a constant feed of glucose and methionlne, has
been reported (27).

GLUCOSE OR CRABTREE EFFECT

I t has been known for a long time that in the cultivation of bakers' yeast there
is a c r i t i c a l glucose concentration above which the substrate Is partlally
metabollsed to ethanol, even In the presence of sufficient dlssolved oxygen to
maintain aerobic metabolism. This phenonemonis known as the glucose or Crabtree
effect and the use of fed-batch culture to avoid this effect has been extensively
researched. The c r i t i c a l concentration at which the partial metabolismto ethanol
occurs has been variously reported (55), ranging from 0.02 to 0.85 mM. I t has
been suggested that these variations are due to differences in the yeast strains
or their physiological conditions. The normal substrates used in industry are
beet or cane molasses and the c o ~ l e x l t y of the sugars together with the lack of
direct on-line substrate sensors has resulted in work being directed at the use
of predictive models and indirect control parameters. Recent work by Dekker (5)
utlllses off-gas analysis to calculate the oxygen uptake rate (OUR) and the
carbon dioxide evolution rate (CER). Using a two-conM)artmentmodel for the cell
and the stoichlometrlc balancing and simple state models for biomass and biomass
production rate, he generates the output equations for the state variables, and
hence an estimation of the values of the process variables using mathematical
f i l t e r i n g techniques. These values can be used to calculate optimal control rates
 FED-BATCH CULTURE TECHNOLOGY 211

for substrate feeding (6, 7).

Another analytical method developed in Japan is the use of a porous Teflon tube
to monitor the ethanol concentration in the medium and using this parameter for
feed-back control (2, 3). Nomba et al (32) used the ethanol off-gas concentration
to feed-back control the glucose supply which was initially controlled by a mass
balancing model. An "advanced control system" was developed by Dalraku et al (2,
3) using the measured ethanol concentration and a comblnation of a PID
(proportional-integral-derivatlve) controller and a simple mathematical model to
allow for self-tuning with increased cell concentration. This report claims good
control, the model providing the initial estimate of the exponential feed rate
and the PID control compensating for errors caused either by system disturbances
or uncertainties in the model.

OTHER FED-BATCH SYSTEMS

With the recent increase in work on plant cell cultures, Dalton (4) reports an
increased specific production rate of total chlorophyll and higher potential
photosynthesis by the use of glucose fed-batch over pure batch culture of
Oclmum basllicum. An enhanced production of chromatophores from
Rhodosplrllllum rubrum was obtained by Smeds and Enfors (47) by increasing the
light intensity during the course of the batch, which they term llght-fed-batch.
Horl et al (29) produced high concentrations of sorbose from sorbltol using
Intermltent feeding of sorbltol to Glucanobacter subox~dans with DO-stat
control, obtaining a concentration of 628 g/L of sorbose after 48 hours.

Fond et al (11, 12) have investigated the acetone-butanol fermentation with

feeding of glucose, x~lose or mixed feeds over a range of feeding rates. They
found the final conversion of sugars into solvents always increased with an
increase in feeding rate.

A study of the production of ethanol from Xylose by Pacl~ysolen. tannophtlus by

Woods and H t l l t s (56) showed an improved y i e l d of ethanol up to 0.41 g/9 or 80~
of theoretical by using slow feedtn9 of xylose, to give an optimum xylose
concentration of 5 to 8 g/L. Concentrations 9rearer than 10 g/L caused ~ y l t t o l
accumulation, w h i l s t below 3 g/L the ethanol was oxtdtsed to acetate. Jefferles
et al (2Z) used lnterlattent feeding of glucose to i n h i b i t the respiration of
ethanol produced by Pach~fsolen tannophylus from xylose, and increased t h e i r
ethanol y i e l d from 0.28 g/g to greater than 0.41 g/g.

Koshtmtzu et al (24) examined the fed-batch fermentation of molasses by

212 B.J. MINIHANE and D.E. BROWN

Saccharo~ces cerevisiae, as used commercially by the Brazilian ethanol plants,

in order to quantify the effect of the inoculum, the feed concentration and the
feeding rate.

CONTROLOF FED-BATCH TECHNIQUES

As indicated above, control systems for fed-batch operations can be divided into
two groups, those with and those without feed-back control. When there is no
feed-back control mechanismbeing u t i l i s e d , the feed rate is changed according to
a pre-determined p r o f i l e , which Yamane(59) has classified as intermitent,
constant, exponentially increasing (38), optimal, or others. If this feeding
p r o f i l e is operated in order to control the growth of the micro-organisms, then a
mathematical model of the process is usually used to describe this p r o f i l e . The
models are often specifically generated to provide a basis for control and can be
divided into either unstructured (phenomenological) or structured (mechanistic)
descriptions of the process. The unstructured models are used to describe overall
observed microbial responses, and do not allow for changes in cell composition as
metabolic actJvlties change. Structured models, on the other hand, a t t e s t to
allow for the cellular mechanisms that affect the overall process. A variety of
structures and degrees of complexity are available. They offer the p o s s i b i l i t y of
accurately predicting the effects of disturbances on the overall process.
Disadvantages include the d i f f i c u l t i e s in determining the kinetic constants and
in the solving of large nu~ers of equations. However, structured models are
becoming more prominent in the operation of computer controlled bioreactors.
Comprehensive general reviews of both unstructured (g, 10, 39, 59) and structured
models are available (18, 25, 39, 59).

When feed-back control is available for fed-batch operation it ideally operates

with a direct signal such as the concentration of the feed substrate in the
medium. Alternatively an indirect feed-back control is possible in which an
observable parameter that can be correlated to the desired feeding rate is
monitored. A direct feed-back control strategy is better than anY other procedure
for minimising deviations,but a lack of suitable sensors has limited the work in
this area. One direct system that has been used by Yamene (57, 58) is the Teflon
tubing ethanol sensor to control the addition of ethanol as a sole carbon source
to a salts medium for the culturing of yeast. The sensor was that developed by
Dalraku et al (2, 3). Ghoul et al (15) have developed an automatic sampler which
they used with a glucose auto-analyser to control a glucose feed to a yeast
fed-batch fermentation. Their system would be suitable for other in-line
measurements as required.
 FED-BATCH CULTURETECHNOLOGY 213

The m s t common situation to date has been the use of indirect measurement
feed-back control. Several parametershave been used, such as dissolved oxygen
(DO), respiratory quotient (RQ), and off-gas analysis to calculate OUR and CER.
As these are indirect parameters, the mathematical correlation with the
organisms' metabolism and substrate uptake must be known. Current work uses
adaptive algorithms and Kalmanf i l t e r i n g techniques (8, 13, 14, 23, 28, 40, 45,
48) to obtain meaningful correlations. Often the feeding p r o f i l e is controlled by
the model directly in a feed-forward mode, and the feed-back control is then used
for correction purposes.

A measurementof the dissolved ovgen has been employedby Renard et al (41) for
the computer controlled feeding of methanol in response to a f a l l in DO. However
more recent work tends to control this type of substrate feeding by maintaining
the DO level at a set-point (DO-star). The production of sorbitol by Mori et al
(29) madeuse of this technique.

The off-gases from a bioreactor are normally used to calculate the OUR and CER,
and from these the RQ. The RQ is extensively used as a control parameter in
bakers' yeast production, as the four types of yeast metabolism are related to
the level of the RQ (55) and the RQ shows a rapid response to changes in
metabolism. Dekker and Voetter (7) have developed two adaptive controllers for
the regulation of the RQ. One is based on the two-compartmentmodel system (5),
and the other is a self-tuning controller. Both of these systems use the OUR as
an inner loop feed-back control parameter. The mass balancing method of Mou and
Cooney (30,31) for controlling the glucose feed for p e n i c i l l i n production also
relies upon an accurate measurement of the CER along with rates of glucose
addition. In the growth phase, CER was found to be a good indicator of the cell
growth rate and total carbon dioxide evolved allowed for an estimate of mycelium
present. The adaptive control strategy adds sufficient carbon to maintain the
desired growth rate, but does not overfeed. At the desired cell concentration,
lower growth rates are achieved by reducedfeeding rates and overall and
instantaneous carbon balances allow the calculation of the specific cell growth
rate.

Carbon dioxide concentration in the exit gas was found to be a good indicator of
the growth of Trlchoderma reesei using cellulose as a main substrate and
feeding celloblose to control the specific growth rate. Wakl et al (54) used a
time-variant set point for carbon dioxide concentration in the exit gas to
control the feeding of the celloblose, pH control has also been used for feed
control purposes. This is possible when the utillsation of a particular medium
component causes a deviation of the pH from a set point. Gottvaldova et al (16)
214 B.J. MINIHANE and D.E. BROWN

used this technique to control a double feed of carbon and nitrogen to achieve
enhanced cellulase production by Trichoderma viride. A further technique that
is possible requires that a measurable motabolite is produced by the microbial
a c t i v i t y . This approach has been used to control Bakers' yeast fermentation where
the production of ethanol is detectable either in the medium using the Teflon
tubing sensor (2,3) or indirectly by the ethanol vapour in the e x i t gas (32). The
major current l i m i t a t i o n in the use of direct feed-back control is the lack of
sensors that can detect specific chemicals in the fermenter medium. Current work
on biosensors (50) that respond to specific chemicals offers the p o s s i b i l i t y of a
major increase in the application of feed-back control in fermentation
technology.

CONCLUSIONS

Fed-batch operation is moving rapidly from an empirical procedure to one based

upon kinetic models and advanced control techniques. An increasing number of
processes are being operated in this way. Advances in computer applications allow
optimisation procedures to be examinedo f f - l i n e , and computer on-line control
using both feed-forward and feed-back control is becoming common practice. A
major l i m i t a t i o n to further application of feed-back control arises from the lack
of available on-line sensors.

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