Biotechnol Lett (2011) 33:1621–1624
DOI 10.1007/s10529-011-0606-8
ORIGINAL RESEARCH PAPER
Industrial production of fructooligosaccharides
by immobilized cells of Aureobasidium pullulans in a packed
bed reactor
K. H. Jung • S. H. Bang • T. K. Oh • H. J. Park
Received: 1 December 2010 / Accepted: 30 March 2011 / Published online: 9 April 2011
Ó Springer Science+Business Media B.V. 2011
Abstract Continuous production of fructooligosac-
charides (FOS) by Aureobasidium pullulans immo-
bilized on calcium alginate beads with a packed bed
was investigated at a plant scale reactor. Optimum
conditions were with 770 g sucrose/l, being fed at
200 l/h at 50°C which gave a productivity of 180 g
FOS/l h. Initial activity was maintained for more than
100 days. The reactor was successfully scaled up to a
production scale of 1.2 m3.
Keywords Cell immobilization

Fructooligosaccharides
Fructosyltransferase

Packed bed reactor
K. H. Jung
Technical Center for Bio-Business Unit, CJ Cheiljedang
Corporation, 92, Kayang-Dong, Kangseo-Ku, Seoul,
Korea
T. K. Oh
Korea Research Institute of Bioscience
and Biotechnology, 115, Yusong, Taejon, Korea
S. H. Bang
H. J. Park (&)
School of Life Science and Biotechnology,
Korea University, 5Ka, Anam-Dong, Seongbuk-Ku,
Seoul, Korea
e-mail: hjpark@korea.ac.kr
Introduction
The application of immobilization techniques to the
enzymatic conversion of sugar is desirable. The
uncharged structure and small size of sugar molecules
minimizes unwanted diffusional restrictions, and a
high substrate concentration prevents contamination
problems in an industrial operation (Chhetham et al.
1985). An example of such a system, which has a
growing industrial importance, is the production of
(fructooligosaccharides (FOS). FOS, in which 1–3
fructose units are bound at the b-2,1 position of
sucrose, are mainly composed of 1-kestose (GF2),
nystose (GF3) and fructofuranosyl nystose (GF4).
FOS which has been used in so-called health food
(Hidaka et al. 1986) is produced from sucrose by the
action of fructosyl transferase (FTase) (EC 2.4.1.9).
In our previous study, conditions for the produc-
tion of FTase from Aureobasidium pullulans were
studied (Jung et al. 1987). Based on enzyme kinetic
studies with various substrates, such as GF, GF2, and
GF3, a disproportionation reaction (viz, GFn ?
GFn ? GFn-1 ? GFn?1) was reported (Jung et al.
1989). Semi-batch production of FOS by immobi-
lized A. pullulans was studied in a stirred tank
bioreactor over 2 months (Yun et al. 1990). From a
process viewpoint, however, a continuous process has
many advantages over a batch or semibatch process.
A packed bed reactor (PBR) can continuously
produce FOS while minimizing the product inhibition
caused by the accumulation of glucose.
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Our previous study of a 1 l PBR reported that

continuous production of FOS employing a reactor
charged with immobilized A. pullulans is optimal at
770 g sucrose/l, at feed rate of 0.2 l/h and 50°C (Yun
et al. 1992). Initial activity was maintained for more
than 100 days. In this study, plant-scale continuous
production of FOS was investigated and the optimal
scale up conditions of 1.2 m3 fermentor, 0.5 m3 in-
house bead forming apparatus and 1.2 m3 PBR were
investigated.
Fig. 1 Schematic diagram of the packed bed reactor (PBR) for
the continuous production of fructooligosaccharides (FOS).
A packed bed reactor, B circulating water bath, C substrate
Materials and methods reservoir, D product reservoir, E peristaltic pump

Cell preparation
Aureobasidium pullulans KFCC 10245 was grown at
28°C for 36 h under 200 rpm in a 1.2 m3 fermentor
containing 800 l growth medium composed of (w/v)
20% sucrose, 2% yeast extract, 0.5% K2HPO4, 0.2%
MgSO4
7H2O and 1.5% NaNO3. Cells were har-
vested by centrifugation and washed twice with
deionized water prior to use.
reactor. The reactor was charged with 0.72 m3
alginate beads. The temperature was kept constant
by circulating hot water through the jacket. Unless
otherwise specified, the reaction was carried out at
770 g sucrose/l at 200 l/h and at 50°C. The pH of the
feed solution remained essentially constant at 6–7
throughout the operation.

Cell immobilization
Results and discussion
The cell suspension, containing 4% (v/v) dry cells,
Batch culture of A. pullulans
was mixed thoroughly with 3% (w/v) food-grade
sodium alginate solution at 1:2 (v/v). The mixture
Batch culture kinetics of A. pullulans in the 1.2 m3
was extruded through No. 25 gauge syringe needles
plant scale fermentor were similar to that reported
to form beads of *2 mm diam. before being dropped
previously (Yun et al. 1990) for 100 l fermentor
into a 0.1 M CaCl2. The beads were cured for 2 h and
culture. A typical time course for the growth of
then hardened overnight at 4°C.
A. pullulans in the growth medium is shown in
Fig. 2a. Both cell concentration and intracellular
Enzyme assay
FTase activity increased almost linearly with time up
to 40 g/l and 200 U/ml, respectively. Also shown in
Fructosyl transferase (FTase) activity was determined
Fig. 2b are sugar concentration profiles. During the
by measuring the released glucose as previously
early period of growth (12 h), sucrose concentration
described (Jung et al. 1987).
declined very rapidly with the accumulation of GF2,
GF3, and GF4. However, these FOS were utilized
Analytical method
slowly toward the end of batch culture. Similar
results were reported during the cultivation of
All reaction products were analyzed by HPLC as
Penicillum expansum (Prata et al. 2010).
previously described (Jung et al. 1989).

Reactor operation Mass production of immobilized cells

A schematic diagram of the PBR is shown in Fig. 1. For the continuous production of FOS, the calcium
Sucrose solution was fed upward continuously to the alginate bead-immobilized cells were mass-produced

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Fig. 2 Batch culture kinetics of Aureobasidium pullulans in
the 1.2 m3 plant-scale fermentor. a Dry cell weight and
intracellular fructosyltransferase activity production; b sugar
concentration profiles (glucose, sucrose, 1-kestose, nystose and
fructofuranosyl nystose)
by an in-house bead forming apparatus. Briefly, a
thousand of needles were evenly positioned on the
bottom of the stainless steel vessel (Fig. 3). The cell-
alginate mixture, previously well mixed, was poured
into the vessel and pressure was applied to drive the
mixture through the needles. The resulting drops
entered the CaCl2 solution and formed calcium
alginate beads. The storage tank contained the calcium
alginate gel was mildly fluidized with air to prevent
bead crush/breakage mediated by hydrostatic pressure
within the tank. Generally, immobilized cell beads
were mass-produced 0.2 m3/h in this apparatus.
Plant-scale optimization of PBR operation
The major operating variables affecting the perfor-
mance of a PBR are flow rate, pH, concentration of
feed, and operating temperature. When scaling up
the PBR to 1.2 m3, the height-to-diameter ratio of the
reactor was kept constant at 3:1 (3.6 m 9 1.2 m). The
1623
Fig. 3 The schematic diagram of the in-house bead forming
apparatus. a Pressure gauge, b mixture inlet and c air inlet
proper design and operation of a PBR were required to
achieve uniform radial temperature within the PBR.
The difference in solution viscosity caused by the
temperature gradient resulted in unbalanced channel-
ing of the solution. Uniform packing of the calcium
alginate beads and proper design of the distributor
were important in preventing the channeling phenom-
enon. Typical reactor dimensions for immobilized
glucose isomerase are 5 m 9 1.5 m (Buchholz and
Seibel 2008).
Plant scale-up results of the PBR
As shown in Fig. 4, when 770 g sucrose/l was
continuously pumped into the reactor at 200 l/h, the
initial activity was maintained for over 100 days
without recharging the immobilized cells. The pro-
ductivity of reactor was 180 g/l h.
Total FOS content was about 57% (w/w) (F: 1%,
G: 27%, GF: 15%, GF2: 32%, GF3: 21%, GF4: 4%).
At the laboratory scale, long-term operational stabil-
ity was reported for FOS production with various
systems: Aspergillus japonicus-immobilized gluten
beads (half-life of 34 days) (Chien et al. 2001),
Pectinex Ultra SP-L (20 days) (Tanriseven and Aslan
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Fig. 4 Operational stability of the 1.2 m3 packed bed reactor
(PBR) for the continuous production of fructooligosaccharides
(FOS). The reaction was carried out in a 0.72 m3 immobilized
beads fed with 770 g sucrose/l at 200 l/h
2005), and a forced-flow membrane reactor system
(half-life of 35 days) (Nishizawa et al. 2000).
Conclusion
The results obtained in the 1.2 m3 PBR operation at
feed rate of 200 l/h using 770 g sucrose/l at 50°C
over 100 days without recharging the immobilized
cells confirm that it can be used for FOS production
in industrial applications.
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Biotechnol Lett (2011) 33:1621–1624
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