Process Biochemistry 35 (1999) 485 – 490

www.elsevier.com/locate/procbio

An integrated fermentation–separation process for the production

of red pigment by Serratia sp. KH-95
Chang-Ho Kim, Seung-Wook Kim, Suk-In Hong *
Department of Chemical Engineering, Korea Uni6ersity, Anam-dong, Sungbuk-ku, Seoul 136 -701, South Korea

Received 22 February 1999; received in revised form 3 May 1999; accepted 10 July 1999

Abstract

Integrated fermentation–separation processes using a polymeric adsorbent were investigated to increase the productivity of red
pigment. Resin HP-20 was chosen for polymeric adsorbent, taking into consideration the adsorption capacity and the desorption
ability. The maximal production of total pigment was obtained when the HP-20 resin was added after 10 h. As the amount of
resin was increased from 5 to 20% (v/v), cell growth gradually decreased. The maximal production of pigment was obtained when
10% (v/v) HP-20 was added and the concentration of pigment contained in the resin was 5.92 g/l. The extractive fermentation was
carried out in bioreactors equipped with extractive columns packed with 5, 10 and 20% (v/v) HP-20. Cell growth decreased
inversely in proportion to the concentration of HP-20 packed into the extractive column. The maximal production of pigment
(6.92 g/l) was obtained with 10% (v/v) HP-20. The bioreactor equipped with an extractive column packed with HP-20 adsorbent
resin showed higher productivity than the conventional batch bioreactor. A 31% increment of pigment production was obtained
in extractive fermentation, compared with the simple batch culture. © 1999 Elsevier Science Ltd. All rights reserved.

Keywords: Serratia sp.; Pigment production; Integrated fermentation – separation process

1. Introduction tion of product in the fermentation broth inhibits fur-

Many synthetic colours, which were popular in the
past because of their stability, low cost and ease of
application, have had their usage drastically curtailed
for reasons of safety. Nevertheless, natural colours,
notwithstanding limitations of choice, adequacy and
stability, are often considered to be safer than synthetic
colours and will be used widely in foods, cosmetics and
pharmaceutics [1,2].
There are a number of natural colours, but only a
few are available in a sufficient quantity to be useful for
the industry because they are directly extracted from
plant flowers, fruits, leaves and roots [1]. It is therefore
advantageous to produce natural colours from microor-
ganisms, and it has been shown that some pigments are
produced from Monascus [3 – 5], Streptomyces [6] and
Serratia [7–10].
A wide range of chemicals can be produced by
microorganisms. In some cases, however, the accumula-
* Corresponding author.
ther production [11–14]. Recently, integrated
fermentation–separation systems have been used suc-
cessfully to reduce end-product inhibition and thus, to
improve overall process efficiency [15–24]. The product
can be removed by a membrane filtration process, by
extraction, by evaporation or by adsorption [15].
Among these techniques, solid adsorbents have been
used by some researchers [20–24]. A potential advan-
tage of solid sorbents over organic solvents as extrac-
tants is the low risk of toxicity when using polymeric
material as sorbents. A wide range of polymeric adsor-
bents have been designed for the removal of organic
material. Diaion HP–20 resin has been used for the
production of esperamicin A1 during the fermentation
of Actinomadura 6errucosospora [22], and amberlite
XAD resins are used to remove anthraquinone pro-
duced by suspension cultures of Chichona ledgeriana
[23].
In previous studies, strain KH-95 producing a high
concentration of prodigiosin-like red pigment was iso-
lated and identified as Serratia sp. based on morpho-

0032-9592/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 2 - 9 5 9 2 ( 9 9 ) 0 0 0 9 1 - 6
486 C.-H. Kim et al. / Process Biochemistry 35 (1999) 485–490
logical and physiological characteristics [25]. Optimal
media and culture condition for the production of
prodigiosin-like red pigment were also established using
this strain [26]. In this study, an integrated fermenta-
tion –separation process using a polymeric adsorbent
was investigated to increase the productivity of red
pigment. The integrated system equipped with an ex-
tractive column packed with adsorbent resin was intro-
duced, and optimal conditions for the process were
investigated.
2. Materials and methods
2.1. Microorganism and culture medium
Serratia sp. KH-95 was maintained on an agar slant
containing 0.2% dextrose, 1.0% bacto peptone, 1.0%
yeast extract, 0.2% K2HPO4 and 1.5% agar [25]. The
basic culture medium consisted of 3.0% casein, 0.1%
K2HPO4, 0.1% MgSO4.7H2O and 0.1% NaCl. The ini-
tial pH of the medium was adjusted to 7.0 before
sterilization.
2.2. Culture conditions
Fermentations were carried out in 500 ml Erlenmeyer
flasks, each containing 50 ml of the culture medium and
in a 5 l bioreactor (working volume; 3 l), operating at
28°C, for 20–24 h. Bioreactors were agitated from 400
to 800 rpm to maintain a proper dissolved oxygen level
and the pH was not controlled.
2.3. Adsorbent
Resins HP-20 (Mitsubishi Chemical Corp., Japan),
SP-850 (Mitsubishi Chemical Corp., Japan) and XAD-
16 (Rohm and Hass) having different characteristics as
adsorbent were used in this study. Prior to use, these
resins were soaked in iso-propyl alcohol and then
washed with several volumes of water. The volume of
resin was measured after complete drying and resin was
added to shake flasks and columns to perform an
integrated fermentation – separation system. For extrac-
tive fermentation, resin was sterilized at 121°C for 40
min.
2.4. Adsorption of pigment to polymeric adsorbents
The binding capacity and adsorption rate of pigment
to each resin were measured with a fixed bed column
containing 20 ml adsorbent. Purified pigment was
added to culture media up to final concentration of 2
g/l and 400 mg/l for the measurement of binding capac-
ity and adsorption rate, respectively. The culture media
containing pigment was passed through the column
from the top and returned to the storage vessel. The
concentration of pigment in the storage vessel was
checked and the binding capacity and the adsorption
rate of pigment to resin were calculated.
2.5. Desorption of pigment from polymeric adsorbents
Desorption ability was determined by elusion with
acidified methanol (pH 2.5), acidified with 0.1 M HCl.
Resins with pigment adsorbed were packed into a fixed
bed column and eluent of volume equivalent to 1–10
volumes of resin was passed through the column. The
concentration of pigment desorbed from the adsorbent
was calculated by measuring the concentration of pig-
ment in the eluent.
2.6. Analyses and measurements
Cell growth was monitored by measuring the optical
density (O.D.) of the culture broth at 600 nm. After
centrifuging the culture broth, the concentrations of red
pigment attached and not attached to cells were mea-
sured separately. The red pigment attached to cells was
extracted with acidified methanol and determined by
measuring the absorbance using a spectrophotometer at
535 nm. The red pigment in the supernatant was diluted
with acidified methanol and the absorbance measured.
The concentration of pigment was calculated from the
standard curve obtained using purified pigment [25].
The casein concentration was analysed by a modified
Biuret method [27].
2.7. Extracti6e fermentation
An integrated fermentation–separation system
equipped with a fixed bed column packed with resin
was designed to remove pigment continuously from
culture broth (Fig. 1). In order to extract pigment
during the fermentation, the culture broth in a bioreac-
tor was passed through the column from the bottom
and then returned to the bioreactor. The recycle rate of
culture broth was 3 l/h.
3. Results and discussion
3.1. Product inhibition
Ju et al. [24] have reported that pigment production
by microoganisms was inhibited by the final products.
Product inhibition was tested by addition of purified
pigment in shake-flask culture. When pigment was
added at concentrations of 0.5, 1.0, 1.5 and 2.0 g/l in
shake-flask culture, the concentration of pigment pro-
duced decreased to 3.95, 3.5, 3.05, 2.65, respectively
(Fig. 2). These data indicate that the pigment produced
by Serratia sp. KH-95 inhibited its further production.
 C.-H. Kim et al. / Process Biochemistry 35 (1999) 485–490
Fig. 1. A schematic diagram of the extractive bioreactor system.
3.2. Selection of adsorbent
Three adsorbents, HP-20, SP-850 and XAD-16 were
tested to reduce the inhibition effect by binding the
pigment efficiently. These were widely used as adsor-
bents in the biotechnological industry and were also
cheap and reusable. Several factors must be taken into
account when choosing an efficient adsorbent including
the adsorption capacity and the desorption ability of
the pigment. The adsorption capacities of adsorbents,
HP-20, SP-850, and XAD-16 to the pigment were 35.7,
39.9 and 28.9 g pigment/l adsorbent, respectively (Table
1). Resin SP-850 exhibited the highest capacity, but the
capacity of XAD-16 was 10 – 15% lower than that of
HP-20 and SP-850.
Desorption of pigment from polymeric adsorbents is
also an important factor and in general the volume of
eluent required affects recovery yield and product pu-
Fig. 2. Effect of the addition of pigment on the production of
pigment in shake flask culture.
487
Table 1
Capacity of adsorbent and adsorption rate of pigment on different
adsorbent resins
Resin Capacity of adsorbent
(g pigment/l adsorbent)
HP-20 35.7
SP-850 39.9
XAD-16 28.9
rity in the purification process. In the case of resin
HP-20 and XAD-16, about 80% of pigment was des-
orbed in both cases when an eluent of eight resin
volumes was passed into the column (Fig. 3). The
desorption ability of SP-850 was 15–20% lower than
that of HP-20 and XAD-16 (Fig. 3). Even though resin
SP-850 has a good adsorption capacity, the desorption
ability was much lower than that of HP-20 and XAD-
16. Moreover, the adsorption capacity of HP-20 was
about 20% higher than that of XAD-16. Resin HP-20
was chosen for further studies, considering the adsorp-
tion capacity and desorption ability.
The adsorption rate of pigment for HP-20 was esti-
mated in order to determine the operating conditions
for extractive fermentation. The adsorption rate was
2.73 g pigment/l adsorbent/h, which was calculated
from the slope of Fig. 4. This value was about 40%
higher than that of the red pigment from Monascus sp.,
reported by Ju et al. [24], when XAD-7 was used as a
resin.
3.3. Effect of the addition time of adsorbent on
producti6ity
Ten percent sterilized HP-20 was added to culture
broth at 0, 5, 10 and 15 h after inoculation, and all
cultures were carried out for a further 20 h. Fig. 5
shows the effect of addition time of HP-20 on produc-
Fig. 3. Desorption of pigment from different adsorbent resins by
elution.
488 C.-H. Kim et al. / Process Biochemistry 35 (1999) 485–490
Fig. 4. Estimation of adsorption rate of pigment for HP-20 resin.
tivity. The addition of adsorbent caused the inhibition
of cell growth. The degree of growth inhibition was
dependent on the addition time of the resin. When resin
HP-20 was added immediately after inoculation, the cell
concentration was about 75% of the control without
resin. As the addition time of the resin was slower, the
cell concentration increased up to 95% of the control.
Each pigment concentration in broth and cell and the
total pigment production that adsorbed in the resin
were measured. The maximal production of total pig-
ment (5.85 g/l) was obtained when the resin was added
at 10 h, the total amount of pigment increasing to more
than 40% compared to that of the control.
Fig. 5 also shows that a considerable concentration
of casein was adsorbed to the resin. The earlier the
resin was added after inoculation, the more casein
adsorbed to the resin. The decrease of cell growth
according to the addition time might be due to the
adsorption of casein onto the resin, thus decreasing the
concentration of amino acids available to the cells.
Fig. 5. Effect of the addition time of HP-20 resin, after inoculation,
on cell growth and pigment production.
Fig. 6. Effect of HP-20 resin content on cell growth and pigment
production.
3.4. Effect of the amount of adsorbent on producti6ity
Fig. 6 shows the effect of amount of adsorbent on
cell growth and pigment production. Resin was added
at 10 h after inoculation. As the amount of resin was
increased from 5 to 20% (v/v), cell growth gradually
decreased. When 20% (v/v) HP-20 was added, 76% cell
growth was obtained compared to the culture without
resin. Maximal pigment production was obtained when
10% (v/v) HP20 was added and the amount of pigment
contained in resin was 5.92 g/l. More casein was ad-
sorbed to the resin when the amount of resin added was
increased (Fig. 6). In the case of 10% (v/v) HP-20 resin,
although the cell concentration was lower when com-
pared to 5% (v/v) HP-20, pigment production in-
creased, presumably by removal of the pigment
produced. This is caused by the reduction of product
inhibition, thus increasing the production of red
pigment.
3.5. Pigment production in a batch culture
In order to investigate the relationship between cell
growth and pigment production, two batch fermenta-
tions in a bioreactor were performed using 2 and 3%
(w/v) casein as substrate (Figs. 7 and 8). When the
initial casein concentration was increased from 20 to 30
g/l, the maximal cell concentration increased to 80%,
but total pigment production only increased by 5%.
About 65% of total pigment produced was present in
the supernatant of the broth and the remainder was
attached to the surface of the cells. Pigment production
increased to 18 h and total pigment concentration
reached 5.15 g/l, although cell growth was ceased at 15
h (Fig. 7). With 3% (w/v) casein (Fig. 8), pigment
production ceased after 18 h and total pigment produc-
tion reached 5.41 g/l. However, cell growth continued
until 24 h. The production of pigment did not increase
 C.-H. Kim et al. / Process Biochemistry 35 (1999) 485–490 489

Fig. 7. Batch culture of Serratia sp. KH-95 in a medium containing Fig. 8. Batch culture of Serratia sp. KH-95 in a medium containing
2.0 % casein, in a bioreactor. 3.0 % casein, in a bioreactor.

Table 2
Extractive fermentation in a bioreactor equipped with an extractive column packed with HP-20 adsorbent resin

Relative resin Growth (O.D.) Pigment concentration Pigment concentration in Amount of pigment Total amount of pigment
volume (%) in cell (g/l) broth (g/l) in resin (g) produceda (g/l)

None 24.8 1.6 3.7 – 5.3

5 22.3 1.55 2.73 4.65 5.83
10 20.5 1.45 2.5 8.9 6.92
20 16.3 0.48 1.5 12.5 6.14

a
(Total pigment produced in cell, broth and resin) } medium volume(3 l).

in proportion to cell growth. Figs. 7 and 8 also show
that the production of pigment ceased when the pig-
ment concentration in broth increased above 3.0 g/l,
irrespective of the casein concentration in the medium.
These results could be explained by the fact that the
accumulation of pigment inhibited its further
production.
3.6. Pigment production in extracti6e fermentation
In previous work [26], it was reported that much of
the pigment was attached to the surface of cells when
the pH of culture broth was maintained below 8.0.
However, when the pH of culture broth was increased
above 8.5, about 70% of total pigment was suspended
in the supernatant of the broth. Therefore, the pH of
the culture broth was not controlled so that the pig-
ment was suspended in the supernatant of the broth for
the extractive fermentation. In shake flask culture, 10%
(v/v) HP-20 showed the best results for pigment pro-
duction in extractive fermentation. There could be some
differences of cell growth and pigment production in a
bioreactor. Extractive fermentations were carried out in
bioreactors equipped with extractive columns packed
with 5, 10 and 20% (v/v) HP-20. The cultivation was
run batchwise for 12 h and then switched to extractive
fermentation. The data for the batch and extractive
fermentation are summarized in Table 2. Maximal cell
growth decreased inversely and in proportion to the
amount of HP-20 packed into the extractive column.
These results were similar to those of extractive fermen-
tation in the shake flask culture with HP-20. Maximal
production of pigment (6.92 g/l) was also obtained with
10% (v/v) of HP20. It was also found that the bioreac-
tor equipped with an extractive column packed with
HP-20 adsorbent resin showed higher productivity than
the conventional batch bioreactor, a 31% increment of
pigment production being obtained in extractive fer-
mentation, compared with the simple batch culture.
The pigment concentrations in broth were kept below
3.0 g/l when the extractive column was used, but the
pigment concentration in broth increased above 3.0 g/l
without the extractive column. From these results, it
was proved that production of pigment increased by the
elimination of pigment produced continuously in broth.
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