JOURNAL OF BIOSCIENCE AND BIOENGINEERING © 2006, The Society for Biotechnology, Japan

Vol. 101, No. 2, 157–161. 2006

DOI: 10.1263/jbb.101.157

Purification and Characterization of Prodigiosin Produced

by Integrated Bioreactor from Serratia sp. KH-95
Min-Jung Song,1 Jungdon Bae,2,3 Dae-Sil Lee,3 Chang-Ho Kim,4
Jun-Seok Kim,5 Seung-Wook Kim,1 and Suk-In Hong1*
Department of Chemical and Biological Engineering, Korea University, Seoul 136-701, Korea,1 School of Life Sciences
and Biotechnology, Korea University, Seoul 136-701, Korea,2 Genome Research Center, Korea Research Institute
of Bioscience and Biotechnology, Daejeon 305-333, Korea,3 Bio M&D, Incheon 400-201, Korea,4 and
Department of Chemical Engineering, Kyonggi University, Kyonggi-do 443-760, Korea5

Received 20 June 2005/Accepted 8 November 2005

To date, prodigiosin and its analogues which have been shown to have anticancer, cytotoxic and
immunosuppressive activities have been isolated from Serratia, Pseudomonas and Streptomyces
species, and chemically synthesized. In a previous study, the red pigment content in Serratia sp.
KH-95 was enhanced using a casein-enriched medium. Recently, an integrated bioreactor with an
internal adsorbent has been developed to increase the production yield and allow easy recovery of
the pigment. Thus, this study focused on both purifying and identifying a single red pigment from
several pigments attached to the adsorbent in an integrated bioreactor. The red pigment was ex-
tracted directly from the internal adsorbent using acidified methanol and phase separation. Sub-
sequently, it was purified by silica gel chromatography and high performance liquid chromato-
graph (HPLC). As a result, pure prodigiosin was identified by structural studies as a pigment.
Also, this downstream procedure that uses the integrated bioreactor can be applied to the direct
production and purification of other prodigiosin analogues and hydrophobic alkaloid compounds
from several microorganisms.

[Key words: integrated bioreactor, internal adsorbent, pigment, prodigiosin, Serratia]

Prodigiosin (5-[(3-methoxy-5-pyrrol-2-ylidene-pyrrol-2-
ylidene)methyl]-2-methyl-3-pentyl-1H-pyrrole) is a red pig-
ment isolated from a few species, such as Serratia, Pseu-
domonas and Streptomyces (1), and is a typical alkaloid com-
pound produced as a secondary metabolite. It has a unique
structure consisting of three pyrrole rings and a pyrrolyl-
pyrromethene skeleton with a C-4 methoxy group (2). Fur-
thermore, several pharmaceutically relevant prodigiosins
(PGs) such as undecylprodigiosin, metacycloprodigiosin,
roseophilin and nonylprodigiosin, in addition to prodigio-
sin, are thought to have potential for antibacterial, antimale-
rial, anticancer, cytotoxic and immunosuppressive activities
(3–7).
The gram-negative bacterium Serratia sp. KH-95 show
similar physiological characteristics to Serratia marcescens
(8), and its red PGs are produced in significant quantities on
a casein-enriched medium (9, 10). However, the production
of red PGs is limited by product inhibition in batch culture,
as shown in other secondary metabolites. Accordingly, to
overcome this limitation, different types of bioreactor have
been designed for the simultaneous production and recovery
of prodigiosin-like pigments (9). Interestingly, when the in-
ternal adsorbent bioreactor (IAB) fabricated by our research
group (11) was used on a laboratory scale, the concentration
* Corresponding author. e-mail: sihong@korea.ac.kr
phone: +82-2-3290-3294 fax: +82-2-926-6102
of red PGs significantly increased and they completely at-
tached to the internal adsorbent as a result of the pigments’
hydrophobicity. It was therefore demonstrated that the pro-
duction and recovery of red PGs using the IAB is a simpler
and more convenient procedure than those using other bio-
reactor types (11). However, the structure of the red PGs
produced by Serratia sp. KH-95 has only been partially
identified to date (10).
Herein, we evaluated a purification methodology of pure
red pigments using the IAB, and identified the major red
pigment, prodigiosin, among several PGs. In addition, we
investigated the effects of casein, cells and prodigiosin on
the adsorbent in the IAB to improve the pigment’s binding
capacity.
MATERIALS AND METHODS
Cell culture and pigment production in an IAB Serratia
sp. KH-95 (KFCC-10970) isolated from soil in Korea was incu-
bated in a preculture medium containing 10 g of peptone, 10 g of
yeast extract, 2 g of K2HPO4 and 10 g of dextrose per liter at 28°C
and shaken at 200 rpm for 12 h in a shaking incubator. The casein-
enriched medium used for the production of red PGs consisted of
20 g of casein, 1.7 g of K2HPO4, 1 g of MgSO4 ⋅7H2O and 1 g of
NaCl per liter. The IAB consisted of two separate compartments to
carry out simultaneous fermentation and adsorption (Fig. 1). The
hydrophobic adsorbent Diaion HP-20 resin polymerized with poly-
styrene and divinylbenzene was purchased from Sigma Chemical

157
158 SONG ET AL.
FIG. 1. Schematic of IAB for production of red PGs from Serratia
sp. KH-95.
(St. Louis, MO, USA). A large open stainless steel grid with a pore
size of 0.5 cm was used as the support of the internal adsorbent.
The grid of the support was fabricated as a hollow cylinder and
welded to a solid stainless steel with eight holes in the bottom. The
inside and outside diameters were 4 cm and 8.5 cm, respectively,
and the height of the support was 5.5 cm. The internal adsorbent
assembly was completed by rolling it in a 50-mesh stainless steel
filter. Half of the adsorbent was packed by the polymeric resin for
expanded bed adsorption and aeration. The internal adsorbent was
fixed on the three baffles in a bioreactor using screws. Fermenta-
tion was operated aerobically in a 2.5-l IAB with a 1 l working vol-
ume and 100 ml of internal adsorbent for 24 h at 28°C. The initial
pH of the medium was adjusted to 7.0 prior to sterilization. Details
of the fermentations are found elsewhere (11). For the adsorption
experiment in the bioreactor, the resin was soaked in 95% (v/v)
propan-2-ol for 12 h. The solvent was then removed by washing
with sufficient distilled water.
Purification of prodigiosin from internal adsorbent
Prodigiosin, among the red PGs attached to the internal adsorbent
in the IAB, was purified as follows: After fermentation, the broth
was drained from the IAB, and then 1 l of a 95% (v/v) acidified
ethanol (pH 3.0) solution was added to the IAB. Attached pig-
ments were extracted by circular desorption using an impeller in
the IAB at 200 rpm for 5 h, and then concentrated and centrifuged.
It was then isolated using phase separation with water and chloro-
form. All red PGs were placed in a down-filled chloroform phase
and then concentrated by evaporation. Prodigiosin was separated
by silica gel column chromatography (2.5 ×30 cm; Kieselgel 60;
Merck, Darmstadt, Germany). It was eluted with a mixture of
n-hexane:ethyl acetate (2 : 1 [v/v]; Rf value of the red pigment by
TLC was 0.13). The concentrated pigment was separated by de-
velopment in a 95 :5 (v/v) mixture of chloroform : methanol using
TLC. Single red prodigiosin (Rf value: 0.43) was then collected
and dissolved in acetone. The pigment (Rf value: 0.83, 120 mg) was
repurified by further TLC (chloroform : methanol : diethyl ether =
6:2: 2), and finally purified by preparative HPLC (Waters Delta
Prep 4000; Waters, Milford, MA, USA) with a Bondapack C18 col-
umn (2.5 × 10 cm). It was isocratically eluted with a mixture of
methanol: water (7 : 3, v/v) that was adjusted to pH 3.0 with 0.1 N
HCl at a flow rate of 20 ml/min.
J. BIOSCI. BIOENG.,
Adsorption of cells, casein and prodigiosin onto adsorbent
A test of cell adsorption onto the HP-20 resin was performed in a
250-ml Erlenmeyer flask at 150 rpm and 28°C. After the cells were
cultivated in a preculture medium for 12 h, they were harvested by
centrifugation at 12,000 rpm for 10 min. The cells (OD600 was 3.0)
were washed twice with a sterilized phosphate buffer (pH 7.0).
Also, pigment-formatted cells (OD600 was 3.0) in a casein-enriched
medium were prepared according to the above method. Sterile
casein (5%, w/v) in distilled water and purified prodigiosin (1 mg
of pigment in 0.1 ml of methanol + 9.9 ml distilled water) were
used for the adsorption studies. A 10% (v/v) HP-20 resin was
added to the three types of solution at different pHs and tempera-
tures.
Analytical methods Casein concentration was determined
using the modified Biuret method (12). The concentration of the
red PGs produced was estimated by measuring the absorbance at
535 nm using an UVIKON 923 Double Beam UV-visible spectro-
photometer (Kontron instruments) in acidified methanol (0.01 N
HCl 4 ml + methanol 96 ml) (13). The molecular mass of the pig-
ment purified was determined using a VG Biotech PLATFORM
electrospray-ionization mass spectrometer (ESI-MS; Fisons, Altrin-
cham, UK). After the pigment sample was dissolved in CDCl3, the
1
H- and 13C-NMR spectra were recorded on a Bruker DRX 300
spectrometer (300 MHz). The chemical shifts were referenced to
an internal TMS (trimethylsilyl) signal. A FT-IR spectrum of the
pigment was recorded with a Perkin-Elmer Spectrum GX1 FT-IR
spectrometer.
The NMR chemical shifts of prodigiosin were as follows: 1H-
NMR (CDCl3, 300 MHz, ppm) δ 0.93 (3H, t, H11″), 1.31 (2H, m,
H9″), 1.34 (2H, m, H10″), 1.59 (2H, m, H8″), 2.44 (2H, t, H7″),
2.58 (3H, s, H6″), 4.04 (3H, s, OCH3), 6.11 (1H, d, H3′), 6.39 (1H,
m, H3), 6.71 (1H, brd, H3″), 6.95 (1H, m, H4), 6.99 (1H, brs, H6′),
7.26 (1H, m, H2), 12.57 (1H, brs, H1), 12.71 (1H, brs, H1′); 13C-
NMR (CDCl3, 75 MHz, ppm) δ 12.69 (C6″), 14.27 (C11″), 22.74
(C10″), 25.58 (C7″), 30.05 (C8″), 31.68 (C9″), 58.95 (OCH3),
93.07 (C3′), 111.97 (C3), 116.29 (C6′), 117.26 (C4), 120.97 (C5′),
122.52 (C5), 125.42 (C2″), 127.21 (C2), 128.67 (C3″), 128.77
(C4″), 147.29 (C5″), 147.98 (C2′), 166.02 (C4′). FT-IR absorption
in KBr was at νmax 3105, 2950, 2928, 2867, 1723, 1644, 1602,
1547, 1508, 1451, 1353, 1252, 1125, 1057, 964, 841 cm–1.
RESULTS AND DISCUSSION
Purification of prodigiosin from an IAB As reported
previously (11), approximately 14.3 g/l red PGs were ob-
tained from the fermentation of the KH-95 strain using the
IAB, and the internal adsorbent tightly bound to the pig-
ments. The circular extraction of the adsorbed pigments was
performed directly using acidified ethanol in the IAB. The
phase separation using chloroform and water was effective
in separating nonpolar pigments from polar compounds such
as nucleotides. However, other nonpolar compounds such as
fatty acids and cell membranes were not removed during
this step. Therefore, several chromatographic procedures
were required to obtain a pure pigment. After development
using TLC, seven red pigment bands were observed at dif-
ferent Rf values. Among these spots, a single red pigment
exhibiting approximately a 15% band intensity, which was
believed to be prodigiosin, was isolated. As shown in the
HPLC profile in Fig. 2A, approximately 120 mg of the sin-
gle pigment from Serratia sp. KH-95 was obtained as a
single compound by HPLC with approximately 95% purity.
However, the overall purification yield of the pigment dur-
VOL. 101, 2006 PRODIGIOSIN PURIFICATION BY INTEGRATED BIOREACTOR 159

FIG. 2. (A) HPLC profile of purified prodigiosin from Serratia sp. KH-95. The purity is 95% as determined by HPLC. (B) UV-visible absorp-
tion spectra of red pigment as function of pH in methanol from Serratia sp. KH-95.

FIG. 3. Electrospray-ionization mass spectrum of red pigment purified from Serratia sp. KH-95. The molecular mass of the red pigment was
determined to be 323.0 Da.

ing the purification was quite low compared with the pro-
ductivity of the red PGs. It has been strongly suggested that
the purification of prodigiosin is affected by the instability
of this pigment in light (14), the formation of several red
PGs in Serratia sp. KH-95, and the complex formation of
this pigment with other compounds in the cell envelope (15).
Pigment identification Prodigiosin, as reported from
S. marcescens (16), had a maximum absorption spectrum at
537–538 nm in 95% ethanol, dependent on pH values. In
this study, the maximum absorption spectrum of prodigiosin
purified from Serratia sp. KH-95 showed similar character-
istics, as shown in Fig. 2B. At pH 2.2 (acid salt; adjusted
using 0.01 N HCl in methanol), it was red and it showed a
maximum absorption at 536 nm, which is identical to that
of prodigiosin hydrochloride (16). Under neutral condition
(pH 7.3), its absorption intensity was decreased and it
changed to pink. However, it was orange and its absorption
spectrum shifted to 470 nm and exhibited a broad range
(400–500 nm) in alkaline condition (pH 9.0). It has been
suggested that the nitrogens of the three conjugated pyrrole
rings are protonated by NaOH (17). The molecular mass of
the sample pigment on ESI-MS was 323.0 Da as m/z 324.0
(M + H)+, as shown in Fig. 3, which corresponds to that of
prodigiosin (C20H25N3O) (5). FT-IR absorption in KBr for
the red pigment was dominated by very strong bands at
2928 cm–1 (aromatic CH), and 1602 cm–1 (aromatic C=C).
Prodigiosin exhibits similar absorptions in CHCl3 at 1630
and 1602 cm–1, except that the relative intensities are re-
versed and the first band is possibly a pyrrolenine (C=N).
The fingerprint region for the red pigment was character-
ized by medium-intensity bands at νmax 1723 (C=O), 1547,
1125 (C–O and C–N), 964 and 817 cm–1. A weak and broad
160 SONG ET AL.
FIG. 4. (A) 1H-NMR and (B) 13C-NMR spectra of prodigiosin in
CDCl3. The signal of all the protons and carbons in the prodigiosin
structure is noted in each spectrum.
absorption for NH was evident at νmax 3105 and 3150 cm–1.
These indicate that this pigment’s pattern is similar to that
of prodigiosin. The positions of each of the proton and
carbon in the prodigiosin structure are indicated on each
1
H-NMR and 13C-NMR spectra, shown in Fig. 4. In the 1H-
NMR spectrum, a chemical shift of the methoxy group in
prodigiosin exhibited δ 4.04 ppm as a singlet, which is quite
different from the 1H-NMR data of a PG derivative with no
methoxy group reported previously (10). In addition, the
chemical shifts (in CDCl3) of NH protons in pyrrole rings
were δ 12.57 (H1) and 12.71 (H1″) ppm. It is therefore con-
cluded that this pigment isolated from the KH-95 strain is
prodigiosin. Also, we think that its purity in several reports
is very low on account of its instability (5, 18–20).
Effect of cells, casein, and pigment on adsorbent In
a previous study (8–11), casein was shown to be a critical
nitrogen and carbon source for the formation of red PGs
from the KH-95 strain. However, when 2% (w/v) casein
was replaced with casitone (pancreatic digest of casein) or
casamino acids (acid hydrolyzed casein) in batch culture,
the maximum cell growth was only slightly decreased
(Table 1), but the red PGs were hardly produced. Thus, it is
possible that red pigment production is closely associated
with the activity of casein hydrolases in the cell. As shown
in Fig. 5A, casein did not adsorb onto the HP-20 resin at any
TABLE 1. Effect of nitrogen and carbon source
on cell growth and production of red PGs
Maximum cell Red PG
Nitrogen and
growth production
carbon source
(g/l) (g/l)
Casein 3.88 4.28
Casitone 3.40 0.08
Casamino acids 3.05 0.08
J. BIOSCI. BIOENG.,
FIG. 5. Adsorption characteristics of casein, cells and prodigiosin
on HP-20 resin. (A) Casein adsorptions at different temperatures, (B)
cell adsorptions in a shaken flask at 28°C and 200 rpm, (C) pigment
adsorptions at different temperatures and (D) prodigiosin adsorptions
at different pH values. C0, Initial concentration of each component; C,
adsorption concentration of each component.
temperature. Indeed, the adsorption of casein might have lit-
tle effect on the adsorption capacity of the resin. Therefore,
we considered that pigment productivity was not decreased
by substrate adsorption during the in situ recovery of the
pigment in the IAB. However, when both casein and the
HP-20 adsorbent were autoclaved, a very small quantity of
red PGs was produced by the inactivated resin. This is
caused by the casein aggregates positioned on the surface
area of the nonpolar polymeric resin at high temperatures.
Moreover, the cells did not adsorb onto the HP-20 resin
(Fig. 5B). However, the pigment-formatted cells adsorbed in
small quantities onto the HP-20 resin, as shown in Fig. 5C.
This suggests that the hydrophobicity of cells is increased
by the formation of red PGs. On the other hand, the adsorp-
tion of prodigiosin was dependent on pH (Fig. 5D). The ini-
tial adsorption rate of prodigiosin increased in the neutral-
pH region and decreased in the alkaline-pH region. Never-
theless, the adsorption capacity of prodigiosin was similar
over time. Consequently, cell adsorption in the IAB is
caused by the hydrophobic PGs bound to the cell. Accord-
ingly, pH is an important factor that affects the initial ad-
sorption of the red PGs. The addition of HP-20 resin to the
cell culture favored the recovery of the hydrophobic pig-
ments produced by bacterial cells.
Conclusions An IAB was introduced to obtain prodi-
giosin of a high purity from Serratia sp. KH-95, and puri-
fication methods, such as extraction and chromatography,
were performed. It is considered that the cells produced sev-
eral red PGs, as evidenced by the TLC results. Overall, this
process can be used for the large-scale production of pro-
digiosin and other alkaloid hydrophobic components from
bacterial cells. The precise spectral data of prodigiosin pre-
VOL. 101, 2006
sented in this report will be helpful in determining its thera-
peutic utility. In addition, other red PGs need to be clarified.
ACKNOWLEDGMENTS
This work was financially supported by an Interdisciplinary
Research Grant from KSEF under project no. R01-2002-000-
00591-0. It was accomplished during Professor Hong’s sabbatical
leave.
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