Biosci. Biotechnol. Biochem.

, 68 (5), 1146–1148, 2004

Note
Isolation of an Exopolysaccharide-producing Bacterium,
Sphingomonas sp. CS101, Which Forms an Unusual Type of Sphingan
Eun-Jung S EO,1; * Sang-Ho Y OO,2; * Ko-Woon O H,1 Jaeho C HA,3
Hyeon Gyu L EE,4 and Cheon-Seok P ARK1; y
1
Department of Food Science and Technology, Kyunghee University, Yongin 449-701, Korea
2
Department of Food Science and Technology, Sejong University, Seoul 143-747, Korea
3
Department of Microbiology, Pusan National University, Busan 609-735, Korea
4
Department of Food Science and Nutrition, Hanyang University, Seoul 133-791, Korea

Received November 26, 2003; Accepted February 16, 2004

An exopolysaccharide-producing Gram negative bac-
terium was isolated and determined to be a Sphingo-
monas sp. (CS101). A sugar composition analysis of an
exopolysaccharide indicated that the Sphingomonas sp.
CS101 secreted an exopolysaccharide composed of
glucose, mannose, fucose, and rhamnose in the ratio of
2.1:1.1:1.0:0.1, suggesting that this exoplysaccharide is
an unusual type of sphingan family. The mean molecu-
lar weight of the exopolysaccharide was determined to
be 4:2  105 Da by size exclusion chromatography
coupled with multi-angle laser-light scattering (SEC/
MALLS) analysis. An exopolysaccharide was produced
up to 17 g/l (pH 7; 30  C) with the optimal medium
condition over 4 days of cultivation.
Key words: Sphingomonas; exopolysaccharide; sphin-
gan
Recently, there is increasing interest in the genus
Sphingomonas, a type of Gram negative bacteria, due to
their great potential for biotechnological applications in
bioremediation, wastewater treatment of xenobiotic
pollutants, and the production of useful exopolysacchar-
ides (EPS).1,2) Among these, EPS has a wide range of
applicability in the cosmetic and pharmaceutical indus-
tries, food technology, oil recovery, and so on.3–5)
Several Sphingomonas strains synthesize a group of
structurally related EPS referred to as sphingans,
including gellan, welan, rhamsan, and S-88. Sphingans
possess a similar linear structure in which an identical
tetrasaccharide (D-glucose, D-glucuronic acid, D-glu-
cose, and L-rhamnose/L-mannose) is repeated as back-
bone.6) The physical properties of sphingans vary
considerably depending on the nature and location of
the side chains and in the presence or absence of certain
acyl groups.7) Generally, the aqueous solution of
y
To whom correspondence should be addressed. Tel: +82-31-201-2631; Fax: +82-31-204-8116; E-mail: cspark@khu.ac.kr
*These two authors contributed equally to this work.
Abbreviations: EPS, exopolysaccharides; MALLS, multi-angle laser-light scattering; TFA, trifluoroacetic acid; HPSEC, high-performance size
exclusion chromatography
sphingan is highly viscous and shows high thermal
stability. Gellan, one of the best known sphingans, has
been used as an agar substitute for microbiological
media and as a gelling agent in the food industry.6,8) We
are particularly interested in isolating an EPS-producing
microorganism that can be used in the biotechnological
industry.
We have isolated 11 bacterial strains (CS101

CS111) forming slimy, viscous, intensively yellow-
pigmented colonies from various bacteria isolated from
soil in South Korea. The strains were incubated in
100 ml of EPS-production (EP) medium containing 20 g
of glucose, 1 g of yeast extract, 1 g of casamino acid,
10 g of Na2 HPO4 , 3 g of KH2 PO4 , 1 g of K2 SO4 , 1 g of

NaCl, 0.2 g of MgSO4 7H2 O, 0.01 g of CaCl2 , and

0.001 g of FeSO4 7H2 O in 1 liter of deionized water.
The cells were incubated at 30  C for 7 days on a rotary
shaking incubator at 250 rpm. The culture broth was
diluted 10-fold with distilled water and then centrifuged
to separate EPS from the cells.9) The crude EPS was
obtained by adding 3 volumes of isopropanol into the
supernatant and keeping it overnight at 4  C. The
precipitate formed during overnight incubation was
recovered by centrifugation at 12,000 g at 4  C. The
purified EPS was washed with 70% ethanol several
times and dried in the oven. After measuring the weights
of dried EPS, one strain (CS101) was selected as the
highest EPS-producer.
The bacterium selected for production of abundant
amounts of EPS was Gram-negative, and the straight
rod-shaped cells that formed intensively viscous, yel-
low-colored colonies. Genotypic identification of isolate
was done by analysis of the 16S rRNA gene with a pair
of universal primers (16S-F, 50 -GAGTTTGATCCTGG-
CTCAG-30 and 16S-R, 50 -AGAAAGGAGGTGATCCA-
GCC-30 ). PCR was performed by Taq DNA polymerase
 Isolation of an Exopolysaccharide-producing Bacterium 1147

using the following protocol: initial denaturation at

94  C for 5 min, followed by 30 cycles of denaturation at
94  C for 1 min, annealing at 55  C for 1 min, and an
extension at 72  C for 1 min with an additional extension
at 72  C for 5 min in the final cycle. The amplified PCR
product was purified with a QIAEX II gel extraction kit
(Qiagen, Hilden, Germany) and sequenced directly
using an Applied Biosystems 373A DNA sequencer
(Perkin-Elmer, Boston, Massachusetts, U.S.A.). The 16S
rRNA sequence was confirmed and analyzed using the
Blast search program (NCBI, Bethesda, Maryland,
U.S.A.). The 16S rRNA sequence of the isolated EPS-
producing strain was most closely related to Sphingo-
monas capsulate (98%) and Sphingomonas subterra-
nean (97%). The identified 16S rRNA sequence of the Fig. 1. TLC Analysis of the Carbohydrate Compositions of EPS
Produced by Sphingomonas sp. CS101.
isolated EPS-producing strain has been deposited in the (A) standards and (B) hydrolyzed EPS. The standards are
GenBank database under accession number AY522503. galacturonic acid (1), glucuronic acid (2), galactose (3), glucose
In addition, phylogenic analysis by the API 20NE (4), arabinose (5), xylose (6), fucose (7), and rhamnose (8). The
identification system (bioMerieux Inc., Durham, North purified EPS was hydrolyzed with 4 M trifluoroacetic acid (TFA) at
Carolina, U.S.A.) revealed that this bacterium belongs to 100  C for 2 h.
Sphingomonas paucimobilis with 91.8% identity. Com-
bining these results, the isolated EPS-producing bacte-
rium was designated as Sphingomonas sp. CS101.
The sugar components of EPS produced by Sphingo-
monas sp. CS101 were examined by thin layer chroma-
tography (TLC) and high performance anion exchange
chromatography (HPAEC).10) The purified EPS was
hydrolyzed with 4 M trifluoroacetic acid (TFA) at 100  C
for 2 h. TFA was then removed by evaporation under N2
gas. The TLC plate was activated in the oven at 110  C
for 30 min. Prepared samples were spotted on the plate
and then developed in a TLC chamber with the solvent
n-propanol:water:triethylamine:30% NH3 = 80:20:0.2:4
(v/v/v/v) at room temperature. The plate was dried and
developed by dipping it rapidly into the solvent with 3 g
of N-(1-naphthyl)-ethylendiamine and 50 ml of concen- Fig. 2. HPAEC Analysis of the Carbohydrate Compositions of EPS
Produced by Sphingomonas sp. CS101.
trated H2 SO4 per liter of methanol. Then the plate was The peaks correspond to fucose (1), rhamnose (2), glucose (3),
dried and placed in the oven at 110  C for 10 min. TLC and mannose (4).
analysis revealed that EPS was likely to be composed of
glucose, arabinose, fucose, and a trivial amount of
rhamnose (Fig. 1). HPAEC analysis was done to confirm
the TLC result. A Dionex Series 4500 HPLC system several Sphingomonas strains, share the same carbohy-
equipped with an analytical column for carbohydrate drate-backbone structure (–L-rhamnose/mannose–D-glu-
detection (CarboPac PA1, Dionex Co., Sunnyvale, cose–D-glucuronic acid–D-glucose–).3) Interestingly, a
California, U.S.A.) and a pulsed electrochemical detec- D-glucuronic acid, which is one of the components of
tor were used. Separation of the various monosacchar- sphingans, was not detected. Instead, D-fucose, which is
ides was achieved isocratically using eluent A (15 mM not normally found in any other sphingans, was present
NaOH) followed by application of a linear gradient from in the EPS produced by Sphingomonas sp. CS101. To
20 up to 40 min using the eluent B containing 600 mM of our knowledge, glucuronic acid is absent only in the
sodium acetate and 150 mM of NaOH. As shown in exopolysaccharide produced by Sphingomonas pauci-
Fig. 2, four monosaccharides appeared in the HPAE mobilis P4, in which the trisacchrides structure (D-
chromatogram. Each peak was designated glucose, glucose–D-glucose–L-rhamnose) is repeated.1) The
mannose, fucose, or rhamnose by comparison with weight-average molecular weight (Mw ) of the EPS
standards. It was found that mannose and arabinose sample was determined using 50 mM of NaNO3 as the
showed the same mobility in the TLC but not in the eluent (0.7 ml/min) for a high-performance size exclu-
HPAEC analysis. The ratios of glucose, mannose, sion chromatography (HPSEC) linked with the MALLS
fucose, and rhamnose were 2.1, 1.1, 1.0, and 0.1, (Multi-angle laser light scattering) detector system
respectively. Sphingans, a group of EPS produced by (Wyatt Technology, Santa Babara, California, U.S.A.).
1148
The molecular weight of the EPS was approximately
4:2  105 Da. This result strongly suggests that EPS
secreted by Sphingomonas sp. CS101 is a new type of
EPS within the sphingan family.
The effect of various factors such as carbon and
nitrogen sources, the carbon/nitrogen ratio, pH, and
temperature on EPS production was investigated to find
the optimal condition for EPS production by Sphingo-
monas sp. CS101. Precultured Sphingomonas sp. CS101
cells were inoculated into the EP media modified by
substituting carbon or nitrogen sources. The cells at each
condition were grown for 5 d at 30  C, then the levels of
dry cell weight and EPS were determined. Maximum
EPS production was obtained at 5% sucrose and 0.25%
yeast extract and 0.25% casamino acid as carbon and
nitrogen sources, respectively. The C/N ratio for
optimal EPS production was 10/1. Although the pH
and temperature did not significantly affect biomass
production over the ranges of pH 4–8 and 25–40  C,
respectively, the production of EPS was drastically
influenced by these two parameters. The highest EPS
production was obtained at pH 7 and 30  C. The batch
fermentation with the optimized medium yielded EPS
production of up to 17.2 g/l in 4-d of cultivation
(Fig. 3).
In conclusion, we have isolated an EPS-producing
bacterium that shows high productivity. The isolated
strain was identified as Sphingomonas sp. by 16S rRNA
analysis, and the EPS composition analysis suggested
that EPS produced by Sphingomonas sp. CS101 is a new
type of EPS within the sphingan family.
20
18
exopolysaccharides(g/l)
16
14
12
10
8
6
4 8)
2
0 0
0 20 40 60
hour
Fig. 3. Production EPS during Batch Culture of Sphingomonas sp.
CS101 in a Medium Containing 50 g of Sucrose, 2.5 g of Yeast
Extract, 2.5 g of Casamino Acid, 10 g of Na2 HPO4 , 3 g of KH2 PO4 ,

1 g of K2 SO4 , 1 g of NaCl, 0.2 g of MgSO4 7H2 O, 0.01 g of CaCl2 ,

and 0.001 g of FeSO4 7H2 O in 1 Liter of Deionized Water.
Temperature and pH were maintained at 30  C and pH 7 with the
aeration rate of 1 vvm.
E.-J. SEO et al.
Acknowledgment
The authors thank Hoa Chau (ERRC/USDA, Wynd-
moor, Pennsylvania, U.S.A.) for technical assistance
HPSEC analysis. This work was supported by Grant
No. R01-2002-000-00207-0 from the Basic Research
Program of the Korean Science and Engineering
Foundation.
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