Bioresource Technology 97 (2006) 18221827

Partial characterization of extracellular polysaccharides from

cyanobacteria

Amit Parikh, Datta Madamwar
Postgraduate Department of Biosciences, Sardar Patel University, Vallabh Vidyanagar 388120, Gujarat, India

Received 7 March 2005; received in revised form 31 August 2005; accepted 3 September 2005
Available online 26 October 2005

Abstract

Four cyanobacterial strains, Cyanothece sp., Oscillatoria sp., Nostoc sp. and Nostoc carneum were studied for physico-chemical charac-
terization of extracellular polysaccharide (EPS) secreted during the controlled growth condition. Hydrolyzed EPSs showed the composi-
tional involvement of four sugar moieties viz. mannose, glucose, xylose and ribose in varying combinations. Infrared spectra of EPSs
showed a speciWc absorbance of OH stretching at 34483400 cm1, asymmetricalsymmetrical CH stretching at 2924 and 2854 cm1
and a bending vibration of CH at 14001380 cm1. Absorbance at 1259 and 1140 cm1 with Cyanothece sp. EPS, indicated the presence
of sulfur containing functional group. Thermal gravimetric analysis and diVerential scanning calorimetric analysis conWrmed the polysac-
charides thermal stability as high as around 250 C. In the presence of 0.1 M NaCl aqueous solution, the intrinsic viscosity of polysaccha-
rides from Oscillatoria sp. and Nostoc sp. decreased 1.6 fold, whereas, 35 fold reduction in intrinsic viscosity was observed with
commercially available guar and xanthan gum.
2005 Elsevier Ltd. All rights reserved.

Keywords: Cyanobacteria; Extracellular polysaccharide; Thermal degradation; Viscosity; Xylose

1. Introduction Polysaccharides are characterized by an extreme struc-

Polysaccharides are renewable resources representing an
important class of polymeric materials of biotechnological
interest, oVering a wide variety of potentially useful prod-
ucts to mankind. Extracellular polysaccharide (EPS) of
microbial origin with a novel functionality, reproducible
physico-chemical properties, stable cost and supply,
became a better alternative to polysaccharides of plant and
algal origin (Selbmann et al., 2002). Microorganisms are
better suited than macroalgae or higher plants, since they
exhibit high growth rate and are more amenable to manip-
ulation of conditions for enhancing growth and/or EPS
production.
* cyanobacterial EPS (Moreno et al., 2000; Bender and
Corresponding author. Tel.: +91 2692 226863; fax: +91 2692 226865/
236475.
E-mail address: datta_madamwar@yahoo.com (D. Madamwar).
tural diversity; as a result they play very diverse roles in
nature. Numerous bacterial polysaccharides are potentially
available, known to be involved in pathogenesis, symbiosis,
bioWlm formation, protection from phagocytic predation
and stress resistance in microorganisms; but relatively few
have been commercially established (Sutherland, 2001;
Vanhaverbeke et al., 2003). Among these microbes, cyano-
bacteria have been known since long as a potential EPS
producer. The presence of proteins, uronic acids, pyruvic
acid, and O-methyl, O-acetyl and sulfate groups emphasizes
the complex nature of cyanobacterial EPS. Various appli-
cations among others such as improvement of water hold-
ing capacity of soil, detoxiWcation of heavy metals and
radionuclides contaminated water and removal of solid
matter from water reservoirs have been proposed for
Phillips, 2004; Freire-Nordi et al., 2005). Despite their ubi-
quitous distribution, a great lack of knowledge exists

0960-8524/$ - see front matter 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2005.09.008
 A. Parikh, D. Madamwar / Bioresource Technology 97 (2006) 18221827
concerning the factors that regulate the EPS synthesis and
the physico-chemical properties of cyanobacterial EPS
(Shah et al., 2000; De Philippis et al., 2001). This prompted
us to study EPS production from cyanobacteria. Though,
structural elucidation of the polysaccharide is a part of gly-
comics (Hirabayashi, 2003), their commercial applicability
is largely depends on to the thermal and viscometric behav-
ior under various environmental conditions.
In our study we isolated three cyanobacterial strains
Oscillatoria sp., Nostoc sp. and Nostoc carneum releasing
polysaccharide during the normal growth process. The
present investigation is aimed at characterizing the physi-
cal and chemical properties of the EPSs produced by these
cyanobacteria to ascertain their industrial exploration. A
comparison of the results was also made with xanthan
and guar gum.
2. Methods
2.1. Microorganisms and EPS production
Oscillatoria sp., Nostoc sp. and N. carneum were isolated
from the soil contaminated with wastewater released by
textile dye manufacturing industries at Vatva Industrial
Estate, Gujarat, India, and maintained in 100 ml Erlen-
meyer Xask containing 50 ml of BG11 medium (Allen,
1968). The cultures were made free of diatoms and green
algae by adding 0.16 mM of cyclohexamide Wnal concentra-
tion for 24 h with incubation in light. A combination of
ampicillin-streptomycin was also used at a Wnal concentra-
tion of 40 g/ml and 100 g/ml respectively to make it free
from bacterial Xora. A marine cyanobacterium Cyanothece
sp. ATCC 51142 was obtained as a gift culture from Prof.
D.O. Hall, Kings College, London, which was maintained
in ASN III medium (Rippka et al., 1979). All the cultures
were incubated at 27 C with light/dark cycle of 12/12 h.
The light intensity was kept to 3 k lux. Axenity of cultures
were checked every fortnight by streaking the cultures on
BG 11/ASN III agar plates. All the chemicals used were of
analytical reagent grade.
EPS production was carried out in 2.0 l Erlenmeyer
Xask containing 600 ml of BG 11/ASN III medium. The
medium was inoculated with 150 ml of inoculum to make
Wnal chlorophyll a concentration to 2.0 mg l1. The cul-
ture was shaken twice daily to provide suYcient mass
transfer of the nutrients or the metabolites secreted by the
cells.
Extraction of chlorophyll a from the cell pellet (culture
centrifuged at 4000g for 10 min) was achieved by cold
methanol (90%) for 10 h under dark at 4 C. The extract
was measured at 665 nm (Tandeau De Marsac and Houm-
ard, 1988) by spectrophotometer (Spectronic 20D, Milton
Roy, New York, USA). Supernatant was used to monitor
EPS production by estimating extracellular soluble carbo-
hydrate content by phenol-sulfuric acid method (Dubois
et al., 1956) using glucose as standard.
1823
2.2. Separation and puriWcation of EPS
Thirty day old cultures were used for the extraction of
EPS. Cells were separated from the growth medium by cen-
trifugation at 15,000g at 15 C for 40 min. The supernatant
was concentrated to one forth volume on magnetic stirrer
at 60 C for 10 h. The polysaccharide from this concen-
trated supernatant was precipitated by gradually adding an
equal volume of cold acetone to the supernatant and kept
at 4 C overnight. The precipitated EPS was redissolved in
Milli Q water (Millipore Co., Milford, MA, USA); this pro-
cess of precipitation and redissolution was repeated. Crude
EPS was further puriWed by dialysis against Milli Q water
for 20 h at 4 C and freeze dried (DURA DRY, FTS Sys-
tems INC., Stone Ridge, NY, USA).
2.3. Sugar analysis and infrared (FT-IR) spectroscopy
Total carbohydrate and protein content of the dried EPS
Xakes was measured by Duboiss and Lowrys method
respectively. The EPSs were hydrolyzed with 2 M triXuoro-
acetic acid (TFA) at 110 C for 90 min and freeze-dried.
Dried hydrolysate was redissolved in Milli Q water and
Wltered through Supor membrane (0.22 m) and was used
for the identiWcation and quantiWcation of the monosac-
charides by HPLC, equipped with RI detector using zorbax
carbohydrate analysis column (Agilent 1100 series, USA)
at 30 C and eluted with the solvent system of aceto-
nitrile:water (75:25, by volume) at 1.4 ml min1 Xow rate.
The FT-IR spectra were measured using Perkin Elmer,
Spectrum GX 2000, Boston, USA and Nicolet 400D, Cali-
fornia, USA instruments. The polysaccharide samples were
pressed into KBr pellets at sample: KBr ratio 1:100. The
spectra were recorded in transmittance mode over the wave
range of 4000400 cm1.
2.4. Thermal and viscometry analysis of EPS
Thermal gravimetric analysis (TGA) and diVerential
scanning calorimetry (DSC) analysis of EPSs and commer-
cial gums was done on Universal V2.6D TA Instruments,
New Castle, USA. Thermograms for TGA and DSC were
obtained in the range of 25550 C and 50450 C respec-
tively, under nitrogen atmosphere at rise of 10 C min1.
Viscosity measurement was performed at 25 C in
Ubbelohde capillary viscometer. A solution of 0.1% (w/v)
EPSs, xanthan and guar gum were prepared in each of
deionized water, 0.1 M NaCl and 0.1 M CaCl2 solution and
the viscosity were measured. The dilutions were made
directly in the viscometer with respective solutions.
3. Results and discussion
All the cultures showing the ability to produce EPS was
released into the medium and it could be separated easily
after precipitation. InXuence of diVerent nutritional factors
on the growth and EPS production by Cyanothece sp.
1824 A. Parikh, D. Madamwar / Bioresource Technology 97 (2006) 18221827

ATCC 51142 was evaluated by Shah et al. (1999) and

observed that higher the growth, the better the EPS produc-
tion, indicating the growth linked EPS production. Identi-
cal behavior of growth and EPS production was also
noticed with Nostoc sp. and Oscillatoria sp. (see Electronic
Annex 1).

3.1. Monosaccharide composition of EPSs

Various organic solvents like methanol, ethyl alcohol,

isopropyl alcohol, acetone and dimethyl sulfoxide were
assessed to precipitate the EPS. Acetone (1 volume) was
best suited for precipitation, giving maximum yield with
compact and Xoating clump of crude EPS, which was fur-
ther saturated overnight at 4 C. Most of the cyanobacterial
EPSs are composed of at least one uronic acid, several neu-
tral sugars (ranging from 2 to 10) in combination with pro-
tein molecules (Otero and Vincenzini, 2003). Total sugar
and protein content of the dried EPS Xakes was calculated
to be 560870 mg g1 and 18.340.09 mg g1 of dried EPS
respectively (Table 1). Upon total acidic hydrolysis of the
obtained EPSs, only four constituent monosaccharides viz.
glucose, mannose, xylose and ribose in varying combina-
tions and ratio were detected by HPLC analysis (Table 1)
(see Electronic Annex 2). This was in contrast with very
heterogeneous composition of other cyanobacterial EPSs
studied by Hu et al. (2003). They have reported the presence
of 612 monosaccharides in EPS, produced by four Wla-
mentous cyanobacteria. The presence of pentoses, which
are usually absent in other polysaccharides of prokaryotic
origin is found to be unique among cyanobacteria (Otero
and Vincenzini, 2003). In the present study xylose was
found to be the common one amongst all. In addition, 6.5
7% ribose found with EPSs of Cyanothece sp. and Oscillato-
ria sp. is rare and distinct among polysaccharide producers.
3.2. Infrared spectroscopy of dried EPSs
Variation in stretching and bending modes of vibration
with single functional group is normally coupled with the
vibration of adjacent group, as well as with the number of
substitution/s taking place on the molecule itself. This leads
to the shifting or overlapping of the peaks of two or more
functional group in the same region of IR spectrum (Silver-
stein and Webster, 1998; de Sousa, 2004). Comparative IR
spectrums of EPS obtained from diVerent cyanobacteria
Fig. 1. Comparative FT-IR spectra of EPSs: (a) Nostoc carneum,
(b) Cyanothece sp.spectra were taken on Nicolet 400D.
are shown in Fig. 1, which shows more complex pattern of
peaks from 2950 to 1200 cm1. The peak centered around
34003448 cm1 is assigned to OH stretching frequency,
weak absorptions near 2924 and 2854 cm1 are attributed
to asymmetrical and symmetrical CH stretching vibra-
tions of aliphatic CH2, weak absorption 14001380 cm1 is
of CH bending of aliphatic CH2. A medium-broad CO
stretching is also observed at 10401074 cm1, CH defor-
mation also contributes at this frequency. Strong absorp-
tion at 1636 cm1 might be due to the stretching vibration
of carboxylate group, which raised a doubt on absence of
uronic acid in hydrolyzed EPS. Degradation of uronic acid
during acid hydrolysis might be one of the reasons that
eliminates its detection during HPLC (De Philippis et al.,
1998). Above absorbance are the most common character-
istics of polysaccharides (Stuart, 2004). However, due to the
merging of several peaks between 800 and 920 cm1 ham-
pered the assessment of the possible linkages taken place
between two monosaccharide molecules. Besides this, IR
spectrum of Cyanothece sp. showed an additional absorp-

Table 1
Total carbohydrate, protein and monosaccharide composition of cyanobacterial EPSs
Organisms Total protein Total carbohydrate Comparative sugar composition of puriWed EPSsa
(mg g1) (mg g1)
Ribose (%) Xylose (%) Glucose (%) Mannose (%)
Oscillatoria sp. 34.4 1.4 700 14 7.0 0.9 19.3 1.0 73.8 2.2
Nostoc sp. 40.1 0.1 685 10 31.7 0.7 68.3 0.5
N. carneum 27.2 0.1 560 10 33.2 1.8 66.8 0.4
Cyanothece sp. 18.3 0.3 870 11 6.6 0.8 16.7 0.7 76.7 1.8
a
Thirty days old cultures were used for EPS extraction and puriWcation. Hydrolyzed EPSs were used for the identiWcation of monosaccharides.
 A. Parikh, D. Madamwar / Bioresource Technology 97 (2006) 18221827 1825

100
2.3

90
1.7

80 1.1

Exo up
70 0.5

-0.1
60
Weight (%)

-0.7
50

Heat Flow (W g-1)

-1.3
40
-1.9
30
-2.5
20
-3.1

10 -3.7

0 -4.3
0.00 50.00 100.00 150.00 200.00 250.00 300.00 350.00 400.00 450.00 500.00 550.00

Temperature (oC)
Oscillatoria sp. Nostoc sp. N. carneum Cyanothece sp. Xanthan Gum Guar Gum
Oscillatoria sp. Nostoc sp. N. carneum Cyanothece sp. Xanthan Gum Guar Gum

Fig. 2. Comparative TG/DSC thermograms of diVerent polysaccharides, at heating rate of 10 C min1: blocks% weight loss in TG analysis, linesDSC
curves.

tion at 1259 and 1140 cm1, which could be attributed to
the presence of sulfate group as SBO and COS (Silver-
stein and Webster, 1998; Stuart, 2004). Presence of sulfur
was further conWrmed by the elemental analysis of EPS.
3.3. Thermal and viscometric behavior of polysaccharides
Besides chemical properties, applicability of polysaccha-
ride is largely dependent on its thermal and rheological
behavior (Marinho-Soriano and Bourret, 2005). Fig. 2
shows the comparative TGA & DSC curve of commercial
gums and the EPSs produced by Cyanothece sp., Oscillato-
ria sp., Nostoc sp. and N. carneum. All the EPSs showed
that the degradation takes place by three well-diVerentiated
steps. Twelve to fourteen percent weight loss from 25
110 C was recorded as the Wrst phase of degradation.
Above this temperature the weight remains constant until a
second degradation process of depolymerization starts at
around 240 C that continues till 300 C for Cyanothece sp.
and 340 C for the rest three EPSs with a major weight loss
of about 41%. Third phase of degradation from 350 to
490 C for Oscillatoria sp. and 350540 C for two Nostoc
strains is also observed, where around 21% weight is lost,
while Cyanothece sp. showed the sequential degradation,
which last beyond 550 C. Slowly, a progressive carbonized
structure is formed as the temperature increased further.
Above 500 C only 15% of the solid residue remains. Analo-
gous degradation pattern is also noticed with xanthan and
guar gum (Table 2), second phase of decomposition (240
360 C) remains the major weight loss producer with 40%

Table 2
Sequential decomposition and thermal kinetics of polysaccharides
Polysaccharide TGA degradationa DSC kineticsa
Phase I Phase II Phase III Melting enthalpy (Hm), Heat of reaction, Activation energy,
J g1 [Endotherm (Tm), C] J g1 [Exotherm, C] kJ mol1
Weight loss, % Weight loss, % Weight loss, %
(Temp., C) (Temp., C) (Temp., C)
Xanthan gum 16.8 (25120) 39.9 (240340) 16.1 (350550) 151.4 (123.31) 93.2 (298.6) 159.9
Guar gum 13.8 (25110) 51.6 (250330) 12.5 (340550) 486.1 (121.67) 192.9 (307.4) 85.2
Oscillatoria sp. 12.1 (25100) 44.7 (240340) 21.3, (350490) 229 (155.48) 73.3 (295.6) 453.9
N. carneum 13.9 (25110) 44.5 (240330) 23.1 (340540) 101.7 (143.50) 129.3 (278.58) 492.8
Nostoc sp. 11.9 (25100) 48.1 (240330) 22.8 (340530) 271.9 (142.19) 72.8 (286.1) 707
Cyanothece sp. 13.1 (25100) 41.6 (240300) 31.1 (310550) First521.6 (151.38)
Second(240)
a
Thermograms were obtained under nitrogen atmosphere at the rise of 10 C min1.
1826 A. Parikh, D. Madamwar / Bioresource Technology 97 (2006) 18221827

and 52% respectively. Remaining decay took place with the Table 3
constant weight reduction as a third phase, which lasted Intrinsic viscosity of the polysaccharides in diVerent aqueous solutions
beyond 550 C. High solid residue content (27%) after Polysaccharide Intrinsic viscositya []
500 C with xanthan gum might be due to its complex Deionized water 0.1 M NaCl solution
molecular conWguration and the presence of cations (Na+, Xanthan gum 55.2 11.0
K+, Ca2+), which acted like a bridge between diVerent Nostoc sp. 18.4 11.7
charged sugar moieties (Shah et al., 2000). Polysaccharide Oscillatoria sp. 12.1 9.8
degradation proceeds in four distinct phases: (1) desorption N. carneum 6.9 5.5
Guar gum 8.5 2.8
of physically absorbed water; (2) removal of structural
a
water (dehydration reactions); (3) depolymerization ac- Viscosity was measured at 25 C on Ubbelohde capillary viscometer.
companied by the rupture of CO and CC bonds in the
ring units resulting in the evolution of CO, CO2 and H2O;
(8.5 dl g1) and the EPSs from diVerent cyanobacterial
(4) formation of polynuclear aromatic and graphitic carbon
strains (Table 3). However, in presence of 0.1 M NaCl
structures (Zamora et al., 2002; Fried, 2000), which stands
viscosity reduced drastically (11 dl g1) with xanthan gum,
true with the present Wndings. Calorimetric analysis of the
while less than 1.6 fold reduction was observed with cyano-
EPSs (Fig. 2) showed an appreciable thermal transition
bacterial EPSs. Acetyl, pyruvic acid and glucuronic acid are
as melting endotherm (Tm) between 140 and 155 C (peak
some of the main components of the side chain in xanthan
value) with Oscillatoria sp. and two Nostoc strains, which
gum that enhanced the monovalent (Na+) ions mediated
is also associated with a melting enthalpy (Hm) of 101
chain cross linking of the polysaccharide molecule. Stability
271 J g1. All polysaccharides were analyzed except Cyano-
of the polymer viscosity in salinity is one of the desired
thece sp., thermal decomposition takes place at around
properties. Constant viscosity of EPSs in presence and
290 C, which supports the decomposition results obtained
absence of NaCl adds advantage to its possible application.
by the TGA analysis. The reason behind the two endother-
mic peaks and the absence of exotherm with Cyanothece sp.
4. Conclusions
EPS is not clear, but might be due to the conformational
changes in sugar molecules at diVerent temperatures. (Shah
Polysaccharide release, though ubiquitous, could be very
et al., 2000). Besides this, activation energy of these decom-
speciWc and diversiWed by evolution in regard to its role and
position processes is highly variable as found with EPSs
chemical composition. Interesting chemical and viscometric
from 453.9 to 707 kJ mol1, a much higher value as com-
properties and thermal stability of EPS may allow further
pared to 159.9 and 85.2 kJ mol1 for xanthan and guar gum
exploration of these organisms as potential polysaccharide
respectively (Table 2).
producers. Further studies need to be conducted to illus-
To gain insight about EPS viscometric behavior; speciWc
trate the detailed rheological and structural conWrmations
viscosity (sp) and reduced viscosity (red) were studied in
of EPSs.
comparison with the viscosity of xanthan and guar gum. In
all the aqueous polysaccharide solutions, reduced viscosity
Acknowledgements
decreased with decreasing EPS concentration. SigniWcant
decline in reduced viscosity is observed with 0.1 M NaCl
This work was supported by the University Grants Com-
(see Electronic Annex 3). In the presence of 0.1 M CaCl2, all
mission, New Delhi. We are thankful to Dr. N.P. Talpada,
the polysaccharides got precipitated, as the cross linking of
Dr. B. Narola and Mr. V. Pandya, Department of Chemis-
polysaccharide chains by bond formation between calcium
try, Sardar Patel University, Vallabh Vidyanagar for their
ions and the carboxylates, ring oxygen and bridging oxygen
special assistance.
of the adjacent chains took place, forming Ca2+-polysac-
charide aggregates (Yang et al., 1999). It has been well
Appendix A. Supplementary data
reported that there is an increase in viscosity at low cation
concentration, which reXects the change in polymer conWg-
Supplementary data associated with this article can be
uration and initial stage of chain aggregation with large
found, in the online version, at doi:10.1016/j.biortech.
amount of trapped solvent. After the maximum compatible
2005.09.008.
aggregation in solution is reached, a rearrangement of the
aggregates in more condensed structures (precipitates) is
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