Carbohydrate Polymers 83 (2011) 852–857
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/carbpol
Characterization of extracellular polymeric substances produced by micro-algae
Avinash Mishra∗, Kumari Kavita, Bhavanath Jha
Discipline of Marine Biotechnology and Ecology, Central Salt and Marine Chemicals Research Institute (CSMCRI), Council of Scientific and Industrial Research (CSIR), G.B. Marg,
Bhavnagar 364021, Gujarat, India
a r t i c l ei n f o
Received 22 July 2010
Received in revised form 25 August 2010
Accepted 26 August 2010
Available online 21 September 2010
a b s t r a c t
Extracellular polymeric substances comprised of average molecule size 1264.354?m, exhibited char-
acteristic diffraction peaks at 6.025◦, 9.675◦, 22.775◦and 28.475◦with d-spacing 14.74755, 9.36297,
3.88747 and 3.11512˚A, respectively. EDX confirms the presence of sulphate (2.7%) and1H NMR reveals
uronic acid, primary amine, aromatic-compounds, halides, aliphatic alkyl and sulfides. EPSs were ther-
mostable upto 270◦C with CIxrd0.12 and CIDSC0.18. The dynamic viscosity is significantly high at pH
3.0 and decreases concomitantly with shear rate, confirming pseudoplastic rheological property. MALDI
TOF–TOF represents a series of masses in linear mode corresponding to mass of pentose and hexose with
ions. The positive ion reflector mode exhibited low mass peaks (m/z) corresponding to oligosaccharide
and higher peaks for polysaccharide consist of different ratio of pentose and hexose associated with ions.
EPSs allow further exploration of D. salina as potential EPSs producer and make it a promising candidate
for biotechnological and industrial exploitation.
© 2010 Elsevier Ltd. All rights reserved.
Extracellular polymeric substances (EPSs) are metabolic prod-
ucts, accumulating on the microbial cell surface, providing
protection to the cells by stabilizing membrane structure against
reserves during starvation. EPSs, a heterogeneous matrix of poly-
mers composed of polysaccharides, proteins, nucleic acids and
(phospho) lipids (McSwain, Irvine, Hausner, & Wilderer, 2005), are
renewable resources representing an important class of biotech-
nological importance. EPSs are observed in bacteria (Freitas et al.,
2009), cyanobacteria (Chi, Su, & Lu, 2007; Parikh & Madamwar,
2006) and marine microorganisms (Satpute, Banat, Dhakephalkar,
from micro-algae Dunaliella salina (Mishra & Jha, 2009) and medic-
inal mushroom Phellinus linteus (Zou, Sun, & Guo, 2006). The
polysaccharides of biological response modifiers can be isolated
Liu, Koon, & Fung, 2006).
Microbial exopolymers have multifarious industrial applica-
tions (Kumar, Mody, & Jha, 2007). These exopolymers are used
in food industries as thickeners and gelling agents to improve
food quality and texture. In pharmaceutical industry, exopoly-
∗Corresponding author. Tel.: +91 278 2567760x626; fax: +91 278 2567562.
E-mail address: firstname.lastname@example.org (A. Mishra).
mers can be used as hydrophilic matrix for controlled release of
drugs, development of bacterial vaccines and to enhance nonspe-
cific immunity. Improvement of water holding capacity of soil,
detoxification of heavy metals and radio nuclides contaminated
water and removal of solid matter from water reservoirs are pro-
posed uses for cyanobacterial EPSs (Bender & Phillips, 2004). In
recent years, interest in the exploitation of valuable EPSs has been
increasing for various industrial applications and the attention
towards exopolymer producing bacteria and cyanobacteria has
ing (Subramanian, Yan, Tyagi, & Surampalli, 2010). The increased
demand of natural polymers for various industrial applications in
past few years has led to sway interest in EPSs production by new
sources and marine microbes (micro-algae and marine microor-
ganism), already used as a source of products of high aggregated
value such as pigments, osmoprotectant, metabolites, fatty acids
and proteins, may also be exploited for the EPSs as biosurfactants
and/or bioemulsifiers (Mishra & Jha, 2009; Satpute et al., 2010).
Micro-algae D. salina is the only eukaryotic photosynthetic
organism can grow in the wide range of hypersaline environment
(Mishra, Mandoli, & Jha, 2008). The ability of cells to survive and
has received considerable attention (Chen & Jiang, 2009) which
make D. salina a perfect candidate for biotechnological exploration
(Tafreshi & Shariati, 2009), molecular farming (Barzegari et al.,
0144-8617/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
A. Mishra et al. / Carbohydrate Polymers 83 (2011) 852–857
in press) and novel industrial applications (Francavilla, Trotta, &
Luque, 2010). Micro-algal (D. salina) extracellular polymeric sub-
stances are a complex mixture of macromolecular polyelectrolytes
containing primary amine-group, halide-group, aliphatic alkyl-
group, aromatic compound, alkyl amine and/or cyclic amine with
polysaccharides (Mishra & Jha, 2009).
acterization of extracellular polymeric substances, produced by
micro-algae D. salina. Despite of ?-carotene, glycerol and other
metabolites, EPSs make Dunaliella more promising candidate to
play an important role in its biotechnological and industrial appli-
cation as the resource of biosurfactants and/or bioemulsifiers.
Emulsifiers from renewable resources have attracted attention and
the emulsifying activity of Dunaliella exopolymer was determined
by its strength in retaining the emulsion. The emulsifying activ-
ity of EPSs was 85.76% retention (Mishra & Jha, 2009), which is
comparatively stable compared to that of other EPSs, produced
by Vibrio harveyi (40% retention; Bramhachari & Dubey, 2006),
Halomonas (25–60% retention) and commercially available surfac-
tants viz. Tween 20 (65%), Tween 80 (60%) and Triton X-100 (65%)
in tetradecane (Mata et al., 2006). The use of biosurfactants and
bioemulsifiers represent a better alternative to overcome the tox-
icity of synthetic compounds as these are less toxic and mostly
makes Dunaliella exopolymer as an alternative of the commercially
available chemical surfactants/emulsifiers.
2. Material and methods
Extracellular polymeric substances (EPSs) were isolated and
purified from axenic culture of D. salina (Mishra et al., 2008).
The emulsification activity, major functional groups analysis using
FT-IR and monosaccharide composition were studied previously
(Mishra & Jha, 2009).
2.1. Energy dispersive X-ray spectroscopy and particle size
Elemental analysis of EPSs was done using energy dispersive
X-ray spectroscopy (EDS or EDX; Oxford Instruments, UK) and par-
Mastersizer 2000, Malvern Ltd., Worcestershire, UK).
2.2. X-ray diffraction (XRD) analysis
tometer (Philips X’pert MPD, The Netherlands) using PW3123/00
curved Ni-filtered CuK? (?=1.54056˚A) radiation generated at
40kV and 30mA with a liquid nitrogen cooled solid-state ger-
manium detector to study the nature of EPSs using slow scan in
2? =2–80◦. The irradiated length and specimen length were 10mm
with receiving slit size of 0.2mm at a 200mm goniometer radius.
sample was mounted on a quartz substrate and intensity peaks of
diffracted X-rays were continuously recorded with scan step time
value of ? was calculated with Bragg’s law (Eq. (1)).
2 sin ?
where ? is half of the scattering angle measured from the incident
Crystallinity index (CIxrd) was calculated from the area under
area i.e. ratio of areas of peaks of crystalline phases to the sum of
areas of crystalline peaks and amorphous profile (Eq. (2), Ricou,
Pinel, & Juhasz, 2005).
2.3. Thermal gravimetric analysis (TGA) and differential scanning
calorimetric (DSC) analysis
TG and DSC analysis of EPSs were carried on Mettler Toledo
and DSC were obtained in the range of 30–400 and 25–600◦C
respectively, under nitrogen atmosphere at rise of 10◦Cmin−1.
TG and DSC analysis were carried out by gradually raising the
temperature, plotting weight (percentage) and heat flow against
and crystallinity of the exopolymers (CIDSC) were calculated by fol-
lowing Eqs. (4) and (5) (Khanna & Kuhn, 1997) respectively.
where A is the frequency factor for the reaction, R is the universal
gas constant, T is the temperature (K), and k is the reaction rate
Ea= −RT ln
where ?H is the enthalpy of transition, K is the calorimetric con-
stant and A is area under the curve.
2.4. Rheological property analysis
milliQ) was carried out on a rheometer (RS1, Haake Instruments,
Karlsruhe, Germany) at different temperature, applied shear rate
and pH with 1ml sample volume. Measurements were carried out
to evaporation at higher temperature, the outer surface of the sam-
in triplicates and slippage of gel due to applied stress was carefully
avoided by selecting appropriate operation parameters.
2.5. Nuclear magnetic resonance (NMR) and scanning electron
The1H NMR spectra of EPSs were obtained in D2O with Bruker
5000rpm. Spectra were measured in the ppm range of 0–10. The
morphology of exopolymer has been observed under a scanning
electron microscope (SEM, LEO series VP1430, Germany) with an
accelerated voltage of 10–20kV.
2.6. MALDI TOF–TOF mass spectroscopy
The EPSs were dissolved in acetonitrile (5% w/v; 1mgml−1),
desalted and mixed with equal volume of matrix ?-Cyano-4-
hydroxycinnamic acid (10mgml−1). MALDI TOF–TOF analysis was
with an Nd-YAG (neodymium-doped yttrium aluminium garnet)
laser (355nm, 200Hz) operated in accelerated voltage (20kV).
Each spectrum was collected in two different modes, linear mode
A. Mishra et al. / Carbohydrate Polymers 83 (2011) 852–857
Elemental EDX microanalysis of EPSs obtained from D. salina. Data are expressed as
both weight and atomic percents.
Element Standard usedWeight%Atomic%
Number of iterations=6.
(150cm) and reflector mode (300cm) as an average of 1400 laser
shots per spectrum. Reproducibility of the spectrum was checked
from 6 spot-sets in each mode and the spectra were analyzed after
centroid and de-isotoping using Data explorer software (Applied
3. Results and discussion
3.1. Energy dispersive X-ray spectroscopy and particle size
Extracellular polymeric substances constituted of particle sizes
ranging from 49.492 (d0.1) to 1634.192 (d0.9)?m with an average
size of 1264.354?m (d0.5) and 0.0651344m2g−1specific surface
areas (Fig. S1). Energy dispersive X-ray spectroscopy (EDS or EDX)
is an analytical technique used for the elemental analysis of a
sample and it is one of the variants of X-ray fluorescence spec-
troscopy. EDX relies on the investigation of a sample through
interactions between electromagnetic radiation and matter, ana-
lyzing X-rays emitted by the matter in response to be hitted with
charged particles (Goldstein et al., 2003). Elemental quantitative
analysis done by EDX revealed the weight and atomic percent-
age of elements present in EPSs (Table 1 and Fig. S2). EDX analysis
confirms the presence of sulphate residue (2.7% w/w), a character-
istic of exopolysaccharides produced by eukaryotic algae (Gudin &
3.2. X-ray diffraction (XRD) analysis
X-ray powder diffraction (XRD) is a rapid analytical technique
The XRD profile of EPSs obtained from D. salina (Figs. 1 and S3)
exhibited the characteristic diffraction peaks at 6.025◦, 9.675◦,
of the EPSs so far and PXRD pattern predict that EPS is amor-
phous in nature with 0.12 crystallinity index. Crystalline parts give
sharp narrow diffraction peaks while amorphous component gives
a broad peak. It is difficult to interpret broad amorphous peaks of
several amorphous polymer in X-ray scattering profile (Shimazu,
Miyazaki, & Ikeda, 2000) and hence the ratio between these inten-
EPSs, obtained from D. salina, are found amorphous in nature with
CIxrd0.12. The 12% crystalline domains act as a reinforcing grid
and improve the performance over a wide range of temperature as
observed in TG and DSC analysis.
3.3. TG and DSC analysis
The applicability of polysaccharides is largely dependent on
its thermal and rheological behavior. Thermogravimetric analy-
Intensity (counts per second)
40 3530 2520 15 105
Degree 2θ (CuKα)
Fig. 1. Representative PXRD profile of EPSs isolated from Dunaliella salina.
sis is a simple analytical technique that measures the weight
loss of a material as a function of temperature. TGA showed that
the degradation in three steps (Parikh & Madamwar, 2006). Fifteen
percent of total EPSs weight loss from 30 to 124◦C was recorded
for phase 1 degradation, thereafter second phase of degradation
(54.6%) was observed with maximum loss at ≥240◦C (Fig. 2(a))
temperature. Phase 1 degradation may be due to water evapora-
tion during heating process while second phase of degradation is
attributable to thermal decomposition same as other study (Parikh
& Madamwar, 2006).
As temperature increases, an amorphous solid will become
less viscous and at a particular temperature the molecules obtain
enough freedom of motion to spontaneously arrange themselves
into a crystalline state, known as the crystallization temperature
(Dean, 1995). This transition from amorphous solid to crystalline
solid is an exothermic process and differential scanning calori-
metric analysis showed a significant thermal transition of EPSs
(Fig. 2(b)). DSC thermogram showed characteristic exothermic
transition of exopolymer with crystallization temperature (Tc)
95.88◦C (onset temperature 59.56◦C) and 475.46mJ latent energy
for crystallization followed by an endothermic transition of melt-
ing. The melting temperature (Tm) of EPSs was found 270.45◦C
(onset temperature 253.52◦C) with 193.06mJ latent energy for
melting. The activation energy (for nth order reaction) of exother-
mic transition was 60.79±0.35kJmol−1while for endothermic
transition, it was found quite higher 292.62±1.56kJmol−1. The
activation energy of EPS isolated from marine cyanobacterium
Cyanothece sp. was found to be 149.6kcalmol−1(Shah, Ray, Garg,
& Madamwar, 2000) while 450–490kJmol−1for Oscillatoria sp.
and Nostoc carneum (Parikh & Madamwar, 2006). EPSs exhib-
ited cross linking characteristics at high temperature ranging
was observed up to high temperature (270◦C). In contrast with
EPSs from DSC thermogram and it may be because of uncertainties
in placing baseline for area integration (Khanna & Kuhn, 1997).
3.4. Rheological property analysis
A. Mishra et al. / Carbohydrate Polymers 83 (2011) 852–857
Fig. 2. Thermogravimetric (a) and DSC (b) thermogram of EPSs obtained from
Dunaliella salina at heating rate of 10◦C.
shear rate, thereafter decline gradually (Fig. 3(a)). In contrast to pH
3.0 (700 shear rate s−1), EPSs showed significant viscosity limit up
to 950 (s−1) shear rate at pH 7.0. Viscosity of EPSs is significantly
high at low pH (3.0) compare to neutral pH (7.0), independent to
pseudoplastic characteristic like halophilic bacterial EPSs, isolated
EPSs at pH 3.0 is because it has been reported that there is an
increase in viscosity at low cation concentration (Parker, Schram,
polymer configuration and initial stage of chain aggregation with
gation in solution is attained, a rearrangement of aggregates in
more condensed structures (precipitates) is likely to occur (Parikh
& Madamwar, 2006; Parker et al., 1996).
Viscosity of EPSs decreased concurrently with temperature at
pH 7.0 and constant shear rate 239 (s−1), however at 52–54◦C, a
54◦C, a sudden increase in viscosity was found at 55◦C leading to
decline thereafter. An increase in viscosity was recorded for EPSs at
pH 3.0 (shear rate 100s−1) with temperature up to 56◦C, after that
extracted from Vibrio alginolyticus, decreased throughout a range
of increasing shear rate, and with an increase in temperature the
1000900 800700600500 400 3002001000
110 1009080 7060 5040 3020100
y (η) mPa s
η) mPa s
1000 900800 700 600
Shear rate (
100 90 80 706050 4030 20 100
Shear Rate 239, pH 7.0
y (η) mPa s
(η) mPa s
Shear Rate 90, pH 7.0
60 50 403020 10
Shear Rate 100, pH 3.0
Fig. 3. Effect of (a) shear rate and (b) temperature on viscosity of EPSs isolated from
viscosity also dropped markedly (Muralidharan & Jayachandran,
2003). They also demonstrated that EPS was unstable at high tem-
decreased with increasing shear rate for exopolysaccharide (agar)
isolated from seaweeds Gracilaria acerosa (Prasad et al., 2007). It
was observed that EPSs obtained from halophilic species were low
in viscosity and pseudoplastic in the behavior (Mata et al., 2008).
3.5. NMR and SEM
The1H NMR spectra of EPSs, obtained from Dunaliella culture,
tional groups (Fig. 4). The ppm 5.1–5.4 and 4.8–4.9 are attributed
to ?- and ?-anomeric carbon of hexose or pentose, respectively. A
spectrum at 4.0ppm is assigned to hydrogen next to the functional
–OH group while uronic acid is observed at 2.3ppm. A stretch-
ing of N–H group is observed at 1.3ppm while alkyl halide group
at 3.1ppm. Spectra at 2.0 and 2.7ppm correspond to the func-
tional group acetyl amine of hexose or pentose sugar moiety. Apart
from polysaccharide functional groups, others functional groups
related to alkene, alkyne, aliphatic, aromatic compound, CH–O,
CH–N and S–H were also observed (Fig. S4). The NMR spectrum
of EPSs confirms the presence of polysaccharides, uronic acid, pri-
mary amine group, aromatic compound, halide group, aliphatic
alkyl group and sulfides. The FT-IR-spectra confirmed the pres-
ence of primary amine-group, aromatic compound, halide-group,
A. Mishra et al. / Carbohydrate Polymers 83 (2011) 852–857
Fig. 4. Proton nuclear magnetic resonance (1H NMR) spectra of EPSs extracted from
aliphatic alkyl-group and polysaccharides, as a resultant presence
of alkyl amine and/or cyclic amine with polysaccharides in EPSs
of D. salina was depicted (Mishra & Jha, 2009). The presence of
acetyl groups renders the EPSs somewhat hydrophobic, which
might also contribute to their emulsifying capacity (Mata et al.,
2006) as observed previously (Mishra & Jha, 2009). The presence
of uronic acids and sulfates confers an overall negative charge and
acidic property to the polymer (Jain, Raghukumar, Tharanathan,
& Bhosle, 2005). Acidic polysaccharides are common in marine
an overall negative charge and acidic property to the exopolymer
and sulfated EPSs are of biotechnological importance. It has been
clear from SEM images (Fig. S5) that exopolymer is compact with
small pore size distribution. Compactness with porosity was also
observed in the EPS extracted from Azotobacter sp. (Gauri, Mandal,
Mondal, Dey, & Pati, 2009).
3.6. MALDI TOF–TOF analysis
Matrix assisted laser desorption–ionization mass spectroscopy
is a convenient method for rapid and sensitive structural analy-
sis of oligosaccharides (Harvey, 1999). The MALDI TOF–TOF mass
spectroscopy was optimized for EPSs and fragmentation peaks are
detected for positive ion mode only. No peaks were observed for
negative ion linear or reflector mode. Linear mode was found suit-
able for the oligosaccharide while positive ion reflector mode for
the polysaccharide analysis. MALDI TOF–TOF mass spectroscopy
of EPSs (Fig. S6) represents a series of masses m/z 177.8892,
to de-protonated hexose sugar and hexose sugar with matrix (size
47.2878 as observed in reflector mode). Besides this, masses m/z
300.2427 and 384.0726 are also observed in positive ion linear mid
(with Mg ion) respectively. The positive ion reflector mode exhib-
ited low mass peak m/z 361.7294 and 662.2907 corresponding to
tively and higher mass peaks at m/z 1101.222, 1651.635, 1939.387
and 2272.089, corresponding to polysaccharides consisting of dif-
ferent ratio of pentose and hexsose sugar associated with ions like
Mg, S, Na, P, etc. present in EPSs, as detected by EDX. It is the first
MALDI TOF–TOF analysis of EPSs so far, whereas MALDI TOF mass
spectroscopy for the bacterial EPS was reported recently (Gauri et
The presence of pentose sugar, which is usually absent in
polysaccharides of prokaryotic origin and quite unique among
cyanobacteria (Parikh & Madamwar, 2006), is remarkable. Pre-
viously, we have detected four constituent monosaccharides
viz. galactose, glucose, xylose and fructose in different com-
binations and ratio by HPLC analysis, after complete acidic
hydrolysis of EPSs (Mishra & Jha, 2009). Monosaccharides detected
in EPSs of Dunaliella can be categorized as aldohexoses (glu-
cose and galactose), ketohexose (fructose) and pentose (xylose)
sugars. Though mass spectroscopy cannot distinguish diastere-
omers but it indicates the number of sugar moiety in an
Extracellular polymeric substances of D. salina can play an
important role in its biotechnological and industrial application.
In this study, EPSs are characterized by advanced analytical meth-
ods and its rheological property is also studied. XRD profiling and
MALDI TOF–TOF analysis for EPSs is the first report so far. XRD pro-
file and interplanar spacing (d-spacing) is the basic characteristic
of a polymer useful to compare or study the nature of EPSs iso-
lated from different sources in future. The EPSs are found thermo
stable up to high temperature (270◦C) which enables it for addi-
tional characteristic for further applications. The exopolymer is
exceptionally attractive for industrial application because of high
viscosity of its solution at acidic pH. The presence of uronic acids
and sulfates confers an overall negative charge and acidic property
to the exopolymer and sulfated EPSs are also of biotechnological
importance. EPSs may allow further exploration of D. salina and
on the biotechnological importance and ecological significance of
the extracellular polymeric substances of D. salina deserve further
Analytical Science Discipline of the institute is duly acknowl-
edged for helping in analysis. The financial support received from
CSIR (NWP-0018), Govt. of India is thankfully acknowledged.
Appendix A. Supplementary data
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