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Colloids and Surfaces B: Biointerfaces 103 (2013) 223230

Contents lists available at SciVerse ScienceDirect

Colloids and Surfaces B: Biointerfaces

journal homepage: www.elsevier.com/locate/colsurfb

Bacterial extracellular polymeric substances and their effect on settlement of

zoospore of Ulva fasciata
Ravindra Pal Singh, Mahendra K. Shukla, Avinash Mishra, C.R.K. Reddy , Bhavanath Jha
Discipline of Marine Biotechnology and Ecology, CSIR-Central Salt and Marine Chemicals Research Institute, Bhavnagar 364 002, India

a r t i c l e

i n f o

Article history:
Received 15 September 2012
Received in revised form 13 October 2012
Accepted 22 October 2012
Available online xxx
Keywords:
Biolm
EPS
MALDI TOFTOF MS
Ulva fasciata
Zoospores

a b s t r a c t
The extracellular polymeric substances (EPSs) secreted by Bacillus exus (GU592213) were estimated to
have the molecular weight of approximately 1528 and 33,686 kDa with the elemental composition of Na,
P, Mg, C, O, Cl and S. The 1 H NMR and FT-IR analysis of EPS conrmed the presence of different aliphatic and
aromatic groups. The EPS was amorphous in nature with an average particle size of 13.969 m (d 0.5) and
roughness of 193 nm. The GCMS analysis has revealed different monosaccharides such as fucose, ribose,
xylose, galactose, mannose and glucose. Oligo and polysaccharides were detected with MALDI TOFTOF
MS. The bacterial EPS for the rst time tested as a natural substratum for settle of zoospores of Ulva fasciata
by incubating for various durations ranging from 2 h to 48 h. The zoospore settlement on EPS coated cover
slips progressively increased with incubation time in axenic cultures over controls. The EPS, thus
investigated in this study was found to facilitate the primary settlement of spores that play crucial role in recruitment of macroalgal communities in coastal environment including intertidal
regions.
2012 Elsevier B.V. All rights reserved.

1. Introduction
Extracellular polymeric substances (EPS) can be referred to as a
network of organic compounds (carbohydrate, proteins and nucleic
acids) bound with cation and/or anion, and can be either loosely
attached to the cell surface or tightly associated with the cells of
producers [1]. EPS helps to hold the marine aggregates and keep
their networks intact that eventually promote aggregate formation and subsequently leads to the biolms formation [2]. Microbial
biolm forming communities provide primary biotic-substrata
for settlement of different fouling prokaryotic and eukaryotic
organisms. The normal morphological growth of many foliaceous
green macroalgae has been reported to be controlled by the
macroalgal associated bacteria [3,4]. Biolm formation is a process of succession following in a sequential pattern. Initially, single
cells attach to the surface and differentiate into complex of closed
microcolonies separated by a network of open water channels [5].
These aggregates are centers of high microbial activity and are presumed to play a signicant role in the carbon cycle [6]. EPS alter
the surface properties of the bacteria themselves to either promote or prevent initial attachment to surface or cell aggregation [7].
Recently, it has been reported that EPS secreted by marine microbes

Corresponding author. Tel.: +91 278 256 5801/256 3805x6140;

fax: +91 278 256 6970/256 7562.
E-mail address: crk@csmcri.org (C.R.K. Reddy).
0927-7765/$ see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.colsurfb.2012.10.037

has enhanced the growth of marine eukaryotic ora [1]. In addition,

the EPS produced by marine bacteria has been utilized as ingredient in food, pharmaceutical and petrochemical industry. It can
also be used as bioremediation agents in environment management
system [8,9].
Although the research on seaweed-bacterial association has
been recently on raise, the ecological signicance of EPS secreted
by epi- and endophytic bacterial communities has not been well
investigated. Further, the bacterial species involved in this process
in many cases have also not been identied. A few studies dealing
with bacterial biolms have reported that they enhanced the settlement of zoospore but the role of EPS in settlement of zoospore
is not understood [3]. In case of fouling organisms such as phytoplankton, benthic algae and larvae, the source material must nd a
suitable surface to settle and adhere to a substratum in a reasonable
time frame, otherwise they will perish without completing their
life cycle processes. Spore settlement is one of the most important
developmental phases in the life cycle of fouling marine organisms
[10]. The previous studies on zoospore colonization on bacterial
biolms have reported that the settlement takes place in three steps
i.e. contact, temporary and irreversible adhesion [11]. Commonly,
the key factor determining the strength of zoospore settlement process in different fouling microorganisms is a sticky material having
either permanent or temporary adhesive properties [12]. Furthermore, zoospores of the marine macroalgae respond to a number of
physicochemical characteristics of substratum including wettability, charge, surface chemistry and topography [12,13].

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Ulva fasciata is one of the most prevalent green alga with worldwide distribution. The propagation of this alga is mainly through
tiny motile asexual zoospores. These spores often settle gregariously in order to form groups or colonies of cells. The biochemical
studies investigated by several researchers across the world have
shown its possible utilization as a promising food supplement [14]
and recently have been projected for its possible utilization in biofuel production [15].
In this study, the EPS produced by an epiphytic marine
bacterium Bacillus exus was studied from the context of physicochemical properties employing a variety of analytical tools and
techniques and, thereafter investigated its possible effect on settlement of zoospore of U. fasciata.

desiccators for overnight. Dried samples were mixed with pyridine and acetic anhydride (1:1, v/v) and reuxed at 100 C. On
appearance of yellow color, mixture was added to ice water and
extracted with ethyl acetate. The extracted solution was washed
with Milli-Q, Na2 CO3 , and saturated CuSO4 to remove excess
acetic anhydride and pyridine. Thereafter, anhydrous Na2 SO4 was
added to remove water from organic layer. Organic layer was
separated and kept in vacuum desiccators for overnight. For gas
chromatography mass spectroscopy (GCMS) analysis, a pinch of
powder was dissolved in 15 ml of dichloromethane, ltered with
Whatmann lter paper and injected into GCMS (Shimadzu, QP2010).
2.3. Chemical properties of EPS

2. Materials and methods

2.1. Growth curve and EPS production
B. exus (NCBI accession number GU592213) an endophytic bacterium, was isolated from U. lactuca [4]. EPS production medium
was prepared according to Singh et al. [9]. Sub samples of 5 ml
aliquots were drawn at every 6 h interval and reading was taken
at 600 nm for growth curve. Three replicates were performed
for each test. The supernatant was obtained in stationary phase
by centrifuging bacterial culture at 15,000 g, 4 C for 30 min.
Thereafter, supernatants were ltered with 0.45 and 0.25 m pore
size (Millipore lters, Bangalore, India) and the resultant bacterial cell pellet were freeze-dried and weighed. Precipitation of
supernatant to obtain EPS and then, obtained EPS was washed
following the protocol of Singh et al. [9]. Thereafter, EPS was collected, dried and dialyzed at 4 C for 24 h against Milli-Q water
for desalting. The desalted EPS was recollected by centrifuging at
15,000 g, 4 C for 30 min and lyophilized. The lyophilized EPS was
stored in pure form for subsequent chemical and physical analysis.
2.2. Determination of molecular mass and their mass analysis
For molecular weight determination, 50 g (2% EPS in Milli-Q,
w/w) EPS was applied to gel permeation chromatography
(GPC, Water Allaince, model 2695) calibrated with ultrahydrogel columns 120 and 500 at 40 C and elution was monitored
by a refractive index detector (2414). The molecular weight of
EPS was calculated with standard Dextran (molecular weight;
5200668,000 kDa) procured from PSS, USA.
The desalted EPS was dissolved in acetonitrile (5%, w/v) and
mixed with equal volume of matrix -cyano-4-hydroxycinnamic
acid (5 mg ml1 ) for matrix assisted laser desorptionionization
(MALDI)-time-of-ight (TOF)TOF mass spectroscopy analysis. It
was performed on an Applied Biosystem 4800 MALDI TOFTOF analyzer and conditions were maintained as previously described [8].
The spectrum obtained from 12 spot-sets in positive ion mode was
taken for mass analysis of EPS. Centroids and de-isotoping spectra
were analyzed using Data explorer software (Applied Biosystem,
USA). Further, monosaccharide contents of EPS were estimated by
alditol-acetate method [9,16]. In brief, the puried EPS (100 mg)
was hydrolyzed in 5 ml of 2 N H2 SO4 in a sealed vial (Teon-lined
cap) at 100 C for 6 h. Thereafter, the acidic solution was neutralized by adding of BaCO3 and sample was concentrated upto
45 ml followed by addition of NaBH4 then reaction mixture was
kept at 25 C for overnight. Subsequently, solution was passed
through cation exchange resin column at elute rate of 5 ml min1
and solution was again concentrated with methanol. This step was
repeated thrice to remove completely borohydride in the form of
methyl borate. The solution was evaporated, dried and kept in

For chemical analyses, lyophilized EPS were hydrolyzed and

processed as previously described [17]. Total carbohydrate, protein
and sulfate contents of EPS were calculated according to anthrone
[18], Bradford [19] and Dodgson & Price methods [20] respectively.
Three replicates were used for each assay. The standard curve of
sulfate was established in the range of 220 g ml1 H2 SO4 . The
protein present in the EPS was analyzed with 1-dimensional 10%
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) according to Laemmli [21]. Protein bands were visualized by
silver staining.
Noise-decoupled 1 H nuclear magnetic resonance (NMR) spectrum was recorded at 200 MHz on a Bruker Avance II 200
spectrometer (Switzerland). About 50 mg of NaOH pellets were
dissolved in 1 ml of D2 O followed by addition of 50 g EPS. The
spectra of the EPS solution were recorded at 25 1 C. The conditions for 1 H NMR were 50005200 accumulations, pulse duration
5.9 s, acquisition time 1.2 s and relaxation delay 6 s. Fourier
transformed-infrared spectroscopy (FT-IR) spectrum was recorded
on Perkin Elmer (Spectrum GX) with a resolution of 4 cm1 in
4000400 cm1 region. For FT-IR analysis, pellets of 6 mg of EPS
were prepared with 500 mg KBr followed by pressing the mixture
into a 16 mm diameter mold.
The inorganic elements of EPS were analyzed with energy dispersive X-ray spectroscopy (EDX; Oxford Instruments, UK). X-rays
emitted by matter in response to bombardment with charged
particles present in the matter was examined using SEM-EDX
[22].
2.4. Physical properties of EPS
An amount of 1 g sample was used for X-ray diffraction
analysis (XRD) and performed on X-ray powder diffractometer
(Philips Xpert MPD, The Netherlands) instrument equipped with
Cona PW3123/00 curved Ni-ltered CuK source ( = 1.54056 A).
ditions were maintained as described previously [8]. The dried EPS
sample was mounted on a quartz substrate and diffraction peaks
were plotted as degrees of 2 value (half of the scattering angle
measured from the incident beam). The peaks of diffracted X-rays
were calculated with Braggs law
d=


2 sin 

crystallinity index (CIxrd ) was calculated according to Ricou, Pinel,

& Juhasz [23].
CIxrd =

Acrystal
Acrystal + Aamorphous

Thermal gravimetric (TG) analysis of EPS was carried with

Mettler Toledo TGA/SDTA System (Greifensee, Switzerland).
Approximately, 5 mg of dried samples was applied to TGA and
thermogram was obtained in the range of 30500 C at rate of

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225

10 C min1 under nitrogen atmosphere. The thermogram was

plotted with weight (percentage) loss vs. temperature. The particle size distributions were measured by laser diffraction (Malvern
Mastersizer 2000, Malvern Ltd., Worcestershire, UK).
The wettability of EPS measured with the DataPhysics Tensiometer of DCAT series connected with Software Module SCAT
32. The contact angle of EPS was calculated according to following
formula:

powder contact angle :
M2
T=
Cp2  cos 
where, T time after contact,  viscosity, C material const., p
density,  surface tension,  contact angle, M mass of liquid
adsorbed on solid. Wettability of EPS was checked against seawater
with three replicates.
2.5. AFM analysis
AFM is a powerful tool to detect topographical variation and
reveal more detailed surface properties than a morphological
image. Glass cover slips were sterilized with mixture of 5 ml of
50% HCl and 5 ml of 50% of H2 SO4 (v/v) for 2 h followed by rinsing with autoclaved Milli-Q water. Thereafter, treated with ethanol
and again washed with autoclaved Milli-Q water and stored in same
for further uses. AFM analysis was carried out with NT-MDT-NTegra
Aura (Russia) in semi-contact mode for EPS coated cover slips and
zoospore settled onto EPS coated cover slips.
2.6. Settlement and adhesion of zoospores on EPS lm
Fertile thalli of U. fasciata were collected from Veraval (20.55
N and 70.20 E) Southern west coast of India during November
2010 and conditions for zoospores released from a single population were maintained according to Callow et al. [24]. The cover
slips (22 mm 22 mm) were submerged in a 50% methanol/50%
HCl (v/v) mixture, concentrated HCl (2 h in each) and followed by
washing thoroughly with autoclaved Milli-Q water for sterilization.
Thereafter, 2 cover slips were submersed in 60 mm sterilized Petri
plates with 4 ml of 1% EPS suspension and kept in desiccator to
form complete dry lm of 0.35 3 mm. There were 7 replicates
of this experiment which were successively used for the zoospore
settlement assay having an incubation time of 2, 6, 24 and 48 h.
About 25 ml aliquots (1.5 106 spore ml1 ) of zoospore suspension
were mixed to 90 mm sterilized Petri plates having 3 replicates of
cover slips coated with EPS and kept in dark at 25 1 C for settlement of zoospores. The control was also considered as cover slips
without coated with EPS. There were different 90 mm sterilized
Petri plates having 3 replicates of cover slips coated with EPS for
different incubation time of interval. Unattached zoospores on coverslips coated with EPS and without EPS were removed by gentle
washing with autoclaved seawater. Zoospores were stained with
dilute carbol fuschin and counted with Olympus inverted microscope (model IX70) tted with DP 72 camera after 2, 6, 24 and 48 h
of incubation time interval. Settled spores on the glass slide were
counted in 15 elds of view located at 100 mm intervals along the
diagonal of each square, starting at the central corner with 3 replicates of each cover slips. The mean number of attached spores per
square centimeter was calculated with 95% condence of one way
ANOVA.
3. Results and discussion
3.1. Growth and optimization of EPS production
Although B. exus showed an overall linear growth up to
36 h, there was an exponential growth between 3 and 36 h, and

Fig. 1. Bacterial growth and EPS produced by B. exus.

thereafter it sharply declined (Fig. 1). The EPS produced by B. exus

was granular in texture and yield varied with duration of time. The
EPS yield was found maximum during the late log phase. Unlike
bacterial growth, the EPS yield showed a linear increase throughout the study and ranged from 7.66 g mg1 to 77.33 g mg1 dry
cell weights at 6 and 48 h, respectively (Fig. 1). While EPS secreted
by another closely related Bacillus licheniformis was ranged from
11.5 g mg1 to 60.2 g mg1 dry cell weight at 6 and 48 h, respectively [9]. The bacterial species of Vibrio also produced maximum
EPS during log phage [8]. The methods employed for extraction
of EPS from microorganisms depends on the nature of the EPS
produced [1,25,26]. However, the amount of EPS produced by the
organism depend on a number of complex controlling factors like
nutrient depletion, cell physiology and changes in growth conditions and/or bacterialseaweed interactions.
3.2. Determination of molecular mass and their analysis
GP chromatogram of puried EPS produced by B. exus showed
two molecular mass peaks corresponding to 1528 and 33,686 kDa
with 2.21 and 1.246 polydispersity, respectively (Fig. S1). GPC
pattern of present EPS was somewhat similar to that of previously reported B. licheniformis having approximately 1540 and
44,565 kDa with 1.92 and 1.41 polydispersity [9]. While, EPS of
dinoagellate, Amphidinium carterae consisted of low molecular
weight approximately 233 and 1354 kDa with 1.195 and 1.107
polydispersity, respectively. However, a single fraction of EPS
with GPC has also been reported from different bacteria. It has
been observed that EPS secreted by Cronobacter sakazakii showed
single chromatographic peak with 3760 kDa approximately with
1.017 polydispersity [27]. Additionally, higher molecular masses
have also been reported for EPS produced by Idiomarina spp.
(1.5 104 1.5 106 kDa) and Bacillus strain B3-15 (6 105 kDa)
[28].
It has been reported that MALDI TOFTOF MS [26] is convenient method for rapid and sensitive structural analysis of oligo and
polysaccharides. MALDI TOFTOF MS spectra of EPS represented
a series of masses in low range mode which correspond to deprotonated pentose (150 m/z), deoxy hexose and hexose (180 m/z)
sugars with inorganic (Na, P, Mg, C, O, Cl and S) and organic groups
(aliphatic and aromatic functional groups). A series of mass peaks
175.1294, 197.1588 and 251.1529 were obtained in low range
mode for monosaccharide of this EPS and deviation from their
actual mass value revealed that sugars were attached with different inorganic (Na, P, Mg, C, O, Cl and S) and organic groups
(aliphatic and aromatic functional groups). Apart from these, a
mass peak 343.2768 m/z was also observed in positive ion mid

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R.P. Singh et al. / Colloids and Surfaces B: Biointerfaces 103 (2013) 223230

Fig. 2. (A) The fragmentation mass peaks of oligo and polysaccharides contained of puried EPS were detected with positive ion mode in MALDI TOFTOF MS analysis and
(B) monosaccharide contained of the puried EPS determined with GCMS analysis.

range mode corresponding to that of hexose and pentose sugars

while 440.4741 m/z peak revealed the presence of disaccharide
(Fig. S2a and S2b). Another mass peak of 679.5378 consisted of
2 hexose and 2 pentose sugars. The mass peaks of 861.8578,
900.4803, 964.5788, 980.7354, 1056.5901, 1110.5988, 1145.7754,
1221.6525, 1316.9216, 1340.5688, 1362.2107, 1375.9656 and
1386.7 reveal the presence of oligosaccharides in the present EPS.
Moreover, higher mass peaks of 4367.53 and 5397.9 revealed
the presence of polysaccharides in EPS (Fig. S2c). In contrast, the
higher molecular mass peaks were not found in EPS produced by
C. sakazakii and Vibrio parahaemolyticus [8,27]. Additionally, hexose (glucose, galactose and mannose), deoxy hexose (fucose) and
pentose sugars (ribose and xylose) were conrmed with GCMS
analysis (Fig. 2B). The amounts of sugars present in EPS were 14.01,
16.43, 3.35, 23.52, 37.09, and 5.6% corresponding to fucose, ribose,
xylose, mannose, galactose and glucose respectively. The monosaccharide compositions of present EPS were found to be apparently
variable from other EPS produced by a number of marine bacteria
such as Flavobacterium, C. sakazakii, V. parahaemolyticus and Pseudoalteromonas sp. [29]. The uronic acid such as galacturonate was
not found in the present EPS while found in the EPS secreted by
Flavobacterium and Pseudoalteromonas species. High contained of
the glucose sugar was present in the EPS of B. licheniformis [9],
Flavobacterium and Pseudoalteromonas sp. [29] while mannose was
in the content of EPS of V. parahaemolyticus [8] and C. sakazakii [27].
In contrast, only two monosaccharides sugars (galactose, 73.13%
and glucose, 26.87%) were observed in the EPS of A. carterae [1]. Similar to A. carterae, present EPS also contained high ratio of galactose
than other sugars. Thus, monosaccharide composition determined
in this study was found to be quite contrasting from those of bacterial and cyanobacterial EPS reported till date [1,8,26,30]. Nielsen
and Jahn [30] described that sugar play an essential role in EPS
synthesis as an activated precursor.

thiocyanate ( SCN) and isothiocyanate ( NCS) groups respectively

[32]. The thiocyanate group was not found in the EPS secreted from
other sources such as V. parahaemolyticus [8], C. sakazakii [27] and
A. carterae [1]. A peak at 1649 cm1 indicated the characteristic
IR absorption of alkenyl stretched (C C) and/or aryl substituted
(C C) of polysaccharides [33]. Absorbance band 1418 cm1 was
representative of functional groups such as methylene and methyl
anti-symmetrical and/or symmetrical bending [32]. The absorption peak at 1138 cm1 was detected due to both sulfate ion and
C-O stretching bend in polysaccharides [31]. The absorption at
996 cm1 was identied as C C and C H vibrations of polysaccharides and nucleic acids [33]. The carbohydrate ring peaks were also
detected at 847 and 620 cm1 . Further, disulde bond (S S) of the
protein was indicated at 620 cm1 (Fig. 3). The FT-IR spectrum of
EPS in this study conrms the presence of various different functional groups as compared to previously reported EPS [1,8,9,26,27].
The 1 H NMR spectrum exhibited various anomeric signals for
chemical shift of functional group present in the EPS [34]. The
chemical shift at 1.01.258 ppm arouses from the methyl protons of 6 deoxy sugars of polysaccharides. The chemical shift of
functional group (R2 CHOR) was observed at 3.24.3 ppm. On the
other hand, stretching of N H group of protein was observed

3.3. Chemical analysis of EPS

FT-IR spectroscopy was used to characterize functional groups
within the EPS and showed a broad peak at 3417 cm1 , a characteristic of polymeric OH stretching [31]. Relatively strong absorption
peaks at 2334, 2184 and 2098 cm1 represented cyanate ion,

Fig. 3. FT-IR analysis of puried EPS produced by B. exus.

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Fig. 4.

H NMR analysis puried EPS produced by B. exus.

at 1.3 ppm while 0.81.2 and 1.11.5 ppm represent the alkanes
and alkenes respectively (Fig. 4). These chemical shift of different groups conrmed glycocalyx nature of EPS produced by B.
exus. Similar characteristic spectral peaks of 1 H NMR were also
observed in biopolymers obtained from different sources including bacteria, diatom and dinoagellate [1,8,9,27]. The ratio of total
carbohydrates, protein and sulfate content in EPS secreted by B.
exus was found to be 62, 23 and 15 mg gl dry cell weight respectively. Protein and sulfate content of the present EPS is higher than
the previously reported EPS from B. licheniformis and C. sakazakii
[9,27]. Nevertheless, the carbohydrate content of EPS was higher
than sulfate and proteins, a feature that corroborates with previous report of Zhenming & Yan [35]. There are also studies wherein
stated that protein content has been higher than carbohydrate
content in EPS [30]. Protein moiety of EPS involved in exopolysaccharides production and/or enhanced the production of EPS [36].
Protein loosely associated to EPS was not well resolved because
of interference with polysaccharides considering the much higher
carbohydrate/protein ratio. Although, Cao & Hu [36] reported that
predominant proteins in bound EPS with micro-organisms were
estimated in the range of 3040 and/or 6090 kDa. In this study,
the 1-dimensional 10% SDS-PAGE revealed that protein content
in EPS has consisted of two different polypeptides chains with
approximately 12 and 42 kDa (Fig. S3). It has been reported that
extracellular matrixes (carbohydrate and protein) play an important role in stabilizing biolm structure by forming electrostatic
bonds with multivalent cations [2]. The previous reports especially
revealed that extracellular protein might have more involved than
polysaccharides in electrostatic bonds inside biolms because it
has relatively high content of negatively charged amino acids and
also help to bind various cation (Na and Mg) [37]. Elemental qualitative and quantitative analysis by SEM-EDX revealed the weight
and atomic percentage of seven elements (S, Na, P, Mg, Cl, C and O)
present in EPS (Table S1). The distribution of cations such as Na and
Mg in the EPS suggest their bonding to negative charge of sulfate
groups and/or thiol group of the protein and thus, better formation of biolm. The sulfate was present as a functional group in
the polysaccharides, conrmed its anionic character in the marine
environment [38]. The cation Mg was not found in the EPS of closely
related other Bacillus species [9] and C. sakazakii [27] while contained the cation Ca which might be played similar role in those EPS.
Moreover, in this study various different masses were observed by
the MALDI TOFTOF MS may be due to pentose and hexose sugars of
EPS attached with different elements that detected with SEM-EDX
(Fig. 2A). The results obtained from MALDI TOFTOF MS, FT-IR, 1 H
NMR and SEM-EDX for present EPS are signicantly different from
the previously reported from other sources [26,39].
3.4. Physical properties of EPS
Particle size distribution is related to owability, moldability,
compressibility and die-lling characteristics of a powder that

227

denes the relative amounts of particles present in the EPS. The

EPS in this study constituted varied particle sizes from 2.856 (d 0.1)
to 40.845 (d 0.9) m with an average size from 12.245 m (d 0.5) to
1.0005 m2 g1 specic surface areas (Fig. S4). While EPS produced
from B. licheniformis was constituted of particle size from 5.274 (d
0.1) to 68.447 (d 0.9) m with an average size from 24.977 m (d
0.5) to 0.63771 m2 g1 specic surface areas [9,26]. It is revealed
that particle size of the present EPS was smaller than previously
reported EPS [9,26]. Besides this, particle size approximately similar to EPS produced by dinoagellate A. carterae [1] while quiet
distinct from those EPS produced from other bacteria such as C.
sakazakii and V. parahaemolyticus [8,27]. The XRD prole of EPS
obtained from B. exus exhibited the characteristic diffraction peaks
at 32.1 , 33.8 , 38.6 and 48.8 with 2.8, 2.7, 2.3 and 1.8 A d-spacing
respectively (Fig. S5). The XRD pattern revealed the amorphous
nature of EPS with CIxrd 0.304 crystallinity area. The ratio between
sharp narrow diffraction and broad peaks were used to calculate
the amounts of crystallinity [40]. XRD pattern of the present EPS
somewhat found similar to previously reported EPS from different
source [9,26] which were conrmed that EPS always amorphous
in nature. TGA showed degradation pattern of EPS from B. exus
that took place in three steps. Initially 6.44% loss in the weight
of EPS was observed between 100 and 200 C then approximately
52.6% and 41% of degradation was observed at 270 C and 400 C
in second and third phase respectively (Fig. S6). This result corroborates with EPS of Bacillus sp. I-450 where degradation was taken
place in three steps [34]. However, Kavita et al. [8] reported that
EPS produced from V. parahaemolyticus followed two degradation
patterns in TGA analysis. High level of carboxyl group in the EPS
increased degradation of rst phase as it is bound to more water
molecules [41]. Additionally, the detection of S O, C O S and carboxyl groups in the present EPS could be responsible for binding of
more water molecules and contributed to more weight loss in the
TGA analysis.
3.5. Atomic force microscopy (AFM) analysis
Physical properties of EPS chain can be determined by measuring parameters like shape, persistent length and end-to-end
distances of the polysaccharide chain using AFM. Now, AFM analysis is extensively being used for the topological characterization
of EPS from different source [1,9,42]. The 3D view of EPS can be
analyzed in live condition using AFM which is being more advantageous over SEM. An AFM micrograph of the 2.0 g ml1 solution
of the EPS following deposition on cover slips showed that cover
slips surface was covered with irregular EPS aggregates of varying dimensions (as represented in two and three dimensional in
Fig. 5A and B, respectively). Although, these compounds appear
nearly homogeneous, closer inspection (Fig. 5C) revealed that each
compound consists of L shaped with 1.5 m length. Recently, pearl
necklace shaped EPS was obtained from mixed culture aerobic
sludge granules [42]. Average roughness was 193 nm and histograms of the AFM demonstrating the topological distribution
pattern of particle of EPS onto cover slips before and after 48 h of
incubation (Fig. S7). Zoospore trapped with EPS was depicted in the
Fig. 5D. The results obtained from GPC collaborated with homogeneous distribution of EPS onto the cover slips and particle size
distribution agreed with AFM.
3.6. Settlement and adhesion of zoospores on EPS lm
It has been reported that settlement of zoospores of Ulva and
Enteromorpha spp. increased toward bacterial biolm [3,24,43,44].
Moreover, it is observed that not only AHLs producing bacterial
biolms but also topology of the co-polymers of different organic
coating enhance or reduce settlement of the Ulva zoospore [45].

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Fig. 5. AFM analysis of [two (A) and three dimensional view (B) before experiment set up] puried EPS. (C) Amplication of the square section from (A) and (D) incubation
of zoospore and EPS after 48 h of incubation. Zoospore stained with carbol fuschin and settled manually counting with Olympus inverted microscope. (E) Control and (F)
incubation of zoospore with EPS.

Ederth et al. [46] prepared methylated-galactoside-terminated

alkanethiol self-assembled monolayers which reduced the settlement of zoospore. Similarly, Cho et al. [47] made uorine-free,
amphiphilic and nonionic surface active block copolymers (SABCs)
with antifouling properties. In addition to this, surface charge
of the co-polymer also enhances or reduces settlement of the
Ulva zoospore. Ederth et al. [48] used cationic oligopeptide selfassembled monolayers (SAMs) for zoospore settlement assay. The
U. linza zoospores interacted strongly with lysine- and argininerich SAMs as compared to acid-washed glass. In another study,
Krishnan et al. [49] used hydrophobic uorinated and hydrophilic
PEGylated block co-polymer and reported that surface wettability
of the material decreased the zoospore settlement as hydrophobic
surface gave strength of adhesion of cells to substrates. Despite,
these many studies have been carried out but effect of the bacterial EPS on the settlement of Ulva zoospore has not been
elucidated yet. Hence, present study provides clear evidence that

U. fasciata zoospores respond to EPS coated cover slip. It was rst

time observed that relative increase in the duration of incubation
with zoospore successively enhanced zoospores settlement. Maximum settlement of zoospore (310,911 per cm2 ) was observed with
EPS coated cover slips after 48 h of incubation over control (41,882
per cm2 ). There were signicant differences (p 0.05) in the effect
of EPS on spore settlement based on one-way ANOVA and the Tukey
HSD test as compared to control (Fig. 5E and F, Fig. 6). Decrease in
mobility of spore was observed after 2 h and after 48 h of incubation nearly all the zoospores were settled down. These spores could
be counted using direct, manual counting rather than image analysis. Contact angle () 90 0.2 of present EPS revealed that EPS
contained hydrophobic as well as hydrophilic moiety. Similarly,
Callow and coworker reported a positive correlation between numbers of spores attached and increasing contact angle (increased in
hydrophobicity). It is reported that most pronounced response of
spore settlement to wettability for the contact angle between 40

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229

Marine Laboratory, UK for his valuable suggestions on zoospore

staining and their settlement assay.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.colsurfb.2012.10.037.
References

Fig. 6. The effect of EPS on zoospore settlement was signicant at p > 0.05 (one way
ANOVA), in comparison to control. Signicant denoted as +.

and 80 which is agreement with our ndings [24,49]. Different

analytical methods such as SEM-EDX, FT-IR and NMR also conrmed that present EPS is charged in nature which further gave
strength to spore settlement.
The present study, for the rst time demonstrated 48 h of incubation as compared to previously report where 6070 min has
been given for zoospore settlement assay [24,4449]. Considering the fact that in their natural environment biofouling bacteria
are continuously secreting EPS by which spore of different fouling
species are settle with respect to different time interval [24,43].
The incubation of zoospore with EPS enhanced settlement might
be due to sticky nature of the former. Present EPS was also rich
in various elements and other polymeric compounds (polysaccharides and protein) as detected with analytical methods. The
organic and inorganic contents of the present EPS may provide
nutrients to phytoplankton and seaweed for their better survival
and growth [1,9]. The presence of hydroxyl and carboxyl groups in
EPS could be responsible for hydrophilic polysaccharide formation
which serves as binding sites for divalent cations [1]. In a natural
marine environment, the nutrients can interact with EPS in order
to increase the rate of elements uptake. It also concentrates the dissolved organic compounds and making them readily available for
microbial growth and their surroundings ora. EPS also facilitates
a mechanism for gliding motility by the adhesion complex of a
range of connector molecules that link the extracellular adhesive
strands through the plasma membrane to an actinmyosin system
[50].
4. Conclusion
The ndings of this study revealed that EPS produced by B.
exus was distinct in terms of chemical composition, size, monosaccharide, oligo and polysaccharide contents, even from those of
produced by closely related bacterial strains. The zoospore settlement assays conducted with EPS coated cover slips revealed higher
colonization of spores than control. The studies exploring the effect
of interaction between settlement of zoospore and bacterial EPS
may provide deeper insights into ecological signicance of such
polymers.

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Acknowledgments
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The nancial support received from the Council of Scientic and Industrial Research (RSP 0016), New Delhi is gratefully
acknowledged. The rst author (Ravindra Pal Singh) gratefully
acknowledges the CSIR, New Delhi (India) for awarding the Senior
Research Fellowship. Special thanks to Dr. Ian Joint, Plymouth

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