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Capsular polysaccharides of cultured phototrophic biofilms
F. Di Pippo
a
; A. Bohn
b
; R. Congestri
a
; R. De Philippis
c
; P. Albertano
a
a
Department of Biology, University of Rome 'Tor Vergata', Rome, Italy
b
Instituto de Tecnologia
Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal
c
Department of Agricultural
Biotechnology, University of Florence, Florence, Italy
First published on: 20 April 2009
To cite this Article Di Pippo, F., Bohn, A., Congestri, R., De Philippis, R. and Albertano, P.(2009) 'Capsular polysaccharides
of cultured phototrophic biofilms', Biofouling, 25: 6, 495 — 504, First published on: 20 April 2009 (iFirst)
To link to this Article: DOI: 10.1080/08927010902914037
URL: http://dx.doi.org/10.1080/08927010902914037
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Capsular polysaccharides of cultured phototrophic biofilms
F. Di Pippo
a
, A. Bohn
b
, R. Congestri
a
, R. De Philippis
c
and P. Albertano
a
*
a
Department of Biology, University of Rome ‘Tor Vergata’, Rome, Italy;
b
Instituto de Tecnologia Quı
´
mica e Biolo
´
gica,
Universidade Nova de Lisboa, Oeiras, Portugal;
c
Department of Agricultural Biotechnology, University of Florence, Florence, Italy
(Received 4 November 2008; final version received 16 March 2009)
Phototrophic biofilm samples from an Italian wastewater treatment plant were studied in microcosm experiments
under varying irradiances, temperatures and flow regimes to assess the effects of environmental variables and
phototrophic biomass on capsular exopolysaccharides (CPS). The results, obtained from circular dichroism
spectroscopy and High Performance Liquid Chromatography, suggest that CPS have a stable spatial conformation
and a complex monosaccharide composition. The total amount present was positively correlated with the biomass of
cyanobacteria and diatoms, and negatively with the biovolume of green algae. The proportion of uronic acids
showed the same correlation with these taxon groups, indicating a potential role of cyanobacteria and diatoms in the
removal of residual nutrients and noxious cations in wastewater treatment. While overall biofilm growth was limited
by low irradiance, high temperature (308C) and low flow velocity (25 l h
71
) yielded the highest phototrophic
biomass, the largest amount of CPS produced, and the highest proportion of carboxylic acids present.
Keywords: phototrophic biofilms; capsular exopolysaccharides; microcosm; circular dichroism; HPLC
Introduction
Aquatic phototrophic biofilms are comprised of photo-
trophs (cyanobacteria and microalgae), and chemo-
trophs (Archaea, bacteria, fungi and protozoa). They
form highly structured communities driven by light as an
energy source and secrete an exopolymeric matrix that
holds the biofilm together (Wimpenny et al. 2000;
Sutherland 2001; Burns et al. 2004). Different from
heterotrophic biofilms, whose structure, growth, dy-
namics and physiology have been extensively studied in
field and in culture (Stoodley et al. 2002; Palmer
and Stoodley 2007), phototrophic biofilms have
received little attention until recently (Wolf et al.
2007). The recent increase of interest in the structure
and functioning of phototrophic biofilm is related to
their ecological importance and their high potential for
biotechnological applications, such as wastewater treat-
ment (Schumacher et al. 2003; Roeselers et al. 2008),
bioremediation (Cohen 2002; Chaillan et al. 2006),
aquaculture (Bender and Phillips 2004) and antifoulant
production (Bhadury and Wright 2004).
The extracellular matrix is a key factor for the
overall functionality of phototrophic biofilms. It is a
highly hydrated system composed of extracellular
polymeric substances (EPS), which mainly comprises
polysaccharides, along with a wide variety of proteins,
glycoproteins, glycolipids and nucleic acids (Stoodley
et al. 2002; Chiovitti et al. 2003; Cogan and Keener
2004). Independent of the composition of EPS
molecules, the matrix typically features a hydrogel-
like structure, which embeds the biofilm cells and
determines the physico-chemical and biological prop-
erties of the whole biofilm (Mayer et al. 1999;
Flemming et al. 2007).
From an ecological point of view, the matrix is the
major factor for biofilm success. Its network of
polymeric chains formed by intra- and intermolecular
linkages provides mechanical stability such that cell
positions are maintained over prolonged periods
(Flemming et al. 2007). In addition, EPS and in
particular capsular polysaccharide s (CPS), are thought
to play an important role in the attachment of cells to
substrata. This allows the formation of stable micro-
consortia with a low expense of energy, as the existence
of a stable micro-environment allows the cells to
metabolise, reproduce and communicate between each
other more efficiently (Decho 2000). In addition to the
advantages of mechanical stabili ty, the matrix also
provides protection against heavy meta ls, other toxic
substances and grazing by predators.
The formation of stable complexes between the
biofilm matrix and metal ions gives the EPS a key role
for a large range of biotechnological applications of
biofilms. The presence of carboxylic groups and other
anionic residues of exopolysaccharides allows the
binding and accumulation of cations (De Philippis
*Corresponding author. Email: albertano@uniroma2.it
Biofouling
Vol. 25, No. 6, August 2009, 495–504
ISSN 0892-7014 print/ISSN 1029-2454 online
Ó 2009 Taylor & Francis
DOI: 10.1080/08927010902914037
http://www.informaworld.com
Downloaded By: [informa internal users] At: 10:11 4 December 2009
and Vincenzini 1998), which pinpoints the potential for
the exploitation of biofilms in the removal of metals
from polluted waters (Mehta and Gaur 2005; De
Philippis et al. 2007) and in wastewater treatment
(Cohen 2002; Bender and Phillips 2004).
The species composition of the microbial biofilm
community is one of the main factors that determines
the composition and production of EPS (Neu 1994;
Bahulikar and Kroth 2008). Chemical studies of the
exopolysaccharides produced by cultured and natural
populations of cyanobacteria and diatoms show that
EPS are mainly complex anionic heteropolymers
formed by different monosaccharides, whose propor-
tions depend on the specific organisms. Because the
biofilm co mmunity itself is a mixture of diverse
microorganisms, the dependence of EPS composition
on the contributing species is very complex (Flemming
et al. 2007). In any case, the crucial role of community
composition should always be considered in studies on
biofilm EPS.
In addition to species composition, exopolymers
are also strongly influenced by environmental factors,
such as irradiance. The influence of irradiance levels on
EPS production by cultured diatoms and cyanobacter-
ia has been studied in some detail, and production has
been closely linked to photosynthesis (Staats et al.
2000; Otero and Vincenzini 2003; Stal and Defarge
2005). On the other hand, there is little information on
the effect of temperature on EPS production in mixed
biofilms containing diatoms (Wolfstein and Stal 2002)
and cyanobacteria (Moreno et al. 1998).
In the present investigation, a study was carried out
of the CPS of phototrophic biofilms grown in a
continuous flow incubator, specifically designed for
studying biofilm development (Zippel and Neu 2005),
using inocula collected from a wastewater treatment
plant (WWTP). In accordance with a series of authors
(Vincenzini et al. 1990; De Philippis and Vincenzini
1998; de Brouwer et al. 2002; Barranguet et al. 2004;
Bellinger et al. 2005; Stal and Defarge 2005) ‘CPS’ is
also used by the current authors to refer to the ‘bound
extracellular carbohydrates’, as the exopolysaccharide
fraction more intimately associated with the cells, as
opposed to the ‘released polysaccharides’ or ‘soluble
extracellular carbohydrates’, the solubilised mucil agi-
nous fraction which is loosely attached to the cell
surface. The effects of light intensity, temperature,
water flow velocity and phototrophic biomass on the
total amount and on the physico-chemical properties
of the CPS were monitored.
Phototrophic biomass was estimated by its total
biovolume, as the latter accounts for the high
variability of shapes and dimensions of different taxa
constituting biofilm communities (Stevenson 1996).
One aim was detecting significant dependencies
between the properties of the CPS (including the
technologically relevant proportion of uronic acids)
and environmental factors and the composition of the
phototrophic component. The results are discussed
with respect to future experimental endeavours to
unravel the complex network behind whole-biofilm
functionality and possibl e optimisation strategies for
the application of phototrophic biofilms in wastewater
treatment.
Materials and methods
Sample collection and inoculum treatment
Biofilm inocula were collected from the Fiumicino
(Rome, Italy) WWTP on four different occasions by
scraping submerged biofilms off the walls of the
sedimentation tank (Table 1). Inocula for the culture
study were obtained according to Guzzon et al. (2005):
the samples were treated to eliminate grazers, and four
aliquots of ca.100 ml homogeneous suspension were
poured into four 5 l High Density Polyethylene (HDPE)
bottles, each containing 3.9 l of the medium. The
medium-inoculum mixture was filtered through a net
(300 mm mesh) and then used to inoculate slides in the
continuous flow incubator (72 h, 100 l h
71
flow rate).
Identification of phototrophs within the inocula
was based on previous studies of biofilms developing in
the sedimentation tank of the Fiumicino WWTP
(Albertano et al. 1999; Congestri et al. 2003, 2005,
2006) and evaluated by light microscope observations.
Microcosm experiments
Inocula were grown in a prototype incubator, devel-
oped by the Department of Inland Water Research,
UFZ Centre for Environmental Research (Magdeburg,
Germany; Zippel and Neu 2005; Zippel et al. 2007).
The incubator consisted of four separate light cham-
bers (LC), featuring photon flux densities of 120
(abbreviated as LC120), 60 (LC60), 30 (LC30) and
15 mmol photon m
72
s
71
(LC15). ‘True light’ lamps
(T-8; AURALIGHT; Sweden) were used as light
source to illuminate the LCs in a 16:8 h light:dark
Table 1. Inoculum sampling period, experimental condi-
tions and labels used in the text of the performed incubator
runs.
Incubator
run Label
Sampling
month
(Year 2004)
Temperature
(8C)
Flow
velocity
(l h
71
)
1 R20.25 April 20 25
2 R20.100 June 20 100
3 R30.25 July 30 25
4 R30.100 September 30 100
496 F. Di Pippo et al.
Downloaded By: [informa internal users] At: 10:11 4 December 2009
cycle. Five litres of medium were pumped through the
incubator over polycarbonate slides serving as an
artificial substratum for biofilm adhesion. In addition
to light intensity, four parameter combinations featur-
ing tw o temperatures (20 and 308C) and two flow rates
(25 and 100 l h
71
) were tested (Table 1). These four
incubator runs are referred to as Runs, abb reviated as
eg R20.25 for the combination of 208C and 25 l h
71
.
Biomass accumulation was estimated by light
transmittance through the biofilm, comparing the
incident light intensity as measured directly under the
lamp with the readings of three subsurface light-sensor
banks, each consisting of three diodes. A linear
relation between subsurface light attenuation and
biomass accumulation has been shown by Zippel
et al. (2007). In this way, growth and development of
the biofilms were monitored over 30 days. In the
present study, biofilm samples were scraped off the
polycarbonate slides for subsequent analyses when
the mature stage, corresponding to an average light
absorption of 90–95%, had been reached.
Extraction and quantification of CPS
Mature biofilms were removed from the slides and
centrifuged at 10,000 rpm for 60 min (J2–21 Beckman
centrifuge) to concentrate the sample. The resulting
pellet, comprising the microorganisms and their
envelopes (namely ‘capsular’ [CPS] or ‘bound’ poly-
saccharides) was re-suspended in distilled water (1:10),
incubated at 808C for 60 min and then centrifuged for
15 min at 10,000 rpm to remove the cells. The CPS
were precipitated in ethanol 100% by centrifugation at
14,000 rpm for 60 min. To further purify the polymers,
the resulting pellet was re-suspen ded in distilled water,
dialysed against EDTA (0.01 M) and NaCl (0.5 M) for
2 h and then against distilled water for 4 days. The
extraction of the CPS was conducted following
Bellezza et al. (2003). The CPS samples were lyophi-
lised and hydrolysed (2N trifluoroacetic acid, 1208C
for 45 min) and their monosaccharide composition
quantified by means of RP-HPLC (reverse phase),
using a Beckman Ultrasphere ODS column according
to Vincenzini et al. (1990). The quantity of CPS
extracts was estimated by the phenol-sulphuric acid
method, using glucose as standard (Dubois 1956).
Circular dichroism analyses
The CPS fractions were analysed by means of circular
dichroism (CD), a qualitative spectroscopic method to
investigate the presence of carboxylic moieties and the
potential conformational transition of polysaccharides
as a function of pH and tempe rature. Measurements
were performed using a Jasco Spectropolarimeter J600,
equipped with quartz cells of 0.5 cm optical length,
using original Jasco software (20 nm min
71
scanning
velocity). Preliminary analyses wer e performed in the
UV spectral region (200–300 nm) in order to verify the
extract purity and subsequently between 200 and
260 nm to detect the presence of carboxylic moieties.
The pH values were measured with the digital
combined pH meter Amel Instruments, Model 334-B,
calibrated with buffer solut ions at pH 4.0 and 8.0. The
pH of the CPS was decreased by adding perchloric acid
(HClO
4
) 0.02 M and increased with NaOH 0.02 M.
The analyses were performed at 20 and 708C,
respectively, using a cell holder connected to a Lauda
M3 thermostat unit.
Cytochemistry
Biofilm fragments wer e stained with Alcian Blue (AB)
and observed with light microscopy (Zeiss Axioskop),
to visualise the presence of carboxylic (AB pH 2.5) and
sulphated (AB pH 0.5) polysaccharides in cyanobac-
teria and microalgae (Albertano and Bellezza 2001).
Phototrophic biomass
Phototrophic biomass was estimated as the biovolume
of individual taxa. Three biofilm replicates of 1 cm
2
were scraped off the slides and fixed in 2% formalde-
hyde in 0.1 M phosphate buffer (pH 7.2) at 48C.
Preparation of samples for biovolume evaluation was
carried out according to Congestri et al. (2006). The
scrapings were sonicated twice for 3 min in a sonic
water bath to disaggregate the samples. Aliquots of
suspensions were diluted in phosphate buffer and
allowed to settle for 24 h in 25 ml counting chambers.
Observations were made with an inverted Zeiss
Axiovert 100 microscope. A Nikon CoolSnap digital
photo-camera was used to acquire optical fields and
digital images. Measu rements of selected morpho-
metric parameters were calculated manually on digital
images using Adobe Photoshop version 7.0. To
estimate the biovolume of single cells standardised
equations proposed for the different cyanobacterial
and algal shapes were used (Hillebrand et al. 1999).
Statistical analyses
Statistical analyses were carried out using the R
language, version 2.7.1 for Mac OS X (R Core
Development Team 2008), using Aabel software
version 2.4.2 (Gigawiz, Tulsa, OK) for graphical
representations. To quantify and describe the mono-
saccharide proportions obtained by High Performance
Liquid Chromatography (HPLC) analysis, the propor-
tions in each experiment were transformed to their
Biofouling 497
Downloaded By: [informa internal users] At: 10:11 4 December 2009
corresponding ranks, with the monosaccharide with
the largest proportion obtaini ng rank 1, and the one
with the least proportion the highest rank. To integrate
the results from all experiments, the mean and the
standard deviation (SD) of the ranks of each mono-
saccharide across the experiments were calculated, and
rounded to integer values, to obtain an overall
ranking.
To calculate the similarities between the mono-
saccharide compositions obtained in each experiment
the centred log-ratios (CLR) of the monosaccharide
proportions were calculated as
CLR
i
¼ log
x
i
GxðÞ
ð1Þ
with G(x) being the geometric mean of the proportions
x
i
, using routines from the R-package compositions
(van de n Boogaart and Tolosana-Delg ado 2008). The
dissimilarity of two co mpositions was quantified by the
Euclidean distance be tween the CLRs of each compo-
sition. On the basis of the resulting dissimilarity
matrix, hierarchical clustering analysis was performed
using the average linkage method.
Correlations were quantified by Pearson’s r, using
log-transformed data and CLRs. Given the presence of
missing data, the effects of the environmental factors
were not assessed by ANOVA. Instead, the relevant
paired differences between data from different LCs and
Runs were computed and discussed. The possibility of
a significant effect was suggested if all observed
differences were clearly different from zero and had
the same, positive or negative, sign.
Results
Phototrophic diversity of the inocula
The diversity of phototrophs within the inocula
collected on the different sampling occasions was
expressed as taxon numbers (Supplementary Table 1)
[Supplementary material is available via a multimedia
link on the online article webpage]. The phototrophic
component consisted primarily of cyanobacteria,
diatoms and green algae, with one xanthophyte and
one euglenophyte. Taxon numbers were generally low
and stable, yielding up to 8 cyanobacterial taxa for
June and July, and 15 diatom taxa in April and
September. Chlorophytes consisted of five (April, June
and July) or six (September) taxa.
Biofilm development
Only biofilms grown under 60 and 120 mmol photon
m
72
s
71
reached the mature stage of development by
day 30. Here, the polycarbonate slides were uniformly
colonised with biofilms of ca. 1 mm thickness,
exhibiting a dark green coloration and seemingly
gelatinous consistency. The cultures grown under
30 mmol photon m
72
s
71
reached the active phase
(average light absorption ¼ 50%) of development in
R20.25 and R30.100, and remained at the initial stage
(average light absorption ¼ 5%) in R20.100 and
R30.25. Cultures grown under 15 mmol photon m
72
s
71
did not show any substantial growth throughout
the 30 day incubation period. As these conditions thus
yielded insufficient biomass for subsequent processing,
all further analyses were limited to samples obtained
from mature LC120 and LC60 cultures.
Amounts of exopolysaccharide
Figure 1 shows the total amount of CPS, expressed as
mg glucose equivalent per cm
72
surface area, ranging
between (47 + 8) 6 10
74
mg cm
72
in R30.100
LC120 and (783 + 46) 6 10
74
mg cm
72
in
R30.25LC60, respectively. Table 2 shows the effects
of environmental parameters on the amount of CPS.
The comparison of the amounts in R20.25 and R30.25
showed an increase of CPS with temperature in both
studied pairs. The other factors yielded no coherent
evidence for an effect on CPS.
Circular dichroism analyses
Supplementary Figure 1 [Supplementary material is
available via a multimedia link on the online article
webpage] shows the CD spectra of the capsular
polysaccharide extracts. In all cases, an ellipticity
typical for carboxyl ic moieties was witnessed between
200 and 250 nm, while no effect was observed above
260 nm, indicating the complete removal of proteins
from the extracts. Variations in pH and temperature
did not elicit clear changes of the CD spectra,
suggesting a stable polymeric conformation of the
CPS.
Figure 1. Concentration of the CPS (10
74
mg cm
72
) for all
analysed samples.
498 F. Di Pippo et al.
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