Journal of Applied Phycology 15: 259–261, 2003.

259
© 2003 Kluwer Academic Publishers. Printed in the Netherlands.

Carbohydrate synthesis by two Navicula strains isolated from benthic and

pelagic mucilages in the Tyrrhenian Sea (Tuscan Archipelago)

Cecilia Faraloni 1, Roberto De Philippis 1,*, Claudio Sili 2 and Massimo Vincenzini 1
1
Dipartimento di Biotecnologie Agrarie, Università degli Studi, Piazzale delle Cascine 24, Firenze, I 50144,
Italy; 2Istituto per lo Studio degli Ecosistemi, CNR, Piazzale delle Cascine 24, Firenze, I 50144, Italy;
*Author for correspondence (e-mail: roberto.dephilippis@unifi.it; phone: +39-055-3288284; fax:
39-055-3288272)
Received 31 July 2002; accepted in revised form 28 December 2002

Key words: Benthic aggregates, Diatoms, EPS, Exocellular polysaccharides, Mucilages, Navicula, Pelagic
aggregates, Tyrrhenian sea

Abstract

Two exopolysaccharide (EPS)-producing strains of the diatom Navicula, were isolated from benthic and pelagic
mucilaginous aggregates sampled in the Tyrrhenian Sea and cultured under laboratory conditions. The amount of
carbohydrate formed over the growth period and on a per cell basis was quite similar. However, the benthic
strain showed a preferential synthesis of the bound (i.e., cellular and capsular) carbohydrate fraction, whereas the
pelagic strain preferentially synthesised soluble, polymeric carbohydrates. The polysaccharides released into the
medium by the two strains showed the same qualitative monosaccharidic composition, being constituted by two
acidic and six neutral sugars. It is suggested that the difference between the benthic and the pelagic strain in the
synthesis of bound or soluble carbohydrates may be related to the different role of these compounds in the par-
ticular habitats of the strains.

Since 1991 there have been frequent occurrence of
conspicuous mucilaginous aggregates in the Tyrrhe-
nian sea, both pelagic and benthic. The pelagic aggre-
gates were usually dominated by photosynthetic mi-
croorganisms, such as microalgae and cyanobacteria,
whereas benthic mucilages showed the additional
presence of clumps of filaments of the algae Acineto-
spora crinita, Chrysonephos lewisii and Nemato-
chrysopsis marina (Sartoni and Sonni 1991; Innamo-
rati 1995). The mucilaginous aggregates contained
large amounts of polysaccharidic material in both
cases, possibly released by the microorganisms en-
trapped in the mucilaginous matrix. Among the pho-
totrophic microorganisms present in these mucilages,
exopolysaccharide (EPS)-producing diatoms have
been frequently found as the dominant component of
the microalgal biocenosis (Innamorati 1995; De Phil-
ippis et al. 2002). A number of studies have been car-
ried out with the aim of identifying the phototrophs
in the mucilaginous aggregates (Innamorati et al.
1993; Golubic et al. 1999), but the physiological fea-
tures of the species occurring in both pelagic and
benthic types have received little study.
The aim of the study reported here was to investi-
gate the possible correlations existing among the
physiological features of two Navicula strains, iso-
lated from benthic and pelagic mucilaginous aggre-
gates sampled in the Tyrrhenian Sea, and their role in
the different type of aggregates.
The two EPS-producing Navicula strains were iso-
lated from enrichment cultures of the benthic and pe-
lagic mucilaginous aggregates sampled near Isola del
Giglio (Tuscan Archipelago, Tyrrhenian Sea). Ac-
cording to the morphological features, the benthic
strain (GP17) was tentatively assigned to N. crypto-
cephala and the pelagic strain (GG3) to N. gracilis.
Axenic, batch cultures of the two strains were carried
out in triplicate at 20 ± 2 °C, with an incident photon
flux of 20 µmol photon m −2 s −1, using f/2 medium
(Guillard and Ryther 1962). Growth was followed by
260

counts of cell density using light microscopy and the

synthesis of both soluble and bound (i.e., cellular and
capsular) carbohydrate fractions was monitored ac-
cording to De Philippis et al. (1996). The EPSs re-
leased by the strains during their photoautotrophic
growth were separated from the culture medium and
their monosaccharidic composition determined by
HPLC analysis (Vincenzini et al. 1990). The anionic
density of the EPSs was determined by the Alcian Blu
method (Bar-Or and Shilo 1987) and the amount of
uronic acids was quantified by the method described
by Galambos (1967).
The two diatoms, growing under laboratory condi-
tions, reached the stationary phase after about 30 days Figure 1. Time course of bound (triangles) and soluble (squares)
carbohydrate fractions in laboratory cultures of benthic (N. crypto-
for strain GP17 and 25 days for strain GG3, with
cephala GP17, closed symbols) and pelagic (N. gracilis GG3, open
maximum cell densities of 2.1 ± 0.3 and 1.4 ± 0.2 × symbols) Navicula strains.
10 6 cell mL −1, respectively. During the growth, the
culture of strain GP17 always showed a higher con- Table 1. Raw composition and anionic density of the exopolysac-
charides produced by N. cryptocephala GP17 and N. gracilis GG3.
tent of total carbohydrates (Figure 1) in comparison
with strain GG3, but, owing to the higher amount of Strain Total carbohydrates (a) Uronic acid (b) Anionic density (c)
cells present in this culture at the stationary phase, the
GP17 47.4 24.2 0.7
mean amount of carbohydrates synthesized per cell GG3 47.4 27.3 1.0
and per day by the two strains, calculated on the
(a) (b) (c)
whole culture period, was quite comparable, being % on crude EPS dry wt; % on total carbohydrates; mg Al-
cian Blue (mg EPS) −1
2.60 ± 0.06 and 2.56 ± 0.05 pg cell −1 day −1, for strain
GP17 and GG3 respectively. However, the two strains
The polysaccharides produced by the two strains
showed a different behavior with regard to the syn-
showed anionic densities of 0.7 and 1.0 mg of Alcian
thesis of the soluble and the bound carbohydrate frac-
Blu per mg of crude EPS, and uronic acid concentra-
tions. Indeed, in the cultures of the benthic strain, the
tions corresponding to 24.2 and 27.3% of the total
bound and the soluble fractions increased at a com-
carbohydrate fraction of the EPSs produced by strain
parable rate, the former fraction always being higher
GP17 and by strain GG3, respectively (Table 1). Both
than the latter one and reaching, at the end of the cul-
EPSs, analysed for their monosaccharidic composi-
tivation period, a value of about 100 mg L −1, in com-
tion, showed the same qualitative composition, being
parison with a value of 80 mg L −1 of the soluble
present two acidic sugars, galacturonic and glucu-
fraction (Figure 1). On the other hand, in the cultures
ronic acid, and six neutral sugars, galactose, arabi-
of the pelagic strain the two carbohydrate fractions
nose, fucose, glucose, mannose and rhamnose (Ta-
initially increased at a comparable rate but, at the sta-
ble 2). The molar percentage of monosaccharides was
tionary phase, the accumulation of the bound fraction
rather similar in the two EPSs, with the exceptions of
stopped whereas the soluble fraction increased up to
mannose and glucose, found in different amounts in
values of about 75–80 mg L −1 (Figure 1). These re-
the polymers synthesised by the two strains. On the
sults point out that the main difference between the
basis of this chemical characterization, the polysac-
benthic and the pelagic Navicula strains is the pref-
charides released by the two Navicula strains ap-
erential synthesis of bound carbohydrates typical of
peared to be rather similar, and comparable to those
the benthic strain. Indeed, this fraction always repre-
produced by other diatoms associated with marine
sented about 60% of the total carbohydrates synthe-
mucilages (Myklestad 1995).
sized by strain GP17, whereas in the cultures of strain
The results suggest that the main difference be-
GG3, the bound fraction decreased from about 70%
tween the benthic and the pelagic strain does not re-
of the total carbohydrates synthesized, during the first
side in the characteristics of the polysaccharide syn-
days of growth, to less than 40%, at the stationary
thesized, but in the different ratio between the bound
phase.
and soluble fractions. This difference suggests that the
 261

Table 2. Monosaccharidic composition (moles %) of the ex- Bar-Or Y. and Shilo M. 1987. Characterization of macromolecular
opolysaccharides produced by N. cryptocephala GP17 and N. gra- flocculants produced by Phormidium sp. strain J-1 and by Ana-
cilis GG3. baenopsis circularis PCC 6720. Appl. envir. Microbiol. 53:
2226–2230.
Strain GlcA GalA Gal Glc Man Ara Fuc Rha Bhosle N.B., Sawant S.S., Garg A. and Wang A.B. 1995. Isolation
and partial chemical analysis of exopolysaccharides from the
GP17 + + 33.0 8.9 3.6 24.1 16.1 14.3
marine fouling diatom Navicula subinsufflata. Bot. mar. 38:
GG3 + + 31.1 4.7 11.3 22.2 16.5 14.2 103–110.
GlcA, glucuronic acid; GalA, galacturonic acid; Gal, galactose; De Philippis R., Faraloni C., Sili C. and Vincenzini M. 2002. Algal
Glc, glucose; Man, mannose; Ara, arabinose; Fuc, fucose; Rha, biocenosis in benthic mucilaginous aggregates of the Tyrrhe-
rhamnose. + = present. nian sea, with emphasis to the exopolysaccahride-producing
microalgal community. Arch. Hydrobiol/Alg. Studies 109 (in
press).
synthesis of carbohydrates plays a different role in the De Philippis R., Sili C. and Vincenzini M. 1996. Response of an
two strains; for the benthic one, the high amount of exopolisaccahride-producing heterocystous cyanobacterium to
bound carbohydrates presumably confers to the cells changes in metabolic carbon flux. J. appl. Phycol. 8: 275–281.
the capability to adhere the benthic surfaces, accord- Galambos J.T. 1967. The reaction of carbazole with carbohydrates.
ing to the previously hypothesized role of EPS-pro- I Effect of borate and sulfamate on the carbazole color of sug-
ars. Analyt. Biochem. 28: 350–356.
ducing diatoms in the building up of primary biofilms Golubic S., Le Campion-Alsumard T. and Campbell S.E. 1999.
(Watherbee et al. 1998) and in the adhesion of the Diversity of marine cyanobacteria. Bullettin de l’Institut ocèan-
mucilages on solid substrates (Bhosle et al. 1995). On ographique, Monaco, n o special 19: 53–76.
the other hand, the prevalence of released carbohy- Guillard R.R. and Ryther J.H. 1962. Studies on marine planktonic
drates observed in the pelagic strain suggests a sig- diatoms. I. Cyclotella nana Hustedt and Detonula confervacea
(Cleve) Gran. Can. J. Microbiol. 8: 229–239.
nificant contribution of this diatom to the formation Innamorati M. 1995. Hyperproduction of mucilages by micro and
of the pelagic mucilages, usually characterized by a macroalgae in the Tyrrhenian Sea. Sci. tot. Environ. 165: 65–
high abundance of mucilaginous particles constituted 81.
by the polysaccharidic exudates of diatoms (Innamo- Innamorati M., Nuccio C., Lazzara L., Mori G., Mussi L. and De
rati 1995; Alldredge 1999). Pol M. 1993. Condizioni trofiche, biomassa e popolamenti fi-
toplanctonici dell’alto Tirreno. In: Nuccio C. (ed.), Regione
toscana, Università di Firenze, Progetto Mare. Ricerca sullo
stato biologico, chimico e fisico dell’alto Tirreno toscano. Dip.
Biol. Veg. Univ., Firenze, pp. 103–156.
Acknowledgements Myklestad S.M. 1995. Release of extracellular products by phy-
toplankton with special emphasis on polysaccharides. Sci. tot.
Environ. 165: 155–164.
This work was supported by the Italian Ministry of Sartoni G. and Sonni C. 1991. Tribonema marinum J. Feldmann e
the Environment, in the framework of the ICRAM Acinetospora crinita nelle formazioni mucillaginose bentoniche
(Istituto Centrale per la Ricerca Scientifica e Tecno- osservate sulle coste toscane nell’estate 1991. Inform. Bot. Ital.
logica Applicata al Mare) project ⬙Processi di for- 23: 23–30.
Watherbee R., Lind J.L., Burke J. and Quatrano R.S. 1998. The first
mazione delle mucillagini nell’Adriatico e nel Tir-
kiss: establishment and control of initial adhesion by raphid
reno⬙. diatoms. J. Phycol. 34: 9–15.
Vincenzini M., De Philippis R., Sili C. and Materassi R. 1990.
Studies on exopolysaccharides release by diazotrophic batch
References cultures of Cyanospira capsulata. Appl. Microbiol. Biotechnol.
34: 392–396.
Alldredge A.L. 1999. The potential role of particulate diatom exu-
dates in forming nuisance mucilaginous scums. Ann. Ist. Su-
per. Sanità 35: 397–400.