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To cite this article: Natascha Staats , Ben De Winder , Lucas Stal & Luuc Mur (1999) Isolation and characterization of
extracellular polysaccharides from the epipelic diatoms Cylindrotheca closterium and Navicula salinarum , European Journal
of Phycology, 34:2, 161-169, DOI: 10.1080/09670269910001736212
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Eur. J. Phycol. (1999), 34 : 161169. Printed in the United Kingdom 161
N A T A S C H A S T A A T S1, B E N D E W I N D E R1*, L U C A S J. S T A L2 A N D L U U C R. M U R1
" Department of Microbiology\ARISE, University of Amsterdam, Nieuwe Achtergracht 127, 1018 WS Amsterdam, The Netherlands
# Netherlands Institute of Ecology, Centre for Estuarine and Coastal Ecology, PO Box 140, 4400 AC Yerseke, The Netherlands
The production and composition of extracellular polymeric substances (EPS) in axenic batch cultures of the benthic marine epipelic
diatoms Navicula salinarum and Cylindrotheca closterium were investigated. EPS was secreted into the medium and the bulk was loosely
associated with the cells. Neither N. salinarum nor C. closterium formed a well-defined polysaccharide capsule. EPS of both N. salinarum
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and C. closterium consisted predominantly of polysaccharide but small quantities of protein were present as well. EPS also contained uronic
acids and SO # groups. Analysis of monosaccharides using gas chromatography showed that for both species glucose and xylose were
%
the main constituents, but several other monosaccharides were present in smaller quantities. Two fractions of EPS were distinguished : a
small amount was secreted into the medium and a second fraction was extracted in water at 30 mC. For both species the two fractions
differed somewhat in composition, indicating that they represented two different types of EPS. The EPS produced by N. salinarum and by
C. closterium differed in their composition. The rate of EPS production in batch culture was highest during the transition from exponential
growth to stationary growth. Negatively charged groups such as uronic acids and sulphated sugars determine the adhesion capacity of
EPS and probably play an important role in the stabilization of intertidal sediments on which these diatoms grow and produce biofilms.
Key words : Cylindrotheca closterium, diatoms, EPS, extracellular polymeric substances, microphytobenthos, Navicula salinarum,
polysaccharide, sediment stabilization
Table 1. Concentration of polysaccharides in culture supernatant from Cylindrotheca closterium and Navicula salinarum, following different
parallel treatments of the cell pellet, and percentage recovery of cellular protein after extraction
C. closterium
Supernatant n.a. n.a. 4n30p0n26 99n41p22n73
Pellet Water 30 3n92p0n34 105n70p6n31
Pellet 0n5 M NaOH 20 4n73p0n27 71n39p9n60
Pellet 1 M NaCl 20 0n68p0n30 98n23p18n99
Pellet 0n1 M EDTA 20 0n58p0n78 107n90p23n68
N. salinarum
Supernatant n.a. n.a. 5n06p1n49 98n19p6n32
Pellet water 30 4n50p1n20 94n94p5n88
Pellet 0n5 M NaOH 20 6n88p1n26 45n49p4n82
Pellet 1 M NaCl 20 0n67p0n61 106n46p10n22
Pellet 0n1 M EDTA 20 2n26p0n47 112n10p11n65
Values are meanpSD of three samples from a late log-phase culture. All pellet treatments were incubated for 1 h.
n.a., not applicable.
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et al., 1967), with a salinity of 33 PSU. The diatoms were optimal procedure, several other treatments of the pellet
cultivated on a substratum of purified sea sand (Merck, remaining after centrifugation had been tested. All
Darmstadt, Germany) in 300 or 1000 ml glass Erlenmeyers extraction procedures were followed by microscopy to
at 20 mC. Cultures were illuminated at an incident check for cell lysis. Damaged or lysed cells could be
irradiance of 60 mol photons m# s", produced by recognized as empty frustules or as cells from which the
fluorescent tubes (Philips TLE 32\33) over a light : dark protoplasm had leaked out. In addition, the cellular content
cycle of 12 : 12 h. of protein was measured before and after extraction to
Cells were harvested either during exponential or establish whether any losses occurred during the ex-
during stationary growth, depending on the experiment. traction. All extracts were obtained by centrifugation for
To harvest the cells the culture was gently shaken by 15 min at 20 000 g. From all extracts polysaccharides were
hand, until all cells were in suspension. Subsequently, the isolated by overnight precipitation in cold (k20 mC) 80 %
sand was allowed to sediment before the cell suspension (v\v) ethanol and quantified with the phenol\H SO
# %
was poured off. Unless stated otherwise, samples were assay (Herbert et al., 1971). In one series of experiments,
taken in triplicate from one culture. Axenity of the cultures cell pellets from late log-phase cultures were extracted in
was checked by plating on agar\medium and by micro- four consecutive steps using commercially available
scopic observation. artificial seawater (Instant Ocean). Extraction lasted for 1 h
at 20 mC. In this experiment, polysaccharides were found
in the culture supernatant (medium) at a concentration of
Isolation of EPS
4n7 and 7n1 mg ml" for C. closterium and N. salinarum,
The cell suspension was centrifuged for 15 min at 20 000 g respectively. The four additional centrifugation steps in
at 10 mC. Centrifugation at a lower temperature resulted in Instant Ocean resulted in very low additional amounts of
cell lysis. The pellet was extracted in water for 1 h at polysaccharides. However, Alcian Blue staining clearly
30 mC. Tap water was used instead of distilled water to revealed that EPS was still associated with the cells.
avoid cell lysis or leakage. This extract was subsequently In another series of experiments, pellets were extracted
centrifuged for 15 min at 20 000 g and 10 mC. Polymers by the following solutions : (i) water, (ii) 0n05 M NaOH,
were isolated from the culture medium as well as from the (iii) 1 M NaCl or (iv) 0.1 M EDTA, either for 1 h at 30 mC
warm water extract by overnight precipitation in cold (i) or for 1 h at 20 mC (iiiv). Extraction with 0n1 M EDTA
(k20 mC) 80 % (v\v) ethanol. Because EPS was present in or 1 M NaCl yielded much less polysaccharide than
small amounts in the culture supernatant, this fraction had extraction in water (Table 1). Extraction with 0n5 M
to be pre-concentrated by ultrafiltration using a 3 kDa cut- NaOH resulted in cell lysis. To distinguish between the
off membrane (Filtron) prior to precipitation in ethanol for effects of temperature and water, pellets were subjected to
compositional analyses. The precipitated EPS was dried extraction for 1 h in water at 20 mC or 30 mC or to
under a flow of nitrogen gas and subsequently stored dry extraction in the growth medium at 30 mC (Table 2).
at k20 mC before analysis. The fraction obtained after Incubation of the cell pellet with medium at 30 mC yielded
centrifugation of a cell suspension was designated non- much less polysaccharide compared with incubation in
attached EPS , while the fraction obtained by extraction in water (Table 2). In water, the elevated temperature of
water for 1 h at 30 mC was designated attached EPS . 30 mC increased the amount of polysaccharides. Extraction
Prior to establishment of these extraction steps as the in water at 70 mC resulted in cell lysis (results not shown).
Characterization of EPS from epipelic diatoms 163
Table 2. Concentration of polysaccharides in culture supernatant from Cylindrotheca closterium and Navicula salinarum and following
different parallel treatments of the cell pellet
Polysaccharide (g ml")
Extraction Extraction Extraction
Fraction solvent time (min) temperature (mC) C. closterium N. salinarum
Hence, incubation of the pellet in water for 1 h at 30 mC 20 mC for 1 h. Glycogen was used as a reference from
resulted in the maximum amount of EPS, leaving the cells which the recovery was calculated. Another batch of
intact (Table 2). Microscopic observation of Alcian Blue- precipitated and non-precipitated carbohydrate was
stained pellets after water extraction showed that all EPS treated with 0.005 units -glucanase (Fluka) per ml acetate
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Table 4. Composition (weight %) of attached and non-attached EPS isolated from late log-phase cultures of Cylindrotheca closterium and
Navicula salinarum
C. closterium N. salinarum
Table 5. Monosaccharide composition (mole %) of attached and non-attached EPS and of intracellular carbohydrates from late log-phase
cultures of Cylindrotheca closterium and Navicula salinarum
C. closterium N. salinarum
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Fig. 3. Changes in concentrations of non-attached EPS (circles) Fig. 4. Changes in concentrations of non-attached EPS (circles)
and attached EPS (diamonds) during batch growth of and attached EPS (diamonds) during batch growth of
Cylindrotheca closterium, expressed on a per cell basis. Cylindrotheca closterium, expressed per unit protein.
damaged the cells. Extraction with water did not damage general storage carbohydrate in diatoms (Darley, 1977 ;
the cells. This was supported by microscopic observations, Myklestad, 1988). Xylose and mannose are important
by the low concentrations of protein found in the extracted components of the cell wall of diatoms. Mannose is also
EPS, and by the fact that there was no loss of protein from known as a compatible solute in diatoms (Paul, 1979).
the pellets. In addition, leakage of carbohydrates from the Monosaccharides occurring exclusively in extracellular
cells due to osmotic stress did not seem to have occurred, fractions were arabinose (both species) and galactose (N.
since virtually all the carbohydrates extracted were salinarum). It is difficult to compare the monosaccharide
polysaccharides, with no low molecular weight (LMW) composition of EPS with that of other diatom species.
carbohydrates present in the extract (data not shown), Mannose, rhamnose and galactose seemed to be the
indicating that release of LMW carbohydrates as osmo- dominant sugars in many cases. There are only a few
lytes had not taken place. Microscopy of samples stained reported studies in which EPS consisted mainly of glucose.
for polysaccharides also confirmed the extraction of EPS. Navicula subinflata produced EPS that contained 94 %
The efficient extraction by water is explained by the glucose (Bhosle et al., 1995). Capsular material from
presence of cations that form bridges with the anionic Nitzschia angularis consisted of glucose with trace amounts
groups of the polymers. It is proposed that water of rhamnose and xylose (Tokuda, 1969). It is clear from
establishes an exchange of ions between the polymers and many studies that monosaccharide composition of diatoms
the water, thereby releasing the bonds of the polymer to varies widely among species (Hoagland et al., 1993). Also,
the cell or other particles (Sutherland, 1980). It is likely it is probable that monosaccharide composition will
that this does not happen to the same extent with NaCl, change with growth status. This remains to be investi-
Downloaded by [179.7.115.156] at 06:46 04 August 2015
components. It is therefore proposed that the cell- D, A.W. (1994). Molecular-scale events influencing the macro-scale
cohesiveness of exopolymers. In Biostabilization of Sediment (Krumbein,
associated EPS is important for sediment binding and
W.E., Paterson, D.M. & Stal, L., editors), 135148. BIS Verlag.
stabilization. Since non-attached EPS was secreted into the E, L.A. & P-H, J.D. (1984). Diatom locomotion. In Progress
medium it will probably be washed out from the sediment in Phycological Research, Vol. 3 (Round, F.E. & Chapman, G., editors),
and consequently cannot contribute to sediment stability. 4788. Biopress, Bristol.
Moreover, much more attached than non-attached EPS E, A., K, H., D, J. & B$ , P. (1984). Glycogen content
and nitrogenase activity in Anabaena variabilis. Arch. Microbiol., 140 :
was produced. Secretion of EPS probably plays a role in 120125.
adhesion to facilitate locomotion (Edgar & Pickett-Heaps, F, M. & F, G.D. (1973). An electron-microscopic dem-
1984 ; Lind et al., 1997 ; Wang et al., 1997). However, the onstration of an acidic polysaccharide involved in the adhesion of a
results presented here and other reports (Bhosle et al., marine bacterium to solid surfaces. J. Gen. Microbiol., 74 : 325334.
H, D., P, P.J. & S, R.E. (1971). Chemical analysis of
1995 ; Sutherland et al., 1998) seem to indicate that, in
microbial cells. In Methods in Microbiology, Vol. 5B (Norris, J.R. &
addition, other factors such as growth phase control the Ribbons, D.W., editors), 209344. Academic Press, London.
amount of EPS produced. The role of nutrient depletion in H, K.D., R, J.R., G, M.R. & R, S.C. (1993).
growth and EPS secretion in cultures of C. closterium is Diatom extracellular polymeric substances : function, fine structure,
currently under investigation. However, more experi- chemistry and physiology. J. Phycol., 29 : 537566.
H, L.A. & P, R.J. (1971). The effect of inorganic nitrogen on
ments with field populations in their natural environment macromolecular synthesis by Thalassiosira fluviatilis Hustedt and Cyclotella
are necessary to confirm whether such factors also control nana Hustedt grown in batch culture. J. Exp. Mar. Biol. Ecol., 6 : 7178.
EPS production in the field. H, A.F., Z, R.G. & D, J.M. (1974). Quantitative
evidence concerning the stabilization of sediments by marine benthic
Downloaded by [179.7.115.156] at 06:46 04 August 2015
T, T.T. & H, K. (1971). Method for determination of the sulfate (1997). Extracellular matrix assembly in diatoms (Bacillariophyceae).
content of glycosaminoglycans. Anal. Biochem., 41 : 471476. II. 2,6-Dichlorobenzonitrile inhibition of motility and stalk production
T, H. (1969). Excretion of carbohydrate by a marine pennate diatom, in the marine diatom Achnanthes longipes. Plant Physiol., 113 :
Nitzschia closterium. Rec. Oceanogr. Works Jap., 10 : 109122. 10711080.
U, G.J.C., P, D.M. & P, R.J. (1995). The W, R., L, J.L., B, J. & Q, R.S. (1998). The first kiss :
measurement of microbial carbohydrate exopolymers from intertidal establishment and control of initial adhesion by raphid diatoms. J. Phycol.,
sediments. Limnol. Oceanogr., 40 : 12431253. 34 : 915.
V S, G.W.M., B, A., B, R.L., V M, F. & W, C. (1988). Bacterial extracellular polysaccharides. Can. J.
M, H.C.P. (1992). Separation of photosystem I and II from the Microbiol., 34 : 415420.
oxychlorobacterium (Prochlorophyta) Prochlorothrix hollandica and as- Y, M.L., D W, B., P, D.M. & S, L.J. (1994).
sociation of chlorophyll binding antennae with photosystem II. Biochem. Comparative structure, primary production and biogenic stabilization of
Biophys. Acta, 1102 : 220228. cohesive and non-cohesive sediments inhabited by microphytobenthos.
W, Y., Lu, J., M, J.-C., G, M.R. & H, K.D. Estuar. Coast. Shelf Sci., 39 : 565582.
Downloaded by [179.7.115.156] at 06:46 04 August 2015