Carbohydrate secretion by phototrophic communities in tidal sediments

ELSEVIER

Journal of Sea Research 42 (1999) 131–146

Carbohydrate secretion by phototrophic communities in tidal sediments B. de Winder a , N. Staats a , L.J. Stal b,Ł , D.M. Paterson c a

Laboratory for Microbiology, ARISE, University of Amsterdam, Nieuwe Achtergracht 127, 1018 WS Amsterdam, Netherlands b Netherlands Institute of Ecology–Centre of Estuarine and Coastal Ecology, P.O. Box 140, 4400 AC Yerseke, Netherlands c SERG, Gatty Marine Laboratory, University of St. Andrews, Fife KY16 8LB, Scotland, UK Received 8 April 1998; accepted 23 March 1999

Abstract Two different benthic phototrophic communities on tidal flats were investigated for their carbohydrate content and distribution. Carbohydrates were analysed as two operationally defined fractions, related to the difficulty of extraction from the sediment matrix. Water-soluble (colloidal) and EDTA-extractable (capsular) carbohydrates were measured in a cyanobacterial mat and a diatom biofilm. The chlorophyll-specific carbohydrate content of the two communities was very different. The diatom biofilm contained up to 100 times more colloidal carbohydrate than the cyanobacterial mat. The concentrations of colloidal carbohydrates in the diatom biofilm correlated with biomass (chlorophyll-a), but this was not the case with the carbohydrate in the EDTA extract. It is proposed that the capsular carbohydrates were probably recalcitrant to mineralisation and therefore accumulated in the sediment. Neither colloidal nor EDTA-extractable carbohydrate in the cyanobacterial mat correlated with chlorophyll-a. This was probably an artefact caused by the fact that approximately 50% of the chlorophyll-a in the mat was attributed to diatoms. The characteristics of extracellular polysaccharides were investigated in laboratory cultures of the dominant organisms. Extracellular polysaccharides of the cyanobacterium Microcoleus chthonoplastes and of the diatom Navicula menisculus did not contain uronic acids. However, carboxylated sugars were found in large quantities in the capsular polysaccharides of the cyanobacterium and were present in equal ratios in the extracellular and capsular carbohydrate of the diatom Cylindrotheca closterium. Both in laboratory model systems of diatom biofilms and in situ, enhanced colloidal carbohydrate production was observed in the light. No light-dependent increase in carbohydrate concentration was found for the cyanobacterial mat. The cyanobacteria formed a mat in which the filamentous organisms entangled sand grains and attached firmly to the substratum. The interparticle spaces were completely occluded by polymers, whereas in the diatom biofilm the organic matrix was less well developed and void spaces could still be discerned. It is conceived that the properties of extracellular polysaccharides influence the stability of the sediment bed.  1999 Elsevier Science B.V. All rights reserved. Keywords: benthic diatoms; cyanobacteria; microbial mat; EPS; carbohydrate secretion

1. Introduction Tidal sediment deposits often support abundant populations of phototrophic microorganisms (Stal et Ł Corresponding

author. Tel.: C31 113 577 497; Fax: C31 113 573616; E-mail: [email protected]

al., 1985; Underwood and Smith, 1998). Such populations may produce macroscopically recognisable microbial mats, often dominated by the cyanobacterium Microcoleus chthonoplastes or by visible biofilms of epipelic diatoms. On cohesive and noncohesive sediments, biological structures are often embedded in a thick mucilaginous matrix of extracel-

1385-1101/99/$ – see front matter  1999 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 5 - 1 1 0 1 ( 9 9 ) 0 0 0 2 1 - 0

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lular polymeric substances (EPS), which may consist of 90% or more of polysaccharides (Hoagland et al., 1993). This EPS has been implicated in the adhesion of microorganisms to sediment particles, thereby influencing the rheology and stability of the sediment bed (Paterson, 1989; Dade et al., 1990; Paterson, 1997; Ruddy et al., 1998a). It has also been shown that polysaccharides are more efficient agents of sediment–particle aggregation than other organic components of the sediment matrix, for instance humic and fulvic acids (Greenland et al., 1961). In the process of matrix formation, the initial step involves the secretion of a polymer to bridge the gap between the particles and the organism (Wetherbee et al., 1998). This allows the organism to adhere to the substratum. Scanning electron microscopy has shown that EPS occurs as a matrix or network of strands in the interparticle voids of the sediment (Chenu and Jaunet, 1992; Chenu, 1993; De´farge et al., 1996). The nature of the polymers visualised in sediments is strongly related to the SEM preparation technique and related sample dehydration (see Paterson, 1995). The polymers forming the matrix should be considered a diffuse medium that permeates the void spaces, and coats the particles. This process alters the size and shape of the grains, changes their potential to adhere to surfaces and affects the geometry of the sediment in such a way that organism–particle, particle–particle, and organism–organism aggregates are formed. The properties of the polymers that determine their capacity to adhere to surfaces depend on the involvement of several types of chemical bonding such as hydrogen bonding between neutral compounds and metal-bridging between ionogenic compounds (Stotzky, 1980; Decho, 1994). Yallop et al. (1994) assumed that the distribution and nature of EPS in the diatom biofilm and the filamentous nature of the cyanobacterial mat underlie the differences in sediment stability and response to physical forcing. Yet LTSEM images from that study also indicate that the infilling of void spaces by organic material was more extreme in the cyanobacterial mat. The present investigation aims to examine the EPS content of cyanobacterial mats in more detail and compare the EPS content of benthic assemblages dominated by cyanobacteria and diatoms and to investigate EPS production by selected representative

species. Special emphasis will be placed on (1) the secretion of extracellular carbohydrate polymers, (2) the nature of the secreted EPS, and (3) the influence of light on the secretion of the EPS by cyanobacteria and diatoms. The implications that EPS secretion might have for sediment stability will be discussed.

2. Material and methods 2.1. Study site The study site was situated on a tidal sandy beach of the Wadden Sea area of the Island of Texel (De Cocksdorp, 53º090 4500 N, 4º120 3000 E), the Netherlands (Fig. 1) (for details, see Yallop et al., 1994). The experimental station, characterised by mats of cyanobacteria, was situated at approximately mean high-water level. The sediment had an average particle size of 257 µm, sorting of 1300 µm and

Fig. 1. Map showing the location of the study site on the island of Texel, The Netherlands.

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skewness of 995 µm. The water content of the station was 20:7 š 0:1% (w=w) with a salinity of 30 psu. The diatom-biofilm station was situated 150 m seawards from the cyanobacterial station. At this lower site, the mean particle size was much smaller (154 µm, sorting of 486 µm, skewness of 1444 µm) and the water content was 23:8 š 0:9% (w=w) with a salinity of 30 psu. At the diatom station, over 20% of the sediment particles were smaller than 63 µm compared to less than 2% at the cyanobacterial station. Both stations were situated in an area of a gently sloping tidal flat. 2.2. Sampling methodology 2.2.1. Low-temperature scanning electron microscopy Sediments were visualised following the lowtemperature scanning electron microscopy (LTSEM) method of Paterson (1995). Sediments were sampled by inserting thin foil planchettes into the surface of the sediment. These were withdrawn and frozen in situ in liquid nitrogen ( 196ºC). The samples were kept frozen under liquid nitrogen until after examination on the specially adapted stage of a SEM (Oxford cryo-systems with Joel 35Fc SEM). Excess surface water was sublimed into vacuum by heat-etching at a temperature of 90ºC. Sediments were prepared for LTSEM for surface visualisation and by freeze-fracture to examine the internal structure of the sediment interface region (Paterson, 1995). 2.2.2. Sediment sampling The microbial mats at the site have been the subject of investigation since 1991 (Krumbein et al., 1994; Yallop et al., 1994). Cells for the recent culture studies were obtained in June and July 1995. Material for LTSEM analysis was obtained in 1991 and 1992 (Krumbein et al., 1994; Yallop et al., 1994). Samples for chemical analyses were taken with a stainless-steel corer (2.4 cm2 ). The top cm of the sediment was sampled and sub-sectioned into 1-mm slices. Five randomly taken cores were pooled for each depth interval to minimise the effects of patchiness. A time-series was initiated, the first samples being taken when the ebb waters had fallen to a depth of approximately 50 cm above the sediment and finished at tidal re-immersion of the station. Sample

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cores were sliced in the field and immediately cooled on ice and subsequently transported to the laboratory where they were stored in a freezer at 20ºC. Samples obtained during culture experiments in the laboratory were lyophilised and stored desiccated in sealed vials at 20ºC. 2.2.3. Mesocosm experiments Small mesocosm experiments were carried out by incubating sediment cores taken in PVC tubes (inner diameter 10 cm, height 15 cm) in thermostatically controlled aquariums. Cores from both stations were allowed to equilibrate overnight in the dark at 15ºC in a simulated natural tidal regime. Subsequently, when the sediment was exposed, randomly chosen parts of the surface of the two cores were kept in the dark while an equal surface area was incubated in the light (200 µmol m 2 s 1 , provided by a 50-W halogen lamp). After 6 h of incubation the cores were sampled as described above. 2.3. Organisms and culture conditions Axenic strains of the diatoms Navicula menisculus and Cylindrotheca closterium were grown in batch culture in Erlenmeyer flasks (250 cm3 ). The bottom of each flask was covered with a layer of acid-washed sea sand (Merck), which provided a substratum for the benthic diatoms. The cultures were grown in Kester medium (Kester et al., 1967) at 20ºC and under continuous light (white fluorescent lamps, Philips TLE 32=33) at a photon flux density of 30 µmol m 2 s 1 . C. closterium was obtained from H. Peletier (RIKZ, Haren, the Netherlands). This diatom was originally isolated from intertidal sediment of the Wadden Sea. N. menisculus was isolated from the experimental site. The cyanobacterium Microcoleus chthonoplastes strain SAG 3192 (Collection of Algae of the University of Go¨ttingen, Germany) was also grown in Erlenmeyer flasks (250 cm3 ) as axenic batch cultures in ASN-III medium (Rippka et al., 1979). This cyanobacterium was originally isolated from a Wadden Sea microbial mat. Cultures of M. chthonoplastes were incubated at 20ºC and under continuous light (white fluorescent lamps, Philips TLE 32=33) at a photon flux density of 30 µmol m 2 s 1 in a Gallenkamp (Sanyo-Gallenkamp, UK) illuminated orbital shaking incubator.

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2.4. Isolation of carbohydrate fractions 2.4.1. Field material The extraction of carbohydrate from intertidal sediments was performed as follows. Weighed lyophilised sediment samples were incubated with a known amount of distilled H2 O (typically 1 cm3 H2 O per 500 mg sediment) for 60 min at 30ºC in a water bath. Carbohydrate remaining in the supernatant after centrifugation for 10 min at 2800 g was referred to as the sediment colloidal fraction (SCF), and is identical to colloidal carbohydrate obtained by the method of Underwood et al. (1995). Subsequently, the remaining pellet was treated with 100 mM EDTA (final concentration), pH 4.0 at 20ºC for at least 4 h. The carbohydrates present in the supernatant after further centrifugation for 10 min at 2800 g were referred to as the sediment capsular fraction (SCAF). 2.4.2. Culture material Cells and culture medium were analysed after 4 to 6 days when the cells entered the late exponential phase of growth. Polymeric carbohydrates found in the supernatant after incubation at 30ºC for 60 min and subsequent centrifugation at 20,000 g at 4ºC for 15 min are referred to as the culture colloidal fraction (CCF). Depending on the organisms the remaining pellets were treated differently. The pellet of the diatom culture was suspended and incubated in water at 70ºC for 30 min. After centrifugation at 20,000 g for 15 min the polysaccharides remaining in the supernatant were referred to as the culture capsular fraction (CCAF). The remaining pellet of Microcoleus chthonoplastes trichomes was suspended in a 0.1 M potassium phosphate buffer, pH 7.2. This suspension was passed three times through a French

Press (4ºC, 80 MPa) and, subsequently, centrifuged at 20,000 g for 15 min. The supernatant was concentrated to a volume of about 10 cm3 by evaporation and subsequently treated with α-amylase and amyloglucosidase to assure the removal of glycogen (De Winder et al., 1990). Carbohydrate polymers in all extracted fractions were precipitated overnight at 4ºC in ice-cold ethanol at a final concentration of 75% (v=v). The precipitate was recovered by centrifugation (15 min, 20,000 g, 4ºC). Chrysolaminaran, the storage carbohydrate of diatoms, does not precipitate in ethanol. Table 1 provides a summary of carbohydrate fraction terminology. 2.5. Analyses 2.5.1. Monosaccharide analysis Freeze-dried extracellular polysaccharide (EPS) fractions of the cultures (CCF and CCAF) were hydrolysed and methylated in 0.6 M methanol=HCl, and trimethylsilylate derivatives were prepared (Chaplin, 1986). Mannitol was used as an internal standard. Samples were injected in split mode on a fused silica WCOT column (25 m by 0.32 mm) coated with CP Sil 5CB mounted in a Chrompack CP9000 gas chromatograph (Chrompack, The Netherlands). Oven temperature was programmed as follows: 10 min at 140ºC, then increasing, at 3ºC min 1 , to a final temperature of 220ºC for 5 min. 2.5.2. Pigments For the characterisation of the phototrophic community, freeze-dried sediment samples were assayed by HPLC (Van der Staay et al., 1992). All other field samples and culture material were extracted overnight in the dark with dimethylformamide (DMFA). Extinction was measured before and after acidifica-

Table 1 Summary of carbohydrate fraction terminology Source

Operational separation

Terminology

Indent.

Culture

Extracted in 30ºC water Extracted in 70ºC water or using French Press a

Colloidal fraction Capsular fraction

CCF CCAF

Sediment

Extracted in water Extracted with EDTA

Colloidal fraction Capsular fraction

SCF SCAF

a CCAF

from diatoms were extracted with water at 70ºC and from M. chthonoplastes by French Press (see Section 2).

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tion. Calculations were performed after Lorenzen and Newton Downs (1986). The values for K and R for DMFA were experimentally derived using purified chlorophyll-a and pheophytin from cyanobacteria and were estimated to be 2.26 and 1.80, respectively. Total carbohydrate was assayed by the phenolsulphuric acid method using glucose as a reference (Chaplin, 1986). Uronic acids were estimated by the meta-dihydroydiphenyl method (Blumenkrantz and Asboe-Hansen, 1973) using galacturonic acid as a reference.

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3. Results

The diatom biofilm developed a much less distinct matrix than the cyanobacterial mat assemblages (Fig. 3). Surface views showed dense assemblages of mainly naviculoid diatoms (Fig. 3A–B), while freeze-fracture of the surface indicated that the diatom biofilm was highly concentrated in the upper 100 mm of the sediment. Polymeric material was clearly visible, but unlike the cyanobacterial assemblage, the sediments were not fully saturated with polymer and void spaces were visible (Fig. 3C– D). Some evidence of other autotrophes was found (Fig. 3E–F) comprising isolated filaments of green algae (cf. Enteromorpha=Monostroma) and isolated bundles of cyanobacterial trichomes located beneath the diatom biofilm (Fig. 3E).

3.1. Visualisation of the sediments by LTSEM

3.2. Depth distribution of pigments

Low-temperature Scanning Electron Microscopy (LTSEM) was performed on samples from the sites to demonstrate the physical nature of the assemblage microstructure of the diatom and cyanobacterial mats (Yallop et al., 1994). While culture isolates were obtained in a different year, these perennial mats have been noted for many seasons and the structure seems stable. The Microcoleus chthonoplastes mat had clearly developed into a thick matrix at the sediment surface. The surface of the sediment was generally infilled with few particles visible (Fig. 2A) forming a coherent layer when the surface was viewed obliquely (Fig. 2B). In areas where the cyanobacterial mat was less developed, particles were more obvious at the surface (Fig. 2C). A fracture face through the cyanobacterial area confirms the observations of Yallop et al. (1994) that the upper interparticle spaces are completely filled and no separate sediment particle could be discerned (Fig. 2D). Lower in the mat, particles could be clearly seen and less organic material was evident (Fig. 2E). The bundle sheaths of Microcoleus filaments were arranged in twisted fibres often comprised of >50 individual trichomes. Freeze-fracture analysis of these bundles showed that the contents were mainly cellular and that EPS coatings were relatively thin (Fig. 2F). Analysis of the surface preparation gave little further information since the polymer layer was continuous, obscuring detail of the surface.

In both communities, the highest concentration of pigment was found in the layer between 0 and 6 mm (Fig. 4). In the diatom biofilm, the maximum pigment concentration was at the sediment surface, decreasing gradually with depth (Fig. 4, bottom). In this community, only pigments characteristic of diatoms were found, indicating that they were the only significant biomass of phototrophic organisms present. This was confirmed by microscopic examination. In the cyanobacterial mat, pigments were concentrated in the top 2 mm (Fig. 4, top). However, this station was shown to contain a mixed assemblage, since a considerable amount of fucoxanthin and other diatom-specific pigments were found. Assuming a ratio of fucoxanthin to chlorophyll-a of 1 : 2, a similar value to that found in the top layers of the diatom biofilm, then 50% of the chlorophyll-a present in the mat of cyanobacteria originates from the diatoms. In both communities, significant amounts of pigment were found at depths below the photic zone. The photic zone is defined as the depth at which light intensity is reduced to 1% of incident intensity, which was reached at a depth of 1.2 mm at both stations, as was measured by fibre-optic light probes (data not shown). Microscopic examination of the phototrophic community in the diatom biofilm showed abundant populations of Navicula spp. (Fig. 3B) and Cylindrotheca closterium. Both epipelic (mostly navicu-

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loids) and epipsammic diatoms were present. Mats of cyanobacteria were dominated by Microcoleus chthonoplastes. It was estimated that this species accounted

for up to 80% of the total biovolume of the cyanobacteria. Other species that were present belonged to the genera Oscillatoria, Lyngbya, and Phormidium.

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When the sediment was exposed, the diatoms migrated to the surface, which was evidenced by the brown coloration. In contrast, no such migration or change in coloration was observed in the mat of cyanobacteria. 3.3. Extracellular carbohydrates The diatom-dominated sediment contained considerably higher amounts of colloidal carbohydrates than the sediment from the cyanobacterial station (Table 2). On a sediment dry-weight basis, the diatom community contained up to 100 times more colloidal carbohydrate than the cyanobacterial assemblage. However, when normalised to chlorophyll-a, as a phototrophic biomass parameter, the difference was considerably smaller. In this case, the diatom community contained about 10 times as much biomass-specific colloidal carbohydrate as the cyanobacteria. The differences in EDTA-extractable carbohydrates were not so large. The diatom community contained 2 to 3 times as much of these polysaccharides. This material was therefore relatively more important in the mat of cyanobacteria. Representatives of the dominant species from each phototrophic community were cultured to characterise the carbohydrates secreted (Table 3). The three organisms that were studied yielded two operationally defined types of carbohydrate. The two species of diatoms secreted most carbohydrate as the CCF. This fraction differed in composition between the two species investigated. In Navicula, glucose was quantitatively the most important with some small amounts of galactose and rhamnose. The colloidal carbohydrate of Cylindrotheca was more complex in composition. Another difference between the two species was the absence of uronic acids in Navicula. A small amount (5% of the total amount

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of EPS) was more firmly associated with the cell and was considered to be capsular (CCAF). In the two species, this capsular material was composed of different monomeric sugars. The most remarkable difference was the absence of uronic acids from the N. menisculus CCAF. The cyanobacterium also secreted EPS into the medium, but most of the extracellular polysaccharide was capsular (CCAF). This material, which accounted for 65% of the total EPS, contained a variety of different monomeric sugars. Uronic acids were found only in the capsular polysaccharides. The colloidal EPS of M. chthonoplastes (CCF) was composed virtually exclusively of glucose with small amounts of galactose. 3.4. Depth distribution of carbohydrates Fig. 5 depicts the typical depth distribution of colloidal carbohydrate and chlorophyll-a in the cyanobacterial mat. This graph shows that both colloidal carbohydrate and chlorophyll decreased with depth but this decrease was more pronounced in the case of carbohydrate, suggesting that the parameters were not directly correlated. The variation of the ratio of colloidal and EDTA-extracted carbohydrates over chlorophyll-a with depth is depicted in Fig. 6. If the ratio is constant over depth, a close coupling between the production of carbohydrates and the biomass of the phototrophic microorganisms may be assumed. This hypothesis was tested for all data (F test, P < 0:05), Table 4). In the diatom community the colloidal carbohydrate fraction correlated well with chlorophyll-a at all depths (Fig. 6B). This relationship was shown in four independent samples (Table 4) (Fig. 6B). In all samples of the diatom community, the EDTAextractable carbohydrates increased with depth (pos-

Fig. 2. Low-temperature scanning electron micrographs of the intertidal sediment dominated by a mixed cyanobacterial=diatom assemblage. (A) Surface view of infilling between surface sediment grains (bar marker 50 µm). (B) An oblique view of the surface showing the confluent nature of the matrix and the lack of protruding grains (bar marker 100 µm). (C) Surface view of an area of sediment where cyanobacteria were less prevalent. The surface grains are more apparent and the infilling is less complete (bar marker 250 µm). (D) A freeze fracture face through the surface region of a cyanobacterial mat. The upper (surface) area is occluded by polymeric material which becomes less evident with depth (bar marker 250 µm). (E) Deeper in the sediment (>2.5 µm), the organic material produced by the biofilm is no longer present and individual grains are clear (bar marker 250 µm). (F) A fracture face through a Microcoleus bundle. The filaments are closely associated with a thin EPS layer both around the individuals and around the sheath (bar marker 2.5 µm).

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Fig. 3. Low-temperature scanning electron micrographs of the intertidal sediment at station 2 (diatom biofilm). (A) Low-power general view of sediment surface showing scattered diatoms on the sediment matrix. Bar marker 200 µm. (B) Detail of an area showing nitzschiod and naviculoid cells on the surface. Bar marker 200 µm. (C) Fracture face through the surface region. Note open pore space and thin surface biofilm. Bar marker 200 µm. (D) Detail of the card-house packing of the sub-surface sediment. (E) Enteromorpha strand among the sediment. (F) Fracture bundle of cyanobacterial filament (cf Oscillatoria sp.). Bar marker 50 µm in (D), (E) and (F).

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ularly the case for colloidal carbohydrates (negative value in Table 4). For EDTA-extractable carbohydrate this is less clear. The colloidal carbohydrate content in the diatom biofilm was found to be highly dynamic (Fig. 7). During exposure of the sediment (low tide) to the light, the amount of colloidal carbohydrates increased dramatically, particularly in the top layer of the sediment. However, the increase of carbohydrates was not only restricted to the euphotic zone. This was evidently due to EPS synthesis and=or secretion by the diatoms that migrate to the surface during exposure. During the subsequent period of inundation most of this carbohydrate disappeared and the concentration returned to its initial level (not shown). 3.5. Effect of light on carbohydrate production

Fig. 4. Depth distribution of the most abundant pigments in the mat of cyanobacteria (top) and in the diatom biofilm (bottom). The amounts of pigments are expressed in µg per g dry weight of sediment.

itive value in Table 4). In the upper 4 mm of the mat of cyanobacteria, the opposite was found (Fig. 6A). The chlorophyll-a-specific amounts of colloidal carbohydrates and the EDTA-extractable carbohydrates were both higher in the top layers of the sediment (Fig. 6A) and decreased with depth. This is particTable 2 A comparison of carbohydrate content in two fractions (SCF D sediment colloidal fraction; SCAF D sediment capsular fraction) of tidal benthic phototrophic communities

Cyanobacteria mat Diatom film

SCF (mg g 1 )

SCAF (mg g 1 )

SCF=chl-a

0.1–0.5 0.5–50

0.5–1.2 1.5–2.5

1–20 2–200

Numbers indicate the range of values for the top 5 mm .n > 20/.

The increase in the concentration of SCF carbohydrate in the diatom biofilm was dependent on light. When a sediment core with a biofilm of diatoms was transferred from the dark to light, a dramatic increase of colloidal carbohydrate was observed after 6 h of incubation. In the control experiment, kept in the darkness, this increase was not observed (Fig. 8). The same experiment was carried out with a mat of cyanobacteria and no increase in colloidal carbohydrate was found (Fig. 9) in the light or in darkness.

4. Discussion 4.1. Assemblage structure and composition Low-temperature scanning electron microscopy is the technique chosen to preserve the structure of hydrated samples (Jeffree and Read, 1991). LTSEM visualisation of the sediment retains structural water and therefore preserves the natural fabric of the matrix, including the EPS polymers of interest in the present study (De´farge et al., 1996; De´farge, 1997). The composition of the two phototrophic communities differed markedly. The biofilm of diatoms occurred on relatively fine-grained sediment, whereas the mat of cyanobacteria developed on much coarser sediment. It has been suggested that cyanobacteria cannot cope with the more mobile and dynamic co-

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Table 3 Composition of the carbohydrates isolated from cultured organisms Total a

Cyanobacteria M. chthonoplastes Diatoms C. closterium N. menisculus

Uronic acid b

Monomer composition c gl

ga

ar

ma

rh

1

1

1

2 1

1 1

2

2

CCF CCAF

35 65

0 100

9 4

1 8

CCF CCAF CCF CCAF

95 5 95 5

50 50 0 0

4 4 8 3

8 1 2

xy

g.a.

6 2

1 1 1

4 6

2

The numbers indicate the percentage of carbohydrate. The sum of colloidal and capsular carbohydrates set as 100%. The monomer composition is expressed as a relative contribution of the specific compound. a Assayed with the phenol-sulphuric acid method using glucose as a reference (Chaplin, 1986). b Assayed with the meta-dihydroydiphenyl method (Blumenkrantz and Asboe-Hansen, 1973) using galacturonic acid as a reference. c Assayed as trimethylsilylate derivatives using gas chromatography (Chaplin, 1986): gl D glucose; ga D galactose; ar D arabinose; ma D mannose; rh D rhamnose; xy D xylose; g.a. D galacturonic acid.

Table 4 Correlation of colloidal- (SCF) and EDTA-extractable (SCAF) carbohydrate with chlorophyll-a in vertical profiles in the diatom biofilm and the mat of cyanobacteria Diatom biofilm

Cyanobacteria mat

samples:

1

2

3

4

1

2

3

4

SCF SCAF

ns 2.21

ns 4.41

ns 2.44

ns 3.32

1.44 ns

1.64 ns

1.48 ns

ns ns

5

6 3.34 3.00

1.86 2.83

The test was carried out for 4 and 6 independent samples in the diatom biofilm and the mat of cyanobacteria, respectively. A positive value indicates an increase of carbohydrate with respect to chlorophyll-a with depth and a negative value indicates a decrease. The value indicates the slope of the line from linear regression carbohydrate over chlorophyll-a with depth. ns indicates a non-significant difference from a constant ratio carbohydrate-over-chlorophyll-a at all depths (F test, P < 0:05).

hesive sediments and develop only in the translucent, fine-grained, non-cohesive quartz sediments (Stal, 1994). However, it should be noted that the microorganisms themselves alter the sediment, including its grain size and porosity (Wachendo¨rfer et al., 1994). A critical point is the relative infilling of the void spaces depicted by LTSEM. The matrix was far more continuous and developed in the cyanobacterial mat than in the diatom biofilm where it was much more restricted and patchy (Figs. 2 and 3). On the basis of the LTSEM images and carbohydrate analysis, it can be concluded that the nature of the material occluding the void can be identified as colloidal carbohydrate for the diatom assemblages (SCF) and as capsular carbohydrate for the cyanobacterial assemblages (SCAF). Examination of fracture surfaces

(e.g. Wachendo¨rfer et al., 1994; Yallop et al., 1994) suggests that the cyanobacterial infilling is more extensive (deeper) and more complete than that produced by diatom assemblages on their own. This conclusion is also supported by the analytical studies conducted. The diatom biofilm contained much more colloidal than capsular carbohydrate, while the opposite was found in the cyanobacterial mat (Table 2). Both microphytobenthic communities formed clearly recognisable, self-sustaining structures that are the result of growth processes rather than of sedimentation. They have been considered analogues to animal or plant tissues by Wachendo¨rfer et al. (1994), who suggests the term ‘parahistology’ as the appropriate technique to study microbial mats and biofilms. Our results confirm the view of Wachendo¨rfer et al.

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Fig. 5. Depth distribution of colloidal carbohydrate (––) and chlorophyll-a (–ž–) concentrations in the cyanobacterial mat. Colloidal carbohydrate is the fraction in the supernatant after water extraction at 30ºC for 60 min. Amounts are expressed in µg per g dry weight of sediment.

(1994) that EPS polymers produced by the microorganisms play a key role for the formation of a matrix, influence the porosity of the sediment, and enhance the deposition of fresh sediments (Underwood and Paterson, 1993). 4.2. Distribution of carbohydrate in the sediment The measurement of carbohydrate fractions showed variations in their distribution and concentration in the two assemblages. In the light, epipelic diatoms migrate to the surface as soon as the sediment is exposed at low tide (Pinckney and Zingmark, 1991). This was also observed in the diatom biofilm investigated in the present study as judged from a change in the colour of the sediment surface from grey to brown. Motility of diatoms is associated with the secretion of polysaccharides (Edgar and Picket-Heaps, 1984). Accumulation of polysaccharides in the diatomaceous sediment was observed 3 h after exposure (Fig. 7). The major

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Fig. 6. Depth distribution of the ratio of colloidal (squares) and EDTA-extractable (circles) carbohydrates to chlorophyll-a in a cyanobacterial mat (A) and a diatom biofilm (B).

increase in SCF was in the surface layers although not exclusively. SFC may be transported below the euphotic depth by diffusion or micro-bioturbation of the surface layers by meiofauna such as harpactecoid copepods and nematodes. Because most of the migration occurred during the first couple of hours after exposure of the sediment (Paterson, 1989), the accumulation of EPS cannot be ascribed exclusively to migration of the diatoms although cell motility may continue on the sediment surface. The rate of accumulation of colloidal EPS in the sediment samples cannot be due solely to growth of the diatoms. In cultures, a maximum growth rate of the diatoms of 1 d 1 was measured (Staats, unpubl. observ.). Hence, this would contribute only little to the five-fold increase in carbohydrate that was observed during the 6-h period of exposure (Fig. 7). It is likely that other factors were responsible for the increase in EPS. For example, the massive accumulation of diatoms at the sediment surface at

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Fig. 7. Depth distribution and time series of contents (mg per g sediment) of colloidal carbohydrates in the top 1 cm of the sediment with a diatom biofilm during a 7-h period of low tide. The first measurement was done at time 14.00 h, which was 30 min before the sediment was exposed. The light period lasted till 23.00 h.

low tide would quickly deplete nutrients and growth would become nutrient-limited (Kromkamp et al., 1995). It has been shown that CO2 can be limiting under high light intensity for dense diatom biofilms (Lorenzen, 1996). Nutrient limitation in phototrophic organisms often results in the over-production of carbohydrate caused by unbalanced growth (OrtegaCalvo and Stal, 1994). Ruddy et al. (1998a,b) report nutrient mass balances from field data of benthic diatoms that also support this hypothesis, and they have developed a model that demonstrates that nutrient limitation in diatom biofilms is very likely to happen. In fact, the energy dissipation that is achieved by secretion of carbohydrate may serve an important function, for instance to protect the diatoms from photo-oxidative damage. The cyanobacterial mat contained much less carbohydrate and did not show such dynamic changes.

An explanation for this could be that nutrient limitation is less likely, because these mats are known to fix nitrogen (Stal et al., 1984). The depth variations of the ratios of carbohydrates to chlorophyll-a in the diatom biofilm and the cyanobacterial mat were notably different (Fig. 6). In the diatom biofilm the ratio of colloidal carbohydrate to chlorophyll-a was constant over depth. This is in agreement with the observations of Underwood and Smith (1998), who demonstrate that EPS concentrations in intertidal mudflats can be predicted from sediment chlorophyll-a. The ratio of EDTA-extractable carbohydrate to chlorophyll-a in the diatom biofilm increased with depth. It is possible that this carbohydrate is more recalcitrant to mineralisation and accumulates to some extent. In the upper 4 mm of the cyanobacterial mat, a strong decrease of colloidal and EDTA-extractable carbohydrate to chlorophyll-a ratio occurred. Below 4 mm these carbohydrate fractions behaved as in the diatom biofilm. This complex behaviour of carbohydrate in the cyanobacterial mat can be explained by the high diatom biomass. On the basis of pigment ratios it can be calculated that about 50% of the chlorophyll-a could be attributed to diatoms. However, the diatoms were relatively more abundant in the surface layers (Fig. 4). This and the much higher chlorophyll-specific carbohydrate content of diatom biofilm (Table 2) would explain the pattern observed. Below 4 mm depth in the cyanobacterial mat the ratio of carbohydrate to chlorophyll-a is largely attributed to cyanobacteria. Hence, as in diatoms, the ratio of colloidal carbohydrate to chlorophyll-a in cyanobacteria seems to be constant, although considerably lower (Table 2). A contributory factor in EPS distribution may be the changes in sediment structure with depth (Taylor and Paterson, 1998). As the bulk density of the sediment increases with depth there is less pore water and hence less colloidal material can accumulate. In addition, bulk density of the sediment also controls light availability, which is particularly important for EPS dynamics in diatom-dominated systems (Figs. 7 and 8). 4.3. Consequences of carbohydrate dynamics for sediment stability The importance of biology in determining the response of sediments to physical forcing is being in-

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Fig. 8. Vertical profiles of the changes in the content of colloidal carbohydrates in the diatom biofilm (A) after incubation for 6 h in the light (200 µmol m 2 s 1 ), and (B) in the dark (control), compared to the initial situation obtained after one night in the dark. The sediments were subject to an artificial tidal cycle in a thermostat aquarium at 15ºC. During the 6-h light period the sediment surface was exposed (low tide).

Fig. 9. Vertical profiles of the changes in the content of colloidal carbohydrates in the mat of cyanobacteria (A) after incubation for 6 h in the light (200 µmol m 2 s 1 ), and (B) in the dark. Further details as in Fig. 8.

creasingly recognised (Wachendo¨rfer et al., 1994; Paterson, 1997). The presence of benthic communities of phototrophic microorganisms is known to cause

an increase in the mechanical stability of the sediment (Neuman et al., 1970; Grant and Gust, 1987). It has been suggested that extracellular polysaccharides

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are responsible for adhesion and aggregate formation and for the increase in the critical threshold for erosion (Paterson, 1989; Dade et al., 1990; Underwood and Paterson, 1993). Also factors that influence biological processes have been shown to alter sediment stability. For example it has been shown that light increases the erosion threshold value for tidal sediments (Paulic et al., 1986; Madsen et al., 1993). These observations strongly suggest that benthic diatoms have a significant impact on the resistance to erosion and the stability of tidal sediments. In other studies, it has been shown that the colloidal fraction of EPS correlates well with the biovolume or biomass of epipelic diatoms (Underwood and Paterson, 1993; Underwood and Smith, 1998; Taylor and Paterson, 1998). As shown in this study, a considerable proportion of the colloidal carbohydrates produced by the diatoms (SCF) are soluble in water and therefore may disappear as soon as the tide comes in. The colloidal carbohydrates may therefore contribute only to a limited extent to sediment stability. However, in shallow water the maximum tidal-flow-induced stress experienced by a mudflat bed occurs shortly after immersion in the flood phase of the cycle (Christie and Dyer, 1998). Thus under non-wave-dominated conditions, the sediment may still be protected by colloidal EPS. In addition to the colloidal carbohydrates (SCF), the EDTA-extractable fraction (SCAF) may have an important role that has not been fully recognised. These carbohydrates contain negatively charged groups and although uronic acids were apparently absent in Navicula menisculus, preliminary evidence suggests that this EPS contains sulphated sugars (Staats, unpubl. observ.). It seems reasonable to assume that the EDTA fractions play a prominent role in the formation of sediment–sediment and sediment–organism interactions. Sutherland (1980) and Geddie and Sutherland (1993) have demonstrated the co-precipitation and gel formation of seawater polysaccharides with metal ions, and a small amount of this carbohydrate gave rise to very strong bonding, especially when they became irreversibly denatured and dehydrated (Foster, 1981). The operational separation between SCF and SCAF has its limitations. The change of polymers between the gel and sol states may be governed by the availability of metal ions and=or desiccation history (Decho, 1994) and therefore have a relatively large influence on sediment stability.

Acknowledgements This investigation was supported by the Netherlands Organisation for Scientific Research (NWO) (BOA research theme ‘The morpho-dynamic and bio-dynamic behaviour of mud in tidal areas’) and by the European Commission Environment and Climate Programme contract EV5V-CT94-0411 (Pro-Mat) and Marine Science and Technology Programme, contract MAS3-CT95-0022 (INTRMUD). The authors would also like to thank Mrs. N. Dijkman for analytical help, Dr. J. Huisman for help on statistics, and Dr. C. George for LTSEM analysis (Fig. 3). This is Publication 2517 of the Centre of Estuarine and Coastal Research, Yerseke, The Netherlands.

References Blumenkrantz, N., Asboe-Hansen, G., 1973. New method for quantitative determination of uronic acids. Anal. Biochem. 54, 484–489. Chaplin, M.F., 1986. Monosaccharides. In: Chaplin, M.F., Kennedy, J.F. (Eds.), Carbohydrate Analysis, a Practical Approach. IRL Press, Oxford, pp. 1–36. Chenu, C., 1993. Clay- or sand-polysaccharide associations as models for the interface between micro-organisms and soil: water related properties and microstructure. Geoderma 56, 143–156. Chenu, C., Jaunet, M., 1992. Cryoscanning electron microscopy of microbial extracellular polysaccharides and their association with minerals. Scanning 14, 360–364. Christie, M.C., Dyer, K., 1998. Measurements of the turbid tidal edge over the Skeffling mudflats. In: Black, K.S., Paterson, D.M., Cramp, A. (Eds.), Sedimentary Processes in the Intertidal Zone. Geol. Soc. London, Spec. Publ. 139, 45–55. Dade, W.B., Davis, J.D., Nichols, P.D., Nowell, A.R.M., Thistle, D., Trexler, M.B., White, D.C., 1990. Effects of bacterial exopolymer adhesion on the entrapment of sand. Geomicrobiol. J. 8, 1–16. Decho, A.W., 1994. Molecular-scale events influencing the macro-scale cohesiveness of exopolymers. In: Krumbein, W.E., Paterson, D.M., Stal, L.J. (Eds.), Biostabilization of Sediment. BIS Verlag, Oldenburg, pp. 135–148. De´farge, C., 1997. Apports du cryo-microscope e´lectronique a` balayage et du microscope e´lectronique a` balayage haute re´solution a` l’e´tude des matie`res organiques et des relations organo-mine´rales naturelles. Exemple des se´diments microbiens actuels. C.R. Acad. Sci. Paris 324, Ser. IIa, 553–561. De´farge, C., Trichet, J., Jaunet, A., Robert, M., Tribble, J., Sansone, F.J., 1996. Texture of microbial sediments revealed by cryo-scanning electron microscopy. J. Sediment. Res. 66, 935–947. De Winder, B., Stal, L.J., Mur, L.R., 1990. Crinalium epipsam-

B. de Winder et al. / Journal of Sea Research 42 (1999) 131–146 mum sp. nov.: a filamentous cyanobacterium with trichomes composed of elliptical cells and containing poly-β-(1,4) glucan (cellulose). J. Gen. Microbiol. 136, 1645–1653. Edgar, L.A., Picket-Heaps, J.D., 1984. Diatom locomotion. Progr. Phycol. Res. 3, 47–88. Foster, R.C., 1981. Polysaccharides in soil fabrics. Science 214, 665–667. Geddie, J.L., Sutherland, I.W., 1993. Uptake of metals by bacterial polysaccharides. J. Appl. Bacteriol. 74, 467–472. Grant, J., Gust, G., 1987. Prediction of coastal sediment stability from photopigment content of mats of purple sulphur bacteria. Nature 330, 244–246. Greenland, D.J., Lindstrom, G.R., Quirk, J.P., 1961. Role of polysaccharides in stabilisation of natural soil aggregates. Nature 191, 1283–1284. Hoagland, K.D., Rosowski, J.R., Gretz, M.R., Roemer, S.C., 1993. Diatom extracellular polymeric substances: function, fine structure, chemistry, and physiology. J. Phycol. 29, 537– 566. Jeffree, C.E., Read, N.D., 1991. Ambient- and low-temperature scanning electron microscopy. In: Hall, J.L., Hawes, C. (Eds.), Electron Microscopy of Plant Cells. Academic Press, London, pp. 313–413. Kester, D.R., Duedall, I.W., Connors, N., Pytkowics, R.M., 1967. Preparation of artificial seawater. Limnol. Oceanogr 12, 176– 179. Kromkamp, J., Peene, J., Van Rijswijk, P., Sandee, A., Goosen, N., 1995. Nutrients, light and primary production by phytoplankton and microphytobenthos in the eutrophic, turbid Westerschelde estuary (The Netherlands). Hydrobiologia 311, 9–19. Krumbein, W.E., Paterson, D.M., Stal, L.J. (Eds.), 1994. Biostabilization of Sediment. BIS Verlag, Oldenburg. Lorenzen, C.J., Newton Downs, J., 1986. The specific absorption coefficients of chlorophyllide a and pheophorbide a in 90% acetone and comments on the fluorometric determinations of chlorophyll and pheopigments. Limnol. Oceanogr. 31, 449– 456. Lorenzen, J., 1996. Begrænsende Faktorer for Mikrofytobentisk Fotosyntese. Unpublished PhD thesis, University of Aarhus. Madsen, K.N., Nilsson, P., Sundback, K., 1993. The influence of benthic microalgae on the stability of a subtidal sediment. J. Exp. Mar. Biol. Ecol. 170, 159–177. Neuman, A.C., Gebelein, C.D., Scoffin, T.P., 1970. The composition, structure and erodibility of subtidal mats, Abaco, Bahamas. J. Sediment. Petrol. 40, 274–297. Ortega-Calvo, J.J., Stal, L.J., 1994. Sulphate-limited growth in the N2 -fixing unicellular cyanobacterium Gloeothece (Nageli) sp PCC 6909. New Phytol. 128, 273–281. Paterson, D.M., 1989. Short-term changes in the erodibility of intertidal cohesive sediments related to the migratory behaviour of epipelic diatoms. Limnol. Oceanogr. 34, 223–234. Paterson, D.M., 1995. The biogenic structure of early sediment fabric visualised by low-temperature scanning electron microscopy. J. Geol. Soc. 152, 131–140. Paterson, D.M., 1997. Biological mediation of sediment erodibility: ecology and physical dynamics. In: Burt, N., Parker, R.,

145

Watts, J. (Eds.), Cohesive Sediments. Wiley, Chichester, pp. 215–229. Paulic, M., Montague, C.L., Mehta, A.J., 1986. Influence of light on sediment erodibility. In: Shen, H.W. (Ed.), Third Int. Symp. River Sedimentation, Mississippi, pp. 1758–1764. Pinckney, J., Zingmark, R.G., 1991. Effects of tidal stage and sun angles on intertidal benthic microalgal productivity. Mar. Ecol. Prog. Ser. 76, 81–89. Rippka, R., Deruelles, J., Waterbury, J.B., Herdman, M., Stanier, R.Y., 1979. Generic assignments strain histories and properties of pure cultures of cyanobacteria. J. Gen. Microbiol. 111, 1– 61. Ruddy, G., Turley, C.M., Jones, T.E.R., 1998a. Ecological interaction and sediment transport on an intertidal mudflat, I. Evidence for a biologically mediated sediment–water interface. In: Black, K.S., Paterson, D.M., Cramp, A. (Eds.), Sedimentary Processes in the Intertidal Zone. Geol. Soc. Spec. Publ. 139, 135–148. Ruddy, G., Turley, C.M., Jones, T.E.R., 1998b. Ecological interaction and sediment transport on an intertidal mudflat, II. An experimental dynamic model of the sediment–water interface. In: Black, K.S., Paterson, D.M., Cramp, A. (Eds.), Sedimentary Processes in the Intertidal Zone. Geol. Soc. Spec. Publ. 139, 149–166. Stal, L.J., 1994. Microbial mats in coastal environments. In: Stal, L.J., Caumette, P. (Eds.), Microbial Mats. Structure, Development and Environmental Significance. NATO ASI Ser. G35, Springer, Heidelberg, pp. 21–32. Stal, L.J., Grossberger, S., Krumbein, W.E., 1984. Nitrogen fixation associated with the cyanobacterial mat of a marine laminated microbial ecosystem. Mar. Biol. 82, 217–224. Stal, L.J., Van Gemerden, H., Krumbein, W.E., 1985. Structure and development of a benthic marine microbial mat. FEMS Microbiol. Ecol. 31, 111–125. Stotzky, G., 1980. Surface interactions between clay minerals and microbes, viruses and soluble organics, and the probable importance of these interactions to the ecology of microbes in soil. In: Berkeley, R.C.W., Lynch, J.M., Melling, J., Rutter, P.R., Vincent, B. (Eds.), Microbial Adhesion to Surfaces. Ellis Horwood, Chichester, pp. 231–247. Sutherland, I.W., 1980. Polysaccharides in the adhesion of marine and freshwater bacteria. In: Berkeley, R.C.W., Lynch, J.M., Melling, J., Rutter, P.R., Vincent, B. (Eds.), Microbial Adhesion to Surfaces. Ellis Horwood, Chichester, pp. 329– 338. Taylor, I.S., Paterson, D.M., 1998. Microspatial variation in carbohydrate concentrations with depth in the upper millimetres of intertidal cohesive sediments. Estuarine Coastal Shelf Sci. 46, 359–370. Underwood, G.J.C., Paterson, D.M., 1993. Seasonal changes in diatom biomass, sediment stability and biogenic stabilisation in the Severn estuary. J. Mar. Biol. Assoc. UK 73, 871–887. Underwood, G.J.C., Smith, D.J., 1998. Predicting epipelic diatom exopolymer concentrations in intertidal sediments from sediment chlorophyll a. Microbiol. Ecol. 35, 116–125. Underwood, G.J.C., Paterson, D.M., Parkes, R.J., 1995. The

146

B. de Winder et al. / Journal of Sea Research 42 (1999) 131–146

measurement of microbial carbohydrate exopolymers from intertidal sediments. Limnol. Oceanogr. 40, 1243–1253. Van der Staay, G.W.M., Brouwer, A., Baard, R.L., Van Mourik, F., Matthijs, H.C.P., 1992. Separation of photosystems I and II from the oxychlorobacterium (prochlorophyte) Prochlorotrix hollandica and association of chlorophyll b binding antennae with photosystem II. Biochim. Biophys. Acta 1102, 220–228. Wachendo¨rfer, V., Krumbein, W.E., Schellnhuber, H.J., 1994. Bacteriogenic porosity of marine sediments — a case of biomorphogenesis of sedimentary rocks. In: Krumbein, W.E.,

Paterson, D.M., Stal, L.J. (Eds.), Biostabilization of Sediment. BIS Verlag, Oldenburg, pp. 203–220. Wetherbee, R., Lind, J.L., Burke, J., Quatrano, R.S., 1998. The first kiss: establishment and control of initial cohesion by raphid diatoms. J. Phycol. 34, 9–15. Yallop, M.L., De Winder, B., Paterson, D.M., Stal, L.J., 1994. Comparative structure, primary production and biogenic stabilisation of cohesive and non-cohesive marine sediments inhabited by microphytobenthos. Estuarine Coastal Shelf Sci. 39, 565–582.