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The production of extracellular carbohydrates by estuarine benthic diatoms: the effects of growth phase and light and dark treatment

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David J. Smith

Department of Biological Sciences, University of Essex, Colchester, United Kingdom

Author for correspondence; Department of Biological Sciences, John Tabor Laboratories, University of Essex, Colchester, Essex, CO4 3SQ, U.K.; email [email protected]; fax 00-44 -206-873416.Search for more papers by this author

Graham J. C. Underwood,

Graham J. C. Underwood

Department of Biological Sciences, University of Essex, Colchester, United Kingdom

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David J. Smith,

Corresponding Author

David J. Smith

Department of Biological Sciences, University of Essex, Colchester, United Kingdom

Graham J. C. Underwood,

Graham J. C. Underwood

Department of Biological Sciences, University of Essex, Colchester, United Kingdom

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First published: 25 December 2001

https://doi.org/10.1046/j.1529-8817.2000.99148.x

Citations: 158

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Abstract

Epipelic diatoms are important constituents of estuarine microphytobenthic biofilms. Field-based investigations have shown that the production of carbohydrates by such taxa is ecologically important. However, limited information exists on the dynamics of carbohydrate production by individual species of epipelic diatoms. The production of low and high molecular weight extracellular carbohydrates in axenic cultures of five species of benthic estuarine diatoms, Cylindrotheca closterium (Ehrenberg), Navicula perminuta (Grun.) in Van Heurck, Nitzschia frustulum (Kütz.) Grunow, Nitzschia sigma (Kütz.) Grunow, and Surirella ovata (Kütz.) Grunow, were investigated. All species produced colloidal (water-soluble) carbohydrates during growth, with maximal production occurring during stationary phase. During logarithmic growth, approximately 20% of extracellular carbohydrates consisted of polymeric material (extracellular polymeric substances [EPS]), but during stationary phase, EPS content increased to 34%–50%. Pyrolysis–mass spectrophotometry analysis showed differences in the composition of EPS produced during logarithmic and stationary phase. All species synthesized glucan as a storage carbohydrate, with maximum glucan accumulation during the transition from log to stationary phase. Short-term labeling with ¹⁴C-bicarbonate found that between 30 and 60% of photoassimilates were released as colloidal carbohydrate, with EPS consisting of approximately 16% of this colloidal fraction. When cells were placed in darkness, EPS production increased, and between 85 and 99% of extracellular carbohydrate produced was polymeric. Glucan reserves were utilized in dark conditions, with significant negative correlations between EPS and glucan for N. perminuta and S. ovata. Under dark conditions, cells continued to produce EPS for up to 3 days, although release of low molecular weight carbohydrates rapidly ceased when cells were dark treated. Three aspects of EPS production have been identified during this investigation: (1) production during rapid growth, which differs in composition from (2) EPS directly produced as a result of photosynthetic overflow during growth limiting conditions and (3) EPS produced for up to 3 days in the dark using intracellular storage reserves (glucans). The ecological implications of these patterns of production and utilization are discussed.

Abbreviations:

EPS: extracellular polymeric substances
Py-MS, pyrolysis: mass spectrophotometry

Epipelic (mud dwelling c.f.Round 1971 diatoms are the dominant microphytobenthos in many intertidal, soft-sediment habitats (Admiraal 1984, Underwood 1994, Smith and Underwood 1998). Microphytobenthic biofilms exhibit high rates of primary production (up to 300 g C·m⁻²·y⁻¹; MacIntyre et al. 1996, Underwood and Kromkamp, 1999 and can contribute up to 50% of estuarine primary production (Underwood and Kromkamp 1999). Epipelic diatoms are motile and migrate through the sediment in response to tidal and diurnal rhythms (Happey-Wood and Jones 1988, Serôdio et al. 1997, Paterson et al. 1998, Smith and Underwood 1998), appearing at the surface of the sediment during periods of emersion and migrating into the sediment when the sediment becomes immersed. Epipelic diatoms move through the production and extrusion of carbohydrate-rich, long-chained heteropolymers (Edgar and Pickett-Heaps 1983, 1984, Hoagland et al. 1993, Lind et al. 1997, Wustman et al. 1997), termed extracellular polymeric substances (EPS c. f.Geesey 1982. As well as playing a functional role in motility, this EPS is important for biogenic stabilization of intertidal and subtidal sediments (Paterson 1989, Sutherland et al. 1998a, b) and as a carbon source for bacteria and invertebrates (Decho 1990, Underwood and Smith 1998).

The extracellular carbohydrate exudates of microphytobenthos can be broadly grouped into low molecular weight, small sugar units and glycollates and larger, increasingly polymeric molecules (EPS). Collectively, this material is termed “colloidal carbohydrate” (Underwood et al. 1995, Smith and Underwood 1998, Sutherland et al. 1998b, Taylor and Paterson 1998, Staats et al. 1999, Underwood et al. 1999), and high concentrations of colloidal carbohydrates can be present on mudflats (typically between 50 and 5000 μg·g⁻¹ sediment; Underwood and Smith 1998. Although other organisms also produce extracellular carbohydrates (notably bacterial exopolymer capsules; Decho 1990, colloidal carbohydrates present in microphytobenthic biofilms are closely related to microalgal biomass and photosynthetic activity (Underwood and Paterson 1993a, b, Underwood et al. 1995, Smith and Underwood 1998, Sutherland et al. 1998b, Taylor and Paterson 1998, Underwood and Smith 1998). The relative composition and production rates of low and high molecular weight components in colloidal carbohydrate varies over tidal cycles and with light intensity (including darkness), nutrient availability, and the taxonomic composition of the biofilm (Underwood 1997, Smith and Underwood 1998, Staats et al. 1999, Taylor et al. 1999). The EPS component of the sediment colloidal carbohydrate pool is usually between 20 and 30% (Underwood et al. 1995, Underwood and Smith 1998). The remaining low molecular weight fractions of colloidal carbohydrate (i.e. the nonpolymeric component) are likely to be a labile form of carbon for sediment-inhabiting bacteria and bacterioplankton (Decho 1990, Smith 1998, Underwood and Smith 1998, Taylor et al. 1999), more so than the long chained, refractory polymeric fraction (EPS) (Sundh 1992, Karner and Rassoulzadegan 1995).

There is extensive literature on the growth dynamics and carbohydrate production of different taxonomic groups of algae, but the majority of these studies have focused on marine planktonic or attached, biofouling species (for a review, see Hoagland et al. 1993. Despite the ecological importance of epipelic species and their associated carbohydrates, there is limited information in the literature regarding the growth dynamics and carbohydrate production of benthic diatom species (Sutherland et al. 1998b, Underwood and Smith 1998, Staats et al. 1999). Whereas some studies have shown that intracellular storage carbohydrate (glucan) is utilized in the synthesis of EPS during periods of darkness and tidal cover (Smith and Underwood 1998, Underwood and Smith 1998), other studies have found EPS production to be a light-dependent process (Staats et al. 2000). The chemical composition and physical characteristics of EPS may also change during different stages of growth, with composition being dependent on the functional properties of the polymers (Decho 1994, Wustman et al. 1997, Staats et al. 1999). Yet, comparison between studies is often complicated by differences in experimental design and extraction procedures, and further field and laboratory investigations of epipelic diatom carbohydrate production are required.

This study examined the production dynamics of both intracellular and extracellular carbohydrate fractions in five different species of epipelic diatom over culture growth cycles and under conditions of continuous light and darkness. Pyrolysis–mass spectrometry (Py-MS) was used to characterize the composition of diatom EPS during different phases of the growth curve. Py-MS generates mass spectra of pyrolyzed material, enabling chemical differences between samples to be detected. This technique has been successfully used to characterize EPS produced by aquatic bacteria (Ford et al. 1991) but has not been applied previously to the analysis of diatom exudates.

Materials and methods

Establishment of experimental axenic monocultures.

Five species of epipelic diatoms; Cylindrotheca closterium (Ehrenberg), Navicula perminuta (Grun.) in Van Heurck, Nitzschia frustulum (Kütz.) Grunow, Nitzschia sigma (Kütz.) Grunow, and Surirella ovata (Kütz.) Grunow, were isolated from lens tissue samples of natural assemblages from Alresford Creek, Essex (51° 50.2′ N, 0° 59.5′ E), a branch of the Colne Estuary (Underwood et al. 1998). Each species was isolated on two separate occasions (“isolates”) to permit interclonal comparisons. Once monocultures had been established by repeated selection and subculturing, a small inoculum of cells (<20) was transferred to conical flasks containing f/2 media (Guillard and Rhyther 1962) at a salinity of 20 (Tropical Marin, Betta Aquatics, Colchester, U.K.) and was incubated at 20° C and a light (30 μmol·m⁻²·s⁻¹)/dark regime of 14 and 10 h, respectively (conditions also used for experiments). Subculturing was conducted each time until mid- to late-logarithmic phase was reached.

Axenic inocula for experimental cultures were made by the addition of diatoms (always <100 cells) to filter-sterilized (cellulose-acetate filters, pore size 0.2 μm; Fisher Scientific) f/2 media containing the antibiotic cocktail “BASH” (penicillin benzypenicilin; Sigma Chemicals Co., St. Louis, Missouri) 63 ng·mL⁻¹ final concentration, streptomycin (streptomycin sulphate; Calibiochem-Novabiochem Co.) 67.5 ng·mL⁻1 final concentration (unit ratio 3.74:1 penicillin:streptomycin). Bacterial contamination was determined by phase-microscopy and growth on nutrient agar plates inoculated with subsamples of experimental cultures and incubated for 2 weeks at 20° C. Any contaminated material was discarded. Once axenic cultures reached mid- to late-logarithmic phase, they were centrifuged at 1810g for 30 min and resuspended in fresh f/2 + BASH (“axenic diatom culture”). This was necessary to ensure minimal cross-contamination of experimental chambers by previously produced extracellular carbohydrates. Under sterile conditions, 4 mL of filter-sterilized f/2 + BASH was added to replicated experimental chambers (“replidishes” 100 × 100 mm, 25 chambers per dish, maximum volume 5 mL·chamber⁻¹; Bibby Sterilin, Stone, U.K.), which were subsequently inoculated with 0.5 mL of axenic diatom culture (see Table 1 for starting cell densities and chl a concentrations).

Table 1. Initial (day 0) cell densities and chl a concentrations in batch cultures of five epipelic diatom species. Means ± SE (n = 5).

Species	1	2	1	2
	Cell densities (mL) Isolate		Chl a (μm·mL⁻¹) Isolate
Cylindrotheca closterium	1400 ± 100	1050 ± 150	0.05 ± 0.01	0.06 ± 0.02
Navicula perminuta	2550 ± 200	2150 ± 70	0.16 ± 0.03	0.11 ± 0.006
Nitzschia frustulum	2500 ± 50	2190 ± 128	0.13 ± 0.01	0.1 ± 0.01
Nitzschia sigma	1500 ± 150	1400 ± 170	0.11 ± 0.02	0.12 ± 0.011
Surirella ovata	1050 ± 100	900 ± 50	0.092 ± 0.021	0.08 ± 0.01

Fractionation of carbohydrates.

Four carbohydrate fractions were measured (colloidal carbohydrate, EPS, glucan [intracellular storage carbohydrate], and “residual” cellular material) by using the following procedures. Culture material was removed from repli-chambers and placed in graduated centrifuge tubes, and the volumes were recorded (this removed> 95% of the cellular and extracellular material; Smith 1998. Samples were thoroughly mixed, and a 1-mL subsample was taken for chl a analysis. An additional 0.25-mL subsample was taken for cell counts using a haemocytometer; this subsample was appropriately diluted or concentrated to give statistically correct counts. Chl a (corrected for phaeopigments) was measured spectrophotometrically after overnight extraction in cold (4° C) methanol, followed by centrifugation (3620g for 15 min) (Underwood et al. 1998).

Colloidal carbohydrate, EPS, and PCP.

The remaining culture material was centrifuged at 3620g for 15 min, and 1 mL of the supernatant containing colloidal carbohydrate (Underwood et al. 1995) was removed and analyzed for carbohydrate with the phenol/H₂SO₄ assay (Dubois et al. 1956). EPS were precipitated from an additional 2 mL of supernatant by addition to cold (4° C) alcohol (Industrial Metholated Spirits [IMS]), resulting in a final concentration of 70% IMS. EPS were allowed to precipitate for 24 h (Underwood et al. 1995). Following centrifugation at 3620g for 15 min, the EPS pellet was resuspended in 3 mL of water of ultra high purity, conductivity <10 μS). One milliliter of this EPS solution was stored at −22° C for Py-MS analysis, whereas the remaining 2 mL was analyzed for carbohydrate using the phenol/H₂SO₄ assay. The percentage of the colloidal fraction that was polymeric (PCP) was calculated for each replicate sample from the concentrations of colloidal and EPS carbohydrate.

Glucan and residual and total carbohydrate.

The pellet of original cellular material was resuspended in 4 mL 0.05 M H₂SO₄, mixed thoroughly, and left for 2 h. The samples were mixed every 30 min, and at the end of the 2 h extraction period they were centrifuged at 3620g for 15 min. The carbohydrate content of this supernatant was termed the glucan fraction and was measured using the phenol/H₂ SO₄ assay (Myklestad and Haug 1972). The resulting pellet after glucan extraction was resuspended in 3 mL 0.05 M H₂SO₄ and mixed thoroughly, and 2 mL was analyzed for residual carbohydrate concentrations. Concentrations obtained from this residual fraction were summed with concentrations of colloidal carbohydrate and glucan to give total carbohydrate concentration. Preliminary measurements found no significant loss of material, with the extraction procedure being> 95% efficient when compared with direct measures of total culture carbohydrates (Smith 1998).

Pyrolysis mass spectrometry.

Analysis of EPS samples (4 μL desiccated) were performed on foils consisting of 50% iron and 50% nickel (Curie point = 530° C) loaded into pyrolysis sample tubes (Horizon Instruments, Heathfield, Surrey, U.K.). The ion mass-to-charge ratio of the samples were determined with Py-MS (Horizon Instruments Py-MS-200X). Curie-point pyrolysis (see Goodacre and Berkeley 1991 for full description) was performed at an equilibrium temperature of 530° C for 4 s in a vacuum. The expansion chamber and collimating tube were heated to 150° C. Ionization of the pyrolysate was by low energy (30 eV) electron impact, and the ions were separated in a quadruple mass spectrometer, scanning the pyrolysate 160 times at 0.2-s intervals from initiation of pyrolysis. Integrated ion counts at unit mass intervals from 51 to 200 Da were stored on a computer.

Experimental design: effects of growth phase on intra- and extracellular carbohydrate concentrations.

Cultures were sampled on six occasions over a full growth curve period. At each sample time, the first being day 0, five replicates were sampled and analyzed as described above. Sampling always occurred at 10.00 h to control for diel variations in carbohydrate concentrations. Sampling intervals and duration of the experiments varied between species to accommodate the different growth rates (see Fig. 1).

Details are in the caption following the image — **Figure 1**
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Chlorophyll a concentrations (▪) and living cells (○) in axenic batch cultures of two separate isolates of (A) *Cylindrotheca closterium*, (B) *Navicula perminuta*, (C) *Nitzschia frustulum*, (D) *Nitzschia sigma*, and (E) *Surirella ovata* grown in f/2 media (salinity 25) subjected to 14-h-light (30 μmol·m⁻²· s⁻¹)/10-h-dark treatment (25/15° C, respectively). Mean values ± SE (n = 5). Where SE is not shown, it was smaller than the symbol.

Experimental design: effects of light and dark treatments on intra- and extracellular carbohydrate production.

Experimental cultures of C. closterium, N. perminuta, and S. ovata were established as described previously and were left to grow to either transitional or early stationary phase depending on their carbohydrate dynamics. Short-term changes in incorporation and allocation of photoassimilated ¹⁴C-labeled bicarbonate were investigated over a 4-h period. Longer term effects of sustained light and dark treatment were measured over a 7-d period.

Incorporation of H¹⁴CO₃ into carbohydrate fractions.

To each of the replicate cultures, 0.5 mL of NaH¹⁴CO₃ (37 KBq·mL⁻¹) was added, and all cultures were incubated in the light (30 ± 5 μmol· m⁻²·s⁻¹) for 1 h. Replicate samples (n = 5) were taken at 30 and 60 min. After this 1-h illuminated assimilation period, 20 culture units (4 × 5) were placed in darkness and the remaining 20 cultures remained illuminated. Sampling occurred at 15 and 30 min, 1, 2, and 3 h after the assimilation period. Before sampling, 0.5 mL of 10% glutaraldehyde (1% v/v final concentration) was added to the culture unit to prevent further assimilation or reallocation of ¹⁴C. Carbohydrate fractions were extracted as outlined above. To remove unincorporated H¹⁴CO₃⁻, all extracts were acidified with 50-μL concentration HCl per 0.5-mL extract and degassed under an airflow for 24 h. Four milliliters of scintillation fluid (Optiphase Safe, Fisher Scientific, Loughborough, U.K.) were added, and samples were counted (Wallace LKb, Turku, Finland with internal quench correction) after 24 h. The following controls were also established: (1) dark controls, which received no light during the assimilation period (control for heterotrophic and anaplerotic uptake); (2) dead controls (1% v/v glutaraldehyde) to control for passive/abiotic uptake); and (3) unlabelled controls to determine background radiation. Values for abiotic and background counts were subtracted from light and dark treated counts during the analyses, but insignificant anaplerotic uptake was found.

Long-term effects of light and dark treatment.

Cultures of C. closterium, N. perminuta, and S. ovata were grown to either transitional or early stationary, and five replicates were sampled for carbohydrates to determine starting concentrations. Replicate cultures were then allocated to either dark or continuous light treatment (7 × 5 replicates for each treatment). Sampling occurred every day (always at 10.00 h for the reasons previously mentioned) for a 7-day-period.

Statistical analyses.

All data were tested (F-test) for heteroscadicity, and where found, statistical analyses were performed on transformed (log N+ 1; Zar 1984 data. Comparisons over time and between species were made by analyses of variance (one- and two-way) followed by post hoc analyses (Tukey test) to establish the significantly different groups. Stationary phase was indicated by no further significant increases in biomass (cell density and chl a concentrations), as determined by one-way ANOVA and post hoc analyses (Tukey test). Raw data derived from Py-MS analyses were analyzed by the Genstat statistical package (Oxford, U.K.), the first stage of analyses being normalization which removed the effect of sampling size. Principle components analyses were performed to reduce the data for canonical variate analyses, the form in which the data are presented.

Results

Effects of growth phase on biomass, carbohydrate concentrations, and carbohydrate production rates.

Although cell densities and chl a concentrations varied between the five species investigated, there were no significant differences between isolates of the same species. Specific growth rates varied significantly with time and, with the exception of Nitzschia sigma (P < 0.05), did not differ between isolates for any of these species studied. Maximum specific growth rates (μ) ranged between 0.51 and 1.07, with Nitzschia frustulum having a significantly (P < 0.001) lower maximum specific growth rate (μ) and generation time than the other four taxa (Table 2). In stationary phase cultures, there were significant differences (P < 0.001) in the concentration of chl a per cell, with a higher chl a:cell content in cultures of Cylindrotheca closterium (5.9 ± 0.30 pg·cell⁻¹) and Surirella ovata (5.2 ± 0.26) and lower ratios in cultures of N. frustulum (3.2 ± 0.18) compared with Navicula perminuta (5.7 ± 0.38 pg·cell⁻¹) and N. sigma (4.0 ± 0.11).

Table 2. Maximum specific growth rates and generation times of five epipelic diatom species grown in axenic batch cultures and subjected to a L:D cycle of 14 h of light (30 μmol·m⁻²·s⁻¹):10 h of dark (25 and 15° C, respectively). Means ± SE (n = 5).

Species	1	2	Mean	1	2	Mean
	μ maximum (in day units) Isolate			G(h) Isolate
Cylindrotheca closterium	0.68 ± 0.07	0.81 ± 0.07	0.72 ± 0.05	25.32 ± 2.63	21.19 ± 1.76	23.25 ± 1.65
Navicula perminuta	0.77 ± 0.06	0.73 ± 0.02	0.74 ± 0.02	21.86 ± 1.47	22.73 ± 0.45	22.3 ± 0.74
Nitzschia frustulum	0.51 ± 0.02	0.53 ± 0.03	0.53 ± 0.02	32.63 ± 1.20	31.83 ± 1.71	32.23 ± 0.99
Nitzschia sigma	1.07 ± 0.04	0.71 ± 0.04	0.89 ± 0.05	15.52 ± 0.63	23.64 ± 1.28	19.58 ± 1.31
Surirella ovata	0.68 ± 0.05	0.71 ± 0.02	0.70 ± 0.02	25.09 ± 2.27	23.39 ± 0.73	24.24 ± 1.16

Total carbohydrate concentrations (Fig. 2) increased over the course of the experiment and were generally highest during stationary phase (ranging between 41.67 ± 2.13 and 177 ± 9.57 μg·mL⁻¹ for S. ovata and C. closterium, respectively). However, in cultures of C. closterium, due to a large decrease in total carbohydrates (of 19 and 11 μg·mL⁻¹·d⁻¹ for the two isolates) during stationary phase, highest concentrations were measured on day 8 (transitional phase).

Colloidal carbohydrate concentrations generally increased over the course of the growth curve for all species (P <0.001) (Fig. 2). There were some significant differences in colloidal carbohydrate concentration during the growth curve between the two isolates of C. closterium and S. ovata (P < 0.001 and P < 0.01, respectively) (Fig. 2). Unlike in the other four taxa, concentrations of colloidal carbohydrate in C. closterium cultures decreased significantly during stationary phase. Maximum production rates (Table 3) did not vary between isolates of the same species, although the time within the growth curve at which maximum production occurred did (i.e. N. perminuta, Fig. 2B). In cultures of N. frustulum and S. ovata, production rates of colloidal carbohydrates were similar through the growth curve, resulting in an approximate linear increase in concentration (r ² = 0.69, 0.62, and 0.81, n = 60, 30, and 30, P < 0.001 for N. frustulum and the two isolates of S. ovata, respectively). This resulted in an average production rate for N. frustulum of 2.8 ± 0.24 μg·mL⁻¹·d⁻¹ (derived from regression coefficient) compared with a maximum rate of 10.1 ± 0.63 μg·μg chl a⁻¹(Table 3) and of 2.68 ± 0.04 μg·mL⁻¹· d⁻¹ for S. ovata. Cultures of C. closterium had maximum production rates (Table 3) during late logarithmic/transitional phase. Maximum rates of production of colloidal carbohydrate in N. sigma cultures occurred during stationary phase (see Fig 2D and Table 3).

Table 3. Maximum production rates of colloidal carbohydrates, EPS, and glucan and maximum utilization rates of glucan in axenic batch cultures of five epipelic diatom species subjected to a L:D cycle of 14 h of light (30 μmol·m⁻²·s⁻¹):10 h of dark (25 and 15° C, respectively). Means ± SE (n = 5).

Species	Maximum production rates (μg glucan equivalent·mg chl⁻¹·d⁻¹)			Maximum production rates (pg glucan equivalent·cell⁻¹·d⁻¹)
Species		Colloidal	EPS	Glucan	Colloidal	EPS	Glucan
Cylindrotheca closterium	8.68 ± 1.06	4.69 ± 0.20	17.64 ± 0.52	54.08 ± 5.70	31.46 ± 1.23	120.18 ± 4.55
Navicular perminuta	15.91 ± 3.58	3.76 ± 0.38	3.91 ± 0.28	128.39 ± 31.32	21.60 ± 2.19	12.89 ± 0.88
Nitzschia frustulum	10.06 ± 0.63	2.03 ± 0.23	2.30 ± 0.37	42.17 ± 2.40	6.31 ± 0.68	6.32 ± 0.98
Nitzschia sigma	16.51 ± 3.04	5.13 ± 0.42	4.59 ± 0.32	61.33 ± 9.87	21.20 ± 1.54	14.90 ± 1.02
Surirella ovata	24.47 ± 1.05	1.64 ± 0.12	3.01 ± 0.24	65.84 ± 3.38	9.48 ± 0.72	15.52 ± 1.16

Maximum production rates of colloidal carbohydrates varied significantly between species (P < 0.01) with N. perminuta having the highest production rate per cell and N. frustulum the lowest (Table 3). Differences in chl a:cell concentrations (see above) meant that colloidal production rates normalized to chl a did not necessarily give the same pattern as rates normalized to cell density (Table 3).

Colloidal carbohydrate extracts contained both polymeric (EPS) and nonpolymeric material. PCP values were significantly higher (P < 0.05) in cultures of N. sigma compared with the other taxa, and for all species there was a significant increase in the PCP during stationary phase (ranging between 34%± 2.8 and 50%± 3.2 for N. perminuta and N. sigma, respectively).

Concentrations of EPS (Fig. 3, I) significantly increased over the growth curve (P < 0.001), with no significant differences between isolates of the same species. EPS concentrations were always highest within stationary phase, when concentrations ranged between 5.7 ± 0.76 and 26.3 ± 1.48 μg·mL for N. frustulum and C. closterium, respectively. There were, however, differences in the pattern of EPS production over the course of the growth curve. EPS concentrations increased linearly with time in cultures of C. closterium and N. perminuta (r = 0.86 and 0.91, respectively, n = 30, P < 0.001). However, EPS concentrations in N. frustulum, N. sigma, and S. ovata cultures remained very low until transitional or early stationary phase, at which time there was a peak in EPS production 1, 3. C. closterium exhibited the highest maximum rate of EPS production (Table 3), with significantly lower maximum production rates by N. frustulum and S. ovata (ANOVA followed by Tukey test, P < 0.05).

The Py-MS ion mass-to-charge ratio spectra derived from logarithmic and stationary phase EPS samples were analyzed using canonical variate analyses, which defines distances between samples and is a form of discriminate analyses (Ford et al. 1991). For all five species of diatoms, Py-MS separated out logarithmic phase EPS (including starting samples and mid-logarithmic and late logarithmic growth phases) from the EPS present during stationary phase (Fig. 3, II). With the exception of N. frustulum (Fig. 3, II, C), the first canonical vector separated EPS samples derived from logarithmic and stationary growth phases. The second canonical vector separated samples derived from cultures of N. frustulum. There was a larger variation in mass-to-charge ratio in samples derived from logarithmic compared with stationary growth phases. In all cases, the first and second canonical vectors explained> 85% of the variation within data sets.

With all five diatom species, concentrations of glucan were highest as cultures entered stationary phase (ranging between 12.6 ± 2.7 and 26.3 ± 1.48 μg·mL⁻¹ for N. sigma and N. perminuta, respectively), after which time concentrations decreased. There was no significant difference between isolates. Maximum glucan production rates (Table 3) generally occurred as cultures were entering stationary phase. C. closterium had significantly higher rates of production than did the other species (P < 0.001 and P < 0.01, respectively). Decreases in glucan concentration tended to correspond to increased EPS concentrations for all species. The maximum utilization rates of glucan were, on average, 52.1 ± 11.1% of maximum glucan production rates.

Effects of light and dark treatments on intra- and extracellular carbohydrate production

The initial chl a concentrations and assimilation of H¹⁴CO₃⁻ (dpm) after the 1-h illuminated “assimilation” period are shown in Table 3. There were no subsequent significant changes in chl a concentrations during the 3-h experiment. After 1 h, between 30 and 60% of the assimilated ¹⁴C was present in the colloidal fraction, the remainder being either glucan (8–22%) or residual within the cell pellet.

On continued illumination, there were significant (all P < 0.001) increases in the ¹⁴C activity of both the colloidal and total carbohydrate fractions for all three species (Figs. 4A and 4D). These increases were linear (r ² colloidal/total: 0.74/0.72 and 0.94/0.94 for C. closterium and N. perminuta, respectively) except for S. ovata in which a plateau was reached. In darkened cultures, there was no significant increase in ¹⁴C activity in the colloidal fraction of C. closterium and N. perminuta(Fig. 4A), whereas with S. ovata, there was a increase during the first 15 min of darkness, after which no further increases occurred (Fig. 4A).

Incorporation by C. closterium of ¹⁴C into EPS was linear both in the light (r ² = 0.78) and in the dark (r ² = 0.51, P < 0.001 in both cases) (Fig. 4B). More activity was present in EPS in the illuminated than in the dark treatment after 3 h, but the percentage of total ¹⁴C in EPS was higher in the dark (Table 4). The increase of ¹⁴C-labelled EPS treatment in the dark accounted for 98% of the observed increase in the colloidal fraction but only 10% of the increase in colloidal in the light (Table 4). With N. perminuta, ¹⁴C-labelled EPS increased significantly in the dark treatments before the increase in light treatment (Fig. 4B). “Dark” EPS production contributed 99% of the observed increases in dark ¹⁴C colloidal activity. After 3 h, ¹⁴C activity was significantly greater in the light compared with the dark EPS fractions. A similar rapid increase in ¹⁴C activity in dark EPS was observed with S. ovata(Fig. 4B). This increase contributed 85% of the increase in dark ¹⁴C colloidal activity (Table 4).

Table 4. Starting chl a concentrations and assimilation of H¹⁴CO₃⁻ (dpm) into carbohydrate fractions of epipelic diatoms subjected to light conditions (30 ± 5 μmol·m⁻²·s⁻¹) for 1 h, followed by 3 h of continual light or dark treatment. Mean values ± SE (n = 5).

Species	chl a (μg·mL)	Carbohydrate fraction	dpm μg·chl a⁻¹·h⁻¹	% of total	Light	Dark
			Assimilation after 1 h light		Allocation of ¹⁴C after a 3-h treatment (% of total)
C. closterium	0.91 ± 0.04	Colloidal	615 ± 58	62 ± 2	46.2 ± 1.7	69.3 ± 3.0
		EPS	98 ± 9	10 ± 1	22.3 ± 2	27.6 ± 1.6
		(contribution of EPS to change in colloidal)	NA		(10%)	(98%)
		Glucan	118 ± 6	12 ± 1	25.2 ± 1.5	7.4 ± 1
		Total	983 ± 81
N. perminuta	1.02 ± 0.09	Colloidal	806 ± 46	60 ± 6	70.6 ± 1.9	47.4 ± 3.9
		EPS	118 ± 9	9 ± 1	13.9 ± 1.2	13.9 ± 1.2
		(contribution of EPS to change in colloidal)	NA		(12%)	(99%)
		Glucan	117 ± 16	8 ± 1	10.6 ± 0.8	10.9 ± 1.7
		Total	1719 ± 254
S. ovata	0.96 ± 0.01	Colloidal	532 ± 69	31 ± 5	36 ± 4	15 ± 2
		EPS	66 ± 7	5 ± 1	10 ± 1	5 ± 1
		(contribution of EPS to change in colloidal)	NA		(42%)	(85%)
		Glucan	338 ± 28	22 ± 4	30 ± 4	16 ± 1
		Total	2258 ± 463

NA, not applicable.

¹⁴C activity in glucan increased significantly in all light treatments to greater levels than present in dark treatments (Fig. 4C). All three diatom species showed an increase in ¹⁴C-glucan in the dark treatments immediately after the assimilation period. This was followed by a decline in labeled glucan in C. closterium and S. ovata (with final glucan ¹⁴C activities> 25% of the total label by the end of the experiment), although glucan activity levels remained constant with N. perminuta (Fig. 4C, Table 3).

Long-term effects

For the three species examined (Fig. 5), total carbohydrate concentrations increased linearly in light-treated cultures (r ² = 0.77, 0.73, and 0.89 for C. closterium, N. perminuta, and S. ovata, respectively, n = 40, P < 0.001) with time (P < 0.001) and were significantly higher than dark-treated cultures (P < 0.001). Total carbohydrate production rates were 16.47 ± 1.48, 8.73 ± 0.83, and 17.88 ± 1.00 μg·mL⁻¹·d⁻¹ for C. closterium, N. perminuta, and S. ovata, respectively. Cultures placed in continuous darkness showed an increase in total carbohydrate concentrations during the first 2 days (8 days after inoculation) in cultures of C. closterium and 3 days (8 days after inoculation) in cultures of N. perminuta; thereafter concentrations did not vary significantly. With S. ovata cultures, there was still a linear increase in total carbohydrate concentration (r ² = 0.71, n = 40, P < 0.001) but only at 4.24 ± 0.44 μg·mL⁻¹·d⁻¹, 25% of the rate predicted from light-treated cultures.

There were linear increases in colloidal concentrations of light-treated cultures (r ² = 0.87, 0.82, and 0.91 in cultures of C. closterium , N. perminuta, and S. ovata, respectively, n = 40, P < 0.001) (production rates of 15.06 ± 0.97, 6.14 ± 0.46, and 10.60 ± 0.54 μg·mL⁻¹·d⁻¹ for C. closterium, N. perminuta, and S. ovata, respectively). In dark-treated cultures of C. closterium and N. perminuta, there was no continuous linear production of colloidal carbohydrates: concentrations only increased significantly in the first 2–3 days of the experiment (8 days after inoculation). Concentration of colloidal carbohydrates of dark-treated cultures of S. ovata did increase linearly (r ² = 0.86, n = 40, P < 0.001), the linear model predicting a production rate of 5.2 ± 0.35 μg·mL⁻¹·d⁻¹, which was approximately half the rate of light production.

PCP was significantly higher in darkened compared with light-treated cultures (P < 0.001 in all cases) (Fig. 5). In darkened cultures, PCP increased significantly with time and was therefore highest at the end of the experiment (65.0 ± 1.2, 44.4 ± 3.6, and 38.6 ± 2.6% for C. closterium, N. perminuta, and S. ovata, respectively). PCP values of light-treated cultures did not vary significantly over the course of the experiment but did between species (P < 0.001) with means values of 35.7 ± 4.47, 22.6 ± 2.1, and 10.7 ± 0.9% for C. closterium, N. perminuta, and S. ovata, respectively.

EPS

In all cases, concentrations of EPS (Fig. 6) increased significantly over the course of the experiment (P < 0.001) and were significantly higher in darkened cultures (P < 0.001). In cultures subjected to continuous light, increases in EPS were linear (r ² = 0.81, 0.69, and 0.79 for C. closterium, N. perminuta, and S. ovata, respectively, n = 40, P < 0.001). Predicted production rates were 7.2 ± 0.6, 1.0 ± 0.1, and 0.8 ± 0.07 μg·mL⁻¹·d⁻¹, respectively. In contrast, concentrations of EPS in darkened cultures of the three species rapidly increased during the first 3–4 days only (8–11 days after inoculation) (r ² = 0.84, 0.87, and 0.94 for cultures of C. closterium, N. perminuta, and S. ovata, respectively, n = 15, P < 0.001) at the higher rates of 16.0 ± 1.61, 6.2 ± 0.57, and 6.8 ± 0.42 μg·mL⁻¹·d⁻¹ in cultures of C. closterium, N. perminuta, and S. ovata, respectively. After this sharp increase, EPS concentrations remained constant.

Glucan concentrations increased over the course of the experiment (Fig. 6) and were significantly higher in light-treated cultures (P < 0.001 in both cases) for all three species investigated. Under illuminated conditions, glucan concentrations in all species increased for a further 3–4 days before declining significantly (P < 0.001) (Fig. 6). There were decreases in glucan concentration in darkened cultures. In darkened cultures of C. closterium, glucan concentrations remained low (see Fig. 6A) and concentrations decreased significantly after 13 days. Glucan decreases in darkened cultures of N. perminuta(Fig. 6B) were linear (r ² = 0.71, n = 40, P < 0.001), and there was a significant negative correlation between EPS and glucan concentrations of darkened cultures (r = −0.60, n = 80, P < 0.001). Glucan concentrations of darkened cultures of S. ovata also decreased during the first 4 days (up to 12 days after inoculation) and then remained at a constant low level for the duration of the experiment (Fig. 6C). There was a negative correlation between EPS and glucan concentrations of dark-treated cultures of S. ovata (r = −0.67, n = 80, P < 0.001).

Discussion

The motility generated through the production of EPS by estuarine epipelic diatoms (Edgar and Pickett-Heaps 1984, Hoagland et al. 1993, Underwood et al. 1995, Lind et al. 1997) is a crucial adaptation to their environment. Light penetration into cohesive sediments is only 100–300 μm (Underwood and Kromkamp 1999), and without the ability to reposition themselves within the photic zone after periods of sediment mixing or deposition, cells would be unable to photosynthesize. Because of the progression of tidal cycles through the diel light curve, estuarine epipelon is also subjected to periods of very limited light availability. These features of the estuarine environment will thus select for the ability both to survive periods of, and move in, conditions of darkness. Therefore, differences between the production of at least some components of epipelic diatom EPS, and the widely accepted overflow mechanism of EPS production during conditions of excess light and nutrient limitation (Fogg 1983), may be expected (Smith and Underwood 1998).

The five species used in this study are all commonly found in intertidal cohesive sediments (Admiraal 1984, Underwood 1994). We isolated fresh clones to avoid the problems of acclimation associated with long-term culture maintenance (Round 1982). In the majority of cases. there was no significant difference between isolates of the same species obtained from the field at different times of the year, suggesting that the growth and carbohydrate dynamics measured are characteristics of the species rather than clones. All five species investigated described a typical growth curve, with a minimal initial lag phase probably because the inoculum consisted of exponentially growing cells (Ramus 1972). The growth rates of benthic diatoms are an important factor influencing their distribution and abundance (Admiraal 1984). It is generally considered that larger species have a slower intrinsic rate of growth than smaller species, which is a consequence of a larger cellular biomass (Williams 1964, Banse 1976). This was not the case in this study, and the majority of species had similar growth rates (Table 2), although exhibiting a range of sizes. The rates reported in this study are in the range of those previously published (Williams 1964, Admiraal et al. 1984, Peletier et al. 1996).

Total carbohydrate concentrations generally matched the growth curves of chl a and cell density. However, the contribution of the different carbohydrate fractions (colloidal, EPS, and glucan) exhibited different patterns. Carbohydrate concentrations were not normalized to biomass because extracellular fractions, such as colloidal and EPS, once produced are, in effect, independent of subsequent changes in biomass. Concentrations of colloidal carbohydrates generally increased throughout the experiments and in three out of five species; these increases were approximately linear (i.e. Nitzschia frustulum, Nitzschia sigma, and Surirella ovata), although biomass increased exponentially. Therefore, colloidal concentrations per unit biomass actually decreased. During stationary phase, however, biomass remained constant while colloidal concentrations continued to increase. The rate of colloidal production also increased in stationary phase cultures. The proportion of EPS present within the colloidal fraction increased through the growth curve, from approximately 20% during logarithmic phase to approximately 40%. Concentrations of EPS increased during the stationary phase of all species investigated, and generally substantial quantities of EPS were produced only during transitional or stationary phase. It has been suggested that increased extracellular carbohydrate release during stationary phase, or during periods of nutrient limitation in the field, is a mechanism of dumping excess carbon from the cell (Angelis et al. 1993, Sutherland et al. 1998b, Staats et al. 2000). Nutrient limitation results in the cessation of protein synthesis while photosynthetic activity continues (Fogg and Thake 1987 and references therein). This excess photoassimilated carbon is not metabolized for growth or reproduction and therefore is excreted in the form of colloidal carbohydrates. The composition of internal metabolites and extracellular products of stationary phase cultures has been found to be different (Allan et al. 1972, Staats et al. 1999), suggesting that cell lysis is not responsible for increased extracellular release. It is possible that nutrient limitation stimulates production of the locomotive polymer (EPS) enabling cells to migrate into a more favorable nutrient environment (Smith 1998). However, increased motility of nutrient-limited cells was not observed during this investigation.

Changes in the nature of the extracellular carbohydrates suggest that during rapid growth, epipelic diatoms excrete material into the surrounding media, of which approximately 80% is nonpolymeric (small poly- and monosaccharides). The presence of approximately 20% polymer in the colloidal carbohydrate fraction from cultures in log phase agrees well with field measurements (Underwood et al. 1995, Smith and Underwood 1998, Underwood and Smith 1998). Colloidal carbohydrate production rates are closely linked to rates of photosynthesis (Smith and Underwood 1998, Staats et al. 2000). Increases in ¹⁴C activity in the short-term continuous light treatments, as well as increases in colloidal carbohydrate concentrations in the 7-day continuous light compared with continuous dark treatment, also indicates a close coupling between this fraction and photosynthesis (Smith and Underwood 1998). In the light treatments, between 36 and 70% of assimilated ¹⁴C was present in the colloidal fraction after 3 h, probably as a result of continuous leaching from the cells. However, increases in ¹⁴C activity in colloidal carbohydrate in the dark could be almost completely accounted for by increases in ¹⁴C-EPS, with almost no low molecular weight ¹⁴C-labeled material being produced. Thus, the production of low molecular weight extracellular carbohydrates by diatoms in active growth appears to be directly dependant on photosynthesis.

When cells entered stationary phase, the dynamics of extracellular carbohydrate production changed. Maximum EPS production rates always occurred during stationary phase (mean rate of five species = 18 pg·cell⁻¹·d⁻¹) with EPS accounting for 16.7% (mean of five species) of the total carbohydrate present. EPS became a greater proportion of the colloidal carbohydrate pool, and the composition of this EPS was different from the EPS produced during log phase (Fig. 3, II). Staats et al. (1999) found a similar pattern in Cylindrotheca closterium, with “non-attached EPS” (their term) produced throughout the growth curve and “attached EPS” produced extensively in stationary phase. The relative monosaccharide composition of these two fractions were different, with non-attached EPS having less glucose and richer in galactose, rhamnose, and xylose than attached EPS. Although it is extremely difficult to quantify specific compounds in Py-MS spectra (Ford et al. 1991), our data show differences in both the composition and rates of production of EPS between logarithmic and stationary phase for all five species of diatoms investigated. It can be hypothesized that the smaller quantities of EPS produced during log phase is “motility EPS” (Lind et al. 1997) being produced virtually continuously by the cells, while the larger quantities produced during stationary phase contain both motility EPS and other polymeric material. It is likely that much of the EPS produced during stationary phase in the light is primarily due to “photosynthetic overflow” (Staats et al. 1999). These hypotheses are currently under investigation.

Glucan is a photosynthetic storage product of diatoms which has been characterized as a β-1-3–linked glucose polymer (Myklestad 1988, Staats et al. 1999, M. J. Boulcott and G. J. C. Underwood, unpublished data). Glucan is utilized for cellular respiration during dark periods (Varum et al. 1986) and daytime (Lancelot and Mathot 1985) or for the synthesis of organic molecules such as protein and EPS (Lancelot and Mathot 1985, Smetacek and Pollehne 1986). The products of glucan catabolism have been implicated in protein synthesis (Myklestad 1988), however, during stationary phase, new protein synthesis is prevented by nutrient limitation. Maximum production rates of glucan were determined during transitional phase (range = 54–128 pg·cell⁻¹·d⁻¹) when between 12 and 42% of total carbohydrates existed as glucan. Increased glucan concentration during early stationary phase has been attributed to the filling of photosynthetic reserves as nutrient limitation prevents protein synthesis (Fogg and Thake 1987, Hawes 1990). Rapid increases in glucan (as a percentage of total carbohydrate) were seen in all three species in the short-term ¹⁴C experiment. As cultures entered stationary phase proper, glucan concentrations decreased (range = 1.4–67.3 pg·cell⁻¹).

Maximum EPS production occurred at approximately the same time as maximum glucan utilization (Myklestad 1977, Janse et al. 1996), and field-based ¹⁴C-labeling studies (Smith and Underwood 1998) have indicated that a proportion of the products of glucan catabolism are utilized for the production of EPS. This hypothesis is supported by the 7-day light and dark treatment experiments, which demonstrated decreasing glucan concentrations, and a rapid increase in EPS concentrations when cells were subjected to dark compared to light treatment. Both Navicula perminuta and S. ovata demonstrated a significant negative correlation between EPS and glucan concentrations when cells were subjected to dark treatment. Although C. closterium showed the same long-term (7 days) pattern, there appears not to be such tight coupling between EPS production and glucan catabolism which may be related to this species existing in both benthic and pelagic environments. Increases in ¹⁴C-labeled EPS during the initial dark period were also seen. This EPS production was almost exclusively (>85%) the only extracellular carbohydrate release occurring during darkness. Corresponding decreases in glucan in dark treatments in the short-term experiments were also seen, although these were not as linear as seen in the longer term experiment. This is probably due to (1) the shorter time period of the ¹⁴C experiments and (2) that after a 1-h assimilation period, much of the intracellular glucan pool will consist of unlabeled (¹²C) glucose. Utilization of this material to generate EPS will not be detectable by the ¹⁴C counts. Time course experiments in natural diatom assemblages suggest an approximate 4-h lag between the peak of carbon assimilation and peak allocation to EPS (Smith and Underwood 1998). Wang et al. (1997) found that 150 μM concentrations of the herbicide 2,6-dichlorobenzonitrile (DCB) prevented motility and stalk production in the diatom Achnanthes longipes, although cells continued to grow and divide. DCB is thought to inhibit the production of β-4–linked glucans. Whether reduced motility in DCB-treated A. longipes is a result of prevention of EPS synthesis itself or a lack of glucan to support that synthesis remains to be determined.

This study has provided evidence that epipelic diatoms can produce EPS during periods (up to 3 days) of darkness. Coincidentally, 3 days was also the period in which Serôdio et al. (1997) showed maintenance of migratory rhythms by undisturbed epipelic diatom biofilms maintained in darkness. In their natural habitat, the endogenous migratory rhythms of vertical migration of estuarine epipelon, and repositioning after burial, require that cells can move in darkness. The occurrence of substantial sediment erosion and deposition events results in live benthic diatoms being found at centimeter depths within sediments (MacIntyre and Cullen 1995). The ability to survive and move back to the surface (via EPS production) under such conditions is essential for cells to survive in such habitats. Heterotrophy has been suggested as a strategy for surviving dark periods (Admiraal 1984), and heterotrophic utilization of EPS by C. closterium and N. perminuta has been observed (N. Staats et al. 2000 and personal observation, respectively). However, in the present study, there was no consistent evidence of heterotrophy, and dark activity appears to be supported primarily by utilization of glucan.

This investigation also has shown consistent patterns of colloidal carbohydrate, EPS, and glucan production and utilization in independent isolates of five epipelic diatom species. Colloidal carbohydrate concentrations were closely related to photosynthetic activity, with increased colloidal production (containing a higher polymer content) occurring in nutrient-limited cultures. Not only did the proportion of EPS present in extracellular extracts change, but PyMS analysis demonstrated that the composition of EPS also varies with growth phase. All five species investigated produced a different type of EPS during logarithmic growth compared with stationary phase. Peaks of EPS production during stationary phase corresponded with glucan depletion. Cells continued to produce EPS for up to 3 days in darkness, with a concomitant decrease in glucan concentrations. Long-term EPS production in the dark will permits cells to survive and move during extended periods of darkness in natural habitats (e.g. due to burial or extended tidal cover). Thus, although colloidal carbohydrate production is heavily light dependant, EPS production by these diatom species is not an obligate, light-dependant process. This is the first systematic study of such processes under controlled culture conditions for epipelic diatom species, the data from which are required to interpret processes occurring in heterogeneous field environments.

Acknowledgments

This work was partly supported by a Ph.D. studentship award (GT4/94/338/P) to D. J. S. by the United Kingdom Natural Environment Research Council.

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Citing Literature

Volume36, Issue2

April 2000

Pages 321-333

The production of extracellular carbohydrates by estuarine benthic diatoms: the effects of growth phase and light and dark treatment