Production and fate of extracellular polymeric substances produced by benthic diatoms and bacteria: A laboratory study

Estuarine, Coastal and Shelf Science 75 (2007) 337e346 www.elsevier.com/locate/ecss

Production and fate of extracellular polymeric substances produced by benthic diatoms and bacteria: A laboratory study M. Lundkvist a,b,*, U. Gangelhof b, J. Lunding b, M.R. Flindt b b

a DHI e Water, Environment, Health. Agern Alle´ 5, 2970 Hørsholm, Denmark Department of Environmental Technology, Biological Institute, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark

Received 1 February 2006; accepted 30 April 2007 Available online 25 July 2007

Abstract It is well known that benthic diatoms and bacteria are able to affect the stability of cohesive sediments. Their production of new extracellular polymeric substances (EPS) increases the erosion threshold and decreases the erosion rate. To predict this build up of biostability in cohesive sediments, it is therefore vital to understand the EPS production rates for both diatoms and bacteria under different ecological conditions. The present study examined the production of EPS as function of light intensity and linked this to biostabilisation. Microbenthos was sampled from a Danish marine embayment at 5-m depth. A 10-day comparison of EPS production under light and under dark conditions showed that the bacterial EPS production hardly changed during the experiment, while the algal EPS production was significantly high already from day 1 and reached a maximum production on day 10. Erosion threshold of natural cohesive sediments was determined in annular flumes after variable consolidation periods under similar light/dark conditions. The evolutions of erosion threshold and EPS concentration correlated well under light conditions, while the development in dark conditions showed no significant correlation, indicating that diatoms are more efficient in sediment stabilisation than bacteria. Further it seems, that the studied biological-sedimentary system needs 2e3 days of acclimatisation before the production of EPS can be well correlated with the increase of sediment stability. Ó 2007 Published by Elsevier Ltd. Keywords: cohesive sediment; EPS production; benthic diatoms; light/dark conditions; photosynthesis; erosion threshold

1. Introduction The stability of cohesive estuarine sediments is important from an ecological point of view in order to ensure an optimal light climate on the sea bottom. Several positive environmental feedback mechanisms are initiated when benthic macrophytes and diatoms become light saturated. The photosynthesis is stimulated, which results in higher nutrient uptake by the benthos that reduces the nutrient loading of the water column. The enhanced growth will also create a stronger biofilm on the sediment surface, which binds sediment particles and increases the overall bed stability against hydrodynamic erosional forces.

* Corresponding author at: DHI e Water, Environment, Health. Agern Alle´ 5, 2970 Hørsholm, Denmark. E-mail address: [email protected] (M. Lundkvist). 0272-7714/$ - see front matter Ó 2007 Published by Elsevier Ltd. doi:10.1016/j.ecss.2007.04.034

Thus less sediment particles are likely to be eroded into suspension and hereby the overall turbidity will be smaller, leading to an improved light climate on the sea bottom (Bergamasco et al., 2003). An improved light climate may not only increase photosynthesis and oxygen production by microorganisms, but also favours the colonisation by rooted vegetation, which stabilises the sediment further and acts as a sediment trap. Besides, this rooted vegetation has a lot of other important ecological roles, for instance it acts as fish nursery in shallow waters. The activity by benthic diatoms and bacteria increases the stability of estuarine cohesive sediments. Both groups excrete extracellular polymeric substances (EPS), which bind sediment particles (Decho, 1990; Heinzelmann and Wallisch, 1991; Decho, 2000) and hereby increase the resistance of the bed against hydrodynamic erosional forces (Paterson, 1989; Madsen et al., 1993; Underwood and Paterson, 1993; Austen et al., 1998; Sutherland et al., 1998). The EPS can be divided in

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two fractions: water-soluble EPS also called colloidal EPS, and EDTA-extractable EPS also called capsular EPS because it forms often resistant capsules around bacteria (Decho, 1990). Diatoms produce EPS during vertical migration in the sediment (Underwood and Smith, 1997; Decho, 1990; Higgins et al., 2000) in order to optimise photosynthesis, nutrient uptake and other similar conditions, which are needed to maintain a good microhabitat (Smith and Underwood, 2000; Staats et al., 2000). Diatom cultures are mainly linked to the mucilage colloidal fraction (Underwood and Smith, 1997), which composition consists mostly of heteropolymers such as glucose, galactose and mannose (Taylor et al., 1999). The bacterial EPS secretion has multiple advantages for the microbe. EPS secures the cell by forming a capsule around it. This capsule is hardly degradable, and it can even protect the cell from being destroyed in the digestion system of deposit feeders (Decho and Lopez, 1993). The EDTA fraction, i.e. the capsular EPS, and the sediment grains are closely bound together, thus the bacteria secure also their local environment or microhabitat by this mean. Lundkvist et al. (2007) compared the influence of diatoms and bacteria on sediment stability. They reported that benthic diatoms were able to increase the erosion threshold by w130% and bacteria by w20% compared to the physical stabilization alone. This means that benthic diatoms in optimal life conditions have a larger impact and are better sediment stabilisers than bacteria. This is in agreement with the research of Black et al. (2002) and Quaresma et al. (2004) who reported similar trends. On the other hand the effect of this biostabilisation of cohesive sediments is very site specific, meaning that diatoms and bacteria are by far not the only organisms living on the bed surface. Andersen (2001) reported that the erodibility increased in summer periods and decreased in winter, due to the seasonal presence or absence of faecal pellets from the mudsnail Hydrobia ulvae on a mudflat in the Danish Wadden Sea. This mudsnail also grazes the diatoms and bioturbates the bed surface (Austen et al., 1998; Andersen and Pejrup, 2002; Andersen et al., 2002). A different biological influence is reported by Amos et al. (2004), who observed bed strength increasing in summer in Venice Lagoon due to fluctuations in water temperature, the presence of microphytobenthos and cyanobacteria, but also due to the abundance of seagrasses. Therefore, an universal ranking of different sediment stabilizing organisms is unlikely to be obtained, due to both temporal and spatial variations. Besides these biological effects, physico-chemical properties and processes also govern the stability of cohesive sediments. The main relevant physical properties are mineralogy, grain size distribution, and water content (Dyer, 1986). The chemical processes are mass attractive forces and interparticular electrostatic bindings (i.e. electrical double layer) around the clay particles, which give rise to the ‘‘sticky’’, cohesive nature of the sediments (Whitehouse et al., 2000). The aim of the present research was to better understand the effect of the studied microbenthos on cohesive sediment stability, by linking the benthic EPS production to photosynthesis and comparing EPS production to biostabilisation. Three different project parts, each with a set of experiments,

were carried out: (1) EPS production under light and dark conditions to differentiate contributions from diatoms and bacteria; (2) flume erosion experiments under similar light and dark conditions to measure the erosion threshold; and (3) a dose-response experiment with photosynthesis under different light intensities to establish the relationship between irradiance and O2-production using Monod kinetics. 2. Materials and methods Throughout the experiment we used sediment, algae inoculum, and seawater sampled in Odense Fjord, Denmark (55 290 9500 N; 10 350 0100 E) at 5e6 m depth in an area with a well-developed diatom biofilm on the seabed. The sediment is cohesive with a mean grain size of w27 mm and an organic content between 5 and 10%. The sediment used in the experiment was sieved using a 2-mm mesh size to remove shells and other larger fragments. The seawater was filtered through Whatman GF-C filters and had a salinity of 20. The inoculum used in the present study was collected from undisturbed sediment cores by gently scraping the top 1e2 mm off the sediment surface using a vacuum system. Afterwards the sampled inocula were placed under light of 100 mmol m2 s1 with addition of nutrients for 12 h. In the text we use experiment or erosion experiment when referring to the entire experiment over the time from day 1 to day 16. Using experimental run, we refer to the procedure carried out in the Miniflumes to determine the critical erosion threshold. 2.1. EPS production (part 1) This part of the experiment was carried out using Kajak tubes, which are acrylic, transparent tubes with a diameter of 50 mm and with height about 400 mm. The sediment used in this experiment was sand with a mean grain size of about 0.2 mm. It had been muffled for 6 h at 550  C. A 50-mm sandy sediment base was created in each Kajak tube, which was then filled with filtered seawater. After this, 10 ml of algae inoculum was added to each tube by pouring the inoculum slurry into the tube and letting it settle as homogeneous muddy layer on top of the sand. One set of tubes was kept in darkness, a second set was exposed to a 12 h/12 h light/dark photoperiod (100 mmol m2 s1). These are similar light conditions than used for the flume erosion experiments. After 0, 1, 2, 5, 10 and 16 days triplicate samples were taken to analyze for colloidal EPS, EDTA extractable EPS, and for Chl a. Six Kajak tubes were used every sample day, 3 from the dark conditions and 3 from the light conditions. They were sliced in the depth segments 0e2, 2e5, 5e10, and 10e20 mm. Between sample days, NH4 and PO4 stock solutions were added in order to avoid nutrient depletion. 2.2. Erosion experiments (part 2) 2.2.1. Flume description Two laboratory Miniflumes were used for the erosion experiments. The Miniflumes are annular flumes made of 2 acrylic

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plastic tubes, one inside the other, fixed onto a base. The flumes are 50 cm in diameter and 36 cm high. The flume channel is 4.5 cm wide, its volume is 21.6 litres and the bed surface area is 0.06 m2. It is closed on the top by a rotating lid. Attached to the lid are six paddles that induce the flow in the flume. The speed of the paddles is controlled by an AC-servo motor with an integrated driver (MAC motor). Two sample ports are situated 15 cm above the base on the outer channel wall. In the first sample port a SeaPointÒ Turbidity Meter (STM) monitors the turbidity 2e3 cm above the sediment surface inside the flume channel by detecting backscattered light from suspended sediment particles. It is interfaced to a PC, which is logging data from the sensor at a rate of 1 Hz. Another sample port for taking water samples is situated on the opposite site of the flume at the same height. The STM output was calibrated against suspended sediment concentration (SSC) measured on water samples. In every velocity step during an experimental run, 200 ml water was sampled from the flume and filtered through pre-weighed Whatman GF-C filters (pore size 1.2 mm), then dried for 24 h at 105  C before dry mass determination. From these measurements a calibration curve was established, which allowed to convert STM output to SSC. 2.2.2. Bed preparation A total of 3.5 litres of sieved sediment were mixed with 0.5 litres filtered seawater to create a mud slurry and then poured into each of the two flumes. The flumes were then filled with 17 litres of filtered seawater carefully poured onto a circular piece of bobble rap protecting the bed from being disturbed. Afterwards 300 ml of algae inoculum was added to both flumes and was let settle on the sediment surface. 2.2.3. Experiment description Two different light conditions were used in the erosion experiment. The first flume was kept with a light intensity of w115 mmol m2 s1 (assumed similar to 100 mmol m2 s1 from experiment part 1) on a 12 h/12 h light/dark photoperiod, which ensured diatom photosynthesis. The second flume was kept in darkness (<3 mmol m2 s1 e assumed to be similar to 0 mmol m2 s1) in order to validate the bacterial contribution to the biostability. The light source was 3 Master SON-T PIA green power 400 W lamps, placed 1 meter above the flumes. The light intensity was measured with a Li-Cor datalogger model LI-1400. After bed preparation the flumes were left to consolidate for 1, 2, 5, and 10 days respectively in between erosion experiments. During these consolidation periods, the flumes were aerated by gently bubbling with air and a constant free stream velocity of 2e3 cm s1 was induced in each flume, which is well below the critical erosion threshold for the sediment used. The water level was maintained at the same level throughout the whole experiment, during experimental runs, but also during consolidation periods. Nutrient growth limitation was avoided by adding NH4 and PO4 stock solutions every second day.

339

The erosion-threshold experimental run was carried out by subjecting the bed to current velocities of increasing magnitude. The turbidity was logged with the STM. Each velocity step had a different time-duration; it continued until the SSC in the water column did not longer increase and the erosion stopped. Once this was observed, the next velocity step was initiated. Different velocity steps were used in each of the erosion experiments; the number depended on how fast the sediment eroded and how much stabilised the sediment surface was. After the last step, the sediment was fully mixed followed by smoothening of the bed surface. Samples were taken during each erosion experimental run to analyse for EPS and Chl a. The samples were frozen at 18  C and analyzed later. 2.2.4. Determination of erosion threshold The sediment stability for each erosion experiment was determined from data series of SSC and bed shear stress. The erosion threshold is expressed as the critical shear stress (tcrit), which was estimated from a scatterplot of SSC versus bed shear stress (Fig. 1). A linear regression was computed using all data points from the erosion start (defined as the first moment with an erosion rate larger than 0.09 g m2 s1) until the SSC stabilisation of the last velocity step. tcrit was defined as the point where this linear regression intersects the x-axis. The erosion rate was calculated for one-minute intervals. 2.3. Dose-response experiment on light intensity and photosynthesis (part 3) The algae inoculum was incubated in 20 ml vials in light intensities of 0, 17, 50, 84, 138, 176, 250, and 341 mmol m2 s1. The setup was prepared by adding 5 ml of well-mixed algae inoculum to each vial and then filling it up with filtered seawater. The content of oxygen before incubation was measured in triplicates using a Unisense oxygen micro sensor electrode. The vials were closed, making sure no air bubbles were trapped inside. After 2 h of incubation, the oxygen level was measured again in triplicates. The oxygen production was calculated as a function of temperature (t0 ¼ 22  C; t2h ranged between 20 and 25  C, with increasing temperature with higher light intensity), salinity and the difference in oxygen concentration over time. 2.4. Measurements of EPS and Chlorophyll a (used in part 1 and 2) Extraction was done on two EPS fractions e the colloidal EPS fraction and the EDTA-extractable fraction. The colloidal fraction was extracted in 3 ml of milli-Q water per 2 ml of sediment placed on a shaking table for 16 h at 20  C. The sample was then centrifuged at 4000 RPM for 15 min, and 2 ml supernatant was designated to the colloidal fraction. To the remaining sample were added 1.7 ml of milli-Q water and 0.3 ml 0.1 M EDTA, giving a final concentration of 10 mM EDTA (Gray and Wilkinson, 1965). The sample was placed again on the shaking table for another 16 h at 20  C. A total of

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340 0,3

Eroded SSC SSC values used to create regression line

SSC (g dw l-1)

0,2

0,1

crit

= 4.60 Pa

0,0

0

4

8

12

Bed stress (Pa) Fig. 1. Determination of erosion threshold using the linear regression line between bed shear stress and SSC. Erosion threshold is defined as the point where the regression line intersects the x-axis.

2 ml of supernatant was provided for the EDTA fraction after centrifugation at 4000 RPM for 15 min. The quantification of the extracted partitions of EPS was done using the phenol/H2SO4 assay (Dubois et al., 1956). The amount of dry weight in the sample was measured by filtration onto pre-dried and pre-weighed Whatman GF-C filters (pore size 1.2 mm) followed by 5 h drying at 105  C. Chl a concentrations were measured in order to validate the algae growth on the sediment surface in experiment part 1 after addition of 2.5 ml of 96% ethanol to each sediment sample to extract the chlorophyll pigment. The samples were placed on a shaking table for 20 h in total darkness (Jespersen and Christoffersen, 1987). The supernatant was analysed in a spectrophotometer at 665 and 750 nm after centrifugation at 3000 RPM for 10 min. The amount of dry weight in the sample was measured by filtration onto pre-dried and pre-weighed Whatman GF-C filters (pore size 1.2 mm) followed by 5 h drying at 105  C. 3. Results 3.1. EPS Production (part 1) The concentration of total EPS (i.e. the colloidal fraction plus the EDTA soluble fraction) in the sediment cores during the 16 days of bioactivity varied significantly between the two experiments with light intensities of 0 and 100 mmol m2 s1. The start concentration of total EPS in the upper 2 mm of the sediment was relative low in both light and dark conditions. With light, it increased after the second day and reached a maximal level of about 1200 mg m2 after about 10 days (Fig. 2A). On the contrary, in the dark conditions the concentrations stayed constant with an EPS amount of about 200 mg m2 throughout the 16 days. A similar pattern was observed for the total EPS production over the entire 20 mm sediment depth. In light conditions, the

concentration started with about 350e400 mg m2; it increased after the first two days to a maximum level of about 1600 mg m2 at day 10 (Fig. 2B). In comparison, the production of total EPS under dark conditions was relative low during the first two days with a concentration similar to the one in the light conditions, and afterwards it remained constant at about 400 mg m2 (Fig. 2B). The colloidal fraction and the EDTA soluble fraction from the light conditions experiment correlate significantly (r ¼ 0.978; p < 0.001) indicating no difference in the production under photosynthetic activity between these two fractions (Fig. 3A). No significant correlation exists between the two EPS fractions in the dark conditions experiment (r ¼ 0.356) (Fig. 3B). Fig. 3A shows that there was an EPS increase when diatoms were photoactive. On the contrary, both EPS factions remained constant in dark conditions, where bacteria were the dominant EPS producers (Fig. 3B). The profiles of EPS concentration below sediment surface show that EPS concentration was highest at the surface and it decreased to a significantly lower level already at 4 mm below the surface (Fig. 4). This applied to both light and dark conditions, although the decrease in the dark experiment was not that strong. The EPS concentration above and below the 4 mm limit increased over time and reached a maximum level after 10e16 days in both the light and dark conditions, however this increase was more pronounced in light conditions. Overall, the production was higher in the photosynthetic active surface, for example after 10 days the difference between light and dark conditions was a factor 6. This difference decreased significantly below 4 mm and the concentrations were in a similar range both in light and in darkness (Fig. 4). The production rate of total EPS under light and dark conditions are calculated as the net production per area per day, for example the EPS amount measured at day 2 minus the amount measured at day 1, divided by the time between the

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2000

A Light

Dark

12

14

Light

Dark

EPS (mg m-2)

1600

1200

800

400

0 0

2

4

6

8

10

16

Period of bed consolidation (day) 2000

B

EPS (mg m-2)

1600

1200

800

400

0 0

2

4

6

8

10

12

14

16

Period of bed consolidation (day) Fig. 2. Development of EPS production during the Kajak tube experiments: (A) in 0e2 mm depth; and (B) in 0e20 mm depth. All values are mean values from triplicates. The standard deviations are also plotted as error bars in panel (A).

measurements, and then normalized for the Kajak tube area (Table 1). Under light conditions, the production rate in the upper 2 mm were lower than the production rate for the full 20 mm sediment profile, except for the rate from day 1 to 2. In dark conditions, the production rate was negative from day 2 until day 16 in the upper 2 mm, while it shifted to positive values from day 5 to day 16 accumulating the values over the full 20 mm sediment. Pearson correlation coefficients (r-values) were calculated between the parameters measured in the whole sediment core, i.e. in 0e20 mm depth (Table 2). Under light conditions, there is a significant correlation between Chl a and total EPS, between Chl a and both fractions of EPS. There also exists a strong correlation between the different EPS fractions. In dark conditions, there is only a significant correlation between EDTA extractable and total EPS. There is nearly no correlation between Chl a and total EPS and further between Chl a and EDTA-EPS fraction. The negative linear correlation between Chl a and

colloidal EPS indicates a negative association between the variables. 3.2. Erosion experiment (part 2) Total EPS concentration was measured on water samples collected in the flumes during the erosion experimental run. It corresponds mainly to EPS from the sediment bed resuspended during the erosion. It correlates significantly with SSC (r ¼ 0.964; p < 0.001). The biofilm is assumed to be fully eroded at the end of the erosion experimental run and all EPS to be in suspension. Therefore the EPS concentration in the water column during the last erosion step was used to compute the EPS concentration per bed area in the biofilm before erosion. In all erosion experiments under light, the total EPS concentration follows the SSC during the erosion experimental run (Fig. 5). The EPS concentration during the last velocity step increased with longer bed consolidation, indicating a production

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342 1200

5

10

15

20

25

30

35

40

0 2

800

4

600

Depth (mm)

EPS (mg m-2)

0

EDTA - Algae

1000

EPS (mg m-2)

A

Colloid - Algae

A

400 200

6 8

Day 0 Day 1 Day 2 Day 5 Day 10 Day 16

10 12

0

0

2

4

6

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16

14

Period of bed consolidation (day) 16 1200

Colloid - Bacteria

B 1000

0

5

10

15

20

25

30

35

40

0

800

2 4

600

Depth (mm)

EPS (mg m-2)

EPS (mg m-2)

B

EDTA - Bacteria

400 200

6 8

Day 0 Day 1 Day 2 Day 5 Day 10 Day 16

10 12

0

0

2

4

6

8

10

12

14

16

14

Period of bed consolidation (day) Fig. 3. The distribution of colloidal EPS and EDTA-soluble EPS during the Kajak tube experiments. (A) Under light conditions; and (B) under dark conditions.

of EPS in the biofilm, particularly from day 2 to day 10. Though, the EPS concentration did not change between day 1 and 2. The erosion threshold decreased from 1.51 Pa on day 1 to 0.85 Pa on day 2. Afterwards the erosion threshold increased to 2.32 Pa on day 5 and further to 7.31 Pa on day 10. The initial decrease of erosion threshold was simultaneous with a stagnation in EPS concentration, while the subsequent increase of erosion threshold was coincident with an increase of EPS concentration. The result from the erosion experiment in darkness shows that the EPS concentration followed, as in the light experiment, the enhanced SSC during the experiment as well as in the experimental run (Fig. 6). The final EPS concentration increased from day 1 to day 2, and decreased again from day 2 to day 10. The erosion threshold decreased from 3.57 Pa on day 1 to 2.94 Pa on day 2 and 0.97 Pa on day 5, before increasing again to 1.85 Pa on day 10. The production rates were more dynamic in the flume experiment compared to the rates in the production experiment in the Kajak tubes, for both the light and dark conditions (Table 3). The production rates were relative high in the first 2e3 days of the flume experiment. In light conditions, the rate decreased after these initial days to a lower, stable level. In the dark setting, the rate also decreased after the first days, actually there was a net degradation of EPS from day 2 to 5, and from day 5 to 10 there was a relatively small net production again.

16

Fig. 4. The production of total EPS at different depth during the Kajak tube experiments. (A) Under light conditions; and (B) under dark conditions.

There are significant correlations between the production rates from experiment part 1 (Kajak tube experiment) and the production rates found during flume experiment part 2 (Table 3). The correlation rate is negative under light conditions (r ¼  0.983; p < 0.05), whereas it is positive under dark conditions (r ¼ 0.999; p < 0.01). 3.3. Dose-response experiment on light intensity and photosynthesis (part 3) Benthic diatom growth can be modelled with Monod kinetics using a formula like: P ¼ Pm $ L/(L  km), where P is the O2 production rate, Pm is the maximum production rate, L is the irradiance, and km is the half saturation constant. To define these parameters, a dose-response curve was produced between the light intensity and the measured phototrophic oxygen Table 1 Production rates of EPS under light and dark conditions (100 and 0 mmol m2 s1, respectively). Unit: mg EPS m2 d1

Day Day Day Day

1e2 2e5 5e10 10e16

Production 0e2 mm

Total production 0e20 mm

Light

Darkness

Light

Darkness

53 105 131 22

65 18 8 5

51 139 158 36

96 12 0.22 1.49

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Table 2 Pearson correlation coefficients (r) between measured parameters. Significant correlations are highlighted: *p < 0.05; **p < 0.01; ***p < 0.001 Light

Chl a Colloidal EPS EDTA EPS

Darkness

Colloidal EPS

EDTA EPS

Total EPS

Colloidal EPS

EDTA EPS

Total EPS

0.782*

0.863* 0.979***

0.834* 0.993*** 0.996***

0.577

0.045 0.356

0.045 0.699 0.917***

production (Fig. 7). Maximum photosynthesis was at light intensity 250 mmol m2 s1. The half saturation constant for oxygen production was estimated from the curve to km z 40 mmol m2 s1, and the maximum production rate to Pm z 1.6 mmol O2 h1. 4. Discussion The EPS production study (part 1) was set up as a parallel experiment to the flume erosion experiment (part 2) to provide a detailed knowledge about the production, the fate and the vertical distribution of EPS over a 16-day period. The two experiments provided production rates, correlations between EPS concentration and erosion thresholds, levels of EPS in the surface sediment and an EPS production in the deeper layers. The dose-response experiment on light intensity and photosynthesis (part 3) was needed to carry out the other experiments without having to high or to low values of irradiance affecting the studied diatom population. 4.1. EPS production (part 1) This experiment shows clearly a much higher EPS production during light conditions than during dark conditions 4000

A

3000

crit = 1.51 Pa

15

2000 10 1000

5

0

0 0

5

10

4000

B

20

EPS SSC

Day 2

15

crit

2000 10 1000

5 0

15

0 0

2

Bed stress (Pa)

2000 10 1000

5 0

0 2

4

Bed stress (Pa)

6

8

4000

D EPS (µg ml-1)

3000

crit = 2.32 Pa

0

8

20

EPS SSC

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15

crit

3000

= 7.31 Pa 2000

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Day 5

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25

3000

= 0.85 Pa

SSC (mg DW l-1)

Day 1

25

SSC (mg DW l-1)

20

EPS SSC

EPS (µg ml-1)

25

EPS (µg ml-1)

(Figs. 2e4), which supports the theory of diatoms producing EPS. This production results either from simple excretion due to disorder in the photosynthesis or from secretion during migration in the sediment matrix (Sutherland et al., 1998). The correlation between Chl a and the EPS concentration (Table 2) further supports the theory of diatoms producing EPS. However, the present study gives no indications whether the diatoms excrete mainly less complex hydrocarbon chains, i.e. colloidal-EPS, or the opposite, namely EDTA-EPS. The production rates of EPS in the sediment in light conditions were initially low, but after 2e3 days the production rate was relatively high with a maximum production of EPS between days 5 and 10 (Table 1). The rate between days 10 and 16 was slightly negative, indicating saturation, mineralization or decrease in production. This applied for the top 2 mm of the sediment core as well as for the whole depth of the core down to 20 mm. The production experiment under dark conditions showed no significant production of EPS during the whole experiment, except a positive and relatively high production rate during the first 2e3 days of incubation (Fig. 2, Table 1). This is most likely due to the vertical migration of diatoms. Before the inocula were added to the Kajak tubes, they were adapted to 12-h cycles of light and darkness. During dark conditions

0 0

5

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Bed stress (Pa)

Fig. 5. Results of the erosion threshold experiment under light conditions. (A) After day 1; (B) after day 2; (C) after day 5; and (D) after day 10.

M. Lundkvist et al. / Estuarine, Coastal and Shelf Science 75 (2007) 337e346 25

EPS (µg ml-1)

15 crit

= 3.57 Pa

2000

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5 0 5

10

Day 2

15

3000 = 2.94 Pa

crit

1000

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2000 1000

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0 5

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EPS (µg ml-1)

EPS (µg ml -1)

= 0.97 Pa

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crit

3000

= 1.85 Pa

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crit

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EPS SSC

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EPS (µg ml-1)

344

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Fig. 6. Results of the erosion threshold experiment under dark conditions. (A) After day 1; (B) after day 2; (C) after day 5; and (D) after day 10.

they migrated in the sediment, searching for light, in order to find a place of optimal irradiance, hereby excreting more EPS. The production rate for the remaining 13 days of the experiment in darkness was first decreasing, and from day 5 on it was slightly increasing at a relatively low level, indicating enhanced bacteria activity in the whole sediment core. At that time, the diatoms did not produce any more, due to lack of photosynthesis. Consequently, EPS concentration decreased in the top 2 mm of the core, indicating that the large amount of material produced in the surface in the first days was still degrading throughout the experiment (Table 1). The assumption of capsular EPS being mainly produced by the bacteria as a capsule surrounding the cell cannot be confirmed from the results on Fig. 3 because the differences between the two EPS fractions (EDTA-EPS and colloidal-EPS) are similar when comparing the dark and light experiments (Fig. 3). However, the EDTA fraction correlates stronger with total EPS (Table 2) than colloidal EPS does in dark conditions. This could indicate that bacteria in the sediment mainly produce this fraction, but in general the total amount of this EPS fraction is smaller in the dark setting than in the light one.

Table 3 Comparison of EPS production rates from experiment EPS production experiment (part 1) and Flume erosion threshold experiment (part 2) under light and dark conditions. Unit: mg EPS m2 d1

Day 1e2 Day 2e5 Day 5e10

Production experiment

Flume experiment

100 mmol m2 s1

115 mmol m2 s1

51 139 158

0 mmol m2 s1 96 12 0.22

708 331 340

<3 mmol m2 s1 1275 142 57

The highest EPS concentration was found in the upper 2 mm of the core (Fig. 4). Yallop et al. (1994) made a similar observation. It is in this surface layer that the diatoms migrate and that the biological activity is the highest. The difference between production in sediment with algae growth (plus bacteria growth) and sediment with mainly bacteria growth is fairly clear in the profiles on Fig. 4. The initial concentrations are considered similar for both experiments but after 16 days the concentrations were much higher in light conditions (Figs. 2 and 4). This outlines that the algae contribution to the net production of EPS develops in the top 2 mm phototrophic zone of the sediment and that deeper layers seem to be relatively unaffected (Fig. 4). So when Fig. 3A shows increasing amounts of EPS during the experiment, this is a result of an increased production in the top 2 mm. Furthermore, the EPS concentration below 2 mm depth, in light conditions shows

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Light intensity (µmol m-2 s-1 ) Fig. 7. Dose-response curve on light intensity and photosynthesis measured as oxygen production by benthic algae population.

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a small tendency of increasing faster than in dark conditions. The bacteria seem to benefit from the higher microphytobenthic production, possibly due to higher amounts of labile organic matter.

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that the biology needs about 2e3 days to acclimatise, to start EPS production, and to bind effectively the sediment particles with newly produced mucus. 4.3. Photosynthesis and light intensity (part 3)

4.2. Influence of EPS production on erosion threshold (part 2) The objective of the erosion experiment is to understand the development of cohesive sediment stability in relation to the production of EPS. The evolution of production rates of EPS in the flumes during the consolidation periods (part 2) was similar to the evolution of EPS production experiment (part 1), except at the beginning of the light experiments. The production rate increased progressively during the Kajak tube experiment, whereas the production rate was highest in the first days of the flume experiment and lower afterwards (Table 3). The level of production rates was higher in the flumes than in the Kajak tube experiment (Table 3). This is probably due to better growth conditions in the flumes. One reason may be the difference in substrate, diatoms coping better on cohesive sediment than on coarser grains. A second reason could be the larger biodiversity, especially of bacteria, in cohesive sediments, which in combination with the larger pool of organic compounds supply the diatoms with nutrients, hereby creating a better microhabitat for diatoms. The effect of EPS stabilizing the sediment has been widely described (Sutherland et al., 1998; Dade et al., 1990; Decho, 1990) and it is also illustrated by this study in light conditions. However, the contributions to sediment stability of the different EPS fractions and their vertical distribution in the sediment are only partially understood. The EDTA fraction correlates significantly with the suspended sediment concentration in the water column in the present study (r ¼ 0.999; p < 0.001; Figs. 5 and 6). This corresponds to the thesis in Decho (1990) saying that the EDTA fraction is linked to the sediment particles and to the cell surfaces. Further, the sediment grains and cells in and on top of the sediment are also linked by the water-soluble colloidal EPS fraction as a slime/mucus web (Decho, 1990; Yallop et al., 1994). But the fact, that this fraction is degraded in water during suspension, means that the interparticular EPS bounds are broken, leaving the sediment weakened after deposition, and with lower colloidal EPS concentration. The present study gives no clear indication whether it is mainly the EDTA or the colloidal EPS fraction that control the overall sediment stability. Contrary to the experiments with light, the development of erosion threshold under dark conditions had no significant correlation with the amount of EPS in the sediment (Fig. 6). Although the amount of EPS increased from day 1 to 2, the erosion threshold decreased (Fig. 6). So even if the bacteria produced EPS during the experiment, they were not able to produce enough to govern the erosion threshold. Both the Kajak tube experiment and the erosion experiment illustrated the fact that EPS is a dynamic pool, which is indirectly adjusted to the surroundings. Comparing the erosion threshold with the EPS concentration in the sediment, it seems

Previous work on optimal growth and light saturation of diatom cultures has resulted in a great variety of half saturations constants, km values, in the range 10e100 mmol m2 s1 and maximal production at 60e2000 mmol m2 s1 (Dodds et al., 1999; Underwood and Kromkamp, 1999; Guarini et al., 2002; Gerbersdorf et al., 2004; Wenchuan et al., 2004). These values are of great importance in the modelling of benthic diatom growth using Monod kinetics and to get better knowledge of the used diatom population. The algae photosynthesis experiment under various light intensities resulted in a classical saturation curve (Fig. 7) for the diatom inoculum adapted for life at 5.5 meters depth in Odense Fjord. The photosynthesis was modelled as a function of light intensity using the Monod saturation kinetics:   mmol O2 production h Light ðmmolm2 s1 Þ ¼ 1:6 Light ðmmolm2 s1 Þ þ 40 mmolm2 s1 valid for 0  light ðmmolm2 s1 Þ  350 The maximum production is at approximately 250 mmol m2 s1, while the half saturation constant is 40 mmol m2 s1, implying that flume experiments should be set up with irradiance of about 200 mmol m2 s1 to have maximum production. 5. Conclusion The production of EPS is an important factor in determining or predicting the development in erosion threshold of cohesive sediment. The present experiments have shown following relevant aspect. (1) When light is present, EPS is mainly produced by microphytobenthos on the sediment surface (top 2 mm). However the biological system needs 2e3 days of adjustment to environmental conditions, before the excreted EPS influences the erosion threshold, i.e. before an EPS increase is correlated with a higher erosion threshold. (2) The net production of EPS by bacteria is constant over time, and does not influence the erosion threshold as much as the net production by microphytobenthos. (3) There is an indication that the EPS production rate is a better proxy for predicting the erosion threshold than the total EPS concentration. The newly produced EPS seems to have a more active bonding efficiency. (4) The dose-response experiment with the studied marine microphytobenthos showed a maximum production at about 250 mmol m2 s1. Fitting a simple model based on Monod

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Kinetics to the results gives a half saturation km of 40 mmol m2 s1. This study provides knowledge of the connection between light conditions, excretion of EPS and the effect on erosion threshold. All theses factors are important in ecological studies of cohesive sediment stability. Acknowledgements This study was supported through a grant (ANS-0413/400) from the Carlsberg Foundation, Copenhagen, Denmark. Furthermore, a special thank to Dr. Urs Neumeier for scientific support as well as for proofreading. References Amos, C.L., Bergamasco, A., Umgiesser, G., Cappucci, S., Cloutier, D., DeNat, L., Flindt, M., Bonardi, M., Cristante, S., 2004. The stability of tidal flats in Venice Lagoon e the results of in-situ measurements using two benthic, annular flumes. Journal of Marine Systems 51, 211e241. Andersen, T.J., 2001. Seasonal variation in erodibility of two temperate, microtidal mudflats. Estuarine, Coastal and Shelf Science 53, 1e12. Andersen, T.J., Pejrup, M., 2002. Biological mediation of the settling velocity of bed material eroded from an intertidal mudflat, the Danish Wadden Sea. Estuarine, Coastal and Shelf Science 54, 737e745. Andersen, T.J., Jensen, K.T., Lund-Hansen, L., Mouritsen, K.N., Pejrup, M., 2002. Enhanced erodibility of fine-grained marine sediments by Hydrobia ulvae. Journal of Sea Research 48, 51e58. Austen, I., Andersen, T.J., Edelvang, K., 1998. The influence of benthic diatoms and invertebrates on the erodibility of an intertidal mudflat, the Danish Wadden Sea. Estuarine, Coastal and Shelf Science 49, 99e111. Bergamasco, A., De Nat, L., Flindt, M.R., Amos, C.L., 2003. Interactions and feedbacks among phytobenthos, hydrodynamics, nutrient cycling and sediment transport in estuarine ecosystems. Continental Shelf Research 23, 1715e1741. Black, K.S., Tolhurst, T.J., Paterson, D.M., Hagerthey, S.E., 2002. Working with natural sediments. Journal of Hydraulic Engineering 128, 2e8. 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 entrainment of sand. Geomicrobiology Journal 8, 1e16. Decho, A.W., 1990. Microbial exopolymer secretions in ocean environments: their role(s) in food webs and marine processes. Oceanography and Marine Biology 28, 73e153. Decho, A.W., 2000. Microbial biofilms in intertidal systems: an overview. Continental Shelf Research 20, 1257e1273. Decho, A.W., Lopez, G.R., 1993. Exopolymer microenvironments of microbial flora: multiple and interactive effects on trophic relationships. Limnology and Oceanography 38, 1633e1645. Dodds, W.K., Briggs, B.J.F., Lowe, R.L., 1999. Photosynthesis-irradiance patterns in benthic microalgae: variations as a function of assemblages thickness and community structure. Journal of Phycology 35, 42e53. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Analytical Chemistry 28, 350e356. Dyer, K.R., 1986. Coastal and Estuarine Sediment Dynamics. Wiley, Chichester, 342 pp. Gerbersdorf, S.U., Meyercordt, J., Meyer-Reil, L.-A., 2004. Microphytobenthic primary production within the flocculent layer, its fractions and

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