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Continental Shelf Research 24 (2004) 16231635 www.elsevier.com/locate/csr

Extracellular polymeric substances: quantication and use in erosion experiments

R.G. Perkinsa,, D.M. Patersona, H. Sunb, J. Watsonb, M.A. Playerb
a

Sediment Ecology Research Group, Gatty Marine Laboratory, St. Andrews University, Fife, Scotland KY16 8LB,UK b Department of Engineering, University of Aberdeen, Kings College, Aberdeen, Scotland AB24 3UE, UK Received 18 August 2003; accepted 4 June 2004 Available online 20 August 2004

Abstract Extracellular polymeric substances (EPS) is a generic term often applied to high molecular weight polymers implicated in the biostabilisation of natural sediments. Quantitative analysis of in situ EPS production rates and sediment contents has usually involved extraction of EPS in saline media prior to precipitation in alcohol and quantication against a glucose standard (phenolsulphuric acid assay). Extracted and synthetic EPS has also been used to create engineered sediments for erosion experiments. This study investigated two steps in the EPS extraction procedure, saline extraction and alcohol precipitation. Comparisons of the effects of different extracted polymers were made in sediment erosion experiments using engineered sediments. Sediment EPS content decreased as the salinity of the extractant increased, with highest values obtained for extraction in fresh water. Potential errors were observed in the quantication of the soluble colloidal polymer fraction when divided into EPS and lower molecular weight polymers (LMW) as used in many studies. In erosion studies, 15 mg kg1 of alcohol (IMS) extracted EPS polymer (in 5 g kg1 IMS precipitate, equivalent to approximately 5 g salt kg1 sediment dry weight) decreased the erosion threshold of cohesive sediments whereas 30 mg kg1 (in 10 g kg1 IMS precipitate, approximately 10 g salt kg1 sediment dry weight) had no effect compared to controls. This could be due to the inuence of EPS on water content: low levels of EPS did not bind but prevented desiccation, lowering sediment stability against controls. At higher EPS content, binding effects balanced water content effects. Salt alone (at 10 g kg1) slightly increased the erosion threshold after a 6-h desiccation period. In comparison, carbohydrates produced without alcohol precipitation (rotary evaporation) increased the erosion threshold at both 0.5 and 1.0 g EPS kg1 dry weight of sediment. It was concluded that the role of microphytobenthic polymers in biostabilisation of sediments is best determined through the study of natural intact sediment samples. r 2004 Elsevier Ltd. All rights reserved.
Keywords: EPS; Extraction; Quantication; Sediment erosion

Corresponding author. Tel.: +44-1334-46-3445; Fax: +44-1334-46-3443

E-mail address: rgp@st-andrews.ac.uk (R.G. Perkins). 0278-4343/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.csr.2004.06.001

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1. Introduction Over the past decade there has been an ongoing search for biological variables that can be used to describe the inuence and extent of biological mediation of sediment stability or erosion potential (Paterson and Black, 1999). Measures used often represent the biological standing stock, such as chlorophyll-a (Hakvoort et al., 1998; Riethmu ller et al., 1998, 2000; Austin et al., 1999), cell number and bio-volume (Madsen et al., 1993), or the extracellular polymeric substances (EPS) secreted from sediment-inhabiting cells (Decho, 1990; Underwood and Paterson, 2003). Given the mechanistic relationship between EPS and sediment stability demonstrated by a number of authors (Dade et al., 1990; Yallop et al., 1994), the latter variable has been most widely used and researched (Underwood and Paterson, 2003, and references therein). However, it has become clear that the methodology used to determine EPS is varied and may actually be in error, depending on the approach used. The term, extracellular polymeric substances, incorporates all polymeric material produced by biota and found external to the cells. What most researchers actually measure is a carbohydrate fraction of the total EPS, along with low molecular weight sugars (not EPS) that are extracted along with the polymeric material (Underwood et al., 1995). They may separate the extractable carbohydrate (low and high molecular weight sugar polymers) into operational fractions by various methods (Underwood and Paterson, 2003). The operational fraction of the colloidal, water-soluble polymeric complex often referred to as EPS, is usually extracted from sediments using saline water, typically with salinity 20 at 20 1C for 20 min in quantication studies (Underwood et al., 1995). Precipitation of polymeric substances can then proceed by addition of alcohol (ethanol or industrial methylated spirit (IMS) at 96% v/v) to the saline extractant solution, with a nal concentration of alcohol of 70% (Underwood et al., 1995) or 80% (Allen et al., 1974). The resultant white precipitate can then be removed by centrifugation and quantied against a glucose standard, using the phenolsulphuric acid assay (Dubois et al., 1956).

Various operationally dened EPS fractions (Underwood et al.,1995; Smith and Underwood, 1998; Perkins et al., 2001; de Brouwer et al., 2002a,b) have been identied as playing a leading role in sediment stability (Paterson, 1989; Yallop et al., 2000; Widdows et al., 2000; Paterson and Hagerthey, 2001; Black et al., 2002; Sun et al., 2002). Results from eld assessments of the effects of EPS on sediment stability have often been inconclusive (Defew et al., 2003) in terms of the prediction of sediment erosion potential. Part of the answer to this problem may be related to the techniques used and partly to the natural variability and complexity of the system. As a response to this natural variability, some studies have developed a new approach in which EPS is extracted and used to create model systems that can be experimentally manipulated to interpret the inuence of EPS under controlled conditions (Dade et al., 1990; Black et al., 2001; Tolhurst et al., 1999; de Brouwer et al., 2002a,b). This approach requires that workers are consistent and condent in the methodology used to extract EPS and also clear with regard to the relationship between the properties of the natural material and the extract. Ideally, the characteristics of the extracted EPS should also reect the properties of the stabilising EPS in the natural environment. In this paper, we examined the factors that affect the extraction of polymers from intertidal sediment where a clear surface microphytobenthic biolm was present. We followed this with a comparison of the affects of various extracts on the erosion of model sediments. The aims of the study were to highlight potential errors in polymer quantication, and difculties in the use of the extracts in engineered sediments for erosion experiments.

2. Methods 2.1. Biochemical characterization of the alcoholextracted polymer precipitate Sediment (30 g) collected from the Eden estuary, Fife, Scotland, UK (561220 N, 021510 W), was mixed in 2 l of saline solution (salinity 20) to extract

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soluble colloidal polymer from the sediment (Underwood et al., 1995). The resulting solution was centrifuged (3000 rpm for 15 min) and the supernatant added to 96% IMS (nal concentration of IMS of 70%; Underwood et al., 1995) and left in darkness (41C for 24 h). The resultant precipitate was separated by centrifugation (3000 rpm for 15 min), the supernatant discarded and the precipitate lyophilised. Qualitative tests for sodium (ame testan orange ame indicating a positive result), and for chloride content (silver iodide testwhite precipitate indicating a positive result) were carried out on the precipitate. Solubility of the compound was investigated in water (salt dissolves easily) and acid (polymer dissolves slowly in acid). The precipitate was added to water and stirred over heat for 48 h and a second sample was added to concentrated nitric acid. The rate of dissolution was monitored. The salt content was also assessed by dissolving 10 g of precipitate in 1 l of ultra-high-purity water, and then measuring the resultant salinity using a refractometer. Finally, the carbohydrate fraction of the material dened as EPS was quantied using the phenolsulphuric acid digest against a glucose standard curve (Dubois et al., 1956).

precipitate, the EPS content of the precipitate and the EPS content of the sediment. 2.3. Effect of IMS concentration on EPS extraction The same methods were applied as above, except that the nal concentrations of IMS used to form the precipitate were 70% (as above) and 80%. Data were analysed as above. 2.4. Effect of sediment weight on EPS extraction The same methods as above were applied, except that the amount of sediment was either 12 or 24 g wet weight (equivalent to 5 and 8 g dry weight of sediment measured by lyophilisation of paired samples). IMS was added for the precipitation step at a nal concentration of 70%. 2.5. Engineered sediments Sediment erosion experiments were carried out on both engineered cohesive and sandy sediments. Cohesive clay sediment was collected from the Eden estuary, and oven dried at 80 1C for 24 h. The dried sediment was ground to a powder and sieved through a 212 mm sieve followed by a 63 mm sieve. The sediment passing through the 63 mm sieve was collected and washed in hydrogen peroxide to reduce organic content, in principal removing the polymer naturally present in the sediment. After washing for 24 h, the sediment was dried and washed three times in ultra-high-purity water. After this, the sediment was re-ground and sieved through a 63 mm sieve and stored in a sealed container. Sand was collected from East Sands, St. Andrews, Fife, Scotland, UK (561200 N, 021460 W), sieved to collect a fraction with particle size range 212250 mm and washed as above in hydrogen peroxide. EPS was extracted from a second sample of surface cohesive sediment (approximate depth 1 cm) collected from the Eden estuary sample site. Surface sediment above the anaerobic zone, rich in surface algal biolm was selected as this was expected to be rich in microphytobenthic EPS. Sediment was re-suspended in saline water (salinity

2.2. Effect of salinity on EPS extraction Sieved and homogenised estuarine sediment collected as described above (15 g) was thoroughly mixed in 150 ml of water with a range of salinity from 0 to 40. After 1 h at 201C, the sediment was removed by centrifugation (3000 g for 15 min) and the supernatant transferred to an acid washed ask. IMS was added to a nal concentration of 70% and the EPS was allowed to precipitate overnight at 4 1C (Underwood et al., 1995). The supernatant was removed by centrifugation (3000 g for 15 min) and the precipitate thoroughly mixed. The wet weight of the precipitate was recorded and the dry weight of a sub-sample determined after evaporation of IMS/water at 80 1C over 24 h. Paired sub-samples of known weight were analysed for EPS content using the phenolsulphuric acid assay (Dubois et al., 1956). Data were then analysed as the total dry weight of

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20) for 24 h. EPS was then precipitated in 70% nal concentration IMS for 24 h (Underwood et al., 1995). The precipitate was centrifuged out of solution and washed gently in UHP water. The precipitate was allowed to partially desiccate between 5 successive washes in order to remove IMS by evaporation. The resultant precipitate (dened as IMS-precipitate) was not lyophilised and was stored in a sealed container in the dark at 4 1C prior to use. Engineered sediments (cohesive samples and sand samples) were created by adding a known weight of sediment to a known weight of articial seawater (salinity 20), to result in a water content of 50% w/w. This sediment was then divided for use in separate experiments. In experiments with IMS-EPS added, a known weight of IMS-precipitate, washed but not lyophilised (hence an equivalent dry weight of precipitate measured in paired sub-samples after measurement of loss of weight on lyophilisation), was added to subsamples of wet sediment and stirred thoroughly prior to use. The IMS-EPS content of the IMSprecipitate was determined by the phenolsulphuric acid assay (Dubois et al., 1956), as described above. EPS is reported as mg glucose equivalents per kg dry weight of sediment (mg gluc. equiv. kg1 sediment dry weight). Three replicate sub-samples were used in all measurements of water content and erosion experiments. Water content was measured after drying a known weight of sediment for 24 h at 801C. The water content was calculated as a percentage of the initial wet weight of sediment. Analysis of the IMS-precipitate (see Results) showed that addition of the IMS precipitate was primarily an addition of salt. As such, 5 and 10 g kg1 IMS-precipitate corresponded to an addition of approximately 5 and 10 g salt kg1 sediment dry weight. Such additions, with a water content of 50%, correspond effectively to pore water salinities of 10 and 20, comparatively low compared sea water of salinity 35, and the increase in salinity in sediment pore water over a low tide emersion period. In addition engineered cohesive sediments were also produced using carbohydrates obtained by rotary evaporation (dened as RE-EPS, though

containing a range of carbohydrates and not simply EPS). RE-EPS was extracted from wet sediment in saline water (salinity 20) as above. This was then slowly rotary evaporated (at 60 1C) to increase the concentration of carbohydrate. The nal RE-EPS concentration was measured by the phenolsulphuric acid assay (Dubois et al., 1956) and a known amount of RE-EPS solution was added to the cleaned sediment/water mixture described above, adjusting the water content to account for the volume of RE-EPS solution added. RE-EPS (all carbohydrates) is reported as g of glucose equivalents per kg dry weight of sediment (g gluc. equiv. kg1 sediment dry weight). 2.6. Effect of EPS and desiccation on sediment erosion Engineered sediment with 50% initial water content and either no added EPS, IMS-precipitate (5 and 10 g kg1 precipitate weight/dry weight sediment, equivalent to 15 and 30 mg kg1 EPS polymer content) and RE-EPS (0.5 and 1.0 g kg1 mixed-polymer/dry weight sediment), were eroded after 0, 60, 120 and 360 min desiccation periods. Desiccation was achieved by surface drying in air assisted by a wick of lens tissue inserted under the sediment sample and passed out into the air to result in loss of water both from the surface and the base of the sediment. Water content was measured at the time of each erosion test using paired sediment samples. Replicated (n 3) erosion experiments were carried out using cleaned cohesive clay sediment (particle size o63 mm) and cleaned sand (212250 mm). Erosion experiments were carried out directly after the desiccation period in a small water tank (10 cm 10 cm 1 cm internal dimensions) constructed from optical quality glass. A small plastic tray holding approximately 4.5 g of wet sediment was inserted into the tank (Sun et al., 2002). The tank was then slowly lled with articial seawater (salinity 20) with minimal disturbance to the sediment surface. The sediment was allowed to stand for a few minutes to allow the loss of any bubbles formed at the surface. Erosion was induced by a cohesive strength meter (CSM,

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Tolhurst et al., 1999) water jet (salinity 20). The CSM jet nozzle was held vertically above the sediment at a distance of 4 cm. Incremental increases in the jet pressure (0.6, 0.8, 1.0, 1.5, 2.5, 3.5, 4.5, 6.0, 8.0, 10.0, 12.0, 14.0, 16.0, 18.0 and 20.0 PSI) were used to stress the sediment and the resultant erosion monitored (SONY DCR PC6E digital video camera, digital zoom of 120, 3 magnication lens, total magnication 360). Erosion was dened as the jet pressure at which bed integrity was lost, clearly removing sediment particles from within the sediment bed, as opposed to erosion of the surface loose oc. 2.7. Effect of salt on sediment erosion The effect of the addition of salt on sediment erosion thresholds was examined using cohesive sediments. Cleaned cohesive sediments (collected from the Eden estuary and cleaned as described above) and with or without salt addition, were eroded using the CSM and optical tank as described. Cleaned sediment was mixed with pure water to a nal water content of 50% weight/ weight. The sample was divided into two, with one half having NaCl added to a nal content equivalent to 10 g salt kg1 dry weight of sediment. The salt was dissolved in the water prior to addition, resulting in a salinity of 20, comparatively low compared to the salinity of seawater, but equivalent to the highest addition of salt added as IMS precipitate described above. Samples (n 3) were allowed to desiccate prior to erosion tests (during which time they were immersed under water in the optical erosion tank), as described above. Erosion threshold was determined as before after 0, 2 and 6 h desiccation period. 2.8. Fine-scale structure of engineered sediment containing polymer Cohesive sediment was prepared as described above and mixed with either an IMS-precipitate solution, a sea-salt solution made from Tropic Marin articial sea-salts, or a cellulose-based synthetic polymer, Blanose (rened carboxy-

methylcellulose, Aqualon (UK) Ltd., Warrington, UK). The nal content of salt or polymer was 10 g kg1, with a water content of 50% weight/weight. Sediment samples were then frozen in liquid nitrogen, (196 1C, 1 atm) prior to examination by low-temperature scanning electron microscopy (LTSEM: JOEL 35CF SEM with a Oxford Instruments CT 1500B). LTSEM was operated following the procedures described by Paterson (1995).

3. Results 3.1. Chemical nature of the alcohol extracted polymer precipitate Initial tests with the alcohol precipitate derived from EPS demonstrated that the bulk of this material was sodium chloride co-precipitated along with polymer. A basic ame test indicated the presence of sodium (strong orange ame), and the presence of chloride was shown by reaction with silver iodide, quickly forming a thick, white precipitate of silver chloride. The saltpolymer precipitate did not completely dissolve in water, despite stirring and gentle warming over 48 h, suggesting the structure was not simply sodium chloride. The material dissolved instantly in concentrated nitric acid, suggesting the precipitate was not a simple polymer. Addition of 10 g of precipitate to 1 l of pure water resulted in a salinity of 10. Analysis of the dried precipitate for EPS content, gave a yield of between 0.2 and 3 mg EPS g1 precipitate dry weight (1.670.37 mg EPS g1, mean7s.e., n 8). 3.2. Effect of salinity on EPS extraction As the salinity of the extractant solution was increased, the EPS content of the IMS-precipitate decreased (Fig. 1A), however the total weight of IMS-precipitate increased (Fig. 1B). Overall, as extractant salinity increased, the EPS content calculated for the sediment decreased, from 640 to 130 mg EPS g1 sediment dry weight (Fig. 1C).

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80000

g EPS /g PPT dw

3.3. Effect of IMS concentration on EPS extraction Increasing IMS concentration from 70% to 80% nal concentration did not signicantly affect either the EPS content of the IMS-precipitate (although at 80%, slightly lower EPS contents were observed at salinities of 0 and 20, Fig. 2A), or the total amount of IMS-precipitate recovered (Fig. 2B). There was also no signicant variation between the calculated EPS content of the sediment for IMS concentrations of 70%and 80% nal concentration (Fig. 2C). 3.4. Effect of sediment weight on EPS extraction Increasing the wet weight of sediment used for extraction resulted in more EPS per unit weight of IMS-precipitate being formed at salinities of 0 and
80000

(A)
60000

70 % 80 %

40000

20000

g PPT dw / g Sed dw

(B)
0.10

0.05

0.00

g EPS / g Sed dw

2400 1800 1200 600 0

(C)

g EPS / g PPT dw

(A)
60000 40000 20000 0

10

20

30

40

50

Salinity

g PPT dw/ g Sed dw

0.12

(B)

Fig. 2. Precipitate IMS extracted EPS (IMS-EPS) content (A), precipitate yield (B) and sediment IMS-EPS content (C) as a function of extractant salinity at 70% and 80% nal concentration of EPS. EPS measured as mg glucose equivalents.

0.08

0.04

0.00

g EPS / g Sed dw

800 600 400 200 0

(C)

20 (Fig. 3A). However, the total amount of IMSprecipitate was less than at the lower sediment weight (Fig. 3B). Overall this resulted in a signicantly lower (F 2;18 8:5; po0:05, post hoc Tukey) sediment EPS content at zero salinity with the greater sediment weight used (Fig. 3C). The sediment EPS content decreased signicantly as salinity increased (F 2;18 179; po0:001, post hoc Tukey).

10

20

30

40

50

3.5. Effect of EPS and desiccation on sediment erosion In control cohesive sediments (no EPS), water content signicantly decreased (F 3;32 8:681; po0:001) from 50% to 23% w/w (Fig. 4) over the 6 h desiccation period. There was no

Salinity

Fig. 1. Precipitate IMS extracted EPS (IMS-EPS) content (A), precipitate yield (B) and sediment IMS-EPS content (C) as a function of extractant salinity. EPS measured as mg glucose equivalents.

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g EPS / g PPT dw

60000 40000 20000 0 0.04 0.03 0.02 0.01 600 500 400 300 200 100 0

(A)

Water Content (% of w.w.)

12g Sed wet wt 24 g sed wet wt

50

No EPS 15 g Kg-1 EPS 30 g Kg-1 EPS

40

g EPS / g Sed dw g PPT dw / g Sed dw

(B)

30

20

100

300

400

Time (minutes)

(C)

Fig. 4. Decrease in water content of cohesive sediment with no added IMS-EPS, 15 mg gluc. equiv. kg1 sediment dry weight added IMS-EPS precipitate (5 g kg1 IMS-precipitate) and 30 mg gluc. equiv. kg1 sediment dry weight added IMS-EPS (10 g kg1 IMS-precipitate), over a 6 h desiccation period.

10

20

30

40

50

10 No EPS 15 IMS-EPS 30 IMS-EPS

(A) A

Salinity
8

Fig. 3. Precipitate IMS extracted EPS (IMS-EPS) content (A), precipitate yield (B) and sediment IMS-EPS content (C) as a function of extractant salinity with 12 and 24 g wet weight of sediment added to the extractant solution. EPS measured as mg glucose equivalents.

signicant decrease in water content for cohesive sediment treatments with added EPS (IMS-precipitate and RE-EPS). Water content was signicantly lower in the controls as compared with EPS treatments (F 2;33 10:493; po0:001). The decrease in water content in the cohesive controls occurred concomitantly with an increase in the erosion threshold (Fig. 5). The erosion threshold increased logarithmically from the minimum measurable (0.6 PSI) CSM jet pressure to 6 PSI in run 1 (Fig. 5A) and 4.4 PSI in run 2 (Fig. 5B) demonstrating the variation in erosion thresholds for cohesive sediment without polymer. The addition of EPS at 15 mg gluc. equiv. kg1 sediment dry weight (5 g kg1 IMS-precipitate and hence approximately 5 g kg1 salts), resulted in an increase in erosion threshold over the desiccation period, however compared to the controls, the erosion thresholds were lower (Fig. 5A). In the presence of 30 mg gluc. equiv. kg1 sediment dry

Jet pressure (PSI)

0 10

No EPS 0.5 RE-EPS 1.0 RE-EPS

(B)

0 -50

50

100

150 300

400

Time (minutes)

Fig. 5. Erosion threshold (CSM jet pressure) for cohesive sediment over a 6 h desiccation period with no added EPS, 15 and 30 mg gluc. equiv. kg1 sediment dry weight added IMSEPS (5 and 10 g kg1 IMS-precipitate, respectively) (A), or 0.5 and 1.0 g gluc. equiv. kg1 sediment dry weight rotary evaporated EPS (B).

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35 30 No EPS 15 IMS-EPS 30 IMS-EPS

weight (10 g kg1 IMS-precipitate and hence approximately 10 g kg1 salts), the erosion threshold was comparable to the cohesive controls (Fig. 5A). In another experiment RE-EPS was added to sediments in contents of 0.5 and 1.0 g gluc. equiv. kg1 sediment dry weight. Addition of 0.5 g gluc. equiv. kg1 sediment dry weight REEPS increased erosion thresholds of cohesive sediments over the desiccation period. Initially erosion thresholds were slightly higher compared to cohesive controls, but increased two-fold at end of the 6 h desiccation period (Fig. 5B). A similar pattern was observed for 1.0 g gluc. equiv. kg1 sediment dry weight RE-EPS, with no signicant difference between the treatments with different RE-EPS concentrations after 6 h desiccation. In both experiments, the increase in erosion threshold over time was signicant (F 3;24 4:359 and 5 .739 for 0.5 and 1.0 g gluc. equiv. kg1 sediment dry weight RE-EPS respectively, po0.05). Water content of sandy sediments decreased signicantly over the 6 h desiccation period (F 3;32 192; po0:001), but did not differ between controls without EPS and those with IMSprecipitate and RE-EPS (Fig. 6). Sand controls without EPS showed no change in erosion threshold over time (a jet pressure of 0.7 PSI eroded the sediment in all tests), and there was no effect of IMS-precipitate at either 15 or 30 mg gluc. equiv. kg1 sediment dry weight. Erosion thresholds for sand sediments (data not shown) increased slightly with addition of RE-EPS, from 0.7 PSI (no added RE-EPS) to 0.970.02 and 0.970.11 PSI at 0.5 and 1.0 g gluc. equiv. kg1 sediment dry weight respectively. However, the erosion threshold then declined back to 0.7 PSI at both RE-EPS contents, after 6 h desiccation. Statistically, these changes in erosion threshold were not signicant over time, or between controls and RE-EPS treatments. When erosion thresholds for the cohesive sediments were plotted as a function of water content (Fig. 7), it was clear that addition of EPS resulted in a greater retention of water in the sediment. The increase in erosion threshold with declining water content was more rapid for EPS treatments compared to controls.

Water Content (%)

25 20 15 10 5 0 0 100 300 400

Time (minutes)

Fig. 6. Decrease in water content of sand sediment with no added IMS-EPS, 15 and 30 mg gluc. equiv. kg1 sediment dry weight added IMS-EPS (5 and 10 g kg1 IMS-precipitate, respectively) over a 6 h desiccation period.

10

Jet pressure (PSI)

15 g Kg IMS-EPS -1 30 g Kg IMS-EPS -1 0.5 g Kg RE-EPS -1 1.0 g Kg RE-EPS No EPS run 1 No EPS run 2

-1

0 20

25

30

35

40

45

50

55

Water Content (%)

Fig. 7. Erosion threshold (CSM jet pressure) for cohesive sediment over a 6 h desiccation period with no added EPS, 15 and 30 mg gluc. equiv. kg1 sediment dry weight added IMSEPS (5 and 10 g kg1 IMS-precipitate, respectively), or 0.5 and 1.0 g gluc. equiv. kg1 sediment dry weight rotary evaporated EPS as a function of water content.

3.6. Effect of salt on sediment erosion The erosion threshold of cleaned cohesive sediment was compared to paired sediment samples with added salt (NaCl, 10 g salt kg1 sediment dry weight). After no dessication period and after 2 h of desiccation, the addition of salt alone had no effect on the erosion threshold of the sediment

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(Fig. 8). However after 6 h desiccation, the erosion threshold signicantly increased with the addition of salt. 3.7. Engineered sediment structure Cohesive engineered sediments with either IMSprecipitate, sea-salt or the industrial cellulose based polymer Blanose, were visualised by lowtemperature scanning electron microscopy. IMSprecipitate and cohesive sediment formed a heterogeneous matrix with strands, plates and lenticular pores attributed to the polymer interaction with the sediment particles (Fig. 9A). A similar appearance of stranding was also observed with a polymer-free, sea-salt and cohesive sediment matrix (Fig. 9B). In contrast, a salt-free polymer (Blanose) and cohesive sediment matrix showed a more homogenous appearance, with a gel-like matrix of polymer and water, in which the sediment particles were imbedded (Fig. 9C).

4. Discussion The precipitate formed during conventional saline/alcohol extraction of IMS-EPS (Underwood et al., 1995) was largely composed of sodium chloride. Forms of bacterial and algal EPS differ

No salt added 10 g Kg-1 salt

Jet pressure (PSI)

0 T0 T2 T6

Time in hours

Fig. 9. Low-temperature scanning electron micrographs (LTSEM) of cleaned cohesive sediments with different polymer additives. (A) Structures of plates and strands observed for IMS-precipitate (5 g kg1); (B) Similar structures observed for sediments with added sea salt solution; (C) Sediment with added Blanose polymer (5 g kg1), showing an absence of any strands or plates. Scale bars all 10 mm.

Fig. 8. Erosion threshold (CSM jet pressure) for cleaned cohesive sediment over a 6 h desiccation period with no added salt and 10 g NaCl kg1 sediment dry weight (mean7s.e., n 3).

greatly, but active charged carboxyl, hydroxyl, phenolic, amine and sulfhydryl binding sites (Liu and Fang, 2002) may facilitate co-precipitation of

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polymer with salt in 70% alcohol, in addition to direct precipitation of polymers (Allen et al., 1974). Direct and co-precipitation is supported by the fact that when the precipitate was replaced in water, not all the precipitate redissolved. The co-precipitation of the polymer with the salt has important implications regarding the quantication of sediment EPS content, as well as the use of this precipitate for creating engineered sediments. The sediment IMS-EPS content was found to be a function of the salinity of the extractant, with higher values obtained at lower salinity (Fig. 1C, 2C and 3C). As a result, it is unclear at which salinity the true EPS content was obtained and that the fraction described as EPS is highly operationally dependent. It may be that differentiation between EPS and LMW carbohydrates is just a gradient by size classication, determined by the extractant salinity, with little biological signicance. The nature of the polymer extracted by this procedure needs to be more fully understood, especially with regard to its role in sediment stability. It is suggested that operational fractionation should be restricted to the soluble colloidal fraction prior to the saline/alcohol precipitation stage (Underwood et al., 1995) or the use of alternative methods including molecular sieves or ultraltration (de Brouwer and Stal, 2001). In addition, the solubility of the polymer may have decreased as the extractant salinity increased. This may be important in sediment stability as it explains why a polymer that is soluble at low salinity may be important in maintaining sediment stability under saline conditions. This suggests that the export of DOC from intertidal sediments may be partially controlled by ood water salinity and rain events. This possibility is worthy of further study. The IMS-EPS content of the precipitate declined as the precipitate weight increased, due to an increase in the salinity of the extractant solution. This has important implications on the use of this precipitate in engineered sediment experiments. This suggests that when sediments of 5 g kg1 IMS-precipitate were produced, the actual EPS content was nearer 1030 mg gluc. equiv. kg1 sediment dry weight (based on the

current study). Thus the role of EPS in sediment stability may have been masked due to the addition of salt, which itself may aid sediment stability through crystal formation during periods of desiccation. Alternatively, where increases in stability were reported, this may indicate a higher binding capacity of the EPS than originally thought and hence a greater role in sediment stability in situ. This will be discussed further with regard to the sediment erosion experiments. It should be noted that the removal of organic material by washing with hydrogen peroxide, would have greatly affected the properties of the engineered sediments compared to natural sediments. This alteration of sediment properties, in addition to problems in extraction and use of benthic polymers highlights potential aws in the use of engineered sediments in erosion studies. Addition of EPS at contents of 1030 mg gluc. equiv. kg1 sediment dry weight are far from excessive. Perkins et al. (2003) reported EPS sediment contents of approximately 3 mg gluc. equiv g1, (3000 mg gluc. equiv. kg1) for cohesive sediment in the Eden estuary, Fife, UK. Perkins et al. (2001) observed an increase in sediment EPS content from approximately 250800 mg gluc. equiv. m2 over a 6-h emersion period. This concentration per unit area of sediment, as in other studies, is not directly comparable to a sediment content used in the experiments described here-in, but demonstrates the comparatively large amounts of polymer that may be found in natural sediments. For a detailed review of EPS and typical production rates see Underwood and Paterson (2003). The importance of a desiccation period (6 h) for the stability of cohesive sediment without polymer was shown by the increase in erosion threshold with time (Fig. 5). This was presumably due to the loss of pore water leading to consolidation and increasing the electrostatic binding between clay particles (Chenu, 1993). The increase in sediment stability was biphasic, with a rapid increase with only a 1% decrease in water content, preceding to a slower increase (Fig. 7). The erosion threshold for cohesive sediment without added polymer varied between

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runs (compare Figs. 5A and B), possibly due to different levels of electrostatic binding due to variation in cleaned sediment. In comparison to controls, the addition of 5 g kg1 IMS-precipitate reduced the erosion threshold over the desiccation period (Fig. 5A), possibly due to the EPS reducing de-watering (Fig. 7). In contrast, 10 g kg1 IMS-precipitate resulted in a comparable erosion threshold to the controls, despite a similar decrease in dewatering. It is suggested that the addition of IMS-precipitate acted to retain water in the sediment, but at 10 g kg1 began to have a stabilising effect. This may have been due to the greater addition of polymer, or due to the heavier salt loading at 10 compared to 5 g kg1 IMS-precipitate. As discussed above, the actual sediment EPS contents were not 5 and 10 g gluc. equiv. kg1sediment dry weight, but 15 and 30 mg gluc. equiv. kg1 sediment dry weight. The salt loading of the two IMSprecipitate additions, was approximately 5 and 10 g salt kg1 sediment dry weight, equivalent to pore water salinities of 10 and 20. This is not a large salt loading compared to natural seawater as stated above. However after 6 h desiccation, 10 g salt kg-1 sediment dry weight resulted in a slight, but signicant increase in the erosion threshold compared to cleaned sediment with no salt addition (Fig. 8). This further complicates the interpretation of polymer binding, but emphasises the negative effect on erosion threshold observed with 5 g kg1 IMS precipitate. In contrast, REEPS increased sediment stability (Fig. 5B) at both 0.5 and 1.0 g gluc. equiv. kg1sediment dry weight, however these were true carbohydrate contents measured by the phenolsulphuric acid assay. However, as stated above the rotary evaporated fraction included a range of carbohydrates, not just EPS, thus the specic role of EPS cannot be identied. The inuence of the water content was important, with both forms of EPS reducing dewatering (Fig. 7) and a biphasic increase in erosion threshold observed as water content decreased. In contrast to the experiments with engineered cohesive sediments, no increase in sediment stability was observed over the desiccation period in sandy sediments, despite a greater decrease in

water content (Fig. 6). This was presumably due to the lack of extensive electrostatic binding compared to clay particles. This is in contrast to experiments with Xanthum gum (a bacterial EPS polymer), as well as natural bacterial EPS (Dade et al., 1990), which have been observed to increase stability in sand (Black et al., 2001; Authors pers. obs.). LTSEM examination of the polymersedimentmatrix (Fig. 9) showed that the strand and plate structures associated with polymer (Paterson, 1990; Tolhurst, 1999), could equally well be explained by the interaction between salts and polymer, with the salts forming crystals due to freeze exclusion during sample preparation. Similar structures were observed for the saltsediment and the polymersaltsediment matrices. Such structures are therefore artefacts, with a true polymer forming a more gel-like matrix with cohesive sediment particles, such as that observed for Blanose, more similar to natural intertidal sediments observed by LTSEM (Authors, pers. obs.). The stabilisation of sediment is probably a complex interaction involving polymers, salt cations and desiccation processes.

5. Conclusions

Quantication of EPS is a function of extractant salinity and alcohol concentration. The operational quantication of microphytobenthic polymers by these techniques should be treated with caution. The use of the saline/alcohol precipitate in sediment erosion experiments is not advised due to the inuence of the high salt loading and the low yield of polymer in the precipitate. Salt should always be removed after polymer extraction and prior to the use in erosion experiments. Rotary evaporated carbohydrate (preferably from a 0 salinity extractant) may be an alternative to alcohol precipitation, although thermal degradation may occur, making comparisons with in situ polymer problematic and the addition of mixed carbohydrates prevents discrimination between active (with respect to stability) and non-active polymers.

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1634 R.G. Perkins et al. / Continental Shelf Research 24 (2004) 16231635 Raffaelli, D., Solan, M., Paterson, D.M., Buck, A.L., Pomfret, J.R. (Eds.), Coastal Zone Topics 5: The Estuaries and Coasts of North-east Scotland. Estuarine Coastal Sciences Association, pp. 6170. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1996. Colorimetric method for determination of sugars and related substances. Analytical Chemistry 28, 350356. Hakvoort, J.H.M., Heineke, M., Heymann, K., Ku hl, H., Riethmu ller, R., Witte, G., 1998. A basis for mapping the erodibility of tidal ats by optical remote sensing. Marine Fresh water Research 49, 867873. Liu, H., Fang, H.H.P., 2002. Characterization of electrostatic binding sites of Extracellular polymers by linear programming analysis of titration data. Biotechnology Bioengineering 80, 806811. Madsen, N.P., Nillson, P., Sundback, K., 1993. The inuence of benthic microalgae and the stabilisation of a subtidal sediment. Journal of Experimental Marine Biology Ecology 170, 159178. Paterson, D.M., 1989. Short-term changes in the erodibility of intertidal cohesive sediments related to the migratory behaviour of epipelic diatoms. Limnology and Oceanography 24, 223234. Paterson, D.M., 1990. The inuence of epipelic diatoms on the erodibility of articial sediment, In: Simola, H. (Ed.), Proceedings of the 10th International Symposium on Living and Fossil Diatoms. Joensuu, 1988. Koenigstein pp. 345355 Paterson, D.M., 1994. Microbiological mediation of sediment structure and behaviour. In: Stal, L.J., Caumette, P. (Eds.), NATO ASI Series Vol. G35. Microbial Mats. Springer, Berlin Heidelberg. Paterson, D.M., 1995. The biogenic structure of early sediment fabric visualised by low-temperature scanning electron microscopy. Journal of the Geological Society 152, 131140. Paterson, D.M., Black, K.S., 1999. Water ow, sediment dynamics, and benthic biology. In: Raffaelii, D., Nedwell, D. (Eds.), Advances in Ecological Research. OUP, Oxford, pp. 155193. Paterson, D.M., Hagerthey, S.E., 2001. Microphytobenthos in contrasting coastal ecosystems: biology and dynamics. In: Reise, K. (Ed.), Ecological Comparisons of Sedimentary Shores. Ecological Studies 151 105125. Perkins, R.G., Underwood, G.J.C., Brotas, V., Jesus, B., Ribeiro, L., Snow, G., 2001. In situ microphytobenthic primary production during low tide emersion in the Tagus estuary, Portugal: production rates, carbon partitioning and vertical migration. Marine Ecology Progress Series 223, 101112. Perkins, R.G., Honeywill, C., Consalvey, M., Austin, H.A., Tolhurst, T.J., Paterson, D.M., 2003. Changes in microphytobenthic chlorophyll-a resulting from sediment compaction due to de-watering: opposing patterns in concentration and content. Continental. Shelf Research. 23, 575586.

 

IMS extracted precipitate acts to retain water in the sediment, which in engineered sediments can cause a decrease in sediment stability. Whilst engineered sediments can be useful in the analysis of sediment erosion, they are not good analogues of natural sediment. The most promising way to investigate the role of polymers in biostabilisation of sediments is through erosion experiments with natural samples with a wide range of polymer and water contents.

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