Extracellular polymeric substances: quantification and use in erosion experiments
Introduction
Over the past decade there has been an ongoing search for biological variables that can be used to describe the influence 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; Riethmüller et al., 2000, Smith and Underwood, 1998; 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 °C for 20 min in quantification 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 final 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 quantified against a glucose standard, using the phenol–sulphuric acid assay (Dubois et al., 1956).
Various operationally defined EPS fractions (Underwood et al.,1995; Smith and Underwood, 1998; Perkins et al., 2001; de Brouwer et al., 2002a, de Brouwer et al., 2002b) have been identified 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 field 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 influence of EPS under controlled conditions (Dade et al., 1990; Black et al., 2001; Tolhurst et al., 1999; de Brouwer et al., 2002a, de Brouwer et al., 2002b). This approach requires that workers are consistent and confident 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 reflect 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 biofilm 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 quantification, and difficulties in the use of the extracts in engineered sediments for erosion experiments.
Section snippets
Biochemical characterization of the alcohol-extracted polymer precipitate
Sediment (30 g) collected from the Eden estuary, Fife, Scotland, UK (56°22′N, 02°51′W), was mixed in 2 l of saline solution (salinity 20) to extract 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 (final concentration of IMS of 70%; Underwood et al., 1995) and left in darkness (4°C for 24 h). The resultant precipitate was separated by centrifugation (3000 rpm for 15 min), the
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 flame test indicated the presence of sodium (strong orange flame), and the presence of chloride was shown by reaction with silver iodide, quickly forming a thick, white precipitate of silver chloride. The salt–polymer precipitate did not completely dissolve in water, despite stirring and gentle warming over 48 h, suggesting the
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 greatly, but active charged carboxyl, hydroxyl, phenolic, amine and sulfhydryl binding sites (Liu and Fang, 2002) may facilitate co-precipitation of 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
Conclusions
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Quantification of EPS is a function of extractant salinity and alcohol concentration. The operational quantification of microphytobenthic polymers by these techniques should be treated with caution.
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The use of the saline/alcohol precipitate in sediment erosion experiments is not advised due to the influence 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.
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Rotary evaporated
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