The measurement of microbial carbohydrate exopolymers from intertidal sediments

Measurements of microbial extracellular polymeric substances (EPS) of natural field populations are required to understand the processes of biogenic stabilization and the microbial ecology of intertidal sediments. EPS in sediments can be measured by the phenol-sulfuric acid assay to measure carbohydrate concentration in sediment samples. We conducted comparative studies of storage and extraction methods for carbohydrate fractions on intertidal estuarine sediments. Measurements of both total and colloidal (material remaining in suspension after aqueous extraction and centrifugation) carbohydrate concentrations were highly dependent on storage conditions, sample size, extraction media, and time. Precipitation (in 70% ethanol) of EPS from saline (25ym) extractions of colloidal carbohydrate (colloidal S) from four different sediment microbial assemblages showed that 20% of colloidal carbohydrate present in extracts consisted of EPS. Concentrations of colloidal S were highly correlated with diatom biomass as determined by chlorophyll a concentrations. Extractions that used 100 mM Na,EDTA as the extracting medium (colloidal EDTA) increased the concentrations of colloidal carbohydrate obtained and, in sediments dominated by bacteria and cyanobacteria, increased the pcrcentagc of EPS in the material to 38%. Uranic acids comprised 20% of the total amount of carbohydrate measured in both total sediment and colloidal-S extracts but increased to 65% in colloidalEDTA extracts, suggesting that colloidal-EDTA extractions remove bacterial capsular EPS and EPS more closely associated with sediment particles. In aquatic habitats, many microorganisms secrete extracellular polymeric substances (EPS) into the surrounding environment. The ability of bacteria to secrete polymeric material is impressive, and it has been calculated that a single bacterium (Azotobacter sp.) can produce enough EPS to coat more than 500 particles (0.4-pm diam) per day (Dade and Nowell 199 1). This polymeric material has variously been described as slime, mucus, mucilage, glycocalyx, and exopolymer. However, the various longchain molecules produced and secreted by microbial metabolism can be described collectively as EPS. Historically, there has been considerable interest in the role of EPS at the cellular level in the avoidance of desiccation, in cell attachment (Savage and Fletcher 1985; Daniel et al. 1987) and locomotion (Edgar and Pickett-Heaps 1984), and in resistance to toxins (Decho 1990). On a larger scale, organic secretion from microbial cells may act as an important vector for carbon cycling and as a carbon source for other microbes and for deposit-feeding invertebrates I Present address: Department of Biological and Chemical Sciences, University of Essex, Colchester, Essex, CO4 3SQ, U.K. * Present address: Gatty Marine Laboratory, The University, St. Andrews, Fife KY 16 8LB, U.K. Acknowledgments We thank B. Cragg, P. Wellsbury, B. de Winder, M. Yallop, and three anonymous reviewers for their comments on the manuscript. This work was funded by NERC grant GR3/8056; this support is gratefully acknowledged. , (Baird and Thistle 1986; Decho and Moriarty 1990; Decho and Lopez 1993). Organic materials in sediments also influence the critical erosion threshold for the entrainment of natural sediments (Dade et al. 1990; Underwood and Paterson 1993a,b; Daborn et al. 1993); for this reason, there is considerable interest in making measurements of EPS concentration in natural sediments. There is no single analytical method that can detect all components of microbial EPS, but a method commonly used to quantify EPS in sediments is the phenol-sulfuric acid assay (Dubois et al. 1956). This calorimetric method is sensitive to a wide range of carbohydrates, including sugars, methylated sugars, and neutral and acidic polysaccharides, which comprise most of the components of microbial EPS (Decho 1990); it is therefore a useful measure to quantify EPS material from sediments. The phenol-sulfuric assay has been used in a number of studies investigating the effects of microbial films on sediment stability (Grant et al. 1986; Madsen et al. 1993; Yallop et al. 1994). Although comparison of the data presented in these publications is desirable, it may not be valid, because there is insufficient information about the effect of different protocols for sample storage and extraction on polymer recovery. Although measurements of sediment carbohydrate concentration with the phcnolsulfuric assay are straightforward, different procedures for sample storage and preparation have been used, including immediate measurement of fresh material (Grant and Gust 1987), wet storage of sediment samples on ice (Grant et al. 1986), storage at 4°C (Liu et al. 1973; Madsen et al. 1993), drying at 105°C (Liu et al. 1973), freezing and