World Journa/ of Microbiology & Biotechnology 11. 481-485 Electron microscopic examination of the extracellular polymeric substances in anaerobic granular biofilms F.A. MacLeod, S.R. Guiot* and J.W. Costerton Scanning electron microscopy revealed that collapsed extracellular polymeric substances (EPS) surrounded bacteria present in granular sludge. Treatment of granular sludge with whole-cell antiserum and staining with polycationic ferritin demonstrated that bacteria were enveloped by extensive EPS. Antibody stabilization permitted a visualiza- tion of the EPS which more closely resembled its natural hydrated state. The BPS was seen to completely fill the intercellular spaces in the microcolonies. Both pure and mixed microcolonies were observed to be enclosed by EPS. The presence of these large amounts of EPS indicates that this extracellular layer is important in maintaining the structural integrity of granular sludge. Key words: Anaerobic granule, electron microscopy, extracellular polymeric substances. Successful operation of upflow anaerobic sludge blanket (UASB) reactors in wastewater treatment depends upon the large accumulation of well-settling bacterial aggregates (De Zeeuw 1988). The aggregates produced in this process generally range from 1 to 3 mm in diam. (Beeftink & Van Den Heuvel 1988; Guiot et al. 1988) and are referred to as bacterial granules. The stability of anaerobic granules is based upon the firm attachment of the cells to each other. The irreversible adhesion of bacterial cells to both inert and living surfaces is mediated by a number of surface structures, including pili (Svanborg-Eden et al. 1977). flagella (Sjoblad & Doltsch 1982), distinct holdfasts (Poindexter 1964) and extracellular polymeric substances (EPS). EPS appear to be ubiquitous in natural ecosystems (Costerton et al. 198ta, b) and their fibrous nature makes them ideal for irreversible adhesion, since it is very unlikely that, once adsorbed, all the fibres would desorb at the same time. Adhesion medi- ated by EPS results in the development of an aggregate in which a large number of cells is bound in a ‘biological cement’. The high stability of the bacterial granules pro- F.A. MacLeod is with the Centrefor Mineral and EnergyTechnology, Natural Resources Canada, Ottawa, Ontario, KlA OGl, Canada. S.R. Guiot is with the Biotechnology Research Institute, National Research Council. 6100 Royalmount Avenue, Montrbal. H4P 2R2. Canada; fax: 514 4966265. J.W. Costerton is with the Centre for Biofilm Engineering, Montana State University, Bozeman. Montana, 59717-0398, USA. ‘Corresponding author. @ 1995 Rapid Science Publishers duced in upflow wastewater treatment systems indicates that bacterial EPS may be involved in the development and maintenance of the granular structure. As bacterial EPS may consist of 99% water (Sutherland 1972) they become severely condensed during the dehydra- tion which is required for examination by standard electron microscopy (Costerton et al. 1981a). To prevent this loss of structure, specific antibodies (Bayer & Thurow 1977; Mackie et al. 1979) have been used to cross link the polymers of the EPS. The application of stains which bind to the EPS, such as ruthenium red (Luft 1971) and polycationic ferritin (Danon et ul. 1972) can then be used to demonstrate the dimensions of the stabilized EPS. In the present study these stabilization techniques have been used to determine the importance of EPS in the structure of granular sludge. Materials and Methods Anaerobic Granules The anaerobic granules used came from a 13.5-1 upflow sludge bed filter (UBF) operated at 35°C and pH 7.2 f 0.1 and fed a synthetic solution containing (g/l): sucrose, 4.4; yeast extract, 0.044; KH,PO,, 0.088; K,HPO,, 0.118; (NHJ2S04, 0.221; KHCO,, 5.0; and NaHCO,, 4.0. The reactor design and operation details are described by Arcand et a!. (1994). The rate of dilution was 3.0 vol/vol. day. A recirculation to feed flow ratio of IO:1 produced a Worldloumal of Micmbmlogy 6 Biotechnology. Vol IL 1995 481