A new method for extraction of extracellular polymeric substances from biofilms and activated sludge suitable for direct quantification of sorbed metals

A method for extraction of extracellular polymeric substances (EPSs) with a dicyclohexyl-18crown-6 ether was developed to determine levels of organic and inorganic contaminants sorbed to EPS. The crown ether selectively binds alkaline and alkaline earth metals but not heavy metals. The effectiveness of the extraction procedure was higher than that of 2 other methods tested and comparable with that of a method based on a cation exchange resin. On average it was possible to extract 20% of the TOC, 12% of the total protein content, and 4% of the total carbohydrate content of sludge or biofilm biomass. Metal sorption studies in activated sludge showed no influence of exposure time on the fractionation of metals within the biomass. Metals sorbed mostly to cellular material. In biofilms 12.2% of the cadmium and 9.1% of the zinc added was found in the EPS. In activated sludge EPS contained only 2.9% zinc. The distribution of metals within the biomass was dose dependent. The percentage of metals found in EPS decreased with increasing metal concentration. This indicates a higher affinity of metals for cellular binding sites. Time course experiments in a rotating biofilm annular reactor, which consisted of an external cylinder with removable slides and an internal solid drum, revealed a gradual change in zinc concentration associated with EPS, although the total zinc concentration in the biomass remained constant. Concurrently, the amount of extractable EPS decreased. This was a consequence of a microbial population shift, with bacterial counts decreasing and algal and fungal biomass increasing. Using confocal laser scanning microscopy and the fluorescent metal complexing agent Newport Green for in situ detection of zinc it was shown that metals were bound to algae and fungi in the latter part of the experiment. The biofilm became more and more heterogeneous coinciding with a decrease in EPS. To summarize, the observed sorption behavior of metals cannot be explained with the conventional paradigm of EPS as hydrophilic gel. Obviously, different binding mechanisms must be invoked to explain the role of EPS in the sorption and removal of toxic substances in activated sludge and biofilm systems. It is important to consider the microbial population to understand differences in sorption in different matrices.