Comparison of extraction methods for quantifying extracellular polymers in biofilms

Five commonly used extraction methods - regular centrifugation, EDTA extraction, ultracentrifugation, steaming extraction and regular centrifugation with formaldehyde (RCF) - were selected to study their effectiveness and repeatability in extracting extracellular polymeric substances (EPS) from aerobic/sulfate reducing and nitrifying/denitrifying biofilm samples. Biofilm EPS extraction yields were represented by carbohydrate and protein concentrations; the amount of cell lysis during the extractions was indicated by DNA concentration. The results showed that analyzing wash waters is essential in quantifying biofilm EPS; the contribution of this step varied from 8-50% of the total carbohydrate yield, depending on the extraction method. Among the extraction methods, the RCF extraction gave the greatest carbohydrate yield, the steaming extraction gave the greatest protein yield, and the other three extraction methods gave approximately equivalent amounts of carbohydrate and proteins for both types of biofilm. DNA in the EPS was 27 times smaller than in the pellets, indicating no significant cell lysis occurred during the extractions.