The mechanism of enhanced sludge floc fracture due to the action of enzymes hydrolysing important structural components, remains a key element in our understanding of how the floc integrity in systems utilising a sulphate-reducing system is compromised. Commercial beta-glucosidase and cellulase as well as proteases were added to methanogenic bioreactor and sulphidogenic bioreactor sludge and the effect of these enzymes on sludge floc size (diameter) distribution was investigated using bright field and differential interference contrast light microscopy. Cellulolytic activity of the enzymes was monitored using standard enzyme assay techniques and Bradford, Somogyi-Nelson, and total carbohydrate assays were performed. Sludge flocs present in the sulphide-rich environment of the sulphidogenic bioreactor were found to be of smaller average diameter (23 mum) than those floes present in the methanogenic bioreactor (average diameter of 40 mum). The addition of commercially available enzymes resulted in an increased rate of matrix hydrolysis, leading to increased rates of floc fracture and smaller flocs. The presence of beta-glucosidase activity and protease, naturally residing within the sludge flocs, was confirmed. The addition of commercial enzymes may be prohibitively costly on a large scale, and ways of increasing the activities of these enzymes naturally residing within the floc matrix were investigated. As the bulk of the extracellular polymeric substances (EPS) was shown to be composed of mainly polysaccharides and protein, it was proposed that, by increasing the activities of the native beta-glucosidases and proteases naturally residing within the floc matrix, the process of floc hydrolysis could be enhanced. Sulphide was shown to increase the activity of these key enzymes, and we propose that this is one of the factors contributing to the accelerated rate of sludge solubilisation under sulphate-reducing conditions. It was also confirmed that this effect of sulphide occurred at the enzymatic level. (C) 2004 Elsevier Inc. All rights reserved.