This study investigated the effect of heat-alkaline treatment (HAT) at pH 11 and 60 degrees C on volatile fatty acid (VFA) production and protein degradation in excess sludge, soluble and insoluble proteins, and pure cultures. In addition, quantification of bacteria present in the sludge was also examined. Experimental results showed that following acid fermentation under pH 7 and 37 degrees C, HAT enhanced VFA production in excess sludge, albumin, and Gram-negative bacteria, but not in casein or Gram-positive bacteria. Protein solubility was therefore found not to be the main criteria for VFA production. In the protein analysis, it was shown that the outer membrane protein (OmpC) of Escherichia coli K12 was resistant to chemical and enzymatic hydrolysis. Gram staining revealed that Gram-negative bacteria were predominant in the activated sludge used in this study. In addition, the bacteria present in the activated sludge comprised only 10% of mixed liquor suspended solids (MLSS) by quantitative PCR. (c) 2012 Elsevier Ltd. All rights reserved.