The major carbohydrate in red macroalgae is agarose, which is composed fp-galactose and 3,6-anhydro-L-galactose (AHG). So far, agarose saccharification was most efficiently achieved by prehydrolysis using a weak acid and subsequent enzymatic hydrolysis into monomeric sugars. However, this process requires rigorous neutralization after acid prehydrolysis, resulting in the formation of salts. Moreover, residual agarotriose is not further hydrolyzed by currently available beta-agarases. In this study, we aimed at eliminating or minimizing the neutralization step by using a low-concentration acid-base buffer such as 20 mM (0.3%, w/v) Tris HCI for hydrothermal pretreatment of agar at 170 degrees C for 10 min. In order to hydrolyze agarotriose, an agarolytic beta-galactosidase acting on odd-numbered agarooligosaccharides, was introduced into the enzymatic saccharification. Using these chemical and enzymatic processes, the monomeric sugar yields of 44.9% for galactose and 44.8% for AHG on the basis of their theoretical maximum amounts from the initial agar were obtained. (C) 2015 Elsevier Ltd. All rights reserved.