BACKGROUND: Brewer's spent grain (BSG) is the most abundant by-product of the brewery industry. The current massive output of BSG requires novel methods of bioconversion into added-value products. Its composition and high biological value, i.e. high protein content, allows for various biotechnological applications. RESULTS: Here, BSG was used as a substrate for submerged cultivation of keratinolytic bacteria, Bacillus cereus PCM 2849 and B. subtilis PCM 2850. The strains biosynthesized proteases to decompose proteins within the substrate and amylases to degrade residual starch. Enzymes degrading structural polysaccharides were also present. Bacillus cereus produced multiple proteases with high activity, predominantly with molecular weight (mw)> 70 kDa, depending on the presence of minerals. Bacillus subtilis biosynthesised proteases of wide mw range, independently of the mineral components. Crude culture fluids of both bacteria exhibited proteolytic activity on a variety of proteins, including keratins. Structural deterioration of the BSG in microbial cultures was observed. CONCLUSION: The BSG was a suitable medium component for the production of bacterial proteases and polysaccharide-degrading enzymes. Two keratinolytic Bacilli, grown in the presence of BSG utilized residual proteins and biosynthesized proteases exhibiting keratinolytic activity. Bacillus cereus dominated in terms of protease production, while B. subtilis presented higher production of amylases, cellulases and xylanases. (C) 2016 Society of Chemical Industry