BACKGROUND: Millions of tons of feather waste from the poultry industry are disposed of annually despite containing a high level of keratin. The aim of this study was to compare the hydrolysis of non-treated feather waste using three different approaches (whole cell microbial digestion, enzymatic and chemical cleavage) and to test the use of hydrolysates as peptone substitutes in a culture medium. RESULTS: Among bacterial isolates, Pseudomonas sp. P5 exhibited the highest keratinolytic activity and efficiency to hydrolyse raw feather material. The hydrolysates contained up to 301 mg L-1 of free amino acids and 6.2 g L-1 of peptides. Hydrolysates obtained by digestion using semi-purified keratinase from Pseudomonas sp. P5 were richer in amino acids (1191 mg L-1, 56% essential ones) but peptides were present in lower amounts (up to 3.3 g L-1). The third approach was feather treatment under mild alkaline conditions. This provided the highest amount of peptides (17.2 g L-1) but a significantly lower level of amino acids, especially the essential ones. CONCLUSIONS: All approaches tested could convert raw feather waste into products of commercial value with proven use in a cultivation medium. The level of peptides, their molecular size and amino acid composition was dependent on the method used. (C) 2016 Society of Chemical Industry.