This work aimed to isolate and characterize organisms able to store polyhydroxyalkanoates (PHAs), from a mixed microbial culture (MMC) selected under feast and famine conditions, using hardwood sulfite spent liquor (HSSL), a by-product of the pulp and paper industry, as the carbon source. The MMC was able to accumulate PHA from the different HSSL carbon components (acetic acid, xylose and lignosulfonates) with a PHA storage content during the reactor operation of 54.2 ± 10.6 % PHB. In order to clarify the MMC composition, isolation was performed from the MMC, using HSSL or its main components. Three isolates were identified: Rhodococcus spp., Pseudomonas spp. and Klebsiella spp. All isolates were able to grow and accumulate PHAs in harsh conditions of HSSL using xylose, the second most abundant sugar in nature. Rhodococcus and Pseudomonas isolates were able to accumulate a homopolymer PHB, whereas Klebsiella accumulated the copolymer P(HB-co-HV), with a composition of 96 % HB and 4 % HV. 16S rDNA clonal analysis was performed to identify the unculturable fraction of the MMC, and members of Novosphingobium spp., Sphingobium spp. and Pleomorphomonas spp., were found to be related to PHA production. The capability of the isolated bacteria to simultaneously consume xylose and accumulate PHAs offers a possibility for further studies and routes for the valorization of xylose-rich by-products. © 2015 Springer-Verlag Berlin Heidelberg and the University of Milan