BACKGROUND: Forest biomass represents a potential low-cost feedstock for polyhydroxyalkanoate (PHA) production. Softwoods, such as Pinus radiata (D. Don) are difficult to digest and require special pretreatments to expose polysaccharides for enzymatic saccharification. Different pretreatments can lead to inhibitor formation that may adversely affect PHA synthesis. RESULTS: Pinus radiata wood chips were subjected to high-temperature mechanical pre-treatment (HTMP) or steam explosion in the presence of sulphur dioxide (SEW) before being enzymatically treated to produce corresponding hydrolysates. Two PHA-producing bacteria Novosphingobium nitrogenifigens Y88(T) and Sphingobium scionense WP01(T) were grown on these hydrolysates. The highest content of PHA was produced by WP01 grown on HTMP hydrolysate (32% w/w PHA, YPHA/glucose = 0.22 and theoretical maximum yield of 46%). Gas chromatography and proton nuclear magnetic resonance spectroscopy indicated that the PHA produced was made up of polyhydroxybutyrate (PHB) with molecular weights ranging from 50-1600 kDa and polydispersities between 4.6 and 8.4. The melting temperature was close to that of pure PHB with percentage crystallinity ranging from 44-64%. CONCLUSION: This is the first report on the production of PHA from softwood hydrolysates derived from enzymatically saccharified pretreated Pinus radiata pulps. (C) 2013 Society of Chemical Industry