Effects of radio frequency (RF) heating as a biomass pretreatment process to generate hydrolysates for polyhydroxybuyrate (PHB) were evaluated in all production steps from pretreatment to enzymatic hydrolysis to fermentation. Switchgrass was pretreated under alkaline conditions with RF-assisted heating (traditional water bath (WB) heating as control) and subsequently enzymatically hydrolyzed. Fermentation was conducted with recombinant Escherichia coli strain for PHB production using the hydrolysates as carbon sources with or without yeast extract (YE) supplemented. Results indicated that the hydrolysates generated through RF pretreatment performed consistently better for PHB production than WB (50% higher PHB levels without YE). YE supplementation (up to 5 g/L) enhanced fermentation under all conditions and diminished the difference among performances of fermentations. When adding 2 g/L YE, PHB production increased by 200% and 80% for WB and RF hydrolysates, respectively. Supplementation of 5 g/L YE brought the final PHB concentration to be very close to each other for all three fermentation conditions. Compared to traditional heating process, the unique heating mechanism of RF generates harsher conditions (under regular pressure) to disrupt the biomass structure more completely and generate more nutrients for bacterial fermentation. RF was therefore proved to be an efficient process for biomass pretreatment. (C) 2016 Elsevier Ltd. All rights reserved.