1.1. Microbial cultures

A laboratory strain of Ralstonia eutropha maintained on agar slant was used in this work. The agar medium contained (per liter): 5 g yeast extract, 5 g peptone, 2.5 g meat extract and 15 g agar. A 16S RNA analysis indicates that the strain has 100% alignment with Ralstonia eutropha H16. The cells were cultivated in 200 mL mineral medium containing: 160 mL glucose mineral solution, 8 mL inoculum, 5-20 mL solutions of residual microbial biomass

hydrolysates, and the rest pre-sterilized water. The glucose mineral solution contained (per liter): 12 g glucose, 2 g NaHzPCk, 2.8 g K2HPO4, 0.5 g MgSO4.7H2O, 1 g (NH4)zSO4 and 1 mL trace solution [21]. The culture solution was shaken in a 500 mL baffled flask at 30 oC and 200 rpm in a rotary incubator for 24 or 48 hours. Cell mass was harvested from 50 mL medium with centrifugation at 5,000 g for 10 min and freeze dried for measurement of cell mass concentration and PHB content.

A large amount of PHB-containing cell mass for biopolyester recovery was produced with a fed-batch culture in a 3L bench-top bioreactor (BioFlo 110, New Brunswick Scientific Co.

NJ). The temperature, pH and dissolved oxygen were controlled at 30 oC, 6.8, and 10% air situation, respectively. A feed solution prepared with a sugar manufacturing byproduct containing about 50% sucrose was introduced into the bioreactor till the cell density reached 130 g/L and PHB concentration 94 g/L (72% PHB of cell mass). The cell mass was harvested with centrifugation and re-suspended in an acidic water (0.2M H2SO4) to make a cell slurry of 278 g dry mass/L. The slurry was reserved for later use.