The dissolution of non-PHA cell mass was monitored by measuring the characteristic absorption of amino acid residues at 280 nm with a UV/VIS spectrophotometer (Beckman Coulter DU530, Fullerton, CA). The concentration of proteins that can be stained in Bradford assay was measured with the spectrophotometer after protein-dye binding [23]. The content of PHB in original cell mass, in sequential treatments, and final product were determined via methanolysis of the biopolyester in methanol (3 wt% sulfuric acid) at 100 oC for 8-10 hours [24]. The 3-hydroxybutyric methyl ester was hydrolyzed into 3-hydroxybutyric acid when the solution pH was raised to 11 with a 10N NaOH solution. The liquid samples were analyzed using an HPLC equipped with a UV detector (Shimadzu, Japan) and an organic acid column (OA-1000, Alltech, Deerfield, IL). The column was maintained at 65 oC and eluted with a water-sulfuric acid solution (pH 2) at 0.8 mL/min. The monomeric acid and crotonic acid, a trace byproduct formed in methanolysis, were detected at 210 nm. For data quality control, the biopolyester was also extracted from the freeze-dried cell mass in hot chloroform followed by precipitation with methanol [21]. The PHB content was calculated from the purified PHB and compared with the results of HPLC analysis.

The purified PHB and non-PHB cell mass were examined with a Nicolet Avatar 370 FTIR spectrometer (Thermo Electron Co., Madison, WI). The solids were pressed on a germanium crystal window of micro-horizontal attenuated total reflectance (ATR) for measurement of single-reflection and absorption of infrared radiation by the specimens. The thermal properties of PHB powder were examined with a differential scanning calorimeter (DSC). A Modulated 2920 instrument (TA Instruments, New Castle, DE) equipped with a refrigerated cooling system was run in heat-cool-heat mode at a rate of 5 oC/min under nitrogen. The selected temperature range was 30oC — 210 oC with sample weights of 4.5 — 5.5 mgs. Images of cell and PHB granules were obtained with an energy­filtering transmission electron microscopy (120 kV LEO 912, Carl Zeiss SMT Inc. MA). The instrument has an in-column electron energy loss spectrometer, allowing analysis of light element in thin sections.