As shown in Figure 1, about 94 wt% of residual microbial biomass was decomposed and hydrolyzed in the acid pretreatment (~44% biomass) and base treatment (~50% biomass). The remaining small amount (~6 wt %) of residual cell mass is most likely mineralized via

oxidation with hypochlorite, a strong oxidation agent. The acid hydrolysates are primarily the cytoplasm proteins released from the damaged cells (Figures 2 and 3). The released biological macromolecules were subjected to further hydrolysis in the thermal acidic solution. The acid hydrolysates solution had a clean brownish color and contained 30-45 g/ of soluble biomass, depending on the density of cell slurry and treatment conditions. The base hydrolysates solution with a dark color contained the hydrolysis products of hydrophobic cell components including lipids and membrane proteins. After centrifugation, the concentration of soluble biomass in the supernatant solution was 30 to 50 g/L. The sequential treatments disrupted and dissolved the structural components so that they could be removed from PHB granules. Equally important, the biomass and biological macromolecules were decomposed into small molecule hydrolysates such as amino acids and organic acids. These hydrolysates could become appropriate substrates that can be assimilated by microbial cells in microbial PHB production.

In addition to the two types of biomass hydrolysates described above, a mixed hydrolysates of residual biomass was generated when the acid pretreatment and base treatment were performed sequentially without solid/liquid separation. It eliminated one operation of solid/liquid separation, but the acid hydrolysates (primarily proteins) were subjected to additional hydrolysis in the base solution. Figure 6 shows the FTIR spectrum of an acid-base hydrolysates. As observed in the IR spectrum of the original cell mass in Figure 4, a major component of the hydrolysates was the amino acids or proteins with infrared radiation absorbance at 1500 to 1700 cm-1 [25]. Another major component in the acid/base hydrolysates had the IR absorbance at 1000 to 1200 cm-1, which was attributed to cell lipids and/or similar compounds. This was confirmed with the spectrum of cell mass extract in acetone. Acetone is a common solvent used to remove hydrophobic lipids, steroids and pigments from PHB-containing cell mass [17]. It does not dissolve and extract PHB and proteins. Based on the observations above, it was concluded that the major cellular components in the acid hydrolysates were derived from cytoplasm proteins and in the base hydrolysates from cell walls, lipids and membrane proteins. In the acid-base hydrolysates, the products were derived from both groups, i. e. amino acids or peptides derived from proteins, and lipids derived from cell walls and membranes. It should be pointed out that the composition of acid-base hydrolysates is not a simple mixture of acid — and base — hydrolysates because the acid hydrolysates were further hydrolyzed in base treatment.

Because of the hydrophobic properties of PHB granules, the residual hydrophobic impurities of cell mass might be attached to the granules and difficult to remove by washing with water. A surfactant such as SDS in the base treatment can remove most of the impurities to a high PHB purity (>99 % w/w). A large portion of the surfactant, however, may be left in the hydrolysates solution and may have an adverse effect on the reuse of the hydrolysates in PHB production.