The biomass pellet was separated from the substrate by centrifugation (6000g for 10 min at 10 C) and
the resulting pellet was washed with acetone and ethanol separately
to remove unwanted materials. The pellet was suspended
in 4% sodium hypochlorite and incubated at room temperature
for 3 h. The resulting mixture was centrifuged (6000g for
10 min at 10 C) and the supernatant was discarded. The pellet
with lysed cells after washing simultaneously with acetone and
ethanol was dissolved in hot chloroform and was passed through
filter paper (0.45 lm pore size) to separate the polymer from cell
debris. PHB was concentrated using Rotary evaporator (Eyela
N-1000) followed by drying to a constant mass. The mass of PHB
was calculated gravimetrically. Final PHB production was quantified
as cell contents (wt.%) and as a concentration (g/l).
Chloroform filtrate obtained was precipitated with cold ethanol
and used for analysis of functional groups, structure, and thermal
properties. Spectrometric analysis was conducted according to
the method described by Law and Slepecky (1960). The extracted
PHB was dissolved in 10 ml of sulfuric acid (36 N), and the resultant
solution was heated at 100 C on a water bath for 10 min.
Addition of sulfuric acid converts the PHB to crotonic acid. The
solution was cooled and the absorbance was measured at
235 nm for determining the PHB concentration against a sulfuric
acid blank. Standard curve was prepared using pure PHB