Poly-B-hydroxybutyrate (PHB): A Biodegradable Polymer of Microbial Origin - Natural Polymers, Biopolymers, Biomaterials, and Their Composites, Blends, and IPNs - page 55

isolatioN aNd PuriFiCatioN oF PhB
solvent extraction
The PHB is soluble in organic solvent; this property leads to the effective and higher
PHB extraction from cells. Several solvents such as ch1oroform, dichloroethane, tri-
chloroethane, ethylene, hexane propanol, and acetone: alcohol have been in use for
isolation and purification of PHB to solubilize the PHB. The treatment of methanol
or acetone to biomass, before the solvent extraction leads to denature low molecular
weight proteins which enhance the purity of PHB.
Sodium hypochlorite was first used by Williamson and Wilkinson (1958) to iso-
late PHB granules from Bacillus cereus. However, sodium hypochlorite digestion of
NPCM resulted in the lysis of cells without affecting the PHB. Sodium hypochlorite
purification method was affecting the sudanophilic properties and molecular weight of
the polymer granules.
The dispersion of chloroform and sodium hypochlorite is also used for the ex-
traction of PHB. By this method, the degradation of PHB by sodium hypochlorite
is slightly reduced but highly inconvenient for analytical purposes, which leads to
improper data analysis. Extraction of PHB with a mixture of hexane and propanol
resulted in poor recovery yield.
The solvent system consisting of 1:1 mixture of ethanol and acetone is known to be
specific for efficient recovery method capable of specifically lysing the NPCM without
affecting PHB. Rawte and Mavikurve (2002) have also reported the usefulness of ac-
etone and ethanol in the extraction of PHA. However, author has reported that ethanol
and acetone (1:1 v/v) is the best recovery method and gives high recovery yields. Table 5
gives the comparative account of recovery yield of PHB with various methods.

table 5. Recovery of PHB with Different Recovery Methods.

Recovery Methods of PHB PHB Recovery Yield (gL -1 ) Reference

Acetone: Alcohol 5.6 Sayyed et al., 2009

Hypochlorite Digestion 1.34 Reusch et al., 1987

Chloroform extraction method 0.63 Choi and Lee, 1997

Dispersion method 1.1 Lee, 1996

An inconvenience for solvent extraction methods is that large volume of solvents
and non-solvents are needed to extract and to precipitate the polymer. However, PHB
recovery by solvent extraction resulted in a pure white very crystalline powder of high
molecular weight.

enzymatic extraction
In 1964, Merrick and Doudoroff isolated PHB granules from B. cereus using lyso-
zymes and deoxyribonuclease to solubilize the peptidoglycans and the nucleic acids
respectively. The enzymatic digestion of cell components usually released the nucleic
acids in the suspension medium which made suspension highly viscous and impossible