Direct observation of polyhydroxyalkanoate chains by atomic force microscopy

Atomic force microscopy in the tapping mode was used to investigate aqueous acetone-treated polyhydroxyalkanoate (PHA) inclusions freshly isolated from a recombinant bacterium. The PHA is a copolymer containing about 95 mol% 3-hydroxybutyrate units while the rests are units of 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydroxydodecanoate. Polymer chains extending to several micrometers in length were observed on glass cover slips upon the evaporation of the aqueous acetone. The polymer chains seem to exist in the form of fibrillar aggregates. The height of the microfibrils was about 1 nm. Upon prolonged standing at ambient conditions, the microfibrils dissociated into finer strands of about 0.5 nm in height. The results suggest that biosynthesized PHA are stored in the inclusions in an amorphous state but with minimal chain entanglement. This is possible because the PHA chains exist in the form of fibrillar aggregates that may be the product of a special biosynthesis mechanism.