The ability of Delftia acidovorans to incorporate a broad range of 3-hydroxyvalerate (3HV) monomers into polyhydroxyalkanoate (PHA) copolymers was evaluated in this study. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] containing 0-90 mol% of 3HV was obtained when a mixture of sodium 3-hydroxybutyrate and sodium valerate was used as the carbon sources. Transmission electron microscopy analysis revealed an interesting aspect of the P(3HB-co-3HV) granules containing high molar ratios of 3HV whereby, the copolymer granules were generally larger than those of poly(3-hydroxybutyrate) [P(3HB)] granules, despite having almost the same cellular PHA contents. The large number of P(3HB-co-3HV) granules occupying almost the entire cell volume did not correspond to a higher amount of polymer by weight. This indicated that the granules of P(3HB-co-3HV) contain polymer chains that are loosely packed and therefore have lower density than P(3HB) granules. It was also interesting to note that a decrease in the length of the side chain from 3HV to 4-hydroxybutyrate (4HB) corresponded to an increase in the density of the respective PHA granules. The presence of longer side chain monomers (3HV) in the PHA structure seem to exhibit steric effects that prevent the polymer chains in the granules from being closely packed. The results reported here have important implications on the maximum ability of bacterial cells to accumulate PHA containing monomers with longer side chain length.
Among the various biomaterials available for tissue engineering and therapeutic applications, microbial polyhydroxyalkanoates (PHAs) offer the most diverse range of thermal and mechanical properties. Of particular interest are the PHAs that contain 4-hydroxybutyrate such as poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB) and poly(4-hydroxybutyrate) [P(4HB)]. These polyesters can only be synthesized by a few types of bacteria, among which Comamonas acidovorans has the most efficient metabolic pathways to channel 4HB monomers. The resulting polyesters are bioabsorbable and are being developed as a new biomaterial for medical applications. By controlling the molar ratio of the monomers, it is possible to produce materials that are as tough and elastic as rubber.
Polyhydroxyalkanoates (PHA) are naturally occurring biopolyesters that have great potential in the medical field. However, the leachables resulting from sterilization process of the biomaterials may exert toxic effect including genetic damage. Here, we demonstrate that although gamma-irradiation of poly(3-hydroxybutyrate-co-50 mol % 4-hydroxybutyrate) [P(3HB-co-4HB)] did not cause any change in the morphology by scanning electron microscopy, there was a significant degradation of this copolymer where the molecular weight was reduced by 37% after sterilization indicating the generation of leachables. Therefore, further investigation on the ability of the extract of this poststerilized copolymer to induce mutagenic effect was performed using Ames test (S. typhimurium strains TA1535 and TA1537) and umu test (S. typhimurium strain TA1535/pSK1002). Additionally, the capability of the extract to induce clastogenic effect was determined using Chinese hamster lung V79 fibroblast cells. Our results showed that with and without the presence of S9 metabolic activation, no mutagenic effects were observed in both Ames and umu tests when treated with P(3HB-co-4HB) extract. Similarly, treatment of P(3HB-co-4HB) extract in V79 fibroblast cells showed no significant production of micronuclei when compared with the positive control (Mitomycin C). Together, these results indicate that leachables of poststerilized P(3HB-co-4HB) cause no mutagenic and clastogenic effects.