Biosynthesis and in vivo depolymerization of intracellular medium-chain-length poly-3-hydroxyalkanoates (mcl-PHA) in Pseudomonas putida Bet001 grown on lauric acid were studied. Highest mcl-PHA fraction (>50 % of total biomass) and cell concentration (8 g L-1 ) were obtained at carbon-to-nitrogen (C/N) ratio 20, starting cell concentration 1 g L-1 , and 48 H fermentation. The mcl-PHA comprised of 3-hydroxyhexanoate (C6 ), 3-hydroxyoctanote (C8 ), 3-hydroxydecanoate (C10 ), and 3-hydroxydodecanoate (C12 ) monomers. In vivo action was studied in a mineral liquid medium without carbon source, and in different buffer solutions with varied pH, molarity, ionic strength, and temperature. The monomer liberation rate reflected the mol percentage distribution of the initial polymer subunit composition. Rate and percentage of in vivo depolymerization were highest in 0.2 M Tris-HCl buffer (pH 9, strength = 0.2 M, 30 °C) at 0.21 g L-1 H-1 and 98.6 ± 1.3 wt%, respectively. There is a congruity vis-à-vis to specific buffer type, molarity, pH, ionic strength, and temperature values for superior in vivo depolymerization activities. Direct products from in vivo depolymerization matched the individual monomeric composition of native mcl-PHA. It points to exo-type reaction for the in vivo process, and potential biological route to chiral molecules.
The biosynthesis of medium-chain-length poly-3-hydroxyalkanoates by Pseudomonas putida Bet001 cultivated on mixed carbon sources was investigated. The mixed carbon sources consisted of heptanoic acid (HA) and oleic acid (OA). A relatively low PHA content at 1.2% (w/w) and 11.4% (w/w) was obtained when HA or OA was used as the sole carbon source. When these fatty acids were supplied as a mixture, PHA content increased threefold. Interestingly, the mixture-derived PHA composed of both odd and even monomer units, namely. 3-hydroxyheptanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydroxydodecanoate and no unsaturated monomer was detected. It is hypothesized that the even-numbered monomers were derived primarily from OA, whereas the odd-numbered monomer was derived from HA. This also points out to an efficient and yet distinct fatty acids metabolism that fed the PHA biosynthesis machinery of this particular microorganism. PHA obtained was elastomeric because melting temperature (Tm ) and crystallinity were absent. It showed good thermal stability with degradation temperature (Td ) ranging from 275.96 to 283.05 °C.