The metabolism and physiology of Pseudomonas putida strain LS46 was investigated using biodiesel-derived waste streams as potential low cost substrates for production of medium chain length polyhydroxyalkanoates (mcl-PHA). Proteomic and trranscriptomic analyses were used to correlate specific gene and gene product expression patterns with differences in phenotypes of mcl-PHA biosynthesis by P. putida LS46. Growth and mcl-PHA content of P. putida LS46 were similar in cultures containing biodiesel-derived waste glycerol versus pure glycerol, and mcl-PHA synthesis occurred during stationary phase after nitrogen concentrations in the medium were exhausted. Waste glycerol cultures contained elevated concentrations of heavy metal ions, such as copper, which induced significant changes in gene expression levels related to heavy metal resistance. Several membrane-bound proteins, such as CusABC efflux and CopAB were identified and putatively play a role in regulating cellular copper concentrations. Cultures containing waste free fatty acids synthesized mcl-PHA throughout the exponential growth phase. Protein expression levels of two mcl-PHA synthases were suppressed during exponential phase growth in waste glycerol cultures, putatively via post-transcriptional regulation. Culture specific expression of monomer supplying proteins (PhaJ1 and PhaG), and sets of fatty acid oxidation enzymes were observed, and may have contributed to differences in the composition of polymers synthesized by P. putida LS46 cultured on the two substrates. Expression levels of the majority of mcl-PHA biosynthesis pathway genes were stable during active polymer synthesis in waste glycerol cultures. However, variations in protein expression levels, and in some cases their corresponding mRNAs, were observed in a number of other metabolic patheays, such as glycerol transportation, partial glycolysis, pyruvate metabolism, the TCA cycle, and fatty acid biosynthesis. These data suggest potential regulatory points that may determine carbon flux during mcl-PHA biosynthesis. Evaluation of identified genetic targets in P. putida LS46 that putatively influence mcl-PHA biosynthesis and monomer composition merit further studies.