Characterization of polyhydroxyalkanoates (PHAs) biosynthesis by isolated Novosphingobium sp THA_AIK7 using crude glycerol

Biodiesel-contaminated wastewater was used to screen for PHAs-producing bacteria by using crude glycerol as the sole carbon source. A gram-negative THA_AIK7 isolate was chosen as a potential PHAs producer. The 16S rRNA phylogeny indicated that THA_AIK7 isolate is a member of Novosphingobium genus which is supported by a bootstrap percentage of 100% with Novosphingobium capsulatum. The 1,487 bp of 16S rRNA gene sequence of THA_AIK7 isolate has been deposited in the GenBank database under the accession number HM031593. Polymer content of 45% cell dry weight was achieved in 72 h with maximum product yield coefficient of 0.29 g PHAs g(-1) glycerol. Transmission electron micrograph results exhibited the PHAs granules accumulated inside the bacterial cell. PHAs polymer production in mineral salt media supplemented with 2% (w/v) of crude glycerol at initial pH 7 was extracted by the sodium hypochlorite method. Polymer film spectrographs from Nuclear magnetic resonance displayed a pattern of signal virtually identical to spectra of commercial PHB. Thermal analysis by Differential scanning calorimeter showed a melting temperature at 179A degrees C. Molecular weight analysis by Gel permeation chromatography showed two main peaks of 133,000 and 700 g mol(-1) with weight-average molecular weight value of 23,800 and number-average molecular weight value of 755. Endotoxin-free of PHAs polymer was preliminarily assessed by a negative result of the gel-clot formation, Pyrotell(A (R)) Single test vial, at sensitivity of 0.25 EU ml(-1). To our knowledge, this is the first reported test of endotoxin-free PHAs naturally produced from gram-negative bacteria which could be used for biomedical application.