Degradation of polyesters by a novel marine Nocardiopsis aegyptia sp. nov.: Application of Plackett-Burman experimental design for the improvement of PHB depolymerase activity

Abstract

This is the first report on the degradation of poly(3-hydroxybutyrate) (PHB), and its copolymers poly(3-hydroxyvalerate) P(3HB-co-10–20% HV) by Nocardiopsis aegyptia, a new species isolated from marine seashore sediments. The strain excreted an extracellular PHB depolymerase and grew efficiently on PHB or its copolymers as the sole carbon sources. The degradation activity was detectable by the formation of a transparent clearing zone around the colony on an agar Petri plate after 25 days, or a clearing depth under the colony in test tubes within 3 weeks. The previous techniques proved that the bacterium was able to assimilate the monomeric components of the shorter alkyl groups of the polymers. Nocardiopsis aegyptia hydrolyzed copolymers 10–20% PHBV more rapidly than the homopolymer PHB. The bacterial degradation of the naturally occurring sheets of poly(3-hydroxybutyrate), and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was observed by scanning electron microscopy (SEM). The samples were degraded at the surface and proceeded to the inner part of the materials. Clear morphological alterations of the polymers were noticed, indicating the degradative capability of the bacterium. Plackett-Burman statistical experimental design has been employed to optimize culture conditions for maximal enzyme activity. The main factors that had significant positive effects on PHB depolymerase activity of Nocardiopsis aegyptia were sodium gluconate, volume of medium/flask and age of inoculum. On the other hand, MgSO₄·7H₂O, KH₂PO₄, K₂HPO₄ and NH₄NO₃ exhibited negative effects. Under optimized culture conditions, the highest activity (0.664 U/mg protein) was achieved in a medium predicted to be near optimum containing (in g/L): PHB, 0.5; C₆H₁₁O₇Na, 7.5; MgSO₄·7H₂O, 0.35; K₂HPO₄, 0.35; NH₄NO₃, 0.5; KH₂PO₄, 0.35; malt extract, 0.5 and prepared with 50% seawater. The medium was inoculated with 1% (v/v) spore suspension of 7 days old culture. Complete clarity of the medium was achieved after 3 days at 30°C.