Characterization and Molecular Study on Polyhydroxybutyrate Produced from Rhodococcus equi

Many soil samples were collected from different places and screened for PHA production by Sudan Black B stain. Twenty isolates had a positive result for Suddan Black B stain. The results were confirmed by staining the viable colonies with the selective stain Nile Red; three isolates gave positive results. One isolate with a more pink fluorescent with Nile Red stain was selected for the present study. This isolate was characterized and identified by biochemical tests as Rhodococcus then the identification was confirmed by 16S rRNA and by blasting the sequence of 16S rRNA to NCBI database, the blast results showed 99% identity to Rhodococcus equi.A one-stage cultivation method was utilized for studying PHA biosynthesis from R. equi using various carbon sources. The composition and the content of biopolymer were quantified by gas chromatography (GC). Besides, Crud Palm Kernel Oil (CPKO 1%) was the effective carbon for PHB production. Cell dry weight was 1.43 g/L and PHB content =38.07 wt%. Nuclear Magnetic Resonance (NMR) analysis confirmed the chemical structure of extracted biopolymer as PHB homopolymer. Moreover the thermal properties of PHB were characterized to include the melting temperature (T) and the glass transition temperature (T ) that reached to 173 C and 2.79 C respectively as determined by differential scanning calorimetry analysis (DSC), while the  ecomposition temperature (Tg) was 276 C as determined by thermogravimetric analysis (TGA). The average molecular weight (d M ) was 642 KD, weight number average mass (M/wM) was 1.72 as determined by the gel permeation chromatography (GPC). nM) was 373 KD and polydispersity (n   The phase contrast light microscope was used to viewing R. equi during PHB production which was characterized by a bright appearance that w m  was increased with time, while fluorescent microscope using Nile Blue A stain illustrated the bacterial cells under best production conditions with the bright orange fluorescent that indicated PHB accumulation inside cells. Transmission Electron Microscope  (TEM) images revealed the presence of PHB granules inside the bacterial cells with white, spherical, ovoid and elongated shape. These granules had different sizes and different numbers about (3-11) granules per cell.The PhaC gene encoded the synthase enzyme which was considered as the key enzyme for PHA biosynthesis, G-D and G-1R primers were used for the amplification of PhaC gene by PCR with a size of 508 bp. The sequence of this gene blast to NCBI database and the results demonstrated 78% similarity  to PhaC gene sequence of Rhodococcus aetherivorans (accession no.CP011341.1). On the other hand, real time PCR technique was used for the evaluation of PhaC gene expression level considering gyrB as a reference gene depending on log phase as a control. It was concluded that the expression level of PhaC gene at lag phase was ~ 1.16 folds, while at the stationary phase was ~2 folds. The PHA synthase enzyme for R. equi was also characterized with optimized production conditions and evaluated with activity 30.83 (U/mg protein). Different formulations of PHB films were prepared to study in vivo degradation in soil for 6 weeks which included: PHB films, PHB-TiO composite films, PHB nanofiber films, PHB-TiO  composite nanofiber films and P(3HB) films, PHB-TiO 2 II2  composite films prepared from ultraviolet light (UV) treatment of  PHB sheets. The results of degradation experiments revealed the significant decrease in molecular weight (Mw), number-average molecular  weight (Mn) and the polydispersity (Mw/Mn) for all films. The weight loss percentage was carried out as a function of degradation which increased with time, but nanofibers of PHB and its composites showed a faster degradation when compared to other films and completely degraded after 3 weeks.The microbial populations in soil at the burying site were increased continuously at each week.  Crack was the major physical changes of all films at each week of degradation experiment. SEM micrographs showed the surface morphology of different PHB film forms before and after biodegradation experiment, which took after 6 weeks. SEM micrographs illustrated various surface changes that included pores, cavities, grooves, incisions, slots and pointers for the growth of microorganisms that secrete PHA depolymerase enzyme which on the whole caused the degradation of all biopolymer films.Nanofibers of PHB and its composites in presence of TiO  were demonstrating more surface changes with the rupture of most of the nanofibers and ultimately
droping the diameter from 500 nm to 400 nm for PHB nanofiber and from 550 nm to 450 nm for PHB-TiO 2 composite nanofiber films.  UV treatment for the PHB sheets and used it in preparing the PHB films and their composite with TiO  caused the increasing of biodegradation in compare to the similar one in the absence of UV treatment.