Bacterial Strain

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Materials and Methods Bacterial Strain Wild type Cupriavidus necator H16 (ATCC 17699) [formerly known as Wautersia eutropha] was used throughout this study. The bacterium was maintained on Nutrient-Rich(NR) agar containing 2 g/L yeast extract, 10 g/L meat extract, 10 g/L peptone and 14 g/3 bacteriological agar powder (Doi et al., 1995). PHA Biosynthesis in 100 L Fermenter PHA biosynthesis was carried out with one-stage fed-batch cultivation in 100 L fermenter. The temperature for this whole experiment was maintained at 30 °C. Starter culture was prepared by inoculating two loopfuls of bacteria cells into 50 mL NR broth and cultivated at 200 rpm at 30 °C. Approximately 1.5 mL (3 % v/v) of the starter culture was inoculated into 7 flasks containing 100 mL NR medium each after 7 h incubation (OD600nm = 5.0). The seed culture of 700 mL was transferred into 6.3 L of fresh NR in a 10 L fermenter ( Labfors, Infors, Switzerland) upon the growth of bacteria cells entered the mid-exponential phase. To initiate PHA biosynthesis, 7 L of the preculture was inoculated into 63 L of MM (Doi et al., 1995) in a 100 L fermenter (Biostat, Germany) after approximately 12 h of cultivation. The agitation rate was increased from 200 to 500 rpm to maintain the level of dissolved O2 at 40 % saturation and the pH was adjusted to 7.0 by using 3 M NaOH. Crude palm kernel oil (CPKO) supplied by Acidchem Int. Ltd. was fed as sole carbon source for P(3HB) production at a maximum concentration of 46 g/L. After 48 h of cultivation, cells were harvested by centrifugation (9370 × g for 5 min) at 4° C. The pellets were then frozen at – 20 °C for 24 h before freeze-drying at 40 oC for three days. Biological Recovery of PHA Test Animal... ... middle of paper ... ...e determined from the DSC endotherm. Thermogravimetric Analysis (TGA) The thermal analysis was carried out using Mettler Toledo Star System. Sample was heated from 50 to 900 °C under a nitrogen atmosphere. Nuclear Magnetic Resonance (NMR) The chemical structure of recovered PHA was determined by NMR spectroscopy at 400 MHz 1H and 300 MHz 13C scans. Approximately 15 mg sample was dissolved in 1 mL of deuterated chloroform (CDCl3). C. Tetramethylsilane (Me4Si) was used as an internal chemical shift reference. Scanning Electron Microscope (SEM) The lyophilized cells and powdered faeces samples were subjected to SEM observation. The samples were mounted on aluminium stubs, sputter-coated with gold for 15 seconds prior to observation under Leo Supra 50 VP Field Emission SEM at an acceleration voltage of 10 kV and magnification of 1000 – 50,000 times.
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