Filter paper no 1

P(3HB-co-4HB) was biosynthesized by Delftia acidovorans JCM 10181 via two-stage cultivation as previously described (Lee et al. 2004 (link)). The bacterial cell was first cultivated in 2× nutrient broth (NB) medium at pH 7.0 supplemented by 1 % w/v of glucose for 24 h. The cell was then harvested and transferred aseptically into nitrogen-free mineral medium (NM) at pH 7.0. NM consisted of 0.37 g/L of K₂HPO₄, 0.58 g/L of KH₂PO₄, 0.1 mM of MgSO₄·7H₂O and 0.1 mL/L trace elements (TE) solution. TE solution consisted of 2.78 g/L of FeSO₄·7H₂O, 1.98 g/L of MnCl₂·4H₂O, 2.81 g/L of CoSO₄·7H₂O, 1.67 g/L of CaCl₂·2H₂O, 0.17 g/L of CuCl₂·2H₂O and 0.29 g/L of ZnSO₄·7H₂O in 0.1 M of HCl. In order to produce copolymer consisting 4HB, 1 % v/v of 1,4-butanediol was added as sole carbon source. The cell was harvested after 48 h and subjected to lyophilisation using freeze-drier (Labconco FreeZone). Gas chromatography (GC) analysis was carried out to determine PHA content and the monomer compositions (Braunegg et al. 1978 (link)). The cell was then stirred in chloroform with concentration of 1 g/150 mL to release P(3HB-co-4HB) from the cell. The cell debris was filtered using Whatman No. 1 filter paper and the polymer in chloroform was concentrated using Eyela rotary evaporator. P(3HB-co-4HB) was further precipitated using methanol by drop-wise method.

The plant samples that were used in this study are listed in Table 2. The samples were purchased from the local Chinese Medical Hall and were first washed with sterile distilled water followed by rinsing with 70% (v/v) ethanol before drying in the oven at 50 °C for three days. The dried samples were ground into fine powder form and were extracted sequentially using hexane [H], chloroform [C] and methanol [M]. The extracts were subsequently filtered through Whatman No. 1 filter paper and the solvent was removed using rotary evaporator (EYELA, Tokyo, Japan). The crude extracts were resuspended with 100% dimethyl sulfoxide (DMSO) at a final concentration of 10 mg/mL and were diluted to a working concentration of 1 mg/mL using ultrapure water prior to be used.