PHB positive strains were further evaluated by shake flask
screening in the nitrogen-deficient MSM medium containing only
0.89 g/L (NH4)2SO4 and 15 g/L glucose or xylose. The isolated
strains were cultivated at 30 C in 100-mL flasks containing
30 mL media on a rotary shaker at 150 rpm for 60 h. PHB was extracted
as described in Section 2.6 and PHB content was measured
as described in Section 2.7.
PHB positive strains were identified by 16S rDNA sequencing.
The genomic DNA was extracted according to the standard protocol
described by Moore et al. (2004). Full-length 16S rDNA sequences
were amplified by PCR using primers 27F (50
-AGA GTT
TGA TCM TGG CTC AG-30
) and 1492R (50
-TAC GGY TAC CTT GTT
ACG ACT T-30
). The PCR products were then separated by agarose
gel electrophoresis, purified using Qiagen PCR Purification Kit
according the manufacturer’s instruction and sequenced by ATI
biotech Pte Ltd., Singapore.