PAO-biofilm taken at day 373 of Stage IV optimisation
"DNA samples were shipped to an external laboratory for 454 pyrosequencing (MR DNA, Molecular Research LP, Texas, USA). The method used for the 454 pyrosequencing was described by Dowd et al. (2008). In brief, HotStart Taq Plus Master Mix (Qiagen, CA, USA) was used for polymerase chain reaction (PCR) amplification of bacterial 16S rRNA genes using the bacterial primer 27F (5¢-AGRGTTTGATCMTGGCTCAG-3¢) and the universal primer 530R (5¢-CCGCNGCNGCTGGCAC-3¢). The thermocycler conditions used included an initial denaturing step at 94°C for 3 min; 28 cycles of denaturation at 94°C for 30 s; annealing at 53°C for 40 s; extension at 72°C for 1 min; and a final extension step at 72°C for 5 min. The amplified products from different samples were mixed in equal concentrations, and purified using Agencourt Ampure beads (Agencourt Bioscience Corporation, MA, USA). Samples were then sequenced using a Roche 454 FLX titanium instrument and reagents, following the manufacturer’s instructions. "