Fastprep 24 apparatus

300 mg of pelleted biomass for each centrifuged sample was used for DNA extraction using the Fast DNA SPIN Kit for Soil and the Fast-Prep24 apparatus (MP Biomedicals, Solon, OH, USA) following the instructions given by the manufacturer. DNA extracts from samples collected in the same bioreactor were merged into a pool. The DNA pool of each bioreactor was divided into 6 subsamples with equal volume for further pyrosequencing analysis.
The primer pair 530F-1100R [21 (link)] was used to amplify 500 bp of the 16S rRNA gene of Bacteria, encompassing the V4-V5-V6 hypervariable regions. Research and Testing Laboratory (Lubbock, Texas, USA) proceeded with pyrosequencing following the procedure described by Dowd et al., 2008 [21 (link)], using the Roche 454 GS-FLX+ apparatus. Amplification of the six subsamples within the same bioreactor DNA pool was assayed under the same PCR conditions but at different annealing temperatures (44 to 49°C), yielding a total of 30 different pyrosequencing datasets in the five different technologies. In this sense the PCR conditions for pyrosequencing were the following: preheating step at 94°C for 3 minutes; 32 cycles at 94°C for 30 seconds, 44–49°C for 40 seconds, and 72°C for 1 minute; elongation at 72°C for 5 minutes.

RNA isolation was performed by the phenol as follows extraction based on a previously described protocol^{56 (link)}: overnight 10 mL cultures were centrifuged at 4 °C and 4816×g for 15 min and immediately used for RNA isolation. After removal of the medium, cells were suspended in 0.5 mL of ice-cold TE buffer (pH 8.0) and kept on ice. All samples were divided into two 2 mL screw-capped tubes containing 0.5 g of zirconium beads, 30 μL of 10% SDS, 30 μL of 3 M sodium acetate (pH 5.2), and 500 μL of Roti-Phenol (pH 4.5−5.0, Carl Roth GmbH). Cells were disrupted using a FastPrep-24 apparatus (MP Biomedicals) at 5500 r.p.m. for 45 s and centrifuged at 4 °C and 9400×g for 5 min. 400 μL of the water phase from each tube was transferred to a new tube, to which 400 μL of chloroform−isoamyl alcohol (Carl Roth GmbH) was added, after which samples were centrifuged at 4 °C and 18,400×g for 3 min. 300 μL of the aqueous phase was transferred to a new tube and mixed with 300 μL of the lysis buffer from the high pure RNA isolation kit (Roche). Subsequently, the rest of the procedure from this kit was performed according to the manufacturer’s protocol, except for the DNase incubation step, which was performed for 45 min. The concentration and integrity of cDNA was determined using Nanodrop-1000 Integrity and concentration of the isolated RNA was checked on a NanoDrop 1000.