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.
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.
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