Bacterial Community Structures are Unique and Resilient in Full-Scale Bioenergy Systems
â??Granular biomass samples were stored at -80 degrees C until processing. For DNA extraction, samples were ground under liquid N2, and a ~100 mg aliquot was suspended in 500 ÂµL solution of 200 mM Tris, 200 mM NaCl, 20 mM EDTA, pH 8. This was combined with 210 microliters 20 percent SDS, 500 ÂµL phenol:chloroform:isoamyl alcohol 25:24:1, and a 500 microliters slurry of 0.1 mm zirconia beads (BioSpec Products, Bartlesville, Oklahoma, USA). The mixture was lysed via bead beating (BioSpec bead beater, 2 min, 22 C) followed by phenol:chloroform:isoamyl alcohol extraction and precipitation with isopropanol. Extracted DNA was quantified using the Quant-iT PicoGreen dsDNA Assay Kit. Bacterial 16S rRNA genes were amplified using primers that were composites of 454 Life Sciences primers B (forward) and A (reverse) with conserved bacterial primers 27F and 338R. Three independent polymerase chain reaction (PCR) amplifications were performed per sample, using HotMaster PCR mix (Eppendorf), 200 ÂµM primer, 10-100 ng template in an Eppendorf thermocycler at 95 C (2 min) followed by 30 cycles of: 95 C (20 s), 52 C (20 s), 65 C (60 s). Replicates were combined, purified using Ampure magnetic purification beads (Agencort), confirmed by gel electrophoresis, and quantified using the Quant-iT PicoGreen dsDNA kit. The purified, barcoded amplicons were pooled at equimolar ratios to a final concentration of 20 ng DNA mL-1. The single pooled amplicon mixture was sequenced at the Cornell University Life Sciences Core Laboratory Center using a Roche 454 FLX pyrosequencer.â?