title: To investigate the denitrifiers living in cassava vinasse
design: Samples were independently homogenized. Genomic DNA was extracted using E.Z.N.A.Soil DNA Kit (OMEGA, USA), according to the manufacturer’s instructions.  The extracted DNA was eluted with 1% agarose gel electrophoresis.  (2) PCR amplification.  For each sample, fungal ITS amplification was performed using the ITS1(5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’- TCCTCCGCTTATTGA-TATGC-3’). PCR reaction mixture (25ml) consisited of 1 unit Taq polymerase, 0.2 mM dNTP, 2.5 ml 10×PCR buffer, 0.2 mM each primer, and 10ng DNA template.  All dilutions were carried out using certified DNA -free PCR water.  The amplification program was as follows: thermal cycling consisted of initial denaturation at 95°C for 2 min followed by 25 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72 °C for 30 s, with a final extension of 5 min at 72°C.  All samples were operated in accordance with formal conditions, triplicate of each sample.  The PCR products from the same sample were mixed and detected by electrophoresis on a 2% agarose gel.  Amplicons were purified using AxyPrep™ DNA Gel Extraction Kit (AXYGEN, USA) according to the manufacturer’s instructions.  (3) Amplicon, quantitation, pooling, and pyrosequencing.  According to electrophoresis analysis, the amplicon DNA concentrations were measured using the QuantiFluorTM-ST Blue fluorescence quantitative systems (Promega co. USA).  Following quantitation, cleaned amplicons were combined in equimolar ratios into a single tube. Pyrosequencing was carried out on a 454 Life Sciences Genome Sequencer FLX Titanium instrument (Roche).