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div>After collection, all samples were refrigerated and transported to the laboratory for immediate analysis. The physicochemical parameters pH, BOD5 (biochemical oxygen demand over 5?days), chemical oxygen demand (COD) and nitrogen as nitrate (N-NO3) and as ammonium (N-NH3) were determined. The pH was determined by pH meter (Digimed DM20; Digimed Instrumenta??o Anal��tica, Brazil). The COD test was performed by the closed reflux method followed by photometric determination, using a COD reactor (Hach Company, Loveland, CO, USA) and visible spectrophotometer (model DR-2500; Hach Company). BOD5, nitrate and ammonium were determined using the potentiometric method with selective electrodes Orion 081010MD, Orion 9707BNWP and Orion 9512HPBNWP, respectively (Hach Company). Total and faecal coliforms were analysed using the Colilert P/A Quanti tray mTOR inhibitor 2000 kit (IDEXX Laboratories, Westbrook, ME, USA) according to the manufacturer��s instructions; results are expressed as most probable number (MPN) in 100?ml?1. The methodologies used to assess the physicochemical parameters and total and faecal coliforms were consistent with the methods described in the Standard Methods for Examination RAD001 of Water and Wastewater (APHA 1998). Three samples (1000?ml) were collected on the same day from influent (raw sewage), a clarifier tank and a chlorine contact tank. Effluent samples were filtered onto a 47-mm-diameter, 0��22-��m nitrocellulose filter (Millipore, MA, USA) and stored at ?80��C PD98059 for later DNA extraction. DNA was extracted using the UltraClean Soil DNA kit (Mo Bio Laboratories, Carlsbad, CA, USA) according to the manufacturer��s protocol. The metagenomic 16S rDNA was amplified by PCR using the primer pair PRBA63f (Marchesi et?al. 1998) and UN518r (?vre?s et?al. 1997) for the domain bacteria. Agarose gel electrophoresis of 150?��l of PCR product was performed prior to purification with the QIAquick gel extraction kit (Qiagen, Hilden, DE) according to the manufacturer��s instructions. Purified amplicons were cloned using the pGEM-T Easy Vector (Promega, WI, USA) according to the manufacturer��s protocol and then transformed into competent DH5-alpha Escherichia coli cells. Ampicillin- and X-Gal (5-bromo-4-chloro-3-indolyl-��-d-galactopyranoside)-amended LB agar was used for the blue-white screening of transformants, which were subject to direct whole-cell PCR to amplify the plasmid insert. Each insert was sequenced using the BigDye terminator system and an ABI-3730 automatic capillary sequencer (Applied Biosystems, CA, USA). The electropherogram sequencing files were processed using the Phred program (Ewing and Green 1988) for base calling and for trimming of vector and low-quality (<20) sequences. The high-quality sequences located between the rRNA primers were used for further analysis. The sequences were chimera-checked using the Mallard program (Ashelford et?al. 2006), and putative chimeras were excluded from further analysis.