title: Whole genome sequencing of a nitrifying consortium
design: The sludge used for inoculating the SBR was obtained from a 3 L continuous reactor which was kept operating for over five years under steady-state nitrification. The initial microbial total protein concentration was 102 mg L-1. In the absence of binary mixture of phenolic compounds (phenol and m-cresol), the medium used for the SBR culture was a mixture of 2 media (A and B). The chemical composition of the medium A was (in g L-1): (NH4)2SO4 (0.72), NH4Cl (0.58), KH2PO4 (0.86), MgSO4 (0.36) and NaCl (0.62). Medium B was (in g L-1): CaCl2.2H2O (0.22) and NaHCO3 (3.22). Medium A and B were pumped at 33 mL min-1. FeSO4.7H2O was daily added into the reactor at 0.01 g L-1. The initial NH4+-N concentration in the SBR was 100 ± 2 mg L-1.  DNA purification was performed by the methods developed by Rojas-Herrera et al (2008). Brevely, 1.5 ml of the consortium was poured into a 1.7 ml microcentrifuge tube with the addition of 1 ml of TEN buffer (100 mM Tris– HCI, 50 mM EDTA, 500 mM NaCl, pH 8.0) and vortexed for 1 min. After centrifugation for 10 min at 10,000 xg at RT, the pellet was resuspended in 1 ml of TEN buffer with 0.2 mg of added lysozyme and incubated for 1 h at 37°C under agitation. Then three cycles of freezing/defrosting were done by incubation 10 min in an ice/alcohol bath and then 5 min at 65°C. 100 l of 20% SDS was added and vortexed for 1 min and incubated for 30 min at RT. After centrifugation for 10 min at 10,000 xg at RT, the supernatant was transferred into a new microcentrifuge tube and 500 l of 5M potassium acetate was added and then incubated at 65°C for 5 min. Tubes were placed in an ice bath for 20 min and centrifuged at 20,000 xg for 30 min at 4°C. The supernatant was transferred into a new microcentrifuge tube and 200 l of a 4% silica suspension was added and incubated at RT for 2 min with gentle agitation. Silica-DNA complex was recovered by centrifugation at 16,000 xg for 2 min at RT and the pellet was washed by adding 1 ml of 70% ethanol. DNA was eluted with 50 l of sterile distilled water and incubated at 55°C for 5 min and recovered by centrifugation at 16,000 xg for 5 min at RT (Rojas-Herrera et al, 2008). DNA samples were quantified with a NanoDropND-1000 spectrophotometer (NanoDrop Technologies,Wilmington, DE, USA).  Pyrosequencing of DNA samples was performed by the Center for Integrated BioSystems at Utah State University (http://www.biosystems.usu.edu) (USA) using a 454 GS FLX Titanium system (Roche, Mannheim, Germany). Unassembled reads were annotated by Metagenome Rapid Annotation using Subsystem Technology (MG-RAST: http://metagenomics.nmpdr.org/), an open-access metagenome curation and analysis platform (Meyer et al, 2008). To analyze the taxonomical structure of the consortium, 16S rRNA gene sequences from all sequences were identified by comparing the reads dataset against the RDP II database (Cole et al, 2009), using recommended parameters (minimum alignment length, 50 bp; E-value cutoffs, 0.01). 16S rRNA gene phylotypes were classified at the family, genus, and species levels based on the respective taxonomic categorizations of the MG-RAST server.  Consortium microbial community metabolic potential to degrade aromatic compounds was determined by carrying functional annotation to metagenomic sequences with subsequent sequence assignment to SEED subsystems.