0 ± 0.3 at the beginning of the experiment and received either an addition of 10 mg NO3–N or an equal volume of distilled water as a control on D30. There were six replicate microcosms for each treatment

(NO3- addition and control). The NO3- addition and distilled water treatments were used because denitrification rate differed in these microcosms (an average of 3.84 ± 0.44 mg N (kg soil)-1 day-1 when NO3- ACY-738 supplier was added and not detected in the microcosms receiving distilled water) [17]. Two replicate soil samples were collected and pooled from each microcosm on D30 approximately 20 hours after the NO3- addition and frozen at −70°C until used for DNA extraction. Soil samples were further pooled by combining 125 mg of soil from two replicate microcosms in the same treatment and then subjecting this pooled soil sample to DNA extraction as described elsewhere [17]. Therefore, there were three replicate DNA samples for each treatment that were used to create two

metagenomes: one for the nitrate treatment (labeled +NO3-) and one for the distilled water treatment (labeled –N). Pyrosequencing Similar to other shotgun metagenomic studies [20, 49–51], DNA was amplified with the illustra Genomiphi V2 amplification kit MK-8931 (GE Healthcare Life Sciences, Inc., Piscataway, NJ) following the manufacturer’s protocol. Two replicate Genomiphi reactions were prepared for each microcosm DNA sample, making six reactions total for each treatment (three replicate microcosm DNA samples × two replicate Genomiphi reactions). The Genomiphi reactions randomly amplified regions of genomic DNA using primers of random sequences and resulted in 8 μg of amplified DNA from the +NO3- sample and the 10 μg of amplified DNA from the –N sample. learn more Because of the use of random primers, these amplified DNA samples potentially

included segments of DNA from all microbial species present in the samples and from regions throughout the microbial genomes. The amplified DNA from Genomiphi reactions was precipitated with sodium acetate and purified with 80% cold ethanol before being sent to Inqaba Biotec (Pretoria, South Africa) for 454 pyrosequencing on a GS-FLX platform. Sequence analysis Because the metagenomes constructed from our microcosms contained DNA reads from multiple species, they were analyzed unassembled using the MG-RAST server [18] and are publicly available with the MG-RAST ID numbers 4445106.3 (+NO3-) and 4445130.3 (−N). Metagenomes are also available through the NCBI site [GenBank: SRP005560]. A BLASTX comparison to a non-redundant protein database was used to match the EGTs in the metagenomes to SEED subsystems [19]. The SEED protein-coding database has been used successfully for comparing shotgun metagenomes to taxonomic [20, 21, 51] and metabolic sequences [20, 21, 49–51] in environmental samples.