The excavated pipe was installed in 1949 and exposed to residential waste. Biomass was removed from the crown (top section of the pipe, TP) and invert (bottom, BP) sections using a sterile
metal spatula by scraping approximately 4 cm2 surface area of each material. Biomass was then transferred to sterile tubes and stored at −20°C. Total DNA was extracted using UltraClean Soil DNA kit following the manufacturer’s instructions (MoBio Laboratories Inc., Solana Beach, CA) and used as a template for the generation of pyrosequencing metagenome libraries. 16S rRNA gene sequence analyses Sequences from Bacteroidetes (n=236), sulfate reducing (n=56) and sulfur oxidizing (n=164) bacteria obtained Selleck INCB024360 from a previous study  were used to develop phylogenetic trees. Briefly, 16S rRNA gene primers 8F and 787R were used to generate community PCR products, which were then cloned using TOPO TA vectors. Clones were sequenced in both directions and assembled using Sequencher software (Gene Codes Corp, Ann Arbor,
MI). Sequences were assigned to specific bacterial groups using MOTHUR v1.19.2 (http://www.mothur.org) with 97% sequence identity as the cut off point for each Operational Taxonomic Unit (OTU). Phylogenetic trees were constructed from the alignments STA-9090 datasheet based on the Maximum Likelihood method and calculated using Tamura-Nei model . MEGA v5.03  was used to build trees using 100 replicates to develop bootstrap confidence values. The Classifier tool of the Ribosomal Database Project II release 10.26  and BLASTn  were used to classify and identify the nearest neighbors. Cluster analysis of wastewater concrete biofilms Cluster analysis based on the transformed (log[x+1]) relative abundance data was eltoprazine used to compare communities associated with different wastewater concrete biofilms. First, we estimated the taxonomic distribution at the genus level of each microbial community from 16S rRNA gene pyrosequences generated in this study and Sanger-chemistry 16S rRNA gene sequences generated in previous studies [7–10]. This information was used to generate Bray-Curtis similarity coefficients of the transformed data
using the software PAST v2.03 . This estimator compares the structures by accounting for the abundance distributions of attributes (e.g. species). Dendrograms indicating relationship of biofilms generated by comparing similarity coefficients estimates among sample sites were calculated using the UPGMA method with the software MEGA v5.03 . Metagenomic studies Pyrosequencing was performed using the 454 Life Sciences GS-FLX Titanium® platform. Prior to sequence analysis we implemented a dereplication pipeline (http://microbiomes.msu.edu/replicates) to identify and remove clusters of artificially replicated sequences, i.e. reads that began at the same position but varied in length or contained a sequencing discrepancy . Filter parameters included a cutoff value of 0.