Total DNA was extracted from all the sediment samples (10 g wet weight) and from the Carrapatos stream bulk water sample (5L) using the UltraClean Mega Prep soil DNA kit and UltraClean Water DNA kit (MoBio Laboratories), respectively, according to the manufacturer's instructions. Quantification and quality of total DNA were determined using the equipment Agilent 2100 Bioanalyzer, again according to the manufacturer's instructions.
Wang et al. (2009 (link)) successfully applied 16S rRNA gene-based approaches using Gallionellaceae-related primers, to describe the FeOB community structure. PCR-DGGE of 16S rRNA gene was performed by a nested PCR using first the Gallionellaceae-related primers set 122F (5′-ATATCGGAACATGTCCGG-3′) and 998R (5′-CTCTGGAAACTTCCTGAC-3′) (Wang et al., 2009 (link)), followed by nested PCR using the primers set 341F/GC (5′-CCTACGGGAGGCAGCAG-3′) and 907R (5′-CCGTCAATTCMTTTGAGTTT-3′), specific for the Bacteria domain (Muyzer et al., 1993 (link)), according to Wang et al. (2011 (link)). The final PCR products were separated by DGGE in a 6% polyacrylamide gel with a vertical gradient of 30–60% of formamide and urea denaturants. The running conditions were 80 V at a constant temperature of 60°C for 18 h.
Wang et al. (2009 (link)) successfully applied 16S rRNA gene-based approaches using Gallionellaceae-related primers, to describe the FeOB community structure. PCR-DGGE of 16S rRNA gene was performed by a nested PCR using first the Gallionellaceae-related primers set 122F (5′-ATATCGGAACATGTCCGG-3′) and 998R (5′-CTCTGGAAACTTCCTGAC-3′) (Wang et al., 2009 (link)), followed by nested PCR using the primers set 341F/GC (5′-CCTACGGGAGGCAGCAG-3′) and 907R (5′-CCGTCAATTCMTTTGAGTTT-3′), specific for the Bacteria domain (Muyzer et al., 1993 (link)), according to Wang et al. (2011 (link)). The final PCR products were separated by DGGE in a 6% polyacrylamide gel with a vertical gradient of 30–60% of formamide and urea denaturants. The running conditions were 80 V at a constant temperature of 60°C for 18 h.