Bacterial diversity and succession along an epigenic cave stream
his study was done to evaluate changes in bacterial communities from a stream in the Cascade Cave system, Carter Caves State Resort Park in Kentucky (United States). Samples from cave sediments, cave water, and from Bio-Traps(R) were acquired from July 2013 through December 2013, from two locations separated by approx. 1.5 km, or approx. 12 hours water flow travel time. At least 500 mL of cave stream water were manually filtered through duplicate 0.22 ?m SterivexTM (PVDF, EMD Millipore) filters. If stream sediments were available to collect, then at least 25 g of sediments were collected aseptically and placed into sterile Falcon tubes; as such, the maximum sediment size collected was >20 mm for any one particle. Standard Bio-trap® samplers baited with 30 g of 2-mm diameter Bio-Sep® beads made of Nomex® composite and powdered activated carbon were obtained from Microbial Insights, Inc. (Knoxville, TN, USA) (www.microbe.com). Bio-traps® were deployed in triplicate at each sampling location in the cave and sampled every. From each Bio-trap®, 2.5 g beads were separated out and frozen until extraction. Samples were also taken from a surface stream location, before the water flowed into the cave.     DNA was extracted from Sterivex filters (0.5 L for each filter) using a method modified from Riemann et al. (2008; Appl Environ Microbiol 74, 503-515) and nucleic acids were precipitated from the suspensions using 25:24:1 phenol:chloroform:isoamyl alcohol (pH 8) twice, and 24:1 chloroform:isoamyl alcohol once, prior to pelleting by centrifugation. MoBio PowerSoil® Extraction kits (MoBio Laboratories, Inc., Carlsbad, CA, USA) were used to extract total nucleic acids in triplicate from 0.25 g from each Bio-Trap® and separately from 0.25 g of sediments at each location. Duplicate (for water) or triplicate (for Bio-sep® beads or sediment) extractions were homogenized prior to purification and amplicon pyrosequencing of single-direction reads generated using multiplexed, bar-coded primers targeting the V1-V3 region of the bacterial of 16S rRNA gene.