|Affiliation(s)||PI||Project period||Funded by|
|DEES||Moser, Duane P.||08/17/2010 - 06/30/2012||Nye County, Nevada|
Task 1. Microbiological Sampling The Contractor will sample for microbiology at approximately 27 wells, determined in consultation with the NWRPO. It is expected that this sampling will occur in conjunction with a separate NWRPO groundwater sampling program. At the same time, Contractor will train NWRPO staff in these sampling techniques. Central to the success of this endeavor will be the development of a sampling manifold system that can be disinfected (or preferably sterilized) to enable a minimum of a least three biological filers (Millipore Sterivex) to be filtered at one time. Through flow on these filters will be monitored and recorded. Once filtration is complete (either after the observation of plugging or the passage of a pre-determined volume of water), filters will be placed on either dry ice or wet ice in the field and stored frozen either at DRI or at the NWRPO. The filtrate will be made available for chemical analysis upon request by the NWRPO. All wells will be sampled for basic microbiology, some at multiple time points, and relevant filters set aside for future analysis. DNA samples held by NWRPO will be maintained at -20oC until such time as they can be transferred to Contractor's lab, where they will be stored at -80oC until final analysis. Prior experience has shown that samples can be stored at -20oC for at least one year and -80oC essentially indefinitely. DNA will be extracted from these filters in Contractor's lab at DRI using the MoBio Soil DNA Kit (MoBio labs, Salano Beach, CA) according to manufacturers' instructions. Extracts thus obtained will be archived frozen at -20oC for at least three years beyond the end of this contract. Wherever possible, Contractor or NWRPO will collect the following field parameters using a Yellow Springs Instrument (or equivalent) multiprobe with flow cell: dissolved oxygen, relative redox potential, temperature, conductivity, and pH. Task 2. Terminal Restriction Fragment Length Polymorphism Analysis (T-RFLP) T-RFLP analysis provides a total microbial community fingerprint. Fluorescently-tagged DNA fragments of diagnostic lengths are separated using a DNA sequencer, and the resulting "ribotypes" represent unique microbes present in the original sample. The fingerprint thus obtained is unique for an individual sample and community profiles can be compared across multiple samples or time-courses (e.g., during well pumping). The specificity of this assay can be fine-tuned (e.g., total life to species level). This method can be performed along with the proposed clone libraries at a low per sample cost. All samples obtained by this project (including multiple time points from certain wells and drilling fluid when available) will be assayed using this technique. Fragment analysis will be performed at the Nevada Genomics Center in Reno, NV, using restriction enzymes already stored there for Contractor's use. Task 3. 16S rRNA gene libraries: In concert with T-RFLP analysis, domain-specific (e.g. bacterial) 16S rRNA clone libraries will be generated. Clones will be screened by unidirectional sequencing reads and representatives of unique types sequenced in their entirety (e.g. 1,500 base pairs). Individual sequencing reads will be assembled using sequencer and clone groups defined using Unifrac and DOTUR software packages. Phylogenetic analyses will be managed within GreenGenes. All required software is in routine use in Contractor's laboratory. Sequences, thus obtained, will be digested in silico to identify major T-RFLP peaks, thus linking the two datasets. Contractor may choose to sequence only from a single direction in the interest of expanding the ability to process samples, but this option will be discussed with NWRPO prior to undertaking. Task 4. Total cell number determination: Cell counts will be determined for all samples using an Advanced Analytical MicroPro 3000 flow cytometer (AATI, Ames, Iowa) currently in routine use in Contractor's lab. This robotic cell counter is able to interrogate up to 20 samples per hour and can record a million events (cells) per minute. The MicroPro can differentiate between live and dead cells and can to some extent reveal major groupings of cells (e.g. differentiate rods from cocci and spores). This instrument is used routinely in Contractor's lab and maintained on other sources of funding. This analysis will be provided at no cost to NWRPO in the interest of improving the publishability of joint datasets.