Proceedings of the South Dakota Academy of Science, Vol. 97 (2018) 129 INVESTIGATING BACTERIAL COMMUNITIES IN A WARM, ALKALINE POOL IN THE SANFORD UNDERGROUND RESEARCH FACILITY USING CULTURE-BASED AND CULTURE-INDEPENDENT METHODS Taylor Liu1 and David J. Bergmann2* 1University of California Berkeley, CA 94720 2Black Hills State University Spearfish, SD 57799 *Corresponding author e-mail: [email protected] ABSTRACT We examined the bacterial communities in a pool of water under a flow- ing rock fracture at a depth of 1470 m in the Sanford Underground Research Facility in Lead, SD. High-throughput sequencing of 16S rDNA indicated that Acidobacteria, Chlorobi, Nitrospirae, Planctomycetes, and Gammaproteobacteria (especially Acidiferribacter) were abundant in pool sediments; while Nitrospirae, Plactomycetes, and Proteobacteria (especially Thiobacillus and Methylococcus) were abundant in pool water. Samples of diluted pool water were mixed with agar media, placed in diffusion chambers surrounded by 0.2 µm pore mem- branes, and incubated in situ within the pool for 19 days. 16S rDNA analysis of media from diffusion chambers indicated Alphaproteobacteria (including Hyphomicrobium), Betaproteobacteria (including Inhella and Methyloversatilis), and Gammaproteobacteria (mainly Pseudomonas) were dominant. Low-nutrient media were used to isolate bacteria from both pool water and media inside diffu- sion chambers. The majority of these isolates were Methyloversatilis, Inhella, and Bacillus. Although the genera isolated were not very abundant in the pool water, the presence of Methylococcus and Methyloversatilis suggests the importance of methane and methanol oxidation in this community. Keywords Bacteria, deep subsurface biome, SURF INTRODUCTION Deep underground rocks and aquifers extending over 1500 m below the earth’s surface constitute a massive region of the biosphere and may contain more pro- karyotic cells than all other habitats (Madigan and Martinko 2006). For example, dense communities of Bacteria and Archaea have been found in aquifers 1500 m 130 Proceedings of the South Dakota Academy of Science, Vol. 97 (2018) deep in basalt, where microbial metabolism involving methanogenesis, acetogen- esis, and sulfate respiration are important (Stevens et al. 1995; Anderson et al. 1998). The Sanford Underground Research Facility (SURF), located in Lead, South Dakota, is one of few sites where deep subsurface microbial environments can directly accessed. SURF is located in the former Homestake gold mine, compris- ing over 590 km of tunnels and extending to a depth of 2460 m. Between the cessation of gold mining in 2003 and the opening of SURF (then known as the Deep Underground Science and Engineering Laboratory) in 2007, pumping of the water accumulating in the lower tunnels of the site was discontinued, and the lower levels of the site (including the 1470 m level) became flooded. At present, water is pumped out to depth of about 1700 m, and the 1470 level is now the site of a number of major physics experiments (Lesko 2011; Sanfordlab). Sediments from the 1470 m level of SURF are rich in chlorite-chamosite [(Fe5Al)(AlSi3) O10(OH)8], annite [KFe3AISi3O10(OH)2], and quartz (SiO2) (Rastogi et al. 2010). The tunnels in SURF provide an environment where air in the tunnels comes in contact with anaerobic fracture water from deep aquifers, making chemoau- trophy and methanotrophy possible. Sediments and drainage water in SURF have been found to contain diverse assemblages of prokaryotes, including ammonia-oxidizing Archaea, sulfide-oxidizing bacteria, and various chemo- heterotrophic bacteria (Waddell et al 2010; Rastogi et al. 2009; Rastogi et al. 2010). Furthermore, SURF contains a wide variety of biofilms, often sustained by fracture water seepage of different redox potentials and chemical composition, supporting diverse microbial communities (Osburn et al. 2014). One difficulty in studying microbial communities is that often less than 1% of microbial species can be cultured on standard media. The cultured species may be minor constituents of their communities, but may be easily isolated because of their rapid growth and their lack of specialized requirements for organic co- factors (Epstein 2013). One way to investigate the taxonomic diversity of micro- bial communities is to use culture-independent techniques involving extraction of microbial DNA, PCR amplification of the 16S rRNA genes, and sequencing PCR amplicons (Madigan et al. 2006). For example, using next-generation 16S rDNA sequencing methods, Osburn et al. (2014) found that samples of water sand biofilms from SURF contained from 0.3 percent to 19% taxa from candi- date phyla such as BRC1, M7, and W53, none of which have been isolated in culture. Despite the difficulties encountered in culturing microbes from environmental samples, it is often desirable to isolate microbes in culture so their phenotypic characteristics can be fully characterized. Recently, the use of diffusion cham- bers, in which environmental samples are initially incubated in situ within small volumes of media surrounded by permeable membranes (diffusion chambers), has enabled a greater diversity of bacterial groups to be isolated in culture from some habitats (Bollmann et al 2006), especially slow-growing bacteria or bacteria requiring certain organic cofactors. During an exploration of the 17 Ledge region of the 1470 m level of SURF, we found an extensive area of microbial biofilms where deep aquifer water from Proceedings of the South Dakota Academy of Science, Vol. 97 (2018) 131 a fracture in the rock flows into an old mine tunnel, forming extensive pools of water. This site was located about 324 m from the ramp to the 1410 m level, about 105 m beyond a flowing borehole known as Thiothrix“ Falls,” and is near the farthest portion of the tunnel which can be safely visited. The pools of water at the site had a temperature about 32 °C, and a pH about 8.8. Because of the unusual physical conditions at the site, we decided to conduct a detailed inves- tigation of the microbial communities in the pool of water, which might harbor novel, uncharacterized taxa of Bacteria and Archaea. In this study, we investigate the bacterial communities in this warm, alkaline pool of water on the 1470 m level of SURF using a variety of techniques: 1) culture-independent analysis of bacterial 16S rDNA, 2) direct culturing on a standard bacteriological medium, and 3) culturing of bacteria using in situ incu- bation of samples in low-nutrient media in diffusion chambers. Analysis of 16S rDNA sequences will allow us to directly evaluate the taxonomic diversity of a possibly unique microbial community. By using the two culture-based techniques of direct isolation and isolation from diffusion chambers, we hope to isolate and culture microbes, including some of those groups not readily cultured by stan- dard bacteriological techniques, for phenotypic analysis and future study. METHODS Location and Sampling. The pool of water sampled in this study is located on the 17 Ledge of the 1470 m level of SURF about 150 m beyond a flowing borehole known as “Thiothrix Falls” which is presently capped by a manifold as part of a NASA biology experiment. The pool is about 1 m wide, a few m long, and 10 cm deep, and is fed by water flowing from a fracture in the rock. The rock in the area consists of the Poorman formation with metamorphic rocks, such as phyllite, predominating (Caddey et al. 1991). For some chemical analyses, unfiltered samples of water were collected with a glass beaker. For other chemical analyses, filtered samples were collected with sterilized #15 silicon tubing (Core-Parmer Instruments Co., IL) connected to a Masterflex E/S Portable Sampler peristaltic pump (Core-Parmer Instruments) with a Sterivex GS 0.2 µm pore cartridge filter (Millipore). Two filter cartridges were used; the first filtering about 3.9 L and the second 2.8 L. Filtered water was collected in 250 ml polyethylene bottles and 40 ml glass vials. Filtered and unfiltered samples were preserved as directed by Mid-Continent Testing Laboratories Rapid City, SD, which conducted chemical analyses within two days of collection. The filter cartridges, containing microbial cells from the pool water, were frozen in dry ice. Samples of sediment from the pool were collected in 50 mL Falcon tubes and frozen on dry ice. Filters and sediment samples were stored at -80 °C until microbial DNA was extracted (see 16S rDNA Library Preparation). Diffusion Chambers.Direct isolation of microbes from the pool water was performed by collecting a water sample in a sterile Falcon tube and taking it to 132 Proceedings of the South Dakota Academy of Science, Vol. 97 (2018) the Black Hills State University Underground Campus cleanroom (BHUC) on the 1470 m level of SURF. The sample was diluted from 10-1 to 10-5 in filtered, autoclaved SURF drainage water (pH 8.5) from the 1470 m level of SURF. Diluted samples were spread across petri plates of 1/10X R2B medium (Reasoner and Geldrich 1985) with 1.5% agar, with 1% ATCC vitamin extract (American Type Culture Collection), and fungicides (100 µg/mL cycloheximide, and 50 µg/ mL Nystatin (Sigma-Aldrich). The plates were then returned to the surface and incubated at 30 °C for two weeks at the Life Sciences building at Black Hills State University, Spearfish, SD. Diffusion chambers for in situ culturing were constructed of sterilized 27 mm (inner diameter) steel washers, 2 mm thick, with 47 mm diameter, 0.2 µm pore size polycarbonate IsoporeTM membrane filters (Millipore, Inc.) covering the upper and lower sides. The lower filter was attached with silicone adhesive, cov- ered in foil, and taken to the pool in SURF. A sample of water was taken from the pool and processed in the BHUC cleanroom. Samples were diluted in filtered, autoclaved SURF drainage water with 1/10X R2B medium. The water samples were diluted to a final concentration of 10-3 in 1/50X R2B medium with 1.0% agar (50 °C), with 1% vitamin extract, and fungicides.