PHYSICS PROJECT 2: PARTICLE TRANSPORT COMPUTER MODELING FOR HPGE SCREENERS

Kara Keeter, BHSU Associate Professor of Physics

Because of their extremely high purity, outstanding energy resolution and reasonable detection efficiency, the low-background HPGe detectors can provide the screening measurements with desired sensitivity in a deep underground setting. To effectively assay material at this level of sensitivity requires that the assay detector itself be exceptionally low in radioactivity and be deployed in a well-shielded and low background environment. Direct cosmic ray backgrounds can be drastically reduced to a negligible level by operating the detector deep underground. In doing so, the local radiation field will be dominated by uranium/thorium (U/Th) rock gammas coming from the mine drifts, gammas from the decay of air-borne radon daughters, as well as reaction induced radioactivity from the surrounding rocks. Backgrounds originating from the detector components, shielding material, and cosmogenic products could be reduced by a stringent low radioactivity material selection program, together with process control to minimize cosmic exposure time on the surface.

Extensive computer simulations are critical in both the design and the use of an HPGe low background counter. Comprehensive simulation packages are used such as GEANT4, a toolkit originally developed by CERN and continuously being developed and improved by a world-wide collaboration of scientists and software engineers, for the simulation of the passage of particles through matter. GEANT4 and other simulation packages such as FLUKA use Monte Carlo techniques to characterize background levels in detectors and to optimize the design of detectors and shielding. The increased sensitivity needs of ultra-low background screeners requires modeling of the detector geometry and shielding and comparing the simulated data to data from long background runs.

Example Student Project: We will train students to use Monte Carlo simulation programs such as GEANT4 and FLUKA to characterize low background detector backgrounds. Such background simulation studies are used to not only understand a detectors specific background for background rejection, but also can be used to further reduce background levels in shielding in an iterative process. Students will be given a specific component (e.g. Pb or Cu shield) of the environment surrounding the HPGe detectors located at the BHUC. Students will be shown how to build the geometry in GEANT and to simulate background radiation affecting the detector signal. Students will be mentored in interpreting the data and in reporting the results with possible recommendations for shielding changes.

BIOLOGY PROJECT 1: THE CONTRIBUTION OF EUKARYOTIC DIVERSITY TO DEEP SUBSURFACE MICROBIAL ECOSYSTEMS

Cynthia Anderson, BHSU Associate Professor of Biology

The diversity of Eukaryotic microbes in subsurface terrestrial ecosystems remains underexplored. While the likelihood of finding Protista and Fungi in the pristine rock strata at any significant depth diminishes due to spatial and nutrient constraints, it is not entirely unreasonable to expect that some endogenous Eukaryotes live and perhaps thrive in the seemingly inhospitable subsurface environment where nutrients are limiting and temperatures increase with depth. Some species of fungi are capable of surviving in extremely nutrient poor rock environments. For example, dematiaceous meristematic fungi have been isolated from rock surfaces (granite, limestone, and calcite), and from stone monuments. It is well documented that fungi play an important role in geomicrobiological transformations, ranging from facilitating syntrophic interactions to aiding rock decay and diagenesis. While prevalent in terrestrial environment on and slightly below the earths surface, these interactions of fungi within the geologic environment of the deep subsurface remain to be explored. Even if endogenous fungi are found to be lacking in the deep subsurface, the role of exogenous fungi in rock decay and diagenesis, as well as biofouling of research infrastructure in the subsurface needs to be understood, especially in light of the establishment of subsurface research infrastructure.

There is no doubt that fungi have been introduced to the subsurface areas of SURF through anthropogenic activities ranging from the mining activities of the past 125 years, to the more recent flooding and dewatering of the mine (past 6 years), to the re-ventilation of the drifts and subsequent construction of the subsurface research infrastructure. Preliminary culture independent environmental genomic data on the Eukaryotic diversity found in a water sample, and a biofilm sample taken from the 4850L indicate the presence of several Eukaryote taxa representing both Protista and Fungi (Fig 3).

The constant influx of ventilation air introduces microbial propagules that can become established on rock surfaces, especially near slow flowing water seeps, and anthropogenic activity during infrastructure construction such as the production of pollutants from fossil fuel run equipment, introduction of wood and other construction materials have introduced organic and inorganic nutrients upon which some of the microbes can feed. Thus it could be hypothesized that the drift walls at the 4850L throughout SURF will continue to harbor a diverse microbial community that includes eukaryotes. The drift wall communities will consist of both aerobic and facultative anaerobic Prokaryotic and Eukaryotic microorganisms some living independently, but most forming complex syntrophic and symbiotic relationships. The prevalence of endemic Eukaryote taxa in SURF is unknown. Furthermore, the influence of exogenous Fungi on the endemic microflora, the level of syntrophy among members of the subsurface microbial communities, the role of endogenous and exogenous Eukaryotic organisms in biogeochemical processes such as rock weathering, and the extent of biogeochemical interactions within the miles of subsurface drifts throughout the former Homestake mine areas are completely unknown.

Example Student Project: The pilot data depicted above was generated from a rDNA amplicon clone library. While informative, the Chao diversity indices estimated that only 45% and 48% of the diversity of the water and biofilm samples was captured. Next Generation sequencing technologies would vastly improve the ability to capture the entire diversity present. Furthermore, comparisons of air, open water, water from deep drill holes, water emanating from drill holes and biofilms would provide us with a much more thorough view of the eukaryotic diversity in this isolated ecosystem and lead to hypotheses about colonization of water and biofilms, and the organismal relationships occurring in the systems. It would also help us to identify those eukaryotic microbes that may actually be endemic to the system and not simply introduced. Water emanating from subsurface drill holes, biofilms, and air from various areas of SURF over varying seasons will be sampled. Culture independent DNA analyses will be employed to explore the eukaryotic microbial diversity. Students will PCR amplify the intergenic spacer region of the rDNA, ITS1-5.8S-ITS2, and construct amplicon libraries for NextGen sequencing and will use bioinformatics data analysis tools to estimate the eukaryotic diversity throughout several sampling areas. Additionally, students will visually survey various substrates throughout SURF, choosing interesting biofilms and instances of colonization to sample in order to attempt to culture fungi that may be colonizing the biofilms and other substrates observed. By collaborating with students working in projects 6 – 9, a deeper understanding of the possible roles of eukaryotic microbes will allow us to further our understanding of their roles in biogeochemical processes as well as their roles in syntrophic relationships in a complex ecosystem.

BIOLOGY PROJECT 2: NEW TECHNIQUES TO ISOLATE NOVEL MICROBIAL GROUPS FROM DEEP UNDERGROUND SEDIMENTS

David Bergmann, BHSU, Professor of Biology

Microbes are abundant in nearly all terrestrial and subsurface habitats on earth, often with over 103-104 species of microbes and over 108 microbial cells per gram of soil [45]. Nonetheless, detailed characterization of the microbes in most habitats has proved challenging, because it is estimated that only about 0.1-15% of most microbes can be isolated from the environment using standard bacteriological techniques. Recent advances in sequencing DNA from environmental samples has greatly expanded our knowledge of the composition and functioning of microbial communities, however, to compile a detailed picture of the phenotypic characteristics of individual microbial species and their interactions with other species in the community requires that these species be isolated and cultured in the laboratory under controlled conditions.

During the past decade, techniques have been developed to improve the diversity of microbes which can be isolated from environmental samples. Some involve placing diluted samples of microbes in membrane-bounded chambers placed in the habitat from which they were derived, so that inorganic ions and organic molecules can freely diffuse into the media. After incubation in situ, the chambers are then opened and micro-colonies transferred individually to fresh media. This technique has enabled the isolation of many microbes which cannot otherwise be cultured. Here, we propose to conduct a detailed investigation of the chemical and biological composition of a poorly-known microbial habitat, the sediments present in the 4850L of SURF, and use this information to isolate novel groups of microbes using in situ culture techniques. From 16s rDNA analysis, sediments in portions of SURF are predicted to have a diverse microbial community, which includes both chemoautotrophic and chemoheterotrophic Bacteria and Archaea.

Example Student Project: To examine the diversity of microbes present in sediments at the 4850L of SURF, students will extract microbial community DNA from slightly moist, well-aerated sediment and perform PCR with universal bacterial 16S rDNA primers. The PCR products will be cloned into a plasmid vector, and plasmids from about 200 clones sequenced using Sanger Big-Dye sequencing. The number of operational taxonomic units (probable species) and search databases to identify taxa present will also be determined. Students will perform in-situ culturing of sediment microbes by diluting sediment samples in gellan gum media containing aqueous sediment extracts and enclosing media in diffusion chambers which are surrounded by 0.2 m nylon membranes. The diffusion chambers will be left in place about 2 weeks in the upper layers of sediment. After the incubation period, the chambers will be opened and examined under a dissecting microscope for microbial micro-colonies, which will be transferred to wells of similar media in 96-well plates. Wells showing growth will be transferred to new media periodically or frozen for long-term storage. Transfer to a completely synthetic medium, whose composition will be determined from the chemical analysis of sediment, will be attempted. To determine if the taxa which were isolated from sediments were the dominant groups originally present in the sediments, students will extract DNA from 100 isolates and the 16S rDNA amplified by PCR as described previously. The PCR products will be purified and sequenced directly. 16S rDNA sequences from isolates will then be compared with those of microbes originally present in the sediments to determine what proportion of common microbes were successfully isolated.

CHEMISTRY PROJECT 1: INDUCTIVELY COUPLED PLASMA MASS SPECTROMETRY FOR RADIOPURITY MEASUREMENTS

Daniel Asunskis, Assistant Professor, Chemistry

Inductively Coupled Plasma Mass Spectrometry (ICP-MS) is a complementary technique to the high purity germanium (HPGe) detectors that are ubiquitously used in underground labs. In the standard ICP-MS technique, liquid samples are pumped into a nebulizer where it is mixed with argon gas to form a fine aerosol and sent into the plasma torch. The molecules of the aerosol are broken into ions by the plasma and sent through the mass spectrometry interface consisting of sampling and skimmer cones and into an ion focusing system which directs the ions into the mass separation device and finally onto the detector. One advantage of ICP-MS over HPGe is in the measurement speed. Once the sample is prepared, ICP-MS takes less than five minutes to analyze one sample, whereas the HPGe detector may take up to a month. Additionally, smaller sample sizes are required with ICP-MS and if laser ablation is utilized, ICP-MS can become a location-specific technique. A disadvantage of ICP-MS is in the preparation of the sample (if laser ablation is not used). A solid sample must be dissolved to a concentration of less than 1%. Preconcentration methods may be used prior to introduction into the ICP-MS.

Example Student Project: Perhaps more important than the ICP-MS itself is the sample preparation required to achieve ultra-trace sensitivities required for low background experiments. The preparation of a sample for ICP-MS analysis requires great care. The sample must be tracked from its origin, through preparation and to the ICP-MS itself. Any step taken along the way could cause contamination of the sample and must be examined carefully. Sample preparation techniques for ICP-MS radiopurity measurements are constantly being researched and refined. The student would work to develop new sample preparation methods with applications to low background physics experiments.

CHEMISTRY PROJECT 2: CHEMICAL CHARACTERIZATION OF UNDERGROUND WATER

Michael Zehfus, BHSU Professor of Chemistry

There are a myriad of unique microenvironments in the shafts and drifts in the SURF complex. The chemical composition composition of the slightly moist sediments from the 4850L must be known to better reproduce this environment in his culturing experiments. Thus one thrust of this project will be to characterize the chemistry of these sediments so he can create a culture medium that more accurately reflects the actual mine environment. It is expected that there will be other microenvironments of potential interest to the biologist involved in this project, and characterizing the chemistry of these other sites will be important to their work as well.

Example Student Project: We will direct our main emphasis towards chemically characterizing water-soluble ions found in sediments. For comparison, we hope to find a stable pool of water nearby so we can compare and contrast the ions found in sediment with the ions found in the pool water. Since the sediments will have little or no free water, our first step will be to suspend the sludge in a known amount of deionized water so we have a dilute aqueous sample to start with. We will start with easy measurements like pH and ORP (Oxidation-Reduction Potential) that can be performed in situ with electrodes using hand-held instruments. Then we will look at some of the more common ions we expect to find like HCO3- , SO42-, Ca2+, Mg2+ and Fe2+ or Fe3+and total alkalinity. While some of these ions can also be measured with various ion-selective electrodes, the electrodes are expensive, so we will instead measure these ions in the lab using various chemical techniques including titrations and photometric reactions. We will also monitor dissolved oxygen and chemical oxygen demand.

CHEMISTRY PROJECT 3: ANALYSIS OF ORGANIC COMPOUNDS PRESENT IN MICROBIAL HABITATS DEEP UNDERGROUND

Katrina Jensen, BHSU Assistant Professor of Chemistry

Microbes produce and consume a diverse array of organic compounds and investigation of the organic content in microbial habitats increases the understanding of the diversity of microbes present in various environments deep underground, as well as the requirements necessary to culture species in the laboratory. In particular, we are interested in detecting one-carbon compounds, such as methanol, and methylamine, used by methylotrophs, as well as more complex organic compounds, such as siderophores, which are produced by microbes to chelate iron(III), and homoserine lactones, which are signaling compounds used by microbes for quorum sensing.

Example Student Project: A student will collect water samples underground and develop assays for the detection of organic compounds. Methanol and methylamine can be detected via derivatization with fluorescent compounds followed by analysis by high-performance liquid chromatography (HPLC)[57]. Siderophore presence can be detected using the chrome azurol sulfonate assay [58]. The chrome azurol iron complex serves as an indicator in this assay, turning from blue to orange in the presence of siderophores, which have a high affinity for Fe3+ and will remove iron from the indicator. Following a positive test for siderophores, individual siderophore compounds shall be analyzed via HPLC. Homoserine lactones will be tested for via extraction with an organic solvent, such as dichloromethane, concentration, and evaluation of the extracts with gas chromatography mass spectrometry [59]. A student working on this project with have the unique opportunity to go underground to collect samples, gain experience developing new analytical chemistry techniques, and collaborate with biologists to improve conditions for culturing diverse forms of microbial life.