Recipient Organization
NAVAJO TECHNICAL COLLEGE
PO BOX 849
CROWNPOINT,NM 87313
Performing Department
Biology
Non Technical Summary
Project Investigators: Dr. Irene Ane Anyangwe (PD), Navajo Technical University; Dr. Palmer Mesumbe Netongo (Co-PD), Navajo Technical University; Dr. Arthur L. Allen, University of Maryland, Eastern Shore, and Dr. Fawzy Hashem, University of Maryland, Eastern Shore. The Navajo nation agricultural soils similar to other soil types adjoining uranium tailings, are a varied mixture of organic and inorganic particles that co-occur to form secondary aggregates. Within and between these aggregates would lie voids or pores comprising mostly air and water, which are ideal ecosystem for bacteria. Generally, this soil ecosystem of the Navajo nation lies in an arid to semi-arid climate in which most areas receive less than 10 inches of annual precipitation. Farming activities depend on the biological soil crust of this environment. This proposal titled, "Pollution properties of uranium tailing reservoirs and their effect on putative extremophiles of contiguous Navajo agricultural soils" is a new application submitted as a New Discovery-Enhanced, Single-function research project. The overall proposal to the National Institute of Food and Agriculture (NIFA), Tribal Colleges Research Grants Program is a collaboration between Navajo Technical University (lead) and the University of Maryland, Eastern Shore (UMES). The overall goal of the project is to determine the diversity of microbial communities and their effect on uranium tailing-littered Navajo farming environments. The proposed research is to determine the bacterial populations that are supported in agricultural soils bordering Navajo Nation uranium tailings and their relationship with known bacterial species having metal-reducing capabilities. The UMES investigators Drs. Arthur Allen and Fawzy Hashem will support the overall project as described in the detailed proposal. Specific responsibilities of Drs. Allen and Fawzy in the project are; 1) facilitate recognition of standard tailings-water interface zones for sample collection in collaboration with NTU Researchers and students, 2) advise and mentor participating NTU students, and 3) provide guidance in the quality of work, clarify expectations regarding publications, and conference presentations. NTU Land Grant Faculty and undergraduate Biology students will implement the project and ensure effective collaboration with University of Maryland, Eastern Shore as a participating institution. The grant opportunity will afford NTU students better prospects for educational achievement by engaging them in laboratory and field research in the natural resource sciences. Ultimately, this project will provide our American Indian student populations with the skills needed to perform laboratory and field analysis of research information, as well as encourage them to undertake domestic field research that would address the food, agricultural and natural resources concern of their tribal communities.
Animal Health Component
30%
Research Effort Categories
Basic
50%
Applied
30%
Developmental
20%
Goals / Objectives
This proposal titled, "Dispersed uranium tailings and their influence on the bacterial community of contiguous Navajo nation agricultural soils" is a new application submitted as a New Discovery-Enhanced, Single-function research project. This proposal to (NIFA), TGRGP is a collaboration between NTU (lead) and the University of Maryland, Eastern Shore (UMES). The overall goal is to determine the diversity of microbial communities and their effect on uranium contaminated Navajo farms. It will also determine bacterial populations habiting these agricultural soils bordering uranium tailings, and relate them to identified metal-reducing species.
Project Methods
Study Area. Water and soil samples from six sampling points within the Navajo Nation will be collected. The unregulated water sources contain uranium exceedances far above accepted water standards (30 μg/L). Sampling points have been selected based on their satisfying the uranium levels dictated by the benchmark uranium water sources, which lie within the vicinity of the farmlands, and often routinely used either for human consumption, for recreational, and agricultural purposes. Chosen fields fall within the vicinity of the following water sources with established high uranium concentration levels: Tohatchi(120 μg/L); Lime-Ride(260 μg/L); Becenti(110 μg/L); Platero(63 μg/L); Pigeon(88.5 μg/L); Red Mesa(700 μg/L).Sampling. Unregulated water samples within the six sampling sites will be collected in summer, when effluents of other seasons do not introduce bacteria from other sources. Triplicates of ground water from the surface and bottom would be collected in sterile 500mL polypropylene screw-capped bottles, and immediately deep-frozen with CO2 and ice, properly labeled and transported to the laboratory within 6 hours. Control water samples will be collected from regulated water sources from uranium-free regions within the Navajo Nation. A 100g soil sample (upper 2cm) will be collected using a sterile scoop in sparsely vegetated areas around the six test points with high uranium levels and control areas.Measurement of uranium. The phosphorescence intensity of uranyl-ions (λ =530) excited by ultraviolet radiation will be measured using Fluorat-02-4M liquid analyzer with optical filters. In this procedure, the concentration of uranium in solution will be measured by the intensity of the fluorescence of the uranyl-ions (λ = 530). Precisely, the solid samples from the soils will be dry-ashed at 450 0C in an oven and dissolved in 2.5M nitric acid saturated with aluminum nitrate. The uranium will be extracted into an organic-phase (ethyl acetate). Then, 10mL of the supernatant solution of the ethyl acetate solution will be destroyed in porcelain crucibles by ignition, and the residues will then be re-dissolved in 0.005% nitric acid.Isolation of Bacteria from soil samples. Soil samples (5g) will be suspended in 30 ml of phosphate buffered saline (PBS) in sterile, labeled, 50ml Falcon tubes, mixed for 1 min and centrifuged at 60 x g for 5min at 4 0C. After transferring 15 ml of the turbid supernatant into new sterile, labeled Falcon tubes, we will sediment all suspended bacteria by re-centrifuging (7,000 x g) for 10min at 40C. We will then recover the bacterial species by suspending the resulting pellet in 10ml PBS, and finally enrich the bacteria species by centrifuging (5,000 x g) the suspended pellet, and adding 1ml sterile PBS to the final pellet. To propagate a large number of the microbes, we will inoculate 0.1ml of the pellet in duplicate sterile tubes with basic medium, LB broth (Gibco). We will also support the most non-fastidious bacteria, and duplicate the sterilized tubes with the enriched medium, "Nutrient Broth" (Alpha Biosciences, FL), a liquid medium for isolating fastidious bacteria. The inoculated culture tubes will be incubated at 37 0C, and observed for macroscopic growth daily for about 6 months before discarding. Given that significantly large percentage of bacteria present in soil samples have never been cultivated or described in the laboratory, we will maximize their culturability by cultivating the inoculum in low nutrient media. Thus, we will plate the LB and Nutrient Broth cultures on dilute nutrient broth (DNB) agar (0.08 g nutrient broth powder [Difco] and 15g of highly purified agar [Fluka impurity-free agar] per liter), which is a low-nutrient medium that allows cultivation of a wide range of soil bacteria (both hard to culture and well characterized groups). This media also supports the growth of typically copiotrophic (nutrient-rich environments) growth, such as Gammaproteo- bacteria, especially E. coli. Finally, we will add 3ml of black India ink (black India waterproof ink; Sanford) and 50 mg of cycloheximide (Sigma-Aldrich) to each liter of the medium to respectively improve the optical contrast and suppression of fungi. Explicitly, we will use two main batches of experiments to prepare and plate these samples. For the first group, we will inoculate 30 replica petri dishes (each containing 25 ml of the DNB agar) with 1: 128 dilutions (in PBS) of the LB and Nutrient Broth cultures as previously described. For the second experiment, we will perform 11 serial dilutions of stock broth cultures ranging from a pure stock to a 1: 2,048 dilutions. We will then inoculate 5 replica petri dishes with each solution. In both experiments, we will inoculate 100μl of the appropriate solution per dish, and vigorously spread the inoculum with 10 to 15 sterile glass beads to enable even distribution of the colonies. After 3 days, 7-days and then at further 7-days interval, we will determine and image the petri dishes containing bacterial colonies with an inverted microscope attached to a standardized camera port (Olympus CKX53), and mark out the colonies of interest.Survival by Colony forming units(CFU) . The control and test samples will be serially diluted and plated on rich medium (RM), plate count agar (PCA), and nutrient agar (NA). We will incubate the plates at 28 0C for 20 days, observe and count the colonies with an Olympus inverted microscope.Identification of bacterial isolates. We will subculture our selected colonies on "dilute nutrient broth solidified with agar" (DNBG) and "VL55 media solidified with gellan mixed with different growth" substances as previously described. We will assess the distinct cultivable microbial diversity of the uranium contaminated arable soils from across the 6 sample sites by targeting the 16S rRNA, a universal bacterial marker. We will amplify a variable region of the bacterial 16S rRNA gene using well established primer set 8F (AGAGTTTGATCCT GGCTCAG-3′) and 531R (ACGCTTGCACCCTCCGTATT-3′). The final concentration of reagents in our 50μl PCR reaction mixture will be made as previously described. We will purify the amplicons using Qiagen's purification kit and submit purified amplicons to Dr. G. Anderson at Lawrence Berkeley National Lab & UC, Berkeley to characterize the microbial diversity using the more rapid and in-depth Phylochip assay.Database similarity searching. We will use pairwise alignment to retrieve biological sequences of arid or semi-arid radiation-resistant extremophiles in databases, based on similarity with sequences of our bacterial community. Briefly, we will submit query sequences of the of bacterial community obtained from the Phylochip analysis, and perform pairwise comparison of this query sequences with all individual sequences in a database.Soil pH levels in relation to bacterial diversity. We will pretreat soils using different soil to water ratio and dissolution periods before pH measurements. We will make it manageable by splitting it into modules and assign students' activities in the modules in which, they have particular expertise. Thus, to analyze the relationship between soil pH and bacteria diversity we will install QIIME in our Mac OS X dedicated to bioinformatics applications.Data Accessibility and Statistical Analysis. Raw sequences would be submitted as a sequence read archive (SRA) to the NCBI. statistical analysis of data will be performed with statistical software Minitab version 19. Significant differences will be considered at P= 0.05 and mean values will be ranked by Tukey's multiple range test when more than two groups are compared with ANOVA. All soil characteristics will be measured three or fourfold, but since not all analysis will be possibly performed on exactly the same sample, we will calculate the mean and standard deviation of each characteristic per sample.