Egbert Schwartz - US grants

A grant has been awarded to Dr. Phillip Danielson at the University of Denver to fund the purchase of a Transgenomic Nucleic Acid Fragment Analysis System. Based on established DNA size separation and mutation detection technology, this system will increase the quality and cost effectiveness of undergraduate/graduate research and
education in molecular biology. Specific research programs that will benefit immediately include National Science Foundation-funded studies to identify novel genes that encode: (1) endocrine hormones in the brain; (2) cytochrome P450 toxin-metabolizing enzymes - which are critical to the control of crop pests and disease-carrying organisms; 3) microbial proteins that can be used to clean up of toxic waste sites contaminated with heavy metals and 4) molecular markers that can be used to identify and track genetic diversity in endangered species - work is conducted in collaboration with the Denver Zoological Gardens and Denver Museum of Science and Nature.

Until recently, the identification of mutations required the laborious screening of hundreds to thousands of genes for subtle variations in DNA sequence. Analysis of a single novel gene by the direct sequence approach currently used, can require a day or more to complete. The Transgenomic WAVE System funded by this grant will reduce the analysis time to 2-4 minutes/sample. The discovery and analysis of genes that encode proteins involved in toxin breakdown, as well as neuropeptides linked to stress is the focus of several research programs. Since these genes often exist as duplicates with subtle but critical differences, it is essential that both copies be isolated. The WAVE system will be used to reduce the potential number of competing non-target gene fragments by precise size fractionation of the initial pool of DNA used for gene amplification reactions. The instrument's mutation detection and fragment capture functions will be used to increase the efficiency with which these related genes are identified and recovered - even where two genes sequences differ by less than 0.5%. In research focused on conservation biology and microbial ecology, the WAVE system will provide an extremely sensitive approach to the analysis of DNA sequence differences among and within species. Finally, the WAVE's high-speed genotyping capabilities will be used to gather gene frequency data from hundreds of samples for large-scale, multi-state conservation genetic projects.

Beyond the benefit to the research activities at the University of Denver, a broad range of
laboratory and classroom-oriented educational goals will be advanced at both the undergraduate and graduate levels. Benefits will be particularly evident in the molecular-oriented laboratory courses that are at the heart of the Bachelor of Science and Bachelor of Arts degrees in Molecular Biology. On a broader level, department-sponsored biotechnology classes offered to high school students and teacher-training workshops that promote hands-on science education at the secondary school level will also be greatly enhanced by providing first-hand experience in one of the most modern methods of genetic analysis. The benefit to high school outreach efforts will immeasurable given that these programs target students in urban and low-income school districts who have traditionally been underrepresented in the natural sciences. In short, acquisition of the
WAVE Nucleic Acid Fragment Analysis System will provide significant and immediate
benefits to education at the high school, undergraduate and graduate levels.

Nitrogen limits plant growth in many soils around the world. Plants compete with microorganisms in the soil for nitrogen. When carbon is abundant in the form of organic matter or dead organisms, and nitrogen is relatively scarce, the microorganisms will take up most of the available nitrogen. In contrast, when microbial growth is limited by carbon, soil microorganisms will increase the amount of nitrogen available to plants by breaking down organic matter that contains nitrogen. At present it is difficult to measure if soil microorganisms are competing with plants for nitrogen or if they are helping plants to grow by supplying nitrogen. Nitrogen atoms occur in a least two forms in the environment. Most of the atoms have 7 protons and 7 neutrons (14N) but a few atoms will consist of 7 protons and 8 neutrons (15N). Schwartz, Hungate, Hart and Dijkstra have found that soil organisms are enriched in 15N relative to nitrogen in the soil solution. They propose that the ratio of 15N to 14N in the microbial biomass indicates if microbial growth is limited by nitrogen or by carbon. If correct, it will be possible to ascertain if plant growth is promoted or restricted by soil organisms. This project will be the first to measure the stable nitrogen isotope composition of the soil microbial biomass, and to test a conceptual model for how microbial 15N enrichment is controlled.

This study may provide a new tool to assay the impact of soil microorganisms on plant productivity. In addition, the project will contribute to education at undergraduate and graduate levels. The principal investigators teach courses in environmental microbiology, general ecology, microbial ecology, ecosystem ecology and forestry. The proposed research will be incorporated into these courses.

Life Science - Biological (61)

One of the most important environmental issues facing the Southwestern U.S. is the availability and quality of water. Water treatment is therefore an increasingly important technology at the forefront of environmental microbiology. To better prepare undergraduate microbiology students to face this challenge, this project adapts and implements a new water quality curriculum in an environmental microbiology laboratory course. In addition, the project engages high school and community college students from around Northern Arizona in water quality laboratory exercises.

Intellectual Merit: The new laboratory curriculum, focused on water quality microbiology, links field experiences at the nearby Rio De Flag Water Reclamation Facility with current molecular and functional laboratory approaches. Students acquire treated and untreated water samples from this facility for assessment of viral abundance, fecal coliform counts and the presence of organic pollutants. Students then enrich for contaminant-degrading organisms and isolate, characterize and sequence a contaminant-degrading organism from treatment facility samples. Student teams also design, conduct, and present experiments focused on removal of coliform bacteria or pollutants using samples from the facility.

Broader Impacts: The project's target audience is in Northern Arizona and is composed of undergraduate students in Flagstaff, community college students at Yavapai Community College, Verde Valley (which serves a substantial population of Navajo and Hopi tribal members) and students at two high schools on the Navajo reservation. The undergraduate students include substantial numbers of ethnic minority students, including Native American students. The collaboration with the Rio De Flag Water Reclamation Facility strengthens ties between the university and the community and provides opportunities for undergraduates to understand the role of environmental microbiology in a critical application.

Abstract
Temperate forest ecosystems play a crucial role in the global carbon cycle. This research seeks to identify mechanisms of carbon stabilization in temperate forest ecosystems of the western United States. Metal complexation and mineral adsorption are key processes controlling soil carbon stabilization. Aluminum, in particular, has been implicated as imposing significant control over soil carbon cycling, but the underlying mechanisms of stabilization are not well known. This study combines field-based sampling of soils from temperate forest ecosystems with controlled laboratory experimentation to elucidate how carbon, aluminum, the soil mineral assemblage and microbes interact to control the biodegradation of natural organic matter in forested ecosystems of the western U.S. Part of this research includes the development and refinement of new analytical tools and approaches for characterizing carbon-mineral-microbe interactions.

This research will demonstrate and quantify the linkage between aluminum and soil carbon biogeochemistry in temperate forest ecosystems. This has significant ramifications for soil carbon cycling and its potential feedback to atmospheric CO2 levels and global climate change. In addition, data from this research will provide the information necessary to include soil mineral variables as parameters in models of regional ecosystem soil carbon dynamics, thereby enhancing estimations of regional soil carbon cycles.

This Integrative Graduate Education and Research Training (IGERT) award supports the establishment of a unique doctoral training program in Integrative Bioscience at Northern Arizona University. The purpose of this program is to provide students with a robust multidisciplinary curriculum and research training that spans disciplines from molecular genetics to ecosystem sciences to spatial and temporal modeling. All students will receive training in each of these areas, so that program graduates will have the skills to address fundamental and applied questions of how genes affect ecosystem function and response. The NAU-IGERT program will provide students with many opportunities to pursue these questions in the context of strong research programs in the College of Engineering and Natural Sciences and the School of Forestry. Students from under-represented minority populations will be actively recruited in order to build on the success of undergraduate minority student programs already in place at Northern Arizona University. Unique aspects of this program will include: 1) multidisciplinary environmental bioscience research with a special emphasis on scaling phenomena, 2) inclusion of molecular methodology and applied statistics coursework in all programs of study, 3) specially designed seminar courses covering scientific ethics, statistics and modeling, student research, and guest speakers from integrative disciplines, 4) unique internships to broaden the graduate experience and enhance connections between the research and the broader community. Specialized internship opportunities will be available working with community colleges, federal agencies, and Native American high schools. The NAU-IGERT program will prepare innovative and creative scientists to become leaders not only in research, but also in science outreach and communication and in environmental problem solving. IGERT is an NSF-wide program intended to meet the challenges of educating U.S. Ph.D. scientists and engineers with the interdisciplinary background, deep knowledge in a chosen discipline, and the technical, professional, and personal skills needed for the career demands of the future. The program is intended to catalyze a cultural change in graduate education by establishing innovative new models for graduate education and training in a fertile environment for collaborative research that transcends traditional disciplinary boundaries.

An award has been made to Northern Arizona University under the direction of Dr. George Koch to acquire an instrument for ecological analysis system for the measurement of various forms of carbon and water vapor in a variety of field sites. A mobile housing will be built to allow them to transport the instrument into locations subject to different environmental conditions. The types and amounts of carbon isotopes that they will measure allow them to test hypotheses on the way carbon accumulates in soils due to fungal and microbial processes of decomposition, to determine the source of carbon in stream food webs, and to test ideas relating to climate change over thousands of years. The investigators will integrate the instruments into their courses and research training for students. The university has a significant Hispanic and Native American student population, and the new instrumentation will be incorporated into research experiences for these students. Graduate students participate in educational activities for local schools and the general public, and the new instrumentation will enhance these outreach efforts.

Ammonia-oxidizing microorganisms in soil are of great scientific interest because their activity directly impacts agricultural yields, water quality, and global climate change. These organisms control the rate of nitrification during which ammonia is converted to nitrate. This often results in nitrogen loss from an ecosystem, because nitrate is leached much more readily than ammonium and nitrate may be converted to gaseous forms of nitrogen, including nitrous oxide, a potent greenhouse gas. Ammonia-oxidizing microorganisms are very difficult to isolate from environmental samples and consequently the environmental parameters that control their growth have not been identified. A new technique, stable isotope probing with 18O-labeled water, has been developed that does not require cultivation of ammonia oxidizers to study their growth in soil. In this new approach the DNA of only growing microorganisms is labeled with 18O from 18O-water added to soil. The 18O-labeled DNA can be separated from non-labeled DNA so that the abundance of DNA sequences unique to ammonia-oxidizers may be quantified in the labeled DNA fraction. This abundance represents the growth of ammonia oxidizers in soil. The impact of nitrogen source, temperature, plant litter, water, pH and soil bulk density, on growth of ammonia oxidizing microorganisms will be investigated in semi-arid soils in northern Arizona, where mean annual precipitation ranges from 141 to 558 mm.

This project will also encompass several important educational initiatives. Native American high school teachers will be offered opportunities to gain research experience in microbiology in order to help them develop curriculum regarding the impact of reclaimed water on the San Francisco Peaks, a sacred site to many tribes in northern Arizona. In addition, an established collaboration with the Center for Science Teaching and Learning at Northern Arizona University will be enhanced by the development of a new course in the Master of Arts in Science Teaching program. The course will focus on how microbiology can be used to teach Arizona content standards in high schools. Finally, each year an undergraduate and a graduate student at Northern Arizona University will be educated in environmental microbiology and will be funded to conduct research.

Northern Arizona University and several Arizona elementary school districts (Mesa Public Schools, Cottonwood-Oak Creek School District, and Flagstaff Unified School District) are working together to build capacity and readiness to provide research based, job-embedded professional learning for elementary teachers that develops science content knowledge, as well as effective pedagogy that meaningfully integrates science with other elementary content learning. Using the MSP-Start, the partnership is building a foundation for this transformation by planning strategies for replication and evaluation of key components of the Valle Imperial Project in Science (VIPS) in El Centro California, and Florida Atlantic University's Science IDEAS. Rather than displacing science instruction to create more time for language arts, these programs purposefully and meaningfully integrate literacy instruction with inquiry-embedded science instruction; results from studies of implementation of the two programs indicate considerable achievement gains in science and writing as well as reading and mathematics. Replication of the Valle Imperial and El Centro projects in diverse schools in Arizona thus has the potential to produce evidence that the strategies learned from these projects can have positive impact on student achievement in reading and math, while at the same time preparing students to be competent in areas of science and technology that are also important in today's global community. During this planning year, the partners are building a common vision, establishing roles and responsibilities, identifying needs, developing and finalizing the research and evaluation plans, and determining the model of implementation. A project evaluator is collecting evidence about the strength and effectiveness of partnership development and completion of planning phase goals, and providing recommendations for improvement as needed to prepare an effective full Targeted Partnership proposal.

Carbon dioxide (CO2) is released to the atmosphere when humans burn oil, coal, and gasoline, and is the major cause of global warming. Soils can store carbon (C), helping counteract rising carbon dioxide, but the future of the soil C sink is uncertain. Will it be converted to soil organic C, which can stay put for thousands of years, or will soil microorganisms convert it back to CO2, returning it to the atmosphere? This is a major uncertainty about the future C sink on land. Recent work suggests a surprising response, called the priming effect, in which adding C to soil boosts the metabolism of microorganisms, causing them to produce even more CO2 than expected. Yet, this phenomenon is variable and very poorly understood. Proposed mechanisms fail to explain what conditions modulate the occurrence and magnitude of the priming response. Preliminary data suggest that the soil mineral assemblage, reflecting the chemical and geological properties of soil, interacts strongly with the soil microbial community to influence the priming effect. This research will test the idea that the priming response depends on interactions between the soil mineral assemblage and the soil microbial community. Thus, this research lies at the interface among geology, biology, and chemistry. The research will investigate priming responses in nine soils, spanning a broad range of climatic and environmental conditions. Laboratory experiments will evaluate how priming responds to variation in the mineral assemblage, and samples from the experiments will be tested for carbon cycling and microbial community characteristics. The work will use state-of-the-art techniques, including in-line isotope-ratio measurements using a cavity ring-down instrument, and new stable isotope probing techniques paired with gene microarrays capable of identifying microorganisms performing specific ecological functions. This project emphasizes integrating research and teaching, will provide interdisciplinary training for undergraduate students at institutions with strong histories of minority enrollment. Students will gain experience with the cutting-edge methods, and with a research field with strong implications for policy decisions surrounding global climate change and carbon management.

The McMurdo Dry Valleys in Antarctica are among the coldest, driest habitats on the planet. Previous research has documented the presence of surprisingly diverse microbial communities in the soils of the Dry Valleys despite these extreme conditions. However, the degree to which these organisms are active is unknown; it is possible that much of this diversity reflects microbes that have blown into this environment that are subsequently preserved in these cold, dry conditions. This research will use modern molecular techniques to answer a fundamental question regarding these communities: which organisms are active and how do they live in such extreme conditions? The research will include manipulations to explore how changes in water, salt and carbon affect the microbial community, to address the role that these organisms play in nutrient cycling in this environment. The results of this work will provide a broader understanding of how life adapts to such extreme conditions as well as the role of dormancy in the life history of microorganisms. Results will be widely disseminated through publications as well as through presentations at national and international meetings; raw data will be made available through a high-profile web-based portal. The research will support two graduate students, two undergraduate research assistants and a postdoctoral fellow. The results will be incorporated into a webinar targeted to secondary and post-secondary educators and a complimentary hands-on class activity kit will be developed and made available to various teacher and outreach organizations.

Healthy soils are essential for maintaining productive natural and agricultural landscapes. Decomposition of dead plant, animal, and microbial materials is a fundamental process that influences ecosystem productivity, the rate of soil erosion, regional hydrology, and the exchange of greenhouse gases with the atmosphere. Because the microbial community plays a key role in decomposition and mineralization, improving the ability to study the microbial processes in soils is a major step towards increased understanding of this essential ecosystem function. The efficiency with which microorganisms process organic carbon compounds for energy production and biosynthesis, or return carbon back to the atmosphere as CO2, is an essential but poorly understood aspect of soil ecology. A change in the carbon use efficiency --- the fraction of substrate used for the production of new microbial biomass in response to increased temperatures or litter input --- will have immediate consequences for soil carbon content, CO2 production, and nutrient cycling. This project will examine changes in the fundamental processes of microbial metabolism from which carbon use efficiency, energy production, and biosynthesis can be calculated. The investigators will use a method that consists of stable isotope labeling of specific C-atoms in various microbial substrates under different conditions of temperature, carbon and nitrogen availability. This study will make use of an existing long-term climate change experiment near Flagstaff, Arizona.

The approach used in this study can be directly applied to other microbial communities, for example communities in marine and freshwater ecosystems and sediments, gastrointestinal communities, communities in environments such as hot springs, and in waste-water treatment plants. Results from this study will improve the representation of soil carbon dynamics in ecosystem models that are used to understand the role of soil processes in the global carbon cycle under current and future climates. This study will also provide opportunities for one graduate student and several undergraduate students to become familiar with state-of-the-art stable isotope techniques, molecular characterization of microbial communities, and interpretation of biochemical processes in soil ecosystems. Finally, results will be incorporated into an undergraduate ecology and ecosystem science curriculum.

Microbial communities are important regulators of organic matter decomposition and nutrient cycling in soils. In arid regions such as Arizona and New Mexico about half of the annual total precipitation occurs during the summer monsoon season when plant growth is rapid and nutrient requirements are high. This study will investigate how monsoon rains and plant growth impact the soil microbial community in these arid regions. Three separate experiments will be conducted to assess how soil microbial biota is influenced by 1) timing of the water addition, 2) degree of moisture, and 3) plant growth. Instantaneous changes in bacterial community composition are expected with watering, while a delayed response is predicted for the fungal community. In plots where moisture is withheld, changes in the structure of both bacterial and fungal communities are predicted to be less pronounced. Finally, plant removal is expected to prevent establishment of many fungal populations and some bacterial strains.

Arid land ecosystems are undergoing change with grasslands being replaced by desert shrubs. Understanding how the soil microbial community responds to seasonal rains is critical to understanding organic matter decomposition and nutrient cycling in desert ecosystems, which in turn, is important for understanding vegetation dynamics. A partnership with a local Native American-serving K-8 school will allow the researchers to bring their research into the classroom, with lessons centering on the three sisters planting scheme -- a traditional Native American companion planting of corn, beans, and squash.

Microbial diversity is vast, and recent discoveries place soils as home to the most diverse of the Earth's microbial communities. The current project will probe a surprising response of microorganisms to changes in soil carbon availability: when new carbon enters soil, especially carbon that is easily assimilated and decomposed by soil microorganisms, a chain reaction occurs leading to the breakdown of older soil carbon, carbon that would otherwise have remained stable. Current theory does not explain this chain reaction. This project will test whether taxonomic biodiversity and the genetic biodiversity it supports - in other words, who is there and what are they doing - can explain this unusual carbon cycling phenomenon. Researchers will use long-term study sites in soils spanning a climatic gradient in Arizona and a variety of molecular, genomic, chemical and analytical approaches. The work will test the idea that parts of the carbon cycle are emergent consequences of interactions among organisms, with biodiversity as a fundamental driver, thereby connecting genes to communities to ecosystems.

The proposed work addresses important questions both in biodiversity science and the global carbon cycle, and is important because soil carbon is a major reservoir, storing about three times the amount of carbon contained in the atmosphere as carbon dioxide. Microbial biodiversity is the biological template upon which much of the carbon cycle unfolds, yet evidence of how diversity alters the soil carbon cycle remains elusive. This project will address this fundamental knowledge gap, generate information applicable to carbon management, train new scientists at the undergraduate, graduate, and post-doctoral levels, and engage the public through outreach activities.

Water is produced in the cells of organisms during respiration. This project seeks to understand the importance of this water for plants and soil microorganisms. This metabolic water is potentially of vital importance when external water supply is low. The project will test the idea that metabolic water is important to plants in withstanding drought and repairing drought-induced damage to the plant water transport system. The project will also test the idea that metabolic water provides a means for soil microbes to sustain activity in dry soils. The project will employ novel techniques based on providing a tracer for metabolic water via a chemically distinct form of oxygen that allows distinguishing metabolic water from water that is not produced by metabolism. The techniques developed in this study to monitor metabolism in complex living tissues have potential applications for studying mitochondrial disorders underlying human disease and cancer detection. Undergraduate students will be active participants in the research, receiving training in microbial and plant ecology, and respiratory metabolism.

Metabolic water is produced during respiratory metabolism in animals, plants, and aerobic microorganisms. Although its vital importance is well recognized in some animals, virtually nothing is known about the physiological or ecological significance of metabolic water in microorganisms and plants. The project hypothesizes that metabolic water contributes importantly to the water balance of soil microorganisms, prolonging activity in drying soils by providing an internal source of water that slows desiccation and delays the onset of dormancy. For plants, it's hypothesized that metabolic water contributes to the recovery of water transport function in embolized vascular cells by providing a local source of water under positive pressure that contributes to refilling of embolized cells. To test these hypotheses, the project uses a suite of innovative observations, experiments, and modeling activities. A key experimental methodology involves incubations in air containing oxygen gas enriched in the stable isotope of oxygen, 18-O, resulting in 18-O enriched metabolic water that can be distinguished from environmental water by its distinctive isotopic composition. The 18-O studies are augmented with two cutting-edge imaging techniques: high-resolution x-ray computed tomography to visualize refilling of embolized vessels, and stable isotope Raman spectroscopy to locate 18-O labeled metabolic water within cells and tissues. New insights from these studies will be transformational in terms of understanding the interaction of central energy metabolism and water relations in plants and microorganisms.

Leaves that fall into streams and rivers in autumn can provide an important food resource for organisms that live in and around them. When leaves enter the water, bacteria and fungi rapidly colonize them and begin to decompose them. The leaves and microbial colonizers provide food for insect larvae that live in the water, which in turn, provide food for fish, and ultimately upon emergence, birds, bats and lizards. This study will test how leaves from different trees impact the food webs of stream and river ecosystems. The researchers will assess the fate of leaves from different tree species to determine whether the leaves are primarily converted into carbon dioxide by the microbial decomposers or whether the leaves are primarily a food resource for insect larvae. Scientists have assumed that leaves that decompose quickly are a better food source for insects and this research challenges that assumption by suggesting that leaves that decompose more slowly, because of their particular chemical composition, are a better food resource for insects. The research will also determine which microbes colonize specific leaf types and how leaf types along with their microbial colonizers affect the nutritional quality for insects that eat them. Understanding how different tree species affect the animals living in and near streams will guide managers to plant trees that are most beneficial. The researchers will also study how the temperature and water quality of streams affects how insects use leaves. This could help land managers understand if certain stream conditions make leaves a better resource for insects.  This research project will train undergraduate and graduate students and work with high school teachers and students. Results will be shared with the governmental and non-governmental organizations that are working to improve stream health.

The researchers will use multiple state-of-the-art techniques to test how different leaf species transfer energy and nutrients to microbes and insects. First, investigators will grow twelve different plant species in the green house using 13C and 15N to label the leaves that will be feed to insects. After the leaf senescence, the researchers will collect the leaves and incubate them in streams in Arizona and New Hampshire, using the stable isotopic signatures to trace the transfer of leaf carbon and nitrogen to microbes and insects. Insects that eat the leaves in the stream will be brought back to the laboratory where they will be analyzed with an isotope ratio mass spectrometer to measure exactly how much of the leaf carbon and nitrogen ends up in their tissues. Second, the researchers will use stable isotope probing and DNA sequencing to study which microbes are a good food source for insects versus which microbes compete with insects by quickly using up the carbon and nitrogen stored in leaves. Third, scientists will construct stream mesocosms to control temperature and water quality to test how stream conditions affect the nutritional quality of leaves.