AAAS/Merck Undergraduate Science Research Program

Colorado State University - Pueblo

Projects

The following projects are available for the AAAS/Merck Summer Program

Project #1    Project #2    Project #3    Project #4    Project #5    Refs

  1. Project #1

    Human Pharmaceuticals Uptake in Crop Plants. Drs Vanden Heuvel (Biology) and Kinney (Chemistry). Biosolids are the solid or semisolid product of municipal wastewater treatment that can then be land applied as an organic rich source of plant nutrients if it meets certain U.S. Environmental Protection Agency (USEPA) regulations. In the United States the USEPA estimates that more than 8 million dry tons of sewage sludge is generated annually with about 50% land applied as biosolid1. Biosolids are known to contain a variety of anthropogenic organic chemicals, including human pharmaceuticals2. In recent years scientists have identified that some common veterinary pharmaceuticals used in livestock production are taken up by crops grown in manure amended soil3,4. In light of such reports and the anthropogenic organic chemical content of biosolids, it is highly warranted to investigate the uptake of these chemicals by crops intended for human consumption grown in biosolid amended soil. Merck/AAAS Scholars will participate in activities to meet the breadth of objectives of this multiyear project, which include:  (1) to optimize a robust analytical method to extract and quantify human pharmaceuticals in select crop plants, using accelerated solvent extraction and liquid chromatography-mass spectrometry, (2) apply the optimized method for the analysis of crops grown hydroponically in a solution fortified with the study pharmaceuticals and in biosolid amended soil, (3) to analyze plant tissues, including the xylem and phloem sap, of the hydroponically and soil grown crops to better understand the mechanism of plant uptake, transport, and storage of the pharmaceuticals, and (4) to place hydroponically and soil grown crops under different nutritional stresses (low nitrogen, low phosphorus, etc.) to examine if the ecological situation plays a role in uptake of pharmaceuticals. back to top


  2. Project #2

    Use of Chemostat Selection for Isolation of Microbes That Carry Out Biocatalysis on Biofuel Waste Streams to Develop Value-Added Products. Drs. Dillon (Chemistry) and D. Caprioglio (Biology).  The commercial viability of a number of biofuels is based, in part, on the revenue from the products of the biofuels plants.  While the actual biofuels is the main product of the plant, the waste stream is usually of low value products such as low grade feed, fertilizer, or burned for energy.  Yet, the waste stream is usually high in a number of organic compounds and these could theoretically be converted to higher value products including chiral materials using low cost microbial biocatalysis5.  The Bio Sun plant in Walsh, Colorado uses Sorghum (important due to the dry conditions in the western US) as the feedstock to produce ethanol.  There is currently no literature on waste stream utilization from this type of feedstock.  This project requests one Merck/AAAS Scholar to work on the isolation of microorganisms that have biocatalytic activity to biofuels waste streams using chemostat (continuous culture) selection.  The products of the biocatalysis of the mixed culture chemostat and isolated individual microbial strains will be separated by physical methods including extraction and gas- or liquid-chromatography and individual components analyzed by chemical techniques including infrared spectrophotometry, NMR, and mass spectral analysis6,7.  Further strain improvements may also be attempted.  The Merck/AAAS Scholar will develop skills in Microbiology, Microbial Physiology, Organic Chemistry, and Analysis.     back to top


  3. Project #3

    Optimization of Cellulase Production of Penicillium fellutanum. Drs. Bonetti (chemistry) and D. Caprioglio (Biology).  Cellulose is the largest source of feedstock for Bioethanol production but, unlike starch (from corn) or sugar (from molasses), the cellulose is not easily available for fermentation.  There is a great deal of interest in development of cheap and highly effective sources of cellulases that could degrade cellulose feedstock for fermentation to ethanol.  In previous work by Dr. Sandra Bonetti’s group, a high activity extracellular cellulase produced by Penicillium fellutanum has been isolated.  This cellulase is most active at 50oC which is of high importance in the pretreatment of the cellulose feedstock.  Preliminary studies indicate that the carbon source has a strong effect on the production of the enzyme.

    The proposed project requests one Merck/AAAS Scholar to optimize the culture conditions for the highest production of the cellulase.  Using a chemostat (continuous culture system), the Merck/AAAS Scholar will be able to vary the culture conditions periodically (allowing the system to equilibrate) and obtaining samples for cellulase activity.  The Scholar will manipulate the system using changing culture conditions such as dissolved oxygen and temperature as well as media composition including carbon source and trace nutrients.  The samples taken at different timepoints during the equilibration will be analyzed for cellulase activity per unit volume. The Merck/AAAS Scholar will receive training in basic microbiology as well as continuous culture of microbes.  The Scholar will also receive training in biochemistry and enzyme analysis.  The Merck/AAAS Scholar will also be involved in strain improvements after optimization of culture conditions.  This would be done by using cellulose as a sole energy source for the chemostat to select for mutants with higher levels of cellulase by the dilution pressure of the chemostat.        back to top


  4. Project #4

    Elucidation of gene expression changes in Frankia after exposure to flavonoids isolated from host plant root exudates. Drs. Vanden Heuvel (Biology) and Lehmpuhl (Chemistry). Frankia, a filamentous nitrogen-fixing soil bacteria (actinomycete) forms a mutualistic actinorhizal relationship with select woody plants. Frankia provide their hosts with a source of fixed nitrogen in root nodules; the host, in turn, provides Frankia with fixed carbon8. Actinorhizal plants rival legumes in the amount of nitrogen they fix on a global scale, yet knowledge of the specific mechanisms that create the symbiotic association between Frankia and actinorhizal plants is limited9. Within the better understood Rhizobium-legume symbiosis, flavonoids have shown to be critical in plant-microbe communication. Although surveys of the Frankia genomes have not found homologous Rhizobium symbiotic genes that respond to flavonoids, actinorhizal plants have shown increased expression of genes involved in polyphenol and flavonoid biosynthesis and metabolism8.

    The Merck/AAAS Scholar in this project will attempt to isolate and identify flavonoids produced by Russian olive (Elaeagnus angustifolia) roots and investigate the how the isolated flavonoids effect gene expression in Frankia. Specifically we will: 1) Isolate flavonoids from root exudates of Russian olive using liquid chromatography and mass spectrometry 2) Expose the Frankia culture Ean1pac (isolated from Russian olive) to the compounds isolated in step 1; and 3) Explore changes in gene expression of coding regions previously identified for their putative role in nodulation using real time PCR (RT-PCR). This study will be the first to attempt to isolate specific components of the root exudates and test whether these compounds change gene expression in Frankia strains. It may serve as an example of a research strategy for testing host-species specificity in Frankia, and shed light on the biochemical processes which allow this mutualism to take place.    back to top

  5. Project #5

    Synthesis and Application of Copper Selective Fluorescent Indicators as Sensors in Bioimaging Studies of Disease Mechanisms. Drs. Smith (Biology) and Druelinger (Chemistry).  The transport, distribution, and homeostasis of copper ions is central to maintaining healthy cellular physiology in a number of body systems such as the liver and brain and it is central in the etiology of a diversity of disorders ranging from cancer to Alzheimer’s disease10.  Despite the wide biological importance of copper fluorescent indicator dyes for imaging the dynamics of the distribution and concentration of this ion, materials analogous to those commercially available for measuring Ca2+, Mg2+, and Zn2+(11) are lacking. Thus the very basic characterization of copper transporter (CTR) kinetics is under-reported.   We propose the synthesis and molecular modification of membrane permeable copper selective fluorescent indicators to image the kinetics of CTR function in living cultured neurons, cerebrovascular endothelial cells, and rodent brain slices.  The Merck/AAAS scholar will be involved in the design and synthesis of target molecules (heterocyclic crown ethers)12,13 serving as fluorescent copper sensors.  Experience will include isolation and purification techniques (chromatography) and spectroscopic characterizations (NMR, MS, UV-VIS).  The student will also develop skills in mammalian cell culture, vivisection of neural tissues, cellular physiology, neuroscience research, fluorescence microscopy, and advanced video microscopy with computerized image analysis. By developing basic tools to examine the kinetics of copper transport with these models we will build a basis to develop more in depth synthesis and biological projects.    back to top

    References

    1.      USEPA, EPA 832-R-06-005, 2006.

    2.      C.A. Kinney et al., Environ. Sci. Technol., 2006, 40, 7207-7215.

    3.      A.B.A Boxall et al., J. Agric. Food Chem., 2006, 54, 2288-2297.

    4.      K. Kumar et al., J. Environ. Qual., 2005, 34, 2082-2085.

    1. Yadzani, S. and Gonzales, R.  (2007) Curr. Opin. Biotechnology 18 213-219
    2. Geladi, P.; Lillhonga, T.; Teuter, L. (2005) Prog. Chemometrics Res. 193-208.
    3. Masohan, A.; Bhatia, V. K.; Tondon, M. (1996) J. Chrom. Sci. 34(12), 550-555.

    8.      Baker, D.D. and C.R. Schwintzer. Introduction. In The Biology of Frankia and Actinorhizal Plants. C.R Schwintzer and J.D. Tjepkema (eds.) Academic Press, New York. 1990. 1-18.

     

    9.      Hocher V et al., New Phytologist, 2006,169, 681–688.

    1. J.Y. Uriu-Adams, C.L. Keen, Mol. Aspects Med. 2006, 26 (4-5), 268-298.
    2. C.R. Woodroofe, R. Masalha, K.R. Barnes, C. J. Frederickson, and S.J. Lippard, Chemistry and Biology, 2004, 11, 1659-1666.
    3. L. Yang, R. McRae, M. Henary, R. Patel, B. Lai, S. Vogt, and C. Fahrni, Proc. of the Natl. Acad. Sciences, 2005, 102, 11179-11184.
    4. L. Zeng, E. Miller, C. Chang, A. Pralle, and E. Isacoff, J. Am. Chem. Soc., 2006, 128(1), 10-16.        back to top