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ARSH AGARWAL, ALLISON BRADFORD, KERRY CHENG, RAMITA DEWAN, ENRIQUE DISLA, ADDISON GOODLEY, NATHAN LIM, LISA LIU, LUCAS PLACE, RAEVATHI RAMADORAI, JAISHRI SHANKAR, MICHAEL WELLEN, DIANE YE, EDWARD YU MENTOR: DR. DAVE TILLEY LIBRARIAN: ROBERT KACKLEY GEMSTONE PROGRAM 03/18/2011 SWAMP Superior Wetlands Against Malicious Pollutants

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Research Problem Agricultural runoff, especially in the spring, leads to high nitrate levels in the Chesapeake Bay Watershed Causes harmful algal blooms Result: Dead zones due to depletion of oxygen and nutrients vital to aquatic wildlife Dead zone: low oxygen area of water

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Research Problem Significance of Project Affects fishing industry, seafood consumers, environmental groups, residents of the Chesapeake Bay Watershed Health of the Chesapeake Bay is vital for maintaining biodiversity

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Overview of Project Goal: to build a wetland that optimally removes nitrates from the Chesapeake Bay and its surrounding waters How? With a constructed wetland! Mostly greenhouse-based experiment in 3 phases Emulate conditions of the Tuckahoe Creek within the greenhouse Questions to answer through literature: Where does the agricultural runoff come from? What plants can we use to remove the nitrates? Can we affect the rate of nitrate removal? How? With what?

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Literature Review Agricultural Runoff One of the largest sources of pollution into the Bay Main sources: fertilizer and manure Plants only absorb up to 18% of nitrogen from fertilizer Up to 35% of nitrogen fertilizer washes into coastal waters and their surrounding bodies of water Nitrates come mostly from chicken manure in agricultural runoff Eutrophication causes harmful algal blooms Eutrophication: steep increase in nutrient concentration in neighboring bodies of water Algal blooms lead to dead zones Constructed wetlands Can remove up to 80% of inflowing nitrates

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Literature Review River Selection Big picture: Chesapeake Bay Not ideal for accessibility, too large a body of water for us to study in such a short time Choptank River largest eastern tributary of the Bay 70% of nitrogen input is from agricultural runoff Still not very accessible for a large group of students with limited funds and transportation Tuckahoe Creek Tuckahoe sub-basin represents 34% of Choptank Watershed More accessible for our team

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The Nitrogen Cycle Image from: www.fao.org

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Literature Review Plant Selection Criteria for plant selection Non-invasive Native to the Chesapeake Bay Watershed Biofuel-capable

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Literature Review Plant Selection Cattail (Typha latifolia) Very commonly researched wetland plant Especially viable as a biofuel Soft-stem Bulrush (Schoenoplectus validus) More effective at denitrification than other comparable plant species. Study: Schoenoplectus is responsible for 90% of all nitrate removal in experimental treatments Switchgrass (Panicum virgatum) One of the most common, effective nitrate-removing plants in the Chesapeake Bay area

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Literature Review Biofuels Why biofuels? To accommodate changing energy and environmental needs Secondary data analysis Cattail Potential ethanol source Can be harvested for cellulose Switchgrass One hectare plot of switchgrass yielded up to 21.0 dry megagrams of biomass Soft-stem bulrush In one study, out of 20 wetland species, soft-stem bulrush ranked second in energy output per unit mass Cross-referenced list of Chesapeake Bay native, non-invasive plants with list of biofuel-capable plants Selected plants seemed to be the best options for research

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Literature Review Organic Factors Why? Increase statistical significance of differences in nitrate removal Three carbon-based factors Glucose Increases nitrate removal rates in artificial wetlands Sawdust Study compared glucose & sawdust glucose ranked first, sawdust ranked second Wheat straw Increases nitrate removal rate for 7 days, then decreases in effectiveness

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Methodology Experimental Design & Setup Take several samples at Tuckahoe Creek Mostly in spring highest nitrate concentration Use highest value of collected samples in greenhouse environment Samples include water and soil Soil samples are necessary to inoculate the greenhouse soil Inoculating soil will allow Tuckahoe-native bacteria to grow in our greenhouse environment Extraneous variables? Realistically, we cannot emulate all elements of the Tuckahoe Creek in the greenhouse. Nitrate concentration, soil composition, & temperature are three elements that we can realistically control

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Methodology Experimental Design & Setup (Phase 1) Goal: find most effective organic factor Use single plant species (cattail) In each microcosm, place one or a combination of organic factors Each microcosm will contain potting soil, top soil, soil from the Tuckahoe Creek (for inoculation), and the experimental variable Inoculating greenhouse soil with Tuckahoe soil will allow Tuckahoe-native bacteria to grow in our greenhouse environment Collect effluent from each microcosm and pour it back over the microcosm once a day for 7 days Measure nitrate concentration of the effluent at the end of the week. Determine which factor or combination of factors per experimental unit most effectively increases nitrate uptake Experimental unit is one bucket

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Methodology Example Diagram of Setup for Phase 1 Note: Phase 2 will look similar, but with different combinations in each bucket the combinations will be of different plants, same organic factor

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Methodology Experimental Design & Setup (Phase 2) Goal: find most effective plant or combination of plants using the organic factor determined in phase 1 Use multiple plant species Place each combination in a microcosm Each microcosm will contain potting soil, top soil, soil from the Tuckahoe Creek (for inoculation), and the experimental variable Collect effluent from each microcosm and pour back over the microcosm once a day for 7 days Standard water analysis will determine water quality Determine which plant or plants (experimental unit) most effectively removes nitrates from water Experimental unit is one bucket

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Methodology Experimental Design & Setup (Phase 3) Goal: apply the results of Phases 1 & 2 to a larger, more wetland-like setting Use the best factor and best combinations of plants Place them in a larger setting (i.e. a mini constructed wetland within the greenhouse) Run experiment for 7 days, flowing water through this larger- scale wetland environment Measure effluent once a day for 7 days to determine nitrate removal efficiency Pending results of 1&2 depends on time

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Methodology Data Collection Data Collection Effluent collected every day for 7 day trial Standard water analysis Includes our variables, plus other details about water quality Mostly within greenhouse Some data collection in the field (Tuckahoe) for samples and testing of environment Six 1-week long trials 7 replicates of each microcosm per trial Total of 42 data points (can assume normal distribution)

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Methodology - Data Analysis Data Analysis Phase 1: Two-factor ANOVA 2 levels 4 treatments Phase 2: Single factor ANOVA, Tukeys Studentized Range 1 level 8 treatments Statistical Analysis Software (SAS) to perform calculations

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Current Progress Finishing experimental setup and design Ironing out the fine details of water collection/measurement/etc Applying for grants Bill James, ACCIAC, Library (submitted), Sea Grant, HHMI Ongoing literature review Tuckahoe Creek visits Soil samples: early March Water samples: late April/early May This is when nitrate concentration is highest Greenhouse space Guaranteed space in the UMD greenhouse until May 2012

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References Anderson, D., & Glibert, P., & Burkholder J. (2002). Harmful algal blooms and eutrophication: Nutrient sources, composition, and consequences. Coastal and Estuarine Research Federation, 24(4), 704-726. Burgin, A., Groffman, P., & Lewis, D. (2010). Factors regulating denitrification in a riparian wetland. Soil Sci. Soc. Am. J., 74(5), 1826-1833. doi: 10.2136/sssaj2009.0463 Fraser, L. H., Carty, S. M., & Steer, D. (2004). A test of four plant species to reduce total nitrogen and total phosphorus from soil leachate in subsurface wetland microcosms. Bioresource Technology, 94(2), 185-192. Hien, T. (2010). Influence of different substrates in wetland soils on denitrification. Water, Air, and Soil Pollution, June 2010, 1-12. doi:10.1007/s11270-010-0498-6 Gray, K. & Serivedhin, T. (2006). Factors affecting denitrification rates in experimental wetlands: Field and laboratory studies. Ecological Engineering, 26, 167-181. Ines, M., Soares, M., & Abeliovich, A. (1998). Wheat straw as substrate for water denitrification. Water Research. 32(12), 3790-3794. Karrh, R., Romano, W., Raves-Golden, R., Tango, P., Garrison, S., Michael, B., Karrh, L. (2007). Maryland tributary strategy Choptank River basin summary report for 1985-2005 Data. Annapolis, MD: Maryland Department of Natural Resources. Rogers, K., Breen, P., & Chick, A. (1991). Nitrogen removal in experimental wetland treatment systems: Evidence for the role of aquatic plants. Research Journal of the Water Pollution Control Federation, 63(7), 9. Staver, L. W., Staver, K. W., & Stevenson, J. C. (1996). Nutrient inputs to the Choptank river estuary: Implications for watershed management. Estuaries, 19(2), 342-358. Wright, L., & Turhollow, A. (2010). Switchgrass selection as a model bioenergy crop: A history of the process. Biomass and Bioenergy, 34(6), 851-868. doi:10.1016/j.biombioe.2010.01.030 Zedler, J. B. (2003). Wetlands at your service: reducing impacts of agriculture at the watershed scale. Frontiers in Ecology and the Environment, 1(2), 65-72. Zhang, B., Shahbazi, A., Wang, L., Diallo, O., & Whitmore, A. (2010). Hot-water pretreatment of cattails for extraction of cellulose. Journal of Industrial Microbiology & Biotechnology, 1-6. doi: 10.1007/s10295-010-0847-x

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Thank you! Many thanks to... Dr. Dave Tilley Dr. Bruce James Brandon Winfrey Dr. Wallace Dr. Thomas Courtenay Barrett Gemstone Program & Staff Robert Kackley

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Any questions?