1. A method for measuring phosphorus nutrient limitation using the oxygen isotopic composition of phosphates Joseph Murray Arizona State University Acknowledgements: Dr. Ariel Anbar Dr. Achim Herrmann Dr. James Elser Arizona Space Grant Symposium April 19 th, 2008

2.  The goal of this project is to develop a new method for detecting nutrient limitation in natural settings  The project is exploring the use of oxygen isotopic signatures in phosphates as a way of measuring phosphorus limitation  Initial research will be on application in freshwater ecology, but future applications have exciting potential

3. P-Limitation: Why does it matter? Phosphorus is a strongly limiting nutrient to primary production in all kinds of ecosystems, including freshwater, terrestrial and marine environments. Phosphorus is also often a limiting component in the process of nitrogen fixation As a limiting nutrient for organisms at the bottom of the food chain, phosphorus helps control the vigor of life in ecosystems all around the planet. Phosphorus is an important nutrient to all forms of life on Earth.

4. Why a new method? Current P-limitation Studies New, oxygen isotope method Growth experiments Involve maintaining living organisms Experiments may last several days to weeks Can take a significant amount of time and work to complete Smaller sample size necessary No need to maintain living samples Faster processing time Less field work required

5. Why Oxygen Isotopes? Alkaline phosphatase enzymeAlkaline phosphatase enzyme Extracellular enzyme that catalyzes the regeneration of P i from P orgExtracellular enzyme that catalyzes the regeneration of P i from P org Equilibrates oxygen isotopic composition of water with that of P iEquilibrates oxygen isotopic composition of water with that of P i Y. Liang, R.E. Blake, 2006. Isotope signature of P i regeneration from P org by monoesterase and UVR PO 4 – H 2 O exchange catalyzed by cell free alkaline phosphatase H 2 18 O + PO 3 16 O  H 2 16 O + PO 3 18 O

6. Organisms often increase production of alkaline phosphatase under P-limiting conditionsOrganisms often increase production of alkaline phosphatase under P-limiting conditions Expected oxygen isotopic composition differences:Expected oxygen isotopic composition differences: P-Limited environmentP-Limited environment  Higher concentration of extracellular alkaline phosphatase  High P i turnover rate  δ 18 O P should be equilibrated with δ 18 O W P-Sufficient environmentP-Sufficient environment  Lower concentration of extracellular alkaline phosphatase  Low P i turnover rate  Would not expect to see δ 18 O P - δ 18 O W equilibration Plan: measure phosphate oxygen isotopic composition of P-limited and P-sufficient organisms to check for this proposed isotopic differencePlan: measure phosphate oxygen isotopic composition of P-limited and P-sufficient organisms to check for this proposed isotopic difference

7. Organic Material Scenedesmus algaeScenedesmus algae P-limitedP-limited P-sufficientP-sufficient Daphnia, fedDaphnia, fed P-limited ScenedesmusP-limited Scenedesmus P-sufficient ScenedesmusP-sufficient Scenedesmus

8. Phosphate extraction and purification Digestion of organic material by UV radiation Precipitation of cerium phosphate using cerium nitrate. Centrifuge. HNO 3 Ion Exchange Chromatography: AG-50 x8 cation exchange resin. HNO 3 Precipitate as silver phosphate using Silver Amine solution.20 M AgNO 3.35 M NH 4 NO 3.74 M NH 4 OH To TC/EA and mass spectrometer Next Slide Ultraviolet Radiation Lamp Indicates procedural steps requiring more investigation

9. Mass Spectroscopy High Temperature Conversion Elemental Analyzer (TC/EA) Ag 3 PO 4 Mass Spectrometer CO Gas Data as 30 CO / 28 CO ratio Measured against reference CO gas, which is normalized against international benzoic acid standards and against internal laboratory standards to ensure accurate data that can be compared to results in other labs around the world. Pyrolysis

10. Preliminary and Expected Results Fractionation observed in both nitrogen and carbon, as well as differing amounts of C, N and P; clearly there are biogeochemical changes occurring. C:N Ratio C:P Ratio δ 15 N δ 13 C Low P Algae 11.31 ≈ 1000 -1.62-12.75 High P Algae 7.19 ≈ 130 -0.75-15.68 δ 18 O -10 K 2 HPO 4 (PO 4 3- source) 0 10 20 18.68 x WaterP-limitedP-sufficient x x x x : predicted x : measured

11. Possible Future Implications  Current P-limitation studies  Analysis of the ecological effects of anthropogenic phosphorus pollution during the past century  Reconstruction of nutrient history throughout longer periods of time

12. Thank You to...  ASU/NASA Space Grant Program  Dr. Achim Herrmann of Barrett, the Honors College  Dr. Ariel Anbar and Dr. Gwyneth Gordon of the W.M. Keck Foundation Laboratory for Environmental Biogeochemistry  Dr. James Elser and Marcia Kyle of the ASU School of Life Sciences  ASU School of Life Sciences Undergraduate Research Program THE END