Joseph Murray Arizona State University

A method for measuringphosphorus nutrient limitation using the oxygen isotopic composition ofphosphates

Joseph MurrayJoseph MurrayArizona State University

Acknowledgements:Acknowledgements:Dr. Ariel AnbarDr. Ariel AnbarDr. Achim HerrmannDr. Achim HerrmannDr. James ElserDr. James Elser

Arizona Space Grant Symposium

April 19th, 2008

The goal of this project is to develop a The goal of this project is to develop a new method for detecting nutrient new method for detecting nutrient limitation in natural settingslimitation in natural settings

The project is exploring the use of The project is exploring the use of oxygen isotopic signatures in phosphates oxygen isotopic signatures in phosphates as a way of measuring phosphorus as a way of measuring phosphorus limitationlimitation

Initial research will be on application in Initial research will be on application in freshwater ecology, but future freshwater ecology, but future applications have exciting potentialapplications have exciting potential

P-Limitation: Why does it matter?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.

Why a new method?Why a new method?

Current P-limitation StudiesCurrent P-limitation Studies New, oxygen isotope methodNew, 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

Why Oxygen Isotopes?Why Oxygen Isotopes?• Alkaline phosphatase enzymeAlkaline phosphatase enzyme

• Extracellular enzyme that catalyzes the regeneration of PExtracellular enzyme that catalyzes the regeneration of P i i

from Pfrom Porgorg

• Equilibrates oxygen isotopic composition of water with Equilibrates oxygen isotopic composition of water with that of Pthat of Pii

Y. Liang, R.E. Blake, 2006. Isotope signature of Pi regeneration from Porg by monoesterase and UVR

PO4 – H2O exchange catalyzed by cell free alkaline phosphatase

HH221818O + POO + PO33

1616O O H H221616O + POO + PO33

1818OO

• Organisms often increase production of alkaline Organisms often increase production of alkaline phosphatase under P-limiting conditionsphosphatase under P-limiting conditions

• Expected oxygen isotopic composition differences:Expected oxygen isotopic composition differences:• P-Limited environmentP-Limited environment

Higher concentration of extracellular alkaline phosphataseHigher concentration of extracellular alkaline phosphatase High PHigh Pi i turnover rateturnover rate

δδ1818OOPP should be equilibrated with should be equilibrated with δδ1818OOWW

• P-Sufficient environmentP-Sufficient environment Lower concentration of extracellular alkaline phosphataseLower concentration of extracellular alkaline phosphatase Low PLow Pi i turnover rateturnover rate

Would not expect to see Would not expect to see δδ1818OOPP - - δδ1818OOW W equilibration equilibration

• Plan: measure phosphate oxygen isotopic composition Plan: measure phosphate oxygen isotopic composition of P-limited and P-sufficient organisms to check for this of P-limited and P-sufficient organisms to check for this proposed isotopic differenceproposed isotopic difference

Organic MaterialOrganic Material

• ScenedesmusScenedesmus algae algae• P-limitedP-limited• P-sufficientP-sufficient

• DaphniaDaphnia, , fedfed

• P-limited P-limited ScenedesmusScenedesmus• P-sufficient P-sufficient ScenedesmusScenedesmus

Phosphate extraction and purificationPhosphate extraction and purification

Digestion of organic material by UV radiation

Precipitation of cerium phosphate using cerium nitrate. Centrifuge.

HNO3

Ion Exchange Chromatography: AG-50 x8 cation exchange resin.

HNO3

Precipitate as silver phosphate using Silver Amine solution

.20 M AgNO3

.35 M NH4NO3

.74 M NH4OH

To TC/EA and mass spectrometer

Next Slide

Ultraviolet Radiation Lamp

Indicates procedural steps requiring more investigation

Mass SpectroscopyMass Spectroscopy

High Temperature High Temperature Conversion Elemental Conversion Elemental

Analyzer (TC/EA)Analyzer (TC/EA)

AgAg33POPO44

Mass SpectrometerMass SpectrometerCO GasCO Gas

•Data as 30CO / 28CO 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.

PyrolysisPyrolysis

Preliminary and Expected ResultsPreliminary 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 RatioC:N Ratio C:P RatioC:P Ratio δδ1515 N N δδ1313 C C

Low P AlgaeLow P Algae 11.3111.31 ≈ ≈ 10001000 -1.62-1.62 -12.75-12.75

High P AlgaeHigh P Algae 7.197.19 ≈ ≈ 130130 -0.75-0.75 -15.68-15.68

δδ181

8 O O

-10K2HPO4

(PO43- source)

0

10

2018.68

x

Water P-limited P-sufficient

xx

xx: predicted

x: measured

Possible Future ImplicationsPossible Future Implications

Current P-limitation studiesCurrent P-limitation studies Analysis of the ecological effects Analysis of the ecological effects

of anthropogenic phosphorus of anthropogenic phosphorus pollution during the past centurypollution during the past century

Reconstruction of nutrient history Reconstruction of nutrient history throughout longer periods of timethroughout longer periods of time

Thank You to...Thank You to...

ASU/NASA Space Grant ProgramASU/NASA Space Grant Program Dr. Achim Herrmann of Barrett, the Honors Dr. Achim Herrmann of Barrett, the Honors

CollegeCollege Dr. Ariel Anbar and Dr. Gwyneth Gordon of the Dr. Ariel Anbar and Dr. Gwyneth Gordon of the

W.M. Keck Foundation Laboratory for W.M. Keck Foundation Laboratory for Environmental BiogeochemistryEnvironmental Biogeochemistry

Dr. James Elser and Marcia Kyle of the ASU Dr. James Elser and Marcia Kyle of the ASU School of Life SciencesSchool of Life Sciences

ASU School of Life Sciences Undergraduate ASU School of Life Sciences Undergraduate Research ProgramResearch Program

THE ENDTHE END