This document summarizes research on applying phosphate isotopes to trace sources and cycling of phosphorus in East Creek, a watershed in the Chesapeake Bay region. It discusses: 1) Phosphorus and high phytate levels in East Creek. Phytate is a major storage form of phosphorus found in plant materials. 2) Measuring oxygen isotopes in phosphate to track the original source of phytate as it is degraded by enzymes. Different enzymes fractionate isotopes in unique ways, allowing identification of active enzymes. 3) Phytate promotes the proliferation of microorganisms that can degrade it. Understanding phytate cycling provides insights into managing phosphorus pollution in

1. Application of phosphate isotopes to detect sources and cycling
of phosphorus in East Creek, a Chesapeake Bay Watershed
Sources and degradation of phytate in East Creek
October 13, 2016
Deb P Jaisi1*, Mingjing Sun1, Jamal Alikhani2, Arash
Massoudieh2, and Ralf Greiner3
1 Plant and Soil Sciences, University of Delaware, Newark, DE, USA
2 Civil Engineering, Catholic University of America, Washington, DC, USA
3 Max Rubner-Institut, Food Technology and Bioprocess Engineering, Karlsruhe, Germany
2013-67019-21373

2. 0
20
40
60
80
100
Waterqualitystandards(%) Chesapeake Bay water quality standards
2002-2012
1. Phosphorus and water quality

3. AL K H C
2. High phosphate and phytate in East Creek
Stout et al. (2016, SSSAJ)

4.  Present in the outer layers of cereal grains and in
the endosperm of legumes and seed oils.
 A major storage form of P and functions as an
essential energy source for the sprouting seed.
3.1. Phytate: Sources
1
4
2
3
5
(1)
(2)
(3)
(4)
(5)
(6)
L
D
6
P
P
P
P
P
P
P Phosphate

5. i) >335 million tons manure/yr generated in the US (Mullins et al., 2005)
ii) Po in manure is dominated by phytate (Turner and Leytem, 2004), even
after phytase addition in animal diets (Pagliari and Laboski, 2012)
iii) Almost all manure ends up in agricultural soils
Source, degradation, and recycling of IPx
Higher role of phytate in Pi release from agricultural soils to open waters
3.2. Phytate: Current state of anthropogenic loading
20 years of continuous manure addition showed no significant phytate build-up (He et al., 2008)

6. 3.3. Phytate:
Renewed interest in biochemistry & environmental chemistry

7. 3.4. Phytate: Nomenclature
1
4
2
3
5
(1)
(2)
(3)
(4)
(5)
(6)
L
D
6
P
P
P
P
P
P
P Phosphate
Bernie Agranoff’s turtle
1
4
2
3
5
(1)
(2)
(3)
(4)
(5)
(6)
L
D
6
P
P
P
P
P
P
P Phosphate
1
4
2
3
5
(1)
(2)
(3)
(4)
(5)
(6)
L
D
6
P
P
P
P
P
P
P Phosphate
OH
OH
OH
OH
OH
1
4
2
3
5
(1)
(2)
(3)
(4)
(5)
(6)
L
D
6
P
P
P
P
P
P
P Phosphate
OH
OH
Ins(1,2,3,4,5,6)P6
D-I(1,2,3,4,5,6)P6
D-I(1,2,5,6)P4
L-I(2,3,4,5)P4
D-I(2)P1
o Same molecular formula: Isomers
o Same formula but not the same connectivity: Constitutional isomers
o Same formula and same connectivity, but not the same: Stereoisomer
o Same formula, same connectivity, and mirror image: Enantiomer
Inositol phosphate (IPx): 63 possible configurations

8. Questions:
 Are phytate degradation products unique for a
particular enzyme?
 Does an enzyme have specific degradation pathway?

9. 4.1. Kinetics of phytate degradation by phosphohydrolase enzymes
 Variable enzyme activity: Acid phosphatase from potato kinetics is slow
 All enzymes can remove ~5 out of 6 phosphate moieties in phytate
Substrate: i) Na-phytate (from rice)
ii) K-phytate (a synthetic product)
Enzyme: i) Wheat phytase
ii) Aspergillus niger phytate
iii) Acid phosphatase from potato
iv) Acid phosphatase from wheat germ

10. 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 34.0 36.0 38.0 40.0 42.0 44.0 46.0 48.0
-0.100
0.125
0.250
0.375
0.500
0.625
0.800
1 - 060215_absorbance #94 w heat 0h UV_VIS_1
2 - 060215_absorbance #98 [modified by anr] w heat 1h UV_VIS_1
3 - 060215_absorbance #100 [modified by anr] w heat 2h UV_VIS_1
4 - 060215_absorbance #101 [modified by anr] w heat 4h UV_VIS_1
5 - 060215_absorbance #102 [modified by anr] w heat 6h UV_VIS_1
6 - 060215_absorbance #103 w heat 8h UV_VIS_1
AU
min
6
5
4
3
2
1
Az
PO4 I(12)P2
I(123)P3
I(126)P3
I(1234)P4
I(1346)P4
I(1256)P4
I(12346)P5
I(12356)P5
I(12456)P5 I(13456)P5
I(123456)P6
 Separation performed on a Dionex DX-500 IC system
 Used CarboPac PA-100 column under a gradient acidic eluent system
 Post-column reaction with Fe [1% Fe(NO3)3∙9H2O]
 Isomers detection in UV range (at 295 nm)
 An in-house IPx reference standard prepared (Chen and Li, 2003)
 Commercial IPx standards used to identity and quantify degradation products
4.2. HPIC separation of inositol phosphates

11. b) Aspergilus niger phytase
c) Acid phosphatase from wheat d) Acid phosphatase from potato
4.3. Intermediate degradation products
a) Wheat phytase

12. 4.4. Enzyme preference to positional PO4 moiety
(Schenk et al., 2013)

13. 4.5. NMR identification of inositol phosphates
1
4
2
3
5
(1)
(2)
(3)
(4)
(5)
(6)
L
D
6
P
P
P
P
P
P
P Phosphate
Turner et al. (2003)
1-D NMR (31P)

14. 1
4
2
3
5
(1)
(2)
(3)
(4)
(5)
(6)
L
D
6
P
P
P
P
P
P
P Phosphate
1
4
2
3
5
(1)
(2)
(3)
(4)
(5)
(6)
L
D
6
P
P
P
P
P
P
P Phosphate
OH
OH
OH
OH
OH
1
4
2
3
5
(1)
(2)
(3)
(4)
(5)
(6)
L
D
6
P
P
P
P
P
P
P Phosphate
OH
OH
D-I(1,2,3,4,5,6)P6
D-I(1,2,5,6)P4
L-I(2,3,4,5)P4
D-I(2)P1
L-I(2)P1
4.5. NMR identification of inositol phosphates

15. 6
6
6
6
a)0 hr
b)1.0 hr
c) 2.5 hrs
d)4.2 hrs
e)24.0 hrs
5b
6
6
66
5b5b
Pi
4a
4a 4a
3a 3a
4b 4a
4b
4a
3a 3a
3b 4a
3b
3b
4a
6
6
6
6
a)0 hr
b)1.0 hr
c)2.5 hrs
d)4.2 hrs
e)24.0 hrs
f) 48.0hrs
5b
6
6
66
5b5b
Pi
4a
4a 4a
3a 3a
4b 4a
4b
4a
3a 3a
3b 4a
3b
3b
4a
5.5 5.0 4.5 4.0 3.5 3.0
Chemical shift, ppm
6
6
6
6
a)0 hr
b)1.0 hr
c)2.5 hrs
d)4.2 hrs
e)24.0 hrs
5b
6
6
66
5b5b
Pi
4a
4a 4a
3a 3a
4b 4a
4b
4a
3a 3a
3b 4a
3b
3b
4a
6
6: I(1,2,3,4,5,6)P6
5a: I(1,2,3,4,5)P5
5b: I(1,2,4,5,6)P5
4a: I(1,2,5,6)P4
4b: I(1,2,3,6)P4
3a: I(1,5,6)P3
2a: I(1,2)P2
1a: I(2)P1
1b: I(X) P1
4.5. NMR identification of inositol phosphates
Wu et al. (2015, SSSAJ) Sun et al. (2016, SSSAJ)
1
4
2
3
5
(1)
(2)
(3)
(4)
(5)
(6)
L
D
6
P
P
P
P
P
P
P Phosphate
OH
OH
OH
OH
OH
D-I(?)P1
?

16. 4.6. NMR identification isomer and stereoisomers
2-D NMR (1H-31P)
1
4
2
3
5
(1)
(2)
(3)
(4)
(5)
(6)
L
D
6
P
P
P
P
P
P
P Phosphate
Murthy (2007)

17. 4.7. Phytate degradation pathways
Sun et al. (2016, SSSAJ)

18. I(1,2,3,4,5,6)P6
D-I(1,2,4,5,6)P5
D-I(1,2,4,5)P4 D-I(1,2,5,6)P4
D-I(1,2,4)P3 D-I(1,2,5)P3 D-I(1,2,6)P3
D-I(1,2)P2
I(2)P1 D-I(1)P1
I(1,2,3,4,5,6)P6
D/L-I(1,2,4,5,6)P5
?
D/L-I(1,2,6)P3
(further dephosphorylation)
I(1,2,3,4,5,6)P6
I(1,3,4,5,6)P5
I(1,3,4,6)P4
D/L-I(1,4,6)P3
(further dephosphorylation,
needs to be confirmed)
I(1,2,3,4,5,6)P6
D/L-I(1,2,3,4,5)P5
D/L-I(1,2,3,4)P4 D/L-I(1,2,4,5)P4 D/L-I(1,3,4,5)P4
I(1,2,3)P3 D/L-I(1,2,6)P3 D/L-I(1,2,5)P3 D/L-I(2,4,5)P3
D/L-I(1,3,4)P3 D/L-I(1,5,6)P3
(further dephosphorylation, see major pathway)
(further dephosphorylation, needs confirmation)
I(1,2,3,4,5,6)P6
I(1,3,4,5,6)P5
D/L-I(1,3,4,5)P4 D/L-I(1,3,4,6)P4
D/L-I(1,3,4)P3 D/L-I(1,5,6)P3 D/L-I(1,4,6)P3
(furhter dephosphorylation, needs confirmation)
A) A. niger phytase
i) Major pathway
ii) Minor pathway-I iii) Minor pathway-II
B) Acid phosphatase (potato)
i) Major pathway
ii) Minor pathway
4.7. Phytate degradation pathways
Sun et al. (2016, SSSAJ)

19. Questions:
 Does an enzyme have unique isotope effect during phytate
degradation?
 Can source and product be connected through a particular
isotope effect?

20. m/z=28
m/z=29
m/z=30
12C16O=28
13C16O=29
12C17O=29
12C18O=30
Ag3PO4
Analyte gas
IRMS
TC/EA EA
GasBench
5.1. Measurement of oxygen isotopes in phosphate moieties in phytate

21. 5.2. Isotope ratios of phosphate moieties in an inositol
1
4
2
3
5
(1)
(2)
(3)
(4)
(5)
(6)
L
D
6
P
P
P
P
P
P
P Phosphate
All phosphate moieties in phytate have the same
isotopic values
Original source of phytate can be tracked from its
partially dephosphorylated products
1
4
2
3
5
(1)
(2)
(3)
(4)
(5)
(6)
L
D
6
P
P
P
P
P
P
P Phosphate
OH
OH
OH
OH
OH
..……….
Progressive degradation
Fractionation factors for all enzymes: positive
and most often distinct
Possibility of identifying active enzyme in the
environment

22. d18Owater =-6 to 0‰
C-O-P bond cleavage
at P-O position
5.3. Bond cleavage and isotope effects
d18Owater
d18Ophosphate
d18Ophosphate
0 10 20 30 40 50
20
25
30
35
40
-20 0 20 40 60 80
10
20
30
40
b)
Slope = 0.01
R2
= 0.62
d18
OP
ofphosphate,0
/00
Incubation time, hrs
a)
d18
Ow
of water, 0
/00
Slope = 0.23
R2
= 0.91
40 80 120 160
12
16
20
24
Slope = 0.01
R2
= 0.71
d18
Op
ofphosphate,0
/00
d18
OO2
of air oxygen, 0
/00
d18Ophosphate =12-18‰
d18Ophytate =18-24‰
Wu et al. (2015, SSSAJ)

23. Questions:
 Does phytate promote proliferation of phytate degrading
microorganisms?
 Can anthropogenic sources of phytate be differentiated from
natural sources?

24. L
K
A
E
H
L
K
H
6.1. Dominance of phytate mineralizing bacteria
Stout et al. (2016, SSSAJ)
AL K H E

25. AL K H E
6.2. Phytase gene expression in water and sediments
ii) b-propeller phytase (BPP) and 16S rRNA
genes
K HL E A K HL E A
Most likely
 Higher rate of phytate degradation in water
than in sediments.
 Presence of phytate promotes the proliferation
of phytate-degrading microorganisms.

26.  Complex phytate degradation pathway/s: Potential source
tracking as well as active enzyme present in the environment.
7. Conclusions
 Coupling phosphate isotopes with HPIC and NMR: Identification
of sources and intermediate degradation products.
 Constrained understanding of the sources and degradation:
address questions on i) anthropogenic and natural loading, ii)
accumulation vs degradation, and iii) impact on water quality.

27. 8. Accomplishments
 6 Invited presentations in US and China (Xiamen University, China; NIGLAS, Chinese
Academy of Sciences; American Chemical Society meeting, Cornell U; Rutgers: U
Vermont):
 1 major federal grant approved; 3 pending
 5 Major media news

28. 8. Accomplishments
Sunendra Joshi (PhD, 2016);
Currently: Postdoc at U Kentucky
Kiran Upreti (MS, 2013);
Currently: PhD student, U Louisiana
Kristi Bear (MS, 2016)
Currently: Soil scientist, USDA-ARS
Qiang Li (PhD, ongoing)
Yuge Bai (MS, 2016)
Currently: PhD student, U Tubingen
Jiying Li (postdoc, 2016)
Currently: U Toronto
Avula Balakrishna (postdoc, 2016)
Currently: Venketaswar U, India
Awet Negusse (BS, 2014)
Evan Analytical, MD
Graduate students Postdoctoral Researchers
Undergraduate student

29. 1. Wu, J., Paudel, P., Sun, M.J., Joshi, S.R., Stout, L.M., Greiner, R. and Jaisi, D.P. Mechanisms and pathways of phytate degradation:
Evidence from d18O of phosphate, HPLC, and 31P NMR spectroscopy. Soil Science Society of America Journal 79, 1615–1628.
2. Li, H. and Jaisi, D.P. An isotope labeling approach to investigate atom exchange during phosphate sorption and desorption. Soil Science
Society of America Journal. 79, 1340–1351.
3. Paudel, P., Negusse, N., and Jaisi, D.P. (2015). Birnessite catalyzed degradation of glyphosate: A mechanistic study aided by kinetics
batch studies and NMR spectroscopy. Soil Science Society of America Journal, 79, 826-837.
4. Joshi, S.R., Kukkadapu, R., Burdige, D., Bowden, M., Sparks, D.L., Jaisi, D.P. Organic matter remineralization predominates phosphorus
cycling in the mid-Bay sediments in the Chesapeake Bay. Environmental Science & Technology 49, 5887-5896.
5. Wang, D., Jin, Y., Jaisi, D. Effect of size selective retention on the co-transport of hydroxyapatite and goethite nanoparticles in saturated
porous media. Environmental Science & Technology 49, 8461–8470.
6. Stout, L.M., Nguyen, T.T and Jaisi, D.P. (2016). Relationship of phytate, phytate mineralizing bacteria, and beta-propeller genes along a
coastal tributary to the Chesapeake Bay. Soil Science Society of America Journal 80, 84–96.
7. Wang, D. , Jin, Y. and Jaisi, D.P. (2015). Effect of size selective retention on the co-transport of hydroxyapatite and goethite nanoparticles
in saturated porous media. Environmental Science & Technology, 49, 8461–8470.
8. Wang, D., Jin, Y. and Jaisi, D.P. (2015). Cotransport of hydroxyapatite nanoparticles and hematite colloids in saturated porous media:
Mechanistic insights from mathematical modeling and phosphate oxygen isotope fractionation. Journal of Contaminant Hydrology, 182,
194–209.
9. Wang, D., Xie, Y., Jaisi, D.P. and Jin, Y. Effects of low-molecular-weight organic acids on the dissolution of hydroxyapatite nanoparticles.
Environmental Science: Nano. DOI: 10.1039/c6en00085a.
10. Li, H., Joshi, S.R. and Jaisi, D.P. (2016). Degradation and isotope source tracking of glyphosate and aminomethylphosphonic acid (AMPA).
Journal of Agricultural and Food Chemistry, 64, 529–538.
11. Sun, M., Alikani, G., Massoudenieh, A, Greiner, R. and Jaisi, D.P. (2016). Phytate degradation by different phosphohydrolase enzymes:
Contrasting kinetics, decay rates, pathways, and isotope effects. Soil Science Society of America Journal (under review).
12. Jaisi, D.P., Blake, R.E., Liang, Y., and Chan, S.J. (2014). Exploration of compound-specific organic-inorganic phosphorus transformation
using stable isotope ratios in phosphate. In “Applied manure and nutrient chemistry for sustainable agriculture and environment” (Editors:
Zhongqi He and Hailin Zhang).
13. Li, W, Joshi, S.R., Hou, G., Burdige, D., Sparks, D.L., and Jaisi, D.P. Characterizing the phosphorus speciation in Chesapeake Bay
sediments using 31P NMR and X-ray absorption fine spectroscopy. Environmental Science & Technology, 49, 203-211.
8. Accomplishments

30. Thank you