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, USA2 Civil Engineering, Catholic University of America, Washington, DC, USA3 Max Rubner-Institut, Food Technology and Bioprocess Engineering, Karlsruhe, Germany

2013-67019-21373

2002-2004

2003-2005

2004-2006

2005-2007

2006-2008

2007-2009

2008-2010

2009-2011

2010-2012

0

20

40

60

80

100Chesapeake Bay water quality standards

2002-2012W

ater

qua

lity s

tand

ards

(%)

1. Phosphorus and water quality

AL K H C

2. High phosphate and phytate in East Creek

Stout et al. (2016, SSSAJ)

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

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)

3.3. Phytate: Renewed interest in biochemistry & environmental chemistry

3.4. Phytate: Nomenclature

Bernie Agranoff’s turtle

OHOH

OH

OHOH

OHOH

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: Isomerso Same formula but not the same connectivity: Constitutional isomerso Same formula and same connectivity, but not the same: Stereoisomero Same formula, same connectivity, and mirror image: Enantiomer

Inositol phosphate (IPx): 63 possible configurations

Questions:

Are phytate degradation products unique for a particular enzyme?

Does an enzyme have specific degradation pathway?

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

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_12 - 060215_absorbance #98 [modif ied by anr] w heat 1h UV_VIS_13 - 060215_absorbance #100 [modif ied by anr] w heat 2h UV_VIS_14 - 060215_absorbance #101 [modif ied by anr] w heat 4h UV_VIS_15 - 060215_absorbance #102 [modif ied by anr] w heat 6h UV_VIS_16 - 060215_absorbance #103 w heat 8h UV_VIS_1AU

min

6

5

4

3

2

1

Az

PO4 I(12)P2

I(123)P3

I(126)P3

I(1234)P4

I(1346)P4I(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

b) Aspergilus niger phytase

c) Acid phosphatase from wheat d) Acid phosphatase from potato

4.3. Intermediate degradation productsa) Wheat phytase

4.4. Enzyme preference to positional PO4 moiety

(Schenk et al., 2013)

4.5. NMR identification of inositol phosphates

Turner et al. (2003)

1-D NMR (31P)

OHOH

OH

OHOH

OHOH

D-I(1,2,3,4,5,6)P6 D-I(1,2,5,6)P4

L-I(2,3,4,5)P4D-I(2)P1

L-I(2)P1

4.5. NMR identification of inositol phosphates

6

66

6

a)0 hr

b)1.0 hr

c) 2.5 hrs

d) 4.2 hrs

e)24.0 hrs

f) 48.0 hrs

5b

6

6

665b5b

Pi

4a

4a 4a

3a 3a

4b 4a4b

4a 3a 3a

3b 4a3b

3b

4a

6

66

6

a)0 hr

b)1.0 hr

c) 2.5 hrs

d)4.2 hrs

e)24.0 hrs

f) 48.0 hrs

5b

6

6

665b5b

Pi

4a

4a 4a

3a 3a

4b 4a4b

4a 3a 3a

3b 4a3b

3b

4a

5.5 5.0 4.5 4.0 3.5 3.0

Chemical shift, ppm

6

66

6

a) 0 hr

b) 1.0 hr

c) 2.5 hrs

d) 4.2 hrs

e)24.0 hrs

f) 48.0 hrs

5b

6

6

665b5b

Pi

4a

4a 4a

3a 3a

4b 4a4b

4a 3a 3a

3b 4a3b

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)

OHOH

OH

OHOH

D-I(?)P1

?

4.6. NMR identification isomer and stereoisomers

2-D NMR (1H-31P)

Murthy (2007)

4.7. Phytate degradation pathways

Sun et al. (2016, SSSAJ)

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)

Questions:

Does an enzyme have unique isotope effect during phytate degradation?

Can source and product be connected through a particular isotope effect?

m/z=28m/z=29

m/z=30

12C16O=2813C16O=2912C17O=2912C18O=30

Ag3PO4

PAg

AgP

Analyte gas

IRMS

TC/EA EA

GasBench

O

5.1. Measurement of oxygen isotopes in phosphate moieties in phytate

5.2. Isotope ratios of phosphate moieties in an inositol

All phosphate moieties in phytate have the same isotopic values

Original source of phytate can be tracked from its partially dephosphorylated products

..……….

Progressive degradation

Fractionation factors for all enzymes: positive and most often distinct

Possibility of identifying active enzyme in the environment

d18Owater =-6 to 0‰

C-O-P bond cleavageat P-O position

5.3. Bond cleavage and isotope effects

d18Owater

d18O

phos

phat

e

1:4 or 25%

d18Ophosphate

0 10 20 30 40 50

20

25

30

35

40

-20 0 20 40 60 80

10

20

30

40b)

Slope = 0.01R2= 0.62

d18O

P o

f pho

spha

te, 0 / 00

Incubation time, hrs

a)

d18Ow of water, 0/00

Slope = 0.23R2= 0.91

40 80 120 160

12

16

20

24

Slope = 0.01R2= 0.71

d18O

p of p

hosp

hate

, 0 / 00

d18OO2 of air oxygen, 0/00

Slope : 23%

Slope : ~0%

d18Ophosphate =12-18‰

d18Ophytate =18-24‰

Wu et al. (2015, SSSAJ)25%0%

Questions:

Does phytate promote proliferation of phytate degrading microorganisms?

Can anthropogenic sources of phytate be differentiated from natural sources?

LK

AEH

LKH

6.1. Dominance of phytate mineralizing bacteria

Stout et al. (2016, SSSAJ)

AL K H E

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.

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.

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

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

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

Thank you