Deuterium is present in all naturally occurring water, and accounts for 1 in 6420 hydrogen atoms. Seemingly insignificant, that ratio is actually quite impactful in life sciences. Pure deuterium oxide (heavy water) has been found to be toxic to eucharyotic organsims, and has been found to be inhibitory in smaller doses. Here I present results that demonstrate that in even smaller doses the concentration of deuterium has a dramatic effect on life processes. I also show that at the molecular level, deuterium has a stabilizing effect. By simply changing the properties of the solvent, there can be dramatic effects that impact a variety of experiments. For the entire story, please refer to my dissertation: https://docs.google.com/document/d/1p9-qqF8oUcwNGJjZG3CoKL2xvIhbqKCFQ7_faegNPIU/edit?usp=sharing

1. The Biophysical Effects
Of
Heavy Water
By Anthony Salvagno

2. History Effects on Life Effects on molecules

3. What is heavy water?
hydrogen deuterium
²H2O
Normal water - H2O Heavy water - D2O
Density 0.998 g/ml Density 1.101 g/ml
Freezing Point 0°C Freezing Point 3.8°C
Boiling Point 100°C Boiling Point 101.4°C
Viscosity 1.002 Viscosity 1.247
pH 7.00 pH 7.43

4. The Biophysical Effects of Heavy Water
Lewis, GN. The biochemistry of water containing hydrogren isotope (1933).

5. “It is not inconceivable that heavy hydrogen,
which exists in small amounts in all natural
water, may actually be essential to some plants
or animals.”
Gilbert N. Lewis
Lewis, GN. The biology of heavy water (1934).

6. Heavy water-life science experimentation
(1933-1970)
Simple organisms Plants Animals
E. coli Tobacco Flies
Yeast Arabidopsis Mice
P. aeruginosa Wheat Dogs
Various pathogens Peas Tadpoles
Paramecium Clover Planaria
Spirogyra Radish Fish
Various algae Kentucky Bluegrass Quail
Humans?

7. Every experiment reported the same results:
1. Pure heavy water is toxic to most organisms
2. Heavy water at lower concentrations inhibits
biological processes
But no one explored Lewis’ hypothesis...

8. ...until now

9. Helen Crumley et al. (1950)

10. 33% D2O
Day 3 Day 7 Day 11

11. What’s the difference?
DI water: 0.0155% D2O
DDW: 0.0000% D2O

12. ...popcorn break!

13. DI water: 0.0155% D2O
DDW: 0.0000% D2O
1% D2O: 1.0000% D2O

14. Tobacco Morphology
DDW DI water tap water

15. lots of seeds in DDW

16. Limitations with tobacco yield experiments with Arabidopsis thaliana
Why use Arabidopsis?
1. Everyone studies Arabidopsis.
2. Arabidopsis grows FAST!

17. Repeating Crumley with Arabidopsis

18. Root phenotypes in DDW

19. Arabidopsis growth after 7 weeks
DDW 10% D2O 60% D2O

20. Arabidopsis growth in pure D2O?

21. Plants and animals cannot
survive in pure heavy water...
...but simple organisms can!

22. Saccharomyces cerevisiae (baker’s yeast) growth in D2O
2.5
99.9% D2O
80% D2O
2.0
60% D2O
Absorbance
1.5 40% D2O
20% D2O
1.0
DI water
0.5
0 1 2 3 4 5
Hours

23. Yeast cellular morphology in D2O
DI YPD 20% D2O YPD 40% D2O YPD
25um
60% D2O YPD 80% D2O YPD 99.9% D2O YPD

24. Yeast in DI water Yeast in 99.9% D2O

25. Yeast in 99.9% D2O

26. Yeast colony morphology
250um
DI YPD 20% D2O YPD 40% D2O YPD
60% D2O YPD 80% D2O YPD 99.9% D2O YPD

27. Escherichia coli exhibits even stranger behavior...

28. E. coli growth in D2O
1.0
99.9% D2O
0.8 60% D2O
30% D2O
Absorbance
0.6
DDW
0.4
DI
0.2
0.0
0 1 2 3 4 5 6 7
Hours

29. E. coli adaptation to D2O
3.0
2.5
Absorbance
2.0
1.5
1.0
0.5
11 25 39
Days

30. Adapted E. coli growth in D2O
3.0
Proj. WT in DI
2.5 D2O cells in DDW
D2O cells in D2O
Absorbance
2.0
1.5
1.0
0.5
0 1 2 3 4 5 6
Hours

31. Adapted E. coli cellular morphology
Adapted cells in DI water Adapted cells in D2O
Normal cells in DI water Normal cells in D2O

32. Adapted E. coli cellular morphology
Normal cells in DI water Adapted cells in D2O
Normal cells in D2O Adapted cells in DI water

33. Summary of deuterium effects on life
1. New phenotypes found in very low deuterium enviornments:
a. Root hairs in tobacco
b. Earlier germination in tobacco
c. Curly roots in arabidopsis
2. Optimal deuterium concentration above normal amounts
a. Tobacco root growth
b. Arabidopsis plant growth
3. D2O adaptation produces new phenotypes
a. E. coli “brainy” colonies
b. E. coli joining
c. Faster growth

34. History Effects on Life Effects on molecules

35. Deuterium chemical effects
covalent bonding hydrogen bonding pH or pD?
DOH

36. These chemical effects can be detected physically!
mass spectroscopy nuclear magnetic resonance
(NMR)
fourier transform cavity ring-down
infrared spectroscopy spectroscopy
(FT-IR) (CRDS)

37. ...evidence of hydrogen-deuterium replacement!
machine use provided by Dr. Krishna
assistance provided by Stephen Myers and Alex Haddad

38. HD exchange
Why should we worry?
In low and high D environments, HD exchange/replacement
can affect experimental results.

39. Growth in <99% D2O

40. To quantify HD replacement, use CRDS.
picarro.com

41. Long-time HD exchange

42. You can also detect bond strength...
...indirectly.

43. Dynamic Light Scattering
Detector
Detector

44. 8 Catalase Aggregation
7
6
Intensity (Millions of Counts)
5
H2O
4
D2O
3
2
1
30 40 50 60 70 80 90
Temp ˚C
machine use provided by Dr. Osinski
assistance provided by Kenji Doering

45. YPD deterioration in D2O
DI water
D2O

46. Even DNA is affected by D2O...

47. The optical tweezers

48. Using optical tweezers to overstretch DNA
tweezer design and development by Pranav Rathi

49. Force (pN)
DNA Length (nm)
Force (pN)
DNA Length (nm)
Force (pN)
DNA Length (nm)

50. Average DNA overstretch force
D2O
75
H2O
Mean Force
Std Error
70
67.6
Force (pN)
65 65.1
60
55
DNA tethers

51. Summary of deuterium effects on molecules
1. D2O stabilizes molecules.
a. Better for single molecule and in-vitro studies
b. It could be used for long term storage of biomolecules.
2. Good probe for solvent effects.
a. Just changing the water affected DNA-DNA interations!
3. HD exchange/replacement:
a. Important to pay attention in low D experiments.
a. Good way to measure local atmospheric isotope ratios.

52. Future work
1. Low D2O concetration effects:
-Deuterium use vs. tolerance
-Lots of potential new discoveries
2. High D2O concentration effects:
-Useful to study rare events
3. Solvent effects:
-Under-utilized in studied of biomolecular interactions
4. HD replacement:
a. Expand studies with CRDS.

53. Ackowledgements
Dr. Steven Koch Dr. Abhaya Datye
-and all of KochLab -NSMS IGERT
Dr. Mary Ann Osley Dr. Maggie Werner-Washburne
-and the entire Osley Lab -IMSD
Dr. Robert Olendorf Dr. Krishna, Dr. Osinski, and
Dr. David Dunlap Dr. Sharpe
Mom and Dad
my brother Jason
All my friends!