Deuterium depleted water affects plant growth and microbial growth. The author conducted experiments repeating a 1950 study on the effect of varying concentrations of deuterium oxide (D2O) on tobacco seed germination. They found that germination rates decreased with increasing D2O concentration, consistent with the original study. The author also grew E. coli and yeast in different water types, including deuterium depleted water and found that growth rates varied. They used spectroscopy techniques like FTIR to analyze the water samples. The author summarized their experiments and results on open notebook science platforms online for reproducibility and collaboration.

1. Effect of Deuterium Depleted
Water on Life
REU: Alexandria Haddad
Mentor: Anthony Salvagno
Advisor: Steve Koch
http://alexhaddadnm.wordpress.com/

2. Open Notebook Science
“ Open Notebook Science is the • Wordpress.com
practice of making the entire – Great place to get started creating an
online notebook. Easy to use, short
primary record of a research project learning curve, and lots of options.
publicly available online as it is • Mindmeister.com
recorded.” – Wonderful tool for project planning
• Google Docs
• Inception • DropBox.com
• Planning
• FigShare.com
• Protocols
– Post all your data sets online
• Equipment
• Slideshare.com
• Data
• Social Media
• Conclusions
– Facebook
• Collaboration – Google +
• Reproducible – Twitter

3. Understanding how water - particularly
Hydrogen isotopes - affect biomolecular
interactions and living cells
We want to better understand how water with varying
amounts of D2O affects life forms.
At what point does D2O become toxic to life?
What is the mechanism of Deuterium/Hydrogen
exchange?
Since D2O occurs naturally, has life evolved to need
it in some amount?

4. Deuterium - Hydrogen Isotope
Common Hydrogen Water is the most abundant resource on
the planet
Vs. Deuterium (Heavy Hydrogen)
Naturally occurring water has about a
17mM (millimolar) concentration of
deuterium
Water (H2O)
Vs. “Heavy” Water (D2O)
 Hydrogen has one proton and one
electron, with an atomic mass 1
 Deuterium has one proton, one
neutron, and one electron with an
atomic mass 2

5. Repeating Crumley
In 1950 Helen A. Crumley et al
performed an experiment
testing plant seed growth in
varying amounts of deuterium
oxide (D2O). They used H2O
and 33%, 66%, and 99% D2O
mixtures with H2O. They
discovered that growth rates
were drastically slower in Influence of ordinary water, 33%, 66%, and 99%
deuterium oxide on tobacco seed germination. Counts
increasing amounts of D2O. made at daily intervals for 39 days.
(From Crumley, Fig 3)
[Original Paper:
http://iweb.tntech.edu/sstedman/JTAS%2025-3.pdf ]

6. Repeating Crumley
EXPERIMENT DETAILS
 Our research was a repeat of the Crumley,  In later experiments we added arabidopsis
et al with the following changes: (mustard) seeds.
 They also used a variety of plant species:  They placed the seeds on wet cloths, we
• Tobacco submerged our seeds in sealed analyslides
• Clover to better control the exchange of
• Radish
• Kentucky bluegrass
deuterium.
 We used two species of Tobacco seeds:  They used 100 seeds per experiment, we
• Havana used around 30 – 40 seeds (the seeds are
• Virginia Gold #1 really small and hard to count).

7. Repeating Crumley
EXPERIMENT DETAILS (CONT)
 They reported their results
in terms of percentage of
germination, but the paper
wasn’t clear how they
calculated the percentages.
Our results are a percentage
of the seeds that germinate.
 The pink box shows a
germinating seed.
typical examples, of no germination, beginning
 The orange box highlights germination, obvious germination, etc
what a non-germinating seed.
All research information… success, failures, and procedures can be found online at:
http://alexhaddadnm.wordpress.com/category/rc-repeated-cromley-experiment/
http://research.iheartanthony.com/tag/d2o-effects-on-life-2/
http://research.iheartanthony.com/category/water-type-effects-on-organism-growth/rc2/?orderby=date&order=ASC

8. D2O – Repeating Crumley (RC)
6 two-week experiments, the RCD experiment took 35 days
RC1 Five water samples: RC5 No Arabidopsis, eight
• DI, DDW water samples as previous
• 33%, 66%, and 99% D2O
RCD Six analslides of D2O
RC2 Eight water samples:
• DI control w/out seeds with tobacco seeds
• DI, DDW RCW Two species of tobacco
• 33% and 66% D2O in DI
• 33% and 66% D2O in DDW
in four purified water types
• 99% D2O (pure D2O) (eight samples):
• CHTM
RC3 Same setup as RC2 • RoDI purified
RC4 Added Arabidopsis • Sigma Molecular Biology pure
• Tissue Culture pure

9. Repeating Crumley
EXPERIMENT DETAILS (CONT)

10. Koch Lab RC Results

11. FTIR Spectroscopy
• Visually (to the naked human eye), all water looks
the same
• Spectroscopically water can be very different:
– Can we notice a difference between the amounts of
D2O in H2O (or vice versa)
– Does DDW absorb D2O naturally over time?
– Isn’t all deionized water the same?
– What about D2O… does it change over time?
• Special thanks to Dr. Sanjay Krishna and Stephen Myers who granted me access to and use of their
laboratory’s FTIR, and graciously trained me on it’s use. Stephen was also especially helpful for
spectroscopic interpretation.

12. FTIR DDW absorption of D2O

13. Gilbert Lewis
• Was the first to purify heavy water
• Was the first to report that tobacco seeds do
not grow in pure D2O
• He hypothesized that life may have evolved a
need deuterium
– No one has explored this question yet!

14. Seeds Grown in DDW
Virginia Gold seeds in DDW Dark Virginia seeds in tap water

15. E.Coli and Yeast Experiments
1. Expectations:
a. That neither organism would grow in 99% D2O
b. DDW and DI growth would be the same
2. Process:
a. Make starter cultures:
a. Make YPD and LB Broth
b. Starter yeast and e.coli colonies
c. Agar Plates and Broth

16. Growing E.Coli [Yeast]
(excerpt from my online notebook)
1. put on gloves – very important not to 5. Dispose of pipette tube in bio-hazard
contaminate myself or the medium bin.
2. get supplies: 6. Remove parafilm from agar plate.
• 10mL tube and pipette
7. Using inoculating loop, get a single
colony of e.coli [yeast] on loop.
• inoculating loop, Green 10 x 1µL 8. Put loop in test tube and swirl for a
• autoclaved test tube few seconds.
• LB broth (pre-made… this is a separate 9. Dispose of loop in bio-hazard bin.
process) [YPD broth] 10. Recover test tube.
• agar plate with e.coli – LB Day 2 batch 11. Place test tube in incubator at 37° C
[agar plate with yeast] [24° C].
3. Remove cover from LB [YPD] broth 12. Re-cover agar plate and seal with
and pipette 10mL of broth into test new parafilm.
tube. 13. Place agar plate and LB [YPD} broth
back in refrigerator.
4. Re-cover test tube and broth.

17. Initial Setup – Nanodrop Readings
We first wanted to get a spectroscopic reading of yeast and e coli grown
in common water.

18. E. Coli Growth Over 4 Hours
at 600 nm
“continuous” data
extrapolated from the 3
separate growth data sets
growth rates of 3 dilutions
of e. coli in LB broth (DI
water)

19. E. Coli Growth in Different Water
Types

20. What I’m taking with me
• Research opportunities available
– Awarded another NSF internship for the summer
• Open Notebook Science and Open Access
– This will be useful in all my future endeavors… even if I
work somewhere that is classified, I now have a solid
skill set and appreciation for keeping an electronic
notebook.
• Development of existing interpersonal skills
• Appreciation and fascination for science that isn’t
in my field of study
• Relationships with some wonderful people …

21. Acknowledgements
Thank you to everyone for providing me with the opportunity to experience
the research field. This has been a wonderful experience for me.
• NSF
• CHTM
• Dr. Marek Osinski
• Linda Bugge
• Dr. Steve Koch
• Anthony Salvagno
Images provided by:
• http://www.etftrends.com/2010/07/water-etfs-play-dwindling-natural-resource/
• http://www.isowater.com/heavy-water/
• Linda Bugge
• Anthony Salvagno

22. 2011/2012 NSF Nanotechnology REU