Effect of Deuterium Depleted Water on Life

REU: Alexandria Haddad Mentor: Anthony Salvagno

Advisor: Steve Koch http://alexhaddadnm.wordpress.com/

Open Notebook Science

“ Open Notebook Science is the practice of making the entire

primary record of a research project publicly available online as it is

recorded.” • Inception • Planning • Protocols • Equipment • Data • Conclusions • Collaboration • Reproducible

• Wordpress.com – Great place to get started creating an

online notebook. Easy to use, short learning curve, and lots of options.

• Mindmeister.com – Wonderful tool for project planning

• Google Docs

• DropBox.com

• FigShare.com – Post all your data sets online

• Slideshare.com

• Social Media – Facebook

– Google +

– Twitter

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?

Deuterium - Hydrogen Isotope

Common Hydrogen Vs. Deuterium (Heavy Hydrogen)

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

Water is the most abundant resource on the planet

Naturally occurring water has about a 17mM (millimolar) concentration of deuterium

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 increasing amounts of D2O.

[Original Paper: http://iweb.tntech.edu/sstedman/JTAS%2025-3.pdf ]

Influence of ordinary water, 33%, 66%, and 99% deuterium oxide on tobacco seed germination. Counts made at daily intervals for 39 days. (From Crumley, Fig 3)

Repeating Crumley EXPERIMENT DETAILS

Our research was a repeat of the Crumley, et al with the following changes:

They also used a variety of plant species: • Tobacco • Clover • Radish • Kentucky bluegrass

We used two species of Tobacco seeds: • Havana • Virginia Gold #1

In later experiments we added arabidopsis (mustard) seeds.

They placed the seeds on wet cloths, we submerged our seeds in sealed analyslides to better control the exchange of deuterium.

They used 100 seeds per experiment, we used around 30 – 40 seeds (the seeds are really small and hard to count).

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.

The orange box highlights 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

typical examples, of no germination, beginning germination, obvious germination, etc

D2O – Repeating Crumley (RC)

RC1 Five water samples: •DI, DDW •33%, 66%, and 99% D2O

RC2 Eight water samples: •DI control w/out seeds •DI, DDW •33% and 66% D2O in DI •33% and 66% D2O in DDW •99% D2O (pure D2O)

RC3 Same setup as RC2

RC4 Added Arabidopsis

RC5 No Arabidopsis, eight water samples as previous

RCD Six analslides of D2O with tobacco seeds

RCW Two species of tobacco in four purified water types (eight samples): •CHTM •RoDI purified • Sigma Molecular Biology pure • Tissue Culture pure

6 two-week experiments, the RCD experiment took 35 days

Repeating Crumley EXPERIMENT DETAILS (CONT)

Koch Lab RC Results

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.

FTIR DDW absorption of D2O

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!

Seeds Grown in DDW

Virginia Gold seeds in DDW Dark Virginia seeds in tap water

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

Growing E.Coli [Yeast] (excerpt from my online notebook)

1. put on gloves – very important not to contaminate myself or the medium

2. get supplies:

• 10mL tube and pipette

• inoculating loop, Green 10 x 1µL

• autoclaved test tube

• LB broth (pre-made… this is a separate process) [YPD broth]

• agar plate with e.coli – LB Day 2 batch [agar plate with yeast]

3. Remove cover from LB [YPD] broth and pipette 10mL of broth into test tube.

4. Re-cover test tube and broth.

5. Dispose of pipette tube in bio-hazard bin.

6. Remove parafilm from agar plate. 7. Using inoculating loop, get a single

colony of e.coli [yeast] on loop. 8. Put loop in test tube and swirl for a

few seconds. 9. Dispose of loop in bio-hazard bin. 10. Recover test tube. 11. Place test tube in incubator at 37° C

[24° C]. 12. Re-cover agar plate and seal with

new parafilm. 13. Place agar plate and LB [YPD} broth

back in refrigerator.

Initial Setup – Nanodrop Readings

We first wanted to get a spectroscopic reading of yeast and e coli grown in common water.

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)

E. Coli Growth in Different Water Types

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 …

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

2011/2012 NSF Nanotechnology REU