KATHARINE MORLEY

she/her | age 16 | Victoria, BC

Bronze Excellence Award, CWSF | Sixth Overall, Vancouver Island Regional Science Fair

Edited by Simran Bhaskar

INTRODUCTION

Due to climate change, a copious amount of record-breaking heat temperatures have been recorded around the world, including Canada. Approximately 55 million people around the world are impacted each year by droughts (World Health Organization, n.d.), which have increased in frequency by nearly one third since 2000 (Rakotoarivony, 2022). As a result of these conditions, communities across the globe have experienced the detrimental effects of drought – this includes food insecurity, which is caused by agricultural loss. In particular, British Columbian farmers have been severely impacted in recent years (Miljure, 2022), which is why I was inspired to investigate techniques to mitigate the effects of drought, with a focus on agriculture.

I was intrigued by a study titled “Ethanol-Mediated Novel Survival Strategy against Drought Stress in Plants” published in Plant and Cell Physiology in late August of 2022 which suggested that ethanol, an alcohol made from biomass, could aid plants in drought resistance (Bashir et al., 2022) - that's right, the solution to food scarcity could come from your wine glass! When plants are faced with drought, their stomata (tiny pores on the outermost layer of a plant’s leaves) close up in order to prevent water from evaporating (Farmer, 2016). However, in this state, plants may not be able to absorb enough CO₂ to perform photosynthesis, a process in which plants convert CO₂, sunlight, and water into oxygen and energy in the form of sugar (Oregon State University, 2008). This can account for why plants naturally produce small amounts of the organic compound ethanol (C₂H₆O) to combat the diminished rate of photosynthesis they exhibit when under water stress. Additionally, this could explain how ethanol reportedly enhances drought tolerance; it helps the plant to produce necessary sugars for energy even while the stomata are closed through the process of alcoholic fermentation (this synthetic pathway is not fully understood by researchers, though) (Tadege et al., 2022). Ethanol also helps to increase sensitivity of drought tolerance genes, which are responsible for making the proteins needed to withstand drought (Nguyen et al., 2017). By augmenting the rate of photosynthesis, it is hypothesized that plants are able to better survive drought conditions than those treated with only water.

The main objective of my experiment was to test how effective ethanol truly is on plant growth under drought conditions. Specifically, I explored how fertilizer could be added to ethanol solutions to maximize their efficacy in affecting plant growth.

METHODS & MATERIALS

The main materials I used included: radish seeds, a grow chamber, liquid fertilizer, denatured ethanol, water, a syringe, a micropipette, and a graduated cylinder. For testing I used a variety of radish called Cherry Belle, a moist soil and cool weather-loving vegetable that can be deleteriously affected by drought (Back to the Roots, n.d.). They were obtained as seeds and cultivated in an Urban Cultivator grow chamber in standard potting soil. The seeds experienced a lighting cycle of 12 hours of light and dark and were situated roughly 1.5 inches apart from each other. An average temperature of 19℃ was maintained, and seeds were watered daily during the pre-testing growth period of 21 days until plants were approximately three-quarters of the way to maturity.

A total of three different ethanol solutions were created. Ethanol was measured with a micropipette while fertilizer was measured with a 10.0 ± 0.1 mL syringe. All solutions were diluted with distilled water in a 1.00 ± 0.05 L graduated cylinder. These were: 0.03% ethanol, 0.6% ProMix liquid fertilizer + 0.03% ethanol, and 0.6% ProMix liquid fertilizer + 0.06% ethanol.

Fertilizer treatment was prepared and applied as follows. Four trays of approximately 15 radish plants were treated with one of the ethanol solutions or water (baseline treatment). For three days, the regular watering system of the grow chamber was halted to allow application of solutions. Next, all substances were suspended for four days; to simulate drought conditions, the seedlings received nothing other than the consistent light and temperature conditions of the chamber. After this, data was recorded.

To test the efficacy of the fertilizers, I used the method of photosynthetic flotation (Exploratorium, n.d.), which measures the rate of photosynthesis of a plant. Seeing as research indicates that ethanol boosts photosynthesis, I can determine the most effective solution based on which group of plants has the highest rate of photosynthesis after treatment (essentially, which plants are most alive). First, I created 10 leaf discs of each treatment type by punching holes in the leaves of the radish seedlings and inserted them into a needleless syringe. After drawing some buffer solution into the syringe (0.2% baking soda solution with dish soap) a vacuum was applied by plugging the opening with my fingertip and pulling the plunger for 10 seconds. I released the plunger abruptly 2-3 times so that gasses could be fully extracted from the leaves. This caused the discs to sink to the bottom of the syringe. The syringe was lightly tapped to expel excess air, and the leaf discs were carefully poured into a clear cup of 150 mL of the buffer solution. The cup was placed approximately 20 cm underneath an LED light and remained there until a minimum of three discs (out of the 10 discs total for each treatment) had floated to the surface of the solution. Each disc was timed and recorded. The faster they reach the surface, the more efficient the rate of photosynthesis is from quicker oxygen production.

RESULTS

No notable visible difference could be ascertained in the appearance of the seedlings after drought conditions had been imposed. The photosynthetic flotation results, however, indicated otherwise.

Fertilizer and 0.03% Ethanol
The first three discs of this solution all reached the surface in well under 6 minutes. An average surfacing time of 213.7 seconds established the highest rate of photosynthesis of the solutions tested.

Water and 0.03% Ethanol
This solution had an average surfacing time of 470 seconds, and all three discs reached the surface within a span of 12 minutes.

Fertilizer and 0.06% Ethanol

This treatment caused the three discs to surface up to 23 minutes in total, with an average surfacing time of 1082.7 seconds.

DISCUSSION

The efficacy of the ethanol solutions ranged from highly successful to less than ideal, as demonstrated by the results.

Fertilizer and 0.03% Ethanol
Out of the three concentrations tested (as well as the baseline: water), the solution consisting of fertilizer and 0.03% ethanol was the most effective at enhancing drought resistance in radishes. It proved to be 77.8% more effective than the water baseline, which had an average surfacing time of 958 seconds. It can also be noted that most of the other 10 leaf discs used in the photosynthetic flotation process for this treatment also surfaced in close proximity to the first three that were recorded for data (in comparison to some of the other concentrations where no more than these three discs even surfaced).

Water and 0.03% Ethanol
The second most effective solution was the one containing water and 0.03% ethanol. It was 50.9% quicker than the plain water treatment. From this result, it can be deduced that the addition of liquid fertilizer in the first solution tested significantly impacted the efficacy, as seen by the 54.5% difference between the two performances.

Fertilizer and 0.06% Ethanol
Both of the 0.03% ethanol solutions (with and without fertilizer) were considerably more effective than the highest concentration of ethanol: 0.06% with fertilizer. This treatment was actually 11.5 % less effective than the water baseline. It could therefore be hypothesized that the higher ethanol concentration caused this decrease, as we have already seen that the use of fertilizer increases efficacy (and it was the only aspect that is different from the other fertilizer solution).

The favorable rate of effectiveness shared by both solutions with the lower concentration of ethanol may suggest a threshold in the amount of ethanol used to maximize efficacy. It is possible that the consistency in efficiency of results of lower quantities of ethanol could reveal a low dosage capability - 0.03% ethanol was approximately 5 times more effective than 0.06% ethanol, with only half the amount!

CONCLUSION

It is clear through these results that ethanol significantly boosted the survival of plants under drought-like conditions as suspected. The solution with fertilizer and 0.03% ethanol demonstrated the highest capacity in improving drought tolerance. It was approximately 5 times more effective than the solution with double the quantity of ethanol (0.06%)! This may indicate a high efficiency capability. This means that lower dosages of the substance may provide just as (or more) significant of an effect as higher dosages - i.e., if used in the real world, only small quantities of ethanol would be required to have the intended efficacy. The impressive rate of effectiveness shared by both solutions with the lower concentration of ethanol (as well as the poor performance of the higher concentration of ethanol) may suggest a threshold in the amount of ethanol used to maximize efficacy.

Additional testing is needed to confirm these conclusions as well as investigate the other possible impacts of ethanol. However, these findings present new opportunities for the development of ethanol in regard to agriculture and improving food security.

ACKNOWLEDGEMENTS

I am incredibly grateful for those involved in my project and would like to thank the following individuals: my mom (support), Ms. Dallin (advising), and Mr. Osbourne (lab advice/materials).

REFERENCES

Back to the Roots. (n.d.). Radish - 'Cherry Belle' – Back to the Roots. Back to the Roots. Retrieved March 13, 2023, from https://backtotheroots.com/products/radish-cherry-belle

Bashir, K., Todaka, D., Sultana Rasheed, Matsui, A., Ahmad, Z., Sako, K., Utsumi, Y., Vu, A. T., Tanaka, M., Takahashi, S., Ishida, J., Tsuboi, Y., Watanabe, S., Kanno, Y., Ando, E., Shin, K.-C., Seito, M., Motegi, H., Sato, M., … Seki, M. (2022, August 25). Ethanol-Mediated Novel Survival Strategy against Drought Stress in Plants. Plant and Cell Physiology, 63(9), 1181–1192. https://academic.oup.com/pcp/article/63/9/1181/6674685?login=false

Bui, L. T., Novi, G., Lombardi, L., Iannuzzi, C., Rossi, J., Santaniello, A., Mensuali, A., Corbineau, F., Giuntoli, B., Perata, P., Zaffagnini, M., & Licausi, F. (2019, February 28). Conservation of ethanol fermentation and its regulation in land plants. Journal of Experimental Botany, 70(6), 1815–1827. https://academic.oup.com/jxb/article/70/6/1815/5376644

Exploratorium. (n.d.). Photosynthetic Floatation: : Biology & Physics Science Activity. Exploratorium. Retrieved October 29, 2023, from https://www.exploratorium.edu/snacks/photosynthetic-floatation

Farmer, S. (2016, April 26). Open or Shut: How Trees Respond to Drought at the Leaf Level. Southern Research Station. Retrieved February 16, 2023, from https://www.srs.fs.usda.gov/compass/2016/04/26/open-or-shut-how-trees-respond-to-dro ught-at-the-leaf-level/

Miljure, B. (2022, October 9). B.C farmers struggle with extreme weather, climate change | CTV News. CTV News Vancouver. https://bc.ctvnews.ca/from-floods-to-drought-unpredictability-of-climate-change-challeng es-b-c-farmers-1.6102886

Nguyen, H. M., Sako, K., Matsui, A., Suzuki, Y., Mostofa, M. G., Ha, C. V., Tanaka, M., Tran, L.-S. P., Habu, Y., & Seki, M. (2017, July 3). Ethanol Enhances High-Salinity Stress Tolerance by Detoxifying Reactive Oxygen Species in Arabidopsis thaliana and Rice. NCBI. Retrieved October 9, 2023, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5494288/

Oregon State University. (2008, January 8). Plant growth and development. OSU Extension Service. Retrieved February 16, 2023, from https://extension.oregonstate.edu/node/115986/printable/print

Rakotoarivony, V. (2022, May 12). World 'at a crossroads' as droughts increase nearly a third in a generation. UN News. Retrieved September 26, 2022, from https://news.un.org/en/story/2022/05/1118142

Tadege, M., Dupuis, I., & Kuhlemeier, C. (2022, October 2). Ethanolic fermentation: new functions for an old pathway. Trends in Plant Science. Retrieved September 23, 2023, from https://www.cell.com/trends/plant-science/fulltext/S1360-1385(99)01450-8?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS1360138599014508%3Fshowall%3Dtrue

World Health Organization. (n.d.). Drought. World Health Organization (WHO). Retrieved September 26, 2022, from

Plastered Plants Defeat Drought: Exploring the Effects of Ethanol on Drought Resistance in Plants