Christopher & Nicholas Kwok

Age 16 | Redwood City, California

Raytheon Technologies Invention Convention Globals First Place | Regeneron International Science & Engineering Fair Fourth Place Grand Award | Raytheon Technologies Invention Convention U.S. Nationals People’s Choice Award | Raytheon Technologies Invention Convention U.S. Nationals | Environmental and Sustainability Industry Award | Raytheon Technologies Invention Convention U.S. Nationals 10th Grade Second Place | California Invention Convention Best In Show Award | Genius Olympiad Silver Medal | California Stockholm Junior Water Prize Third Place | Penn Climate Venture Prize Third Place | Yerba Buena Plant Society Award

Edited by Danlin Zeng

VIEW PDF

Ocean acidification adversely shifts Earth's oceans towards pH-neutral conditions. The global crisis has left coral reefs unlivable, jeopardizing marine biodiversity and resulting in economic devastation. This study pioneers an ecological solution to ocean acidification. The research chemically modified enzymes in algae to mitigate ocean acidification by increasing pH, reversing the interconversion between carbon dioxide and water, and dissociating ions of carbonic acid. The experiments aimed to increase the hydrogenase enzymes with carotene and decrease the carbonic anhydrase enzymes with phytic acid and iron. Hydrogenase enzymes catalyze the reversible oxidation of molecular hydrogen. Carbonic anhydrase enzymes catalyze the interconversion between carbon dioxide and water and the dissociated ions of carbonic acid. The investigation discovered that 6 grams of carotene with 4 grams of phytic acid performed the modifications. After testing the novel strain of algae for 30 days, the chaetomorpha increased the hydrogen gas concentration by 0.5 ppm, reducing the bicarbonate concentration by 47. This raised the total pH by 2. The project invented chaeto biofilters to implement the algae in oceanic habitats. The devices require no electricity and are cost-effective, viable solutions for governments.

INTRODUCTION

Purpose
Documentation of the ocean's acidity originates from the creation of the pH scale in 1909 by Søren Peter Lauritz Sørensen (Umair Irfan, 2021). In 2003, the first biological study was published regarding ocean acidification. The articles conceptualized and confirmed the global phenomenon, leading to the first annual "Ocean in a High CO2 World" conference in 2004 (United Nations Educational, Scientific and Cultural Organization, 2021). Ocean acidification refers to the ongoing decline in pH. By absorbing the atmospheric carbon dioxide, an increased concentration of hydrogen ions combines with carbonate to form bicarbonate ions. As a result, fewer carbonate ions exist. This concerns marine organisms such as oysters, clams, corals, and calcareous plankton that need carbonate to construct their shells, skeletons, and other calcium structures (National Oceanic and Atmospheric Administration, 2021).

As humans exploit fossil fuels, the progression of ocean acidification has increased the waters' acidity by 26% since 1850, a change that has been ten times faster than any time in the last 55 million years (National Oceanic and Atmospheric Administration, 2021). The pH difference has led to 1,800 reefs across 41 countries with the evaluation that only 5% can provide for their lucrative byproducts (Sarah Gibbens, 2021). As the crisis festers, over 4,000 fish species are at risk, jeopardizing 25% of marine life (Francis Staub, 2021). With unprecedented trends, the consequences of ocean acidification are becoming more irreversible and tethered to larger social and economic impacts worldwide.

For many nations, the ocean has become a contributing source of livelihoods, gross domestic product, and sustainable development (United Nations Conference on Trade and Development, 2021). Today, the fisheries and aquaculture industry supports nearly 12% of the planet's population, with 870 million people depending on the ocean for income (Our Ocean, 2021). On the other hand, the Maldives, British Virgin Islands, Macao, and Aruba rely on tourists attracted to their coastal beaches, marine biodiversity, and scuba diving opportunities to generate revenue (Natasha Frost, 2021). However, as ocean acidification escalates, economic impact projects a US$100 billion loss by 2100 for the mollusk and fish market and a A$5.4 billion plummet to the Australian economy for their tourism around the Great Barrier Reef (CoastAdapt, 2021). As of the century, more countries and ordinary civilians have become increasingly vulnerable to the devastating effects of ocean acidification.

Ocean acidification remains an unsolved existential crisis (Sarah Gibbens, 2021). Solutions include plans regarding carbon emissions or over-ambitious coral restoration operations. However, these approaches are long-term projects that require decades for change. For instance, governmental operations to farm aquatic plants have been pursued. Artificial seagrass meadows in the ocean have given corals an 18% boost in growth by absorbing dissolved carbon dioxide. However, outplanting and land-based nurseries strengthening coral resilience require substantial funding and labor-intensive efforts that cannot be replicated to preserve large oceanic habitats (Nicola Jones, 2021).

For the research, the green macroalgae, chaetomorpha, was selected for its biological abundance, sustainable cultivation, and ability to organically neutralize ocean acidification. The alga is classified as a Chlorophyta species, which consists of single tubular strands of cells, allowing for fast growth compared to most algae. Additionally, a lack of turpins prohibits the algae's ability to impede coral growth, eliminating the possibility of outcompeting organisms for nutrients after implementation (Hui Huang, 2021).

Hypothesis
The hypothesis regards increasing the activity of hydrogenase enzymes and decreasing the carbonic anhydrase enzymes in chaetomorpha to mitigate ocean acidification. By utilizing carotene, phytic acid, and iron to chemically modify enzymes in chaetomorpha, the study aims to produce a novel strain of algae that combat ocean acidification by increasing pH, reversing the interconversion between carbon dioxide and water, and dissociating ions of carbonic acid. To implement the modified chaetomorpha, the research intends to engineer chaeto biofilters.

Background Information
The first objective is to increase the activity of hydrogenase enzymes with carotene, which catalyzes the reversible oxidation of molecular hydrogen. Organisms utilize the enzyme to convert hydrogen ions to hydrogen gas during anaerobic respiration. In fact, hydrogenase enzymes found in algae are among the most efficient hydrogen-generating biocatalysts and use low-potential electrons from the photosynthetic light reaction (Hideaki Ogata, 2021). The utilization of carotene is to transfigure the algae's pigmentation to become a carotenoid, absorbing marine light more readily. In return, this amplifies the hydrogenase enzymes, reducing the concentration of harmful hydrogen ions and protecting carbonate ions (NASA Science, 2021).

The second objective is to decrease the activity of carbonic anhydrase enzymes with phytic acid and iron. This enzyme catalyzes the interconversion between carbon dioxide and water and the dissociated ions of carbonic acid (i.e. bicarbonate and hydrogen ions). The enzyme is accountable for absorbing the atmosphere's carbon dioxide and dissolving it into the ocean (Mondem Reddy, 2021). Since the enzyme is a metalloenzyme of zinc, phytic acid and iron can inhibit zinc intake by binding to the nutrients before absorption (Bo Lönnerdal, 2021).

MATERIALS & METHODS

Step 1) Preparation | The Experimental Method
The experimental setup consisted of one large polycarbonate container, which served as the culture system, followed by four smaller aquariums. The suspension of a light-emitting diode strip light equally distributed the needed photosynthetic active radiation to all the habitats. Then, each aquarium was filled with saltwater and dosed 2.2g of artificial nutrients and 4.9g of fertilizer every week.

Step 2) Setup | The Culture System
The culture system was a 12.38 quart container that acted as a habitat for the chaetomorpha to continuously grow, being the source of unmodified, healthy algae for each phase. This aquarium contained a heater, air pump, and a sponge filter to expedite reproduction. Initially, two cups of chaetomorpha were released. The organism grew for one month to harvest the sufficient amount of algae before the first experiment.

Step 3) Phase 1 | Increasing the Activity of Hydrogenase Enzymes with Carotene
Phase 1 dissolved 0g, 2g, 4g, and 6g of beta carotene 1% powder into separate aquariums with 4.5 ounces of chaetomorpha. Phase 1 lasted for four weeks. Twice a week, the hydrogen gas concentration was measured with a hydrogen test reagent kit that quantifies the hydrogen (in parts per million) in the aquarium. The goal was to identify the algae that produced the most hydrogen gas, which indicates the carotene is competent in amplifying the hydrogenase enzymes.

Step 4) Phase 2a | Decreasing the Activity of Carbonic Anhydrase Enzymes with Phytic Acid and Iron
Phase 2a dissolved 0g, 2g, 4g, and 6g of inositol hexaphosphate powder (phytic acid) into separate aquariums with 4.5 ounces of chaetomorpha. This phase took four weeks. Twice a week, litmus test strips measure the bicarbonates in the water. The goal was to identify the amount of phytic acid needed to reduce the bicarbonate concentration, which signifies that the interconversion between carbon dioxide and water has stopped.

Step 5) Phase 2b | Decreasing the Activity of Carbonic Anhydrase Enzymes with Iron
Likewise, Phase 2b dissolved 0g, 2g, 4g, and 6g of ferrous gluconate powder (iron) into separate aquariums and identically followed the experimentation of Phase 2b. Phase 1, Phase 2a, and Phase 2b were repeated three times for three experimental trials.

Step 6) Phase 3a | Combining the Results of Phase 1 and Phase 2
Phase 3a began with six 4.5 quart aquariums. As a repeat of Step 1, each aquarium received adequate lighting, saltwater, doses of fertilizers and nutrients, and 4.5 ounces of chaetomorpha. Using the results from Phase 1 and Phase 2, the combination of each powder's optimal quantities produced the new strain of algae in three aquariums, leaving the rest as controls. However, between Phase 2a and Phase 2b, the chemical reactant that inhibited the carbonic anhydrase enzymes the best was used. Ultimately, the algae was given two weeks to absorb the chemicals before formal investigation of the modified chaetomorpha.

Step 7) Phase 3b | Investigating the New Strain of Algae
The final step of the project was to evaluate whether, after exposing the algae to the reactants for two weeks, the modified chaetomorpha would retain the desired properties and continue the results within a new environment. This trial lasted for 30 days. Every five days, the hydrogen gas concentration, bicarbonate concentration, and total pH level was measured in each aquarium. The research wanted the new strain of algae to be sustainable and preserve the enzyme modifications even after the powders are withdrawn.

To implement the modified chaetomorpha, chaeto biofilters were invented to contain the novel strain of algae. By melting holes around a 4 gallon Sterilite container, a prototype was engineered. Using zip ties, recycled water bottles were attached for the flotation mechanism. To test the invention, chaeto biofilters were deployed in local lagoons and the San Francisco Bay to monitor the algae's survival in the biofilter's conditions.

RESULTS

Phase 1 | Increasing the Activity of Hydrogenase Enzymes

**Figure 1:** Investigating the amount of carotene to increase hydrogenase enzymes.

According to Figure 1, 6g of carotene altered the algae's pigmentation, accelerated the hydrogenase enzymes, and increased the hydrogen gas concentration most significantly. With 6 grams of carotene, the hydrogen gas concentration quadrupled to an average 0.85 parts per million compared to the control, which remained at 0.2 parts per million. This result was incomparable to 2 grams and 4 grams of carotene, producing a 0.1 to 0.4 parts per million increase.

Phase 2 | Decreasing the Activity of Carbonic Anhydrase Enzymes

**Figure 2:** Investigating the amount of phytic acid to decrease carbonic anhydrase enzymes.

**Figure 3:** Investigating the amount of iron to decrease carbonic anhydrase enzymes.

Between Figure 2 and Figure 3, 4 grams of phytic acid inhibited the carbonic anhydrase enzymes the most among the two chemical reactants. The control consistently ended with a bicarbonate concentration of 170 milligrams. From Phase 2a, 2 grams of phytic acid resulted in a 40 to 60 milligram bicarbonate reduction, 4 grams of phytic acid resulted in a 90 milligram reduction, and 6 grams increased the bicarbonate concentration by 30 to 40 milligrams. In contrast, the iron was insufficient in reducing the bicarbonate concentration, only inducing a 50 milligram reduction at most with 6 grams of iron.

Phase 3 | Investigating the New Strain of Algae

**Figure 4:** Investigating the new strain of algae in retaining the effects of carotene.

**Figure 5:** Investigating the new strain of algae in retaining the effects of phytic acid.

**Figure 6:** Investigating the new strain of algae with stabilizing the pH.

After modifying the chaetomorpha in Phase 3a with 6 grams of carotene and 4 grams of phytic acid, Phase 3b demonstrated significant results after 30 days. According to Figure 4, the modified chaetomorpha increased the hydrogen gas concentration by 0.5 parts per million (from 0.3 parts per million to 0.8 parts per million). The graph suggests that the carotene has become a catalyst for the hydrogenase enzymes and converted the harmful hydrogen ions by expediting hydrogen gas production until days 20 and 25 when the process climaxed. In Figure 5, the novel strain of algae reduced the bicarbonate concentration by 47% through a 90 milligram reduction. Illustrated in the graph, there was a steady decrease in bicarbonates until days 20 and 25, where all the aquariums plateaued at a constant bicarbonate concentration of 100 milligrams. Ultimately, in Figure 6, the modified chaetomorpha increased the pH by 2 (from 7.5 to 9.5) after 30 days. In correlation with the increased hydrogen gas concentration, the pH surged towards basic conditions.

DISCUSSION

Interpretation
In summary, the investigation discovered that 6g of carotene with 4g of phytic acid performed the enzyme modifications. Debriefing the science, 6g of carotene transfigured the algae's pigmentation to become similar to a carotenoid, absorbing marine light more readily. The increased sensitivity to light was parallelled with the amplified hydrogenase enzyme activity, which uses low-potential electrons from the photosynthetic light reaction to produce hydrogen gas and prevent the formation of adverse bicarbonate ions.

Phase 2 suggested that iron minimally reduced the bicarbonate concentration compared to the phytic acid. Phytic acid was superior due to its ability to immediately prevent the algae's zinc absorption by binding to the nutrient before consumption. The 4 grams of phytic acid optimized the inhibition of zinc absorption that ceased the carbonic anhydrase enzymes. However, 6 grams of phytic acid killed the chaetomorpha, which drastically increased the bicarbonate concentration because it completely stopped the algae's absorption of minerals such as iron and magnesium.

This was detrimental as algae needs small amounts of iron to be healthy and produce chlorophyll, which gives the organism oxygen (Dan Brennan, 2021).

Analysis
Overall, the novel strain of algae increased the waters' pH by 2. Considering the pH scale ranges from 0 to 14 and a 0.1 pH increase in the ocean is equivalent to extracting all the dissolved carbon dioxide since the Industrial Revolution, this result was substantial. As the world's dependence on fossil fuels surpasses biocapacity and 30% of the atmospheric carbon dioxide will end up in the ocean, the 2 pH increase would be imperative (SLO Active, 2021).

Applicability
Chaeto biofilters could be implemented in endangered oceanic habitats to infinitely absorb the billions of gallons of carbon dioxide produced by humans annually. Chaetomorpha can thrive in nutrient-poor regions, making the technology sustainable and resilient in extreme conditions and various environments. Subsequently, the composition of cells in chaetomorpha allows for speedy cultivation for immediate operations. Additionally, carotene and phytic acid both originate from organic sources considered as environmentally friendly chemicals. With the algae's potential to stabilize the ocean's chemistry by 2 pH, the biofilters can be instituted in marine ecosystems to preserve planet Earth while preventing the catastrophic relapses of ocean acidification.

To test the invention, the temporary installation of chaeto biofilters in three local lagoons before the San Francisco Bay was performed. At these sites, 48-hour time lapse videos reveal the functionality and durability of the biofilter's design. After preliminary testing, tweaks to the invention improved its practicality. Significant additions included a Trackimo GPS tracker and an anchor for easy detection and maintenance purposes. Furthermore, the filtration holes were made smaller to ensure the modified chaetomorpha could not escape and no external organisms could enter. Preventing the foreign organism (chaetomorpha) from escaping was critical as it could become invasive to ecosystems and lead to environmental concerns.

Chaeto biofilters are floating devices that contain the modified chaetomorpha. After implementation in oceanic habitats, the invention acts as a filter, impeding the acidification process in its proximity without electricity. The device features holes for water flow, recycled

bottles for flotation, an anchor, and a GPS tracker for easy maintenance. With a clear international demand, the market for the chaeto biofilters consists of non-governmental conservation organizations and government contracts.

Due to the simplistic design of the chaeto biofilter, the technology is scalable, feasible, and cost-effective for governments and environmental agencies anywhere. The 4 gallon prototype would be scaled to 2,000 gallon biofilters, capable of filtering 3.415 million gallons of saltwater in a 6 to 8 week period. Composed of various recycled materials, each chaeto biofilter is contrived from high-density polyethylene, an environmentally safe plastic that does not leach chemicals, durable to prevent microplastics, and heat resistant. With limited parts and few technical components, the chaeto biofilters are the low-cost solution to ocean acidification. The durable, mobile nature of the device makes the chaeto biofilter convenient and adjustable, allowing installation in remote locations.

conclusion

In conclusion, the initial hypothesis of modifying the hydrogenase and carbonic anhydrase enzymes in chaetomorpha to mitigate ocean acidification was proven correct and successful. From this investigation, several conclusions were fashioned: (1) The modification of the hydrogenase and carbonic anhydrase enzymes was completed naturally, a process that eliminates expensive technology and can be reproduced in the developing world. (2) A mixture of 6 grams of carotene and 4 grams of phytic acid is adequate to produce the novel strain of algae capable of mitigating ocean acidification by increasing pH, reversing the interconversion between carbon dioxide and water, and dissociating ions of carbonic acid. (3) The modified chaetomorpha yielded a 2 pH increase that will be critical in stabilizing the ocean's water chemistry and preserving marine biodiversity. (4) Unlike conventional solutions to ocean acidification, such as renewables or coral restoration, chaeto biofilters are immediate, cost-effective, and ecological solutions for governments that repurpose accessible materials and biological chemicals/organisms. (5) The simplistic design of the chaeto biofilter could be universally replicated to accommodate size adjustments, budgets, and environments.

ACKNOWLEDGEMENTS

Christopher Kwok and Nicholas Kwok are sincerely appreciative of their parents for funding the project.

REFERENCES

Brennan, Dan. "Foods High in Phytic Acid." Nourish by WebMD, Jan 2, 2021. https://www.webmd.com/diet/foods-high-in-phytic-acid#:~:text=Phytic%20acid%20is%20found%20in,%2C%20linseeds%2C%20and%20sunflower%20seeds.&text=Nuts%20naturally%20contain%20a%20high%20amount%20of%20phytic%20acid
CoastAdapt. "Ocean acidification and its effects." CoastAdapt, Jan 12, 2021. https://coastadapt.com.au/ocean-acidification-and-its-effects
Food and Agriculture Organization of the United Nations. "Fishery and Aquaculture Country Profiles." Food and Agriculture Organization of the United Nations, Jan 28, 2021. http://www.fao.org/fishery/facp/CHN/en
Frost, Natasha. "These are the countries most reliant on your tourism dollars." Quartz, Jan 22, 2021. https://qz.com/1724042/the-countries-most-reliant-on-tourism-for-gdp/#:~:text=The%20Maldives%2C%20located%20in%20the,gross%20domestic%20product%20from%20tourism
Gibbens, Sarah. "Scientists are trying to save coral reefs. Here's what's working." National Geographic, Jan 6, 2021. https://www.nationalgeographic.com/science/article/scientists-work-to-save-coral-reefs-climate-change-marine-parks
Huang, Hui. "Chaetomorpha." ScienceDirect, Jan 2, 2021. https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/chaetomorpha
Irfan, Umair. "S. P. L. Sørensen invented the pH scale by experimenting with beer." Vox, Jan 13, 2021. https://www.vox.com/2018/5/29/17404820/spl-sorensen-google-doodle-ph-scale
Jones, Nicola. "How Growing Sea Plants Can Help Slow Ocean Acidification." Yale School of the Environment, Jan 6, 2021. https://e360.yale.edu/features/kelp_seagrass_slow_ocean_acidification_netarts
Lönnerdal, Bo. "Dietary Factors Influencing Zinc Absorption." Oxford Academic, Jan 2, 2021. https://academic.oup.com/jn/article/130/5/1378S/4686381
Medical News Today. "All you need to know about beta carotene." Medical News Today, Jan 2, 2021. https://www.medicalnewstoday.com/articles/252758#_noHeaderPrefixedContent
NASA Science. "Ocean Color." NASA Science, Jan 10, 2021. https://science.nasa.gov/earth-science/oceanography/living-ocean/ocean-color#:~:text=The%20most%20important%20light%2Dabsorbing,photosynthesis
National Oceanic and Atmospheric Administration. "Ocean acidification." National Oceanic and Atmospheric Administration, Jan 3, 2021. https://www.noaa.gov/education/resource-collections/ocean-coasts/ocean-acidification
National Oceanic and Atmospheric Administration. "What is Ocean Acidification?" National Oceanic and Atmospheric Administration, Jan 3, 2021. https://oceanservice.noaa.gov/facts/acidification.html
National Ocean Policy Coalition. "Oceans Impact the Economy." National Ocean Policy Coalition, Jan 27, 2021. http://oceanpolicy.com/about-our-oceans/oceans-impact-the-economy/
Ogata, Hideaki, et al. "Hydrogenase." ScienceDirect, Jan 2, 2021. https://www.sciencedirect.com/topics/medicine-and-dentistry/hydrogenase
Our Ocean. "Sustainable Fisheries." Our Ocean, Jan 22, 2021. http://ourocean2016.org/sustainable-fisheries#:~:text=The%20livelihoods%20of%2010%2D12,depend%20on%20fisheries%20and%20aquaculture
Reddy, Mondem, et al. "Carbonic Anhydrase." ScienceDirect, Jan 2, 2021. https://www.sciencedirect.com/topics/materials-science/carbonic-anhydrase
Royal Society of Chemistry. "Iron." Royal Society of Chemistry, Jan 2, 2021. https://www.rsc.org/periodic-table/element/26/iron#:~:text=Iron%20is%20the%20fourth%20most,such%20as%20magnetite%20and%20taconite
SLO Active. "How our reefs are under threat and what we can do about it." SLO Active, Jan 7, 2021. https://sloactive.com/coral-reef-crisis-guide/#:~:text=However%2C%20coral%20reefs%20are%20currently,over%20the%20next%2030%20years
Staub, Francis. "How the world is coming together to save coral reefs." World Economic Forum, Jan 6, 2021. https://www.weforum.org/agenda/2020/12/how-the-world-is-coming-together-to-save-coral-reefs/
United Nations. "Climate Action." United Nations, Jan 6, 2021. https://www.un.org/en/climatechange/science/key-findings
United Nations Conference on Trade and Development. "Oceans Economy and Fisheries." United Nations Conference on Trade and Development, Jan 27, 2021. https://unctad.org/topic/trade-and-environment/oceans-economy
United Nations Educational, Scientific and Cultural Organization. "Ocean acidification: summary for policymakers; third Symposium on the Ocean in a High-CO2 World." United Nations Educational, Scientific and Cultural Organization, Jan 5, 2021. https://unesdoc.unesco.org/ark:/48223/pf0000224724

Modifying the Activity of Hydrogenase and Carbonic Anhydrase Enzymes in Chaetomorpha to Mitigate Ocean Acidification