Manipulating Mutant Chlamydomonas reinhardtii strains in Hydrogen production: A Novel Source of Renewable Energy

Part of the Young Naturalist Awards Curriculum Collection.

by Kriti, Grade 10, California - 2013 YNA Winner


Hydrogen fuel is a promising new source of green energy. Several scientists and companies are currently using photosynthesis to produce clean hydrogen using the green algae Chlamydomonas reinhardtiito. Clean hydrogen can be used as energy in replacement of fossil fuels. However, currently, algal hydrogen production isn’t economically viable due to the algae’s low light utilization. To improve light utilization, light penetration in algae cultures needs to be enhanced, and there can be many ways to do it.

Chlamydomonas reinhardtii (strain CC-125) has an eyespot and exhibits phototaxis, i.e., it moves toward light to perform photosynthesis. Because of phototaxis, the algae closest to the light collect near it and form a “cloud” that blocks light for the other algae, which then can’t perform photosynthesis. I used the Chlamydomonas reinhardtii mutant CC-1101, which lacks an eyespot, to test the effect of the absence of phototaxis on hydrogen production. Another way to possibly increase hydrogen production is to use algae mutants that have less chlorophyll. I used the strain CC-4170; it contains the gene tla1, which has only 115 molecules of chlorophyll (compared with 222 chlorophyll molecules in CC-125) and a light utilization efficiency of 10%.

In a previous experiment, I tested two methods of producing hydrogen from Chlamydomonas reinhardtii—by sulfur deprivation and by copper enrichment of the algae’s environment. I tested three copper concentrations—0.8 ppm, 1.0 ppm and 1.6 ppm—and a sulfur-free environment. Copper enrichment of 0.8 ppm was best at producing hydrogen on a continuous basis. A sulfur-free environment produced the most hydrogen, but also killed the algae within a few days of deprivation.

This study focuses on improving the photosynthetic efficiency of this process. I tested whether mutants with special properties improve the algae’s light utilization efficiency, resulting in better hydrogen production. I tested two mutants—CC-1101 and CC-4170, with CC-125 wild type as a control—for their light utilization and hydrogen-production capabilities under 0.8 ppm copper-enriched and sulfur-deprived conditions. I chose the 0.8 ppm copper-enriched culture because it was the best culture medium from the last study.

I found that CC-1101 was the worst in producing hydrogen, meaning than algae need an eyespot to function properly. Therefore, the CC-1101 results were eliminated from some of the analysis. Excluding CC-1101, the less green (with less chlorophyll) algae CC-4170 had the least decline in light intensity through its culture, and also produced the most hydrogen. This study can improve commercial hydrogen photobioreactors, making algal hydrogen economically viable.

Introduction and Question

With the world facing an energy crisis, scientists are searching for a clean and renewable source of energy. One alternative energy source is hydrogen, which can be used as a fuel. The use of hydrogen greatly reduces carbon dioxide emissions, but hydrogen has to be produced from fossil fuels, which emit carbon dioxide. So, in reality, hydrogen as a fuel doesn’t completely solve the carbon dioxide emission problem. But a new idea is emerging: using algae to make clean hydrogen, which can be used as energy instead of fossil fuels. In fact, the U.S. Department of Energy estimates that if algae fuel replaced all the petroleum in the U.S., it would require only a 40,000-square-kilometer algae farm to produce this energy.

I initially become interested in this topic when I stumbled across an article that discussed a new combination: algae and energy. My interest was sparked, and I was curious. Within a few days, I had read most of the research papers relating to the topic, and in the following months I had experimented and read, then experimented and read again. I was hooked. And here, from the research papers and graphs cluttering my desk, a question was sparked and my project was born.

In a previous experiment, I tested two methods of producing hydrogen from the algae Chlamydomonas reinhardtii—by sulfur deprivation and by copper enrichment of the algae’s environment. I tested three copper concentrations—0.8 ppm, 1.0 ppm and 1.6 ppm—and a sulfur-free environment. I found that 0.8 ppm copper enrichment was the best at producing hydrogen on a continuous basis. The sulfur-free environment produced the most hydrogen overall, but it eventually killed the algae; this is because sulfur is needed to make proteins that algae need to survive.

This study expanded upon those results. In this research, I subjected the algae to sulfur-free and 0.8 ppm copper-enriched environments. In addition to using the normal strain (wild type) of Chlamydomonas reinhardtii (strain CC-125), I tested two of its mutants—CC-1101 and CC-4170—for their hydrogen production capabilities. My question was: Are C. reinhardtii mutants better at producing hydrogen than the naturally occurring species in copper-enriched or sulfur-deprived media? I hypothesized that  CC-4170 and CC-1101 would produce more hydrogen than strain CC-125.


Copper Enrichment and Sulfur Deprivation

From previous research, I knew the biological processes behind copper enrichment and sulfur deprivation, the two methods for making algae produce hydrogen. Sulfur deprivation in algae acts as a switch from the production of oxygen to hydrogen. Sulfur is a valuable nutrient that helps the algae produce proteins and amino acids to make the enzymes that permit photosynthesis to occur. When these enzymes can no longer be produced due to sulfur deprivation, photosynthesis shuts down in the algae. Once the algae stop photosynthesizing, they use the oxygen in their environment via respiration. The environment becomes anaerobic, setting the stage for hydrogenase, which uses water oxidation and the catabolism of starch to produce hydrogen.

The other method for producing hydrogen is copper enrichment. When copper is added to the algae’s environment, their ability to produce hydrogen is blocked. Copper impedes the ability of the algae to do photosynthesis by preventing Photosystem II synthesis to function. This happens because the copper ions disrupt the Nac2 gene required for Photosystem II synthesis. Thus, the same effect occurs as in the sulfur-deficient method, with the hydrogenase enzyme again producing hydrogen.

The Strains

Chlamydomonas reinhardtii (strainCC-125) is a green algae that has a large chloroplast for photosynthesis and an “eyespot” that senses light. C. reinhardtii can grow on inorganic salts in the light and photosynthesize. But under certain conditions—namely, copper addition or sulfur deprivation—the algae can switch from producing oxygen to producing hydrogen. The strain CC-125 has a light utilization efficiency of 3% out of an ideal 30%, and has 222 chlorophyll molecules.

The strain CC-1101, a mutant of the original Chlamydomonas reinhardtii strain, contains the same properties as CC-125—except for the fact that it lacks an eyespot. Because of this property, CC-1101 cannot sense and doesn’t gravitate toward light. The strain CC-1101 has a gene called eye 1 that gives it this property. The strain CC-4170 is also a mutant of the original strain of Chlamydomonas reinhardtii (CC-125).CC-4170 contains the same properties as CC-125, except that it contains the gene tla1. Because of this gene, CC-4170 only has about 115 molecules of chlorophyll and a higher light utilization efficiency of 10%.

Chlorophyll Antennae

The following picture shows sunlight utilization efficiency in algae (CC-125 and CC-1101) with a regular antennae size (regular amount of chlorophyll pigments). In this case, the cells at the surface of the culture over-absorb the incoming sunlight (they absorb more than can be utilized by photosynthesis) and dissipate most of it as heat.


The picture below shows sunlight utilization efficiency in algae (CC-4170) with a truncated antennae size (reduced number of chlorophyll pigments). In this case, the cells at the surface absorb less light due to less chlorophyll. Hence, the light can penetrate to the algae deeper in the culture.




My experiment tested three algae strains—CC-125, CC-1101 and CC-4170—and their performances in sulfur-free and 0.8 ppm copper-enriched environments. The independent variables


were the algal strains and the culture media; the dependent variables were the hydrogen produced and the light intensity through each bottle; and the controlled variables were the amount of algae and solutions in each bottle, as well as the time and light given to each bottle.

I used six water bottles (with spouts) and labeled them: CC-125 Copper, CC-125 Sulfur, CC-1101 Copper, CC-1101 Sulfur, CC-4170 Copper and CC-4170 Sulfur. I added sulfur-free and 0.8 ppm copper solutions to the bottles, as appropriate, along with an equal amount of algae in each bottle with their respective strains. I assembled an airtight apparatus to make the algae environment anaerobic.


After five days, I took off the apparatus and fitted balloons onto the water bottle spouts, which would be used to collect the gas the algae produced.

After 12 days, I observed the bottles again. The balloons were filling up with gas. Carefully pinching the balloons, I took them off the bottle spout. I measured the amount of hydrogen gas produced using a graduated cylinder and a burning splinter. At the beginning and the end of the experiment, I measured the light intensity of each bottle using a light meter and homemade “light box.” The experiment was repeated.

Data and Results

I tested six cultures, half in copper-enriched conditions and the other half in sulfur-deprived conditions. My results showed that sulfur-deprived CC-4170 produced the most hydrogen, followed by copper-enriched CC-4170, sulfur-deprived CC-125, copper-enriched CC-125, sulfur-deprived CC-1101 and copper-enriched CC-1101. The decline in light intensity was the most for copper-enriched CC-125 (78%) and the least for sulfur-deprived CC-1101 (58%). In the following graphs, CE represents copper-enriched cultures and SD represents sulfur-deprived cultures.

I performed two trials and averaged the data.

Type of Mutant Light Intensity Day 0 (Lux) Light Intensity Day 12 (Lux)

Percentage Decline

in light intensity

Hydrogen Produced (mL)
Trail 1 Trail 2 Average Trail 1 Trail 2 Average Trail 1 Trail 2 Trail 3
CC125 CE 4830 4790 4810 1020 1060 1040 78% 16 14 15
CC1101 CE 4760 4800 4780 1980 1900 1940 59% 4 5.5 5
CC4170 CE 4750 4830 4790 1540 1560 1550 68% 27 29 28
CC125 SD 4900 4920 4910 1750 1850 1800 63% 23 27 25
CC1101 SD 4610 4690 4650 1970 1930 1950 58% 8 12 10
CC4170 SD 4790 4810 4800 1940 2000 1970 59% 31 33 32

This graph shows the light intensity drop (in Lux) for the strains from Day 0 to Day 12. As shown in the graph, the light intensity started out as high on Day 0, then went down by Day 12:


This following graph shows the hydrogen produced for each of the strains in Trail 1 and Trail 2, as well as the average (in blue) of both the data points.


As exhibited by the graph below, CC-1101 SD, although producing the second least amount of hydrogen, had the greatest light intensity of all the bottles


A graph detailing the average hydrogen produced in all the cultures follows:


Analysis and Observations

I noticed that as light passed through the algae bottles, its intensity declined. The decline was the most for copper-enriched CC-125 and the least for sulfur-deprived CC-1101. When the experiment started out, the algae were just beginning to grow, so the bottles were not too green. But as the algae multiplied and the bottles became a darker color, the algae started forming dense clouds and the light intensity passing through each bottle dropped.

I also saw that the amount of hydrogen produced by strain CC-1101 was lower than expected. I believe this is because it lacks an eyespot, which is needed for the normal functioning of the algae. Without the eyespot, the algae CC-1101 might have died, since it was not able to actively engage in photosynthesis without its eyespot. This could explain the high light intensities in the CC-1101 cultures; when algae die, they lose their green color, which allows more light to pass through them. It also might explain their low hydrogen production rate, which, naturally, would not have been much if the producers were slowly dying.

I noticed that the hydrogen produced in each of the strains’ sulfur-deprived environments was higher than the amount produced in the copper-enriched counterparts. This was expected because of the results of my prior experiment. It also explains why the copper-enriched cultures were thriving at the end of the experiment, while the sulfur-deprived algae were slowly dying. Furthermore, it ties back into the fact that in the light-intensity comparison graph, the sulfur-deprived cultures had a greater light intensity passing through them in all the media and strains. This could mean that the algae were already dying near the end of the experiment, and their light intensities were affected.

Since the copper-enriched and sulfur-deprived results were different, I created two additional graphs—not including strain CC-1101 because its results were an outlier—to show separately the hydrogen produced by CC-125 and CC-4170 in copper-enriched and sulfur-deprived media. These graphs show that for algae strains with eyespots, as the light passing through the culture media decreases, hydrogen production also decreases. CC-4170 was the best mutant. It produced 87% and 28% more hydrogen as compared to CC-125 in copper-enriched and sulfur-deprived media, respectively.


Here’s a comparison between the average hydrogen produced by CC-125 CE and CC-4170 CE. The copper-enriched data points for each strain are comparatively less than their sulfur-deprived counterparts, but the trend can still be seen:CC-4170, with less chlorophyll, did better than wild type C. reinhardtii.


It’s important to notice that CC-4170 produced 28% more hydrogen in the sulfur-deprived culture, as well as 87% more hydrogen in a copper-enriched culture than CC-125:


Conclusion and Application

The results of this research are very exciting, as they promise a novel method of efficiently and inexpensively producing hydrogen from algae. Strain CC-4170, which has less chlorophyll, let more light pass through it and produced more hydrogen than CC-125. CC-1101, which lacked an eyespot and performed poorly. I think this is because an eyespot is needed for Chlamydomonas reinhardtii to function properly. As I expected, the mutants in the sulfur-deprived medium produced more hydrogen, but by the end of the experiment they began to die. The algae in the copper-enriched medium produced less hydrogen but remained healthy.

This research can be used in powering cars and fuel cells. With a Chlamydomonas reinhardtii mutant as the power supplier, clean energy can be produced in photosynthetic bioreactors, a step toward easing global warming. For example, in a real-life algal tubular racetrack photobioreactor, sunlight is supplied from above, so only the algae at the top receive light. They absorb the light with their chlorophyll but only use a small amount and give off the rest of the light as heat dissipation. With smaller amounts of chlorophyll in the algae (as in CC-4170), the algae at the top absorb less light, hence giving more light to the algae at the bottom and sides of the photobioreactor. This would enable more hydrogen to be produced with minimum light loss, increasing the overall efficiency of the process.

Project Expansion

I did not have access to a lab, so if I were to do this experiment again, I would use better lab equipment to measure the hydrogen and light intensity more accurately. I would also test different algae strains, not just Chlamydomonas reinhardtii, for hydrogen production. I would experiment with different materials, not just copper and sulfur, and test different photobioreactors to see which one is the best.


I would like to thank my parents for helping me procure the algae strains, and my school and teachers for supporting me.


Amos, Wade A. “Updated Cost Analysis of Photobiological Hydrogen Production from Chlamydomonas reinhardtii Green Algae.” Golden, CO: National Renewable Energy Laboratory, January 2004.

“Biological hydrogen production.” Wikipedia. Retrieved from the World Wide Web on 28 Jan 2011.

Chlamydomonas reinhardtii.”Wikipedia. Retrieved from the World Wide Web on 12 Apr 2011 from

“Hydrogen economy.” Wikipedia. Retrieved from the World Wide Web on 9 Feb 2011.

“Photobiological Production of Hydrogen (Fact Sheet).” Golden, CO: National Renewable Energy Laboratory, November 2007.

Demazel, Delphine. “Use of Algae as an Energy Source.” Nordic Folke Center, August and September 2008. Retrieved from the World Wide Web on 2 May 2011.

Gartner, John. “Algae: Power Plant of the Future?” Wired,19 Aug 2002. Retrieved from the World Wide Web on 15 Feb 2011.

Ghirardi, Maria L. “Biological Systems for Hydrogen Photoproduction.” Golden, CO: National Renewable Energy Laboratory, 2005.

Greenbaum, E., and J.W. Lee. “Photosynthetic Hydrogen and Oxygen Production by Green Algae.” Oak Ridge, TN: Oak Ridge National Laboratory, 2008.

Happe, Thomans, and Tasios Melis.Trails of Green Alga Hydrogen Research: From Hans Gaffron to New Frontiers.Berkeley, CA: University of California Press, 2004.

Melis, Tasios. “Maximizing Photosynthetic Effects and Hydrogen Production in Microalgal Cultures.” Department of Energy Hydrogen Program, 2004.

Melis, Tasios. “Maximizing Light Utilization Efficiency and Hydrogen Production in Microalgal Cultures.” Department of Energy Hydrogen Program, 2007.

Melis, Tasios. “Maximizing Light Utilization Efficiency and Hydrogen Production in Microalgal Cultures.” Department of Energy Hydrogen Program, 2008.