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Establishing new breeding methods for improving oil production using algae

Researchers use neutron radiation to boost algae oil production, offering a promising solution for reducing greenhouse gases and producing renewable biofuels.

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According to the Intergovernmental Panel on Climate Change (IPCC) Sixth Assessment Report, “It is unequivocal that human influence has warmed the atmosphere, ocean and land.” Therefore, greenhouse gases such as CO2 emitted from human activities must be urgently reduced. Energy-saving technologies are being researched and developed to address this issue, and renewable energy sources, such as wind and solar power, are being promoted. Another solution gaining attention is to utilise algae, which, like plants, perform photosynthesis. There are several reasons for this focus, but specifically, algae grow faster than plants and do not require arable land.

Algae can potentially contribute to reducing greenhouse gases in multiple ways, including biofuel production, as they can produce oil for biofuel feedstock. Although biofuel produced from algal oil results in CO2 emissions, this CO2 is considered to be derived from the CO2 absorbed by the algae to produce biofuel. Therefore, the environmental impact is considered to be smaller than when using fossil fuels (Figure 1). However, commercial biofuel production from algal oil faces several challenges, with high production costs being a significant hurdle. One possible solution to this issue is to enhance the oil production capacity of each algal cell, thereby improving overall productivity.

A new algal breeding method using neutron beams increases oil accumulation in biofuel strains by up to 1.3 times. This method could also address various global warming-related social issues.

Sousuke Imamura
Figure 1. Environmentally friendly biofuels.
Photosynthetic organisms such as algae produce oil that can be used as a feedstock for biofuel, utilizing carbon derived from atmospheric carbon dioxide. When biofuel is used, carbon dioxide is emitted but is then reabsorbed by photosynthetic organisms. Therefore, biofuel is considered to have a lower environmental impact than fossil fuels.
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Why did we attempt to breed algae using neutron beams?

One method to increase the oil production of algae is through breeding technologies. Breeding involves altering the genetic sequence to change the characteristics of an organism. Methods to alter genetic sequences include crossbreeding and exposing organisms to mutagens, such as radiation and carcinogenic substances. Combined with methods that can specifically select desired traits, these techniques have produced the improved varieties of crops, vegetables, and fruits that we consume today. Therefore, breeding technology can be considered essential for supporting our food supply. Efforts in algae breeding focus on targeting specific genes and modifying their sequences.

However, growing these genetically modified algae in outdoor pool cultivation systems encounters strict regulations and significant challenges. In contrast, algae with enhanced traits, such as increased oil production due to mutagen-induced genetic changes, typically face fewer regulatory restrictions. As a result, breeding through mutagenesis is seen as an effective approach for developing these algae varieties.

Previously, attempts were made to alter the genetic sequences of algae using radiation such as gamma rays and X-rays. However, this method is not very effective for many algal cells since those types of radiation have difficulty penetrating water-containing substances like culture media. In response, NTT and Euglena Co., Ltd. have focused on the characteristics of neutron rays, which can easily penetrate water-containing substances such as culture media (Figure 2).

Figure 2. Research flow for establishing algae breeding technology using neutron radiation.
Step 1: Irradiate the algae with neutron beams. Step 2: Analyze the relationship between the neutron radiation dose and changes in the genetic sequence. This enables optimal neutron radiation conditions to be identified. Step 3: Isolate only the cells with the desired traits from the algae population irradiated under optimal conditions. In this study, the goal was to obtain cells with improved oil production.
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NTT possesses a lot of knowledge about neutron radiation, accumulated from testing errors caused by cosmic-ray-derived neutrons in semiconductors used in ground-based communication devices. On the other hand, Euglena Co., Ltd. has technology to evaluate the oil production capacity of algal cells. Leveraging the strengths of both companies, a research project to establish algae breeding technology using neutron radiation was initiated in 2022.

Establishing the world’s first algal breeding technology using neutron beam irradiation

To establish algae breeding technology using neutron radiation, it was necessary to clarify the relationship between the absorption dose of neutron radiation suitable for breeding and the efficiency of genetic sequence changes. While a weak neutron dose does not introduce significant changes, a too-strong dose introduces many changes, potentially causing cell death.

The induction of genetic sequence changes was assessed using a unicellular alga (scientific name: Cyanidioschyzon merolae). The mechanism involved introducing a genetic sequence change into a gene responsible for nucleic acid synthesis, allowing the alga to grow on an agar medium containing a growth-inhibiting drug. Analysis revealed that genetic sequence changes are effectively induced when the alga is irradiated with a neutron dose adjusted to approximately 20 Gy.

We applied the identified optimal neutron irradiation conditions to Euglena (scientific name: Euglena gracilis), a practical strain used for producing oil as raw materials for biofuels (equivalent to jet fuel and diesel fuel), attempting to obtain cells with increased oil production. A fluorescent dye that specifically stains oils were added to the neutron-irradiated cells and strains were selected based on the strength of the fluorescence emitted by each cell. As a result, we successfully obtained four strains with 1.2 to 1.3 times higher oil production than the parent strain (Figure 3). These results demonstrate the world’s first successful algal breeding using neutron irradiation to induce genetic sequences.

Figure 3. Isolation of algae strains with enhanced oil production.
Applying the optimal neutron radiation conditions elucidated in this study to Euglena gracilis, four improved strains with increased oil production were selected. The graph compares the oil accumulation of the four bred strains to the original strain.
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Future perspectives based on this research achievement

Our research results show that neutron-induced breeding can be applied to two types of algae. Moving forward, NTT will use these results further to breed algae strains with enhanced CO2 absorption capacity and analyse the underlying genes responsible for these traits.

Additionally, NTT will investigate the effectiveness of expanding the application range of this technology to algae species beyond the two initially studied. By improving and producing algae strains tailored to various purposes, we aim to offer solutions to climate change-related challenges, such as reducing greenhouse gases, producing energy resources, and creating agricultural, forestry, and fishery feed.

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Journal reference

Imamura, S., Yamada, K., Takebe, H., Kiuchi, R., Iwashita, H., Toyokawa, C., … & Takaya, K. (2024). Optimal conditions of algal breeding using neutral beam and applying it to breed Euglena gracilis strains with improved lipid accumulation. Scientific Reports, 14(1), 14716. https://doi.org/10.1038/s41598-024-65175-1

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Dr. Sousuke Imamura is a senior distinguished researcher at NTT Space Environment and Energy Laboratories. He received his Ph.D. from Tokyo University of Agriculture and Technology in 2005. He worked as a JSPS Research Fellow at The University of Tokyo from 2005 to 2009. He joined Chuo University as an assistant professor in 2009 and was appointed associate professor at the Tokyo Institute of Technology in 2012. In 2019, he was a visiting professor at the Institute for Plant Biochemistry, Heinrich Heine University. In March 2021, he joined NTT Space Environment and Energy Laboratories. His research interests include the regeneration of the global environment using photosynthetic organisms, especially algae, with molecular biology approaches.

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Dr. Koji Yamada is the head of the Advanced Science Research Institute at Euglena Co. Ltd. He received his Ph.D. from the University of Tokyo in 2009. He worked as a project assistant professor at the University of Tokyo from 2009 to 2012 and as a special postdoctoral researcher at RIKEN from 2012 to 2014 before joining Euglena Co. Ltd. in February 2014. He specialises in molecular genetics, and his current primary research focus is the development and practical application of algal breeding techniques.

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