Key biofuel-producing microalgae thought to be a single species are actually three

When a global pandemic forced former graduate student Devon Boland, Ph.D., out of the lab and into computers, he uncovered a world of hidden differences in the long-studied species Botryoccocus braunii and discovered that it wasn’t just one species, but three.

Botryococcus braunii was first discovered in the mid-1800s. Technically speaking, it is a plant, it undergoes photosynthesis and, most interestingly for researchers, produces large quantities of hydrocarbons that can be used as a renewable source of fuel.

Until now, it was thought to be a single species made up of three races: A, B and L, each of which produces slightly different types of oils. But after discovering a dramatic 20 to 30 percent genetic difference between each race, a team of researchers at Texas A&M AgriLife has proposed a new classification, fulfilling every biologist’s dream of naming species.

“As a graduate student, you read papers that all say the same thing, which is that this is one species with three chemical races, and you take it in,” said Boland, first author of the study showing the genomic comparisons. “You start thinking that must be true. No one has found otherwise, and all these scientists have had much longer careers than I have — I’m just a kid.”

“But I ended up proposing names for a species that were accepted for publication, which I never thought would happen.”

Half necessity, half circumstance

Before coming to Texas A&M, Boland devoted his undergraduate research to “core” biochemistry research in areas such as protein engineering. His senior thesis was to focus on the production process used by Botryococcus braunii to synthesize its unique hydrocarbons.

But when the COVID-19 pandemic hit, Boland worried about losing time on his thesis and how it might delay his graduation.

In response, Tim Devarenne, Ph.D., associate director of undergraduate programs and associate professor in the Department of Biochemistry and Biophysics in the Texas A&M College of Agriculture and Life Sciences, suggested that Boland take the opportunity to delve into genetic data and bioinformatics.

“Having the genome of the organism you are interested in mapped is always ideal in research because it makes it easier for you to find the genes and work to determine their functions,” Devarenne said.

Another former graduate student in the lab, Daniel Browne, had sequenced and assembled the genome of race B. During one of Devarenne and Boland’s weekly meetings, Devarenne proposed that they try to do the same thing with races A and L.

“It had a double benefit,” Boland said. “We were able to do something that had never been done before, and it could help us better understand hydrocarbon biosynthesis.”

Although the breeds are virtually indistinguishable under a microscope, Boland said there has been debate over whether they are different species. They were interested to see if a genomic study could shed light on the issue.

In addition to Devarenne, Boland and Browne, the research team included Ivette Cornejo Corona, Ph.D., a postdoctoral researcher in Devarenne’s lab; John Mullet, Ph.D., another researcher and professor in the Department of Biochemistry and Biophysics; Rebecca Murphy, Ph.D., a former graduate student in Mullet’s lab; and a longtime collaborator on Botryococcus studies, Shigeru Okada, Ph.D., a professor at the University of Tokyo in Japan.

Genetic analysis

Although Botryococcus is commonly studied for its hydrocarbon production, sequencing its genome has proven difficult.

Boland, now an adjunct research scientist at the Texas A&M Institute for Genome Sciences and Society, said the thick, oily medium in which cells live makes DNA extraction and isolation difficult.

Nevertheless, the team was determined to analyze the genomes to see how similar the genes and proteins involved in each breed’s biofuel production processes were.

But after reconstructing the genomes and using supercomputers at Texas A&M’s High-Performance Research Computing Center to perform genomic comparisons, Boland said it became clear that these organisms were not the same species.

“Everywhere we looked, things were different,” he said.

Ultimately, the researchers said, about 1 in 5 genes were unique to each of the Botryococcus races. To put that 20 percent difference in perspective, the genetic difference between humans and chimpanzees, our closest evolutionary relative, is less than 2 percent.

After some further validation, Boland and Devarenne set about reclassifying the Botryococcus races. Boland said the team spent months working on different names.

They kept race B with its original name, Botryococcus braunii, to preserve its history and renamed race A to Botryococcus alkenealis and race L to Botryococcus lycopadienor, signifying the type of hydrocarbons each produces.

What makes a species?

In recent times, biologists have placed more emphasis on genes and genomes when it comes to classifying organisms.

But even with all the evidence to support considering these Botryococcus algae as distinct species, Devarenne said what really makes a species is general acceptance by the scientific community.

After publishing their study in PLOS ONEDevarenne shared the team’s findings with more than 100 other researchers who study the organisms in their own labs.

“How we define different species may not change much in how these organisms are used in research,” he said. “But it is important for scientific understanding to understand how we think about the relationships between these organisms and with all other species.”

Boland said he and Devarenne published their work in an open-access journal so other scientists could build on their work. The species’ complete genomes are also available on the National Center for Biotechnology Information website.

“It was important to us that the information be made publicly available as soon as it was ready to be published,” he said. “Science is a community affair. The ultimate goal is always to further our collective knowledge, and I think that’s what we’ve accomplished here.”