Better Living Through Algae Biotechnology

ABOVE: Microalgae are a diverse group of organisms with underexplored metabolic abilities. © istock.com, Elif Bayraktar

At present, Earth is the only planet known to be capable of supporting life. But despite the fact that humanity cannot reasonably exist anywhere else, the way that humans currently live on this planet is simply not sustainable in the long term. From resource-intensive agriculture and pollution to climate change and global biodiversity loss, humans must fundamentally alter their relationships with the environment to ensure that Earth stays within certain planetary boundaries and remains habitable for our species.^1–3

As the famed British naturalist David Attenborough noted, “In our hands now lies not only our own future but that of all other living creatures with whom we share the Earth.”⁴

While there’s no silver bullet for all of humanity’s sustainability problems, some researchers believe that microalgae—tiny photosynthetic eukaryotes found in nearly every aquatic environment on the planet—might be an important piece of the solution. As scientists delve into algal genomes and metabolic pathways, they are identifying and figuring out how to enhance the remarkable abilities of these tiny creatures. They found algae that can fix nitrogen, produce lipids and proteins for food and bioplastics, and recover nutrients from wastewater; applying these algal abilities at scale could have important implications for fossil fuel usage, agriculture, pollution, and conservation.^5–8

The Biofuel Bubble

In the 2000s and early 2010s, algal biofuels were touted as the fuel of the future; companies even promised to produce “fuel from thin air,” using only water, sunlight and CO₂. Researchers knew that many species of algae produced energy-dense lipids that could be converted to biofuels, and teams around the world began to explore how to supercharge this lipid production. 

Scientists also understood that algae upregulated lipid biosynthesis in response to certain kinds of stressors, including nitrogen restriction.⁹ In the long term, however, algae cannot continue growing without nitrogen. Researchers hoped that elucidating the mechanism underlying stress-induced lipid production could help them engineer algae that made ample amounts of lipids even when they were not stressed. 

A research team at Yale University characterized the transcriptomes of Neochloris oleoabundans algal cells under nitrogen-rich and nitrogen-poor conditions, finding several pathways that were associated with greater production of triacylglyceride, a biodiesel precursor.¹⁰ Other groups identified genes that could be overexpressed to increase fatty acid accumulation in a diatom—a type of algae encased in a cell wall made of silica—called Phaeodactylum tricornutum.^11,12 

The blueprint that nature gives us is that eukaryotes acquire nitrogen fixation by endosymbiosis.

 —Ellen Yeh, Stanford University

Researchers from the Massachusetts Institute of Technology and the National Renewable Energy Laboratory even explored algae as a sustainable strategy for manufacturing hydrogen for fuel. Using a bioengineered fusion protein, they managed to shift the balance of photosynthetic output, coaxing algal thylakoids—the tiny, energy-harvesting sacs inside of chloroplasts—to turn less of the sun’s energy into sugar and put more energy into producing hydrogen.¹³

Despite promising preliminary work, the reality was that cultivating enough algae to make a dent in the enormous global demand for fuel would require more than just water, sunlight, and CO₂. Researchers brought up concerns that algae’s nutrient demands and the energy required to run the facilities and process algal products into usable fuel would be prohibitively expensive.¹⁴

Stephen Mayfield, a molecular biologist at the California Center for Algae Biotechnology, initially believed in the promise of algae biofuels—even co-founding the startup Sapphire Energy.    

“But then in about 2012, it occurred to me, ‘Wait a minute, we're never really going to make algae biofuels,’” said Mayfield. “Because fuel is the lowest value product that you make. Everything is more valuable than fuel.” Furthermore, the cost of other renewables such as wind and solar keeps falling, making these more attractive options.¹⁵ But algae—a group that contains tens of thousands of different species—is not a one-trick pony. Researchers took what they had learned about algal genomes and metabolic pathways, and the strategies to manipulate them, and began exploring whether algae’s unique biological capabilities could be applied in other ways to help decrease humanity’s negative effect on Earth.

Power Plants

While burning fossil fuels likely constitutes one of the biggest threats to the planet’s continued habitability, the agricultural systems required to feed billions of humans are also an important component. “Agriculture is such a burden on the environment,” said Elizabeth Hann, a biologist at the Wyss Institute. There are many different components of this burden: Agriculture requires huge amounts of fresh water, and in turn creates water polluted with pesticides and nutrients, which can be devastating to aquatic ecosystems. Agriculture turns forests—hotspots of biodiversity and important carbon-sequestering ecosystems—into vast spaces of monocultures. “There’s a huge need to change our food systems,” said Hann.

Researchers are now exploring how algae might help humans create a more sustainable food system. Algae have been around for a long time—possibly as long as a billion years—so they have had plenty of time to evolve certain interesting abilities, some of which might help scientists engineer better crop plants.¹⁶ For example, researchers from Aix-Marseille University are studying the biochemical pathways underlying algae’s ability to concentrate CO₂, which enhances photosynthetic efficiency.¹⁷

Other researchers, including Stanford University microbiologist Ellen Yeh, explore how the intracellular mechanisms at work within certain algal species could be applied to the problem of nitrogen fixation. Yeh spent more than a decade studying malaria before pivoting into algal biology research. At first glance, it might seem that pond scum does not have much in common with a parasite that thrives inside the human body, but Yeh said that these two microscopic critters are more similar than they first appear. Apicomplexan parasites, which include Plasmodium and Toxoplasma, evolved from a free-living, photosynthetic ancestor.¹⁸ These ancestors had organelles called chloroplasts, which enabled them to carry out photosynthesis; in Plasmodium, these chloroplasts evolved into non-photosynthetic plastids called apicoplasts. For years, Yeh explored the function of these evolutionary relics and how they might be targeted by a new generation of anti-malarial therapies. 

Studying Plasmodium and the apicoplast made Yeh curious about algal sister lineages. “I think [this research] really opened up for me the world of non-model [organisms] as well,” said Yeh. “I like that they break all the textbook rules of biology; anything that I was taught in cell biology, there was always an exception in malaria … It really got me on the idea that we were really missing a lot of microbial diversity.” 

At the same time, Yeh watched climate change become an increasingly urgent issue. “I felt like biologists should be able to participate more in helping to solve those problems,” said Yeh. “Particularly because most of carbon cycling and nitrogen cycling is through organisms—through biological mechanisms.”

So, when the pandemic hit and life screeched to a halt, Yeh had time to consider how she might apply her expertise to this planetary problem. 

Some organelles such as chloroplasts and mitochondria are thought to be remnants of an ancient endosymbiosis.¹⁹ Yeh gained plenty of experience with unusual endosymbiotic organelles during her work on the apicoplast, so she applied her skills to study an unusual endosymbiont present in algae. 

While diatoms—now classified in the genus Epithemia—that house nitrogen-fixing endosymbionts were reported in 1980, this odd couple had received relatively little attention from scientists in the following decades.²⁰ Yeh knew that the nitrogen requirements of crop plants were a major environmental concern: nitrogen-polluted runoff creates massive dead zones in rivers, lakes, and oceans across the planet.²¹

Bioengineers around the world have made valiant attempts to endow cereal crops including wheat, maize, and rice with nitrogen-fixation abilities, but have not yet been successful.²² According to Yeh, there are a few different reasons why this might be an especially tricky problem to solve, even with the sophisticated genetic engineering tools available today. First, for an organism to build the nitrogenase enzyme, which converts nitrogen available in the atmosphere (N₂) to a form that can be used by plants (ammonia, or NH₃), it needs several different genes that encode the protein’s multiple subunits as well as assembly factors to piece together these subunits into a functional protein.²³ This means that researchers not only have to add multiple genes to an organism’s genome, their expression must be properly coordinated to produce sufficient amounts of the functional enzyme.

Furthermore, said Yeh, “Nitrogenase is very, very sensitive to oxygen. So, if you put that into a photosynthetic cell, that's a problem because the chloroplast produces oxygen. [Nitrogenase] also has very high energy demands … So, you really need to be able to couple it to cell respiration.”

For these reasons, incorporating nitrogen-fixation abilities directly into a plant genome is a tall order—a problem that even the great experimenter of all time, evolution, has not yet solved. “It's like billions of years of experiments were being run, but no one was keeping the lab notebook, so you only see the end results,” said Yeh.

“If you look in the natural world, you don't find eukaryotes that have acquired nitrogenase genes, even though I think there are many, many opportunities to do so, [since] DNA really does flow fairly freely between bacteria and eukaryotes,” she continued. “The blueprint that nature gives us is that eukaryotes acquire nitrogen fixation by endosymbiosis.”

Thus, studying the relationship between photosynthetic algae and their nitrogen-fixing endosymbionts could give scientists new strategies to engineer crops that can pull nitrogen from the air. To study these algae, however, Yeh first had to find them. Unlike bacterial cultures, most algae don’t remain viable after freezing, and none of the algae scientists that Yeh contacted had maintained continuous cultures of the Epithemia algae that she was looking for. 

Although there are relatively few known eukaryotic species with nitrogen-fixing endosymbionts, they are extremely widespread in freshwater ecosystems throughout the world. “[One of] the graduate students took a hike and decided to just grab some water from a random creek that she passed by,” said Yeh. They cultured these samples in nitrogen-free medium, and when some of the wells turned brown, they researchers knew they had a hit: an algal species that could fix nitrogen from the air. They named these organisms Epithemia clementina, for their resemblance to microscopic orange wedges.

Now, Yeh is exploring how a nitrogen-fixing bacterium might make the transition from independent living to life as an endosymbiont, including the genetic modifications needed to swap resources with the host or protect its nitrogenase enzymes from the host’s oxygen production.²⁴

“Then you can imagine how we could use genetic engineering to do the same thing in the lab,” said Yeh. “The hard part, of course, is that we have lots of tools like CRISPR for genetic engineering organisms, changing their genomes. But physically taking a bacterial cell and putting it inside a eukaryotic cell is not something we have as many tools for. One of the big steps that we're really working on is figuring out how to do that in the lab.” 

While some research groups explore ways that algal biology could help improve the crops that currently exist, others think that microalgae itself could be an important future food source: Many edible species are around 40 percent protein and others contain a high percentage of lipids, including in-demand omega-3 fatty acids.²⁵ Some companies already sell algae-based meat alternatives and cooking oils. Another company has genetically modified the fatty acid-producing algae Prototheca moriformis with the lysophosphatidic acid acyltransferase enzyme to manufacture analogs of the fats found in human milk for potential use in infant formula.²⁶ 

Algae: The Next Green Revolution It’s no secret that human activities can be detrimental to planetary health and biodiversity; agriculture, manufacturing, and the simple acts of daily life use up natural resources, burn fossil fuels, and pollute water systems. Now, researchers study how algae, a diverse group of ancient unicellular organisms, might provide new approaches for sustainable living.

1) Food production Algae could provide proteins for alternative meat and lipids for cooking oils and omega-3 fatty acids. 2) Crop improvement Special abilities possessed by certain algae species, like nitrogen fixation and enhanced photosynthesis efficiency, could be engineered into staple crop plants. 3) Water treatment Algae could reduce nutrient pollution in wastewater at the same time as recovering minerals like phosphorus, which could be reused as a component of fertilizer. 4) Other products Algae could produce more sustainable versions of organic compounds, such as squalene, that are currently harvested from the marine environment, or make ingredients for biodegradable plastics.

Trash into Treasure

While fertilizer runoff is a major factor in promoting harmful algal blooms and hypoxic dead zones, it is far from the only way that anthropogenic pollution damages aquatic ecosystems. The production of nutrient-laden wastewaters is almost an inevitability of human habitation: sewage treatment plants, livestock operations, urban stormwaters, food processing plants, and industrial aquaculture all contribute to eutrophication.²⁷

In natural water bodies, these nutrients are damaging largely because they promote excessive algal growth. Sometimes the damage is caused by the deadly toxins that some algal species produce, but overgrowth of normally-benign algae can also wreak havoc. After a massive bloom, the algae die and the microbes that decompose their tiny corpses deplete the water’s dissolved oxygen, which in turn kills fish, crustaceans, and bivalves. 

But what if this nutrient-polluted water could be used to grow useful types of algae, wondered Peter Lammers, a microbiologist at Arizona State University. His previous research on algal biofuels had taught him that these tiny creatures can produce complicated lipids, proteins, and carbohydrates. So, he reasoned, “Why not couple legally mandated water clean up to the production of novel and valuable biomolecules?”

Why not couple legally mandated water clean up to the production of novel and valuable biomolecules?

 —Peter Lammers, Arizona State University

While initially it may be hard to view wastewater as a valuable resource, Lammers sees great possibility. For example, in a recent collaboration with bioengineer Kyle Lauersen at King Abdullah University of Science and Technology, Lammers metabolically engineered a strain of Cyanidioschyzon merolae algae to produce a ketocarotenoid called astaxanthin.²⁸ Dietary carotenoids, including astaxanthin, are necessary for the vibrant pinkish-orange of wild salmon flesh; pure astaxanthin, which costs thousands of dollars per kilogram, is used as an animal feed additive to give farmed salmon the appropriate color that customers prefer.²⁹ These algae, said Lammers, could be used to treat wastewater from salmon farms, reducing ecological damage, while at the same time producing this valuable compound.   

There are naturally-occurring algae that produce astaxanthin, so why did the researchers need to genetically engineer C. merolae? Because this species has abilities that few other algae—and indeed few other living creatures—possess. C. merolae, forged in the volcanic fields of southern Italy, thrives in scorchingly hot and blisteringly acidic environments. This, said Lammers, helps prevent unwanted visitors.

“If there's a single lesson that we've learned, it’s that basically there is no algae out there that isn't vulnerable to competitors, pathogens, and grazers,” he said. “There are all kinds of protozoa and viruses and fungi that will take down a large culture. And if you keep growing the same thing in the same place—the first year, you're always going to do great. The second year, [the yield] goes down, and the third year it goes down again. The environment has all of these pests, and if you lay out a brunch table for them, they come to feed … But by using extremophiles, we greatly limit that process.”

While reducing pollution from fish farms is undoubtedly ecologically important, it is likely not game-changing in terms of climate. So, Lammers also wants to put algae to work on a larger scale; he hopes that another species of extremophile algae called Galdieria sulphuraria could help make a dent in greenhouse gas emissions by recovering nutrients, such as nitrogen and phosphorous, from wastewater. 

Today, most crops depend on synthetic fertilizers for nitrogen, phosphorous, and potassium. Producing and using these synthetic fertilizers, however, may account for as much as five percent of global greenhouse gas emissions.²⁹ In part, this is due to burning natural gas to convert atmospheric N₂ to ammonia, the form plants can use. “Then, in most wastewater treatment facilities, we reverse that process,” said Lammers. “We take the ammonia or nitrate, and we turn that back into N₂ … so there's negative greenhouse gas impacts on both ends of that nitrogen cycle. That’s why we want to circularize this process.”

Lammers and Nirmala Khandan, an environmental engineer at New Mexico State University, have recently developed a pilot-scale G. sulphuraria-based water treatment system.³⁰ Algae are housed in a bioreactor, which traps heat from the sun, and fed a diet of nutrient-rich sewage that is pH-adjusted to their acidic preferences. In less than seven days, they reduced wastewater nitrogen and phosphorous concentrations to meet wastewater discharge standards. This was only the beginning, however. 

The algae are then collected and processed with hydrothermal liquefaction, which involves heating and pressurizing the biomass to kill any pathogens and produce bio-crude oil and biochar, a charcoal-like material, along with leftover liquids. Components of the biochar can then be combined with the liquids and magnesium chloride to produce struvite, a mineral containing phosphorous, nitrogen, and magnesium that can be used as fertilizer. Although this is a complicated process, the researchers estimated that the value of the oil and struvite was enough to compensate for the costs of running the system. 

A World of Possibility

This may be only the tip of the iceberg of algal abilities, however. “More than 60 percent of the genes in the diatom genome basically have no reference whatsoever with any other gene in any other organism,” said Michele Fabris, a molecular biologist at the University of Southern Denmark. “There’s so many novel functions that we have no idea about.”

Even Chlamydomonas reinhardtii, arguably one of the most extensively studied algal species, still contains many mysteries concerning what its genes actually do. Hann said that algal mutant libraries—in which a piece of DNA is randomly inserted into the genome, disrupting the function of whatever gene it is inserted into—can provide a more complete picture of genetic function. “We use that library to screen for mutants that have either increased lipid content or decreased lipid content,” said Hann. Studying what happens when a gene is “broken” is an effective, if time-intensive, way to understand a gene’s role in the functioning of the organism.

It's surprising how much biological diversity and metabolic diversity is out there in the algae field.

 —Michele Fabris, University of Southern Denmark

There are many potential applications to explore. Mayfield uses algae-derived compounds to make rapidly biodegradable plastics to help combat rampant plastic pollution.⁷ Researchers at Kyoto University have tinkered with the genome of a green algae species to accumulate squalene, a compound widely used in cosmetics and vaccines—one of the major natural sources of squalene today involves the unsustainable harvest of livers from deep-sea sharks.^31,32

Advances in genome sequencing and algae-compatible genetic engineering techniques mean that researchers may be able to not only up- or down-regulate certain metabolic pathways, but also create algae with entirely new abilities. As Fabris noted, plants are very good at producing high-value compounds that are used in pharmaceuticals, colorants, flavorings, and fragrances, but they are often only produced by a tiny fraction of the plant cells, perhaps those in the flowers, fruit, or seeds.³³ “A huge amount of plant biomass is required to harvest these compounds,” he said.

While yeasts or bacteria can be engineered to produce some of these compounds, in other cases, researchers run into difficulties. “The metabolic setup of yeast and bacteria can be pretty different from that of plants,” said Fabris. “Some microalgae—even though they are distinct from plants—might have a more similar setup, and be able to use similar metabolic pathways.”

Fabris’ research team is currently evaluating different species of microalgae to determine which are best suited for this type of production. They are also working on genetic engineering strategies to coax them to produce monoterpenes and sesquiterpenes. This diverse group contains compounds that are used as flavors and fragrances, and some that undergo further processing to produce chemotherapy medications.³⁴  

The process of engineering a strain of algae, growing it into large cultures, measuring the amount of the desired molecules it produces—and repeating ad nauseum to optimize production—is extremely time-consuming. To expedite the process, Fabris is developing an algal biosensor.³⁵ “We want to have something that works at the single cell level, something that detects the amount of the molecule that is produced … and provides a rapid and easy readout so that we can quickly go back to the drawing board if we need to adjust something," he said.

Scientists believe there are still many discoveries to be made in this underexplored group of organisms and foresee a bright future for algal biology. “It's surprising how much biological diversity and metabolic diversity is out there in the algae field,” said Fabris. “So, there might be other species that are ideally suited for each biotechnological or industrial application, we just need to find it and understand it and perhaps also optimize it.”