The New Feedstock Frontier

Cutting-edge research and development has the potential to turn conventional wisdom on its head and revolutionize how, and from where, plant oils are obtained.
By Ron Kotrba | May 17, 2018

Detractors have long argued that one of the greatest limiting factors to biodiesel growth is feedstock availability, but what if convention were tossed aside and the once-impossible became completely feasible? Soybean oil, for instance, provides half of the feedstock for U.S. biodiesel production.

Modern farming practices and improved crop varieties have led to incremental increases in per-acre yields over time, but what if scientists could tap into the genetic makeup of the beans and remove the inhibitor that caps oil production at 20 percent? Or how about if researchers could redirect oil production to grow significant quantities of harvestable oil not in seeds but in vegetative stalks and leaves? Work at Brookhaven National Laboratory and schools such as the University of Illinois Urbana-Champaign and the University of Florida are pioneering such research to make the impossible a reality.

Scientists at BNL led by biochemist John Shanklin have identified an enzyme—ACCase—central to determining the rate of oil production in oilseeds. The enzyme is made of four subunits, all of which are necessary for the enzyme to function. With all four subunits in place, ACCase drives the first step in fatty acid synthesis. Previous work by Shanklin in 2012 revealed that when plant cells were fed a short-term excess of fatty acids, a “feedback loop” inhibited this enzyme, slowing oil production. When fatty acid concentrations dropped within two days, the enzyme and oil production would turn back on—but a longer-term excess of fatty acids would permanently disable the enzyme.

At the time, scientists knew of multiple ways to inhibit the enzyme, but none explained the irreversible inhibition observed. When University of Missouri researchers in 2016 discovered an inactive version of one of the four enzyme subunits, Shanklin thought the inactive subunit taking one of the active subunit’s place might be at play and sought to test this hypothesis. It turns out cells with combinations of the disabled genes didn’t turn off oil production. In effect, disabling the inactive subunits turned off the off-switch.

“We had been studying what happens when the plant senses it has made too much fatty acid,” Shanklin tells Biodiesel Magazine. “Just like the thermostat in your house, when the temperature gets to its high point, the furnace  turns off.” He says living systems use homeostasis, which is a set point not unlike a thermostat in a house used to control temperature. “We tried to figure out how this thermostat worked in plants, so when it has too much of something it stops producing it,” Shanklin explains. “Our hypothesis was that this new component”—aptly named BADC, for biotin-attachment-domain-containing proteins—“could be a candidate. We identified a mutant and showed how plants that did not contain BADC made more fatty acids. The BADC protein was the thermostat.”

One might expect wild plants being fed excess fatty acids would limit oil production, but the BNL team was surprised to see that, even under normal conditions, the enzyme driving oil production was much more active in plant cells with the disabled genes than in the wild, unmodified variety. “That means that, even under normal conditions, inactive subunits are putting the brakes on ACCase, reducing its activity and limiting oil production,” Shanklin says.

The work was performed on Arabidopsis due to its rapid growth and small stature. “Soybeans take a whole season to grow,” Shanklin says. “It’s like how human geneticists work on fruit flies because it’s much faster. Arabidopsis grows very rapidly, and you can grow thousands of plants in a small space. It’s an oilseed about nine-inches high compared to canola, which is about three feet high.”

Shanklin says this research, recently published in Plant Physiology, will soon move into the hands of seed companies. They will take their own elite lines, the specific properties of which they know intimately, and make manipulations themselves for in-depth study. “Whenever you change something like this, you have to check whether it’s changing agronomic properties,” he says. “There has to be field testing. We provide the intellectual foundation upon which others will build.” Shanklin postulates that this development could provide a 20 to 30 percent increase in oil yields for crops such as soybeans. “That would be a guess,” he says. “Forty percent would be a big jump.”

Shanklin says he wears two hats in this type of work. “The first is as a basic scientific researcher, understanding the fundamental mechanisms of oil production in plants—the end of the story in that role is discovery of new mechanisms, which points the way to strategies to improve oil yields in commercial crops. But I also wear a second hat and participate in a large project with partners such as the universities of Illinois and Florida, which is an applied science project taking very productive crops like sugarcane and trying to rewire them metabolically to produce oil.”

Earlier this year the U.S. DOE awarded the University of Illinois a $10.6 million, five-year grant to transform two productive nonoilseed crops—a variety of sugarcane called energycane and miscanthus—into high-oil-yielding crops that could revolutionize the plant oil industry. The new research project is called ROGUE, which stands for Renewable Oil Generated with Ultra-productive Energycane.

Shanklin says he received a call from Steve Long at University of Illinois in 2012. “With my knowledge of lipids, he asked me if there was a way to rewire sugarcane to produce more oil,” Shanklin says. “We are co-principal investigators on ROGUE.” The work utilizes computer models that project these two crops can achieve 20 percent oil, a major increase from natural levels of less than a tenth of 1 percent. “If fully successful, these crops could produce as much as 15 times more biodiesel per unit of land compared to soybeans, a food crop that currently produces half of our nation’s biodiesel,” says Long, who leads the project’s efforts at the Carl R. Woese Institute for Genomic Biology at University of Illinois.

In oilseeds—as the name suggests—oil accumulates in the seeds, whereas in vegetative or biomass crops such as energycane or miscanthus, oil is produced but is then metabolized. “What we learned before this project is, in sugarcane, if we regulated [oil] synthesis and inhibit consumption, we get oil accumulation,” Long tells Biodiesel Magazine. “We added a protein found in rapeseed, which coats the oil and ‘hides’ it from the plant so it’s not metabolized—that was the strategy we took.”

In earlier work, the researchers were able to demonstrate 8 percent triglyceride accumulation in sugarcane leaves. “If we add the fatty acids, this is more like 12 percent,” Long says. ROGUE is targeting accumulation in the stems rather than the leaves. Long says this oil is much the same as oil accumulated in oilseed crops such as soy or canola. “It’s really very similar to soy oil or other temperate oilseeds,” he says.

Long says one aspect of the ROGUE project is modifying oil synthesis in the stems for accumulation. “We’re identifying gene promoters that target oil accumulation in the stem,” he says. “Oil requires more energy than sugar, so we’re also boosting photosynthesis, and our work there would have relevance for other C4 crops. In this project, however, we’re only applying this to energycane and miscanthus.” The actual genetic transformation work is being performed  at University of Florida.

“They’re one of the most experienced places in the world doing this,” he says. “We’ve shown we can accumulate oil in vegetative tissue like leaves, but now we want to get oil in the stem—and that’s probably one of biggest challenges presently.” Why the stems? Because that’s where 80 percent of the biomass is, “so that’s where you want the oil,” Long says. “Furthermore, the technology is in place to harvest and process the stems—that’s what sugarcane processors do. To process the leaves, you would need an organic solvent to get the oil out. And it wouldn’t achieve the high numbers needed to make this attractive.”

Long discusses the patented method to extract the oil from the energycane stems. “It’s based on technology used in sugarcane processing,” he says. “The stems are crushed with hot-water washes to get out sugar, but this also removes any oil, which floats.” He says the mixture is then centrifuged, much like what’s used in corn ethanol plants to remove distillers corn oil. “The technologies exist to do all of this,” Long says. “Energycane is grown the same way as sugarcane and processed the same way. Then a centrifuge can be used to get the oil out after the crush.”

The commercial potential for grasses is “really huge,” Shanklin says. “Our goal in ROGUE, for instance, is to eventually get to 15 to 20 percent oil by dry weight.” He says even if the research led to commercialization and extraction of energycane yielding just a tenth of their goal—1.5 percent oil in the stem—this would yield on a per-acre basis the same amount of oil as canola. “If we realize our goal of 15 to 20 percent, then 15 percent oil content in one acre of this grass would be equivalent to the amount of oil from 10 acres of canola,” Shanklin says. “And at the end of it, there’s still a whole lot of sugarcane stems left that can be dissolved and made into ethanol or used to fire the plant.”

Targeting grassy crops such as energycane or miscanthus that can be grown on marginal lands is important to Long, Shanklin and this work. “The Southeast seems to be our best target,” Long says. Shanklin agrees. “There’s a lot of marginal land in the Southeast U.S. currently not used for commercial production of anything,” Shanklin says. “This land could easily be converted to grow ‘oilcane’ without impacting food supply. If we can use this land that is otherwise fallow at moment, we completely avoid the food vs. fuel problem. That’s something I’m very excited about—creating new sources of renewable fuel with a very low carbon footprint on land that is fallow. Owners can make money from the land and it doesn’t have to compete with food production.”

As recently as 10 years ago, scientists thought oil was only produced in seeds and that there was a reason for this. “The big surprise is plants can tolerate high levels of oil production in any tissue, it’s just that evolution selected production of oil in seeds because it’s the most energy-dense form of storage,” Shanklin says. “Because they don’t make much oil in the leaves, we thought there was a biological reason for this. But to our surprise the reason is that it’s just not necessary. When we try to make oil in leaves, we can make high levels without any real detriment to any other function.”

Shanklin says we are on the verge of extremely exciting developments in the next decade. “I think it’s fair to say the potential for this research is enormous for boosting the biodiesel industry in terms of volumes produced, and its significance in reducing fossil fuel use.”

Author: Ron Kotrba
Editor, Biodiesel Magazine
218-745-8347
rkotrba@bbiinternational.com

 
 
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