Leaving on a Bio-Jet Plane

One hundred and ten years ago when Wilbur Wright piloted the first U.S. passenger flight by taking employee Charles Furnas along for a ride, it’s unlikely that either of them ever dreamed of jet liners, let alone jets that use fuel made by yeast from switchgrass.

Today, the airline industry is committed to having at least 1 percent of its jet fuel, roughly 600 million gallons a year, sourced from petroleum alternatives, and the U.S. Department of Defense is looking to biojet fuel as a way to diversify its fuel supplies to lessen our dependence on foreign oil.

Like other transportation industries, aviation is concerned about the amount of greenhouse gases it contributes to the atmosphere, especially because aviation is among the fastest growing sectors globally in terms of both economics and emissions. The airlines have set a goal of carbon-neutral growth by 2020 and thereafter. This will be accomplished by increased efficiencies, sourcing fuels with a low carbon footprint, and purchasing carbon credits. By 2050, the industry is also seeking to halve its 2005-level carbon dioxide emissions.

A direct way for the airlines to cut carbon dioxide emissions would be to blend or substitute petroleum aviation fuel with biojet fuel. But the technology for biojet and renewable jet fuels lags behind fossil fuel alternatives for cars and trucks by at least a decade. Biojet (or renewable jet) fuels are produced from plant sources. These fuels are interchangeable with conventional aviation fuel, but the two types of fuel are typically blended (for example, half and half).

Removing fundamental barriers to cost-effective renewable or biojet fuel is key to limiting carbon dioxide emissions, given that other primary power sources like electric batteries and hydrogen fuel cells are unlikely to work for passenger jets.

Now, ARS scientists have successfully advanced the prospects of biojet fuel with two major leaps forward: Newly identified yeasts and an ARS-developed switchgrass with superior biofuel and agricultural traits. One of the research team’s additional successes was to find a new scheme to bioprocess switchgrass into biojet fuel, one with clear potential to be commercially feasible.

“We have come up with a system to start out with a pile of switchgrass and conveniently bioprocess it into a refinable oil for a second-generation biojet fuel that could be directly mixed into conventional aviation fuel,” says ARS chemical engineer Patricia Slininger. She is in the Bioenergy Research Unit of the ARS National Center for Agricultural Utilization Research (NCAUR) in Peoria, Illinois.

First-generation biofuels are those made from food crops, for example, ethanol from corn. Second-generation, or advanced, biofuels are made from cellulose, and possibly hemicellulose. These fibrous materials come from the stalks and leaves of bioenergy plants, like switchgrass, or from the residual or nonfood parts of food crops. Woody plants, like poplar, are another source.

While biofuel is harder to produce with second-generation sources, their use does overcome the concerns of possibly diverting food supplies or raising food costs. It also broadens the plant material (biomass) available for converting to biofuel.

In place of typical yeasts that ferment switchgrass into sugars to make ethanol—the conventional pathway to a biofuel—Slininger and her team are using completely different yeasts that convert switchgrass sugars directly into lipids, or fats. These fats have a chemical profile that very closely resembles common household vegetable oil—and biodiesel or biojet fuel.

“We knew there were reports in the scientific literature of ‘oily yeasts’ with some capabilities to produce fats from sugars. The question was, how efficiently could they do it, starting with the plant fiber of switchgrass?” Slininger says.

“NCAUR also happens to be home to the ARS Culture Collection, one of the world’s largest collections of fungi, which includes the yeasts. So we asked the yeast collection curator, Cletus Kurtzman, whose knowledge of yeast properties and traits was beyond encyclopedic, to point us in the right direction. Otherwise we might still be checking out strains,” Slininger adds. (Kurtzman, who was inducted into the ARS Hall of Fame in 2016, passed away November 27, 2017.)

The researchers screened about 75 yeast strains. These yeasts had been collected from all over the world, and the best producers came from Russian soil. From the start, Slininger and ARS chemical engineer Bruce Dien were after yeasts that had unique abilities, such as thriving in the presence of growth inhibitors produced during pretreatment and producing exceptionally high amounts of oily fat.

All cellulosic or fibrous plant material has to be pretreated to make its sugars available for fermentation by yeast, Slininger explains. The standard pretreatment for switchgrass is hot dilute acid followed by enzymes to extract the sugars on which the yeast cells feed, grow, and store oil globules. Less common is pretreating with ammonia—which is a base rather than an acid—followed by enzymes that convert the plant fibers to sugars. While possibly more expensive, it has the advantage of not generating the growth inhibitors that the dilute acid pretreatment does.

Slininger and Dien screened for yeasts that performed well after either acid or base pretreatment.

“We applied for patents on the five top fat-producing yeast species, covering those that did best in acid or base conditions and those that could do well under both conditions,” Slininger says.

Two yeasts, Lipomyces tetrasporus and L. kononenkoae, produced more than 10 grams of oily fat per liter of pretreated switchgrass. But the researchers weren’t going to leave it to the yeasts alone to work as the fermenting factory. They needed to reach a production point far above 10 grams to get within reach of interest to the biofuel industry.

They pursued an improved process to reach ultra-high fat accumulation. The method took advantage of the fact that yeast cells require nitrogen for growth, but when nitrogen is in short supply compared to the amount of sugars, the cells make and store fat instead of making more yeast cells.

Based on that, Slininger and Dien devised a multistage fermentation procedure with a “yeast cell fattening step” that greatly amplified the oil concentration. In the first stage, the pretreated switchgrass was enriched with nitrogen to support abundant yeast cell production. Then the large yeast population was transferred to a second stage in which the switchgrass is rich in sugars, but unsupplemented in nitrogen, a condition that leads to increased lipid production.

“The final super-oil-enriched yeast broths had concentrations of 28-29 grams of oily fat per liter,” Dien says. “That’s an amazing yield increase of 270 percent better than any previous work, and it is a level that is of commercial interest. With yeasts, theoretically up to 190 gallons of oil per acre could be produced from switchgrass. In contrast, soybeans, the common source for biodiesel, only produce 68 gallons of oil per acre.”

The researchers note that this kind of success wouldn’t be possible with just any switchgrass.

This work was done with Liberty switchgrass, a variety developed and released by ARS in 2013. Bred for outstanding biomass production and winter hardiness, Liberty far exceeds the potential bioethanol production of other popular switchgrass cultivars, such as Summer or Kanlow.

“Liberty produces exceptional amounts of biomass, which can be used for producing ethanol or oils for biodiesel and biojet fuel. It can grow on marginal land that can’t be used for growing food crops and in northern areas such as Michigan and Minnesota, where farmers welcome having an alternative crop,” Dien says.

Slininger and Dien are still working on improving their process and selecting a yeast that can convert switchgrass more efficiently.

“There is actually a pretty simple way to screen for the yeast cells that have the best individual ability. Each yeast cell stores the oily fat it makes internally,” Slininger explains. “Fat globules are more buoyant than water. This property means we can design an aqueous solution to mix with the cells. Then, after centrifuging the mix, we can pull off the ones that float higher as better converters.”

As they refine conditions, the researchers may return to the ARS Culture Collection to screen for other yeasts with other abilities or let natural selection play a role as cultures are challenged with specific operating conditions that will guide future strain evolution.

“Next, we really need a partner to begin looking into scaling up the technology, to begin looking towards seeing a new industry emerge. We’ve done what ARS does best—open the doors. Now industry needs to grab hold,” Slininger adds.

Not ARS’s First Time

Research that creates the basis for new biobased products or overcomes barriers to a blossoming bioprocessing industry is one of ARS’s specialties. ARS scientists played that role when the modern biodiesel industry was just beginning in 2001.

The industry was in need of a critical advance to put biodiesel on the road to being a mainstream auto fuel. In the first biodiesel formulations, temperatures near or below freezing caused waxy crystals to form that could plug fuel lines and block fuel filters. ARS scientists developed a fuel additive that improved biodiesel's ability to start up engines in cold weather.

With that fix in place, biodiesel made from soybean oil became a competitive fuel, with the added advantage of being able to cut unburned hydrocarbon emissions by 67 percent compared to petroleum-based diesel—not to mention cutting the country’s dependence on foreign oil.

Mixing petroleum diesel with 20 percent biodiesel reduces carbon dioxide emissions by 16 percent, carbon monoxide emissions by 20 percent, particulate matter by 22 percent, and sulfates by 20 percent.

ARS researchers also helped the fledgling biodiesel industry strengthen its wings by developing needed tools for the industry, such as an easy, rapid method to guarantee precision and quality control when manufacturing different blends of biodiesel and petroleum diesel.

In 2016, the U.S. biodiesel market was a record high 2.8 billion gallons, according to the U.S. Environmental Protection Agency.

In 2017, a handful of commercial airliners flew well publicized pilot flights with biojet fuels.

Imagine what the Wright brothers would have had to say.—By J. Kim Kaplan, ARS Office of Communications.