Increasing the production of biofuels like ethanol could be a significant step toward lowering the global consumption of fossil fuels. However, ethanol production is hampered in large part by its reliance on corn, which is not grown in sufficient quantities to meet a significant portion of the United States’ fuel needs.
To try to broaden the potential impact of biofuels, a team of MIT engineers has now discovered a way to produce such fuels using a broader range of non-food feedstocks. Feedstocks such as straw and woody plants are currently difficult to use for biofuel production because they must first be broken down to fermentable sugars, a process that produces numerous byproducts that are toxic to yeast, the microbes most commonly used to produce biofuels.
The MIT researchers devised a method to avoid the toxicity, making it possible to produce biofuels from these much more abundant sources. They also demonstrated that this tolerance can be engineered into yeast strains used to produce other chemicals, potentially allowing “cellulosic” woody plant material to be used as a source for biodiesel or bioplastics.
“What we really want to do is open up cellulose feedstocks to almost any product and take advantage of the sheer abundance that cellulose provides,” explains Felix Lam, an MIT research associate, and the study’s lead author.
Researchers found a way to reduce cellulosic feedstocks’ toxicity to yeast, making it feasible to use these abundant feedstocks to produce ethanol, biodiesel, or bioplastics.
The paper’s senior authors are Gregory Stephanopoulos, the Willard Henry Dow Professor in Chemical Engineering, and Gerald Fink, the Margaret and Herman Sokol Professor at the Whitehead Institute of Biomedical Research and the American Cancer Society Professor of Genetics in MIT’s Department of Biology.
Boosting tolerance
Currently, approximately 40% of the corn harvest in the United States is used to make ethanol. Because corn is primarily a food crop that requires a lot of water and fertilizer, plant material known as cellulosic biomass is seen as an appealing, noncompeting source of renewable fuels and chemicals.
According to a US Department of Energy study, this biomass, which includes many types of straw and parts of the corn plant that are typically unused, could amount to more than 1 billion tons of material per year, enough to replace 30 to 50 percent of the petroleum used for transportation.
However, there are two major barriers to using cellulosic biomass: first, cellulose must be liberated from the woody lignin, and then the cellulose must be further broken down into simple sugars that yeast can use. The particularly aggressive preprocessing required produces aldehydes, which are highly reactive and can kill yeast cells.
To overcome this, MIT researchers expanded on a technique they developed several years ago to improve yeast cells’ tolerance to a wide range of alcohols, which are also toxic to yeast in large quantities. In that study, they discovered that spiking the bioreactor with specific compounds that strengthen the yeast membrane helped the yeast survive much longer in high ethanol concentrations. Using this method, they were able to increase the traditional fuel ethanol yield of a high-performing yeast strain by about 80%.
The researchers engineered yeast in their new study so that it could convert the cellulosic byproduct aldehydes into alcohols, allowing them to use the alcohol tolerance strategy they had already developed. They tested several naturally occurring enzymes that perform this reaction from various yeast species and found one that worked best. They then used directed evolution to improve it even further.
“This enzyme converts aldehydes into alcohols, and we’ve shown that using the other methods we’ve developed, yeast can be made a lot more tolerant of alcohols as a class than it is of aldehydes,” Stephanopoulos says.
Yeast is typically inefficient at producing ethanol from toxic cellulosic feedstocks; however, when the researchers expressed this top-performing enzyme and spiked the reactor with membrane-strengthening additives, the strain more than tripled its cellulosic ethanol production, reaching levels comparable to traditional corn ethanol.
Abundant feedstocks
The researchers demonstrated that high ethanol yields could be obtained using five different cellulosic feedstocks, including switchgrass, wheat straw, and corn stover (the leaves, stalks, and husks left behind after the corn is harvested).
“With our engineered strain, you can essentially get maximum cellulosic fermentation from all of these normally very toxic feedstocks,” Lam says. “The great thing about this is that it doesn’t matter if your corn residues aren’t great one season. You can use energy straws instead, or if straws aren’t readily available, you can use some sort of pulpy, woody residue.”
The researchers also inserted their aldehyde-to-ethanol enzyme into a yeast strain engineered to produce lactic acid, a precursor to bioplastics. This strain, like ethanol, was able to produce the same amount of lactic acid from cellulosic materials as it did from corn.
This demonstration suggests that it may be possible to engineer aldehyde tolerance into yeast strains that produce other products like diesel. Biodiesels have the potential to have a significant impact on industries such as heavy trucking, shipping, and aviation, which lack an emission-free alternative such as electrification and rely heavily on fossil fuel.
“We now have a tolerance module that can be bolted on to almost any type of production pathway,” Stephanopoulos says. “Our goal is to apply this technology to other organisms that are better suited to producing these heavy fuels, such as oils, diesel, and jet fuel.” The research was supported by the Department of Energy and the National Institutes of Health in the United States.
