Yeast may be essential to producing ethanol through fermentation, but for years, biofuel production has been constrained by the fact that heat and ethanol itself can be deadly or damaging to yeast at high levels.

Recently, researchers at Chalmers University of Technology in Gothenburg, Sweden, and the Massachusetts Institute of Technology have found ways to ameliorate both of these production problems. They published their research yesterday in the journal Science.

Jens Nielsen, a professor of systems biology at Chalmers University of Technology, was among the researchers interested in improving yeast's heat tolerance.

Finding a way to make yeast better able to withstand heat would make ethanol production cheaper. With current industrial yeast strains, ethanol producers have to cool the yeast fermentation to 30 degrees Celsius (86 degrees Fahrenheit). While this may be an optimal temperature for the yeast, it isn't for the producers. The enzymes needed to break down the sugars and starches work better around 40 C (104 F), and the cooling process adds to the cost of production.

Instead of trying to manipulate specific genes involved in heat tolerance, Nielsen and his colleagues used a process called adaptive laboratory evolution.

"We said, 'Why don't we try to do nature's approach and try to do an evolution of this?'" he said.

They exposed three yeast cultivations with numerous yeast strains to temperatures around 40 C for extended periods and observed what genetic changes emerged.

After three months and more than 300 generations, the yeast abruptly started to grow well in all of the cultivations at the higher temperature.

"It's really Darwinian survival of the fittest. We found quite a large number of new mutations," Nielsen said.

The one mutation that was consistent among different heat-tolerant strains was a single point mutation that produced a different type of sterol in the cell membranes, called fecosterol.

'A very novel discovery'
Normally, the cell membranes contain ergosterol, similar to the cholesterol found in human and animal cells. Unlike ergosterol, fecosterol has a bent shape and is similar to the sterol-like molecules that protect some types of bacteria and plants against higher temperatures. More importantly, the mutation that produced fecosterol was inheritable and it did not appear to have any negative side effects on the yeast's ability to grow or reproduce, according to Nielsen.

"This is a very novel discovery," he said. "We can also transfer this trait in industrial yeast strains and use it for large-scale ethanol production."

Across the Atlantic Ocean, at MIT and the Whitehead Institute of Biomedical Research, a second team of researchers has been working on a way to make yeast more ethanol-tolerant.

The researchers found that by adding potassium chloride and potassium hydroxide to the medium growing the yeast, they could improve its alcohol tolerance and extend the amount of time that individual cells could produce ethanol.

By doing this, the researchers increased the concentration of ethanol they produced by around 80 percent, according to Gregory Stephanopoulos, a professor of chemical engineering at MIT and one of the authors of the study.

"Toxicity is probably the single most important problem in cost-effective biofuels production," he said.

The increased alcohol tolerance can be traced back to the medium's influence on the electrochemical membrane gradients across the cell membranes. When yeast cells are exposed to a lot of ethanol, the alcohol makes the cell membrane more porous. The proton and potassium pumps were better able to compensate for the damage to the cell membrane when the medium was enhanced with the potassium chloride and potassium hydroxide.

Not only that, the yeast were able to consume all of the available glucose, according to the study.

Potential production savings
The researchers tested the medium on a wide variety of yeast strains.

"The effect is equally strong in industrial strains and lab strains, and the effect is almost identical," Stephanopoulos said.

The electrochemical gradient was further enhanced when the researchers boosted the expression of genes related to the potassium and proton pumps.

They developed the idea of altering the yeasts' medium based on their previous research showing that more heat-tolerant yeast had higher expression of phosphate-related genes. When they introduced potassium phosphate to the medium in which they were growing the yeast, they saw "extraordinary results" when they compared ethanol yields.

Initially, it wasn't clear whether it was the potassium or the phosphate that was driving the increased production. Eventually, the researchers figured out that the added potassium was enhancing the cell membrane's potassium pump, while the phosphate was increasing the pH of the medium.

The results extended to other alcohols that are also toxic to yeast.

"The more we understand about why a molecule is toxic and methods that will make these organisms more tolerant, the more people will get ideas about how to attack other, more severe problems of toxicity," Stephanopoulos said.

Stephanopoulos' lab is now experimenting with growing yeast in different types of media to see whether they can further reduce the toxicity effects of ethanol and other alcohols.

The researchers at both universities are confident that their results are transferable from the laboratory to commercial ethanol production. That could, in turn, translate into savings for the U.S. ethanol fuel industry, which produced 13.3 billion gallons of ethanol in 2013 and 7 billion gallons in the first half of this year, according to the Energy Information Administration.

The two methods could even be used together to create a yeast strain that is both heat and alcohol-tolerant, Nielson said.

"The two could easily be combined; I don't see any technical hindrance to combining the two approaches," he said.

Reprinted from Climatewire with permission from Environment & Energy Publishing, LLC., 202-628-6500