Some Like It Hot

Yeast species L. Orentalis. Credit: Steve Ruizin

In the hunt for more sustainable biofuels, microbes derived directly from crop environments are heating up the chase. Recent research led by Professor John Taylor at UC Berkeley’s Energy Biosciences Institute (EBI) has shown that fungal and yeast strains taken from hot decomposing plant matter will thrive at high temperatures, enabling higher yielding industrial processes for cellulosic ethanol production.

Taylor, who is a professor in the Department of Plant and Microbial Biology (PMB), has made his career studying fungal evolution, focusing heavily on the organization of fungal populations in the environment. Now, he is using his expertise to take on the challenge of rising atmospheric carbon dioxide (CO2) levels and global warming. Taylor had been troubled for some time by the environmental effects of modern society’s dependence on fossil fuels. When EBI was established at UC Berkeley in 2007, he saw it as an opportunity to take action. “After years of talking about CO2, I almost felt slimy not to get involved,” he says today. Together with other EBI scientists, Taylor has since been working on a particularly difficult problem in advanced biofuel production: the conversion of cellulose obtained from sustainable crops into clean burning ethanol.

Cellulose is the long-sugar polymer that forms the rigid scaffolding of plant cell walls. Thus, while it stores an enormous amount of energy, it is also highly robust. For this reason, making biofuel from cellulose is far more challenging than conventional methods, which start with sugars and starches directly. The sturdy polymers must first be broken down before sugars can be converted to ethanol. State-of-the-art industrial processes already exist to digest cellulose chains into sugars using enzymes that are secreted by the fungus Trichoderma reesei. Yeast can then ferment these sugars into ethanol. However, low fuel yields have prevented these processes from being cost-effective alternatives to current fuels.

Drs. Bruns and Taylor next to a crop of Mescanthus. Credit: John Taylor

Taylor’s initial study at EBI focused on finding new fungi that could break down cellulose better than existing industrial strains. “We suspected that the environment would provide much more efficient strains.” Along with fellow PMB professor Tom Bruns, Taylor collected samples from farms at the University of Illinois and sugarcane plantations in Louisiana by isolating fungi that grew on powdered Miscanthus giganteus, a promising crop for biofuel production. To increase the chances of isolating useful environmental fungi, Taylor employed a classical ecological sampling technique called “dilution to extinction.” Before introducing the samples into the growth medium used for isolation, they were diluted extensively. “By diluting to the point where each tube can have at most one living fungal cell we avoid selecting for fast growing species,” Taylor says of his approach. This process allows him to find strains that are less abundant or slower growing, and it is these rare strains that are often the most exciting. Indeed, many of the fungi isolated from Miscanthus released more sugars from the plant tissue than Trichoderma, confirming the potential for identifying useful fungal strains from the environment.

After the success of this first sampling excursion, Taylor worked with EBI on identifying new fungal targets to improve biofuel production. In practice, industrial biomass conversion and fermentation suffer from contamination by other microbes that can ruin both raw materials and equipment. An attractive remedy to this problem is to perform the process at elevated temperatures where potential contaminants cannot grow. However, in order for such a process to work, the active fungi must be resistant to these elevated temperatures. To find fungi compatible with this technique, Taylor, Bruns and their team again looked to nature, isolating cellulose-degrading fungi from high temperature environments. These microbes are named thermophiles: literally “heat-loving” organisms. Taylor’s team sampled from active compost piles, which are often extremely hot due to the energy released from decaying plant matter. “We were sampling from sites where you couldn’t reach your arm in up to your elbow without getting burned,” Taylor describes. The fungi isolated from this set of samples can now be used to produce enzymes for high temperature cellulose degradation processes.

To complete the process, Taylor and his team also sought thermophilic yeasts for conversion of sugars to ethanol. Fortunately, a unique farming practice at the original sampling sight in Louisiana provided another ideal high-temperature environment. After extracting sugar from sugarcane, farmers discard the fibrous waste, called bagasse, in large piles where it breaks down slowly. Much like compost piles, the bagasse gets quite hot as it decomposes. By sampling from these piles, Taylor found two promising species of yeast, Kluyveromyces marxianus and Issatchenkia orientalis, both capable of growing at high temperatures. Eventually, he hopes these yeasts can be used for more efficient fermentations.

Though the fungi already isolated could complete a novel high-temperature cellulosic ethanol process, Taylor is not yet satisfied. Moving forward, he hopes to sequence the genomes and perform association analysis on variants of his newly discovered strains isolated from a variety of locations. This approach involves correlating temperature-sensitivity and fermentation efficiency to specific parts of the fungal genome. In this way, Taylor hopes to identify genes that could be useful for engineering microbes optimized for biofuel production. Thanks to his new projects, Taylor is no longer feeling slimy. “As a basic biologist, it’s been nice to work on something that has a direct application.” With any luck, his continuing research will play an important role in advancing the next generation of biofuels from the laboratory to your gas tank.

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