Extreme Makeover-Nature Edition

Sandia researchers are looking to biology in earth's extreme environments to help solve the cellulosic ethanol puzzle. Their enzyme studies may provide the key needed to spark an industry.
By Mike Janes
Buried beneath a sulfurous cauldron in European seas lies a class of microorganisms known as "extremophiles," so named because of the extreme environmental conditions in which they live and thrive. Perhaps almost as radical is the idea that these organisms and their associated enzymes could somehow unlock the key to a new transportation economy based on a renewable biofuel-cellulosic ethanol.

That's the concept behind an internally funded research program, now in its second year, at Sandia National Laboratories. Sandia is a multiprogram laboratory operated by Sandia Corp., a Lockheed Martin company, for the U.S. DOE's National Nuclear Security Administration. Sandia has major research and development responsibilities in national security, energy and environmental technologies, and economic competitiveness.

As researchers search for ways to cheaply and efficiently process cellulosic biomass for the production of cellulosic ethanol, the Sandia project aims to successfully demonstrate various computational tools and enzyme engineering methods that will make extreme enzymes relevant to the technical debate.

Blake Simmons, a chemical engineer and project lead at Sandia's Livermore, Calif., site, says the primary hurdle preventing cellulosic ethanol from becoming a viable transportation fuel is not the availability of cellulosic biomass, but rather its efficient and cost-effective processing. "Production is not a concern," Simmons says. "More than a billion tons of biomass is estimated to be created each year in the timber and agricultural industries, as well as a variety of grasses and potential energy crops. Unfortunately, you can't just take a tree trunk, stick it into an enzymatic reactor, and ferment the sugar produced into ethanol with any kind of efficiency. The process of turning certain lignocellulosic materials into ethanol is very difficult and costly."

That process typically involves several pretreatment steps that break up cellulosic material into easily converted polymers, according to Simmons.

Continuing with the tree trunk analogy, Simmons says the laborious process typically begins by chopping the biomass to reduce its size and then delivering it into a dilute acid pretreatment reactor. The reactor would then break down the biomass into cellulose, hemicellulose and lignin. The hemicellulose and cellulose polymers released from the biomass must go through additional processing and acid neutralization before the final product is recovered and placed back into an enzymatic reactor to deconstruct the polymers into fermentable sugars. Not exactly swift and efficient, Simmons says. It's also very costly.

Using Nature's Own Extreme Enzymes
Enter enzymes isolated from extremophiles, which may solve this vexing processing riddle. Simmons says Sandia's current biological object of interest is Sulfolobus solfataricus, an organism whose extreme enzymes were isolated and discovered years ago by the German researcher Georg Lipps. Sulfolobus expresses cellulase enzymes that are known to exist in organisms that prosper in sulfuric acid environments and, through an inexplicable quirk of nature, efficiently break down cellulose into sugars.

"Biology generally likes sugar since it offers an easy energy intermediate that can be converted into some usable output," Simmons says. The Sandia team members are apparently among a handful of researchers looking at enzymes expressed by Sulfolobus and manipulating them in the laboratory with the objective of processing biomass into cellulosic ethanol, he adds.

Extreme enzymes can be found in a variety of locations, including hot springs, gold mines and even within the rust found under a leaking hot water heater, Simmons says.

While other researchers are examining common biomass sources and attempting to express their enzymes at higher temperatures and lower pH, Sandia has, in effect, taken the opposite approach.

"Instead of trying to create an extremozyme from sources that live in rather benign environmental conditions, why not just manipulate a real one isolated from its natural state?" Simmons says. Sandia has brought the DNA that produces these extreme enzymes into the lab, where researchers employ a technique called "site-directed mutagenesis" to manipulate and optimize the enzymes' genetic sequence in hopes of improving performance, he says. These mutations are identified using computational modeling techniques that compare the structure and sequence of the extremozymes with their more benign counterparts to identify key genetic sequences of interest.

"The ultimate dream-and it's only a dream right now-would be to take a poplar tree, put it into a tank, let it sit for three days, then come back and watch as the ethanol comes pouring out of the spigot," Simmons says. "Though we're probably decades away from that, this project aims to consolidate the pretreatment steps and get us one step closer to realizing that vision."

Ethanol Products the Same, Starting Material Vastly Different
The benefits of developing biomass-to-ethanol technology are well-known, says Grant Heffelfinger, senior manager for molecular and computational biosciences at Sandia's Albuquerque, N.M., site and the lab's lead on biofuels programs. He points to increased national energy security, reduction in greenhouse gas emissions, use of renewable resources and other oft-cited advantages. "But corn ethanol must compete with food markets, leaving lignocellulosic ethanol as the fuel most likely to make the most meaningful short-term impact in reducing gasoline's stranglehold on the transportation sector," Heffelfinger says.

Although the end product with cellulosic ethanol and corn ethanol is the same, Simmons points out the difference is in the complexity of the starting material. While corn is a simple, starch-based material that is easily processed into fermentable sugars, cellulosic biomass consists of a cellulose polymer wrapped within a complex vascular structure of lignin, hemicellulose and other components.

"Because lignocellulosic biomass is such a multifaceted material, we need to have a fundamental understanding of how it works," Simmons says. While various industry researchers are investigating new technologies and facilities that will allow for the processing of cellulosic biomass into ethanol, Simmons says he and his Sandia colleagues are hopeful that their method can be efficiently and cheaply integrated with current and future pretreatment steps. "We believe extremophile enzymes-and the technology that demonstrates how to use them-can be a very powerful resource for the research and industrial community to draw upon," he says.

Research Aimed at Commercial Partnerships
Simmons recently presented his team's preliminary findings from the extremophile project at the 4th World Congress on Industrial Biotechnology & Bioprocessing. The team hopes to publish more advanced findings soon and is finalizing several proposals that could lead to further funding. Simmons says the lab would be open to conducting collaborative research and development with other commercial partners or research entities, or to licensing its research capabilities.

This and other efforts at Sandia National Laboratories are expected to be a vital component of the Joint Bio-Energy Institute, a multilab/university effort to bring a U.S. DOE-funded bioresearch facility to the San Francisco Bay area. Sandia is planning a key role in that facility, which will focus on cost-effective, biologically based renewable energy sources to reduce U.S. dependence on fossil fuels.

"We believe the use of enzyme engineering to enable the next generation of ethanol biorefineries, with a focus on extremophile enzymes, is a realistic and achievable goal," Simmons says. "But we need others to believe, too."

Universities, industry or other institutions interested in partnering with Sandia on biofuels studies or other areas of research can contact Carrie Burchard, Sandia business development, at clburch@sandia.gov or (925) 294-1213.

Mike Janes is a media relations officer at Sandia National Laboratories' Livermore, Calif., site. Reach him at mejanes@sandia.gov or (925) 294-2447.