Biofuels on tap

Buried beneath a sulfurous cauldron in European seas lies a class of microbes known as extremophiles, so named because of the extreme environment in which they live and thrive. In the hot, acidic neighborhoods of undersea volcanic vents, some of the world’s most unusual forms of life make their homes. Not surprisingly, such hardscrabble organisms possess enzymes — the chemical workhorses of cells — with similarly unique and useful abilities: They break down cellulose into sugars with remarkable efficiency.

Biochemist Joanne Volponi prepares enzyme samples for assaying.

Such enzymes are interesting to researchers studying ways to make biofuels from plant materials — called lignocellulosic ethanol — cheaply. If the enzymes can be enlisted to do much of the chemical deconstruction work, they might unlock a new transportation economy based on ethanol derived from candidate plants other than corn.*

A Sandia group led by Blake Simmons is among a handful of U.S. research teams studying extreme enzymes’ utility for ethanol production. Sponsored by Sandia’s program, the project will demonstrate computational modeling tools and enzyme engineering methods that could lead to more efficient biofuel processing techniques. The effort is in its second year.

Waste as fuel

Lignocellulose, one of the most abundant renewable organic materials on Earth, is a mix of complex sugars and lignin that gives strength and structure to plant cell walls. To produce fuel ethanol, lignocellulose is, with the help of enzymes or acids, broken down and converted to glucose, or sugar. The glucose is then fermented to produce ethanol and carbon dioxide.**

Because of their chemical simplicity, starchy plants such as corn are the easiest to convert to biofuel. Corn, however, is an important source of food and agricultural feed, so large scale production of ethanol from corn is not seen as a long-term solution to the world’s burgeoning fuel demands.***

Ideally other sources of lignocellulose, such as the billion-plus tons of biomass created annually as byproducts of the timber and agricultural industries, could be converted to fuel instead. This could alleviate the economic pressure on the corn industry (and perhaps lower corn and meat prices) imposed by the growing demand for corn-derived fuel ethanol.

The primary hurdle preventing plant-derived ethanol from becoming a viable transportation fuel is not the availability of biomass, but rather its efficient and cost-effective processing, says Simmons.

Deep-sea hydrothermal vent
(Courtesy of U.S. National Oceanic and Atmospheric Administration)

“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,” he says. “The process of turning certain lignocellulosic materials into ethanol is very difficult and costly.” That process typically involves several pretreatment steps that break up the initial lignocellulosic material into easily converted biopolymers, such as cellulose, for eventual fermentation.

Quirky microbes

Enzymes isolated from extremophiles might help solve this processing problem. Such microbes — from an ancient branch of microbial life, the archaea, discovered by scientists in 1977 — can be found in a variety of places, including hot springs, deep-sea heat vents, gold mines, and within the rust found under a leaking hot water heater. Some can live without sunlight or carbon as food, and instead survive on sulfur, hydrogen, and other materials that normal organisms can’t metabolize.

While other researchers are examining common biomass sources and attempting to express their enzymes at higher temperatures and lowered 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?” asks Simmons.

Sandia’s current microbe of interest is Sulfolobus solfataricus, an organism that prospers in high temperature sulfuric acid environments and expresses enzymes that efficiently break down cellulose into sugars.

Using samples of the DNA that produces these extreme enzymes, the researchers are modifying the enzymes’ genetic sequence with the goal of improving performance.

Candidate mutations are identified through computational modeling performed at Sandia that compares the structure and sequence of the extremozymes with their more benign counterparts to identify key genetic patterns.

The computational modeling has helped the researchers focus on those predicted changes to the genetic sequence that, if a matching enzyme was created in the lab, would have the best chances of improving performance.

“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,” says Simmons. “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.”

Realistic goal

Modeling is helping researchers identify beneficial changes to an enzyme’s genetic sequence.

While various researchers are investigating new technologies and facilities that will allow for processing cellulosic biomass into ethanol, Simmons and his team are hopeful that their method can be efficiently and cheaply integrated with current and future pretreatment steps.

“We believe the use of enzyme engineering to enable the next generation of ethanol bio-refineries, with a focus on improving enzymes isolated from extremophiles, is a realistic and achievable goal,” he says.

The project is a component of the Joint Bio- Energy Institute (JBEI), a partnership of three national laboratories (including Sandia/California) and three research universities in the San Francisco Bay Area, funded by the U.S. Department of Energy’s Office of Science.

JBEI researchers will tackle key scientific problems that hinder the cost-effective conversion of lignocellulose into biofuels and other important chemicals. They also will develop the tools and infrastructure to accelerate future biofuel research and production efforts, and help move new technologies into the commercial sector. Sandia’s JBEI focus will be cost-effective,biologically based renewable energy sources to reduce U.S. dependence on fossil fuels. The Lab’s capabilities in enzyme engineering, systems biology, membrane transport, protein expression, and hyperspectral imaging are expected to contribute to the JBEI mission.

For more information about JBEI, visit www.jbei.org.