A study of the microbes that allow cows to digest grass could lead to better ways of making cellulosic biofuels.

Biofuels made from agricultural waste, sawdust, and prairie grass promise to be more economical than biofuels derived from corn, sugarcane, and other food crops.

The first step in cellulosic biofuels is converting tough plant materials made of cellulose and lignin into sugars that can then be fermented to make fuels. But this is expensive and currently requires a large quantity of enzymes to break down cellulose. “We’re talking truckloads,” says Frances Arnold, a professor of chemical engineering at Caltech who was not involved with the cow research. “We need a two- to fivefold reduction in the cost of enzymes,” she says.

In contrast, the microbes that live in the part of the bovine digestive tract called the rumen have been turning cellulose into sugar efficiently for millions of years. Researchers hope a new database of  28,000 genes sequenced from microbes involved in bovine digestion will help engineers come up with new enzymes, and bring down the cost of making cellulosic biofuels.

So far, manufacturers have brought down the costs of making cellulolytic enzymes mostly by changing processing methods. Another approach would be to make enzymes that work faster or work under different conditions, such as extreme temperatures, that might facilitate the breakdown of plant matter. “To begin to lower the costs of making cellulosic biofuels, we need new enzymes that do more,” says Eddy Rubin, director of the U.S. Department of Energy Joint Genome Institute. Rubin led the cow-microbe study.

The trouble is that an estimated 99.9 percent of all microbes on earth, including those in cow rumen, cannot be grown in culture in the lab. So bioprospectors looking for natural microbial enzymes with industrial promise have had a very limited pool of material to work with. Fortunately, new gene-sequencing technologies are changing that, allowing researchers to discover microbial enzymes by looking in their genes. Without having to grow microbes in the lab, researchers can sequence all the genetic material present in an entire ecosystem, then screen this data for genes of interest. This type of research is called metagenomics.

Rubin’s group started their search for better cellulolytic enzymes by studying termites in 2007. Microbes living in termite guts ferment woody roughage into sugars. The trouble with termites, Rubin says, was that “it was hard to get much material to work with” because termite guts are small. The studies didn’t generate many of the full-length genes needed to make working enzymes.

The cow rumen can hold over 150 liters of digesting food—giving the researchers plenty of material to work with. Cows are particularly advantageous test subjects for this type of study for another reason. Agricultural scientists have developed a system for putting a window-covered opening into cows’ rumens. It’s literally possible to look into the stomach through this window, called a fistula, and to put experimental samples inside and then draw them out. Working with researchers at the University of Illinois who have cows fitted with fistulas on campus, the Joint Genome Institute researchers put bags of switchgrass into the cow stomachs, let them sit for 48 hours, and took them back out again. Microbes found adhering to the switchgrass were presumed to be involved in fermenting it.

The researchers then separated out the microbes, broke them down, and sequenced all the genetic material they found. The researchers were able to produce a tremendous amount of data about the genes, and some of the genomes, found in the cow rumen. They found about 250 billion base-pairs worth of genes, about 10 times more than make up the human genome.

The challenge was then to interpret all that data. Using the high-performance computing facilities at California’s Lawrence Berkeley National Lab, the group compared the cow-microbe sequences with a database of sequences known to code for enzymes that break down carbohydrates. This led to a pool of 28,000 genes for further study. The researchers then used lab bacteria to make the proteins coded by 90 of these genes, and tested their functionality. About half of them were able to break down cellulosic materials.

In addition to the 28,000 genes identified, the researchers were able to reassemble the genomes of several previously undiscovered microbial species. To test this part of the study, they isolated a single unculturable bacterial cell from the rumen samples, and sequenced its genome. It matched one of the ones they had assembled. “This biological reality check makes us quite confident,” says Rubin.

How the new database will be used isn’t clear. “It’s an encyclopedia for people to pull out,” says Rubin.

Previous efforts to come up with new cellulytic enzymes haven’t resulted in much. There are two ways to do it. One is to try to make the enzymes more active. Another way, one that Rubin and Caltech’s Arnold both consider more promising, is to find or make enzymes that are not only more active, but also work under extreme conditions that might help facilitate the breakdown of tough plants, such as high-temperature, high-salt solutions–conditions that destabilize today’s enzymes.

Meanwhile, researchers are applying metagenomic analysis to other microbial communities that ferment tough plants. David Weiner, head of research and development at the enzyme company Verenium, says his company already has termite-microbe enzymes in its product-evaluation pipeline. The company was involved with the earlier termite studies done by the Joint Genome Institute researchers and has developed a platform to speed up the testing of new enzymes. Weiner says the company is also looking for enzyme genes in other ruminants, including zebras, and in samples taken from, for example, decomposing logs. Verenium sold part of its celullolytic enzyme business to BP last fall.