Among the hundreds of thousands of species including various orchids, toucans and tropical fish that flourish in the sultry climate of Costa Rica, one group of insects holds the attention of scientists and technology developers with a stake in the biofuels industry. Wood-boring termites abound in the lush foliage of this Central American country. But it’s not the termites themselves that people are betting on—it’s the microbes within the guts of these segmented cellulose-feasters that may hold a key to toppling the barriers to commercial-scale cellulosic ethanol production. “Being a general microbial ecologist, it’s just a rule of thumb that if there’s some job going on you can put your money on it that bacteria are doing that job,” says Phil Hugenholtz, head of the Microbial Ecology Program at the U.S. DOE Joint Genome Institute. The JGI based in Walnut Creek, Calif., was created to unite the expertise and resources in genome mapping, DNA sequencing, technology development and information sciences pioneered at the DOE genome centers at Lawrence Berkeley National Laboratory, Lawrence Livermore National Laboratory and Los Alamos National Laboratory. For some time, the role of bacteria in metabolizing the wood ingested by termites was highly unclear. However, in a recent issue of the journal Nature, Hugenholtz along with other researchers from universities, private industry and the DOE report that the bacteria seem to be doing a great deal of the work. “These bacteria are absolutely loaded with hydrolytic enzymes principally for cellulose and xylan [the main component of hemicellulose] decomposition,” he says. “This is good news.”

Community Sequencing
All termites serve as vehicles for various microbial passengers. These microscopic organisms are more than just hitching a ride, however; the relationships forged between these two diverse groups of organisms are necessary and beneficial. Termites provide microbes with a protected, nutritious place to live and in return, the microbes make enzymes able to crack the intricate cage of cellulose, hemicellulose and lignin that form the cell walls of plant material. Breaking this armor releases sugars that can be fermented into a host of products including ethanol. In the termite gut, microbes convert these sugars into other compounds that drive the metabolism of the termite. This mutually beneficial association has been well-documented in a group of insects dubbed the “lower” termites. These insects carry unicellular microbes called flagellates in their guts. Flagellates have long been known to produce cellulases and hemicellulases, hydrolytic enzymes that disassemble woody materials.

“Higher” termites, on the other hand, don’t harbor flagellates. Instead, these insects are packed with bacteria. Although higher termites are the most abundant and diverse of all the termites, very little is known about how or even if the bacteria residing in their intestinal tracts play any role in degrading cellulose. “Most of the research done on termite microbiology in the past 20 years or so dealt with lower termites,” explains Falk Warnecke of JGI’s Microbial Ecology Program.

One way to find out if these termite-gut bacteria do play a role is to isolate them, grow them in the laboratory, sequence their genetic information and see if there are any genes that code for hydrolytic enzymes. “Only a fraction of the organisms in the natural world can be studied like this,” Hugenholtz says. That fraction doesn’t include the bacterial species associated with the termite gut. “When you have these organisms that grow on plates and you sequence them and characterize their enzymes you only see a skewed picture of all the enzymes that are out there in nature,” he explains. To get around this, Jared Leadbetter, an environmental microbiologist at the California Institute of Technology proposed to sequence the entire community of termite-gut microbes. This method is termed metagenomics and allows researchers to study all DNA from a particular environment rather than the genetic contribution of a single cell or organism.

Leadbetter initiated the project by applying to the Community Sequencing Program at JGI. The CSP is a DOE-funded program designed to allow genomic sequencing of systems of relevance to the agency’s missions including those associated with global carbon cycling, alternative energy production and bioremediation. Leadbetter’s proposal involved examining the microbial members of the hindgut pouch—the largest part of the termite gut—of higher termites that belong to the genus Nasutitermes. “This was a fairly risky project when we proposed it,” Leadbetter says. “In these abundant tropical termites, there was no compelling evidence that microbes play direct roles in cellulose degradation.” Once the proposal was accepted, the fun began.

Termite-Gut Bacteria
Early on in the project, several other groups joined the Leadbetter team including microbial ecologists at JGI, researchers at Verenium Corp. and INBio, the National Biodiversity Institute of Costa Rica. The first step was to find termites. “Termites are very small so one termite is not enough for doing the genome sequencing,” Warnecke says. The termite hindgut consists of a viscous fluid with a toothpaste-like consistency. Although it’s the largest intestinal compartment, the volume of the hindgut liquid from a single termite would only form a pinhead-sized dot if placed on a piece of wax paper. “We needed a couple of hundred termites,” Warnecke explains.

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