Termite-Tailored Cellulosic Ethanol

The diverse ecosystems that mark the landscape of Costa Rica, ranging from lowland rain forests to cloud forests, lakes and rivers, are a hot spot for life. For scientists, this treasure trove of biodiversity represents yet to be discovered insights into medicine, species diversity and for some, the commercialization and development of enzymes for cellulosic ethanol production.
By Jessica Ebert | February 05, 2008
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.

To that end the team headed to Central America. "Verenium has agreements with the Costa Rican government and has established a lab there so they had the infrastructure and permits for sampling termites," Warnecke says. "Besides, everyone knows that Costa Rica is a hot spot of biodiversity so there are many termite species."

On the team's first trip to the country they found a nest on the trunk of a tree that housed hundreds of termites belonging to a species of Nasutitermes. The nest was about the size of a football and the walls were paper thin like a wasp nest. "We knew that all the individuals in this nest would be coming from the same queen," Warnecke says. "So we were sure we weren't comparing apples and pears."

The scientists pooled the hindgut contents from a couple of hundred termites, extracted the DNA from the community as a whole and sequenced a large number of genetic fragments. By comparing these sequences with other DNA fragments of known sequence and functionality, the team could determine the types of bacteria present as well as the genes these bacteria carried. Overall, about 300 different bacterial species were identified along with more than 500 genes linked to cellulose and hemicellulose degradation. "These enzymes are consistent with the diet of this termite species," Hugenholtz explains. Nasutitermes species are foragers. They climb out of the nest, scurry across the ground and feed on dead wood from several different species with a range of plant cell wall compositions. "That's why they're loaded with this enzyme artillery to break down these different plant cell wall types," he adds.

While the DNA analysis was going on, Verenium was isolating proteins directly from the hindgut fluid, sequencing those molecules and testing them for activity. "From that you could tell definitely that the genes weren't just there they were being expressed and producing the enzymes," Hugenholtz says. "This was confirmed for quite a few cellulases and xylanases and other enzymes involved in the process."

Raymond Orbach, DOE's under secretary for science has described the termite and its bacterial cargo as a remarkable machine. "Termites can digest a frightening amount of wood in a very short time, as anyone who has had termites in their house is painfully aware," he says. "Instead of using harsh chemicals or excess heat to do so, termites employ an array of specialized microbes in their hindguts to break down the cell walls of plant material and catalyze the digestion process. Industrial-scale DNA sequencing by DOE JGI was key to identifying the genetic structures that comprise the tools that termites use. Our task now is to discover the metabolic pathways generated by these structures to figure out how nature digests plant materials. We can then synthesize the novel enzymes discovered through this project to accelerate the delivery of the next generation of cellulosic biofuels."
That is what Verenium continues to work on. The challenge lies in finding the mixture of enzymes that work optimally for specific feedstocks and certain pretreatments. "We are currently testing our cocktails of enzymes on a variety of feedstocks and a variety of pretreatments to determine how well they perform," says Kevin Gray, director of biofuels at Verenium.

Jessica Ebert is an Ethanol Producer Magazine staff writer. Reach her at [email protected] or (701) 738-4962.