Microbial Tricks for the Cellulosic Ethanol Trade

In two recently reported research projects, scientists describe how they have taken cues from microbes that may ultimately lead to more efficient and cost-competitive production of biofuels.
By Jessica Ebert | July 08, 2008
Malcolm Brown Jr. and David Nobles Jr., molecular geneticists and microbiologists at the University of Texas, Austin, liken their approach to scientific exploration to the way the "Car Talk" brothers on National Public Radio seem to approach mechanical maladies, with quick wit, good humor and an expansive knowledge of their respective subjects. Although the puzzles may be different"Click and Clack" may try to solve a mysterious "clunk" that rings out each time someone tries to make a left turn in their '99 Jeep Cherokee while Brown and Nobles tinker with the genetics of bacteria to make the organisms produce cellulosethe objective is the same: increased efficiency and security, and reduced costs.

For Brown, the search for the solution to the cellulose biosynthesis puzzle has been ongoing for 40 years. "I've used many different living organisms as test organisms from cotton to trees to algae to even bacteria," he explains. The most prolific cellulose producer that Brown's team has ever worked with is the bacterium, Acetobacter xylinum, more commonly known as the vinegar bacterium because it's used to make vinegar. "It has given us probably the most information on how cellulose is made by any living organism," Brown says.

The potential of the vinegar bacterium as a new source of cellulose was only made more attractive by the fact that the cellulose secreted by the organism is a pure form of the molecule minus hemicellulose and lignin, which makes the biomass of wood and other plants so difficult to initially break down. However, a challenge to the large-scale production of cellulose by A. xylinum is that the bacterium requires a sugar such as glucose as an energy source to grow and subsequently make the molecule.

One way to overcome this challenge would be to colocate with a sugar mill, which could provide a rich supply of sugar to the microbes. This wasn't an option for Brown's team so they chose to search for other microbes that could grow and produce cellulose without being fed sugar. Photosynthesizers, or organisms that get their energy from the sun, were the obvious choice. "That's when I turned to David Nobles, who was then my graduate student and I said, could you check and see if there is cellulose in the cyanobacteria,'" Brown explains.

Cyanobacteria can be found in diverse habitats ranging from deserts to the oceans. They can survive exposure to high levels of radiation and can fix nitrogen from the atmosphere. Cyanobacteria are often referred to as blue-green algae but unlike their eukaryotic namesakes, these microbes multiply much more quickly.

In 2001, the Brown team published a paper in the journal Plant Physiology based on Nobles' work that described the machinery for cellulose production that certain species of cyanobacteria carried in their genes. "Until that time no one had any really good evidence that cyanobacteria could produce cellulose," Brown explains. "This study validated the fact that they make cellulose."

It turns out that the cellulose produced by cyanobacteria is incorporated into a gooey protective shell that encapsulates the cells. Unfortunately for Brown and Nobles, this naturally produced cellulose has some drawbacks including it's crystalline structure, which is difficult to break apart, the other polysaccharides that incorporate with the cellulose, and the small amounts of the molecule that the bacteria are able to make. Instead of trying to improve the outcome of cellulose production in cyanobacteria by modifiying the genome, Nobles took a strain of cyanobacterium that doesn't naturally produce cellulose and inserted the genes for cellulose production from A. xylinum.

At first, Brown and Nobles were disappointed in the results because the gel-like cellulose produced by the cyanobacterium didn't look like the molecule made by the vinegar bacterium. That's when the team had a "eureka" moment; the noncrystalline, low-molecular weight cellulose was easier to break down into glucose than the typical crystalline product made by A. xylinum. "That's when we started thinking about this source of cellulose as a feedstock for biofuel," Brown says.

Under the right growth conditions, the transgenic cyanobacterium also makes glucose and sucrose, two sugars that can be used directly to make ethanol. These molecules, along with the cellulose, are released directly into the liquid broth or medium used to grow the cells and can be easily separated from the microbial cells themselves. "What's exciting about this is that it can be done nondestructively. We don't have to kill the cells to get the products," Nobles explains. "It eliminates the need for harvesting the cells, it eliminates the need for extracting a product, and in the case of the sugars, it eliminates the need for any digestion or treatment to get the sugars. The idea is that some of the most costly steps in the production of biofuels from conventional crops or even from algae can be circumvented."

Brown and Nobles are currently working on moving this research out of the lab and into small-scale outdoor experiments. The two have formed a company called Phykotek Inc. and aim to commercialize the process on many different scales, Brown says. Ultimately, the cyanobacteria will be raised in large, salt-water ponds. "If you can use salt water and nonarable land, then it's a noncompetitive issue with food and water," Nobles explains. In addition, since cyanobacteria can take nitrogen from the air and convert it into a form that the microbe can use to grow, there's no need for fertilization.

"Here we have a microbe that takes the terawatt energy from sunlight and converts it into all these useful products in a place where it's never been done before. What better idea can one have?" Brown says. He and Nobles are initially planning on being a feedstock provider for the ethanol industry since the infrastructure is in place. "If we can get ethanol producers to start thinking about other sources and how to integrate those, I think that'll be a win-win situation," Brown says. "There will be many avenues to become completely energy independent, and we want to be part of the overall effort."

Concentrating on Corn
Spearheading a second avenue of discovery within that portfolio of approaches is a team of scientists from Michigan State University. While the Texas researchers pioneer the effort to find new sources of cellulose, the MSU researchers are engineering corn to be a better biofuels feedstock. Led by Mariam Sticklen, a crop scientist and molecular geneticist, the scientists recently reported the development of a line of corn that harbors microbial enzymes for converting plant cellulose into simple sugars for ethanol production.

Only in the past few years has Sticklen focused her energy on this type of research. "Because the cost of gasoline was not as high as it is now, it was more basic research," she explains. "Could one produce these materials from microbes in plants while maintaining the function of the enzyme? In 2000, we realized this is something we should take seriously, so we dropped some of our other projects to focus on this research."

Since that time, the team has developed two corn varieties, dubbed Spartan I and Spartan II. Each line carries a gene that directs the production of an enzyme for breaking down cellulose. The Spartan I variety, for instance, expresses an endoglucanase, which was isolated from a bacterium found living in a hot spring. The enzyme cuts long chains of cellulose into shorter fragments. In Spartan II, an exoglucanase isolated from a soil fungus was inserted into the corn genome. This molecule degrades the shorter fragments of cellulose into dimers, which contain two glucose units. The newest generation of Spartan corn, Spartan III, carries the genes that code for the enzymes in Spartan I and Spartan II and an enzyme that breaks dimers into simple sugars.

"The third [line] I consider the most interesting," Sticklen says. The third enzyme in this line came from a microbe that resides in the rumen of African cows. "We selected a cow from Africa because they only eat grass and must have a good system for the conversion of cellulose to energy," she explains.

The researchers made numerous modifications to the gene sequence of this microbial enzyme, which is called cellobiase, so it could be expressed in corn and perform its dimer-degrading function. "It's similar to an electrician wiring a house for heating or cooling or for electricity," Sticklen explains. "You have a lot of wiring. You have to synthesize switches. It even has zoning. There are different elements that control this gene at a level that can be produced by plants."

In addition, the genes are engineered so that each enzyme is stored in a different part of the plant cell. The cellobiase, for example, can be found in a specialized compartment of the cells in the leaves and stalks of the plant. "We didn't want the enzyme produced in the pollen or roots so we developed this system, and specifically made the enzyme only inside the leaves and stalks," Sticklen explains. The special compartment within the cells of these plant parts is called the vacuole and serves as the garbage can for the cell. "Whatever the cell doesn't need it dumps into the vacuole," she explains.

The cellobiase collects in the vacuole as the corn grows and develops. It can be extracted along with the other two enzymes when the plant is harvested and ground. At that point, the enzymes can be separated from the fibrous parts of the plant and can be applied to pretreated biomass to break it down into simple sugars for ethanol production.

Sticklen says that MSU is getting close to finalizing a deal with a seed company for the Spartan varieties. Meanwhile, her team continues to work on new Spartan lines engineered with better enzymes and reduced pretreatment requirements. "The work we're doing in my laboratory is to bring down the cost of biofuels," she says. "That's the whole idea."

Jessica Ebert is an Ethanol Producer Magazine freelance writer. Reach her at jebertserp@yahoo.com.