Unlike ethanol, isobutanol can be blended at any ratio with gasoline and should eliminate the need for dedicated infrastructure in tanks or vehicles. Plus, it may be possible to use isobutanol directly in current engines without modification.

—James Liao

Higher alcohols such as isobutanol are better candidates for gasoline replacement because they have an energy density, octane value and Reid vapor pressure that is much closer to gasoline, Liao said.

While cellulosic biomass like corn stover and switchgrass is abundant and cheap, it is much more difficult to utilize than corn and sugar cane. This is due in large part because of recalcitrance, or a plant’s natural defenses to being chemically dismantled. Adding to the complexity and cost is the need for several steps—pretreatment, enzyme treatment and fermentation—resulting in a process that is more costly than a method that combines biomass utilization and the fermentation of sugars to biofuel into a single process.

To make the conversion possible, Liao and postdoctoral researcher Wendy Higashide of UCLA and Yongchao Li and Yunfeng Yang of Oak Ridge National Laboratory (ORNL) developed a strain of Clostridium cellulolyticum, a native cellulose-degrading microbe, that could synthesize isobutanol directly from cellulose. This work was on Liao’s earlier work at UCLA in building a synthetic pathway for isobutanol production.

Producing biofuels directly from cellulose, known as consolidated bioprocessing, is believed to reduce costs substantially compared to a process in which cellulose degradation and fermentation to fuel are accomplished in separate steps. Here we present a metabolic engineering example to develop a Clostridium cellulolyticum strain for isobutanol synthesis directly from cellulose. This strategy exploits the host’s natural cellulolytic activity and the amino acid biosynthetic pathway and diverts its 2-keto acid intermediates for alcohol synthesis. Specifically, we have demonstrated the first isobutanol production to approximately 660 mg/L from crystalline cellulose using this microorganism.

—Higashide et al.

While some Clostridium species produce butanol, these organisms typically do not digest cellulose directly. Other Clostridium species digest cellulose but do not produce butanol. None produce isobutanol, an isomer of butanol.

In nature, no microorganisms have been identified that possess all of the characteristics necessary for the ideal consolidated bioprocessing strain, so we knew we had to genetically engineer a strain for this purpose.

—Yongchao Li

While there were many possible microbial candidates, the research team ultimately chose Clostridium cellulolyticum, which was originally isolated from decayed grass. The researchers noted that their strategy exploits the host’s natural cellulolytic activity and the amino acid biosynthetic pathway and diverts its intermediates to produce higher alcohol than ethanol.

The researchers also noted that Clostridium cellulolyticum has been genetically engineered to improve ethanol production, and this has led to additional more detailed research. Clostridium cellulolyticum has a sequenced genome available via DOE’s Joint Genome Institute. This proof of concept research sets the stage for studies that will likely involve genetic manipulation of other consolidated bioprocessing microorganisms.

This work was supported in part by BESC at ORNL and by UCLA-DOE Institute for Genomics and Proteomics.

BESC is one of three DOE Bioenergy Research Centers established by the DOE’s Office of Science in 2007. The centers support multidisciplinary, multi-institutional research teams pursuing the fundamental scientific breakthroughs needed to make production of cellulosic biofuels, or biofuels from nonfood plant fiber, cost-effective on a national scale. The centers are led by ORNL, Lawrence Berkeley National Laboratory and the University of Wisconsin-Madison in partnership with Michigan State University.

Resources

• Wendy Higashide, Yongchao Li, Yunfeng Yang, and James C. Liao (2011) Metabolic Engineering of Clostridium Cellulolyticum for Isobutanol Production from Cellulose. Appl. Environ. Microbiol. doi: 10.1128/AEM.02454-10