Qi BioEnergy

Butanol offers many advantages as a substitute for gasoline because of higher energy content and higher hydrophobicity. Typically, 1-butanol is produced by Clostridium in a mixed-product fermentation.  To facilitate strain improvement for specificity and productivity, we engineered a synthetic pathway in Escherichia coli and demonstrated the production of 1-butanol from this non-native user-friendly host.  Alternative genes and competing pathway deletions were evaluated for 1-butanol production. Results show promise for using E. coli for 1-butanol production.

In a push to find better biofuels to reduce gasoline consumption and lower greenhouse-gas emissions, scientists have genetically engineered E. coli that is highly efficient in producing butanol, a promising new type of biofuel. The new technology could speed up the development of butanol biofuels into a cost-effective alternative to ethanol.

While ethanol is the main biofuel on the market today, energy firms are increasingly looking to alternatives such as butanol. “It has many attractive properties,” says Jim McMillan, manager of biorefining process R&D at the National Renewable Energy Laboratory’s National Bioenergy Center, in Golden, CO. Because butanol packs more energy per gallon than ethanol does, cars running on butanol get better mileage. And, unlike ethanol, it doesn’t mix with water, so it can be shipped in existing petroleum pipelines without causing problems.

A number of research groups are engineering microbes that can convert sugar from various feedstocks into butanol. Most of these groups rely on the bacterium Clostridium acetobutylicum, which naturally makes a form of butanol called 1-butanol. “But Clostridium is not easy to deal with,” says James Liao, a chemical engineer at the University of California, Los Angeles. “It grows slowly, it’s very fastidious, and it’s not easy to genetically manipulate.” Despite decades of tinkering by scientists, the microbe still can’t produce enough butanol to make it economically viable as a transportation fuel, Liao says.

Instead, he and his colleagues turned to E. coli. Although the bacterium does not produce butanol naturally, it is easy to modify and grows fast. Instead of tweaking the pathway that the microbes employ for fermenting sugar into alcohol, Liao reasoned that he could program E. coli to produce butanol by diverting some of the microorganism’s metabolites into alcohol production. These metabolites, called keto acids, are involved in the synthesis of amino acids, the building blocks of proteins.

To make butanol from keto acids, the researchers inserted two different nonnative genes into E. coli. The first gene came from a microbe commonly used in the production of cheese. The gene codes for an enzyme that converts keto acids into aldehydes. The second gene, derived from yeast, codes for an enzyme that converts aldehydes into butanol.

Initially, when linked together in E. coli, the two genes allowed the microbe to produce small amounts of butanol. With further genetic modifications, Liao was able to dramatically increase the efficiency of the process. For instance, deleting certain genes and boosting the activity of others increased the amount of keto acids available for conversion into butanol. With all the combined manipulations, the engineered microbes achieved an efficiency high enough for industrial use, says Liao.

Gevo, a biofuels startup based in Pasadena, CA, has acquired an exclusive license to commercialize Liao’s technology. (Liao is on the company’s scientific advisory board.) “It’s a real breakthrough,” says Mathew Peters, Gevo’s chief scientific officer. Not only did Liao improve the efficiency of the process, but he also designed his microbes to produce a particular form of butanol called isobutanol. “We believe isobutanol is a superior fuel,” says Peters. Compared with 1-butanol, isobutanol has a higher octane number, which reduces knocking in the vehicle’s engine.

What’s more, the biochemical pathway Liao designed for making isobutanol can be transferred to other microbes. In addition to investigating E. coli, Gevo is looking at different microorganisms that could be modified in the same way. “We’re interested in any organism that will make the process cheaper,” says Peters.

Gevo isn’t alone in its pursuit of a better butanol-producing bug. In June 2006, BP and DuPont joined efforts to develop butanol.

Last June, BP and DuPont, along with Associated British Foods, announced their plans to build a biobutanol pilot plant at an existing BP site in England. The plant, which will use sugar beet as a feedstock, is expected to begin operations in 2009, with the ultimate goal of commercializing butanol after 2010.

According to Peters, Gevo plans to make a decision by the end of the year on whether to go ahead with its own plans to build a butanol plant. In the meantime, certain technological hurdles still need to be overcome to make butanol cost competitive, he says. Mainly, the microbes need to get faster at producing butanol, and their tolerance to isobutanol, which is toxic to the organisms, must improve. Still, Peters expects Gevo to resolve these issues in the coming months.

Gevo has developed a proprietary process technology to enhance productivity and lower product separation costs. Our process models predict that the economical production of butanol and other fuels is comparable in cost to current ethanol production.

Butanol is a superior fuel and chemical than ethanol. It also has higher energy content than ethanol and can be produced from agricultural crops and crop residues such as lignocellulosic materials. In a study, it has been identified that raw material cost influences butanol production price significantly. Hence, it was considered that use of economically available raw materials would make production of butanol economically attractive. For this reason, these studies were focused on production of butanol from wheat straw using a microbial culture known as Clostridium beijerinckii. In these studies, it has been demonstrated that butanol could be produced from wheat straw hydrolyzed to monomeric sugars using dilute acid and enzymes. Hydrolysis of wheat straw to sugars (glucose, xylose, mannose, arabinose, and galactose), their conversion to butanol, and butanol recovery were achieved in a single reactor (single step). In this process, hydrolysis efficiency and butanol productivity were improved. Successful production of butanol from lignocellulosic materials would benefit U.S. farmers and the U.S. public. Development of such an alternative fuel and chemical is essential since gas prices have been rising steadily.Technical Abstract: In these studies, Clostridium beijerinckii P260 was used to produce butanol (acetone butanol ethanol, or ABE) from wheat straw (WS) hydrolyzate in a fed-batch reactor. It has been demonstrated that simultaneous hydrolysis of WS to achieve 100% hydrolysis to simple sugars (to the extent achievable under present conditions) and fermentation to butanol is possible. In addition to WS, the reactor was fed with a sugar solution containing glucose, xylose, arabinose, galactose, and mannose. The culture utilized all of the above sugars. It was noticed that near the end of fermentation (286-533 h), the culture had difficulties utilizing xylose. As a result of supplemental sugar feed to the reactor, ABE productivity was improved by 16% as compared to previous studies. In our previous experiment on simultaneous saccharification of WS and fermentation to butanol, a productivity of 0.31 gL**-1h**-1 was observed, while in the present studies a productivity of 0.36 gL**-1h**-1 was observed. It should be noted that a productivity of 0.77 gL**-1h**-1 was observed when the culture was highly active. The fed-batch fermentation was operated for 533 h. It should be noted that C. beijerinckii P260 can be used to produce butanol from WS in integrated fermentations.