Final Report: Second-Generation Isobutanol Producing Biocatalyst

EPA Contract Number: EPD09023
Title: Second-Generation Isobutanol Producing Biocatalyst
Investigators: Urano, Jun
Small Business: Gevo, Inc.
EPA Contact: Richards, April
Phase: I
Project Period: February 1, 2009 through July 31, 2009
Project Amount: $70,000
RFA: Small Business Innovation Research (SBIR) - Phase I (2009) RFA Text |  Recipients Lists
Research Category: Small Business Innovation Research (SBIR)


Isobutanol as a Second-Generation Biofuel:
There is a need for the development of an economically viable and environmentally friendly fuel alternative. This need is precipitated by the current political instability in oil-producing nations, concerns about global warming, and a call for indigenous energy resources. Ethanol has potential as a first-generation biofuel; isobutanol, however, is an advanced second-generation biofuel with superior performance and economics. 

A lifecycle inventory (LCI) based on the conversion of corn stover to one of Gevo’s isobutanol-based products, isooctane, was performed using the Argonne National Laboratory’s Greenhouse Gases, Regulated Emissions and Energy Use in Transportation (GREET) model developed by Michael Wang, et al. (Wang M, et al. Environ Res Lett 2007;2(2):024001 (13pp)), which showed that isooctane produced and combusted in light duty vehicles is expected to reduce greenhouse gas emissions by 94.0 percent and fossil energy use by 97.3 percent compared to the production and use of conventional and reformulated gasoline. 

While ethanol is a viable first-generation biofuel, isobutanol brings superior performance and economic value. Isobutanol has similar physical and chemical properties to gasoline; it has a high octane number and 25 percent greater energy density compared to ethanol. Isobutanol’s most valuable property is its low Reid Vapor Pressure (RVP) blend value with other gasoline components. Gasoline is typically targeted to the range of 7 –  10 for RVP. Isobutantol blend RVP is 3.8 – 5.2, which is similar to the premium value gasoline component alkylate with an RVP of 4 – 5. In contrast, ethanol has an RVP blend value of 18 – 22, which results in additional costs for refiners.
Gevo has developed and demonstrated the technology to convert isobutanol into aliphatic and aromatic hydrocarbons using known chemistry and existing refinery infrastructure. Isobutylene is produced by the dehydration of isobutanol over an acidic catalyst and then further reacted to product mixtures of longer chain aliphatic hydrocarbons. A portion of this material is reacted separately to form high density aromatic compounds. Hydrogen gas, a byproduct of the aromatization reaction, is used to remove unsaturated bonds in the aliphatic material. The hydrocarbons then are blended in proportions that can meet all ASTM standards for fuels. Isooctane is a dimer of dehydrated isobutanol and is a major component of the premium value alkylates, a key gasoline component. A trimer of the isobutylene (dehydrated isobutanol) is a jet fuel blend stock. A polymer of four and five isobutylenes can make a diesel blend stock.
Current market projections place the market for butanol at 1.7 byn gallons and the market for isobutanol at ~ 175 myn gallons. Current end user markets include paints, coating and inks, adhesives and sealants, metal cleaning and other industrial cleaning products, plasticizers and other chemical intermediates. In addition, when isobutanol is converted to isobutylene it can serve as the precursor molecule for numerous high-value products such as terephthalic acid for the production of PET and other polymers.
Microbial Production of Isobutanol:
The isobutanol biosynthetic pathway utilizes pyruvate and reducing equivalents (NADH) derived from glycolysis. Each molecule of glucose generates two moles of pyruvate and two moles of NADH. The five enzymes that carry out the conversion of two moles of pyruvate to one mole of isobutanol are:  (1)acetolactate synthase (ALS; EC, (2) ketol-acid reductoisomerase (KARI; EC, (3) dihydroxy-acid dehydratase (DHAD; EC4.2.1.9), (4) detoisovalerate decarboxylase (KIVD; EC, and (5) alcohol dehydrogenase (ADH; EC or
Gevo has licensed technology from Prof. J. Liao (UCLA) for the production of isobutanol based on the above pathway (Atsumi S, et al. Nature 2008:451(7174):86). Gevo has been able to further develop isobutanol production in E. coli and has achieved commercially viable performance. Yeasts, however, exhibit higher tolerance to isobutanol. Yeasts also provide additional advantages. Yeast biocatalysts have a long history of accepted industrial use in dry mills and in lignocellulosic processes. The resulting dry distillers' grains have been approved for cattle feed. Furthermore, yeasts exhibit higher tolerance to toxic compounds found in corn and lignocellulosic feedstocks. It is thus the goal of this work to engineer a commercially viable yeast biocatalyst for the production of isobutanol.

Summary/Accomplishments (Outputs/Outcomes):

With the aid of EPA’s SBIR Program, Gevo launched a project to identify rate-limiting steps in the isobutanol pathway in yeast. This effort resulted in the development of genetic and biochemical assays to characterize each of the enzymes involved in the production of isobutanol in yeast. Using these tools, rate-limiting steps of the isobutanol pathway in yeast were identified.


Gevo has identified the rate-limiting steps in the isobutanol pathway in yeast.  During the process, numerous assays that will aid future development of a yeast biocatalyst for use in the commercial production of isobutanol as the next generation biofuel were developed and optimized.


Gevo plans to develop the yeast biocatalyst for the commercial production of isobutanol. The development of a yeast biocatalyst is an integral part of Gevo’s “retro-fit” model for the commercial production of isobutanol. Ethanol, another industrial alcohol, is produced using yeast in a well designed, energy efficient fermentation-based process that has steadily evolved over the past 30 years. Gevo intends to retro-fit existing ethanol plants to produce isobutanol. To do so, the biocatalyst that Gevo will use must be able to work efficiently in an anaerobic, low-sterility environment and be able to provide similar productivity, titer, and yield to be commercially viable. By retrofitting existing plants, Gevo is able to reduce its initial capital investment by ~ 85 percent compared to capital costs for a new “green-field site.” This results in a substantial reduction in commercial risk from a financing perspective and allows Gevo to leverage up capital by at least five-fold. In other words, Gevo can “retro-fit” ~ 7 plants for the same amount of capital that it would cost to build 1 new one and would have ~ 550 million gallons/year of isobutanol to bring to market compared to the 80 million gallons/year that could be produced in one plant. The end result is an efficient way to utilize capital investment to its fullest potential while reducing risk to the lenders.
Isobutanol has properties similar to gasoline and can be commercialized directly as a fuel.
Isobutanol can be commercialized as a fuel oxygenate. Isobutanol can be used in gasoline to meet the reforumlated gasoline (RFG) requirement established by the EPA. In addition, the low RVP of isobutanol in gasoline blendstock also will facilitate the achievement of the Renewable Fuel Standard (RFS) requirements, also established by the EPA.

Supplemental Keywords:

small business, SBIR, EPA, isobutanol, biocatalyst, biofuel, fuel infrastructure, reformulated gasoline, RFG, renewable fuel standards, RFD, energy density, gasoline, gasoline refining industry, gasoline market, imported oil, greenhouse gas emissions, GHG, host microorganism, biotechnology, biological isobutanol production