Butanol fuel

may be used as a fuel in an internal combustion engine. It is more similar to gasoline than it is to ethanol. A C4-hydrocarbon, butanol is a drop-in fuel and thus works in vehicles designed for use with gasoline without modification.
Both n-butanol and isobutanol have been studied as possible fuels. Both can be produced from biomass as well as from fossil fuels. The chemical properties depend on the isomer, not on the production method.

Genetically modified organisms

Obtaining higher yields of butanol involves manipulation of the metabolic networks using metabolic engineering and genetic engineering. While significant progress has been made, fermentation pathways for producing butanol remain inefficient. Titer and yields are low and separation is very expensive. As such, microbial production of butanol is not cost-competitive relative to petroleum-derived butanol.
Although unproven commercially, combining electrochemical and microbial production methods may offer a way to produce butanol from sustainable sources.

''Escherichia coli''

Escherichia coli, or E. coli, is a Gram-negative, rod-shaped bacterium. E. coli is the microorganism most likely to move on to commercial production of isobutanol. In its engineered form, E. coli produces the highest yields of isobutanol of any microorganism. Methods such as elementary mode analysis have been used to improve the metabolic efficiency of E. coli so that larger quantities of isobutanol may be produced. E. coli is an ideal isobutanol bio-synthesizer for several reasons:

E. coli is an organism for which several tools of genetic manipulation exist, and it is an organism for which an extensive body of scientific literature exists. This wealth of knowledge allows E. coli to be easily modified by scientists.
E. coli has the capacity to use lignocellulose in the synthesis of isobutanol. The use of lignocellulose prevents E. coli from using plant matter meant for human consumption, and prevents any food-fuel price relationship which would occur from the biosynthesis of isobutanol by E. coli.
Genetic modification has been used to broaden the scope of lignocellulose which can be used by E. coli. This has made E. coli a useful and diverse isobutanol bio-synthesizer.

The primary drawback of E. coli is that it is susceptible to bacteriophages when being grown. This susceptibility could potentially shut down entire bioreactors. Furthermore, the native reaction pathway for isobutanol in E. coli functions optimally at a limited concentration of isobutanol in the cell. To minimize the sensitivity of E. coli in high concentrations, mutants of the enzymes involved in synthesis can be generated by random mutagenesis. By chance, some mutants may prove to be more tolerant of isobutanol which will enhance the overall yield of the synthesis.

''Clostridia''

n-Butanol can be produced by fermentation of biomass by the A.B.E. process using Clostridium acetobutylicum, Clostridium beijerinckii. C. acetobutylicum was once used for the production of acetone from starch. The butanol was a by-product of fermentation. The feedstocks for biobutanol are the same as those for ethanol: energy crops such as sugar beets, sugar cane, corn grain, wheat and cassava, prospective non-food energy crops such as switchgrass and even guayule in North America, as well as agricultural byproducts such as bagasse, straw and corn stalks. According to DuPont, existing bioethanol plants can cost-effectively be retrofitted to biobutanol production. Additionally, butanol production from biomass and agricultural byproducts could be more efficient than ethanol or methanol production.
A strain of Clostridium can convert nearly any form of cellulose into butanol even in the presence of oxygen.
A strain of Clostridium cellulolyticum, a native cellulose-degrading microbe, affords isobutanol directly from cellulose.
A combination of succinate and ethanol can be fermented to produce butyrate by utilizing the metabolic pathways present in Clostridium kluyveri. Succinate is an intermediate of the TCA cycle, which metabolizes glucose. Anaerobic bacteria such as Clostridium acetobutylicum and Clostridium saccharobutylicum also contain these pathways. Succinate is first activated and then reduced by a two-step reaction to give 4-hydroxybutyrate, which is then metabolized further to crotonyl-coenzyme A. Crotonyl-CoA is then converted to butyrate. The genes corresponding to these butanol production pathways from Clostridium were cloned to E. coli.

Cyanobacteria

are a phylum of photosynthetic bacteria. They are suited for isobutanol biosynthesis when genetically engineered to produce isobutanol and its corresponding aldehydes. Isobutanol-producing species of cyanobacteria offer several advantages as biofuel synthesizers:

Cyanobacteria grow faster than plants and also absorb sunlight more efficiently than plants. This means they can be replenished at a faster rate than the plant matter used for other biofuel biosynthesizers.
Cyanobacteria can be grown on non-arable land. This prevents competition between food sources and fuel sources.
The supplements necessary for the growth of cyanobacteria are CO₂, H₂O, and sunlight. This presents two advantages:
* Because CO₂ is derived from the atmosphere, cyanobacteria do not need plant matter to synthesize isobutanol. Since plant matter is not used by this method of isobutanol production, the necessity to source plant matter from food sources and create a food-fuel price relationship is avoided.
* Because CO₂ is absorbed from the atmosphere by cyanobacteria, the possibility of bioremediation exists.

The primary drawbacks of cyanobacteria are:

They are sensitive to environmental conditions when being grown. Cyanobacteria suffer greatly from sunlight of inappropriate wavelength and intensity, CO₂ of inappropriate concentration, or H₂O of inappropriate salinity, though a wealth of cyanobacteria are able to grow in brackish and marine waters. These factors are generally hard to control, and present a major obstacle in cyanobacterial production of isobutanol.
Cyanobacteria bioreactors require high energy to operate. Cultures require constant mixing, and the harvesting of biosynthetic products is energy-intensive. This reduces the efficiency of isobutanol production via cyanobacteria.

Cyanobacteria can be re-engineered to increase their butanol production, showing the importance of ATP and cofactor driving forces as a design principle in pathway engineering. Many organisms have the capacity to produce butanol utilizing an acetyl-CoA dependent pathway. The main problem with this pathway is the first reaction involving the condensation of two acetyl-CoA molecules to acetoacetyl-CoA. This reaction is thermodynamically unfavorable due to the positive Gibbs free energy associated with it.

''Bacillus subtilis''

Bacillus subtilis is a gram-positive rod-shaped bacteria. Bacillus subtilis offers many of the same advantages and disadvantages of E. coli, but it is less prominently used and does not produce isobutanol in quantities as large as E. coli. Similar to E. coli, B. subtilis is capable of producing isobutanol from lignocellulose, and is easily manipulated by common genetic techniques. Elementary mode analysis has also been used to improve the isobutanol-synthesis metabolic pathway used by B. subtilis, leading to higher yields of isobutanol being produced.

''Saccharomyces cerevisiae''

Saccharomyces cerevisiae, or S. cerevisiae, is a species of yeast. It naturally produces isobutanol in small quantities via its valine biosynthetic pathway. S. cerevisiae is an ideal candidate for isobutanol biofuel production for several reasons:

S. cerevisiae can be grown at low pH levels, helping prevent contamination during growth in industrial bioreactors.
S. cerevisiae cannot be affected by bacteriophages because it is a eukaryote.
Extensive scientific knowledge about S. cerevisiae and its biology already exists.

Overexpression of the enzymes in the valine biosynthetic pathway of S. cerevisiae has been used to improve isobutanol yields. S. cerevisiae, however, has proved difficult to work with because of its inherent biology:

As a eukaryote, S. cerevisiae is genetically more complex than E. coli or B. subtilis, and is harder to genetically manipulate as a result.
S. cerevisiae has the natural ability to produce ethanol. This natural ability can "overpower" and consequently inhibit isobutanol production by S. cerevisiae.
S. cerevisiae cannot use five-carbon sugars to produce isobutanol. The inability to use five-carbon sugars restricts S. cerevisiae from using lignocellulose, and means S. cerevisiae must use plant matter intended for human consumption to produce isobutanol. This results in an unfavorable food/fuel price relationship when isobutanol is produced by S. cerevisiae.
''Ralstonia eutropha''

Cupriavidus necator is a Gram-negative soil bacterium of the class Betaproteobacteria. It is capable of indirectly converting electrical energy into isobutanol. This conversion is completed in several steps:

Anodes are placed in a mixture of H₂O and CO₂.
An electric current is run through the anodes, and through an electrochemical process H₂O and CO₂ are combined to synthesize formic acid.
A culture of C. necator is kept within the H₂O and CO₂ mixture.
The culture of C. necator then converts formic acid from the mixture into isobutanol.
The biosynthesized isobutanol is then separated from the mixture, and can be used as a biofuel.