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=Isobutanol=

Second Generation Biofuel
Isobutanol can be used as a biofuel substitute for gasoline in the current petroleum infrastructure. Isobutanol has not yet been put into mainstream use as a biofuel and would serve as a replacement for ethanol. Ethanol is a first generation biofuel, and is used primarily as a gasoline additive in the petroleum infrastructure. Isobutanol is a second generation biofuel with several qualities that resolve issues presented by ethanol.

Isobutanol has four qualities which make it an attractive biofuel :


 * Isobutanol has a relatively high energy density, 98% of that of gasoline


 * Isobutanol does not readily absorb water from air, preventing the corrosion of engines and pipelines


 * Isobutanol can be mixed at any proportion with gasoline, meaning the fuel can "drop into" the existing petroleum infrastructure as a replacement fuel or major addivitive


 * When biosynthesized by particular microorganisms, isobutanol can be produced from plant matter not connected to food supplies, preventing a fuel-price/food-price relationship

Biosynthesis of Isobutanol
The biosynthetic pathway used to produce isobutanol was first discovered in species of bacteria from the genus Clostridium. This pathway has been genetically engineered into several species of microorganisms which are more easily manipulated by current scientific methods than microorganisms of the genus Clostridium. Although these engineered organisms are capable of producing isobutanol, they have not yet achieved the ability to produce isobutanol in quantities large enough for commercial use. These organisms are being moved toward commercialization through genetic modifications which allow higher yields of isobutanol. The organisms being pursued as commercial isobutanol producers are listed below:

Cyanobacteria
Cyanobacteria, are a phylum of photosynthetic bacteria. Cyanobacteria are suited for isobutanol biosynthesis when genetically engineered to produce isobutanol. 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 (land not used for farming) . This prevents space 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 (in other organisms which synthesize isobutanol, plant matter is the source of the carbon necessary to synthetically assemble 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 (in the form of Cyanobacteria removing excess CO2 from the atmosphere) exists.

The primary drawbacks of Cyanobacteria are:
 * Cyanobacteria are sensitive to environmental conditions when being grown. Cyanobacteria sufferer greatly from sunlight of inappropriate wavelength and intensity, CO2 of inappropriate concentration, or H2O of inappropriate salinity. These factors are generally hard to control, and present a major obstacle in cyanbacterial 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.

Escherichia coli
Escherichia coli, or E. coli, is a Gram-negative, rod-shaped bacteria. 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 (waste plant matter leftover from agriculture) 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.

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, Bacillus 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 Bacillus subtilis, leading to higher yields of isobutanol being produced.

Saccharomyces cerevisiae
Saccharomyces cerevisiae, or S. cerevisiae is a species of yeast. S. cerevisiae 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. cerevisiaeis 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
Ralstonia eutropha is a gram-negative soil bacterium of the betaproteobacteria class. Ralstonia eutropha is capable of converting electrical energy into isobutanol. This conversion is completed in several steps:
 * Anodes are placed in a mixture of H2O and CO2.
 * An electrical current is run through the anodes, and through an electrochemical process H2O and CO2 are combined to synthesize formic acid.
 * A culture of Ralstonia eutropha (composed of a strain tolerant to electricity) is kept within the H2O and CO2 mixture.
 * The culture of Ralstonia eutropha 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.

This method of isobutanol production offers a way to chemically store energy produced from sustainable sources.