13TSB_CRD: HIGH PRODUCTIVITY HOMOFERMENTATIVE PROCESS for BUTANOL (HIPHOP)

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The acetone-butanol-ethanol (ABE) fermentation uses anaerobic bacteria from the genus Clostridium to ferment sugars orstarch to solvent mixtures in a typical A:B:E ratio of 3:6:1. Historically, the fermentation was used to manufacture solventsand chemicals, but fell out of favour when the oil industry developed cheaper ways to make these chemicals. With growingconcerns about oil security and global warming, the ABE fermentation is now undergoing a massive revival. Today, butanolis the preferred product, since it can be used as a biofuel, a solvent and an intermediate to manufacture a wide range ofchemicals.At present, butanol fermentations are inefficient because the accumulating butanol poisons the bacteria, ultimately causingthe fermentation to stop. Furthermore, butanol-producing Clostridium species are genetically unstable, so the fermentationcan only be run for a short time before shutting down and restarting from fresh cells. There are also problems withrecovering the butanol, because the product stream is a dilute mixture of butanol, acetone and ethanol in water. Distillationprovides the only easy way to recover the products, but requires a lot of energy.We will use synthetic biology to produce new Clostridium strains that produce butanol without forming acetone and ethanol- homofermentative strains. Scientists at Green Biologics have already developed homofermentative mutants usingtraditional mutagenesis techniques and have sequenced their DNA, to identify the mutated genes. In this project, we willselect the most important mutations and recreate them in a commercial production strain to develop a genetically stable,high productivity butanol-producing organism.The new organisms will produce much cleaner product streams, allowing development of new separation processes, basedon liquid-liquid extraction. This involves mixing the growing culture with a water-immiscible solvent that dissolves thebutanol more efficiently than water. As a result, the butanol will transfer into the solvent phase, which can easily beseparated by allowing the two immiscible liquid phases to settle out (like oil and water). This provides a very neat way tosolve problems with butanol toxicity, because the butanol is removed from the immediate environment surrounding thecells, so the cells are not exposed to the poisonous product. This allows butanol production to continue until the solventphase is saturated, so that the cells can form very high butanol concentrations.In situ solvent extraction depends on finding a water-immiscible liquid that is not only a good solvent for butanol but is alsonot poisonous to the cells. Most conventional solvents struggle to extract butanol from water and are just as poisonous asbutanol itself. However, scientists at the University of Nottingham have discovered that a new class of solvents called ionicliquids (ILs) can extract butanol from water and are not poisonous to living cells. ILs are made from salts that are molten atroom temperature, and so are non-volatile, unlike conventional solvents. Therefore, the butanol can easily be recovered,simply by separating the IL phase and then boiling off the butanol, leaving the IL behind for re-use.Overall, this project brings together synthetic biology and innovative bioseparations to develop a single-product, highproductivity butanol fermentation, together with a simple, low energy process for product purification. The last part of theproject will bring these technologies together to develop a continuous process for butanol production with stable operationover long periods. This process will exploit the genetic stability of the new, engineered strains, the simplified butanolseparation and the relief of product inhibition by in situ butanol recovery. The new process will provide significantly greaterproductivity than conventional batch fermentations, thus transforming the economics of butanol production