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

The acetone-butanol-ethanol (ABE) fermentation uses anaerobic bacteria from the genus Clostridium to ferment sugars or
starch to solvent mixtures in a typical A:B:E ratio of 3:6:1. Historically, the fermentation was used to manufacture solvents
and chemicals, but fell out of favour when the oil industry developed cheaper ways to make these chemicals. With growing
concerns about oil security and global warming, the ABE fermentation is now undergoing a massive revival. Today, butanol
is the preferred product, since it can be used as a biofuel, a solvent and an intermediate to manufacture a wide range of
chemicals.
At present, butanol fermentations are inefficient because the accumulating butanol poisons the bacteria, ultimately causing
the fermentation to stop. Furthermore, butanol-producing Clostridium species are genetically unstable, so the fermentation
can only be run for a short time before shutting down and restarting from fresh cells. There are also problems with
recovering the butanol, because the product stream is a dilute mixture of butanol, acetone and ethanol in water. Distillation
provides 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 using
traditional mutagenesis techniques and have sequenced their DNA, to identify the mutated genes. In this project, we will
select 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, based
on liquid-liquid extraction. This involves mixing the growing culture with a water-immiscible solvent that dissolves the
butanol more efficiently than water. As a result, the butanol will transfer into the solvent phase, which can easily be
separated by allowing the two immiscible liquid phases to settle out (like oil and water). This provides a very neat way to
solve problems with butanol toxicity, because the butanol is removed from the immediate environment surrounding the
cells, so the cells are not exposed to the poisonous product. This allows butanol production to continue until the solvent
phase 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 also
not poisonous to the cells. Most conventional solvents struggle to extract butanol from water and are just as poisonous as
butanol itself. However, scientists at the University of Nottingham have discovered that a new class of solvents called ionic
liquids (ILs) can extract butanol from water and are not poisonous to living cells. ILs are made from salts that are molten at
room 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, high
productivity butanol fermentation, together with a simple, low energy process for product purification. The last part of the
project will bring these technologies together to develop a continuous process for butanol production with stable operation
over long periods. This process will exploit the genetic stability of the new, engineered strains, the simplified butanol
separation and the relief of product inhibition by in situ butanol recovery. The new process will provide significantly greater
productivity than conventional batch fermentations, thus transforming the economics of butanol production

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The primary impact of this project will be within the chemicals and fuels industry, and will result in revenue generation for
the UK. Biobutanol is an attractive biofuel due to its high energy density and excellent blending characteristics with both
diesel and gasoline, and the scope for upgrading to drop-in jet fuel. Biobutanol could also provide an alternative to
synthetic, oil-derived butanol, as a valuable platform chemical for a variety of intermediates, polymers, coatings, plastics
and solvents. The fermentation feedstocks are renewable and the biological process results in lower energy use and green
house gas (GHG) emissions than the petrochemical process. Not surprisingly, biobutanol has been targeted by the UK
Industrial Biotech Leadership Forum as a strategically important renewable chemical for the UK.
Virtually all 1-butanol today is synthetic and produced from petroleum derived propylene, with a current price of $1800/t. An
advanced fermentation route to produce biobutanol is an attractive option since it offers significantly lower production costs;
for example, GBL's current production costs are approximately $1500/t on a molasses based feedstock. Success in this
project would reduce the costs to <$1200/t, whilst also decreasing the capital costs and financing required to service debt
on a plant.The route to commercialisation will be via GBL, with IP sharing between GBL and UoN, and revenue sharing based on
royalties. GBL has the skills and experience to deliver competitive, capital efficient production of biobutanol and other C4
chemicals. GBL maintains offices and laboratories for molecular biology, microbiology and fermentation in the UK and has
a pilot plant facility, labs and offices in Columbus, Ohio, US. GBL has developed proprietary fermentation technology using
solventogenic Clostridia for the production of biobutanol from a variety of renewable feedstocks including cellulosics such
as forest and crop residues. GBL operates globally in China, North America, India and Brazil. Therefore, it is eminently
sensible for the industrial partner to take full responsibility for commercialising the results of the project.
In addition to economic impact, the link between Nottingham and GBL will result in the output of trained people, directly
from the project and the associated CASE student who will start in year 2. The work at Nottingham will also provide benefit
for wider undergraduate, masters and PhD training programmes in the form of case studies for lectures, topics for
engineering design projects and subjects for research projects. There is also scope for GBL to deliver lectures and to cosupervise
undergraduate design and research projects. Therefore, the project will have a significant impact on training the
next generation of development scientists and plant operators.
The project will result in environmental benefits, via significant reductions in both energy and GHG emissions (>85%)
compared with synthetic butanol manufacturing, and the ability to use cellulosic feedstocks. The delivery of an economic
and sustainable cellulosic biofuel fermentation process is strategically important for the UK and Europe. Production of low
cost and sustainable biofuels and renewable chemicals will create jobs and help meet our renewable obligations and GHG
reduction targets. In addition, alternative uses of wood and woody residues will make a significant contribution to the new
clean tech economy.