membranes and membrane transport in industrial...
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Membranes and Membrane Transport in Industrial BiotechnologyMarch 2016Contact:Preben [email protected]
+44 (0) 1235 438992
• A UK (Oxford based) Industrial Biotech
Company
• Focus on biobutanol
• Core expertise is microbial strain
development & fermentation
• Transforming conventional ABE
fermentation and driving production costs
• Developing attractive routes to global
market (chemicals & biofuels)
• GB has experienced/highly qualified team
(>100)
• Operate globally
About Green BiologicsBuilding a renewable chemicals business
© Green Biologics Ltd. 2014 Private and Confidential
Personal backgroundWhere Engineering and Biology meets…?
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• M.Sc., and Ph.D. in
biochemical engineering,
1988-1997.
• Microbial physiologist,
chemical engineer, or
neither.
Story lineor rather circles
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HistoryEnergetics
Membranes
GeopoliticsAvoiding the indigo disaster
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In 1897 19000 tons of indigo was produced in the world, all natural. In
1902 17000 tons of synthetic indigo was produced by the German
chemical industry.
Would a similar transformation happen for rubber ?
Chaim WeizmannAdaptive laboratory evolution
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One year of adaptive laboratory evolution generated the acetone butanol
production strain which was used for the next 15-20 years.
Almost identical strains isolated from 3 different continents.
Horton HeathPilot scale
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During the experimental period at Poole, before work was commenced in
Toronto, there had been about ten fermentations carried through. Seven
of these were failures, which were discharged on to the neighbouring
heath, and by their subsequent activities brought the process and those
responsible for it into grave disrepute in the vicinity (Speakman 1919)
British Acetones TorontoProduction scale
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Microbiologist Speakman and
Carter.
‘You can have my distillery’, and with these words in 1915–16 Colonel
Gooderham generated the second largest fermentation process in he
world.
The fermentation plant. Now
converted to a leisure district.
PublickerLast new butanol plant build in the US
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• Weizmann patent expired in
1936.
• Two new plants build in 1936
in the US.
• The Publicker plant in
employed 1400 m3 Horton
spheres as fermenters.
Microbiology TodayAcetone production
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• Early history summarised,
and referenced.
• Microbiology Today, May
74-77. 2014.
• Please read and cite.
MembranesWhat are they good for
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Membranes provide a controlled environment, avoid dilution of
metabolites and enzymes, and provides a concentration gradient.
Membrane transportType of transport
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Diffusion, facilitated diffusion, symport, antiport, ATP dependent
transporters, and even transport within the plasma-membrane.
Membrane transportDiffusion
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Small hydrophobic compounds (alcohols and VOA), water, and
gases diffuses over the plasma-membrane.
Cytoplasma Extracellular
Co
nce
ntr
atio
n
Butanol transportDiffusion of some sort
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Butanol concentration is always higher at the site of production.
The cost of butanol removal depends on the concentration of
butanol in the broth.
Cytoplasma Extracellular
Butanol
ButanolSugar Butyaldehyde
MembraneStability
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Eukaryotes, which has a marge membrane area around the cytoplasma,
stabilises the membranes with cholesterol.
The ethanol tolerant Z. mobilis uses hopanoids to stabilise the plasma-
membrane.
Other ways includes modifying the plasma-lipids, and/or having longer
chain fatty acids
200 ns time course of plasma-membrane to 12 g/L butanol (Hinks et al
2015)
MembraneStability
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Is the plasma-membrane pressed against the peptidoglucan or can the
plasma-membrane stability be improved by more plasma-membrane
peptidoglucan connectors or more rafting?
MembraneStability and thickness
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Acetic acid/acetate is a common organic acid in a mixed culture and
acetic acid is a well known membrane potential un-coupler.
Extensive lactate production quickly lower the extracellular pH and
thereby toxifies acetate and butyrate.
Lactic acid bacteria have a membrane fatty acid chain length of 22
carbons, which should results in a smaller acetic acid diffusion rate.
Cytoplasma Extracellular
Acetic acid <-> Acetate- + H+
pKa = 4.7
Lactic acid <-> Lactate- + H+
pKa = 3.7
Acetate- + H+ <-> Acetic acid
Butanol transportMembrane stability
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A thicker plasma-membrane will result in more stability, but a
larger gradient will be needed in order to obtain the same specific
butanol export rate.
Cytoplasma Extracellular
Butanol
ExtracellularCytoplasma
Butanol
ButanolIntracellular effects
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Solventogenesis in C. acetobutylicum have been shown to co-
incite with up-regulation of cytosolic chaperones, which refolds
mis-folded proteins.
Cytoplasma Extracellular
Butanol
ButanolProduction host
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Lactic acid bacteria are very tolerant to external supplied butanol, and has
historically often been identified as ABE-fermentation contaminating
microbes, but they will be very poor production strains because of the
intracellular butanol concentration needed for export.
ButanolMorphology
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Low surface area to volume High surface area to volume
Medium surface area to volume
ButanolMorphology
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Low surface area to volume High surface area to volume
Medium surface area to volume
Cell volume is proportional to intracellular butanol production capacity.
Cell surface is membrane area.
Surface to volume ratio is proportional to butanol membrane gradient.
Bu
tan
ol p
rod
uctio
n c
ap
acity.
ButanolMorphology
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Low surface area to volume High surface area to volume
Medium surface area to volume
Yeast could be a bad choice as a host strain for butanol production.
Clostridium is a Goldilock organisms for butanol production as surface area
to volume increases as cells progresses further into solventogenesis.
Bu
tan
ol p
rod
uctio
n c
ap
acity.
ClostridiumAnaerobes vs. aerobe
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Oxygen provides extra energy,
and this drives ATP production
with significant heat loss.
Anaerobes conserve the maximum
amount of energy in ATP and products
and does not loose it as heat
n-Butanol
• Four different reduction reactions from 2 acetyl-CoA to n-butanol, i.e.– 2 reductions per acetyl-CoA
• The saturation of a carbon-carbon double bond is energetically “too” easy
2 Steps away
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Acetyl-CoA
Acetyl-CoA
n-Butanol+
ketone -> alcohol
carboxy group-> aldehyde
aldehyde > alcohol
carbon double bond -> carbon single bond
Butyryl-CoA dehydrogenase
• Pure use of 1 NADH would make the reaction irreversible.
• The extra reducing power of 1 NADH is combined with another NADH to produce the strong reducing agent reduced ferredoxin.
• Conversion of the ferredoxin to NADH via the Rnf-complex will generate another 0.55 ATP per glucose.
• Total ATP produced from glucose to n-butanol is 3.10 ATP/glucose, much more than yeast ethanol
Electron bifurcation
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The Rnf complex
• First discovered in in Rhodobacter capsulatus in 1993.
• Studied intensively in “Clostridia” by the groups of Müller/Bückel in Germany since 2007.
• Energy generating mode (2 H+) : transfer of electrons from reduced ferredoxin to oxidised NAD+.
• Redox upgrading model : formation of reduced ferredoxin using pmf.
Connecting with the proton motive force, pmf
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Michael Köpke et al. PNAS 2010;107:13087-13092
80 years later
Acknowledgement
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All the people who provided my with the graphics and pictures.
University of York,
Gavin Thomas
University of Lincoln
Alan Goddard
Lorna Lancaster
Manuela Mura
And all the ones I have forgotten.
ButanolFacilitated butanol diffusion
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Falkirk lock
ButanolMorphology
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Low surface area to volume High surface area to volume
Medium surface area to volume
Bu
tan
ol p
rod
uctio
n c
ap
acity.
Introduction/up-regulation of a butanol facilitator protein decreases
the surface area to volume effect on butanol production capacity.
ButanolButanol facilitator protein
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The potential butanol facilitator protein most likely belong to the
aquaporin/GlpF family.