Membranes and Membrane Transport in Industrial...

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Membranes and Membrane Transport in Industrial BiotechnologyMarch 2016Contact:Preben Krabbenpreben.krabben@greenbiologics.com

+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

Personal backgroundWhere Engineering and Biology meets…?

Private & Confidential 3

• M.Sc., and Ph.D. in

biochemical engineering,

1988-1997.

• Microbial physiologist,

chemical engineer, or

neither.

Story lineor rather circles

HistoryEnergetics

Membranes

GeopoliticsAvoiding the indigo disaster

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

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

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

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

• Weizmann patent expired in

• Two new plants build in 1936

in the US.

• The Publicker plant in

employed 1400 m3 Horton

spheres as fermenters.

Microbiology TodayAcetone production

• Early history summarised,

and referenced.

• Microbiology Today, May

74-77. 2014.

• Please read and cite.

MembranesWhat are they good for

Membranes provide a controlled environment, avoid dilution of

metabolites and enzymes, and provides a concentration gradient.

Membrane transportType of transport

Diffusion, facilitated diffusion, symport, antiport, ATP dependent

transporters, and even transport within the plasma-membrane.

Membrane transportDiffusion

Small hydrophobic compounds (alcohols and VOA), water, and

gases diffuses over the plasma-membrane.

Cytoplasma Extracellular

Butanol transportDiffusion of some sort

Butanol concentration is always higher at the site of production.

The cost of butanol removal depends on the concentration of

butanol in the broth.

Butanol

ButanolSugar Butyaldehyde

MembraneStability

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

MembraneStability

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

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.

Acetic acid <-> Acetate- + H+

pKa = 4.7

Lactic acid <-> Lactate- + H+

pKa = 3.7

Acetate- + H+ <-> Acetic acid

Butanol transportMembrane stability

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.

Butanol

ExtracellularCytoplasma

Butanol

ButanolIntracellular effects

Solventogenesis in C. acetobutylicum have been shown to co-

incite with up-regulation of cytosolic chaperones, which refolds

mis-folded proteins.

Butanol

ButanolProduction host

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

Low surface area to volume High surface area to volume

Medium surface area to volume

ButanolMorphology

Cell volume is proportional to intracellular butanol production capacity.

Cell surface is membrane area.

Surface to volume ratio is proportional to butanol membrane gradient.

acity.

ButanolMorphology

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.

acity.

ClostridiumAnaerobes vs. aerobe

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

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

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

Michael Köpke et al. PNAS 2010;107:13087-13092

80 years later

Acknowledgement

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

Falkirk lock

ButanolMorphology

acity.

Introduction/up-regulation of a butanol facilitator protein decreases

the surface area to volume effect on butanol production capacity.

ButanolButanol facilitator protein

The potential butanol facilitator protein most likely belong to the

aquaporin/GlpF family.

Membranes and Membrane Transport in Industrial...

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