vinnova programme: industrial biotechnology for the

VINNOVA programme:Industrial Biotechnology for the production

of fine and platform chemicals

Patrick Adlercreutz, Dept. of Biotechnology, Lund University

Rajni Hatti-Kaul, Dept. of Biotechnology, Lund University

Development of process technology for immobilized biocatalysts.

• Cambrex Karlskoga• Perstorp Specialty Chemicals• GE Healthcare• Protista International• Lunds Universitet• SIK

Project partners

Demonstration processes

• Chiral amines• Dihydroxyaceton (DHA)• 3-Hydroxypropionaldehyd (3-HPA)

Development of processes with immobilized whole cells

Purification and immobilisation of transaminase

• GE Healthcare supports• Other supports and methods

Chiral amines• Important bulding blocks in

pharmaceuticals• Chemical process

– High environmental impact– Difficult to reach high enough purity

• Enzymatic process– Cambrex has good enzymes– Need for process development

Enzymatic routes to chiral amines

NH2 ONH2

O NH2

2 +

pyruvate L-alanine

Resolution

(S)-transaminase

acetoneisopropylamine

(S)-transaminase

Asymmetric synthesis

(R)

(S)

Immobilization of transaminase-containing cells

Calciumalginategel

Titaniumoxide

Cell immobilization on titanium oxide

Titanium oxide particles Titanium oxide with cells

Transaminase immobilization. Effect of cell loading

0%

20%

40%

60%

80%

100%

120%

0 0.5 1 1.5 2

Loading (g cells / g carrier)

Res

idua

l act

ivity

(%)

TiOX

Ca- alginate

Transaminase immobilization. Operational stability

0%

20%

40%

60%

80%

100%

0 2 4 6 8 10

Batch no

Res

idua

l act

ivity

(%)

Titanium oxide

Ca-alginate

Dihydroxyacetone

• Acetic acid bacteria catalyse selective oxidation reactions

• Gluconobacter oxydans is a typical example

OH

OH

OH

OH

OH

O+ 0,5 O2 + H2O

Glycerol dehydrogenase

DHA processLimitations

• Inhibition/inactivation by glycerol• Inhibition/inactivation by DHA• Oxygen supply

Oxygen supply to cells in spherical particles

Oxygen is only present in the outer part of the sphere

The particle size has a big impact on the effectiveness factor

Production of 3-hydroxypropionaldehyde

Glyceroldehydratase

Glycerol

3-Hydroxypropionaldehyde

• Precursor for industrial chemicals - 1,3-propanediol, acrolein, acrylic acid, polymers

• Food preservative

Current mode of production

Propylene Acrolein

Ethylene

Hydration

* Product separation * Low product yield * Acrolein toxic

hydroformylation

Ethylene oxide3-HPA

Production Problems

Strategies for production

1. Fermentative production using Lactobacillus reuteri

• Reuterin, the antibacterial compound produced by L. reuteri is 3-HPA, HPA hydrate and HPA dimer

• Prevent the production of side-products- Metabolic engineering-Selectively extracting the product during fermentation

Glycerol 1,3-Propanediol

Glycerol

dehydratase

Propanediol

oxidoreductase

x2H2O

2. Biocatalytic process

• Recombinant glycerol dehydratase gene expressed in Escherichia coli

• High cell density reactor

• Continuous process

Strategies for production

Industrial biotechnology for the Industrial biotechnology for the production of platform chemicalsproduction of platform chemicals

20082008--20132013

RajniRajni HattiHatti--KaulKaul

Organic acids Organic acids are important building are important building blocks for large blocks for large number number of of chemicals used by Swedish industrychemicals used by Swedish industry

Biodiesel and oleochemicals

Organic acids as platform chemicalsOrganic acids as platform chemicals

• Important building blocks for C3-C6 chemistry• Biotechnology (anaerobic fermentation) – an important

mode for production• Bottlenecks in fermentative production

– Organic acid toxicity– Nutrient costs– Side products– Downstream processing resulting in waste build up

• Integration of biotech process downstream with the chemicals production

PlatformPlatform chemicalschemicals in this in this projectproject

• Propionic acid

• 3-Hydroxypropionic acid

Methacrylic acid

1,3-propanediol Acrylic acid

Amongst top 20 value added chemicals from biomass

OurOur planplan• Develop economically competitive process technology–

based on biotechnological production of organic acids

• Integrate biotechnology with the chemical processes for production of secondary chemicals

• Integrate production of bioenergy from fermentation residues

• Environmental and economic assessment to determinesustainability

• Set up a demonstration process

• Propionic acid•3-Hydroxypropionate

GlucoseGlycerol

•1,3-propanediol•Acrylic acid•Methacrylic acid

Biodiesel

•Coatings•Thermoplastics

•Polymers •etc. etc.

Platform chemicals

Secondary chemicals

RapeseedBarley residue

Biogas

residues

Fermentation

Anaerobicdigestion

PerstorpLyckeby

Indienz

PerstorpBona

Göteborgsenergi

Danisco

LU

Potential for Potential for improvementimprovement in in environmentenvironment

Platform organisms

• Propionibacterium sp.

• Lactobacillus sp. (native or engineered)

• Escherichia coli (metabolically engineered)

Microbial Production of Microbial Production of PropionicPropionic AcidAcid

Glycerol

Dihydroxyacetone phosphate

Phosphoenol pyruvate

Pyruvate

Oxaloacetate

Malate

Succinate

Propionate

Acetate

0.5 Glucose

NAD+

NADH2

ATPADP

ADP

ATP

ADPATP

ADPATP

NADH2NAD+ADP

ATP

Acetyl-CoA

2ATP 2ADP

NAD+NADH2

CO2

NADH2NAD+

NAD+

NADH2

• Reached productivity of 2-3 g l-1h-1 from glycerol using high cell density reactor

• Scale up being planned

• Enzymatic pretreatment of barley residues studied

• Process for production of methacrylic acid being set up

LCA of LCA of PropionicPropionic AcidAcid

• Swedish average electricity• Steam/heat produced with 75% wood

chips and 25% LPG• 100 g glycerol/l, 90% of max

conversion• Productivity is 2 g/l*h• Sterilization with steam• Multistage evaporation for separation

0

5

10

15

20

25

30

35

40

45

Bio-based Propionic acid Fossil-based Propionic acid

MJ/

kg

Process

Raw material

Separation

Fermentation

Potatoe juice

Glycerol

0

500

1000

1500

2000

2500

3000

3500

4000

Bio-based Propionic acid Fossil-based Propionic acid

g C

O2

eq/k

g

Product

Process

Raw material

Separation

Fermentation

Potatoe juice

Glycerol

0

1

2

3

4

5

6

7

Bio-based Propionic acid Fossil-based Propionic acid

g PO

43- e

q/kg

Process

Raw material

Separation

Fermentation

Potatoe juice

Glycerol

Primary energy consumption

Global warming potential

Eutrophication potential

Glucose

Pyruvate

Acetyl-CoA Malonyl-CoA Malonic acid

3-oxopropionic acid

Oxaloacetic acid Propionic acid Propionyl-CoA

Acroloyl-CoALactic acid Lactoloyl-CoA

α-alanine β-alanine

Aspartic acid

Succinic acid

MetabolicMetabolic pathwayspathways for 3for 3--HP HP productionproduction

Glycerol

3-hydroxy-propionaldehyde

3-hydroxypropionyl phosphate

3-HP3-hydroxypropionyl-CoA

Poly(3-hydroxy-propionic acid)

Glucose

Pyruvate

Acetyl-CoA Malonyl-CoA Malonic acid

3-oxopropionic acid

Oxaloacetic acid Propionic acid Propionyl-CoA

Acroloyl-CoALactic acid Lactoloyl-CoA

α-alanine β-alanine

Aspartic acid

Glycerol

3-hydroxy-propionaldehyde

3-hydroxypropionyl phosphate

3-HP

Succinic acid

3-hydroxypropionyl-CoA

MetabolicMetabolic pathwayspathways for 3for 3--HP HP productionproduction

Poly(3-hydroxy-propionic acid)

1)

Glycerol

3-hydroxy-propionaldehyde

3-hydroxypropionyl phosphate

3-HP3-hydroxypropionyl-CoA

Poly(3-hydroxy-propionic acid)

Glucose

Pyruvate

Acetyl-CoA Malonyl-CoA Malonic acid

3-oxopropionic acid

Oxaloacetic acid Propionic acid Propionyl-CoA

Acroloyl-CoALactic acid Lactoloyl-CoA

α-alanine β-alanine

Aspartic acid

Succinic acid

MetabolicMetabolic pathwayspathways for 3for 3--HP HP productionproduction

2)

Glucose

Pyruvate

Acetyl-CoA Malonyl-CoA Malonic acid

3-oxopropionic acid

Oxaloacetic acid Propionic acid Propionyl-CoA

Acroloyl-CoALactic acid Lactoloyl-CoA

α-alanine β-alanine

Aspartic acid

Succinic acid

MetabolicMetabolic pathwayspathways for 3for 3--HP HP productionproduction

Glycerol

3-hydroxy-propionaldehyde

3-hydroxypropionyl phosphate

3-HP3-hydroxypropionyl-CoA

Poly(3-hydroxy-propionic acid)

3)

Glucose

Pyruvate

Acetyl-CoA Malonyl-CoA Malonic acid

3-oxopropionic acid

Oxaloacetic acid Propionic acid Propionyl-CoA

Acroloyl-CoALactic acid Lactoloyl-CoA

α-alanine β-alanine

Aspartic acid

Succinic acid

MetabolicMetabolic pathwayspathways for 3for 3--HP HP productionproduction

Glycerol

3-hydroxypropionyl phosphate

3-HP3-hydroxypropionyl-CoA

Poly(3-hydroxy-propionic acid)

3-hydroxy-propionaldehyde

4)

Glycerol

3-hydroxy-propionaldehyde

3-hydroxypropionyl phosphate

3-HP3-hydroxypropionyl-CoA

Poly(3-hydroxy-propionic acid)

Glucose

Pyruvate

Acetyl-CoA Malonyl-CoA Malonic acid

3-oxopropionic acid

Oxaloacetic acid Propionic acid Propionyl-CoA

Acroloyl-CoALactic acid Lactoloyl-CoA

α-alanine β-alanine

Aspartic acid

Succinic acid

Metabolic pathways for 3-HP production

5)

Glycerol

3-hydroxy-propionaldehyde

3-hydroxypropionyl phosphate

3-HP3-hydroxypropionyl-CoA

Poly(3-hydroxy-propionic acid)

Glucose

Pyruvate

Acetyl-CoA Malonyl-CoA Malonic acid

3-oxopropionic acid

Oxaloacetic acid Propionic acid Propionyl-CoA

Acroloyl-CoALactic acid Lactoloyl-CoA

α-alanine β-alanine

Aspartic acid

Succinic acid

MetabolicMetabolic pathwayspathways for 3for 3--HP HP productionproduction6)

Glycerol

3-hydroxy-propionaldehyde

3-hydroxypropionyl phosphate

3-HP3-hydroxypropionyl-CoA

Poly(3-hydroxy-propionic acid)

Glucose

Pyruvate

Acetyl-CoA Malonyl-CoA Malonic acid

3-oxopropionic acid

Oxaloacetic acid Propionic acid Propionyl-CoA

Acroloyl-CoALactic acid Lactoloyl-CoA

α-alanine β-alanine

Aspartic acid

Succinic acid

MetabolicMetabolic pathwayspathways for 3for 3--HP HP productionproduction

7)

Glycerol

3-hydroxy-propionaldehyde

3-hydroxypropionyl phosphate

3-HP3-hydroxypropionyl-CoA

Poly(3-hydroxy-propionic acid)

Glucose

Pyruvate

Acetyl-CoA Malonyl-CoA Malonic acid

3-oxopropionic acid

Oxaloacetic acid Propionic acid Propionyl-CoA

Acroloyl-CoALactic acid Lactoloyl-CoA

α-alanine β-alanine

Aspartic acid

Succinic acid

MetabolicMetabolic pathwayspathways for 3for 3--HP HP productionproduction

ThankThank you for your you for your attentionattention

Putting the care into chemicals