Green conversions and processing

Enzymes and Green Chemistry, University of Campinas

November 2017, Elinor L. Scott

From resource to product

▪ The biorefinery

● Pre-treatments

● Extraction and isolation of

biomolecules

▪ The reaction

● The (bio)catalyst

● Temperature and pressure

● Conversion and selectivity

▪ Separation of product

● Distillation

● Extraction etc...

2

A B C+

Often consider these separately. Important....but

Processes influences choices

3

A B C+

Biorefinery affects feed quality for use in a reaction and

subsequent down stream processing

These factors will affect the overall process technology,

efficiency and success

▪ Synergistic effect

● Choices in one step can have a positive effect on the next

● Able to combine multiple steps in less unit operations

▪ Opportunities to use less energy and chemicals

● Improve efficiency

Knowledge and process integration from

resource to product

4

So where are we and what do we need to do?

Biorefinery

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Green Marine

Lipid

Sugar

Lignocellulose

Current approaches in biorefinery of

biomass - Lignocellulose

▪ Before enzyme conversion opening of the structure is required

● Mechanical

● changes in crystallinity and particle size

● Thermo-chemical

● Use of steam, acid and base

6

Hemicellulose

Lignin

Cellulose

Plant cell wall

Plant

Pre-treatment

Sulphuric acid

Biomass

High pressuresteam

Low pressuresteam

Treated Biomass

High pressure reactor

Explosive decompression

What have we learnt?

▪ Techniques can lead to use of lignocellulose as a glucose feedstock

But....

▪ Current limitations

Robust structure require “effort” to make is usable

“Heavy” use of chemicals and energy and effect on costs

Production of side products and inhibitors and dealing with that

12

So how can we tackle challenges?

▪ More understanding of plant growth and cellulose chemistry

● If we identify key factors how it is produced in nature...more specific (milder) deconstruction can be used

● Development of new pre-treatment and hydrolysis strategies

13

Current approaches in biorefinery of

biomass – Green (and marine)

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e.g.

(also contains dissolved, proteins, amino acids, sugars, organic acids)

What have we learnt?

15

▪ Techniques to isolate some compounds from aqueous (juicey) streams can be achieved to a certain level

But....

▪ Current limitations

Low /not optimal protein recovery

• From juices and solids

Addition of chemicals

• Base, acid

Quality of protein

What have we learnt (2)?

▪ Isolation/extraction of proteins is inefficient

● Also other molecules in aqueous fractions

● Inefficient use of resources

In general there is an issue in dealing with dilute heterogeneous resources

Dealing Large volumes of water

Low concentrations of various dissolved molecules

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So how can we tackle challenges?

▪ Simplify the media using microorganisms

● Conversion to one product

● Product is insoluble

● E.g. Polyhydroxyalkonates

● E.g. Cyanophycin

▪ Use other specific conversions to change chemical and physical properties and integrate with separation

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PHB (Polyhydroxybutyrate)

▪ Plasticicumulans acidivorans utilise VFAs to produce PHB

▪ Use of waste water

● Water is cleaned and PHB is produced

▪ Over 80% of dry weight in PHB

L. Marang et al. Bioresour. Technol., 142, 232, 2013.

PHB as an intermediate to industrial

chemicals

▪ Reaction occurs via thermolysis followed by esterification

▪ Use of intracellular material

● Reduces costs and carbon footprine

PHB as an intermediate to industrial

chemicals

▪ PHB can be used as an intermediate to obtain biobased monomers from waste water

▪ Intracellular PHA can also be used

● Reduction in costs

● Reduction in carbon footprint

Conclusion PHB to chemicals

1. Spekreijse J., Ortega J.H., Sanders J.P.M., Bitter J.H., Scott, E.L. (2016) Conversion of polyhydroxyalkanoates to methyl crotonate using whole cells Bioresource

Technology, 211, 267-272

2. Spekreijse J., Le Notre J., Sanders J.P.M. Scott, E.L. (2015) Conversion of polyhydroxybutyrate (PHB) to methyl crotonate for the production of biobased monomers J.

App. Polym. Sci., 132(35), 42462-4270

3. Spekreijse J., Ohrstrom L., Sanders J.P.M., Bitter J.H., Scott, E.L. (2016) Mechanochemical immobilisation of methathesis catalysts in a metal-organic framework

Chemistry- A European Journal, 22(43), 15437-15443

4. Spekreijse J., Sanders J.P.M., Bitter J.H., Scott, E.L. (2016) The future of ethenolysis in biobased chemistry ChemSusChem, 10(3), 470-482

Cyanophycin

Polymer: aspartic acid backbone and arginine side chains

Mainly in cyanobacteria as nitrogen and energy reserve

Granule: 35% (wt/wt)

Steinle et al

Agentschap.NL, WUR (NL), University of Munster (D), AVEBE (NL),

Cosun (NL), ECN (NL)

Konst et al

Könst, P.M.; Franssen, M.C.R.; Scott, E.L.; Sanders, J.P.M. (2011) Stabilization and immobilization of Trypanosoma brucei ornithine decarboxylase for the biobased

production of 1,4-diaminobutane Green Chem. 13 (5), 1167 - 1174.

Könst, P.M.; Scott, E.L.; Franssen, M.C.R.; Sanders, J.P.M. (2011) Acid and Base Catalyzed Hydrolysis of Cyanophycin for the Biobased Production of Nitrogen Containing

Chemicals Journal of Biobased Materials and Bioenergy 5 (1), 102 - 108.

Könst, P.M.; Turras, P.M.C.C.D.; Franssen, M.C.R.; Scott, E.L.; Sanders, J.P.M. (2010)

Stabilized and Immobilized Bacillus subtilis Arginase for the Biobased Production of Nitrogen-Containing Chemicals Advanced Synthesis and Catalysis 352 (9), 1493 - 1502.

Amino acids

▪ Useful functionality....

● R group interesting

● Amine and carboxylic acid groups present

▪ Decarboxylation reactions

● To amines

▪ Oxidative decarboxylation

● to nitriles

Industrial chemicals in less steps

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Why is that interesting?

▪ Less steps...potential to use less energy

▪Assume a typical production route to 1 tonne of a chemical

● Energy for production and use of co-reagent and for the carbon backbone of the chemical (e.g. ethylene, propylene etc…)

● Use of ca. 60 GJ per tonne product

▪What is 60 GJ’s and what does it look like?

60 GJ

63 000 x

OR

300 000 people (population Utrecht) cycling for

10 minutes

Some reactions...Glutamic acid

Diaminobutane

Acrylonitrile

N-Methylpyrrolidone

N-Vinylpyrrolidone

Glutamic acid

H2N COOH

COOH

Some reactions...Glutamic acid

▪ Some steps use enzymatic conversions under ambient conditions

● Use of decarboxylase to make intermediate

● Excellent conversion and selectivity

● Immobilisation of enzyme improves stability and reuse

▪ Other reactions use stoichiometric amounts of reagents and requires cooling

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acrylonitrile

Glutamic acid – what we learnt

▪ Some reactions work best using enzymes

● Decarboxylation (glutamic acid to aminobutyric acid)

▪ Other reactions works with chemistry...but some with problems

● Oxidative decarboxylation (glutamic acid to cyanopropanoic acid) to nitriles requires stoichiometric amounts of reagents (hypochlorite) and cooling

▪ How to solve?

● Use enzymes

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Amino acids flexible building

block...oxidative decarboxylation

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Plastics

Agrochemicals

Solvents

Pharmaceuticals

Adhesives

isobutyronitrile

Vanadium ChloroPerOxidase (VCPO)

H2O2 + H + + X - H2O + HOX X -: Cl -, Br -, I -

From amino acids to nitriles using VCPO:

i. Production of HOBr:

ii. Oxidative decarboxylation:

HOBr: Br +, Br 2, Br 3 -, VCPO-Br

H 2O 2 + H + + Br - H 2O + HOBr

2 equiv. HOBr- CO2

- 2 Br -

Oxidative decarboxylation with enzymes –

what we learnt

▪ This works well with some amino acids and not with others

● Possible to control reactivity as a function of amino acid structure and bromide concentration

● We have a handle on this now

▪ Still rely on hydrogen peroxide as oxidant

● Can we use molecular oxygen?

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H2O2 – the Anthraquinone Process

Conc.

30 - 70%

In situ production of H2O2

< 3% (wt-%)

Nice (bio)chemistry....but use of single amino acids

Integrated conversion and separation –

charge difference

▪ Mixture of compounds of comparable charge behaviour are unable to be separated by electrodialysis

▪ Selective reaction of a compound in the mixture to new molecule with a different charge.

● Product separation from the rest of the mixture

A(0) B(+)

▪ Why is this useful?

● Combined conversions and separation/isolation of product in

aqueous, dilute, heterogeneous mixtures

● If the product is a (intermediate) chemical for the chemical

industry, biorefinery and conversion becomes more attractive

Phenylalanine (Phe) and Serine (Ser)

▪ At pH ca.7 Phe and Ser are neutral

Ser (0) Ethanolamine (+)

Phe (0) Cinnamic acid (-)

pKa 9.50

-CO2

-NH3

Use of serine

decarboxylase enzyme

Use of phenylalanine

ammonia lyase enzyme

Integrated conversion and separation

CEM

-

-

-

-

-

AEM

+

+

+

+

+

CEM

-

-

-

-

-

AEM

+

+

+

+

+

+

Anode

-

Cathode

Teng, Y.; Witte-van Dijk S. C. M.; Scott, E.L.; Sanders, J.P.M. (2015) Simultaneous decarboxylation of L-serine and deamination of L-phenylalanine as a means to aid the separation of neutral amino acids New Biotechnol. http://dx.doi.org/10.1016/j.nbt.2015.04.006Teng, Y.; Scott, E.L.; Zeeland, A.N.T. van; Sanders, J.P.M. (2011) The use of L-lysine decarboxylase as a means to separate amino acids by electrodialysis Green Chem. 13 (3), 624 - 630. Teng, Y.; Scott, E.L.; Sanders, J.P.M. (2014) The Selective Conversion of Glutamic Acid in Amino Acid Mixtures Using Glutamate Decarboxylase - A Means of Separating Amino Acids for Synthesizing Biobased Chemicals Biotechnology Progress 30(3):681-8

Integrated conversion and separation

▪ Cinnamic acid can be used as a pre-cursor for biobasedstyrene and acrylic acid synthesis

● metathesis of cinnamic acid (ester) with ethene

▪ Ethanolamine is used in gas scrubbing of flue gas and as an intermediate for a number of pharma, fine and bulk chemical products

OR

O

OR

O12.5 mol% catalyst

ethene (1 bar)

DCM, 40 oC, 24 h

+

Spekreijse, J.; Le Notre, J.; van Haveren, J.; Scott, E.L.; Sanders, J.P.M. (2012) Simultaneous production of biobased styrene and acrylates using ethenolysis

Green Chem., 14, 2747

Chemistry and/or enzymatic conversions?

▪ From the examples...

● Sometimes one and sometimes the other

▪ How to decide?

● Is my molecule largely present in an aqueous environment?

● Is the conversion possible by either an enzymatic conversion or is it limited by chemistry?

● Can I use the best of both worlds to overcome isolation problems?

● Do I need to develop new catalysts

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Conclusions

▪ A more holistic approach on the whole chain from resource to product could prevent the need for some separation steps

▪ We have learnt a lot about processing and converting biomass

● But there are limitations

▪ Conventional processing, conversions, separation can be used to some extent

● Improvement by combining disciplines

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Advantages PHB as intermediate

▪ Insoluble in water

● Concentrating C from dilute media

▪ More defined

● Instead of mixture of VFAs

From resource to product

▪ Biorefinery

▪ Conversion

▪ Separation

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Reactivity of glutamic and aspartic acid

45Aspartic acid (Asp) conversion into 2-cyanopropanoic acid (AspCN) by vanadium

chloroperoxidase (VCPO) at 10 mM NaBr. Reaction rate of Asp is 0.12 mM/min.

aspartic acid, 2-cyanoacetic acid, malonic acid.

Figure 1. Conversion of a) glutamic acid (Glu) into 3-cyanopropanoic acid (GluCN) and b) aspartic acid (Asp) into

2-cyanoacetic acid (AspCN) by vanadium chloroperoxidase at 0.5 mM NaBr. Reaction rate of Glu is 0.15

mM/min.

Increase Br+...

increase conversion

Thoughts on conversion and separation

▪ Conversion to useful product while changing chemical or physical property is a useful tool.

● Not only applicable to change in charge difference

● Also a change in boiling point/vapour pressure

valine isobutylamine (food and flavour)

● Also a change in solubility

Bpt 63oC

Aspartic acid

4.5g/L H2O@STP

Glutamic acid

8.6g/L H2O@STP

Aspartic acid Pyroglutamic acid

50g/L H2O@STP

Cosmetics and chemical

intermediate

Δ

Conclusions

▪ Some things work...others dont

▪ Bioconversions and chemical conversions synergistic in the path from resource to product

▪ Thinking outside the box of your reaction can lead to surprising and useful solutions

▪ Working with others is better than working alone

▪ We’ve still got alot to do!

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