Engineering Scalable Biocatalytic Processes John M Woodley Department of Chemical and Biochemical Engineering Technical University of Denmark (DTU) DK-2800 Lyngby Denmark

Background

The Next Fifty Years 6-7 x 5-6 x 3.5 x 7 x Increase Increase Global GDP growth (in constant dollars) 5-6 x Production capacity for most commodities (steel, chemicals, lumber, etc.) 3.5 x Energy demand 7 x Electricity demand Increase Water demand Increase GHG emissions Siirola (2012) Proc 11th Symp PSE (Ed Karimi and Srinivasan) 1

Features of BioProcesses Effective conversion of sustainable (cheap, available, renewable) feedstocks Operate under mild conditions Renewable catalyst Woodley et al (2013) Chem Eng Res Des 91, 2029

Types of Bioreaction Fermentation A+   A  B   B Microbial biocatalysis A+   A  B   B A   A  B   B A  B Enzymatic biocatalysis

Types of Product from BioProcesses Cost High cost small molecules e.g. pharmaceuticals (e.g. sitagliptin) High cost large molecules e.g. biopharmaceuticals (e.g. human growth hormone) Molecular weight Low cost small molecules e.g. bulk chemicals (e.g. lactic acid) Low cost large molecules e.g. industrial enzymes (e.g. amylase)

Fermentation Process Structure Separation 2 Fermenter Separation 1 F P2 C

Challenges with Fermentation for Chemical Production Diversion of carbon to make cells and by-products, limits yield Conversion rate limited by growth, limited productivity Product toxicity, limits concentration

Biocatalysis

Optimization of Biocatalyst Production and Conversion Fermentation Biocatalyst production Biocatalysis Conversion Separate growth and conversion Increased productivity by adding more biocatalyst

Increase Biocatalyst Yield Fermentation Biocatalysis Recycle (whole-cell) biocatalyst

Features of Microbial Biocatalysis Independent growth and conversion Yield of reaction is set by the reaction (not growth) Increased space-time yield (productivity) by addition of more biocatalyst Potential to recycle biocatalyst Marshall and Woodley (1995) Nature Biotech 13, 1072 Woodley (2006) Adv Appl Microbiol 60, 1

Reduce Cellular Constraints Fermentation Enzyme isolation Biocatalysis Use (immobilized) enzyme to achieve high biocatalyst concentration

Features of Enzyme Catalysis Exquisite selectivity Operate under mild conditions Enzyme from renewable resource Ability to alter enzyme properties via protein engineering Pollard and Woodley (2007) Trends Biotechnol 25, 66 Woodley (2008) Trends Biotechnol 26, 321 Sheldon and Woodley (2017) Chem Rev. DOI:10.1021/acs.chemrev.7b00203

Biocatalytic Process Structure Separation 2 R Reactor Separation 1 P B Lima-Ramos et al (2014) Green Proc Synth 3, 195

1. Multi-step Catalysis

Biocatalysts for Multiple Steps France et al (2017) ACS Catalysis 7, 710

2. Chemo-enzymatic Catalysis

Combining Biocatalysis and Heterogeneous Inorganic Catalysis Vennestrøm et al (2010) ChemCatChem 2, 249

Bio-petrochemicals

DTU - Novozymes Collaboration Step 1 Step 2 Step 3 Bottles made of Polyethylene terephthalate (PET) Step 1: enzyme catalysed Step 2 & 3 : cataylzed by inorganic catalysts Boisen et al (2009) Chem Engng Res Dev 87, 1318 NOVOZYMES PRESENTATION NOVOZYMES A/S 21

HMF as a Platform Chemical

DTU - Novozymes HMF Process Solvent Solvent separation HMF recovery G  F F  HMF Isomerization Dehydration HMF (crude) Water, G, “Other sugars” Water, G, “Other sugars” Mixing Glucose syrup Abbreviations F: Fructose G: Glucose HMF: Hydroxymethylfurfural

3. Continuous Catalytic Processes

Michaelis-Menten Kinetics

Reactor Modelling Kinetics for reactors obeying Michaelis-Menten kinetics: Batch XS0 + Km(ln(1/(1 - X))) = kEt/V Continuous plug-flow XS0 + Km(ln(1/(1 - X))) = kE/Q Continuous flow stirred tank XS0 + Km(X/(1 - X)) = kE/Q

Biocatalytic Flow Reactors Andrade et al (2014) Org Lett 16, 6092

Multiple Stirred Tanks

Agitated Cell Reactor Toftgaard Pedersen et al Biotech Bioeng 114, 1222

Segmented Flow Reactor Multi-phase options with organic solvent and aqueous phase

Tube-in-Tube Reactor Teflon AF-2400 Tubing Inner Radius: 0.3 mm Tubing Thickness: 0.1 mm Reactor Length: 0.5 m Residence Times: 1-20 s Flow Rate: 0.4-8.5 mL.min-1 Yang and Jensen (2013) Org Process Res Dev 17, 927

Mechanism and Rate Law  

Tube-in-Tube Reactor (TiTR) for Enzyme Kinetic Analysis Ringborg et al (2017) ChemCatChem 9, 3285

Comparison of TiTR with BSTR (1 atm) Ringborg et al (2017) ChemCatChem 9, 3285

TiTR at Varying Pressure Ringborg et al (2017) ChemCatChem 9, 3285

Role of Kinetics: Guiding Protein Engineering Improved process Improved biocatalyst Kinetic analysis Woodley (2017) Phil Trans R Soc A. DOI:19.1098/rsta.2017.0062

Final Comments Biocatalysis looks attractive for large scale processes due to: excellent economic parameters (scalable) ability to create new catalytic pathways (multi-step, chemo-enzymatic) ability to operate processes continuously At DTU we are always open to collaboration.

Special Thanks to Collaborators Industries Universities BASF (D) University of Manchester (UK) Novozymes (DK) University of Stuttgart (UK) DSM (NL) University of Groningen (NL) c-LEcta (D) TU Graz (A) Sigma Aldrich Chemie (CH) UAB (ES) Novo Nordisk (DK) InnoSyn (NL) Lundbeck (DK) Enzymicals (D) Biosyntia (DK)

Contact Professor John M Woodley DTU Chemical Engineering jw@kt.dtu.dk +45 4525 2885