CO 2 Fixation by Anaerobic Non- Photosynthetic Mixotrophy for - PowerPoint PPT Presentation

CO 2 Fixation by Anaerobic Non- Photosynthetic Mixotrophy for Improved Carbon Conversion Emily E. Crawford, John R. Phillips, Pradeep C. Munasinghe, Carrissa A. Wiedel, Biniam Maru, Ilana Aldor, & Shawn W. Jones, Terry Papoutsakis & Bryan Tracy ARPA-E Carbon Optimized Bioconversion Sept 26, 2019 1

Bio-economy Conundrum Glucose is • ~50% Carbon • ~50% Oxygen • Carbon = = $$$ • Oxygen = = -$$$ Nearly all molecules in industry have a carbon backbone with little to no oxygen. How does conventional fermentation deal with this? 2 Proprietary and confidential information of White Dog Labs, Inc

Bio-economy Conundrum Glucose Ethanol Carbon Dioxide Fermentation densifies energy and sacrifices 1/3 of the carbon to get rid of oxygen. 3 Proprietary and confidential information of White Dog Labs, Inc

Conventional Ethanol Glucose Ethanol Carbon Dioxide Fermentation sacrifices 33% of the carbon to get rid of oxygen and densify energy into ethanol. Is there a way to improve carbon yield? 4 Proprietary and confidential information of White Dog Labs, Inc

Fermentation Plus Chemical Reductant Glucose Ethanol Water + � Hydrogen 6 x 100% carbon yield with water as only by-product 5 Proprietary and confidential information of White Dog Labs, Inc

Clostridial Mixotrophy (MixoFerm) at its Core Solar Biomass H 2 Biomass Fossil fuel Fossil fuel Waste streams H 2 /CO/CO 2 H 2 CO 2 Biomass Reducing equivalents are Hexose/Pentose Glycolysis [H] WLP used to fix CO 2 through the Wood-Ljungdahl Pathway Cell mass Acetyl-CoA Acetyl-CoA (WLP) Acetogen host Metabolite Fast, AG, Schmidt, ED, Jones, SW, & Tracy, BT. 2015. Curr Opin Biotechnol 33:60-72. � Jones, SW, et al . 2016. Nat Comm 7:12800. � 6 Proprietary and confidential information of White Dog Labs, Inc

Clostridial Mixotrophy In Video MixoFerm organisms consume sugar and CO 2 thus increasing yield MixoFerm™ fermentation – note the lack of bubbles Conventional fermentation – note the bubbles due to gas evolution 7 Proprietary and confidential information of White Dog Labs, Inc

Spectrum of Mixotrophic Processes No No Only Only Carbohydrate Carbohydrate Carbohydrate Carbohydrate Carbohydrate " Carbohydrate ! Carbohydrate ! CO/CO 2 /H 2 ! CO/CO 2 /H 2 ! Carbohydrate ! Carbohydrate ! H 2 ! CO/CO 2 /H 2 ! Gas Fermentation ! MixoFermPlus™ ! Conventional 
 MixoFermPlus™ ! MixoFermPlus™ ! MixoFerm™ ! (ATP limited) ! (Redox limited) " (Carb - enhanced) ! (H 2 - enhanced) ! (Syngas - enhanced) ! Metabolite ! Metabolite ! Metabolite ! Metabolite ! Metabolite ! CO 2 ! CO 2 ! CO 2 ! Metabolite " CO 2 " H 2 " CO 2 ! (product) ! (product) ! (product) ! (product) ! (product) ! (product) " No Gas No Gas Only Gas Only Gas 8 Proprietary and confidential information of White Dog Labs, Inc

Array of Feedstock Combinations Carbohydrates CO 2 Reducing Gases Reformed CO 2 1 st Reformed 2 nd Gasified Gasified Natural gas Gen Sugars Glycerin emissions Landfill Gen Sugars Acid whey Biomass Coal gas Reducing gases Carbohydrates Clostridial Mixotrophy � Diversity of feedstock use provides synergies across industries 9 Proprietary and confidential information of White Dog Labs, Inc

Starch Industry Motivation for Mixotrophy USDA Agricultural Projections to 2023 http://www.ers.usda.gov/publications/oce-usda-agricultural-projections/oce141.aspx � 10 Proprietary and confidential information of White Dog Labs, Inc

ClearMash™ Technology Built pilot system – 3 years of operation Applied the technology to corn mash clarification WDL adopted an existing production technology Low CapEx clarification technology • <$3M project cost for production of >50,000 MT clarified starch per year • Sterilizable, readily customized • Starch generation upwards of 30wt% solids • Resistant to major process disruptions ClearMash™ - enabling <$0.08/lb starch production 11 Proprietary and confidential information of White Dog Labs, Inc

Starch Industry Motivation for Mixotrophy • Attractive sugar economics today • Robust supply and highly supportive supply chain with strong lobbying capabilities • Trillions of dollars of already invested capital However • Sacrifice of biodiversity • Through practices that are not so carbon neutral (corn itself exhibits 25% of the petro-CO 2 intensity) • Still mandated markets that lead to tremendous business uncertainty • Even at $0.08/lb sugar, it still struggles to compete with $60/barrel oil 12 Proprietary and confidential information of White Dog Labs, Inc

2 nd Generation Sugar Motivation for Mixotrophy Cellulosic Hydrolysis Cellulosic Hydrolysis � Biomass � Pre-treatment Pre-treatment � liberates cellulose from the lignin Status � Status “seal” technical issues technical issues � & expensive & expensive � Poet operating at partial capacity � Poet operating at partial capacity Abengoa Abengoa and DuPont sold prior to startup and DuPont sold prior to startup � Cellulose � Hydrolysis � Hydrolysis Convert cellulose to sugar with enzymes � Sugar � Fermentation - Fermentation - Conventional � technology � 13 Proprietary and confidential information of White Dog Labs, Inc

55M gallon per year corn ethanol Corn ethanol CapEx ~$1.75/gallon Cellulosic ethanol CapEx >$10/ gallon Cost largely associated with biomass destruction, i.e., sugar production Cellulosic sugars are 20-40M gallon more expensive or valuable than commonly per year perceived cellulosic ethanol 14 Proprietary and confidential information of White Dog Labs, Inc

Industrial Gas Motivation for Mixotrophy Co-located Production <$1/kg H 2 ~$1.1M/ 1,000Nm 3 /hr Pipeline ~$1,000Nm 3 /hr H 2 / 1M gal $1 – 1.2/kg H 2 EtOH 15 Proprietary and confidential information of White Dog Labs, Inc

Industrial Gas Motivation for Mixotrophy - SCALE 1M gallons of annual ethanol production requires 1,000Nm 3 /hr of H 2 Potential Today 18 Billion Gallons Ethanol 18 Billion Gallons Ethanol 4.3 billion bushels of corn 6.5 billion bushels of corn + 120 steam methane reformers 16 Proprietary and confidential information of White Dog Labs, Inc

Industrial Gas Motivation for Mixotrophy - SCALE 2 nd Generation Opportunity 20 Billion Gallons Ethanol 20B gallons * $10/gallon EtOH = $200B in capital 13.2B gallons * $10/gallon + 6.8B gallons * $1.1B/B gallon EtOH = $139B in capital >30% reduction in capital required & Normalized for energy, H 2 comparable cost to glucose is $0.054/lb à recall, I claim starch can be $0.08/lb from corn 17 Proprietary and confidential information of White Dog Labs, Inc

MixoFerm™ at its core (cellular level) Solar Biomass MixoFerm™ with gas H 2 Biomass Fossil fuel Fossil fuel addition Waste streams Gases can provide H 2 /CO/CO 2 more reductant H 2 CO 2 Biomass Reducing equivalents [H] Hexose/Pentose Glycolysis [H] WLP are used to fix CO 2 through the Wood-Ljungdahl Cell mass Acetyl-CoA Acetyl-CoA Pathway (WLP) Acetogen host Metabolite Fast, AG, Schmidt, ED, Jones, SW, & Tracy, BT. 2015. Curr Opin Biotechnol 33:60-72. � Jones, SW, et al . 2016. Nat Comm 7:12800. � 18 Proprietary and confidential information of White Dog Labs, Inc

Energetic Requirements Comparison 19 Proprietary and confidential information of White Dog Labs, Inc

Demonstration of MixoFerm Conventional MixoFerm™ 20 Proprietary and confidential information of White Dog Labs, Inc

Demonstration of MixoFerm Plus Broad host range, which we have since curated >20 unique MixoFerm strains Conventional MixoFerm™ MixoFerm™ Plus Syngas 21 Proprietary and confidential information of White Dog Labs, Inc

MixoFerm – Catabolite Repression? Grew Clostridium ljungdahlii on 12 C fructose and 13 C syngas mixture. Any 13 C-labeled metabolite had to come from syngas utilization. Syngas is consumed simultaneously with fructose at high incorporation levels – actually preferred Jones, SW, et al . 2016. Nat Comm 7:12800. � 22 Proprietary and confidential information of White Dog Labs, Inc

H 2 MixoFerm Plus for CO 2 Capture & EtOH H 2 addition to C. autoethanogenum • Effectively captured all CO 2 • Shifted metabolism to reduced product formation • Achieved ~65wt% yield of EtOH on consumed fructose 23 Proprietary and confidential information of White Dog Labs, Inc

Benefits of Mixotrophy Key ratio to benefits of Increase in yield of NAD(P)H/Acetyl-CoA MixoFerm™ over Example molecules MixoFerm™ is: ratio standard fermentation NAD(P)H to Acetyl-CoA 0 51% Acetic acid or Acetone 0.5 35% Isopropanol 1.0 22% Butyric acid Focus of first 1.5 11% 2,3-Butanediol demonstration of MixoFerm™ 2 2% Ethanol or n-Butanol 3 0% Propionic acid 24 Proprietary and confidential information of White Dog Labs, Inc

Acetone Production No known acetogen naturally produces acetone. Acetogen host: C. ljungdahlii Sugar CO 2 CO 2 WLP Pyruvate Acetolactate Acetoin ALS ALDC 2[H] SADH/ CO 2 + 2[H] PFOR 2,3BDH 4[H] 2,3-Butanediol Acetate Acetyl-CoA Ethanol AAD AK & PTA Köpke, M., et. al. 2014. Appl Environ Microbiol 80:3394-3403. 25 Proprietary and confidential information of White Dog Labs, Inc