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

ARPA-E Carbon Optimized Bioconversion

Sept 26, 2019 1

CO2 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

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Glucose is

• ~50% Carbon
• ~50% Oxygen
• Carbon = = $$$
• Oxygen = = -$$$

Bio-economy Conundrum

Nearly all molecules in industry have a carbon backbone with little to no oxygen. How does conventional fermentation deal with this?

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Bio-economy Conundrum

Fermentation densifies energy and sacrifices 1/3 of the carbon to get rid of oxygen.

Ethanol Carbon Dioxide Glucose

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Conventional Ethanol

Fermentation sacrifices 33% of the carbon to get rid of oxygen and densify energy into ethanol. Ethanol Carbon Dioxide Glucose Is there a way to improve carbon yield?

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Fermentation Plus Chemical Reductant

100% carbon yield with water as only by-product

+

Ethanol Water Glucose Hydrogen

6 x

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Clostridial Mixotrophy (MixoFerm) at its Core

Hexose/Pentose Acetyl-CoA CO2 [H] Cell mass

Metabolite

H2 WLP Acetyl-CoA H2/CO/CO2 Glycolysis Biomass H2 Solar Biomass Fossil fuel Waste streams Biomass Fossil fuel Acetogen host

Reducing equivalents are used to fix CO2 through the Wood-Ljungdahl Pathway (WLP)

Jones, SW, et al. 2016. Nat Comm 7:12800. Fast, AG, Schmidt, ED, Jones, SW, & Tracy, BT. 2015. Curr Opin Biotechnol 33:60-72.

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Clostridial Mixotrophy In Video

MixoFerm organisms consume sugar and CO2 thus increasing yield

Conventional fermentation – note the bubbles due to gas evolution MixoFerm™ fermentation – note the lack of bubbles

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Spectrum of Mixotrophic Processes

MixoFermPlus™!

(H2 - enhanced)! Metabolite! (product)! Carbohydrate! H2!

MixoFermPlus™!

(Syngas - enhanced)! CO/CO2/H2! Metabolite! (product)! CO2! Carbohydrate!

MixoFermPlus™!

(Carb - enhanced)! CO/CO2/H2! Carbohydrate! Metabolite! (product)! CO2!

Gas Fermentation!

(ATP limited)! CO/CO2/H2! Metabolite! (product)! CO2!

Conventional


(Redox limited)" Carbohydrate" CO2" H2" Metabolite" (product)"

MixoFerm™!

Metabolite! (product)! CO2! Carbohydrate!

Only Only Carbohydrate Carbohydrate No No Carbohydrate Carbohydrate Only Gas Only Gas No Gas No Gas

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Array of Feedstock Combinations

1st Gen Sugars 2nd Gen Sugars Glycerin

Carbohydrates

Acid whey Gasified Biomass Reformed Natural gas Gasified Coal

Reducing Gases

Reformed Landfill gas

CO2

emissions

CO2

Reducing gases Carbohydrates

Clostridial Mixotrophy

Diversity of feedstock use provides synergies across industries

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Starch Industry Motivation for Mixotrophy

USDA Agricultural Projections to 2023 http://www.ers.usda.gov/publications/oce-usda-agricultural-projections/oce141.aspx

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ClearMash™ Technology

ClearMash™ - enabling <$0.08/lb starch production

WDL adopted an existing production technology Applied the technology to corn mash clarification Built pilot system – 3 years of operation

• <$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

Low CapEx clarification technology

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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-CO2 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

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2nd Generation Sugar Motivation for Mixotrophy

Cellulosic Hydrolysis Cellulosic Hydrolysis

Biomass Cellulose Sugar Pre-treatment Pre-treatment liberates cellulose from the lignin “seal” technical issues technical issues & expensive & expensive Hydrolysis Hydrolysis Convert cellulose to sugar with enzymes Fermentation - Fermentation - Conventional technology

Status Status

Poet operating at partial capacity Poet operating at partial capacity Abengoa Abengoa and DuPont sold prior to startup and DuPont sold prior to startup

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55M gallon per year corn ethanol 20-40M gallon per year cellulosic ethanol

Corn ethanol CapEx ~$1.75/gallon

Cellulosic ethanol CapEx >$10/ gallon

Cellulosic sugars are more expensive or valuable than commonly perceived Cost largely associated with biomass destruction, i.e., sugar production

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Industrial Gas Motivation for Mixotrophy

Pipeline $1 – 1.2/kg H2 Co-located Production <$1/kg H2 ~$1.1M/ 1,000Nm3/hr ~$1,000Nm3/hr H2 / 1M gal EtOH

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Industrial Gas Motivation for Mixotrophy - SCALE

1M gallons of annual ethanol production requires 1,000Nm3/hr of H2

Today 18 Billion Gallons Ethanol 6.5 billion bushels of corn Potential 18 Billion Gallons Ethanol 4.3 billion bushels of corn + 120 steam methane reformers

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Industrial Gas Motivation for Mixotrophy - SCALE 2nd 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, H2 comparable cost to glucose is $0.054/lb à recall, I claim starch can be $0.08/lb from corn

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MixoFerm™ at its core (cellular level)

Hexose/Pentose Acetyl-CoA CO2 [H] Cell mass

Metabolite

H2 WLP Acetyl-CoA H2/CO/CO2 Glycolysis Biomass H2 Solar Biomass Fossil fuel Waste streams Biomass Fossil fuel Acetogen host

Reducing equivalents [H] are used to fix CO2 through the Wood-Ljungdahl Pathway (WLP) MixoFerm™ with gas addition Gases can provide more reductant

Jones, SW, et al. 2016. Nat Comm 7:12800. Fast, AG, Schmidt, ED, Jones, SW, & Tracy, BT. 2015. Curr Opin Biotechnol 33:60-72.

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Energetic Requirements Comparison

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Demonstration of MixoFerm

Conventional MixoFerm™

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Broad host range, which we have since curated >20 unique MixoFerm strains

Conventional MixoFerm™ MixoFerm™ Plus Syngas

Demonstration of MixoFerm Plus

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MixoFerm – Catabolite Repression?

Jones, SW, et al. 2016. Nat Comm 7:12800.

Grew Clostridium ljungdahlii on 12C fructose and 13C syngas mixture. Any 13C-labeled metabolite had to come from syngas utilization.

Syngas is consumed simultaneously with fructose at high incorporation levels – actually preferred

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H2 MixoFerm Plus for CO2 Capture & EtOH

H2 addition to C. autoethanogenum

• Effectively captured all CO2
• Shifted metabolism to

reduced product formation

• Achieved ~65wt% yield of

EtOH on consumed fructose

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Benefits of Mixotrophy

Key ratio to benefits of MixoFerm™ is: NAD(P)H to Acetyl-CoA

NAD(P)H/Acetyl-CoA ratio Increase in yield of MixoFerm™ over standard fermentation Example molecules 51% Acetic acid or Acetone 0.5 35% Isopropanol 1.0 22% Butyric acid 1.5 11% 2,3-Butanediol 2 2% Ethanol or n-Butanol 3 0% Propionic acid

Focus of first demonstration of MixoFerm™

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Acetone Production

Sugar Pyruvate Acetyl-CoA

CO2 + 2[H]

WLP Acetate

PFOR AK & PTA

Acetogen host: C. ljungdahlii Acetolactate Acetoin 2,3-Butanediol

CO2 ALS CO2 ALDC 2[H] SADH/ 2,3BDH

Ethanol

4[H] AAD

No known acetogen naturally produces acetone.

Köpke, M., et. al. 2014. Appl Environ Microbiol 80:3394-3403.

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Acetone Production

Sugar Pyruvate Acetyl-CoA Acetoacetyl-CoA Acetoacetate Acetone

CO2 CO2 + 2[H]

WLP Acetate

PFOR THL CoAT ADC AK & PTA

thlCAC ctfACAC ctfBCAC adcCAC Ω Ppta Acetogen host: C. ljungdahlii

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Acetone Production

Strain #1 largely produced side-products

Sugar Pyruvate Acetyl-CoA Acetoacetyl-CoA Acetoacetate Isopropanol Acetone 3-Hydroxybutyrate

CO2 CO2 + 2[H]

WLP Acetate

2[H] 2[H] PFOR THL CoAT ADC SADH/ 2,3BDH SADH AK & PTA

101wt% yield total Acetogen host: C. ljungdahlii

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Acetone Production

Sugar Pyruvate Acetyl-CoA Acetoacetyl-CoA Acetoacetate Isopropanol Acetone 3-Hydroxybutyrate

CO2 CO2 + 2[H]

WLP Acetate

2[H] 2[H] PFOR THL CoAT ADC SADH/ 2,3BDH SADH AK & PTA

thlCAC ctfACAC ctfBCAC adcCAC Ω Ppta Acetogen host: C. ljungdahlii

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Acetone Production

Strain #2 mostly eliminated side-products

Sugar Pyruvate Acetyl-CoA Acetoacetyl-CoA Acetoacetate Isopropanol Acetone 3-Hydroxybutyrate

CO2 CO2 + 2[H]

WLP Acetate

2[H] 2[H] PFOR THL CoAT ADC SADH/ 2,3BDH SADH AK & PTA

80wt% yield total Acetogen host: C. ljungdahlii

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Acetone Production

Sugar Pyruvate Acetyl-CoA Acetoacetyl-CoA Acetoacetate Isopropanol Acetone 3-Hydroxybutyrate

CO2 CO2 + 2[H]

WLP Acetate

2[H] 2[H] PFOR THL CoAT ADC SADH/ 2,3BDH SADH AK & PTA

thlCKL ctfACAC ctfBCAC adcCAC Ω Ppta Acetogen host: C. ljungdahlii

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Acetone Production

Strain #3 produced acetone >95% of theoretical maximum

Sugar Pyruvate Acetyl-CoA Acetoacetyl-CoA Acetoacetate Isopropanol Acetone 3-Hydroxybutyrate

CO2 CO2 + 2[H]

WLP Acetate

2[H] 2[H] PFOR THL CoAT ADC SADH/ 2,3BDH SADH AK & PTA

55wt% yield total

Jones, SW, et al. 2016. Nat Comm 7:12800.

Acetogen host: C. ljungdahlii

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Batch Fermentation

Productivity too low for commercial production

Strain #3

• C. ljungdahlii ΔSADH (pTCtA-Ckl)

Batch fermentation on fructose

Parameter Batch fermentation Yield (wt%) 43 Productivity (g/L/hr) 0.05 Titer (g/L) 6

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Continuous Fermentation

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Continuous Fermentation

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Continuous Fermentation

1 10 100 2 4 6 8 10 12 14 20 40 60 80 100 120 140

OD(600nm) Concentration (g/L) Continuous Fermentation Time (hrs)

Acetone pathway Acetate OD600

• nsum

0% 5% 10% 15% 20% 25% 30% 35% 40% 45% 50% 1 2 3 4 5 6 20 40 60 80 100 120 140

Yield (g/g)

Volumetric Rate of Fructose Consumption or Metabolite Production (g/L/hr)

Continuous Fermentation Time (hrs)

Fructose Acetone pathway Acetate Acetone pathway yield

Acetone pathway products:

• Acetone – 11 g/L
• IPA – 1 g/L
• 3-HB – 1 g/L

Steady production phase (~80 hrs)

Substrate was Fructose

Jones, SW, et al. 2016. Nat Comm 7:12800.

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Glucose Utilization

Glucose! Fructose! Glucose-6P! Fructose-6P! Fructose-1,6P! Glyceraldehyde-3P! Glycerone-P! Pyruvate! Fructose-1P!

Extracellular! Intracellular!

Fructose-specific PTS!

(CLJU_c20590)!

Glucose-specific PTS Glucose-specific PTS! Glucose-6P isomerase (CLJU_c37130)! 6-Phosphofructokinase (CLJU_c03250)! Fructose-biphosphate aldolase (CLJU_c00660)! 1-Phosphofructokinase (CLJU_c20600)!

Missing a glucose transport system!

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Glucose Utilization

Glucose! Fructose! Glucose-6P! Fructose-6P! Fructose-1,6P! Glyceraldehyde-3P! Glycerone-P! Pyruvate! Fructose-1P!

Extracellular! Intracellular!

Fructose-specific PTS!

(CLJU_c20590)!

Glucose-specific PTS Glucose-specific PTS! Glucose-6P isomerase (CLJU_c37130)! 6-Phosphofructokinase (CLJU_c03250)! Fructose-biphosphate aldolase (CLJU_c00660)! 1-Phosphofructokinase (CLJU_c20600)!

Missing a glucose transport system!

Different transport genes tested:

• PTS gene from C. acetobutylicum

(CA_C0570)

• PTS gene from C. saccharobutylicum

(CLSA_c10070)

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Glucose Utilization

Enabled glucose utilization through engineering and evolution

Plasmid expression enabled glucose utilization, though at a significantly slower rate.

0.0 2.0 4.0 6.0 8.0 10.0 12.0 24 48 72 96 120 144 168 192 216 240 Conc (g/L) Time (hr) Clj (pB6) - Fructose Clj (pB6) - Glucose Clj (pB6-CAC) - Glucose Clj (pB6-CSB) - Glucose

Gene from C. saccharobutylicum was integrated into the genome and the strain was adaptively evolved in a chemostat

0.0 2.0 4.0 6.0 8.0 10.0 24 48 72 96 120 144 168 Conc (g/L) Time (hr) Clj WT - Fructose Clj WT - Glucose CljG - Gluocse

WT fructose consumption rate: 0.074 g/L/hr CljG glucose consumption rate: 0.056 g/L/hr

CljG now consumes glucose at a rate 75% of fructose rate.

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Other Substrate Utilization

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Various Gas Substrate Utilization

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Summary

• Clostridial mixotrophy works – evolution did us a favor
• Robust opportunity to couple known gas production technology from fossil

sources with known fermentation technology

• New industries are motivated and will serve an important, strategic role in

development, scale-up and commercial deployment CHALLENGES!

• Gas delivery in MixoFerm Plus – plug and play with Lanzatech

experience?

• Optimal gas composition based on economics and preference of
• rganisms
• Capital efficient solutions for renewably source H2 or syngas mixtures
• Improved –omics and organism engineering tools

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Acknowledgements

Entire WDL team

University of Delaware collaborators:

• Ellinor Carlson
• Alan Fast
• E. Terry Papoutsakis
• Jennifer Au
• Maciek Antoniewicz

Air Liquide collaborators:

• Ilana Aldor
• M Sundar