Non-photosynthetic Biological CO2 Fixation

Dr. Benjamin M. Woolston

Postdoctoral Associate in Prof. Greg Stephanopoulos’ Lab

MIT Department of Chemical Engineering

Developing a Research Agenda for Utilization of Gaseous Carbon Waste StreamsNational AcademiesTuesday, March 6th, 2018

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Sources of electrons for biological CO2 fixation

Solar Biomass Sugars ProductsCO2 Hydrolysis Fermentation

E. coliS. cerevisiaePhotosynthesis

<1% efficiency

Solar ElectricityCOHCOOHCH3OH

ProductsCO2

ElectrocatalysisPhotovoltaics~20% efficiency

Fermentation

TechnologicalMaturity

++++

Solar ProductsPhotosynthetic fermentationAlgae, cyanobacteriaCO2

++

Solar Electricity H2 Products

H2O

ElectrolysisGas Fermentation

Photovoltaics~20% efficiency

AcetogensKnallgas Bacteria

CO2

+

+

Photosynthetic CO2 Fixation

Non-photosynthetic CO2 Fixation TechnologicalMaturity

Solar Electricity

CO2

Microbial Electrosynthesis

ProductsAcetogens

+

Waste Industrial Gases CO, CO2, H2Gas Fermentation

Products ++

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Advantages of biological gas fermentation

• Biology provides specificity, avoiding the need to separate and sell a wide range of products

• “Dirty” gas streams poison traditional catalysts, but not syngas-fermenting microbes

• Feedstock flexibility

• Comparatively low CapEx, allows monetization of smaller point sources

Kibby C et. al. 2013. Catal. Today 215:131–141.Perego et.al. Catalysis Today 142.1 (2009): 9-16.

Typical FT Product Distribution

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Major Players in Non-photosynthetic CO2 Fixation

Acetogens Knallgas Bacteria

Anaerobic microbesHigh electron efficiency of CO2 fixation: ~92%

Relatively lower rates: 6.2 g Acetate L-1 hr-1, μ = 0.05 hr-1

Low titer: ~30 g L-1

Aerobic microbesHigh rates: 1.6 g PHB L-1 hr-1, μ = 0.42 hr-1

Lower efficiency of CO2 fixation pathway: ~27%High titer: 62 g L-1 (intracellular)

4H2 + 2CO2 CH3COOH + 2H2O (ΔG0’ = -95 kJ/mol) 2H2 + O2 2H2O (ΔG0’ = -477 kJ/mol)

Kantzow, et al. J. Biotechnol. 212 (2015) 11–18 Ishizaki, et al. Appl. Microbiol. Biotechnol. 57 (2001), 6–12.

Rate & TiterEfficiency

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CO2, H2, CO, CH2O2, CH3OH

Acetate (+ATP)Ethanol

Products

Ac-CoA

• Feedstock flexibility• High electron efficiency• Commercial interest• Drivers: Preliminary genetic tools, native

ethanol production

Acetogenic CO2 fixation with the Wood-Ljungdahl Pathway

Wood-Ljungdahl Pathway

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Technical Challenge: H2/CO Mass Transfer

• Efficient feedstock utilization requires high mass transfer rates to deliver substrate to microbe

• CO and H2 poorly soluble

• Reactor design for high mass transfer rate benefits from well-established literature and industrial practice

Source: LanzaTech Presentation https://www.energy.gov/sites/prod/files/2017/07/f35/BETO_2017WTE-Workshop_SeanSimpson-LanzaTech.pdf

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Technical Challenge: Genetic Tools and Metabolic Understanding

Genetic Tools

• Plasmids and transformation procedures (Kopke, 2010)

• Gene knockouts (Leang, 2013)• CRISPR-cas9 knockouts (Huang,

2016)• Temperature-sensitive replicons

(Molitor, 2016)• CRISPRi (Woolston, 2018)

Metabolic Understanding

• Discovery of Flavin-based electron bifurcation (Thauer, 2008)

• First GSM of acetogen (Nagarajan, 2013)

• Transcriptomics (2013-2016)• Proteomics (Richter, 2016)

Technologies for metabolic engineering in acetogens are new, but developing rapidly

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Technical Challenge: Low ATP Yield Constrains Portfolio of Target Molecules

0%

20%

40%

60%

80%

100%

$0.00

$1.00

$2.00

$3.00

$4.00

$5.00

Eth

anol

3HB

(tes

B)

3HB

(ptb

/buk

)

But

yrat

e

But

anol

But

anol

(AO

R)

2,3,

BD

O

Lact

ate

4HB

1,4

BD

O

Prod

uct S

elec

tivity

Hyd

roge

n C

ost (

$/kg

)

Maximum H2 Price ($/kg) Product Molar Selectivity

ATP Produced ½ 0 1 1 0 1 -1 - ½ 0 0

Acetyl-CoA

CO2 CO2

COCH3-THF

AcetateATP

ptaack

WL~0.5 ATP

Product

4H2 + 2CO2 CH3COOH + 2H2O (ΔG0’ = -95 kJ/mol)

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Overcoming ATP limitation in gas fermentationSeparate metabolic capabilities of multiple strains

Two-stage bioreactor system optimizes division of labor, and intracellular product overcomes challenge with dilute product stream

Hu P, Chakraborty S, Kumar A, Woolston BM, Liu H, Emerson D and Stephanopoulos G. PNAS. 2016 (113) 3773-3778

Bench-Scale Metrics18 g L-1 Lipids (C16-C18)0.19 g L-1 hr-1

Energetic efficiency: 10%

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Overcoming ATP limitation in gas fermentationBolster ATP production through additional supplements

Emerson D, Woolston BM, et al., and Stephanopoulos G. (2018) Submitted

Supplemented with 15 mM nitrate, μ = 0.08 hr-1, Y = 0.48No supplement, μ = 0.04 hr-1, Y = 0.11

Nitrate respiration improves growth and yield

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Overcoming ATP limitation in gas fermentationBolster ATP production through additional supplements

Valgepea, et al. and Marcellin. 2017. Met. Eng 41:202-211

Arginine eliminates autotrophic acetate production in C. autoethanogenum

“Mixotrophy”Co-feeding of fructose enhances CO2 fixation

Jones et al. and Papoutsakis. 2016. Nat Comm. DOI: 10.1038/ncomms12800

Eventual solution will be decided by process economic considerations

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Major Players in Non-photosynthetic CO2 Fixation

Acetogens Knallgas Bacteria

Anaerobic microbesHigh electron efficiency of CO2 fixation: ~92%

Relatively lower rates: 6.2 g Acetate L-1 hr-1, μ = 0.05 hr-1

Low titer: ~30 g L-1

Aerobic microbesHigh rates: 1.6 g PHB L-1 hr-1, μ = 0.42 hr-1

Lower efficiency of CO2 fixation pathway: ~27%High titer: 62 g L-1 (intracellular)

4H2 + 2CO2 CH3COOH + 2H2O (ΔG0’ = -95 kJ/mol) 2H2 + O2 2H2O (ΔG0’ = -477 kJ/mol)

Kantzow, et al. J. Biotechnol. 212 (2015) 11–18 Ishizaki, et al. Appl. Microbiol. Biotechnol. 57 (2001), 6–12.

Rate & TiterEfficiency

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Knallgas Bacteria Technical Challenges

• Explosive Gas Mixtures: Maintaining O2 below explosion limit leads to drop in productivity

• Use Calvin Cycle for CO2 fixation: Lower efficiency

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New pathways for improved CO2 fixation performanceSynthetic biology is allowing us to design new (non-natural) CO2 fixation pathways

Schwander, et al. and Erb. J. Science (2016) 354, 900-904

CETCH

• De Novo designed pathway for CO2 fixation

• in vitro rate similar to those measured in CBB pathway

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Microbial Electrosynthesis for CO2 Fixation

Aryal et. al. Green Chem., 2017, 19, 5748

Anaerobic microbes (acetogens) grown on cathode; accept electrons directly

Bypasses need for “intermediate” electron carrier

Technical Challenges

• Lack of mechanistic understanding of microbe-cathode interface

• Scale-up. Best rates ~0.13 g Acetate L-1 hr-1

Curr. Opinion. in Biotech. 2016 (42) 225-233

Compare to 6.2 g Acetate L-1 hr-1

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Summary

• Non-photosynthetic CO2 fixation using H2, CO is an attractive technology for converting CO2 to products with high specificity, bypassing some of the challenges of photosynthesis

• Major remaining technical challenges are overcoming energy limitations to improve rate and access higher-value chemicals, and dilute product streams

Questions?