non-photosynthetic biological co2 fixation
TRANSCRIPT
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?