E. hydro Express Streamlining Bacterial Production of Hydrogen Gas - - PowerPoint PPT Presentation

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E. hydro Express Streamlining Bacterial Production of Hydrogen Gas - - PowerPoint PPT Presentation

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E. hydro Express Streamlining Bacterial Production of Hydrogen Gas H 2 H 2 H 2 University of California, Merced iGEM 2012 Paul Barghouth 2 , John Flicker 1 , Israel Juarez-Contreras 2 , Marwin Ko 2 , Norman Luong 2 , Rumman Razzak 1 , Sunny Seth

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SLIDE 1

University of California, Merced iGEM 2012

Paul Barghouth2, John Flicker1, Israel Juarez-Contreras2, Marwin Ko2, Norman Luong2, Rumman Razzak1, Sunny Seth1, Michael Urner1,2, Duc Vo1, Catherine Vu1, Yale Yuen2, Nosherwan Zahid1, Marcos E. García-Ojeda1

  • 1. School of Natural Science, University of California, Merced
  • 2. School of Engineering, University of California, Merced
  • E. hydro Express

Streamlining Bacterial Production of Hydrogen Gas

H2

H2

H2
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SLIDE 2

Foundation

2012

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SLIDE 3

Global Need for Energy

  • Fossil fuel resources

– Finite, non-renewable – Harmful carbon byproducts

Other biofuels?

http://www.homepagedaily.com/Pages/article10410-dwindling-fossil-fuels-and-our-food-system.aspx

http://youevolving.wordpress.com/tag/fossil-fuels/

Background

flickr user aero nerd. http://www.motherjones.com/blue-marble/2009/06/climate-bill-biofuel-boondoggle

Veziroglu TN and Sahin S. (2008) 21st Century’s energy: Hydrogen energy system. Energy Conversion and Management 49 : 1820–1831.

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SLIDE 4

Hydrogen as Renewable Energy Source

Benefits

– Efficient: fuel + energy carrier – Reduces dependency on petroleum – Produced domestically – Clean energy

Challenges

  • High fuel cost, low availability
  • Most hydrogen gas produced by thermochemical

reformation of fossil fuels  still emit greenhouse gases

http://www.hydrogen.co.uk/

Background

Veziroglu TN and Sahin S. (2008) 21st Century’s energy: Hydrogen energy system. Energy Conversion and Management 49 : 1820–1831. Jensen J et al. (2011) Hydrogen Implementing Agreement: Hydrogen. International Energy Agency, IEA CERT Workshop: 1-23. Spormann AM et al. (2005) Metabolic Engineering of Hydrogen Production in Cyanobacterial Heterocysts. Stanford: GCEP Technical Report: 1-2.
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SLIDE 5
  • Goal:

Exploit fermentative capabilities in E. coli to produce H2

  • Strategy:

Knockouts & Insertions New Metabolic Pathway

H2 Project Overview

Biohydrogen Gas Production via Fermentation

H2

H2

H 2 Lee D et al. (2011) Dark fermentation on hydrogen production: pure culture. Bioresource Technology 102: 8393-8402. Toshinari M et al. (2008) Metabolic engineering to enhance bacterial hydrogen production. Microbial Biotechnology 1(1): 30-39. Hallenbeck PC and Benemann JR. (2002) Biological hydrogen production; fundamentals and limiting processes. International Journal of Hydrogen Energy 27: 1185-1193.
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SLIDE 6

2 NAD+, 2ADP, 2Pi

2 NADH, 2 ATP, 2 H+

Design & Strategy

Glycolysis Produces H+ and NADH

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SLIDE 7

2 Pyruvate H+

2 CO2 + Pyruvate decarboxylase 2 NAD+

2 NADH + 2 H+

2 Acetyl-CoA Acetaldehyde dehydrogenase

Design & Strategy

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SLIDE 8

FO Replenishes NAD+ and Contributes to H2 Production

NADH NAD+ Ferredoxinox Ferredoxinred H2 H+

Hydrogenase Ferredoxin

  • xidoreductase

Design & Strategy

FO gene missing in

  • E. coli
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SLIDE 9

Overview of Fermentation in E. coli

Project Overview

Mixed Acid Fermentation

Source: Cortassa S et al. (2002) An Introduction to Metabolic and Cellular Engineering. Singapore: World Scientific: 1-34.

Design & Strategy

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SLIDE 10

Design & Strategy Targeted Pathway for Modification

Source: Cortassa S et al. (2002) An Introduction to Metabolic and Cellular Engineering. Singapore: World Scientific: 1-34.

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SLIDE 11

Modifications to E. Coli Fermentation Pathway

Design & Strategy

Mixed Acid Fermentation

+ Acetaldehyde dehydrogenase + Pyruvate Decarboxylase

Source: Cortassa S et al. (2002) An Introduction to Metabolic and Cellular Engineering. Singapore: World Scientific: 1-34.

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SLIDE 12

Pyruvate Acetyl-CoA Acetaldehyde Ethanol Formate Lactate 1 2

H+ 2 NAD+ 2 NADH + 2 H+

3

H2

4

Design & Strategy

Overview of Engineered H2 Production Pathway

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SLIDE 13
  • E. coli W

Methylophilales bacterium Acetaldehyde dehydrogenase (AD) Ferredoxin Oxidoreductase (FO)

Gibson Assembly

Insert Design

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SLIDE 14
  • E. coli FMJ39

ldhA pflB adhE Insert Knockout PD ldhA FO pflB AD (mhpF) H2 Plasmid Insertion H2 H2 H2 H2

Plasmid Insertion

PD

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SLIDE 15

PCR Cloning of Parts

Results

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SLIDE 16

Successful Transformation

  • f FMJ39

Results

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Next Steps

  • Transduction to knock out last gene (adhE)
  • Ensure correct inserted sequences
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SLIDE 18

Testing for Hydrogen Gas

Setup to measure the hydrogen gas

Next Steps

Source: Toshinari M et al. (2008) Metabolic engineering to enhance bacterial hydrogen production. Microbial Biotechnology 1(1): 30-39.

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SLIDE 19

Future Projects & Applications

Cellulose Glucose

Future Directions

http://commons.wikimedia.org/wiki/File:Sunlight_Through_Leaves.jpg

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SLIDE 20

Acknowledgements

  • School of Natural Sciences &

School of Engineering, University of California, Merced

  • ASUCM
  • Dr. Giovannoni,

Oregon State University

  • University of California, Davis
  • Yale University
  • Advisors:

– Dr. Marcos E. Garcia-Ojeda – Dr. Wei-Chun Chin

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SLIDE 21
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SLIDE 22

Thank You!