Augusto & Jessica's Research Proposal Information

We want to engineer a microorganism or virus that will improve either:

• sewage water treatment/waste degradation (probably this)
• biofuel synthesis

Biofuel Synthesis

Social Context

• why biofuels are important, etc etc

Processing Background

1. Biomass Handling: break the biomass into smaller, more manageable pieces
2. Biomass Pretreatment: dilute sulfuric acid is used to break hemicellulose into its component sugars

• Pentoses
  • xylose
  • arabinose
• Hexoses
  • mannose
  • galactose
  • glucose

3. Cellulose Hydrolysis: remaining cellulose is hydrolyzed to glucose
4. Glucose Fermentation: C₆H₁₂O₆ + 2P_i + 2ADP → 2CH₃CH₂OH + 2CO₂ + 2 ATP
5. Pentose Fermentation: xylose (most common pentose) is fermented using Zymomonas mobilis or another genetically engineered bacteria
6. EtOH recovery: EtOH is purified and water is removed
7. Lignin utilization: lignin can be used to produce energy for the biofuel production process

Problems

1. Cellulose's crystal structure makes it difficult to hydrolyze into its constituent sugars. More efficient enzymes must be produced to break down cellulose
2. No organism is capable of breaking down all five sugars contained in the biomass. An organism must be designed with this capability in order to increase the efficiency of the fermentation reaction

Design Ideas

• Build on the pre-existing Zymomonas mobilis to include ability to ferment other pentoses/hexoses
  • Add enzymes
  • Add sugar receptors
• Try to increase cellulase efficiency in pre-existing cellulase-producing cells
• Combine cellulase and fermentation capabilities into one cell
• So we could really try to do all of these things in a multi-step process, i.e., first add arabinose transport function, test to ensure it works, then add arabinose fermentation function...

References

US DOE Biomass Program http://www1.eere.energy.gov/biomass/abcs_biofuels.html#prod

Proposal Outline

(Slide 1)

• Overview
  • Background
  • Methods
  • Predicted Outcomes
  • Necessary Resources
  • Societal Impact

• Background
  • Societal Context (Slide 2)
    • Necessity of biofuels as a new energy source
  • Scientific Context (Slide 3)
    • Limitations of natural bacteria in cellulose digestion and fermentation
    • Explanation of modifications to Zymomonas mobilis and short coming of this system

• Statement of Research Purpose and Goals (Slide 3)
  • To engineer a bacteria to efficiently degrade cellulose and ferment cellulose and hemicellulose component sugars

• Methods
  • This will be a multi-stage project
    • Stage 1: improve pentose fermatation (Slide 4)
      • Add arabinose membrane transport proteins
      • Add xylose membrane transport proteins
    • Stage 2: add cellulose digestion capabilities to Zymomonas mobilis (Slide 5)
      • Add cellulase genes
      • Up-regulate cellulase gene (stonger promoter, RBS, etc)
    • Stage 3: Add mannose and galactose fermentation (Slide 6)
      • Add genes necesary for mannose and galactose fermentation
      • Need to add transport proteins?

• Predicted Outcomes (Slide 7)
  • Hopefully, we will produce a bacteria that is able to degrade cellulose and ferment cellulose (and hemicellulose) components
  • Even if not every stage of the project is successful, successful completion of of any stage will result in a more stream-lined and efficient method of producing biofuel from biomass.

• Necessary Resources (Slide 8)
  • $125,000 per person working on this x 2 scientists = $250,000 ???
  • This assumes we already have the lab setup...

• Societal Impact (Slide 9)
  • Improve ability to produce biofuels
    • If they are easier and cheaper to produce, they will be more widely used
  • Could replace fossil fuels
  • Reduce green house gas emissions

Sewage Water Treatment

Scientific Background

• Bacteria are commonly used indicators of the effectiveness of wastewater treatment
  • May not accurately indicate the effectiveness of removing viruses and protozoa
• Bacteriophages have been proposed to be potential indicators of viruses
• sulphite-reducing bacteria have been proposed to be potential indicators of viruses

Social Context

• under-treated sewage is the main source of pathogens in water
• Estimated that 80% of infectious diseases (world-wide) may be water-related
  • Diarrheal diseases linked to water kill 2 million children and cause 900 million episodes of illness each year

Random Thoughts

• If we design a bacteria, perhaps utilize a protein degradation switch that will allow the bacteria to be destroyed at will
• We could try to make a virus that is highly effective in killing bacteria, but also has properties that allow it to easily be removed (i.e. charged coat?)

Project Ideas

• • Parts that could use some BE

1. Secondary treatment: is either fixed film or suspended growth.

Types of systems and procedures used:

• • Activated sludge, we could do some denitrifications
  • Aerated basins
  • Filter beds
  • Membrane biological reactors
  • Rotating contractors

2. Tertiary treatment:

• • Removal of minerals and disinfection
    • Nitrogen removing bacteria
    • Phosphorus removing bacteria
    • Disinfection with: choro, uv, o3. There are problems with all of them... use phage?

3. Sludge treatment and disposal

• • Treatment done by Digestion, composting

Some Ideas:

• • Removing chemicals more effectively
  • Using phage to disinfect
  • Cheap purification?

References

Gaussier H, Yang Q et al. Building a virus from scratch: assembly of an infectious virus using purified components in a rigorously defined biochemical assay system. Journal of Molecular Biology 2004; 357:1154-66

Kim DY and Rhe YH. Biodegradation of microbial and synthetic polyesters by fungi. Applications of Microbiology Biotechnology 61:300-308

Lucena F, Duran AE, et al. Reduction of bacterial indicators and bacteriophages infecting faecal bacteria in primary and secondary wastewater treatments. Journal of Applied Microbiology 2004; 97:1069-1076

Grilly C, Stricker J, Pang WL, Bennett MR, Hasty J. A synthetic gene network for tuning protein degradation in Saccharomyces cerevisiae . Molecular Systems Biology 2007; 3:127