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Joshua Demeo , Yin Wong, Cuiwen He, and Yang Liu May 31, 2012. Background . Biofuels are currently produced from carbohydrates and lipids in feedstock Problems: The algae based schemes have limited efficiency due to feedstock cultures having to be starved

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Joshua demeo yin wong cuiwen he and yang liu may 31 2012

Joshua Demeo, Yin Wong, Cuiwen He, and Yang Liu

May 31, 2012


Background
Background

  • Biofuels are currently produced from carbohydrates and lipids in feedstock

    • Problems:

      • The algae based schemes have limited efficiency due to feedstock cultures having to be starved

        • Results in lipid feedstocks with less cell growth and less total CO2 fixation

      • All current schemes result in the accumulation of protein by-products

        • There are currently no ways in which to convert proteins into liquid fuels

        • Normally used as animal feed

          • Feed markets lack the infrastructure to absorb the increasing number of protein-by-products

      • Reduced nitrogen (ammonia) is not recycled

        • Increase in the amount of nitrous oxide produced (from bacteria in waste, fertilizer, or cultivating soil)

        • Future crops must be supplemented with nitrogen (ammonia)

          • Requires the energy-intensive and environmentally unfriendly Haber-Bosch process


Ethanol fermentation corn
Ethanol Fermentation (Corn)

  • Corn is the main feedstock for producing ethanol in the United States

    • Issues related to ethanol production:

      • High area of land usage and land nutrient depletion

      • Requires vehicles that use fossil fuels to harvest the crops

      • Large excess of biomass that is unusable (insoluble carbohydrates, proteins, etc.)


Haber bosch process
Haber-Bosch Process

  • Nitrogen fixation reaction of nitrogen gas and hydrogen gas to produce ammonia

    • Catalyzed by enriched iron or ruthenium

    • Performed under 150-250 bar and temperatures between 300 and 550 oC

    • Forms CO2 during conversion of CH4 to H2

    • Removes nitrogen from the environment

  • Sustains one-third of the earth’s population from the fertilizer created


Protein as a feedstock to make biofuels
Protein as a feedstock to make biofuels?

  • Importance:

    • Deamination of amino acids would complete the nitrogen loop

      • Problem: limited by thermodynamic reversibility and biological regulation that favors anabolism

    • Amino acids could act as a carbon source to create biofuels in the form of alcohols

      • Problem: biological regulation and competing metabolic pathways

    • Proteins are the dominate fraction in industrial fermentation residues and fast-growing photosynthetic microorganisms

      • Maximize growth and CO2 fixation in feedstocksvs lipid and carbohydrate production


Summary
Summary

  • Applied metabolic engineering to generate E. coli that can deaminate protein hyrdrolysates

    • Completed the nitrogen loop

      • Created an irreversible metabolic force to drive deamination reactions to completion

        • Ammonia can be harvested/recycled

    • Created biofuels from amino acids

      • Createdthree exogenous transamination and deamination cycles

        • Enabled conversion of proteins to C4 and C5alcohols at 56% of theoretical yield

          • Able to produce as high as 4,035 mg/l of alcohols from biomass containing ~22g/l of amino acids


Wild-type E. coli w/

Isobutanol synthesis pathway

Yield: 2.3% of theoretical yield

E. Coli can grow well in yeast extract or mixtures of 20 amino acids, but the utilization of amino acids is incomplete.


Improvement on amino acid utilization
Improvement on amino acid utilization

NTG

NTG

NTG

Wild Type

(JCL16)

Mutant

(YH19)


Inhibition of ai 2 re uptake
Inhibition of AI-2 re-uptake

luxS

AI-2

Met

lsrABCD





Biofuel production by yh83
Biofuel production by YH83

Identified by GC-MS and quantified by GC-FID

C2: Ethanol (3%)

C4: Isobutanol (50%)

C5: C5 alcohols (47%)


What happen to the rest of the amino acids
What happen to the rest of the amino acids?

Pseudomonas can grow on the six left over AA: Lys, Tyr, Phe, Trp, Met and His, and convert them to 20 amino acids.


Proposed protein based biorefinery scheme
Proposed protein-based biorefinery scheme


Using algal and bacterial proteins as a feedstock
Using algal and bacterial proteins as a feedstock

Algal biomass mixture includes C. vulgaris, P. purpureum, S.platensis and S.elongatus.

C. vulgaris- green algae

P. purpureum-red algae

S. platensis-green blue algae

S.elongatus-cyanobacterium

Algae and bacteria were grew and collected, digested with protease and used as feedstock for biofuel production with YH83.


Significance
Significance

  • Engineering strategies focus  carbon flux (previous)

Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels

ShotaAtsumi, TaizoHanai & James C. Liao

Nature 451, 86-89(3 January 2008)


Significance1
Significance

  • Engineering strategies focus  carbon flux (previous)

Metabolic engineering of Saccharomycescerevisiaefor the production of n-butanol

Steen et al.Microbial Cell Factories 2008 7:36


Significance2
Significance

  • Engineering strategies focus  carbon flux (previous)

Microbial production of fatty-acid-derived fuels and chemicals from plant biomass

Eric J. Steen, Yisheng Kang, Gregory Bokinsky, ZhihaoHu, Andreas Schirmer, Amy McClure, Stephen B. del Cardayre & Jay D. Keasling

Nature 463, 559-562(28 January 2010)


Significance3
Significance

  • Engineering strategies focus  carbon flux (previous)

Synthesis of Transportation Fuels from Biomass:  Chemistry, Catalysts, and Engineering

George W. Huber, Sara Iborra, and AvelinoCorma

Chem. Rev., 2006, 106 (9), pp 4044–4098


Significance4
Significance

  • Engineering strategies focus  nitrogen flux (this paper)

This paper:

Conversion of proteins into biofuels by engineering nitrogen flux

Yi-XinHuo, KwangMyung Cho, Jimmy G Lafontaine Rivera, Emma Monte, Claire R Shen, Yajun Yan & James C Liao

Nature Biotechnology 29, 346–351 (2011)


Energy-intensive Haber-Bosch process limits biofuel efficiency

Removal of dependence

Biofuels from protein

Jonathan R Mielenz

Nature Biotechnology 29, 327–328 (2011)


Significance5
Significance

  • More advantages of this approach:

    • Higher theoretical yield (long chain alcohol production)

    • Protein more readily hydrolyzes

    • Oligopeptides but not oligosaccharides can be utilized

    • No microbial growth impeding by-products generated

    • Can produce a liquid fuel/bulk chemicals/pharmaceutical intermediates

    • Using fast growing microbes with high protein content

      X photobioreactors

    • Nitrogen can be recycled  for other usage


Challenges
Challenges

  • Large scale algal production and harvesting

  • Product purification

  • Nitrogen recycling


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