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Fermentative production of butanol – challenges and solutions

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  • Fermentative production of butanol challenges and solutions

    Peter Drre Sao Paulo, July 25, 2012

  • Industrial use of butanol

    bulk chemical precursor for production of

    - acrylate and methacrylate esters

    - glycol esters

    - butyl acetate

    - butylamines

    - amino resins

    used for production of adhesives/scalants, alkaloids, antibiotics, camphor, deicing fluid,

    dental products, detergents, elastomers, electronics, emulsifiers, eye makeup, fibres

    flocculants, flotation aids (e.g. butyl xanthate), hard-surface cleaners, hormones and

    vitamins, hydraulic and brake fluids, industrial coatings, lipsticks, nail care products,

    odorant standard, paints, paint thinners, perfumes, pesticides, plastics, printing ink,

    resins, safety glass, shaving and personal hygiene products, surface coatings, super

    absorbents, synthetic fruit flavoring, textiles, as mobile phases in paper and thin-layer

    chromatography, as oiladditive, as well as for leather and paperfinishing

  • Advantages of butanol as a biofuel over ethanol

    1. Can be blended in any concentration with gasoline (ethanol only

    up to 85 %). 2. No modification of existing car engines required.

    3. Has lower vapor pressure and is thus safer to handle.

    4. Since it is not hygroscopic, blending is already possible in refinery.

    5. Less corrosive. Complete existing infrastructure (tanks, pipelines,

    pumps, filling stations, etc.) can be used.

    6. Energy content is higher, resulting in a higher mileage/gasoline

    blend ratio.

    7. Dibutyl ether derivative has the potential for a diesel fuel.

  • Clostridium acetobutylicum -

    biotechnological and political impact

    (courtesy by H. Hippe)

    -Isolation by Weizmann (between 1912 and 1914) in an research

    project aiming at producing synthetic rubber from fermentation

    products

    -Large-scale fermentation of acetone for ammunition production

    during World War I

    -Balfour Declaration in 1917

    -New production plants in Canada and the U.S.

    -Stop of production after armistice in November 1918

    -Introduction of prohibition in the U.S. in 1920, as a consequence

    shortage of amyl alcohol, a solvent for laquers

    -Henry Fords assembly line automobile production required large

    amounts of solvents for laquers

    -Reopening of plants for butanol production (app. 2/3 of the world

    market)

    -After 1950, decline of the fermantation due to cheaper crude oil

    prices

    -Closure of last plants in South Africa (1982) and China (2004)

  • NCP, South Africa: 12 fermenters, working volume of 90,000 l each ( Jones, in: Clostridia (Bahl, Drre, eds.))

  • Carbohydrates

    Acid formation

    Solvent formation Toxin synthesis

    Clostridial form

    Forespore

    Spore

    Spore maturation

    Vegetative cells

  • Metabolism of Clostridium acetobutylicum

    Hexose

    Acetyl-CoA

    Acetoacetyl-CoA

    Butyryl-CoA Butyrate

    Acetate Ethanol

    Acetone

    Adc

    Butanol

    AdhE

    CtfA/B

  • Genes encoding solventogenic enzymes in

    C. acetobutylicum

    2000 4000 6000

    orf5solBorfLadhEctfActfBadc

    1000200030004000

    adhE2 adcSadcR

    500 1000150020002500

    bdhB bdhA

    megaplasmid

    chromosome

  • Regulation of solvent formation

    At the level of transcription:

    Induction/repression controlled by known

    transcription factors (Spo0A-P, CcpA, CodY)

    as well as novel ones (AdcR/AdcS),

    Posttranscriptional:

    mRNA processing of the sol operon transcript

    Posttranslational:

    Protein modification of acetoacetate decarboxylase

  • Distribution of regulator binding sites

  • Genes encoding solventogenic enzymes are induced

    by the master regulator Spo0A~ P

    Ravagnani et al., Spo0A directly controls the swithch from acid to solvent production in

    solvent-forming clostridia.

    Mol. Microbiol. 37, 1172-1185, 2000

    Most data stemming from investigation of C. beijerinckii.

    Harris et al., Northern, morphological, and fermentation analysis of spo0A inactivation

    and overexpression in Clostridium acetobutylicum ATCC824.

    J. Bacteriol. 184, 3586-3597, 2002

    Data stemming from investigation of C. acetobutylicum.

    Problem solved, next question! Or?

  • acetone production after growth on MES medium

    0

    5

    10

    15

    20

    25

    30

    0 100 200 300 400

    acet

    on

    e c

    on

    cen

    trat

    ion

    [m

    M]

    time [h]

    WT

    DadcS 1

    Dspo0A

    wt

    adcS73s::intron

    spo0A462s::intron

    0

    10

    20

    30

    40

    50

    60

    70

    80

    90

    0 100 200 300 400

    bu

    tan

    ol c

    on

    cen

    trat

    ion

    [m

    M]

    time [h]

    butanol production after growth on MES medium

    Growth of C. acetobutylicum WT and mutants on glucose

  • 0

    10

    20

    30

    40

    50

    60

    70

    0 100 200 300 400

    bu

    tan

    ol c

    on

    cen

    trat

    ion

    [m

    M]

    time [h]

    0

    0,5

    1

    1,5

    2

    2,5

    3

    3,5

    4

    0 100 200 300 400

    acet

    on

    e c

    on

    cen

    trat

    ion

    [m

    M]

    time [h]

    WT 1

    DadcS 1

    Dspo0A 2

    wt

    adcS73s::intro

    n spo0A462s::intron

    3.5

    2.5

    1.5

    0.5

    Growth of C. acetobutylicum WT and mutants on glucose

    plus glycerol

    acetone production after growth on MES medium butanol production after growth on MES medium

  • Regulator binding sites upstream of the sol promoter

  • Effect of solB overexpression on solventogenesis

  • 200 nt

    Northern blot experiments

    for verifying solB presence under

    physiological conditions

  • Transcriptome of the sol operon region

    solventogenic phase

    acidogenic phase

  • A

    T

    A

    A

    T

    A

    A

    A

    G

    T

    C

    T

    T

    C

    A

    G

    A

    T

    G

    T

    T

    T

    A

    A

    T

    T

    C

    C T A G

    Primer extension experiments

    for identification of solB transcription

    start point

  • 1 TAAGATATAG CTTCTTTTAT GTAGTATTAT TTCAGAAGTC TACAAATTAA GTTTATATTT

    61 AGACCCTGGG GTGTAACTAT AGTATTTAAT ATTGGTACTA TTAATTAGGG TTATATATAC

    121 TAGAACTTAT CATGGTAAAC ATAAATATAA ACTCAATTCT ATTTATGCTC CTATAAAATT

    181 TTATAATATA GGAAAACTGC TAAATGTAAA TTATACGTTT ACATTTAGCA GTTTATTTT

    Length 195 b

    Promoter -35- AAGATA (consensus TTGACA)

    -10- TATTAT (consensus TATAAT)

    Starting point nt 39

    Terminator stem loop structure

    (rho-independent terminator)

    Integration site nt 149

    -35 -10 149 39

    Features of solB

  • Secondary structure of solB transcript

  • Putative mechanism of action of solB transcript

  • Approaches to improve biological butanol

    production

    1. Cloning of genes encoding enzymes required for butanol

    formation into new host (e. g. E. coli)

    This will include all enzymes that convert acetyl-

    CoA into butanol (thiolase, 3-hydroxybutyryl-CoA

    dehydrogenase, crotonase, butyryl-CoA dehydro-

    genase, butyraldehyde dehydrogenase, butanol

    dehydrogenase). Important: ETF proteins!

    Potential problems:

    - expression of enzymes from an anaerobe

    - solvent tolerance

  • 2. Tailor-made strain of Clostridium acetobutylicum,

    producing only butanol, H2, and CO2

    This will be possible by targeted knock-outs of the lactate

    dehydrogenase, 2-acetolactate synthase, acetoacetate

    decarboxylase, phosphotransacetylase, and phosphotrans-

    butyrylase genes, preventing lactate, acetoin, acetone,

    acetate, and butyrate formation.

    In addition, deregulatory mutations are required to prevent

    development of metabolic bottlenecks.

  • Fermentation substrates

    Sugar and starch are excellent substrates for Clostridium

    acetobutylicum.

    Problems:

    1. Limitation of arable land, biofuels from biomass will only

    represent a fraction of the total fuel required.

    However, this will have a substantial effect on greenhouse

    gas emissions.

    2. Ethical problem of competition between nutrition and

    biofuels.

    Possible solutions: Conversion of lignocellulose to sugars or

    transfer of butanol production feature to syngas-using bacteria

  • Commercial Cellulosic Butanol Production

    Cheaper feedstocks & more efficient fermentation technology required to

    improve economics.

    Laihe Rockley Bio-Chemicals resolved to restart operation of their biobutanol plant

    with biomass 150K tonne/year plant in NE China (corn belt)

    Largest biobutanol plant in the world

    Developed hydrolysis technology for generating

    sugars from corn residues

    Transitioning (with help grom GBL) from corn

    starch to corn residues (bagasse, stover, and

    shells).

    Green Biologics Ltd., Abingdon, UK:

    Industrial technology leader in butanol fermentation

  • Characteristics:

    Gram-positive

    obli

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