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Production of an acetone-butanol-ethanol mixture from Clostridium acetobutylicum and its conversion to high-value biofuels

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Abstract

Clostridium acetobutylicum is a bacterial species that ferments sugar to a mixture of organic solvents (acetone, butanol and ethanol). This protocol delineates a methodology to combine solventogenic clostridial fermentation and chemical catalysis via extractive fermentation for the production of biofuel blendstocks. Extractive fermentation of C. acetobutylicum is operated in fed-batch mode with a concentrated feed solution (500 grams per liter glucose and 50 grams per liter yeast extract) for 60 h, producing in excess of 40 g of solvents (acetone, butanol and ethanol) between the completely immiscible extractant and aqueous phases of the bioreactor. After distillation of the extractant phase, the acetone, butanol and ethanol mixture is upgraded to long-chain ketones over a palladium-hydrotalcite (Pd-HT) catalyst. This reaction is generally carried out in batch with a high-pressure Q-tube for 20 h at 250 °C. Following this protocol enables the production of ∼0.5 g of high-value biofuel precursors from a 1.7-g portion of fermentation solvents.

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Figure 1: Process flow diagram for extractive fermentation coupled with heterogeneous catalysis to produce biofuel blendstocks.
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Figure 4: Monitoring fermentation growth and culture stability with absorbance at 600 nm.
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Figure 7: Major metabolite concentrations in both phases of a C. acetobutylicum extractive fermentation.

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References

  1. Durre, P. New insights and novel developments in clostridial acetone/butanol/isopropanol fermentation. Appl. Microbiol. Biotechnol. 49, 639–648 (1998).

    Article  CAS  Google Scholar 

  2. Durre, P. Fermentative butanol production bulk chemical and biofuel. Ann. N Y Acad. Sci. 1125, 353–362 (2008).

    Article  Google Scholar 

  3. Jones, D.T. & Woods, D.R. Acetone-butanol fermentation revisited. Microbiol. Rev. 50, 484–524 (1986).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Green, E.M. Fermentative production of butanol-the industrial perspective. Curr. Opin. Biotechnol. 22, 337–343 (2011).

    Article  CAS  Google Scholar 

  5. Huber, G.W., Cortright, R.D. & Dumesic, J.A. Renewable alkanes by aqueous-phase reforming of biomass-derived oxygenates. Angew. Chem. Int. Ed. Engl. 43, 1549–1551 (2004).

    Article  CAS  Google Scholar 

  6. Dellomonaco, C., Clomburg, J.M., Miller, E.N. & Gonzalez, R. Engineered reversal of the β-oxidation cycle for the synthesis of fuels and chemicals. Nature 476, 355–360 (2011).

    Article  CAS  Google Scholar 

  7. Atsumi, S., Hanai, T. & Liao, J.C. Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Nature 451, 86–90 (2008).

    Article  CAS  Google Scholar 

  8. Blanch, H.W. Bioprocessing for biofuels. Curr. Opin. Biotechnol. 23, 390–395 (2012).

    Article  CAS  Google Scholar 

  9. Tracy, B.P., Jones, S.W., Fast, A.G., Indurthi, D.C. & Papoutsakis, E.T. Clostridia: the importance of their exceptional substrate and metabolite diversity for biofuel and biorefinery applications. Curr. Opin. Biotechnol. 23, 1–18 (2011).

    Article  Google Scholar 

  10. Ni, Y. & Sun, Z. Recent progress on industrial fermentative production of acetone–butanol–ethanol by Clostridium acetobutylicum in China. Appl. Microbiol. Biotechnol. 83, 415–423 (2009).

    Article  CAS  Google Scholar 

  11. Jang, Y.-S . et al. Enhanced butanol production obtained by reinforcing the direct. mBio 3, 1–9 (2012).

    Article  Google Scholar 

  12. Harris, L.M., Desai, R.P., Welker, N.E. & Papoutsakis, E.T. Characterization of recombinant strains of the Clostridium acetobutylicum butyrate kinase inactivation mutant: need for new phenomenological models for solventogenesis and butanol inhibition? Biotechnol. Bioeng. 67, 1–11 (2000).

    Article  CAS  Google Scholar 

  13. Bormann, S. et al. Engineering Clostridium acetobutylicum for production of kerosene and diesel blendstock precursors. Metab. Eng. 25, 124–130 (2014).

    Article  CAS  Google Scholar 

  14. Anbarasan, P. et al. Integration of chemical catalysis with extractive fermentation to produce fuels. Nature 491, 235–239 (2012).

    Article  CAS  Google Scholar 

  15. Roffler, S.R., Blanch, H.W. & Wilke, C.R. In situ extractive fermentation of acetone and butanol. Biotechnol. Bioeng. 31, 135–143 (1988).

    Article  CAS  Google Scholar 

  16. Sreekumar, S., Baer, Z.C., Gross, E. & Padmanaban, S. Chemocatalytic upgrading of tailored fermentation products toward biodiesel. ChemSusChem 7, 2445–2448 (2014).

    Article  CAS  Google Scholar 

  17. Schwartz, T.J., Neill, B.J.O., Shanks, B.H. & Dumesic, J.A. Bridging the chemical and biological catalysis gap: challenges and outlooks for producing sustainable chemicals. ACS Catal. 4, 2060–2069 (2014).

    Article  CAS  Google Scholar 

  18. Fisher, K. Neues Verfahren zur maßanalytischen Bestimmung des Wassergehaltes von Flüssigkeiten und festen Körpern. Angew. Chem. Int. Ed. Engl. 48, 394–396 (1935).

    Article  Google Scholar 

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Acknowledgements

This work was funded by the Energy Biosciences Institute.

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Authors and Affiliations

  1. Department of Chemistry, University of California, Berkeley, California, USA

    Sanil Sreekumar, Anbarasan Pazhamalai, Gorkem Gunbas, Adam Grippo & F Dean Toste

  2. Energy Biosciences Institute, University of California, Berkeley, California, USA

    Sanil Sreekumar, Zachary C Baer, Anbarasan Pazhamalai, Gorkem Gunbas, Adam Grippo, Harvey W Blanch, Douglas S Clark & F Dean Toste

  3. Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California, USA

    Zachary C Baer, Harvey W Blanch & Douglas S Clark

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  1. Sanil Sreekumar
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  2. Zachary C Baer
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  3. Anbarasan Pazhamalai
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  4. Gorkem Gunbas
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  5. Adam Grippo
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  6. Harvey W Blanch
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  7. Douglas S Clark
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  8. F Dean Toste
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Contributions

S.S. and Z.C.B. contributed equally to this work. S.S., P.A., G.G. and A.G. performed experiments on the chemical catalysts, Z.C.B. optimized the fermentation and extractive processes. All authors contributed to the conception of the experiments, discussion of the results and preparation of the manuscript.

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Correspondence to Harvey W Blanch, Douglas S Clark or F Dean Toste.

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The authors declare no competing financial interests.

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Sreekumar, S., Baer, Z., Pazhamalai, A. et al. Production of an acetone-butanol-ethanol mixture from Clostridium acetobutylicum and its conversion to high-value biofuels. Nat Protoc 10, 528–537 (2015). https://doi.org/10.1038/nprot.2015.029

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