A thermophilic ionic liquid-tolerant cellulase cocktail for the production of cellulosic biofuels

Joshua I Park; Eric J Steen; Helcio Burd; Sophia S Evans; Alyssa M Redding-Johnson; Tanveer Batth; Peter I Benke; Patrik D'haeseleer; Ning Sun; Kenneth L Sale; Jay D Keasling; Taek Soon Lee; Christopher J Petzold; Aindrila Mukhopadhyay; Steven W Singer; Blake A Simmons; John M Gladden

doi:10.1371/journal.pone.0037010

A thermophilic ionic liquid-tolerant cellulase cocktail for the production of cellulosic biofuels

Joshua I Park, Eric J Steen, Helcio Burd, Sophia S Evans, Alyssa M Redding-Johnson, Tanveer Batth, Peter I Benke, Patrik D'haeseleer, Ning Sun, Kenneth L Sale, Jay D Keasling, Taek Soon Lee, Christopher J Petzold, Aindrila Mukhopadhyay, Steven W Singer, Blake A Simmons, John M Gladden

93 Citations (Scopus)

Abstract

Generation of biofuels from sugars in lignocellulosic biomass is a promising alternative to liquid fossil fuels, but efficient and inexpensive bioprocessing configurations must be developed to make this technology commercially viable. One of the major barriers to commercialization is the recalcitrance of plant cell wall polysaccharides to enzymatic hydrolysis. Biomass pretreatment with ionic liquids (ILs) enables efficient saccharification of biomass, but residual ILs inhibit both saccharification and microbial fuel production, requiring extensive washing after IL pretreatment. Pretreatment itself can also produce biomass-derived inhibitory compounds that reduce microbial fuel production. Therefore, there are multiple points in the process from biomass to biofuel production that must be interrogated and optimized to maximize fuel production. Here, we report the development of an IL-tolerant cellulase cocktail by combining thermophilic bacterial glycoside hydrolases produced by a mixed consortia with recombinant glycoside hydrolases. This enzymatic cocktail saccharifies IL-pretreated biomass at higher temperatures and in the presence of much higher IL concentrations than commercial fungal cocktails. Sugars obtained from saccharification of IL-pretreated switchgrass using this cocktail can be converted into biodiesel (fatty acid ethyl-esters or FAEEs) by a metabolically engineered strain of E. coli. During these studies, we found that this biodiesel-producing E. coli strain was sensitive to ILs and inhibitors released by saccharification. This cocktail will enable the development of novel biomass to biofuel bioprocessing configurations that may overcome some of the barriers to production of inexpensive cellulosic biofuels.

Original language	English
Journal	PLoS ONE
Volume	7
Issue number	5
Pages (from-to)	e37010
ISSN	1932-6203
DOIs	https://doi.org/10.1371/journal.pone.0037010
Publication status	Published - 23 May 2012

Keywords

Biofuels
Biotechnology/methods
Cellulases/metabolism
Escherichia coli/metabolism
Glycoside Hydrolases
Ionic Liquids/metabolism
Lignin/metabolism
Paenibacillus/genetics
Panicum/chemistry
Proteomics
Rhodothermus/genetics
Sequence Analysis, DNA
Temperature
Thermus thermophilus/genetics

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Park, J. I., Steen, E. J., Burd, H., Evans, S. S., Redding-Johnson, A. M., Batth, T., Benke, P. I., D'haeseleer, P., Sun, N., Sale, K. L., Keasling, J. D., Lee, T. S., Petzold, C. J., Mukhopadhyay, A., Singer, S. W., Simmons, B. A., & Gladden, J. M. (2012). A thermophilic ionic liquid-tolerant cellulase cocktail for the production of cellulosic biofuels. PLoS ONE, 7(5), e37010. https://doi.org/10.1371/journal.pone.0037010

Park, JI, Steen, EJ, Burd, H, Evans, SS, Redding-Johnson, AM, Batth, T, Benke, PI, D'haeseleer, P, Sun, N, Sale, KL, Keasling, JD, Lee, TS, Petzold, CJ, Mukhopadhyay, A, Singer, SW, Simmons, BA & Gladden, JM 2012, 'A thermophilic ionic liquid-tolerant cellulase cocktail for the production of cellulosic biofuels', PLoS ONE, vol. 7, no. 5, pp. e37010. https://doi.org/10.1371/journal.pone.0037010

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title = "A thermophilic ionic liquid-tolerant cellulase cocktail for the production of cellulosic biofuels",

abstract = "Generation of biofuels from sugars in lignocellulosic biomass is a promising alternative to liquid fossil fuels, but efficient and inexpensive bioprocessing configurations must be developed to make this technology commercially viable. One of the major barriers to commercialization is the recalcitrance of plant cell wall polysaccharides to enzymatic hydrolysis. Biomass pretreatment with ionic liquids (ILs) enables efficient saccharification of biomass, but residual ILs inhibit both saccharification and microbial fuel production, requiring extensive washing after IL pretreatment. Pretreatment itself can also produce biomass-derived inhibitory compounds that reduce microbial fuel production. Therefore, there are multiple points in the process from biomass to biofuel production that must be interrogated and optimized to maximize fuel production. Here, we report the development of an IL-tolerant cellulase cocktail by combining thermophilic bacterial glycoside hydrolases produced by a mixed consortia with recombinant glycoside hydrolases. This enzymatic cocktail saccharifies IL-pretreated biomass at higher temperatures and in the presence of much higher IL concentrations than commercial fungal cocktails. Sugars obtained from saccharification of IL-pretreated switchgrass using this cocktail can be converted into biodiesel (fatty acid ethyl-esters or FAEEs) by a metabolically engineered strain of E. coli. During these studies, we found that this biodiesel-producing E. coli strain was sensitive to ILs and inhibitors released by saccharification. This cocktail will enable the development of novel biomass to biofuel bioprocessing configurations that may overcome some of the barriers to production of inexpensive cellulosic biofuels.",

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T1 - A thermophilic ionic liquid-tolerant cellulase cocktail for the production of cellulosic biofuels

AU - Park, Joshua I

AU - Steen, Eric J

AU - Burd, Helcio

AU - Evans, Sophia S

AU - Redding-Johnson, Alyssa M

AU - Batth, Tanveer

AU - Benke, Peter I

AU - D'haeseleer, Patrik

AU - Sun, Ning

AU - Sale, Kenneth L

AU - Keasling, Jay D

AU - Lee, Taek Soon

AU - Petzold, Christopher J

AU - Mukhopadhyay, Aindrila

AU - Singer, Steven W

AU - Simmons, Blake A

AU - Gladden, John M

PY - 2012/5/23

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N2 - Generation of biofuels from sugars in lignocellulosic biomass is a promising alternative to liquid fossil fuels, but efficient and inexpensive bioprocessing configurations must be developed to make this technology commercially viable. One of the major barriers to commercialization is the recalcitrance of plant cell wall polysaccharides to enzymatic hydrolysis. Biomass pretreatment with ionic liquids (ILs) enables efficient saccharification of biomass, but residual ILs inhibit both saccharification and microbial fuel production, requiring extensive washing after IL pretreatment. Pretreatment itself can also produce biomass-derived inhibitory compounds that reduce microbial fuel production. Therefore, there are multiple points in the process from biomass to biofuel production that must be interrogated and optimized to maximize fuel production. Here, we report the development of an IL-tolerant cellulase cocktail by combining thermophilic bacterial glycoside hydrolases produced by a mixed consortia with recombinant glycoside hydrolases. This enzymatic cocktail saccharifies IL-pretreated biomass at higher temperatures and in the presence of much higher IL concentrations than commercial fungal cocktails. Sugars obtained from saccharification of IL-pretreated switchgrass using this cocktail can be converted into biodiesel (fatty acid ethyl-esters or FAEEs) by a metabolically engineered strain of E. coli. During these studies, we found that this biodiesel-producing E. coli strain was sensitive to ILs and inhibitors released by saccharification. This cocktail will enable the development of novel biomass to biofuel bioprocessing configurations that may overcome some of the barriers to production of inexpensive cellulosic biofuels.

AB - Generation of biofuels from sugars in lignocellulosic biomass is a promising alternative to liquid fossil fuels, but efficient and inexpensive bioprocessing configurations must be developed to make this technology commercially viable. One of the major barriers to commercialization is the recalcitrance of plant cell wall polysaccharides to enzymatic hydrolysis. Biomass pretreatment with ionic liquids (ILs) enables efficient saccharification of biomass, but residual ILs inhibit both saccharification and microbial fuel production, requiring extensive washing after IL pretreatment. Pretreatment itself can also produce biomass-derived inhibitory compounds that reduce microbial fuel production. Therefore, there are multiple points in the process from biomass to biofuel production that must be interrogated and optimized to maximize fuel production. Here, we report the development of an IL-tolerant cellulase cocktail by combining thermophilic bacterial glycoside hydrolases produced by a mixed consortia with recombinant glycoside hydrolases. This enzymatic cocktail saccharifies IL-pretreated biomass at higher temperatures and in the presence of much higher IL concentrations than commercial fungal cocktails. Sugars obtained from saccharification of IL-pretreated switchgrass using this cocktail can be converted into biodiesel (fatty acid ethyl-esters or FAEEs) by a metabolically engineered strain of E. coli. During these studies, we found that this biodiesel-producing E. coli strain was sensitive to ILs and inhibitors released by saccharification. This cocktail will enable the development of novel biomass to biofuel bioprocessing configurations that may overcome some of the barriers to production of inexpensive cellulosic biofuels.

KW - Biofuels

KW - Biotechnology/methods

KW - Cellulases/metabolism

KW - Escherichia coli/metabolism

KW - Glycoside Hydrolases

KW - Ionic Liquids/metabolism

KW - Lignin/metabolism

KW - Paenibacillus/genetics

KW - Panicum/chemistry

KW - Proteomics

KW - Rhodothermus/genetics

KW - Sequence Analysis, DNA

KW - Temperature

KW - Thermus thermophilus/genetics

U2 - 10.1371/journal.pone.0037010

DO - 10.1371/journal.pone.0037010

M3 - Journal article

C2 - 22649505

SN - 1932-6203

VL - 7

SP - e37010

JO - PLoS ONE

JF - PLoS ONE

IS - 5

ER -

A thermophilic ionic liquid-tolerant cellulase cocktail for the production of cellulosic biofuels

Abstract

Keywords

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