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Title: Multiple levers for overcoming the recalcitrance of lignocellulosic biomass

Abstract

The recalcitrance of cellulosic biomass is widely recognized as a key barrier to cost-effective biological processing to fuels and chemicals, but the relative impacts of physical, chemical and genetic interventions to improve biomass processing singly and in combination have yet to be evaluated systematically. Solubilization of plant cell walls can be enhanced by non-biological augmentation including physical cotreatment and thermochemical pretreatment, the choice of biocatalyst, the choice of plant feedstock, genetic engineering of plants, and choosing feedstocks that are less recalcitrant natural variants. A two-tiered combinatoric investigation of lignocellulosic biomass deconstruction was undertaken with three biocatalysts (Clostridium thermocellum, Caldicellulosiruptor bescii, Novozymes Cellic(R) Ctec2 and Htec2), three transgenic switchgrass plant lines (COMT, MYB4, GAUT4) and their respective nontransgenic controls, two Populus natural variants, and augmentation of biological attack using either mechanical cotreatment or cosolvent-enhanced lignocellulosic fractionation (CELF) pretreatment. In the absence of augmentation and under the conditions tested, increased total carbohydrate solubilization (TCS) was observed for 8 of the 9 combinations of switchgrass modifications and biocatalysts tested, and statistically significant for five of the combinations. Here, our results indicate that recalcitrance is not a trait determined by the feedstock only, but instead is coequally determined by the choice of biocatalyst. TCSmore » with C. thermocellum was significantly higher than with the other two biocatalysts. Both CELF pretreatment and cotreatment via continuous ball milling enabled TCS in excess of 90%. Based on our results as well as literature studies, it appears that some form of non-biological augmentation will likely be necessary for the foreseeable future to achieve high TCS for most cellulosic feedstocks. However, our results show that this need not necessarily involve thermochemical processing, and need not necessarily occur prior to biological conversion. Under the conditions tested, the relative magnitude of TCS increase was augmentation > biocatalyst choice > plant choice > plant modification > plant natural variants. In the presence of augmentation, plant modification, plant natural variation, and plant choice exhibited a small, statistically non-significant impact on TCS.« less

Authors:
 [1];  [1];  [2];  [3];  [4];  [5];  [5];  [6];  [7];  [7];  [8];  [9];  [9];  [4];  [4];  [2];  [10];  [10];  [10];  [2] more »;  [4];  [1] « less
  1. Dartmouth College, Hanover, NH (United States). Thayer School of Engineering; Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). BioEnergy Science Center (BESC)
  2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). BioEnergy Science Center (BESC). Bourns College of Engineering, Dept. of Chemical and Environmental Engineering and Center for Environmental Research and Technology
  3. Dartmouth College, Hanover, NH (United States). Thayer School of Engineering
  4. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). BioEnergy Science Center (BESC) and Biosciences Division
  5. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). BioEnergy Science Center (BESC); Noble Research Inst. Ardmore, OK (United States). Genomics Division
  6. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). BioEnergy Science Center (BESC); Univ. of North Texas, Denton, TX (United States). Dept. of Biological Sciences
  7. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). BioEnergy Science Center (BESC); Univ. of Georgia, Athens, GA (United States). Complex Carbohydrate Research Center
  8. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). BioEnergy Science Center (BESC) ; Noble Research Inst. Ardmore, OK (United States). Genomics Division
  9. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). BioEnergy Science Center (BESC); Univ. of Tennessee, Knoxville, TN (United States). Dept. of Plant Sciences
  10. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). BioEnergy Science Center (BESC); National Renewable Energy Lab. (NREL), Golden, CO (United States). Bioenergy Science and Technology
Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1493693
Alternate Identifier(s):
OSTI ID: 1501677
Report Number(s):
NREL/JA-2700-73226
Journal ID: ISSN 1754-6834
Grant/Contract Number:  
AC36-08GO28308; AC05-00OR22725
Resource Type:
Accepted Manuscript
Journal Name:
Biotechnology for Biofuels
Additional Journal Information:
Journal Volume: 12; Journal Issue: 1; Journal ID: ISSN 1754-6834
Publisher:
BioMed Central
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; biomass deconstruction; recalcitrance; transgenic switchgrass; Populus natural variants; Clostridium thermocellum; Caldicellulosiruptor bescii; cotreatment; CELF; fungal cellulase

Citation Formats

Holwerda, Evert K., Worthen, Robert S., Kothari, Ninad, Lasky, Ronald C., Davison, Brian H., Fu, Chunxiang, Wang, Zeng-Yu, Dixon, Richard A., Biswal, Ajaya K., Mohnen, Debra, Nelson, Richard S., Baxter, Holly L., Mazarei, Mitra, Muchero, Wellington, Tuskan, Gerald A., Cai, Charles M., Gjersing, Erica E., Davis, Mark F., Himmel, Michael E., Wyman, Charles E., Gilna, Paul, and Lynd, Lee R. Multiple levers for overcoming the recalcitrance of lignocellulosic biomass. United States: N. p., 2019. Web. doi:10.1186/s13068-019-1353-7.
Holwerda, Evert K., Worthen, Robert S., Kothari, Ninad, Lasky, Ronald C., Davison, Brian H., Fu, Chunxiang, Wang, Zeng-Yu, Dixon, Richard A., Biswal, Ajaya K., Mohnen, Debra, Nelson, Richard S., Baxter, Holly L., Mazarei, Mitra, Muchero, Wellington, Tuskan, Gerald A., Cai, Charles M., Gjersing, Erica E., Davis, Mark F., Himmel, Michael E., Wyman, Charles E., Gilna, Paul, & Lynd, Lee R. Multiple levers for overcoming the recalcitrance of lignocellulosic biomass. United States. doi:10.1186/s13068-019-1353-7.
Holwerda, Evert K., Worthen, Robert S., Kothari, Ninad, Lasky, Ronald C., Davison, Brian H., Fu, Chunxiang, Wang, Zeng-Yu, Dixon, Richard A., Biswal, Ajaya K., Mohnen, Debra, Nelson, Richard S., Baxter, Holly L., Mazarei, Mitra, Muchero, Wellington, Tuskan, Gerald A., Cai, Charles M., Gjersing, Erica E., Davis, Mark F., Himmel, Michael E., Wyman, Charles E., Gilna, Paul, and Lynd, Lee R. Thu . "Multiple levers for overcoming the recalcitrance of lignocellulosic biomass". United States. doi:10.1186/s13068-019-1353-7. https://www.osti.gov/servlets/purl/1493693.
@article{osti_1493693,
title = {Multiple levers for overcoming the recalcitrance of lignocellulosic biomass},
author = {Holwerda, Evert K. and Worthen, Robert S. and Kothari, Ninad and Lasky, Ronald C. and Davison, Brian H. and Fu, Chunxiang and Wang, Zeng-Yu and Dixon, Richard A. and Biswal, Ajaya K. and Mohnen, Debra and Nelson, Richard S. and Baxter, Holly L. and Mazarei, Mitra and Muchero, Wellington and Tuskan, Gerald A. and Cai, Charles M. and Gjersing, Erica E. and Davis, Mark F. and Himmel, Michael E. and Wyman, Charles E. and Gilna, Paul and Lynd, Lee R.},
abstractNote = {The recalcitrance of cellulosic biomass is widely recognized as a key barrier to cost-effective biological processing to fuels and chemicals, but the relative impacts of physical, chemical and genetic interventions to improve biomass processing singly and in combination have yet to be evaluated systematically. Solubilization of plant cell walls can be enhanced by non-biological augmentation including physical cotreatment and thermochemical pretreatment, the choice of biocatalyst, the choice of plant feedstock, genetic engineering of plants, and choosing feedstocks that are less recalcitrant natural variants. A two-tiered combinatoric investigation of lignocellulosic biomass deconstruction was undertaken with three biocatalysts (Clostridium thermocellum, Caldicellulosiruptor bescii, Novozymes Cellic(R) Ctec2 and Htec2), three transgenic switchgrass plant lines (COMT, MYB4, GAUT4) and their respective nontransgenic controls, two Populus natural variants, and augmentation of biological attack using either mechanical cotreatment or cosolvent-enhanced lignocellulosic fractionation (CELF) pretreatment. In the absence of augmentation and under the conditions tested, increased total carbohydrate solubilization (TCS) was observed for 8 of the 9 combinations of switchgrass modifications and biocatalysts tested, and statistically significant for five of the combinations. Here, our results indicate that recalcitrance is not a trait determined by the feedstock only, but instead is coequally determined by the choice of biocatalyst. TCS with C. thermocellum was significantly higher than with the other two biocatalysts. Both CELF pretreatment and cotreatment via continuous ball milling enabled TCS in excess of 90%. Based on our results as well as literature studies, it appears that some form of non-biological augmentation will likely be necessary for the foreseeable future to achieve high TCS for most cellulosic feedstocks. However, our results show that this need not necessarily involve thermochemical processing, and need not necessarily occur prior to biological conversion. Under the conditions tested, the relative magnitude of TCS increase was augmentation > biocatalyst choice > plant choice > plant modification > plant natural variants. In the presence of augmentation, plant modification, plant natural variation, and plant choice exhibited a small, statistically non-significant impact on TCS.},
doi = {10.1186/s13068-019-1353-7},
journal = {Biotechnology for Biofuels},
number = 1,
volume = 12,
place = {United States},
year = {2019},
month = {1}
}

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Figures / Tables:

Fig. 1 Fig. 1: Fractional total carbohydrate solubilization for three transgenic switchgrass lines and their controls mediated by three different biocatalysts. Fungal cellulase was loaded at 5 mg/g solids and in a 9:1 ratio for Ctec2 and Htec2. Red bars show solubilization for the control plant lines (−) and blue bars showmore » solubilization for the transgenic switchgrass lines (+). Initial solids concentrations were based on equal glucan loadings, and fermentations were done in duplicate. Solubilization results are after 120 h of incubation. Error bars represent one standard deviation and are based on biological replicates. Both COMT and MYB4 represent modifications in the lignin pathway, and GAUT4 represents modification in the pectin pathway. An asterisk (*) indicates that the difference in solubilization between transgenic and control plant lines was statistically significant at p ≤ 0.05. Details of the statistical analysis are presented in Additional file 2: Table S3« less

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Works referenced in this record:

Fungal cellulases and complexed cellulosomal enzymes exhibit synergistic mechanisms in cellulose deconstruction
journal, January 2013

  • Resch, Michael G.; Donohoe, Bryon S.; Baker, John O.
  • Energy & Environmental Science, Vol. 6, Issue 6
  • DOI: 10.1039/c3ee00019b

Two-year field analysis of reduced recalcitrance transgenic switchgrass
journal, April 2014

  • Baxter, Holly L.; Mazarei, Mitra; Labbe, Nicole
  • Plant Biotechnology Journal, Vol. 12, Issue 7
  • DOI: 10.1111/pbi.12195

Fungal Cellulases
journal, January 2015

  • Payne, Christina M.; Knott, Brandon C.; Mayes, Heather B.
  • Chemical Reviews, Vol. 115, Issue 3
  • DOI: 10.1021/cr500351c

Natural genetic variability reduces recalcitrance in poplar
journal, May 2016

  • Bhagia, Samarthya; Muchero, Wellington; Kumar, Rajeev
  • Biotechnology for Biofuels, Vol. 9, Issue 1
  • DOI: 10.1186/s13068-016-0521-2

Functional characterization of the switchgrass (Panicum virgatum) R2R3-MYB transcription factor PvMYB4 for improvement of lignocellulosic feedstocks
journal, October 2011


Microbial Cellulose Utilization: Fundamentals and Biotechnology
journal, September 2002

  • Lynd, L. R.; Weimer, P. J.; van Zyl, W. H.
  • Microbiology and Molecular Biology Reviews, Vol. 66, Issue 3, p. 506-577
  • DOI: 10.1128/MMBR.66.3.506-577.2002

Genetic manipulation of lignin reduces recalcitrance and improves ethanol production from switchgrass
journal, February 2011

  • Fu, Chunxiang; Mielenz, Jonathan R.; Xiao, Xirong
  • Proceedings of the National Academy of Sciences, Vol. 108, Issue 9, p. 3803-3808
  • DOI: 10.1073/pnas.1100310108

ENVIRONMENT: Sustainable Development of the Agricultural Bio-Economy
journal, June 2007


Biological lignocellulose solubilization: comparative evaluation of biocatalysts and enhancement via cotreatment
journal, January 2016

  • Paye, Julie M. D.; Guseva, Anna; Hammer, Sarah K.
  • Biotechnology for Biofuels, Vol. 9, Issue 1
  • DOI: 10.1186/s13068-015-0412-y

Co-solvent Pretreatment Reduces Costly Enzyme Requirements for High Sugar and Ethanol Yields from Lignocellulosic Biomass
journal, February 2015


Enzyme-microbe synergy during cellulose hydrolysis by Clostridium thermocellum
journal, October 2006

  • Lu, Y.; Zhang, Y. -H. P.; Lynd, L. R.
  • Proceedings of the National Academy of Sciences, Vol. 103, Issue 44
  • DOI: 10.1073/pnas.0605381103

Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a plant cell wall pectin biosynthetic homogalacturonan:galacturonosyltransferase complex
journal, November 2011

  • Atmodjo, M. A.; Sakuragi, Y.; Zhu, X.
  • Proceedings of the National Academy of Sciences, Vol. 108, Issue 50
  • DOI: 10.1073/pnas.1112816108

Winter rye as a bioenergy feedstock: impact of crop maturity on composition, biological solubilization and potential revenue
journal, January 2015


Designed for deconstruction - poplar trees altered in cell wall lignification improve the efficacy of bioethanol production
journal, January 2012


Development and evaluation of methods to infer biosynthesis and substrate consumption in cultures of cellulolytic microorganisms
journal, April 2013

  • Holwerda, Evert K.; Ellis, Lucas D.; Lynd, Lee R.
  • Biotechnology and Bioengineering, Vol. 110, Issue 9
  • DOI: 10.1002/bit.24915

Optimization of Hydrothermal Pretreatment of Hardwood and Softwood Lignocellulosic Residues for Selective Hemicellulose Recovery and Improved Cellulose Enzymatic Hydrolysis
journal, August 2016

  • Nitsos, Christos K.; Choli-Papadopoulou, Theodora; Matis, Konstantinos A.
  • ACS Sustainable Chemistry & Engineering, Vol. 4, Issue 9
  • DOI: 10.1021/acssuschemeng.6b00535

Bioenergy and African transformation
journal, January 2015


Continually emerging mechanistic complexity of the multi-enzyme cellulosome complex
journal, June 2017

  • Smith, Steven P.; Bayer, Edward A.; Czjzek, Mirjam
  • Current Opinion in Structural Biology, Vol. 44
  • DOI: 10.1016/j.sbi.2017.03.009

Revealing Nature's Cellulase Diversity The Digestion Mechanism of Caldicellulosiruptor bescii CelA
journal, December 2013


Dramatic performance of Clostridium thermocellum explained by its wide range of cellulase modalities
journal, February 2016


ORIGINAL RESEARCH: Lignocellulose recalcitrance screening by integrated high-throughput hydrothermal pretreatment and enzymatic saccharification
journal, April 2010

  • Selig, Michael J.; Tucker, Melvin P.; Sykes, Robert W.
  • Industrial Biotechnology, Vol. 6, Issue 2
  • DOI: 10.1089/ind.2010.0009

Enhanced characteristics of genetically modified switchgrass (Panicum virgatum L.) for high biofuel production
journal, January 2013

  • Shen, Hui; Poovaiah, Charleson R.; Ziebell, Angela
  • Biotechnology for Biofuels, Vol. 6, Issue 1
  • DOI: 10.1186/1754-6834-6-71

Cellulosic ethanol: status and innovation
journal, June 2017


Enhancing digestibility and ethanol yield of Populus wood via expression of an engineered monolignol 4-O-methyltransferase
journal, June 2016

  • Cai, Yuanheng; Zhang, Kewei; Kim, Hoon
  • Nature Communications, Vol. 7, Issue 1
  • DOI: 10.1038/ncomms11989

Biomass Recalcitrance: Engineering Plants and Enzymes for Biofuels Production
journal, February 2007

  • Himmel, M. E.; Ding, S.-Y.; Johnson, D. K.
  • Science, Vol. 315, Issue 5813, p. 804-807
  • DOI: 10.1126/science.1137016

The need for biofuels as part of a low carbon energy future
journal, June 2015

  • Fulton, Lewis M.; Lynd, Lee R.; Körner, Alexander
  • Biofuels, Bioproducts and Biorefining, Vol. 9, Issue 5
  • DOI: 10.1002/bbb.1559

Revisiting cellulase production and redefining current strategies based on major challenges
journal, March 2016

  • Kuhad, Ramesh Chander; Deswal, Deepa; Sharma, Sonia
  • Renewable and Sustainable Energy Reviews, Vol. 55
  • DOI: 10.1016/j.rser.2015.10.132

Population genomics of Populus trichocarpa identifies signatures of selection and adaptive trait associations
journal, August 2014

  • Evans, Luke M.; Slavov, Gancho T.; Rodgers-Melnick, Eli
  • Nature Genetics, Vol. 46, Issue 10
  • DOI: 10.1038/ng.3075

Accelerated growth of the sugarcane, sugar, and ethanol sectors in Brazil (2000–2008): Effects on municipal gross domestic product per capita in the south-central region
journal, August 2016

  • Moraes, Márcia Azanha Ferraz Dias de; Bacchi, Mírian Rumenos Piedade; Caldarelli, Carlos Eduardo
  • Biomass and Bioenergy, Vol. 91
  • DOI: 10.1016/j.biombioe.2016.05.004

A comparative study of the enzymatic hydrolysis of acid-pretreated white pine and mixed hardwood
journal, December 1984

  • Grethlein, H. E.; Allen, D. C.; Converse, A. O.
  • Biotechnology and Bioengineering, Vol. 26, Issue 12
  • DOI: 10.1002/bit.260261215

High-resolution genetic mapping of allelic variants associated with cell wall chemistry in Populus
journal, January 2015


Improved saccharification and ethanol yield from field-grown transgenic poplar deficient in cinnamoyl-CoA reductase
journal, December 2013

  • Van Acker, R.; Leple, J. -C.; Aerts, D.
  • Proceedings of the National Academy of Sciences, Vol. 111, Issue 2
  • DOI: 10.1073/pnas.1321673111

Field Evaluation of Transgenic Switchgrass Plants Overexpressing PvMYB4 for Reduced Biomass Recalcitrance
journal, January 2015

  • Baxter, Holly L.; Poovaiah, Charleson R.; Yee, Kelsey L.
  • BioEnergy Research, Vol. 8, Issue 3
  • DOI: 10.1007/s12155-014-9570-1

CELF pretreatment of corn stover boosts ethanol titers and yields from high solids SSF with low enzyme loadings
journal, January 2016

  • Nguyen, Thanh Yen; Cai, Charles M.; Osman, Omar
  • Green Chemistry, Vol. 18, Issue 6
  • DOI: 10.1039/C5GC01977J

Monolignol Ferulate Transferase Introduces Chemically Labile Linkages into the Lignin Backbone
journal, April 2014


Comparative material balances around pretreatment technologies for the conversion of switchgrass to soluble sugars
journal, December 2011


A defined growth medium with very low background carbon for culturing Clostridium thermocellum
journal, February 2012

  • Holwerda, Evert K.; Hirst, Kyle D.; Lynd, Lee R.
  • Journal of Industrial Microbiology & Biotechnology, Vol. 39, Issue 6
  • DOI: 10.1007/s10295-012-1091-3

The cellulosome and cellulose degradation by anaerobic bacteria
journal, September 2001


Cellulosomes: bacterial nanomachines for dismantling plant polysaccharides
journal, December 2016

  • Artzi, Lior; Bayer, Edward A.; Moraïs, Sarah
  • Nature Reviews Microbiology, Vol. 15, Issue 2
  • DOI: 10.1038/nrmicro.2016.164

A Genomics Approach to Deciphering Lignin Biosynthesis in Switchgrass
journal, November 2013


Perennial grasslands enhance biodiversity and multiple ecosystem services in bioenergy landscapes
journal, January 2014

  • Werling, B. P.; Dickson, T. L.; Isaacs, R.
  • Proceedings of the National Academy of Sciences, Vol. 111, Issue 4
  • DOI: 10.1073/pnas.1309492111

    Figures/Tables have been extracted from DOE-funded journal article accepted manuscripts.