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Title: Lignin Exhibits Recalcitrance-Associated Features Following the Consolidated Bioprocessing of Populus trichocarpa Natural Variants

Abstract

Because cellulosic ethanol production remains cost-prohibitive„ advances in consolidated bioprocessing (CBP) have been directed towards lifting this restriction. CBP reduces the need for added enzymes and can potentially slash ethanol production costs through process integration. Clostridium thermocellum, a CBP microorganism, organizes its enzymes in a multi-enzyme complex - a stark contrast to fungal enzymes. Nonetheless, recalcitrance may limit the extent of biomass deconstruction. Here in this study, six Populus were treated with C. thermocellum (ATCC 27405) and characterized to determine structural changes that resulted from CBP. The 2D HSQC NMR spectra of lignin-enriched residues revealed that higher S/G ratio (2.6) and fewer carbon-carbon interunit linkages (generally 2–5%) were present in the top performing poplar. Furthermore, cellulose degree of polymerization data suggests that C. thermocellum likely circumvents long chain cellulose, while cellulose crystallinity and hemicellulose molecular weight data do not provide a direct indication of features connected to recalcitrance. Hence, C. thermocellum is similarly impacted by the proposed lignin properties that negatively impact biomass deconstruction using fungal enzymes.

Authors:
 [1];  [2]; ORCiD logo [2]; ORCiD logo [2]; ORCiD logo [3]; ORCiD logo [3];  [2]; ORCiD logo [2]; ORCiD logo [2];  [2]; ORCiD logo [4]
  1. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). BioEnergy Science Center; Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division; Georgia Inst. of Technology, Atlanta, GA (United States). Dept. of Chemistry and Biochemistry
  2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). BioEnergy Science Center; Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division
  3. Univ. of Tennessee, Knoxville, TN (United States). Center for Renewable Carbon at Wildlife, and Fisheries, Dept. of Chemical and Biomolecular Engineering & Department of Forestry; Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). BioEnergy Science Center; Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division
  4. Univ. of Tennessee, Knoxville, TN (United States). Center for Renewable Carbon at Wildlife, and Fisheries, Dept. of Chemical and Biomolecular Engineering & Department of Forestry; Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division
Publication Date:
Research Org.:
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:
1410909
Grant/Contract Number:
AC05-00OR22725
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Chemistry Select
Additional Journal Information:
Journal Volume: 2; Journal Issue: 33; Journal ID: ISSN 2365-6549
Publisher:
Wiley
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; cellulosic ethanol; Clostridium thermocellum; lignin; Populus trichocarpa; recalcitrance

Citation Formats

Akinosho, Hannah, Yee, Kelsey, Rodriguez, Miguel, Muchero, Wellington, Yoo, Chang Geun, Li, Mi, Thompson, Olivia, Pu, Yunqiao Joseph, Brown, Steven D., Mielenz, Johnathan R., and Ragauskas, Arthur J. Lignin Exhibits Recalcitrance-Associated Features Following the Consolidated Bioprocessing of Populus trichocarpa Natural Variants. United States: N. p., 2017. Web. doi:10.1002/slct.201701572.
Akinosho, Hannah, Yee, Kelsey, Rodriguez, Miguel, Muchero, Wellington, Yoo, Chang Geun, Li, Mi, Thompson, Olivia, Pu, Yunqiao Joseph, Brown, Steven D., Mielenz, Johnathan R., & Ragauskas, Arthur J. Lignin Exhibits Recalcitrance-Associated Features Following the Consolidated Bioprocessing of Populus trichocarpa Natural Variants. United States. doi:10.1002/slct.201701572.
Akinosho, Hannah, Yee, Kelsey, Rodriguez, Miguel, Muchero, Wellington, Yoo, Chang Geun, Li, Mi, Thompson, Olivia, Pu, Yunqiao Joseph, Brown, Steven D., Mielenz, Johnathan R., and Ragauskas, Arthur J. 2017. "Lignin Exhibits Recalcitrance-Associated Features Following the Consolidated Bioprocessing of Populus trichocarpa Natural Variants". United States. doi:10.1002/slct.201701572.
@article{osti_1410909,
title = {Lignin Exhibits Recalcitrance-Associated Features Following the Consolidated Bioprocessing of Populus trichocarpa Natural Variants},
author = {Akinosho, Hannah and Yee, Kelsey and Rodriguez, Miguel and Muchero, Wellington and Yoo, Chang Geun and Li, Mi and Thompson, Olivia and Pu, Yunqiao Joseph and Brown, Steven D. and Mielenz, Johnathan R. and Ragauskas, Arthur J.},
abstractNote = {Because cellulosic ethanol production remains cost-prohibitive„ advances in consolidated bioprocessing (CBP) have been directed towards lifting this restriction. CBP reduces the need for added enzymes and can potentially slash ethanol production costs through process integration. Clostridium thermocellum, a CBP microorganism, organizes its enzymes in a multi-enzyme complex - a stark contrast to fungal enzymes. Nonetheless, recalcitrance may limit the extent of biomass deconstruction. Here in this study, six Populus were treated with C. thermocellum (ATCC 27405) and characterized to determine structural changes that resulted from CBP. The 2D HSQC NMR spectra of lignin-enriched residues revealed that higher S/G ratio (2.6) and fewer carbon-carbon interunit linkages (generally 2–5%) were present in the top performing poplar. Furthermore, cellulose degree of polymerization data suggests that C. thermocellum likely circumvents long chain cellulose, while cellulose crystallinity and hemicellulose molecular weight data do not provide a direct indication of features connected to recalcitrance. Hence, C. thermocellum is similarly impacted by the proposed lignin properties that negatively impact biomass deconstruction using fungal enzymes.},
doi = {10.1002/slct.201701572},
journal = {Chemistry Select},
number = 33,
volume = 2,
place = {United States},
year = 2017,
month =
}

Journal Article:
Free Publicly Available Full Text
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  • The bacterium Clostridium thermocellum offers a distinct and integrated approach to ethanol production through consolidated bioprocessing (CBP). The Simons’ stain technique, which assays the accessibility of lignocellulosic biomass, has been traditionally applied to fungal cellulase systems; however, its application to CBP has not been fully explored. For this reason, the structural properties of eight Populus trichocarpa with either high or low biomass densities were compared in this paper to determine bioconversion differences during separate hydrolysis and fermentation (SHF) and CBP with C. thermocellum. Simons’ staining generally identifies low density poplar as more accessible than high density poplar. Additionally, low densitymore » P. trichocarpa generally contained less Klason lignin than high density poplar. SHF and CBP treatments consistently identified BESC-7 (high density, low accessibility, low surface roughness) as a low ethanol yielding biomass and GW-9914 (low density, high accessibility, high surface roughness) as a high ethanol yielding biomass. Upon further investigation, BESC-7 also contained a high Klason lignin content (~25%), while GW-9914 had a low lignin content (~20%). Cellulose degree of polymerization (DP) measurements exhibited a weak linear correlation with accessibility (r 2 = 0.17). Finally, therefore, the ethanol yields were correlated with accessibility and lignin content extremes but not cellulose DP.« less
  • Background: Higher ratios of syringyl-to-guaiacyl (S/G) lignin components of Populus were shown to improve sugar release by enzymatic hydrolysis using commercial blends. Cellulolytic microbes are often robust biomass hydrolyzers and may offer cost advantages; however, it is unknown whether their activity can also be significantly influenced by the ratio of different monolignol types in Populus biomass. Hydrolysis and fermentation of autoclaved, but otherwise not pretreated Populus trichocarpa by Clostridium thermocellum ATCC 27405 was compared using feedstocks that had similar carbohydrate and total lignin contents but differed in S/G ratios. Results: Populus with an S/G ratio of 2.1 was converted moremore » rapidly and to a greater extent compared to similar biomass that had a ratio of 1.2. For either microbes or commercial enzymes, an approximate 50% relative difference in total solids solubilization was measured for both biomasses, which suggests that the differences and limitations in the microbial breakdown of lignocellulose may be largely from the enzymatic hydrolytic process. Unexpectedly, the reduction in glucan content per gram solid in the residual microbially processed biomass was similar (17–18%) irrespective of S/G ratio, pointing to a similar mechanism of solubilization that proceeded at different rates. Fermentation metabolome testing did not reveal the release of known biomass-derived alcohol and aldehyde inhibitors that could explain observed differences in microbial hydrolytic activity. Biomass-derived p-hydroxybenzoic acid was up to ninefold higher in low S/G ratio biomass fermentations, but was not found to be inhibitory in subsequent test fermentations. Cellulose crystallinity and degree of polymerization did not vary between Populus lines and had minor changes after fermentation. However, lignin molecular weights and cellulose accessibility determined by Simons’ staining were positively correlated to the S/G content. Conclusions: Higher S/G ratios in Populus biomass lead to longer and more linear lignin chains and greater access to surface cellulosic content by microbe-bound enzymatic complexes. Substrate access limitation is suggested as a primary bottleneck in solubilization of minimally processed Populus, which has important implications for microbial deconstruction of lignocellulose biomass. Our findings will allow others to examine different Populus lines and to test if similar observations are possible for other plant species.« less
  • Background: Higher ratios of syringyl-to-guaiacyl (S/G) lignin components of Populus were shown to improve sugar release by enzymatic hydrolysis using commercial blends. Cellulolytic microbes are often robust biomass hydrolyzers and may offer cost advantages, however, it is unknown whether their activity can also be significantly influenced by the ratio of different monolignol types in Populus biomass. Hydrolysis and fermentation of autoclaved but otherwise not pretreated Populus trichocarpa by Clostridium thermocellum ATCC 27405 was compared using feedstocks that had similar carbohydrate and total lignin contents but differed in S/G ratios. Results: Populus with an S/G ratio of 2.1 was converted moremore » rapidly and to a greater extent compared to similar biomass that had a ratio of 1.2. For either microbes or commercial enzymes, an approximate 50% relative difference in total solids solubilization was measured for both biomasses, which suggests that the differences and limitations in the microbial breakdown of lignocellulose may be largely from the enzymatic hydrolytic process. Unexpectedly, the reduction in glucan content per gram solid in the residual microbially processed biomass was similar (17 18 %) irrespective of S/G ratio, pointing to a similar mechanism of solubilization that proceeded at different rates. Fermentation metabolome testing did not reveal the release of known biomass-derived alcohol and aldehyde inhibitors that could explain observed differences in microbial hydrolytic activity. Biomass-derived p-hydroxybenzoic acid was up to nine-fold higher in low S/G ratio biomass fermentations, but was not found to be inhibitory in subsequent test fermentations. Cellulose crystallinity and degree of polymerization did not vary between Populus lines and had minor changes after fermentation. However, lignin molecular weights and cellulose accessibility determined by Simons staining were positively correlated to the S/G content. Conclusions: Higher S/G ratios in Populus biomass lead to longer and more linear lignin chains and greater access to surface cellulosic content by microbe-bound enzymatic complexes. Substrate access limitation is suggested as a primary bottleneck in solubilization of minimally processed Populus, which has important implications for microbial deconstruction of lignocellulose biomass. Our findings will allow others to examine different Populus lines and to test if similar observations are possible for other plant species.« less
  • During consolidated bioprocessing (CBP), Clostridium thermocellum hydrolyzes several plant cell wall components. Cellulose hydrolysis, specifically, liberates sugars for fermentation, which generates ethanol, acetate, hydrogen, and other products. While several studies indicate that C. thermocellum hydrolyzes carbohydrates in biomass, the structural changes to lignin during CBP remain unclear. In this paper, the whole plant cell walls of untreated and C. thermocellum-treated Populus trichocarpa were characterized using NMR and FTIR. The results suggest that C. thermocellum reduces the β-O-4 linkage content and increases the lignin S/G ratio. Finally, this investigation indicates that C. thermocellum not only modifies lignin in order to accessmore » cellulose but also leaves behind a suitable lignin substrate for value-added applications in the cellulosic ethanol production scheme.« less