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Title: Integrated omics analyses reveal the details of metabolic adaptation of Clostridium thermocellum to lignocellulose-derived growth inhibitors released during the deconstruction of switchgrass

Abstract

Clostridium thermocellum is capable of solubilizing and converting lignocellulosic biomass into ethanol. Though much of the work-to-date has centered on characterizing the organism s metabolism during growth on model cellulosic substrates, such as cellobiose, Avicel, or filter paper, it is vitally important to understand it metabolizes more complex, lignocellulosic substrates to identify relevant industrial bottlenecks that could undermine efficient biofuel production. To this end, we have examined a time course progression of C. thermocellum grown on switchgrass to assess the metabolic and protein changes that occur during the conversion of plant biomass to ethanol. The most striking feature of the metabolome was the observed accumulation of long-chain, branched fatty acids over time, implying an adaptive restructuring of C. thermocellum s cellular membrane as the culture progresses. This is likely a response to the gradual build-up of lignocellulose-derived inhibitory compounds detected as the organism deconstructs the switchgrass to access the embedded cellulose and includes 4-hydroxybenzoic acid, vanillic acid, ferulic acid, p-coumaric acid and vanillin. Corroborating the metabolomics data, proteomic analysis revealed a corresponding time-dependent increase in enzymes involved in the interconversion of branched amino acids valine, leucine and isoleucine to iso- and anteiso-fatty acid precursors. Furthermore, the metabolic accumulation of hemicellulose-derivedmore » sugars and sugar-alcohols concomitant with increased abundance of enzymes involved in C5 sugar metabolism / the pentose phosphate pathway, indicate that C. thermocellum either shifts glycolytic intermediates to alternate pathways to modulate overall carbon flux or is simply a response to C5 sugar metabolite pools that build during lignocellulose deconstruction.« less

Authors:
 [1];  [2];  [2];  [3];  [2];  [2];  [2];  [4];  [1];  [2];  [4];  [1]
  1. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States)
  2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
  3. Dow Chemical Company, Midland, MI (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
  4. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Arkansas, Fayetteville, AR (United States)
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1340445
Grant/Contract Number:
AC05-00OR22725
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Biotechnology for Biofuels
Additional Journal Information:
Journal Volume: 10; Journal Issue: 14; Journal ID: ISSN 1754-6834
Publisher:
BioMed Central
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; Clostridium thermocellum; Switchgrass; Lignocellulosic; Biofuel; Ethanol; Mass spectrometry; Proteomics; Metabolomics; Transcriptomics; Cellulosome

Citation Formats

Poudel, Suresh, Giannone, Richard J., Rodriguez, Jr., Miguel, Raman, Babu, Martin, Madhavi Z., Engle, Nancy L., Mielenz, Jonathan R., Nookaew, Intawat, Brown, Steven D., Tschaplinski, Timothy J., Ussery, David W., and Hettich, Robert L. Integrated omics analyses reveal the details of metabolic adaptation of Clostridium thermocellum to lignocellulose-derived growth inhibitors released during the deconstruction of switchgrass. United States: N. p., 2017. Web. doi:10.1186/s13068-016-0697-5.
Poudel, Suresh, Giannone, Richard J., Rodriguez, Jr., Miguel, Raman, Babu, Martin, Madhavi Z., Engle, Nancy L., Mielenz, Jonathan R., Nookaew, Intawat, Brown, Steven D., Tschaplinski, Timothy J., Ussery, David W., & Hettich, Robert L. Integrated omics analyses reveal the details of metabolic adaptation of Clostridium thermocellum to lignocellulose-derived growth inhibitors released during the deconstruction of switchgrass. United States. doi:10.1186/s13068-016-0697-5.
Poudel, Suresh, Giannone, Richard J., Rodriguez, Jr., Miguel, Raman, Babu, Martin, Madhavi Z., Engle, Nancy L., Mielenz, Jonathan R., Nookaew, Intawat, Brown, Steven D., Tschaplinski, Timothy J., Ussery, David W., and Hettich, Robert L. Tue . "Integrated omics analyses reveal the details of metabolic adaptation of Clostridium thermocellum to lignocellulose-derived growth inhibitors released during the deconstruction of switchgrass". United States. doi:10.1186/s13068-016-0697-5. https://www.osti.gov/servlets/purl/1340445.
@article{osti_1340445,
title = {Integrated omics analyses reveal the details of metabolic adaptation of Clostridium thermocellum to lignocellulose-derived growth inhibitors released during the deconstruction of switchgrass},
author = {Poudel, Suresh and Giannone, Richard J. and Rodriguez, Jr., Miguel and Raman, Babu and Martin, Madhavi Z. and Engle, Nancy L. and Mielenz, Jonathan R. and Nookaew, Intawat and Brown, Steven D. and Tschaplinski, Timothy J. and Ussery, David W. and Hettich, Robert L.},
abstractNote = {Clostridium thermocellum is capable of solubilizing and converting lignocellulosic biomass into ethanol. Though much of the work-to-date has centered on characterizing the organism s metabolism during growth on model cellulosic substrates, such as cellobiose, Avicel, or filter paper, it is vitally important to understand it metabolizes more complex, lignocellulosic substrates to identify relevant industrial bottlenecks that could undermine efficient biofuel production. To this end, we have examined a time course progression of C. thermocellum grown on switchgrass to assess the metabolic and protein changes that occur during the conversion of plant biomass to ethanol. The most striking feature of the metabolome was the observed accumulation of long-chain, branched fatty acids over time, implying an adaptive restructuring of C. thermocellum s cellular membrane as the culture progresses. This is likely a response to the gradual build-up of lignocellulose-derived inhibitory compounds detected as the organism deconstructs the switchgrass to access the embedded cellulose and includes 4-hydroxybenzoic acid, vanillic acid, ferulic acid, p-coumaric acid and vanillin. Corroborating the metabolomics data, proteomic analysis revealed a corresponding time-dependent increase in enzymes involved in the interconversion of branched amino acids valine, leucine and isoleucine to iso- and anteiso-fatty acid precursors. Furthermore, the metabolic accumulation of hemicellulose-derived sugars and sugar-alcohols concomitant with increased abundance of enzymes involved in C5 sugar metabolism / the pentose phosphate pathway, indicate that C. thermocellum either shifts glycolytic intermediates to alternate pathways to modulate overall carbon flux or is simply a response to C5 sugar metabolite pools that build during lignocellulose deconstruction.},
doi = {10.1186/s13068-016-0697-5},
journal = {Biotechnology for Biofuels},
number = 14,
volume = 10,
place = {United States},
year = {Tue Jan 10 00:00:00 EST 2017},
month = {Tue Jan 10 00:00:00 EST 2017}
}

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Cited by: 8works
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  • Background The thermophilic anaerobe Clostridium thermocellum is a candidate consolidated bioprocessing (CBP) biocatalyst for cellulosic ethanol production. The aim of this study was to investigate C. thermocellum genes required to ferment biomass substrates and to conduct a robust comparison of DNA microarray and RNA sequencing (RNA-seq) analytical platforms. Results C. thermocellum ATCC 27405 fermentations were conducted with a 5 g/L solid substrate loading of either pretreated switchgrass or Populus. Quantitative saccharification and inductively coupled plasma emission spectroscopy (ICP-ES) for elemental analysis revealed composition differences between biomass substrates, which may have influenced growth and transcriptomic profiles. High quality RNA was preparedmore » for C. thermocellum grown on solid substrates and transcriptome profiles were obtained for two time points during active growth (12 hours and 37 hours postinoculation). A comparison of two transcriptomic analytical techniques, microarray and RNA-seq, was performed and the data analyzed for statistical significance. Large expression differences for cellulosomal genes were not observed. We updated gene predictions for the strain and a small novel gene, Cthe_3383, with a putative AgrD peptide quorum sensing function was among the most highly expressed genes. RNAseq data also supported different small regulatory RNA predictions over others. The DNA microarray gave a greater number (2,351) of significant genes relative to RNA-seq (280 genes when normalized by the kernel density mean of M component (KDMM) method) in an analysis of variance (ANOVA) testing method with a 5 % false discovery rate (FDR). When a 2-fold difference in expression threshold was applied, 73 genes were significantly differentially expressed in common between the two techniques. Sulfate and phosphate uptake/utilization genes, along with genes for a putative efflux pump system were some of the most differentially regulated transcripts when profiles for C. thermocellum grown on either pretreated switchgrass or Populus were compared. Conclusions Our results suggest that a high degree of agreement in differential gene expression measurements between transcriptomic platforms is possible, but choosing an appropriate normalization regime is essential.« less
  • Milling during lignocellulosic fermentation, henceforth referred to as cotreatment, is investigated as an alternative to thermochemical pretreatment as a means of enhancing biological solubilization of lignocellulose. We investigate the impact of milling on soluble substrate fermentation by Clostridium thermocellum with comparison to yeast, document solubilization for fermentation of senescent switchgrass with and without ball milling, and characterize residual solids. Soluble substrate fermentation by C. thermocellum proceeded readily in the presence of continuous ball milling but was completely arrested for yeast. Total fractional carbohydrate solubilization achieved after fermentation of senescent switchgrass by C. thermocellum for 5 days was 0.45 without cotreatmentmore » or pretreatment, 0.81 with hydrothermal pretreatment (200 degrees C, 15 minutes, severity 4.2), and 0.88 with cotreatment. Acetate and ethanol were the main fermentation products, and were produced at similar ratios with and without cotreatment. Analysis of solid residues was undertaken using molecular beam mass spectrometry (PyMBMS) and solid-state nuclear magnetic resonance spectroscopy (NMR) in order to provide insight into changes in plant cell walls during processing via various modes. The structure of lignin present in residual solids remaining after fermentation with cotreatment appeared to change little, with substantially greater changes observed for hydrothermal pretreatment - particularly with respect to formation of C-C bonds. The observation of high solubilization with little apparent modification of the residue is consistent with cotreatment enhancing solubilization primarily by increasing the access of saccharolytic enzymes to the feedstock, and C. thermocellum being able to attack all the major linkages in cellulosic biomass provided that these linkages are accessible.« less
  • Consolidated bioprocessing (CBP) by anaerobes, such as Clostridium thermocellum, which combine enzyme production, hydrolysis, and fermentation are promising alternatives to historical economic challenges of using fungal enzymes for biological conversion of lignocellulosic biomass. However, limited research has integrated CBP with real pretreated biomass, and understanding how pretreatment impacts subsequent deconstruction by CBP vs. fungal enzymes can provide valuable insights into CBP and suggest other novel biomass deconstruction strategies. This study focused on determining the effect of pretreatment by dilute sulfuric acid alone (DA) and with tetrahydrofuran (THF) addition via co-solvent-enhanced lignocellulosic fractionation (CELF) on deconstruction of corn stover and Populusmore » with much different recalcitrance by C. thermocellum vs. fungal enzymes and changes in pretreated biomass related to these differences.« less
  • Consolidated bioprocessing (CBP) by anaerobes, such as Clostridium thermocellum, which combine enzyme production, hydrolysis, and fermentation are promising alternatives to historical economic challenges of using fungal enzymes for biological conversion of lignocellulosic biomass. However, limited research has integrated CBP with real pretreated biomass, and understanding how pretreatment impacts subsequent deconstruction by CBP vs. fungal enzymes can provide valuable insights into CBP and suggest other novel biomass deconstruction strategies. This study focused on determining the effect of pretreatment by dilute sulfuric acid alone (DA) and with tetrahydrofuran (THF) addition via co-solvent-enhanced lignocellulosic fractionation (CELF) on deconstruction of corn stover and Populusmore » with much different recalcitrance by C. thermocellum vs. fungal enzymes and changes in pretreated biomass related to these differences.« less