Elucidating the Structural Changes to Populus Lignin during

Letter pubs.acs.org/journal/ascecg

Elucidating the Structural Changes to Populus Lignin during Consolidated Bioprocessing with Clostridium thermocellum Hannah O. Akinosho,†,‡,⊥ Chang Geun Yoo,‡,§,⊥ Alexandru Dumitrache,‡ Jace Natzke,‡ Wellington Muchero,‡ Steven D. Brown,‡ and Arthur J. Ragauskas*,‡,§,∥ †

School of Chemistry and Biochemistry and Renewable Bioproducts Institute, Georgia Institute of Technology, 901 Atlantic Drive, Atlanta, Georgia 30332, United States ‡ BioSciences Division, BioEnergy Science Center, Oak Ridge National Laboratory, One Bethel Valley Road, BioEnergy Science Center, Building 1520, Oak Ridge, Tennessee 37831, United States § UT-ORNL Joint Institute for Biological Sciences, Oak Ridge National Laboratory, One Bethel Valley Road, BioEnergy Science Center, Building 1520, Oak Ridge, Tennessee 37831, United States ∥ Department of Chemical and Biomolecular Engineering; Department of Forestry, Wildlife, and Fisheries; and Center for Renewable Carbon, University of Tennessee, 1512 Middle Drive, Knoxville, Tennessee 37996, United States S Supporting Information *

ABSTRACT: 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 study, 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. This investigation indicates that C. thermocellum not only modifies lignin in order to access cellulose but also leaves behind a suitable lignin substrate for value-added applications in the cellulosic ethanol production scheme. KEYWORDS: Consolidated bioprocessing, Lignin characterization, Clostridium thermocellum, NMR, FTIR

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INTRODUCTION Consolidated bioprocessing (CBP) has often been regarded as an effective and potentially revolutionary process for the commercialization of cellulosic ethanol.1−4 CBP microorganisms address challenges associated with enzyme production5 and biomass recalcitrance6 that plagued ethanol production from fungal cellulases. Clostridium thermocellum, a thermophilic and anaerobic CBP microorganism, produces cellulosomes and small amounts of free cellulases to deconstruct the plant cell wall architecture. C. thermocellum’s cellulosomes, or multienzymes complexes, encourage synergistic relationships between enzymes and contribute to its high rates of cellulose hydrolysis.7,8 C. thermocellum utilizes cellulose-derived hydrolysis products and carries out fermentation to generate ethanol, hydrogen, acetate, and other organic acids. While C. thermocellum possesses one of the fastest known rates of cellulose hydrolysis,8 recalcitrance and low ethanol yields are its leading challenges to industrial-scale ethanol production. Approaches such as genetic engineering of microbes9−12 and plants13 as well as the metabolic adaptation of wild-type strains14 have provided major advances in ethanol yields from C. thermocellum. In contrast, recalcitrance during CBP with C. © 2017 American Chemical Society

thermocellum is poorly understood and requires biomass characterization studies to understand changes to biomass during CBP. A few investigations have documented changes to cellulose15,16 and hemicellulose17 during CBP, and the current understanding of the structural changes to lignin during CBP remain in its infancy.18 Lignin is a key contributor to biomass recalcitrance with fungal cellulases. It inhibits enzyme accessibility and reduces enzyme activity by nonproductive enzyme adsorption.19 Likewise, lignin influences biomass conversion during CBP,20 and the interactions between lignin and CBP microorganisms suggest that lignin content and structure affect CBP.13,18,21 Because the enzymes typically used for ethanol production are cellulases isolated from fungi, they are specific to cellulose hydrolysis and do not catalyze changes to lignin.22 In contrast, C. thermocellum contains at least 70 enzymes23 that alter plant cell wall components, often to reduce recalcitrance. Pectinanses,24 chitinases,25 cellulases,26 amylases,27 β-glucaReceived: May 11, 2017 Revised: June 6, 2017 Published: July 20, 2017 7486

DOI: 10.1021/acssuschemeng.7b01203 ACS Sustainable Chem. Eng. 2017, 5, 7486−7491

Letter

ACS Sustainable Chemistry & Engineering

Figure 1. 2D HSQC 1H−13C NMR spectra of the whole-cell wall for the control (BESC-102 and BESC-922) and CBP-treated (BESC-102T and BESC922T) P. trichocarpa.

nases,28 and hemicellulases17,29 exemplify the many enzymes available to C. thermocellum to modify the plant cell wall components and access cellulose. Although lignin is a major cell wall component and known contributor to recalcitrance, C. thermocellum contains no known lignin-modifying enzymes, whose presence would be especially useful in accessing cellulose. Populus trichocarpa, a fast-growing and high-yielding hardwood, is a genetically diverse and competitive feedstock for ethanol production.30 Its genetic diversity results in natural variants that vary in recalcitrance.31 Furthermore, the genetic modification of P. trichocarpa has been studied to reduce recalcitrance as its entire genome has been sequenced.32 In this study, to verify the static nature of lignin isolated from P. trichocarpa during CBP, the lignin structure was studied using two spectroscopic (NMR and FTIR) techniques.

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d6), and hexamethylphosphoramide (HMPA-d18) used in this study were purchased from VWR, Sigma-Aldrich, or Armar Chemicals. Consolidated Bioprocessing (CBP). Consolidated bioprocessing was conducted with autoclaved and milled (0.84 mm screen) P. trichocarpa. Sterile MTC media (50 mL culture volume) were prepared for each sample under anaerobic conditions. Freshly prepared C. thermocellum ATCC 27405 was used at a 10% (v/v) inoculum in 1 L Sartorius BIOSTAT QPlus bioreactors (Sartorius Stedum Biotech, Göttingen, Germany). CBP was conducted at a 20 g/L (dry basis) biomass loading for 5 days at 60 °C and 200 rpm. The resulting fermentation mixture was centrifuged to separate the solid residues from the spent fermentation broth. The residuals were washed with Milli-Q water several times to remove bacterial cells and freeze-dried prior to NMR and FTIR analysis. Cell removal was confirmed with the absence of the characteristic amino acid peaks34 in the FTIR spectra, and the comparison of the FTIR spectra of the control poplar with the CBP treated poplar. Whole-Cell Wall Nuclear Magnetic Resonance (NMR) Analysis. The samples were Soxhlet-extracted with a mixture of ethanol/ toluene (1:2, v/v) for 8 h to remove extractives. The extractives-free samples were air-dried and ball-milled using a planetary ball mill (Retsch PM 100) spinning at 600 rpm with zirconium dioxide (ZrO2) vessels (50 mL) containing ZrO2 ball bearings (10 mm × 10 mm) for 2 h and 30 min (5 min grinding and 5 min break) for whole-cell wall NMR analysis. The ball-milled whole-cell wall samples (∼50 mg) were placed in a 5 mm

EXPERIMENTAL SECTION

Materials. The stem disks of two natural variants of four-year-old Populus trichocarpa, which were harvested from Clatskanie, OR, were supplied by Oak Ridge National Laboratory.33 Chemical compositions of both natural variants, BESC-102 and BESC-922 (also known as HARA 26-2), before and after CBP were discussed in a previous study.18 All chemicals including ethanol, toluene, dimethyl sulfoxide (DMSO7487

DOI: 10.1021/acssuschemeng.7b01203 ACS Sustainable Chem. Eng. 2017, 5, 7486−7491

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ACS Sustainable Chemistry & Engineering NMR tubes and dissolved with DMSO-d6/HMPA-d18 (4:1, v/v, ∼ 0.5 mL). The biomass and NMR solvents were vortexed and placed in a sonicator for 1−2 h before the analysis. NMR spectra were measured at 298 K using a Bruker Avance III 400 MHz console equipped with a 5 mm Broadband Observe (BBO) probe. Two-dimensional (2D) 1H−13C heteronuclear single quantum coherence (HSQC) spectra were collected using a Bruker standard pulse sequence (“hsqcetgpsi2”). The parameters for the measurements are as follows: spectral width of 11 ppm in F2 (1H) with 2048 data points and 190 ppm in F1 (13C) with 256 data points, 64 scans (NS), and 1 s interscan delay (D1). Volume integration of contours in NMR spectra was carried out using Bruker’s TopSpin 3.5 software. For quantitating lignin subunits, the S2/6, S′2/6, G2, and PB2/6 contours were used. Interunit linkages of lignin were also measured with the α-position of each unit (β-O-4, β-5, and β-β) as described in our previous study.35 Fourier Transform Infrared (FTIR) Analysis. Lignin-enriched residues, which were prepared according to a previous study,18 were analyzed with a PerkinElmer 100 FTIR spectrometer (Wellesley, MA) with a universal attenuated total reflection (ATR) accessory. FTIR spectra were acquired by averaging 32 scans and 4 cm−1 resolution from 4000 to 800 cm−1. The S/G index was measured by normalizing the spectra to 1505 cm−1 and calculated by dividing the peak intensity at 1328 cm−1 (C−O in syringyl lignin) by the peak intensity at 1233 cm−1 (C−O in guaiacyl lignin) as discussed in a previous study.36

Table 1. Quantitative Comparison of Lignin S/G Ratio, Hydroxycinnamates (p-hydroxybenzoate, PB), and Inter-Unit Linkages As Determined by Whole-Cell Wall NMR Analysis Aromatic region

Aliphatic region

Sample

S/G

PB (%)

β-O-4 (%)

β-5 (%)

β-β (%)

BESC-102 BESC-102T BESC-922 BESC-922T

0.96 1.00 2.69 2.95

13 12 4 2

66 59 68 60

1.3 1.5 2.2 0.8

1.5 1.1 4.8 4.3

both natural variants. Besides the aromatic compounds, the β-O4 linkage represented the largest relative proportions (66−68% over total lignin subunits) among lignin interunit linkages in the untreated P. trichocarpa. The β-O-4 content in both P. trichocarpa natural variants decreased by 7−8% after CBP. With the exception of the β-5 content in BESC 922, relative proportions of the carbon−carbon linkages, such as β-5 and β-β did not show remarkable changes compared to β-O-4 content after CBP. In case of BESC 922, more than half of β-5 linkage was removed, while the change in β-β content was not significant after CBP. Lignin S/G Index As Determined by FTIR Analysis. In previous studies, Fourier transform infrared (FTIR) spectroscopy has been applied to observe lignin structural changes with fungal cellulases.38,39 It was found that the carbonyl peak intensities in lignin’s fingerprint region, 1800−600 cm−1, decreased as lignin-degrading enzymatic activities ensued.38,39 Attenuated total reflectance (ATR)-FTIR was conducted with lignin-enriched residues from untreated and CBP-treated P. trichocarpa. The peak intensities, which were normalized to 1505 cm−1, are compared in Figure 2.

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RESULTS AND DISCUSSION Structural Information on Lignin by 2D 1H−13C HSQC NMR Analysis. In the previous study, the total solid solubilization and fermentation of P. trichocarpa with C. thermocellum in the CBP were reported, and the effects of lignin properties to biomass conversion were briefly discussed.18 For a better understanding of lignin structural changes during CBP with C. thermocellum, lignins in two natural variants (BESC-102 and BESC-922) of P. trichocarpa before and after the CBP were characterized using nuclear magnetic resonance (NMR) and Fourier transform infrared (FTIR). The performance of C. thermocellum was comparable as confirmed with the previous results18 before the characterization. NMR analysis was conducted to analyze the structural properties of lignins in the P. trichocarpa whole plant cell wall. The two-dimensional heteronuclear single quantum coherence (2D HSQC) 1H−13C NMR analysis was performed with the whole-cell wall dissolved in DMSO-d6/HMPA-d18 (4:1, v/v). Figure 1 presents the 2D HSQC 1H−13C NMR spectra of the whole-cell wall for the control (BESC-102 and BESC-922) and CBP-treated (BESC102T and BESC-922T) P. trichocarpa. Qualitative analysis identified syringyl (S), guaiacyl (G), and hydroxycinnamates (p-hydroxybenzoate) in the aromatic regions of the NMR spectra. Also, the relative contents of S and G lignins were determined by integration at the S2/6 and G2 contours, which occurred at approximately δC/δH 104.0/6.66 and 111.2/6.98 ppm, respectively.37 The p-hydroxybenzoate (PB) content was measured with the PB2/6 contour at 131/7.63 ppm.37 The lignin interunit linkages were also observed in the aliphatic region of the NMR spectra. The cross peaks for β-aryl ether (β-O-4), resinols (β−β), and phenylcoumaran (β-5) were quantified at the αposition of each linkage at δC/δH 72.2/4.90, 87.0/5.38, and 85.0/ 4.64 ppm, respectively.37 Table 1 displays the semiquantitative analysis of lignin S/G ratio, PB content, and the three major interunit linkage contents in the P. trichocarpa examined in this study. Lignin S/G ratios increased in both natural variants following CBP. In particular, the P. trichocarpa with a higher initial lignin S/G ratio (BESC922) showed a more notable increase in lignin S/G ratio from 2.69 to 2.95. The PB content experienced a 1−2% decrease with

Figure 2. FTIR spectra of two P. trichocarpa before and after CBP with C. thermocellum.

The ratio of peak intensities at 1328 and 1233 cm−1, which represent C−O in syringyl lignin and C−O in guaiacyl lignin, were used to calculate an alternative index for S/G ratios.36 Similar to the 2D NMR experiments, the S/G index for BESC102 (1.08) was lower than that of BESC-922 (1.11). In BESC102, the index was essentially unchanged between lignin from control and CBP-treated samples, as the difference may have been too small for FTIR to detect. BESC-922’s S/G index increased from 1.11 to 1.15 following CBP. In this study, both NMR and FTIR analysis results indicated similar trends in lignin S/G ratio due to CBP. 7488

DOI: 10.1021/acssuschemeng.7b01203 ACS Sustainable Chem. Eng. 2017, 5, 7486−7491

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ACS Sustainable Chemistry & Engineering Structural Changes to Lignin by C. thermocellum during CBP. The observed transformations in lignin reported in this and our previous study18 suggest that C. thermocellum induces structural changes to lignin. If enzymes catalyzed these changes (i.e., increased S/G ratio, decreased β-O-4, and decreased β-5), they are different from the fungal enzymes that catalyze similar changes to lignin structure. Wei et al. studied gene expression in C. thermocellum during CBP on pretreated yellow poplar and found that the genes that encoded lignin degradation for enzymes such as lignin peroxidase (LiP), laccase, or manganese peroxidase (MnP) were not identified from transcriptomic profiling.40 The absence of these enzymes does not negate the presence of other lignin modifying enzymes in C. thermocellum that are specific to bacteria. Similar to C. thermocellum, P. anantis Sd-1 hydrolyzes cellulose, hemicellulose, and lignin. Additionally, transcriptomics revealed that the enzymes involved in lignin degradation were not LiPs, laccases, or MnPs; however, P. anantis Sd-1 did possess a class of enzymes that were specific to bacteria.40 Microbial β-etherases represent one class of microbe-specific enzymes that remove β-O-4 bonds in lignin41,42 and could be partially responsible for enzymes for the lignin alteration observed in this study. However, additional studies are needed to identify these and other enzymes involved in lignin modification by C. thermocellum. Phenolic groups have been recognized as a common initiation site for the enzymatic depolymerization of lignin.43 Additionally, steric effects and substituent properties largely influence the ease of phenolic radical generation.44 In this study, the increase in lignin S/G ratio was attributed to the removal of G lignin, whose phenolic group is less sterically hindered than S lignin. The relatively reduced peak integration intensities, as determined by HSQC, in G lignin following CBP further support this idea. C. thermocellum may also remove PB during biomass deconstruction, according to the data collected during this study. PB’s unhindered phenolic group renders it a viable starting site for chemical reactions; however, the strong electron withdrawing substituent at the para position on the aromatic ring may negatively impact its reactivity. This investigation indicates that lignin depolymerization and/ or restructuring occurs during CBP. Lignin depolymerization would be beneficial to CBP as it has been known to improve cellulase accessibility to cellulose.45 Lignin restructuring could also be beneficial to biomass deconstruction. For example, high molecular weight lignin has been found to donate electrons to lytic polysaccharide monooxygenases (LPMO),46 a group of copper-dependent enzymes that are present in fungi and C. thermocellum and are involved in glycosidic bond cleavage, to promote hydrolysis. Structural changes to lignin were observed during pH controlled CBP; however, additional research is still necessary to pinpoint lignin-degrading enzymes. Nonetheless, this heteropolymer remains a hindrance to CBP. Lignin Valorization Following CBP. Besides glucose, the core substrate of current cellulosic ethanol, lignin has several growing outlets for biomass valorization.47 Various studies have proposed using lignin for biomass-derived materials, chemicals, and fuels using catalysis,48 enzymes,49 microorganisms,50,51 and pyrolysis52 to achieve the desired end products. For example, the microorganism Rhodococci consumed lignin as a carbon source to produce lipids that are valuable for fuel production.53,54 An engineered Pseudomonas was also employed to convert ligninderived monomers to cis,cis-muconic acid, which is an intermediate for commodity chemicals.55 These microorganisms preferred to convert aromatic monomers and oligomers. In this

study, C. thermocellum in CBP were found to partially change the lignin structures in biomass. The structures of lignin, whether derived from raw biomass, a pretreatment, or cellulosic ethanol production, influence its suitability for future processing. Oftentimes, it is desirable that lignin possesses certain structural features such as high S/G ratio or high β-O-4 contents that facilitate subsequent processing.56 The resultant lignin from CBP is desirable for a number of applications. Industrial-scale ethanol production from CBP will warrant lignin valorization to maximize the profitability of the major biomass components. Accordingly, lignin structure following CBP will dictate the ease and choice of processing. Therefore, lignin structure is expected to be reasonably preserved during CBP, which has important implications for β-O-4 content and S/G ratio. Several recent studies into pyrolysis and its product heterogeneity have employed β-O-4 model compounds,57−61 which indicate that these bonds are of particular interest in lignin upgrading. In other lignin valorization applications, chemical conversions commonly target the β-O-4 linkage.62−65 Populus lignin after CBP still contained ≥59% β-O4 content over total lignin subunits in this investigation, which renders lignin from CBP especially relevant to valorization. Higher lignin S/G ratios have been associated with higher glass transition temperatures, greater cross-linking, and higher molecular weights in lignin, which promotes its function in carbon fibers.66 In this study, the lignin S/G ratios did not exhibit significant differences after CBP.

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CONCLUSIONS In summary, the effects of C. thermocellum on the P. trichocarpa lignin structure were discussed using 2D HSQC NMR and FTIR analyses. The alteration of lignin, such as lignin S/G ratio and βO-4 content, during CBP indicated that C. thermocellum presumably interacts with lignin in the biomass. Accordingly, the CBP-treated lignin without significant modification of structural properties possesses properties that improve its attractiveness for valorization.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b01203. Experimental details. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +1-865-974-2042. ORCID

Arthur J. Ragauskas: 0000-0002-3536-554X Author Contributions ⊥

H.O.A. and C.G.Y. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We greatly appreciate the Renewable BioProducts Institute at Georgia Institute of Technology for their financial support of H.O.A. through the RBI Fellowship for graduate studies. Funding provided by The BioEnergy Science Center a U.S. Department of Energy Bioenergy Research Center supported by 7489

DOI: 10.1021/acssuschemeng.7b01203 ACS Sustainable Chem. Eng. 2017, 5, 7486−7491

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ACS Sustainable Chemistry & Engineering

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the Office of Biological and Environmental Research in the DOE Office of Science. This manuscript has been authored by UTBattelle, LLC under Contract No. DE-AC05-00OR22725 with the U.S. Department of Energy.

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DOI: 10.1021/acssuschemeng.7b01203 ACS Sustainable Chem. Eng. 2017, 5, 7486−7491

Letter

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DOI: 10.1021/acssuschemeng.7b01203 ACS Sustainable Chem. Eng. 2017, 5, 7486−7491