G Ratio in Biomass Recalcitrance of Populus

Subscriber access provided by READING UNIV

Article

Significance of lignin S/G ratio in biomass recalcitrance of Populus trichocarpa variants for bioethanol production Chang Geun Yoo, Alexandru Dumitrache, Wellington Muchero, Jace Natzke, Hannah O. Akinosho, Mi Li, Robert Sykes, Steven D. Brown, Brian H. Davison, Gerald A. Tuskan, Yunqiao Pu, and Arthur Jonas Ragauskas ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b03586 • Publication Date (Web): 11 Dec 2017 Downloaded from http://pubs.acs.org on December 16, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Sustainable Chemistry & Engineering is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sustainable Chemistry & Engineering

Significance of lignin S/G ratio in biomass recalcitrance of Populus trichocarpa variants for bioethanol production Chang Geun Yoo,†,‡,⊥ Alexandru Dumitrache,†,⊥ Wellington Muchero,† Jace Natzke,† Hannah Akinosho,§ Mi Li,†,‡ Robert W. Sykes,∇ Steven D. Brown,† Brian Davison,† Gerald A. Tuskan,† Yunqiao Pu,†,‡ Arthur J. Ragauskas*,†,‡,¶ †

BioEnergy Science Center & Center for Bioenergy Innovation, Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA. ‡ UT-ORNL Joint Institute for Biological Science, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA. § School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, USA. ∇ National Renewable Energy Laboratory, US Department of Energy, Golden, CO 80401, USA. ¶ Department of Chemical and Biomolecular Engineering & Center for Renewable Carbon, Department of Forestry, Wildlife, and Fisheries, University of Tennessee, Knoxville, TN 37996, USA. ⊥ *

These authors contributed equally to this work. Email: [email protected]

Mailing address†: 1 Bethel Valley Road, BioEnergy Science Center, Building 1520, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA ‡ Mailing address : 1 Bethel Valley Road, BioEnergy Science Center, Building 1520, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA § Mailing address : 901 Atlantic Drive, Georgia Institute of Technology, Atlanta, GA 30332, USA ∇ Mailing address : 15013 Denver West Parkway, National Renewable Energy Laboratory, Golden, CO 80401, USA ¶ Mailing address : 1512 Middle Drive, University of Tennessee, Knoxville, TN 37996, USA Note: This manuscript has been authored by UT-Battelle, LLC under contract no. DEAC05-00OR22725 with the U.S. Department of Energy. The publisher, by accepting the article for publication, acknowledges that the United States Government retains a nonexclusive, paid-up, irrevocable, world-wide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in Accordance with the DOE Public Access Plan (http://energy.gov/downloads/doe-public-access-plan).

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Lignin S/G ratio has been investigated as an important factor in biomass recalcitrance to bioethanol production. Due to the complexity and variety of biomass, recalcitrance was also reportedly influenced by several other factors such as total lignin content, degree of cellulose polymerization, etc. In addition, the effect of S/G ratio on biomass conversion is not uniform across plant species. Herein, 11 Populus trichocarpa natural variants grown under the same conditions with similar total lignin content were selected to minimize the effects of other factors. The lignin S/G ratio of the selected P. trichocarpa natural variants showed negative correlations with p-hydroxybenzoate (PB) and β-β linkage contents, while it had positive ones with β-O-4 linkage, lignin molecular weight, and ethanol production. This study showed the importance of lignin S/G ratio as an independent recalcitrance factor that may aid future energy crop engineering and biomass conversion strategies.

Keywords: lignin S/G ratio, Populus, bioethanol, NMR, Py-MBMS

Introduction Biochemicals and bioenergy derived from plant biomass have become a focus of intense research in recent years. Populus trichocarpa (P. trichocarpa) is considered one of the promising plant feedstocks for bioconversion to bioethanol due to its carbohydrate-rich cell wall, relative rapid growth, easy propagation and genetic manipulation.1, 2 Extracting carbohydrates from biomass and their conversion to fermentable saccharides remain major challenges in the biofuels industry. To date, thermochemical pretreatment of the


Page 2 of 23

Page 3 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


biomass coupled with enzymatic hydrolysis is most commonly applied to overcome these barriers. While developments in pretreatment methods significantly removed or reduced the plant cell wall recalcitrance, these pre-conversion strategies are still costly. In an effort to bring this cost down, researchers are modifying the biomass for less inherent recalcitrance. To aid this effort, the underlying mechanisms of cell wall recalcitrance need to be better understood. Lignin, a highly-branched heterogeneous biopolymer in the plant cell wall, is the main component responsible for the observed biomass recalcitrance to enzymatic and microbial deconstruction. In the cell wall, it provides a hydrophobic surface that aids in water transport and defense against biotic and abiotic stresses.3 Studies of lignin biosynthesis have led to a greater understanding of how monolignol precursors are formed and by what mechanism lignin polymerization occurs.4 Lignin consists of three main monolignols: p-hydroxyphenyl (H), syringyl (S) and guaiacyl (G) propane units. The S and G units interact together to form the backbone of the polymer via a labile arylglycerol-β-aryl ether (β-O-4) bond.5 Other carbon-carbon bonds such as β-β, 5-5 and β-5 linkages are also formed between these lignin units and are considered to have influences on biomass degradation.6 Studer et al. reported that the ratio of lignin S/G is likely important and can range from 1.0-3.0 in undomesticated P. trichocarpa.7 Dumitrache et al. discussed that a high S/G ratio may lead to larger linear chains, as shown by increased lignin molecular weight, which creates less interference in carbohydrate deconstruction.8 Huntley et al. also reported significant increases in pulping efficiency in the C4H-F5H transformed poplar mutant with high S/G ratio.9 However, the association of lignin S/G ratio with its recalcitrance is still not fully understood. For



instance, in alfalfa, the S/G ratio did not show strong correlations with recalcitrance because of high proportion of H unit,10 while it did in poplar and maize.11, 12 Also, it may be argued that other factors, such as total lignin content, may be a larger contributing factor to the observed results. A strong negative correlation between total lignin content and enzymatic carbohydrate release was already confirmed,10, 13, 14 for the reported range of 16-28% total lignin content in Populus.7 Therefore, in the current study, to better determine whether S/G ratio is indeed a driving force behind the observed recalcitrance of P. trichocarpa, eleven natural variant samples containing similar total lignin contents (17%-19%; Table S1), yet varying S/G ratios were selected. Lignin S/G ratios were obtained by nuclear magnetic resonance (NMR) and pyrolysis molecular beam mass spectrometry (Py-MBMS) analyses. Structural characteristics of each lignin were also analyzed by NMR and the data was then correlated to the S/G ratios with other ligninrelated properties. Ethanol yield from each poplar variant obtained by separate hydrolysis and fermentation (SHF) was used to understand the correlation between the lignin S/G ratio and biomass recalcitrance to bioconversion.

Experimental Section Biomass preparation: As significant differences in plant physiological behavior between field and greenhouse was pointed out in previous studies15, we recognized the importance of assessing field-grown specimens. For this reason, four year old natural P. trichocarpa variants grown the same field at Clatskanie, OR (46°6′ 11′′ N 123°12′ 13′′ W) were selected. Information of field establishment, growth conditions, and post harvest handling was described in a previous study.16 Each P. trichocarpa stem was debarked, Wiley-


Page 4 of 23

Page 5 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


milled and screened to 0.84 mm. The screened samples were washed with water and sterilized by autoclave at 121 °C for 20 min during media preparation.

Lignin sample preparation for NMR and GPC analyses: Each Populus sample was extracted using ethanol/toluene mixture (1:2, v/v) for 8 h and air-dried for 24h. The extractives-free sample was ball-milled at 580 rpm using a Retsch Ball Mill PM 100 with 10 ZrO2 balls for 2 h and 30 min. The ball-milled sample was hydrolyzed with a mixture of Cellic® CTec-2 and HTec-2 (Novozymes) in the sodium acetate buffer solution (pH 4.8) at 50 °C and 170 rpm for 48h. The same enzymatic hydrolysis was conducted twice, and the residues were isolated and treated by protease for 24h. After the protease treatment, the solid residues, called lignin-enrich residues, were separated using a centrifuge and freeze-dried for NMR analysis. For the analysis of molecular weights, lignin-enrich residue was purified further by dioxane extraction. Dioxane extraction was conducted with 96% dioxane at room temperature for 48 h. The extracted lignin in dioxane was recovered through rotary evaporation at 40 °C followed by freeze-drying. The recovered dioxane-extractable lignins were acetylated by pyridine/acetic anhydride mixture (1:1, v/v) at room temperature for 24h. After the acetylation of lignins, the reaction mixtures were quenched with ethanol and the solvents were removed by rotary evaporation at 40 °C for gel permeation chromatography (GPC) analysis.

NMR analysis: Structural characteristics of lignins in the 11 P. trichocarpa natural variants were analyzed by nuclear magnetic resonance (NMR) analysis. About 50 mg of lignin-enrich residues was dissolved in the NMR solvent (DMSO-d6, 0.4 – 0.5 mL). The



analysis was conducted using a Bruker Avance III 400-MHz spectroscopy equipped with a 5-mm Broadband Observe probe (5-mm BBO 400MHz W1 with Z-gradient probe, Bruker). Two-dimensional (2D) 1H–13C heteronuclear single quantum coherence (HSQC) spectra of each Populus sample were detected by a Bruker standard pulse sequence (‘hsqcetgpsi2’) with the following parameters: 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 0.5 s interscan delay (D1). Data processing was carried out using Bruker’s TopSpin 3.5pl6 software.

Py-MBMS analysis: Lignin S/G ratio and total lignin content were analyzed by pyrolysis molecular beam mass spectrometry (Py-MBMS) as described in the previous study.17

GPC analysis: The weight average molecular weights and number average molecular weights of lignin samples were analyzed by GPC system using Agilent 1200 HPLC system with Waters Styragel columns (HR1, HR4, and HR) and UV detector. The acetylated lignins, as described in the previous section, were dissolved in tetrahydrofuran (THF) and filtered by 0.45 µm membrane filter for GPC analysis. The peaks for lignin molecular weights were detected at 270 nm, and THF was used as a mobile phase with 1.0 mL/min flow rate. The relative values of lignin molecular weights were calculated with external calibration curve obtained with polystyrene standards.

Ethanol fermentation: Separate hydrolysis and fermentation (SHF) was conducted as previously detailed.18 In short, biological triplicate samples of each poplar variant, at 5%


Page 6 of 23

Page 7 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(w/v) solids loading, were hydrolyzed for 5 days at 50 °C with a mixture of hydrolytic enzymes (Cellic® Ctec2, Cellic® Htec2 and beta-glucosidase Novozymes 188), then fermented for 3 days at 35 oC with Saccharomyces cerevisiae D5α. Once fermentation finished, the aqueous samples were analyzed by high performance liquid chromatography (HPLC, LaChrom Elite™, Hitachi High Technologies America Inc.) equipped with refractive index (L-2490) and Aminex HPX-87H column (Bio-Rad Laboratories Inc., Hercules, CA) to measure the ethanol amounts. The analysis was conducted at 60 °C and 0.5 mL/min flow rate with 5.0 mM H2SO4 as a mobile phase.

Statistical analysis: Where appropriate, the linear relationship between continuous variables (e.g., S/G ratio, ethanol yield, etc.) was expressed with the Pearson coefficient. Origin Pro (OriginLabs, MA) was used to calculate both the Pearson coefficient and the statistical test for the significance of these correlations, expressed as p-value.

Results and discussion The influence of biomass chemical composition19-22 and its structural features7, 21 on plant cell wall recalcitrance has been reported in numerous studies. In particular, the general negative impact of lignin in the plant cell wall towards biofuels production was well acknowledged.23,

24

Among many of the lignin-related factors, lignin S/G ratio was

introduced as one of the indicators of biomass recalcitrance.7,

9

However, the use of

various engineered crops or pretreatments methods in these studies have broadly altered key biomass properties, such as chemical compositions (e.g. total lignin content);11, 25 and therefore, the influence of lignin S/G ratio as a single factor on biomass conversion was



Page 8 of 23

not clearly confirmed. In this study, P. trichocarpa specimens were grown under the same environment condition (common garden at Clatskanie, OR) and growing period (4 years). Eleven natural variants with similar total lignin content (18.5 ± 0.17%) were selected to understand the role of lignin S/G ratio in biomass recalcitrance. Diverse analytical methods including Py-GCMS, NMR, thioacidolysis, FT-IR and PyMBMS have been introduced to evaluate lignin S/G ratio.26, 27 Among these methods, PyMBMS and NMR, were used to analyze lignin S/G ratio in this study. Although the scales of lignin S/G ratios were different in each analysis (Table 1 and Figure 1), both methods showed a very comparable ranking of the 11 samples by S/G ratio with strong positive linear correlation (Pearson coefficient = 0.834, p-value = 0.001). For example, lignin S/G ratios measured by Py-MBMS were 1.32 to 1.99, while the ratio by NMR ranged from 1.52 to 3.85. In both analysis methods, BESC-77, BESC-838, and BESC-28 had the three lowest S/G ratios and SLMD-28-1, BESC-803, HARC-26-2, and SLMC28-2 had the four highest ratios.

Table 1. Lignin S/G ratios of P. trichocarpa natural variants using Py-MBMS and NMR analysis methods. Sample Name

Lignin S/G ratio by Py-MBMS

BESC-77 1.60 (0.02) BESC-838 1.52 (0.00) BESC-28 1.32 (0.03) BESC-53 1.63 (0.01) BESC-893 1.62 (0.02) KLNG-20-3 1.74 (0.05) BESC-291 1.79 (0.03) SLMD-28-1 1.84 (0.02) BESC-803 1.82 (0.02) HARC-26-2 1.85 (0.02) SLMC-28-2 1.99 (0.04) Note. (value) = standard deviation


Lignin S/G ratio by NMR 1.52 (0.06) 1.63 (0.15) 1.91 (0.05) 2.04 (0.05) 2.36 (0.05) 2.26 (0.11) 2.57 (0.08) 2.58 (0.01) 2.79 (0.12) 3.09 (0.04) 3.85 (0.28)

Page 9 of 23

2.1 2 Py-MBMS S/G ratio

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Pearson coefficient = 0.834 (p-value = 0.001)

1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.00

1.50

2.00

2.50 NMR S/G ratio

3.00

3.50

4.00

Figure 1. Correlation of lignin S/G ratios of P. trichocarpa natural variants by NMR analysis. Table 2. Quantitative information of lignin subunits (S and G units), p-hydroxybenzoate (PB), and inter-unit linkages (β-O-4, β-5, and β-β) as determined by NMR analysis Sample

BESC-77 BESC-838 BESC-28 BESC-53 BESC-893 KLNG-20-3 BESC-291 SLMD-28-1 BESC-803 HARC-26-2

S/G S [%]a G [%]a PB [%]a β-O-4 [%]a β-5 [%]a β-β [%]a 60.26 39.75 9.70 59.84 2.83 4.88 1.52 (1.04) (1.04) (6.29) (0.71) (2.19) (0.06) (1.02) 1.63 61.88 38.13 4.77 60.40 2.74 5.98 (0.15) (2.17) (2.17) (1.27) (7.52) (0.02) (0.69) 1.91 65.63 34.38 3.25 60.60 2.55 6.19 (0.05) (0.59) (0.59) (0.74) (6.82) (0.04) (1.47) 2.04 67.10 32.91 9.18 58.92 1.35 4.68 (0.05) (0.54) (0.54) (2.88) (10.13) (0.57) (0.70) 69.31 30.70 4.53 58.88 1.65 5.68 2.26 (0.11) (1.07) (1.07) (0.71) (3.91) (0.34) (0.87) 2.36 70.24 29.77 3.69 60.33 1.83 5.65 (0.05) (0.49) (0.49) (0.01) (7.96) (1.15) (0.78) 2.57 71.99 28.02 2.33 64.24 1.31 5.09 (0.08) (0.67) (0.67) (1.34) (9.57) (0.40) (0.60) 2.58 72.08 27.92 0.90 60.95 1.92 5.99 (0.01) (0.11) (0.11) (0.04) (4.31) (0.69) (0.97) 2.79 73.57 26.44 0.00 65.96 1.51 5.67 (0.12) (0.84) (0.84) (0.00) (8.01) (0.41) (0.97) 75.55 24.45 3.18 64.79 1.46 6.79 3.09 (0.23) (0.23) (0.55) (7.37) (0.57) (0.30) (0.04)



SLMC-28-2

3.85 (0.28)

79.32 (1.18)

20.69 (1.18)

2.02 (0.71)

66.59 (5.15)

Page 10 of 23

1.29 (0.35)

4.56 (0.74)

Note. a Content (%) expressed as a fraction of total lignin subunits (S+G); (value) = standard deviation NMR analysis provides further structural information of lignins in the P. trichocarpa natural variants including lignin subunits, p-hydroxybenzoate, and inter-unit linkages (Figure 2). Qualitative and semi-quantitative information of lignin inter-unit linkages were analyzed in the aliphatic region between at 50.0-90.0/2.5-6.0 ppm. The contours for three major inter-unit linkages in P. trichocarpa, β–aryl ether (β-O-4), resinols (β-β), and phenylcoumaran (β-5) were quantified with the α-position of each linkage at 72.0/4.89, 87.1/5.50, and 85.0/4.67 ppm, respectively. Aromatic poplar lignins are mainly composed of lignin subunits (S and G) and phydroxybenzoate (PB). The information of lignin S and G units and PB unit were analyzed in the aromatic regions of NMR spectra. Quantification of S and G units was conducted by integrating S2/6, oxidized S2/6 (S’2/6), and G2 contours at 104.3/6.71, 106.5/7.32, and 111.1/6.96 ppm, respectively. The PB2/6 contour at 131.2/7.69 ppm was also used for quantifying the content over total lignin subunits (S+G). Table 2 shows semi-quantitative information of 11 P. trichocarpa natural variant lignins. The S/G ratios of BESC-77, BESC-838, and BESC-28 were lower than 2.0, while those of HARC-26-2 and SLMC-28-2 were higher than 3.0. Interestingly, as lignin S/G ratio increased, the PB content in the natural variants decreased (Table 2 and Figure 3). PB is known as a free phenolic pendant units on lignin, attached to the γ position of lignin side chains.28, 29 Morreel et al. also found that p-hydroxybenzoate is a precursor for polymerization of poplar lignin.30 Lu et al. reported that sinapyl p-hydroxybenzoate is a precursor of lignification.31 In the


Page 11 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


previous studies, an analogous hydroxycinnamates, p-coumarate, in bamboo and maize lignins predominantly acylated S unit,32-34 while, PB in the natural poplar variants showed a negative correlation with their S/G ratio in this study.



Cβ

Methoxyl

High lignin S/G ratio

Bβ

F1 [ppm]

Low lignin S/G ratio

F1 [ppm]

Aliphatic Regions Cβ

Methoxyl

HO HO

O 4 1

OMe OMe

O

A. -aryl ether ( -O-4)

60

Xγ

60

Xγ

HO G

HO

O 4 1

OMe OMe

O

-O-4-G

A-G.

Aα

Aα

70

HO MeO

70

S

HO

O 4 1

Cγ

Cγ

OMe OMe

O

A-S. -O-4-S

80

80

OH 5

A-Gβ

A-Gβ

OMe

4

O 1 O OH

Cα

Bα

Cα

Bα

A-Sβ

A-Sβ

B. phenylcoumaran ( -5)

O

5.5

5.0

4.5

4.0

3.5

5.5

3.0 F2 [ppm]

5.0

4.5

4.0

3.5

3.0 F2 [ppm]

1 O

OMe

MeO

O 1

Aromatic Regions Low lignin S/G ratio

S2/6

F1 [ppm]

O

F1 [ppm]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 23

High lignin S/G ratio

S2/6

C. resinol ( - )

OH

1 2

6

OMe

110

G2 G5+PB3/5

110

G2

O

X. cinnamyl alcohol

1

G5+PB3/5

2

6 MeO

G6

G6

120

OMe O

120

R

S. syringyl 1 2

6 5

OMe O

130

PB2/6

130

PB2/6

R

G. guaiacyl

O

O 1

6

2

5

3 O

R

PB. p-hydroxybenzoate

7.5

7.0

6.5

F2 [ppm]

7.5

7.0

6.5

F2 [ppm]

Figure 2. Aromatic and aliphatic regions in 2D HSQC NMR spectra of P. trichocarpa natural variants: high lignin S/G ratio (BESC-1544: 3.85) and low lignin S/G ratio (BESC-77: 1.52)


Page 13 of 23

12 Person coefficient = - 0.630 (p-value = 0.038)

10 PB Content [%]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


8 6 4 2 0 1.20

1.70

2.20

2.70 NMR S/G ratio

3.20

3.70

4.20

Figure 3. Correlation of lignin S/G ratio and p-hydroxybenzoate (PB) content of P. trichocarpa natural variants Content of lignin linkages is also a crucial lignin-related characteristic. In the P. trichocarpa natural variants, β-aryl ether linkage ranged from 58.88 to 66.59% over total lignin subunits. In the plant cell wall, β-O-4 bonds were reported as the largest proportion of lignin linkage in biomass, and they were relatively labile compared to other carbon-carbon bonds (e.g., β-β, β-5, 5-5, and others) via pretreatments.35, 36 Specifically, β-O-4 linkage is the major linkage of lignin and was well correlated with lignin S/G ratio in hardwood.5, 37 The other two major linkages in the natural variants, β-5 and β-β, ranged at 1.29 – 2.83% and 4.56 – 6.79%, respectively. Figure 4 shows the correlations between lignin S/G ratio and these lignin inter-unit linkages. Relatively high correlations were observed with βO-4 linkage content (Pearson coefficient = 0.816) and β-5 linkage content (Pearson coefficient = -0.757). Our results indicated that lignin S/G ratio had a positive



correlation with the content of β-O-4 linkage. Associations of carbon-carbon linkages (β-5 and β-β linkage) with S and G units were also discussed in previous studies.7,

38

In P. trichocarpa natural variants, the lignin S/G ratio negatively

correlated with the β-5 linkage content.

68 67

3.0

Person coefficient = 0.816 (p-value = 0.002) 2.5

β-5 Content [%]

66

β-O-4 Content [%]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 23

65 64 63 62

2.0

1.5

1.0

61 60

0.5

59 58

1.20

Person coefficient = - 0.757 (p-value = 0.007)

0.0

2.20 3.20 NMR S/G ratio

4.20

1.20


4.20

Figure 4. Correlation of lignin S/G ratio and β-O-4 linkage and β-5 linkage contents of P. trichocarpa natural variants To complement the aforementioned structural information, molecular weights of lignin in P. trichocarpa natural variants were analyzed. Weight-average molecular weight (Mw) and number-average molecular weight (Mn) were measured by GPC analysis. The Mw of the natural variants ranged from 8,310 to 11,240 and Mn from 3,490 to 4,770 (Figure 5a). A positive correlation was observed between Mw with the lignin S/G ratio with a linear coefficient of 0.472 (p-value = 0.142) (Figure 5b). The correlation improved when


Page 15 of 23

associating Mw with the lignin S/G values measured by Py-MBMS with a coefficient of 0.635 (p-value = 0.036) (Figure S1). A positive correlation between Mw and lignin S/G ratio implies that increased S content leads to larger lignin polymerization fragments.

(a) Average Molecular Weight [g/mol]

12000

Mn

Mw

10000 8000 6000 4000 2000 0

(b) 11500 11000 Lignin molecular weight [g/mol]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


10500 10000 9500

Person coefficient = 0.472 (p-value = 0.142)

9000 8500 8000 7500 7000 1.20

1.70

2.20



3.70

4.20


Figure 5. (a) Lignin molecular weights (Mw and Mn) and (b) Correlation of lignin molecular weight of P. trichocarpa natural variants and lignin S/G ratio by NMR analysis.

Lastly, lignin S/G ratio was evaluated as a factor for biomass conversion. Each P. trichocarpa natural variant was converted to ethanol by SHF with commercial enzymes and S. cerevisiae yeast. Yields of ethanol from P. trichocarpa natural variants ranged from 12.44 to 32.70 mg/g biomass (Table 3). In Figure 6 and S2, ethanol production from Populus was well correlated with lignin S/G ratio. Populus samples with higher lignin S/G ratio had higher ethanol yield. Considering that lignin S/G ratio also correlated with other lignin characteristics such as inter-unit linkages and lignin molecular weights, we infer that compositional variation in lignin leads to structural differentiation that ultimately impacts solubilization to sugars; therefore, lignin S/G content may serve as an index to anticipate biomass recalcitrance for conversion into ethanol.

Table 3. Ethanol production of P. trichocarpa natural variants Sample

Ethanol [mg/g biomass]

BESC-77 BESC-893 BESC-28 BESC-838 BESC-53 KLNG-20-3 BESC-291 BESC-803 SLMD-28-1 SLMC-28-2 HARC-26-2 Note. (value) = standard deviation

12.44 (1.34) 18.13 (0.86) 19.17 (0.06) 20.43 (1.32) 21.13 (1.13) 22.57 (1.52) 23.52 (0.23) 24.49 (0.01) 24.50 (0.79) 25.67 (0.38) 32.70 (0.38)


Page 16 of 23

Page 17 of 23

35

Ethanol yield [mg/g biomass]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


30

Person coefficient = 0.750 (p-value = 0.008)

25

20

15

10

5 1.20

1.70

2.20

2.70 NMR S/G ratio

3.20

3.70

4.20

Figure 6. Correlation of lignin S/G ratio and ethanol production of P. trichocarpa natural variants Conclusion The study provides evidence to the principle that lignin composition correlated with structural differentiation and resulted in a predictable change to biomass conversion. In P. trichocarpa, higher lignin S/G ratio led to lower contents of p-hydroxybenzoate and resinols, and higher β-aryl ether content and lignin molecular weight. This phenotype was more susceptible to carbohydrate solubilization by mixed fungal enzymes and may aid future strategies to engineer low recalcitrance transgenic poplar for bioethanol production. Furthermore, our finding provides insights that can help motivate the development of lignin targeting transgenic lines. ASSOCIATED CONTENT



Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: xxx. Correlation of lignin molecular weight of P. trichocarpa natural variants and lignin S/G ratio by Py-MBMS analysis; Correlation of ethanol production of P. trichocarpa natural variants and lignin S/G ratio by Py-MBMS analysis; Lignin contents of P. trichocarpa natural variants

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Phone: +1-865-974-2042.

ORCID Chang Geun Yoo: 0000-0002-6179-2414 Alexandru Dumitrache: 0000-6359-0779 Yunqiao Pu: 0000-0003-2554-1447 Mi Li: 0000-0001-7523-1266 Brian Davison: 0000-0002-7408-3609 Wellington Muchero: 0000-0002-0200-9856 Steven D. Brown: 0000-0002-9281-3898 Gerald A. Tuskan: 0000-0003-0106-1289 Arthur J. Ragauskas: 0000-0002-3536-554X Author Contributions ⊥

C.G.Y. and A.D. contributed equally to this work.

Notes The authors declare no competing financial interest.


Page 18 of 23

Page 19 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Acknowledgements This manuscript has been authored by UT-Battelle, LLC under Contract No. DE-AC0500OR22725 with the U.S. Department of Energy (DOE). This study was supported and performed as part of the BioEnergy Science Center (BESC) and the Center for Bioenergy Innovation (CBI). The BESC and CBI are U.S. DOE Bioenergy Research Centers supported by the Office of Biological and Environmental Research in the DOE Office of Science.

REFERENCES 1.

2.

3. 4.

5.

6.

7.

8.

9.

Tuskan, G.; DiFazio, S.; Teichmann, T., Poplar genomics is getting popular: The impact of the poplar genome project on tree research, Plant Biol. 2004, 7 (01), 24. Tuskan, G. A.; Difazio, S.; Jansson, S.; Bohlmann, J.; Grigoriev, I.; Hellsten, U.; Putnam, N.; Ralph, S.; Rombauts, S.; Salamov, A., The genome of black cottonwood, Populus trichocarpa (Torr. & Gray), Science 2006, 313 (5793), 1596-1604. Sarkanen, K. V.; Ludwig, C. H., Liguins. Occurrence, formation, structure, and reactions, New York.; Wiley-Interscience, 1971. Ralph, J.; Lundquist, K.; Brunow, G.; Lu, F.; Kim, H.; Schatz, P. F.; Marita, J. M.; Hatfield, R. D.; Ralph, S. A.; Christensen, J. H., Lignins: Natural polymers from oxidative coupling of 4-hydroxyphenyl-propanoids, Phytochem. Rev. 2004, 3 (1-2), 29-60. Santos, R. B.; Capanema, E. A.; Balakshin, M. Y.; Chang, H.-m.; Jameel, H., Lignin structural variation in hardwood species, J. Agric. Food Chem. 2012, 60 (19), 4923-4930. Kishimoto, T.; Chiba, W.; Saito, K.; Fukushima, K.; Uraki, Y.; Ubukata, M., Influence of syringyl to guaiacyl ratio on the structure of natural and synthetic lignins, J. Agric. Food Chem. 2009, 58 (2), 895-901. Studer, M. H.; DeMartini, J. D.; Davis, M. F.; Sykes, R. W.; Davison, B.; Keller, M.; Tuskan, G. A.; Wyman, C. E., Lignin content in natural Populus variants affects sugar release, Proc. Natl. Acad. Sci. U.S.A. 2011, 108 (15), 6300-6305. Dumitrache, A.; Akinosho, H.; Rodriguez, M.; Meng, X.; Yoo, C. G.; Natzke, J.; Engle, N. L.; Sykes, R. W.; Tschaplinski, T. J.; Muchero, W., Consolidated bioprocessing of Populus using Clostridium (Ruminiclostridium) thermocellum: a case study on the impact of lignin composition and structure, Biotechnol. Biofuels 2016, 9 (1), 31. Huntley, S. K.; Ellis, D.; Gilbert, M.; Chapple, C.; Mansfield, S. D., Significant increases in pulping efficiency in C4H-F5H-transformed poplars: Improved



10. 11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

chemical savings and reduced environmental toxins, J. Agric. Food Chem. 2003, 51 (21), 6178-6183. Chen, F.; Dixon, R. A., Lignin modification improves fermentable sugar yields for biofuel production, Nat. Biotechnol. 2007, 25 (7), 759-761. Davison, B. H.; Drescher, S. R.; Tuskan, G. A.; Davis, M. F.; Nghiem, N. P., Variation of S/G ratio and lignin content in a Populus family influences the release of xylose by dilute acid hydrolysis, Appl. Biochem. Biotechnol. 2006, 129132 427-435. Fontaine, A.-S.; Bout, S.; Barrière, Y.; Vermerris, W., Variation in cell wall composition among forage maize (Zea mays L.) inbred lines and its impact on digestibility: analysis of neutral detergent fiber composition by pyrolysis-gas chromatography-mass spectrometry, J. Agric. Food Chem. 2003, 2003, 51 (27), 8080-8087. Van Acker, R.; Leplé, J.-C.; Aerts, D.; Storme, V.; Goeminne, G.; Ivens, B.; Légée, F.; Lapierre, C.; Piens, K.; Van Montagu, M. C., Improved saccharification and ethanol yield from field-grown transgenic poplar deficient in cinnamoyl-CoA reductase, Proc. Natl. Acad. Sci. U.S.A. 2014, 111 (2), 845-850. Yu, Z.; Jameel, H.; Chang, H.-m.; Park, S., The effect of delignification of forest biomass on enzymatic hydrolysis, Bioresour. Technol. 2011, 102 (19), 90839089. Voelker, S. L.; Lachenbruch, B.; Meinzer, F. C.; Jourdes, M.; Ki, C.; Patten, A. M.; Davin, L. B.; Lewis, N. G.; Tuskan, G. A.; Gunter, L., Antisense downregulation of 4CL expression alters lignification, tree growth, and saccharification potential of field-grown poplar, Plant Physiol. 2010, 154 (2), 874-886. Muchero, W.; Guo, J.; DiFazio, S. P.; Chen, J.-G.; Ranjan, P.; Slavov, G. T.; Gunter, L. E.; Jawdy, S.; Bryan, A. C.; Sykes, R., High-resolution genetic mapping of allelic variants associated with cell wall chemistry in Populus, BMC genomics 2015, 16 (1), 24. Sykes, R.; Yung, M.; Novaes, E.; Kirst, M.; Peter, G.; Davis, M., in Biofuels. Methods in Molecular Biology (Methods and Protocols), ed. Mielenz, J., Humana Press, Totowa, NJ, 2009, vol. 581, pp. 169-183. Dumitrache, A.; Natzke, J.; Rodriguez, M.; Yee, K. L.; Thompson, O. A.; Poovaiah, C. R.; Shen, H.; Mazarei, M.; Baxter, H. L.; Fu, C., Transgenic switchgrass (Panicum virgatum L.) targeted for reduced recalcitrance to bioconversion: a 2‐year comparative analysis of field‐grown lines modified for target gene or genetic element expression, Plant Biotechnol. J. 2017, 15 (6), 688697. Himmel, M. E.; Ding, S.-Y.; Johnson, D. K.; Adney, W. S.; Nimlos, M. R.; Brady, J. W.; Foust, T. D., Biomass recalcitrance: engineering plants and enzymes for biofuels production, Science 2007, 315 (5813), 804-807. Zhao, X.; Zhang, L.; Liu, D., Biomass recalcitrance. Part I: the chemical compositions and physical structures affecting the enzymatic hydrolysis of lignocellulose, Biofuels, Bioprod. Biorefin. 2012, 6 (4), 465-482. DeMartini, J. D.; Pattathil, S.; Miller, J. S.; Li, H.; Hahn, M. G.; Wyman, C. E., Investigating plant cell wall components that affect biomass recalcitrance in poplar and switchgrass, Energy Environ. Sci. 2013, 6 (3), 898-909.


Page 20 of 23

Page 21 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


22.

23.

24. 25.

26.

27.

28.

29.

30.

31.

32. 33.

34.

35.

Li, X.; Ximenes, E.; Kim, Y.; Slininger, M.; Meilan, R.; Ladisch, M.; Chapple, C., Lignin monomer composition affects Arabidopsis cell-wall degradability after liquid hot water pretreatment, Biotechnol. Biofuels 2010, 3 (1), 27. Zeng, Y.; Zhao, S.; Yang, S.; Ding, S.-Y., Lignin plays a negative role in the biochemical process for producing lignocellulosic biofuels, Curr. Opin. Biotechnol. 2014, 27 38-45. Pan, X., Role of functional groups in lignin inhibition of enzymatic hydrolysis of cellulose to glucose, J. Biobased Mater. Bio. 2008, 2 (1), 25-32. Fu, C.; Mielenz, J. R.; Xiao, X.; Ge, Y.; Hamilton, C. Y.; Rodriguez, M.; Chen, F.; Foston, M.; Ragauskas, A.; Bouton, J., Genetic manipulation of lignin reduces recalcitrance and improves ethanol production from switchgrass, Proc. Natl. Acad. Sci. U.S.A. 2011, 108 (9), 3803-3808. Lupoi, J. S.; Singh, S.; Parthasarathi, R.; Simmons, B. A.; Henry, R. J., Recent innovations in analytical methods for the qualitative and quantitative assessment of lignin, Renew. Sust. Energy Rev. 2015, 49 871-906. Lupoi, J. S.; Singh, S.; Simmons, B. A.; Henry, R. J., Assessment of lignocellulosic biomass using analytical spectroscopy: an evolution to highthroughput techniques, BioEnergy Res. 2014, 7 (1), 1-23. Ralph, J.; Akiyama, T.; Coleman, H. D.; Mansfield, S. D., Effects on lignin structure of coumarate 3-hydroxylase downregulation in poplar, BioEnergy Res. 2012, 5 (4), 1009-1019. Ralph, J.; Lu, F., The DFRC method for lignin analysis. Part 6. A modified method to determine acetate regiochemistry on native and isolated lignins, J. Agric. Food Chem 1998, 46 4616-4619. Morreel, K.; Ralph, J.; Kim, H.; Lu, F.; Goeminne, G.; Ralph, S.; Messens, E.; Boerjan, W., Profiling of oligolignols reveals monolignol coupling conditions in lignifying poplar xylem, Plant Physiol. 2004, 136 (3), 3537-3549. Lu, F.; Ralph, J.; Morreel, K.; Messens, E.; Boerjan, W., Preparation and relevance of a cross-coupling product between sinapyl alcohol and sinapyl phydroxybenzoate, Org. Biomol. Chem. 2004, 2 (20), 2888-2890. Lu, F.; Ralph, J., Detection and determination of p-coumaroylated units in lignins, J. Agric. Food Chem. 1999, 47 (5), 1988-1992. Grabber, J. H.; Quideau, S.; Ralph, J., p-Coumaroylated syringyl units in maize lignin: Implications for β-ether cleavage by thioacidolysis, Phytochemistry 1996, 43 (6), 1189-1194. Meyermans, H.; Morreel, K.; Lapierre, C.; Pollet, B.; De Bruyn, A.; Busson, R.; Herdewijn, P.; Devreese, B.; Van Beeumen, J.; Marita, J. M., Modifications in lignin and accumulation of phenolic glucosides in poplar xylem upon downregulation of caffeoyl-coenzyme A O-methyltransferase, an enzyme involved in lignin biosynthesis, J. Biol. Chem. 2000, 275 (47), 36899-36909. Saito, K.; Kaiho, A.; Sakai, R.; Nishimura, H.; Okada, H.; Watanabe, T., Characterization of the interunit bonds of lignin oligomers released by acidcatalyzed selective solvolysis of Cryptomeria japonica and Eucalyptus globulus woods via thioacidolysis and 2D-NMR, J. Agric. Food Chem. 2016, 64 (48), 9152-9160.



36.

37.

38.

Samuel, R.; Cao, S.; Das, B. K.; Hu, F.; Pu, Y.; Ragauskas, A. J., Investigation of the fate of poplar lignin during autohydrolysis pretreatment to understand the biomass recalcitrance, RSC Adv. 2013, 3 (16), 5305-5309. Akiyama, T.; Goto, H.; Nawawi, D. S.; Syafii, W.; Matsumoto, Y.; Meshitsuka, G., Erythro/threo ratio of β-O-4 structures as an important structural characteristic of lignin. Part 4: Variation in the erythro/threo ratio in softwood and hardwood lignins and its relation to syringyl/guaiacyl ratio, Holzforschung 2005, 59 (3), 276-281. Zeng, J.; Helms, G. L.; Gao, X.; Chen, S., Quantification of wheat straw lignin structure by comprehensive NMR analysis, J. Agric. Food Chem. 2013, 61 (46), 10848-10857.


Page 22 of 23

Page 23 of 23

Table of Contents (TOC) Graphic: Synopsis: Impact of lignin S/G ratio in Populus trichocarpa on its structural characteristics and bioethanol production was understood by NMR and Py-MBMS analyses. Abstract Graphic

F1 [ppm]

1 2

6

S MeO

OMe O

R

110 1 2

6

G 5

OMe O

R

120

O

O 1

6

PB O

7.5

2

130

3

5

R

7.0

6.5

F2 [ppm]

BE S BE C-7 SC 7 BE 893 SC BE - 2 SC 8 BE 838 KL SCNG 53 BE 20SC 3 BE - 2 9 S 1 SL C-8 M 03 D SL -28 M -1 C HA -28 RC -2 -2 62

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



G Ratio in Biomass Recalcitrance of Populus

Recommend Documents