Integration of renewable deep eutectic solventswith engineered biomass to achieve aclosed-loop biorefineryKwang Ho Kima,b,1, Aymerick Eudesc,d, Keunhong Jeonge, Chang Geun Yoof, Chang Soo Kima,and Arthur Ragauskasg,h,i,j

aClean Energy Research Center, Korea Institute of Science and Technology, Seoul 02702, Republic of Korea; bDepartment of Wood Science, University ofBritish Columbia, Vancouver, BC, V6T 1Z4, Canada; cFeedstocks Division, Joint BioEnergy Institute, Emeryville, CA 94608; dEnvironmental Genomics andSystems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; eDepartment of Chemistry, Korea Military Academy, Seoul 01805,Republic of Korea; fDepartment of Paper and Bioprocess Engineering, State University of New York College of Environmental Science and Forestry, Syracuse,NY 13210; gCenter for Bioenergy Innovation, University of Tennessee–Oak Ridge National Laboratory Joint Institute for Biological Science, Oak Ridge, TN37831; hBiosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831; iDepartment of Chemical and Biomolecular Engineering, University ofTennessee, Knoxville, TN 37996; and jCenter for Renewable Carbon, Department of Forestry, Wildlife, and Fisheries, University of Tennessee, Institute ofAgriculture, Knoxville, TN 37996

Edited by Alexis T. Bell, University of California, Berkeley, CA, and approved June 3, 2019 (received for review March 17, 2019)

Despite the enormous potential shown by recent biorefineries, thecurrent bioeconomy still encounters multifaceted challenges. Todevelop a sustainable biorefinery in the future, multidisciplinaryresearch will be essential to tackle technical difficulties. Herein, weleveraged a known plant genetic engineering approach thatresults in aldehyde-rich lignin via down-regulation of cinnamylalcohol dehydrogenase (CAD) and disruption of monolignol bio-synthesis. We also report on renewable deep eutectic solvents(DESs) synthesized from phenolic aldehydes that can be obtainedfrom CAD mutant biomass. The transgenic Arabidopsis thalianaCAD mutant was pretreated with the DESs and showed a twofoldincrease in the yield of fermentable sugars compared with wildtype (WT) upon enzymatic saccharification. Integrated use of low-recalcitrance engineered biomass, characterized by its aldehyde-type lignin subunits, in combination with a DES-based pretreat-ment, was found to be an effective approach for producing a highyield of sugars typically used for cellulosic biofuels and biobasedchemicals. This study demonstrates that integration of renewableDES with plant genetic engineering is a promising strategy in de-veloping a closed-loop process.

green solvent | bioenergy | cinnamyl alcohol dehydrogenase |lignocellulosic biomass

The modern lignocellulosic biorefinery strives to develop newprocesses and products to achieve a sustainable energy fu-

ture. Although renewable fuels from lignocellulosic biomasshave proven to be alternatives to fossil fuels, innovative tech-nologies are still required to build economically viable processesfor converting biomass to fuels, chemicals, and materials (1).Recent efforts to overcome such barriers include (i) developing afeedstock-agnostic biomass pretreatment, (ii) engineering mi-croorganisms that can catabolize both monosaccharides andlignin, and (iii) understanding biosynthesis of plant cell walls todevelop engineered biomass with improved properties for biofuelsand bioproducts. In lignocellulosic biomass-to-ethanol processes,researchers have endeavored to develop a biocompatible andscalable biomass pretreatment process, design new microbialstrains that can convert both pentose and hexose with enhancedresistance to inhibitors, and engineer feedstocks to provide highyields of sugars and readily processable lignin.In contrast to first-generation ethanol, which has been studied

in great depth and is considered to be mature, the production ofcellulosic ethanol from lignocellulosic biomass still requiresovercoming technical and economic hurdles. In particular, ligninrepresents one of the primary factors contributing to the re-calcitrance of biomass as its presence restricts enzymatic hy-drolysis by nonproductive binding enzymes (2). Although recent

studies have unlocked lignin’s potential for various applications(3), it is still one of the most challenging biopolymers to workwith because of its inherent recalcitrant structural characteristics.In this regard, reducing the total amount of lignin in lignocel-lulosic biomass has been a widely adopted strategy to improvesaccharification and the extractability of biomass components(4). In addition, there have been many recent efforts to alterlignin monomeric composition in plants to render lignin moreamenable to extraction or chemical depolymerization. For ex-ample, by targeting the monolignol biosynthetic pathway (Fig. 1),genetic down-regulation of caffeic acid 3-O-methyltransferase(COMT) (5), hydroxycinnamoyl-CoA shikimate hydroxycinnamoyltransferase (HCT) (6), cinnamoyl-CoA reductase (CCR) (7), fer-ulate 5-hydroxylase (F5H) (8, 9), caffeoyl shikimate esterase (CSE)(10), and cinnamyl alcohol dehydrogenase (CAD) (11) alter lignincontent and/or composition, thus reducing biomass recalcitranceand resulting in an enhancement of saccharification efficiency.Recently, introducing an exotic feruloyl-CoAmonolignol transferase

Significance

Deep eutectic solvents (DESs) have gained increasing attentiondue to their application-friendly properties, including universalsolvating capabilities and wide tunability. Additionally, ease ofsynthesis and broad availability from inexpensive chemicalcomponents could render DESs more versatile solvents forbiomass pretreatment, as compared with traditional ionic liq-uids. Because the long-term success of the biorefinery dependson the development of sustainable processes to convert lig-nocellulosics into biofuels, DESs derived from renewablesources such as lignin are highly desirable. We herein presentour innovative process that integrates the use of low-recalcitrantengineered biomass with its pretreatment using lignin-derivedDESs. The promising results described by near-theoretical sugaryield demonstrate the effectiveness of the integrated process,opening up opportunities toward a sustainable and circularbioeconomy.

Author contributions: K.H.K. and A.E. designed research; K.H.K., A.E., K.J., and C.G.Y.performed research; K.H.K., K.J., C.G.Y., C.S.K., and A.R. analyzed data; and K.H.K.,A.E., K.J., C.G.Y., C.S.K., and A.R. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.1To whom correspondence may be addressed. Email: kwanghokim@kist.re.kr.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1904636116/-/DCSupplemental.

Published online June 24, 2019.

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(FMT) gene in poplar resulted in the production of monolignolferulate ester conjugates that are incorporated into lignin (“Ziplignin”) (12). Structurally altered lignin with ester linkages intransgenics was found to be less recalcitrant, which led to im-proved cell wall digestibility and liberated more monosaccha-rides from biomass compared with the wild type (WT) (12, 13).Such strategic modification of lignin structure can certainly en-hance its overall processability by lowering the energy andchemicals required for biomass conversion (13). Herein, a pre-viously characterized CAD-deficient transgenic Arabidopsis linewas used as a feedstock. CAD is an enzyme that catalyzes the laststep of the lignin monomer biosynthetic pathway, convertingp-coumaryl aldehyde, coniferyl aldehyde, and sinapyl aldehydeto p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol,respectively (Fig. 1). Thus, in a CAD mutant expression system,cinnamaldehydes in the cell wall participate in the lignifica-tion process instead of conventional monolignols, producingmainly coniferyl aldehyde- and sinapyl aldehyde-derived ligninunits (14).Recently, deep eutectic solvents (DESs) have been introduced

to various biomass processes. DES is a solvent system formedfrom a eutectic mixture of Lewis or Brønsted acids and bases(15). Like ionic liquids (ILs), DESs exhibit desirable chemicaland physical properties such as low vapor pressure, tunability,and high thermal stability (16). Also, choline chloride (ChCl)-based DESs have drawn significant attention due to their abilityto solvate a wide range of compounds including metal oxides(17), CO2 (18), and lignin (19, 20). Furthermore, DESs are verysimple to synthesize, biodegradable, and cheaper to preparecompared with conventional ILs (15, 21). Although ChCl-based

DESs are typically hygroscopic, they are relatively unreactivewith water, making DESs versatile solvents (22).DESs are typically prepared from the complexation of qua-

ternary ammonium salts (e.g., ChCl) and hydrogen bond donors(HBDs) including amide (e.g., urea), alcohols (e.g., glycerol),and carboxylic acids (e.g., lactic acid and oxalic acid). ChClhas been widely used as a hydrogen bond acceptor due to itscapability of forming an intermolecular hydrogen bond, bio-compatibility, and relative inexpensiveness (23, 24). It is alsonoteworthy that ChCl can be produced biologically, although thecurrent industrial production of ChCl is via reaction of trime-thylamine hydrochloride with ethylene oxide (25). DESs havebeen widely employed as reagents for biomass pretreatment (19,26–28). More recently, renewable DESs have been preparedfrom lignin-derived phenolic compounds and proven to be ef-fective in biomass pretreatment, especially when pretreated withp-coumaric acid-derived DES (24). More importantly, a closed-loop biorefinery concept was also proposed by using lignin-derivedDESs (24).In this work, we report the integration of renewable DESs with

biomass genetic engineering to move closer toward achieving aclosed-loop biorefinery (Fig. 2). Previously, Socha et al. (29)described a closed-loop process using ILs synthesized with ligninand hemicellulose derived aromatic aldehydes. Down-regulationof CAD genes results in the formation of an abnormal and po-tentially valuable aldehyde-rich lignin (4, 14). As a proof-of-concept,biomass from CAD–down-regulated plants is pretreated with DESsderived from phenolic aldehydes. Then, the residual ligninrecovered after pretreatment can be further processed to ex-tract phenolic aldehydes for the production of renewable

Fig. 1. The main pathway involved in monolignol biosynthesis. The three monolignol precursors shown with a gray background accumulate upon down-regulation of the CAD genes. PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate-CoA ligase; HCT, hydroxycinnamoyl-CoAshikimate hydroxycinnamoyl transferase; C3H, coumarate 3-hydroxylase; CSE, caffeoyl shikimate esterase; CCR, cinnamoyl-CoA reductase; CCOMT, caffeoyl-CoA O-methyltransferase; F5H, ferulate 5-hydroxylase; COMT, caffeic acid O-methyltransferase; CAD, cinnamyl alcohol dehydrogenase.

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biomass-based DESs. The synthesized DESs can refill thepretreatment reactor while released monomeric sugars can beconverted into biofuels and other value-added products. It isbelieved that the integrated strategy introduced in the presentwork could provide future biorefineries with a closed-loopprocess to achieve a sustainable bioeconomy.

ResultsNMR Analysis of Lignin Structures in WT and CAD Mutant Biomass.We first characterized the lignin structures in Arabidopsis WTand the CAD double mutant (cad-c cad-d) (30) using 2D het-eronuclear single-quantum coherence spectroscopy (HSQC)NMR analysis. Fig. 3 shows partial HSQC spectra of ligninsextracted from the WT and CAD transgenic line. The HSQCspectra were divided broadly into aromatic (δH/δC 6.0–8.0/90–160), aliphatic (δH/δC 2.5–6.0/50–90), and aldehyde regions(δH/δC 9.0-10.0/184-196) and analyzed separately. In the aromaticregions of the lignin from the WT (Fig. 3A), typical guaiacyl (G)and syringyl (S) units and cinnamyl alcohol-end groups (sub-structure X1) were observed. In addition, naturally occurringaldehyde units [cinnamaldehydes (X2) and benzaldehydes (X3)]were also present in the WT lignin, although signal intensitieswere relatively lower. In contrast, the CADmutant showed a verylow level of typical G/S lignins (Fig. 3B). Instead, new signalsfrom uncommon guaiacyl (G′) and syringyl (S′) units derivedfrom polymerization of coniferyl aldehyde and sinapyl aldehydewere observed, respectively (31, 32). Also, correlations fromcinnamaldehydes (X2) and benzaldehydes (X3) appeared in thespectra from the CAD mutant. The recession of the normal Gand S units is obvious and the signals from G′ and S′ units aredominant, which is also demonstrated by the semiquantitativeanalysis. As can be seen in Fig. 3B, the HSQC contour intensityratio of the typical G and S units from the mutant only accountsfor 11% of the total lignin aromatics observed, while G′ and S′account for 80%. The S/G ratio in the WT was determined to be0.31, which was nearly identical to the S′/G′ and (S + S′)/(G + G′)ratios in the CAD mutant.In the aliphatic regions of the WT, as expected, typical lignin

side-chain hydrogen and carbon resonances were identified. Thisincludes β–O–4 aryl ether (I), β–5 phenylcoumaran (II), and β–βresinol (III) structures. As for the CAD mutant lignin isolates,the HSQC spectra differed markedly from the WT lignin. Thecommon lignin interunit linkages such as β–O–4, β–5, and β–β (I,II, and III) were not detected. The lower molecular weight oflignin in the CAD mutant compared with that of the WT (SIAppendix, Fig. S1) can be explained by this observation. In-terestingly, some unique aldehyde signals derived from the po-

lymerization of hydroxycinnamaldehyde appeared. As previouslyreported, the presence of the aldehydes-derived 8–O–4 (I′), 8–5(II′), and 8–8 (III′) substructures are evident from the analysis ofthe HSQC spectra (31, 32). The application of NMR spectros-copy clearly provides useful structural information on ligninsfrom both the WT and CAD mutant.

DES Synthesis from Lignin-Derived Phenolic Aldehydes. As an initialscreen test, vanillin (VAN) and 4-hydroxybenzaldehyde (HBA)were tested as an HBD at three different molar ratios (2:1, 1:1,and 1:2) with ChCl. As shown in SI Appendix, Fig. S2, both ChCl-VAN and ChCl-HBA formed clear and homogeneous DESs at1:2 molar ratio, respectively. A representation of the possiblecomplex of the ChCl-VAN and ChCl-HBA is given in Scheme 1.A strong hydrogen bond between ChCl and phenolic aldehydeformed under the synthesis conditions, which resulted in thedepressed melting point of the mixture (24). It has been dem-onstrated that HBDs capable of forming strong hydrogen bondswith a chloride ion can exhibit a significant depression of themelting point (33). As shown in Table 1, the eutectic point ofeach DES is significantly reduced. The decrease in the meltingpoint of ChCl-HBA was more dramatic than that of ChCl-VAN.It is believed that the phenolic hydroxyl group plays a crucial rolein forming a strong hydrogen bond between ChCl and the phe-nolic compound for DES formation. Based on the computationalanalysis, a chloride ion tends to strongly interact with the phe-nolic hydroxyl group upon the DES synthesis (SI Appendix, Fig.S3). The presence of a methoxy group in the ortho position waspreviously reported to interrupt the formation of a DES by sterichindrance (24), which resulted in relatively less depression ofmelting point of the eutectic mixture.

Biomass Pretreatment Using DESs and Enzymatic Saccharification.Two DESs synthesized from lignin-derived phenolic aldehydeswere tested for biomass pretreatment. The pretreatment of WTand CAD mutant was conducted at 80 °C, which is a relativelymild condition considering that the organosolv pretreatment ofbiomass is typically carried out at 100–250 °C (34). In addition tothe efficiency of DESs as pretreatment solvents, the impact ofmild DES pretreatment severity on plant cell wall modificationsis discussed.After pretreatment, the residual DES was washed out, and the

solid was recovered for compositional analysis to determine theefficacy of each DES in terms of lignin removal. Compositionalanalysis of pretreated samples indicates that DES pretreatmentof the CAD mutant removed a substantial amount of lignin (SIAppendix, Table S1). Lignin removals from WT biomass were

Fig. 2. A closed-loop biorefinery that can be achieved by integration of renewable deep eutectic solvent (DES) from lignin-derived phenolic aldehydes withengineered biomass.

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∼5% and 10% when pretreated with ChCl-VAN and ChCl-HBA, respectively, and those from CAD mutant significantlyincreased to 25% and 32%, respectively. Previously, the ligninremoval was reported to be 6.5% and 10.1% from corncob whenpretreated with ChCl-glycerol and ChCl-urea at 80 °C, re-

spectively (26). Clearly, more lignin was removed from biomassof the CAD mutant compared with WT, regardless of the DESused for pretreatment.It has been reported that lignins from CAD-deficient biomass

are more readily solubilized and extracted in alkaline conditions

Fig. 3. HSQC spectra of isolated lignins from Arabidopsis WT (A) and transgenic CAD mutant (B). Main structures of conventional lignin subunits (C) andunique lignin subunits from CAD mutant (D).

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due to their higher content of free-phenolic end groups (11, 35,36). Based on the principles of hydrogen bond interaction, DEScould provide a mild acid-base catalysis mechanism that initiatesthe cleavage of aryl-ether linkages, leading to lignin separationfrom the biomass (20). Also, lignin removal by DESs is possiblycorrelated to the hydrogen bond basicity of the constituent part(29, 37). In addition, gel permeation chromatography (GPC)analysis determined that the lignin macromolecule in the CADmutant sample has lower molecular weight compared with theWT (SI Appendix, Fig. S1). This also suggests that the reduceddegree of polymerization of lignins from CAD mutant could renderthem more prone to solubilization in DES (38), resulting in greaterremoval during pretreatment.Interestingly, in both samples, ChCl-HBA showed higher

pretreatment performance regarding lignin removal thanChCl-VAN. It is speculated that the interaction betweenChCl-HBA and lignin is relatively stronger than ChCl-VANand lignin complexes, which led to higher lignin removal frombiomass. The pretreated biomass was then subjected to en-zymatic hydrolysis to analyze the digestibility of polysaccha-rides. Fig. 4 shows the sugar yields after ChCl-VAN andChCl-HBA pretreatment of biomass from both WT andCAD mutant followed by enzymatic hydrolysis. A significantincrease in saccharification yield after DES pretreatment wasapparent in transgenic biomass. Yields of glucose releasedfrom WT were 10.0 and 10.5 wt% (based on initial dried bio-mass) when pretreated with ChCl-VAN and ChCl-HBA, re-spectively. Regardless of the DESs employed, glucose yieldsalmost doubled in the case of the CADmutant. The glucose yieldfrom biomass of the transgenic plant was 19.4 and 20.8 wt% withChCl-VAN and ChCl-HBA, respectively. The higher di-gestibility of the CAD mutant results from the strategic down-regulation of CAD genes involved in lignin biosynthesis. Thisresult is in accordance with previous saccharification resultsobtained from CAD–down-regulated poplar (+39.6–52.0%glucose and +34.2–63.8% xylose after 62.5 mM NaOH pre-treatment at 90 °C) (11), switchgrass (+15.0–35.0% glucoseafter 1.5% H2SO4 pretreatment at 121 °C) (39), alfalfa

(+16.0% total sugars after 1.5% H2SO4 pretreatment at130 °C) (40), and Brachypodium (+44.0–46.0% total sugarsafter 0.5 M NaOH pretreatment at 90 °C) (41). Clearly, theuse of DESs as reagents for biomass pretreatment under mildtemperature resulted in comparable amount of sugar withoutusing any conventional acid or base catalyst. Glucan conver-sions were also calculated based on the biomass compositionalanalysis (SI Appendix, Table S1). Taking into account theslightly higher glucan content in the raw CAD mutant biomass(∼11.7%), the glucan conversion to glucose was significantlyhigher from CAD mutant compared with the WT (70% vs. 40%with ChCl-HBA and 66% vs. 38% with ChCl-VAN). It is notedthat the enzyme mixture used in this work primarily consists ofcellulase, and that overall xylose yield was relatively low. How-ever, the yields of xylose from CAD mutant were considerablyhigher than those from WT. When pretreated with ChCl-VAN,the xylose yield increased from 2.0% (WT) to 7.9% (CAD mu-tant). Also, the pretreatment with ChCl-HBA yielded 8.5% xy-lose from the transgenic biomass, which is more than fourfoldincrease compared with WT. The discrepancy in xylose yieldcould be attributed to the enhanced enzyme accessibility to xylanchains in the CAD mutant because of higher lignin removal byDES-assisted pretreatment.

The Fate of Aldehyde-Rich Lignin. The CAD mutant released morefermentable sugars than the corresponding WT control whenpretreated with ChCl-VAN and ChCl-HBA, which could be at-tributed to its modified lignin. Thus, it was hypothesized thatDESs effectively remove CAD mutant lignin from the wholebiomass structure during pretreatment. To prove this hypothesis,solid residues recovered after pretreatment followed by enzymatichydrolysis were characterized by 2D HSQC NMR analysis. Asshown in SI Appendix, Fig. S4, each residual solid obtained from the2 DES pretreatments did not show peaks of phenolic aldehydes,which were observed in the raw material. SI Appendix, Fig. S4 shows2D HSQC NMR spectra of the residual lignin obtained from the 2DES pretreatments. Although the lignin residue from the ChCl-HBA pretreatment shows weak correlations from benzaldehydes

Scheme. 1. Possible complex formation in ChCl-VAN and ChCl-HBA.

Table 1. Two DESs used for biomass pretreatment

*From Sigma-Aldrich.†Determined in a convection oven; heated until clear and homogeneous liquid reforms with temperatureinterval of 5 °C and holding 30 min at each temperature.

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(X3, δH/δC 9.7/191), most of the cross-peaks from aldehydesdisappeared. This indicates that DESs effectively removedaldehyde-derived lignin substructures during pretreatment.Also, it reveals that the structures in the aldehyde-rich ligninfrom the CAD mutant are more chemically reactive than thosefound in normal lignin. To further confirm this hypothesis,density functional theory (DFT)-based computational studywas conducted to calculate a kinetic quantity of lignin structure(29). Model dimers typically found in lignin from WT (β–O–4)and CAD mutant (8–O–4) were used to understand thechemical reactivity. Fig. 5 illustrates the optimized geometryand electrophilicity index of β–O–4 and 8–O–4 dilignols. Anelectrophilicity index, which determines a quantitative classifi-cation of the global electrophilic nature of a molecule (42), wascalculated by the global hardness and electronic chemical po-tential. As shown in this figure, the electrophilicity index ofβ–O–4 is 1.88 eV, whereas that of the 8–O–4 model is 3.00 eV.It is obvious that 8–O–4 dimer has a higher electrophilicityindex, indicating that this structure is chemically more reactivethan the normal β–O–4 structure (43). The results from HSQCNMR analysis and computational study support the hypothesisthat lignin in CAD mutant biomass is more amenable to pro-cessing, thus being readily removed by DES-based pretreatmentin comparison with WT.

Recovery of Phenolic Aldehydes. As hypothesized earlier, DESswere successfully synthesized using phenolic aldehydes andproved to be effective for biomass pretreatment. Another ex-periment was carried out to demonstrate that lignin from CAD-deficient plants can supply more phenolic aldehydes for de-

signing DESs. Simply, both WT and CAD mutant biomass werehydrothermally depolymerized at 200 °C without catalyst. Theresulting liquid fraction was analyzed by gas chromatography andthree primary phenolic aldehydes were identified and quantified(see SI Appendix, Fig. S5 for gas chromatograms). Fig. 6 showsthe amount of three phenolic aldehydes obtained from WT andCAD mutant. As shown, the overall yield of phenolic aldehydesproduced from transgenic biomass was significantly higher thanthat from WT (+317%). For individual compounds, the yield ofvanillin was 1,345 μg/g lignin from WT, while the CAD mutantyielded 4,142 μg/g lignin. Additionally, CAD mutant producedover 3,000 μg/g lignin of syringyl aldehyde and coniferyl alde-hyde, respectively, whereas WT biomass yielded only a smallamount of those phenolic aldehydes. A simple hydrothermalcracking of lignin that has aldehyde-rich structures (i.e., as inCAD-deficient plants) was found to be effective at extractingphenolic aldehydes, although more in-depth tests are required tofind optimal reaction conditions to maximize yields.

DiscussionAltering the fundamental composition of lignin has proven to beeffective to make biomass more processable during pretreat-ments. The strategic down-regulation of CAD during lignifica-tion results in aldehyde-rich lignin units. As observed from theHSQC NMR analysis, the lignin extracted from CAD mutantshows a significant amount of aldehyde-derived substructures (8–O–4, 8–5, and 8–8) and cinnamaldehyde- and benzaldehyde-endunits. In this work, VAN and HBA were used as representativephenolic aldehydes because the CAD mutant has potential forproducing such compounds upon depolymerization. The pre-treatment of WT and CAD mutant using lignin-derived DESsrevealed that lignin removal was more significant from biomassof CAD-deficient plants, which resulted in higher sugar releaseupon enzymatic saccharification.The improved sugar yield obtained from CAD transgenic

biomass is attributed to several factors, which include (i) theslight decrease in total lignin amount in biomass cell walls re-ducing the overall physical barrier; (ii) significant structuralmodification in lignin that may loosen the interactions betweenlignin and carbohydrates, thus enhancing the accessibility ofenzymes (44–46); and (iii) the higher reactivity of aldehyde-richunits in lignin that are chemically more amenable. Consideringthat only a modest reduction of total lignin content was observedin the CAD transgenic biomass, the effect associated with lignincontent is likely to be marginal. Instead, the higher concentrationof phenolic aldehydes due to CAD down-regulation reduces theinterconnectivity within the macromolecular structure (45). Also,altering lignin composition and the corresponding lignin structures

Fig. 5. Optimized geometry of β–O–4 dimeric model compounds from WT(A), 8–O–4 dimeric model compounds from CAD mutant (B), and electro-philicity index as a descriptor of chemical reactivity.

Fig. 4. Sugar yield from raw biomass (A) and glucan conversion (B) for WTand CAD mutant pretreated with ChCl-VAN and ChCl-HBA, respectively.

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with increased reactivity are likely to reduce cell wall recalci-trance and to result in higher sugar yield.DESs synthesized from phenolic aldehydes were found to be

effective at biomass pretreatment. Interestingly, ChCl-HBA wasmore effective than ChCl-VAN for lignin removal during pre-treatment and sugar release upon saccharification, which is likelyattributed to the strong intermolecular interaction in ChCl-HBAcompared with ChCl-VAN. Although more supporting param-eters (e.g., Kamlet–Taft solvent parameters) are required tocompare the pretreatment efficiency of DESs, a better pre-treatment performance is anticipated for ChCl-HBA consideringits larger depression of melting point compared with that fromChCl-VAN. The strategic down-regulation of CAD during lig-nification produced biomass with aldehyde-rich lignin, whichhas the potential to serve as feedstock for the production ofaldehyde-derived DESs or other value-added chemicals. Thesynthesized DESs from phenolic aldehydes proved to be prom-ising solvents for biomass pretreatment resulting in a high yieldof fermentable sugars under mild conditions.There have been reports on enhanced saccharification from

CAD mutant biomasses (11, 39–41). It is challenging to directlycompare the results from these different studies with the presentstudy because the biomass is derived from different plant speciesthat have various levels of both lignin and CAD deficiency.However, it would be interesting to test and scale-up DES-basedpretreatment process with biomass from CAD-deficient bio-energy crops such as poplar (11), bm1 maize (47), bm6 sorghum(48), and switchgrass (49). It is worth noting that, unlike certainmutants affected in other steps of the lignin biosynthetic path-way, several CAD mutants do not show any visible phenotype orbiomass yield penalty. This is the case for greenhouse-grownswitchgrass (39, 49) and field-grown corn (50, 51), poplar (52),and sorghum (53), although genotype-dependent variations havebeen observed for the latter (54, 55). It is also important thatDES-based catalyst-free pretreatment of transgenic biomasscould lower chemical use and energy inputs typically required forthe production of intermediate sugars, which can be furtherprocessed into value-added products. Although the computa-tional study proved higher reactivity of CAD lignin, more in-depth studies are necessary to obtain a more comprehensiveunderstanding of the effect of compositional alteration of lignin.In actual chemical systems, many atoms concertedly interact withother compounds, contributing to the stability of the whole re-action system (56). Also, the effect of a solvent can be significantin electrophile interactions. Despite some technical challenges,this study clearly demonstrates the potential of developingsustainable biorefinery benefiting from looped production ofrenewable DESs.Taken together, down-regulation of CAD in biomass produces

lignin with aldehyde-rich units. This increases the overall sac-

charification efficiency and gives the potential for producingphenolic aldehyde-derived DESs, which can be used for biomasspretreatment. Integration of renewable DESs with biomass ge-netic engineering is a step closer toward a closed-loop bio-refinery and developing a sustainable energy future.

Materials and MethodsBiomass Material. Arabidopsis thaliana (ecotype Wassilewskija) seeds fromWT and CAD double mutant (cad-c and cad-d) (14) were germinated directlyon soil. Growing conditions were 14-h light/day at 100 μmol·m−2·s−1, 22 °C,and 55% humidity. For analyses, stems from mature senesced dried plantsharvested without siliques and leaves were ball-milled to a fine powderusing a Mixer Mill MM 400 (Retsch) and stainless-steel balls for 2 min. Allchemicals used in this work were purchased from Sigma-Aldrich and usedwithout any purification.

DES Synthesis. DESs used in this work were synthesized using vanillin (ChCl-VAN) and 4-hydroxybenzaldehyde (ChCl-HBA), respectively (Table 1). ChCland two hydrogen-bonding donor molecules were mixed in the three dif-ferent molar ratios (2:1, 1:1, and 1:2), respectively, and heated to 100 °C withcontinuous stirring until a clear liquid was formed. As shown in SI Appendix,Fig. S2, both ChCl-VAN and ChCl-HBA formed a homogeneous DES only atthe 1:2 (ChCl:HBD) molar ratio under the synthesis conditions tested in thiswork. 1H and 13C NMR spectra of VAN, HBA, ChCl, ChCl-VAN, and ChCl-HBAwere obtained on an AVANCE III HD 800-MHz instrument in D2O (SI Ap-pendix, Fig. S6).

ChCl-VAN. 1H NMR: 3.22 (s, 9H), 3.54 (t, 2H), 3.87 (m, 6H), 4.09 (m, 2H),6.97 (m, 2H), 7.34 (m, 2H), 7.44 (m, 2H), 9.62 (s, 2H); 13C NMR: 54.00 (3C),55.71 (1C), 55.90 (2C), 67.55 (1C), 111.30 (2C), 115.45 (2C), 127.54 (2C), 129.10(2C), 147.92 (2C), 152.31 (2C), 194.73 (2C).

ChCl-HBA. 1H NMR: 3.22 (s. 9H), 3.54 (t, 2H), 4.07 (m, 2H), 7.03 (m, 4H),7.88 (m, 4H), 9.73 (s, 2H); 13C NMR: 54.01 (3C), 55.71 (1C), 67.55 (1C), 116.15(4C), 128.84 (2C), 133.20 (4C), 162.67 (2C), 194.99 (2C).

Pretreatment and Enzymatic Saccharification. For DES-mediated biomasspretreatment, ∼5 wt% biomass solution was prepared by mixing 0.20 g ofbiomass with 3.80 g of each DES in a 20-mL pressure tube (Ace Glass). Al-though typical solids loading in biomass pretreatment is 10 wt% or greater,5 wt% solids loading was used in this work for the purpose of demonstratinga closed-loop biorefinery. Pretreatment of each biomass with two differentDESs was conducted in an oil bath at 80 °C for 3 h. After pretreatment,biomass was transferred to a 15-mL centrifuge tube and washed with 50 mL(10 mL × 5) of an ethanol/DI water mixture [2:1 (vol/vol)] to remove anyresidual DESs, and the solid fraction was completely dried for digestibilityassays. For saccharification of the pretreated biomass, enzymatic hydrolysiswas performed using a commercial enzyme blend Cellic CTec2 (Sigma-Aldrich). Briefly, 5 mL of 50 mM citrate buffer (pH 5.0) was added to thepretreated biomass with an enzyme dosage of 10 mg protein per grambiomass. Protein content of the enzyme mixture used in this study was de-termined from the ninhydrin-based assay (161.5 mg/mL). The hydrolysis wasconducted in a rotating hybridization oven at 50 °C for 72 h.

Analytical Methods. After saccharification, the hydrolysate was separatedfrom the substrate and filtered through a 0.45-μm syringe filter. Glucose andxylose release was measured using a YL 9100 high-performance liquidchromatography (Young-Lin) equipped with a refractive index detector anda Bio-Rad Aminex HPX-87H ion-exchange column. The mobile phase usedwas 4 mM H2SO4 at a constant flow rate of 0.6 mL/min, and the columntemperature was set at 60 °C.

To elucidate the structural characteristics of lignin, a 2D HSQC NMRanalysis was conducted with biomass samples. Before the NMR analysis,cellulolytic enzyme lignin (CEL) was isolated as described in the previous study(57). In brief, extractives-free biomass was prepared from the ball-milledsamples using Soxhlet extraction with a toluene-ethanol mixture [2:1 (vol/vol)]for 12 h followed by water extraction for an additional 8 h. The extractives-freesamples were ball-milled using a Retsch Ball Mill PM 100 in a ZrO2 jar (50-mLinternal volume) with 10 ZrO2 balls. The ball milling was conducted at 600 rpmat a frequency of 5 min with 5-min pauses in-between for 2.5 h. The ball-milledsamples were treated with a mixture of Cellic CTec2 in the sodium acetatebuffer solution (pH 4.8) at 50 °C and 200 rpm for 48 h. The residual solids werecentrifuged and treated with fresh enzyme (CTec2) one more time under thesame conditions. The residual solids after two-step enzymatic hydrolysis werewashed with DI water and freeze-dried. The dried solid residues were extractedwith dioxane [96% (vol/vol)] for 24 h. The extracted supernatant was collected

Fig. 6. The amount of phenolic aldehydes released from hydrothermaldepolymerization of WT and CAD mutant lignin.

13822 | www.pnas.org/cgi/doi/10.1073/pnas.1904636116 Kim et al.

and dioxane extraction was repeated. Dioxane and water in the collected su-pernatant were evaporated by rotary evaporator at 45 °C, and the recoveredlignins (CEL) were freeze-dried for the further analysis. The prepared ligninsamples were dissolved in a 5-mm NMR tube with DMSO-d6. The NMR analysiswas performed using a Bruker Avance III HD 500-MHz spectrometer equippedwith a N2 Cryoprobe (BBO 1H and 19F-5 mm) with the following acquisitionparameters: spectra width 12 ppm in F2 (1H) dimension with 1,024 time ofdomain (acquisition time, 85.2 ms), 220 ppm in F1 (13C) dimension with256 time of domain (acquisition time, 6.1 ms), a 1.0-s delay, a 1JC–H of 145 Hz,and 128 scans. HSQC experiments were carried out with a Bruker pulse se-quence (hsqcetgpspsi2.2). Assignment of the HSQC spectra is described else-where (11, 30).

Molecular-weight distribution of lignin was analyzed by GPC. The analysiswas conducted using the Agilent GPC SECurity 1200 system equipped withthree Walters Styragel columns (HR1, HR2, and HR6) and an UV detector(270 nm). The analysis was conducted with tetrahydrofuran as mobile phaseat 1.0 mL/min. Polystyrene standards were used for calibration. PolymerStandards Service WinGPC Unity software was used for data collection andprocessing. All lignin samples were acetylated using acetic anhydride andpyridine [1:1 (vol/vol)] at 60 °C for 2 h before analysis.

Hydrothermal Depolymerization. Hydrothermal depolymerization of WT andCAD mutant was performed in a 50-mL batch Parr reactor (Parr InstrumentCompany). For the test, 0.40 g of each biomass sample was placed in thereactor and 25 mL of deionized water was then added. The reactor wassealed, purged, and pressurized to 300 psi with He. The reaction mixture washeated and maintained at 200 °C for 1 h under continuous stirring. Afterreaction, the reactor was cooled in an ice bath. The resulting solution wastransferred into a separatory funnel with ethyl acetate (25 mL) added. Themixture was vigorously mixed, and the organic layer was separated. The waterphase was extracted once more with ethyl acetate (25 mL). The combinedethyl acetate fractions were dried over sodium sulfate, and the filtrate wasevaporated under reduced pressure. Approximately 50 mg of the productswere then dissolved in 2 mL of ethyl acetate and used for gas chromatog-raphy (GC) analysis. Identification and quantification of the phenolic alde-hydes were conducted using an Agilent 7820A GC equipped with 5975 massspectrometry detector. The capillary column used was an Agilent HP-Innowax (30 m × 0.25 mm × 0.25 μm). Injection temperature was 250 °Cand oven temperature was programmed to hold at 70 °C for 5 min, rampto 260 °C at 5 °C/min, and then hold for additional 5 min. In this work,three phenolic aldehydes were identified and quantified, namely vanillin,syringaldehyde, and coniferylaldehyde.

Computational Analysis. Considering the complex structure of lignin, com-putational studies typically use lignin model compounds to gain mechanisticunderstanding. Therefore, lignin model compounds with representative in-terunit linkages, such as a β–O–4 dimer, are widely used because it accountsfor 50–80% of whole interunit structures. In this work, a β–O–4 dimer [1-(4-hydroxyphenyl)-2-phenoxypropane-1,3-diol] from normal lignin and the

corresponding 8–O–4 dimer [3-(4-hydroxyphenyl)-2-phenoxyacrylaldehyde]from lignin of the CAD mutant were used for mechanistic study. For ge-ometry optimizations, two model dimers were analyzed using DFT withthe M06-2X hybrid exchange-correlation functional method and the 6-31+G(2d,2p) basis set in Gaussian 09 program (58). In this work, the DFT-based global reactivity descriptor, electrophilicity index (ω) was calculatedusing the electronegativity (χ) and the chemical hardness (η) to compare thereactivity of chemical system (29, 43, 56, 59). Equations for the calculation ofthe electronegativity (χ) and the hardness (η) are given as follows:

χ =−EHOMO + ELUMO

2, [1]

η=ELUMO − EHOMO

2, [2]

where EHOMO and ELUMO are the energies of the highest occupied and lowestunoccupied molecular orbitals, respectively. The electrophilicity index isdefined as follows:

ω=χ2

2η. [3]

ACKNOWLEDGMENTS. This work is supported, in part, by the Korea Instituteof Science and Technology–The University of British Columbia Biorefineryon-site laboratory project. In addition, support was provided in part, byUT-Battelle, LLC, under Contract DE-AC05-00OR22725 with the U.S. Depart-ment of Energy. This study was supported and performed, in part, as part ofthe Center for Bioenergy Innovation (CBI). CBI is a U.S. Department of En-ergy (DOE) Bioenergy Research Centers supported by the Office of Biologicaland Environmental Research in the DOE Office of Science. This work waspart of the DOE Joint BioEnergy Institute (http://www.jbei.org) supported bythe U.S. DOE, Office of Science, Office of Biological and Environmental Re-search, through Contract DE-AC02-05CH11231 between Lawrence BerkeleyNational Laboratory and the U.S. DOE. The publisher, by accepting the arti-cle for publication, acknowledges that the U.S. Government retains a non-exclusive, paid-up, irrevocable, worldwide license to reproduce the publishedform of this manuscript, or allow others to do so, for U.S. Government pur-poses. The DOEwill provide public access to these results of federally sponsoredresearch in accord with the DOE Public Access Plan (https://www.energy.gov/downloads/doe-public-access-plan). The views and opinions of the authorsexpressed herein do not necessarily state or reflect those of the U.S. Govern-ment or any agency thereof. The views and opinions of the authors expressedherein do not necessarily state or reflect those of the U.S. Government or anyagency thereof. Neither the U.S. Government nor any agency thereof, nor anyof their employees, makes any warranty, expressed or implied, or assumesany legal liability or responsibility for the accuracy, completeness, or useful-ness of any information, apparatus, product, or process disclosed, or repre-sents that its use would not infringe privately owned rights. We thank JieWu of the Department of Wood Science at the University of British Columbiafor measuring the protein content. The NIH Shared Instrumentation Grant1S10OD012254 for the 800-MHz NMR spectrometer is also acknowledged.

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