Ammonia Pretreatment of Corn Stover Enables ... - ACS Publications

Research Article pubs.acs.org/journal/ascecg

Ammonia Pretreatment of Corn Stover Enables Facile Lignin Extraction Ashutosh Mittal,*,† Rui Katahira,‡ Bryon S. Donohoe,† Sivakumar Pattathil,§,∥ Sindhu Kandemkavil,§ Michelle L. Reed,‡ Mary J. Biddy,‡ and Gregg T. Beckham*,‡ Biosciences Center and ‡National Bioenergy Center, National Renewable Energy Laboratory, 15013 Denver West Parkway, Golden, Colorado 80401, United States § BioEnergy Science Center and Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens, Georgia 30602, United States Downloaded via KAOHSIUNG MEDICAL UNIV on July 1, 2018 at 13:48:46 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

S Supporting Information *

ABSTRACT: Thermochemical pretreatment of lignocellulose is often employed to render polysaccharides more digestible by carbohydrate-active enzymes to maximize sugar yields. The fate of lignin during pretreatment, however, is highly dependent on the chemistry employed and must be considered in cases where lignin valorization is targeted alongside sugar conversionan important feature of future biorefinery development. Here, a two-step process is demonstrated in which anhydrous ammonia (AA) pretreatment is followed by mild NaOH extraction on corn stover to solubilize and fractionate lignin. As known, AA pretreatment simultaneously alters the structure of cellulose with enhanced digestibility while redistributing lignin. The AA-pretreated residue is then extracted with dilute NaOH at mild conditions to maximize lignin separation, resulting in a digestible carbohydrate-rich solid fraction and a solubilized lignin stream. Lignin removal of more than 65% with over 84% carbohydrate retention is achieved after mild NaOH extraction of AA-pretreated corn stover with 0.1 M NaOH at 25 °C. Two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy of the AA-pretreated residue shows that ammonolysis of ester bonds occurs to partially liberate hydroxycinnamic acids, and the AA-pretreated/NaOH-extracted residue exhibits a global reduction of all lignin moieties caused by reduced lignin content. A significant reduction (∼70%) in the weight-average molecular weight (Mw) of extracted lignin is also achieved. Imaging of AA-pretreated/NaOH extracted residues show extensive delamination and disappearance of coalesced lignin globules from within the secondary cell walls. Glycome profiling analyses demonstrates ultrastructural level cell wall modifications induced by AA pretreatment and NaOH extraction, resulting in enhanced extractability of hemicellulosic glycans, indicating enhanced polysaccharide accessibility. The glucose and xylose yields from enzymatic hydrolysis of AA-pretreated/NaOH-extracted corn stover were higher by ∼80% and ∼60%, respectively, compared to untreated corn stover at 1% solids loadings. For digestions at 20% solids, a benefit of NaOH extraction is realized in achieving ∼150 g/L of total monomeric sugars (glucose, xylose, and arabinose) in the enzymatic hydrolysates from AA-pretreated/NaOH-extracted corn stover. Overall, this process enables facile lignin extraction in tandem with a leading thermochemical pretreatment approach, demonstrating excellent retention of highly digestible polysaccharides in the solid phase and a highly depolymerized, soluble lignin-rich stream. KEYWORDS: Ligninocellulose, Fractionation, Ammonia, Pretreatment, Biofuels

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INTRODUCTION

area, degree of polymerization, and porosity influence the rate and extent of saccharification.3,4 Chemical pretreatment is thus

For biochemical conversion of biomass to fuels and chemicals, thermochemical pretreatment is commonly utilized to overcome biomass recalcitrance and render polysaccharides more accessible to cellulolytic and hemicellulolytic enzymes for producing sugars.1,2 The crystalline morphology of cellulose coupled with covalent cross-linking between carbohydrates and lignin provides a defensive structural framework to plant cell walls, consequently making it resistant to biological deconstruction via lytic enzymes. Moreover, the structural properties of the biomass substrate, such as cellulose crystallinity, surface © 2017 American Chemical Society

often viewed as an essential step required to modify biomass structure to enhance cellulose accessibility to cellulase enzymes thereby increasing fermentable sugar yields. Multiple types of chemistries, including dilute acid, alkaline, ammonia, and Received: November 29, 2016 Revised: January 22, 2017 Published: February 9, 2017 2544

DOI: 10.1021/acssuschemeng.6b02892 ACS Sustainable Chem. Eng. 2017, 5, 2544−2561

Research Article

ACS Sustainable Chemistry & Engineering

ammonolysis.40,41 Lignin−carbohydrate complex (LCC) ester/ether linkages between lignin and hemicellulose moieties are considered to pose a major obstruction to saccharification.40 The cleavage of these ester linkages results in the formation of phenolic amides such as coumaroyl and feruloyl amides,42 and the hydroxycinnamic acids p-coumaric and ferulic acid41 that are extracted under mild process conditions thereby enabling selective extraction of up to 50% of the lignin from lignocellulosic biomass. Based on the excellent solvent and extraction properties of anhydrous liquid ammonia, it has been used to extract biomass components (lignin and hemicellulose) from lignocellulosic biomass for over six decades.28,43 Yan and Purves extracted lignin from different wood species under various operational conditions varying extraction temperature from 25 to 100 °C, extraction time from 3 to 65 h and liquid ammonia to biomass ratio of 15:1 under completely dry and with 7% moisture in biomass.28 They reported 25−30% and 52% of lignin removal from various hardwoods and rye straw, respectively. Bludworth and Knopf extracted yellow poplar wood with supercritical ammonia−water mixtures and were able to extract about 70% of the lignin, 50% of hemicellulose, and 15% of the cellulose initially present in the wood in 1 h using 20 wt % water in ammonia at 272 atm and 200 °C.44 In ammonia pretreatment, whether biomass components are extracted or not depend on how ammonia is recovered from the reactor. If ammonia is recovered in the gaseous phase from the head space of the reactor, it results in the pretreated substrate with altered structure (cellulose IIII) but with minimal change in biomass composition due to reprecipitation of the solubilized fraction on pretreated residue as ammonia is evaporated. Alternatively, ammonia can be extracted from the reactor in the liquid phase by applying external pressure exceeding the vapor pressure of ammonia at a given temperature resulting in the extraction of solubilized biomass components along with the recovered liquid ammonia. Recently, Sousa and co-workers extracted corn stover by implementing this approach and were able to extract up to 45% of the lignin with near-quantitative retention of all the polysaccharides utilizing a single-stage extractive ammonia process.19 In the present work, an alternative two-step process is explored wherein a combination of anhydrous ammonia (AA) pretreatment followed by a mild NaOH extraction is performed on corn stover with the objective of maximizing separation of lignin from carbohydrate fraction (cellulose and hemicellulose) to utilize each subsequent fraction more effectively for the production of fuels and chemicals. In the first step, AA pretreatment is investigated at various conditions utilizing the ability of liquid ammonia to simultaneously alter the structure of biomass to a form with enhanced cellulose accessibility (cellulose IIII) while promoting relocalization of lignin in the pretreated residue. In the second step, the AA pretreated residue is extracted with sodium hydroxide under mild conditions to maximize separation of lignin from biomass resulting in a carbohydrate rich fraction suitable for efficient enzymatic hydrolysis. The advantages of utilizing a two-step process are (1) simplifying ammonia recovery and thus improving its recyclability and (2) performing solid−liquid separation under ambient conditions. To assess the potential utility of this pretreatment process, the mass closure for each of the three primary components, lignin, hemicellulose, and cellulose at each pretreatment step are examined followed by the characterization of the physical and chemical properties of

hydrothermal pretreatments, along with many others, have been developed over many decades of research.1,5 Lignin valorization is increasingly being recognized as an essential component of biorefineries to achieve economic viability.6−8 For example, Vardon et al. predicted that lignin conversion to adipic acid could contribute nearly a $2/gasolinegallon equivalent reduction in the minimum fuel selling price to a biorefinery producing hydrocarbon biofuels from biomass sugars, and the conversion technology to accomplish this has been recently proposed.9−11 However, as lignin composition varies as a function of feedstock and is often quite reactive in the harsh chemical environments of pretreatment, consideration must be given to the combined effect of both feedstock and pretreatment chemistry on lignin vis-à-vis the anticipated lignin conversion approach. In this vein, dilute acid, hydrothermal, and acid−based organosolv pretreatments, some of which are now being deployed at the industrial scale, have long been known to produce more condensed lignin via aryl-ether bond cleavage and carbon−carbon bond formation due to coupling between reactive intermediates.12,13 This in turn produces lignin that is more difficult to depolymerize to monomeric compounds, thus hindering valorization strategies that rely on lignin depolymerization. Instead, alkaline-based pretreatments offer the potential to solubilize and redistribute lignin, or even extract lignin directly when using sodium hydroxide or other similar base catalysts.14,15 Besides the use of strong base catalysts, high pH pretreatment with weak bases has also been examined in detail. In particular, ammonia-based pretreatments16−19 have been extensively used to redistribute lignin and alter the molecular structure of biomass to make it more amenable to enzymatic hydrolysis. The main benefits of using ammonia over other alkaline catalysts in biomass pretreatment are rapidity and uniformity of biomass swelling with liquid ammonia and cost effectiveness in ammonia recyclability making ammonia-based pretreatment to be economically attractive.18,20,21 AFEX pretreatment,22−24 which utilizes ammonia as a catalyst, is among one of the few pretreatments that has been scaled up and successfully demonstrated at up to a 1 t scale.25,26 Liquid ammonia, which is a much more effective solvent than water for many organic substances,27,28 has been known and used for over 80 years as a swelling agent of cellulose as well as for altering cellulose morphology.29,30 Ammonia penetrates both amorphous and crystalline regions of cellulose and interacts with the hydroxyl groups in cellulose resulting in the swelling of cellulose by replacing OH···O hydrogen bonds with OH···N bonds to form a disordered structure called ammonia− cellulose complex.29−33 As the ammonia is allowed to evaporate, rearrangement of hydrogen bonds within the cellulose lattice occurs resulting in a completely new structure designated as cellulose IIII.34−37 Igarashi et al.38 and Chundawat et al.39 both reported that cellulose IIII exhibits enhanced hydrolysis by the reducing-end specific cellobiohydrolase Cel7A producing cellobiose at rates more than 5 times higher than from cellulose I. These authors concluded that supercritical ammonia treatment activates crystalline cellulose for hydrolysis by Cel7A. It has been postulated, therefore, that cellulose IIII should have enhanced cellulose accessibility and chemical reactivity.38,39 Besides altering the structure of cellulose I to the more reactive cellulose IIII form, treatment of biomass with ammonia also results in the cleavage of ester-linked ferulate and coumarate linkages that exist in grass cell walls via 2545


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Figure 1. (A) Yield and (B) composition of the untreated corn stover (raw CS) and AA pretreated solids are shown as a function of pretreatment temperature. AA pretreatment was conducted at 25−130 °C with ammonia loading of 3 g anhydrous NH3/g dry corn stover. The error bars shown are the standard deviation for the analyses conducted in triplicate.

Figure 2. Yield of AA-pretreated solids followed by NaOH extraction shown as a function of AA pretreatment temperature. AA pretreatment was conducted at 25−130 °C with ammonia loading of 3 g anhydrous NH3/g dry corn stover. NaOH extractions were conducted with 0.1 M NaOH at 10% solids loading at (A) 25 and (B) 75 °C. The error bars show the standard deviation for the analyses conducted in triplicate.

residual pretreated solids obtained with AA pretreatment conducted at various temperatures are shown in Figure 1. The yield data show that the amounts of solids recovered after pretreatment ranged between 94 and 96% with no effect of pretreatment temperature on solids yield (Figure 1A). The effects of AA pretreatment on the composition of the pretreated solid residue as a function of pretreatment temperature are shown in Figure 1B. The compositional analysis of the untreated corn stover is shown for reference, which contained 36.3 ± 2.3% glucan, 34.2 ± 4.0% hemicelluloses (26.4% xylan, 5.3% arabinan, and 2.5% acetyl), 18.5 ± 1.6% lignin, and 11.3% others (5.5% ash and 5.8% water extractives). As can be seen in Figure 1B, all the major constituents of AA-pretreated solids such as glucan, xylan, lignin, arabinan, and galactan remained almost unchanged for pretreatment up to 60 °C. However, for pretreatment above 100 °C, a slight decrease in xylan and lignin content was observed which suggests that under these conditions a small fraction of xylan and lignin are removed when ammonia is released from the reactor. Acetate is almost completely removed even at the lowest pretreatment temperature of 25

the pretreated residue and extracted lignin-rich stream with various techniques.

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RESULTS AND DISCUSSION Anhydrous Ammonia (AA) Pretreatment. The amount of mass solubilized after the AA pretreatment was denoted by Mrem whereas the degree of solid phase recovery was denoted by SY, defined as solid residue yield (grams solid recovered after pretreatment per 100 g of untreated oven dry corn stover) in eq 1: SY = 100 − M rem

(1)

Where, Mrem is the total mass solubilized or removed as described in eq 2: M rem =

M rcs − M pcs M rcs

× 100%

(2)

Where Mrcs is the OD mass of the untreated raw corn stover and Mpcs is the OD mass of the pretreated corn stover. The residual dry solids yield and compositional analyses of the 2546


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Figure 3. Percent yields of lignin, hemicellulose, and cellulose remaining in the AA-pretreated/NaOH-extracted corn stover solids shown as a function of NaOH loading and extraction temperature. (A) Effect of NaOH concentration at extraction conducted at 75 °C. (B) Effect of NaOH extraction temperature at NaOH loading of 0.1 M NaOH. AA pretreatment was conducted at 130 °C with ammonia loading of 3 g anhydrous NH3/ g dry corn stover. The control sample represents untreated corn stover extracted with 0.1 M NaOH at 75 °C. The error bars shown are the standard deviation for the analyses conducted in triplicate.

°C resulting from the cleavage of ester bonds under alkaline conditions. The extractives fraction increases with increasing pretreatment temperature reaching 10.6 and 19.4% for pretreatment at 100 and 130 °C, respectively. NaOH Extraction of AA-Pretreated Corn Stover. Since the primary objective of this work was to evaluate separation of lignin from the carbohydrate fraction (cellulose and hemicellulose) of corn stover to utilize each fraction more effectively, the AA-pretreated residue (obtained at various pretreatment temperatures ranging from 25−130 °C) was extracted with dilute NaOH at mild conditions to separate lignin from carbohydrate fraction. Sodium hydroxide extractions were conducted with 0.025−0.1 M NaOH at 10% solids loading at temperatures ranging from 25−75 °C for 2 h. Additionally, AA pretreated corn stover obtained at 130 °C was also extracted with water at 75 °C to evaluate and compare the lignin separation efficacy of this process where only water is used as an extracting solvent. The residual dry solids yield of the extracted solids obtained after AA pretreatment followed by NaOH extraction at 25 and 75 °C are calculated using eq 1 and are provided in Figure 2. The yield of AA-pretreated corn stover obtained at 130 °C (with no NaOH extraction) is shown for reference. The yield data show that the amount of solids recovered after NaOH extraction decreased steadily and ranged between 86−74% and 83−68% for the extractions conducted with 0.1 M NaOH at 25 and 75 °C, respectively, as the temperature of AA pretreatment is increased from 25 to 130 °C. The data clearly indicate that the AA pretreatment temperature has more influence on the solids yields than the NaOH extraction temperature. The data also show that the yield obtained after extraction with water was 77% compared to 68% obtained with the 0.1 M NaOH extraction. This suggests that water is quite effective in extracting a substantial fraction of extractable material, though not to the same extent as the NaOH-based extractions. Component Yield in NaOH Extracted Solids. To separate the effect of AA pretreatment from the effect of NaOH extraction on component fractionation, NaOH extraction was also conducted on untreated corn stover with 0.1 M NaOH at 75 °C. NaOH extraction of untreated corn stover resulted in lignin removal of 25% while cellulose and

hemicellulose removal were 9% and 14%, respectively (see control in Figure 3). The effects of NaOH loading (Figure 3A) and extraction temperature (Figure 3B) on the solubilization of lignin, hemicellulose, and cellulose on AA-pretreated corn stover (obtained at AA-pretreatment of 130 °C) are shown in Figure 3. The dashed lines are shown to evaluate the AApretreatment and NaOH-extraction conditions where 75% and 35% fraction of hemicellulose (red) and lignin (green) were retained in the AA-pretreated/NaOH-extracted residue. Both lignin and hemicellulose removal increased with increasing NaOH loading (Figure 3A) and extraction temperature (Figure 3B). Figure 3A shows that at 0.025 M NaOH loading, lignin removal was 47% while cellulose and hemicellulose removal were 6% and 30%, respectively. At 0.05 M NaOH loading, lignin removal was 54% while cellulose and hemicellulose removal were 6% and 33%, respectively. At further increasing NaOH loading to 0.1 M, lignin removal was 70% while cellulose and hemicellulose removal were 9% and 35%, respectively. For the extraction conducted with water at 75 °C, lignin removal was 27% while no cellulose removal was observed and hemicellulose removal was 37%. It is interesting to see up to 27% of lignin removal with water extraction, however, it also results in the removal of large amount of hemicellulose fraction (37%) while leaving the majority of lignin still present in the extracted residue. Clearly, NaOHbased extraction is much more effective in extracting lignin than water extraction. Figure 3B shows that for extraction conducted at 25 °C, lignin removal was 65% while cellulose and hemicellulose removal were 6% and 25%, respectively. For extraction conducted at 50 °C, lignin removal was 67% while cellulose and hemicellulose removal were 8% and 33%, respectively. Upon further increasing the extraction temperature to 75 °C, lignin removal increased to 70% while cellulose and hemicellulose removal were 9% and 35%, respectively. These results clearly show that lignin extraction increased linearly with increasing NaOH loading resulting in 70% of lignin removal with 0.1 M NaOH at 75 °C whereas no significant effect of extraction temperature was observed on lignin extraction. Next, the effect of AA pretreatment temperature on the solubilization of lignin and retention of hemicellulose and 2547


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Figure 4. Percent yields of lignin, hemicellulose, and cellulose remaining in the AA-pretreated/NaOH extracted corn stover solids shown as a function of AA pretreatment temperature. NaOH extractions were conducted at 0.1 M NaOH at (A) 25 and (B) 75 °C. AA pretreatment was conducted at 25−130 °C with ammonia loading of 3 g anhydrous NH3/g dry corn stover. The error bars shown are the standard deviation for the analyses conducted in triplicate.

syringyl and guaiacyl units and their ratio also changed only slightly. The Cα peaks in ferulate and p-coumarate units at δc/ δH 144−145/7.3−7.8 disappeared and corresponding amides (feruloyl amide and p-coumaryl amide) peaks appeared at δc/ δH 139−141/7.3−7.6, indicating that most of ester groups in ferulate and p-coumarate remaining in the AA-pretreated solid residue were substituted to an amino group (−NH2) under AA pretreatment at 130 °C. In the solid residues obtained with AA pretreatment followed by NaOH extraction at 25 °C (Figure 5C), the amount of β-O-4 units decreased dramatically. Feruloyl amide peaks disappeared although p-coumaryl amide peaks still remained to some extent. This is probably because most of feruloyl amide and components of p-coumaryl amide were removed to NaOH aqueous soluble fraction. In the solid residues obtained with AA pretreatment followed by NaOH extraction at 75 °C (Figure 5D), p-coumaryl amide peaks also remained after 0.1 M NaOH extraction at 75 °C. This is probably because in corn stover lignin the p-coumarate units are more abundant and relatively more stable than the ferulate units in the alkaline conditions that prevail under the extraction temperatures of 25−75 °C investigated here, and possibly due to a more condensed structure in the p-coumarate units than in the ferulate units. Interestingly, most of the syringyl and guaiacyl units still remain in the solid residue obtained after NaOH extraction at 25 °C; however, a large portion of the syringyl and guaiacyl units were solubilized in the solid residue obtained after extraction at 75 °C. Overall, NMR spectroscopy results of the AA-pretreated residue shows that ammonolysis of ester bonds occurs to partially liberate the hydroxycinnamic acids, whereas the AA-pretreated/NaOH-extracted residue exhibited reduction of all lignin moieties caused by a significant reduction (60−65%) in the lignin content of this residue. It is known that some pretreatment chemistries produce more condensed lignin via aryl-ether bond cleavage and carbon−carbon bond formation due to coupling between reactive intermediates.12,13 This in turn produces lignin that is more difficult to depolymerize to monomeric compounds, thus preventing effective valorization strategies that rely on lignin depolymerization. In this vein, mild NaOH extraction of AApretreated corn stover is performed with the objective of maximizing separation of lignin from the carbohydrates. To

cellulose followed by NaOH extraction (0.1 M NaOH) was evaluated at 25 and 75 °C (Figure 4). Figure 4A shows that for AA pretreatment conducted at 25 °C followed by NaOH extraction, lignin removal was 26% while cellulose and hemicellulose removal were 8% and 13%, respectively. At AA pretreatment conducted at 60 °C, lignin removal was 28% while cellulose and hemicellulose removal were 6% and 9%, respectively. For AA pretreatment conducted at 100 °C, lignin removal was 56% while cellulose and hemicellulose removal were 6% and 19%, respectively. Upon further increasing AA pretreatment temperature to 130 °C, lignin removal was 65% while cellulose and hemicellulose removal were 9% and 25%, respectively. Figure 4B shows a similar trend of increasing lignin and hemicellulose solubilization with increasing AA pretreatment temperature with only a modest increase in the extraction yield of lignin after increasing NaOH extraction temperature to 75 °C. It is important to note that increasing the NaOH extraction from 25 to 75 °C results in only a 5% increase (65−70%) in lignin extraction but also results in increased solubilization of cellulose and hemicellulose from 6% to 9% and 25% to 35%, respectively. It is evident from Figures 3 and 4 that AA pretreatment temperature plays a significant role in the effectiveness of lignin extraction whereas NaOH loading and extraction temperature provide flexibility to tailor the process and optimize the lignin extraction and carbohydrate retention efficiency. 2D NMR. To understand chemical structural changes in lignin from AA pretreatment and AA pretreatment followed by NaOH extraction, pretreated solid residues were analyzed by 2D-NMR. Heteronuclear single quantum coherence (HSQC) spectra of whole corn stover, AA-pretreated (130 °C), and AApretreated/NaOH-extracted solids obtained at 25 and 75 °C are shown in Figure 5. In the untreated corn stover (Figure 5A), the β-O-4 unit is predominant with the presence of other linkages such as resinol and phenylcoumaran in small amounts. The ratio of syringyl to guaiacyl units is nearly 1.0, and ferulate and p-coumarate units are also present. After AA pretreatment (Figure 5B), the β-O-4 units decrease only slightly, indicating that aryl-ether bonds mostly remain intact after AA treatment. The same phenomenon was observed during AFEX and extractive ammonia pretreatments.19,45 The peak area of both 2548


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Figure 5. HSQC NMR spectra of (A) untreated raw corn stover, (B) AA-pretreated corn stover at 130 °C for 1 h, (C) AA-pretreated/NaOH extracted corn stover (0.1 M-NaOH, 25 °C), and (D) AA-pretreated/NaOH extracted corn stover (0.1 M-NaOH, 75 °C).

that aryl-ether bonds mostly remain intact in soluble lignin obtained after extraction of AA-pretreated corn stover conducted with either dilute NaOH or water alone at mild conditions. As the β-O-4 linkage is the most abundant linkage in lignin, and since the lignin extracted with the process demonstrated here preserves these linkages, it suggests that this lignin is suitable for depolymerization to monomeric compounds, thus enabling effective valorization strategies. GPC Analysis of Soluble Lignin Removed during AA Treatment. The properties of the lignin fraction, especially molecular weight (MW) distribution, are also of keen interest to gauge its potential to be converted into target products. As such, GPC was conducted on the freeze-dried soluble fractions obtained after H2O or NaOH extractions from AA-pretreated corn stover at 130 °C. The molecular weight distributions of the lignin (which is the primary biomass component that absorbs in the UV range) in the soluble fractions and ballmilled lignin are summarized in Figure 7 and their weightaverage MW (Mw) and polydispersity (PD) results are given in Table 1. The GPC profile in the control sample (ball-milled lignin) exhibited a minor peak between ∼200−600 Da, which is

better understand chemical structural changes that occur in lignin extracted with either dilute NaOH or water alone at mild conditions, 2D-NMR was performed on soluble lignin obtained in H2O extractives obtained at 75 °C and NaOH extractives obtained with 0.1 M NaOH at 25 and 75 °C from AApretreated corn stover (130 °C), and these are compared with ball-milled lignin from corn stover (Figure 6). There were specific peaks of ferulate, p-coumarate, and tricin in native corn stover lignin (Figure 6A). After ammonia treatment, however, these peaks disappeared and feruloyl amide and p-coumaryl amide peaks appeared (Figure 6B). In the 0.1 M-NaOH extractives obtained at both 25 and 75 °C (Figure 6C and D), feruloyl amide peaks as well as p-coumaryl amide peak were observed, although no feruloyl amide peaks appeared in the solid residue obtained after extraction with 0.1 M-NaOH (Figure 5C and D). This indicates that feruloyl amide unit was more fully solubilized in the base condition than the p-coumaryl amide unit. Peaks of Cα and Cβ in S type β-O-4 unit (Aα and Aβ) also appeared at δc/δH 70/5.0 and 86/4.2 suggesting that a large portion of β-O-4 linkage was not cleaved but solubilized after both ammonia treatment and NaOH extraction, indicating 2549


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Figure 6. HSQC NMR spectra of (A) ball milled lignin from corn stover, (B) H2O extractives from AA-pretreated corn stover at 130 °C for 1 h, (C) 0.1 M-NaOH extractives at 25 °C from AA-pretreated corn stover, and (D) 0.1 M-NaOH extractives at 75 °C from AA-pretreated corn stover.

Table 1. Impact of AA Pretreatment and NaOH Extraction on the Molecular Weight of Extracted Lignin lignin fraction

Mn

Mw

PD

(A) CS ball-milled lignin (B) H2O extracted at 75 °C (C) NaOH extracted at 25 °C (D) NaOH extracted at 75 °C

1700 420 470 490

6400 780 1600 1800

3.8 1.9 3.3 3.6

MW (HMW) lignin. Both H2O and NaOH extracted fractions exhibited a bimodal distribution in GPC profile: a highintensity narrow peak between ∼150−400 Da representing a significant increase in monomeric and LMW species, followed by a low-intensity peak between ∼500−10 000 Da representing a dramatic reduction in HMW lignin when compared to the control sample. The general trend for both MW and PD for extracted lignin is 0.1 M-NaOH/75 °C > 0.1 M-NaOH/25 °C > H2O/75 °C (Table 1). The H2O-extracted lignin exhibited the lowest Mw = 780 Da and a PD of 1.9 with large monomer/ dimer peaks between 250−450 Da and a small portion of products between 500−4000 Da. The NaOH-extracted lignin obtained at both 25 and 75 °C exhibited a roughly similar trend

Figure 7. GPC chromatograms of corn stover ball milled lignin, H2O extractives from AA-pretreated CS, 0.1 M-NaOH extractives at 25 °C from AA-pretreated CS, and 0.1 M-NaOH extractives at 75 °C from AA-pretreated CS. AA pretreatment was conducted at 130 °C for 1 h.

attributed to monomeric and low MW (LMW) species, followed by a wide peak up to 40 000 Da representing high 2550


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ACS Sustainable Chemistry & Engineering to that of H2O-extracted lignin with a difference of an additional minor monomer/dimer peak at 100−250 Da and a relatively larger portion of products between 500−10 000 Da. Clearly, the presence of LMW compounds in the extracted fraction shows the effectiveness of this process in depolymerizing HMW lignin into monomeric, dimeric, and oligomeric lignin species in the aqueous fraction which could be converted to value added products. Crystallinity of Pretreated Solids. X-ray diffraction was used to examine and compare the changes in the cellulose crystallinity index (CrI) of untreated, AA-pretreated, and AApretreated/NaOH-extracted corn stover samples. Figure 8 shows the X-ray diffractograms for corn stover treated with AA under various experimental conditions (i.e., AA pretreatment temperature, NaOH loading, and extraction temperature). The diffractograms show that under all conditions cellulose I was completely converted to cellulose IIII; however, the treatment conditions had an effect on the crystallinity of the cellulose IIII. In Figure 8, the distinction between cellulose I and cellulose IIII can be made from the 200 peak which shifts from a 2θ value of 23 to 21° as cellulose I is converted to cellulose IIII at all treatment temperatures. It is interesting to note that after NaOH extraction of AA-pretreated samples obtained at AA pretreatment of 25 and 60 °C, a fraction of cellulose IIII transitioned to cellulose I which is consistent with earlier findings.33 However, the cellulose IIII formed at higher AA pretreatment temperatures, i.e., at above 100 °C is much more stable and retained its crystalline form after NaOH extraction (Figure 8C) which is consistent with an earlier report.37 The data on the degree of crystallinity for untreated, AApretreated, and AA-pretreated/NaOH-extracted corn stover samples are provided in Table S1, Supporting Information (SI) which is calculated using eq 3. From Table S1, it can be noted that increasing the final temperature to which corn stover was heated during ammonia treatment resulted in cellulose IIII samples with higher CrI. For example, liquid ammonia treatment at a final temperature of 130 °C for 1 h produced a cellulose IIII sample with a CrI of 54%, which is much higher than the CrI of the starting cellulose I (36%). It is known that addition of AA to cellulose I (biomass) results in the formation of less ordered ammonia-cellulose complex which organizes into cellulose IIII upon ammonia removal in the absence of water.29,46 Moreover, the crystallinity and the degree of conversion to cellulose IIII is highly dependent on the pressure and temperature of AA pretreatment with highly crystalline cellulose IIII is formed at AA pretreatment above critical temperature of ammonia.37 From Table S1 it can be clearly noted that CrI of corn stover increased with increasing AA pretreatment temperature and its value decreased slightly after NaOH extraction of samples pretreated at 25 and 60 °C; however, no change in CrI was observed after NaOH extraction of corn stover pretreated with AA above 100 °C. Multiscale Microscopic Characterization Reveals Patterns of Deconstruction with Increasing AA Pretreatment Severity. Surface examination of the AA-pretreated corn stover particles by stereomicroscopy showed subtle changes in color and particle behavior. All of the AA-pretreated stover particles became a more uniform dull gray-brown color relative to the range of shades of tan and brown of the untreated material (Figure S1). All of the AA-pretreated/NaOH-extracted samples were lighter in color (Figure S1) as expected, denoting lignin extraction. The samples pretreated at 130 °C displayed a

Figure 8. X-ray diffraction of untreated and AA-pretreated corn stover showing the effects of (A) increased AA pretreatment temperature, (B) NaOH extraction of AA-pretreated corn stover pretreated at 25− 100 °C, and (C) NaOH extraction of AA-pretreated corn stover pretreated at 130 °C. AA pretreatment was conducted at 25−130 °C with NH3 loading of 3 g anhydrous NH3/g dry corn stover. NaOH extractions were conducted with 0.025−0.1 M NaOH at 10% solids loading at temperatures ranging from 25−75 °C.

slightly increased tendency to form clumps (Figures S1 asterisks). There is some evidence for an increase in very small particles (fines), but no overall dramatic reduction in particle size. By confocal scanning laser microcopy (CSLM) the untreated cell walls displayed relatively uniform, intact tissue structure (Figures 9A, 10A). Even at the higher severities and either with or without NaOH extraction, there is no evidence for extensive disjoining of cells. The tissues remain largely intact. In the AApretreated samples at 130 °C (Figures 9C and 10D and E), there is an increase in cell wall fragments appearing in the cell 2551


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Figure 9. Confocal scanning laser micrographs (A−C) and transmission electron micrographs (D−F) of corn stover samples pretreated with anhydrous ammonia at 100 and 130 °C with ammonia loading of 3 g anhydrous NH3/g dry corn stover.

Figure 10. Confocal scanning laser micrographs (A−E) and transmission electron micrographs (F−J) of corn stover samples pretreated with anhydrous ammonia at 100 and 130 °C with ammonia loading of 3 g anhydrous NH3/g dry corn stover followed by NaOH extraction conducted at 0.1 M NaOH loading at 25 and 75 °C.

NaOH extraction was the disappearance of coalesced lignin globules. This extraction of relocalized lignin from within the secondary cell walls, left behind cleared zones of delamination (Figure 10J arrows) and a highly irregular, scalloped surface (Figure 10J arrowheads). Glycome Profiling Analyses Reveal Cell Wall Ultrastructural Modifications Induced by AA Pretreatment and AA/NaOH-Extraction Pretreatment Regimes in Corn Stover Biomass Residues. Glycome profiling analyses were conducted on untreated, AA-pretreated, and AA-pretreated/ NaOH-extracted corn stover residues (Figure 11) for monitoring changes in the composition, extractability, and integrity of most major noncellulosic matrix cell wall glycans that are induced by various pretreatment processes employed here.

lumen and the lumenal cell wall surface appears rough and less uniform in many cells (Figures 9C arrows and 10D and E arrows). The AA-pretreated cells at 130 °C have also lost their shape, with wavy walls indicating that the walls have become less rigid. At the light microcopy scale, there are no dramatic differences between the AA-pretreated or AA-pretreated/ NaOH-extracted samples. Transmission electron micrographs (TEM) of the untreated samples show secondary cell walls with uniform density and fine texture (Figures 9D and 10F). AA-pretreated cell walls at 100 °C display some evidence for delamination and surface changes. Delamination is much more extensive in the AApretreated cell walls at 130 °C (Figure 9F arrows), and these walls showed evidence of coalesced lignin (Figure 9F arrowheads). The most consistent and unique feature of the cell walls exposed to AA pretreatment at 130 °C followed by 2552


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Figure 11. Glycome profiling analyses of untreated, AA-pretreated, and AA-pretreated/NaOH-extracted corn stover residues. Cell wall materials (AIR) were isolated from various biomass residues as explained in the Materials and Method section. The cell wall materials were subjected the sequential extractions using increasingly harsh reagents. The extracts were screened with a comprehensive suite of cell wall glycan directed mAbs to monitor epitope structures in most major noncellulosic matrix cell wall glycans. The glycan specificities of the suite of mAbs employed are depicted on the right-hand side panel. Amounts of carbohydrate materials recovered per extraction steps are depicted as a bar graph on the top panel. The strength of mAb binding is represented as a heatmap with a color scheme with bright yellow representing the highest binding strength (highest epitope abundance), red, medium (medium epitope abundance), and black, no binding (least epitope abundance).

Xylan epitopes were significantly abundant in all cell wall extracts except the oxalate extract. In the carbonate extract, xylan epitopes were recognized by xylan-6 groups of mAbs whereas two mAbs (CCRC-M160 and CCRC-M137) from the xylan-7 group were absent. In all subsequent harsher extracts (1 M KOH through 4 M KOHPC) unsubstituted and substituted xylan epitopes that are recognized by the xylan-4 through 7 groups of mAbs were abundantly present. Xylan epitopes recognized by most xylan-3 group of mAbs were observed in chlorite and 4 M KOHPC extracts. However, the epitope detected CCRC-M114, a xylan-3 mAb was present in all extracts except the oxalate extract. Homogalacturonan (a pectin component) backbone epitopes were primarily detected in carbonate, chlorite, and 4 M KOHPC extracts. Again, rhamnogalacturonan-1 backbone epitopes were present in higher proportion in chlorite and 4 M KOHPC extracts. Pectic-arabinogalactan epitopes that are recognized by RG-I/ AG group of mAbs were detected in all extracts with significantly higher abundance noted in oxlate, carbonate, chlorite, and 4 M KOHPC extracts. Similarly, arabinogalactan

The glycome profile of untreated corn stover revealed the overall noncellulosic cell wall matrix glycan compositions and their extractability patterns and the results obtained wellsubstantiated previously reported corn stover biomass glycome profiles.47 During extractions, the highest amount of carbohydrate material was recovered during the 1 M KOH extraction step (see the top bar graph in Figure 11). The predominant component of this 1 M KOH extract was xylan as indicated by the significantly higher binding of xylan-3 through xylan-7 groups of monoclonal antibodies (mAbs; recognizing xylan epitopes representative of both substituted and unsubstituted regions of xylans). Xyloglucan epitopes were detected only in 4 M KOH and 4 M KOHPC extracts and both nonfucosylated (indicated by the binding of NON-FUC-XG-1 through 4 groups of mAbs) and fucosylated xyloglucans (indicated by the binding of FUC-XG group of mAbs) epitopes were detected in these extracts. Interestingly, and in agreement with previous reports,47 significantly reduced abundance of xyloglucans epitopes that are recognized by NON-FUC-XG-5 group of mAbs was observed in the cell walls of untreated corn stover. 2553


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Figure 12. Glucose and xylose yields obtained after enzymatic hydrolysis of AA pretreatment of corn stover at temperatures ranging from 25−130 °C. Enzymatic hydrolysis was performed at 1% solids with 20 mg (16 mg CTec3 + 4 mg HTec3)/g glucan. The error bars show the standard deviation for the analyses conducted in triplicate.

increased pectin extractability in all pretreated residues. Such increase in abundance of pectin and pectic arabinogalactan epitopes was more pronounced in AA pretreatments at 100 and 130 °C. Overall, these results demonstrate that enhanced extractability of hemicellulosic glycans such as xyloglucans and xylans and pectic and pectic arabinogalactan components is induced by AA pretreatment and these effects are highest at pretreatments conducted at 100 and 130 °C. These results are in agreement with previous results on biomass residues subjected to ammonia based pretreatment methods such as AFEX where enhanced extractability of hemicellulosic glycans and pectic polysaccharides occur as a pretreatment induced effect in diverse plant biomass including corn stover.47 The next set of glycome profiling analyses were conducted on AA-pretreated corn stover at 100 and 130 °C temperatures subjected to an NaOH extraction (0.1 M) at two different temperatures, 25 and 75 °C. Overall, the glycome profiles of these residues were largely similar to those of AA-pretreated residues at 100 and 130 °C exhibiting significantly enhanced extractability of hemicellulosic glcyans and pectic components. In general, a marginal increase in the amounts of carbohydrate materials recovered was noted in these residues in alkaline extracts (1 M KOH, 4 M KOH, and 4 M KOHPC) compared to respective AA-pretreated residues at 100 and 130 °C suggesting additional biomass modifications induced by the NaOH extraction step. Interestingly, the chlorite extracts (where lignin associated matrix glycans are released) from NH3-100 °C/NaOH 75 °C and NH3-130 °C/NaOH 75 °C residues exhibited a significant reduction in the abundance of xylan epitopes recognized by the xylan-6 group of mAbs, indicating a reduction in lignin content of AA-pretreated/ NaOH-extracted corn stover residues, as corroborated by data presented above. Enzymatic Hydrolysis of AA-Pretreated Corn Stover. A key driver for pretreatment processes is the ability to produce highly digestible polysaccharides. As such, enzymatic hydrolysis of untreated and AA-pretreated corn stover was performed at 1% solids loading using CTec3 at 16 mg protein/g of glucan. CTec3 was supplemented with HTec3 at 4 mg protein/g of glucan to digest hemicellulose and enhance cellulose accessibility for the cellulases. The glucose and xylose yields obtained after enzymatic hydrolysis of untreated and AA-

epitopes that are detected by various AG groups of mAbs were primarily present only in oxlate, carbonate, chlorite, and 4 M KOHPC extracts. Thus, glycome profiling analyses demonstrate the overall composition and extractability of major noncellulosic cell wall glycans in untreated corn stover biomass allowing further comparative studies with corresponding glycome profiles of biomass residues subjected to various AA and AA/NaOH-extraction pretreatments. Glycome profiles of AA-pretreated and AA-pretreated/ NaOH-extracted corn stover residues exhibited differences in comparison to untreated residues (Figure 11). First, pretreated residues obtained after AA pretreatment for 1 h at increasing pretreatment temperatures (25, 60, 100, and 130 °C) were analyzed with glycome profiling. Several changes were apparent in glycome profiles of all these pretreated residues. An increased abundance of xyloglucan epitopes was observed in 1 M KOH extracts from all AA-pretreated residues. The most dramatic differences were noted in the extractability of the xylan epitopes. In particular, the xylan epitopes were extracted out even in the least harsh step, such as the oxalate extract from AA-pretreated corn stover obtained at lowest pretreatment temperature of 25 °C. Additionally, the oxalate extracted abundances of xylan epitopes increased with the increase in the AA pretreatment temperatures. It is noteworthy that the xylan epitopes recognized by all the xylan-6 groups of mAbs were not completely extracted in oxalate and carbonate extracts at AA pretreatment temperatures of 25 °C (where all the xylan-6 recognized epitopes were absent) and 60 °C (where epitopes recognized by selected mAbs of the xylan-6 group were absent). However, at pretreatment temperatures of 100 and 130 °C, a significantly higher abundance of xylan epitopes (of both unsubstituted and substituted xylans) recognized by all xylan-3 through 7 groups of mAbs was observed. This was further substantiated by the higher amounts of carbohydrate materials recovered in the oxalate extracts in these residues (see the bar graphs on top of Figure 11). Increased abundances of pectic backbone epitopes (as recognized by HG-backbone-1 and RG-I backbone groups of mAbs), pectin, and pectic-arabinogalactan epitopes (indicated by the increased binding of linseed mucilage RG-I and RG-I/AG groups of mAbs) were also apparent in two least harsh extractions, especially in oxalate extracts in the case of all AA-pretreated samples suggesting 2554


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Figure 13. Glucose and xylose yields obtained after enzymatic hydrolysis of AA-pretreated corn stover (100 and 130 °C) and AA-pretreated/NaOHextracted corn stover obtained after extraction with 0.025−0.1 M NaOH at 25−75 °C at 1% solids. (A and B) Glucose and xylose yields, respectively, obtained with 20 mg (16 mg CTec3 + 4 mg HTec3)/g glucan. (C and D) Glucose and xylose yields, respectively, obtained with 7.5 mg (5 mg CTec3 + 2.5 mg HTec3)/g glucan. (E and F) Glucose and xylose yields, respectively, obtained with 4.5 mg (3 mg CTec3 + 1.5 mg HTec3)/g glucan. The error bars show the standard deviation for the analyses conducted in triplicate.

Interestingly, no significant increase in glucose and xylose yields was observed by increasing the pretreatment time to 3 h at 25 °C. For corn stover pretreated at 60 °C, the maximum glucose and xylose yields of 78 and 63%, respectively, were obtained which is a 4-fold increase when compared to the untreated corn stover. For corn stover pretreated at 100 °C, a high glucose and xylose yield of 75 and 53%, respectively, was obtained in just 16 h, which increased to 85 and 63%, respectively in 72 h with no considerable increase thereafter. Surprisingly, no increase in glucose and xylose yields was observed by increasing the pretreatment temperature to 130 °C. Overall, high glucose and xylose yields obtained for AApretreated corn stover can be attributed to its cellulose IIII

pretreated corn stover are shown in Figure 12. The extent of glucan and xylan conversion was calculated based on the release of cellobiose, glucose, and xylose during hydrolysis. As expected, the untreated corn stover showed poor susceptibility to enzymatic hydrolysis, reaching only up to 23 and 17% yields for glucose and xylose, respectively. On the other hand, the effect of pretreatment temperature on enzymatic hydrolysis of AA-pretreated corn stover was clearly observed with higher glucose and xylose yields being obtained for corn stover pretreated at higher temperature. For example, the maximum glucose and xylose yields of 45 and 31%, respectively, were obtained for corn stover pretreated 25 °C which is a 2-fold increase when compared to the untreated corn stover. 2555


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Figure 14. Glucose and xylose yields obtained after enzymatic hydrolysis of AA-pretreated corn stover (100 and 130 °C) and AA-pretreated/NaOHextracted corn stover (0.1 M NaOH, 75 °C) at 20% solids. The error bars show the standard deviation for the analyses conducted in triplicate.

72 h and plateauing thereafter with the maximum monomeric sugar yields being achieved for AA-pretreated/NaOH-extracted corn stover obtained with 0.1 M NaOH at 75 °C. The effect of NaOH extraction conditions was more obvious on glucose and xylose yields at this enzyme loading where glucose and xylose yields for AA-pretreated/NaOH-extracted corn stover were consistently higher by about 20−25% and 10%, respectively throughout the digestion period of 120 h when compared to AA-pretreated only corn stover (Figure 13C and D). For an enzyme loading of 4.5 mg/g of glucan, the glucose and xylose yields increased linearly with hydrolysis time and reached to maximum yields at a digestion period of 72−96 h (Figure 13E and F). It is interesting to note that even at a modest enzyme loading of 4.5 mg/g of glucan, the glucose and xylose yield approaching 100% and 75% of theoretical were obtained. It is well-known that nonspecific binding of cellulases to lignin results in the loss of cellulase activity, decreasing the overall yields of cellulose hydrolysis.48−50 The effect of NaOH extraction on enzymatic hydrolysis of AA-pretreated corn stover was clearly observed with significantly higher glucose and xylose yields being obtained for AA-pretreated/NaOHextracted corn stover (0.1 M NaOH, 75 °C) even at a modest enzyme loading of 4.5 mg per gram of glucan possibly by diminishing the inhibitory action of lignin on cellulases activity due to the removal of a significant amount of lignin from the AA-pretreated corn stover. High Solids Enzymatic Hydrolysis of AA-Pretreated/ NaOH-Extracted Corn Stover. High solids loading during enzymatic hydrolysis offers industrial advantages such as reduced reactor size, high sugar concentration in fermentation, less energy-intensive distillation of fuel products, and reduced water consumption. Therefore, industrially relevant high solids enzymatic hydrolysis of AA-pretreated/NaOH-extracted corn stover was performed. Digestions were performed in duplicate in 2 mL vials at 20% solids loading, 50 °C, and 130 rpm for digestion periods of 72 and 120 h. CTec3 and Htec3 were added at 16 and 4 mg protein per gram of glucan. The glucose and xylose yields are shown in Figure 14. For AA-pretreated corn stover pretreated with AA at both 100 and 130 °C the glucose yield were 60−62% after 72 h digestion period (Figure 13A). After 120 h the glucose yield reached 88−90% for both AA-pretreated residues. The effect of NaOH extraction on

structure formed after AA pretreatment (Figure 8A). It is important to mention that corn stover pretreated at 100 and 130 °C showed maximum glucan and xylan conversions also had the highest crystallinity (Table S1, SI). Moreover, a trend can be seen between increasing crystallinity and high digestibility of AA-pretreated samples. This could be attributed to increased deconstruction of plant cell walls obtained at increased severity of pretreatment which is evident from the wavy walls of the pretreated cells obtained with AA pretreatment at 130 °C indicating that the walls have become less rigid (Figure 9C). Enzymatic Hydrolysis of AA-Pretreated/NaOH-Extracted Corn Stover. Since AA pretreatment temperatures of 100 and 130 °C showed high digestibility (Figure 12), AApretreated/NaOH-extracted corn stover obtained under various NaOH extraction conditions (0.025−0.1 M NaOH, 25−75 °C) from AA pretreatment temperatures at 100 and 130 °C were evaluated for enzymatic digestibility. Additionally, enzyme digestibility of AA-pretreated/NaOH-extracted corn stover was studied in more detail as a function of digestion time and by varying enzyme loading from 4.5−20 mg/g of glucan. The glucose and xylose yields obtained after enzymatic hydrolysis are shown in Figure 13. For an enzyme loading of 20 mg/g of glucan, AA-pretreated/NaOH-extracted corn stover showed high glucose yields ranging between 50−64% for residues obtained under various extraction conditions even at 4 h digestion period. At 16 h, the glucose yield increased to 88− 96%, which further increased to 96−100% at 24 h and plateaued thereafter (Figure 12A). The xylose yield showed similar trend as glucose yield except the maximum xylose yield obtained was about 85% in 72 h (Figure 12B). Two observations are made from Figure 13A and B: first, both AA-pretreated/NaOH-extracted and AA-pretreated corn stover showed two regions of the digestion curve. An initial region of rapid increase in glucose and xylose yields reaching the maximum yield in the first 24 h and plateauing thereafter. Second, the glucose and xylose yields for AA-pretreated/ NaOH-extracted corn stover were consistently higher by about 20% and 15%, respectively, throughout the digestion period of 120 h when compared to AA-pretreated only corn stover. For an enzyme loading of 7.5 mg/g of glucan, both glucose and xylose yields increased steadily reaching to a maximum at 2556


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ides. Enzymatic hydrolysis of AA-pretreated/NaOH-extracted corn stover showed very high glucan and xylan conversion yields approaching 100 and 75% for glucose and xylose, respectively with CTec3 and Htec3 at a modest enzyme loading of 4.5 mg/g of glucan after 72 h digestion period at 1% solids loadings. Overall, this simple two-step pretreatment process results in simultaneously altering the structure of biomass to a form with enhanced cellulose accessibility (cellulose IIII) while promoting the depolymerization and subsequent solubilzation of a large portion of lignin in the aqueous fraction which could be converted to value added products to aid the overall economics of the lignocellulosic biorefinery.51,54

enzymatic hydrolysis was clearly observed with over 20% higher glucose yields being obtained after 72 h compared to AApretreated only corn stover. However, after 120 h this difference diminished as the glucose yield for both AApretreated and AA-pretreated/NaOH-extracted corn stover also reached to 90%. The xylose yield were 60−65% for both AApretreated and AA-pretreated/NaOH-extracted corn stover after 72h and reached to 72−80% in 120h (Figure 13B). It is interesting to note that for both 72 and 120 h digestions, no effect of NaOH extraction was seen on xylose yield. In fact, slightly lower xylose yields were observed for AA-pretreated corn stover at 130 °C followed by NaOH extraction. From similar glucose and xylose yields being obtained for both AA-pretreated and AA-pretreated/NaOH-extracted corn stover, one might conclude that there is no apparent benefit of adding a NaOH extraction step after AA pretreatment. However, it is important to emphasize that since the total carbohydrate content of AA-pretreated/NaOH-extracted corn stover (75−80%) is 30% higher than the AA-pretreated only corn stover (55−60%), in spite of reaching a similar level glucan and xylan conversions, the total monomeric sugars (glucose, xylose and arabinose) in the enzymatic hydrolyzates obtained with AA-pretreated/NaOH-extracted corn stover were about 150 g/L which were 30% higher than 115 g/L obtained with AA-pretreated only corn stover that would result in a similar increase in ethanol yield and titer assuming similar ethanol yields are realized during fermentation of monomeric sugars obtained via enzymatic hydrolysis of both AA-pretreated and AA-pretreated/NaOH-extracted corn stover. Moreover, the extracted lignin obtained after NaOH extraction of AApretreated corn stover exhibits a highly reduced molecular weight distribution, and subsequent solubilization of a large portion of lignin in the aqueous fraction, which could be valorized to value-added products through emerging biological approaches.9,51−53

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MATERIALS AND METHODS

Raw Material. Air-dried corn stover from Idaho National Laboratory (Lot no. 4) was milled to 2 mm particle size and was used as is. The compositional analysis of the untreated corn stover showed that it contained 36.3 ± 2.3% glucan, 34.2 ± 4.0% hemicelluloses (26.4% xylan, 5.3% arabinan, and 2.5% acetyl), 18.5 ± 1.6% lignin, and 11.3% others (5.5% ash and 5.8% water extractives). Anhydrous ammonia was purchased from General Air (Denver, CO) and sodium hydroxide was purchased from SigmaAldrich. Anhydrous Ammonia (AA) Pretreatment. Anhydrous ammonia (AA) pretreatment of corn stover was conducted in a Parr reactor with ammonia loading of 3 g anhydrous NH3/g dry corn stover according to the method previously reported.34 Briefly, about 3 to 5 g of corn stover (oven dry equivalent weight) was placed in a stainless steel reaction vessel (PARR Instrument Co., IL, Model 4714). The reaction vessel was clamped shut, weighed, and then chilled in a dry ice/ acetone bath (−75 °C) to facilitate transfer of liquid ammonia into the reactor at atmospheric pressure. Anhydrous liquid ammonia was added slowly to the reaction vessel in the ratio of ∼3 g NH3/g dry CS using a stainless steel transfer tube from a liquid ammonia cylinder equipped with an eductor tube. After adding ammonia, the vessel was immediately weighed and then cooled in the dry ice/acetone bath for 15 min. For a 25 °C treatment, the corn stover was initially treated at −75 °C for 15 min, and then the vessel was immersed in a water bath maintained at 25 °C for a reaction period of either 60 or 90 min. After completion of the desired reaction period and prior to its removal from the water bath, the treatment was terminated by immediately depressurizing the vessel in a ventilated hood. The AApretreated corn stover was removed from the vessel and left in the hood overnight until all ammonia had evaporated. For treatment at elevated temperatures, i.e., 60−130 °C, the corn stover was initially treated at −75 °C for 15 min then the vessel was maintained at 25 °C for 30 min prior to subjecting the vessel to a final heat treatment of 1 h in a preheated fluidized sand bath (Techne Inc., Burlington, NJ) maintained at the desired reaction temperature (60, 100, or 130 °C). After concluding the final heat treatment and prior to its removal from the sand bath, the reaction vessel was depressurized by allowing the ammonia to leak out in a ventilated hood. After releasing ammonia the vessel was cooled in a water bath maintained at 25 °C. The ammoniatreated corn stover was removed from the vessel and left in the hood overnight until all the ammonia had evaporated. To determine the structural carbohydrates and lignin components of the pretreated solids, a water extraction was performed on the samples to remove any nonstructural components. A typical automated solvent extraction55 was not performed because of concerns that the high pressure would remove lignin in addition to nonstructural sugars. An adapted extraction was developed based on previous internal work where 100 mg of sample was weighed into 15 mL conical BD Falcon tubes (352196, Thermo Fisher Scientific) and 10 mL of nanopure water was added into each tube, capped, and vortexed. Samples were then placed in a counter top shaker flask incubator (Innova 4000, New Brunswick Scientific, Enfield, CT) set at 21 °C for 4 h and 200 rpm. Samples were then removed, vortexed, and transferred to preweighed 12 mL empty fritted solid phase extraction (SPE) tubes (54223-U, Supelco). The

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CONCLUSIONS Tandem AA pretreatment and mild NaOH extraction readily separates the carbohydrate (cellulose and hemicellulose) and lignin fractions in corn stover under mild process conditions. AA pretreatment conducted at 130 °C followed by mild sodium hydroxide extraction (0.1 M NaOH, 25 °C) resulted in more than 65% lignin removal with over 84% of carbohydrate remaining in the extracted residue. 2D-NMR spectroscopy indicates that the combination of AA pretreatment followed by mild NaOH extraction (0.1 M NaOH at 25−75 °C) results in the ammonolysis of ester bonds of p-coumarate and ferulate units in lignin. The GPC analysis suggests that the lignin fractions obtained after NaOH extraction of AA-pretreated corn stover resulted in large monomer/dimer peaks with 250−450 Da and solubilization of lignin including feruloyl amide moieties. Electron microscopy of the cell walls treated with AA at 100 and 130 °C display delamination and surface changes and evidence of coalesced lignin. The cell walls exposed to AA at 130 °C followed by NaOH extraction revealed the disappearance of coalesced lignin globules from within the secondary cell wall and a highly irregular, scalloped surface. Glycome profiling results clearly demonstrated ultrastructural level cell wall modifications induced by AA pretreatment followed by NaOH extraction resulting in a significantly enhanced extractability of hemicellulosic glycans such as xyloglucans and xylans and pectic and pectic arabinogalactan components indicating enhanced accessibility of polysacchar2557


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ACS Sustainable Chemistry & Engineering SPE tubes were attached to a 24-place filtration manifold (57250-U, Supelco) equipped with stopcocks and attached to a house vacuum system. The filtrate was collected in a falcon tube and analyzed for nonstructural sugars by HPLC.56 Sample tubes were then washed with nanopure water until all solid material had been transferred to the corresponding SPE tube. Any biomass clinging to the tube sides was washed down with nanopure water to ensure all biomass was at the bottom of the tube. Once washed, the entire SPE tube was placed in a 40 °C vacuum oven and dried for a minimum of 2 days. After drying, the moisture content of the biomass sample was assumed to be 0% and the SPE tube was then weighed. Mass loss of the solid sample was then calculated, using eq 3:

Rigaku (Tokyo, Japan) Ultima IV diffractometer with Cu Kα radiation having a wavelength λ(Kα1) = 0.15406 nm generated at 40 kV and 44 mA. The diffraction intensities of freeze-dried samples placed on a quartz substrate were measured in the range of 8−42° 2θ using a step size of 0.02° at a rate of 2°/min. The crystallinity indexes (CrI) of the cellulose samples were calculated according to the method described by Segal et al.58 using eq 4:

CrI =

I200 − IAm I200

(4)

where I200 and IAm are the maximum and minimum intensity of diffraction at approximately 2θ = 22.4−22.5° and 2θ = 18.0−19.0°, respectively. HSQC NMR. Heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectra were acquired for untreated, AA-pretreated, and AA-pretreated/NaOH-extracted corn stover samples. Each sample (0.2 g) was ball milled by a planetary ball milled using a Retsch PM100 mill fitted with a 50 mL ZrO2 grinding jar and 10 mm × 10 mm ball bearings, set at 600 rpm. A ball milled sample (80 mg) was suspended into 0.5 mL of d6-DMSO/d5-pyridine (4:1, v/v) in an NMR tube. The mixture was sonicated for 5 h until the gel became homogeneous.45 Spectra were acquired at 40 °C on a Bruker 400 MHz spectrometer using a BBO probe with Z gradient. Spectra were acquired with 1024 points and a sweep width of 15 ppm in the F2 (1H) dimension and 512 points and 220 ppm of sweep width in the F1 (13C) dimension. The DMSO peak was used as an internal reference (δH 2.5, δC 39.51 ppm). Peak assignment was performed according to the literature.19,45,59 Gel Permeation Chromatography (GPC) Analysis. To determine the molecular weight distribution of the lignin in the AApretreated and AA-pretreated/NaOH-extracted corn stover, 20−50 mg of freeze-dried samples were derivatized and analyzed by GPC. Each sample was acetylated in a mixture of pyridine (0.5 mL) and acetic anhydride (0.5 mL) at 40 °C for 24 h with stirring. The reaction was terminated by addition of methanol (0.2 mL). The acetylation reagents were removed by evaporation under a stream of nitrogen gas. The samples were further dried in a vacuum oven at room temperature overnight. The dried, acetylated lignin samples were dissolved in tetrahydrofuran (THF, Baker HPLC grade). The dissolved samples were filtered (0.45 μm nylon membrane syringe filters) before GPC analysis. GPC analysis was performed using an Agilent HPLC with 3 GPC columns (Polymer Laboratories, 300 mm × 7.5 mm) packed with polystyrene-divinylbenzene copolymer gel (10 μm beads) having nominal pore diameters of 104, 103, and 50 Å. The eluent was THF and the flow rate 1.0 mL/min. An injection volume of 25 μL was used. The HPLC was attached to a diode array detector measuring absorbance at 260 nm (bandwidth 80 nm). Retention time was converted into molecular weight (MW) by applying a calibration curve established using polystyrene standards of known molecular weight (1 × 106 to 580 Da) plus toluene (92 Da). Stereomicroscopy. Whole pieces of milled, untreated, AApretreated, and AA-pretreated/NaOH-extracted corn stover samples were examined without further processing. Images were captured on a Nikon SMZ1500 stereomicroscope and captured with a Nikon DS-Fi1 CCD camera operated by a Nikon Digital Sight system (Nikon Instruments, Melville, NY). Fiji (ImageJ) was used to adjust brightness and white balance images. Microwave Processing. Untreated, AA-pretreated, and AA-pretreated/NaOH-extracted corn stover tissue was processed using microwave processing as described previously.60 Briefly, samples were fixed 2 × 6 min (with variable power) in 2.5% gluteraldehyde buffered in 0.1 M sodium cacodylate buffer (EMS, Hatfield, PS) under vacuum. The samples were dehydrated by treating with increasing concentrations of ethanol and heating in a specially outfitted microwave oven for 1 min each dilution (i.e., 15%, 30%, 60%, 90%, and 3 × 100% ethanol). After dehydration, the samples were infiltrated with LR White resin (EMS, Hatfield, PA) by incubating at room temperature (RT) for several hours to overnight in increasing concentrations of resin (15%, 30%, 60%, 90%, 3 × 100% resin,

⎛ ODW sample − ODW extracted sample ⎞ % Mass Loss = ⎜ ⎟ × 100 ODW sample ⎝ ⎠ (3) The compositional analysis of the pretreated and washed solids was conducted at 1/6 the scale of the standard NREL Laboratory Analytical Procedures (LAPs).57 NaOH Extraction of AA-Pretreated Corn Stover. Sodium hydroxide extraction of AA pretreated corn stover was performed on 2−3 g of AA pretreated corn stover in 125 mL Erlenmeyer shake flasks with 0.025−0.1 M NaOH at 10% solids loading at temperatures ranging from 25−75 °C and 150 rpm for 2 h (Scheme 1). After

Scheme 1. AA Pretreatment and NaOH Extraction Procedure Used in This Study for Separation of the Lignin and Carbohydrate Fractions

extraction, the insoluble material was separated by centrifugation at 4000 rpm for 10 min. The separated solid was mixed with 30 mL of DI water and the obtained slurry was centrifuged again at 4000 rpm for 10 min. This step was repeated twice, and the two wash fractions obtained after these steps were mixed with the supernatant obtained after first step of centrifugation. Both soluble and insoluble fractions obtained after centrifugation were neutralized followed by freeze-drying to determine the total solids of each fraction. The compositional analysis of the pretreated solids was conducted at 1/6 the scale of the standard NREL LAPs.57 X-ray Diffraction Measurements. The crystallinity indexes (CrI) of untreated, AA-pretreated, and AA-pretreated/NaOH-extracted corn stover samples were measured by X-ray diffraction (XRD) using a 2558



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diluted in ethanol). The samples were transferred to capsules and the resin polymerized by heating to 60 °C overnight. Confocal Scanning Laser Microscopy (CSLM). LR White embedded samples were sectioned to 300 nm with Diatome diamond knife on a Leica EM UTC ultramicrotome (Leica, Wetzlar, Germany). Semithin sectioned samples were positioned on glass microscope slides and stained with 0.1% acriflavine. Images were captured using a 40X 1.4NA Plan Fluor lens on a Nikon C1 Plus microscope (Nikon, Tokyo, Japan), equipped with the Nikon C1 confocal system. Samples were excited with a 488 nm laser and fluorescence detected at 530 nm. A Z-axis stack of images was collected then projected onto a single plane. Transmission Electron Microscopy (TEM). LR White embedded samples were sectioned to 60 nm with Diatome diamond knife on a Leica EM UTC ultramicrotome (Leica, Wetzlar, Germany). Thin sections were collected on 0.5% Formvar coated slot grids (SPI Supplies, West Chester, PA). Grids were poststained for 6 min with 2% aqueous uranyl acetate and 3 min with 1% KMnO4. Images were taken with a 4 mega-pixel Gatan UltraScan 1000 camera (Gatan, Pleasanton, CA) on a FEI Tecnai G2 20 Twin 200 kV LaB6 TEM (FEI, Hilsboro, OR). Enzymatic Hydrolysis. Enzymatic hydrolysis of untreated, AApretreated, and AA-pretreated/NaOH-extracted corn stover samples was performed in duplicate in 2 mL polypropylene cryovials (Simport−T308-2A) at 1% or 20% solids loading, at 50 °C for a digestion period of 120 h. CTec3 was added at 16 mg protein per gram of glucan to evaluate the initial hydrolysis rate and the extent of maximum conversion of glucan after 120 h. CTec3 was supplemented with HTec3 at 4 mg protein per gram of glucan to reduce the physical barrier of hemicelluloses and enhance cellulose accessibility to cellulases. The total volume of the saccharification slurries after adding enzymes and 50 mM citrate buffer (pH 4.8) was 2 mL for 1% solids and 0.4−1 mL for 20% solids. Digestions at 1% solids were continuously mixed by inversion at 10−12/min intervals whereas digestions at 20% solids were continuously mixed in a horizontal shaker at 130 rpm to avoid the separation of solids from liquid. To determine the progress of cellulose conversion at 1% solids, a 0.1 mL aliquot of the well mixed slurries was taken at predetermined time points starting with 4, 16, and 24 h and diluted to 1 mL with DI water and filtered and stored at 4 °C for HPLC analysis. Thereafter samples were removed every 24 h until 120 h. For the digestions performed at 20% solids, the entire well mixed slurry was taken at final time points of 72 or 120 h and diluted 10-fold with DI water and filtered and stored at 4 °C for HPLC analysis. HPLC Analysis. The digestion samples taken at each time point were immediately filtered through a 0.2 μm filter and then refrigerated until subjected to monomeric sugar analysis. The monomeric sugars (glucose and xylose) yield was measured by HPLC using Aminex 87H column maintained at 55 °C using standard protocols and method.56 The mobile phase used was 0.2 μm filtered 0.01 N sulfuric acid solution at a flow rate of 0.6 mL/min. The sample injection volume was 10 μL, and the run time, 26 min. Glycome Profiling Analyses. Alcohol insoluble residues (AIRs) were prepared from various corn stover biomass residues as described previously.61 Glycome profiling of these AIR preps was performed using the method described in the work of Pattathil et al.61 In brief, glycome profiling involved preparation of cell wall extracts using increasingly harsh reagents (ammonium oxalate, sodium carbonate, 1 M KOH, 4 M KOH, chlorite, and 4 M KOHPC) and subsequent enzyme-linked immunoabsorbent assay (ELISA) screening of these extracts using a comprehensive suite of plant cell wall glycan-directed monoclonal antibodies (mAbs). The suite of mAbs were obtained from laboratory stocks (CCRC, JIM, and MAC series) at the Complex Carbohydrate Research Center (available through CarboSource Services; http://www.carbosource.net) or from BioSupplies (Australia) (BG1, LAMP).

Research Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.6b02892. Crystallinity index (CrI) of untreated, ammonia-treated, and ammonia-treated/NaOH-extracted corn stover obtained under various pretreatment conditions. Stereoscope micrographs of ammonia-treated and ammonia treated/NaOH-extracted corn stover (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (A.M.). *E-mail: [email protected] (G.B.). ORCID

Gregg T. Beckham: 0000-0002-3480-212X Present Address

∥ S.P.: Mascoma LLC (Lallemand Inc.), 67 Etna Road, Lebanon, NH 03766, USA.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Justin Sluiter and Courtney Payne for their assistance in developing the extraction protocol for the compositional analysis of AA-pretreated corn stover. We thank the U.S. Department of Energy Bioenergy Technologies Office (DOEBETO) for funding this work via Contract No. DE-AC3608GO28308 with the National Renewable Energy Laboratory. For the glycome profiling work, we acknowledge the BioEnergy Science Center (BESC) administered by Oak Ridge National Laboratory and funded by a grant (DE-AC05-00OR22725) from the Office of Biological and Environmental Research, Office of Science, United States, Department of Energy. The generation of the CCRC series of plant cell wall glycan-directed monoclonal antibodies used in this work was supported by the NSF Plant Genome Program (DBI-0421683 and IOS0923992). The U.S. Government retains and the publisher, by accepting the article for publication, acknowledges that the U.S. Government retains a nonexclusive, paid up, irrevocable, worldwide license to publish or reproduce the published form of this work, or allow others to do so, for U.S. Government purposes.

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REFERENCES

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