BiomassâWater Interaction and Its Correlations with Enzymatic

Research Article pubs.acs.org/journal/ascecg

Biomass−Water Interaction and Its Correlations with Enzymatic Hydrolysis of Steam-Exploded Corn Stover Zhi-Hua Liu†,‡ and Hong-Zhang Chen*,† †

State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China ‡ University of Chinese Academy of Sciences, Beijing 100190, China S Supporting Information *

ABSTRACT: Author: Biomass−water interaction is a main factor affecting the enzymatic hydrolysis process. The interactions between corn stover and water and their correlations with enzymatic hydrolysis performance were investigated. The peak height of the main water pool was lower, peak width at half-height was bigger, and T2 relaxation time was shorter in steam-exploded corn stover (SECS) than in untreated corn stover (UCS). The relations between the total peak area of water pools and the moisture content can be well expressed by exponential models. Steam explosion enhanced corn stover−water interactions due to an increase in specific surface area, pore volume, average pore diameter, porosity, and oxygen to carbon ratio. Small particle size strengthened UCS−water interactions while slightly weakening SECS−water interactions. In enzymatic hydrolysis, constrained water was released before 36 h, which was consistent with the increase in glucan conversion. The release of constrained water and increase in glucan conversion facilitated each other. Steam explosion enhanced the interactions between SECS and water (mainly bound water), which increased the accessibility of carbohydrates to enzymes. Therefore, the release of constrained water before 36 h and the increase in SECS−water interactions on the surface of SECS facilitated the enzymatic hydrolysis efficiency. KEYWORDS: Biomass−water interactions, Steam explosion, Enzymatic hydrolysis, Water pool, Time domain nuclear magnetic resonance (TD-NMR), Particle size

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INTRODUCTION Decreasing petroleum supplies, increasing greenhouse gas emissions, and growing societal security demands urgently call for sustainable energy resources.1−3 Second-generation bioethanol has the potential to contribute to resolve these problems.4−6 Generally, pretreatment and enzymatic hydrolysis are two major unit operations in bioethanol production. Due to the recalcitrance of lignocellulosic biomass, pretreatment is a crucial step for breaking down the lignin−carbohydrate complex structures and increasing the accessible surface area of carbohydrates to enzymes.7−9 Steam explosion (SE) is one of the most effective pretreatments due to the potentials of lowering environmental impact and lessening hazardous chemicals use. SE depolymerizes hemicellulose, melts lignin partly, and disrupts cellulose fibers, which facilitate the enzymatic hydrolysis of carbohydrates.6,10,11 Water certainly plays a crucial role in the pretreatment and enzymatic hydrolysis of lignocellulosic biomass.12−14 Water is the medium through which catalysts diffuse to and products diffuse away from the reaction sites.15 Most importantly, water is also a catalyst in SE and a reactant in enzymatic hydrolysis of glycosidic bonds within carbohydrates. Additionally, the adsorbed water on the surface of the lignocellulosic biomass is © XXXX American Chemical Society

related to the ad-/desorption of enzymes and hence the reaction rate of enzymatic hydrolysis.12,14,16 Lignocellulosic biomass has complex physical structures and multiple chemical compositions, which determine the states and locations of water. In the range from microscale to macroscale, there are several “water pools” in the mixture of lignocellulosic biomass and water. Primary bound water is constrained by interacting with carbohydrates (mainly glucan) via hydrogen bonds.14−17 Secondary bound water, also known as thin film water, is located in a porous structure with confined spaces and bonded to the hydrophilic interface.14−17 Capillary bound water is constrained by capillary forces within cell wall lumens and micro/macropores.14−17 Free water is not restricted by any solvents or solids. Water interacted with solid substances is generally called constrained water, and this phenomenon is named water constraint.15,16 The constrained water bound in a plant cell wall matrix shows quite different properties compared with free water. So far, little research has been done to study the change of biomass−water interactions by SE and their correlations with subsequent enzymatic hydrolysis. Received: October 14, 2015 Revised: December 15, 2015

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DOI: 10.1021/acssuschemeng.5b01303 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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ACS Sustainable Chemistry & Engineering

particle sizes using standard sieves between 10 and 140 mesh. As for the composition analysis and other experiments, samples were milled and passed through a screen of 10 mesh. The sieved particles were stored in sealed plastic bags for further use. Enzymatic Hydrolysis Experiment. Cellulase preparation Cellic CTec2 was a generous gift from Novozymes (China) Investment Co., Ltd. (Beijing, China). Enzyme activity was analyzed according to the laboratory analysis protocol (Measurement of Cellulase Activities) of the National Renewable Energy Laboratory, Colorado, U.S.A.23,24 Cellulase and β-glucosidase activity was determined using Whatman No.1 filter paper strips and cellobiose as substrates, respectively. The procedure took use of 1.0 mL of citrate buffer (50 mM, pH 4.8), 0.5 mL of each enzyme dilution, and a 50-mg filter paper strip or a 5.0 g/L cellobiose. The mixture was incubated at 50 °C with 1.0 h for cellulase activity and 0.5 h for β-glucosidase activity. Filter paper activity (FPU) of cellulase was 108 FPU/mL. Cellobiase activity of β-glucosidase was 1290 CBU/mL. As for enzymatic hydrolysis, SECS was diluted to different solid loadings in a 50 mM sodium citrate buffer (pH 4.8). The experiment was performed at an enzyme loading of 10 FPU/g glucan with a reaction mixture of 200 g in 1.0-L Erlenmeyer flasks under the conditions of 50 °C, 200 rpm, and 120 h. For enzymatic hydrolysis kinetics, the enzymatic hydrolysis slurry was mixed well, and samplings of 5.0 g of slurry were conducted at 12, 24, 36, 48, and 72 h. NMR Measurement. TD-NMR analyses were done using a NIUMAN NMI20-Analyst with a 0.05 T permanent magnet (2 MHz proton resonance frequency), which consisted of a radio frequency electronic cabinet, a gradient electronic cabinet, a magnet cabinet, an industrial personal computer system, and a constant temperature system operating at 32 °C (NIUMAN, Shanghai, China). The determination of spin−spin transverse, T2, relaxation times was conducted using Carr− Purcell−Meiboom−Gill (CPMG) sequence. A total of 4000 echoes were collected with a pulse separation of 0.08 ms. The magnetization decay curves were analyzed using NMR Application and Analysis Software Ver 1.0 to determine the discrete values for T2. In order to study the interactions between SECS and water compared with those between UCS and water, 0.3 g of sample (DW) was adjusted to different moisture contents by adding deionized water and then determined by TD-NMR. As for the assignment of water pools distribution in enzymatic hydrolysis, the enzymatic hydrolysis slurry was mixed well, and 2.4 g of slurry was sampled and determined by TDNMR at different hydrolysis time points. A water pool, whose peak area is more than 50% of the total peak area, is defined as the main water pool. Analysis Methods and Calculations. Sugars and acetyl groups were analyzed by HPLC (Agilent 1200, U.S.A.) equipped with a refractive index detector and a Bio-Rad Aminex HPX-87H column. The column temperature was 65 °C, and the mobile phase (5 mM H2SO4) flow rate was 0.6 mL/min. Composition analysis of corn stover was conducted according to the laboratory analysis protocol of National Renewable Energy Laboratory, Colorado, U.S.A. The weights of UCS and SECS were analyzed by analytical balance (Sartorius AG, Goettingen, Germany). Compositions of UCS and SECS are given in Table 1. The pore structure of UCS/SECS was determined by a mercury intrusion porosimetry (Auto Pore IV 9500, Micromeritics instrument, Norcross, U.S.A.). Glucan conversion in enzymatic hydrolysis was calculated as follows:

Nuclear magnetic resonance spectroscopy is one of the most powerful techniques used to study the states of water under a variety of conditions and solid substances.16,18,19 Time-domain nuclear magnetic resonance (TD-NMR) is a rapid noninvasive and nondestructive analytical technique. Spin−spin (T2) NMR relaxation times, depending on the environment of hydrogen nuclei in the sample, are generally used to study the water within solid substrates. TD-NMR can be used to assess the states and locations of water in lignocellulosic biomass.15,18 When the mixture of lignocellulosic biomass and water is subjected to a brief pulse of an external magnetic field, more tightly bound water highly interacting with surrounding nuclei will give shorter spin−spin relaxation times (shorter T2) than free water (longer T2). Previous studies had confirmed that solid samples with multiple water pools corresponding to different constrained water showed different multiple T2 relaxation times, and thus, the proportion of water in each pool can be determined.18−20 TD-NMR can also be applied as a probe to assess the property changes of lignocellulosic biomass because the changes of water behavior reflect the modifications of chemical compositions and physical structures.12,21 Furthermore, TD-NMR is an efficient tool for investigating the pore-space structure of lignocellulosic biomass by characterizing the states of water.3,17 Thus, TD-NMR can be used as a qualitative and also a quantitative determination method of water in lignocellulosic biomass. This work investigated the changes of corn stover−water interactions by SE and their correlations with the performance of enzymatic hydrolysis. The states and locations of water associated with corn stover before and after SE were analyzed. The fiber saturation point of corn stover before and after SE was determined, and the mathematical model of corn stover−water interactions was established. The states and locations of water in enzymatic hydrolysis slurry and their correlations with the efficiency of enzymatic hydrolysis were also analyzed at increased solid loading. Additionally, process conditions (including solid loading, particle size, and enzymatic hydrolysis time) that might affect water pools distribution were systematically evaluated. The physicochemical properties of corn stover before and after SE were determined to reveal the potential mechanisms of the effects of water pools distribution on the enzymatic hydrolysis performance.

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MATERIALS AND METHODS

Steam Explosion (SE) Experiment and Samples Preparation. Corn stover was kindly provided by the Chinese Academy of Agricultural Sciences (CAAS) in Beijing, China. After harvest, corn stover was air-dried to the moisture content of less than 5% (w/w) and then stored for composition analysis and further use. Prior to SE, corn stover was manually cut into 2.0 cm particles and adjusted to 30% (w/w) moisture content using deionized water. The conditions were the optimal ones in our previous study. During SE, 1.0 kg corn stover (dry weight, DW) was loaded into a 20-L steam explosion reactor connected with a steam generator (Weihai Automatic Control Co. Ltd., China). Corn stover was heated by high temperature steam supplied by the steam generator and then cooked at 1.5 MPa (198 °C). After 6 min residence time, corn stover was instantaneously exploded into a reception tank. After SE, steam-exploded corn stover (SECS) was separated from the pretreated liquor fraction by vacuum filtration and washed with about 15 L of water until the free glucose concentration in washing stream was less than 0.2 g/L. SECS was then stored at 4 °C for further use. As for different particles preparation, untreated corn stover (UCS) and SECS samples were air-dried to less than 5% (w/w) moisture content and then ground by a high speed knife mill (GS-05, KJYCJX, China) for a period of 30 s. The milled samples were sieved to different

Glucan conversion (%) = (Glucose concentration in hydrolyzate × volume of hydrolyzate × 162/180)/Glucan in steam‐exploded corn stover × 100%

(1)

Glucose productivity (GP) = (GCi − GCj) × GSECS × 180/162/[V × (i − j)]

(2)

−1 −1

where GP is glucose productivity, g L h ; GC is glucan conversion, %; GSECS is glucan in SECS, g; V is the volume of enzymatic hydrolysis B


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Composition analysis of corn stover, SE, and enzymatic hydrolysis were performed in duplicate. TD-NMR was also conducted in duplicate. Error bars in the tables and figures represent the standard deviation of the replicates. For all significance tests, a student t-test was used requiring a probability of p < 0.05 to be significant.

Table 1. Compositions of Untreated and Steam-Exploded Corn Stovera component (%, dry weight)

UCS

SECS

glucan xylan araban acetyl acid-insoluble lignin acid-soluble lignin extractives ash total

31.2 (1.2) 18.6 (0.6) 3.1 (0.2) 4.2 (0.3) 15.4 (0.5) 1.2 (0.1) 15.9 (0.7) 4.6 (0.2) 94.2

56.2 (1.7) 9.7 (1.0) 1.0 (0.1) − 24.3 (0.5) − − 3.1 (0.1) 95.2

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RESULTS AND DISCUSSION Fiber Saturation Point of Untreated and SteamExploded Corn Stover. The fiber saturation point (FSP) is a key metric assessing the interactions between lignocellulosic biomass and water.16,22 FSP is also a main factor closely related to the ad-/desorption of enzymes on lignocellulosic biomass in enzymatic hydrolysis.12,22 FSP is based on the concept that a certain (and repeatable) amount of water is chemically bound to glucan and other substances in lignocellulosic biomass.22,25 When the moisture content is below FSP, the existing water within lignocellulosic biomass belongs to bound water. When the moisture content is above FSP, liquid water begins to accumulate

a

UCS, untreated corn stover; SECS, steam-exploded corn stover. Standard deviation in parentheses is representative of two replicates.

hydrolyzate, L; i = 12, 24, 36, 48, 72 h and j = 0, 12, 24, 36, 48 h are enzymatic hydrolysis times, h.

Figure 1. Water pools distribution (T2 relaxation time) and fiber saturation point of untreated (A1 and A2) and steam-exploded (B1 and B2) corn stover below 50% moisture content. UCS is untreated corn stover and SECS is steam-exploded corn stover. “0.3 g UCS+0.03 g Water” stands for the mixture of 0.3 g of UCS and 0.03 g of water. C


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Figure 2. Water pools distribution (T2 relaxation time) of untreated (A1, A2, and A3) and steam-exploded (B1, B2, and B3) corn stover above 50% moisture content. UCS is untreated corn stover, and SECS is steam-exploded corn stover.

hydrolysis.25 Therefore, an increase in FSP (mainly bound water) by SE increased the accessibility of carbohydrates to water and enzymes.13,16,25 Mathematical Model of Corn Stover−Water Interactions. In order to investigate corn stover−water interactions under different moisture contents, water pools distribution was determined by TD-NMR (Figures 1 and 2 and Table S1). The peak height and peak area of water pools in UCS and SECS increased with an increase in moisture content, respectively. As shown in Figure 1, the T2 relaxation time of each water pool in UCS and SECS increased with an increase in moisture content. The observations of extended relaxation times with an increase in moisture content may be due to a decrease in solid-to-water ratio and an increase in interactions between water molecules, which would be more frequent at higher moisture content. It is interesting to note that UCS had two peaks below FSP, while SECS had one (Figure 1A and B). The possible reason was that the ordered and rigid structure of UCS made the contaction between glucan and water difficult. Part of the water entered into the inner portion of the cellulose fibers, and the other part of the

in the cell wall lumens and micro/macropores of lignocellulosic biomass, indicating the transition of water pool and the change of water physical state. Furthermore, above FSP, the swelling of the plant cell wall ceases, and then, the strength of the plant cell wall hardly changes.16,22,25 Previous reports suggested that the FSP of lignocellulosic biomass can be analyzed approximately by NMR.16,22,26 As shown in Figure 1A1 and A2, the transition of water pools in UCS occurred under the conditions of 0.3 g of UCS and 0.09 g of water, which corresponded to 23% moisture content, while the transition of water pools in SECS occurred under the conditions of 0.3 g of SECS and 0.21 g of water, which corresponded to 41% moisture content. The T2 relaxation time was well below 100 ms, which meant that there was no capillary water (Figure 1). Results suggested that the FSP of corn stover was obviously increased by SE. The increase in FSP of SECS should be due to the increase in glucan content (Table 1), disruption of cell wall structure and cellulose fibers, and increase in accessible surface area of cellulose.6,27 A previous study reported that an increase in water retention value of the biomass should be helpful for biomass susceptibility to enzymatic D


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Table 2. Exponential Model Fitting Results for Relations between Total Peak Area of Water Pools in Untreated and SteamExploded Corn Stover and Moisture Contenta

a

samples

A

standard error 1

T0

standard error 2

C0

standard error 3

R2

MUCS MSECS MDTPA

122.5 105.3 87.5

9.8 13.6 12.4

−19.7 −18.9 −22.6

0.36 0.49 2.2

192.6 127.3 −1.3

20.7 81.6 0.5

0.9990 0.9999 0.9338

DTPA, decreased total peak area; UCS, untreated corn stover; SECS, steam-exploded corn stover.

water was adsorbed to the surface of the cellulose fibers. As for SECS, the ordered and rigid structure was obviously disrupted, and cellulose fibers were separated and exposed by SE, generating more accessible surface area of cellulose fibers to water. At the same moisture content, the T2 relaxation time of each water pool in SECS was shorter than that in UCS (Table S1). Lower T2 relaxation time presented stronger interactions between lignocellulosic biomass and water. These results indicated that the interactions between SECS and water were stronger than those between UCS and water below 50% moisture content. As shown in Figures 1 and 2, water pool 1, water pool 2, and water pool 3, which responded to primary bound water, secondary bound water, and capillary water, respectively, appeared sequentially with an increase in moisture content. This result should be due to the fact that as water continued to be added, UCS/SECS−water interactions both on the inside of and on the surface of cellulose fibers eventually reached an equilibrium state, and then, water began to accumulate in micropores and macropores of UCS/SECS. Interestingly, the peak height of the main water pool in SECS (pool 2 in Figure 2B1, pool 2 in Figure 2B2, and pool 3 in Figure 2B3) was lower than that in UCS (pool 2 in Figure 2A1, pool 3 in Figure 2A2, and pool 3 in Figure 2A3) under the same moisture content, respectively, while the peak width at half-height (Wh/2) showed the opposite trend. Furthermore, the T2 relaxation time of the main water pool in SECS was shorter than that in UCS. It should be noticed that the peak height of water pool 3 in UCS was about 6−7 times higher than that of water pool 2 beyond 87.5% of moisture content (Figure 2A3), while the peak height of water pool 3 in SECS was only 1.5−2.3 times higher than that of water pool 2 (Figure 2B3). The states and locations of water in SECS obviously changed by SE compared with those in UCS. Thus, the lower peak height, bigger peak width at half-height (Wh/2), and shorter T2 relaxation time of the main water pool in SECS suggested that the interactions between SECS and water were also stronger than those between UCS and water above 50% moisture content. The relations between the total peak area of water pools and moisture content should reflect the interactions between lignocellulosic biomass and water. The results showed that the total peak area of water pools in both UCS and SECS had a positive correlation with the increase in moisture content, and the following exponential model was used to express such relation in the present study: M (t ) = A × exp( −t /T0) + C0

MSECS (t ) = 105.3 × exp(t /18.9) + 127.3

Figure 3. Fitting model for the total peak area of water pools in untreated or steam-exploded corn stover with an increase in moisture content (A) and for the decreased total peak area of water pools in steam-exploded corn stover compared with untreated corn stover (B). UCS is untreated corn stover, and SECS is steam-exploded corn stover.

The correlation coefficient R2 was 0.9990 for UCS−water interactions and 0.9999 for SECS−water interactions, which suggested that the exponential model can well express the relations between the total peak area of water pools in UCS/ SECS and the moisture content. Compared with that in UCS, the decreased total peak area (DTPA) of water pools in SECS was also fitted with moisture content by an exponential model (Figure 3B). The correlation coefficient R2 was 0.9338, and the fitting model is given as follows:

(3)

where M is total peak area; t is moisture content, %; A, T0, and C0 are constants. The fitting results for the total peak area of water pools in UCS/SECS with an increase in moisture content are given in Table 2, and the fitting model is given as follows (Figure 3A): MUCS (t ) = 122.5 × exp(t /19.7) + 192.6

(5)

MDTPA (t ) = 87.5 × exp(t /22.6) − 1.3

(4) E

(6)


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Figure 4. Water pools distribution of untreated (A) and steam-exploded (B) corn stover under different screening meshes. “−10 m ∼ +20 m” stands for the particles that was passed 10 mesh and collected on 20 mesh during screening.

interactions between UCS and water. These results suggested that smaller particle size should facilitate the efficiency of enzymatic hydrolysis, but it also consumed more energy for size reduction in the handling process of lignocellulosic biomass. There were four water pools below 60 mesh and three water pools beyond 60 mesh. The peak area of the main water pool (water pool 4) accounted for above 54% of the total peak area below 60 mesh, while the peak area of the main water pool (water pool 3) accounted for above 89% of total peak area beyond 60 mesh (Table S2). Thus, a critical point for the transition of the water pool in UCS particles should be around 60 mesh. All of the above results showed that smaller particle size significantly enhanced the interactions between UCS and water. As for SECS (Figure 4B), the water pool transition from pool 3 to pool 4 in UCS was not observed in SECS with an increase in particle size. Interestingly, the peak height and peak width at halfheight (Wh/2) of water pool 3 hardly changed with the particle size increasing from 140 mesh to 60 mesh. However, the peak height of water pool 3 in SECS decreased, and the peak width at half-height (Wh/2) became bigger with particle size increasing from 60 mesh to 10 mesh. Particle size below 60 mesh obviously changed the peak height and the peak width at half-height (Wh/ 2) of water pools in SECS, which showed the same trends as those in UCS. The T2 relaxation times of water pool 1, water pool 2, and water pool 3 hardly changed with particle size increasing from 140 mesh to 60 mesh and then slightly extended with particle size increasing from 60 mesh to 10 mesh (Table S2). Results suggested that particle size hardly affected the T2 relaxation time of the water pools in SECS, which was different than that in UCS. T2 relaxation time was a key factor assessing the interactions between lignocellulosic biomass and water. Therefore, smaller SECS particles had slightly lower interactions with water compared with bigger ones. These results clearly suggested that SE significantly eliminated the effects of particle size on water pools distribution of SECS compared with UCS. The main reason for this result was that compared with UCS, SECS became more homogeneous by the autohydrolysis and mechanical tearing effect of SE.7,27 The screening may not be

Results showed that the total peak area of SECS−water interactions obviously decreased under low moisture content compared with that of UCS−water interactions. Results suggested that SECS−water interactions were stronger than UCS−water interactions especially under lower moisture content. The reason may be that SE modified the physical structures and altered the chemical composition of corn stover and hence enhanced the interactions between SECS and water. Most of the water in SECS presented as bound water under lower moisture content. With an increase in moisture content, the interactions between water molecules became significant, resulting in the approximate total peak area of water pools for UCS and SECS. In conclusion, the relations between the total peak area of water pools in UCS/SECS and moisture content can be well expressed by the exponential model. The interactions between UCS/SECS and water were stronger under lower moisture content. Water Pools Distribution in Different Biomass Particle Sizes. Particle size is a main factor affecting the locations and states of water and the conversion process of lignocellulosic biomass.27,28 Water pools distributions in UCS and SECS with different screening meshes were determined (Figure 4 and Table S2). As for UCS (Figure 4A), water transited from pool 3 to pool 4 with an increase in particle size. The peak width at half-height (Wh/2) of the main water pool increased, and the T2 relaxation time of the main water pool extended with an increase in particle size. The T2 relaxation times of water pool 1 and water pool 2 also extended with an increase in particle size (Table S2). All of these results suggested that smaller particle size resulted in stronger interactions between UCS and water within the present scope of particle size. The reason for this phenomenon should be that smaller particles have higher specific surface area, which should increase the accessibility of carbohydrates to water. Size reduction disrupted the well-organized plant cell wall and should expose hydrophilic groups, leading to stronger interactions between UCS and water.27,28 Additionally, UCS was heterogeneous, and the compositions and structures of UCS particles after screening were also different,28 which obviously affected the F


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Figure 5. Water pools transition with the enzymatic hydrolysis progression of steam-exploded corn stover at different solid loadings. “1% SL” stands for 1% (w/w) solid loading. “96 h” stands for 96 h of enzymatic hydrolysis.

affected by heterogeneity, and thus, the compositions and structures of different SECS particles after screening should be

approximate except for particle size. As for particle size, the porous structures should be further disrupted by milling for G


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ACS Sustainable Chemistry & Engineering smaller particles compared with those for bigger particles.27−29 Thus, the above results implied that smaller particle size slightly weakened the interactions between SECS and water. Interestingly, the peak heights of the main water pool in SECS at different particle sizes were obviously lower than those in UCS, while the peak widths at half-height (Wh/2) were bigger. Most importantly, the T2 relaxation times of water pools in SECS at different particle sizes were shorter than those in UCS. The shorter T2 relaxation time meant stronger interactions between lignocellulosic biomass and water. As a result, the lower peak heights, bigger peak widths at half-height (Wh/2), and shorter T2 relaxation times of water pools in SECS at different particle sizes also suggested that SE enhanced the interactions between SECS and water compared with those between UCS and water. Water Pools Transition in Enzymatic Hydrolysis. The states and locations of water in SECS reflected the mobility of water, which should affect the performance of enzymatic hydrolysis. The interactions between SECS and water can be considered as a metric to characterize the efficiency of enzymatic hydrolysis. The water pools distribution in enzymatic hydrolysis at different solid loadings was determined (Figures 5 and 6). Interestingly, the peak height of the main water pool (mainly capillary water) decreased, and the peak width at half-height (Wh/2) of the main water pool increased with solid loading increasing from 1% to 30% at a specific hydrolysis time (Figures 5 and 6D). The T2 relaxation times of all water pools became shorter with solid loading increasing from 1% to 30%, and the T2 relaxation time of the main water pool was below 100 ms beyond 18% solid loading (Figures 5 and 6A, C, and E). Results suggested that the interactions between SECS and water became stronger at higher solid loading, implying the reduced mobility of water. The reason for those phenomena was that water-to-solid ratio reduced with an increase in solid loading, and most of the capillary water at lower solid loading transformed into bound water at higher solid loading, resulting in stronger interactions between SECS and water. Previous reports also confirmed that each water pool in pretreated solids was constrained to a different extent, and water constraint became serious at high solid loading.12,30 The T2 relaxation time analysis of water in cellulose suspensions demonstrated that an increase in solids content led to an increase in the physical constraint of water, which ultimately increased diffusion resistance and decreased the performance of enzymatic hydrolysis.18 Water constraint (mainly capillary water) led to poor diffusion efficiency of enzymes and products at high solid loading, which should be reduced or avoided in order to achieve high efficiency of enzymatic hydrolysis. The T2 relaxation time of water pool 1 hardly changed at 1− 18% solid loading with hydrolysis progression except at 6% solid loading for 12 h (Figure 6A), while it slightly increased at 24% and 30% solid loadings before 12 h and then maintained at a certain value. However, the peak area proportion of water pool 1 at all solid loadings significantly decreased with hydrolysis progression especially before 36 h. The main reason was that water pool 1 mainly corresponded to primary bound water, which was constrained by glucan through hydrogen bonds. With hydrolysis progression, glucan was converted into glucose and then dissolved into the hydrolysate, and thus, the water content of water pool 1 decreased. A previous study also confirmed that the states and locations of water within the cellulose fibers changed with hydrolysis progression due to the structural changes of cellulose.16 The T2 relaxation time of water pool 2 hardly changed at 1−6% solid loading with hydrolysis

Figure 6. T2 relaxation times and peak heights of water pools with the enzymatic hydrolysis progression of steam-exploded corn stover at different solid loadings. “Pool 1 peak area of total peak area” means the ratio of pool 1 peak area to total pool peak area. “1% SL” stands for 1% (w/w) solid loading.

progression except at 6% solid loading for 12 h, while it increased before 12 h at 12−30% solid loading. It should be noted that the peak height of water pool 2 obviously increased before 36 h at 1−18% solid loading, and it then hardly changed. The peak height of water pool 2 increased with hydrolysis progression at 24% and 30% solid loadings. Additionally, the peak width at half-height (Wh/2) of water pool 2 decreased at all solid loadings with hydrolysis progression (Figure 5). Higher solid loading led to lower peak height and bigger peak width at H


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Figure 7. Enzymatic hydrolysis kinetics (A) of steam-exploded corn stover and glucose productivity (B) in enzymatic hydrolysis at different solid loadings. Enzymatic hydrolysis conditions: enzyme loading of 10 FPU/g glucan, 50 °C, 200 rpm, and 72 h.

Table 3. Physicochemical Property of Untreated and Steam-Exploded Corn Stovera property 2

specific surface area (m /g) pore volume (cm3/g) average pore diameter (nm) porosity (%) crystallinity index (%) surface atomic composition C1 C2 C3 O/C

SE conditions

UCS

SECS

refs

1.5 MPa (198 °C) for 6 min 200 °C for 5 min 1.5 MPa (198 °C) for 6 min 200 °C for 10 min 1.5 MPa (198 °C) for 6 min 200 °C for 10 min 1.5 MPa (198 °C) for 6 min 1.7 MPa for 1 min 1.5 MPa for 5 min 190 °C, 5 min, 3% SO2

1.2 1.0 0.23 0.29 14.2 15.1 71.5 73.3 41.5

2.9 2.3 3.21 3.52 38.6 42.5 78.9 76.0 43.8

this study 27 this study 34 this study 34 this study 35 34 37

62.0 29.2 8.0 0.33

49.9 41.9 8.2 0.45

a

SE, steam explosion; UCS, untreated corn stover; SECS, steam-exploded corn stover. C1 stands for the class of carbon that corresponds to carbon atoms bonded to carbon or hydrogen (C−C). C2 stands for the class of carbon atoms bonded to single noncarbonyl oxygen (C−O). C3 stands for the class of carbon atoms bonded to a carbonyl or two noncarbonyls (CO or O−C−O). O/C stands for oxygen to carbon ratio.

half-height (Wh/2). The main reason was that water pool 2 mainly corresponded to capillary water, which was restricted by the presence of micropores and macropores. Most of the glucan and xylan were hydrolyzed and dissolved into hydrolysate with hydrolysis progression at low solid loading, which resulted in further disruption of SECS and hence released the constrained water. Enzymatic hydrolysis at high solid loading had lower water content and sugars conversion, resulting in a lower peak height and shorter T2 relaxation time of water pool 2. As for water pool 3, the T2 relaxation time increased before 24 h at 12−30% solid loading, and then, it hardly changed with hydrolysis progression, suggesting that water pool 3 was also released before 24 h. The peak area proportion of water pool 3 hardly changed at 12% and 18% solid loadings, while it increased before 24 h at 24% and 30% solid loadings. Results indicated that enzymatic hydrolysis at 24% and 30% solid loadings obviously affected the peak area of water pool 3. From the above results, the main water pool in SECS was

obviously released before 36 h in enzymatic hydrolysis especially at low solid loading. Enzymatic hydrolysis Kinetics of Steam-Exploded Corn Stover. Enzymatic hydrolysis kinetics can be used as a tool to reveal the dissolution process of carbohydrates. Enzymatic hydrolysis kinetics of SECS under different solid loadings is given in Figure 7. Glucan conversion obviously increased before 36 h and then slightly increased with hydrolysis progression (Figure 7A). Compared with higher solid loading, lower solid loading obtained higher glucan conversion at each hydrolysis time point. The main reason was that water constraint became serious with an increase in solid loading, and thus, the mass transfer efficiency was obviously reduced.30,31 For example, the efficiency of enzymes diffusing to and products diffusing away from the reaction sites was reduced. Another reason should be that the products’ (such as monosaccharides, cellobiose, and oligosaccharide) feedback inhibition effect became serious at high solid loading.31,32 Additionally, the decreasing adsorption of enzymes I


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increase in glucan−water interactions in SECS (Tables 1 and 3).36 A previous study also reported that the glucan hydroxyl groups in wheat straw leaves after hydrothermal pretreatment were more accessible to water.13 As for surface atomic composition, C1 content of SECS decreased by 19.5% compared with that of UCS, while C2 content increased by 43.5% and C3 content increased by 2.5%.37 The oxygen to carbon ratio (O/C) of SECS increased by 36% compared with that of UCS.37 The increase in C2 and C3 contents and O/C in SECS facilitated the interactions between SECS and water. FT-IR spectra showed that SE enhanced the intensities of peaks at 3406 cm−1 attributed to O−H stretching and 1244 cm−1 attributed to C−O stretching (guaiacyl units) in SECS.27,38 Additionally, SE increased the intensities of peaks at 1167 and 1116 cm−1 arising from C−O antisymmetric bridge stretching and C−OH skeletal vibration, respectively, and at 1055 cm−1 attributed to C−O−C stretching typical of glucan and xylan in SECS.27,38 These results of surface atomic composition also indicated that the interactions of C−O, CO, O−C−O, C−OH, and C−O−C with H2O by a hydrogen bond (mainly bound water) should be significantly increased on the surface of SECS. Zhang et al. confirmed that there was a correlation between the accessibility of glucan to water and to enzymes.13 Enzymatic hydrolysis of wheat straw after hydrothermal pretreatment can be indicated by the accessibility of the hydroxyl groups to water, which meant that water can be considered as a metric to assess the glucan accessibility in lignocellulosic biomass.13 Tsuchida et al. also reported that stronger interaction of pretreated bagasse and water was consistent with better enzymes accessibility to cellulose and higher efficiency of enzymatic hydrolysis.39 Therefore, the interactions between SECS and water especially for bound water were enhanced by SE, which facilitated the adsorption of enzymes to the surface of glucan and the efficiency of enzymatic hydrolysis.13,16,25 As for enzymatic hydrolysis, the states and locations of water on the surface of SECS (mainly bound water) were closely related to the ad-/desorption of enzymes, and mobility of water inside and outside of the pores of SECS (mainly capillary water) affected the diffusion of enzymes and products.14,16,18 In the enzymatic hydrolysis of SECS, the constrained water obviously released before 36 h and then slightly changed with hydrolysis progression, which was consistent with an increase in glucan conversion. The release of constrained water was beneficial to the increase in mass transfer efficiency. These results indicated that the release of constrained water and the increase in glucan conversion facilitated each other especially before 36 h. A previous study confirmed that during the initial enzymatic hydrolysis of cellulose, the action of enzymes disrupted and loosened the cellulose, introducing more water into the structure and providing better access to enzymes.16 The results of enzymatic hydrolysis also showed that water constraint became serious withan increase in solid loading, resulting in the decrease in water mobility and mass transfer efficiency. These results should lead to low glucan conversion in enzymatic hydrolysis at high solid loading. For example, at high solid loading (30%), constrained water in SECS hardly released with hydrolysis progression, and glucan conversion was correspondingly low. Thus, how to reduce or avoid water constraint should be a main challenge for the improvement of enzymatic hydrolysis performance at high solid loading. From all the above results, the release of constrained water before 36 h and the increase in SECS−water interactions on the surface of SECS increased the efficiency of enzymatic hydrolysis.

at high solid loading should also contribute to low conversion of enzymatic hydrolysis.33 Glucose productivity rapidly decreased before 36 h and then slightly decreased with hydrolysis progression (Figure 7B). The reason was that most of the glucan was dissolved into hydrolyzate before 36 h, and the glucan content in SECS decreased. The crystalline cellulose and steric hindrance effect of lignin further led to the reduction of glucan conversion and hence low glucose productivity. Interestingly, glucose productivity increased with solid loading increasing from 1% to 18% at each hydrolysis time point, but it was approximate at 18%, 24%, and 30% solid loadings. The reason was that the increased glucose productivity should be the balance of solid loading and glucan conversion, and thus, a suitable solid loading and an ideal glucan conversion should result in high glucose productivity. Solid loading beyond 24% should not further increase glucose productivity due to strong water constraint, poor mass transfer efficiency, and product’ feedback inhibition effect. The increase in glucan conversion with hydrolysis progression under different solid loadings showed a similar trend to the increase in the peak height of main water pool (Figures 5, 6, and 7). These results suggested that the release of constrained water improved the efficiency of enzymatic hydrolysis, while an increase in glucan conversion further facilitated the release of constrained water. Water constraint was a key factor affecting the efficiency of enzymatic hydrolysis. On the one hand, the flow states and locations of water (mainly capillary water) obviously affect the mass transfer efficiency.14,18 On the other hand, the interactions between carbohydrates (glucan and xylan) and water (mainly bound water) should significantly affect the ad-/ desorption, slide and jump, and activity of enzymes.12,16,19 Biomass−Water Interactions in Steam Explosion and Enzymatic Hydrolysis Processes. Biomass−water interactions affected the conversion efficiency of lignocellulosic biomass. The states and locations of water should be determined by the pore properties (such as specific surface area, pore volume, average pore diameter, and porosity) and the surface features (such as atomic composition and groups) of lignocellulosic biomass. As shown in Table 3, the specific surface area of SECS was 2.4 times higher than that of UCS in the present study. A previous study reported that the specific surface area of corn stover increased by 130% after SE.27 Results indicated that SE obviously increased the surface area of interaction between SECS and water. The pore volume and average pore diameter of SECS were 14.0 and 2.7 times higher than that of UCS, respectively. A previous study reported that the pore volume and average pore diameter of SECS were 12.2 and 2.8 times higher than that of UCS, respectively.34 The porosity of SECS increased by 10.3% compared with that of UCS. A previous study reported that the porosity of corn stover increased by 3.7% after SE.35 The change in pore properties of corn stover by SE in the present study was consistent with previous studies.6,27,34 These results should be due to the fact that the lignin−carbohydrate complex structures of corn stover were disrupted, hemicellulose was partly removed, lignin was melted, and cellulose fibers were separated by SE.6,27,34 Although the increase in average pore diameter reduced water constraint by capillary forces (mainly capillary water), the modifications of surface area increased the amount of bound water in SECS compared with that in UCS. Glucan more easily interacted with water with a hydrogen bond, and such interaction was further enhanced by its crystalline structure. The glucan content and crystallinity index of SECS increased by about 80% and 5.5% compared with that of UCS, respectively, leading to an J


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ASSOCIATED CONTENT

S Supporting Information *

This material is available free of charge via the Internet. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.5b01303. Two tables. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +86-10-82544982. Fax: +86-1082627071. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National High Technology Research and Development Program of China (863 Program, SS2012AA022502) and the Open Funding Project of the National Key Laboratory of Biochemical Engineering (2013KF-01).

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