deacetylation on enzymatic hydrolysis of corn stalk

b i o m a s s a n d b i o e n e r g y 7 1 ( 2 0 1 4 ) 2 9 4 e2 9 8

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Effect of acetylation/deacetylation on enzymatic hydrolysis of corn stalk Wei Jiang 1, Huadong Peng 1, Hongqiang Li, Jian Xu* National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China

article info

abstract

Article history:

Effect of degree of acetylation (DA) within corn stalk (CS) on the enzymatic hydrolysis was

Received 10 June 2014

investigated. By acetylation and deacetylation treatment, CS with different DA from 0.34%

Received in revised form

to 11.20% was prepared. The analysis of chemical composition by two-step acid hydrolysis

24 September 2014

and characteristic functional groups via FTIR implied that the structure of the three major

Accepted 25 September 2014

components (cellulose, hemicellulose and lignin) in the treated CS was not changed

Available online 14 October 2014

noticeably. The correlation between enzymatic yield and DA was navigated well by the nonlinear curve fit with Adj. R-Square greater than 0.90. The DA was proved to be a crucial

Keywords:

barrier to the digestibility of CS. The efficiency of the enzymatic hydrolysis was negatively

Corn stalk

correlated with DA value. © 2014 Elsevier Ltd. All rights reserved.

Deacetylation Acetyl group Enzymatic hydrolysis FTIR

1.

Introduction

Although a rapid progress has been made on enzymatic hydrolysis of lignocelluloses, mechanisms correlating the deconstruction of plant cell wall matrix with its components and structures are not yet fully clarified. The objective of understanding the effect of the key structural features within biomass on the hydrolysis process of polysaccharides is beneficial to reducing the treatment severity in various kinds of physical, chemical and biological pretreatments, which are often used to damage the natural recalcitrance of lignocelluloses [1,2]. Some structural features significantly affecting the enzymatic hydrolysis efficiency have been identified, such as degree of acetylation (DA), lignin content, crystalline index, specific surface area, degree of polymerization, etc [3,4].

As one of the most important structural features of biomass, DA has attracted a lot of attention since 1990. By investigating the effect of DA on the enzymatic digestibility of acetylated oatspelts xylans with DA from 0.26 to 1.67 mol acetyl groups per mole of anhydro-xylose units, Mitchell et al. [5] found that the enzymatic digestibility was dramatically affected by DA. Rivard et al. [6] prepared the cellulose, starch, xylan samples with various DA and observed significant reductions in anaerobic biodegradability and enzymatic hydrolysis at substitution levels between 1.2 and 1.7. Kong et al. [7] deacetylated wood by various alkali metal hydroxide solutions at different alkali/wood ratios. It was found that the sugar yield in enzymatic hydrolysis was directly associated with acetyl groups, but not the swelling feature. Chang and Holtzapple [8] reported that acetyl groups were less important than lignin content and crystallinity index in biomass bioconversion. Later in 2006, Kim and Holtzapple [3] found

* Corresponding author. Tel./fax: þ86 10 8254 4852. E-mail addresses: [email protected], [email protected] (J. Xu). 1 Both authors contributed equally to this work. http://dx.doi.org/10.1016/j.biombioe.2014.09.028 0961-9534/© 2014 Elsevier Ltd. All rights reserved.

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Table 1 e Scheme of deacetylation/acetylation process on CS. Sample A1 A2 A3 A4 Raw D1 D2 D3 D4

Solid to liquid ratio 1: 1: 1: 1: e 1: 1: 1: 1:

Acetic anhydride (mL)

NaOH (mL)

Water (mL)

Time (h)

Temperature ( C)

200 200 200 200 e e e e e

e e e e e 4 16 48 80

0 0 0 0 e 196 184 152 120

0.5 2 4 8 e 24 24 24 24

90 90 90 90 e 25 25 25 25

20 20 20 20 20 20 20 20

A1~A4: acetylation with acetic anhydride; D1~D4: deacetylation with 1% NaOH.

that acetyl groups could be removed much easier than lignin. Selig et al. [1] observed that the acetyl groups and xylan removal can enhance not only the initial hydrolysis rates of xylan and glucan, but also the overall conversion extent. Although progresses obtained from the above studies could improve the understanding of the effect of acetyl groups on the bioconversion, more detailed mechanism on how the acetyl groups affected the whole enzymatic hydrolysis process still needs to be further investigated. In the present work, not only deacetylation was carried out on CS as other studies did, but also acetylation was included to prepare samples with different gradients of DA in order to investigate the relationship between acetylation degree and efficiency of biomass enzymolysis. Analysis of chemical composition and characteristic functional groups were used to reflect variation of major structures of CS in the deacetylation/acetylation process. Enzymatic hydrolysis was employed to testify that whether DA was crucial to the bioconversion process or not.

The treated CS from both processes was then washed with tap water until the eluant was neutral. It was then dried at 45  C before being milled to 1 mm for the following experiments. The acetylated CS was labeled as A1, A2, A3, A4, respectively, based on different treatment time. The deacetylated CS was named as D1, D2, D3, D4, according to the NaOH concentration employed.

2.3.

Enzymatic hydrolysis

The detailed procedure of enzymatic hydrolysis was the same as in our previous study [9]. The acetylated/deacetylated CS of 2 g (Dry Matter) was employed in the enzymatic hydrolysis. Cellulase purchased from the Zesheng Bioengineering Technology Co., Ltd. was the same as used in Li and Xu's study [10]. The filter paper activity (FPA) and b-glucanase activity were 46.65 ± 0.85 IU FPA cm3 and 4.97 ± 0.14 IU cm3, respectively. The enzymatic hydrolysis yield was calculated according to Eq. (1). YE ð%Þ ¼ CG  VE  ð9=10Þ=MC 100

2.

Material and methods

2.1.

Feedstock preparation

Maize (Zea mays L., 1# Jiyuan) was grown from May to October, 2011 at Yanqing County (115 970 E Longitude, 40 450 N Latitude) of Beijing, China. After maize ear was harvested, corn stalk (CS) was collected and air-dried, which was then milled to  2 mm with a grinder (RT-34S, Beijing Kun Jie Yu Cheng Machinery Co., Ltd.). To avoid the effect of monosaccharides remained in the CS on the analysis, the milled CS was washed thoroughly with tap water. It was then dried at 45  C overnight and sealed into plastic bags for the following experiments.

2.2.

Acetylation/deacetylation process

In both acetylation and deacetylation process, 10 g dried CS was used. As shown in Table 1, acetylation was carried out as follows: Dried CS was mixed with the acetic anhydride as the set solid to liquid ratio (SLR) of 1: 20, 150 rpm at 90  C for 0.5 h, 2 h, 4 h, 8 h. Deacetylation process: Dried CS was mixed with different amount of NaOH as the set SLR of 1: 20, 150 rpm at 25  C for 24 h. The final NaOH concentration was 0.005, 0.02, 0.06, 0.1 mol L1, respectively.

(1)

YE: Enzymatic yield of the theoretical, %; CG: Concentration of glucose, g L1; VE: Volume of the enzymatic reaction mixture, L; MC: Mass of cellulose in treated or raw CS, g; (9/10): Conversion coefficient of glucose to cellulose.

2.4.

Analytical methods

2.4.1.

Chemical composition

The chemical composition of treated/raw CS was analyzed according to the procedure from the National Renewable Energy Laboratory (NREL) [11]. The structural polysaccharides of CS were broke down into sugar monomers by a two-step sulfuric acid hydrolysis process. The concentration of sugar monomers, acetic acid were quantified by HPLC (Agilent 1260 Infinity, USA) via a refractive detector. A Hi-Plex H column (300  7.7 mm) was used at 65  C with H2SO4 of 0.005 mol L1 as the eluant at a flow rate of 0.6 cm3 min1. The Klason lignin was determined gravimetrically by subtracting the ash content from the solid residue obtained from acid hydrolysis. The content of acetyl groups was regarded as one component of the CS which was calculated based on Eq. (2). DAð%Þ ¼ MA =ðMP or MR Þ100

(2)

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MA: Mass of acetic acid in treated or raw CS, g; MP or MR: Mass of treated or raw CS, g.

2.4.2.

obtained under the condition that the major components of CS were not altered obviously.

FTIR analysis

3.2.

The treated/raw CS was mixed and grinded with KBr (100: 1, w/w) in an agate mortar. The characteristic functional groups of CS were analyzed by an FT/IR-600 Plus spectrometer (JASCO Corp., Tokyo, Japan) using an average of 32 scans over the range between 400 cm1 and 4000 cm1 with a spectral resolution of 1 cm1 [12].

3.

Results and discussion

3.1.

Chemical composition

To investigate the variation on chemical composition of CS in acetylation/deacetylation process, the contents of the three major components (cellulose, hemicellulose and Klason lignin) and degree of acetylation (DA) were analyzed and shown in Fig. 1. After acetylation with different treatment time (0.5 h, 2 h, 4 h, 8 h), the DA of A1, A2, A3, A4 was 3.65%, 4.22%, 10.85%, 11.20%, respectively. The maximum DA was obtained from A4 with 8 h treatment, which was 4.02 times more than that of raw CS. As Kong et al.'s study [7] has proved that deacetylation degree was confined to base to wood ratio instead of the base concentration, the acetylation should be subject to the mass ratio of acetic anhydride to CS. By employing NaOH with different concentration as the deacetylation reagent, the DA value of 2.19%, 1.79%, 0.96%, 0.34% was achieved for D1, D2, D3, D4, respectively. As can be seen from the DA value of the deacetylated samples (D1~D4), it can be concluded that the amount of NaOH used in the treatment for D4 was adequate for removing almost all the acetyl groups. Among the 9 samples (A1~A4, D1~D4, raw CS), the cellulose content was very close ranging from 34.81% to 41.61%. There was no significant variation observed on the content of hemicellulose and Klason lignin for all the samples, ranging from 20.77% to 26.77% and 13.38%e16.01%, respectively. Therefore, the CS samples prepared with different DA were

Chemical composition of CS (%)

Hemicellulose

Cellulose

9

60

6

40

3

20

0

0

A1

A2

A3

A4

Raw D1

D2

D3

FTIR was employed to observe the variation on the characteristic functional groups of CS in the acetylation/deacetylation process. Characteristic absorption peaks for acetyl groups were near 1730 cm1, 1372 cm1, and 1237 cm1 delegated for C]O, eCeCH3, eCeOe, respectively [13,14]. In comparison with the three major absorption peaks of raw CS (Fig. 2A), absorption intensity of the samples treated by acetylation was enhanced. It was consistent with the chemical composition analysis that the DA of A1~A4 was higher than that of the raw CS. Moreover, the increase of the three absorption peaks was positively related with the DA value. The deacetylated samples (D1~D4) displayed a similar trend as A1~A4 that relative higher DA value showed stronger absorption intensity of the peak that delegated to acetyl groups (Fig. 2B), and vice versa. It should be pointed out that the DA of D1, D2 and raw CS were not obviously different from each other, and thus, their FTIR curves were very close. While with extremely lower DA, D4 showed much notable reduction on characteristic absorption peak than that of the CS. In addition, the main characteristics of CS are attributed to the presence of cellulose, hemicellulose, and lignin. The peak at 1740-1750 cm1 was delegated to the unconjuagted C]O stretch in hemicellulose. The bands at 1423 cm1, 1243 cm1 were representative for the symmetric deformation of CH2 and CeOeC chain in cellulose, respectively. The band at 1160e1170 cm1 was in connection with the asymmetric deformation of CeOeC of the cellulose and hemicellulose [14,15]. Two types of lignin (guaiacyl and syringyl lignin) exist in CS. The lignin characteristic peaks were attributed to the bands at 1214 cm1, 1268 cm1 (CeO of guaiacyl ring), 1315 cm1 (CeO of syringyl ring), and 1502e1606 cm1 (aromatic skeletal vibration) [13,16]. The band intensities of the treated CS listed above were in comparable with that of raw CS. It can thus be concluded that the chemical structures of the three major components in CS were not changed substantially in the acetylation/deacetylation process.

3.3.

12

Degree of acetylation (%)

Klason lignin

80

D4

Fig. 1 e Chemical composition and degree of acetylation of CS A1~A4: CS acetylated with acetic anhydride; Raw: Raw CS; D1~D4: CS deacetylated by NaOH.

Variation on characteristic functional groups

Enzymatic hydrolysis of acetylated/deacetylated CS

The enzymatic hydrolysis was carried out to investigate the digestibility of CS with different DA value which is shown in Fig. 3. The A4 with the maximum DA of 11.20% showed a minimum enzymatic yield (YE) of 6.2% after 120 h, lower than that of raw CS which was 18.49%. Cellulose in D4 with a minimum DA of 0.34% was enzymatically converted to glucose with YE of 39.99%, more than two time higher than that of raw CS. The YE of A3 and A4 were close which were the lowest in all the samples. This might be due to their higher and similar DA of 10.85% and 11.20%, respectively. Similarly, the A1, A2, Raw, D1, D2 with a close DA displayed the similar EY. It can be seen from Fig. 2 that the samples treated by the acetylation (A1~A4) showed much lower EY than that from the samples prepared by deacetylation (D1~D4). It can be concluded that the existence of acetyl groups showed negative effects on the digestibility of CS.

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b i o m a s s a n d b i o e n e r g y 7 1 ( 2 0 1 4 ) 2 9 4 e2 9 8

140

T%

120 100 80 A1

(A)

60

A2 A4

1730 A3

1372

1730

1372

1237

Raw

100

T%

80 60 40 D1

(B)

20

D2 D4

4000

3200

D3

1237

Raw

2400

1600

800

Wavenumber (cm-1)

Fig. 2 e FTIR spectra of acetylated/deacetylated CS.

To further clarify the effect of DA on the enzymatic hydrolysis, the EY at 17 h, 24 h were selected to be over fitted with DA values. The Adj. R-Square of nonlinear curve fit was 0.94 and 0.91 for the enzymatic hydrolysis of 17 h and 24 h, respectively, indicating that the model chosen above was reasonable to navigate the relationship between DA and EY (Fig. 4). The similar curve correlating EY with DA has been reported [17], but lack of the related model to explain it. As DA reduced, EY was susceptible negatively to the variation of DA, showing that acetyl groups at high concentration could inhibit the enzymatic hydrolysis process partly or even completely, which was consistent with the results observed by Mitchell et al. [5] and Rivard et al. [6]. Two possible reasons might be

45 40

A1 A4 D2

A2 Raw D3

30 25 20 15 10 5 0 24

48

Conclusions

In order to make the present study universally applicable, corn stover with different DA was prepared by both acetylation and deacetylation and used to explore the effect of DA on the process of enzymatic hydrolysis. The analysis of chemical

40

A3 D1 D4

35

0

4.

Enzymatic hydrolysis ratio (%,w/w)

Enzymatic yield (%, of the theoretical)

50

responsible: (1) Hydroxyl groups of cellulose were substituted by the acetyl groups which hindered the binding formation of cellulase and cellulose through hydrogen bonds; (2) Acetyl groups changed the hydrophobicity of cellulose and increased the dimension (diameter) of the cellulose chain [18].

72

96

120

Time (h) Fig. 3 e Enzymatic hydrolysis on acetylated/deacetylated CS.

35 30

Model

NewFunction2 (User)

Equation

y=A*exp(-B*x)

Reduced Chi-Sqr

10.02971 0.90929

Adj. R-Square

8.21929 0.93513 Value

25 20

Standard Error

H

A

36.88538

H

B

0.35924

3.52506 0.05993

I

A

38.99634

3.09057

I

B

0.33131

0.04763

15 10

17h 24h

5 0 0

2

4

6

8

10

12

Ratio of Acetylation (%,w/w) Fig. 4 e Correlation between degree of acetylation and enzymatic hydrolysis yield.

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composition and FTIR showed that the major components of CS except DA were not varied obviously in acetylation/ deacetylation process. The nonlinear curve fit was proved to be reasonable in the predication of the relationship between enzymatic yield and DA. The DA was verified to be a crucial obstacle in the bioconversion process. CS with lowest DA gave the highest enzymatic conversion. The results found in the present study will be beneficial in guiding the design of the new biomass pretreatment regarding the appropriate deacetylation.

Acknowledgments The work was financially supported by the National Natural Science Foundation of China (No. 21276259), the National Basic Research Program of China (973 Program: 2011CB707401), the National High Technology Research and Development Program of China (863 Program: 2012AA022301), and the 100 Talents Program of the Chinese Academy of Sciences.

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