Electron beam irradiation to enhance enzymatic saccharification of alkali soaked Artemisia ordosica used for production of biofuels

Accepted Manuscript Title: Electron beam irradiation to enhance enzymatic saccharification of alkali soaked Artemisia ordosica used for production of biofuels Authors: Yulin Xiang, Yuxiu Xiang, Lipeng Wang PII: DOI: Reference:

S2213-3437(17)30362-7 http://dx.doi.org/doi:10.1016/j.jece.2017.07.058 JECE 1773

To appear in: Received date: Revised date: Accepted date:

5-5-2017 29-6-2017 23-7-2017

Please cite this article as: Yulin Xiang, Yuxiu Xiang, Lipeng Wang, Electron beam irradiation to enhance enzymatic saccharification of alkali soaked Artemisia ordosica used for production of biofuels, Journal of Environmental Chemical Engineeringhttp://dx.doi.org/10.1016/j.jece.2017.07.058 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Electron beam irradiation to enhance enzymatic saccharification of alkali soaked Artemisia ordosica used for production of biofuels

Yulin Xiang1*, Yuxiu Xiang2, Lipeng Wang1

1

College of Chemistry and Chemical Engineering, Yulin University, Yulin 719000 Shaanxi Province, China;

2

Department of Management Engineering, Qiqihar Institute of Engineering, Qiqihar 161005 Heilongjiang

Province, China

Highlights: 1. Alkali presoaking with EBI on A. ordosica enzymatic hydrolysis. 2. Optimal factors were 1.5%, 8%, 24 h and 7.5 kGy for reducing sugar yield. 3. Reducing sugar yield increased by 1136.75% after alkali soaked-EBI pretreatment. 4. A. ordosica with alkali soaked-EBI pretreatment is promising for enzymolysis.

Abstract Artemisia ordosica, a promising biofuel production material, was pretreated by alkali (Ca(OH)2) soaking-electron beam irradiation (EBI) to improve enzymatic digestibility. After 72 h enzymatic hydrolysis, alkali soaking-EBI pretreated sample showed high reducing sugar yield. The content of lignin and hemicellulose in Artemisia ordosica decreased after alkali soaking-EBI pretreatment. SEM observation showed that the Artemisia ordosica surface with alkali soaking-EBI pretreatment suffered serious erosion. The conditions of alkali soaking-EBI pretreatment and enzymatic hydrolysis were optimized. Under the optimal conditions, the reducing sugar yield of Artemisia ordosica

*

Corresponding author E-mail: [email protected]; Phone: +8613720699281 1

achieved 520.67 mg/g, which increased by 1136.75% comparing to the untreated control group. The Artemisia ordosica with alkali (Ca(OH)2) soaking-EBI pretreatment is a promising potential for biofuel production. Keywords: Artemisia ordosica; Alkali soaking; Electron beam irradiation; Enzymatic hydrolysis; Lignocellulosic biomass 1. Introduction Sustainable biofuels (e.g. biogas, cellulosic ethanol, biodiesel, etc.) produced from renewable biomasses are being exploited as alternative energy sources due to the exhaustion of fossil energy resources, increasing fuel costs, and serious environmental pollution problems caused by the use of fossil fuels [1]. Lignocellulosic biomass is considered a powerful natural resource for the production of biofuels [2]. The biofuels can be produced from various lignocellulosic biomasses, including trees, grasses, as well as different kinds of wastes and residues of plant origin from yard trimmings, agroindustrial residues, and paper and food industry wastes etc [3]. These lignocellulosic biomasses consist of lignin, cellulose and hemicellulose in different ratios [4]. Hemicellulose and cellulose are the target substrates in the conversion process for biomass, whereas lignin, being a barrier for the efficient conversion of lignocellulosic biomass, is hardly depolymerized by only hydrolases [5]. Different types of pretreatment methods, such as steam explosion pretreatment [6], hot compressed water pretreatment [7], organic solvent pretreatment [8], dilute acid pretreatment [9], alkali pretreatment [10], ionic liquids (ILs) pretreatment [11], and irradiation pretreatment methods [12], have been proposed for the improvement of enzymatic hydrolysis of lignocellulosic biomass. Among the pretreatment methods, 2

irradiation pretreatment method (especially electron beam irradiation) is considered to be one of the most promising pretreatment method due to its prominence for biomass delignification, cellulose crystallinity reduction and sugar solubilization [13-15]. Major changes that electron beam irradiation bring about in the cellulose are, decrease in the crystallinity and molecular weight and increase in the surface area [14]. Structural changes caused by the high energy electron beam irradiation in the lignocellulosic polymer network facilitate the enzymatic saccharification [15]. Preliminary research studies have evidenced improvements in the enzymatic saccharification of cellulosic material after electron beam irradiation treatment. It is also important to emphasize that the effectiveness of the electron beam irradiation pretreatment on the rate of enzymatic hydrolysis depends on the nature of the biomass with respect to energy delivered, sources and concentration of enzymes used. Furthermore, this method also has many other advantages as below: mild temperature, short treatment time, and less undesirable inhibitory byproducts than chemical pretreatment methods etc [13]. Due to the efficiency of electron beam irradiation, a high dose rate can be achieved at shorter time period with good utilization of energy and development of indigenous industrial scale electron beam irradiation facilities has opened up opportunities for researchers. On the other hand, Jin et al. (2016)[16] has reported that hemicellulose and lignin were solubilized more significantly in an alkaline environment than that of in the other surroundings. Corn stover was pretreated by NaOH (80℃), and the enzymatic hydrolysis efficiency was obviously enhanced [17]. Hydrothermal- mechanochemical pretreatment of eucalyptus at 170℃ with Ca(OH)2 solution showed a glucose yield of 90% [18]. Willow sawdust was pretreated by fungal-NaOH, and microstructure of the 3

lignocellulosic biomass was improved significantly [4]. However, enhancing enzymatic saccharification efficiency using electron beam irradiation-alkaline pretreatment method has not been reported. Artemisia ordosica is a perennial subshrub biomass. It has especially easy survival, fast growth, strong drought tolerance, and is planted as a green plant in desert. It has many uses, such as phytoremediation, landscaping, wind-break and sand-fixing function etc [19]. However, using A. ordosica to produce second generation biofuels has not been reported. Therefore, the objectives of this work were to investigate the influences of the alkali soaking-electron beam irradiation pretreatment on the physicochemical characteristics of A. ordosica; to study the effects of the alkali soaking-electron beam irradiation pretreatment on the reducing sugar yield; and to optimize the operating condition of enzymatic saccharification of A. ordosica. 2. Materials and methods 2.1. Collection and preparation of materials A. ordosica from Mu Us desert, China was collected. The sample (moisture content: 89.68%) was naturally dried (temperature: 22±3 ℃; humidity: 40±5%) for 2 weeks, then the dried sample was crushed and sieved (40 mesh). The obtained sample sealed in plastic bags was stored at room temperature. Ca(OH)2 (chemical pure) was purchased from Comio Chemical Reagent Co., Ltd., Tianjin, China. Cellulase was purchased from Choi Biotechnology Co., Ltd., Ningxia, China, which was generated by Acremonium cellulolyticus, and its activity is 1,040 μ/g (pH 5.7). Enzyme activity was measured by IUPAC method, and filter paper size was 1×1 cm2 square piece. 2.2. Alkali soaking-based electron beam irradiation pretreatment 4

Prior to EBI, 10 g prepared sample was soaked in 200 mL alkali solution with a dosage of 0.5% (w/v) Ca(OH)2 for 24 h. A control group was prepared using the same water (deionized water) in the same liquid to solid ratio to investigate the influence of moisture if any. After the soaking, the sample was instantly sealed in polystyrene bags before EBI pretreatment. EBI experiments of alkali soaked A. ordosica were conducted at the electron beam facility with 1.5 MeV of electrons energy and 6 m/min conveyor stream velocity (Changchun Yi Fu Irradiation Accelerator Ltd., China). The sample was placed in a Pyrex tray (the width: 2.5-5.5 cm, and about 320 g each absorbed dose). The selected doses were in the range from 0 kGy to 10 kGy. Samples were scanned uniformly by 10 mm diameter beam over 1 m length of the conveyor (speed 1 m/min) delivering a dose of 20±2 Gy per pass. Selected doses were achieved by calculating the number of passes and were confirmed by radio-chromatic films. 2.3. Enzymatic hydrolysis The pretreated A. ordosica of 0.5 g was soaked in 10 mL of 0.055 mol /L (pH 4.8) citrate buffer in a 50 mL flask, and then cellulase was added into the flask. Enzymatic hydrolysis was carried out in a shaking incubator at 180 rpm and 50 ℃ for 50 h. The sample was taken at stated time interval for the measure of reducing sugar using DNS method [20]. 2.4. Analysis methods The chemical composition of A. ordosica was analyzed by an automatic fiber analyzer (ANKOM 2000i, USA). The structure variations of A. ordosica were observed using a scanning electron microscope (SEM, Quanta-600, FEI) before and after pretreatment. The specific surface area (SSA) of A. ordosica was measured by the surface 5

area and pore size analyzer (ASAP-2000, MIC, USA), and calculated according to Brunauer, Ennett and Teller (BET) mothod [14]. FT-IR spectra of pretreated/untreated A. ordosica was recorded by a Fourier Transform Spectrometer (IR Prestige-21). It was used to study the component changes of A. ordosica before and after pretreatment. The wavenumber range is 4000 to 500 cm-1 using 100 scans at 4 cm-1 resolution. The mass ratio of A. ordosica (10 mg) and KBr was 100:1. The crystallinity of A. ordosica was measured by an X-ray diffractometer (D/MAX-2400) using Cu kα radiation source carried out at 30 mA and 40 kV, and the sample was scanned over a 2θ range from 5° to 40° with a step size of 0.2° and a scan speed of 2°/s. The crystalline index (CrI) was obtained by Eq. (1) [21]:

(1) Where I002 represents the diffraction intensity of crystalline structure at 2θ=22.6°; Iam represents the diffraction of the amorphous portion at 2θ=18.0°. 2.5. Data analysis Each experiment was performed in triplicate. In order to minimize the systematic error, all measurements were replicated 3 times. The data were analyzed using the Origin 8.0 software. 3. Results and discussion 3.1. Composition change before and after pretreatment of A. ordosica The chemical component content of several frequently-used lignocellulosic biomass and A. ordosica are shown in Table 1. The cellulose content of A. ordosica was higher 6

(53.1%) than some frequently-used bioenergy biomass. During enzymatic hydrolysis, the reducing sugars yields increased with the cellulose content increase [22]. Therefore, the high cellulose content in A. ordosica is propitious to bioenergy production. Table 1 Chemical composition of frequently-used lignocellulosic biomass and A. ordosica. Materials

Willow

Catalpa

Hybrid napier grass

Switchgrass

Corn stove

A. ordosica

Lignin (%) Cellulose (%) Hemicellulose (%)

28.7 35.6

15.8 51.2

15 36

18.4 35.4

19.3 38.7

15.9 53.1

21.5

16.0

28

31

21.7

15.3

Maria et al., 2016 [4]

Jin et al., 2016 [16]

Karthika et al., 2013 [15]

Smith et al., 2014 [23]

Zhao ＆ Xia, 2009 [17]

This study

Reference 60

Cellulose Hemicellulose Lignin

50

g /100 g TS

40 30 20 10 0 No treated

Ca(OH)2

EBI

EBI+Ca(OH)2

3 4 Fig. 1. The 1effect of2 different pretreatments on the chemical composition of A. ordosica (10 g prepared sample, 200 ml alkali solution with a dosage of 0.5% (w/v) Ca(OH)2, soaking time of 24 h; 5 kGy EBI).

Fig. 1 shows the effect of different pretreatments (alkali soaking pretreatment and/or EBI pretreatment) on the fractionation of A. ordosica in terms of lignin, hemicellulose and cellulose. The values of lignin, hemicellulose and cellulose are presented per g of 100 g initial total solids (100 gTS). It can be seen that the weight loss after combined alkali soaking with EBI pretreatment of A. ordosica was the highest, followed by alkali soaking and EBI pretreatment alone. The percentage lignin degradation for A. ordosica was 21.6% (from 15.9 ± 0.3 to 12.7 ± 0.0 g/100 gTS) due to the combined pretreatment with alkali soaking and EBI, and lignin degradation (8.0% and 8.6%) was also observed during EBI 7

and alkali soaking pretreatment alone (from 15.9 ± 0.3 to 14.8 ± 0.1 g/ 100 gTS, and from 15.9 ± 0.3 to 14.3 ± 0.5 g/100 gTS), respectively. The fact that EBI or alkali soaking pretreatment affects lignin degradation has been also confirmed by other researchers [24-26]. A significant increase of the lignin degradation extent using combination pretreatment method was also observed by Soo et al. (2015) [27], who investigated the combination of acid or alkali (H2SO4 and NH3·H2O) pretreatment with EBI pretreatment on Miscanthus sinensis. The respective fractions of hemicellulose and cellulose degradation were 45.8 and 10.6% for combined alkali soaking with EBI pretreatment, 28.7 and 4.6% for EBI pretreatment alone, and 35.0 and 7.0% for EBI pretreatment alone. To summarize, the effect of these pretreatment methods on cellulose of A. ordosica was slight. As anticipated, the hemicellulose and lignin degradation efficiency was higher for combined alkali soaking and EBI pretreatment, when compared with alkali soaking or EBI pretreatment alone. Additionally, the combination of alkali soaking and EBI pretreatment resulted in higher cellulose and lignin degradation, especially hemicellulose degradation, which could be attributed to higher molecular degradation efficiency for the combination pretreatment method. Meanwhile, other researchers also found that the hemicellulose was more easily removed [28, 29]. The degradation of hemicellulose improved the enzymatic hydrolysis owing to the structure change of biomass [30]. 3.2. Effect of pretreatment on A. ordosica characterization

8

40

a

12

CrI -☆- SSA

10

30

2

SSA(m /g)

CrI(%)

8 6

20

4 10 2 0 No treated

Ca(OH)2

0 EBI+Ca(OH)2

EBI

c

Transmittance(%)

1596

Ca(OH)2

1507

EBR+Ca(OH)2 EBR No treated 3043 1738

4000

3500

3000

2500

2000

1242 1158 896

1500

1000

500

Wave number(cm-1)

Fig.2. The changes in characterizations of untreated and irradiated HPS. (a: CrI and SSA; b:XRD; c:FT-IR. 10 g prepared sample, 200 mL alkali solution with a dosage of 0.5% (w/v) Ca(OH)2, soaking time of 24 h; 5 kGy EBI). The crystallinity and specific surface area are key factors to assess the effects of a pretreatment method on the hydrolysis of biomass [14, 31]. CrI (crystallinity index) is the ratio of crystalline cellulose of lignocellulosic biomass. X-ray measurement of CrI is the best method to investigate the influence of pretreatment on biomass crystallinity [14]. Fig. 2a presented the changes of CrI and SSA (specific surface area) of untreated and pretreated A. ordosica. As seen from Fig. 2a, compared with the untreated sample, the CrI values were negative after EBI pretreatment alone. This indicated that crystalline structure of A. ordosica was disrupted to some extent in this pretreatment. The results were similar to the findings of Liu et al. (2015) [14]. While the CrI increased after combined alkali soaking with EBI pretreatment or alkali soaking pretreatment alone. Comparing to the raw A. ordosica, the CrI of samples pretreated by alkali soaking-EBI and alkali soaking alone 9

increased by 6.44% and 19.63%, respectively. Other studies also found increase of CrI, and the efficiency of enzymatic hydrolysis increased accordingly. The CrI of catalpa sawdust increased by 25.71%, and reducing sugar yield increased significantly after thermo-Ca(OH)2 pretreatment [16]. The CrI of water-soaked rice straw increased slightly after EBI pretreatment, and glucose yields from the pretreated rice straw after 120 h of hydrolysis reached 70.4% [32]. XRD image of untreated and pretreated A. ordosica was shown in Fig.2b. The peaks for all curves appeared at 2θ=17.3°, 22.45°, 35.44°, which represented respectively diffraction peaks of 101, 002 and 040 crystalline surfaces for the cellulose of A. ordosica [33]. It can be seen that no matter untreated or pretreated A. ordosica, their 2θ of the most powerful peak remained unchanged. The result indicated that the polymorphic form of cellulose was not changed under the different pretreatments despite the CrI varying. Compared to the untreated sample, peak intensities presented different increase levels at the samples pretreated by alkali soaking-EBI and alkali soaking alone, while peak intensities decreased at the sample pretreated by EBI alone, especially peak intensity of 002 crystalline surface. The results showed that the relative content of crystallization region decreased under irradiation pretreatment, and increased under alkali soaking pretreatment. The results were also in accordance with the change trends of CrI under different pretreatment. Similar findings were reported by other researchers [34, 35]. It can be seen that the SSA was increased after the pretreatments. The SSA after combined alkali soaking with EBI pretreatment of A. ordosica was the highest (11.66±0.4 m2/g), followed by EBI pretreatment alone (7.56±0.2 m2/g), and alkali soaking pretreatment alone (4.80±0.3 m2/g). Therefore, the combination pretreatment presented an excellent effect on SSA of A. ordosica, and the influence on CrI of A. ordosica is also 10

significant. Otherwise, alkali soaking pretreatment alone showed great influence on its CrI, but little influence on SSA of A. ordosica. EBI pretreatment alone showed great influence on its SSA of A. ordosica, but the CrI slightly decreased after EBI pretreatment alone. Liu et al. (2015) reported that irradiation pretreatment on microcrystalline cellulose showed a significant influence on its SSA, but little effect on its CrI value. Alkaline pretreatment possessed great effect on CrI of sample, but little effect on its SSA [14, 16, 4, 36]. The FT-IR spectroscopy has been widely used for monitoring the chemical and structural changes of lignocellulosic feedstocks after various pretreatments [15]. FTIR image of untreated and pretreated A. ordosica was shown in Fig.2c. Fig.2c showed major peaks at 3043, 1738, 1596, 1507, 1242, 1158 and 896 cm-1 in untreated and pretreated A. ordosica. Decrease in the intensity of characteristic peak was observed in the prominent IR bands at 1738 cm-1(C=O stretching vibration), 1595 cm-1 (carbon skeleton vibration of benzene ring, Lignin), 1507 cm-1 (carbon skeleton vibration of aromatic ring, Lignin), 1242 cm-1 (Ar-O stretching vibration, Lignin), 1158 cm-1 (C-O-C stretching vibration, cellulose and hemicellulose), 896 cm-1 (Anomeric carbon-C1 vibration frequency, Polysaccharide). Slight shifts in many characteristic bands are also showed for the pretreated samples. It indicated that the structure of A. ordosica occurred change under the pretreatment conditions [34]. This founding is consistent with the CrI result of Fig.2a. The research about lignocellulosic structure indicated that in addition to amorphous and crystalline regions, lignocellulosic polymer also possesses an intermediate paracrystalline phase, and the pretreatments are helpful to the transformation between different forms (crystalline and amorphous) of the polymer [37]. Such transformations were favorable to 11

the enzymatic hydrolysis of recalcitrant lignocellulosic structure [15, 38]. 3.3. Enzymatic hydrolysis of pretreated A. ordosica 160

Reducing sugar yield(mg/g)

140 120 100

Control EBI Ca(OH)2

80

EBI+Ca(OH)2

60 40 20 0

0

10

20

30

40

50

Time(h)

Fig.3. Reducing sugar yield of A. ordosica before and after pretreatment. (solid content of 5% (w/v), alkali dosage of 0.5% (w/v), soaking time of 24 h; 5 kGy EBI, enzyme loading of 25 FPU/g, and enzymatic hydrolysis time of 50 h). The purpose of biomass pretreatment is improving its enzymatic efficiency. The enzymatic hydrolysis results of pretreated A. ordosica are shown in Fig. 3. Obviously, the reducing sugar yield of pretreated samples was higher than that of control, especially that of the samples pretreated by alkali soaking-EBI and alkali soaking alone, and reached 156.3 and 138.5 mg/g, respectively. The lignin and hemicellulose were major barriers to enzymatic hydrolysis, and the alkali soaking-EBI pretreatment degraded and solubilized plentiful of hemicellulose and lignin (as shown in fig.1) to improve enzymatic hydrolysis of the biomass. The hemicellulose was an essential part of cellulose-hemicelluloses-lignin network and improved the network stability [16]. However, the stability of hemicellulose was weaker than that of cellulose. After alkali soaking-EBI pretreatment, the hemicellulose of A. ordosica was firstly solubilized, causing destruction of the network structure. Simultaneously, another obstacle was removed by alkaline pretreatment, namely lignin. Delignification has been reported as the main method for alkaline pretreatment to 12

enhance the enzymatic hydrolysis of biomass [39]. The sodium hydroxide pretreatment of rapeseed straw showed 42.3% delignification yields for 40 min of treatment under high voltage electrical discharges (204-814 kJ/kg) [40]. The napier grass pretreated by sodium hydroxide showed more than 84% lignin loss and the high glucan conversion rate (94%) was reached by enzymatic hydrolysis [41]. Combining the results of Fig. 1 and Fig. 2, it was found that the degradation of hemicellulose and lignin might be the primary causes for enhancing reducing sugar yield of alkali soaking-EBI pretreated samples. 3.4. Optimization of alkali soaking-electron beam irradiation pretreatment a

b 250

absorbed dose:7.5kGy solid content: 8% (w/v) presoaked time: 20 h

200

Reducing sugar yield (mg/g)

Reducing sugar yield (mg/g)

250

150 100 50 0

0.25

0.50

0.75

1.00

1.25

1.50

1.75

absorbed dose:7.5kGy solid content: 8% (w/v) Ca(OH)2 dosage: 1.5%

200 150 100 50 0

2.00

Ca(OH)2 dosage(%)

0

4

8

12

16

20

24

28

32

36

40

Presoaked time(h)

300

c

absorbed dose:7.5kGy; Ca(OH)2 dosage: 1.5% presoaked time: 24 h

Reducing sugar yield (mg/g)

Reducing sugar yield (mg/g)

250

250

200 150 100 50 0

0

2

4

6

8

Solid content (% (w/v))

10

12

200

d presoaked time:24 h solid content: 8% (w/v) Ca(OH)2 dosage: 1.5%

150 100 50 0

0

2

4 6 Absorbed dose (kGy)

8

10

Fig.4. Effect of pretreatment conditions of A. ordosica on reducing sugar yield (enzyme loading of 25 FPU/g, and enzymatic hydrolysis time of 50 h). The results of reducing sugar yield after three different pretreatments showed that the alkali soaking-EBI pretreatment was the best for reducing sugar yield from A. ordosica. Therefore, it is necessary to investigate the optimal operating conditions of alkali soaking-EBI pretreatment. Fig.4. presents the effects of different pretreatment conditions 13

of A. ordosica on reducing sugar yield. Fig. 4a shows the enzymatic hydrolysis of alkali soaking-EBI pretreated A. ordosica with different Ca(OH)2 dosages. The enzymatic hydrolysis efficiency first increased and then decreased with the increase of Ca(OH)2 dosage (from 0.25 to 2% (w/v)), when the Ca(OH)2 dosage was 1.5%, the enzymatic hydrolysis efficiency achieved the maximum (244.33 mg/g). Accordingly, the Ca(OH)2 dosage of 1.5% was chosen for the following researches. Catalpa sawdust was pretreated by thermo-Ca(OH)2 and the reducing sugar yield was increased to about 225 mg/g at 100 ℃ when the Ca(OH)2 dosage was 1.75% [16]. Fig. 4b presents the reducing sugar yield as a function of the presoaked time. The reducing sugar yield increased along with the increase of presoaked time until the presoaked time reached to 24 h, and then stopped increasing. The maximum reducing sugar yield was 258.93 mg/g. This variation tendency was similar with the research on acid and alkali presoaked-EBI pretreated hybrid napier grass [15]. Therefore, the presoaked time of 24 h was chosen as the optimal parameter. Fig. 4c presents the effect of solid content of A. ordosica on the reducing sugar yield. The reducing sugar yield reduced with solid content increasing. The decrease rate was first slight from 1% to 8%, then the decrease rate was significant from 8% to 11%. Lower solid content produced higher reducing sugar yield, which was similar with the researches from water soaked-EBI pretreated rice straw [32] and thermo-Ca(OH)2 pretreated catalpa sawdust [16]. In order to enhance the pretreatment efficiency, the solid content of 8% was chosen. Fig. 4d shows the effect of absorbed dose on the reducing sugar yield of A. ordosica. 14

It was clearly showed that the reducing sugar yield (from 41.51 mg/g to 261.66 mg/g) increased with the increase of absorbed dose from 0 to 7.5 kGy. It might be attributed to higher SSA and depolymerization of A. ordosica treated by irradiation at 7.5 kGy, which supplied a broad surface area and lots of cellulose chain terminals for accessibility of cellulase [14, 42]. However, the reducing sugar yield decreased from 261.66 mg/g to 226.21 mg/g with the further increase of absorbed dose from 7.5 kGy to 10 kGy. This decrease of enzymatic hydrolysis might be caused by the accumulation of by-products inhibitors in high absorbed dose of EBI pretreatment process, such as, oligosaccharides and formic acid, and even brown pigment [14, 43]. 3.5. Optimization of enzymatic hydrolysis and Mechanism analysis

Reducing sugar yield(mg/g)

700 25FPU/g 50FPU/g 75FPU/g 100FPU/g 125FPU/g 150FPU/g 175FPU/g

600 500 400 300 200 100 0

0

20

40

60

80

100

Time(h)

Fig. 5. Optimization of enzymatic hydrolysis of A. ordosica (Pretreatment conditions: Ca(OH)2 dosage of 1.5%, solid content of 8%, presoaked time of 24 h, absorbed dose of 7.5 kGy). Based on the above studies, Ca(OH)2 dosage of 1.5%, solid content of 8%, presoaked time of 24 h, and absorbed dose of 7.5 kGy were chosen. Under these conditions, the optimum enzymatic hydrolysis time and enzyme loading were investigated. Fig. 5 shows the 108 h enzymatic hydrolysis of Ca(OH)2 soaking-EBI pretreated A. ordosica with different enzyme loadings. As it is shown on Fig. 5, the reducing sugar yield 15

from pretreated A. ordosica increased with the increase of enzymatic hydrolysis time until 72 h, and then increased insignificantly. Thus, the hydrolysis time of 72 h was selected for the following experiments. At 72 h, the reducing sugar yield increased from 214.11 to 523.89 mg/g with an enzyme loading range of 25-175 FPU/g. However, when the enzyme loading increased from 150 to 175 FPU/g, the increase of the reducing sugar yield was not obvious. It revealed that the cellulose sites were saturated at a cellulase loading of 150 FPU/g [34]. Therefore, Ca(OH)2 dosage of 1.5%, solid content of 8%, presoaked time of 24 h, absorbed dose of 7.5 kGy, enzymatic hydrolysis time of 72 h, and enzyme loading of 150 FPU/g were chosen for improving enzymatic saccharification of A. ordosica. Under these conditions, the reducing sugar yield reached 520.67 mg/g, and was higher than reported in the previous works [34, 3, 16]. The reducing sugar yield of the control sample (without pretreatment, enzymatic hydrolysis time of 72 h, and enzyme loading of 150 FPU/g) was 42.10 mg/g, the reducing sugar yield of the EBI treatment group (7.5 kGy, other conditions were the same as the control group) was 63.55 mg/g, and the reducing sugar yield of the alkali soaking treatment group (Ca(OH)2 dosage of 1.5%, solid content of 8%, presoaked time of 24 h, other conditions were the same as the control group) was 188.23 mg/g. The reducing sugar yield under the optimal conditions increased by 1136.75%, 719.24% and 176.59% comparing to the control, the EBI treatment group and the alkali soaking treatment group, respectively. Consequently, alkali (Ca(OH)2) soaking-EBI pretreatment for improving enzymatic saccharification of A. ordosica shows a promising potential for biofuel production.

16

Fig. 6. SEM images of A. ordosica (a: raw A. ordosica, b: A. ordosica after combined alkali soaking with EBI pretreatment, c: A. ordosica after enzymatic hydrolysis; Pretreatment conditions: Ca(OH)2 dosage of 1.5%, solid content of 8%, presoaked time of 24 h, absorbed dose of 7.5 kGy; enzymatic hydrolysis conditions: enzyme loading of 150 FPU/g, enzymatic hydrolysis time of 72 h). Fig. 6 shows that A. ordosica structures before and after treatment. The SEM images illustrated that the A. ordosica appearances before and after treatment were distinctly different. The lignocellulosic structure of raw A. ordosica was comparatively tight and coherent. After alkali (Ca(OH)2) soaking-EBI pretreatment, the lignocellulosic structure of A. ordosica was disfigured, and became loose, leading to increase of surface area and porosity. While the lignocellulosic structure of A. ordosica after enzymatic hydrolysis was further damaged, and cellulose and hemicellulose in the raw material were fully degraded. Thus, the reducing sugar yield was significantly increased. enzyme hydrolyzate

→ of A. ordosica

Transmittance(%)

→ glucose solution 2966.73

3321.38 1051.66 1633.45

4000

3500

3000

2500

2000

1500

1000

Wave number(cm-1)

Fig. 7. FT-IR of enzyme hydrolyzate of A. ordosica under the optimal conditions and glucose solution. 17

Fig. 7 shows that FT-IR images of enzyme hydrolyzate of A. ordosica and glucose solution. As seen from Fig. 7, The FT-IR images of the enzyme hydrolyzate of A. ordosica and glucose solution were closely similar. Characteristic peaks from enzyme hydrolyzate of A. ordosica were observed at 3321.38 cm-1(-OH stretching vibration), 2966.73 cm-1 (C-H stretching vibration), 1633.45 cm-1 (C=O stretching vibration), 1051.66 cm-1 (C-O stretching vibration), and these peaks are all characteristic peaks of glucose molecules. The results indicated that enzyme hydrolyzates of A. ordosica were mainly glucose-dominated reducing sugars. In addition, fermentation inhibitory compounds from the combined process (such as formic acid, acetic acid and levulinic acid) did not occur in Fig. 7.

Fig. 8. Predicted mechanism of electron beam irradiation to enhance enzymatic saccharification of alkali soaked Artemisia ordosica. The lignin and hemicellulose both were main barriers to valid enzymatic hydrolysis, and the hemicellulose was a major part of cellulose-hemicelluloses-lignin network and promoted the stability of the network structure [16]. But the hemicellulose was not as stable as cellulose. After alkali soaking pretreatment, the hemicellulose of the Artemisia ordosica was firstly damaged, leading to the structure destroy of 18

cellulose-hemicelluloses-lignin network. In this work, hemicellulose and lignin could be partly destroyed and improve accessibility of the enzymes to the inner structure of the Artemisia ordosica (Fig. 8). Thus, the enzymatic hydrolysis of Artemisia ordosica is improved. On the other hand, electron beam irradiation can split water molecules into OH radical, hydrated electron, and so on (especially alkaline conditions) [44]. Irradiation  H 2O   OH ,H , H 3O  , eaq

OH radical is strong oxidation, and the network are degraded further when exposed to OH radical, and more celluloses were released into the aqueous phase, and the accessibility of enzymes onto cellulose increased. Therefore, the reducing sugar yields increased. 4. Conclusions The A. ordosica was a promising biomass for biofuel production, and the alkali (Ca(OH)2) soaking-EBI pretreatment could effectively enhance enzymatic hydrolysis of A. ordosica. The reducing sugar yield achieved 520.67 mg/g under the optimal conditions: Ca(OH)2 dosage of 1.5%, solid content of 8%, presoaked time of 24 h, absorbed dose of 7.5 kGy, enzymatic hydrolysis time of 72 h, and enzyme loading of 150 FPU/g. Any effective pretreatment method should be environmentally friendly, sustainable and fast reliable. A vast scope exists for utilizing EBI+Ca(OH)2 or modifications thereof for employing as an effective method for the lignocellulolsic biomass. Though concerns over the cost and effectiveness of the EBI pretreatment persist, other factors such as future demands for chemicals and fuel and environmental sustainability shall not limit the research efforts on utilizing EBI+Ca(OH)2 considering a series of value added products that can be produced by effective pretreatment of cellulosic materials. Acknowledgement 19

The authors are grateful for the funding and support provided by the following projects: The National Natural Science gold project, Shaanxi provincial science and technology department foster industrialization projects (15JF035). The Scientific Research Starting Foundation for high-level professionals in Yulin University of China (No. 12GK04). References [1] S.K. Bauer, L.S. Grotz, E.B. Connelly, L.M. Colosi, Reevaluation of the global warming impacts of algae-derived biofuels to account for possible contributions of nitrous oxide, Bioresour. Technol. 218(2016)196-201. [2] K. Zaafouri, A.B.H. Trabelsi, S. Krichah, A. Ouerghi, A. Aydi, C.A. Claumann, Z.A. Wust, S. Naoui, L. Bergaoui, M. Hamdi, Enhancement of biofuels production by means of co-pyrolysis of Posidonia oceanica (L.) and frying oil wastes: Experimental study and process modeling, Bioresour. Technol. 207(2016)387-398. [3] K. Zhang, Z.J. Pei, D.H. Wang, Organic solvent pretreatment of lignocellulosic biomass for biofuels and biochemicals: A review, Bioresour. Technol. 199(2016) 21-33. [4] M. Alexandropoulou, G. Antonopoulou, E. Fragkou, I. Ntaikou, G. Lyberatos, Fungal pretreatment of willow sawdust and its combination with alkaline treatment for enhancing biogas production, J. Environ. Manage. http://dx.doi.org/10.1016/j.jenvman. 2016.04.006. [5] C. Sawatdeenarunat, D. Nguyen, K.C. Surendra, S. Shrestha, K. Rajendran, H. Oechsner, L. Xie, S.K. Khana, Anaerobic biorefinery: Current status, challenges, and opportunities, Bioresour. Technol. 215(2016)304-313. 20

[6] Z.H. Liu, H.Z. Chen, Periodic peristalsis enhancing the high solids enzymatic hydrolysis performance of steam exploded corn stover biomass, Biomass

Bioenergy

93(2016)13-24. [7] R.Z. Mohd, H. Satoshi, F. Shinji, A.H. Mohd, Combined pretreatment with hot compressed water and wet disk milling opened up oil palm biomass structure resulting in enhanced enzymatic digestibility, Bioresour. Technol. 193(2015)128-134. [8] H.L. Yu, Y.Z. You, F.H. Lei, Z.G. Liu, W.M. Zhang, J.X. Jiang, Comparative study of alkaline hydrogen peroxide and organosolv pretreatments of sugarcane bagasse to improve the overall sugar yield, Bioresour. Technol. 187(2015)161-166. [9] B.Y. Chen, B.C. Zhao, M.F. Li, Q.Y. Liu, R.C. Sun, Fractionation of rapeseed straw by hydrothermal/dilute acid pretreatment combined with alkali post-treatment for improving its enzymatic hydrolysis, Bioresour. Technol. 225(2017)127-133. [10] Z. Ling, S. Chen, X. Zhang, F. Xu, Exploring crystalline-structural variations of cellulose during alkaline pretreatment for enhanced enzymatic hydrolysis, Bioresour. Technol. http://dx.doi.org/10.1016/j.biortech.2016.10.064. [11] Z.Q. Pang, W.K. Lyu, C.H. Dong, H.X. Li, G.H. Yang, High selective delignification using oxidative ionic liquid pretreatment at mild conditions for efficient enzymatic hydrolysis of lignocellulose, Bioresour. Technol. 214(2016)96-101. [12] Y.N. Yin, J.L. Wang, Enhancement of enzymatic hydrolysis of wheat straw by gamma irradiation–alkaline pretreatment, Radiat. Phys. Chem. 123(2016)63-67. [13] Y. Liu, H. Zhou, L.Y. Wang, S.H. Wang, L.H. Fan, Improving Saccharomyces cerevisiae growth against lignocellulose-derived inhibitors as well as maximizing ethanol production by a combination proposal of γ-irradiation pretreatment with in situ 21

detoxification, Chemical Engineering Journal 287(2016)302 -312. [14] Y. Liu, H. Zhou, S.H. Wang, K.Q. Wang, X.J. Su, Comparison of γ-irradiation with other pretreatments followed with simultaneous saccharification and fermentation on bioconversion of microcrystalline cellulose for bioethanol production, Bioresour. Technol. 182(2015)289-295. [15] K. Karthika, A.B. Arun, J.S. Melo, K.C. Mittal, K. Mukesh, P.D. Rekha, Hydrolysis of acid and alkali presoaked lignocellulosic biomass exposed to electron beam irradiation, Bioresour. Technol. 129(2013)646-649. [16] S.G. Jin, G.M. Zhang, P.Y. Zhang, F. Li, S.Y. Fan, J. Li, Thermo-chemical pretreatment and enzymatic hydrolysis for enhancing saccharification of catalpa sawdust, Bioresour. Technol. 205(2016)34-39. [17] J. Zhao, L.M. Xia, Simultaneous saccharification and fermentation of alkalinepretreated corn stover to ethanol using a recombinant yeast strain, Fuel Processing Technology 90(2009)1193-1197. [18] M. Ishiguro, T. Endo, Effect of the addition of calcium hydroxide on the hydrothermal-mechanochemical treatment of Eucalyptus, Bioresour. Technol. 177 (2015)298-301. [19] J.W. Hou, Q.Y. Suo, H. Liang, X.Q. Han, C.T. Liu, Effects of carbonization temperature on artemisia ordosica biochar morphology and chemical properties, Soils 46(5)(2014)814-818. [20] R. Hu, L. Lin, T. Liu, P. Ouyang, B. He, S. Liu, Reducing sugar content in hemicellulose hydrolysate by DNS method: a revisit, J. Biobased Mater. Bioenergy 2(2008)156-161. 22

[21] L. Segal, J.J. Creely, A.E. Martin, C.M. Conrad, An empirical method for estimating the degree of crystallinity of native cellulose using the X-ray diffractometer, Text. Res. J. 29(1959)786-794. [22] I. Pancha, K. Chokshi, R. Maurya, S. Bhattacharya, P. Bachani, S. Mishra, Comparative evaluation of chemical and enzymatic saccharification of mixotrophically grown de-oiled microalgal biomass for reducing sugar production, Bioresour. Technol. 204(2016)9-16. [23] S. Sundar, N. Scott Bergey, S.C. Lucia, S. Arthur, D. Mark, Electron beam pretreatment of switchgrass to enhance enzymatic hydrolysis to produce sugars for biofuels, Carbohydrate Polymers 100(2014)195-201. [24] K. Karthika, A.B. Arun, P.D. Rekha, Enzymatic hydrolysis and characterization of lignocellulosic biomass exposed to electron beam irradiation, Carbohydrate Polymers 90(2)(2012)1038-1045. [25] C. Karunanithy, K. Muthukumarappan, Optimization of alkali soaking and extrusion pretreatment of prairie cord grass for maximum sugar recovery by enzymatic hydrolysis, Biochemical Engineering Journal 54(2)(2011)71-82. [26] J.K. Ko, J.S. Bak, M.W. Jung, H.J. Lee, I.G. Choi, T.H. Kim, K.H. Kim, Ethanol production from rice straw using optimized aqueous-ammonia soaking pretreatment and simultaneous saccharification and fermentation processes, Bioresour. Technol. 100(19)(2009)4374-4380. [27] S.J. Yang, H.Y. Yoo, H.S. Choi, J.H. Lee, C. Park, S.W. Kim, Enhancement of enzymatic digestibility of Miscanthus by electron beam irradiation and chemical combined treatments for bioethanol production, Chemical Engineering Journal 23

275(2015)227-234. [28] P. Li, D. Cai, C.W. Zhang, S.F. Li, P.Y. Qin, C.J. Chen, Y. Wang, Z. Wang, Comparison of two-stage acid-alkali and alkali-acid pretreatments on enzymatic saccharification ability of the sweet sorghum fiber and their physicochemical characterizations, Bioresour. Technol. 221(2016)636-644. [29] Q. Li, Y. Gao, H.S. Wang, B. Li, C. Liu, G. Yu, X.D. Mu, Comparison of different alkali-based pretreatments of corn stover for improving enzymatic saccharification, Bioresour. Technol. 125(2012)193-199. [30] M.E. Himmel, S. Ding, D.K. Johnson, W.S. Adney, M.R. Nimlos, J.W. Brady, T. D. Foust, Biomass recalcitrance: engineering plants and enzymes for biofuels production, Science 315(2007)804-807. [31] Uju, M. Goto, N. Kamiya, Powerful peracetic acid-ionic liquid pretreatment process for the efficient chemical hydrolysis of lignocellulosic biomass, Bioresour. Technol. 214(2016)487-495. [32] J. S. Bak, Electron beam irradiation enhances the digestibility and fermentation yield of water-soaked lignocellulosic biomass, Biotechnology Reports 4(2014)30-33. [33] Y.L. Xiang, Y.X. Xiang, L.P. Wang, Cobalt-60 gamma-ray irradiation pretreatment and sludge protein for enhancing enzymatic saccharification of hybrid poplar sawdust, Bioresour. Technol. 221(2016)9-14. [34] J. Lin, G.J. Zhao, L.X. Meng, Z.P. Li, Analysis of decayed wood by fungi with X-ray diffractometry and fourier transform infrared spectroscopy, Spectroscopy Spectral Analysis 30(6)(2010)1674-1677. [35] J.S. Bak, J. K. Ko, Y. H. Han, B. C. Lee, I.G. Choi, K.H. Kim, Improved enzymatic 24

hydrolysis yield of rice straw using electron beam irradiation pretreatment, Bioresour. Technol. 100(3)(2009)1285-1290. [36] H.W. Ren, N. Xu, J.P. Li, T. Zhang, Chemical pretreatment improving effect of enzymatic saccharification of distillers grains biomass, Transactions of the chinese society of agricultural engineering 30(16)(2014)239-246. [37] G. Cheng, P. Varanasi, C. Li, H. Liu, Y.B. Melnichenko, B.A. Simmons, Transition of cellulose crystalline structure and surface morphology of biomass as a function of ionic liquid pretreatment and its relation to enzymatic hydrolysis, Bio Macromolecules 12(2011)933-941. [38] F. Shi, H.J. Xiang, Y.F. Li, Combined pretreatment using ozonolysis and ball milling to improve enzymatic saccharification of corn straw, Bioresour. Technol. 179(2015)444-451. [39] G.J.M. Rocha, C. Martin, F.N. Vinicius, E.O. Gomez, A.R. Goncalves, Mass balance of pilot-scale pretreatment of sugarcane bagasse by steam explosion followed by alkaline delignification, Bioresour. Technol. 111 (2012) 447-452. [40] M. Brahim, S. El Kantar, N. Boussetta, N. Grimi, N. Brosse, E. Vorobiev, Delignification of rapeseed straw using innovative chemo-physical pretreatments, Biomass Bioenergy 95(2016)92-98. [41] P. Phitsuwan, K. Sakka, K. Ratanakhanokchai, Structural changes and enzymatic response of Napier grass (Pennisetum purpureum) stem induced by alkaline pretreatment, Bioresour. Technol. 218(2016)247-256. [42] S. Sun, X. Cao, S. Sun, F. Xu, X. Song, R.C. Sun, G. Jones, Improving the enzymatic hydrolysis of thermo-mechanical fiber from Eucalyptus urophylla by a combination of 25

hydrothermal pretreatment and alkali fractionation, Biotechnol Biofuels 7(1) (2014)1-12. [43] R. Kont, M. Kurasin, H. Teugjas, P. Vaeljamaee, Strong cellulase inhibitors from the hydrothermal pretreatment of wheat straw, Biotechnol Biofuels 6(1)(2013) 29-29. [44] J. Chen, H. Zhang, Z. Mi, Y. Bai, Application of accelerator electron beam irradiation technology to wastewater treatment, Industrial Water Treatment 34(3)(2014)1-6.

26