Effect of Four Types of Chemical Pretreatment on Enzymatic Hydrolysis by SEM, XRD and FTIR Analysis

JIN Shu-guang, ZHANG Guang-ming, ZHANG Pan-yue*,

ZHOU Jin-chi3, GAO Yong-wei3, SHI Jun-na3

Effect of Four Types of Chemical Pretreatment on Enzymatic Hydrolysis by SEM, XRD and FTIR Analysis

JIN Shu-guang1, ZHANG Guang-ming2, ZHANG Pan-yue1*,

ZHOU Jin-chi3, GAO Yong-wei3, SHI Jun-na3

1. Beijing Key Lab for Source Control Technology of Water Pollution, Beijing Forestry University, Beijing 100083, China 2. School of Environment and Resource, Renmin University of China, Beijing 100872, China 3. Analysis Test Center, Beijing Forestry University, Beijing 100083, China

Catalpa sawdust was respectively pretreated by NaOH, Ca(OH)2, H2SO4and HCl solution, and the enzymatic hydrolysis of catalpa sawdust was significantly enhanced by alkaline pretreatments. In order to investigate the mechanisms of pretreatment of catalpa sawdust, the characteristics of catalpa sawdust before and after pretreatments were analyzed by scanning electron microscopy (SEM), X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy. It was found that the surface of catalpa sawdust was disrupted by four kinds of chemical pretreatment, and the pretreatment with Ca(OH)2solution resulted in the most serious damage. The XRD results showed that part of amorphous regions was damaged by alkaline pretreatments, which led to a relative increase of crystallinity Index (CrI) of catalpa sawdust; while the CrI of catalpa sawdust was insignificantly influenced by acid pretreatments. The FTIR analysis displayed that the molecular structures of hemicellulose and lignin of catalpa sawdust were damaged in different degrees by four types of pretreatment. The significant improvement of enzymatic hydrolysis of catalpa sawdust after alkaline pretreatment might be attributed to the effective delignification of alkaline.

Chemical pretreatment; Catalpa sawdust; SEM; XRD; FTIR

Introduction

With the rapid development of economy, a large number of fossil fuel has been consumed by human. The consumption of fossil fuel have caused a series of problems including environment pollution and energy crisis[1-2]. Facing this challenges, lignocellulosic biomass has been regarded as an important resource for second-generation biofuel[3]. Among numerous lignocellulosic biomasses, wood processing residual shows high production, easy availability, and high cellulose content. Therefore, the wood processing residual has good potential for biofuel production. According to statistic, more than a third of raw wood has become wastes during wood processing[4]. Catalpa is one traditional tree species extensively used in the wood processing due to easy drying, little shrinkage, and insect resistance. Therefore, huge amount of wood processing residual of catalpa has been produced[5].

Efficiency of direct conversion of the lignocellulosic biomass to biofuel is usually low, so pretreatment of lignocellulosic biomass is required[6]. The pretreatment methods commonly include physical pretreatment, chemical pretreatment, biological pretreatment, or combined physic-chemical pretreatment, etc[7-8]. In these pretreatment methods, the chemical pretreatment attracts much attention in research and practical application because of its simple operation and low cost[9].

In this paper, NaOH, Ca(OH)2, H2SO4and HCl solutions were used to pretreat the catalpa sawdust. The structure, crystallinity, and chemical construction of catalpa sawdust before and after pretreatment were analyzed by scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared (FTIR) spectroscopy. The information may clarify the mechanisms of chemical pretreatment of catalpa sawdust, and provide theoretical basis for further research and practical application.

1 Experimental

1.1 Materials and reagents

Catalpa sawdust was collected from a furniture factory in Beijing. The sawdust was air-dried away from light for two weeks, graded and sieved through 20 mesh sieve. KBr (spectrum pure reagent) was purchased from Shanghai Macklin Biochemical Co., Ltd. NaOH, Ca(OH)2, H2SO4, and HCl (analytical reagent) were bought from Beijing Chemical Industry Group CO., LTD. The concentration of pretreatment solution was 0.5%.

1.2 Pretreatment of catalpa sawdust

1.3 Enzymatic hydrolysis of catalpa sawdust

Enzymatic hydrolysis of catalpa sawdust was carried out with the enzyme generated byTrichodermaviride(Beijing Lanyi Chemical Co., Ltd). The catalpa sawdust of 0.5 g and cellulase of 30 FPU·g-1were added in citrate buffer (0.05 mol·L-1, pH 4.8). Enzymatic hydrolysis of samples was conducted in a shaking incubator at 50 ℃ and 100 r·min-1for 48 h. After 48-hour enzymatic hydrolysis, the supernatant was taken for determination of reducing sugar.

1.4 Analytical methods

Reducing sugar was analyzed by DNS method[10]. Structure of catalpa sawdust was observed by scanning electron microscopy (S-3400N II, Hitachi, Japan). Crystallinity of samples was determined by X-ray diffractometer (D8 Advance, Bruker, Germany). Specimens were scanned at 6 s/step from 2θ=5°～40° with a step of 0.2°. Crystallinity index (CrI) of catalpa sawdust was calculated by the Segal method, shown in Eq. (1)[11]

(1)

whereI002is the diffraction intensity of crystalline structure (2θ=22.6°), andIamorphousis the diffraction intensity of amorphous fraction (2θ=18.0°). The functional group change in catalpa sawdust was recorded from 4 000 to 400 cm-1by a FTIR spectrometer (VERTEX 70, Bruker, Germany).

2 Results and discussion

2.1 Effect of chemical pretreatment on enzymatic hydrolysis of catalpa sawdust

Swales认为，“体裁”是交际事件的一种分类，人们在生活中按照特定目的和特定程式运用语言在生活中办事[7](P45-58)。Bhatia根据Swales的观点将“体裁”进一步概括为：“体裁”是一种可辨认的交际事件，内部结构特征鲜明且高度约定俗成[8](P13-16)。以上学者的定义都表明体裁分析注重话语生成过程对于交际目的实现的影响和制约，语篇的内容和程式都受到体裁的制约。元旦社论也是一种内部结构鲜明、可辨认的、高度约定俗成的交际事件，有其特殊的交际目的，是一种特定的体裁，具有既定的语步结构。我们运用体裁分析的方法，将元旦社论划分为几个服务于同一交际目的不同语步，并进行了统计(见表2)。

The reducing sugar yield of catalpa sawdust before and after four kinds of pretreatment is shown in Fig.1. The reducing sugar yield was significantly improved by two kinds of alkaline pretreatment, and the Ca(OH)2pretreatment showed a higher reducing sugar yield than NaOH pretreatment. The reducing sugar yield of control was 38.45 mg·g-1, which increased by 2.4 and 2.8 times by NaOH and Ca(OH)2pretreatment respectively. However, the reducing sugar yield of samples pretreated by H2SO4and HCl decreased to 28.66 and 29.94 mg·g-1, respectively.

Fig.1 Reducing sugar yield of catalpa sawdust with different pretreatment methods after 48 h enzymatic hydrolysis (Pretreatment condition: solid content 5%, solution concentration 0.5%, time 1 h, temperature 100 ℃. Enzymatic hydrolysis conditions: 50 ℃, 100 r·min-1, 30 FPU·g-1, buffer pH 4.8)

2.2 Microstructure of catalpa sawdust

Fig.2 shows the microstructure of catalpa sawdust before and after pretreatment by SEM. The surface of unpretreated catalpa sawdust was compact and smooth without any erosion traces. However, the surface of pretreated catalpa sawdust showed jagged erosion traces and was stripped in different degrees. Moreover, the erosion degree by alkaline pretreatments was more serious than that by acid pretreatments, including erosion area and degree. The Ca(OH)2solution eroded the surface of catalpa sawdust most seriously. The microstructure change of catalpa sawdust could affect the contact area and degree between enzyme then biomass, and further affect the enzymatic hydrolysis of catalpa sawdust.

Fig.2 SEM images of catalpa sawdust pretreated with different chemical solutions (a) Control, (b) NaOH, (c) Ca(OH)2, (d) H2SO4, (e) HCl (Pretreatment condition: solid content 5%, solution concentration 0.5%, time 1 h, temperature 100 ℃)

2.3 XRD study of catalpa sawdust

Fig.3 shows the XRD spectra of catalpa sawdust before and after pretreatment. Unpretreated and pretreated catalpa sawdust showed similar XRD spectra. Obvious characteristic peaks of cellulose were observed, including the peak around 2θ=22° and the peak around 2θ=18°. The cellulose structure of catalpa sawdust was slightly damaged by acid pretreatments. The calculated crystallinity index (CrI) changed insignificantly. On the other hand, the absorption intensity of catalpa sawdust obviously changed after alkaline pretreatment. The area of crystalline region increased and that of amorphous region decreased after NaOH pretreatment. The calculated CrI increased from 30.7% to 38.6%. The possible reason was that NaOH removed a part of amorphous material, leading to a relatively high cellulose CrI. The absorption intensity of catalpa sawdust obviously decreased after Ca(OH)2pretreatment and the crystalline region and the amorphous region were both damaged. The CrI increased to 39.2%. Therefore, alkaline pretreatments changed the cellulose crystal structure of catalpa sawdust, which benefited the subsequent enzymatic hydrolysis[12].

Fig.3 X-ray diffraction spectra of catalpa sawdust pretreated with different chemical solutions (Pretreatment condition: solid content 5%, solution concentration 0.5%, time 1 h, temperature 100 ℃)

2.4 FTIR investigation of catalpa sawdust

Fig.4 FTIR spectra of catalpa sawdust pretreated with different chemical solutions (Pretreatment condition: solid content 5%, solution concentration 0.5%, time 1 h, temperature 100 ℃)

3 Conclusions

(1)Alkaline pretreatment could significantly improve the enzymatic hydrolysis efficiency of catalpa sawdust. The best pretreatment effect was obtained by Ca(OH)2pretreatment, and the reducing sugar yield was increased by 2.8 times. However, the reducing sugar yield of acid pretreated sample was decreased. Ca(OH)2pretreatment method was a kind of effective and simple method for catalpa sawdust.

(2) The integrity of surface on catalpa sawdust was damaged after all four types of pretreatments. The damage degree of Ca(OH)2pretreated sample was the most obvious.

(3) The XRD spectra showed that the catalpa sawdust CrI was increased after alkaline pretreatments. The reason might be the removal of a part of amorphous materials. However, acid pretreatments did not affect the CrI of catalpa sawdust.

(4) The FTIR spectra displayed that hemicellulose and lignin were both damaged after pretreatments. Alkaline removed lignin and acid removed hemicellulose. The effective removal of lignin might be the main reason of the significant enhancement of enzymatic efficiency after alkaline pretreatment.

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利用SEM, XRD和FTIR研究四种化学预处理对楸木木屑酶解的影响

金曙光1, 张光明2, 张盼月1*, 周金池3, 高永伟3, 史军娜3

1. 北京林业大学水体污染源控制技术北京市重点实验室，北京 100083 2. 中国人民大学环境学院，北京 100872 3. 北京林业大学公共分析测试中心，北京 100083

利用NaOH，Ca(OH)2，H2SO4和HCl四种溶液对楸木木屑进行化学预处理，发现两种碱预处理均能显著提高楸木木屑的酶解效率。采用扫描电镜(SEM), X射线衍射(XRD)和傅里叶红外光谱(FTIR)对四种化学预处理后的楸木木屑进行观察和分析。SEM观察发现，四种化学预处理对楸木木屑的纤维表面均有不同程度的破坏与侵蚀，其中Ca(OH)2的破坏效果最为明显。XRD谱图表明，碱预处理对楸木木屑纤维素的非晶体结构产生破坏，导致其结晶度升高，而经过酸预处理后楸木木屑纤维素的结晶度没有明显变化。FTIR谱图显示，酸碱预处理对楸木木屑中半纤维素和木质素的分子结构均有不同程度的破坏，碱预处理过程中木质素的高效溶出可能是楸木木屑酶解效率显著提高的主要原因。

化学预处理； 楸木木屑； 扫描电镜； X射线衍射； 傅里叶红外光谱

2015-02-16，

2015-06-15)

Foundation item： National Nature Science Foundation Issues (51178047, 51578068)

10.3964/j.issn.1000-0593(2016)06-1966-05

Received： 2015-02-16; accepted： 2015-06-15

Biography： JIN Shu-guang, (1982—), doctor candidate in Beijing Forestry University e-mail: shugguangjin@126.com *Corresponding author e-mail: panyue_zhang@bjfu.edu.cn