**3. Results and discussion**

#### **3.1. FTIR analysis**

250-425 um. Finally, the particles were dried at 105℃ until mass variability of samples was less

6 Sustainable Degradation of Lignocellulosic Biomass - Techniques, Applications and Commercialization

Bamboo particles were pretreated using a microwave accelerated reaction system. 2% of sulfuric acid (w/w of H2SO4/bamboo) and 10% sodium hydrate (w/w of NaOH/bamboo) were used in the pretreated process. About 3g bamboo particles were mixed with the solvent in a 100 mL vessel. The mass ratio of liquor-to-bamboo was about 8:1. The vessel with samples was positioned at the centre of a rotating circular ceramic plate in the microwave oven for pre‐ treatment at the power level of 400 W. The temperature was raised to 180 ℃ (target tempera‐ ture) in about 10 min and maintained for an additional 30min. After the pretreatment, a few minutes were allowed for the temperature to drop down below 50 ℃, and then pretreated substrate and spent liquor were then separated by filtration. The substrate was washed using distilled water until pH value of washed liquor was about 7. Each experiment was carried out

The functional group difference of untreated and pretreated samples was analyzed by means of FTIR-spectrometer (Bruker, Bremen, Germany). The concentration of the sample in the tablets was constant of 1 mg/400 mg KBr. Scans were run at a resolution of 4cm-1 and each sample consisted of 64 scans recorded in absorbance units from 3800 to 750cm-1. The spectra were ATR and baseline corrected and the spectra analyzed for carbonyl bands using relative indices. A minimum of two samples were tested, and data from the first run was used when it was shown to be in accordance with the second run. The experimental data could be directly obtained though FTIR-spectrometer, but they were analyzed by using Origin 8.0 software.

XRD test of untreated and pretreated bamboo samples was carried out using an X-ray diffractometer (Diffraktometer D5000, Siemens, Germany) with an X-ray generator and a Co target (λ=0.1729 nm) at a scanning speed of 3º/min, and the data were recorded every 0.02º (2θ) for the angle range of 2θ=5-45º. The cellulose crystallinity (CrI) was calculated based on

Where, I002 was the overall intensity of the peak at 2θ about 22º and Iam was the intensity of the

( ) CrI = I -I /I002 002 am (1)

than 0.2%.

**2.2. Pretreatment**

**2.3. Property test**

**1.** FTIR test

**2.** XRD test

formula (1):

baseline at 2θ about 18º.

in three times and average results were reported.

The functional groups of untreated and pretreated bamboo were shown in the FTIR spectra presented in Figure 2. For untreated bamboo, there was a strong broad O-H stretching absorbance at 3350cm-1. The absorbance at 2910 cm-1 was a prominent C-H stretching. In the fingerprint region (from 1800 to 800 cm-1), some important information on various functional groups presented in untreated bamboo. The absorbance at 1740 cm-1 was attributed to C=O stretching vibration in hemicelluloses. The bands from 1600 to 1450 cm-1 was due to aromatic skeletal vibration in lignin. The absorbance at 1370cm-1 was C-H bending of cellulose or hemicelluloses, and that of 1230cm-1 was C-O stretching of phenolic hydroxyl group in the lignin. The absorbance at 1160cm-1 and 1030 cm-1 was respectively attributed to C-O-C stretching of cellulose or hemicelluloses and C-O stretching of cellulose, hemicelluloses or lignin [16].

The chemical group difference of bamboo-H2SO4 and bamboo was showed in Figure 3. It was very obvious that the number of most chemical groups on the bamboo-H2SO4 sample surface was more than that of untreated bamboo surface, except for absorption of C=O stretching vibration. The information is very important, which confirms the removal of hemicelluloses of bamboo. The main feature of difference between hemicelluloses and cellulose is that hemicelluloses has branches with short lateral chains consisting of different sugars. These monosaccharides include pentoses (xylose, rhamnose, and arabinose), hexoses (glucose, mannose, and galactose), and uronic acids (e.g., 4-omethylglucuronic, D-glucuronic, and Dgalactouronic acids) [29]. Hemicelluloses is known to coat the cellulose microfibrils in the plant cell wall, forming a physical barrier to access by hydrolytic enzymes. Removal of hemicellu‐ loses from the microfibrils is believed to expose the cellulose surface and to increase the enzymatic hydrolysis of cellulose [17, 18]. Dilute-acid pretreatment is a main method for the selective fractionation of hemicelluloses from biomass. Both cellulose and hemicelluloses components can also be hydrolysed using dilute-acid catalysed processes but in this case a two step-hydrolysis is required. The difference between two steps is mainly the operational temperature, which is high in the second step (generally around 230-240 ℃) [19, 20].

The chemical group difference of bamboo-NaOH and bamboo was showed in Figure 4. It was found that the number of all chemical groups on the bamboo-NaOH sample surface was less than that of bamboo surface. This indicated the removal of hemicelluloses and lignin of bamboo. Alkaline pre-treatments are very effective for lignin solubilisation exhibiting only minor cellulose and slightly higher hemicelluloses solubilisation [21]. Generally, alkaline pretreatments include wet oxidation and the ammonia. The wet oxidation was used in this research. Wet oxidation is defined as pretreatment process including oxygen and water at elevated temperatures and pressure, promoting the oxidation of lignin and decomposing it to CO2, H2O and carboxylic acids [22, 23]. The hemicellulosic sugars remain mainly in the oligomeric form, and although there is a low formation of furan-aldehydes, a significant formation of carboxylic acids still exists [24]. It is well known that lignin confers integrity and structural rigidity on the plant cell wall. There is several information that cellulolytic enzymes are adsorbed non-specifically on the lignin fraction of lignocelluloses even in the absence of a carbohydrate-binding module [25, 26, 27]. Therefore, the increase in the initial hydrolysis rate of cellulose and hemicelluloses should be due in part to the decreasing number of non-specific binding sites on lignin, making more enzyme available for hydrolysis [28].

3500 3000 2500 2000 1500 1000

Difference of bamboo-NaOH and bamboo

Characteristics of Moso Bamboo with Chemical Pretreatment

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9

Wave length (cm-2)

The untreated and pretreated bamboo was determined using an X-ray generator in this study. The XRD results were shown in the Figure 5. The cellulose crystallinity was calculated according to formulas (1). The cellulose crystallinity of bamboo, bamboo-H2SO4 and bamboo-NaOH samples were shown in Figure 6. It was obvious that the cellulose crystallinity of pretreated bamboo was greater than that of bamboo. Biomass materials exhibit two types of cellulose crystallinity: absolute and relative crystallinity. The relative crystallinity was used in this research. The relative crystallinity of bamboo, bamboo-H2SO4 and bamboo-NaOH samples was 44.4%, 49.8% and 55.2%, respectively. This phenomenon further confirmed that some compositions of bamboo were removed in the pretreatment process. It also expressed that the NaOH pretreatment was better than H2SO4 pretreatment. This can be explained based on substrate content of different pretreatment. It was found from Table 2 that the substrate content

of NaOH pretreatment (64.1%) was lower than that of H2SO4 pretreatment (70.3%).

Bamboo is composed of cellulose, hemicelluloses, lignin and a remaining smaller part comprising extractives and minerals. The cellulose and hemicelluloses typically comprise up to 70% of the bamboo and are the substrates for second generation ethanol production. The microbial conversion of the hemicelluloses fraction, either in the monomeric form or in the oligomeric form, is essential for increasing fuel ethanol yields from bamboo. Unlike cellulose, hemicelluloses are not chemically homogeneous and different hydrolytic technologies and various biological and non-biological pre-treatment options are available both for fractionation or solubilisation of hemicelluloses from lignocellulosic materials [21]. Depending on the process and conditions used during pre-treatment, hemicelluloses sugars may be degraded to weak acids and furan derivatives which potentially act as microbial inhibitors during the

**Figure 4.** The FTIR spectra difference of untreated and sodium hydrate pretreated bamboo


Absorbance

**3.2. XRD analysis**

**Figure 2.** The FTIR spectra of untreated and pretreated bamboo

**Figure 3.** The FTIR spectra difference of untreated and sulfuric acid pretreated bamboo

**Figure 4.** The FTIR spectra difference of untreated and sodium hydrate pretreated bamboo

#### **3.2. XRD analysis**

are adsorbed non-specifically on the lignin fraction of lignocelluloses even in the absence of a carbohydrate-binding module [25, 26, 27]. Therefore, the increase in the initial hydrolysis rate of cellulose and hemicelluloses should be due in part to the decreasing number of non-specific

3500 3000 2500 2000 1500 1000

Difference of bamboo-H2SO4 and bamboo

3500 3000 2500 2000 1500 1000

)

Wave length (cm-2

**Figure 3.** The FTIR spectra difference of untreated and sulfuric acid pretreated bamboo

)

Bamboo Bamboo-H2SO4 Bamboo-NaOH

Wave length (cm-2

binding sites on lignin, making more enzyme available for hydrolysis [28].

8 Sustainable Degradation of Lignocellulosic Biomass - Techniques, Applications and Commercialization

0.0

**Figure 2.** The FTIR spectra of untreated and pretreated bamboo


0.00

0.05

0.10

Absorbance

0.15

0.20

0.1

0.2

Absorbance

0.3

0.4

0.5

The untreated and pretreated bamboo was determined using an X-ray generator in this study. The XRD results were shown in the Figure 5. The cellulose crystallinity was calculated according to formulas (1). The cellulose crystallinity of bamboo, bamboo-H2SO4 and bamboo-NaOH samples were shown in Figure 6. It was obvious that the cellulose crystallinity of pretreated bamboo was greater than that of bamboo. Biomass materials exhibit two types of cellulose crystallinity: absolute and relative crystallinity. The relative crystallinity was used in this research. The relative crystallinity of bamboo, bamboo-H2SO4 and bamboo-NaOH samples was 44.4%, 49.8% and 55.2%, respectively. This phenomenon further confirmed that some compositions of bamboo were removed in the pretreatment process. It also expressed that the NaOH pretreatment was better than H2SO4 pretreatment. This can be explained based on substrate content of different pretreatment. It was found from Table 2 that the substrate content of NaOH pretreatment (64.1%) was lower than that of H2SO4 pretreatment (70.3%).

Bamboo is composed of cellulose, hemicelluloses, lignin and a remaining smaller part comprising extractives and minerals. The cellulose and hemicelluloses typically comprise up to 70% of the bamboo and are the substrates for second generation ethanol production. The microbial conversion of the hemicelluloses fraction, either in the monomeric form or in the oligomeric form, is essential for increasing fuel ethanol yields from bamboo. Unlike cellulose, hemicelluloses are not chemically homogeneous and different hydrolytic technologies and various biological and non-biological pre-treatment options are available both for fractionation or solubilisation of hemicelluloses from lignocellulosic materials [21]. Depending on the process and conditions used during pre-treatment, hemicelluloses sugars may be degraded to weak acids and furan derivatives which potentially act as microbial inhibitors during the fermentation step to ethanol. For fuel ethanol production, hemicelluloses are commonly removed during the initial stage of biomass processing aiming to reduce structural constraints for further enzymatic cellulose hydrolysis. Lignin affects the enzymatic hydrolysis of ligno‐ cellulosic biomass because it forms a physical barrier to attack by enzymes. The removal of hemicelluloses and lignin through pretreatment is very helpful to make the cellulose accessible to hydrolysis for conversion to fuels.

**Pretreated method**

**4. Conclusions**

**Table 2.** The substrate content of pretreated bamboo

substrate hydrolysis, biofuels synthesis, etc.

**Acknowledgements**

1632012002).

**Author details**

Zhijia Liu and Benhua Fei\*

\*Address all correspondence to: Feibenhua@icbr.ac.cn

International Centre for Bamboo and Rattan, Beijing, China

**Bamboo mass (g)**

Untreated Pretreated

**H2SO4** 3.015 2.119 70.3

**NaOH** 3.017 1.934 64.1

It can be concluded from this research that hemicelluloses and lignin of bamboo are removed by H2SO4 and NaOH pretreatment. The number of most chemical groups on the bamboo-H2SO4 sample surface is more than that of bamboo surface, except for absorption of C=O stretching vibration, which indicates hemicelluloses removal of bamboo through H2SO4 pretreatment. The number of all chemical groups on the bamboo-NaOH sample surface is less than that of bamboo surface, which expresses hemicelluloses and lignin removal of bamboo through NaOH pretreatment. The relative crystallinity of bamboo-H2SO4 and bamboo-NaOH samples is 49.8% and 55.2%, respectively. The substrate content of NaOH pretreatment (64.1%) is lower than that of H2SO4 pretreatment (70.3%).This phenomenon further confirms that NaOH pretreatment is better than H2SO4 pretreatment. This research represents an initial stage in the study of bamboo bioethanol and may provide guidelines for further research, such as

This research was financially supported by '12th Five Years Plan-Study on manufacturing technology of functional bamboo (rattan)-based materials' (Grant No. 2012BAD54G01) and 'Basic Scientific Research Funds of International Centre for Bamboo and Rattan' (Grant No.

**Substrate content (%)**

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Characteristics of Moso Bamboo with Chemical Pretreatment

**Figure 5.** XRD curve of untreated and pretreated bamboo

**Figure 6.** The cellulose crystallinity of untreated and pretreated bamboo


**Table 2.** The substrate content of pretreated bamboo
