**3. Results and discussion**

The structural changes for each biomass sample: thanaka heartwood, sugarcane bagasse and rice straw of (a) raw sample (b) acid-treated sample (c) base-treated sample and (d) cellulose fiber were shown in **Figures 4**–**6** respectively. The broad peaks at 3400 cm−1 in all samples were due to the stretching vibrations of O-H groups of water contents. The peak at 2900 cm−1 was due to C-H stretching. From the comparison of the presented FTIR spectra, the peaks around 1510 cm−1and 1520 cm−1 showed the presence of lignin and lignocellulose in the initial raw sample. Moreover, the band at around 1200 cm−1 disappeared in treated fiber which was attributed to the removal of hemicelluloses. The changes of the bands in the region from 1423 cm−1 to 1080 cm−1 were due to the removal of lignin and hemicelluloses [36, 37]. This is a clear indication that the amount of lignin from the raw sample was successfully reduced by the chemical treatments.

The surface morphologies of biomass raw samples and their pretreated samples were shown in SEM images of thanaka heartwood in **Figure 7**, sugarcane bagasse in *Comparative Study of Cellulose Hydrogel Films Prepared from Various Biomass Wastes DOI: http://dx.doi.org/10.5772/intechopen.99215*

#### **Figure 4.**

*FTIR spectra of (a) thanaka heartwood (b) acid-treated (c) base-treated samples and (d) cellulose fiber.*

**Figure 5.**

*FTIR spectra of (a) sugarcane bagasse (b) acid-treated (c) base-treated samples and (d) cellulose fiber.*

**Figure 8** and rice straw sample in **Figure 9**. Morphological changes in the raw, acid and base pretreated samples were clearly seen in these SEM images. The fibrous patterns of the plant cell wall appeared in the raw samples shown in **Figures 7(a), 8(a)**, and **9(a)**. **Figures 7(b), 8(b), 9(b)** and **7(c), 8(c), 9(c)** represented the distorted structure of lignin and hemicellulose of plant cell wall structure due to pretreatment of acid and base. Pretreatment of NaOH tended to decrease the lignin and hemicellulose which in turn increased the content of cellulose. It also resulted in an increase in cellulose accessibility shown in **Figures 7(d), 8(d)** and **9(d)**.

Water content of the hydrogel films was determined by weighing dry and wet samples by the following procedure. Disk samples with 5 mm diameter were cut from cast films and dried in a vacuum oven for 24 h and weighed (W0). Then, samples were immersed in distilled water for 36 h. After that, films were removed from the water and wrapped with filtered paper in order to remove excess water and weighed again (W1). Finally, the equilibrium water content was calculated from the wet (W1) and dried (W0) hydrogel films. For each sample, four independent measurements were done and averaged by the following formula: Water content (%) = (W1–W0)/W0 x 100. According to the calculation, water content was 165.5%

**Figure 6.** *FTIR spectra of (a) rice straw raw (b) acid-treated samples (c) base-treated and (d) cellulose fiber.*

#### **Figure 7.**

*Comparative SEM images of (a) thanaka heartwood (b) acid-treated (c) base-treated samples and (d) cellulose fiber.*

for THCF, 188.47% for SBCF film and 168.63% for RSCF film. Thus, the films have water retainable property which will be the formation of the interaction of hydrogen bonding networks of the resultant cellulose in the hydrogel films.

XRD measurement was carried out to evaluate the effect of treatment conditions on the crystalline structure of raw samples, treated fibers and cellulose fibers. The patterns of (a) to (d) exhibited typical crystalline lattice of cellulose with peaks at 22.3° and 16.4° [38, 39]. This cellulose crystalline can be found in natural

*Comparative Study of Cellulose Hydrogel Films Prepared from Various Biomass Wastes DOI: http://dx.doi.org/10.5772/intechopen.99215*

#### **Figure 8.**

*Comparative SEM images of (a) sugarcane bagasse (b) acid-treated (c) base-treated samples and (d) cellulose fiber.*

#### **Figure 9.**

*Comparative SEM images of (a) rice straw (b) acid-treated (c) base-treated samples and (d) cellulose fiber.*

plant cellulose. **Figure 10** showed the XRD patterns of thanaka heartwood and the treated fibers. In the crystallinity of thanaka heartwood and cellulose fibers, the peak ratio showed that thanaka sample and pre-treated fibers were at 68.5%, 82.1%, 84.2%, and 85.4%, respectively. **Figure 11** showed the XRD patterns of the bagasse

#### **Figure 10.**

*XRD patterns of (a) thanaka heartwood (b) acid-treated (c) base-treated and (d) cellulose fiber.*

#### **Figure 11.**

*XRD patterns of (a) sugar cane bagasse (b) acid-treated (c) base-treated and (d) cellulose fiber.*

and the purified fibers. The crystallinity indexes of sugar cane bagasse raw sample, acid-treated sample, base-treated sample and cellulose fiber were 44.1%, 58.8%, 59.1% and 60.2% respectively. **Figure 12** showed the XRD patterns of rice straw and the purified fibers. The crystallinity indexes of rice straw, acid-treated sample, base-treated sample and cellulose fiber were 71.43%, 75.7%, 76.25% and 78.57%, respectively. The increment of the crystallinity in the pre-treated fibers was due to the removal of hemicelluloses and lignin by sodium hydroxide and sodium hypochloride treatment, indicating especially higher purity of cellulose fibers.

Viscoelasticity measurements indicated the relationship between storage elastic modulus G', loss elastic modulus G" and strain for the hydrogel films. **Figure 13** showed the viscoelastic data for (a) THCF films (b) SBCF films and (c) RSCF films. It was noted that the deformation of THCF occurred at G'= 8 x 104 and G"= 8 x 103 Pa at 1 x 10−2 to 9% strain, SBCF at G'= 7 x 104 and G"= 6.5 x 103 Pa at 1 x 10−2 to 35.71% strain, the RSCF film at G'= 8 x 104 and G"= 7.5 x 103 Pa at 1 x 10−2 to 12.45% strain [40]. The loss elastic modulus (G") of SBCF film was lower than that of the others. This meant that the elastic nature was low and deformation was high in SBCF film. The crossover point of G' and G" meant fracture of materials or inability to follow deformation due to the rigid polymer network. In the prepared

*Comparative Study of Cellulose Hydrogel Films Prepared from Various Biomass Wastes DOI: http://dx.doi.org/10.5772/intechopen.99215*

**Figure 12.**

*XRD patterns of (a) rice straw raw (b) acid-treated (c) base-treated and (d) cellulose fiber.*

#### **Figure 13.**

*Viscoelasticity of (a) thanaka heartwood cellulose hydrogel film (THCF), (b) sugarcane bagasse cellulose hydrogels films (SBCF) and (c) rice straw cellulose hydrogel film (RSCF).*

cellulose hydrogel, cross points of G' and G" shifted towards a low strain range in order of strength of cellulose fiber. The crossover point of THCF film was found to be 9% and that of RSCF was 12.45% strain. In the case of SBCF, the G' and G" values overlapped at 35.71% strain. It seemed that the SBCF film showed a very soft and less elastic nature than the other. Based on the comparison of viscoelasticity measurement for these three films, THCF film was the strongest and most elastic compared to the other two films as a consequence of its rigid polymer network of hardwood resource.

**Table 1** showed the measurements of inhibition zones of cellulose solutions. The inhibition zone appearing around the agar-well was measured; an indication of the presence of antimicrobial activity. The measurable zone diameter, including the well diameter showed the degree of antimicrobial activity. The well diameter was 10 mm in this experiment. The larger the inhibition zone diameter showed stronger antimicrobial activity on the test organisms. According to the results, cellulose solution samples showed an inhibition zone more than the DMAc/LiCl solution, noting the DMAc solvent had no antimicrobial activity. It was found that all cellulose solutions showed antimicrobial activity and TH cellulose solution exhibited high activity against all test microorganisms (diameter of inhibition zone ranging 12–18 mm). On grounds of the antimicrobial activity results and water retaining properties, cellulose hydrogel films prepared from plant biomass waste can be applied as wound dressing, medical plaster and facial mask for biomedical applications.

#### **Table 1.**

*Antimicrobial activity results for test samples.*


**9. Pollutant-induced apoptosis and necrosis of invertebrate immunocytes**

Apoptosis, the programmed cell death, is functionally involved in developmental and immunological processes of organisms. Moreover, apoptosis helps in the process of removal of damaged cells and thus is considered an important machinery of survival of host animal in polluted environment. It is involved in specific cell signaling mechanism, which is yet to be studied in invertebrates in detail. However, pesticides and other environmental xenobiotics are reported to affect the apoptotic and necrotic pathways of the immunoactive cells of invertebrates (Figure 7). Current scientific reports suggest apoptosis as an effective biomarker of aquatic pollution. Apoptosis presented changes in the morphological characteristics of cells including membrane blebbing, nuclear condensation, cytoplasmic shrinkage and membrane asymmetry. Kiss (2010) reported the translocation of phosphatidylserine from inner leaflet of the plasma membrane to the outer leaftet, which was considered a hallmark of apoptosis [38]. Ray et al. (2013b) reported the apoptotic and necrotic cell deaths of haemocytes of *B. benga‐ lensis* and *L. marginalis* under the sublethal exposures of cypermethrin and fenvalerate, respectively, employing flow cytometry [2]. According to them, cypermethrin and fenvalerate treatment yielded decrease in the percentage of apoptotic and necrotic haemocyte morpho‐ types of *B. bengalensis* and *L. marginalis.* Pyrethroid-induced apoptosis of molluscan haemo‐ cytes is considered as impairment of immunological status of *B. bengalensis* and *L. marginalis.*

Figure 7: Immunofluorescent detection of apoptosis exposed to sublethal (3000 ppm/ 7 days) concentrations of washing soda. (a) and represent positive staining by Annexin V of hemocytes of pond snail, V-FITC and propidium iodide respectively, (c) pond B. bengalensis FITC **Figure 7.** Immunofluorescent detection of apoptosis of haemocytes of pond snail, *B. bengalensis* exposed to sublethal (3000 ppm/7 days) concentrations of washing soda. (a) and (b) represent positive staining by Annexin V-FITC and pro‐ pidium iodide, respectively; (c) depicts co-localization of fluorescent signals.

and (b)

P.