PLA in pellet form was purchased from Titan chemical, Pasir Gudang, Malaysia. SGF were collected from rural area of Jamalpur district in Bangladesh. The as-received SGF were cut carefully to separate the outer mat core from the inner fiber core. Only the outer mat core was used in this study. Single fibers were separated by hand until fine particulate fibers were obtained. Promising chemical modifiers like sodium hydroxide (NaOH) for alkalization, acetic acid (CH₃COOH), acetic anhydride [(CH₃CO)₂O] and sulphuric acid (H₂SO₄) for acetylation, and benzoyl chloride (C₆H₅COCl) and Ethanol (C₂H₅OH) for benzoylation were procured for treatment of SGF. 1,4-dioxane was purchased from the chemical market in Dhaka for dissolving PLA. All chemical compounds were the products of Merck, Germany.

Water intakes of the composites were measured according to ASTM: C-67-91. The test specimens were cut in a size of 6 cm length, 2 cm width and 0.5 cm thickness. The cut samples were kept in an oven at 80˚C for 24 h. It was taken out from the oven and immediately weighed. Let this weight be W_i. The samples were then immersed in distilled water of 23˚C and kept for 24 h. It was taken out from the water, wiped by a cloth, dried in air and then weighed. Let this weight be W_f. Then the amount of water intake was calculated by the following formula:

Water intake (%) = [ W f − W i W f ] × 100 (1)

The above procedures were repeated for 2 days to 30 days for all samples. It is noteworthy that the cut sides of the samples were coated with araldite to prevent from penetrating water into the sample.

The degradation under soil of the composites was performed according to ASTM: G-160. For this method, the samples were kept in an oven at 80˚C for 24 h. It was taken out from the oven and immediately weighed. Let this weight be w_i. The samples were then buried under soil kept in a pot. The pot was covered with a plastic net and exposed to atmospheric conditions for 7 days. Readings were taken of the changes in the weight loss of the samples at intervals of 7 days for approximately one month. To determine the weight loss the specimen of each sample was taken out from the soil, wiped by a dry cloth or tissue paper, quickly washed with cold water and dried in an oven at 80˚C to a constant weight. Let this weight be w_f. Then the percentage of weight loss was calculated by the following formula:

Weight loss (%) = [ w i − w f w i ] × 100 (2)

Fourier Transform Infrared (FTIR) spectra of the samples were recorded at room temperature by using a double beam IR spectrophotometer (model: Frontier, FT-IR/NIR Spectrometer, PerkinElmer, Japan) in the wave number range of 650 - 4000 cm⁻¹. For these measurements, the samples were crushed for recording the attenuated total reflectance (ATR)-FTIR spectra in the transmittance (%) mode.

The Bruker D8 ADVANCE XRD was used for taking pattern of X-ray diffraction (XRD). In this machine, a high voltage power supply (35 kV, 20 mA) was used to generate X-ray radiation. The composites were finely ground to prepare the disc specimen of same thickness for each category of samples. 1 g chopped samples were compressed in a cylindrical mold with a pressure of 1 MPa. The samples were step-wise scanned by XRD over the operational range of scattering angle between 10˚ to 30˚, with a step of 0.02˚, using the CuK_α radiation of wavelength λ = 1.5406 Å. The data were recorded in terms of the diffracted X-ray intensity (I) versus 2θ. The relative intensity is calculated by the following equation:

I = I T F P C I U F P C (3)

Compressive strength (CS) tests of PLAF, UFPC and differently treated fiber reinforced PLA composites (TFPCs) were performed by a universal testing machine (UTM) (model: H10KS, Hounsfield, UK) following ASTM: D-638-98 at a crosshead speed of 1 mm/min, keeping a gauge length of 10 mm. The CS values were calculated by the following equation:

Compressive strength, CS = P/A (4)

where P = Maximum load applied to the sample, A = Area of the sample (for the four-sided specimen, A = LW, where L is the length and W is the width of the sample).

The fractured samples of PLAF, UFPC and TFPCs obtained from the compressive strength tests were examined using a field emission scanning electron microscope (FESEM) (model: JSM-7600F, Jeol, Japan) at an acceleration potential of 20 kV. The fractured surfaces of the specimens were sputter-coated with a thin layer of platinum using a JFC-1600 auto fine coater.

Thermo gravimetric analyzer (TGA) coupled with a differential thermal analyzer (DTA) (model: EXSTAR, TG/DTA 6300, Seiko, Japan) was used in this study. The samples were heated from room temperature up to 600˚C in a nitrogen atmosphere at a heating rate of 20˚C/min. TGA is used primarily for determining thermal stability of samples. Data are recorded as thermograms of weight versus temperature.

Antibacterial activities of UFPC, ALT-FPC, ACT-FPC and BCT-FPC were measured against Gram-negative bacteria Escherichia coli and Gram-positive bacteria Staphylococcus aureus with agar diffusion method by Kirby-Bauer, 1985 [30] .

Cytotoxic effect of the samples was also examined. Vero cell line, kidney epithelial cells extracted from an African green monkey, was maintained in Dulbecco’s modified eagles’ medium (DMEM) containing 1% penicillin-streptomycin (1:1) and 0.2% gentamycin and 10% fetal bovine serum (FBS). Cells (3.7 × 10⁴/250 µl) were seeded onto 48-well plates and incubated at 37˚C + 5% CO₂. Next day, autoclaved samples were added to each well. Cytotoxicity was examined under an inverted light microscope after 72 h of incubation. Duplicate wells were used for each sample.

Figure 1(a) and Figure 1(b) show the variation of water intake (WI) on soaking time for PLAF, UFPC and different TFPCs. WI increases rapidly at the initial stage and nearly levels off after 10 days for UFPC and after 15 days for TFPCs. These results indicate that the absorption of water is slower for TFPCs than UFPC. WI is more in UFPC followed by BCT-FPC, ACT-FPC, ALT-FPC for both 5 and 10 wt% treated fiber reinforced composites, and PLAF. Although it is observed that WI value of 5TFPCs is higher than that of 10TFPCs. Untreated SGF is hydrophilic in nature and as a result WI is found to be more in UFPC than that of TFPCs and PLAF. After treatment, the hydrophilicity of the treated fiber decreases as well as a good adhesion of fiber with polymer matrix is established. Consequently WI of TFPCs decreases, as also reported elsewhere [31] . The maximum WI obtained in this study is 43.76 wt% for UFPC.

The degradation of composites has been evaluated by measuring the weight loss of samples, which have been soil buried in outdoor for 28 days. Figure 2(a) and Figure 2(b) illustrate the weight loss (WL) of PLAF, UFPC and different TFPCs as a function of the degradation time. As represented, all composites show an increased degradation rate during the buried process. This is likely a result of the penetration of moisture into the composites, causing the hydrolysis of surfaces and interfaces [32] . After 28 days coverage, the WL of samples reaches 36.06 wt% for PLAF, 40.98 wt% for UFPC, and 44.92, 47.44 and 45.84 wt% for 5ALT-FPC, 5ACT-FPC and 5BCT-FPC, respectively. Moreover, the WL is found 50.49, 47.75 and 49.44 wt% for 10ALT-FPC, 10ACT-FPC and 10BCT-FPC, respectively. These results suggest that the degradability of composites is slightly higher than that of PLAF. Apparently, the degradability of composites as compared to that of the PLAF is not swift but is slow, which is expected from bio-composites so that they can substitute the non-degradable composite materials [25] .

The ATR-FTIR spectra of the PLAF, UFPC, ALT-FPC, ACT-FPC and BCT-FPC are shown in Figure 3(a) and Figure 3(b), and Figure 4(a) and Figure 4(b) for the treatment concentration of 5 and 10 wt%, respectively. There is no notable

variation in the FTIR spectra of Figure 3(a). The peaks at the range of 1000 - 1300 cm⁻¹ from the skeletal stretching vibrations may include the C−C axial elongation and C−N stretching of amines of the samples. On the other hand, there are some variations in the spectra of Figure 3(b). The main transmittance peaks in this study have been identified. The first notable variation is found at the peak for 5ACT-FPC as shown by an arrow at 2153 cm⁻¹, which corresponds to the characteristic C=O stretching vibration. The peak at 2849 cm⁻¹ for 5ACT-FPC and 2853 cm⁻¹ for 5BCT-FPC are the characteristic bands for the C−H stretching vibration. The transmittance peak at 2928 cm⁻¹ of 5BCT-FPC may be belong to the O−H stretching vibration of linkage of carboxylic acid in lignin or ester group in hemicellulose. In Figure 4(a), there is also any remarkable variation is absent. But in Figure 4(b), a clear change is notable in the transmittance peak for 10ACT-FPC. The sharp peak at 2153 cm⁻¹ of 5ACT-FPC [Figure 3(b)] is removed at the stage of 10ACT-FPC [Figure 4(b)] indicates removing of −C=O stretching vibration band.

From the above stated experiment of FTIR, especially from Figure 3(b) and Figure 4(b) it is notable that the acetic anhydride and benzoyl chloride may have interacted chemically with SGF.

The XRD patterns of PLAF, UFPC, ALT-FPC, ACT-FPC and BCT-FPC are illustrated in Figure 5(a) and Figure 5(b) for the treatment concentration of 5 and 10 wt%, respectively. The two sharp diffraction peaks of PLAF and composites come from the component PLA at the scattering angle 2θ = 16.50˚ to 18.80˚. The very small XRD peak of treated SGF appears at 22.30˚ as indicated by arrow. The position of first peak is shifted to slightly higher angle in alkali treated sample in both 5 and 10 wt%, indicating a decrease in interplanar spacing of PLA crystal. The relative intensities have been estimated to be I₁ = 1.57, I₂ = 1.02, and I₃ = 1.08 for 5TFPCs, and I₄ = 1.62, I₅ = 1.61, and I₆ = 1.32 for 10TFPCs, respectively. Thus, it is clear that the intensity of the first and second peaks increases by fiber treatment, suggesting an increase in crystallinity of the modified composites. The results also suggest that a close packing takes place in the PLA crystal possibly by the crystal nucleation effect of cellulose molecules after removal of hemicellulose, lignin, pectin, etc. by different treatments. These results are similar to the reported results [33] . Among these samples, the crystallinity is better for 5ALT-FPC than others.

The compressive strength (CS) test results for PLAF, UFPC, and 5 and 10TFPCs are shown in Figure 6(a) and Figure 6(b), respectively. In all of the samples the strength of UFPC is the least of value, which is 7.61 MPa. On the other hand, the results indicate that there is a notable increase in CS of composites with the treatment of fibers. The CS values for 5ALT-FPC, 5ACT-FPC and 5BCT-FPC are 8.35, 8.59 and 10.06 MPa, respectively, and for 10ALT-FPC, 10ACT-FPC and 10BCT-FPC are 9.7, 9.97 and 9.89 MPa, respectively. The highest strength is found for 5BCT-FPC having the value of 10.06 MPa.

In all of the composites, the CS value is found to increase by 10% - 35% after chemical treatment. This increase can be attributed to the reduction in the

porosity of foam type PLA and an enhancement in mechanical bond strength [34] . It is observed that the formation of cracks is extended in the specimens without fibers. However, the increase in CS due to SGF inclusion can be attributed to the improvement in the mechanical bond strength between the fibers and matrix where the fibers contribute to delay of micro-crack formation and stop their propagation afterwards up to a certain extent of fibers volume fraction [35] [36] .

Figure 7 illustrates FESEM micrograph of the fractured surface of (a) PLAF and (b) UFPC. The surfaces of both images are not fairly smooth. The porosity of PLA increases when it is made PLAF. Besides, a lot of flaws and voids are seen on the surface of PLAF. In Figure 7(b), the image of UFPC shows lot of weak interfaces, resulting poor adhesion between fiber and matrix components. Huge pores are also found on the surface of UFPC.

Figure 8(a), Figure 8(b) and Figure 8(c) represent FESEM micrographs of the fractured surfaces of 5ALT-FPC, 5ACT-FPC and 5BCT-FPC, respectively. Figure 8(a) shows that the impact between PLA matrix and SGF is moderate. It is clear that the surface of the 5ALT-FPC is slightly rough but smoother than that of PLAF and UFPC. In Figure 8(b) and Figure 8(c), it is remarkable that

the interfacial adhesion between matrix and SGF powder is found to be improved. In Figure 8(c), it is seen that the surface is a little bit rough and some cracks are observed.

Figure 9(a), Figure 9(b) and Figure 9(c) represent the FESEM images of fractured surfaces of 10ALT-FPC, 10ACT-FPC and 10BCT-FPC, respectively. Figure 9(a) shows that the impact between fiber and matrix is good. It is clear that the surface of the 10ALT-FPC is smoother than 5ALT-FPC. In Figure 9(b) and Figure 9(c), it is clearly seen that the interfacial adhesion between PLA matrix and SGF powder of 10TFPCs are also found to be improved than that of 5TFPCs.

Figure 10(a) and Figure 10(b) illustrate the thermograms for TGA of the PLAF, UFPC, and TFPCs for the treatment concentration of 5 and 10 wt%, investigated in a temperature range of 35˚C - 600˚C. The on-set degradation for all samples occurs at higher temperature, precisely after 280˚C. The TGA sharp-fall occurs at different temperatures, depending on the samples. However, the

temperature at 50% weight loss is considered as the thermally stable temperature for the sample [37] . The on-set temperature (T_o), the stable temperature (T_S), and the end-set temperature (T_e) values evaluated from the TGA curves of the samples are introduced in Table 1.

Figure 11(a) and Figure 11(b) depict the DTA thermograms of PLAF, UFPC, and different TFPCs for 5 and 10 wt% chemical concentrations, investigated in a temperature range of 35˚C - 600˚C. The starting points of first endothermic

From the results presented in Table 1, it is clearly seen that the sample 10BCT-FPC is thermally more stable than others.

peak of DTA for all samples indicate the melting temperature (T_m) of the samples. The starting points of second endothermic peak correspond to the degradation temperatures (T_d) of the samples. The DTA indicates that the T_m value of 5ALT-FPC is more than others, but the T_d value of 10BCT-FPC is 404.41˚C, which appears as the highest value observed among the composites. The T_m and T_d values for different samples are inserted in Table 2.

The observed results strongly suggest that the benzoyl chloride treated fiber reinforced PLA composites are thermally more stable than others and the chemical treatments of SGF result in slow thermal decomposition. This may be connected to the different decomposition behaviors of the molecules of the differently treated SGF. The decomposition of the parent molecule, benzoyl chloride, is highly condition-dependent with the sample heating rate and temperature of decomposition playing a preponderant role in the course of the decomposition [38] . The degradation of SGF reinforced PLA composites have been ascribed by the dissociation of C−C chain bonds along with H-abstraction at the site of dissociation [39] .

Figure 12 shows the antibacterial activities of the UFPC, 5ALT-FPC, 5ACT-FPC and 5BCT-FPC composites. Evaluation of antibacterial activities was carried out through dynamic shake flask method accompanying with plate count agar technique (Kirby-Bauer agar diffusion) using (a) E. coli and (b) S. aureus (Reference: FARL-72) as testing bacteria. Antibacterial properties of materials were accounted by comparative analysis of viable cell count from treated and untreated composites by means of bactericidal behavior. We have found that the antibacterial activity has decreased with the treatment of SGF. It is observed that there is no zone of inhibition surrounded the sample disc. Moreover, the used chemicals couldn’t import antimicrobial properties to the composites. So, none of the samples have any antibacterial effect on E. coli and S. aureus.

Figure 13 shows the cytotoxic effect of only treated samples like 5ALT-FPC, 5ACT-FPC and 5BCT-FPC composites. All of the samples were tested on the category of Vero cell (non-cancer cell) line. The results collected after 72 h from the time of sample placement. The test results are summarized in Table 3.

The protocols in this research describe the growth and maintenance of Vero cells using DMEM as the culture medium. The DMEM is a very common culture medium, but a variety of other media can also be successfully used with Vero cells. Depending on the application, it may be desired or necessary to count the number of cells (i.e., if a specific number of cells need to be analyzed, plated, etc.). The concentration of cells in suspension (following trypsin treatment) was determined using a hemacytometer. In this Figure, the black shadows indicated the amount of death cells and the light colors indicated the survivor cells. The images proof that the prepared samples are not harmful for non-cancer cell of human body.