BIBECHANA Vol. 20, No. 1, April 2023, 27–35 ISSN 2091-0762 (Print), 2382-5340 (Online) Journal homepage: http://nepjol.info/index.php/BIBECHANA Publisher:Dept. of Phys., Mahendra Morang A. M. Campus (Tribhuvan University)Biratnagar A systematic study on material properties of water retted Sterculia and Bauhinia fiber Krishna Prasad Kandel, Girja Mani Aryal, Sishir Achyarya KC Mahesh Kumar Joshi, Bipeen Dahal, Bhanu Bhakta Neupane∗ 1Central Department of Chemistry, Tribhuvan University, Kathmandu 44613, Nepal ∗Corresponding author. Email: newbhanu@gmail.com Abstract Lignocellulose biomass forms an important component of traditional and next generation com- posite materials. To obtain desired properties, the biomass needs to be chemo–mechanically processed at different levels. The raw lignocellulose fiber obtained from sterculia villosa (Roxb.) and Bauhinia vahlii is traditionally believed to have high water stability; and therefore used in rural areas of South Asian regions to secure objects submerged under water. In this research, we systematically studied several material properties of raw Sterculia and Bauhinia fiber sam- ples retted for 0, 20, 30 and 55 days (n=8). Water retting resulted in significant decrease in lignin and extractives content (p<0.05) and increase in cellulose content. Fiber bundle strength of Sterculia fiber increased with retting time (R2= 0.7) but Bauhinia fiber did not show significant change (p>0.05). Interestingly, water retting resulted in increased thermal stability in both fiber types. These findings suggested that the fiber studied have excellent water stability. The observed trend in mechanical and thermal properties could have resulted from crystallinity change and/or nominal fiber damage as supported by XRD and SEM imaging data; respectively. These findings suggested that Sterculia and Bauhinia fiber biomass could be an important component of biodegradable composite materials which are intended for high wetting and/or humid conditions. Keywords Lignocellulose, Water retting, Fiber processing, Cellulose fiber, Mechanical strength. Article information Manuscript received: January 26, 2023; Accepted: March 28, 2023 DOI https://doi.org/10.3126/bibechana.v20i1.51793 This work is licensed under the Creative Commons CC BY-NC License. https://creativecommons. org/licenses/by-nc/4.0/ 1 Introduction Lignocellulose biomass is one of cheap, environ- mentally friendly, and most abundant terrestrial biomass. The biomass can be obtained from leaf, seed, fruit, and bast portion of different plant species or other plant derived wastages following different processing methods [1–5]. Bast fibers are the soft woody fiber obtained from phloem tis- sues of plant stem. The fibers are usually long and strong and form an important components of next generation materials and finding applications in waste water treatment, biomedical applications, agro and automotive industries [6–10]. Cellulose 27 http://nepjol.info/index.php/BIBECHANA newbhanu@gmail.com https://doi.org/10.3126/bibechana.v20i1.51793 https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ Krishna Prasad Kandel et al./ BIBECHANA 20 (2023) 27-35 28 nano–composite fibers are also being explored in fabrication of fabrics having self-cleaning, antimi- crobial, and UV-protection properties [11–13]. In natural state, bast fibers are cemented to the ad- jacent tissues by different gummy or non–cellulosic components such as wax, pectin, and lignin. To yield fiber of desired properties, the gummy com- ponents needs to be removed following different chemo-mechanical methods. Retting is one of the most important degumming methods. Several ret- ting types are in practice such as chemical ret- ting, enzymatic retting, water retting, dew ret- ting, gel retting, ribbon retting, or their combina- tions [14–17]. Retting, in general, removes non- cellulosic components so that fiber bundles par- tially separate. To get fiber of optimal strength retting conditions needs to be optimized [1]. In wa- ter retting, fiber bundles are submerged in slow or stagnant water in a pond or tank normally for 2–3 weeks [15,18]. Ground or tap water contain anaero- bic bacterial and or fungal colonies that produce en- zymes capable of hydrolyzing gummy or fiber bind- ing components such lignin and pectin with release of galacturonic acid and sugar as major by-products [1,19,20]. This results in partial removal of gummy components so that fiber bundles partially separate. The retting conditions, such as retting time, micro- bial type and load, and fiber type determine the fiber quality. Water retting results in putrid wa- ter and many pollutants. However, this is one of the cost effective methods and yields fibers having excellent length uniformity and strength [15, 21]. Retting can also be carried out by using enzyme such as pectinase, xylanases in controlled condi- tion. Although enzymatic retting is faster (12–24 hours) and does not produce unnecessary pollu- tants, it results in fiber of low strength [1,18]. Ret- ting can also be done mechanically using decortica- tor to break fiber to fiber bonding. Mechanical ret- ting is rapid but results fiber of low quality. Also, high cost limits it application in resource limited settings. As a result, traditional water retting is still being explored as viable option for obtaining fibers of various lengths and excellent strength [22]. The fiber biomass obtained from Sterculia villosa (Roxb.) and Bauhinia vahlii plant species is be- lieved to have excellent water stability; and there- fore traditionally being used at local levels to secure objects submerged in water or in high humid con- ditions. It would be interesting to explore material properties of fiber water retted for different time period. In this study, two fiber types were retted in tank water for 0, 20, 30, and 55 days. Retting efficiency, change in content of major chemical com- ponents, fiber strength, and thermal stability of the fiber samples were systematically investigated. We also bridge the observed end properties with the morphological and crystallinity change. Finally, a brief discussion on the further implications of the research is provided. 2 Materials and methods 2.1 Materials The Sterculia Villosa (Roxb.) and Bauhinia Vahlii plant stems ( 1 m long and 3 cm wide) were col- lected from Digam, Gulmi, Nepal (hilly region, 1250 masl). Bast fiber biomass was separated from the wet stem following traditional method. The stems were gently beaten around one end with a wooden log and pulled mechanically with hands. Outer cuticular layer was removed from biomass using a stainless steel knife (Figure 1). The samples were sun dried to remove excessive moisture and moved to lab for further study. 2.2 Methods 2.2.1 Water retting Water retting was performed following literature re- ported methods with slight modifications [23, 24]. Bast fiber samples were divided into four groups of 100 g each. Samples were well rinsed with tap water and gently crushed to loosen the bundles. The sam- ples were immersed in separate buckets containing 4 L of tap water (pH 7.2±1, conductivity 9± 1 µS). The samples were left open under natural condi- tions (temperature 20–300C and humidity 50–70%) for 20, 30 and 55 days. To minimize the excessive growth of microorganism and fouling, talk water was one third diluted in every 10 days. After each retting time, sample was washed with distilled wa- ter and oven dried at 105±20C. From the initial and final weights, weight loss% or retting efficiency was calculated. For convenience, Sterculia samples retted for 0, 20, 30, and 55 days hereunder named as S0, S20, S30, and S55 and the Bauhinia samples B0, B20, B30, and B55. Until further characteriza- tion these samples were stored in air tight plastic bag in dark. A simple schematics of experimental design used in this work is provided in figure 1. 2.2.2 Chemical analysis Estimation of major chemical components viz. ex- tractives, lignin, hemicellulose, cellulose and ash content in the water retted samples (S0, S20, S30, S55 and B0, B20, B30 and B55) was performed gravimetrically following the previously reported methods [25–28]. Triplicate measurements were made in all the samples for each chemical compo- nent. For estimation of lignin, 1.000 g of extrac- tive free dry sample was treated with 72% H2SO4 (1:12.5 m/v ratio) for two hours at room temper- ature. The top solvent was removed with several Krishna Prasad Kandel et al./ BIBECHANA 20 (2023) 27-35 29 washings with distilled water. The residue was dried at 105 0C till constant weight was obtained. Form the known initial and final weights, lignin con- tent was obtained. To determine the hemicellulose 1.000 g of extractive free dry biomass was boiled in 150 mL of 0.5 mol/L NaOH for 4 hours. The treated fiber content was washed several times with distilled water to neutral pH and oven dried at 1050C until constant weigh was obtained. Hemicellulose con- tent in % was obtained from the initial and final weights of the biomass. Finally, cellulose content was obtained by subtracting the collective content of extractive, lignin and hemicellulose from 100. Figure 1: Schematic outline of the experimental design used in this work. 2.2.3 Mechanical strength of fiber Water retted fiber samples were preconditioned for 24 hours at 23±1 0C and 65 % relative humid- ity [29]. A fiber bundle strength tester (TSI instru- ments) with a maximum load capacity of 7 kg was used to determine fiber strength. Flat fiber bundles were inserted into the Pressley Jaw (gauze length 15 mm) and then loaded into the tester. Load was applied at a continuous rate of 1 kg/sec until the fiber bundle breaks. The load at fiber break was recorded. The broken fiber pieces was removed from the Pressley Jaw and weighed in an analytical bal- ance at nearest accuracy of 0.0001. The fiber bundle strength (breaking tenacity) in g/tex was obtained from known values of breaking load, grammage, and gauge length. To achieve statistical significant re- sult, 25 measurements were taken for each sample type. 2.2.4 XRD, TGA, and SEM measurements XRD data were measured in 2θ range 5–300 using a X-ray diffractometer (Rigaku, UK) The voltage, scan rate and step size were 40 kV, 0.020/min, 0.020; respectively. A monochromatic from Cu–Kα line (λ= 1.540 A) was used as X-ray source. Thermo- grametric data of the fiber samples was measured using thermogravimetric analyzer (Perkin Elmer TGA–7). 7–10 mg of fiber sample was heated in an alumina crucible in nitrogen atmosphere (flow rate 80 mL/min) at the rate of 10 oC/minute. Data collection range, weighing accuracy and pre- cision were 26-600 0C, 0.1 µg, and ± 2 0C; respec- tively. Scanning electron microscopic images were obtained using field emission electron microscope (Carl Zeiss, Supra 40VP) at the accelerating volt- age of 15 kV. The collected images were imported in ImageJ (NIH, USA) for further analysis. 2.2.5 Statistical analysis Descriptive statistical parameters such as mean, maximum, minimum, standard deviation and con- fidence intervals were calculated using Origin Pro (Origin lab, USA). To test the significance differ- ence between the data, whenever relevant, t-test was performed. 3 Results and Discussion 3.1 Gravimetric analysis The % weight loss in Sterculia fiber retted for 20, 30, and 55 days was found to be 10.5, 15.2, and 17.5 %; respectively. The corresponding numbers for Bauhina fiber were 4.1, 5, 8.1%; respectively. These data suggested that Sterculia fiber has higher retting efficiency than Bauhinia. Since retting con- ditions were same for both fiber types, the difference is retting efficiency could be linked to fiber origin; that is difference in chemical composition and fiber to fiber bonding. Krishna Prasad Kandel et al./ BIBECHANA 20 (2023) 27-35 30 Figure 2: Analysis of major chemical components in the fiber samples. (A) Extractive. (B) Lignin. (C) Hemicellulose. (D) Cellulose. The blue and red data points correspond to Sterculia and Bauhinia; respectively. The dotted lines are the linear fit to the experimental data and R2 values greater than 0.5 are only shown. Figure 3: Mechnical strength of the fiber samples. The dotted lines are the liner fit to the experimental data points. The error bars are the 95% CI values to the mean (n=25. The extractive, lignin, hemicellulose, and cel- lulose contents in the fiber samples for two fiber types in reported in figure 2. As expected, water retting resulted in decrease in extractive content with increase in retting time (Figure 2A). In the 55 days retting period, the extractive content de- creased significantly from 13 to 1.3 % (p<0.05) in the Sterculia fiber. The corresponding decrease in Bauhinia fiber was from 13.6 to 8.7 %. The weight loss could have resulted from the removal of pectin and waxy materials due to antimicrobial action. In the 55 days period, a significant decrease in lignin (p<0.05) content from 21.9 to 15.5 % was also ob- served in Sterculia (Figure 2B). The weight loss in retting depends on type of microorganism, water composition, retting time, and fiber types [4, 19]. So it is not surprising to see the observed differ- ence in weight loss in Sterculia and Bauhinia fibers. Hemicellulose content in both sample types did not change significantly (Figure 2D). In the 55 days retting period hemicellulose changed from 28.6 to 33.2 % in the Sterculia fiber. The corresponding Krishna Prasad Kandel et al./ BIBECHANA 20 (2023) 27-35 31 Figure 4: XRD data for the fiber samples. (A) XRD data of Sterculia samples retted for 0 (S0), 30 (S30) and 55 (S55) days. (B) XRD data of Bauhinia samples retted for 0 (B0), 30 (B30) and 55 (B55) days. Data are overlaid vertically for easy comparison. Numbers in parentheses indicate the reflection planes. change in Bauhinia fiber was from 31.2 to 33.6 %. In the same time period, cellulose content in Ster- culia fiber increased significantly (Figure 2C) from 36.5% to 50% (p<0.05). A nominal change from 27 to 30% was observed in Bauhinia fiber. We also measured ash content in fibers retted for 0 and 55 days. The corresponding values for Sterculia and Bauhinia were 6.7 and 4.7% and 6.2 and 4.4 %; re- spectively. The decrease in ash content could be due to partial loss of inorganic minerals and other im- purities on water retting. As expected, ash change in ash content showed excellent negative correlation (r= -0.9) with extractive content. 3.2 Mechanical strength The tenacity data for all the samples is provide in figure 3. In the 55 days period, the break- ing tenacity of Sterculia fiber increased signifi- cantly (p<0.05) from 18.3±1.6 (mean ±95% CI) to 26.9±2.7 g/tex (R2=0.73). In the same pe- riod, mechanical strength of Bauhinia fiber did not change significantly (p>0.05). The increase in fiber strength in Sterculia could be due to more loss in lignin from inter-fibrillar region that can lead in increased reorganization of cellulose chain thereby increasing crystallinity (later section) and strength [28]. 3.3 XRD data We measured XRD data of selected sterculia and Bauhinia samples (Figure 4A and B). The XRD data of all the samples resemble to that of typ- ical cellulose material. Cellulose fiber in natural form contains amorphous and crystalline regions. A broad peak at 2θ value of 15.50 orginates form the crystalline planes (1-10) and (110) and the strong peak at 2× value of ∼21.50 from (200) plane. The broad contribuation underlying the crystalline peaks is known to orignate from amorphous scat- tering [30]. We used deconvolution method in the 2θ range of 5–300 to get information on crystallinity index (CI) [30]. CI = ( At −Aam At ) 100% (1) where, Aam and At are the integrated intensity amorphous phase and total intensity of crystalline and amorphous phases; respectively. The crys- tallinity index in Sterculia samples retted for 0, 30, and 55 days was found to be 44%, 48% and 50%; respectively. A good positive correlation (r=+0.85) between crystallinity and strength was obtained. Crystallinity index increases on water retting is also reported for hemp fibers [31]. The increase in crys- tallinity index could be due to removal of lignin form inter–fibrillar region that can result in close packing of cellulose chains [28]. Crystallinity index in Bauhinia samples retted for 0, 30,and 55 days was found to be 55%, 54% and 54%; respectively. Krishna Prasad Kandel et al./ BIBECHANA 20 (2023) 27-35 32 Figure 5: TGA and DTG data for the fiber samples. (A), (B) TGA and DTG for Sterculia water retted for 0 (S0), 30 (S30), and 55 (S55) days. (C), (D) TGA and DTG for Bauhinia water retted for 0 (B0), 30 (B30), and 55 (B55) days. A poor positive poor correlation (r= +.5) was be- tween the crystallinity change and strength was ob- served. This could be due to lower loss of lignin form inter–fibrillar region; consistent with data re- ported in figure 2B. A small peak shift in the XRD data could be due to change in relative contribution of amorphous and crystalline phases and or change in crystallite size. 3.4 Thermal properties In both samples the weight loss below 1000C (Fig- ure 5A and C) is due to loss of moisture and or low volatile impurities [28, 32–34]. The weight loss between 100 to 3000C is mainly due to hemicel- lulose; as hemicellulose is known to be less stable than cellulose (Figure 5A and C). The weight loss in 300–4000C range is mainly due to degradation of glycosidic bonding in cellulose that creates organic molecules such alkenes and additional derivatives of hydrocarbons. The weight loss beyond 4000C is mainly due lignin (5A and C) [35–37]. The main DTG peak for Sterculia shifts from 3400C to 3500C in water retted samples. This suggested that wa- ter retting results in increase in thermal stability (Figure 5B). It could be due increase of cellulose content and change in crystalline properties; as re- ported previously. The strong DTG cellulose peak in water retted Sterculia samples (S30 and S55) is consistent with increase of cellulose on water ret- ting (figure 2D). In Bauhinia, DTG peak shifts from 3500C to 3550C in water retted samples (Figure 5C). This suggested that thermal stability of wa- ter retted Bauhinia does not change significantly on water retting. 3.5 SEM imaging Imaging of selected samples (6 out of 8) was per- formed to see any change in fiber surface morphol- ogy. A close comparison of images of the Stercu- lia fiber retted for 0, 30, and 55 days (Figure 6A, B, and C) reveals that gummy materials are sig- nificantly removed from the fiber surface and fiber bundles are partially separated. This observation is consistent with the wet loss study. Similar change is observed in Bauhinia fiber (Frames D, E, and F). 4 Conclusion To summarize, water retting efficiency and ligno- cellulosic content of Sterculia villosa and Bauhinia vahlii fiber retted for different periods was mea- sured. In Sterculia fiber, weight loss increased sig- nificantly with retting time; which is consistent with reduction of lignin, ash and extractive con- tents. The change was less significant in Bauhinina fiber suggesting lower retting efficiency. In around Krishna Prasad Kandel et al./ BIBECHANA 20 (2023) 27-35 33 Figure 6: SEM images. A, B, and C are the images of Sterculia fiber retted for 0 (S0), 30 (S30), and 55 (S55) days; respectively. D, E, and F are the corresponding images for Bauhinia fiber. Scale bar of 100 µm are provided in A (also applicable for B and C) and D (also applicable for E and F). 2 months retting period, mechanical strength of Sterculia fiber increased significantly but that of Bauhinia did not change. Thermal stability of both fiber increased on water retting. These observations are also supported by XRD and SEM imaging data. These findings suggested that both fiber have ex- cellent water stability and can be explored as an important component in fabricating cellulose based composite materials that are intended for high wet- ting or humid conditions. References [1] M.T. Paridah, A.B. Basher, S. SaifulAzry, and Z. 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Introduction Materials and methods Materials Methods Water retting Chemical analysis Mechanical strength of fiber XRD, TGA, and SEM measurements Statistical analysis Results and Discussion Gravimetric analysis Mechanical strength XRD data Thermal properties SEM imaging Conclusion