DOI: 10.3303/CET2292109 Paper Received: 14 January 2022; Revised: 26 March 2022; Accepted: 30 April 2022 Please cite this article as: Taokaew S., Thienchaimongkol J., Nakson N., Kobayashi T., 2022, Valorisation of Okara Waste as an Alternative Nitrogen Source in the Biosynthesis of Nanocellulose, Chemical Engineering Transactions, 92, 649-654 DOI:10.3303/CET2292109 CHEMICAL ENGINEERING TRANSACTIONS VOL. 92, 2022 A publication of The Italian Association of Chemical Engineering Online at www.cetjournal.it Guest Editors: Rubens Maciel Filho, Eliseo Ranzi, Leonardo Tognotti Copyright © 2022, AIDIC Servizi S.r.l. ISBN 978-88-95608-90-7; ISSN 2283-9216 Valorisation of okara Waste as an Alternative Nitrogen Source in the Biosynthesis of Nanocellulose Siriporn Taokaew*, Jirath Thienchaimongkol, Nawachon Nakson, Takaomi Kobayashi Department of Materials Science and Technology, Nagaoka University of Technology, Nagaoka, Niigata, Japan t.siriporn@mst.nagaokaut.ac.jp Cellulose nanofibers biosynthesized by bacteria (BC) possess outstanding properties such as non-toxicity, high purity, high liquid holding capacity, high crystallinity, and high tensile strength. It is a more eco-friendly, bio- based material compared to cellulose isolated from plants via extraction/purification processes in which harsh chemicals are used. However, the low yield of BC limits the widespread production and usage of BC nanofibers. Hence, the purpose of this research was to study the effects of four different culture media, various carbon and nitrogen sources, initial pH, and static/dynamic cultivation conditions on BC yield. Suitable culture media formulations and cultivation conditions of cellulose-producing bacteria (Gluconacetobacter xylinus) were assessed. Moreover, BC production was examined in a modified Hestrin-Shramm (HS) medium by replacing the nitrogen source ingredients (yeast extract and peptone) with okara, an agricultural by-product from industrial soybean production. Nanocellulose yield and content in a hydrogel-like pellicle were compared. As a result, high BC pellicle yield (100-350 g/L) and nanocellulose content (3-12 mg/cm3) were observed in HS and Zhou media at pH 5.0 under static and combined static-dynamic conditions by adding mannitol and corn steep powder as carbon and nitrogen sources, respectively. When okara was used as an alternative nitrogen source, the resultant BC exhibited high crystallinity (90%) and high molecular weight (7-9106 g/mol) over the range of tested concentrations. The highest BC pellicle yield (85 g/L) and cellulose content (3.4 mg/cm3) were obtained when the culture was supplemented with 1 %w/v freeze-dried okara. The valorisation of non-valued okara waste through nanocellulose production can alleviate economic constraints around BC production and can simultaneously provide enhanced sustainability. 1. Introduction Cellulose, a common polysaccharide in nature, is mainly found in cell walls to strengthen the structure of plants. It contains other natural impurities such as lignin and hemicellulose (Klemm et al., 2005). Chemical treatments to remove these impurities are required, which create several environmental problems (Ng et al., 2015). Consequently, there is an interest in reducing or replacing demand of plant cellulose with an alternative biocellulose synthesized by bacteria in an eco-friendly one-step production, so-called bacterial nanocellulose (BC) (Almeida et al., 2021; Klemm et al., 2005). The chemical structure of BC is analogous to that of plant cellulose i.e. many D-glucose units bound by 1,4--glycosidic bonds, but has superior properties such as high cellulose purity, an ultrafine nanofiber network, high surface area, and high-water holding capacity (Joseph et al., 2020; Taokaew et al., 2016). Due to an abundance of hydroxyl groups bonded inter and intramolecularly between parallel linear chains and adjacent glucose units, BC possesses highly crystallinity resulting in high tensile strength and these hydroxyl groups are available for modification and functionalization (Peter, 2021). These properties allow BC to be employed as a base material in foods, papers, medical uses, pharmaceutics, electronics, fuel cells, and other applications (Taokaew et al., 2016). However, the low yield of BC production limits its industrial applications. Therefore, various recent efforts have focused on increasing production of BC by optimizing the bacterial cultivation condition; for instance, carbon sources (mono- and disaccharides), nitrogen sources (proteins), minerals, pH, and air/oxygen content (Andriani et al., 2020). The use of wastes e.g. fruit residues (Amorim et al., 2019) and corn steep liquor (Aragão et al., 2020) as alternative nutrient sources to produce BC have also been explored. The application of okara, an agricultural 649 waste from industrial soybean production, as a nitrogen source for G. xylinus is rarely reported in scientific literatures. Hence, utilization of okara in the biosynthesis is the novel aspect of this study. Not only is the environmental impact minimized, and economic value gained, but also, the costly commercial protein in the bacterial culture is substituted. Commonly, the nitrogen source is expensive relative to the carbon source e.g. sugar. However, other studies focused on the carbon source to produce nanocellulose but retaining an expensive nitrogen source. This study aims to biosynthesize BC in various culture media by adjusting carbon source, nitrogen source, and pH. The effects of cultivation conditions including static, dynamic, and the combination of both were also assessed. An optimum condition was applied in the okara experiment. The resultant BC was characterized in terms of yield, cellulose content, purity, crystallinity, molecular weight, morphology, and viscoelastic properties. 2. Experimental methods 2.1 Materials Fresh okara (Nikko Food Cooperation, Japan) was purchased from a local food market in Niigata prefecture, Japan. The okara was freeze-dried using a freeze dryer (Eyela FDU-1200, Japan) for 24 h prior to use in the experiment. Gluconacetobacter xylinus BPR2001 ATCC® 700178 (G. xylinus) was purchased from American Type Culture Collection (ATCC). Peptone and yeast extract were purchased from Becton, Dickinson and Company (BD), USA. Corn steep powder (CSP) was purchased from Oriental Yeast company, Japan. The 99% purity-grade dimethylacetamide (DMAc) was purchased from Nacalai Tesque Inc, Japan. Other chemicals used in this study were purchased from Wako Pure Chemical Industries, Ltd, Japan. 2.2 Cultivation of G. xylinus To prepare the inoculum, G. xylinus was incubated in sterilized HS culture medium containing 2 %w/v glucose, 0.5 %w/v yeast extract, 0.5 %w/v peptone, 0.27 %w/v Na2HPO4, and 0.115 %w/v (COOH)CH2COOHH2O (citric acidH2O) using coconut water (Malee, Malee Group PCL, Thailand) as a solvent. The pH value of culture media was adjusted to pH 5 using acetic acid before sterilization at 121 oC for 15 min. This cell suspension was incubated at 30 oC for 7 days for using as an inoculum in the experiment. The four different culture media, which were yeast extract/glucose/calcium carbonate (YGC), HS, Yamanaka (Y), and Zhou (Z), having different ingredients (Table 1), were inoculated with 10 %v/v inoculum and incubated at 30 oC under a static condition for 7 days. Effects of various carbon (glucose, sucrose, fructose, and mannitol) and nitrogen (CSP, peptone, tryptone, and yeast extract) sources, initial pH (4-6.5), and static/dynamic conditions were evaluated in Z medium. The dynamic condition was performed in a horizontally shaking incubator at 160 rpm. The combined static and dynamic condition was also performed in a shaking incubator at 0 and 160 rpm for 3.5 days of each. Thereafter, peptone and yeast extract in HS medium were replaced by okara at the concentration of 0-3 %w/v using deionized (DI) water as a solvent. The medium (100 mL) was inoculated with 10 %v/v of the inoculum, and the cultivation was performed in a static condition. All the experiments were conducted at 30 oC. After 7 days, the hydrogel-like BC pellicles floating on the air-liquid medium interface were harvested and purified by washing with running tap water, rinsing with DI water, immersing in 1 %w/v NaOH for 24 h at room temperature (253 oC), and neutralizing with DI water. The samples were named as “BCx”, in which x indicated concentrations of the freeze-dried okara supplemented in the culture media. Table 1: Formulations of bacterial cell culture media in water (%w/v) YGC HS Y Z Glucose Yeast extract Peptone CSP CaCO3 Na2HPO4 (COOH)CH2COOHH2O (NH4)2SO4 KH2PO4 MgSO47H2O 5 0.5 - - 1.25 - - - - - 2 0.5 0.5 - - 0.27 0.115 - - - 5 0.5 - - - - - 0.5 0.3 0.005 2 - - 2 - - - 0.4 0.2 0.04 650 2.3 Characterization Yield and nanocellulose content were calculated according to Eq(1) and Eq(2), respectively. 𝑌𝑖𝑒𝑙𝑑 = 𝑊𝐵𝐶 𝑝𝑒𝑙𝑙𝑖𝑐𝑙𝑒 𝑉𝑀𝑒𝑑𝑖𝑎 (1) 𝐶𝑒𝑙𝑙𝑢𝑙𝑜𝑠𝑒 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 = 𝑊𝐵𝐶 𝑉𝐵𝐶 𝑝𝑒𝑙𝑙𝑖𝑐𝑙𝑒 (2) where WBC pellicle and WBC are weights of hydrogel-like BC pellicle and freeze-dried BC, respectively. Vmedia and VBC pellicle are volumes of cell culture medium and BC pellicle, respectively. Analysis of elemental composition of the samples was performed by X-Ray Fluorescence spectrometer (XRF, Rigaku ZSX Primus II, Japan) using ZSX software to quantify the purity according to total mass of carbon and nitrogen elements. This spectrometer contains a 30-keV and 100-mA (Rh 4.0 kW) X-ray tube, providing the detection of diverse elements of the Periodic Table at 2 of 63o. Prior to characterization, pressed pellet used for measurement were prepared by press machine under a pressure of 500 kgf/cm2. X-ray diffractograms were obtained using X-Ray Diffractometer (XRD, Rigaku Smart Lab 3 kW, Japan) under operation conditions of 40 kV and 30 mA with Cu-K radiation. The relative intensity was recorded in steps of 0.01o and speed of 3.0o/min. Percent of crystallinity was determined by Eq(3). % 𝑐𝑟𝑦𝑠𝑡𝑎𝑙𝑙𝑖𝑛𝑖𝑡𝑦 = 𝐼𝑐𝑟𝑦𝑠−𝐼𝑎𝑚 𝐼𝑐𝑟𝑦𝑠 × 100 (3) where Icrys denotes the maximum intensity of 200 crystalline planes and Iam denotes the minimum intensity of amorphous diffraction at ~18o (Nam et al., 2016). The crystallite size was obtained by Rigaku PDXL2 software. Molecular weights of BC were determined using size exclusion chromatography equipped with refractive index detector (RID-10A, Shimadzu, Japan). Sample solution was prepared by dissolving in DMAc containing concentrated dehydrated LiCl at room temperature until the completed dissolution was observed. Thereafter, the sample was diluted to 0.1 %w/w of sample and 1 %w/w of LiCl using DMAc, and filtered through 0.45-m membrane before injecting to an injector (7725i Rheodyne, Sigma-Aldrich, Germany). The sample was fed at 1 mL/min into chromatographic column (KD-806 M, Shodex, USA) with the temperature controlled at 40 oC. The molecular weight of the samples was determined by comparing with those of the polystyrene standards. Microscopic images of the samples were visualized by Scanning Electron Microscopy (SEM Hitachi TM3030 Plus, Japan). Freeze-dried BC was coated with a thin layer of gold using a gold sputter under a high-vacuum condition for 50 s before observation. The topographical information on the sample surface was then observed at a voltage of 15 kV using Charge-up reduction observation and Secondary Electron (SE) modes. The viscoelastic property of the hydrogel-like BC pellicles was acquired using a shear rheometer (Anton Paar Rheoplus, Austria). The pellicle was placed on the lower plate while temperature was controlled at 25 oC. Complex modulus (G*) values were measured using a parallel plate having a diameter of 25 mm (PP25) at the frequency of 1 Hz and strains of 0.01-100%. Figure 1: Yield of hydrogel-like BC pellicle (grey bar) and nanocellulose content in the pellicle (blue bar) after cultivating G. xylinus in different cell media (a), carbon sources (b), nitrogen sources (c), initial pH values of the cell media (d), and static/dynamic conditions (e). 651 3. Results and discussion 3.1 Optimization of culture media, carbon and nitrogen sources, pH, and static/dynamic conditions Cultivation system of cellulose-producing bacteria, G. xylinus, was preliminarily optimized and assessed in terms of yield of BC pellicles and cellulose content in the pellicles. The yield and cellulose content were low, when YGC and Y media were used (Figure 1a), despite containing a high carbon source (5 %w/v glucose as listed in Table 1). In HS and Z culture media containing lower carbon source (2 %w/v) but higher nitrogen source (1-2 %w/v), yield and cellulose content were higher. This demonstrated that nitrogen source was critically important in cellulose biosynthesis. As seen in Figure 1b, the glucose-containing medium yielded the lowest cellulose production, whereas approximately three-time higher yields were obtained by using fructose and mannitol. This was because the pH of the media dropped to pH 3 on the second and seventh days of the cultivation, when glucose and sucrose were used as a carbon source (data not shown).This lower pH, more acidic, was a consequence of gluconic acid produced by metabolism of G. xylinus (Liu et al., 2015). In contrast, the pH of the media containing fructose and mannitol remained 4-5 throughout the culture period. As seen in Figure 1d, the optimal pH suitable for cellulose biosynthesis was pH 5. In Figure 1c, among various nitrogen sources, yield and cellulose content did not show an apparent difference. This might strengthen the case for potential replacement with protein from waste sources. In Figure 1e, the combined static and dynamic condition resulted the highest yield. However, higher cellulose content was observed in static condition (4 mg/cm3) than in other conditions (3 mg/cm3). Although the dynamic system enhanced solubility of oxygen/air into the culture medium preferable for aerobic bacteria like G. xylinus, the cellulose production was relatively lower since the motive cell media induced formation of spontaneous cellulose-nonproducing mutant cells, which dominated in the culture (Krystynowicz et al., 2002). From this preliminary optimization, HS media at pH 5 under a static condition was adopted for further study using okara as a nitrogen source. According to the economic potential of the nanocellulose production (5.5 US$/g), sucrose was supplemented in HS media since it was cheaper (1.14 US$/kg) comparing with other tested carbon sources. 3.2 Effects of okara as a nitrogen source in nanocellulose biosynthesis Concentrations of okara in the culture media influenced BC production. Okara at the concentration of 1% in the culture medium yielded the highest production of nanocellulose (sample BC1) as shown in Table 2, which doubled the yield of BC0 produced in an absence of okara in the medium. At higher okara concentrations, the increased medium viscosity was observed, which might not be suitable for motile bacteria resulting in lower cellulose production. Hence, this suggested that the optimal concentration of okara in the culture medium was 1%. The cellulose content in all BC samples reached similar values of about 3 mg/cm3, in spite of the high pellicle yield of BC1. This could be explained by the higher thickness of BC1 providing a higher volume of BC pellicle. In comparison with other nitrogen sources as described in Figure 1c, cellulose content was slightly lower (1-3 mg/cm3), when okara was added in the cell culture medium. Elemental analysis was performed to assess sample purity. The result showed that all BC samples had a high purity of ca. 100% quantified by total mass percentages of carbon (45%) and oxygen (55%), which were the major elements in carbohydrate molecules. For crystallinities of BC samples as summarized in Table 2, BC0.5, BC1, BC2, and BC3 exhibited crystalline material with crystallinity of about 90%, whereas BC0 possessed lower crystallinity (70%). This was caused by the difference in orientation of the polymer chains in the samples and incomplete growth of crystallite during the polymerization/regeneration process (Castro et al., 2011; Shanshan et al., 2012). In relation to % crystallinity, the increase in crystallite size at 2 of 16o from 63 to 76 nm was noticed as the okara concentrations increased from 0 to 0.5 and 1% (BC0.5, and BC1), and slightly dropped to 68 nm when the okara concentrations increased to 2 and 3% (BC2 and BC3). Table 2: Yield of BC pellicle, cellulose content in BC pellicle, purity, crystallinity, crystallite size at 2 of 16.8o, weight average molecular weight (MW) and polydispersity index (PDI) Yield (g/L) Cellulose content (mg/cm3) Purity (%) Crystallinity (%) Crystallite size (nm) MW (106 g/mol) PDI BC0 BC0.5 BC1 BC2 BC3 43.61.1 57.15.2 84.76.7 72.75.9 61.11.6 3.00.0 3.30.3 3.40.2 3.20.0 3.00.0 99.90.1 99.90.0 99.80.0 99.80.1 99.80.1 70.4 89.7 91.2 89.4 90.8 63.2 70.6 76.8 68.3 68.4 5.80.9 6.82.3 9.40.9 7.80.0 7.552.0 2.3 2.3 2.3 2.2 2.9 652 Figure 2: SEM images of freeze-dried BC samples at the magnification of 10000. Scale bars represent 10 m The biosynthesis of cellulose in 0% okara resulted in a relatively low molecular weight of BC0, which appeared to be almost half of molecular weight of BC1. The polydispersity index (PDI) indicating the breadth of molecular weight distribution of the polydisperse polymer i.e. cellulose was in the intermediate range of about 2 for all the samples. This meant that the weight average molecular weights were two times higher than the number average molecular weights. SEM images in Figure 2 visualize nanofibrous cellulose (100 nm) with differences in density relating to the yield (Table 2). The lowest yield of BC0 brought about a loose fiber network with large vacant spaces between fibers. Unlike BC0, the high compactness of finer cellulose fibers with a uniform size of BC1 having the highest cellulose content was observed. SEM results additionally confirmed that nanocellulose biosynthesis was achieved using okara as a nitrogen source in the culture system of G. xylinus. This was the consequence of protein in okara containing various essential amino acids such as glutamic acid, aspartic acid, serine, glycine, tyrosine, alanine, proline, valine, lysine, isoleucine, and phenylalanine, supporting cell growth and bacterial products (Taokaew et al., 2022). These SEM results, revealing the interior nanofiber density and arrangement in each BC sample, explained the observed impact resistance against an external shear force. Viscoelastic properties of the hydrogel-like BC pellicles were characterized in terms of complex shear modulus (G*) and tangent phase angle (Tan ) tested over the entire range of strains from 0.01 to 100% strain as seen in Figure 3. At strains ranging from 0.01-0.1%, all the moduli of BC samples were rather constant, and then gradually decreased to nearly 0 at the 100% strain due to the deformation of the BC pellicle (Figure 3a). As considered before the deformation occurred (in such constant region), G* of BC1 pellicles (24 kPa) was almost five times higher as compared to that of BC0 (5 kPa) due to the yield of BC1 being nearly twice that of BC0. The other samples, BC0.5, BC2, and BC3, had G* of 16, 20 and 17 kPa, respectively. In Figure 3b, Tan  of greater than 1 indicated the phase change or deformation caused by the applied shear force. BC0 and BC1 deformed at 1 and 3% strains, respectively, while other samples deformed at about 2%. This meant that BC0 exhibited a lower resistance to deformation against the external shear force, than BC biosynthesized in the culture medium containing okara. Figure 3: Complex shear modulus (a) and tangent phase angle (b) of hydrogel-like BC pellicles 4. Conclusions Difference in formulations of cell culture media, carbon sources, nitrogen sources, initial pH values of the culture media, and static/dynamic conditions resulted different nanocellulose yield. The optimized cultivation system consisted of Hestrin-Shramm medium, sucrose as a carbon source, pH 5, and a static condition. The effects of nitrogen source type were not pronounced compared with the effects of different carbon sources. Okara waste was a promising nitrogen source for biosynthesis of high purity nanocellulose by Gluconacetobactor xylinus. The various concentrations of okara supplemented in the culture medium influenced the nanocellulose properties. With the okara as a nitrogen source, higher yield, crystallinity, molecular weight, and better viscoelastic properties were achieved, as compared to the cultivation without okara. Therefore, valorisation of the non-valued okara waste in the biosynthesis of nanocellulose let to enhanced opportunity for economic profit and had enhanced sustainability. 653 Acknowledgments This work was supported by Nagaoka University of Technology and Funds for the Development of Human Resources in Science and Technology "Initiative for the implementation of diversity research environment", the Ministry of Education, Culture, Sports, Science, and Technology (MEXT). References Almeida T., Silvestre A.J.D., Vilela C., Freire C.S.R., 2021, Bacterial Nanocellulose toward Green Cosmetics: Recent Progresses and Challenges, International Journal of Molecular Sciences, 22, 1-25. https://doi.org/10.3390/ijms22062836. Amorim J.D.P., Costa A.F.S., Galdino C.J.S., Vinhas G.M., Santos E.M.S., Sarubbo L.A., 2019, Bacterial cellulose production using fruit residues as substract to industrial application, Chemical Engineering Transactions, 74, 1165-1170. https://doi.org/10.3303/CET1974195. Andriani D., Apriyana A.Y., Karina M., 2020, The optimization of bacterial cellulose production and its applications: a review, Cellulose, 27, 6747-6766. https://doi.org/10.1007/s10570-020-03273-9. Aragão J.V.S., Costa A.F.S., Silva G.L., Silva S.M., Macêdo J.S., Galdino C.J.S., Milanez V.F.A., Sarubbo L.A., 2020, Analysis of the environmental life cycle of bacterial cellulose production, Chemical Engineering Transactions, 79, 445-450. https://doi.org/10.3303/CET2079075. Castro C., Zuluaga R., Putaux J.-L., Caro G., Mondragon I., Gañán P., 2011, Structural characterization of bacterial cellulose produced by Gluconacetobacter swingsii sp. from Colombian agroindustrial wastes, Carbohydrate Polymers, 84, 96-102. https://doi.org/10.1016/j.carbpol.2010.10.072. Joseph B., K S.V., Sabu C., Kalarikkal N., Thomas S., 2020, Cellulose nanocomposites: Fabrication and biomedical applications, Journal of Bioresources and Bioproducts, 5, 223-237. https://doi.org/10.1016/j.jobab.2020.10.001. Klemm D., Heublein B., Fink H.P., Bohn A., 2005, Cellulose: Fascinating biopolymer and sustainable raw material, Angewandte Chemie - International Edition, 44, 3358-3393. https://doi.org/10.1002/anie.200460587. Krystynowicz A., Czaja W., Wiktorowska-Jezierska A., Gonçalves-Miśkiewicz M., Turkiewicz M., Bielecki S., 2002, Factors affecting the yield and properties of bacterial cellulose, Journal of Industrial Microbiology and Biotechnology, 29, 189-195. https://doi.org/10.1038/sj.jim.7000303. Liu M., Zhong C., Wu X.-Y., Wei Y.-Q., Bo T., Han P.-P., Jia S.-R., 2015, Metabolomic profiling coupled with metabolic network reveals differences in Gluconacetobacter xylinus from static and agitated cultures, Biochemical Engineering Journal, 101, 85-98. https://doi.org/10.1016/j.bej.2015.05.002. Nam S., French A.D., Condon B.D., Concha M., 2016, Segal crystallinity index revisited by the simulation of X- ray diffraction patterns of cotton cellulose Iβ and cellulose II, Carbohydrate Polymers, 135, 1-9. https://doi.org/10.1016/j.carbpol.2015.08.035. Ng H.M., Sin L.T., Tee T.T., Bee S.T., Hui D., Low C.Y., Rahmat A.R., 2015, Extraction of cellulose nanocrystals from plant sources for application as reinforcing agent in polymers, Composites Part B: Engineering, 75, 176-200. https://doi.org/10.1016/j.compositesb.2015.01.008. Peter Z., 2021, Order in cellulosics: Historical review of crystal structure research on cellulose, Carbohydrate Polymers, 254, 1-17. https://doi.org/10.1016/j.carbpol.2020.117417. Shanshan G., Jianqing W., Zhengwei J., 2012, Preparation of cellulose films from solution of bacterial cellulose in NMMO, Carbohydrate Polymers, 87, 1020-1025. https://doi.org/10.1016/j.carbpol.2011.06.040. Taokaew S., Nakson N., Zhang X., Kongklieng P., Kobayashi T., 2022, Biotransformation of okara extracted protein to nanocellulose and chitin by Gluconacetobacter xylinus and Bacillus pumilus, Bioresource Technology Reports, 17, 1-8. https://doi.org/10.1016/j.biteb.2021.100904. Taokaew S., Phisalaphong M., Newby B.M.Z. 2016. Bacterial cellulose: Biosyntheses, modifications, and applications. in: Applied Environmental Materials Science for Sustainability, pp. 255-283. https://doi.org/10.4018/978-1-5225-1971-3.ch012. 654 https://doi.org/10.3390/ijms22062836 https://doi.org/10.3303/CET1974195 https://doi.org/10.1007/s10570-020-03273-9 https://doi.org/10.3303/CET2079075 https://doi.org/10.1016/j.carbpol.2010.10.072 https://doi.org/10.1016/j.jobab.2020.10.001 https://doi.org/10.1002/anie.200460587 https://doi.org/10.1038/sj.jim.7000303 https://doi.org/10.1016/j.bej.2015.05.002 https://doi.org/10.1016/j.carbpol.2015.08.035 https://doi.org/10.1016/j.compositesb.2015.01.008 https://doi.org/10.1016/j.carbpol.2020.117417 https://doi.org/10.1016/j.carbpol.2011.06.040 https://doi.org/10.1016/j.biteb.2021.100904 https://doi.org/10.4018/978-1-5225-1971-3.ch012 241Taokaew.pdf Valorisation of okara Waste as an Alternative Nitrogen Source in the Biosynthesis of Nanocellulose