Valorisation of Chinese hickory shell as novel sources for the e�cient production of xylooligosaccharides

Chinese hickory shell, a by-product of the food industry, is still not utilized and urgent to develop sustainable technologies for its valorization. This research focuses on the systematical evaluation of degraded products and xylooligosaccharide production with high yield from the shell via hydrothermal process. The results indicated that the pretreatment condition strongly affected the chemical structures and compositions of the liquid fraction. The maximum yield of XOS (55.3 wt%) with limitation of by-products formation was achieved at 160 o C for 2 h. High temperature (220 o C) and short time (0.5 h) contributed to hydrolysis of xylooligosaccharide with high DP to yield 37.5 wt% xylooligosaccharide with DP from 2 to 6. xylooligosaccharide obtained mainly consisted of xylan with branches according to the HSQC NMR analysis. Overall, the production of XOS with a high yield from food waste will facilitate the valorization of food waste in the biore�nery industry.


Introduction
Chinese hickory (Carya.Cathayensis) is a famous nut due to its nutritional values.More than 15,000 ha of Chinese hickory trees are cultivated in the Zhejiang and Anhui province of China (Sharma et al., 2020).Recently, it has been reported that kernel, leaves, and green husk have abundant nutraceutical components (Feng, Wang, Belwal, Li, & Luo, 2020).However, the hickory shell is a by-product produced in large quantities after the cracking and shelling process.Furthermore, hickory shell as waste would pollute the environment (Nita, Zhang, Dentzer, & Matei Ghimbeu, 2021).As far as we know, there is no report that developing and using sustainable and green strategy is used for high value-added utilization of hickory shell.Therefore, how to utilize the hickory shell into value-added chemicals, materials or biofuels is urgent and promising.
Similar to other biomass, hickory shell husk has high hemicelluloses, cellulose and lignin contents.However, the differences in the structure and reactivity of these major components make the fractionation and utilization of hickory shell (Gao, Remón, & Matharu, 2021;Yuan et al., 2019).In general, cellulose can be hydrolyzed to fermentable sugar for further biofuel production.At the same time, hemicelluloses were used as feedstock to produce gels, lms, coating, and prebiotics in the biomedical, food and pharmaceutical elds (Amorim, Silvério, Prather, & Rodrigues, 2019;Bian, Dong, et al., 2019;Bian, Luo, et al., 2019).However, it also has high biomass recalcitrance, which resulted in the di cult utilization of lignocellulose (Chan et al., 2020).Consequently, lignocellulosic biomass pretreatment is a prerequisite step for fractionation and utilization of hickory shell to overcome this challenge.Recently, various methods, including ultrasonic, enzymatic, microwave, hydrothermal and a combination of these methods have been applied to lignocellulosic biomass for fractionation of soluble components (Liu et al., 2019;Silveira et al., 2015).According to previous studies, hydrothermal pretreatment is considered the economic and sustainable strategy that can promote reducing sugar and simultaneously producing value-added biochemicals, including hemicelluloses, oligosaccharides and furfural (Wang et al., 2016;X. Xiao et al., 2013).Besides, there are no acid, alkali, organic solvents, and catalysts introduced during hydrothermal pretreatment, which is in accord with the principle of Green Chemistry (Zimmerman, Anastas, Erythropel, & Leitner, 2020).
During hydrothermal pretreatment, value-added chemicals, such as xylooligosaccharides (XOS) derived from hemicelluloses, can be produced (Monteiro et al., 2021).Recently, the residues after pretreatment have been extensive investigations, but the lignocellulose is still underutilized to prepare XOS (Liguori & Faraco, 2016;Otieno & Ahring, 2012).Therefore, exploiting waste by industries to produce XOS is very promising from the perspective of economics and environment.Recent researches showed that XOS are non-digestible saccharides with prebiotic action (Bindels, Delzenne, Cani, & Walter, 2015;Cunningham et al., 2021;Swanson et al., 2020).Generally, sugarcane bagasse and corncob are used as feedstocks to prepare XOS (Amorim et al., 2019;Poletto et al., 2020).In addition, other lignocellulosic biomass, such as bamboo, brewery residues, also can produce XOS (Monteiro et al., 2021;X. Xiao et al., 2013).Hickory shell waste from Chinese hickory is the part of the volume of lignocellulosic biomass available and consists of ca.30% hemicellulose.However, the production of XOS from this waste has not beguen reported until now.
Based on the reported research, acid, alkaline, and hydrothermal pretreatment are the main strategies for the fractionation of hemicelluloses and the production of XOS (Amorim et al., 2019;Poletto et al., 2020).Among these methodologies, hydrothermal pretreatment was gaining popularity relative and was considered an economically feasible method to produce signi cant amounts of XOS with a low degree of polymerization (DP) and inhibit the depolymerization of XOS (Gullón et al., 2018;Rico et al., 2018).In the industry, the XOS with DP between 2-20 obtained only from hydrothermal treatment can be suitable for prebiotic applications (Food & Drug Administration, 2019).Besides, hydrothermal pretreatment can catalyze the transformation of hemicelluloses during the reaction, while cellulose and lignin would not be degraded (Singh et al., 2019).
According to the lignocellulosic composition and the hydrothermal conditions in the production of XOS, the yield and DP of XOS are different.For example, 73.5 wt% of the xylan can be released from chestnut when the hydrothermal pretreatment was 180 o C, and the DP of XOS was related low (4.43) (Rico et al., 2018), while 45 wt% hemicelluloses of hazelnut shell were converted into XOS with DP ranging from 2 to 6 (Buruiana, Gómez, Vizireanu, & Garrote, 2017).When the hydrothermal pretreatment temperature further increased, the DP of XOS produced from peanut shell and corn stover ranged from 2 to 17 and 6 to 15, respectively (Surek & Buyukkileci, 2017).Although XOS can be obtained from various biomass, undesirable compounds, such as furfural, 5-hydroxymethylfurfural (HMF) and water-soluble lignin, were also produced during the hydrothermal pretreatment (Amorim et al., 2019).Therefore, how to produce XOS with a high yield from the hickory shell via hydrothermal pretreatment and avoid the formation of toxic product is much more signi cant and urgent.
Based on the above considerations, this research aims to exploit the hydrothermal pretreatment for the production of XOS from the hickory shell for the revelation of this industrial waste.Meanwhile, the constitutional changes of the hickory shell by characterization of the dissolved components (XOS, monosaccharides and water-soluble lignin) were systemically evaluated to understand the fractionation and degradation of the hemicelluloses in hickory shell during the hydrothermal pretreatment.The effects of hydrothermal pretreatment on the transformation of hemicelluloses were investigated by highperformance anion-exchange liquid chromatography (HAPEC), high-performance liquid chromatography (HPLC) as well as two-dimension heteronuclear single-quantum coherence (2D-HSQC) to reveal the XOS formation from the hickory shell.Besides, the characteristics of soluble products were systemically elucidated.This research would provide valuable results in the commercial exploitation of hickory shell for industrial production of biochemicals from hickory shell waste.

Materials
The raw Chinese hickory harvested in 2019 from a local farm in Zhejiang Province, China.After drying, the kernel of Chinese hickory was removed, and the shell was ground to obtain a 20-80 mesh fraction for pretreatment.The raw Chinese hickory shell consisted of 38.94% cellulose, 29.92% hemicelluloses, 29.62% lignin and 8.24 % moisture according to the NREL method (Sluiter, et al., 2008).

hydrothermal pretreatment for production of XOS
The pretreatment experiments of Chinese hickory shell were carried out in a hydrothermal reactor with a stir bar.Typically, the reactor was charged with a suspension of 3.0 g Chinese hickory shell in 30 mL deionized water.Then the reactor was heated to the desired temperature under continuous stirring at 400 rpm within the desired time.After the reaction was completed, the mixed liquor was separated into residual solid and ltrate by XOS-rich ltration via thoroughly washing with deionized water.Finally, the residual solid and XOS-rich ltrate were dried using a lyophilizer.Besides, the XOS-rich ltrate was also stored in a refrigerator for further analysis.

Characterization of original and residual Chinese hickory shell
The lignocellulosic compositions (%, w/w) of the original and residual Chinese hickory shell were determined according to the NREL standard analytical method (Sluiter, et al., 2008).

Characterization of the XOS-rich ltrate
The amount of monosaccharides and XOS in the XOS-rich ltrate were measured by an HPAEC (Dionex ICS-3000, Thermal) with equipped a Carbopac PA-29 column according to our previous report (Shen, Wang, Huang, Wen, & Sun, 2016).The amounts of HMF, furfural, lactic acid, levulinic acid, acetic acid and formic acid were analyzed by HPLC (Agilent 1200) according to the method reported (Shen et al., 2016).In addition, the structure information of XOS-rich ltrate was measured by 2D-HSQC NMR (Shen et al., 2016).

Results And Discussion
In this work, the Chinese hickory shell was pretreated with deionized water at solid/liquid of 1:10 and pretreatment temperatures of 140, 160, 180, 200 and 220 o C for 0.5, 1, 2 and 4 h, respectively.In general, hydrothermal pretreatment can deconstruct lignocellulose and break the biomass recalcitrance, which fractionated the lignocellulose into water-insoluble solid and water-soluble liquid fraction (Monteiro et al., 2021;Wang et al., 2016).Water-insoluble solid mainly consisted of cellulose and lignin.Due to the reduction of biomass recalcitrance, cellulose can be readily hydrolyzed into glucose, and the left lignin can further be used for the production of lignin-based resin.The water-soluble liquid fraction was mainly composed of hemicelluloses-based monosaccharide and XOS.Meanwhile, by-products were also generated during the hydrothermal process, especially in harsh reaction conditions.As a result, the liquid fraction is very complicated.Consequently, it should need to qualitatively and quantitatively analyze the liquid fraction to promote the utilization of Chinese hickory shell.

The yield of the solid and dried liquid fraction
Since complicated by-products would be formed, which was terrible for the utilization of hemicelluloses, the ultimate goal is that most of the hemicelluloses in lignocellulose were released and converted into XOS, but not further into furfural, levulinic acid, formic acid and other by-products.Herein, the yield of solid and dried liquid fraction after pretreatment was investigated.
As shown in Fig. 1a and Table S1, the total yield of solid and dried liquid fraction is strongly affected by the reaction conditions.The higher the reaction temperature was, the longer reaction time was, the total yield was much declined from 98.73 to 63.93%, which mainly was ascribed to the factor that hemicelluloses and amorphous cellulose were severely degraded into by-products (furfural, HMF, levulinic acid, lactic acid, acetic acid and formic acid) under harsh reaction conditions (Wang et al., 2016).When the pretreatment condition was further harsher (temperature 220 o C, time 4 h), the by-products would be condensed to form of humin and precipitate in the solid fraction, improving the yield of solid fraction.
Generally, the hydrolysis products in the liquid fraction were mainly XOS and monosaccharides.Figure 1b and Table S1 show the yields of dried liquid fraction as the pretreatment temperature and time increased during the hydrothermal process.Under mild temperature (≤ 180 o C), a long time would promote the yield of dried liquid fraction.Meanwhile, the water-soluble components would be further degraded into small molecule under harsh reaction conditions (≥ 180 o C), which decreased the yield of dried liquid fraction.At 200 o C for 1 h, the yield of the dried liquid fraction can be up to 21.27 wt%.However, under high temperature, part lignin in the lignocellulose also would be dissociated into the water, which improved the yield of dried liquid fraction.On the other hand, lignin introduced XOS-rich liquid fraction will increase the di culty of separation and puri cation of XOS.Therefore, preventing dissociation of lignin and degradation of XOS and improving the release of hemicelluloses is a challenge during biomass pretreatment.

composition analysis of original and pretreated Chinese hickory shell
To understand the mechanism of hemicelluloses release and lignin dissociation during hydrothermal pretreatment, the chemical composition of the raw and pretreated Chinese hickory shell was determined by the NREL standard process (Sluiter, et al., 2008).Figure 2 and Table S2 show the chemical composition and recovery yield of the shell with different pretreatment conditions.The chemical composition of the raw Chinese hickory shell was 38.94% cellulose, 29.92% hemicelluloses, and 29.62% lignin.As compared to lignin and cellulose, hemicelluloses are much more readily to be release and degraded from lignocellulose.After pretreatment, the contents of hemicelluloses in the pretreated shell rapidly decreased, especially in severe conditions.This was attributed to the factor that the water autoionization can form the hydronium ions (H 3 O + ), which resulted in the degradation of hemicelluloses by selective cleavage of glycosidic linkages (Shen et al., 2016).Simultaneously O-acetyl group also was hydrolyzed to form acetic acid, which further promoted the degradation of hemicelluloses (Huang et al., 2019).As shown in Fig. 2a, high temperature or long time can be conducive to release most hemicelluloses from the lignocellulosic shell, which indicated hydrothermal pretreatment could e ciently remove hemicelluloses from plant cells.Unlike hemicelluloses, cellulose content rstly increased from 38.94% to ca. 50% due to the removal of hemicelluloses.While further elevated temperature or time, cellulose content sharply decreased, suggesting that some amorphous cellulose was degraded into glucose, HMF and other chemicals under harsh conditions (Bian, Luo, et al., 2019).Besides, it was observed that the content of lignin increased as the temperature and time elevated.The reason for this increase is that most hemicelluloses and part cellulose were degraded under harsh pretreatment condition (Lin et al., 2020).Notably, the lignin recovery in the pretreated shell was over 100% under the severe condition as shown in Table S2, suggesting that condensation between carbohydrate-derived products and lignin led to the accumulation of pseudo-lignin at harsh conditions, which improve the content of lignin (Shen et al., 2016).The above results indicated that hydrothermal pretreatment could e ciently fractionate the lignocellulose and break biomass recalcitrance, especially when most hemicelluloses was released into liquid, favoring the production of XOS.

Monosaccharide and XOS analysis of the dried liquid fractions
During hydrothermal pretreatment, the release of hemicelluloses was triggered by the hydronium ion formed in situ by autoionization of water and further heightened by organic acids produced from the degradation of hemicelluloses (Ibbett, Gaddipati, Greetham, Hill, & Tucker, 2014).Generally, the liquid fraction generated from hydrothermal pretreatment mainly consisted of the products degraded from hemicelluloses, including oligosaccharides, monosaccharides and furfural (Amorim et al., 2019).During the hydrothermal process, the hemicelluloses were stepwisely converted into oligosaccharides via hydrolysis of glycosidic bonds and further degraded into monosaccharides, which would also be reacted to form by-products via dehydration reaction (Sun, Wen, Sun, & Sun, 2015).This work tried to produce XOS with a high yield and prevent further degradation via optimized reaction conditions.
In this study, the liquid fraction obtained from hydrothermal pretreatment was rich in XOS.Therefore, the amount of XOS with different DP was also measured.As shown in Fig. 3, the effects of the reaction temperature and time in the hydrothermal pretreatment on the yield and composition of XOS from Chinese hickory shell were compared.As can be seen, the total yield of XOS increased slowly as pretreatment time prolonged at 140 o C. When pretreatment temperature elevated to 160 o C, the yield increased sharply to the maximum value (165.49g/kg raw material) when time prolonged to 2 h.Still, too long time would make further degradation of XOS into monosaccharide and other by-products, which con rmed that properly prolonging pretreatment time was necessary for the production of XOS with a high yield at 160 o C during the hydrothermal process.As expected, when the pretreatment temperature was further increased to 220 o C, the yield of XOS constantly decreased, especially prolonging time to 4 h at 220 o C, almost of XOS was degraded into xylose or other products, which was attributed to the factor that high temperature decreased the viscosity of the mixture and simultaneously increased the ionic mobility resulting in releasing more hydronium ions for degradation and dehydration of hemicelluloses into the xylose and other small organic molecules (Savage, 1999).Besides, it was observed that the contents of high-DP XOS were high at mild conditions (low temperature and short time).While the condition became harsh, the proportion of XOS with DP ranging 2 to 6 increased due to the hydrolysis of XOS with high DP.The maximum yield of XOS with DP ranging 2 to 6 was 112.27 g/kg raw material at 220 o C for 0.5 h.Taken together, the optimum condition for the production of XOS with wide DP was at 160 o C for 2 h, while 220 o C and 0.5 h is the best condition for preparation of XOS with DP ranging 2-6.
Figure 4 also illustrated that the monosaccharide was obtained at various pretreatment conditions.Arabinose, galactose, glucose, xylose, mannose, galacturonic acid and glucuronic acid were observed in the liquid fraction, and xylose was the primary product after hydrothermal pretreatment due to the high content of xylan in the raw material.Arabinose and galactose had a relatively high yield under mild reaction conditions.As the temperature increased and time prolonged, xylose was the primary product since the XOS obtained from hemicelluloses was further hydrolyze into xylose under high temperature and a long time which was in agreement with the quantitative analysis of XOS.At the same time, cellulose also was be degraded into glucose.In general, as shown in Fig. 4, high temperature and long time would promote the hydrolysis of hemicelluloses and cellulose into pentose and hexose.Still, the yield of pentose and hexose was lower than that of XOS under the best condition for the production of XOS.

By-products distribution of the liquid fraction
Monosaccharide obtained from hemicelluloses during the hydrothermal process would be further degraded into furfural, HMF and carboxylic acids (formic, acetic, levulinic and lactic acid) (Sun et al., 2015).Generally, the acetyl groups of hemicelluloses were cleaved to produce acetic acid under the hydrothermal process (Bian, Dong, et al., 2019).Meanwhile, the pentose was dehydrated into furfural and was further degraded into formic acid under acidic condition, and hexoses (glucose, galactose and mannose) can be transformed into HMF and further degraded into levulinic and formic acids (Shen et al., 2016).
As shown in Fig. 5, furfural, HMF, formic acid, acetic acid, levulinic acid, and lactic acid were the main products in the liquid fraction.The yield of acetic acid (63.91 g/kg raw material) and furfural (84.73 g/kg raw material) were much higher than other by-products (< 40 g/kg raw material) at the harsh pretreatment condition.However, with the maximum yield of XOS with wide DP obtained at 160 o C for 2h, the yield of acetic acid was only 16.21 g/kg raw material, which can prevent the degradation of XOS.Besides, the low yield of acetic acid (18.06 g/kg raw material) also can be found in the best condition of the yield of XOS with DP ranging 2 to 6. Interestingly, the formation of all by-products was strongly related to the pretreatment condition, that is, the yield of by-products increased as the temperature elevated and time prolonged, which were in good agreement with the previous reports (Nitsos, Matis, & Triantafyllidis, 2013;Sun et al., 2015;Wang et al., 2016).As can be seen in Fig. 5, it found that the hydrothermal process under the higher temperature at a long time favored the dehydration of pentose and hexose as compared to that formed at other mild reaction conditions.As compared to the maximum yield of XOS (165.49g/kg raw material) at 160 o C for 2h, the amount of the by-products in the liquid fraction was at a quite low level (the amount of formic acid, acetic acid, lactic acid, levulinic acid, furfural, and HMF were 2. 70, 16.21, 1.59, 1.05, 5.01, 2.50, respectively), which indicated hydrothermal pretreatment is an e cient strategy with fewer by-products for lignocellulosic Chinese hickory shell to prepare XOS with high yield.

Structural analysis of XOS in liquid fraction
To further obtain the detailed information on the various structures features and changes of the dried liquid fraction under the 140, 160, 220 o C for 2 h and retention of 0.5, 2, 4 h at 160 o C, 2D-HSQC NMR was carried out to analyze the substructures and chemical compositions.The cross-signals assignments in the HSQC spectra of carbohydrates and lignin are according to the previous reports and listed in Table S3 ( Shen et al., 2019;Sun et al., 2015;Wang et al., 2016).In addition, typical structures of lignocellulose found in the liquid fraction are shown in Figure S2b to better analyze the NMR spectra of the liquid fraction.
Besides the signals of carbohydrate, water-soluble lignin also can be observed.As can be seen, the peaks from ferulates (FA), p-coumarates (PCE), guaiacyl (G), syringyl (S), and oxidized syringyl (S') units are obvious in the HSQC spectra (Figure S2) (Shen et al., 2016;Shen et al., 2019).After hydrothermal treatment, the value of S/G of the liquid fraction decreased as the pretreatment severity increased, suggesting the S units were much easily degraded under hydrothermal process at the high severity, which was in agreement with the previous reports (Nitsos et al., 2013;Sun et al., 2015;Wang et al., 2016).
Besides the peaks of typical lignin structure, some condensed signal for lignin also observed under severe conditions.Although lignin can be dissociated from Chinese hickory shell and released into liquid fraction, the content is much less than that XOS.Besides, it was also found that water-soluble lignin was rich in phenolic hydroxyl group, which gave water-soluble lignin good antioxidation.Therefore, there's reason to believe that XOS with some water-soluble lignin has much better performance in the eld of prebiotics (Dong et al., 2020).

Process mass balance
In this work, the maximum yield of XOS with wide DP and DP ranging DP 2 to 6 was achieved at 160 o C for 2h and 220 o C for 0.5 h with a relatively low yield of monosaccharide and by-products.Excessively prolonged time or increased temperature sharply decreased the yield of XOS and improved the amount of monosaccharide and by-products.Herein, a process mass balance of the hydrothermal pretreatment was developed for the two pretreatment conditions (160 o C for 2h and 220 o C for 0.5 h).(Fig. 6).Process yields of products in the solid and liquid fractions were normalized to a common basis of 100 kg raw Chinese hickory shell as the starting material.After hydrothermal pretreatment, liquid and solid fractions were separated by ltration.Under medium condition (160 o C for 2h), hemicelluloses were converted into XOS from lignocellulose and were limited to further degrade into monosaccharide and by-products, cellulose and lignin still remain in the solid lignocellulose.In this condition, it was found that 16.55 kg XOS with wide DP can be obtained, while only a small amount of lignin was observed in the dried liquid fraction.Further increased temperature to 220 o C, most hemicelluloses were removed from lignocellulose.
Meanwhile, some lignin is also dissociated into the liquid fraction.Shorted time can prevent hydrolysis of oligosaccharide at high temperature.Under this pretreatment condition (220 o C for 0.5 h), 11.22 kg XOS ranging DP 2 to 6 from 100 kg raw material can be observed in the hydrothermal liquid.Besides, other more degraded products appeared in the liquid fraction due to the severe degradation of hemicellulose under high pretreatment temperature.These results showed that a relatively mild temperature and long time or a high temperature and short time were preferable for the production XOS.

Conclusions
In this work, the Chinese hickory shell, a by-product of the food industry, was pretreated via the hydrothermal process.To understand the structural changes of hemicelluloses from lignocellulose to liquid fraction and obtain XOS with high yield, a detailed and systematic evaluation of solid and liquid fractions was conducted for the hydrothermal pretreatment of Chinese hickory shell at various temperatures and different time.The research showed that the pretreatment severity strongly affects the side chain constituents and backbone of xylan.With an increase of pretreatment temperature or time, the polysaccharides were hydrolyzed into oligomers and monomer, and further degraded.Moderate temperature (160 o C) and relatively long time were preferred for the production XOS with wide DP, the maximum yield can be up to 165.5 g/kg raw material with a relatively low level of xylose and other degraded products, while relatively high temperature (220) and short time (0.5 h) was in favor for preparation of XOS with DP from 2 to 6, the maximum yield was 112.2 g/kg raw material.The structural characteristics of the dried liquid fraction were also comprehensively elucidated by 2D-HSQC NMR.It was also observed that the XOS in the liquid fraction has a backbone of (1→4)-linked β-D-xylopyranosyl xylan decorated with branches.Meanwhile, lignin in the lignocellulose can be dissociated, degraded, and condensed.In short, the comprehensive evaluation of the liquid fraction during hydrothermal pretreatment of Chinese hickory shell will facilitate the valorization of food wastes in the biore nery industry, especially the production of hemicellulose into XOS.By-products in the liquid fraction after hydrothermal pretreatment.