Production of xylooligosaccharides and monosaccharides from switchgrass by FeCl3 hydrolysis combined with sodium perborate pretreatment

doi:10.21203/rs.3.rs-2221046/v1

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Research Article

Production of xylooligosaccharides and monosaccharides from switchgrass by FeCl₃ hydrolysis combined with sodium perborate pretreatment

https://doi.org/10.21203/rs.3.rs-2221046/v1

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published 31 Jan, 2023

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The production of xylooligosaccharides (XOS) using metal salt-catalyzed hydrolysis has the advantages of rapid reaction and mild conditions. At present, no metal salt has been reported that can be used to produce XOS from switchgrass. In this study, the productions of XOS from switchgrass using the hydrolysis of FeCl₃ and that of FeCl₂ were compared with each other, and the residual lignin in FeCl₃-hydrolyzed switchgrass was removed using sodium perborate (SPB) for producing monosaccharides. The highest XOS (X2-X6) of 40.7% was obtained from switchgrass using the hydrolysis of 25 mM FeCl₃ at 160°C for 50 min. The optimized conditions for the SPB pretreatment of FeCl₃-hydrolyzed switchgrass were 8% (w/v) SPB, 70°C, and 4 h, which removed 75.5% of lignin and retained 90.5% of glucan. After the hydrolysis of switchgrass’s solid residue using cellulase, 87.5% glucose and 95.0% xylose were obtained. The results suggest that the hydrolysis of FeCl₃ combined with SPB pretreatment is an efficient novel method for producing XOS and monosaccharides from switchgrass under relatively mild conditions.

Switchgrass

Xylooligosaccharides

FeCl3 hydrolysis

Sodium perborate pretreatment

Monosaccharides

Among many functional oligosaccharides, xylooligosaccharides (XOS) have received much attention as a new ingredient in food, feed, and pharmaceutical industries [1–3]. This is due to their multiple biological and medical activities, including regulating intestinal flora, boosting immunity, reducing allergies, and preventing colon cancer [4–6].

Currently, acid hydrolysis, xylanase hydrolysis and autohydrolysis are considered effective strategies for the production of XOS from lignocellulosic materials. Among them, acid hydrolysis inevitably causes corrosion of various pieces of equipment [7, 8]. Moreover, it excessively degrades carbohydrates to generate by-products, which is detrimental to the subsequent purification of XOS [9]. Although the XOS obtained using the hydrolysis of xylanase is characterized by high purity, low by-products and environment-friendliness [10, 11], the natural barrier of lignocellulosic materials hinders effective hydrolysis of xylanase and usually requires the removal of lignin from lignocellulosic materials, thus greatly increasing the production cost of XOS [12]. In contrast, the simple and economical auto-hydrolysis not only exhibits the advantages of fast reaction and few by-products, but also effectively overcomes the problems of equipment corrosion and chemical residues [13]. Unfortunately, the harsh reaction requirements (high temperature and pressure) of autohydrolysis limit further development of autohydrolysis [14, 15]. It has been shown that the addition of a small amount of metal salts during autohydrolysis can greattly reduce the temperature and pressure required for the reaction [16–18]. Therefore, the catalysis of autohydrolysis using metal salts may be more suitable for producing XOS from lignocellulosic materials than a single self-hydrolysis.

In previous studies, metal salts are often used to depolymerize hemicelluloses into chemicals such as xylose and furfural, or for removing xylan to promote the release of fermentable sugars [16, 19]. Romero et al. (2018) optimized the FeCl₃ pretreatment conditions using response surface methodology and a glucose yield of 70% was obtained from rapeseed straw (12%, w/v) using 0.25 mM FeCl₃ at 138°C [20]. Tang et al. (2021) reported that FeCl₃ and AlCl₃ can remove 33.8% and 36.5% of xylan from wheat straw [21], respectively, indicating that FeCl₃ has the potential to catalyze the depolymerization of xylan, and might be suitable for the preparation of XOS from lignocelluloses. In addition, both the FeCl₃ and XOS are feed additives, and therefore, after the production of XOS from lignocellulosic materials using hydrolysis of FeCl₃, the hydrolysates (including XOS and FeCl₃) could promise as a direct commercial product, which can provide higher economic benefits to producers. Therefore, FeCl₃ might be a good metal salt to catalyze the degradation of xylan in lignocelluloses into XOS. However, FeCl₃ used for producing XOS from lignocellulosic materials has not been reported in literature.

Switchgrass (Panicum virgatum L. Poaceae) is a low-cost, high-yielding energy crop and has high contents of glucan and xylan. Due to these characteristics, switchgrass is a good candidate for producing XOS and monosaccharides. In this work, the productions of XOS from switchgrass using the hydrolysis of FeCl₃ and FeCl₂ under different conditions were compared with each other. Then, after the hydrolysis of FeCl₃, the solid residue of switchgrass was pretreated using sodium perborate (SPB) for its saccharification by cellulase. The aim of this work is to propose an efficient novel method for producing XOS and monosaccharides from switchgrass under relatively mild conditions.

2.1 Materials

Switchgrass (60-mesh, ≤ 0.25 mm) collected from Yangling, Shaanxi, China, was naturally dried. The chemical composition of switchgrass (32.9% glucan, 20.2% xylan, and 22.8% acid-insoluble lignin, and 3.0% acid-soluble lignin) was determined following the procedure given by the National Renewable Energy Laboratory (NREL) [22].

The cellulase (Cellic CTec2, Novozymes A/S, Bagsvaerd, Denmark) activity was 123.0 FPU/mL. All of the chemicals utilized in this study including metal chlorides (FeCl₃•6H₂O and FeCl₂•4H₂O) and sodium perborate (NaBO₃•4H₂O) were analytically pure.

2.2 XOS production using the hydrolysis of FeCl₃ and FeCl₂

Switchgrass (5 g) hydrolyzed by 25–100 mM FeCl₃ or FeCl₂ was placed in the steel reactor (HT-200H-316L, Anhui Kemi, Anhui, China). The hydrolysis temperature (140–180°C) and time (50–70 min) were controlled using a super-constant temperature oil bath (HH-SC, Changzhou Huayi Instruments, Jiangsu, China). After FeCl₃ or FeCl₂ hydrolysis, the steel reactor was rinsed using cold water. When the reactor was completely cooled, the solid residue of FeCl₃ or FeCl₂-hydrolyzed switchgrass and the supernatant were separated using filtration. The obtained supernatant was collected for analyzing the concentrations of XOS and monosaccharides (glucose and xylose). The solid residue was collected in a sealed bag for determining the chemical composition and for subsequent SPB pretreatment.

2.3 Sodium perborate pretreatment

The pretreatment of FeCl₃-hydrolyzed switchgrass residue (5 g) using 6–8% (w/v) SPB was performed in a flask (250 ml). The pretreatment temperature (60–80°C) and time (3–5 h) were controlled using a digital thermostatic magnetic stirrer (B11-3, Sile Instruments, Shanghai, China). After the reaction was completed, the solid residue of SPB-pretreated switchgrass and the supernatant were separated using filtration for enzymatic hydrolysis experiments.

2.4 Enzymatic hydrolysis

The hydrolysis of 0.04 g of raw, FeCl₃-hydrolyzed and SPB-pretreated switchgrass residue (2%, w/v) using cellulase was performed in centrifuge tubes (10 mL each). The centrifuge tubes were placed in a shaking incubator (50°C, 200 rpm, and 72 h). The cellulase loading was 5–20 FPU/g dry mass (DM). After hydrolysis, the centrifuge tubes were placed in a high temperature environment (10 min). Finally, the obtained supernatant was collected for analyzing the concentrations of monosaccharides (glucose and xylose).

2.5 Analytical methods

Chemical compositions of FeCl₃-hydrolyzed and SPB-pretreated switchgrass samples were analyzed following the procedure given by NREL [22]. The recoveries of switchgrass solid residue and chemical contents were calculated based on the study of Seesuriyachan et al. (2017) [23].

The concentrations of monosaccharides (glucose and xylose) in the supernatant were determined using high-performance liquid chromatography (HPLC, Agilent 1260) with an Aminex HPX-87H column (Bio-Rad, Hercules, CA) and a refractive index detector (RID). The mobile phase was 5 mM H₂SO₄ (0.5 mL/min). Furthermore, XOS (DP 2–6) were analyzed using a high-performance anion exchange chromatograph (HPAEC-PAD, Dionex ICS-3000, USA) with a pulsedamperometric detector (Dionex ICS5000) and a CarboPac™ PA200 anion exchange column. The mobile phases were 500 mM CH₃COONa and 100 mM NaOH (0.3 mL/min). The XOS (DP2-6) standards were analytically pure.

All reactions were carried out three times, and all measurements were conducted twice to ensure the reproducibility of all results.

2.6 Calculations

The percentage recovery and percentage removals of glucan, xylan and lignin are calculated using Equations (1) and (2), respectively [23].

$$\text{Soild recovery (%)}=\frac{\text{W}\text{after reaction }\text{(g)}}{\text{W}\text{before reaction }\text{(g)}}\text{×100 (1)}$$

where Wbefore reaction and Wafter reaction represent the dry mass of switchgrass before and after the reaction, respectively.

$$\text{ }\text{Removal (%)}=(1-\frac{\text{W}\text{content after reaction }\text{(g)}}{\text{W}\text{content before reaction }\text{(g)}}\text{)}\text{ }\text{×100 (2)}$$

where Wcontent after reaction and Wcontent after reaction represent the chemical compositions of switchgrass before and after the reaction, respectively.

The yeilds of XOS (DP 2–6) and xylose from the hydrolysis liquids using metal salts are calculated using Equations (3) and (4), respectively [24].

$$\text{XOS yield (%)=}\frac{\text{XOS (DP 2-6) in hydrolysis}\text{ }\text{liquids (g)}}{\text{Xylan in sample (g)}}\text{×100 (3)}$$

$$\text{Xylose yield (%)}=\frac{\text{ Xylose in hydrolysis}\text{ }\text{liquids (g)}}{\text{Xylan in sample (g)}}\text{×100 (4)}$$

The yields of glucose and xylose after cellulase hydrolysis are calculated by Equations (5) and (6), respectively [25].

$$\text{Glucose yield (%)=}\frac{\text{Glucose in supernatant after enzymolysis (g) × 0.9}}{\text{Glucan content in switchgrass residues (g)}}\text{×100 (5)}$$

$$\text{Xylose yield (%) }=\frac{\text{Xylose in supernatant after enzymolysis (g) × 0.88}}{\text{Xylan content in }\text{switchgrass }\text{residues (g)}}\text{×100 (6)}$$

where ‘0.9’ represents the conversion factor for glucose and glucan, while ‘0.88’ represents the conversion factor for xylose and xylan.

3.1 XOS production by FeCl₃ and FeCl₂ hydrolysis

The productions of XOS from switchgrass using the hydrolysis of FeCl₃ and FeCl₂ under different conditions are shown in Fig. 1. When the concentration of FeCl₃ was increased from 25 to 100 mM, the yield of XOS greatly decreased from 40.7–1.0% at 160°C and 50 min. It was also found that the 40.7% XOS contained 15.7% xylobiose (X2), 10.8% xylotriose (X3), 6.5% xylotetraose (X4), 4.7% xylopentaose (X5), and 3.0% xylohexaose (X6) (Fig. 1A). The data here demonstrated that increasing the dosage of FeCl₃ to 100 mM was detrimental to the release of XOS. In contrast, when the FeCl₂ concentration was 25 mM, a low XOS yield of 6.6% was obtained at 160°C for 50 min. As the FeCl₂ concentration was increased to 100 mM, the XOS yield increased to 39.9%, and contained 11.3% X2, 9.7% X3, 7.1% X4, 6.3% X5, and 5.5% X6 (Fig. 1D), indicating that under the tested conditions, increasing the dosage of FeCl₂ was beneficial to the release of XOS. Therefore, FeCl₃ showed more potential for XOS production due to its low concentration. This might be due to the reason that FeCl₃ can greatly reduce the activation energy required for the depolymerization of xylan [26, 27].

Interestingly, the effects of FeCl₃ and FeCl₂ on the production of XOS from switchgrass were consistent within a certain time (30–70 min) or temperature (140–180°C) range. Under constant conditions (25 mM FeCl₃ and 160°C), the yield of XOS was found reduce to 29.5% as hydrolysis time was increased from 50 to 70 min (Fig. 1B). Similarly, 29.4% XOS was obtained from switchgrass after100 mM FeCl₂ at 160°C for 70 min (Fig. 1E). The results showed that almost similar yields of XOS were obtained after the hydrolysis of switchgrass using FeCl₃ and FeCl₂ for 50 min. Under constant conditions (25 mM FeCl₃ and 50 min), the XOS yield increased from 8.3–40.7% (Fig. 1C) as hydrolysis temperature was increased from 140 to 160°C. A similar increase in XOS yield (from 8.4–39.9%) was also found after the hydrolysis of switchgrass at 140–160°C for 50 min with 100 mM FeCl₂ (Fig. 1F). However, with further increase in hydrolysis temperature to 180°C, the XOS yields of switchgrass hydrolyzed by FeCl₃ and FeCl₂ decreased significantly to less than 5% (Fig. 1C and F). Obviously, the higher temperature (180°C) was detrimental to the release of XOS from switchgrass through the hydrolysis of FeCl₃ or FeCl₂. In contrast, FeCl₃ hydrolysis of switchgrass had a great potential to produce XOS, particularly, the yield of X2 (15.7%) and X3(10.8%) is increased (Fig. 1). The optimal conditions for XOS production from switchgrass were 25 mM FeCl₃ at 160°C for 50 min.

3.2 Xylose production in FeCl₃ and FeCl₂ hydrolysis

The productions of xylose from switchgrass using FeCl₃ and FeCl₂ hydrolysis were determined (Table 1). Moreover, xylose/XOS ratio was used to evaluate the relative purity of XOS in the hydrolysates. A high value of xylose/XOS ratio represented a low purity. When switchgrass was hydrolyzed using 25 mM FeCl₃ at 160°C for 50 min, the obtained xylose yield was 38.5%, whereas the ratio of xylose/XOS was 0.9. Additionally, after the switchgrass was hydrolyzed by 100 mM FeCl₂ at 160°C for 50 min, 19.2% of xylose yield was obtained and the ratio of xylose/XOS was 0.5 (Table. 1). Obviously, under the same conditions (160°C and 50 min), the hydrolysis using FeCl₃ produced more xylose than the hydrolysis of FeCl₂. The results showed that the hydrolysis of switchgrass using FeCl₂ produced a higher purity XOS than that using FeCl₃. However, the yields of X2 (11.3%) and X3 (9.7%) in the supernatant of FeCl₂-hydrolyzed switchgrass were low (Fig. 1). Significantly, after increasing the hydrolysis temperature to 180°C, a decrease in xylose yield (from 38.5–1.6% or 19.2–7.4%) was observed in the hydrolysis of switchgrass by FeCl₃ or FeCl₂ (Table 1), indicating that high hydrolysis temperature reduces the content of xylose. The decrease in xylose yield might have been caused by further degradation of xylose into furfural under high-temperature conditions [17, 28].

Table 1

Yields of xylose and ratio of xylose/XOS obtained from switchgrass by 25–100 mM FeCl₃ or FeCl₂ hydrolysis at 140–180°C for 30–70 min.
FeCl₃/FeCl₂ hydrolysis		Severity factor	Xylose (%)	Xylose/XOS
Reaction parameters		(Log R₀)
None	0 mM-160°C-50 min	2.77	4.4 ± 0.1	0.8 ± 0.0
FeCl₃	25 mM-160°C-50 min	2.77	38.5 ± 0.7	0.9 ± 0.0
	50 mM-160°C-50 min	2.77	61.3 ± 1.1	21.9 ± 0.1
	100 mM-160°C-50 min	2.77	59.8 ± 0.9	66.4 ± 0.3
	25 mM-160°C-30 min	3.24	17.1 ± 0.5	0.9 ± 0.0
	25 mM-160°C-70 min	3.61	26.3 ± 1.7	0.9 ± 0.2
	25 mM-140°C-50 min	2.88	1.4 ± 0.2	0.2 ± 0.0
	25 mM-180°C-50 min	4.05	1.6 ± 0.2	0.4 ± 0.0
FeCl₂	25 mM-160°C-50 min	2.77	7.9 ± 0.6	1.2 ± 0.0
	50 mM-160°C-50 min	2.77	9.1 ± 1.0	0.4 ± 0.0
	100 mM-160°C-50 min	2.77	19.2 ± 1.6	0.5 ± 0.0
	100 mM-160°C-30 min	3.24	6.9 ± 1.1	0.5 ± 0.0
	100 mM-160°C-70 min	3.61	11.8 ± 0.7	0.4 ± 0.1
	100 mM-140°C-50 min	2.88	7.0 ± 0.4	0.8 ± 0.0
	100 mM-180°C-50 min	4.05	7.4 ± 0.9	2.5 ± 0.0

3.3 Chemical composition of FeCl₃-hydrolyzed switchgrass

Almost 100% xylan removal was obtained using FeCl₃ hydrolysis (100 mM FeCl₃, 160°C and 50 min) of switchgrass (Table 2), whereas FeCl₃-hydrolyzed switchgrass contained 52.4% glucan, 1.1% xylan and 45.0% lignin. Zhang et al. (2017) obtained nearly 100% xylan removal using FeCl₃ hydrolysis (100 mM FeCl_3, 170°C and 30 min) from sugarcane bagasse [29], which supported the results presented in Table 2. The removal of large amounts of xylan increased the relative contents of glucose and lignin in FeCl₃-hydrolyzed switchgrass, which might be beneficial to the enzymatic digestibility of switchgrass carbohydrates.

Table 2

Chemical compositions of switchgrass before and after FeCl₃ hydrolysis. The FeCl₃ hydrolysis were conducted by 25–100 mM FeCl₃ at 160–180°C for 30–70 min.
FeCl₃ hydrolysis	Glucan	Xylan	Acid	Acid	Solid	Removal
Reaction parameters	(%)	(%)	insoluble	soluble	Recovery	Glucan	Xylan	Lignin
			Lignin (%)	Lignin (%)	(%)	(%)	(%)	(%)
Raw	32.9 ± 1.0	20.2 ± 1.2	22.8 ± 0.2	3.0 ± 0.0	-	-	-	-
0 mM-160°C-50 min	38.6 ± 0.4	13.9 ± 0.3	26.6 ± 0.8	1.9 ± 0.0	77.1	9.6	47.2	15.3
25 mM-160°C-50 min	50.7 ± 0.7	7.2 ± 0.6	35.5 ± 1.2	1.6 ± 0.1	57.0	11.8	80.9	18.3
50 mM-160°C-50 min	55.1 ± 0.3	3.0 ± 0.2	40.4 ± 0.6	1.5 ± 0.0	51.3	14.5	92.3	16.7
100 mM-160°C-50 min	52.4 ± 0.4	1.1 ± 0.0	45.0 ± 0.4	1.5 ± 0.0	47.3	25.0	97.5	15.0
25 mM-160°C-30 min	47.3 ± 0.5	21.0 ± 0.7	33.0 ± 0.7	1.9 ± 0.0	62.1	10.5	36.2	16.7
25 mM-160°C-70 min	53.4 ± 1.0	4.1 ± 0.2	38.5 ± 0.8	1.3 ± 0.0	52.9	14.1	89.2	19.0
25 mM-140°C-50 min	43.8 ± 0.1	19.7 ± 0.5	29.5 ± 0.5	2.1 ± 0.0	69.5	7.9	31.9	15.0
25 mM-180°C-50 min	54.5 ± 0.5	2.3 ± 0.1	39.1 ± 0.9	1.8 ± 0.1	51.3	14.5	93.6	20.0

After FeCl₃ hydrolysis under optimal conditions (25 mM FeCl₃, 160°C, and 50 min), 80.9% of xylan removal and 88.2% of glucan retention were observed, whereas the obtained FeCl₃-hydrolyzed switchgrass contained 50.7% glucan, 7.2% xylan and 37.1% lignin (Table 2). Such differences in the removals of glucan and xylan might be due to the fact that xylan was more readily depolymerized than glucan during the hydrolysis of lignocellulose [30]. Although after FeCl₃ hydrolysis, a large amount of xylan was removed, a large amount of residual lignin (37.1%) in switchgrass could compete with lignocellulosic materials for cellulase attachment sites in the hydrolysis. Therefore, an efficient pretreatment was necessary to reduce the lignin content in FeCl₃-hydrolyzed switchgrass.

3.4 Effect of SPB pretreatment on the chemical composition of switchgrass

It is well known that SPB can be used for dental bleaching [31, 32]. Its application for the pretreatment of lignocellulosic materials is rarely reported in literature. Guo et al. (2022) reported that SPB pretreatment (8% SPB, 80°C, and 4 h ) can remove 45.76% of lignin from rice [32]. In this work, after SPB pretreatment (6–8% SPB, 60–80°C and 3–5 h ), the total lignin in FeCl₃-hydrolyzed switchgrass was clearly decreased from 37.1% to 14.4–20.8%, and more than 61.2% lignin removal was obtained (Table 3). The retentions of glucan and xylan were more than 84.8% and 76.3%, respectively. When the SPB concentration was increased from 6–8% (70°C, and 3 h), the removal of lignin increassed from 65.1–70.0%, whereas the retention of glucan increased from 92.3–91.3%, respectively (Table 3). After the SPB concentration was further increased to 10%, the removal of lignin only slightly increased to 72.5%. However, the retention of glucan was less than 90%, indicating that higher SPB concentration was not beneficial to retaining glucan. Therefore, 8% SPB was considered as the optimal concentration, while SPB-pretreated switchgrass (8% SPB, 70°C, and 3 h) contained 70.7% glucan, 9.4% xylan and 17.0% lignin. When the temperature was increased from 70°C to 80°C, the removal of lignin increased to its maximum value (76.2%) with the least glucan retention (84.8%). When the pretreatment time was increased from 3 to 4 h, the removal of lignin rapidly increased (65.1–75.5%), while the retention of glucan decreased (92.3–90.5%). However, when the time was further increased to 4 h, the removal of lignin and the retention of glucan decreased to 73.9% and 90.0%, respectively (Table 3). These results showed that further increase in temperature or time could enhance the degradation of hemicelluloses in switchgrass [33]. Therefore, the optimal conditions for SPB pretreatment were 8% SPB at 70°C for 4 h, whereas the SPB-pretreated switchgrass contained 72.9% glucan, 9.9% xylan and 14.4% lignin. The high content of glucan (72.9%) was suitable for the production of highly concentrated monosaccharide solutions after the hydrolysis at high substrate concentrations, which facilitated the subsequent ethanol fermentation to obtain higher ethanol concentrations, thus reducing the cost of ethanol [9].

Table 3

Chemical compositions of switchgrass before and after SPB pretreatment. The SPB pretreatment were conducted by 6–10% (w/v) SPB at 60–80°C for 3–5 h.
SPB pretreatment	Glucan	Xylan	Acid	Acid	Solid	Removal
Reaction parameters	(%)	(%)	insoluble	soluble	Recovery	Glucan	Xylan	Lignin
			Lignin (%)	Lignin (%)	(%)	(%)	(%)	(%)
FeCl₃-hydrolyzed	50.7 ± 0.7	7.2 ± 0.6	35.5 ± 1.2	1.6 ± 0.1	-	-	-	-
switchgrass
6%-70°C-3 h	68.7 ± 0.3	9.4 ± 0.2	17.3 ± 0.0	1.6 ± 0.0	68.1	7.7	10.2	65.1
8%-70°C-3 h	70.7 ± 0.9	9.4 ± 0.5	15.2 ± 0.1	1.8 ± 0.0	65.5	8.7	14.0	70.0
10%-70°C-3 h	70.5 ± 0.8	8.5 ± 0.1	14.0 ± 0.3	1.8 ± 0.0	64.5	10.4	23.4	72.5
8%-60°C-3 h	66.9 ± 0.3	9.2 ± 0.1	19.0 ± 0.6	1.8 ± 0.0	69.2	8.8	11.5	61.2
8%-80°C-3 h	73.2 ± 0.4	9.4 ± 0.1	13.3 ± 0.8	1.7 ± 0.0	58.8	15.2	22.6	76.2
8%-70°C-4 h	72.9 ± 0.8	9.9 ± 0.3	12.6 ± 0.7	1.8 ± 0.0	63.0	9.5	12.9	75.5
8%-70°C-5 h	74.9 ± 1.2	9.0 ± 0.2	14.0 ± 1.0	1.9 ± 0.0	61.0	10.0	23.7	73.9

In short, SPB pretreatment is an effective strategy to remove lignin from FeCl₃-hydrolyzed switchgrass. During the SPB pretreatment, the reaction between SPB and lignin in the substrate might be due to the generation of sodium borate and hydrogen peroxide, and a large amount of superoxide anion and hydrogen peroxide anion preferentially reacted with the electron-deficient part of lignin, resulting in a high removal of lignin in FeCl₃-hydrolyzed switchgrass [34]. After SPB pretreatment, the lignin content in the SPB-pretreated substrate greatly decreased, which might improve the enzymatic hydrolyzability of the substrate.

3.5 Monosaccharides production from switchgrass samples

The untreated, FeCl₃-hydrolyzed and SPB-pretreated switchgrass samples were hydrolyzed using cellulase at different loadings of 5, 15, and 20 FPU/g DM for 48 h (Fig. 2). Only 27.1% glucose and 12.7% xylose were obtained from the raw switchgrass using cellulase (20 FPU/g DM) hydrolysis (Fig. 2A and B). The low monosaccharide yields might be due to the stubborn structure of switchgrass, which obstructed the hydrolysis of cellulase [35].

When the cellulase loading was increased from 5 to 20 FPU/g DM, the glucose and xylose yields of FeCl₃-hydrolyzed switchgrass increased to 41.1–54.0% and 47.6–65.9% (Fig. 2A and B), respectively. In the hydrolysis of switchgrass using FeCl₃, the high removal of xylan (80.9%) and lignin (8.3%) (Table 2) could lead to an increased porosity of the material, which could facilitate the adsorption of cellulases, to some extent, increase the hydrolyzability of FeCl₃-hydrolyzed switchgrass [36]. However, a high lignin content of 37.1% in FeCl₃-hydrolyzed switchgrass limited the enzymatic hydrolysis of the substrate (Table 2), whereas the hydrolysis yield with a high enzyme loading (20 FPU/g DM) was still unsatisfactory (Fig. 2A and B).

After the FeCl₃-hydrolyzed switchgrass was pretreated using SPB (8% SPB, 70°C, and 4 h), the yields of glucose and xylose rapidly increased from 49.8–72.2% and from 57.9–73.2%, respectively, with the relatively low loading of cellulase (10 FPU/g DM) (Fig. 2A and B). A higher enzymatic hydrolysis efficiency was due to the high removal of lignin (75.5%) (Table 3), which lead to more cellulose exposure from lignocellulosic materials and reduced the ineffective adsorption of cellulase on SPB-pretreated substrate [33]. When the cellulase loading was increased to 20 FPU/g DM, 85.6% glucose and 90.0% xylose were obtained.

The effects of cellulase loading (5–20 FPU/g DM) and hydrolysis time (12–72 h) on enzyme hydrolysis of SPB-pretreated switchgrass using cellulase were studied (Fig. 3). Relatively satisfactory enzymatic hydrolysis yields (74.6% glucose and 75.7% xylose) were obtained after the cellulase (10 FPU/g DM) hydrolysis for 72 h. When cellulase loading was changed to 20 FPU/g DM, the highest glucose (87.5%) and xylose (95.0%) were obtained at 72 h (Fig. 3A and B). However, the residual of lignin (14.4%) (Table 3) in SPB-pretreated switchgrass could still adsorb some of the cellulase, which increased the enzymatic loading [37, 38]. In general, 87.5% glucose and 95% xylose were obtained from SPB-pretreated switchgrass using cellulase (20 FPU/g DM) hydrolysis for 72 h. In order to further increase the enzyme’s hydrolysis efficiency of SPB-pretreated substrate with a low enzyme loading, the addition of surfactants could be performed in the saccharification of SPB-pretreated switchgrass to increase the stability of cellulase [39]. In addition, some lignin on the substrate surface could be removed through alkali incubation to improve the attachment sites for cellulase [40], thus increasing enzyme’s hydrolysis efficiency for the SPB-pretreated switchgrass.

3.6 Mass balance

A total mass balance for 1000 g switchgrass after FeCl₃ hydrolysis combined with SPB pretreatment is presented in Fig. 4. Switchgrass (1000 g) was hydrolyzed using 25 mM FeCl₃ at 160°C for 50 min. As a result, 82.2 g XOS and 77.8 g xylose were obtained. If FeCl₃-hydrolyzed switchgrass (570.2 g) was hydrolyzed using cellulase (20 FPU/g DM) for 48 h, 156.1 g glucose and 27.1 g xylose were obtained. However, after FeCl₃-hydrolyzed switchgrass was pretreated by 8% SPB at 70°C for 4 h, 234.7 g glucose and 33.8 g xylose were obtained from the SPB-pretreated switchgrass (359.2 g) by cellulase hydrolysis for 72 h. In general, after the FeCl₃ hydrolysis (25 mM FeCl₃, 160°C, and 50 min) combined with SPB pretreatment (8% SPB, 70°C, and 4 h) and cellulase hydrolysis (20 FPU/g DM, and 72 h), 82.2 g XOS and 346.3 g monosaccharides (234.7 g glucose, and 111.6 g xylose) were recovered from 1000 g switchgrass.

Compared with previous reports, a lower yield of XOS (38.6%) was obtained than this work (40.7%) from poplar by 5% acetic acid at 170°C for 30 min [41]. In addition, a higher yield of XOS (42.7%) was obtained from poplar after 2% lactic acid hydrolysis under the same conditions [42]. However, FeCl₃ hydrolysis showed lower temperature (160°C) and concentration (25 mM), which may be more conducive to the industrial production of XOS. It is obvious that the combination of FeCl₃ hydrolysis and SPB pretreatment was effective for producing XOS and monosaccharides from switchgrass. Of course, the cost assessment for the production of XOS and monosaccharides from switchgrass needs to be performed in future work. It will also be interesting to investigate the effects of other metal salts on the production of XOS from switchgrass. Furthermore, the application of FeCl₃ hydrolysis combined with SPB pretreatment of other lignocellulosic materials for XOS and monosaccharides production needs to be evaluated in the future.

In this work, FeCl₃ was found to be more efficient than FeCl₂ for producing XOS from switchgrass. The highest XOS (DP2-6) yield of 40.7% was obtained using FeCl₃ hydrolysis under the conditions of 25 mM FeCl₃, 160°C and 50 min. A high lignin removal of 75.5% was obtained using SPB pretreatment of FeCl₃-hydrolyzed switchgrass and a satisfied hydrolysis yield (87.5% glucose and 95.0% xylose) of SPB-pretreated switchgrass was obtained using cellulase hydrolysis. Finally, FeCl₃ hydrolysis combined with SPB pretreatment of 1000 g of switchgrass produced 82.2 g of XOS and 234.7 g of glucose. This work provides a novel method to overcome the stubborn structure of switchgrass to efficiently produce XOS and monosaccharides under relatively mild conditions.

CRediT authorship contribution statement

Kaikai Gao: Methodology, Writing - original draft, Data curation. Yu Chen: Conceptualization, Resources. Hanxing Wang: Investigation, Software. Xiyu Quan: Investigation. Jie Chu: Supervision. Junhua Zhang: Supervision, Funding acquisition, Writing - review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the National Natural Science Foundation of China, China (No. 31670598).

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Production of xylooligosaccharides and monosaccharides from switchgrass by FeCl₃ hydrolysis combined with sodium perborate pretreatment

Status:

Journal Publication

Version 1

Abstract

Figures

1 Introduction

2 Materials And Methods

2.1 Materials

2.2 XOS production using the hydrolysis of FeCl₃ and FeCl₂

2.3 Sodium perborate pretreatment

2.4 Enzymatic hydrolysis

2.5 Analytical methods

2.6 Calculations

3 Results And Discussion

3.1 XOS production by FeCl₃ and FeCl₂ hydrolysis

3.2 Xylose production in FeCl₃ and FeCl₂ hydrolysis

3.3 Chemical composition of FeCl₃-hydrolyzed switchgrass

3.4 Effect of SPB pretreatment on the chemical composition of switchgrass

3.5 Monosaccharides production from switchgrass samples

3.6 Mass balance

4 Conclusions

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1

Production of xylooligosaccharides and monosaccharides from switchgrass by FeCl3 hydrolysis combined with sodium perborate pretreatment

Status:

Journal Publication

Version 1

Abstract

Figures

1 Introduction

2 Materials And Methods

2.1 Materials

2.2 XOS production using the hydrolysis of FeCl3 and FeCl2

2.3 Sodium perborate pretreatment

2.4 Enzymatic hydrolysis

2.5 Analytical methods

2.6 Calculations

3 Results And Discussion

3.1 XOS production by FeCl3 and FeCl2 hydrolysis

3.2 Xylose production in FeCl3 and FeCl2 hydrolysis

3.3 Chemical composition of FeCl3-hydrolyzed switchgrass

3.4 Effect of SPB pretreatment on the chemical composition of switchgrass

3.5 Monosaccharides production from switchgrass samples

3.6 Mass balance

4 Conclusions

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1

Production of xylooligosaccharides and monosaccharides from switchgrass by FeCl₃ hydrolysis combined with sodium perborate pretreatment

2.2 XOS production using the hydrolysis of FeCl₃ and FeCl₂

3.1 XOS production by FeCl₃ and FeCl₂ hydrolysis

3.2 Xylose production in FeCl₃ and FeCl₂ hydrolysis

3.3 Chemical composition of FeCl₃-hydrolyzed switchgrass