Polyploid Miscanthus Lutarioriparius: A Sustainable and Scalable Biomass Feedstock for Cellulose Nanocrystal Preparation in Biorefinery

Abstract

In this study, polyploid Miscanthus lutarioriparius (PML) was introduced as a new sustainable and scalable source for cellulose nanocrystal (CNC). The agronomic traits of PML were significantly different from Miscanthus × giganteus (MG), but their chemical components and physical features were similar. Notably, a remarkable co-extraction of hemicellulose, lignin and ash and non-crystalline cellulose was observed during crude cellulose isolation from PML than it from MG by modified alkaline peroxide pretreatment. In addition, subjecting crude cellulose of PML and MG biomass to sulfuric acid hydrolysis provided high-quality CNC. The analysis of particle size distribution, zeta potential, crystalline index, the degree of polymerization, SEM and yield potential suggested that the CNC extracted from PML showed higher stability, processability and productivity than that from MG. Therefore, it provides a new theoretical basis for the applications of CNC prepared by PML and MG. The results also revealed potential genetic approaches for Miscanthus spp. to enhance biomass and CNC yield.

1. Introduction

Lignocellulose is a promising feedstock for biorefinery, which mainly consists of ligin, hemicellulose and cellulose. The chemical components of lignocellulosic biomass make them a substrate of enormous biotechnological value [1]. Several of the polysaccharides possessing interesting physical and biological properties have been applied in biotechnology products or are presently being widely investigated (i.e., hyaluronic acid, alginate, chitosan) [2]. With the development of cellulose research, cellulose nanocrystal (CNC) separated from lignocellulose has emerged as a promising material and a major focus in nanomaterial research [3].

Cellulose nanocrystal is a polymer produced by the hydrolysis of cellulose. CNCs are rod-like particles with a highly crystalline structure, high aspect ratio, large surface area, unique tensile strength (0.8–10 GPa), low density and high Young’s modulus (100–170 GPa) [3]. Advantages in the use of CNC are related not only to their useful, unsurpassed, physical features, but also to their biodegradability, renewability, sustainability, abundance and high biocompatibility. Some authors have proposed that nanotechnology will change our lives in profound ways, allowing engineers to devise more efficient ways of meeting human needs [4,5]. It is believed that nanotechnology has the potential to completely change lignocellulose products and the biomass “industry” through improvements in the products and by designing new applications of biomass-derived materials with different properties [6,7]. CNC is posited as being a high potential material for the multi-industries of food [8], packaging [9], (bio)sensors [10], and medicine [11].

Various materials with a high content of cellulose could be used to prepare CNC, such as wood [12], bamboo [13], crop residue [14], and bagasse [15]. However, the geometrical characteristics of CNC (i.e., shape, particle size, aldehyde and carboxyl groups) would significantly vary according to different raw materials and reaction conditions [16]. Although woody materials are an optimal feedstock for CNC production, such reserves are steadily declining [17]. The continuous supply of woody materials for the CNC industry might not be environmentally friendly, and may not be feasible in the coming decades. Therefore, the shortage of ideal feedstock is a bottleneck for the industrial application of CNC.

Miscanthuslutarioriparius is a high-yield perennial lignocellulosic crop endemic to China and is widely distributed along Hunan, Hubei, Zhejiang, Anhui, Jiangsu, Jiangxi, Henan, Shanghai province. It is a promising second-generation energy crop with high stress resistance, a wide propagation range, stable genetic properties, and is widely grown as a marginal crop [18]. It has been evaluated that M. lutarioriparius could produce an annual biomass of 28.37 ton/ha in alkaline land [19]. Meanwhile, M. lutarioriparius is regarded as a good biomass feedstock, because its stem has a high content of lignocellulose and a low content of ash [20]. Several processes and products have been reported that utilize M. lutarioriparius as a raw material for industrial applications. These include bioenergy [21], paper making [19] and building materials [22]. In addition, prior studies found that polyploid M. lutarioriparia (PML) has the characteristics of thick stems, high biomass yield, and tolerance to floods and droughts, and so has greater development potential than diploids [4,22].

In Miscanthus spp., there exists another high biomass potential polyploidy called Miscanthus × giganteus (MG) [23]. Experimental bases for studying the biomass potential of triploid MG in northern Europe have been established successively in Denmark, Germany, Ireland, and the United Kingdom [24]. In 1993, Greef and Deuter speculated that MG was a triploid and was hybridized by M. sacchariflorus (tetraploid) and M. sinensis (diploid). MG is now a widely cultivated perennial energy crop in Europe which is used for electricity generation and bioethanol production [23,24]. However, the CNC feedstock potentials of both MG and PML have not been evaluated yet. Considering that perennial energy grass is a resource for biorefinery, it is necessary to assess the biomass potentials of MG and PML for CNC production. Therefore, the objectives of this study are (ⅰ) to investigate the differences in agronomic properties, (ⅱ) the physical properties (i.e., Crystalline, degree of polymerization) between MG and PML, and (ⅲ) to explore their potentials for preparing CNC.

2. Materials and Methods

2.1. Sampling and Measurements

The study site is located at “The Nursery of Miscanthus Germpalsms” in Hunan Agricultural University (113°4′50.12400″ E, 28°11′5.67600″ N). In 2020, nine plants of PML and MG were grown in plots (1.5 m × 1.5 m) arranged in a randomized complete block design, with 11 replicates. Aboveground biomass in each plot was harvested manually and weighed to determine dry biomass weight (DBW), panicle height (PH), stem number (SN), stem diameter (SD), moisture content (MC), leaf length (LL), leaf width (LW), node number (NN) and leaf/stem ratio (L/S). The definitions of the characteristics are as follows. DBW: The dry weight per plant, measured using an electronic scale and expressed in kg. PH: The distance from the ground to the top of the panicle, expressed in cm. SN: The number of stems of the plant. SD: The diameter of the middle of the last segment of the stem, measured using vernier calipers and expressed in cm. MC: The difference between the weight of the plant at the time of sampling and the weight of the plant after drying, expressed as a percentage. LL: The length from leaf base to leaf tip, expressed in cm. LW: The length at the widest part of the leaf, expressed in cm. NN: The number of internodes of plant. L/S: The ratio of leaf weight (g) to stem weight (g) per plant. All samples were dried under 90 °C for 3 days. Dried samples were grounded to powder, and then sieved through a combined −80 mesh (0.180 mm × 0.180 mm) screen. Sieved dry samples as raw biomass were sealed in sealing bags and preserved at room temperature [25].

2.2. Flow Cytometry

First, 20 mg of sieved young leaves of the PML was added to 1 mL of Galbraith’s buffer (containing 45 mmol/L MgCl₂, 30 mmol/L sodium citrate, and 20 mmol/L 3-(n-morphinol) propanulinic acid (pH 7.0). Then, 0.1% Triton X-100 and 50 μg/mL RNase were added, and the supernatant was discarded by centrifugation. The cell suspension was obtained by adding 500 μL Galbraith’s buffer to the precipitate and shaking the mixture. The propidium iodide solution was dropped into the suspension until a final concentration of 50 μg/mL, and then the mixture was stained on ice for 30 min against light. At least 10,000 cells were collected from each sample to be tested, and this was repeated three times for each sample. The genome size was determined using Epics XL flow cytometry (Epics XL, Beckman Coulter Co., Ltd., Indianapolis, IN, USA). The fluorescence of the FL3 channel was collected by blue light excitation at 488 nm, and the emission fluorescence intensity of propidium iodide was detected using Tree Star FlowJo software [26].

2.3. Sample Pretreatment

Crude cellulose was extracted by the modified alkaline peroxide pretreatment according to Gabriel et al. [27] with minor modifications. In this study, we replaced 10% NaOH with 20% NaOH and 7.5% H₂O₂ with 20% H₂O₂. The first step of pretreatment was alkaline hydrolysis. Raw biomass was placed in sealed beaker 20% NaOH at the liquid to solid ratio 10:1, and oil-bathed at 100 °C for 1 h, 140 °C for 30 min, and 160 °C for 30 min to obtain the pulp. Obtained pulp was washed with distilled water to a neutral pH. For the next step, the pulp was washed by 20% H₂O₂ at liquid to solid ratio of 10:1, under 90 °C for 3 h to remove lignin. The solid and liquid were centrifugated to obtain precipitate. The precipitate was oven-dried and preserved with sealed plastic bags under room temperature. The yield of crude cellulose was calculated according to Equation (1).

CCY = (W_c × (1 − m_c))/(W_b × (1 − m_b)) × 100 [%](1)

where, CCY is crude cellulose yield (%); W_c is the weight of crude cellulose (g); m_c is the moisture content of crude cellulose (%); W_b is the weight of biomass feedstock (g); and m_b is the moisture content of biomass feedstock (%).

2.4. CNC Preparation

The CNC was prepared using a sulfuric acid hydrolysis method as introduced by Lunardi et al. [3] with minor modifications. The crude cellulose was hydrolyzed with 56% H₂SO₄, at a liquid to solid ratio of 20:1, under a temperature of 61 °C for 40 min to obtain the CNC. Upon expiration of the reaction time, the hydrolysis was stopped by fourfold dilution with distilled water and cooling of the suspension to room temperature. The hydrolyzed cellulose solution was dialyzed with periodic water replacement to achieve a neutral pH. The dialyzed cellulose solution was subjected to ultrasonic treatment with an ultrasonic disintegrator (KS-1000KDE, Kushan Jielimei Ultrasonic Instrument Co., Ltd., Kunshan, China) at 40 kHz, 300 W for 15 min, following filtration using 1 micrometer aperture filter paper. Filter liquor was transferred to a 150 mL volumetric flask and the volume was set with distilled water. A CNC solution of 1 mL was transferred from the volumetric flask to a beaker and diluted fiftyfold with distilled water, for the determination of CNC size. The remaining solution in the flask was frozen in a refrigerator at −20 °C for 12 h, then dried in a freezer dryer (LC-12N-80C, Shanghai Lichenbangxi Instrument Science Technology Co., Ltd., Shanghai, China). The freeze-dried CNC was preserved using sealed plastic bags. The yield of CNC was calculated according to Equation (2).

CNCY = W_n / (W_c × (1 − m_c)) × 100 [%](2)

where, CNCY is crude cellulose yield (%); W_n is the weight of CNC (g);.m_c is the moisture content of crude cellulose (%); and W_c is the weight of crude cellulose (g).

2.5. Biomass Feedstock Chemical Contents Measurement

Soluble contents of PML and MG were extracted from raw biomass and crude cellulose using 75% ethyl alcohol, and after six cycles, the value was found to be the difference between the weight of the biomass feedstock and the weight of the biomass feedstock after extraction. The soluble content was calculated according to Equation (3).

SC = (W_ap× (1 − m_ap) / (W_b × (1 − m_b)) × 100 [%](3)

where, SC is soluble content (%); W_ap is the weight of biomass feedstock or crude cellulose after extraction (g); m_ap is the moisture content of biomass feedstock or crude cellulose (%); W_b is the weight of biomass feedstock (g); and m_b is the moisture content of biomass feedstock (%).

Ash content was determined using a muffle furnace (SX-4-10, Tianjin Taisite Instrument Co., Ltd., Tianjin, China) with 15 mL ceramic crucibles [28]. The ash content was calculated according to Equation (4).

AC = (W_ar − W_r) / (W_b × (1 − m_b)) × 100 [%](4)

where, AC is ash content (%); W_ar is the weight of crucible after calcination (g); W_r is the weight of crucible (g); W_b is the weight of biomass feedstock (g); and m_b is the moisture content of biomass feedstock (%).

A two-step sulfuric acid hydrolysis process was used to extract cellulose, hemicellulose and lignin [25]. Structural carbohydrates (i.e., glucose, xylose, and arabinose) were measured using an HPLC system (LC-40, SHIMADZU Co., Ltd., Kyoto, Japan) equipped with an Aminex HPX-87H chromatography column (300 mm × 7.8 mm, particle size 9 μm, Bio-Rad Laboratories, Hercules, CA, USA) and a refractive index detector (RID 20A, SHIMADZU Co., Ltd., Kyoto, Japan). The lignin content was measured using a UV-VIS spectrometer (A590, Aoyi Instrument Shanghai Co., Ltd., Shangha, China) and the same muffle furnace mentioned above. The cellulose content and hemicellulose content were calculated according to Equation (5).

(5)$Y=\frac{C\times e\times V\times X}{1000\times W\times \left(1-b\right)\times f}\times 100 \left[\%\right]$

where, Y is cellulose content (%) or hemicellulose content (%); C is the concentration of fiber polysaccharides (mg/mL); e is the mass conversion coefficient of polysaccharides into monosaccharides by dehydration; V is the hydrolysate volume (mL); X is the extraction residue rate (%); W is the weight of sample (g); b is the moisture content of sample (%); and f is the recovery coefficient of chromatographic correction standard sample.

The lignin content is calculated according to Equation (6).

(6)$L=\frac{U\times V\times n\times X}{1000\times \epsilon \times W\times \left(1-b\right)}+\frac{\left(W_L\right)\times X}{W\times \left(1-b\right)}\times 100 \left[\%\right]$

where L is lignin content (%); U is the absorbance of neutral hydrolysate at 320 nm; e is the mass conversion coefficient of polysaccharides into monosaccharides by dehydration; V is the hydrolysate volume (mL); n is the dilution ratio of hydrolysate; X is the extraction residue rate (%); b is the moisture content of sample (%); ε is the Light absorption rate of hydrolysate (L/(g·cm)); and W_L is the weight of precipitation (g).

The purity is determined by the cellulose content of CNC.

2.6. CNC Physical Property Measurement

The cellulose crystallinity index was detected with the X-ray diffraction (XRD) method using an X-ray diffractometer (XRD-6000, SHIMADZU Co., Ltd., Kyoto, Japan) [29]. The raw biomass power was laid on the glass sample holder and detected under plateau conditions. Ni-filtered Cu Ka radiation (k = 0.15406 nm) was generated at a voltage of 40 kV and a current of 30 mA and scanned at a speed of 5°/min from 10° to 35°. The Crl was estimated using the intensity of the 200 peak (I₂₀₀, h = 22.5°) and the intensity at the minimum point between the 200 and 110 peaks (I_am, h = 18.5°).

CrI = (I₂₀₀ − I_am)/I₂₀₀ × 100 [%](7)

where CrI is the cellulose crystallinity index; I₂₀₀ is the intensity of the peak at 22.5°; and I_am is the intensity of the minimum between the 200 and 110 peaks at 18.5°.

The degree of polymerization (DP) was determined with the Ubbelohde Viscosity Test [30]. Copper ammonia solution (double hydroxide ethylenediamine copper solution:distilled water (V/V) = 1:1) was used to determine the DP. Since the viscosity of the copper ammonia solution is easily affected by temperature, the value of the measured polymerization degree generally decreased with the increased in temperature to ensure the reaction temperature was strictly controlled at 25°C. The zeta potential and particle size distribution of CNC were measured using nanometer particle size meter (ZETASIZER NANO ZS, Malvern Panalytical, Ltd., Birmingham, British). The morphology of CNC was observed with a scanning electron microscope (SEM) (ZEISS Sigma 300, Carl Zeiss AG, Oberkochen, Germany) and a digital camera (Canon EOS M50, Tokyo, Japan).

2.7. Statistical Analysis

Word Processing System (WPS) Office software (v2021) was used to calculate the percentages of soluble content, cellulose content, hemicellulose content, lignin content and ash content of different samples and to draw the corresponding bar graphs. SPSS 25.0 software was used for data and variance analysis, and one-way ANOVA was used to test the difference between the contents of each component, where statistical significance was defined as p < 0.01. Origin 2021 was used to draw the chemical composition and physical properties in different samples.

3. Results and Discussion

3.1. Chromosome Number Identification of PML

Confirming the chromosome ploidy of PML was the first step before comparing PML and MG in this study. Table 1 provides the flow cytometry results of PML and diploid M. lutarioriparius. Significantly, the mean X value and DNA content of PML were 285.5 and 6.3, respectively, which is about 1.5 times of that of diploid M. lutarioriparius. Since the number of chromosomes of diploid M. lutarioriparius is 38 (2×), the number of chromosomes of triploid M. lutarioriparius could be 57 (3×). The result proved that the PML was triploid which was the same for MG. The implication of this result was that all comparisons as shown below removed the effect of ploidy differences between PML and MG.

3.2. Significant Differences in Agronomic Traits between PML and MG

MG, an allotriploid, has been considered as an ideal biomass feedstock for the commercial production of nanocellulose due to high annual biomass yields and cellulose content [31]. In the present study, a natural PML was screened from a nationwide collection, and investigated as a new domesticable and scalable source biomass resource for CNC production, which had exhibited an even higher annual biomass yield than MG in previous study (23.5 vs. 14.1 t/ha) [32].

Apart from annual biomass yield, the employed PML significantly differed from MG in terms of other agronomic traits. Summary statistics of agronomic traits for PML and MG are presented in Figure 1. Compared with MG, PML exhibited higher values in terms of its node number, stem diameter, leaf width and leaf length. By contrast, its values of dry biomass weigh per plant, leaf/stem ratio, panicle height and stem number were lower than MG. Overall, PML is a perennial grass that primarily grows in wetlands along the middle and lower reaches of the Yangtze River, the significant differences in agronomic traits of PML and MG were closely related to their different growth environments and survival strategies [33].

3.3. Comparison of Chemical Components and Physical Features of PML and MG

The compositional analysis of raw biomass of PML and MG used are presented in Figure 2. The PML raw biomass consisted of 41.0 ± 0.1% cellulose, 22.4 ± 0.3% hemicellulose, 22.0 ± 0.3% lignin, 7.9 ± 0.6% soluble content and 5.3 ± 0.1% ash, so that the lignocellulosic polymers content was over 85.0%. The ANOVA of chemical component contents indicated that there was no significant different between PML and MG. In addition, both PML and MG contained more than 40% cellulose, suggesting a higher potential for nanocellulose production compared with common agricultural wastes (rice straw, wheat straw and corn stalk, etc) and bagasse [34]. Moreover, as the major chemical component inhibiting crude cellulose isolation, the lignin content of PML raw biomass was lower than woody biomass [35]. Meanwhile, the soluble content and ash of PML raw biomass was significantly lower than agricultural waste, indicating its higher utilization rate of raw biomass for biofuel, biomaterials and biochemicals [36].

Since nanocellulose was isolated from the crystalline region of cellulose in plant cell walls, the crystalline index of CrI and DP were considered as the main factors affecting its nanocellulose potential [37]. As shown in Figure 3, the CrI values of the raw biomass of PML and MG were 56.5% and 57.1%, respectively. Additionally, the DP values of the raw biomass of PML and MG were 639 and 618, respectively. Specifically, the CrI and DP values of PML and MG were higher than those of Miscanthus sinensis, Miscanthus floridulus, Miscanthus sacchariflorus, and Miscanthus lutarioriparius [30]. Hence, the results indicate that both PML and MG possessed great potential for cellulose derivatives compared to other Miscanthus species.

3.4. Isolation of Crude Cellulose from Raw Biomass of PML and MG by Modified Alkaline Peroxide Pretreatment

Alkaline peroxide pretreatment has proven to be an excellent method for the separation of crude cellulose from a variety of lignocellulosic biomass [27]. In this study, a modified alkaline peroxide pretreatment was employed to extract crude cellulose from PML and MG. Notably, the cellulose contents of PML and MG increased sharply from 41.0% and 40.9% of raw biomass to 95.9% and 89.9% of crude cellulose, respectively (Figure 4). These results were significantly higher than the cellulose content of crude cellulose extracted from other biomasses by alkaline peroxide pretreatment [27,29]. By contrast, the non-cellulosic polymers (hemicellulose and lignin) of both PML and MG were heavily co-extracted during pretreatment, which could lead to increases in CrI and/or lignocellulose deconstruction [29]. Therefore, the results indicated that the modified alkaline peroxide pretreatment was an effective method by which to isolate crude cellulose from lignocellulose.

To confirm the effects of the isolation process on cellulose structure, the CrI and DP of crude cellulose were detected in this study (Figure 5). Interestingly, the CrI values of crude cellulose extracted from PML (72.4%) and MG (69.8%) were significantly higher than those of their raw biomass, respectively. Meanwhile, the DP values of crude cellulose in PML and MG were less than 1/8 and 1/6 of their raw biomass, respectively. Together, these results provided important insights in that the modified alkaline peroxide pretreatment had exhibited great efficiency in removal of non-cellulosic polymers, as well as the non-crystalline cellulose. Besides, the ideal biomass feedstock of nanocellulose could be identified with high levels of cellulose content, CrI values, low levels of non-cellulosic polymers content and DP values [30,38]. Therefore, the crude cellulose of PML exhibited a greater potential for CNC preparation compared with that of MG.

3.5. Comparative Analysis of CNC from Crude Cellulose of PML and MG Extracted by Sulfuric Acid Hydrolysis

As the only commercialized technology, sulfuric acid hydrolysis was further applied to isolate CNC from the crude cellulose of PML and MG in this study [39]. Table 2 provides the purity of CNC prepared by PML and MG. Notably, while the CNC purity of MG was slightly lower than that of PML, it was much higher than that of other biomass in previous studies [3,4,6,7,31,34]. Moreover, Figure 6 provides the results obtained from the preliminary analysis of zeta potential and particle size distribution of CNC. Specifically, CNC extracted from PML has a higher zeta potential and more uniform particle size distribution than CNC extracted from MG. Previous studies suggested that the higher the zeta potential of CNC, the more likely the suspension is to be stable as the charged particles repel each other, and this force overcomes the natural tendency to aggregate [40]. In addition, the particle size uniformity of CNC was an important factor affecting their processability to further prepare advanced materials [38]. Hence, the CNC extracted from PML showed higher stability and processability than that from MG.

The CrI and DP values of CNC extracted from PML and MG were measured in this study (Figure 7). After sulfuric acid hydrolysis, the CrI values of CNC extracted from PML and MG increased slightly to 75.1% and 74.7%, respectively. The results indicate that the non-cellulose polymers and non-crystalline cellulose were further co-extracted from crude cellulose during the sulfuric acid hydrolysis process, which could lead to improvement of CNC purity. Meanwhile, the DP values of CNC extracted from PML and MG decreased to 56 and 45, respectively. According to the data, we could infer that an internal degradation occurred in the crystalline regions of cellulose during sulfuric acid hydrolysis, which could lead to the further shrinkage of cellulose particles to the nanoscale [41].

For an intuitive comparison of the differences in CNC between PML and MG, a series of morphological observations were conducted step by step in the present study. In Figure 8 and Figure 9 an obvious particle size reduction in PML and MG raw biomass is observable after modified alkaline peroxide pretreatment, whereas the color of MG crude cellulose was found to be deeper than that of PML.This result could be explained by the fact that the co-extraction of amorphous substances that occurred in PML raw biomass was more significant than in MG raw biomass, which would theoretically significantly improve the yield and purity of its CNCs (Figure 4, Figure 8D and Figure 9D). The SEM images further confirmed that the CNCs of both PML and MG reached the nanoscale, but their particle shapes were significantly different. To be specific, the CNC of PML exhibited a standard crystal shape, while the CNCs of MG were irregularly shaped and composed of spherical particles (Figure 8). The reason for this is not clear but it may have something to do with the different natural forms of cellulose in PML and MG raw biomass.

In addition, these CNC, with a regular shape and high crystalline index, have great potential as filler and performance-enhanced materials [4,17]. For example, many polyer (Polylactic acid, xylan, chitosan) complexes with CNC have enhanced mechanical and barrier properties [35,38]. Raghav et al. [3] combined phosphorylated CNCs with drugs to prepare sustained-release carriers [3]. Xie et al. [6] observed an improvement in the tensile strength and oxygen permeability with the addition of CNC to food packaging films.

3.6. The Yield Potential of Crude Cellulose and CNC

Table 3 provides the yield potential of crude cellulose and CNC extraction from PML and MG. Briefly, the crude cellulose yield of PML was slightly lower than that of MG, but its CNC yield was slightly higher than that of MG. A possible explanation for this might be that the CrI values of PML crude cellulose are significantly higher than that of MG crude cellulose, indicating that the content of crystalline cellulose in PML crude cellulose is higher than that in MG crude cellulose (Figure 5). According to our previous study, the dry matter yield of PML and MG were 23.5 t/ha and 14.1 t/ha, respectively [32], which means the 2.4 t and 1.3 t of CNCs can be produced per hectare. Therefore, the results suggested that PML shows greater potential for industrialized CNC preparation compared to MG.

4. Conclusions

In this study, a natural autotriploid Miscanthus lutarioriparius was firstly and successfully used for CNC preparation via modified alkaline peroxide pretreatment followed by sulfuric acid hydrolysis. Firstly, the agronomic traits of PML were significantly different from those of MG, but their chemical composition and physical features were similar. In addition, a significantly better co-extraction of non-cellulosic polymers and non-crystalline cellulose was obtained by modified alkaline peroxide pretreatment with PML than with MG. As expected, comparative analyses of particle size distribution, zeta potential, CrI, the DP, SEM and yield potential suggest that the CNC extracted from PML showed higher stability, processability and productivity than that from MG. These results provide a new competitive biomass feedstock for CNC preparation and new insight into the integrated utilization of PML for biorefinery.

Author Contributions

Conceptualization: S.W. and M.L.; methodology: S.W.; resources: Z.C. and Z.Y.; investigation: Y.I.; data curation, S.W.; writing—original draft preparation, S.W. and M.L.; writing—review and editing S.X. and T.F.; supervision, M.L.; project administration, Z.Y. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

We express our deepest gratitude to Hunan Engineering Laboratory of Miscanthus Ecological Applications of Hunan province for providing support and permission to conduct this research. We are thankful to Weiming Liu, Jie Li, Yao Li, Yancen He, Weihong Du and other colleagues for their assistance in data collection.

Conflicts of Interest

The authors declare that they have no competing interest.

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures and Tables

Figure 1. Agronomic traits of polyploid Miscanthus lutarioriparius and Miscanthus × giganteus. Dry biomass weight (DBW), leaf/stem ratio (L/S), Panicle height (PH), Stem number (SN), Leaf length (LL), leaf width (LW), stem diameter (SD), Node number (NN).

Figure 2. Chemical components of lignocellulosic biomass in polyploid Miscanthus lutarioriparius and Miscanthus × giganteus. Soluble content (SC), Cellulose content (CC), Hemicellulose content (HC), Lignin content (LC), Ash content (AC). Vertical bar are standard deviation (n = 2).

Figure 3. Crystalline index and degree of polymerization of lignocellulosic biomass in polyploid Miscanthus lutarioriparius and Miscanthus × giganteus. Vertical bar are standard deviation (n = 3).

Figure 4. Chemical components of crude cellulose in polyploid Miscanthus lutarioriparius and Miscanthus × giganteus. * Significant difference at p ≤ 0.05, ** Significant difference at p ≤ 0.01, *** Significant difference at p ≤ 0.001. Vertical bar are standard deviation (n= 2).

Figure 5. Crystalline index and degree of polymerization of crude cellulose in polyploid Miscanthus lutarioriparius and Miscanthus × giganteus. ** Significant difference at p ≤ 0.01, *** Significant difference at p ≤ 0.001. Vertical bar are standard deviation (n = 3).

Figure 6. Particle size distribution, zeta potential of CNC in polyploid Miscanthus lutarioriparius and Miscanthus × giganteus.

Figure 7. Crystalline index and degree of polymerization of CNC in polyploid Miscanthus lutarioriparius and Miscanthus × giganteus.* Significant difference at p ≤ 0.05. Vertical bar are standard deviation (n = 3).

Figure 8. Morphological observation of the raw biomass (A), crude cellulose (B), and the CNC (C,D) of polyploid Miscanthus lutarioriparius.

Figure 9. Morphological observation of the raw biomass (A), crude cellulose (B), and the CNC (C,D) of Miscanthus × giganteus.

Table 1

Chromosome ploidy of Polyploid Miscantus lutarioriparius.

Species	X-Mean	DNA Content (pg)	Chromosomes Number
Diploid Miscantus lutarioriparius	199.4	4.4	2n
Polyploid Miscantus lutarioriparius	283.5	6.3	3n

Table 2

The purity of CNCs extracted bisulfuric acid hydrolysis.

Species	Purity (%)
Polyploid Miscanthus lutarioriparius	95.6
Miscanthus × giganteus	95.0

Table 3

Yield potential of crude cellulose and CNCs.

Production	Species	Yield (%, Raw Biomass)
Crude cellulose	Polyploid Miscanthus lutarioriparius	32.4
Crude cellulose	Miscanthus × giganteus	33.7
CNCs	Polyploid Miscanthus lutarioriparius	10.1
CNCs	Miscanthus × giganteus	9.9

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