Recalcitrance of Corn Straw Significantly Correlated to the Heterogeneity in Physicochemical Properties of Cell Wall


 Background: Tissue heterogeneity significantly influences the overall saccharification efficiency of plant biomass. However, the mechanisms of specific organ or tissue recalcitrance to enzymatic deconstruction are generally complicated and unclear. A multidimensional analysis for the four parts from twelve corn cultivars was conducted.Results: The results showed that leaf, sheath, pith and rind of corn straw exhibited remarkable heterogeneity in chemical composition, physical structure and cell type, which resulted in the different recalcitrance to enzymatic hydrolysis. High values of lignin, hemicelluloses, degree of polymerization and crystallinity index were critical for the increased recalcitrance, while high value of neutral detergent soluble and pore size generated weak recalcitrance. Interestingly, pore traits of cell wall, especial for microcosmic interface structure, seemed to be a crucial factor that correlated to cellulase adsorption and further affected saccharification.Conclusions: Tissue heterogeneity in physical-chemical traits of cell wall influences the overall saccharification efficiency of biomass. Furthermore, the holistic outlook of cell wall interface is indispensable to understand the recalcitrance and promote the biomass conversion.


Background
The growing energy demands have caused the exhaustion of fossil fuel resources and severe environmental issues, which lead to the increasing attention towards clean alternative energy [1]. Bioethanol produced from plant biomass is commonly blended with gasoline in the transportation sector, which is deemed as one of the most promising renewable fuel, devoting environmentally friendly and renewable advantages [2]. Governments and energy corporations around the world are actively boosting the development of cellulosic ethanol to replace traditional fossil fuel [3]. Conversion of cellulose to glucose is deemed as the core step in cellulosic ethanol production. Although enzymatic sacchari cation of cellulose to attain the increased sugar generation is more attractive than other industrial conversion, pretreatment and sacchari cation enzymes occupy 30-50% of the total operational costs, resulting in the unfeasibility of cellulosic ethanol in a large scale [4]. Series of physicochemical factors that hinder the enzymatic sacchari cation of cellulose are deemed as recalcitrance, which are referred as (but are not limited to) composition (consisting of cellulose, lignin, hemicelluloses and pectin), polymer properties (such as lignin monomer components, cellulose crystalline index and degree of polymerization), and interactive network among these polymers [5]. Assessing and destroying the recalcitrance are prerequisite to circumvent the technical and economic obstacle in recent cellulosic ethanol production.
Chemical composition and physical structure of different plant biomass vary obviously, depending on their origin, organ type, tissue trait, architecture hierarchy, stage of development, etc, which further cause heterogeneity of the recalcitrance. Researches show that heterogeneity and molecular of the speci c tissue forming the plant are responsible for the differences in sacchari cation performance [6]. In terms of hemicelluloses and lignin, the amount, distribution and their decoration pattern add complexity to the origins and tissues of plant biomass [7]. Thus, a consensus can be reached that the high heterogeneity of plant biomass leads to the signi cant complexity of recalcitrance. A further complication is that the complexity commonly causes empirically implemented enzymatic sacchari cation processes and drastic pretreatment for recalcitrance more than necessary. It is seemed to be di cult to systematically compare these qualities to evaluate the intrinsic recalcitrance among different feedstocks due to the heterogeneity, especially for different studies. Hence, this immense heterogeneity is considered as the chief reason why the precise mechanisms of recalcitrance are still ambiguous and also closely in uence the optimal choice of pretreatment and enzymatic hydrolysis strategy.
Dried plant biomass is primarily composed of cell wall. Several authors have suggested that enzymatic sacchari cation processes correlate to the properties of cell wall including of the relative abundances of composition, ne proportion, their interconnecting the cell wall components into different matrices, and multiple structures [8]. In essence, sacchari cation of cellulose is enzymatic hydrolysis of cell wall in plant, exhibiting as glucose release from the cellulose. The basic types of cells among various plants are overall similar. Varied behaviors of different cell types in enzymatic hydrolysis re ect the tissue-speci c recalcitrance that in uenced by the nature of the cell wall. Thus, comprehending the resistant factors with viewpoint of cell wall might provide a deep insight into the recalcitrance of plant biomass. Allowing the enzyme access to the cell wall is the primary step in enzymatic hydrolysis of cellulose, which signi cantly in uences the latter sacchari cation process. However, the susceptibility of different cell wall, especially the accessibility of bio-interface to enzyme is still unclear.
Corn is the main agricultural crop on the earth. Corn straw (CS) is the main residue of corn. Based on the desirable agronomic and biochemical properties in terms of high biomass yield and cellulose content, and C4 carbon xation, CS has attracted huge attention among researchers for cellulosic ethanol production. Furthermore, CS was especially suitable model to clarify tissue-speci c recalcitrance for its greater diversity of cells than other plant biomass. From visual angle, it was easily found that leaf, stem sheath, stem rind and stem pith had different morphologies. Although the cellulosic ethanol produced from CS had been studied by many researchers, little investigation concerning the relevance of enzymatic sacchari cation to heterogeneity in composition and architecture of speci c organs or tissues had been reported. In an effort to obtain fundamental knowledge about the origins of recalcitrance of CS to deconstruction, we obtained series of corn cultivars with different phenotype and set out to describe cell wall traits and analyze their impacts on sacchari cation e ciency. This paper will focus on understanding the relationship between composition and assembly of cell wall domains and recalcitrance, which will also help in selecting of corn varieties with improved biore ning capabilities and setting downstream pretreatment.

Sacchari cation Performance
The 144 reducing sugar yield values of the four parts from twelve corn cultivars were used to generate a heat map contained in a matrix with legend color bar. Dendrograms were selected to describe the similarity between clusters. Hierarchical clustering analysis con rmed the initial idea that some cultivars necessarily had a comparable performance in sacchari cation (Fig. 1). Additionally, the reducing sugar yield was not always consistent to the macroscopic phenotypes. The general varying patterns for reducing sugar yield among the same parts within the different cultivar was similar, suggesting that biomass sacchari cation obviously correlated to the speci c organs or tissues (Fig. 2). The highest reducing sugar yield was obtained from pith, and followed by leaf, rind and sheath in sequence, despite the difference between the pith and leaf was not signi cant. Specially, the reducing sugar yield of pith ranged from 21.47% to 38.96%, whereas ranged from 17.1% to 27.43 % among sheath.
Enzymatic digestibility expressed as reducing sugar yield can be used to re ect the recalcitrance. Higher enzymatic digestibility indicates weaker recalcitrance [9]. Brans Using Subcritical Wate ACS Sustainable Chem. Eng. 2020, 8, 7192−7204). The outermost sheath was the most recalcitrant part, whereas the middle rind was less recalcitrant and the inner pith was the least recalcitrant. The leaf was the least recalcitrant part except for the pith. Hence, recalcitrance exhibits obvious heterogeneity among organs or tissues. Similar trends were also observed in sorghum stem and sugar cane. Costa suggests that the recalcitrance of internode in sugar cane becomes weak gradually from the outer to the inner parts [10]. Jung reports that recalcitrance of cell wall correlates to the tissue-speci c distribution of lignin; furthermore, highly ligni ed vascular bundles are more degradable than the less ligni ed parenchyma [11]. Li reports that different parts of sorghum with structural or chemical variations generate special recalcitrance that required different pretreatments [12]. This lack of agreement among leaf, sheath, rind and pith indicated that the complication of cell wall assembly, which were translated dictate recalcitrance to sugar release. Thus, the relationships between the enzymatic digestibility and physiochemical characteristics of cell wall were further studied.

Associations between Cell Wall Properties and Their Impact on Enzymatic Digestibility
Representative DK and YH were chosen because they were expected to exhibit extreme and contrasting phenotypes. DK had the highest biomass and height among the twelve cultivars, yet, YH represented the opposite phenotypes (the lowest biomass and height). Physical-chemical properties were determined for each leaf, sheath, rind and pith, and used to evaluate the in uence of cell wall traits on enzymatic digestibility. To avoid concealing of recalcitrance differences, no biomass pretreatment was used. The results showed that enzymatic digestibility differed signi cantly (P<0.05) for various parts across DK and YH, except for pith (table S2). Overall, enzymatic digestibility of leaf, rind and pith were signi cantly higher in DK than in YH (P < 0.01). Yet, enzymatic digestibility of sheath did not differ signi cantly (P=0.225) between the two cultivars. Detailed information concerning the different physicalchemical properties and signi cance analysis was provided in table S3 and S4. The overall varying patterns for physical-chemical properties within the same part were similar. The crystallinity index, degree of polymerization, and ash values were higher in rind and sheath than in pith and leaf. Sheath contained the least content of neutral detergent soluble, whereas, leaf showed the highest content of neutral detergent soluble, due to the large amount of soluble sugars and protein. Similar trend is also observed in sorghum stem [13]. Higher crystallite dimension and DB/DO values were in pith or leaf. Moreover, higher cellulose, hemicelluloses and lignin values were in rind or sheath.
A correlation matrix was performed to evaluate how the enzymatic digestibility was associated with cell wall properties (Fig. 3). The results showed that the content of cellulose and neutral detergent soluble positively impacted the enzymatic digestibility. The positive effect of cellulose was not surprising for it probably re ected a greater availability of substrate for enzymatic hydrolysis. Higher content of neutral detergent soluble commonly indicated the lower relative content of other substance that negatively correlated to the enzymatic digestibility. The in uences of ash and crystallite dimension among the subset of samples on enzymatic digestibility were not statistically. Strangely, the negative impact of hemicelluloses content was also not statistically signi cant. This may be in uenced by a generated spot of xylose. The negative impact of Lignin, degree of polymerization and crystallinity index values on enzymatic hydrolysis were accordant with the recalcitrance model reported in the previous literature [14,15]. The collective data con rmed that characteristics of cell wall from different organs or tissues were related to the varied recalcitrance.
Interestingly, The DO/DB had a positive effect on enzymatic digestibility. Arantes suggests that catalytic cleavage of the cellulose is not the actual rate-limiting step but rather the limited accessibility of the cellulose chains to the enzyme within the substrate matrix [16]. Enzyme accesses to the cellulose core needs to be in intimate and prolonged contact. Pore properties involving in volume, speci c surface, tortuosity, size and fractal dimension were all affected accessibility of cellulose. So pore characteristics and size distribution of various samples were determined.
Desirable Pore Traits Cell Wall for Enzymatic Digestibility Generally, the leaf had the highest average pore diameter, whereas the pith had the highest total pore area and porosity (Table 1). These results were signi cantly positively correlated with enzymatic digestibility, indicating that higher pore size and more pore number could facilitate enzymatic hydrolysis. Differential intrusion volume vs. diameter was also plotted (Fig. 4). The pore size distribution trend of DK and YH was similar. Plant biomass could be deemed as special porous material, consisting of highly ordered porous structure at various levels, such as cell lumen, inter cellular spaces, pit in cell wall, and space among chemical polymers. Arantes suggests that pore size distribution is mainly responsible for the e ciency of enzymatic hydrolysis [17]. Ishizawa deems that the digestibility of cellulose signi cantly correlats to the volume accessible [18]. Sun suggests that the speci c structure of pore not the pore volume impacts the e ciency of enzymatic hydrolysis [19]. A large fraction of the pore volume might be ascribed to pores with small size. Pores with size bigger than 5.1 nm are su cient for accessibility of cellulase, but the small pore might form the mass transfer resistance, thus hindering the enzymatic hydrolysis [20]. Pore size instead of volume could accurately represent the relationship between the porous structure and enzymatic digestibility. Multiple architectures of cell wall, especially for the interface make the glycosyl hydrolase more di cult attack the plant cell wall.

Morphology Variation and Cellulase Adsorption
Allowing the enzyme easier access to the cellulose core might be the primary step to improve enzymatic digestibility. The labeled cellulase with the speci c function of recognizing and adsorbing on cellulose was selected to observe the accessibility of different cell. The imaging results displayed that all the parts of CS gave the complete structure after sectioning, except for the sheath (Fig. 5) Epidermic cells with special structure encrusted with waxes and cutin mainly envelop leaves, rind and sheath. The cuticle is overlaid by wax deposits that play a role in resistance to microbes and insects, and water retention in the nature [21]. Fibers and dermal tissues together provided rigidity, which was signi cantly resistant to enzyme adsorption. This revealed the complicated inter-relationship that the heterogeneity in cell type of CS signi cantly affected the rankings of cellulase adsorption also correlated to enzymatic digestibility. Thus, the recalcitrance of CS varied according to heterogeneity of physicochemical properties that was correlated to cell type. Furthermore, the different parts of CS may require various selections of pretreatment conditions and enzymatic hydrolysis strategy, due to its heterogeneity.

Conclusion
The compiled data revealed that the heterogeneity in structure and physicochemical properties of cell wall were distinct among organs or tissues of CS, and signi cantly correlated to the recalcitrance. Critical factors of cell wall had obvious impacts on cellulase adsorption that furthermore determined enzymatic digestion. Among a set of variables, pore size was shown as the predominant necessity that affected the access of cellulase to polysaccharides within the cell wall. Thus, a holistic view of microscopic interface of cell wall was proposed, considering that concrete variable have interactive impacts on cellulase adsorption depending on overall interface structure assembly. Furthermore, a combination of many variables might contribute to sacchari cation e ciency, indicating that biore nery should be an optimal choice to better exploit heterogeneity and optimize biomass ows.

Materials
Twelve corn (Zea mays L.) cultivars with different macroscopic phenotypes including dry weight, height and diameter were grown in experimental eld of Helin, Inner Mongolia, China (Table S1). The total aboveground biomass was harvested for this experiment when it was mature. The fresh corn straw was separated into two organs including leaf and stalk. The stalk was further dissected into three different tissues including rind, sheath and pith by hand, based on their macroscopic appearance. After that the sample was washed by tap water and air dried. The tested samples were random mixed for sampling uniformity and further divided into two fractions for different analysis requirement. One fraction was ground to powder with size ranging from 0.84 to 0.42 mm through high-speed rotary cutting mill (BJ-300A, Baijie, China). The other fraction was sectioned and used for microscopy. Cellulase (Trichoderma vride G) was bought from Shanghai Yuanye Co., Ltd. The activity of cellulase presenting a lter paper unit of 110.2 U/g was obtained following the classic procedure described by Ghose [22]. Cellulases (Trichoderma reesei ATCC 26921) bought from Sigma-Aldrich and labelled DyLight 633™ provided by Thermo Fisher were used for cell wall staining. All other reagents were analytic grade and obtained from Shengkang Biotechnology Company (Huhhot, China) unless otherwise noted.

Assessment of enzymatic digestibility
Enzymatic digestibility was determined via cellulase mediated hydrolysis and quantitation of reducing sugars generated from cellulose and hemicelluloses. The enzymatic hydrolysis was performed in 50 mM sodium citrate (pH 4.8) with solid consistency of 5% (w/w), enzyme loading of 20 FPU/g and at 50 °C for 48 h. The reducing sugars were assayed according to the 3, 5-dinitrosalicylic acid method described by Miller [23]. The Eq. 1 was used to calculate the reducing sugar yield.
Reducing sugar yield (%) = (m g ×0.9×100)/ (m H-Cel+ m Cel ) Where m g represented amount (g) of the generated reducing sugars during enzymatic hydrolysis, 0.9 was the converting factor, and m H-Cel and m Cel represented weight of hemicelluloses and cellulose in the tested sample (g), respectively.

Chemical composition analysis
Chemical characterization analysis was performed according to a protocol recommended by Van Soest [24]. Various dry samples were treated by neutral buffered detergent solution, hydrochloric acid (2 M) and sulfuric acid (7.34 M) to determine neutral detergent ber (NDF), acid detergent ber (ADF), and acid detergent lignin (ADL) in sequence. The ash content was assayed by heating the ADL at 550 º C for 180 min. The contents of neutral detergent soluble (NDS), hemicelluloses, cellulose, and lignin were calculated by subtracting the corresponding fractions from the initial weight, NDF, ADF, ADL and ash.

Crystallinity analysis
The powder X-ray diffractometer (X'Pert PRO, PANalytical, Netherlands) was used to measure the Segal crystallinity index (CrI) and crystallite dimension (CD). Scans were performed in triplicate at 1 º /min from 5 to 40 º 2θ with a step size of 0.02 º . The CrI was calculated from the XRD patterns based on the empirical peak-height method (Eq. 2) [25]. The dimensions of the crystallites in various samples were determined through Scherrer method (Eq. 3).
CrI = (I total -I amor )/I total (2) I total was the diffraction intensity of major peak at 2θ between 22 º and 23 º for cellulose . I amor was the diffraction intensity of the minima between the major peak and secondary peaks.
Where K was a constant value of 0.94; λ was the X-ray wavelength of 0.1542 nm; β was peak width of diffraction band at the half maximum height; τ represented the crystallite dimension; and θ was the Bragg angle tted from the major peak by Inc Jade 6.5 software.
Degree of polymerization determination . The cellulase labelling procedure was carried out as the manufacturer's instructions adopted by Donaldson with a slightly modi cation [29]. Concretely, the freeze dried cellulase powder was dissolved in borate-phosphate buffered saline (0.05 M, pH 7.5) and mixed with DyLight 633™ in the tube covered with aluminum foil at ambient temperature (around 25 °C ) for 1 h with gentle shaking [30]. And then the mixture was passed through the supplied resin and centrifuged multiple times (8000 rpm, 15 min) to separate the unlabeled uorophore. The puri ed labelled cellulase was kept at 4 °C avoiding light. Cellulase (the given average molecular weight of 65 kDa), with uorophore to protein molar ratio of 7.48:1, was used to label the section of samples. The distribution of the label cellulase on cell wall was described by confocal laser scanning microscopy (LSM-710, ZEISS, Germany), using the identical instrument settings: 10×/1.40 NA Plan-Apochromatic objective lens, pinhole size of 1 AU, 405 nm and 633 nm sequential excitation, and 410-480 nm (blue lignin auto uorescence), and 650-750 (red DyLight 633™ labelled cellulase uorescence) emission. Lignin uorescence and labelled enzyme were visualized by sequential excitation, without any signi cant bleed-through of signals. All images were shown under the uniform condition with optimal intensity.

Statistical analysis
Heat map and dendrogram were plotted using the original "heatmap" function shipped with R installation, based on the hierarchical clustering results of reducing sugar yield. Biological triplicates were selected for each corn cultivar. Chemical and physical analysis was carried out in technical triplicates to ensure reproducibility. The data was expressed as the mean of triplicates ± standard deviation (SD). Statistical analysis was carried out using the SPSS statistical software with one-way ANOVA. The results were considered statistically signi cant, when p value of the differences was lower than 5% at the 95% con dence interval.