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Anatomic and Ultrastructural Characteristics of Different Regions of Sugar Cane Internodes Which Affect Their Response to AlkalineSulfite Pretreatment and Material Recalcitrance Fernanda M. Mendes, Mariana B. Fonseca, André Ferraz, and Adriane M. F. Milagres* Departamento de Biotecnologia, Escola de Engenharia de Lorena, Universidade de São Paulo, 12602-810 Lorena, SP, Brazil ABSTRACT: Sugar cane internodes have great morphological heterogeneity and different cell types that respond in varied ways to pretreatment processes. At the outermost region of the internode there are epithelial cells and a ring of cortical cells. Toward the central part of the internode there are a number of vascular bundles surrounded by parenchyma cells. The innermost regions are richer in parenchyma cells. The sugar cane internode regions were evaluated according to their response to alkaline-sulfite pretreatment and the consequences for material recalcitrance. The experimental data set included 4 sugar cane hybrids dissected into 3 different internode regions that were pretreated for 5 different reaction times, totaling 60 samples. Cellular ultraviolet microspectrophotometric evaluation of the samples suggested that in the thin cell walls (parenchyma and vessels) the hydroxycinnamic acids were accessible to the pretreatment reagents, whereas in the thicker fiber cell walls lignin and hydroxycinnamic acids were more resistant. The outermost regions were the most resistant to lignin and hemicellulose removal. Enzymatic hydrolysis of the pretreated samples indicated that the outermost fraction and the rind were recalcitrant regions, whereas the pith−rind interface was less recalcitrant. The parenchyma-rich pith−rind interface region benefitted not only from delignification during pretreatment but also from hydroxycinnamic acid removal. By contrast, the most external fractions required a longer pretreatment time to overcome the recalcitrance because it was necessary to remove significant amounts of lignin and hydroxycinnamic acids from the samples.

1. INTRODUCTION Sugar cane and other grasses constitute an abundant and sustainable source of polysaccharides that can be converted into biofuels and other sugar-derived products.1 Nevertheless, the efficient enzymatic conversion of polysaccharides into monomers is hindered by many physicochemical, ultrastructural, and compositional characteristics of the lignocellulosic materials.2,3 Grasses are anatomically heterogeneous because the internodes contain a dense epidermis, cortical cells, variedsize vascular bundles, and parenchyma cells.4−6 The vascular bundles include vessels and fibers and a limited number of nonlignified phloem cells. Parenchyma cells can occupy 30− 70% of the internode volume depending on the grass, but only a small percentage of the internodes dry mass.7,8 In sugar cane, parenchyma cells are numerous, with an average diameter of 60 μm and thin cell walls (1.7 μm). The vessel elements are also wide cells, with an average diameter of 80 and 2.7 μm cell walls. The fibers represent the main source of dry mass in the material because they are numerous narrow cells (20 μm) with thick cell walls (4 μm).8,9 Such anatomic diversity suggests that different cell types would respond in varied ways to a pretreatment process. For example, the thin-cell walls of poorly lignified parenchyma should promptly react to a chemical pretreatment, while the thick and lignified fibers should resist for a longer time during the same process. These types of varied responses create the possibility that a component released from parenchyma cell walls decompose in the reaction medium before a similar component is released from the fiber cell wall. Previous studies evaluating industrially relevant grass lignocellulosic materials, such as corn stover10−12 and sugar cane,13−15 demonstrated varied recalcitrance along different © XXXX American Chemical Society

regions and tissues of the plant stalks. The epidermis and periphery of the internodes (rind region) were significantly more recalcitrant than the innermost region (pith). It is noteworthy that in addition to cellulose, hemicellulose, and lignin, the secondary cell wall of grasses is characterized by the presence of hydroxycinnamic acids that play a significant role in cross-linking wall polymers into a cohesive network.16 Ferulic acid is reported to cross-link hemicellulose and lignin, whereas p-coumaric acid mostly esterifies lignin.17 The topochemical distribution of hydroxycinamic acids and lignin on the cell wall of such materials is an additional source of heterogeneity. For example, UV-microspectrophotometry (UMSP) indicated that sugar cane vessels are the most lignified cell walls, followed by fibers and parenchyma.13,14 Parenchyma cell walls present a UV maximal absorbance at 310 nm, which is characteristic of hydroxycinnamic acids instead of lignin.13 The aim of the present work was to relate the anatomic and ultrastructural characteristics of different regions of sugar cane internodes with their responses to alkaline-sulfite pretreatment and the consequences for material recalcitrance to hydrolytic enzymes. The lignin and hydroxycinnamic acid distribution in different cell types, revealed by UMSP, was also considered to be a source of the microheterogeneity of the samples. The experimental data set included four different experimental sugar cane hybrids that present varied recalcitrance as a consequence of varied chemical composition.14,18−20 The alkaline-sulfite pretreatment process selected for this work is known to Received: November 2, 2015 Revised: December 15, 2015

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DOI: 10.1021/acs.energyfuels.5b02569 Energy Fuels XXXX, XXX, XXX−XXX

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Table 1. Chemical Composition (%, g of component/100 g of dry material) of Three Different Sugarcane Internode Fractions Pretreated with an Alkaline-Sulfite Process

applying a vacuum to the samples contained in a Buchner flask for 30 min, and then, the liquor was displaced into the flask and an additional 15 min of vacuum was applied. The impregnated biomass was cooked at 121 °C for varied periods, from 30 to 120 min. The cooked material was filtered through filter paper, and the liquor was discarded. The retained solids were washed with 2 L of water and stored for subsequent experiments. Subsamples used for UV-microspectrophotometry (UMSP) were prepared from pretreated materials by dissecting 5 mm × 1 mm × 1 mm fragments that were hand cut in a longitudinal direction and storing them at 4 °C under water containing 0.01% sodium azide. The remaining pretreated materials were air-dried and milled to pass through a 0.84 mm screen. 2.3. Determination of the Chemical Composition of the Samples. Approximately 1 g of the milled sample was extracted with 95% ethanol for 6 h in a Soxhlet apparatus. The ethanol-extracted milled samples were hydrolyzed with 72% (w/w) sulfuric acid at 30 °C for 1 h (3 mL acid and 300 mg sample) as described previously.25 The acid hydrolysate was diluted with 79 mL of water, and the mixture was autoclaved at 121 °C for 1 h. The residual material was cooled and filtered through a porous glass filter (number 3). The solids were dried to a constant weight at 105 °C and assessed as the insoluble Klason lignin component. The concentration of soluble Klason lignin in the aqueous fraction was determined by measuring the absorbance of the solute at 205 nm, using the value of 105 L·cm−1·g−1 as the absorptivity of soluble lignin. The concentrations of the monomeric sugars in the soluble fraction were determined by HPLC using an HPX87H column at 45 °C and an elution rate of 0.6 mL·min−1 with 5 mmol·L−1 sulfuric acid. Sugars were detected in a temperature-controlled refractive index detector at 35 °C. Glucose, xylose, arabinose, and acetic acid were used as external calibration standards. No corrections were performed regarding the sugar degradation reactions that take place during acid hydrolysis. The factors used to convert sugar monomers to anhydromonomers were 0.90 for glucose and 0.88 for xylose and arabinose. The acetyl content was calculated as the acetic acid content multiplied by 0.72. The sum of the anhydromonomers calculated from

partially remove lignin and hemicellulose, while the residual lignin becomes sulfonated and more hydrophilic. Thus, the alkaline-sulfite process makes the lignocellulosic matrix weaker, without harming polysaccharide yields.21−23 This pretreatment showed great results of enzymatic digestion in sugar cane bagasse.20,22

2. MATERIALS AND METHODS 2.1. Raw Material and Biomass Preparation. Four experimental sugar cane hybrids, coded as H89, H58, H146, and H140, were selected from the breeding program developed by RIDESA, which is associated with the Federal University of Viçosa, Viçosa, MG, Brazil.24 Mature plants (16-month old) of four hybrids were obtained as previously described.14 Internodes 3−7 (from the plant base) were separated from the stalks. These internodes were cut into 25 mm circular pieces following the longitudinal axis of the stalks. The 25 mm pieces were then cut from the periphery to the center as follows: (a) the first external 2 mm containing the epidermis, cortical cells, and part of the outermost rind (outermost fraction); (b) the diameter of the remaining material was measured and divided into three equal segments, which were dissected from the periphery to the internode center corresponding to the rind, pith−rind interface, and pith fractions. The samples were stored at −18 °C until used. Microscopic evaluation of the samples demonstrated that all sugar cane tissues were maintained after thawing the samples. 2.2. Biomass Pretreatment. The internode segments were thawed, the excess water was drained, and the samples were weighed to ensure that there was 20 g of biomass (oven dry basis) for each pretreatment reaction. The crude material subjected to the alkalinesulfite pretreatment contained originally 75% of water. Approximately, half of the dry matter was composed of lignocellulosic biomass and the other half of sucrose.18 The samples were impregnated with 200 mL of alkaline-sulfite liquor containing 5% NaOH and 10% Na2SO3 (g of chemicals/100 g of dry biomass). Impregnation was performed by B

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Energy & Fuels xylose, arabinose and acetyl were reported as the xylan content. This procedure was conducted in triplicate. The glucan, hemicelluloses and total lignin contents varied by 3.8%, 2.3%, and 2.7% of the average values, respectively. 2.4. UMSP Evaluation of the Samples. The sample segments were left for 24 h in water at 4 °C to remove sodium azide. The samples were then dehydrated in a graded series of acetone and embedded in Spurr’s epoxy resin26 formulated to the standard hardness of the blocks (Spurr Low-Viscosity Embedding Kit, EM0300, SIGMA-Aldrich, St. Louis, MO, USA). Transversal sections (1 μm) were cut from these samples with a LEICA EM-UC7 ultramicrotome fitted with a diamond knife (4 mm-Histo, DIATOME, Switzerland) and transferred to quartz microscope slides. UV spectra were recorded from 1 μm2 areas focused on the secondary wall of the selected cell types with a ZEISS Axio Imager/ J&M microspectrophotometer (ZEISS/J&M, Germany). At least 20 spectra from 250 to 400 nm were recorded from each individual cell type (i.e., vessel, fiber, and parenchyma). 2.5. Enzymatic Digestion of the Samples. The enzymatic hydrolysis experiments were performed using a mixture of commercial enzyme preparations (i.e., cellulases from Trichoderma reeseiSIGMA C2730and β-glucosidases from Aspergillus nigerSIGMA C6105) at a dosage of 10 FPU·g−1 of substrate and 20 IU β-glucosidase·g−1 of substrate in all of the experiments. The hydrolysis reaction was performed in 50 mL centrifuge tubes containing 200 mg of milled substrate (dry weight basis), the enzyme mixture, and 10 mL of 50 mmol·L−1 sodium acetate buffer, pH 4.8, containing 0.01% (w/v) sodium azide. The flasks were incubated at 45 °C with reciprocal agitation at 120 cycles per min. The reaction was stopped at defined periods from 8 to 72 h by heating the flask to 100 °C for 5 min; the material was then centrifuged at 3400g for 15 min. The soluble fractions were assayed for glucose and xylose by HPLC (Waters Corporation, Millford, USA) using the same HPLC procedure described for chemical characterization of the samples. The cellulose and xylan conversion levels reported in the text refer to the average conversion of the polysaccharides to their monomers.

data set. The pretreated samples were enriched in glucans, whereas the lignin contents decreased along pretreatment times. Xylans were partially degraded and dissolved during alkaline-sulfite pretreatments;31−33 however, considering the extensive lignin removal during the process, the pretreated solids were enriched in polysaccharide contents, including xylan. Independently of the studied hybrid or internode region, data from Table 1 corroborate that the alkaline-sulfite pretreatment is selective for delignification.31,32 The delignification levels calculated for short and long pretreatment periods are shown in Table 2. The longest pretreatment periods (120 Table 2. Initial (30 min Pretreatment) and Maximal Delignification (120 min Pretreatment) Levels of Different Internode Regions from Sugar Cane Hybrids Pretreated with an Alkaline-Sulfite Process Delignification Level (%) outermost fraction

rind

pith−rind interface

hybrid

30 min

120 min

30 min

120 min

30 min

120 min

89 58 146 140

6 30 24 13

30 32 30 31

43 72 59 59

67 71 63 65

67 74 67 58

65 79 76 73

min) provided maximal delignification. The delignification levels in the outermost fractions were limited to ca. 30% after 120 min of pretreatment, independent of the hybrid, whereas the rind and the pith-rind interfaces were delignified to ca. 70% with the same pretreatment period. Short pretreatment periods (30 min) were useful for discriminating among hybrids. For example, the outermost fraction of the H58 was delignified to almost 30% in the first 30 min of the reaction, whereas the delignification of the H89 was limited to 6% in the same period. The rind and the pith−rind interfaces of the H58 were also promptly delignified, reaching 72% and 74% in the first 30 min of the reaction, respectively. The lignin removal within the cell walls of different cell types was monitored by UMSP. Absorption spectra (between 240 and 400 nm) were recorded from the secondary cell walls of fibers, vessels and parenchyma located in the different internode regions. All of the evaluated fibers and vessels presented UV absorption spectra with two bands: one at 280− 285 nm, which was assigned to the lignin moieties, and another at 310−320 nm, corresponding to hydroxycinnamic acids, corroborating previous work.13,14,34 Parenchyma cells presented only a dominant band at 315 nm, which suggested that hydroxycinnamic acids were the predominant aromatic components in this cell type, corroborating the previous work of Costa et al.14 Additional data were obtained by mapping lignin removal during the alkaline-sulfite pretreatment of the varied internode regions, cell types, and different hybrids. Data compilation indicated that the UV-absorption intensities of the cell walls in untreated hybrids increased from the pith−rind interface toward the outermost fraction. These data were consistent with the cytological observations of numerous vascular bundles in the internode periphery4,14,29 as well as the high lignin contents detected in this internode region (Table 1). Illustrative UV-absorption data for the 3 main cell types contained in the rind region are shown in Table 3. Colored labels varying from light yellow to red were used to facilitate

3. RESULTS AND DISCUSSION 3.1. Pretreatment of DiffErent Sugar Cane Internode Regions. Grass monocotyledons, such as sugar cane and maize, comprise heterogeneous lignocellulosic materials.4,10,14,27 Cytological investigation of sugar cane indicates that the internode periphery contains epithelial cells, cortical cells, and several vascular bundles, whereas the innermost regions contain numerous parenchyma cells surrounding some small vascular bundles.4,14,28,29 The innermost regions present low recalcitrance and do not require pretreatment for subsequent enzymatic digestion.13,14,27,30 To test whether different periphery internode regions from sugar cane respond in various ways to pretreatment and enzymatic digestion, we dissected four different internode fractions corresponding to the pith, pith-rind interface, rind, and outermost fraction. The last three most external fractions were selected for pretreatments prior to saccharification. The experimental plan included 4 different sugar cane hybrids with varied chemical compositions18 to provide a large data set composed of 12 samples pretreated for 5 different reaction times, lead to a total of 60 samples under evaluation. The studied hybrids were chosen because they contrast for chemical composition, especially for lignin content (H140 > H146 = H58 > H89, with total Klason lignin of 21.5 ± 0.2%, 18.6 ± 0.1%, and 16.8 ± 0.1%, respectively).18 The chemical composition of untreated and alkaline-sulfite treated samples is shown in Table 1. This table employs a set of colored labels (heat map) varying from blue for low values to red for high values facilitating pattern recognition in the large C

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Table 3. UV-Spectrophotometric Evaluation of Different Cell Types from the Rind Region of Different Sugarcane Hybrids Pretreated with an Alkaline-Sulfite Process

Table 4. Glucan Conversion to Glucose in Different Sugar Cane Internode Regions Pretreated with an Alkaline-Sulfite Process with Various Reaction Timesa

a

Four different sugar cane hybrids were pretreated and hydrolyzed by commercial enzymes for periods varying from 8 to 72 h.

almost zero in most of the hybrids after the first 30 min of pretreatment; however, the absorbance at 285 nm diminished gradually with pretreatment times. These data indicated that hydroxycinnamic acids, but not lignin, were promptly removed from the vessel cell walls. In fibers, the UV-absorption at both wavelengths (315 and 285 nm) decreased gradually with pretreatment times in most of the hybrids.

pattern recognition in this large data set. Irrespective of the studied hybrid, the UV-absorption intensities of the parenchyma cells decreased significantly after short pretreatment periods, either at 285 or 315 nm. The first 30 min of pretreatment were enough to decrease the original UVabsorption intensities of the parenchyma (ranging from 0.14 to 0.28, depending on the hybrid) to values close to zero. In vessel cell walls, the UV-absorption at 315 nm also decreased to D

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Table 5. Xylan Conversion to Xylose in Different Sugar Cane Internode Regions Pretreated with an Alkaline-Sulfite Process with Various Reaction Timesa

a

Four different sugar cane hybrids were pretreated and hydrolyzed by commercial enzymes for periods varying from 8 to 72 h.

UV data from the three cell types suggested that in the thin cell walls (parenchyma and vessels)9 the hydroxycinnamic acids were accessible to the pretreatment reagents, whereas in the thicker fiber cell walls they were more resistant. Hydroxycinnamic acids can be linked to the lignocellulose backbone through ester linkages at the Cγ-carbon of the phenylpropanoid side chain, presenting free phenols at the C-4 position of the aromatic ring.17,35 Another hydroxycinnamic acids linkage pattern include the typical ester linkage and the aromatic C-4 oxygen etherified to lignin.17 The ester linkage should be promptly cleaved in the alkaline reaction media employed in the pretreatments. In contrast, the hydroxycinnamic acids ether-linked to lignin would require a longer pretreatment period to be released, with a similar resistance to that presented by lignin. Data considered altogether indicate that the esterlinked/phenol-free hydroxycinnamic acid structures predominate in parenchyma and vessel cell walls. Lignin-etherified hydroxycinnamic acids, in contrast, would predominate in fiber cell walls. However, it is not possible to rule out that the removal of hydroxycinnamic acids from fibers would also be limited due to the thicker cell walls of this cell type compared to the vessels and the parenchyma.4,9 The vessels and fibers retained part of the original UVabsorption at 285 nm with pretreatment. This behavior was especially noted for the hybrids 89, 146, and 140. In the case of hybrid 58, UV-absorption at 285 nm decreased significantly with pretreatment, reaching values as low as 0.01−0.06 (Table 3), asserting that this hybrid seems to be suitable for short pretreatment periods. 3.2. Enzymatic Digestion of Sugar Cane Fractions Pretreated with the Alkaline-Sulfite Process. Untreated and pretreated sugar cane internode fractions from different sugar cane hybrids were subjected to enzymatic hydrolysis by commercial enzymes (Tables 4 and 5). Colored labels from blue to red were also used to facilitate pattern recognition of

the levels of glucan and xylan conversions obtained from these samples. The pith−rind interfaces from hybrids H89 and H58 were susceptible to direct enzymatic hydrolysis (without pretreatment) because the glucan to glucose conversion levels of these samples reached 71% and 58% after 72 h of digestion, respectively. The same internode regions of the H146 and H140 were more recalcitrant, reaching only 40% and 31% glucan conversion after 72 h of enzymatic digestion, respectively. The untreated rind and outermost fractions were more recalcitrant in most of the studied hybrids, except for the rind from H89, which reached 34% glucan conversion after 72 h of enzymatic hydrolysis. These data corroborate that the lesslignified-parenchyma-rich innermost regions of grasses are less recalcitrant, especially in low-lignin content plants.11,14,30 However, the periphery regions of the internodes require some pretreatment to be efficiently converted into monosaccharides. The alkaline-sulfite pretreatment of the pith-rind interface provided highly digestible substrates. For example, 30 min of pretreatment of the less recalcitrant hybrids H89 and H58 were enough for the almost complete conversion of glucan to glucose in the first 8 h of enzymatic digestion (Table 4). In the case of the more recalcitrant hybrids (H146 and H140), longer pretreatment periods and longer hydrolysis times were necessary to provide complete conversion of glucan to glucose. Comparison of the initial enzymatic hydrolysis rates calculated for the 30 min pretreated materials demonstrated that the pithrind interface hydrolyzed at 10−12% h−1 in the less recalcitrant hybrids H89 and H58. The rind fractions of the same hybrids were also hydrolyzed at high rates (8−10% h−1) with short pretreatment periods. However, the rind fractions of the H146 and H140 as well as the outermost fractions of all of the hybrids presented initial enzymatic hydrolysis rates lower than 6% h−1 (Figure 1). E

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required mild pretreatment and short enzymatic hydrolysis times to reach efficient polysaccharide conversion to sugar monomers. Moreover, the responses to pretreatment and enzymatic digestion were critically affected depending on the internode region and the type of sugar cane hybrid. The outermost fractions, which included some vascular bundles plus epithelial and cortical cells, were significantly more recalcitrant than the other internode regions, independent of the studied sugar cane hybrid. The data created a center of attention for a new design in sugar cane processing for the subsequent enzymatic hydrolysis of the polysaccharides. It is possible to envisage that the sucrose-poor outermost fractions could be peeled and used, for example, for burning and steam/electricity generation. The remaining stalk could then be processed, requiring less energy in the sucrose extraction process, providing a more suitable bagasse for subsequent pretreatment and enzymatic hydrolysis.

Figure 1. Initial glucan conversion rates (up to 4 h reaction) during enzymatic hydrolysis of different sugar cane regions recovered from varied hybrids pretreated for 30 min with an alkaline-sulfite process.



The rind of the H89 and H58 required at least 30 min of pretreatment and 24 h of enzymatic digestion to reach glucan conversion levels higher than 85%. For H146 and H140, even longer pretreatment and/or enzymatic digestion times were necessary to provide high glucan conversion levels. The outermost fractions were considerably more recalcitrant, even after pretreatment. This region from the less recalcitrant hybrids (H58 and H89) required at least 90−120 min pretreatment and 48−72 h of enzymatic digestion to reach high glucan conversion levels (Table 4). For H146 and H140, 120 min of pretreatment and 72 h of enzymatic hydrolysis increased the glucan conversion levels only to 62% and 64%, respectively. Xylan conversion to xylose followed the same behavior as discussed for cellulose hydrolysis (Table 5). However, the maximal xylan conversions attained lower values compared to cellulose conversion mainly because the commercial enzymatic preparations are not well-balanced for hemicellulases.36 The improvement of enzymatic polysaccharide conversion by alkaline-sulfite pretreatment has been assigned to lignin removal and its sulfonation.23,32,36 The residual lignin contents in the pith-rind interface of all of the hybrids and the rind fractions of the H89 and H58 were relatively low after pretreatment (Table 1), which explains the high efficiency of the enzymatic hydrolysis of these samples. In contrast, the rind of H146 and H140 and especially the outermost fractions of all of the hybrids retained high lignin contents even after long pretreatment periods, which correlated with the high recalcitrance of these samples. Hydroxycinnamic acids could also explain part of the recalcitrance in grasses.5,18 Data obtained in the present work showed that the alkali labile hydroxycinnamic acids present in the parenchyma and vessel cell walls were promptly removed at the initial phase of the alkaline-sulfite pretreatment (Table 3). Therefore, the parenchyma-rich pith−rind interface region benefited not only from delignification but also from hydroxycinnamic acid removal. In contrast, the most external fractions required longer pretreatment times to overcome the recalcitrance because it was necessary to remove significant amounts of lignin and also etherified hydroxycinnamic acids.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +55(12)31595019. Author Contributions

F.M.M., A.F., and A.M.F.M. designed the experiments. F.M.M. and M.B.F. performed the experiments and analyzed the data. All of the authors discussed the results and implications and commented on the manuscript at all stages. All of the authors read and approved the final manuscript. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS The authors would like to thank J. M. Silva for technical assistance. This work was supported by FAPESP (contracts 08/ 56256-5, 11/50535-2, and 14/06923-6). F.M.M. and M.B.F. thank FAPESP for their student fellowships.



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