Chemical and morphological characterization of sugarcane bagasse submitted to a delignification process for enhanced enzymatic digestibility

Abstract

Background: In recent years, biorefining of lignocellulosic biomass to produce multi-products such as ethanol and other biomaterials has become a dynamic research area. Pretreatment technologies that fractionate sugarcane bagasse are essential for the successful use of this feedstock in ethanol production. In this paper, we investigate modifications in the morphology and chemical composition of sugarcane bagasse submitted to a two-step treatment, using diluted acid followed by a delignification process with increasing sodium hydroxide concentrations. Detailed chemical and morphological characterization of the samples after each pretreatment condition, studied by high performance liquid chromatography, solid-state nuclear magnetic resonance, diffuse reflectance Fourier transformed infrared spectroscopy and scanning electron microscopy, is reported, together with sample crystallinity and enzymatic digestibility.

Results: Chemical composition analysis performed on samples obtained after different pretreatment conditions showed that up to 96% and 85% of hemicellulose and lignin fractions, respectively, were removed by this two-step method when sodium hydroxide concentrations of 1% (m/v) or higher were used. The efficient lignin removal resulted in an enhanced hydrolysis yield reaching values around 100%. Considering the cellulose loss due to the pretreatment (maximum of 30%, depending on the process), the total cellulose conversion increases significantly from 22.0% (value for the untreated bagasse) to 72.4%. The delignification process, with consequent increase in the cellulose to lignin ratio, is also clearly observed by nuclear magnetic resonance and diffuse reflectance Fourier transformed infrared spectroscopy experiments. We also demonstrated that the morphological changes contributing to this remarkable improvement occur as a consequence of lignin removal from the sample. Bagasse unstructuring is favored by the loss of cohesion between neighboring cell walls, as well as by changes in the inner cell wall structure, such as damaging, hole formation and loss of mechanical resistance, facilitating liquid and enzyme access to crystalline cellulose.

Conclusions: The results presented herewith show the efficiency of the proposed method for improving the enzymatic digestibility of sugarcane bagasse and provide understanding of the pretreatment action mechanism. Combining the different techniques applied in this work warranted thorough information about the undergoing morphological and chemical changes and was an efficient approach to understand the morphological effects resulting from sample delignification and its influence on the enhanced hydrolysis results.

Figure 1

Remaining fractions of lignocellulosic components in bagasse samples after pretreatment steps.

Figure 2

CPMAS-TOSS NMR spectra of sugarcane bagasse. (a) Untreated; (b) bagasse treated with acid (H₂SO₄ 1.0%) and (c) bagasse treated with acid and NaOH 4.0%. The spectra were normalized by the intensity of line 10 (C1 carbon of cellulose). CPMAS-TOSS: cross polarization under magic angle spinning with total suppression of spinning sidebands; NMR: nuclear magnetic resonance.

Figure 3

13C NMR spectra of the lyophilized hydrolysate obtained by treating the sugarcane bagasse with acid and NaOH 4.0%.(a) CPMAS-TOSS; (b) CPMAS-TOSS NMR with dipolar dephasing; (c) CPMAS-TOSS NMR with CSA filter. The absolute intensities are arbitrary. CPMAS-TOSS: cross polarization under magic angle spinning with total suppression of spinning sidebands; CSA: chemical shift anisotropy; NMR: nuclear magnetic resonance.

Figure 4

13C CPMAS-TOSS spectra of bagasse samples treated with different NaOH concentrations. (a) Remaining solid fraction and (b) the corresponding lyophilized hydrolysate. The inset in (a) is a zoom of the 110 ppm to 120 ppm region. Spectra in (a) were normalized by the intensity of line 10 (C1 carbon of cellulose) and in (b) by the intensity of the line at 56.2 ppm (lignin methoxy). CPMAS-TOSS: cross polarization under magic angle spinning with total suppression of spinning sidebands.

Figure 5

Surface images of the untreated sugarcane bagasse obtained by scanning electron microscopy. (a) General view of the sample showing fibers and pith (arrows F and P); (b) amplifications on the fiber surface, with parallel stripes covered by residues; (c) amplification on the pith; and (d) amplifications on the fiber surface, with parallel stripes covered by residues. Samples underwent roll and knife milling.

Figure 6

Scanning electron microscopy images obtained from fractures of untreated sugarcane bagasse. (a) General view of a bark fracture; and (b) fracture of the stalk core, both showing conducting vessels surrounded by sclerenchyma (arrow S) and embedded in parenchyma (arrow P); (c) amplification on parenchyma and (d) on sclerenchyma cells. Samples underwent roll milling only.

Figure 7

Scanning electron microscopy surface images of the sugarcane bagasse treated with acid. (a) General view of the sample showing fibers (mainly) and pith; (b) amplification on the fiber surface (dashed area in (a)), showing more exposed parallel stripes. Samples underwent roll and knife milling before the treatment.

Figure 8

Scanning electron microscopy surface images of the sugarcane bagasse sample treated with alkaline solutions. (a) NaOH 0.5% with bundles starting to come apart; (b) and (c) NaOH 2%, showing unstructured and unattached bundles; and (d) NaOH 4%, showing individual fibers. Samples underwent roll and knife milling before the treatment.

Figure 9

Scanning electron microscopy fracture images of the sugarcane bagasse sample treated with alkaline solutions. (a) NaOH 1%, showing the damaged surface of the cell wall; (b) NaOH 1%, with collapsed cell walls; (c) NaOH 0.5%, cell wall unstructured by peeling off radial layers. Samples underwent roll milling only.

Figure 10

Crystallinity index (%) calculated for each sample as a function of the cellulose amount (%). The error bars are standard deviations from the average values of duplicate determinations.

Figure 11

Total cellulose conversion for untreated sugarcane bagasse and bagasse after acid and acid/alkali treatments after 24 hours, 48 hours and 72 hours hydrolysis. The error bars are standard deviations from the average values of duplicate determinations.