Nondestructive, real-time determination and visualization of cellulose, hemicellulose and lignin by luminescent oligothiophenes

Abstract

Enabling technologies for efficient use of the bio-based feedstock are crucial to the replacement of oil-based products. We investigated the feasibility of luminescent conjugated oligothiophenes (LCOs) for non-destructive, rapid detection and quality assessment of lignocellulosic components in complex biomass matrices. A cationic pentameric oligothiophene denoted p-HTEA (pentamer hydrogen thiophene ethyl amine) showed unique binding affinities to cellulose, lignin, hemicelluloses, and cellulose nanofibrils in crystal, liquid and paper form. We exploited this finding using spectrofluorometric methods and fluorescence confocal laser scanning microscopy, for sensitive, simultaneous determination of the structural and compositional complexities of native lignocellulosic biomass. With exceptional photostability, p-HTEA is also demonstrated as a dynamic sensor for real-time monitoring of enzymatic cellulose degradation in cellulolysis. These results demonstrate the use of p-HTEA as a non-destructive tool for the determination of cellulose, hemicellulose and lignin in complex biomass matrices, thereby aiding in the optimization of biomass-converting technologies.

Figure 1. Optical detection of cellulose and cellulose nanofibrils by p-HTEA.

(a) Structure of p-HTEA. (b,c) Excitation spectra of cellulose in the absence and presence of p-HTEA. Spectra were collected at λ_Ex = 300–500 nm, and λ_Em = 545 nm of (b) M. cellulose (5 mg/ml), (c) p-HTEA (dashed), and M. cellulose mixed with p-HTEA (solid). (d) Correlation of the fluorescence signal (RFU) from p-HTEA + M. cellulose, recorded at λ_Ex = 447 nm, λ_Em = 545 nm, to varying M. cellulose concentrations. Error bars = SD, n = 3. (e–g) Scanning electron micrographs of cellulosic materials. (e) M. cellulose (f) pulp cellulose and (g) cellulose nanofibrils. Scale bars (e,f) = 100 μm, (g) = 10 μm. (h–j) Normalized excitation spectra of p-HTEA bound to cellulosic materials. Spectra were collected at λ_Ex = 300–500 nm and λ_Em = 545 nm of p-HTEA (dashed) or p-HTEA mixed with (h) M. cellulose, (i) pulp cellulose, (j) cellulose nanofibrils (solid). Normalized relative fluorescence units (RFU) = each data point represented as a percentage of the largest emitted fluorescence of the excitation spectra.

Figure 2. Binding of p-HTEA to lignin enables optical identification of lignin in mixed samples.

(a) Excitation spectra of lignin in ^DH₂O (solid) and ^DH₂O alone (dashed) collected at λ_Ex = 300–500 nm, λ_Em = 545 nm. (b) Correlation between intrinsic fluorescence signals at λ_Ex 339 nm, λ_Em 545 nm, and lignin concentration. (c) Normalized excitation spectra of p-HTEA in the absence (dashed) and presence of lignin (solid). (d) Correlation between p-HTEA fluorescence signals at λ_max-p-HTEA (λ_Ex 387 nm, λ_Em 545 nm) and lignin concentration. (e) Normalized excitation spectra of p-HTEA bound to cellulose before (dashed) and after (solid) addition of lignin. (f) Correlation between fluorescence signals recorded at λ_{max-cellulose} (λ_Ex 444 nm, λ_Em 545 nm) of p-HTEA pre-bound to cellulose with different added concentrations of lignin. (g) Excitation spectra of p-HTEA + lignin (dashed) and p-HTEA + lignin + cellulose (solid). (h) Excitation spectra of intrinsic fluorescence of lignin (dashed) and lignin + cellulose (solid). All panels show the mean of triplicates from one out of three experimental repeats.

Figure 3. p-HTEA-based optical identification of hemicellulose in mixed samples.

(a–c) Excitation spectra of (a) O-acetylgalactoglucomannan (solid), (b) galactomannan (solid) and (c) xylan (solid) using ^DH₂O (dashed) as a control. (d–f) Excitation spectra of p-HTEA bound to (d) O-acetylgalactoglucomannan (solid), (e) galactomannan (solid) and (f) xylan (solid) and from p-HTEA alone (dashed). (g–i) Correlation of the signal at λ_max in (d–f, solid lines) with increasing concentrations of each hemicellulose in the presence (solid) and absence (dashed) of p-HTEA. (g) λ_{max p-HTEA-O-acetylgalactoglucomannan} (λ_Ex 414 nm, λ_Em 545 nm) correlated to the concentration of O-acetylgalactoglucomannan (GGM), (h) λ_{max p-HTEA-galactomannan} (λ_Ex 414 nm, λ_Em 545 nm) to the concentration of galactomannan, and (i) λ_{max p-HTEA-xylan} (λ_Ex 375 nm, λ_Em 545 nm) to the concentration of xylan. (j–l) RFU (λ_{max p-HTEA-cellulose,} λ_Ex 444 nm, λ_Em 545 nm) from p-HTEA with increasing cellulose concentrations in the presence (solid line) and absence (dashed line) of (j) O-acetylgalactoglucomannan, (k) galactomannan, and (l) xylan.

Figure 4. Spectral quality assessment of cellulose during the pulping process.

Fluorescence signal from p-HTEA mixed with cellulose in acidic and alkaline conditions. Spectra were recorded at λ_Ex 445 nm, λ_Em 530 nm. Signal (ΔRFU = RFU _{Bound p-HTEA}–RFU _{Unbound p-HTEA}) from (a) p-HTEA bound to cellulose at pH 4, pH 7.4 and pH 10. (b–d) Signal from p-HTEA at increasing concentrations of cellulose (solid) at (b) pH 4, (c) pH 7.4 and (d) pH 10. Linear regression is shown as dashed line. (e) Cartoon illustrating the continuous cooking process, presenting key stages for p-HTEA quality analysis of cellulose and by-products. (f–h) Excitation spectra of p-HTEA mixed with (f) process liquor (solid), (g) lignosulphonate (solid), and (h) Kraft lignin (solid), compared to spectra of p-HTEA alone (dashed), and the basal fluorescence of each sample (dotted). (i,j) Excitation spectra of p-HTEA mixed with (i) unbleached (solid) and (j) bleached (solid) pulp, compared to spectra of p-HTEA alone (dashed), and the basal fluorescence of each sample (dotted). Photographs show the characteristic colours of unbleached (brownish) and bleached (white) pulp.

Figure 5. p-HTEA enables combined fluorescence imaging and spectrometric analysis of cellulose materials.

Phase contrast (left) and fluorescence confocal microscopy (right) of (a) M. cellulose, (c) pulp cellulose, (e) cellulose nanofibrils, (g) paper made of cellulose nanofibrils, and (i) lignin stained with p-HTEA. In confocal microscopy, excitation at 473 nm and bandwidth filters detecting 490–530 nm were applied. Corresponding unstained controls are shown in Supplementary Fig. 2. Scale bar = 200 μm. Normalized excitation spectra of p-HTEA mixed with (b) M. cellulose, (d) pulp cellulose, (f) cellulose nanofibrils, (h) paper made of cellulose nanofibrils, and (j) lignin (solid). Spectra of the pure materials (dotted) and of p-HTEA (dashed) are shown for comparison.

Figure 6. Cellulolysis recorded in real-time by fluorescence monitoring.

(a) Fluorescence signal from p-HTEA mixed with M. cellulose in the presence (solid) and absence (dashed) of cellulase. Background signal is monitored in samples containing cellulase and p-HTEA, but no cellulose (dotted line). (b) Enzymatic digestion of paper made of cellulose nanofibrils. Decreased fluorescence occurs as cellulase digests the p-HTEA-stained paper (solid), whereas signal remains constant in the absence of enzyme (dashed). (c) Rapid degradation of p-HTEA-stained suspension of cellulose nanofibrils occurs when increasing units of cellulase (right to left 0, 0.5, 1, 2, 4, 8 U/ml) is added. (d) Linear regression analysis of each assay in (c) reveal linear correlation between reaction rate and enzyme units.