Lignin extraction and recovery in hydrothermal pretreatment of bamboo

A significant amount of lignin and hemicellulose are dissolved in the hydrothermal treatment of biomass. The hemicellulose can be recovered and utilized for value-added products. The dissolved lignin can undergo depolymerization and condensation reactions, and interferes with the separation and purification process for hemicellulose recovery. This paper investigated the behavior of the lignin extracted from the hydrothermal pretreatment of bamboo and its contributions to the physical characteristics of the hydrolysate. It was found that the turbidity of the hydrolysate was strongly associated with the lignin fragments and suspended long chain hemicelluloses. As the lignin depolymerization and condensation reactions occurred simultaneously, the dissolved lignin fractions in the hydrolysate increased first and then decreased. The molecular weight (MW) of the dissolved lignin fragments ranged from 3342 ~ 5611 g/mol, with mainly guaiacyl (G) and syringyl (S) unit in the structure.


INTRODUCTION
3][4][5][6] Therefore, the hydrolysate solution is considerably complex; particles, colloidal substances and solubles are involved in the system.Dissolved and colloidal polymers are mainly originated from the carbohydrate and lignin reactions via acid-catalytic hydrolysis.Depending on the pretreatment severity, monosaccharide dehydration reactions also take place, with furfural and 5hydroxymethyl furfural as the main producst of the dehydration reactions of hexose and pentose, respectively. 7Besides, some soluble organics such as acetic acid, methanol, furans, and aromatics were also found in the solution.18 phenolic monomers had been detected in the hydrolysate from CEL (cellulolytic enzymatic lignin) hydrothermal pretreatment, p-hydroxybenzoic acid was also observed to be the primary phenol; additional trace amounts of phenolic compounds also existed in the hydrolysate. 8Among those complicated components, lignin and its derivatives are viewed mainly as negatively charged colloidal particles.Gaining a thorough understanding of the chemical/physical process occurring at the hydrothermal pretreatment and of all the component interactions is difficult due to its complexity.The previous study found that hemicelluloses dissolved in the solution not only by means of solubilization, 9 but also by diffusion or transportation to some extent.
With respect to lignin, a portion of the lignin degraded via hemolytic/acidolytic depolymerization of aryl ether linkage, and therefore small quantities of lignin fragments dissolved into the hydrolysate liquor.Evidently, only the lignin with low MW could be dissolved into the hydrolysate liquor; however, the low MW lignin exhibited extremely high reactivity towards the acid-catalysed condensation reactions. 6he low MW lignin could be condensed and precipitated out from the liquor in the hydrothermal pretreatment.Several studies focused on the determination of the residual lignin in the solid substrate, and it is widely accepted that the β-O-4 linkage cleaved and thus promoted lignin dissolution. 4,9 arbonium ions formed under acidic conditions are the key intermediates that initiated lignin depolymerization or condensation; both the depolymerization and condensation reactions occurred more or less simultaneously. 10The cleavage of β-O-4 linkage might result into lignin fractionation and therefore may be responsible for the decrease of the lignin content of the solid substrate; the condensation reaction between the soluble lignin fragments could result in the lignin precipitating from the hydrolysate and account for the later increase of lignin content.It was found from the hydrothermal pretreatment of wood meal that the extracted lignin after pretreatment had higher carbon content, but lower hydrogen and oxygen content than the wood lignin.Moreover, the methoxyl content was also found to be lower than the raw lignin.The condensation reaction between the lignin fragments might be responsible for these differences. 11The reaction severities, such as acidity, could shift the reaction dominance from one mechanism to another.
Using 2-naphthol may effectively inhibit lignin repolymerization and had been suggested for dissolving pulp production. 10,12 emicelluloses utilization and value-added product conversion would largely benefit the effectiveness and competitiveness of the entire ORIGINAL PAPER bio-fuel production process.However, further utilization of the dissolved sugar degradation product is hindered due to the uncontrollable precipitation of lignin-rich compounds during handling and processing of the hydrolysate.
In this paper, lignin depolymerization and condensation reactions, and their effects on the physical characteristics of the hydrolysate during hydrothermal pretreatment were investigated.The goal was to gain more understanding on the lignin reactions and its dissolution and precipitation.

Isolation of dissolved lignin fragments from hydrolysate
Hydrothermal pretreatments were conducted in an oil-bathed digester at 170 ℃ and a solid to liquid ratio 1:3.The detailed process conditions were the same as described in the literature. 13,14 fter the hydrothermal pretreatment, the hydrolysate was collected by filtering the content with a 200 mesh bag.] The lignin was washed with deionized water three times to remove soluble oligosaccharides.The lignin precipitate was then freeze-dried for further analyses.The hydrolysate solutions were labeled as S-1, S-2, S-3, S-4 and S-5, and the lignin fractions from the hydrolysates were labeled as L-1, L-2, L-3, L-4 and L-5, to represent the pretreatment time of 10, 30, 90, 150 and 240 min respectively.The lignin yield was measured in grams contained per liters of hydrolysate (g/L).

Characterization of hydrolysate
The pH of the hydrolysate was directly measured with a pH meter.The solids content of the hydrolysate (%) was determined by drying it at 105 ℃ to constant weight (>6 h).
A turbidity meter (QZ201) was used to determine the turbidity of the hydrolysate.Prior to the test, the hydrolysate solution was diluted 10 times (10X) with water.After the lignin was precipitated out by acidification at pH=2, the turbidity of the hydrolysate solution was measured again.
The average particle size in the hydrolysate solution was determined by a laser particle size analyzer (Zetasizer Nano-ZS, Malvern, England), while the morphology of the particles was observed by TEM (Transmission Electron Microscopy, JEM1010, Japan).

Characterization of dissolved lignin
Molecular weight of the dissolved lignin was determined by GPC (gel permeation chromatography).The lignin was first acetylated with acetic anhydride/pyridine to improve its solubility in THF (tetrahydrofuran) prior to the GPC analyses.The procedure was as follows.150 mg of dried lignin was dissolved in a 15 mL mixture of anhydride/pyridine (anhydride: pridine=2:1, v/v) and stirred at room temperature for 72 h.Thereafter, the acetylated lignin was precipitated out by adding 200 mL diethyl ether, dried under vacuum, and then dissolved in THF at 10 mg/mL concentration.The GPC analyses were carried out on a Water GPC2414 system with 3 analytical columns in series (Waters Styrage HR2, Waters Styrage HR3 and Waters Styrage HR4), and a refractive index detector (Water 2414, RI).The mobile phase was THF, with a flow rate of 1.0 mL/min at 40 ℃.The sample solution was filtered through a 0.45 μm membrane, with polystyrene as the calibration standard.
FT-IR (Fourier-transform infrared) spectra of the lignin samples were obtained with a Thermo Nicolet Avatar 380 FT-IR.The samples were palletized with KBr powder, and the spectrum was recorded in the range of 4000-500 cm -1 .
The 1 H-NMR and 13C-NMR spectra were acquired with a Bruker Advance III 500 MHz spectrometer operating at a frequency of 100.6 MHz.Prior to NMR analysis, the lignin samples (~70 mg) were dissolved in deuterated dimethyl sulfoxide (DMSO-d 6 0.5 mL) at 50 ℃ with ultrasonic treatment.

Lignin extraction
Carbohydrate oligomers and lignin are the major components dissolved in the process.It was observed in Table 1 that the solid content of the hydrolysate increased in the initial 90 min and then decreased as the pretreatment continued.Carbohydrate and lignin degradation and dissolution gave rise to the sharp increase of solid content.However, as the hydrothermal pretreatment continued, the solid content started to decrease, probably due to lignin precipitation and adsorption on bamboo chips and production of volatile degradation products (e.g.furfural and hydroxymethyl furfural).These results are different from those reported in a previous study, in which the lignin content was found to decrease first and increase. 13he increase of lignin dissolution may be attributed to the diffusion and depolymerization of lignin in the hydrothermal pre-treatment, and the decrease of lignin dissolution may be explained by lignin condensation and precipitation.It has been reported that the lignin dissolved in the hydrolysate exhibited extremely high reactivity towards acid-catalyzed condensation reactions, which inevitably led to formation of sticky precipitates during the subsequent pretreatment under the prevailing acid conditions. 6

Physical characteristics of the hydrolysate
The physical characteristics of the hydrolysate are more dependent on the components in the solution.As the hydrothermal pre-treatment proceeded, the organic acid released from hemicelluloses degradation lowered the pH, as shown in Table 1.The degradation products, such as furfural, hydroxymethyl furfural and phenols caused the color of the hydrolysate, as shown in Fig. 1.

Fig. 1 Photographs of the hydrolysate before (S) and after acidification (aS)
As shown in Table 1, turbidity of the hydrolysate initially increased and subsequently decreased to a low level.The same trend was found for the extraction yields for lignin and hemicelluloses.It is believed that suspended substances and colloidal particles were the main factors affecting the turbidity.As mentioned above, lignin and its derivatives were the main colloidal particles which were negatively charged and could be precipitated by acidification.In addition, the insoluble long chain hemicelluloses might also contribute to the turbidity.Part of the long chain hemicelluloses fractions might be suspended as colloidal particles in the solution, 14 contributing to the turbidity.
While lignin colloids were negatively charged, they could be flocculated with the addition of acid and cationic polymer.After most of the lignin was precipitated out by acidification, the turbidity decreased substantially (Table 1 and Fig. 1), except for the hydrolysate sample of S-2.The hydrolysate had a high turbidity even after the removal lignin by acidification.This may be due to the presence of long chain hemicelluloses in the hydrolysate, which is supported by that fact that the hemicelluloses from the S-2 hydrolysate had more insoluble hemicelluloses fractions.This is also in agreement with a previous study that demonstrated the presence of high MW hemicelluloses in the hydrolysate. 14herefore, it is clear that the turbidity of the hydrolysate was determined by both the lignin fragments and the insoluble long chain hemicelluloses.
The particle size of the hydrolysate was determined by a laser particle size analyzer, and the average particle size was found to be around 500 nm.The shape of the particles was further observed by TEM (Fig. 2).Some irregular particles were observed in the initial pretreatment samples; whereas the hydrolysates from the later stage of pretreatment had round shape particles.As the hydrothermal pre-treatment progressed, carbohydrate degradation and peeling off from LCC linkage and render the lignin more hydrophobic.In water of high temperature, the hydrophobic lignin tends to form sphere shapes to minimize the surface area.However, when the pretreatment time exceeded 240 min, the spheres disappeared almost completely.So another possibility was that the spheres were pseudo-lignin from carbohydrate degradation which degraded and dissolved completely in the extended hydrothermal treatment. 9Fig. 2 TEM micrographs of the hydrolysate (a: S-1; b: S-2; c1, c2: S-4)

M W of lignin isolated from the hydrolysate
The MW results were listed in Table 2.The Mw of the lignin fragments ranged from 3342 to 5611 g/mol, the lignin samples corresponding to the initial 10 min had the highest value, 5611 g/mol.Afterward, the cleavage of the lignin units decreased the M W . Whereas, condensation reaction occurred between the soluble lignin fragments at higher reaction severity, leading eventually to the formation of insoluble lignin and leaving low M W lignin in the hydrolysate.The Mw and Mn of the lignin fragments (L-4) decreased to 3342 and 2645 g/mol, and PDI decreased to 1.26.Some of the dissolved lignin had a great propensity to settle out during cooling or storage.As the charge of the dissolved lignin decreased, its stability (solubility/dispersibilty) depended largely on the temperature, pH and viscosity of the hydrolysate, as well as the presence of long chain hemicelluloses.

FT-IR Analyses
The finger-print part of the FT-IR spectra of the lignin samples were shown in Fig. 3.The bands at 1605, 1515 and 1425 cm -1 typically corresponding to aromatic ring vibrations of phenyl-propane (C9) skeleton and the C-H deformation combined with aromatic ring vibration at 1460 cm -1 . 20The typical G (guaiacyl) and S (syringyl) bands presented at 1270, 1125, 1325, 1118 and 835 cm -1 (Tejado et al., 2007); meanwhile the peak at 1383 cm -1 exhibited the phenolic hydroxyl group of the lignin fragments.Some of the phenolic hydroxyl groups might originate from the cleavage of the β-O-4 linkage.
Additionally, the occurrence of the bands at 1027 and 1080 cm -1 probably indicated the existence of the polysaccharide in the lignin samples 15 .The gradual decrease of the two bands was generally attributed to the degradation of polysaccharide.This fact might suggest that it was impossible to separate pure lignin from hydrolysate by acidification; some hemicelluloses or LCC could precipitate together with lignin.The relative intensities of the bands at 1605, 1460, 1425 and 1027 cm -1 to band at 1510 cm -1 changed frequently responding to the four samples referring the progressive structural changes.The L-1 and L-2 samples showed the bonds at 1655 cm -1 was assigned to the conjugated carbonyl groups of the lignin.As the condensation reaction proceeded, the conjugated carbonyl groups of residual lignin fragments decreased.
The FT-IR spectrum analyses suggested that lignin fragments had the structures of G and S units; some hemicelluloses fractions or LCC were presented in the lignin samples.The conjugated carbonyl groups of the residual lignin decreased, indicating the condensation reactions of lignin in the hydrothermal pre-treatment.

NMR analyses
The 1 H NMR spectra of the lignin fractions L-3 and L-4 are shown in Fig. 4a.The signals between 6.0 and 8.0 ppm belonged to aromatic protons in lignin units, whereas those between 0.8 and 1.5 ppm are assigned to the aliphatic moiety in the lignin. 16Specifically, the signals at ~7.1 and ~6.7 ppm are attributed to aromatic protons in the G and S units, respectively.The absence of the signal at ~7.6 ppm revealed that dissolved lignin contained little to no H (p-hydroxy phenyl) units.Meanwhile it was visible that the signal at 6.7 ppm was more pronounced than that at 7.1 ppm, suggesting that there were more protons from the S units than from the G units.The decrease signal at 4.9 ppm of the L-4 revealed the degradation of hemicelluloses and little amount of hemicelluloses were connected to lignin.Protons in -OCH 3 gave two strong signals at 3.7 and 3.4 ppm, respectively.The strong signals at 2.4 and 1.90 ppm were attributed to protons in the acetyls connected to the benzene ring and the aliphatic side chains, respectively.The relative intensity of the signal at 2.4 ppm to the signal at 1.9 ppm increased substantially, further suggesting the cleavage of linkage of sugar unit.As observed in Fig. 4b, the G and S units in the lignin fragments could be identified by the peaks in the regions δ125-110 and δ109-103 ppm, respectively. 17The relative increase of the signal at 148.4 ppm (G/C 3 ) and decrease of the signal at 152.4 ppm (ES/C 3,5 , esterify S) might probably be accounted for through the methoxyl reaction in the ES units; 15 namely, the ES units were converted in G units because of the subtraction of methoxyl under acid medium.Lignin with ES unit had a great tendency to condense with each other and precipitate out, leaving dissolved lignin with more G units.
Weak signals which appeared at 75.5, 74.0, 63.1, 62.9 and 61.5 ppm were assigned to the D-xylose unit, indicating the presence of hemicelluloses.The NMR results support the conclusions from FT-IR analyses, showing that the G and S units were the primary structure of the dissolved lignin, and a small amount of hemicelluloses was presented in the lignin samples.Condensation pathways dominated the lignin reactions in the later stage of the hydrothermal pretreatment, and drastically changed the structure of the dissolved lignin.

Fig. 3
Fig.3FT-IR spectra of the lignin isolated from thehydrolysate

Table 1
Characteristics of the hydrolysate solutions

Table 2
Molecular weight of the lignin from the hydrolysate