Biogas potential of organosolv pretreated wheat straw as mono and co-substrate: substrate synergy and microbial dynamics.

Anaerobic digestion (AD) technology can potentially address the gap between energy demand and supply playing a crucial role in the production of sustainable energy from utilization of biogenic waste materials as feedstock. The biogas production from anaerobic digestion is primarily influenced by the chemical compositions and biodegradability of the feedstock. Organosolv-steam explosion offers a constructive approach as a promising pretreatment method for the fractionation of lignocellulosic biomasses delivering high cellulose content.This study showed how synergetic co-digestion serves to overcome the challenges of mono-digestion's low efficiency. Particularly, the study evaluated the digestibility of organosolv-steam pretreated wheat straw (WSOSOL) in mono as well as co-digesting substrate with cheese whey (CW) and brewery spent grains (BSG). The highest methane yield was attained with co-digestion of WSOSOL + CW (338 mL/gVS) representing an enhanced biogas output of 1-1.15 times greater than its mono digestion. An ammonium production was favored under co-digestion strategy accounting for 921 mg/L from WSOSOL + BSG. Metagenomic study was conducted to determine the predominant bacteria and archaea, as well as its variations in their populations and their functional contributions during the AD process. The Firmicutes have been identified as playing a significant role in the hydrolysis process and the initial stages of AD. An enrichment of the most prevalent archaea genera enriched were Methanobacterium, Methanothrix, and Methanosarsina. Reactors digesting simpler substrate CW followed the acetoclastic, while digesting more complex substrates like BSG and WSOSOL followed the hydrogenotrophic pathway for biomethane production. To regulate the process for an enhanced AD process to maximize CH4, a comprehensive understanding of microbial communities is beneficial.

Combination of alkaline biodiesel-derived crude glycerol pretreated corn stover with dilute acid pretreated water hyacinth for highly-efficient single cell oil production by oleaginous yeast Cutaneotrichosporon oleaginosum.

Single cell oil (SCO) prepared from biodiesel-derived crude glycerol (BCG) and lignocellulosic biomass (LCB) via oleaginous yeasts is an intriguing alternative precursor of biodiesel. Here, a novel strategy combining alkaline BCG pretreated corn stover and dilute acid pretreated water hyacinth for SCO overproduction was developed. The mixed pretreatment liquors (MPLs) were naturally neutralized and adjusted to a proper carbon-to-nitrogen ratio beneficial for SCO overproduction by Cutaneotrichosporon oleaginosum. The toxicity of inhibitors was relieved by dilution detoxification. The enzymatic hydrolysate of solid fractions was suitable for SCO production either separately or simultaneously with MPLs. Fed-batch fermentation of the MPLs resulted in high cell mass, SCO content, and SCO titer of 80.7 g/L, 75.7 %, and 61.1 g/L, respectively. The fatty acid profiles of SCOs implied high-quality biodiesel characteristics. This study offers a novel BCG&LCB-to-SCO route integrating BCG-based pretreatment and BCG/LCB hydrolysates co-utilization, which provides a cost-effective technical route for micro-biodiesel production.

Polymer properties of softwood organosolv lignins produced in two different reactor systems.

Lignin, the second most abundant biopolymer on earth and with a predominantly aromatic structure, has the potential to be a raw material for valuable chemicals and other bio-based chemicals. In industry, lignin is underutilized by being used mostly as a fuel for producing thermal energy. Valorization of lignin requires knowledge of the structure and different linkages in the isolated lignin, making the study of structure of lignin important. In this article, lignin samples isolated from two types of reactors (autoclave reactor and displacement reactor) were analyzed by FT-IR, size exclusion chromatography, thermogravimetric analysis (TGA), and Py-GC-MS. The average molecular mass of the organosolv lignins isolated from the autoclave reactor decreased at higher severities, and FT-IR showed an increase in free phenolic content with increasing severity. Except for molecular mass and molecular mass dispersity, there were only minor differences between lignins isolated from the autoclave reactor and lignins isolated from the displacement reactor. Carbohydrate analysis, Py-GC-MS and TGA showed that the lignin isolated using either of the reactor systems is of high purity, suggesting that organosolv lignin is a good candidate for valorization.

Biovalorization of brewer's spent grain as single-cell protein through coupling organosolv pretreatment and fungal cultivation.

Brewer's spent grain (BSG) is a clean byproduct from the food sector, comprising 85% of the brewing process solid byproducts. BSG is mainly used as low-quality animal feed and often ends up in landfills due to its short shelf life. However, considering its abundant availability and high nutritional content, BSG holds the potential for biorefineries to produce valuable products. The recalcitrant nature of BSG poses a challenge, requiring pretreatment steps. Therefore, this study focused on valorizing BSG obtained from organosolv pretreatment by producing food- and feed-grade single-cell protein (SCP). The BSG was subject to organosolv pretreatment at 180C for 2 h with 50% v/v ethanol as solvent. Filamentous fungi N. intermedia and A. oryzae were cultivated on as-received and different fractions of organosolv-treated BSG to evaluate the effect of factors such as pretreatment, fungal strain, pretreated fraction content, and substrate loading on fungal biomass yield, biomass composition (protein content), and metabolite production. A. oryzae cultivation on all tested substrates yielded 7%-40% more biomass than N. intermedia. Cultivating A. oryzae on organosolv liquor resulted in the highest biomass protein content (44.8% ± 0.7%) with a fungal biomass concentration of 5.1 g/L. A three-fold increase in the substrate loading increased the ethanol-to-substrate yield by 50%, while protein content was decreased by 23%. Finally, a biorefinery concept was proposed to integrate the organosolv pretreatment of BSG with fungal cultivation for maximum yield of SCP while obtaining other products such as lignin and ethanol, providing a sustainable rout for managing BSG.

Modified 1,4-butanediol organosolv pretreatment on hardwoodandsoftwood for efficient coproduction of fermentable sugars and lignin antioxidants.

In this work, 1,4-butanediol (BDO) organosolv pretreatment was modified with different additives for efficient coproduction of fermentable sugars and lignin antioxidants from hardwood poplar and softwood masson pine. It was found additives more greatly improved pretreatment efficacy of softwood than hardwood. 3-hydroxy-2-naphthoic acid (HNA) addition introduced hydrophilic acid groups to lignin structure, while 2-naphthol-7-sulphonate (NS) addition promoted lignin removal, both improving cellulose accessibility for enzymatic hydrolysis. Consequently, BDO pretreatment with 90 mM acid and 2-naphthol-7-sulphonate addition achieved near complete cellulose hydrolysis (97.98%) and maximized sugar yield of 88.23% from masson pine at 2% cellulose and 20 FPU/g enzyme loading. More importantly, the recovered lignin possessed great antioxidant activity (RSI = 2.48), due to increased phenolic OH groups, reduced aliphatic OH groups and molecular weight. Results indicated the modified BDO pretreatment could significantly improve enzymatic saccharification of highly-recalcitrant softwood, while enabling coproduction of high-performance lignin antioxidants for full biomass utilization.

Optimization of lignin extraction from bamboo by ultrasound-assisted organosolv pretreatment.

For a sustainable biorefinery, reduction in the recalcitrance of lignocellulosic biomass is very crucial for the efficient utilization of each fraction. The present work investigated an integrated pretreatment method to recover high-quality lignin along with the cellulose-rich pulp. An optimization study employing response surface methodology investigated the synergistic effects of ultrasound and organosolv pretreatment from Bambusa tulda (bamboo). The optimal condition (180 °C, 55 min, and 30 min sonication) resulted in 65.81 ± 2.40% of lignin yield with 95.37 ± 1.17% purity. A reduction in 7.85% yield and 1.54% purity of lignin with organosolv pretreatment highlighted the efficacy of sonication in lignin extraction. Ultrasound resulted in homolytic cleavage of the lignin-carbohydrate bond that enhanced delignification and increase the cellulose crystallinity. NMR, FTIR, GPC, and TGA of lignin suggested the superiority of sonication in maintaining lignin quality. A significant amount of ß-O-4 linkages in extracted lignin is favorable for its subsequent valorization.

Comparative study of acid- and alkali-catalyzed 1,4-butanediol pretreatment for co-production of fermentable sugars and value-added lignin compounds.

BACKGROUND: Organosolv pretreatment is one of the most efficient methods for delignification and boosting biomass saccharification. As compared to typical ethanol organosolv pretreatments, 1,4-butanediol (BDO) organosolv pretreatment is a high-boiling-point solvent pretreatment, which can generate low pressure in the reactor during high temperature cooking that improves the operation safety. Although several studies showed that organosolv pretreatment can lead to effective delignification and enhancement in glucan hydrolysis, there has been no studies on acid- and alkali-catalyzed BDO pretreatment, as well as their comparison on promoting biomass saccharification and lignin utilization. RESULTS: It was shown that BDO organosolv pretreatment was more effective in removing lignin from poplar as compared with typical ethanol organosolv pretreatment under the same pretreatment conditions. HCl-BDO pretreatment with 40 mM acid loading led to 82.04% of original lignin removed from biomass, as compared to the lignin removal of 59.66% in HCl-Ethanol pretreatment. Besides, acid-catalyzed BDO pretreatment was more effective in improving the enzymatic digestibility of poplar than alkali-catalyzed BDO pretreatment. As a result, HCl-BDO with acid loading of 40 mM provided a good enzymatic digestibility of cellulose (91.16%) and the maximum sugar yield of 79.41% from original woody biomass. The linear correlations between physicochemical structure (e.g., fiber swelling, cellulose crystallinity, crystallite size, surface lignin coverage and cellulose accessibility) changes of BDO pretreated poplar and enzymatic hydrolysis were plotted to figure out the main factors that influenced biomass saccharification. Moreover, acid-catalyzed BDO pretreatment mainly brought about the phenolic hydroxyl (PhOH) groups formation in lignin structure, while alkali-catalyzed BDO pretreatment mostly led to the lower molecular weight of lignin. CONCLUSIONS: Results indicated that the acid-catalyzed BDO organosolv pretreatment could significantly improve enzymatic digestibility of the highly recalcitrant woody biomass. The great enzymatic hydrolysis of glucan resulted from increased cellulose accessibility, which mostly associated with the higher degree of delignification and hemicellulose solubilization, as well as the more increase in fiber swelling. Besides, lignin was recovered from the organic solvent, which could be used as natural antioxidants. The formation of phenolic hydroxyl groups in lignin structure and the lower molecular weight of lignin contributed to its greater radical scavenging capacity.

Heterotrophic Cultivation of the Cyanobacterium Pseudanabaena sp. on Forest Biomass Hydrolysates toward Sustainable Biodiesel Production.

Environmental pollution, greenhouse gas emissions, depletion of fossil fuels, and a growing population have sparked a search for new and renewable energy sources such as biodiesel. The use of waste or residues as substrates for microbial growth can favor the implementation of a biorefinery concept with reduced environmental footprint. Cyanobacteria constitute microorganisms with enhanced ability to use industrial effluents, wastewaters, forest residues for growth, and concomitant production of added-value compounds. In this study, a recently isolated cyanobacterium strain of Pseudanabaena sp. was cultivated on hydrolysates from pretreated forest biomass (silver birch and Norway spruce), and the production of biodiesel-grade lipids was assessed. Optimizing carbon source concentration and the (C/N) carbon-to-nitrogen ratio resulted in 66.45% w/w lipid content when microalgae were grown on glucose, compared to 62.95% and 63.79% w/w when grown on spruce and birch hydrolysate, respectively. Importantly, the lipid profile was suitable for the production of high-quality biodiesel. The present study demonstrates how this new cyanobacterial strain could be used as a biofactory, converting residual resources into green biofuel.

A new l-cysteine-assisted glycerol organosolv pretreatment for improved enzymatic hydrolysis of corn stover.

Deconstruction of lignocellulose via efficient pretreatment is crucial for producing fermentable sugars. In this study, effects of glycerol organosolv pretreatment (GOP) on main chemical composition of corn stover were investigated. Results indicate that the residual corn stover after 80 wt% glycerol pretreatment (at 220 °C for 0.5 h) yielded 75.97 % glucose and 78.21 % xylose after enzymatic hydrolysis, which were enhanced by 3.39- and 6.08-fold compared to the untreated corn stover. Subsequently, an l-cysteine-assisted GOP was proposed with higher yields of glucose (86.20 %) and xylose (91.13 %). When pretreating corn stover with 80 wt% glycerol containing 0.07 wt% l-cysteine at 220 °C for 0.5 h, higher fermentable sugars of 26.08 g were produced from 100 g feedstock after enzymolysis. Intrinsic mechanisms of the proposed pretreatment for enhancing enzymatic digestibility were elucidated by physiochemical characterization technologies and techno-economic analysis was also studied. This study provides guidance for fermentable sugars production from renewable lignocellulose.

Aqueous ethanol organosolv process for the valorization of Brewer's spent grain (BSG).

Brewers spent grain (BSG), the main solid byproduct of brewing, is annually generated by ca 37 million tons worldwide, which due to limited application, mostly ends up in landfills. This study aims to separate BSG's fractions (lignin, cellulose, and hemicellulose) by ethanol organosolv pretreatment. Lignin-rich fractions were recovered using a two-step separation technique. The effects of temperature, retention time, and ethanol concentration on the quantity and quality of fractions were studied. The temperature considerably impacted the quality and quantity of obtained fractions, while other parameter effects greatly depended on the temperature. Substantial hemicellulose removal (90 %) along with lignin removal (56 %) and recovery (57 %) were obtained at 180 °C. The highest lignin purity (95 %) was obtained at the pretreatment conditions of 180 °C, 120 min, and 50 % ethanol concentration. This work provides an alternative route for BSG utilization, mitigating its environmental impact while enhancing the economy of a brewery.

Morphology of lignin structures on fiber surfaces after organosolv pretreatment.

The redeposition of lignin to the fiber surface after organosolv pretreatment was studied using two different reactor types. Results from the conventional autoclave reactor suggest that redeposition occurs during the cooling down stage. Redeposited particles appeared to be spherical in shape. The size and population density of the particles depends on the concentration of organosolv lignin in the cooking liquor, which is consistent with the hypothesis that reprecipitation of lignin occurs when the system is cooled down. The use of a displacement reactor showed that displacing the spent cooking liquor with fresh cooking liquor helps in reducing the redeposition and the inclusion of a washing stage with fresh cooking liquor reduced the reprecipitation of lignin, particularly on the outer fiber surfaces. Redeposition of lignin was still observed on regions that were less accessible to washing liquid, such as fiber lumens, suggesting that complete prevention of redeposition was not achieved.

Fed-batch anaerobic digestion of raw and pretreated hazelnut skin over long-term operation.

This study provided important insights on the anaerobic digestion (AD) of hazelnut skin (HS) by operating a fed-batch AD reactor over 240 days and focusing on several factors impacting the process in the long term. An efficient reactor configuration was proposed to increase the substrate load while reducing the solid retention time during the fed-batch AD of HS. Raw HS produced maximally 19.29 mL CH4/g VSadd/d. Polyphenols accumulated in the reactor and the use of NaOH to adjust the pH likely inhibited AD. Maceration and methanol-organosolv pretreatments were, thus, used to remove polyphenols from HS (i.e. 82 and 97%, respectively) and improve HS biodegradation. Additionally, organosolv pretreatment removed 9% of the lignin. The organosolv-pretreated HS showed an increment in methane potential of 21%, while macerated HS produced less methane than the raw substrate, probably due to the loss of non-structural sugars during maceration.

Comparative study on the hydrogenolysis performance of solid residues from different bamboo pretreatments.

Both alkaline organosolv and formaldehyde stabilization pretreatment can yield high-quality lignin by preventing condensation. For the hydrogenolysis of the pretreated solid residues, the highest yield of C2-C4 chemicals was 66.8% under alkaline organosolv pretreatment for 60 min. Specifically, the crimped fibers and residual lignin and hemicellulose increased the surface roughness of the residue by 40.6%, the crystallinity index decreased to 44.4%, and the crystal size was reduced to 2.15 nm, which in turn promoted hydrogenolysis of the residue. However, the increase of crystallinity and crystal size and the decrease in surface roughness of the formaldehyde stabilization pretreatment residue greatly hindered the conversion of polysaccharides. In addition, residual formaldehyde on the residue may also inhibit catalyst activity. Overall, this study provides novel perspectives on the full utilization of biomass, as well as new insights into the conversion of polysaccharides.

Sustainable bioconversion of potato peel wastes into ethanol and biogas using organosolv pretreatment.

Potato processing industries generate considerable amounts of residues, i.e., potato peel wastes (PPW). Valorization of PPW for bioethanol and biogas production via a biorefining process was investigated in this study. Organosolv pretreatment was performed on the PPW using 50-75% (v/v) ethanol solution at 120-180 °C with/without the presence of 1% (w/w) H2SO4 (as a catalyst). After the pretreatment, the solvent, i.e., ethanol, was recovered by distillation. Catalyzed organosolv pretreatment using 50% (v/v) ethanol at 120 °C followed by enzymatic hydrolysis resulted in a high hydrolysate yield of 539.8 g glucose/kg dry PPW that was successfully fermented to 224.2 g ethanol/kg dry PPW. To recover more energy, the liquid fraction of the pretreatment remained after solvent recovery and the unhydrolyzed solids that remained from the enzymatic hydrolysis were anaerobically digested. From each kg of dry PPW, the anaerobic digestion produced 57.9 L biomethane. Thus, the biorefinery comprising ethanolic organosolv pretreatment, solvent recovery, enzymatic hydrolysis, ethanolic fermentation, and anaerobic digestion of residues was produced 8112 kJ energy per kg of dry PPW.

Glycerol organosolv pretreatment can unlock lignocellulosic biomass for production of fermentable sugars: Present situation and challenges.

The complex structure of lignocellulosic biomass forms the recalcitrance to prevent the embedded holo-cellulosic sugars from undergoing the biodegradation. Therefore, a pretreatment is often required for an efficient enzymatic lignocellulosic hydrolysis. Recently, glycerol organosolv (GO) pretreatment is revealed potent in selective deconstruction of various lignocellulosic biomass and effective improvement of enzymatic hydrolysis. Evidently, the GO pretreatment is capable to modify the structure of dissolved components by glycerolysis, i.e., by trans-glycosylation onto glyceryl glycosides and by hydroxylation grafting onto glyceryl lignin. Such modifications tend to protect these main components against excessive degradation, which can be mainly responsible for the obviously less fermentation inhibitors arising in the GO pretreatment. This pretreatment can provide opportunities for valorization of emerging lignocellulosic biorefinery with production of value-added biochemicals. Recent advances in GO pretreatment of lignocellulosic biomass followed by enzymatic hydrolysis are reviewed, and perspectives are made for addressing remaining challenges.

Organosolv Fractionation of Birch Sawdust: Establishing a Lignin-First Biorefinery.

The use of residual biomass for bioconversions makes it possible to decrease the output of fossil-based chemicals and pursue a greener economy. While the use of lignocellulosic material as sustainable feedstock has been tried at pilot scale, industrial production is not yet economically feasible, requiring further technology and feedstock optimization. The aim of this study was to examine the feasibility of replacing woodchips with residual sawdust in biorefinery applications. Woodchips can be used in value-added processes such as paper pulp production, whereas sawdust is currently used mainly for combustion. The main advantages of sawdust are its large supply and a particle size sufficiently small for the pretreatment process. Whereas, the main challenge is the higher complexity of the lignocellulosic biomass, as it can contain small amounts of bark and cambium. Here, we studied the fractionation of birch sawdust by organosolv pretreatment at two different temperatures and for two different durations. We evaluated the efficiency of fractionation into the three main fractions: lignin, cellulose, and hemicellulose. The cellulose content in pretreated biomass was as high as 69.2%, which was nearly double the amount in untreated biomass. The obtained lignin was of high purity, with a maximum 4.5% of contaminating sugars. Subsequent evaluation of the susceptibility of pretreated solids to enzymatic saccharification revealed glucose yields ranging from 75% to 90% after 48 h but reaching 100.0% under the best conditions. In summary, birch sawdust can be successfully utilized as a feedstock for organosolv fractionation and replace woodchips to simplify and lower the costs of biorefinery processes.

Efficiency enhancement of a new cellulase cocktail at low enzyme loading for high solid digestion of alkali catalyzed atmospheric glycerol organosolvent pre-treated sugarcane bagasse.

The acquisition during biomass saccharification of elevated levels of fermentable sugars with lower cellulase concentration is central to ensuring an economically viable and industrially relevant hydrolytic process. Thus, using a new cellulase preparation (LT4) at low cellulase loading (2 mg protein/g dried substrate), this study assessed the possible boosting effect of integrating accessory enzymes and additives on high-solids hydrolysis of sugarcane bagasse via fed-batch feeding. Hydrolysis which commenced with initial 8% solids loading and subsequent substrate feeding of 4% solids at 6 h, 18 h, and 24 h respectively, proved optimal for the 20% high-solids saccharification producing 158 g/L total sugars and 83% glucose yield after 72 h with the combined optimized additives and accessory enzymes. The results obtained indicate that the integration of accessory enzymes and additives offers a benignant approach to minimizing the enzyme load and cost of high solids saccharification of lignocellulosic heteropolymers while also boosting enzyme hydrolytic performance.

Organosolv pretreatment assisted by carbocation scavenger to mitigate surface barrier effect of lignin for improving biomass saccharification and utilization.

BACKGROUND: Ethanol organosolv (EOS) pretreatment is one of the most efficient methods for boosting biomass saccharification as it can achieve an efficient fractionation of three major constituents in lignocellulose. However, lignin repolymerization often occurs in acid EOS pretreatment, which impairs subsequent enzymatic hydrolysis. This study investigated acid EOS pretreatment assisted by carbocation scavenger (2-naphthol, 2-naphthol-7-sulfonate, mannitol and syringic acid) to improve biomass fractionation, coproduction of fermentable sugars and lignin adsorbents. In addition, surface barrier effect of lignin on cellulose hydrolysis was isolated from unproductive binding effect of lignin, and the analyses of surface chemistry, surface morphology and surface area were carried out to reveal the lignin inhibition mitigating effect of various additives. RESULTS: Four different additives all helped mitigate lignin inhibition on cellulose hydrolysis in particular diminishing surface barrier effect, among which 2-naphthol-7-sulfonate showed the best performance in improving pretreatment efficacy, while mannitol and syringic acid could serve as novel green additives. Through the addition of 2-naphthol-7-sulfonate, selective lignin removal was increased up to 76%, while cellulose hydrolysis yield was improved by 85%. As a result, 35.78 kg cellulose and 16.63 kg hemicellulose from 100 kg poplar could be released and recovered as fermentable sugars, corresponding to a sugar yield of 78%. Moreover, 22.56 kg ethanol organosolv lignin and 17.53 kg enzymatic hydrolysis residue could be recovered as lignin adsorbents for textile dye removal, with the adsorption capacities of 45.87 and 103.09 mg g-1, respectively. CONCLUSIONS: Results in this work indicated proper additives could give rise to the form of less repolymerized surface lignin, which would decrease the unproductive binding of cellulase enzymes to surface lignin. Besides, the supplementation of additives (NS, MT and SA) resulted in a simultaneously increased surface area and decreased lignin coverage. All these factors contributed to the diminished surface barrier effect of lignin, thereby improving the ease of enzymatic hydrolysis of cellulose. The biorefinery process based on acidic EOS pretreatment assisted by carbocation scavenger was proved to enable the coproduction of fermentable sugars and lignin adsorbents, allowing the holistic utilization of lignocellulosic biomass for a sustainable biorefinery.

Organosolv Pretreatment of Sorghum Stalks Using Glycerol.

Glycerol is a promising low-cost solvent for biomass pretreatment since a large amount of glycerol is generated as a by-product in the biodiesel industry. Pretreatment is a method of disintegration of the recalcitrant structure of biomass to enhance the accessibility of cellulose and hemicelluloses to enzymes for complete saccharification. During pretreatment, glycerol breaks the lignin carbohydrate complex and selectively solubilizes lignin. Thus, the glycerol pretreatment improves the accessibility of cellulose to cellulases leading to higher sugar yields. The glycerol pretreatment is carried out at high temperature (>190 °C) to disintegrate the structure of biomass. The glycerol pretreatment in the presence of acid or base catalyst such as H2SO4 or NaOH results in lower pretreatment temperature and higher glucan hydrolysis. This chapter describes the methodology to carry out glycerol pretreatment of sorghum biomass with or without acid/alkali as catalyst and the basic calculations to determine the efficiency of the pretreatment.

The effects of mild Lewis acids-catalyzed ethanol pretreatment on the structural variations of lignin and cellulose conversion in balsa wood.

Ethanol organosolv pretreatment is a green and effective deconstruction process for main components in lignocellulose biomass. Herein, balsa wood was firstly subjected to a modified ethanol/water solution (EWS) pretreatment with different Lewis acids catalysts (AlCl3, CuCl2, FeCl3) at 140-180 °C. The delignification ratios and structural characteristics of the dissociated lignin, enzymatic hydrolysis of cellulose in the pretreated substrates as well as the degradation products from hemicellulose during the pretreatment process were comprehensively investigated. Results showed that dissociation and depolymerization of lignin fragments was robust in AlCl3-catalyzed pretreatment than those by CuCl2 and FeCl3-catalyzed pretreatment. In detail, the results showed that the optimal delignification ratio and removal of the hemicelluloses occurred in AlCl3-catalyzed pretreatment. Moreover, the structural characterizations of lignin fractions by 2D-HSQC, 31P NMR and GPC also revealed that the obtained lignin has the advantages of small and homogeneous molecules as well as abundant functional groups. As a result of adequate removal of hemicellulose and lignin, the enzymatic digestibility of cellulose in the pretreated residue was significantly elevated. In short, the above findings are also in line with the concept of maximizing the utilization of bioresources, which will be beneficial for value-added applications of balsa wood in the biorefinery.