Enzymatic Saccharification Of Cellulose Research Articles

Heterologous expression and characterization of novel GH12 β-glucanase and AA10 lytic polysaccharide monooxygenase from Streptomyces megaspores and their synergistic action in cellulose saccharification

BackgroundThe combination of cellulase and lytic polysaccharide monooxygenase (LPMO) is known to boost enzymatic saccharification of cellulose. Although the synergy between cellulases (GH5, 6 or 7) and LPMOs (AA9) has been extensively studied, the interplay between other glycoside hydrolase and LPMO families remains poorly understood.ResultsIn this study, two cellulolytic enzyme-encoding genes SmBglu12A and SmLpmo10A from Streptomyces megaspores were identified and heterologously expressed in Escherichia coli. The recombinant SmBglu12A is a non-typical endo-β-1,4-glucanase that preferentially hydrolyzed β-1,3-1,4-glucans and slightly hydrolyzed β-1,4-glucans and belongs to GH12 family. The recombinant SmLpmo10A belongs to a C1-oxidizing cellulose-active LPMO that catalyzed the oxidation of phosphoric acid swollen cellulose to produce celloaldonic acids. Moreover, individual SmBglu12A and SmLpmo10A were both active on barley β-1,3-1,4-glucan, lichenan, sodium carboxymethyl cellulose, phosphoric acid swollen cellulose, as well as Avicel. Furthermore, the combination of SmBglu12A and SmLpmo10A enhanced enzymatic saccharification of phosphoric acid swollen cellulose by improving the native and oxidized cello-oligosaccharides yields.ConclusionsThese results proved for the first time that the AA10 LPMO was able to boost the catalytic efficiency of GH12 glycoside hydrolases on cellulosic substrates, providing another novel combination of glycoside hydrolase and LPMO for cellulose enzymatic saccharification.

Using citric acid to suppress lignin repolymerization in the organosolv pretreatment of corn stalk

The esterification reactions that occur between citric acid (CA) and lignin on hydroxyl groups of side chains can inhibit the formation of reactive intermediates in acid-catalyzed pretreatments. The effects of CA on dilute hydrochloric acid (1.0%) pretreatments of corn stalk in hydrated 1,4–dioxane were investigated with sodium hypophosphite (0.5%) as the catalyst at 95 °C for 3.5 h. The resulting cellulose recovery (62.5–80.9%) and lignin removal (72.5–85.0%) after adding CA were significantly higher than those in the pretreatment without CA. Water content influenced pretreatment and delignification when CA was added into the treatment systems. The highest yield of total reducing sugar (97.1%) was achieved during the enzymatic hydrolysis of cellulose-rich materials obtained from the pretreatment with 11.4% water content. Meanwhile, β–O–4′ in the extracted lignin increased from 22.4/100Ar to 41.2/100Ar with increasing water content from 11.4% to 25.9%. These findings implied that CA as a stabilization agent could inhibit the repolymerization of degraded lignin fragments due to the esterification between CA and lignin and then improved the enzymatic saccharification of cellulose.

Immobilization of Cellulase on Zinc Oxide Deposited on Zeolite Pellets for Enzymatic Saccharification of Cellulose

The consumption of fossil fuels to fulfill the global energy demand can cause global warming issues. Renewable energy, i.e., bioethanol, from lignocellulosic biomass, is a promising source of alternative energy to fossil fuels. The conversion of lignocellulosic biomass into bioethanol requires the release of fermentable sugars during the saccharification process using cellulase. However, the utilization of this enzyme on an industrial scale is not feasible due to its difficult separation, instability, and high cost. Here, we present a method for cellulase immobilization on functionalized zinc oxide prepared from either zinc nitrate hexahydrate (ZnO(I)) or zinc acetate dihydrate (ZnO(II)) solutions on zeolite (ZEO) pellets. The immobilized cellulase on ZnO-ZEO structures was characterized by scanning electron microscopy, Xray diffraction spectroscopy, and Fourier transform infrared spectroscopy. The immobilization efficiencies of immobilized cellulase either on ZnO(I)-ZEO or ZnO(II)-ZEO were determined as 58.17 ± 0.75% and 55.51 ± 0.81%, respectively. The immobilized cellulase on ZnO-ZEO was capable of catalyzing microcrystalline cellulose breakdown, releasing reducing sugars. The immobilized cellulase on these structures could be recycled up to four repetitive runs. Based on kinetic data, both the Michaelis constants (Km) and maximum reaction velocity (Vmax) of the immobilized cellulase on the ZnO-ZEO structures were lower than those of free cellulase. This suggests that immobilized cellulase has a higher affinity toward the substrate, but a lower reaction rate than the free enzyme.

Comparison of solid and liquid fractions of pretreated Norway spruce as reductants in LPMO-supported saccharification of cellulose.

The role of lignin in enzymatic saccharification of cellulose involving lytic polysaccharide monooxygenase (LPMO) was investigated in experiments with the solid and liquid fractions of pretreated Norway spruce from a biorefinery demonstration plant using hydrothermal pretreatment and impregnation with sulfur dioxide. Pretreated biomass before and after enzymatic saccharification was characterized using HPAEC, HPLC, Py-GC/MS, 2D-HSQC NMR, FTIR, and SEM. Chemical characterization indicated that relatively harsh pretreatment conditions resulted in that the solid phase contained no or very little hemicellulose but considerable amounts of pseudo-lignin, and that the liquid phase contained a relatively high concentration (∼5g/L) of lignin-derived phenolics. As judged from reactions continuously supplied with either air or nitrogen gas, lignin and lignin fragments from both the solid and the liquid phases efficiently served as reductants in LPMO-supported saccharification. When air was used to promote LPMO activity, the enzymatic conversion of cellulose after 72h was 25% higher in reactions with pretreated solids and buffer, and 14% higher in reactions with pretreatment liquid and microcrystalline cellulose. Research in this area is useful for designing efficient saccharification steps in biochemical conversion of lignocellulosic biomass.

Tailored one-pot lignocellulose fractionation to maximize biorefinery toward controllable producing lignin nanoparticles and facilitating enzymatic hydrolysis

To achieve the global bio-based recycling economy with lignocellulosic biomass as feedstock, the additional value must be gained from both carbohydrates and lignin. In this study, a tailored one-pot lignocellulose fractionation, which was driven by ultrafast and recyclable microwave-assisted ternary deep eutectic solvent (DES, choline chloride: ethylene glycol: AlCl3) pretreatment, was developed to significantly reduce the natural “biomass recalcitrance”, promoting the production of lignin nanoparticles and facilitating enzymatic saccharification of cellulose. Confocal Raman microscopy (CRM) was applied to visually monitor the delignification in different cell walls during the DES pretreatments. Under the optimal condition (110 °C, 10 min), the glucose yield of the pretreated poplar via enzymatic saccharification reached 95.4 %, which was 5.8 times higher than that of the control. Advanced NMR characterizations of the DES lignin and control lignin fractions demonstrated that lignin polymers in the poplar wood had undergone the following structural changes, such as dehydration, depolymerization, condensation, and modification. Results showed that the regenerated DES lignin were nano-sized lignin, exhibiting the low molecular weights, narrow distribution (1.28<PDI<1.38) with controllable size (<100 nm), and excellent antioxidant activity. In short, the tailored one-pot DES fractionation will improve the enzymatic digestibility and facilitate the lignin valorization, which will further promote the economic and overall benefits of biorefinery.

A hydrotrope pretreatment for stabilized lignin extraction and high titer ethanol production

The biomass pretreatment strategies using organic acids facilitate lignin removal and enhance the enzymatic digestion of cellulose. However, lignin always suffers a severe and irreversible condensation. The newly generated C–C bonds dramatically affect its further upgrading. In this study, we used a recyclable hydrotrope (p-Toluenessulfonic acid, p-TsOH) to dissolve lignin under mild condition and stabilized lignin with a quenching agent (formaldehyde, FA) during extraction, achieving both value-added lignin extraction and efficient enzymatic saccharification of cellulose. Approximately 63.7% of lignin was dissolved by 80% (wt. %) p-TsOH with 1.5% FA addition at 80 °C, 30 min. The obtained lignin was characterized by FTIR spectroscopy, TGA, 2D HSQC NMR spectroscopy, and GPC. The results indicated that the extracted lignin exhibited excellent properties, such as light color, a low molecular weight (Mw, 5371 g/mol), and a narrow polydispersity (Mw/Mn, 1.63). The pretreated substrate was converted to ethanol via a quasi-simultaneous saccharification and fermentation process (Q-SSF). After fermentation of 60 h, the ethanol concentration reached 38.7 ± 3.3 g/L which was equivalent to a theoretical ethanol yield of 82.9 ± 2.2% based on the glucan content, while the residual glucose concentration was only 4.69 ± 1.4 g/L. In short, this pretreatment strategy protected lignin to form new C–C linkages and improved the enzymatic saccharification of glucan for high-titer ethanol production.Graphical

Saccharification of Used Paper Cup for Ethanol Production by Yeast Isolate

The main aim of this work was to develop an effective method for pretreatment and enzymatic saccharification of cellulose and hemicellulose components into fermentable sugars, as well as fermentation of the hydrolysate to bioethanol by new yeast isolates. YAJ-2M (3.2%), YCJ-1 (3.4%) and YAJ-2M 11 (5.6%) produced maximum ethanol. UV- induction responsible for genetic improvement of yeast isolates for ethanol production. Pretreatment of paper with alkali- acid reduced the lignin content from 25% to 14.3%. Maximum liberation of reducing sugars (168.7 μg/ml) was attained at 50°C for 96 hours at 150 rpm using enzyme dose of 20 U/g in alkali-acid (sodium hydroxide-orthophosphoric acid) pretreated paper cup. Fermentation of hydrolysate paper by yeast isolates (YAJ-1 M2 11 mutant) at 30°C, pH 4.7, 150 rpm and resulted in 1% ethanol after 96 h. Present investigation was found mutant YAJ-1 M2 11 produced highest ethanol concentration in hydrolysed paper than other tested mutant.

Hydrothermal Pretreatment of Lignocellulosic Feedstocks to Facilitate Biochemical Conversion.

Biochemical conversion of lignocellulosic feedstocks to advanced biofuels and other bio-based commodities typically includes physical diminution, hydrothermal pretreatment, enzymatic saccharification, and valorization of sugars and hydrolysis lignin. This approach is also known as a sugar-platform process. The goal of the pretreatment is to facilitate the ensuing enzymatic saccharification of cellulose, which is otherwise impractical due to the recalcitrance of lignocellulosic feedstocks. This review focuses on hydrothermal pretreatment in comparison to alternative pretreatment methods, biomass properties and recalcitrance, reaction conditions and chemistry of hydrothermal pretreatment, methodology for characterization of pretreatment processes and pretreated materials, and how pretreatment affects subsequent process steps, such as enzymatic saccharification and microbial fermentation. Biochemical conversion based on hydrothermal pretreatment of lignocellulosic feedstocks has emerged as a technology of high industrial relevance and as an area where advances in modern industrial biotechnology become useful for reducing environmental problems and the dependence on fossil resources.

Hydrothermal Pretreatment of Water-Extracted and Aqueous Ethanol-Extracted Quinoa Stalks for Enzymatic Saccharification of Cellulose

Auto-catalyzed hydrothermal pretreatment (A-HTP) and sulfuric-acid-catalyzed hydrothermal pretreatment (SA-HTP) were applied to quinoa stalks in order to reduce their recalcitrance towards enzymatic saccharification. Prior to pretreatment, quinoa stalks were extracted with either water or a 50:50 (v/v) ethanol–water mixture for removing saponins. Extraction with water or aqueous ethanol, respectively, led to removal of 52 and 75% (w/w) of the saponins contained in the raw material. Preliminary extraction of quinoa stalks allowed for a lower overall severity during pretreatment, and it led to an increase of glucan recovery in the pretreated solids (above 90%) compared with that of non-extracted quinoa stalks (73–74%). Furthermore, preliminary extraction resulted in enhanced hydrolysis of hemicelluloses and lower by-product formation during pretreatment. The enhancement of hemicelluloses hydrolysis by pre-extraction was more noticeable for SA-HTP than for A-HTP. As a result of the pretreatment, glucan susceptibility towards enzymatic hydrolysis was remarkably improved, and the overall conversion values were higher for the pre-extracted materials (up to 83%) than for the non-extracted ones (64–69%). Higher overall conversion was achieved for the aqueous ethanol-extracted quinoa stalks (72–83%) than for the water-extracted material (65–74%).

Differential effects of inorganic salts on cellulase kinetics in enzymatic saccharification of cellulose and lignocellulosic biomass.

Inorganic salt pretreatment of lignocellulosic biomass has proven to be an efficient way to increase the efficiency of enzymatic saccharification. However, it is not clear that this improvement is the result of modification of the lignocellulosic substrate after pretreatment, or removal of inhibitor, or enhancement of cellulase or a combination of these events. Therefore, this study aimed to analyze the effects of inorganic salts on kinetics of cellulase enzymes (celluclast 1.5L and accellerase 1500). Two substrates rich in cellulose content [carboxymethylcellulose (CMC), avicel (AV)] and lignocellulose substrate [sugarcane bagasse (SB)] were considered. The enzymatic saccharification was carried with and without the addition of inorganic salts (NaCl and KCl) at 0.5M and 1.0M concentration. The kinetic parameters, Km and Vm, were determined to mechanically understand the pattern of inhibition and enhancement of inorganic salts on enzymatic saccharification. The kinetics parameters of celluclast 1.5L and accellerase 1500 for hydrolysis of CMC and AV with NaCl showed uncompetitive inhibition. Whereas, influences of KCl on both cellulase were differentiated to function in inhibition or enhancement modes when challenged with different substrates. On the other hand, enzymatic hydrolysis efficiencies of SB using both cellulases were enhanced under addition of NaCl and KCl, by increasing Vm of celluclast 1.5L from 0.303 to 0.635mg/mLmin (0.5M KCl) and accellerase 1500 from 0.383 to 0.719mg/mLmin (1.0M NaCl). The details of kinetic analysis in this work revealed the mechanism of inorganic salts on cellulase kinetics to be involved in substrate modification and removal of inhibitor.

Comparison of Corn Stover Pretreatments with Lewis Acid Catalyzed Choline Chloride, Glycerol and Choline Chloride-Glycerol Deep Eutectic Solvent.

Herein, corn stover (CS) was pretreated by less corrosive lewis acid FeCl3 acidified solutions of neat and aqueous deep eutectic solvent (DES), aqueous ChCl and glycerol at 120 °C for 4 h with single FeCl3 pretreatment as control. It was unexpected that acidified solutions of both ChCl and glycerol were found to be more efficient at removing lignin and xylan, leading to higher enzymatic digestibility of pretreated CS than acidified DES. Comparatively, acidified ChCl solution exhibited better pretreatment performance than acidified glycerol solution. In addition, 20 wt% water in DES dramatically reduced the capability of DES for delignification and xylan removal and subsequent enzymatic cellulose saccharification of pretreated CS. Correlation analysis showed that enzymatic saccharification of pretreated CS was highly correlated to delignification and cellulose crystallinity, but lowly correlated to xylan removal. Recyclability experiments of different acidified pretreatment solutions showed progressive decrease in the pretreatment performance with increasing recycling runs. After four cycles, the smallest decrease in enzymatic cellulose conversion (22.07%) was observed from acidified neat DES pretreatment, while the largest decrease (43.80%) was from acidified ChCl pretreatment. Those findings would provide useful information for biomass processing with ChCl, glycerol and ChCl-glycerol DES.

In situ measurements of oxidation\u2013reduction potential and hydrogen peroxide concentration as tools for revealing LPMO inactivation during enzymatic saccharification of cellulose

BackgroundBiochemical conversion of lignocellulosic biomass to simple sugars at commercial scale is hampered by the high cost of saccharifying enzymes. Lytic polysaccharide monooxygenases (LPMOs) may hold the key to overcome economic barriers. Recent studies have shown that controlled activation of LPMOs by a continuous H2O2 supply can boost saccharification yields, while overdosing H2O2 may lead to enzyme inactivation and reduce overall sugar yields. While following LPMO action by ex situ analysis of LPMO products confirms enzyme inactivation, currently no preventive measures are available to intervene before complete inactivation.ResultsHere, we carried out enzymatic saccharification of the model cellulose Avicel with an LPMO-containing enzyme preparation (Cellic CTec3) and H2O2 feed at 1 L bioreactor scale and followed the oxidation–reduction potential and H2O2 concentration in situ with corresponding electrode probes. The rate of oxidation of the reductant as well as the estimation of the amount of H2O2 consumed by LPMOs indicate that, in addition to oxidative depolymerization of cellulose, LPMOs consume H2O2 in a futile non-catalytic cycle, and that inactivation of LPMOs happens gradually and starts long before the accumulation of LPMO-generated oxidative products comes to a halt.ConclusionOur results indicate that, in this model system, the collapse of the LPMO-catalyzed reaction may be predicted by the rate of oxidation of the reductant, the accumulation of H2O2 in the reactor or, indirectly, by a clear increase in the oxidation–reduction potential. Being able to monitor the state of the LPMO activity in situ may help maximizing the benefit of LPMO action during saccharification. Overcoming enzyme inactivation could allow improving overall saccharification yields beyond the state of the art while lowering LPMO and, potentially, cellulase loads, both of which would have beneficial consequences on process economics.

Characteristics of SSSF of rice straw and mass transfer of ethanol in a granular packed bed with N2 sparging

In this work, a solid simultaneous saccharification and fermentation (SSSF) system with N2 stripping was constructed for ethanol production, and the glucose inhibition to enzymatic saccharification of cellulose and the ethanol inhibition to anaerobic fermentation of yeast cells could be relieved. First, effects of flow rate of carrier gas and initial yeast concentration on mass transfer of ethanol and performance of SSSF were experimentally investigated. It is found that the peaks of total ethanol amount (6.32 g), cellulose consumption amount (13.29 g), ethanol yield (0.48 g/g substrate) and change rate of porosity of the packed bed (57.45 %) were obtained at an initial yeast concentration of 2% and a N2 flow rate of 30 mL/min. The results reveal that mass transfer efficiency during SSSF could be improved at an appropriate N2 flow rate and an optimal initial yeast concentration. Then, a novel kinetic model was established to simulate mass transfer of ethanol during SSSF. The data predicted by the model were in good accordance with the experimental results, which shows that the established model could reasonably reflect the characteristics of ethanol mass transfer in the packed bed. This work demonstrates that the produced ethanol could be effectively stripped out of the packed bed by convection of the sparged N2, leading to improving performances of enzymatic hydrolysis and ethanol fermentation.

Co-production of biofuels and value-added compounds from industrial Eucalyptus globulus bark residues using hydrothermal treatment

In this work, hydrothermal treatment was assessed for the fractionation of industrial Eucalyptus globulus bark residue (EBR) to obtain biofuels and value-added compounds (such as oligosaccharides and phenolic compounds) in separated streams. Hydrothermal treatment was evaluated under non-isothermal regimen in the range of maximum temperature (Tmax) of 177–228 °C or severities (S0) between 2.76 and 4.25. The highest oligosaccharides concentration (17.5 g/L) was achieved at S0 of 3.69, corresponding to hemicellulose recovery of 77.30%. Under all severities evaluated in this work, over 90.94% and 84.17% of cellulose and lignin remained in the solid phase, respectively. The increase of S0 improved 4.38-fold the enzymatic saccharification of cellulose, the highest glucose yield (84%) being achieved at S0 of 4.04. Considering the maximal recovery of polysaccharides as glucose and oligosaccharides from the liquid and solid phases, S0 of 4.04 was selected for bioethanol production using high solid loadings and following different strategies (simultaneous saccharification and fermentation – SSF and pre-saccharification and simultaneous saccharification and fermentation – PSSF). The utilization of 15% hydrothermally pretreated EBR without nutrient supplementation resulted in 26 g/L of ethanol, independently of the strategy used. An increase up to 17.5% solids and employing nutrient supplementation enabled the production of 38 g/L (or 4.8% v/v) of ethanol by PSSF.

Optimization of Culture Conditions and Reaction Parameters of β-glucosidase From a New Isolate of Bacillus subtilis (B1)

Introduction: The increased need for a considerable β-glucosidase activity, especially in the enzymatic saccharification of cellulose for bioenergy, has strongly stimulated the identification of effective β-glucosidase producing microbes. This study was conducted to optimize culture condition for β-glucosidase production from the identified new isolate of Bacillus subtilis (B1) and to find out an ideal condition for β-glucosidase activity.Materials and Methods: For β-glucosidase production, the bacterium was cultivated in a basal medium. The culture condition was optimized at several pH, different temperatures, varying cultivation periods, and various substrate concentrations. Finally, the activity of the β-glucosidase enzyme was investigated at different incubation periods, pH, temperatures, metal ions, and various percentages of methanol. The activity of β-glucosidase was measured by the capability of crude enzyme to convert pNPG (p-nitrophenyl-β-D glucopyranoside) into yellow product PNP (p-nitrophenol).Results: Cellulolytic bacterial strain B. subtilis (B1) showed high potentiality for β-glucosidase production at a pH of 7.0 after 24 hours incubation at 40°C. The highest level of enzyme production was achieved when 3% of CMC was provided in the culture medium. Optimum reaction conditions for β-glucosidase activity were shown to be 10 minutes, 60°C and at pH 7. Salts like Magnesium Sulfate (MgSO4), Calcium Chloride (CaCl2), and Manganese Sulfate (MnSO4) positively influenced the activity where NaCl and KCl had negative effects. The presence of methanol (80%) appreciably enhanced the activity of enzyme.Conclusions: Complete saccharification of different industrial processes can be augmented by using this novel β-glucosidase produced by B. subtilis strain isolated from effluent of biogas plant.

The synergy between LPMOs and cellulases in enzymatic saccharification of cellulose is both enzyme- and substrate-dependent.

The synergistic effects between cellulases and lytic polysaccharide monooxygenases (LPMOs) were investigated systematically in terms of their degree of synergy (DS) on amorphous and crystalline cellulose. Synergy curves were obtained for enzyme pairs containing a cellulase from Trichoderma reesei (Cel6A and Cel7A) and three LPMOs from Thermoascus aurantiacus (TaAA9A), Lentinus similis (LsAA9A) and Thielavia terrestris (TtAA9E). The synergistic experiments showed that the three LPMOs significantly improved the hydrolytic efficiency of Cel6A, on both cellulosic substrates; a more pronounced effect being seen for TtAA9E on amorphous cellulose at low cellulase:LPMO ratios. In contrast, the highly processive, reducing-end acting Cel7A synergised with the C1-C4 oxidising LPMOs, TaAA9A and LsAA9A, but was inhibited by the presence of C1-oxidizing TtAA9E. The degree of synergy exhibited by the cellulase-LPMO mixtures was enzyme- and substrate-specific. The observed Cel7A inhibition, rather than synergy, by the C1-oxidizing LPMO, TtAA9E, warrants further investigations.

Effect of alkaline pretreatments on the enzymatic hydrolysis of wheat straw.

Lignocellulosic materials are mainly consisted of lignin, cellulose, and hemicellulose. Lignin is recognized as the main obstacle for the enzymatic saccharification of cellulose towards the fermentable sugars' production. Hence, the removal of lignin from the lignocellulosic feedstock is beneficial for reducing the recalcitrance of lignocellulose for enzymatic attack. For this purpose, various different alkaline pretreatments were examined in order to study their effect on the enzymatic saccharification of wheat straw, as a typical lignocellulosic material. Results revealed that the alkaline pretreatments promoted delignification reactions. Regarding the removal of lignin, the most efficient pretreatments were alkaline treatment with hydrogen peroxide 10% and NaOH 2% autoclave with delignification efficiencies of 89.60% and 84.86% respectively. X-ray diffraction analysis was performed to enlighten the structural changes of raw and pretreated materials. The higher the delignification of the raw material, the higher the conversion of cellulose during enzymatic saccharification. In all cases after enzymatic saccharification, the cellulosic conversion was much higher (32-77%) than the untreated wheat straw (8.6%). After undergoing alkaline peroxide 10% pretreatment and cellulase treatment, 99% of the initial raw straw was eventually solubilized. Thus, wheat straw could be considered as an ideal material for the production of glucose with proper pretreatments and effective enzymatic hydrolysis.

Lignin prepared from different alkaline pretreated sugarcane bagasse and its effect on enzymatic hydrolysis

Sugarcane bagasse (SCB) was pretreated by sodium hydroxide (SH), alkaline ethanol (AE) and alkaline hydrogen peroxide (AHP), and the solid residue with similar lignin content after different pretreatment was selected. Enzymatic saccharification with different substrates was performed at 2%, 10%, and 20% (w/v) solid loading. After that, the lignin from different substrate was extracted and its structure was characterized. Furthermore, the adsorption capability of isolated lignin to cellulase and its effect on enzymatic hydrolysis were studied. The results showed that, as the substrate content increased from 2% (w/v) to 20% (w/v), the glucose yield after digestion of SH, AE, and AHP pretreated SCB reduced by 41.8%, 35.4%, and 28.7%, respectively. The inhibitory effect of different prepared lignin on the digestibility of Avicel was as follows, SH pretreated lignin (SHL) > cellulase enzymatic hydrolysis lignin (CEL) > AE pretreated lignin (AEL) > AHP pretreated lignin (AHPL), which exhibited positive correlation with its non-productive adsorption capability to cellulase. Lignin samples with low negative zeta potential, high hydrophobicity and high ration of syringyl (S) to guaiacyl (G) were unfavorable for the enzymatic saccharification of cellulose.

Overcoming the drying-induced pore closure of APMP poplar fibers in old newsprint by surfactant treatment to promote enzymatic hydrolysis of the cellulose

This study analyzed the hornification properties of the old newsprint fiber and its effects on the enzymatic hydrolysis and investigated the strategy for improving the hydrolysis efficiency. The results showed both of heating and vacuum can dry the fibers and cause hornification, which resulted in a significant decrease in the pore volume and the enzymatic saccharification efficiencies. A good linear relationship was presented between the enzymatic digestibility and the pore volume of the drying-induced hornified fibers from the two drying methods. The high content of lignin in this kind of hornified fiber aggravated the inhibition of hornification on the enzymatic saccharification of cellulose through physical barriers blocking cellulase to contact the partial surface of hornified cellulose. Moreover, the strategy of using surfactants to enhance enzymatic hydrolysis was preliminarily explored. The results showed that enzymatic saccharification of the hornified lignin-containing fiber was significantly improved after the addition of surfactants. The increase in glucose yield reached the maximum value of 7.15 g/L after adding PEG 8000. Anionic surfactants exhibited the similar effects but weaker than the non-ionic ones. And the most effective time of the surfactants applied to hornified fibers was postponed from 6 to 12 h as the fiber hornification degree increased. This study provides a meaningful complement for the research work of enzymatic hydrolysis of recycled fiber.

An optimum combined severity factor improves both the enzymatic saccharification yield and the functional lignin structure

Liquid hot water (LHW) pretreatment has become an interesting alternative among the known pretreatment approaches. In this study, an extended combined severity factor (CSF) as LHW pretreatment severity was used to make a balance between the enzymatic hydrolysis of cellulose and the potential application value of lignin. The results, mainly from the degradation of carbohydrates, the enzymatic saccharification of cellulose, and the characterization of lignin by FTIR, TGA, 31P NMR, and 2D HSQC NMR, demonstrated that a CSF of approximately 8.37 was the optimum pretreatment severity to facilitate the cellulose enzymatic hydrolysis and obtain functional lignin. Hemicellulose was removed for > 74.7% with a negligible glucose concentration of 1.19 g/L. The pretreated substrates were subsequently enzymatically hydrolyzed reaching a saccharification yield of 83.92%. The obtained lignin exhibited excellent structural properties, such as a higher hydroxyl (OH) content (2.34 mmol/g of aliphatic hydroxyl and 2.43 mmol/g of phenolic OH) and more β-O-4 aryl ether linkages (27.86%). In a word, this study hence provides useful guidance for the enzymatic saccharification of cellulose and the extraction of functional lignin using LHW pretreatment.