Comparison of Alkaline Sulfite Pretreatment and Acid Sulfite Pretreatment with Low Chemical Loading in Saccharification of Poplar.

Sulfite pretreatment is a productive process for lignin dissolution in lignocelluloses and to reduce the hydrophobicity of lignin by sulfonation, thus promoting the hydrolyzability of the substrate. Previously, sulfite pretreatment needs high dosages of chemicals and thus results in the high cost of the pretreatment and the great pressure of environmental pollution. To overcome these problems, it was crucial to research whether alkaline sulfite pretreatment (ALS) and acid sulfite pretreatment (ACS) with low chemical loading could enhance the saccharification of poplar. In this work, the results indicated that with low loading of chemicals in sulfite pretreatment, ALS pretreatment (1.6% Na2SO3 and 0.5% NaOH) at 180 °C removed more lignin, resulted in lower hydrophobicity and higher cellulase adsorption capacity of poplar than ACS pretreatment (1.6% Na2SO3 and 0.5% H2SO4) at 180 °C. A satisfying glucose yield of 84.9% and a xylose yield of 76.0% were obtained from poplar after ALS pretreatment with 1.6% Na2SO3 and 0.5% NaOH at 180 °C for 1 h using 10 FPU cellulase/g dry matter, saving sodium sulfite by 60.0% compared to the loading of sulfite in traditional sulfite pretreatment. The strategy developed in this work reduced chemical loading and cellulase loading in alkali sulfite pretreatment for the saccharification of poplar.

Combination of hydrothermal and chemi-mechanical pretreatments to enhance enzymatic hydrolysis of poplar branches and insights on cellulase adsorption.

An integration of different pretreatments is important to overcome recalcitrance and realize efficient bioconversion of lignocellulosic biomass. This study aims at the effects of combination of hydrothermal pretreatment and different chemi-mechanical pretreatments on enzymatic hydrolysis, and understanding the enzymes adsorption mechanism. The combination of hydrothermal and chemi-mechanical pretreatments effectively improved the enzymatic hydrolysis of poplar substrates, in which the enzymatic hydrolysis of substrates pretreated by hydrothermal pretreatment + Fenton pretreatment + mechanical refining (HFM) was the highest (92.39% of glucose conversion yield, and 20.88 g/L of glucose concentration). The substrates' main characteristics were obviously changed after combined pretreatments, such as swelling ability and specific surface area of substrates were increased. The Langmuir adsorption model (R2 > 0.98) and pseudo second-order adsorption kinetic model (R2≈1) were well suitable to describe the adsorption of enzymes on substrates, meanwhile the adsorption mechanism was summarized.

Carboxylated and quaternized lignin enhanced enzymatic hydrolysis of lignocellulose treated by p-toluenesulfonic acid due to improving enzyme activity.

Modificated lignins can affect enzymatic hydrolysis efficiency (EHE) because of changing physicochemical properties of lignin. In this study, carboxylated and quaternized lignin (CQL) and hydroxymethylated lignin (HML) were prepared to explore the effect of lignin modification on cellulase adsorption and EHE of p-toluenesulfonic acid treated corn stover (PCS). The results showed that CQL enhanced EHE of PCS due to the higher ß-glucosidase (ß-GL) activity, resulting from the formation of CQL-ß-GL complexes with a lower binding free energy and the improvement of ß-GL conformation made by the binding of CQL and ß-GL. However, the drop in EHE due to the addition of HML was consequent on ß-GL deactivation that was because the binding site of HML and ß-GL overlapped with the carbohydrate binding domain of ß-GL, causing the decrease in ß-GL activity compared with CQL. This study would help deeply elucidate the effect of modified lignins on EHE and cellulase adsorption.

Impact of bagasse lignin-carbohydrate complexes structural changes on cellulase adsorption behavior.

Pretreatment technology has attracted much attention as an effective method for the conversion of sugarcane bagasse into biochemicals. However, residual lignin-carbohydrate complexes (LCC) can negatively impact the subsequent enzymatic hydrolysis of bagasse. In this work, the changes in bagasse LCC after pretreatment with hot water and dilute acid were characterized by component analysis, 13C NMR and 1H-13C HSQC NMR to reveal the correlation between LCC structure and cellulase adsorption. A real-time dynamic model of LCC affecting adsorption of cellulase was constructed using a quartz crystal microbalance (QCM-D). The QCM-D results demonstrated that cellulase exhibited different adsorption characteristics on different LCCs. For example, the maximum adsorption capacities for cellulase onto the raw material LCC (RW-LCC), hot water pretreated LCC (LHW-LCC), and dilute acid pretreated LCC (AP-LCC) at 4 °C were 29.0 ng/cm2, 94.9 ng/cm2 and 129.8 ng/cm2, respectively. In addition, the adsorption rate constants for cellulase on RM-LCC, LHW-LCC and AP-LCC at 4 °C were 0.09, 0.14 and 0.19, respectively.

Lignin extracted by γ-valerolactone/water from corn stover improves cellulose enzymatic hydrolysis.

The impact of lignin extracted from γ-valerolactone/water (GVL/H2O) pretreatment of corn stover on the enzymatic hydrolysis of cellulose was investigated. Two lignin samples were separated and named as GL25 and GL75 according to the amounts of sulfuric acid (25 mM and 75 mM) used in the GVL/H2O pretreatment. With the addition of 2 g/L of GL25 and GL75, the glucan conversion of enzymatic hydrolysis of Avicel improved markedly from 28.0% to 37.4% and 31.3%, respectively. Moreover, the improvement of glucan conversion increased when increasing the loadings of GL25 and GL75. A similar observation was made when GVL/H2O pretreated corn stover was the substrate. The results of the cellulase adsorption experiments showed that the GLs had a lower maximum cellulase adsorption capacity and binding strength compared to that of acid-insoluble lignin. Further structural characterization of the GLs revealed that they had a low zeta-potential and hydrophobicity, but a high Syringyl/Guaiacyl ratio.

Exploring why sodium lignosulfonate influenced enzymatic hydrolysis efficiency of cellulose from the perspective of substrate-enzyme adsorption.

BACKGROUND: Cellulase adsorbed on cellulose is productive and helpful to produce reducing sugars in enzymatic hydrolysis of lignocellulose; however, cellulase adsorbed on lignin is non-productive. Increasing productive adsorption of cellulase on cellulose would be beneficial in improving enzymatic hydrolysis. Adding lignin that was more hydrophilic in hydrolysis system could increase productive adsorption and promote hydrolysis. However, the effect mechanism is still worth exploring further. In this study, lignosulfonate (LS), a type of hydrophilic lignin, was used to study its effect on cellulosic hydrolysis. RESULTS: The effect of LS on the enzymatic hydrolysis of pure cellulose (Avicel) and lignocellulose [dilute acid (DA) treated sugarcane bagasse (SCB)] was investigated by analyzing enzymatic hydrolysis efficiency, productive and non-productive cellulase adsorptions, zeta potential and particle size distribution of substrates. The result showed that after adding LS, the productive cellulase adsorption on Avicel reduced. Adding LS to Avicel suspension could form the Avicel-LS complexes. The particles were charged more negatively and the average particle size was smaller than Avicel before adding LS. In addition, adding LS to cellulase solution formed the LS-cellulase complexes. For DA-SCB, adding LS decreased the non-productive cellulase adsorption on DA-SCB from 3.92 to 2.99 mg/g lignin and increased the productive adsorption of cellulase on DA-SCB from 2.00 to 3.44 mg/g cellulose. Besides, the addition of LS promoted the formation of LS-lignin complexes and LS-cellulase complexes, and the complexes had more negative charges and smaller average sizes than DA-SCB lignin and cellulase particles before adding LS. CONCLUSIONS: In this study, LS inhibited Avicel's hydrolysis, but enhanced DA-SCB's hydrolysis. This stemmed from the fact that LS could bind cellulase and Avicel, and occupied the binding sites of cellulase and Avicel. Thus, a decreased productive adsorption of cellulase on Avicel arose. Regarding DA-SCB, adding LS, which enhanced hydrolysis efficiency of DA-SCB, increased the electrostatic repulsion between DA-SCB lignin and cellulase, and therefore, decreased non-productive adsorption of cellulase on DA-SCB lignin and enhanced productive adsorption of cellulase on DA-SCB cellulose.

An aggregated understanding of cellulase adsorption and hydrolysis for ball-milled cellulose.

This study evaluated the effects of physicochemical properties of a series of ball-milled cellulose on cellulase adsorption and glucose yield. The relationship between cellulase adsorption and initial hydrolysis rate was also discussed. We found that hydrophobicity and surface charge are the key factors affecting cellulase adsorption on ball-milled cellulose. The results demonstrated that glucose yield had a positive correlation with specific surface area, while showed a negative correlation with particle size, degree of polymerization and crystallinity. Among these properties, specific surface area and crystallinity are the key factors affecting glucose yield. As ball milling progressed, cellulose showed lower enzyme adsorption capacity/amount of bound enzyme during initial stage of hydrolysis, but had higher initial hydrolysis rate. The enhanced rate is attributed to the fact that the amorphous region produced by ball milling reduces the free energy required for decrystallization thus increases the catalytic efficiency of the bound enzyme.

Enhanced enzymatic hydrolysis of cellulose from waste paper fibers by cationic polymers addition.

Cationic polymers (cationic polyacrylamide (CPAM), polyethyleneimine (PEI) or cationic starch (CS)) were used to enhance the enzymatic hydrolysis of waste paper fibers (WPFs) at 15% (w/w) solids concentration. Results showed that 0.05 g/L PEI, CPAM and CS resulted in 72.5%, 65.9% and 59.7% conversion of WPFs, increased by 15.4%, 8.8% and 2.6%, respectively, compared with control (57.1%). PEI was shown to have a larger effect than CPAM and CS, and generate a total sugar concentration of 73.9 g/L. Improvement in hydrolysis with cationic polymer addition is attributed to increased cellulase adsorption on cellulose through electrostatic attraction, rather than enhancement of cellulase activity. A patching/ bridging mechanism of cationic polymer enhancement of cellulose adsorption in cellulose is hypothesized. PEI exhibited maximum cellulose binding for polymers examined and appears to promote binding through a patching mechanism. CPAM and CS adsorbed a relatively low cellulase through bridging mechanism. In addition, enzyme loading could be reduced by addition of cationic polymers to obtain the same glucose yield, especially when PEI was used.

Mechanisms of the inhibition of enzymatic hydrolysis of waste pulp fibers by calcium carbonate and the influence of nonionic surfactant for mitigation.

Recycled paper mills produce large quantities of fibrous rejects and fines which are usually sent to landfills as solid waste. These cellulosic materials can be enzymatically hydrolyzed into sugars for the production of biofuels and biomaterials. Paper mill wastes also contain large amounts of calcium carbonate which inhibits cellulase activity. The calcium carbonate (30%, w/w) decreased 40-60% of sugar yield of unbleached softwood kraft pulp. The prime mechanisms for this are by pH variation, competitive and non-productive binding, and aggregation effect. Addition of acetic acid (pH adjustment) increased the sugar production from 19 to 22 g/L of paper mill waste fibers. Strong affinity of enzyme-calcium carbonate decreased free enzyme in solution and hindered sugar production. Electrostatic and hydrogen bond interactions are mainly possible mechanism of enzyme-calcium carbonate adsorption. The application of the nonionic surfactant Tween 80 alleviated the non-productive binding of enzyme with the higher affinity on calcium carbonate. Dissociated calcium ion also inhibited the hydrolysis by aggregation of enzyme.

Effect of bisulfite treatment on composition, structure, enzymatic hydrolysis and cellulase adsorption profiles of sugarcane bagasse.

The effect of sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) on composition, structure, enzymatic hydrolysis and cellulase adsorption profiles of sugarcane bagasse (SCB) was investigated. SPORL gave a higher SCB hydrolysis yield (85.33%) compared to dilute acid pretreatment (DA) (64.39%). The SEM pictures showed that SPORL SCB structure became more disordered and looser, suggesting SPORL SCB was more accessible to cellulase. The zeta potential of SPORL SCB suspension (-21.89mV) was significantly different from that of DA SCB (-12.87mV), which demonstrated the lignin in SPORL SCB was more hydrophilic. With regard to cellulase adsorption profiles, SPORL SCB had a lower non-productive adsorption (14.87mg/glignin) and a higher productive adsorption (37.67 mg/gcarbohydrate) compared with DA SCB (17.05mg/glignin; 25.79mg/gcarbohydrate). These results indicated that SPORL SCB had better accessibility to cellulase and the higher productive cellulase adsorption of SPORL SCB had improved hydrolysis.

Comparison of organosolv and hydrotropic pretreatments of eucalyptus for enhancing enzymatic saccharification.

The objective of this study was to investigate the effects of organosolv and hydrotropic pretreatments on improving enzymatic hydrolysis of eucalyptus. The chemical composition of the fiber surface was analyzed using X-ray photoelectron spectroscopy (XPS) to determine the surface characteristics of pretreated eucalyptus. Other than the significant decrease of surface coverage by lignin, hydrotropic pretreatment was more effective in removing the lignin and xylose from fiber cell walls than organosolv pretreatment. The restriction of acetyl and phenolic groups in pretreated substrates was typically eliminated by hydrotropic pretreatments. Moreover, fiber structure and morphology after pretreatments were more suitable for enzymatic hydrolysis. Cellulase adsorption capacity was notably improved by hydrotropic pretreatment, which indicating the better enzyme accessibility of cellulose in pretreated substrates. Eventually, higher glucose yield was obtained with hydrotropic pretreatment. In addition, the precipitated lignin as an important by-product of pretreatments was characterized by Fourier transforms infrared spectroscopy (FTIR) also.

Lignin triggers irreversible cellulase loss during pretreated lignocellulosic biomass saccharification.

BACKGROUND: Non-productive binding of enzymes to lignin is thought to impede the saccharification efficiency of pretreated lignocellulosic biomass to fermentable sugars. Due to a lack of suitable analytical techniques that track binding of individual enzymes within complex protein mixtures and the difficulty in distinguishing the contribution of productive (binding to specific glycans) versus non-productive (binding to lignin) binding of cellulases to lignocellulose, there is currently a poor understanding of individual enzyme adsorption to lignin during the time course of pretreated biomass saccharification. RESULTS: In this study, we have utilized an FPLC (fast protein liquid chromatography)-based methodology to quantify free Trichoderma reesei cellulases (namely CBH I, CBH II, and EG I) concentration within a complex hydrolyzate mixture during the varying time course of biomass saccharification. Three pretreated corn stover (CS) samples were included in this study: Ammonia Fiber Expansion(a) (AFEX™-CS), dilute acid (DA-CS), and ionic liquid (IL-CS) pretreatments. The relative fraction of bound individual cellulases varied depending not only on the pretreated biomass type (and lignin abundance) but also on the type of cellulase. Acid pretreated biomass had the highest levels of non-recoverable cellulases, while ionic liquid pretreated biomass had the highest overall cellulase recovery. CBH II has the lowest thermal stability among the three T. reesei cellulases tested. By preparing recombinant family 1 carbohydrate binding module (CBM) fusion proteins, we have shown that family 1 CBMs are highly implicated in the non-productive binding of full-length T. reesei cellulases to lignin. CONCLUSIONS: Our findings aid in further understanding the complex mechanisms of non-productive binding of cellulases to pretreated lignocellulosic biomass. Developing optimized pretreatment processes with reduced or modified lignin content to minimize non-productive enzyme binding or engineering pretreatment-specific, low-lignin binding cellulases will improve enzyme specific activity, facilitate enzyme recycling, and thereby permit production of cheaper biofuels.

Improvement of the enzymatic hydrolysis of furfural residues by pretreatment with combined green liquor and ethanol organosolv.

Furfural residues (FRs) were pretreated with ethanol and a green liquor (GL) catalyst to produce fermentable sugar. Anthraquinone (AQ) was used as an auxiliary reagent to improve delignification and reduce cellulose decomposition. The results showed that 42.7% of lignin was removed and 96.5% of cellulose was recovered from substrates pretreated with 1.0 mL GL/g of dry substrate and 0.4% (w/w) AQ at 140°C for 1h. Compared with raw material, ethanol-GL pretreatment of FRs increased the glucose yield from 69.0% to 85.9% after 96 h hydrolysis with 18 FPU/g-cellulose for cellulase, 27 CBU/g-cellulose for ß-glucosidase. The Brauner-Emmett-Teller surface area was reduced during pretreatment, which did not inhibit the enzymatic hydrolysis. Owing to the reduced surface area, the unproductive binding of cellulase to lignin was decreased, thus improving the enzymatic hydrolysis. The degree of polymerization of cellulose from FRs was too low to be a key factor for improving enzymatic hydrolysis.

Simultaneous production of cellulase and reducing sugar through modification of compositional and structural characteristic of sugarcane bagasse.

This study examined the potential of untreated and alkali-pretreated sugarcane bagasse (SCB) in cellulase, reducing sugar (RS) and fungal biomass production via solid state fermentation (SSF) using Pycnoporus sanguineus. The impact of the composition, structure and cellulase adsorption ability of SCB on the production of cellulase, RS and fungal biomass was investigated. From the morphological and compositional analyses, untreated SCB has relatively more structural changes with a higher percentage of depolymerisation on the cellulose, hemicellulose and lignin content compared to alkali-pretreated SCB. Thus, untreated SCB favoured the production of cellulase and fungal biomass whereas alkali-pretreated SCB yielded a higher amount of RS. The composition and morphology of untreated SCB did not encourage RS production and this suggested that RS produced during SSF might be consumed in a faster rate by the more abundantly grown fungus. Besides that, alkali-pretreated SCB with higher cellulase adsorption ability could have adsorbed the cellulase produced and resulted in a lower cellulase titre. In short, the production of specific bioproducts via SSF is dependent on the structure and composition of the substrate applied.

Enzymatic hydrolysis, adsorption, and recycling during hydrolysis of bagasse sulfite pulp.

The high costs of enzymatic hydrolysis along with the high enzyme dosage are often considered as the major bottlenecks in lignocellulosic bioconversion. This study investigated the hydrolysis efficiency, cellulase adsorption and enzyme recycling during the hydrolysis of bagasse sulfite pulp (BSP). After 48 h of hydrolysis, more than 70% of the cellulose was hydrolyzed, while the protein concentration and cellulase activity in solution remained 31% and 17% of the initial value, respectively. The cellulase adsorption on the fresh BSP was better fitted by a Sips model, suggesting the occurrence of a multilayer adsorption at low cellulase concentration and monolayer adsorption at high concentration on the BSP surfaces. Desorption profile studies showed that the optimum desorption condition was at pH 4.8 and 40 °C. Moreover, considering the limited ability to desorption, directly empolying the bound enzyme with residual substrate is more effective method to recover cellulase during the hydrolysis of BSP.