Ionic Liquid Pretreated Biomass Research Articles

Charge-Tunable μ-Gelzymes Achieving Cellulose Affinity under Synergistic Ionic Liquid-Pretreated Biomass in Situ Saccharification

Charge-Tunable μ-Gelzymes Achieving Cellulose Affinity under Synergistic Ionic Liquid-Pretreated Biomass <i>in Situ</i> Saccharification

A novel sequential ultrasound-rhamnolipid assisted [EMIM]OAc pretreatment for enhanced valorization of invasive Cogongrass to bioethanol

Cogongrass, one of the most invasive pest species worldwide, was considered as a promising bioresource for second-generation ethanol production. For the first time, it was successfully valorized to bioethanol using a novel sequential ionic liquid pretreatment assisted by synergistic effects of ultrasound irradiation and biosurfactant impregnation. The biomass was sonicated (50 and 100 W, 20 kHz) for 15 and 30 min, suspended in rhamnolipid solution (1, 2, and 3% w/w), and finally pretreated with 1-ethyl-3-methylimidazole acetate at 120 °C. Enzymatic digestibility of the regenerated biomass substantially enhanced by ~ 1.2-fold, upon a fast and low-intensity sonication (15 min, 50 W) followed by impregnation with the diluted rhamnolipid solution (2% w/w). Accordingly, non-isothermal simultaneous saccharification and fermentation of the pretreated Cogongrass by fungus M. hiemalis led to a maximum ethanol yield of ~ 195 (g/kg dry substrate), which was almost 1.6-fold higher than that obtained from the conventionally ionic liquid pretreated biomass. Comprehensive semi-quantitative analyses of the biomass together with FTIR and FESEM measurements revealed major improvements in surface area, surface hydrophilicity, pore sizes and volumes, cellulose crystallinity, and morphological features following the pretreatment. The findings may also provide a promising approach for enhanced recycling of the expensive ionic liquid and lignin in a biorefinery framework; however, more investigation should be dedicated to this subject.

Investigation of Rheological Behavior of Untreated and Microwave-Assisted Alkaline Pretreated Sugarcane Straw for Biofuel Production

Biomass slurries, such as those subjected to microwave-assisted alkaline (MAA) pretreatment, will become common substrates for the production of biofuels in the future. While the rheology of acid and ionic liquid pretreated biomass is known, the rheology of alkaline pretreated biomass is not yet reported; hence, the goal of this study was to establish the rheological characteristics of untreated and MAA pretreated sugarcane straw (SC). Using rotational rheometry and rheological models, the rheology of SC slurries was assessed as a function of particle size and insoluble solids concentration. In the range of 5–17% insoluble solids concentration, the slurries were consistently pseudo-plastic (n = 0.33 ± 0.02), possessed yield stress with their flow accurately described by the Casson rheological model (R2 = 0.98–0.99). The apparent viscosity and yield stress increased by two orders of magnitude with an increase in insoluble solids concentration. Essentially, MAA pretreated slurries exhibited significantly higher values of apparent viscosity and yield stress than the untreated ones, in the range of 55–80% and 21–63% for particle sizes of < 63 µm (P63) and 90–180 µm (P90), respectively. Pretreated P63 samples exhibited higher apparent viscosity and yield stress than the P90. On the other hand, for untreated samples, P63 samples had a reduced apparent viscosity than P90 samples. The results of this study definitively reveal that MAA significantly increases the shear rate dependent shear viscosity values along with the yield stress of biomass slurries. This will, therefore, serve as a benchmark for characterizing other MAA pretreated biomass slurries to guide the design of industrial-scale production equipment.

Lignin triggers irreversible cellulase loss during pretreated lignocellulosic biomass saccharification.

BackgroundNon-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.ResultsIn 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 Expansiona (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.ConclusionsOur 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.

Improved Activity of a Thermophilic Cellulase, Cel5A, from Thermotoga maritima on Ionic Liquid Pretreated Switchgrass

Ionic liquid pretreatment of biomass has been shown to greatly reduce the recalcitrance of lignocellulosic biomass, resulting in improved sugar yields after enzymatic saccharification. However, even under these improved saccharification conditions the cost of enzymes still represents a significant proportion of the total cost of producing sugars and ultimately fuels from lignocellulosic biomass. Much of the high cost of enzymes is due to the low catalytic efficiency and stability of lignocellulolytic enzymes, especially cellulases, under conditions that include high temperatures and the presence of residual pretreatment chemicals, such as acids, organic solvents, bases, or ionic liquids. Improving the efficiency of the saccharification process on ionic liquid pretreated biomass will facilitate reduced enzyme loading and cost. Thermophilic cellulases have been shown to be stable and active in ionic liquids but their activity is typically at lower levels. Cel5A_Tma, a thermophilic endoglucanase from Thermotoga maritima, is highly active on cellulosic substrates and is stable in ionic liquid environments. Here, our motivation was to engineer mutants of Cel5A_Tma with higher activity on 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) pretreated biomass. We developed a robotic platform to screen a random mutagenesis library of Cel5A_Tma. Twelve mutants with 25–42% improvement in specific activity on carboxymethyl cellulose and up to 30% improvement on ionic-liquid pretreated switchgrass were successfully isolated and characterized from a library of twenty thousand variants. Interestingly, most of the mutations in the improved variants are located distally to the active site on the protein surface and are not directly involved with substrate binding.

Critical cellulase and hemicellulase activities for hydrolysis of ionic liquid pretreated biomass

Critical cellulase and hemicellulase activities are identified for hydrolysis of ionic liquid (IL) pretreated poplar and switchgrass; hemicellulase rich substrates with largely amorphous cellulose. Enzymes from Aspergillus nidulans were expressed and purified: an endoglucanase (EG) a cellobiohydrolase (CBH), an endoxylanase (EX) and an acetylxylan esterase (AXE). β-Xylosidase (βX) from Selenomonas ruminantium and a commercial β-glucosidase (βG) from Novozyme 188 were admixed with the A. nidulans enzymes. Statistical analysis indicates that βG and βX activities are significant for both glucose and xylose yields for the two substrates. EG is a significant factor for glucan hydrolysis while EX is significant for xylan hydrolysis of the substrates. The CBH, which has activity on crystalline cellulose and negligible activity on amorphous cellulose, was not a significant factor in glucan hydrolysis. EX is significant in glucan hydrolysis for poplar. The addition of AXE significantly improves xylan hydrolysis for poplar but not switchgrass.

Saccharification of ionic liquid pretreated biomass with commercial enzyme mixtures

The performance of Spezyme CP, a commercial cellulase system, and Primafast Luna CL, a textile bio-finishing enzyme, on hydrolysis of substrates following ionic liquid pretreatment was evaluated. Ionic liquid pretreatment of lignocellulosic biomass produces amorphous cellulose with little residual crystallinity and enhances its saccharification. The high crystallinity of native cellulose poses an impediment to enzyme hydrolysis of the cellulosic portion of biomass. Target substrates included poplar, switchgrass and Avicel, a highly crystalline cellulose substrate. Spezyme was found to hydrolyze glucan and xylan components completely in 24 h with modest enzyme loadings. Primafast was found to principally hydrolyze glucan components of biomass. Xylanases added to the Primafast enzyme mixture appear to act synergistically to improve glucan hydrolysis with the poplar substrate.

Visualization of biomass solubilization and cellulose regeneration during ionic liquid pretreatment of switchgrass

Auto-fluorescent mapping of plant cell walls was used to visualize cellulose and lignin in pristine switchgrass (Panicum virgatum) stems to determine the mechanisms of biomass dissolution during ionic liquid pretreatment. The addition of ground switchgrass to the ionic liquid 1-n-ethyl-3-methylimidazolium acetate resulted in the disruption and solubilization of the plant cell wall at mild temperatures. Swelling of the plant cell wall, attributed to disruption of inter- and intramolecular hydrogen bonding between cellulose fibrils and lignin, followed by complete dissolution of biomass, was observed without using imaging techniques that require staining, embedding, and processing of biomass. Subsequent cellulose regeneration via the addition of an anti-solvent, such as water, was observed in situ and provided direct evidence of significant rejection of lignin from the recovered polysaccharides. This observation was confirmed by chemical analysis of the regenerated cellulose. In comparison to untreated biomass, ionic liquid pretreated biomass produces cellulose that is efficiently hydrolyzed with commercial cellulase cocktail with high sugar yields over a relatively short time interval.