Effect of oxygen delignification operating parameters on downstream enzymatic hydrolysis of softwood substrates

Enhanced oxygen delignification of softwood pulp samples (taken upstream and downstream of a commercial oxygen delignification unit) improved the initial rate of enzymatic saccharification and overall yield of monomeric sugars by 62-82% and 76-80%, respectively. Laboratory-scale experiments were used to examine the effect of a broad range of operating parameters (temperature, time, caustic concentration, and oxygen partial pressure) on the effectiveness of oxygen delignification. Using empirical models, kappa number (residual lignin content) was found to effectively predict final conversion to monomeric sugars. Application of oxygen delignification to sulfite mill knots resulted in smaller (20-25%) reduction in lignin content. However, using a combination of oxygen delignification and particle size reduction, up to 80% of the carbohydrate in the reject knots could be converted to fermentable sugars.     

Steam pretreatment of lignocellulosic material for enhanced enzymatic hydrolysis

Pretreatment methods were compared with steam explosion, and differing views on the relative importance of mechanical and chemical effects were outlined. Hydrolysis was desirable; pyrolysis was undesirable. The effects of initial moisture content on steam consumption, mechanism and rate of heat transfer, pentosan solubilization, and subsequent glucose yield were summarized. The insignificant effect, after treatment at 240 degrees C, of 90% pressure bleed-down before explosion on subsequent simultaneous saccharification and fermentation (SSF) yields was described. Treatment at 190 degrees C with complete bleed-down (no explosion), when compared with that at 240 degrees C with explosion from full pressure, showed at least as good solubilizatoin of pentosan, enzymatic hydrolysis, and SSF but showed greater pentosan destruction for the same degree of pentosan removal. Water washing of unexploded steamed aspenwood chips was at least as efficient as that of similarly treated but exploded chips. Scanning electron micrographs of unexploded chips showed extensive rupturing of vessel pit membranes and other morphological features associated with steam-exploded wood. Neither the explosion nor the high temperatures (above 190 degrees C) are necessary.     

纤维素酶与木质纤维素生物降解转化的研究进展

利用纤维素酶将预处理后的秸秆降解成可发酵性单糖,然后发酵生产所需的液体燃料及化工产品的技术,对于我国解决能源、环境、人口就业等难题有着巨大的积极影响。在木质纤维素生物降解转化工艺中,减少纤维素酶用量及提高酶解效率是降低木质纤维素降解成本的关键。纤维素酶系和木质纤维素酶水解技术的改进需要深入了解纤维素酶系统的组成及其协同作用、纤维素酶的结构与功能以及纤维素酶的生产技术。将就以上几个方面的研究进展进行讨论,并深入探讨了纤维素酶糖化能力的评价方法。     

The effect of delignification of forest biomass on enzymatic hydrolysis

The effect of delignification methods on enzymatic hydrolysis of forest biomass was investigated using softwood and hardwood that were pretreated at an alkaline condition followed by sodium chlorite or ozone delignification. Both delignifications improved enzymatic hydrolysis especially for softwood, while pretreatment alone was found effective for hardwood. High enzymatic conversion was achieved by sodium chlorite delignification when the lignin content was reduced to 15%, which is corresponding to 0.30-0.35 g/g accessible pore volume, and further delignification showed a marginal effect. Sample crystallinity index increased with lignin removal, but it did not show a correlation with the overall carbohydrate conversion of enzymatic hydrolysis.     

The effect of shaking regime on the rate and extent of enzymatic hydrolysis of cellulose

In an attempt to elucidate the effect of mixing on the rate and extent of enzymatic hydrolysis of cellulosic substrates, alpha-cellulose was hydrolysed using a commercial cellulase preparation at varying levels of substrate concentration (2.5,5 and 7.5% (w/v)) and by using three shaking regimes: continuous at low-speed (25 rpm), continuous at high-speed (150 rpm) and an intermittent regime comprised of high and low-speed shaking intervals. The continuous, high-speed shaking produced the highest conversion yields, whereas the intermittent and low-speed shaking regimes resulted in lower conversions. After 72 h, at all shaking regimes (150 rpm,25 rpm and intermittent), using a low substrate concentration (2.5%) produced conversion yields (82,79 and 80%) higher than those obtained at high (7.5%) substrate concentration (68,63 and 68%). As the substrate concentration increased, the conversion yields at intermittent shaking gradually approached those resulting from high-speed shaking. Thus, it appears that intermittent shaking could be a beneficial process option as it can reduce the mixing energy requirements while producing reasonably high conversion yields.     

Organosolv pretreatment of lignocellulosic biomass for enzymatic hydrolysis

Production of ethanol by bioconversion of lignocellulosic biomass has attracted much interest in recent years. However, the pretreatment process for increasing the enzymatic digestibility of cellulose has become a key step in commercialized production of cellulosic ethanol. During the last decades, many pretreatment processes have been developed for decreasing the biomass recalcitrance, but only a few of them seem to be promising. From the point of view for integrated utilization of lignocellulosic biomass, organosolv pretreatment provides a pathway for biorefining of biomass. This review presents the progress of organosolv pretreatment of lignocellulosic biomass in recent decades, especially on alcohol, organic acid, organic peracid and acetone pretreatments, and corresponding action mechanisms. Evaluation and prospect of organosolv pretreatment were performed. Finally, some recommendations for future investigation of this pretreatment method were given.     

Epidemic based modeling of enzymatic hydrolysis of lignocellulosic biomass

An epidemic based model was developed to describe the enzymatic hydrolysis of a lignocellulosic biomass, dilute sulfuric acid pretreated corn stover. The process of substrate getting adsorbed and digested by enzyme was simulated as susceptibles getting infected by viruses and becoming removed and recovered. This model simplified the dynamic enzyme “infection” process and the catalysis of cellulose into a two‐parameter controlled, enzyme behavior guided mechanism. Furthermore, the model incorporates the adsorption block by lignin and inhibition effects on cellulose catalysis. The model satisfactorily predicted the enzyme adsorption and hydrolysis, negative role of lignin, and inhibition effects over hydrolysis for a broad range of substrate and enzyme loadings. Sensitivity analysis was performed to evaluate the incorporation of lignin and other inhibition effects. Our model will be a useful tool for evaluating the effects of parameters during hydrolysis and guide a design strategy for continuous hydrolysis and the associated process control. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1021–1028, 2014     

High-throughput microplate technique for enzymatic hydrolysis of lignocellulosic biomass

Several factors will influence the viability of a biochemical platform for manufacturing lignocellulosic based fuels and chemicals, for example, genetically engineering energy crops, reducing pre-treatment severity, and minimizing enzyme loading. Past research on biomass conversion has focused largely on acid based pre-treatment technologies that fractionate lignin and hemicellulose from cellulose. However, for alkaline based (e.g., AFEX) and other lower severity pre-treatments it becomes critical to co-hydrolyze cellulose and hemicellulose using an optimized enzyme cocktail. Lignocellulosics are appropriate substrates to assess hydrolytic activity of enzyme mixtures compared to conventional unrealistic substrates (e.g., filter paper, chromogenic, and fluorigenic compounds) for studying synergistic hydrolysis. However, there are few, if any, high-throughput lignocellulosic digestibility analytical platforms for optimizing biomass conversion. The 96-well Biomass Conversion Research Lab (BCRL) microplate method is a high-throughput assay to study digestibility of lignocellulosic biomass as a function of biomass composition, pre-treatment severity, and enzyme composition. The most suitable method for delivering milled biomass to the microplate was through multi-pipetting slurry suspensions. A rapid bio-enzymatic, spectrophotometric assay was used to determine fermentable sugars. The entire procedure was automated using a robotic pipetting workstation. Several parameters that affect hydrolysis in the microplate were studied and optimized (i.e., particle size reduction, slurry solids concentration, glucan loading, mass transfer issues, and time period for hydrolysis). The microplate method was optimized for crystalline cellulose (Avicel) and ammonia fiber expansion (AFEX) pre-treated corn stover.     

High-throughput enzymatic hydrolysis of lignocellulosic biomass via in-situ regeneration

The high cost of lignocellulolytic enzymes is one of the main barriers towards the development of economically competitive biorefineries. Enzyme engineering can be used to significantly increase the production rate as well as specific activity of enzymes. However, the success of enzyme optimization efforts is currently limited by a lack of robust high-throughput (HTP) cellulase screening platforms for insoluble pretreated lignocellulosic substrates. We have developed a cost-effective microplate based HTP enzyme-screening platform for ionic liquid (IL) pretreated lignocellulose. By performing in-situ biomass regeneration in micro-volumes, we can volumetrically meter biomass (sub-mg loading) and also precisely control the amount of residual IL for engineering novel IL-tolerant cellulases. Our platform only requires straightforward liquid-handling steps and allows the integration of biomass regeneration, washing, saccharification, and imaging steps in a single microtiter plate. The proposed method can be used to screen individual cellulases as well as to develop novel cellulase cocktails.     

Effect of particle size on the rate of enzymatic hydrolysis of cellulose

The effect of particle size on enzymatic hydrolysis of cellulose has been investigated. The average size of microcrystalline cotton cellulose has been reduced to submicron scale by using a media mill. The milled products were further subjected to hydrolysis using cellulase. High cellulose concentration (7%) appeared to retard the size reduction and resulted in greater particles and smaller specific surface areas than those at low concentration (3%) with the same milling time. Initial rate method was employed to explore the rate of enzymatic hydrolysis of cellulose. The production rate of cellobiose was increased at least 5-folds due to the size reduction. The yield of glucose was also significantly increased depending upon the ratio of enzyme to substrate. A high glucose yield (60%) was obtained in 10-h hydrolysis when the average particle size was in submicron scale.     

Optimized delignification of wood‐derived lignocellulosics for improved enzymatic hydrolysis

One of the major bottlenecks in the bioconversion of lignocelluosic feedstocks to liquid ethanol is the recalcitrance of residue following pretreatment, specifically softwood derived residues. Peroxide delignification has previously been shown to effectively aid in the removal of condensed lignaceous moieties from substrates following pretreatment, and thereby improve the hydrolyzability of the polymeric carbohydrates to their monomeric constituents. Despite the effectiveness of peroxide, drawbacks in this system still remain, as the concentration of peroxide required for adequate hydrolysis performance is currently uneconomical. In an attempt to improve the efficacy of the delignification process, we evaluated other post‐treatment operations and concurrently attempted to limit the decomposition of peroxide loading; with the over arching aim to improve the efficiency of the bioconversion process. By employing several peroxide stabilizers and pre‐chelating the steam exploded recalcitrant substrates, we were able to substantially improve the delignification treatment of Douglas‐fir wood chips, and to reduce peroxide loading by more than 50% without negative effects on the hydrolysis rates and yield. Biotechnol. Bioeng. 2010;106: 884–893. © 2010 Wiley Periodicals, Inc.     

A rapid microassay to evaluate enzymatic hydrolysis of lignocellulosic substrates

Current attempts to produce ethanol from lignocellulosic biomass are focused on the optimization of pretreatment to reduce substrate recalcitrance and the improvement of enzymes for hydrolysis of the cellulose and hemicellulose components to produce fermentable sugars. Research aimed at optimizing both aspects of the bioconversion process involves assessment of the effects of multiple variables on enzyme efficiency, resulting in large factorial experiments with intensive assay requirements. A rapid assay for lignocellulose hydrolysis has been developed to address this need. Pretreated lignocellulose is formed into handsheets, which are then used to prepare small disks that are easily dispensed into microtiter plates. The hydrolysis of cellulose to glucose is estimated using an enzyme-coupled spectrophotometric assay. Using disks prepared from ethanol organosolv pretreated yellow poplar, it is shown that the assay generates data comparable with those produced by hydrolysis of pretreated yellow poplar pulp in Erlenmeyer flasks, followed by HPLC analysis of glucose. The assay shows considerable time and cost benefits over the standard assay protocol and is applicable to a broad range of lignocellulosic substrates.     

Enhanced enzymatic hydrolysis of lignocellulosic biomass pretreated by low-pressure steam autohydrolysis

Summary The use of low-pressure steam autohydrolysis in the pretreatment of corn stover and hybrid poplar has been assessed. In terms of yield of prehydrolyzed solids, minimal by-product formation and extent of subsequent enzymatic saccharification, the results of low-pressure steam pretreatment were found to be as good as or better than those reported for more severe pretreatment processes. Almost complete saccharification of the cellulose in the prehydrolyzed biomass solids was obtained within 24h with a commercial cellulase preparation — Celluclast. The presence of grinding elements (glass beads) during the enzymatic hydrolysis was found to increase the extent of saccharification by 40% to 50% over controls without any grinding elements.     

Fuzzy logic feedback control for fed-batch enzymatic hydrolysis of lignocellulosic biomass

A fuzzy logic feedback control system was developed for process monitoring and feeding control in fed-batch enzymatic hydrolysis of a lignocellulosic biomass, dilute acid-pretreated corn stover. Digested glucose from hydrolysis reaction was assigned as input while doser feeding time and speed of pretreated biomass were responses from fuzzy logic control system. Membership functions for these three variables and rule-base were created based on batch hydrolysis data. The system response was first tested in LabVIEW environment then the performance was evaluated through real-time hydrolysis reaction. The feeding operations were determined timely by fuzzy logic control system and efficient responses were shown to plateau phases during hydrolysis. Feeding of proper amount of cellulose and maintaining solids content was well balanced. Fuzzy logic proved to be a robust and effective online feeding control tool for fed-batch enzymatic hydrolysis.     

Fungal delignification of lignocellulosic biomass improves the saccharification of cellulosics

The biological delignification of lignocellulosic feedstocks, Prosopis juliflora and Lantana camara was carried out with Pycnoporus cinnabarinus, a white rot fungus, at different scales under solid-state fermentation (SSF) and the fungal treated substrates were evaluated for their acid and enzymatic saccharification. The fungal fermentation at 10.0 g substrate level optimally delignified the P. juliflora by 11.89% and L. camara by 8.36%, and enriched their holocellulose content by 3.32 and 4.87%, respectively, after 15 days. The fungal delignification when scaled up from 10.0 g to 75.0, 200.0 and 500.0 g substrate level, the fungus degraded about 7.69–10.08% lignin in P. juliflora and 6.89–7.31% in L. camara, and eventually enhanced the holocellulose content by 2.90–3.97 and 4.25–4.61%, respectively. Furthermore, when the fungal fermented L. camara and P. juliflora was hydrolysed with dilute sulphuric acid, the sugar release was increased by 21.4-42.4% and the phenolics content in hydrolysate was decreased by 18.46 and 19.88%, as compared to the unfermented substrate acid hydrolysis, respectively. The reduction of phenolics in acid hydrolysates of fungal treated substrates decreased the amount of detoxifying material (activated charcoal) by 25.0–33.0% as compared to the amount required to reduce almost the same level of phenolics from unfermented substrate hydrolysates. Moreover, an increment of 21.1–25.1% sugar release was obtained when fungal treated substrates were enzymatically hydrolysed as compared to the hydrolysis of unfermented substrates. This study clearly shows that fungal delignification holds potential in utilizing plant residues for the production of sugars and biofuels.     

Phospholipases. I. Effect ofn-alkanols on the rate of enzymatic hydrolysis of egg phosphatidylcholine

Summary The rate of hydrolysis of unsonicated liposomes of egg lecithin by phospholipase A (from bee venom and Russell viper venom) and phospholipase C (fromBacillus cereus andClostridium welchii) is markedly dependent on the nature and concentration of a variety of added alcohols. Typical plots of rate against alcohol concentration are bell-shaped. The maximum rate and the alcohol concentration at which it is achieved are alcohol-specific. In a homologous series ofn-alkanols, the maximal rates increase and the optimal concentrations decrease as the chain length is increased from C₄ to C₈. For longer alcohols (C₉ to C₁₂), progressively higher concentrations are required to elicit maximal activation. The optimal activating concentrationsC for C₄ to C₈ n-alkanols obey the relationshipp C=a logP _octanol+constant [cf. Hansch & Dunn,J. Pharm. Sci. 61:1 (1972)], suggesting that the alcohol-activating effect is a consequence of their incorporation into the liposomes with resultant modification of liposomal structure.     

Interdependence between lignocellulosic biomasses,enzymatic hydrolysis and yeast cell factories in biorefineries

Biorefineries have a pivotal role in the bioeconomy scenario for the transition from fossil-based processes towards more sustainable ones relying on renewable resources. Lignocellulose is a prominent feedstock since its abundance and relatively low cost. Microorganisms are often protagonists of biorefineries, as they contribute both to the enzymatic degradation of lignocellulose complex polymers and to the fermentative conversion of the hydrolyzed biomasses into fine and bulk chemicals. Enzymes have therefore become crucial for the development of sustainable biorefineries, being able to provide nutrients to cells from lignocellulose. Enzymatic hydrolysis can be performed by a portfolio of natural enzymes that degrade lignocellulose, often combined into cocktails. As enzymes can be deployed in different operative settings, such as separate hydrolysis and fermentation (SHF) or simultaneous saccharification and fermentation (SSF), their characteristics need to be combined with microbial ones to maximize the process. We therefore reviewed how the optimization of lignocellulose enzymatic hydrolysis can ameliorate bioethanol production when Saccharomyces cerevisiae is used as cell factory. Expanding beyond biofuels, enzymatic cocktail optimization can also be pivotal to unlock the potential of non-Saccharomyces yeasts, which, thanks to broader substrate utilization, inhibitor resistance and peculiar metabolism, can widen the array of feedstocks and products of biorefineries.     

Recent advances in understanding the role of cellulose accessibility in enzymatic hydrolysis of lignocellulosic substrates

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