Analysis of solids resulted from dilute-acid pretreatment of lignocellulosic biomass

Summary Lignocellulosic biomass materials were pretreated with dilute sulfuric acid, followed by analysis of partially and extensively washed pretreated solids. It is demonstrated that it is of crucial importance to perform extensive washing for accurate carbohydrate analyses to be obtained. Klason lignin, acid-soluble lignin, and ash analyses were not significantly affected by the extent of washing.     

Modeling of countercurrent shrinking-bed reactor in dilute-acid total-hydrolysis of lignocellulosic biomass

A mathematical model was developed for a countercurrent shrinking-bed reactor to investigate its performance in dilute-acid pretreatment/hydrolysis of lignocellulosic biomass. The results indicate that bed shrinking provides a positive effect on both hemicellulose and cellulose hydrolysis resulting high yield and product concentration. The effect of bed shrinking is more profound on cellulose hydrolysis than on hemicellulose hydrolysis. With dilute sulfuric acid (0.08 wt%) and with optimal adjustment of other operating parameters, the model predicts that near quantitative recovery of hemicellulose sugars is feasible. It further predicts that 80–90% yield with 2–4 wt% product concentration is attainable from hydrolysis of hardwood cellulose. The model also indicates that acid concentration and temperatures acutely affect the reactor performance in cellulose hydrolysis. In contrast, hemicellulose hydrolysis is less sensitive to acid concentration and temperature allowing broader latitude in operating conditions.     

Effects of thermo-chemical pre-treatment on anaerobic biodegradability and hydrolysis of lignocellulosic biomass

The effects of different thermo-chemical pre-treatment methods were determined on the biodegradability and hydrolysis rate of lignocellulosic biomass. Three plant species, hay, straw and bracken were thermo-chemically pre-treated with calcium hydroxide, ammonium carbonate and maleic acid. After pre-treatment, the plant material was anaerobically digested in batch bottles under mesophilic conditions for 40 days. From the pre-treatment and subsequent anaerobic digestion experiments, it was concluded that when the lignin content of the plant material is high, thermo-chemical pre-treatments have a positive effect on the biodegradability of the substrate. Calcium hydroxide pre-treatment improves the biodegradability of lignocellulosic biomass, especially for high lignin content substrates, like bracken. Maleic acid generates the highest percentage of dissolved COD during pre-treatment. Ammonium pre-treatment only showed a clear effect on biodegradability for straw.     

Conversion of bark-rich biomass mixture into fermentable sugar by two-stage dilute acid-catalyzed hydrolysis

Despite high availability and low cost, bark has not actively been considered as a biomass feedstock for producing bio-based products due to its high content of extractives and lignin. In this study, to investigate the feasibility of utilizing bark-rich sawmill residues for producing value-added materials, the mixed Hemlock hog fuel/pin chips (85:15 by dry weight) from a local sawmill were converted into fermentable sugar by two-stage dilute sulfuric acid-catalyzed hydrolysis. Combining the sugar yields from the first-stage (190 degrees C for 150 s with 1.1% acid) and second-stage (210 degrees C for 115 s with 2.5% acid) hydrolyses, which aimed to maximize the recovery of mannose/galactose and glucose, respectively, 13.6 g of glucose (46% theoretical maximum), 10.5 g of mannose and galactose (98% theoretical maximum), and 2.8 g of xylose (85% theoretical maximum) were obtained per 100 g of the original dry feedstock.     

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。     

Enzymatic hydrolysis of lignocellulosic biomass from Onopordum nervosum

Some properties of the cellulolytic complex obtained from Trichoderma reesei QM 9414 grown on Solka floc as carbon source and its ability to hydrolyze the lignocellulosic biomass of Onopordum nervosum Boiss were studied. The optimum enzyme activity was found at temperatures between 50 and 55 degrees C and pH ranging from 4.3 to 4.8. Hydrolysis of 4-nitropnenyl-beta-D-glucopyranoside (4-NPG) and cellobiose by the beta-glucosidase of the complex, showed competitive inhibition by glucose with a K(i) value of 0.8 mM for 4-NPG and 2. 56 mM for cellobiose. Enzymatic hydrolysis yield of Onopordum nervosum, evaluated as glucose production after 48 h, showed a threefold increase by pretreating the lignocellulosic substrate with alkali. When the loss of glucose incurred by de pretreatment was taken into account, a 160% increase in the final cellulose to glucose conversion was found to be due to the pretreatment.     

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.     

Compatible ionic liquid-cellulases system for hydrolysis of lignocellulosic biomass

Ionic liquids (ILs) have been increasingly recognized as novel solvents for dissolution and pretreatment of cellulose. However, cellulases are inactivated in the presence of ILs, even when present at low concentrations. To more fully exploit the benefits of ILs it is critical to develop a compatible IL‐cellulases system in which the IL is able to effectively solubilize and activate the lignocellulosic biomass, and the cellulases possess high stability and activity. In this study, we investigated the stability and activity of a commercially available cellulases mixture in the presence of different concentrations of 1‐ethyl‐3‐methylimidazolium acetate ([Emim][OAc]). A mixture of cellulases and β‐glucosidase (Celluclast1.5L, from Trichoderma reesei, and Novozyme188, from Aspergillus niger, respectively) retained 77% and 65% of its original activity after being pre‐incubated in 15% and 20% (w/v) IL solutions, respectively, at 50°C for 3 h. The cellulases mixture also retained high activity in 15% [Emim][OAc] to hydrolyze Avicel, a model substrate for cellulose analysis, with conversion efficiency of approximately 91%. Notably, the presence of different amounts of yellow poplar lignin did not interfere significantly with the enzymatic hydrolysis of Avicel. Using this IL‐cellulase system (15% [Emim][OAc]), the saccharification of yellow poplar biomass was also significantly improved (33%) compared to the untreated control (3%) during the first hour of enzymatic hydrolysis. Together, these findings provide compelling evidence that [Emim][OAc] was compatible with the cellulase mixture, and this compatible IL‐cellulases system is promising for efficient activation and hydrolysis of native biomass to produce biofuels and co‐products from the individual biomass components. Bioeng. 2011; 108:1042–1048. © 2010 Wiley Periodicals, Inc.     

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.     

Catalytic hydrolysis of lignocellulosic biomass into 5-hydroxymethylfurfural in ionic liquid

Production of 5-hydroxymethylfurfural (HMF) from cellulose catalyzed by solid acids and metal chlorides was studied in the 1-butyl-3-methylimidazolium chloride ([BMIM]Cl) under microwave irradiation. Among the applied catalysts, the use of CrCl₃/LiCl resulted in the highest yield of HMF. The effects of catalyst dosage (mole ratio of catalyst to glucose units in the feedstock) and reaction temperature on HMF yields were investigated to obtain optimal process conditions. With the 1:1 mol ratio of catalyst to glucose unit, the HMF yield reached 62.3% at 160 °C for 10 min. Untreated wheat straw was also investigated as feedstock to produce HMF for the practical use of raw biomass, in which the HMF yield was comparable to that from pure cellulose. After the extraction of HMF, [BMIM]Cl and CrCl₃/LiCl could be reused and exhibited no activity loss after three successive runs.     

Investigating the role of mechanics in lignocellulosic biomass degradation during hydrolysis

Enzymes and mechanics play major roles in lignocellulosic biomass deconstruction in biorefineries by catalyzing chemical cleavage or inducing physical breakdown of biomass, respectively. At industrially relevant substrate concentrations mechanical agitation is also a driving force for mass transfer as well as agglomeration of elongated biomass particles. Contrary to the physically induced particle attrition, which typically facilitates feedstock handling, particle agglomeration tends to hinder mass transfer and in the worst case induces processing difficulties like pipe blockage. Understanding the complex interplay between mechanical agitation and enzymatic degradation during hydrolysis is therefore critical and was the aim of this study. Particle size analyses revealed that neither mechanical agitation alone nor enzymatic treatment without mechanical agitation had any noteworthy effect on flax fiber attrition. Similarly, successive treatment, where mechanical agitation was either preceded or proceeded by enzymatic hydrolysis, did not induce any substantial segmentation of flax fibers. Simultaneous enzymatic and mechanical treatment on the other hand was found to promote fast fiber shortening. Higher hydrolysis yields, however, were obtained from nonagitated samples after prolonged enzymatic treatment, indicating that mechanical agitation in the long run reduces activity of the cellulolytic enzymes. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2754, 2019.     

Calculating sugar yields in high solids hydrolysis of biomass

Calculation of true sugar yields in high solids enzymatic hydrolysis of biomass is challenging due to the varying liquid density and liquid volume resulting from solid solubilization. Ignoring these changes in yield calculations can lead to significant errors. In this paper, a mathematical method was developed for the estimation of liquid volume change and thereafter the sugar yield. The information needed in the calculations include the compositions of the substrate, initial solids loading, initial liquid density, and sugar concentrations before and after hydrolysis. All of these variables are measurable with conventional laboratory procedures. This method was validated experimentally for enzymatic hydrolysis of dilute sulfuric acid pretreated corn stover at solid loadings up to 23% (w/w). The maximum relative error of predicted glucose yield from the true value was less than 4%. Compared to other methods reported in the literature, this method is relatively easy to use and provides good accuracy.     

Pretreatment of lignocellulosic biomass using ultrasound aiming at obtaining fermentable sugar

This study aims to evaluate the activity of the cellulase enzyme forward the use of ultrasound technology in different conditions of temperature, pH and exposure time, as well, to match the steps of pretreatment and enzymatic hydrolysis in one step. A central composite design (CCRD) and response surface analysis were used to evaluate the effect of ultrasound power, temperature and pH on enzyme activity. Optimum condition in the studied range was 30% for ultrasound power, pH 4.6 and 50?°C, yielding an enzyme activity of 15.5 UPF/mL. From this, we carried out kinetics of enzymatic hydrolysis on filter paper and bagasse malt, in optimized conditions. Total reducing sugars (TRS) were 3.85 and 0.46?mg/mL when the filter paper and bagasse malt were used as substrate, respectively. Ultrasound showed to be a good technology to increase the enzyme activity aiming to intensify enzymatic processes.     

Co‐hydrolysis of lignocellulosic biomass for microbial lipid accumulation

The herbaceous perennial energy crops miscanthus, giant reed, and switchgrass, along with the annual crop residue corn stover, were evaluated for their bioconversion potential. A co‐hydrolysis process, which applied dilute acid pretreatment, directly followed by enzymatic saccharification without detoxification and liquid–solid separation between these two steps was implemented to convert lignocellulose into monomeric sugars (glucose and xylose). A factorial experiment in a randomized block design was employed to optimize the co‐hydrolysis process. Under the optimal reaction conditions, corn stover exhibited the greatest total sugar yield (glucose + xylose) at 0.545 g g^?1 dry biomass at 83.3% of the theoretical yield, followed by switch grass (0.44 g g^?1 dry biomass, 65.8% of theoretical yield), giant reed (0.355 g g^?1 dry biomass, 64.7% of theoretical yield), and miscanthus (0.349 g g^?1 dry biomass, 58.1% of theoretical yield). The influence of combined severity factor on the susceptibility of pretreated substrates to enzymatic hydrolysis was clearly discernible, showing that co‐hydrolysis is a technically feasible approach to release sugars from lignocellulosic biomass. The oleaginous fungus Mortierella isabellina was selected and applied to the co‐hydrolysate mediums to accumulate fungal lipids due to its capability of utilizing both C5 and C6 sugars. Fungal cultivations grown on the co‐hydrolysates exhibited comparable cell mass and lipid production to the synthetic medium with pure glucose and xylose. These results elucidated that combining fungal fermentation and co‐hydrolysis to accumulate lipids could have the potential to enhance the utilization efficiency of lignocellulosic biomass for advanced biofuels production. Biotechnol. Bioeng. 2013; 110: 1039–1049. © 2012 Wiley Periodicals, Inc.     

Enzymatic hydrolysis of lignocellulosic biomass from low to high solids loading

Solid state enzymatic hydrolysis (SSEH) has many advantages, such as higher sugar concentration, lower operating costs, and less energy input. It should be a potential approach for the industrial application of lignocellulosic ethanol. The purpose of this work is to review the enzymatic hydrolysis of lignocellulosic biomass from low to high solids loading and introduce its both challenges and perspectives. The limitations of SSEH, including inhibition effects, water constraint, and rheology characteristic, are summarized firstly. Various strategies for overcoming these limitations are proposed correspondingly. Fed batch process and its feeding strategy to improve the SSEH efficiency are then discussed. Finally, several intensification methods, hydrolysis reactor, and pilot‐ and demonstration‐scale operations of SSEH are described. In‐depth analysis of main limitations and development of novel intensification methods and reactors should provide an effective way to achieve large‐scale implementation of SSEH.     

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.     

Binding characteristics of Trichoderma reesei cellulases on untreated, ammonia fiber expansion (AFEX), and dilute-acid pretreated lignocellulosic biomass

Studying the binding properties of cellulases to lignocellulosic substrates is critical to achieving a fundamental understanding of plant cell wall saccharification. Lignin auto-fluorescence and degradation products formed during pretreatment impede accurate quantification of individual glycosyl hydrolases (GH) binding to pretreated cell walls. A high-throughput fast protein liquid chromatography (HT-FPLC)-based method has been developed to quantify cellobiohydrolase I (CBH I or Cel7A), cellobiohydrolase II (CBH II or Cel6A), and endoglucanase I (EG I or Cel7B) present in hydrolyzates of untreated, ammonia fiber expansion (AFEX), and dilute-acid pretreated corn stover (CS). This method can accurately quantify individual enzymes present in complex binary and ternary protein mixtures without interference from plant cell wall-derived components. The binding isotherms for CBH I, CBH II, and EG I were obtained after incubation for 2 h at 4 °C. Both AFEX and dilute acid pretreatment resulted in increased cellulase binding compared with untreated CS. Cooperative binding of CBH I and/or CBH II in the presence of EG I was observed only for AFEX treated CS. Competitive binding between enzymes was found for certain other enzyme-substrate combinations over the protein loading range tested (i.e., 25-450 mg/g glucan). Langmuir single-site adsorption model was fitted to the binding isotherm data to estimate total available binding sites E(bm) (mg/g glucan) and association constant K(a) (L/mg). Our results clearly demonstrate that the characteristics of cellulase binding depend not only on the enzyme GH family but also on the type of pretreatment method employed.