Quantitative assessment of the enzymatic degradation of amorphous cellulose by using a quartz crystal microbalance with dissipation monitoring

The systematic evaluation of the degradation of an amorphous cellulose film by a monocomponent endoglucanase (EG I) by using a quartz crystal microbalance with dissipation monitoring (QCM-D) identified several important aspects relevant to the study the kinetics of cellulose degradation by enzymes. It was demonstrated that, to properly evaluate the mechanism of action, steady state conditions in the experimental set up need to be reached. Rinsing or diluting the enzyme, as well as concentration of the enzyme, can have a pronounced effect on the hydrolysis. Quantification of the actual hydrolysis was carried out by measuring the film thickness reduction by atomic force microscopy after the enzymatic treatment. The values correlated well with the frequency data obtained by QCM-D measurement for corresponding films. This demonstrated that the evaluation of hydrolysis by QCM-D can be done quantitatively. Tuning of the initial thickness of films enabled variation of the volume of substrate available for hydrolysis which was then utilized in establishing a correlation between substrate volume and hydrolytic activity of EG I as measured by QCM-D. It was shown that, although the amount of substrate affects the absolute rate of hydrolysis, the relative rate of hydrolysis does not depend on the initial amount of substrate in steady state system. With this experimental setup it was also possible to demonstrate the impact of concentration on crowding of enzyme and subsequent hydrolysis efficiency. This effort also shows the action of EG I on a fully amorphous substrate as observed by QCM-D. The enzyme was shown to work uniformly within the whole volume of swollen film, however being unable to fully degrade the amorphous film.     

To understand the superior hydrolytic activity after polymorphic conversion from cellulose I to II from the adsorption behaviors of enzymes

The role of the cellulose ultrastructure on the relationship between cellulase binding and activity is not clear yet. In this article, a quartz crystal microbalance with dissipation (QCM-D) was employed to monitor the interactions between a given cellulase and the cellulose substrates with varied polymorphs of pure cellulose I and II and the intermediate state (I/II). Initially, cellulose nanocrystals (CNCs) with polymorphs of cellulose I, I/II and II were prepared and spin-coated on QCM sensors. The cellulose substrates’ crystallinity degree was examined by XRD, and morphology was detected by AFM. Then, a commercial cellulase from Trichoderma reesei was used to test the adsorption and hydrolysis of cellulose substrates with polymorphs of I, I/II and II, respectively. The results revealed that in the enzyme adsorption and desorption process at a temperature of 15 °C, CNC-II had the lowest adsorption capacity with a total adsorption mass of 179 ng cm^?2 but the highest reversible binding ratio of 33.7%; for comparison, the values were 235 ng cm^?2 versus 25.6% and 207 ng cm^?2 versus 26.9% for CNC-I and -I/II, respectively. And the conformation of adlayers on CNC-I, -I/II and -II derived from the QCM data became softer and softer in turn. On the other hand, CNC-II exhibited the best enzymatic hydrolytic ability among three substrates when enzymatic hydrolysis experiments were conducted at 45 °C. The results indicated that polymorphic conversion from I to II changes the affinity between the enzyme and cellulose surface; CNC-II has the lowest affinity to the enzyme, but the softer conformation of the adsorbed enzyme layer, and the more reversible adsorption may facilitate its hydrolytic activity. This article gives a perspective from the adsorption dynamics and conformation of the adsorbed enzyme layer, helping to understand the superior hydrolytic activity of cellulose with polymorph II. Thus, there is a potential of polymorphic conversion in the reduction of enzyme dosage and cost in the enzymatic hydrolysis process.     

Structural Insights into Rice Straw Pretreated by Hot-Compressed Water in Relation to Enzymatic Hydrolysis

Pretreatment-induced structural alteration is critical in influencing the rate and extent of enzymatic saccharification of lignocellulosic biomass. The present work has investigated structural features of rice straw pretreated by hot-compressed water (HCW) from 140 to 240 °C for 10 or 30 min and enzymatic hydrolysis profiles of pretreated rice straw. Compositional profiles of pretreated rice straw were examined to offer the basis for structural changes. The wide-angle X-ray diffraction analysis revealed possible modification in crystalline microstructure of cellulose and the severity-dependent variation of crystallinity. The specific surface area (SSA) of pretreated samples was able to achieve more than 10-fold of that of the raw material and was in linear relationship with the removal of acetyl groups and xylan. The glucose yield by enzymatic hydrolysis of pretreated materials correlated linearly with the SSA increase and the dissolution of acetyl and xylan. A quantitatively intrinsic relationship was suggested to exist between enzymatic hydrolysis and the extraction of hemicellulose components in hydrothermally treated rice straw, and SSA was considered one important structural parameter signaling the efficiency of enzymatic digestibility in HCW-treated materials in which hemicellulose removal and lignin redistribution happened.     

Evaluation and Characterization of Forage Sorghum as Feedstock for Fermentable Sugar Production

Sorghum is a tropical grass grown primarily in semiarid and drier parts of the world, especially areas too dry for corn. Sorghum production also leaves about 58 million tons of by-products composed mainly of cellulose, hemicellulose, and lignin. The low lignin content of some forage sorghums such as brown midrib makes them more digestible for ethanol production. Successful use of biomass for biofuel production depends on not only pretreatment methods and efficient processing conditions but also physical and chemical properties of the biomass. In this study, four varieties of forage sorghum (stems and leaves) were characterized and evaluated as feedstock for fermentable sugar production. Fourier transform infrared spectroscopy and X-ray diffraction were used to determine changes in structure and chemical composition of forage sorghum before and after pretreatment and the enzymatic hydrolysis process. Forage sorghums with a low syringyl/guaiacyl ratio in their lignin structure were easy to hydrolyze after pretreatment despite the initial lignin content. Enzymatic hydrolysis was also more effective for forage sorghums with a low crystallinity index and easily transformed crystalline cellulose to amorphous cellulose, despite initial cellulose content. Up to 72% hexose yield and 94% pentose yield were obtained using modified steam explosion with 2% sulfuric acid at 140 °C for 30 min and enzymatic hydrolysis with cellulase (15 filter per unit (FPU)/g cellulose) and β-glucosidase (50 cellobiose units (CBU)/g cellulose).     

Preparation of Langmuir/Blodgett-cellulose Surfaces by Using Horizontal Dipping Procedure. Application for Polyelectrolyte Adsorption Studies Performed with QCM-D

A method of preparing model cellulose surfaces by the Langmuir–Blodgett (LB) technique with horizontal dipping procedure has been developed. The primary aim for the use of these surfaces was adsorption studies performed with the quartz crystal microbalance with dissipation (QCM-D) instrument. Hydrophobised cellulose (trimethylsilyl cellulose, TMSC) was deposited on the hydrophobic, polystyrene-coated QCM-D crystal. After 15 dipping cycles, the TMSC film fully covers the crystal surface. TMSC can easily be hydrolysed back to cellulose with acid hydrolysis. With this method a smooth, rigid, thin and reproducible cellulose film was obtained. Its morphology, coverage, chemical composition and wetting was further characterised using atomic force microscopy (AFM), X-Ray photoelectron spectroscopy (XPS), and contact angle measurements. The swelling behaviour and the stability of the cellulose film in aqueous solutions at different ionic strengths were studied using the QCM-D instrument. The swelling/deswelling properties of the cellulose film were those expected of polyelectrolytes with low charge density; some swelling occurred in pure water and the swelling decreased when the ionic strength was increased. No significant layer softening was detected during the swelling. The effect of electrolyte concentration and polymer charge density on the adsorption of cationic polyelectrolytes on the cellulose surface was also investigated. At low electrolyte concentration less of the highly charged PDADMAC was adsorbed as compared to low charged C-PAM. The adsorbed amount of PDADMAC increased with increasing ionic strength and a more compact layer was formed while the effect of electrolyte concentration on the adsorption of C-PAM was not as pronounced.     

Effects of hemicellulose and lignin on enzymatic hydrolysis of cellulose from dairy manure

This study focused on the effect of hemicellulose and lignin on enzymatic hydrolysis of dairy manure and hydrolysis process optimization to improve sugar yield. It was found that hemicellulose and lignin in dairy manure, similar to their role in other lignocellulosic material, were major resistive factors to enzymatic hydrolysis and that the removal of either of them, or for best performance, both of them, improved the enzymatic hydrolysis of manure cellulose. This result combined with scanning electron microscope (SEM) pictures further proved that the accessibility of cellulose to cellulase was the most important feature to the hydrolysis. Quantitatively, fed-batch enzymatic hydrolysis of fiber without lignin and hemicellulose had a high glucose yield of 52% with respect to the glucose concentration of 17 g/L at a total enzyme loading of 1300 FPU/L and reaction time of 160 h, which was better than corresponding batch enzymatic hydrolysis.     

Enzymatic hydrolysis of native cellulose nanofibrils and other cellulose model films: effect of surface structure

Model films of native cellulose nanofibrils, which contain both crystalline cellulose I and amorphous domains, were used to investigate the dynamics and activities of cellulase enzymes. The enzyme binding and degradation of nanofibril films were compared with those for other films of cellulose, namely, Langmuir-Schaefer and spin-coated regenerated cellulose, as well as cellulose nanocrystal cast films. Quartz crystal microbalance with dissipation (QCM-D) was used to monitor the changes in frequency and energy dissipation during incubation at varying enzyme concentrations and experimental temperatures. Structural and morphological changes of the cellulose films upon incubation with enzymes were evaluated by using atomic force microscopy. The QCM-D results revealed that the rate of enzymatic degradation of the nanofibril films was much faster compared to the other types of cellulosic films. Higher enzyme loads did not dramatically increase the already fast degradation rate. Real-time measurements of the coupled contributions of enzyme binding and hydrolytic reactions were fitted to an empirical model that closely described the cellulase activities. The hydrolytic potential of the cellulase mixture was found to be considerably affected by the nature of the substrates, especially their crystallinity and morphology. The implications of these observations are discussed in this report.     

Understanding factors that limit enzymatic hydrolysis of biomass

Spectroscopic characterization of both untreated and treated material is being performed in order to determine changes in the biomass and the effects of pretreatment on crystallinity, lignin content, selected chemical bonds, and depolymerization of hemicellulose and lignin. The methods used are X-ray diffraction for determination of cellulose crystallinity (CrI); diffusive reflectance infrared (DRIFT) for changes in C-C and C-O bonds; and fluorescence to determine lignin content. Changes in spectral characteristics and crystallinity are statistically correlated with enzymatic hydrolysis results to identify and better understand the fundamental features of biomass that govern its enzymatic conversion to monomeric sugars. Models of the hydrolysis initial rate and 72 h extent of conversion were developed and evaluated. Results show that the hydrolysis initial rate is most influenced by the cellulose crystallinity, while lignin content most influences the extent of hydrolysis at 72 h. However, it should be noted that in this study only crystallinity, lignin, and selected chemical bonds were used as inputs to the models. The incorporation of additional parameters that affect the hydrolysis, like pore volume and size and surface area accessibility, would improve the predictive capability of the models.     

Evaluation of wet oxidation pretreatment for enzymatic hydrolysis of softwood

The wet oxidation pretreatment (water, oxygen, elevated temperature, and pressure) of softwood (Picea abies) was investigated for enhancing enzymatic hydrolysis. The pretreatment was preliminarily optimized. Six different combinations of reaction time, temperature, and pH were applied, and the compositions of solid and liquid fractions were analyzed. The solid fraction after wet oxidation contained 58–64% cellulose, 2–16% hemicellulose, and 24–30% lignin. The pretreatment series gave information about the roles of lignin and hemicellulose in the enzymatic hydrolysis. The temperature of the pretreatment, the residual hemicellulose content of the substrate, and the type of the commercial cellulase preparation used were the most important factors affecting the enzymatic hydrolysis. The highest sugar yield in a 72-h hydrolysis, 79% of theoretical, was obtained using a pretreatment of 200°C for 10 min at neutral pH.     

Influence of Homogenization Treatment on Physicochemical Properties and Enzymatic Hydrolysis Rate of Pure Cellulose Fibers

The aim of this study is to compare the effect of different homogenization treatments on the physicochemical properties and the hydrolysis rate of a pure bleached cellulose. Results obtained show that homogenization treatments improve the enzymatic hydrolysis rate of the cellulose fibers by 25 to 100 %, depending of the homogenization treatment applied. Characterization of the samples showed also that homogenization had an impact on some physicochemical properties of the cellulose. For moderate treatment intensities (pressure below 500 b and degree of homogenization below 25), an increase of water retention values (WRV) that correlated to the increase of the hydrolysis rate was highlighted. Result also showed that the overall crystallinity of the cellulose properties appeared not to be impacted by the homogenization treatment. For higher treatment intensities, homogenized cellulose samples developed a stable tridimentional network that contributes to decrease cellulase mobility and slowdown the hydrolysis process.     

Pretreatment with ammonia water for enzymatic hydrolysis of corn husk, bagasse, and switchgrass

Bagasse, corn husk, and switchgrass were pretreated with ammonia water to enhance enzymatic hydrolysis. The sample (2 g) was mixed with 1–6 mL ammonia water (25–28% ammonia) and autoclaved at 120°C for 20 min. After treatment, the product was vacuum-dried to remove ammonia gas. The dried solid could be used immediately in the enzymatic hydrolysis without washing. The enzymatic hydrolysis was effectively improved with more than 0.5 and 1 mL ammonia water/g for corn husk and bagasse, respectively. In bagasse, glucose, xylose, and xylobiose were the main products. The adsorption of CMCase and xylanase was related to the initial rate of enzymatic hydrolysis. In corn husks, arabinoxylan extracted by pretreatment was substantially unhydrolyzed because of the high ratio of arabinose to xylose (0.6). The carbohydrate yields from cellulose and hemicellulose were 72.9% and 82.4% in bagasse, and 86.2% and 91.9% in corn husk, respectively. The ammonia/water pretreatment also benefited from switchgrass (Miscanthus sinensis and Solidago altissima L.) hydrolysis.     

Pretreatment of corn stover with acidic electrolyzed water and FeCl3 leads to enhanced enzymatic hydrolysis

Pretreatment has been viewed as the most efficient strategy for lignocellulosic biomass-to-fermentable sugars conversion. In this study a novel pretreatment with acidic electrolyzed water (AEW) and FeCl₃ was proposed and tested to deconstruct the recalcitrance of corn stover and enhance its subsequent cellulose-to-sugar conversion. The effects of AEW pH and FeCl₃ concentration on hemicellulose degradation were investigated, and the results showed the highest hemicellulose removal (93.40 %) and recovery (93.04 %) were achieved at AEW pH 2.30 and FeCl₃ concentration 0.05 mol/L. Further research on the properties of AEW solutions with FeCl₃, including their pH, ORP, and DO revealed the synergistic effects of strong acidity and high oxidizing capacity of the solution could boost hemicellulose breakup and enhance the enzymatic hydrolysis of cellulose (92.00 %) by removing most of hemicellulose and increasing the accessibility and digestibility of cellulose. Therefore, these studies prove AEW coupled with FeCl₃ pretreatment is an effective and promising approach in biomass-to-biofuel process.     

Pretreatment of corn stover using wet oxidation to enhance enzymatic digestibility

Corn stover is an abundant, promising raw material for fuel ethanol production. Although it has a high cellulose content, without pretreatment it resists enzymatic hydrolysis, like most lignocellulosic materials. Wet oxidation (water, oxygen, mild alkali or acid, elevated temperature and pressure) was investigated to enhance the enzymatic digestibility of corn stover. Six different combinations of reaction temperature, time, and pH were applied. The best conditions (60g/L of corn stover, 195°C, 15 min, 12 bar O₂, 2 g/L of Na₂CO₃) increased the enzymatic conversion of corn stover four times, compared to untreated material. Under these conditions 60% of hemicellulose and 30% of lignin were solubilized, whereas 90% of cellulose remained in the solid fraction. After 24-h hydrolysis at 50°C using 25 filter paper units (FPU)/g of dry matter (DM) biomass, the achieved conversion of cellulose to glucose was about 85%. Decreasing the hydrolysis temperature to 40°C increased hydrolysis time from 24 to 72 h. Decreasing the enzyme loading to 5 FPU/g of DM biomass slightly decreased the enzymatic conversion from 83.4 to 71%. Thus, enzyme loading can be reduced without significantly affecting the efficiency of hydrolysis, an important economical aspect.     

Pretreatment of Reed by Wet Oxidation and Subsequent Utilization of the Pretreated Fibers for Ethanol Production

Common reed (Phragmites australis) is often recognized as a promising source of renewable energy. However, it is among the least characterized crops from the bioethanol perspective. Although one third of reed dry matter is cellulose, without pretreatment, it resists enzymatic hydrolysis like lignocelluloses usually do. In the present study, wet oxidation was investigated as the pretreatment method to enhance the enzymatic digestibility of reed cellulose to soluble sugars and thus improve the convertibility of reed to ethanol. The most effective treatment increased the digestibility of reed cellulose by cellulases more than three times compared to the untreated control. During this wet oxidation, 51.7% of the hemicellulose and 58.3% of the lignin were solubilized, whereas 87.1% of the cellulose remained in the solids. After enzymatic hydrolysis of pretreated fibers from the same treatment, the conversion of cellulose to glucose was 82.4%. Simultaneous saccharification and fermentation of pretreated solids resulted in a final ethanol concentration as high as 8.7 g/L, yielding 73% of the theoretical.     

The Effect of Alkaline Addition in Hydrothermal Pretreatment of Empty Fruit Bunches on Enzymatic Hydrolysis Efficiencies

In general, lignocellulosic biomass contains three major components, namely lignin, hemicellulose and cellulose which are the polymers of C5 and C6 sugars. Thus, there is potential to utilize of this biomass for bioethanol production. The hydrolysis of cellulose into glucose was difficult due to the more fibrous nature and thus inhibit enzyme penetration into the cellulose. In order to solve this problem, hydrothermal pretreatment can be used for breaking the bonds within the lignin structure and increase the accessibility of enzyme into the cellulose. In this study, the effect of chemical addition, sodium hydroxide (NaOH) and calcium oxide (CaO) in hydrothermal pretreatment at 180 °C and 30 minutes reaction time of palm oil empty fruit bunches (EFB) on the enzymatic hydrolysis efficiencies was investigated. The enzymatic hydrolysis of hydrothermally pretreated EFB give the highest concentration of glucose at 0.67 g/L while the hydrothermally pretreated of EFB in the presence of NaOH gives the lowest glucose concentration 0.45 g/L.     

Bioconversion of mixed solids waste to ethanol

A mixed solids waste (MSW) feedstock, comprising construction lumber waste (35% oven-dry basis), alm ond treeprunings (20%), wheat straw (20%), office waste paper (12.5%), and newsprint (12.5%), was converted to ethanol via dilute-acid pretreatment followed by enzymatic hydrolysis and yeast fermentation. The MSW was pretreated with dilute sulfuricacid (0.4% w/w) at 210°C for 3 min in a 4-L stea mexplosion reactor, then washed with water to recover the solubilized hemicellulose. The digestibility of water-washed, pretreated MSW was 90% in batch enzymatic hydrolysis at 66 FPU/g cellulose. Using an enzyme-recycle bioreactor system, greater than 90% cellulose hydrolysis was achieved at a net enzyme loading of about 10 FPU/g cellulose. Enzyme recycling using mebrane filtration and a fed-batch fermentation technique is a promising option for significantly reducing the cost of enzyme in cellulose hydrolysis. The hexosesugars were readily fermentable using a Saccharomyces cerevisiae yeast strain that was adapted to the hydrolysate. Solid residue after enzyme digestion was subjected to various furnace experiments designed to assess the fouling and slagging characteristics. Results of these analyses suggest the residue to be of a low to moderate slagging and fouling type if burned by itself.     

Comparison of hot-water extraction and steam treatment for production of high purity-grade dissolving pulp from green bamboo

The performance of hot-water extraction (HWE) and steam treatment (ST), followed by kraft pulping were compared for production of high purity-grade dissolving pulp from green bamboo. With the same prehydrolysis intensity (represented by the P-factor), the fractionation efficiency of HWE is far lower than that of ST. Because of lower removal of non-cellulosic components, the solid residue from HWE (even at approximately double the prehydrolysis intensity, P-factor = 1,379) required more active alkali (AA) during kraft pulping to obtain a cellulose purity equivalent to that achieved by the ST (P-factor = 756)-kraft process. To reach equivalent hemicellulose removal, HWE required more severe intensity than ST. However, FTIR and SEM characterizations of solid residue confirmed that intensified HWE resulted in significant lignin condensation. Antagonistic effects of hemicellulose removal and lignin condensation extent on subsequent kraft pulping were therefore more apparent in HWE than that in ST. Under the same kraft pulping conditions, lignin condensation from a severely intensified HWE process (P-factor = 2,020) caused greater cellulose yield and viscosity loss than that found for ST. Finally, at a given residual pentosan or lignin content, the cellulose yields from all HWE-kraft pulps were about 3 % lower than those from ST-kraft pulps. Consequently, based on an optimally setup chlorine dioxide bleaching stage, a cellulosic pulp with alpha-cellulose content of 97.6 % and viscosity of 927 mL/g was successfully produced from a ST-kraft pulp (P-factor = 756, AA = 19 %).     

Effect of alkaline ultrasonic pretreatment on crystalline morphology and enzymatic hydrolysis of cellulose

In this study, ultrasound-assisted alkaline pretreatment is developed to evaluate the morphological and structural changes that occur during pretreatment of cellulose, and its effect on glucose production via enzymatic hydrolysis. The pretreated samples were characterized using scanning electron microscopy, infrared spectroscopy, and X-ray diffraction to understand the change in surface morphology, crystallinity and the fraction of cellulose Iβ and cellulose II. The combined pretreatment led to a great disruption of cellulose particles along with the formation of large pores and partial fibrillation. The effects of ultrasound irradiation time (2, 4 h), NaOH concentration (1–10 wt%), initial particle size (20–180 μm) and initial degree of polymerization (DP) of cellulose on structural changes and glucose yields were evaluated. The alkaline ultrasonic pretreatment resulted in a significant decrease in particle size of cellulose, besides significantly reducing the treatment time and NaOH concentration required to achieve a low crystallinity of cellulose. More than 2.5 times improvement in glucose yield was observed with 10 wt% NaOH and 4 h of sonication, compared to untreated samples. The glucose yields increased with increase in initial particle size of cellulose, while DP had no effect on glucose yields. The glucose yields exhibited an increasing tendency with increase in cellulose II fraction as a result of combined pretreatment.     

Optimizing Phosphoric Acid plus Hydrogen Peroxide (PHP) Pretreatment on Wheat Straw by Response Surface Method for Enzymatic Saccharification

Wheat straw was pretreated by phosphoric acid plus hydrogen peroxide (PHP), in which temperature, time, and H₃PO₄ proportion for pretreatment were investigated by using response surface method. Results indicated that hemicellulose and lignin removal positively responded to the increase of pretreatment temperature, H₃PO₄ proportion, and time. H₃PO₄ proportion was the most important variable to control cellulose recovery, followed by pretreatment temperature and time. Moreover, these three variables all negatively related to cellulose recovery. Increasing H₃PO₄ proportion can improve enzymatic hydrolysis; however, reduction on cellulose recovery results in decrease of glucose yield. Extra high temperature or long time for pretreatment was not beneficial to enzymatic hydrolysis and glucose yield. Based on the criterion for minimizing H₃PO₄ usage and maximizing glucose yield, the optimized pretreatment conditions was 40 °C, 2.0 h, and H₃PO₄ proportion of 70.2 % (H₂O₂ proportion of 5.2 %), by which glucose yielded 299 mg/g wheat straw (946.2 mg/g cellulose) after 72-h enzymatic hydrolysis.