Enhanced Xylanase Performance in the Hydrolysis of Lignocellulosic Materials by Surfactants and Non-catalytic Protein

Addition of additives has been confirmed to increase cellulase performance in the hydrolysis of lignocellulosic materials. In the hydrolysis of xylan-containing lignocellulosic biomass, xylanase can synergistically enhance the performance of cellulase. However, the role of additives in xylan hydrolysis by xylanase is not yet clear. In this work, with the presence of additives (bovine serum albumin, poly(ethylene glycol), and Tween), the hydrolysis of isolated xylan and the xylan in corn stover increased to different extents. Additives increased free xylanase in supernatants in the hydrolysis with xylanase, indicating the reduction of the adsorption of xylanase on corn stover and insoluble xylan. Enhanced hydrolysis of Avicel and corn stover by additives suggested that besides the prevention of unproductive binding of xylanase to lignin by additives, reducing the adsorption of xylanase on substrates was also contributed to enzymatic hydrolysis. The increment of xylanase activity by additives suggests that the additives were activators of xylanase. The results of this work indicate that the supplementation of additives could improve xylanase performance, synergistically enhanced the cellulose hydrolysis, and beneficial for the recycling of xylanase.     

Microbial Lipid Production from Corn Stover via Mortierella isabellina

Microbial lipid is a promising source of oil to produce biofuel if it can be generated from lignocellulosic materials. Mortierella isabellina is a filamentous fungal species featuring high content of oil in its cell biomass. In this work, M. isabellina was studied for lipid production from corn stover. The experimental results showed that M. isabellina could grow on different kinds of carbon sources including xylose and acetate, and the lipid content reached to 35 % at C/N ratio of 20. With dilution, M. isabellina could endure inhibition effects by dilute acid pretreatment of corn stover (0.3 g/L furfural, 1.2 g/L HMF, and 1 g/L 4-hydroxybenozic acid) and the strain formed pellets in the cell cultivations. An integrated process was developed combining the dilute acid pretreatment, cellulase hydrolysis, and cell cultivation for M. isabellina to convert corn stover to oil containing fungal biomass. With 7.5 % pretreated biomass solid loading ratio, the final lipid yield from sugar in pretreated biomass was 40 % and the final lipid concentration of the culture reached to 6.46 g/L.     

Effect of corn stover concentration on rheological characteristics

Corn stover, a well-known example of lignocellulosic biomass, is a potential renewable feed for bioethanol production. Dilute sulfuric acid pretreatment removes hemicellulose and makes the cellulose more susceptible to bacterial digestion. The rheologic properties of corn stover pretreated in such a manner were studied. The Power Law parameters were sensitive to corn stover suspension concentration becoming more non-Newtonian with slope n, ranging from 0.92 to 0.05 between 5 and 30% solids. The Casson and the Power Law models described the experimental data with correlation coefficients ranging from 0.90 to 0.99 and 0.85 to 0.99, respectively. The yield stress predicted by direct data extrapolation and by the Herschel-Bulkley model was similar for each concentration of corn stover tested.     

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 and Fractionation of Corn Stover by Soaking In Ethanol and Aqueous Ammonia

A new process for pretreatment of lignocellulosic biomass, designated the soaking in ethanol and aqueous ammonia (SEAA) process, was developed to improve hemicellulose preservation in solid form. In the SEAA process, an aqueous ammonia solution containing ethanol is used. Corn stover was treated with 15 wt.% ammonia at 1:9 solid–liquid ratio (by weight) at 60 °C for 24 h with ethanol added at 1, 5, 20, and 49 wt.% (balance was water). The extents by which xylan was solubilized with no ethanol and with ethanol added at 1, 5, 20, and 49 wt.% of the total liquid were 17.2%, 16.7%, 14.5%, 10.4%, and 6.3% of the original xylan, respectively. Thus, at the highest ethanol concentration used the loss of hemicellulose to the liquid phase was reduced by 63%. The digestibility of glucan and xylan in the pretreated corn stover samples by cellulase was not affected by ethanol addition of up to 20 wt.%. The enzymatic digestibility of the corn stover treated with 49 wt.% ethanol added was lower than the digestibility of the sample treated with no ethanol addition. Thus, based on these results, 20 wt.% was found to be the optimum ethanol concentration for use in the SEAA process for pretreatment of corn stover.     

Relative fermentability of lignocellulosic diluteacid prehydrolysates

The relative toxicity of the combined nonxylose components in prehydrolysates derived from three different lignocellulosic biomass feedstocks was determined. One woody (poplar) and two herbaceous (corn stover and switchgrass) feedstocks were dilute-acid pretreated under temperature and acid conditions chosen to optimize xylose recovery in the liquid prehydrolysate; xylose yields averaged 96,89,and 87% of theoretical for switchgrass,corn stover,and poplar,respectively. Prehydrolysates from each feedstock were neutralized,adjusted to equivalent xylose concentrations,and bioassayed for toxicity,using a standardized fermentation protocol withPichia stipitis NRRL 11545. Full time-courses for ethanol production (30-60 h) clearly illustrate the distinct inhibitory effects of prehydrolysates from different feedstocks. The relative toxicity of the prehydrolysates,ranked in order of decreasing toxicity,is poplar-derived prehydrolysates > switchgrass-derived prehydrolysates > corn stover-derived prehydrolysates. The inhibition of ethanol production appeared to be the result of a general slowdown of yeast metabolism,rather than the result of the production of alternative, nonethanol end products. Ethanol yields averaged 74,83,and 88% of control values for poplar,switchgrass,and corn stover prehydrolysates, respectively. Volumetric ethanol productivities (g ethanol L/h) averaged 32,70,and 102% of control values for poplar,switchgrass,and corn stover prehydrolysates,respectively. Ethanol productivities correlated closely with acetate concentrations in the prehydrolysates; however, regression lines correlating acetate concentrations and ethanol productivities were found to be feedstock-dependent. Oregon State University Agricultural Experiment Station Technical Publication Number 11114     

The effect of organic nitrogenous compound content and different pretreatments on agricultural lignocellulosic biomass characterization methods

Considering the diverse compounds contained in lignocellulosic biomasses, especially in agricultural biomass, and the complexity of the different chemical and physical structural connections between these compounds, appropriate analytical methods must be used to characterize these biomasses. In this study, two extraction processes and two analytical methods were used to characterize the main compounds in corn stover residue before and after different pretreatments. The results of this study suggest that pretreatment and organic nitrogenous compound content greatly affect the most common extraction processes and analytical methods used for biomass characterization. For raw corn stover biomass residue, the extraction process using neutral detergent fiber solvent removed more extractives and more organic nitrogenous compounds (18.5 and 2.4%, respectively) than the combination of extractions using water and ethanol (13.4% of extractives and 1.8% of organic nitrogenous compounds). This study also showed that the different pretreatments improve the efficiency of both extraction processes and that these processes seem to be more effective with the neutral detergent solvent extraction than the combination of extractions with water and ethanol. Organic nitrogenous compounds represent a significant proportion of agricultural biomasses; therefore, insufficient organic nitrogenous compound extraction can cause interference with different characterization methods. It was observed that, for the same analytical method and with the raw biomass, the value of insoluble lignin after the extractions with water and ethanol was higher than that obtained after the extraction with the neutral detergent solvent 16.1 and 13.2%, respectively. This can be explained by the fact that there are more organic nitrogenous compounds in the samples after the extraction with water and ethanol than in the samples after the extraction with the neutral detergent solvent. This result was also observed for the pretreated samples. These results, therefore, show that the extraction step is very important because it can remove more organic nitrogenous compounds before the main compounds are characterized (lignin, carbohydrates).     

Fungal Cellulase/Xylanase Production and Corresponding Hydrolysis Using Pretreated Corn Stover as Substrates

Three pretreated corn stover (ammonia fiber expansion, dilute acid, and dilute alkali) were used as carbon source to culture Trichoderma reesei Rut C-30 for cellulase and xylanase production. The results indicated that the cultures on ammonia fiber expansion and alkali pretreated corn stover had better enzyme production than the acid pretreated ones. The consequent enzymatic hydrolysis was performed applying fungal enzymes on pretreated corn stover samples. Tukey’s statistical comparisons exhibited that there were significant differences on enzymatic hydrolysis among different combination of fungal enzymes and pretreated corn stover. The higher sugar yields were achieved by the enzymatic hydrolysis of dilute alkali pretreated corn stover.     

Impacts of delignification and hot water pretreatment on the water induced cell wall swelling behavior of grasses and its relation to cellulolytic enzyme hydrolysis and binding

The relationships between biomass composition, water retention value (WRV), settling volume and enzymatic glucose yield and enzyme binding is investigated in this work by employing grasses pretreated with combinations of alkaline hydrogen peroxide (AHP) delignification and liquid hot water pretreatment that result in significant alterations of cell wall properties and subsequent enzymatic hydrolysis yields. Specifically, these cell wall treatments are performed on corn stover and switchgrass to generate material with a range of lignin (6–35 %) and xylan (2–28 %) contents as well as a range of other properties such as carboxylic acid content, water binding affinity and swellability. It was determined that WRV and settling volume are predictors of glucose yield (R² = 0.900 and 0.895 respectively) over the range of materials and treatment conditions used. It was also observed that mild AHP delignification can result in threefold increases in the WRV. Dynamic vapor sorption isotherms demonstrated that AHP-delignified corn stover exhibited an increased affinity for water sorption from the vapor phase relative to untreated corn stover. These results indicate that these water properties may be useful proxies for biomass susceptibility to enzymatic deconstruction.     

Quantitative ^31p NMR Analysis of Lignocellulosic Materials in Ionic Liquids

In an effort to utilize NMR technique for the analysis of the structural components of wood and biomasses such as lignin, we made a comparative study of lignin contents of several types of lignocellulosic materials both before and after enzymatic hydrolyses. To this end, Eucalyptus Globulus, Norway spruce thermomechanical pulp(TMP) corn stover and Eucalyptus kraft pulp were ball-milled and treated with cellulase to prepare materials with high lignin contents. These materials were analyzed via 31P NMR in ionic liquids. The results show that the contents of various functional groups for the lignocellulosic materials after enzymatic hydrolyses are generally in agreement with those from their corresponding enzymatic mild acidolysis liginins(EMALs), indicating that 31P NMR analysis of lignocellulosic materials in ionic liquids is a promising method for quantitative characterization of lignocellulosic materials.     

Influence of ionic-liquid incubation temperature on changes in cellulose structure,biomass composition,and enzymatic digestibility

Varying ionic liquid, 1-ethyl 3-methyl imidazolium acetate, pretreatment incubation temperature on lignocellulosic biomass substrates, corn stover, switchgrass and poplar, can have dramatic effects on the enzymatic digestibility of the resultant regenerated biomass. In order to delineate the chemical and physical changes resulting from the pretreatment process and correlate changes with enzymatic digestibility, X-ray powder and fiber diffraction, ¹³C cross polarization/magic angle spinning nuclear magnetic resonance spectroscopy, and compositional analysis was completed on poplar, corn stover and switchgrass samples. Optimal pretreatment incubation temperatures were most closely associated with the retention of amorphous substrates upon drying of regenerated biomass. Maximal glucan to glucose conversion for 24 h enzyme hydrolysis was observed for corn stover, switchgrass and poplar at ionic liquid incubation temperatures of 100, 110 and 120 °C, respectively. We hypothesize that effective pretreatment temperatures must attain lignin redistribution and retention of xylan for optimal enzyme digestibility.     

Pretreatment of Corn Stover with Twin-Screw Extrusion Followed by Enzymatic Saccharification

Pretreatment of biomass before subjecting it to enzyme saccharification is crucial with regards to facilitating access of enzyme to biomass. Extrusion, as a continuous and cost-effective pretreatment method, combines heating with high shear and mixing opening cell walls at the microscopic scale, thus largely increasing the specific surface area (SSA) of biomass for enzyme adsorption. The objective of this study was to examine the effect of extrusion as a pretreatment method and the underlying factors ruling the improvement of sugar yields. The optimum glucose, xylose, and combined sugar recoveries were 48.79%, 24.98%, and 40.07%, respectively, at 27.5% moisture content and 80 rpm screw speed. These yields were 2.2, 6.6, and 2.6 times higher than those for untreated corn stover. X-ray diffraction analysis showed that the crystallinity index was not a good indicator of sugar yield. However, scanning electron microscopy showed that the cellulose network was exposed due to the destruction of the lignin sheath. The Langmuir adsorption model was shown to be an effective tool for the estimation of the SSA of corn stover. The SSA of pretreated samples was significantly amplified over the control, revealing that extrusion can open the cell wall at the microscopic scale, which was especially favorable on sugar yields.     

Enzymatic production of xylooligosaccharides from corn stover and corn cobs treated with aqueous ammonia

A novel method of producing food-grade xylooligosaccharides from corn stover and corn cobs was investigated. The process starts with pretreatment of feedstock in aqueous ammonia, which results delignified and xylan-rich substrate. The pretreated substrates are subjected to enzymatic hydrolysis of xylan using endoxylanase for production of xylooligosaccharides. The conventional enzyme-based method involves extraction of xylan with a strong alkaline solution to form a liquid intermediate containing soluble xylan. This intermediate is heavily contaminated with various extraneous components. A costly purification step is therefore required before enzymatic hydrolysis. In the present method, xylan is obtained in solid form after pretreatment. Water-washing is all that is required for enzymatic hydrolysis of this material. The complex step of purifying soluble xylan from contaminant is essentially eliminated. Refining of xylooligosaccharides to food-grade is accomplished by charcoal adsorption followed by ethanol elution. Xylanlytic hydrolysis of the pretreated corn stover yielded glucan-rich residue that is easily digestible by cellulase enzyme. The digestibility of the residue reached 86% with enzyme loading of 10 filter paper units/g-glucan. As a feedstock for xylooligosaccharides production, corn cobs are superior to corn stover because of high xylan content and high packing density. The high packing density of corn cobs reduces water input and eventually raises the product concentration.     

Direct and simultaneous determination of representative byproducts in a lignocellulosic hydrolysate of corn stover via gas chromatography-mass spectrometry with a Deans switch

Pretreatment is one of the most important steps in producing fuel ethanol from lignocellulosic biomass. Simple, fast and accurate quantification of byproducts in lignocellulosic hydrolysates is critical to optimize the pretreatment procedures, but still a challenge. In this paper, a new GC-MS (SIM) method based on a Deans switch has been developed for the determination of byproducts in a corn stover hydrolysate. The Deans switch was incorporated into a hardware system that facilitated the direct aqueous injection (DAI) on GC-MS system. Simultaneous chromatographic separation and quantification of 18 byproducts including four aliphatic acids, five furan derivatives, four phenolic compounds and five others were achieved within 45 min. The detection limits of the presented method for various byproducts were in the range of 0.007-0.832 mg/L. The within-day and between-day precisions of the method were less than 6.0% (RSD, n=6). The accuracy of the method was confirmed with recoveries of 86-128%. A lignocellulosic hydrolysate sample of corn stover was successfully analyzed using this method, with aliphatic acids and furan derivatives accounting for 89.15% of the selected total byproducts.     

Enhancing the Enzymatic Hydrolysis of Corn Stover by an Integrated Wet-milling and Alkali Pretreatment

An integrated wet-milling and alkali pretreatment was applied to corn stover prior to enzymatic hydrolysis. The effects of NaOH concentration in the pretreatment on crystalline structure, chemical composition, and reducing-sugar yield of corn stover were investigated, and the mechanism of increasing reducing-sugar yield by the pretreatment was discussed. The experimental results showed that the crystalline structure of corn stover was disrupted, and lignin was removed, while cellulose and hemicellulose were retained in corn stover by the pretreatment with 1% NaOH in 1 h. The reducing-sugar yield from the pretreated corn stovers increased from 20.2% to 46.7% when the NaOH concentration increased from 0% to 1%. The 1% NaOH pretreated corn stover had a holocellulose conversion of 55.1%. The increase in reducing-sugar yield was related to the crystalline structure disruption and delignification of corn stover. It was clarified that the pretreatment significantly enhanced the conversion of cellulose and hemicellulose in the corn stover to sugars.     

Improved multivariate calibration models for corn stover feedstock and dilute-acid pretreated corn stover

We have studied rapid calibration models to predict the composition of a variety of biomass feedstocks by correlating near-infrared (NIR) spectroscopic data to compositional data produced using traditional wet chemical analysis techniques. The rapid calibration models are developed using multivariate statistical analysis of the spectroscopic and wet chemical data. This work discusses the latest versions of the NIR calibration models for corn stover feedstock and dilute-acid pretreated corn stover. Measures of the calibration precision and uncertainty are presented. No statistically significant differences (p = 0.05) are seen between NIR calibration models built using different mathematical pretreatments. Finally, two common algorithms for building NIR calibration models are compared; no statistically significant differences (p = 0.05) are seen for the major constituents glucan, xylan, and lignin, but the algorithms did produce different predictions for total extractives. A single calibration model combining the corn stover feedstock and dilute-acid pretreated corn stover samples gave less satisfactory predictions than the separate models.     

Chemical pretreatments of corn stover for enhancing enzymatic digestibility

Corn stover, the most abundant agricultural residue in Hungary, is a potential raw material for the production of fuel ethanol as a result of its high content of carbohydrates, but a pretreatment is required for its efficient hydrolysis. In this article, we describe the results using various chemicals such as dilute H₂SO₄, HCl, and NaOH separately as well as consecutively under relative mild conditions (120°C, 1h). Pretreatment with 5% H₂SO₄ or 5% HCl solubilized 85% of the hemicellulose fraction, but the enzymatic conversion of pretreated materials increased only two times compared to the untreated corn stover. Applying acidic pretreatment following a 1-d soaking in base achieved enzymatic conversion that was nearly the theoretical maximum (95.7%). Pretreatment with 10% NaOH decreased the lignin fraction >95%, increased the enzymatic conversion more than four times, and gave a 79.4% enzymatic conversion. However, by increasing the reaction time, the enzymatic degradability could also be increased significantly, using a less concentrated base. When the time of pretreatment was increased three times (0.5% NaOH at 120°C), the amount of total released sugars was 47.9 g from 100 g (dry matter) of untreated corn stover.     

Effects of temperature and moisture on dilute-acid steam explosion pretreatment of corn stover and cellulase enzyme digestibility

Corn stover is emerging as a viable feedstock for producing bioethanol from renewable resources. Dilute-acid pretreatment of corn stover can solubilize a significant portion of the hemicellulosic component and enhance the enzymatic digestibility of the remaining cellulose for fermentation into ethanol. In this study, dilute H₂SO₄ pretreatment of corn stover was performed in a steam explosion reactor at 160°C, 180°C, and 190°C, approx 1 wt% H₂SO₄, and 70-s to 840-s residence times. The combined severity (Log₁₀ [R _o ] - pH), an expression relating pH, temperature, and residence time of pretreatment, ranged from 1.8 to 2.4. Soluble xylose yields varied from 63 to 77% of theoretical from pretreatments of corn stover at 160 and 180°C. However, yields >90% of theoretical were found with dilute-acid pretreatments at 190°C. A narrower range of higher combined severities was required for pretreatment to obtain high soluble xylose yields when the moisture content of the acid-impregnated feedstock was increased from 55 to 63 wt%. Simultaneous saccharification and fermentation (SSF) of washed solids from corn stover pretreated at 190°C, using an enzyme loading of 15 filter paper units (FPU)/g of cellulose, gave ethanol yields in excess of 85%. Similar SSF ethanol yields were found using washed solid residues from 160 and 180°C pretreatments at similar combined severities but required a higher enzyme loading of approx 25 FPU/g of cellulose.     

Shrinking-bed model for percolation process applied to dilute-acid pretreatment/hydrolysis of cellulosic biomass

For many lignocellulosic substrates, hemicellulose is biphasic upon dilute-acid hydrolysis, which led to a modified percolation process employing simulated two-stage reverse-flow. This process has been proven to attain substantially higher sugar yields and concentrations over the conventional single-stage percolation process. The dilute-acid pretreatment of biomass solubilizes the hemicellulose fraction in the solid biomass, leaving less solid biomass in the reactor and reducing the bed. Therefore, a bed-shrinking mathematic kinetic model was developed to describe the two-stage reverse-flow reactor operated for hydrolyzing biphasic substrates, including hemicellulose, in corn cob/stover mixture (CCSM). The simulation indicates that the shrinking-bed operation increases the sugar yield by about 5%, compared to the nonshrinking bed operation in which 1 reactor volume of liquid passes through the reactor (i.e.,t = 1.0). A simulated optimal run further reveals that the fast portion of hemicellulose is almost completely hydrolyzed in the first stage, and the slow portion of hemicellulose is hydrolyzed in the second stage. Under optimal conditions, the bed shrank 27% (a near-maximum value), and a sugar yield over 95% was attained.     

Enhancement of Enzymatic Hydrolysis and Klason Lignin Removal of Corn Stover Using Photocatalyst-Assisted Ammonia Pretreatment

Photocatalyst-assisted ammonia pretreatment was explored to improve lignin removal of the lignocellulosic biomass for effective sugar conversion. Corn stover was treated with 5.0–12.5 wt.% ammonium hydroxide, two different photocatalysts (TiO₂ and ZnO) in the presence of molecular oxygen in a batch reactor at 60 °C. Various solid-to-liquid ratios (1:20–1:50) were also tested. Ammonia pretreatment assisted by TiO₂-catalyzed photo-degradation removed 70 % of Klason lignin under the optimum condition (12.5 % ammonium hydroxide, 60 °C, 24 h, solid/liquid?=?1:20, photocatalyst/biomass?=?1:10 with oxygen atmosphere). The enzymatic digestibilities of pretreated corn stover were 85 % for glucan and 75 % for xylan with NH₃-TiO₂-treated solid and 82 % for glucan and 77 % for xylan with NH₃-ZnO-treated solid with 15 filter paper units/g-glucan of cellulase and 30 cellobiase units/g-glucan of β-glucosidase, a 2–13 % improvement over ammonia pretreatment alone.