cellulase and 5-hydroxymethylfurfural

Caffeic acid (3,4-dihydroxycinnamic acid) serves as a building block for thermoplastics and a precursor for biologically active compounds and was recently produced from glucose by microbial fermentation. To produce caffeic acid from inedible cellulose, separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) reactions were compared using kraft pulp as lignocellulosic feedstock. Here, a tyrosine-overproducing Escherichia coli strain was metabolically engineered to produce caffeic acid from glucose by introducing the genes encoding a 4-hydroxyphenyllactate 3-hydroxylase (hpaBC) from Pseudomonas aeruginosa and tyrosine ammonia lyase (fevV) from Streptomyces sp. WK-5344. Using the resulting recombinant strain, the maximum yield of caffeic acid in SSF (233 mg/L) far exceeded that by SHF (37.9 mg/L). In the SSF with low cellulase loads (≤2.5 filter paper unit/g glucan), caffeic acid production was markedly increased, while almost no glucose accumulation was detected, indicating that the E. coli cells experienced glucose limitation in this culture condition. Caffeic acid yield was also negatively correlated with the glucose concentration in the fermentation medium. In SHF, the formation of by-product acetate and the accumulation of potential fermentation inhibitors increased significantly with kraft pulp hydrolysate than filter paper hydrolysate. The combination of these inhibitors had synergistic effects on caffeic acid fermentation at low concentrations. With lower loads of cellulase in SSF, less potential fermentation inhibitors (furfural, 5-hydroxymethyfurfural, and 4-hydroxylbenzoic acid) accumulated in the medium. These observations suggest that glucose limitation in SSF is crucial for improving caffeic acid yield, owing to reduced by-product formation and fermentation inhibitor accumulation.

Topics: Acetates; Ammonia-Lyases; Biomass; Caffeic Acids; Cellulase; Culture Media; Escherichia coli; Fermentation; Furaldehyde; Glucose; Hydrogen-Ion Concentration; Hydrolysis; Lignin; Metabolic Engineering; Pseudomonas aeruginosa; Recombinant Proteins; Streptomyces

The aim of this work was to investigate the kinetics of multiple chemicals in acid bisulfite pretreatment and the relationship between total sugar yields and pretreatment factors (temperature and time). The results showed Saeman model accurately fitted the pretreatment process. According to this kinetic model, a maximum hemicellulose hydrolysis yield was achieved at a treatment time of 75min with a temperature of 145°C. Meantime, the concentrations of acetic acid, hydroxymethylfurfural (HMF), and furfural were 1.54, 0.60, and 1.15gL

Topics: Acetic Acid; Carbohydrates; Cellulase; Furaldehyde; Hydrolysis; Kinetics; Lignin; Models, Theoretical; Pseudotsuga; Sulfites; Temperature

In the storage of Radix Ophiopogonis, browning often happens to cause potential risk with regard to safety. Previously few reports investigate the browning of Radix Ophiopogonis. In this research, the causes and mechanisms of the browning of Radix Ophiopogonis were preliminarily elucidated. Content determination by high-performance liquid chromatography (HPLC) and spectrophotometry, enzyme activity determination by colorimetry, and morphological observation by electron microscopy were performed in the present study. Uniform design and three-dimensional response surfaces were applied to investigate the relationship between browning and storage factors. The cortex cell wall of browned Radix Ophiopogonis was ruptured. Compared with the normal Radix Ophiopogonis, cellulase and polyphenol oxidase enzymes were activated, the levels of 5-hydroxymethylfurfural (5-HMF), total sugars, and reducing sugars were increased, while the levels of polysaccharides and methylophiopogonanone A were decreased in browned Radix Ophiopogonis. The relationship between the storage factors and degree of browning (Y) could be described by following correlation equation: Y = - 0.625 4 + 0.020 84 × X3 + 0.001 514 × X1 × X2 - 0.000 964 4 × X2 × X3. Accompanied with browning under storage conditions, the chemical composition of Radix Ophiopogonis was altered. Following the activation of cellulase, the rupture of the cortex cell wall and the outflow of cell substances flowed out, which caused the Radix Ophiopogonis tissue to become soft and sticky. The main causes of the browning were the production of 5-HMF, the activation of polyphenol oxidase, Maillard reactions and enzymatic browning. Browning could be effectively prevented when the air relative humidity (HR), temperature, and moisture content were under 25% RH, 12 °C and 18%, respectively.

Topics: Carbohydrates; Catechol Oxidase; Cell Wall; Cellulase; Chromatography, High Pressure Liquid; Food Storage; Furaldehyde; Humidity; Maillard Reaction; Ophiopogon; Temperature

A renewable raw material, rice straw is pretreated for biorefinery usage. Solution-state two-dimensional (2D) 1H-13 C hetero-nuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectroscopy, was used to analyze 13 cultivars of rice straw before and after dilute acid pretreatment, to characterize general changes in the lignin and polysaccharide components. Intensities of most (15 of 16) peaks related to lignin aromatic regions, such as p-coumarate, guaiacyl, syringyl, p-hydroxyphenyl, and cinnamyl alcohol, and methoxyl, increased or remained unchanged after pretreatment. In contrast, intensities of most (11 of 13) peaks related to lignin aliphatic linkages or ferulate decreased. Decreased heterogeneity in the intensities of three peaks related to cellulose components in acid-insoluble residues resulted in similar glucose yield (0.45-0.59 g/g-dry biomass). Starch-derived components showed positive correlations (r = 0.71 to 0.96) with glucose, 5-hydroxymethylfurfural (5-HMF), and formate concentrations in the liquid hydrolysates, and negative correlations (r = -0.95 to -0.97) with xylose concentration and acid-insoluble residue yield. These results showed the fate of lignin and polysaccharide components by pretreatment, suggesting that lignin aromatic regions and cellulose components were retained in the acid insoluble residues and starch-derived components were transformed into glucose, 5-HMF, and formate in the liquid hydrolysate.

Topics: Acids; Biomass; Carbon-13 Magnetic Resonance Spectroscopy; Cellulase; Formates; Furaldehyde; Glucose; Hydrolysis; Lignin; Oryza; Plant Stems; Polysaccharides; Solutions

A novel conversion process using steam explosion combined with enzymatic digestibility was exploited to increase sugar yield. Results showed that glucan and xylan recovery decreased with the increase of holding temperature and residence time in SE, respectively, while glucan and xylan conversion exhibited an opposite trend. The optimal conditions of steam explosion were 160 °C and 48 min, under which glucan and xylan recovery was 93.4% and 71.6%, respectively. Glucan and xylan conversion at 18% solid loading by periodic peristalsis increased by 3.4-5.8% and 4.5-6.2%, respectively, compared with that by water baths shaker. In the whole process, glucose, xylose and total sugar yield reached to 77.3%, 62.8% and 72.3%, respectively. The yield of hydroxymethyl furfural, furfural and lignin-derived products was 6.3 × 10(-2), 7.5 × 10(-2) and less than 3.7 × 10(-2) g/100 g feedstock, respectively. This novel conversion process increased sugar recovery, reduced degradation products formation, improved digestibility efficiency, and hence increased sugar yield.

Topics: Biomass; Biotechnology; Cellulase; Furaldehyde; Glucans; Glucose; Kinetics; Lignin; Pressure; Steam; Temperature; Waste Products; Water; Xylans; Xylose; Zea mays

Steam refining of non-debarked spruce forest residues was investigated as pretreatment for enzymatic hydrolysis as well as for biogas production. Pretreatment conditions were varied in the range of 190-220 °C, 5-10 min and 0-3.7% SO₂ according to a statistical design. For both applications highest product yields were predicted at 220 °C and 2.4% SO₂, whereas the reaction time had only a minor influence. The conformity of the model results allows the conclusion that enzymatic hydrolysis is a suitable test method to evaluate the degradability of lignocellulosic biomass in the biogas process. In control experiments under optimal conditions the results of the model were verified. The yield of total monomeric carbohydrates after enzymatic hydrolysis was equivalent to 55% of all theoretically available polysaccharides. The corresponding biogas yield from the pretreated wood amounted to 304 mL/gODM. Furthermore, furans produced under optimal process conditions showed no inhibitory effect on biogas production. It can be concluded that steam refining opens the structure of wood, thus improving the enzymatic hydrolysis of the polysaccharides to fermentable monomeric sugars and subsequently enabling a higher and faster production of biogas. Anaerobic fermentation of pretreated wood is a serious alternative to alcoholic fermentation especially when low quality wood grades and residues are used. Anaerobic digestion should be further investigated in order to diversify the biorefinery options for lignocellulosic materials.

Topics: beta-Glucosidase; Biofuels; Cellulase; Furaldehyde; Hydrolysis; Models, Statistical; Picea; Sulfur Dioxide; Temperature; Time Factors; Trees

Pretreatment is essential in the production of alcohol from lignocellulosic material. In order to increase enzymatic sugar release and bioethanol production, thermal, dilute acid, dilute basic and alkaline peroxide pretreatments were applied to wheat straw. Compositional changes in pretreated solid fractions and sugars and possible inhibitory compounds released in liquid fractions were analysed. SEM analysis showed structural changes after pretreatments. Enzymatic hydrolysis and fermentation by Pichia stipitis of unwashed and washed samples from each pretreatment were performed so as to compare sugar and ethanol yields. The effect of the main inhibitors found in hydrolysates (formic acid, acetic acid, 5-hydroxymethylfurfural and furfural) was first studied through ethanol fermentations of model media and then compared to real hydrolysates. Hydrolysates of washed alkaline peroxide pretreated biomass provided the highest sugar concentrations, 31.82g/L glucose, and 13.75g/L xylose, their fermentation yielding promising results, with ethanol concentrations reaching 17.37g/L.

Topics: Acetic Acid; beta-Glucosidase; Biotechnology; Carbohydrates; Cellulase; Chemical Fractionation; Ethanol; Fermentation; Formates; Furaldehyde; Glucose; Hydrolysis; Pichia; Solubility; Solutions; Triticum; Waste Products; Xylose

Sugarcane bagasse was pretreated with ozone to increase lignocellulosic material digestibility. Bagasse was ozonated in a fixed bed reactor at room temperature, and the effect of the two major parameters, ozone concentration and sample moisture, was studied. Acid insoluble and total lignin decreased whereas acid soluble lignin increased in all experiments. Pretreatment barely attacked carbohydrates, with cellulose and xylan recovery rates being >92%. Ozonolysis increased fermentable carbohydrate release considerably during enzymatic hydrolysis. Glucose and xylose yields increased from 6.64% and 2.05%, for raw bagasse, to 41.79% and 52.44% under the best experimental conditions. Only xylitol, lactic, formic and acetic acid degradation compounds were found, with neither furfural nor HMF (5-hydroxymethylfurfural) being detected. Washing detoxification provided inhibitor removal percentages above 85%, increasing glucose hydrolysis, but decreasing xylose yield by xylan solubilization. SEM analysis showed structural changes after ozonization and washing.

Topics: Acids; Biotechnology; Cellulase; Cellulose; Furaldehyde; Glucose; Hydrolysis; Lignin; Ozone; Saccharum; Solubility; Time Factors

In order to improve the efficiency of distillers' grains converting to ethanol, 13 dilute solution explosions were evaluated based on the optimization of pure water explosion. To decrease residual inhibitor content, the exploded slurry was dried at 105°C. Using a 1.1 mol/L butanone solution explosion, with the explosion temperature set at 160°C (pressure at 1.9 MPa), the residence time at 10 min, and the dried distillers' grains-to-water ratio at 1:2 (w/w), the yields of total sugar, glucose, and xylose were 86%, 89%, and 84% (w/w), respectively, and the ethanol yield was 25.3 g/100 g distillers' grains dry matter. Moreover, the eight other reagent solution explosions improved the efficiency of enzymatic hydrolysis, and of simultaneous saccharification and co-fermentation, and the residual contents of furfural, 5-hydroxymethylfurfural, and acetic acid decreased to an acceptable concentration range after detoxification by drying. The results suggested that compared with pure water explosions, the use of volatile solutions lowered the explosive temperature and improved the sugar yield. This study offers a reference for the further study of lignocellulosic materials with higher starch and hemicelluloses contents as raw materials for converting biomass to bioethanol.

Topics: beta-Glucosidase; Biofuels; Biomass; Bioreactors; Candida; Cellulase; Desiccation; Edible Grain; Endo-1,4-beta Xylanases; Ethanol; Explosions; Fermentation; Furaldehyde; Glucose; Hydrolysis; Lignin; Saccharomyces cerevisiae; Temperature; Water; Xylose

Soybean hulls were subjected to thermo-mechanical extrusion pretreatment at various in-barrel moisture contents and screw speeds. Extrusion degraded the lignocellulosic structure and enhanced enzymatic hydrolysis of soybean hulls, with up to 155% increase in glucose yield as compared to untreated substrate. Greater glucose yields were observed at higher in-barrel moistures (45% and 50%) and lower screw speed (280 and 350 rpm). Maximum 74% cellulose to glucose conversion resulted from using a two-enzyme cocktail consisting of cellulase and β-glucosidase. Conversion increased to 87% when a three-enzyme cocktail having a cell wall degrading enzyme complex was used for hydrolysis. Fermentation inhibitors, such as furfural, 5-(hydroxymethyl)-2-furaldehyde (HMF), and acetic acid, were found in the extrusion pretreated soybean hulls and hydrolysate. However, their concentrations were below the known thresholds for inhibition. Fermentation of hydrolysate by Saccharomyces cerevisiae led to high yields of ethanol, with concentration ranging from 13.04 to 15.44 g/L.

Topics: beta-Glucosidase; Biomass; Cellulase; Cellulose; Enzyme Inhibitors; Ethanol; Fermentation; Furaldehyde; Glucose; Glycine max; Humidity; Hydrolysis; Pressure; Saccharomyces cerevisiae; Waste Products

The concept of reaction severity, which combines residence time and temperature, is often used in the pulp and paper and biorefining industries. The influence of corn stover pretreatment severity on yield of sugar and major degradation products and subsequent effects on enzymatic cellulose hydrolysis was investigated. The pretreatment residence time and temperature, combined into the severity factor (Log R(o)), were varied with constant acid concentration. With increasing severity, increasing concentrations of furfural and 5-hydroxymethylfurfural (5-HMF) coincided with decreasing yields of oligosaccharides. With further increase in severity factor, the concentrations of furans decreased, while the formation of formic acid and lactic acid increased. For example, from severity 3.87 to 4.32, xylose decreased from 6.39 to 5.26 mg/mL, while furfural increased from 1.04 to 1.33 mg/mL; as the severity was further increased to 4.42, furfural diminished to 1.23 mg/mL as formate rose from 0.62 to 1.83 mg/mL. The effects of dilute acid hydrolyzate, acetic acid, and lignin, in particular, on enzymatic hydrolysis were investigated with a rapid microassay method. The microplate method gave considerable time and cost savings compared to the traditional assay protocol, and it is applicable to a broad range of lignocellulosic substrates.

Topics: Acids; Aspergillus niger; beta-Glucosidase; Cellulase; Cellulose; Enzyme Inhibitors; Furaldehyde; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Lignin; Plant Leaves; Plant Stems; Polysaccharides; Temperature; Trichoderma; Zea mays

Dilute sulfuric acid was used to pretreat coastal Bermuda grass at high temperature prior to enzymatic hydrolysis. After both pretreatment and enzymatic hydrolysis processes, the highest yield of total sugars (combined xylose and glucose) was 97% of the theoretical value. The prehydrolyzate liquor was analyzed for inhibitory compounds (furfural, hydroxymethylfurfural (HMF)) in order to assess potential risk for inhibition during the following fermentation. Accounting for the formation of the inhibitory compounds, a pretreatment with 1.2% acid at 140 °C for 30 min with a total sugar yield of 94% of the theoretical value may be more favorable for fermentation. From this study, it can be concluded that dilute sulfuric acid pretreatment can be successfully applied to coastal Bermuda grass to achieve high yields of monomeric glucose and xylose with acceptable levels of inhibitory compound formation.

Topics: Analysis of Variance; beta-Glucosidase; Biomass; Carbohydrates; Cellulase; Computer Simulation; Cynodon; Fermentation; Furaldehyde; Glucose; Hot Temperature; Hydrolysis; Lignin; Oxidation-Reduction; Seawater; Sulfuric Acids; Xylose

Untreated and hydrothermally treated sorghum bagasse (SB) was hydrolyzed to simple sugars by the synergistic action of cellulases and hemicellulases produced by the fungi Fusarium oxysporum and Neurospora crassa. Synergism between the two lignocellulolytic systems was maximized with the application of higher fraction of N. crassa enzymes. Hydrothermolysis of SB was studied at a wide range of treatment times and temperatures. At intense pretreatment conditions (210 degrees C for 20 min; logR(0)=4.54), the residual hemicellulose percentage was 17.45%, while formation of inhibitory products, 5-hydromethyl-furfural (HMF), furfural, acetic and formic acid, (0.21, 0.51, 3.36 and 1.80 g/l, respectively) remained in acceptable levels. Maximum conversion of cellulose and total polysaccharides of the untreated SB were 23.18% and 18.79%, respectively. Combining hydrothermal treatment and enzymatic hydrolysis of released oligosaccharides and insoluble solids resulted in improvement of cellulose (approximately 15% increase) and total polysaccharides (two fold) hydrolysis compared to that of untreated SB.

Topics: Biotechnology; Carbohydrates; Cellulase; Cellulose; Chromatography, Ion Exchange; Enzymes; Fermentation; Furaldehyde; Fusarium; Glycoside Hydrolases; Hydrolysis; Neurospora; Sorghum; Temperature; Water