cellulase and formic-acid

Anaerobic fungi (phylum Neocallimastigomycota), an early branching family of fungi, are commonly encountered in the digestive tract of mammalian herbivores. To date, isolates from ten described genera have been reported, and several novel taxonomic groupings are detected using culture-independent molecular methods. Anaerobic fungi are recognized as playing key roles in the decomposition of lignocellulose (up to 50% of the ingested and untreated lignocellulose), with their physical penetration and extracellular enzymatical secretion of an unbiased diverse repertoire of cell-wall-degrading enzymes. The secreted cell-wall-degrading enzymes of anaerobic fungi include both free enzymes and extracellular multi-enzyme complexes called cellulosomes, both of which have potential as fiber degraders in industries. In addition, anaerobic fungi can provide large amounts of substrates such as hydrogen, formate, and acetate for their co-cultured methanogens. Consequently, large amounts of methane can be produced. And thus, it is promising to use the co-culture of anaerobic fungi and methanogens in the biogas process to intensify the biogas yield owing to the efficient and robust degradation of recalcitrant biomass by anaerobic fungi and improved methane production from co-cultures of anaerobic fungi and methanogens.

Topics: Acetic Acid; Anaerobiosis; Biodegradation, Environmental; Biofuels; Biomass; Biotechnology; Cellulase; Cellulosomes; Coculture Techniques; Cotton Fiber; Euryarchaeota; Fermentation; Formates; Fungi; Hydrogen; Lignin; Methane; Neocallimastigomycota; Polysaccharides; Substrate Specificity

Biomass pretreatment is an essential strategy to overcome biomass recalcitrance and promote lignocellulosic bioconversion. Here, a reusable organic solvent system (formic acid-methanesulfonic acid) was explored to pretreat poplar under a mild temperature (below 100 °C). The results showed that the co-solvent system could extract basically complete hemicelluloses and part of lignin with original cellulose retained in the pretreated substrates. Meanwhile, sulfonic acid groups were introduced into lignin structure remained in the substrates. The glucose conversion yield of the substrates with a higher concentration of sulfonic acid groups (13.2 mmol/kg) reached 45.9 % by reducing the hydrophobic interaction between lignin and cellulase, showing 89.3 % improvement compared with that of the substrates treated with single formic acid. This progressive study aimed to develop a new strategy to realize sulfonation and promote enzymatic hydrolysis of substrates by using mild organic solvent pretreatment.

Topics: Biomass; Cellulase; Hydrolysis; Lignin; Populus; Solvents; Sulfonic Acids

This study investigated the process intensification strategies for the pretreatment of Radiata Pine with the green deep eutectic solvent (DES) system composed of benzyltrimethylammonium chloride/formic acid (BTMAC/FA). The results showed that DES pretreatment drastically improved the delignification and hemicelluloses-removal capacity. The conducted process acceptably remained most of the cellulose in pretreated biomass (88.3%-91.8%). Benefiting from the overcoming of recalcitrance, the enzymatic digestibility of pretreated residues reached 92.4%. The efficient conversion was mainly ascribed to the lignin and hemicelluloses co-extraction. Meanwhile, the lignin yield and enzymatic saccharification was still largely maintained after five reuses. Further structural characteristics of lignin nanoparticles revealed that the lignin possessed high purity (95.19-97.51%), medium molecular weight (9600 to 6495 g/mol), and low polydispersity (ca 2.0), which was attributed to cleavage of ether bonds in lignin as well as linkages between lignin and hemicelluloses. Overall, this study illustrated that DES pretreatment was promising to achieve an efficient fractionation of woody biomass into fermentable glucose and high-quality lignin.

Topics: Cellulase; Formates; Hydrolysis; Lignin; Molecular Weight; Nanoparticles; Particle Size; Pinus; Quaternary Ammonium Compounds; Solvents

Topics: Acids; Alkalies; Carbohydrate Metabolism; Cellulase; Cellulose; Formates; Furans; Glucose; Hydrolysis; Phenols; Polyethylene Glycols; Saccharum; Xylose

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

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

Formiline pretreatment pertains to a biomass fractionation process. In the present work, Formiline-pretreated sugarcane bagasse was hydrolyzed with cellulases by batch and multi-step fed-batch processes at 20% solid loading. For wet pulp, after 144 h incubation with cellulase loading of 10 FPU/g dry solid, fed-batch process obtained ~150 g/L glucose and ~80% glucan conversion, while batch process obtained ~130 g/L glucose with corresponding ~70% glucan conversion. Solid loading could be further increased to 30% for the acetone-dried pulp. By fed-batch hydrolysis of the dried pulp in pH 4.8 buffer solution, glucose concentration could be 247.3±1.6 g/L with corresponding 86.1±0.6% glucan conversion. The enzymatic hydrolyzates could be well converted to ethanol by a subsequent fermentation using Saccharomices cerevisiae with ethanol titer of 60-70 g/L. Batch and fed-batch SSF indicated that Formiline-pretreated substrate showed excellent fermentability. The final ethanol concentration was 80 g/L with corresponding 82.7% of theoretical yield.

Topics: Acetone; Batch Cell Culture Techniques; Carbohydrate Metabolism; Carbohydrates; Cellulase; Cellulose; Ethanol; Fermentation; Formates; Glucose; Hydrolysis; Saccharum

A lignocellulose pretreatment process was developed with formic acid delignification (FAD) followed by alkaline deformylation (AD), which was termed as Formiline process. In FAD, more than 80% of lignin and hemicellulose were removed, but cellulose formylation also happened. Formic acid concentration (FAC) was the most important factor affecting delignification and cellulose formylation. Increasing FAC could enhance degree of delignification but also increased cellulose formylation. The presence of formyl group could inhibit the enzymatic hydrolysis of cellulose; however, removing formyl group with a small loading of alkali well recovered cellulose digestibility. The spent liquor could be directly recycled for delignification thus significantly decreasing energy consumption in solvent recovery. The Formiline-pretreated substrates showed an excellent enzymatic digestibility and could be very well converted to ethanol by simultaneous saccharafication and fermentation (SSF). The final ethanol concentrations were 55.4 and 80.1g/L respectively at initial solid consistencies of 15% and 20%.

Topics: Alkalies; beta-Glucosidase; Biotechnology; Carbohydrate Metabolism; Cellulase; Cellulose; Chemical Fractionation; Ethanol; Fermentation; Formates; Lignin; Recycling; Saccharomyces cerevisiae; Saccharum; Sulfuric Acids; Time Factors; Water

In recent years, growing attention has been focused on the use of lignocellulosic biomass as a feedstock for the production of ethanol, a possible renewable alternative to fossil fuels. Several pretreatment processes have been developed for decreasing the biomass recalcitrance, but only a few of them seem to be promising. In this study, effect of various organic solvents and organic acids on the pretreatment of sugarcane bagasse was studied. Among the different organic acids and organic solvents tested, formic acid was found to be effective. Optimization of process parameters for formic acid pretreatment was carried out. The structural changes before and after pretreatment was investigated by scanning electron microscopy, X-ray diffraction (XRD), and Fourier transform infrared (FTIR) analysis. The X-ray diffraction profile showed that the degree of crystallinity was more for pretreated biomass than that of untreated. The FTIR spectra shown at the stretching of hydrogen bonds of pretreated sugarcane bagasse arose at higher number. It also revealed that the cellulose content in the solid residue increased because the hemicelluloses fraction in raw materials was released by acid hydrolytic reaction.

Topics: Biofuels; Biomass; Cellulase; Cellulose; Ethanol; Fermentation; Formates; Hydrolysis; Microscopy, Electron, Scanning; Saccharum; Spectroscopy, Fourier Transform Infrared; X-Ray Diffraction

We evaluated the effects of cellulase (from Trichoderma longibrachiatum) application rates on neutral detergent fiber (NDF) concentration and fermentation products of orchardgrass (Dactylis glomerata L.) and alfalfa (Medicago sativa L.) silages harvested with decreasing dry matter (DM) digestibility. Additionally, the impacts of inoculant (Lactobacillus plantarum and Pediococcus cerevisiae), pectinase (from Aspergillus niger), or formic acid on silage composition were studied. Forages wilted to a DM content of about 320 g/kg were ensiled in laboratory silos for 60 d. Cellulase, combined with inoculant, was applied at 2, 10, and 20 ml/kg of herbage (at least 2500 IU/ml). Cellulase at 10 ml/kg was also applied alone or in combination with pectinase and inoculant or formic acid. The NDF concentration of orchardgrass silage decreased with increasing cellulase up to 20 ml/kg, at which NDF content was decreased by 30%. The NDF concentration of alfalfa silage decreased with increasing cellulase application up to 10 ml/kg, at which NDF content was decreased by 13%. Immature plants were more responsive to cellulase treatment than mature plants. Cellulase at 2 ml/kg combined with inoculant improved fermentation characteristics of the silages but generally, there was no effect on silage fermentation by higher cellulase applications, resulting in an accumulation of sugar. The improved fermentation of orchardgrass treated with cellulase and inoculant was mostly related to the effect of inoculant, whereas cellulase alone improved fermentation characteristics of alfalfa silage and this effect was enhanced by addition of inoculant. Decreased NDF and increased sugar concentrations did not improve the in vitro DM digestibility of cellulase-treated silages.

Topics: Aspergillus niger; Bacteria; Cellulase; Dietary Fiber; Digestion; Fermentation; Formates; Hydrogen-Ion Concentration; Lactic Acid; Lactobacillus; Medicago sativa; Pediococcus; Plant Proteins; Poaceae; Polygalacturonase; Silage

The objectives of this study were to determine the effect of a cellulase (from Trichoderma longibrachiatum) alone or combined with a bacterial inoculant (Lactobacillus plantarum and Pediococcus cerevisiae) or formic acid on composition, intake, and digestibility of orchardgrass (Dactylis glomerata L.) and alfalfa (Medicago sativa L.) silages. Orchardgrass and alfalfa were harvested at the early heading stage and at the early bloom stage of maturity and wilted to approximately 22 and 32% DM, respectively. Forages were then ensiled in 100-L sealed barrels for at least 60 d before they were fed to lambs. Silage treated with cellulase had lower (P < .001) pH and lower (P < .001) acetic acid and NH3 N concentrations than untreated silage of both plant species and a higher (P = .004) lactic acid concentration than the control treatment of alfalfa silage. Fermentation characteristics of cellulase-treated silages, especially of alfalfa, were further enhanced by use of inoculant. Formic acid addition increased (P < .001), reducing sugar concentration of cellulase-treated orchardgrass and alfalfa silage by 90 and 154%, respectively, and decreased (P < .001) NH3 N concentration of cellulase-treated alfalfa silage by 19%. Averaged across plant species, cellulase, combined with inoculant or formic acid, resulted in 8 and 13% greater (P = .03) DMI, respectively, than the control silage. Extensive enzymatic cell-wall degradation during ensiling decreased (P = .003) NDF intake of cellulase-treated orchardgrass silage by 25% and decreased (P = .001) cellulose intake by 23%, when averaged across plant species. Addition of formic acid increased (P = .003) NDF intake of cellulase-treated orchardgrass silage by 19%. Averaged across species, cellulase application decreased (P < .05) silage NDF digestibility by 18%. Greater sugar and lower acetic acid, NH3 N, and NDF concentrations resulted in greater DMI of cellulase-treated silage than of control silage, when cellulase was combined with formic acid or inoculant.

Topics: Animal Feed; Animals; Cellulase; Digestion; Formates; Lactobacillus; Male; Medicago sativa; Pediococcus; Poaceae; Sheep; Trichoderma

The objectives of this study were to determine the effects of cellulase (from Trichoderma longibrachiatum) combined with formic acid, applied before ensiling, on the subsequent concentration and composition of the cell wall and on the extent and rate of in situ cell-wall digestion of orchardgrass (Dactylis glomerata L.) and alfalfa (Medicago sativa L.). Treated and control forages of both plant species were ensiled for at least 60 d before being ruminally digested by two fistulated cows. Analyses of NDF, ADF, and acid detergent lignin were conducted sequentially on original and digested samples. Data were fitted with a first-order, nonlinear model to estimate extents and rates of digestion of potentially digestible NDF, cellulose, and hemicellulose. The concentration of indigestible residue and the discrete lag time before digestion were also determined for the cell-wall components. After ensiling, the mean NDF concentration of treated silages was 19% lower than that of control silages; the effect was greater for orchardgrass than for alfalfa. The extent of digestion of NDF, cellulose, and hemicellulose, respectively, was 33, 37, and 27% lower for treated silages than for control silages. Treatment effects on the extent of digestion varied between plant species. Cellulose from treated orchardgrass was digested 19% more slowly than cellulose from the control silage. Indigestible residue concentrations of NDF, cellulose, and hemicellulose, respectively, were 7, 8, and 7% lower in treated silages than in control silages. Thus, extensive cell-wall degradation by cellulase during ensiling resulted in less digestible cell-wall material for ruminal digestion but greater total cell-wall degradation, including that during ensiling and ruminal incubation, especially during early digestion in the rumen.

Topics: Cell Wall; Cellulase; Formates; Kinetics; Lignin; Medicago sativa; Poaceae; Silage; Solubility; Trichoderma