Enzymatic hydrolysis of steam-exploded and ethanol organosolv-pretreated douglas-fir by novel and commercial fungal cellulases

Softwood residues are the most abundant feedstock available for bioconversion in many northern countries. However, the high costs for delignification and enzymatic hydrolysis currently deter commercialization of softwood bioconversion processes. This study evaluates the abilities of two novel fungal preparations (MSUBC1 and MSUBC2) and two commercial cellulase preparations (TR1 and TR2) to hydrolyze cellulose in Douglas-fir pretreated by steam explosion or ethanol organosolv process. MSUBC1 showed significantly better performance than the other preparations on both lignocellulosic substrates. In particular, MSUBC1 achieved >76% cellulose conversion for hydrolysis of steam-exploded Douglas-fir (～44% lignin) after 72 h at low enzyme loading (10 filter paper units/g of cellulose) and without β-glucosidase supplementation.     

Preparation of Arabinoxylan and its Sorption on Bacterial Cellulose During Cultivation

The sorption of arabinoxylan (AX) on bacterial cellulose was investigated by adding AX to the culture medium of Gluconacetobacter xylinus. The starting AX material was produced by alkaline extraction of oat spelts. To investigate the impact of varying AX quality, the residual lignin was reduced by ClO₂ bleaching. Furthermore, bleached and unbleached xylans were subjected to xylanase hydrolysis in order to produce fractions of varying molar mass. Of all samples only the water soluble fractions were used for sorption experiments. A reduced molar mass resulted in a lower sorption of AX to the cellulose, while the lignin content increased the sorption of AX on bacterial cellulose. The sorption of AX resulted in a reduction of bacterial cellulose crystallinity and cellulose Iα content. In combined treatments of AX with xyloglucan and β-glucan no synergistic effect of those polysaccharides on the AX sorption was found.     

Novel approach of corn fiber utilization

The corn wet milling process produces a 10% (w/w of the processed corn) byproduct called corn fiber, which is utilized worldwide as a low-value feedstock for cattle. The aim of this study was to find a higher value use of corn fiber. The main fractions of corn fiber are: 20% starch, 40% hemicellulose, 14% cellulose, and 14% protein. Extraction of the highly valuable, cholesterol-lowering corn fiber oil is not feasible owing to its low (2% w/w) concentration in the fiber. The developed technology is based on simple and inexpensive procedures, like washing with hot water, dilute acid hydrolysis at 120°C, enzymatic hydrolysis of cellulose, screening, drying, and extraction. The main fractions are sharply separated in the order of starch, hemicellulose, cellulose, lipoprotein, and lignin). The lipoprotein fraction adds up to 10% of the original dry corn fiber, and contains 45% corn fiber oil, thus yielding more oil than direct extraction of the fiber. It is concluded that the defined method makes the extraction of the corn fiber oil economically feasible. The fractionation process also significantly increases the yield of cholesterol-lowering substances (sterols and sterolesters). At the same time clear and utilizable fractions of monosaccharides, protein, and lignin are produced.     

Cellulose solvent-based pretreatment for corn stover and avicel: concentrated phosphoric acid versus ionic liquid [BMIM]Cl

Since cellulose accessibility has become more recognized as the major substrate characteristic limiting hydrolysis rates and glucan digestibilities, cellulose solvent-based lignocellulose pretreatments have gained attention. In this study, we employed cellulose solvent- and organic solvent-based lignocellulose fractionation using two cellulose solvents: concentrated phosphoric acid [~85?% (w/w) H₃PO₄] and an ionic liquid Butyl-3-methylimidazolium chloride ([BMIM]Cl). Enzymatic glucan digestibilities of concentrated phosphoric acid- and [BMIM]Cl-pretreated corn stover were 96 and 55?% after 72?h at five filter paper units of cellulase per gram of glucan, respectively. Regenerated amorphous cellulose by concentrated phosphoric acid and [BMIM]Cl had digestibilities of 100 and 92?%, respectively. Our results suggested that differences in enzymatic glucan digestibilities of concentrated phosphoric acid- and [BMIM]Cl-pretreated corn stover were attributed to combinatory factors. These results provide insights into mechanisms of cellulose solvent-based pretreatment and effects of residual cellulose solvents and lignin on enzymatic cellulose hydrolysis.     

Novel approach of corn fiber utilization

The corn wet milling process produces a 10% (w/w of the processed corn) byproduct called corn fiber, which is utilized worldwide as a low-value feedstock for cattle. The aim of this study was to find a higher value use of corn fiber. The main fractions of corn fiber are: 20% starch, 40% hemicellulose, 14% cellulose, and 14% protein. Extraction of the highly valuable, cholesterol-lowering corn fiber oil is not feasible owing to its low (2% w/w) concentration in the fiber. The developed technology is based on simple and inexpensive procedures, like washing with hot water, dilute acid hydrolysis at 120 degrees C, enzymatic hydrolysis of cellulose, screening, drying, and extraction. The main fractions are sharply separated in the order of starch, hemicellulose, cellulose, lipoprotein, and lignin). The lipoprotein fraction adds up to 10% of the original dry corn fiber, and contains 45% corn fiber oil, thus yielding more oil than direct extraction of the fiber. It is concluded that the defined method makes the extraction of the corn fiber oil economically feasible. The fractionation process also significantly increases the yield of cholesterol-lowering substances (sterols and sterolesters). At the same time clear and utilizable fractions of monosaccharides, protein, and lignin are produced.     

Cellulase adsorption and an evaluation of enzyme recycle during hydrolysis of steam-exploded softwood residues

The sugar yield and enzyme adsorption profile obtained during the hydrolysis of SO₂-catalyzed steam-exploded Douglas-fir and posttreated steamexploded Douglas-fir substrates were determined. After hot alkali peroxide posttreatment, the rates and yield of hydrolysis attained from the posttreated Douglas-fir were significantly higher, even at lower enzyme loadings, than those obtained with the corresponding steam-exploded Douglas-fir. The enzymatic adsorption profiles observed during hydrolysis of the two substrates were significantly different. Ultrafiltration was employed to recover enzyme in solution (supernatant) and reused in subsequent hydrolysis reactions with added, fresh substrate. These recycle findings suggested that the enzyme remained relatively active for three rounds of recycle. It is likely that enzyme recovery and reuse during the hydrolysis of posttreated softwood substrates could lead to reductions in the need for the addition of fresh enzyme during softwood-based bioconversion processes.     

The Challenging Measurement of Protein in Complex Biomass-Derived Samples

Measurement of the protein content in samples from production of lignocellulosic bioethanol is an important tool when studying the adsorption of cellulases. Several methods have been used for this, and after reviewing the literature, we concluded that one of the most promising assays for simple and fast protein measurement on this type of samples was the ninhydrin assay. This method has also been used widely for this purpose, but with two different methods for protein hydrolysis prior to the assay—alkaline or acidic hydrolysis. In samples containing glucose or ethanol, there was significant interference from these compounds when using acid hydrolysis, which was not the case when using the alkaline hydrolysis. We evaluated the interference from glucose, cellulose, xylose, xylan, lignin and ethanol on protein determination of BSA, Accellerase® 1500 and Cellic® CTec2. The experiments demonstrated that the presence of cellulose, lignin and glucose (above 50 g/kg) could significantly affect the results of the assay. Comparison of analyses performed with the ninhydrin assay and with a CN analyser revealed that there was good agreement between these two analytical methods, but care has to be taken when applying the ninhydrin assay. If used correctly, the ninhydrin assay can be used as a fast method to evaluate the adsorption of cellulases to lignin.     

Enhanced enzymatic hydrolysis of steam-exploded douglas fir wood by alkali-oxygen post-treatment

Good enzymatic hydrolysis of steam-exploded Douglas fir wood (SEDW) cannot be achieved owing to the very high lignin content (>40%) that remains associated with this substrate. Thus, in this study, we investigated the use of alkali-oxygen treatment as a posttreatment to delignify SEDW and also considered the enzymatic hydrolyzability of the delignified SEDW. The results showed that under optimized conditions of 15% NaOH, 5% consistency, 110°C, and 3h, approx84% of the lignin in SEDW could be removed. The resulting delignified SEDW had good hydrolyzability, and cellulose-to-glucose conversion yields of over 90 and 100% could be achieved within 48 h with 20 and 40 filter paper units/g of cellulose enzyme loadings, respectively. It was also indicated that severe conditions, such as high NaOH concentration and high temperature, should not be utilized in oxygen delignification of SEDW in order to avoid extensive condensation of lignin and significant degradation of cellulose.     

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 %).     

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.     

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).     

Cellulose hydrolysis catalyzed by highly acidic lignin-derived carbonaceous catalyst synthesized via hydrothermal carbonization

Acidic carbonaceous solids were synthesized from mass pine alkali lignin via hydrothermal carbonization followed by sulfonation. Hydrothermal carbonization of lignin in the presence of acrylic acid (LAHC-SO₃H) provided many more carboxylic groups than that in the absence of acrylic acid, allowing subsequent sulfonation to produce a highly active and stable catalyst for cellulose hydrolysis in the [BMIM]Cl-H₂O solvent system. The hydrochar and catalyst were characterized using field emission scanning electron microscopy, X-ray diffractometer, X-ray photoelectron spectroscopy, thermal gravimetric analysis, Fourier transform infrared spectrometer, Brunauer–Emmett–Teller and acid–base titration. Results showed that a high acid content of 5.48 mmol/g, including carboxylic group (2.85 mmol/g), phenolic hydroxyl group (1.05 mmol/g) and sulfonic acid group (1.58 mmol/g), contributed significantly to the highly efficient hydrolysis of cellulose. Further, it was found that addition of trace water in [BMIM]Cl was favorable to cellulose hydrolysis. The highest yield (75.4%) of total reducing sugar (TRS) obtained in [BMIM]Cl-H₂O at a mass ratio of 100:1 was more than twice that (36.1%) achieved in [BMIM]Cl without water; the corresponding reaction conditions were 50 mg of microcrystalline cellulose, 30 mg of catalyst, 1.0 g of [BMIM]Cl, 10 mg of H₂O, reaction temperature of 130 °C and reaction time of 2 h. Furthermore, the TRS yield with 5 cycles for LAHC-SO₃H was higher than 68.1%, and the catalytic activity of catalyst could be fully recovered (74.0% of TRS yield) easily by regeneration.     

Pretreatment of softwood by acid-catalyzed steam explosion followed by alkali extraction

A process for converting lignocellulosic biomass to ethanol hydrolyzes the hemicellulosic fraction to soluble sugars (i.e., pretreatment), followed by acid- or enzyme-catalyzed hydrolysis of the cellulosic fraction. Enzymatic hydrolysis may be improved by using an alkali to extract a fraction of the lignin from the pretreated material. The removal of the lignin may increase the accessibility of the cellulose to enzymatic attack, and thus improve overall economics of the process, if the alkali-treated material can still be effectively converted to ethanol. Pretreated Douglas fir produced by a sulfuric-acid-catalyzed steam explosion was treated with NaOH, NH₄OH, and lime to extract some of the lignin. The treated material, along with an untreated control sample, was tested by an enzymatic-digestion procedure, and converted to ethanol by simultaneous saccharification and fermentation using a glucose-fermenting yeast. NaOH was most effective at removing lignin (removed 29%), followed by NH4OH and lime. However, the susceptibility of the treated material to enzymatic digestion was lower than the control and decreased with increasing lignin removal. Ethanol production was similar for the control and NaOH-treated material, and lower for NH₄OH- and lime-treated material.     

Experimental and molecular simulating study on promoting electrolyte-immersed mechanical properties of cellulose/lignin separator for lithium-ion battery

Lithium-ion batteries have been developing intensively and earn an unprecedented reputation, yet advanced performance and safety issue still require considerable investigation. Separator is vital to comprehensive properties of batteries, where the mechanical properties are key to breaking through of new-type separator. Unfortunately, electrolyte submersion has caused damage to strength of cellulose separator. Whereupon, in this work, cellulose separator is optimized by introducing lignin particles to promote electrolyte-immersed mechanical strength. Experiments are conducted concerning surface morphology, contact angle, porosity, electrolyte uptake, mechanical properties and electrochemical performance. Molecular simulation is implemented to explore the mechanism of tensile behavior of cellulose and lignin subjected to electrolyte solvents. Experimental results confirm positive effect of lignin addition in improving mechanical properties and simultaneously maintaining impressive electrochemical performance of the cellulose/lignin composites separators. Besides, lignin addition amount of 2.5% and 5% is recommended to achieve promising overall properties. Molecular simulation has successfully unveiled that weakening of cellulose separator submerged in electrolyte is resulted by the deformed cellulose amorphous region and the promoting effect of adding lignin is contributed from the new hydrogen bonds generated between cellulose and lignin molecules. Hopefully, this work provides novel insight on preparing remarkable separator and mechanism of materials behavior.     

The effect of chemical and physical characteristics of spruce SEW pulps on enzymatic hydrolysis

Conifers, which are the most abundant biomass species in Nordic countries, USA, Canada and Russia, exhibit strong resistance towards depolymerization by cellulolytic enzymes. At present, it is still not possible to isolate a single structural feature which would govern the rate and degree of enzymatic hydrolysis. On the other hand, the forest residues alone represent an important potential for biochemical production of biofuels. In this study, the effect of substrate properties on the enzymatic hydrolysis of softwood was studied. Stem wood spruce chips were fractionated by SO₂–ethanol–water (SEW) treatment to produce pulps of varying composition by applying different operating conditions. The SEW technology efficiently fractionates different types of lignocellulosic biomass by rapidly dissolving hemicelluloses and lignin. Cellulose remains fully in the solid residue which is then treated by enzymes to release glucose. The differences in enzymatic digestibility of the spruce SEW pulp fibers were interpreted in terms of their chemical and physical characteristics. A strong correlation between the residual lignin content of SEW pulp and enzymatic digestibility was observed whereas cellulose degree of polymerization and hemicellulose content of pulp were not as important. For the pulps containing about 1.5 % (w/w) lignin, 90 % enzymatic digestibility was achieved at 10 FPU enzyme charge and 24 h of hydrolysis time.     

Purification of cellulosic pulp by hot water extraction

Hot water extraction (HWE) of pulp in a flow-through reactor was evaluated as a method to purify paper-grade pulps. About 50–80 % of the xylan and up to 50 % of the lignin in unbleached birch Kraft pulp was extracted by the HWE without losses in cellulose yield. The residual xylan content in the extracted pulps was predominantly too high for dissolving-grade applications, but some of the pulps with a xylan content of 5–7 % might still be suitable as rayon-grade pulps. Increasing extraction temperature lowered the xylan content at which cellulose yield started to decrease. Furthermore, at any given xylan content, increasing extraction temperature resulted in cellulosic pulp with higher degree of polymerization. The extracted xylan was recovered almost quantitatively as xylo-oligosaccharides. The results suggest that HWEs at elevated temperatures may be applied to purify cellulosic pulps, preferably containing a low xylan content, and to recover the extracted sugars.     

Biological pretreatment of lignocellulose for enzymatic hydrolysis of cellulose

Pretreatment of lignocellulosic materials is considered as the rate-limiting step in an economically feasible process for enzymatic hydrolysis of cellulose. Biological delignification techniques have not been developed as intensively as physical and chemical methods. However, white-rot fungi are effective degraders of lignin, and some of them even preferentially remove lignin from wood compared with carbohydrates, and therefore might be suitable for biological pretreatment of lignocellulose. White-rot fungi were cultivated on wheat straw and the residue was hydrolyzed withTrichoderma reesei cellulase. Of nineteen fungi examined,Pleurotus ostreatus, Pleurotus sp. 535,Pycnoporus cinnabarinus 115,Ischnoderma benzoinum 108,Phanerochaete sordida 37,Phlebia radiata 79, and two unidentified fungi were found suitable for pretreatment of straw: the yields of reducing sugars and glucose based on original straw were markedly better compared with uninoculated straw, and these fungi also gave better results thanPolyporus versicolor, a nonselective reference fungus (Cowling, 1961). In the best cases the efficiency of the biological pretreatment was comparable with that of alkali treatment (2% NaOH, 0.4 g NaOH/g straw, 10 min at 115‡C), but the fungal treatment resulted in a higher proportion of glucose in the hydrolyzates. Combined fungal and (strong) alkali treatment did not give better results than alkali or fungal treatment alone. When culture flasks were periodically flushed with oxygen the treatment time could be reduced by about 1 wk with the two fungi,P. sordida 37 andP. cinnabarinus 115, tested. The effect of oxygen in pretreatment reflected the effect of oxygen in the degradation of¹⁴C-lignin of poplar wood to¹⁴CO₂ by these fungi (Hatakka and Uusi-Rauva, 1983). The economic feasibility of the biological pretreatment process is poor due to the long cultivation times needed. The best results were obtained with the longest treatment time studied, which was 5 wk. However, the rapid progress in the field of biological lignin degradation may help to accelerate the delignification process, and also find factors that favor lignin degradation, but suppress the utilization of carbohydrates.     

On the mechanism of the unwanted acetylation of polysaccharides by 1,3-dialkylimidazolium acetate ionic liquids: part 2—the impact of lignin on the kinetics of cellulose acetylation

Cellulose acetylation has been reported as a side reaction of cellulose treatment with the ionic liquid 1-ethyl-3-methylimidazolium acetate ([EMIm][OAc]) (Karatzos et al. in Cellulose 19:307–312, 2012) and other 1,3-dialkylimidazolium acetate ionic liquids. 1-Acetylimidazole (AcIm), an [EMIm][OAc] impurity, has been found to be the actual acetylating agent (Zweckmair et al. in Cellulose 22:3583–3596, 2015), and the degree of acetylation was relatively low, below a DS of approx. 0.1%. Higher degrees of cellulose acetylation (DS > 10%) have been observed when the entire wood was mixed with [EMIm][OAc] instead of cellulosic pulp only (Abushammala et al. in Carbohydr Polym 134:609–616, 2015). In this paper, we explore the impact of wood constituents, mainly lignin, on cellulose acetylation using AcIm. The results demonstrate that lignin itself can be readily acetylated upon mixing with AcIm, and—noteworthy—that lignin presence significantly accelerates cellulose acetylation. The initial rate of cellulose acetylation by AcIm increased from 1.8 to 4.7%/h when only 1% of lignin, based on cellulose mass, was added. A mechanistic study employing cellulose and lignin model compounds showed lignin to be more susceptible to acetylation than cellulose and to act as an intermediate acetyl group source for further cellulose acetylation in a catalytic scenario.     

Modification of cellulose fibres with organosilanes: Under what conditions does coupling occur?

Different cellulose substrates and organosolv lignin were treated heterogeneously with organic solutions of trialkoxysilanes bearing variable function on the forth substituent. It was shown unambiguously for the first time that Si-OR does not react with the hydroxy groups of cellulose even at high temperature, whereas it condenses with the phenolic OH of lignin. The addition of moisture to these systems induces the partial hydrolysis of the siloxane moieties and the ensuing silanol groups can then react with the cellulose OH, but only at high temperature. Using the latter systems and a siloxane bearing a polymerizable function, it was possible to attach poly(methylmethacrylate) chains to the surface of cellulose fibres through a two-step procedure.     

Affinity of Hemicellulose for Cellulose Produced by Acetobacter Xylinum

Cellulose composites were produced by culturing Acetobacter aceti subsp. xylinum (ATCC 53524, agitation tolerant strain) under shaking and agitating conditions in the presence of 2% pine or beech Björkman lignin-carbohydrate complexes (LCCs) or six different types of hemicellulosic polysaccharides including glucuronoxylan, glucomannan, O-acetyl-glucuronoxylan, arabinoglucuronoxylan, arabinogalactan and xyloglucan. Hemicellulosic polysaccharide contents in cellulose composites were similar in spite of the differences in culture, shaking and agitating conditions. On the basis of hemicellulosic polysaccharide contents and X-ray diffraction patterns after extraction by dilute NaOH solution, glucomannan family polysaccharides were found to have the highest affinity to bacterial cellulose. Composites with neutral and acidic LCCs were resistant against alkali while high lability of their delignified carbohydrates against alkali indicates the importance of lignin for formation of cellulose-hemicellulose-lignin framework of plant secondary cell-walls.