Biorefining of softwoods using ethanol organosolv pulping: preliminary evaluation of process streams for manufacture of fuel-grade ethanol and co-products

Pulps with residual lignin ranging from 6.4-27.4% (w/w) were prepared from mixed softwoods using a proprietary biorefining technology (the Lignol process) based on aqueous ethanol organosolv extraction. The pulps were evaluated for bioconversion using enzymatic hydrolysis of the cellulose fraction to glucose and subsequent fermentation to ethanol. All pulps were readily hydrolyzed without further delignification. More than 90% of the cellulose in low lignin pulps (< or =18.4% residual lignin) was hydrolyzed to glucose in 48 h using an enzyme loading of 20 filter paper units/g cellulose. Cellulose in a high lignin pulp (27.4% residual lignin) was hydrolyzed to >90% conversion within 48 h using 40 filter paper units/g. The pulps performed well in both sequential and simultaneous saccharification and fermentation trials indicating an absence of metabolic inhibitors. Chemical and physical analyses showed that lignin extracted during organosolv pulping of softwood is a suitable feedstock for production of lignin-based adhesives and other products due to its high purity, low molecular weight, and abundance of reactive groups. Additional co-products may be derived from the hemicellulose sugars and furfural recovered from the water-soluble stream.     

Analysis of lignin–carbohydrate and lignin–lignin linkages after hydrolase treatment of xylan–lignin,glucomannan–lignin and glucan–lignin complexes from spruce wood

Xylan–lignin (XL), glucomannan–lignin (GML) and glucan–lignin (GL) complexes were isolated from spruce wood, hydrolyzed with xylanase or endoglucanase/β-glucosidase, and analyzed by analytical pyrolysis and 2D-NMR. The enzymatic hydrolysis removed most of the polysaccharide moieties in the complexes, and the lignin content and relative abundance of lignin–carbohydrate linkages increased. Analytical pyrolysis confirmed the action of the enzymatic hydrolysis, with strong decreases of levoglucosane and other carbohydrate-derived products. Unexpectedly it also revealed that the hydrolase treatment alters the pattern of lignin breakdown products, resulting in higher amounts of coniferyl alcohol. From the anomeric carbohydrate signals in the 2D-NMR spectra, phenyl glycoside linkages (undetectable in the original complexes) could be identified in the hydrolyzed GML complex. Lower amounts of glucuronosyl and benzyl ether linkages were also observed after the hydrolysis. From the 2D-NMR spectra of the hydrolyzed complexes, it was concluded that the lignin in GML is less condensed than in XL due to its higher content in β-O-4′ ether substructures (62 % of side chains in GML vs 53 % in XL) accompanied by more coniferyl alcohol end units (16 vs 13 %). In contrast, the XL lignin has more pinoresinols (11 vs 6 %) and dibenzodioxocins (9 vs 2 %) than the GML (and both have ~13 % phenylcoumarans and 1 % spirodienones). Direct 2D-NMR analysis of the hydrolyzed GL complex was not possible due to its low solubility. However, after sample acetylation, an even less condensed lignin than in the GML complex was found (with up to 72 % β-O-4′ substructures and only 1 % pinoresinols). The study provides evidence for the existence of structurally different lignins associated to hemicelluloses (xylan and glucomannan) and cellulose in spruce wood and, at the same time, offers information on some of the chemical linkages between the above polymers.     

Coniferyl ferulate incorporation into lignin enhances the alkaline delignification and enzymatic degradation of cell walls

Incorporating ester interunit linkages into lignin could facilitate fiber delignification and utilization. In model studies with maize cell walls, we examined how partial substitution of coniferyl alcohol (a normal monolignol) with coniferyl ferulate (an ester conjugate from lignan biosynthesis) alters the formation and alkaline extractability of lignin and the enzymatic hydrolysis of structural polysaccharides. Coniferyl ferulate moderately reduced lignification and cell-wall ferulate copolymerization with monolignols. Incorporation of coniferyl ferulate increased lignin extractability by up to 2-fold in aqueous NaOH, providing an avenue for producing fiber with less noncellulosic and lignin contamination or of delignifying at lower temperatures. Cell walls lignified with coniferyl ferulate were more readily hydrolyzed with fibrolytic enzymes, both with and without alkaline pretreatment. Based on our results, bioengineering of plants to incorporate coniferyl ferulate into lignin should enhance lignocellulosic biomass saccharification and particularly pulping for paper production.     

Carbohydrate derived‐pseudo‐lignin can retard cellulose biological conversion

Dilute acid as well as water only (hydrothermal) pretreatments often lead to a significant hemicellulose loss to soluble furans and insoluble degradation products, collectively termed as chars and/or pseudo‐lignin. In order to understand the factors contributing to reducing sugar yields from pretreated biomass and the possible influence of hemicellulose derived pseudo‐lignin on cellulose conversion at the moderate to low enzyme loadings necessary for favorable economics, dilute acid pretreatment of Avicel cellulose alone and mixed with beechwood xylan or xylose was performed at various severities. Following pretreatment, the solids were enzymatically hydrolyzed and characterized for chemical composition and physical properties by NMR, FT‐IR, and SEM imaging. It was found that hemicelluloses (xylan) derived‐pseudo‐lignin was formed at even moderate severities and that these insoluble degradation products can significantly retard cellulose hydrolysis. Furthermore, although low severity (CSF ～ 1.94) dilute acid pretreatment of a xylan–Avicel mixture hydrolyzed most of the xylan (98%) and produced negligible amounts of pseudo‐lignin, enzymatic conversion of cellulose dropped significantly (>25%) compared to cellulose pretreated alone at the same conditions. The drop in cellulose conversion was higher than realized for cellulase inhibition by xylooligomers reported previously. Plausible mechanisms are discussed to explain the observed reductions in cellulose conversions. Biotechnol. Bioeng. 2013; 110: 737–753. © 2012 Wiley Periodicals, Inc.     

Enzymatic hydrolysis of chemithermomechanically pretreated sugarcane bagasse and samples with reduced initial lignin content

Chemithermomechanical (CTM) processing was used to pretreat sugarcane bagasse with the aim of increasing cell wall accessibility to hydrolytic enzymes. Yields of the pretreated samples were in the range of 75-94%. Disk refining and alkaline-CTM and alkaline/sulfite-CTM pretreatments yielded pretreated materials with 21.7, 17.8, and 15.3% of lignin, respectively. Hemicellulose content was also decreased to some extent. Fibers of the pretreated materials presented some external fibrillation, fiber curling, increased swelling, and high water retention capacity. Cellulose conversion of the alkaline-CTM- and alkaline/sulfite-CTM-pretreated samples reached 50 and 85%, respectively, after 96 h of enzymatic hydrolysis. Two samples with low initial lignin content were also evaluated after the mildest alkaline-CTM pretreatment. One sample was a partially delignified mill-processed bagasse. The other was a sugarcane hybrid selected in a breeding program. Samples with lower initial lignin content were hydrolyzed considerably faster in the first 24 h of enzymatic digestion. For example, enzymatic hydrolysis of the sample with the lowest initial lignin content (14.2%) reached 64% cellulose conversion after only 24 h of hydrolysis when compared with the 30% observed for the mill-processed bagasse containing an initial lignin content of 24.4%.     

Cellulase stimulation during biodegradation of lignocellulosic residues at increased biomass loading

Microbial degradation of lignocellulosic biomass is primarily affected by the composition and structure of biomass, as well as enzyme activities that are influenced by the presence of in-process degradation products. This study focuses on the latter, and demonstrates that cellulase activity of Neurospora discreta is stimulated in the presence of in-process soluble lignin degradation products. Two types of biomass - cocopeat and sugarcane bagasse, with contrasting lignin content and cellulose structure were tested at two biomass loadings each. At the higher biomass loading, cocopeat showed the highest amount of hydrolyzed cellulose and cellulase activity, despite its low cellulose content and recalcitrant cellulose structure. A strong positive correlation was revealed between the amount of in-process degraded lignin and cellulase activity, indicating a stimulatory effect on cellulase, which contradicts most previous literature. Furthermore, the causal relationship between the amount of degraded lignin and cellulase activity was established in a model system of commercial cellulase and standard soluble lignin. This work could pave the way for using biomass loading as a process lever to enhance cellulose hydrolysis in microbial conversion of lignocellulosic biomass.     

The laccase-catalyzed modification of lignin for enzymatic hydrolysis

The efficient use of cellulases in the hydrolysis of pretreated lignocellulosic biomass is limited due to the presence of lignin. Lignin is known to bind hydrolytic enzymes nonspecifically, thereby reducing their action on carbohydrate substrates. The composition and location of residual lignin therefore seem to be important for optimizing the enzymatic hydrolysis of lignocellulosic substrates. The use of lignin-modifying enzymes such as laccase may have potential in the modification or partial removal of lignin from the biomass. In this study, the effect of lignin modification by laccase on the hydrolysis of pretreated spruce (Picea abies) and giant reed (Arundo donax) was evaluated. The substrates were first treated with laccase and then hydrolyzed with commercial cellulases. Laccase modification improved the hydrolysis yield of spruce by 12%, but surprisingly had an adverse effect on giant reed, reducing the hydrolysis yield by 17%. The binding properties of cellulases on the untreated and laccase-treated lignins were further studied using isolated lignins. The laccase treatment reduced the binding of enzymes on modified spruce lignin, whereas with giant reed, the amount of bound proteins increased after laccase treatment. Further understanding of the reactions of laccase on lignin will help to control the unspecific-binding of cellulases on lignocellulosic substrates.     

The laccase-catalyzed modification of lignin for enzymatic hydrolysis

The efficient use of cellulases in the hydrolysis of pretreated lignocellulosic biomass is limited due to the presence of lignin. Lignin is known to bind hydrolytic enzymes nonspecifically, thereby reducing their action on carbohydrate substrates. The composition and location of residual lignin therefore seem to be important for optimizing the enzymatic hydrolysis of lignocellulosic substrates. The use of lignin-modifying enzymes such as laccase may have potential in the modification or partial removal of lignin from the biomass. In this study, the effect of lignin modification by laccase on the hydrolysis of pretreated spruce (Picea abies) and giant reed (Arundo donax) was evaluated. The substrates were first treated with laccase and then hydrolyzed with commercial cellulases. Laccase modification improved the hydrolysis yield of spruce by 12%, but surprisingly had an adverse effect on giant reed, reducing the hydrolysis yield by 17%. The binding properties of cellulases on the untreated and laccase-treated lignins were further studied using isolated lignins. The laccase treatment reduced the binding of enzymes on modified spruce lignin, whereas with giant reed, the amount of bound proteins increased after laccase treatment. Further understanding of the reactions of laccase on lignin will help to control the unspecific-binding of cellulases on lignocellulosic substrates.     

Effects of cellulose crystallinity, hemicellulose, and lignin on the enzymatic hydrolysis of Miscanthus sinensis to monosaccharides

The effects of cellulose crystallinity, hemicellulose, and lignin on the enzymatic hydrolysis of Miscanthus sinensis to monosaccharides were investigated. A air-dried biomass was ground by ball-milling, and the powder was separated into four fractions by passage through a series of sieves with mesh sizes 250-355 microm, 150-250 microm, 63-150 microm, and <63 microm. Each fraction was hydrolyzed with commercially available cellulase and beta-glucosidase. The yield of monosaccharides increased as the crystallinity of the substrate decreased. The addition of xylanase increased the yield of both pentoses and glucose. Delignification by the sodium chlorite method improved the initial rate of hydrolysis by cellulolytic enzymes significantly, resulting in a higher yield of monosaccharides as compared with that for untreated samples. When delignified M. sinensis was hydrolyzed with cellulase, beta-glucosidase, and xylanase, hemicellulose was hydrolyzed completely into monosaccharides, and the conversion rate of glucan to glucose was 90.6%.     

Bioconversion of Phenolic Monomers of Lignin and Lignin-Containing Substrates by the Basidiomycete <Emphasis Type="Italic">Lentinus tigrinus</Emphasis>

The bioconversion of phenolic monomers of lignin (veratrol, vanillin, and vanillyl alcohol), hydrolyzed lignin, and sodium lignosulfonate (a product of the chemical modification of native lignin) by the basidiomycete Lentinus tigrinus was studied. It was found that the growth of the fungi on lignin monomer compounds is suppressed. A noticeable growth of the fungal biomass was observed only on the technical substrate sodium lignosulfonate. A comprehensive physicochemical study of the products of microbial transformation of sodium lignosulfonate was performed. It was established that the main direction of lignin bioconversion is oxidative condensation to form humic substances. In this case, depolymerization of the phenolic skeleton of lignin to monomeric phenol derivatives did not occur. The aromatic carbon atoms of the phenolic skeleton, unlike the carbon atoms of polysaccharides, were not involved in the fungal biomass growth. The observed growth of the fungus on the technical substrate sodium lignosulfonate can be explained by the presence of admixtures of oligomeric polysaccharides hemicellulose and cellulose, which can be used by the fungus as a carbon source.     

Fractionation of sugarcane bagasse by hydrothermal treatment

Hydrothermal treatment of sugarcane bagasse was conducted using a semi-batch reactor to develop a new biomass fractionation method that has low impact in the environment. A continuously increasing temperature was used in this treatment. It was found that hemicellulose and lignin could be mainly extracted as a water-soluble fraction at 200-230 degrees C, while the cellulose fraction was hydrolyzed at higher temperatures (230-280 degrees C) or recovered as solid residue from this treatment. Detailed analyses of the solid residue indicated that the crystal structure and the chemical composition of the residue were in good accordance with those of untreated crystalline cellulose. These experimental and analytical findings show that this method is promising for removal of hemicellulose and lignin from woody biomass without any catalyst and organic solvent.     

Binding-site Analysis of the Ether Linkages between Lignin and Hemicelluloses in Lignin–Carbohydrate Complexes by DDQ-Oxidation

Lignin-carbohydrate complexes (LCC; nor-C-1-M, com-C-1-A) isolated from normal and compression woods of Pinus densiflora were hydrolyzed with two types of cellulase preparations, and the hydrolyzates formed were fractionated by adsorption chromatography on polyvinyl gel into water-soluble materials and LCC fragments. To elucidate the binding sites between the lignin and carbohydrate, the cellulase-degraded LCC fragments were subjected to acetylation, and then oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), which was confirmed to oxidatively cleave the benzyl ether linkages between the lignin and carbohydrate. The DDQ-oxidized fraction was then methylated by the method of Prehm, hydrolyzed, reduced and acetylated. A GC-MS analysis of the methylated sugar revealed that alditol acetates from 6-O-methyl mannose, 6-O-methyl galactose, 6-O-methyl glucose and a small amount of their 2-O- or 3-O-methyl isomers existed in both methylated fractions. 2-O-Methyl xylose and 3-O-methyl xylose were also identified in the fraction from the acidic LCC (com-C-1-A). These results led to the conclusion that acetylglucomannan and β-1,4-galactan were preferably bound to the lignin at C-6 position of the hexoses, and that arabinoglucuronoxylan did likewise at the C-2 and C-3 positions of xylose units.     

Correlating the effect of pretreatment on the enzymatic hydrolysis of straw

Avicell, Alkali-treated straw cellulose (ATSC), and wheat straw were ball-milled to reduce crystallinity; wheat straw was delignified by hot (120 degrees C) sodium hydroxide solutions of various concentrations. The physically and chemically pretreated cellulosic materials were hydrolyzed by the cellulases of Fusarium oxysporum strain F3. Enzymic hydrolysis data were fitted by the hyperbolic correlation of Holtzapple, which involves two kinetic parameters, the maximum conversion (X(max)), and the enzymic hydrolysis time corresponding to 50% of X(max) (t(1/2)). An empirical correlation between X(max) and cellulose crystallinity, lignin content, and degree of delignification has been found under our experimental conditions. Complete cellulose hydrolysis is shown to be possible at less than 60% crystallinity indices or less than 10% lignin content.     

Phosphorylation of lignin peroxidases from Phanerochaete chrysosporium. Identification of mannose 6-phosphate

Many of the extracellular lignin-degrading peroxidases from the wood-degrading fungus Phanerochaete chrysosporium are phosphorylated. Immunoprecipitation of the extracellular fluid of cultures grown with H2K32PO4 with a polyclonal antibody raised against one of the lignin peroxidase isozymes, H8 (pI 3.5), revealed the incorporation of H2K32PO4 into lignin peroxidases. Analyses of the purified isozymes from labeled cultures by isoelectric focusing showed that, in addition to isozyme H8, lignin peroxidase isozymes H2 (pI 4.4), H6 (pI 3.7), and H10 (pI 3.3) are also phosphorylated. These analyses also showed that lignin peroxidase isozyme H1 (pI 4.7) and manganese-dependent peroxidase isozymes H3 (pI 4.9) and H4 (pI 4.5) are not phosphorylated. Phosphate quantitation indicated the presence of one molecule of phosphate/molecule of enzyme for all of the phosphorylated isozymes. To locate the site of phosphorylation, one-dimensional phosphoamino acid analysis was performed with hydrolyzed 32P-protein. However, phosphotyrosine, phosphoserine, and phosphothreonine could not be identified. Coupled enzyme assays of acid hydrolysate indicated the presence of mannose 6-phosphate as the phosphorylated component on the lignin peroxidase isozymes. Digestion of the isozymes with N-glycanase released the phosphate component, indicating that the mannose 6-phosphate is contained on an asparagine-linked oligosaccharide.     

An accurate and non-labor intensive method for the determination of syringyl to guaiacyl ratio in lignin

The syringyl to guaiacyl (S:G) ratio of hardwood lignin has long been identified as a significant parameter in delignification processes and more recent results have shown that it is also important in determining the amount of ethanol that can be obtained from fermentation of hydrolyzed wood. Acidolysis of Klason or acid insoluble lignin in dioxane/water/HCl was being investigated when syringyl and guaiacyl nuclei with a diketone-containing sidechain were observed as the major products. The area ratio of the two gas chromatogram peaks appeared to be indicative of the S:G ratio. After optimization of the method the relative standard deviation was found to be in the range of 0.3–3.76% for Klason lignin from a wide range of Eucalyptus grandis grown in South Africa. The method was then compared to nitrobenzene oxidation (NBO) using 13 poplars in a double-blind study. The respective S:G ratios were used to calculate percentages of S units and when these values were plotted against each other a linear correlation was obtained with a slope of approximately 1.0 (R² = 0.86). The largest discrepancy for any poplar was 6.9% (62% vs. 58% S units). Both methods convincingly demonstrated a significant decrease in lignin content with an increase in the S:G ratio. Discussion is presented on a series of reaction that could lead to the formation of the two diketones.     

玉米秸秆不同部位的组成及其对预处理的影响

不同玉米秸秆部位的成分组成及分布对预处理和酶解影响显著。研究表明:韧皮部与髓芯的成分相近,但叶子的差异较大,其木聚糖和总糖的质量分数最高,分别为29.48%和66.15%,而木质素的质量分数最低,因而叶子更容易预处理。玉米秸秆在稀酸预处理过程中可回收96.9%葡聚糖和50.0%～70.0%木聚糖,其中50.0%～60.0%木聚糖水解成木糖溶出;不同部位的木聚糖损失率与初始的木聚糖含量正相关;经稀酸预处理后,叶子中葡聚糖的质量分数最高,达72.40%,叶子和髓芯易于被纤维素酶水解生成葡萄糖,而韧皮部困难。不同部位的酶解得率与自身的葡聚糖含量正相关,与酸不溶木质素含量负相关,同时受原料的物理结构、葡聚糖和木质素大分子的化学组成等影响。     

Microbial utilization of levoglucosan in wood pyrolysate as a carbon and energy source

The Waterloo Fast Pyrolysis Process (WFPP) can produce an organic liquid high in levoglucosan (1, 6-anhydro-beta-D-glucopyranose) content from suitably pretreated lignocellulosics. A variety of fungi and yeasts were screened for their ability to utilize and ferment this organic liquid. To enhance its fermentability, the pyrolysis tar was posttreated in three different ways: (1) an aqueous extract (lignin removed); (2) activated charcoal treated (lignin and aromatics removed); and (3) acid hydrolysate (lignin and aromatics removed with the levoglucosan hydrolyzed to glucose). Four fungal strains were examined. None grew in the aqueous extract, but all grew equally well in both the activated charcoal treated and the acid hydrolysate, suggesting that the aromatic species were inhibitory to growth. Seven yeast species were examined, two of which did not grow on any of the extracts. Five of the yeast strains grew well on both the aqueous extract as well as the activated charcoal extract. The hydrolysate was optimal in terms of biomass yield and ethanol production. Ethanol yields on the hydrolysate were comparable or better than those on glucose. Ethanol was also produced in the aqueous extract and activated charcoal-treated substrate, but yields were considerably lower than on the hydrolysate or glucose. It is apparent that a wood pyrolysate maximized for levoglucosan can serve as a fermentable substrate, although postpyrolysis clean-up appears necessary. (c) 1993 John Wiley & Sons, Inc.     

Fast and efficient alkaline peroxide treatment to enhance the enzymatic digestibility of steam-exploded softwood substrates.

The enzymatic digestibility of steam-exploded Douglas-fir wood chips (steam exploded at 195 degrees C, 4.5 min, and 4.5% (w/w) SO(2)) was significantly improved using an optimized alkaline peroxide treatment. Best hydrolysis yields were attained when the steam-exploded material was post-treated with 1% hydrogen peroxide at pH 11.5 and 80 degrees C for 45 min. This alkaline peroxide treatment was applied directly to the water-washed, steam-exploded material eliminating the need for independent alkali treatment with 0.4% NaOH, which has been traditionally used to post-treat wood samples to try to remove residual lignin. Approximately 90% of the lignin in the original wood was solubilized by this novel procedure, leaving a cellulose-rich residue that was completely hydrolyzed within 48 h, using an enzyme loading of 10 FPU/g cellulose. About 82% of the originally available polysaccharide components of the wood could be recovered. The 18% of the carbohydrate that was not recovered was lost primarily to sugar degradation during steam explosion.     

Fractionation of wheat straw by atmospheric acetic acid process

Fractionation of wheat straw was investigated using an atmospheric acetic acid process. Under the typical conditions of 90% (v/v) aqueous AcOH, 4% H(2)SO(4) (w/w, on straw), ratio of liquor to straw (L/S) 10 (v/w), pulping temperature 105 degrees C, and pulping time 3h, wheat straw was fractionated to pulp (cellulose), lignin and monosaccharides mainly from hemicellulose with yields of approximately 50%, 15% and 35%, respectively. Acetic acid pulp from the straw had an acceptable strength for paper and could be bleached to a high brightness over 85% with a short bleaching sequence. Acetic acid pulp was also a potential feedstock for fuels and chemicals. The acetic acid process separated pentose and hexose in wheat straw to a large extent. Most of the pentose (xylan) was dissolved, whereas the hexose (glucan) remained in the pulp. Approximately 30% of carbohydrates in wheat straw were hydrolyzed to monosaccharides during acetic acid pulping, of which xylose accounted for 70% and glucose for 12%. The acetic acid lignin from wheat straw showed relatively lower molecular weight and fusibility, which made the lignin a promising raw material for many products, such as adhesive and molded products.     

The lignin present in steam pretreated softwood binds enzymes and limits cellulose accessibility

The influence of cellulose accessibility and protein loading on the efficiency of enzymatic hydrolysis of steam pretreated Douglas-fir was assessed. It was apparent that the lignin component significantly influences the swelling/accessibility of cellulose as at low protein loadings (5 FPU/g cellulose), only 16% of the cellulose present in the steam pretreated softwood was hydrolyzed while almost complete hydrolysis was achieved with the delignified substrate. When lignin (isolated from steam pretreated Douglas-fir) was added back in the same proportions it was originally found to the highly accessible and swollen, delignified steam pretreated softwood and to a cellulose control such as Avicel, the hydrolysis yields decreased by 9 and 46%, respectively. However, when higher enzyme loadings were employed, the greater availability of the enzyme could overcome the limitations imposed by both the lignin’s restrictions on cellulose accessibility and direct binding of the enzymes, resulting in a near complete hydrolysis of the cellulose.