Saccharification Potential of Transgenic Greenhouse- and Field-Grown Aspen Engineered for Reduced Xylan Acetylation

High acetylation of xylan in hardwoods decreases their value as biorefinery feedstocks. To counter this problem, we have constitutively suppressed RWA genes encoding acetyl-CoA transporters using the 35S promoter, or constitutively and wood-specifically (using the WP promoter) expressed fungal acetyl xylan esterases of families CE1 (AnAXE1) and CE5 (HjAXE), to reduce acetylation in hybrid aspen. All these transformations improved the saccharification of wood from greenhouse-grown trees. Here, we describe the chemical properties and saccharification potential of the resulting lines grown in a five-year field trial, and one type of them (WP:AnAXE1) in greenhouse conditions. Chemically, the lignocellulose of the field- and greenhouse-field-grown plants slightly differed, but the reductions in acetylation and saccharification improvement of engineered trees were largely maintained in the field. The main novel phenotypic observation in the field was higher lignification in lines with the WP promoter than those with the 35S promoter. Following growth in the field, saccharification glucose yields were higher from most transformed lines than from wild-type (WT) plants with no pretreatment, but there was no improvement in saccharification with acid pretreatment. Thus, acid pretreatment removes most recalcitrance caused by acetylation. We found a complex relationship between acetylation and glucose yields in saccharification without pretreatment, suggesting that other variables, for example, the acetylation pattern, affect recalcitrance. Bigger gains in glucose yields were observed in lines with the 35S promoter than in those with the WP promoter, possibly due to their lower lignin content. However, better lignocellulose saccharification of these lines was offset by a growth penalty and their glucose yield per tree was lower. In a comparison of the best lines with each construct, WP:AnAXE1 provided the highest glucose yield per tree from saccharification, with and without pretreatment, WP:HjAXE yields were similar to those of WT plants, and yields of lines with other constructs were lower. These results show that lignocellulose properties of field-grown trees can be improved by reducing cell wall acetylation using various approaches, but some affect productivity in the field. Thus, better understanding of molecular and physiological consequences of deacetylation is needed to obtain quantitatively better results

Fractionated Lignosulfonates for Laccase-Catalyzed Oxygen-Scavenging Films and Coatings

Lignin derivatives have potential as antioxidants in advanced packaging materials through their ability to scavenge oxygen in reactions catalyzed by phenol-oxidizing enzymes, such as laccase. The effects of size fractionation of lignosulfonates on laccase-catalyzed reactions were investigated in experiments with aqueous solutions, films, and coated paperboard. Four industrial lignosulfonate preparations were compared: Feed (unfractionated), Prod (5–60 kDa enriched), Conc (≥60 kDa enriched), and Perm (≤60 kDa enriched). Extraction of lignosulfonates from films showed that the enzymic reaction increased the average molecular weight from <10,000 to up to 66,000. The enzymatic reaction resulted in an increase in the water contact angle of the films from the range 25–49° to 56–81°. The four preparations showed relatively small differences with regard to their ability to scavenge oxygen in aqueous solution and in experiments with coated paperboards in sealed chambers. Coatings with lignosulfonates where the contents of low-molecular weight material had been reduced (i.e., Prod and Conc) showed improved water resistance after the enzymic reaction. Thus, in both aqueous and solid media, fractionation of lignosulfonates had little effect on oxygen scavenging, but fractionation was beneficial for other reasons, such as improved cross-linking resulting in higher molecular weight and superior water resistance

Fractionated lignosulfonates for laccase-catalyzed oxygen-scavenging films and coatings

Lignin derivatives have potential as antioxidants in advanced packaging materials through their ability to scavenge oxygen in reactions catalyzed by phenol-oxidizing enzymes, such as laccase. The effects of size fractionation of lignosulfonates on laccase-catalyzed reactions were investigated in experiments with aqueous solutions, films, and coated paperboard. Four industrial lignosulfonate preparations were compared: Feed (unfractionated), Prod (5–60 kDa enriched), Conc (≥60 kDa enriched), and Perm (≤60 kDa enriched). Extraction of lignosulfonates from films showed that the enzymic reaction increased the average molecular weight from <10,000 to up to 66,000. The enzymatic reaction resulted in an increase in the water contact angle of the films from the range 25–49◦ to 56–81◦. The four preparations showed relatively small differences with regard to their ability to scavenge oxygen in aqueous solution and in experiments with coated paperboards in sealed chambers. Coatings with lignosulfonates where the contents of low-molecular weight material had been reduced (i.e., Prod and Conc) showed improved water resistance after the enzymic reaction. Thus, in both aqueous and solid media, fractionation of lignosulfonates had little effect on oxygen scavenging, but fractionation was beneficial for other reasons, such as improved cross-linking resulting in higher molecular weight and superior water resistance

Comparison of [HSO4](-), [Cl](-) and [MeCO2](-) as anions in pretreatment of aspen and spruce with imidazolium-based ionic liquids

Background: Ionic liquids (ILs) draw attention as green solvents for pretreatment of lignocellulose before enzymatic saccharification. Imidazolium-based ILs with different anionic constituents ([HSO4]−, [Cl]−, [MeCO2]−) were compared with regard to pretreatment of wood from aspen and spruce. The objective was to elucidate how the choice of anionic constituent affected the suitability of using the IL for pretreatment of hardwood, such as aspen, and softwood, such as spruce. The investigation covered a thorough analysis of the mass balance of the IL pretreatments, the effects of pretreatment on the cell wall structure as assessed by fluorescence microscopy, and the effects of pretreatment on the susceptibility to enzymatic saccharification. Torrefied aspen and spruce were included in the comparison for assessing how shifting contents of hemicelluloses and Klason lignin affected the susceptibility of the wood to IL pretreatment and enzymatic saccharification. Results: The glucose yield after IL pretreatment increased in the order [Cl]− < [HSO4]− < [MeCO2]− for aspen, but in the order [HSO4]− < [Cl]− < [MeCO2]− for spruce. For both aspen and spruce, removal of hemicelluloses and lignin increased in the order [Cl]− < [MeCO2]− < [HSO4]−. Fluorescence microscopy indicated increasingly disordered cell wall structure following the order [HSO4]− < [Cl]− < [MeCO2]−. Torrefaction of aspen converted xylan to pseudo-lignin and changed the glucose yield order to [HSO4]− < [Cl]− < [MeCO2]−. Conclusions: The acidity of [HSO4]− caused extensive hydrolysis of xylan, which facilitated pretreatment of xylan-rich hardwood. Apart from that, the degree of removal of hemicelluloses and lignin did not correspond well with the improvement of the enzymatic saccharification. Taken together, the saccharification results were found to mainly reflect (i) the different capacities of the ILs to disorder the cell wall structure, (ii) the recalcitrance caused by high xylan content, and (iii) the capacity of the [HSO4]−-based IL to hydrolyze xylan.Bio4Energ

Chemical and structural factors influencing enzymatic saccharification of wood from aspen, birch and spruce

The susceptibility of untreated and sulfuric-acid-pretreated aspen, birch, and spruce to analytical enzymatic saccharification was studied in relation to their chemical composition and physical-structural features. The analytical data collected covered the mass fractions of lignin, carbohydrates, and extractives, the release of acetic acid, formic acid, and uronic acids by acid and alkaline hydrolysis, crystallinity and crystallite size, syringyl: guaiacyl (S:G) ratio of lignin, cellulose accessibility, FTIR spectra, images from SEM and fluorescence microscopy, and susceptibility to enzymatic saccharification using enzyme mixtures with and without supplementary xylanase.In the absence of pretreatment the mass fraction yield of Glc on the original dry wood in the analytical enzymatic saccharification increased in the order birch (16 g kg−1) < spruce (35 g kg−1) < aspen (150 g kg−1). After acid pretreatment, the order changed to spruce (170 g kg−1) < aspen (290 g kg−1), birch (290 g kg−1). The relatively high recalcitrance of untreated birch was not possible to relate to mass fraction of lignin, S:G ratio, cellulose crystallinity, or mass fraction of acetyl, but rather to structural features, such as a more compact surface structure with high density and low cellulose accessibility. The relatively high sugar yields from both untreated and pretreated aspen suggest that aspen wood is well suited as feedstock for production of liquid biofuels and green chemicals in forest-based biorefineries.Bio4Energ

Evaluation of Oxalate Decarboxylase and Oxalate Oxidase for Industrial Applications.

Increased recirculation of process water has given rise to problems with formation of calcium oxalate incrusts (scaling) in the pulp and paper industry and in forest biorefineries. The potential in using oxalate decarboxylase from Aspergillus niger for oxalic acid removal in industrial bleaching plant filtrates containing oxalic acid was examined and compared with barley oxalate oxidase. Ten different filtrates from chemical pulping were selected for the evaluation. Oxalate decarboxylase degraded oxalic acid faster than oxalate oxidase in eight of the filtrates, while oxalate oxidase performed better in one filtrate. One of the filtrates inhibited both enzymes. The potential inhibitory effect of selected compounds on the enzymatic activity was tested. Oxalate decarboxylase was more sensitive than oxalate oxidase to hydrogen peroxide. Oxalate decarboxylase was not as sensitive to chlorate and chlorite as oxalate oxidase. Up to 4 mM chlorate ions, the highest concentration tested, had no inhibitory effect on oxalate decarboxylase. Analysis of the filtrates suggests that high concentrations of chlorate present in some of the filtrates were responsible for the higher sensitivity of oxalate oxidase in these filtrates. Oxalate decarboxylase was thus a better choice than oxalate oxidase for treatment of filtrates from chlorine dioxide bleaching

Evaluation of four ionic liquids for pretreatment of lignocellulosic biomass.

BACKGROUND: Lignocellulosic biomass is highly recalcitrant and various pretreatment techniques are needed to facilitate its effective enzymatic hydrolysis to produce sugars for further conversion to bio-based chemicals. Ionic liquids (ILs) are of interest in pretreatment because of their potential to dissolve lignocellulosic materials including crystalline cellulose. RESULTS: Four imidazolium-based ionic liquids (ILs) ([C=C2C1im][MeCO2], [C4C1im][MeCO2], [C4C1im][Cl], and [C4C1im][HSO4]) well known for their capability to dissolve lignocellulosic species were synthesized and then used for pretreatment of substrates prior to enzymatic hydrolysis. In order to achieve a broad evaluation, seven cellulosic, hemicellulosic and lignocellulosic substrates, crystalline as well as amorphous, were selected. The lignocellulosic substrates included hybrid aspen and Norway spruce. The monosaccharides in the enzymatic hydrolysate were determined using high-performance anion-exchange chromatography. The best results, as judged by the saccharification efficiency, were achieved with [C4C1im][Cl] for cellulosic substrates and with the acetate-based ILs for hybrid aspen and Norway spruce. After pretreatment with acetate-based ILs, the conversion to glucose of glucan in recalcitrant softwood lignocellulose reached similar levels as obtained with pure crystalline and amorphous cellulosic substrates. IL pretreatment of lignocellulose resulted in sugar yields comparable with that obtained with acidic pretreatment. Heterogeneous dissolution with [C4C1im][HSO4] gave promising results with aspen, the less recalcitrant of the two types of lignocellulose included in the investigation. CONCLUSIONS: The ability of ILs to dissolve lignocellulosic biomass under gentle conditions and with little or no by-product formation contributes to making them highly interesting alternatives for pretreatment in processes where high product yields are of critical importance

Comparison of lignin derivatives as substrates for laccase-catalyzed scavenging of oxygen in coatings and films

Background: Lignin derivatives are phenylpropanoid biopolymers derived from pulping and biorefinery processes. The possibility to utilize lignin derivatives from different types of processes in advanced enzyme-catalyzed oxygen-scavenging systems intended for active packaging was explored. Laccase-catalyzed oxidation of alkali lignin (LA), hydrolytic lignin (LH), organosolv lignin (LO), and lignosulfonates (LS) was compared using oxygen-scavenging coatings and films in liquid and gas phase systems. Results: When coatings containing lignin derivatives and laccase were immersed in a buffered aqueous solution, the oxygen-scavenging capability increased in the order LO < LH < LA < LS. Experiments with coatings containing laccase and LO, LH or LA incubated in oxygen-containing gas in air-tight chambers and at a relative humidity (RH) of 100% showed that paperboard coated with LO and laccase reduced the oxygen content from 1.0% to 0.4% during a four-day period, which was far better than the results obtained with LA or LH. LO-containing coatings incubated at 92% RH also displayed activity, with a decrease in oxygen from 1.0% to 0.7% during a four-day period. The oxygen scavenging was not related to the content of free phenolic hydroxyl groups, which increased in the order LO < LS < LH < LA. LO and LS were selected for further studies and films containing starch, clay, glycerol, laccase and LO or LS were characterized using gel permeation chromatograpy, dynamic mechanical analysis, and wet stability. Conclusions: The investigation shows that different lignin derivatives exhibit widely different properties as a part of active coatings and films. Results indicate that LS and LO were most suitable for the application studied and differences between them were attributed to a higher degree of laccase-catalyzed cross-linking of LS than of LO. Inclusion in active-packaging systems offers a new way to utilize some types of lignin derivatives from biorefining processes

Analysis, pretreatment and enzymatic saccharification of different fractions of Scots pine

Background: Forestry residues consisting of softwood are a major lignocellulosic resource for production of liquid biofuels. Scots pine, a commercially important forest tree, was fractionated into seven fractions of chips: juvenile heartwood, mature heartwood, juvenile sapwood, mature sapwood, bark, top parts, and knotwood. The different fractions were characterized analytically with regard to chemical composition and susceptibility to dilute-acid pretreatment and enzymatic saccharification. Results: All fractions were characterized by a high glucan content (38-43%) and a high content of other carbohydrates (11-14% mannan, 2-4% galactan) that generate easily convertible hexose sugars, and by a low content of inorganic material (0.2-0.9% ash). The lignin content was relatively uniform (27-32%) and the syringyl-guaiacyl ratio of the different fractions were within the range 0.021-0.025. The knotwood had a high content of extractives (9%) compared to the other fractions. The effects of pretreatment and enzymatic saccharification were relatively similar, but without pretreatment the bark fraction was considerably more susceptible to enzymatic saccharification. Conclusions: Since sawn timber is a main product from softwood species such as Scots pine, it is an important issue whether different parts of the tree are equally suitable for bioconversion processes. The investigation shows that bioconversion of Scots pine is facilitated by that most of the different fractions exhibit relatively similar properties with regard to chemical composition and susceptibility to techniques used for bioconversion of woody biomass