Flexure wood formation via growth reprogramming in hybrid aspen involves jasmonates and polyamines and transcriptional changes resembling tension wood development.

Stem bending in trees induces flexure wood but its properties and development are poorly understood. Here, we investigated the effects of low-intensity multidirectional stem flexing on growth and wood properties of hybrid aspen, and on its transcriptomic and hormonal responses. Glasshouse-grown trees were either kept stationary or subjected to several daily shakes for 5 wk, after which the transcriptomes and hormones were analyzed in the cambial region and developing wood tissues, and the wood properties were analyzed by physical, chemical and microscopy techniques. Shaking increased primary and secondary growth and altered wood differentiation by stimulating gelatinous-fiber formation, reducing secondary wall thickness, changing matrix polysaccharides and increasing cellulose, G- and H-lignin contents, cell wall porosity and saccharification yields. Wood-forming tissues exhibited elevated jasmonate, polyamine, ethylene and brassinosteroids and reduced abscisic acid and gibberellin signaling. Transcriptional responses resembled those during tension wood formation but not opposite wood formation and revealed several thigmomorphogenesis-related genes as well as novel gene networks including FLA and XTH genes encoding plasma membrane-bound proteins. Low-intensity stem flexing stimulates growth and induces wood having improved biorefinery properties through molecular and hormonal pathways similar to thigmomorphogenesis in herbaceous plants and largely overlapping with the tension wood program of hardwoods.

Field testing of transgenic aspen from large greenhouse screening identifies unexpected winners.

Trees constitute promising renewable feedstocks for biorefinery using biochemical conversion, but their recalcitrance restricts their attractiveness for the industry. To obtain trees with reduced recalcitrance, large-scale genetic engineering experiments were performed in hybrid aspen blindly targeting genes expressed during wood formation and 32 lines representing seven constructs were selected for characterization in the field. Here we report phenotypes of five-year old trees considering 49 traits related to growth and wood properties. The best performing construct considering growth and glucose yield in saccharification with acid pretreatment had suppressed expression of the gene encoding an uncharacterized 2-oxoglutarate-dependent dioxygenase (2OGD). It showed minor changes in wood chemistry but increased nanoporosity and glucose conversion. Suppressed levels of SUCROSE SYNTHASE, (SuSy), CINNAMATE 4-HYDROXYLASE (C4H) and increased levels of GTPase activating protein for ADP-ribosylation factor ZAC led to significant growth reductions and anatomical abnormalities. However, C4H and SuSy constructs greatly improved glucose yields in saccharification without and with pretreatment, respectively. Traits associated with high glucose yields were different for saccharification with and without pretreatment. While carbohydrates, phenolics and tension wood contents positively impacted the yields without pretreatment and growth, lignin content and S/G ratio were negative factors, the yields with pretreatment positively correlated with S lignin and negatively with carbohydrate contents. The genotypes with high glucose yields had increased nanoporosity and mGlcA/Xyl ratio, and some had shorter polymers extractable with subcritical water compared to wild-type. The pilot-scale industrial-like pretreatment of best-performing 2OGD construct confirmed its superior sugar yields, supporting our strategy.

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.

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.

Downregulating aspen xylan biosynthetic GT43 genes in developing wood stimulates growth via reprograming of the transcriptome.

Xylan is one of the main compounds determining wood properties in hardwood species. The xylan backbone is thought to be synthesized by a synthase complex comprising two members of the GT43 family. We downregulated all GT43 genes in hybrid aspen (Populus tremula × tremuloides) to understand their involvement in xylan biosynthesis. All three clades of the GT43 family were targeted for downregulation using RNA interference individually or in different combinations, either constitutively or specifically in developing wood. Simultaneous downregulation in developing wood of the B (IRX9) and C (IRX14) clades resulted in reduced xylan Xyl content relative to reducing end sequence, supporting their role in xylan backbone biosynthesis. This was accompanied by a higher lignocellulose saccharification efficiency. Unexpectedly, GT43 suppression in developing wood led to an overall growth stimulation, xylem cell wall thinning and a shift in cellulose orientation. Transcriptome profiling of these transgenic lines indicated that cell cycling was stimulated and secondary wall biosynthesis was repressed. We suggest that the reduced xylan elongation is sensed by the cell wall integrity surveying mechanism in developing wood. Our results show that wood-specific suppression of xylan-biosynthetic GT43 genes activates signaling responses, leading to increased growth and improved lignocellulose saccharification.

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.

Tolerance of the nanocellulose-producing bacterium Gluconacetobacter xylinus to lignocellulose-derived acids and aldehydes.

Lignocellulosic biomass serves as a potential alternative feedstock for production of bacterial nanocellulose (BNC), a high-value-added product of bacteria such as Gluconacetobacter xylinus. The tolerance of G. xylinus to lignocellulose-derived inhibitors (formic acid, acetic acid, levulinic acid, furfural, and 5-hydroxymethylfurfural) was investigated. Whereas 100 mM formic acid completely suppressed the metabolism of G. xylinus, 250 mM of either acetic acid or levulinic acid still allowed glucose metabolism and BNC production to occur. Complete suppression of glucose utilization and BNC production was observed after inclusion of 20 and 30 mM furfural and 5-hydroxymethylfurfural, respectively. The bacterium oxidized furfural and 5-hydroxymethylfurfural to furoic acid and 5-hydroxymethyl-2-furoic acid, respectively. The highest yields observed were 88% for furoic acid/furfural and 76% for 5-hydroxymethyl-2-furoic acid/5-hydroxymethylfurfural. These results are the first demonstration of the capability of G. xylinus to tolerate lignocellulose-derived inhibitors and to convert furan aldehydes.

Effects of aromatic compounds on the production of bacterial nanocellulose by Gluconacetobacter xylinus.

BACKGROUND: Bacterial cellulose (BC) is a polymeric nanostructured fibrillar network produced by certain microorganisms, principally Gluconacetobacter xylinus. BC has a great potential of application in many fields. Lignocellulosic biomass has been investigated as a cost-effective feedstock for BC production through pretreatment and hydrolysis. It is well known that detoxification of lignocellulosic hydrolysates may be required to achieve efficient production of BC. Recent results suggest that phenolic compounds contribute to the inhibition of G. xylinus. However, very little is known about the effect on G. xylinus of specific lignocellulose-derived inhibitors. In this study, the inhibitory effects of four phenolic model compounds (coniferyl aldehyde, ferulic acid, vanillin and 4-hydroxybenzoic acid) on the growth of G. xylinus, the pH of the culture medium, and the production of BC were investigated in detail. The stability of the phenolics in the bacterial cultures was investigated and the main bioconversion products were identified and quantified. RESULTS: Coniferyl aldehyde was the most potent inhibitor, followed by vanillin, ferulic acid, and 4-hydroxybenzoic acid. There was no BC produced even with coniferyl aldehyde concentrations as low as 2 mM. Vanillin displayed a negative effect on the bacteria and when the vanillin concentration was raised to 2.5 mM the volumetric yield of BC decreased to ~40% of that obtained in control medium without inhibitors. The phenolic acids, ferulic acid and 4-hydroxybenzoic acid, showed almost no toxic effects when less than 2.5 mM. The bacterial cultures oxidized coniferyl aldehyde to ferulic acid with a yield of up to 81%. Vanillin was reduced to vanillyl alcohol with a yield of up to 80%. CONCLUSIONS: This is the first investigation of the effect of specific phenolics on the production of BC by G. xylinus, and is also the first demonstration of the ability of G. xylinus to convert phenolic compounds. This study gives a better understanding of how phenolic compounds and G. xylinus cultures are affected by each other. Investigations in this area are useful for elucidating the mechanism behind inhibition of G. xylinus in lignocellulosic hydrolysates and for understanding how production of BC using lignocellulosic feedstocks can be performed in an efficient way.

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.

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.

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.

Production of bacterial cellulose and enzyme from waste fiber sludge.

BACKGROUND: Bacterial cellulose (BC) is a highly crystalline and mechanically stable nanopolymer, which has excellent potential as a material in many novel applications, especially if it can be produced in large amounts from an inexpensive feedstock. Waste fiber sludge, a residue with little or no value, originates from pulp mills and lignocellulosic biorefineries. A high cellulose and low lignin content contributes to making the fiber sludge suitable for bioconversion, even without a thermochemical pretreatment step. In this study, the possibility to combine production of BC and hydrolytic enzymes from fiber sludge was investigated. The BC was characterized using field-emission scanning electron microscopy and X-ray diffraction analysis, and its mechanical properties were investigated. RESULTS: Bacterial cellulose and enzymes were produced through sequential fermentations with the bacterium Gluconacetobacter xylinus and the filamentous fungus Trichoderma reesei. Fiber sludges from sulfate (SAFS) and sulfite (SIFS) processes were hydrolyzed enzymatically without prior thermochemical pretreatment and the resulting hydrolysates were used for BC production. The highest volumetric yields of BC from SAFS and SIFS were 11 and 10 g/L (DW), respectively. The BC yield on initial sugar in hydrolysate-based medium reached 0.3 g/g after seven days of cultivation. The tensile strength of wet BC from hydrolysate medium was about 0.04 MPa compared to about 0.03 MPa for BC from a glucose-based reference medium, while the crystallinity was slightly lower for BC from hydrolysate cultures. The spent hydrolysates were used for production of cellulase with T. reesei. The cellulase activity (CMCase activity) in spent SAFS and SIFS hydrolysates reached 5.2 U/mL (87 nkat/mL), which was similar to the activity level obtained in a reference medium containing equal amounts of reducing sugar. CONCLUSIONS: It was shown that waste fiber sludge is a suitable raw material for production of bacterial cellulose and enzymes through sequential fermentation. The concept studied offers efficient utilization of the various components in fiber sludge hydrolysates and affords a possibility to combine production of two high value-added products using residual streams from pulp mills and biorefineries. Cellulase produced in this manner could tentatively be used to hydrolyze fresh fiber sludge to obtain medium suitable for production of BC in the same biorefinery.

Oxygen-scavenging coatings and films based on lignosulfonates and laccase.

Laccase and lignosulfonates were included in coating colors and embedded in latex-based or starch-based films and coatings on foil or board. After 6 days at 23 °C and 100% relative humidity, the oxygen content in airtight chambers decreased from 1.0% (synthetic gas consisting of 99% N(2) and 1% O(2)) to 0.3% in the presence of board coated with lignosulfonate and laccase, while the oxygen content remained unchanged in control experiments without enzyme. The water stability of lignosulfonate-containing latex-based coatings and starch-based films was improved after laccase-catalyzed oxidation of lignosulfonates, which indicates polymerization to products with lower solubility in water. Furthermore, the E' modulus of starch-based films increased with 30%, which indicates laccase-catalyzed polymerization of lignosulfonates resulting in increased stiffness of the film. The results suggest that laccases and lignosulfonates can be used as an oxygen-scavenging system in active packaging and that enzyme-catalyzed polymerization of lignosulfonates contributes to improved water stability and mechanical properties.

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.