The effect of nitrogen source and levels on hybrid aspen tree physiology and wood formation.

Nitrogen can be taken up by trees in the form of nitrate, ammonium and amino acids, but the influence of the different forms on tree growth and development is poorly understood in angiosperm species like Populus. We studied the effects of both organic and inorganic forms of nitrogen on growth and wood formation of hybrid aspen trees in experimental conditions that allowed growth under four distinct steady-state nitrogen levels. Increased nitrogen availability had a positive influence on biomass accumulation and the radial dimensions of both xylem vessels and fibers, and a negative influence on wood density. An optimal level of nitrogen availability was identified where increases in biomass accumulation outweighed decreases in wood density. None of these responses depended on the source of nitrogen except for shoot biomass accumulation, which was stimulated more by treatments complemented with nitrate than by ammonium alone or the organic source arginine. The most striking difference between the nitrogen sources was the effect on lignin composition, whereby the abundance of H-type lignin increased only in the presence of nitrate. The differential effect of nitrate is possibly related to the well-known role of nitrate as a signaling compound. RNA-sequencing revealed that while the lignin-biosynthetic genes did not significantly (FDR <0.01) respond to added NO3 - , the expression of several laccases, catalysing lignin polymerization, was dependent on N-availability. These results reveal a unique role of nitrate in wood formation and contribute to the knowledge basis for decision-making in utilizing hybrid aspen as a bioresource.

In vitro evaluation of the application of an optimized xylanase cocktail for improved monogastric feed digestibility.

Xylanases from glycoside hydrolase (GH) families 10 and 11 are common feed additives for broiler chicken diets due to their catalytic activity on the nonstarch polysaccharide xylan. This study investigated the potential of an optimized binary GH10 and GH11 xylanase cocktail to mitigate the antinutritional effects of xylan on the digestibility of locally sourced chicken feed. Immunofluorescence visualization of the activity of the xylanase cocktail on xylan in the yellow corn of the feed showed a substantial collapse in the morphology of cell walls. Secondly, the reduction in the viscosity of the digesta of the feed by the cocktail showed an effective degradation of the soluble fraction of xylan. Analysis of the xylan degradation products from broiler feeds by the xylanase cocktail showed that xylotriose and xylopentaose were the major xylooligosaccharides (XOS) produced. In vitro evaluation of the prebiotic potential of these XOS showed that they improved the growth of the beneficial bacteria Streptococcus thermophilus and Lactobacillus bulgaricus. The antibacterial activity of broths from XOS-supplemented probiotic cultures showed a suppressive effect on the growth of the extraintestinal infectious bacterium Klebsiella pneumoniae. Supplementing the xylanase cocktail in cereal animal feeds attenuated xylan's antinutritional effects by reducing digesta viscosity and releasing entrapped nutrients. Furthermore, the production of prebiotic XOS promoted the growth of beneficial bacteria while inhibiting the growth of pathogens. Based on these effects of the xylanase cocktail on the feed, improved growth performance and better feed conversion can potentially be achieved during poultry rearing.

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.

LPMO-supported saccharification of biomass: effects of continuous aeration of reaction mixtures with variable fractions of water-insoluble solids and cellulolytic enzymes.

BACKGROUND: High substrate concentrations and high sugar yields are important aspects of enzymatic saccharification of lignocellulosic substrates. The benefit of supporting the catalytic action of lytic polysaccharide monooxygenase (LPMO) through continuous aeration of slurries of pretreated softwood was weighed against problems associated with increasing substrate content (quantitated as WIS, water-insoluble solids, in the range 12.5-17.5%), and was compared to the beneficial effect on the saccharification reaction achieved by increasing the enzyme preparation (Cellic CTec3) loadings. Aerated reactions were compared to reactions supplied with N2 to assess the contribution of LPMO to the saccharification reactions. Analysis using 13C NMR spectroscopy, XRD, Simons' staining, BET analysis, and SEM analysis was used to gain further insights into the effects of the cellulolytic enzymes on the substrate under different reaction conditions. RESULTS: Although glucose production after 72 h was higher at 17.5% WIS than at 12.5% WIS, glucan conversion decreased with 24% (air) and 17% (N2). Compared to reactions with N2, the average increases in glucose production for aerated reactions were 91% (12.5% WIS), 70% (15.0% WIS), and 67% (17.5% WIS). Improvements in glucan conversion through aeration were larger (55-86%) than the negative effects of increasing WIS content. For reactions with 12.5% WIS, increased enzyme dosage with 50% improved glucan conversion with 25-30% for air and N2, whereas improvements with double enzyme dosage were 30% (N2) and 39% (air). Structural analyses of the solid fractions revealed that the enzymatic reaction, particularly with aeration, created increased surface area (BET analysis), increased disorder (SEM analysis), decreased crystallinity (XRD), and increased dye adsorption based on the cellulose content (Simons' staining). CONCLUSIONS: The gains in glucan conversion with aeration were larger than the decreases observed due to increased substrate content, resulting in higher glucan conversion when using aeration at the highest WIS value than when using N2 at the lowest WIS value. The increase in glucan conversion with double enzyme preparation dosage was smaller than the increase achieved with aeration. The results demonstrate the potential in using proper aeration to exploit the inherent capacity of LPMO in enzymatic saccharification of lignocellulosic substrates and provide detailed information about the characteristics of the substrate after interaction with cellulolytic enzymes.

Impact of xylan on field productivity and wood saccharification properties in aspen.

Xylan that comprises roughly 25% of hardwood biomass is undesirable in biorefinery applications involving saccharification and fermentation. Efforts to reduce xylan levels have therefore been made in many species, usually resulting in improved saccharification. However, such modified plants have not yet been tested under field conditions. Here we evaluate the field performance of transgenic hybrid aspen lines with reduced xylan levels and assess their usefulness as short-rotation feedstocks for biorefineries. Three types of transgenic lines were tested in four-year field tests with RNAi constructs targeting either Populus GT43 clades B and C (GT43BC) corresponding to Arabidopsis clades IRX9 and IRX14, respectively, involved in xylan backbone biosynthesis, GATL1.1 corresponding to AtGALT1 involved in xylan reducing end sequence biosynthesis, or ASPR1 encoding an atypical aspartate protease. Their productivity, wood quality traits, and saccharification efficiency were analyzed. The only lines differing significantly from the wild type with respect to growth and biotic stress resistance were the ASPR1 lines, whose stems were roughly 10% shorter and narrower and leaves showed increased arthropod damage. GT43BC lines exhibited no growth advantage in the field despite their superior growth in greenhouse experiments. Wood from the ASPR1 and GT43BC lines had slightly reduced density due to thinner cell walls and, in the case of ASPR1, larger cell diameters. The xylan was less extractable by alkali but more hydrolysable by acid, had increased glucuronosylation, and its content was reduced in all three types of transgenic lines. The hemicellulose size distribution in the GALT1.1 and ASPR1 lines was skewed towards higher molecular mass compared to the wild type. These results provide experimental evidence that GATL1.1 functions in xylan biosynthesis and suggest that ASPR1 may regulate this process. In saccharification without pretreatment, lines of all three constructs provided 8-11% higher average glucose yields than wild-type plants. In saccharification with acid pretreatment, the GT43BC construct provided a 10% yield increase on average. The best transgenic lines of each construct are thus predicted to modestly outperform the wild type in terms of glucose yields per hectare. The field evaluation of transgenic xylan-reduced aspen represents an important step towards more productive feedstocks for biorefineries.

Conditioning of pretreated birch by liquid-liquid organic extractions to improve yeast fermentability and enzymatic digestibility.

By-products from hydrothermal pretreatment of lignocellulosic biomass inhibit enzymatic saccharification and microbial fermentation. Three long-chain organic extractants (Alamine 336, Aliquat 336 and Cyanex 921) were compared to two conventional organic solvents (ethyl acetate and xylene) with regard to conditioning of birch wood pretreatment liquid (BWPL) for improved fermentation and saccharification. In the fermentation experiments, extraction with Cyanex 921 resulted in the best ethanol yield, 0.34 ± 0.02 g g-1 on initial fermentable sugars. Extraction with xylene also resulted in a relatively high yield, 0.29 ± 0.02 g g-1, while cultures consisting of untreated BWPL and BWPL treated with the other extractants exhibited no ethanol formation. Aliquat 336 was most efficient with regard to removing by-products, but the residual Aliquat after the extraction was toxic to yeast cells. Enzymatic digestibility increased by 19-33% after extraction with the long-chain organic extractants. The investigation demonstrates that conditioning with long-chain organic extractants has the potential to relieve inhibition of both enzymes and microbes.

Genetic markers and tree properties predicting wood biorefining potential in aspen (Populus tremula) bioenergy feedstock.

BACKGROUND: Wood represents the majority of the biomass on land and constitutes a renewable source of biofuels and other bioproducts. However, wood is recalcitrant to bioconversion, raising a need for feedstock improvement in production of, for instance, biofuels. We investigated the properties of wood that affect bioconversion, as well as the underlying genetics, to help identify superior tree feedstocks for biorefining. RESULTS: We recorded 65 wood-related and growth traits in a population of 113 natural aspen genotypes from Sweden ( https://doi.org/10.5061/dryad.gtht76hrd ). These traits included three growth and field performance traits, 20 traits for wood chemical composition, 17 traits for wood anatomy and structure, and 25 wood saccharification traits as indicators of bioconversion potential. Glucose release after saccharification with acidic pretreatment correlated positively with tree stem height and diameter and the carbohydrate content of the wood, and negatively with the content of lignin and the hemicellulose sugar units. Most of these traits displayed extensive natural variation within the aspen population and high broad-sense heritability, supporting their potential in genetic improvement of feedstocks towards improved bioconversion. Finally, a genome-wide association study (GWAS) revealed 13 genetic loci for saccharification yield (on a whole-tree-biomass basis), with six of them intersecting with associations for either height or stem diameter of the trees. CONCLUSIONS: The simple growth traits of stem height and diameter were identified as good predictors of wood saccharification yield in aspen trees. GWAS elucidated the underlying genetics, revealing putative genetic markers for bioconversion of bioenergy tree feedstocks.

Biopolymer production from biomass produced by Nordic microalgae grown in wastewater.

Biomass from four different Nordic microalgal species, grown in BG-11 medium or synthetic wastewater (SWW), was explored as inexpensive carbohydrate-rich feedstock for polyhydroxybutyrate (PHB) production via microbial fermentation. Thermochemical pre-treatment (acid treatment followed by autoclavation) with 2% hydrochloric acid or 1% sulphuric acid (v/v) was used to maximize sugar yield prior to fermentation. Pre-treatment resulted in ∼5-fold higher sugar yield compared to the control. The sugar-rich hydrolysate was used as carbon source for the PHB-producing extremophilic bacterium Halomonas halophila. Maximal PHB production was achieved with hydrolysate of Chlorococcum sp. (MC-1) grown on BG-11 medium (0.27 ± 0.05 g PHB/ g DW), followed by hydrolysate derived from Desmodesmus sp. (RUC-2) grown on SWW (0.24 ± 0.05 g PHB/ g DW). Nordic microalgal biomass grown on wastewater therefore can be used as cheap feedstock for sustainable bioplastic production. This research highlights the potential of Nordic microalgae to develop a biobased economy.

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.

Xylan glucuronic acid side chains fix suberin-like aliphatic compounds to wood cell walls.

Wood is the most important repository of assimilated carbon in the biosphere, in the form of large polymers (cellulose, hemicelluloses including glucuronoxylan, and lignin) that interactively form a composite, together with soluble extractives including phenolic and aliphatic compounds. Molecular interactions among these compounds are not fully understood. We have targeted the expression of a fungal α-glucuronidase to the wood cell wall of aspen (Populus tremula L. × tremuloides Michx.) and Arabidopsis (Arabidopsis thaliana (L.) Heynh), to decrease contents of the 4-O-methyl glucuronopyranose acid (mGlcA) substituent of xylan, to elucidate mGlcA's functions. The enzyme affected the content of aliphatic insoluble cell wall components having composition similar to suberin, which required mGlcA for binding to cell walls. Such suberin-like compounds have been previously identified in decayed wood, but here, we show their presence in healthy wood of both hardwood and softwood species. By contrast, γ-ester bonds between mGlcA and lignin were insensitive to cell wall-localized α-glucuronidase, supporting the intracellular formation of these bonds. These findings challenge the current view of the wood cell wall composition and reveal a novel function of mGlcA substituent of xylan in fastening of suberin-like compounds to cell wall. They also suggest an intracellular initiation of lignin-carbohydrate complex assembly.

Comparison of solid and liquid fractions of pretreated Norway spruce as reductants in LPMO-supported saccharification of cellulose.

The role of lignin in enzymatic saccharification of cellulose involving lytic polysaccharide monooxygenase (LPMO) was investigated in experiments with the solid and liquid fractions of pretreated Norway spruce from a biorefinery demonstration plant using hydrothermal pretreatment and impregnation with sulfur dioxide. Pretreated biomass before and after enzymatic saccharification was characterized using HPAEC, HPLC, Py-GC/MS, 2D-HSQC NMR, FTIR, and SEM. Chemical characterization indicated that relatively harsh pretreatment conditions resulted in that the solid phase contained no or very little hemicellulose but considerable amounts of pseudo-lignin, and that the liquid phase contained a relatively high concentration (∼5 g/L) of lignin-derived phenolics. As judged from reactions continuously supplied with either air or nitrogen gas, lignin and lignin fragments from both the solid and the liquid phases efficiently served as reductants in LPMO-supported saccharification. When air was used to promote LPMO activity, the enzymatic conversion of cellulose after 72 h was 25% higher in reactions with pretreated solids and buffer, and 14% higher in reactions with pretreatment liquid and microcrystalline cellulose. Research in this area is useful for designing efficient saccharification steps in biochemical conversion of lignocellulosic biomass.

Valorization of hydrolysis lignin from a spruce-based biorefinery by applying γ-valerolactone treatment.

Hydrolysis lignin, i.e., the hydrolysis residue of cellulosic ethanol plants, was extracted with the green solvent γ-valerolactone (GVL). Treatments at 170-210 °C were performed with either non-acidified GVL/water mixtures (NA-GVL) or with mixtures containing sulfuric acid (SA-GVL). SA-GVL treatment at 210 °C resulted in the highest lignin solubilization (64% (w/w) of initial content), and 76% of the solubilized mass was regenerated by water-induced precipitation. Regenerated lignins were characterized through compositional analysis with sulfuric acid, as well as using pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS), high-performance size-exclusion chromatography (HPSEC), solid-state cross-polarization/magic angle spinning 13C nuclear magnetic resonance (CP/MAS 13C NMR) spectroscopy, 1H-13C heteronuclear single-quantum coherence NMR (HSQC NMR), and Fourier-transform infrared (FTIR) spectroscopy. The characterization revealed that the main difference between regenerated lignins was their molecular weight. Molecular weight averages increased with treatment temperature, and they were higher and had broader distribution for SA-GVL lignins than for NA-GVL lignins.

Hydrothermal Pretreatment of Lignocellulosic Feedstocks to Facilitate Biochemical Conversion.

Biochemical conversion of lignocellulosic feedstocks to advanced biofuels and other bio-based commodities typically includes physical diminution, hydrothermal pretreatment, enzymatic saccharification, and valorization of sugars and hydrolysis lignin. This approach is also known as a sugar-platform process. The goal of the pretreatment is to facilitate the ensuing enzymatic saccharification of cellulose, which is otherwise impractical due to the recalcitrance of lignocellulosic feedstocks. This review focuses on hydrothermal pretreatment in comparison to alternative pretreatment methods, biomass properties and recalcitrance, reaction conditions and chemistry of hydrothermal pretreatment, methodology for characterization of pretreatment processes and pretreated materials, and how pretreatment affects subsequent process steps, such as enzymatic saccharification and microbial fermentation. Biochemical conversion based on hydrothermal pretreatment of lignocellulosic feedstocks has emerged as a technology of high industrial relevance and as an area where advances in modern industrial biotechnology become useful for reducing environmental problems and the dependence on fossil resources.

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.

Overexpression of vesicle-associated membrane protein PttVAP27-17 as a tool to improve biomass production and the overall saccharification yields in Populus trees.

BACKGROUND: Bioconversion of wood into bioproducts and biofuels is hindered by the recalcitrance of woody raw material to bioprocesses such as enzymatic saccharification. Targeted modification of the chemical composition of the feedstock can improve saccharification but this gain is often abrogated by concomitant reduction in tree growth. RESULTS: In this study, we report on transgenic hybrid aspen (Populus tremula × tremuloides) lines that showed potential to increase biomass production both in the greenhouse and after 5 years of growth in the field. The transgenic lines carried an overexpression construct for Populus tremula × tremuloides vesicle-associated membrane protein (VAMP)-associated protein PttVAP27-17 that was selected from a gene-mining program for novel regulators of wood formation. Analytical-scale enzymatic saccharification without any pretreatment revealed for all greenhouse-grown transgenic lines, compared to the wild type, a 20-44% increase in the glucose yield per dry weight after enzymatic saccharification, even though it was statistically significant only for one line. The glucose yield after enzymatic saccharification with a prior hydrothermal pretreatment step with sulfuric acid was not increased in the greenhouse-grown transgenic trees on a dry-weight basis, but increased by 26-50% when calculated on a whole biomass basis in comparison to the wild-type control. Tendencies to increased glucose yields by up to 24% were present on a whole tree biomass basis after acidic pretreatment and enzymatic saccharification also in the transgenic trees grown for 5 years on the field when compared to the wild-type control. CONCLUSIONS: The results demonstrate the usefulness of gene-mining programs to identify novel genes with the potential to improve biofuel production in tree biotechnology programs. Furthermore, multi-omic analyses, including transcriptomic, proteomic and metabolomic analyses, performed here provide a toolbox for future studies on the function of VAP27 proteins in plants.

Effects of redox environment on hydrothermal pretreatment of lignocellulosic biomass under acidic conditions.

The effects of the redox environment on acidic hydrothermal pretreatment were investigated in experiments with sugarcane bagasse (190 °C, 14 min) and Norway spruce (205 °C, 5 min). To modulate the redox environment, pretreatment was performed without gas addition, with N2, or with O2. Analyses covered pretreated solids, pretreatment liquids, condensates, enzymatic digestibility, and inhibitory effects of pretreatment liquids on yeast. Addition of gas, especially O2, resulted in increased severity, as reflected by up to 18 percent units lower recoveries of pretreated solids, up to 31 percent units lower glucan recoveries, improved hemicellulose removal, formation of pseudo-lignin, improved overall glucan conversion, and increased concentrations of several microbial inhibitors. Some inhibitors, such as formaldehyde and coniferyl aldehyde, did not, however, follow that pattern. TAC (Total Aromatic Content) values reflected inhibitory effects of pretreatment liquids. This study demonstrates how gas addition can be used to modulate the severity of acidic hydrothermal pretreatment.

Effects of Biosurfactants on Enzymatic Saccharification and Fermentation of Pretreated Softwood.

The enzymatic hydrolysis of cellulose is inhibited by non-productive adsorption of cellulases to lignin, and that is particularly problematic with lignin-rich materials such as softwood. Although conventional surfactants alleviate non-productive adsorption, using biosurfactants in softwood hydrolysis has not been reported. In this study, the effects of four biosurfactants, namely horse-chestnut escin, Pseudomonas aeruginosa rhamnolipid, and saponins from red and white quinoa varieties, on the enzymatic saccharification of steam-pretreated spruce were investigated. The used biosurfactants improved hydrolysis, and the best-performing one was escin, which led to cellulose conversions above 90%, decreased by around two-thirds lignin inhibition of Avicel hydrolysis, and improved hydrolysis of pretreated spruce by 24%. Red quinoa saponins (RQS) addition resulted in cellulose conversions above 80%, which was around 16% higher than without biosurfactants, and it was more effective than adding rhamnolipid or white quinoa saponins. Cellulose conversion improved with the increase in RQS addition up to 6 g/100 g biomass, but no significant changes were observed above that dosage. Although saponins are known to inhibit yeast growth, no inhibition of Saccharomyces cerevisiae fermentation of hydrolysates produced with RQS addition was detected. This study shows the potential of biosurfactants for enhancing the enzymatic hydrolysis of steam-pretreated softwood.

Hybrid Aspen Expressing a Carbohydrate Esterase Family 5 Acetyl Xylan Esterase Under Control of a Wood-Specific Promoter Shows Improved Saccharification.

Fast-growing broad-leaf tree species can serve as feedstocks for production of bio-based chemicals and fuels through biochemical conversion of wood to monosaccharides. This conversion is hampered by the xylan acetylation pattern. To reduce xylan acetylation in the wood, the Hypocrea jecorina acetyl xylan esterase (HjAXE) from carbohydrate esterase (CE) family 5 was expressed in hybrid aspen under the control of the wood-specific PtGT43B promoter and targeted to the secretory pathway. The enzyme was predicted to deacetylate polymeric xylan in the vicinity of cellulose due to the presence of a cellulose-binding module. Cell-wall-bound protein fractions from developing wood of transgenic plants were capable of releasing acetyl from finely ground wood powder, indicative of active AXE present in cell walls of these plants, whereas no such activity was detected in wild-type plants. The transgenic lines grew in height and diameter as well as wild-type trees, whereas their internodes were slightly shorter, indicating higher leaf production. The average acetyl content in the wood of these lines was reduced by 13%, mainly due to reductions in di-acetylated xylose units, and in C-2 and C-3 mono-acetylated xylose units. Analysis of soluble cell wall polysaccharides revealed a 4% reduction in the fraction of xylose units and an 18% increase in the fraction of glucose units, whereas the contents of cellulose and lignin were not affected. Enzymatic saccharification of wood from transgenic plants resulted in 27% higher glucose yield than for wild-type plants. Brunauer-Emmett-Teller (BET) analysis and Simons' staining pointed toward larger surface area and improved cellulose accessibility for wood from transgenic plants compared to wood from wild-type plants, which could be achieved by HjAXE deacetylating xylan bound to cellulose. The results show that CE5 family can serve as a source of enzymes for in planta reduction of recalcitrance to saccharification.