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.

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.

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.

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.

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.

A multi-omics approach reveals function of Secretory Carrier-Associated Membrane Proteins in wood formation of​ ​​Populus​​ ​trees.

BACKGROUND: Secretory Carrier-Associated Membrane Proteins (SCAMPs) are highly conserved 32-38 kDa proteins that are involved in membrane trafficking. A systems approach was taken to elucidate function of SCAMPs in wood formation of Populus trees. Phenotypic and multi-omics analyses were performed in woody tissues of transgenic Populus trees carrying an RNAi construct for Populus tremula x tremuloides SCAMP3 (PttSCAMP3; Potri.019G104000). RESULTS: The woody tissues of the transgenic trees displayed increased amounts of both polysaccharides and lignin oligomers, indicating increased deposition of both the carbohydrate and lignin components of the secondary cell walls. This coincided with a tendency towards increased wood density as well as significantly increased thickness of the suberized cork in the transgenic lines. Multivariate OnPLS (orthogonal projections to latent structures) modeling of five different omics datasets (the transcriptome, proteome, GC-MS metabolome, LC-MS metabolome and pyrolysis-GC/MS metabolome) collected from the secondary xylem tissues of the stem revealed systemic variation in the different variables in the transgenic lines, including changes that correlated with the changes in the secondary cell wall composition. The OnPLS model also identified a rather large number of proteins that were more abundant in the transgenic lines than in the wild type. Several of these were related to secretion and/or endocytosis as well as both primary and secondary cell wall biosynthesis. CONCLUSIONS: Populus SCAMP proteins were shown to influence accumulation of secondary cell wall components, including polysaccharides and phenolic compounds, in the woody tissues of Populus tree stems. Our multi-omics analyses combined with the OnPLS modelling suggest that this function is mediated by changes in membrane trafficking to fine-tune the abundance of cell wall precursors and/or proteins involved in cell wall biosynthesis and transport. The data provides a multi-level source of information for future studies on the function of the SCAMP proteins in plant stem tissues.

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.

Cytosolic invertase contributes to the supply of substrate for cellulose biosynthesis in developing wood.

Carbon for cellulose biosynthesis is derived from sucrose. Cellulose is synthesized from uridine 5'-diphosphoglucose (UDP-glucose), but the enzyme(s) responsible for the initial sucrose cleavage and the source of UDP-glucose for cellulose biosynthesis in developing wood have not been defined. We investigated the role of CYTOSOLIC INVERTASEs (CINs) during wood formation in hybrid aspen (Populus tremula × tremuloides) and characterized transgenic lines with reduced CIN activity during secondary cell wall biosynthesis. Suppression of CIN activity by 38-55% led to a 9-13% reduction in crystalline cellulose. The changes in cellulose were reflected in reduced diameter of acid-insoluble cellulose microfibrils and increased glucose release from wood upon enzymatic digestion of cellulose. Reduced CIN activity decreased the amount of the cellulose biosynthesis precursor UDP-glucose in developing wood, pointing to the likely cause of the cellulose phenotype. The findings suggest that CIN activity has an important role in the cellulose biosynthesis of trees, and indicate that cellulose biosynthesis in wood relies on a quantifiable UDP-glucose pool. The results also introduce a concept of altering cellulose microfibril properties by modifying substrate supply to cellulose biosynthesis.

Downregulation of RWA genes in hybrid aspen affects xylan acetylation and wood saccharification.

High acetylation of angiosperm wood hinders its conversion to sugars by glycoside hydrolases, subsequent ethanol fermentation and (hence) its use for biofuel production. We studied the REDUCED WALL ACETYLATION (RWA) gene family of the hardwood model Populus to evaluate its potential for improving saccharification. The family has two clades, AB and CD, containing two genes each. All four genes are expressed in developing wood but only RWA-A and -B are activated by master switches of the secondary cell wall PtNST1 and PtMYB21. Histochemical analysis of promoter::GUS lines in hybrid aspen (Populus tremula × tremuloides) showed activation of RWA-A and -B promoters in the secondary wall formation zone, while RWA-C and -D promoter activity was diffuse. Ectopic downregulation of either clade reduced wood xylan and xyloglucan acetylation. Suppressing both clades simultaneously using the wood-specific promoter reduced wood acetylation by 25% and decreased acetylation at position 2 of Xylp in the dimethyl sulfoxide-extracted xylan. This did not affect plant growth but decreased xylose and increased glucose contents in the noncellulosic monosaccharide fraction, and increased glucose and xylose yields of wood enzymatic hydrolysis without pretreatment. Both RWA clades regulate wood xylan acetylation in aspen and are promising targets to improve wood saccharification.

Profiling of Saccharomyces cerevisiae transcription factors for engineering the resistance of yeast to lignocellulose-derived inhibitors in biomass conversion.

BACKGROUND: Yeast transcription factors (TFs) involved in the regulation of multidrug resistance (MDR) were investigated in experiments with deletion mutants, transformants overexpressing synthetic genes encoding TFs, and toxic concentrations of lignocellulose-derived substances added to cultures as complex mixtures or as specific compounds, viz. coniferyl aldehyde, 5-hydroxymethylfurfural, and furfural. RESULTS: In the presence of complex mixtures of toxic substances from spruce wood, transformants overexpressing YAP1 and STB5, TFs involved in oxidative stress response, exhibited enhanced relative growth rates amounting to 4.589 ± 0.261 and 1.455 ± 0.185, respectively. Other TFs identified as important for resistance included DAL81, GZF3, LEU3, PUT3, and WAR1. Potential overlapping functions of YAP1 and STB5 were investigated in experiments with permutations of deletions and overexpression of the two genes. YAP1 complemented STB5 with respect to resistance to 5-hydroxymethylfurfural, but had a distinct role with regard to resistance to coniferyl aldehyde as deletion of YAP1 rendered the cell incapable of resisting coniferyl aldehyde even if STB5 was overexpressed. CONCLUSIONS: We have investigated 30 deletion mutants and eight transformants overexpressing MDR transcription factors with regard to the roles the transcription factors play in the resistance to toxic concentrations of lignocellulose-derived substances. This work provides an overview of the involvement of thirty transcription factors in the resistance to lignocellulose-derived substances, shows distinct and complementary roles played by YAP1 and STB5, and offers directions for the engineering of robust yeast strains for fermentation processes based on lignocellulosic feedstocks.

Comparison of tolerance of four bacterial nanocellulose-producing strains to lignocellulose-derived inhibitors.

BACKGROUND: Through pretreatment and enzymatic saccharification lignocellulosic biomass has great potential as a low-cost feedstock for production of bacterial nanocellulose (BNC), a high value-added microbial product, but inhibitors formed during pretreatment remain challenging. In this study, the tolerance to lignocellulose-derived inhibitors of three new BNC-producing strains were compared to that of Komagataeibacter xylinus ATCC 23770. Inhibitors studied included furan aldehydes (furfural and 5-hydroxymethylfurfural) and phenolic compounds (coniferyl aldehyde and vanillin). The performance of the four strains in the presence and absence of the inhibitors was assessed using static cultures, and their capability to convert inhibitors by oxidation and reduction was analyzed. RESULTS: Although two of the new strains were more sensitive than ATCC 23770 to furan aldehydes, one of the new strains showed superior resistance to both furan aldehydes and phenols, and also displayed high volumetric BNC yield (up to 14.78 ± 0.43 g/L) and high BNC yield on consumed sugar (0.59 ± 0.02 g/g). The inhibitors were oxidized and/or reduced by the strains to be less toxic. The four strains exhibited strong similarities with regard to predominant bioconversion products from the inhibitors, but displayed different capacity to convert the inhibitors, which may be related to the differences in inhibitor tolerance. CONCLUSIONS: This investigation provides information on different performance of four BNC-producing strains in the presence of lignocellulose-derived inhibitors. The results will be of benefit to the selection of more suitable strains for utilization of lignocellulosics in the process of BNC-production.

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.

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.

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.

Production of cellulosic ethanol and enzyme from waste fiber sludge using SSF, recycling of hydrolytic enzymes and yeast, and recombinant cellulase-producing Aspergillus niger.

Bioethanol and enzymes were produced from fiber sludges through sequential microbial cultivations. After a first simultaneous saccharification and fermentation (SSF) with yeast, the bioethanol concentrations of sulfate and sulfite fiber sludges were 45.6 and 64.7 g/L, respectively. The second SSF, which included fresh fiber sludges and recycled yeast and enzymes from the first SSF, resulted in ethanol concentrations of 38.3 g/L for sulfate fiber sludge and 24.4 g/L for sulfite fiber sludge. Aspergillus niger carrying the endoglucanase-encoding Cel7B gene of Trichoderma reesei was grown in the spent fiber sludge hydrolysates. The cellulase activities obtained with spent hydrolysates of sulfate and sulfite fiber sludges were 2,700 and 2,900 nkat/mL, respectively. The high cellulase activities produced by using stillage and the significant ethanol concentrations produced in the second SSF suggest that onsite enzyme production and recycling of enzyme are realistic concepts that warrant further attention.

Comparison of methods for detoxification of spruce hydrolysate for bacterial cellulose production.

BACKGROUND: Bacterial cellulose (BC) is a nanostructured material with unique properties and wide applicability. In order to decrease the production cost of bacterial cellulose, lignocellulose-based media have considerable potential as alternative cost-effective feedstocks. However, pretreatment and enzymatic hydrolysis of lignocellulose to sugars also generate fermentation inhibitors. Detoxification of lignocellulosic hydrolysates is needed to achieve efficient production of BC. In this investigation, different methods for detoxification of spruce hydrolysate prior to production of BC were compared with respect to effects on potential inhibitors and fermentable sugars, sugar consumption, BC yield, and cell viability. The objectives were to identify efficient detoxification methods and to achieve a better understanding of the role played by different inhibitors in lignocellulosic hydrolysates. RESULTS: In a first series of experiments, the detoxification methods investigated included treatments with activated charcoal, alkali [sodium hydroxide, calcium hydroxide (overliming), and ammonium hydroxide], anion and cation ion-exchange resins, and reducing agents (sodium sulfite and sodium dithionite). A second series of detoxification experiments included enzymatic treatments (laccase and peroxidase). The potential inhibitors studied included aliphatic acids, furan aldehydes, and phenolic compounds. The best effects in the first series of detoxification experiments were achieved with activated charcoal and anion exchanger. After detoxification with activated charcoal the BC yield was 8.2 g/L, while it was 7.5 g/L in a reference medium without inhibitors. Treatments with anion exchanger at pH 10 and pH 5.5 gave a BC yield of 7.9 g/L and 6.3 g/L, respectively. The first series of experiments suggested that there was a relationship between the BC yield and phenolic inhibitors. Therefore, the second series of detoxification experiments focused on treatments with phenol-oxidizing enzymes. The BC yield in the laccase-detoxified hydrolysate reached 5.0-5.5 g/L after 14 days cultivation, which demonstrated the important inhibitory role played by phenolic compounds. CONCLUSIONS: The investigation shows that detoxification methods that efficiently remove phenolics benefit bacterial growth and BC production. Negative effects of salts could not be excluded and the osmotolerance of Gluconacetobacter xylinus needs to be further investigated in the future. Combinations of detoxification methods that efficiently decrease the concentration of inhibitors remain as an interesting option.

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.