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.

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.

Bioconversion of Waste Fiber Sludge to Bacterial Nanocellulose and Use for Reinforcement of CTMP Paper Sheets.

Utilization of bacterial nanocellulose (BNC) for large-scale applications is restricted by low productivity in static cultures and by the high cost of the medium. Fiber sludge, a waste stream from pulp and paper mills, was enzymatically hydrolyzed to sugar, which was used for the production of BNC by the submerged cultivation of Komagataeibacter xylinus. Compared with a synthetic glucose-based medium, the productivity of purified BNC from the fiber sludge hydrolysate using shake-flasks was enhanced from 0.11 to 0.17 g/(L × d), although the average viscometric degree of polymerization (DPv) decreased from 6760 to 6050. The cultivation conditions used in stirred-tank reactors (STRs), including the stirring speed, the airflow, and the pH, were also investigated. Using STRs, the BNC productivity in fiber-sludge medium was increased to 0.32 g/(L × d) and the DPv was increased to 6650. BNC produced from the fiber sludge hydrolysate was used as an additive in papermaking based on the chemithermomechanical pulp (CTMP) of birch. The introduction of BNC resulted in a significant enhancement of the mechanical strength of the paper sheets. With 10% (w/w) BNC in the CTMP/BNC mixture, the tear resistance was enhanced by 140%. SEM images showed that the BNC cross-linked and covered the surface of the CTMP fibers, resulting in enhanced mechanical strength.

Ozone detoxification of steam-pretreated Norway spruce.

BACKGROUND: Pretreatment of lignocellulose for biochemical conversion commonly results in formation of by-products that inhibit microorganisms and cellulolytic enzymes. To make bioconversion processes more efficient, inhibition problems can be alleviated through conditioning. Ozone is currently commercially employed in pulp and paper production for bleaching, as it offers the desirable capability to disrupt unsaturated bonds in lignin through an ionic reaction known as ozonolysis. Ozonolysis is more selective towards lignin than cellulose, for instance, when compared to other oxidative treatment methods, such as Fenton's reagent. Ozone may thus have desirable properties for conditioning of pretreated lignocellulose without concomitant degradation of cellulose or sugars. Ozone treatment of SO2-impregnated steam-pretreated Norway spruce was explored as a potential approach to decrease inhibition of yeast and cellulolytic enzymes. This novel approach was furthermore compared to some of the most effective methods for conditioning of pretreated lignocellulose, i.e., treatment with alkali and sodium dithionite. RESULTS: Low dosages of ozone decreased the total contents of phenolics to about half of the initial value and improved the fermentability. Increasing ozone dosages led to almost proportional increase in the contents of total acids, including formic acid, which ultimately led to poor fermentability at higher ozone dosages. The decrease of the contents of furfural and 5-hydroxymethylfurfural was inversely proportional (R (2) > 0.99) to the duration of the ozone treatment, but exhibited no connection with the fermentability. Ozone detoxification was compared with other detoxification methods and was superior to treatment with Fenton's reagent, which exhibited no positive effect on fermentability. However, ozone detoxification was less efficient than treatment with alkali or sodium dithionite. High ozone dosages decreased the inhibition of cellulolytic enzymes as the glucose yield was improved with 13 % compared to that of an untreated control. CONCLUSIONS: Low dosages of ozone were beneficial for the fermentation of steam-pretreated Norway spruce, while high dosages decreased the inhibition of cellulolytic enzymes by soluble components in the pretreatment liquid. While clearly of interest for conditioning of lignocellulosic hydrolysates, future challenges include finding conditions that provide beneficial effects both with regard to enzymatic saccharification and microbial fermentation.

Techno-economic evaluation of conditioning with sodium sulfite for bioethanol production from softwood.

Conditioning with reducing agents allows alleviation of inhibition of biocatalytic processes by toxic by-products generated during biomass pretreatment, without necessitating the introduction of a separate process step. In this work, conditioning of steam-pretreated spruce with sodium sulfite made it possible to lower the yeast and enzyme dosages in simultaneous saccharification and fermentation (SSF) to 1g/L and 5FPU/g WIS, respectively. Techno-economic evaluation indicates that the cost of sodium sulfite can be offset by benefits resulting from a reduction of either the yeast load by 0.68g/L or the enzyme load by 1FPU/g WIS. As those thresholds were surpassed, inclusion of conditioning can be justified. Another potential benefit results from shortening the SSF time, which would allow reducing the bioreactor volume and result in capital savings. Sodium sulfite conditioning emerges as an opportunity to lower the financial uncertainty and compensate the overall investment risk for commercializing a softwood-to-ethanol process.

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.

Bioconversion of lignocellulose: inhibitors and detoxification.

Bioconversion of lignocellulose by microbial fermentation is typically preceded by an acidic thermochemical pretreatment step designed to facilitate enzymatic hydrolysis of cellulose. Substances formed during the pretreatment of the lignocellulosic feedstock inhibit enzymatic hydrolysis as well as microbial fermentation steps. This review focuses on inhibitors from lignocellulosic feedstocks and how conditioning of slurries and hydrolysates can be used to alleviate inhibition problems. Novel developments in the area include chemical in-situ detoxification by using reducing agents, and methods that improve the performance of both enzymatic and microbial biocatalysts.

Reducing agents improve enzymatic hydrolysis of cellulosic substrates in the presence of pretreatment liquid.

Enzymatic hydrolysis of pretreated lignocellulosic substrates has emerged as an interesting option to produce sugars that can be converted to liquid biofuels and other commodities using microbial biocatalysts. Lignocellulosic substrates are pretreated to make them more accessible to cellulolytic enzymes, but the pretreatment liquid partially inhibits subsequent enzymatic hydrolysis. The presence of pretreatment liquid from Norway spruce resulted in a 63% decrease in the enzymatic saccharification of Avicel compared to when the reaction was performed in a buffered aqueous solution. The addition of 15mM of a reducing agent (hydrogen sulfite, dithionite, or dithiothreitol) to reaction mixtures with the pretreatment liquid resulted in up to 54% improvement of the saccharification efficiency. When the reducing agents were added to reaction mixtures without pretreatment liquid, there was a 13-39% decrease in saccharification efficiency. In the presence of pretreatment liquid, the addition of 15mM dithionite to Avicel, α-cellulose or filter cake of pretreated spruce wood resulted in improvements between 25 and 33%. Positive effects (6-17%) of reducing agents were also observed in experiments with carboxymethyl cellulose and 2-hydroxyethyl cellulose. The approach to add reducing agents appears useful for facilitating the utilization of enzymes to convert cellulosic substrates in industrial processes.

Effect of sulfur oxyanions on lignocellulose-derived fermentation inhibitors.

Recent results show that treatments with reducing agents, including the sulfur oxyanions dithionite and hydrogen sulfite, efficiently improve the fermentability of inhibitory lignocellulose hydrolysates, and that the treatments are effective when the reducing agents are added in situ into the fermentation vessel at low temperature. In the present investigation, dithionite was added to medium with model inhibitors (coniferyl aldehyde, furfural, 5-hydroxymethylfurfural, or acetic acid) and the effects on the fermentability with yeast were studied. Addition of 10 mM dithionite to medium containing 2.5 mM coniferyl aldehyde resulted in a nine-fold increase in the glucose consumption rate and a three-fold increase in the ethanol yield. To investigate the mechanism behind the positive effects of adding sulfur oxyanions, mixtures containing 2.5 mM of a model inhibitor (an aromatic compound, a furan aldehyde, or an aliphatic acid) and 15 mM dithionite or hydrogen sulfite were analyzed using mass spectrometry (MS). The results of the analyses, which were performed by using UHPLC-ESI-TOF-MS and UHPLC-LTQ/Orbitrap-MS/MS, indicate that the positive effects of sulfur oxyanions are primarily due to their capability to react with and sulfonate inhibitory aromatic compounds and furan aldehydes at low temperature and slightly acidic pH (such as 25°C and pH 5.5).

Improving the fermentability of enzymatic hydrolysates of lignocellulose through chemical in-situ detoxification with reducing agents.

Inhibitory lignocellulose hydrolysates were treated with the reducing agents dithionite and sulfite to achieve improved fermentability. Addition of these reducing agents (in the concentration range 5.0-17.5 mM) to enzymatic hydrolysates of spruce wood or sugarcane bagasse improved processes based on both SHF (simultaneous hydrolysis and fermentation) and SSF (simultaneous saccharification and fermentation). The approach was exemplified in ethanolic fermentations with Saccharomyces cerevisiae and by using hydrolysates with sugar concentrations>100 g/L (for SHF) and with 10% dry-matter content (for SSF). In the SHF experiments, treatments with dithionite raised the ethanol productivities of the spruce hydrolysate from 0.2 to 2.5 g×L(-1)×h(-1) and of the bagasse hydrolysate from 0.9 to 3.9 g×L(-1)×h(-1), values even higher than those of fermentations with reference sugar solutions without inhibitors. Benefits of the approach include that the addition of the reducing agent can be made in-situ directly in the fermentation vessel, that the treatment can be performed at a temperature and pH suitable for fermentation, and that the treatment results in dramatically improved fermentability without degradation of fermentable sugars. The many benefits and the simplicity of the approach offer a new way to achieve more efficient manufacture of fermentation products from lignocellulose hydrolysates.

Biorefining of wood: combined production of ethanol and xylanase from waste fiber sludge.

The possibility to utilize fiber sludge, waste fibers from pulp mills and lignocellulose-based biorefineries, for combined production of liquid biofuel and biocatalysts was investigated. Without pretreatment, fiber sludge was hydrolyzed enzymatically to monosaccharides, mainly glucose and xylose. In the first of two sequential fermentation steps, the fiber sludge hydrolysate was fermented to cellulosic ethanol with the yeast Saccharomyces cerevisiae. Although the final ethanol yields were similar, the ethanol productivity after 9.5 h was 3.3 g/l/h for the fiber sludge hydrolysate compared with only 2.2 g/l/h for a reference fermentation with similar sugar content. In the second fermentation step, the spent fiber sludge hydrolysate (the stillage obtained after distillation) was used as growth medium for recombinant Aspergillus niger expressing the xylanase-encoding Trichoderma reesei (Hypocrea jecorina) xyn2 gene. The xylanase activity obtained with the spent fiber sludge hydrolysate (8,500 nkat/ml) was higher than that obtained in a standard medium with similar monosaccharide content (1,400 nkat/ml). Analyses based on deglycosylation with N-glycosidase F suggest that the main part of the recombinant xylanase was unglycosylated and had molecular mass of 20.7 kDa, while a minor part had N-linked glycosylation and molecular mass of 23.6 kDa. Chemical analyses of the growth medium showed that important carbon sources in the spent fiber sludge hydrolysate included xylose, small aliphatic acids, and oligosaccharides. The results show the potential of converting waste fiber sludge to liquid biofuel and enzymes as coproducts in lignocellulose-based biorefineries.

Cellulase production from spent lignocellulose hydrolysates by recombinant Aspergillus niger.

A recombinant Aspergillus niger strain expressing the Hypocrea jecorina endoglucanase Cel7B was grown on spent hydrolysates (stillage) from sugarcane bagasse and spruce wood. The spent hydrolysates served as excellent growth media for the Cel7B-producing strain, A. niger D15[egI], which displayed higher endoglucanase activities in the spent hydrolysates than in standard medium with a comparable monosaccharide content (e.g., 2,100 nkat/ml in spent bagasse hydrolysate compared to 480 nkat/ml in standard glucose-based medium). In addition, A. niger D15[egI] was also able to consume or convert other lignocellulose-derived compounds, such as acetic acid, furan aldehydes, and phenolic compounds, which are recognized as inhibitors of yeast during ethanolic fermentation. The results indicate that enzymes can be produced from the stillage stream as a high-value coproduct in second-generation bioethanol plants in a way that also facilitates recirculation of process water.

The potential in bioethanol production from waste fiber sludges in pulp mill-based biorefineries.

Industrial production of bioethanol from fibers that are unusable for pulp production in pulp mills offers an approach to product diversification and more efficient exploitation of the raw material. In an attempt to utilize fibers flowing to the biological waste treatment, selected fiber sludges from three different pulp mills were collected, chemically analyzed, enzymatically hydrolyzed, and fermented for bioethanol production. Another aim was to produce solid residues with higher heat values than those of the original fiber sludges to gain a better fuel for combustion. The glucan content ranged between 32 and 66% of the dry matter. The lignin content varied considerably (1-25%), as did the content of wood extractives (0.2-5.8%). Hydrolysates obtained using enzymatic hydrolysis were found to be readily fermentable using Saccharomyces cerevisiae. Hydrolysis resulted in improved heat values compared with corresponding untreated fiber sludges. Oligomeric xylan fragments in the solid residue obtained after enzymatic hydrolysis were identified using matrix-assisted laser desorption ionization-time of flight and their potential as a new product of a pulp mill-based biorefinery is discussed.

Dilute sulfuric acid pretreatment of agricultural and agro-industrial residues for ethanol production.

The potential of dilute-acid prehydrolysis as a pretreatment method for sugarcane bagasse, rice hulls, peanut shells, and cassava stalks was investigated. The prehydrolysis was performed at 122 degrees C during 20, 40, or 60 min using 2% H(2)SO(4) at a solid-to-liquid ratio of 1:10. Sugar formation increased with increasing reaction time. Xylose, glucose, arabinose, and galactose were detected in all of the prehydrolysates, whereas mannose was found only in the prehydrolysates of peanut shells and cassava stalks. The hemicelluloses of bagasse were hydrolyzed to a high-extent yielding concentrations of xylose and arabinose of 19.1 and 2.2 g/L, respectively, and a xylan conversion of more than 80%. High-glucose concentrations (26-33.5 g/L) were found in the prehydrolysates of rice hulls, probably because of hydrolysis of starch of grain remains in the hulls. Peanut shells and cassava stalks rendered low amounts of sugars on prehydrolysis, indicating that the conditions were not severe enough to hydrolyze the hemicelluloses in these materials quantitatively. All prehydrolysates were readily fermentable by Saccharomyces cerevisiae. The dilute-acid prehydrolysis resulted in a 2.7- to 3.7-fold increase of the enzymatic convertibility of bagasse, but was not efficient for improving the enzymatic hydrolysis of peanut shells, cassava stalks, or rice hulls.

Optimal conditions for alkaline detoxification of dilute-acid lignocellulose hydrolysates.

Alkaline detoxification strongly improves the fermentability of dilute-acid hydrolysates in the production of bioethanol from lignocellulose with Saccharomyces cerevisiae. New experiments were performed with NH4OH and NaOH to define optimal conditions for detoxification and make a comparison with Ca(OH)2 treatment feasible. As too harsh conditions lead to sugar degradation, the detoxification treatments were evaluated through the balanced ethanol yield, which takes both the ethanol production and the loss of fermentable sugars into account. The optimization treatments were performed as factorial experiments with 3-h duration and varying pH and temperature. Optimal conditions were found roughly in an area around pH 9.0/60 degrees C for NH4OH treatment and in a narrow area stretching from pH 9.0/80 degrees C to pH 12.0/30 degrees C for NaOH treatment. By optimizing treatment with NH4OH, NaOH, and Ca(OH)2, it was possible to find conditions that resulted in a fermentability that was equal or better than that of a reference fermentation of a synthetic sugar solution without inhibitors, regardless of the type of alkali used. The considerable difference in the amount of precipitate generated after treatment with different types of alkali appears critical for industrial implementation.

Ammonium hydroxide detoxification of spruce acid hydrolysates.

When dilute-acid hydrolysates from spruce are fermented to produce ethanol, detoxification is required to make the hydrolysates fermentable at reasonable rates. Treatment with alkali, usually by overliming, is one of the most efficient approaches. Several nutrients, such as ammonium and phosphate, are added to the hydrolysates prior to fermentation. We investigated the use of NH4OH for simultaneous detoxification and addition of nitrogen source. Treatment with NH4OH compared favorably with Ca(OH)2, Mg(OH)2, Ba(OH)2, and NaOH to improve fermentability using Saccharomyces cerevisiae. Analysis of monosaccharides, furan aldehydes, phenols, and aliphatic acids was performed after the different treatments. The NH4OH treatments, performed at pH 10.0, resulted in a substantial decrease in the concentrations of furfural and hydroxymethylfurfural. Under the conditions studied, NH4OH treatments gave better results than Ca(OH)2 treatments. The addition of an extra nitrogen source in the form of NH4Cl at pH 5.5 did not result in any improvement in fermentability that was comparable to NH4OH treatments at alkaline conditions. The addition of CaCl2 or NH4Cl at pH 5.5 after treatment with NH4OH or Ca(OH)2 resulted in poorer fermentability, and the negative effects were attributed to salt stress. The results strongly suggest that the highly positive effects of NH4OH treatments are owing to chemical conversions rather than stimulation of the yeast cells by ammonium ions during the fermentation.

Critical conditions for improved fermentability during overliming of acid hydrolysates from spruce.

Bioethanol can be produced from wood via acid hydrolysis, but detoxification is needed to achieve good fermentability. Overliming was investigated in a factorial designed experiment, in which pH and temperature were varied. Degradation of inhibitory furan aldehydes was more extensive compared to monosaccharides. Too harsh conditions led to massive degradation of sugars and formation of inhibiting acids and phenols. The ethanol productivity and yield after optimal overliming reached levels exceeding reference fermentations of pure glucose. A novel metric, the balanced ethanol yield, which takes both ethanol production and losses of fermentable sugars into account, was introduced and showed the optimal conditions within the investigated range. The findings allow process technical and economical considerations to govern the choice of conditions for overliming.