Stella Rovio

Inhibitory effect of lignin during cellulose bioconversion: The effect of lignin chemistry on non-productive enzyme adsorption

The effect of lignin as an inhibitory biopolymer for the enzymatic hydrolysis of lignocellulosic biomass was studied; specially addressing the role of lignin in non-productive enzyme adsorption. Botanical origin and biomass pre-treatment give rise to differences in lignin structure and the effect of these differences on enzyme binding and inhibition were elucidated. Lignin was isolated from steam explosion (SE) pre-treated and non-treated spruce and wheat straw and used for the preparation of ultrathin films for enzyme binding studies. Binding of Trichoderma reesei Cel7A (CBHI) and the corresponding Cel7A-core, lacking the linker and the cellulose-binding domain, to the lignin films was monitored using a quartz crystal microbalance (QCM). SE pre-treatment altered the lignin structure, leading to increased enzyme adsorption. Thus, the positive effect of SE pre-treatment, opening the cell wall matrix to make polysaccharides more accessible, may be compromised by the structural changes of lignin that increase non-productive enzyme adsorption.

Determination of monosaccharide composition in plant fiber materials by capillary zone electrophoresis.

The neutral sugar composition of acid hydrolyzed extracts of cellulose fiber samples, i.e. oat spelt, wheat straw, thermomechanica pulp (TMP) made of spruce, aspen stemwood, and bleached birch kraft pulp, was determined by a new capillary zone electrophoresis (CZE) method employing an alkaline background electrolyte. The method relies on in-capillary reaction and direct UV detection at wavelength 270 nm. Neutral carbohydrates D-(+)-galactose, D-(+)-glucose, L-rhamnose, D-(+)-mannose, D-(-)-arabinose, and D-(+)-xylose were simultaneously separated. The calibration plots were linear over a range from 10 to 150 mg/L for D-(+)-galactose, L-rhamnose, D-(+)-mannose, and D-(-)-arabinose and from 50 to 400mg/L for D-(+)-glucose and D-(+)-xylose. Relative standard deviations (RSDs) of peak areas during a 5-day analysis period varied from 3.3% for galactose to 11.8% for rhamnose. RSDs of migration times varied between 0.3 and 0.7%. The detection limit (at S/N 3) was 5mg/L for each monosaccharide. The results obtained by CZE agreed well with results obtained by high-performance anion-exchange chromatography. Glucose and xylose were the two predominant monosaccharides in the plants, except in the spruce TMP sample where glucose and mannose dominated.

Solvent extraction as a means of preparing homogeneous lignin fractions

Abstract Sequential extraction method was applied to lignins from hardwood and softwood isolated by kraft and VTT organosolv processes. Solvent extraction was found to fractionate lignin according to the molecular weight: small molecular weight lignin is dissolved in the organic solvents and the lignin with higher molecular weight is enriched into the residue. Isolated acetone fractions of lignin are more homogeneous with narrow molecular weight distributions. Based on the 31P NMR results, both total hydroxyl content and the content of phenolic hydroxyl units are higher in the acetone fraction than in the residue. Pyrolysis-GC/MS of all lignins showed that p-hydroxy phenols are enriched to the residues. Preferential dissolution of syringyl type lignin in acetone was observed for hardwood kraft lignin, whereas the opposite behavior was seen for the hardwood organosolv lignin. Glass transition temperatures of all acetone soluble fractions were notably low compared to starting and residue lignins, which gives possibilities for future applications as a material with specific properties.

Partial enzymatic hydrolysis of microcrystalline cellulose in ionic liquids by Trichoderma reesei endoglucanases

The enzymatic hydrolysis of cellulose in ionic liquid (IL) containing systems has recently received a lot of interest as a pretreatment in biomass conversion to liquid biofuels and chemicals. In this paper we present a study in which the activity and action of two Trichoderma reesei endoglucanases, Cel7B and Cel5A, were evaluated in aqueous solutions containing 0–90% (v/v) of the ionic liquids 1,3-dimethylimidazolium dimethylphosphate or 1-ethyl-3-methylimidazolium acetate, using microcrystalline cellulose (Avicell) as a model substrate. The degree of hydrolysis was analysed by capillary electrophoresis of the hydrolysates and gel permeation chromatography of the remaining cellulose residues. Both of the employed ionic liquids severely inactivated the T. reesei endoglucanases. Only traces of soluble oligosaccharides were present in hydrolysis mixtures containing 40% (v/v) or more of ionic liquids. The employed ILs were found to have a basic impact on the hydrolysis environment, but it could be concluded that the basicity of the ILs was not the only reason for the cellulase inactivation. The effect of an IL on the cellulose binding module in Cel5A was evaluated by comparing the hydrolysis yields of the intact Cel5A and the Cel5A core lacking the cellulose binding module. In this study the cellulose binding module was found to be the most ionic liquid sensitive part of the enzymes used. Comparative data from the partial hydrolysis of an ionic liquid regenerated cellulose is also reported.

Determination of the carboxylic acids in acidic and basic process samples by capillary zone electrophoresis

Capillary zone electrophoresis (CZE) with indirect UV detection was used in developing a method for the simultaneous determination of inorganic anions, aliphatic and heterocyclic organic acids in various processed samples. The analytes were determined simultaneously in 10 min using an electrolyte containing 20mM 2,3-pyrazine dicarboxylic acid, 65mM tricine, 2mM BaCl(2), 0.5mM cetyltrimethylammonium bromide, and 2M urea at pH 8.06. Linear plots for the analytes were obtained in the concentration range of 2-150 mg L(-1). Relative standard deviations (RSDs) of peak areas during a 3-day analysis period varied from 5.5% for glycolate to 9.5% for oxalate. RSDs of migration times varied between 0.4% and 1.1%. The detection limit (at S/N 3) was 1 mg L(-1) for all the analytes studied. The proposed method was successfully demonstrated for the determination of carboxylic acids in eight oxygen treated samples of commercial softwood and hardwood kraft lignin and two red wine samples of Pinot Noir grapes. In the kraft lignin samples the concentrations of carboxylic acids correspond to the oxidation time. The acid concentrations of wine varied considerable.

Impact of Steam Explosion on the Wheat Straw Lignin Structure Studied by Solution-State Nuclear Magnetic Resonance and Density Functional Methods

Chemical changes of lignin induced by the steam explosion (SE) process were elucidated. Wheat straw was studied as the raw material, and lignins were isolated by the enzymatic mild acidolysis lignin (EMAL) procedure before and after the SE treatment for analyses mainly by two-dimensional (2D) [heteronuclear single-quantum coherence (HSQC) and heteronuclear multiple-bond correlation (HMBC)] and (31)P nuclear magnetic resonance (NMR). The β-O-4 structures were found to be homolytically cleaved, followed by recoupling to β-5 linkages. The homolytic cleavage/recoupling reactions were also studied by computational methods, which verified their thermodynamic feasibility. The presence of the tricin bound to wheat straw lignin was confirmed, and it was shown to participate in lignin reactions during the SE treatment. The preferred homolytic β-O-4 cleavage reaction was calculated to follow bond dissociation energies: G-O-G (guaiacyl) (69.7 kcal/mol) > G-O-S (syringyl) (68.4 kcal/mol) > G-O-T (tricin) (67.0 kcal/mol).

On‐line measurement of pulp water anions by capillary electrophoresis with fast sequential sampling and dynamic solvent feeding

This paper demonstrates the use of the first on-line capillary electrophoresis instrument with continuously flowing electrolyte solutions and sequentially applied sample introduction. The system was used in on-line determination of chloride, sulfate, and carbonate concentrations of process waters at a pulp mill. The module constructed for on-line feeding of the solvents is described. Separations were performed in a pyromellitic acid-hexamethonium hydroxide-triethanol amine mixture at pH7.7 with indirect-UV detection at 254 nm. Two standard mixtures were used to show the repeatability of the analysis and to estimate the accuracy of the on-line system. The analytes could be separated within 240 s. Sampling from a pulp machine could be accomplished on-line at 6-minute intervals. On-line tests gave a limit of detection and a limit of determination in the low mg/L range.

Sustainability of cellulose dissolution and regeneration in 1,5-diazabicyclo[4.3.0]non-5-enium acetate: a batch simulation of the IONCELL-F process

The recyclability of 1,5-diazabicyclo[4.3.0]non-5-enium acetate ([DBNH][OAc]), as a direct dissolution solvent for cellulose, was evaluated during laboratory scale recycling trials. The main objective was to simulate the conditions of a spinning bath from a Lyocell-type air-gap spinning process, called the IONCELL-F process. The saline solution was then concentrated, recycled and reused as many times as possible before cellulose dissolution was no longer possible. The chemical compositions of the ionic liquid and pulp were recorded throughout the experiments. The results of the experiments showed that [DBNH][OAc] can be recycled from aqueous media with an average recovery rate of 95.6 wt% using basic laboratory equipment, without any further process intensification or optimisation. The recycling of the ionic liquid did not change the chemical composition or degree of polymerisation of the recovered pulp but the colour of the regenerated pulps gradually darkened as the recycling times increased. The ionic liquid was found to hydrolyse 6.0–13.6 mol% per cycle, under these conditions. The build-up of the hydrolysis product, 3-(aminopropyl)-2-pyrrolidonium acetate, killed the dissolution feature at between 30.6–45.6 wt% hydrolysis product. The enzymatic digestibility of the regenerated pulp samples was studied with both a monocomponent endoglucanase and a cellulase mixture. The amount of residual [DBNH][OAc] in the regenerated pulps was determined, by both NMR and capillary electrophoresis. Although hydrolysis of the ionic liquid occurs, this study clearly shows potential for industrial application, with appropriate process equipment and recycling conditions.

Analysis of mono- and oligosaccharides in ionic liquid containing matrices

Ionic liquids (ILs), that is, salts with melting points <100°C, have recently attracted a lot of attention in biomass processing due to their ability to dissolve lignocellulosics. In this work, we studied how two imidazolium-based, hydrophilic, cellulose dissolving ionic liquids 1,3-dimethylimidazolium dimethylphosphate [DMIM]DMP and 1-ethyl-3-methylimidazolium acetate [EMIM]AcO affect the usually employed analytical methods for mono- and oligosaccharides, typical products from hydrolytic treatments of biomass. HPLC methods were severely hampered by the presence of ILs with loss of separation power and severe baseline problems, making their use for saccharide quantification extremely challenging. Problems in DNS photometric assay and chromatography were also encountered at high ionic liquid concentrations and many capillary electrophoresis (CE) methods did not allow an efficient analysis of saccharides in these matrices. In this paper we describe an optimized CE method with pre-column derivatization for the qualitative and quantitative analysis of mono- and oligosaccharides in sample matrices containing moderate (20-40% (v/v)) concentrations of ILs. The IL content and type in the sample matrix was found to affect both peak shape and quantification parameters. Generally, the presence of high IL concentrations (≥20% (v/v)) had a dampening effect on the detection of the analytes. IL in lower concentrations of <20% (v/v) was, however, found to improve peak shape and/or separation in some cases. The optimized CE method has good sensitivity in moderate concentrations of the ionic liquids used, with limits of detection of 5mg/L for cellooligomers up to the size of cellotetraose and 5-20mg/L for cellopentaose and cellohexaose, depending on the matrix. The method was used for analysing the action of a commercial β-glucosidase in ILs and for analysing saccharides in the IL containing hydrolysates from the hydrolysis of microcrystalline cellulose with Trichoderma reesei endoglucanase Cel5A. According to the results, [DMIM]DMP and [EMIM]AcO] showed clear differences in enzyme inactivation.

Lignin oxidation mechanisms under oxygen delignification conditions. Part 2: Advanced methods for the detailed characterization of lignin oxidation mechanisms

Abstract Advanced analysis methods have been developed to follow the reactions of lignin during alkaline oxygen delignification conditions more comprehensively than before. This aim was attained by monitoring both the lignin macromolecule and the dissolved reaction products. Softwood (SW) and hardwood (HW) kraft spent liquor lignins were studied as substrates under various reaction conditions. The decrease in the contents of different types of free phenolic hydroxyl groups and the concurrent formation of carboxylic acids was followed by 31P NMR of the phosphitylated products. In addition, the formation of acidic degradation products with low molecular weight was determined by capillary zone electrophoresis (CE). This way, it was possible to distinguish the carboxylic acids bound to the lignin macromolecule from the cleaved reaction products, even if they partly co-precipitated during sample preparation. Peak deconvolution was applied to get information on syringyl type phenolic structures and on C(5) condensed guaiacyl structures in hardwood lignin. Pyrolysis-GC/MS was applied to provide additional information about the distribution of guaiacyl/syringyl/p-hydroxyphenyl (G/S/H) type lignin subunits, as well as changes in the phenylpropane side chain.