Claus Felby

Cell-wall structural changes in wheat straw pretreated for bioethanol production.

BackgroundPretreatment is an essential step in the enzymatic hydrolysis of biomass and subsequent production of bioethanol. Recent results indicate that only a mild pretreatment is necessary in an industrial, economically feasible system. The Integrated Biomass Utilisation System hydrothermal pretreatment process has previously been shown to be effective in preparing wheat straw for these processes without the application of additional chemicals. In the current work, the effect of the pretreatment on the straw cell-wall matrix and its components are characterised microscopically (atomic force microscopy and scanning electron microscopy) and spectroscopically (attenuated total reflectance Fourier transform infrared spectroscopy) in order to understand this increase in digestibility.ResultsThe hydrothermal pretreatment does not degrade the fibrillar structure of cellulose but causes profound lignin re-localisation. Results from the current work indicate that wax has been removed and hemicellulose has been partially removed. Similar changes were found in wheat straw pretreated by steam explosion.ConclusionResults indicate that hydrothermal pretreatment increases the digestibility by increasing the accessibility of the cellulose through a re-localisation of lignin and a partial removal of hemicellulose, rather than by disruption of the cell wall.

Production and effect of aldonic acids during enzymatic hydrolysis of lignocellulose at high dry matter content

BackgroundThe recent discovery of accessory proteins that boost cellulose hydrolysis has increased the economical and technical efficiency of processing cellulose to bioethanol. Oxidative enzymes (e.g. GH61) present in new commercial enzyme preparations have shown to increase cellulose conversion yields. When using pure cellulose substrates it has been determined that both oxidized and unoxidized cellodextrin products are formed. We report the effect of oxidative activity in a commercial enzyme mix (Cellic CTec2) upon overall hydrolysis, formation of oxidized products and impact on β-glucosidase activity. The experiments were done at high solids loadings using a lignocellulosic substrate simulating commercially relevant conditions.ResultsThe Cellic CTec2 contained oxidative enzymes which produce gluconic acid from lignocellulose. Both gluconic and cellobionic acid were produced during hydrolysis of pretreated wheat straw at 30% WIS. Up to 4% of released glucose was oxidized into gluconic acid using Cellic CTec2, whereas no oxidized products were detected when using an earlier cellulase preparation Celluclast/Novozym188. However, the cellulose conversion yield was 25% lower using Celluclast/Novozym188 compared to Cellic CTec2. Despite the advantage of the oxidative enzymes, it was shown that aldonic acids could be problematic to the hydrolytic enzymes. Hydrolysis experiments revealed that cellobionic acid was hydrolyzed by β-glucosidase at a rate almost 10-fold lower than for cellobiose, and the formed gluconic acid was an inhibitor of the β-glucosidase.Interestingly, the level of gluconic acid varied significantly with temperature. At 50°C (SHF conditions) 35% less gluconic acid was produced compared to at 33°C (SSF conditions). We also found that in the presence of lignin, no reducing agent was needed for the function of the oxidative enzymes.ConclusionsThe presence of oxidative enzymes in Cellic CTec2 led to the formation of cellobionic and gluconic acid during hydrolysis of pretreated wheat straw and filter paper. Gluconic acid was a stronger inhibitor of β-glucosidase than glucose. The formation of oxidized products decreased as the hydrolysis temperature was increased from 33° to 50°C. Despite end-product inhibition, the oxidative cleavage of the cellulose chains has a synergistic effect upon the overall hydrolysis of cellulose as the sugar yield increased compared to using an enzyme preparation without oxidative activity.

Biomass for energy in the European Union - a review of bioenergy resource assessments

This paper reviews recent literature on bioenergy potentials in conjunction with available biomass conversion technologies. The geographical scope is the European Union, which has set a course for long term development of its energy supply from the current dependence on fossil resources to a dominance of renewable resources. A cornerstone in European energy policies and strategies is biomass and bioenergy. The annual demand for biomass for energy is estimated to increase from the current level of 5.7 EJ to 10.0 EJ in 2020. Assessments of bioenergy potentials vary substantially due to methodological inconsistency and assumptions applied by individual authors. Forest biomass, agricultural residues and energy crops constitute the three major sources of biomass for energy, with the latter probably developing into the most important source over the 21st century. Land use and the changes thereof is a key issue in sustainable bioenergy production as land availability is an ultimately limiting factor.

Enhanced Auto Adhesion of Wood Fibers Using Phenol Oxidases

Enzymatic oxidation of lignin phenolic hydroxyl groups can enhance the level of auto adhesion between wood fibers. Previous investigations within the area have not taken the adhesive effect of proteins and carbohydrates in the enzyme solution into consideration, and part of the claimed enzyme generated adhesion may merely be a result of the adhesive effect of the proteins and carbohydrates in the enzyme solution. In this paper the enzyme laccase is used to oxidize beech (Fagus sylvatica) wood fibers. Laccase catalyzes a one-electron oxidation of phenolic hydroxyl groups while reducing oxygen, yielding phenoxy radicals and water. Wet and dry process fiberboards are made from enzyme treated and untreated beech wood fibers. The enzyme treated fiberboards have significantly higher modulus of rupture and modulus of elasticity as well as better dimensional stability. No effect of the protein and carbohydrate content in the enzyme solution upon the mechanical properties and dimensional stability can be found. The bonding effect is caused by the catalytic effect of the enzyme only. Electron spin resonance spectroscopy of beech wood fibers shows that a considerable amount of the laccase generated radicals is stabilized in the lignin polymer. and the bonding mechanism appears to be associated with reactions of these free radicals. The physico-mechanical parameters of the enzyme treated fiberboards have not been optimized in the present work. Hence, strength improvements may be obtained simply by optimizing the process parameters. The possibility of producing a medium density fiberboard without the use of synthetic adhesives appears to be promising. The use of oxidative enzymes in processing and manufacturing of wood-based materials may yield new and more environmentally safe products.

Pilot-scale production of fiberboards made by laccase oxidized wood fibers: board properties and evidence for cross-linking of lignin

Abstract Oxidoreductases can be applied for bonding of fiberboards, particle boards, paper boards and kraft-liner boards. In this work we report on pilot-scale production of laccase bonded fiberboards made from fibers of beech ( Fagus sylvatica ). Dioxane extractable lignin from fibers and boards are isolated and the molecular mass estimated by gel permeation chromatography. The strength properties of the enzyme bonded boards are comparable to boards bonded by an urea–formaldehyde adhesive, whereas the dimensional stability properties of the enzyme bonded boards are not at the same level. Wax treatment of the fibers, in order to improve dimensional stability of boards, is not compatible with the enzyme treatment. Cross-linking of the lignin can be observed in enzyme treated fibers and boards. Hot pressing of enzyme treated fibers results in a substantial cross-linking of lignin in boards. The enzymatic bonding effect may be caused by covalent bonds between fibers or an adhesive effect of polymerized loosely associated lignin. Laccase catalyzed bonding requires higher pressing temperatures and longer pressing times, and the concept may not be economically feasible as it is. However, it shows promise and possibilities in the use of oxidative enzymes for industrial bonding and modification of lignin.

Identification and quantification of radical reaction intermediates by electron spin resonance spectrometry of laccase-catalyzed oxidation of wood fibers from beech (Fagus sylvatica)

Abstract During laccase-catalyzed oxidation of beech wood fibers in an aqueous suspension, phenoxy radicals were detected in steady-state concentrations by electron-spin resonance (ESR) spectrometry of the suspension liquid, suggesting that colloidal lignin functions as a mediator between laccase and the fiber lignin matrix. Phenoxy radicals were observed directly, whereas ESR spin-trapping techniques gave no evidence for reduced oxygen species, such as the superoxide or hydroxyl radical. A reaction mechanism involving parallel direct oxidation of the lignin on fiber surfaces and a phenol/phenoxy cyclic mediator process in the suspension liquid could accordingly describe laccase-catalyzed oxidation of beech wood fibers. Cytochrome c assays for detection of superoxide in systems involving lignin oxidized by oxidoreductases should be used with caution, as cytochrome c may be reduced by species other than superoxide.

Characterization of lignin during oxidative and hydrothermal pre-treatment processes of wheat straw and corn stover.

The objective of the study was to characterize and map changes in lignin during hydrothermal and wet explosion pre-treatments of wheat straw and corn stover. Chemical composition, microscopic (atomic force microscopy and scanning electron microscopy) and spectroscopic (attenuated total reflectance Fourier transform infrared spectroscopy, ATR-FTIR) analyses were performed. Results showed that both pre-treatments improved the cellulose and lignin content with substantial removal of hemicellulose in the pre-treated biomasses. These values were slightly higher for hydrothermal compared to wet explosion pre-treatment. ATR-FTIR analyses also confirmed these results. Microscopic analysis showed that pre-treatments affected the biomass by partial difibration. Lignin deposition on the surface of the hydrothermally pre-treated fibre was very distinct while severe loss of fibril integrity was noticed with wet exploded fibre. The present study thus revealed that the lignin cannot be removed by the studied pre-treatments. However, both pre-treatments improved the accessibility of the biomass towards enzymatic hydrolysis.

Effects of thinning and soil properties on accumulation of carbon, nitrogen and phosphorus in the forest floor of Norway spruce stands

Abstract Area-based sampling was carried out to investigate the effect of thinning and soil properties on accumulation of forest floor carbon (C), nitrogen (N) and phosphorus (P) in Norway spruce (Picea abies (L.) Karst.) stands in Denmark. Four thinning intensities (unthinned, and about 83%, 67% and 50% of unthinned basal area) were investigated at three sites in Denmark: a calcareous, relatively nutrient rich soil with a sandy loam/loam texture and two soils with low to intermediate nutritional status and sandy loam and loamy sand textures, respectively. The effect of thinning on accumulated carbon and nitrogen was significant at two of the investigated sites. Accumulated phosphorus was significantly affected by thinning at one of these two sitesand at the third site. Accumulated carbon and phosphorus were negatively linearly correlated with thinning intensity. pH tended to be highest and C N and C P ratios tended to be lowest in the heaviest thinned plots. It is hypothesized that the differences in accumulation may be due to a more favourable microclimate and substrate for saprophytic organisms in the most heavily thinned plots. However, the differences between sites were greater than differences between thinning intensities. The accumulation of carbon, nitrogen and phosphorus in the forest floors was much higher at the two less fertile sites with loamy sand and sandy loam than at the relatively fertile site with sandy loam/loam. Significant differences in pH and in C N and C P ratios at the three sites indicate that the amounts of available nutrients influence the mineralization pattern. In addition, at the site with the greatest forest floor root density, competition for nutrients and moisture between mycorrhiza-infected roots and free-living saprophytic decomposers may be co-responsible for the large amounts of accumulated carbon, nitrogen and phosphorus.

Determining Yields in High Solids Enzymatic Hydrolysis of Biomass

As technologies for utilizing biomass for fuel and chemical production continue to improve, enzymatic hydrolysis can be run at still higher solids concentrations. For hydrolyses that initially contain little or no free water (10–40% total solids, w/w), the saccharification of insoluble polymers into soluble sugars involves changes of volume, density, and proportion of insoluble solids. This poses a new challenge when determining the degree of hydrolysis (conversion yield). Experiments have shown that calculating the yield from the resulting sugar concentration in the supernatant of the slurry and using the assumed initial volume leads to significant overestimations of the yield. By measuring the proportion of insoluble solids in the slurry as well as the sugar concentration and specific gravity of the aqueous phase, it is possible to precisely calculate the degree of conversion. The discrepancies between the different ways of calculating yields are demonstrated along with a nonlaborious method for approximating yields in high solids hydrolysis.

Pretreatment and enzymatic hydrolysis of wheat straw (Triticum aestivum L.) – The impact of lignin relocation and plant tissues on enzymatic accessibility

Wheat straw is a potential feedstock for bioethanol production. This paper investigates tissues from whole internode sections subjected to hydrothermal pretreatment at 185°C and subsequent enzymatic hydrolysis up to 144 h. Analyses revealed an increase in surface lignin as hydrolysis progressed, which could be coupled to the gradual decrease in hydrolysis rate over time. The data support the hypothesis of lignin extraction from the cell wall matrix during pretreatment and deposition as droplets upon cooling. These droplets are assumed to accumulate during enzymatic hydrolysis. Additionally, after 144 h of enzymatic hydrolysis the cortex had vanished, exposing the heavier lignified vascular tissue. Accumulation of lignin droplets and exposure of residual lignin could be part of the explanation for the decreasing hydrolysis rate. Flattening of macrofibrils after pretreatment together with more indentations on the surfaces was also observed, possibly caused by a proposed synergistic effect of cellobiohydrolases and endoglucanases.