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The main objectives of this project were to investigate the application of micro-organisms and their enzymes in removing lignin from straw and other non-woody materials which could be used in paper manufacture. This item is based on the consolidated report produced after three years work. In the last year the work was focused mainly on the optimization of experimental conditions for modification and removal of lignin from wheat straw and straw pulp using isolated enzymes or enzyme mediator systems. This strategy had been shown to work with wood pulps, and degradation of non phenolic lignin model systems has been reported. The use of the mediator appears to improve penetrability of the substrate and increase the oxidation capabilities of the enzymes are improved by the presence of the mediator.

The objective of the project is to optimize enzymatic treatments for straw delignification - through an interdisciplinary approach that will take into account gramineous peculiarities both in tissue anatomy and chemical structure of cell wall polymers - in order to provide a biotechnological solution (increasing paper productivity by improved dewatering, and decreasing lignin in effluents) to some of the main drawbacks of straw for pulp production, and to reduce simultaneously the costs of energy and chemicals used in pulp manufacture that will be combined with biological means. The above interdisciplinary approach will include the following tasks:

1. Testing and improving strains of selected species of ligninolytic fungi for preferential degradation of straw lignin

2. Identification and production of the fungal enzymes that could contribute to lignin removal, including those acting on phenolic and non-phenolic lignin moieties, and those splitting linkages with other straw polymers

3. Enzymatic treatment of straw by the individual enzymes and by enzyme mixtures, which will exhibit synergistic effects (e.g. ligninolytic enzymes assisted by enzymes providing co-substrates or preventing repolymerization of degradation products)

4. Evaluation of the efficiency of the above enzymatic treatments in terms of the modifications of lignin composition and its relationships with other cell-wall polymers

5. Monitoring histological changes produced by enzymes in order to verify the degradation of undesirable tissues (e.g. Parenchymatic cells included in the fine fraction of pulp) and the preservation of fibre structure

6. Testing pulping properties of the enzyme-treated straw, compared with fungus-treated straw and semichemical pulp, in combination with other pulping treatments and with special emphasis on the reductions in energy and chemicals and the decrease in the amount of lignin-derived products in effluents.
Among the most interesting results were those showing the modification of cell wall lignin by enzymatic treatment of wheat straw with Mn oxidising peroxidase (from Pleurotus eryngii in the presence of Mn2+ and H2O2), which resulted in preferential degradation of phenolic units (90% decrease at the highest enzyme dose used) on the basis of Py GC MS/pmy. A positive effect seen with Tween 80 on pulp treatment with Mn oxidising peroxidase (from Phlebia radiata), suggested a peroxidation type reaction. A decrease in lignin content (kappa number) of soda pretreated straw was shown to result from the enzymatic treatment with xylanase from Aspergillus niger. Various structural modifications were observed under microscopic examination of wheat straw tissues degraded by ligninolytic fungi, including fibre separation, which provided material with good properties for pulp production. The localisation of enzymes involved in lignin degradation (Mn oxidising peroxidase, laccase and aryl alcohol oxidase) during straw treatment with fungi was established using antibody gold complexes and transmission electron microscopy giving evidence for a strong defibrillation of secondary wall and enzyme penetration (shown by gold immunolocalisation). An improvement of straw pulp mechanical properties (including 20% increase of tensile index) was found after treatment with enzyme cocktail containing Mn oxidising peroxidase and aryl alcohol oxidase and natural fungal metabolites.


An extension has been requested to enable further analyses to be completed during 1997 to finish the comparison of lignin modification by the different enzyme and enzyme mediator systems as well as providing further information on ultrastructural and immunolocalisation studies during straw pulp treatment with fungal laccase, optimization of cooking conditions, and evaluation of subsequent papermaking properties of wheat straw treated with fungi under solid-state fermentation conditions as well as final evaluation of refining and mechanical properties of straw, pretreated straw and pulp treated with Mn oxidising peroxidase and laccase containing enzymatic "cocktails" supplemented with mediators. In the meantime a number of interim conclusions have been reached.


- The establishment of analytical methodologies to study lignin modification by fungi and enzymes (Py GC MS), the correlation analysis between laboratory and papermaking parameters, the selection of fungal strains, and the ultrastructural characterisation of wheat straw tissues were among the most important results obtained during the first year of the project.

- The identification and characterisation of ligninolytic enzymes produced by fungi during lignin degradation under simultaneous saccharification/fermentation as well as optimization of enzyme production and purification together with the chemical and ultrastructural characterisation of wheat straw treated with fungi and the production of polyclonal antibodies used in immunolocalisation were significant results from the second year of the project.

- In the third year evidence for enzymatic modification of lignin in wheat straw cell wall (using enzymes or enzyme mediator systems) was obtained using these techniques and new analytical methods were developed for absolute quantitation of pyrolysis products and for laboratory evaluation of small samples, and used to show improved mechanical properties in straw pulp treated with enzyme "cocktails" and mediators.

- Complete analyses of samples obtained during 1996 will enable a general comparison of lignin modification and cell wall alteration by the different enzymes and enzyme mediator systems, providing a final evaluation of fungally and enzymatically treated wheat straw and straw pulp samples related to cooking, refining and papermaking parameters.
This project has investigated in detail the use of various biological treatments, based on either growing fungi or isolated enzymes, in the preparation of paper pulp from straw. Straw, in common with other fibrous lignocellulosic materials, is made up of the cellulose components which form the bulk of the final paper, as well as hemicellulose (carbohydrate) and lignin (phenolic) materials which are removed and/or bleached during processing. Conventionally pulping uses alkali salts and may use strong chemical bleaches based on chlorine, which produces polluting effluents and environmental concern. The enzyme approach should provide less degraded by-product streams and reduced environmental impact. However, although many fungi produce enzymes which breakdown hemicellulose and some produce lignin degrading systems, considerable development and optimisation is required to make such approaches commercially viable. As indicated here, this groups has made significant advances in understanding of the mechanisms and possibilities.


These included laccase (laccase ABTS, 2 2' azinobis (3 ethylbenzothiazoline 6 sulphonate) and laccase HBT, hydroxybenzotriazol) and peroxidase (peroxidase Mn2+, peroxidase Tween 80 and peroxidase veratryl alcohol) systems. The effect of different parameters (including enzyme and mediator type and concentration, reaction pH, source of electron acceptor, substrate pre-treatment, reaction time, and other process parameters were investigated. The performance of natural mediators synthesized by the fungi were also investigated also, by treating wheat straw or straw pulp with culture liquids (containing fungal metabolites). The need to analysis the samples used to optimise treatment conditions led to the development of a set of original laboratory tests for evaluation, in small samples, of parameters of interest in pulp and paper manufacture. These included pyrolysis linked to gas chromatography and mass spectroscopy (Py-GC-MS), especially after sample permethylation (pmy), which enabled discrimination of phenolic and non phenolic lignin units. Furthermore, an original method for absolute quantitation of pyrolysis products, based on the use of tri t butylbenzene as internal standard, was optimised and applied to the analysis of enzyme treated samples.

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