AIR Project on Biological Delignification of
Non -Woody Materials
by Ángel T. Martínez
Department of Molecular
Microbiology
Centro de Investigaciones Biológicas (CIB)
Consejo Superior de Investigaciones Científicas (CSIC)
Velázquez 144, E-28006 Madrid, Spain
Tel: +34 91 561 18 00 or +34 91 565 45 62 (EXT. 4407)
Fax: +34 91 562 75 18
E-mail: atmartinez@cib.csic.es
Homepage: http://www.cib.csic.es/~lignina/lignina_en.html
Project Summary: The project was initiated with a fungal screening to identify strains producing a selective removal of lignin from wheat straw. This was based on both lignin mineralization and solubilization activity (estimated with lignin-radiolabeled straw), and microscopic examination and chemical analyses of substrates treated with fungi under solid-state fermentation (SSF) conditions. The four fungi selected - i.e., strains of Phlebia radiata, Pleurotus eryngii, Pleurotus pulmonarius and Poria (synonym: Ceriporiopsis) subvermispora - were considered as model organisms for: i) biological delignification of wheat straw (under SSF conditions); and ii) identification of the enzymes involved in wheat lignin degradation. They exhibit high ligninolytic activity, remove wheat lignin preferentially with respect to cellulose, and microscopy studies revealed a selective degradation pattern resulting in individualization of sclerenchymatic and xylematic fibers (used in paper pulp manufacture). Since different pieces of evidence suggest that xylan degradation facilitate lignin removal from lignocellulosic substrates, a strong xylanolytic strain of Aspergillus niger was included also in the studies (for both straw SSF and xylanase production).
Simultaneously to the fungal selection, wheat straw (as a model for non-woody materials with a potential interest in paper pulp manufacture) was characterized using ultrastructural and chemical techniques. In the course of these studies, several new analytical methodologies were developed to obtain additional information when applied to the biologically-treated samples. In this way, antibodies directed against different types of synthetic lignin - constituted by condensed and non-condensed H (p-hydroxyphenylpropanoid), G (guaiacylpropanoid) and S (syringylpropanoid) units - were obtained to be used in immunolocalization studies. On the other hand, two modifications of standard analytical pyrolysis methods were developed: i) absolute quantitation using the internal standard 1,3,5-tri-tert-butylbenzene and new response factors; ii) separate analysis of phenolic and non-phenolic phenylpropanoid compounds (lignin and p-hydroxycinnamic acids) in wheat straw by sample permethylation (followed in both cases by single ion quantitation in gas chromatography-mass spectrometry). In this way, it was possible to properly monitor the alteration degree of lignin after fungal transformation of the substrate.
In spite of standard refining and papermaking tests of biologically-delignified substrates were programmed for the last phase of the project, the development of laboratory tests predicting the above properties after analysis of small samples (as those obtained during optimization of enzymatic treatments) was also included in the workplan. Some infrared bands appeared among the most valuable parameters to forecast papermaking properties, and useful pyrolysis marker-compounds were also identified. Multiple correlation studies using a large set of straw samples (including those from alkaline cooking under different conditions and preliminary treatments with fungi and enzymes) showed that results from standard papermaking tests could be satisfactorily defined as polynomial functions of laboratory parameters (such as amount of different straw fractions, intensity of infrared bands or results from thermogravimetric analysis). Moreover, this information was complemented with a set of original methods for laboratory preparation and analysis (including color, opacity, infrared spectroscopy, mechanical resistance and porosity) of microhandsheets, which was later developed and checked with straw and pulp samples treated with the above fungi and the different enzymes cited below.
As described in the workplan, the strategy to define enzymatic treatments for straw delignification was based on the identification of the enzymes involved in wheat lignin degradation by the four fungi selected for their high ligninolytic capabilities. This study included: i) direct estimation of enzymatic activities in extracts from delignified straw; ii) chromatographic separation of the extract proteins followed by activity tests; and iii) enzyme immunolocalization (by transmission-electron and fluorescence microscopy) using antibodies against lignocellulose-degrading enzymes purified from liquid cultures. The immunolocalization techniques showed the production of manganese peroxidase (MnP), laccase and xylanase (as well the H2O2-producing enzyme aryl-alcohol oxidase) during fungal treatment of wheat straw. These studies also revealed differences in enzyme penetration in the plant cell wall, as well as preferential removal of different types of lignins (evidenced by the specific anti-lignin antibodies). In the studies with Phlebia radiata (and Phanerochaete chrysosporium, which was included as a reference ligninolytic fungus) production of lignin peroxidase (LiP) on a lignocellulosic substrate was demonstrated for the first time (activity detected only after Sepharose®-Q chromatography of the extract proteins). However, both quantitative estimation of enzymes and immunolocalization results suggested that a different peroxidase - Mn-oxidizing peroxidases (MnP) - was more directly involved in lignin degradation. Similar results concerning MnP production were obtained with Pleurotus species. It is necessary to mention that the latter fungi, as well as Poria subvermispora, lack LiP (proteins and genes). Moreover, the strong stimulation of lignin mineralization observed after addition of Mn2+ to SSF cultures (including in vivo 14C-labeled lignin), also supported MnP involvement in lignin degradation (via Mn3+ chelates) by Pleurotus species. When Pleurotus eryngii and Pleurotus pulmonarius MnP were purified and characterized, they were found different from typical MnP (such as those produced by Phlebia radiata or Phanerochaete chrysosporium) since exhibited Mn-independent activity on aromatic substrates. Because laccase, as well as xylanase activities were found in straw extracts and localized in degraded straw, both enzymes were selected together with MnP for the enzymatic delignification of wheat straw.
In order to treat straw substrates, the above enzymes were produced and purified from cultures of Phlebia radiata, Pleurotus eryngii and Aspergillus niger. Some culture parameters for xylanase production by the latter fungus were optimized, and laccase and Phlebia radiata peroxidase were isolated by already available protocols. Moreover, both production conditions (using peptone-containing liquid media) and purification strategy were optimized for the new MnP of Pleurotus eryngii. Subsequently, different physicochemical, spectroscopic and catalytic properties of this enzyme were determined. It has been proposed that ligninolytic enzymes are not able to attack directly lignin in the intact plant cell-wall due to steric hindrances and that simple redox mediators are involved at least in the initial stages of delignification. Because preliminary experiments including laccase and MnP mediators yielded promising results, the enzyme/mediator strategy was adopted in the present project. To facilitate the action of the enzyme/mediator couples, different substrate pretreatments were investigated (including the use of chopped and milled straw, straw after mild soda extraction, crude and refined soda pulp from wheat straw, and isolated wheat-straw lignin). These substrates were treated with different enzyme/mediator systems, including: MnP/Mn2+/H2O2, MnP/Mn2+/Tween-80, laccase/HBT and laccase/ABTS and compared with controls without enzyme or without mediators or cofactors.
Chemical and ultrastructural modification as a result of fungal treatment of lignocellulosic materials has been demonstrated with different ligninolytic fungi (as described for those used in the present project). However, in the course of the above enzyme/mediator treatments of straw substrates we obtained some of the first evidences on the capabilities of fungal enzymes for in vitro modification of lignin in the lignocellulosic matrix. Although lignin is responsible for tissue structure and resistance, it does not represent a major fraction in wheat straw, and the alteration of other straw constituents during biological delignification should be as low as possible to maintain the substrate characteristics for paper pulp production. Therefore, the modifications caused by ligninolytic enzymes are not easily evidenced by spectroscopic or other analytical techniques applied to the whole lignocellulosic substrate, although minor changes in FTIR and NMR peaks suggested lowered lignin content and S/G ratio. However, wheat lignin alteration by enzymes (including changes in lignin content composition) was especially evident when pyrolysis-based analyses were performed. Moreover, a separate analysis of lignin phenolic and nonphenolic moieties was possible leading to establish that the enzymatic attack to grass lignin is initiated at the phenolic units (20-30% of phenylpropanoid compounds in wheat straw). This is a typical characteristic of the laccase/mediator systems. At similar enzyme dose, the MnP/Mn2+-based systems produced more moderate decrease of phenolic content but some of them caused the highest alteration of the substrates in terms of H:G:S ratio (and up to 90% decrease of phenolic units was attained at higher enzyme/mediator doses). On the contrary, the formation of new hydroxylated phenylpropanoid compounds (identified by analytical pyrolysis) was one of the most noticeable among the effects of MnP/Mn2+ /Tween-80 system (acting via lipid-derived free radicals). It is necessary to mention also that a decrease of lignin content (kappa number) was obtained after xylanase treatment of soda-pretreated straw. The capabilities of lignocellulose-degrading enzymes to modify lignin in straw substrates were confirmed by electron microscopy and immunolocalization technologies showing defibrillation of secondary wall and enzyme penetration during straw pulp treatment with MnP and laccase-based enzyme/mediator systems (including that based on Tween-80 peroxidation).
Finally, some refining, cooking and papermaking features of straw substrates treated with enzymes or fungal cultures were comparatively evaluated. The refining tests revealed a decrease in the energy necessary to produce biomechanical pulps after straw treatment with fungi (attaining 70% energy saving). The mechanical properties of the handsheets obtained were similar to those from mechanical pulps obtained with higher energy consumption (but worst than those from semichemical pulp). In order to improve mechanical properties maintaining biopulping advantages, biological pretreatment was combined with soda cooking using shorter contact times. In this way, the quality of the paper obtained was increased, being refining energy and cooking costs lower than those of semichemical pulping. For preliminary evaluation of different enzyme/mediator systems and substrate pretreatments, the above-described set of laboratory parameters was applied to predict papermaking properties. The interpretation of the whole set of data suggested that MnP can attack wheat-straw lignin via Mn3+-chelates as well as via lipid peroxidation, and that laccase treatment in presence of mediators and followed by a chelator extraction improved some substrate properties. The above results were confirmed in the course of larger scale treatments of wheat straw and straw pulp with crude laccase and MnP (supplemented with mediators). The results from sample evaluation suggested that some process improvement can be obtained using laccase/ABTS as a pretreatment before soda cooking of wheat straw (this resulted in lower energy consumption for the same refining degree, and reduced total suspended-solids and chemical oxygen demand in black liquors). However, the most promising result was attained with the MnP/Mn2+ system as a post-treatment to improve characteristics of crude or refined pulp. In the latter case, better mechanical properties were obtained, including increases of tensile indices. Moreover, the improvements obtained after enzymatic treatment of crude pulp included the possibility of more than 20% energy saving to obtain 60oSR refined pulp, with similar papermaking properties.
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Author: Ángel
T. Martínez |
Docmaster: Sonia Centeno |