AGRE-0047 Biopulping and Biobleaching

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ECLAIR Cluster II - Lignocellulose : Paper/Pulp : Pulping : Wood (Lignocellulose)

Biobleaching of kraft pulp with A niger: Time course of xylanase production (triangle), and its effect on kappa number (square) (measure of lignin content) on Eucalyptus pulp obtained from Portucel (Eixo, Portugal).

SUMMARY

This project investigated the feasibility of producing paper pulp from various raw materials, including eucalyptus chips and straw, using biological methods as substitutes for the alkali and chemical (chlorine) bleaches used at present. To produce pulp (release the cellulose fibres) from the plant cell wall material, the bulk of the hemicellulose and lignin components must be removed and residual lignin discoloured. A number of different types of filamentous fungi which grow on dead plant material produce enzymes which are capable of breaking down hemicellulose and lignin without adversely affecting the cellulose fibres. A detailed investigation has matched some of these organisms and enzymes they produce with the various stages of pulping and bleaching. At the same time various reactors have been developed for these stages. Areas of commercial potential have been identified.

INTRODUCTION

Paper pulp may be produced from a wide range of fibrous or woody (lignocellulosic) plant materials. At present most pulp is produced using chemicals such as alkali, to free the required cellulose fibres from the other components (hemicellulose and lignin), and bleach (such as chlorine), to reduce the colour due to residual lignin. A number of microorganisms, including filamentous fungi (Ascomycetes and Basidiomycetes) that live on decaying plant material, produce enzymes which break down hemicellulose (hemicellulase) and, in some cases, lignin. In theory, it should be possible to use enzymes from such organisms to develop appropriate biocatalysts for use in the two major phases (pulping and bleaching) of paper manufacture; in other words to substitute enzymes for chemicals in what can then be described as biopulping and biobleaching processes. Investigations into lignin removal from within the wood matrix have formed an essential part of this project in order to produce an environmentally compatible biotechnology, with reduced energy costs and reduced dependency on potentially noxious chemicals. The actual problems in developing such processes are complex since both the raw materials which might be used (wood, fibre crops or agricultural residues - straw) and the mixture of enzymes produced by a particular fungus are highly variable. Hence, there is a need to match raw materials and enzyme systems.

OBJECTIVES

The main objectives included identification and isolation of suitable microorganisms; production of mutants of these with enhanced ability for lignin biodegradation; and then characterisation of their enzyme complex involved in lignin degradation. Further work was carried out to compare enzymatic and non-enzymatic methods of generating hydrogen peroxide as the bleaching agent. Further objectives were the design of a reactor for application of the biological systems for biopulping of wood chips and straw, as well as investigation of appropriate enzymatic solutions for the biobleaching of Eucalyptus Kraft pulp and determination of the characteristics of the bleached pulp, followed by pilot scale studies for technical and economical evaluation of an integrated biobleaching process.

ACTIVITIES

Microorganisms, including those from public and private collections, were screened in order to identify the most appropriate microbial species for lignin degradation and lignin removal from solution (decolorisation) coupled with a limited ability to degrade cellulose (to avoid degradation of the cellulose fibres). Various systems were adopted which were matched to the raw material under consideration: Pleurotus species for biopulping of cereal straw; and Phanerochaete chrysosporium, P. magnolia, Pycnoporus cinnabarinus, Aspergillus and Penicillium for the pulping of Eucalyptus wood. Enzymatic systems were searched for the production of hydrogen peroxide, lignin molecule attack (LiP, MnP, and laccase), hemicellulases and other complementary systems. Production of enzymes was optimised at laboratory scale. Lignin peroxidase and manganese peroxidases from Phanerochaete chrysosporium were produced on air-lift type reactors, while good values for xylanase from Aspergillus niger were obtained from stirred tank reactors. It is known that hydrogen peroxide production is needed for the peroxidase mechanism for lignin degradation. Therefore, a non-enzymatic system would be beneficial in cases where hydrogen peroxide production was limiting. Hence, an electrochemical device was designed and used, coupled with the enzyme reactors, for in situ continuous hydrogen peroxide generation. Although bleaching of Eucalyptus pulp was aided by the use of a simple enzymatic hemicellulolytic preparation from A. niger, general experiments showed that the use of single enzymatic systems was not sufficient for pulping, which required the use of whole organisms. Biopulping of straw in sterile conditions required the design and operation of a solid state fermentation reactor with good mixing and oxygen transfer ability, since oxygen is a very important nutrient for growth of fungi and lignin degradation. Inoculation and colonisation of straw were also important aspects to take into consideration for a quick and full culture development. Eucalyptus was found to be a more difficult material to biopulp using growing fungi, since in chip form it was resistant to fungal attack. Xylanases were found to be of benefit for bleaching of pulps where laboratory application is straight forward, and industrial scale-up does not seem to require significant investment costs or pose technological limitations. Pulps produced in this way were subject to chemical, physical and, sometimes, ultrastructural analysis in order to evaluate the biological action and the commercial properties of the products. Finally one lignin peroxidase gene was cloned. Its expression in Aspergillus niger is being investigated.

CONCLUSIONS AND BENEFITS

Biopulping of cereal straw with Pleurotus sp. could reduce significantly the energy required for refining of mechanical pulp. Similar observations were made in respect of refining of Eucalyptus pulp pre-treated with different fungi. New reactors were developed as prototypes for solid state microbial pulping and non-enzymatic continuous hydrogen peroxide delivery to air-lift reactors for peroxidase production.

EXPLOITATION

Bioprocessing for use in the paper and pulp industries must be developed as part of a fully integrated processes which takes account of the economics of its introduction and the environmental impact of the whole process. Adoption of the actual processes has to be decided on a case by case analysis into aspects related to the use of peroxidases and auxiliary systems, scaling up, molecular biology and reactor technology in discussion with the industry.

PARTICIPANTS

INETI, Departamento de Biotecnologia (Portugal); University of Westminster, School of Biological & Health Sciences (UK); TNO Nutrition & Food Research (Netherlands); CSIS, Centro de Investigaciones Biologicas (Spain); SAICA, Sociedad Anonima Industrias de la Celulosa Aragonesa (Spain) and UNAHE, Universidad de Alcalá de Henares, Dept. of Microbiology y Parasitologia (Spain).