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Extremozymes for wood based building blocks: From pulp mill to board and insulation products

Periodic Reporting for period 1 - WoodZymes (Extremozymes for wood based building blocks: From pulp mill to board and insulation products)

Reporting period: 2018-06-01 to 2019-11-30

One of the main challenges faced by the wood industry is to attain the full exploitation of all wood components. During kraft pulping, the predominant technology to produce paper pulp from wood, the majority of lignin and many hemicelluloses are partially extracted under strong alkaline conditions, captured in the spent black liquors and burnt in the recovery boiler to generate heat for the mill. WoodZymes aims to valorise these underutilized lignin and hemicellulose fractions by supplying the wood and paper pulp industries with new extremophilic enzymes able to work at the high temperature and pH of the kraft process. The goal is to obtain bio-based building blocks through extremozyme-assisted depolymerization of kraft lignin and delignification and bleaching of kraft pulps. The obtained lignin-derived phenols will be used as precursors of resins in the manufacture of medium-density fibreboards (MDF) or as components of insulation polyurethane (PU) foams to replace oil-derived chemicals, and the hemicellulose-derived sugars as additives in papermaking. A techno- economic and environmental impact evaluation of the new materials and processes will be performed.

The WoodZymes consortium covers the whole value chain, from feed-stock suppliers to end-users, being able to demonstrate the extremozyme-based technology at the pilot scale. It comprises: i) world-leading companies of the pulp & paper (The Navigator Company and Fibre Excellence), fibreboard (FINSA) and PU foam (Soprema) sectors; ii) a biotech SME commercializing industrial enzymes (MetGen); and iii) reputed research institutes (CSIC: CIB, IATA and IRNAS) and technological centres (CTP, FCBA and RAIZ) of the wood, cellulose, lignin and enzyme sectors.
New extremophilic enzymes have been identified by in silico genome screening or improved by protein engineering, produced in heterologous hosts, and their impact on kraft lignin or xylan demonstrated. Specifically, several extremophilic fungal laccases were developed by directed evolution at CIB; IATA discovered new bacterial xylanases with outstanding extremophilic activity; and MetGen identified several bacterial laccase candidates and developed new bacterial xylanase prototypes with extremophilic properties. Existing MetZyme® products were supplied by MetGen as benchmarking material and for application testing activities. Expression up-scale trials of the new developed enzymes were conducted in MetGen’s ENZINE® E.coli platform, while CIB set up the conditions for expression of fungal laccases in Pichia pastoris to be scaled up by MetGen.

CTP successfully produced kraft lignins from the black liquors of softwood, hardwood and eucalyptus kraft pulp mills. All lignins were fully characterized by CTP and IRNAS. 2D-NMR analyses showed clear differences both in lignin units and in linkage distribution. Lignins were processes utilising METNIN™ enzymatic fractionation technology yielding lignin oligomers with distinct Mw and varying physicochemical properties. These fractions have been tested in MDF and PU foam manufacture. MetGen’s most prominent extremophilic laccases and xylanases have been also assayed in delignification and bleaching of kraft pulps by CTP and RAIZ. Enzymatic pre-treatment of softwood and eucalyptus kraft pulps prior to both steps gave the best (although still slight) improvements. In the coming months new extremozymes developed in the project will be assayed to enhance these results. The highest sugar content, mainly xylose, was found in the effluent of enzymatic pre-bleaching test. Yet, it seems not sufficient. Hemicellulose extraction from bleached pulps is being carried out as an alternative approach to obtain higher sugar yields.

One end-use case of WoodZymes is the use of extremozymes and resin bio-equivalents in MDF manufacture. Lignin-phenol-formaldehyde (LPF) resole resins with different lignin to phenol substitution levels were formulated and evaluated by FCBA, aiming to reduce the content on oil-based chemicals. Chemical analyses of these resins (IRNAS) assisted in selecting the best formulation conditions and confirmed the crosslinking of lignin to formaldehyde and to phenol. The addition of a CIB alkalophilic laccase to the formulations enhanced resin’s viscosity 4-fold and reduced the gel time 30%. Finally, up-scaled production of LPF resin and pilot wood-based panelboard production were validated. On the other hand, treatment of the chips with extremophilic laccases might facilitate fibre separation from the wooden matrix or pre-activate the fibre surface, resulting in higher bonding potential. FCBA has evaluated the action of a laccase developed by CIB on washed chips before defibering, resulting in a positive impact in the MDF obtained due to better cohesion of the treated fibres.

Another WoodZymes end-use case is to use lignin and lignin-derived phenols as bio-equivalents of fossil polyols in polyurethane (PU) foam manufacture. Various samples of kraft lignins and METNIN™ lignin fractions have been oxypropylated to obtain polyols which have been used in the formulations of rigid foams at SOPREMA. First foaming tests showed comparable behaviour to standard formulation for some of the lignin-PU foams.

As regards sugar-based paper additives, the solubility of the hemicelluloses obtained from pulp must be improved in order to allow their adsorption onto fibres before refining. For that, two chemical modification methods were evaluated, obtaining in both cases totally soluble hemicelluloses. The latter are being used as refining additives to reduce energy consumption while improving the paper physical properties.

An advisory group was created with the industrial partners to complete the technical requirements for environmental and socio-economic assessment of: i) use of enzymes and lignin-based adhesives in MDF production (FINSA); ii) production of lignin polyols for PU foams (Soprema); and iii) use of pulp sugars as paper additives for paper making (Fibre Excellence and Navigator); all comprising the whole value chain: extraction, conversion, modification and valorisation. Data collection has started to define the reference scenarios in MDF and PU foam production to be compared with WoodZymes approaches.
Enzymatic biocatalysts are expected to make an important contribution in the wood conversion sector. Considering the extreme conditions used in the current industrial processes, the discovery or design of extremophilic enzymes via protein engineering highlights the relevance of the new biocatalysts developed here as a step forward towards turning kraft pulp mills into wood biorefineries.

The knowhow and technology developed by R&T partners enabling the exploitation of underutilized wood fractions, in collaboration with feedstock providers and end-users, allows seamless integration of products from biomass conversion to the production process. WoodZymes approach interconnects pulp and paper industry, valorising black liquors and extracting hemicelluloses from kraft pulp, with fibreboard and PU foams manufacture, reducing the need for fossil-based chemicals, thus contributing to reduce the carbon-footprint of these industries. In addition to environmental benefits, a large stable off-take of wood-based feedstock will create stable jobs and incomes for rural areas and improve the viability of the agricultural and forest based sectors.