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Eco-friendly new products from enzymatically modified industrial lignins
i n d u s t r i a l c r o p s a n d p r o d u c t s 2 7 ( 2 0 0 8 ) 189–195
available at www.sciencedirect.com
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Eco-friendly new products from enzymatically modified industrial lignins G. Sena-Martins ∗ , E. Almeida-Vara, J.C. Duarte Department of Biotecnology, National Institute of Engineering and Industrial Innovation, Estrada do Pac¸o do Lumiar, No. 22, Ed. E, P-1649-038 Lisbon, Portugal
a r t i c l e
i n f o
a b s t r a c t
Keywords:
Industrial lignins are by-products from the pulp and paper industry, as well as from other
Industrial lignins
biomass-based industries. They are non-toxic, potentially of high value, inexpensive and
Enzyme
available in large amounts. They possess highly reactive locations that can be enzymatically
Binders
modified to develop new and environmentally friendly products.
Co-polymerizates Coatings Paintings
The oxidative enzymes produced by ligninolytic fungi are the catalysts that have mostly been used for the up-grading of these new technologies. This paper aims to provide a general picture of the variety of new and eco-friendly products that have recently been produced through enzyme-based technologies and using industrial lignins as raw materials, namely for the production of lignin-based copolymers by grafting, binders for wood composites, chelating agents, compositions for treating porous materials, coatings and paintings. In addition, it introduces fundamental aspects related to the enzymes used to modify the lignin structure to the interested readers that are not familiar with this field of research. © 2007 Elsevier B.V. All rights reserved.
1.
Introduction
Prevention, reduction and elimination, as far as possible, of environmental contamination stands out as a major issue on the agenda for the 21st century. In addition, the consumers’ demand for products complying with consumers’ health, safety and environmental requirements is steadily increasing. As a result, worldwide intensive R&D is occurring aiming at cleaner industrial practices, more eco-friendly, biodegradable and non-toxic products, a higher industrial usage of renewable and more natural raw materials and, finally, to improve technologies for by-products up-grading, contributing thus for waste minimisation. Industrial lignins are by-products from the pulp and paper industry, as well as from other biomass-based industries. They are non-toxic, potentially of high value, inexpen-
∗
sive and available in large amounts. Their macromolecular structure presents a high heterogeneity, which is caused by variations in lignin composition, size, cross-linking and functional groups due to differences in raw material, pulping and isolation conditions (Gosselink et al., 2004a). These by-materials possess highly reactive locations that can be surprisingly modified through a selection of chemical, physical and/or enzymatic reactions, which gives them a great potential for their exploitation as industrial raw materials. A particularly promising solution for up-grading industrial lignins is through biotechnology, once it will be possible to minimize the problems associated with a both profitable and environmentally sound industrial production. In fact, extensive research efforts have been dedicated to evaluate the possibilities offered by enzymes to enhance production effi-
Corresponding author. Tel.: +351 210 924 600/1x4206; fax: +351 217 166 966. E-mail address: [email protected] (G. Sena-Martins). 0926-6690/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.indcrop.2007.07.016
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i n d u s t r i a l c r o p s a n d p r o d u c t s 2 7 ( 2 0 0 8 ) 189–195
ciency and concomitantly obtain final products presenting higher quality and biodegradability. This paper aims to provide a general picture of the variety of new and eco-friendly products developed through enzymebased technologies using industrial lignins as raw materials. In addition, it introduces fundamental aspects related to the enzymes used to modify the lignin structure to some of the interested readers that are not familiar with this field of research.
2. Industrial lignins available as raw materials The cheapest and most abundant sources of industrial lignins are, until now, the materials derived from leftovers in pulp and paper processes. These lignins present a great variety of functional groups, some of them, depending on the genetic origin of lignin (ethers of various types, primary and secondary hydroxyl groups, carbonyl groups, carboxyl and ester functions, etc.), while others are due to the applied pulping sequences and extraction processes. For example, the material derived from the sulphite and the kraft processes, respectively the lignosulphonates (LS) and kraft lignins, present sulphur-containing groups such as thiols and sulphuric acids. Besides these already well-established LS and kraft lignins, a large variety of other industrial lignin types are available nowadays or on the way to become available. This is mainly due to the implementation of new pulping sequences, to the use of other non-woody pulp raw materials, and also to the recent technologies that use lignocellulosics for other proposals (e.g., ethanol production). But there is still a very small market for the large quantities of industrial lignins produced (only about 2% of the lignins available from the pulp and paper industry are commercialised), being the rest of it burned to generate energy and to recover chemicals. The existing markets are either for very low value products (LS mainly in dispersing and binding applications) or limited to very narrow market segments (high quality dispersants from chemically modified kraft lignin) (Gosselink et al., 2004b). Recently, progress has been made in the use of these lignins as feedstock for novel chemicals (Boudet et al., 2003). However, there is lack of a wider range of value-added applications, which is mainly caused by the low-purity standards, heterogeneity, smell and colour problems of the existing commercial lignins (Gosselink et al., 2004a,b). But, the great efforts that are presently done to develop new techniques for lignin modification and to increase the sensitiveness of the analytical methods used to follow-up those modifications, will soon give rise to technically more useful materials and are expected to overcome this situation.
3. Enzymatic approaches for industrial lignins modification Application of new technologies based on enzymatic catalysis brings numerous advantages to industrial processes, resulting in cleaner and more environmental friendly processes
and products. This is mainly due to the high selectivity and efficiency of enzymatic catalysts, to the mild operating conditions required, to a broad range of substrates and their ability to react under adverse conditions (e.g., high temperatures, extreme pH values), among others. But the effective use of enzymes may be hampered by some peculiar properties of the enzymatic proteins, such as their non-reusability unless conveniently immobilized, and their high sensitivity to several denaturating agents and to the presence of adverse sensory ´ et al., 2002). Nevertheless, the or toxicological effects (Duran undoubting advantages offered by enzymes justify the intensive research that has been done in this field for the last three decades. For the technical processes requiring modifications of the lignin structure, research has focused on the oxidative enzymes produced by ligninolytic fungi. In nature, lignin is rotted by a combined action of different biological and chemical factors. The initial steps of lignin biodegradation consist in introducing new functional groups into its macromolecular structure by oxidative enzymes, which render lignin more susceptible towards its subsequent degradation by other enzymes. There are a wide variety of fungi and bacteria that can degrade lignin through a battery of different enzymes, but the so-called white-rot fungi (WRF) are the only known organisms that can completely break down the lignin molecule to CO2 and H2 O (Table 1). They owe their name to the specific bleaching process that occurs during the fungal degradation of wood (ten Have and Teunissen, 2001). Lignin degradation by fungi has been subject of intensive research for more than half a century now. Until the early 1980s several studies reported the breakdown of lignin when it was added to WR fungal cultivations. But it was only in 1983 that three laboratories independently (Glenn et al., 1983; Shimada and Higuchi, 1983; Tien and Kirk, 1983) reported that the lignin degradation observed in the culture broth of the WRF Phanerochaete chrysosporium was due to an enzymatic process, with the discovery of ligninase, an H2 O2 -dependent enzyme (now referred to as lignin peroxidase—LiP). Later on, it was found that some of the peroxidase activity detected in the extracellular fluid of P. chrysosporium was dependent on Mn2+ , leading to the discovery of manganese peroxidase (MnP) (Glenn and Gold, 1985). Since then, considerable research has been done in this field, with the subsequent purification of several peroxidases isoenzymes, finding of new other ligninolytic enzymes like laccases, molecular characterization of a large number of peroxidases and laccases, detection of peroxidases in ligninolytic cultures of other WRF (namely, Bjerkandera, Trametes, Pleurotus, Phlebia, Ceriporiopsis species, etc.), studies on ligninolytic enzymes catalytic cycles and on the role of low molecular weight compounds (mediators). Different WRF possess different lignin-degrading systems, comprising extracellular enzymes like the lignin oxidative enzymes and the H2 O2 -generating enzymes, and low molecular weight cofactors (e.g., oxalate, veratryl alcohol, Mn2+ , etc.). Table 2 summarizes the ligninolytic enzymes and their substrates and reactions. The main extracellular enzymes participating in lignin degradation are the heme-containing lignin peroxidase (ligninase, LiP, EC 1.11.1.14) and manganese peroxidase (MnP, EC 1.11.1.13) and the Cu-containing laccase (benzenediol:oxygen
Streptomyces, Nocardia, Pseudomonas Limited lignin degradation Actinomycetes or Myxobacteroa
Poria, Polyporus, Chaetomium, Paecilomyces, Fusarium
Mainly softwood, aquatic environment, wood with preserving chemicals, plant litter Sapwood, water-saturated wood, wood at late stage of decomposition, plant litter Basidiomycotina, Ascomycotina or Deuteromycotina
Brown-rot fungi, soft-rot fungi Bacteria
Phanerochaete, Phlebia, Trametes Mainly hardwood
Lignin mineralization, selective or non-selective delignification Lignin modification, Limited lignin degradation Basidiomycotina (Ascomycotina) White-rot fungi
Subdivision Organism
Table 1 – Lignin-degrading organisms (Tuomela et al., 2000)
Lignin degradation
Environment
Genera (e.g.)
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oxidoreductase, EC 1.10.3.2). A new group of ligninolytic hemecontaining peroxidases, combining structural and functional properties of the LiPs and MnPs, are the versatile peroxidases (VPs). The enzymes involved in H2 O2 production, such as glyoxal oxidase and aryl alcohol oxidase (EC 1.1.3.7), are also considered to belong to the ligninolytic system (Lankinen, 2004). The yielding of free radical species as a result of peroxidases and laccases activity is of utmost importance from the industrial point of view. These radicals greatly increase the reactivity of the lignin molecules, leading to further polymerisations in a random non-enzymatic way to form three-dimensional polymers of higher Mw and with a variety of new linkages. As a result, a wide variety of new materials with distinct properties can be obtained. In Fig. 1 is presented the LiP and MnP catalysis and mediation as proposed by Cameron et al. (2000), and how the radical species are generated.
4. Scale-up of ligninolytic enzymes production Due to their highly promising industrial potential, production of ligninolytic enzymes has been targeted since their discovery and isolation. But the levels of active enzyme production are very low, rendering very expensive their use for industrial applications. During the 1980s research mainly focused on peroxidases (mainly LiP) that were thought to be the key-enzymes for lignin degradation. However, the difficulties associated with the fast decay of hydrogen peroxide, indispensable for LiP and MnP catalysis, and the inactivation of LiP and MnP for certain amounts of H2 O2 , turned over research emphasis on the use of laccases, that only need O2 for the oxidation of their reduced stage and are produced in slightly higher amounts. Once ligninolytic enzymes are produced by WRF in relatively low amounts, recombinant DNA technology for strain improvement and heterologous protein production is a suitable approach for this problem. Heterologous expression is also a requisite for structure–function studies in order to ´ obtain protein variants by site-direct mutagenisis (PerezBoada et al., 2002) and also to solve the difficulties encountered in purification of the referred enzymes, e.g., MnP isoenzymes from Ceriporiopsis subvermispora (Larrondo et al., 2001). The ability of filamentous fungi (in particular Ascomycetes and Deuteromycetes) to secrete large quantities of protein, and the existence of well-developed transformation and expression systems make them very suitable hosts for expression of heterologous genes. Following the initial focus put on peroxidases (both LiP and MnP) several attempts were tried in order to produce LiP and MnP recombinant proteins, using several homologous and heterologous host systems, but with limited success (reviewed in Conesa et al., 2002; Mart´ınez, 2002). In what concerns laccases, and despite the intensive research developed, the molecular basis of laccase-catalyzed reaction is still partially unknown (Kiiskinen and Saloheimo, 2004). In order to determine the function of laccases and to produce them heterologously in large quantities, several
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Table 2 – Ligninolytic enzymes and their main reactions (Lankinen, 2004) Enzyme and abbreviation
Cofactor
Substrate, mediator
Reaction
Lignin peroxidase (LiP) Manganese peroxidase (MnP)
H2 O2 H2 O2
Veratryl alcohol Mn, organic acids as chelators, thiols, unsaturated fatty acids
Versatile peroxidase (VP)
H2 O2
Laccase
O2
Mn, Veratryl alcohol, compounds similar to LiP and MnP Phenols, mediators, e.g., hydroxybenzotriazole or ABTS Glyoxal, methyl glyoxal Aromatic alcohols (anisyl, veratryl alcohol) Many organic compounds
Aromatic ring oxidized to cation radical Mn(II) oxidized to Mn(III), chelated Mn(II) oxidizes phenolic compounds to phenoxyl radicals, other reactions in the presence of additional compounds Mn(II) oxidized to Mn (III), oxidation of phenolic and non-phenolic compounds, and dyes
Glyoxal oxidase (GLOX) Aryl alcohol oxidase (AAO) Other H2 O2 -producing enzymes
laccases genes have been cloned, especially the genes of basidiomycetes like Phlebia radiata, Cryptococcus neoformans, Pleurotous ostreatus, Trametes versicolor, Trametes vilosa, Pycnoporous cinnabarinus and Coprinus cireneus (cited in Kiiskinen and Saloheimo, 2004). Also a significant number of studies were carried out to express laccases genes in fungal hosts like Saccharommyces cerevisiae, Trichoderma reesei, Aspergillus oryzae, Pichia pastoris, Aspergillus sojae and Aspergillus niger (cited in Kiiskinen and Saloheimo, 2004). In spite of the fact that active excreted recombinant laccase could be achieved in several of the heterologous systems developed, the levels of produced recombinant laccase are still too low for industrial purposes. In fact, recombinant fungal proteins production is always encountered with the lack of specific knowledge about the pathway secretion in filamentous fungi, emphasizing the research needed in this area in order to clarify this bottleneck problem. Even so, there are already several laccases commercially available, namely from Novozymes (Denmark), Sigma, SynectiQ Corp. (Dover, NJ, USA) and Fluka. To conclude, and in order to succeed in implementing enzyme-based technologies, it is imperative to remove some of the undesirable constraints referred above related to the industrial production of enzymes. Special emphasis should be done on production levels, costs and in multiplying the number of enzymes commercially available.
5. Applications of enzymatically modified industrial lignins Despite the difficulties related both to the low quality of the existing commercial industrial lignins and the high cost and low availability of commercial enzymes, important progress has been done with respect to the development of new, added-value, and environment friendly enzyme-based products. Although there were a significant number of publications in this field in recent years, we will only refer to the applications presenting more novelty and higher potential for future exploitation.
5.1.
Lignin copolymers
The grafting of industrial lignins to synthetic polymers for preparing a new class of engineering plastics is probably the
Phenols are oxidized to phenoxyl radicals, other reactions in the presence of mediators Glyoxal oxidized to glyoxal acid, H2 O2 production Aromatic alcohols oxidized to aldehydes, H2 O2 production O2 reduced to H2 O2
most frequent application found in the literature. Examples of the published results encountered are (1) the copolymerisation of straw pulp lignin with cresol, using horseradish peroxidase as polymerisation catalytic enzyme. The resulting copolymers can be used as replacement for normal phenolic resins for different proposals (Liu et al., 1999) and (2) the copolymerisation of different lignins (organosolv lignin obtained from delignification of beech and spruce pulp, Indulin AT and a synthetic hydroxypropylated lignin) with vanilic acid, diisocyanate and acrylamide catalysed by laccase (Milstein ¨ et al., 1994; Mai et al., 2000; Huttermann et al., 2001a). The co-polymerizates obtained offer the potential to prepare new engineering materials. An enhanced biodegradability of the new products, considerably higher graft efficiency and a better molecular weight control of the reaction products were obtained. Nevertheless, in order to improve the yields of enzymatically synthesized copolymerizates, it is necessary to clarify the exact mechanisms once the enzymatic cross-linking (homopolymerization) of lignin is a competitive reaction to grafting. Process scale-up and the finding of new marketable products for the synthetic products obtained are also essential.
5.2.
Binders for wood composites
Production of wood composites, such as fibre- or particleboards, follows a basic process: solid wood is fragmented into small strands, chips or fibres; then they are supplemented with a binder and pressed to form a wood-like structure again. Using this approach, anisotropy of wood is reduced and wood of small dimensions or recycled timber can be converted into a useful product. Among the commercialised wood binders, the mostly used are urea-formaldehyde resins, phenol-formaldehyde resins and melamine-formaldehyde resins. However, the formaldehyde-based resins release a harmful amount of formaldehyde during the manufacturing and use of wood composites. Therefore, it is urgent to develop formaldehydefree wood binders. Several approaches to use mostly water-insoluble lignins with the addition of small amounts of petrochemical resins as binders for wood composites, under ¨ laccase catalysed reactions have been developed (Huttermann et al., 2001a).
i n d u s t r i a l c r o p s a n d p r o d u c t s 2 7 ( 2 0 0 8 ) 189–195
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Fig. 1
5.3.
Chelating complexes
Gonc¸alves and Benar (2001) treated a lignin obtained from sugarcane acetosolv pulping with a polyphenoloxidase. An increase in the number of hydroxyl and carbonyl groups after
the enzymatic treatment was detected. Thus, the chelating capacity of treated lignin was improved, being 110% higher than in the non-treated acetosolv lignin. The chelating complexes obtained can either be used for removing heavy metals in pulps or to treat effluents containing heavy metals. A bet-
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i n d u s t r i a l c r o p s a n d p r o d u c t s 2 7 ( 2 0 0 8 ) 189–195
Fig. 2
ter characterization of the macromolecular products obtained must be done and, according to the properties found, their application can be directed to other proposes.
5.4. Intermediate product for manufacturing highly reactive reagents A highly reactive intermediate product can be obtained through the treatment of several technical lignins (LS, kraft lignin, acetosolv lignin, etc.) with laccases, in the presence of different oxidation agents (O2 , air or chemical oxidation agents). The propose for the development of this intermediate product is, when it will further react with other lignin derivatives, to obtain polymers to be used in composite materials from plant fibres, in waterproof papers and cardboards or even in thermosetting plastics. It presents numerous advantages, as being easily isolated and ability to be stored for long peri¨ ods of time while its reactivity remains intact (Huttermann et al., 2001b).
5.5.
Compositions for treating porous materials
Porous articles can be treated with an enzyme catalyst, which macromolecularizes a phenolic and/or an aromatic amine compound inside them, in order to give or increase properties, such as strength, flame resistance, antibacterial and antiseptic properties, adhesiveness, chemical agent-slow-releasing properties, colouring properties, dimension stability, crack resistance, deodorizing properties, humidity controlling properties, moisture conditioning properties, surface smoothness, ion exchangeability, among many others as described in the Patent registered by Echigo and Ohno (2003). The phenolic compounds and aromatic amine compounds, i.e. the targets of macromolecularization, may be any compound (including lignin, lignosulfonic acid, etc.) that can be oxidized by the enzymes presenting peroxidase activity.
5.6.
Coatings
Coatings can be prepared through the enzymatic polymerisation of lignin-based compositions. They can be used
for protection, beautifying or treating lignocellulosics, wood or paper-based products. This method allows coating an object using an environmental safe process comprising only enzymes (like catechol oxidase, laccase, peroxidases) and lignin derivatives (either LS, kraft and organosolv lignins). Other components can be added to accelerate lignin polymerisation, such as the laccase mediator ABTS (2,2 -azino-bis, 3-ethylbenzthiazoline-6-sulfonic acid), lignin copolymerisation agents in case of waterproof coatings, etc. (Bolle and Aehle, 2001).
5.7.
Paintings
Paintings, including protective paintings, can be prepared through the enzymatic polymerisation of lignin-based compositions: a mixture containing a solution of lignin (LS, kraft and organosolv lignins) plus a dye or a pigment, and the enzyme (catechol oxidase, laccase, and peroxidase) is incubated under conditions and sufficient length of time to reach the desired viscosity. Then the mixture is spread on an article and subject to proper conditions and time to form a painting on its surface (Bolle and Aehle, 2000).
5.8.
Polymer-template complex
A LS is used to form a polymer-template complex through enzymatic polymerisation. The reaction mixture, including a monomer, a template (a micelle, a borate-containing electrolyte, or a LS) and an enzyme (peroxidases, such as horseradish peroxidase) is incubated under proper conditions to allow the monomer to align along the template and polymerise, forming a polymer-template complex (Fig. 2). Once such complex possesses exceptional electrical and optical stability, water solubility and processibility, it can be used for diverse applications such as light-weight energy storage devices (e.g., rechargeable batteries), electrolytic capacitors, anti-static and anti-corrosive coatings for smart windows, and biological sensors (Samuelson et al., 2004).
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6.
Conclusions
Regardless of the problems related to the quality of the industrial lignins and to the cost and availability of the enzymes that are commercialised, important progress has been done with respect to the development of new, added value, and environmentally friendly enzyme-based products. The examples here presented possess a significant potential for further exploitation, and thus will enlarge the range of marketable applications. But firstly, it is important to clarify the exact mechanisms of the enzymatic synthesis of these products to increase the production yields, to scale-up the developed technologies and to enhance the characterization, quality and biodegradability of the final products. In addition, other industrial lignins and other ligninolytic enzymes should be tried. In the near future, science will globally provide new biocatalysts, alternative ways of using already known enzymes, new metabolites and clearly novel metabolic pathways will be discovered. The toolkit of enzymes that are usable in biotechnology will be enriched and more cost-effective technologies will be available, promoting the use of renewable resources for a more clean and sustainable environment.
Acknowledgements The authors would like to thank the European Commission for the financial support of the Thematic Network called EUROLIGNIN, with contract no. G1RT-CT-2002-05088, within the competitive and sustainable growth programme (CORDIS, 2002).
references
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Eco-friendly new products from enzymatically modified industrial lignins G. Sena-Martins ∗ , E. Almeida-Vara, J.C. Duarte Department of Biotecnology, National Institute of Engineering and Industrial Innovation, Estrada do Pac¸o do Lumiar, No. 22, Ed. E, P-1649-038 Lisbon, Portugal
a r t i c l e
i n f o
a b s t r a c t
Keywords:
Industrial lignins are by-products from the pulp and paper industry, as well as from other
Industrial lignins
biomass-based industries. They are non-toxic, potentially of high value, inexpensive and
Enzyme
available in large amounts. They possess highly reactive locations that can be enzymatically
Binders
modified to develop new and environmentally friendly products.
Co-polymerizates Coatings Paintings
The oxidative enzymes produced by ligninolytic fungi are the catalysts that have mostly been used for the up-grading of these new technologies. This paper aims to provide a general picture of the variety of new and eco-friendly products that have recently been produced through enzyme-based technologies and using industrial lignins as raw materials, namely for the production of lignin-based copolymers by grafting, binders for wood composites, chelating agents, compositions for treating porous materials, coatings and paintings. In addition, it introduces fundamental aspects related to the enzymes used to modify the lignin structure to the interested readers that are not familiar with this field of research. © 2007 Elsevier B.V. All rights reserved.
1.
Introduction
Prevention, reduction and elimination, as far as possible, of environmental contamination stands out as a major issue on the agenda for the 21st century. In addition, the consumers’ demand for products complying with consumers’ health, safety and environmental requirements is steadily increasing. As a result, worldwide intensive R&D is occurring aiming at cleaner industrial practices, more eco-friendly, biodegradable and non-toxic products, a higher industrial usage of renewable and more natural raw materials and, finally, to improve technologies for by-products up-grading, contributing thus for waste minimisation. Industrial lignins are by-products from the pulp and paper industry, as well as from other biomass-based industries. They are non-toxic, potentially of high value, inexpen-
∗
sive and available in large amounts. Their macromolecular structure presents a high heterogeneity, which is caused by variations in lignin composition, size, cross-linking and functional groups due to differences in raw material, pulping and isolation conditions (Gosselink et al., 2004a). These by-materials possess highly reactive locations that can be surprisingly modified through a selection of chemical, physical and/or enzymatic reactions, which gives them a great potential for their exploitation as industrial raw materials. A particularly promising solution for up-grading industrial lignins is through biotechnology, once it will be possible to minimize the problems associated with a both profitable and environmentally sound industrial production. In fact, extensive research efforts have been dedicated to evaluate the possibilities offered by enzymes to enhance production effi-
Corresponding author. Tel.: +351 210 924 600/1x4206; fax: +351 217 166 966. E-mail address: [email protected] (G. Sena-Martins). 0926-6690/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.indcrop.2007.07.016
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ciency and concomitantly obtain final products presenting higher quality and biodegradability. This paper aims to provide a general picture of the variety of new and eco-friendly products developed through enzymebased technologies using industrial lignins as raw materials. In addition, it introduces fundamental aspects related to the enzymes used to modify the lignin structure to some of the interested readers that are not familiar with this field of research.
2. Industrial lignins available as raw materials The cheapest and most abundant sources of industrial lignins are, until now, the materials derived from leftovers in pulp and paper processes. These lignins present a great variety of functional groups, some of them, depending on the genetic origin of lignin (ethers of various types, primary and secondary hydroxyl groups, carbonyl groups, carboxyl and ester functions, etc.), while others are due to the applied pulping sequences and extraction processes. For example, the material derived from the sulphite and the kraft processes, respectively the lignosulphonates (LS) and kraft lignins, present sulphur-containing groups such as thiols and sulphuric acids. Besides these already well-established LS and kraft lignins, a large variety of other industrial lignin types are available nowadays or on the way to become available. This is mainly due to the implementation of new pulping sequences, to the use of other non-woody pulp raw materials, and also to the recent technologies that use lignocellulosics for other proposals (e.g., ethanol production). But there is still a very small market for the large quantities of industrial lignins produced (only about 2% of the lignins available from the pulp and paper industry are commercialised), being the rest of it burned to generate energy and to recover chemicals. The existing markets are either for very low value products (LS mainly in dispersing and binding applications) or limited to very narrow market segments (high quality dispersants from chemically modified kraft lignin) (Gosselink et al., 2004b). Recently, progress has been made in the use of these lignins as feedstock for novel chemicals (Boudet et al., 2003). However, there is lack of a wider range of value-added applications, which is mainly caused by the low-purity standards, heterogeneity, smell and colour problems of the existing commercial lignins (Gosselink et al., 2004a,b). But, the great efforts that are presently done to develop new techniques for lignin modification and to increase the sensitiveness of the analytical methods used to follow-up those modifications, will soon give rise to technically more useful materials and are expected to overcome this situation.
3. Enzymatic approaches for industrial lignins modification Application of new technologies based on enzymatic catalysis brings numerous advantages to industrial processes, resulting in cleaner and more environmental friendly processes
and products. This is mainly due to the high selectivity and efficiency of enzymatic catalysts, to the mild operating conditions required, to a broad range of substrates and their ability to react under adverse conditions (e.g., high temperatures, extreme pH values), among others. But the effective use of enzymes may be hampered by some peculiar properties of the enzymatic proteins, such as their non-reusability unless conveniently immobilized, and their high sensitivity to several denaturating agents and to the presence of adverse sensory ´ et al., 2002). Nevertheless, the or toxicological effects (Duran undoubting advantages offered by enzymes justify the intensive research that has been done in this field for the last three decades. For the technical processes requiring modifications of the lignin structure, research has focused on the oxidative enzymes produced by ligninolytic fungi. In nature, lignin is rotted by a combined action of different biological and chemical factors. The initial steps of lignin biodegradation consist in introducing new functional groups into its macromolecular structure by oxidative enzymes, which render lignin more susceptible towards its subsequent degradation by other enzymes. There are a wide variety of fungi and bacteria that can degrade lignin through a battery of different enzymes, but the so-called white-rot fungi (WRF) are the only known organisms that can completely break down the lignin molecule to CO2 and H2 O (Table 1). They owe their name to the specific bleaching process that occurs during the fungal degradation of wood (ten Have and Teunissen, 2001). Lignin degradation by fungi has been subject of intensive research for more than half a century now. Until the early 1980s several studies reported the breakdown of lignin when it was added to WR fungal cultivations. But it was only in 1983 that three laboratories independently (Glenn et al., 1983; Shimada and Higuchi, 1983; Tien and Kirk, 1983) reported that the lignin degradation observed in the culture broth of the WRF Phanerochaete chrysosporium was due to an enzymatic process, with the discovery of ligninase, an H2 O2 -dependent enzyme (now referred to as lignin peroxidase—LiP). Later on, it was found that some of the peroxidase activity detected in the extracellular fluid of P. chrysosporium was dependent on Mn2+ , leading to the discovery of manganese peroxidase (MnP) (Glenn and Gold, 1985). Since then, considerable research has been done in this field, with the subsequent purification of several peroxidases isoenzymes, finding of new other ligninolytic enzymes like laccases, molecular characterization of a large number of peroxidases and laccases, detection of peroxidases in ligninolytic cultures of other WRF (namely, Bjerkandera, Trametes, Pleurotus, Phlebia, Ceriporiopsis species, etc.), studies on ligninolytic enzymes catalytic cycles and on the role of low molecular weight compounds (mediators). Different WRF possess different lignin-degrading systems, comprising extracellular enzymes like the lignin oxidative enzymes and the H2 O2 -generating enzymes, and low molecular weight cofactors (e.g., oxalate, veratryl alcohol, Mn2+ , etc.). Table 2 summarizes the ligninolytic enzymes and their substrates and reactions. The main extracellular enzymes participating in lignin degradation are the heme-containing lignin peroxidase (ligninase, LiP, EC 1.11.1.14) and manganese peroxidase (MnP, EC 1.11.1.13) and the Cu-containing laccase (benzenediol:oxygen
Streptomyces, Nocardia, Pseudomonas Limited lignin degradation Actinomycetes or Myxobacteroa
Poria, Polyporus, Chaetomium, Paecilomyces, Fusarium
Mainly softwood, aquatic environment, wood with preserving chemicals, plant litter Sapwood, water-saturated wood, wood at late stage of decomposition, plant litter Basidiomycotina, Ascomycotina or Deuteromycotina
Brown-rot fungi, soft-rot fungi Bacteria
Phanerochaete, Phlebia, Trametes Mainly hardwood
Lignin mineralization, selective or non-selective delignification Lignin modification, Limited lignin degradation Basidiomycotina (Ascomycotina) White-rot fungi
Subdivision Organism
Table 1 – Lignin-degrading organisms (Tuomela et al., 2000)
Lignin degradation
Environment
Genera (e.g.)
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oxidoreductase, EC 1.10.3.2). A new group of ligninolytic hemecontaining peroxidases, combining structural and functional properties of the LiPs and MnPs, are the versatile peroxidases (VPs). The enzymes involved in H2 O2 production, such as glyoxal oxidase and aryl alcohol oxidase (EC 1.1.3.7), are also considered to belong to the ligninolytic system (Lankinen, 2004). The yielding of free radical species as a result of peroxidases and laccases activity is of utmost importance from the industrial point of view. These radicals greatly increase the reactivity of the lignin molecules, leading to further polymerisations in a random non-enzymatic way to form three-dimensional polymers of higher Mw and with a variety of new linkages. As a result, a wide variety of new materials with distinct properties can be obtained. In Fig. 1 is presented the LiP and MnP catalysis and mediation as proposed by Cameron et al. (2000), and how the radical species are generated.
4. Scale-up of ligninolytic enzymes production Due to their highly promising industrial potential, production of ligninolytic enzymes has been targeted since their discovery and isolation. But the levels of active enzyme production are very low, rendering very expensive their use for industrial applications. During the 1980s research mainly focused on peroxidases (mainly LiP) that were thought to be the key-enzymes for lignin degradation. However, the difficulties associated with the fast decay of hydrogen peroxide, indispensable for LiP and MnP catalysis, and the inactivation of LiP and MnP for certain amounts of H2 O2 , turned over research emphasis on the use of laccases, that only need O2 for the oxidation of their reduced stage and are produced in slightly higher amounts. Once ligninolytic enzymes are produced by WRF in relatively low amounts, recombinant DNA technology for strain improvement and heterologous protein production is a suitable approach for this problem. Heterologous expression is also a requisite for structure–function studies in order to ´ obtain protein variants by site-direct mutagenisis (PerezBoada et al., 2002) and also to solve the difficulties encountered in purification of the referred enzymes, e.g., MnP isoenzymes from Ceriporiopsis subvermispora (Larrondo et al., 2001). The ability of filamentous fungi (in particular Ascomycetes and Deuteromycetes) to secrete large quantities of protein, and the existence of well-developed transformation and expression systems make them very suitable hosts for expression of heterologous genes. Following the initial focus put on peroxidases (both LiP and MnP) several attempts were tried in order to produce LiP and MnP recombinant proteins, using several homologous and heterologous host systems, but with limited success (reviewed in Conesa et al., 2002; Mart´ınez, 2002). In what concerns laccases, and despite the intensive research developed, the molecular basis of laccase-catalyzed reaction is still partially unknown (Kiiskinen and Saloheimo, 2004). In order to determine the function of laccases and to produce them heterologously in large quantities, several
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Table 2 – Ligninolytic enzymes and their main reactions (Lankinen, 2004) Enzyme and abbreviation
Cofactor
Substrate, mediator
Reaction
Lignin peroxidase (LiP) Manganese peroxidase (MnP)
H2 O2 H2 O2
Veratryl alcohol Mn, organic acids as chelators, thiols, unsaturated fatty acids
Versatile peroxidase (VP)
H2 O2
Laccase
O2
Mn, Veratryl alcohol, compounds similar to LiP and MnP Phenols, mediators, e.g., hydroxybenzotriazole or ABTS Glyoxal, methyl glyoxal Aromatic alcohols (anisyl, veratryl alcohol) Many organic compounds
Aromatic ring oxidized to cation radical Mn(II) oxidized to Mn(III), chelated Mn(II) oxidizes phenolic compounds to phenoxyl radicals, other reactions in the presence of additional compounds Mn(II) oxidized to Mn (III), oxidation of phenolic and non-phenolic compounds, and dyes
Glyoxal oxidase (GLOX) Aryl alcohol oxidase (AAO) Other H2 O2 -producing enzymes
laccases genes have been cloned, especially the genes of basidiomycetes like Phlebia radiata, Cryptococcus neoformans, Pleurotous ostreatus, Trametes versicolor, Trametes vilosa, Pycnoporous cinnabarinus and Coprinus cireneus (cited in Kiiskinen and Saloheimo, 2004). Also a significant number of studies were carried out to express laccases genes in fungal hosts like Saccharommyces cerevisiae, Trichoderma reesei, Aspergillus oryzae, Pichia pastoris, Aspergillus sojae and Aspergillus niger (cited in Kiiskinen and Saloheimo, 2004). In spite of the fact that active excreted recombinant laccase could be achieved in several of the heterologous systems developed, the levels of produced recombinant laccase are still too low for industrial purposes. In fact, recombinant fungal proteins production is always encountered with the lack of specific knowledge about the pathway secretion in filamentous fungi, emphasizing the research needed in this area in order to clarify this bottleneck problem. Even so, there are already several laccases commercially available, namely from Novozymes (Denmark), Sigma, SynectiQ Corp. (Dover, NJ, USA) and Fluka. To conclude, and in order to succeed in implementing enzyme-based technologies, it is imperative to remove some of the undesirable constraints referred above related to the industrial production of enzymes. Special emphasis should be done on production levels, costs and in multiplying the number of enzymes commercially available.
5. Applications of enzymatically modified industrial lignins Despite the difficulties related both to the low quality of the existing commercial industrial lignins and the high cost and low availability of commercial enzymes, important progress has been done with respect to the development of new, added-value, and environment friendly enzyme-based products. Although there were a significant number of publications in this field in recent years, we will only refer to the applications presenting more novelty and higher potential for future exploitation.
5.1.
Lignin copolymers
The grafting of industrial lignins to synthetic polymers for preparing a new class of engineering plastics is probably the
Phenols are oxidized to phenoxyl radicals, other reactions in the presence of mediators Glyoxal oxidized to glyoxal acid, H2 O2 production Aromatic alcohols oxidized to aldehydes, H2 O2 production O2 reduced to H2 O2
most frequent application found in the literature. Examples of the published results encountered are (1) the copolymerisation of straw pulp lignin with cresol, using horseradish peroxidase as polymerisation catalytic enzyme. The resulting copolymers can be used as replacement for normal phenolic resins for different proposals (Liu et al., 1999) and (2) the copolymerisation of different lignins (organosolv lignin obtained from delignification of beech and spruce pulp, Indulin AT and a synthetic hydroxypropylated lignin) with vanilic acid, diisocyanate and acrylamide catalysed by laccase (Milstein ¨ et al., 1994; Mai et al., 2000; Huttermann et al., 2001a). The co-polymerizates obtained offer the potential to prepare new engineering materials. An enhanced biodegradability of the new products, considerably higher graft efficiency and a better molecular weight control of the reaction products were obtained. Nevertheless, in order to improve the yields of enzymatically synthesized copolymerizates, it is necessary to clarify the exact mechanisms once the enzymatic cross-linking (homopolymerization) of lignin is a competitive reaction to grafting. Process scale-up and the finding of new marketable products for the synthetic products obtained are also essential.
5.2.
Binders for wood composites
Production of wood composites, such as fibre- or particleboards, follows a basic process: solid wood is fragmented into small strands, chips or fibres; then they are supplemented with a binder and pressed to form a wood-like structure again. Using this approach, anisotropy of wood is reduced and wood of small dimensions or recycled timber can be converted into a useful product. Among the commercialised wood binders, the mostly used are urea-formaldehyde resins, phenol-formaldehyde resins and melamine-formaldehyde resins. However, the formaldehyde-based resins release a harmful amount of formaldehyde during the manufacturing and use of wood composites. Therefore, it is urgent to develop formaldehydefree wood binders. Several approaches to use mostly water-insoluble lignins with the addition of small amounts of petrochemical resins as binders for wood composites, under ¨ laccase catalysed reactions have been developed (Huttermann et al., 2001a).
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Fig. 1
5.3.
Chelating complexes
Gonc¸alves and Benar (2001) treated a lignin obtained from sugarcane acetosolv pulping with a polyphenoloxidase. An increase in the number of hydroxyl and carbonyl groups after
the enzymatic treatment was detected. Thus, the chelating capacity of treated lignin was improved, being 110% higher than in the non-treated acetosolv lignin. The chelating complexes obtained can either be used for removing heavy metals in pulps or to treat effluents containing heavy metals. A bet-
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Fig. 2
ter characterization of the macromolecular products obtained must be done and, according to the properties found, their application can be directed to other proposes.
5.4. Intermediate product for manufacturing highly reactive reagents A highly reactive intermediate product can be obtained through the treatment of several technical lignins (LS, kraft lignin, acetosolv lignin, etc.) with laccases, in the presence of different oxidation agents (O2 , air or chemical oxidation agents). The propose for the development of this intermediate product is, when it will further react with other lignin derivatives, to obtain polymers to be used in composite materials from plant fibres, in waterproof papers and cardboards or even in thermosetting plastics. It presents numerous advantages, as being easily isolated and ability to be stored for long peri¨ ods of time while its reactivity remains intact (Huttermann et al., 2001b).
5.5.
Compositions for treating porous materials
Porous articles can be treated with an enzyme catalyst, which macromolecularizes a phenolic and/or an aromatic amine compound inside them, in order to give or increase properties, such as strength, flame resistance, antibacterial and antiseptic properties, adhesiveness, chemical agent-slow-releasing properties, colouring properties, dimension stability, crack resistance, deodorizing properties, humidity controlling properties, moisture conditioning properties, surface smoothness, ion exchangeability, among many others as described in the Patent registered by Echigo and Ohno (2003). The phenolic compounds and aromatic amine compounds, i.e. the targets of macromolecularization, may be any compound (including lignin, lignosulfonic acid, etc.) that can be oxidized by the enzymes presenting peroxidase activity.
5.6.
Coatings
Coatings can be prepared through the enzymatic polymerisation of lignin-based compositions. They can be used
for protection, beautifying or treating lignocellulosics, wood or paper-based products. This method allows coating an object using an environmental safe process comprising only enzymes (like catechol oxidase, laccase, peroxidases) and lignin derivatives (either LS, kraft and organosolv lignins). Other components can be added to accelerate lignin polymerisation, such as the laccase mediator ABTS (2,2 -azino-bis, 3-ethylbenzthiazoline-6-sulfonic acid), lignin copolymerisation agents in case of waterproof coatings, etc. (Bolle and Aehle, 2001).
5.7.
Paintings
Paintings, including protective paintings, can be prepared through the enzymatic polymerisation of lignin-based compositions: a mixture containing a solution of lignin (LS, kraft and organosolv lignins) plus a dye or a pigment, and the enzyme (catechol oxidase, laccase, and peroxidase) is incubated under conditions and sufficient length of time to reach the desired viscosity. Then the mixture is spread on an article and subject to proper conditions and time to form a painting on its surface (Bolle and Aehle, 2000).
5.8.
Polymer-template complex
A LS is used to form a polymer-template complex through enzymatic polymerisation. The reaction mixture, including a monomer, a template (a micelle, a borate-containing electrolyte, or a LS) and an enzyme (peroxidases, such as horseradish peroxidase) is incubated under proper conditions to allow the monomer to align along the template and polymerise, forming a polymer-template complex (Fig. 2). Once such complex possesses exceptional electrical and optical stability, water solubility and processibility, it can be used for diverse applications such as light-weight energy storage devices (e.g., rechargeable batteries), electrolytic capacitors, anti-static and anti-corrosive coatings for smart windows, and biological sensors (Samuelson et al., 2004).
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6.
Conclusions
Regardless of the problems related to the quality of the industrial lignins and to the cost and availability of the enzymes that are commercialised, important progress has been done with respect to the development of new, added value, and environmentally friendly enzyme-based products. The examples here presented possess a significant potential for further exploitation, and thus will enlarge the range of marketable applications. But firstly, it is important to clarify the exact mechanisms of the enzymatic synthesis of these products to increase the production yields, to scale-up the developed technologies and to enhance the characterization, quality and biodegradability of the final products. In addition, other industrial lignins and other ligninolytic enzymes should be tried. In the near future, science will globally provide new biocatalysts, alternative ways of using already known enzymes, new metabolites and clearly novel metabolic pathways will be discovered. The toolkit of enzymes that are usable in biotechnology will be enriched and more cost-effective technologies will be available, promoting the use of renewable resources for a more clean and sustainable environment.
Acknowledgements The authors would like to thank the European Commission for the financial support of the Thematic Network called EUROLIGNIN, with contract no. G1RT-CT-2002-05088, within the competitive and sustainable growth programme (CORDIS, 2002).
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