**5.2 Biocomposites from fibres using enzyme and lignin**

The study of enzymatic systems to activate the cellulosic fibres is a green alternative approach to other modifications for preparation of biocomposites and very well scientifically studied in last four decades to improve the surface, chemical, morphological and thermal properties of natural fibres as reinforcement materials. The enzymes offer an inexpensive and ecofriendly attractive option to improve the surfaces of natural fibres for composites.

Laccases (EC1.10.3.1) are multinuclear copper oxidases often called 'blue' oxidases that catalyze the oxidation of a wide range of susbstrates including phenols. Fungal laccases (benzenediol:oxygen oxidoreductase, EC1.10.3.2) are obtained from extractives of various fungal strains as an extracellular product. This enzyme is produced extensively in higher plants and fungi. The enzyme is produced by different genera of ascomycetes [132–134]; deuteromycetes [135, 136] and mainly from basidiomycetes [137]. The production and purification of biotechnological enzymes have been reviewed extensively [138–145] due to its overwhelming response. The first laccase was obtained from a Japanese lacquer tree (Rhus vernicifera), since then new fungal laccases from *Trametes versicolor*, *Polyporus pinsitus*, *Rhizocotonia solani* and from Ascomycetes *Myceliophthora* thermophila and *Sccytalidium thermophilum* etc. were obtained and studied extensively [142, 143, 145–159].

Commercially, laccases have been used for delignification of wood, production of ethyl alcohol and identification of morphine and codeine etc. among the various applications [142, 160–170]. Various delignification processes using fungi have been developed by the scientific community successfully. These enzymes were considered to be capable of Cα-Cβ cleavage of the side chain of lignin models and it was suggested that the enzymes participate in lignin degradation [171, 172]. The white rot fungi especially basidiomycetes degrade lignin in natural system more robustly than other organisms. They completely degrade lignin to carbon dioxide and water. The lignin degradation by white rot fungi was extensively studied earlier on *Phanerochaete chrysoporium* and *Sporotrichum pulverulentum* [173, 174]. The

#### *Opportunity of Non-Wood Forest Products in Biocomposites DOI: http://dx.doi.org/10.5772/intechopen.97825*

enzymes lignin peroxidase and manganese peroxidase were the first isolated from the *Phanerochaete chrysoporium* fungus culture. Lignin peroxidase is capable of ionizing non-phenolic aromatic substrates as an oxidizing peroxidase and produce aryl cation radicals [175, 176], whereas, manganese peroxidase does not degrade the non-phenolic parts of lignin in wood [177]. Further, it was observed that laccases exhibit strong catalytic activity and biotechnological applications in the bleaching of kraft pulp by depolymerising and solubilising lignin in the presence of mediator compounds [168, 178–182]. These mediator compounds were called laccase mediator systems (LMS). A number of possible mediator compounds have been searched and described for enhancing the activity of enzymes but mostly the ABTS (2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) and HBT (1-hydroxybenzotriazole) (**Figure 2e** and **f**), a derivative of benzotriazole have been used as the mediator systems [173, 183]. The oxidation of benzyl alcohols with ABTS2+ (**Figures 3** and **4**) have been experimentally proved by [182–184]. The reaction mechanism of lignin degradation with LMS is complex due to the complex nature of LMS and various reactive centres on the lignin molecule. The simple reaction mechanism was postulated by Freudenreich [185] on the non-phenolic lignin model compound, veratryl alcohol and suggested a possible mechanism of delignification of residual lignin. The scientific kinetic studies of veratryl alcohol and benzyl alcohols have also been studied in laccase mediator systems [178, 184, 186, 187].

Laccases have been found to possess catalytic ability not only to degrade lignin and in delignification process for applications in biobleaching process but also observed as their involvement in the *in vivo* lignin biosynthesis and possibly in lignification woody tissues in higher plants [188–196]. The approach of oxidation and polymerisation of lignin by the enzymes [161, 162, 197] was advanced to another biotechnological application for compounded materials using wood fibres, lignin and enzyme laccase. The high tensile strength of the woody system or biocomposites is produced by the cellulose fibres and the pressure strength is produced by the lignin matrix which is a cementing material polymerised *in situ* and held cellulose fibres together resulting in high strength to the natural composites. Cellulose and hemicelluloses (**Figure 5a**) are macromolecules composed of sugar molecules. Cellulose is composed of only glucose molecules having β-1-4 linkage (**Figure 5b**), whereas hemicelluloses composed of different sugar monomers aligned in a definite fashion. Cellulose formation by a single glucose molecule in plant cell requires four enzymes and biosynthesis of lignin composed of phenol units utilizes peroxidases and phenoloxidases (Laccases) [198–201]. The monomeric units of lignin comprised of coumaryl alcohol (H-lignin) present in grasses and agriculture crops, coniferyl alcohol (G-lignin) present in all species, dominant in conifers and syringyl alcohol (S-lignin) present in hard wood species up to 40% (**Figure 6**).

The production of composites emanates the same basic principle as the formation of natural wood: wood is processed and fragmented into fibres and small pieces as per need of the required composites. Fibres, isolated from soft or hardwood, fibre bearing species or agriculture wastes, are embedded into a matrix. The matrix or binder may be a urea-formaldehyde, phenol-formaldehyde, resorcinolformaldehyde, isocynates or in a combination as per requirement of the composites.

**Figure 3.** *Laccase-catalyzed oxidation of veratryl alcohol in the presence of a mediator.*

**Figure 4.** *Oxidation of ABTS by Laccase enzyme (blue colour,* λ*max753nm).*

The postulated theories of delignification of lignin, lignifications of woody tissues, activation of the surface of fibres possessing lignin, by the peroxidases enzymes have been successfully applied to prepare green biocomposites. The use of enzyme for bonding in the wood was first suggested by Nimz [202]. In continuum, several scientific communities have been engaged in producing biocomposites using laccase peroxidase enzyme. Wood fibres are incubated for a certain time with phenoloxidas laccase enzyme and lignin crust on the fibre surface gets activated and oxidized. Activated fibres are compressed by operating standard operating conditions of pressure, temperature etc, and binderless fibre boards may be prepared as per standards [170, 200, 201, 203–207]. The utilization of peroxidases in production of biocomposites was also applied to fibres in last two decades [208]. Cellulose fiber enzyme

*Opportunity of Non-Wood Forest Products in Biocomposites DOI: http://dx.doi.org/10.5772/intechopen.97825*

#### **Figure 5.**

*(a) Schematic diagram of cellulose and hemicelluloses in cellulose microfibrils arrangement, blue lines: cellulose; red line: hemicelluloses (b) Chemical Structure of cellulose.*

composites [207], hemp fibre reinforced composites using enzyme and chelators [209], polypropylene composites using abaca fibre [210], sisal fibre/phenolic resin composites [211], laccase-treated kenaf fibre reinforced composites with polypropylene and maleic anhydride grafted polypropylene as coupling agents [212], rubber wood fibreboards [205], laccase-mediated grafting dodecyl gallate (DG) on the jute fiber composites [213], banana/polypropylene composites [214, 215], coconut fibre composites [216], natural fiber medium density fibreboard [217], jute polypropylene composites [218, 219], flax fibre epoxy Composites [220, 221] were successfully prepared and studied for increasing mechanical properties and interfacial adhesion of the biocomposites. These all studies indicated that enzymes have the potential ability to modify the surface properties of fibres as being utilized in production of biocomposites. The formation of biocomposites has been shown in graphical representation (**Figure 7**) [163, 165, 200, 201, 203, 204, 222–225].

### **6. Opportunities and future perspectives**

In this chapter, we have underlined and discussed the different sources of natural fibres, their properties and the effect of treatments on natural fibres, etc. and further their effective use as reinforcement for polymer composite materials. Natural fibres are lucrative and worthwhile option for biocomposites. However, limitations such as poor thermal stability, moisture absorption and poor compatibility with polymeric matrices are challenges that need to be resolved.

There are a large number of fibres obtained from the natural resources; intriguingly only few of these fibres have been studied in detail for reinforcement of bio-composite materials and other industrial and traditional applications. In the present chapter popular natural fibres have been discussed as reinforced composites materials with combination of synthetic and natural polymers as modified matrix. Among the most popular natural fibres; flax, jute, hemp, sisal, ramie, and kenaf fibres were extensively studied and employed in different applications as reinforced

#### **Figure 6.**

*Monomeric units of Lignin (a) Coumaryl alcohol (H-lignin) (b) Coniferyl alcohol (G-lignin) (c) Syringyl alcohol (S-lignin).*

materials. But due to environmental and economic concern other fibres from natural resources such as pine, bagasse, pineapple leaf, coir, oil palm, banana, and agriculture residues are acquiring interest for various value added applications due to their inherent and diverse physical properties. Merits and demerits of the natural fibres and their inherent properties mainly influence the mechanical properties of bio-composites due to interfacial adhesion between the fibre and synthetic matrix.

Variability in natural fibres such as processing conditions, fibre diameter and length, lumen diameter, presence of other compounds such as amount of lignin and hemicelluloses needs to be standardising for the processing of particular fibres. High qualities of fibres are required to increase the potential of fibres as reinforcement materials. Maleated coupling agents are extensively used to enhance the composites strength using fibre as reinforcement material. These agents are used as

*Opportunity of Non-Wood Forest Products in Biocomposites DOI: http://dx.doi.org/10.5772/intechopen.97825*

#### **Figure 7.**

*Graphical representation of Biocomposites.*

couplers and bind synthetic matrix and functional surface of fibres and economical in processing. Further, using couplers the strength of fibres increased which lead to increase interfacial adhesion between two dissimilar components. However, maleated couplers illustrate superior performances with polypropylenes, polylactic acid and other polyolefins etc. Further, scientific inputs are required to improve the strength of biocomposites using maleated couplings by incorporating varied fibres.

The diverse ranges of fibres are required to investigate the quantification of residual lignin on the fiber surface and optimization of fiber isolation parameters since during the processing of fibres the amount of residual lignin may be different to the fibres isolated from the same resource. Further, the constituents and structure of lignin is also different in the fibres sourced material viz. soft and hard woods and vegetable crops and agriculture residues. Therefore, proper attention is required to investigate the activation of fibre surface and binder in the biocomposites similar to natural composites. In continuum, diverse range of appropriate laccase mediator systems (LMS) needs attention for biocomposites.

Green composites may be a suitable alternative for petroleum-based synthetic non-environment friendly materials by using enzymes especially 'laccases' one of the most ancient and efficient enzymes with promising future applications. The high reduction potential of laccases has led the vast industrial applicability, despite this, laccase potentialities are not fully exploited due to large-scale production, cost and efficiency. Systemetic progress has been made over the last three decades to enhance the utilization of laccase enzyme for various biotechnological applications and it is expected that laccases will be able to compete with other processes of bio-composites. Thus, scientific efforts are need of the hour in order to achieve the economical production of the biocatalyst, development of optimum production conditions like pH, temperature, medium composition and efficient mediator systems and further utilization in lignin activation of fibres.

In view of the above discussion, the following activities may be expedient in bio-composites development from natural resources.

• Identification and search of new fibres with better inherent properties and compatibility to the synthetic matrices.

