(535f) Utilization of Lignin Previously Depolymerized From WHITE-Rot FUNGI to Obtain Phenolic Compounds

NATALIACANO₁, ÁNGELA RUÍZ₂, CAMILO SUÁREZ₃

1Ingeniera Biológica, estudiante maestría Medio ambiente y Desarrollo, Universidad Nacional de Colombia Sede Medellín, nacanol@unal.edu.co, 2,3 Docente Escuela de Procesos y Energía, Universidad Nacional de Colombia Sede Medellín, aaruiz@unal.edu.co , camisua@gmail.com

Abstract: The white-rot fungi play an important role in the depolymerization of lignin because they produce a series of oxidative extracellular enzymes that combining efficiently are capable of completely degrades lignin to CO₂ and H₂O by biological means. The products of enzymatic depolymerization of lignin can be used as intermediates for the production of molecules with aromatic rings in its structure (eg, phenolic compounds).

Given that the lignin is the only renewable resource in the world that contains aromatic compounds and is available in large volumes, has the ability to introduce chemical functionality of these compounds to give a wide industrial application. It is necessary to do research on these processes of degradation from both standpoints: biological and traditional chemical synthesis. Additionally, comparing the lignin with other phenolic compounds, the first exists as a less toxic raw materials and lower price. These phenolics compounds constitute a large number of products and services highly demanded by society.

Keywords: Depolymerized, lignin, laccase, phenolic compounds, white rot fungi

Lignin is the third most abundant natural polymer found in nature, after cellulose and hemicellulose. It is estimated that the amount of lignin in the earth is 300 million metric tons, with an annual biosynthesis production of 20 million metric tons [1]. Its mass content depends on the vegetable species origin. In wood, lignin content varies between 19 and 35% [2].

Lignin is an amorphous polyphenolic material,generated by the copolymerization of three monomers phenyl-propanoic: alcohol coniferyl, alcohol sinaphylco, alcohol and p-coumaryl [3][4]. This three-dimensional polymer is highly branched with a variety of functional groups: phenolic hydroxyl, aliphatic hydroxyl, methoxyl, carbonyl, carboxyl and sulfonates, which provide active sites for chemical and biological interactions [5].

This material comes mainly from industrial processes such as: paper industry (50 million tons of solid per year) [6], and bioethanol production processes. This availability arouses great interest about lignin use, in order to take economic and environmental advantages.

However, the potential use of lignin is far from being exploited and most of the production is burned for energy and chemical recycling process. Only a limited number (1-2%) has been used for different applications [7].

Although lignin is the largest renewable source of aromatic polymers in nature, one of the reasons of its limited use is that lignin is a compound chemically recalcitrant to degradation by most microorganisms, because its complex heterogeneous structure. However, the white-rot fungi produce a range of extracellular oxidative enzymes (being the most common laccases and peroxidases), which are capable of combining efficiently depolymerize lignin completely to CO₂ and H₂O [8]. These types of fungi are involved in the degradation of most chemical structures that compose wood, including cellulose, even when lignin is the main compound degraded [9].

One of the advantages that white rot fungus has is the fact that these microorganisms are extremely efficient at using nitrogen, because in poor condition of it, the strain can use it, especially for the production of extracellular enzymes and essential components of the cell, [10].

Within lignollitic extracellular enzymes, there are two extracellular peroxidases: lignin peroxidase (LiP) and manganese peroxidase (MnP) that generate H₂O₂ in the system, and there are fungi that secrete a combination of peroxidases and oxidases [11]. Within oxidases that produce these fungi are laccases which does not need H₂O₂ to act.

Laccase is a polyphenol (p-diphenol oxidase), it can be inducible or constitutive and intracellular or extracellular depending on growing conditions, which catalyzes the oxidation of a variety of aromatic compounds such as mono, di and polyphenols, aminophenols, and diamines using molecular oxygen as an electron acceptor, reducing it to water [12]. This extracellular glycoprotein has four copper atoms in the oxidation state, giving them a blue color, also has the ability to interact with a redox mediator such as ABTS (2.2 '-azinobis (3 - etilbencenotiazolin 6- sulfonate) for oxidize nonphenolic compounds, which are not substrates of laccases [13]. It also degrades recalcitrant phenols in an oxidative process involving the mediator and the substrate [12]. i.e, the ABTS acts as an activator or co-oxidant of enzyme and is characterized by be a synthetic aromatic compound with nitrogen substitutions and is oxidized by laccase obtaining a stable radical cation ABTS⁺ by an interaction mechanism enzyme-mediator still unknown [13].

Thus, this enzyme can act as an oxidant-donor from an electron able to penetrate and react within the inner regions of the lignin polymer. The result is the depolymerization of ligning, by biological activity of the laccase enzyme on this material.

The products of enzymatic depolymerization of lignin can be used as intermediates for the production of molecules with aromatic rings in its structure (eg, phenolic compounds).

Given that the lignin is the only renewable resource in the world that contains aromatic compounds and is available in large volumes, has the ability to introduce chemical functionality of these compounds to give a wide industrial application. It is necessary to do research on these processes of degradation from both standpoints: biological and traditional chemical synthesis. Additionally, comparing the lignin with other phenolic compounds, the first exists as a less toxic raw materials and lower price.

This way, this study aimed  the isolation of the white-rot fungi from soils of Urabá - Colombia(With potencial lignolytic) with the purpose of determining the ability of these strains to produce the enzyme of interest (laccase) under a variety of culture mediums referenced in the literature for this purpose, by testing coloring cualitative 2,2’-azino-bis(3-etilbenzotiazolin-6- ácido sulfónico) (ABTS). The enzymatic activity was measured and the time of the highest laccase activity under specific conditions of fermentation was determined

After obtaining the enzyme cocktail, it was applied on commercial lignin for identification and quantification of phenolic compounds, formed from the biological pathway of lignin degradation by the enzymatic action of laccases. These kind of phenolic compounds constitute a large number of products and services highly demanded by society (Phenolic resin for industrial abrasives, manufacture of alkyl phenols to additives for lubricating oils, industry Adhesives such as wood and shoe-, Resins for decorative and industrial laminates, insulating varnishes drivers, conservative cosmetic additives, Manufacture of dyes), therefore, this source is considered as an attractive economic and environmental alternative .

The methodology has been organized based on the approach of the overall. The design of the project is done so in 3 stages of development, first  tests are performed with enzymes extracted from white-rot fungi (laccases) to determine, by quantitative and qualitative methods, enzymatic activity of these enzymes on lignin (using spectrophotometric technique). Second stage is the depolymerization of lignin and characterization of products formed from the biological degradation of lignin (GC-MS technique) and the third stage faces the industrial application of the products obtained.

REFERENCES

[1] CRESTINI, C., SERMANNI, G.G. AND ARGYROPOULOS D.S. Structural Modifications Induced During Biodegradation of Wheat Lignin by Lentinula edodes: Bioorganic & Medicinal Chemistry. 6, 967- 973,1998.

[2] DENCE, C.W. and LIN, S.Y. General structural features of lignin in: Lin, S.Y., Dence, C.W. (Eds.), Methods in Lignin Chemistry, Springer-Verlag, Berlin Heidelberg, 3–6. 1992.

[3] WANG, M., LEITCH, M. AND XU, C. Synthesis of phenol–formaldehyde resol resins using organosolv pine lignins: European Polymer. 45, 3380–3388, 2009.

[4] JOLIVETA, C., GUILLET, B., KARROUMB, M., ANDREUX, F., BERNOUXD, M. AND ARROUAYSA, D. Les phénols de la lignine et le 13C, traceurs de l’origine des matières organiques du sol: Sciences de la Terre et des planètes / Earth and Planetary Sciences, 651–657, 2001.

[5] VÁZQUEZ, G., ANTORRENA, G., GONZÁLEZ & J. AND MAYOR, J. lignin-phenol-formaldehyde adhesives for exterior grade plywoods: Bioresource Technology . 51,187-192, 1995.

[6] GOSSELINK, R.J.A., DE JONG, E., GURAN, B. AND ABÄCHERLI A. Co-ordination network for lignin-Standardization, production and applications adapted to market requirements (EUROLIGNIN): Industrial Crops and Products. 20,121-129, 2004.

[7] LORA, J.H. AND GLASSER, W.G. Recent application of lignin: A sustainable alternative to nonrenewable materials. J. Polym. Environ. 10, 39-48, 2002.

[8] HAKALAA, T.K., LUNDELLA, T., GALKINA, S., MAIJALAA, P., KALKKINENB, N. AND HATAKKAA, A. Manganese peroxidases, laccases and oxalic acid from the selective white-rot fungus Physisporinus rivulosus grown on spruce wood chips: Enzyme and Microbial Technology. 36, 461–468, 2005.

[9] DOWSON, C.G., RAYNER, A.D.M. AND BODDY, L. Foraging patterns of Phallus impudicus, Phanerochaete laevis and Steccherinum fimbriatum between discontinuous resource units in soil: FEMS Microbiology Letters. 53, 291-298, 1998.

[10] DEACON, J.W. En: Introducción a la Micología Moderna (Editorial Limusa.Primera edición). México, 13-41, 1993.

[11] PERI, F.H. AND GOLD, M.H. Manganese regulation of manganese peroxidase expession and lignin degradation by the white-rot fungus Dichomitus squalens: Appl. Environ. Microbiol. 57,2240-2245, 1991.

[12] RODAKIEWICZ-NOWAK, J. Phenols oxidizing enzymes in water-restricted media: Topics in Catalysis. 11, 419-434, 2000.

[13] BOURBONNAIS, R. and PAICE, M. G. Oxidation of non-phenolic substrates An expanded role for laccase in lignin biodegradation: FEBS Letters. 267, 99-106, 1990.