*2.3.3 Lignin-derived aromatic compounds breaking down by microorganisms*

Low molecular weight aromatic compounds are obtained after fungal lignin depolymerization, such as guaiacol, coniferyl alcohol, p-coumarate, ferulate, protocatechuate, p-hydroxybenzoate, and vanillate [57]. Bacteria have the enzymatic machinery to metabolize-derived aromatic compounds that could allow the generation of value-added products such as flavors, polymer building blocks, and energy storage compounds (**Figure 1**). *R. opacus* DSM 1068 and PD630 strains were able to convert lignin into triacylglycerols under nitrogen-limiting conditions [58].

*P. paucimobilis* is able to metabolize β-aryl ether lignin dimer compounds to yield vanillic acid [59]. In the catecholic compounds production, *O*-demethylation is an essential process with ring cleavage catalyzed by dioxygenase [60]. *Sphingobium* is a bacterial genus characterized for the catabolism of lignin-derived aromatic compounds sp. being able to produce protocatechuate/gallate and 3-*O*-methylgallate [61]. While *Ralstonia eutropha* strain H16 was able to synthesize the biopolyester

**43**

*Getting Environmentally Friendly and High Added-Value Products from Lignocellulosic Waste*

polyhydroxyalkanoate (PHA) from lignin derivates [62], *Pandorea* sp. ISTKB converts lignin and its derivates into a value-added product PHA [63].

Fungal and bacterial lignin degraders (BLD) depolymerize the lignocellulosic residues, thus obtaining hemicellulose and cellulose that can be used to produce biocomposites or biofuels and lignin-derived aromatic compounds which can be

Biological lignin degradation process does not involve high temperatures and pressures and does not generate any undesirable products. However, it is a timeconsuming process, and there is not an accurate control on it [64]. Long time is necessary to achieve microbial lignin degradation that can range from 10 to 100 days, which is not suitable for commercial applications [28]. Several efforts have been made to engineer microorganisms in order to be more efficient to metabolize lignin-derived compounds with remarkable biotechnological applications, such as pretreatment of lignocellulosics, pulping and bleaching in the paper industry, and decolorization in the textile industry [49]. *Yarrowia lipolytica* was transformed with laccases genes from *Pycnoporus cinnabarinus* offering an efficient model for the engineering of laccases with industrial applications [65]. A dye-decolorizing peroxidase from *P. putida* MET94 strain was engineered to enhance 100-fold the catalytic efficiency when oxidizing phenolic lignin model substrates [66]. On the other hand, multi-copy recombinant *Pichia pastoris* strain expressed lignin peroxidase from *P. chrysosporium* reaching a maximum activity after 12 h induction [67]. Systems among ligninolytic microorganisms and enzymes demonstrate an enormous poten-

The application of enzymes is an attractive alternative due to its shortened time, improved yield, and simple processing [69]. The most common enzymes used to break down lignin are peroxidase and laccase, catalyzing lignin oxidation. Among the most studied peroxidases are lignin peroxidases and manganese-dependent peroxidases. These enzymes degrade lignin randomly converting the phenolic group to free radicals, which lead to lignin depolymerization [70]. Fungal peroxidase from *P. ostreatus* shown lignin degradation at 30°C and pH 4 yielding 2,6-dimethoxy-1,4-benzoquinone, benzoic acid, butyl phthalate, and bis(2-ethylhexyl) phthalate [71]. Laccase can be isolated from fungi and bacteria; it is able to oxidize phenolic compounds; however, it can cooperate with mediators (small molecules able to transfer an electron) to degrade nonphenolic compounds [72]. In spite fungal laccases are selected, not only bacterial laccases have higher thermostability and an extended pH range of use but also they represent a good alternative to lignin depolymerization [73]. The company MetGen Oy has designed the enzyme MetZyme® LIGNO™, a genetically laccase of bacteria origin that can perform its activity in extremely alkaline pH and at elevated temperatures [74]. The enzyme immobilization has also been attempted to improve product separation and catalyzation because the enzymes can be made reusable through techniques such as cross-linking of enzymes, immobilization onto nanomaterials, or entrapping on beads [75]. Laccases from *Fomes fomentarius* and *T. versicolor* were cross-linked showing higher catalytic efficiency, stabilities, and high reusability compared with the free laccase [76]. Other efforts have been made to design multienzyme biocatalysts to improve stability and efficiency of lignin degradation. Co-immobilization of laccase and horseradish peroxidase by cross-

linking maintains their activity and improves enzyme stability [77].

*DOI: http://dx.doi.org/10.5772/intechopen.93645*

transformed by bacteria to value-added bioproducts.

*2.3.4 Challenges in microbial lignin degradation*

tial to enhance the lignin degradation [68].

*2.3.5 Purified enzymes*

**Figure 1.** *Biorefinery based on microbial pretreatment in lignocellulosic residues.*

#### *Getting Environmentally Friendly and High Added-Value Products from Lignocellulosic Waste DOI: http://dx.doi.org/10.5772/intechopen.93645*

polyhydroxyalkanoate (PHA) from lignin derivates [62], *Pandorea* sp. ISTKB converts lignin and its derivates into a value-added product PHA [63].

Fungal and bacterial lignin degraders (BLD) depolymerize the lignocellulosic residues, thus obtaining hemicellulose and cellulose that can be used to produce biocomposites or biofuels and lignin-derived aromatic compounds which can be transformed by bacteria to value-added bioproducts.

### *2.3.4 Challenges in microbial lignin degradation*

Biological lignin degradation process does not involve high temperatures and pressures and does not generate any undesirable products. However, it is a timeconsuming process, and there is not an accurate control on it [64]. Long time is necessary to achieve microbial lignin degradation that can range from 10 to 100 days, which is not suitable for commercial applications [28]. Several efforts have been made to engineer microorganisms in order to be more efficient to metabolize lignin-derived compounds with remarkable biotechnological applications, such as pretreatment of lignocellulosics, pulping and bleaching in the paper industry, and decolorization in the textile industry [49]. *Yarrowia lipolytica* was transformed with laccases genes from *Pycnoporus cinnabarinus* offering an efficient model for the engineering of laccases with industrial applications [65]. A dye-decolorizing peroxidase from *P. putida* MET94 strain was engineered to enhance 100-fold the catalytic efficiency when oxidizing phenolic lignin model substrates [66]. On the other hand, multi-copy recombinant *Pichia pastoris* strain expressed lignin peroxidase from *P. chrysosporium* reaching a maximum activity after 12 h induction [67]. Systems among ligninolytic microorganisms and enzymes demonstrate an enormous potential to enhance the lignin degradation [68].

#### *2.3.5 Purified enzymes*

*Biotechnological Applications of Biomass*

being the first bacterial laccases identified in *Azospirillum lipoferum* [54]. Many soil bacteria, actinobacteria, and α-, β-, and γ-proteobacteria have shown bacterial laccase genes [55]. A higher laccase production was reached by the bacteria *Streptomyces* sp. KS1025A compared with white-rot fungi in reduced time [56].

Low molecular weight aromatic compounds are obtained after fungal lignin depolymerization, such as guaiacol, coniferyl alcohol, p-coumarate, ferulate, protocatechuate, p-hydroxybenzoate, and vanillate [57]. Bacteria have the enzymatic machinery to metabolize-derived aromatic compounds that could allow the generation of value-added products such as flavors, polymer building blocks, and energy storage compounds (**Figure 1**). *R. opacus* DSM 1068 and PD630 strains were able to

*P. paucimobilis* is able to metabolize β-aryl ether lignin dimer compounds to yield vanillic acid [59]. In the catecholic compounds production, *O*-demethylation is an essential process with ring cleavage catalyzed by dioxygenase [60]. *Sphingobium* is a bacterial genus characterized for the catabolism of lignin-derived aromatic compounds sp. being able to produce protocatechuate/gallate and 3-*O*-methylgallate [61]. While *Ralstonia eutropha* strain H16 was able to synthesize the biopolyester

*2.3.3 Lignin-derived aromatic compounds breaking down by microorganisms*

convert lignin into triacylglycerols under nitrogen-limiting conditions [58].

**42**

**Figure 1.**

*Biorefinery based on microbial pretreatment in lignocellulosic residues.*

The application of enzymes is an attractive alternative due to its shortened time, improved yield, and simple processing [69]. The most common enzymes used to break down lignin are peroxidase and laccase, catalyzing lignin oxidation. Among the most studied peroxidases are lignin peroxidases and manganese-dependent peroxidases. These enzymes degrade lignin randomly converting the phenolic group to free radicals, which lead to lignin depolymerization [70]. Fungal peroxidase from *P. ostreatus* shown lignin degradation at 30°C and pH 4 yielding 2,6-dimethoxy-1,4-benzoquinone, benzoic acid, butyl phthalate, and bis(2-ethylhexyl) phthalate [71]. Laccase can be isolated from fungi and bacteria; it is able to oxidize phenolic compounds; however, it can cooperate with mediators (small molecules able to transfer an electron) to degrade nonphenolic compounds [72]. In spite fungal laccases are selected, not only bacterial laccases have higher thermostability and an extended pH range of use but also they represent a good alternative to lignin depolymerization [73]. The company MetGen Oy has designed the enzyme MetZyme® LIGNO™, a genetically laccase of bacteria origin that can perform its activity in extremely alkaline pH and at elevated temperatures [74]. The enzyme immobilization has also been attempted to improve product separation and catalyzation because the enzymes can be made reusable through techniques such as cross-linking of enzymes, immobilization onto nanomaterials, or entrapping on beads [75]. Laccases from *Fomes fomentarius* and *T. versicolor* were cross-linked showing higher catalytic efficiency, stabilities, and high reusability compared with the free laccase [76]. Other efforts have been made to design multienzyme biocatalysts to improve stability and efficiency of lignin degradation. Co-immobilization of laccase and horseradish peroxidase by crosslinking maintains their activity and improves enzyme stability [77].
