Appl Microbiol Biotechnol DOI 10.1007/s00253-014-5810-8

MINI-REVIEW

Laccase applications in biofuels production: current status and future prospects Tukayi Kudanga & Marilize Le Roes-Hill

Received: 21 February 2014 / Revised: 30 April 2014 / Accepted: 1 May 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract The desire to reduce dependence on the ever diminishing fossil fuel reserves coupled with the impetus towards green energy has seen increased research in biofuels as alternative sources of energy. Lignocellulose materials are one of the most promising feedstocks for advanced biofuels production. However, their utilisation is dependent on the efficient hydrolysis of polysaccharides, which in part is dependent on cost-effective and benign pretreatment of biomass to remove or modify lignin and release or expose sugars to hydrolytic enzymes. Laccase is one of the enzymes that are being investigated not only for potential use as pretreatment agents in biofuel production, mainly as a delignifying enzyme, but also as a biotechnological tool for removal of inhibitors (mainly phenolic) of subsequent enzymatic processes. The current review discusses the major advances in the application of laccase as a potential pretreatment strategy, the underlying principles as well as directions for future research in the search for better enzyme-based technologies for biofuel production. Future perspectives could include synergy between enzymes that may be required for optimal results and the adoption of the biorefinery concept in line with the move towards the global implementation of the bioeconomy strategy.

Keywords Laccase . Lignocellulose . Pretreatment . Biofuels

T. Kudanga (*) : M. Le Roes-Hill Biocatalysis and Technical Biology Research Group, Cape Peninsula University of Technology, PO Box 1906, Bellville 7535, South Africa e-mail: [email protected] T. Kudanga e-mail: [email protected]

Introduction Physical and chemical processes have been, and continue to be, an integral aspect of modern life. This is mainly due to their utility in the conversion of raw materials into valueadded products widely available on the market. However, in recent years, there has been a growing interest in the use of bio-based processes, which is attributable to the increasing evidence of their advantages as alternatives to physicochemical processes and in some cases, the fact that they may be the only viable option. Unlike physico-chemical processes, the bio-based processes are considered more efficient, costeffective, environmentally friendly and sustainable; mostly because they are not overly reliant on non-renewable resources such as fossil fuels. A combination of punitive action and legislations introduced to improve protection of human health and the environment, pressure from environmental protection lobbyists and conscious proactive efforts, have seen an upsurge in the search for alternative biotechnological processes. Consequently, biotechnology, and in particular biocatalysis, is now established as an important option in many applications. The main success factors for biocatalytic routes include the selectivity of the catalyst, the efficiency of the process (Burton et al. 2002), potential reusability of the enzymes and their biodegradability (once no longer needed), and general environmental considerations (Pollard and Woodley 2007). Thus, many industries are currently pursuing enzymatic approaches for the development of green chemistry technologies. Laccases are among the enzymes which are being intensively investigated for various biotechnological applications. Laccase (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) is a multi-copper-containing enzyme which performs one-electron oxidation of various substrates such as diphenols, methoxy-substituted monophenols, as well as aromatic and aliphatic amines to form radicals and at the same

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time reduces molecular oxygen to water (Kudanga et al. 2011a). Laccase-generated radicals undergo a number of enzymatic or non-enzymatic reactions, which have implications in many industries. Coupling of the radicals to form oligomers has been applied in organic synthesis, whereas polymerisation to form insoluble compounds (which can be easily removed by filtration or sedimentation) has been utilised in bioremediation. The radicals can also participate in the degradation of complex polymers by cleavage of covalent bonds especially alkyl–aryl bonds (sometimes in the presence of mediators), releasing monomers (Breen and Singleton 1999) and in the ring cleavage of aromatic compounds (Claus et al. 2002; Durán and Esposito 2000; Kawai et al. 1988b). These processes have potential application in the degradation of lignin and xenobiotic compounds and therefore have implication in the paper and pulp industry, textile industry, biofuel industry and in bioremediation, among others. The broad substrate range of laccase, which is further widened by the use of a laccase-mediator system (LMS), allows the enzyme to be applied in many industries. Currently, the main applications are in the pulp and paper industry, textile industry and in bioremediation while application potential has been demonstrated for the food industry, pharmaceutical industry, organic synthesis, lignocellulose modification, biofuels production

and diagnostic purposes (Burton and Le Roes-Hill 2008). While most of these applications have been reviewed (Table 1), the potential of the enzyme in biofuel production has not been comprehensively reviewed in recent articles. Recent reviews on the pretreatment strategies have focused mainly on physico-chemical processes (Mood et al. 2013; Bensah and Mensah 2013; Talebnia et al. 2010; Alvira et al. 2010; Hendriks and Zeeman 2009; Mosier et al. 2005) while biotechnological processes have been confined to detoxification processes in the broad sense of application of all potentially relevant enzymes and whole cells (Parawira and Tekere 2011). The main advantages of using enzymes over whole cells include the possibility of using higher temperatures which are not necessarily optimal for the growth of the microorganisms, high catalytic efficiencies of the enzymes and low utility costs associated with the use of mild conditions. However, there are some disadvantages which include prolonged incubation periods which may be required, high enzyme production costs despite the possibility of recombinant production of laccases (Parawira and Tekere 2011) and missed opportunity of using a combination of enzymes available in whole cells. In nature, various enzymes (laccases and peroxidases) produced by whole cells are involved in the degradation of lignin, cumulatively generating unstable

Table 1 Reviewed applications of laccases Application

Main reactions

Pulp and paper industry

Oxidative delignification (mainly using LMS)

Relevant reviews

Arora and Sharma (2010), Bourbonnais et al. (2001), Breen and Singleton (1999), Cañas and Camarero (2010), Kunamneni et al. (2008b), Mayer and Staples (2002), Nyanhongo et al. (2007), Rodríguez-Couto and TocaHerrera (2006), Thurston (1994) and Widsten and Kandelbauer (2008) Textile industry Oxidative delignification (mainly using LMS) and Arora and Sharma (2010), Kudanga et al. (2011b), Kunamneni oxidative degradation et al. (2008b) and Rodríguez-Couto and Toca-Herrera (2006) Food industry Coupling/crosslinking reactions Brijwani et al. (2010), Kudanga et al. (2011b), Kunamneni et al. (2008b), Minussi et al. (2002) and Osma et al. (2010) Wood products and modification Coupling and grafting and co-polymerisation Kudanga et al. (2011b) and Widsten and Kandelbauer (2008) of lignocellulose materials Bioremediation Oxidative degradation (usually in the presence Arora and Sharma (2010), Cañas and Camarero (2010), Durán of mediators); coupling coupled with and Esposito (2000), Durán et al. (2002), Kudanga et al. polymerisation, demethylation or (2011a, b), Kunamneni et al. (2008b), Majeau et al. (2010), dechlorination; and oxidative cleavage Mayer and Staples (2002) Nyanhongo et al. (2007), Rodríguez-Couto and Toca-Herrera (2006) and Strong and Claus (2011) Organic synthesis Oxidative oligomerisation Durán et al. (2002), Kunamneni et al. (2008a, b), Mikolasch and Schauer (2009); Riva (2006), Rodríguez-Couto and Toca-Herrera (2006) and Witayakran and Ragauskas (2009) Pharmaceutical industry Oxidative oligomerisation Arora and Sharma (2010), Kudanga et al. (2011b), Kunamneni et al. (2008b), Mikolasch and Schauer (2009) and Nyanhongo et al. (2007) Biosensors and other Oxidation Arora and Sharma (2010), Durán et al. (2002), Kunamneni bio-analytical purposes et al. (2008a), Mayer and Staples (2002), Nyanhongo et al. (2007) and Rodríguez-Couto and Toca-Herrera (2006)

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radicals that can attack the lignin component in a process often referred to as enzymatic combustion (Pérez et al. 2002). While other oxidase enzymes such as lignin peroxidases, manganese peroxidases and versatile peroxidases can also play a key role in lignin biodegradation and therefore biofuels production, these enzymes do, however, require the presence of cofactors, e.g. H2O2 and Mn(II) to catalyse the reaction, which makes the process expensive and environmentally unfriendly. On the contrary, laccases are essentially ‘green’ catalysts; they require only air (oxygen) as co-substrate, producing water as the only by-product. Therefore the main aim of this article is to provide a focused comprehensive review of the application potential of laccases in the production of biofuels, mainly as a delignifying enzyme to facilitate access of hydrolytic enzymes to carbohydrate moieties and as a detoxifying enzyme for removal of inhibitory phenolic compounds from lignocellulose hydrolysates, as well as providing the mode of action of the enzyme in each case. In addition, the review provides directions for future research as a way of highlighting future prospects of the enzyme in biofuels production.

Laccase: sources, structure and function Laccase was first isolated from sap of the Japanese lacquer tree Rhus vernicifera (Yoshida 1883) and is now widely regarded as a ubiquitous enzyme which has been isolated from higher plants (Dwivedi et al. 2011), insects (Kramer et al. 2001), invertebrates, specifically oysters, mussels and other bivalves (Luna-Acosta et al. 2010), archaea (Uthandi et al. 2010), prokaryotes (Claus 2003) and is widespread in fungi (Baldrian 2006). Structurally, laccase is a glycosylated monomer or homodimer and generally laccase of fungal or bacterial origin is less glycosylated (10–25 %) than those of plant origin. The carbohydrate component is believed to enhance stability of the enzyme, and its main building units are glucose, hexoamines, fucose, mannose, arabinose and galactose. Each functional laccase monomeric unit consists of four copper atoms namely type 1 copper (T1), type 2 copper (T2) and two type 3 copper (T3) atoms. The T1 copper is the site where oneelectron oxidation of substrates takes place and therefore acts as the primary electron acceptor. The two T3 copper atoms and T2 copper form a trinulear site where molecular oxygen is reduced to water. It is widely believed that the axial ligand in the T1 copper influences its redox potential. Recently, a second group of laccases with two copper atoms per molecule which work in concert with pyrolloquinoline quinone (PQQ) factor has been reported (Rogalski and Leonowicz 2004). These laccases show high N-terminus amino acid sequence homology with blue laccases but do not show the typical spectroscopic and paramagnetic properties of blue laccases (Leontievsky et al. 1997a). They are yellow-brown in colour

and are now frequently referred to as yellow laccases. The oxidation state of the copper in their active site appears to be different from that of blue laccases and there have been reports that, unlike blue laccases, they can oxidise non-phenolic lignin models and veratryl alcohol in the absence of mediators (Leontievsky et al. 1997b; Giardina et al. 2010). They are therefore interesting candidates in the search for laccases for use in lignin degradation with potential for biofuel production. A third type of laccase, the white laccase, has been identified in various fungal strains (Giardina et al. 2010). The white laccase from Trametes hirsuta, as in the case of the yellow laccases, is able to oxidise complex dyes in the absence of mediators and therefore also show potential for application in biofuel production. The three-dimensional structure of fungal laccases has been elucidated by a number of researchers and has been reviewed by Morozova et al. (2007a). Generally, the structure is organised as monomeric units consisting of three sequentially arranged domains of a barrel-type structure. Recently, it was confirmed that fungal laccases show no evidence of higherorder oligomeric assemblies in the crystal lattice and essentially exist as monomers (Ge et al. 2010). The T1 copper site of laccases is located in the third domain, and the tri-nuclear cluster T2/T3 is located between the first and third domains while the amino acid residues of the second and third domains are involved in formation of the substrate-binding pocket (Morozova et al. 2007a). Although laccases are typically three-domain monomeric enzymes, a small four-copper laccase was identified from Streptomyces coelicolor A3(2) (Machczynski et al. 2004) and its three-dimensional structure shows that it consists of only two domains, which are similar to domains 1 and 3 of the classical fungal and plant laccases (Skálová et al. 2009). Unlike typical laccases, this two domain laccase named small laccase, because of its size, is active in the oligomeric form as dimers (Machczynski et al. 2004) or more frequently trimers (Skálová et al. 2009). The trimeric organisation of the enzyme and relative proximity of the type 1 copper ions bring the three substrate binding sites close in space effectively forming a shallow trimeric substrate binding site (Skálová et al. 2009). This organisation highlights the importance of a stable trimeric structure since the active sites are placed between the individual protein chains. Laccase-catalysed oxidation proceeds through the direct interaction with the substrate or indirectly via the oxidation of a substrate by a laccase oxidised mediator. With direct oxidation the substrate is oxidised by one electron oxidation at the T1 copper atom to form an unstable reactive radical. After four cycles of one-electron oxidation, the electrons are transferred to the trinuclear centre where they reduce one oxygen molecule to two molecules of water (Kudanga et al. 2011b; Riva 2006). However, some potential substrates cannot be oxidised directly because they are either too large to enter the laccase active site or they have a higher redox

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potential than the enzyme. Such substrates can be oxidised using a LMS in which a small, low redox potential substrate is first oxidised by laccase and then its radical can be utilised for the oxidation of complex or high redox potential substrates. The LMS could be particularly important in the use of blue laccases in biofuel production due to high redox potential of substrates in particular the non-phenolic moieties, and inaccessibility of active site by complex lignocellulosic substrates. However, many laccase mediators are either toxic or expensive, which has necessitated the search for natural mediators.

Search for natural mediators As mentioned previously, the group of laccases often referred to as the blue laccases, typically require the presence of small compounds (mediators) to oxidise compounds that are considered to be atypical substrates for laccases, e.g. complex biopolymers such as lignin (Desai and Nityanand 2011; Kunamneni et al. 2008b). The molecule 2,2′-azino-bis-(3ethyl-benzothizoline-6-sulphonic acid) (ABTS) was the first mediator described and was used in the application of laccases in the bio-bleaching of wood pulps (Madhavi and Lele 2009). With the development of the LMS, laccases have been successfully applied in the oxidation of aromatic methyl groups, benzyl alcohols, as well as in bioremediation such as the removal of pesticides, polycyclic aromatic hydrocarbons (PAH), explosives and chemical warfare agents, and in the bleaching of various textile dyes (Madhavi and Lele 2009; Torres-Duarte et al. 2009; Trovaslet-Leroy et al. 2010). It is therefore conceivable that the LMS have huge potential in biofuels production. There are two main categories of mediators: synthetic and natural (Fig. 1a, b) (Cañas and Camarero 2010). To date, more than 100 synthetic mediators have been identified and their effects on laccase activity described. Among these, ABTS, 1hydroxybenzotriazole (HBT), violuric acid (VIO), Nhydroxyphthalimide (HPI), N-hydroxyacetanilide (NHA), 2,2,6,6,-tetramethyl-1-piperidinyloxy-free radical (TEMPO), phenothiazines and other heterocycles have been extensively studied (Kunamneni et al. 2008b). As pointed out earlier, these synthetic mediators have two major drawbacks: (1) They are expensive and therefore not feasible for application at industrial scale, and (2) They can cause the formation of toxic derivatives that may result in the inactivation of laccases. There has therefore been an increased interest in the identification and utilisation of cheaper, eco-friendly natural mediators (Kunamneni et al. 2008b), with the main focus of identifying natural mediators from renewable sources (such as industrial by-products) and from plants (Johannes and Majcherczyk 2000; Moldes et al. 2008; Vila et al. 2011).

Natural mediators include 4-hydroxybenzoic acid, 4hydroxybenzyl alcohol, aniline, phenol and phenolic compounds derived from lignin degradation such as acetosyringone, acetovanillone, p-coumaric acid, ferulic acid, syringaldehyde and vanillin (Kunamneni et al. 2008b). It has been determined that natural mediators act very similar to synthetic mediators and sometimes have a greater effect (Fillat et al. 2010). Lignin-derived phenolic mediators have successfully been applied to fungal laccase reactions involving dye decolourisation, delignification of paper pulp from wood and non-wood fibres, delignification of flax pulp and kenaf pulp, delignification of Eucalyptus wood chips, removal of lipophilic extractives and the oxidation of PAH (Andreu and Vidal 2011; Fillat et al. 2010; Rico et al. 2014). However, one potential drawback is that in some instances, an opposite effect can be achieved when using natural mediators vs synthetic mediators, e.g. Vila et al. (2011) reported on a laccase that acted as a polymerising agent in the presence of natural mediators, while acting as a delignifying agent in the presence of a synthetic mediator. Aracri et al. (2009) reported a similar result in their study on the bleaching of pulp and effluents obtained from sisal pulp. In this study, natural mediators resulted in laccase-mediated coupling reactions rather than bleaching observed in the presence of synthetic mediators. This could compromise the use of the mediators in biofuel production. Such mediators may be useful in laccase-assisted biografting applications (Aracri et al. 2009). Indeed, we have observed that lignin model compounds guaiacylglycerol βguaiacyl ether (erol) and syringylglycerol β-guaiacyl ether acted as mediators during laccase assisted coupling of fluorophenols to the models (Kudanga et al. 2010c).

Lignin: the target component in laccase-assisted biofuel production Lignin is one of the three major components of lignocellose materials comprising (18–35 %); the other ones being cellulose (40–50 %) and hemicellulose (15–25 %). It is thus the second most abundant organic polymer on Earth, representing a huge reserve of non-fossil carbon which at the moment is heavily underutilised. Structurally, lignin is a heterogeneous, optically inactive, three-dimensional polymer of hydroxylated and methoxylated phenylpropane units, linked in an irregular manner through oxidative coupling to form ether and C-C bonds. It is the binding agent which holds cells together, giving rigidity to the cell. Its complex and hydrophobic nature and ability to act as an encrusting agent on and around the carbohydrate fraction, limits enzymatic hydrolysis of the carbohydrate fraction hence the need for pretreatment when converting biomass to fuels such as bioethanol (Asgher et al. 2014) (Fig. 2). Apart from lignin content, other prominent

Appl Microbiol Biotechnol Fig. 1 Examples of natural (a) and synthetic (b) laccase mediators. HPI Nhydroxyphthalimide, TEMPO 2,2′,6,6′-tetramethyl-piperidineN-oxyl, SPP-m 1-(3′sulphophenyl)-3-methylpyrazolone-5, HBT 1hydroxybenzotriazole, ABTS 2,2′-azino-bis-(3-ethylbenzothiazoline-6 sulphonic acid), NHA N-hydroxyacetanilide

a COCH 3

MeO

OMe

HO

OH

CH 2OH

OH

O

H

OH

OH

Vanillyl alcohol

O

OMe

OMe

OH

OH

HO

MeO

OMe

OH

Gallic acid

Acetosyringone

COOH

H

O

Syringic acid

Vanillin

COCH 3

OH

OH

O

O

OH

NH 2

OH

MeO

OMe

OMe OH

OH

p-coumaric acid

OH

3-hydroxyanthranilic acid Catechol

Syringaldehyde Acetovanillone CHO

OMe

OH

MeO MeO

OMe OH

MeO

OH

OH

2,6-dimethylphenol

O

HO

OCH 2CH 3

OMe

MeO

2,4,6-trimethylphenol Ethyl vanillin

Sinapic acid OH

OH O

O HO

MeO

OH

O

OH

HO MeO

4-hydroxybenzoic acid

Ferulic acid

Cinnamic acid

OH

Coniferyl alcohol

b

H3C

O N OH

O

N

H3C

N

CH3 N

H3C

OH

H

CH3

N

O

O

O

N

N

OH

O

H

SO3H

TEMPO

HPI

SPP-m

Violuric acid OH

H 3C

N

O

N N

N

OH

OH

OH

HBT

NHA

S O O

O

O

HO S

O

S

O

N N

N

+

S

OH S

Phenol red

O N

H3C

CH3

ABTS

lignin-related factors that impact biomass conversion may include lignin composition, its chemical structures and lig nin-ca rb ohydrate co mplex (LCC) linkage s in

lignocellulosic biomass (Pu et al. 2013). There are three monolignol monomers; p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol (Fig. 3a–c), which are connected

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Fig. 2 Lignocellulose model structure, delignification strategies, and subsequent steps in the production of bioethanol (Adapted from Du et al. 2013b; Asgher et al. 2014 with permission from ACS Publications and Elsevier)

through radical coupling to form inter-unit linkages such as βO-4, β-5, 5-5, β-β, 5-O-4 and the more recently discovered dibenzodioxocin 5-5-O-4 (Karhunen et al. 1995a, b) (Fig. 3d– k). The first comprehensive model of softwood lignin structure was developed by Sakakibara (1980) based upon the degradation products formed from lignin samples as a result of hydrolysis in aqueous dioxane and catalytic hydrogenolysis. According to the Sakakibara model, the most prevalent interunit linkages were thought to be the β-O-4 alkyl aryl ether, 5-5 biphenyl, β-5 and β1 alkyl arene and α-O-4 benzyl aryl ether linkages. However, recent developments have shown that there are very low levels, if any of β-1 and non-cyclic α-O-4 linkages (Kilpeläinen et al. 1994; Ede and Kilpeläinen 1995)

and that many of the 5-5-biphenyl inter-unit linkages are etherified with phenylpropanoid units to form 5-5-O-4 dibenzodioxocin structures (Karhunen et al. 1995a, b). Consequently, a new improved model, which took into account the new knowledge and the proposed structures involved in the formation of lignin-carbohydrate complexes, was developed (Chen and Sarkanen 2003, 2010) (Fig. 4). Lignin carbohydrate complexes are formed when lignin is covalently linked to hemicellulose. Xylan, glucan and glucomannan residues appear to be the major interface between lignin and the carbohydrate components (Du 2013). For example spruce wood was found to consist of 49.5 % glucanlignin, 30.9 % glucomannan–lignin and 12.0 % xylan–lignin (Du 2013) while benzyl ether, γ-ester and phenyl glycoside

Appl Microbiol Biotechnol Fig. 3 Lignin precursor molecules (a–c) and common linkages in lignin (d–k)

LCC bonds have been reported as the predominant linkages between lignin and carbohydrates (Du 2013; Balakshin et al. 2011). An interesting observation on gymnosperm lignin is that the lignin residues that are bound primarily to xylans are composed almost exclusively of β-O-4 alkyl aryl ether substructures while those which are predominantly attached to glucomannans, are thought to encompass all the other substructures with a lower proportion of β-O-4 ethers (Chen and Sarkanen 2010) (Fig. 4). However, recent research on spruce wood has shown that lignin in glucomannan lignin has a higher content of β-O-4 ether substructures (62 %) than in xylan–lignin complexes (53 %) (Du et al. 2014). It has been suggested that it is not just the lignin content that effects recalcitrance of lignocellulosic materials, but rather and possibly more importantly, the integration of lignin and polysaccharides within the cell wall, and their associations with one another and with other wall components (DeMartini et al. 2011). Therefore the cleavage of the linkages between lignin and carbohydrates in LCCs should be an important consideration in the pretreatment of biomass for biofuel production.

Application of laccases in biofuel production The use of second-generation biofuels such as biogas and bioethanol is generally regarded as ‘greener’ because they are produced from sustainable feedstocks. Biogas is produced in an anaerobic process in which organic matter is converted into methane and carbon dioxide. The process involves four main phases namely pretreatment, hydrolysis, acidogenesis and acetogenesis, and methane fermentation. In the first phase, pretreatment is performed to improve the hydrolysis yield and consequently total methane yield. The hydrolysis step, which is carried out by obligate anaerobes such as Bacteroides spp., Clostridium spp. and facultative bacteria such as Streptococcus spp., involves enzymatic breakdown of insoluble organic material and higher molecular mass compounds such as lipids, polysaccharides, proteins, fats and nucleic acids into soluble organic materials such as monosaccharides, amino acids and other simple organic compounds. In the third step, primary fermenting bacteria (obligate and facultative anaerobes) convert the products of hydrolysis into volatile fatty acids, hydrogen and alcohols (acidogenesis). Volatile fatty acids longer than two carbon atoms and alcohols longer than one carbon atom cannot be utilised by

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Fig. 4 A model of the structural features of gymnosperm lignin and its association with hemicellulose components to form lignin-carbohydrate complexes (Chen and Sarkanen 2010). Reprinted with permission from Elsevier

methanogenic bacteria and must be converted to acetic acid, hydrogen and carbon dioxide (acetogenesis) by obligate hydrogen-producing secondary fermenting bacteria (Bruni 2010). In the final phase, acetic acid, hydrogen, carbon dioxide and other one-carbon compounds such as formate and methanol are converted into methane and carbon dioxide (biogas) by obligate anaerobic methanogenic bacteria (acetate utilisers such as Methanosarcina spp. and Methanothrix spp. and hydrogen and formate utilising species of genera such as Methanobacterium and Methanococcus). Bioethanol production involves sequential steps of pretreatment (for the same reasons as in biogas production), enzymatic hydrolysis, fermentation of monomeric sugars to ethanol and product separation by distillation (not necessary in biogas production since the methane escapes from the water phase) (Bruni 2010) (Fig. 2).

Currently, biogas and bioethanol only contribute a minor fraction to fuel products from the energy industry. In recent years the demand for biogas has been increasing as the world seeks ways to reduce consumption of fossil fuels and substituting firewood in rural households. This is driven by the need to respond to climate change and to shift the prime resource base from non-renewable to renewable feedstocks, an aspect clearly highlighted in the global bio-economy strategy. Consequently, research efforts to increase output of biogas are being intensified in the hope that it may one day be a leading energy source. A number of factors that limit the hydrolysis of lignocellulose materials and therefore reduce biofuel yields have been identified (Alvira et al. 2010; Hendriks and Zeeman 2009). One of the factors is the lignin component which limits the rate and extent of enzymatic hydrolysis by shielding the digestible parts of the substrate

Appl Microbiol Biotechnol

from enzymes (Ding et al. 2012; Jung et al. 2000; Chang and Holtzapple 2000) or by binding cellulolytic enzymes nonproductively (Kumar et al. 2012; Palonen et al. 2004; Esteghlalian et al. 2001). A number of physical and chemical pretreatment processes are being considered to minimise the effect of lignin for example removal or breakdown of the lignin component in order to expose the carbohydrate component to hydrolytic enzymes or hydrolysis of hemicellulose to release monomeric sugars and soluble oligomers from the cell wall matrix into the hydrolysate, leaving the lignin with the residual substrate for subsequent removal after the hydrolysis of cellulose or even after distillation. These include mechanical treatment, thermal treatment, acid treatment, alkali treatment, oxidative treatment with hydrogen peroxide or peracetic acid, ammonia treatment, carbon dioxide treatment and using various combinations of these physico-chemical processes. However, these processes are in most cases unattractive as they fail to meet the basic pretreatment requirements such as avoiding the degradation or loss of carbohydrates, avoiding the formation of by-products which are inhibitory to the subsequent hydrolysis and fermentation processes, and being cost-effective (Sun and Cheng 2002; Talebnia et al. 2010). For example, acid treatment causes enzyme inhibition, and condensation and precipitation of solubilised lignin components which decreases digestibility (Hendriks and Zeeman 2009). In addition, acid treatment has a risk in the formation of volatile degradation products and this carbon is lost during conversion to ethanol (not a major issue in methane production, as volatile products can be converted to methane). Steam pretreatment causes production of furfural, hydroxy-methylfurfural and soluble phenolic compounds which inhibit ethanol or biogas production. Alkali treatment can cause solubilisation, redistribution and condensation of lignin and modifications in the crystalline state of the cellulose which counteract the positive effects of lignin removal and cellulose swelling (Gregg and Saddler 1996). Oxidative pretreatment causes solubilisation of lignin which inhibits conversion of the cellulose to ethanol or methane, and also causes loss of sugars due to non-selective oxidation. Ammonia treatment can only be carried out at ambient temperatures for several days while carbon dioxide treatment is carried out at high temperatures of up to 200 °C. In general, these physico-chemical processes are energy intensive, uneconomical and environmentally unfriendly. In light of this, enzymatic approaches, using lignin oxidising or delignifying enzymes are being considered for the pretreatment of lignocellulosic materials either alone or in combination with other pretreatment methods. Laccase is one enzyme which is being intensively investigated for the pretreatment of lignocellulosic materials in bioethanol or biogas production since it is one of the most common ligninolytic enzymes. Table 2 summarises some of the research output in the application of laccases in biogas and bioethanol production. As shown in Table 2, a number of substrates have been

used as feedstock for second generation biofuels production. Composition of lignocellulosic materials of the starting materials varies depending on the plant species, the age and growth conditions of the plant material and how the material has been processed, e.g. lignin content is higher in soft wood than in hard wood, wheat straw and switchgrass. Access to the sugarrich cellulose and hemicellulose components would therefore require varying degrees of treatment. In nature, synergism between different sets of enzymes (oxidative and hydrolytic) is required for the effective breakdown of lignocellulose. There are, however, certain factors that limit the enzymatic hydrolysis of lignocellulose, for example, type of raw material, pretreatment of the raw material (e.g. drying could collapse cell walls and pores thereby limiting the binding ability of the enzymes), end-product inhibition, depletion of accessible degradable parts, enzyme inactivation and unproductive binding or entrapment of cellulases. Laccase increase fermentability of lignocellulosic materials mainly through lignin degradation (Tabka et al. 2006). Delignification by laccases is often carried out in combination with mediators due to the low redox potential of laccases and the complexity and size of lignocellulose materials. Laccase alone can oxidise only the easily oxidisable phenolic units at the surface of the substrate which often constitute less than 10 % of the polymer. LMS generate radicals which are able to extensively cleave covalent bonds in lignin as they can oxidise both phenolic and non-phenolic lignin components. Recent research efforts have been dedicated to understanding the mechanism of oxidative cleavage of non-phenolic lignin and LCCs using LMS. Building on earlier findings (Fabbrini et al. 2002; Kawai et al. 1988a, 2002; Camarero et al. 1997), the mechanism of enzymatic cleavage of LCCs using Pycnoporus cinnabarinus laccase-HBT system was elucidated (Du et al. 2013b) (Fig. 2). The LMS is able to oxidise the Cα carbon to form oxidised G and S structures (structure I in Fig. 2). This can be followed by a nonenzymatic reaction in which O2 attacks the carbon-centered radical intermediates, forming unstable structures IIa which undergo a number of reactions such as Cα oxidation (to produce structure III), Cα–Cβ cleavage, and aromatic ring cleavage. An alternative route involves structure IIa abstracting a proton to form IIb before undergoing Cα–Cβ cleavage to form structures IV and V. Recently, the mechanism of LMS-catalysed delignification using the natural mediator methyl syringate was elucidated through the use of lignin markers which showed shortened side chains and increased syringyl-to-guaiacyl ratio which suggests preferential removal of guaiacyl units over syringyl units (Rico et al. 2014). However, the most noticeable modification observed was the formation of Cα-oxidised syringyl lignin units during the enzymatic treatment which indicates a Cα-oxidation mechanism of degradation. Mechanisms of oxidation of lignin using LMS-based on other mediators have been reviewed (Morozova et al. 2007b). However, some of the mechanisms

Lignin peroxidase; manganese peroxidase; β-glucosidase (whole cells used) Whole cells used (other enzymes such as lignin peroxidase, xylanase, cellulose, β-glucosidase present) Steam explosion; no mediator

Alone or with HBT

P. cinnabarinus

P. cinnabarinus

P. cinnabarinus

Modified strains of Cerrena unicolor

Trametes versicolor immobilised

Pycnoporus sp. SYBC-L3

Streptomyces griseorubens ssr38

P. cinnabarinus and Trametes villosa

T. villosa

Trametes hirsuta

Ganoderma lucidum Yarrowia lipolytica

Wheat straw slurries

Wheat straw

Wheat straw

Cotton gin trash

Wheat straw

Switchgrass

Paddy straw

Wheat straw

Eucalypt (Eucalyptus globulus) and elephant grass (Pennisetum purpureum)

Perennial weed Parthenium sp.

Sugar cane bagasse Rice straw (Oryza sativa L.)

Steam explosion Alone or with mediators; mild acid/steam explosion

Whole cells use (other lignolytic enzymes present)

With mediator 3,5-dimethoxy-4hydroxybenzonitrile alone or combined with ultrasonication, hot water treatment Ethanol organosolv process

Steam explosion

Steam explosion; no mediator

Steam explosion; no mediator

Laccase alone or with methyl syringate as mediator

Myceliophthora thermophila

Eucalyptus

Combination treatment/mediator

Source of laccase

Substrate

Table 2 Applications of laccases in the production of bioethanol and biogas

Delignification Removal of phenolic compounds

Delignification

Delignification

Polymerisation and removal of phenolics

Delignification

Adsorption; polymerisation; oligomerisation and removal of phenolics Delignification; modification of biomass (increased porosity)

Delignification and modification of cellulose structure

Polymerisation and removal of phenolics Removal of phenolic compounds

Polymerisation and reduction of phenolic content

Delignification

Mode of action

Reduction of phenolic content by up to 94 %; shortening of lag phase of fermentation yeast Kluyveromyces marxianus; enhanced ethanol concentration (increase of up to ~13X) Up to 48 % lignin reduction with HBT; 5 % reduction without mediator; increase in ethanol production (over 4 g L−1 in 17 h in eucalypt and 2 g L−1 in 17 h in elephant grass); no significant increase without mediator Improved rate of sugar release (up to 18 times); 5 fold higher saccharification efficiency; 52.65 higher availability of holocellulosea ~75 % increase in glucose equivalents Up to 75 % removal of phenolic compounds (with HBT as mediator); Increased saccharification yield (90.4 % with mediator; 68 % laccase only; 52.5 %)

30 % reduction in lignin content after 36 days; 50 % improvement in enzymatic hydrolysisa 25 % of depolymerised recovered; high saccharification efficiency of 97.8 %a

Up to 50 % lignin removal accompanied by approximately 40 % increase in glucose and xylose yields after enzymatic hydrolysis. Laccase alone removed up to 20 % of lignin Prolonged hydrolysis time from 48 to 144 h; reduced lag phase and increased viability of Saccharomyces cerevisiae F12; boosted f ermentation (yield, 22 g/L ethanol) without adding extra nitrogen Increased substrate loading up to 12 % (w/v); increased ethanol concentration (16.7 g/L) Up to 73 % removal of phenolic compounds; increased fermentation yeast cell viability and ethanol yield 10 % decrease in lignin and up to 23 % cellulose conversion and 31.6 % ethanol yield after combination treatment of ultrasonication, hot water and LMS Improvement of ethanol productivity from 0.13 to 0.17 g L−1 h−1

Positive effects observed

Sitarz et al. (2013) Lee et al. (2012)

Rana et al. (2013)

Gutiérrez et al. (2012)

Moreno et al. (2012)

Saritha et al. (2013)

Liu et al. (2013)

Ludwig et al. (2013)

Plácido et al. (2013)

Moreno et al. (2013b)

Moreno et al. (2013a)

Moreno et al. (2013c)

Rico et al. (2014)

Reference

Appl Microbiol Biotechnol

Sclerotium sp.

Trametes sp.

T. versicolor

Trametes sp. AH28-2 heterologously expressed in Trichoderma reesei C. unicolor and T. hirsuta

Wheat straw

Newspaper

Corn stover

Corncob residue

Commercial Novozyme 51003 Phellinus robustus, Pleurotus eryngii, Irpex lacteus and Poria subvermispora Pleurotus ostreatus

Manure

None

Ethanol and hot water extraction; violuric acid as mediator

Trametes hirsuta

Cyathus stercoreus

Trametes versicolor

Pycnoporus sanguineus H275

Steam-pretreated softwood

Sugarcane bagasse

Willow pretreated with steam and SO2 Wheat straw and corn stover

N-hydroxy-N-phenylacetamide (NHA) and N-acetoxy-Nphenylacetamide Acid hydrolysis

Steam explosion

Coriolopsis rigida and T. villosa

Wheat straw

Wheat bran as mediator

Delignification

Polymerisation and removal of phenolic compounds Detoxification through removal of phenolic compounds

Delignification (partial)

Polymerisation of phenolic compounds

Delignification

Delignification

Ultrasonic and H2O2

Ceriporiopsis subvermispora ATCC 90467, CZ-3 and CBS 347.63

Delignification

Delignification

Delignification

Lignin modification

Delignification (probable; not investigated)

Delignification

Loosening of lignin-carbohydrate complex Delignification/lignin modification; de-inking

Removal of phenolic compounds

Mode of action

Mild alkali treatment

None

Substrate crushed to powder

Impregnated with SO2 gas and steam pretreated (spruce); steam treated (giant reed)

1-HBT; cellulase and hemicellulase mixture Cellulase

None

Japanese cedar wood

Rice hull

Wheat straw

Pleurotus sp.

Ricinus communis

Spruce (Picea abies) and giant reed (Arundo donax)

Steam explosion

Coltricia perennis Commercial Novozym 51003

Rice straw (O. sativa L.)

Steam explosion

Combination treatment/mediator

Source of laccase

Substrate

Table 2 (continued)

97.8 % klason lignin removed from 1 g wheat straw and 19.98 % increase in Somogyi carbohydrate (fermentable sugar)

77.5 % reduction in total phenolics; 1.7-fold increase in ethanol yield Rapid consumption of glucose and increased ethanol productivity

44 % lignin degraded, net yield of total sugars increased (~12–36.7 %); glucose content increase (~10–38.9 %) 74–76 % decrease of β-O-4 aryl ether linkages; methane yield increased from 6 to 9 % in absence of mediator to 25 % of theoretical yield with mediator Reduction of the toxic effect of phenolic compounds, improved fermentability of hydrolysates 13 % (using laccase alone) or 21 % (using LMS) increase in sugar yield

Improved the hydrolysis yield of spruce by 12 %; reduced hydrolysis yield of giant reed by 17 %; reduced binding of enzymes on modified spruce lignin (opposite effect on giant reed) Maximum delignification 85.69 %; reducing sugar yield increased 2.68-fold Increased the methane yield by (19.8±0.4 m3 CH4 (t WW)−1) Ethanol yield increased by 62 %

Increase in ethanol concentration (0.28 % (v/v) in laccase treated vs 0 % in absence of laccase) Downstream cellulose hydrolysis was improved 7 % Increase in reducing sugar yields by up to 71.6 %

Removal of phenolic compound (76 % with C. perennis; 52 % with commercial laccase); Increased saccharification yield by 48 and 28 %, respectively 16.8 % increase in cellulose conversion

Positive effects observed

Lu et al. (2010)

Jönsson et al. (1998)

Chandel et al. (2007)

Palonen and Viikari (2004)

Jurado et al. (2009)

Amirta et al. (2006)

Yu et al. (2009)

Salvachúa et al. (2011)

Mukhopadhyay et al. (2011) Bruni et al. (2010)

Moilanen et al. (2011)

Zhang et al. (2012)

Chen et al. (2012)

Nakanishi et al. (2012)

Qiu and Chen (2012)

Kalyani et al. (2012)

Reference

Appl Microbiol Biotechnol

Not stated Pleurotus sp.

Myceliopthera thermophilae (NS51003 Novozymes A/S)

Basidiomycete Euc-1

Saccharomyces cerevisiae transformants (with laccase gene) T. versicolor, Bjerkandera adusta, Ganoderma applanatum and Phlebia rufa

Cotton stalk Ricinus communis

Wheat straw

Wheat straw

Spruce hydrolysate supplemented with coniferyl aldehyde Wheat straw

b

a

Rhus vernificera

Rice straw

Delignification

None

Laccase activity was not determined

Result includes effect of other enzymes since whole cells were used

Polymerisation and detoxification

Delignification

Lignin oxidation

Not specified (possible combination of delignification and detoxification) Polymerisation of lignin residues Delignification

None

None

None

Alkali treatment None

None

None

Lentinus edodesb

Mushroom logs

Delignification

Detoxification

None

Ureibacillus thermosphaericus

Waste house wood

Mode of action

Combination treatment/mediator

Source of laccase

Substrate

Table 2 (continued)

43 % decrease in lignin content

Glucose yield increased by 5.45 % after 72 h 85.69 % delignification, 2.68-fold increase in yield of reducing sugars Increase in O2 consumption ascribed to lignin oxidation and therefore chemical alteration of lignin Percentage of Klason lignin present decreased from 20 to 13 %; 4-fold increase in substrate saccharification; ratio of cellulose/lignin increased from 2.7 to 5.9 Removal of coniferyl aldehyde; ethanol produced only by transformants

Reduced sugar loss when compared with overliming, removal of toxic compounds Decrease in lignin content from 21.07 % to 18.78 %; higher total sugar and ethanol yield Improved monosaccharide production by 29.13 %

Positive effects observed

Dinis et al. (2009)

Larsson et al. (2001)

Dias et al. (2010)

Haykir (2009) Mukhopadhyay et al. (2011) Kaparaju and Felby (2010)

Chang et al. (2011)

Lee et al. (2008)

Okuda et al. (2008)b

Reference

Appl Microbiol Biotechnol

Appl Microbiol Biotechnol

were not well known at the time and were therefore presented as suggestions or possible mechanisms. Besides delignification, laccase or LMS can improve biofuels production through changes in the structure of the lignocellulose microfiber which modify properties such as porosity, surface area, and hydrophobicity resulting in the reduction of unproductive binding of cellulases (Moilanen et al. 2011). For example, it has been observed that laccase-treatment of steam pretreated spruce resulted in an increase in acidic groups which would indicate a decrease in lignin hydrophobicity and an increase in negative surface charge (Palonen and Viikari 2004). This could result in electrostatic repulsion toward cellulases which subsequently decreases the unproductive binding of cellulases. It is worth mentioning, however, that lignin degradation using LMS can also be accompanied by the release of inhibitory mono-aromatic phenolic compounds in the medium that decreases activity of cellulases and other hydrolases (Gamble et al. 2000). Similarly, thermochemical pretreatment steps such as acid and steam explosion typically lead to the production of furans, phenols and weak acids which can inhibit the activity of cellulases and sugar fermenting organisms. Treating the steam exploded material is costly, requires special separation equipment (filtration) and generates large amounts of wastewater. Subsequently, research efforts are now also being directed towards detoxification of hydrolysates through laccase-catalysed removal of phenolic compounds and other inhibitory compounds such as furans and weak acids (Moreno et al. 2013a, c, 2012; Kolb et al. 2012; Parawira and Tekere 2011; Jönsson et al. 1998). The process involves laccase-catalysed oxidation of the compounds to unstable radicals which couple to form oligomeric and polymeric compounds which are less toxic to enzymes required for subsequent processes than the corresponding monomeric units. The reaction mechanism is well characterised. For example, we have shown recently that laccase-catalysed oxidation of the phenolic molecule ferulic acid led to coupling of the radicals through β-5 and β-β coupling to form dimers (Adelakun et al. 2012) and the reaction also produces oligomers and polymers. Polymerisation of phenolic compounds is so typical of laccases that we have observed this phenomenon in virtually all the coupling reactions we carried out with simple phenolic compounds and oligomeric lignin models (Kudanga et al. 2009, 2010a, b, c; Widsten et al. 2010). Although physicochemical methods are also efficient at removing phenolics, they also remove compounds such as acetic acid (Chandel et al. 2007) which are important in biogas production. Some oxidised mediators such as oxidised N-hydroxy-Nphenylacetamide can also inhibit the cellulases required for subsequent hydrolysis (Palonen and Viikari 2004). The effect of such inhibitors can be reduced by using excess substrate (probably by offering sites for the radical attacks) or by using acetylated mediators which are slowly released by a lipase in

situ (Palonen and Viikari 2004). Recently a system that uses immobilised laccase for detoxifying lignocelluloses hydrolysates was developed (Ludwig et al. 2013). By immobilising laccase onto a hydrophobic carrier, compounds that could not be removed by free laccase (such as furfural) could be fully removed while incorporation of an anion exchanger as a subsequent step led to the reduction of lignocellulose degradation products such as HMF, formic acid, levulinic acid and acetic acid by 90, 62, 39 and 20 %, respectively before fermentation for the production of ethanol (Ludwig et al. 2013). Combining laccase treatment with other treatment methods usually results in higher yields than the individual methods alone (Bruni et al. 2010). Laccase treatment can be accomplished using free purified laccase or whole cells which produce the enzyme. However, where whole cells are used it is usually not easy to find a direct correlation among enzyme production, lignin degradation, and sugar yield (Salvachúa et al. 2011; Yamagishi et al. 2011) because oxidative lignin degradation is a nonspecific process where other extracellular ligninolytic enzymes and low molecular-weight extracellular oxidant compounds (such as Mn3+ and oxygen free radicals) generated during the process, participate (Guillén et al. 2000; Hammel et al. 2002). It is also worth noting the work of Zhang et al. (2012) in which Trametes sp. AH28-2 laccase gene lacA fused to cellobiohydrolase I signal peptide coding sequence was heterologously expressed in Trichoderma reesei resulting in a huge improvement in yield of reducing sugars. It was proposed that the improvement in cellulolytic activity could be due to the modification of the lignin surface which decreased the unproductive binding of cellulases to lignin and/or partial breakdown of lignin structure by laccase which increased access of cellullase to cellulose and/or possible synergistic effects in which depolymerisation of cellulose and of lignin could accelerate each other (Zhang et al. 2012; Westermark and Eriksson 1974). Generally, there have been few research outputs on laccasemediated delignification for the direct purpose of bioethanol or biogas production. However, most delignification processes catalysed by laccase and LMS either for research purposes or for the purposes of biopulping as recently reviewed (Cañas and Camarero 2010; Martínez et al. 2009) and reported (Eugenio et al. 2010a, b; Fillat et al. 2010; Crestini et al. 2011; Martín-Sampedro et al. 2011a, b; Moldes et al. 2008, 2010; Du et al. 2013a, b), could be adapted for biogas or bioethanol production. In related uses, laccases have been used for de-inking newspapers which were subsequently used for bioethanol production (Kuhad et al. 2010; Nakanishi et al. 2012). Although there was no ethanol production using newspapers which were not treated with laccase, Nakanishi et al. (2012) did not observe significant evidence of delignification in laccase-treated newspapers. They proposed that laccasemediated de-inking and change in the structure of biomass exposed the cellulose in newspaper to cellulolytic enzymes.

Appl Microbiol Biotechnol

Conclusion and directions for future research Across the globe, countries have implemented, defined or are working towards the development of a bio-economy strategy. The need for feasible and sustainable bio-based processes underpins a large part of the bio-economy approach. The application of laccases in biofuel production is currently based on our understanding of the role of laccase and laccasemediator systems in lignocellulose degradation or modification. Designing bio-based processes around what happens in nature, e.g. understanding the synergy between all the enzymes involved in the breakdown of lignocellulosics, and the application of this knowledge in the design of new biobased processes will greatly contribute towards more efficient processes. For example it may be better to target the LCC with a cocktail of enzymes (e.g. laccases and hemicellulases) that degrade the lignin and hemicellulose components and possibly the benzyl ether, γ-ester and phenyl glycoside linkages between lignin and carbohydrates. The success recorded by Chen et al. (2012) emphasises the need for more research in this area. A different approach would be the heterologous expression of laccase in for example a host that has hemicellulase capacity in a similar way to experiments carried out by Zhang et al. (2012) in which they successfully expressed Trametes sp. AH28-2 laccase in the cellulolytic fungus T. reesei. There is also a continued need for the discovery of natural mediators that can be applied in a LMS for the cleavage of lignin linkages, as well as a need for the discovery of novel enzymes with new and interesting activities (e.g. the activities observed for the yellow and white laccases). Even though there may be many advantages to the LMS (e.g. they can be used for woody and non-woody lignocellulose materials where they can have direct action on lignin), there are certain disadvantages, e.g. the generation of by-products that can lead to the inactivation of laccases and other enzymes such as xylanases when using HBT as mediator. However, another synthetic mediator NHA is much less toxic as mediator (Woolridge 2014) while the bulk of natural mediators produce much better results in mixed enzyme systems. However, in general, mediators add cost when compared with using laccase alone and a cost–benefit analysis should be considered before choosing a laccase only or a LMS. The application of metagenomic techniques in mining enzymes from microbial communities (Xing et al. 2012) which has become more feasible due to advances in sequencer technology and metagenomic sequencing should also be considered in the search for novel laccases. Other related approaches include site-directed mutagenesis to improve activity of laccases and application of new bioinformatics tools such as the Laccase Engineering Database (LccED) (Sirim et al. 2011), that, for example, have allowed researchers to design better laccases. Such technologies can be adapted in the search for better

pretreatment enzyme systems that can be applied in biobased processes for the production of biofuels. Acknowledgments Financial support from the National Research Foundation (NRF)—South Africa and the Cape Peninsula University of Technology Research Funding (URF) is gratefully acknowledged. Any opinion, findings and conclusions or recommendations expressed in this material are those of the author(s) and therefore the NRF does not accept any liability in regard thereto.

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Laccase applications in biofuels production: current status and future prospects.

The desire to reduce dependence on the ever diminishing fossil fuel reserves coupled with the impetus towards green energy has seen increased research...
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