As a first note, different fungi may require different sources of carbon and nitrogen to fully release their laccase expression potential. Under submerged conditions, Hariharan and Nambisan (2012) tested many sources of carbon including glucose, sucrose, starch, maltose, and lactose. Their results suggested that glucose and sucrose enhanced the enzyme expression, but other carbon sources contributed to activity decrease. These results are consistent with those recently unveiled by other researchers, where glucose effectively promoted laccase activity (Schneider et al., 2019; Marin et al., 2020). Furthermore, Schneider et al. (2019) found that the secretion of laccase was related to the nitrogen source in the media, with casein being a better enzyme promoter than peptone. In the same sense, Lentinus strigosus 1566 showed highest laccase activity in a peptone-yeast extract medium supplemented with galactose, arabinose, and xylose, while glucose, sucrose, or maltose decreased its activity (Myasoedova et al., 2015). These authors also found that glucose slightly increased laccase activity compared to malt dextrin, whereas fructose decreased laccase production. As for sucrose and glycerol, they lowered laccase activity yield but substitution by maltose had no effects on laccase production. Overall, diverse carbon sources have a significant role in laccase production. Determining the best carbon source is the first step towards optimal growth medium design and eventually optimal laccase production.

Typically, culture media are supplemented with organic or inorganic nitrogen sources. Depending upon these two forms, different levels of laccase expression and activity can be observed with the same strain and from one strain to another. A direct positive correlation between peptone concentration and biomass development and laccase activity increase was observed in a culture of Coriolopsis gallica 142 strain (Mikiashvili et al., 2006; Elisashvili et al., 2017). However, at a certain threshold, the subsequent increase in peptone concentration led to an opposite effect on the activity. In the aforementioned study, nitrogen sources such as peptone, yeast extract, beef extract, ammonium sulphate, ammonium nitrate, and urea, were also tested for laccase production. The authors found that beef extract was the best nitrogen source for highest activity expression after 120 h of incubation (Hariharan and Nambisan, 2012). Previously, Zerva et al. (2017) studied the comparative influence of five different nitrogen sources including diammonium tartrate, potassium nitrate, ammonium nitrate, yeast extract and corn steep liquor (CSL) on laccase expression by Pleurotus citrinopileatus and Irpex lacteus. It was observed that both species developed highest biomass and laccase activities in samples supplemented with CSL. Besides, inorganic nitrogen sources were found to promote less fungal growth. In another study, Chauhan (2019) obtained a similar result with Grammothele fuligo cultured in glucose-based medium, where inorganic nitrogen sources tested failed to promote abundant biomass and further to secrete laccase. The fermentation of P. ostreatus Pl 22 strain using different nitrogen sources showed that yeast extract increased laccase activity by almost six-fold in comparison with ammonium sulfate (Karp et al., 2015). Mikiashvili et al. (2006) determined that ammonium sulfate and ammonium nitrate were good sources of nitrogen for laccase production by Trametes multicolor. Besides their individual effects, the Carbon/Nitrogen (C/N) ratio can significantly influence the synthesis and secretion of fungal laccase (Rivera-Hoyos et al., 2013; Elisashvili et al., 2018). Globally, depending upon the strains, low or high C/N ratio can alternately improve or decrease the production (Elisashvili et al., 2018). Interestingly, Yang et al. (2016) determined that the combination of high concentrations of carbon and nitrogen led to higher production of laccase from Cerrena sp.

In summary, a wide range of nitrogen sources has been studied and can induce diverse effects on laccase production, hence there is considerable uncertainty regarding the selection of the optimal nitrogen concentration for laccase production (Elisashvili et al., 2018).

Lignin degradation metabolites and metals naturally present in the environment can act as promoters of fungal laccase production. In a laboratory context, phenolic and aromatic compounds, especially those structurally related to lignin (Furukawa et al., 2014; Pollegioni et al., 2015; Elisashvili et al., 2017), and metals such as copper, manganese, cadmium, and magnesium can play an important role in laccase production (Valle et al., 2014; Martani et al., 2017; Lallawmsanga et al., 2019). However, these compounds have also been depicted to be playing dual roles as they can act as inducer or repressor, depending notably on their concentration, the media composition, the fungal species, and the enzyme tested (Elisashvili et al., 2017). Under submerged fermentation, hydroquinone was found to cause an increase in laccase production by T. versicolor, whereas C. unicolor rather decreased laccase activity (Elisashvili et al., 2010). Under laboratory conditions, compounds such as 2,5-xylidine, guaiacol, veratryl alcohol (VA) and catechol are often used as laccase inducers (Krull et al., 2013; Martani et al., 2017). In a submerged fermentation of T. multicolor 511, VA and guaiacol enhanced laccase specific activity by two-fold (Mikiashvili et al., 2006). Similarly, gallic acid (1 mM), tartaric acid (20 mM), and citric acid (20 mM) could elevate laccase activity (Chang and Chang, 2016). It was also observed that among several organic inducers, ethanol and guaiacol induced laccase production by Lentinus crinitus while pyrogallol, veratryl alcohol, xylidine, and vanillin were ineffective (Valle et al., 2014). It was also determined that the induction of laccase activity by ethanol was concentration-dependent, as concentrations of 1% v/v and 3% v/v have increased Ganoderma lucidum laccase activity production by 6.5 and 14 times compared to the control, repectively. However, with up to 5% v/v ethanol, the activity reached only 10 times that of the control, showing that the correlation of activity induction with ethanol concentration was positive up to a certain level, beyond which the ethanol concentration could be less effective in increasing laccase activity (Manavalan et al., 2013). Resveratrol, tannic acid, and guaiacol were found to be the best laccase inducers in a culture of C. gallica, however, 2–2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and gallic were ineffective (Xu et al., 2016) under the same fermentation conditions. On the contrary, laccase activity was increased in Cerrena sp. HYB07 fermentation by ABTS and guaiacol, though other aromatic compounds had no significant effects (Yang et al., 2016).

Several inorganics can modulate laccase expression. In general, trace metallic elements at high concentrations can be toxic to ligninolytic fungi growth and repress their laccase expression. Besides, it was demonstrated early on that tolerance to high concentrations of trace metallic elements can largely be species dependent (Giller et al., 1998; Valle et al., 2014). Meanwhile, some metallic compounds such as Cu, Mn, Co, and Zn, present at low concentrations in the culture medium are essential for fungal growth and biological functions (Baldrian, 2003; Asif et al., 2017). Among microelements, copper is largely used as an inducer in enzyme production. The positive correlation between laccase production and copper, often added to the media as copper sulfate, has been well described in previous studies (Valle et al., 2014; Karp et al., 2015; Chang and Chang, 2016; Vrsanska et al., 2016; Yang et al., 2016; Zhu et al., 2016; Schneider et al., 2019). Moreover, the influence of copper on laccase expression is likely to be magnified or minimized concomitantly with high or low nitrogen concentration, respectively (Valle et al., 2014). However, under certain conditions, the negative effect of copper has also been highlighted (Dao et al., 2019). Thus, as reported by Martani et al. (2017), the overall influence of copper on laccase production depends on its concentration in the culture medium, the microbial strains involved, and the presence of other components in the medium.

In addition to the design of the nutritional environment, operational factors such as temperature, pH, time, agitation rate, and dissolved oxygen can significantly influence the fungal growth and enzyme production.

Temperature does not correlate significantly with fungal growth rate and biomass development (Martani et al., 2017). However, it importantly influences the potential and the level of laccase activity expressed, as revealed by several studies. Schneider et al. (2019) showed that laccase activity of Marasmiellus palmivorus VE111 was maximum at 28°C and decreased when this temperature was either lowered or raised by 5°C. The decrease of laccase activity below or above 28°C was explained by the reduction of expression of some genes involved in the transcription of this enzyme (Rivera-Hoyos et al., 2013; Schneider et al., 2019). A previous study on M. palmivorus LA1 laccase secretion under SSF using pineapple leaf as substrate led to a similar conclusion (Chenthamarakshan et al., 2017). Hariharan and Nambisan (2012) found that 27°C was the best temperature for laccase production by Ganoderma lucidum under SSF, while temperatures lower than 23°C or higher than 33°C led to a significant reduction in enzyme production. Yet, Chang and Chang (2016) determined 30°C as the optimum temperature for laccase production from Pleurotus eryngii, under submerged conditions.

The pH can have an important influence on fungal growth and thereby on laccase expression. According to previous studies, highly acidic or basic media negatively affect fungal growth and laccase activity, and this can be noticed either under SSF or SmF. Chang and Chang (2016) noted an increase of laccase activity of P. eryngii between pH 2 and 5, before its decrease in the 5–9 pH-range. In another study, Chenthamarakshan et al. (2017) determined that pH 5 was the optimum for best growth of M. palmivorus LA1 on pineapple leaf for laccase secretion and maximum activity. In the same vein, pH 5 was determined as optimal for production of laccase from G. lucidum under SSF, after an optimization process (Hariharan and Nambisan, 2012) while Zerva et al. (2017) got the best results at pH 5 and 6 with Pleurotus citrinopileatus and Irpex lacteus strains using supplemented olive mill wastewater as culture medium. For Schneider et al. (2019), pH 4 and below or pH 8 and above led to laccase activity decrease, whereas it reached maximum activity at pH 7.

Incubation time for an enzyme to reach maximum activity expression varies from one strain to another and according to fermentation conditions. In general, microorganisms are characterized by a period of acclimation followed by growth and biomass production accompanying the substrate consumption. Overall, thanks to the ready availability of nutrients, the culture period for enzyme production in SmF is generally shorter than that of SSF (Wang et al., 2019). The Ganoderma lucidum 447 culture for enzyme production in olive mill by-products medium achieved highest laccase activity after 6 days, i.e. earlier than with other fungi tested in the same study. In contrast, Cerrena unicolor 302 attained maximum laccase activity after 2 weeks of fermentation (Elisashvili et al., 2017). A 2-week period was also the cultivation time necessary for Ganoderma applanatum with rice bran as media to achieve maximal laccase activity (Wang et al., 2019). The culture of Coriolus versicolor on sweet sorghum bagasse in SSF supplemented with CuSO₄, gallic acid and syringic acid produced maximum laccase activity within 16 days (Mishra et al., 2017). Under SmF, P. citrinopileatus and I. lacteus produced highest laccase activity in 10 and 24 days of cultivation in olive mil wastewater, respectively (Zerva et al., 2017), however Cerrena consors took much more time (30 days) for the laccase activity peak in a 50% olive mill wastewater (Mann et al., 2015).

Under submerged fermentation conditions, the availability and transfer of oxygen is essential for fungal growth. As mentioned earlier, mycelial uncontrolled expansion can limit oxygen transfer (Krull et al., 2013; Silvério et al., 2013). To promote oxygen transfer, it is important that the culture must remain continuously under shaking conditions. This was corroborated by Domingos et al. (2017) who found that unshaken culture resulted in incomplete sugar consumption partially due to lack of proper oxygen transfer. In another study, Schneider et al. (2019) have analyzed the influence of the concentration of dissolved oxygen on enzymatic activity from Marasmiellus palmivorus VE111 strain. Thus, in general, it is proved that increased laccase activity is directly related to DO concentration.

The monitoring of agitation has shown a positive correlation between biomass growth and agitation rate. However, above a certain threshold, agitation can lead to a negative effect on biomass growth and enzyme expression. In fact, under excessive agitation, hydrodynamic shear stress on biomass can result in changes in its morphology, leading to subsequent enzyme under-expression (Zerva et al., 2017).

Entrapment is identified as the simplest immobilization technique in which enzyme molecules disperse into a porous solid matrix; hence no direct attachment may be formed between carrier and enzyme (Fernández-Fernández et al., 2013; Karthik et al., 2021). Alginate, collagen, silicon rubber, gelatin, carrageenan, polyurethane, polyacrylamide, and polyvinyl alcohol with styryl pyridinium groups are solid matrices that can be used for enzyme entrapment (Dayaram and Dasgupta, 2008; Phetsom et al., 2009; Fernández-Fernández et al., 2013). Enzyme entrapment can be carried out in two steps: first enzyme molecules are dispersed into monomer solution, and then a polymerization process ensues which maintains enzyme molecules trapped (Karthik et al., 2021). Entrapment technology could increase laccase stability considerably and it can be helpful to avoid enzyme denaturation. Despite its benefits, this method has some limitations which restrict its application. One such issue is enzyme leakage which can be significant when a support with a large pore size is used.

In the adsorption immobilization technique, the enzyme is linked to the carrier through weak interactions (Sirisha et al., 2016). Based on the types of weak forces, adsorption immobilization can be divided into two categories, namely ionic attachment (electrostatic interaction is dominant) and physical attachment (mainly through van der Waals forces, hydrophobic interactions or hydrogen bond formation) (Karthik et al., 2021; Zhou et al., 2021). Compared with other techniques, adsorption methodology is recognized as a simple and low-cost procedure for enzyme immobilization (Fernández-Fernández et al., 2013). Despite its benefits, the amount of enzyme leakage in this method is high, therefore the application of adsorption immobilization for long-term processes or processes with varying operational conditions is not recommended (Zhou et al., 2021). pH, ionic strength of the solution and solid support surface area are three factors that should be considered during adsorption immobilization (Rekuć et al., 2008; Huajun et al., 2009; Xu et al., 2009; Forde et al., 2010).

Covalent binding is considered as the most reliable method for industrial application (Fernández-Fernández et al., 2013). In this methodology, strong bonds are formed between non-essential amino acids at the surface of enzymes and carrier chemical groups. Due to the formation of these strong bonds between supports and enzymes, the amount of leakage decreases significantly (Hernandez and Fernandez-Lafuente, 2011; Zdarta et al., 2018b). Based on the functional groups on the supports, various reagents could be implemented to prepare the support for covalent immobilization. For supports with hydroxyl groups, cyanogen bromide (CNBr) and carbonyl diimidazole (CDI) are recommended (Karthik et al., 2021). For supports with carboxyl groups, zero length reagents such as EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride), NHS (N-hydroxysulfosuccinimide), and EDC coupling with Sulfo-NHS are recommended (Hermanson, 2013). In addition to these reagents, ionic liquids have been frequently used in enzyme immobilization as they are eco-friendly solvent media (Hermanson, 2013). However, selection of ionic liquid types is a key step since cation or anion changes in such a liquid could affect activity, structure and enzyme stability (Hermanson, 2013). The possibility of laccase immobilization on magnetic nanoparticles was also investigated (Qiu et al., 2020). In this study, the surface of magnetic nanoparticles was modified with an amino-functionalized ionic liquid. Through surface modification with 3-(chloropropyl) trimethoxysilane (CPTMO) and (3-aminopropyl) trimethoxysilane (APTES), laccase was covalently immobilized on the surface (Qiu et al., 2020). Stability-wise, the biocatalyst could maintain around 70% of its initial activity after six cycles (Qiu et al., 2020). In the context of magnetic supports, bioinspired magnetic particles bearing laccase (laccase-biotitania, lac-bioTiO₂) were applied for the efficient removal of bisphenol A, 17α-ethinylestradiol and diclofenac in a mixture of six model endocrine disrupting compounds (EDCs) and retained 90% of activity after five reaction cycles and 60% after 10 cycles (Ardao et al., 2015).

Cross-linking of enzyme aggregates is a carrier-free insolubilization procedure in which multifunctional or bifunctional reagents are implemented to assist enzyme cross-linking into a unified structure with no added carriers (Mateo et al., 2004; Zhou et al., 2021). Since, in this methodology, enzymes act as their own solid supports, this procedure is also called a self-immobilization technique (Karthik et al., 2021). Among different cross-linker reagents such as diiminoesters, diisocyanates, and diamines activated by carbodiimide, the best-known is glutaraldehyde (GA) as it is inexpensive, widely available, and easy to manipulate (Fernández-Fernández et al., 2013; Xiang et al., 2018). However, currently this cross-linker is raising potential toxicity concerns (Yang et al., 2019). This method is highly dependent on pH which includes Schiff’s base formation and Michael-type 1,4 in addition to α, β-unsaturated aldehyde moieties (Migneault et al., 2004). There are two kinds of enzyme cross-linking techniques, namely formation of cross-linking enzyme crystals (CLEC), and of cross-linking aggregates (CLEA) (Ba et al., 2013). In CLEA (Schoevaart et al. 2004), first enzyme molecules are clustered in chemical precipitant solutions such as acetone, ammonium sulfate or ethanol and subsequently a cross-linking reaction completes the process, as initially demonstrated with laccase CLEA by Cabana et al. (2007) and then by others (Matijošyte et al., 2010; Nguyen et al., 2017). CLEC techniques demonstrate good stability and promising activity, however for this process high purity of enzyme is required (Sirisha et al., 2016). Finally, cross-linking with the concomitant enzyme immobilization on an inert porous support may confer additional stability. For istance, Nair et al. (2013) described the deactivation of free and immobilized enzymes during their incubation at 45, 55, 65 and 75°C at pH 5 in absence of electron-donor substrate by periodically measuring the residual activity with ABTS as a substrate. An apparent higher stability of immobilized laccase was evidenced with greater half-lives for the immobilized laccase than soluble laccase. Table 2 presents indicative properties of enzyme immobilization techniques applicable to laccases.

Solid support particle size plays a significant role in the success of immobilization. In industrial applications, large particles may be handled better than small ones (Santos et al., 2015). Nanoporous gold supports were employed to study the effect of particle size on laccase immobilization (Huajun et al., 2009). The results obtained from three different particle size samples demonstrated that the larger particle size support had the ability to keep more enzyme on its surface due to laccase accessibility to inner pore structures (Huajun et al., 2009). However, having larger support particles could have some drawbacks as well. Large particles could enhance diffusional limitations which could, in turn, affect negatively the enzyme activity (Bortone et al., 2014). In the case of the substrate, if its consumption rate by the enzyme is higher than its diffusion rate, there is a possibility of the enzyme located at the support core not receiving any substrate and therefore the biocatalyst’s apparent enzyme activity could decrease (Lortie and André, 1991; Boniello et al., 2010). At the same time, even though nanoparticles present handling issues, the diffusion problems can be prevented by the use of nanoparticles instead of microparticles and for non-porous supports the enzyme is always exposed to the substrate (Bilal and Iqbal, 2019; Bilal et al., 2020). Moreover, to produce effective multipoint covalent immobilization on nanoparticles, epoxy, glyoxyl or divinylsulfone activated nanoparticles can be used (Bilal and Iqbal, 2019; Bilal et al., 2020).

There is a connection between pore size and surface area in which larger pores result in a lower specific area. Specific surface area determines the amount of enzyme that could be loaded over the carrier (Di Cosimo et al., 2013). From an economics perspective, a larger specific surface area could result in a higher amount of enzyme that could be loaded over the support (Santos et al., 2015). Pore diameter determines the size of the enzyme which could be immobilized over the solid support. Importantly, the size of the pore should be big enough to allow the new enzyme molecules to enter in the support (Hudson et al., 2008). In general, the diameter of the pore should be four to five fold larger than the enzyme’s molecule size (Hanefeld et al., 2009). According to a comprehensive analysis of 182 experiments with emphasis on the effect of pore size and surface area on enzyme immobilization, a general trend emerged: higher surface area would result in higher enzyme load on the support (Bayne et al., 2013). However, this general trend for pore size was divided into three ranges in which for the supports with pore size less than 10 nm, the amount of loading is less (apparently due to physical restrictions in accessing the augmenting surface inherent in this pore diameter range), for the supports with pore size between 10 and 100 nm, the amount of enzyme loading tends to be constant (possibly due to protein–protein interaction blocking pores and restricting access to the higher surface area available at lower pore diameters), and for supports with pore size higher than 100 nm, the amount of enzyme loading per unit mass would decline due to a parallel reduction in available surface area (Bayne et al., 2013). Thus, upon a critical analysis even if the surface area is larger for solid supports with small pores the possibility of enzyme loading is lower. Moreover, there was no clear trend between pore characteristics and retention of catalytic activity (Bayne et al., 2013).

The existence of functional groups on the solid supports is another factor that controls enzyme-support interactions (Santos et al., 2015). Favorable functional groups on the solid support are essential to ensure that strong multiple interactions would occur between enzyme, binding agent, and support leading to decreased leakage (Pandey et al., 2020). While the density of active groups on the solid support is crucial, the nature of functional groups is also critical. Most active groups are stable and do not require further consideration (Garcia-Galan et al., 2011). However, covalent immobilization merits further analysis (Garcia-Galan et al., 2011). An ideal functional group for successful covalent immobilization should have the following properties:

- Allow reaction between enzyme and support with low steric hindrances (Mateo et al., 2005);

Support inertness could affect both immobilization and the substrate on which immobilized laccase is expected to act (Daronch et al., 2020). Commonly, a solid support should maintain its physical integrity and be inert after immobilization to avoid interfering with desired reactions (Ba et al., 2013). Polysaccharide matrices such as agarose and cellulose beads, carbonaceous materials, as well as silica compounds are considered as inert solid supports (Santos et al., 2015). Mechanical properties of solid supports are highly dependent on the process use intended for the immobilized laccase (Garcia-Galan et al., 2011). For instance, in a fixed-bed reactor, the solid support should have high rigidity to tolerate high pressure (Santos et al., 2015), hence silica materials, carbon-based materials, and inorganic oxides are recommended (Kim et al., 2008; Tartaj, 2011; Hartmann and Kostrov, 2013). However, the situation would be different in a stirred-tank reactor (Santos et al., 2015)where, instead of mineral materials, more flexible compounds such as agarose beads, cellulose beads, and lentikats can be used (Grazu et al., 2006; Cárdenas-Fernández et al., 2012; Lam et al., 2012).

Besides the above-mentioned properties, the ideal solid support should be low cost and eco-friendly (not increasing operation cost and generating environmental problems), with high affinity toward the enzyme to be amenable to regeneration (Ba et al., 2013; Daronch et al., 2020). Table 3 categorizes three major types of support materials used for immobilization and their specific properties.

The study of biochar’s composition has revealed that, depending on the feedstocks and pyrolysis conditions, this material can present incompletely degraded lignocellulosic biomass and nitrogen-content residues such amine groups (see below). Furthermore, functionalization can introduce new chemical groups to the biochar structure. These elements make biochar a complementary source among the common carbonaceous nutrients provided in culture media for laccase production WRF. Besides, the large specific area and pore size, and the existence of specific chemical groups on biochar surface favor its adsorptive capacity, which can also be related to the molecular size of the enzyme (Rajapaksha et al., 2016; Li et al., 2018; Fernandez-Sanroman et al., 2020; Pandey et al., 2020). Several studies have reported the successful enhancement of laccase production and immobilization on biochar either by adsorption or covalent bonds (Lonappan et al., 2018a; Li et al., 2018; Fernandez-Sanroman et al., 2020; Pandey et al., 2020; Imam et al., 2021). A summary of such studies is shown in Table 4.

In a biochar-based medium for laccase production, laccase can adsorb onto biochar or some of its components can be released in the culture medium and absorbed by the fungus. In both cases, as discussed in other sections, these organic and inorganic components in the biochar exert regulatory actions on laccase production, either as promoting or inhibiting agents (Giller et al., 1998). Due to its physicochemical characteristics, i.e., its high porosity and hydrophobicity (Taskin et al., 2019b), biochar can demonstrate high affinity for organic and inorganic contaminants (Taskin et al., 2019b; Fernandez-Sanroman et al., 2020). This property allows its use as a sorbent of organic or inorganic pollutants for soil amendments (Taskin et al., 2019b). Biochar has also been used in wastewater as additive/support media during anaerobic digestion, filtration matrix for the removal of suspended matter, heavy metals, or pathogens (Madadi and Bester, 2021).

The presence in biochar of bioavailable organic components like hydrophilic compounds and thermally labile fractions (Rombolà et al., 2016), adsorbed volatile organic compounds (Spokas et al., 2011), and polycyclic aromatic hydrocarbons (Buss et al., 2015) is well established. Many inorganic compounds including essential elements for the improvement of fungal laccase production such as Cu, Mn, or Fe have also been found in the biochar structure (see below). On the other hand, some of these compounds are potentially toxic and can be detrimental to laccase production or immobilization (Singh et al., 2010; Zhang G. et al., 2018). In some cases, it all depends on biochar level in culture media (Taskin et al., 2019b). Ultimately, the use of biochar as a substrate for laccase production or immobilization remains an open question.

Regarding carbon-based stimulation of WRF enzyme production, Liu et al. (2019) investigated the impact of single-walled carbon nanotubes, graphene and oxidized graphene (graphene oxide, GO) on the extracellular LME activities of a Cladosporium sp. strain, using a SmF with basal medium made of peptone and yeast extracts. It was found that, among the three carbon-based materials tested, single-walled carbon nanotubes and graphene increased laccase production, while GO caused a slight decrease in laccase activity (Liu et al., 2019). The effects on laccase expression of two carbon-based materials, i.e., biochar (BC) and hydrochar (HC) prepared from four feedstocks were also studied using T. versicolor, P. ostreatus and P. eryngii strains (Taskin et al., 2019a). At two different doses (0.4 and 2% w/v), the two materials significantly stimulated laccase production and increased its activity for T. versicolor and P. eryngii strains, but P. ostreatus did not release any detectable laccase. Hence, BC from red spruce pellets at 0.4% w/v and HC from urban pruning residues at 2% w/v have promoted T. versicolor laccase activity by 6.4 and 21-fold with respect to the controls, respectively. Similarly, BC from vine pruning residues at 0.4% w/v and HC from urban pruning residues at 2% w/v induced a 6.4- and 21-fold increase in P. eryngii laccase activity over controls, respectively. Despite the promoting impacts of BC on laccase production, some inhibitory effects were noticed in connection with higher doses of BC (2%, w/v) in laccase expression by T. versicolor and P. ostreatus (Taskin et al., 2019a). On the other hand, Ascough et al. (2010) previously found depressive effects of BC at concentrations as low as 0.5% (w/v) on the growth of P. pulmonarius and T. versicolor. As for the effects of microelements such as Cu, Fe and Mn, Taskin et al. (2019a) could relate laccase expression induction to high levels of Fe (about 4.3 mM) and Mn (2.5 mM) in BC. In contrast, the absence of Mn, coupled with the presence of As, Pb, and Cl at relatively high levels, may have contributed to the decrease of laccase expression by P. ostreatus at both BC doses.

Lonappan et al. (2018a) immobilized laccase on BC from three different feedstocks, i.e., pine wood (BC-PW), pig manure (BC-PM) and almond shell (BC-AS) produced in different pyrolysis conditions, for diclofenac elimination. The specific surface areas of the three BCs, determined using the Brunauer, Emmett, and Teller (BET) method were 14.1 m² g⁻¹ (BC-PW), 46.1 m² g⁻¹ (BC-PM) and 17 m² g⁻¹ (BC-AS), respectively. The BCs exhibited different surface texture, morphology, surface chemistry and functional groups. In addition, they demonstrated good results in covalent laccase immobilization, with BC-PM being the best immobilization support, mostly due to its higher specific area. In a similar study, two BCs prepared from maple (MB) and spruce (SB) were used as supports for laccase immobilization and for chlorinated biphenyl removal in wastewater (Li et al., 2018). FT-IR, SEM and BET analyses showed a honeycomb structure in the MB with a specific area of 613.6 m²g⁻¹ and pore volume 0.695 cm³g⁻¹ while SB exhibited 86.3 m²g⁻¹ specific area and 0.065 cm³g⁻¹ pore volume. Maple-based BC displayed the higher immobilization yield (Li et al., 2018).

As mentioned earlier, several studies have demonstrated the potential of ethanol to induce laccase production (Meza et al., 2005; Manavalan et al., 2013; Valle et al., 2014, 2015). Furthermore, due to its antimicrobial activity, ethanol has also been used as inactivating agent of competing fungal strains (Peters et al., 2013; Lucas et al., 2017) and other undesired microorganisms. In addition, ethanol is a safe, stable, and affordable solvent that can easily permeate the BC structure. Therefore, ethanol-based sterilization of BC and the subsequent use of the soaked BC as a substrate and carrier for laccase production and immobilization may be considered as an attractive means of enhancing the expression of specific fungal laccases. More generally, BC could be soaked in inducer solutions (e.g., copper containing solution) to serve as a complete culture medium of laccase production.

Carbon nanotubes (CNTs) or buckytubes are hollow cylinders in which carbon atoms are located in hexagonal arrangements (Assi et al., 2021). Since CNT materials are formed from graphene sheets, they demonstrate similar properties to graphene materials like thermal and chemical stability, high tensile strength, and biocompatibility (Karthik et al., 2021). However, graphene atoms are in a two-dimensional arrangement while carbon atoms of CNTs are in a one-dimensional arrangement (Anzar et al., 2020). Moreover, CNTs exhibit radical breathing mode (RBM) in Raman spectrum which is unique to CNTs in comparison with other carbon systems, where all of the carbon atoms move in the radial direction synchronously thus generating an effect similar to breathing (Lei et al., 2011). CNTs can be formed through three different methods, i.e., arc discharge method, laser ablation method and chemical vapor deposition procedure. Commonly, two forms of CNTs can be developed: single wall carbon nanotubes (SWCNTs), and multiple wall carbon nanotube (MWCNTs).

- Single-walled carbon nanotubes:

SWCNTs may be developed from a single graphene sheet rolling upon itself (1–2 nm diameter) (Sabzehmeidani et al., 2021). SWCNTs were first reported in 1993 (Karthik et al., 2021). They have unique properties such as strong covalent bonding, one-dimensional structure, and nanometer size (Liu et al., 2015). Based on how graphene sheets are rolled up, two forms of SWCNTs can be obtained: a zigzag structure, and an armchair structure (Shoukat and Khan, 2021).

- Multi-walled carbon nanotubes:

MWCNTs are prepared by rolling up multiple layers of graphene sheets on themselves (Ali et al., 2021). Based on the number of graphene tubes being rolled up, MWCNT diameter varies from 2 to 50 nm (Ibrahim, 2013). The simplest form of MWCNT is a double-walled carbon nanotube (DWCNT) (Karthik et al., 2021).

Recently, studies on enzyme immobilization over CNTs have increased rapidly since these materials have high surface area, capability of enhanced enzyme loading, and low mass transfer hindrances. For instance, in a study conducted by Xu et al. (2015) laccase was immobilized on a novel composite membrane (polyvinyl alcohol/chitosan/MWCNTs) (Xu et al., 2015). The immobilization was completed through surface modification of the membrane with glutaraldehyde. The final product was shown to maintain 80% of initial laccase activity after seven cycles of operation. As mentioned previously, industrial application of nanoparticles due to their small sizes could be challenging, especially their handling in the environmental arena. Most studies in the field of enzyme immobilization on graphene and carbon nanotubes are related to biosensor applications. In addition, plasma based treatment/production of CNTs may result in better immobilization/loading of laccase. Plasma based treatments are non-polluting in nature and can provide a wide range of functional groups (Ruelle et al., 2011). To the best of our knowledge, this technique has not been used for the immobilization of laccase on plasma treated CNTs. However, Othman et al. (2016) used MWCNTs synthesized using plasma enhanced chemical vapor deposition for the immobilization of laccase (Othman et al., 2016)

Activated carbon (AC) denotes amorphous carbonaceous materials with good chemical and physical characteristics (Barroso Bogeat, 2021). Its high surface area (600–1300 m² g⁻¹) with large number of contact sites makes activated carbon a valuable support for enzyme immobilization (Karthik et al., 2021). Previous studies have demonstrated that natural activated carbon or functionalized activated carbon with HCl could act as a support in laccase immobilization (Sirisha et al., 2016). Recently mesoporous activated carbon with large contact sites has been using for laccase immobilization as well as acid protease and acid lipases immobilization (Ganesh Kumar et al., 2010; Datta et al., 2013). In a study, activated carbon fibers modified with dopamine was utilized as a support for laccase obtained from Aspergillus sp. immobilization (Zhang C. et al., 2018). The results indicated that the biocatalyst had the capability of maintaining its activity (around 60% of initial laccase activity) after six cycles of operation while free laccase only kept 40% of initial activity after the same number of operations (Zhang C. et al., 2018). Table 4 presented various studies of immobilization of enzymes on carbon based materials.

Kinetic parameters such as Km, Vmax and the catalytic efficiency kcat/Km determine the catalytic action of enzymes. These parameters can vary considerably depending on the types of enzymes, support materials and process conditions. The Michaelis constant (Km) expresses the affinity of the laccase to the substrate. Vmax is the maximum reaction rate. Low apparent Vmax can result from mass transfer limitations and reduction in enzyme–substrate affinity after immobilization (Gahlout et al., 2017). The Vmax/Km ratio reflects the catalytic efficiency of the enzyme-substrate system. Some values of kinetic parameters related to free laccase and its immobilized counterparts formed using different techniques and carriers are reported in Table 6.

A number of studies have been performed using immobilized laccases for the biotransformation of organic contaminants. Most of these studies have been conducted using synthetic wastewater, however a few of them also involved real wastewater, at laboratory or pilot scale. Due to the immobilized enzymes’ overall stability over free enzymes and their recyclability, they generally exhibited higher removal. Table 7 summarizes some of the very recent studies on the application of immobilized laccase for emerging contaminant removal.

BC sources can be divided into two categories, i.e., BCs produced from lignocellulosic materials and BCs produced from non-lignocellulosic materials (Stella Mary et al., 2016; Karim et al., 2019). Lignocellulosic biochars can be divided into three different subcategories namely: wood (hardwood or softwood), crop waste, and grass and leaves (Ippolito et al., 2020). Non-lignocellulosic biochars mainly come from sewage sludge, manure, and algae (Ippolito et al., 2020; Pandey et al., 2020). From lignocellulosic sources, corn, wheat straw, and rice/husk straw are commonly used (Ippolito et al., 2020). Regarding non-lignocellulosic sources poultry, pig, and cattle manure are the most common sources for biochar production (Ippolito et al., 2020). Feedstock significantly affect the carbon content, surface area, and functional groups of final products (Novak et al., 2019). Normally carbon content is proportionally related to biomass lignin content. Biochars produced from wood feedstock demonstrates higher carbon content compared to other sources (Wang et al., 2016). Biochars produced from manure normally have higher content of N, S, and P (Ippolito et al., 2020). In the terms of surface area, lignocellulosic biochars have higher surface and among different sources, wood-based biochar represent higher surface area (Lehmann and Joseph, 2009; Weber and Quicker, 2018). Biochar produced from manure usually have low surface area due to structural cracking or micropore blockage (Ahmad et al., 2014; Ippolito et al., 2017). Regarding functional groups, normally lignocellulosic biochars exhibit content of hydroxyl and carboxyl bonds on their surface (Pandey et al., 2020). However, manure-based biochars demonstrate amine groups on their structures (Leng et al., 2019). The amine content on biochars obtained from different biomasses is followed the pattern in order of wood biochars<crop biochars<grass biochars<manure biochars (Ippolito et al., 2020).

There are two kinds of pyrolysis, slow and fast. During slow pyrolysis, low temperature heating rate (0.01^–2 Cs⁻¹) would be implemented (Sohi et al., 2009). However, temperature heating rate would be higher than 2°Cs⁻¹ in fast pyrolysis. Pyrolysis type would affect surface area and average particle size (Ippolito et al., 2020). Biochar produced through fast pyrolysis usually have higher surface area compared to biochars produced with slow pyrolysis; however, fast pyrolysis biochars demonstrate lower average particle size compared to slow pyrolysis biochars (Asadullah et al., 2010; Qambrani et al., 2017).

Temperature is considered as a significant parameter that affects biochar physiochemical properties. Biochar porosity and surface area would crucially change by pyrolysis temperature variation. Generally, at higher temperature, larger pore volume and surface area would be expected (Mendonça et al., 2017; Weber and Quicker, 2018). Pyrolysis temperature could also affect the content of functional groups and aromatic structure of biochar. Biochar produced at temperature above 500°C demonstrate lower amount of O- and H-containing functional groups (Janu et al., 2021). However, biochars produced below 500°C exhibits higher O- containing functional groups (Janu et al., 2021). For instance, Li X. et al. (2013) studied how variation in pyrolysis temperature could affect biochar properties. The obtained results from two-dimensional (2D) ¹³C nuclear magnetic resonance (NMR) demonstrated the lower aromaticity ratio (H/C) and lower polarity (O/C and (O+N)/C ratios. This could happen because at higher temperature, the carbon content would increase while H, N, and O contents would decrease (Li X. et al., 2013).

In the physical activation approach, no chemical agents are implemented, and this methodology is considered as an economical and simple approach (Rajapaksha et al., 2016). Physical activation of biochar involves the use of gases such steam, CO₂, and ozone at temperatures above 700°C (Jimenez-Cordero et al., 2015; Shen et al., 2015; Shim et al., 2015). This modification can be summarized into two steps: first biochar surface area is increased through modification of its unstructured parts and second its crystalline-C formation is improved (Jung and Kim, 2014, 2014; Cha et al., 2016). Park et al. (2016) studied the effect of steam modification on BC surface. In this study BC was produced from P. tenera at 500°C and steam modification was carried out at 700°C for 1 h. The results confirmed that while the surface area of untreated BC was close to zero, that of treated BC increased to 22 m² g⁻¹ (Park et al., 2016).

During chemical modification, BC is mixed with a chemical agent and through dehydration and oxidation, its properties can change (Xiang et al., 2020). Despite its drawback such as the high cost of chemicals, and inability to recover and reuse such chemical agents, this method has a higher efficiency compared to physical activation (Cha et al., 2016). Chemical treatment of BC is achieved using strong acids such as H₃PO₄, HCl, and H₂SO_4, and strong bases such as KOH, NaOH, and NH₃ (Cha et al., 2016; Zhang C. et al., 2020; Pandey et al., 2020).

Acid treatments normally promote the emergence of oxygen-containing functional groups together with increasing surface area (Rajapaksha et al., 2016). In a study of covalent laccase immobilization on modified BC Lonappan et al. (2018b) used raw BC from pinewood, pig manure, and almond shell. Through BC modification with citric acid, more carboxylic groups were observed on its surface compared to untreated BC (Lonappan et al., 2018b).

BC alkalinization enhances non-polarity with increasing surface area and functional group content. Jin et al., 2014) studied the effects of KOH on the BC produced from municipal solid wastes (Jin et al., 2014). FTIR analysis demonstrated that the number of hydroxyl and carboxyl groups on the surface of treated BC was increased (Jin et al., 2014). In addition, surface area was increased from 14.4 m² g⁻¹ for raw BC to 49.1 m² g⁻¹ for treated BC (Jin et al., 2014).