Keywords

7.1 Introduction

White-rot basidiomycetes (WRB) belong to one of the most diverse and important groups of living organisms because of their role in ecosystem function and unique ability to degrade and completely metabolize all wood polymers due to their capability to synthesize required hydrolytic and oxidative enzymes. Lignin degradation is an oxidative process catalyzed by extracellular and non-specific lignin-modifying enzymes (LME) composed of laccase (EC 1.10.3.2), manganese peroxidase (MnP, EC 1.11.1.13), lignin peroxidase (LiP, EC1.11.1.14), and versatile peroxidase (VP, EC1.11.1.16) with the assistance of several auxiliary enzymes. Some WRB secrete several different LME, while others produce only one or two of them, usually as a set of isoenzymes encoded by multiple genes within one fungal species (Lundell et al. 2010; Piscitelli et al. 2011; Janusz et al. 2013; Peng et al. 2018).

Among various industrial enzymes, LME have attracted tremendous attention due to the wide range of industrial and biotechnological applications (Yadav and Yadav 2015; Mäkelä et al. 2016; Chowdhary et al. 2019). However, the wide implementation of LME at the industrial scale is delayed because of too high enzyme cost due to the relatively low yield in the hundreds of explored strains belonging to various taxonomic groups. To solve this problem, the exploitation of molecular biology approaches was widely considered. Various techniques have been utilized to modify enzymes for industrial purposes. Artificial intelligence and computational tools have provided better results for modification and utilization of enzymes (Kumar et al. 2019; Dixit et al. 2019). Nevertheless, although many industrial enzymes (amylases, cellulases, etc.) are efficiently overproduced in heterologous systems, high expression of LME by recombinant organisms has not been yet obtained (Mekmouche et al. 2014). The highest yields of laccase were obtained when the enzyme from Moniliophthora roreri and Pycnoporus cinnabarinus was expressed in Pichia pastoris (281 U/mL) (Bronikowski et al. 2017). At the same time, several wild strains of WRB overproducing individual LME have been discovered (Galhaup et al. 2002; Revankar and Lele 2006; Elisashvili et al. 2014; Coconi-Linares et al. 2014; Kachlishvili et al. 2014; Schneider et al. 2019).

To increase LME yields, various approaches and strategies such as exploitation of cheap plant raw materials as growth substrates, optimization of fermentation media and cultivation conditions, development of better bioprocess technologies have been widely exploited (Elisashvili and Kachlishvili 2009; Piscitelli et al. 2011; Li et al. 2016; Park et al. 2015; Martani et al. 2017). Literature date evidence that the synthesis and secretion of LME and their isoenzymes are affected by many factors, but the effects of these factors vary among the fungal species (Terrón et al. 2004; Xiao et al. 2006; Piscitelli et al. 2011; Yang et al. 2013; Bertrand et al. 2013, 2014). The results and advances in strain engineering, transcriptional regulation in response to environmental conditions, and biotechnological applications of LME have been comprehensively and excellently reviewed (Yadav and Yadav 2015; Mäkelä et al. 2016; Martinez et al. 2017; Chowdhary et al. 2019), but an exhaustive overview of the basic aspects of modulating enzyme activity through alterations of the cultivation conditions is still lacking in the literature. In this chapter, we summarize recent literature reports and our data on the physiological features of LME production by WRB, focusing on the diversity, common characteristics, and unique properties of individual fungi as well as on several approaches and strategies that provide enhanced (or reduced) secretion of laccases and peroxidases.

7.1.1 Screening of LME Producers

Screening of WRB species is an important initial stage for selecting promising LME producers. It aims to discover novel fungal strains (1) producing high yield and (2) the right set of LME, (3) exhibiting enhanced physical, chemical, and catalytic properties, and (4) suitable for specific industrial applications. Moreover, LME of some basidiomycetes, such as Phanerochaete chrysosporium (Kirk and Farrell 1987), are formed during secondary metabolism; therefore, it is important to detect fungi that efficiently produce LME during primary metabolism.

A countless WRB belonging to different taxonomic groups have been screened to identify industrially significant producers of LME, but only a few were able to produce encouraging amounts of target enzymes (Galhaup et al. 2002; Revankar and Lele 2006; Songulashvili et al. 2012; Kachlishvili et al. 2014), MnP and LiP (Kapich et al. 2004; Singh and Chen 2008; Elisashvili and Kachlishvili 2009; Coconi-Linares et al. 2014). It is worth noting that in some cases the direct comparison and correct assessment of WRB biosynthetic potential and enzyme yields are difficult due to the differences in media composition and fermentation conditions as well as the use of various methods and analyses to evaluate the LME activity.

Sergentani et al. (2016) evaluated the enzyme activity of twenty-eight strains in liquid cultures with wheat bran as a suitable growth substrate. Laccase activity (0.12–30.14 U/mL) was detected in the culture supernatants of all strains except Hydnum repandum, Pholiota adiposa, and Pycnoporus cinnabarinus. Neither LiP nor MnP was detected even in Trametes spp., although these fungi are known producers of MnP and LiP (Levin et al. 2010; Kachlishvili et al. 2018). Recently, Kinnunen et al. (2017) performed an automated and miniaturized screening and comparison of 53 species of basidiomycetes for LME production in their cultivation in liquid mineral, soy, peptone, and solid-state oat husk media. Among the tested fungi, MnP (96%) and laccase (92%) producers were the most widespread. Strains of Phlebia radiate and Trametes ochracea showed the highest LiP activity; P. radiate appeared to be the best candidate for VP production in soy liquid medium. In this study, the highest enzyme activities were produced on lignocellulose-containing media. By contrast, low laccase activity (0.01–1.97 U/mL) was revealed in 34 basidiomycetes screened in the stationary cultivation using malt extract broth without lignocellulose (Bodke et al. 2012), while only 5 of 21 endophyte fungi were able to produce low laccase activity (0.01–0.04 U/mL) but not peroxidase activity in modified Kirk’s liquid medium (Fillat et al. 2016). There is other evidence that not all WRB species readily express LME activity in a defined medium (Schlosser et al. 1997; Kapich et al. 2004; Elisashvili and Kachlishvili 2009).

Therefore, in our screening studies, we simultaneously evaluated the LME activity of fungi during their cultivation in media containing mandarin peels and glycerin, which provide abundant fungal growth and significant enzyme production (Elisashvili and Kachlishvili 2009; Kachlishvili et al. 2014, 2018; Elisashvili et al. 2017). These studies revealed several features that should be considered during screening experiments. Firstly, in concordance with available literature data, our results clearly showed a wide intra- and interspecies diversity in the ability of WRB to produce LME. Thus, in the submerged fermentation of mandarin peelings, the laccase activity of Ganoderma spp. varied from 2.0 U/m/L to 75.4 U/mL, while the MnP activity of Trametes spp. strains ranged from 0 to 0.9 U/mL. Secondly, the WRB manifested different responses to the used carbon sources. Among them, Cerrena spp., Coriolopsis gallica, Pseudotrametes gibbosa, and T. versicolor produced significant laccase activity in synthetic medium, whereas the presence of lignocellulosic material was a prerequisite for the enzyme production by Ganoderma spp., Phlebia radiata, Pycnoporus coccineus, and Trametes ochracea. Likewise, 13 fungal strains secreted laccase (0.2–9.4 U/mL) in the 1% glycerol-containing medium, but supplementation of this medium with 20 g/L milled mandarin peels 2 to 22-fold enhanced their laccase activity (to 1.2–38.3 U/mL) (Elisashvili et al. 2017). Moreover, only Coriolopsis gallica was capable to express LiP activity in the synthetic medium, whereas in the lignocellulose-based medium all fungi, with the exclusion of Merulius tremellosus produced appreciable levels of this enzyme. In another work (Kachlishvili et al. 2018), the substitution of glycerol with mandarin peels two- to tenfold increased the laccase activity of Trametes spp., although T. versicolor 775 better produced this enzyme in the synthetic medium. Besides, the use of mandarin peels as the fungal growth substrate stimulated or even induced MnP and LiP production. On the whole, an analysis of literature data shows that for the greatest production of individual LME by specific WRB, a certain composition of the nutrient medium is required, and the use of an inappropriate medium may lead to non-detection of promising enzyme producer.

7.1.2 Effect of Lignocellulosic Substrates

Understanding the mechanisms of regulation of the individual LMEs synthesis under specific growth conditions and elucidating the cultivation conditions ensuring their predominant production is a critical necessity. In particular, to ensure abundant fungal growth and efficient production of LME, it is important to select a suitable lignocellulosic substrate rich in readily available carbohydrates, nitrogen, trace elements, and inducers for the synthesis of target enzymes (Winquist et al. 2008; Elisashvili et al. 2009; Kachlishvili et al. 2018). Thus, a study conducted by Mikiashvili et al. (2006) with Pleurotus ostreatus 98 showed that substitution of glucose with mandarin peels 40-fold and 18-fold increased the fungus laccase and MnP activities, respectively. Likewise, the cultivation of P. radiata 79 in low-nitrogen defined medium with milled alder as sole carbon source induced tenfold production of LiP and increased MnP activity, in comparison to the glucose-supplemented cultures (Mäkelä et al. 2013). Laccase and MnP activities were the highest in cultures of the P. radiata grown on birch wood (Villavicencio et al. 2020). On liquid ME medium, however, the production of laccase by P. radiata during 4 weeks of cultivation was very low, although distinct MnP activities were produced by the fungus. In another study, when the basal medium including 20 g/L glucose as a carbon source was supplemented by 10 g/L of orange peel, tea, bagasse, and corn cobs, the laccase production by P. ostreatus was improved 9-, 5-, 2-, and 1.1-fold, respectively (Zhao et al. 2017). It is worth noting that the proliferation capacity of the culture with orange peel extract was 0.5-fold higher than that of control. The more orange peel (ranged from 0 to 10 g/L) was added to the culture with glucose, the better was the fungus growth and enzyme production.

Undoubtedly, lignocellulosic biomasses differ greatly in their chemical composition, physical, mechanical, and other properties affecting fungal metabolism and productivity. For example, the tested residues provided equally good growth of C. unicolor and Phellinus robustus in their submerged fermentation, but the fungal laccase activity varied from 15.7 U/mL to 151.6 U/mL and from 0.9 U/mL to 8.4 U/mL, respectively (Elisashvili and Kachlishvili 2009). Wheat bran and residue after ethanol production appeared to be the best growth substrates for laccase production by C. unicolor, whereas mandarin peels and kiwi and walnut pericarp favored enzyme secretion by P. robustus. Simultaneously, as compared with wheat bran, the fermentation of kiwi residue stimulated MnP secretion by P. robustus, whereas walnut pericarp 12-fold augmented activity of this enzyme in C. unicolor.

Coffee husk (CH) and citric pulp pellet (CP) from an orange juice industry used as a carbon source for the submerged cultivation of Lentinus crinitus without an additional nitrogen source provided the secretion of 33.4 and 29.1 U/mL laccase activity, respectively(Almeida et al. 2018). Since CH contains caffeine, polyphenols, and tannins, while CP contains polyphenols (mainly flavonoids) soluble in water, the authors assumed that these compounds induced the laccase production by L. crinitus. Nevertheless, no MnP or LiP was detected in the fungus cultivation. On the contrary, Conceição et al. (2017) reported the production of MnP but not laccase in the cultivation of L. crinitus on a solid medium with barley and cassava residues (1:1). The stimulating effect of water-soluble aromatic compounds obtained from lignocellulosic substrates on the synthesis of LME has been shown by many authors (Crestini et al. 1996; Kapich et al. 2004; Adekunle et al. 2017). The induction of laccase isoforms by aqueous extracts from softwood and hardwood was shown in T. versicolor HEMIM-9; specifically, the pine, cedar, and oak extracts addition to the fungal cultures increased 4.6-, 4.5-, and 3.7-fold, respectively, the fungus laccase activity (Bertrand et al. 2014). The authors suggested that not only the concentration of phenolic compounds in aqueous extracts but also the origin or composition of the extract may influence the induction of laccase. Moreover, native isoelectric focusing of laccases that were isolated from the control and the induced cultures revealed differences in isoform number (from 3 to 6) and pI values. According to the researchers, some isoforms of T. versicolor HEMIM-9 are downregulated, while others are upregulated in the presence of the tested inducers.

The available results suggest that kind and composition of lignocellulosic materials determine the set and yield of LME produced by the WRB. Thus, Janusz et al. (2018) compared the transcriptomes of C. unicolor FCL139 grown in the solid-state growth conditions on birch, ash, maple sawdust with those of fungus grown on mineral medium. It was found that the expression of MnP XLOC_004360 was upregulated during the fungus growth on maple sawdust, the expression of the MnP XLOC_004631 was specifically induced in the fungus cultivation on ash and maple but it was downregulated during C. unicolor growth on the birch medium. Analyses of laccase transcripts (XLOC_008955) revealed their reduced amounts in the fungus cultivation on the birch and ash sawdust-containing media.

It should be noted that the composition of some lignocellulosic substrates may not correspond to the nutritive demands of the fungus; then nutrient medium requires supplementation with an additional carbon source. Thus, the laccase activity of P. ostreatus in the fermentation of rice bran and other plant raw materials appeared to be lower as compared with that in the control culture with glucose (2.2 U/mL) (Selvaraj et al. 2014). However, the combinations of rice bran with sugarcane bagasse, corn stalks, or orange peelings showed laccase activity of 3.24 U/mL, 2.96 U/mL, and 2.6 U/mL, respectively, whereas supplementation of these media with 0.2 g glucose further increased laccase activity to 6.47 U/mL, 4.41 U/mL, and 5.71 U/mL, respectively.

Overall, the literature data demonstrate a clear regulatory role of individual lignocellulosic materials in LME activity expression. Undoubtedly, the chemical composition and other characteristics of lignocellulosic biomass play a decisive role in the production of LME; in particular, phenol-containing plant materials, such as coffee husks and by-products of citrus processing, provide the highest production of enzymes. However, the deconstruction of complex lignocellulosic substrates requires the participation of a consortium of various enzymes acting in synergy to provide the microorganism with the necessary nutrients. Therefore, LME producers simultaneously expressing high hydrolytic activity may be more attractive for the successful production of target enzymes during lignocellulose fermentation.

7.1.3 Effect of Carbon Source

Among the medium components, carbon sources and concentrations are the most important factors determining the rate and degree of fungal biomass accumulation, displaying diverse effects on the LME production, depending on the fungal strain (Galhaup et al. 2002; Elisashvili et al. 2002; Stajić et al. 2006; Elisashvili and Kachlishvili 2009; Piscitelli et al. 2011). For example, in the submerged cultivation of Trametes pubescens in the presence of glucose, the sugar was rapidly metabolized and resulted in high laccase activity, but active enzyme secretion by the fungus was observed when the glucose concentration in the growth medium decreased to a certain low concentration (Galhaup et al. 2002). Slowly utilized lactose and cellulose resulted in poor laccase production. Likewise, laccase activity obtained in the cultivation of Pleurotus sajor-caju in media containing 0.5 g/L fructose or glucose was 12-fold higher than that obtained with lactose (Bettin et al. 2008). On the contrary, exactly lactose ensured the maximum laccase secretion by P. gibbosa (Elisashvili and Kachlishvili 2009). Interestingly, starch appeared to be the best carbon source for the laccase production by WRBWR-1 fourfold increasing enzyme activity compared with fructose-containing medium (Revankar and Lele 2006). Myasoedova et al. (2015) observed the highest laccase activity of Lentinus strigosus 1566 when peptone-yeast extract medium was supplemented with galactose, arabinose, and xylose at a final concentration of 20 g/L. The presence of glucose, sucrose, or maltose in the medium led to a decreased laccase activity. The researchers demonstrated the selectivity of the L. strigosus 1566 towards mono- and disaccharides synthesizing different sets of laccase isozymes.

Early observations established that the production of LME in some fungi occurs in response to carbon depletion (Kirk and Farrell 1987). Then it was shown that individual laccase isozymes of basidiomycete I-62 (Mansur et al. 1998), T. pubescens (Galhaup et al. 2002), and Trametes sp. AH28-2 (Xiao et al. 2006) are differentially regulated by carbon and synthesis of some of them is subjected to catabolite repression by glucose or other easily metabolizable carbon sources. Moreover, many fungi such as P. coccineus (Kachlishvili et al. 2016), species of genus Ganoderma (Songulashvili et al. 2012; Elisashvili and Kachlishvili 2009) produce very low laccase activity in defined media containing glucose or glycerol. Therefore, in our study, to elucidate if there is a mechanism of catabolite repression of LME synthesis the mandarin squeeze-based medium (control) was supplemented with glucose before inoculation and after 4 days of the submerged fermentation (middle of logarithmic phase of growth). In addition to P. coccineus and Ganoderma lucidum, Trametes trogii and T. versicolor, which produce marked laccase activity in the presence of glucose, were studied for comparison. Laccase activity of P. coccineus in the control medium gradually increased to 4.2 U/mL peaking on day 5 (Fig. 7.1a). Supplementation of the control medium with 0.5% and 1% glucose delayed laccase secretion and gave lower enzyme activities after 3 days of the fermentation (2.1 and 1.0 U/mL vs. 2.6 U/mL in the control). However, subsequently, laccase activity rapidly increased to 5.6 and 7.5 U/mL, respectively, on day 8 significantly exceeding that in the control medium owing to higher biomass accumulation. When 0.5% glucose was added to the growing fungal culture no increase in enzyme activity was observed during 4 days of cultivation, then the enzyme secretion resumed. In the cultivation of G. lucidum, almost no delay in the laccase production was observed due to the supplementation of glucose in the initial medium, but enzyme activity was completely suppressed during 1 day when 0.5% glucose was added to the growing culture (Fig. 7.1b). However, without a transcriptomic analysis, the data obtained are insufficient to confirm the presence of catabolite repression of laccase synthesis in P. coccineus and G. lucidum, since the growth of exactly these fungi in the presence of glucose was accompanied by particularly strong acidification of the medium, which of course negatively affected the synthesis and activity of laccase. Finally, unlike these fungi, T. trogii (Fig. 7.1c) and T. versicolor (Fig. 7.1d) responded to the addition of glucose to the control medium by an accelerated secretion of laccase activity. This means the absence of catabolite repression in the tested Trametes species and using an additional carbon source is the right strategy to increase the yield of target enzyme.

Fig. 7.1
figure 1

The effect of glucose addition in the production of laccase during the submerged fermentation of mandarin squeeze by four basidiomycetes species. (a) Pycnoporus cinnabarinus 811; (b) Ganoderma lucidum 447; (c) Trametes trogii 146; (d) Trametes versicolor 159. 1 medium containing 40 g/L mandarin squeeze (control); 2 control + 0.5% glucose; 3 control + 1% glucose; 4 control + 0.5% glucose after 4 days of cultivation

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It is interesting that Cerrena sp. HYB07 showed especially distinctive features since very high carbon concentration (malt dextrin, 60 g/L) in the fermentation medium with a high concentration of nitrogen was beneficial for laccase production (280 U/mL) (Yang et al. 2016). Substitution of malt dextrin by glucose slightly increased laccase yield, whereas fructose decreased laccase production by 60%. Moreover, disaccharide maltose did not affect laccase yields, sucrose lowered laccase yield by 15.5%, whereas glycerol threefold decreased the fungus enzyme activity. At the same time, Coriolopsis gallica T906 expressed the highest laccase activity in medium with glycerol and ammonium dihydrogen phosphate as carbon and nitrogen sources, respectively (Xu et al. 2016).

Recently, Schneider et al. (2018) investigated LME activity of Marasmiellus palmivorus VE111 in media containing glucose, sucrose, or glycerol and showed that the medium prepared with glucose ensured the highest laccase and MnP activities. It is interesting that the effect of the carbon source on laccase secretion significantly depended on nitrogen source (casein was better than peptone). Usually, media that provided higher fungal biomass resulted in higher enzymatic activity, medium prepared with sucrose and casein ensured the best growth of the mushroom, but not the highest laccase activity. In our study with Trametes multicolor, the lowest laccase activity was detected in the fungus cultivation in the sodium gluconate-based medium (Kachlishvili et al. 2018). It turned out that mannitol, as well as cellobiose and xylose, were the best carbon sources for laccase production by T. multicolor but increased enzyme activities were not due to higher biomasses accumulated in the presence of these carbohydrates. In particular, calculation of the specific laccase activity showed that mannitol, cellobiose, and xylose favored enzyme production by the fungus, increasing more than two- to threefold the specific laccase activity compared with the control medium. Likewise, mannitol more than fourfold increased the T. multicolor MnP activity compared to the control medium. However, unlike laccase, it did not promote the secretion of MnP, since the specific activity of MnP in the medium with mannitol decreased 1.5-fold compared with the control medium.

These and many other observations indicate that some carbohydrates can modulate LME expression in WRB; therefore, to maximize the synthesis of LME, it is necessary to find out the carbon source optimal for the fungal growth and the target enzyme production. Yet, to correctly assess fungal enzymatic potential, it is necessary to take into account the amount of biomass accumulated in the presence of a specific carbon source. Moreover, in fungal cultures, the metabolism of various carbohydrates is accompanied by varying degrees of acidification of the nutrient medium. In turn, media with different pH differently affect both the synthesis of a target enzyme and the stability of the already synthesized enzyme.

It is clear that a carbon source, especially, in the form of lignocellulosic biomass, plays a principal role in modulation of basidiomycetes LME activity, but for each enzyme producer, fungus-specific carbohydrate ensuring the highest enzyme activity must be established. Some published data on the laccase activity of WRB, given in Table 7.1, show their diversity in their requirements for a carbon source to achieve maximum laccase activity.

Table 7.1 WRB laccase activity (ABTS) under submerged cultivation conditions
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7.1.4 Effect of Nitrogen Source

Many studies verified the effect of organic and inorganic nitrogen sources in the fermentation medium on the WRB enzyme activity to create the best nutritional conditions for the maximum production of LME. Thus, the assessment of the effect of different nitrogen sources (20 mM as nitrogen) on the LME production by T. multicolor was assessed in the submerged fermentation of mandarin peels (Kachlishvili et al. 2018). The lowest activities of laccase, MnP, and LiP were revealed in the medium with potassium nitrate. This nitrogen source and yeast extract provided even lower specific laccase activity (2.4 and 2.9 U/mg, respectively) compared with that in the control medium. On the contrary, casein hydrolysate favored the laccase production with the highest productivity (12.3 U/mg), obviously, due to available aromatic amino acids. Likewise, the casein hydrolysate ensured the highest specific activity of MnP activity increasing almost twofold specific activity of this enzyme compared with that in the control medium.

It is considered that compared with organic nitrogen sources inorganic ones provide lower enzyme yields despite sufficient biomass accumulation (Piscitelli et al. 2011). Indeed, laccase activity was not observed with any inorganic nitrogen source in the cultivation of Grammothele fuligo in glucose-based medium although it was detected in the control medium containing ammonium chloride as a nitrogen source in experiments with various trace elements (Chauhan 2019). It is useful to note that in this study, the enzyme activity was measured only after 12 days of fungus cultivation. At the same time, the fungus showed maximum LiP activity with ammonium chloride and MnP activity with ammonium acetate. In experiments with T. multicolor, ammonium sulfate and ammonium nitrate were suitable sources of nitrogen for the production of laccase and MnP, respectively (Kachlishvili et al. 2018). Besides, ammonium sulfate provided two times higher LiP activity compared with that in the control medium, although the calculations showed it was achieved due to the higher fungal biomass. Moreover, the highest values of laccase activity were observed for P. eryngii 616 and P. ostreatus 493 when (NH4)2SO4 and NH4Cl, respectively, were supplemented to the medium (Stajić et al. 2006). Likewise, inorganic nitrogen supported high laccase activity in the cultures of Pleurotus dryinus, Pleurotus tuberregium, and Pleurotus pulmonarius (Kachlishvili et al. 2006; Stajić et al. 2006), but organic nitrogen was favorable for high production of MnP in the culture of P. ostreatus 108 (Mikiashvili et al. 2006). These and many other data indicate the diversity of the nutritional needs of fungi for nitrogen sources necessary to express their enzymatic potential and also evidence that the nitrogen source optimal for one type of enzyme does not necessarily contribute to the production of another.

The highest value of Coriolopsis gallica T906 laccase activity was obtained with ammonium dihydrogen phosphate as nitrogen sources (Xu et al. 2016). Ammonium sulfate and casein were also good sources of nitrogen. Under the solid-state fermentation (SSF) of peach waste by Pleurotus eryngii supplementation of medium with 4.0 g/L ammonium nitrate resulted in 12.8 times higher laccase activity than in control culture (Akpinar and Urek 2017). Among nitrogen sources tested in the submerged fermentation of coffee husk (CH) and citric pulp pellet (CP) by Lentinus crinitus, only urea favored laccase production in both media, while the addition of yeast extract in CH medium and sodium nitrate in CP medium decreased enzyme activity as compared with control medium (Almeida et al. 2018). Interestingly, the urea concentration of 0.7 g/L was sufficient for maximum laccase production in CH medium and the enzyme production was completely inhibited at the urea concentration of 11.2 g/L whereas exactly this concentration of urea provided the highest laccase activity in the CP medium. The authors suggested that the effect of nitrogen on the laccase production by L. crinitus depends on the carbon substrate in the culture medium.

The production of fungal LME depends not only on the chemical nature but also on the concentration of nitrogen. In P. chrysosporium synthesis of MnP and LiP is suppressed by high-nitrogen concentrations in the medium and it is induced only during secondary metabolism in response to nitrogen limitation (Kirk and Farrell 1987). However, in the fermentation of lignocellulosic substrates, even a high concentration of organic nitrogen did not inhibit enzyme production in P. chrysosporium ME-446, but instead stimulated it (Kapich et al. 2004). Laccase, MnP, and LiP activities of Coriolopsis polyzona reached significantly higher values under low-nitrogen (2.2 mM) compared with high-nitrogen (20 mM) conditions (Jaouani et al. 2006). On the contrary, a tenfold increase in laccase production of Ganoderma lucidum was observed when nitrogen was increased from 2.4 to 24 mM (D’Souza et al. 1999). Interestingly, in the cultivation of P. dryinus only laccase activity increased with nitrogen concentration, whereas the MnP production was not affected (Kachlishvili et al. 2006). Especially distinctive features showed Cerrena sp. HYB07 since high carbon (malt dextrin, 60 g/L) and nitrogen (peptone, 10 g/L and ammonium tartrate, 1.6 g/L) concentrations in the fermentation medium provided the highest laccase production (280 U/mL), whereas low-nitrogen concentrations reduced laccase yields to 5.3 U/mL (Yang et al. 2016).

Finally, some above-mentioned and other studies indicate that the ratio of carbon to nitrogen (C/N) is important for the maximum production of the LME. Thus, a high carbon–nitrogen ratio favored laccase productions by Trametes gallica (Dong et al. 2005); in contrast, high laccase activity of P. ostreatus was observed with low carbon–nitrogen ratio (Hou et al. 2004). These data show a wide variety in the nitrogen requirements of WRB for the production of LME as well as some uncertainty regarding the selection of the optimal nitrogen concentration for enzyme production.

7.2 Aromatic Compounds

The effect of a wide variety of aromatic or phenolic compounds, especially of structurally related to lignin compounds, in the production of LME by WRB has been extensively studied (Eggert et al. 1996; Elisashvili and Kachlishvili 2009; Cambria et al. 2011; Bertrand et al. 2017; Daly et al. 2020). 2,5-Xylidine was the most widely used as a potent inducer of laccase synthesis (Elisashvili et al. 2002; Revankar and Lele 2006; Birhanlı and Yeşilada 2017; Lallawmsanga et al. 2019). However, the laccase production by T. versicolor increased twofold when veratryl alcohol or guaiacol was used instead of 2,5-xylidine (Lee et al. 1999). At the same time, in Lentinus strigosus veratryl alcohol insignificantly increased the laccase synthesis, whereas 2,6-dimethylphenol promoted eightfold compared to the control (Myasoedova et al. 2008). In the submerged fermentation of mandarin squeeze by T. multicolor 511, veratryl alcohol and guaiacol twofold increased the specific laccase activity compared with the control medium, guaiacol fourfold increased the fungus MnP activity, while veratryl alcohol favored LiP secretion (Kachlishvili et al. 2018). Kumari et al. (2019) observed a differential effect of inducers on the secretion of LME by Stereum ostrea in a defined medium containing chlorpyrifos. Gallic acid induced maximum secretion of laccase and MnP; however, the highest production of LiP was recorded in the presence of veratryl alcohol. Interestingly, induction with 0.5 mM resveratrol showed the best laccase activity of C. gallica, followed by tannic acid and guaiacol, while ABTS and gallic acid did not affect laccase production (Xu et al. 2016). On the contrary, ABTS and guaiacol increased the Cerrena sp. HYB07 laccase production by 40.1 and 26.7%, respectively, whereas other aromatic compounds did not significantly affect enzyme production (Yang et al. 2016). It is interesting that in another strain of C. unicolor VKMF-3196 laccase production needed Cu2+ ions, but not aromatic compounds (Lisova et al. 2010). These results suggest that the effect of inducer for laccase production depends on the species and/or strains of the tested fungus.

In the early work, Soden and Dobson (2001) showed that 300 μM xylidine, 100 μM 1-hydroxybenzotriazole, 100 μM ferulic acid, and 500 μM veratric acid resulted in 21-, 16-, 10-, and 5-fold increases of laccase activity of P. sajor-caju, respectively, compared to the basal culture with no additions. Since one of the functions of fungal laccases is the polymerization of aromatic compounds formed during the degradation of lignin these authors, similarly to Eggert et al. (1996), proposed that laccase induction is a protective reaction of fungal culture to avoid the toxic effects of these compounds. Moreover, it turned out that in some WRB different laccase isoenzyme genes responded differently to the same aromatic inducer. Thus, in Trametes sp. AH28-2 o-toluidine induced expression of the laccase A gene, while 3,5-dihydroxytoluene favored laccase B production (Xiao et al. 2006). Guaiacol and p-coumaric acid selectively induced lcc1 and lcc2 expression in Trametes sp. I-62 but ferulic acid induced lcc3 expression (Terrón et al. 2004). In the cultivation of P. ostreatus HAUCC 162 ferulic, vanillic, coumaric, and cinnamic acids increased the expression of lacc8 and lacc11 genes, but not lacc1 and lacc2 (Zhuo et al. 2017).

Besides fungal species peculiarities, the effect of aromatic compounds on LME production depends on inducer concentration and adjusting the concentration of phenolic/aromatic compounds necessary for individual fungi is essential. In particular, a gradual increase in the concentration of ABTS from 0 to 0.05 mM and 2,5-xylidine from 0 to 1 mM in the glucose-yeast extract basal medium (SBM) increased T. versicolor ATCC 200801 laccase activity from 0.6 U/mL to 4.76 U/mL and 2.87 U/mL, respectively (Birhanlı and Yeşilada 2017). Higher concentrations of both compounds significantly decreased the enzyme yields. It is worth noting that under the same cultivation conditions, no induction of laccase production was observed in SBM with 0.025–0.5 mM syringaldazine.

Finally, Lallawmsanga et al. (2019) showed that the effect of aromatic compounds may depend on nutrient medium composition. Thus, the addition of xylidine in MEB medium (malt extract broth) resulted in 2.8-fold induction of laccase activity, while a 1.9-fold increase was observed when xylidine was added to potato dextrose broth medium (PDB). In contrast, the use of CuSO4 as an inducer resulted in higher laccase activity in PDB than in MEB.

7.3 Effect of Microelements

The regulating effect of metal ions on LME activity and gene expression in WRB is well established (Piscitelli et al. 2011; Janusz et al. 2013). Copper is the most important and commonly used metal ion for laccase production. However, the effect of metal ions may vary depending on their concentration and fungal species/strain physiological peculiarities. Thus, in the cultivation of P. coccineus, the addition of 0.2 mM CuSO4 to potato dextrose broth significantly upregulated the extracellular laccase activity in five strains of P. coccineus, whereas two strains did not respond to Cu2+ (Park et al. 2015). Supplementation of glucose-yeast extract medium with 0.5 mM Cu2+ increased laccase activity of T. versicolor ATCC 200801 from 0.60 to 10.25 U/mL (Birhanlı and Yeşilada 2017). Higher concentrations of copper decreased the enzyme yield with complete inhibition of laccase secretion at 5 mM Cu2+. Supplementation of glucose-peptone-yeast extract medium with as high as 0.5–4 mM CuSO4 led to a 13- to 22-fold increase in laccase activity of L. strigosus 1566 when compared with the control (Myasoedova et al. 2015). At the same time, low concentrations of copper ions (10–50 μM) were sufficient to 1.1 to 2.6-fold increase in the laccase activity of G. lucidum in comparison with control medium (Liu et al. 2020). When the concentration of added copper ions was increased to 60–80 μM, the laccase activity decreased. The optimal copper dose for the maximum laccase activity for C. unicolor C-139 in the glucose and L-asparagine containing culture was found to be only 10 μM (Janusz et al. 2007). Copper concentrations increase from 25 μM to 300 μM Cu2+ repressed laccase production (up to 90%) with no effect on fungal growth. The authors showed that the copper supplementation mode was also important. Adding 10 μM Cu2+ to the culture medium after 3 days and repeating on the 6th and 9th day of the fungus cultivation led to the greatest activity of laccase. Likewise, the time-dependent effect of copper was observed in P. ostreatus ACCC 52857 (Zhu et al. 2016). The laccase activity dramatically increased (more than 80-fold) upon the addition of copper to the medium from the 6th to the 9th day of cultivation. It is interesting that unlike C. unicolor C-139, the stimulating effect of even 100 times higher concentration of copper on laccase secretion was revealed in the cultivation of C. unicolor CBS 117347 (Kachlishvili et al. 2014).

Many studies proved that metal ions may control the expression of individual isozymes through activation of metal responsive elements (Janusz et al. 2013). Thus, the addition of 0.15 mM copper sulfate to the potato dextrose medium increased production (up to 500-fold) of P. ostreatus POXA1b laccase isoform, while POXA1w isoform was not affected (Palmieri et al. 2000). Klonowska et al. (2001) reported that Marasmius quercophilus produced only one laccase (LacI) in liquid medium with malt extract. Supplementation of the same medium with CuSO4 induced three other isoforms, increasing the total activity 10 times. Concentrations of 0.05–1 mM Cu2+ induced Lac1 and Lac2 of Coprinus comatus, while Lac3 was strongly induced by the addition of 3 mM Cu2+ (Lu and Ding 2010).

The effect of metal ions other than Cu2+ on the LME activity was also widely studied. The addition of Mn2+ to the cultures of C. subvermispora (Manubens et al. 2007), P. chrysosporium (Brown et al. 1991), T. versicolor (Johansson et al. 2002), and several other WRB is required for the stimulation of MnP production. According to Brown et al. (1991), manganese is directly involved in the regulation of the mnp gene transcription through a mechanism specific for the growth stage and depending on the concentration. Manganese was a highly potent laccase inducer in C. comatus (Lu and Ding 2010). Supplemented to the cultures in the range of 0.05–0.8 mM, Mn2+ markedly increased the overall laccase activity and caused the synthesis of additional Lac2 and Lac3 isoforms compared with un-supplemented cultures. Increase of CuSO4 and MnSO4 concentrations from 1 to 3 mM as inducers for LME production by Marasmiellus palmivorus VE111 in the medium composed of 0.5% glucose, 0.18% casein, and potato broth led to increased production of the enzymes relative to the control (Schneider et al. 2019). Moreover, the presence of a third isoform in the zymogram of laccase was revealed after the fungus cultivation with inducers. For MnP, only 3 mM MnSO4 significantly induced enzyme production over the control, 2 mM CuSO4 also provided greater enzymatic activity as compared to the control medium, while veratryl alcohol rather decreased the fungus MnP activity.

Testing of the effects of eight microelements on Grammothele fuligo growth and enzyme production revealed that the fungus did not show any laccase activity with Ca2+, Mn2+, and Zn2+ and no MnP activity with Co2+ and Fe2+, but it showed three LME activity at different concentration of B3+, Cu2+, and Mo6+ (Chauhan 2019). No effect on laccase production by P. ostreatus ACCC 52857was observed after the addition to the medium of K+, Na+, Mn2+, Mg2+, Ca2+, Zn2+, Fe2+, and Fe3+, whereas Cd2+, Pb2+, and Cu2+ caused a three- to sixfold increase in laccase activity (Zhu et al. 2016). Finally, it should be noted that the same metal ion may have the opposite effect depending on species or enzyme type. For example, Mn2+ induced laccase gene transcription in P. sajor-caju (Soden and Dobson 2001), but inhibited gene transcription in C. subvermispora (Manubens et al. 2007). Fe2+ significantly increased laccase activity in P. eryngii (Stajić and Vukojević 2011), while it inhibited laccase production in G. lucidum (Murugesan et al. 2009). Moreover, Mn2+ stimulated lacc3 and lacc12 genes transcription in P. ostreatus HAUCC, but decreased transcription of lacc2 and lacc4 (Zhuo et al. 2017). In the submerged fermentation of mandarin squeezes, an augmentation in copper concentration from 0 to 0.5 mM increased 18-fold the activity of T. multicolor laccase but did not significantly affect the activity of MnP and LiP, which indicated that the inductive effect of copper was specific for laccase (Kachlishvili et al. 2018). It is interesting that iron ions at the concentration of 0.1 mM increased almost eightfold both volumetric and specific activity of laccase compared with the control medium. In the same culture, increase in the concentration of manganese from 0 to 0.5 mM only slightly increased the activity of T. multicolor laccase, decreased the activity of the fungus LiP, but this metal 13–14 times specifically increased the volume and specific activity of MnP when it was added to the control medium at a concentration of 0.5 mm.

Several studies revealed the synergistic effect of different inducing agents on LME production. Manubens et al. (2007) found a synergistic effect between Mn2+ and syringic acid on laccase production in C. subvermispora. Laccase activity of P. ostreatus HAUCC 162 increased from 205.5 U/L in the presence of Fe2+ alone to 250 and 363 U/L due to the addition of 0.5 mM vanillic or ferulic acid, respectively (Zhuo et al. 2017). In our study with C. unicolor CBS 117347, copper and xylidine added separately accelerated laccase secretion and 1.8-fold improved laccase yield. An additive effect and fourfold increase of laccase activity were observed upon supplementation of copper and xylidine to the medium simultaneously before inoculation. Likewise, supplementation of glucose-yeast extract basal medium (SBM) with 0.5 mM Cu2+ increased laccase activity of T. versicolor ATCC 200801 from 0.60 to 10.25 U/mL (Birhanlı and Yeşilada 2017). Enzyme production was enhanced considerably (to 33.61 U/mL) due to the synergistic effect of 1 mM 2,5-xylidine and 0.5 mM Cu. Interestingly, no induction of laccase production was observed in SBM with 0.025–0.5 mM syringaldazine alone, but laccase activity reached 22.23 U/mL in medium containing simultaneously 0.5 mM Cu and 0.1 mM syringaldazine. However, conflicting results were obtained when optimal concentrations of caffeic acid and Mn2+ were added to C. comatus culture, resulting in a decrease in total extracellular laccase activity compared to a culture supplemented with caffeic acid only (Lu and Ding 2010). Nevertheless, the supplementation of nutrient medium with different microelements is a suitable and effective tool to regulate the production of individual LME.

7.4 Effect of Co-cultivation

Co-cultivation of WRB may be a promising strategy to significantly improve LME production. Previous studies have shown that laccase activity of L. edodes (Savoie et al. 1998), C. unicolor (Elisashvili et al. 2014), P. ostreatus, T. versicolor, and several other basidiomycete species (Baldrian 2004) significantly increased in their co-cultivation with Trichoderma sp. The mixed cultivation of Gongronella sp. W5 with Panus rudis (Wei et al. 2010) and Shiraia bambusicola and Phoma sp. BZJ6 (Du et al. 2017) caused a 25-fold and 9.2-fold increase, respectively, of dual cultures laccase activity as compared with those in their monocultures.

Few studies were performed in the co-cultivation of basidiomycetes. Among four fungi tested, high stimulation of laccase production was observed only in the co-cultivation of P. ostreatus with C. subvermispora, while MnP activity was stimulated in the co-culture with both C. subvermispora and Physisporinus rivulosus and Western blotting revealed proteins from both fungi (Chi et al. 2007). However, in the co-cultivation of C. subvermispora with P. rivulosus low MnP activity was detected and the protein pattern resembled that of P. rivulosus, indicating that this fungus suppressed C. subvermispora growth. Similarly, among six WRB cultured in pairs for LME production, Hypoxylon fragiforme inhibited the expression of LME during co-cultivation with P. ostreatus, Dichomitus squalens inhibited MnP expression by P. ostreatus or Phlebia radiata (Qi-he et al. 2011). At the same time, the co-culture of P. radiata with D. squalens showed the maximum specific laccase activity but the co-culture of P. radiata with P. ostreatus was beneficial for MnP and LiP expression.

In our work, we attempted to improve LME production through interspecific interaction of the best enzyme producer C. unicolor 305 with 5 different WRB species in the submerged fermentation of mandarin peels. Moreover, their growth and interactions were simultaneously verified on agar plates using the same medium composition. On the solid medium, a distance between inoculated fungal colonies was 1 and 4 cm. None of the dual cultures demonstrated invasion or replacement over 10 days after the creation of confrontation zones. All pairs inoculated at a distance of 4 cm formatted delineated barrages in mycelial contact zones although without brown pigmentation. Among closely inoculated fungi, only C. unicolor 305 and L. betulina 141 formed a faintly visible barrage.

The results presented in Table 7.2 show several distinctive features of the tested cultures. Firstly, like the above-mentioned data, the enzymatic activity of the dual cultures depended on the fungal species combination. Co-cultivation of C. unicolor 305 with L. betulina 141 and T. versicolor 159 on both solid and liquid media was the most beneficial for the laccase production and their synergistic interaction led to a much higher enzyme activity as compared with those produced by either monoculture. Secondly, it turned out that the cultivation method strongly affected the laccase activity of individual fungal pairs. Thus, pairs of C. unicolor 305 + P. lecometei 903 and P. coccineus 310 + T. hirsuta 82 demonstrated an upregulation of laccase activity when replacing SSF with submerged liquid fermentation. On the contrary, interspecies interaction of C. unicolor 305 + P. coccineus 310 and P. coccineus 310 + T. versicolor 159 on the solid medium promoted laccase production, whereas in the submerged fermentation their co-cultivation inhibited laccase activity. Also, it should be noted that in monocultures grown on a solid medium, the laccase activity of the tested fungi differed less than three times, while in submerged cultures the enzyme activity varied from 4.3 to 140.3 U/mL. Thirdly, although no evident regularities were found between a distance of inoculation and enzyme activity values our findings indicate that in some pairs (for example, P. coccineus310 + T. versicolor 159) the distance between inoculated colonies can determine the outcomes of the interaction. Fourthly, no stimulation of MnP activity was observed in this study; on the contrary, a combination of C. unicolor 305 with other fungi 3 to 12-fold downregulated MnP activity of this fungus. Finally, the interaction of only live fungi is required to observe an increased outcome since the cultivation of C. unicolor 305 with a thermally inactivated mycelium of T. versicolor 159 did not increase laccase activity. Overall, it is clear that the process of interspecies interaction is complex and its result may depend not only on the particular microbe’s combination, but also on physiological peculiarities and enzymatic systems of individual competitors, nutritional conditions in dual culture, and space they occupy. Compared with the SSF, in the submerged fermentation, mycelium of the dual culture has permanent access to the available nutrients, but the degree of fungal competition is closely related to the initial concentration of the nutrients. Undoubtedly, individual competitors' inoculum size, ratio, time, and sequence of their inoculation should be considered in the co-cultivation study since these parameters can significantly affect enzyme activities. An understanding of the peculiarities and mechanisms of interspecies interaction of WRB is essential for the development of improved technologies of LME production.

Table 7.2 Enzymatic activity of mono- and dual cultures of WRB in fermentation of mandarin squeeze on agar plates and in submerged cultures
Full size table

7.5 Conclusion

LME constitute one of the most important groups of versatile enzymes for industrial applications. Owing to the progress in recent research, screening of potent enzyme producers, genetic engineering, omics-based data, understanding of physiological and transcriptional regulation of individual LME synthesis, the production of these enzymes was maximized. Nevertheless, there are still many limitations that need to be overcome, and further research is needed, in particular, using various genetic methods and the recombinant technologies to improve the production of LME and to obtain these biocatalysts in quantity and quality, satisfying various fields of application. The use of cheap lignocellulosic materials, metals, and aromatic inducers, clarification of the molecular mechanisms triggering and regulating LME expression in a response to different stimuli may boost additional production of LME at low cost. Besides, elucidation of the factors that impede the production of enzymes is also one of the most challenging tasks. Mixed cultivation, as well as the development of process design in the lignocellulose SSF, should be considered as encouraging and cost-efficient strategies.