Coculture of white rot fungi enhance laccase activity and its dye decolorization capacity

Fungal cocultures can promote complex interactions that result in physiological and biochemical alterations that favor the synergic and more efficient action of extracellular enzymes such as laccase. Thus, coculture can be used as a strategy to increase enzymatic activity, dye degradation, and bioremediation of textile effluents. This study aimed to evaluate the coculture effect of Lentinus crinitus, Pleurotus ostreatus, Pycnoporus sanguineus, and Trametes polyzona on laccase activity, mycelial biomass production, and in vitro decolorization of azo, anthraquinone, and triphenylmethane dyes. The species were cultivated in liquid medium in monoculture and coculture in paired combinations for 15 days to determine the laccase activity and produced mycelial biomass. The enzymatic extracts of fungal cultivations were used in decolorization tests of reactive blue 220 (RB220), malachite green (MG), and remazol brilliant blue R (RBBR). Pleurotus-Trametes, Lentinus-Pleurotus, and Lentinus-Trametes cocultures increase laccase activity compared to respective monocultures. Lentinus-Pycnoporus, Lentinus-Trametes, Lentinus-Pleurotus, and PleurotusTrametes cocultures stimulate mycelial biomass production in relation to their respective monocultures. The enzymatic extracts of monocultures and cocultures promoted the decolorization of all dyes. RB220 dye presented fast decolorization. In 24 h, all extracts reached maximum decolorization and the greatest color reduction percentage was 90% for Pleurotus-Trametes coculture extract. Pleurotus-Trametes extract also increased the decolorization of MG and RBBR dyes when compared to their respective monocultures in 48 Research, Society and Development, v. 9, n. 11, e88191110643, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i11.10643 3 h and 72 h, respectively. However, RBBR dye presented the greatest resistance to decolorization.


Introduction
In nature, fungi grow with other microorganisms and can establish symbiotic or competitive interactions. Concurrent interactions among fungi, mainly basidiomycetes, are common, competing for space/territory and nutrients. This interaction can be divided into two types: the interference competition and exploratory competition (Boddy, 2000). In interference competition, one species totally inhibits the other's growth while in exploratory competition there is partial inhibition by reduction of available resources. During exploratory competition, fungi can present morphological alterations in the mycelial branching with the formation of barriers that act as a defensive response to the invasion of other mycelia (Boddy, 2000). These morphological alterations of the mycelium can induce metabolic changes with the production of specific secondary metabolites (Hiscox et al., 2010).
Laccases (benzenediol: oxygen oxidoreductases, EC 1.10.3.2) catalyze the oxidation of a wide range of phenolic compounds to the corresponding free radicals using molecular oxygen as electron acceptor (Martínková et al., 2016). Usually, the use of redox mediators can extend the substrate range of laccases to non-phenolic compounds, polycyclic aromatic hydrocarbons, and dyes (Rivera-Hoyos et al., 2013). Redox mediators are expensive and toxic compounds and laccases that can cause substrate oxidation without the presence of mediators are of special interest to biotechnological applications (Husain & Husain 2008). Laccases are produced by basidiomycetes during the secondary metabolism, frequently induced by stresses Development, v. 9, n. 11, e88191110643, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i11.10643 5 and limited levels of nutrients (Wesenberg et al., 2003). Laccase production during interactions among basidiomycetes has been believed to be a response to stress, representing a defensive action against the mycelial invasion and/or competition for nutrients (Score et al., 1997;Zhang et al., 2006). Cocultures may induce the enzymatic production or cause high levels of stress, which impair production (Chi et al., 2007). Therefore, combination of species and strains in cultivations are decisive to induce a positive effect on enzyme activity such as laccase.
Laccases have potential for industrial applications as in food, paper, cellulose, and textile industries, and in bioremediation (Sharma et al., 2018). The growth estimate for the world enzyme market is 6.7% a year, and 11 billion dollars in 2020 (Kumar et al., 2014).
Thus, alternatives that aim to increase enzyme production for biotechnological applications to meet the growing demand in industrial and biotechnological processes are needed (Chander et al., 2004).
A lot of studies on fungal interactions are carried out to understand the general ecological aspects; however, it is important to understand these interactions in order to produce enzymes, mainly the ones that degrade dyes (Chi et al., 2007). The increasing utilization of dyes by several industrial sectors provided an increment in the production of colorful effluents, mostly due to dyes lost during the productive process (Vikrant et al., 2018).
The utilization of conventional chemical and physical methods to decolorize effluents is not always efficient and may generate additional residues that also demand treatment (Sen et al., 2016). Fungal laccases have shown their capacity to degrade dyes from several chemical classes (Cardoso et al., 2018) and could represent an eco-friendly alternative to conventional treatments. Therefore, the axenic fungal coculture can be a viable method to increase enzymatic activity compared to the ones obtained by axenic monoculture (Bader et al., 2010;Chan-Cupul et al., 2016). Thus, this study aimed to evaluate the laccase activity, mycelial biomass production, and the in vitro decolorization of synthetic dyes by laccase obtained by mono and coculture of basidiomycetes.

Microorganisms and inoculum production
The species utilized in this study and their respective identifiers' GenBank accession numbers are Lentinus crinitus U9-1 (MG211674), Pleurotus ostreatus U2-11 (KJ010860), Pycnoporus sanguineus U13-4 (MG211680), and Trametes polyzona U16-5 (MG211678). All strains belong to the culture collection of the Graduate Program of Biotechnology Applied to Agriculture of Paranaense University. The inoculum was produced by mycelial biomass growth in malt-extract-agar medium (MEA, 20 g L -1 ) at 28 ± 1 °C for seven days in the dark.
MEA disks (6 mm diameter) with mycelium without sectioning were utilized as inoculum for the coculture.

Coculture in liquid medium
The species were cultivated in conical flasks (250 mL) containing 100 mL of malt extract liquid 20 g L -1 previously autoclaved at 121 °C for 20 min (Valle et al., 2014). Paired combinations of the species were inoculated using three MEA disks containing mycelium of each species. The material was kept static at 28 ± 1 °C in the dark for 15 days (Marim et al., 2018). A monoculture of each fungus was used as control. At the end of the cultivation, the mycelial biomass was separated from the cultivation medium by filtration, and the filtrated was utilized to determine laccase activity. The produced mycelial biomass was kept in a stove with air circulation at 60 °C until constant mass to determine mycelial biomass.
The experimental design was completely randomized and all assays were carried out with three replicates. The results were evaluated by analysis of variance (ANOVA) and the significant differences among the arithmetical averages (p ≤ 0.05) were determined by Scott-Knott test.

Manganese peroxidase assay
The oxidation of MnSO4 at room temperature was used to determinate manganese peroxidase (MnP) activity (Wariishi et al., 1992). MnSO4 (10 mM) in sodium malonate buffer (50 mM, pH 4.5) was mixed with liquid cultivation and 0.5 mM hydrogen peroxide. The oxidation was monitored by absorbance increase at 270 nm (ε = 11,590 M −1 cm −1 ) caused by the complex formed by Mn 3+ ion with malonate. MnP activity was expressed in international units (U) defined as the amount of enzyme that oxidizes 1 μmol MnSO4 per minute.

Lignin peroxidase assay
Lignin peroxidase (LiP) activity was determined by the oxidation of methylene blue at room temperature (Magalhães et al., 1996). Methylene blue (1.2 mM) in sodium tartrate buffer (0.5 M; pH 4) was mixed with liquid cultivation, and 2.7 mM hydrogen peroxide. The oxidation was monitored by absorbance increase at 664 nm (ε = 52.400 M −1 cm −1 ) caused by methylene blue conversion to azure-C. LiP activity was expressed in international units (U) defined as the amount of enzyme that oxidizes 1 μmol methylene blue per minute.

In vitro decolorization of synthetic dyes by enzymatic extract
The color reduction of different classes of dyes was determined according to Cardoso et al. (2018). Azo dye reactive blue 220 (RB220), anthraquinone remazol brilliant blue R (RBBR), and triphenylmethane malachite green (MG) dyes were diluted in sodium acetate buffer (100 mM, pH 5), filtered (0.22-μm porous filter), and used in the assays. Aliquots of cultivation medium obtained on the 15 th cultivation day were mixed with dye solutions in sufficient volume to obtain dye concentration of 0.1 mg mL -1 (mass/volume) in all assays.

Statistical analysis
The research was based on quantitative methods developed with an experimental approach (Pereira et al., 2018). The assays had a completely randomized design (CRD) with three replicates. The results were evaluated using ANOVA, and significant differences among arithmetic means were determined by the Scott-Knott test at 5% of probability.
crinitus (Table 1 and Figure 4). In 72 h of decolorization, the enzymatic extract from all cultivations had the same decolorization capacity (p ≤ 0.05) ranging from 72 to 90% indicating the efficiency of the process and that coculture did not affect the decolorization capacity of RB220 (Table 1).    Figure 4). Overall, the coculture extract was better than the respective monocultures, mainly for Pleurotus-Trametes and except for Lentinus-Pycnoporus (Table 1).
RBBR dye was the most resistant to decolorization. For enzymatic extract of Lentinus-Trametes coculture the decolorization was 30% for RBBR in 24 h, whereas for RB220 it was 88% and MG 75% (Table 1 and Figure 4). Pleurotus-Trametes coculture presented the greatest (p ≤ 0.05) RBBR decolorization in 72 h (Table 1) compared to the respective monocultures and also had the greatest laccase activity (Figure 1). However, P. sanguineus monoculture, which had the greatest (p ≤ 0.05) laccase activity (Figure 1) presented decolorization of 31% of RBBR in72 h. In general, the enzymatic extracts presented maximum (p ≤ 0.05) decolorization in 48 h, varying from 17% for P. ostreatus to 72% for carbon/nitrogen source (Fernández-Fueyo et al., 2014). Lignocellulose carbon sources can favor peroxidases activity and the malt extract medium used in our study (~60% reducing sugars) may have been a hindrance to ligninolytic peroxidase activity (Mali et al., 2017).
However, laccases are associated with a defense response to competition and oxidative stress of cocultures, which can explain their greater activity in the cultivation conditions evaluated in our study (Giardina et al., 2010).
Two of three cocultures containing P. ostreatus promoted an expressive increase in laccase activity but P. ostreatus monoculture showed the smallest laccase activity among monocultures. Its presence in coculture with L. crinitus or T. polyzona seems to favor laccase activity. Our results are in accordance with Verma & Madamwar (2002) who demonstrated that P. ostreatus coculture with Phanerochaete chrysosporium resulted in higher ligninolytic activity than the respective monocultures. Chi et al. (2007) also reported that P. ostreatus During fungal coculture, fungi can compete for space and nutrients which can provoke oxidative stress, resulting in physiological and biochemical adaptations that can favor the synergic action of extracellular enzymes (Luo et al., 2017). The greater laccase activity during competitive interactions has been believed to be a response to stress, representing a defensive action against the mycelial invasion and the competition by nutrients (Zhang et al., 2006). On the other hand, cocultures can cause high levels of stress that negatively affect enzyme expression (Wesenberg et al., 2003). However, positive effects on the enzymatic activity depend on the species kept in coculture and the cultivation conditions (Chi et al., 2007). In our study, although most interactions did not increase laccase activity, there was still enzyme production and it suggests that interactions among the evaluated species could increase laccase activity if other cultivation conditions were optimized such as the presence of lignocellulosic substrate or laccase inducers (Mali et al., 2017).
Mycelial biomass production can be a parameter in the evaluation of growth and fungus biological efficiency as an indication of its capacity to convert nutrients in cells (Yang et al., 2013). However, the effects of basidiomycete cocultures have been little explored. Lentinus-Pycnoporus, Lentinus-Trametes, and Pleurotus-Trametes cocultures presented the greatest increases in mycelial biomass. Our results are in accordance with the ones by Kumari & Naraian (2016) that reported an increase in mycelial biomass during P. florida and R. solani Development, v. 9, n. 11, e88191110643, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i11.10643 16 cocultures. The increase in mycelial biomass production in cocultures can be attributed to competitive interaction among fungi that promotes the increase of several enzymes. It is believed that a greater expression of amounts and variety of enzymes may favor the most efficient utilization of the substrate by the combined and/or synergic action of produced enzymes stimulating the mycelial biomass growth (Dwivedi et al., 2011).
Enzymatic extracts of all evaluated species, whether in monoculture or coculture, were able to decolorize the dyes, and decolorization efficiency varied according to the dye class (MG > RB220 > RBBR) and according to the species combination. It is important to note that the enzymatic extracts obtained in our study had laccase as the predominant enzyme and the decolorization reactions were maintained without hydrogen peroxide addition which excludes any other peroxidase activity. It implies that the decolorization occurred mainly, if not only, due to laccase activity.
In our study the level of laccase activity in the enzymatic extract apparently did not affect decolorization since P. sanguineus, the greatest producer of laccase, was cultivated alone and produced the greatest decolorization of MG in 72 h (94%) but produced one of the lowest decolorization of RBBR in 24 h (30%) and reasonable decolorization of RB220 in 24 h (86%). The same was observed with the coculture Trametes-Pycnoporus whose laccase activity was the greatest one, but the decolorization of RB220 and MG in 24 h was 87% and 88%, respectively. Our results are in accordance with Moreira-Neto et al. (2013) that reported similar decolorization rate with basidiomycetes with very different laccase activity.
In our study the combination of Pleurotus-Pycnoporus in coculture was more efficient in decolorizing the triphenylmethane dye MG achieving 94% of color reduction in 48 h. Our results are in accordance to the ones found by Kumari & Naraian (2016) who also reported that the enzymatic extract of P. florida and R. solani cocultivation was more efficient to decolorize triphenylmethane brilliant green dye than the extract of monocultures, reaching up 98% of decolorization when the dye was used at lower concentration.
The coculture of Pleurotus-Trametes promoted the greatest decolorization of azo dye RB220 and the anthraquinone dye RBBR among all cultivations. Krishnamoorthy et al. (2018) isolated fungi and bacteria from soil contaminated with azo dyes and reported that the coculture of two ascomycetes, Dichotomomyces cejpii (current name Aspergillus cejpii) and Phoma tropica (current name Allophoma tropica), produced the greatest decolorization rates of azo dyes assessed during four days (12 to 73%). On the other hand, Przystaś et al. (2013) reported the low efficiency of P. ostreatus and Gloeophyllum odoratum cocultures to decolorize the azo dye Evans blue and the triphenylmethane dye brilliant green. Instead, the authors reported the color change and increase in the absorbance of decolorization reactions that were attributed to the interaction among strains metabolites and dyes and/or stress connected with presence of another strain in cultivation. In our study we also observed an increase in absorbance during the RBBR decolorization reactions of all the cultivations, particularly after 48 h. It suggests that the increase in absorbance could be due to the generation of intermediary metabolites or incomplete degradation of dye because after 72 h absorbance decreases (Eichlerová et al., 2007).
The enzymatic extracts of monoculture and cocultures promote the decolorization of all dyes.
The enzymatic extract of Pleurotus-Trametes coculture is the most efficient to decolorize RBBR dye. All cocultures are efficient to decolorize MG and RB220. The enzymatic extracts of P. ostreatus and T. polyzona axenic monocultures are not effective to decolorize MG and RBBR, but they are effective to decolorize RB220. Enzymatic extracts of L. crinitus and P. sanguineus monocultures are effective to decolorize RB220 and MG, but not to decolorize RBBR. RBBR is decolorized efficiently by enzymes only produced by Pleurotus-Trametes coculture and it is not effective for the other mono or cocultures. Laccase from different basidiomycetes produced during coculture in liquid cultivation medium is an option to decolorize dyes with different chemical structures and has potential for biotechnological applications in bioremediation. Further studies are needed to evaluate differences in the inoculation of cocultivations as well as to evaluate the decolorization of other textile dyes.