Improvement of laccase production by the Co-culture Pleurotus florida and Rhodotorula mucilaginosa in submerged fermentation culture

Introduction

Laccase (benzenediol: oxygen oxidoreductase, EC 1.10. 3.2) is a phenol-oxidizing enzyme that oxidizes a large variety of organic substrates including aromatic and phenolic compounds with the reduction of oxygen (1). Due to its multiple physiological functions such as detoxification, lignin degradation, plant pathogenesis, and stress defense, laccase is broadly used in various biotechnological processes, including textile bleaching (2), pulps delignification (3), effluent detoxification (4), and in various food industry processes (5).

Laccases are commonly distributed in bacteria, higher plants, and insects; however, fungi, particularly basidiomycetes, are a more important source. Under natural conditions, low constitutive extracellular laccases from white-rot fungi cannot relieve the practical demands of industrial biotechnology. Therefore, it is crucial to improve the productivity process for potential industrial and biotechnological applications (6).

Enhancing fungal laccase production has been obtained by optimizing different nutritional (mainly carbon and nitrogen sources) and physicochemical (temperature, pH, yeast cell concentration, and agitation rates) conditions (7). Much consideration has also been performed for laccase enhancement by different inducers such as phenolic compounds (8), copper (9), and ethanol (10). Oxidative stress is also proposed as a mechanism for laccase overproduction by an inducer (11).

The cultivation of white-rot fungi with different microorganisms (mixed fermentation) is of interest for their ability to induce enzyme activity (12). Enhanced laccase production was effectively reported by white-rot fungi during interaction with Trichoderma spp. or its culture metabolite (13, 14) and yeasts (15). Co-culture with yeasts led to glucose deprivation, which caused the overproduction of Ganoderma lucidum (12), Pleurotus eryngii var. ferulae (16), and T. versicolor (11) laccase. The purpose of this study was to increase laccase production by Pleurotus florida, a commercial edible oyster mushroom by R. mucilaginosa, as a biotechnologically important yeast. In addition, we identified the effect of yeast cell concentration and time of R. mucilaginosa inoculation in the co-cultured media.

Materials and Methods

Chemicals and Media: ABTS (>98.0%), potato dextrose agar (PDA), and potato dextrose broth (PDB) media were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Organisms: P. florida strain 285 (gau.1999.511) and R. mucilaginosa (gau.1400.222) were kindly provided by the culture collection of the Department of Plant Protection, Gorgan University of Agricultural Sciences and Natural Resources. The fungal strains were maintained on PDA slants at 5-7 °C in the dark.

Culture Conditions: The submerged fermentation (SmF) was used for the determination of maximum laccase production at 0-20 day intervals (17). The cultures were performed in 250 mL flasks with 50 mL of PDB medium. The SmF cultures were inoculated with four fungal agar discs (5 mm, diameter) of the 8-day culture and incubated at 25±2 °C, in the dark for 14 days.

Co-culture of P. florida with yeast and extraction of yeast cells: The 48-h-old culture of R. mucilaginosa in PDB was used for inoculation of P. florida cultures. The yeast cell concentrations at 10³, 10⁵, and 10⁷ CFU mL^-1 were added into 1-, 3-, 5- and 8-day cultures of P. florida to estimate the effect of inoculation size and time on the improvement of laccase production. Yeast cells were collected by centrifugation and their number was determined using a hemocytometer. To investigate the effect of temperature on R. mucilaginosa cells or its metabolites for laccase enhancement, yeast cells were exposed to different temperatures for 1 h, including 121 ^◦C (autoclaved) and 70 ^◦C, and 3% of the suspension (v/v) was added into the P. florida culture (18).

Biochemical Analyses: The laccase activity was assessed by the oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonnic acid) (ABTS) as the substrate. A reaction mixture containing 0.5 mM ABTS, 2.8 mL sodium acetate buffer 0.1 M (pH 4.5), and 100 μL of culture supernatant was incubated for 5 min. The absorbance was determined at 436 nm and one unit (U) of enzyme activity was defined as one μmol of substrate that is oxidized per min (19).

Statistical Analysis: All the experimental data were analyzed as factorial using a randomized complete design with four replications. A two-way ANOVA was used to analyze the data, and differences between means were compared using Duncan’s multiple range tests at P≤0.05. Regression models were also used to predict the influence of the independent variable, namely temperatures on laccase production in P. florida cultures. All statistical analyses were performed using R 4.2.1 statistical software.

 Results

Analysis of variance revealed significant differences in the laccase production by P. florida on different days (P≤0.05, Table 1). The laccase production by P. florida dramatically increased at various time intervals (Fig. 1). The highest laccase activity in the PDB medium (2.88 U mL⁻¹) was obtained on the 14^th day of cultivation and became partially constant thereafter. The relationship between time (x) and maximum laccase activity (y) was described by

y=-0.0101x² + 0.3881x – 0.7937 model (adjusted R-squared = 0.9738 and p<0.001). Considering the results, the effects of R. mucilaginosa on laccase activity were screened on the 14^th day. Laccase activity was not detected in R. mucilaginosa cultures.

Table 1- Results of Variance Analysis (ANOVA) of Incubation Time (days) on Pleurotus florida Laccase Activity (U mL^-1)

Table 2- Results of Variance Analysis (ANOVA) of Inoculation Time and Rhodotorula mucilaginosa Cell Concentrations on Laccase Production in Pleurotus florida-yeast Cultures

Fig. 1- Time course (days) of laccase activity (U mL^-1) of Pleurotus florida cultivated in potato dextrose broth medium. The columns with the same letter are not significantly different (P < 0.05)

The significant influence of inoculation time (Fig. 2A) and the yeast cell concentration (Fig. 2B) of R. mucilaginosa, as a single factor, and their interactions were observed in the co-culture interaction of P. florida and yeast. A day after the cultivation of P. florida with R. mucilaginosa, the laccase by P. florida on the 14^th day was significantly suppressed (Fig. 2A). The limited laccase activities of P. florida were almost observed for yeast inoculation after 1 day at all yeast cell concentrations (Fig. 2C). Inoculation of yeast 5 days after P. florida cultivation led to obtaining higher laccase activity than other days of cultivation. Maximum laccase activity was determined in P. florida culture which was inoculated by yeast after 5 days at the yeast cell concentration of 10⁵ CFU mL^-1, with 9.2 ±0.52 U mL^-1 of laccase production on day 14 of P. florida cultivation (Fig. 2C). However, an increase in laccase activity was detected after 8 days of P. florida cultivation, the increase was not as high as inoculation of the P. florida culture after 5 days (Fig. 2A). These results propose that the predominance of P. florida in co-culture was involved in enhancing the laccase activity.

Analysis of variance revealed that the impact of treatments was significant on the laccase production by P. florida (P≤0.05, Table 2). According to the yeast cell concentration of R. mucilaginosa, the laccase production in submerged co-cultivation was significantly divided into three levels (Fig. 2B). When the yeast cell concentration in P. florida culture was adjusted to 10⁵, and the increasing of laccase production was observed on day 14 of cultivation. In contrast, when the yeast cell concentration was adjusted to 10³ CFU mL^-1, the laccase production of P. florida significantly decreased, as compared to 10⁵ or 10⁷ CFU mL^-1 of yeast cell concentrations (Fig. 2B). The highest amount of laccase production was concerned with 10⁵ CFU mL^-1 of R. mucilaginosa at the 5-day cultures of P. florida (Fig. 2C).

Fig. 2- Mean comparisons of single-factor (a and b) and interaction (c) effects on laccase enhancement on the 14^th day in Pleurotus florida cultures as affected by the infection time (dates after P. florida cultivation, A) and yeast cell concentrations (B) of Rhodotorula mucilaginosa. The columns with the same letter are not significantly different (P < 0.05)

Analysis of variance revealed that laccase enhancement in Pleurotus florida cultures by Rhodotorula mucilaginosa sterilized cells at different temperatures was significant (P≤0.05, Table 3). As shown in Fig. 3, the addition of autoclaved (121 ^◦C) R. mucilaginosa cells to P. florida culture did not significantly enhance laccase production, although yeast cells under the lower temperature (70 ^◦C) had a stimulatory effect on laccase activity. This suggested that the live cells of R. mucilaginosa were necessary for enhancing laccase production in co-culture and their stimulatory compounds were temperature-sensitive.

Table 3- Results of Variance Analysis of Laccase Enhancement in Pleurotus florida Cultures as Affected by Rhodotorula mucilaginosa Sterilized Cells at Different Temperatures

Fig. 3- The effect of sterilized Rhodotorula mucilaginosa at different temperatures (121 and 70 ^◦C) on laccase production in Pleurotus florida cultures. Three percent (v/v) of sterilized cells were added to the P. florida cultures

Discussion and Conclusion

Laccases have a leading position among the commercially produced industrial enzymes in the global market. Therefore, the extent of enzyme production is important for industrial and environmental applications. Microbial enzymes are widely used as cost-efficient and eco-friendly alternatives for chemical processing in different industries and bioremediation. Therefore, the global demand for microbial enzymes is drastically increasing. The incorporation of several nutritional and inductive substances can increase microbial laccase production (9, 20). Therefore, various safe and low-cost methods to enhance laccase production were determined by some researchers. The results of this study indicated the improvement of laccase activity during P. florida-R. mucilaginosa interaction, yielding up to 4.5 times activity as compared with control (P. florida alone). The interaction of white-rot fungi with bacteria or other fungi for enhancing laccase production has already been investigated by several authors. Enhancement of white-rot fungi laccase production was strongly influenced by co-culturing of Funalia floccose with Penicillium commune (21); Lentinula edodes with Trichoderma strains (22, 23) Pleurotus eryngii var. ferulae with several yeasts (16); Pleurotus ostreatus with Bacillus subtilis (24), Trichoderma spp. (24, 25, 26) or Penicillium spp. (24); Trametes sp. with Lentinus crinitus (27), Paecilomyces carneus (28), Sporidiobolus pararoseus (29), Trichoderma spp. (30, 14) or Penicillium spp. (24) and Trychophyton rubrum with Trichoderma spp. (31).

Our results proposed the consideration of the effect of yeast cell concentration and time on the production of laccase in co-culture, which indicated the importance of the growth balance of the P. florida against R. mucilaginosa. This indicates enough competitive dominance of P. florida in co-culture is needed for the enhancement of laccase production.

Our results showed the higher laccase activities of P. florida culture when inoculated with R. mucilaginosa in the 5-day culture than those of the 8-day culture, and the inoculation of P. florida cultures after 1 day of cultivation suppressed the laccase production. Likewise, Jung (31) showed the higher laccase production of T. rubrum when culture inoculation occurred with T. longibrachiatum after 5 days of cultivation. The best time for a significant laccase production of Trametes maxima with P. carneus was also suggested in the 3-day culture of T. maxima by Cupul et al. (28).

The results of our experiment showed the 10⁵ CFU mL^-1 of R. mucilaginosa for enhancing the laccase activity by P. florida. Cupul et al. (28) emphasized the importance of the number of mycelial disks of P. carneus for enhancing the T. maxima laccase production in T. maxima- Paecilomyces carneus co-cultivation. Velázquez-Cedeno et al. (14) also showed the relatively low spore concentration in the interaction of P. ostreatus with T. longibrachiatum to colonize liquid or solid medium. These results are in agreement with Jung (31) on the effect of the spore concentration of T. longibrachiatum on laccase production in cultures of T. rubrum.

These results indicated that laccase production was increased mainly by the live cells of microbial inducers as supported by Baldrian (24), who reported no laccase induction by the addition of sterilized T. harzianum culture in Pleurotus ostreatus cultures. Also, the results of Jung (14) showed more enhancement of laccase production of the live mycelium of T. longibrachiatum than its culture extract or autoclaved mycelium by T. rubrum in co-culture. On the other hand, Mata et al. (32) proposed a significantly increased in laccase production of Plurotus pulmonarius cultures by inoculation of lytic enzymes of the Trichoderma sp. Also, Savoie and Mata (33, 34) suggested that laccase production of L. edodes was affected by Trichoderma cell wall degrading enzymes. The results are in agreement with Zhang et al. (30) on some thermal-resistant stimulators of Trichoderma sp. that can induce laccase production of Trametes sp. The results of Guo et al. (16) determine the thermal-sensitive compounds from yeasts that act strongly to induce laccase activity of P. eryngii var. ferulae. Considering limited work on the enhancement of yeasts on the laccase production of white-rot fungi, our results also showed the presence of temperature-sensitive stimulatory compounds in yeast cells.

The present study explains a partial analysis of the co-culture on the laccase activity in P. florida. In the present study, the yeast, R. mucilaginosa, effectively enhanced the laccase production in a co-cultivation with P. florida. The responses of the laccase production could be affected by the inoculation time (after P. florida cultivation) and yeast cell concentration of R. mucilaginosa. The interactions need the live stimulator cells and the improvement of laccase production did not occur with yeast-autoclaved cells.

Conflict of Interest

The authors declare that there is no conflict of interest.

 Acknowledgment

We appreciate the financial support of this study funded by the Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran.