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Han, M.-L., An, Q., Ma, K.-Y., An, W.-N., Hao, W.-Y., Liu, M.-Y., Shi, W.-Y., Yang, J., and Bian, L.-S. (2021). "A comparative study on the laccase activity of four basidiomycete fungi with different lignocellulosic residues via solid-state fermentation," BioResources 16(2), 3017-3031.

Abstract

The laccase producing abilities of four Basidiomycete fungi species were compared using solid-state fermentation using four different lignocellulosic residues. The biosynthetic potential of the Basidiomycetes was highly dependent on the type of fungi. In general, the laccase secreting ability of Cerrena unicolor Han 849 was greater than Lenzites betulinus Han 851, Stropharia rugosoannulata Han 1321, and Auricularia heimuer Han 1333. The maximum laccase production of C. unicolor Han 849 was approximately 11.25, 122.26, and 15.27 times higher than L. betulinus Han 851, S. rugosoannulata Han 1321 and A. heimuer Han 1333, respectively. Different species of fungi had a preference in lignocellulosic residues. The presence of Firmiana platanifolia was conducive to secreting laccase via C. unicolor Han 849 during solid-state fermentation. A continuous and stable laccase production via C. unicolor Han 849 was an obvious advantage of solid-state fermentation with any of the four lignocellulosic residues used. The maximum laccase production of C. unicolor Han 849 using Firmiana platanifolia was approximately 2.12, 1.68, and 6.13 times higher than Populus beijingensis, Sorghum bicolor, and Oryza sativa, respectively. These findings will be helpful for developing new productivity strains in industrial applications and selecting suitable lignocellulosic residues for laccase production.


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A Comparative Study on the Laccase Activity of Four Basidiomycete Fungi with Different Lignocellulosic Residues via Solid-state Fermentation

Mei-Ling Han,a,c Qi An,a,b,* Kai-Yue Ma,a Wen-Ning An,a Wen-Yao Hao,a Meng-Yi Liu,a Wen-Yi Shi,a Jing Yang,a and Lu-Sen Bian d

The laccase producing abilities of four Basidiomycete fungi species were compared using solid-state fermentation using four different lignocellulosic residues. The biosynthetic potential of the Basidiomycetes was highly dependent on the type of fungi. In general, the laccase secreting ability of Cerrena unicolor Han 849 was greater than Lenzites betulinus Han 851, Stropharia rugosoannulata Han 1321, and Auricularia heimuer Han 1333. The maximum laccase production of C. unicolor Han 849 was approximately 11.25, 122.26, and 15.27 times higher than L. betulinus Han 851, S. rugosoannulata Han 1321 and A. heimuer Han 1333, respectively. Different species of fungi had a preference in lignocellulosic residues. The presence of Firmiana platanifolia was conducive to secreting laccase via C. unicolor Han 849 during solid-state fermentation. A continuous and stable laccase production via C. unicolor Han 849 was an obvious advantage of solid-state fermentation with any of the four lignocellulosic residues used. The maximum laccase production of C. unicolor Han 849 using Firmiana platanifolia was approximately 2.12, 1.68, and 6.13 times higher than Populus beijingensis, Sorghum bicolor, and Oryza sativa, respectively. These findings will be helpful for developing new productivity strains in industrial applications and selecting suitable lignocellulosic residues for laccase production.

Keywords: Basidiomycete Fungi; Laccase activity; Lignocellulosic residues; Solid-state fermentation

Contact information: a: College of Life Science, Langfang Normal University, Langfang, Hebei 065000 China; b: Technical Innovation Center for Utilization of Edible and Medicinal Fungi in Hebei Province, Langfang, Hebei 065000 China; c: Edible and Medicinal Fungi Research and Development Center of Universities/Colleges in Hebei Province, Langfang, Hebei 065000 China; d: Experimental Centre of Forestry in North China, Chinese Academy of Forestry, Beijing 102300 China;

* Corresponding author: fungiqian@yahoo.com

INTRODUCTION

The application of enzymes in order to degrade pollutants has attracted a large amount of attention in the past few decades. Laccases (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) are a group of blue multicopper oxidases that were first isolated from Rhus vernicifera (Yoshida 1883) and are widely present in plants, fungi, bacteria, and insects. Laccases can catalyze the oxidation of a wide variety of phenolic and nonphenolic lignin-related compounds, causing the reduction of molecular oxygen to water (Li et al. 2011; Yang et al. 2015; Zerva et al. 2019). Fungal laccases are best known for their role in lignin degradation, but they serve many other roles involving various aspects of multiple fields (An et al. 2018). Since they are energy-saving and environmentally friendly, laccases have potential applications in numerous industrial and biotechnological fields, e.g., wine and juice stabilization, fiber properties improvement, biosensor development, detoxification of environmental pollutants, dye decolorization, bio-synthesis, and bioremediation (Bilal et al. 2019; Deska and Kończak 2019; An et al. 2020a,b; Han et al. 2020). However, their application to biotechnological processes has been limited due to high production costs, which results in low enzyme activity and low yields. Increasing amounts of attention have been paid to studies demonstrating effective laccase production strategies associated with increased activity and reduced costs (Akpinar and Urek 2017; Chenthamarakshan et al. 2017; An et al. 2018; Singh and Arya 2019).

Among the various fungal phyla, basidiomycete fungi produce different kinds of extracellular oxidoreductases including laccases, peroxidases (manganese peroxidase and lignin peroxidase), and oxidases that generate H2O2 (Shrestha et al. 2016). Laccase production is highest in white rot fungi (Basidiomycetes) (Wang et al. 2019). Commonly used fermentation methods are submerged fermentation and solid-state fermentation. In the past, submerged fermentation (SF) was the most commonly used technology for the production of most enzymes, including laccase. The advantage of submerged fermentation is the homogeneous distribution of nutrients, which can result in full contact by the cultured microorganisms and therefore the full absorption of the nutrients. However, it also causes the dilution of the enzyme. There has been a trend during the past decade towards the increased use of solid-state fermentation (SSF) for the production of certain enzymes (Xin and Geng 2011; Nguyen et al. 2018). During solid-state fermentation, the microorganism is grown in the near or complete absence of free water with an natural substrate as solid support (Jaramillo et al. 2017). Therefore, SSF more closely simulates the natural environment of the microorganism. In addition, SSF has numerous advantages over SF, e.g., lower energy consumption and higher product recovery (Jiang et al. 2017). However, disadvantages for SSF have been observed during laccase production, which included difficulties in scaling up and large batch-to-batch variation.

Laccase is generally produced at low concentrations by fungi grown on basal media (Songulashvili et al. 2011), but relatively higher concentrations are obtained by the addition of various supplements or altering other factors, e.g., pH, temperature, and included aromatic compounds (Dominguez et al. 2007; Janusz et al. 2015; Metreveli et al. 2017; An et al. 2018; Filipe et al. 2019; An et al. 2020a,b; Atilano-Camino et al. 2020; Rajavat et al. 2020). Metal ions, e.g., Cd2+, Ag+, Hg2+, Mn2+, and Cu2+, are often used as additional supplements (Mäkela et al. 2013; An et al. 2016a, 2020a). Among the various metal ions, Cu2+ is considered to be an excellent inducer at certain concentrations. An et al. (2016) reported that in general, the presence of copper could increase the laccase activity obtained from strains belonging to the Flammulina genus. An et al. (2020a) reported that a final ion concentration of 2 mM could increase the laccase activity obtained from Pleurotus ostreatus CCEF 89. A combination of copper and manganese ions as the inducer for enhancing the laccase activity of P. ostreatus was shown to be superior to the use of single copper ions or manganese ions as inducers. The substrates used for SSF are typically lignocellulosic wastes needed for microorganism growth, enzyme production, and metabolite synthesis (Hatvani and Mécs 2001). Fungi, particularly white rot fungi, have a strong ability to degrade lignins and other cellulosic substances. Many research groups have attempted to improve laccase production by screening fungal strains based on the choice of lignocellulosic waste and optimization of the medium (Tišma et al. 2012; Soumya et al. 2016). Approximately 200 billion tons of agricultural wastes are generated each year all over the world (Ren et al. 2009). Lignocellulose, the major form of agricultural waste, is regarded as a low-cost nutrient substitute for laccase production in SSF systems in comparison with other complex nutrient sources (Huang et al. 2017). Lignocellulosic biomass, e.g., agro-industrial residues (straw, corncob, sugar cane bagasse, cottonseed hull, and corn stover) and forestry materials (leaves and sawdust), are principally made up of cellulose, hemicellulose, and lignins. A large majority of agricultural waste is used as livestock feed, fuel, and in paper production, or burned or left to rot, which contributes to environmental pollution and resource waste. Therefore, the efficient bioconversion of lignocellulose is an important goal in terms of agricultural waste resource utilization. It is a better method for producing laccase via culture fungus with lignocellulosic wastes. In addition, laccase activity is greatly dependent on the type of fungal species or strain (An et al. 2016, 2018; Han et al. 2018, 2020). The capacity of producing laccase from different fungi is different, so it is necessary to compare the laccase production of different species (Agrawal et al. 2018; An et al. 2018, 2020a,b).

Most of the previous studies in this area were focused on the laccase production secreted by the fungal genera Pleurotus and Trametes. However, the development of new productive strains is a persistent topic. Meanwhile, using inexpensive lignocellulosic wastes to ferment productive strains to produce laccase is a popular method. However, it is extremely necessary to evaluate the laccase activity of new productive strains using various lignocellulosic wastes. Under the circumstances, the present work deals with the evaluation of the laccase producing ability of four strains belonging to different species via solid-state fermentation using various lignocellulosic residues.

EXPERIMENTAL

Materials

Microorganisms

The four tested Basidiomycete strains (Han 849, Han 851, Han 1321, and Han 1333) were maintained on malt extract agar (MEA) medium (composed of 10 g/L of glucose, 20 g/L of malt extract, 3 g/L of KH2PO4, and 20 g/L of agar) at 4 °C at the College of Life Science, Langfang Normal University. Two wild strains (Han 849 and Han 851) were isolated from the Wulingshan National Nature Reserve, Xinglong county, Chengde city, Hebei province, China. One cultivated strain (Han 1321) was purchased from a market and isolated, and another cultivated strain (Han 1333) was donated by farmers from the Heilongjiang province, China. The fungus was reactivated in Petri dishes containing a complete yeast medium (CYM) (composed of 20 g/L of glucose, 2 g/L of peptone, 2 g/L of yeast extract, 0.5 g/L of MgSO4·7H2O, 1 g/L of K2HPO4·3H2O, 0.46 g/L of KH2PO4, and 20 g/L of agar) at 26 °C for 9 d.

Lignocellulosic materials

Populus beijingensis was obtained from the Langfang Normal University (Hebei, China). Firmiana platanifolia was also obtained from Langfang. Sorghum bicolor and Oryza sativa were kindly provided by farmers in Chengde city, Hebei province, China. All of the lignocellulosic residue samples were chopped into small pieces before being air-dried and ground. The particle size of the lignocellulosic residues was between 20 mesh and 60 mesh.

Methods

Inoculum preparation

Inoculants with a diameter of 5 mm were made with a hole punch from reactivated fungus in Petri dishes. Then, 5 inoculants were placed in 250 mL flasks containing 100 mL of CYM medium (composed of 20 g/L of glucose, 2 g/L of peptone, 2 g/L of yeast extract, 0.5 g/L of MgSO4·7H2O, 1 g/L of K2HPO4·3H2O, and 0.46 g/L of KH2PO4) and cultured on a rotary shaker at temperature of 26 °C and 150 rpm. After 8 d, the mycelial pellets were harvested and homogenized with a laboratory blender for 2 min at 5000 rpm. The resulting suspension was used as an inoculum.

Culture conditions

The solid-state fermentation (SSF) processes of the Populus beijingensis, Firmiana platanifolia, Sorghum bicolor, and Oryza sativa wastes were performed individually at a temperature of 26 °C in 250 mL flasks containing 3 g of the substrate with 12 mL of deionized water. The Erlenmeyer flasks containing the substrate and deionized water were autoclaved at 121 °C for 30 min. When it cooled to room temperature, 3 mL of inoculum was added to inoculate each flask. All flasks were incubated at 26 °C.

Extracellular laccase was extracted with 100 mL of 50 mM acetate-sodium acetate buffer (a pH 5.5). The extractions were performed on a rotary shaker at a temperature of 10 °C and a speed of 150 rpm for 4 h (Han et al. 2020). The extracted liquid was filtered with Whatman No. 1 filter paper and then centrifuged at a temperature of 4 °C with a speed of 12000 rpm for 20 min. The supernatant was the crude enzyme liquid used for measuring the enzyme activity.

Enzyme activities assays

Laccase activity was assayed via the changes in the absorbance at 415 nm, which is related to the rate of oxidation of 1 mM 2,2’-azinobis-[3-ethyltiazoline-6-sulfonate] (ABTS). The reaction mixture was referred to in Han et al. (2020) and measured using an iMarkTM microplate absorbance reader (Bio-Rad, Hercules, CA). One unit of enzyme activity was defined as the amount of enzyme forming 1 µmol of ABTS+ per min (Ɛ415 = 3.16 × 104 M-1 cm-1).

The mean values were taken from three independent experiments and the standard deviations for the experiments were less than ± 10% of the mean values.

Statistical analysis

Two-way analysis of variance between the lignocellulosic residues and the different species was performed according to Han et al. (2020), using SPSS software (PROC GLM, version 22.0, IBM, Armonk, NY). All statistical figures were generated using the program Origin 2016 (OriginLab Corporation, Northampton, MA).

Genomic DNA extraction, polymerase chain reaction, and sequencing

Mycelia for the DNA extraction were grown on CYM for 9 d, and the samples were obtained from the surfaces of the CYM via scraping. The total genomic DNA of the mycelia was extracted via a cetyltrimethylammonium bromide rapid plant genome extraction kit (Aidlab Biotechnologies Co., Ltd., Beijing, China) according to the instructions provided by the manufacturer with some modifications (An et al. 2016; Han et al. 2016; An et al. 2020a). The method of amplifying the ITS regions and polymerase chain reaction (PCR) cycling schedule for ITS was in accordance with a study by Han et al. (2016). The PCR products were purified and sequenced at Beijing Genomics Institute (Beijing, China). The newly generated sequence was deposited in GenBank.

RESULTS AND DISCUSSION

Molecular Biological Results

The four tested basidiomycete strains (Han 849, Han 851, Han 1321, and Han 1333) were identified via molecular biology as Cerrena unicolor, Lenzites betulinus, Stropharia rugosoannulata, and Auricularia heimuer. The corresponding GenBank numbers were MW467890, MW467891, MW467892, and MW467893, respectively.

Laccase Activity from Different Lignocellulosic Residues

Recent studies have shown that lignocellulosic residues stimulate enzyme production in basidiomycetes (Pinar et al. 2017; Gupta and Jana 2018; Huang et al. 2019; Leite et al. 2019; Sadeghian-Abadi et al. 2019; Atilano-Camino et al. 2020; Han et al. 2020). Moreover, lignocellulosic residues are suitable for solid-state fermentation and submerged fermentation. Previous studies on the laccase production of fungi grown on lignocellulosic residues have primarily used common lignocellulosic materials, e.g., poplar wood, cottonseed hull, tree leaves, corncob, and coffee shells (An et al. 2020b; Han et al. 2020). However, there is a large variety of lignocellulose residues. Finding new suitable lignocellulosic residues for the growth of fungi in order to produce laccase is also important for solid-state fermentation. In this study, Populus beijingensis, Firmiana platanifolia, Sorghum bicolor, and Oryza sativa were used to stimulate the tested fungi grown into secreting laccase. As shown in Table 1, the effect of the lignocellulosic residues on the laccase production of four Basidiomycete fungi strains was significant (p-value was less than 0.001) throughout the fermentation stage.

In terms of Cerrena unicolor Han 849, laccase activity was detected in all lignocellulosic residues on day 1 (as shown in Figs. 1 through 4). The laccase activity of C. unicolor Han 849 with Oryza sativa, Sorghum bicolor, Firmiana platanifolia and Populus beijingensis residues was 35.56 U/L ± 1.81 U/L, 26.92 U/L ± 0.87 U/L, 121.86 U/L ± 3.48 U/L, and 45.11 U/L ± 1.96 U/L, respectively, on day 1. Compared to the other three lignocellulosic residues, C. unicolor Han 849 secreted higher laccase activity with Firmiana platanifolia from day 1 (as shown in Fig. 3). The laccase activity of C. unicolor Han 849 with Oryza sativa, Sorghum bicolor, Firmiana platanifolia, and Populus beijingensis residues ranged from 25.12 U/L ± 0.97 U/L to 102.17 U/L ± 3.55 U/L, 26.92 U/L ± 0.87 U/L to 371.71 U/L ± 5.69 U/L, 121.86 U/L ± 3.48 U/L to 625.98 U/L ± 24.08 U/L, and 45.11 U/L ± 1.96 U/L to 295.96 U/L ± 4.85 U/L, respectively (as shown in Figs. 1 through 4). The laccase activity of C. unicolor Han 849 with Firmiana platanifolia was maintained at a high level during the entire fermentation stage (as shown in Fig. 3). The maximum laccase activity using Firmiana platanifolia was higher than Oryza sativa, Sorghum bicolor, and Populus beijingensis, approximately 6.13, 1.68, and 2.12 times as much (as shown in Table 2). The time to reach maximum laccase activity for C. unicolor Han 849 with Oryza sativa, Sorghum bicolor, Firmiana platanifolia, and Populus beijingensis was 6 d, 6 d, 7 d, and 10 d, respectively (as shown in Table 2). The time to reach maximum laccase activity for soft lignocellulosic residues (Oryza sativa and Sorghum bicolor) was slightly sooner than for hard wood residues (Firmiana platanifolia and Populus beijingensis). Han et al. (2020) reported that the time to reach maximum laccase activity using Pleurotus ostreatus CY 568 with poplar sawdust was earlier than when using corncob, while the time to reach maximum laccase activity using P. ostreatus CCEF 99 with corncob was earlier than when using poplar sawdust. An et al. (2020b) reported that the time to reach and the value of the maximum laccase activity from some of P. ostreatus and F. velutipes strains using corncob were earlier and higher, respectively, than those strains using poplar wood. Therefore, different species or different strains of the same species have different preferences for lignocellulosic residues. Based on C. unicolor Han 849, generally speaking, Firmiana platanifolia was more suitable for C. unicolor Han 849 in terms of producing laccase. For Lenzites betulinus Han 851, no laccase activity was detected when using Sorghum bicolor on the 1st day (as shown in Figs. 1 through 4). The laccase activity of L. betulinus Han 851 using Oryza sativa, Sorghum bicolor, Firmiana platanifolia, and Populus beijingensis was 5.22 U/L ± 0.17 U/L, 0 U/L ± 0 U/L, 3.32 ± 0.30 U/L, and 5.83 U/L ± 0.17 U/L, respectively, on day 1. Furthermore, laccase activity was first detected in Sorghum bicolor on the day 5 and was very low (as shown in Fig. 2). The maximum laccase activity for L. betulinus Han 851 using Oryza sativa (55.66 U/L ± 3.60 U/L on day 5) was higher than Sorghum bicolor (3.62 U/L ± 0.30 U/L on day 7), Firmiana platanifolia (6.73 U/L ± 0.35 U/L on day 10), and Populus beijingensis (21.20 U/L ± 1.42 U/L on day 8), 15.38, 8.27, and 2.63 times greater, respectively (as shown in Table 2). In general, Oryza sativa was more suitable for L. betulinus Han 851 in terms of producing laccase according to the value and the time to reach maximum laccase activity. For Stropharia rugosoannulata Han 1321, the laccase activity was barely detected when using Populus beijingensis on day 1 (as shown in Figs. 1 through 4). The laccase activity of S. rugosoannulata Han 1321 when using Sorghum bicolor was undetected during the entire fermentation stage (as shown in Fig. 2). The maximum laccase activity for S. rugosoannulata Han 1321 using Populus beijingensis, Firmiana platanifolia, and Oryza sativa was 3.11 U/L ± 0.17 U/L, 1.61 U/L ± 0.17 U/L, and 5.12 U/L ± 0.30 U/L, respectively (as shown in Table 2). Relatively speaking, Oryza sativa was more suitable for S. rugosoannulata Han 1321 in term of producing laccase. For Auricularia heimuer Han 1333, the laccase activity when using Oryza sativa, Firmiana platanifolia, and Populus beijingensis ranged from 7.13 U/L ± 0.46 U/L to 40.99 U/L ± 2.09 U/L, 1.00 U/L ± 0.17 U/L to 15.07 U/L ± 0.80 U/L, and 0.30 U/L ± 0 U/L to 11.05 U/L ± 0.17 U/L, respectively (as shown in Figs. 1, 3, and 4). No laccase activity was detected when using Sorghum bicolor (as shown in Fig. 2). Therefore, Oryza sativa was more suitable for A. heimuer Han 1333 in terms of secreting laccase.

Many species from the Basidiomycetes genus are excellent laccase producers, e.g., Trametes trogii and Pleurotus ostreatus. Bacteria and actinomycetes also have ability to secrete laccase, but the activity and the yield of the secreted laccase are lower. The activity of laccase secreted by Actinobacteria strains using Olive pomace as substrate is between 5.63 x 10-3 U/mL and 2.15 x 10-3 U/mL (Medouni-Haroune et al. 2017). Therefore, most of the studies related to laccase have focused on Basidiomycetes. Lakhtar et al. (2010) reported a laccase activity of 0.2 U/mL via the cultivation of Lentinula edodes with olive mill wastewater. The maximum laccase activities obtained from SSF cultures of Trametes trogii and T. versicolor using Corylus maxima, Zea mays and Triticum sativum were 384 U/L and 68 U/L, 198 U/L and 387 U/L, and 244 U/L and 215 U/L, respectively (Birhanli and Yesilada 2013). The maximum laccase activity for Flammulina velutipes CCMSSC 05331 using cottonseed hull, corncob, and poplar wood was 68.11 U/L ± 1.09 U/L, 50.13 U/L ± 3.68 U/L, and 39.98 U/L ± 0.17 U/L, respectively (An et al. 2020b). In this study, the maximum laccase activity of C. unicolor Han 849 using Firmiana platanifolia was 625.98 U/L ± 24.08 U/L (as shown in Fig. 3); therefore, C. unicolor Han 849 showed a strong laccase secretion ability with Firmiana platanifolia compared to the other lignocellulosic residues. The maximum laccase activity of Coriolopsis trogii or T. versicolor using walnut shells was higher than using wheat straw (Birhanli and Yeşilada 2013). The time to reach of maximum laccase activity for strains in genus Pleurotus and Flammulina when using cottonseed hull was earlier than using corncob or poplar wood, except for P. ostreatus CY 568 and CCMSSC 00406 (An et al. 2020b).

Fig. 1. The laccase activity from Han 849, Han 851, Han 1321, and Han 1333 grown on Oryza sativa. The average values were calculated from individual measurements from three parallel cultures of the corresponding strains.

Fig. 2. The laccase activity from Han 849, Han 851, Han 1321, and Han 1333 grown on Sorghum bicolor. The average values were calculated from individual measurements from three parallel cultures of the corresponding strains.

Fig. 3. The laccase activity from Han 849, Han 851, Han 1321, and Han 1333 grown on Firmiana platanifolia. The average values were calculated from individual measurements from three parallel cultures of the corresponding strains.

The laccase activity obtained from P. ostreatus CCEF 99 and CY 568 using sawdust as a substrate was sometimes higher than using a corncob substrate, but sometimes lower than using a corncob substrate in a solid-state fermentation process (Han et al. 2020). The results of this study were similar to previous studies, which indicated that there was no complete correlation between lignin content and laccase activity. Of course, the difference of lignocellulosic residues and the adaptability of fungi to different lignocellulosic residues will lead to great differences in laccase production time and yield.

Fig. 4. The laccase activity from Han 849, Han 851, Han 1321 and Han 1333 grown on Populus beijingensis. The average values were calculated from individual measurements from three parallel cultures of the corresponding strains.

Laccase Activity in Different Fungi

Previously studies on improving laccase activity have primarily focused on the effects of different physico-chemical parameters, e.g., substrate ratio, inoculum age, inoculum size, pH, and temperature (Elissetche et al. 2007; Vibha and Negi 2018; Gupta and Jana 2019; Unuofin et al. 2019a,b; Han et al. 2020). However, different species or different strains of the same species are an important factor affecting laccase activity (Lamia et al. 2017; Huang et al. 2019; An et al. 2020a,b; Han et al. 2020). It is important to analyze the capacity of laccase production from fungi among different genera for the development of new productive strains as well as providing more valuable strains for industrial production. In this study, using four different Basidiomycete fungi strains had a significant (p-value is less than 0.001) effect on laccase activities throughout the fermentation stage (as shown in Table 1). Cerrena unicolor Han 849, Lenzites betulinus Han 851, Stropharia rugosoannulata Han 1321, and Auricularia heimuer Han 1333 displayed their unique ability of secreting laccase (Figs. 1 through 4).

The laccase activity from Cerrena unicolor Han 849 with Oryza sativa was approximately 6.81 and 4.99 times greater than the activity from Lenzites betulinus Han 851 and Auricularia heimuer Han 1333, respectively, on day 1, while the laccase activity of Stropharia rugosoannulata Han 1321 with Oryza sativa was undetected on day 1 (as shown in Figs. 1 through 4). In addition, the maximum laccase activity of C. unicolor Han 849 on Oryza sativa was 102.17 U/L ± 3.55 U/L, which is approximately 1.84, 19.96, and 2.49 times greater than L. betulinus Han 851, S. rugosoannulata Han 1321, and A. heimuer Han 1333, respectively (as shown in Table 2). The laccase activity from C. unicolor Han 849 with Oryza sativa was higher than L. betulinus Han 851, S. rugosoannulata Han 1321, and A. heimuer Han 1333 during the entire fermentation process (as shown in Fig. 1). The laccase activity of L. betulinus Han 851 and A. heimuer Han 1333 was roughly equivalent, and the laccase activity from S. rugosoannulata Han 1321 was extremely low. Different from Oryza sativa, C. unicolor Han 849 showed a high laccase activity when using Sorghum bicolor, while the other three species showed extremely low or even undetectable laccase activity levels during the entire fermentation process (as shown in Fig. 2). The maximum laccase activity of C. unicolor Han 849 using Sorghum bicolor was 371.71 U/L ± 5.69 U/L, which was approximately 102.68 times greater than L. betulinus Han 851 (as shown in Table 2). Both S. rugosoannulata Han 1321 and A. heimuer Han 1333 had undetected laccase activity. The laccase activity trends of the four species using Firmiana platanifolia were similar to the trends of Sorghum bicolor (as shown in Fig. 3). As such, C. unicolor Han 849 showed a superior laccase secreting capacity with Firmiana platanifolia and a maximum laccase activity of 625.98 U/L ± 24.08 U/L (as shown in Table 2). The maximum laccase activity using Firmiana platanifolia was approximately 93.01, 388.81, and 41.54 times greater than L. betulinus Han 851, S. rugosoannulata Han 1321, and A. heimuer Han 1333, respectively. The laccase activity of the four species using Populus beijingensis was similar to the trends of the other three lignocellulosic residues. (as shown in Fig. 4). The maximum laccase activity of C. unicolor Han 849 using Populus beijingensis was 295.96 U/L ± 4.85 U/L, which was approximately 13.96, 95.16, and 26.78 times greater than L. betulinus Han 851, S. rugosoannulata Han 1321, and A. heimuer Han 1333, respectively. Only C. unicolor Han 849 maintained a high level of laccase activity; the other species all maintained a low level of laccase activity (as shown in Fig. 4). An et al. (2020b) reported that the maximum laccase activity for P. ostreatus CY 568 grown on cottonseed hull was approximately 1.21, 1.47, 1.84, 11.33, 12.57, 12.82, and 13.26 times greater than P. ostreatus CCEF 89, P. ostreatus CCMSSC 00322, P. ostreatus CCMSSC 00406, F. velutipes CCMSSC 00114, F. velutipes CCMSSC 00118, F. velutipes CCMSSC 05317, and F. velutipes CCMSSC 05331, respectively. The maximum laccase activity of P. ostreatus CCEF 89 with corncob was approximately 1.14, 1.89, 4.46, 11.46, 12.00, 12.49, and 13.95 times greater than P. ostreatus CY 568, P. ostreatus CCMSSC 00322, P. ostreatus CCMSSC 00406, F. velutipes CCMSSC 00114, F. velutipes CCMSSC 00118, F. velutipes CCMSSC 05317, and F. velutipes CCMSSC 05331, respectively (An et al. 2020b). Therefore, it was found that different strains have substrate bias and their laccase secretion capacities are different. In this study, L. betulinus Han 851 and A. heimuer Han 1333 showed a more obvious preference for Oryza sativa (as shown in Fig. 1). However, C. unicolor Han 849 preferred Firmiana platanifolia. Han et al. (2020) reported that a continuous and stable laccase production from P. ostreatus was an extremely important advantage when using solid-state fermentation with poplar sawdust. Similarly, a continuous and stable laccase production from C. unicolor Han 849 was obvious advantage when using solid-state fermentation with any of the four lignocellulosic residues used in this study (as shown in Figs. 1 through 4). In general, the laccase secreting ability of C. unicolor Han 849 was stronger than the other three tested species, and the laccase secreting ability of S. rugosoannulata Han 1321 was extremely poor in this study.

CONCLUSIONS

  1. The biosynthetic potential of Basidiomycetes was highly dependent on the species of fungi. In general, the laccase secreting ability of C. unicolor Han 849 was stronger than L. betulinus Han 851, S. rugosoannulata Han 1321, and A. heimuer Han 1333. In addition, the laccase secreting ability of S. rugosoannulata Han 1321 was extremely poor.
  2. The biosynthetic potential of Basidiomycetes was highly dependent on the lignocellulosic residues.
  3. Different species of fungi had a preference in lignocellulosic residues. Cerrena unicolor Han 849 preferred Firmiana platanifolia, while L. betulinus Han 851 and A. heimuer Han 1333 showed a more obvious preference for Oryza sativa.
  4. The continuous and stable laccase production from C. unicolor Han 849 was an obvious advantage of solid-state fermentation when using any of the four lignocellulosic residues tested in this study.
  5. The presence of Firmiana platanifolia was conducive to C. unicolor Han 849 secreting laccase during solid-state fermentation. The maximum laccase production of C. unicolor Han 849 grown on Firmiana platanifolia was approximately 2.12, 1.68, and 6.13 times greater than Populus beijingensis, Sorghum bicolor, and Oryza sativa, respectively.

ACKNOWLEDGMENTS

This research was supported by the National Natural Science Foundation of China (No. 31900009), the Funds for Undergraduate Innovation and Entrepreneurship Training Project in Langfang Normal University (No. S202010100020), the Fundamental Research Funds for the Universities in Hebei Province (No. JYQ201901), the Top-notch Youth Project of Langfang City, and the Top-notch Youth Project of Colleges and Universities in Hebei Province (No. BJ2019007).

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Article submitted: January 16, 2021; Peer review completed: February 28, 2021; Revised version received and accepted: March 1, 2021; Published: March 5, 2021.

DOI: 10.15376/biores.16.2.3017-3031