Abstract
The laccase activity of three cultivated Pleurotus strains was investigated using different lignocellulosic biomasses for solid-state fermentation. The lignocellulosic biomasses were Sophora japonica, Salix babylonica, Populus beijingensis, and Pinus tabuliformis, which were selected because of their laccase producing ability and low cost. The maximum laccase activities from P. ostreatus Han 1189, P. citrinopileatus Han 1192, and P. eryngii Han 1205 using Sophora japonica were 768.74 U/L ± 11.25 U/L, 144.77 U/L ± 9.70 U/L, and 200.42 U/L ± 6.85 U/L, respectively. The three species of Pleurotus showed consistency in having a preference of hardwood to secret laccase under solid-state fermentation when facing hardwood and softwood as substrates. Furthermore, the presence of Sophora japonica was beneficial to improving the laccase activity for three Pleurotus strains. The capacity of the laccase secretion of P. ostreatus Han 1189 was superior to the capacity of P. citrinopileatus Han 1192 and P. eryngii Han 1205. The discovery was helpful for obtaining new high-producing strains and suitable wood materials, as well as expanding these high-producing strains for industrial applications to obtain high laccase activity, high laccase yield, and low cost laccase.
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Effect of Different Lignocellulosic Biomasses on Laccase Production by Pleurotus Species
Mei-Ling Han,a,# Xue-Qing Li,a,# Chun-Dan Zhang,a Mei-Xue Li,a Meng-Han Zhang,a Min An,a Xin-Ying Dou,a Tian-Xin Zhang,a Xun-You Yan,a,c Lu-Sen Bian,b and Qi An a,c,*
The laccase activity of three cultivated Pleurotus strains was investigated using different lignocellulosic biomasses for solid-state fermentation. The lignocellulosic biomasses were Sophora japonica, Salix babylonica, Populus beijingensis, and Pinus tabuliformis, which were selected because of their laccase producing ability and low cost. The maximum laccase activities from P. ostreatus Han 1189, P. citrinopileatus Han 1192, and P. eryngii Han 1205 using Sophora japonica were 768.74 U/L ± 11.25 U/L, 144.77 U/L ± 9.70 U/L, and 200.42 U/L ± 6.85 U/L, respectively. The three species of Pleurotus showed consistency in having a preference of hardwood to secret laccase under solid-state fermentation when facing hardwood and softwood as substrates. Furthermore, the presence of Sophora japonica was beneficial to improving the laccase activity for three Pleurotus strains. The capacity of the laccase secretion of P. ostreatus Han 1189 was superior to the capacity of P. citrinopileatus Han 1192 and P. eryngii Han 1205. The discovery was helpful for obtaining new high-producing strains and suitable wood materials, as well as expanding these high-producing strains for industrial applications to obtain high laccase activity, high laccase yield, and low cost laccase.
DOI: 10.15376/biores.17.3.4921-4936
Keywords: Pleurotus species; Solid-state fermentation; Lignocellulosic biomass; Substrate preference
Contact information: a: College of Life Science, Langfang Normal University, Langfang, Hebei 065000 China; b: Experimental Centre of Forestry in North China, Chinese Academy of Forestry, Beijing 102300 China; c: Technical Innovation Center for Utilization of Edible and Medicinal Fungi in Hebei Province, Langfang, Hebei 065000 China; #Mei-Ling Han and Xue-Qing Li contributed equally to this article;
* Corresponding author: fungiqian@163.com
INTRODUCTION
Currently, the primary sources (80% to 88%) of the world energy supply are fossil fuels, e.g., crude oil, coal, and natural gas, which are non-renewable, limited, and costly (Erakhrumen 2014; Taha et al. 2015). Meanwhile, the demand for and amount consumed of energy is still rising, as it is necessity for human life (Yousef et al. 2021a,b). Using fossil fuels, e.g., gasoline, as a primary source of energy has environmental impacts, including air, water, and soil pollution, as well as negative effects on public health (Galbe and Zacchi 2002; Taha et al. 2015). The methods of obtaining sustainable bioenergy from biomaterials, e.g., bioethanol, not only can reduce reliance on fossil fuels, but the final product may also have a greater economical and net performance. As such, lignocellulosic biomass, the most abundant renewable material on earth, has attracted considerable attention because of its ability to be converted into other products (Kumar et al. 2016; Gaikwad and Meshram 2020; An et al. 2021c; Galic et al. 2021; Han et al. 2021b). Fungi cultured with lignocellulosic biomass can produce fruiting bodies for sale or eating and produce various other products, e.g., enzymes and sterol. Furthermore, numerous enzymes related to lignocellulosic degradation could be secreted by these fungi, e.g., ligninolytic enzymes, cellulase, and hemicellulose. Among the ligninolytic enzymes, laccase, as a long since discovered enzyme, has been widely studied in different research fields.
Laccase (EC 1.10.3.2), also called p-diphenol or dioxygen oxidoreductase, was first described in Toxicodendron vernicifluum, which was primitively named Rhus vernicifera; it was subsequently found in bacteria, fungi, and some insects (Zerva et al. 2019; Kumar et al. 2021; Khatami et al. 2022). Laccase displays a wide substrate specificity, along with the simultaneous reduction of molecular oxygen to water (Garrido-Bazán et al. 2016; Hernández et al. 2017). Based on this, laccase could be applied in many fields related to biotechnology, environmental protection, and medicine, e.g., lignin degradation, biocatalysis, bioremediation and detoxification, food and beverages, detergents, sensors and biofuel cells, biopolymers, nanobiotechnology, and biomedicine (Baldrian 2006; Kunamneni et al. 2008; Chauhan et al. 2017; Mate and Alcalde 2017; Yang et al. 2017; Agrawal et al. 2018; Munk et al. 2018; Navas et al. 2019; Unuofin et al. 2019; Zerva et al. 2019; Xu et al. 2020). However, the widespread application of laccase in numerous aspects of biotechnological processes has been limited due to low laccase activity, low laccase yields, and the high laccase costs (Cardona et al. 2010; Wang et al. 2019). Effective laccase production strategies have attracted increasing research attention to improve activity and reduce cost (Akpinar and Urek 2017; Chenthamarakshan et al. 2017).
Currently, laccase is primarily secreted by bacteria and fungi. Laccase products are primarily higher-plant laccase and fungal laccase. Producing laccase via fungi is primarily concentrated in white-rot fungi of the Aspergillus and Trichoderma genus. This is likely because the laccase production is highest in white-rot fungi. Fungal species are also important factors affecting enzyme activity. Therefore, it is meaningful to develop a new enzyme-producing strain belonging to white rot fungi. Another important factor affecting enzyme activity is the fermentation method. At present, the primary fermentation methods include submerged fermentation, solid-state fermentation, and solid fermentation combined with submerged fermentation (An et al. 2016; Dey et al. 2016). Previously, submerged fermentation (SF) has been the most common approach for producing most enzymes, including laccase. Submerged fermentation provides a uniform distribution of nutrients, allowing adequate exposure and absorption of nutrients by cultured microorganisms. However, the disadvantage of SF is higher energy consumption. Under this circumstance, there has been a trend towards an increasing use of solid state fermentation (SSF) to produce certain enzymes over the past decade (Xin and Geng 2011; Nguyen et al. 2018; An et al. 2021a; An et al. 2021b; Liu et al. 2022). Compared with SF, the desired microorganism grown on solid lignocellulosic biomass with the absence of free water is closer to their natural environment in the wild (Jaramillo et al. 2017). In addition, being a simpler operating technique, there is lower energy consumption and lower dilution of the enzymes, which are the major advantages of SSF. Lignocellulosic biomass is the commonly substrate used for fungi growth. The selection of different types of lignocellulosic materials and the optimization of the media are the methods that many research groups have used to improve laccase activity (Tisma et al. 2012; Soumya et al. 2016).
Previous research had studied laccase secreted by numerous fungi cultivated on lignocellulosic biomass, e.g., Coriolopsis gallica on sawdust waste, and Ganoderma lucidum on rice husks, rice straw, and sunflower seed hulls (Daâssi et al. 2016; Postemsky et al. 2017; Zhang et al. 2018). In addition, many studies indicated that Pleurotus ostreatus is also an excellent producer of laccase (Karp et al. 2015; An et al. 2018; Brugnari et al. 2018; Song et al. 2020; Perez-Montiel et al. 2021; Cruz-Vazquez et al. 2022; Duran-Sequeda et al. 2022). Under these circumstances, it is important to evaluate the laccase-producing ability of different species belonging to the Pleurotus genus for developing new strains for enzyme production. In the present study, the laccase activities of three cultivated Pleurotus sp. strains, grown on lignocellulosic biomass, were reported. The lignocellulosic biomass used was all wood materials with a high lignin content. The results were helpful for obtaining new high-producing strains and suitable wood materials, as well as expanding high-producing strain for industrial application to obtain high laccase activity, high laccase yield, and low cost laccase.
EXPERIMENTAL
Materials
Microorganisms
Three cultivated strains, Han 1189, Han 1192, and Han 1205, from the genus of Pleurotus, were purchased from a local market in China (Langfang city, Hebei province, China). All strains were isolated and purified on complete yeast medium (CYM) and maintained at the college of life science, Langfang Normal University.
Lignocellulosic biomass
Four different lignocellulosic biomasses (Sophora japonica, Salix babylonica, Populus beijingensis, and Pinus tabuliformis) were collected from Langfang city, then cut into small pieces, and air-dried. All the small pieces of lignocellulosic biomass were subjected to grinding and milling to a fine powder with a particle size between 20- and 60-mesh using a FZ102 micro plant grinding machine (Tianjin Taisite Instrument Co., Ltd.). These were kept in a dry environment for further use as the nutrient substance for fungi growth and crude enzyme induction.
Methods
Fungal culture and inoculums preparation
To perform the activation of the fungus, three cultivated Pleurotus strains were transferred and grown on new CYM agar plates at a temperature of 26 °C for 7 d. Five 5-mm fungal mycelium disks, which were excised from the CYM agar plates, were transferred into 250 mL flasks containing 100 mL of malt extract agar (MEA) liquid medium. All flasks were maintained in a rotary shaker at 150 rpm at a temperature of 26 °C. Mycelial pellets were harvested after 7 d and mixed with a laboratory blender (Ningbo Xinzhi Biotechnology Co., Ltd.) for 2 min at 8000 rpm. These suspensions would act as the inoculum.
Induction laccase production of fungi
First, 2 g of the milled lignocellulosic biomass was moistened with 10 mL of basal solution (MgSO4·7H2O 0.5 g/L, K2HPO4 1 g/L, and KH2PO4 0.46 g/L) in a 250 mL Erlenmeyer flasks and autoclaved at a temperature of 121 °C for 30 min. After cooling, 3 mL of inoculum was added into the flasks and all cultivations were carried out at a temperature of 26 °C while undergoing agitation (at 150 rpm).
Enzyme activity
First, 100 mL of acetate-sodium acetate buffer (50 mM, at a pH of 5.5) was added into each of the flasks containing a fermentation substrate at different time intervals. Then, these flasks were transferred into a rotary shaker to perform the extraction (at a temperature of 10 °C, at 120 rpm, for 4 h) (An et al. 2021a). To determine the extracellular laccase activities, the lignocellulosic biomass and mycelium soaked into the culture fluid were removed via filter paper, and the filtered liquid was centrifuged (at 12000 rpm for 15 min at a temperature of 4 ℃). The supernatant obtained after centrifugation was preserved at a temperature of -80 ℃ and used for subsequent determination of the laccase activity.
The laccase activity was assayed with 2,2’-azinobis-[3-ethyltiazoline-6-sulfonate] (ABTS) as the substrate, which was referred to the description previously by Han et al. (2021b). The assay mixture (300 μL) was prepared with 190 μL of 50 mM acetate-sodium acetate buffer (a pH of 4.2), 100 μL of 1 mM ABTS, and 10 μL of the appropriately diluted enzyme sample. Then, the reaction mixture was measured using an iMarkTM microplate absorbance reader (Bio-Rad, Hercules, CA). For calculating the laccase activity molar extinction coefficient of the oxidized ABTS at 415 nm, Ɛ = 3.16 × 104 M-1 cm-1, was used. The laccase activity was expressed in Units, where one unit was defined as the amount of laccase required to oxidize 1 μmol of ABTS per min.
Statistical analysis
Analyses of variance (ANOVA) between the fungi and lignocellulosic biomass were performed using the SPSS statistical program. The colored figures were created by Origin 2016.
Nucleic acid (fungal DNA) extraction and PCR amplification
Genomic DNA of three Pleurotus strains was collected using a cetyltrimethyl-ammonium bromide (CTAB) rapid plant genome extraction kit-DN14. The method for obtaining the mycelium was outlined in Han et al. (2016) and Han et al. (2021a). In addition, ITS1 and ITS4 primers (for Han 1192) and ITS5 and ITS4 primers (for Han 1189 and Han 1205) were used for PCR amplification in a thermocycler (Applied Biosystems, Waltham, MA) (White et al. 1990). The PCR products were detected via electrophoresis, and the qualified products were sent to Beijing Genomics Institute (Beijing, China) for sequencing. The sequenced data was edited and processed using BioEdit software, and then blasted and identified on the National Centre for Biotechnology Information website (NCBI, http://www.ncbi.nlm.gov/). Finally, these new internal transcribed spacer (ITS) sequences of Han 1189, Han 1192, and Han 1205 were submitted to the GenBank website, and the GenBank numbers were ON197668, ON197669, and ON197670, respectively.
RESULTS AND DISCUSSION
Identification of the Three Cultivated Pleurotus Strains
The taxonomic status of strains Han 1189, Han 1192, and Han 1205, as identified via ITS sequence, were Pleurotus ostreatus, Pleurotus citrinopileatus, and Pleurotus eryngii, respectively.
Results of the Statistical Analysis
The fungi and lignocellulosic biomass significantly affected the laccase activity (p-value less than 0.001) at different times of cultivation (Table 2). Meanwhile, the interaction of the fungi and lignocellulosic biomass significantly affected the laccase activity during the whole fermentation process (p-value less than 0.001) (Table 2).
Table 1. Effects of the Fungi, Lignocellulosic Biomass, and the Interactions of Fungi and Lignocellulosic Biomass on Laccase Activity (Two-way ANOVA)
Effect of Various Lignocellulosic Biomasses on Laccase Activity
Microorganisms capable of degrading lignins usually secrete extracellular lignin-degrading enzymes (Martani et al. 2017). Lignocellulosic biomass is a valuable substrate for laccase production. Abundant studies have provided evidence that lignins can stimulate laccase production (Liu et al. 2013; Adekunle et al. 2017; Mishra et al. 2017; Palazzolo et al. 2019; Liu et al. 2022). However, selecting a suitable lignocellulose to stimulate laccase production by fungi will be one of the important methods for efficient laccase production using solid state fermentation (Elisashvili et al. 2008; An et al. 2021a,c; Han et al. 2021b).
At the beginning of the fermentation process, the laccase activities of Pleurotus ostreatus Han 1189 with Pinus tabuliformis, Sophora japonica, Salix babylonica, and Populus beijingensis were 5.83 U/L ± 0.70 U/L, 27.93 U/L ± 2.01 U/L, 16.38 U/L ± 0.92 U/L, and 14.67 U/L ± 0.17 U/L, respectively (as shown in Fig. 1). Interestingly, the laccase activity detected in broad-leaved trees (Sophora japonica, Salix babylonica, and Populus beijingensis) was higher than the laccase activity in coniferous trees (Pinus tabuliformis). Therefore, Sophora japonica may be a suitable lignocellulosic biomass for stimulating P. ostreatus Han 1189 to secret laccase due to the laccase activity detected at the 1st day. The maximum laccase activity of P. ostreatus Han 1189 with Sophora japonica was 768.74 U/L ± 11.25 U/L on the 7th day, nearly 8.25-fold, 4.67-fold, and 3.82-fold higher than the laccase activity of Pinus tabuliformis (93.13 U/L ± 3.65 U/L, 6th day), Salix babylonica (164.46 U/L ± 3.32 U/L, 8th day), and Populus beijingensis (201.13 U/L ± 16.92 U/L, 8th day) (as shown in Table 2). Clearly, broad-leaved trees were more helpful for P. ostreatus Han 1189 in terms of improving laccase activity. However, the laccase activity detected on 10th day was 308.12 U/L ± 28.43 U/L, which was approximately 7.82-fold, 513.53-fold, and 34.08-fold higher than the laccase activity of Pinus tabuliformis (39.38 U/L ± 0.92 U/L), Salix babylonica (0.60 U/L ± 0 U/L), and Populus beijingensis (9.04 U/L ± 1.09 U/L) (Fig. 1). Furthermore, the laccase activity value of Sophora japonica was greater than 300 U/L from the 3rd day to the 10th day of fermentation (Fig. 1). This seems to indicate that a relatively stable and continuous laccase activity can be obtained with this lignocellulosic biomass. Obviously, broad-leaved trees, especially Sophora japonica, were more suitable for Pleurotus ostreatus Han 1189 in terms of secreting laccase.
Fig. 1. Laccase activity of Pleurotus ostreatus Han 1189 grown on Pinus tabuliformis, Sophora japonica, Salix babylonica, and Populus beijingensis using solid-state fermentation
Table 2. Maximum Laccase Activity, Lignocellulosic Biomass, and Occurrence Time of Cultivated Pleurotus sp. Strains
Note: Data is presented as the mean ± standard deviation for biological triplicates and is expressed as U/L
For the 1st day, the laccase activity value from Pleurotus citrinopileatus Han 1192 could be measured for Pinus tabuliformis (4.32 U/L ± 0.17 U/L), Salix babylonica (14.37 U/L ± 0.35 U/L), and Populus beijingensis (10.95 U/L ± 0.97 U/L), and unmeasured on Sophora japonica (Fig. 2). The maximum laccase activity from P. citrinopileatus Han 1192 was 33.35 U/L ± 1.52 U/L (7th day), 144.77 U/L ± 9.70 U/L (8th day), 102.57 U/L ± 3.47 U/L (5th day), and 134.82 U/L ± 5.39 U/L (8th day) for Pinus tabuliformis, Sophora japonica, Salix babylonica, and Populus beijingensis, respectively (Table 2). In other words, the maximum laccase activity for Sophora japonica, Salix babylonica, and Populus beijingensis was approximately 4.34-fold, 3.08-fold, and 4.04-fold higher than the laccase activity for Pinus tabuliformis. However, broad-leaved trees were more conducive to the secretion of laccase for P. citrinopileatus Han 1192. Interestingly, the highest laccase activity of the tested four lignocellulosic biomasses was still present for Sophora japonica, although no laccase activity was detected for Sophora japonica at the 1st day. In addition, the laccase activity level from P. citrinopileatus Han 1192 with Pinus tabuliformis was low throughout the whole fermentation stage.
Fig. 2. Laccase activity of Pleurotus citrinopileatus Han 1192 grown on Pinus tabuliformis, Sophora japonica, Salix babylonica, and Populus beijingensis using solid-state fermentation
On the 1st day, the laccase activity of Pleurotus eryngii Han 1205 for Pinus tabuliformis, Sophora japonica, Salix babylonica, and Populus beijingensis was 2.91 U/L ± 0.17 U/L, 10.95 U/L ± 0.87 U/L, 15.47 U/L ± 0.76 U/L, and 11.05 U/L ± 0.87 U/L, respectively (as shown in Fig. 3). Therefore, the laccase activity for Sophora japonica, Salix babylonica, and Populus beijingensis was approximately 3.76-fold, 5.32-fold, and 3.80-fold greater than Pinus tabuliformis, respectively. The maximum laccase activities value for P. eryngii Han 1205 with Pinus tabuliformis, Sophora japonica, Salix babylonica, and Populus beijingensis were 38.18 U/L ± 1.66 U/L, 200.42 U/L ± 6.85 U/L, 130.30 U/L ± 8.79 U/L, and 117.54 U/L ± 4.35 U/L, respectively, and its time of occurrence was the 5th day, 9th day, 6th day, and 6th day, respectively (as shown in Table 2). In conclusion, the maximum laccase activity of P. eryngii Han 1205 using Sophora japonica, Salix babylonica, and Populus beijingensis was approximately 5.25-fold, 3.41-fold, and 3.08-fold higher than the laccase activity for Pinus tabuliformis. As such, it was not hard to see that broad-leaved trees were more useful for P. eryngii Han 1205 in terms of secreting laccase. Furthermore, Sophora japonica was beneficial for P. eryngii Han 1205 in terms of secreting laccase.
Previous studies indicated that laccase activity may be adversely affected by an excessive lignin content. Gómez et al. (2005) found that the laccase activity from cultures with barley bran (1799.6 U/L) was approximately 2-fold higher than the laccase activity from cultures with chestnut shell (959.8 U/L). Chen et al. (2011) evaluated the laccase activity from seven plant species under solid-state fermentation and observed maximal laccase activity (10,700 IU/g) in cultures with rice straw (lignin 10% to 15% w/w). In addition, the tested lignocellulosic biomass belonged to hardwood stems (Sophora japonica, Salix babylonica, and Populus beijingensis) and softwood stems (Pinus tabuliformis). The lignin content of hardwood stems is 18% to 25%, while the lignin content of softwood stems is 25% to 35% (Malherbe and Cloete 2002; Howard et al. 2003; Sánchez 2009). As such, the present study also indicated that excessive lignin content was a disadvantage in terms of fungi secreting laccase, because the laccase activity from hardwood stems was higher than the laccase activity from softwood stems. Han et al. (2021b) found that maximum laccase activity of Cerrena unicolor Han 849, Lenzites betulina Han 851, and Schizophyllum commune Han 881 with Firmiana platanifolia was 552.34 U/L ± 49.14 U/L, 309.72 U/L ± 12.53 U/L and 5.22 U/L ± 0.35 U/L, which was higher than the laccase activity for Pinus tabuliformis (223.53 U/L ± 21.06 U/L, 36.57 U/L ± 3.39 U/L, and 1.51 U/L ± 0.00 U/L), respectively. A similar phenomenon appeared in this study. An et al. (2021a) indicated that the presence of cottonseed hull and Populus beijingensis were useful for improving the laccase activity of P. ostreatus CY 568. However, this study found that the presence of Sophora japonica was more beneficial in terms of enhancing the laccase activity than Populus beijingensis. In conclusion, broad-leaved trees, especially Sophora japonica, were more suitable for the three cultivated strains of Pleurotus in terms of secreting laccase.
Fig. 3. Laccase activity of Pleurotus eryngii Han 1205 grown on Pinus tabuliformis, Sophora japonica, Salix babylonica, and Populus beijingensis using solid-state fermentation
Effect of Different Fungi on Laccase Activity
Numerous studies have sought to find new high-laccase producing strains (Chen et al. 2011; Xu et al. 2016; Sun et al. 2020; Han et al. 2021b; Khatami et al. 2022). Generally, screening laccase-producing strains focuses on three primary methods, i.e., different species with far different taxonomic status, different species belonging to one genus, and different strains from one species. Janusz et al. (2015) studied the laccase activity of various strains of Flammulina velutipes and found that the laccase producing ability of the different strains was considerably different. Huang et al. (2019) analyzed the ability of a number of edible fungi to degrade crop straw and found that different edible fungi had different abilities to secrete enzymes. As such, developing new laccase producing strains is still one of the important methods for producing high levels of low-cost laccase.
In terms of the laccase activity of Pinus tabuliformis, the laccase activities from Pleurotus ostreatus Han 1189, P. citrinopileatus Han 1192, and P. eryngii Han 1205 were 5.83 U/L ± 0.70 U/L, 4.32 U/L ± 0.17 U/L and 2.91 U/L ± 0.17 U/L on the 1st day (as shown in Fig. 1 through Fig. 3). On the 1st day, the laccase activities of P. ostreatus Han 1189, P. citrinopileatus Han 1192, and P. eryngii Han 1205 with Sophora japonica were 27.93 U/L ± 2.01 U/L, 0 U/L ± 0 U/L and 10.95 U/L ± 0.87 U/L, respectively. Obviously, the laccase activity of P. ostreatus Han 1189 with Pinus tabuliformis or Sophora japonica was higher than the laccase activity of P. citrinopileatus Han 1192 or P. eryngii Han 1205. Similarly, the same phenomenon occurred when Salix babylonica was used as a substrate (as shown in Fig. 1 through Fig. 3). The laccase activity of P. ostreatus Han 1189 was lower than the laccase activity of the other two species when Populus beijingensis was used as a substrate.
The maximum laccase activity values of P. ostreatus Han 1189, P. citrinopileatus Han 1192, and P. eryngii Han 1205 with Pinus tabuliformis were 93.13 U/L ± 3.65 U/L, 33.35 U/L ± 1.52 U/L, and 38.18 U/L ± 1.66 U/L, respectively (Table 2). Therefore, the maximum laccase activity of P. ostreatus Han 1189 was approximately 2.79-fold and 2.44-fold higher than the laccase activities of P. citrinopileatus Han 1192 and P. eryngii Han 1205, respectively. Similarly, the maximum laccase activity values for P. ostreatus Han 1189 with Sophora japonica was 768.74 ± 11.25 U/L, which was approximately 5.31-fold and 3.84-fold higher than the laccase activities of P. citrinopileatus Han 1192 (144.77 U/L ± 9.70 U/L) and P. eryngii Han 1205 (200.42 U/L ± 6.85 U/L), respectively. Furthermore, the laccase activity of P. ostreatus Han 1189 from day 3 to day 10 of fermentation was higher than the maximum laccase value of P. citrinopileatus Han 1192 and P. eryngii Han 1205. This also reflected that P. ostreatus Han 1189 had the advantages of stable laccase production and high activity compared to P. citrinopileatus Han 1192 and P. eryngii Han 1205. The maximum laccase activities of P. ostreatus Han 1189 with Salix babylonica and Populus beijingensis were 164.46 U/L ± 3.32 U/L and 201.13 U/L ± 16.92 U/L, respectively, which were greater than the laccase activities of P. citrinopileatus Han 1192 (102.57 U/L ± 3.47 U/L and 134.82 U/L ± 5.39 U/L) and P. eryngii Han 1205 (130.30 U/L ± 8.79 U/L and 117.54 U/L ± 4.35 U/L), respectively. Therefore, the laccase secretion of P. ostreatus Han 1189 was greater than the secretions from P. citrinopileatus Han 1192 and P. eryngii Han 1205.
The laccase activities of Pleurotus ostreatus IBB 8, P. ostreatus 2175, and P. tuberregium IBB 624 were 7 ± 0.7 or 7 U/flask ± 0.8 U/flask, 15 ± 1.4 or 12 U/flask ± 1.2 U/flask, ands 20 ± 1.8 or 10 U/flask ± 1.0 U/flask, respectively, when grown on tree leaves or wheat straw (Elisashvili et al. 2008). An et al. (2020) found that the laccase activity of P. ostreatus CCEF 89 was 748.24 U/L ± 9.53 U/L, 699.12 U/L ± 44.91 U/L, and 509.75 U/L ± 15.43 U/L using submerged fermentation with cottonseed hull, corncob, and poplar wood, respectively. The laccase activity of P. ostreatus CY 568 and P. ostreatus CCEF 99 for sawdust and corncob was 353.83 U/L ± 11.94 U/L and 440.73 U/L ± 8.36 U/L, and 548.72 U/L ± 19.59 U/L and 286.12 U/L ± 25.80 U/L, respectively, when using solid-state fermentation (Han et al. 2020). Therefore, the laccase secretion ability of P. ostreatus Han 1189 was better than the other P. ostreatus strains in previous studies.
CONCLUSIONS
- Three cultivated strains, belonging to the Pleurotus genus, showed a uniform preference for hardwood in terms of secreting laccase under solid-state fermentation conditions when facing hardwood and softwood as substrates.
- The presence of Sophora japonica was beneficial in terms of improving the laccase activity for Pleurotus ostreatus Han 1189, Pleurotus citrinopileatus Han 1192, and Pleurotus eryngii Han 1205.
- The laccase secretion capacity of P. ostreatus Han 1189 was superior to the laccase secretion capacity of P. citrinopileatus Han 1192 and P. eryngii Han 1205.
ACKNOWLEDGMENTS
The work was funded by the Science and Technology Project of Hebei Education Department (Grant No. BJK2022052), the National Natural Science Foundation of China (Grant No. 31900009), the Special Project of Cultivating Scientific and Technological Innovation Ability of College and Middle School Students (Grant No. S202110100009), the Fundamental Research Funds for the Universities in Hebei Province (Grant No. JYQ201901), and the Top-notch Youth Project of Langfang City, China.
REFERENCES CITED
Adekunle, A. E., Guo, C., and Liu, C.-Z. (2017). “Lignin-enhanced laccase production from Trametes versicolor,” Waste and Biomass Valorization 8(4), 1061-1066. DOI: 10.1007/s12649-016-9680-4
Agrawal, K., Chaturvedi, V., and Verma, P. (2018). “Fungal laccase discovered but yet undiscovered,” Bioresources and Bioprocessing 5, 1-12. DOI: 10.1186/s40643-018-0190-z
Akpinar, M., and Urek, R. O. (2017). “Induction of fungal laccase production under solid state bioprocessing of new agroindustrial waste and its application on dye decolorization,” 3 Biotech 7, 1-10. DOI: 10.1007/s13205-017-0742-5
An, Q., Li, C.-S., Yang, J., Chen, S.-Y., Ma, K.-Y., Wu, Z., Bian, L.-S., and Han, M.-L. (2021a). “Evaluation of laccase production by two white-rot fungi using solid-state fermentation with different agricultural and forestry residues,” BioResources 16(3), 5287-5300. DOI: 10.15376/biores.16.3.5287-5300
An, Q., Liu, Z.-Y., Wang, C.-R., Yang, J., Chen, S.-Y., Chen, X., Zhang, Y.-J., Bian, L. S., and Han, M.-L. (2021b). “Laccase activity from Pleurotus ostreatus and Flammulina velutipes strains grown on agro- and forestry residues by solid-state fermentation,” BioResources 16(4), 7336-7353. DOI: 10.15376/biores.16.4.7337-7354
An, Q., Ma, H.-F., Han, M.-L., Si, J., and Dai, Y.-C. (2018). “Effects of different induction media as inducers on laccase activities of Pleurotus ostreatus strains in submerged fermentation,” BioResources 13(1), 1143-1156. DOI: 10.15376/biores.13.1.1143-1156
An, Q., Qiao, J., Bian, L., Han, M., Yan, X., Liu, Z., and Xie, C. (2020). “Comparative study on laccase activity of white rot fungi under submerged fermentation with different lignocellulosic wastes,” BioResources 15(4), 9166-9179. DOI: 10.15376/biores.15.4.9166-9179
An, Q., Wu, X.-J., Han, M.-L., Cui, B.-K., He, S.-H., Dai, Y.-C., and Si, J. (2016). “Sequential solid-state and submerged cultivation of white rot fungus Pleurotus ostreatus on lignocellulosic biomass for the activity of lignocellulolytic enzymes,” BioResources 11(4), 8791-8805. DOI: 10.15376/biores.11.4.8791-8805
Baldrian, P. (2006). “Fungal laccases-occurrence and properties,” FEMS Microbiology Reviews 30(2), 215-242. DOI: 10.1111/j.1574-4976.2005.00010.x
Brugnari, T., Pereira, M. G., Bubna, G. A., Freitas, E. N. d., Contato, A. G., Corrêa, R. C. G., Castoldi, R., Souza, C. G. M. d., Polizeli, M. d. L. T. d. M., Bracht, A., et al. (2018). “A highly reusable MANAE-agarose-immobilized Pleurotus ostreatus laccase for degradation of bisphenol A,” Science of The Total Environment 634, 1346-1351. DOI: 10.1016/j.scitotenv.2018.04.051
Cardona, C. A., Quintero, J. A., and Paz, I. C. (2010). “Production of bioethanol from sugarcane bagasse: Status and perspectives,” Bioresource Technology 101(13), 4754-4766. DOI: 10.1016/j.biortech.2009.10.097
Chauhan, P. S., Goradia, B., and Saxena, A. (2017). “Bacterial laccase: Recent update on production, properties and industrial applications,” 3 Biotech 7, 1-20. DOI: 10.1007/s13205-017-0955-7
Chen, H.-Y., Xue, D.-S., Feng, X.-Y., and Yao, S.-J. (2011). “Screening and production of ligninolytic enzyme by a marine-derived fungal Pestalotiopsis sp J63,” Applied Biochemistry and Biotechnology 165(7-8), 1754-1769. DOI: 10.1007/s12010-011-9392-y
Chenthamarakshan, A., Parambayil, N., Miziriya, N., Soumya, P. S., Lakshmi, M. S. K., Ramgopal, A., Dileep, A., and Nambisan, P. (2017). “Optimization of laccase production from Marasmiellus palmivorus LA1 by Taguchi method of design of experiments,” BMC Biotechnology 17, 1-10. DOI: 10.1186/s12896-017-0333-x
Cruz-Vazquez, A., Tomasini, A., Armas-Tizapantzi, A., Marcial-Quino, J., and Montiel-Gonzalez, A. M. (2022). “Extracellular proteases and laccases produced by Pleurotus ostreatus PoB: The effects of proteases on laccase activity,” International Microbiology in press, 1-8. DOI: 10.1007/s10123-022-00238-9
Daâssi, D., Zouari-Mechichi, H., Frikha, F., Rodríguez-Couto, S., Nasri, M., and Mechichi, T. (2016). “Sawdust waste as a low-cost support-substrate for laccases production and adsorbent for azo dyes decolorization,” Journal of Environmental Health Science and Engineering 14, 1-12. DOI: 10.1186/s40201-016-0244-0
Dey, T. B., Chakraborty, S., Jain, K. K., Sharma, A., and Kuhad, R. C. (2016). “Antioxidant phenolics and their microbial production by submerged and solid state fermentation process: A review,” Trends in Food Science & Technology 53, 60-74. DOI: 10.1016/j.tifs.2016.04.007
Duran-Sequeda, D., Suspes, D., Maestre, E., Alfaro, M., Perez, G., Ramirez, L., Pisabarro, A. G., and Sierra, R. (2022). “Effect of nutritional factors and copper on the regulation of laccase enzyme production in Pleurotus ostreatus,” Journal of Fungi 8(1), 1-21. DOI: 10.3390/jof8010007
Elisashvili, V., Penninckx, M., Kachlishvili, E., Tsiklauri, N., Metreveli, E., Kharziani, T., and Kvesitadze, G. (2008). “Lentinus edodes and Pleurotus species lignocellulolytic enzymes activity in submerged and solid-state fermentation of lignocellulosic wastes of different composition,” Bioresource Technology 99(3), 457-462. DOI: 10.1016/j.biortech.2007.01.011
Erakhrumen, A. A. (2014). “Growing pertinence of bioenergy in formal/informal global energy schemes: Necessity for optimising awareness strategies and increased investments in renewable energy technologies,” Renewable and Sustainable Energy Reviews 31, 305-311. DOI: 10.1016/j.rser.2013.11.034
Gaikwad, A., and Meshram, A. (2020). “Effect of particle size and mixing on the laccase-mediated pretreatment of lignocellulosic biomass for enhanced saccharification of cellulose,” Chemical Engineering Communications 207(12), 1696-1706. DOI: 10.1080/00986445.2019.1680364
Galbe, M., and Zacchi, G. (2002). “A review of the production of ethanol from softwood,” Applied Microbiology and Biotechnology 59(6), 618-628. DOI: 10.1007/s00253-002-1058-9
Galic, M., Stajic, M., Vukojevic, J., and Cilerdzic, J. (2021). “Obtaining cellulose-available raw materials by pretreatment of common agro-forestry residues with Pleurotus spp.,” Frontiers in Bioengineering and Biotechnology 9, article 720473. DOI: 10.3389/fbioe.2021.720473
Garrido-Bazán, V., Téllez-Téllez, M., Herrera-Estrella, A., Díaz-Godínez, G., Nava-Galicia, S., Villalobos-López, M. Á., Arroyo-Becerra, A., and Bibbins-Martinez, M. (2016). “Effect of textile dyes on activity and differential regulation of laccase genes from Pleurotus ostreatus grown in submerged fermentation,” AMB Express 6, 1-9. DOI: 10.1186/s13568-016-0263-3
Gómez, J., Pazos, M., Couto, S. R., and Sanromán, M. A. (2005). “Chestnut shell and barley bran as potential substrates for laccase production by Coriolopsis rigida under solid-state conditions,” Journal of Food Engineering 68(3), 315-319. DOI: 10.1016/j.jfoodeng.2004.06.005
Han, M.-L., Chen, Y.-Y., Shen, L.-L., Song, J., Vlasák, J., Dai, Y.-C., and Cui, B.-K. (2016). “Taxonomy and phylogeny of the brown-rot fungi: Fomitopsis and its related genera,” Fungal Diversity 80(1), 343-373. DOI: 10.1007/s13225-016-0364-y
Han, M.-L., An, Q., He, S.-F., Zhang, X.-L., Zhang, M.-H., Gao, X.-H., Wu, Q., and Bian, L.-S. (2020). “Solid-state fermentation on poplar sawdust and corncob wastes for lignocellulolytic enzymes by different Pleurotus ostreatus strains,” BioResources 15(3), 4982-4995. DOI: 10.15376/biores.15.3.4982-4995
Han, M.-L., Bian, L.-S., Zhang, Y., Zhu, M., and An, Q. (2021a). “Pseudolagarobasidium baiyunshanense sp. nov. from China inferred from morphological and sequence analyses,” Phytotaxa 483(2), 169-176. DOI: 10.11646/phytotaxa.483.2.9
Han, M.-L., Yang, J., Liu, Z.-Y., Wang, C.-R., Chen, S.-Y., Han, N., Hao, W.-Y., An, Q., and Dai, Y.-C. (2021b). “Evaluation of laccase activities by three newly isolated fungal species in submerged fermentation with single or mixed lignocellulosic wastes,” Frontiers in Microbiology 12, 1-11. DOI: 10.3389/fmicb.2021.682679
Hernández, C., Silva, A. M. F. D., Ziarelli, F., Perraud-Gaime, I., Gutiérrez-Rivera, B., García-Pérez, J. A., and Alarcon, E. (2017). “Laccase induction by synthetic dyes in Pycnoporus sanguineus and their possible use for sugar cane bagasse delignification,” Applied Microbiology and Biotechnology 101(3), 1189-1201. DOI: 1007/s00253-016-7890-0
Howard, R. L., Abotsi, E., Rensburg, E. L. J. v., and Howard, S. (2003). “Lignocellulose biotechnology: Issues of bioconversion and enzyme production,” African Journal of Biotechnology 2(12), 602-619. DOI: 10.5897/AJB2003.000-1115
Huang, L., Sun, N., Ban, L., Wang, Y., and Yang, H. (2019). “Ability of different edible fungi to degrade crop straw,” AMB Express 9, 1-6. DOI: 10.1186/s13568-018-0731-z
Janusz, G., Czuryło, A., Frąc, M., Rola, B., Sulej, J., Pawlik, A., Siwulski, M., and Rogalski, J. (2015). “Laccase production and metabolic diversity among Flammulina velutipes strains,” World Journal of Microbiology and Biotechnology 31(1), 121-133. DOI: 10.1007/s11274-014-1769-y
Jaramillo, A. C., Cobas, M., Hormaza, A., and Sanroman, M. Á. (2017). “Degradation of adsorbed azo dye by solid-state fermentation: Improvement of culture conditions, a kinetic study, and rotating drum bioreactor performance,” Water, Air, & Soil Pollution 228(6), 1-14. DOI: 10.1007/s11270-017-3389-2
Karp, S. G., Faraco, V., Amore, A., Letti, L. A. J., Soccol, V. T., and Soccol, C. R. (2015). “Statistical optimization of laccase production and delignification of sugarcane bagasse by Pleurotus ostreatus in solid-state fermentation,” BioMed Research International 2015, 1-9. DOI: 10.1155/2015/181204
Khatami, S. H., Vakili, O., Movahedpour, A., Ghesmati, Z., Ghasemi, H., and Taheri-Anganeh, M. (2022). “Laccase: Various types and applications,” Biotechnology and Applied Biochemistry in press, 1-15. DOI: 10.1002/bab.2313
Kumar, R., Tabatabaei, M., Karimi, K., and Horváth, I. S. (2016). “Recent updates on lignocellulosic biomass derived ethanol – A review,” Biofuel Research Journal 3(1), 347-356. DOI: 10.18331/BRJ2016.3.1.4
Kumar, A., Singh, A. K., Bilal, M., and Chandra, R. (2021). “Sustainable production of thermostable laccase from agro-residues waste by Bacillus aquimaris AKRC02,” Catalysis Letters in press, 1-17. DOI: 10.1007/s10562-021-03753-y
Kunamneni, A., Plou, F. J., Ballesteros, A., and Alcalde, M. (2008). “Laccases and their applications: A patent review,” Recent Patents on Biotechnology 2(1), 10-24. DOI: 10.2174/187220808783330965
Liu, J. Y., Wang, M. L., Tonnis, B., Habteselassie, M., Liao, X., and Huang, Q. (2013). “Fungal pretreatment of switchgrass for improved saccharification and simultaneous enzyme production,” Bioresource Technology 135, 39-45. DOI: 10.1016/j.biortech.2012.10.095
Liu, W.-X., Zhao, M.-Y., Li, M.-X., Li, X.-Q., Zhang, T.-X., Chen, X., Yan, X.-Y., Bian, L.-S., An, Q., Li, W.-J., et al. (2022). “Laccase activities from three white-rot fungal species isolated from their native habitat in north China using solid-state fermentation with lignocellulosic biomass,” BioResources 17(1), 1533-1550. DOI: 10.15376/biores.17.1.1533-1550
Malherbe, S., and Cloete, T. E. (2002). “Lignocellulose biodegradation: Fundamentals and applications,” Reviews in Environmental Science and Biotechnology 1, 105-114. DOI: 10.1023/A:1020858910646
Martani, F., Beltrametti, F., Porro, D., Branduardi, P., and Lotti, M. (2017). “The importance of fermentative conditions for the biotechnological production of lignin modifying enzymes from white-rot fungi,” FEMS Microbiology Letters 364(13), 1-18. DOI: 10.1093/femsle/fnx134
Mate, D. M., and Alcalde, M. (2017). “Laccase: A multi-purpose biocatalyst at the forefront of biotechnology,” Microbial Biotechnology 10(6), 1457-1467. DOI: 10.1111/1751-7915.12422
Mishra, V., Jana, A. K., Jana, M. M., and Gupta, A. (2017). “Enhancement in multiple ligninolytic enzymes production for optimized lignin degradation and selectivity in fungal pretreatment of sweet sorghum bagasse,” Bioresource Technology 236, 49-59. DOI: 10.1016/j.biortech.2017.03.148
Munk, L., Andersen, M. L., and Meyer, A. S. (2018). “Influence of mediators on laccase catalyzed radical formation in lignin,” Enzyme and Microbial Technology 116, 48-56. DOI: 10.1016/j.enzmictec.2018.05.009
Navas, L. E., Martínez, F. D., Taverna, M. E., Fetherolf, M. M., Eltis, L. D., Nicolau, V., Estenoz, D., Campos, E., Benintende, G. B., and Berretta, M. F. (2019). “A thermostable laccase from Thermus sp. 2.9 and its potential for delignification of Eucalyptus biomass,” AMB Express 9, 1-10. DOI: 10.1186/s13568-019-0748-y
Nguyen, K. A., Wikee, S., and Lumyong, S. (2018). “Brief review: Lignocellulolytic enzymes from polypores for efficient utilization of biomass,” Mycosphere 9(6), 1073-1088. DOI: 10.5943/mycosphere/9/6/2
Palazzolo, M. A., Postemsky, P. D., and Kurina-Sanz, M. (2019). “From agrowaste to tool: Biotechnological characterization and application of Ganoderma lucidum E47 laccase in dye decolorization,” 3 Biotech 9(6), 1-7. DOI: 10.1007/s13205-019-1744-2
Perez-Montiel, G., Torres-Garcia, J. L., Juarez-Santacruz, L., Cortes-Espinosa, D. V., Rubio-Pina, J., and Ahuactzin-Perez, M. (2021). “Pleurotus ostreatus growth and laccase enzymes production during the degradation process of bisphenol a,” Biotecnia 23(2), 39-46. DOI: 10.18633/biotecnia.v23i2.1357
Postemsky, P. D., Bidegain, M. A., González-Matute, R., Figlas, N. D., and Cubitto, M. A. (2017). “Pilot-scale bioconversion of rice and sunflower agro-residues into medicinal mushrooms and laccase enzymes through solid-state fermentation with Ganoderma lucidum,” Bioresource Technology 231, 85-93. DOI: 10.1016/j.biortech.2017.01.064
Sánchez, C. (2009). “Lignocellulosic residues: Biodegradation and bioconversion by fungi,” Biotechnology Advances 27(2), 185-194. DOI: 10.1016/j.biotechadv.2008.11.001
Song, Q., Deng, X., and Song, R.-Q. (2020). “Expression of Pleurotus ostreatus laccase gene in Pichia pastoris and its degradation of corn stover lignin,” Microorganisms 8(4), 1-14. DOI: 10.3390/microorganisms8040601
Soumya, P. S., Lakshmi, M. S. K., and Nambisan, P. (2016). “Application of response surface methodology for the optimization of laccase production from Pleurotus ostreatus by solid state fermentation on pineapple leaf substrate,” Journal of Scientific & Industrial Research 75(5), 306-314.
Sun, K., Cheng, X., Yu, J., Chen, L., Wei, J., Chen, W., Wang, J., Li, S., Liu, Q., and Si, Y. (2020). “Isolation of Trametes hirsuta La-7 with high laccase-productivity and its application in metabolism of 17 beta-estradiol,” Environmental Pollution 263(Part B),1-11. DOI: 10.1016/j.envpol.2020.114381
Taha, M., Shahsavari, E., Al-Hothaly, K., Mouradov, A., Smith, A. T., Ball, A. S., and Adetutu, E. M. (2015). “Enhanced biological straw saccharification through coculturing of lignocellulose-degrading microorganisms,” Applied Biochemistry and Biotechnology 175(8), 3709-3728. DOI: 10.1007/s12010-015-1539-9
Tišma, M., Žnidaršič-Plazl, P., Vasić-Rački, Đ., and Zelić, B. (2012). “Optimization of laccase production by Trametes versicolor cultivated on industrial waste,” Applied Biochemistry and Biotechnology 166, 36-46. DOI: 10.1007/s12010-011-9401-1
Unuofin, J. O., Okoh, A. I., and Nwodo, U. U. (2019). “Aptitude of oxidative enzymes for treatment of wastewater pollutants: a laccase perspective,” Molecules 24(11), 1-36. DOI: 10.3390/molecules24112064
Wang, F., Xu, L., Zhao, L., Ding, Z., Ma, H., and Terry, N. (2019). “Fungal laccase production from lignocellulosic agricultural wastes by solid-state fermentation: A review,” Microorganisms 7(12), 1-25. DOI: 10.3390/microorganisms7120665
White, T. J., Bruns, T., Lee, S., and Taylor, J. (1990). “Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics,” in: PCR Protocols: A Guide to Methods and Applications, M. A., Innis, D. H. Gelfand, J. J. Sninsky, and T. J., White (ed.), Academic Press, Cambridge, MA, pp. 315-322.
Xin, F., and Geng, A. (2011). “Utilization of horticultural waste for laccase production by Trametes versicolor under solid-state fermentation,” Applied Biochemistry and Biotechnology 163(2), 235-246. DOI: 10.1007/s12010-010-9033-x
Xu, S., Wang, F., Fu, Y., Li, D., Sun, X., Li, C., Song, B., and Li, Y. (2020). “Effects of mixed agro-residues (corn crop waste) on lignin-degrading enzyme activities, growth, and quality of Lentinula edodes,” RSC Advances 10(17), 9798-9807. DOI: 10.1039/c9ra10405d
Xu, X., Feng, L., Han, Z., Luo, S., Wu, A., and Xie, J. (2016). “Selection of high laccase-producing Coriolopsis gallica strain T906: Mutation breeding, strain characterization, and features of the extracellular laccases,” Journal of Microbiology and Biotechnology 26(9), 1570-1578. DOI: 10.4014/jmb.1604.04011
Yang, J., Li, W., Ng, T. B., Deng, X., Lin, J., and Ye, X. (2017). “Laccases: Production, expression regulation, and applications in pharmaceutical biodegradation,” Frontiers in Microbiology 8, 1-24. DOI: 10.3389/fmicb.2017.00832
Yousef, S., Eimontas, J., Zakarauskas, K., Striugas, N., and Mohamed, A. (2021a). “A new strategy for using lint-microfibers generated from clothes dryer as a sustainable source of renewable energy,” Science of The Total Environment 762, 1-12. DOI: 10.1016/j.scitotenv.2020.143107
Yousef, S., Kuliešiene, N., Sakalauskaitė, S., Nenartavičius, T., and Daugelavičius, R. (2021b). “Sustainable green strategy for recovery of glucose from end-of-life euro banknotes,” Waste Management 123, 23-32. DOI: 10.1016/j.wasman.2021.01.007
Zerva, A., Simić, S., Topakas, E., and Nikodinovic-Runic, J. (2019). “Applications of microbial laccases: patent review of the past decade (2009-2019),” Catalysts 9(12), 1-26. DOI: 10.3390/catal9121023
Zhang, W., Wu, S., Cai, L., Liu, X., Wu, H., Xin, F., Zhang, M., and Jiang, M. (2018). “Improved treatment and utilization of rice straw by Coprinopsis cinerea,” Applied Biochemistry and Biotechnology 184(2), 616-629. DOI: 10.1007/s12010-017-2579-0
Article submitted: April 12, 2022; Peer review completed: June 26, 2022; Revisions accepted: July 5, 2022; Published: July 7, 2022.
DOI: 10.15376/biores.17.3.4921-4936