Identification of a Fomitopsis pinicola from Xiaoxing’an Mountains and Optimization of Cellulase Activity

Abstract

Brown-rot fungi are large fungi that can decompose the cell walls of wood; they are notable for their secretion of diverse and complex enzymes that synergistically hydrolyze natural wood cellulose molecules. Fomitopsis pinicola (F. pinicola) is a brown-rot fungus of interest for its ability to break down the cellulose in wood efficiently. In this study, through a combination of rDNA-ITS analysis and morphological observation, the wood decay pathogen infecting Korean pine (Pinus koraiensis Siebold and Zucc.) was identified. Endoglucanase (CMCase) and β-glucosidase were quantified using the DNS (3,5-Dinitrosalicylic acid) method, and the cellulase activity was optimized using a single-factor method and orthogonal test. The results revealed that the wood-decaying fungus NE1 identified was Fomitopsis pinicola with the ITS accession number OQ880566.1. The highest cellulase activity of the strain reached 116.94 U/mL under the condition of an initial pH of 6.0, lactose 15 g·L⁻¹, KH₂PO₄ 0.5 g·L⁻¹, NH₄NO₃ 15 g·L⁻¹, MgSO₄ 0.5 g·L⁻¹, VB₁ 0.4 g·L⁻¹, inoculated two 5 mm fungal cakes in 80 mL medium volume cultured 28 °C for 5 days. This laid a foundation for improving the degradation rate of cellulose and biotransformation research, as well as exploring the degradation of cellulose by brown rot fungi.

1. Introduction

Wood-decaying fungi are an important factor leading to wood disintegration and decay [1]. These fungi can be categorized into brown-rot fungi, white-rot fungi, and soft-rot fungi, depending on the type of decay [2].

Korean pine (Pinus koraiensis Siebold and Zucc.) is a Pinaceae evergreen tree that is a valuable key protected wild plant in China with broad application potential [3]. However, under favorable conditions, wood-decaying fungi can infest Korean pine, leading to decay, diminished material quality, and economic losses in the protection and utilization of forest resources [4,5].

Cellulase is primarily composed of endoglucanases (CMCase), exoglucanases, and β-glucosidases, which can be produced by a wide range of microorganisms such as bacteria, actinomycetes, and fungi [6]. Different microbial taxa produce different enzyme systems to break down cellulose, with fungi being the most capable of breaking down cellulose. Among fungi, wood-decaying fungi are notable for their secretion of diverse and complex enzymes that synergistically hydrolyze natural wood cellulose molecules [7]. Fomitopsis pinicola is a brown-rot fungus widely distributed in the forest, which has attracted much attention because it can effectively decompose the cellulose in wood [8].

Cellulase has been widely used in industrial production [9]. For instance, the addition of cellulase in white wine production enhances output and maximizes raw material utilization [10]. In the textile industry, cellulase improves the effectiveness of detergents and enhances textile quality [11]. In the paper industry, cellulase is used to improve pulp properties, reduce energy consumption, and increase paper quality and yield [12]. Cellulases play a pivotal role in biofuel production, particularly in the manufacturing of bioethanol. By converting cellulose from plants into sugars, cellulases provide the necessary feedstock for the fermentation process, enabling efficient conversion of biomass into ethanol. Compared to traditional fossil fuels, the production and utilization of bioethanol can effectively reduce reliance on fossil fuels, thereby mitigating energy crises and environmental pollution issues [13]. In recent years, with in-depth research into the industrial applications of cellulase, the optimization of cellulase activity has garnered widespread attention [14]. The activity and stability of cellulase are critical determinants of its application efficiency. A high activity ensures rapid and efficient conversion of cellulose. Therefore, enhancing the activity of cellulase has become a key focus of research. Saravanan et al. adopted a response surface methodology to optimize the production conditions of cellulase enzymes, such as carbon source, fermentation temperature, and pH value, which were effectively adjusted and significantly improved enzyme yield [15]. Lin et al. successfully improved the cellulase activity of the endogenous fungus Colletotrichum gloeosporioides by single-factor optimization of the carbon source, nitrogen source, fermentation temperature, and pH in liquid culture [16]. Adriana et al. isolated and molecularly identified basidiomycete fungi from the Boyacá Forest, screening them for lignin and cellulose-degrading enzyme activities. Several white-rot fungi with biotechnological potential were identified, and their lignocellulolytic enzyme activity was explored [17]. At present, extensive research has been conducted on the mechanism of action, isolation and screening, biodegradation, enzyme composition, and application of cellulases. In biodegradation, there is a continuous search for cellulases that can degrade cellulose efficiently [18]. However, the yield of cellulase is influenced not only by inherent factors in fungi but also by external factors such as culture medium composition and conditions [19]. While significant research has focused on cellulase production in white-rot fungi, less attention has been given to the important brown-rot fungi [20,21].

The objective of this study is to identify the wood decay pathogens infecting Korean pine using molecular biology and morphological identification methods. Additionally, the cellulase production activity of the identified brown-rot fungus is determined and optimized through DNS, single-factor method, and orthogonal tests to establish the optimal culture conditions for cellulase production. These research findings provide a foundation for improving the study of cellulose degradation rate and biotransformation. Moreover, they offer new fungal resources for cellulose degradation research by fungi and laid the foundation for the study of cellulose degradation by brown-rot fungi.

2. Materials

The fruiting bodies of wood-decaying fungus were collected from the trunks of Korean pine (Pinus koraiensis Siebold and Zucc.) in Xiaoxing’an Mountains (37°42′–47′ N, 128°52′–17′ E), Heilongjiang Province, China, we named the strain to be NE1. This strain was deposited in the Biophysics Laboratory, College of Science, Northeast Forestry University.

3. Methods

3.1. Molecular Biological Identification

3.1.1. DNA Extraction

The hyphae of the fruiting bodies specimens were cultivated on the PDA culture medium. The inoculated plates were then placed in a 28 °C for 7 days incubator for cultivation. Genomic DNA extraction was performed on the internal tissues of these fruiting bodies with the Sangon SK8259 Genomic DNA Extraction Kit (Sangon Biotech Co., Ltd., Shanghai, China). The ITS region was amplified with the universal primers ITS1 and ITS4 [22,23]. A final reaction total volume of 25 µL, comprising 0.5 µL of genomic DNA, 2.5 µL of 5x PCR buffer, 0.5 µL each of F-primer and R-primer (10 µM each), along with dNTP mix by 1 µL (2.5 mM), 0.2 µL of Taq DNA polymerase, and the remainder was distilled water. The PCR procedure started with a 4-min initial denaturation at 94 °C, thereafter 30 cycles consisting of denaturation for 45 s at 94 °C, annealing for 45 s at 55 °C, extension for 1 min at 72 °C, and concluded with a final extension phase at 72 °C for 10 min. In order to view PCR products by agarose gel electrophoresis at a concentration of 1% (w/v). The concentration of the extracted DNA was determined with a UV-visible spectrophotometer. The PCR product was sequenced by Sangon Co., Ltd.

3.1.2. Phylogenetic Analysis

The obtained sequences were compared with reference sequences in NCBI GenBank via BLAST searches to spot highly similar ones [24]. Then, phylogenetic trees were built using MEGA 7.0 to analyze the genetic relationships among them.

3.2. Identification of Morphology

In the field, we collected the fruiting bodies of wood-decaying fungi and documented their external morphologies using a Canon EOS M3 camera (Canon, Tokyo, Japan) in Figure 1. The primary identification of NE1 was based on the morphological characteristics of both the fruit bodies and the mycelium observed under a ZEISS Primo Star iLED microscope (Carl Zeiss Co., Ltd., Oberkochen, Germany). Specifically, strain NE1 was inoculated on PDA and culture incubated at 28 °C for a period of 7 days. Mycelium from the nascent zone at the edge of the colony was taken for observation.

3.3. Preparation of Glucose Standard Curves

The DNS method was employed to measure reducing sugars and determine the enzymatic activity of endoglucanase (CMCase) and β-glucosidase. This method relies on the reaction between the DNS reagent and reducing sugars at a specific temperature and set of conditions, resulting in the formation of a red compound with a proportional color gradient. The intensity of the solution color after the reaction corresponds to the amount of reducing sugar present. To prepare the glucose standard solution, 0.1 g of glucose (pre-dried at 105 °C until reaching a constant weight) was precisely weighed and then dissolved in distilled water, ultimately bringing the solution to a total volume of 100 mL. Subsequently, various volumes of this glucose solution (0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL) were pipetted into 25 mL stoppered colorimetric tubes, each accompanied by corresponding volumes of distilled water (2.0 mL, 1.8 mL, 1.6 mL, 1.4 mL, 1.2 mL, and 1.0 mL), as specified in the methodology [25,26]. After thorough mixing, 1.5 mL of the DNS solution was added to each tube. The mixture was shaken well, heated in a boiling water bath for 5 min, and then cooled to room temperature. A glucose standard curve was constructed, and the linear regression equation and regression coefficient were calculated.

3.4. Extraction of Crude Enzyme Solution

The isolated strain was inoculated on a PDA solid medium at 28 °C until the plate was fully colonized. To prepare fungal cakes, a 5 mm diameter hole punch was used to transfer the mycelium from the PDA plate. After 9 days of expanded culture, four fungal cakes from the outermost ring of the colony were inoculated into 100 mL of enzyme-producing liquid fermentation medium (CMC-Na 5 g·L⁻¹, KH₂PO₄ 0.5 g·L⁻¹, yeast powder 10 g·L⁻¹, MgSO₄ 0.5 g·L⁻¹, VB₁ 0.4 g·L⁻¹) [27,28]. The inoculated medium was then incubated in a shaking bed at 28 °C with a speed of 160 r/min. After the fermentation period, the liquid fermentation broth was subjected to centrifugation at 12,000 r/min for 15 min at 4 °C. The supernatant, obtained using a pipetting gun, served as the crude enzyme solution.

3.5. Determination of the Cellulase Activity

Cellulase activity unit (U/mL) refers to the quantity of enzyme needed to yield 1 µg of glucose per minute at 50 °C within a 1 mL solution of crude enzyme. It is expressed as X (U/mL). The calculation formula for cellulase activity is as follows:

(1)$\mathrm{X}=\left(\mathrm{A}\times \mathrm{n}\times 1000\right)/\mathrm{V}\times \mathrm{T}$

where A stands for the glucose content (mg) determined by the OD value on the standard curve, n stands for dilution multiple of the crude enzyme solution used in the experiment, 1000 is the conversion factor of glucose units from mg to µg, V is the reaction volume (mL), and T is the time required for participating in the reaction to be converted into 1 min.

3.5.1. Determination of Endoglucanase (CMCase)

To determine the cellulase activity, we diluted 1 mL of crude enzyme extract and mixed it with 2 mL of 1% CMC-Na solution (prepared in citric acid buffer at pH 5.0). The boiled enzyme solution was used as the control group. After a 30 min water bath at 50 °C, we added 1.5 mL DNS immediately. Following a 5-min bath, we removed the sample to cool and adjusted the volume to 25 mL. We measured the absorbance of the solution at a wavelength of 520 nm using a Multiskan SkyHigh microplate reader (Thermo Fisher Scientific Co., Ltd, Waltham, MA, USA) [29].

3.5.2. Determination of the β-Glucosidase Activity

To measure the β-glucosidase enzyme activity, 2 mL of a buffered solution containing 0.5% salicylate citrate was added to a centrifuge tube, then 1 mL of enzyme solution was added. A boiled enzyme solution was used as the control group. The subsequent steps were the same as mentioned above.

3.6. Optimization of Cellulase Production Conditions

After reviewing the literature and previous studies, it was discovered that multiple factors, such as carbon source, inoculum amount, nitrogen source, initial pH, and medium volume, can affect cellulase activity [30,31]. In this study, a single-factor approach was employed to investigate how different factors impact the enzyme production of wood brown-rot fungi strain NE1. Furthermore, the cellulase activity of a brown-rot fungus was optimized using an orthogonal test table, which allowed for the systematic investigation of the combined effects of these factors on enzyme production.

3.6.1. Optimal Initial Reaction pH

The pH can effectively influence the growth of the strain [32]. To investigate the effect of different initial pH (pH at the beginning of the experiment) values on the cellulase activity in Fomitopsis pinicola and determine the optimal pH value, this study conducted a single-factor optimization experiment using a liquid fermentation medium for enzyme production. The initial pH values tested were pH 5.0, pH 5.5, pH 6.0, and pH 6.5 [33]. A total of 4 pieces of 5 mm fungal cakes were inoculated medium volume 100 mL shake flask (rotation speed of 150 r/min) cultured at 28 °C for 5 days. To measure the enzyme activity, we used the standard enzyme activity measurement method, replicated each treatment three times, and statistically analyzed the results with SPSS 27.0 software.

3.6.2. Different Carbon Sources

The carbon source is a fundamental component of the medium and serves as the source of material and energy for microbial growth [34]. In this experiment, a liquid fermentation medium was used as the substrate, and the optimal initial pH value was selected after single-factor optimization. The culture was incubated at 150 r/min for 5 days, and glucose, sucrose, lactose, starch, and CMC-Na were selected as different carbon sources to determine the activities producing CMCase and β-glucosidase of Fomitopsis pinicola [35].

3.6.3. Different Nitrogen Source

To assess the impact of nitrogen sources on enzyme activities, we measured the activities of CMCase and β-glucosidase using various nitrogen sources (yeast powder, peptone, ammonium tartrate, ammonium nitrate, sodium nitrate) under the aforementioned conditions.

3.6.4. Orthogonal Experimental Design

To find the optimal cellulase production culture conditions for Fomitopsis pinicola, we used an orthogonal experimental design based on single-factor optimization. We used a four-factor, three-level experimental design with three parallel samples per group and conducted statistical analysis of the results using SPSS software.

The effect of four combinations of factors at different carbon source concentrations (A), nitrogen source concentration (B), medium volume (C), and inoculum amount (D) on cellulase production activity was investigated by designing the factor levels according to L₉(3⁴) Table 1 [31].

4. Results and Discussions

4.1. Molecular Biology Identification Results

The DNA quality and yield were evaluated using UV-2802S UV-vis spectrophotometry (ABOUT US Co., Ltd, Shanghai, China)and gel electrophoresis. The OD260/OD280 ratio was determined to be 1.91, and the DNA concentration was measured to be 191.88 ug/uL. The detection results confirmed the high purity of the DNA.

The ITS sequences were homologated in the NCBI database. The phylogenetic tree was constructed by the NJ (Neighbor-Joining) with MEGA 7.0, setting the bootstrap value to 1000 and Ganoderma applanatum as the outgroup. As shown in Figure 1, strain NE1 and F. pinicola (Fomitopsis pinicola), OR468709, have the closest genetic relationship and converge in the same branch. The ITS sequence of strains NE1 has been deposited in the NCBI under the accession number OQ880566.1. On the basis of the BLAST comparison, NE1 was identified as Fomitopsis pinicola, belonging to the taxonomic ranks of Basidiomycota, Agaricomycetes, Polyporales, Coriolaceae, and Fomitopsis.

4.2. Morphological Identification Results

The fruiting body NE1 exhibits a sessile laterally grows at the base of Korean pine trunks. Its characteristic is a flattened semi-spherical shape with a smooth surface and a width of approximately 18 cm. The upper surface appears reddish brown, with thin edges and milky white edge bands. The back presents a milky yellow to light yellow–brown color, with tiny pores on the lower surface. The flesh of the fungus is woody and appears white or pale yellow (Figure 2). The strain demonstrates rapid growth, displaying white colonies on both sides of the culture medium. The mycelium grows evenly, closely adhering to the inner surface of the medium, and the edge slightly rises to form a felt-like texture. It emits a slight mushroom aroma. After 5 weeks, the front side of the colony undergoes a slight browning, while the back of the culture medium turns slightly yellow (Figure 3). As depicted in Figure 4, the mycelium is colorless and transparent and has a diameter of about 4 µm. Locking associations appear at the intersections or dense areas of the mycelium.

4.3. Cellulase Production Optimization Results

4.3.1. The Initial pH on Cellulase Activity

The different initial pH values on the cellulase activity of the identified wood-decaying fungi were explored using a single-factor optimization approach. The strains were subjected to fermentation culture under various initial pH conditions, and these data were analyzed using SPSS software, as depicted in Figure 5.

The results revealed that the tallest activity of CMCase was observed at an initial pH of 6.0. In the range of initial pH 5.0–6.0, the enzyme production gradually increased with increasing pH. However, the activity of CMCase decreased when the initial pH exceeded 6.0. This indicates that the strain exhibits optimal growth and higher cellulase activity in acidic environments. The initial pH is an important environmental factor for the growth of fungal strains, and the selection of an appropriate initial pH can affect the growth rate, enzyme activity, and yield of microorganisms. CMCase usually exhibits optimal activity under acidic conditions. For example, Trichoderma, Aspergillus, Penicillium, and some other common fungal genera usually produce the highest CMCase activity in the initial pH range of 4.0–6.0 [36]. For many fungi, acidic conditions are more favorable for cellulase production and help maximize the activation and promotion of cellulase activity [37]. Therefore, the optimal initial pH for strain NE1 was selected to be 6.0.

4.3.2. Carbon Sources on Cellulase Activity

A single-factor optimization approach was employed to optimize the carbon source from the fermentation medium, and the experimental results are presented in Figure 6. The findings revealed that among the six carbon sources tested, lactose yielded the highest activity of both cellulases under the same experimental conditions. This suggests that lactose is the most favorable carbon source for the production of CMCase and β-glucosidase, exhibiting a clear advantage over the other carbon sources. Lactose was chosen as a carbon source, which has an inducing effect on certain fungi and may improve the production of specific cellulases. However, CMC-Na is a positive inducer of cellulase production, which improves strain growth and promotes cellulase production. However, the efficiency of CMC-Na degradation by Fomitopsis pinicola may be lower, and it may take a longer time to exert its advantages. As a relatively stable carbon source, sucrose may provide continuous carbon supplements in long-term culture and contribute to the long-term enzyme production process. However, Fomitopsis pinicola may have limited utilization ability of sucrose, resulting in a lower enzyme production efficiency. After evaluation, lactose was determined as the optimal carbon source for producing cellulase by strain NE1.

4.3.3. Nitrogen Sources on Cellulase Activity

Based on the best initial pH value and the best carbon source obtained from the above experiments, the nitrogen source required for fermentation and culture was optimized. The experimental results are shown in Figure 7. It was found that when the nitrogen source was ammonium nitrate, the enzyme activity of CMCase was the highest, and when the nitrogen source was ammonium tartrate, the enzyme activity of β-glucosidase was the highest. This indicates that ammonium nitrate promotes CMCase production, while ammonium tartrate enhances β-glucosidase production. However, during the actual degradation of cellulose, CMCase is required to break down the chemical bonds of cellulose before β-glucosidase can play its role in degradation. Therefore, lactose was determined as the best carbon source for producing cellulase by strain NE1.

4.3.4. Effect of Orthogonal Experimental Optimization on Cellulase Production by Fomitopsis pinicola

SPSS software was used to design a four-factor, three-level orthogonal experimental table. The four factors were different carbon source concentrations (A), different nitrogen source concentrations (B), different loading volumes (C), and different inoculum volumes (D). Different concentrations of carbon sources may affect the growth rate and metabolite formation of microorganisms, which in turn affects cellulase activity.

Cellulase is a key enzyme that decomposes cellulose into simple sugars that can be fermented into biofuels or converted into other valuable products and plays a pivotal role in improving the efficiency of the bioenergy production process. Fomitopsis pinicola is a widely distributed brown-rot fungus found in forests, gaining attention for its ability to secrete various complex enzymes that synergistically and efficiently hydrolyze natural wood cellulose molecules. This paper discusses how to improve the yield and activity of cellulases of Fomitopsis pinicola by optimizing fermentation conditions, such as pH, temperature, and concentration of nutrients. The change in nitrogen source concentration directly affects the growth status and metabolic activity of microorganisms, thereby affecting enzyme production and activity. An appropriate inoculation amount can ensure the rapid growth of microorganisms in the initial fermentation stage, and too much or too little inoculation amount may lead to fermentation failure or decreased enzyme activity.

Each factor is set to three different levels. In Table 2, the A factor is set to three levels: A1, A2, and A3; the B factor is set to three levels: B1, B2, and B3; so are other factors (C and D). The three levels are three repetitions of each factor; that is to say, the level of each factor is 3, which is indicated by 1, 2, and 3, respectively, in the table. There are nine possible combinations between the levels of various factors. The values of K1, K2, K3, and R are used to measure the results of orthogonal experimental analysis, and they are used to measure the influence of the values of factor levels on the experimental results. Using an intuitive analysis method, by comparing the mean K of four factors and three levels, the maximum average value of each factor was obtained, and the optimal scheme for producing cellulase was determined to be A₃B₃C₂D₁, with the highest CMCase enzyme activity. Through the R values analysis, we determined the order of influence of the four factors on different variables as B > D > A > C; that is, the concentration of nitrogen source and inoculation amount have the greatest influence on the yield of NE1 endoglucanase, followed by the concentration of carbon source and loading volume. The strain’s cellulase activity peaks under specific conditions of initial pH 6.0, lactose 15 g·L⁻¹, KH₂PO₄ 0.5 g·L⁻¹, ammonium nitrate 15 g·L⁻¹, MgSO₄ 0.5 g·L⁻¹, VB₁ 0.4 g·L⁻¹, with two pieces of 5 mm fungal cakes inoculated in 80 mL medium cultivated at 28 °C of 5 days.

The cellulase activity is generally highest at 50 °C, but this temperature is not suitable for the growth of Fomitopsis pinicola. Therefore, in order to ensure the normal growth of the red-edge layered pore fungus NE1 and the highest cellulase activity, 28 °C can be used as the optimal culture temperature. Improving microbial growth efficiency, reducing energy consumption and production costs, and lowering the optimal activity temperature of CMCase can make the industrial application of cellulase more efficient, economical, and sustainable [38,39]. Deswal et al. showed the highest CMCase yield of the newly isolated Fomitopsis sp. strain RCK2010 under solid-state fermentation conditions with an initial pH of 5.5, a water ratio of 1:3.5 (solid–liquid ratio), a carbon source of wheat bran, a nitrogen source of urea, and an amino acid of glutamic acid [40]. We optimized the liquid fermentation medium for Fomitopsis pinicola, which is cheap and easy to formulate, more suitable for mass production, and has the highest CMCase production at an initial pH of 6.0, which indicates that acidic conditions are more conducive to the growth of Fomitopsis pinicola and the production of CMCase. When using CMC-Na and wheat bran WB as co-substrates, the activity of CMCase from the Penicillium oxalicum P07 strain was significantly increased to 5.66 U/mL at pH 5.0 and 37 °C, producing cellulase efficiently. We optimized for different carbon sources with single-factor tests; lactose as a carbon source yielded higher cellulase activity than CMC-Na. Using lactose as the carbon source, the cellulase activity of the Fomitopsis pinicola strain was optimized to 116.94 U/mL. Fomitopsis pinicola NE1 is a natural strain capable of producing cellulase more efficiently, and the optimization of the culture medium formula is simpler. The strain NE1 is stable and adaptable, possessing the potential to be developed for industrial production of cellulase [41]. At present, in the study of the wood-decaying fungus optimizing fermentation conditions of culture medium, more scholars are committed to optimizing the cellulase activity of Trichoderma reesei. Compared with related studies, it was found that the cellulase activity of Fomitopsis pinicola under acidic conditions may be significantly better than that of Trichoderma reesei, while Trichoderma reesei maintained a relatively stable activity in a wide pH range [42,43]. This difference shows that Fomitopsis pinicola and Trichoderma reesei have their own advantages in different industrial application scenarios. We optimized the cellulase activity of Fomitopsis pinicola, which can be used in combination with Trichoderma reesei to show a certain synergistic degradation effect, so as to achieve more efficient and economical industrial application in the field of biomass conversion.

5. Conclusions

The diversity and high efficiency of cellulase secreted by Fomitopsis pinicola make it a potential microbial source for the industrial production of cellulase. This study optimized the cellulase activity of a strain of Fomitopsis pinicola NE1 identified to obtain the optimal cellulase production conditions. The cellulase activity of the strain was the highest under the condition of initial pH 6.0, lactose 15 g·L⁻¹, KH₂PO₄ 0.5 g·L⁻¹, ammonium nitrate 15 g·L⁻¹, MgSO₄ 0.5 g·L⁻¹, VB₁ 0.4 g·L⁻¹, inoculating two pieces of 5 mm fungal cakes in 80 mL medium volume cultivation at 28 °C for 5 days. These research findings provide a foundation for improving the study of cellulose degradation rate and biotransformation. Moreover, they offer new fungal resources for cellulose degradation research by fungi and laid the foundation for the study of cellulose degradation by brown-rot fungi.

Author Contributions

Conceptualization, J.S.; Funding acquisition, Y.C. and D.Q.; Methodology, H.Y. and S.G.-Z.; Project administration, Y.C. and D.Q.; Supervision, Y.C. and D.Q.; Visualization, J.S. and H.Y.; Writing—original draft, J.S.; Writing—review and editing, J.S., H.Y., S.G.-Z., Y.C. and D.Q. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

Data is contained within the article.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

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Figures and Tables

Figure 1. Phylogenetic tree of the NE1.

Figure 2. Observation of morphological characteristics. (a) Front; (b) Back.

Figure 3. Hyphal growth of NE1 strain on PDA medium. (a) Front; (b) Back.

Figure 4. Photo of hyphae of wood-decaying fungi NE1 observed with optical microscope.

Figure 5. Different initial pH on cellulase activity by NE1 (p [less than] 0.05). (a, b, c, d indicate significant differences between groups. Different letters indicate significant differences between groups, while the same letters indicate no significant differences between groups.)

Figure 6. Different carbon sources on cellulase activity by NE1 (p [less than] 0.05). (a, b, c, d, e indicate significant differences between groups. Different letters indicate significant differences between groups, while the same letters indicate no significant differences between groups).

Figure 7. Different nitrogen sources on cellulase activity by NE1 (p [less than] 0.05). Different letters indicate significant differences between groups, while the same letters indicate no significant differences between groups.

Table 1

Orthogonal experimental design.

Levels	Factors
	A//g/L	B//g/L	C//mL	D//pcs
1	5	5	60	2
2	10	10	80	4
3	15	15	100	6

Where A stands for different carbon sources, B stands for different source concentrations, C stands for different medium volumes, and D stands for different inoculum amounts.

Table 2

Table of orthogonal experimental design.

No.	A	B	C	D	U/mL
1	1	1	1	1	20.94
2	1	2	2	2	80.73
3	1	3	3	3	31.86
4	2	1	2	3	19.65
5	2	2	3	1	81.17
6	2	3	1	2	105.89
7	3	1	3	2	26.63
8	3	2	1	3	30.56
9	3	3	2	1	116.94
K1	44.51	22.41	52.46	73.19
K2	68.9	64.15	72.44	71.08
K3	85.46	84.89	46.55	27.35
R	40.95	62.48	25.89	43.59

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