A white rot strain with high lignin degradation activity and its application

Abstract

The present invention discloses a white-rot strain with high lignin degradation activity and its applications in pulp production and wastewater decolorization. The white-rot strain is Trametes hirsuta XY1CGMCC No. 40195. This strain can selectively degrade lignin and has the characteristics of highly active secretion of laccase (Lac), manganese peroxidase (Mnp), and lignin peroxidase (Lip). This strain also has the characteristics of low amylase and low xylanase activities. After eucalyptus wood is directly treated with the fermentation broth of this Trametes hirsuta strain without chemical treatment methods such as acid or alkali, high pulp yield can be obtained for the eucalyptus wood chips, the quality of the prepared rough pulp is improved, and thus the flexibility of the paper made from the pulp is increased and the whiteness is enhanced. When the fermentation broth of this strain is used to treat papermaking wastewater, the decolorization rate reaches more than 80%.

Description

Technical Field

[0001] The present invention relates to a white rot strain with high lignin-degrading enzyme activity and its applications in biological pulping and wastewater decolorization. Background Art

[0002] Pulping refers to the process of forming papermaking fiber raw materials, which is carried out by breaking the chemical bonds of woody raw materials or other raw materials (such as non-wood, grass, and agricultural residues). During the pulping process, unwanted lignin components should be removed as much as possible to avoid the decomposition of cellulose, and chemical pulping methods are generally used. In chemical pulping, the raw materials are cooked with appropriate chemicals at high temperature and high pressure in an aqueous solution to dissolve the lignin. There are three methods for dissolving lignin in chemical pulping, including the alkaline method, the sulfate method, and the sulfite method. The substances that play a role in dissolving lignin in the alkaline method, the sulfate method, and the sulfite method are sodium hydroxide, sodium hydroxide plus sodium sulfide, and sulfurous acid, respectively. Chemical pulping is the link in the entire papermaking process that generates the most pollutants.

[0003] Biological pulping is considered an environmentally friendly and economically viable method for delignifying lignocellulosic raw materials because biological pretreatment reduces the amount of chemicals used in the pulping process and is considered a potential alternative to traditional pulping. Before pulping, white rot fungi are used to pretreat lignocellulosic raw materials. When the fungi grow on the raw materials, they produce lignin-degrading enzymes such as lignin peroxidase, laccase, and manganese peroxidase, thereby selectively degrading lignin while retaining cellulose. The prerequisite for pulping is the removal of lignin and hemicellulose under the combined action of hemicellulase and lignin-degrading enzymes. Summary of the Invention

[0004] Based on this, the object of the present invention is to provide a white rot fungus with high lignin-degrading enzyme activity. This white rot fungus can produce a large amount of lignin-degrading enzymes while producing relatively low levels of cellulase, making it more suitable for biological pulping. Moreover, after the wood is degraded by the white rot fungus, due to the oxidation and loss of lignin, a bleaching effect is produced, and finally white fibrous wood is formed. The white rot fungus of the present invention can fully decompose the lignin in the middle layer, separating intact cells into fibers. This selectively delignifies to form concentrated cellulose. The extracellular oxidases produced by the white rot fungus of the present invention include manganese peroxidase (MnP), lignin peroxidase (LiP), and laccase, and these enzymes participate in the degradation of lignin in wood. The white rot fungus of the present invention has the following characteristics:

[0005] 1) It should have a relatively fast growth rate;

[0006] 2) It should not produce pigments, which would have an adverse effect on the brightness of the pulp;

[0007] 3) It has high lignin-degrading enzyme activity and produces lignin-degrading enzymes as early as possible.

[0008] Based on this, the present invention utilizes the effect of white-rot fungi in degrading lignin to treat wastewater and make it purified before flowing into the environment, reducing the damage to the environment and avoiding the defects of a large amount of lignin and its related derivatives in the wastewater of traditional papermaking industry.

[0009] The white-rot fungi provided by the present invention are derived from the fruiting bodies collected from the ancient trees in the Summer Palace, separated and purified, and subcultured in the laboratory. Since they are directly separated from nature, their activity is relatively high.

[0010] Specifically, the white-rot fungus isolated from the ancient tree provided by the invention is the Trametes hirsuta strain, named Trametes hirsuta XY1. In the present invention, its species is identified by ITS primers.

[0011] The Trametes hirsuta XY1 is obtained by collecting the fruiting bodies from the ancient trees in the Summer Palace, separating and purifying them. Its taxonomic name is Trametes hirsuta. It was deposited in the China General Microbiological Culture Collection Center (Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing) on May 26, 2022, and the deposit number is CGMCC No. 40195.

[0012] Application of the above Trametes hirsuta XY1 CGMCC No. 40195 strain in biopulping.

[0013] The present invention also provides the application of the Trametes hirsuta XY1 CGMCC No. 40195 strain in lignin degradation, especially in the selective degradation of lignin.

[0014] The present invention also provides the application of the Trametes hirsuta XY1 CGMCC No. 40195 strain in the production of laccase, manganese peroxidase and / or lignin peroxidase.

[0015] The present invention also provides the application of the Trametes hirsuta XY1 CGMCC No. 40195 strain in the decolorization of wastewater in the papermaking process.

[0016] The present invention also claims to protect a biopulping method, in which the pretreatment fungus is Trametes hirsuta XY1 CGMCC No. 40195.

[0017] The strain obtained by the present invention has the characteristics of fast growth and reproduction, and its mycelia are white without variegation. This strain can selectively degrade lignin and has high laccase, manganese peroxidase, and lignin peroxidase activities. It also has the characteristics of low amylase and xylanase activities. After eucalyptus is directly treated with the fermentation broth of this Trametes hirsuta strain without chemical treatment methods such as acid or alkali, the eucalyptus chips can obtain a high pulp yield, the quality of the prepared crude pulp is improved, and the flexibility and whiteness of the paper made from this pulp are increased. The tensile index, tear index, burst index, breaking length, and folding endurance of the pulp prepared by pretreating wood with this strain are all significantly improved, significantly improving the strength and brightness of the paper. The fermentation broth of this strain is used to treat papermaking wastewater, and the decolorization rate reaches more than 80%. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 For the fruiting bodies and culture characteristics on PDA medium of Trametes hirsuta XY1 CGMCC No.40195 and other white rot fungus controls YH9-4 and YH17-6, a and b are the growth states of the fruiting body and mycelial colonies of XY1 on the PDA medium respectively; c and d are the growth states of the fruiting body and mycelial colonies of YH9-4 on the PDA medium respectively; e and f are the growth states of the fruiting body and mycelial colonies of YH17-6 on the PDA medium respectively.

[0019] Figure 2 For the color development pictures of the primary screening of Trametes hirsuta XY1 CGMCC No.40195 and controls YH9-4 and YH17-6 on guaiacol medium, a and b are the mycelial colonies and color development states of XY1 on the guaiacol medium respectively; c and d are the mycelial colonies and color development states of YH9-4 on the guaiacol medium respectively; e and f are the mycelial colonies and color development states of YH17-6 on the guaiacol medium respectively.

[0020] Figure 3 For the laccase secretion activity curves of Trametes hirsuta XY1 CGMCC No.40195 and controls YH9-4 and YH17-6 during the 8th to 13th days of shaking culture in liquid culture.

[0021] Figure 4 For the manganese peroxidase secretion activity curves of Trametes hirsuta XY1 CGMCC No.40195 and controls YH9-4 and YH17-6 during the 8th to 13th days of shaking culture in liquid culture.

[0022] Figure 5Secretion activity curves of lignin peroxidase of Trametes hirsuta XY1 CGMCC No.40195 and controls YH9-4 and YH17-6 during the 8th to 13th days of liquid culture shaking culture.

[0023] Figure 6 Secretion activity curves of total lignin-degrading enzymes of Trametes hirsuta XY1 CGMCC No.40195 and controls YH9-4 and YH17-6 during the 8th to 13th days of liquid culture shaking culture.

[0024] Figure 7 Secretion activity curves of amylase of Trametes hirsuta XY1 CGMCC No.40195 and controls YH9-4 and YH17-6 during the 8th to 13th days of liquid culture shaking culture.

[0025] Figure 8 Secretion activity curves of hemicellulase of Trametes hirsuta XY1 CGMCC No.40195 and controls YH9-4 and YH17-6 during the 8th to 13th days of liquid culture shaking culture.

[0026] Figure 9 Before and after the decolorization of wastewater by the fermentation broth of Trametes hirsuta XY1 CGMCC No.40195. Detailed implementation methods

[0027] The methods in the following examples are all conventional methods unless otherwise specified.

[0028] Example 1: Isolation, purification and preliminary screening of growth characteristics of Trametes hirsuta XY1 CGMCC No.40195 strain

[0029] 1), Strain isolation and purification

[0030] Collect the fungal fruiting bodies on the Prunus persica in the Summer Palace. After surface disinfection on the laboratory aseptic workbench, cut a small amount of fruiting body tissue for isolation and purification to obtain three strains of bacteria, XY1, YH9-4 and YH17-6. Figure 1 In a, c, and e are photos of the fruiting bodies collected from the three strains of fungi.

[0031] 2) PDA culture characteristics and growth rate tests of the three strains of fungi

[0032] The purified strains of XY1, YH9-4, and YH17-6 were cultured on PDA medium for 5 to 10 days. Agar discs with a diameter of 5 mm were punched out and inoculated into petri dishes filled with PDA medium. The dishes were placed in an incubator at a temperature of 28°C and a relative humidity of 80% for cultivation. The colony growth characteristics of each fungus on the PDA medium were observed. The growth of mycelia, colony morphology, and changes in the color of mycelia and colonies on different media were measured and observed. The diameter of the colonies was measured and recorded, and the daily colony growth rate was calculated (colony growth rate = total growth amount / number of growth days) (Table 1).

[0033] Table 1 Basic growth characteristics of three strains of fungi

[0034]

[0035] The daily growth rates of the three strains XY1, YH9-4, and YH17-6 were 16.5 cm, 22.81 cm, and 17.35 cm respectively, showing relatively fast growth. Their mycelia were all white and free of variegated colors on the PDA medium, meeting the basic requirements of having a relatively fast growth rate and not producing variegated pigments. Figure 1 b, d, and f are the cultivation characteristics of the purified products of the three strains of fungi on the PDA medium.

[0036] 3) Guaiacol primary screening

[0037] The production of laccase is a basic characteristic of biological pulping strains. To test whether the strains produce laccase, the guaiacol plate method was used in the laboratory. The medium composition was 100 μL of guaiacol added to every 400 mL of PDA medium, sterilized at 121°C for 30 min, and poured into petri dishes after sterilization. Agar discs with a diameter of 5 mm were punched out from the edge of the colonies cultured in the PDA petri dishes and inoculated into the center of the medium, with three replicates for each strain. After culturing at 27°C for 4 - 5 days, the color change circle was observed. Figure 2 As shown in the color development of the three strains, all had red color circles, indicating that all three strains secrete laccase.

[0038] 4) Mass loss rate test

[0039] Refer to the standards LY / T 1283-2011 and GB / T 13942.1-2009. First, prepare the PDA medium, then inoculate the three strains of bacteria onto this medium and culture for 7-10 days. Then transfer them to the sand-based medium for culture. After the mycelium has covered the feeding wood, place the same group of specimens into a sealed container containing filter paper soaked with 5 mL of distilled water, and place it in a steam sterilizer. Steam at 105±2°C for 30 minutes. After cooling, under sterile conditions, place the poplar wood specimens with the wide surface in contact with the feeding wood covered with mycelium in the culture flask on the feeding wood. Place 1 specimen on each piece of feeding wood, and place 2 pieces of feeding wood and 2 specimens in each culture flask. Place the culture flasks containing the specimens in the culture room, maintain the temperature at 28±2°C, and the relative humidity at 75-85%. Let the test bacteria infect for 12 weeks. Do not turn on the light at other times except when it is necessary to turn on the light for inspection. After infection, take out the specimens from the culture flasks, carefully scrape off the mycelium and impurities on the surface of the specimens, place the specimens in a forced-air drying oven, dry them to a constant weight at 103±2°C, and then weigh each specimen one by one, accurate to 0.01 g. Calculate the mass loss rate of the test blocks before and after culture.

[0040] Table 2 Mass loss rate of poplar wood infected by three strains of fungi

[0041] Strain Mass loss rate (%) XY1 70.92 YH9-4 6.35 YH17-6 10.36

[0042] Table 2 shows that XY1 causes a 70.92% mass loss rate in poplar wood, while YH9-4 and YH17-6 only cause mass loss rates of 6.35% and 10.36% in poplar wood respectively. It is preliminarily judged that XY1 has higher lignin-degrading enzyme activity.

[0043] 4) Molecular identification of the strain Trametes hirsuta XY1

[0044] Use the universal primers ITS4 and ITS5 to amplify the ITS1-5.8S-ITS2 segment sequence of XY1 (sequence 1 in the sequence list). Send the product to Shanghai Bioengineering Technology Service Co., Ltd. for sequencing, and perform sequence alignment on the obtained sequence information through the TrichoKEY software to identify the strain type.

[0045] The sequence 1 in the sequence list is as follows:

[0046] acctgcggaa ggatcattaa cgagttttga aatgggttgt tgctggcctt ccgaggcatg 60

[0047] tgcacgccct gctcatccac tctacacctg tgcacttact gtaggttggc gtgggtttct 120

[0048] agcctccggg tttggaagca ttctgccggc ctatgtacac tacaaactct taaagtatca 180

[0049] gaatgtaaac gcgtctaacg catcttaata caactttcag caacggatct cttggctctc 240

[0050] gcatcgatga agaacgcagc gaaatgcgat aagtaatgtg aattgcagaa ttcagtgaat 300

[0051] catcgaatct ttgaacgcac cttgcgctcc ttggtattcc gaggagcatg cctgtttgag 360

[0052] tgtcatgaaa ttctcaaccc ataaatcctt gtgatctatg ggcttggatt tggaggcttg 420

[0053] ctggccctag cggtcggctc ctcttgaatg cattagcttg attccgtgcg gatcggctct 480

[0054] cagtgtgata attgtctacg ctgtgaccgt gaagcgtttt ggcaagcttc taaccgtcca 540

[0055] ttaggacaat tttataacat ctgacctcaa atcaggtagg actacccgct gaacttaagc 600

[0056] atatcaataa ggcggagga 619

[0057] Through the identification of the morphological characteristics of the fruiting body and molecular sequence characteristics above, XY1 was identified as Trametes hirsuta, named Trametes hirsuta XY1, and was deposited in the China General Microbiological Culture Collection Center (abbreviated as CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing) on June 8, 2022, with the deposit number of CGMCC No. 40195.

[0058] It was further identified that YH9-4 was Ceriporia lacereta and YH17-6 was Irpex lacteus. Figure 1 Among them, a, c, and e are respectively the fruiting body photos of the three strains of fungi collected.

[0059] Example 2: Determination of the Secretion Activity of Lignin-Degrading Enzyme System of Trametes hirsuta XY1 CGMCC No. 40195

[0060] 1) Preparation of the crude enzyme solution of Trametes hirsuta XY1 CGMCC No. 40195

[0061] Medium preparation:

[0062] Liquid medium (g / L): PD 26 g; tryptone 10 g; WYS (trace element solution) 100 ml.

[0063] Composition of the trace element solution: NTA 1.5 g; MgSO 4 ·7H 2 O 3.0 g,; MnSO 4 0.5 g; NaCl 1.0 g; FeSO 4 ·7H 2 O 0.1 g,; CoCl 2 0.1 g,; ZnSO 4 ·7H 2 O 0.1 g; CuSO 4 ·5H 2 O 0.1 g,; KAl(SO 4 ) 2 ·12H 2 O 10 mg,; H 3 BO 3 10 mg,; Na 2 MoO 4 ·2H 2 O 10 mg, distilled water 800 ml, adjust the pH value to 6.5 with NaOH, make up the volume to 1000 ml, and then sterilize it in a 121 °C autoclave for 15 minutes for standby.

[0064] After the strain was activated on the PDA medium, a mycelial plug with a diameter of 5 mm was punched at the edge of the colony and inoculated into the liquid medium, and then cultured in a constant temperature shaker at 29 °C and 160 r / min. The enzyme activity was measured every day starting from the 4th day.

[0065] 2) Laccase Activity Test of Trametes hirsuta XY1 CGMCC No.40195 Strain

[0066] The laccase (Lac) activity was determined by the ABTS method. Using ABTS as the substrate, the total reaction system was 3 ml, including 2700 μl of 0.1 mol / L sodium acetate, 100 μl of crude enzyme solution, and 200 μL of 1 mmol / L ABTS. The reaction was carried out in a water bath at 37°C for 5 min. Measure the change in absorbance of the reaction system at a wavelength of 420 nm. Define the amount of enzyme required to increase the absorbance value by 0.1 per minute in the reaction system as one enzyme activity unit (U). The molar extinction coefficient of ABTS at 420 nm is 36000 L / (mol·cm).

[0067] Figure 3 It shows that the laccase activity of the XY1 crude enzyme solution began to increase significantly from the 8th day, reaching 1032.6 U / ml. It continued to rise on the 9th and 10th days, reaching the highest on the 10th day, with 1438.8 U / ml, and then remained between 1200 - 1350 U / ml. However, the activities of YH9 - 4 and YH17 - 6 were always relatively low. The activity of YH17 - 6 only began to increase on the 12th day and was only 554.4 U / ml on the 13th day. The laccase activity of XY1 was significantly higher than that of YH9 - 4 and YH17 - 6, and the time to start secreting a large amount of laccase was also the earliest, starting on the 8th day.

[0068] 3) Manganese Peroxidase (Mnp) Activity Test of Trametes hirsuta XY1 CGMCC No.40195 Strain

[0069] The Mnp activity was determined by referring to the 2,6 - DMP method. The activity of manganese peroxidase was measured using 2,6 - DMP as the substrate. The reaction system was 3.3 mL of 0.1 mol / L tartaric acid - sodium tartrate buffer (pH = 4.5), 0.1 mL each of 40 mmol / L MnSO 4 and 40 mmol / L 2,6 - DMP solution. Add 0.1 mL of 4 mmol / L H 2 O 2 and 0.4 mL of the enzyme supernatant to initiate the reaction. Incubate in a 30°C water bath for 3 min and measure the change in absorbance at λ = 469 nm within 3 min. The extinction coefficient of the product formed by the oxidation of DMP is ε = 55000 (mol / L)-1cm-1. Define the amount of enzyme that oxidizes 1 μmol DMP per minute as one enzyme activity unit (U).

[0070] Figure 4Results showed that the manganese peroxidase activities of XY1 and YH9-4 started to increase on the 8th day, reaching 1143.6 U / ml for XY1 and 620.23 U / ml for YH9-4, and remained at this level in the following days. The manganese peroxidase activity of YH17-6 started to increase on the 12th day and only reached 320.5 U / ml on the 13th day. It was shown that the manganese peroxidase activity of XY1 was significantly higher than that of YH17-6 and was twice that of YH9-4, and the time when XY1 started to secrete a large amount of laccase was also the earliest, starting from the 8th day.

[0071] 4) Activity test of lignin peroxidase (Lip) of Trametes hirsuta XY1 CGMCC No.40195 strain

[0072] The Lip enzyme activity was determined with reference to the VA method, and the lignin peroxidase activity was determined using veratryl alcohol (VA) as the substrate. The reaction system was 1 mL of 50 mmol / L tartaric acid-sodium tartrate buffer (pH = 3.0), 500 μL of 10 mmol / L VA solution, and 1 mL of the enzyme supernatant. After incubation in a 30 °C water bath for 10 min, 20 μL of 2 O 2 solution was added. The absorbance change at λ = 310 nm was measured within the first 3 min of the reaction. The amount of enzyme that oxidized 1 μmol of VA per minute was defined as one enzyme activity unit (U). In the calculation, ε = 9300 (mol / L)-1cm-1.

[0073] Figure 5 Results showed that the lignin peroxidase activities of the three strains started to increase on the 8th day, reaching 1293 U / ml for XY1, 1380.6 U / ml for YH9-4, and 1032.6 U / ml for YH17-6. They remained at their respective levels in the following days. It was shown that the lignin peroxidase activity of XY1 was comparable to that of YH9-4 and higher than that of YH17-6.

[0074] Adding the activities of the three enzymes, the lignin-degrading enzyme activities starting from the 8th day are shown in Table 3 and Figure 6 .

[0075] Table 3 Lignin-degrading enzyme activities (U / ml) of crude enzyme solutions of three white-rot fungal strains from the 8th day to the 13th day

[0076] Days / Strain XY1 YH9-4 YH17-6 8d 3469.20 2027.91 1149.86 9d 3822.43 2078.49 1066.20 10d 3903.17 2135.03 1110.51 11d 3756.60 2157.26 1097.91 12d 3689.74 2070.94 1543.20 13d 3879.94 2141.31 1881.69

[0077] Table 3 and Figure 6 Results showed that the lignin-degrading enzyme activity of Trametes hirsuta XY1 CGMCC No.40195 maintained a relatively high level starting from the 8th day, far exceeding those of YH9-4 and YH17-6 strains, and was more than twice that of common strains.

[0078] Example 3: Determination of Amylase Secretion Activity of Trametes hirsuta XY1 Strain (CGMCC No.40195)

[0079] Cellulase is a general term for a series of enzymes that can hydrolyze cellulose into glucose or cellobiose composed of two glucose molecules. Cellulase mainly includes three categories according to different functions: endoglucanase, exoglucanase, and β-glucosidase. Amylase belongs to the hydrolase class and is a general term for a class of enzymes that catalyze starch, glycogen, and dextrin. Amylase can break the α-1,4-glycosidic bond and α-1,6-glycosidic bond in starch, thereby hydrolyzing starch into oligosaccharides or monosaccharide molecules. The amylase that hydrolyzes the α-1,4-glycosidic bond is liquefying amylase, and the amylase that hydrolyzes the α-1,6-glycosidic bond is saccharifying amylase. The amylase activity is measured using a 1% starch solution as the substrate. Reagents such as DNS solution are water-bathed with the substrate, and then the absorbance at 540 nm is detected using a spectrophotometer to measure the activity of five hydrolases. The enzyme activity unit is the amount of enzyme required to produce 1 μmol of glucose in 1 minute.

[0080] Specific steps:

[0081] 1. Detection of endoglucanase: CMC-Na method

[0082] 1) Preparation of glucose standard curve

[0083] Prepare test tubes and add glucose standard solutions with a concentration of 1 mg / ml, distilled water, and DNS reagent according to Table 1 as follows:

[0084] Table 4 Solution Preparation

[0085]

[0086] 2) Add 1.6 ml of sodium carboxymethyl cellulose and 0.4 ml of enzyme solution, incubate in a water bath at 50 °C for 3 min, keep warm for 30 min, add 2 ml of 3,5-dinitrosalicylic acid (DNS color reagent), boil in a water bath for 5 min, and measure the absorbance value at 540 nm after cooling.

[0087] 3) Calculate the enzyme activity

[0088] 2. Measurement of filter paper enzyme (FPA) activity:

[0089] Enzyme solution, buffer solution, Whatman filter paper, substitute into the formula (glucose standard curve)

[0090] Figure 7It is shown that the amylase content of Trametes hirsuta XY1 CGMCC No.40195 is lower than that of common white rot fungi. The secretion of YH9-4 reaches more than 460 at the highest, and the secretion of the patented strain has always remained low. This characteristic is beneficial to improving the pulp yield during the pulping process.

[0091] Example 4: Determination of the xylanase secretion activity of Trametes hirsuta XY1 CGMCC No.40195 strain

[0092] Due to the diversity of monosaccharide composition and the high degree of branching in the structure of hemicellulose, the types of hemicellulase systems are more complex than those of cellulases. Completely and thoroughly degrading hemicellulose also requires the cooperation of multiple enzymes. At present, the research on hemicellulases mainly focuses on xylanases. The xylanase activity is determined using a 1% xylan solution as the substrate. Reagents such as DNS solution are heated in a water bath with the substrate, and then the absorbance at 540 nm is detected using a spectrophotometer to determine the activity of five hydrolases. The enzyme activity unit is the amount of enzyme required to produce 1 μmol of glucose in 1 min. Specific steps:

[0093] 1) Drawing of the xylose standard concentration curve

[0094] Measure 0 - 0.5 ml of the xylose standard solution and make up to 2 ml, add 2 ml of DNS, boil in a water bath for 5 min, make up the volume to 10 ml, and measure the OD at 540 nm

[0095] 2) Take 1.8 ml of 0.5% xylan solution, 0.2 ml of enzyme solution, heat in a water bath at 50 °C for 10 min, add 2 ml of DNS, boil in a water bath for 5 min, make up the volume to 10 ml, and measure the OD at 540 nm

[0096] Figure 8 It is shown that the xylanase content of Trametes hirsuta XY1 CGMCC No.40195 is lower than that of common white rot fungi. The secretion of YH17-6 reaches nearly 100 U / ml or more at the highest, and the secretion of the XY1 strain has always remained low. High lignin-degrading enzyme activity, low hemicellulase activity, and low cellulase activity are beneficial to improving the pulp yield during the pulping process.

[0097] Example 5: Fiber pulping test of Eucalyptus urophylla × Eucalyptus grandis treated with Trametes hirsuta XY1 CGMCC No.40195 strain

[0098] The wood chips were pretreated by alkali pretreatment (control) and biological pre-hydrolysis. The reaction temperature for pretreatment was 90 - 180 °C, the alkali concentration was 1% - 14%, the heating-up time was 2 h, the holding time was 2 h, and the liquor-to-wood ratio was 1:4 - 1:6. The reaction temperature for biological pretreatment was 45 °C, the heating-up time was 90 min, the holding time was 120 min, and the liquor-to-wood ratio was 1:5 - 1:10. After the pretreatment reaction, the extract was separated from the wood chips and used for colorimetric determination. The wood chips after pretreatment were washed repeatedly with distilled water and soaked for 24 h, during which the water was changed frequently. After the soaking solution became colorless, they were taken out and air-dried; then they were rinsed with distilled water and air-dried. The pulp yield differences are shown in Table 5.

[0099] Table 5 Comparison of pulp yield differences between the two pretreatments

[0100] Treatment Pulping yield (%) Kappa number Viscosity / ml / g Degree of polymerization Alkaline pretreatment (control) 47.00 22 855 1255 Biological pretreatment 58.79 20 890 1330

[0101] The pulp yield of biological pretreatment increased compared with that of alkali treatment, from 47% to 58.79%. The Kappa numbers were not very different and were both relatively low, indicating that it was relatively easy to form pulp; the viscosity values and degrees of polymerization were slightly higher, reflecting that the mechanical strength and processing adaptability of the pulp were improved.

[0102] The test results of the paper produced by sending the pulp to the factory showed that: for the paper made from the biological pulp treated with the strain Trametes hirsuta XY1 CGMCC No.40195, the tensile index, burst index, and folding endurance increased by 27.5%, 18.9%, and 45% respectively, and the tear index decreased by 15%.

[0103] Example 5. Decolorization test of the fermentation broth of the strain Trametes hirsuta XY1 CGMCC No.40195

[0104] The mycelia cultured for 8 d at 30 °C were transferred to an improved PDA medium containing several glass beads. After culturing at 200 r / min and 30 °C for 4 d, they were centrifuged (n = 8000 r / min, 10 min), and the mycelia were rinsed 3 - 4 times with sterile water and homogenized in the bleaching wastewater medium for standby. Then, the homogenized mycelia suspension was inoculated into a triangular flask containing 90 mL of sterilized bleaching wastewater medium at 10% and sealed with four layers of gauze, and cultured on a shaker (n = 150 r / min, 35 °C). The treated bleaching wastewater was filtered by a glass filtering crucible, and the filtrate was centrifuged again (n = 8000 r / min, 10 min), the pH was adjusted to 7.6, and the absorbance at 465 nm was measured with a distilled water reference in a 721 spectrophotometer.

[0105] The results showed that the decolorization rate of the fermentation broth for the wastewater was 82.7% ( Figure 9 ).

Claims

1. A strain of Trametes hirsuta characterized in that Name: Trametes gallica ( Trametes hirsuta ), XY1, with the preservation number of CGMCC No. 40195 at the General Microbiology Center of China Committee for Culture Collection of Microorganisms.

2. Use of the Trametes gallica ( Trametes hirsuta ) strain XY1 in biological pulping.

3. A biological pulping method, wherein the pretreatment fungus uses Trametes gallica ( Trametes hirsuta ) XY1.

4. Use of strain XY1 of Trametes gallica described in claim 1 in lignin degradation. Trametes hirsuta ​ 5. Use of Trametes gallica ( Trametes hirsuta ) strain XY1 in the production of laccase, manganese peroxidase and lignin peroxidase.

6. Use of the Trametes gallica ( Trametes hirsuta ) strain XY1 in decolorizing wastewater in the papermaking process.

Images