Strain S-LWZ20190614-6 of *Veperus spp.* and its application in the degradation of lignin and bisphenol S

Abstract

This invention belongs to the field of microbial degradation technology. This invention provides a strain of *Phlebiaacerina* S-LWZ20190614-6 (CGMCC No. 40196) and its application in the degradation of lignin and bisphenol S. This strain was deposited on June 6, 2022, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 40196, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Under PDB conditions, the degradation rate of strain S-LWZ20190614-6 reached 99.6% after 7 days; under Kirk conditions, the degradation rate reached 96.2% after 7 days.

Description

Technical Field

[0001] This invention belongs to the field of microbial degradation technology. Background Technology

[0002] Lignocellulose is one of the most abundant renewable energy sources in nature and the largest natural reserve of aromatic compounds, with a global annual production of 150 billion tons, including 6 billion tons of straw alone. Lignocellulose raw materials are mainly composed of lignin, cellulose, and hemicellulose. Hemicellulose is tightly linked to lignin molecules through covalent and hydrogen bonds, making the natural lignocellulose structure very robust and highly resistant to enzymatic and microbial hydrolysis. However, the complex structure of lignin hinders the enzymatic hydrolysis of cellulose and hemicellulose to produce fermentable sugars and high-value-added products. Therefore, the effective removal of lignin significantly impacts the efficient utilization of cellulose and hemicellulose.

[0003] The main reason why straw is difficult to degrade naturally is that lignocellulose, a major component of plant cell walls, is difficult to degrade naturally and efficiently. As an important renewable resource, straw is of great significance for its rational resource utilization.

[0004] White-rot fungi are a major type of lignin-degrading microorganism. Fungi in nature degrade lignin by producing laccase, lignin peroxidase, and manganese peroxidase. Using white-rot fungi to pretreat straw can disrupt the lignin structure of straw. Therefore, screening for highly efficient white-rot fungi that degrade lignin is very important for the rapid degradation of straw lignin.

[0005] Bisphenol S (BPS) is an important alternative to bisphenol A (BPA). As an additive, BPS is commonly used in everyday products such as polycarbonate plastics, adhesives, canned foods, baby bottles, beverage packaging, banknotes, cardboard boxes, flyers, newspapers, and thermal paper receipts. However, recent studies have shown that BPS has similar toxicity to BPA. In recent years, four new BPS analogues have been discovered in thermal paper and ordinary paper products, and all four have been shown to possess one or more of the aforementioned toxicities. This indicates that BPS analogues are not entirely safe.

[0006] Studies have shown that bisphenol S (BPS) has been detected at high concentrations in water and sediment samples in my country. Research indicates that BPS is also a compound with potential endocrine effects, and its estrogenic effects may be comparable to those of bisphenol A (BPA). Due to its high stability, traditional water treatment technologies struggle to remove BPS, leading to its widespread detection in various water bodies, soils, and even human urine. Given the numerous negative effects of BPS, current technologies struggle to safely and rapidly degrade it; therefore, there is an urgent need to develop an efficient and environmentally friendly method for its degradation. Summary of the Invention

[0007] In view of this, the present invention provides a strain of Phlebia acerina Peck S-LWZ20190614-6 CGMCC No.40196.

[0008] The present invention also provides a microbial inoculant, including Phlebia acerina S-LWZ20190614-6 CGMCC No.40196.

[0009] The present invention provides a composition comprising Phlebia acerina S-LWZ20190614-6 CGMCC No.40196.

[0010] Finally, this invention provides the application of Phlebia acerina Peck S-LWZ20190614-6 in the degradation of lignin and bisphenol S.

[0011] Furthermore, in a specific embodiment of the present invention, the lignin is present in straw or wood.

[0012] Furthermore, in a specific embodiment of the present invention, the straw is wheat straw, corn straw, or white bamboo straw.

[0013] Furthermore, in a specific embodiment of the present invention, the wood is beech or pine.

[0014] The strain S-LWZ20190614-6 provided by this invention achieved a degradation rate of 99.6% after 7 days under PDB conditions and 96.2% after 7 days under Kirk conditions. Attached Figure Description

[0015] Figure 1 This is a comparison chart of the lignin degradation rates of strain S-LWZ20190614-6 on wheat straw powder, corn straw powder, beech wood powder, white bamboo powder, and pine wood powder.

[0016] Figure 2 This is a diagram showing the degradation of bisphenol S by strain S-LWZ20190614-6. Detailed Implementation

[0017] Example 1

[0018] 1. Acquisition, isolation, and identification of bacterial strains

[0019] The strain was collected on June 14, 2019, from the Chebaling National Nature Reserve in Shaoguan City, Guangdong Province. It was obtained from fresh fruiting bodies growing on decaying forest wood using the tissue isolation method and named strain S-LWZ20190614-6. After purification, it was cultured at 28℃ for 10 days.

[0020] DNA was extracted from S-LWZ20190614-6 using a rapid fungal genome extraction kit. Its ITS sequence was obtained by PCR amplification, and its sequence information is shown in SEQ ID NO.1 in the sequence listing. By comparing with the BLAST database of NCBI and constructing a phylogenetic tree, combined with morphological analysis, the strain was identified as Phlebia acerina.

[0021] Phlebia acerina S-LWZ20190614-6 was deposited on June 6, 2022, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 40196. The center is located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0022] 2. Passage cultivation:

[0023] Phlebia acerina S-LWZ20190614-6, which was stored in a 4°C refrigerator, was passaged on potato dextrose agar (PDA) medium and incubated at 30°C for 3-6 days.

[0024] The same experiment was conducted using the model white-rot fungus *Phanerochaete chrysosporium* Burds. as a control group.

[0025] 3. Degradation of lignin

[0026] (1) Static incubation:

[0027] To prepare culture media for wheat straw powder, corn straw powder, beech wood powder, white bamboo powder, and pine wood powder, weigh 0.5g of the corresponding wood powder or straw powder, spread it evenly, and dispense it into Erlenmeyer flasks. Add 1.25mL of deionized water and autoclave at 121℃ for 15min. Take 10mm diameter mycelial blocks from PDA medium that has been cultured for 3 days and inoculate one block into each of the following media: wheat straw powder, corn straw powder, beech wood powder, white bamboo powder, and pine wood powder. Incubate these media at 30℃ for 20 days.

[0028] (2) Determination of lignin content by the sulfuric acid method (Klason method)

[0029] The culture media (wheat straw powder medium, corn straw powder medium, beech wood powder medium, white bamboo powder medium, and pine wood powder medium) were dried together with the bacterial cells in an oven at 105℃ for 1.5 hours, weighed, and the drying and weighing were repeated until a constant weight was achieved.

[0030] Measure 10 mL of 72% concentrated sulfuric acid and add it to each culture medium. React for 1.5 h, stirring several times during the reaction to ensure a complete reaction.

[0031] Measure 382 mL of deionized water, rinse the mixed liquid in the culture medium several times, and transfer it to a 500 mL Erlenmeyer flask. Seal the flask with aluminum foil and heat it in an autoclave for 60 min. While the heated acidic washing solution is still hot, filter it through glass fiber filter paper with a pore size of 1 μm that has been dried to constant weight. Dry the filter paper and filter residue to constant weight and record the result.

[0032] After diluting the filtrate, its absorbance was measured at 205 nm using a UV spectrophotometer.

[0033] After summarizing the above data and comparing it with the blank group, the degradation rate of lignin can be calculated (Table 1). Figure 2 ).

[0034] Table 1. Lignin degradation rates (%) of *Phlebia acerina* S-LWZ20190614-6 and *Phanerochaete chrysosporium* on different culture media.

[0035]

[0036] 4. Degradation of bisphenol S (BPS)

[0037] (1) Pre-culture

[0038] 1) Static incubation in potato dextrose liquid medium (PDB):

[0039] Weigh 6g of PDB and dissolve it in 250mL of pure water.

[0040] 10 mL of PDB was transferred into each of the 24 100 mL Erlenmeyer flasks, and then the Erlenmeyer flasks containing PDB were placed in an autoclave and sterilized at 121 °C for 15 min.

[0041] 2) Static incubation in Kirk medium (250 mL):

[0042] Weigh 2.5g glucose, 0.05525g ammonium tartrate, 0.41g anhydrous sodium acetate, and 25mL Kirk salt solution to prepare 200mL Kirk medium.

[0043] Adjust the pH to 4.5 using 1 mol / L HCl and 1 mol / L NaOH.

[0044] Transfer 10 mL of Kirk medium to each of the 24 100 mL Erlenmeyer flasks, and then place the Erlenmeyer flasks containing Kirk medium into an autoclave and sterilize at 121 °C for 15 min.

[0045] 3) Take a 10mm diameter bacterial block from the PDA medium after 6 days of culture, add 2 bacterial blocks to each conical flask in the PDB and Kirk, and place them in a 30℃ incubator for static culture for 6 days.

[0046] (2) Degradation experiment

[0047] After 6 days of pre-culture, 100 μL of 10 mM BPS dissolved in DMSO (final concentration in the culture medium is 0.1 mM) was added to Erlenmeyer flasks containing 10 mL PDB and Kirk, respectively, and the culture time was 0 to 7 days.

[0048] After the required number of days of incubation, remove the sample, add 20 mL of acetone, and then add bisphenol F (BPF) as an internal standard compound.

[0049] The mixture was homogenized, filtered, evaporated to dryness by rotary evaporation, dissolved in 10 mL of chromatographic methanol, filtered through a 0.22 mm filter membrane, and the contents of BPS and BPF were determined by high performance liquid chromatography. The concentration of BPS was calculated by standard curve (Table 2). Figure 2 The degradation rate of BPS was calculated by comparing it with that at day 0. As shown in the figure above, strain S-LWZ20190614-6 achieved a degradation rate of 99.6% after 7 days under PDB conditions, and 96.2% under Kirk conditions.

[0050] Table 2. BPS concentration (mM) of Phlebia acerina S-LWZ20190614-6 after treatment with PDB and Kirk media supplemented with BPS for 0-7 days.

[0051]

[0052] The strain S-LWZ20190614-6 provided by this invention achieves a degradation rate of 99.6% after 7 days under PDB conditions and 96.2% after 7 days under Kirk conditions.

Claims

1. *Acer buergerianum* ( Phlebia acerina S-LWZ20190614-6, its accession number is CGMCC No.40196.

2. A microbial inoculant, characterized in that, Including the *Acer buergerianum* as described in claim 1 (… Phlebia acerina )S-LWZ20190614-6.

3. A composition capable of degrading lignin and bisphenol S, characterized in that, Including the *Acer buergerianum* as described in claim 1 (… Phlebia acerina )S-LWZ20190614-6.

4. The *Acer buergerianum* as described in claim 1 (… Phlebia acerina Application of S-LWZ20190614-6 in the degradation of lignin.

5. The *Acer buergerianum* according to claim 4 (… Phlebia acerina The application of S-LWZ20190614-6 in the degradation of lignin is characterized by, The lignin is present in straw or wood.

6. The *Acer buergerianum* according to claim 5 (… Phlebia acerina The application of S-LWZ20190614-6 in the degradation of lignin is characterized by, The straw is wheat straw, corn straw, or white bamboo straw.

7. The *Acer buergerianum* according to claim 5 (… Phlebia acerina The application of S-LWZ20190614-6 in the degradation of lignin is characterized by, The wood is beech or pine.

8. The *Acer buergerianum* as described in claim 1 (… Phlebia acerina Application of S-LWZ20190614-6 in the degradation of bisphenol S.

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