A strain of Aspergillus kefeigensis F25 capable of degrading lignin and cellulose at low temperature and its application

Abstract

The present invention belongs to the field of microorganisms, and specifically discloses an Aspergillus fusca F25 strain capable of degrading lignin and cellulose at low temperature and an application thereof; the Aspergillus fusca F25 strain is deposited in the General Microbiology Center of China Microorganism Culture Collection Committee, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the deposit date is January 25, 2024, and the deposit number is CGMCC No. 41052; the Aspergillus fusca F25 strain has the ability to degrade lignin and cellulose at low temperature, and can be used for the degradation of agricultural and forestry wastes at low temperature. Furthermore, the present invention also finds that the Aspergillus fusca F25 strain is combined with Geotrichum candidum and Acinetobacter to form a composite bacterial agent, which can produce a synergistic effect and produce a better effect of degrading agricultural and forestry wastes.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and specifically discloses an Aspergillus keifelder F25 strain capable of degrading lignin and cellulose at low temperature and application thereof. Background Art

[0002] my country is a major agricultural country and also a major producer of agricultural and forestry waste. According to statistics, my country's straw production in 2021 was about 900 million tons (Han Zhe, Pan Kai. Straw return to the field and its application in vegetable production [J]. Agriculture and Technology, 2022, 42(20): 61-64.), but the comprehensive utilization rate was low. It is reported that the proportion of straw burning in Heilongjiang Province and Jilin Province reached 21.0% and 23.2% (Wu Xiaosha, Li Zaixing, Huang Yali, et al. Screening and identification of high-temperature lignocellulose degrading bacteria and their composting application [J]. Anhui Agricultural Science, 2021, 49(20): 68-71.). Extensive disposal methods such as discarding or burning not only cause serious pollution to the environment, but also lead to waste of biomass resources.

[0003] At present, domestic and foreign countries mainly adopt various methods such as fertilizer, feed, fuel, raw material, and base material to comprehensively process and utilize straw (Li Peng. Performance evaluation of agricultural waste recycling and research on industrial development mechanism [D]. Huazhong Agricultural University, 2014.), and its utilization rate is 60%, 18%, 8.5%, 0.9% and 0.7% respectively (Shi Zuliang, Jia Tao, Wang Yajing, et al. Current status of comprehensive utilization of crop straw in my country and estimation of carbon emissions from incineration [J]. Chinese Agricultural Resources and Zoning, 2017, 38(09): 32-37.). Fertilizerization is to use straw and other agricultural and forestry wastes such as livestock and poultry manure for aerobic composting and fermentation, and then use it as organic fertilizer to improve soil fertility and increase production and income. my country has been promoting the return of straw to the fields for a long time, which can increase the content of soil organic matter. However, due to the slow degradation rate after straw is returned to the fields and the high C\N, it competes with grain for nitrogen and leads to an increase in crop diseases and pests. Therefore, aerobic composting is still the main way to utilize straw resources.

[0004] The presence of lignin in the cell walls of straw limits the efficiency of straw biodegradation, especially in northern my country, where the autumn and winter are long and the temperature is low, which hinders the straw composting and fermentation process, prolongs the composting cycle, and limits the use of straw as fertilizer. Therefore, screening microbial strains that can efficiently degrade lignocellulose in low-temperature environments and improving the efficiency of straw lignocellulose degradation in low-temperature scenarios are of great practical significance for more efficient straw processing, reducing resource waste, and promoting green development.

[0005] It is reported that domestic and foreign scholars have screened out more than 200 strains of efficient lignocellulose degradation strains (Yang Qian, Li Weizun, Ju Guanting, et al. Research progress on microbial degradation of lignocellulose biomass solid waste [J]. Microbiology Bulletin, 2015, 42(08): 1569-1583.), among which aerobic bacteria Pseudomonas (Zhang Erhong. Screening of efficient cellulase strains and construction of bacterial community [D]. Shihezi University, 2015.), thermoacid bacteria (Zhang Liang, Lin Ning, Du Qian, et al. Research on production of organic fertilizer and fertilizer efficiency of crop straw high temperature composting [J]. Guizhou Agricultural Science, 2015, 43(07): 91-96.) and Bacillus sp. (Zhang Yang, Li Ying, Ai Shiqi, et al. Cellulose decomposition ability and enzyme activity characteristics of artificially constructed simplified composite bacterial system based on WSC-9 [J]. Journal of Heilongjiang Bayi Agricultural University, 2016, 28(02): 80-84.), anaerobic pyrolytic cellulose bacteria (Zhang Erhong. Screening of efficient cellulase strains and construction of bacterial community [D]. Shihezi University, 2015.) and Clostridium (Wang Zhenchuan, Xie Guixian, Liu Qiang, et al. Effect of adding rice straw to pig manure on the maturity process and material changes of high-temperature composting [J]. Agricultural Modernization Research, 2015, 36(02): 309-314.) are all major lignocellulose degrading strains. Whether a strain can efficiently degrade lignocellulose mainly depends on its enzyme production ability. In the process of microbial degradation of lignin, enzyme activity is greatly affected by temperature factors. Under low temperature conditions, enzyme activity is easily reduced or inactivated (Gou Changlong. Screening of low-temperature cellulose-degrading bacteria and application of composite bacterial systems in cow dung composting [D]. Jilin Agricultural University, 2014.). At present, there have been a lot of studies on microbial degradation of cellulose under low temperature conditions (Zhang Mengjun, Qiu Chenhao, Chai Liwei, et al. Screening, identification and fermentation optimization of low-temperature fungi with high efficiency in cellulose degradation [J]. Microbiological Bulletin, 2019, 46(10): 2494-2503. Kang Zongjing, Yuan Nan, Wang Yingyan, et al. Fungal community structure and characteristics of low-temperature cellulose-degrading fungi in Ruoergai Plateau wetlands [J]. Soil Bulletin, 2017, 48(4): 830-836.), but there are relatively few studies on low-temperature lignin-degrading bacteria. Summary of the invention

[0006] In view of the above problems, the present invention discloses an Aspergillus keifenesii F25 strain capable of degrading lignin and cellulose at low temperature and application thereof.

[0007] The present invention includes the following technical solutions:

[0008] A polymorpha F25 strain that can degrade lignin and cellulose at low temperature. The polymorpha F25 strain is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the deposit date is January 25, 2024, and the deposit number is: CGMCC No.41052.

[0009] Furthermore, the present invention discloses the application of the above-mentioned Aspergillus kafei-like F25 strain in the degradation of agricultural and forestry wastes.

[0010] Furthermore, the present invention also discloses a method for screening the above-mentioned Aspergillus kaifei-like F25 strain, comprising the following steps: 1) sampling: taking feces from herbivorous animals in Beijing Zoo;

[0011] 2) Enrichment and acclimatization: the sample is placed in acclimatization medium for acclimatization;

[0012] 3) Isolation and purification: dilute the acclimation solution and add it to the screening medium to select the colonies with good growth and purify the single colonies;

[0013] 4) Preliminary screening: Preliminary screening is carried out based on the ability of the strain to produce laccase Lac, lignin peroxidase LiP, manganese peroxidase MnP and cellulase;

[0014] 5) Rescreening: The strains obtained in the initial screening were further subjected to 5°C low-temperature screening, lignin degradation rate determination, ligninase activity determination, cellulase activity determination, hemicellulase activity determination and filter paper disintegration test to obtain the final strains;

[0015] 6) Identification: The final strain is subjected to morphological and molecular identification.

[0016] The invention also discloses a composite bacterial agent, which comprises the above-mentioned Aspergillus niger F25 strain.

[0017] Furthermore, the above-mentioned composite bacterial agent also includes Geotrichum candidum and Acinetobacter.

[0018] Preferably, in the above-mentioned composite bacterial agent, the Geotrichum candidum is preferably the Geotrichum candidum K10 strain, which is preserved in the General Microbiology Center of China Microbiological Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; preservation date January 25, 2024, preservation number: CGMCC No.41053.

[0019] Preferably, in the above-mentioned composite bacterial agent, the Acinetobacter is preferably Acinetobacter F16 strain, which is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the deposit date is January 25, 2024, and the deposit number is: CGMCC No.29760.

[0020] Furthermore, in the above-mentioned composite bacterial agent, the optimal bacterial ratio of F16:F25:K10 is 3:2:1.

[0021] The invention discloses application of the composite bacterial agent in the degradation of agricultural and forestry wastes.

[0022] Furthermore, in the above application in the degradation of agricultural and forestry wastes, the agricultural and forestry wastes are preferably agricultural and forestry wastes derived from poplar leaves.

[0023] The present invention has the following beneficial effects:

[0024] The present invention discloses an Aspergillus kefei-like F25 strain capable of degrading lignin and cellulose at low temperature and an application thereof. The Aspergillus kefei-like F25 strain has the ability to degrade lignin and cellulose at low temperature and can be used for degrading agricultural and forestry wastes at low temperature. Furthermore, the present invention also finds that the Aspergillus kefei-like F25 strain is combined with Geotrichum candidum and Acinetobacter to form a composite bacterial agent, which can produce a synergistic effect and produce a better effect of degrading agricultural and forestry wastes.

[0025] Biomaterial deposit information:

[0026] The Acinetobacter F16 strain is deposited in the General Microbiology Center of the China Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the deposit date is January 25, 2024, and the deposit number is CGMCC No. 29760.

[0027] The Aspergillus fumigatus F25 strain is deposited in the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the deposit date is January 25, 2024, and the deposit number is CGMCC No.41052.

[0028] The Geotrichum candidum K10 strain is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the deposit date is January 25, 2024, and the deposit number is: CGMCC No.41053. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a schematic diagram of the overall screening and identification technology route of the present invention;

[0030] Figure 2 This is the result of the guaiacol plate colorimetric test;

[0031] Figure 3 It is the result of MnCl2 plate color development test;

[0032] Figure 4 This is the result of the Coomassie brilliant blue plate decolorization test;

[0033] Figure 5 This is the result of Congo red staining test;

[0034] Figure 6 is the bacterial growth curve at 5°C;

[0035] Figure 7 is the fungal growth curve at 5°C;

[0036] Figure 8 is the standard curve of alkaline lignin;

[0037] Fig. 9 is the degradation rate of alkaline lignin at 20 days;

[0038] Fig.10 To test the Lac enzyme activity of each bacteria;

[0039] Fig.11 To test the LiP enzyme activity of each bacteria;

[0040] Fig.12 To test the MnP enzyme activity of each bacteria;

[0041] Fig.13 To test the cellulase activity of each bacteria;

[0042] Fig.14 To test the xylanase activity of each fungus;

[0043] Fig.15 The maximum enzyme activity of each strain is summarized;

[0044] Fig.16 It is the filter paper disintegration test for each bacteria;

[0045] Fig.17 This is the Gram staining image of three bacterial strains under oil microscope;

[0046] Fig.18 The morphological pictures of two fungi under microscope;

[0047] Fig.19 For the construction of developmental trees;

[0048] Fig. 20 This is a graph showing the effect of different bacteria on enzyme activity;

[0049] Fig.21 This is the growth curve of the composite bacterial agent in the degradation of agricultural and forestry wastes;

[0050] Fig. 22 This is a graph showing the pH changes of the culture medium during the degradation of agricultural and forestry wastes;

[0051] Fig.23 This is a comparison chart of the weight loss rates of four types of agricultural and forestry wastes at 15 days;

[0052] Fig.24 Scanning electron microscope images of different agricultural and forestry wastes after degradation (3000×, the left picture is the experimental group and the right picture is the control group). DETAILED DESCRIPTION

[0053] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. The following embodiments and features in the embodiments can be combined with each other without conflict.

[0054] The detailed description of the embodiments of the present invention provided below is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0055] like Figure 1 Shown is a schematic diagram of the overall screening and identification technology route of the present invention.

[0056] Test materials

[0057] Feces of herbivorous animals in Beijing Zoo (obtained F10, F16, F21 and F25), dead branches and leaves of peach trees (obtained K10 and K14), and compost soil samples (obtained D10).

[0058] Culture medium

[0059] Acclimation medium: alkaline lignin 3g, (NH4)2SO4 2g, NaCl 1.5g, KH2PO4 1g, K2HPO4 0.3g, MgSO4·7H2O 0.2g, add water to 1000mL, pH 7.0

[0060] LB medium: 5.0 g yeast powder, 10.0 g tryptone, 10.0 g NaCl, add water to 1000 mL, pH 7.0

[0061] PDA medium: 5.0 g potato powder, 20.0 g glucose, 0.1 g chloramphenicol (to inhibit bacterial growth), 20.0 g agar, add water to 1000 mL, pH 7.0

[0062] LB-guaiacol medium: 5.0 g yeast powder, 10.0 g tryptone, 10.0 g NaCl, 0.4 ml guaiacol, 20.0 g agar, add water to 1000 mL, pH 7.0

[0063] LB-aniline blue medium: 5.0 g yeast powder, 10.0 g tryptone, 10.0 g NaCl, 0.1 g aniline blue, 20.0 g agar, add water to 1000 mL, pH 7.0

[0064] LB-manganese chloride medium: 5.0 g yeast powder, 10.0 g tryptone, 10.0 g NaCl, 0.15 g MnCL2·4H2O2, 20.0 g agar, add water to 1000 mL, pH 7.0

[0065] LB-Coomassie Brilliant Blue Medium: 5.0 g yeast powder, 10.0 g tryptone, 10.0 g NaCl, 1.0 g Coomassie Brilliant Blue, 20.0 g agar, add water to 1000 mL, pH 7.0

[0066] PDA-guaiacol medium: 5.0 g potato powder, 20.0 g glucose, 20.0 g agar, add water to 1000 mL, pH 7.0

[0067] PDA-aniline blue medium: 5.0 g potato powder, 20.0 g glucose, 20.0 g agar, add water to 1000 mL, pH 7.0

[0068] PDA-Coomassie Brilliant Blue Medium: 5.0 g potato powder, 20.0 g glucose, 20.0 g agar, add water to 1000 mL, pH 7.0

[0069] PDA-manganese chloride medium: potato powder 5.0g, glucose 20.0g, agar 20.0g, add water to 1000mL, pH 7.0

[0070] CMC-Na medium: CMC-Na 5.0-10g, (NH4)2SO41.0g, Na2HPO40.9g, MgSO4·7H2O0.5g, NaCl 0.5g, agar powder 20.0g, add water to 1000mL, pH 7.0

[0071] Lignin degradation medium: alkaline lignin 2g, (NH4)2SO4 2g, NaCl 1.5g, KH2PO4 1g, K2HPO4 1g, MgSO4·7H2O 0.2g, add water to 1000mL, pH 7.0

[0072] Filter paper disintegration medium: filter paper (1×4cm, 3 strips per bottle), (NH4)2SO4 2.0g, KH2PO4 1g, NaCl 0.5g, MgSO4·7H2O 0.5g, NaNO3 1.0g, peptone 0.5g, yeast powder 0.5g, add water to 1000mL, pH 7.0

[0073] Agricultural and forestry waste degradation medium: 20g of agricultural and forestry waste powder (after washing the soil on the surface of agricultural and forestry waste, drying to constant weight, and crushing to 42 mesh), 30g of NaCl, 10g of ammonium sulfate, 0.5g of yeast extract powder, add water to 1000mL, pH 7.0

[0074] Reagents

[0075] Congo red indicator, NaCl decolorizing solution, 0.5mmol / LABTS, 100mmol / L acetic acid-sodium acetate buffer, 10mmol / L resveratrol solution, 250mmol / L tartaric acid-sodium tartrate buffer, 10mmol / L H2O2, 1.6mmol / LMnSO4 solution, 2.5mL 50mmol / L succinic acid-sodium succinate buffer, 0.1% glucose standard solution, 1% xylan solution, 3,5-dinitrosalicylic acid (DNS), 2X SanTaq Fast PCR Mix, 27F universal primer, 1492R universal primer, ITS1 universal primer, ITS4 universal primer, 10XTAE buffer, DNA molecular weight standard Marker, 4S Elred nucleic acid dye, 18% hydrochloric acid.

[0076] Example 1

[0077] Acclimation and screening of low-temperature lignocellulose-degrading bacteria

[0078] 1) Enrichment and domestication

[0079] Put 150mL of acclimation medium into a 250mL Erlenmeyer flask, sterilize at 121℃ for 15min, cool to room temperature, add 10g of sample aseptically, and culture at 20℃, 160r / min for 2d. Culture at 15℃ for 2d, then cool to 10℃, continue culture for 2d, then heat to 15℃ and 20℃ for 2d each, repeat the temperature rise and fall cycle 2 times, and ensure that the acclimation liquid is not less than 150mL during the acclimation period.

[0080] 2) Separation and purification

[0081] Pipette 100 μL of degradation bacteria acclimation solution, add it to a test tube containing 900 μL of sterile water, mix well, and set it to 10 -2 Dilution. Then draw 100 μL of solution into a new test tube, add 900 μL of sterile water, and set it to 10-3 dilution. And so on, we get 10 -4 , 10 -5 , 10 -6 , 10 -7 diluent.

[0082] Select 10 -3 , 10 -4 , 10 -5 , 10 -6 Dilution liquid, each 100 μL was pipetted into screening culture medium, and cultured at a constant temperature of 15 ℃. When colonies grew in the culture medium, strains with good growth and different morphologies were selected for streak culture. Repeat 3 times to obtain purified single colonies.

[0083] 3) Initial screening

[0084] 1) Guaiacol plate colorimetric experiment - the ability of strains to produce Lac [Qi Yeqiang, 2021]

[0085] 2) Aniline blue plate decolorization experiment - the ability of strains to produce LiP and MnP [Qi Yeqiang, 2021]

[0086] 3) Manganese chloride plate colorimetric test - the ability of strains to produce MnP [Qi Yeqiang, 2021]

[0087] 4) Congo red staining experiment - the ability of strains to produce cellulase [Bai Changsheng, 2022]

[0088] 5) Coomassie Brilliant Blue Plate Decolorization Test - Cellulase Production Ability of the Strain

[0089] The obtained single bacteria, bacteria and yeast were spotted on LB-Coomassie Brilliant Blue medium, and the fungi were spotted on PDA-Coomassie Brilliant Blue medium. The medium was cultured in a constant temperature incubator at 15°C. When a transparent circle appeared around it, its diameter was recorded and the Hc value (Hc=D / d, i.e., the ratio of the transparent circle diameter to the colony diameter) was measured. Three replicates were set for each strain.

[0090] 4) Rescreening

[0091] 41) 15℃ low temperature screening

[0092] The selected bacteria were inoculated into LB liquid medium, and the fungi were inoculated into PDA liquid medium, and cultured at 5°C, 160 r / min with shaking until OD 600 reached 1. The bacterial solution was re-inoculated into LB and PDA liquid medium at 1% inoculum, and cultured at 5°C, 160 r / min with shaking.

[0093] 1) Bacterial growth curve determination (turbidimetric method) [Chen Yuqi, 2020]

[0094] 2) Fungal growth curve determination (dry weight method)

[0095] Each fungus strain was inoculated with 7 bottles of liquid culture medium. Starting from the 3rd day, one bottle of liquid culture medium was taken every 24 hours, the fungus bodies were filtered out with filter paper, and then the filter paper and the fungus were placed in a 100℃ oven to dry.

[0096] Set up a blank group to measure the weight loss of the filter paper during the drying process. Take 3 pieces of filter paper as the blank group, wet them with deionized water, place them in a 100℃ oven, and dry them for the same time as the experimental group.

[0097] The growth curve was drawn using the dry weight of the strain every 24 hours after 3 days until the curve flattened out.

[0098] 42) Determination of lignin degradation rate

[0099] The alkaline lignin determination method was used [He Pengliang, 2021].

[0100] 43) Determination of ligninase activity

[0101] The strain obtained in the initial screening was made into a seed suspension, inoculated into the enzyme production medium at an inoculum of 3%, and cultured at a constant temperature of 15°C and 160r / min with shaking. Then, 5 mL of the bacterial solution was taken into a centrifuge tube at 3, 6, 9, 12, 15, and 18 days, centrifuged at 10000r / min for 8 minutes, and the supernatant was taken to determine the enzyme activity. The crude enzyme solution in a boiling water bath for 10 minutes was used as a control.

[0102] 1) Lac enzyme activity determination - using the ABTS method [Yu Hao, 2022].

[0103] 2) Lip enzyme activity determination - using resveratrol method [Yu Hao, 2022].

[0104] 3) Mnp enzyme activity determination - using the DMP method [Zhang Sainan, 2022].

[0105] Cellulase activity assay

[0106] The glucose content method [Zhang Bizhou, 2020] was used to react the cellulase activity.

[0107] 44) Hemicellulase activity assay

[0108] The xylan content method [Zhang Bizhou, 2020] was used to react hemicellulase activity.

[0109] 45) Filter paper disintegration

[0110] The bacterial solution was inoculated into the filter paper disintegration medium at a 3% inoculum volume, and cultured at 15°C and 160 r / min for 20 days. The disintegration of the filter paper strips was observed at 10 and 20 days.

[0111] Example 2

[0112] Strain identification

[0113] 1) Morphological identification

[0114] Bacteria were observed under an oil immersion lens after Gram staining, and fungi were observed directly under a microscope.

[0115] 2) Molecular identification

[0116] Bacterial 16S rDNA and fungal ITS DNA were amplified by PCR, and the products were sent to Bioengineering for sequencing. The obtained sequences were compared with the NCBI database by Blast, and the gene sequences with higher homology were selected to construct a phylogenetic tree using MEGAX software, and comparative analysis was performed based on the phylogenetic tree.

[0117] The K10 sequence is SEQ ID No 1:

[0118]

[0119]

[0120] The sequence of F16 is SEQ ID No 2:

[0121]

[0122]

[0123] The sequence of F25 is SEQ ID No. 3:

[0124]

[0125] Example 3

[0126] Construction of composite bacterial strain

[0127] 1) Antagonism assay

[0128] Bacteria were inoculated on LB medium using the cross-streaking method, and fungi were inoculated on LB medium using the dot-seeding method. After culturing at 15°C for 3 days, the growth of colonies at the intersections was observed, and composite bacterial agents were prepared for strains without antagonism.

[0129] 2) Determination of enzyme activity of compound bacterial agent

[0130] According to the strains screened in the re-screening that have certain degradation abilities for lignin, cellulose, and hemicellulose, four combinations were obtained: F25+F16+F10, F25+F16+K10, F25+F16+K14, and all (F25+F16+F10+K10+K14). The inoculation amount was 3% of the total volume and the enzyme activity of the composite bacteria was determined. The composite bacteria with the best enzyme production effect were screened out, and the determination method was consistent with Example 1.

[0131] Table 1 Grouping of lignocellulose degradation ability of strains in the repeated screening

[0132]

[0133]

[0134] 3) Enzyme production optimization

[0135] 31) Optimal bacteria ratio

[0136] The bacteria ratio was set to 29 levels as shown in Table 2. Other conditions were 15°C, 160 r / min, carbon source, nitrogen source, salinity, inoculation size, and pH, all of which were optimal. Five enzyme activities were measured and repeated three times to determine the optimal bacteria ratio.

[0137] Table 2 Bacterial ratio of compound bacteria

[0138]

[0139] 4) Indoor agricultural and forestry waste degradation

[0140] The composite bacteria were inoculated at 3% in the agricultural and forestry waste degradation medium of poplar leaves, poplar branches, peach branches and rice, and cultured at 15℃ and 160r / min for 15 days. The agricultural and forestry waste degradation medium without bacteria was used as the blank group, and the determination method was the same as that of the experimental group.

[0141] 41) Growth curve

[0142] The OD value of the agricultural and forestry waste degradation culture medium at 600 nm was measured every 3 days, with 3 replicates each time, and a growth curve was drawn.

[0143] 42) pH

[0144] The pH of the agricultural and forestry waste degradation medium was measured every 3 days, with 3 replicates each time.

[0145] 43) Weight loss rate

[0146] The weight loss rate was measured every 3 days, with 3 replicates each time. The waste was washed repeatedly with dilute hydrochloric acid to remove the bacteria and CaCO3 precipitation attached to the waste, and then dried at 100℃ to constant weight, and the degradation rate of the waste was calculated.

[0147] Agricultural and forestry waste degradation rate Y = (m0-m1) / m0×100%

[0148] m0 is the weight of agricultural and forestry waste before degradation (g); m1 is the weight of agricultural and forestry waste after degradation (g).

[0149] 44) Scanning electron microscope

[0150] The agricultural and forestry wastes at 0 days and 15 days old were scanned by electron microscope to compare the degree of damage of the agricultural and forestry wastes after 15 days of degradation.

[0151] Example 4

[0152] Test results

[0153] 1. Initial screening

[0154] 1) Guaiacol plate color test, the results are shown in Figure 2 .

[0155] 2) MnCl2 plate color test, the results are shown in Figure 3

[0156] 3) Coomassie brilliant blue plate decolorization test, the results are shown in Figure 4

[0157] 4) Congo red staining test, the results are shown in Figure 5

[0158] By enriching and acclimating the samples at 15°C, separating and purifying, 93 strains were obtained. These 93 strains were respectively applied on 5 primary screening media to obtain the following Figure 2-5 In the guaiacol plate color test, brown circles appeared in F16 and F26, and brown circles appeared in F25 in the MnCl2 plate color test. No strains with transparent circles appeared in the aniline blue decolorization test. In the Coomassie brilliant blue decolorization test, transparent circles appeared in K17, K28-1, F9-2, F11-1, and D10. In the Congo red staining test, 21 strains showed decolorization reactions. According to the measured D / d values ​​(as shown in Table 3), the strains with better decolorization were selected: F9-1, D10 green (1), D10 white (1), K14, F10, F18, W1, F21, and F4.

[0159] Based on the results of the five primary screening tests, F21, F26, K10, K14, F10, F25, F16, and D10 were selected for rescreening tests.

[0160] Table 3 Congo red staining test D / d ratio results

[0161]

[0162]

[0163] 2. Rescreening

[0164] 1) 5℃ low temperature screening

[0165] The 8 strains obtained from the initial screening were rescreened at 5°C to select strains that can grow at lower temperatures. Figure 6 and Figure 7 It can be seen that F26, K10, K14, F10, F16, F21, and F25 can also grow at 5°C, and these 7 strains were used for subsequent rescreening tests.

[0166] 2) Determination of lignin degradation rate

[0167] Figure 8 Correlation coefficient R 2 The value of is greater than 0.99, so the reliability is high, and the alkaline lignin content can be calculated. Fig. 9 After 20 days of cultivation, the degradation rates of alkaline lignin by K14 and F16 were the highest at 33.00% and 28.86%, respectively, while the degradation rates of the other strains were between 6.00 and 19.58%.

[0168] 3. Enzyme Activity Assay

[0169] 1) Lac enzyme activity

[0170] The Lac enzyme activity was determined by the ABTS method. Fig.10 It can be seen that among the 7 strains, F25 has the highest enzyme activity, with an enzyme activity of 3.62U at 3d, but the enzyme activity dropped to 0U at 4d, and the stability is poor. The second highest enzyme activity is K10, which reaches a maximum of 3.33U at 9d. Compared with F25, K10 has better stability in producing Lac enzymes, producing Lac enzymes from 2-14d. F10 and F16 produce enzyme activity at 2d and 3d respectively and last until 18d, and both reach the highest values ​​of 1.79U and 1.48U at 9d. K14 has low enzyme activity before 12d, and reaches a peak of 1.29U at 15d. The peak enzyme activities of F21 and F26 are not high, and the sustainability is poor.

[0171] 2) LiP enzyme activity

[0172] The resveratrol method was used to determine the LiP enzyme activity. Fig.11 It can be seen that among the 7 strains, F10 has the highest enzyme activity, reaching 57.92U at 2d, and the enzyme activity decreased to 0U at 9d. The changes in the Lip enzyme activity of K14, F16, K10, F21, F26, and F25 were not obvious, and the enzyme activity was low.

[0173] 3) MnP enzyme activity

[0174] The MnP enzyme activity was determined by the DMP method. Fig.12 It can be seen that among the 7 strains, F25 has the highest enzyme activity, which is 12.30U. However, this strain produces MnP enzyme at 10d, and the enzyme production time is relatively late, reaching the peak at 15d, and the enzyme activity disappears at 18d, and the enzyme production time is also short. Although the highest enzyme activity values ​​of K10 are similar to those of F10, F26, and F21, all between 5.13-6.81U, K10 reaches the peak faster than other strains, and produces MnP enzyme from 1-18d, with excellent enzyme production continuity. K14 and F16 have low MnP enzyme activities.

[0175] 4) Cellulase activity

[0176] The cellulase activity was determined by the glucose content method. Fig.13 It can be seen that the enzyme activities of F25, F16 and F21 among the 7 strains are better than those of the other 4 strains, all reaching peak values ​​at 2 days and lasting for a long period, but the peak value of F16 is higher than that of F25 and F21, with the highest enzyme activity of 5.46U / mL. The enzyme activity of K10, F10, F26 and K14 did not fluctuate significantly, and the enzyme activity was relatively low.

[0177] 5) Hemicellulase activity

[0178] Hemicellulase activity was determined by the xylan content method. Fig.14 It can be seen that among the 7 strains, F25 and F21 have the highest enzyme activities, reaching a peak at 4 days, with enzyme activities of 3.26 and 3.23 U / mL respectively, and the enzyme activities continued to drop to 0 U / mL after 6 days. The next highest enzyme activities are F10, K14 and F26, with maximum enzyme activities between 1.58-1.68 U / mL, but F10 can produce xylanase from 1 to 9 days, so the enzyme production period of F10 is relatively long. The enzyme activities of K10 and F16 do not fluctuate significantly, and the enzyme activities are relatively low. Lac, LiP, and MnP enzymes are all enzymes that degrade lignin. Fig.15 It can be seen that the strains with better lignin degradation ability are F25, F10, and K10, the strain with better cellulose degradation ability is F16, and the strain with better hemicellulose degradation ability is F25.

[0179] 4. Filter paper disintegration test

[0180] Filter paper contains lignin, cellulose and hemicellulose, so the degree of filter paper disintegration can reflect the ability of the strain to degrade lignin.

[0181] like Fig.16As shown in the figure, under the same conditions, no strain was added to the blank group. It can be seen that none of the three filter paper strips were damaged or broken, and only slight paper scraps floated on the water surface. Compared with the blank group, the most obvious disintegration effect of filter paper strips was F21 and F25. At 10 days, all three filter paper strips of F25 were broken, and two filter paper strips of F21 were broken, and one was obviously damaged. At 20 days, all three filter paper strips of F25 and F21 were broken into smaller paper segments. Next were K10 and F16. When one filter paper of K10 was obviously damaged at 10 days, there was no obvious change in F16. At 20 days, two filter paper strips of K10 and F16 were broken, and the paper segment of K10 was smaller. F26, F10 and K14 did not change at 10 days and 20 days.

[0182] Based on the above, the strains with better lignin degradation ability were selected as F25, F10, K10 and K14, the strain with better cellulose degradation ability was F16, and the strain with better hemicellulose degradation ability was F25. These five strains were subjected to subsequent experiments.

[0183] 5. Identification of bacterial species

[0184] 1) Morphological identification

[0185] The results of Gram staining of the three bacterial strains are as follows: Fig.17 F16, spherical, Gram-negative bacteria. After staining the two fungi, the results were as follows Fig.18 As shown in the figure, the spores of K10 are oblong and cylindrical with blunt ends. The hyphae of F25 have no septa and rhizoids, and the spores are spherical.

[0186] 2) Molecular identification

[0187] The sequences obtained by sequencing were aligned by Blast on NCBI, and then the developmental tree was constructed using MEGAX software. The results are as follows Fig.19 The identification results showed that F16 was Acinetobacter, F25 was Aspergillus parakaife, and K10 was Geotrichum candidum, as shown in Table 4.

[0188] Table 4 Summary of species of degradation strains

[0189]

[0190] Example 5

[0191] Construction of composite bacterial agent

[0192] The orthogonal method was used to develop the composite microbial agent (the specific process is omitted). The results showed that the combinations with better Lac enzyme production ability in the comprehensive test group were F25+F16+K14 and F25+F16+K10, the combinations with better LiP enzyme production ability were F25+F16+K10, the combinations with better MnP enzyme production ability were F25+F16+K10, the combinations with better cellulase production ability were F25+F16+F10 and F25+F16+K10, and the ability to produce hemicellulase was average. Therefore, the combination F25+F16+K10 with better comprehensive ability was selected for subsequent experiments.

[0193] The ratio of the composite bacteria F16:F25:K10 was adjusted to 1:1:4, 1:2:3, 1:3:2, 1:4:1, 2:1:3, 2:2:2, 3:1:2, 3:2:1, 4:1:1, and the other conditions were the same as those in Example 3. The changes in the activities of the five enzymes in the composite bacteria were continuously measured within 6 days. The results are shown in FIG. Fig. 20 The redder the color, the higher the enzyme activity. The bacterial solution ratio is a volume ratio, and the bacterial concentrations of each bacterial solution are adjusted to be the same before compounding.

[0194] Depend on Fig. 20 It can be seen that the color of LiP in the ratio 1:1:4 is red. The color of LiP, MnP and Lac in the ratio 1:2:3 is red. The color of Lac in the ratio 1:3:2 is red. The color of MnP and cellulase in the ratio 1:4:1 is red. The color of no red in the ratio 2:1:3. The color of LiP and MnP in the ratio 2:2:2 is red. The color of Lac and cellulase in the ratio 2:3:1 is red. The color of LiP and Lac in the ratio 3:1:2 is red. The color of MnP, Lac, cellulase and hemicellulase in the ratio 3:2:1 and the ratio 4:1:1 are all red. From the perspective of the types of enzyme activity optimized by different bacterial ratios, the types of enzyme activity optimized by the ratio 3:2:1 and the ratio 4:1:1 are more. From the effect of different bacterial ratios on optimizing the same enzyme activity, the ratios of 1:1:4 and 1:2:3 have better optimization effects on LiP, 1:2:3, 2:2:2 and 3:2:1 have better optimization effects on MnP, 3:2:1 has better optimization effects on Lac, 1:4:1, 3:2:1 and 4:1:1 have better optimization effects on cellulase, and 4:1:1 has better optimization effects on xylanase. In summary, the optimal bacterial ratio of the composite bacterial agent F16:F25:K10 is 3:2:1.

[0195] Example 6

[0196] Indoor agricultural and forestry waste degradation experiment

[0197] 1) Growth curve

[0198] Fig.21 It is the absorbance value (600nm) measured every 3 days of the composite bacterial system, which can indicate the growth of the bacterial system. Fig.21 It can be seen that the growth curves of poplar branches, peach branches, poplar leaves, and rice composite microbial agents were basically the same before 9 days, all of which rose slowly. After 9 days, the growth curve of poplar leaves grew rapidly, reaching a peak at 12 days, and then rapidly declined, indicating that it entered a weak period due to the reduction of substrates in the late growth period. The growth curves of the other three agricultural and forestry wastes changed slowly, indicating that peach branches, poplar branches, and rice were difficult to be used by composite microbial agents as the only carbon source.

[0199] 2) Fig. 22 These are the test groups after the four types of agricultural and forestry wastes were inoculated with composite bacteria and their corresponding blank groups. The pH of the bacterial solution changed every 3 days. The dynamic changes in pH value illustrate the regulation of the bacterial system.

[0200] Depend on Fig. 22 It can be seen that the pH of the three experimental groups of peach branches, poplar leaves and rice continued to rise before 6 days, and finally stabilized at neutral. The initial pH of the culture medium of poplar branches was relatively acidic. After 15 days of culture, although the pH of the culture medium was not neutral, it also increased. Although the pH value of the corresponding control group remained acidic, there was no obvious change.

[0201] 3) Weight loss rate test

[0202] Fig.23 is the change in degradation rate of agricultural and forestry waste with the increase of cultivation days, Fig.23 It can be seen that the degradation rate of agricultural and forestry waste increases with the increase of cultivation time, and the degradation rate of agricultural and forestry waste with the addition of exogenous bacteria is always significantly higher than that of the control group. After 15 days of cultivation, the degradation rates of agricultural and forestry waste of peach branches, poplar leaves, poplar branches, and rice reached 23%, 42%, 39%, and 26%, respectively, and the corresponding degradation rates of the control group were 8%, 14%, 13%, and 9%. The degradation rate of agricultural and forestry waste by the composite bacteria system was about 3 times that of the control group. Throughout the process, the degradation rate of poplar leaves was always higher than that of other groups, and the improvement rate was significant, indicating that the degradation effect of poplar leaves was better.

[0203] 4) Electron microscopy observation

[0204] Fig.24The following are the images of different agricultural and forestry wastes after degradation under scanning electron microscope (3000×, the left picture is the test group, the right picture is the control group). The commercial strain Phanerochaete chrysosporium Burdsall was used as the control group. The scanning electron microscope results show that after 15 days of degradation, the agricultural and forestry wastes in the control group have a relatively complete morphology, orderly fiber tissue, smooth surface, and no obvious damage. After inoculation with the composite bacterial agent, the fiber tissue surface of peach branches (3-12-B), rice (3-12-C), and poplar branches (3-12-D) is rough and arranged in a messy manner, with obvious fractures. The surface of the peach branches is loose and porous, showing a honeycomb shape. The surface morphology of the poplar leaves (3-12-A) is the most severely damaged, with some parts in the form of fine strips and some in the form of fragments, arranged in a messy manner. This result shows that the composite bacterial agent can destroy the lignocellulose structure of the above four agricultural and forestry wastes, and has a better degradation effect on poplar leaves.

[0205] Finally, it should be noted that the protection scope of the present invention is not limited to the above embodiments. Ordinary technicians in this field should understand that they can still modify the technical solutions recorded in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents; and these modifications or replacements will fall within the protection scope of the present invention.

Claims

1. A strain of Aspergillus niger that can degrade lignin and cellulose at low temperature ( Aspergillus keveioides ) F25 strain, characterized in that The Aspergillus fusiformis F25 strain is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the deposit date is January 25, 2024, and the deposit number is: CGMCC No.41052.

2. Use of the Aspergillus fumigatus F25 strain as claimed in claim 1 in the degradation of agricultural and forestry wastes.

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