A composite bacterial agent capable of degrading lignin and cellulose at low temperature and its application

Abstract

The invention belongs to the field of microorganisms, and specifically discloses a composite bacterial agent capable of degrading lignin and cellulose at low temperature and application thereof; the bacterial agent consists of Geotrichum candidum K10, Aspergillus pseudokaife F25 and Acinetobacter F16, and the preservation numbers thereof are CGMCC No.41053, CGMCC No.41052 and CGMCC No.29760 respectively; each bacterium in the bacterial agent has a synergistic effect, and the whole bacterial agent has the ability to degrade lignin and cellulose at low temperature, and can be used for degrading agricultural and forestry wastes at low temperature.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and specifically discloses a composite bacterial agent capable of degrading lignin and cellulose at low temperature and application thereof. Background Art

[0002] According to statistics, my country's straw production in 2021 was about 900 million tons (Han Zhe, Pan Kai. Returning straw 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 will lead to a 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 the utilization rates are 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 as organic fertilizer to improve soil fertility and increase production and income. Returning straw to the field can increase the content of soil organic matter, but because the degradation rate of straw after returning to the field is slow and the C\N is high, it competes with grain for nitrogen and leads to an increase in crop diseases and insect 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 a Geotrichum candidum K10 strain capable of degrading lignin and cellulose at low temperature and application thereof.

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

[0008] A composite bacterial agent capable of degrading lignin and cellulose at low temperature; the bacterial agent is composed of Geotrichum candidum K10, Aspergillus pseudokaife F25 and Acinetobacter F16, and the deposit numbers thereof are respectively:

[0009] 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.

[0010] 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.

[0011] 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.

[0012] Furthermore, the present invention discloses the application of the composite bacterial agent in the degradation of agricultural and forestry wastes.

[0013] Furthermore, the present invention also discloses a method for screening the strains in the composite bacterial agent, comprising the following steps:

[0014] 1) Sampling: animal feces and peach tree branches and leaves from Beijing Zoo;

[0015] 2) Enrichment and acclimatization: the sample is placed in acclimatization medium for acclimatization;

[0016] 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;

[0017] 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;

[0018] 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;

[0019] 6) Identification: The final strain is subjected to morphological and molecular identification.

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

[0021] Furthermore, the above-mentioned composite bacterial agent is used in the degradation of agricultural and forestry wastes, wherein the agricultural and forestry wastes come from poplar leaves.

[0022] The present invention has the following beneficial effects:

[0023] The invention discloses a composite bacterial agent capable of degrading lignin and cellulose at low temperature and application thereof; the bacterial agent is composed of Geotrichum candidum K10, Aspergillus pseudokaficifolius F25 and Acinetobacter F16, and the composite bacterial agent composed of the Geotrichum candidum K10, Aspergillus polydodecene F25 and Acinetobacter F16 strains has the ability to degrade lignin and cellulose at low temperature, and can be used for fermentation and degradation of agricultural and forestry wastes at low temperature.

[0024] Biomaterial deposit information:

[0025] 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.

[0026] 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.

[0027] 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

[0028] Figure 1 It is a schematic diagram of the overall screening and identification technology route of the present invention;

[0029] Figure 2 This is the Gram staining image of three bacterial strains under oil microscope;

[0030] Figure 3 The morphological pictures of two fungi under microscope;

[0031] Figure 4 For the construction of developmental trees;

[0032] Figure 5 This is a graph of the antagonism test of degrading bacteria;

[0033] Figure 6 It is the Lac enzyme activity test of each bacterial agent;

[0034] Figure 7 It is the LiP enzyme activity test of each bacterial agent;

[0035] Figure 8 To test the MnP enzyme activity of each bacterial agent;

[0036] Fig. 9To test the cellulase activity of each bacterial agent;

[0037] Fig.10 To test the xylanase activity of each bacterial agent;

[0038] Fig.11 The figure shows the effect of different carbon sources on enzyme activity;

[0039] Fig.12 The figure shows the effect of different nitrogen sources on enzyme activity;

[0040] Fig.13 The effect of different salinities on enzyme activity is shown in the figure;

[0041] Fig.14 This is the effect of different inoculation amounts on enzyme activity;

[0042] Fig.15 The effect of different temperatures on enzyme activity;

[0043] Fig.16 The effect of different pH on enzyme activity;

[0044] Fig.17 This is a graph showing the effect of different bacteria on enzyme activity;

[0045] Fig.18 This is the growth curve of the composite bacterial agent in the degradation of agricultural and forestry wastes;

[0046] Fig.19 This is a graph showing the pH changes of the culture medium during the degradation of agricultural and forestry wastes;

[0047] Fig. 20 This is a comparison chart of the weight loss rates of four types of agricultural and forestry wastes at 15 days;

[0048] Fig.21 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

[0049] 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.

[0050] 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.

[0051] like Figure 1 Shown is a schematic diagram of the overall screening and identification technology route of the present invention.

[0052] Test materials

[0053] 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).

[0054] Culture medium

[0055] Acclimation medium: 3 g alkaline lignin, (NH 4 ) 2 SO 4 2g, NaCl 1.5g, KH 2 PO 4 1g, K 2 HPO 4 0.3 g, MgSO 4 7H 2 O 0.2g, add water to 1000mL, pH 7.0

[0056] LB medium: 5.0 g yeast powder, 10.0 g tryptone, 10.0 g NaCl, add water to 1000 mL, pH 7.0

[0057] 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

[0058] 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

[0059] 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

[0060] LB-manganese chloride medium: yeast powder 5.0g, tryptone 10.0g, NaCl 10.0g, MnCl 2 ·4H 2 O 2 0.15g, agar 20.0g, add water to 1000mL, pH 7.0

[0061] 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

[0062] PDA-guaiacol medium: 5.0 g potato powder, 20.0 g glucose, 20.0 g agar, add water to 1000 mL, pH 7.0

[0063] PDA-aniline blue medium: potato powder 5.0g, glucose 20.0g, agar 20.0g, add water to 1000mL, pH 7.0

[0064] 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

[0065] PDA-manganese chloride medium: potato powder 5.0g, glucose 20.0g, agar 20.0g, add water to 1000mL, pH 7.0

[0066] CMC-Na medium: CMC-Na 5.0-10 g, (NH 4 ) 2 SO 4 1.0g, Na 2 HPO 4 0.9 g, MgSO 4 7H 2 O0.5g, NaCl 0.5g, agar powder 20.0g, add water to 1000mL, pH 7.0

[0067] Lignin degradation medium: alkaline lignin 2g, (NH 4 ) 2 SO 4 2 g, NaCl 1.5 g, KH 2 PO 4 1g, K 2 HPO 4 1g, MgSO 4 7H 2 O 0.2g, add water to 1000mL, pH 7.0

[0068] Filter paper disintegration medium: filter paper (1×4 cm, 3 strips per bottle), (NH 4 ) 2 SO 4 2.0g, KH 2 PO 4 1g, NaCl 0.5g, MgSO4 7H 2 O 0.5g, NaNO 3 1.0g, peptone 0.5g, yeast powder 0.5g, add water to 1000mL, pH 7.0

[0069] 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

[0070] Reagents

[0071] Congo red indicator, NaCl decolorizing solution, 0.5mmol / L ABTS, 100mmol / L acetic acid-sodium acetate buffer, 10mmol / L resveratrol solution, 250mmol / L tartaric acid-sodium tartrate buffer, 10mmol / L H 2 O 2 、1.6mmol / LMnSO 4 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 Gelred nucleic acid dye, 18% hydrochloric acid.

[0072] Example 1

[0073] Acclimation and screening of low-temperature lignocellulose-degrading bacteria

[0074] 1) Enrichment and domestication

[0075] 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.

[0076] 2) Separation and purification

[0077] 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 -2Dilution. 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.

[0078] 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.

[0079] 3) Initial screening

[0080] 1) Guaiacol plate colorimetric experiment - the ability of strains to produce Lac [Qi Yeqiang, 2021]

[0081] 2) Aniline blue plate decolorization experiment - the ability of strains to produce LiP and MnP [Qi Yeqiang, 2021]

[0082] 3) Manganese chloride plate colorimetric test - the ability of strains to produce MnP [Qi Yeqiang, 2021]

[0083] 4) Congo red staining experiment - the ability of strains to produce cellulase [Bai Changsheng, 2022]

[0084] 5) Coomassie Brilliant Blue Plate Decolorization Test - Cellulase Production Ability of the Strain

[0085] 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.

[0086] 4) Rescreening

[0087] 41) 15℃ low temperature screening

[0088] 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.

[0089] 1) Bacterial growth curve determination (turbidimetric method) [Chen Yuqi, 2020]

[0090] 2) Fungal growth curve determination (dry weight method)

[0091] 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.

[0092] 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.

[0093] The growth curve was drawn using the dry weight of the strain every 24 hours after 3 days until the curve flattened out.

[0094] 42) Determination of lignin degradation rate

[0095] The alkaline lignin determination method was used [He Pengliang, 2021].

[0096] 43) Determination of ligninase activity

[0097] 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.

[0098] 1) Lac enzyme activity determination - using the ABTS method [Yu Hao, 2022].

[0099] 2) Lip enzyme activity determination - using resveratrol method [Yu Hao, 2022].

[0100] 3) Mnp enzyme activity determination - using the DMP method [Zhang Sainan, 2022].

[0101] Cellulase activity assay

[0102] The glucose content method [Zhang Bizhou, 2020] was used to react the cellulase activity.

[0103] 44) Hemicellulase activity assay

[0104] The xylan content method [Zhang Bizhou, 2020] was used to react hemicellulase activity.

[0105] 45) Filter paper disintegration

[0106] 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.

[0107] Example 2

[0108] Strain identification

[0109] 1) Morphological identification

[0110] Bacteria were observed under an oil immersion lens after Gram staining, and fungi were observed directly under a microscope.

[0111] 2) Molecular identification

[0112] Bacterial 16S rDNA and fungal ISDNA 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.

[0113] The K10 sequence is SEQ ID No 1:

[0114]

[0115] The sequence of F16 is SEQ ID No 2:

[0116]

[0117] The F25 sequence is SEQ ID No. 3:

[0118]

[0119]

[0120] Example 3

[0121] Construction of composite bacterial strain

[0122] 1) Antagonism assay

[0123] 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.

[0124] 2) Determination of enzyme activity of compound bacterial agent

[0125] 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.

[0126] Table 1 Grouping of lignocellulose degradation ability of strains in the repeated screening

[0127]

[0128] 3) Enzyme production optimization

[0129] 31) Optimal carbon source

[0130] The carbon source was set to 5 levels of glucose, yeast powder, sucrose, reed straw powder and mixture. Other conditions were 15°C, 160r / min, inoculation amount of 3%, nitrogen source of peptone, salinity of 1%, and pH of 7.0. Five enzyme activities were measured and repeated 3 times to determine the optimal carbon source.

[0131] 32) Optimal nitrogen source

[0132] The nitrogen source was set to 5 levels of peptone, urea, ammonium sulfate, ammonium nitrate, and mixed. Other conditions were 15°C, 160 r / min, 3% inoculation amount, yeast powder as carbon source, 1% salinity, and pH 7.0. Five enzyme activities were measured and repeated 3 times to determine the optimal nitrogen source.

[0133] 33) Optimum salinity

[0134] The salinity was set to 5 levels of 1, 2, 3, 4, and 5%. Other conditions were 15°C, 160 r / min, 3% inoculation, yeast powder as carbon source, peptone as nitrogen source, and pH 7.0. The activities of 5 enzymes were measured and repeated 3 times to determine the optimal salinity.

[0135] 34) Optimum pH

[0136] The pH was set to 5.0, 6.0, 7.0, 8.0, and 9.0. The other conditions were 15°C, 160 r / min, 3% inoculation, and the carbon source, nitrogen source, and salinity were all optimal. Five enzyme activities were measured and repeated 3 times to determine the optimal pH.

[0137] 35) Optimum temperature

[0138] The temperature was set to 6 levels of 5, 15, 25, 35, 45, and 55°C. Other conditions were 160 r / min, 3% inoculation, and the carbon source, nitrogen source, salinity, and pH were all optimal. Five enzyme activities were measured and repeated 3 times to determine the optimal temperature.

[0139] 36) Optimal inoculum size

[0140] The inoculation amount was set to 5 levels of 1, 2, 3, 4, and 5%. Other conditions were 15°C, 160 r / min, carbon source, nitrogen source, salinity, and pH, all of which were optimal for measuring 5 enzyme activities. The test was repeated 3 times to determine the optimal inoculation amount.

[0141] 37) Optimal Bacteria Ratio

[0142] 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.

[0143] Table 2 Bacterial ratio of compound bacteria

[0144]

[0145]

[0146] 4) Indoor agricultural and forestry waste degradation

[0147] 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.

[0148] 41) Growth curve

[0149] 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.

[0150] 42) pH

[0151] The pH of the agricultural and forestry waste degradation medium was measured every 3 days, with 3 replicates each time.

[0152] 43) Weight loss rate

[0153] The weight loss rate was measured every 3 days, with 3 replicates each time. The samples were repeatedly washed with dilute hydrochloric acid to remove the bacteria and CaCO attached to the agricultural and forestry waste. 3 The sediment was dried at 100℃ to constant weight, and the degradation rate of agricultural and forestry waste was calculated.

[0154] Degradation rate of agricultural and forestry waste Y = (m 0 -m1 ) / m 0 ×100%

[0155] m 0 Weight of agricultural and forestry waste before degradation (g); m 1 Weight of agricultural and forestry waste after degradation (g).

[0156] 44) Scanning electron microscope

[0157] 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.

[0158] Example 4

[0159] Test results

[0160] 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.

[0161] 1. Identification of bacterial species

[0162] 1) Morphological identification

[0163] The results of Gram staining of the three bacterial strains are as follows: Figure 2 F16, spherical, Gram-negative bacteria. After staining the two fungi, the results were as follows Figure 3 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.

[0164] 2) Molecular identification

[0165] 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 Figure 4 The identification results showed that F16 was Acinetobacter, K14 was Bacillus glutamicum, and K10 was Geotrichum candidum, as shown in Table 4.

[0166] Table 4 Summary of species of degradation strains

[0167]

[0168] 2. Construction of composite bacterial agents

[0169] 1) Antagonism test

[0170] The 7 degradation strains were subjected to plate antagonism experiments. If there was no inhibition zone between the bacteria, it indicated that there was no antagonism between them. Figure 5 It can be seen that all 7 strains have no antagonistic effect.

[0171] Phanerochaete chrysosporium has a good degradation effect on lignocellulose, so the commercial bacterial agent of Phanerochaete chrysosporium was used as a control group for comparison with the test group, and the test group was compounded according to Table 1.

[0172] 2) Lac enzyme activity test

[0173] The Lac enzyme activity was determined by the ABTS method. Figure 6 It can be seen that the ability of the four test groups to produce Lac enzymes is stronger than that of Phanerochaete chrysosporium, and the enzyme activity is between 1.42-3.76U, reaching a peak at 2 days. Among them, the enzyme activity of F25+F16+K14 is the highest at 3.76U, and the enzyme production stability of F25+F16+K10 is the best.

[0174] 3) LiP enzyme activity test

[0175] The LiP enzyme activity was determined by the resveratrol method. Figure 7 It can be seen that the ability of the four experimental groups to produce LiP enzymes is stronger than that of Phanerochaete chrysosporium, among which F25+F16+K10 has the highest enzyme activity, reaching 47.41 U at 5 days. The changes in the activity of LiP enzymes in other experimental groups were not obvious, and the enzyme activity was relatively low.

[0176] 4) MnP enzyme activity test

[0177] The MnP enzyme activity was determined by the DMP method. Figure 8 It can be seen that the MnP enzyme activity of the four test groups is better than that of Phanerochaete chrysosporium. F25+F16+K10 and F25+F16+F10 have the highest enzyme activity, but F25+F16+K10 has a long enzyme production cycle and good stability, reaching a peak of 14.36U at 6 days, and F25+F16+F10 reaches a maximum enzyme activity of 14.37U at 5 days, but the enzyme activity of this strain drops rapidly after the peak. The ability of the other two groups to produce MnP enzymes is average, and the floating changes are small.

[0178] 5) Cellulase activity test

[0179] The cellulase activity was determined by the glucose content method. Fig. 9 It can be seen that the ability of the four test groups to produce cellulase is significantly better than that of Phanerochaete chrysosporium. The enzyme activity of F25+F16+F10 is the highest at 4.47U / mL, and the ability to produce cellulase is stable from 1 to 6 days. Among the other three groups, F25+F16+K10 has the highest enzyme activity, which is 4.05U / mL.

[0180] 6) Hemicellulase activity test

[0181] Hemicellulase activity was determined by the xylan content method. Fig.10It can be seen that the ability of Phanerochaete chrysosporium to produce xylanase is better than that of the test group, with a peak enzyme activity of 2.02U / mL. The peak enzyme activity of F25+F16+K10 in the test group reached 1.52U / mL, but the enzyme activity only lasted for 2 days, with poor sustainability. The maximum enzyme activity of the other three groups ranged from 0.68 to 0.94U / mL.

[0182] In the comprehensive test group, the combinations with better Lac enzyme production ability are F25+F16+K14 and F25+F16+K10, the combinations with better LiP enzyme production ability are F25+F16+K10, the combinations with better MnP enzyme production ability are F25+F16+K10, the combinations with better cellulase production ability are F25+F16+F10 and F25+F16+K10, and the ability to produce hemicellulase is average. Therefore, the combination F25+F16+K10 with better comprehensive ability was selected for subsequent tests.

[0183] 3. Enzyme production optimization

[0184] 1) Determination of the optimal carbon source

[0185] Yeast powder, sucrose, glucose, reed, and C mixture were used as the sole carbon source of the culture medium, and other conditions were the same as those in Example 3. The changes in the activities of the five enzymes in the composite bacterial agent were continuously measured within 6 days. The results are shown in Fig.11 The redder the color, the higher the enzyme activity.

[0186] Depend on Fig.11 It can be seen that the colors corresponding to yeast powder are red for LiP, Lac, and cellulase. The colors corresponding to sucrose are all blue, indicating that sucrose cannot optimize the composite bacterial agent. The color corresponding to glucose is red for xylanase, the color corresponding to reed is red for LiP, and the color corresponding to C mix is ​​red for LiP and cellulase. From the perspective of the types of enzyme activities optimized by different carbon sources, yeast powder optimizes more types of enzyme activities. From the perspective of the effect of different carbon sources on optimizing the same enzyme activity, yeast powder has better effects on LiP, Lac, and cellulose. Therefore, the most suitable carbon source is yeast powder.

[0187] 2) Determination of the optimal nitrogen source

[0188] With peptone, urea, ammonium sulfate, ammonium nitrate, and N mixed as the only nitrogen source of the culture medium, and other conditions consistent with those in Example 3, the changes in the activities of the five enzymes in the composite bacterial agent were continuously measured within 6 days. Fig.12 The redder the color, the higher the enzyme activity.

[0189] Depend on Fig.12It can be seen that in the corresponding colors of peptone, LiP and cellulase are red, and Lac is light blue. The corresponding colors of urea are all blue, indicating that urea cannot optimize the composite bacterial agent. In the corresponding colors of ammonium sulfate, LiP and Lac are red, and MnP, cellulase and xylanase are light blue. In the corresponding colors of ammonium nitrate, LiP and Lac are red. In the corresponding colors of C mix, LiP is red, and Lac, cellulase and xylanase are light blue. From the perspective of the types of enzyme activity optimized by different nitrogen sources, there are more types of enzyme activity optimized by peptone, ammonium sulfate and ammonium nitrate. From the perspective of the effect of different nitrogen sources on optimizing the same enzyme activity, peptone has a better optimization effect on LiP and cellulase, and ammonium sulfate has a better optimization effect on LiP, MnP, Lac and xylanase. Therefore, the optimal nitrogen source is ammonium sulfate.

[0190] 3) Determination of the optimum salinity

[0191] The salt concentration of the culture medium was 1%, 2%, 3%, 4%, and 5%, respectively. The other conditions were the same as those in Example 3. The changes in the activities of the five enzymes in the composite bacterial agent were continuously measured within 6 days. Fig.13 The redder the color, the higher the enzyme activity.

[0192] Depend on Fig.13 It can be seen that the color corresponding to the 1% salt concentration is red for LiP and cellulase, and light blue for Lac. The color corresponding to the 2% salt concentration is red for LiP, Lac and cellulase. The color corresponding to the 3% salt concentration is red for LiP, Lac and cellulase, and light blue for xylanase. The color corresponding to the 4% salt concentration is red for LiP, Lac and cellulase. The color corresponding to the 5% salt concentration is red for LiP, Lac and cellulase. From the perspective of the types of enzyme activities optimized by different salinities, the types of enzyme activities optimized by the 3% salt concentration are more. From the perspective of the effect of optimizing the same enzyme activity at different salinities, the 1% salt concentration has a better optimization effect on LiP, and the 3% salt concentration has a better optimization effect on MnP, Lac, cellulase and xylanase. So the optimal salinity is 3%.

[0193] 4) Determination of the optimal inoculum size

[0194] The inoculation amount of the bacterial solution was 1%, 2%, 3%, 4%, and 5%, respectively. The other conditions were the same as those in Example 3. The changes in the activities of the five enzymes in the composite bacterial agent were continuously measured within 6 days. The results are shown in FIG. Fig.14 The redder the color, the higher the enzyme activity.

[0195] Depend on Fig.14It can be seen that the color corresponding to the 1% inoculation amount is red for cellulase. The color corresponding to the 2% inoculation amount is red for all cellulases. The color corresponding to the 3% inoculation amount is red for LiP, Lac, cellulase and hemicellulase. The color corresponding to the 4% inoculation amount is red for MnP and cellulase. The color corresponding to the 5% salt concentration is red for cellulase. From the perspective of the types of enzyme activities optimized by different inoculation amounts, the types of enzyme activities optimized by the 3% inoculation amount are more. From the perspective of the effect of different inoculation amounts on optimizing the same enzyme activity, the 3% inoculation amount has a better effect on optimizing LiP, Lac, cellulase and xylanase, and the 4% inoculation amount has a better effect on optimizing MnP and cellulase. Based on the above, the optimal inoculation amount is 3%.

[0196] 5) Determination of the optimum temperature

[0197] The culture temperatures were set at 5°C, 15°C, 25°C, 35°C, 45°C, and 55°C, respectively. Other conditions were the same as those in Example 3. The changes in the activities of the five enzymes in the composite bacterial agent were continuously measured within 6 days. Fig.15 The redder the color, the higher the enzyme activity.

[0198] Depend on Fig.15 It can be seen that LiP and Lac are red in the color corresponding to 5℃. LiP, Lac, cellulase and hemicellulose are all red in the color corresponding to 15℃. LiP and Lac are red in the color corresponding to 25℃, and cellulase is light blue. LiP and Lac are red in the color corresponding to 35℃, and cellulase is light blue. The colors corresponding to 45℃ and 55℃ are both dark blue. The culture medium at 45℃ and 55℃ did not become turbid or produce mycelium after 6 days of cultivation, indicating that 45℃ and 55℃ will inhibit the growth of the composite bacterial agent. From the perspective of the types of enzyme activity optimized at different temperatures, there are more types of enzyme activity optimized at 15℃. From the perspective of the effect of optimizing the same enzyme activity at different temperatures, 15℃ has better optimization effects on LiP, cellulase and xylanase, and 25℃ has better optimization effects on Lac and MnP. Based on the above, the optimal temperature is 15℃.

[0199] 6) Determination of the optimal pH

[0200] Adjust the pH of the culture medium to 5, 6, 7, 8, and 9 respectively. Keep other conditions the same as in 2.2.4.4.6. Continuously measure the changes in the activities of the five enzymes in the composite inoculant within 6 days. The redder the color, the higher the enzyme activity.

[0201] Depend on Fig.16It can be seen that the color corresponding to pH5 is red for LiP and cellulase. The color corresponding to pH6 is red for LiP, Lac and cellulase. The color corresponding to pH7 is red for LiP, MnP, Lac and cellulase. The color corresponding to pH8 is red for LiP and cellulase. The color corresponding to pH9 is red for LiP. From the perspective of the types of enzyme activities optimized by different pH, there are more types of enzyme activities optimized by pH7. From the perspective of the effect of optimizing the same enzyme activity by different pH, pH7 has a good optimization effect on LiP, MnP, Lac, cellulase and xylanase, so the optimal pH is 7.

[0202] 7) Determination of the optimal bacterial ratio

[0203] 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.16 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.

[0204] Depend on Fig.17 It can be seen that the color corresponding to the ratio of 1:1:4 is red for LiP. The color corresponding to the ratio of 1:2:3 is red for LiP, MnP and Lac. The color corresponding to the ratio of 1:3:2 is red for Lac. The color corresponding to the ratio of 1:4:1 is red for MnP and cellulase. The color corresponding to the ratio of 2:1:3 is not red. The color corresponding to the ratio of 2:2:2 is red for LiP and MnP. The color corresponding to the ratio of 2:3:1 is red for Lac and cellulase. The color corresponding to the ratio of 3:1:2 is red for LiP and Lac. The color corresponding to the ratio of 3:2:1 and the ratio of 4:1:1 is red for MnP, Lac, cellulase and hemicellulase. From the perspective of the types of enzyme activity optimized by different bacterial ratios, the types of enzyme activity optimized by the ratio of 3:2:1 and the ratio of 4:1:1 are more. From the effect of different bacterial ratios on optimizing the same enzyme activity, the ratio of 1:1:4 and 1:2:3 had better optimization effects on LiP, 1:2:3, 2:2:2 and 3:2:1 had better optimization effects on MnP, 3:2:1 had better optimization effects on Lac, 1:4:1, 3:2:1 and 4:1:1 had better optimization effects on cellulase, and 4:1:1 had better optimization effects on xylanase. In summary, the optimal bacterial ratio of the composite bacterial agent F16:F25:K10 was 3:2:1.

[0205] 4. Indoor agricultural and forestry waste degradation experiment

[0206] 1) Growth curve

[0207] Fig.18 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.18 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.

[0208] 2) Fig.19 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.

[0209] Depend on Fig.19 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.

[0210] 3) Weight loss rate test

[0211] Fig. 20 is the change in degradation rate of agricultural and forestry waste with the increase of cultivation days, Fig. 20 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.

[0212] 4) Electron microscopy observation

[0213] Fig.21The 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.

[0214] 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 composite bacterial agent for low temperature degradation of lignin and cellulose, characterized in that: The bacterial agent is composed of Geotrichum candidum K10, Aspergillus pseudokaife F25 and Acinetobacter F16, and their deposit numbers are respectively: Acinetobacter F16 strain, deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; deposit date: January 25, 2024, deposit number: CGMCC No. 29760; Aspergillus spp. F25 strain, deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; deposit date: January 25, 2024, deposit number: CGMCC No.41052; The Geotrichum candidum K10 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.41053; Among them, the bacterial liquid ratio of the composite bacteria F16:F25:K10 is 3:2:1, and the bacterial liquid ratio is a volume ratio.

2. Use of the composite bacterial agent as claimed in claim 1 in the degradation of agricultural and forestry wastes.

3. The use according to claim 2, characterized in that: The agricultural and forestry waste comes from poplar leaves.

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