WO2021196693A1 - 一种草酸青霉菌sdf-25及其应用 - Google Patents

一种草酸青霉菌sdf-25及其应用 Download PDF

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WO2021196693A1
WO2021196693A1 PCT/CN2020/133526 CN2020133526W WO2021196693A1 WO 2021196693 A1 WO2021196693 A1 WO 2021196693A1 CN 2020133526 W CN2020133526 W CN 2020133526W WO 2021196693 A1 WO2021196693 A1 WO 2021196693A1
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sdf
penicillium oxalicum
strain
culture
inoculant
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黄亚丽
邢慧珍
黄媛媛
张丽
李再兴
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河北科技大学
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    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
    • C05F17/10—Addition or removal of substances other than water or air to or from the material during the treatment
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F11/00—Other organic fertilisers
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
    • C05F17/20—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation using specific microorganisms or substances, e.g. enzymes, for activating or stimulating the treatment
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/80—Penicillium
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/141—Feedstock
    • Y02P20/145—Feedstock the feedstock being materials of biological origin

Definitions

  • This scheme relates to Penicillium oxalicum SDF-25 and its application, and belongs to the field of environmental microorganisms.
  • Straw-degrading microorganisms are the indispensable driving force for the straw degradation process.
  • the degradation efficiency of straw is low and the required time is long.
  • the natural degradation of straw cannot meet the needs of crop planting. Therefore, in order to accelerate the decomposition of straw in the soil, it is necessary to add efficient straw degrading bacteria. Since 1912, there have been reports of cellulose-degrading bacteria isolated from soil, and then a large number of cellulose-degrading bacteria have been screened and applied at home and abroad.
  • the isolated and enriched straw degrading microorganisms include filamentous fungi that can degrade rice straw cellulose at 28-37°C. After 15 days of cultivation at 28°C, the degradation rate of corn straw can reach 44.8%.
  • the resulting mold and the FPase produced are 5.02. U/mL and CMCase activity is 4.2 U/mL cellulase production strain Trichoderma reesei RUT-C30.
  • the existing research on cellulose-degrading bacteria mainly focuses on normal temperature and high-temperature environments, and there are relatively few studies on low-temperature cellulose-degrading bacteria. Especially when the soil temperature is lower than 16°C, the degradation effect of microorganisms on straw is very poor.
  • North China has a temperate monsoon climate, with high temperature and rain in summer and cold and dry winter.
  • the annual average temperature is around 8-13°C and the annual precipitation is around 400-1000mm.
  • the development and application of low-temperature straw-degrading inoculants suitable for this climatic feature are of great significance for promoting the rapid decay of corn stalks and returning to the field, improving soil fertility and the effective utilization of biomass resources.
  • this solution provides a Penicillium oxalicum SDF-25 and its application to improve the microorganisms’ low temperature environment Lower the degradation efficiency of straw.
  • the first aspect of this solution provides a strain of Penicillium oxalicum SDF-25, the strain deposit number is CGMCC No. 19272, this strain belongs to Penicillium oxalicum (Penicillium oxalicum), and was deposited in the Chinese Collection of Microorganisms on January 16, 2020 The General Microbiology Center of the Management Committee, the preservation address is located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing.
  • the second aspect of this solution provides the application of the Penicillium oxalicum SDF-25 in straw degradation.
  • the third aspect of this solution provides an inoculant prepared by the Penicillium oxalicum SDF-25, which includes a bacterial adsorption substrate and Penicillium oxalicum SDF-25 powder.
  • the bacterial adsorption substrate is light calcium carbonate.
  • Light calcium carbonate as a bacterial adsorption matrix can make the freezer storage time of live Penicillium oxalicum SDF-25 bacteria reach more than one year.
  • the number of viable cells of the Penicillium oxalicum SDF-25 per gram of the inoculant is ⁇ 2 ⁇ 10 8 cfu/g.
  • the fourth aspect of this solution provides a method for preparing the inoculant, which includes the following process steps:
  • the method of strain activation in step a is: streaking the cryo-preserved Penicillium oxalicum SDF-25 strain on a PDA plate, and incubating at 10-20°C for 12-24 hours.
  • the liquid culture method in step b is: inoculate the hyphae of Penicillium oxalicum SDF-25 activated in step a in an Erlenmeyer flask containing PD liquid medium at 10-20°C, Incubate on a shaker at 150-250r/min for 24-48h.
  • the method for fermentation of the seed liquid in step c is: inoculate the culture liquid of Penicillium oxalicum SDF-25 obtained in step b into a liquid enzyme production medium at 10-20°C, 150-250r /min fermentation culture for 2-3 days; the inoculation volume of the Penicillium oxalicum SDF-25 culture solution accounts for 5-10% of the volume of the enzyme production medium.
  • the method of expanding culture in step d is: inoculating the fermentation broth of Penicillium oxalicum SDF-25 obtained in step c into a seed tank containing PD liquid medium at 10-20°C , 150-200r/min fermentation for 24-48h; the inoculation volume of the fermentation broth of Penicillium oxalicum SDF-25 accounts for 2-5% of the volume of the PD liquid medium.
  • the SDF-25 strain of Penicillium oxalicum provided by this program can grow normally at 6-37°C, and the activity of carboxymethyl cellulase is highest at 10-16°C. It was inoculated into a liquid enzyme-producing medium with a pH of 5-10 at a 2% inoculum, and cultured at 10-16°C, the enzyme activity produced by it could reach up to 993.3U/mL. And Penicillium oxalicum SDF-25 can still grow and produce enzymes at a low temperature of 6°C.
  • the Penicillium oxalicum SDF-25 provided by this program was inoculated into a straw degradation medium at an inoculum of 2%, and after 15 days of culture at 16°C, the degradation rate of corn stalks was as high as 48.6%; under the same conditions The degradation rate can reach 40% after incubation at 10°C for 15 days. It shows that the Penicillium oxalicum SDF-25 provided by this scheme has the ability to grow normally and synthesize highly active cellulase under low temperature conditions, and has great potential to promote the rapid return of straw to the field in the autumn and winter low temperature seasons.
  • step e the preparation method of the inoculant prepared by Penicillium oxalicum SDF-25 provided by this scheme, the obtained inoculant can quickly adapt to the low temperature environment and grow rapidly in the straw, accelerate the straw degradation and increase the straw degradation rate.
  • step e the adsorption matrix is added and dried, and the method of adsorbing strain spores by the matrix is adopted, which can ensure the activity of the bacteria in the inoculant.
  • Fig. 1 is a bar graph of the degradation rate of corn stover by the four strains re-screened in Example 1 of this scheme;
  • Figure 2 is a morphological diagram of the colony of Penicillium oxalicum SDF-25 on PDA medium in Example 1 of this scheme;
  • Figure 3 is a morphological diagram of the conidiophore of Penicillium oxalicum SDF-25 in Example 1 of the present scheme;
  • Figure 4 is a morphological diagram of the conidia of Penicillium oxalicum SDF-25 in Example 1 of the present scheme
  • Figure 5 is a phylogenetic tree of Penicillium oxalicum SDF-25 constructed based on the similarity of ITS gene sequence in Example 1 of this scheme;
  • Figure 6 is a bar graph of the enzyme activity of Penicillium oxalicum SDF-25 under different inoculation amounts in Example 1 of this scheme;
  • Figure 7 is a bar graph of the enzyme activity of Penicillium oxalicum SDF-25 under different pH conditions in Example 1 of the present scheme;
  • Figure 8 is a bar graph of the enzyme activity of Penicillium oxalicum SDF-25 under different temperature conditions in Example 1 of the present scheme;
  • Figure 9 is a bar graph of the straw degradation rate of Penicillium oxalicum SDF-25 under different inoculation amounts in Example 1 of this scheme;
  • Figure 10 is a bar graph of the straw degradation rate of Penicillium oxalicum SDF-25 under different pH conditions in Example 1 of the present scheme;
  • Figure 11 is a bar graph of the straw degradation rate of Penicillium oxalicum SDF-25 under different temperature conditions in Example 1 of this scheme.
  • the samples were collected from the soil in cold climate areas such as Mongolia, Heilongjiang, and Bashang, Hebei. After removing impurities such as animal and plant residues on the soil surface, the samples were placed in pre-sterilized sterilization bags and transported back to the experiment in an ice box Store in a refrigerator at 4°C.
  • PD medium 200g potato, 20g glucose, constant volume to 1000mL.
  • PDA medium add 3 mL of gentamicin sulfate injection and 15-20 agar to 1000 mL of PD medium g, is the PDA medium.
  • Cellulose-Congo Red Medium Sodium Carboxymethyl Cellulose (CMC-Na) 5g, (NH4) 2 SO 4 2g, KH 2 PO 4 1g, MgSO 4 •7H 2 0 0.5g, NaCl 0.5g, NaN0 3 1g, peptone 0.5g, yeast powder 0.5g, Congo red 0.2g, agar 20g, pH 7.0, dilute to 1000mL.
  • Liquid enzyme production medium 200g potato, 20g glucose, CMC-Na 10g, dilute to 1000mL.
  • Corn stalk culture medium corn stalks (crushed through a 40-mesh sieve, dried to constant weight at 50°C) 2g, nutrient solution 30mL; among them, nutrient solution: urea 3.0g, (NH 4 ) 2 SO 4 6.0g, peptone 3.0g , CaCl 2 0.1g, MgSO 4 •7H 2 O 0.5g, K 2 HPO 4 1.0g, NaCl 0.1g, FeS0 4 •7H 2 O 0.05g, MnSO 4 •7H 2 O 0.016g, ZnSO 4 •7H 2 O 0.014g, CoCl 2 •6H 2 O 0.037g, distilled water 1000 mL.
  • Table 1 The size of the hydrolysis circle of the strain on the cellulose-Congo red medium
  • D hydrolysis circle diameter
  • d colony diameter
  • the 13 strains obtained by the preliminary screening were activated on the PDA plate, the fresh hyphae were picked from the inoculation loop and cultured in PD medium at 16°C, 200r/min for 48h, and the inoculum volume was 2% of the inoculum volume and inoculated into 50mL of liquid.
  • the enzyme-producing medium 16°C, 200r/min constant temperature shaking culture liquid fermentation culture, 3 days later, sample 10mL in a centrifuge tube, centrifuge at 10000r/min for 10min, and the supernatant is the crude enzyme solution.
  • the 3,5-dinitrosalicylic acid method was used to determine the carboxymethyl cellulase (CMCase) activity of the strain.
  • the re-screening results are shown in Table 2.
  • the CMCase activities of 13 strains were 233.3 U/mL and 703.3 Between U/mL. Among them, the carboxymethyl cellulase (CMCase) activity of strain SDF-25 was the highest, followed by SDF-4, SDF-15 and SDF-22.
  • the CMCase enzyme activities of these four strains were 600.3 U/mL and 565.3 respectively. U/mL, 636.3U/mL, 703.3U/mL, significantly higher than the cellulase activity of the other 9 strains.
  • the 4 strains obtained by the re-screening were activated on the PDA plate, and fresh hyphae were picked from the inoculation loop and placed in the PD medium at 16°C, 200 Cultivate for 48h at r/min, add 2% of the inoculum volume to corn stalk culture medium (control plus equal amount of sterile water), incubate at a constant temperature of 16°C, 15 days later, filter with filter paper to collect the stalks, distilled water repeatedly Wash 3 times, bake at 80°C to constant weight, and calculate the weight loss rate. Each treatment was repeated 3 times.
  • the measurement results are shown in Figure 1.
  • the straw degradation rates of SDF-4, SDF-15, SDF-22, and SDF-25 were 29.4%, 25.7%, 24.6%, and 33.6% respectively at 15 days.
  • 4 The degradation rate of low temperature straw of each strain was significantly higher than that of the control, and the increase rate of degradation rate compared with the control was 93.4%, 69.1%, 61.8% and 121%, respectively.
  • SDF-25 strain is more helpful to the degradation of corn stalks and has strong practical application value.
  • the cellulase activity and corn stalk degradation rate of SDF-25 strain were both the highest, which was significantly higher than the straw degradation rate of the other three strains.
  • Strain SDF-25 was cultured on PDA medium at 16°C. It was initially a white, nearly circular colony. After 5 days, it gradually turned to turquoise. The hyphae became dense, with concentric rings and neat edges. The surface of the colony was powdery and accompanied. The hyphal villi appear with transparent secretion, as shown in Figure 2; the conidiophore of the fungus is observed under a microscope. The conidiophore of the fungus has branched many times, showing a dispersed or tandem spore chain, as shown in Figure 3. As shown; after the spore chain matures, it falls off to form a single spore, the conidia are nearly round, smooth, and the size is 2.2-3.0 ⁇ m, as shown in Figure 4. According to the characteristics of the colony morphology and conidia structure, with reference to the "Fungus Identification Manual" and "Chinese Fungi Chronicles" for comparative analysis, the strain SDF-25 can be preliminarily determined as Penicillium.
  • ITS4 5'-TCCTCCGCTTATTGATATGC-3'
  • ITS6 5'-GAAGGTGAAGTCGTAACAAGG-3'
  • PCR reaction system 25 ⁇ L: 10 ⁇ bufer 2 ⁇ L, DNA template 2 ⁇ L, dNTP 2 ⁇ L, primers 1 ⁇ L each, r-Tap enzyme 0.2 ⁇ L, ddH 2 O 11.8 ⁇ L
  • reaction conditions 94°C 5 min; 94°C 1 min, 53°C 1 min, 72°C 1 min, 33 cycles; 72°C 10 min.
  • the amplified products were sequenced by Shenggong Bioengineering (Shanghai) Co., Ltd. Center for Biotechnology, NCBI) database for BLAST comparison, and finally with the help of MEGA 5.1 Neighbor-Joining method to construct a phylogenetic tree to determine the phylogenetic status of the strain.
  • strain SDF-25 was used as a template for PCR amplification of the ITS sequence, and the rDNA fragment obtained by sequencing was 616 bp (SEQ ID NO: 3).
  • the sequencing results were compared with the NCBI database by BLAST and the Neighbor-Joining method of MEGA 5.1 was used to construct a phylogenetic tree.
  • the strains SDF-25 and P. oxalicum_strain (accession number: MH911357.1) and P. oxalicum_strain were found (Accession number: MK163534.1) and other strains are all in the same branch.
  • the strain SDF-25 can be determined to be Penicillium oxalicum (P. oxalicum ), and the accession number obtained after submitting the sequence is MN841785.
  • the inoculum, the initial pH and temperature of the medium were optimized in sequence (the rest of the culture conditions remained unchanged), and the enzyme-producing activity of the SDF-25 strain after 3 days was measured.
  • the SDF-25 bacterial solution was connected to the liquid enzyme production medium according to different inoculum amounts, cultured with shaking at 16°C for 72 hours, and centrifuged to take the supernatant to determine the enzyme activity of the crude enzyme solution.
  • the results are shown in Figure 6, the inoculation amount has a significant effect on the enzyme activity. As the inoculation amount increases, the enzyme activity first increases and then decreases. When the inoculation amount is 2%, the enzyme activity is the highest, which is 644.5 U/mL. If the inoculum is too low or too high, the enzyme activity will be significantly affected.
  • the results of studying the enzyme production characteristics of strain SDF-25 at different temperatures are shown in Figure 8.
  • the enzyme activity first increases and then decreases with the increase of the culture temperature. At a temperature of 10°C, the enzyme activity reaches the maximum value of 993.3 U/mL , 16 °C followed by 28 °C and 37 °C enzyme activity significantly reduced, and SDF-25 can still grow and produce enzymes at a low temperature of 6 °C, so it shows that the SDF-25 strain belongs to cold-tolerant bacteria.
  • the strain SDF-25 was inoculated into the straw degradation medium, and the inoculum amount, the initial pH and temperature of the medium were optimized in turn (the rest of the culture conditions remained unchanged), after standing for 15 days, the straw degradation of the strain was determined according to the method shown in 1.4 Rate. Choose 1%, 2%, 3%, 4% for the inoculum; choose 5, 6, 7, 8, 9, 10 for the initial pH; choose 6°C, 10°C, 16°C, 28°C, and 37°C for the temperature.
  • the strain SDF-25 was inoculated into the straw degradation medium according to different inoculation amounts, and after standing for 15 days, the results are shown in Figure 9. As the inoculation amount increases, the degradation rate first increases and then decreases. The inoculation amount is The degradation rate is highest at 2%, which is 45.6%.
  • the straw degradation rate of strain SDF-25 at different culture temperatures is shown in Figure 11. When the temperature is 16°C, the straw degradation rate reaches 44.9%; when the temperature is 10°C, it can also reach 39.5%, indicating that SDF-25 The strain has a strong ability to degrade straw at low temperature.
  • the SDF-25 strain was preserved in the General Microbiology Center of the China Microbial Species Collection Management Committee, and the strain obtained was CGMCC No. 19272, which belongs to Penicillium oxalicum .
  • the inoculant was prepared by using the Penicillium oxalicum SDF-25 selected in Example 1.
  • the inoculant includes an adsorption matrix and Penicillium oxalicum SDF-25 powder.
  • the preparation method of the inoculant specifically includes the following process steps:
  • Activated strain streak the SDF-25 strain of Penicillium oxalicum stored at -80°C on a PDA plate and incubate at 16°C for 24h.
  • Liquid culture Inoculate the SDF-25 hyphae of Penicillium oxalicum activated in step a with an inoculating loop in an Erlenmeyer flask containing PD liquid medium, and culture for 48 hours at 16°C and 200r/min on a shaker.
  • Fermentation of seed liquid inoculate the culture liquid of Penicillium oxalicum SDF-25 obtained in step b in a 10L seed tank, and ferment at 16°C and 200r/min for 2 days; the fermentation medium is PD medium and culture of Penicillium oxalicum SDF-25 The inoculation volume of the solution is 5% of the volume of the PB liquid medium.
  • Expanded culture Inoculate the seed liquid of Penicillium oxalicum SDF-25 obtained by fermentation in step c into PD liquid medium, and ferment for 24h at 16°C and 200r/min; the inoculum volume of seed liquid of Penicillium oxalicum SDF-25 accounts for PB 3% of the volume of the liquid medium.
  • the Penicillium oxalicum SDF-25 inoculum obtained in Example 2 was used for field degradation of corn stalks.
  • the specific degradation method is: mixing SDF-25 inoculum with crushed straw at an inoculum of 0.5%, and then loading it into 1 700g (dry weight) of straw is placed in a sandbag with a length of 0.8 meters and a hole diameter of 0.12mm.
  • the straw without SDF was used as the blank control, and the normal temperature micro-strain was used as the positive control.
  • SDF-25 strain of Penicillium oxalicum selected by this scheme has strong straw degradation ability and high enzyme activity under low temperature conditions, and can adapt to a wide range of growth temperatures, especially in cold northern areas and areas with large temperature differences between day and night. The straw is degraded.

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Abstract

提供了一种草酸青霉菌SDF-25菌株及其应用，所述草酸青霉菌SDF-25的菌株保藏号为CGMCC No.19272，所述草酸青霉菌SDF-25可应用于秸秆降解，草酸青霉菌SDF-25菌株在6-37℃均可正常生长，在10-16℃可分泌大量的羧甲基纤维素酶，在6℃的低温时仍能产酶，酶活力最高可达到993.3U/mL。

Description

一种草酸青霉菌SDF-25及其应用
本方案要求于2020年04月03日提交的中国专利申请CN202010258362.6的优先权。在先申请的公开内容通过整体引用并入本方案。
技术领域
本方案涉及草酸青霉菌SDF-25及其应用，属于环境微生物领域。
 背景技术
我国农作物秸秆年产量超过8亿吨，是一种重要的生物质资源，所以秸秆能否有效利用直接影响现代农业的可持续发展。秸秆还田是秸秆利用的有效方式，对降低土壤水势、提高土壤温度和水解酶的活性、提高土壤有机质含量和增强土壤微生物功能多样性等方面具有重要的作用。然而，对于我国华北地区来说，由于光热资源不足造成玉米/小麦种植茬口紧、小麦种植季气温低、玉米秸秆还田后不能较快降解，导致小麦出苗、扎根困难，越冬受阻。
秸秆降解微生物是秸秆降解过程必不可少的原动力，但是由于土壤原生态环境下秸秆降解微生物数量较少，使得秸秆的降解效率低、所需时间长。在作物茬口紧张的情况下秸秆的自然降解不能够满足农作物种植的需求，因此为了加速秸秆在土壤中的腐解，需要进行高效秸秆降解菌的添加。自1912年就有从土壤中分离出纤维素降解菌的报道，之后国内外进行了大量纤维素降解菌的筛选和应用。目前分离富集的秸秆降解微生物有能在28-37℃降解稻秆纤维素的丝状真菌、28℃培养15d使玉米秸秆降解率达44.8%得霉菌以及产生的FPase为5.02 U/mL和CMCase活力为4.2 U/mL的纤维素酶生产菌株里氏木霉RUT-C30。然而现有对纤维素降解菌的研究主要集中在常温和高温环境，对低温降解纤维素菌的研究相对较少。尤其在土壤温度低于16℃时，微生物对秸秆的降解效果很差。
我国华北地区主要为温带季风气候，夏季高温多雨，冬季寒冷干燥，年平均气温在8-13℃左右，年降水量在400-1000mm左右。适合这种气候特点的低温秸秆降解菌剂的研制及应用对促进玉米秸秆快腐还田、提高土壤肥力和生物质资源的有效利用具有重要的意义。
 技术问题
针对现有秸秆降解微生物低温环境对秸秆降解效率低、繁殖速度慢、纤维素酶的产量和活性低的问题，本方案提供一种草酸青霉菌SDF-25及其应用，以提高微生物在低温环境下对秸秆的降解效率。
 技术解决方案
本方案的第一方面提供一种草酸青霉菌SDF-25，菌株保藏号为CGMCC No.19272，该菌株属于草酸青霉（ Penicillium oxalicum），于2020年01月16日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏地址位于北京市朝阳区北辰西路1号院3号中国科学院微生物研究所。
本方案的第二方面提供所述的草酸青霉菌SDF-25在秸秆降解中的应用。
本方案的第三方面提供所述草酸青霉菌SDF-25制备的接种剂，包括菌体吸附基质和草酸青霉菌SDF-25菌粉。
作为本方案的一个实施例，所述菌体吸附基质为轻质碳酸钙。
轻质碳酸钙作为菌体吸附基质可使草酸青霉菌SDF-25活菌的冷冻保存时间达到一年以上。
作为本方案的一个实施例，每克所述接种剂中所述草酸青霉菌SDF-25的活菌数≥2×10 8cfu/g。
本方案的第四方面提供所述接种剂的制备方法，包括如下工艺步骤：
a、活化菌种；
b、液体培养；
c、种子液发酵；
d、扩大培养；
e、离心去除上清，加入吸附基质并干燥。
作为本方案的一个实施例，所述步骤a中的菌种活化方法为：将冷冻保存的草酸青霉菌SDF-25菌株划线接种于PDA平板上，10-20℃培养12-24h。
作为本方案的一个实施例，所述步骤b中液体培养方法为：将步骤a中活化的草酸青霉菌SDF-25菌丝接种于装有PD液体培养基的三角瓶中，10-20℃、150-250r/min摇床培养24-48h。
作为本方案的一个实施例，所述步骤c中的种子液发酵方法为：将步骤b得到的草酸青霉菌SDF-25培养液接种于液体产酶培养基中，10-20℃、150-250r/min发酵培养2-3d；所述草酸青霉菌SDF-25培养液的接种体积占产酶培养基体积的5-10%。
作为本方案的一个实施例，所述步骤d中扩大培养的方法为：将步骤c中得到的草酸青霉菌SDF-25发酵液接种于装有PD液体培养基的种子罐中，10-20℃、150-200r/min发酵24-48h；所述草酸青霉菌SDF-25发酵液的接种体积占PD液体培养基体积的2-5%。
 有益效果
与现有技术相比，本方案具有如下优点：
（1）本方案提供的草酸青霉菌SDF-25菌株在6-37℃均可正常生长，且在10-16℃羧甲基纤维素酶活性最高。将其以2%的接种量接种到pH为5-10的液体产酶培养基中，10-16℃下培养，其产生的酶活力最高可达到993.3U/mL。而且草酸青霉菌SDF-25在6℃的低温时仍能生长并产酶。
（2）将本方案提供的草酸青霉菌SDF-25以2%的接种量接种在秸秆降解培养基中，16℃培养15d后，经检测其对玉米秸秆的降解率高达48.6%；相同条件下10℃培养15d后降解率也能达到40%。说明本方案提供的草酸青霉菌SDF-25具有低温条件下正常生长及合成高活性纤维素酶的能力，具有促进秋冬低温季节的秸秆快速还田巨大潜力。
（3）本方案提供的草酸青霉菌SDF-25制备的接种剂的制备方法，得到的接种剂可快速适应低温环境并在秸秆中快速生长，加快秸秆降解以及提高秸秆降解速率。步骤e中加入吸附基质并干燥采用了基质吸附菌株孢子的方法，可以保证接种剂中菌体的活性。
 附图说明
图1是本方案实施例1中复筛的4种菌株对玉米秸秆的降解率的柱形图；
图2是本方案实施例1中草酸青霉菌SDF-25在PDA培养基上菌落的形态图；
图3是本方案实施例1中草酸青霉菌SDF-25的分生孢子梗的形态图；
图4是本方案实施例1中草酸青霉菌SDF-25的分生孢子的形态图；
图5是本方案实施例1中基于ITS基因序列相似性构建的草酸青霉菌SDF-25的系统发育树；
图6是本方案实施例1中不同接种量下草酸青霉菌SDF-25的酶活力柱形图；
图7是本方案实施例1中不同pH条件下草酸青霉菌SDF-25的酶活力柱形图；
图8是本方案实施例1中不同温度条件下草酸青霉菌SDF-25的酶活力柱形图；
图9是本方案实施例1中不同接种量下草酸青霉菌SDF-25的秸秆降解率柱形图；
图10是本方案实施例1中不同pH条件下草酸青霉菌SDF-25的秸秆降解率柱形图；
图11是本方案实施例1中不同温度条件下草酸青霉菌SDF-25的秸秆降解率柱形图。
本方案的实施方式
实施例1
草酸青霉菌SDF-25的筛选过程
1.1样品与培养基
1.1.1样品
样品采集自我国内蒙、黑龙江、河北坝上等气候冷凉地区的土壤，去除土壤表面的动植物残体等杂质后，将样品置入事先灭好菌的灭菌袋中，冰盒运回实验室，4℃冰箱保存。
1.1.2培养基
PD培养基：马铃薯200g，葡萄糖20g，定容至1000mL。
PDA培养基：在1000mL的PD培养基中加硫酸庆大霉素注射液3mL，琼脂15-20 g，即为PDA培养基。
纤维素-刚果红培养基：羧甲基纤维素钠（CMC-Na）5g、（NH4） 2SO 4 2g、KH 2PO 4 1g、MgSO 4•7H 20 0.5g、NaCl 0.5g、NaN0 3 1g、蛋白胨0.5g、酵母粉0.5g、刚果红0.2g、琼脂20g，pH7.0，定容至1000mL。
液体产酶培养基：马铃薯200g、葡萄糖20g、CMC-Na 10g，定容至1000mL。
玉米秸秆培养基：玉米秸秆（粉碎过40目筛，50℃烘干至恒重）2g，营养液30mL；其中，营养液：尿素3.0g、（NH 4） 2SO 4 6.0g，蛋白胨3.0g，CaCl 2 0.1g、MgSO 4•7H 2O 0.5g、K 2HPO 4 1.0g、NaCl 0.1g、FeS0 4•7H 2O 0.05g、MnSO 4•7H 2O 0.016g、ZnSO 4•7H 2O 0.014g、CoCl 2•6H 2O 0.037g，蒸馏水1000 mL。
1.2分离和纯化
称取土样5g置于45mL的无菌水中，在振荡器上200r/min充分振荡30min，然后用灭菌生离盐水逐级稀释为10 -2、10 -3、10 -4倍，分别吸取100μL稀释梯度为10 -2、10 -3、10 -4的土壤稀释液涂布在PDA加庆大霉素培养基上，每个稀释度3个平板。将平板置于16℃恒温培养箱中培养3d后挑取不同形态的菌落，进行3次划线纯化。挑取培养菌落接种于PDA试管斜面培养基上，16℃培养至长满斜面，4℃保存备用。
1.3低温纤维素降解菌的筛选
1.3.1初筛
将保存的低温菌株接种到PDA平板上进行活化，然后用直径为5mm的打孔器打取菌落边缘生长旺盛的菌块，接种于纤维素-刚果红培养基上，16℃恒温培养3d，观察菌落附近有无水解圈，测量、记录菌落和水解圈直径，计算D/d值，每个菌株3次重复。
通过低温PDA培养基分离，共纯化获得16℃快速生长菌株40株，依次编号为SDF-1至SDF-40，经纤维素-刚果红培养基初筛，其中21株具有纤维素降解能力，13株菌的水解圈与菌落直径比值（D/d）均大于2.0，如表1所示。
表1菌株在纤维素-刚果红培养基上的水解圈大小
菌株编号 D(mm) d(mm) D/d
SDF-2 21.5 ± 0.71 9.0± 0.00 2.388 ± 0.07
SDF-3 21.5 ± 3.54 10.0 ± 1.41 2.146 ± 0.05
SDF-4 41.5 ± 10.61 17.0 ± 4.24 2.439 ± 0.01
SDF-9 15.2 ± 0.35 7.2 ± 0.35 2.105 ± 0.05
SDF-13 21.5 ± 0.71 10.0 ± 1.41 2.166 ± 0.23
SDF-15 27.0 ± 4.24 11.5 ± 2.12 2.354 ± 0.06
SDF-17 23.0 ± 1.41 10.5 ± 0.70 2.191 ± 0.01
SDF-18 30.6 ± 5.46 12.8 ± 1.92 2.391 ± 0.22
SDF-21 24.3 ± 0.58 9.6 ± 0.57 2.522 ± 0.13
SDF-22 25.5 ± 0.71 12.2 ± 0.35 2.081 ± 0.01
SDF-25 25.5 ± 0.71 11.7 ± 0.35 2.170 ± 0.00
SDF-30 29.5 ± 2.12 13.0 ± 1.41 2.273 ± 0.08
SDF-31 39.0 ± 1.41 17.2 ± 1.06 2.262 ± 0.05
其中，D：水解圈直径；d：菌落直径。
1.3.2复筛
将初筛得到的13株菌株在PDA平板上活化，接种环挑取新鲜菌丝于PD培养基中，16℃，200r/min培养48h，按接种体积为2%的接种量接种于50mL的液体产酶培养基中，16℃，200r/min恒温振荡培养液体发酵培养，3d后取样10mL于离心管中，10000r/min离心10min，上清液即为粗酶液。用3,5-二硝基水杨酸法（DNS法）对菌株的羧甲基纤维素酶（CMCase）活性进行测定。取1.0mL的粗酶液加入4.0mL CMC缓冲液，在50℃的水浴锅中反应20min取出，然后加入3.0mL DNS显色液沸水浴显色10min后冷却，490nm处比色，测其OD值，与标准葡萄糖曲线对照，由OD值计算出葡萄糖量（m 1）。对照管中粗酶液在水浴20min后再加入，测得粗酶液的葡萄糖量（m 2）。通过公式计算纤维素分解菌在上述条件下的酶活力（单位：U/mL）:U=（m1-m2）/20×稀释倍数。
复筛结果如表2所示，13株菌株的CMCase活性在233.3U/mL和703.3 U/mL之间。其中以菌株SDF-25的羧甲基纤维素酶（CMCase）活性最高，之后依次为SDF-4、SDF-15和SDF-22，这4个菌株的CMCase酶活力分别为600.3U/mL，565.3U/mL，636.3U/mL，703.3U/mL，显著高于其他9个菌株的纤维素酶活性。
表2菌株的纤维素酶活性
菌株编号 CMC酶活(U/mL) 菌株编号 CMC酶活(U/mL)
SDF-2 362.7 ± 0.35i SDF-18 383.3 ± 0.16 h
SDF-3 416.7 ± 0.70 f SDF-21 233.3 ± 0.54 m
SDF-4 600.3 ± 0.37 c SDF-22 636.3 ± 0.67 b
SDF-9 388.3 ± 0.12g SDF-25 703.3 ± 0.23 a
SDF-13 285.0 ± 0.32 k SDF-30 281.7 ± 0.19 l
SDF-15 565.3 ± 0.18 d SDF-31 486.7 ± 0.21 e
SDF-17 313.3 ± 0.62 j    
1.4复筛菌株在不同温度下的生长情况
将直径为5 mm的SDF-25、SDF-4、SDF-15和SDF-22菌块分别接种到PDA培养基上，每种菌株分别置于6℃、10℃、16℃、28℃、37℃的恒温培养箱中静置培养7d，测定并记录各菌株在不同温度下的生长情况。
采用菌株生长速率测定法，比较了4株低温秸秆降解真菌在6℃、10℃、16℃、28℃、37℃培养条件下的生长速率，由表3可得，SDF-25菌株16℃生长速率最高，在6-37℃均可正常生长，其具有低温适应性以及具有促进秋冬低温季节的秸秆快速还田潜力。
表3菌株低温生长速度mm/d
菌株编号 6℃ 10℃ 16℃ 28℃ 37℃
SDF-4 0.714 1.171 3.224 3.128 3.083
SDF-15 0.714 1.114 3.112 3.015 3.117
SDF-22 0.714 1.785 3.267 3.243 3.216
SDF-25 1.071 2.314 3.392 3.341 3.329
1.5菌株的秸秆降解率测定
将复筛得到的4种菌株在PDA平板上活化，接种环挑取新鲜菌丝于PD培养基中，16℃，200 r /min培养48h，按接种体积为2%的接种量接入玉米秸秆培养基中（对照加等量无菌水），16℃下恒温培养，15d后，用滤纸过滤发收集秸秆，蒸馏水反复清洗3次，80℃烘至恒重，计算失重率。每个处理3次重复。
测定结果如图1所示，15d时SDF-4、SDF-15、SDF-22、SDF-25的秸秆降解率分别为29.4%、25.7%、24.6%、33.6%，与空白对照相比，4个菌株的低温秸秆降解率均显著高于对照，降解率比对照的增加幅度分别为93.4%、69.1%、61.8%和121%。说明SDF-25菌株更有助于玉米秸秆的降解，具有较强的实际应用价值。其中SDF-25菌株的纤维素酶活力和玉米秸秆降解率均最高，显著高于其他3株菌的秸秆降解率。
1.6菌株鉴定
1.6.1形态学观察
将直径为5 mm的SDF-25菌株菌块接种到PDA培养基上，16℃培养，观察菌落生长状况及其形态特征；采用插片培养法进行菌株分生孢子梗形态的观察；用无菌镊子夹取适量培养好的菌丝于载玻片上，拨动菌丝使其均匀散开，盖上盖玻片置于显微镜下观察分生孢子的显微特点。
菌株SDF-25在PDA培养基上16℃培养，初始为白色、近圆形菌落，5d后逐渐变为青绿色，菌丝变稠密，有同心环纹，边缘齐整，菌落表面呈粉末状并伴随菌丝绒毛，出现透明分泌液，如图2所示；在显微镜下观察该菌的分生孢子梗，该菌分生孢子梗多次分枝，呈分散状或串联的孢子链，如图3所示；孢子链成熟后脱落形成单个孢子，分生孢子呈近圆形，光滑，大小为2.2−3.0 μm，如图4所示。根据菌落形态和分生孢子结构特征，参照《真菌鉴定手册》和《中国真菌志》进行比对分析，可初步确定菌株SDF-25为青霉属。
1.6.2分子生物学鉴定
采用真菌基因组DNA提取试剂盒（北京索莱宝科技有限公司）提取SDF-25菌株的DNA；利用真菌通用引物ITS4（5’-TCCTCCGCTTATTGATATGC-3’）和ITS6（5’-GAAGGTGAAGTCGTAACAAGG-3’）扩增菌株的ITS区域rDNA序列；PCR反应体系(25μL)：10×bufer 2μL、DNA模板2μL、dNTP 2μL、引物各1μL、r-Tap酶0.2μL、ddH 2O 11.8μL，反应条件：94℃ 5 min；94℃ 1 min，53℃ 1 min，72℃ 1 min，33个循环；72℃ 10 min。
扩增产物由生工生物工程(上海)股份有限公司测序，测序结果在美国生物技术研究中心（National Center for Biotechnology，NCBI）数据库中进行BLAST比对，最后借助MEGA 5.1的Neighbor-Joining法构建系统发育树，确定该菌株的系统发育学地位。
以菌株SDF-25的DNA为模板进行ITS序列的PCR扩增，测序获得ITS的rDNA片段的长度为616bp（SEQ ID NO:3）。测序结果与NCBI数据库进行BLAST比对并利用MEGA 5.1的Neighbor-Joining法构建系统发育树，如图5所示，发现菌株SDF-25与 P. oxalicum_strain(accession number: MH911357.1)、 P.oxalicum_strain(accession number:MK163534.1)等菌株均处于同一分支。综合菌落特征及系统发育分析，可以确定菌株SDF-25为草酸青霉( P. oxalicum)，提交序列后获得的登录号为MN841785。
1.7SDF-25菌株的产酶特性研究
将菌株SDF-25接种到50 mL产酶培养基中，依次对接种量、培养基初始pH及温度进行优化（其余培养条件不变），测定SDF-25菌株3d后的产酶活情况。接种量分别选择1%、2%、3%、4%；初始pH分别选择5、6、7、8、9、10；温度分别选择6℃、10℃、16℃、28℃、37℃。
将SDF-25菌液按照不同接种量接入到液体产酶培养基中，16℃下振荡培养72h，离心取上清测定粗酶液酶活力。结果如图6所示，接种量对酶活的影响比较显著，随着接种量的增加酶活呈现出先增加后降低的趋势，在接种量为2%时酶活最高，为644.5U/mL。接种量过低或过高都会显著影响酶活力。
不同的初始pH对菌株SDF-25产酶特性的研究结果如图7所示，在不同的初始pH值下，SDF-25的羧甲基纤维素酶活性也不同，说明初始pH值对菌株的产酶能力有明显的影响，菌株SDF-25的酶活性随着pH的增加呈现出先增加后降低的趋势，在初始pH为7时酶活力达到最高点，为655.7U/mL。且该菌在偏酸环境下的产酶活性高于偏碱环境下的产酶活性。
对不同温度下菌株SDF-25的产酶特性进行研究结果如图8所示，酶活力随培养温度的升高先增大后减小，在温度为10℃时，酶活力达到最大值993.3U/mL，16℃其次，28℃和37℃酶活显著降低，而且在6℃低温时SDF-25仍能生长并产酶，因此说明SDF-25菌株属于耐冷菌。
1.8SDF-25对玉米秸秆降解率的研究
将菌株SDF-25接种到秸秆降解培养基中，依次对接种量、培养基初始pH及温度进行优化（其余培养条件不变），静置培养15d后，按1.4所示方法测定菌株的秸秆降解率。接种量分别选择1%、2%、3%、4%；初始pH分别选择5、6、7、8、9、10；温度分别选择6℃、10℃、16℃、28℃、37℃。
将菌株SDF-25按不同接种量接种在秸秆降解培养基中，静置培养15d后，结果如图9所示，随着接种量的增加降解率呈现出先增加后降低的趋势，在接种量为2%时降解率最高，为45.6%。
不同的初始pH对菌株SDF-25的秸秆降解结果如图10所示，随着pH的增加降解率呈现出先增加后降低的趋势，在初始pH为7降解率最高，为43.2%，其次为pH为6时的降解率。分析不同初始pH处理的秸秆降解率，发现SDF-25菌株在偏酸条件下比偏碱条件下的降解率高。
不同培养温度对菌株SDF-25的秸秆降解率，如图11所示，在温度为16℃时，秸秆降解率达到44.9%；在温度为10℃时，也能达到39.5%，说明SDF-25菌株具有较强的低温秸秆降解能力。
将SDF-25菌株保藏于中国微生物菌种保藏管理委员会普通微生物中心，获得的菌株保藏号为CGMCC No.19272，该菌株属于草酸青霉（ Penicillium oxalicum）。
实施例2
利用实施例1中筛选出来的草酸青霉菌SDF-25制备接种剂，该接种剂包括吸附基质和草酸青霉菌SDF-25菌粉，接种剂的制备方法具体包括以下工艺步骤：
a、活化菌种：将-80℃保存的草酸青霉菌SDF-25菌株划线接种于PDA平板上，16℃培养24h。
b、液体培养：用接种环将步骤a中活化的草酸青霉菌SDF-25菌丝接种于装有PD液体培养基的三角瓶中，16℃、200r/min摇床培养48h。
c、种子液发酵：将步骤b得到的草酸青霉菌SDF-25培养液接种于10L种子罐中，16℃、200r/min发酵2d；发酵培养基为PD培养基，草酸青霉菌SDF-25培养液的接种体积占PB液体培养基体积的5%。
d、扩大培养：将步骤c发酵得到的草酸青霉菌SDF-25种子液接种于PD液体培养基中，16℃、200r/min发酵24h；其中草酸青霉菌SDF-25种子液的接种体积占PB液体培养基体积的3%。
e、将发酵液进行离心去除上清并收集孢子，将步骤d扩大培养得到的菌液进行离心，收集孢子，向收集的孢子液中加入轻质碳酸钙并进行干燥，获得草酸青霉菌SDF-25菌剂。
实施例3
将实施例2中获得的草酸青霉菌SDF-25菌剂，用于玉米秸秆的田间降解，具体降解方法为：将SDF-25菌剂按0.5%的接种量与粉碎秸秆混合，之后装入1米长、0.8米宽、孔径0.12mm的沙袋中，每个沙袋中装入700g（干重）的秸秆。以未添加SDF菌剂秸秆为空白对照，以常温微菌株为阳性对照。将沙袋于小麦种植季节埋入小麦种植田中。于埋入土壤中15d时取出，冲洗后称量秸秆干重，计算秸秆的降解率达到48.6%。说明本方案筛选得到的草酸青霉菌SDF-25菌株在低温条件下具有较强的秸秆降解能力和较高产酶活性，适应生长的温度范围广泛，尤其可以适用于北方寒冷地区及昼夜温差大的地区的秸秆降解。

Claims (10)

  1. 一种草酸青霉菌SDF-25，其特征在于：菌株保藏号为CGMCC No.19272。
  2. 权利要求1所述的草酸青霉菌SDF-25在秸秆降解中的应用。
  3. 权利要求1所述的草酸青霉菌SDF-25制备的接种剂，其特征在于：包括吸附基质和草酸青霉菌SDF-25菌粉。
  4. 如权利要求3所述的草酸青霉菌SDF-25制备的接种剂，其特征在于：所述吸附基质为轻质碳酸钙。
  5. 如权利要求3所述的草酸青霉菌SDF-25制备的接种剂，其特征在于：每克所述接种剂中所述草酸青霉菌SDF-25的活菌数≥2×10 8cfu/g。
  6. 权利要求3-5任一项所述的接种剂的制备方法，其特征在于：包括如下工艺步骤：
    a、活化菌种；
    b、液体培养；
    c、种子液发酵；
    d、扩大培养；
    e、离心去除上清，添加吸附基质并干燥。
  7. 如权利要求6所述的接种剂制备方法，其特征在于：所述步骤a中的菌种活化方法为：将冷冻保存的草酸青霉菌SDF-25菌株划线接种于PDA平板上，10-20℃培养12-24h。
  8. 如权利要求6所述的接种剂制备方法，其特征在于：所述步骤b中液体培养方法为：将步骤a中活化的草酸青霉菌SDF-25菌丝接种于装有PD液体培养基的三角瓶中，10-20℃、150-250r/min摇床培养24-48h。
  9. 如权利要求6所述的接种剂制备方法，其特征在于：所述步骤c中的种子液发酵方法为：将步骤b得到的草酸青霉菌SDF-25培养液接种于装有PD液体培养基的种子罐中，10-20℃、150-250r/min发酵培养2-3d；所述草酸青霉菌SDF-25培养液的接种体积占产酶培养基体积的5-10%。
  10. 如权利要求6所述的接种剂制备方法，其特征在于：所述步骤d中扩大培养的方法为：将步骤c中得到的草酸青霉菌SDF-25发酵液接种于PD液体培养基中，10-20℃、150-200r/min发酵24-48h；所述草酸青霉菌SDF-25发酵液的接种体积占PD液体培养基体积的2-5%。
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