A mixed bacterial fermentation agent and its application in fermenting biological substrates

Abstract

The present invention belongs to the technical field of biological substrates, and specifically relates to a mixed bacterial fermenting agent and its application in fermenting biological substrates. The fermenting agent is composed of fermenting agent A and fermenting agent B; the fermenting agent A is a fermented powder of Trichoderma harzianum Ta97 from Africa; the fermenting agent B is a fermented powder of Arthrobacter ureafaciens Fp64. The two bacterial agents added to the special biological substrate for strawberries in the present invention cooperate to play a role in dynamically regulating rhizosphere phosphorus, which is beneficial to the establishment of a healthy rhizosphere microecology. While having a significant growth-promoting effect on strawberries, it significantly reduces phosphorus loss and achieves a purification effect. In addition, Arthrobacter ureafaciens Fp64 also has high IAA activity. This substrate has a significant growth-promoting effect on strawberries.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological substrates, and particularly relates to a mixed bacterial ferment and its application in fermenting biological substrates. Background Art

[0002] According to statistics, China has become the world's largest producer and consumer of strawberries. Due to its high nutritional value, delicious flavor, and high economic value, the planting area and output of strawberries have both shown an upward trend as a whole. Strawberries are also important crops for protected cultivation in China, especially in the northern regions with relatively low temperatures, and the scale of the protected strawberry industry has been increasing year by year. In protected agriculture, the substrate is the most important basic material. Especially in strawberry cultivation, the cultivation substrate is indispensable from seedling raising to fruit reproduction.

[0003] Conventional substrates are composed of nutrients (including peat, etc.) and auxiliary materials with functions such as air permeability and water retention (including vermiculite, coconut coir, perlite, etc.). Among them, peat belongs to non-renewable natural resources. With the increasing demand, its exploitation has caused serious damage to nature and has been strictly controlled, while the cost has increased significantly. The fermented products of straw and cow dung in agricultural waste are good substitutes for peat. Under reasonable utilization, it can not only reduce the production cost of the substrate but also promote the agricultural carbon cycle, with good application prospects. Among them, corn straw is the crop straw with the largest stock in China, and cow dung is also the main livestock and poultry breeding waste. The mixed compost fermentation of straw and cow dung to prepare the substrate can effectively balance the carbon-nitrogen ratio, promote the decomposition of materials, and regulate the porosity and water content compared with only straw fermentation or only cow dung fermentation. However, at present, the production mainly relies on natural decomposition of compost, and the decomposition rate is limited by the lignin degradation rate in the polymeric crystal structure of lignocellulose. The fermentation and decomposition time is as long as 2 - 3 months, resulting in unstable compost quality. In addition, since strawberries have fibrous roots and relatively shallow root depths, they have strict requirements for the porosity and bulk density of the cultivation substrate, and the phosphorus applied in general substrate products is easily fixed, resulting in phosphorus deficiency. To address these problems, there is an urgent need for a strawberry-specific substrate product with precise matching. Summary of the Invention

[0004] Aiming at the technical defects and other problems existing in the existing strawberry substrate products in the prior art, one of the purposes of the present invention is to provide a mixed bacterial ferment.

[0005] The present invention also provides an application of a mixed bacterial ferment in fermenting biological substrates, and the prepared biological substrate has functions of phosphorus regulation, microecological improvement, and growth promotion.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a mixed bacterial fermenter, which is composed of fermenter A and fermenter B; the fermenter A is a fermentation powder of Trichoderma harzianum Ta97 from Africa, and the preservation number is CGMCC No. 19930; the fermenter B is a fermentation powder of Arthrobacter ureafaciens Fp64, and the preservation number is CGMCC No. 15644.

[0008] The present invention also provides an application of the above-mentioned mixed bacterial fermenter in the fermentation preparation of a biological substrate.

[0009] Furthermore, the preparation process of the biological substrate specifically includes the following steps:

[0010] (1) Mix corn straw and washed cow dung in proportion, and adjust the water content to obtain compost fermentation raw materials;

[0011] (2) Conduct primary fermentation on the compost fermentation raw materials to obtain primary fermentation products;

[0012] (3) Add the fermentation powder of Trichoderma harzianum Ta97 to the primary fermentation products for secondary fermentation to obtain secondary fermentation products;

[0013] (4) Add the fermentation powder of Arthrobacter ureafaciens Fp64 to the secondary fermentation products to obtain mixture A;

[0014] (5) Mix vermiculite, coconut coir and perlite evenly to obtain mixture B;

[0015] (6) Mix mixture A and mixture B in proportion to obtain the biological substrate.

[0016] Furthermore, in step (1), the particle size of the corn straw is 1 - 2 cm; the volume ratio of the corn straw to the washed cow dung is 2.5 - 4.5 : 1; the water content is adjusted to 30% - 40%.

[0017] Furthermore, in step (2), the specific process of the primary fermentation is: place the compost fermentation raw materials in a trough-type fermentation workshop for fermentation, the height of the materials is more than 2 meters, turn and toss once every 3 - 4 days, the time above 60°C is 7 days, when the temperature is between 60°C and 70°C, maintain intermittent ventilation, turn the pile immediately when the temperature is higher than 70°C, and continue ventilation to obtain the materials to be fermented, then continue to turn and toss once every 2 - 3 days, with intermittent ventilation, and maintain the temperature at 40°C - 50°C for 10 days to obtain the primary fermentation materials.

[0018] Furthermore, in step (3), the total number of colonies of the fermentation powder of Trichoderma harzianum Ta97 is 1×10 9cfu / g; The weight ratio of the Trichoderma afroharzianum Ta97 fermentation powder to the primary fermentation product is 1:10000; The process of the secondary fermentation is: turning and throwing once every 1 day, intermittent ventilation, and maintaining at 25°C to 35°C for 10 days.

[0019] Further, in step (4), the total number of colonies of the Arthrobacter ureafaciens Fp64 fermentation powder is 6×10 9 cfu / g; The weight ratio of the Arthrobacter ureafaciens Fp64 fermentation powder to the secondary fermentation product is 1:1000.

[0020] Further, in step (5), the vermiculite, coconut coir and perlite are in a volume ratio of 5 - 6 : 2 - 3 : 1 - 2.

[0021] Further, in step (6), the volume ratio of the mixture A and the mixture B is 2 - 5 : 1 - 2.

[0022] The present invention also provides an application of the biological substrate prepared by fermentation using the above method as a special substrate for strawberry cultivation.

[0023] In addition to the biological characteristics of the lignin-degrading enzyme recorded in the patent ZL202011309961.2, the above-mentioned Trichoderma afroharzianum Ta97 also has an efficient phosphorus-solubilizing effect, and the phosphorus-solubilizing amount reaches 525.26 ± 22.70 mg / L in 7 days.

[0024] In addition to the polyphosphorus recorded in the patent ZL201810547376.2, the above-mentioned Arthrobacter ureafaciens Fp64 also has a high IAA-producing ability, and the IAA yield reaches 255.94 ± 13.25 mg / L.

[0025] Adopting the technical solution of the present invention, compared with the known public technology, it has the following beneficial effects:

[0026] (1) The preparation process of the special substrate for strawberries in the present invention innovatively adopts a tank-type segmented fermentation process, which respectively realizes disinfection (60 - 70°C), enrichment of the background degradation flora (40 - 50°C) and rapid composting fermentation (25 - 40°C), and the composting fermentation can be completed within 32 days at most, greatly shortening the composting time compared with the existing technology which is generally 2 - 3 months;

[0027] (2) The strawberry - specific biological substrate of the present invention uses corn straw - water - washed cow dung fermentate to replace traditional peat. By using highly efficient lignocellulose - degrading bacterial agents and precisely regulating fermentation parameters, a fermentate with a certain amount of short fibers is obtained through trough - type composting fermentation, and then a substrate that meets the growth requirements of strawberries in terms of key indicators such as porosity and bulk density is prepared. Compared with the general substrate using peat, on the one hand, the present invention is more low - carbon and environmentally friendly. More importantly, it can adjust the porosity according to the characteristics of crop roots, has better adaptability for strawberries, a high diversity of enriched microorganisms, and has a good micro - ecological growth environment;

[0028] (3) Since the growth of strawberries is sensitive to the demand for phosphorus, a large amount of phosphate fertilizer is easily converted into an insoluble state after being applied, but it is also easily leached away by water. Among the two bacterial agents added to the strawberry - specific biological substrate of the present invention, Trichoderma harzianum Ta97 from Africa has a highly efficient phosphorus - solubilizing effect, and Arthrobacter ureafaciens Fp64 has a phosphorus - accumulating effect. The fixed phosphate fertilizer is activated through the phosphorus - solubilizing effect, and the excess active phosphorus is stored through the phosphorus - accumulating effect. The cooperation of the two bacteria plays a role in dynamically regulating the phosphorus in the rhizosphere, which is beneficial to the establishment of a healthy rhizosphere micro - ecology. While having a significant growth - promoting effect on strawberries, it significantly reduces phosphorus loss and achieves a purification effect. In addition, Arthrobacter ureafaciens Fp64 also has high IAA activity. This substrate has a significant growth - promoting effect on strawberries.

[0029] (I) Arthrobacter ureafaciens Fp64

[0030] Preservation time: April 26, 2018,

[0031] Preservation unit: China General Microbiological Culture Collection Center,

[0032] Preservation number: CGMCC No. 15644,

[0033] Address of the preservation unit: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing 100101

[0034] Taxonomic name: Arthrobacter ureafaciens Arthrobacter ureafaciens

[0035] (II) Trichoderma harzianum Ta97

[0036] Preservation time: July 14, 2020,

[0037] Preservation unit: China General Microbiological Culture Collection Center,

[0038] Preservation number: CGMCC No. 19930,

[0039] Depository address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing 100101, Postcode: 100101

[0040] Taxonomic name: Trichoderma harzianum from Africa Trichoderma afroharzianum 。 Detailed implementation manners

[0041] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below in conjunction with specific embodiments Example 1

[0042] Determination of phosphorus solubilization amount of Trichoderma harzianum Ta97 from Africa

[0043] Activate the Ta97 strain on a PDA solid plate to prepare a conidia suspension. Take 10 8 conidia / mL and inoculate it into a liquid phosphorus-solubilizing medium (in 1 L of the medium, it contains: 10 g of glucose, 0.5 g of (NH4)2SO4, 0.3 g of NaCl, 0.3 g of KCl, 0.3 g of MgSO4·7H2O, 0.03 g of FeSO4·7H2O, 0.03 g of MnSO4·H2O, 5 g of Ca3(PO4)2, adjust the pH to 7.0) at an inoculation amount of 1%, and culture it at 26°C ± 0.5°C and 130 rpm for 7 days to obtain a fermentation broth. The control is a liquid phosphorus-solubilizing medium without the Trichoderma conidia suspension, with 3 biological replicates. Centrifuge the fermentation broth at 4°C and 10000 rpm for 5 min, take the supernatant and dilute it to an appropriate multiple, and use the molybdenum antimony anti-colorimetric method to measure the soluble phosphorus content. The calculation formula is: Y = m×15.91×A, where Y is the soluble phosphorus content (mg / L) in the fermentation broth, m is the dilution multiple, and A is the absorbance value at 660 nm. After detection, the phosphorus solubilization amount of Trichoderma harzianum Ta97 in 7 days is 525.26 ± 22.70 mg / L. Example 2

[0044] Determination of IAA production of Arthrobacter ureafaciens Fp64

[0045] Activate the Fp64 strain on an LB solid plate and shake-culture it in an LB liquid medium at 30°C and 160 rpm to obtain a seed liquid (OD 600= 0.6). The seed liquid was inoculated into the IAA-producing liquid medium at an inoculation amount of 1% (the 1L medium contained: 10 g of glucose, 1.0 g of (NH4)2SO4, 2.0 g of K2HPO4, 0.5 g of MgSO4·7H2O, 0.1 g of NaCl, 0.5 g of yeast extract, 0.5 g of CaCO3, 0.5 g of L-tryptophan, and the pH was adjusted to 7.0), and cultured at 30 °C and 160 rpm for 72 h. The IAA-producing liquid medium without inoculating the strain was used as a control, with 3 biological replicates. The fermentation broth was centrifuged at 4 °C and 12,000 rpm for 20 min, and the supernatant was taken. The content of IAA in the fermentation supernatant was detected by GC-MS. After detection, the IAA yield of Arthrobacter ureafaciens Fp64 was 255.94 ± 13.25 mg / L. Example 3

[0046] The preparation process of the special biological substrate for strawberries of the present invention specifically includes the following steps:

[0047] Step 1: Mix corn straw with a length of 1 cm and washed cow dung in a ratio of 3:1, and adjust the water content to 30% to obtain the compost fermentation raw material.

[0048] Step 2: Place the compost fermentation raw material in a trough-type fermentation workshop for fermentation. The height of the material is more than 2 meters, and it is turned over and thrown once every 3 days. The time above 60 °C is 7 days. When the temperature is between 60 °C and 70 °C, intermittent ventilation is maintained. When the temperature is higher than 70 °C, the pile is immediately turned over and continuous ventilation is carried out. The material to be fermented is obtained.

[0049] Step 3: Continue to turn over and throw once every 2 days, with intermittent ventilation, and keep the temperature at 40 - 50 °C for 10 days to obtain the primary fermentation material.

[0050] Step 4: The Trichoderma agent is the fermentation powder of Trichoderma afroharzianum Ta97 with 1×10 9 cfu / g, and the preservation number is CGMCC No. 19930. The Trichoderma agent and the primary fermentation product are mixed evenly at a weight ratio of 1:10,000, and turned over and thrown once every 1 day, with intermittent ventilation, and kept at 25 °C - 35 °C for 10 days to obtain the secondary fermentation product.

[0051] Step 5: The Arthrobacter agent is the fermentation powder of Arthrobacter ureafaciens Fp64 with 6×10 9 cfu / g, and the preservation number is CGMCC No. 15644. The Arthrobacter agent and the secondary fermentation product are mixed evenly at a weight ratio of 1:1000 to obtain the mixture A. The vermiculite, coconut fiber and perlite are mixed evenly at a volume ratio of 5:2:1 to obtain the mixture B. The mixture B is added to the mixture A, and the volume ratio of the mixture A to the mixture B is 2:1 and mixed evenly to prepare the special biological substrate for strawberries.

[0052] The physicochemical and biological indicators of the special biological substrate for strawberries prepared in this example are as follows: particle size 13.52 ± 2.32 mm, water content 46.25 ± 6.23%, total porosity 69.25 ± 5.25 %, bulk density 0.25 ± 0.01 g / cm 3 , available phosphorus content 230.42 ± 24.62 mg / kg, organic matter content 77.32% ± 4.42%, EC value 0.5, pH 5.8 ± 0.4, concentration of Trichoderma afroharzianum Ta97 5×10 8 cfu / g, concentration of Arthrobacter ureafaciens Fp64 6×10 6 cfu / g.

[0053] The special biological substrate for strawberries prepared in this example requires 27 days for fermentation and a total of 28 days for substrate preparation. Example 4

[0054] The preparation process of the special biological substrate for strawberries of the present invention specifically includes the following steps:

[0055] Step 1: Mix corn straw cut into 2 cm lengths and washed cow dung in proportion, and adjust the water content to 40% to obtain the raw material for compost fermentation;

[0056] Step 2: Place the raw material for compost fermentation in a trough-type fermentation workshop for fermentation. The height of the material is more than 2 meters, and it is turned over and aerated every 4 days. The time above 60°C is 7 days. When the temperature is between 60°C and 70°C, intermittent ventilation is maintained. When the temperature is higher than 70°C, it is immediately turned over and continuous ventilation is maintained. The material to be fermented is obtained.

[0057] Step 3: Continue to turn over and aerate every 3 days, and maintain intermittent ventilation. Keep the temperature at 40 - 50°C for 10 days to obtain the primary fermented material.

[0058] Step 4: The Trichoderma agent is a fermentation powder of Trichoderma afroharzianum Ta97 with a concentration of 1×10 9 cfu / g, and the preservation number is CGMCC No. 19930. The Trichoderma agent and the primary fermentation product are mixed evenly at a weight ratio of 1:10000, turned over and aerated every 2 days, and maintained at 25°C - 40°C for 15 days to obtain the secondary fermentation product.

[0059] Step 5: The Arthrobacter agent is 6×10 9Arthrobacter ureafaciens Fp64 fermentation powder with a cfu / g of 6×10⁶, preservation number CGMCC No. 15644. The Arthrobacter agent and the secondary fermentation product are mixed evenly at a weight ratio of 1:1000 to obtain mixture A. Vermiculite, coconut fiber and perlite are mixed evenly at a volume ratio of 6:3:2 to obtain mixture B. Mixture B is added to mixture A, and mixture A and mixture B are mixed evenly at a volume ratio of 5:2 to prepare a special biological substrate for strawberries.

[0060] The special biological substrate for strawberries prepared in this example has the following physical, chemical and biological indicators: particle size 12.01 ± 3.90 mm, water content 51.04 ± 7.91%, total porosity 71.18 ± 9.55 %, bulk density 0.29 ± 0.01 g / cm 3 , available phosphorus content 250.02 ± 45.84 mg / kg, organic matter content 78.37% ± 8.73%, EC value 0.6, pH 6.2 ± 0.1, concentration of Trichoderma afroharzianum Ta97 4×10 8 cfu / g, concentration of Arthrobacter ureafaciens Fp64 6×10 6 cfu / g.

[0061] The special biological substrate for strawberries prepared in this example requires 32 days for fermentation and a total of 33 days for substrate preparation.

[0062] Comparative Example 1

[0063] Replace the Trichoderma afroharzianum Ta97 fermentation powder in step 4 of Example 4 with diatomaceous earth without any live bacteria, and the remaining steps are the same as in Example 4 to obtain the substrate of Comparative Example 1.

[0064] Comparative Example 2

[0065] Replace the Arthrobacter ureafaciens Fp64 fermentation powder in step 5 of Example 4 with diatomaceous earth without any live bacteria, and the remaining steps are the same as in Example 4 to obtain the substrate of Comparative Example 2.

[0066] Comparative Example 3

[0067] Step 4 in Example 4 is replaced with:

[0068] Step 4. The Trichoderma agent is Trichoderma afroharzianum Ta97 fermentation powder with a cfu / g of 1×10⁶, preservation number CGMCC No. 19930. The Trichoderma agent and the primary fermentation product are mixed evenly at a weight ratio of 1:10000. The Arthrobacter agent is 6×10 9 cfu / g. 9Arthrobacter ureafaciens Fp64 fermentation powder with cfu / g, preservation number CGMCC No. 15644, the Arthrobacter agent and the primary fermentation product are mixed evenly at a weight ratio of 1:1000. It is turned and tossed every 2 days, with intermittent ventilation, and maintained at 25°C to 40°C for 15 days to obtain the secondary fermentation product.

[0069] Step 5 in Example 4 is replaced with:

[0070] Step 5, a mixture of vermiculite, coconut coir and perlite evenly mixed at a volume ratio of 6:3:2, the mixture is added to the secondary fermentation product, and the volume ratio of the secondary fermentation product to the mixed material is 5:2 and mixed evenly.

[0071] The remaining steps are the same as in Example 4 to obtain the substrate of Comparative Example 3.

[0072] Effect verification test

[0073] (I) Using the strawberry-specific biological substrates prepared in Example 3 and Example 4 as treatments, the substrates prepared in Comparative Example 1, Comparative Example 2, and Comparative Example 3, and the commercially available strawberry-specific substrate purchased (the volume ratio of peat, coconut coir, vermiculite, and perlite is 8:2:4:1) as controls, strawberries are planted in a greenhouse. The strawberry variety is Snow White, and all treatments are placed in the same environment and planted and managed uniformly. During the growth process of strawberries, 30 days after transplantation, 20 strawberry seedlings are randomly selected from each treatment to measure the growth indicators. After the strawberries are mature, the number of fruits per plant and the single fruit weight are counted, the soluble sugar and vitamin C contents are detected, and the average value and difference statistics are calculated. The specific results are shown in Table 1:

[0074] Table 1 Promoting growth effect of strawberry-specific biological substrate on strawberries ( p <0.05)

[0075]

[0076] The strawberry-specific biological substrate of the present invention has a significant promoting growth effect on strawberries and significantly improves the flavor quality of strawberries.

[0077] (II) Take the rhizosphere soil of strawberries from each treatment and control in the examples and comparative examples, extract total DNA, and use 16S amplicon high-throughput sequencing to analyze the bacterial flora diversity. The results are shown in Table 2:

[0078] Table 2 Improvement effect of strawberry-specific biological substrate on strawberry rhizosphere microbial diversity ( p <0.05)

[0079]

[0080] The special biological substrate for strawberries of the present invention significantly improves the diversity of the strawberry rhizosphere flora and improves the strawberry rhizosphere microecological environment.

Claims

1. Application of a mixed bacterial ferment in the fermentation preparation of a special biological substrate for strawberry cultivation, characterized in that, The mixed starter is composed of starter A and starter B; the starter A is Trichoderma harzianum Ta97 fermentation powder ( Trichoderma afroharzianum ), with the preservation number of CGMCC No. 19930; the starter B is Arthrobacter ureafaciens Fp64 fermentation powder ( Arthrobacter ureafaciens ), with the preservation number of CGMCC No. 15644; Specifically, it includes the following steps: (1) Mix corn straw and washed cow dung in proportion and adjust the water content to obtain the compost fermentation raw material; (2) Conduct primary fermentation on the compost fermentation raw material to obtain the primary fermentation product; (3) Add the fermentation powder of Trichoderma afroharzianum Ta97 to the primary fermentation product and conduct secondary fermentation to obtain the secondary fermentation product; (4) Add the fermentation powder of Arthrobacter ureafaciens Fp64 to the secondary fermentation product to obtain mixture A; (5) Mix vermiculite, coconut coir and perlite evenly to obtain mixture B; (6) Mix mixture A and mixture B in proportion to obtain the biological substrate.

2. The application according to claim 1, wherein In step (1), the particle size of the corn straw is 1 - 2 cm; the volume ratio of the corn straw to the washed cow dung is 2.5 - 4.5 : 1; the water content is adjusted to 30 - 40%.

3. The application according to claim 1, wherein In step (2), the specific process of the primary fermentation is as follows: Place the compost fermentation raw material in a trough - type fermentation workshop for fermentation, with the material height above 2 meters, turn and toss once every 3 - 4 days, the time at a temperature above 60°C is 7 days. When the temperature is between 60 - 70°C, maintain intermittent ventilation. When the temperature is above 70°C, turn the pile immediately and keep continuous ventilation to obtain the material to be fermented. Then continue to turn and toss once every 2 - 3 days, with intermittent ventilation, and keep the temperature at 40 - 50°C for 10 days to obtain the primary fermentation material.

4. The application according to claim 1, characterized in that In step (3), the total number of colonies of the Trichoderma harzianum Ta97 fermentation powder is 1×10 9 cfu / g; the weight ratio of the Trichoderma harzianum Ta97 fermentation powder to the primary fermentation product is 1:10000; the process of the secondary fermentation is: turning and throwing once every 1 day, intermittent ventilation, and maintaining at 25-35°C for 10 days.

5. The application according to claim 1, wherein In step (4), the total number of colonies of the Arthrobacter ureafaciens Fp64 fermentation powder is 6×10 9 cfu / g; the weight ratio of the Arthrobacter ureafaciens Fp64 fermentation powder to the secondary fermentation product is 1:1000.

6. The application according to claim 1, wherein In step (5), the vermiculite, coconut coir and perlite are mixed in a volume ratio of 5 - 6 : 2 - 3 : 1 - 2.

7. The application according to any one of claims 1-6, characterized in that, In step (6), the volume ratio of mixture A and mixture B is 2 - 5 : 1 - 2.

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