A co-culture system of paenibacillus polymyxa and hyphomicrobium hydropicin and application thereof

Abstract

The application discloses application of Paenibacillus polymyxa in improving yield of pyrroloquinoline quinone (PQQ), and application of a living silk microorganism in improving nitrogen fixation capacity of Paenibacillus polymyxa. The application also discloses a co-cultivation system with high PQQ yield. The application further discloses the living silk microorganism with a preservation number of CCTCC NO: M 20241604. The application further discloses Paenibacillus polymyxa with a preservation number of CCTCC NO: M 20241603. The application further discloses a fermentation liquor prepared by using the co-cultivation system. The application further discloses application of the co-cultivation system and the fermentation liquor. The application can promote yield of PQQ produced by the living silk microorganism and improve nitrogenase activity of Paenibacillus polymyxa simultaneously in a bidirectional way by co-cultivating and fermenting the living silk microorganism and Paenibacillus polymyxa, and the co-cultivation fermentation liquor can improve crop nitrogen fixation, stress resistance, growth and yield.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, and particularly relates to a co-culture system of bacillus polymyxa and hyphomicrobium and application thereof. BACKGROUND

[0002] Pyrroloquinoline quinone (PQQ) is the third coenzyme discovered after nicotinamide and riboflavin, which participates in electron transfer in redox reactions and has special biological activity and physiological function. Studies have shown that PQQ, as an important coenzyme and biological catalyst, is widely distributed in various biological cells and has biological functions such as scavenging free radicals, repairing cell oxidative damage, and improving the growth and metabolism of organisms. PQQ has shown significant effects on promoting plant growth and improving stress resistance. According to many domestic and foreign studies, PQQ can significantly enhance the survival ability of plants in extreme environments by activating the activity of antioxidant enzymes. At the same time, in the case of cold stress, PQQ can significantly reduce the adverse effects of low temperature environment on cucumber seedlings by improving the activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX) and effectively preventing the decrease of glutathione (GSH) content. In addition, during the vigorous physiological metabolism and peak of small flower degeneration of winter wheat, spraying PQQ can significantly improve the chlorophyll content in leaves, and then improve the leaf photosynthetic rate and reduce the spike flower abortion. At the same time, low-concentration PQQ in the modes of coating, seed dressing and spraying can improve the synergistic supply of organic and inorganic nutrients of plants to improve their stress resistance and disease resistance. The mechanism may be related to the direct participation of PQQ in DNA chain break repair caused by stress and the antioxidant effect of the plant itself, which indirectly improves the tolerance of plants to stress.

[0003] In addition, PQQ is also an important plant growth promoter, which can accelerate the synthesis of auxins and cytokinins by promoting the metabolism of plant cells, thereby shortening the plant growth lag phase and effectively improving the plant growth and production capacity. Studies have found that PQQ can significantly increase the content of growth-regulating substances such as indole acetic acid in tobacco seedling leaves and roots through leaf spraying or root irrigation fertilization. Spraying PQQ during the booting stage of winter wheat can effectively increase the chlorophyll content in the leaves, thereby improving the photosynthetic rate and enhancing the activities of nitrate reductase and glutathione transferase; this not only improves the organic and inorganic nutrient supply of winter wheat plants, but also regulates the physiological metabolism of the plants, reduces the abortion of small flowers, and improves the seed setting rate. At the same time, PQQ has significant antioxidant capacity, which can protect plant cells from oxidative damage and enhance the stress resistance of plants, so that they can maintain normal growth and yield under adverse conditions; studies have shown that PQQ can effectively increase the number of filled grains per panicle and the seed setting rate of rice, and reduce the number of empty grains per panicle, which has a positive promoting effect on rice yield. This is mainly due to the positive regulation of PQQ on plant growth during the booting, flowering and grain filling periods, which can enhance the photosynthetic intensity of plants, promote nutrient transfer to grains, prolong the functional period of leaves, and increase the grain number and weight, thereby achieving yield increase. In addition to rice, the yield-increasing effect of PQQ on other crops is also expected. Some studies have also shown that PQQ can promote the absorption of organic phosphorus in the soil by crops, increase the availability of phosphate in the soil, and thus promote the growth and yield of crops. In addition, PQQ also acts as a coenzyme for dehydrogenase of plant growth-promoting rhizobacteria (PGPR), which helps to acidify the soil environment and convert insoluble phosphorus into soluble phosphorus, which is more easily absorbed and utilized by plants.

[0004] Hyphomicrobium vulgare is a microorganism that can produce PQQ; however, the synthesis rate of PQQ by Hyphomicrobium vulgare single strain fermentation is slow, the yield is not high, and it is difficult to scale up production, so it is necessary to improve the PQQ yield. SUMMARY

[0005] Based on the technical problems existing in the background art, the present application provides a co-culture system of Paenibacillus polymyxa and Hyphomicrobium vulgare and its application. The co-culture fermentation system of Paenibacillus polymyxa and Hyphomicrobium vulgare discovered by the present application can bidirectionally promote the yield of PQQ produced by Hyphomicrobium vulgare and enhance the nitrogenase activity of Paenibacillus polymyxa. The obtained co-culture fermentation broth provides a new effective way to enhance the nitrogen fixation, stress resistance and production capacity of crops, and is easy to operate, easy to scale up production and application, and has a wide application prospect.

[0006] The present application provides an application of Paenibacillus polymyxa in improving the yield of pyrroloquinoline quinone.

[0007] Preferably, the application in improving the production of pyrrolquinoline quinone of Lechevalieria.

[0008] Preferably, the Paenibacillus polymyxa is Paenibacillus polymyxa (X136001) with the preservation number of CCTCC NO: M 20241603. Paenibacillus polymyxa

[0009] Preferably, the Lechevalieria is Lechevalieria (X119001) with the preservation number of CCTCC NO: M 20241604. Hyphomicrobium vulgare

[0010] The application further provides an application of Lechevalieria in improving the nitrogen fixation capacity of Paenibacillus polymyxa.

[0011] Preferably, the application in improving the nitrogenase activity of Paenibacillus polymyxa.

[0012] Preferably, the Paenibacillus polymyxa is Paenibacillus polymyxa (X136001) with the preservation number of CCTCC NO: M 20241603. Paenibacillus polymyxa

[0013] Preferably, the Lechevalieria is Lechevalieria (X119001) with the preservation number of CCTCC NO: M 20241604. Hyphomicrobium vulgare

[0014] The application further provides a co-culture system with high pyrrolquinoline quinone production, comprising: co-culturing Paenibacillus polymyxa and Lechevalieria.

[0015] Preferably, the Paenibacillus polymyxa is Paenibacillus polymyxa (X136001) with the preservation number of CCTCC NO: M 20241603. Paenibacillus polymyxa

[0016] Preferably, the Lechevalieria is Lechevalieria (X119001) with the preservation number of CCTCC NO: M 20241604. Hyphomicrobium vulgare

[0017] The application further provides Lechevalieria (X119001) with the preservation number of CCTCC NO: M 20241604. Hyphomicrobium vulgare The application further provides Paenibacillus polymyxa (X136001) with the preservation number of CCTCC NO: M 20241603.

[0018] Paenibacillus polymyxa The application further provides a fermentation liquor prepared by using the co-culture system.

[0019] The application further provides a fermentation liquor prepared by using the co-culture system.​​​​​​​

[0020] The application further provides a preparation method of the fermentation liquor, comprising the following steps: co-culturing Paenibacillus polymyxa and Hyphomicrobium in a fermentation medium to obtain the fermentation liquor.

[0021] Preferably, the fermentation medium comprises at least one of an assimilable carbon source and an assimilable nitrogen source.

[0022] Preferably, the assimilable carbon source is one of formic acid and a mixture of formic acid and substance A, methanol and methylamine, wherein the substance A is at least selected from one or more of the following: methylamine, ethanol, glycerol, acetic acid, mannitol, starch, malt dextrin, glucose, sucrose, lactose, maltose, molasses, soybean oil and sorbitol.

[0023] Preferably, the assimilable nitrogen source is at least selected from one or more of the following: yeast extract powder, yeast powder, yeast paste, soybean lecithin, soybean meal, cottonseed meal, peanut meal, gluten meal, corn steep liquor dry powder, soybean meal, peptone, urea, ammonium salt and nitrate.

[0024] The application further provides application of the co-culturing system and the fermentation liquor in improving the stress resistance, growth rate and yield of crops.

[0025] Preferably, the crops include food crops and economic crops.

[0026] The application finds that in the co-culturing system, Paenibacillus polymyxa can significantly promote Hyphomicrobium to express pyrroloquinoline quinone and improve the yield of pyrroloquinoline quinone, and the application also finds that Hyphomicrobium can also enhance the nitrogenase activity of Paenibacillus polymyxa; the obtained co-culturing fermentation liquor can be used as an agricultural green input in the planting process of crops such as wheat and corn to improve the nitrogen fixation, stress resistance, growth rate and yield of crops; the application solves the problems of slow synthesis rate, low yield and difficult large-scale production of pyrroloquinoline quinone in the single-bacterium fermentation of Hyphomicrobium; and the nitrogenase activity of Paenibacillus polymyxa can be improved at the same time, thereby providing a new effective way for enhancing the nitrogen fixation, stress resistance and production capacity of crops.

[0027] The application screens specific Hyphomicrobium and Paenibacillus polymyxa, and the co-culturing fermentation system of the two has high pyrroloquinoline quinone synthesis efficiency, significantly increased yield and significantly improved nitrogenase activity, is easy to operate, easy to produce and apply on a large scale, has wide application prospects, and the fermentation liquor can be used in crop planting. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 A plate colony photo of Paenibacillus polymyxa (P. Paenibacillus polymyxa ) X136001 strain.

[0029] Figure 2 For the silk microbacteria Hyphomicrobium vulgare X119001 strain plate colony photos.

[0030] Figure 3 For the PQQ content HPLC detection spectrum of the fermentation liquor obtained in Comparative Example 1 and Example 2, wherein A is Comparative Example 1, and B is Example 2.

[0031] Figure 4 For the yield results of pyrroloquinoline quinone in the fermentation liquor of Comparative Example 1 and Example 2, wherein the single-strain fermentation group is Comparative Example 1, and the co-culture fermentation group is Example 2.

[0032] Figure 5 For the nitrogenase activity of the strain cells in the single-strain fermentation of Paenibacillus polymyxa, the single-strain fermentation of silk microbacteria, and the co-culture fermentation of the two strains. DETAILED DESCRIPTION

[0033] The application provides application of Paenibacillus polymyxa in improving yield of pyrroloquinoline quinone.

[0034] Preferably, the application is in improving yield of pyrroloquinoline quinone of silk microbacteria.

[0035] Preferably, the Paenibacillus polymyxa is Paenibacillus polymyxa (X136001) with a preservation number of CCTCC NO: M 20241603. Paenibacillus polymyxa

[0036] Preferably, the silk microbacteria is silk microbacteria (X119001) with a preservation number of CCTCC NO: M 20241604. Hyphomicrobium vulgare

[0037] The application further provides application of silk microbacteria in improving nitrogen fixation capacity of Paenibacillus polymyxa.

[0038] Preferably, the application is in improving nitrogenase activity of Paenibacillus polymyxa.

[0039] Preferably, the Paenibacillus polymyxa is Paenibacillus polymyxa (X136001) with a preservation number of CCTCC NO: M 20241603. Paenibacillus polymyxa Preferably, the silk microbacteria is silk microbacteria (X119001) with a preservation number of CCTCC NO: M 20241604.

[0040] Hyphomicrobium vulgare

[0041] The application further provides a co-culture system with high yield of pyrroloquinoline quinone, which comprises: co-culture of Paenibacillus polymyxa and silk microbacteria.​​​​

[0042] Preferably, the Paenibacillus polymyxa is Paenibacillus polymyxa (X136001) with a preservation number of CCTCC NO: M 20241603. Paenibacillus polymyxa

[0043] Preferably, the Siderocapsa silvisoli is Siderocapsa silvisoli (X119001) with a preservation number of CCTCC NO: M 20241604. Hyphomicrobium vulgare

[0044] The present application also provides a Siderocapsa silvisoli (X119001) with a preservation number of CCTCC NO: M 20241604. Hyphomicrobium vulgare The present application also provides a Paenibacillus polymyxa (X136001) with a preservation number of CCTCC NO: M 20241603.

[0045] Paenibacillus polymyxa The present application also provides a Paenibacillus polymyxa (X136001) with a preservation number of CCTCC NO: M 20241603.

[0046] The present application screens Paenibacillus polymyxa (X136001) and Siderocapsa silvisoli (X119001) from the soil of farmland in Wangzhuang Village, Lanban Town, E'qiao District, Suzhou City, Anhui Province, and deposits them in China Center for Type Culture Collection (CCTCC) on July 18, 2024, which is located in Wuhan University, Wuhan, China. Paenibacillus polymyxa Hyphomicrobium vulgare The preservation number of Paenibacillus polymyxa (X136001) is CCTCC NO: M 20241603, and the preservation number of Siderocapsa silvisoli (X119001) is CCTCC NO: M 20241604. Paenibacillus polymyxa Hyphomicrobium vulgare The preservation number of Paenibacillus polymyxa (X136001) is CCTCC NO: M 20241603, and the preservation number of Siderocapsa silvisoli (X119001) is CCTCC NO: M 20241604.

[0047] Traditional microbial fermentation methods mainly rely on a single strain to synthesize target products or obtain live bacterial products, but this method often increases the metabolic burden of the strain due to the introduction of key coenzyme factors and target metabolic product precursors, thereby affecting the yield of the target product.

[0048] In view of the above problems, the inventors accidentally found that co-culture fermentation of Paenibacillus polymyxa and Siderocapsa silvisoli can simultaneously bidirectionally promote the yield of PQQ produced by Siderocapsa silvisoli and enhance the nitrogenase activity of Paenibacillus polymyxa.

[0049] In particular, the PQQ yield of the co-culture system of Paenibacillus polymyxa X136001 and Siderocapsa silvisoli X119001 is significantly increased compared to the single culture of Siderocapsa silvisoli X119001, and the nitrogenase activity is significantly increased compared to the single culture of Paenibacillus polymyxa X136001.

[0050] ​​​​​The mechanism of the simultaneous bidirectional promotion of the production of PQQ by Hyphomicrobium sp. and the improvement of the nitrogenase activity of Paenibacillus polymyxa by the co-culture fermentation of Paenibacillus polymyxa and Hyphomicrobium sp. involves multiple interactions and synergistic effects: first, Paenibacillus polymyxa can produce some hydrolytic enzymes capable of decomposing complex organic matter, thereby providing more available nutrients for Hyphomicrobium sp., thereby enhancing the growth and metabolic capacity of Hyphomicrobium sp.; at the same time, Paenibacillus polymyxa can also adjust the pH value, temperature and other environmental factors of the co-culture system to create more suitable growth conditions for Hyphomicrobium sp., and the optimization of these environmental factors helps Hyphomicrobium sp. to better exert its potential for PQQ fermentation; at the same time, the PQQ synthesized by Hyphomicrobium sp. in the co-culture system can also act as a coenzyme to promote the growth and reproduction, metabolic activity and expression of functional genes such as nitrogenase structural genes of Paenibacillus polymyxa; in addition, in the co-culture process, Paenibacillus polymyxa and Hyphomicrobium sp. can also recognize and coordinate the growth and metabolic activity of each other through the quorum sensing mechanism, thereby achieving the improvement of PQQ production and nitrogen fixation capacity, which provides a new effective way for improving crop nitrogen fixation, stress resistance and production capacity.

[0051] The present application also provides a fermentation broth prepared by the co-culture system.

[0052] The present application also provides a preparation method of the fermentation broth, which comprises the following steps: co-culturing Paenibacillus polymyxa and Hyphomicrobium sp. in a fermentation medium to obtain the fermentation broth.

[0053] Preferably, the fermentation medium comprises at least one of an assimilable carbon source and an assimilable nitrogen source.

[0054] Preferably, the assimilable carbon source is one of a mixture of formic acid and substance A, methanol and methylamine, wherein the substance A is at least selected from one or more combinations of methylamine, ethanol, glycerol, acetic acid, mannitol, starch, malt dextrin, glucose, sucrose, lactose, maltose, molasses, soybean oil and sorbitol.

[0055] Preferably, the assimilable nitrogen source is at least selected from one or more combinations of yeast extract powder, yeast powder, yeast paste, soybean lecithin, soybean cake powder, cottonseed cake powder, peanut cake powder, gluten powder, corn syrup dry powder, soybean meal, peptone, urea, ammonium salt and nitrate.

[0056] More preferably, the fermentation medium contains methanol, and the content of methanol is 1.5-2.5 wt%, more preferably 2 wt%.

[0057] The Paenibacillus polymyxa and Hyphomicrobium sp. are activated and seed liquid cultured respectively, and then inoculated into the fermentation medium together.

[0058] The activation culture medium of the said Lysinibacillus sphaericus and Paenibacillus polymyxa is the same.

[0059] The formula of the activation culture medium comprises: NaCl 0.5~1.5wt%, yeast powder 0.4~0.6wt%, tryptone 0.5~1.5wt%, and the rest is water, pH=7.0.

[0060] The preferred formula of the activation culture medium comprises: NaCl 1wt%, yeast powder 0.5wt%, tryptone 1wt%, and the rest is water, pH=7.0.

[0061] The seed liquid culture medium of the said Lysinibacillus sphaericus and Paenibacillus polymyxa is the same.

[0062] The formula of the seed liquid culture medium comprises: sucrose 2~4wt%, yeast powder 1.2~1.6wt%, NaH2PO40.03~0.05wt%, Na2HPO40.01~0.015wt%, sodium chloride 0.3~0.4wt%, MgSO4·7H2O 0.05~0.07wt%, antifoam agent 0.08~0.12wt%, and the rest is water.

[0063] The preferred formula of the seed liquid culture medium comprises: sucrose 3wt%, yeast powder 1.4wt%, NaH2PO40.04wt%, Na2HPO40.012wt%, sodium chloride 0.35wt%, MgSO4·7H2O 0.06wt%, antifoam agent 0.1wt%, and the rest is water.

[0064] The formula of the fermentation culture medium comprises: (NH4)2SO40.2~0.4wt%, KH2PO40.1~0.2wt%, Na2HPO40.2~0.4wt%, MgSO4·7H2O 0.05~0.15wt%, trace element solution 0.05~0.1wt%, methanol 1.5~2.5wt%, and the rest is water.

[0065] The preferred formula of the fermentation culture medium comprises: (NH4)2SO40.3wt%, KH2PO40.14wt%, Na2HPO40.3wt%, MgSO4·7H2O 0.1wt%, trace element solution 0.07wt%, methanol 2wt%, and the rest is water.

[0066] The formula of the trace element solution comprises: FeSO4·7H2O 7~8 g, ZnSO4·7H2O 22~23 g, MnSO4·4~5H2O 4~5 g, CuSO4·5H2O 0.7~0.8 g, NaCl 1~2 g, (NH4)6Mo7O 24• 4H2O 25~35 mg, KI 25~35 mg, CoCl2-6H2O 25~35 mg, H3BO3 25~35 mg, CaCl2-2H2O 28~32 g, water 1000 mL.

[0067] Preferably, the trace element solution has the following formula: FeSO4-7H2O 7.5 g, ZnSO4-7H2O 22.5 g, MnSO4-4-5H2O 4.5 g, CuSO4-5H2O 0.75 g, NaCl 1.5 g, (NH4)6Mo7O 24 • 4H2O 30 mg, KI 30 mg, CoCl2-6H2O 30 mg, H3BO3 30 mg, CaCl2-2H2O 30 g, water 1000 mL.

[0068] The trace element solution is filtered through a 0.22 μm filter before use.

[0069] The activation medium, seed medium and fermentation medium are sterilized. The water can be sterile water or purified water.

[0070] The activation conditions for the living Lysinibacillus sphaericus and Paenibacillus polymyxa are the same, i.e., the activation temperature is 28~32°C, preferably 30°C; the rotation speed is 100~300 rpm, preferably 200 rpm; and the activation time is 10~15 h, preferably 12 h.

[0071] The seed culture conditions for the living Lysinibacillus sphaericus are as follows: the culture temperature is 28~32°C, preferably 30°C; the inoculation amount is 4~6%, preferably 5%; the initial rotation speed is 50~150 rpm, preferably 100 rpm; the pH of the medium is adjusted to 6.5~7.0, preferably 6.8, by using ammonia water during the culture; the dissolved oxygen is >10% during the culture; the aeration amount is ≤1:1, preferably 0.5~1:1 during the culture; and the culture is continued until the concentration of the Lysinibacillus sphaericus reaches 10 8 The seed culture can be used in the fermentation.

[0072] The seed culture conditions for the Paenibacillus polymyxa are as follows: the culture temperature is 28~32°C, preferably 30°C; the inoculation amount is 8~12%, preferably 10%; the initial rotation speed is 100~200 rpm, preferably 150 rpm; the pH of the medium is adjusted to 7.0~7.2, preferably 7.1, by using NaOH during the culture; the dissolved oxygen is >40% during the culture; the aeration amount is ≤1:1, preferably 0.5~1:1 during the culture; and the culture is continued until the concentration of the Paenibacillus polymyxa reaches 10 8As a backup, it can be transferred into the fermentation broth, and the culture time is preferably 10-12 h.

[0073] The fermentation temperature is 28-32 DEG C, preferably 30 DEG C, the initial rotation speed is 50-150 rpm, preferably 100 rpm, the pH of the fermentation broth is controlled to 6.5-7.0 by adding ammonia water, preferably 6.8, the dissolved oxygen is greater than 10% during the co-culture fermentation, the aeration amount is less than or equal to 1:1, preferably 0.5-1:1, and the fermentation is ended when the methanol consumption rate significantly slows down, preferably for 160-300 h, more preferably for 240 h.

[0074] During the co-culture fermentation, the methanol concentration in the fermentation broth can be adjusted by adding 50% methanol solution to control the methanol concentration in the fermentation broth in the range of 0.05-0.1 wt%.

[0075] During the co-culture fermentation, the seed liquid is composed of the silk microbacterial seed liquid and the bacillus polymyxa seed liquid in a volume ratio of 1:1, and the seed liquid inoculation amount is 4-6%, preferably 5%.

[0076] The co-culture conditions of the silk microbacteria and the bacillus polymyxa are optimized, the PQQ yield is improved, and the nitrogen fixation capacity is improved.

[0077] In the co-culture system, the cell growth and metabolism process is stable, and the fermentation production is taken as an example in a 20 L fermentation tank, methanol is used as a carbon source, and 50% methanol solution is added during the fermentation to maintain the methanol concentration in the range of 0.05-0.1 wt%, and the total feeding volume of the methanol solution in a fermentation cycle is 50% of the initial fermentation liquid volume, and the methanol consumption amount in different batches has little difference; further scaling up to 100 L fermentation tank production, the total feeding volume of the methanol solution in a fermentation cycle is 50% of the initial fermentation liquid volume, and no significant change is shown.

[0078] During the co-culture fermentation, the yield of pyrroloquinoline quinone in the co-culture fermentation broth is monitored by regularly sampling and using a liquid chromatograph.

[0079] The pretreatment step of the fermentation broth includes: taking 2 mL of the fermentation broth, centrifuging at 10000 r for 20 min to obtain the fermentation supernatant.

[0080] The detection method of the liquid chromatograph was as follows: the chromatographic column was YMC-Pack A-302 ODS (4.6 mm × 150 mm, 5 μm); the mobile phase was 0.1 M CH3COOH aqueous solution: 0.1 M CH3COONH4 aqueous solution = 30:70 (v / v), pH=5.1; a UV detector was used with a detection wavelength of 259 nm; the column oven temperature was 40 ℃; isocratic elution was used with a mobile phase flow rate of 1.5 mL / min and an injection volume of 20 μL.

[0081] Nitrogenase activity was detected by acetylene reduction method.

[0082] This invention also proposes the application of the above-mentioned co-culture system and fermentation broth in improving crop stress resistance and increasing crop yield.

[0083] This invention involves the co-culturing of *Bacillus polymyxa* and *Microphytes filamentosa*. This co-culturing system can improve the stress resistance of crops and increase crop yields. By optimizing the co-culturing conditions of *Microphytes filamentosa* and *Bacillus polymyxa*, this invention increases the yield of pyrroloquinoline quinone and the activity of nitrogenase, while reducing production costs. Applying the co-culture fermentation broth to increase crop yields has improved the growth rate and yield of wheat and corn, showing broad application prospects. This invention is simple, easy to operate, and suitable for large-scale production and application.

[0084] Preferably, crops include food crops and cash crops.

[0085] The aforementioned food crops can include cereal crops, tuber crops, legume crops, etc.

[0086] Cereal crops such as wheat, rice, and corn; tuber crops such as sweet potatoes and potatoes; legume crops such as soybeans, broad beans, peas, and mung beans.

[0087] The aforementioned economic crops can include fiber crops, oil crops, sugar crops, beverage crops, spice crops, condiment crops, medicinal crops, dye crops, ornamental crops, and fruits.

[0088] Fiber crops such as cotton and hemp; oil crops such as sesame and peanuts; sugar crops such as sugarcane and sugar beets.

[0089] The above co-culture system and fermentation broth can be applied to crop leaves, roots, and stems.

[0090] The technical solution of the present invention will now be described in detail through specific embodiments.

[0091] Example 1

[0092] 1. Screening and isolation of Bacillus polymyxa X136001 and Microfibrillariae X119001

[0093] Take the soil sample from 0.5 m below the surface of farmland in Wangzhuang village, Lanban town, E'qiao district, Suzhou city, Anhui province, dilute the soil sample with physiological saline, gradient dilute and coat on the activated culture medium (the formula of the activated culture medium includes: NaCl 1wt%, yeast powder 0.5wt%, tryptone 1wt%, the rest is water, pH=7.0), culture under the condition of 30°C and 200 rpm, wait for single colony to grow on the plate, pick the single colony and inoculate into the seed liquid culture medium (the formula of the seed liquid culture medium includes: sucrose 3wt%, yeast extract 1.4wt%, NaH2PO4 0.04wt%, Na2HPO4 0.012wt%, sodium chloride 0.35wt%, MgSO4·7H2O 0.06wt%, antifoam 0.1wt%, the rest is water), culture under the condition of 30°C and initial rotation speed of 100 rpm, and in the process of culture, adjust the pH of the seed liquid culture medium to 6.8 with ammonia water, keep the dissolved oxygen >10%, the aeration amount is 0.5~1:1, and the culture is ended when the strain concentration reaches 10 8 Then, the PQQ content and nitrogenase activity in the supernatant of the culture solution are determined, and the PQQ-producing strain and the strain with relatively high nitrogenase activity are screened.

[0094] 2. Strain identification

[0095] Take the two single strains obtained in step 1 to describe the morphological characteristics and identify the species, and the results are as follows:

[0096] (1) The morphological characteristics of Paenibacillus polymyxa X136001 are shown in Figure 1 A, 1B, and it can be seen that the colony is rod-shaped after the strain is cultured on the solid plate medium at 30°C for 12 h.

[0097] (2) The morphological characteristics of Hyphomicrobium X119001 are shown in Figure 2 A, 2B, and it can be seen that the colony is short rod-shaped after the strain is cultured on the solid plate medium at 30°C for 12 h.

[0098] (3) The 16S rRNA gene sequence of the above-mentioned Paenibacillus polymyxa X136001 and Hyphomicrobium X119001 is determined, and the homology comparison and analysis results with the gene sequence in the GenBank database are carried out to determine the species of the bacteria from the molecular biology level.

[0099] Finally, one strain is determined as Paenibacillus polymyxa, which is named as Paenibacillus polymyxa (PQQ-1) Paenibacillus polymyxaX136001 was deposited on July 18, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20241603.

[0100] The other strain belongs to the genus Microphyte, and it is named Microphyte. Hyphomicrobium vulgare X119001 was deposited on July 18, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20241604.

[0101] Example 2

[0102] A method for preparing a co-culture fermentation broth of Bacillus polymyxa and filamentous microorganisms includes the following steps:

[0103] Take polymyxin Bacillus (Bacillus polymyxinus) stored in glycerol cryovials at -80℃. Paenibacillus polymyxa X136001, Raw Silk Microbes Hyphomicrobium vulgare For X119001, take 20 μL of each sample and inoculate them into 100 mL of activation medium (the activation medium consists of: NaCl 1 wt%, yeast extract 0.5 wt%, tryptone 1 wt%, and the remainder is water, pH=7.0). Activate to OD at 30℃ using a shaker (200 rpm). 600 Activation ends when the value is 0.4.

[0104] Take the activated culture medium as described above and inoculate it into seed culture medium (the formula of seed culture medium includes: 3wt% sucrose, 1.4wt% yeast extract, 0.04wt% NaH2PO4, 0.012wt% Na2HPO4, 0.35wt% sodium chloride, 0.06wt% MgSO4·7H2O, 0.1wt% defoamer, and the balance being water) for cultivation.

[0105] The inoculum size of the raw silk microbacterium X119001 was 5%, and it was cultured at 30℃ and an initial rotation speed of 100 rpm. During the culture, the pH was adjusted to 6.8 with saturated ammonia water, dissolved oxygen was maintained at >10%, and the aeration rate was 0.5~1:1. The culture was carried out until the biomass reached 10. 8 Then, the seed culture of raw silk microorganism X119001 was obtained and used for later use;

[0106] The inoculum size of *Bacillus polymyxa* X136001 was 5%, and the culture was carried out at 30℃ and an initial agitation speed of 150 rpm. During the culture, the pH was adjusted to 7.1 with sodium hydroxide aqueous solution, dissolved oxygen was maintained at >40%, and the aeration rate was 0.5~1:1. The culture was carried out until the biomass reached 10⁶.8 Afterwards, Paenibacillus polymyxa X136001 seed liquid was obtained for standby use.

[0107] The seed liquid of Microspora X119001 and the seed liquid of Paenibacillus polymyxa X136001 were mixed at a volume ratio of 1:1 to form a seed liquid, and the total inoculation amount was 5% which was inoculated into the co-culture fermentation medium (the formula of the fermentation medium included: (NH4)2SO40.3wt%, KH2PO40.14wt%, Na2HPO40.3wt%, MgSO4·7H2O 0.1wt%, trace element solution 0.07wt%, methanol 2wt%, and the rest was water), and the culture was carried out at 30°C and the initial rotation speed was 100 rpm, and during the culture process, the pH was adjusted to 6.8 with saturated ammonia water, the dissolved oxygen was kept >10%, the aeration amount was 0.5~1:1, and during the culture process, 50% methanol aqueous solution was supplemented to control the methanol concentration in the fermentation broth to be 0.05~0.1wt%, and when the methanol consumption rate significantly slowed down, the fermentation was ended, and the fermentation time was 240h, and the co-culture fermentation broth of Paenibacillus polymyxa and Microspora was obtained.

[0108] Comparative Example 1

[0109] The seed liquid of Microspora X119001 of Example 2 was fermented alone, and the fermentation conditions were the same as those of the co-culture fermentation, and the fermentation broth was obtained.

[0110] During the co-culture fermentation process, the yield of pyrroloquinoline quinone (PQQ) in the co-culture fermentation broth was monitored by taking samples regularly and using a liquid chromatograph. 2mL of the fermentation broth was centrifuged at 10000r for 20min by a centrifuge to obtain the fermentation supernatant, which was detected by a liquid chromatograph. And compared with the fermentation broth of Comparative Example 1. The results are shown in Figures 3-4 .

[0111] Figure 3 The HPLC detection spectrum of the PQQ content of the fermentation broth obtained in Comparative Example 1 and Example 2, wherein A is Comparative Example 1, and B is Example 2.

[0112] Figure 4 The yield of pyrroloquinoline quinone in the fermentation broth of Comparative Example 1 and Example 2, wherein the single strain fermentation group is Comparative Example 1, and the co-culture fermentation group is Example 2.

[0113] From Figures 3-4It can be seen that: compared with the fermentation of L. silvestris X119001 alone, the co-culture fermentation of L. silvestris X119001 and P. polymyxa X136001 can significantly improve the yield of pyrroloquinoline quinone (PQQ); among them, when the fermentation time is 164 h, the PQQ yield of the single strain fermentation group is 626.4 mg / L, and the PQQ yield of the co-culture fermentation group can reach 1514.5 mg / L; when the fermentation time continues to 240 h, the PQQ yield of the co-culture fermentation group is still higher than that of the single strain fermentation group.

[0114] Example 3

[0115] Take the seed liquid of L. silvestris X119001, the seed liquid of P. polymyxa X136001, and the mixed seed liquid (consisting of the seed liquid of L. silvestris X119001 and the seed liquid of P. polymyxa X136001 in a volume ratio of 1:1) of Example 2, respectively inoculate 20 mL of fresh co-culture fermentation medium at a inoculation amount of 1%, and culture at 30°C, 160 rpm for 8 h. Collect the bacterial liquid, and then detect the nitrogenase activity of each group by the acetylene reduction method, respectively. The results are shown in Figure 5 .

[0116] Figure 5 The nitrogenase activity of the strain cells in the fermentation process of P. polymyxa single strain, L. silvestris single strain, and co-culture of the two strains.

[0117] From Figure 5 It can be seen that: when P. polymyxa X136001 is fermented alone, the nitrogenase activity of the bacterial cells is 358 nmol C2H4 / mg protein hr; when L. silvestris X119001 is fermented alone, the nitrogenase activity is close to 0; when the two strains are co-cultured, the nitrogenase activity of the strain can be as high as 556 nmol C2H4 / mg protein hr; thus, co-culture fermentation of the two strains can significantly improve the nitrogenase activity and thus play a better nitrogen fixation function.

[0118] Example 4 Wheat stress resistance influence experiment

[0119] In 2021-2022, in the agricultural science and technology demonstration and display base of Suzhou City E Bridge District, using Huacheng 3366 as the test variety, the wheat stress resistance influence experiment was carried out, and the specific steps included:

[0120] Take the co-culture fermentation liquid of P. polymyxa and L. silvestris obtained in Example 2, and the L. silvestris single culture fermentation liquid obtained in Comparative Example 1, respectively dilute 1000 times with water as bacterial fertilizer, and respectively spray the bacterial fertilizer on the leaves of wheat seedlings (spraying amount is 22.5 g / m 2) 10 h after, placed in 4℃ low temperature incubator for 3 days, then sample determination of superoxide dismutase (SOD) activity, peroxidase (POD) activity and malondialdehyde (MDA) content, each group set 3 times parallel sample; and set spray bacteria fertilizer 10 h after, at normal temperature (25℃) for 3 days as control group. The test results are shown in Table 1.

[0121] Table 1 Results of wheat resistance influence experiment

[0122]

[0123] As can be seen from Table 1, application of co-culture fermentation liquor can significantly improve the cold resistance of wheat seedlings, and the activities of superoxide dismutase and peroxidase in wheat seedlings are increased by 22.65-70.53% and 22.34-46.37% respectively, and the content of malondialdehyde is reduced by 26.12-35.26%, which is significantly better than the control group; the application of single culture fermentation liquor is better than the control group, but the effect is significantly lower than that of co-culture fermentation liquor.

[0124] Example 5 Influence on wheat net photosynthetic rate

[0125] In 2022-2023, in Lingbi County Yonghao Planting Professional Cooperative, with Huacheng 3366 as the test variety, an experiment on the influence of wheat net photosynthetic rate was carried out, and the specific steps included:

[0126] Take the co-culture fermentation liquor of Paenibacillus polymyxa and X. camusii obtained in Example 2 and the single culture fermentation liquor of X. camusii obtained in Comparative Example 1, dilute them 1000 times with water as bacterial fertilizer, and spray them on the leaves of wheat at the seedling stage, jointing stage (10 days after turning green) and filling stage (15 days after flowering) (spraying amount is 15 kg / mu), respectively. Measure the net photosynthetic rate of flag leaves at the heading stage, 10 days and 20 days after flowering, respectively. The sample size is 20 flag leaves each time, and each group sets 3 parallel samples. And use water to spray in the same way as the control group. The test results are shown in Table 2.

[0127] Table 2 Results of wheat net photosynthetic rate influence

[0128]

[0129] As can be seen from Table 2, application of co-culture fermentation liquor can significantly improve the net photosynthetic rate of wheat flag leaves, and the net photosynthetic rate of wheat flag leaves at the heading stage, 10 days and 20 days after flowering is increased by 6.65-18.35%, 7.31-18.39% and 5.51-21.27% respectively compared with the control group; the net photosynthetic rate of flag leaves sprayed with single culture fermentation liquor is better than that of the control group, but the effect is significantly lower than that of co-culture fermentation liquor.

[0130] Effect on wheat yield

[0131] In 2022-2023, in Lingbi County Yonghao Planting Professional Cooperative, with Xinnong 920 as the test wheat variety, an experiment on the effect on wheat yield was carried out, and the specific steps included:

[0132] The co-culture fermentation broth of Paenibacillus polymyxa and Hyphomicrobium obtained in Example 2 and the single culture fermentation broth of Hyphomicrobium obtained in Comparative Example 1 were diluted with water by 700, 1000, 1500 times respectively, and were recorded as microbial fertilizer 1, microbial fertilizer 2, microbial fertilizer 3 in turn, and water was used as the control group. At the wheat seedling stage (fertilizer amount was 10 kg / mu), booting stage (fertilizer amount was 20 kg / mu), and filling stage (fertilizer amount was 20 kg / mu), they were uniformly sprayed on the leaf surface. Each group was set up 3 parallel samples, and randomly arranged. At the harvest stage, the effective ear number, ear grain number, and thousand grain weight were investigated, and other management was consistent. The results are shown in Table 3.

[0133] Table 3 Results of the effect on wheat yield

[0134]

[0135] As can be seen from Table 3, the co-culture fermentation broth with different dilution multiples has a promoting effect on the ear grain number per plant, thousand grain weight, and yield of wheat. Compared with the control group, the ear grain number per plant increased by 1.46-5.96%, the thousand grain weight increased by 2.30-3.60%, and the yield increased by 15.30-35.60 kg / mu. The single culture fermentation broth was better than the control group, but the effect was significantly lower than that of the co-culture fermentation broth.

[0136] Example 7 Effect on corn yield

[0137] In 2021-2022, in Suzhou City E'qiao District Agricultural Science and Technology Demonstration and Display Base, with Zhengdan 958 as the test corn variety, an experiment on the effect on corn yield was carried out, and the specific steps included:

[0138] The co-culture fermentation broth of Paenibacillus polymyxa and Hyphomicrobium obtained in Example 2 and the single culture fermentation broth of Hyphomicrobium obtained in Comparative Example 1 were diluted with water by 700, 1000, 1500 times respectively, and were recorded as microbial fertilizer 1, microbial fertilizer 2, microbial fertilizer 3 in turn, and water was used as the control group. At the corn large trumpet stage (fertilizer amount was 20 kg / mu), filling stage (fertilizer amount was 20 kg / mu), they were uniformly sprayed on the leaf surface. Each group was set up 3 parallel samples, and randomly arranged. At the harvest stage, the effective ear number, ear grain number, and thousand grain weight were investigated, and other management was consistent. The results are shown in Table 4.

[0139] Table 4 Results of the effect on corn yield

[0140]

[0141] As can be seen from Table 4: applying different dilution multiples of the co-culture fermentation liquor has a certain promoting effect on the ear grain number, thousand kernel weight and yield of corn, compared with the control group, the single plant ear grain number increases by 3.65-7.59%, the thousand kernel weight increases by 0.26-0.96%, and the yield increases by 34.76-95.28 kg / mu; applying the single culture fermentation liquor, the corn yield is better than that of the control group, but is significantly lower than the effect of the co-culture fermentation liquor.

[0142] Example 8: Effect on soybean yield

[0143] In 2023, a soybean yield experiment was conducted in the agricultural science and technology demonstration and display base in E'qiao District, Suzhou City, with Zhonghuang 13 as the test soybean variety. The specific steps include:

[0144] Take the co-culture fermentation liquor of Paenibacillus polymyxa and Hyphomicrobium obtained in Example 2, and the single culture fermentation liquor of Hyphomicrobium obtained in Comparative Example 1, and dilute them with water by 700, 1000 and 1500 times respectively, and mark them as microbial fertilizer 1, microbial fertilizer 2 and microbial fertilizer 3 in turn, and take water as the control group. Spray them evenly on the leaf surface at the soybean seedling stage (fertilizer amount is 10 kg / mu), flowering and podding stage (fertilizer amount is 20 kg / mu), and grain filling stage (fertilizer amount is 20 kg / mu). Each group sets 3 parallel samples, and arranges them randomly. Investigate the harvested plant number, single plant pod number and single plant grain number at the harvest stage, and determine the hundred kernel weight. The other treatments are consistent. The results are shown in Table 5.

[0145] Table 5: Results of soybean yield effect

[0146]

[0147] As can be seen from Table 5: applying different dilution multiples of the co-culture fermentation liquor has a certain promoting effect on the single plant pod number, single plant grain number, hundred kernel weight and yield of soybean, compared with the control group, the single plant pod number increases by 2.64-5.66-7.68-7.98%, the single plant grain number increases by 2.38-8.91%, the hundred kernel weight increases by 6.56-3.03-9.22-6.37%, the yield increases by 10.17-24.2-32.3 kg / mu, and the yield increase rate is 10.85-14.48%; in terms of yield and constituent factors, the co-culture fermentation liquor has obvious effect on increasing the yield of soybean, and the optimal dilution multiple is 1000-1500 times; the experiment also shows that the co-culture fermentation liquor can significantly improve the yield of soybean; applying the single culture fermentation liquor, the yield of soybean is better than that of the control group, but is significantly lower than the effect of the co-culture fermentation liquor.

[0148] The above Examples 4-8 are all measured according to the "National Grain High Yield Creation Yield Acceptance Method (Trial)" of the Ministry of Agriculture and Rural Affairs.

[0149] The above merely describes preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art, according to the technical solution and inventive concept of the present application, makes equivalent replacement or change within the technical range disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. Application of a polymyxa bacillus in increasing the yield of pyrroloquinoline quinone in silk-producing microbes; The polymyxobacterium is a polymyxobacterium (Bacillus polymyxobinus). Paenibacillus polymyxa X136001, its accession number is CCTCC NO: M 20241603; The silk microbes are silk microbes. Hyphomicrobium vulgare X119001, its accession number is CCTCCNO: M 20241604.

2. Application of a type of filamentous microbacterium in enhancing the nitrogen-fixing ability of Bacillus polymyxa; The polymyxobacterium is a polymyxobacterium (Bacillus polymyxobinus). Paenibacillus polymyxa X136001, its accession number is CCTCC NO: M 20241603; The silk microbes are silk microbes. Hyphomicrobium vulgare X119001, its accession number is CCTCCNO: M 20241604.

3. The application according to claim 2, characterized in that, Application in enhancing nitrogenase activity in Bacillus polymyxa.

4. A type of silk microbacterium Hyphomicrobium vulgare X119001, its accession number is CCTCC NO: M20241604.

5. A polymyxin Bacillus ( Paenibacillus polymyxa X136001, its accession number is CCTCC NO: M 20241603.

6. A method for preparing fermentation broth, characterized in that, The process includes the following steps: co-culturing and fermenting Bacillus polymyxa and microfibrils in a fermentation medium to obtain a fermentation broth; The polymyxobacterium is a polymyxobacterium (Bacillus polymyxobinus). Paenibacillus polymyxa X136001, its accession number is CCTCC NO: M 20241603; The silk microbes are silk microbes. Hyphomicrobium vulgare X119001, its accession number is CCTCCNO: M 20241604.

7. The method for preparing fermentation broth according to claim 6, characterized in that, The fermentation medium contains at least one of a carbon source and a nitrogen source.

8. The method for preparing fermentation broth according to claim 7, characterized in that, The carbon source is methanol.

9. The method for preparing fermentation broth according to claim 7, characterized in that, The nitrogen source is selected from at least one or a combination of yeast extract powder, yeast powder, yeast paste, soybean lecithin, soybean meal powder, cottonseed meal powder, peanut meal powder, gluten meal, corn steep liquor powder, soybean meal, peptone, urea, ammonium salt, and nitrate.

10. A fermentation broth, characterized in that, Fermentation broth prepared according to any one of claims 6-9.

11. The use of the fermentation broth as described in claim 10 in improving the cold resistance and / or net photosynthetic rate of wheat.

12. An application of the fermentation broth as described in claim 10 in increasing crop yield, wherein, The crops are wheat, corn, or soybeans.

Images