Paenibacillus polymyxa and hyphomicrobium co-culture system and application

Abstract

The invention discloses an application of paenibacillus polymyxa in improving the yield of pyrroloquinoline quinone, and relates to an application of hyphomicrobium in improving the nitrogen fixation capability of the paenibacillus polymyxa. The invention also discloses a co-culture system with high pyrroloquinoline quinone yield. The invention further discloses the hyphomicrobium, and the preservation number of the hyphomicrobium is CCTCC (China Center For Type Culture Collection) NO: M 20241604. The invention further discloses the paenibacillus polymyxa, and the preservation number of the paenibacillus polymyxa is CCTCC (China Center for Type Culture Collection) NO: M 20241603. The invention also discloses a fermentation broth prepared by using the co-culture system. The invention further discloses application of the co-culture system and the fermentation liquor. The paenibacillus polymyxa and the hyphomicrobe are co-cultured and fermented, so that the yield of PQQ produced by the hyphomicrobe can be bidirectionally promoted at the same time, and the nitrogenase activity of the paenibacillus polymyxa can be improved; the co-culture fermentation broth can improve nitrogen fixation, stress resistance, growth and yield of crops.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to a co-culture system of Paenibacillus polymyxa and Hyphomicrobium and its application. Background Art

[0002] Pyrroloquinoline quinone (PQQ) is the third coenzyme discovered after nicotinamide and riboflavin, participates in electron transfer in oxidation-reduction reactions, and has special biological activities and physiological functions. Research shows that PQQ, as an important coenzyme and biocatalyst, is widely distributed in various biological cells, and has biological functions such as scavenging free radicals, repairing oxidative damage to cells, and improving the growth and metabolic levels of organisms. PQQ has shown remarkable effects on promoting plant growth and improving stress resistance. According to many studies at home and abroad, 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 cucumber seedlings suffering from cold stress, PQQ can greatly reduce the adverse effects of low-temperature environment by increasing the activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX), and effectively preventing the decrease in the content of glutathione (GSH). In addition, during the booting stage of winter wheat when physiological metabolism is vigorous and the peak of floret degeneration occurs, spraying PQQ can significantly increase the chlorophyll content in leaves, thereby increasing the photosynthetic rate of leaves and reducing the abortion of florets in spikes. At the same time, low-concentration PQQ can improve the coordinated supply of organic and inorganic nutrients of plants in the forms of coating, seed dressing, spraying, etc., so as to improve their stress resistance and disease resistance; the mechanism may be related to PQQ directly participating in the repair of DNA strand breaks caused by stress and the antioxidant effect of the plant itself, indirectly improving the plant's tolerance to stress.

[0003] In addition, PQQ is also an important plant growth promoter. By promoting the metabolism of plant cells and accelerating the synthesis of auxin and cytokinin, it shortens the plant growth stagnation period and effectively improves the plant growth and production capacity. Research has found that when tobacco seedlings are treated by spraying on leaves or applying fertilizer through irrigation to roots, PQQ significantly increases the content of growth regulatory substances such as indole acetic acid in leaves and roots. During the booting stage of winter wheat, spraying PQQ can effectively increase the chlorophyll content in leaves, thereby enhancing the photosynthetic rate and increasing the activities of nitrate reductase and glutamic-pyruvic transaminase. This not only improves the organic and inorganic nutrient supply of winter wheat plants, but also regulates the physiological metabolism of the plants, reduces floret abortion, and increases the seed setting rate. At the same time, PQQ has significant antioxidant capacity, which can protect plant cells from oxidative damage, enhance the stress resistance of plants, and enable them to maintain normal growth and yield under adverse conditions. Research shows 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 increase. This is mainly due to the positive regulatory effect of PQQ on plant growth during the booting, flowering, and filling periods. It can enhance the photosynthesis intensity of plants, promote the transfer of nutrients to grains, extend the leaf functional period, and increase the filling rate, thereby increasing the number and weight of grains and achieving yield increase. In addition to rice, the yield increase effect of PQQ on other crops is also worth expecting. Some studies also show 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 increase of crops. In addition, PQQ also serves as a coenzyme of the dehydrogenase of Plant Growth-Promoting Rhizobacteria (PGPR), which helps to acidify the soil environment and convert insoluble phosphorus into soluble phosphorus, making it more easily absorbed and utilized by plants.

[0004] Hyphomicrobium is a microorganism that can produce PQQ; however, the synthesis rate of PQQ by single-strain fermentation of Hyphomicrobium is slow and the yield is not high, making it difficult to carry out large-scale production, and it is necessary to increase its PQQ yield. Summary of the Invention

[0005] Based on the technical problems existing in the background technology, the present invention proposes a co-culture system and application of Paenibacillus polymyxa and Hyphomicrobium; the co-culture fermentation system of Paenibacillus polymyxa and Hyphomicrobium discovered by the present invention can bidirectionally promote and increase the yield of PQQ produced by Hyphomicrobium and enhance the nitrogenase activity of Paenibacillus polymyxa; the obtained co-culture fermentation broth provides a new and effective way for enhancing the nitrogen fixation, stress resistance, and production capacity of crops, and has convenient operation, is easy to carry out large-scale production and application, and has a wide application prospect.

[0006] The present invention proposes an application of Paenibacillus polymyxa in increasing the yield of pyrroloquinoline quinone.

[0007] Preferably, it is used in increasing the production of pyrroloquinoline quinone by Hyphomicrobium.

[0008] Preferably, the Paenibacillus polymyxa is Paenibacillus polymyxa X136001, and its preservation number is CCTCC NO: M 20241603.

[0009] Preferably, the Hyphomicrobium is Hyphomicrobium vulgare X119001, and its preservation number is CCTCC NO: M 20241604.

[0010] The present invention also provides an application of Hyphomicrobium in increasing the nitrogen fixation ability of Paenibacillus polymyxa.

[0011] Preferably, it is used in increasing the activity of nitrogenase of Paenibacillus polymyxa.

[0012] Preferably, the Paenibacillus polymyxa is Paenibacillus polymyxa X136001, and its preservation number is CCTCC NO: M 20241603.

[0013] Preferably, the Hyphomicrobium is Hyphomicrobium vulgare X119001, and its preservation number is CCTCC NO: M 20241604.

[0014] The present invention also provides a co - culture system with high pyrroloquinoline quinone production, comprising: co - culturing Paenibacillus polymyxa and Hyphomicrobium.

[0015] Preferably, the Paenibacillus polymyxa is Paenibacillus polymyxa X136001, and its preservation number is CCTCC NO: M 20241603.

[0016] Preferably, the Hyphomicrobium is Hyphomicrobium vulgare X119001, and its preservation number is CCTCC NO: M 20241604.

[0017] The present invention also provides a Hyphomicrobium vulgare X119001, and its preservation number is CCTCC NO: M 20241604.

[0018] The present invention also provides a Paenibacillus polymyxa X136001, with the deposit number of CCTCC NO: M 20241603.

[0019] The present invention also provides a fermentation broth prepared by using the above co-culture system.

[0020] The present invention also provides a method for preparing the above fermentation broth, which includes the following steps: co-culturing and fermenting Paenibacillus polymyxa and Hyphomicrobium in a fermentation medium to obtain the fermentation broth.

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

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

[0023] Preferably, the assimilable nitrogen source is at least selected from one or a combination of more of yeast extract powder, yeast powder, yeast extract, soybean lecithin, soybean cake powder, cottonseed cake powder, peanut cake powder, gluten powder, dried corn steep liquor, soybean meal, peptone, urea, ammonium salts, and nitrates.

[0024] The present invention also provides the applications of the above co-culture system and the above fermentation broth in improving the stress resistance of crops, increasing the growth rate of crops, and increasing the yield of crops.

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

[0026] The present invention discovers that in the co-culture system, Paenibacillus polymyxa can significantly promote the expression of pyrroloquinoline quinone by Hyphomicrobium and increase its yield. Moreover, the present invention also discovers that Hyphomicrobium can simultaneously enhance the nitrogenase activity of Paenibacillus polymyxa. Applying the obtained co-culture fermentation broth as an agricultural green input during the planting process of crops such as wheat and corn can improve the nitrogen fixation, stress resistance, growth rate, and yield of the crops. The present invention solves the problems that the synthesis rate of pyrroloquinoline quinone is slow and the yield is not high during the single-strain fermentation of Hyphomicrobium, making it difficult to carry out large-scale production. And at the same time, it can improve the nitrogenase activity of Paenibacillus polymyxa, providing a new and effective way to enhance the nitrogen fixation, stress resistance, and production capacity of crops.

[0027] Moreover, the present invention has screened out specific Hyphomicrobium and Paenibacillus polymyxa. The co-culture fermentation system of the two has a high synthesis efficiency of pyrroloquinoline quinone, a significant increase in yield, a significant enhancement in nitrogenase activity, convenient operation, easy large-scale production and application, and broad application prospects. Its fermentation broth can be used in crop planting. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a plate colony photograph of Paenibacillus polymyxa strain X136001.

[0029] Figure 2 It is a plate colony photograph of Hyphomicrobium vulgare strain X119001.

[0030] Figure 3 It is an HPLC detection spectrum of the PQQ content in the fermentation broths obtained in Comparative Example 1 and Example 2. Among them, A is Comparative Example 1 and B is Example 2.

[0031] Figure 4 It is the yield result of pyrroloquinoline quinone in the fermentation broths of Comparative Example 1 and Example 2. Among them, the single-strain fermentation group is Comparative Example 1, and the co-culture fermentation group is Example 2.

[0032] Figure 5 It is the nitrogenase activity of the strain cells during the single-strain fermentation of Paenibacillus polymyxa, the single-strain fermentation of Hyphomicrobium, and the co-culture fermentation of the two strains. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0033] The present invention provides an application of Paenibacillus polymyxa in increasing the yield of pyrroloquinoline quinone.

[0034] Preferably, it is an application in increasing the yield of pyrroloquinoline quinone of Hyphomicrobium.

[0035] Preferably, the Paenibacillus polymyxa is Paenibacillus polymyxa X136001, and its deposit number is CCTCC NO: M 20241603.

[0036] Preferably, the Hyphomicrobium is Hyphomicrobium vulgare X119001, and its deposit number is CCTCC NO: M 20241604.

[0037] The present invention also provides an application of Hyphomicrobium in enhancing the nitrogen fixation ability of Paenibacillus polymyxa.

[0038] Preferably, it is an application in enhancing the nitrogenase activity of Paenibacillus polymyxa.

[0039] Preferably, the Paenibacillus polymyxa is Paenibacillus polymyxa X136001, and its preservation number is CCTCC NO: M 20241603.

[0040] Preferably, the Hyphomicrobium vulgare is Hyphomicrobium vulgare X119001, and its preservation number is CCTCC NO: M 20241604.

[0041] The present invention also provides a co-culture system with a high pyrroloquinoline quinone yield, including: co-culturing Paenibacillus polymyxa and Hyphomicrobium vulgare.

[0042] Preferably, the Paenibacillus polymyxa is Paenibacillus polymyxa X136001, and its preservation number is CCTCC NO: M 20241603.

[0043] Preferably, the Hyphomicrobium vulgare is Hyphomicrobium vulgare X119001, and its preservation number is CCTCC NO: M 20241604.

[0044] The present invention also provides a Hyphomicrobium vulgare X119001, and its preservation number is CCTCC NO: M 20241604.

[0045] The present invention also provides a Paenibacillus polymyxa X136001, and its preservation number is CCTCC NO: M 20241603.

[0046] The present invention screened Paenibacillus polymyxa X136001 and Hyphomicrobium vulgare X119001 from the soil of the farmland in Wangzhuang Village, Langgan Town, Yongqiao District, Suzhou City, Anhui Province, and deposited them at the China Center for Type Culture Collection (CCTCC) on July 18, 2024. The deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China. The preservation number of Paenibacillus polymyxa X136001 is CCTCC NO: M 20241603, and the preservation number of Hyphomicrobium vulgare X119001 is CCTCC NO: M20241604.

[0047] Traditional microbial fermentation methods mainly rely on single strains to synthesize target products or obtain live microbial products. However, this method often increases the metabolic burden of the strains due to the introduction of key coenzyme factors and precursors of target metabolites, thus affecting the yield of target products.

[0048] In response to the above problems, the inventors unexpectedly discovered in their research that the co-culture fermentation of Paenibacillus polymyxa and Hyphomicrobium vulgare can simultaneously and bidirectionally promote the increase in the yield of PQQ produced by Hyphomicrobium vulgare and enhance the nitrogenase activity of Paenibacillus polymyxa.

[0049] In particular, the PQQ yield of the co-culture system of Paenibacillus polymyxa X136001 and Hyphomicrobium vulgare X119001 was significantly increased compared to that of Hyphomicrobium vulgare X119001 cultured alone, and the nitrogenase activity was significantly increased compared to that of Paenibacillus polymyxa X136001 cultured alone.

[0050] The mechanism by which the co-culture fermentation of Paenibacillus polymyxa and Hyphomicrobium vulgare can simultaneously and bidirectionally promote the yield of PQQ produced by Hyphomicrobium vulgare and improve the nitrogenase activity of Paenibacillus polymyxa involves multiple aspects of interaction and synergy: First, Paenibacillus polymyxa may produce some hydrolases that can decompose complex organic substances, providing more available nutrients for Hyphomicrobium vulgare, thereby enhancing its growth and metabolic capabilities; at the same time, Paenibacillus polymyxa may also create more suitable growth conditions for Hyphomicrobium vulgare by regulating environmental factors such as the pH value and temperature of the co-culture system. The optimization of these environmental factors helps Hyphomicrobium vulgare better exert its potential for fermenting and producing PQQ; at the same time, PQQ synthesized by Hyphomicrobium vulgare in the co-culture system can also act as a coenzyme to promote the growth, reproduction, metabolic activities of Paenibacillus polymyxa and the expression of functional genes such as the nitrogenase structural gene; in addition, during the co-culture process, Paenibacillus polymyxa and Hyphomicrobium vulgare can also interact through the quorum sensing mechanism, enabling the two microorganisms to recognize each other, coordinate their growth and metabolic activities, thereby achieving an increase in PQQ yield and nitrogen fixation ability, providing a new and effective way to improve the nitrogen fixation, stress resistance, and production capacity of crops.

[0051] The present invention also provides a fermentation broth prepared using the above co-culture system.

[0052] The present invention also provides a method for preparing the above fermentation broth, comprising the following steps: co-culturing and fermenting Paenibacillus polymyxa and Hyphomicrobium vulgare in a fermentation medium to obtain a fermentation broth.

[0053] Preferably, the fermentation medium contains 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, where substance A is at least selected from one or a combination of more of methylamine, ethanol, glycerol, acetic acid, mannitol, starch, maltodextrin, glucose, sucrose, lactose, maltose, molasses, soybean oil, and sorbitol.

[0055] Preferably, the assimilable nitrogen source is at least selected from one or a combination of more of yeast extract powder, yeast powder, yeast extract, soy lecithin, soybean cake powder, cottonseed cake powder, peanut cake powder, gluten powder, corn steep liquor dry powder, soybean meal, peptone, urea, ammonium salts, and nitrates.

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

[0057] After the above Paenibacillus polymyxa and Hyphomicrobium are activated and cultured in the seed liquid, they are then inoculated into the fermentation medium together.

[0058] The activation media of the Hyphomicrobium and Paenibacillus polymyxa are the same.

[0059] The formula of the activation medium includes: 0.5 - 1.5 wt% NaCl, 0.4 - 0.6 wt% yeast powder, 0.5 - 1.5 wt% tryptone, with the balance being water, pH = 7.0.

[0060] Preferably, the formula of the activation medium includes: 1 wt% NaCl, 0.5 wt% yeast powder, 1 wt% tryptone, with the balance being water, pH = 7.0.

[0061] The seed liquid media of the Hyphomicrobium and Paenibacillus polymyxa are the same.

[0062] The formula of the seed liquid medium includes: 2 - 4 wt% sucrose, 1.2 - 1.6 wt% yeast extract powder, 0.03 - 0.05 wt% NaH2PO4, 0.01 - 0.015 wt% Na2HPO4, 0.3 - 0.4 wt% sodium chloride, 0.05 - 0.07 wt% MgSO4·7H2O, 0.08 - 0.12 wt% antifoaming agent, with the balance being water.

[0063] Preferably, the formula of the seed liquid medium includes: 3 wt% sucrose, 1.4 wt% yeast extract powder, 0.04 wt% NaH2PO4, 0.012 wt% Na2HPO4, 0.35 wt% sodium chloride, 0.06 wt% MgSO4·7H2O, 0.1 wt% antifoaming agent, with the balance being water.

[0064] The formulation of the above fermentation medium includes: (NH4)2SO4 0.2 - 0.4 wt%, KH2PO4 0.1 - 0.2 wt%, Na2HPO4 0.2 - 0.4 wt%, MgSO4·7H2O 0.05 - 0.15 wt%, trace element solution 0.05 - 0.1 wt%, methanol 1.5 - 2.5 wt%, and the balance is water.

[0065] Preferably, the formulation of the above fermentation medium includes: (NH4)2SO4 0.3 wt%, KH2PO4 0.14 wt%, Na2HPO4 0.3 wt%, MgSO4·7H2O 0.1 wt%, trace element solution 0.07 wt%, methanol 2 wt%, and the balance is water.

[0066] The formulation of the above trace element solution includes: 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, and water 1000 mL.

[0067] Preferably, the formulation of the above trace element solution includes: 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, and water 1000 mL.

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

[0069] The above activation medium, seed liquid medium, and fermentation medium are all sterilized. The above water can be sterile water, purified water, etc.

[0070] The condition parameters for the activation culture of the above Hyphomicrobium and Paenibacillus polymyxa are the same, which are: the activation temperature is 28 - 32 °C, preferably 30 °C; the activation rotation speed is 100 - 300 rpm, preferably 200 rpm; the activation culture time is 10 - 15 h, preferably 12 h.

[0071] The condition parameters for the seed culture of Hyphomicrobium 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 during culture is 50 - 150 rpm, preferably 100 rpm; during the culture process, the pH of the culture medium is adjusted to 6.5 - 7.0 with ammonia water, preferably pH = 6.8; during the culture process, the dissolved oxygen > 10%; during the culture process, the ventilation rate ≤ 1:1, preferably the ventilation rate is 0.5 - 1:1; culture until the concentration of Hyphomicrobium reaches 10 8 and then reserve for use, which can be transferred into the fermentation broth.

[0072] The condition parameters for the seed culture of 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 during culture is 100 - 200 rpm, preferably 150 rpm; during the culture process, the pH of the culture medium is adjusted to 7.0 - 7.2 with NaOH, preferably pH = 7.1; during the culture process, the dissolved oxygen > 40%; during the culture process, the ventilation rate ≤ 1:1, preferably the ventilation rate is 0.5 - 1:1; culture until the concentration of Paenibacillus polymyxa reaches 10 8 and then reserve for use, which can be transferred into the fermentation broth, and the preferred culture time is 10 - 12 h.

[0073] The condition parameters for the co - culture fermentation are as follows: the fermentation temperature is 28 - 32°C, preferably 30°C; the initial rotation speed during fermentation is 50 - 150 rpm, preferably 100 rpm; during the co - culture fermentation process, the pH of the fermentation broth is controlled at 6.5 - 7.0 with ammonia water, preferably pH = 6.8; during the co - culture fermentation process, the dissolved oxygen > 10%; during the co - culture fermentation process, the ventilation rate ≤ 1:1, preferably the ventilation rate is 0.5 - 1:1; end the fermentation when the methanol consumption rate becomes significantly slower, preferably the fermentation time is 160 - 300 h, more preferably 240 h.

[0074] During the co - culture fermentation process, the methanol concentration in the fermentation broth can be adjusted by adding a 50% methanol aqueous solution by volume, so that the methanol concentration in the fermentation broth is controlled within the range of 0.05 - 0.1 wt%.

[0075] During the above - mentioned co - culture fermentation, the seed liquid is composed of Hyphomicrobium seed liquid and Paenibacillus polymyxa seed liquid in a volume ratio of 1:1; during the co - culture fermentation, the inoculation amount of the seed liquid is 4 - 6%, preferably 5%.

[0076] The present invention improves the production of PQQ and enhances the nitrogen - fixing ability by optimizing the co - culture conditions of Hyphomicrobium and Paenibacillus polymyxa.

[0077] In the co-culture system of the present invention, the growth and metabolism processes of the bacterial cells are stable. Taking the small-scale fermentation production in a 20 L fermenter as an example, methanol is used as the carbon source, and a 50% methanol aqueous solution is added during the fermentation process to maintain the methanol concentration at 0.05 - 0.1 wt%. After statistics, the total feeding volume of the methanol aqueous solution within one fermentation cycle is 50% of the initial fermentation broth volume, and the difference in methanol consumption between different batches is small. Further scaling up to production using a 100 L fermenter, after statistics, the total feeding volume of the methanol aqueous solution within one fermentation cycle is 50% of the initial fermentation broth volume, without showing significant changes.

[0078] During the co-culture fermentation process, samples are taken regularly, and a liquid chromatograph is used to monitor the production of pyrroloquinoline quinone in the co-culture fermentation broth. After the fermentation broth is pretreated, it is monitored using a liquid chromatograph.

[0079] The pretreatment steps of the fermentation broth include: taking 2 mL of the fermentation broth and centrifuging it at 10,000 r for 20 min to obtain the fermentation supernatant.

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

[0081] The activity of nitrogenase is detected by the acetylene reduction method.

[0082] The present invention also proposes the application of the above co-culture system and the above fermentation broth in improving the stress resistance of crops and increasing crop yields.

[0083] The present invention co-cultures Paenibacillus polymyxa and Hyphomicrobium vulgare. This co-culture system can improve the stress resistance of crops and increase crop yields. By optimizing the co-culture conditions of Hyphomicrobium vulgare and Paenibacillus polymyxa, the production of pyrroloquinoline quinone and the activity of nitrogenase are improved, and the production cost is reduced. Applying the co-culture fermentation broth to crop yield increase has improved the growth rate and yield of wheat and corn, and has broad application prospects. The present invention is simple and easy to implement, convenient to operate, and suitable for large-scale production and application.

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

[0085] The above food crops can be cereal crops, tuber crops, leguminous crops, etc.

[0086] Cereal crops such as wheat, rice, corn, etc.; tuber crops such as sweet potato, potato, etc.; leguminous crops such as soybean, broad bean, pea, mung bean, etc.

[0087] The above-mentioned economic crops can be fiber crops, oil crops, sugar crops, beverage crops, spice crops, seasoning crops, medicinal crops, dye crops, ornamental crops, fruits, etc.

[0088] Fiber crops such as cotton, hemp, etc.; oil crops such as sesame, peanut, etc.; sugar crops such as sugarcane, beet, etc.

[0089] The above-mentioned co-culture system and fermentation broth can be applied to parts such as crop leaves, roots and rhizomes.

[0090] Next, the technical solution of the present invention will be described in detail through specific examples.

[0091] Example 1

[0092] 1. Screening and isolation of Paenibacillus polymyxa X136001 and Hyphomicrobium X119001

[0093] Take a soil sample at a depth of 0.5 m below the surface of the farmland in Wangzhuang Village, Langgan Town, Yongqiao District, Suzhou City, Anhui Province. After diluting the soil sample with normal saline, gradient dilute and spread it on an activation medium (the formula of the activation medium includes: 1 wt% NaCl, 0.5 wt% yeast powder, 1 wt% tryptone, the balance is water, pH = 7.0); culture at 30 °C and a rotation speed of 200 rpm. Wait for single colonies to grow on the plate, pick the single colonies and inoculate them into a seed liquid medium (the formula of the seed liquid medium includes: 3 wt% sucrose, 1.4 wt% yeast extract powder, 0.04 wt% NaH2PO4, 0.012 wt% Na2HPO4, 0.35 wt% sodium chloride, 0.06 wt% MgSO4·7H2O, 0.1 wt% antifoaming agent, the balance is water), culture at 30 °C and an initial rotation speed of 100 rpm. During the culture process, adjust the pH of the seed liquid medium to 6.8 with ammonia water, keep the dissolved oxygen > 10%, the ventilation volume is 0.5 - 1:1, and culture until the strain concentration reaches 10 8 When the culture ends; then measure the PQQ content and nitrogenase activity in the supernatant of the culture solution; screen out the strains producing PQQ and the strains with relatively high nitrogenase activity.

[0094] 2. Identification of strains

[0095] Take the 2 single strains obtained in step 1 for morphological characteristic description and species identification, and the results are as follows:

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

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

[0098] (3) The 16S rRNA gene sequences of the above Paenibacillus polymyxa X136001 and Hyphomicrobium X119001 were determined, and the homology comparison was carried out with the gene sequences in the GenBank database and the results were analyzed to determine the genus of the bacteria at the molecular biology level.

[0099] Finally, one strain was determined to be of the genus Paenibacillus, named Paenibacillus polymyxa X136001, and was deposited in the China Center for Type Culture Collection (CCTCC) on July 18, 2024. The deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, and the deposit number is CCTCC NO: M20241603.

[0100] The other strain was of the genus Hyphomicrobium, named Hyphomicrobium vulgare X119001, and was deposited in the China Center for Type Culture Collection (CCTCC) on July 18, 2024. The deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, and the deposit number is CCTCC NO: M20241604.

[0101] The gene sequence of 16S rRNA of Paenibacillus polymyxa X136001 is as shown in SEQ ID NO.1.

[0102] The gene sequence of 16S rRNA of Hyphomicrobium vulgare X119001 is as shown in SEQ ID NO.2.

[0103] Example 2

[0104] A method for preparing a co-culture fermentation broth of Paenibacillus polymyxa and Hyphomicrobium includes the following steps:

[0105] Paenibacillus polymyxa X136001 and Hyphomicrobium vulgare X119001 preserved at -80°C in glycerol cryotubes were sampled at 20 μL each and inoculated into 100 mL of activation medium (the formula of the activation medium includes: 1 wt% NaCl, 0.5 wt% yeast powder, 1 wt% tryptone, the balance being water, pH = 7.0), and activated on a shaker at 30°C (rotation speed 200 rpm) until 600 the OD reaches 0.4, and the activation ends;

[0106] The above-activated media were each taken and inoculated into seed liquid medium (the formula of the seed liquid medium includes: 3 wt% sucrose, 1.4 wt% yeast extract powder, 0.04 wt% NaH2PO4, 0.012 wt% Na2HPO4, 0.35 wt% sodium chloride, 0.06 wt% MgSO4·7H2O, 0.1 wt% antifoaming agent, the balance being water) for culture;

[0107] Among them, the inoculation amount of Hyphomicrobium vulgare X119001 was 5%, and it was cultured at 30°C with an initial rotation speed of 100 rpm. During the culture process, the pH was adjusted to 6.8 with saturated ammonia water, the dissolved oxygen was maintained > 10%, and the ventilation volume was 0.5 - 1:1. After culturing until the biomass reached 10 8 a seed liquid of Hyphomicrobium vulgare X119001 was obtained and reserved;

[0108] The inoculation amount of Paenibacillus polymyxa X136001 was 5%, and it was cultured at 30°C with an initial rotation speed of 150 rpm. During the culture process, the pH was adjusted to 7.1 with an aqueous sodium hydroxide solution, the dissolved oxygen was maintained > 40%, and the ventilation volume was 0.5 - 1:1. After culturing until the biomass reached 10 8 a seed liquid of Paenibacillus polymyxa X136001 was obtained and reserved;

[0109] Take the seed liquid of Hyphomicrobium X119001 and Paenibacillus polymyxa X136001 in a volume ratio of 1:1 to form a seed liquid, and inoculate it into the co-culture fermentation medium at a total inoculation amount of 5% (the formula of the fermentation medium includes: (NH4)2SO4 0.3 wt%, KH2PO4 0.14 wt%, Na2HPO4 0.3 wt%, MgSO4·7H2O 0.1 wt%, trace element solution 0.07 wt%, methanol 2 wt%, and the balance is water). Cultivate at 30 °C with an initial rotation speed of 100 rpm. During the cultivation process, adjust the pH to 6.8 with saturated ammonia water, keep the dissolved oxygen > 10%, and the ventilation volume is 0.5 - 1:1. And during the cultivation process, add a 50% methanol aqueous solution by volume, and control the methanol concentration in the fermentation broth to be 0.05 - 0.1 wt%. When the methanol consumption rate becomes significantly slower, end the fermentation. The fermentation time is 240 h to obtain the co-culture fermentation broth of Paenibacillus polymyxa and Hyphomicrobium.

[0110] Comparative Example 1

[0111] Take the seed liquid of Hyphomicrobium X119001 in Example 2 and ferment it alone under the same fermentation conditions as the co-culture fermentation conditions to obtain a fermentation broth.

[0112] During the co-culture fermentation process, by regularly sampling and using a liquid chromatograph to monitor the production of pyrroloquinoline quinone (PQQ) in the co-culture fermentation broth. Take 2 mL of the fermentation broth, centrifuge it at 10000 r for 20 min by a centrifuge to obtain the fermentation supernatant, and detect it with a liquid chromatograph. And compare it with the fermentation broth of Comparative Example 1. The results are as Figures 3-4 shown.

[0113] Figure 3 It is the HPLC detection spectrum of the PQQ content in the fermentation broths obtained from Comparative Example 1 and Example 2. Among them, A is Comparative Example 1, and B is Example 2.

[0114] Figure 4 It is the production results of pyrroloquinoline quinone in the fermentation broths of Comparative Example 1 and Example 2. Among them, the single-strain fermentation group is Comparative Example 1, and the co-culture fermentation group is Example 2.

[0115] It can be seen from Figures 3-4 that compared with the single fermentation of Hyphomicrobium X119001, the co-culture fermentation of Hyphomicrobium X119001 and Paenibacillus polymyxa X136001 can significantly increase the production of pyrroloquinoline quinone (PQQ); among them, when the fermentation time is 164 h, the PQQ production of the single-strain fermentation group is 626.4 mg / L, while the PQQ production of the co-culture fermentation group can reach 1514.5 mg / L; when the fermentation time continues to 240 h, the PQQ production of the co-culture fermentation group is still higher than that of the single-strain fermentation group.

[0116] Example 3

[0117] Take the seed liquid of Hyphomicrobium X119001, the seed liquid of Paenibacillus polymyxa X136001, and the mixed seed liquid (composed of the seed liquid of Hyphomicrobium X119001 and the seed liquid of Paenibacillus polymyxa X136001 in Example 2 at a volume ratio of 1:1), and inoculate them into 20 mL of fresh co-culture fermentation medium at an inoculation amount of 1% respectively. Cultivate for 8 h under the conditions of 30 °C and 160 rpm, collect the bacterial liquid respectively, and then detect the nitrogenase activity of each group according to the acetylene reduction method. The results are as Figure 5 shown.

[0118] Figure 5 The nitrogenase activities of the bacterial cells during the fermentation of Paenibacillus polymyxa single strain, Hyphomicrobium single strain, and the co-culture fermentation of the two strains.

[0119] It can be seen from Figure 5 that when Paenibacillus polymyxa X136001 is fermented and cultured alone, the nitrogenase activity of the bacterial cells is 358 nmol C2H4 / mg protein hr; when Hyphomicrobium X119001 is fermented alone, the nitrogenase activity is close to 0; when the two are co-cultured and fermented, the nitrogenase activity of the strain can reach as high as 556 nmol C2H4 / mg protein hr; thus, it can be seen that the co-culture fermentation of the two can significantly improve the nitrogenase activity, thereby playing a better nitrogen fixation function.

[0120] Experiment on the effect of Example 4 on wheat stress resistance

[0121] From 2021 to 2022, in the agricultural science and technology demonstration and display base in Yongqiao District, Suzhou City, using Huacheng 3366 as the test variety, an experiment on the effect of wheat stress resistance was carried out. The specific steps include:

[0122] Take 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. Dilute them 1000 times with water as bacterial fertilizers respectively, and spray the bacterial fertilizers on the wheat leaves at the seedling stage (the spraying amount is 22.5 g / m 2 ). After 10 h, place them in a low-temperature incubator at 4 °C for 3 days, and then take samples to measure the activities of superoxide dismutase (SOD), peroxidase (POD), and the content of malondialdehyde (MDA). Set 3 parallel samples for each group; and set a control group by placing them at room temperature (25 °C) for 3 days after spraying the bacterial fertilizer for 10 h. The test results are shown in Table 1.

[0123] Table 1 Results of the experiment on the effect of wheat stress resistance

[0124]

[0125] As can be seen from Table 1, applying the co-culture fermentation broth can significantly improve the cold resistance of wheat seedlings, increasing the activities of superoxide dismutase and peroxidase in wheat seedlings by 22.65 - 70.53% and 22.34 - 46.37% respectively, and reducing the malondialdehyde content by 26.12 - 35.26%. Its effect is significantly better than that of the control group. Applying the single-culture fermentation broth has a better cold resistance effect on wheat seedlings than the control group, but is significantly lower than that of the co-culture fermentation broth.

[0126] Example 5 Effect on the net photosynthetic rate of wheat

[0127] This was carried out at Yonghao Planting Professional Cooperative in Lingbi County from 2022 to 2023. Using Huacheng 3366 as the test variety, an experiment on the effect of the net photosynthetic rate of wheat was conducted. The specific steps included:

[0128] Take 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, and dilute them 1000 times with water as bacterial fertilizers respectively. Spray them on the wheat leaves at the seedling stage, jointing stage (10 days after turning green), and filling stage (15 days after flowering) of wheat (the spraying amount is 15 kg / mu). Measure the net photosynthetic rate of the flag leaves at the heading stage, 10 days after flowering, and 20 days after flowering respectively. The sample size for each measurement is 20 flag leaves, and 3 parallel samples are set for each group. Spray with clear water in the same way as a control group. The test results are shown in Table 2.

[0129] Table 2 Results of the effect on the net photosynthetic rate of wheat

[0130]

[0131] As can be seen from Table 2, applying the co-culture fermentation broth can significantly improve the net photosynthetic rate of wheat flag leaves, increasing the net photosynthetic rate of wheat flag leaves at the heading stage, 10 days after flowering, and 20 days after flowering 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 the flag leaves with the single-culture fermentation broth is better than that of the control group, but significantly lower than that of the co-culture fermentation broth.

[0132] Example 6 Effect on the wheat yield

[0133] This was carried out at Yonghao Planting Professional Cooperative in Lingbi County from 2022 to 2023. Using Xinong 920 as the test wheat variety, an experiment on the effect of wheat yield was conducted. The specific steps included:

[0134] Take 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, and dilute them 700, 1000, and 1500 times with water respectively, which are denoted as bacterial fertilizer 1, bacterial fertilizer 2, and bacterial fertilizer 3 in sequence. Use clear water as the control group, and spray them evenly on the leaves during the seedling stage of wheat (fertilizer application rate: 10 kg / mu), booting stage (fertilizer application rate: 20 kg / mu), and filling stage (fertilizer application rate: 20 kg / mu). Set 3 parallel samples in each group and arrange them randomly. Investigate the effective ear number and grain number per ear at the harvest stage, and measure the 1000-grain weight. Other management measures are the same. The results are shown in Table 3.

[0135] Table 3 Results of the impact on wheat yield

[0136]

[0137] It can be seen from Table 3 that applying the co-culture fermentation broth with different dilution multiples has a promoting effect on the number of grains per ear per plant, 1000-grain weight, and yield of wheat. Compared with the control group, the number of grains per ear per plant increases by 1.46 - 5.96%, the 1000-grain weight increases by 2.30 - 3.60%, and the yield increases by 15.30 - 35.60 kg / mu. When applying the single-culture fermentation broth, the wheat yield is better than that of the control group, but significantly lower than that of the co-culture fermentation broth.

[0138] Impact on the yield of corn in Example 7

[0139] This was carried out at the agricultural science and technology demonstration and display base in Yongqiao District, Suzhou City from 2021 to 2022. Using Zhengdan 958 as the test corn variety, an experiment on the impact of corn yield was conducted. The specific steps are as follows:

[0140] Take 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, and dilute them 700, 1000, and 1500 times with water respectively, which are denoted as bacterial fertilizer 1, bacterial fertilizer 2, and bacterial fertilizer 3 in sequence. Use clear water as the control group, and spray them evenly on the leaves during the large flare stage of corn (fertilizer application rate: 20 kg / mu) and filling stage (fertilizer application rate: 20 kg / mu). Set 3 parallel samples in each group and arrange them randomly. Investigate the effective ear number and grain number per ear at the harvest stage, and measure the 1000-grain weight. Other management measures are the same. The results are shown in Table 4.

[0141] Table 4 Results of the impact on corn yield

[0142]

[0143] As can be seen from Table 4, applying the co-culture fermentation broth at different dilution multiples has a certain promoting effect on the number of grains per ear, 1000-grain weight, and yield of corn. Compared with the control group, the number of grains per ear per plant increased by 3.65 - 7.59%, the 1000-grain weight increased by 0.26 - 0.96%, and the yield increased by 34.76 - 95.28 kg / mu. When applying the single-culture fermentation broth, the corn yield was better than that of the control group, but significantly lower than that of the co-culture fermentation broth.

[0144] Effect on the Yield of Soybeans in Example 8

[0145] In 2023, an experiment on the effect of soybean yield was carried out at the agricultural science and technology demonstration and display base in Yongqiao District, Suzhou City, using Zhonghuang 13 as the test soybean variety. The specific steps were as follows:

[0146] Take 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. Dilute them with water 700, 1000, and 1500 times respectively, and record them as bacterial fertilizers 1, 2, and 3 in sequence. Use clear water as the control group, and evenly spray them on the leaves during the seedling stage (fertilizer application rate: 10 kg / mu), flowering and pod-setting stage (fertilizer application rate: 20 kg / mu), and grain-filling stage (fertilizer application rate: 20 kg / mu) of soybeans. Set 3 parallel samples in each group and arrange them randomly. At the harvest stage, investigate the number of harvested plants, the number of pods per plant, and the number of grains per plant, and measure the 100-grain weight. Other management among treatments was the same. The results are shown in Table 5.

[0147] Table 5 Results of the Effect on Soybean Yield

[0148]

[0149]

[0150] As can be seen from Table 5, applying the co-culture fermentation broth at different dilution multiples has a certain promoting effect on the number of pods per plant, the number of grains per plant, 100-grain weight, and yield of soybeans. Compared with the control group, the number of pods per plant increased by 2.645.66 - 7.687.98%, the number of grains per plant increased by 2.38 - 8.91%, the 100-grain weight increased by 6.563.03 - 9.226.37%, the yield increased by 10.1724.2 - 32.3 kg / mu, and the yield increase rate was 10.85 - 14.48%. Considering the comprehensive yield and component factors, the co-culture fermentation broth has an obvious effect on increasing soybean yield, and the optimal dilution multiple is 1000 - 1500 times. The experiment also shows that the co-culture fermentation broth can significantly increase soybean yield. When applying the single-culture fermentation broth, the soybean yield is better than that of the control group, but significantly lower than that of the co-culture fermentation broth.

[0151] The above Examples 4 - 8 were all carried out according to the yield measurement method in the "Measures for Yield Measurement and Acceptance of High-yield Grain Creation in the Whole Country (Trial)" of the Ministry of Agriculture and Rural Affairs.

[0152] The above are only the preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, making equivalent substitutions or changes, shall be covered by the protection scope of the present invention.

Claims

1. Application of Bacillus polymyxa in increasing the yield of pyrroloquinoline quinone.

2. The application according to claim 1, characterized in that: Application in improving the pyrroloquinoline quinone yield of Hyphomicrobium; preferably, the polymyxa Paenibacillus is polymyxa Paenibacillus (Paenibacillus polymyxa) X136001, and its preservation number is CCTCC NO: M 20241603; preferably, the Hyphomicrobium is Hyphomicrobium vulgare (Hyphomicrobium vulgare) X119001, and its preservation number is CCTCC NO: M 20241604.

3. Application of a hyphae microorganism in improving the nitrogen fixation ability of Bacillus polymyxa.

4. The use according to claim 3, characterized in that: Application in improving the nitrogenase activity of Paenibacillus polymyxa; preferably, the Paenibacillus polymyxa is Paenibacillus polymyxa X136001, and its preservation number is CCTCC NO: M 20241603; preferably, the Hyphomicrobium vulgare is Hyphomicrobium vulgare X119001, and its preservation number is CCTCC NO: M 20241604.

5. A co-cultivation system with high pyrroloquinoline quinone production, characterized in that: include: Paenibacillus polymyxa and Hyphomicrobium vulgare are co-cultured; preferably, the Paenibacillus polymyxa is Paenibacillus polymyxa (Paenibacilluspolymyxa) X136001, and its preservation number is CCTCC NO: M 20241603; preferably, the Hyphomicrobium vulgare is Hyphomicrobium vulgare (Hyphomicrobium vulgare) X119001, and its preservation number is CCTCC NO: M 20241604.

6. A Hyphomicrobium vulgare X119001, whose deposit number is CCTCCNO: M20241604.

7. A Paenibacillus polymyxa X136001, whose deposit number is CCTCC NO: M20241603.

8. A fermentation broth prepared using the co-cultivation system according to claim 5.

9. A method for preparing the fermentation liquid as claimed in claim 8, characterized in that: The method comprises the following steps: co-culturing and fermenting Paenibacillus polymyxa and Mycelium spp. in a fermentation medium to obtain a fermentation liquid; preferably, the fermentation medium contains at least one of an assimilable carbon source and an assimilable nitrogen source; preferably, the assimilable carbon source is a mixture of formic acid and substance A, methanol, and methylamine, wherein substance A is at least selected from one or a combination of methylamine, ethanol, glycerol, acetic acid, mannitol, starch, maltodextrin, glucose, sucrose, lactose, maltose, molasses, soybean oil, and sorbitol; preferably, the assimilable nitrogen source is at least selected from one or a combination of yeast extract powder, yeast powder, yeast paste, soybean lecithin, soybean cake powder, cottonseed cake powder, peanut cake powder, gluten powder, corn steep liquor dry powder, soybean meal, peptone, urea, ammonium salt, and nitrate.

10. Use of the co-cultivation system according to claim 5 and the fermentation liquid according to claim 8 in improving the stress resistance of crops, increasing the growth rate of crops, and increasing the yield of crops; preferably, the crops include food crops and cash crops.

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