Acinetobacter calcoaceticus and application thereof

Abstract

The application belongs to the technical field of microorganisms, and particularly relates to a strain of Acinetobacter calcoaceticus and application thereof. The application discloses a strain of Acinetobacter calcoaceticus CDWB36, the preservation number of which is CCTCC NO:M2023964, FL04, and the strain is preserved in the China Center for Type Culture Collection on June 8, 2023, and the preservation address is Wuhan, Wuhan University, China. The Acinetobacter calcoaceticus CDWB36 can produce PQQ after fermentation culture, in addition, the bacterial agent containing PQQ produced by the Acinetobacter calcoaceticus CDWB36 after fermentation can prevent and treat pepper bacterial leaf spot, can be used for biocontrol agent, can relieve drought stress of plants, can promote growth, and can be used as a fertilizer additive.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microorganisms, and particularly relates to a strain of Acinetobacter calcoaceticus and application thereof. BACKGROUND

[0002] Acinetobacter belongs to the family Moraxellaceae of the order Pseudomonadales, is a kind of microorganism widely distributed in soil and having salt tolerance and growth promotion functions, and is divided into six species, namely, A. calcoaceticus, A. lwoffi, A. baumanii, A. haemolytius, A. junii and A. johnsonii.

[0003] Researches show that A. calcoaceticus plays an important role in improving plant stress resistance, promoting growth, degrading diesel, repairing heavy metals in soil and degrading petroleum hydrocarbons. For example, CN103060241A discloses a strain of A. calcoaceticus for promoting growth of walnut rhizosphere, which can promote growth of walnut; CN104946570A discloses a copper-tolerant A. calcoaceticus KW3 capable of removing copper ions in copper mine wastewater; and CN110791461A discloses a strain of A. calcoaceticus capable of effectively controlling and reducing the concentration of chromium metal COD and aniline in wastewater.

[0004] PQQ is a new coenzyme, is the third coenzyme found in membrane-bound bacterial dehydrogenase after FMN (FAD) and MAD (NADP), and is called the fourteenth vitamin in the world medical field. It has become possible to produce PQQ by microbial fermentation. For example, CN113337433A discloses a Pseudomonas producing PQQ, and CN113337432A discloses a methylotrophic bacterium producing PQQ.

[0005] However, there is no mature technical solution reported for producing PQQ by using A. calcoaceticus, and in addition, it has great market value to find a strain of A. calcoaceticus capable of producing PQQ and improving plant stress resistance and promoting plant growth. SUMMARY

[0006] To solve the above technical problems, the application provides the following technical solutions.

[0007] The application provides an Acinetobacter calcoaceticus CDWB36, which is preserved in the China Center for Type Culture Collection (CCTCC NO: M2023964, FL04) on June 8, 2023, and has an evolutionary tree as shown in the figure. Figure 1 The 16S rRNA nucleotide sequence of the Acinetobacter calcoaceticus CDWB36 is shown as SEQ ID NO: 1.

[0008] The application of the Acinetobacter calcoaceticus CDWB36 in the fermentation production of PQQ.

[0009] A method for producing PQQ, comprising: using the Acinetobacter calcoaceticus CDWB36 to perform fermentation production of PQQ.

[0010] A method for producing a PQQ-containing microbial agent, comprising the following steps:

[0011] (1) activating the Acinetobacter calcoaceticus CDWB36 in an LB liquid culture medium to obtain a seed liquid;

[0012] (2) inoculating the seed liquid obtained in step (1) into a fermentation culture medium to perform fermentation culture, and obtaining a PQQ-containing microbial agent.

[0013] Preferably, the formula of the LB liquid culture medium comprises: 8-12 g / L of tryptone, 4-6 g / L of yeast extract, 9-11 g / L of sodium chloride, 14-16 g / L of agar powder, and pH 7.0±0.2; the formula of the fermentation culture medium is: 8-12 g / L of a carbon source, 3-5 g / L of a nitrogen source, 1.5-2.5 g / L of Na2HPO4, 1.2-1.6 g / L of KH2PO4, 0.8-1.2 g / L of MgSO4·7H2O, and 0.42-0.43 g / L of a trace element solution, and pH 6.5-7.0; the trace element solution comprises: 75-85 mg / L of FeSO4·7H2O, 22-23 mg / L of ZnSO4·7H2O, 0.3-0.4 mg / L of KI, 2.5-3.5 mg / L of H3BO3, 4-6 mg / L of CuSO4, 14-16 mg / L of NaCl, and 200-400 mg / L of CaCl2·2H2O.

[0014] Further, the carbon source is at least one of yeast powder, glucose, sucrose, maltose, and galactose.

[0015] Further, the nitrogen source is one of anhydrous ammonium sulfate, L-glutamic acid, L-tyrosine, and peptone, or a mixed nitrogen source consisting of anhydrous ammonium sulfate, L-glutamic acid, and L-tyrosine.

[0016] Further preferably, the formula of the LB liquid medium comprises: 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of sodium chloride, 15 g / L of agar powder, pH 7.0; the formula of the fermentation medium is: 10 g / L of carbon source, 4 g / L of nitrogen source, 2 g / L of Na2HPO4, 1.4 g / L of KH2PO4, 1 g / L of MgSO4·7H2O, 0.4258 g / L of trace element solution, pH 6.5; the carbon source is glucose; the nitrogen source is a mixed nitrogen source consisting of anhydrous ammonium sulfate, L-glutamic acid, and L-tyrosine in a mass ratio of 2:1:1; the trace element solution comprises: 80 mg / L of FeSO4·7H2O, 22.5 mg / L of ZnSO4·7H2O, 0.3 mg / L of KI, 3 mg / L of H3BO3, 5 mg / L of CuSO4, 15 mg / L of NaCl, and 400 mg / L of CaCl2·2H2O.

[0017] Preferably, the inoculation amount in step (2) is 0.5-1%; the fermentation culture conditions are: temperature 28-30℃, time 7d.

[0018] The PQQ-containing microbial agent produced according to the method.

[0019] The PQQ-containing microbial agent comprises the Acinetobacter calcoaceticus CDWB36 and the fermentation broth, wherein the effective viable cell count of the Acinetobacter calcoaceticus CDWB36 is more than 10 9 CFU / ml, and the concentration of PQQ in the fermentation broth is more than 4 mg / L.

[0020] Preferably, in the PQQ-containing microbial agent, the effective viable cell count of the Acinetobacter calcoaceticus CDWB36 is 2.0-2.5*10 10 CFU / ml, and the concentration of PQQ in the fermentation broth is more than 60 mg / L.

[0021] The PQQ-containing microbial agent in the preparation of a biocontrol microbial agent.

[0022] The PQQ-containing bacterial agent in improving the stress resistance of plants.

[0023] The PQQ-containing bacterial agent in promoting the growth of plants.

[0024] The PQQ-containing bacterial agent in preparing a fertilizer additive.

[0025] The PQQ-containing bacterial agent is diluted 200-300 times for spraying application and diluted 100-150 times for root irrigation application.

[0026] Advantages of the present application:

[0027] The Acinetobacter calcoaceticus CDWB36 of the present application can produce PQQ through fermentation culture, in addition, the PQQ-containing bacterial agent produced by the fermentation culture of the Acinetobacter calcoaceticus CDWB36 can prevent and control pepper bacterial leaf spot, is used for biocontrol agent, can also relieve plant drought stress, promote growth, and is used as a fertilizer additive. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Phylogenetic analysis of Acinetobacter calcoaceticus CDWB36;

[0029] Figure 2 Photos of morphological characteristics of Acinetobacter calcoaceticus CDWB36;

[0030] Figure 3 Photos of morphological characteristics of Acinetobacter calcoaceticus CDWB36;

[0031] Figure 4 Culture of Acinetobacter calcoaceticus CDWB36 and HPLC results of PQQ standard;

[0032] Figure 5 Effects of applying PQQ-containing bacterial agent on the growth of peppers;

[0033] Figure 6 Effects of applying PQQ-containing bacterial agent on the root development of peppers;

[0034] Figure 7 Effects of applying PQQ-containing bacterial agent on the root development of peppers. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0036] As a specific embodiment of the present application, it relates to a strain of Acinetobacter calcoaceticus CDWB36, which has a preservation number of CCTCC NO: M2023964, FL04, is preserved in the China Center for Type Culture Collection, and was preserved on June 8, 2023, at Wuhan, Wuhan University, China. The strain can be used for fermentation production of PQQ. The phylogenetic tree analysis of the Acinetobacter calcoaceticus CDWB36 is shown in Figure 1 . The morphological characteristics of the strain are shown in Figure 2 , specifically, the OLYMPUS CX33 microscope is used for observation, and it can be observed that the strain is short and thick rod or spherical rod; the colony morphological characteristics of the strain are shown in Figure 3 , and it can be observed that the colony is milky white, flat and opaque.

[0037] The Acinetobacter calcoaceticus CDWB36 is sequenced and identified by Beijing Qianke Biological Technology Co., Ltd. The 16s rRNA gene sequence determination result is shown as SEQ ID NO: 1.

[0038] In the following examples, the Acinetobacter calcoaceticus is Acinetobacter calcoaceticus CDWB36; the formula of the LB liquid culture medium is: 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of sodium chloride, 15 g / L of agar powder, dissolved with water, finally constant volume to 1000 mL, pH 7.0±0.2, high pressure sterilization (121℃, 30 min).

[0039] Example 1: Optimization scheme for fermentation of PQQ by Acinetobacter calcoaceticus

[0040] 1. Configuration of basic culture medium

[0041] Table 1: Formula of basic culture medium

[0042]

[0043]

[0044] Table 2 Formulation of trace elements

[0045] Composition of trace element solution Content mg / L FeSO4·7H2O 80 ZnSO4·7H2O 22.5 KI 0.3 H3BO3 3 [CuSO4] 5 NaCl 15 CaCl2·2H2O 300

[0046] 2. Determining the type of carbon source

[0047] Acinetobacter calcoaceticus was cultured using LB liquid medium, the seed liquid OD 600 = 0.8, the concentration was 2.3 x 10 4 cfu / mL, and then on the basis of the same basic medium formulation, the temperature was 28°C, the pH was 7, the shaking speed was 180 rpm / min, the bacterial inoculation amount was 1%, the nitrogen source was added: proteose peptone 4 g / L, and the carbon source was 10 g / L. The effect of the type of carbon source on the fermentation of PQQ by Acinetobacter calcoaceticus was explored, and the fermentation was carried out for 7 days. The results are shown in Table 3. When yeast powder was used as the carbon source, the PQQ concentration produced by Acinetobacter calcoaceticus was the highest, so yeast powder was selected as the carbon source.

[0048] Table 3 Effect of different types of carbon sources on the PQQ concentration produced by Acinetobacter calcoaceticus

[0049]

[0050] 2.1 Determining the content of the carbon source

[0051] Acinetobacter calcoaceticus was cultured using LB liquid medium, the seed liquid OD 600 = 0.8, the concentration was 2.3 x 10 4 cfu / mL; and then on the basis of the same basic medium formulation, the temperature was 28°C, the pH was 7, the shaking speed was 180 rpm / min, the bacterial inoculation amount was 1%, the nitrogen source was added: proteose peptone 4 g / L, and the carbon source was yeast powder. The effect of the content of yeast powder on the fermentation of PQQ by Acinetobacter calcoaceticus was explored, and the fermentation was carried out for 7 days. The results are shown in Table 4. When the content of yeast powder was 10 g / L, the PQQ concentration produced by Acinetobacter calcoaceticus was the highest, so the addition amount of yeast powder was selected to be 10 g / L.

[0052] Table 4 Effect of different contents of yeast powder on the PQQ concentration produced by Acinetobacter calcoaceticus

[0053] Treatment Yeast powder content g / L PQQ yield (mg / L) T1 5 16.92±2.70 T2 10 24.52±1.04 T3 15 21.93±0.42 T4 20 18.56±0.96 T5 25 15.32±0.33

[0054] 3. Determining the type of nitrogen source

[0055] Acinetobacter calcoaceticus was cultured using LB liquid medium, the seed liquid OD 600 = 0.8, the concentration was 2.3 x 10 4cfu / mL; then on the basis of the same basic medium formula, temperature 28℃, pH 7, shaking speed 180 rpm / min, bacterial inoculation amount 1%, adding carbon source yeast powder 10 / L, nitrogen source content 4 g / L, exploring the influence of different types of nitrogen source on the fermentation of PQQ by Acinetobacter calcoaceticus, and the fermentation lasted for 7 days. The results are shown in Table 5. When the mixed nitrogen source (ammonium sulfate: glutamic acid: tyrosine = 2:1:1) is used as the nitrogen source, the PQQ concentration produced by Acinetobacter calcoaceticus is the highest. Therefore, the mixed nitrogen source (ammonium sulfate: glutamic acid: tyrosine = 2:1:1) is selected as the nitrogen source.

[0056] Table 5 Influence of different types of nitrogen source on PQQ concentration produced by Acinetobacter calcoaceticus

[0057]

[0058] 3.1 Determining the content of nitrogen source

[0059] Acinetobacter calcoaceticus was cultured using LB liquid medium, and the seed liquid OD 600 = 0.8, with a concentration of 2.3 x 10 4 cfu / mL; then on the basis of the same basic medium formula, temperature 28℃, pH 7, shaking speed 180 rpm / min, bacterial inoculation amount 1%, adding carbon source yeast powder 10 / L, nitrogen source content 4 g / L, exploring the influence of different types of nitrogen source on the fermentation of PQQ by Acinetobacter calcoaceticus, and the fermentation lasted for 7 days. The results are shown in Table 5. When the mixed nitrogen source (ammonium sulfate: glutamic acid: tyrosine = 2:1:1) is used as the nitrogen source, the PQQ concentration produced by Acinetobacter calcoaceticus is the highest. Therefore, the mixed nitrogen source (ammonium sulfate: glutamic acid: tyrosine = 2:1:1) is selected as the nitrogen source.

[0060] Table 6 Influence of different content of nitrogen source on PQQ concentration produced by Acinetobacter calcoaceticus

[0061] Treatment Mixed nitrogen source content g / L PQQ yield (mg / L) T1 4 43.65±0.18 T2 8 36.57±0.33 T3 12 40.34±0.73 T4 16 29.66±1.20 T5 16 21.61±0.20

[0062] 4. Determining the pH value during fermentation culture

[0063] Acinetobacter calcoaceticus was cultured using LB liquid medium, and the seed liquid OD 600 = 0.8, with a concentration of 2.3 x 10 4 cfu / mL; then on the basis of the same basic medium formula, temperature 28℃, shaking speed 180 rpm / min, bacterial inoculation amount 1%, yeast powder content 10 g / L, mixed nitrogen source (ammonium sulfate: glutamic acid: tyrosine = 2:1:1) 4 g / L, setting different pH fermentation experiments, and the fermentation lasted for 7 days. The results are shown in Table 7. When the pH is 6.5, the PQQ concentration produced by Acinetobacter calcoaceticus is the highest.

[0064] Table 7 Effect of pH on PQQ concentration produced by Acinetobacter calcoaceticus

[0065] Treatment pH PQQ yield (mg / L) T1 6 39.21±4.02 T2 6.5 45.17±0.53 T3 7 43.65±0.18 T4 7.5 37.25±3.72 T5 8 37.35±0.68

[0066] 5. Determining different inoculation bacterial amounts

[0067] Acinetobacter calcoaceticus was cultured using LB liquid medium, the seed liquid OD 600 = 0.8, with a concentration of 2.3 x 10 4 cfu / mL; then on the basis of the same basic medium formula, at a temperature of 28°C, pH 6.5, a shaking speed of 180 rpm / min, with a yeast powder content of 10 g / L, and mixed nitrogen source (ammonium sulfate: glutamic acid: tyrosine = 2: 1: 1) 4 g / L, different bacterial inoculation amounts were set for fermentation experiments, and the fermentation was carried out for 7 days. The results are shown in Table 8. When the inoculation amount was 0.5%, the PQQ concentration produced by Acinetobacter calcoaceticus was the highest.

[0068] Table 8 Effect of different inoculation amounts on PQQ concentration produced by Acinetobacter calcoaceticus

[0069]

[0070]

[0071] 6. Determining different temperatures

[0072] Acinetobacter calcoaceticus was cultured using LB liquid medium, the seed liquid OD 600 = 0.8, with a concentration of 2.3 x 10 4 cfu / mL; then on the basis of the same basic medium formula, at pH 6.5, a shaking speed of 180 rpm / min, with a bacterial inoculation amount of 0.5%, a yeast powder content of 10 g / L, and mixed nitrogen source (ammonium sulfate: glutamic acid: tyrosine = 2: 1: 1) 4 g / L, different fermentation temperatures were set for fermentation experiments, and the fermentation was carried out for 7 days. The results are shown in Table 9. When the temperature was 28°C, the PQQ concentration produced by Acinetobacter calcoaceticus was the highest.

[0073] Table 9 Effect of different fermentation temperatures on PQQ concentration produced by Acinetobacter calcoaceticus

[0074] Treatment Temperature (°C) PQQ yield (mg / L) T1 28 59.43±2.63 T2 30 55.97±0.58 T3 32 53.32±0.94 T4 34 53.09±0.16 T5 36 50.12±0.53

[0075] 7. Determining magnesium sulfate content

[0076] Acinetobacter calcoaceticus was cultured using LB liquid medium, the seed liquid OD 600 = 0.8, with a concentration of 2.3 x 10 4cfu / mL; the other components of the basic medium were kept constant, and different magnesium sulfate contents were set for the fermentation experiment, pH 6.5, shaking speed 180 rpm / min, bacterial inoculation amount 0.5% as the basis, yeast powder content 10 g / L, mixed nitrogen source (ammonium sulfate: glutamic acid: tyrosine = 2: 1: 1) 4 g / L, culture temperature 28°C as the basis, fermentation for 7d, and the results are shown in Table 10. The PQQ concentration produced by Acinetobacter calcoaceticus was the highest when the magnesium sulfate content was 1 g / L.

[0077] Table 10 Effect of different magnesium sulfate contents on PQQ concentration produced by Acinetobacter calcoaceticus

[0078] Treatment Magnesium sulfate (g / L) PQQ yield (mg / L) T1 0.5 56.00±1.54 T2 1 59.43±2.63 T3 1.5 57.15±1.83 T4 2 56.57±1.64 T5 2.5 55.85±1.10

[0079] 8. Determination of calcium chloride content

[0080] Acinetobacter calcoaceticus was cultured using LB liquid medium, the seed liquid OD 600 = 0.8, and the concentration was 2.3 x 10 4 cfu / mL; the other components of the basic medium were kept constant, and different calcium chloride contents were set for the fermentation experiment, pH 6.5, shaking speed 180 rpm / min, bacterial inoculation amount 0.5% as the basis, yeast powder content 10 g / L, mixed nitrogen source (ammonium sulfate: glutamic acid: tyrosine = 2: 1: 1) 4 g / L, culture temperature 28°C as the basis, fermentation for 7d, and the results are shown in Table 11. The PQQ concentration produced by Acinetobacter calcoaceticus was the highest when the calcium chloride content was 0.4 g / L.

[0081] Table 11 Effect of different magnesium sulfate contents on PQQ concentration produced by Acinetobacter calcoaceticus

[0082] Treatment Calcium chloride (g / L) PQQ yield (mg / L) T1 0.01 43.87±1.66 T2 0.1 46.08±1.10 T3 0.2 54.99±1.54 T4 0.4 61.48±1.64 T5 0.6 55.24±1.11

[0083] Example 2 Preparation of PQQ-containing microbial agent

[0084] Acinetobacter calcoaceticus was cultured using LB liquid medium, the seed liquid OD 600 = 0.8, and the concentration was 2.3 x 10 4 cfu / mL; on the basis of the medium optimized in Example 1, pH 6.5, shaking speed 180 rpm / min, bacterial inoculation amount 0.5% as the basis, yeast powder content 10 g / L, mixed nitrogen source (ammonium sulfate: glutamic acid: tyrosine = 2: 1: 1) 4 g / L, 28°C shaking culture for 7d, to obtain a PQQ-containing microbial agent.

[0085] The PQQ-containing bacterial agent prepared in this example and the PQQ standard sample were used as test samples, and the PQQ was determined by spectroscopy. PQQ standard samples with different mass concentrations (1, 2, 5, 10, 20, 25, 50, and 100 mg / L) were set, and the enzyme-labeled instrument detection showed good linear relationship, and the standard curve is shown in Figure 4 The PQQ-containing bacterial agent was centrifuged at 8000 r / min for 5 min, the supernatant was filtered with a 0.22 μm water-based membrane, and then the filtrate was subjected to spectroscopy test. The absorbance value was measured, and then the absorbance value of the culture medium was deducted. According to the standard curve, the concentration of PQQ was 61.48 mg / L.

[0086] It was detected that the effective viable count of Acinetobacter calcoaceticus in the PQQ-containing bacterial agent was 2.5*10 10 CFU / ml.

[0087] Example 3 Verification of the prevention and treatment effect of the PQQ-containing bacterial agent on pepper bacterial leaf spot

[0088] Healthy seedlings (peppers) with good growth and uniform size were selected, transplanted into pots containing 2.0 kg of substrate soil, one plant per pot, and conventionally managed in a 28°C greenhouse. Two treatments were set up in the test:

[0089] Bacterial agent treatment: 3 days after transplanting, each plant was irrigated with 100 mL of the PQQ-containing bacterial agent prepared in Example 2, and 10 8 cfu / mL of Pseudomonas syringae bacterial liquid 10 mL was irrigated into the roots of each plant 2 days later.

[0090] Water control: 3 days after transplanting, each plant was irrigated with 100 mL of water, and 10 8 cfu / mL of Pseudomonas syringae bacterial liquid 10 mL was irrigated into the roots of each plant 2 days later.

[0091] There were 12 plants per treatment, and each treatment was repeated 3 times. After 35 days of planting, the incidence rate was counted, and the results are shown in Table 10.

[0092] Table 10 Prevention and treatment effect of different treatments on pepper bacterial leaf spot

[0093] Treatment Number of diseased plants Total number Disease incidence (%) Fungicide treatment 3 36 8.33 Water control 32 36 88.89

[0094] As can be seen from the results in Table 10, the treatment group applied with the PQQ-containing bacterial agent can effectively prevent and treat the pepper bacterial leaf spot caused by Pseudomonas syringae, with an incidence rate of 8.33%, which is significantly lower than the incidence rate of 88.89% of the control group.

[0095] Example 4 Application of the PQQ-containing bacterial agent as a fertilizer additive in promoting plant growth

[0096] Select the healthy seedlings (peppers) with good growth and uniform size, transplant to pots containing 2.0 kg of substrate soil, 1 plant per pot, and manage them in a 28 °C greenhouse. Before the flowering and fruiting stage of the peppers, the plants are subjected to the following four treatments:

[0097] Fungicide treatment group 1: each plant is irrigated with 100 mL of the PQQ-containing fungicide prepared in Example 2, and the soil moisture content is controlled at 30-40%.

[0098] Fungicide treatment group 2: the PQQ-containing fungicide prepared in Example 2 is used as a fertilizer additive mixed with the fertilizer, and the soil moisture content is controlled at 75-80%.

[0099] Control group 1: the soil moisture content is controlled at 30-40%.

[0100] Control group 2: the same amount of fertilizer as in the fungicide treatment group 2 is applied without adding the PQQ-containing fungicide, and the soil moisture content is controlled at 75-80%.

[0101] Each treatment has 10 plants, and each treatment is repeated 3 times. After 21 days of planting, the growth and yield are counted, and the results are shown in Table 11.

[0102] Table 11 Growth and yield of peppers under different treatments

[0103]

[0104]

[0105] As shown by the results in Table 11, the PQQ-containing fungicide not only alleviates the impact of drought stress on the growth of peppers; when the PQQ-containing fungicide is mixed with the fertilizer and used as a fertilizer additive, it can significantly improve the yield of peppers.

[0106] In addition, the PQQ-containing fungicide prepared in Example 2 is diluted 100 times and sprayed on the leaves and roots of the peppers every 7 days. The experimental group and the control group are sprayed with the same amount. After 21 days of spraying, it is observed that the PQQ-containing fungicide can promote the growth of the peppers in the experimental group (especially the root development), and the results are shown in Table 12. Figures 5-7 Table 12 Growth and yield of peppers under different treatments

Claims

1. A strain of Acinetobacter calcoaceticus (A. calcoaceticus) CDWB36, characterized in that, Acinetobacter calcoaceticus ) CDWB36, characterized in that, The Acinetobacter calcoaceticus (A. Acinetobacter calcoaceticus ) CDWB36 has a preservation number of CCTCC NO: M2023964, is preserved in the China Center for Type Culture Collection, and was preserved on June 8, 2023, in Wuhan, Wuhan University, China; and the 16S rRNA nucleotide sequence of the Acinetobacter calcoaceticus (A. Acinetobacter calcoaceticus ) CDWB36 is shown as SEQ ID NO:

1.

2. A strain of Acinetobacter calcoaceticus (A. calcoaceticus) CDWB36 as claimed in claim 1 for use in the fermentative production of PQQ. Acinetobacter calcoaceticus ) CDWB36 in the fermentative production of PQQ.

3. A method of producing PQQ, comprising: The acinetobacter calcoaceticus of claim 1 is used to ferment PQQ. Acinetobacter calcoaceticus ) CDWB36 to ferment PQQ.

4. A method for producing a PQQ-containing microbial agent, comprising the following steps: (1) The Acinetobacter calcoaceticus (A. calcoaceticus) CDWB36 of claim 1 is activated in LB liquid medium to obtain a seed liquid; Acinetobacter calcoaceticus ) (2) inoculating the seed liquid obtained in step (1) into a fermentation medium to perform fermentation culture, and obtaining a PQQ-containing microbial agent.

5. The method of claim 4, wherein, The formula of the LB liquid culture medium comprises: 8-12 g / L of tryptone, 4-6 g / L of yeast extract, 9-11 g / L of sodium chloride, 14-16 g / L of agar powder, and pH 7.0±0.2; the formula of the fermentation medium is: 8-12 g / L of a carbon source, 3-5 g / L of a nitrogen source, 1.5-2.5 g / L of Na2HPO4, 1.2-1.6 g / L of KH2PO4, 0.8-1.2 g / L of MgSO4·7H2O, and 0.42-0.43 g / L of a trace element solution, and pH 6.5-7.0; the trace element solution comprises: 75-85 mg / L of FeSO4·7H2O, 22-23 mg / L of ZnSO4·7H2O, 0.3-0.4 mg / L of KI, 2.5-3.5 mg / L of H3BO3, 4-6 mg / L of CuSO4, 14-16 mg / L of NaCl, and 200-400 mg / L of CaCl2·2H2O; the carbon source is at least one of yeast powder, glucose, sucrose, maltose, and galactose; and the nitrogen source is one of anhydrous ammonium sulfate, L-glutamic acid, L-tyrosine, and peptone or a mixed nitrogen source composed of anhydrous ammonium sulfate, L-glutamic acid, and L-tyrosine.

6. The method according to claim 4 or 5, characterized in that, The formula of the LB liquid culture medium comprises: 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of sodium chloride, 15 g / L of agar powder, and pH 7.0; the formula of the fermentation medium is: 10 g / L of a carbon source, 4 g / L of a nitrogen source, 2 g / L of Na2HPO4, 1.4 g / L of KH2PO4, 1 g / L of MgSO4·7H2O, and 0.4258 g / L of a trace element solution, and pH 6.5; the carbon source is glucose; the nitrogen source is a mixed nitrogen source composed of anhydrous ammonium sulfate, L-glutamic acid, and L-tyrosine at a mass ratio of 2:1:1; and the trace element solution comprises: 80 mg / L of FeSO4·7H2O, 22.5 mg / L of ZnSO4·7H2O, 0.3 mg / L of KI, 3 mg / L of H3BO3, 5 mg / L of CuSO4, 15 mg / L of NaCl, and 400 mg / L of CaCl2·2H2O.

7. The method of claim 4, wherein, The inoculation amount in step (2) is 0.5-1%; the fermentation culture conditions are: temperature is 28-30℃, time is 7d.

8. A PQQ-containing bacterial agent produced by the method according to any one of claims 4 to 7, characterized in that, In the bacterial agent containing PQQ, Acinetobacter calcoaceticus ( Acinetobacter calcoaceticus )The effective viable count of CDWB36 is 10 9 CFU / ml or above, and the concentration of PQQ produced is above 4 mg / L.

9. The PQQ-containing bacterial agent according to claim 8, characterized by the fact that, In the bacterial agent containing PQQ, Acinetobacter calcoaceticus ( Acinetobacter calcoaceticus )The effective viable count of CDWB36 is 2.0-2.5*10 10 CFU / ml, and the concentration of PQQ was above 60 mg / L.

10. The PQQ-containing bacterial agent of claim 8 or 9 or the PQQ-containing bacterial agent produced by the method of any one of claims 4-7 for use in at least one of the following: 1) preparing a biocontrol agent; 2) improving the stress resistance of peppers; 3) promoting the growth of peppers; 4) preparing a fertilizer additive.

11. Use according to claim 10, characterized in that, The PQQ-containing bacterial agent is used by spraying with dilution of 200-300 times or by root irrigation with dilution of 100-150 times.

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