Isoschiff bud botrytis cinerea with potassium hyperaccumulation capacity and application of isoschiff bud botrytis cinerea

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to botrytis isoschiff bud yeast with potassium hyperaccumulation capacity and application of the botrytis isoschiff bud yeast. Aiming at the problem that a strain capable of enriching potassium ions in wastewater is lacked in the prior art, the invention provides the botrytis isoschizochytrium JK-1 which is preserved in the general microbiological center of the China Committee for Culture Collection of Microorganisms on January 12, 2026, and the preservation number of the botrytis isoschizochytrium JK-1 is CGMCC (China General Microbiological Culture Collection Center) No.39121. The invention further provides a preparation method of the botrytis isoschizochytrium JK-1. The strain JK-1 is found for the first time, has the characteristic of potassium hyperaccumulation, not only can purify biogas slurry, but also can be used as a biological pump to concentrate and recover potassium resources.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of *Botrytis cinerea* with potassium hyperaccumulation ability and its application. Background Technology

[0002] Currently, there is a lack of effective and low-cost methods for recovering potassium ions from wastewater.

[0003] Utilizing microorganisms to transform wastewater into single-cell protein (SCP) is an effective way to achieve the resource utilization of wastewater (such as biogas slurry). However, existing yeast strains used for wastewater treatment (such as Saccharomyces cerevisiae and Candida utilis) often have the following drawbacks: insufficient tolerance to high concentrations of ammonia nitrogen and complex inhibitors in wastewater such as biogas slurry, resulting in slow growth; lack of efficient enrichment mechanisms for specific mineral potassium ions, leading to the risk of high salt content in the treated biogas slurry; and difficulty in low-cost separation of potassium ions enriched within the cells, resulting in excessively high ash content in the produced SCP, affecting feed quality.

[0004] Therefore, screening a new strain that possesses the characteristic of "excess potassium uptake" has significant industrial application value. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of *Botrytis cinerea* with potassium hyperaccumulation ability and its applications.

[0006] To achieve the above-mentioned objectives, the technical solution adopted in this invention is: Blastobotrys allociferrii JK-1, which was deposited on January 12, 2026 at the China General Microbiological Culture Collection Center, with the accession number CGMCC No. 39121.

[0007] Accordingly, bacterial preparations made using the aforementioned Botrytis cinerea JK-1, or bacterial preparations containing the aforementioned Botrytis cinerea JK-1.

[0008] Accordingly, the application of *Botrytis cinerea* JK-1 or the bacterial preparation in the treatment of potassium-containing wastewater. The wastewater treatment aims to reduce the ammonia nitrogen concentration in the wastewater and / or reduce the potassium ion concentration in the potassium-containing wastewater.

[0009] Accordingly, the application of the Isochrysis budding yeast JK-1 or the bacterial preparation in the enrichment of potassium ions involves inoculating the Isochrysis budding yeast JK-1 or the bacterial preparation into an environment to be enriched with potassium ions, and controlling the ambient temperature at 20-33°C.

[0010] Accordingly, the application of *Botrytis cinerea* JK-1 or the bacterial preparation in potassium ion transfer includes the following steps:

[0011] (1) Inoculate the isosievorous budding botrytis cinerea JK-1 or the bacterial preparation into an environment rich in potassium ions to enrich potassium ions, and control the ambient temperature to be 20-33℃.

[0012] (2) Inoculate the potassium-enriched Botrytis cinerea JK-1 or the bacterial preparation into an environment where the potassium concentration needs to be increased, and control the ambient temperature to be 0℃~6℃.

[0013] This invention offers the following advantages: The strain provided by this invention is a newly discovered *Botrytis cinerea* strain with "potassium hyperaccumulation" characteristics. This strain can not only purify biogas slurry but also act as a "biological pump" to concentrate and recover potassium resources. Using this strain to treat potassium-containing wastewater, such as biogas slurry, the potassium ion removal rate is as high as 96% or more, and the ammonia nitrogen removal rate is as high as 96%, significantly reducing the pollution load of wastewater. The strain JK-1 provided by this invention can rapidly grow in high-concentration biogas slurry (ammonia nitrogen concentration > 3000 mg / L) using biogas slurry as the sole nitrogen source / main nutrient source. Under normal temperature and aerobic conditions, strain JK-1 exhibits extremely strong active potassium ion transport capacity and strong ammonia nitrogen absorption capacity. Attached Figure Description

[0014] Figure 1 This is a colony morphology diagram of strain JK-1;

[0015] Figure 2 This is an optical microscope image of strain JK-1;

[0016] Figure 3 This is a schematic diagram of the growth curve of strain JK-1;

[0017] Figure 4 This is a schematic diagram of the overlapping regions of the molecular identification characteristic sequences of strain JK-1. Detailed Implementation

[0018] This invention provides a strain of *Blastobotrysallociferrii* JK-1 with potassium hyperaccumulation characteristics, which was deposited on January 12, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 39121. The characteristic sequence of strain JK-1 is shown in SEQ ID NO: 1. In this characteristic sequence, 1–1795 bp is the 18S-SSU sequence, 1744–2354 bp is the ITS sequence, 2335–2954 bp is the 26S sequence, and 1956–5996 bp is the 28S-LSU sequence.

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the data obtained are all average values ​​obtained after at least three repetitions, and each repetition yields valid data.

[0020] Example 1: Acquisition and Identification of Strains

[0021] 1. Screening of strains (verification of biogas slurry tolerance)

[0022] Chicken manure biogas slurry from Shandong (sterilized at high temperature and with added glucose at a concentration of 40 g / L) was extracted and inoculated onto YPD solid medium using the streak plate method. The medium was incubated at 30℃ for 48 h, and intact, independent yeast colonies (numbered JK-1) were picked. These colonies were then inoculated into 50 mL of biogas slurry medium (diluted to an ammonia nitrogen concentration of 2500 mg / L, with added glucose to a concentration of 40 g / L, and sterilized by steam at 115℃ for 15 min in a 250 mL Erlenmeyer flask) and incubated with shaking in a constant temperature shaking incubator for 5 days (180 rpm, 30℃).

[0023] After the culture was completed, the culture was transferred to a 50 mL centrifuge tube for centrifugation (8000 rpm, 5 min, 25 ℃). It was observed that the biomass of the sedimentary bacteria was significantly increased compared with the initial inoculation, which confirmed that JK-1 can tolerate harsh environments such as high ammonia nitrogen in biogas slurry and can use ammonia nitrogen in biogas slurry as a nitrogen source and glucose as a carbon source for growth and reproduction.

[0024] 2. Identification

[0025] The colonies of strain JK-1 on YPD plates are milky white, with wrinkled surfaces and irregular edges, and the colony morphology is as follows. Figure 1As shown; under a microscope, JK-1 cells are spherical, ellipsoidal, or lemon-shaped, and reproduce through budding, forming pseudohyphae, such as... Figure 2 As shown. The optimal growth temperature for this strain is 25–33℃, and the optimal growth pH is 3.2–8.0. It can utilize one or a mixture of glucose, acetic acid, lactic acid, xylose, sucrose, molasses, etc., as carbon sources.

[0026] The growth curve of strain JK-1 in biogas slurry medium (biogas slurry diluted to an ammonia nitrogen concentration of 1900 mg / L, glucose added to a concentration of 10 g / L, and sterilized by steam at 115°C for 15 min in a 250 mL Erlenmeyer flask) is as follows: Figure 3 As shown.

[0027] Genomic DNA was extracted from strain JK-1 and sequenced. The results are shown in Tables 1-3. Figure 4 As shown. Figure 4 This indicates the relationships between DNA sequences involved in the molecular identification of strain JK-1 (BLAST database alignment).

[0028] Table 1 18S-SSU Comparison

[0029]

[0030] Table 2 28S-LSU Comparison

[0031]

[0032] Table 3 ITS Comparison

[0033]

[0034] The results showed that the DNA sequence within the sequencing alignment range included 5996 base pairs, with 8 repetitions on the chromosome, 1 incomplete repetition, 5 complete and identical repetitions, and the remaining 3 repetitions containing only one mutated base. Strain JK-1 showed over 99% homology with *Blastobotrys allociferrii*. Based on the combined physiological and biochemical characteristics, strain JK-1 was identified as *Blastobotrys allociferrii*.

[0035] Example 2: Demonstration of the effect of strain JK-1 in removing potassium from biogas slurry

[0036] Complete, independent yeast colonies were picked from streak plates used to activate strain JK-1 and inoculated into YPD liquid medium (250 mL Erlenmeyer flasks). The culture was then incubated in a constant-temperature shaking incubator for 24 h to obtain the seed culture. The seed culture was then inoculated into biogas slurry medium (prepared as in Example 1, but the ultrafiltered biogas slurry was diluted to 1 / 3 of its original concentration, resulting in a potassium ion concentration of 1440 mg / L, and glucose was added to a concentration of 10 g / L) for tank fermentation. During fermentation, the glucose concentration was maintained at 10 g / L, and the pH was maintained at 4.0 ± 0.1 using 4 M NaOH and 4 M H₂SO₄. The total fermentation volume was approximately 8 L, the inoculum size was 10% (v / v), and the fermentation ran for 8 days.

[0037] After fermentation, the fermentation broth was transferred to 50 mL centrifuge tubes and centrifuged (8000 rpm, 5 min, 25℃). The potassium ion concentration and ammonia nitrogen concentration of the fermentation supernatant were determined using flame atomic absorption spectrophotometry (GB / T 11904-1989 Determination of Potassium and Sodium in Water) and Nessler's reagent colorimetric method (GB / T 7479-1987 Determination of Ammonium in Water). The results showed that the potassium ion concentration decreased from 1440.00 mg / L to 48.01 mg / L, and the ammonia nitrogen concentration decreased from 1570 mg / L to 55 mg / L. The potassium ion absorption rate exceeded 96%, and the ammonia nitrogen absorption rate exceeded 96%. These phenomena remained stable in repeated experiments.

[0038] A control group was also set up: the control group was inoculated with equal amounts of Debaryomyces hansenii and Candida utilis, both of which are tolerant of biogas slurry environments and produce single-cell protein. All other conditions were the same. The potassium ion concentration in the supernatant and the biomass were measured. Each group was set up with 3 replicates, and the results were averaged. The results are shown in Table 4.

[0039] Table 4. Comparison of potassium ion concentration and biomass changes in each group

[0040]

[0041] The results showed that the potassium ion concentration in the supernatant of group JK-1 decreased to about 48 mg / L, and the potassium ion removal rate reached more than 96%; the potassium ion concentration in the cells increased to 32.74 mg / g; and the cell biomass increased significantly, while Hansenula d'Barry yeast and Candida utilis did not show the same phenomenon.

[0042] Example 3: Demonstration of potassium ion transport capacity of strain JK-1

[0043] The strain JK-1 cultured in Example 2 was placed in a low-temperature (0℃~6℃, 4℃ used in this example) liquid environment (including but not limited to a low-potassium ion liquid environment of biogas slurry after fermentation; in this example, biogas slurry diluted 3 times was used, with a potassium ion concentration of 503.3 mg / L after dilution) for cold stress treatment to induce the release of intracellular potassium ions into the extracellular environment. After the addition, the cell CDW = 32.03 g / L. The potassium ion concentration in the environment and in the strain cells before and after cold stress treatment was measured. Each group was set up with 3 replicates, and the results were averaged. The results are shown in Table 5.

[0044] Table 5 Comparison of potassium ion concentration changes in each group

[0045]

[0046] The results showed that cold stress treatment significantly increased the extracellular potassium ion concentration from the baseline level before treatment, and this characteristic of strain JK-1 can be used to achieve rapid, efficient and low-cost potassium ion transfer.

[0047] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, alterations, substitutions, or variations made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.

Claims

1. *Blastobotrys allociferrii* JK-1, characterized by: The *Botrytis cinerea* JK-1 was deposited on January 12, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 39121.

2. A bacterial preparation made using Botrytis cinerea JK-1 as described in claim 1, or a bacterial preparation containing Botrytis cinerea JK-1 as described in claim 1.

3. The application of the *Botrytis cinerea* JK-1 of claim 1 or the bacterial preparation of claim 2 in the treatment of potassium-containing wastewater.

4. The application according to claim 3, characterized in that: The wastewater treatment involves reducing the ammonia nitrogen concentration in potassium-containing wastewater and / or reducing the potassium ion concentration in potassium-containing wastewater.

5. The application of the *Botrytis cinerea* JK-1 of claim 1 or the bacterial preparation of claim 2 in the enrichment of potassium ions, characterized in that: The *Botrytis cinerea* JK-1 or the bacterial preparation is inoculated into an environment rich in potassium ions, and the ambient temperature is controlled at 20–33°C.

6. The application of the *Botrytis cinerea* JK-1 of claim 1 or the bacterial preparation of claim 2 in potassium ion transfer, characterized in that: The application includes the following steps: (1) Inoculate the *Botrytis cinerea* JK-1 or the bacterial preparation into a potassium-rich environment to enrich potassium ions, and control the ambient temperature to be 20-33°C. (2) Inoculate the potassium-enriched Botrytis cinerea JK-1 or the bacterial preparation into an environment where the potassium concentration needs to be increased, and control the ambient temperature to be 0℃~6℃.

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