Cold-tolerant oligophototrophic bacteria and application thereof

Abstract

The application discloses a Paucibacter psychrotolerans capable of degrading microcystins and application thereof. The Paucibacter psychrotolerans provided by the application is Paucibacter psychrotolerans 2WFC-1, and the preservation number thereof in the China General Microbiological Culture Collection Center is CGMCC No. 24793. The strain 2WFC-1 belongs to a new species of the Paucibacter genus, can degrade microcystins, and is different from other Paucibacter in that it can grow at a temperature ranging from 4 DEG C to 37 DEG C, has great potential for degrading microcystins in a low-temperature water environment, and has important application prospects in water ecological restoration, seasonal water pollution treatment and drinking water purification.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a cold-resistant oligotrophic bacillus that degrades microcystin and its applications. Background Technology

[0002] Microcystins (MCs) are a class of algal toxins produced by cyanobacteria. During algal blooms, the large amounts of cyanobacteria covering the water surface release various microcystins into the water. Studies have shown that microcystins are hepatotoxic cyclic peptide toxins, primarily targeting the liver in animals and causing liver damage. Skin contact with water containing microcystins during bathing, swimming, and other water recreational activities can cause allergic reactions in sensitive areas; small amounts ingested can cause acute gastroenteritis; long-term consumption can lead to non-alcoholic fatty liver disease by interfering with lipid metabolism, further inducing liver cancer. Furthermore, these toxins are quite stable due to their cyclic structure and multiple double bonds, making them difficult to degrade. They are resistant to high and low temperatures, and are highly soluble in water yet difficult to precipitate, meaning they are largely not adsorbed by sediments and suspended solids in water. Therefore, they pose a serious threat to both animals and humans. Given the increasingly severe problem of algal bloom pollution, research on the degradation of microcystins is urgently needed. Currently, the main methods for treating microcystin in the aquatic environment include activated carbon adsorption, photodegradation and photocatalytic oxidation, chemical oxidation, membrane filtration, and biodegradation. Compared with the aforementioned physical and chemical treatment methods, microbial degradation technology has advantages such as thorough degradation, low cost, no secondary pollution, good safety, and benefits for ecological restoration, and is likely to become one of the most suitable methods for solving large-scale algal toxin pollution.

[0003] Oligotrophobic bacteria ( Paucibacter ) is in the bacterial domain β -Proteobacteria ( Betaproteobacteria A group of bacteria. In 2005, Rapala et al. discovered and effectively described *Oligoceps eutrophus* (a type of bacteria). Paucibacter toxinivorans This is a typical species of the genus, with strain 2C20 possessing the ability to degrade microcystin. Currently, this genus contains only two validly published species: *Oligococcus pyogenes* (a type of microcystin). Paucibacter toxinivorans , DOI: 10.1099 / ijs.0.63599-0) and oligotrophic oligotrophic bacteria ( Paucibacter oligotrophusThe genus *Oligocactus* has only five published articles (DOI: 10.1099 / ijsem.0.001931, DOI: 10.1099 / ijsem.0.001931) regarding its ability to degrade microcystin. There are also only five other reports on its function in degrading microcystin in both domestic and international publications (DOI: 10.3390 / toxins13040265, 10.1016 / j.envpol.2022.119079, 10.1099 / ijs.0.63599-0, org / 10.3390 / toxins8110318, 10.13227 / j.hjkx.2014.01.045). Therefore, actively exploring and discovering new resources of *Oligocactus* species in nature and utilizing their ability to degrade microcystin for water pollution remediation is of great significance for improving the water environment of inland lakes and the health of drinking water for the people, developing highly efficient microbial agents for water pollution remediation, and promoting the steady and healthy development of my country's economy. Summary of the Invention

[0004] The purpose of this invention is to provide an oligotrophic bacillus capable of degrading microcystin, a microbial preparation containing the oligotrophic bacillus, and its applications.

[0005] This invention provides a cold-resistant oligotrophic bacillus ( Paucibacter psychrotolerans The strain number is 2WFC-1, and its accession number at the China General Microbiological Culture Collection Center is CGMCC No. 24793.

[0006] The present invention also provides a microbial preparation, wherein the active ingredient of the microbial preparation includes the aforementioned psychrophilic oligotrophic bacillus or a culture of the aforementioned psychrophilic oligotrophic bacillus, wherein the culture of the psychrophilic oligotrophic bacillus is a substance in a culture container obtained by culturing the aforementioned psychrophilic oligotrophic bacillus in a microbial culture medium.

[0007] The culture medium is either an inorganic salt culture medium or an R2A culture medium.

[0008] The application of the aforementioned psychrophilic oligotrophic bacilli or the aforementioned microbial preparations in the degradation of microcystin toxins should also be within the scope of protection of this invention.

[0009] The method for degrading microcystin is a microbial degradation method.

[0010] The application of the aforementioned psychrophilic oligotrophic bacillus or the aforementioned microbial preparation in aquatic ecological restoration and / or water pollution control should also be within the scope of protection of this invention.

[0011] The aquatic ecosystem to be restored is an aquatic ecosystem containing microcystin, and the water pollution is microcystin pollution.

[0012] The aforementioned water ecological restoration and / or water pollution control refers to low-temperature water ecological restoration and / or low-temperature water pollution control. Here, low temperature refers to a temperature range above 4℃ and below 15℃.

[0013] The application of the aforementioned psychrophilic oligotrophic bacteria or the aforementioned microbial preparation in drinking water purification should also be within the scope of protection of this invention.

[0014] The application of the psychrophilic oligotrophic bacillus or the microbial preparation in drinking water purification refers to the use of psychrophilic oligotrophic bacillus or the microbial preparation to purify microcystin toxins present in drinking water. In order to ensure the safety of drinking water, it can be used in combination with other methods or substances for purifying drinking water.

[0015] The present invention provides a method for degrading microcystin, comprising the step of adding the aforementioned cold-resistant oligotrophic bacillus or the aforementioned microbial preparation to a body of water containing microcystin.

[0016] Compared with the prior art, the present invention has the following advantages:

[0017] 1. Psychrophilic oligotrophic bacillus 2WFC-1 is a new species of oligotrophic bacillus that is different from other microcystin-degrading bacteria;

[0018] 2. The psychrophilic oligotrophic bacillus 2WFC-1 can grow in a temperature range of 4℃ to 37℃, which is significantly wider than other reported oligotrophic bacillus strains (15℃ to 37℃), especially showing growth advantage at low temperatures above 4℃.

[0019] Therefore, the psychrophilic oligotrophic bacillus 2WFC-1 disclosed in this invention can be used to degrade microcystin in water and has a broader temperature adaptability. It has great potential for the degradation of microcystin in low-temperature water environments and has important application prospects in water ecological restoration, seasonal water pollution control and drinking water purification.

[0020] Preservation Instructions

[0021] Strain name: Psychrophilic oligotrophic bacillus

[0022] Latin name: Paucibacter psychrotolerans

[0023] Strain number: 2WFC-1

[0024] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0025] Collection institution abbreviation: CGMCC

[0026] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0027] Deposit date: March 3, 2022

[0028] CGMCC Registration Number: 24793 Attached Figure Description

[0029] Figure 1 This is a phylogenetic tree of strain 2WFC-1 of the present invention based on the 16S rRNA gene sequence.

[0030] Figure 2 This is a transmission electron microscope image of strain 2WFC-1 of the present invention.

[0031] Figure 3 This is a comparison diagram of the growth status of strain 2WFC-1 of the present invention at 4℃.

[0032] Figure 4 This is a graph showing the microcystin degradation rate of strain 2WFC-1 of the present invention. Detailed Implementation

[0033] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0035] Example 1. Isolation, screening and identification of strain 2WFC-1

[0036] 1. Isolation and screening of strain 2WFC-1

[0037] The strain 2WFC-1 of this invention was isolated from a spring water sample from Xinjiang Uygur Autonomous Region. R2A medium was used for isolation, with water as the solvent. The solute composition was as follows (g·L⁻¹). -1 ): Glucose 0.5, soluble starch 0.5, peptone 0.5, yeast extract 0.5, acid-hydrolyzed casein 0.5, sodium pyruvate 0.3, dipotassium hydrogen phosphate 0.3, magnesium sulfate heptahydrate 0.05, agar 15, pH 7.2.

[0038] Isolation and screening method: Cells were collected by filtering spring water samples through a 0.22-micron filter membrane and then resuspended in physiological saline. 200 μL of the cell suspension was plated onto R2A agar plates and incubated at 30 °C for 2-3 weeks. Colonies with good growth, intact bacterial growth, and diverse morphological characteristics were selected and streaked onto R2A plates for purification, yielding different pure strains. These strains were preserved using two methods: freeze-drying with 10% (v / v) skim milk and cryopreservation with 15% (v / v) glycerol in liquid nitrogen. One of the obtained strains was designated 2WFC-1, which is the psychrophilic oligotrophic bacillus described in this application. Paucibacter psychrotolerans 2WFC-1, with accession number CGMCC No. 24793, hereinafter referred to as strain 2WFC-1.

[0039] 2. Phylogenetic analysis of strain 2WFC-1

[0040] Genomic DNA was extracted from strain 2WFC-1 of this invention, and its 16S rRNA gene sequence was amplified, cloned, and sequenced (as shown in Sequence 1 of the sequence listing). The obtained sequence was compared online in an internationally authoritative bacterial taxonomy database (http: / / www.ezbiocloud.net / ) (DOI: 10.1099 / ijs.0.038075-0). The results showed that strain 2WFC-1 of this invention had the highest similarity to species of the genus *Oligobacterium*, with the most pairwise sequence similarity represented by the following strains: Paucibacter oligotrophus CHU3 T (98.49% similarity) Paucibacter toxinivorans 2C20 T (98.12%). Therefore, the highest similarity of the 16S rRNA gene sequence of strain 2WFC-1 of this invention with known bacterial species is 98.49%, which is significantly lower than the species-defining threshold of 98.7% (DOI:10.1099 / ijsem.0.002516). To further clarify the phylogenetic position of the strain, a phylogenetic tree was constructed using the 16S rRNA gene sequences of representative strains from all species of the genus *Oligobacterium*, as shown below. Figure 1 As shown, strain 2WFC-1 of the present invention forms a single branch and clusters in the genus Oligotrophobicus, which fully demonstrates that it is a new species of the genus Oligotrophobicus.

[0041] 3. Detection of physiological and biochemical characteristics of strain 2WFC-1

[0042] 3.1 After growing strain 2WFC-1 in R2A medium at 30℃ for 3 days, the color, size, and morphology of the bacterial colony were observed visually. Cell morphology of the strain was observed using a transmission electron microscope. The results are as follows: Figure 2As shown, strain 2WFC-1 is a Gram-negative bacterium, a facultative anaerobe, with short rod-shaped cells, 1.5-2.0 μm long and 0.7-1.0 μm wide, and moves by single-ended flagella.

[0043] 3.2 The temperature range for the growth of strain 2WFC-1 was determined in R2A liquid medium (Haibo Biotechnology, HB0167-2). The growth temperature range was 4, 10, 20, 25, 30, 37, 45, and 50 °C. Physiological and biochemical functions of the strain were assessed using the API 20E, API 20NE, and API ZYM assay kits manufactured by bioMérieux (France) and the GEN III assay system manufactured by Biolog (USA). Other physiological characteristics of the strain, including Gram staining properties, motility, oxygen requirement, catalase activity, and casein hydrolysis activity, were determined according to the *Handbook of Systematic Identification of Common Bacteria*.

[0044] Identification results showed that strain 2WFC-1 is a Gram-negative, facultative anaerobic bacterium with short rod-shaped cells, 1.5–2.0 μm long and 0.7–1.0 μm wide, and moves via unilateral flagella (see...). Figure 2 The strain's growth tolerance range is 4-37℃. Figure 3 ).Depend on Figure 3 As can be seen, compared with the clear blank control medium, strain 2WFC-1 showed continuous cell growth at 4℃, 10℃, and 20℃, with a large number of cells causing the medium to become turbid. This indicates that strain 2WFC-1 can grow and reproduce at 4℃, while other reported Oligotrophobic bacteria strains have a temperature growth range of only 15 to 37℃ (DOI: 10.1099 / ijs.0.63599-0, 10.1099 / ijsem.0.001931, 10.3390 / toxins13040265, 10.1016 / j.envpol.2022 .119079, org / 10.3390 / toxins8110318, 10.13227 / j.hjkx.2014.01.045, 10.1007 / s00203-018-1494-2).

[0045] The strain can produce tryptophan deaminase, gelatinase, and arginine dihydrolase; it does not produce... β- galactosidase, trypsin and β - Glucosidase. The physiological and biochemical differences between strain 2WFC-1 and representative published strains of the closely related *Oligobacterium* genus are shown in Table 1.

[0046] Table 1. Differences in physiological and biochemical characteristics between strain 2WFC-1 and representative strains of the genus *Oligobacterium*.

[0047]

[0048] Note: In the table, + indicates positive and - indicates negative.

[0049] As shown in Table 1, the strain 2WFC-1 of this invention differs significantly from the published oligotrophic bacteria strains in some physiological and biochemical characteristics.

[0050] 4. Detection of cytochemical characteristics of strain 2WFC-1

[0051] The cytochemical components of strain J1A816, including fatty acids and quinone types, were detected by gas chromatography (6890; Hewlett Packard) and HPLC. Fatty acid composition is shown in Table 2. The main fatty acid of strain 2WFC-1 is C. 16:1 ω7c / C 16:1 ω6c and C 16:0 It accounts for 63.58% of the total content. The main respiratory quinone type in the respiratory chain is coenzyme Q (Q-8) with 8 isoprene side chains.

[0052] Table 2. Differences in fatty acid characteristics between strain 2WFC-1 and representative strains of closely related oligotrophic bacteria.

[0053]

[0054] Note: "-" in the table indicates not detected; general characteristic components refer to 2 or 3 fatty acid components that cannot be separated by the gas chromatography MIDI system. General characteristic 2 includes C 12:0 aldehyde (unknown ingredient), C 16:1 iso I / C 14:0 3-OH; Key characteristic 3 includes C 16:1 ω 7 c / C 16:1 ω 6 c ; Summary features 8 include C 18:1 ω 7 c / C 18:1 ω 6 c .

[0055] Detection of microcystin degradation capacity of strain 2WFC-1

[0056] In this embodiment, the degradation rate of microcystin of strain 2WFC-1 was calculated using liquid chromatography.

[0057] 1. First, the standard curve for microcystin concentration was constructed: Microcystin standard solutions with concentration gradients of 100, 200, 400, 800, and 1000 μg / L were prepared, wherein the microcystin was a methanol solution of microcystin (MC-LR) (Alta, 1ST9122-10M). High-performance liquid chromatography (HPLC) was used to detect the concentration and peak area, and a standard curve was plotted with concentration on the x-axis and peak area on the y-axis. HPLC detection conditions: mobile phase was 40% acetonitrile dissolved in 0.05% trifluoroacetic acid aqueous solution; flow rate was 1.0 mL / min; UV detection wavelength was 238 nm; injection volume was 20 μL; and a C18 reversed-phase column was used.

[0058] 2. Degradation capacity detection of the strain: 500 μL of bacterial suspension (1.0 OD concentration) was inoculated into 4.5 mL of inorganic salt liquid medium containing 1000 μg / L microcystin and cultured at 30℃ and 150 rpm on a shaker. Three replicates were performed (the average result was taken), and a blank control group (containing 1000 μg / L microcystin liquid medium) was included. Every hour during the culture process, 500 μL of the culture medium was sampled, filtered, and the concentration of microcystin in the culture medium was calculated using high-performance liquid chromatography (HPLC). The results are shown below. Figure 4 As shown. Inorganic salt culture medium composition (g·L) -1 ): Magnesium sulfate heptahydrate 1.0, potassium dihydrogen phosphate 0.5, dipotassium hydrogen phosphate 4.0, sodium chloride 1.0, calcium chloride 0.02, ferrous sulfite 0.005, zinc chloride 0.005, manganese chloride tetrahydrate 0.005, copper chloride 0.0005.

[0059] Formula for calculating microcystin degradation rate: Total degradation rate of strain = Degradation toxin concentration / Time;

[0060] Unit degradation rate = Degradation toxin concentration / Cell number / Time.

[0061] Cell counts were obtained using colony counting methods.

[0062] The microcystin degradation rate of strain 2WFC-1 of this invention was calculated to be 0.0225 mg / L / h, with a unit degradation rate of 30 fg / cell / h. The degradation rate is as follows: Figure 4 As shown.

[0063] Depend on Figure 4 As can be seen, compared with the blank control group, the concentration of microcystin in the inorganic salt culture medium inoculated with strain 2WFC-1 decreased significantly with increasing culture time, indicating that strain 2WFC-1 of the present invention has the ability to degrade microcystin.

[0064] In summary, strain 2WFC-1 of this invention differs significantly from existing oligotrophic bacilli in genotype, phenotype, physiological and biochemical properties, and cytochemistry, thus belonging to a new species of the genus Oligotrophic Bacilli. Furthermore, this strain possesses the ability to degrade microcystin; and unlike existing oligotrophic bacilli, it can grow in low-temperature environments above 4°C, hence it is named *Cryopharyngotrophic Bacilli*. Paucibacter psychrotolerans Therefore, the strain WFC-1 of this invention can be used to degrade microcystins in water, and has a broader temperature adaptability. It has great potential for the degradation of microcystins in low-temperature water environments and has important application prospects in water ecological restoration, seasonal water pollution control and drinking water purification.

[0065] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Psychrophilic oligotrophic bacteria ( Paucibacter psychrotolerans The strain number is 2WFC-1, and its accession number at the China General Microbiological Culture Collection Center is CGMCC No. 24793.

2. A microbial preparation, wherein the active ingredient of the microbial preparation comprises the psychrophilic oligotrophic bacillus of claim 1 or a culture of the psychrophilic oligotrophic bacillus of claim 1, wherein the culture of the psychrophilic oligotrophic bacillus is a substance in a culture container obtained by culturing the psychrophilic oligotrophic bacillus of claim 1 in a microbial culture medium.

3. The microbial preparation according to claim 2, wherein, The culture medium is an inorganic salt culture medium or an R2A culture medium.

4. The use of the psychrophilic oligotrophic bacillus of claim 1 or the microbial preparation of claim 2 in the degradation of microcystin.

5. In the application according to claim 4, the method for degrading microcystin is a microbial degradation method.

6. The application of the psychrophilic oligotrophic bacillus of claim 1 or the microbial preparation of claim 2 in aquatic ecological restoration and / or water pollution control.

7. In the application according to claim 6, the aquatic ecosystem to be restored is an aquatic ecosystem containing microcystin, and the water pollution is microcystin pollution.

8. The application according to claim 6, wherein the water ecological restoration and / or water pollution control is low-temperature water ecological restoration and / or low-temperature water pollution control.

9. The application of the psychrophilic oligotrophic bacillus of claim 1 or the microbial preparation of claim 2 in drinking water purification.

10. A method for degrading microcystin, characterized in that, Add the cold-resistant oligotrophic bacillus of claim 1 or the microbial preparation of claim 2 to water containing microcystin.

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