Streptomyces sp. TH05 derived from Taihu Lake sediment and application of streptomyces sp. TH05

Abstract

The invention discloses streptomyces sp. TH05 sourced from Taihu Lake sediment and application of the streptomyces sp. TH05, and belongs to the technical field of microorganisms. The invention aims to provide a microbial cell with a microcystis aeruginosa function. The invention provides a strain of streptomyces sp. TH05, which is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC NO.32816, and the preservation time is November 27, 2024. The method is used for treating water pollution and preventing algal bloom.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a strain of Streptomyces sp. TH05 derived from Taihu Lake sediments and an application thereof. Background Art

[0002] Microcystis aeruginosa is an algae belonging to the phylum Cyanobacteria, class Chrococophorales, family Chrococophoraceae, and genus Microcystis. It grows mostly in lakes, ponds, and other water bodies rich in organic matter, and lives a phytoplankton life. Microcystis aeruginosa will reproduce in large numbers in eutrophic water bodies, forming cyanobacterial blooms, which will reduce the transparency of the water body, affect the photosynthesis of underwater plants, and thus destroy the balance of the entire aquatic ecosystem. Its large-scale reproduction will inhibit the growth of other phytoplankton, making the phytoplankton community structure uniform, reducing biodiversity, affecting the stability and function of the aquatic ecosystem, consuming dissolved oxygen, and causing fish hypoxia: Microcystis aeruginosa will breathe at night, consuming a large amount of dissolved oxygen in the water body, which is easy to cause water hypoxia, causing fish to float and even flood the pond and die. Its large-scale reproduction will pollute drinking water sources such as lakes and rivers, causing the water source to have odor and color changes, and increasing the difficulty and cost of water treatment. Therefore, a microorganism that effectively inhibits the reproduction of Microcystis aeruginosa is sought for biological control of Microcystis aeruginosa. Summary of the invention

[0003] The purpose of the present invention is to provide a microbial cell with the function of dissolving Microcystis aeruginosa.

[0004] The present invention provides a strain of Streptomyces salinarius, named as Streptomyces sp. TH05, which is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration with a deposit number of CGMCCNO.32816 and a deposit date of November 27, 2024.

[0005] The present invention provides a microbial preparation containing the above-mentioned Streptomyces sp. TH05.

[0006] The invention provides a fermentation liquid of Streptomyces sp. TH05. The fermentation liquid is obtained after Streptomyces sp. TH05 is cultured in Gao's No. 1 liquid culture medium for 5 days.

[0007] The present invention provides a cell-free filtrate prepared by the Streptomyces sp. TH05. After the Streptomyces sp. TH05 is cultured in Gao's No. 1 liquid culture medium for 5 days, the fermentation broth is centrifuged at 12000 rpm / min for 10 minutes, the supernatant is aspirated and filtered through a 0.22 μm filter membrane to obtain the cell-free filtrate.

[0008] The present invention provides an application of the Streptomyces sp. TH05, the microbial preparation, the fermentation broth or the sterile filtrate in inhibiting the growth of Microcystis aeruginosa.

[0009] The present invention provides an application of the Streptomyces sp. TH05, the microbial preparation, the fermentation broth or the sterile filtrate in improving the MDA concentration and SOD enzyme activity of Microcystis aeruginosa.

[0010] The present invention provides a method for dissolving Microcystis aeruginosa, wherein the Streptomyces sp. TH05, the microbial preparation, the fermentation liquid or the sterile liquid are added into an aqueous solution containing Microcystis aeruginosa.

[0011] Further defined, the lighting conditions are 24 hours dark, 24 hours light, or 14 hours light / 10 hours dark.

[0012] It is further defined that the reaction time is 9 days.

[0013] It is further defined that the Streptomyces sp. TH05, the microbial preparation, the fermentation liquid or the sterile liquid is added at a volume fraction of 5%, and the content of Microcystis aeruginosa is 50 mL.

[0014] Beneficial effects:

[0015] [Biological Deposit Information]: A strain of Streptomyces salinarius, named Streptomyces sp.TH05, is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, located at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCC NO.32816 and the deposit date November 27, 2024. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The colony morphology of strain TH05 on different culture media; A, B, C, D, E, and F represent the colony morphology of strain TH05 after 7 days of culture on Gao's medium No. 1, glucose-asparagine medium, nutrient agar medium, ISP2 medium, ISP3 medium, and ISP5 medium, respectively;

[0017] Figure 2 is the 16S rRNA gene phylogenetic tree of strain TH05 and similar strains (NJ), Bootstrap values ​​are based on 1000 replicates; only values ​​≥50% are shown, Bar, 0.002 substitution per nucleotide position;

[0018] Figure 3The diagram shows the inhibitory effect of strain TH05 on Microcystis aeruginosa; Figure A is the control group, and Figure B is the experimental group;

[0019] Figure 4 The results of the algicidal rate of strain TH05 on Microcystis aeruginosa at different co-cultivation times;

[0020] Figure 5 This is the result diagram of the effect of different light conditions on the chlorophyll content of Microcystis aeruginosa;

[0021] Figure 6 The results of the algicidal rate of the fermentation broth, cell-free filtrate and bacterial suspension of strain TH05 on Microcystis aeruginosa are shown in the figure;

[0022] Figure 7 The following are scanning electron microscopic images of the destruction of Microcystis cells by the fermentation liquid of strain TH05, wherein A, B, and C are scanning electron microscopic images of the control group on day 0, day 1, and day 3, respectively, and D, E, and F are scanning electron microscopic images of the treatment with TH05 fermentation liquid on day 0, day 1, and day 3, respectively;

[0023] Figure 8 The algae-lysing effect of the fermentation liquid of strain TH05 treated at different temperatures on Microcystis aeruginosa;

[0024] Fig. 9 The algae-lysing effect of the fermentation broth of strain TH05 after treatment with different pH values ​​on Microcystis aeruginosa. DETAILED DESCRIPTION

[0025] Gao's liquid medium No. 1 (soluble starch 2%, NaCl 0.05%, MgSO4·7H2O 0.05%, K2HPO4·3H2O0.05%, KNO3 0.1%, FeSO4·7H2O 0.001%, pH adjusted to 7.2, sterilized at 121℃ for 25min)

[0026] Example 1. Isolation of a strain of Streptomyces sp. (Streptomyces salinarius) TH05

[0027] Separation: The collected Taihu sediment samples were naturally air-dried, ground and weighed, 5 g was placed in a conical flask containing 45 mL of sterile water, sterile glass beads were added, mixed and placed on a shaker, and shaken at 28°C and 180 rpm / min for 30 min, placed on a clean bench and allowed to stand, the supernatant was aspirated and diluted with sterile water, and 10 -2 -10 -4150 μL of the dilution was spread on a modified Gao's agar medium (the medium composition was: soluble starch 20 g, NaCl 0.5 g, KNO3 1 g, K2HPO4·3H2O 0.5 g, MgSO4·7H2O 0.5 g, FeSO4·7H2O 0.01g, 1000mL of distilled water, 20g of agar, adjust the pH to 7.2, sterilize at 121℃ for 25min, when the culture medium is cooled to about 50℃, add 20mg / L nalidixic acid and 50mg / L nystatin), seal with sealing film, place in a 28℃ constant temperature incubator and culture upside down for 7d, pick colonies of different morphologies and streak on ISP2 culture medium (the culture medium is made of 4g yeast extract powder, 4g glucose, 10g malt extract powder, 20g agar powder, 1000mL of distilled water, adjust the pH to 7.2, and sterilize at 121℃ for 25min), and preserve the purified actinomycetes with 20% glycerol.

[0028] Observation of morphological characteristics of strain TH05: The strain TH05 was inoculated on Gao's agar No. 1 plates, glucose asparagine plates (the culture medium consisted of 10 g glucose, 1 g L-asparagine, 1 g K2HPO4, 0.001 g FeSO4·7H2O, 0.001 g MnCl2·4H2O, 0.001 g ZnSO4·7H2O, 1000 mL distilled water, 20 g agar, the pH value was adjusted to 7.2, and sterilized at 121°C for 25 min), nutrient agar plates (the culture medium consisted of 10 g peptone, 3 g beef powder, 5 g sodium chloride, 1000 mL distilled water, 20 g agar, the pH value was adjusted to 7.3, and sterilized at 121°C for 25 min), ISP2 plates, and ISP3 plates (the culture medium consisted of 20 g oatmeal powder, 1 g NaCl, 0.001 g FeSO4·7H2O, 0.001 g MnCl2·4H2O, 0.001 g ZnSO4·7H2O, 1000 mL distilled water, 20 g agar, the pH value was adjusted to 7.3, and sterilized at 121°C for 25 min). 0.001g, MnCl2·4H2O0.001g, ZnSO4·7H2O 0.001g, distilled water 1000mL, agar 20g, pH value adjusted to 7.2, sterilized at 121℃ for 25min) and ISP5 medium plates (the culture medium was composed of glycerol 10g, L-asparagine 1g, K2HPO41g, FeSO4·7H2O0.001g, MnCl2·4H2O 0.001g, ZnSO4·7H2O 0.001g, distilled water 1000mL, agar 20g, pH value adjusted to 7.2, sterilized at 121℃ for 25min), placed in a constant temperature incubator at 28℃ for 7d. After the cultivation was completed, the characteristics of the colonies on each plate were observed.

[0029] The results showed that the colony surface on Gao's agar medium was white and the back was yellow-brown. The colony diameter was 5-6 mm, round, slightly raised in the center, and wrinkled around. No pigment was produced ( Figure 1A); On the glucose-asparagine medium, the colony surface is white in the center, gray at the edge, light yellow on the back, 7-8 mm in diameter, and does not produce pigment ( Figure 1 B); On nutrient agar medium, the colony surface is light ivory, the back is yellow-brown, the diameter is 7-8mm, the edge is irregular, the colony surface is wrinkled, and no pigment is produced ( Figure 1 C); The colonies on ISP2 medium are white with a grayish tint, the back is light brown, the diameter is 8-9 mm, the colonies have obvious wrinkles, and do not produce pigment ( Figure 1 D); On ISP3 medium, the colony surface is gray with red edges, the back is purple, the colony diameter is 6-7mm, and the edges are relatively flat ( Figure 1 E); On ISP5 medium, the colonies are pink on both the surface and back, 4-5 mm in diameter, with irregular edges and no pigment ( Figure 1 F).

[0030] The physiological and biochemical characteristics of strain TH05 were studied, and the identification method was carried out according to the method in "Modern Microbiology Experimental Technology" (author: Zhu Xufen, publisher: Zhejiang University Press, publication time: August 2011). The results showed that methyl red, VP determination, hydrogen sulfide test, oxidase, gelatinase, and amylase reactions were negative, and it could not utilize raffinose, sorbitol, phenylalanine, and valine. The esculin hydrolysis test and cellulase reaction were positive, and the strain could utilize glucose, fructose, arabinose, rhamnose, galactose, mannose, aspartic acid, tyrosine, proline, serine, arginine, and threonine. (Table 1).

[0031] Table 1 Physiological and biochemical identification results of strains

[0032]

[0033] 2. Molecular Identification of Strain

[0034] The genomic DNA of algae-lytic actinomycetes TH05 was extracted using the Ezup column bacterial genomic DNA extraction kit (Shanghai Biotech Co., Ltd.). The specific operation process was carried out according to the kit instructions. The concentration and purity of the extracted DNA were determined using a micro-spectrophotometer NanoDrop One. The universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′, SEQ ID NO.1) and 1492R (5′-TACGGCTACCTTGTTACGACTT-3′, SEQ ID NO.2) were used to perform PCR amplification of algae-lytic actinomycetes TH05. The amplification conditions were: 98°C pre-denaturation for 2.5 min; 98°C denaturation for 5 s, 55°C annealing for 10 s, 72°C extension for 25 s, 35 cycles; 72°C extension for 2 min. The PCR products were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing after electrophoresis detection. The measured 16S rDNA sequences were uploaded to the EzBioCloud (http: / / www.ezbiocloud.net / identify) database for comparison analysis. The phylogenetic tree was constructed using the neighbor-joining method (NJ) using MEGAX software. Figure 2 The results showed that strain TH05 was closely related to strains of the genus Streptomyces and to Streptomyces salinarius SS06011. T It has the highest similarity, with a 16S rDNA sequence similarity of 99.85%. It was identified as Streptomyces salinarius and named Streptomyces salinarius TH05.

[0035] 16S rDNA: (SEQ ID NO.3)

[0036]

[0037] Example 2. Algicidal function of Streptomyces salinarius TH05

[0038] The strain TH05 was inoculated into Gao's No. 1 liquid culture medium and cultured at 30°C with a shaker at 200 rpm for 48 hours. Microcystis aeruginosa FACHB 905 was purchased from the freshwater algae seed bank of the Chinese Academy of Sciences, and the Microcystis aeruginosa FACHB 905 was transferred into BG11 culture medium (formula see Table 2), 2000 lx white light, light: dark = 14 hours: 10 hours, cultured at 25°C, and manually shaken three times a day.

[0039] Table 2 BG11 medium formula

[0040]

[0041] Co-culture the fermentation liquid of strain TH05 with Microcystis aeruginosa FACHB-905 cultured to the logarithmic phase. 4. Take 1 mL of algae liquid from the treatment group and the blank group, centrifuge at 12,000 rpm for 10 min, discard the supernatant, place the chlorophyll precipitate in a -80°C refrigerator, freeze and thaw once every 30 min, repeat three times in total; blow the frozen chlorophyll precipitate into a centrifuge tube containing 5 mL of 95% ethanol, place the centrifuge tube at -4°C for 12 h, centrifuge at 5,500 rpm for 10 min, aspirate the supernatant into a cuvette, and use a spectrophotometer to measure the corresponding OD values ​​at 635 nm, 645 nm, 663 nm, and 750 nm. Chlorophyll content calculation formula:

[0042]

[0043] V1: supernatant volume (5 mL); V: sample volume (1 mL); δ: cuvette optical path (1 cm)

[0044] The algae lysis rate was calculated by the measured chlorophyll content. The algae lysis rate was calculated using the following formula: Algae lysis rate (%) = [(C0-C) / C0] × 100%, where C0 is the concentration of chlorophyll a (Chl-a) at the initial stage, and C is the concentration of Chl-a at the end of the experiment. Chlorophyll a determination method:

[0045] The algicidal rate of strain TH05 on Microcystis aeruginosa FACHB-905 increased over time ( Figure 4 ), the algicidal rate of the strain on Microcystis aeruginosa FACHB-905 was 12.46% on the first day. On the seventh day of co-cultivation, obvious yellowing occurred ( Figure 3 ), the algae-lysis rate was 78.85% on the 7th day and 92.71% on the 8th day.

[0046] Example 3. Effect of light on the algalytic activity of strain TH05

[0047] The fermentation liquid of strain TH05 was inoculated into the culture liquid (1-2×10 cells / mL) of Microcystis aeruginosa FACHB-905 in the logarithmic growth phase at a ratio of 5% (v / v), and cultured under three different conditions: dark condition (24 hours of darkness), full light condition (24 hours of light), and light / dark cycle condition (14 hours of light / 10 hours of darkness). At the same time, 5% (v / v) of sterilized Gao's No. 1 liquid culture medium was added to the FACHB-905 culture liquid as a blank control. After 9 days of co-cultivation, the anti-algae activity was measured.

[0048] The results showed that under completely dark conditions, the chlorophyll contents of algal cells in the treatment group and control group with the fermentation broth of strain TH05 were 613.23 μg / L and 470.05 μg / L, respectively. Figure 5 ), the chlorophyll content decreased by 23.35%; under full light conditions, the chlorophyll content of the treatment group decreased by 6.41% compared with the control group; under light-dark cycle conditions, the chlorophyll content of algal cells in the treatment group decreased by 90.30% compared with the control group.

[0049] Example 4. Study on the algicidal pattern of TH05

[0050] To explore the algicidal pattern of TH05:

[0051] Fermentation broth: The strain TH05 was cultured in Gao's liquid medium No. 1 for 5 days to obtain the fermentation broth;

[0052] Cell-free filtrate: After strain TH05 was cultured in Gao's liquid medium No. 1 for 5 days, a fermentation broth was obtained, and the fermentation broth was centrifuged at 1200 rpm / min for 10 minutes. The supernatant was aspirated and filtered through a 0.22 μm filter membrane to obtain a cell-free filtrate.

[0053] Bacterial suspension: The mycelial pellet was washed three times with sterilized BG11 medium and suspended in an equal volume of BG11 medium to prepare a bacterial suspension.

[0054] 5% (v / v) untreated fermentation broth, cell-free filtrate and bacterial suspension were added to the culture medium of Microcystis aeruginosa FACHB 905, respectively, and placed in a light incubator at 25°C, 2000 lx white light, light: dark = 14 h: 10 h, and manually shaken three times a day. After culturing for 7 days, the algicidal activity was determined.

[0055] The results showed that actinomycete cultures treated in different ways showed different algicidal activities against Microcystis aeruginosa FACHB-905 ( Figure 6 ). After 7 days, the algicidal rates of TH05 fermentation broth, bacterial suspension and cell-free filtrate on Microcystis aeruginosa FACHB-905 were 84.31%, 70.24% and 66.38%, respectively. Both the cell-free filtrate and bacterial suspension of strain TH05 could effectively inhibit the growth of Microcystis aeruginosa, indicating that strain TH05 can exert algicidal effects by both direct algicidal and indirect algicidal methods.

[0056] Example 5. Effect of strain TH05 on the cell morphology of Microcystis aeruginosa FACHB 905

[0057] 1. Treat Microcystis aeruginosa with the fermentation liquid of strain TH05 for 0, 1, and 3 days, take 10 mL of the algae liquid, centrifuge at 8000 rpm / min and 4°C for 10 minutes to collect the algae cells. The algae cells were fixed with 2.5% glutaraldehyde solution overnight and washed three times with phosphate buffer (PBS, 50 mM, pH = 7.4). The fixed cells were dehydrated with different gradient ethanol (30%, 50%, 70%, 90% and 100%), immersed in isoamyl alcohol for 4 hours, and finally dried at the critical point of carbon dioxide and observed using a scanning electron microscope.

[0058] In the control group, the cells of Microcystis aeruginosa showed a normal round shape, smooth surface, intact cell membrane, and no signs of damage ( Figure 7 AC). Figure 7 D-7F showed that algal cells were gradually entangled by strain TH05, which could inhibit algae by direct contact.

[0059] Example 6. Effect of temperature on algae dissolution rate

[0060] The fermentation broth of strain TH05 was treated in a water bath at 0, 50, 75, and 100°C for 1h, respectively. The fermentation broth placed at room temperature (about 25°C) for 1h was used as a control group. The treated fermentation broth was then placed at room temperature (25°C). After the fermentation broth temperature returned to room temperature, the treated fermentation broth was inoculated into Microcystis aeruginosa in the logarithmic growth phase at a volume fraction of 5%, 2000lx white light, light: dark = 14h:10h, 25°C, and manually shaken three times a day. After 7 days of cultivation, the chlorophyll a concentration of the algae liquid was measured, and the algae inhibition rate was calculated.

[0061] The results showed that after being treated at different temperatures, the fermentation liquid of strain TH05 had a certain effect on the algae-lysing effect of Microcystis aeruginosa. The algae-lysing rate of the 0℃ and 25℃ treatment groups remained above 80%, while after being treated at high temperatures such as 75℃ and 100℃, the algae-lysing rate dropped to 67.48% and 55.44%, respectively. Figure 8 ).

[0062] Example 7. Effect of pH on algae lysis rate

[0063] The fermentation broth of strain TH05 (initial pH is about 7) was adjusted to pH 5, 6, 8 and 9 with 1 mol / L sodium hydroxide and 1 mol / L hydrochloric acid solution, and the untreated fermentation broth was used as the control group. After maintaining at each pH for 1 hour, the pH was adjusted to the initial value. The treated fermentation broth was inoculated into the logarithmic growth phase of Microcystis aeruginosa at a volume fraction of 5%, 2000lx white light, light: dark = 14 hours: 10 hours, 25°C culture, manual shaking three times a day, and the concentration of chlorophyll a in the algae broth of Microcystis aeruginosa was measured on the 7th day, and the algalysis rate was calculated.

[0064] The results showed that the algae-lysing rate of the fermentation broth of strain TH05 decreased slightly after being treated with different acids and alkalis, but the difference was not significant.

Claims

1. A strain of Streptomyces salinarius, characterized in that: It was named Streptomyces sp. TH05 and deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number CGMCCNO.32816 and the deposit date November 27, 2024.

2. A microbial preparation containing the Streptomyces sp. TH05 according to claim 1.

3. A fermentation broth of Streptomyces sp. TH05, characterized in that: The strain TH05 was cultured in Gao's liquid medium No. 1 for 5 days to obtain the fermentation broth.

4. A cell-free filtrate prepared from Streptomyces sp. TH05 according to claim 1, characterized in that: After strain TH05 was cultured in Gao's liquid medium No. 1 for 5 days, the fermentation broth was centrifuged at 12000 rpm / min for 10 minutes, and the supernatant was aspirated and filtered through a 0.22 μm filter membrane to obtain a cell-free filtrate.

5. Use of the Streptomyces sp. TH05 according to claim 1, the microbial preparation according to claim 2, the fermentation broth according to claim 3 or the sterile filtrate according to claim 4 in inhibiting the growth of Microcystis aeruginosa.

6. Use of the Streptomyces sp. TH05 according to claim 1, the microbial preparation according to claim 2, the fermentation broth according to claim 3 or the cell-free filtrate according to claim 4 in increasing the MDA concentration and SOD enzyme activity of Microcystis aeruginosa.

7. A method for dissolving Microcystis aeruginosa, characterized in that: The Streptomyces sp. TH05 according to claim 1, the microbial preparation according to claim 2, the fermentation broth according to claim 3 or the cell-free filtrate according to claim 4 is added to an aqueous solution containing Microcystis aeruginosa.

8. The method according to claim 7, characterized in that Light conditions were 24 h dark, 24 h light, or 14 h light / 10 h dark.

9. The method according to claim 7, characterized in that: The reaction time is 9 days.

10. The method according to claim 7, characterized in that TH05 according to claim 1, the microbial preparation according to claim 2, the fermentation broth according to claim 3 or the cell-free filtrate according to claim 4 are added at a volume fraction of 5%, and the content of Microcystis aeruginosa is 50 mL.

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