A strain of Bacillus marinus and its application in degrading organic pollutants in high-salinity environments

Abstract

The present invention discloses a strain of Bacillus marinus and its application. The Bacillus marinus is named strain HN14, and its taxonomic name is Pontibacillus chungwhensis. It is preserved in the Guangdong Microbial Culture Collection Center, and its preservation number is GDMCC No. 62925. The strain HN14 has the ability to degrade a variety of organic pollutants, including steroid hormones (estradiol, testosterone) and polycyclic aromatic hydrocarbons (phenanthrene, pyrene, benzo[a]pyrene). The strain HN14 can still maintain efficient degradation of estradiol at a salinity of 25%, providing a new strain option for the remediation of high-salinity environmental pollution.

Description

Technical Field

[0001] The present invention belongs to the field of microorganisms, and particularly relates to a Bacillus marinus and its application in degrading organic pollutants in a high-salinity environment. Background Art

[0002] Among numerous environmental endocrine disruptors, steroid hormones with a wide pollution range, strong teratogenicity, and high resistance to degradation have the strongest interference activity. Steroid hormones appear and persist in the aquatic ecosystem, especially at relatively high concentrations during flood tides. Since steroid hormones can adsorb to fine sediments in small river estuaries, especially in the lower estuaries and around estuaries with relatively high salinity, this causes the accumulation of steroid hormones in the bottom waters of estuaries. And when disturbed by tidal currents, it becomes the main source of steroid hormones entering the estuary, and the concentration of steroid hormones increases with the increase in salinity. Thus, the increasing amount of trace steroid hormone pollutants in the aquatic environment and their related consequences have caused serious environmental problems globally.

[0003] Bioremediation technology based on microbial degradation is considered the most effective and environmentally friendly remediation method. However, the practical application of steroid hormone-degrading microorganisms is still limited due to their unstable degradation efficiency and limited tolerance. Many isolated steroid hormone-degrading strains have shown high efficiency in laboratory tests, but it is difficult for them to survive and maintain their degradation ability in complex environments. Many factors in the real environment, such as salinity, osmotic pressure, pH, other carbon sources, and co-existing pollutants, will cause different stresses on microorganisms and affect their survival and degradation. Microbial communities are used because of their high degradation, stability, and strong environmental adaptability, but uncontrollable changes in community structure may lead to a significant decrease in degradation efficiency. It has become an urgent problem to expect microbial strains to have stable degradation efficiency and great stress tolerance under any circumstances, especially the environmental adaptability and degradation stability under high-salinity conditions. Summary of the Invention

[0004] The object of the present invention is to provide a Bacillus marinus and its application in degrading organic pollutants in a high-salinity environment, so as to solve one or more technical problems existing in the prior art and provide at least one beneficial option or create conditions.

[0005] The first aspect of the present invention provides a strain of Bacillus chungwhensis. The Bacillus chungwhensis is named as the HN14 strain, with a taxonomic name of Pontibacillus chungwhensis, and is deposited in the Guangdong Microbial Culture Collection Center, with a deposit number of GDMCC No. 62925, and a deposit address of 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province, and a deposit date of October 26, 2022. The HN14 strain was isolated from the surface sediments of the Dongzhaigang National Mangrove Nature Reserve in Hainan; it is rod-shaped and Gram-positive; the colony characteristics are round, moist, raised surface, and neatly edged yellow colonies.

[0006] In some embodiments of the present invention, the Bacillus subtilis has a 16SrDNA gene fragment with a sequence as shown in SEQ ID No:1.

[0007] The second aspect of the present invention provides the use of the HN14 strain in degrading steroid hormones. The HN14 strain has been verified by experiments to have a good steroid hormone degradation effect.

[0008] In some embodiments of the present invention, the degradation process is carried out under environmental conditions with a salinity of 10 to 25%. The algae used to degrade estrogen in the prior art are mainly Chlorella. However, Chlorella is mainly distributed in freshwater waters, and there are fewer individuals under natural conditions. It is difficult to reproduce in large quantities in the lower estuaries and around the estuaries with high salinity. The HN14 strain is Bacillus marineus, which can survive and reproduce in a high-salinity environment and can be used to degrade steroid hormones in water bodies under high-salinity environmental conditions.

[0009] In some embodiments of the present invention, the steroid hormone includes estradiol (E2) or testosterone (T). Estradiol and testosterone are widely present as pollutants in the environment. After entering the organism, they can disrupt normal endocrine function and change the intracellular signaling process of the body during development and adulthood, thereby causing various lesions of the reproductive, immune, nervous and other systems. It has been verified that the HN14 strain still has a good estradiol and testosterone degradation effect in a high-salt environment.

[0010] In some embodiments of the present invention, the culture medium components for cultivating the HN14 strain for degrading steroid hormones are: (NH4) 2 SO 4 , 1.0g; Na 2 HPO 4 , 0.8g; KH 2 PO 4 , 0.2g; MgSO 4 7H 2 O, 0.2 g; CaCl 2 ·2H2 O, 0.1 g; FeCl 3 ·6H 2 O, 5 mg; (NH4) 6 Mo 7 O 24 ·4H 2 O, 1 mg; distilled water 1000 mL; steroid hormone 20 - 200 mg; pH 7.0. The medium needs to be sterilized at 121 °C for 20 min before use.

[0011] The third aspect of the present invention provides the application of the HN14 strain in degrading polycyclic aromatic hydrocarbons. The HN14 strain has been experimentally verified to have good polycyclic aromatic hydrocarbon degradation effect.

[0012] In some embodiments of the present invention, the degradation process is carried out under environmental conditions with a salinity of 10 - 25%.

[0013] In some embodiments of the present invention, the polycyclic aromatic hydrocarbons include at least one of phenanthrene, pyrene, and benzo(a)pyrene.

[0014] In some embodiments of the present invention, the medium components for culturing the HN14 strain for degrading polycyclic aromatic hydrocarbons are: (NH4) 2 SO 4 ,1.0 g; Na 2 HPO 4 ,0.8 g; KH 2 PO 4 ,0.2 g; MgSO 4 ·7H 2 O, 0.2 g; CaCl 2 ·2H 2 O, 0.1 g; FeCl 3 ·6H 2 O, 5 mg; (NH4) 6 Mo 7 O 24 ·4H 2 O, 1 mg; distilled water 1000 mL; polycyclic aromatic hydrocarbons 20 mg; pH 7.0. The medium needs to be sterilized at 121 °C for 20 min before use.

[0015] Beneficial effects: A strain of HN14 (Pontibacillus chungwhensis) with the ability to degrade organic pollutants was isolated in this invention, providing a new germplasm resource for the remediation of polluted environments. The HN14 strain has the ability to degrade a variety of organic pollutants, including steroid hormones (estradiol, testosterone) and polycyclic aromatic hydrocarbons (phenanthrene, pyrene, benzo[a]pyrene). The HN14 strain can still maintain efficient degradation of estradiol at a salinity of 25%, providing a new strain option for the remediation of high-salinity environmental pollution. Brief Description of the Drawings

[0016] Figure 1 is a photograph of the HN14 strain, (a) is a plate colony map, and (b) is a scanning electron microscope image;

[0017] Figure 2 is a bar graph of the degradation rates of the HN14 strain for different organic pollutants;

[0018] Figure 3 is a line graph of the degradation efficiency of the HN14 strain for steroid hormones, (a) for estradiol and (b) for testosterone;

[0019] Figure 4 is a bar graph of the degradation rates of the HN14 strain for different concentrations of estradiol;

[0020] Figure 5 is a line graph of the degradation efficiency of estradiol by the HN14 strain under salinity gradient conditions;

[0021] Figure 6 is a LC-MS detection map of the HN14 strain during the degradation of estradiol, (a) for the intermediate metabolite estrone and (b) for the intermediate metabolite 4-hydroxyestrone.

[0022] Figure 7 is an HPLC detection map of the HN14 strain during the degradation of estradiol. Detailed Embodiments

[0023] The concept and technical effects of this invention will be clearly and completely described below in combination with the embodiments to fully understand the purpose, features, and effects of this invention. Obviously, the described embodiments are only a part of the embodiments of this invention, not all of them. Based on the embodiments of this invention, other embodiments obtained by those skilled in the art without creative efforts fall within the scope of protection of this invention.

[0024] Example 1, Obtaining the HN14 Strain

[0025] The isolation and screening process of the HN14 strain is as follows:

[0026] 1) Scrape the sediment from the surface layer of the national-level mangrove forest in Hainan Dongzhai Port, weigh 5 g of the sediment into an inorganic salt medium with a benzo[a]pyrene concentration of 20 mg / L, culture it on a shaker at 25 °C for 25 days, then aspirate 5 mL and transfer it to a new inorganic salt medium containing benzo[a]pyrene, and repeat the stress culture conditions for continuous subculture 5 times to achieve the enrichment of degrading bacteria;

[0027] 2) Take 10 mL of the finally enriched culture solution in step 1), and use the multiple dilution method to dilute it by a factor of 10 -3 ~10 -6 times, and then spread it on an inorganic salt solid plate with estradiol as the sole carbon source, and culture it in an incubator at 25 °C until colonies grow;

[0028] 3) Pick the grown colonies and streak them on a 2216E solid plate to obtain a single strain (the composition of the 2216E solid medium is: yeast extract, 1 g; tryptone, 5 g; K 2 HPO 4 , 0.074 g; Fe 2 (SO 4 ) 3 , 0.002 g; agar powder, 15 g; dissolved in 1 L of aged seawater, pH 7.6 - 7.8, sterilized at 121 °C for 20 min), to obtain the strain Bacillus marinus - HN14. It can be seen from Figure 1 (a) that the colony characteristics of the HN14 strain cultured on a 2216E plate for two days are: round, moist, convex on the surface, and yellow colonies with neat edges. It can be seen from Figure 1 (b) that the strain of the HN14 strain is rod-shaped.

[0029] Example 2, Degradation efficiency test of the HN14 strain

[0030] Cultivate the HN14 strain in a 2216E medium until the OD value reaches 0.8, centrifuge to collect the cells, wash them three times with a liquid inorganic salt medium, and then inoculate them into an inorganic salt medium containing different organic pollutants ((NH4) 2 SO 4 , 1.0 g; Na 2 HPO 4 , 0.8 g; KH 2 PO 4 , 0.2 g; MgSO 4 ·7H 2 O, 0.2 g; CaCl 2 ·2H 2 O, 0.1 g; FeCl 3 ·6H 2 O, 5 mg; (NH4) 6 Mo 7 O 24· 4H 2 O, 1 mg; distilled water 1000 mL; pH 7.0; polycyclic aromatic hydrocarbons or steroid hormones 20 mg), cultured on a shaker at 25 °C and 150 rpm. The culture medium containing steroid hormones was cultured for 9 days, and the culture medium containing polycyclic aromatic hydrocarbons was cultured for 25 days. The control group was an inorganic salt medium supplemented with only the same concentration of substrate. The culture solution was extracted with ethyl acetate, and the residual amount of organic pollutants was determined by HPLC; the results are as Figure 2 shown. The degradation rates of HN14 strain for 20 mg / L estradiol, testosterone, phenanthrene, pyrene, benzo[a]pyrene were 80.26%, 75.82%, 93.04%, 19.65%, 40.15% respectively.

[0031] Example 3, Growth and Degradation Efficiency Test of HN14 Strain on Steroid Hormones

[0032] The HN14 strain was cultured in 2216E medium until the OD value reached 0.8, and the cells were collected by centrifugation. After washing three times with liquid inorganic salt medium, the cells were inoculated into inorganic salt media containing estradiol and testosterone respectively ((NH4) 2 SO 4 , 1.0 g; Na 2 HPO 4 , 0.8 g; KH 2 PO 4 , 0.2 g; MgSO 4 ·7H 2 O, 0.2 g; CaCl 2 ·2H 2 O, 0.1 g; FeCl 3 ·6H 2 O, 5 mg; (NH4) 6 Mo 7 O 24 ·4H 2 O, 1 mg; distilled water 1000 mL; pH 7.0; steroid hormones 20 mg), cultured on a shaker at 25 °C and 150 rpm for 1 day, 3 days, 5 days, 7 days and 9 days respectively, and the OD600 value at different times was measured. The control group was an inorganic salt medium supplemented with only the same concentration of substrate. All samples were extracted with ethyl acetate from the culture solution, and the residual amount of organic pollutants was determined by HPLC; the results are as Figure 3 shown. The degradation efficiency of HN14 strain for 20 mg / L estradiol and testosterone increased continuously with the extension of time, and reached more than 95% at about 1 day.

[0033] Example 4, Degradation Efficiency Test of HN14 Strain on Different Concentrations of Estradiol

[0034] Take the fresh strain of HN14, inoculate it into 2216E liquid medium and culture until the OD value reaches 0.8. Collect the bacterial cells and wash them three times with inorganic salt medium. Pipette an equal volume of bacterial liquid into 50 mL of inorganic salt medium containing 20 mg / L, 50 mg / L, 100 mg / L, and 200 mg / L of estradiol as the carbon source and energy source, and culture in a shaker at 25 °C and 150 rpm for 9 days. Use HPLC to measure and calculate the degradation rate of estradiol; the results are as Figure 4 shown. It shows that for 20 mg / L of estradiol, the degradation rate of the HN14 strain reaches 95.28%; for high-concentration estradiol of 50 - 200 mg / L, the degradation rate also reaches about 92.24%, reflecting the characteristic of the HN14 strain to degrade high-concentration substrates.

[0035] Example 5, Estradiol Degradation Efficiency Test of HN14 Strain under Different Salt Concentrations

[0036] Pick a single colony from the fresh plate culture of the HN14 strain and inoculate it into 2216E liquid medium. Culture in a shaker at 25 °C until the OD600 value reaches 0.8. Centrifuge at 5000 rpm to collect the bacterial cells and wash them three times with inorganic salt medium. Pipette an equal volume of bacterial liquid into inorganic salt medium with 20 mg / L of estradiol as the sole carbon source and salt concentrations of 1%, 3%, 5%, 7%, 10%, 15%, 20%, and 25%, and culture in a shaker at 25 °C for 9 days. Use HPLC to measure the residual amount of estradiol. The results are as Figure 5 shown. The HN14 strain shows wide applicability and stable estradiol degradation efficiency in the salinity range of 1% to 10%, maintaining above 90%; when the salinity of the culture increases to 10%, the HN14 strain shows great adaptability. Even under the condition of 15% salinity, the degradation efficiency of the HN14 strain for estradiol remains relatively stable, with a degradation rate of 74.2%; even when the salinity of the culture reaches 25%, the HN14 strain still maintains the ability to degrade estradiol, and the degradation rate can reach more than 40%. These results indicate that the HN14 strain has wide adaptability to high-salt conditions and maintains stable degradation efficiency for estradiol, suggesting its good stress tolerance and degradation stability in extreme environments such as high salt, and has potential application prospects in the bioremediation of estradiol pollution. Use GC-MS to measure the intermediate metabolites of estradiol degraded by the HN14 strain, and the results are as Figure 6 shown, indicating that the HN14 strain can convert estradiol into estrone and 4-hydroxyestrone. By tracking and monitoring (HPLC chromatograms of the samples on days 0, 1, and 3), it is found that the sample mainly containing estradiol reduces estradiol to a lower level after one day, and a large amount of estrone is detected at the same time. As time extends, the content of estrone also gradually decreases, as Figure 7 shown.

[0037] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those of ordinary skill in the art. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A strain of Pontibacillus chungwhensis Characterized in that The Bacillus marinus is named as strain HN14, and its taxonomic name Pontibacillus chungwhensis , is deposited in the Guangdong Microbial Culture Collection Center with the deposit number of GDMCC No. 62925, and the deposit date is October 26, 2022.

2. Use of the Pontibacillus chungwhensis according to claim 1 in degrading steroid hormones, Characterized in that The steroid hormones include estradiol or testosterone.

3. Use of the Pontibacillus chungwhensis according to claim 2 in degrading steroid hormones, Characterized in that The degradation process is carried out under environmental conditions with a salinity of 10-25%.

4. Use of the Pontibacillus chungwhensis according to claim 3 in degrading steroid hormones, Characterized in that The culture medium components for culturing the Bacillus marinus are: (NH4) 2 SO 4 , 1.0 g; Na 2 HPO 4 , 0.8 g; KH 2 PO 4 , 0.2 g; MgSO 4 ·7H 2 O, 0.2 g; CaCl 2 ·2H 2 O, 0.1 g; FeCl 3 ·6H 2 O, 5 mg; (NH4) 6 Mo 7 O 24 ·4H 2 O, 1 mg; distilled water 1000 mL; steroid hormone 20 - 200 mg; pH 7.0; the steroid hormone includes estradiol or testosterone.

5. Use of the Pontibacillus chungwhensis according to claim 1 in degrading polycyclic aromatic hydrocarbons, Characterized in that The polycyclic aromatic hydrocarbons include at least one of phenanthrene, pyrene, and benzo[a]pyrene.

6. Use of the Pontibacillus chungwhensis according to claim 5 in degrading polycyclic aromatic hydrocarbons, Characterized in that The degradation process is carried out under environmental conditions with a salinity of 10-25%.

7. Use of the Pontibacillus chungwhensis according to claim 6 in degrading polycyclic aromatic hydrocarbons, Characterized in that The culture medium components for culturing the Bacillus marinus are: (NH4) 2 SO 4 , 1.0 g; Na 2 HPO 4 , 0.8 g; KH 2 PO 4 , 0.2 g; MgSO 4 ·7H 2 O, 0.2 g; CaCl 2 ·2H 2 O, 0.1 g; FeCl 3 ·6H 2 O, 5 mg; (NH4) 6 Mo 7 O 24 ·4H 2 O, 1 mg; distilled water 1000 mL; polycyclic aromatic hydrocarbons 20 mg; pH 7.0; the polycyclic aromatic hydrocarbons are selected from at least one of phenanthrene, pyrene, and benzo(a)pyrene.

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