BACILLUS METHYLOTROPHICUS STRAIN AND USE THEREOF FOR DEGRADING MICORPOLLUTANT IN ENVIRONMENT

Abstract

The present invention discloses a Bacillus methylotrophicus strain named Bacillus methylotrophicus BP1.1, which was deposited in China Center for Type Culture Collection under Deposit No. CCTCC M 20191078 on Dec. 20, 2019. The present invention further discloses the use of the Bacillus methylotrophicus strain for degrading benzophenone ultraviolet sunscreens. By domesticating the activated sludge of the domestic sewage treatment plant step-by-step, the present invention provides a Bacillus methylotrophicus BP1.1 strain which has high efficiency in removing benzophenone ultraviolet sunscreens in water environment.

Claims

1. A Bacillus methylotrophicus strain, wherein the strain is named as Bacillus methylotrophicus BP1.1, and deposited in China Center for Type Culture Collection under Deposit No. CCTCC M 20191078 on Dec. 20, 2019.

2-9. (canceled)

10. A method for degrading a micropollutant in an environment, comprising: using a Bacillus methylotrophicus strain, wherein the strain is named as Bacillus methylotrophicus BP1.1, and deposited in China Center for Type Culture Collection under Deposit No. CCTCC M 20191078 on Dec. 20, 2019.

11. The method according to claim 10, wherein the micropollutant is a benzophenone ultraviolet sunscreen, and the benzophenone ultraviolet sunscreen is 2,4-dihydroxybenzophenone.

12. The method according to claim 11, wherein the Bacillus methylotrophicus strain degrades the benzophenone ultraviolet sunscreen in an aerobic condition.

13. The method according to claim 10, wherein the Bacillus methylotrophicus BP1.1 strain is inoculated into an expansion culture medium, and incubated in an aerobic condition at 28-30° C. for 24-48 h to obtain a BP1.1 expansion product, and the expanded culture product is inoculated into a wastewater containing a benzophenone ultraviolet sunscreen for treatment.

14. The method according to claim 13, wherein the inoculation amount of Bacillus methylotrophicus BP1.1 in the wastewater is 1%.sub.o-2%.sub.o.

15. The method according to claim 13, wherein the mass concentration of the benzophenone ultraviolet sunscreen in the wastewater is 10 mg/L.

16. The method according to claim 13, wherein the temperature of the wastewater is 20-30° C., and the pH of the wastewater is 7.3-8.0.

17. The method according to claim 13, wherein the expansion culture medium is one of a LB culture medium, an inorganic salt culture medium or a beef peptone culture medium.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0012] FIG. 1 illustrated a microscopic morphology of gram-stained Bacillus methylotrophicus BP1.1 in Example 2 of the present invention;

[0013] FIG. 2 illustrated the phylogenetic tree of Bacillus methylotrophicus BP1.1 in Example 2 of the present invention;

[0014] FIG. 3 illustrated the degradation rate of 2,4-dihydroxybenzophenone by Bacillus methylotrophicus BP1.1 in Example 3 of the present invention.

DESCRIPTION OF EMBODIMENTS

[0015] The technical solutions of the present invention are further described in detail below in conjunction with drawings and specific embodiments.

Example 1

[0016] In the present invention, Bacillus methylotrophicus BP1.1 was obtained from activated sludge at aerobic stage of a domestic sewage treatment plant in Nanjing.

[0017] The specific procedures are as follows:

[0018] (1) Activated sludge was obtained at aerobic stage from a domestic sewage treatment plant in Nanjing;

[0019] (2) 250 mL of the sludge-water mixture was added into a conical flask, stirred with a magnetic stirrer at room temperature, and added with 5 mg of 2,4-dihydroxybenzophenone every day; the supernatant was removed before each addition and was detected for the target pollutant 2,4-dihydroxybenzophenone, and the mixture was filled up to the volume with tap water; the strain was continuously acclimated until the target pollutant was not detected in the solution;

[0020] (3) the sludge obtained in step (2) was gradient diluted and spread on an inorganic salt culture medium containing 2,4-dihydroxybenzophenone, and incubated for 3-5 days at 30° C. to get individual colonies; the mass concentration of 2,4-dihydroxybenzophenone in the inorganic salt culture medium was 50 mg/L;

[0021] (4) individual colonies in step (3) was selected, isolated by streaking on an inclined plane, and preserved to obtain an isolated strain.

[0022] The culture medium for expanding the isolated strain was one of LB culture medium, inorganic salt culture medium or beef peptone culture medium.

[0023] The LB culture medium comprised the following components by mass per liter: 5.0 g of yeast powder, 10 g of sodium chloride and 10 g of peptone, pH 7.0-7.5.

[0024] The inorganic salt culture medium comprised the following components by mass per liter: 10-50 mg of 2,4-dihydroxybenzophenone, 1.6 g of sodium hydroxide and 6.8 g of monopotassium phosphate, pH 7.3-7.5. The Bacillus methylotrophicus BP1.1 strain can grow and propagate using 2,4-dihydroxybenzophenone as the only carbon source.

[0025] The beef peptone culture medium comprised the following components by mass per liter: 3 g of beef, 10 g of peptone and 5 g of sodium chloride, pH 7.2-7.6.

[0026] The Bacillus methylotrophicus BP1.1 strain obtained in step (4) was inoculated into an expansion culture medium with an inoculation amount of 1% (volume fraction) for expansion, and was incubated under aerobic conditions at 28-30° C. for 24-48 h to get a BP1.1 expansion culture product.

Example 2

[0027] The strain selected in Example 1 was identified.

[0028] In Example 1 of the present invention, a BP1.1 strain with the best degradation effect and the fastest growth rate was obtained by screening. The BP1.1 was identified as Bacillus methylotrophicus.

[0029] The Bacillus methylotrophicus BP1.1 strain grew well in LB culture medium under aerobic conditions at 30° C. The colony had a round shape, a diameter of 0.2-1 mm, a light pink color, an opaque appearance and a dry and smooth surface. The stain was gram-negative, and demonstrated a short-rod shape under a microscope.

[0030] The complete sequence of the 16S rRNA of the BP1.1 strain obtained by PCR amplification and Sanger sequencing is as follows (SEQ ID NO. 1):

TABLE-US-00001 agggggcggggcggcgtgctatacatgcaagtcgagcggacagatggga gcttgctccctgatgttagcggcggacgggtgagtaacacgtgggtaac ctgcctgtaagactgggataactccgggaaaccggggctaataccggat ggttgtctgaaccgcatggttcagacataaaaggtggcttcggctacca cttacagatggacccgcggcgcattagctagttggtgaggtaacggctc accaaggcgacgatgcgtagccgacctgagagggtgatcggccacactg ggactgagacacggcccagactcctacgggaggcagcagtagggaatct tccgcaatggacgaaagtctgacggagcaacgccgcgtgagtgatgaag gttttcggatcgtaaagctctgttgttagggaagaacaagtgccgttca aatagggcggcaccttgacggtacctaaccagaaagccacggctaacta cgtgccagcagccgcggtaatacgtaggtggcaagcgttgtccggaatt attgggcgtaaagggctcgcaggcggtttcttaagtctgatgtgaaagc ccccggctcaaccggggagggtcattggaaactggggaacttgagtgca gaagaggagagtggaattccacgtgtagcggtgaaatgcgtagagatgt ggaggaacaccagtggcgaaggcgactctaggtagtaactgacgctgag gagcgaaagcgtggggagcgaacaggattagataccaggtagtccacgc cgtaaacgatgagtgctaagtgttagggggtttccgccccttagtgagc agctaacgcattaagcactccgcctggggagtacggtcgcaagactgaa actcaaaggaattgacgggggcccgcacaagcggtggagcatgtggttt aattcgaagcaacgcgaagaaccttaccaggtcttgacatcctctgaca atcctagagataggacgtccccttcgggggcagagtgacaggtggtgca tggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacg agcgcaacccttgatcttagttgccagcattcagttgggcactctaagg tgactgccggtgacaaaccggaggaaggtggggatgacgtcaaatcatc atgccccttatgacctgggctacacacgtgctacaatggacagaacaaa gggcagcgaaaccgcgaggttaagccaatcccacaaatagttctcagtt cggatcgcagtctgcaactcgactgcgtgaagctggaatcgctagtaat cgcggatcagcatgccgcggtgaatacgttcccgggccttgtacacacc gcccgtcacaccacgagagtttgtaacacccgaagtcggtgaggtaacc tttatggagccagccgccgaaggtgaacccggagttg

[0031] The sequencing result of BP1.1 was submitted to GenBank for BLAST alignment, and strain sequences with more than 98% similarity to base sequences of BP1.1 were selected from the BLAST results. Clustal W multiple sequence alignment was performed, and a phylogenetic tree was established using MAGE 7.0, indicating that the genetic relationship between BP1.1 and Bacillus methylotrophicus was the nearest. Thus the strain BP1.1 was identified as Bacillus methylotrophicus.

Example 3

[0032] The degradation rate of the pollutant 2,4-dihydroxybenzophenone by the Bacillus methylotrophicus BP1.1 strain was measured. The method comprised the following steps:

[0033] (1) the expansion culture of BP1.1 was centrifuged at 6000 rpm for 10 min, and the supernatant was removed to get the strain;

[0034] (2) the strain obtained in step (1) was resuspended and inoculated into a water solution containing 2,4-dihydroxybenzophenone with an inoculation amount of 1.8%.sub.o; the mass concentration of 2,4-dihydroxybenzophenone in the solution was 10 mg/L; a group without adding BP1.1 was set as a control;

[0035] (3) the control group and the treatment group in step (2) were shaken on a shaker under aerobic conditions at 150 rpm and 28° C.;

[0036] (4) the concentration of remaining pollutant in the treatment and control group of step (3) were regularly sampled and measured. The results were shown in FIG. 3.

[0037] As shown in FIG. 3, the BP1.1 strain can efficiently degrade 2,4-dihydroxybenzophenone in water. 2,4-Dihydroxybenzophenone at 10 mg/L could be completely degraded within 6 h. In the first 2 h of treatment, the BP1.1 strain had a lower degradation rate due to the adaptability period in the degradation system; after the adaptation (2-5 h), the degradation process came into a rapid stage; in the late degradation stage (5-6 h), the degradation rate gradually became slower due to the decrease of substrate concentration.

[0038] By domesticating the activated sludge at aerobic stage of a domestic sewage treatment plant step-by-step, the present invention provides a Bacillus methylotrophicus BP1.1 strain with high removal capacity for benzophenone ultraviolet sunscreens. The biological degradation rate of benzophenone ultraviolet sunscreens by the Bacillus methylotrophicus strain under aerobic conditions may reach 100%, and it can significantly shorten the time for degradation.