WO2021138924A1 - 一种甲基芽孢杆菌及其在降解环境中微污染物方面的应用 - Google Patents
一种甲基芽孢杆菌及其在降解环境中微污染物方面的应用 Download PDFInfo
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- 241000563903 Bacillus velezensis Species 0.000 title claims abstract description 30
- 239000003344 environmental pollutant Substances 0.000 title claims description 18
- 230000000593 degrading effect Effects 0.000 title abstract description 3
- 239000012965 benzophenone Substances 0.000 claims abstract description 27
- 239000000516 sunscreening agent Substances 0.000 claims abstract description 22
- RWCCWEUUXYIKHB-UHFFFAOYSA-N benzophenone Chemical compound C=1C=CC=CC=1C(=O)C1=CC=CC=C1 RWCCWEUUXYIKHB-UHFFFAOYSA-N 0.000 claims abstract description 20
- 230000000475 sunscreen effect Effects 0.000 claims abstract description 18
- 230000015556 catabolic process Effects 0.000 claims description 22
- 238000006731 degradation reaction Methods 0.000 claims description 22
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 16
- 239000010865 sewage Substances 0.000 claims description 16
- ZXDDPOHVAMWLBH-UHFFFAOYSA-N 2,4-Dihydroxybenzophenone Chemical compound OC1=CC(O)=CC=C1C(=O)C1=CC=CC=C1 ZXDDPOHVAMWLBH-UHFFFAOYSA-N 0.000 claims description 11
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 7
- 241000894006 Bacteria Species 0.000 claims description 5
- 239000001888 Peptone Substances 0.000 claims description 5
- 108010080698 Peptones Proteins 0.000 claims description 5
- 238000011081 inoculation Methods 0.000 claims description 5
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 5
- 235000019319 peptone Nutrition 0.000 claims description 5
- 235000015278 beef Nutrition 0.000 claims description 4
- 241000589323 Methylobacterium Species 0.000 claims 2
- 239000002609 medium Substances 0.000 description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 150000008366 benzophenones Chemical class 0.000 description 7
- 239000010802 sludge Substances 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000006065 biodegradation reaction Methods 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000003794 Gram staining Methods 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000007613 environmental effect Effects 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 231100000719 pollutant Toxicity 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000011987 methylation Effects 0.000 description 2
- 238000007069 methylation reaction Methods 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- -1 2,4-dihydroxybenzophenone compound Chemical class 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 231100000693 bioaccumulation Toxicity 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 230000033558 biomineral tissue development Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000002354 daily effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 231100000507 endocrine disrupting Toxicity 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 150000004702 methyl esters Chemical class 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 239000008239 natural water Substances 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 239000002957 persistent organic pollutant Substances 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000007480 sanger sequencing Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000037072 sun protection Effects 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 230000037303 wrinkles Effects 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/02—Aerobic processes
- C02F3/12—Activated sludge processes
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/32—Hydrocarbons, e.g. oil
- C02F2101/327—Polyaromatic Hydrocarbons [PAH's]
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/34—Organic compounds containing oxygen
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/34—Organic compounds containing oxygen
- C02F2101/345—Phenols
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/02—Aerobic processes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W10/00—Technologies for wastewater treatment
- Y02W10/10—Biological treatment of water, waste water, or sewage
Definitions
- the present invention relates to a bacillus methylation, and also relates to the application of the above-mentioned bacillus methylation in degrading benzophenone sunscreens in water, and belongs to the field of sewage treatment.
- Benzophenones (BPs) UV sunscreens have been widely used in personal care products because of their high safety, good sun protection effect and low price. Because most UV sunscreens have good durability, with the increase in daily use, benzophenone UV sunscreens continuously enter the environmental water body, but the urban sewage treatment plant cannot completely remove them, and will eventually enter Rivers and lakes continue to accumulate, and it is more and more common to detect UV sunscreens in water environments. Among them, benzophenone ultraviolet sunscreens are lipophilic substances, have endocrine disrupting effects and bioaccumulation, and are widely present in the environment, becoming an emerging micro-pollutant.
- Photochemical conversion is an important conversion pathway of UV sunscreens in natural water.
- the photochemical conversion reaction rate and pathway are affected by the pH and dissolved substances of the water environment, and BPs can produce more eco-toxic metabolites under light.
- Advanced oxidation technology is to degrade benzophenone with the characteristics of strong oxidizing substance-hydroxyl free HO ⁇ group produced in the reaction process.
- the most reported technologies currently include: UV/H 2 O 2 , O 3 / H 2 O 2 , UV/O 3 , Fenton, etc., the chemical reagents added in the advanced oxidation reaction and some of the products produced during the conversion process all have potential environmental risks.
- Microbial degradation is the only way for the final mineralization of organic matter in nature, and it is the key to determining the environmental fate of organic pollutants.
- the technical problem to be solved by the present invention is to provide a microorganism-Bacillus methylate which can rapidly degrade benzophenone ultraviolet sunscreen in an aqueous environment under aerobic conditions.
- the methyl bacillus described in the present invention is named Bacillus methylotrophicus BP1.1, classified as Bacillus methylotrophicus, and the strain number is BP1.1. It has been deposited in China The Type Culture Collection, the deposit number is CCTCC M 20191078, and the deposit date is December 20, 2019.
- the above-mentioned strain Bacillus methylotrophicus BP1.1, grows well in LB medium under aerobic conditions at 30°C. The colonies are round with a diameter of 0.2 ⁇ 1mm, and the colonies are slightly pink and opaque. , The surface is dry and wrinkle-free. Gram staining was positive, and the cell shape was short rod-shaped under microscope observation.
- the technical problem to be solved by the present invention is to provide the application of the above-mentioned methyl bacillus in the degradation of micro-pollutants in the environment.
- the micro-pollutant is a benzophenone ultraviolet sunscreen; the benzophenone ultraviolet sunscreen is 2,4-dihydroxybenzophenone (BP-1).
- the application is as follows: inoculate Bacillus methylotrophicus BP1.1 in an expansion medium according to the inoculation amount of 1%, aerobic culture at 28 ⁇ 30°C for 24 ⁇ 48h, after cultivation, BP1.1 expansion is obtained The culture product is then inoculated into the sewage containing benzophenone ultraviolet sunscreen for treatment.
- the inoculation amount of Bacillus methylotrophicus BP1.1 in the sewage is 1 ⁇ to 2 ⁇ , and the mass concentration of benzophenone ultraviolet sunscreen in the sewage is 10mg/L.
- the temperature of the sewage containing benzophenone ultraviolet sunscreen agent is 20-30°C, preferably 28°C, and the pH value is 7.3-8.0, preferably 7.4. Under these conditions, it is beneficial to accelerate Bacillus methylarum. (Bacillus methylotrophicus) BP1.1 degradation rate of benzophenones in sewage.
- the biodegradation rate of the benzophenone UV sunscreen agent under aerobic conditions of the Bacillus methyls of the present invention can reach 100%, and its degradation time is significantly shortened; the present invention uses biological methods to rapidly and efficiently degrade sewage
- the emerging micro-pollutant BP-1 is of great significance to the treatment of emerging micro-pollutants in sewage.
- Figure 1 is a morphological diagram of Bacillus methylotrophicus (Bacillus methylotrophicus) BP1.1 under a Gram staining microscope in Example 2 of the present invention
- Example 2 is a phylogenetic tree of Bacillus methylotrophicus (Bacillus methylotrophicus) BP1.1 in Example 2 of the present invention
- Fig. 3 is a graph showing the degradation rate of 2,4-dihydroxybenzophenone by Bacillus methylotrophicus BP1.1 in Example 3 of the present invention.
- Bacillus methylotrophicus BP1.1 is screened and obtained from the activated sludge of the biological aerobic section of a domestic sewage treatment plant in Nanjing.
- Activated sludge is taken from the biological aerobic section of a domestic sewage treatment plant in Nanjing;
- step (3) Take out the sludge obtained in step (2), apply the sludge to the inorganic salt medium containing 2,4-dihydroxybenzophenone after gradient dilution, and cultivate it statically at 30°C 3-5 days, get a single colony; the mass concentration of 2,4-dihydroxybenzophenone in the inorganic salt medium is 50mg/L;
- step (3) Pick the single colony in step (3) after streaking and purification, and streaking on the slant for storage to obtain a pure strain.
- the medium used for the expanded culture of pure strains is one of LB medium, inorganic salt medium or beef extract peptone medium.
- the LB medium includes the following quality components: 5.0 g of yeast powder, 10 g of sodium chloride and 10 g of peptone per liter of water, and the pH of the medium is 7.0-7.5.
- the Bacillus methylotrophicus BP1.1 of the present invention can grow and reproduce using 2,4-dihydroxybenzophenone compound as the sole carbon source.
- the beef extract peptone medium includes the following components: 3g beef, 10g peptone and 5g sodium chloride per liter of water, and the pH of the medium is 7.2-7.6.
- step (4) Inoculate the Bacillus methylotrophicus BP1.1 obtained in step (4) (inoculation amount is 1% (volume fraction)) in an expanded medium for expansion, and aerobic culture at 28-30°C for 24 ⁇ After 48h, the expanded product of BP1.1 was obtained after culture.
- Example 1 The strains screened in Example 1 were identified.
- Example 1 of the present invention the strain BP1.1 with the best degradation effect and the fastest growth rate was obtained by screening. Among them, BP1.1 was identified as Bacillus methylotrophicus.
- Bacillus methylotrophicus (Bacillus methylotrophicus) BP1.1 grows well under aerobic conditions at 30°C in LB medium. The colonies are round with a diameter of 0.2 ⁇ 1mm. The colonies are slightly pink in color, opaque, and the surface is dry. No wrinkles. Gram staining was positive, and the cell shape was short rod-shaped under microscope observation.
- BP1.1 sequencing results were submitted to GenBank for BLAST comparison. From the BLAST results, strains with a base sequence similarity of more than 98% to BP1.1 were selected, and multiple series alignments were performed through Clustal W, and a phylogenetic tree was constructed using MAGE7.0 BP1.1 is closely related to Bacillus methylotrophicus (Bacillus methylotrophicus), so the strain BP1.1 is identified as Bacillus methylotrophicus.
- Bacillus methylotrophicus BP1.1 measures the degradation rate of pollutant 2,4-dihydroxybenzophenone, and the specific steps are as follows:
- step (2) Take the bacteria obtained in step (1) according to 1.8 ⁇ (mass fraction) inoculation amount, break up, add to the aqueous solution containing 2,4-dihydroxybenzophenone, the 2,4-dihydroxy
- the mass concentration of benzophenone is 10mg/L; and the treatment group without BP1.1 is used as a control;
- step (3) Put the control group and the treatment group in step (2) into a shaker, and react under aerobic conditions at 150 rpm and 28°C;
- BP1.1 can efficiently degrade 2,4-dihydroxybenzophenone in water, and 10mg/L 2,4-dihydroxybenzophenone can be completely degraded within 6h.
- the strain BP1.1 has a certain adaptability time in the degradation system.
- the degradation rate of BP1.1 in the water is relatively slow.
- the adaptation is completed (2 ⁇ 5h)
- due to the decrease of the substrate concentration the degradation rate gradually slows down.
- the invention obtains Bacillus methylotrophicus (Bacillus methylotrophicus) BP1.1 with high efficiency in removing benzophenone ultraviolet sunscreens through the step-by-step domestication of the activated sludge in the aerobic section of the domestic sewage plant. Under aerobic conditions, the biodegradation rate of the benzophenone ultraviolet sunscreen agent of the bacillus methyl ester of the present invention can reach 100%, and the biodegradation time is significantly shortened.
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Abstract
提供了一种甲基芽孢杆菌（Bacillus methylotrophicus）BP1.1，该菌株于2019年12月20日保藏于中国典型培养物保藏中心，保藏编号为CCTCC M 20191078。还公开了该甲基芽孢杆菌在降解二苯甲酮类紫外防晒剂中的应用。
Description
本发明涉及一种甲基芽孢杆菌，还涉及上述甲基芽孢杆菌在降解水中二苯甲酮类防晒剂中的应用，属于污水处理领域。
二苯甲酮类(BPs)紫外防晒剂因其具有安全性高、防晒效果好且价格低廉等特点，已被广泛应用于个人护理品中。由于大部分紫外防晒剂持久性好，随着日常使用量的增加，二苯甲酮类紫外防晒剂源源不断地进入到环境水体中，城市污水处理厂却不能将其完全去除，最终会汇入江河湖泊并不断累积，在水环境中检测到紫外防晒剂也越来越常见。其中，二苯甲酮类紫外防晒剂是亲脂性物质，具有内分泌干扰效应和生物积累性，并在环境中广泛存在，成为一种新兴微污染物。
光化学转化是紫外防晒剂在天然水体中的重要转化途径，其光化学转化反应速率及途径受水环境的pH、溶解性物质等影响，且BPs光照下可产生生态毒性更大的代谢产物。高级氧化技术是利用在反应过程中产生的强氧化性物质——羟基自由HO·基为特性对二苯甲酮进行降解，目前报道最多的技术主要包括：UV/H
2O
2、O
3/H
2O
2、UV/O
3、Fenton等，高级氧化反应所投加的化学试剂及其在转化过程中产生的部分产物均存在潜在的环境风险。微生物降解作用是自然界有机物最终矿化的唯一途径，是决定有机污染物环境归宿的关键。由于BPs的生物可降解率不高，导致对BPs的微生物降解研究相对较少。最新研究发现，活性污泥在好氧条件下BPs的生物降解率可达到60％，但是降解时间较长，一般为10～20d。
发明内容
本发明所要解决的技术问题是提供一种能够在好氧条件下快速降解水环境中二苯甲酮类紫外防晒剂的微生物——甲基芽孢杆菌。
技术方案：本发明所述的甲基芽孢杆菌命名为甲基芽孢杆菌(Bacillus methylotrophicus)BP1.1，分类命名为甲基芽孢杆菌(Bacillus methylotrophicus)，菌株号为BP1.1，现已保藏于中国典型培养物保藏中心，保藏编号为CCTCC M 20191078，保藏日前为2019年12月20日。
上述菌株——甲基芽孢杆菌(Bacillus methylotrophicus)BP1.1，在LB培养基中，30℃好氧条件下生长良好，菌落呈圆形，直径大小0.2～1mm，菌落颜色略带淡粉色，不透明，表面干燥，无褶皱。革兰氏染色呈阳性，显微镜观察下细胞形状为短杆状。
本发明还要解决的技术问题是提供上述甲基芽孢杆菌在降解环境中微污染物方面 的应用。
所述微污染物为二苯甲酮类紫外防晒剂；二苯甲酮类紫外防晒剂为2,4-二羟基二苯甲酮(BP-1)。
所述的应用为：将甲基芽孢杆菌(Bacillus methylotrophicus)BP1.1按接种量1％接种于扩大培养基中，于28～30℃下好氧培养24～48h，培养后得到BP1.1扩培产物，然后将扩培产物接种于含二苯甲酮类紫外防晒剂污水中进行处理。污水中甲基芽孢杆菌(Bacillus methylotrophicus)BP1.1的接种量为1‰～2‰，污水中二苯甲酮类紫外防晒剂的质量浓度为10mg/L。
菌体应用时，含二苯甲酮类紫外防晒剂的污水的温度为20～30℃，优选为28℃，pH值为7.3～8.0，优选为7.4，此条件下有利于加快甲基芽孢杆菌(Bacillus methylotrophicus)BP1.1对污水中二苯甲酮类物质的降解速率。
有益效果：本发明的甲基芽孢杆菌在好氧条件下对二苯甲酮类紫外防晒剂的生物降解率可达到100％，且显著缩短其降解时间；本发明利用生物法快速高效降解污水中新兴微污染物BP-1，对污水中新兴微污染物的处理有重要意义。
图1为本发明实施例2中甲基芽孢杆菌(Bacillus methylotrophicus)BP1.1革兰氏染色显微镜下形态图；
图2为本发明实施例2中甲基芽孢杆菌(Bacillus methylotrophicus)BP1.1系统发育树；
图3为本发明实施例3中甲基芽孢杆菌(Bacillus methylotrophicus)BP1.1对2,4-二羟基二苯甲酮降解速率图。
以下结合附图和具体实施例对本发明的技术方案做进一步说明。
实施例1
本发明中，是从南京某生活污水处理厂生物好氧段活性污泥中筛选获得的甲基芽孢杆菌(Bacillus methylotrophicus)BP1.1。
具体过程如下：
(1)从南京某生活污水处理厂生物好氧段取活性污泥；
(2)将得到的泥水混合物取250mL盛装于锥形瓶中，在磁力搅拌器上常温搅拌，每天向其中加入5mg 2,4-二羟基二苯甲酮；每次加药前倒掉上部分上清液(检测上清液中是否含有目标污染物-2,4-二羟基二苯甲酮)，然后用自来水补足，如此驯化直至溶液 中检测不到目标污染物，则完成驯化；
(3)取出步骤(2)中得到的污泥，将污泥经过梯度稀释后，分别涂布于含2,4-二羟基二苯甲酮的无机盐培养基中，30℃条件下静态培养3～5d，获得单菌落；无机盐培养基中2,4-二羟基二苯甲酮的质量浓度为50mg/L；
(4)挑取步骤(3)中的单菌落划线纯化后，于斜面上划线保存，得到纯菌株。
纯菌株的扩大培养采用的培养基为LB培养基、无机盐培养基或牛肉膏蛋白胨培养基中的一种。
LB培养基包括如下质量的组分：每升水中含酵母粉5.0g、氯化钠10g和蛋白胨10g，培养基的pH＝7.0～7.5。
无机盐培养基包括如下质量的组分：每升水中含2,4-二羟基二苯甲酮10～50mg、氢氧化钠1.6g和磷酸二氢钾6.8g，培养基的pH＝7.3～7.5。本发明的甲基芽孢杆菌(Bacillus methylotrophicus)BP1.1能够以2,4-二羟基二苯甲酮化合物作为唯一碳源进行生长繁殖。
牛肉膏蛋白胨培养基包括如下质量的组分：每升水中含牛肉3g、蛋白胨10g和氯化钠5g，培养基的pH＝7.2～7.6。
将步骤(4)得到的甲基芽孢杆菌(Bacillus methylotrophicus)BP1.1接种(接种量为1％(体积分数))于扩大培养基中进行扩培，于28～30℃下好氧培养24～48h，培养后得到BP1.1的扩培产物。
实施例2
对实施例1筛选的菌种进行鉴定。
本发明实施例1中，筛选得到降解效果最佳、生长速度最快的菌株BP1.1。其中，经鉴定，BP1.1为甲基芽孢杆菌(Bacillus methylotrophicus)。
甲基芽孢杆菌(Bacillus methylotrophicus)BP1.1，在LB培养基中，30℃好氧条件下生长良好，菌落呈圆形，直径大小0.2～1mm，菌落颜色略带淡粉色，不透明，表面干燥，无褶皱。革兰氏染色呈阳性，显微镜观察下细胞形状为短杆状。
通过PCR扩增和Sanger法测序，得到BP1.1菌株16S rRNA全序列如下(SEQ ID NO.1)：
BP1.1测序结果提交GenBank进行BLAST比较，从BLAST结果中挑选与BP1.1碱基序列相似度98％以上的菌株序列，通过Clustal W进行多重系列比对，并利用MAGE 7.0构建系统发育树得到BP1.1与甲基芽孢杆菌(Bacillus methylotrophicus)亲缘关系最近，因此鉴定菌株BP1.1为甲基芽孢杆菌。
实施例3
本发明甲基芽孢杆菌(Bacillus methylotrophicus)BP1.1对污染物-2,4-二羟基二苯甲酮降解速率进行测定，具体步骤如下：
(1)将BP1.1经过扩大培养后菌液于6000rpm下离心10min，倒掉上清液，得到菌体；
(2)取步骤(1)中得到的菌体按1.8‰(质量分数)接种量，打散，加入含2,4-二羟基二苯甲酮的水溶液中，水溶液中2,4-二羟基二苯甲酮的质量浓度为10mg/L；并以不加BP1.1的处理组做对照；
(3)将步骤(2)中的对照组与处理组放入摇床中，好氧条件下，在150rpm，28℃下振荡反应；
(4)定时取样检测步骤(3)中处理组与对照组中污染物的剩余浓度，结果见图3。
如图3所示，BP1.1能够高效降解水中2,4-二羟基二苯甲酮，10mg/L的2,4-二羟基二苯甲酮在6h内可被完全降解。处理前期2h内，菌株BP1.1在降解体系中有一定的适应性时间，此阶段水中BP1.1降解速率较慢，适应完成后(2～5h)，进入到一个快速降解阶段；降解后期(5～6h)，由于底物浓度的降低，降解速率逐步变缓。
本发明通过对生活污水厂好氧段的活性污泥逐级驯化后，获得对二苯甲酮类紫外防晒剂有高效去除效率的甲基芽孢杆菌(Bacillus methylotrophicus)BP1.1。本发明的甲基 芽孢杆菌在好氧条件下对二苯甲酮类紫外防晒剂的生物降解率可达到100％，且显著缩短了生物降解时间。
Claims (9)
- 一种甲基芽孢杆菌，其特征在于：命名为甲基芽孢杆菌(Bacillus methylotrophicus)BP1.1，于2019年12月20日保藏于中国典型培养物保藏中心，保藏编号为CCTCC M 20191078。
- 权利要求1所述的甲基芽孢杆菌在降解环境中微污染物方面的应用。
- 根据权利要求2所述的甲基芽孢杆菌在降解环境中微污染物方面的应用，其特征在于：所述微污染物为二苯甲酮类紫外防晒剂；二苯甲酮类紫外防晒剂为2，4-二羟基二苯甲酮。
- 根据权利要求3所述的甲基芽孢杆菌在降解环境中微污染物方面的应用，其特征在于：甲基芽孢杆菌降解二苯甲酮类紫外防晒剂在好氧条件下进行。
- 根据权利要求2所述的甲基芽孢杆菌在降解环境中微污染物方面的应用，其特征在于：将甲基芽孢杆菌(Bacillus methylotrophicus)BP1.1接种于扩大培养基中，于28～30℃下好氧培养24～48h，培养后得到BP1.1扩培产物；将扩培产物接种于含二苯甲酮类紫外防晒剂污水中进行处理。
- 根据权利要求5所述的甲基芽孢杆菌在降解环境中微污染物方面的应用，其特征在于：污水中的甲基芽孢杆菌(Bacillus methylotrophicus)BP1.1接种量为1‰～2‰。
- 根据权利要求5所述的甲基芽孢杆菌在降解环境中微污染物方面的应用，其特征在于：污水中二苯甲酮类紫外防晒剂的质量浓度为10mg/L。
- 根据权利要求5所述的甲基芽孢杆菌在降解环境中微污染物方面的应用，其特征在于：菌体应用时，污水的温度为20～30℃，pH值为7.3～8.0。
- 根据权利要求5所述的甲基芽孢杆菌在降解环境中微污染物方面的应用，其特征在于：扩大培养基为LB培养基、无机盐培养基或牛肉膏蛋白胨培养基中的一种。
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