WO2024130870A1 - 一株具有邻苯二甲酸酯降解能力的谷氨酸杆菌a4及其应用 - Google Patents

一株具有邻苯二甲酸酯降解能力的谷氨酸杆菌a4及其应用 Download PDF

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WO2024130870A1
WO2024130870A1 PCT/CN2023/082500 CN2023082500W WO2024130870A1 WO 2024130870 A1 WO2024130870 A1 WO 2024130870A1 CN 2023082500 W CN2023082500 W CN 2023082500W WO 2024130870 A1 WO2024130870 A1 WO 2024130870A1
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phthalate
strain
glutamicibacter
preparation
phthalates
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French (fr)
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高彦征
王建
陆雯逸
张帅
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南京农业大学
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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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
    • B09C—RECLAMATION OF CONTAMINATED SOIL
    • B09C1/00—Reclamation of contaminated soil
    • B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
    • 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
    • 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
    • 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
    • C12R2001/265—Micrococcus
    • C12R2001/28—Micrococcus glutamicus ; Corynebacterium glutamicum

Definitions

  • the invention relates to the technical field of biological treatment of environmental pollutants, and in particular to a strain of Bacillus glutamicum A4 with phthalate degradation ability and application thereof.
  • Phthalic Acid Esters are one of the most widely used plasticizers and the most widely studied environmental pollutants with endocrine disrupting properties. Phthalic Acid Esters are widely used in industries such as plastics, paints, and cosmetics. In 2018, global plastic product production increased by nearly 359 million tons, of which 30% came from China. Among the large amount of plastic waste, only 9% of the waste is recycled, and 79% of the waste ends up in landfills or directly enters the environment, causing plastic fragments to accumulate rapidly. Microplastics (MPs) contain a large number of harmful additives, such as plasticizers.
  • PAEs are additives that improve the flexibility of plastic products and are a commonly used plasticizer.
  • PAEs are carcinogenic, teratogenic, and mutagenic.
  • PAEs inhibit the activity of microorganisms in the soil, such as interfering with bacterial communities and reducing urease activity, and enter the food chain through enrichment, further threatening human health. Therefore, PAEs are listed as restricted organic pollutants in many countries. The impact of PAEs on ecology and human health has attracted people's attention and has become a hot topic in environmental science and environmental ecology.
  • the degradation process of PAEs in the natural environment mainly involves hydrolysis, photodegradation and microbial degradation, but in the natural environment, hydrolysis and photodegradation are weak and the degradation rate is slow.
  • researchers have isolated strains that can degrade phthalate pollutants such as DMP, DEHP and DBP in the environment.
  • degrading bacteria have the ability to efficiently degrade only one type of phthalate, and germplasm resources of degrading bacteria that can efficiently degrade both short-chain PAEs (such as DMP, DEP, etc.) and long-chain PAEs (such as DBP, BBP, etc.) are rarely found.
  • short-chain PAEs such as DMP, DEP, etc.
  • long-chain PAEs such as DBP, BBP, etc.
  • the technical problem solved by the present invention is that most of the reported degrading bacteria have high efficiency in degrading only one type of phthalate, while germplasm resources of degrading bacteria that can degrade both short-chain PAEs and long-chain PAEs are rarely found.
  • the invention provides a Glutamicibacter sp. A4 strain having phthalate degradation ability, which is deposited in China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC M20221850.
  • CTCC China Center for Type Culture Collection
  • the phthalates are: dimethyl phthalate, diethyl phthalate, dibutyl phthalate, butyl benzyl phthalate.
  • Glutamicibacter sp. A4 can efficiently degrade both short-chain phthalates (dimethyl phthalate, diethyl phthalate) and long-chain phthalates (dibutyl phthalate, butyl benzyl phthalate).
  • the present invention also provides an application of the glutamicum strain A4 having the ability to degrade phthalates, namely, application in preparing a preparation for degrading phthalates.
  • the phthalate is dimethyl phthalate and/or diethyl phthalate and/or dibutyl phthalate and/or butyl benzyl phthalate.
  • Preparations for degrading phthalates can efficiently degrade both short-chain phthalates (dimethyl phthalate, diethyl phthalate) and long-chain phthalates (dibutyl phthalate, butyl benzyl phthalate).
  • the preparation process of the preparation is:
  • SB1 transfer 500 ⁇ L of glutamicum strain A4 into 100 ml of LB medium, culture at 30°C and 150 rpm for 24 h, and then centrifuge at 8000 rcf for 5 min to obtain the cultured bacterial liquid;
  • OD 600 value refers to the absorbance value of a solution at a wavelength of 600nm.
  • the above application is to activate Glutamicibacter sp. A4 for 10-16 hours, shake culture to the logarithmic phase, collect the bacteria, adjust the bacterial content according to actual needs and prepare a bacterial suspension, and inoculate the prepared bacterial suspension into water or soil contaminated by phthalates.
  • the application process of the preparation is: the bacterial suspension is inoculated into a medium contaminated with phthalates.
  • the above medium is: water or soil.
  • the preparation can be applied to both contaminated water and contaminated soil.
  • the dosage of the preparation is: 5% to 20% of the mass of the medium.
  • the present invention provides a glutamicum strain Glutamicibacter sp. A4 and its application in degrading phthalates, the glutamicum strain Glutamicibacter sp. A4 can use dimethyl phthalate, diethyl phthalate, dibutyl phthalate and butyl benzyl phthalate as the sole carbon source and energy source for growth and reproduction, and under pure culture conditions, the bacteria can almost completely degrade 20 mg/L of mixed phthalates (containing 20 mg/L of dimethyl phthalate, diethyl phthalate, dibutyl phthalate and butyl benzyl phthalate, respectively) in an inorganic salt culture medium within 48 hours, and the degradation
  • Figure 1 shows the growth morphology of Glutamicibacter sp. A4 cultured on LB medium for 5 days in Example 2;
  • Figure 2 is a scanning electron microscope image of Glutamicibacter sp. A4 in Example 2;
  • FIG3 is a schematic diagram of the phylogenetic tree of 16S rDNA of Glutamicibacter sp. A4 in Example 2;
  • FIG. 4 is a diagram showing the degradation effect of Glutamicibacter sp. A4 on phthalates in Example 5.
  • Example 1 This example provides a Glutamicibacter sp. A4 strain having phthalate degradation ability, which is deposited in China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC M20221850 and a deposit address of Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei province.
  • CTCC China Center for Type Culture Collection
  • Glutamicibacter sp. A4 was isolated and enriched from the contaminated soil of the archway of Nanjing Agricultural University in Nanjing through enrichment culture.
  • SEQ ID NO.1 The gene sequence of the above-mentioned Glutamicibacter sp. A4 is shown in SEQ ID NO.1, which is:
  • Example 2 This example is a method for preparing a glutamicum strain Glutamicibacter sp. A4 having phthalate degradation ability, based on the glutamicum strain Glutamicibacter sp. A4 of Example 1, comprising the following steps:
  • strain A4 is transferred to LB solid culture medium and cultured inverted at an ambient temperature of 30°C for 5 days to observe the colony morphology of strain A4.
  • the content of phthalates in the inorganic salt liquid culture medium after the first transfer is 5 mg/L, which means that the content of dimethyl phthalate, diethyl phthalate, dibutyl phthalate, and butyl benzyl phthalate are all 5 mg/L.
  • the content of phthalates in the inorganic salt liquid culture medium after the second transfer is 10 mg/L, which means that the content of dimethyl phthalate, diethyl phthalate, dibutyl phthalate, and butyl benzyl phthalate are all 10 mg/L.
  • the content of phthalates in the inorganic salt liquid culture medium after the third transfer is 20 mg/L, which means that the content of dimethyl phthalate, diethyl phthalate, dibutyl phthalate, and butyl benzyl phthalate are all 20 mg/L.
  • the content of phthalates in the inorganic salt liquid culture medium after the fourth transfer is 40 mg/L, which means that the content of dimethyl phthalate, diethyl phthalate, dibutyl phthalate, and butyl benzyl phthalate are all 40 mg/L.
  • the content of phthalates in the inorganic salt liquid culture medium after the fifth transfer is 80 mg/L, which means that the content of dimethyl phthalate, diethyl phthalate, dibutyl phthalate, and butyl benzyl phthalate are all 80 mg/L.
  • the A4 strain was streaked and cultured on LB solid medium for 5 days.
  • the colonies were light yellow, round, mucous and opaque, with irregular edges, raised surfaces, and smooth and moist surfaces.
  • the specific morphology is shown in FIG1 .
  • the morphological feature of the structure is an ellipse when observed under a scanning electron microscope.
  • the specific morphological feature is shown in FIG. 2 .
  • the inorganic salt liquid culture medium is an inorganic salt culture solution
  • the components of the inorganic salt culture solution include: (NH 4 ) 2 SO 4 with a concentration of 1.5 g/L, KH 2 PO 4 with a concentration of 0.5 g/L, K 2 HPO 4 ⁇ 3H 2 O with a concentration of 1.91 g/L, NaCl with a concentration of 0.5 g/L, and MgSO4 ⁇ 7H2O with a concentration of 0.2 g/L.
  • the pH value of the inorganic salt culture solution is 7.0.
  • the inorganic salt solid medium is obtained by adding 1.5% (W/V) agar powder to the inorganic salt liquid medium.
  • SA1 add ultrapure water to 5.0 g yeast extract, 10.0 g tryptone, and 10.0 g sodium chloride to 1 L, adjust the pH to 7.0, and sterilize at 121° C. for 20 minutes to obtain LB liquid culture medium;
  • the 16S rDNA sequence of strain A4 was compared with the 16S rDNA sequences of other registered bacterial strains using the BLAST program on the official website of NCBI (http://www.ncbi.nlm.nih.gov/). The results showed that the strain had the highest similarity with Glutamicibacter sp., with a homology rate of 99%.
  • the strain obtained by screening in this example was identified as Glutamicibacter and named Glutamicibacter sp. A4.
  • Example 3 This example is a method for preparing a Glutamicibacter sp. A4 strain having phthalate degradation ability, which is different from Example 1 in that:
  • step S1-3 the culture medium in the inorganic salt liquid culture medium after five transfers is diluted by 10 4 .
  • step S1-3 the cells were incubated upside down at an ambient temperature of 30°C for 2 days.
  • step S2-1 the A4 strain purified on the LB solid medium was inoculated into the LB liquid medium for 13 hours for activation.
  • step S2-2 the mixture was centrifuged at 8000 rpm for 4 min and allowed to stand at 4°C for 13 h.
  • step S2-3 the sample was finally fixed with 1% osmium acid solution for 1.5 h.
  • step S2-5 the sample is infiltrated with pure super epoxy resin for 14 h.
  • step S2-6 the sections are then stained with a 1% lead citrate solution and a 50% ethanol saturated solution of uranyl acetate for 8 minutes each.
  • Example 4 This example is a method for preparing a Glutamicibacter sp. A4 strain having phthalate degradation ability, which is different from Example 1 in that:
  • step S1-3 the cells were incubated upside down at an ambient temperature of 30°C for 3 days.
  • step S2-3 the sample was finally fixed with 1% osmium acid solution for 2 h.
  • step S2-1 the A4 strain purified on LB solid medium was inoculated into LB liquid medium for 16 hours for activation.
  • step S2-2 the mixture was centrifuged at 8000 rpm for 5 min and allowed to stand at 4°C for 16 h.
  • step S2-3 the sample was finally fixed with 1% osmium acid solution for 2 h.
  • step S2-5 the sample is infiltrated with pure super epoxy resin for 16 h.
  • step S2-6 the sections are then stained with a 1% lead citrate solution and a 50% ethanol saturated uranyl acetate solution for 10 minutes each.
  • Example 5 This example is an application of a glutamicum strain Glutamicibacter sp. A4 having the ability to degrade phthalates. Based on the glutamicum strain Glutamicibacter sp. A4 in Example 1, the glutamicum strain Glutamicibacter sp. A4 is used to prepare a preparation for degrading phthalates.
  • the preparation process of the preparation is:
  • SB1 transfer 500 ⁇ L of glutamicum strain A4 into 100 ml of LB medium, culture at 30°C and 150 rpm for 24 h, and then centrifuge at 8000 rcf for 5 min to obtain the cultured bacterial liquid;
  • This embodiment only provides a preparation method of the preparation.
  • Glutamicibacter sp. A4 is activated and shaken for 10 to 16 hours to the logarithmic phase, the bacteria are collected, the bacterial content is adjusted according to actual needs and a bacterial suspension is prepared, and the prepared bacterial suspension is then inoculated into water or soil contaminated with phthalates.
  • the method for applying the preparation is: the preparation is introduced into water contaminated by phthalates, and the amount of the preparation applied is 15% of the mass of the water.
  • 1 mL of the above bacterial suspension was transferred to 19 mL of MSM culture medium containing 20 mg/L PAEs (i.e., 20 mg/L DMP, 20 mg/L DEP, 20 mg/L DBP, and 20 mg/L BBP), with no inoculation as the control, and the pH was adjusted to 7.0, with three replicates per group.
  • 20 mg/L PAEs i.e., 20 mg/L DMP, 20 mg/L DEP, 20 mg/L DBP, and 20 mg/L BBP
  • Chromatographic conditions LC-20AT high performance liquid chromatograph (equipped with SPD-2A ultraviolet detector) was used. The detection time was 40 min, and the injection volume of the injection system was 20 ⁇ L.
  • the separation system used acetonitrile-water as the mobile phase, the initial flow rate was 1.0 mL/min, and gradient elution was used to separate PAEs.
  • the chromatographic column was a ⁇ 4.6 ⁇ 250 mm Inertsil ODS-P liquid chromatography column, and the column temperature was 40°C.
  • the detection system used an ultraviolet detector and turned on the dual wavelength detection mode, which were 205 nm and 225 nm respectively.
  • the degradation effect of Glutamicibacter sp. A4 on four mixed PAEs is shown in Figure 4.
  • the bacteria has a significant degradation effect on the four PAEs under 3-day shaking culture.
  • short-chain PAEs such as DMP and DEP
  • the degradation rate of A4 on these two PAEs can exceed 95% on the first day, and the degradation rate on the second day is more than 98%.
  • the degradation rate of long-chain PAEs (such as DBP and BBP) is relatively slow, and the degradation rate of DBP can reach 90% on the first day, and the degradation rate of BBP is about 70%, but the degradation rate of BBP on the second day is about 80%.
  • DMP is used as the abbreviation for phthalate
  • DEP is used as the abbreviation for diethyl phthalate
  • DBP is used as the abbreviation for dibutyl phthalate
  • BBP is used as the abbreviation for butyl benzyl phthalate.
  • Example 6 This example is an application of Glutamicibacter sp. A4 having phthalate degradation ability, which is different from Example 5 in that:
  • the application method of the preparation is: inoculate the preparation into the soil contaminated by phthalates, and the application amount of the preparation is 5% of the soil mass.
  • Example 7 This example is an application of Glutamicibacter sp. A4 having phthalate degradation ability, which is different from Example 5 in that:
  • the application method of the preparation is: inoculate the preparation into the soil contaminated by phthalates, and the application amount of the preparation is 20% of the soil mass.

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Abstract

提供了一种具有邻苯二甲酸酯降解能力的谷氨酸杆菌株A4及其应用，属于环境污染物生物处理技术领域。谷氨酸杆菌株A4在中国典型培养物保藏中心（CCTCC）保藏，其保藏编号为CCTCC M20221850。谷氨酸杆菌株A4用于制备降解邻苯二甲酸酯的制剂，对苯二甲酸二甲酯、邻苯二甲酸二乙酯、邻苯二甲酸二丁酯、邻苯二甲酸丁基苄酯具有高效的降解能力。

Description

一株具有邻苯二甲酸酯降解能力的谷氨酸杆菌A4及其应用 技术领域
本发明涉及环境污染物生物处理技术领域，具体是涉及一株具有邻苯二甲酸酯降解能力的谷氨酸杆菌A4及其应用。
 背景技术
邻苯二甲酸酯（PhthalicAcidEsters，PAEs）是应用最广泛的增塑剂之一，也是研究最为广泛的具有内分泌干扰特性的一种环境污染物，邻苯二甲酸酯被广泛应用于塑料、油漆、化妆品等行业。2018年，全球塑料制品产量增加了近3.59亿吨，其中30%来自中国。在大量的塑料垃圾中，只有9%的垃圾被循环利用，79%的垃圾最终进入到垃圾填埋场或直接进入到环境中，导致塑料碎片迅速堆积。微塑料（MPs）含有大量的有害添加剂，比如增塑剂。由于其较大的比表面积和较强的疏水性，重金属、抗生素、农药和其他持久性有机污染物会吸附在微塑料的表面，使得土壤受到威胁，并对人类健康产生不利的影响。随着微塑料的增加，这些污染物影响到了生态系统的一些功能，比如土壤微生物活性和养分的循环等。PAEs是一种提高塑料制品柔韧性的添加剂，是一种常用的增塑剂。但是，PAEs具有致癌性、致畸性和致突变性的危害。当释放到环境中时，PAEs会抑制土壤中微生物的活性，例如干扰细菌群落，降低脲酶活性，并通过富集作用进入食物链，进一步威胁人类的健康。因此，PAEs在许多国家被列为限制性有机污染物。PAEs对生态和人类健康的影响引起了人们的关注，成为了环境科学和环境生态学的一个热点。
PAEs在自然环境中的降解过程主要经过水解、光降解以及微生物降解，但在自然环境中，水解和光降解较弱并且降解速度缓慢。目前，学者们已经在环境中分离出了可降解DMP、DEHP和DBP等邻苯二甲酸酯污染物的菌株。
但是，大部分已报道的降解菌仅对一种邻苯二甲酸酯具有高效降解能力，而既能高效降解短链PAEs（例如DMP、DEP等），又能降解长链PAEs（例如DBP、BBP等）的降解菌的种质资源很少被发现。
 技术问题
本发明解决的技术问题是：大部分已报道的降解菌仅对一种邻苯二甲酸酯具有高效降解能力，而既能高效降解短链PAEs，又能降解长链PAEs的降解菌的种质资源很少被发现。
 技术解决方案
为解决上述问题，本发明的技术方案如下：
本发明提供一种具有邻苯二甲酸酯降解能力的谷氨酸杆菌株 Glutamicibacter sp. A4，在中国典型培养物保藏中心（CCTCC）保藏，其保藏编号为CCTCC M20221850。
进一步地，邻苯二甲酸酯为：邻苯二甲酸二甲酯、邻苯二甲酸二乙酯、邻苯二甲酸二丁酯、邻苯二甲酸丁基苄酯。
说明：谷氨酸杆菌株 Glutamicibacter sp. A4既能高效降解短链邻苯二甲酸酯（邻苯二甲酸二甲酯、邻苯二甲酸二乙酯），又能降解长链邻苯二甲酸酯（邻苯二甲酸二丁酯、邻苯二甲酸丁基苄酯）。
本发明还提供了一种具有邻苯二甲酸酯降解能力的谷氨酸杆菌株A4的应用，即用于制备降解邻苯二甲酸酯的制剂。
优选地，邻苯二甲酸酯为邻苯二甲酸二甲酯和/或邻苯二甲酸二乙酯和/或邻苯二甲酸二丁酯和/或邻苯二甲酸丁基苄酯。
说明：降解邻苯二甲酸酯的制剂既能高效降解短链邻苯二甲酸酯（邻苯二甲酸二甲酯、邻苯二甲酸二乙酯），又能降解长链邻苯二甲酸酯（邻苯二甲酸二丁酯、邻苯二甲酸丁基苄酯）。
优选地，制剂的制备过程为：
SB1、将500μL谷氨酸杆菌株A4转接到100ml的LB培养基中，在30℃、150rpm环境下培养24h再8000rcf离心5min，得到培养后的菌液；
SB2、菌液通过MSM清洗两次后将细菌的OD 600值调整到1.0作为菌种悬液，并在4℃环境温度下暂存备用。
上述过程中，OD 600值指的是某种溶液在600nm波长处的吸光值。
说明：上述应用是将谷氨酸杆菌 Glutamicibacter sp. A4经过10~16h活化、震荡培养至对数期，收集菌体，根据实际需要调节菌体含量并制成菌悬液，将制得的菌悬液接入受邻苯二甲酸酯污染的水或土壤中。
优选地，制剂的应用过程为：菌悬液接入受邻苯二甲酸酯污染的介质中。
说明：制剂的应用方式较为简单，对于反应的环境要求较低。
优选地，上述介质为：水、土壤。
说明：制剂既能够应用于受到污染的水中，也能应用于受到污染的土壤中。
进一步优选地，制剂的施用量为：介质质量的5%~20%。
说明：将介质质量的5%~20%的制剂接入后的72小时内，四种邻苯二甲酸酯几乎完全降解，短链邻苯二甲酸酯降解率均超过98%，长链邻苯二甲酸酯的降解率也能达到75%以上，说明制剂对四种PAEs具有高效的降解能力。
 有益效果
本发明的有益效果是：本发明提供谷氨酸杆菌株 Glutamicibacter sp. A4及其在降解邻苯二甲酸酯中的应用，谷氨酸杆菌株 Glutamicibacter sp. A4能够利用邻苯二甲酸二甲酯、邻苯二甲酸二乙酯、邻苯二甲酸二丁酯和邻苯二甲酸丁基苄酯作为唯一碳源和能源进行生长繁殖，在纯培养条件下该菌48h可将无机盐培养基中20 mg/L的混合邻苯二甲酸酯（分别包含20mg/L邻苯二甲酸二甲酯、邻苯二甲酸二乙酯、邻苯二甲酸二丁酯和邻苯二甲酸丁基苄酯）几乎降解完全，降解
附图说明
图1为实施例2中 Glutamicibacter sp. A4在LB培养基上培养5天的生长形态；
图2为实施例2中 Glutamicibacter sp. A4的扫描电镜图；
图3为实施例2中 Glutamicibacter sp. A4的16S rDNA的系统发育树示意图；
图4为实施例5中 Glutamicibacter sp. A4对邻苯二甲酸酯的降解效果图。
本发明的实施方式
为了使本发明的目的、技术方案和优点更加清楚，下面将结合附图对本发明作进一步地详细描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其它实施例，都属于本发明保护的范围。
在本发明实施例中使用的术语是仅仅出于描述特定实施例的目的，而非旨在限制本发明。在本发明实施例和所附权利要求书中所使用的单数形式的“一种”、“所述”和“该”也旨在包括多数形式，除非上下文清楚地表示其他含义，“多种”一般包含至少两种。
实施例1：本实施例提供一种具有邻苯二甲酸酯降解能力的谷氨酸杆菌株 Glutamicibacter sp. A4，在中国典型培养物保藏中心（CCTCC）保藏，其保藏编号为CCTCC M20221850，保藏地址：湖北省武汉市武昌区八一路珞珈山。
上述谷氨酸杆菌株 Glutamicibacter sp. A4是通过富集培养从南京市南京农业大学牌楼污染土中分离并富集获得。
上述谷氨酸杆菌株 Glutamicibacter sp. A4的基因序列如SEQ ID NO.1所示，为：
GGAGTGGCGGGGTGCTTACACATGCAGTCGAACGATGAAGCCCAGCTTGCTGGGTGGATTAGTGGCGAACGGGTGAGTAACACGTGAGTAACCTGCCCCCGACTCTGGGATAAGCCCGGGAAACTGGGTCTAATACCGGATATGACCTCGCACCGCATGGTGCGGGGTGGAAAGATTTATCGGTGGGGGATGGACTCGCGGCCTATCAGCTTGTTGGTGAGGTAATGGCTCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGTAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTATCCGGATTTATTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCTGCCGTGAAAGTCCGAGGCTCAACCTCGGATCTGCGGTGGGTACGGGCAGACTAGAGTGATGTAGGGGAGACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGATGGCGAAGGCAGGTCTCTGGGCATTTACTGACGCTGAGGAGCGAAAGCATGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGTTGGGCACTAGGTGTGGGGGACATTCCACGTTTTCCGCGCCGTAGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCAAGGCTTGACATGTGCCAGACCGCTTCAGAGATGGGGTTTCCCTTCGGGGCTGGTTCACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGTTCCATGTTGCCAGCACGTAGTGGTGGGGACTCATGGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGTCTTGGGCTTCACGCATGCTACAATGGCCGGTACAATGGGTTGCGATACTGTGAGGTGGAGCTAATCCCTAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCACGAAAGTTGGTAACACCCGAAGCCGATGGCCTAACCACCTTGTGTGGGGGGAGTCGTCGAAGTGACGCCGTT。
实施例2：本实施例为一种具有邻苯二甲酸酯降解能力的谷氨酸杆菌株 Glutamicibacter sp. A4的制备方法，基于实施例1的谷氨酸杆菌株 Glutamicibacter sp. A4，包括以下步骤：
S1、分离与培养菌株，包括以下步骤：
S1-1、称取约5g供试土壤加入到250mL锥形瓶中，加入100mL超纯水，置于30℃、150rpm摇床中避光振荡培养8h后取出，静置两小时，得到的上清液即为富集的初始土著微生物；
S1-2、取5mL上清液转接至95mL含邻苯二甲酸酯的无机盐液体培养基中，所述无机盐液体培养基中邻苯二甲酸酯含量为5mg/L，在30℃、150rpm条件下摇床振荡培养5天后，按照取5mL上清液的转接量对无机盐液体培养基进行连续富集培养，并调整无机盐液体培养基中邻苯二甲酸酯的含量，上述过程为一次转接，转接5次，其中，第一次转接后无机盐液体培养基中邻苯二甲酸酯的含量为5mg/L，第二次转接后无机盐液体培养基中邻苯二甲酸酯的含量为10mg/L，第三次转接后无机盐液体培养基中邻苯二甲酸酯的含量为20mg/L，第四次转接后无机盐液体培养基中邻苯二甲酸酯的含量为40mg/L，第五次转接后无机盐液体培养基中邻苯二甲酸酯的含量为80mg/L；
S1-3、将转接5次后的无机盐液体培养基中培养液稀释10 3，再将稀释后的培养液涂布于邻苯二甲酸酯含量为20mg/L的无机盐固体培养基上，在30℃环境温度下倒置培养1天；
S1-4、待无机盐固体培养基上长出单菌落后，挑取单菌落多次划线纯化，分离获得一株细菌，并编号为A4菌株，再将A4菌株转接于LB固体培养基上，在30℃环境温度下倒置培养5天，观察A4菌株的菌落形态。
上述步骤S1-2中，第一次转接后无机盐液体培养基中邻苯二甲酸酯的含量为5mg/L，指的是邻苯二甲酸二甲酯、邻苯二甲酸二乙酯、邻苯二甲酸二丁酯、邻苯二甲酸丁基苄酯的含量均为5mg/L。
上述步骤S1-2中，第二次转接后无机盐液体培养基中邻苯二甲酸酯的含量为10mg/L，指的是邻苯二甲酸二甲酯、邻苯二甲酸二乙酯、邻苯二甲酸二丁酯、邻苯二甲酸丁基苄酯的含量均为10mg/L。
上述步骤S1-2中，第三次转接后无机盐液体培养基中邻苯二甲酸酯的含量为20mg/L，指的是邻苯二甲酸二甲酯、邻苯二甲酸二乙酯、邻苯二甲酸二丁酯、邻苯二甲酸丁基苄酯的含量均为20mg/L。
上述步骤S1-2中，第四次转接后无机盐液体培养基中邻苯二甲酸酯的含量为40mg/L，指的是邻苯二甲酸二甲酯、邻苯二甲酸二乙酯、邻苯二甲酸二丁酯、邻苯二甲酸丁基苄酯的含量均为40mg/L。
上述步骤S1-2中，第五次转接后无机盐液体培养基中邻苯二甲酸酯的含量为80mg/L，指的是邻苯二甲酸二甲酯、邻苯二甲酸二乙酯、邻苯二甲酸二丁酯、邻苯二甲酸丁基苄酯的含量均为80mg/L。
本实施例中，A4菌株菌株在LB固体培养基上划线培养5天，菌落呈淡黄色，圆形，黏质不透明，边缘不整齐，表面隆起，湿润光滑，其具体形态见图1。
S2、扫描电镜观察鉴定菌株，包括以下步骤：
S2-1、将LB固体培养基上纯化后的A4菌株接入LB液体培养基中10~16h活化；
S2-2、吸取LB液体培养基中1mL菌液，再经过经8000rpm离心3min，去上清液，加入已灭菌的MSM培养基洗菌3次，在获取的菌体沉淀中加入1mL 2.5%（v/v）的戊二醛混匀，并在4℃环境温度下静置10h；
S2-3、将上一步静置过夜的溶液离心后倒掉上清液，再通过0.1M、pH为7.0的PBS缓冲液洗菌3次，每次洗菌的持续时间为15min，最后通过1%的锇酸溶液固定样品1h；
S2-4、倒掉锇酸废液，通过0.1M、pH为7.0的PBS缓冲液漂洗样品3次，每次漂洗的持续时间为15min，再通过梯度浓度的乙醇溶液对样品进行脱水处理，所述梯度浓度为30%、50%、70%、80%、90%和95%五种浓度，每种浓度的乙醇溶液处理时间均为15min，最后使用100%的乙醇对对样品脱水处理20min；
S2-5、通过纯丙酮对样品进行渗透处理，处理时间为20min，通过体积比为1：1的super环氧树脂与丙酮混合液渗透样品1h，通过体积比为3：1的super环氧树脂与丙酮混合液渗透样品3h，再通过纯super环氧树脂渗透样品12h，将经过渗透处理的样品包埋起来，得到包埋后的样品；
S2-6、将包埋后的样品超薄切片机中切片，获得厚度为70~90nm的切片，再将切片先后经铅浓度为1%的柠檬酸铅溶液和醋酸双氧铀50%乙醇饱和溶液各染色5min，将染色后的切片晾干后，通过透射电镜对切片进行观察。
本实施例中，扫描电镜观察其形态特征为椭圆状，其具体形态特征如图2所示。
上述步骤S1和步骤S2中：
无机盐液体培养基为无机盐培养液，无机盐培养液成分包括：浓度为1.5g/L的（NH 4） 2SO 4、浓度为0.5g/L的KH 2PO 4、浓度为1.91g/L的K 2HPO 4·3H 2O、浓度为0.5g/L的NaCl、浓度为0.2g/L的MgSO4·7H2O，无机盐培养液的pH值为7.0。
无机盐固体培养基通过向无机盐液体培养基中加入1.5%（W/V）琼脂粉得到。
LB液体培养基及LB固体培养基的制备过程为：
SA1、在5.0g酵母提取物、10.0g胰化蛋白胨、10.0g氯化钠中加入超纯水至1L，调节pH至7.0，在121℃温度环境下灭菌20分钟，得到LB液体培养基；
SA2、在LB液体培养基的基础上，加入1.5%（W/V）琼脂粉，得到LB固体培养基。
S3、菌株的16S rDNA分子鉴定，包括以下步骤：
S3-1、提取切片中菌株的总DNA，通过细菌16S rDNA通用引物对菌株基因组进行PCR扩增，得到PCR产物；
S3-2、PCR产物经测序，将测序结果与GenBank中已报道的16S rDNA序列进行同源性比对，并选取相关菌种做进化树分析鉴定，确定菌株种属。
上述步骤中，进化树分析的结果如图3所示，菌株A4的16S rDNA序列通过NCBI官方网站（http：//www.ncbi.nlm.nih.gov/）的BLAST程序与其他已登录细菌菌株16S rDNA序列进行比对，结果表明该菌株与 Glutamicibacter sp.相似性最高，同源率达99%。
因此，本实施例筛选获得的菌株鉴定为谷氨酸杆菌（ Glutamicibacter），命名为 Glutamicibacter sp. A4。
实施例3：本实施例为一种具有邻苯二甲酸酯降解能力的谷氨酸杆菌株 Glutamicibacter sp. A4的制备方法，与实施例1的区别之处在于：
步骤S1-3中，将转接5次后的无机盐液体培养基中培养液稀释10 4。
步骤S1-3中，在30℃环境温度下倒置培养2天。
步骤S2-1中，将LB固体培养基上纯化后的A4菌株接入LB液体培养基中13h活化。
步骤S2-2中，经过经8000rpm离心4min，在4℃环境温度下静置13h。
步骤S2-3中，最后通过1%的锇酸溶液固定样品1.5h。
步骤S2-5中，通过纯super环氧树脂渗透样品14h。
步骤S2-6中，再将切片先后经铅浓度为1%的柠檬酸铅溶液和醋酸双氧铀50%乙醇饱和溶液各染色8min。
实施例4：本实施例为一种具有邻苯二甲酸酯降解能力的谷氨酸杆菌株 Glutamicibacter sp. A4的制备方法，与实施例1的区别之处在于：
步骤S1-3中，在30℃环境温度下倒置培养3天。
步骤S2-3中，最后通过1%的锇酸溶液固定样品2h。
步骤S2-1中，将LB固体培养基上纯化后的A4菌株接入LB液体培养基中16h活化。
步骤S2-2中，经过经8000rpm离心5min，在4℃环境温度下静置16h。
步骤S2-3中，最后通过1%的锇酸溶液固定样品2h。
步骤S2-5中，通过纯super环氧树脂渗透样品16h。
步骤S2-6中，再将切片先后经铅浓度为1%的柠檬酸铅溶液和醋酸双氧铀50%乙醇饱和溶液各染色10min。
实施例5：本实施例为一种具有邻苯二甲酸酯降解能力的谷氨酸杆菌株 Glutamicibacter sp. A4的应用，基于实施例1的谷氨酸杆菌株 Glutamicibacter sp. A4，将谷氨酸杆菌株 Glutamicibacter sp. A4用于制备降解邻苯二甲酸酯的制剂。
制剂的制备过程为：
SB1、将500μL谷氨酸杆菌株A4转接到100ml的LB培养基中，在30℃、150rpm环境下培养24h再8000rcf离心5min，得到培养后的菌液；
SB2、菌液通过MSM清洗两次后将细菌的OD 600值调整到1.0作为菌种悬液，并在4℃环境温度下暂存备用。
本实施例仅给出了制剂的一种制备方法，在实际应用中，则是将谷氨酸杆菌 Glutamicibacter sp. A4经过10~16h活化、震荡培养至对数期，收集菌体，根据实际需要调节菌体含量并制成菌悬液，再将制得的菌悬液接入受邻苯二甲酸酯污染的水或土壤中。
本实施例中，制剂的施用方法为：将制剂接入受邻苯二甲酸酯污染的水中，制剂的施用量为水质量的15%。
Glutamicibacter sp. A4菌的降解性能测定：
向含有20 mg/L浓度PAEs（即含20 mg/L DMP、20 mg/L DEP、20 mg/L DBP和20 mg/L BBP）的19mL MSM培养液中转接上述菌悬液1mL，以不接菌作为对照，并调节pH为7.0，每组三个重复。在30℃，150rpm恒温摇床培养48h，分别于1、3、6、12、24、48、72h取样，向取出的锥形瓶中加入40mL色谱纯甲醇，水浴超声震荡1h；超声结束后涡旋摇匀，将上清液用0.22μm有机相滤膜过滤并转入2mL棕色液相小瓶，用高效液相色谱仪进行检测。
色谱条件：采用LC-20AT高效液相色谱仪（配有SPD-2A紫外检测器）。检测时间为40min，进样系统的进样量为20μL；分离系统以乙腈-水为流动相、初始流速为1.0mL/min，采用梯度洗脱的方式分离PAEs，色谱柱为Φ4.6×250mm Inertsil ODS-P液相色谱柱，柱温为40℃；检测系统采用紫外检测器检测、开启双波长检测模式，分别为205nm和225nm。
Glutamicibacter sp. A4菌对四种混合PAEs的降解效果如图4所示，该菌在3天振荡培养下对四种PAEs具有显著的降解效果。特别是针对短链PAEs（如DMP、DEP），A4对这两种PAEs在第一天的降解率均能超过95%，第2天降解率超过98%。而对长链PAEs（如DBP、BBP）的降解率速度相对较慢，对DBP的降解率第一天能达到90%，对BBP的降解率在70%左右，但第二天对BBP的降解率达到80%左右。在培养至72h时，四种PAEs几乎降解完全，短链PAEs降解率均超过98%，长链PAEs的降解率也能达到75%以上，说明 Glutamicibacter sp. A4菌对四种PAEs具有高效的降解能力。
上述性能测定过程中，DMP作为邻苯二甲酸酯的缩写，DEP作为邻苯二甲酸二乙酯的缩写，DBP作为邻苯二甲酸二丁酯的缩写，BBP作为邻苯二甲酸丁基苄酯的缩写。
实施例6：本实施例为一种具有邻苯二甲酸酯降解能力的谷氨酸杆菌株 Glutamicibacter sp. A4的应用，与实施例5的区别在于：
制剂的施用方法为：将制剂接入受邻苯二甲酸酯污染的土壤中，制剂的施用量为土壤质量的5%。
实施例7：本实施例为一种具有邻苯二甲酸酯降解能力的谷氨酸杆菌株 Glutamicibacter sp. A4的应用，与实施例5的区别在于：
制剂的施用方法为：将制剂接入受邻苯二甲酸酯污染的土壤中，制剂的施用量为土壤质量的20%。

Claims (7)

  1. 一种具有邻苯二甲酸酯降解能力的谷氨酸杆菌株A4，其特征在于，在中国典型培养物保藏中心（CCTCC）保藏，其保藏编号为CCTCC M20221850。
  2. 如权利要求1所述的一种具有邻苯二甲酸酯降解能力的谷氨酸杆菌株A4，其特征在于，所述邻苯二甲酸酯为：邻苯二甲酸二甲酯、邻苯二甲酸二乙酯、邻苯二甲酸二丁酯、邻苯二甲酸丁基苄酯。
  3. 一种具有邻苯二甲酸酯降解能力的谷氨酸杆菌株A4的应用，其特征在于，用于制备降解邻苯二甲酸酯的制剂。
  4. 如权利要求3所述的应用，其特征在于，所述邻苯二甲酸酯为邻苯二甲酸二甲酯和/或邻苯二甲酸二乙酯和/或邻苯二甲酸二丁酯和/或邻苯二甲酸丁基苄酯。
  5. 如权利要求4所述的应用，其特征在于，所述制剂的制备过程为：
    SB1、将500μL谷氨酸杆菌株A4转接到100ml的LB培养基中，在30℃、150rpm环境下培养24h再8000rcf离心5min，得到培养后的菌液；
    SB2、所述菌液通过MSM清洗两次后将细菌的OD 600值调整到1.0，得到制剂，并在4℃环境温度下暂存备用。
  6. 如权利要求5所述的应用，其特征在于，所述制剂的施用方法为：将制剂接入受邻苯二甲酸酯污染的介质中。
  7. 如权利要求5所述的应用，其特征在于，所述介质为：水、土壤。
PCT/CN2023/082500 2022-12-24 2023-03-20 一株具有邻苯二甲酸酯降解能力的谷氨酸杆菌a4及其应用 WO2024130870A1 (zh)

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