WO2021135237A1 - 一株弗留明拜叶林克氏菌菌株及其在砷氧化中的应用 - Google Patents

一株弗留明拜叶林克氏菌菌株及其在砷氧化中的应用 Download PDF

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WO2021135237A1
WO2021135237A1 PCT/CN2020/108071 CN2020108071W WO2021135237A1 WO 2021135237 A1 WO2021135237 A1 WO 2021135237A1 CN 2020108071 W CN2020108071 W CN 2020108071W WO 2021135237 A1 WO2021135237 A1 WO 2021135237A1
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arsenic
iii
beijerinckia
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孙蔚旻
孙晓旭
李宝琴
黄裕青
孔天乐
王小雨
张苗苗
裘浪
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广东省生态环境技术研究所
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Priority to US17/790,406 priority Critical patent/US20230055742A1/en
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    • C12N1/20—Bacteria; Culture media therefor
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    • B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
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  • the present invention belongs to the field of environmental microorganisms, and specifically relates to a strain of Rinckella fruminensis, which can oxidize arsenic, thereby reducing the adverse effects of arsenic on the environment.
  • Arsenic is a typical human carcinogen with relatively high toxicity.
  • treatment methods for arsenic-contaminated soil at home and abroad include physical, chemical and bioremediation methods.
  • Electrical repair technology, soil replacement method and other physical methods have a large amount of work, time-consuming, and high processing cost; chemical methods such as solidification/stabilization method and soil leaching method have a narrow range of use, strong limitations, and are likely to cause other environmental risks; plants
  • the remediation method has a long treatment cycle, and the growth of plants in different regions makes it difficult to standardize this technology; at the same time, the above methods are difficult to promote in the treatment of large areas of arsenic-contaminated soil.
  • Microbial remediation is a method to reduce the toxicity of pollutants in the soil by using indigenous microorganisms in the natural environment or artificially added functional microorganisms to produce redox, absorption, and degradation of pollutants. It is safe, efficient, low-cost, and environmentally friendly. It has the advantages of friendliness and large-scale application. It is the most promising new technology for environmental restoration.
  • Arsenic in the natural environment mainly exists in two valences: As(V) and As(III). Under most environmental conditions, the toxicity of As(III) is 25-60 times that of As(V). Therefore, if the more toxic As(III) can be oxidized into the less toxic As(V), the migration and toxicity of arsenic can be reduced, which has practical environmental remediation significance.
  • Microorganisms in nature are widely involved in the geochemical cycle of arsenic. Microorganisms are highly adaptable to arsenic. Some microorganisms can even oxidize As(III) to obtain energy for growth and oxidize As(III) in the environment. As (V), reducing the toxicity of arsenic. At the same time, the oxidation of arsenic driven by microorganisms can significantly increase the rate of arsenic oxidation. Therefore, microbial-driven arsenic oxidation can be used to alleviate the problem of arsenic pollution in natural habitats.
  • the oxidation process of arsenic driven by microorganisms can reduce the toxicity and migration ability of arsenic in the environment, and play an important role in the remediation of arsenic pollution.
  • the arsenic-oxidizing bacteria found in the current study mainly belong to the genus Pseudomonas
  • the purpose of the present invention is to provide a strain of Beijerinckia fluminesis AS-56, which has the function of oxidizing arsenic and can oxidize the more toxic As(III) into less toxic As(V), which greatly reduces the toxicity of arsenic in the environment, can be applied to the remediation of arsenic polluted environments such as mining areas.
  • strain AS-56 The process of screening and isolation of strain AS-56 is as follows: collect arsenic-contaminated soil from the tailings of Realgar Mine, Shimen County, Hunan province, culture in liquid medium for one week, then take the culture solution and spread it evenly on solid medium and place it in a biochemical incubator Cultivate at 30°C. After the bacteria have grown, pick out colonies of different forms to be separated by streaking, and continuously separate and purify to obtain a single colony. This colony is a high-efficiency arsenic-oxidizing bacteria with trivalent arsenic oxidation ability.
  • the strain in the single colony is rod-shaped, gram-negative, and has a size of 0.1-0.3 ⁇ 0.06-1.0 ⁇ m, and the bacteria have no motility. After identification with Biolog, it was found that in the metabolism and tolerance tests of 94 different substances, the strain can metabolize 43 carbon sources such as acetic acid, D-gluconic acid, and D-maltose. 8 kinds of substances including tetrazolium and tetrazolium show tolerance.
  • strain AS-56 of the present invention has the ability to oxidize arsenic and can oxidize As(III) to As(V);
  • the strain AS-56 of the present invention can oxidize all As(III) into As(V);
  • strain AS-56 of the present invention can be used to repair arsenic-contaminated soil, and can oxidize As(III) into As(V).
  • the present invention has the following advantages and effects:
  • the present invention has discovered a strain of Beijerinckia fluminesis AS-56 with arsenic oxidation ability, which can oxidize As(III) into As(V) with lower toxicity, and can oxidize As(III) into As(V) with lower toxicity.
  • the 0.15g/L As(III) solution is completely oxidized to As(V), which greatly reduces the toxicity of arsenic. Therefore, this strain has broad application prospects in environmental remediation.
  • Figure 1 is a scanning electron micrograph of Beijerinckia fluminesis AS-56 strain.
  • Figure 2 is the phylogenetic analysis of Beijerinckia fluminesis AS-56 strain.
  • Figure 3 is a graph showing the oxidation of As(III) by Beijerinckia fluminesis AS-56 strain.
  • the soil sample was collected from realgar tailings in Shimen County, Hunan province on January 5, 2018. 1.5g of arsenic-contaminated soil sample was taken into a 50mL Erlenmeyer flask, and 15mL liquid medium was added.
  • the specific composition is: 20g/L sucrose, 1.0g/LK 2 HPO 4 , 0.5g/L MgSO 4 , 0.5g/L NaCl, 0.1g/L FeSO 4 , 0.005g/L MoNa 2 O 4 , 2.0g/L CaCO 3 . After inoculation, it was sealed with a sterile aerobic membrane and placed in a shaker at 30°C and 150 rpm for shaking culture.
  • Example 1 The single colony obtained in Example 1 was observed by scanning electron microscopy, and the bacteria of the strain was rod-shaped and belonged to Brevibacterium, as shown in Figure 1.
  • Gram staining of the strain is as follows: drop a drop of water on a clean glass slide, use an inoculation loop to pick the bacteria and spread it evenly in the water, put the glass slide near the flame of an alcohol lamp, evaporate the water, and add oxalic acid Stain with ammonium crystal violet for 1 minute, rinse with distilled water, cover the coated surface with iodine solution and dye for about 1 minute. After washing, use absorbent paper to absorb water, add a few drops of 95% alcohol, and gently shake the glass slide to decolorize. Wash with water after 20 seconds , Absorb the water, add safranin staining solution to dye for 1 minute, rinse with distilled water, dry, and inspect under microscope. The staining result was red, and the strain was determined to be Gram-negative.
  • Biolog microbial identification steps are: after confirming that the Gram stain of the strain is negative, pick a single purified colony and inoculate it into the Biolog special inoculum GN/GP-IF to make a bacterial suspension with a certain cell concentration, and then use a pipette Transfer the bacterial suspension to the 96 wells of the Biolog GEN III microwell identification plate according to 150 ⁇ L per well, mark them and place them in the 30°C constant temperature biochemical incubator for 4-6h and 16-24h, read on the reader The metabolic fingerprint characteristics of the strain are compared in the Biolog database to find the closest result.
  • Biolog database comparison steps are: open Biolog software, set the culture time, strain name, strain number, strain type and other parameters, wipe the bottom of the cultured identification plate with a clean paper towel, and put it in the reader. A-1 hole is located at the upper left , Click “Read This” to read and get the identification result, see Table 1.
  • the bacterial genomic DNA extraction kit was used to extract DNA from the single colony obtained in Example 1, and then the 16S rRNA gene universal primer pair F27(5'agagtttgatcmtggctcag3')(SEQ.ID.NO.1) and 1492R(5'ggytaccttgttacgactt3) ') (SEQ.ID.NO.2) was amplified and sequenced, the obtained DNA sequence (SEQ.ID.NO.3) was input into GenBank, and all the sequences in the database were compared and analyzed with the Blastn program.
  • a phylogenetic tree was constructed using the 16S rRNA gene sequence (as shown in Figure 2), and it was found that the 16S rRNA and Beijerinckia fluminesis of a single colony in Example 1 clustered in a cluster, and the similarity reached 99%.
  • Example 1 Beijerinckia fluminesis (Beijerinckia fluminesis), named as Beijerinckia fluminesis AS-56.
  • Example 3 The oxidation performance of strain AS-56 to As(III)
  • Inoculation group Scrape half of the AS-56 colony on the surface of the plate in the solid medium and inoculate it into a 50mL vial, add 25mL liquid medium, the medium composition: 1.5g/L KH 2 PO 4 , 10.55g/ L Na 2 HPO 4 ⁇ 12H 2 O, 0.3g/L NH 4 Cl, 0.1g/L MgCl 2 , 0.672g/L NaHCO 3 , 0.13g/L NaAsO 2 . After inoculation, it was sealed with a rubber stopper, placed in a shaker at 30°C, and cultured with shaking at 150 rpm.
  • Non-inoculated group add 25mL liquid medium to a 50mL vial.
  • the medium composition is: 1.5g/L KH 2 PO 4 , 10.55g/L Na 2 HPO 4 ⁇ 12H 2 O, 0.3g/L NH 4 Cl , 0.1g/L MgCl 2 , 0.672g/L NaHCO 3 , 0.13g/L NaAsO 2 .
  • the As(III) concentration in the non-inoculated medium, the As(III) concentration is basically maintained at about 0.14g/L, and the presence of As(V) is not detected; while in the inoculated medium, the solution The concentration of As(III) continuously decreased from 0.15g/L to 0g/L, while the concentration of As(V) increased from 0g/L to about 0.15g/L, indicating that the arsenic oxidation reaction occurred from the access of strains.
  • the strain Beijerinckia fluminesis AS-56 is added to the medium containing arsenic, which can oxidize all As(III) to As(V).

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Abstract

提供了一株弗留明拜叶林克氏菌菌株及其在砷氧化中的应用，所述的菌株是AS-56，已于2019年12月5日保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO：M 20191014。所述弗留明拜叶林克氏菌（Beijerinckia fluminesis）菌株AS-56能把As（III）氧化为毒性较低的As（V），能将0.15g/L的As（III）溶液全部氧化成As（V），大大降低砷的毒性，因此该菌株在环境修复方面具有广泛的应用前景。

Description

一株弗留明拜叶林克氏菌菌株及其在砷氧化中的应用 技术领域
本发明属于环境微生物领域，具体涉及一株弗留明拜叶林克氏菌菌株，该菌株可氧化砷，从而降低砷对环境的不利影响。
背景技术
近年来，随着矿冶、化工、农药等行业的发展，我国湖南、广东、贵州和云南等省份的部分地区土壤砷污染十分严重。
砷是一种典型的人体致癌物，毒性较大。目前，国内外砷污染土壤的治理方法包括物理、化学和生物修复方法。电动修复技术、换土法等物理方法工程量大、耗时长、处理费用高；固化/稳定化法、土壤淋洗法等化学方法使用范围窄、局限性强，且容易引起其他环境风险；植物修复法治理周期长，植物在不同区域条件的生长差异使此技术难以形成标准化；同时，以上方法均在大面积砷污染土壤的治理中难以推广。
微生物修复是通过自然环境中的土著微生物或者人为添加的功能微生物，使污染物产生氧化还原、吸收、降解等作用，以此降低土壤中污染物毒性的方法，具有安全、高效、低成本、环境友好、能大面积应用等优点，是最具发展和应用前景的环境修复新技术。
自然环境中的砷主要以两种化合价存在：As(V)和As(III)，在大多数环境条件下，As(III)的毒性是As(V)的25～60倍。因此，如果能将毒性较强的As(III)氧化成毒性较弱的As(V)，可以减少砷的迁移性与毒性，具有实际的环境修复意义。
自然界中的微生物广泛地参与了砷的地球化学循环，微生物对砷的适应性极强，有的微生物甚至可以通过氧化As(III)来获取能量以供生长，使环境中的As(III)氧化成As(V)，减轻了砷的毒性。同时，微生物驱动的砷氧化作用，能够明显提高砷的氧化速度。因此，可以利用微生物驱动的砷氧化作用，来减轻自然生境中的砷污染问题。
微生物驱动的砷的氧化过程，能够降低砷在环境中的毒性和迁移能力，在砷污染修复方面起到重要作用。目前研究发现的砷氧化菌主要属于假单胞菌属
(Pseudomonas)、硫单胞菌属(Thiomonas)、芽孢杆菌属(Bacillus)、无色杆菌属(Achromobacter)等，至今暂未发现拜叶林克氏菌属(Beijerinckia)氧化砷的报道。
发明内容
本发明的目的在于提供一株弗留明拜叶林克氏菌(Beijerinckia fluminesis)菌株AS-56，该菌株具有氧化砷的功能，可以将毒性较强的As(III)氧化为毒性较低的As(V)，大大降低了砷在环境中的毒性，可应用于矿区等砷污染环境的修复。
本发明的目的通过下述技术方案实现：
一株弗留明拜叶林克氏菌(Beijerinckia fluminesis)菌株AS-56，已于2019年12月5日保藏于中国典型培养物保藏中心(地址：中国湖北省武汉市武汉大学内)，保藏编号为CCTCC NO：M 20191014。
菌株AS-56的筛选、分离过程为：采集湖南省石门县雄黄矿区尾矿砷污染土壤，在液体培养基中培养一周后，取培养液均匀涂布于固体培养基上，置于生化培养箱30℃培养，待菌长出后挑取不同形态的菌落采用划线法进行分离，连续分离纯化得到单一菌落，此菌落为具有三价砷氧化能力的高效砷氧化菌。
该单一菌落中的菌株为杆状，革兰氏阴性，大小为0.1-0.3×0.06-1.0μm，细菌不具有运动性。采用Biolog鉴定后发现，在94种不同物质的代谢性和耐受性测试中，该菌株能代谢乙酸、D-葡糖酸、D-麦芽糖等43种碳源，对1％NaCl、醋竹桃霉素、四唑紫等8种物质显示出耐受性。
结合16S rRNA等相关生物信息学鉴定结果，确定该菌株是弗留明拜叶林克氏菌(Beijerinckia fluminesis)，命名为弗留明拜叶林克氏菌AS-56。
利用高效液相色谱-氢化物发生-原子荧光(HPLC-HG-AFS)技术定量分析，发现本发明的菌株AS-56具有砷氧化能力，能够将As(III)氧化成As(V)；
尤其地，在含有0.15g/L As(III)的溶液中，本发明的菌株AS-56能够将其中的As(III)全部氧化为As(V)；
尤其地，本发明的菌株AS-56能够用于修复砷污染土壤，能够将其中的As(III)氧化成As(V)。
本发明相对于现有技术具有如下的优点及效果：
本发明发现了一株具有砷氧化能力的弗留明拜叶林克氏菌(Beijerinckia fluminesis)菌株AS-56，该菌株能把As(III)氧化为毒性较低的As(V)，能将0.15g/L的As(III)溶液全部氧化成As(V)，大大降低砷的毒性，因此该菌株在环境修复方面具有广泛的应用前景。
附图说明
图1是Beijerinckia fluminesis AS-56菌株的扫描电镜图。
图2是Beijerinckia fluminesis AS-56菌株的系统发育分析。
图3是Beijerinckia fluminesis AS-56菌株氧化As(III)的曲线图。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
实施例1：菌株的分离、纯化
(1)土壤样品采集于2018年1月5日湖南省石门县雄黄尾矿，取1.5g砷污染土壤样品于50mL锥形瓶中，加入15mL液体培养基，具体组成为：20g/L蔗糖，1.0g/L K 2HPO 4，0.5g/L MgSO 4，0.5g/L NaCl，0.1g/L FeSO 4，0.005g/L MoNa 2O 4，2.0g/L CaCO 3。接种后采用灭菌好氧膜封口，放置于30℃150rpm摇床中振荡培养。
(2)培养一周后，取20μL培养液均匀涂布于固体培养基上(每升培养基加15g琼脂，其它成分同上述液体培养基)，置于生化培养箱30℃培养，待菌长出后挑取不同形态的菌落采用划线法进行分离、纯化，划线接种于固体培养基，重复此步骤四次得到单一菌落，放4℃冰箱待用并用瓷珠菌种保存管保存于-80℃。
实施例2：菌株的鉴定
(1)生理生化性质的鉴定
对实施例1得到的单一菌落进行扫描电镜观察，该菌株菌体为杆状，属于短杆菌，见图1。
对该菌株做革兰氏染色，具体步骤为：在干净的载玻片上滴一滴水，用接种环挑取菌体均匀涂布于水中，将玻片靠近酒精灯火焰，蒸干水分，加入草酸 铵结晶紫染1分钟，蒸馏水冲洗，加碘液覆盖涂面染约1分钟，水洗后用吸水纸吸去水分，加95％酒精数滴，并轻轻摇动玻片进行脱色，20秒后水洗，吸去水分，加入蕃红染色液染色1分钟，蒸馏水冲洗，干燥，镜检。染色结果为呈红色，确定菌株为革兰氏阴性。
Biolog微生物鉴定步骤为：确定菌株革兰氏染色呈阴性结果后，挑取单个纯化菌落接种到Biolog专用接种液GN/GP-IF中，制成一定细胞浓度的菌悬液，然后用移液枪将菌悬液按每孔150μL转接Biolog GEN Ⅲ微孔鉴定板的96个孔内，做好标记然后放到30℃恒温生化培养箱培养4-6h和16-24h时在读数仪上读取菌株的代谢指纹特征，在Biolog数据库中进行相似性比对，查找最接近的结果。
Biolog数据库比对步骤为：打开Biolog软件，设置好培养时间、菌株名称、菌株编号、菌株类型等参数，用干净纸巾擦拭培养好的鉴定板底部，放入读数仪，A-1孔位于左上方，点击“Read This”进行读数，得出鉴定结果，见表1。
由表1可以看出，在94种不同物质的代谢性和耐受性测试中，第1-9列为代谢性试验，第10-12列为耐受性试验，结果显示，该菌株能代谢乙酸、D-葡糖酸、D-麦芽糖等43种碳源，对1％NaCl、醋竹桃霉素、四唑紫等8种物质显示出耐受性。
(2)分子生物学鉴定
采用细菌基因组DNA抽提试剂盒对实施例1得到的单一菌落进行DNA提取，然后采用16S rRNA基因通用引物对F27(5'agagtttgatcmtggctcag3')(SEQ.ID.NO.1)和1492R(5'ggytaccttgttacgactt3')(SEQ.ID.NO.2)进行扩增并测序，将获得的DNA序列(SEQ.ID.NO.3)输入GenBank，以Blastn程序对数据库中的所有序列进行比较分析。
利用16S rRNA基因序列构建了系统发育树(如图2所示)，发现实施例1单一菌落的16S rRNA与Beijerinckia fluminesis聚在一簇，相似度达到99％。
综合上述两方面的结果，确定实施例1分离得到的菌株是弗留明拜叶林克氏菌(Beijerinckia fluminesis)，命名为弗留明拜叶林克氏菌AS-56。
实施例3：菌株AS-56对As(III)的氧化性能
As(III)的氧化性能研究设置了接菌组和不接菌组，具体操作如下：
接菌组：在固体培养基中刮取半个平板表面的AS-56菌落接种到50mL西 林瓶中，加入25mL液体培养基，培养基组成为：1.5g/L KH 2PO 4，10.55g/L Na 2HPO 4·12H 2O，0.3g/L NH 4Cl，0.1g/L MgCl 2，0.672g/L NaHCO 3，0.13g/L NaAsO 2。接种后采用橡胶塞封口，置于30℃摇床中，以150rpm震荡培养。于0h，6h，12h，24h，48h分别取一次样品，并过滤保存于4℃备用；将样品稀释500倍后，用高效液相色谱-氢化物发生-原子荧光(HPLC-HG-AFS)测As(Ⅲ)和As(Ⅴ)浓度。
不接菌组：向50mL西林瓶中加入25mL液体培养基，培养基组成为：1.5g/L KH 2PO 4，10.55g/L Na 2HPO 4·12H 2O，0.3g/L NH 4Cl，0.1g/L MgCl 2，0.672g/L NaHCO 3，0.13g/L NaAsO 2。接种后采用橡胶塞封口，置于30℃摇床中，以150rpm震荡培养。于0h，6h，12h，24h，48h分别取一次样品，并过滤保存于4℃备用；将样品稀释500倍后，用高效液相色谱-氢化物发生-原子荧光(HPLC-HG-AFS)测As(Ⅲ)和As(Ⅴ)浓度。
将接菌组和不接菌组的As(Ⅲ)和As(Ⅴ)浓度绘制砷氧化曲线图，如图3所示。图3中：X轴为时间(h)，取样时间为0h，6h，12h，24h，48h；Y轴为砷浓度(g/L)。
从图3可知，在不接菌的培养基中，As(III)浓度基本维持在0.14g/L左右，也检测不到As(V)的存在；而在接菌的培养基中，溶液中As(III)浓度从0.15g/L不断下降至0g/L，而As(V)浓度则从0g/L上升至0.15g/L左右，表明砷氧化反应的发生来自于菌株的接入，在含砷的培养基中接入菌株Beijerinckia fluminesis AS-56，可以将全部的As(III)氧化为As(V)。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
Figure PCTCN2020108071-appb-000001
Figure PCTCN2020108071-appb-000002
Figure PCTCN2020108071-appb-000003
Figure PCTCN2020108071-appb-000004

Claims (4)

  1. 一株弗留明拜叶林克氏菌(Beijerinckia fluminesis)菌株AS-56，其特征在于：已于2019年12月5日保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO：M 20191014。
  2. 权利要求1所述的弗留明拜叶林克氏菌菌株AS-56在将As(III)氧化成As(V)中的应用。
  3. 根据权利要求2所述的应用，其特征在于：在含有0.15g/L As(III)的溶液中，所述的菌株AS-56将As(III)氧化为As(V)。
  4. 根据权利要求2所述的应用，其特征在于：所述的菌株AS-56用于修复砷污染土壤。
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