WO2020107823A1 - 一种细菌原位修复砷污染土壤的方法 - Google Patents

一种细菌原位修复砷污染土壤的方法 Download PDF

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WO2020107823A1
WO2020107823A1 PCT/CN2019/088224 CN2019088224W WO2020107823A1 WO 2020107823 A1 WO2020107823 A1 WO 2020107823A1 CN 2019088224 W CN2019088224 W CN 2019088224W WO 2020107823 A1 WO2020107823 A1 WO 2020107823A1
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arsenic
soil
bacteria
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王华伟
吕紫娟
王亚楠
孙英杰
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Qingdao University of Technology
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    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
    • B09C—RECLAMATION OF CONTAMINATED SOIL
    • B09C2101/00—In situ

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  • the invention belongs to soil remediation treatment technology; in particular, it relates to a method for bacteria to repair arsenic-contaminated soil in situ.
  • Arsenic is a highly toxic metal that can cause a variety of diseases and cancers, posing a serious threat to human health. Human activities such as mineral processing, fossil fuel combustion, and arsenic-containing pesticides have caused serious arsenic pollution in the soil. As arsenic can accumulate in crops such as rice, the consumption of arsenic-contaminated rice may pose serious health risks to local residents.
  • Plant remediation technology is one of the most environmentally friendly soil remediation technologies.
  • the phytoremediation technology is suitable for soils with large contaminated areas and lightly contaminated soils. It has the characteristics of low investment and long repair cycle. Studies have shown that most arsenic-enriched plants (such as centipede grass) are not suitable for growing in northern my country and are difficult to promote.
  • the microbial remediation of arsenic-contaminated soil is to change the bioavailability of arsenic through microbial redox, adsorption, methylation, precipitation and other biochemical reactions of arsenic to achieve the purpose of remediation of arsenic-contaminated soil.
  • the microbial remediation technology of arsenic contaminated soil mainly includes two types: one is biosorption technology, and the other is biooxidation reduction technology.
  • Bioremediation technology is through the fixation of the cell walls of microorganisms.
  • the cell walls of many types of microorganisms such as bacteria, algae and fungi have the ability to combine with metal ions.
  • Biological redox technology uses microorganisms to change the valence state and activity of ions in arsenic-contaminated soil to form intolerant compounds, so as to reduce the toxicity of arsenic in soil and reduce soil pollution.
  • the main problem with the above-mentioned microbial remediation technology is that the remediation capacity for arsenic is very limited, and it is extremely prone to migration. In view of this, some new technologies need to be developed to improve the remediation effect of arsenic in soil.
  • amorphous iron-manganese composite oxide can effectively fix arsenic in soil.
  • These iron-manganese composite oxides usually have the characteristics of relatively large area and strong adsorption capacity, and can effectively adsorb arsenic in soil.
  • the artificially synthesized iron-manganese composite oxide has the disadvantages of high cost and large dosage (>5%).
  • microbial-induced iron-manganese composite oxides are usually in benign environmental conditions (such as neutral pH conditions and low energy consumption), and have the advantages of low biological toxicity and low cost.
  • the manganese composite oxide induced by these microorganisms mainly exists in the arsenic pollution remediation, and its application in the in-situ remediation of arsenic-contaminated soil has not yet been seen.
  • An object of the present invention is to provide a method for bacteria to repair arsenic-contaminated soil in situ, including the following steps:
  • the selected bacterial strain was P. putida strain MnB1, and the bacteria was inoculated into the Pseudomonas putida enrichment medium at a transfer volume of 2-10% by volume, and was shaken at a level of 15-35 degrees Celsius. Under (100-180rpm), aerobic enrichment culture for 1-5 days; the medium components are yeast powder 0.3-0.8g, hydrolyzed casein 0.2-0.8g, glucose 0.3-0.8g, calcium chloride 0.1- 0.4g, magnesium sulfate 0.1-0.6g, trace elements 1-5mL, deionized water 1 liter;
  • the bacterial strain is from the American Type Bacteria Collection Center (ATCC), ATCC deposit number 23483.
  • the method is simple in process, convenient in operation, low in processing cost, large in processing range, and free from secondary pollution.
  • Figure 1 Schematic diagram of transmission electron microscope of in-situ induced iron-manganese composite oxide in Example 1;
  • Figure 2 Schematic diagram of TCLP arsenic content before and after repair in Example 1;
  • Figure 3 Schematic diagram of the morphological changes of arsenic before and after repairing arsenic in Example 1;
  • Figure 4 Schematic diagram of TCLP arsenic content before and after repair in Example 2;
  • Figure 5 Schematic diagram of the morphological changes of arsenic before and after repairing arsenic in Example 2;
  • Figure 6 Schematic diagram of the change of TCLP arsenic content before and after repair in Example 3.
  • Figure 7 Schematic diagram of the morphological changes of arsenic before and after repairing arsenic in Example 3;
  • Pseudomonas putida P. putida strain MnB1 bacterial strain was selected, and the bacteria was inoculated into the Pseudomonas putida enrichment medium at a switching volume of 2% by volume, and shaken at 30 degrees Celsius (120rpm), Aerobic enrichment culture for 3 days;
  • the medium composition is yeast extract 0.5g, hydrolyzed casein 0.5g, glucose 0.5g, calcium chloride 0.29g, magnesium sulfate 0.5g, trace elements 1mL, deionized water 1 liter;
  • step (1) Add 50mL of the Pseudomonas putida strains enriched in step (1) and 1g of manganese carbonate, 1g of ferrous ammonium sulfate, 0.5g of sodium citrate, and 0.08g of yeast extract powder to 50g of arsenic contaminated soil, add water to adjust the moisture It is 65%, stirred for 20 minutes, and then placed at room temperature (30 degrees Celsius), keeping the soil under aerobic/microaerobic conditions for 3 weeks.
  • the iron-manganese composite oxide induced by bacteria is mainly an amorphous iron-manganese mineral.
  • Figure 2 the arsenic content of solid waste in and out toxicity (TCLP) after remediation is significantly reduced, from 118.2 ⁇ g/L when unrepaired to 74.7 ⁇ g/L, and the remediation efficiency is 36.8%.
  • the results of morphological analysis of arsenic in the soil showed ( Figure 3) that the exchangeable arsenic was 4.4% when it was not repaired, and the percentage of exchangeable arsenic after repair was reduced to 0.45%, and the residue state was increased from 69.69% to 78.38. %.
  • Pseudomonas putida P. putida strain MnB1 bacterial strain was selected, and the bacteria was inoculated into the Pseudomonas putida enrichment medium at a transfer volume of 10% by volume, and shaken at 25 degrees Celsius (150rpm), Aerobic enrichment culture for 5 days;
  • the medium components are yeast extract powder 0.8g, hydrolyzed casein 0.8g, glucose 0.8g, calcium chloride 0.4g, magnesium sulfate 0.2g, trace elements 1mL, and deionized water 1L ;
  • step (1) Add 100mL of Pseudomonas putida strains enriched in step (1) and 2.5g of manganese carbonate, 2g of ferrous ammonium sulfate, 2g of sodium citrate, and 0.4g of yeast extract powder to 50g of arsenic contaminated soil, add water to adjust the water content It is 70%, stirred for 10 minutes, and then placed at room temperature (40 degrees Celsius), keeping the soil under aerobic/microaerobic conditions and cultivating for 6 weeks.
  • Pseudomonas putida P. putida strain MnB1 bacterial strain was selected, and the bacteria was inoculated into the Pseudomonas putida enrichment medium at a transfer volume of 3% by volume, and then shaken at 35 degrees Celsius (150rpm) , Aerobic enrichment culture for 2 days;
  • the medium components are 0.45g yeast extract powder, 0.45g hydrolyzed casein, 0.35g glucose, 0.2g calcium chloride, 0.3g magnesium sulfate, 1mL trace elements, deionized water 1 Rise;
  • step (1) enriched Pseudomonas putida strain and 1.5g of manganese carbonate, 1g of ferrous ammonium sulfate, 1.5g of sodium citrate, and 0.34g of yeast extract powder to 50g of arsenic contaminated soil, add water to adjust the water content The rate was 65%, stirred for 10 minutes, and then placed at room temperature (30 degrees Celsius), keeping the soil under aerobic/microaerobic conditions and cultivating for 4 weeks.
  • the method of the present invention can achieve the effect of remediation of arsenic-contaminated soil.
  • P. putida P. putida strain MnB1 oxidizes the ferric iron and manganese in the medium into high-priced biologically active iron-manganese composite oxides.
  • active iron-manganese composite oxides are A good soil remediation agent can effectively reduce the exchangeable arsenic in the soil, reduce the mobility and bioavailability of arsenic in the soil, and thus repair the arsenic in the soil in situ.
  • the method has the advantages of simple process, convenient operation, low processing cost and no secondary pollution.

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  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Health & Medical Sciences (AREA)
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  • Mycology (AREA)
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Abstract

一种细菌原位修复砷污染土壤的方法，该方法选取恶臭假单胞菌(Pseudomonas putida strain MnB1，ATCC 23483)，将该菌种加入到砷污染土壤中，在细菌的作用下该菌种会以土壤中二价铁、锰为电子供体，诱导形成活性铁锰复合氧化物同时原位修复酸化土壤中的砷。在细菌的作用下，可以将土壤中可交换态的砷转化为残渣态的砷，降低砷在土壤中的迁移和生物有效性，原位固定土壤中的砷。该工艺操作简单，处理成本低，适用范围广，无二次污染。

Description

一种细菌原位修复砷污染土壤的方法 技术领域
本发明属于土壤修复处理技术；具体涉及一种细菌原位修复砷污染土壤的方法。
技术背景
砷(As)是一种高毒类金属，会引起多种疾病和癌症，对人体健康具有严重威胁。由于矿产加工、化石燃料燃烧及含砷农药等人类活动，导致土壤严重的砷污染。由于砷可以在水稻等农作物中累积，食用砷污染的大米可能对当地居民造成严重的健康风险。
砷污染土壤修复技术主要有物理-化学修复法、植物修复法和微生物修复法三大类。传统的物理-化学修复技术通常投加量大、成本高、易产生二次污染，不适用大面积砷污染土壤的修复。
植物修复技术是最环保的土壤修复技术之一。植物修复技术适用于污染面积大，轻度污染的土壤，具有投资少、修复周比长等特点。研究表明，大多数砷富集植物(如蜈蚣草)并不适宜在我国北方生长，难以推广应用。
砷污染土壤的微生物修复作用是通过微生物对砷的氧化还原、吸附、甲基化、沉淀等生化反应来改变砷的生物有效性，来实现修复砷污染土壤的目的。砷污染土壤的微生物修复技术主要包括两种：一个是生物吸附技术，另一个是生物氧化还原技术。生物修复技术是通过微生物细胞壁的固定作用，细菌、藻类、真菌等多类微生物的细胞壁都具有与金属离子结合的能力。生物氧化还原技术是利用微生物可以改变砷污染土壤中神的离子价态和活性，使其形成不容的化合物，从而达到降低土壤中砷的毒性，减轻土壤污染的目的。上述微生物修复技术存在的主要问题是对砷修复能力十分有限，极易发生迁移。鉴于此，需要发展一些新技术提高土壤中的砷修复效果。
研究表明，无定型的铁锰复合氧化物能够有效固定土壤中砷。这些铁锰复合氧化物通常具有比较面积大、吸附能力强等特点，能够有效吸附土壤中的砷。但人工合成的铁锰复合氧化物具有成本高、投加量大(>5％)等缺点。与化学合成的铁锰复合氧化物相比，微生物诱导产生的铁锰复合氧化物通常在良性环境条件下(如中性pH条件和能源消耗低)，而且具有低生物毒性和低成本等优势。目前，这些微生物诱导产生的锰复合氧化物修复砷污染主要存在于水体，还未见其在砷污染土壤的原位修复中应用。
发明内容
本发明的目的在于，提供一种细菌原位修复砷污染土壤的方法，包括以下步骤：
1)细菌菌株的富集培养：
选用的细菌菌株为恶臭假单胞菌P.putida strain MnB1，将该细菌按体积比2-10％的转接量接种于恶臭假单胞菌富集培养基中，于15-35摄氏度水平震荡下(100-180rpm)，好氧富集培养1-5天；所述培养基成分为酵母浸粉0.3-0.8g，水解酪蛋白0.2-0.8g，葡萄糖0.3-0.8g，氯化钙0.1-0.4g，硫酸镁0.1-0.6g，微量元素1-5mL，去离子水1升；
所述细菌菌株来自于美国模式菌收集中心(ATCC)，ATCC保藏号23483。
2)修复过程
将步骤(1)富集好的恶臭假单胞菌菌株(液固比mL/g＝1:1至20:1)以及碳酸锰(质量比0.2-5％)、硫酸亚铁铵(质量比0.05-4％)、柠檬酸钠(质量比0.05-4％)、酵母浸粉(质量比0.01-2％)添加到砷污染土壤中，加水调节含水率为50-70％，搅拌5-30分钟，然后置于室温条件下(10-40摄氏度)，保持土壤为好氧/微好氧条件下培养2-6周，让细菌增长繁殖并诱导产生活性铁锰复合氧化物，同时有效固定土壤中的砷，将可交换的砷转化为残渣态而降低土壤中砷的可迁移性，实现原位修复砷污染土壤。
有益效果：
本发明通过美国模式菌种收集中心购买的能够将二价铁、锰异化氧化的恶臭假单胞菌P.putida strain MnB1，将该菌在富集培养基中富集培养，按液固比mL/g＝1:1至20:1的接种量投加到砷污染土壤中，并添加该菌株生长所必需的碳源；以二价铁、锰为电子供体，细菌在土壤中诱导产生铁锰复合氧化物，同时有效固定土壤中的砷，将土壤中可交换态的砷转化为残渣态，降低土壤中砷的可迁移性和生物有效性，从而实现原位砷污染土壤的原位修复；该方法工艺简单、操作方便、处理成本低、处理范围大、无二次污染。
附图说明
图1：实施例1原位诱导的铁锰复合氧化物透射电镜示意图；
图2：实施例1修复前后TCLP砷含量变化示意图；
图3：实施例1修复砷前后砷的存在形态变化示意图；
图4：实施例2修复前后TCLP砷含量变化示意图；
图5：实施例2修复砷前后砷的存在形态变化示意图；
图6：实施例3修复前后TCLP砷含量变化示意图；
图7：实施例3修复砷前后砷的存在形态变化示意图；
具体实施方式
以下结合实施例对本发明作进一步说明，而非限制本发明。
实施案例1
1)细菌菌株的富集培养：
选用恶臭假单胞菌P.putida strain MnB1的细菌菌株，将该细菌按体积比2％的转接量接种于恶臭假单胞菌富集培养基中，于30摄氏度水平震荡下(120rpm)，好氧富集培养3天；所述培养基成分为酵母膏0.5g，水解酪蛋白0.5g，葡萄糖0.5g，氯化钙0.29g，硫酸镁0.5g，微量元素1mL，去离子水1升；
2)修复过程
将50mL步骤(1)富集好的恶臭假单胞菌菌株以及碳酸锰1g、硫酸亚铁铵1g、柠檬酸钠0.5g、酵母浸粉0.08g添加到50g砷污染土壤中，加水调节含水率为65％，搅拌20分钟，然后置于室温条件下(30摄氏度)，保持土壤为好氧/微好氧条件下培养3周。
由附图1可知，细菌诱导的铁锰复合氧化物主要为无定型的铁锰矿物。由附图2可知，修复后固体废物进出毒性(TCLP)砷含量明显降低，由未修复时的118.2μg/L降至74.7μg/L，其修复效率为36.8％。土壤中砷的形态分析结果表明(附图3)，未修复时可交换态砷为4.4％，而修复后可交换态砷的百分比降至0.45％，残渣态由未修复的69.69％增加至78.38％。
实施案例2
1)细菌菌株的富集培养：
选用恶臭假单胞菌P.putida strain MnB1的细菌菌株，将该细菌按体积比10％的转接量接种于恶臭假单胞菌富集培养基中，于25摄氏度水平震荡下(150rpm)，好氧富集培养5天；所述培养基成分为酵母浸粉0.8g，水解酪蛋白0.8g，葡萄糖0.8g，氯化钙0.4g，硫酸镁0.2g，微量元素1mL，去离子水1升；
2)修复过程
将100mL步骤(1)富集好的恶臭假单胞菌菌株以及碳酸锰2.5g、硫酸亚铁铵2g、柠檬酸钠2g、酵母浸粉0.4g添加到50g砷污染土壤中，加水调节含水率为70％，搅拌10分钟，然后置于室温条件下(40摄氏度)，保持土壤为好氧/微好氧条件下培养6周。
由附图4可知，修复后TCLP砷含量明显降低，由未修复时的118.2μg/L降至20.2μg/L，其修复效率为82.9％。土壤中砷的形态分析结果表明(附图5)，未修复时可交换态砷为4.4％，而修复后可交换态砷的百分比降至0.15％，残渣态由未修复的69.69％增加至81.27％。
实施案例3
1)细菌菌株的富集培养：
选用恶臭假单胞菌P.putida strain MnB1的细菌菌株，将该细菌按体积比3％的转接量接种于恶臭假单胞菌富集培养基中，然后于35摄氏度水平震荡下(150rpm)，好氧富集培养2天；所述培养基成分为酵母浸粉0.45g，水解酪蛋白0.45g，葡萄糖0.35g，氯化钙0.2g，硫酸镁0.3g，微量元素1mL，去离子水1升；
2)修复过程
将65mL步骤(1)富集好的恶臭假单胞菌菌株以及碳酸锰1.5g、硫酸亚铁铵1g、柠檬酸钠1.5g、酵母浸粉0.34g添加到50g砷污染土壤中，加水调节含水率为65％，搅拌10分钟，然后置于室温条件下(30摄氏度)，保持土壤为好氧/微好氧条件下培养4周。
由附图6可知，修复后TCLP砷含量明显降低，由未修复时的118.2μg/L降至60.2μg/L，其修复效率为49.1％。土壤中砷的形态分析结果表明(附图7)，未修复时可交换态砷为4.4％，而修复后可交换态砷的百分比降至0.24％，残渣态由未修复的69.69％增加至80.34％。
从上述实施例可以看出：通过本发明所述方法可达到修复砷污染土壤的效果。在好氧/微好氧条件下，恶臭假单胞菌P.putida strain MnB1将培养基中二价铁、锰氧化为高价的生物活性铁锰复合氧化物，这些活性的铁锰复合氧化物是一种良好的土壤修复剂，能够有效降低土壤中可交换态的砷，降低土壤中砷的可迁移性和生物有效性，从而原位修复土壤中的砷。该方法工艺简单、操作方便、处理成本低、无二次污染等优点。

Claims (3)

  1. 一种细菌原位修复砷污染土壤的方法，其特征在于；包括以下步骤：
    1)细菌菌株的富集培养：
    选用的细菌菌株为恶臭假单胞菌P.putida strain MnB1，将该细菌按体积比2-10％的转接量接种于恶臭假单胞菌富集培养基中，于15-35摄氏度水平震荡下(100-180rpm)，好氧富集培养1-5天；
    2)修复过程
    将步骤(1)富集好的恶臭假单胞菌菌株(液固比mL/g＝1:1至20:1)以及碳酸锰(质量比0.2-5％)、硫酸亚铁铵(质量比0.05-4％)、柠檬酸钠(质量比0.05-4％)、酵母浸粉(质量比0.01-2％)等添加到砷污染土壤中，加水调节含水率为50-70％，搅拌5-30分钟，然后置于室温条件下(10-40摄氏度)，保持土壤为好氧/微好氧条件下培养2-6周，让细菌增长繁殖并诱导产生活性铁锰复合氧化物，同时有效固定土壤中的砷，将可交换的砷转化为残渣态。
  2. 如权利要求1所述的细菌原位修复砷污染土壤的方法，其特征在于；所述细菌菌株来自于美国模式菌收集中心(ATCC)，ATCC保藏号23483。
  3. 如权利要求1所述的细菌原位修复砷污染土壤的方法，其特征在于；步骤1)中的培养基成分为酵母浸粉0.3-0.8g，水解酪蛋白0.2-0.8g，葡萄糖0.3-0.8g，氯化钙0.1-0.4g，硫酸镁0.1-0.6g，微量元素1-5mL，去离子水1升。
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