WO2019218412A1 - 一种原位修复受污染沉积物的固定化菌剂及制法与应用 - Google Patents

一种原位修复受污染沉积物的固定化菌剂及制法与应用 Download PDF

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WO2019218412A1
WO2019218412A1 PCT/CN2018/090736 CN2018090736W WO2019218412A1 WO 2019218412 A1 WO2019218412 A1 WO 2019218412A1 CN 2018090736 W CN2018090736 W CN 2018090736W WO 2019218412 A1 WO2019218412 A1 WO 2019218412A1
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hangjintu
microbial agent
immobilized microbial
immobilized
culture
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PCT/CN2018/090736
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French (fr)
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霍守亮
张靖天
席北斗
马春子
李小闯
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中国环境科学研究院
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Publication of WO2019218412A1 publication Critical patent/WO2019218412A1/zh
Priority to US17/039,883 priority Critical patent/US11993527B2/en

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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
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C02F3/341—Consortia of bacteria
    • 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
    • C02F11/00—Treatment of sludge; Devices therefor
    • C02F11/008—Sludge treatment by fixation or solidification
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F11/00—Treatment of sludge; Devices therefor
    • C02F11/02—Biological treatment
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
    • C02F2103/007—Contaminated open waterways, rivers, lakes or ponds

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  • the invention belongs to the field of water ecological restoration, and in particular relates to an immobilized microbial agent for repairing contaminated sediments in situ.
  • the invention also relates to a process for the preparation of the above materials.
  • the invention also relates to the use of the above materials for the in situ repair of contaminated deposits.
  • In-situ coverage and repair techniques separate the sediment from the upper body by placing one or more layers of cover on the surface of the sediment, while using the chemical properties of the filler itself to interact with contaminants in the sediment to achieve removal. Contaminants and the purpose of preventing nutrients from being released from the sediment to the water.
  • the in-situ coverage technology is mainly applied to some water bodies with low fluidity and weak hydrodynamic conditions.
  • the natural materials such as sand and gravel were used to physically isolate the contaminated sediments and water bodies, and then slowly developed into modified clay and bentonite.
  • Artificial zeolite, ash, geotextile and other materials with certain adsorption properties and low price can effectively reduce the content of organic matter in sediments.
  • the development of immobilized microbial agents has made microbial remediation of contaminated sediments a hot topic.
  • the strain is loaded with zeolite and coated with flocculant and then put into oil-contaminated waters.
  • the oil degradation efficiency of most stations is above 50% (CN106698672A); using river sand and fixed high-efficiency nitrifying bacteria and denitrifying bacteria
  • the bioactive multilayer coating synergistically with natural zeolites reduces the release of contaminants into the water body (CN102531197A).
  • the immobilized carrier used in the above technique only serves to adsorb bacteria, and cannot participate in the degradation process of microorganisms on organic substances, so that the degradation efficiency of the pollutants is limited.
  • the immobilized microbial agent for in situ remediation of contaminated sediment provided by the present invention is composed of Hangjintu 2# load-conducting microorganism, and is obtained by the following method:
  • the microbial strains are S. sulphide, Reductive genus and Shewanella.
  • the particle size of the Hangjintu 2# particulate filler is less than 0.15 mm.
  • the immobilized microbial agent 15-20 g of Hangjintu 2# is added per 30 ml of the bacterial liquid to obtain an immobilized microbial agent having a cell adsorption rate of ⁇ 90%.
  • the microbial strains are S. sulphide, Reductive genus and Shewanella.
  • the particle size of the Hangjintu 2# particulate filler is less than 0.15 mm.
  • the pretreatment method of Hangjintu 2# is: after grinding the Jinjintu 2# through a 100 mesh sieve, adding H 2 SO 4 to acidify at 90 ° C, adjusting the pH to 7.5 with an alkali solution, and removing the product by centrifugation. The supernatant was washed with water, dried at 80-90 ° C, and fired at 700-800 ° C to obtain a particulate filler having a particle diameter of less than 0.15 mm.
  • the expanded culture of the conductive microbial strain is:
  • the liquid medium is: 20g/L NaCl, 0.77g/L KCl, 0.25g/L NH 4 Cl, 0.1g/L KH 2 PO 4 , 0.2g/L MgSO 4 ⁇ 7H 2 O, 1% DL vitamin, 1 %DL mineral and 2.0g/L NaHCO 3 , the electron acceptor is 12.25g/L ferric citrate, and the electron donor is 0.6g/L acetic acid;
  • the oxygen in the liquid medium before sterilization is removed with N 2 :CO 2 in a volume ratio of 80:20, and then sterilized at 121 ° C for 15 min.
  • the immobilized microbial agent of the invention can be used for in situ repairing the contaminated sediment: the immobilized microbial agent is inoculated into the sediment at a ratio of 0.5% to 5%, and the lake water filtered through the 0.45 ⁇ m filter membrane is used, and the natural Bio-Earth Battery Effect to degrade contaminants in sediments.
  • the microorganisms can use the Fe 2 O 3 as an electron acceptor to complete the extracellular electron transfer process, accelerate the reaction rate, and effectively improve the removal effect of pollutants.
  • FIG. 1 is a process flow of an immobilized microbial agent of the present invention.
  • Figure 2 is a graph of TN removal rate versus time in sediment.
  • Figure 3 is a graph of TOC removal rate versus time in sediment.
  • the preparation method of the immobilized microbial agent of the invention comprises the following steps:
  • the microorganisms to be screened include Geobacter sulfurreducens strain DL1 (ATCC 52573), Geobacter metallireducens (ATCC 53774), and Shewanella oneidensis strain MR-1 (ATCC 700500).
  • the immobilized microbial agent to be prepared is inoculated into the sediment at a ratio of 0.5% to 5%, and the lake water filtered through the 0.45 ⁇ m filter membrane is added to decompose the contaminants in the sediment by the natural bio-terraine battery effect.
  • the used Hangjintu 2# was crushed and ground with a mortar and sieved through a 100 mesh sieve. 200 g of the ground Hangjintu 2# was weighed, 2000 ml of 2.5 mol/L H 2 SO 4 was added , and acidified in a water bath at 90 ° C for 2 h. A pH of 7.5 was adjusted by adding a 5 mol/L NaOH solution. The mixture was poured into a centrifuge tube, centrifuged at 8000 rpm for 10 min, the supernatant was decanted, washed with pure water, and centrifuged again, 3-4 times.
  • the precipitate was placed in an oven and dried at 90 ° C for 2 h, and then placed in a muffle furnace at 750 ° C for 3 h at a high temperature to finally obtain a particulate filler having a particle diameter of less than 0.15 mm of about 190 g.
  • three kinds of bacteria capable of using iron oxides, humus and the like as electron acceptors were selected from more than 30 kinds of extracellular electrogenic strains, and the selected strains included Geobacter sulfurreducens strain DL1, ATCC52573. It is labeled as T1), Geobacter metallireducens (ATCC 53774, designated as T2), and Shewanella oneidensis strain MR-1 (ATCC 700500, designated as T3).
  • the plate is pure, slanted, and expanded.
  • the colonies were picked from the plate by an inoculating loop into a 500 mL Erlenmeyer flask containing 200 mL of liquid medium, and cultured at 30 ° C, 200 r/min until the cell density reached 10 8 /mL. The above is stopped and ready for use.
  • the composition of the liquid medium is 20g/L NaCl, 0.77g/L KCl, 0.25g/L NH 4 Cl, 0.1g/L KH 2 PO 4 , 0.2g/L MgSO 4 ⁇ 7H 2 O, 1% DL vitamin, 1% DL mineral and 2.0g/L NaHCO 3 , the electron acceptor is 12.25g/L ferric citrate, the electron donor is 0.6g/L acetic acid, and the pH is adjusted between 7.0-7.5.
  • the fermenter is made of borosilicate glass, which can withstand large pressure. It is wrapped with tin foil to protect it from light.
  • the fermenter has a volume of 2.5L, and 1.5L liquid medium is added to it, and N 2 :CO 2 (80:20) is used. The volume ratio was ventilated for 15 min. After the end of the aeration, the cans were sealed with a butyl rubber stopper and sterilized at 121 ° C for 20 min.
  • the prepared Hangjin soil filler is mixed with the bacterial liquid in different proportions, and the specific ratio is shown in Table 1.
  • the mixture was shaken under anaerobic conditions for 24 hours in a shaker, and after standing for a period of time, the supernatant was decanted, and the immobilized bacteria having a cell adsorption rate of ⁇ 90% was finally obtained by screening.
  • the adsorption rate of Hangjintu 2# was the highest.
  • the adsorption rate of T1 was 93.8%
  • the adsorption rate to T2 was 92.2%
  • the adsorption rate to T3 was 91.3%.
  • the adsorption rate no longer increases.
  • the prepared immobilized microbial agent is inoculated into the sediment at a ratio of 0.5% to 5%, and the lake water filtered through the 0.45 ⁇ m filter membrane is added to degrade the pollution in the sediment by using the natural bio-terraine battery effect. Things.
  • Collect lake sediments and lake water with high nutrient content in the sediment filter the sediment with a stainless steel sieve with a pore size of 1 mm, remove coarse particles such as dead branches, leaves and gravel, and filter the lake water with 0.45 ⁇ m filter to remove the suspension.
  • Things and algae In five 7.5-liter jars (caliber 10.4 cm, height 34 cm, diameter 20.7 cm), 2.5 kg of sediment (filling height of about 8 cm) and 2.2 L of lake water (water body height of about 7 cm) were added, respectively. The climate was cultured at 30 ° C for 30 days.
  • the immobilized fungi were added to the sediments in an amount of 0.0 g, 12.5 g, 25.0 g, 50.0 g, and 125.0 g (inoculation ratios were 0.0%, 0.5%, 1%, 2%, and 5, respectively). %), stir well with a glass rod. After the end of the reaction cycle, sediments of 0-2 cm, 2-4 cm and 4 cm were collected with a sterile sampler, and the contents of TN, TP and TOC in the sediment were determined.
  • the sediments TN, TP and TOC of the five jars were between 5461-5643 mg/kg, 1560-1723 mg/kg and 15.34-17.56 g/kg, respectively, due to the bacterial liquid itself.
  • the TP content of sediments at 0-2 cm depth is between 1496-1619 mg/kg
  • the TP content of sediments at 2-4 cm depth is between 1502-1622 mg/kg
  • the TP content of sediments below 4 cm is 1500-1624 mg.
  • the TP content of sediments at different depths changes little, mainly because a small part of phosphorus is released into the overlying water by the exchange between sediment and water, in the condition of anoxic
  • the lower conductive microorganisms are capable of decomposing organic phosphorus in the sediment into inorganic phosphorus, but do not reduce the total amount of phosphorus in the sediment.
  • the TN removal rate of the blank group (0.00% inoculation ratio) was between 5.38% and 12.34%, which indicates that the microorganisms in the sediment itself can also be in hypoxia during the culture.
  • the organic nitrogen compounds in the sediment are decomposed and denitrification reaction occurs to reduce the TN content of the sediment.
  • the TN removal rate of sediment increased gradually.
  • the inoculation ratio was 5%
  • the TN content of sediment decreased the fastest.
  • the sediment TN was between 3419-4280 mg/kg, and the TN removal rate was 24.15. Between % and 39.42%.
  • the TN content of the 0-2cm sediment was the lowest, significantly lower than the TN content of the sediment below 2-4cm and 4cm, indicating that the conductive microorganisms in the sediment in the surface layer can utilize the sediment in a shorter distance.
  • the organic matter reacts with the dissolved oxygen in the overlying water to increase the degradation efficiency of the surface sediment TN.
  • the TOC content of the sediment was also reduced, showing a similar change law to the sediment TN.
  • the TOC reduction of sediment was the largest, and the reduction rate was 22.39%-35.98%, which was reduced from 17.56g/kg before inoculation to 11.24-13.63g/kg.
  • the material for in situ remediation of contaminated sediments of the present invention can effectively improve the removal effect of carbon and nitrogen in sediments, and is suitable for the repair of contaminated sediments.

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Abstract

提供了一种固定化菌剂及其制备方法和该固定化菌剂在原位修复受污染沉积物中的应用。该固定化菌剂由杭锦土2#负载导电微生物构成，通过下述方法步骤得到：1)杭锦土2#经过预处理得到颗粒态填料；2)将导电微生物菌种扩大培养至待接种菌液，按比例添加步骤1预处理的杭锦土2#，厌氧条件下混合，静置后去除上清液，得到固定化菌剂。所述导电微生物菌种为硫还原地杆菌、金属还原地杆菌和希瓦氏菌。

Description

一种原位修复受污染沉积物的固定化菌剂及制法与应用 技术领域
本发明属于水生态修复领域，具体地涉及一种原位修复受污染沉积物的固定化菌剂。
本发明还涉及上述材料的制备方法。
本发明还涉及上述材料在原位修复受污染沉积物方面的应用。
背景技术
近些年来随着国家对生态环境保护工作的重视，特别是湖长制、河长制的实施有效地推动了水域岸线管理、水污染防治、水环境治理等工作的开展。对于湖泊生态***而言，外源输入的营养物进入水体后除部分被降解和吸收外，大部分富集于沉积物中，并在适当的条件下在水体与沉积物中迁移、转化，因而湖泊的治理既要关注水环境质量的改善，也要关注沉积物中污染物的去除。国内的一些湖泊沉积物治理通常采用底泥疏浚的方式，短期内能够很好地改善水质效果，但对底泥的生境破坏较大，很难恢复。
原位覆盖和修复技术通过在底泥表面铺放一层或多层覆盖物，将底泥与上层水体隔离开来，同时利用填料本身的化学性质与底泥中的污染物相互作用，达到去除污染物并阻止营养物由底泥向水体释放的目的。
原位覆盖技术主要应用于一些流动性不大、水动力条件较弱的水体中，最初采用沙子、砂砾等天然材料物理隔离受污染沉积物和水体，后来慢慢发展到改性粘土、膨润土、人工沸石、灰渣、土工织物等具有一定吸附性能、价格低廉的材料，能够有效降低沉积物中有机物的含量。例如，以湖泊沉积物为原料，辅以沸石和聚合氯化铝改性、造粒和焙烧等过程控制沉积物内源磷负荷(CN106277673A)；或采用磁性颗粒活性炭作为覆盖材料固定沉积物中持久性有机污染物(CN105923963A)。
近年来固定化微生物菌剂的发展，使得微生物修复受污染沉积物成为 了研究热点。例如，将菌株经沸石负载并用絮凝剂包覆后投入受石油污染的海域，一个月后大部分站位石油降解效率在50％以上(CN106698672A)；采用河沙和固定高效硝化细菌和反硝化细菌、天然沸石协同作用的生物活性多层覆盖削减沉积物中污染物向水体的释放(CN102531197A)。然而，上述技术所采用的固定化载体仅起到吸附菌体的作用，并不能参与到微生物对有机物的降解过程中，使得污染物的降解效率受到限制。
2007年科学家们首次在海洋沉积物中发现了天然生物地球电池效应，即微生物在沉积物厌氧区域氧化有机碳、硫化物等电子供体，产生的电子经胞外介体“长距离”传输至好氧区，从而与上覆水中的氧气等电子受体发生空间上隔离的还原反应的过程。这一胞外电子传递过程主要是通过微生物纳米导线和细胞色素c来实现的，具有类似结构的微生物主要是地杆菌和希瓦氏菌。然而，如何将这一机理在实际水环境中进行应用，促进多种污染物的降解，是当前环境微生物和环境工程研究者面临的重要挑战。
发明内容
本发明的目的是提供一种原位修复受污染沉积物的固定化菌剂。
本发明的又一目的是提供一种上述材料的制备方法。
为实现上述目的，本发明提供的原位修复受污染沉积物的固定化菌剂，由杭锦土2#负载导电微生物构成，通过下述方法得到：
1)杭锦土2#经过预处理得到颗粒态填料；
2)将导电微生物菌种扩大培养至待接种菌液，按比例添加步骤1预处理的杭锦土2#，厌氧条件下混合，静置后去除上清液，得到固定化菌剂；所述导电微生物菌种为硫还原地杆菌、金属还原地杆菌和希瓦氏菌。
所述的固定化菌剂中，杭锦土2#颗粒填料的粒径小于0.15mm。
所述的固定化菌剂中，每30ml菌液添加15-20g杭锦土2#，得到菌体吸附率≥90％的固定化菌剂。
本发明提供的制备上述固定化菌剂的方法：
1)杭锦土2#经过预处理得到颗粒态填料；
2)将导电微生物菌种扩大培养至待接种菌液，按比例添加步骤1预处理的杭锦土2#，厌氧条件下混合，静置后去除上清液，得到固定化菌剂； 所述导电微生物菌种为硫还原地杆菌、金属还原地杆菌和希瓦氏菌。
所述的制备方法中，杭锦土2#颗粒填料的粒径小于0.15mm。
所述的制备方法中，每30ml菌液添加15-20g杭锦土2#，得到菌体吸附率≥90％的固定化菌剂。
所述的制备方法中，杭锦土2#的预处理方法是：将杭锦土2#研磨后过100目筛，加入H 2SO 4于90℃下酸化，碱溶液调节pH值至7.5，将产物离心去除上清液，水洗涤，于80-90℃烘干，700-800℃灼烧，得到粒径小于0.15mm的颗粒填料。
所述的制备方法中，导电微生物菌种的扩大培养是：
1)在厌氧的无菌环境下将硫还原地杆菌、金属还原地杆菌和希瓦氏菌三种原始菌株分别挑涂在培养基平板上，置入厌氧培养箱中于25-30℃下培养，并验证目标菌的纯度；
2)将验证后的单菌落接种到固体培养基斜面上，25-30℃培养，在厌氧的无菌环境中接种到液体培养基中，25-30℃、200r/min培养；
液体培养基为：20g/L NaCl、0.77g/L KCl、0.25g/L NH 4Cl、0.1g/L KH 2PO 4、0.2g/L MgSO 4·7H 2O、1％DL维生素、1％DL矿物和2.0g/L NaHCO 3，电子受体为12.25g/L柠檬酸铁，电子供体为0.6g/L乙酸；
3)将上述三种菌液分别以2％-6％的接种量同时接种在发酵罐中厌氧培养，培养条件为：pH值在7.0-7.5，温度为25-30℃，振荡速度为150r/min，至菌体细胞密度达到10 8个/mL以上，培养结束。
所述的制备方法中，导电微生物菌种扩大培养前，用体积比80:20的N 2:CO 2除去灭菌前液体培养基中的氧气后，再于121℃灭菌15min。
本发明的固定化菌剂可以用于原位修复受污染沉积物：将固定化菌剂以0.5％-5％的比例接种于沉积物中，加入经0.45μm滤膜过滤后的湖水，利用天然生物地球电池效应来降解沉积物中的污染物。
本发明的显著效果是：
将其应用于受污染沉积物中，能够提高沉积物中希瓦氏菌和地杆菌的丰度，强化对有机污染物的去除。同时，采用杭锦土2#作为载体，使得微生物能够利用其中的Fe 2O 3作为电子受体完成胞外电子传递过程，加快了反应速率，有效地提高了污染物的去除效果。
附图说明
图1是本发明固定化菌剂的工艺流程。
图2是沉积物中TN去除率随时间变化曲线图。
图3是沉积物中TOC去除率随时间变化曲线图。
具体实施方式
以下对本发明作详细说明。
本发明固定化菌剂的制备方法，包括如下步骤：
1)将杭锦土2#进行预处理：用研钵将杭锦土2#粗碎、研磨后直接过100目筛，以1:10(质量：体积)比例用2.5mol/L H 2SO 4在90℃下酸化2h，再加入5mol/L NaOH溶液调节pH值至7.5，将泥水混合物离心，倒去上清液，用纯水洗涤3-4次，在90℃下烘干，放入马弗炉中于750℃下高温灼烧3h后待用，最终得到粒径小于0.15mm的颗粒填料。
2)菌株的筛选：所筛选的微生物包括硫还原地杆菌(Geobacter sulfurreducens strain DL1,ATCC52573)、金属还原地杆菌(Geobacter metallireducens,ATCC53774)、希瓦氏菌(Shewanella oneidensis strain MR-1，ATCC700500)。
3)菌株的分纯和培养：在厌氧的无菌环境下将冻存的原始菌株挑涂在灭菌干燥的培养基平板上，反复划线，并置入厌氧培养箱中在30℃下培养，24h后通过观察有无杂菌，以验证目标菌的纯度。用灭菌的接种环挑取验证正确的单菌落一环，接种到固体培养基斜面上，反复划线，30℃下培养72h后备用。将上述斜面上的菌种，在厌氧的无菌环境中，接种到液体培养基中，30℃，200r/min下培养。
4)菌株的混合：将上述3种菌液分别以2％-6％的接种量同时接种在发酵罐中厌氧培养。培养条件为：pH值在7.0-7.5之间，温度为30℃，振荡速度为150r/min。至菌体细胞密度达到10 8个/mL以上，培养结束。
5)固定化菌剂的吸附：将所制得的杭锦土填料以一定比例与菌液混合，在厌氧条件下，摇床中振荡24h，静置一段时间后倒掉上清液，制得菌体吸附率≥90％的固定化菌剂。
其工艺流程可参阅图1。
本发明固定化菌剂在受污染沉积物中的应用方法：
即将制备好的固定化菌剂以0.5％-5％的比例接种于沉积物中，加入经0.45μm滤膜过滤后的湖水，利用天然生物地球电池效应来降解沉积物中的污染物。
下面结合附图和具体实施例对本发明进行详细描述，然而本发明的范围不以具体实施方式为限。
实施例1
1)杭锦土2#负载基体的制备
将购置的杭锦土2#用研钵粗碎、研磨后过100目筛，称取200g研磨后的杭锦土2#，加入2000ml 2.5mol/L H 2SO 4，在水浴锅中90℃下酸化2h，再加入5mol/L NaOH溶液调节pH值至7.5。将混合液倒入离心管中，在8000rpm下离心10min，倒去上清液，用纯水洗涤后再次离心，反复3-4次。将沉淀物放入烘箱中在90℃下烘干2h，然后放入马弗炉中于750℃下高温灼烧3h，最终得到质量约为190g的粒径小于0.15mm的颗粒填料。
2)菌株的筛选
本实施例从30余种胞外产电菌种中筛选出3种能够以铁氧化物、腐殖质等为电子受体的细菌，所选定的菌株包括硫还原地杆菌(Geobacter sulfurreducens strain DL1,ATCC52573，记为T1)、金属还原地杆菌(Geobacter metallireducens,ATCC53774，记为T2)、希瓦氏菌(Shewanella oneidensis strain MR-1，ATCC700500，记为T3)。
3)菌株的平板分纯、斜面培养、扩大培养
①在厌氧的无菌环境中，将冻存的三种原始菌株分别以灭菌接种环接种到灭菌后的LB固体培养基平板上，反复划线，在厌氧环境中30℃下培养24h，观察有无杂菌，以验证目标菌的纯度。
其中，LB固体培养基平板制备方法为：分别称取10g胰蛋白胨、5g酵母提取物、10g氯化钠搅拌溶解于1000ml纯水中，搅拌均匀后用NaOH调节pH值为7.4。取100ml LB培养基向其中加入1.5g琼脂粉，煮沸溶解后，于121℃下灭菌20min后降至60℃以下，以每15ml倒入并平铺于培养皿(d＝9cm)底部，冷却后得到固体培养基平板。
②在厌氧的无菌环境中，将验证正确的单菌落以灭菌后的接种环挑取 一环，接种到LB固体培养基斜面上，反复划线，30℃下培养72h后备用。
③在厌氧的无菌环境中，用接种环将菌落从平板上挑选至装有200mL液体培养基的500mL锥形瓶中，30℃，200r/min下培养到细胞密度达到10 8个/mL以上即停止培养，备用。
液体培养基的成分为20g/L NaCl、0.77g/L KCl、0.25g/L NH 4Cl、0.1g/L KH 2PO 4、0.2g/L MgSO 4·7H 2O、1％DL维生素、1％DL矿物和2.0g/L NaHCO 3，电子受体为12.25g/L柠檬酸铁，电子供体为0.6g/L乙酸，调节pH值在7.0-7.5之间。
4)菌株的混合：在厌氧无菌环境下，将上述3种菌液分别以2％-6％的接种量同时接种在发酵罐的液体培养基中，在水浴振荡器中扩大培养。培养条件为：温度为30℃，振荡速度为150r/min。培养至菌体细胞密度达到10 8个/mL以上。
发酵罐材质为高硼硅玻璃，能够承受较大的压力，四周用锡纸包裹以避光；发酵罐体积为2.5L，向其中加入1.5L液体培养基，并用N 2：CO 2(80:20，体积比)通气15min，通气结束后罐口用丁基橡胶塞封口，在121℃下灭菌20min。
5)固定化菌剂的吸附：
本实施例采用不同的比例将所制得的杭锦土填料与菌液混合，具体比例见表1。将混合液在厌氧条件下，摇床中振荡24h，静置一段时间后倒掉上清液，通过筛选最终制得菌体吸附率≥90％的固定化菌剂。
由表1可以看出，各组实验中，杭锦土2#对三种菌液的吸附率都类似。随着杭锦土2#添加量的增加，杭锦土2#对三种菌液的吸附率增加，其中，第7组中杭锦土2#对三种菌液的吸附率最高。即将30ml菌液与15g杭锦土2#混合，杭锦土2#占比为33.33％(菌液与杭锦土2#混合比例为2:1)时，厌氧振荡24h后，杭锦土2#的吸附率最高，对T1的吸附率为93.8％、对T2的吸附率为92.2％、对T3的吸附率为91.3％。此后杭锦土2#投加量再增大时，吸附率不再增大。
6)固定化菌剂的接种：
本实施例中将制备好的固定化菌剂以0.5％-5％的比例接种于沉积物中，加入经0.45μm滤膜过滤后的湖水，利用天然生物地球电池效应来降 解沉积物中的污染物。
采集沉积物中营养物含量较高的湖泊沉积物和湖水，将沉积物用孔径为1mm的不锈钢筛过滤，去除枯枝、树叶、碎石等粗颗粒物，将湖水用0.45μm滤膜过滤去除悬浮物和藻类。在5个7.5L的广口瓶(口径10.4cm、高度34cm、直径20.7cm)中，分别加入2.5kg沉积物(填充高度约为8cm)及2.2L湖水(水体高度约为7cm)，在人工气候室内30℃下敞口培养30d。在加入湖水前，向沉积物中分别添加质量为0.0g、12.5g、25.0g、50.0g和125.0g的固定化菌剂(接种比例分别为0.0％、0.5％、1％、2％和5％)，用玻璃棒搅拌均匀。反应周期结束后，用无菌采样器分别采集0-2cm、2-4cm及4cm以下的沉积物，测定沉积物中TN、TP、TOC含量。
结果：
如表2所示，接种初期，5个广口瓶中沉积物TN、TP和TOC分别在5461-5643mg/kg、1560-1723mg/kg和15.34-17.56g/kg之间，由于菌液本身含有一定的C、N、P，随着接种比例的增加，沉积物中TN、TP、TOC含量略有增加。
经过30d培养后，0-2cm深度沉积物TP含量在1496-1619mg/kg之间，2-4cm深度沉积物TP含量在1502-1622mg/kg之间，4cm以下深度沉积物TP含量在1500-1624mg/kg之间，与第0d相比，不同深度下沉积物TP含量变化较小，这主要是因为除少部分磷经过沉积物与水体之间的交换作用释放到上覆水体外，在缺氧条件下导电微生物能够将沉积物中有机磷分解为无机磷，但并不会削减沉积物中磷的总量。
经过30d培养后沉积物TN和TOC呈现不同程度的下降，结果分别如图2和图3所示。
如图2所示，经过30d的培养，空白组(接种比例为0.00％)沉积物TN去除率在5.38％-12.34％之间，这说明培养过程中沉积物本身具有的微生物也能够在缺氧条件下分解利用沉积物中有机氮化合物，并发生反硝化反应从而降低沉积物TN含量。随着接种比例的增加，沉积物TN去除率逐渐增大，接种比例为5％时，沉积物TN含量下降最快，30d后沉积物TN在3419-4280mg/kg之间，TN去除率在24.15％-39.42％之间。每个接种比例下，0-2cm沉积物TN含量最低，明显低于2-4cm和4cm以下沉积 物TN含量，这说明位于表层的沉积物中导电微生物能够在较短的距离内利用沉积物中有机物与上覆水中溶解氧发生氧化还原反应，从而提高了表层沉积物TN的降解效率。如图3所示，经过30d的培养，沉积物TOC含量也得到了一定的削减，呈现出与沉积物TN类似的变化规律。接种比例为5％时，沉积物TOC削减量最大，削减率为22.39％-35.98％，由接种前的17.56g/kg降至11.24-13.63g/kg。
结论：从上述实施例中可以看出，本发明的原位修复受污染沉积物的材料能够有效地提高沉积物中碳、氮的去除效果，适用于受污染沉积物的修复。
表1 杭锦土2#对混合菌液的吸附作用
Figure PCTCN2018090736-appb-000001
表2 接种前不同接种比例下沉积物中TN、TP、TOC含量
组别 接种比例 TN(mg/kg) TP(mg/kg) TOC(mg/kg)
1 0.00％ 5461 1560 15.34
2 0.50％ 5495 1585 15.75
3 1.00％ 5526 1609 16.38
4 2.00％ 5580 1646 17.05
5 5.00％ 5643 1723 17.56

Claims (10)

  1. 一种原位修复受污染沉积物的固定化菌剂，由杭锦土2#负载导电微生物构成，通过下述方法得到：
    1)杭锦土2#经过预处理得到颗粒态填料；
    2)将导电微生物菌种扩大培养至待接种菌液，按比例添加步骤1预处理的杭锦土2#，厌氧条件下混合，静置后去除上清液，得到固定化菌剂；所述导电微生物菌种为硫还原地杆菌、金属还原地杆菌和希瓦氏菌。
  2. 根据权利要求1所述的固定化菌剂，其中，杭锦土2#颗粒填料的粒径小于0.15mm。
  3. 根据权利要求1所述的固定化菌剂，其中，每30ml菌液添加15-20g杭锦土2#，得到菌体吸附率≥90％的固定化菌剂。
  4. 权利要求1所述固定化菌剂的制备方法：
    1)杭锦土2#经过预处理得到颗粒态填料；
    2)将导电微生物菌种扩大培养至待接种菌液，按比例添加步骤1预处理的杭锦土2#，厌氧条件下混合，静置后去除上清液，得到固定化菌剂；所述导电微生物菌种为硫还原地杆菌、金属还原地杆菌和希瓦氏菌。
  5. 根据权利要求4所述的制备方法，其中，杭锦土2#颗粒填料的粒径小于0.15mm。
  6. 根据权利要求4所述的制备方法，其中，每30ml菌液添加15-20g杭锦土2#，得到菌体吸附率≥90％的固定化菌剂。
  7. 根据权利要求4所述的制备方法，其中，杭锦土2#的预处理方法是：将杭锦土2#研磨后过100目筛，加入H 2SO 4于90℃下酸化，碱溶液调节pH值至7.5，将产物离心去除上清液，水洗涤，于80-90℃烘干，700-800℃灼烧，得到粒径小于0.15mm的颗粒填料。
  8. 根据权利要求4所述的制备方法，其中，导电微生物菌种的扩大培养是：
    1)在厌氧的无菌环境下将硫还原地杆菌、金属还原地杆菌和希瓦氏菌三种原始菌株分别挑涂在培养基平板上，置入厌氧培养箱中于25-30℃下培养，并验证目标菌的纯度；
    2)将验证后的单菌落接种到固体培养基斜面上，25-30℃培养，在厌氧的无菌环境中接种到液体培养基中，25-30℃、200r/min培养；
    液体培养基为：20g/L NaCl、0.77g/L KCl、0.25g/L NH 4Cl、0.1g/L KH 2PO 4、0.2g/L MgSO 4·7H 2O、1％DL维生素、1％DL矿物和2.0g/L NaHCO 3，电子受体为12.25g/L柠檬酸铁，电子供体为0.6g/L乙酸；
    3)将上述三种菌液分别以2％-6％的接种量同时接种在发酵罐中厌氧培养，培养条件为：pH值在7.0-7.5，温度为25-30℃，振荡速度为150r/min，至菌体细胞密度达到10 8个/mL以上，培养结束。
  9. 根据权利要求8所述的制备方法，其中，导电微生物菌种扩大培养前，用体积比80:20的N 2:CO 2除去灭菌前液体培养基中的氧气后，再于121℃灭菌15min。
  10. 权利要求1所述固定化菌剂在原位修复受污染沉积物的应用：
    将固定化菌剂以0.5％-5％的比例接种于沉积物中，加入经0.45μm滤膜过滤后的湖水，利用天然生物地球电池效应来降解沉积物中的污染物。
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