WO2021077531A1 - 一种高效石油降解菌tdyn1t及其应用 - Google Patents

一种高效石油降解菌tdyn1t及其应用 Download PDF

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WO2021077531A1
WO2021077531A1 PCT/CN2019/120938 CN2019120938W WO2021077531A1 WO 2021077531 A1 WO2021077531 A1 WO 2021077531A1 CN 2019120938 W CN2019120938 W CN 2019120938W WO 2021077531 A1 WO2021077531 A1 WO 2021077531A1
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petroleum
tdyn1t
bacteria
soil
efficiency
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French (fr)
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钟磊
田静楠
陈冠益
田姝
孙于茹
武文竹
马文超
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天津大学
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Priority to AU2019460845A priority Critical patent/AU2019460845B2/en
Priority to KR1020217001927A priority patent/KR102525855B1/ko
Priority to US17/285,060 priority patent/US20230286026A1/en
Priority to EP19940165.4A priority patent/EP4050094B1/en
Publication of WO2021077531A1 publication Critical patent/WO2021077531A1/zh

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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
    • 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
    • B09B—DISPOSAL OF SOLID WASTE NOT OTHERWISE PROVIDED FOR
    • B09B3/00—Destroying solid waste or transforming solid waste into something useful or harmless
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
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    • C12N2523/00—Culture process characterised by temperature
    • 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

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  • the invention belongs to the technical field of microbial remediation, and relates to a high-efficiency petroleum degrading bacterium TDYN1T and its application.
  • Petroleum is one of the most important energy sources for centuries, and its consumption is increasing with the development of society and the improvement of people's living standards, and the scale of exploitation continues to expand.
  • oil spills often occur, causing serious harm to the ecological environment and human health.
  • the ground crude oil produced in the process of oil extraction has become the main source of soil pollution.
  • the world's total oil production is about 3 ⁇ 10 10 tons per year, and about 8 million tons of petroleum pollutants enter the environment, most of which enter the soil.
  • the hazards of petroleum hydrocarbons entering the soil are mainly in three aspects: 1) A large amount of petroleum sludge is produced, which has stable physical and chemical properties, is difficult to remove and has a long residual time, changes the physical and chemical properties of the soil and the structure of organic matter, and destroys the soil structure and soil microbes. The living environment of plants; 2) Some highly mobile petroleum hydrocarbons (such as benzene, toluene, xylene, etc.) enter the soil and reach the underground aquifer with soil moisture, thereby polluting the groundwater; 3) Some highly volatile petroleum hydrocarbons enter the soil The soil will volatilize and diffuse into the air, which will affect the air quality; in turn, it will affect human health through the soil, water and atmospheric environment.
  • the current methods of dealing with petroleum-contaminated soil mainly include physical, chemical and bioremediation technologies.
  • the physical remediation technology is mainly to achieve the purpose of soil remediation through the physical migration of petroleum hydrocarbon pollutants, and is widely used in heavily polluted soils such as oil sludge; Wang Zhenguo and others have applied gas phase extraction to achieve a petroleum hydrocarbon removal rate of 88%, and its remediation effect is good and There is no secondary pollution, but its repair cost is high and it is mainly suitable for heavily polluted soil.
  • the chemical remediation technology is to inject chemical oxidants into the soil and use its oxidative properties to remediate petroleum-contaminated soil; Lu et al.
  • H2O2 and Fe3+-EDTA complexes to treat petroleum-contaminated soil to reduce the oil mass ratio in the soil from 14800mg/kg to 2300mg /kg, the repair effect is good and the cost is low, but chemical agents are prone to secondary pollution to the soil.
  • the degradation rate of existing petroleum degrading bacteria is mostly between 60% and 80%, and the low repair efficiency has become the technical bottleneck for the further development and application of this technology; therefore, the discovery of new and efficient petroleum hydrocarbon degrading bacteria is important for improving microbial soil remediation. The prospects are significant.
  • the purpose of the present invention is to overcome the insufficient degradation rate of petroleum degrading bacteria in the prior art, and provide a high-efficiency petroleum degrading bacteria TDYN1T.
  • the second object of the present invention is to provide an application of high-efficiency petroleum degrading bacteria TDYN1T.
  • a high-efficiency oil-degrading bacterium TDYN1T its classification and name is Falsochrobactrum sp. It is deposited in the General Microbiology Center of China Microbial Culture Collection Management Committee, and the preservation number is CGMCC No.18061.
  • the high-efficiency petroleum-degrading bacterium TDYN1T of the present invention is used in petroleum-containing soil to degrade, and the effect is good, especially when the petroleum is degraded under the conditions of 30 ⁇ 1°C and pH 7-7.4, the degradation efficiency is high.
  • Figure 1 shows the morphology of the strain.
  • Figure 2 is a scanning electron micrograph of the strain.
  • Figure 3 shows the degradation curve of strain Falsochrobactrum sp. TDYN1T with temperature.
  • Figure 4 shows the degradation curve of strain Falsochrobactrum sp. TDYN1T over time.
  • Figure 5 shows the degradation curve of strain Falsochrobactrum sp. TDYN1T with pH.
  • Figure 6 shows the petroleum standard curve in the petroleum degradation performance test experiment.
  • a sample of oily soil was taken from somewhere in Tianjin Dagang Oilfield, stored at 4°C, and transported back to the laboratory.
  • Inorganic salt medium KH 2 PO 4 1g, K 2 HPO 4 0.5g, NaCl 10g, (NH 4 ) 2 SO 4 1.5g, anhydrous CaCl 2 0.1g, FeSO 4 ⁇ 7H 2 O 0.01g, MgSO 4 0.2 g. Add distilled water to 1000 mL, pH 7.0-7.4, and sterilize at 121°C for 20 minutes.
  • LB (Luria-Bertani) liquid medium beef extract 3g, peptone 10g, NaCl 5g, add distilled water to 1000mL, pH 7.0-7.4, and sterilize at 121°C for 20 minutes.
  • Slant storage medium and LB solid plate medium beef extract 5g, peptone 10g, NaCl 5g, agar 20g, distilled and distilled water to 1000mL, pH 7.0-7.4, sterilized at 121°C for 20 minutes, and taken out at 70-80°C.
  • Material crude oil, petroleum ether (extractant), LB liquid medium, inorganic salt medium
  • the process is carried out in a sterile environment.
  • the inoculated solution is heated at 10°C, 15°C, 20°C, 25°C, 30°C, respectively.
  • the process is carried out in a sterile environment.
  • the inoculated solution is shaken at a constant temperature of 180 rpm at 30°C for 5d, 10d, 15d. , 20d, 25d, 30d. Set up three sets of repeatability tests at the same time.
  • the process is all in In a sterile environment, the inoculated solution was shaken at a constant temperature of 180 rpm at 30°C for 7 days. Set up three sets of repeatability tests at the same time.
  • the degraded solution was extracted twice with 10 mL petroleum ether. The total supernatant was taken to measure the absorbance and compared with the standard curve to determine the degradation rate.
  • the 16S rDNA gene sequence analysis method was used to identify the genus and species of the isolated and screened Falsochrobactrum sp. strain TDYN1T.
  • the 16S rDNA gene sequence (SEQ ID NO.1) has 98% homology with the Falsochrobactrum sp. strain HN4 sequence in the NCBI database.
  • the General Microbiology Center of the Chinese Microbial Culture Collection and Management Committee is entrusted to conduct biochemical identification, and its biochemical characteristics are similar to those of Falsochrobactrum sp.
  • the .type strain HN4 is more consistent. Comprehensive molecular biology and biochemical identification results, Falsochrobactrum sp. TDYN1T was identified as Falsochrobactrum sp.
  • the present invention uses traditional methods to isolate and domesticate petroleum-degrading bacteria.
  • the high-efficiency petroleum-degrading bacteria TDYN1T screened from the soil of Tianjin Dagang Oilfield is in the form of short rods. After 7 days of petroleum degradation, the degradation rate is as high as 90%.
  • the high-efficiency oil-degrading bacteria TDYN1T strain is classified and named Falsochrobactrum sp. It is non-sporogenic, Gram-negative, and Corynebacterium.
  • Colony characteristics inoculated on LB solid plate medium and cultured at 30°C for 24 hours.
  • the colony is milky white, smooth, flat, and has neat edges; it is gram-negative, and the bacteria are short rod-shaped. See Figure 1 and Figure 2.
  • Oxidase activity Put a piece of filter paper on a clean petri dish, drop an equal amount of 1% dimethyl p-phenylenediamine hydrochloride aqueous solution and 1% a-phenolphthalein alcohol (95%) solution (mix) . The dripping amount is appropriate to make the filter paper wet.
  • Use a platinum wire inoculation loop nickel-chromium wire is not available) to pick a small amount of the lawn that has been cultured for 18-24 hours, and smear it on the filter paper. (The mushroom moss smeared within 10s is rose red or blue as positive; 10s-60s color development is a delayed reaction; after 60s, color development is still treated as negative).
  • Sugar fermentation test According to the difference in the ability of bacteria to decompose and utilize sugar, it shows whether to produce acid and gas as the basis for identification.
  • the specific operation is to add the indicator bromomethyl violet (B.C.P indicator, the pH is below 5.2 to yellow, and the pH is above 6.8 to purple), which can be judged according to the color change of the indicator after culturing. Whether to produce gas or not, you can put an inverted Dulbecco tube in the fermentation medium to observe.
  • Test tube marking blank control is marked for each sugar fermentation.
  • Inoculation culture Aseptically inoculate a small amount of bacterial lawn to each corresponding test tube, and the blank control of each sugar fermentation is not inoculated with bacteria. Put the Dulbecco tube upside down into the test tube, place it in a constant temperature at 30°C, and observe the results at 24h, 36h and 72h respectively.
  • the bacteria can use glucose and lactose, but both produce acid but not gas.
  • Reagent preparation Dissolve 20g potassium iodide in 50ml water, and add mercury iodide pellets to this solution until the solution is saturated (about 32g), then add 460ml water and 134g potassium hydroxide, and store the supernatant in a dark bottle In the spare.
  • Inoculation Inoculate with fresh strains and cultivate at suitable temperature for 1, 3, 5 days. 4) Observation of results: Take a small amount of culture medium, add a few drops of reagent, and a yellowish-brown precipitate is positive.
  • indole medium peptone 10g, NaCl 5g, add distilled water to 1000ml, pH 7.2-7.4, and sterilize with moist heat at 121°C for 20 min.
  • Test tube labeling Put the indole medium into the corresponding test tube and mark the blank control.
  • Inoculation and culture Use aseptic technique to inoculate a small amount of bacterial lawn into the corresponding test tube, leave a tube as a blank control without inoculation, and place it in a 30°C incubator for 24-48 hours.
  • the ether presents a rose red color, which is a positive reaction in the indole test, and the record is indicated by "+”; if there is no indole present, the ether presents the original color, which is a negative reaction in the indole test, and the record is indicated by "-" .
  • the high oil decomposing bacteria TDYN1T Accession No. CGMCC No.18061 logarithmic growth phase of cell suspension After fixing the toner obtained grass density of 10 8 bacteria / g repair agents, repair agents and the oily soil 10kg ( Tianjin Dagang Oilfield soil) mixed with 3% addition mass, reacted for 10 days under the conditions of 30°C and pH 7.2, and the oil degradation efficiency was 81%.
  • the high oil decomposing bacteria TDYN1T Accession No. CGMCC No.18061 logarithmic growth phase of cell suspension After fixing the toner obtained grass density of 10 8 bacteria / g repair agents, repair agents and the oily soil 10kg ( Tianjin Dagang Oilfield soil) mixed with 3% added mass, reacted for 15 days under the conditions of 29°C and pH 7, and the oil degradation efficiency is 85%.

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Abstract

提供了一种石油降解菌（Falsochrobactrum sp.）TDYN1T及其应用，该菌株保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号：CGMCC No.18061。所述菌株可以降解土壤中的石油，特别是在30±1℃，pH 7-7.4的条件下，降解效率较高。

Description

一种高效石油降解菌TDYN1T及其应用 技术领域
本发明属于微生物修复技术领域，涉及一种高效石油降解菌TDYN1T及其应用。
背景技术
石油是人类最主要的能源之一，其消耗量随着社会的发展与人们生活水平的提高与日俱增，开采规模不断扩大。但是在石油的开采、运输和使用过程中，频繁漏油事故经常发生，给生态环境和人类健康造成了严重危害。特别是石油开采过程中产生的落地原油，已成为土壤污染的主要来源。据统计，2005年至今，每年世界石油总产量约3×10 10吨，约有800万吨的石油污染物进入环境，大部分进入到了土壤中。
石油烃进入土壤后的危害主要在三方面：1)产生大量的石油污泥，其物理化学性质稳定、难以去除且残留时间长，改变土壤的理化性质和有机质结构，破坏土壤结构及土壤微生物和植物的生存环境；2)部分迁移性强的石油烃(如苯、甲苯、二甲苯等)进入土壤后会随土壤水分到达地下含水层，从而污染地下水；3)部分挥发性强的石油烃进入土壤后会向空气挥发扩散，从而影响空气质量；进而通过土壤、水和大气环境影响人体健康。
当前处理石油污染土壤的方法主要有物理、化学和生物修复技术。物理修复技术主要是通过对石油烃污染物的物理迁移达到土壤修复目的，在油泥类等重度污染土壤应用广泛；王贞国等应用气相抽提法使石油烃去除率达到88％，其修复效果良好且无二次污染，但其修复成本较高且主要适用于重度污染土壤。化学修复技术则是通过向土壤中注入化学氧化剂，利用其氧化性修复石油污染土壤；Lu等利用H2O2和Fe3+-EDTA复合体对石油污染土壤进行处理使土壤中油质量比由14800mg/kg降低到2300mg/kg，修复效果好且成本较低，但化学药剂容易对土壤产生二次污染。
近年来，随着微生物技术的发展，利用原位土壤筛选高效降解菌株修复石油烃污染技术逐渐兴起，因其修复效率高，成本低廉和环境友好的特点成为未来土壤修复行业的发展趋势。已知降解石油的微生物共有70属200余种，其中细菌28个属包括假单胞菌属、不动杆菌属、无色杆菌属等，霉菌30个属包括青霉菌、曲霉菌、镰孢霉菌等，酵母菌12个属包括假丝酵母属、红酵母属等。目前，已有石油降解菌的降解率大多在60％-80％之间，修复效率不高成为该技术进一步发展和应用的技术瓶颈；因此挖掘新型高效石油烃降解菌，对于提升微生物土壤修复的前景意义重大。
发明内容
本发明的目的是克服现有技术存在的石油降解菌降解率不高的不足，提供一种高效石油降解菌TDYN1T。
本发明的第二个目的是提供一种高效石油降解菌TDYN1T的应用。
本发明的技术方案概述如下：
一种高效石油降解菌TDYN1T，其分类命名为Falsochrobactrum sp.保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号CGMCC No.18061。
上述一种高效石油降解菌TDYN1T降解土壤中石油的应用。
上述应用优选：将高效石油降解菌TDYN1T保藏编号CGMCC No.18061对数生长期的菌液用草碳粉固定后得到菌密度为10 8个/g的修复菌剂,将所述修复菌剂与10kg含油土壤混合，添加质量为3％，在30±1℃，pH 7-7.4条件下反应。
本发明的优点：
含有石油土壤中采用本发明的一种高效石油降解菌TDYN1T降解，效果较好，特别在30±1℃，pH 7-7.4条件下石油降解，降解效率高。
附图说明
图1为菌株形态图。
图2为菌株扫描电镜照片。
图3为菌株Falsochrobactrum sp.TDYN1T随温度的降解曲线。
图4为菌株Falsochrobactrum sp.TDYN1T随时间的降解曲线。
图5为菌株Falsochrobactrum sp.TDYN1T随pH的降解曲线。
图6为石油降解性能测试实验中的石油标准曲线。
具体实施方式
针对天津市含油土壤污染情况，从天津大港油田土壤筛选出的一株高效石油降解菌，并进一步研究其降解能力，以期应用于石油污染土壤。
下面通过具体实施方案叙述本发明方法。除非特别说明，本发明中所用的技术手段均为本领域技术人员所公知的方法。另外，实施方案应理解为说明性的，而非限制本发明的范围，本发明的实质和范围仅由权利要求书所限定。对于本领域技术人员而言，在不背离本发明实质和范围的前提下，对这些实施方案中的各种改变或改动也属于本发明的保护范围。
实施例1
1.材料准备
从天津大港油田某处取含油土壤样品，4℃保存，运回实验室。
2.实验用培养基
无机盐培养基：KH 2PO 4 1g、K 2HPO 4 0.5g、NaCl 10g、(NH 4) 2SO 4 1.5g、无水CaCl 2 0.1g、FeSO 4·7H 2O 0.01g、MgSO 4 0.2g、加蒸馏水至1000mL，pH 7.0-7.4，121℃灭菌20min。
LB(Luria-Bertani)液体培养基：牛肉膏3g、蛋白胨10g、NaCl 5g、加蒸馏水至1000mL，pH 7.0-7.4，121℃灭菌20min。
斜面保存培养基和LB固体平板培养基：牛肉膏5g、蛋白胨10g、NaCl 5g、琼脂20g、蒸馏加蒸馏水至1000mL，pH 7.0-7.4，121℃灭菌20min，70-80℃取出。
3.实验仪器和设备
恒温振荡器
电热恒温培养箱
高压灭菌锅
紫外可见分光光度计
超净工作台
4.菌株的分离筛选与驯化
(1)取步骤1的含油土壤10g放入装有100mL LB液体培养基的锥形瓶中，在30±1℃、180rmp的恒温要床上进行震荡，富集培养24h；
(2)24h后取2mL培养液用涂布棒涂布到LB固体平板培养基上，放入恒温箱中30±1℃倒置培养7d；
(3)待菌落长出后，观察菌落形态，用无菌接种环从上述LB固体平板培养基中挑选形态差距大的菌落，采用平板划线分离法接种于LB固体平板培养基上，置于恒温培养箱中30±1℃倒置培养24h，待菌落长出后挑取单菌落，并以同样的方法接种至LB固体平板培养基。重复上述划线分离过程，直至形成形态单一的纯化菌落。将单菌落在斜面保存培养基上4℃保存备用。使用前需在LB液体培养基中活化。
(4)对上述纯化菌落分别进行石油降解试验，取3ml菌液接种在含有0.1g原油的100ml无机盐培养基中，在30℃、180rmp、pH 7.2条件下降解7天，用石油醚萃取剩余原油，取上清液稀释，紫外分光光度法测定其吸光度，对照标准曲线(见步骤5)判断菌株降解率。实验过程挑选出一株高效石油降解菌Falsochrobactrum sp.TDYN1T并对其进行降解条件测定。详细参考步骤5。
5.高效石油降解菌Falsochrobactrum sp.TDYN1T石油降解性能测试实验
(1)材料：原油，石油醚(萃取剂)，LB液体培养基，无机盐培养基
(2)方法：紫外分光光度法
(3)步骤：
1)石油标准曲线的配制
将0.1g石油用石油醚溶液定容到100mL容量瓶中，经多次稀释配置成25mg/L的石油-石油醚溶液，在此浓度下确定最大吸收波长256nm。同时配置浓度为10mg/L、20mg/L、30mg/L、40mg/L、50mg/L、60mg/L、70mg/L、80mg/L、90mg/L、100mg/L的石油-石油醚标准溶液，并在最大吸收波长处测定吸光度同时绘制标准曲线。(见图6)
2)石油降解
i.菌株在不同温度下降解效果
将活化后的菌液3mL接种到含0.1g石油的100mL无机盐培养基中，过程均在无菌环境下进行，将接种后的溶液分别在10℃、15℃、20℃、25℃、30℃、35℃、40℃、45℃、50℃条件下180rmp恒温振荡7d。同时设置三组重复性试验。
Figure PCTCN2019120938-appb-000001
见图3。
ii.菌株在不同时间下降解效果
将活化后的石油降解菌接种3mL到含0.1g石油的100mL无机盐培养基中，过程均在无菌环境下进行，将接种后的溶液分别在30℃条件下180rmp恒温振荡5d、10d、15d、20d、25d、30d。同时设置三组重复性试验。
Figure PCTCN2019120938-appb-000002
见图4。
iii.将石油降解菌活化后接种3mL到含0.1g石油的100mL无机盐培养基中，无机盐培养基在灭菌前调pH分别为1、3、5、7、9、11，过程均在无菌环境下进行，将接种后的溶液180rmp、30℃恒温振荡7天。同时设置三组重复性试验。
pH 1 3 5 7 9 11
降解率(％) 2 4 20 90 70 50
见图5。
石油降解率的测定
降解后的溶液用10mL石油醚萃取重复两次，取总上清液测定吸光度并对比标准曲线确定降解率。
6.菌株形态图，见图1。
7.菌株的扫描电镜观察，见图2。
8.菌株的鉴定
采用16S rDNA基因序列分析方法，对分离筛选的Falsochrobactrum sp.型菌株TDYN1T进行属种鉴定。16S rDNA基因序列(SEQ ID NO.1)与NCBI数据库中Falsochrobactrum sp.型菌株HN4序列同源性达98％，委托中国微生物菌种保藏管理委员会普通微生物中心进行生化鉴定，其生化特征与Falsochrobactrum sp.型菌株HN4较为一致。综合分子生物学及生化鉴定结果，Falsochrobactrum sp.TDYN1T鉴定为Falsochrobactrum sp.
Falsochrobactrum sp.TDYN1T基因全序列：(SEQ ID NO.1)
Figure PCTCN2019120938-appb-000003
9.结论：本发明采用传统方法分离驯化石油降解菌，从天津大港油田土壤筛选出的高效石油降解菌TDYN1T，呈短棒状，经7天石油降解，降解率高达90％以上。
10.菌株的保存：
高效石油降解菌TDYN1T菌株分类命名Falsochrobactrum sp.为非孢子形成、革兰氏阴性、短棒菌。
保藏日期：2019年7月2日，
保藏单位：保藏于中国微生物菌种保藏管理委员会普通微生物中心，
保藏地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所。
保藏编号：CGMCC No.18061。并存活。
实施例2
高效石油降解菌TDYN1T的特点：
1.菌落特征：接种于LB固体平板培养基上30℃培养24h，菌落乳白色，光滑，平坦，边缘整齐；革兰氏阴性，菌体呈短棒状。见图1、图2。
2.氧化酶活性：在洁净的培养皿上放上一张滤纸，滴上等量的1％盐酸二甲基对苯二胺水溶液和1％a-酚酞酒精(95％)溶液(混匀)。滴入量以使滤纸湿润为适宜，用白 金丝接种环(不可用镍铬丝)挑取少量培养了18-24h的菌苔，涂抹于滤纸上。(在10s内涂抹的菌苔呈现玫瑰红或者蓝色为阳性；10s-60s显色为延迟反应；在60s后显色仍按阴性处理)。
试验观察：在15s出现显色反应，呈阳性反应。
3.过氧化氢酶活性：
1)试剂配制：3％-10％过氧化氢。
2)接种与结果观察：将24h培养的斜面菌种，以铂丝接种环取一小环涂抹于已滴有3％过氧化氢的载玻片上，静置1-3min观察，有气泡产生，如有气泡产生则为阳性，无气泡为阴性。试验观察：有气泡产生，呈阳性反应。
4.糖发酵试验：根据细菌分解利用糖能力的差异表现出是否产酸产气作鉴定的依据。具体操作是可在糖发酵培养基中加入指示剂溴甲基紫(B.C.P指示剂，其pH在5.2以下呈黄色，pH在6.8以上呈紫色)经过培养后根据指示剂的颜色变化来判断。是否产气，可在发酵培养基中放入倒置杜氏小管观察。
1)糖发酵培养基的制作：称取蛋白胨2g和NaCl 5g溶解于热水中，调节pH至7.4，加入K 2HPO 4 0.2g，加蒸馏水至1000ml，再加溴麝香草酚蓝1％水溶液3ml(先用少量95％乙醇溶解后，再加水配成1％水溶液)。取两份上述制备溶液分别加入葡萄糖和乳糖，分装试管，装量4-5cm高，并倒放入一杜氏小管(管口向下，管内充满培养液)，115℃条件下湿热灭菌20min。灭菌时注意适当延长煮沸时间，尽量把冷空气排尽以使杜氏小管内不残存气泡。
2)试管标记：每种糖发酵均标记空白对照。
3)接种培养：无菌操作分别接种少量菌苔至各相应试管，每种糖发酵的空白对照均不接种细菌。将杜氏小管倒置放入试管中，置30℃恒温培养，分别在24h，36h和72h观察结果。
4)观察记录：与对照管比较，若接种管培养液保持原有颜色，其反应结果为阴性，表明该细菌不能利用该种糖，记录用“-”表示；如果培养液成黄色，反应结果为阳性，表明该菌能够分解该种糖类产酸，记录用“+”表示。培养液中的杜氏小管内有气泡为阳性反应，表明该细菌能分解该种糖类产酸并产气，记录用“+”表示，如杜氏小管内没有气泡为阴性反应，记录用“-”表示。
试验观察：该细菌能利用葡萄糖及乳糖，但均产酸不产气。
5.产氨试验：
1)产氨培养基的制作：蛋白胨5g、加蒸馏水至1000ml、pH 7.2，分装试管，121℃灭菌15-20min。
2)试剂配制：将20g碘化钾溶于50ml水中，并在此溶液中加碘化汞小粒至溶饱和为止(约32g)此后再加460ml水和134g氢氧化钾，将上清液贮于暗色瓶中备用。
3)接种：以新鲜菌种接种，置适温培养1，3，5天。4)结果观察：取培养液少 许，加入试剂数滴，出现黄褐色沉淀为阳性。
试验观察：出现黄褐色沉淀，呈阳性反应。
6.吲哚试验：
1)吲哚培养基的制作：蛋白胨10g，NaCl 5g，加蒸馏水至1000ml，pH7.2-7.4，121℃湿热灭菌20min。
2)吲哚试剂配制:对二甲苯氨基苯甲醛8g、乙醇(95％)760ml、浓HCl 160ml。
3)试管标记:将吲哚培养基装入相应试管，并标记空白对照。
4)接种培养：以无菌操作技术分别接种少量菌苔到相应试管中，留一管做空白对照不接种，置30℃恒温箱中培养24-48小时。
5)观察记录：取出培养液，沿管壁缓缓加入3-5ml的吲哚试剂于培养液表面，在液层界面发生红色，即为阳性反应。若颜色不明显，可加4-5滴乙醚至培养液，摇动，使乙醚分散于液体中，将培养液静置片刻，待乙醚浮至液面后再加吲哚试剂。如培养液中有吲哚时，吲哚可被提取在乙醚层中，浓缩的吲哚和试剂反应，则颜色明显。如有吲哚存在，乙醚呈现玫瑰红色，此为吲哚试验阳性反应，记录用“+”表示；如没有吲哚存在，乙醚呈现原色，此为吲哚试验阴性反应，记录用“-”表示。
试验观察：在界面发生红色，呈阳性反应。
总结高效降解菌TDYN1T特点如下表：
Figure PCTCN2019120938-appb-000004
实施例3
高效石油降解菌TDYN1T降解土壤中石油的应用
将高效石油降解菌TDYN1T保藏编号CGMCC No.18061对数生长期的菌液用草碳粉固定后得到菌密度为10 8个/g的修复菌剂,将所述修复菌剂与10kg含油土壤(天津大港油田土壤)混合，添加质量为3％，在30℃，pH 7.2条件下反应10天，石油降解效率为81％。
实施例4
高效石油降解菌TDYN1T降解土壤中石油的应用
将高效石油降解菌TDYN1T保藏编号CGMCC No.18061对数生长期的菌液用草碳粉固定后得到菌密度为10 8个/g的修复菌剂,将所述修复菌剂与10kg含油土壤(天津大港油田土壤)混合，添加质量为3％，在29℃，pH 7条件下反应15天，石油降解效率为85％。
实施例5
高效石油降解菌TDYN1T降解土壤中石油的应用
将高效石油降解菌TDYN1T保藏编号CGMCC No.18061对数生长期的菌液用草碳粉固定后得到菌密度为10 8个/g的修复菌剂,将所述修复菌剂与10kg含油土壤(天津大港油田土壤)混合，添加质量为3％，在31℃，pH＝7.4条件下反应12天，石油降解效率为83％。

Claims (3)

  1. 一种高效石油降解菌TDYN1T，其分类命名为Falsochrobactrum sp.保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号CGMCC No.18061。
  2. 根据权利要求1所述的一种高效石油降解菌TDYN1T降解土壤中石油的应用。
  3. 根据权利要求2所述的应用，其特征是包括如下步骤：将高效石油降解菌TDYN1T保藏编号CGMCC No.18061对数生长期的菌液用草碳粉固定后得到菌密度为10 8个/g的修复菌剂,将所述修复菌剂与10kg含油土壤混合，添加质量为3％，在30±1℃，pH 7-7.4条件下反应。
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CN115612630A (zh) * 2021-07-13 2023-01-17 中国石油天然气股份有限公司 Fe(III)还原菌株、其培养方法、缩膨制剂及应用
CN115612630B (zh) * 2021-07-13 2023-12-22 中国石油天然气股份有限公司 Fe(III)还原菌株、其培养方法、缩膨制剂及应用
CN113943677A (zh) * 2021-11-03 2022-01-18 华中农业大学 一株降解甲基汞的赫尔曼亚特兰大杆菌及其应用

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