CN101624576A - Mycobacterium cosmeticum with capacity of degrading benzene compound and application thereof - Google Patents

Abstract

The invention provides a mycobacterium cosmeticum byf-4 with the capacity of degrading a benzene compound and an application thereof. The mycobacterium cosmeticum byf-4 is stored in Chinese typical culture storage center which has an address: Wuhan University Wuhan China, 430074, a storage date: Oct. 29, 2008 and a storage number: CCTCC No. M 208180. The invention has the advantages that the strain can rapidly degrade various benzene compounds with high efficiency and has an important meaning to the engineering practice of biologically purifying contaminants caused by the benzene compounds and the derivatives thereof.

Description

Translated fromChinese

具有降解苯系化合物能力的分枝杆菌及其应用Mycobacteria with ability to degrade benzene series compounds and application thereof

(一)技术领域(1) Technical field

本发明涉及一株具有降解苯系化合物能力的分枝杆菌(Mycobacterium cosmeticum)byf-4及其应用。The invention relates to a strain of mycobacterium (Mycobacterium cosmeticum) byf-4 capable of degrading benzene series compounds and its application.

(二)背景技术(2) Background technology

苯系化合物是一类易挥发的单环芳香类化合物，包括苯、甲苯、乙苯、二甲苯等，简称为BTEX，广泛应用于石油、化工、农药、纺织、造纸、油漆、制鞋等工业的生产过程，是环境中分布较广的一类有毒化合物。BTEX具有“三致效应”，被许多国家列入优先控制污染物，且已被确认为强致癌物质。研发废水、废气中BTEX的污染控制技术显得十分重要和迫切。目前，生物降解技术是降解这类物质的最有效方法之一。Benzene compounds are a class of volatile monocyclic aromatic compounds, including benzene, toluene, ethylbenzene, xylene, etc., referred to as BTEX, widely used in petroleum, chemical, pesticide, textile, papermaking, paint, shoemaking and other industries It is a kind of toxic compound widely distributed in the environment. BTEX has "three effects", has been listed as a priority pollutant by many countries, and has been confirmed as a strong carcinogen. It is very important and urgent to develop BTEX pollution control technology in wastewater and waste gas. Currently, biodegradation technology is one of the most effective ways to degrade such substances.

采用生物法净化废水、废气中BTEX等有毒化合物的关键之一便是获得具有高效降解BTEX能力的优良菌株。人们已经分离出了多株单环芳香类化合物降解菌，主要有假单胞菌(Pseudomonas)、不动杆菌(Acinetobacter)、红球菌(Rhodococcus)、罗尔斯通氏菌(Ralstonia)、产碱杆菌(Alcaligenes)、黄杆菌属(Flavobacterium)、孢瓶霉属真菌(Cladophialophora)、诺卡氏菌(Nocardia)等。这些降解菌株仅能降解某一种或两种苯系化合物，且大多数菌株的降解效率有待进一步提高，而对BTEX混合物中各物质均能实现高效降解的菌株并不多见。One of the keys to using biological methods to purify toxic compounds such as BTEX in wastewater and waste gas is to obtain excellent strains with high-efficiency degradation of BTEX. Many strains of single-ring aromatic compounds degrading bacteria have been isolated, mainly Pseudomonas, Acinetobacter, Rhodococcus, Ralstonia, alkagenes Alcaligenes, Flavobacterium, Cladophialophora, Nocardia, etc. These degrading strains can only degrade one or two kinds of benzene compounds, and the degradation efficiency of most strains needs to be further improved, while the strains that can efficiently degrade all substances in the BTEX mixture are rare.

(三)发明内容(3) Contents of the invention

本发明的目的是提供一株高效、快速降解苯系化合物的菌种及其应用。The purpose of the present invention is to provide a highly efficient and fast degrading benzene series strain and its application.

本发明采用的技术方案是：The technical scheme adopted in the present invention is:

具有降解苯系化合物能力的分枝杆菌(Mycobacterium cosmeticum)byf-4，保藏于中国典型培养物保藏中心，地址：中国武汉武汉大学，430074，保藏日期：2008年10月29日，保藏编号：CCTCC No：M 208180。本发明的降解菌株可实现高效降解BTEX混合物，因此，在实际应用中可发挥更重要的作用。经检索，目前未有在好氧条件下应用Mycobacteriumcosmeticum降解BTEX的报道，因此该降解菌的发现对BTEX类污染物生物净化的工程实践具有重要意义。Mycobacterium cosmeticum byf-4 with the ability to degrade benzene series compounds, preserved in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, 430074, preservation date: October 29, 2008, preservation number: CCTCC No: M 208180. The degrading bacterial strain of the present invention can efficiently degrade the BTEX mixture, therefore, it can play a more important role in practical applications. After retrieval, there is currently no report on the application of Mycobacteriumcosmeticum to degrade BTEX under aerobic conditions. Therefore, the discovery of the degrading bacteria is of great significance to the engineering practice of biological purification of BTEX pollutants.

所述Mycobacterium cosmeticum byf-4(CCTCC No：M 208180)来源于中国石化浙江镇海炼化污水处理厂曝气池中的活性污泥中，经驯化、分离、纯化获得。The Mycobacterium cosmeticum byf-4 (CCTCC No: M 208180) is derived from the activated sludge in the aeration tank of the Sinopec Zhejiang Zhenhai Refining and Chemical Wastewater Treatment Plant, and is obtained through domestication, separation and purification.

该菌株在固体培养基中呈淡乳白色，圆形，边缘整齐，光滑湿润，直径为0.5～1mm；菌体的形态为短杆菌，长度在2μm左右；无芽孢；好氧，革兰氏染色阴性，接触氧化酶阴性，能以BTEX混合物或其中的单一物质为唯一碳源，其生理生化特征如表1所示。Mycobacterium cosmeticumbyf-4CCTCC No：M 208180的16S rDNA序列如下：The strain is light milky white in solid medium, round, with neat edges, smooth and moist, with a diameter of 0.5-1mm; the shape of the bacteria is short bacillus, with a length of about 2μm; no spores; aerobic, Gram staining negative , contact oxidase negative, can use BTEX mixture or a single substance in it as the sole carbon source, and its physiological and biochemical characteristics are shown in Table 1. The 16S rDNA sequence of Mycobacterium cosmeticumbyf-4CCTCC No: M 208180 is as follows:

ttaacacatgcaagtcgaacggaaaggcccttcggggtgctcgagtggcgaacgggtgagtaacacgtgggtgatctgccctgcactttgggataagcctgggaaactgggtctaataccgaataggactccggccttcatgggttggggtggaaagcttttgcggtgtgggatgggcccgcggcctatcagcttgttggtggggtaatggcctaccaaggcgacgacgggtagccggcctgagagggtgaccggccacactgggactgagatacggcccagactcctacgggaggcagcagtggggaatattgcacaatgggcgcaagcctgatgcagcgacgccgcgtgagggatgacggccttcgggttgtaaacctctttcagcacagacgaagcgcaagtgacggtatgtgcagaagaaggaccggccaactacgtgccagcagccgcggtaatacgtagggtccgagcgttgtccggaattactgggcgtaaagagctcgtaggtggtttgtcgcgttgttcgtgaaaactcacagcttaactgtgggcgtgcgggcgatacgggcagactggagtactgcaggggagactggaattcctggtgtagcggtggaatgcgcagatatcaggaggaacaccggtggcgaaggcgggtctctgggcagtaactgacgctgaggagcgaaagcgtggggagcgaacaggattagataccctggtagtccacgccgtaaacggtgggtactaggtgtgggtttccttccttgggatccgtgccgtagctaacgcattaagtaccccgcctggggagtacggccgcaaggctaaaactcaaaggaattgacgggggcccgcacaagcggcggagcatgtggattaattcgatgcaacgcgaagaaccttacctgggtttgacatgcacaggacgctggtagagatatcagttcccttgtggcctgtgtgcaggtggtgcatggctgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttgtcctatgttgccagcgggttatgccggggactcgtaggagactgccggggtcaactcggaggaaggtggggatgacgtcaagtcatcatgccccttatgtccagggcttcacacatgctacaatggccggtacaaagggctgcgatgccgtgaggtggagcgaatccttgtaaagccggtctcagttcggatcggggtctgcaactcgaccccgtgaagtcggagtcgctagtaatcgcagatcagcaacgctgcggtgaatacgttcccgggccttgtacacaccgcccgtcacgtcatgaaagtcggtaacacccgaagccggtggcctaaccccttgtgggagggagccgtcgaaggtgggatcggcgattgggacgaagtcgaacaattaacacatgcaagtcgaacggaaaggcccttcggggtgctcgagtggcgaacgggtgagtaacacgtgggtgatctgccctgcactttgggataagcctgggaaactgggtctaataccgaataggactccggccttcatgggttggggtggaaagcttttgcggtgtgggatgggcccgcggcctatcagcttgttggtggggtaatggcctaccaaggcgacgacgggtagccggcctgagagggtgaccggccacactgggactgagatacggcccagactcctacgggaggcagcagtggggaatattgcacaatgggcgcaagcctgatgcagcgacgccgcgtgagggatgacggccttcgggttgtaaacctctttcagcacagacgaagcgcaagtgacggtatgtgcagaagaaggaccggccaactacgtgccagcagccgcggtaatacgtagggtccgagcgttgtccggaattactgggcgtaaagagctcgtaggtggtttgtcgcgttgttcgtgaaaactcacagcttaactgtgggcgtgcgggcgatacgggcagactggagtactgcaggggagactggaattcctggtgtagcggtggaatgcgcagatatcaggaggaacaccggtggcgaaggcgggtctctgggcagtaactgacgctgaggagcgaaagcgtggggagcgaacaggattagataccctggtagtccacgccgtaaacggtgggtactaggtgtgggtttccttccttgggatccgtgccgtagctaacgcattaagtaccccgcctggggagtacggccgcaaggctaaaactcaaaggaattgacgggggcccgcacaagcggcggagcatgtggattaattcgatgcaacgcgaagaaccttacctgggtttgacatgcacaggacgctggtagagatatcagttcccttgtggcctgtgtgcaggtggtgcatggctgtcgtcagctcgtgt cgtgagatgttgggttaagtcccgcaacgagcgcaacccttgtcctatgttgccagcgggttatgccggggactcgtaggagactgccggggtcaactcggaggaaggtggggatgacgtcaagtcatcatgccccttatgtccagggcttcacacatgctacaatggccggtacaaagggctgcgatgccgtgaggtggagcgaatccttgtaaagccggtctcagttcggatcggggtctgcaactcgaccccgtgaagtcggagtcgctagtaatcgcagatcagcaacgctgcggtgaatacgttcccgggccttgtacacaccgcccgtcacgtcatgaaagtcggtaacacccgaagccggtggcctaaccccttgtgggagggagccgtcgaaggtgggatcggcgattgggacgaagtcgaacaa

该降解菌株生理生化性能见表1：The physiological and biochemical properties of the degrading strain are shown in Table 1:

表1：Mycobacterium cosmeticum  byf-4的生理生化特征Table 1: Physiological and biochemical characteristics of Mycobacterium cosmeticum byf-4

  实验项目 experimental project  结果 result  M.R(甲基红)M.R (methyl red)  --  V.P实验V.P experiment  --  淀粉水解酶Amylolytic enzyme  --  吲哚实验Indole experiment  ++  硝酸盐还原Nitrate reduction  ++  明胶实验Gelatin experiment  ++  H₂S实验H₂ S experiment  --  葡萄糖Glucose  ++  乳糖Lactose  --

附注：“+”表示能利用或反应阳性；“-”表示不能利用或反应阴性本发明还涉及所述的分枝杆菌byf-4在降解苯系化合物及其衍生物中的应用。Remarks: "+" means it can be used or the reaction is positive; "-" means it cannot be used or the reaction is negative. The present invention also relates to the application of the mycobacterium byf-4 in degrading benzene compounds and their derivatives.

所述分枝杆菌byf-4可用于降解工业废水或废气中的苯系化合物及其衍生物。用于降解废水中的苯系化合物时，按照本领域常规方法将分枝杆菌byf-4接种至含有苯系化合物的废水中，在适宜分支杆菌生长的条件下进行培养即可；用于降解废气中的苯系化合物时，按照本领域常规方法，在适宜分支杆菌生长的条件下，将废气通入分枝杆菌byf-4的菌液中进行处理即可。The mycobacterium byf-4 can be used to degrade benzene series compounds and derivatives thereof in industrial waste water or waste gas. When used to degrade benzene series compounds in wastewater, inoculate mycobacterium byf-4 into wastewater containing benzene series compounds according to conventional methods in the art, and cultivate them under conditions suitable for the growth of mycobacteria; for the degradation of waste gas In the case of benzene series compounds, according to conventional methods in the field, under conditions suitable for the growth of mycobacteria, the exhaust gas can be passed into the bacterial liquid of mycobacterium byf-4 for treatment.

具体的，所述应用为：将分枝杆菌byf-4菌体悬浮于含有苯系化合物及其衍生物的废水中，在15～35℃、pH5～9条件下培养，降解废水中的苯系化合物及其衍生物。Specifically, the application is: suspending mycobacterium byf-4 in wastewater containing benzene series compounds and their derivatives, cultivating them at 15-35°C and pH 5-9, and degrading the benzene series compounds in wastewater. compounds and their derivatives.

优选的，所述应用为：将分枝杆菌byf-4菌体悬浮于含有苯系化合物及其衍生物的废水中，在30℃、pH7条件下培养，降解废水中的苯系化合物及其衍生物。Preferably, the application is: suspending mycobacterium byf-4 cells in wastewater containing benzene series compounds and their derivatives, cultivating them at 30°C and pH7, and degrading the benzene series compounds and their derivatives in the wastewater things.

所述苯系化合物优选为下列之一或其中两种以上的混合物：苯、甲苯、乙苯、邻二甲苯。The benzene series compound is preferably one of the following or a mixture of two or more thereof: benzene, toluene, ethylbenzene, ortho-xylene.

本发明的有益效果主要体现在：本发明菌株可高效、快速降解各类苯系化合物，对于苯系化合物及其衍生物引起的污染物的生物净化的工程实践具有重要意义。The beneficial effect of the present invention is mainly reflected in that the bacterial strain of the present invention can efficiently and rapidly degrade various benzene compounds, and is of great significance to the engineering practice of biological purification of pollutants caused by benzene compounds and their derivatives.

(四)附图说明(4) Description of drawings

图1为Mycobacterium cosmeticum byf-4显微镜和扫描、透射电镜照片(a，革兰氏染色显微镜图片；b，扫描电镜图片；c，透射电镜图片)；Figure 1 is the microscope, scanning and transmission electron micrographs of Mycobacterium cosmeticum byf-4 (a, Gram staining microscope picture; b, scanning electron microscope picture; c, transmission electron microscope picture);

图2为Mycobacterium cosmeticum byf-4的系统发育树图；Fig. 2 is the phylogenetic tree diagram of Mycobacterium cosmeticum byf-4;

图3为不同温度下菌Mycobacterium cosmeticum byf-4对BTEX的降解曲线图；Fig. 3 is the degradation curve of bacteria Mycobacterium cosmeticum byf-4 to BTEX under different temperatures;

图4为不同pH下菌Mycobacterium cosmeticum byf-4对BTEX的降解曲线；Fig. 4 is the degradation curve of bacteria Mycobacterium cosmeticum byf-4 to BTEX under different pH;

图5为菌Mycobacterium cosmeticum byf-4对不同初始浓度的苯的降解曲线图；Fig. 5 is the degradation curve graph of bacterium Mycobacterium cosmeticum byf-4 to the benzene of different initial concentration;

图6为菌Mycobacterium cosmeticum byf-4对不同初始浓度的甲苯的降解曲线图；Fig. 6 is the degradation curve of bacteria Mycobacterium cosmeticum byf-4 to different initial concentrations of toluene;

图7为菌Mycobacterium cosmeticum byf-4对不同初始浓度的乙苯的降解曲线图；Fig. 7 is the degradation curve graph of bacteria Mycobacterium cosmeticum byf-4 to the ethylbenzene of different initial concentrations;

图8为菌Mycobacterium cosmeticum byf-4对不同初始浓度的邻二甲苯的降解曲线图；Fig. 8 is the degradation curve graph of bacteria Mycobacterium cosmeticum byf-4 to the o-xylene of different initial concentrations;

图9为菌Mycobacterium cosmeticum byf-4对混合底物的降解曲线图。Fig. 9 is a graph showing the degradation curve of bacteria Mycobacterium cosmeticum byf-4 to mixed substrates.

(五)具体实施方式(5) Specific implementation methods

下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此(如未特别说明，实施例中百分比浓度均指质量百分比浓度)：The present invention is further described below in conjunction with specific embodiment, but protection scope of the present invention is not limited thereto (if not specified, percentage concentration all refers to mass percent concentration in the embodiment):

实施例1：Mycobacterium cosmeticum byf-4的分离、纯化及其鉴定1.Mycobacterium cosmeticum byf-4的分离及纯化Example 1: Isolation, purification and identification of Mycobacterium cosmeticum byf-4 1.Isolation and purification of Mycobacterium cosmeticum byf-4

Mycobacterium cosmeticum byf-4是从浙江镇海炼化污水处理厂曝气池的活性污泥中取样，经驯化、分离及纯化得到的一株革兰氏阴性菌。具体步骤如下：Mycobacterium cosmeticum byf-4 is a Gram-negative bacterium that was domesticated, isolated and purified from the activated sludge in the aeration tank of Zhejiang Zhenhai Refining and Chemical Wastewater Treatment Plant. Specific steps are as follows:

(1)污泥定向驯化(1) Directed domestication of sludge

取浙江镇海炼化污水处理厂曝气池的活性污泥。活性污泥进行苯系化合物(BTEX)定向驯化，每5天为一驯化周期，驯化3个月，直至活性污泥对苯系化合物实现稳定高效降解。Activated sludge from the aeration tank of Zhejiang Zhenhai Refining and Chemical Wastewater Treatment Plant was taken. The activated sludge was subjected to directional domestication of benzene series compounds (BTEX), and every 5 days was a domestication cycle, and the domestication period was 3 months until the activated sludge achieved stable and efficient degradation of benzene series compounds.

(2)菌株的筛选与纯化(2) Screening and purification of bacterial strains

利用苯系物降解菌筛选培养基对活性污泥进行菌株筛选。苯系物降解菌筛选培养基配制：KH₂PO₄，0.5g；K₂HPO₄，0.5g；NaCl，0.01g；NH₄Cl，1g；MgCl₂·6H₂O，0.2g；CaCl₂，0.02g；MnSO₄，0.339mg；ZnSO₄，0.428mg；钼酸铵，0.347mg；琼脂，15g；蒸馏水，1000ml；pH 7.2～7.4。苯系物另外分别添加：苯，200mg/l；甲苯，200mg/l；乙苯，200mg/l；邻二甲苯：200mg/l。The benzene series degrading bacteria screening medium was used to screen the activated sludge strains. Preparation of screening medium for benzene series degrading bacteria: KH₂ PO₄ , 0.5g; K₂ HPO₄ , 0.5g; NaCl, 0.01g; NH₄ Cl, 1g; MgCl₂ 6H₂ O, 0.2g; CaCl₂ , 0.02g; MnSO₄ , 0.339mg; ZnSO₄ , 0.428mg; Ammonium molybdate, 0.347mg; Agar, 15g; Distilled water, 1000ml; pH 7.2～7.4. Benzene series were additionally added separately: benzene, 200 mg/l; toluene, 200 mg/l; ethylbenzene, 200 mg/l; o-xylene: 200 mg/l.

按照菌株分离纯化的常规方法，在恒温培养箱28℃培养2～3天，挑取平板上长出的单菌落，得到多株纯菌落。According to the conventional method of isolation and purification of bacterial strains, culture in a constant temperature incubator at 28°C for 2 to 3 days, pick single colonies growing on the plate, and obtain multiple pure colonies.

将筛选到的多株苯系物降解菌在含有苯系化合物为唯一碳源的无机盐培养基中考察菌株对苯系化合物的去除效果，通过同空白实验的对照，结果得到了一株高效降解苯系化合物的菌株byf-4。所用的培养基成分为：苯0.1g/L，甲苯0.1g/L，乙苯0.1g/L，邻二甲苯0.1g/L，NH₄Cl 0.1g/L，MgCl₂·6H₂O 0.08g/L，K₂HPO₄0.25g/L，CaCl₂·2H₂O 0.1g/L，溶剂为水，pH 7.2～7.4。The screened multiple strains of benzene series degrading bacteria were tested in the inorganic salt medium containing benzene series compounds as the only carbon source to investigate the removal effect of the strains on benzene series compounds, and compared with the blank experiment, the result was a highly efficient degradation strain Benzene compound strain byf-4. The medium components used are: benzene 0.1g/L, toluene 0.1g/L, ethylbenzene 0.1g/L, o-xylene 0.1g/L, NH₄ Cl 0.1g/L, MgCl₂ 6H₂ O 0.08g /L, K₂ HPO₄ 0.25g/L, CaCl₂ ·2H₂ O 0.1g/L, solvent is water, pH 7.2-7.4.

2.Mycobacterium cosmeticum byf-4的16S rDNA鉴定2. Identification of 16S rDNA of Mycobacterium cosmeticum byf-4

通过16S rRNA序列分析和Biolog微生物鉴定系统鉴定，确定byf-4菌为Mycobacterium cosmeticum。具体步骤如下：Through 16S rRNA sequence analysis and Biolog microbial identification system identification, the byf-4 bacteria were identified as Mycobacterium cosmeticum. Specific steps are as follows:

采用3S柱离心式环境样品DNA回收试剂盒(V2.2，上海申能博彩生物科技有限公司)提取Mycobacterium cosmeticum byf-4的DNA，4℃保存。然后作为PCR反应的模板，设计引物，扩增Mycobacteriumcosmeticum byf-4的全序列DNA，上下游引物序列分别如下：The DNA of Mycobacterium cosmeticum byf-4 was extracted with a 3S column centrifugal environmental sample DNA recovery kit (V2.2, Shanghai Shenergy Biotechnology Co., Ltd.), and stored at 4°C. Then as a template for the PCR reaction, primers were designed to amplify the full-sequence DNA of Mycobacteriumcosmeticum byf-4, and the sequences of the upstream and downstream primers were as follows:

BSF8/20：5′-AGAGT TTGAT CCTGG CTCAG-3′BSF8/20: 5′-AGAGT TTGAT CCTGG CTCAG-3′

BSR1541/20：5′-AAGGA GGTGA TCCAGCCGCA-3′BSR1541/20: 5′-AAGGA GGTGA TCCAGCCGCA-3′

PCR反应程序设定为：先94℃预变性4min；然后94℃变性1min，59℃退火1min，72℃延伸1.5min，循环35个周期；然后72℃延伸10min；最后4℃保持10min。将PCR产物进行测序(上海英骏)，测序结果见SEQID No.1。The PCR reaction program was set as follows: pre-denaturation at 94°C for 4 min; then denaturation at 94°C for 1 min, annealing at 59°C for 1 min, extension at 72°C for 1.5 min, and a cycle of 35 cycles; extension at 72°C for 10 min; and finally hold at 4°C for 10 min. The PCR product was sequenced (Shanghai Yingjun), and the sequencing result is shown in SEQID No.1.

将byf-4的16S rDNA序列上传到Genbank，获得Genbank的登录号FJ169473，同时同Genbank中的基因序列进行同源性比较，发现其属于Mycobacterium属，然后通过系统发育树的建立，byf-4可能为Mycobacterium cosmeticum，图2为该菌的系统发育树图，图1为该菌的显微镜和扫描、透射电镜照片。为了进一步确定鉴定结果，通过各项生理生化实验以及Biolog微生物鉴定系统的分析，最终确定byf-4为Mycobacterium cosmeticum。Uploaded the 16S rDNA sequence of byf-4 to Genbank, obtained the Genbank accession number FJ169473, and compared the homology with the gene sequence in Genbank, and found that it belongs to the genus Mycobacterium, and then through the establishment of the phylogenetic tree, byf-4 may be It is Mycobacterium cosmeticum, Fig. 2 is the phylogenetic tree of the bacterium, and Fig. 1 is the microscope, scanning and transmission electron micrographs of the bacterium. In order to further confirm the identification results, through the analysis of various physiological and biochemical experiments and the Biolog microbial identification system, byf-4 was finally determined to be Mycobacterium cosmeticum.

实施例2：Mycobacterium cosmeticum byf-4对BTEX生物降解性能检测Embodiment 2: Detection of Mycobacterium cosmeticum byf-4 to BTEX biodegradation performance

1.不同温度下菌Mycobacterium cosmeticum byf-4对BTEX的生物降解特性1. Biodegradation characteristics of Mycobacterium cosmeticum byf-4 on BTEX at different temperatures

在不同温度下实施Mycobacterium cosmeticum byf-4对BTEX的降解实验，发现其在25～35℃具有较高的降解苯系化合物的能力，从实际应用角度看，30℃为最为适宜的温度，此时去除率最高，具体实施步骤如下：The degradation experiment of Mycobacterium cosmeticum byf-4 on BTEX was carried out at different temperatures, and it was found that it has a high ability to degrade benzene compounds at 25-35 ° C. From the perspective of practical application, 30 ° C is the most suitable temperature. At this time The removal rate is the highest, and the specific implementation steps are as follows:

在含有苯系化合物为唯一碳源的无机培养基(终浓度组成为：苯50mg/L、甲苯50mg/L、乙苯50mg/L、邻二甲苯50mg/L，NH₄Cl 0.1g/L，MgCl₂·6H₂O 0.08g/L，K₂HPO₄0.25g/L，CaCl₂·2H₂O 0.1g/L，溶剂为水，pH 7.2～7.4)加入新鲜培养的byf-4的菌悬液，OD₆₀₀为0.2，接种量为1ml/50ml，分取50ml培养基分装至5个250ml的盐水瓶中，分别置于15℃、20℃、25℃、30℃、35℃的摇床中160rpm培养；另外准备5个同样的未接菌的培养基分别置于对应的温度同时培养，作为空白对照。培养48h后，取样气相检测苯系化合物的浓度，同时测定菌株的菌密度(OD₆₀₀)，绘制苯系物去除率曲线图。In the inorganic medium containing benzene compounds as the only carbon source (final concentration composition: benzene 50mg/L, toluene 50mg/L, ethylbenzene 50mg/L, o-xylene 50mg/L, NH₄ Cl 0.1g/L, MgCl₂ 6H₂ O 0.08g/L, K₂ HPO₄ 0.25g/L, CaCl₂ 2H₂ O 0.1g/L, solvent is water, pH 7.2～7.4) add fresh cultured byf-4 bacterial suspension solution, OD₆₀₀ is 0.2, the inoculum size is 1ml/50ml, divide 50ml of medium into five 250ml saline bottles, and place them in shakers at 15°C, 20°C, 25°C, 30°C, and 35°C respectively culture medium at 160rpm; in addition, prepare 5 identical uninoculated culture media and place them at corresponding temperatures for simultaneous culture as a blank control. After cultivating for 48 hours, the gas phase was taken to detect the concentration of benzene series compounds, and the bacterial density (OD₆₀₀ ) of the strain was measured at the same time, and the removal rate curve of benzene series compounds was drawn.

结果如图3所示，表明在温度为15℃时，菌株对苯系化合物的去除率较低。随着温度的逐渐提高，菌株对苯系化合物的去除率也随之提高；在温度为25～35℃时为该菌株较为适宜生长的温度，菌株的生长和对苯系物的去除率都达到了较高的值；温度30℃时达到了最大值，48h对苯系物的去除率分别为苯，98.2％；甲苯，95.6％；乙苯，78.3％；邻二甲苯，68.4％。随着温度的进一步提高，菌株的生长和降解能力开始下降。The results are shown in Figure 3, indicating that when the temperature was 15°C, the strains had a low removal rate of benzene compounds. As the temperature gradually increases, the removal rate of the strain to benzene compounds also increases; when the temperature is 25-35°C, it is a more suitable temperature for the growth of the strain, and the growth of the strain and the removal rate of the benzene compounds both reach A higher value was reached; the maximum value was reached when the temperature was 30°C, and the removal rates of benzene series in 48 hours were benzene, 98.2%; toluene, 95.6%; ethylbenzene, 78.3%; o-xylene, 68.4%. With the further increase of temperature, the growth and degradation ability of the strain began to decline.

2.不同pH下菌Mycobacterium cosmeticum byf-4对BTEX的降解特性2. Degradation characteristics of Mycobacterium cosmeticum byf-4 on BTEX at different pH

在不同pH下实施Mycobacterium cosmeticum byf-4对BTEX的降解实验，结果表明其最佳的生长降解pH值为7，具体实施方案如下：Implement Mycobacterium cosmeticum byf-4 to the degradation experiment of BTEX under different pH, the result shows that its optimal growth and degradation pH value is 7, and specific embodiment is as follows:

在5个250ml的盐水瓶中加入50ml等量的无机盐培养基，苯系化合物作为唯一碳源，培养基终浓度组成同上，分别调节pH至5、6、7、8、9；同时准备5瓶相同的培养基作为空白对照。均接种1ml新鲜培养的byf-4菌悬液，OD₆₀₀为0.2，在30℃，160rpm下培养。分别在0h，48h取样，检测培养基中的苯系化合物浓度变化和菌株的菌密度(OD₆₀₀)，绘制苯系物去除率曲线图。Add 50ml of the same amount of inorganic salt medium to five 250ml saline bottles, benzene compounds as the only carbon source, the final concentration of the medium is the same as above, and adjust the pH to 5, 6, 7, 8, 9 respectively; prepare 5 The same culture medium was used as blank control. All were inoculated with 1ml of freshly cultured byf-4 bacterial suspension, with an_OD600 of 0.2, and cultured at 30°C and 160rpm. Samples were taken at 0h and 48h to detect the concentration of benzene series compounds in the medium and the bacterial density (OD₆₀₀ ) of the strains, and draw the curve of the removal rate of benzene series compounds.

结果如图4所示，在pH为5、9时，byf-4对苯系物去除效率较差。在pH为5的培养液中，溶液较澄清，同空白溶液一样，表明菌株生长十分不好。在pH为5～9的条件下，细菌都能以苯系化合物为唯一碳源生长，只是生长和降解速率有所不同，在pH为时，菌株byf-4的生长和降解速率最快，对苯、甲苯、乙苯、邻二甲苯48h的去除率分别为97.4％、95.8％、86.5％、60.5％。The results are shown in Figure 4. When the pH is 5 and 9, the removal efficiency of byf-4 for benzene series is poor. In the culture solution with a pH of 5, the solution is clearer, same as the blank solution, indicating that the growth of the strain is very poor. Under the condition of pH 5-9, the bacteria can grow with benzene compounds as the only carbon source, but the growth and degradation rates are different. When the pH is 5, the growth and degradation rates of the strain byf-4 are the fastest. The removal rates of benzene, toluene, ethylbenzene, and o-xylene for 48 hours were 97.4%, 95.8%, 86.5%, and 60.5%, respectively.

3.菌Mycobacterium cosmeticum byf-4对不同初始浓度的BTEX的降解情况3. Degradation of Mycobacterium cosmeticum byf-4 to different initial concentrations of BTEX

在不同苯系物初始浓度下，实施byf-4对苯、甲苯、乙苯和邻二甲苯四种苯系物的降解，结果发现byf-4对苯、甲苯、乙苯和邻二甲苯的耐受浓度为500mg/l、300mg/l、125mg/l、200mg/l。具体实施方案如下：Under different initial concentrations of benzene series, the degradation of four benzene series by byf-4 to benzene, toluene, ethylbenzene and o-xylene was carried out, and it was found that byf-4 was resistant to benzene, toluene, ethylbenzene and o-xylene Acceptable concentrations are 500mg/l, 300mg/l, 125mg/l, 200mg/l. The specific implementation plan is as follows:

在250ml的盐水瓶中加入50ml的无机盐培养基(除碳源外其他组分终浓度为：NH₄Cl 0.1g/L，MgCl₂·6H₂O 0.08g/L，K₂HPO₄0.25g/L，CaCl₂·2H₂O 0.1g/L，溶剂为水，pH 7.2～7.4)，苯系化合物作为唯一碳源。苯的终浓度分别为25、50、150、300、500mg/l；甲苯的终浓度分别为25、50、150、200、300mg/l；乙苯的终浓度分别为25、50、75、100、125mg/l；邻二甲苯的终浓度分别为25、50、100、150、200mg/l。分别接种等量1mlOD₆₀₀为0.2的菌悬液，30℃，160rpm培养，间隔一段时间取样，绘制byf-4对苯系物的降解曲线图。Add 50ml of inorganic salt medium to a 250ml saline bottle (final concentration of other components except carbon source: NH₄ Cl 0.1g/L, MgCl₂ 6H₂ O 0.08g/L, K₂ HPO₄ 0.25g /L, CaCl₂ ·2H₂ O 0.1g/L, solvent is water, pH 7.2~7.4), benzene compounds as the only carbon source. The final concentrations of benzene are 25, 50, 150, 300, 500 mg/l; the final concentrations of toluene are 25, 50, 150, 200, 300 mg/l; the final concentrations of ethylbenzene are 25, 50, 75, 100 , 125mg/l; the final concentration of o-xylene is 25, 50, 100, 150, 200mg/l respectively. Inoculate an equal amount of 1ml of bacterial suspension with_OD600 of 0.2, incubate at 30°C and 160rpm, take samples at intervals, and draw the degradation curve of byf-4 to benzene series.

从图5～图8可以看出在苯浓度低于500mg/l时，byf-4可快速地降解苯，细菌生长良好，而高于500mg/l时，菌株受到强烈抑制；由于受甲苯在水中的溶解度限制，甲苯的最高浓度只做到300mg/l，当甲苯浓度在300mg/l以内时，byf-4都可快速地降解甲苯，细菌生长良好；byf-4对乙苯、邻二甲苯的降解速度要慢于苯和甲苯，当乙苯、邻二甲苯浓度分别大于125mg/l、150mg/l时，byf-4的生长降解受到抑制，不能高效降解。From Figure 5 to Figure 8, it can be seen that when the benzene concentration is lower than 500mg/l, byf-4 can rapidly degrade benzene, and the bacteria grow well, and when it is higher than 500mg/l, the bacterial strain is strongly inhibited; due to the influence of toluene in water The solubility limit of toluene, the highest concentration of toluene is only 300mg/l, when the toluene concentration is within 300mg/l, byf-4 can quickly degrade toluene, and the bacteria grow well; The degradation rate is slower than that of benzene and toluene. When the concentrations of ethylbenzene and o-xylene are greater than 125mg/l and 150mg/l respectively, the growth and degradation of byf-4 are inhibited and cannot be efficiently degraded.

4.菌Mycobacterium cosmeticum byf-4对混合底物的降解情况4. Degradation of mixed substrates by Mycobacterium cosmeticum byf-4

在以苯、甲苯、乙苯、邻二甲苯四种苯系物为混合底物，实施菌Mycobacterium cosmeticum byf-4对BTEX的降解特性，结果表明其对四种物质的降解难易程度为邻二甲苯＞乙苯＞苯＞甲苯。具体实施方案如下：Using benzene, toluene, ethylbenzene, and o-xylene as four benzene series as mixed substrates, the degradation characteristics of Mycobacterium cosmeticum byf-4 to BTEX were carried out. Toluene>Ethylbenzene>Benzene>Toluene. The specific implementation plan is as follows:

在250ml的盐水瓶中加入50ml的无机盐培养基(终浓度组成为：苯50mg/L、甲苯50mg/L、乙苯50mg/L、邻二甲苯50mg/L，NH₄Cl 0.1g/L，MgCl₂·6H₂O 0.08g/L，K₂HPO₄0.25g/L，CaCl₂·2H₂O 0.1g/L，溶剂为水，pH 7.2～7.4)，苯系化合物作为唯一碳源，总浓度分别都为50mg/L。接种1ml OD₆₀₀为0.2的菌悬液，30℃，160rpm培养。每隔一段时间取样，检测培养基中的苯系化合物浓度和菌密度(OD₆₀₀)，直到降解完全。绘制苯系物的降解曲线和菌株生长图。Add 50ml of inorganic salt medium (final concentration: benzene 50mg/L, toluene 50mg/L, ethylbenzene 50mg/L, o-xylene 50mg/L, NH₄ Cl 0.1g/L, MgCl₂ 6H₂ O 0.08g/L, K₂ HPO₄ 0.25g/L, CaCl₂ 2H₂ O 0.1g/L, solvent is water, pH 7.2～7.4), benzene compounds as the only carbon source, total The concentration is 50mg/L respectively. Inoculate 1ml of bacterial suspension with OD₆₀₀ of 0.2, culture at 30°C, 160rpm. Samples were taken at intervals to detect the concentration of benzene series compounds and bacterial density (OD₆₀₀ ) in the culture medium until the degradation was complete. Draw the degradation curve and strain growth chart of benzene series.

结果如图9所示，在降解初期，菌株对苯系物的降解有5～10h左右的延滞期，同时菌株生长缓慢；在15h后，菌株开始适应该环境，进入对数生长期，苯系物被快速降解，从图中可以看出，byf-4对甲苯的去除速率要明显快于苯、乙苯和邻二甲苯；在35h时，甲苯被降解完全，苯、乙苯、邻二甲苯先后在41h、45h、50h被降解完。菌株的菌密度在30～45h达到最大值，50h后菌株进入衰退期。The results are shown in Figure 9. At the initial stage of degradation, the strain had a lag period of about 5-10 hours for the degradation of benzene series, and at the same time the strain grew slowly; after 15 hours, the strain began to adapt to the environment and entered the logarithmic growth phase. It can be seen from the figure that the removal rate of toluene by byf-4 is significantly faster than that of benzene, ethylbenzene and o-xylene; at 35h, toluene is completely degraded, while benzene, ethylbenzene, and o-xylene It was degraded at 41h, 45h, and 50h successively. The bacterial density of the strain reached the maximum at 30-45 hours, and the strain entered the decay period after 50 hours.

SEQUENCE LISTINGSEQUENCE LISTING

<110>浙江工业大学<110> Zhejiang University of Technology

<120>具有降解苯系化合物能力的分枝杆菌byf-4及其应用<120> Mycobacterium byf-4 with the ability to degrade benzene series compounds and its application

<130><130>

<160>3<160>3

<170>PatentIn version 3.2<170>PatentIn version 3.2

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<213>Mycobacterium cosmeticum<213>Mycobacterium cosmeticum

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taacacgtgg gtgatctgcc ctgcactttg ggataagcct gggaaactgg gtctaatacc  120taacacgtgg gtgatctgcc ctgcactttg ggataagcct gggaaactgg gtctaatacc 120

gaataggact ccggccttca tgggttgggg tggaaagctt ttgcggtgtg ggatgggccc  180gaataggact ccggccttca tgggttgggg tggaaagctt ttgcggtgtg ggatgggccc 180

gcggcctatc agcttgttgg tggggtaatg gcctaccaag gcgacgacgg gtagccggcc  240gcggcctatc agcttgttgg tggggtaatg gcctaccaag gcgacgacgg gtagccggcc 240

tgagagggtg accggccaca ctgggactga gatacggccc agactcctac gggaggcagc  300tgagagggtg accggccaca ctgggactga gatacggccc agactcctac gggaggcagc 300

agtggggaat attgcacaat gggcgcaagc ctgatgcagc gacgccgcgt gagggatgac  360agtggggaat attgcacaat gggcgcaagc ctgatgcagc gacgccgcgt gagggatgac 360

ggccttcggg ttgtaaacct ctttcagcac agacgaagcg caagtgacgg tatgtgcaga  420ggccttcggg ttgtaaacct ctttcagcac agacgaagcg caagtgacgg tatgtgcaga 420

agaaggaccg gccaactacg tgccagcagc cgcggtaata cgtagggtcc gagcgttgtc  480agaaggaccg gccaactacg tgccagcagc cgcggtaata cgtagggtcc gagcgttgtc 480

cggaattact gggcgtaaag agctcgtagg tggtttgtcg cgttgttcgt gaaaactcac  540cggaattact gggcgtaaag agctcgtagg tggtttgtcg cgttgttcgt gaaaactcac 540

agcttaactg tgggcgtgcg ggcgatacgg gcagactgga gtactgcagg ggagactgga  600agcttaactg tgggcgtgcg ggcgatacgg gcagactgga gtactgcagg ggagactgga 600

attcctggtg tagcggtgga atgcgcagat atcaggagga acaccggtgg cgaaggcggg  660attcctggtg tagcggtgga atgcgcagat atcaggagga aacccggtgg cgaaggcggg 660

tctctgggca gtaactgacg ctgaggagcg aaagcgtggg gagcgaacag gattagatac  720tctctgggca gtaactgacg ctgaggagcg aaagcgtggg gagcgaacag gattagatac 720

cctggtagtc cacgccgtaa acggtgggta ctaggtgtgg gtttccttcc ttgggatccg  780cctggtagtc cacgccgtaa acggtgggta ctaggtgtgg gtttccttcc ttgggatccg 780

tgccgtagct aacgcattaa gtaccccgcc tggggagtac ggccgcaagg ctaaaactca  840tgccgtagct aacgcattaa gtaccccgcc tggggagtac ggccgcaagg ctaaaactca 840

aaggaattga cgggggcccg cacaagcggc ggagcatgtg gattaattcg atgcaacgcg  900aaggaattga cgggggcccg cacaagcggc ggagcatgtg gattaattcg atgcaacgcg 900

aagaacctta cctgggtttg acatgcacag gacgctggta gagatatcag ttcccttgtg  960aagaacctta cctgggtttg acatgcacag gacgctggta gagatatcag ttcccttgtg 960

gcctgtgtgc aggtggtgca tggctgtcgt cagctcgtgt cgtgagatgt tgggttaagt  1020gcctgtgtgc aggtggtgca tggctgtcgt cagctcgtgt cgtgagatgt tgggttaagt 1020

cccgcaacga gcgcaaccct tgtcctatgt tgccagcggg ttatgccggg gactcgtagg  1080cccgcaacga gcgcaaccct tgtcctatgt tgccagcggg ttatgccggg gactcgtagg 1080

agactgccgg ggtcaactcg gaggaaggtg gggatgacgt caagtcatca tgccccttat  1140agactgccgg ggtcaactcg gaggaaggtg gggatgacgt caagtcatca tgccccttat 1140

gtccagggct tcacacatgc tacaatggcc ggtacaaagg gctgcgatgc cgtgaggtgg  1200gtccagggct tcacacatgc tacaatggcc ggtacaaagg gctgcgatgc cgtgaggtgg 1200

agcgaatcct tgtaaagccg gtctcagttc ggatcggggt ctgcaactcg accccgtgaa  1260agcgaatcct tgtaaagccg gtctcagttc ggatcggggt ctgcaactcg accccgtgaa 1260

gtcggagtcg ctagtaatcg cagatcagca acgctgcggt gaatacgttc ccgggccttg  1320gtcggagtcg ctagtaatcg cagatcagca acgctgcggt gaatacgttc ccgggccttg 1320

tacacaccgc ccgtcacgtc atgaaagtcg gtaacacccg aagccggtgg cctaacccct  1380tacacaccgc ccgtcacgtc atgaaagtcg gtaacacccg aagccggtgg cctaacccct 1380

tgtgggaggg agccgtcgaa ggtgggatcg gcgattggga cgaagtcgaa caa         1433tgtggggaggg agccgtcgaa ggtgggatcg gcgattggga cgaagtcgaa caa 1433

<210>2<210>2

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<212>DNA<212>DNA

<213>Unknown<213>Unknown

<220><220>

<223>人工序列<223> Artificial sequence

<400>2<400>2

agagtttgat cctggctcag    20agagtttgat cctggctcag 20

<210>3<210>3

<211>20<211>20

<212>DNA<212>DNA

<213>Unknown<213>Unknown

<220><220>

<223>人工序列<223> Artificial sequence

<400>3<400>3

aaggaggtga tccagccgca    20aaggaggtga tccagccgca 20

Claims (6)

1. mycobacterium (Mycobacterium cosmeticum) byf-4 that has the degrading benzene compounds ability, be preserved in Chinese typical culture collection center, address: Chinese Wuhan Wuhan University, 430074, preservation date: on October 29th, 2008, deposit number: CCTCC No:M208180.

2. the application of mycobacterium byf-4 as claimed in claim 1 in degrading benzene compounds and derivative thereof.

3. application as claimed in claim 2 is characterized in that described mycobacterium byf-4 be used for the degrading benzene compounds and the derivative thereof of trade effluent or waste gas.

4. application as claimed in claim 2, it is characterized in that described being applied as: mycobacterium byf-4 thalline is suspended in the waste water that contains benzene compounds and derivative thereof, under 15～35 ℃, pH5～9 conditions, cultivate benzene compounds in the degrading waste water and derivative thereof.

5. application as claimed in claim 4, it is characterized in that described being applied as: mycobacterium byf-4 thalline is suspended in the waste water that contains benzene compounds and derivative thereof, under 30 ℃, pH7 condition, cultivate benzene compounds in the degrading waste water and derivative thereof.

6. as the described application of one of claim 2～5, it is characterized in that described benzene compounds is one of following or wherein two or more mixture: benzene, toluene, ethylbenzene, o-Xylol.

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