CN116463254A - 蒙氏假单胞菌sd-2及其在降解有机污染物中的应用 - Google Patents
蒙氏假单胞菌sd-2及其在降解有机污染物中的应用 Download PDFInfo
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Abstract
本发明公开了一种蒙氏假单胞菌SD‑2及其在降解有机污染物中的应用,本发明所述的蒙氏假单胞菌SD‑2生长环境温和,易扩大培养,能将乙醇完全降解为无机物(CO2、H2O)和细胞生物质,实现完全矿化,且对于400mg/L以内的乙醇的去除率高达100%。而且,该蒙氏假单胞菌对于厨余垃圾常见的臭气成分(如乙醛)具有高效的降解能力,且能够承受较高浓度的污染物。
Description
(一)技术领域
本发明涉及一种蒙氏假单胞菌SD-2及其在降解有机污染物中的应用。
(二)背景技术
乙醇是有机化合物、醇类的一种,被通常称为酒精。乙醇在常温常压下是一种易燃易挥发,且具有特殊香味、略带刺激的无色透明液体,与甲醚是同分异构体。与水混溶、可溶于乙醚、氯仿、甘油、甲醛等多数的有机溶剂。乙醇是一种常用的燃料、溶剂和消毒剂,在国防工业、医疗卫生、有机合成、食品工业、工农业生产中都有广泛的用途,也常被用于有机合成。
乙醇是一种易挥发的物质,低毒、具有一定刺激性。但由于乙醇易挥发,遇明火、高热以及与氧化剂接触后发生化学反应均会燃烧。同时,乙醇作为一种良好的溶剂,能够溶解众多的有机物和无机物,具有还原性,会被氧化形成乙醛。乙醇具有成瘾性及致癌性,但乙醇并不是直接导致癌症的物质,而是致癌物质普遍溶于乙醇。在生产过程中,长期接触高浓度的乙醇引起鼻、眼、粘膜刺激症状等;长期酗酒可引起多发性神经病、慢性胃炎、脂肪肝、肝硬化等。
在厨余垃圾降解初期中,乙醇是产生的典型污染物之一,因此研究环境中乙醇的高效降解对人类的生活环境的改善是很有必要的。通过文献检索发现陆晨晨等在2011年在酸败的酒糟中筛选得到一株有较高乙醇降解能力的巴士醋杆菌(Acetobacterpasteurianus)Ⅶ,但是在研究中所用的液体发酵培养液中含有葡萄糖等生长碳源。未发现有关蒙氏假单胞菌以乙醇为唯一碳源来实现高效降解的报道。
(三)发明内容
本发明目的是提供一种蒙氏假单胞菌(Pseudomonas monteilii)SD-2及其在降解有机污染物中的应用,该菌株生长环境温和,易扩大培养,能够高效、有力地去除有机污染物,特别是能以乙醇为唯一碳源生长,对乙醇具有较强的降解能力。该降解菌的发现对于厨余垃圾、酿酒行业废水废气中乙醇等常见污染物的高效净化有重要意义。
本发明采用的技术方案是:
本发明提供一种菌株-蒙氏假单胞菌(Pseudomonas monteilii)SD-2,保藏于中国典型培养物保藏中心,保藏编号:CCTCC NO:M2023241,保藏日期:2023年03月03日,地址:中国,武汉,武汉大学,430072。
本发明蒙氏假单胞杆菌SD-2的基本特征为:菌落呈白色、不透光,易挑取,菌苔沿划线生长;电镜下形态为椭圆形、有鞭毛,好氧。
本发明还提供一种所述蒙氏假单胞杆菌SD-2在降解有机污染物中的应用,具体所述的应用是将蒙氏假单胞杆菌SD-2接种至pH=5-9(优选pH=6-8)、含有机污染物的无机盐培养液中,在25-35℃、100-200rpm条件(优选30℃、160rpm)下进行培养,实现有机污染物的降解;所述有机污染物包括乙醇、乙醛、乙酸、甲醇。
所述无机盐培养液中有机污染物的初始浓度为315.6-613.2mg/L,优选400mg/L。
所述蒙氏假单胞杆菌SD-2以静息细胞形式加入,所述无机盐培养液中,静息细胞加入量以菌体干重计为10-100mg/L,优选50mg/L。
所述无机盐培养液组成为:K2HPO4·3H2O 0.942g/L、KH2PO4 0.234g/L、NaNO31.7g/L、NH4Cl 0.98g/L、MgCl·6H2O 0.2033g/L、CaCl·2H2O 0.11g/L、FeCl3 0.0162g/L、微量元素母液5ml/L,溶液去离子水,pH 7.0;其中微量元素母液组成:CuSO4·5H2O 0.02g/L、FeSO4·7H2O 1.0g/L、MnSO4·4H2O 0.02g/L、CoCl·6H2O 0.02g/L、H3BO3 0.014g/L、ZnSO4·7H2O 0.10g/L,溶剂为去离子水。
本发明蒙氏假单胞菌SD-2以静息细胞形式接种,所述静息细胞按如下步骤制备:
(1)斜面培养:将蒙氏假单胞菌SD-2接种于斜面LB固体培养基,30℃培养24~36h,获得斜面菌体;所述LB固体培养基终浓度组成为:NaCl 10g/L、蛋白胨10g/L、酵母粉5g/L、琼脂18~20g/L,溶剂为去离子水,pH值自然;
(2)扩大培养:用接种环挑取步骤(1)获得的斜面菌体接种至LB液体培养基中,30℃培养24~36h,获得扩大培养液,离心,收集湿菌体,灭菌的去离子水洗涤,获得蒙氏假单胞杆菌SD-2静息细胞;所述LB液体培养基终浓度组成为:NaCl 10g/L、蛋白胨10g/L、酵母粉5g/L,溶剂为去离子水,pH值自然。
与现有技术相比,本发明有益效果主要体现在:
本发明提供的蒙氏假单胞菌SD-2取自某厨余垃圾处理厂污水处理站曝气池和厨余垃圾臭气处理设施生物段循环段,对于乙醇等有机污染物具有高效的降解效果,可以较为完全地将污染物转化成CO2、H2O等无害的物质;同时,该菌株也能不同程度地降解其他的乙醛、乙酸等厨余臭气中常见的污染物,因而在厨余垃圾堆肥产生的臭气处理中的生物净化技术中有广阔的应用前景。该菌株生长环境温和,易扩大培养。
本发明所述的蒙氏假单胞菌SD-2能将乙醇完全降解为无机物(CO2、H2O)和细胞生物质,实现完全矿化,且对于400mg/L以内的乙醇的去除率高达100%。而且,该蒙氏假单胞菌对于厨余垃圾常见的臭气成分(如乙醛)具有高效的降解能力,且能够承受较高浓度的污染物。
(四)附图说明
图1为菌株SD-2在LB培养基上菌落形态照片。
图2为菌株SD-2的透射电子显微镜照片。
图3为菌株SD-2的系统发育树图。
图4为蒙氏假单胞菌SD-2对于48h内不同浓度乙醇的降解曲线。
图5为蒙氏假单胞菌SD-2在48h内在不同pH对于400mg/L乙醇的降解效果。
(五)具体实施方式
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:
除非特别说明,下述实施例中使用的试剂原料为常规市购或商业途径获得的生化试剂原料,使用的实验仪器均为实验室常规仪器,除非特别说明,下述实施例中使用的方法和设备为本领域常规使用的方法和设备。
所述无机盐培养液组成为:K2HPO4·3H2O 0.942g/L、KH2PO4 0.234g/L、NaNO31.7g/L、NH4Cl 0.98g/L、MgCl·6H2O 0.2033g/L、CaCl·2H2O 0.11g/L、FeCl3 0.0162g/L、微量元素母液5ml/L,溶液去离子水,pH 7.0;其中微量元素母液组成:CuSO4·5H2O0.02g/L、FeSO4·7H2O 1.0g/L、MnSO4·4H2O 0.02g/L、CoCl·6H2O 0.02g/L、H3BO30.014g/L、ZnSO4·7H2O 0.10g/L,溶剂为去离子水。
所述LB固体培养基终浓度组成为:NaCl 10g/L、胰蛋白胨10g/L、酵母粉5g/L、琼脂18g-20g/L,pH自然,溶剂为去离子水。
所述LB液体培养基终浓度组成为:NaCl 10g/L、胰蛋白胨10g/L、酵母粉5g/L,pH自然,溶剂为去离子水。
实施例1:蒙氏假单胞菌(Pseudomonas monteilii)SD-2的分离、纯化及其鉴定。
1、蒙氏假单胞菌SD-2的分离、纯化。
蒙氏假单胞菌SD-2是从某厨余垃圾处理站曝气池活性污泥和厨余垃圾臭气处理设施的生物段循环液,具体步骤如下:
初筛:从现场采集含活性污泥的水样500ml,将水样放置于2L的量杯中,加入上述的无机盐培养液1.5L,以乙醇为碳源作为唯一碳源,控制浓度100mg/L,在室温下驯化15天,每天添加乙醇以提供足够的碳源,每隔三天更换无机培养液保证有氮源。
复筛:在250ml的摇瓶中加入50mL无机盐培养液,并加5mL初筛驯化中的上清液水样和浓度50mg/L的乙醇,在30℃进行富集培养,待乙醇浓度为初始加入浓度的50%时,从中取出5mL富集液于50mL新鲜无机盐培养液中,加入相同量的乙醇(使其浓度为50mg/L),重复上述富集过程5次后,将最后一次富集液用无菌水稀释1500倍后划线LB固体培养基,30℃培养,选择单菌落划线接种至LB固体培养基,30℃培养24h,菌落形态照片见图1。将单菌落加入无机盐培养液,并加入终浓度100mg/L乙醇作为唯一的碳源及能源,30℃培养24h,筛选能够生长的菌株,得到目的菌株SD-2,通过透射电子显微镜确定其形态(图2)。
2、菌株SD-2的鉴定
通过1 6S r R N A序列分析和生理生化实验鉴定,确定该菌株SD-2为Pseudomonas monteilii,具体步骤如下:
采用PrepMan Ultra Kits核酸萃取剂提取菌株SD-2的DNA,4℃保存。采用细菌通用引物(正向引物:5’-AGAGTTTGATCCTGGCTCAG-3’;反向引物:5’-GGTTACCTTGTTACGACTT-3’)在ABI公司PCR扩增仪中进行扩增。采用Applied Biosystems 3500基因分析仪对PCR纯化后产物进行核酸测序。测序工作由浙江天科高新技术发展有限公司完成。菌株的16SrDNA序列如下(Genebank登录号为OQ457019),SEQ ID NO.1:
AGTCGAGCGGATGACGGGAGCTTGCTCCTTGATTCAGCGGCGGACGGGTGAGTAATGCCTAGGAATCTGCCTGGTAGTGGGGGACAACGTTTCGAAAGGAACGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTTGCGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGGGGTAATGGCTCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCAGTAAGTTAATACCTTGCTGTTTTGACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTTGTTAAGTTGGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTGGCAAGCT AGAGTACGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGAATCCTTGAGATTTTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGCCTTGACATGCAGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCTGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTTATGGTGGGCACTCTAAGGAGACTGCCGGTGACAACCCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCTGGGCTACACACGTGCTACAATGGTCGGTACAGAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCTCACAAATCCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTGCACCAGAAGTAGCTAGT。
菌株SD-2对梅里埃GN卡上62种碳源的利用能力:利用梅里埃全自动鉴定仪考察菌株对62种不同碳源的代谢情况(委托给浙江天科高新技术发展有限公司(原浙江省微生物研究所))。鉴定结果如表1所示。经梅里埃全自动鉴定仪VITEK生化反应,菌株SD-2可较强利用13种碳源,对其它49种碳源不能利用。
表1菌株SD-2梅里埃全自动鉴定仪VITEK生化反应结果(GN卡)
表注:+,阳性反应:-:阴性反应
通过对菌株SD-2的形态特征及生理生化特征进行研究和分析,同时将该菌序列同NCBI数据库中的基因序列进行Blast对比,结合16s RNA同源性分析构建系统发生树(如图3),从而确定菌株SD-2为Pseudomonas monteilii,命名为蒙氏假单胞菌(Pseudomonasmonteilii)SD-2,保藏于中国典型培养物保藏中心,保藏编号:CCTCC NO:M2023241,保藏日期:2023年03月03日,地址:中国,武汉,武汉大学,430072。
实施例2、蒙氏假单胞菌SD-2静息细胞的获得。
1、斜面培养:
将蒙氏假单胞菌SD-2接种至LB液体培养基中,在30℃,160rpm下培养24~36h,再将活化的细菌划线于固体LB平板,30℃培养箱培养,取单菌落继续平板画线以检测细菌的纯度,进行LB试管斜面常规(4℃)保存。
2、扩大培养
将步骤1中的斜面菌体接种至LB液体培养基中,在30℃,160rpm下培养24~36h,获得的扩大培养液,离心,收集湿菌体,灭菌的去离子水洗涤,获得蒙氏假单胞菌SD-2静息细胞。
实施例3:蒙氏假单胞菌SD-2对不同浓度的乙醇的降解性能的检测。
将无机盐培养液分装在体积均为250ml的摇瓶中,每瓶50ml,110℃灭菌40min。灭菌结束后室温放置2d,确定无杂菌生长。加入终浓度达到50mg/L(以细胞干重记)实施例2方法获得的静息细胞,然后加入乙醇作为唯一的碳源使其终浓度分别为315.6、394.5、473.4、552.3、631.2mg/L,摇瓶密封后,30℃,160rpm摇床培养,并做不加细菌的空白对照。定时测定摇瓶中残留的乙醇浓度,绘制菌株在不同的初始浓度乙醇随着时间变化的去除率曲线,结果见图4所示。结果表明,当乙醇浓度低于400mg/L,菌株SD-2可以快速地降解所有添加的底物。
采用福立9790II气相色谱仪检测气相中乙醇的浓度。色谱柱为KB-5(30m×0.32mm×0.5μm)。进样口、检测器(FID)和柱温分别为120、200和80℃,辅助炉温度为100℃,分流比为100:1。氢气流量为30mL/min,气流为300mL/min,载气氮气流量为30mL/min,气体注入量为1mL。
实施例4:蒙氏假单胞菌SD-2在不同初始pH环境下对于400mg/L的乙醇的降解性能检测。
用1mol/L NaOH水溶液或1mol/L HCl水溶液调节无机盐培养液为不同pH值(5.0、6.0、7.0、8.0、9.0),在初始乙醇浓度为400mg/L的条件下接入实施例2方法制备的蒙氏假单胞菌SD-2静息细胞,使各平行样中的初始细胞干重为50mg/L。将样品于30℃,160rpm恒温摇床里振荡培养,并做不加细菌的空白对照。定时测定摇瓶中残留的乙醇浓度,绘制菌株不同pH环境下乙醇随着时间变化的去除率曲线以及在48h内不同pH对于400mg/L的乙醇的降解率,结果见图5所示。结果表明,在各pH下,蒙氏假单胞菌SD-2均能降解乙醇,且在pH为6-8时对乙醇的降解效果最好。
实施例5:蒙氏假单胞菌SD-2对不同碳源底物的降解能力
在实际应用中,不仅仅存在乙醇这一种有机污染物,厨余垃圾臭气一般包含多种挥发性有机废气。因此,研究蒙氏假单胞菌SD-2对其他底物的降解效果很有必要,采用实施例3实验操作,初始细胞干重为50mg/L,在pH值为7.0,将碳源改为初始浓度100mg/L的甲醇、D-柠檬烯、乙酸、乙醛、二硫化碳、氨水、二硫化碳和甲硫醇,其他操作同实施例3,降解效果如表2所示。
表2不同碳源降解效果
注:“+”有降解能力;“-”无降解能力。
Claims (8)
1.蒙氏假单胞菌(Pseudomonas monteilii)SD-2,保藏于中国典型培养物保藏中心,保藏编号:CCTCC NO:M2023241,保藏日期:2023年03月03日,地址:中国,武汉,武汉大学,430072。
2.一种权利要求1所述蒙氏假单胞杆菌SD-2在降解有机污染物中的应用。
3.如权利要求2所述的应用,其特征在于,所述的应用是将蒙氏假单胞杆菌SD-2接种至pH=5-9、含有机污染物的无机盐培养液中,在25-35℃、100-200rpm条件下进行培养,实现有机污染物的降解。
4.如权利要求3所述的应用,其特征在于,所述有机污染物包括乙醇、乙醛、乙酸、甲醇。
5.如权利要求3所述的应用,其特征在于,所述无机盐培养液中有机污染物的初始浓度为315.6-613.2mg/L。
6.如权利要求3所述的应用,其特征在于,所述蒙氏假单胞杆菌SD-2以静息细胞形式加入,所述无机盐培养液中静息细胞加入量以菌体干重计为10-100mg/L。
7.如权利要求3所述的应用,其特征在于,所述无机盐培养液组成为:K2HPO4·3H2O0.942g/L、KH2PO4 0.234g/L、NaNO3 1.7g/L、NH4Cl 0.98g/L、MgCl·6H2O 0.2033g/L、CaCl·2H2O 0.11g/L、FeCl3 0.0162g/L、微量元素母液5ml/L,溶液去离子水,pH 7.0;其中微量元素母液组成:CuSO4·5H2O 0.02g/L、FeSO4·7H2O 1.0g/L、MnSO4·4H2O 0.02g/L、CoCl·6H2O 0.02g/L、H3BO3 0.014g/L、ZnSO4·7H2O 0.10g/L,溶剂为去离子水。
8.如权利要求3所述的应用,其特征在于,所述蒙氏假单胞菌SD-2以静息细胞形式接种,所述静息细胞按如下步骤制备:
(1)斜面培养:将蒙氏假单胞菌SD-2接种于斜面LB固体培养基,30℃培养24~36h,获得斜面菌体;所述LB固体培养基终浓度组成为:NaCl 10g/L、蛋白胨10g/L、酵母粉5g/L、琼脂18~20g/L,溶剂为去离子水,pH值自然;
(2)扩大培养:用接种环挑取步骤(1)获得的斜面菌体接种至LB液体培养基中,30℃培养24~36h,获得扩大培养液,离心,收集湿菌体,灭菌的去离子水洗涤,获得蒙氏假单胞杆菌SD-2静息细胞;所述LB液体培养基终浓度组成为:NaCl 10g/L、蛋白胨10g/L、酵母粉5g/L,溶剂为去离子水,pH值自然。
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