CN111205997A - 一种甲基芽孢杆菌及其在降解环境中微污染物方面的应用 - Google Patents
一种甲基芽孢杆菌及其在降解环境中微污染物方面的应用 Download PDFInfo
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Abstract
本发明公开了一种甲基芽孢杆菌,其命名为甲基芽孢杆菌(Bacillus methylotrophicus)BP1.1,于2019年12月20日保藏于中国典型培养物保藏中心,保藏编号为CCTCC M 20191078。本发明还公开了上述甲基芽孢杆菌在降解二苯甲酮类紫外防晒剂中的应用。本发明通过对生活污水厂活性污泥逐级驯化后,获得对二苯甲酮类紫外防晒剂有高效去除效率的甲基芽孢杆菌(Bacillus methylotrophicus)BP1.1,该甲基芽孢杆菌(Bacillus methylotrophicus)BP1.1能够有效去除水环境中的二苯甲酮类紫外防晒剂。
Description
技术领域
本发明涉及一种甲基芽孢杆菌,还涉及上述甲基芽孢杆菌在降解水中二苯甲酮类防晒剂中的应用,属于污水处理领域。
背景技术
二苯甲酮类(BPs)紫外防晒剂因其具有安全性高、防晒效果好且价格低廉等特点,已被广泛应用于个人护理品中。由于大部分紫外防晒剂持久性好,随着日常使用量的增加,二苯甲酮类紫外防晒剂源源不断地进入到环境水体中,城市污水处理厂却不能将其完全去除,最终会汇入江河湖泊并不断累积,在水环境中检测到紫外防晒剂也越来越常见。其中,二苯甲酮类紫外防晒剂是亲脂性物质,具有内分泌干扰效应和生物积累性,并在环境中广泛存在,成为一种新兴微污染物。
光化学转化是紫外防晒剂在天然水体中的重要转化途径,其光化学转化反应速率及途径受水环境的pH、溶解性物质等影响,且BPs光照下可产生生态毒性更大的代谢产物。高级氧化技术是利用在反应过程中产生的强氧化性物质——羟基自由HO·基为特性对二苯甲酮进行降解,目前报道最多的技术主要包括:UV/H2O2、O3/H2O2、UV/O3、Fenton 等,高级氧化反应所投加的化学试剂及其在转化过程中产生的部分产物均存在潜在的环境风险。微生物降解作用是自然界有机物最终矿化的唯一途径,是决定有机污染物环境归宿的关键。由于BPs的生物可降解率不高,导致对BPs的微生物降解研究相对较少。最新研究发现,活性污泥在好氧条件下BPs的生物降解率可达到60%,但是降解时间较长,一般为10~20d。
发明内容
本发明所要解决的技术问题是提供一种能够在好氧条件下快速降解水环境中二苯甲酮类紫外防晒剂的微生物——甲基芽孢杆菌。
技术方案:本发明所述的甲基芽孢杆菌命名为甲基芽孢杆菌(Bacillusmethylotrophicus)BP1.1,分类命名为甲基芽孢杆菌(Bacillus methylotrophicus),菌株号为BP1.1,现已保藏于位于中国武汉的中国典型培养物保藏中心,保藏编号为CCTCC M20191078,保藏日期为2019年12月20日。
上述菌株——甲基芽孢杆菌(Bacillus methylotrophicus)BP1.1,在LB培养基中, 30℃好氧条件下生长良好,菌落呈圆形,直径大小0.2~1mm,菌落颜色略带淡粉色,不透明,表面干燥,无褶皱。革兰氏染色呈阳性,显微镜观察下细胞形状为短杆状。
本发明还要解决的技术问题是提供上述甲基芽孢杆菌在降解环境中微污染物方面的应用。
所述微污染物为二苯甲酮类紫外防晒剂;二苯甲酮类紫外防晒剂为2,4-二羟基二苯甲酮(BP-1)。
所述的应用为:将甲基芽孢杆菌(Bacillus methylotrophicus)BP1.1按接种量1%接种于扩大培养基中,于28~30℃下好氧培养24~48h,培养后得到BP1.1扩培产物,然后将扩培产物接种于含二苯甲酮类紫外防晒剂污水中进行处理。污水中甲基芽孢杆菌(Bacillus methylotrophicus)BP1.1的接种量为1‰~2‰,污水中二苯甲酮类紫外防晒剂的质量浓度为10mg/L。
菌体应用时,含二苯甲酮类紫外防晒剂的污水的温度为20~30℃,优选为28℃,pH值为7.3~8.0,优选为7.4,此条件下有利于加快甲基芽孢杆菌(Bacillusmethylotrophicus)BP1.1对污水中二苯甲酮类物质的降解速率。
有益效果:本发明的甲基芽孢杆菌在好氧条件下对二苯甲酮类紫外防晒剂的生物降解率可达到100%,且显著缩短其降解时间;本发明利用生物法快速高效降解污水中新兴微污染物BP-1,对污水中新兴微污染物的处理有重要意义。
附图说明
图1为本发明实施例2中甲基芽孢杆菌(Bacillus methylotrophicus)BP1.1革兰氏染色显微镜下形态图;
图2为本发明实施例2中甲基芽孢杆菌(Bacillus methylotrophicus)BP1.1系统发育树;
图3为本发明实施例3中甲基芽孢杆菌(Bacillus methylotrophicus)BP1.1对2,4-二羟基二苯甲酮降解速率图。
具体实施方式
以下结合附图和具体实施例对本发明的技术方案做进一步说明。
实施例1
本发明中,是从南京某生活污水处理厂生物好氧段活性污泥中筛选获得的甲基芽孢杆菌(Bacillus methylotrophicus)BP1.1。
具体过程如下:
(1)从南京某生活污水处理厂生物好氧段取活性污泥;
(2)将得到的泥水混合物取250mL盛装于锥形瓶中,在磁力搅拌器上常温搅拌,每天向其中加入5mg 2,4-二羟基二苯甲酮;每次加药前倒掉上部分上清液(检测上清液中是否含有目标污染物-2,4-二羟基二苯甲酮),然后用自来水补足,如此驯化直至溶液中检测不到目标污染物,则完成驯化;
(3)取出步骤(2)中得到的污泥,将污泥经过梯度稀释后,分别涂布于含2,4-二羟基二苯甲酮的无机盐培养基中,30℃条件下静态培养3~5d,获得单菌落;无机盐培养基中2,4-二羟基二苯甲酮的质量浓度为50mg/L;
(4)挑取步骤(3)中的单菌落划线纯化后,于斜面上划线保存,得到纯菌株。
纯菌株的扩大培养采用的培养基为LB培养基、无机盐培养基或牛肉膏蛋白胨培养基中的一种。
LB培养基包括如下质量的组分:每升水中含酵母粉5.0g、氯化钠10g和蛋白胨10g,培养基的pH=7.0~7.5。
无机盐培养基包括如下质量的组分:每升水中含2,4-二羟基二苯甲酮10~50mg、氢氧化钠1.6g和磷酸二氢钾6.8g,培养基的pH=7.3~7.5。本发明的甲基芽孢杆菌(Bacillus methylotrophicus)BP1.1能够以2,4-二羟基二苯甲酮化合物作为唯一碳源进行生长繁殖。
牛肉膏蛋白胨培养基包括如下质量的组分:每升水中含牛肉3g、蛋白胨10g和氯化钠5g,培养基的pH=7.2~7.6。
将步骤(4)得到的甲基芽孢杆菌(Bacillus methylotrophicus)BP1.1接种(接种量为1%(体积分数))于扩大培养基中进行扩培,于28~30℃下好氧培养24~48h,培养后得到BP1.1的扩培产物。
实施例2
对实施例1筛选的菌种进行鉴定。
本发明实施例1中,筛选得到降解效果最佳、生长速度最快的菌株BP1.1。其中,经鉴定,BP1.1为甲基芽孢杆菌(Bacillus methylotrophicus)。
甲基芽孢杆菌(Bacillus methylotrophicus)BP1.1,在LB培养基中,30℃好氧条件下生长良好,菌落呈圆形,直径大小0.2~1mm,菌落颜色略带淡粉色,不透明,表面干燥,无褶皱。革兰氏染色呈阳性,显微镜观察下细胞形状为短杆状。
通过PCR扩增和Sanger法测序,得到BP1.1菌株16S rRNA全序列如下(SEQ IDNO.1):
BP1.1测序结果提交GenBank进行BLAST比较,从BLAST结果中挑选与BP1.1 碱基序列相似度98%以上的菌株序列,通过ClustalW进行多重系列比对,并利用MAGE 7.0构建系统发育树得到BP1.1与甲基芽孢杆菌(Bacillus methylotrophicus)亲缘关系最近,因此鉴定菌株BP1.1为甲基芽孢杆菌。
实施例3
本发明甲基芽孢杆菌(Bacillus methylotrophicus)BP1.1对污染物-2,4-二羟基二苯甲酮降解速率进行测定,具体步骤如下:
(1)将BP1.1经过扩大培养后菌液于6000rpm下离心10min,倒掉上清液,得到菌体;
(2)取步骤(1)中得到的菌体按1.8‰(质量分数)接种量,打散,加入含2,4- 二羟基二苯甲酮的水溶液中,水溶液中2,4-二羟基二苯甲酮的质量浓度为10mg/L;并以不加BP1.1的处理组做对照;
(3)将步骤(2)中的对照组与处理组放入摇床中,好氧条件下,在150rpm,28℃下振荡反应;
(4)定时取样检测步骤(3)中处理组与对照组中污染物的剩余浓度,结果见图3。
如图3所示,BP1.1能够高效降解水中2,4-二羟基二苯甲酮,10mg/L的2,4-二羟基二苯甲酮在6h内可被完全降解。处理前期2h内,菌株BP1.1在降解体系中有一定的适应性时间,此阶段水中BP1.1降解速率较慢,适应完成后(2~5h),进入到一个快速降解阶段;降解后期(5~6h),由于底物浓度的降低,降解速率逐步变缓。
本发明通过对生活污水厂好氧段的活性污泥逐级驯化后,获得对二苯甲酮类紫外防晒剂有高效去除效率的甲基芽孢杆菌(Bacillus methylotrophicus)BP1.1。本发明的甲基芽孢杆菌在好氧条件下对二苯甲酮类紫外防晒剂的生物降解率可达到100%,且显著缩短了生物降解时间。
序列表
<110> 南京大学
<120> 一种甲基芽孢杆菌及其在降解环境中微污染物方面的应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1463
<212> DNA
<213> 甲基芽孢杆菌(Bacillus methylotrophicus)
<400> 1
agggggcggg gcggcgtgct atacatgcaa gtcgagcgga cagatgggag cttgctccct 60
gatgttagcg gcggacgggt gagtaacacg tgggtaacct gcctgtaaga ctgggataac 120
tccgggaaac cggggctaat accggatggt tgtctgaacc gcatggttca gacataaaag 180
gtggcttcgg ctaccactta cagatggacc cgcggcgcat tagctagttg gtgaggtaac 240
ggctcaccaa ggcgacgatg cgtagccgac ctgagagggt gatcggccac actgggactg 300
agacacggcc cagactccta cgggaggcag cagtagggaa tcttccgcaa tggacgaaag 360
tctgacggag caacgccgcg tgagtgatga aggttttcgg atcgtaaagc tctgttgtta 420
gggaagaaca agtgccgttc aaatagggcg gcaccttgac ggtacctaac cagaaagcca 480
cggctaacta cgtgccagca gccgcggtaa tacgtaggtg gcaagcgttg tccggaatta 540
ttgggcgtaa agggctcgca ggcggtttct taagtctgat gtgaaagccc ccggctcaac 600
cggggagggt cattggaaac tggggaactt gagtgcagaa gaggagagtg gaattccacg 660
tgtagcggtg aaatgcgtag agatgtggag gaacaccagt ggcgaaggcg actctctggt 720
ctgtaactga cgctgaggag cgaaagcgtg gggagcgaac aggattagat accctggtag 780
tccacgccgt aaacgatgag tgctaagtgt tagggggttt ccgcccctta gtgctgcagc 840
taacgcatta agcactccgc ctggggagta cggtcgcaag actgaaactc aaaggaattg 900
acgggggccc gcacaagcgg tggagcatgt ggtttaattc gaagcaacgc gaagaacctt 960
accaggtctt gacatcctct gacaatccta gagataggac gtccccttcg ggggcagagt 1020
gacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa 1080
cgagcgcaac ccttgatctt agttgccagc attcagttgg gcactctaag gtgactgccg 1140
gtgacaaacc ggaggaaggt ggggatgacg tcaaatcatc atgcccctta tgacctgggc 1200
tacacacgtg ctacaatgga cagaacaaag ggcagcgaaa ccgcgaggtt aagccaatcc 1260
cacaaatctg ttctcagttc ggatcgcagt ctgcaactcg actgcgtgaa gctggaatcg 1320
ctagtaatcg cggatcagca tgccgcggtg aatacgttcc cgggccttgt acacaccgcc 1380
cgtcacacca cgagagtttg taacacccga agtcggtgag gtaaccttta tggagccagc 1440
cgccgaaggt gaacccggag ttg 1463
Claims (9)
1.一种甲基芽孢杆菌,其特征在于:命名为甲基芽孢杆菌(Bacillusmethylotrophicus)BP1.1,于2019年12月20日保藏于中国典型培养物保藏中心,保藏编号为CCTCC M 20191078。
2.权利要求1所述的甲基芽孢杆菌在降解环境中微污染物方面的应用。
3.根据权利要求2所述的甲基芽孢杆菌在降解环境中微污染物方面的应用,其特征在于:所述微污染物为二苯甲酮类紫外防晒剂;二苯甲酮类紫外防晒剂为2,4-二羟基二苯甲酮。
4.根据权利要求3所述的甲基芽孢杆菌在降解环境中微污染物方面的应用,其特征在于:甲基芽孢杆菌降解二苯甲酮类紫外防晒剂在好氧条件下进行。
5.根据权利要求2所述的甲基芽孢杆菌在降解环境中微污染物方面的应用,其特征在于:将甲基芽孢杆菌(Bacillus methylotrophicus)BP1.1接种于扩大培养基中,于28~30℃下好氧培养24~48h,培养后得到BP1.1扩培产物;将扩培产物接种于含二苯甲酮类紫外防晒剂污水中进行处理。
6.根据权利要求5所述的甲基芽孢杆菌在降解环境中微污染物方面的应用,其特征在于:污水中的甲基芽孢杆菌(Bacillus methylotrophicus)BP1.1接种量为1%0~2‰。
7.根据权利要求5所述的甲基芽孢杆菌在降解环境中微污染物方面的应用,其特征在于:污水中二苯甲酮类紫外防晒剂的质量浓度为10mg/L。
8.根据权利要求5所述的甲基芽孢杆菌在降解环境中微污染物方面的应用,其特征在于:菌体应用时,污水的温度为20~30℃,pH值为7.3~8.0。
9.根据权利要求5所述的甲基芽孢杆菌在降解环境中微污染物方面的应用,其特征在于:扩大培养基为LB培养基、无机盐培养基或牛肉膏蛋白胨培养基中的一种。
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