CN116640681A - 一株拉贝洛尔降解菌及其应用 - Google Patents
一株拉贝洛尔降解菌及其应用 Download PDFInfo
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Abstract
本发明公开了一株拉贝洛尔降解菌及其应用。该拉贝洛尔降解菌为芽孢杆菌(Bacillussp.)QF12,其保藏编号为:GDMCC No:62562。所述的芽孢杆菌QF12在拉贝洛尔为环境水体相关的污染浓度0.5mg/L时,27~30℃,pH 7~8的条件下能够快速降解拉贝洛尔,降解率达99%以上。因此,本发明提供的芽孢杆菌QF12对拉贝洛尔的降解速率快,可用于降解拉贝洛尔,为含有拉贝洛尔的城市污水及医疗废水处理提供了一种新的种质资源。
Description
技术领域
本发明属于环境有机污染物生物处理技术领域,具体涉及一株拉贝洛尔高效降解菌及其应用。
背景技术
拉贝洛尔是一种兼有α和β肾上腺素能受体阻滞剂作用的降血压药物,能放缓窦性心律,减少外周血管阻力,并对妊娠高血压综合征有特殊疗效,广泛用于轻度至重度高血压和心绞痛的治疗。目前,全球现有高血压患者12.8亿人,其中中国有高血压患者3.85亿人(马丽媛等,2022)。庞大的高血压患者群体导致降压药拉贝洛尔的长期和大量使用。大部分拉贝洛尔使用以后不能被患者完全吸收,其中60%左右以原药形式随患者代谢物进入城市污水管网(Salem et al.,2012)。但是,现有城市污水处理系统对拉贝洛尔的去除效果有限,导致其以原药或人体代谢产物的形式持续排放并残留在受纳环境。调查发现,降血压药物拉贝洛尔在世界各地的污水处理厂进水、污水处理厂出水及地表水中均普遍检出,其浓度可达数10μg/L(Salem et al.,2012;Serrano et al.,2016)。水环境中存在的拉贝洛尔能造成斑马鱼胚胎发育异常(Bittner et al.,2018),长期暴露还能对鱼、小鼠和人的肝脏造成损伤(Yang et al.,2021)。因此,水环境中残留的拉贝洛尔对生态环境和人类健康构成潜在威胁,研究水环境中拉贝洛尔的消减技术十分必要。
微生物降解是环境中β受体阻滞剂降血压药物污染物自然消减的一种重要途径,也是最经济绿色的方法之一。城市污水处理厂、医院废水和地表水等环境水体中频繁持续地检出拉贝洛尔等β受体阻滞剂降血压药物,表明现有污水处理工艺对该类污染物的去除效果有限,有必要开发针对性更强,更高效和适用性更大的微生物降解去除方法。
发明内容
本发明旨在克服现有去除α和β肾上腺素能受体阻滞剂拉贝洛尔所存在的技术空白,提供一株拉贝洛尔高效降解菌,该菌株可快速降解拉贝洛尔。
本发明的拉贝洛尔高效降解菌,为芽孢杆菌(Bacillus sp.)QF12,其于2022年6月24日保藏于广东省微生物菌种保藏中心(GDMCC),地址:广东省广州市先烈中路100号大院59号楼5楼广东省科学院微生物研究所,邮编:510070,保藏编号:GDMCC No:62562。
本发明的第二个目的是提供一种生物制剂,包含上述的芽孢杆菌(Bacillus sp.)QF12。
优选的,所述的生物制剂还包含能延长菌株活性时间的辅料,或其它生物制剂可接受的辅料。
本发明的第三个目的是提供上述的芽孢杆菌(Bacillus sp.)QF12或生物制剂在降解拉贝洛尔中的应用。优选的,是在降解污染水体环境中的拉贝洛尔的应用。
优选的,所述的污染水体环境包括医疗废水和生活污水。
优选的,所述的降解适用的温度条件为30℃;适用的酸碱条件为pH 7.4;适用的培养条件是:在无机盐培养基中碳源为拉贝洛尔,或碳氮源为拉贝洛尔与其他共代谢底物,所述其他共代谢底物为蛋白胨。
本发明的第四个目的是提供一种降解拉贝洛尔的方法,将上述的芽孢杆菌(Bacillus sp.)QF12或生物制剂投放于拉贝洛尔污染水体环境中。优选的,所述的污染水体环境包括医疗废水和生活污水。
本发明具有以下技术优势:
(1)本发明提供的芽孢杆菌(Bacillus sp.)QF12能够降解拉贝洛尔。该芽孢杆菌(Bacillussp.)QF12在拉贝洛尔为环境水体相关的污染浓度0.5mg/L时,27~30℃,pH 7~8的条件下能够快速降解拉贝洛尔,降解率达99%以上。
(2)本发明提供的芽孢杆菌(Bacillus sp.)QF12对拉贝洛尔的降解速率快,可用于降解拉贝洛尔,为含有拉贝洛尔的城市污水及医疗废水处理提供了一种新的种质资源。
本发明的芽孢杆菌(Bacillus sp.)QF12于2022年6月24日保藏于广东省微生物菌种保藏中心(GDMCC),地址:广东省广州市先烈中路100号大院59号楼5楼广东省科学院微生物研究所,邮编:510070,保藏编号:GDMCC No:62562。
附图说明
图1为芽孢杆菌(Bacillus sp.)QF12在MH琼脂培养基上生长的菌落图。
图2为芽孢杆菌(Bacillus sp.)QF12在MH肉汤培养基中,以30℃,180rpm条件培养时的生长曲线。
图3为在MH肉汤培养基中芽孢杆菌(Bacillus sp.)QF12对拉贝洛尔的降解效果。
具体实施方式
以下实施例是对本发明的进一步说明,而不是对本发明的限制。
实施例1:芽孢杆菌(Bacillus sp.)QF12的筛选及鉴定
前期以广东省某大型生活污水处理厂的好氧池活性污泥为菌源,经人工富集培养、有目的性地分离纯化得到5种单菌。本发明在此基础上,筛选出一株拉贝洛尔的高效降解菌,具体实施过程如下:
取30个50mL玻璃试管,分别加入20mL灭菌后的MH肉汤培养基,其中15个作为实验组分别加入5种已培养至对数期的单菌,每种单菌各3个平行,另取15个三角瓶不加细菌作为对照组。向实验组和对照组中分别加入拉贝洛尔,使其最终浓度为0.5mg/L。将实验组和对照组的试管用活塞封口并用锡纸包好避光,放入摇床,于30℃,180rpm恒温培养,分别于0h,32h,40h,48h,56h,64h,72h,80h取样上机检测,根据肉汤中拉贝洛尔的残留量,筛选出具有降解拉贝洛尔能力的单菌。5种课题组现存细菌中共有2种可以降解拉贝洛尔,其中处于对数期的菌株QF12在刚添加拉贝洛尔的T0时刻能少量降解拉贝洛尔,且在60小时内已将拉贝洛尔降解完全,具有降解速率优势。
将菌株QF12送往上海生工生物有限公司,进行16S rDNA鉴定:16S引物序列为:27F:5'-AGAGTTTGATCMTGGCTCAG-3';1492R:5'-TACGGYTACCTTGTTACGACTT-3',以细菌总DNA为模板,PCR反应程序如下:95℃变性5min,95℃30s,55℃30s,72℃60s,共34个循环;72℃10min,12℃保存。将得到的16S rDNA序列(如SEQ ID NO.1所示)登录GenBank,进行Blast同源比对,经比对发现,菌株QF12与Bacillus cereus的同源性能够达到100%。
16S rDNA序列如SEQ ID NO.1所示,具体为:
GGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGC
TTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAACGGTTTTATGAGATTAGCTCCACCTCG
CGGTCTTGCAGCTCTTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATG
ATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTTAATG
ATGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTG
ACGACAACCATGCACCACCTGTCACTCTGCTCCCGAAGGAGAAGCCCTATCTCTAGGGTTTTCAGA
GGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGC
GGGCCCCCGTCAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGGAGTGCTTAATGCG
TTAACTTCAGCACTAAAGGGCGGAAACCCTCTAACACTTAGCACTCATCGTTTACGGCGTGGACTAC
CAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGTGTCAGTTACAGACCAGAAAGTC
GCCTTCGCCACTGGTGTTCCTCCATATCTCTACGCATTTCACCGCTACACATGGAATTCCACTTTCCT
CTTCTGCACTCAAGTCTCCCAGTTTCCAATGACCCTCCACGGTTGAGCCGTGGGCTTTCACATCAGA
CTTAAGAAACCACCTGCGCGCGCTTTACGCCCAATAATTCCGGATAACGCTTGCCACCTACGTATTA
CCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCAGCTTATTC
AACTAGCACTTGTTCTTCCCTAACAACAGAGTTTTACGACCCGAAAGCCTTCATCACTCACGCGGC
GTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGC
CGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTTGCCTTGGTGAGCC
GTTACCTCACCAACTAGCTAATGCGACGCGGGTCCATCCATAAGTGACAGCCGAAGCCGCCTTTCAA
TTTCGAACCATGCGGTTCAAAATGTTATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAGTCTTA
TGGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACTTCATAAGAGCAAGCTCTTAATCCATTCGCTCGACTTGCATGTATTA。
菌株QF12在鉴定培养基(MH肉汤培养基)纯培养条件下,菌落呈圆形(图1),表面光滑,乳状,边缘呈锯齿状,呈乳白色,中间颜色深,边缘颜色浅。在MH肉汤培养基中,以30℃,180rpm条件培养时的生长曲线如图2所示。进行系列生理生化试验,结果为:溶菌酶耐性试验阳性,硝酸盐还原反应阳性,过氧化氢酶试验阳性,明胶水解试验阴性;淀粉水解试验阳性,西蒙氏柠檬酸盐试验阴性,V-P试验阳性,葡萄糖试验阳性,甘露醇试验阴性,动力学培养基试验阳性,3%过氧化氢试验阳性,革兰氏试验阳性。
将菌株QF12的16S rDNA基因序列结合其形态及其他生理生化指标进行分析,初步鉴定菌株QF12属于Bacillus sp.,将其命名为:芽孢杆菌(Bacillus sp.)QF12,其于2022年6月24日保藏于广东省微生物菌种保藏中心(GDMCC),地址:广东省广州市先烈中路100号大院59号楼5楼广东省科学院微生物研究所,邮编:510070,保藏编号:GDMCC No:62562。
实施例2:芽孢杆菌(Bacillus sp.)QF12对水体中拉贝洛尔的降解研究
将150mL含有拉贝洛尔的MH肉汤培养基置于250mL洁净锥形瓶中,使拉贝洛尔最终浓度为0.5mg/L,锥形瓶均分成两组,分别为有菌实验组和无菌对照组:(1)一组添加芽孢杆菌(Bacillus sp.)QF12,设置为有菌实验组;(2)另一组不添加芽孢杆菌(Bacillus sp.)QF12,灭菌后加入浓度为质量分数0.5%的叠氮化钠抑菌,设置为无菌对照组。所有锥形瓶均用可透空气的封口膜封口,外壁包裹锡纸避光,置于摇床中(30℃,180rpm)培养;实验设置3个平行,连续取样测定培养液中拉贝洛尔的残留浓度,分析芽孢杆菌(Bacillus sp.)QF12对拉贝洛尔的降解能力,结果如图3所示。由图3可见,芽孢杆菌(Bacillus sp.)QF12可在60小时内完全降解0.5mg/L拉贝洛尔,未添加菌株的对照组中拉贝洛尔浓度基本没变化。本实施例充分说明了芽孢杆菌(Bacillus sp.)QF12对水体中存在的拉贝洛尔具有良好的降解效果。
以上仅是本发明的优选实施方式,应当指出的是,上述优选实施方式不应视为对本发明的限制,本发明的保护范围应当以权利要求所限定的范围为准。对于本技术领域的普通技术人员来说,在不脱离本发明的精神和范围内,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.芽孢杆菌(Bacillus sp.)QF12,其保藏编号为:GDMCC No:62562。
2.一种生物制剂,其特征在于,包含权利要求1所述的芽孢杆菌(Bacillus sp.)QF12。
3.根据权利要求2所述的生物制剂,其特征在于,所述的生物制剂还包含能延长菌株活性时间的辅料,或其它生物制剂可接受的辅料。
4.权利要求1所述的芽孢杆菌(Bacillus sp.)QF12或权利要求2所述的生物制剂在降解拉贝洛尔中的应用。
5.根据权利要求4所述的应用,其特征在于,是在降解污染水体环境中的拉贝洛尔的应用。
6.根据权利要求4所述的应用,其特征在于,所述的污染水体环境包括医疗废水和生活污水。
7.根据权利要求4所述的应用,其特征在于,所述的降解适用的温度条件为30℃。
8.根据权利要求4所述的应用,其特征在于,所述的降解适用的酸碱条件为pH 7.4。
9.一种降解拉贝洛尔的方法,其特征在于,将权利要求1所述的芽孢杆菌(Bacillussp.)QF12或权利要求2所述的生物制剂投放于拉贝洛尔污染水体环境中。
10.根据权利要求9所述的方法,其特征在于,所述的污染水体环境包括医疗废水和生活污水。
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