CN116790407B - 降解2,4-dnt和2,4-dnt-3-sa的高地芽孢杆菌d47及其应用 - Google Patents
降解2,4-dnt和2,4-dnt-3-sa的高地芽孢杆菌d47及其应用 Download PDFInfo
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- RMBFBMJGBANMMK-UHFFFAOYSA-N 2,4-dinitrotoluene Chemical compound CC1=CC=C([N+]([O-])=O)C=C1[N+]([O-])=O RMBFBMJGBANMMK-UHFFFAOYSA-N 0.000 title claims abstract description 27
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- 239000002689 soil Substances 0.000 claims abstract description 33
- 241000193830 Bacillus <bacterium> Species 0.000 claims abstract description 15
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- 239000003795 chemical substances by application Substances 0.000 claims abstract description 3
- 239000007788 liquid Substances 0.000 claims description 24
- 239000001963 growth medium Substances 0.000 claims description 15
- 241001150381 Bacillus altitudinis Species 0.000 claims description 14
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- DDYKUIIMQBQEAZ-UHFFFAOYSA-N (2,4-dinitrophenyl)methanesulfonic acid Chemical compound OS(=O)(=O)CC1=CC=C([N+]([O-])=O)C=C1[N+]([O-])=O DDYKUIIMQBQEAZ-UHFFFAOYSA-N 0.000 description 4
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Abstract
本发明涉及一株降解2,4‑DNT和2,4‑DNT‑3‑SA的高地芽孢杆菌D47及其应用,属于微生物工程领域,菌株于2023年2月27日保藏于中国普通微生物菌种保藏管理中心,保藏号为CGMCC26705。本发明还提供含有所述高地芽孢杆菌D47土壤处理剂。将上述菌株培养至对数生长期,分别接种到2,4‑DNT‑3‑SA浓度为500mg·kg‑1的污染土壤和2,4‑DNT浓度为50mg·kg‑1的污染土壤中,经过1‑4天处理后,2,4‑二硝基甲苯及2,4‑二硝基甲苯‑3‑磺酸盐的去除率均达到100%。该菌株对硝基化合物类有机污染土壤修复中具有很好的应用。
Description
技术领域
本发明属于微生物工程领域,具体地涉及一株降解2,4-DNT和2,4-DNT-3-SA的高地芽孢杆菌D47及其应用。
背景技术
2,4-二硝基甲苯(Dinitrotoluen,2,4-DNT)和2,4-二硝基甲苯-3-磺酸盐(Dinitrotoluene sulfonates,2,4-DNT-3-SA)还是***(Trinitrotoluene,TNT)生产过程中产生的“TNT红水”的主要污染成分。先前由于管理不当导致含2,4-DNT和2,4-DNT-3-SA的废水泄漏到土壤中,使生态环境遭受严重破坏,并威胁周边居民的身体健康。传统的物理与化学修复技术不仅成本高昂,而且还会对环境造成二次破坏,不适合2,4-DNT/DNT-3-SA污染土壤的规模化修复。
生物修复法是利用土壤生物(植物、动物和微生物)吸附、转化和降解土壤中的有机污染物,从而降低土壤中有机污染物含量,并转化为低/无毒害物质的过程。其中,微生物修复是指利用微生物代谢清除污染物的方式,是一种环境友好且成本较低的方法,可以利用微生物有效地去除土壤和水体中的污染物。目前,已经分离鉴定出多种可以用于降解硝基甲苯类污染物的微生物。例如,从硝基甲苯类污染场地中分离出能降解DNT的菌种Burkholderia sp.R34、Phanerochaete chrysosporium.S65和Alcaligenes sp.JS867等,最近也有研究报道从硝基甲苯类污染场地中分离出一株能代谢DNTS的恶臭假单胞杆菌Pseudomonas sp.X5。
但是,尚未有研究指出在土壤中分离出可以降解2,4-二硝基甲苯(2,4-DNT)和2,4-二硝基甲苯磺酸盐(2,4-DNT-3-SA)的微生物。
发明内容
本发明的目的在于解决“TNT红水”污染土壤治理问题,本发明提供了一种新的微生物种质资源,从土壤中分离出一株可以有效降解“TNT红水”污染土壤中主要污染物2,4-二硝基甲苯(2,4-DNT)又可降解2,4-二硝基甲苯磺酸盐(2,4-DNT-3-SA)。
本发明的技术方案为:
本发明公开了一株降解2,4-DNT和2,4-DNT-3-SA的高地芽孢杆菌D47(Bacillusaltitudinis),2023年2月27日保藏于中国普通微生物菌种保藏管理中心,保藏号为CGMCC26705,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。
本发明所提供的菌株Bacillus altitudinis D47从甘肃省白银市“TNT红水”污染土壤中分离、纯化、筛选得到。菌落呈黄色,圆形***,革兰氏染色阴性。显微镜下观察呈杆状形态,且在生长环境不适宜条件下能呈芽孢形态,该菌株最适生长温度为37℃,最适生长pH为7。上述菌株的16S rRNA基因序列特征,采用分析法将序列与数据库进行比对分析,发现该菌株属于高地芽孢杆菌属(Bacillus sp.),其DNA序列表如SEQ ID NO.1所示。
本发明还提供一种土壤处理剂,所述土壤处理剂中含有高地芽孢杆菌D47。
本发明还提供上述菌株的应用,具体方法为:高地芽孢杆菌D47菌液活化至对数生长期,将菌液使用MSM基础盐液体培养基按照1:1稀释后接种于含有2,4-DNT或2,4-DNT-3-S的污染土壤中,按照液土质量比为2:5,温度37℃,pH为7进行处理。
本发明与现有技术相比的有益效果:
本发明菌株能够有效果降解土壤中的2,4-DNT或2,4-DNT-3-S的污染物,将对数生长期的高地芽孢杆菌D47菌液按照液土比2:5的比例接种到2,4-DNT或2,4-DNT-3-S污染的土壤中,在2-4天降解率达到100%。
附图说明
图1为菌株Bacillus altitudinis D47的菌体形态图;
图2为不同温度对Bacillus altitudinis D47菌株的生长影响;
图3为不同pH对Bacillus altitudinis D47菌株的生长影响;
图4为Bacillus altitudinis D47对2,4-二硝基甲苯磺酸盐的降解率;
图5为Bacillus altitudinis D47对2,4-二硝基甲苯的降解率。
具体实施方式
实施例所用试剂如下:
MSM基础盐培养基:3.06g·L-1Na2HPO4·12H2O,0.76g·L-1KH2PO4,0.20g·L- 1MgSO4·7H2O,0.25g·L-1CaCl2,10mL·L-1微量元素溶液。
微量元素溶液:CuSO40.05 g,MnSO40.05 g,FeSO4.7H2O 0.05g,去离子水定容至50mL。
LB液体培养基:胰蛋白胨10g,酵母提取物5g,氯化钠10g,蒸馏水定容至1000mL。121℃灭菌15min。
LB固体培养基LB液体培养基中加入18g·L-1琼脂,121℃灭菌15min后取出倒置平板,冷却凝固后备用。
实施例1:降解2,4-DNT-3-SA菌株筛选鉴定。
称量50-100g“TNT红水”污染土壤(取自甘肃省白银市军工厂附近)于250mL三角瓶中,加入100mL液体LB培养基,于37℃恒温振荡摇床中振荡培养两个小时,使红土与水充分混匀,将滤纸过滤后的红水均匀的涂布在含有2,4-DNT-3-SA的固体LB培养基上,培养至长出肉眼可见的菌株。菌落呈黄色,圆形***,革兰氏染色阴性。显微镜下观察呈杆状形态,且在生长环境不适宜条件下能呈芽孢形态,如图1所示。高地芽孢杆菌D47(Bacillusaltitudinis),2023年2月27日保藏于中国普通微生物菌种保藏管理中心,保藏号为CGMCC26705,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。
挑取单克隆于液体LB培养基中过夜培养,再以1:100的比例接种于含2,4-DNT-3-SA的液体LB培养基中培养,剩余菌液加入甘油,保存于-80℃冰箱中。
用液相色谱仪(HPLC)检测培养基中2,4-DNT-3-SA的降解量,筛选高效降解DNTS的菌株。
将筛选出的高效降解DNTS的菌株取100μL送擎科生物科技公司进行16S rRNA测序分析。将测得的16S rDNA基因序列通过Blast进行相似序列搜索,将菌株的基因序列与Genbank中的16S rDNA进行比对,利用最相似序列确定其***发育地位。***进化分析结果表明,D47菌株与已有报道的高地芽孢杆菌Bacillus altitudinis 11-1-1最相近,其次是高地芽孢杆菌Bacillus altitudinis BIM-B-63,因此D47菌株为芽孢杆菌属的高地芽孢杆菌,最终我们将筛选获得的高效降解DNTS菌株命名为Bacillus altitudinis D47。
16S rDNA序列如下:
gctggctctt gttcgacttc ccccaatcat ctgccccacc ttcggcggctggctccataaaggttacctc accg acttcg ggtgttgcaa actctcgtgg tgtgacgggcggtgtgtacaaggcccggga acgtattcac cgcggcatgc tgatccgcga ttactagcgattccagcttcacgcagtcga gttgcagact gcgatccgaa ctgagaacag atttgtgg gattggctaaaccttgcggtct cgcagccctttgttctgtcc attgtagcac gtgtgtagcccaggtcataaggggcat gat gatttgacgt catccccacc ttcctccggt ttgtcaccggcagtcaccttagagtgccca actgaatgct ggcaa ctaag atcaagggtt gcgctcgttgcgggacttaacccaacatct cacgacacga gctgacgaca accatgcacc acctgtcactctgtccccgaagggaaagcc ctatctctag ggttgtcaga ggatgtcaag acctggtaag gttcttcgcgttgcttcgaattaaaccaca tgctccaccg cttgtgcggg cccccgtcaattcctttgagtttcagtcttgcgacc gtac tccccaggcg gagtgcttaa tgcgttagct gcagcactaaggggcggaaaccccctaacacttagcactc at cgtttacg gcgtggacta ccagggtatctaatcctgtt cgctccccacgctttcgctc ctcagcgtca gttacagacc agagagtcgccttcgccact ggtgttcctc cacatctctacgcatttcac cgctacacgt ggaattccactctcctcttc tgcactcaag tttcccagtttccaatgaccctccccggtt gagccgggggctttcacatc agacttaaga aaccgc ctgc gagcccttta cgcccaataattccggacaacgcttgccac ctacgtatta ccgcggctgc tggcacgtag tt agccgtgg ctttctggttaggtaccgtc aaggtgcaag cagttactcttgcacttgtt cttccctaac aacag agctt tacgatccgaaaaccttcat cactcacgcg gcgttgctcc gtcagacttt cgtccattgcggaagattcc ctactgc tgcctcccgtagg agtctgggcc gtgtctcagt cccagtgtggccgatcaccc tctcaggtcg gct acgcatcgtcgccttgg tgagccgtta cctcaccaactagctaatgc gccgcgggtc catctgtaag tgacagccgaaaccgt cttt catccttgaa ccatgcggtt caaggaacta tccggtatta gctccggttt cccggagttatcccagtcttacag gcaggt tacccacgtg ttactcaccc gtccgccgct aacatccgggagcaagctcccttctgtccg ctcgactgca gtatagcacg ccgccc.
实施例2:降解菌株基本特征描述。
将-80℃冰箱里保存的2,4-DNT-3-SA高效降解菌株接种于500μL液体LB培养基中,于37℃恒温振荡摇床中培养4-6h,当菌液明显变浑浊时,再将适量菌液分别接入含有50mL液体LB培养基的100mL三角瓶中,使初始OD600达到0.01,每种温度下设置三个重复,分别置于16℃、28℃、37℃和50℃恒温振荡摇床中,180rpm培养12h,取样稀释后,检测OD600值,通过比较12h后的OD600值确定2,4-DNT-3-S高效降解菌株的最适生长温度。如图2所示,B.altitudinis D47菌株最适宜生长温度为37℃。另外,28℃和50℃下,该菌株也能够呈现显著生长,但16℃下,则生长非常缓慢。
将-80℃冰箱里保存的2,4-DNT-3-S高效降解菌株接种于500μL液体LB培养基中,于37℃恒温振荡摇床中培养4-6h,当菌液明显变浑浊时,再将适量菌液分别接种于pH值为4、5、6、7、8、9、10的含有50mL液体LB培养基的100mL三角瓶中,使初始OD600值达到0.01,每种pH值下设置三个重复,置于37℃、180rpm恒温振荡摇床中,培养12h后,取样稀释后,检测OD600值,通过比较12h后的OD600值,确定2,4-DNT-3-S高效降解菌株的最适生长pH值。从图3所示,B.altitudinis D47菌株适宜生长的pH值区间为pH 5-9,最适生长pH值为7。
如图3所示,缺少结果描述,比如各pH值情况下的菌株生长对比情况,得出菌株的最适生长pH值为7。
实施例3:Bacillus altitudinis D47对2,4-二硝基甲苯磺酸盐的降解率。
取未污染土壤,风干研磨、过1mm筛,备用。称取一定量的2,4-DNT-3-SA溶于丙酮中。在通风橱中,向上述土壤中均匀喷洒含有2,4-DNT-3-SA的丙酮溶液,并搅拌均匀。使土壤浓度为2,4-DNT-3-SA的浓度为500mg·kg-1;在通风橱中使其自然风干2天。
菌株活化:从-80℃冰箱取出甘油中保存的2,4-DNT-3-S微生物降解菌株,接种于500μL液体LB培养基中,于37℃恒温振荡摇床中培养4-6h至对数生长期,收集菌体,使用等比例添加MSM基础盐液体培养基重悬菌体。
2,4-DNT-3-SA处理:按照液土质量比比2:5的比例接种至上述土壤中。放置于37℃恒温培养箱中培养,每隔24h取样。用液相色谱仪(HPLC)检测2,4-DNT-3-SA的含量。降解曲线如图4所示,对2,4-DNT-3-SA的降解在第4天达到100%。
实施例4:Bacillus altitudinis D47对2,4-二硝基甲苯的降解率
取未污染土壤,风干研磨、过1mm筛,备用。称取一定量的2,4-DNT溶于丙酮中。在通风橱中,向上述土壤中均匀喷洒含有2,4-DNT的丙酮溶液,并搅拌均匀。使土壤浓度为2,4-DNT的浓度为50mg·kg-1;在通风橱中使其自然风干2天。
菌株活化:从-80℃冰箱取出甘油中保存的2,4-DNT微生物降解菌株,接种于500μL液体LB培养基中,于37℃恒温振荡摇床中培养4-6h至对数生长期,收集菌体,使用等比例添加MSM基础盐液体培养重悬菌体。
2,4-DNT处理:按照液土质量比比2:5的比例接种至上述土壤中。放置于37℃恒温培养箱中培养,每隔24h取样。用液相色谱仪(HPLC)检测2,4-DNT的含量。降解曲线如图5所示,对2,4-DNT的降解在第2天达到100%。
Claims (3)
1.一株降解2,4-DNT和2,4-DNT-3-SA的高地芽孢杆菌D47,其特征在于,所述高地芽孢杆菌(Bacillus altitudinis)D47于2023年2月27日保藏于中国普通微生物菌种保藏管理中心,保藏号为CGMCC 26705,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。
2.一种土壤处理剂,其特征在于,所述土壤处理剂中含有权利要求1所述高地芽孢杆菌D47。
3.权利要求1所述高地芽孢杆菌D47的应用,其特征在于,所述应用具体方法为将高地芽孢杆菌D47菌液活化至对数生长期,将菌液使用MSM基础盐液体培养基按照1:1稀释后接种于含有2,4-DNT或2,4-DNT-3-S的污染土壤中,按照液土质量比为2:5,温度37℃,pH为7进行处理。
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