CN109097296A - 一种具有降解一甲胺能力的蜡状芽孢杆菌及其应用 - Google Patents
一种具有降解一甲胺能力的蜡状芽孢杆菌及其应用 Download PDFInfo
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- BAVYZALUXZFZLV-UHFFFAOYSA-N Methylamine Chemical compound NC BAVYZALUXZFZLV-UHFFFAOYSA-N 0.000 title claims abstract description 104
- 241000193830 Bacillus <bacterium> Species 0.000 title claims abstract description 53
- 230000015556 catabolic process Effects 0.000 title claims abstract description 40
- 238000006731 degradation reaction Methods 0.000 title claims abstract description 40
- 241000193755 Bacillus cereus Species 0.000 claims abstract description 15
- 238000005067 remediation Methods 0.000 claims abstract description 8
- 238000004321 preservation Methods 0.000 claims abstract description 5
- 108020004465 16S ribosomal RNA Proteins 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
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- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 description 2
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 2
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- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 2
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- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 1
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Abstract
本发明公开了一种具有降解一甲胺能力的蜡状芽孢杆菌及其在环境修复中的应用。本发明通过对广州市垃圾填埋场渗滤液进行分离纯化，筛选出了一株对含氮恶臭污染物一甲胺具有良好降解能力的蜡状芽孢杆菌，并将此蜡状芽孢杆菌命名为蜡状芽孢杆菌(Bacillus cereus)GDUTAN3，保藏在中国典型培养物保藏中心，保藏地址为湖北省武汉市武汉大学；保藏编号为CCTCC NO.：M 2018229。保藏日期为2018年4月23日。该蜡状芽孢杆菌GDUTAN3呈革兰氏阳性，杆状，菌落形态为圆形，白色，不透明，菌落直径为2～3mm。本发明所得到的蜡状芽孢杆菌GDUTAN3对环境中恶臭污染物甲胺降解效果较好，可应用于环境修复。在甲胺浓度为5～130mg/L条件下降解96h，降解率可达94.8％以上。
Description
技术领域
本发明涉及一种微生物技术，尤其涉及一种具有降解一甲胺能力的蜡状芽孢杆菌及其应用。
背景技术
有机胺类化合物是仅次于有机硫化物的恶臭污染物，是农药、染料和医药等化工行业的原料或中间体，在相关化工企业排放的废水和废气中常常被检测出，且他们大多具有高毒性、可迁移性和生物蓄积性等特点，对人体健康造成较大影响。因此，近年来有机胺类化合物在环境中的行为已引起了广泛关注。
而一甲胺(Methylamine，CH3NH2)是一种重要的脂肪胺，是氨的烃基衍生物。是最基本的有机类化工材料之一，其主要用于合成橡胶的硫化功能促进剂、药类、驱虫产品、印染产品、活性剂等。其也是常见的有机含氮恶臭污染物，在较低浓度时就具有强烈的鱼臭味(嗅阈值：0.021ppm)。另外，液态或气态的一甲胺类有刺激性和腐蚀性，暴露于一甲胺时可导致人体和动物的皮肤灼伤、眼部不适、呼吸道刺激等症状，严重者可导致死亡。因此对水体和大气中的一甲胺进行去除将对人体健康、环境保护、实现生态自然的可持续发展有着非常重要的意义。
目前，国内外对环境中恶臭污染物一甲胺的主要去除技术包括化学治理法中的燃烧法、吸收法等和物理治理法中的吸附法、冷凝法、掩蔽法等技术。这些技术各有优点。但总体来说这些技术的缺点是处理费用高、易产生二次污染等。而生物法处理恶臭技术由于其处理成本较低，无二次污染，可操作性强等优点，正好填补了这一部分的空缺，已成为目前治理恶臭污染物的主流技术，市场前景也逐步被看好。据报道，目前已有一部分研究者通过接种混合菌种对一甲胺进行降解研究。然而，使用分离培养新的单一菌种对一甲胺进行降解研究的文献报道却非常有限，因此筛选出高效率、低成本的一甲胺降解菌在恶臭有机含氮废气的净化方面具有非常重要的意义。该蜡状芽孢杆菌是一种革兰氏阳性菌、杆状，在固体培养基表面，菌体可形成白色、圆形、边缘不整齐，表面粗糙的菌落。迄今为止，还没有见到有关使用蜡状芽孢杆菌降解一甲胺的相关报道及专利。
发明内容
为了克服现有技术的不足，本发明的目的之一在于提供一种具有降解一甲胺能力的蜡状芽孢杆菌。该蜡状芽孢杆菌GDUTAN3属于蜡状芽孢杆菌属的一个新变种，具有优异的甲胺降解能力，能降解环境中的甲胺，且甲胺降解率高。
本发明的目的之二在于提供一种具有降解一甲胺能力的蜡状芽孢杆菌在环境修复中的应用。
本发明的目的之一采用如下技术方案实现：提供一种具有甲胺降解能力的蜡状芽孢杆菌，该菌株名称为蜡状芽孢杆菌(Bacillus cereus)GDUTAN3，保藏在中国典型培养物保藏中心，保藏地址为湖北省武汉市武昌区八一路299号武汉大学；保藏编号为 CCTCCNO.：M 2018229，保藏日期为2018年04月23日。
本发明的所述具有降解含氮恶臭污染物一甲胺能力的蜡状芽孢杆菌的形态特性如下：
(a)采用常规的细菌生理生化鉴定方法和电子显微镜观察，所筛选的蜡状芽孢杆菌的细胞染色为革兰氏阳性，杆状。
(b)菌落的形态特性为：在LB固体培养基培养24h后，菌落形态为圆形，白色，不透明，菌落直径为2～3mm。
本发明的所述具有甲胺降解能力的蜡状芽孢杆菌的主要的生理生化特征如下表1所示：
表1蜡状芽孢杆菌的主要的生理生化特征
本发明的所述具有甲胺降解能力的蜡状芽孢杆菌的16S rDNA序列如SEQ ID NO：1所示。
通过16S rDNA序列比对分析，发现本发明的菌株与Bacillus cereus TRB66 的同源性高达99％。通过结合菌体形态特征、生长条件、生理生化鉴定结果确定蜡状芽孢杆菌(Bacillus cereus)GDUTAN3属于蜡状芽孢杆菌属的一个新变种，命名为蜡状芽孢杆菌(Bacillus cereus)GDUTAN3。
本发明的目的之二采用如下技术方案实现：提供所述的具有甲胺降解能力的蜡状芽孢杆菌在环境修复中的应用。
进一步地，本发明的具有甲胺降解能力的蜡状芽孢杆菌在环境修复的应用中，能够降解环境中的甲胺。
进一步地，所述环境包括大气、水体或者土壤。
相比现有技术，本发明的有益效果在于：
(1)本发明的菌株是首次从广东省广州市某垃圾填埋场的垃圾渗滤液中筛选得到具有降解甲胺能力的蜡状芽孢杆菌GDUTAN3。
(2)本发明的蜡状芽孢杆菌GDUTAN3具有高效降解甲胺的能力，在底物浓度为5～130mg/L条件下降解96h，降解率可达94.8％。
附图说明
图1是本发明的蜡状芽孢杆菌GDUTAN3在电子显微镜下的形态图。
具体实施方式
下面，结合附图以及具体实施方式，对本发明做进一步描述，需要说明的是，在不相冲突的前提下，以下描述的各实施例之间或各技术特征之间可以任意组合形成新的实施例。
实施例1
一株具有甲胺降解能力的蜡状芽孢杆菌，该菌株的名称为蜡状芽孢杆菌(Bacillus cereus)GDUTAN3，保藏在中国典型培养物保藏中心，保藏地址为湖北省武汉市武昌区八一路299号武汉大学校内；保藏编号为CCTCC NO：M 2018229，保藏日期为2018年04月23日。
本实施例的蜡状芽孢杆菌GDUTAN3分离筛选自广东省广州市某垃圾填埋场渗滤液。其分离纯化方法如下：所用的驯化培养基为无机盐培养基g/L(每1000 mL无机盐培养基含K2HPO4·3H2O 1.2g，KH2PO4 1.2g，NH4Cl 0.4g， MgSO4·7H2O 0.2g，FeSO4·7H2O 0.01g，1mL微量元素液(g/L)：CaCl2·2H2O 0.2 g，MnSO4·4H2O 0.2g，CuSO4·2H2O 0.01g，ZnSO4·7H2O 0.2g，CoCl2·6H2O 0.09 g，Na2MoO4·2H2O 0.12g，H3BO3 0.006g)。首先取1mL垃圾渗滤液稀释100倍，接种至营养肉汤中，在摇床温度为37℃、转速150rpm条件下好氧培养1天。取经富集的菌液1mL，接种在含甲胺的无机盐营养液中，在摇床温度为37℃、转速150rpm条件下好氧培养5天后以10％接种量移入下一浓度驯化，底物驯化梯度分别为：10，20，50，100mg/L。将终浓度驯化液做10-1～10-6的稀释，分别取稀释10-5和10-6倍的稀释液200μL涂布到以甲胺为唯一碳源的固体琼脂平板(含甲胺的固体培养基为：每升上述无机盐培养基中加入琼脂18g和甲胺 4mg)上，放入35℃恒温箱中培养3天，挑选不同形态单菌落进行甲胺降解率的测定，选择降解率最高的菌种进行纯化。
降解率的测定：在甲胺生物降解的过程中定时取样通过分光光度法测定降解率。降解率(％)＝(初始浓度-终浓度)/初始浓度。
分光光度法测定甲胺的浓度：分别取一定量的甲胺降解液于10mL比色管中，以吸收液(0.01M HCl)稀释至2.0mL，分别加入缓冲液(80mL蒸馏水内溶解4.08g磷酸二氢钾、1.6g硼砂，再加入5.0M NaOH溶液6.35mL，用水稀释至100mL)4.0mL，重氮盐溶液(10mL对硝基苯胺盐酸溶液中加入亚硝酸钠溶液1.0mL，混匀)0.4mL，摇匀，放置40min，再加入5M NaOH溶液1.0 mL，混匀，放置20min，于波长510nm处比色定量。
将纯化得到的菌落进行鉴定，鉴定结果如下：
(1)菌体的形态特性：
a.采用常规的细菌生理生化鉴定方法和电子显微镜观察，所筛选的蜡状芽孢杆菌的细胞染色为革兰氏阳性；在电子显微镜下观察，其形态为杆状，菌体大小为(0.5～2.5)μm×(1.2～5)μm，周生鞭毛，如图1所示；
b.菌落的形态特性：在LB固体培养基培养24h后，菌落边缘整齐，圆形，乳白色，不透明，菌落直径为5～7mm；
c.主要的生理生化特征如表2所示：
表2蜡状芽孢杆菌的各项生理生化特征
上述结果表明本发明所筛选的细菌与蜡状芽孢杆菌属的生理生化特性很相似。
(2)提取细菌基因组DNA，采用细菌的16S rDNA通用引物：
上游引物：F27(5'-AGTTTGATCMTGGCTCAG-3')
下游引物:R1492(5'-GGTTACCTTGTTACGACTT-3')
扩增其16S rDNA全部基因，测序结果如如SEQ ID NO：1所示：
taagagcttg ctcttatgaa gttagcggcg gacgggtgag taacacgtgg gtaacctgcccataagactg ggataactcc gggaaaccgg ggctaatacc ggataacatt ttgaaccgca tggttcgaaattgaaaggcg gcttcggctg tcacttatgg atggacccgc gtcgcattag ctagttggtg aggtaacggctcaccaaggc aacgatgcgt agccgacctg agagggtgat cggccacact gggactgaga cacggcccagactcctacgg gaggcagcag tagggaatct tccgcaatgg acgaaagtct gacggagcaa cgccgcgtgagtgatgaagg ctttcgggtc gtaaaactct gttgttaggg aagaacaagt gctagttgaa taagctggcaccttgacggt acctaaccag aaagccacgg ctaactacgt gccagcagcc gcggtaatac gtaggtggcaagcgttatcc ggaattattg ggcgtaaagc gcgcgcaggt ggtttcttaa gtctgatgtg aaagcccacggctcaaccgt ggagggtcat tggaaactgg gagacttgag tgcagaagag gaaagtggaa ttccatgtgtagcggtgaaa tgcgtagaga tatggaggaa caccagtggc gaaggcgact ttctggtctg taactgacactgaggcgcga aagcgtgggg agcaaacagg attagatacc ctggtagtcc acgccgtaaa cgatgagtgctaagtgttag agggtttccg ccctttagtg ctgaagttaa cgcattaagc actccgcctg gggagtacggccgcaaggct gaaactcaaa ggaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgaagcaacgcgaa gaaccttacc aggtcttgac atcctctgaa aaccctagag atagggcttc tccttcgggagcagagtgac aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacgagcgcaaccct tgatcttagt tgccatcatt aagttgggca ctctaaggtg actgccggtg acaaaccggaggaaggtggg gatgacgtca aatcatcatg ccccttatga cctgggctac acacgtgcta caatggacggtacaaagagc tgcaagaccg cgaggtggag ctaatctcat aaaaccgttc tcagttcgga ttgtaggctgcaactcgcct acatgaagct ggaatcgcta gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgggccttgtaca caccgcccgt cacaccacga gagtttgtaa cacccgaagt cgg
将SEQ ID NO：1中所示的长为1373bp的16S rDNA基因序列与Genbank 中已登录的基因序列进行比对分析发现菌株与Bacillus cereus TRB66的同源性高达99％。
综合上述的生理生化特性、16S rDNA基因序列结果，本发明所筛选的细菌应归属蜡状芽孢杆菌属的一个新变种，命名为蜡状芽孢杆菌(Bacillus cereus) GDUTAN3。
该蜡状芽孢杆菌(Bacillus cereus)GDUTAN3于2018年04月23日保藏于中国典型培养物保藏中心(CCTCC)，保藏地址为湖北省武汉市武昌区八一路299 号武汉大学校内，保藏编号为CCTCC NO.：M 2018229。
实施例2
本实施例为蜡状芽孢杆菌(Bacillus cereus)GDUTAN3在环境修复中的应用，可降解环境中的甲胺。其中环境包括大气、水体或者土壤。
下面对本发明筛选得到的蜡状芽孢杆菌(Bacillus cereus)GDUTAN3的甲胺降解能力进行试验验证的方法如下：
根据降解实验需要，配置无机盐培养基，往300mL的血清瓶中加入无机盐溶液100mL(每100mL无机盐溶液含：K2HPO4·3H2O 0.12g，KH2PO4 0.12g， NH4Cl 0.04g，MgSO4·7H2O 0.02g，FeSO4·7H2O 0.001g，CaCl2·2H2O 0.02g， MnSO4·4H2O 0.02g，CuSO4·2H2O0.001g，ZnSO4·7H2O 0.02g，CoCl2·6H2O 0.009g，Na2MoO4·2H2O 0.012g，H3BO3 0.0006g，双蒸水100mL)，于121℃高压灭菌30min。首先，将筛选所得的具有甲胺降解能力的蜡状芽孢杆菌 GDUTAN3在营养肉汤培养基中(牛肉膏3.0g/L、蛋白胨10.0g/L、NaCl 5.0g/L) 30℃，100rpm的摇床中活化菌体24h，菌液离心，收集菌体，并用磷酸盐缓冲液洗涤三次，并用10mL无机盐溶液重悬，并向含有不同浓度的甲胺的100mL 无机盐溶液中接种1.0mL菌液，其中甲胺的浓度分别为5mg/L、10mg/L、 40mg/L、70mg/L、100mg/L和130mg/L，其中无机盐的pH值为7，在30℃100 rpm的摇床反应96h时，定时取样通过分光光度法测定降解率。降解率的测定同实施例1，结果如表3所示：
表3蜡状芽孢杆菌GDUTAN3对不同初始浓度的甲胺的降解率
从表3中可以看出，本发明筛选得到的蜡状芽孢杆菌GDUTAN3在该条件下对甲胺的降解能力最高可达到94.8％。
上述实施方式仅为本发明的优选实施方式，不能以此来限定本发明保护的范围，本领域的技术人员在本发明的基础上所做的任何非实质性的变化及替换均属于本发明所要求保护的范围。
序列表
<110> 广州紫科环保科技股份有限公司
<120> 一种具有降解含氮恶臭污染物能力的蜡状芽孢杆菌及其应用
<130> 广州紫科环保科技股份有限公司
<141> 2018-07-13
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1373
<212> DNA
<213> 蜡状芽孢杆菌(Bacillus cereus GDUTAN3)
<400> 1
taagagcttg ctcttatgaa gttagcggcg gacgggtgag taacacgtgg gtaacctgcc 60
cataagactg ggataactcc gggaaaccgg ggctaatacc ggataacatt ttgaaccgca 120
tggttcgaaa ttgaaaggcg gcttcggctg tcacttatgg atggacccgc gtcgcattag 180
ctagttggtg aggtaacggc tcaccaaggc aacgatgcgt agccgacctg agagggtgat 240
cggccacact gggactgaga cacggcccag actcctacgg gaggcagcag tagggaatct 300
tccgcaatgg acgaaagtct gacggagcaa cgccgcgtga gtgatgaagg ctttcgggtc 360
gtaaaactct gttgttaggg aagaacaagt gctagttgaa taagctggca ccttgacggt 420
acctaaccag aaagccacgg ctaactacgt gccagcagcc gcggtaatac gtaggtggca 480
agcgttatcc ggaattattg ggcgtaaagc gcgcgcaggt ggtttcttaa gtctgatgtg 540
aaagcccacg gctcaaccgt ggagggtcat tggaaactgg gagacttgag tgcagaagag 600
gaaagtggaa ttccatgtgt agcggtgaaa tgcgtagaga tatggaggaa caccagtggc 660
gaaggcgact ttctggtctg taactgacac tgaggcgcga aagcgtgggg agcaaacagg 720
attagatacc ctggtagtcc acgccgtaaa cgatgagtgc taagtgttag agggtttccg 780
ccctttagtg ctgaagttaa cgcattaagc actccgcctg gggagtacgg ccgcaaggct 840
gaaactcaaa ggaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgaa 900
gcaacgcgaa gaaccttacc aggtcttgac atcctctgaa aaccctagag atagggcttc 960
tccttcggga gcagagtgac aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt 1020
tgggttaagt cccgcaacga gcgcaaccct tgatcttagt tgccatcatt aagttgggca 1080
ctctaaggtg actgccggtg acaaaccgga ggaaggtggg gatgacgtca aatcatcatg 1140
ccccttatga cctgggctac acacgtgcta caatggacgg tacaaagagc tgcaagaccg 1200
cgaggtggag ctaatctcat aaaaccgttc tcagttcgga ttgtaggctg caactcgcct 1260
acatgaagct ggaatcgcta gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg 1320
gccttgtaca caccgcccgt cacaccacga gagtttgtaa cacccgaagt cgg 1373
Claims (5)
1.一种具有甲胺降解能力的蜡状芽孢杆菌，其特征在于，该菌株名称为蜡状芽孢杆菌(Bacillus cereus)GDUTAN3，保藏在中国典型培养物保藏中心，保藏地址为湖北省武汉市武汉大学；保藏编号为CCTCC NO.：2018229。保藏日期为2018年4月23日。
2.如权利要求1所述的具有甲胺降解能力的蜡状芽孢杆菌，其特征在于，所述蜡状芽孢杆菌(Bacillus cereus)GDUTAN3的16S rDNA序列如SEQ ID NO：1所示。
3.权利要求1所述的具有甲胺降解能力的蜡状芽孢杆菌在环境修复中的应用。
4.如权利要求3所述的具有甲胺降解能力的蜡状芽孢杆菌在环境修复中的应用，其特征在于，所述蜡状芽孢杆菌(Bacillus cereus)GDUTAN3能够降解环境中的甲胺。
5.如权利要求3或4所述的具有甲胺降解能力的蜡状芽孢杆菌在环境修复中的应用，其特征在于，所述环境为大气、水体或者土壤。
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