CN116814511A - 具有高亚硝酸盐耐受和还原能力的甲烷氧化菌及其应用 - Google Patents
具有高亚硝酸盐耐受和还原能力的甲烷氧化菌及其应用 Download PDFInfo
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- 241000894006 Bacteria Species 0.000 title claims abstract description 39
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 claims abstract description 72
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- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims abstract description 5
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 abstract description 10
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- 229910021591 Copper(I) chloride Inorganic materials 0.000 description 2
- 229910002651 NO3 Inorganic materials 0.000 description 2
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
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Abstract
本发明公开了一株具有高亚硝酸盐耐受和还原能力的甲烷氧化菌及其应用。该甲烷氧化菌的名称为甲基单胞菌(Methylomonas sp.)HYX‑M1,保藏编号为GDMCC No:63477,于2023年5月18日保藏于位于广州市先烈中路100号大院59号楼5楼广东省科学院微生物研究所的广东省微生物菌种保藏中心。该甲烷氧化菌能够在不同亚硝酸盐作为氮源的条件下进行高效甲烷氧化过程,同时去除水体中的亚硝酸盐;而且在高浓度亚硝酸盐的环境下,能生长繁殖,从而能用于含亚硝酸盐水体中亚硝酸盐和甲烷的处理。
Description
技术领域
本发明属于微生物领域,特别涉及一株具有高亚硝酸盐耐受和还原能力的甲烷氧化菌及其应用。
背景技术
甲烷是仅次于二氧化碳的温室气体,近30年来,大气中甲烷含量已经增加了近5倍。2020年大气甲烷浓度已高达1893.4ppb,对全球温室效应的贡献率高达 25%。废水处理过程是在厌氧环境中,废水中的有机物经过微生物作用会被厌氧分解产生大量的甲烷。同时,在化肥制造、钢铁生产、火药制造等工业废水中含有高浓度的亚硝酸盐。一般来说,亚硝酸盐对微生物具有较强的毒害能力,较高浓度的亚硝酸盐会抑制甲烷氧化菌的生理活性,导致甲烷氧化效果的减弱。因此,有必要获得具有高亚硝酸盐耐受和还原能力的甲烷氧化菌,帮助处理高亚硝酸盐浓度的工业废水。
发明内容
本发明的首要目的在于克服现有技术的缺点与不足,提供一株具有高亚硝酸盐耐受和还原能力的甲烷氧化菌。
本发明的另一目的在于提供上述具有高亚硝酸盐耐受和还原能力的甲烷氧化菌的应用。
本发明的目的通过下述技术方案实现:一株具有高亚硝酸盐耐受和还原能力的甲烷氧化菌,名称为甲基单胞菌(Methylomonas sp.)HYX-M1,保藏编号为GDMCC No:63477,于2023年5月18日保藏于位于广州市先烈中路100号大院59号楼5楼广东省科学院微生物研究所的广东省微生物菌种保藏中心。
上述具有高亚硝酸盐耐受和还原能力的甲烷氧化菌在甲烷氧化和/或去除亚硝酸盐中的应用;特别适合于在亚硝酸盐存在环境下对甲烷进行氧化。
所述的亚硝酸盐存在环境优选为含亚硝酸盐水体;更优选为亚硝酸盐浓度为2mM以上的水体;最优选为亚硝酸盐浓度为4~6mM的水体。
所述的水体优选为工业废水。
本发明相对于现有技术具有如下的优点及效果:
本发明提供了一株具有高亚硝酸盐耐受和还原能力的甲烷氧化菌,能够在不同亚硝酸盐作为氮源的条件下进行高效甲烷氧化过程,同时去除水体中的亚硝酸盐;而且在高浓度亚硝酸盐的环境下,能生长繁殖。
附图说明
图1是甲烷氧化菌的菌体形态照片图。
图2是不同浓度的亚硝酸盐对甲基单胞菌甲烷氧化的影响结果图;其中,a是硝酸盐浓度为4 mM的结果,b是亚硝酸盐浓度为6 mM的结果。
图3是甲基单胞菌对亚硝酸盐的利用的检测结果图;其中,a是亚硝酸盐浓度为4mM的结果,b是亚硝酸盐浓度为6 mM的结果。
图4是甲基单胞菌在不同亚硝酸盐条件下的生长情况图。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。
实施例1:
1)甲烷氧化菌的富集培养
对珠江三角洲湿地土壤进行预孵育培养。取10 g新鲜土壤于125 mL西林瓶中,再用丁基橡胶和铝盖密封。添加约25%(v/v)的甲烷(25 mL)作为底物,在30 ℃暗室静态培养。期间用气体进样针取0.2 mL瓶内气体,利用高效气相色谱(Agilent 7820A , USA)检测甲烷浓度,待瓶中甲烷不再消耗后,取3 mL孵育土壤接种到25 mL硝酸盐无机盐(NMS)培养基,重复三次。
NMS培养基制备过程如下:用电子天平分别称量0.2 g MgSO4·7H2O、0.14 gCaCl2·6H2O和1.0 g KNO3,并用注射器取50 mL磷酸缓冲液(5.44 g/L KH2PO4、5.68 g/LNa2HPO4)和2 mL微量元素液(每升含 1.0 g Na2-EDTA、2.0 g FeSO4·7H2O、0.8 g ZnSO4·7H2O、0.03 g MnCl2·4H2O、0.03 g H3BO3、0.2 g CoCl2·6H2O、0.6 g CuCl2·2H2O、0.02 gNiCl2·6H2O和0.05 g Na2MoO4·2H2O),添加到去离子水中,混匀后定容到1 L,加氢氧化钠调节pH值至7.0,取25 mL培养基分装到125 mL 西林瓶,丁基胶塞密封,再用铝盖封口,于121℃灭菌20 min,得到NMS培养基。
2)甲烷氧化菌的分离鉴定:
使用Hungate滚管技术分离甲烷氧化菌,在25 mL厌氧管中,取上述0.1 mL甲烷氧化菌富集物转接到5 mL 含2%(w/v)的琼脂NMS培养基中,添加约25%甲烷(5 mL),在30℃暗室条件下静止培养5天。挑取单菌落到5 mL液体培养基,重复滚管、挑单菌落的步骤3次,获得纯化后的菌株,命名为HYX-M1。期间用气体进样针取0.2 mL管内气体,利用高效气相色谱(Agilent 7820A , USA)检测甲烷浓度来确定甲烷去除效果。
取菌液作为DNA模板进行PCR扩增。PCR 反应体系组成(50µL):10×ExTaq buffer(含 Mg2+)5.0 µL,dNTPs(2.5 mM) 12.5 µL,引物27F/1492R(10 µM)各1.0 µL,DNA模板 1.0µL,无菌超纯水9.5 µL。PCR反应条件为:94 ℃预变性 2 min;94 ℃变性 30 s、55 ℃退火30 s、72 ℃延伸1 min,30个循环;72 ℃终延伸10 min。
27F:5’-AGAGTTTGATCCTGGCTCAG-3’;
1492R:5’-GGYTACCTTGTTACGACTT-3’。
测序得到的16S rDNA序列为:
TGCACCATGGCGGGATGCTTAACACATGCAAGTCGAACGCTGATAAGGTGCTTGCACCTTTGATGAGTGGCGGACGGGTGAGTAATGCATAGGAATCTGCCTATTAGTGGGGGATAACGTGGGGAAACTCACGCTAATACCGCATACGATCTACGGATGAAAGCAGGGGACCTTTTGGCCTTGCGCTAATAGATGAGCCTATGTCGGATTAGCTAGTTGGTAGGGTAAAGGCCTACCAAGGCGACGATCCGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCAATACCGCGTGTGTGAAGAAGGCCTGAGGGTTGTAAAGCACTTTCAATGGGAAGGAATACCTATCGGTTAATACCCGGTAGACTGACATTACCCATACAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGTAGGCGGTTTTTTAAGTCAGATGTGAAAGCCCTGGGCTTAACCTGGGAACTGCATTTGATACTGGAAAACTAGAGTTGAGTAGAGGAGAGTGGAATTTCAGGTGTAGCGGTGAAATGCGTAGAGATCTGAAGGAACACCAGTGGCGAAGGCGGCTCTCTGGACTCAAACTGACGCTGAGGTACGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAACCGTTGGGCGCTTTAAGTGCTTAGTGGTGGAGCTAACGTATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACCCTTGACATCCAGAGAATCTGTTAGAGATAGCGGAGTGCCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTATCCTTAGTTGCCAGCGGTTCGGCCGGGAACTCTAGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTATGGGTAGGGCTACACACGTGCTACAATGGCCGGTACAGAGGGTTGCGATCTCGCGAGAGCAAGCTAATCCCAAAAAGCCGGTCTTAGTCCGGATTGCAGTCTGCAACTCGACTGCATGAAGTCGGAATCGCTAGTAATCGCAGATCAGAATGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGTTTAACCTTCGGGAGGGCGCTACCACTTTGATTCCGG。
3)甲烷氧化菌的形态学观察:
扫描电镜观察:取2 mL菌液,5000 r/min离心3 min,弃上清;加入2 mL0.1 mol/LPBS缓冲液,重悬后5000 r/min离心3 min,重复清洗3遍;再加入1 mL 2 .5% 戊二醛磷酸缓冲液(pH7.2),于4℃固定过夜;次日,用0.15%的戊二醛磷酸缓冲液冲洗;然后依次用30%、50%、70%、90%和无水乙醇进行梯度脱水,每次脱水15 min后于5000 r/min离心3 min;再加入2 mL叔丁醇置换乙醇,5000 r/min离心3 min,弃掉大部分上清,留少量上清液 用来重悬菌体,吸取0.1 mL菌液滴加到洁净盖玻片上,于超净工作台吹干;经镀金后,在扫描电子显微镜下观察菌体形态。
菌株HYX-M1在厌氧管管壁上的菌落形态如图1所示,为边缘整齐的圆形,且菌落表面较光滑,呈粉色不透明状。菌株HYX-M1在扫描电镜下为短杆状。
4)甲烷氧化菌的分离鉴定:
将筛选出的菌株16S rRNA测序结果在NCBI数据库进行Nucleotide BLAST序列比对,结果显示该菌与Methylomonas rhizoryzaestrain GJ1有99.86%的序列相似性,结合理化特性和菌体形态特征,初步判断为甲烷氧化菌(Methylomonas),命名为甲基单胞菌(Methylomonas sp.)HYX-M1,保藏编号为GDMCC No:63477,于2023年5月18日保藏于位于广州市先烈中路100号大院59号楼5楼广东省科学院微生物研究所的广东省微生物菌种保藏中心。
实施例2:
1)营养液的制备:
首先用NMS培养液进行甲基单胞菌富集培养,培养至甲烷氧化菌的浓度为0.05~0.06 mg/mL,获得甲烷氧化菌种子液。
NMS培养液的成分为:0.2 g MgSO4•7H2O、0.14 g CaCl2·6H2O,并用注射器取50mL磷酸缓冲液 (5.44 g/L KH2PO4、5.68 g/L Na2HPO4) 和2 mL微量元素液(溶质为:1.0 g/L Na2-EDTA、2.0 g/L FeSO4•7H2O、0.8 g/L ZnSO4•7H2O、0.03 g/L MnCl2•4H2O、0.03 g/LH3BO3、0.2 g/L CoCl2•6H2O、0.6 g/L CuCl2•2H2O、0.02 g/L NiCl2•6H2O和0.05 g/LNa2MoO4•2H2O,溶剂为去离子水),添加到去离子水中,混匀后定容到1 L,加氢氧化钠调节pH值至6.8,制备得到NMS培养基。取25 ml培养基分装到125 mL西林瓶,丁基胶塞密封,再用铝盖封口,于121℃灭菌20 min。
2)甲烷氧化菌活性检验:
在超净工作台中,取1 mL甲烷氧化菌Methylomonas sp. HYX-M1和1 mL甲烷氧化菌Methylomonas sp. LW 13(德国微生物菌种保藏中心,编号为DSM 24493)分别接种到上述25 mL培养基中,再加入亚硝酸盐浓缩液(100mM亚硝酸钠),使培养基中亚硝酸盐的最终浓度分别为4 mM和6 mM,并且每个组按25 %(v/v)(甲烷体积/顶空体积)添加量加入25 mL甲烷,每个处理设3个重复,在30 ℃黑暗条件下静止培养96 h,期间利用高效气相色谱仪(Agilent 7820A, USA)检测甲烷浓度来确定甲烷氧化活力。
3)甲烷氧化菌亚硝酸盐还原检验:
在超净工作台中,取1 mL甲烷氧化菌HYX-M1接种到上述25 mL培养基中,同时加入亚硝酸盐浓缩液,使培养基中亚硝酸盐的最终浓度分别为4 mM和6 mM,并且每个组按25 %(v/v)(甲烷体积/顶空体积)添加量加入25 mL甲烷,每个处理设3个重复,在30 ℃黑暗条件下静止培养96 h,期间利用离子色谱(ICS-90, USA)检测亚硝酸盐浓度。
4)甲烷氧化菌活性检验:
在超净工作台中,取1 mL甲烷氧化菌Methylomonas sp. HYX-M1/甲烷氧化菌Methylomonas sp. LW 13和1 mL亚硝酸盐浓缩液接种到上述25 mL培养基中,使培养基中亚硝酸盐的最终浓度分别为4 mM和6 mM,并且每个组按25 %(v/v)(甲烷体积/顶空体积)添加量加入25 mL甲烷,每个处理设3个重复,在30 ℃黑暗条件下静止培养96 h,期间利用紫外分光光度计检测菌体的生长情况。
甲烷氧化菌活性检验的结果如图2所示。从图2中可以看出,在4 mM和6mM亚硝酸盐作为氮源的条件下,与甲烷氧化菌Methylomonas sp. LW 13 相比,Methylomonas sp.HYX-M1菌株可快速降解甲烷,96 h的甲烷氧化量分别为6.21和5.52 mM,能够很好得起到降低甲烷浓度的效果。且培养基成分容易获得,使用量少,成本低。且菌株的稳定性好,培养简单,可控性强。
甲烷氧化菌亚硝酸盐还原检验的结果如图3所示。从图3中可以看出,在4 mM和6mM亚硝酸盐作为氮源的条件下,与甲烷氧化菌Methylomonas sp. LW 13 相比,Methylomonas sp. HYX-M1菌株可快速消耗亚硝酸盐,96 h的亚硝酸盐去除量分别为2.76和1.31 mM,能够很好得起到降低水环境中亚硝酸盐的效果。且培养基成分容易获得,使用量少,成本低。且菌株的稳定性好,培养简单,可控性强。
甲烷氧化菌活性检验的结果如图4所示。从图4中可以看出,在4 mM和6mM亚硝酸盐作为氮源的条件下,甲烷氧化菌Methylomonas sp. LW 13 几乎不生长,Methylomonas sp.HYX-M1菌株生长较为快速,96 h的菌体的OD600均可达到0.5左右,对亚硝酸盐有较好的耐受能力。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (7)
1.一株具有高亚硝酸盐耐受和还原能力的甲烷氧化菌,其特征在于:所述的甲烷氧化菌的名称为甲基单胞菌(Methylomonas sp.)HYX-M1,保藏编号为GDMCC No:63477,于2023年5月18日保藏于位于广州市先烈中路100号大院59号楼5楼广东省科学院微生物研究所的广东省微生物菌种保藏中心。
2.权利要求1所述的具有高亚硝酸盐耐受和还原能力的甲烷氧化菌在甲烷氧化和/或去除亚硝酸盐中的应用。
3.根据权利要求2所述的应用,其特征在于:所述的具有高亚硝酸盐耐受和还原能力的甲烷氧化菌在亚硝酸盐存在环境下对甲烷进行氧化。
4.根据权利要求3所述的应用,其特征在于:所述的亚硝酸盐存在环境为含亚硝酸盐水体。
5.根据权利要求4所述的应用,其特征在于:所述的含亚硝酸盐水体为亚硝酸盐浓度是2mM以上的水体。
6.根据权利要求5所述的应用,其特征在于:所述的含亚硝酸盐水体为亚硝酸盐浓度是4~6mM的水体。
7.根据权利要求4~6任一项所述的应用,其特征在于:所述的水体为工业废水。
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