CN109055282B - 一株肺炎克雷伯氏菌新菌株及其分离方法和应用 - Google Patents
一株肺炎克雷伯氏菌新菌株及其分离方法和应用 Download PDFInfo
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Abstract
本发明公开了一株肺炎克雷伯氏菌新菌株及其分离方法和应用,所述新菌株从造纸污水处理厂曝气池活性污泥中分离、筛选得到,保藏在中国普通微生物菌种保藏管理中心,保藏名称为,ZS‑01,保藏号是16041,保藏日期为2018年6月30日。所述菌株特性及性能:(1)特性:在苯酚MedA固体培养基上形成的菌落为较短粗的杆菌,大小0.5~0.8×1~2μm,单独或成双排列,形成淡黄色菌落,质地均匀且不透明;在MPYE固体培养基上形成较大的灰白色粘性菌落;无鞭毛,有荚膜,菌株属肠杆菌科,为革兰氏染色阴性的粗短杆菌。(2)性能:具有一定的苯酚降解能力;对苯酚具有较高的耐受性。其优点是:能够降解较高浓度的苯酚,为有效处理较高浓度的含酚废水提供了新菌源。
Description
技术领域
本发明属于生物工程技术领域,涉及一株肺炎克雷伯氏菌新菌株及其分离方法和应用。
背景技术
苯酚存在于提炼厂、石化制造等各种工业废水中。苯酚也是煤气化和液化过程,凝析油流中主要的有机组分。制药厂,原木加工厂,造纸厂,制塑厂等的工业废水中也含有苯酚。未经处理的含苯酚工业废水不能排放到开放水域,否则会使饮用水和食品加工用水产生异味。环境保护署制定了一个排放标准:地表水中苯酚的含量低于十亿分之一。含酚工业废水污染面积广、危害作用大,对居民饮食、鱼类养殖及农作物灌溉都会带来严重危害。水体中苯酚浓度高于50ppb对水生生物是有毒害的,血液中4.7-130mg/100mL苯酚是致死的,苯酚会损害神经系统及主要器官包括脾、胰和肝,持久摄入10-240mg/L浓度的苯酚会引起嘴唇发炎酸痛、腹泻、黑色尿液及视力受损。
苯酚是一种具有特殊气味的无色针状晶体,有毒,苯酚分子由一个羟基直接连在苯环上构成。由于苯环的稳定性,这样的结构几乎不会转化为酮式结构。酚羟基的氧原子采用sp2杂化,提供一对孤电子与苯环的6个碳原子共同形成离域键。大π键加强了烯醇的酸性,羟基的推电子效应又加强了O-H键的极性,因此苯酚中羟基的氢可以电离出来。
目前对于苯酚的处理方法主要分为3大类,即化学方法、物理方法和生物方法,微生物法降解苯酚,该方法条件温和,特异性强,且不涉及二次污染等问题,是环境友好型处理方法。因此,微生物法降解苯酚这一思路吸引了国内外学者的广泛关注。
发明内容
本发明的目的在于提供一株肺炎克雷伯氏菌新菌株及其分离方法和应用。
为了达到上述目的,本发明采用以下技术方案予以实现:
本发明公开了一种肺炎克雷伯氏菌菌株,该肺炎克雷伯氏菌菌株命名为Klebsiella Pneumoniae sp,保藏于中国普通微生物菌种保藏管理中心,菌种保藏号为CGMCC No.16041,保藏日期为为2018年7月2日。
优选地,所述肺炎克雷伯氏菌菌株在苯酚MedA固体培养基上培养时,形成粗短的黄色杆菌菌落,大小为0.5~0.8×1~2μm,单独或成双排列,质地均匀且不透明。
优选地,所述肺炎克雷伯氏菌菌株在MPYE固体培养基上培养时,形成较大的灰白色粘性菌落;无鞭毛,有荚膜,为革兰氏染色阴性的粗短杆菌。
优选地,该肺炎克雷伯氏菌菌株在30℃~35℃,pH为7时生长状态良好。
本发明还公开了上述的肺炎克雷伯氏菌菌株的分离方法,包括以下步骤:
1)取样:取造纸污水处理厂曝气池活性污泥,注入灭菌且苯酚作为唯一碳源的培养液中,制成菌液;
2)富集:将步骤1)制得的菌种按10%的接种量接种到100mL MPYE培养液中,于35℃、200r/min的条件下摇床培养,培养期间观测溶液浑浊情况,取上清液制成富集菌液;
3)初筛:将步骤2)的富集菌液取15mL加入装有135mL的MedA培养基锥形瓶中,每组3个平行样,置于35℃恒温摇床中,200r/min培养,待溶液浑浊后逐级接入高浓度苯酚含量的MedA培养基中,进行菌种的逐级筛选驯化,初步筛选得到细菌;
4)复筛:将步骤3)初步筛选得到的细菌在MedA固体培养基上进行平板涂布,然后置于35℃的恒温培养箱中培养;在超洁净工作台中进行多次平板划线后获取得到分离的纯种细菌,即肺炎克雷伯氏菌菌株。
优选地,MPYE培养基的配方如下,1000mL MPYE培养基中含有:蛋白胨3g、酵母膏3g、1M Mgcl2 1.6mL、1M Cacl2 1.6mL、余量为纯水。
优选地,MedA培养基的制备方法如下:配制1000mL MedA培养基,取(NH4)2SO40.4g、2%的天门冬氨酸2mL、10%的NaCl 10mL、氮川三乙酸0.2g、MgSO4·7H2O 0.59g、无水CaCl2 0.05036g、FeSO4·7H2O 251.98mg、(NH4)6Mo24·4H2O 0.186mg、ZnSO4·7H2O0.5475g、EDTA 125mg、H3BO4 5.7mg、MnSO4·H2O 77mg、CuSO4·5H2O 19.6mg、Co(NO3)2·6H2O12.4mg、余量为蒸馏水;装到1000mL蓝盖玻璃瓶中,封口压盖,放入Yamato高压灭菌锅121℃高压灭菌30min;取出后放入超洁净工作台紫外杀菌30min后,待温度降到室温,加入盐酸硫胺0.5mg、烟酸1mg、生物素0.25μL、K2HPO4 1.7418g、KH2PO41.361g,最后加入800mg~2500mg不同梯度的苯酚制成不同苯酚梯度MedA培养基。
本发明还公开了上述的肺炎克雷伯氏菌菌株在降解含苯酚废水中的应用。
优选地,所述肺炎克雷伯氏菌菌株能够降解浓度在2500mg/L以下的含苯酚废水。
与现有技术相比,本发明具有以下有益效果:
本发明的新菌株从造纸污水处理厂曝气池活性污泥中分离、筛选得到,保藏在中国普通微生物菌种保藏管理中心,保藏名称为,ZS-01,保藏号是16041,保藏日期为2018年6月30日。所述菌株特性及性能:单独或成双排列,形成淡黄色菌落,质地均匀且不透明;在MPYE固体培养基上形成较大的灰白色粘性菌落;无鞭毛,有荚膜,菌株属肠杆菌科,为革兰氏染色阴性的粗短杆菌。本发明的菌株具有一定的苯酚降解能力;对苯酚具有较高的耐受性,为有效处理较高浓度的含酚废水提供了新菌源。
附图说明
图1为本发明的肺炎克雷伯氏菌ZS01在苯酚为碳源的MedA固体培养基上的形态;
图2为本发明的肺炎克雷伯氏菌ZS01在MPYE固体培养基上的形态图;
图3为本发明的肺炎克雷伯氏菌ZS01的系统发育树。
菌株保藏
肺炎克雷伯氏菌新菌株命名为ZS-01,保藏在中国普通微生物菌种保藏管理中心,地址是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,保藏名称为,ZS-01,分类命名为Klebsiella Pneumoniae sp,保藏号是16041,保藏日期为为2018年7月2日。
具体实施方式
为了使本技术领域的人员更好地理解本发明方案,下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分的实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都应当属于本发明保护的范围。此外,术语“包括”和“具有”以及他们的任何变形,意图在于覆盖不排他的包含,例如,包含了一系列步骤或单元的过程、方法、系统、产品或设备不必限于清楚地列出的那些步骤或单元,而是可包括没有清楚地列出的或对于这些过程、方法、产品或设备固有的其它步骤或单元。
下面结合附图对本发明做进一步详细描述:
实施例1:本发明菌株的分离与鉴定
(1)取样:直接选取造纸污水处理厂曝气池活性污泥,将活性污泥用桶带回实验室,取少许活性污泥的泥水混合物注入灭菌且苯酚作为唯一碳源的培养液中,放入锥形瓶中制成菌液;
(2)富集:配置1000mL MPYE培养基:蛋白胨3g、酵母膏3g、1M Mgcl21.6mL、1M Cacl21.6mL、余量为纯水;每500mL MPYE培养基分装到500mL蓝盖玻璃瓶中,封口压盖,放入Yamato高压灭菌锅121℃高压灭菌30min;取出后放入超洁净工作台紫外杀菌30min后,步骤1的菌种按接种量10%接种到含有100mL MPYE培养液的250mL锥形瓶中,置于35℃,200r/min摇床中培养,培养期间观测溶液浑浊情况,取上清液制成富集菌液;
(3)初筛:配置1000mL MedA培养基:(NH4)2SO4 0.4g、2%的天门冬氨酸2mL、10%的NaCl 10mL、氮川三乙酸0.2g、MgSO4·7H2O 0.59g、无水CaCl2 0.05036g、FeSO4·7H2O251.98mg、(NH4)6Mo24·4H2O 0.186mg、ZnSO4·7H2O 0.5475g、EDTA 125mg、H3BO4 5.7mg、MnSO4·H2O 77mg、CuSO4·5H2O 19.6mg、Co(NO3)2·6H2O 12.4mg、余量为蒸馏水;装到1000mL蓝盖玻璃瓶中,封口压盖,放入Yamato高压灭菌锅121℃高压灭菌30min;取出后放入超洁净工作台紫外杀菌30min后,待温度降到室温,加入盐酸硫胺0.5mg、烟酸1mg、生物素0.25μL、K2HPO4 1.7418g、KH2PO4 1.361g、最后加入800mg-2500mg不同梯度的苯酚制成不同苯酚梯度MedA培养基;将步骤2的富集菌液取15mL加入装有135mL的MedA培养基锥形瓶中,每组3个平行样,置于35℃恒温摇床中,200r/min培养,待溶液浑浊后逐级接入高浓度苯酚含量的MedA培养基中,进行菌种的逐级筛选驯化。
(4)复筛:将步骤3用高浓度苯酚含量的MedA培养基筛选驯化的细菌在MedA固体培养基上进行平板涂布,置于35℃的恒温培养箱中培养;在超洁净工作台中进行多次平板划线后获取得到分离的纯种细菌。
以上菌种筛选过程均需在有氧条件下进行。
ZS01在苯酚为碳源的MedA固体培养基上的形态如图1所示,在MPYE固体培养基上的形态如图2所示。
(5)菌种鉴定
新菌株ZS-01 16S rDNA基因的PCR扩增和序列分析
以ZS-01 DNA为模板,用16S rDNA基因的通用引物进行PCR扩增,PCR产物进行凝胶电泳检测,将扩增片段回收、测序,用MEGA 4.0软件包中的Kimura-2-parameter法计算遗传距离,用Neighbor-Joining法构建系统发育树,重复抽样1000次分析系统树各分枝的置信度。并与GenBank中的序列进行比对,与肺炎克雷伯氏菌的同源性最高,为99%,如图3所示。菌株ZS-01的16S rDNA的长度为1396bp,其序列详见核苷酸序列表,如SEQ.ID.NO.1所示,。
实施例2:新菌株ZS-01性能
(1)菌种苯酚降解特性
苯酚含量测定及标准曲线制作采用4-氨基安替吡林法。
苯酚降解率由下式计算:
苯酚降解率(%)=(A0-As)/A0×100%
其中,A0--不接种的空白参照试验中相应的苯酚含量
As--接过菌种的待测样液的苯酚含量
(2)菌株ZS-01对苯酚的最优降解条件
实验方法:将不同条件下接种ZS-01的培养液,置于35℃,200r/min的恒温摇床中研究该菌株的苯酚降解性能。利用4-氨基安替吡林法测定不同时间段的苯酚降解率。
实验结果:菌株的最佳苯酚降降解条件是接种量为OD600=0.8时,接种30%,培养液pH=7.5时,且L-谷氨酸浓度为4.5mmol/L时;菌株ZS-01在30h对1000mg/L的苯酚降解率可达到100%。
通过以上实例表明,新菌株ZS-01具有较高的苯酚耐受阈,且具有较高的苯酚降解性能,为有效处理较高浓度的含酚废水提供了新途径。
以上内容仅为说明本发明的技术思想,不能以此限定本发明的保护范围,凡是按照本发明提出的技术思想,在技术方案基础上所做的任何改动,均落入本发明权利要求书的保护范围之内。
序列表
<120> 一株肺炎克雷伯氏菌新菌株及其分离方法和应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1396
<212> DNA
<213> 肺炎克雷伯氏菌菌株(Klebsiella pneumoniae)
<400> 1
gcaagtcgag cggtagcaca gagagcttgc tctcgggtga cgagcggcgg acgggtgagt 60
aatgtctggg aaactgcctg atggaggggg ataactactg gaaacggtag ctaataccgc 120
ataacgtcgc aagaccaaag tgggggacct tcgggcctca tgccatcaga tgtgcccaga 180
tgggattagc tagtaggtgg ggtaacggct cacctaggcg acgatcccta gctggtctga 240
gaggatgacc agccacactg gaactgagac acggtccaga ctcctacggg aggcagcagt 300
ggggaatatt gcacaatggg cgcaagcctg atgcagccat gccgcgtgtg tgaagaaggc 360
cttcgggttg taaagcactt tcagcgggga ggaaggcgtt aaggttaata accttggcga 420
ttgacgttac ccgcagaaga agcaccggct aactccgtgc cagcagccgc ggtaatacgg 480
agggtgcaag cgttaatcgg aattactggg cgtaaagcgc acgcaggcgg tctgtcaagt 540
cggatgtgaa atccccgggc tcaacctggg aactgcattc gaaactggca ggctagagtc 600
ttgtagaggg gggtagaatt ccaggtgtag cggtgaaatg cgtagagatc tggaggaata 660
ccggtggcga aggcggcccc ctggacaaag actgacgctc aggtgcgaaa gcgtggggag 720
caaacaggat tagataccct ggtagtccac gccgtaaacg atgtcgattt ggaggttgtg 780
cccttgaggc gtggcttccg gagctaacgc gttaaatcga ccgcctgggg agtacggccg 840
caaggttaaa actcaaatga attgacgggg gcccgcacaa gcggtggagc atgtggttta 900
attcgatgca acgcgaagaa ccttacctgg tcttgacatc cacagaactt agcagagatg 960
gattggtgcc ttcgggaact gtgagacagg tgctgcatgg ctgtcgtcag ctcgtgttgt 1020
gaaatgttgg gttaagtccc gcaacgagcg caacccttat cctttgttgc cagcggtccg 1080
gccgggaact caaaggagac tgccagtgat aaactggagg aaggtgggga tgacgtcaag 1140
tcatcatggc ccttacgacc agggctacac acgtgctaca atggcatata caaagagaag 1200
cgacctcgcg agagcaagcg gacctcataa agtatgtcgt agtccggatt ggagtctgca 1260
actcgactcc atgaagtcgg aatcgctagt aatcgtagat cagaatgcta cggtgaatac 1320
gttcccgggc cttgtacaca ccgcccgtca caccatggga gtgggttgca aaagaagtag 1380
gtagcttaac cttcgg 1396
Claims (2)
1.一种能够降解苯酚的肺炎克雷伯氏菌菌株,其特征在于,该肺炎克雷伯氏菌菌株命名为Klebsiella Pneumoniae,保藏于中国普通微生物菌种保藏管理中心,菌种保藏号为CGMCC No.16041,保藏日期为2018年7月2日,该肺炎克雷伯氏菌菌株在30℃~35℃,pH为7时生长状态良好。
2.权利要求1所述的肺炎克雷伯氏菌菌株在降解含苯酚废水中的应用。
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