CN1313598C - 一种降解对氯代硝基苯的睾丸酮丛毛单胞菌及其用途 - Google Patents
一种降解对氯代硝基苯的睾丸酮丛毛单胞菌及其用途 Download PDFInfo
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- CN1313598C CN1313598C CNB2003101143377A CN200310114337A CN1313598C CN 1313598 C CN1313598 C CN 1313598C CN B2003101143377 A CNB2003101143377 A CN B2003101143377A CN 200310114337 A CN200310114337 A CN 200310114337A CN 1313598 C CN1313598 C CN 1313598C
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- chloro
- nitrobenzol
- comamonas testosteroni
- strain
- degraded
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明涉及一株睾丸酮丛毛单胞菌(Comamonas testosteroni)CNB1CGMCC No.1028菌株,该菌株的生长细胞、细胞悬浮液、固定化细胞均可以降解对氯代硝基苯;该菌株能以对氯代硝基苯为唯一碳源、氮源和能源生长,同时将对氯代硝基苯完全降解利用。该菌株在以对氯代硝基苯为唯一碳源、氮源和能源生长时,先将硝基还原成羟胺,然后在菌株产生的2-氨基酚1,6-双加氧酶的作用下,通过间位开环的方式,将2-氨基-5-氯苯酚开环成烃类,从而进一步被降解利用。该菌株适用于生产对氯代硝基苯的工业废水的生物处理和对氯代硝基苯污染土壤的生物修复(生物整治)。
Description
技术领域
本发明属于涉及降解对氯代硝基苯类芳香族化合物的生物技术领域。具体地说,本发明涉及一株睾丸酮丛毛单胞菌(Comamonas testosteroni)CNB1 CGMCC No.1028菌株,该菌株的生长细胞、细胞悬浮液、固定化细胞菌可以降解对氯代硝基苯;该菌株能以对氯代硝基苯为唯一碳源、氮源和能源生长,并将对氯代硝基苯完全降解利用。该菌株在以对氯代硝基苯为唯一碳源、氮源和能源生长时,先将硝基还原成羟胺,然后在菌株产生的2-氨基酚1,6-双加氧酶的作用下,通过间位开环的方式,将2-氨基-5-氯苯酚开环成烃类,从而进一步被降解利用。该菌株适用于生产对氯代硝基苯的工业废水的生物处理和对氯代硝基苯污染土壤的生物修复(生物整治)。
技术背景
氯代硝基苯类化合物(Chloronitrobenzenes,CNBs)作为重要的化工中间体被广泛用于生产和制造橡胶、染料、农药、医用药品等,以对氯硝基苯为例,我国2000年产量为12万吨。氯代硝基苯等芳香族化合物具有多种途径进入环境成为污染物,例如残留在生产过程中排放的废水,或者由环境中含芳香环化合物的不彻底降解和转化而来,例如硝基苯胺和氯代硝基苯胺是农药、染料、颜料等不彻底分解产生的;我国自来水供应多采用氯化消毒,氯代硝基苯是检测到的氯化消毒副产物之一。欧共体早已宣布氯代硝基苯是一种特别有害的化合物且在环境中难以降解,其危害包括引起人和动物的高铁血红蛋白血症(Methemoglobinemia),并且是一种诱变剂和致癌剂,五氯硝基苯破坏肺、肝、肾的功能,并直接损害神经系统。因此,研究氯代硝基苯类化合物的生物降解对于保护环境及提高人类健康具有重要意义。
目前,国内外就氯代苯类或者硝基苯类有机物的生物降解进行了大量研究,而对芳环上同时存在氯原子和硝基的氯代硝基苯类化合物的研究报道则较少,其主要原因是当氯原子和硝基同时存在时,由于二者的吸电子特性,导致芳环裂解更加困难。许多能够降解氯代苯或硝基苯等芳烃衍生物的菌种并不能降解对氯硝基苯。迄今,除了国外个别报道之外,单一菌种或混合菌种对氯硝基苯的降解能力、降解性质等所知甚少。因此,研究氯代硝基苯生物降解途径、开发微生物菌种资源有着重要的意义。
发明内容
本发明的目的在于针对氯代硝基苯类芳香族化合物污染对环境造成的严重危害,提供了一种丛毛单胞菌CNB1和该菌用于治理工业废水的生物治理技术,该菌是具有降解对氯硝基苯的丛毛单胞菌,在处理工业废水和治理环境污染中可以应用。具体地说,涉及了一株睾丸酮丛毛单胞菌(Comamonas testosteroni)CNB1 CGMCC No.1028菌株,该菌株的生长细胞、细胞悬浮液、固定化细胞菌可以降解对氯代硝基苯;该菌株能在含碳源、氮源或以对氯代硝基苯为为唯一碳源、氮源和能源的培养基生长,并将对氯代硝基苯完全降解利用。该菌株在以对氯代硝基苯为碳源、氮源和能源生长时,先将硝基还原成羟胺,然后在菌株产生的2-氨基酚1,6-双加氧酶的作用下,通过间位开环的方式,将2-氨基-5-氯苯酚开环成烃类,从而进一步被降解利用。该菌株适用于生产对氯代硝基苯的工业废水的生物处理和对氯代硝基苯污染土壤的生物修复(生物整治)。
本发明提供的睾丸酮丛毛单胞菌(Comamonas testosteroni)CNB1菌株,于2003年11月04日,在北京保藏于“中国微生物菌种保藏管理委员会普通微生物中心”,其保藏号为CGMCC No.1028。
该菌具有以下特征:(1)菌落特征:在LB平板上培养2天的菌落大小位直径1~2mm,菌落呈圆形,表面光滑,边缘整齐,凸起,无色。(2)睾丸酮丛毛单胞菌(Comamonas testosteroni)CNB1 CGMCC No.1028的细胞形态特征:适宜于pH 6~11范围内常温培养,细胞为短杆状,极毛2根,革兰式染色阴性,细胞大小为0.45μm×1.5μm。(3)睾丸酮丛毛单胞菌(Comamonas testosteroni)CNB1 CGMCC No.1028的生理生化特征:好氧生长,积累PHB,接触酶阴性,氧化酶阳性,不产生荧光色素,不水解淀粉和酪素,不液化明胶,能利用柠檬酸盐和丙二酸盐,不能利用葡萄糖、木糖、核糖、L-阿拉伯糖、海藻糖、棉籽糖、松三糖、甘露醇、甘露糖、乳糖、果糖、鼠李糖、山梨醇、蔗糖、蜜二糖、麦芽糖、纤维二糖、L-精氨酸、β-丙氨酸、酒石酸盐和乙醇;能利用L-缬氨酸、L-丙氨酸、L-脯氨酸、L-苯丙氨酸、组氨酸和葡萄糖酸钠。(4)睾丸酮丛毛单胞菌(Comamonas testosteroni)CNB1 CGMCC No.1028的16S rRNA基因序列特征:其16S rDNA具有如序列表所示的核苷酸序列,在GenBank中的登录号为AY291591。
参照《Bergey’s Mannual of Systematic Bacteriology》Vol.VIII的内容,根据其形态特征和生理生化特征,以及根据其16S rRNA基因序列在GenBank中的搜索结果,鉴定该菌(CGMCC No.1028)为睾丸酮丛毛单胞菌属(Comamonas testosteroni)。
该菌具有较高的降解对氯硝基苯的能力,并且还可以降解邻苯二酚、氯苯和苯胺等芳烃的衍生物。用Comamonas testosteroni CNB1 CGMCC No.1028生长细胞进行降解试验的结果表明,降解200mg/L的对氯硝基苯时,经过20h的培养,OD600可达0.12,对氯硝基苯的降解率达87%。该菌的最佳生长和降解条件为:28℃,pH 9.0。
睾丸酮丛毛单胞菌(Comamonas testosteroni)CNB1 CGMCC No.1028也可以在以部分芳烃的衍生物,如:邻苯二酚、氯苯、苯胺和对氯代硝基苯为唯一碳源的无机盐基础培养基中培养。应用前如在以对氯硝基苯为底物的基础培养基中驯化则效果更好,菌株在20~40℃之间的一适当温度下,进行好氧生长。
本发明提供的CGMCC No.1028菌能在碳源、氮源营养培养基,如:普通牛肉汁、LB、营养琼脂中培养或在以对氯硝基苯为为唯一碳源、氮源和能源的无机盐基础培养基中生长并降解利用对氯硝基苯,所述无机盐基础培养基组成:磷酸氢二钠1g,磷酸二氢钾0.5g,MgSO4·7H2O 0.03g,微量元素溶液5mL,对氯硝基苯200mg,蒸馏水1000mL,pH7.0。固体培养基中加入15g/L的琼脂。121℃灭菌30min。
附图说明:
图1Comamonas testosteroni CNB1 CGMCC No.1028以对氯硝基苯为唯一碳源、氮源和能源生长的生长和降解曲线
图2Comamonas testosteroni CNB1 CGMCC No.1028在不同温度下的生长曲线
图3Comamonas testosteroni CNB1 CGMCC No.1028在不同pH条件下的生长曲线
具体实施方案
为了更好地理解本发明,通过一下实施实例予以进一步说明,但并非对本发明的限定。
实施例1:睾丸酮丛毛单胞菌Comamonas testosteroni CNB1 CGMCC No.1028的培养及生物学特征。
睾丸酮丛毛单胞菌Comamonas testosteroni CNB1 CGMCC No.1028接种于无机盐培养基中,培养基组成:磷酸氢二钠1g,磷酸二氢钾0.5g,MgSO4·7H2O 0.03g,微量元素溶液5mL,对氯硝基苯200mg,蒸馏水1000mL,pH 7.0,固体培养基中加入15g/L的琼脂。121℃灭菌30min。于28-30℃、130rpm的摇床上振荡培养。该菌具有以下特征:(1)在LB平板上培养2天的菌落大小为直径1~2mm,菌落呈圆形,表面光滑,边缘整齐,凸起,无色。(2)睾丸酮丛毛单胞菌(Comamonastestosteroni)CNB1 CGMCC No.1028的细胞形态特征:适宜于pH 6~11范围内常温培养,细胞为短杆状,极毛2根,革兰式染色阴性,细胞大小为0.45μm×1.5μm。(3)睾丸酮丛毛单胞菌(Comamonastestosteroni)CNB1 CGMCC No.1028的生理生化特征:好氧生长,积累PHB,接触酶阴性,氧化酶阳性,不产生荧光色素,不水解淀粉和酪素,不液化明胶,能利用柠檬酸盐和丙二酸盐,不能利用葡萄糖、木糖、核糖、L-阿拉伯糖、海藻糖、棉籽糖、松三糖、甘露醇、甘露糖、乳糖、果糖、鼠李糖、山梨醇、蔗糖、蜜二糖、麦芽糖、纤维二糖、L-精氨酸、β-丙氨酸、酒石酸盐和乙醇;能利用L-缬氨酸、L-丙氨酸、L-脯氨酸、L-苯丙氨酸、组氨酸和葡萄糖酸钠。
参照《Bergey’s Mannual of Systematic Bacteriology》Vol.VIII的内容,根据其形态特征和生理生化特征,以及根据其16S rRNA基因序列在GenBank中的搜索结果,鉴定该菌(CGMCC No.1028)为睾丸酮丛毛单胞菌属(Comamonas testosteroni)。
睾丸酮丛毛单胞菌(Comamonas testosteroni)CNB1 CGMCC No.1028可以在营养培养基,如:普通牛肉汁、LB、营养琼脂中培养,也可以在以部分芳烃的衍生物,如:邻苯二酚、氯苯、苯胺和对氯代硝基苯为唯一碳源的基础培养基中培养。应用前如在以对氯硝基苯为底物的基础培养基中驯化则效果更好,菌株在20~40℃之间的一适当温度下,进行好氧生长。
该菌具有较高的降解对氯硝基苯的能力,并且还可以降解邻苯二酚、氯苯和苯胺等芳烃的衍生物。用Comamonas testosteroni CNB1 CGMCC No.1028生长细胞进行降解试验的结果表明,降解200mg/L的对氯硝基苯时,经过20h的培养,OD600可达0.12,对氯硝基苯的降解率达87%。该菌的最佳生长和降解条件为:28℃,pH 9.0。
本发明提供的CGMCC No.1028菌能以对氯硝基苯为唯一碳源、氮源和能源的无机盐基础培养基中生长并降解利用对氯硝基苯,所述无机盐基础培养基组成:磷酸氢二钠1g,磷酸二氢钾0.5g,MgSO4·7H2O 0.03g,微量元素溶液5mL,对氯硝基苯200mg,蒸馏水1000mL,pH7.0。固体培养基中加入15g/L的琼脂。121℃灭菌30min。
CGMCC No.1028所产生的对氯硝基苯硝基还原酶的酶活测定方法为:用紫外分光光度计检测340nm处由于NADPH氧化成NADP+而引起的光吸收值的减少值,计算出还原酶的活力。反应体系包括0.3μmol对氯硝基苯,0.3μmol NADPH,50mmol/L磷酸盐缓冲液1mL,含有0.3~0.9mg蛋白的细胞提取液,酶促反应总体积为3mL。NADPH的摩尔消光系数ε=6.22mmol-1cm-1。1个单位(U)的酶活力定义为每分钟转化1nmole的NADPH所需的酶量。
CGMCC No.1028所产生的2-氨基酚1,6-双加氧酶的酶活测定方法为:2-氨基苯酚的反应产物2-氨基粘康酸半醛在380nm处有最大吸收峰,紫外分光光度计测定380nm处的光吸收值的增加量,可测得双加氧酶的酶比活。反应体系包括0.3μmol 2-氨基苯酚,50mmol/L磷酸盐缓冲液1mL,含有0.3~0.9mg蛋白的细胞提取液,酶促反应总体积为3mL。2-氨基粘康酸半醛的摩尔消光系数ε=15.1mmol-1cm-1。1个单位(U)的酶活力定义为每分钟生成1nmol的2-氨基粘康酸半醛所需的酶量。
实施例2:Comamonas testosteroni CNB1 CGMCC No.1028的16S rRNA基因的PCR扩增和序列测定
Comamonas testosteroni CNB1 CGMCC No.1028接种于LB平板,从平板中直接挑取一环菌体,加入100μL无菌重蒸水中,漩涡混匀后,沸水浴2min,12000r/min离心5min,上清液直接用于PCR。用于16SrDNA扩增的PCR反应的引物为一对通用引物。正向引物Pf:5’-AGAGTTTGATCCTGGCTCAG-3’;反向引物Pr:5’-ACGGCTACCTTGTTACGACT-3’,分别对应于大肠杆菌的16S rRNA基因的8-27和1495-1514碱基。PCR反应体系(50μL)为:10×buffer 5μL、25mmol/L MgCl24μL、10mmol/LdNTPs 1μL、30pmol/L引物各1μL、BSA 0.5μL、ddH2O.37μL、TaqDNA酶0.4μL。PCR反应条件为:95℃4min,95℃1min,48℃1min,72℃1min,30个循环;72℃10min,4℃保存。PCR产物的纯化和测序由上海申能博彩生物科技有限公司完成。16S rDNA基因序列长度为1412bp,在GenBank中的登录号为AY291591,与菌株Comamonastestosteroni isolate Q10的16S rDNA(登录号为AF519533)序列的相似性为99%。其16S rDNA的核苷酸序列如序列表所示。
实施例3:本发明提供的Comamonas testosteroni CNB1 CGMCC No.1028对对氯硝基苯的降解
预先在无机盐培养基上培养的Comamonas testosteroni CNB1CGMCCNo.1028菌株培养液(OD600=0.2)按1%的接入量接种于含有对氯硝基苯(20mg/L)的培养基中,在30℃,130r/min的摇床上振荡培养,定时取样测定菌体生长、对氯硝基苯含量和氯离子浓度,结果如图1所示。图1表明,CNB1菌的生长和对氯硝基苯的降解都有个短暂的迟缓期,然后进入对数期,到第20小时达到最高生长量,此时降解率达87%,并且对氯硝基苯摩尔数的减少与氯离子摩尔数的增加相当。
实施例4:温度对Comamonas testosteroni CNB1 CGMCC No.1028降解对氯硝基苯的影响
预先在无机盐培养基上培养的Comamonas testosteroni CNB1CGMCCNo.1028培养液(OD600=0.2)按1%的接入量接种于含有对氯硝基苯(20mg/L)的培养基中,在130rpm的摇床,不同的温度条件下振荡培养1天,测定细胞的生长和氯离子释放,如图2所示。图2的结果表明,以对氯代硝基苯为唯一碳源和氮源培养,CNB1菌株细胞生长和对氯硝基苯的降解受温度变化的影响,在28℃左右达到最高的生长水平和最高的底物降解率,高于或低于此温度时细胞的生长和对氯硝基苯的降解都受到影响。
实施例5:培养基初始pH对Comamonas testosteroni CNB1 CGMCC No.1028降解对氯硝基苯的影响
预先在无机盐培养基上培养的CNB1菌株培养液(OD600=0.2)按1%的接入量接种于含有对氯硝基苯(200mg/L)的不同pH的培养基中,在30℃,130rpm的摇床上振荡培养1天,结果见图3。由图3可见,菌株CNB1是比较适合在碱性的条件下生长,同时,在碱性的条件下的底物降解率也比较好,而且它的生长和降解随pH的变化是一致的,pH 9.0是细胞生长和对氯硝基苯降解的最佳pH。
一种降解对氯代硝基苯的睾丸酮丛毛单胞菌及其用途
睾丸酮丛毛单胞菌(Comamonas testosteroni)CNB1 CGMCC No.1028的16S rDNA核苷酸序列
序列表
<110>中国科学院微生物研究所
<120>一株降解对氯代硝基苯的睾丸酮丛毛单胞菌CNB1及其用途
<130>
<160>1
<170>PatentIn version 3.1
<210>1
<211>1368
<212>DNA
<213>Comamonas testosteroni CNB1
<400>1
gtcctagtag tgggggataa ctactcgaaa gagtagctaa taccgcatga gatctacgga 60
tgaaagcagg ggaccttcgg gccttgtgct actagagcgg ctgatggcag attaggtagt 120
tggtggggta aaggcttacc aagcctgcga tctgtagctg gtctgagagg acgaccagcc 180
acactgggac tgagacacgg cccagactcc tacgggaggc agcagtgggg aattttggac 240
aatgggcgaa agcctgatcc agcaatgccg cgtgcaggat gaaggccctc gggttgtaaa 300
ctgcttttgt acggaacgaa aagcctgggg ctaatatccc cgggtcatga cggtaccgta 360
agaataagca ccggctaact acgtgccagc agccgcggta atacgtaggg tgcaagcgtt 420
aatcggaatt ctgggcgtaa agcgtgcgca ggcggttttg taagacaatg gtgaaatccc 480
cgggctcaac ctgggaactg ccattgtgac tgcaaggcta gagtgcggca gagggggatg 540
gaattccgcg tgtagcagtg aaatgcgtag atatgcggag gaacaccgat ggcgaaggca 600
atcccctggg cctgcactga cgctcatgca cgaaagcgtg gggagcaaac aggattagat 660
accctggtag tccacgccct aaacgatgtc aactggttgt tgggtcttaa ctgactcagt 720
aacgaagcta acgcgtgaag ttgaccgcct ggggagtacg gccgcaaggt tgaaactcaa 780
aggaattgac ggggacccgc acaagcggtg gatgatgtgg tttaattcga tgcaacgcga 840
aaaaccttac ccacctttga catggcagga acttaccaga gatggtttgg tgctcgaaag 900
agaacctgca cacaggtgct gcatggctgt cgtcagctcg tgtcgtgaga tgttgggtta 960
agtcccgcaa cgagcgcaac ccttgccatt agttgctaca ttcagttgag cactctaatg 1020
ggactgccgg tgacaaaccg gaggaaggtg gggatgacgt caagtcctca tggcccttat 1080
aggtggggct acacacgtca tacaatggct ggtacaaagg gttgccaacc cgcgaggggg 1140
agctaatccc ataaagccag tcgtagtccg gatcgcagtc tgcaactcga ctgcgtgaag 1200
tcggaatcgc tagtaatcgt ggatcagaat gtcacggtga atacgttccc gggtcttgta 1260
cacaccgccc gtcacaccat gggagcgggt ctcgccagaa gtaggtagcc taaccgcaag 1320
gagggcgctt accacggcgg ggttcgtgac tggggtgaag tcgtaacgxx tcccxxxaaa 1380
aaxtttxgat cctggctcax tcgtaacaag gt 1412
Claims (4)
1.一种具有降解对氯代硝基苯类芳香族化合物能力的睾丸酮丛毛单胞菌(Comamonas testosteroni)CNB1 CGMCC No.1028,其16S rDNA具有如SEQ ID NO:1所示的核苷酸序列。
2.一种培养权利要求1的睾丸酮丛毛单胞菌的培养基,包括普通牛肉汁,LB,营养琼脂培养基或以部分芳烃的衍生物或对氯代硝基苯为唯一碳源的无机盐基础培养基,所述部分芳烃的衍生物选自邻苯二酚、氯苯或苯胺。
3.权利要求2的培养基,其中所述无机盐基础培养基组成:磷酸氢二钠1g,磷酸二氢钾0.5g,MgSO4·7H2O 0.03g,微量元素溶液5mL,对氯硝基苯200mg,蒸馏水1000mL,pH7.0。
4.根据权利要求1所述的睾丸酮丛毛单胞菌在治理对氯代硝基苯类芳香族化合物的污染中的应用。
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CN101348772B (zh) * | 2008-02-19 | 2010-09-08 | 南京大学 | 水生丛毛单胞菌属菌株及其在废水生物脱氮中的应用 |
CN101947380B (zh) * | 2010-08-31 | 2012-08-01 | 天津工业大学 | 一种对苯二甲酸二乙酯的降解方法 |
CN103773705A (zh) * | 2012-10-22 | 2014-05-07 | 天津工业生物技术研究所 | 一种耐高浓度硫酸铵的睾丸酮丛毛单胞菌f2及其在废水脱氮中的应用 |
CN105385451B (zh) * | 2015-12-10 | 2018-12-28 | 湖南美鑫隆生态环保科技有限公司 | 一种腐殖酸铁与微生物复合土壤污染修复剂及其制备方法 |
CN105624063A (zh) * | 2016-02-04 | 2016-06-01 | 云南圣清环保科技有限公司 | 一种用于处理污泥的高效混合菌群 |
CN108048375B (zh) * | 2018-02-06 | 2020-08-04 | 北京大学 | 一种含油污泥中石油烃类的降解菌株jn6及其应用 |
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一株硝基苯高效降解菌的筛选及其降解特性 蔡邦成等,环境科学与技术,第26卷第4期 2003 * |
痰液培养中分离出睾丸酮丛毛单胞菌 周庭银等,上海医学检验杂志,第16卷第5期 2001 * |
痰液培养中分离出睾丸酮丛毛单胞菌 周庭银等,上海医学检验杂志,第16卷第5期 2001;一株硝基苯高效降解菌的筛选及其降解特性 蔡邦成等,环境科学与技术,第26卷第4期 2003 * |
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