CN114196589A - 食醚红球菌zhc及其在降解丙烯酸甲酯中的应用 - Google Patents
食醚红球菌zhc及其在降解丙烯酸甲酯中的应用 Download PDFInfo
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Abstract
本发明公开了一种食醚红球菌ZHC及其在降解丙烯酸甲酯中的应用，所述的应用是将食醚红球菌ZHC经扩大培养获得的静息细胞加入pH＝6‑9、含丙烯酸甲酯的无机培养液中，在25‑35℃、150‑200rpm条件下进行培养，实现丙烯酸甲酯的降解。本发明食醚红球菌ZHC对于丙烯酸甲酯具有较好地降解效果，去除浓度范围95‑475mg/L，可以较为完全地把丙烯酸甲酯转化为CO2、H2O等无害物质，因而在工业废气废水的生物净化中具有广阔的应用前景。因此，该食醚红球菌ZHC对于丙烯酸甲酯具有高效的降解能力，且能承受较高浓度的污染物。
Description
(一)技术领域
本发明涉及一种食醚红球菌(Rhodococcus aetherivorans)ZHC及其在降解丙烯酸甲酯污染物中的应用。
(二)背景技术
酯是酸(羧酸或无机含氧酸)与醇起反应生成的一类有机化合物。低级的酯是有香气的挥发性液体，高级的酯是蜡状固体或很稠的液体。几种高级的酯是脂肪的主要成分。酯难溶于水，易溶于乙醇等有机溶剂，其中低分子量的酯类是无色、易挥发的气体。低分子量的酯可用作溶剂，分子量较大的酯是良好的增塑剂。如甲基丙烯酸甲酯是制造有机玻璃(聚甲基丙烯酸甲酯)的单体；聚酯树脂主要用于纤维和油漆工业，也可制成压塑粉；许多带有支链的醇形成的酯是优良的润滑油。酯还用于香料、香精、化妆品、肥皂和药品等工业。小分子酯类经排放挥发进入大气环境后，对眼及呼吸道产生刺激作用，高浓度则会产生急性中毒。
丙烯酸甲酯是一种无色易挥发液体，是重要的精细化工单体及有机合成中间体。广泛用于涂料、纺织、制革、黏合剂、建材、造纸等行业。但是丙烯酸甲酯有催泪性，对呼吸系统和皮肤有刺激性，属全身性毒物。侵入途径有：吸入、食入、经皮吸收。健康危害：高浓度接触，引起流涎、眼及呼吸道的刺激症状，严重者口唇发白、呼吸困难、痉挛，因肺水肿而死亡。误服急性中毒者，出现口腔、胃、食管腐蚀症状，伴有虚脱、呼吸困难、躁动等。长期接触可致皮肤损害，亦可致肺、肝、皮肤病变。
因此研究环境中丙烯酸甲酯的高效降解对人类健康很有必要，通过文献检索，未发现有关食醚红球菌以丙烯酸甲酯为唯一碳源来实现丙烯酸甲酯高效降解的报道。本发明的食醚红球菌(Rhodococcus aetherivorans)ZHC能降解丙烯酸甲酯，并且生长环境温和，易扩大培养，目前，暂无有降解丙烯酸甲酯降解菌的报道。该降解菌的发现对于工业废水废气中酯类污染物的高效净化具有重要意义。
(三)发明内容
本发明目的是提供一种食醚红球菌ZHC及其在降解丙烯酸甲酯中的应用，该菌株具有高效、去除能力强的具有丙烯酸甲酯降解能力，解决了物理化学方法在处理丙烯酸甲酯时效果不佳、不经济的局限性，减少了能耗并解决了二次污染问题。
本发明采用的技术方案是：
本发明提供一株丙烯酸甲酯降解新菌株——食醚红球菌(Rhodococcusaetherivorans)ZHC，保藏于中国典型培养物保藏中心，保藏日期：2021年7月5日，保藏编号：CCTCC NO:M2021816，地址：中国，武汉，武汉大学，430072。
本发明食醚红球菌ZHC的基本特征为：菌落橙黄色，边缘规则，不透明，表面光滑，凸起。透射电镜下观察该菌体的形态为球菌，无鞭毛，革兰氏染色阳性。
本发明还提供一种食醚红球菌ZHC在降解丙烯酸甲酯中的应用，具体所述的应用是将食醚红球菌ZHC经扩大培养获得的静息细胞加入pH＝6-9、含丙烯酸甲酯的无机培养液中，在25-35℃、150-200rpm条件下进行培养，实现丙烯酸甲酯的降解。
进一步，所述无机盐培养液中，静息细胞加入量以菌体干重计为15-45mg/L，优选30mg/L，丙烯酸甲酯的初始浓度为95-475mg/L，优选95-190mg/L，更优选190mg/L。
进一步，所述无机盐培养液组成为：KH2PO4 0.234g/L、K2HPO4·3H2O 0.942g/L、NaNO3 1.7g/L、NH4Cl 0.98g/L、MgCl2·6H2O 0.2033g/L、CaCl2·2H2O 0.0111g/L、FeCl30.0162g/L、微量元素5mL/L，pH 6-8，溶剂为去离子水；所述微量元素组成为ZnCl20.088g/L、MnCl2·4H2O 0.060g/L、KI 0.01g/L、Na2MoO4·2H2O 0.1g/L、H3BO3 0.05g/L，溶剂为去离子水。
进一步，优选培养条件为30℃、160rpm反应72h。pH优选为6-8，更优选pH为7。
进一步，所述食醚红球菌ZHC按如下步骤制备：
(1)斜面培养：
将食醚红球菌ZHC接种至LB液体培养基中，在30℃，160rpm下培养2d，使保藏的细菌活化复苏，再将活化的细菌画线于固体LB平板30℃培养箱培养，取单菌落继续平板画线以检测细菌的纯度，得到可常规保持(4℃)的细菌斜面，该细菌斜面需要每三个月转接一次，以保证菌种的活性；LB固体培养基组成：5g/L酵母膏，10g/L NaCl，10g/L蛋白胨，20g/L琼脂，pH自然，溶剂为去离子水。
(2)扩大培养
将步骤(1)斜面菌体接种至LB液体培养基中，在30℃，160rpm下培养12h，获得扩大培养液，离心(优选8000rpm离心10min)，收集湿菌体，无机盐培养液洗涤，获得食醚红球菌ZHC静息细胞；LB液体培养基组成：5g/L酵母膏，10g/L NaCl，10g/L蛋白胨，pH自然，溶剂为去离子水。
与现有技术相比，本发明有益效果体现在：
本发明提供的食醚红球菌ZHC取自污水厂污泥，对于丙烯酸甲酯具有较好地降解效果，可以较为完全地把丙烯酸甲酯转化为CO2、H2O等无害物质，因而在工业废气废水的生物净化中具有广阔的应用前景。
本发明所述的食醚红球菌ZHC能将丙烯酸甲酯完全降解，去除浓度范围95-475mg/L。食醚红球菌ZHC能够在72h内完全降解浓度低于200mg/L丙烯酸甲酯。因此，该食醚红球菌ZHC对于丙烯酸甲酯具有高效的降解能力，且能承受较高浓度的污染物。
(四)附图说明
图1为食醚红球菌ZHC在LB培养基上菌落形态照片。
图2为食醚红球菌ZHC的透射电子显微镜照片。
图3为食醚红球菌ZHC的系统发育树图。
图4为食醚红球菌ZHC对丙烯酸甲酯的降解率，(a)为食醚红球菌ZHC在72h内对不同浓度丙烯酸甲酯的降解率，(b)为食醚红球菌ZHC在48h内在不同pH下对190mg/L丙烯酸甲酯的降解率。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。
本发明实施例所述无机盐培养液组成为：KH2PO4 0.234g/L、K2HPO4·3H2O 0.942g/L、NaNO3 1.7g/L、NH4Cl 0.98g/L、MgCl2·6H2O 0.2033g/L、CaCl2·2H2O 0.0111g/L、FeCl30.0162g/L、微量元素5mL/L，pH 6-8，溶剂为去离子水；所述微量元素组成为FeSO4·7H2O1mg/L、CuSO4·5H2O 0.02mg/L、CoCl2·6H2O 0.02mg/L、MnSO4·4H2O 0.1mg/L、Na2MoO4·2H2O 0.02mg/L、H3BO3 0.02mg/L，溶剂为去离子水。
无机盐固体培养基是在无机盐培养液中添加20g/L琼脂。
LB固体培养基组成：5g/L酵母膏，10g/L NaCl，10g/L蛋白胨，20g/L琼脂，pH自然，溶剂为去离子水。
LB液体培养基组成：5g/L酵母膏，10g/L NaCl，10g/L蛋白胨，pH自然，溶剂为去离子水。
所述室温为25-30℃。
实施例1：Rhodococcus aetherivorans ZHC的分离、纯化及其鉴定。
1、Rhodococcus aetherivorans ZHC的分离及纯化。
(1)菌株筛选
Rhodococcus aetherivorans ZHC是从活性污泥中驯化、分离得到的一株革兰氏阳性菌，具体步骤如下：
在330mL摇瓶中加入50mL无机盐培养液，并加入10mL取自某污水厂活性污泥(MLVSS 75mg/L)，再加入终浓度95mg/L的丙烯酸甲酯，30℃、160rpm下进行富集培养，待丙烯酸甲酯浓度为初始加入的50％时，从中取出5mL富集液接种于50mL新鲜无机盐培养液中，加入相同量的丙烯酸甲酯使其终浓度95mg/L，重复上述富集过程5次后，将最后一次富集液用无机盐培养液梯度稀释10、100、1000倍，分别涂布无机盐固体培养基，30℃培养3天，选择单菌落再次接种至LB固体培养基画线纯化，30℃培养3天，见图1。挑取单菌落接种至含终浓度95mg/L丙烯酸甲酯作为唯一的碳源及能源的无机盐固体培养基，30℃、160rpm培养3天，得到菌株ZHC，通过透射电子显微镜确定其形态(图2)。
(2)菌株ZHC特征：菌落橙黄色，边缘规则，不透明，表面光滑，凸起。透射电镜下观察该菌体的形态为球菌，无鞭毛，革兰氏染色阳性。
2、菌株ZHC的鉴定
通过16S rRNA序列分析和生理生化实验鉴定，确定该菌株为Rhodococcusaetherivorans，具体步骤如下：
(1)16S rRNA序列分析
采用Ezup柱式细菌基因组DNA抽提试剂盒提取和纯化菌株ZHC的DNA，4℃保存。用细菌的通用引物对纯化的DNA进行PCR扩增，引物分别为27F(AGAGTTTGATCCTGGCTCAG)和1492R(GGTTACCTTGTTACGACTT)，PCR反应程序设定为94℃预变性4min，然后94℃变性45s，55℃退火45s，72℃延伸1min，循环30个周期，最后72℃修复延伸10min。将PCR产物纯化回收后进行测序(上海美吉生物医药科技有限公司)，16S rRNA(核苷酸序列如SEQ ID NO.1所示)测序结果上传至NCBI，获得登录号OK036739，同时将该序列同NCBI数据库中的基因序列进行Blast对比。发现其属于Rhodococcus属，与Rhodococcus aetherivorans strain DSM44752、Rhodococcus zopfii strain DSM44108和Rhodococcus coprophilus strain DSM43347具有99％的同源性。从结果中选取8株Rhodococcus具有代表性的菌株，以16S rRNA基因序列同源性为基础，采用MEGA6.0软件构建系统发育树，如图3。
16S rRNA基因序列
(2)生理生化实验
菌株ZHC对梅里埃CBC卡上41种碳源的利用能力。
利用梅里埃全自动鉴定仪考察菌株ZHC对41中不同碳源的代谢情况(委托给浙江天科高新技术发展有限公司(原浙江省微生物研究所))。鉴定结果如表1所示。经梅里埃全自动鉴定仪VITEK生化反应，菌株ZHC可较强利用11种碳源，对其他37种碳源不能利用。
表1菌株ZHC梅里埃全自动鉴定仪VITEK生化反应结果(CBC卡)
表注：+，阳性反应；-：阴性反应
通过遗传距离及16S rRNA序列对比，结合菌落特征和生理生化特征，将菌株ZHC鉴定为Rhodococcus aetherivorans，命名为食醚红球菌(Rhodococcus aetherivorans)ZHC，保藏于中国典型培养物保藏中心，保藏日期：2021年7月5日，保藏编号：CCTCC NO:M2021816，地址：中国，武汉，武汉大学，430072。
实施例2、食醚红球菌ZHC静息细胞的获得
①斜面培养：
将食醚红球菌ZHC接种至LB液体培养基中，在30℃，160rpm下培养2d，再将活化的细菌画线于固体LB平板30℃培养箱培养，取单菌落继续平板画线以检测细菌的纯度，进行LB试管斜面常规(4℃)保存。
②扩大培养
将步骤①斜面菌体接种至LB液体培养基中，在30℃，160rpm下培养12h，获得扩大培养液，8000rpm离心10min，收集湿菌体，无机盐培养液洗涤，获得食醚红球菌ZHC静息细胞。
实施例3：食醚红球菌ZHC对不同浓度丙烯酸甲酯的降解性能检测。
将无机盐培养液分装于体积均为330mL的摇瓶中，每瓶50mL，110℃灭菌40min。灭菌结束后室温放置2d，确定无杂菌生长。加入终浓度30mg/L(以细胞干重记)实施例2方法获得的静息细胞，然后加入丙烯酸甲酯作为唯一碳源使其终浓度分别为95、190、285、380、475mg/L，摇瓶用聚四氟乙烯瓶塞密封后，30℃，160rpm摇床培养，并做不加细菌的空白对照。采用气相色谱测量，使用检测器为火焰离子化检测器，色谱柱型号为Agilent 19091N-213(30m×320um×0.5um)，每次使用1mL气体进样针进样，每次进样1mL。气相色谱条件为：色谱柱温90℃，进样口温度210℃，检测器温度250℃，高纯氮气为流动相，流量为16.6mL/min，柱流量0.9mL/min。定时取样，测定摇瓶中残留的丙烯酸甲酯浓度，绘制菌株72h对于不同初始浓度丙烯酸甲酯的去除率曲线，结果见图4中(a)所示。结果表明，当丙烯酸甲酯浓度低于190mg/L时，菌株ZHC可以在72h内快速的降解所添加的底物，降解率达100％。
实施例4：食醚红球菌ZHC在不同pH下降解190mg/L丙烯酸甲酯的降解性能测试。
将无机盐培养液分装于体积均为330mL的摇瓶中，每瓶50mL，调节pH，pH设置为：4、5、6、7、8、9。110℃灭菌40min。灭菌结束后室温放置2d，确定无杂菌生长。加入终浓度30mg/L(以细胞干重记)实施例2方法获得的静息细胞，然后加入丙烯酸甲酯作为唯一碳源使其终浓度均为190mg/L，摇瓶用聚四氟乙烯瓶塞密封后，30℃，160rpm摇床培养，并做不加细菌的空白对照。定时取样，采用实施例3方法测定摇瓶中残留的丙烯酸甲酯浓度，绘制菌株48h对于不同pH下，同一初始浓度丙烯酸甲酯的去除率曲线，结果见图4中(b)所示。结果表明，当pH＝7菌株ZHC可以快速的降解所添加的底物。
虽然本发明已以实施例公开如上，但其并非用以限定本发明的保护范围，任何熟悉该技术的技术人员，在不脱离本发明的构思和范围内所作的更改与润饰，均应属于本发明的保护范围。
序列表
<110> 浙江工业大学
<120> 食醚红球菌ZHC及其在降解丙烯酸甲酯中的应用
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<170> SIPOSequenceListing 1.0
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<212> DNA
<213> 食醚红球菌(Rhodococcus aetherivorans)
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cggctccctc cccgaggggt taggccccgg cttcgggtgt taccgacttt catgacgtga 60
cgggcggtgt gtacaaggcc cgggaacgta ttcaccgcag cgttgctgat ctgcgattac 120
tagcgactcc gacttcacgg ggtcgagttg cagaccccga tccgaactga gaccggcttt 180
aagggattcg ctccacctcg cggtatcgca gccctctgta ccggccattg tagcatgtgt 240
gaagccctgg acataagggg catgatgact tgacgtcgtc cccaccttcc tccgagttga 300
ccccggcagt ctcctgcgag tccccaaccg aattgctggc aacacaggac aagggttgcg 360
ctcgttgcgg gacttaaccc aacatctcac gacacgagct gacgacagcc atgcaccacc 420
tgtacaccga ccacaaggga aaccccatct ctggggcggt ccggtgtatg tcaaacccag 480
gtaaggttct tcgcgttgca tcgaattaat ccacatgctc cgccgcttgt gcgggccccc 540
gtcaattcct ttgagtttta gccttgcggc cgtactcccc aggcggggcg cttaatgcgt 600
tagctacggc acggatcccg tggaaggaaa cccacaccta gcgcccaccg tttacggcgt 660
ggactaccag ggtatctaat cctgttcgct acccacgctt tcgctcctca gcgtcagtta 720
ctgcccagag acccgccttc gccaccggtg ttcctcctga tatctgcgca tttcaccgct 780
acaccaggaa ttccagtctc ccctgcagta ctcaagtctg cccgtatcgc ctgcaagccc 840
gcagttgagc tgcgggtttt cacagacgac gcgacaaacc gcctacgagc tctttacgcc 900
cagtaattcc ggacaacgct cgcaccctac gtattaccgc ggctgctggc acgtagttgg 960
ccggtgcttc ttctgtacct accgtcactc gcgcttcgtc ggtactgaaa gaggtttaca 1020
acccgaaggc cgtcatccct cacgcggcgt cgctgcatca ggcttgcgcc cattgtgcaa 1080
tattccccac tgctgcctcc cgtaggagtc tgggccgtgt ctcagtccca gtgtggccgg 1140
tcgccctctc aggccggcta cccgtcgtcg ccttggtggg ccgttacccc accaacaagc 1200
tgataggccg cgggcccatc ctgcaccgga aaacctttcc accccggaac atgcatcccg 1260
aggtcctatc cggtattaga cccagtttcc caggcttatc ccgaagtgca gggcagatca 1320
cccacgtgta ctcacccgtt cgccactaat ccacccagca agctgggctc atcgtcgact 1380
gcaggtacc 1389
Claims (7)
1.食醚红球菌(Rhodococcus aetherivorans)ZHC，保藏于中国典型培养物保藏中心，保藏日期：2021年7月5日，保藏编号：CCTCC NO:M2021816，地址：中国，武汉，武汉大学，430072。
2.一种权利要求1所述食醚红球菌ZHC在降解丙烯酸甲酯中的应用。
3.如权利要求2所述的应用，其特征在于所述的应用是将食醚红球菌ZHC经扩大培养获得的静息细胞加入含丙烯酸甲酯的pH6-9无机培养液中，在25-35℃、150-200rpm条件下进行培养，实现丙烯酸甲酯的降解。
4.如权利要求3所述的应用，其特征在于所述无机盐培养液中，静息细胞加入量以菌体干重计为15-45mg/L，丙烯酸甲酯的初始浓度为95-475mg/L。
5.如权利要求3所述的应用，其特征在于所述无机盐培养液组成为：KH2PO4 0.234g/L、K2HPO4·3H2O 0.942g/L、NaNO3 1.7g/L、NH4Cl 0.98g/L、MgCl2·6H2O 0.2033g/L、CaCl2·2H2O 0.0111g/L、FeCl3 0.0162g/L、微量元素5mL/L，pH 6-8，溶剂为去离子水；所述微量元素组成为ZnCl2 0.088g/L、MnCl2·4H2O 0.060g/L、KI 0.01g/L、Na2MoO4·2H2O0.1g/L、H3BO30.05g/L，溶剂为去离子水。
6.如权利要求3所述的应用，其特征在于所述培养条件为30℃、160rpm。
7.如权利要求3所述的应用，其特征在于所述食醚红球菌ZHC按如下步骤制备：
(1)斜面培养：
将食醚红球菌ZHC接种至LB液体培养基中，在30℃，160rpm下培养2d，再将活化的细菌画线于固体LB平板，30℃培养箱培养，得到斜面菌体；LB固体培养基组成：5g/L酵母膏，10g/L NaCl，10g/L蛋白胨，20g/L琼脂，pH自然，溶剂为去离子水；
(2)扩大培养
将步骤(1)斜面菌体接种至LB液体培养基中，在30℃，160rpm下培养12h，获得扩大培养液，离心，收集湿菌体，无机盐培养液洗涤，获得食醚红球菌ZHC静息细胞；LB液体培养基组成：5g/L酵母膏，10g/L NaCl，10g/L蛋白胨，pH自然，溶剂为去离子水。
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| CN116179402B (zh) * | 2022-09-06 | 2024-01-30 | 南京工业大学 | 一株类胡萝卜素合成菌株及其应用 |
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