CN112592867A - 兼具六价铬还原和活性黑5降解的人苍白杆菌及其应用 - Google Patents
兼具六价铬还原和活性黑5降解的人苍白杆菌及其应用 Download PDFInfo
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Abstract
本发明属于微生物菌剂技术领域，具体是涉及到兼具六价铬还原和活性黑5降解的人苍白杆菌及其应用，所述人苍白杆菌为苍白杆菌Ochrobactrum anthropic S1，保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO：M 2020461，本申请的人苍白杆菌能同时进行六价铬还原和活性黑5降解，具有较好的Cr污染和活性黑5污染处理前景。
Description
技术领域
本发明属于微生物菌剂技术领域，具体是涉及到兼具六价铬还原和活性黑5降解的人苍白杆菌及其应用。
背景技术
我国是世界上最早使用染料的国家之一，也是世界染料生产数量、消费数量和贸易数量最多的国家。染料应用范围广泛，常用纺织、印染、皮革和造纸等多个行业。在工业应用过程中，染料利用率较低，常有约15％的染料不能被利用而排放到环境中。据报道，我国仅纺织行业每年废水排放量高达18.4亿吨，每年大约有2000-3000t的染料随废水进入环境。含有偶氮双键(-N＝＝N-)的染料统称为偶氮染料，占纺织业染料使用量的70％左右，全球每年产生约10000种结构不同的偶氮染料，活性黑5是一种典型的双偶氮活性染料，其广泛用于工业生产中。所用原料包括多种已知的致癌物和诱变剂，且染料经分解后会产生有毒的中间产物，其多种物质是欧盟所禁用的，如果长期与人体接触，可能与人体分泌代谢物发生氧化还原反应，进而生成致癌的芳香胺化合物，增加患癌症的概率。另一方面，染料废水高色度不仅影响美观，同时其也能遮住光线，降低水体的透光率，影响水中植物的光合作用。
有报道显示，希瓦氏菌、肠杆菌等对于活性黑5具有较强的降解能力。
铬(Cr)是一种广泛应用于工业生产的重金属元素，常应用于电镀、制革、染色等行业。我国是世界铬盐生产大国，据统计2015年我国工业废水中六价铬(Cr(Ⅵ))及总铬(Cr)的排放量高达为70.4吨和188.6吨。水体中的Cr(Ⅵ)有剧毒，其属于美国环境保护署的十七类致癌物质化合物之一。自然环境中，Cr主要以Cr(Ⅵ)与Cr(Ⅲ)形式稳定存在。Cr(Ⅲ)的性质较稳定，毒性较小；Cr(Ⅵ)具有强氧化性和腐蚀性，研究表明Cr(Ⅵ)的毒性是Cr(Ⅲ)毒性的100倍。Cr(Ⅵ)氧化物和硫酸根、磷酸根的分子结构类似，能进入细胞与细胞内的还原性物质发生氧化还原反应，产生大量的自由基。其可与胞内的大分子物质结合，扰乱机体的新陈代谢。还能干扰线粒体的正常活动，抑制ATP的合成，诱导细胞凋亡。因此，Cr(Ⅵ)具有很强的致癌性和诱变性，可导致人体的肾、肝脏和脾等多种器官的功能损伤。
然而，铜和铬等金属常用于染料颜色的固定，所以含六价铬(Cr(Ⅵ))的铬酸盐常常存在纺织、染色、皮革废水中与染料共存，而染料和重金属污染物本身都具有毒性，对微生物具有抑制致死作用，因此，微生物处理含有活性黑5和Cr(Ⅵ)的废水时效率会大大降低。目前，针对含活性黑5或Cr(Ⅵ)废水治理的物理法、化学法、生物法及不同方法联合应用已经较为普遍，但即能处理活性黑5又可以处理Cr(Ⅵ)污染的微生物却鲜见报道。所以寻求能处理染料和Cr(Ⅵ)复合污染的微生物对解决环境污染问题具有极其重要的实际意义。
发明内容
本发明要解决的技术问题是提供一种兼具六价铬还原和活性黑5降解的人苍白杆菌及其应用，减少其他手段的配合使用，降低处理成本，提高经济效益。
本发明的内容包括一株兼具六价铬还原和活性黑5降解的人苍白杆菌，所述人苍白杆菌为苍白杆菌Ochrobactrum anthropic S1，保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO：M 2020461。
所述人苍白杆菌能兼具六价铬还原和活性黑5降解。具体步骤为，将所述人苍白杆菌加入含有六价铬和活性黑5的污水中，添加适量培养基，实现六价铬还原和活性黑5降解。
所述污水的温度为35-40℃，所述污水的pH为7。
所述能降解活性黑5和还原六价铬的人苍白杆菌S1筛选自广州东莞某纺织印染厂活性污泥中，样品在无菌收集后，经低温运送至实验室进行下一阶段研究工作。
在实验室环境中：利用厌氧反应器对活性污泥进行进一步驯化，驯化后的原液进行梯度稀释，以10-6为稀释终浓度，均匀涂布于LB固体平板上，并倒置于30℃恒温培养箱培养约12h。经反复分离纯化，最终得到能降解活性黑5和还原六价铬的单菌S1。提取S1的基因组DNA并扩增它的16S r DNA片段，并在NCBI上选取高相似度序列，再用MEGA 7.0构建系统发育树。NCBI比对发现其16s r DNA序列与Ochrobactrum anthropic MW6相似性为99.93％，系统进化树也表明其与Ochrobactrum sp.及为相近，因此命名为Ochrobactrumanthropic S1。此菌株已于2020年9月1日保藏于中国典型培养物保藏中心，地址：中国武汉武汉大学，邮编：430072，保藏中心保藏编号为CCTCC M 2020461。
在LB固体平板上挑取Ochrobactrum anthropic S1的单菌落接种于LB液体培养基中过夜培养，即为S1种子液。将10mL种子液与90mL灭菌培养基混合，然后在此培养基中加入干燥后至恒重的重铬酸钾粉末，使体系中的Cr(Ⅵ)浓度为20mg/L，并在30℃，180rpm条件下培养。同样接种比例另一份加入活性黑5粉末，使其浓度为50mg/L，并在30℃，静止条件下培养，每12h取样分析Cr(Ⅵ)和活性黑5的含量。
经实验验证：Ochrobactrum anthropic S1在pH 7.0，温度30℃，含Cr(Ⅵ)浓度为20mg/L的培养基中，180rpm培养3d，对Cr(Ⅵ)去除率达80.6％。
经实验验证：Ochrobactrum anthropic S1在pH 7.0，温度30℃，含活性黑5浓度为50mg/L的培养基中，静止培养3d，对活性黑5去除率达100％。
菌株Ochrobactrum anthropic S1在35-40℃表现出较好的去除效果。其中活性黑5去除在35℃最佳，Cr(Ⅵ)的去除在40℃最佳。
本发明的有益效果是，本发明的能降解活性黑5和还原六价铬的菌株Ochrobactrum anthropic S1分离及应用有效填补了该方面研究的空白，并为实际污染环境的治理工作提供了有效的理论依据，展现出巨大的实际应用前景。
附图说明
图1为本发明的菌株Ochrobactrum anthropic S1的系统发育分析树。
图2为本发明的菌株Ochrobactrum anthropic S1不同时间对活性黑5及Cr(Ⅵ)的去除率。
图3为本发明的菌株Ochrobactrum anthropic S1对活性黑5和Cr(Ⅵ)混合污染的去除率。
图4为本发明的菌株Ochrobactrum anthropic S1对含活性黑5和Cr(Ⅵ)的实际污水的去除率。
具体实施方式
实施例1：本发明的降解活性黑5和还原六价铬菌株Ochrobactrum anthropic S1形态特征
将单菌落划线接种至LB固体培养基中，将平板倒置于恒温培养箱内，30℃培养12h。该菌革兰氏染色呈阴性，专性需氧，菌落光滑呈圆形，中央凸起且明亮半透明。
实施例2：本发明的降解活性黑5和还原六价铬菌株Ochrobactrum anthropic S1的筛选与系统发育学鉴定
(1)广州东莞纺织印染厂采集的活性污泥样品，低温寄送至实验室，以供进一步筛选分析目的菌株。
(2)在实验室环境中：利用厌氧反应器对活性污泥进行进一步驯化，驯化后的原液进行梯度稀释，以10-6为稀释终浓度，均匀涂布于LB固体平板上，并倒置于30℃恒温培养箱培养12h。经反复分离纯化，最终得到能降解活性黑5和还原六价铬的单菌株S1。
(3)提取S1的基因组DNA并扩增它的16S r DNA片段，并在NCBI上选取高相似度序列，再用MEGA 7.0构建系统发育树。NCBI比对结果表明其16s r DNA序列与Ochrobactrumanthropic MW6相似性为99.93％，系统进化树再一次表明其属于Ochrobactrum sp.，因此命名为Ochrobactrum anthropic S1，如图1所示。
实施例3：本发明的降解活性黑5和还原六价铬菌株Ochrobactrum anthropic S1对活性黑5和六价铬的去除效果
(1)配制LB液体培养基，并在LB固体平板上挑取Ochrobactrum anthropic S1的单菌落接种于LB液体培养基中，过夜培养，即为S1种子液。
(2)将10mL种子液与90mL灭菌培养基混合，然后在此培养基中加入干燥后至恒重的重铬酸钾粉末，使体系中的Cr(Ⅵ)浓度为20mg/L，并在30℃，180rpm条件下培养。每12h取样分析剩余Cr(Ⅵ)的含量。结果显示Ochrobactrum anthropic S1对Cr(Ⅵ)去除率达80.6％。
(3)将10mL种子液与90mL灭菌培养基混合，然后在此培养基中加入活性黑5粉末，使其浓度为50mg/L，并在30℃，静止条件下培养，每12h取样分析剩余活性黑5的含量。结果显示Ochrobactrum anthropic S1对活性黑5去除率高达100％。结果如图2所示。
实施例4：本发明的降解活性黑5和还原六价铬菌株Ochrobactrum anthropic S1同时去除活性黑5和还原Cr(Ⅵ)的效果
(1)配制LB液体培养基，并在LB固体平板上挑取菌株Ochrobactrum anthropic S1的单菌落接种于LB液体培养基中，过夜培养，即为S1种子液。
(2)将10mL种子液与90mL灭菌培养基混合，然后加入活性黑5粉末和重铬酸钾粉末，设定Cr(Ⅵ)的浓度为20mg/L，活性黑5的浓度为50mg/L，并在30℃，静止条件下培养，每12h取样分析剩余活性黑5和Cr(Ⅵ)的含量。
(3)该实验结果显示菌株Ochrobactrum anthropic S1可同时去除65.9％Cr(Ⅵ)和86％的活性黑5，结果如图3所示。
上述LB固体培养基组分为：胰蛋白胨10g，酵母浸粉5g，氯化钠10g，琼脂粉15g/L，去离子水1L，pH6.9～7.1。LB液体培养基组分为：胰蛋白胨10g，酵母浸粉5g，氯化钠10g，去离子水1L，pH6.9～7.1。
实施例5：本发明的降解活性黑5和还原六价铬菌株Ochrobactrum anthropic S1处理实际污水同时去除活性黑5和还原Cr(Ⅵ)的效果
(1)量取9L的实际污水置于反应器中，并在LB固体平板上挑取菌株Ochrobactrumanthropic S1的单菌落接种于LB液体培养基中，过夜培养，即为S1种子液。
(2)将1L种子液与9L从东莞某印染厂取得的实际污水混合，并在30℃静止条件下培养，每12h取样分析剩余活性黑5和Cr(Ⅵ)的含量。
(3)该实验结果显示菌株Ochrobactrum anthropic S1可同时去除44％Cr(Ⅵ)和72％的活性黑5，结果如图4所示。
对比例1：对比例1和实施例3相比，区别在于选取的人苍白杆菌为一般的人苍白杆菌，其他和实施例3相同。试验结果显示，其对Cr(Ⅵ)去除率达78.5％，对活性黑5去除率高达70.2％。
对比例2：对比例2和实施例4相比，区别在于选取的人苍白杆菌一般的人苍白杆菌，其他和实施例4相同。试验结果显示，其可同时去除58.7％Cr(Ⅵ)和49.6％的活性黑5。
Claims (5)
1.一株兼具六价铬还原和活性黑5降解的人苍白杆菌，其特征是，所述人苍白杆菌为苍白杆菌Ochrobactrum anthropic S1，保藏于中国典型培养物保藏中心，保藏编号为CCTCCNO：M 2020461。
2.一种如权利要求1所述的人苍白杆菌的应用，其特征是，所述人苍白杆菌能兼具六价铬还原和活性黑5降解。
3.如权利要求2所述的人苍白杆菌的应用，其特征是，将所述人苍白杆菌加入含有六价铬和活性黑5的污水中，添加适量培养基，实现六价铬还原和活性黑5降解。
4.如权利要求3所述的人苍白杆菌的应用，其特征是，所述污水的温度为20-45℃。
5.如权利要求3所述的人苍白杆菌的应用，其特征是，所述污水的pH为7-11。
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| CN101418266A (zh) * | 2007-10-23 | 2009-04-29 | 中国科学院福建物质结构研究所 | 苍白杆菌cts-325及其培养方法以及还原六价铬的应用 |
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