CN107177532A - 一种有机氮类污染物降解菌剂及其应用 - Google Patents
一种有机氮类污染物降解菌剂及其应用 Download PDFInfo
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Abstract
本发明公开了一种有机氮类污染物降解菌剂及其应用。该菌株分类命名为索诺拉沙漠芽孢杆菌（Bacillus sonorensis）DX‑5，该菌已保藏于中国微生物菌种保存管理委员会普通微生物中心，保藏编号为：CGMCC No.14161，其生物学特性为短杆状，革兰氏阳性，氧化酶阴性，氯化钠耐受浓度为3%，能够厌氧生长，生长温度范围为15℃‑55℃。所述索诺拉沙漠芽孢杆菌（Bacillus sonorensis）可用于制备含含氮杂化化合物废水处理微生物菌剂。
Description
技术领域
本发明属于微生物领域，具体涉及一株有机氮类污染物降解菌剂------索诺拉沙漠芽孢杆菌（Bacillus sonorensis）DX-5及其应用。
背景技术
有机氮化合物广泛应用于石油、食品和医药工业，大都是有毒且难降解的有机物，不易受代谢过程的破坏，属于污染面广的难降解有机物，对生态系统和人类健康会产生潜在的长期危害。目前有机氮化合物（包括吡啶、喹啉、吡咯、咔唑及其衍生物等）被广泛用于石油、焦化、制药、染料和橡胶合成过程，随生产废水进入环境。其中吡啶和喹啉是典型的含氮杂环化合物，而且也是重要的工业污染物。吡啶对不同生物体表现出广泛的毒性，具有致畸变特性和毒性，对健康具有潜在的危害性，吡啶美国环境保护署（United StatesEnvironmental Protection Agency，USEPA）列为优先控制物质之一。
2-氯-5-氯甲基吡啶（2-chloro-chloromethylpyridine，简称CCMP）是新型高效农药吡虫啉的关键中间体，也是这一类农药分子中最具杀虫活性的部分，而且通过它的烷基化反应、与杂环N-H的缩合反应以及氨化后与1-硝基-2，2-双(硫甲基)乙烯的反应，还可制备一系列新的杀虫杀螨剂，是合成农药和医药的重要中间体。2-氯-5-氯甲基吡啶具有较强的致敏性和刺激性，随吡虫啉生产废水进入环境会对生态和人体健康造成严重危害。微生物降解具有成本低、效率高、无二次污染、生态恢复性好等优点，已在很多方面得到应用，所以筛选出CCMP降解菌对于处理吡虫啉生产废水具有重大意义。
发明内容
本发明提供一株有机氮类污染物降解菌剂-索诺拉沙漠芽孢杆菌（Bacillus sonorensis）DX-5，及其在降解含氮杂化化合物中的应用。
本发明提供的索诺拉沙漠芽孢杆菌（Bacillus sonorensis）DX-5，该菌株已于2017年5月15日保藏于中国微生物菌种保存管理委员会普通微生物中心，地址：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，邮编100101，保藏号为CGMCC No.14161。所述索诺拉沙漠芽孢杆菌（Bacillus sonorensis）DX-5是一株革兰氏染色阳性菌，短杆状，氧化酶阴性，氯化钠耐受浓度为3%，能够厌氧生长，生长温度范围为15℃-55℃。
所述的有机氮类污染物降解菌在降解有机氮类污染物中的应用
所述索诺拉沙漠芽孢杆菌（Bacillus sonorensis）DX-5在降解2-氯-5-氯甲基吡啶（CCMP）中应用。
含有所述索诺拉沙漠芽孢杆菌DX-5的菌剂，菌剂浓度为2.5-5.0×108cfu/ml。
所述索诺拉沙漠芽孢杆菌DX-5菌剂的生产方法，包括：斜面种－摇瓶种－种子罐－生产罐－产品，所述产品的包装剂型为液体菌剂或固体吸附菌剂。
所述菌剂的生产方法，具体为：
1）将索诺拉沙漠芽孢杆菌DX-5的试管种接种于添加CCMP的LB培养基中，振荡培养至对数期；
2）将步骤1）培养好的菌种按10％的接种量接种入500升种子罐，培养至对数生长期，即种子液；
3）将步骤2）所述种子液按10％的接种量接入生产罐进行发酵培养，生产罐所用培养基与种子罐培养基相同，发酵完成后的培养液即为菌剂；
步骤3）所述菌剂直接用塑料包装桶或包装瓶分装成液体剂型，或采用泥炭吸附用包装袋分装成固体菌剂剂型。
步骤1）中所述的LB培养基配方为：NaCl 10.00g/L,蛋白胨10.00g/L,酵母粉5.00g/L,CCMP 100mg/L，pH7.0。
步骤2）所述的种子罐所用的培养基配方为：CCMP 100mg/L，葡萄糖8g/L，（NH4）2SO4 1g/L，K2HPO4 2g/L，MgSO40.5g/L，NaCl 1g/L%，CaCO30.5g/L，酵母膏2g/L，pH值7.2-7.5。
步骤2）所述种子罐和步骤3）所述的生产罐的培养过程中，无菌空气的通气量为1：0.6-1.2，搅拌速度为180-240转/分，培养温度为30-35℃，步骤2）和3）的整个工艺流程培养时间为48-60小时。
有益效果：
本发明提供的索诺拉沙漠芽孢杆菌（Bacillus sonorensis）DX-5可以在24h内降解100mg/LCCMP。将其应用于吡虫啉生产废水中可有效提高废水的可生化性（B/C比），在接种的24h内，吡虫啉生产废水的B/C比可从0.09上升至0.58。
附图说明
图1索诺拉沙漠芽孢杆菌（Bacillus sonorensis）DX-5在LB固体培养基生长的菌落照片；
图2索诺拉沙漠芽孢杆菌（Bacillus sonorensis）DX-5利用CCMP的生长降解曲线；
图3不同温度索诺拉沙漠芽孢杆菌（Bacillus sonorensis）DX-5降解CCMP的降解情况；
图4不同pH对索诺拉沙漠芽孢杆菌（Bacillus sonorensis）DX-5降解CCMP的影响；
图5DX-5菌剂对吡虫啉生产废水的降解效果。
具体实施方式
实施例1 CCMP降解菌DX-5的筛选及分离
取从生产吡虫啉的某农药厂生产车间的废水处理池中得到的活性污泥3.0 ml加入100ml CCMP浓度为100 mg/L的含有吡啶的无机盐培养基中，其配方为：每升含100 mg/L CCMP，1.50g K2HPO4，0.50g KH2PO4，0.20g MgSO4×7H2O、1.00g NaCl，1.00g (NH4)2SO4，pH 7.0。于160 r/min，30℃条件下振荡培养，每隔24h按5%的接种量转接到新鲜的无机盐培养基中，连续转接5次。
取上面得到的富集菌液1.0 ml，加入9.0 ml无菌水中，配成10-1的富集液，再吸取1.0 ml配好的10-1的富集液加入9.0 ml无菌水中，充分混匀配成10-2的富集液，以此类推，对富集液进行梯度稀释。以此类推，对富集液进行梯度稀释。吸取各梯度的稀释液0.1 ml涂布于含CCMP的浓度为100 mg/L无机盐固体培养基上，30℃培养24 h。24 h后从以上的无机盐固体培养基上挑取单菌落，于3.0 mL的LB液体培养基中培养24 h，LB液体培养基配方为：NaCl 10.00 g/L,蛋白胨10.00 g/L,酵母粉5.00 g/L, pH 7.0于8000 r/min的条件下离心2 min，倒去上清液，加入3.0 mL的无菌水摇匀，仍于8000 r/min的条件下离心2min，按此方法用无菌水洗两遍后，加入3.0 ml无菌水重悬该菌。吸取1.0 mL该菌液加入100 ml CCMP浓度为100 mg/L的无机盐液体培养基中，于160 r/min，30℃条件下振荡培养24h后，用高效液相色谱测其降解效果。通过上述方法筛选到一株对CCMP降解率最高的菌株，命名为DX-5，DX-5在LB固体培养基上的菌落形态为，淡黄色，表面光滑（见附图1）。利用高盐法提取CCMP降解菌株DX-5的基因组，利用16S通用引物PCR获得16SrDNA序列，由南京金斯瑞测序公司获得序列信息，在NCBI上利用Blast在GenBank中与其他的16SrDNA序列进行同源性比较，同时通过构建系统进化树将菌株鉴定为DX-5为索诺拉沙漠芽孢杆菌。
实施例2菌株DX-5以CCMP为碳源生长及对CCMP的降解作用
将DX-5单菌落接种于20ml液体LB培养基（NaCl 10.00g/L,蛋白胨10.00g/L,酵母粉5.00g/L,CCMP 100mg/L，pH7.0）中，于160 r/min，30℃条件下振荡培养至OD600=1.0，在含100 mg/L CCMP的无机盐培养基（每升含100 mg/L CCMP，1.50g K2HPO4，0.50g KH2PO4，0.20g MgSO4×7H2O、1.00g NaCl，1.00g (NH4)2SO4，pH 7.0）中，按5%的接种量接种DX-5，于30℃振荡培养，每隔2h取样测定600nm处的吸光度以及CCMP浓度。由图2可以看出，菌株DX-5可以在24 h内完全降解浓度为100 mg/L的CCMP，同时菌株生长量明显，OD600由接种时的0.05增加至0.6左右。
实施例3索诺拉沙漠芽孢杆菌DX-5的摇瓶降解实验
3.1 温度对DX-5降解效率的影响
在含100 mg/LCCMP的无机盐培养基中，其配方为：每升含100 mg/L CCMP，1.50 gK2HPO4、0.50gKH2PO4、0.20gMgSO4×7H2O、1.00gNaCl、1.00g (NH4)2SO4，pH 7.0，置于温度分别为15℃、20℃、25℃、30℃、35℃、40℃的摇床培养，24小时取样测定CCMP浓度，结果见图3。结果由图3可以看出，菌株降解CCMP的最适温度为30℃；当温度范围为25-35℃范围内，菌株对CCMP的降解率均可超过80%。
3.2 pH对DX-5降解效率的影响
pH 值是影响微生物生长和生存的另一个重要环境因素，微生物生长有一个适宜的pH值范围，超过适宜生长的pH值范围，大多数微生物都不能很好人的生长。
在含100 mg/L CCMP的无机盐培养基中，其配方为：每升含100 mg/L CCMP，1.50 gK2HPO4、0.50gKH2PO4、0.20gMgSO4×7H2O、1.00gNaCl、1.00g (NH4)2SO4，pH 7.0，按5%的接种量接种DX-5于培养液中，pH值分别调至5.0、6.0、7.0、8.0、9.0、1.0，于30℃振荡培养，24小时取样测定。结果由图4可以看出，pH过高或过低都会影响DX-5对CCMP的降解，最优pH范围为7。
实施例4 DX-5菌剂在吡虫啉生产废水处理中的应用
DX-5菌剂的制备：
1）将索诺拉沙漠芽孢杆菌DX-5接种于添加CCMP的LB培养基（NaCl 10.00g/L,蛋白胨10.00g/L,酵母粉5.00g/L，2-氯-5氯-甲基吡啶100mg/L，pH7.0）中，30℃振荡培养至对数期；
2）将步骤1）培养好的菌种按10％的接种量接种入种子罐（2-氯-5氯-甲基吡啶100mg/L，葡萄糖8g/L，（NH4）2SO4 1g/L，K2HPO4 2g/L，MgSO40.5g/L，NaCl 1g/L%，CaCO30.5g/L，酵母膏2g/L，pH值7.2-7.5），培养至对数生长期，即种子液；
3）将步骤2）所述种子液按10％的接种量接入生产罐进行发酵培养，生产罐所用培养基与种子罐培养基相同，无菌空气的通气量为1：0.6-1.2，搅拌速度为180-240转/分，培养温度为30-35℃，步骤2）和步骤3）的整个工艺流程培养时间为48-60小时，发酵完成后的培养液即为菌剂；所述菌剂直接分装成液体剂型，或采用泥炭吸附用包装袋分装成固体菌剂剂型。
将DX-5菌剂投加至装有100ml吡虫啉生产废水的250ml三角瓶中，调pH为6-7，按1%的接种量接种DX-5菌剂，每隔2h取样监测CCMP浓度以及废水的可生化性。由图5可以看出，在接种的24h内，吡虫啉生产废水的B/C比可从0.09上升至0.58。
Claims (9)
1.一株有机氮类污染物降解菌，分类命名为索诺拉沙漠芽孢杆菌（Bacillus sonorensis）DX-5，该菌已保藏于中国微生物菌种保存管理委员会普通微生物中心，保藏编号为：CGMCC No.14161。
2.权利要求1所述的有机氮类污染物降解菌在降解有机氮类污染物中的应用。
3.权利要求1所述的有机氮类污染物降解菌在降解2-氯-5氯-甲基吡啶中的应用。
4.一种有机氮类污染物降解菌剂，含有权利要求1所述的索诺拉沙漠芽孢杆菌DX-5。
5.权利要求4所述菌剂的制备方法，其特征在于，包括如下步骤：
1）将索诺拉沙漠芽孢杆菌DX-5接种于添加CCMP的LB培养基中，振荡培养至对数期；
2）将步骤1）培养好的菌种按10％的接种量接种入种子罐，培养至对数生长期，即种子液；
3）将步骤2）所述种子液按10％的接种量接入生产罐进行发酵培养，生产罐所用培养基与种子罐培养基相同，发酵完成后的培养液即为菌剂；所述菌剂直接分装成液体剂型，或采用泥炭吸附用包装袋分装成固体菌剂剂型。
6.根据权利要求5所述的制备方法，其特征在于，步骤1）中所述的LB培养基配方为：NaCl 10.00g/L,蛋白胨10.00g/L,酵母粉5.00g/L，2-氯-5氯-甲基吡啶100mg/L，pH7.0。
7.根据权利要求5所述的制备方法，其特征在于，步骤2）中所述的种子罐所用的培养基配方为：2-氯-5氯-甲基吡啶100mg/L，葡萄糖8g/L，（NH4）2SO4 1g/L，K2HPO4 2g/L，MgSO40.5g/L，NaCl 1g/L%，CaCO30.5g/L，酵母膏2g/L，pH值7.2-7.5。
8.根据权利要求5所述的制备方法，其特征在于，步骤2）所述种子罐和步骤3）所述的生产罐的培养过程中，无菌空气的通气量为1：0.6-1.2，搅拌速度为180-240转/分，培养温度为30-35℃。
9.权利要求1所述的有机氮类污染物降解菌或者权利要求4所述的菌剂在吡虫啉生产废水处理中的应用。
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