分枝杆菌TZh51菌株的分离鉴定及其生物修复污染土壤的特性

Isolation, identification of a pyrene-degrading Mycobacterium sp. strain TZh51and its characteristics for contaminated soil bioremediation

摘要:

【目的】为获得降解芘的微生物菌株，并用其生物修复被多环芳烃污染的土壤。【方法】芘降解菌的分离采用平板升华法。根据表型观察、生理生化特性和16S rDNA的序列同源性分析，对菌株进行分类学鉴定。通过活菌计数、HPLC测定多环芳烃的残留量，研究菌株在固体、液体无机盐培养基以及在污染土壤中降解多环芳烃(polycyclic aromatic hydrocarbons, PAHs)的能力。【结果】分离到4株能降解芘的菌株TZh51、TZh52、TG42和TG52。实验结果表明，TZh51降解PAHs的能力强于其余3株菌。TZh51被鉴定为分枝杆菌属（Mycobacterium sp.），但与已发表的分枝杆菌菌株M11为不同的种。TZh51接种在芘膜的固体无机盐培养基上，测定获得最大芘降解量的条件是培养温度为35℃和芘膜厚度为130 ng/mm2。在芘浓度为50、100 mg/L的液体无机盐培养基中培养，6天时TZh51的芘降解率分别达到91.9%、71.8%，10天时菌体数量分别达到最大值为2.0、6.0×108 cfu/mL；TZh51降解芘的效果强于M11。在种植作物的处理中，到第6周时TZh51的菌体数量达到每克干土含7.2×108 个菌落数，到第8周时菲、荧蒽和芘的降解率分别达到91.4%、86.9%和85.8%；【结论】TZh51具有很强降解PAHs的能力；另外，TZh51与作物联合生物修复污染土壤的效果明显。

Abstract:

[Objective] To obtain pyrene-degrading microorganism strains, and to use them for bioremediation of polycyclic aromatic hydrocarbons-contaminated soil. [Methods] pyrene-degrading strains were isolated by agar plate subliming method. They were identified based on morphological observation, physiological and biochemical characteristics, and on analysis of their 16S rDNA gene sequence homology. Their ability to degrade PAHs in solid and liquid mineral salt medium, and in PAHs-contaminated soil, was studied by counting their live cells and measuring residual PAHs’ quantity by HPLC. [Results] Four pyrene-degrading strains TZh51, TZh52, TG42 and TG52 were isolated. The results indicated that TZh51’s ability to degrade PAHs was stronger than the other three strains. TZh51 was identified as Mycobacterium sp.; however, it did not belong to the same species with the reported Mycobacterium sp. strain M11. Our results indicated that the effects on maximal pyrene-degrading quantity were incubation temperature at 35℃and pyrene film thickness for 130ng/mm2 when TZh51 was incubated on solid mineral salt medium coated with pyrene films. When TZh51 was incu-bated in liquid mineral salt medium containing 50 and 100 mg/L pyrene, 91.9% and 71.8% pyrene was degraded after 6 days. On the10th day, maximal vital cells reached 2.0×108 and 6.0×108 cfu/mL, respectively. TZh51’s ability to degrade pyrene was stronger than strain M11. Moreover, TZh51’s vital cells reached 7.2×108 cfu/g dry soil on the 6th week and 91.4% phenanthrene, 86.9% fluoranthrene and 85.8% pyrene was degraded after 8 weeks, when both TZh51 and plants were used for bioremediation of PAHs-contaminated soil. [Conclusion] TZh51 had strong ability to degrade PAHs. In addition, combined bioremediation of TZh51 and plants was effective for PAHs-contaminated soil bioremediation.