内生贪铜菌的分离鉴定及其对铜污染含羞草生长的影响

Isolation and identification of Cupriavidus sp.HXC-8 and its effects on growth of Mimosa pudica contaminated with copper

【目的】研究贪铜菌及其与含羞草(Mimosa pudica)形成的共生体对重金属铜污染的抗性,为重金属污染环境的微生物修复提供参考,丰富功能菌株资源库。【方法】自含羞草根瘤中分离得到菌株HXC-8,扩增该菌株的16S rDNA序列,对其进行鉴定,并对其进行生理生化特性测定,研究菌株HXC-8对重金属铜的耐受性及去除率。此外,设置接菌组(含羞草种子在HXC-8菌悬液中浸泡6 h、自然阴干1 h)和未接菌组(含羞草种子在蒸馏水中浸泡6 h、自然阴干1 h),将2组含羞草种子在Cu^(2+)质量浓度分别为0,50,100,150,200,250,300,350,400 mg/L的混合营养液中培养,分析菌株HXC-8对铜胁迫下含羞草种子萌发和幼苗生长的影响。【结果】16S rDNA基因序列分析结果显示,菌株HXC-8为贪铜菌属(Cupriavidus sp.)细菌,将其命名为“贪铜菌HXC-8”。生理生化特性测定结果显示,贪铜菌HXC-8为革兰氏阴性菌,能分泌活性较高的H_(2)O_(2)酶和淀粉酶,不产生纤维素酶,不适合在肉汁培养基上生长,在糖代谢过程中无混合酸发酵,属于快生型内生菌。随着Cu^(2+)质量浓度的升高,贪铜菌HXC-8的生长受到了抑制,该菌对Cu^(2+)的耐受量是0~150 mg/L。当Cu^(2+)质量浓度为25~150 mg/L时,随着菌液中Cu^(2+)质量浓度的增大,贪铜菌HXC-8对铜的去除率逐渐降低,其中Cu^(2+)质量浓度为25 mg/L时,贪铜菌HXC-8对铜的去除率最高,为97.93%。在0~400 mg/L Cu^(2+)胁迫下,随Cu^(2+)质量浓度的升高,未接菌组和接菌组含羞草种子萌发率均无显著变化,但种子活力指数明显下降;未接菌组和接菌组含羞草幼苗鲜质量、根长、茎长及株高均明显降低。当Cu^(2+)质量浓度为0~300 mg/L时,接菌组与未接菌组含羞草幼苗鲜质量、根长、茎长、株高均无显著差异;当Cu^(2+)质量浓度为350~400 mg/L时,接菌组幼苗以上指标均明显大于未接菌组。【结论】贪铜菌HXC-8对铜具有一定的耐受性,能够降低环境中游离态的Cu^(2+),可减缓重金属铜对含羞草生长的胁迫。

【Objective】This study investigated the resistance of Cupriavidus sp.and their symbionts with Mimosa pudica to heavy metals copper to provide references for microbial remediation of heavy metal contaminated environment and enriching resources of functional strains.【Method】A strain of“HXC-8”was isolated from root nodules of Mimosa pudica,and its 16S rDNA genetic sequence was determined and identified.The physiological and biochemical characteristics of HXC-8 were determined,and its tolerance and copper(Cu^(2+))removal rate were examined.Inoculated(M.pudica seeds soaked in HXC-8 bacterial suspension for six hours and dried in natural shade for one hour)and un-inoculated(M.pudica seeds soaked in distilled water for six hours and dried in natural shade for one hour)groups of seeds were cultured in mixed nutrient solution with Cu^(2+)mass concentrations of 0,50,100,150,200,250,300,350 and 400 mg/L.The effects of HXC-8 on seed germination and seedling growth of M.pudica under different Cu^(2+)stress were then examined.【Result】The 16S rDNA gene sequence analysis showed that HXC-8 was Cupriavidus sp.,and it was named as Cupriavidus sp.HXC-8.The physiological and biochemical test showed that HXC-8 was gram-negative,could secrete catalase and amylase with high activity,but it could not produce cellulose and not suitable for growth on gravy medium.It had no mixed acid fermentation in sugar metabolism,belonging to fast-growing endophytes.With the increase of Cu^(2+)mass concentration,the growth of HXC-8 was inhibited with tolerance range of 0-150 mg/L.With the increase of Cu^(2+)mass concentrations from 25 to 150 mg/L,the removal rate of Cu^(2+)gradually decreased and the highest removal rate was 97.93%at 25 mg/L.Under the concentrations of 0-400 mg/L,germination percentage of M.pudica seeds had no significant change in both inoculated and un-inoculated groups,while seed vigor,fresh weight of seedling,stem length,and plant height decreased significantly.Under the concentrations of 0-300 mg/L,there was no differences in fresh weight,root length,stem length and plant height of seedlings between inoculated and un-inoculated groups.With Cu^(2+)mass concentrations of 350 to 400 mg/L,seedling growth index of inoculated group was significantly greater than that of un-inoculated group.【Conclusion】Cupriavidus sp.HXC-8 showed certain tolerance to copper and it could reduce free Cu^(2+)and alleviate the inhibition of copper on growth of M.pudica.