冬虫夏草产地土壤中耐砷细菌的分离、鉴定及耐砷能力测定

Isolation, Identification and Arsenic Tolerance of Arsenic-Resistant Soil Bacteria of Cordyceps sinensis

目的分析冬虫夏草产地土壤中的耐砷细菌,为研究冬虫夏草砷超富集机制提供依据。方法对五个冬虫夏草产地土壤中耐砷细菌进行分离、纯化,筛选出一批耐砷细菌,对其中两株优势菌株XB和XNQ进行形态、生理生化、16S rRNA鉴定,并利用硝酸银溶液(AgNO3)与三价砷As(Ⅲ)、五价砷As(Ⅴ)的颜色反应,检测两株细菌对砷的氧化还原性,最后对菌株的耐砷能力进行测定。结果通过菌落形态特征分析,菌株XB和XNQ均为革兰氏阴性菌,主要脂肪酸均为C18:1ω9c、C16:0、Summed Feature 3(C16:1ω7c/C16:1ω6c)和C12:0;通过16S rRNA基因的序列分析,菌株XB的16S序列与已知种Acinetobacter guillouiae的序列APOS01000028相似性为99.72%,菌株XNQ与已知种Acinetobacter nosocomialis的序列APOP01000014相似性为99.86%;定性硝酸银染色实验证明菌株XB、XNQ具有砷还原性,都是As(Ⅴ)还原细菌;菌株XB和XNQ对As(Ⅲ)的耐受性为20 mmol·L-1,对As(Ⅴ)的耐受性为70 mmol·L-1。结论分离鉴定的两株优势细菌XB和XNQ分别为不动杆菌属的A.guillouiae和A.nosocomialis,均为革兰氏阴性菌,为砷还原细菌,对As(Ⅲ)和As(Ⅴ)的耐受性相同,对As(Ⅴ)的耐受性略高于As(Ⅲ)。

Objective To analyze the arsenic-tolerant bacteria in the soil of Cordyceps sinensis(Ophiocordyceps sinensis)and to provide a basis for studying the mechanism of arsenic enrichment in C.sinensis.Methods A group of arsenic-resistant soil bacteria of C.sinensis were isolated and purified from five producing areas.Two dominant bacteria XB and XNQ were identified by morphological,physiological and biochemical,16 S rRNA and nitric acid.Silver solution(AgNO3)was used to detect the redox of arsenic of XB and XNQ strains.Finally,gradually increase As(Ⅲ)and As(Ⅴ)to detect the arsenic tolerance of two strains.Results By colony morphological characteristics,both XB and XNQ strains were Gram-negative bacteria,and the main fatty acids of which were C18:1ω9 c,C16:0,Summed Feature 3(C16:1ω7 c/C16:1ω6 c)and C12:0.Sequence analysis of 16 S rRNA indicated that the most similarity sequence of strain XB was APOS01000028 of Acinetobacter guillouiae,and the identity was 99.72%,while the most similarity sequence of strain XNQ was APOP01000014 of Acinetobacter nosocomialis,and the identity was 99.86%.Qualitative silver nitrate staining indicated that both XB and XNQ strains have arsenic reducing properties and are As(Ⅴ)reducing bacteria.Strains XB and XNQ have the same tolerance to As(Ⅲ)and As(Ⅴ),and the value is 20 mmol·L-1 and 70 mmol·L-1,respectively.Conclusion The XB strain is A.guillouiae and the XNQ strain is A.nosocomialis,both of which is Gram-negative arsenic-reducing bacteria with the same arsenic tolerance of As(Ⅲ)and As(Ⅴ)and with higher tolerance to As(Ⅴ)than As(Ⅲ).