孔雀石绿脱色菌恶臭假单胞菌菌株M6的分离、鉴定及其生长特性研究

Isolation, Identification and Growth Characteristics of Pseudomonas putida Strain M6 with Malachite Green Decolorization

从养殖池污泥中分离了6株对孔雀石绿具有脱色能力的菌株,经过进一步在孔雀石绿营养肉汤中富集培养及其脱色率的比较,筛选出对孔雀石绿具有较强脱色能力的优良菌株M6。菌株M6在30°C、150r/min条件下对孔雀石绿的脱色率为97.14%,通过革兰氏染色、电镜对其形态进行了观察,用ATB细菌鉴定仪对其进行了生理生化鉴定;通过对其16SrDNA序列进行PCR扩增和测序,与NCBI中收录的与其同源性较高的菌株进行了聚类分析并构建了系统发育树。此外,对其生长特性也进行了研究。实验结果表明,菌株M6革兰氏染色阴性,杆形,端生一根鞭毛,大小约为0.45μm×0.84μm,在孔雀石绿营养琼脂平板上形成的菌落特征为圆形、浅蓝色、粘稠、不易挑取;菌株M6的16SrDNA序列与GenBank基因库中假单胞菌属的细菌菌株的16SrDNA序列有98%~99%的高度同源性,菌株M6与恶臭假单胞菌OW-16(登录号:DQ112328.1)的亲缘关系最近。结合传统的形态与生理生化特性鉴定以及16SrDNA序列分析鉴定的结果,判定菌株M6为恶臭假单胞菌(Pseudomonasputida)(登录号:EU348741.1)。此外,菌株M6在30°C、150r/min条件下摇床振荡培养的生长曲线为:0h^4h为生长延迟期,4h^64h为对数生长期,64h^80h为稳定期,80h以后为衰亡期;其最适生长pH值为7,最适生长温度为30°C,在转速为50r/min^250r/min条件下,其浓度随转速的增加而增大。

Six bacterial strains with malachite green decolorization ability were isolated from a sediment of aquaculture pond, and strain M6 was selected by further enrichment culture in nutrition broth with malachite green and decolorization rate comparison. The decolorization rate of strain M6 to malachite green was 97.14% in the conditon of 30℃ and 150 r/min, and its morphology was observed by gram stain and electron microscopy, its physiological and biochemical characteristic was studied by ATB bacteria identification instrument for identification of bacteria, and its 16S rDNA sequence was determined following PCR amplifi- cation, the sequence was aligned and the phylogenic tree was instructed with those bacterial strains of high identity with strain M6. In addition, its growth characteristics was also studied. The experimental results showed that strain M6 was gram negative and bacilliform with a flagellum at one end. Its size was 0.45μm ×0.84 μm. Its colony produced on common agar plate appeared as round, light blue, dense, hard to choose; 16S rDNA sequence of strain M6 had high identity of 98%-99% with Pseudomonas sp. located in GenBank and strain M6 had the most close relative relation to Pseudomonas putida OW-16 （Locus number： DQ112328.1）. Combined the results of the traditional morphological, physiological, biochemical characteristics and 16S rDNA sequence analysis, strain M6 was identified as Pseudomonas putida （Locus number： EU348741.1）. Additionally, its growth curve in the condition of 30℃ and 150 r/min was as follows： lag phase was 0-4 h, log phase was 4 h-64 h, stationary phase was 64 h-80 h, decline phase was after 80 h. Its best growth conditions were pH 7 and 30℃,and in the rotational speed of 50 r/min to 250 r/min. Its concen- tration increased with the increase in rotational speed.