Culture-Independent Analysis of Bacterial Fuel Contamination Provides Insight into the Level of Concordance with the Standard Industry Practice of Aerobic Cultivation
Culture-Independent Analysis of Bacterial Fuel Contamination Provides Insight into the Level of Concordance with the Standard Industry Practice of Aerobic Cultivation
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DOI：
10.1128/aem.02317-10
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发表时间：
2011-05
影响因子：
4.4
通讯作者：
Judith White;J. Gilbert;G. Hill;E. Hill;Susan M. Huse;A. Weightman;E. Mahenthiralingam
中科院分区：
文献类型：
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作者：
Judith White;J. Gilbert;G. Hill;E. Hill;Susan M. Huse;A. Weightman;E. Mahenthiralingam
摘要污染燃料中的细菌多样性尚未用不依赖培养的方法进行系统研究。燃料工业依赖于基于表型培养的污染物识别，这可能缺乏准确性，并忽视了难以培养的分类群。通过工业需氧培养、16S rRNA基因测序和菌株基因分型，从54个燃料样品中收集到152个独特的污染菌株，其中假单胞菌(21%)、伯克霍尔德氏菌(7%)和芽孢杆菌(7%)为优势种。对15个样品进行变性梯度凝胶电泳法(DGGE)分析，结果表明变形杆菌和菌丝是最丰富的类群。对4个燃料样品进行16S rRNA V6基因焦糖测序时，β蛋白细菌(42.8%)和伽马蛋白细菌(30.6%)构成了最大比例的READ；最丰富的属是海洋杆菌(15.4%；JW57)、无色杆菌(41.6%；JW63)、伯克霍尔德氏菌(80.7%；JW76)和盐单胞菌(66.2%；JW78)，这些都是DGGE观察到的。而焦糖测序鉴定的梭状芽胞杆菌(38.5%)和变形杆菌(11.1%)在DGGE和好氧培养中均未检出。基因分型发现了三个相同的菌株：(I)假单胞菌。在一个工业现场从两个不同的柴油储罐中回收的菌株；(Ii)一株红树林杆菌。从一个炼油厂的3个生物柴油罐中分离到一株伯克霍尔德氏菌；以及(Iii)在两个无关的汽车柴油样本中存在一株伯克霍尔德氏菌。总体而言，燃料污染物的好氧培养回收了广泛代表目前存在的门和纲的分离物，但由于过度代表某些组的成员，如假单胞菌，缺乏准确性。
ABSTRACT Bacterial diversity in contaminated fuels has not been systematically investigated using cultivation-independent methods. The fuel industry relies on phenotypic cultivation-based contaminant identification, which may lack accuracy and neglect difficult-to-culture taxa. By the use of industry practice aerobic cultivation, 16S rRNA gene sequencing, and strain genotyping, a collection of 152 unique contaminant isolates from 54 fuel samples was assembled, and a dominance of Pseudomonas (21%), Burkholderia (7%), and Bacillus (7%) was demonstrated. Denaturing gradient gel electrophoresis (DGGE) of 15 samples revealed Proteobacteria and Firmicutes to be the most abundant phyla. When 16S rRNA V6 gene pyrosequencing of four selected fuel samples (indicated by “JW”) was performed, Betaproteobacteria (42.8%) and Gammaproteobacteria (30.6%) formed the largest proportion of reads; the most abundant genera were Marinobacter (15.4%; JW57), Achromobacter (41.6%; JW63), Burkholderia (80.7%; JW76), and Halomonas (66.2%; JW78), all of which were also observed by DGGE. However, the Clostridia (38.5%) and Deltaproteobacteria (11.1%) identified by pyrosequencing in sample JW57 were not observed by DGGE or aerobic culture. Genotyping revealed three instances where identical strains were found: (i) a Pseudomonas sp. strain recovered from 2 different diesel fuel tanks at a single industrial site; (ii) a Mangroveibacter sp. strain isolated from 3 biodiesel tanks at a single refinery site; and (iii) a Burkholderia vietnamiensis strain present in two unrelated automotive diesel samples. Overall, aerobic cultivation of fuel contaminants recovered isolates broadly representative of the phyla and classes present but lacked accuracy by overrepresenting members of certain groups such as Pseudomonas.