Open AccessCulture-Independent Analysis of Bacterial Fuel Contamination Provides Insight into the Level of Concordance with the Standard Industry Practice of Aerobic Cultivation
Author(s) -
Judith White,
Jack A. Gilbert,
G C Hill,
E. G. Hill,
Susan M. Huse,
Andrew J. Weightman,
Eshwar Mahenthiralingam
Publication year - 2011
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.02317-10
Subject(s) - betaproteobacteria , gammaproteobacteria , biology , stenotrophomonas , temperature gradient gel electrophoresis , microbiology and biotechnology , firmicutes , clostridia , proteobacteria , achromobacter , 16s ribosomal rna , actinobacteria , pseudomonas , food science , bacteria , genetics
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 ofPseudomonas (21%),Burkholderia (7%), andBacillus (7%) was demonstrated. Denaturing gradient gel electrophoresis (DGGE) of 15 samples revealedProteobacteria andFirmicutes 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%) andGammaproteobacteria (30.6%) formed the largest proportion of reads; the most abundant genera wereMarinobacter (15.4%; JW57),Achromobacter (41.6%; JW63),Burkholderia (80.7%; JW76), andHalomonas (66.2%; JW78), all of which were also observed by DGGE. However, theClostridia (38.5%) andDeltaproteobacteria (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) aPseudomonas sp. strain recovered from 2 different diesel fuel tanks at a single industrial site; (ii) aMangroveibacter sp. strain isolated from 3 biodiesel tanks at a single refinery site; and (iii) aBurkholderia 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 asPseudomonas .