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.

Microbial contamination and growth in distillate fuels has been described for seventy years. The consequences have ranged from fouling of filters and injectors, to engine malfunction and damage, fuel gauge malfunctions and aggravated corrosion of engines, fuel tanks, equipment and facilities. The types of microbes present vary with the differences in fuel composition and differences in storage and use conditions. Anti-microbial strategies have traditionally included prevention by ‘good housekeeping’ and ‘fire-brigade’ applications of biocides when there are operational problems. Since 2002, first the aviation industry and later fuel suppliers and some militaries, have used simple on-site microbiological tests to monitor fuel and fuel systems and use the results to take remedial actions before operational problems occur. This paper will review our latest knowledge of microbially influenced corrosion and of the new anti-microbial strategies which are being successfully implemented to prevent it.

THE OLD MAXIM, THAT ANY TEST result is only as good as the sample, is never more true than for analysis of samples drawn for the investigation of microbiological contamination in fuels and fuel systems. Probably more than any other fuel contamination type, microbial contamination will tend to have a highly heterogeneous dispersion that is likely to be in a continual state of change. There may be changes in the overall numbers of microbes present, their viability (and culturability), the relative numbers of the predominant types (genera and species) and the amounts of microbial biomass present [1]. These changes may be due to the microbial activity itself or as a consequence of tank or system operating activities. It is thus apparent that both the timing of sampling operations and selection of appropriate sampling points need careful consideration and planning. In order that those conducting the analyses can put the best possible interpretation on the results obtained, as much information as possible about the sampling needs to be conveyed to the testing laboratory.

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