Culture-Independent Analysis of Bacterial Fuel Contamination Provides Insight into the Level of Concordance with the Standard Industry Practice of Aerobic Cultivation

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.

Rapid Detection of Emerging Pathogens and Loss of Microbial Diversity Associated with Severe Lung Disease in Cystic Fibrosis

“…Briefly, PCRs were set up with 1 l of extracted sputum DNA (approximately to 20 to 40 ng) and 10 pmol of each RISA PCR primer (1406F, TGYACACACCGCCCGT, and 23SR, GGGTTBCCCCATTCRG) (16). RISA amplicons (2 l of amplified DNA) were separated by microfluidics (Agilent 2100 Bioanalyzer; Agilent Technologies, California, USA), and their profiles were analyzed using Gelcompar II (Applied Maths, SintMartens Latem, Belgium) and clustered based on their similarity as described previously (21). DNA from pure bacterial cultures of reference CF pathogen species was used to generate control ITS amplicons for putative pathogen identification based on size correlation.…”

Growth of Paecilomyces variotii in B0 (diesel), B100 (biodiesel) and B7 (blend), degradation and molecular detection

“…Biodegradability of biodiesel compared to mineral diesel it is a problem specially when occurs microbial contamination during storage which may lead to blocking of pipelines and filters, affecting the final quality of the fuel and corrosion of the tanks (Bento and Gaylarde, 2001;Bento et al, 2004Bento et al, , 2006Passman and Dobranick, 2005;Bücker et al, 2011;White et al, 2011;Cazarolli et al, 2012Cazarolli et al, , 2014Zimmer et al, 2013;Passman, 2013).…”

“…A concentration of only 1% water in a storage system is sufficient for the growth of aerobic and anaerobic bacteria and yeasts, as well as for the development of fungal biomass at the oil/water interface (Gaylarde et al, 1999;Chesneau, 2000;Bento and Gaylarde, 2001;Bento et al, 2004;Bücker et al, 2011;Sørensen et al, 2011;Cazarolli et al, 2012Cazarolli et al, , 2014Zimmer et al, 2013;Passman, 2013). Numerous microorganisms have been isolated from fuels (Atlas, 1981;Gaylarde et al, 1999;Bento and Gaylarde, 2001;Bento et al, 2004;Bücker et al, 2011;White et al, 2011). The fungi most frequently and considered as deteriogenics in diesel include Hormoconis resinae, Aspergillus fumigatus, Paecilomyces variotii, Penicillium sp., Rhodotorula glutinis and Candida silvicola (Bento andGaylarde, 1996, 2001;Bento, 1999).…”

“…Many factors, such as the presence of water in the bottom of the tanks during storage, have been cited as increasing microbial growth in the systems and can lead to blocking of pipelines and filters, affecting the final quality of the fuel and corrosion of the tanks (Bento and Gaylarde, 2001;Bento et al, 2004;Bücker et al, 2011;White et al, 2011;Cazarolli et al, 2012Cazarolli et al, , 2014Zimmer et al, 2013;Passman, 2013). A concentration of only 1% water in a storage system is sufficient for the growth of aerobic and anaerobic bacteria and yeasts, as well as for the development of fungal biomass at the oil/water interface (Gaylarde et al, 1999;Chesneau, 2000;Bento and Gaylarde, 2001;Bento et al, 2004;Bücker et al, 2011;Sørensen et al, 2011;Cazarolli et al, 2012Cazarolli et al, , 2014Zimmer et al, 2013;Passman, 2013).…”

“…Recognized institutions linked to the fuel market, such as the International Air Transport Association (IATA), the Institute of Petroleum (IP) in the UK and ASTM in the U.S.A., established methodologies and values for diagnosis of an acceptable condition and alert of microbial contamination in fuel. According to White et al (2011) the fuel industry relies on phenotypic cultivation-based identification to monitor the level of contamination, which may lack accuracy and neglect difficult-to-culture taxa. Molecular methods like polymerase chain reaction (PCR) have been developed for detection of fungi with several advantages compared to those of conventional techniques, such as high specificity and sensitivity (Busch and Nitschko, 1999).…”