Fuel biodegradation and molecular characterization of microbial biofilms in stored diesel/biodiesel blend B10 and the effect of biocide Francielle Bücker a, * , Cristiane Santos Barbosa a , Patrícia D € orr Quadros a , Mariana Krüger Bueno a , Paula Fiori a , Chun te Huang b , Ana Paula Guedes Frazzon c , Marco Flores Ferr ~ ao b , Fl  avio Anast  acio de Oliveira Camargo d ,F  atima Menezes Bento a a Fuels and Biofuels Biodeterioration Laboratory (LAB-BIO), Department of Microbiology, Immunologyand Parasitology, Federal University of Rio Grande of Sul, Sarmento Leite Street # 500, Porto Alegre, RS, Brazil b Department of Inorganic Chemistry, Federal University of Rio Grande of Sul, Bento Gonçalves Avenue # 9500, Porto Alegre, Brazil c Department of Microbiology, Immunology and Parasitology, Federal University of Rio Grande do Sul, Rua Sarmento Leite, N  500, Porto Alegre, RS, Brazil d Department of Soil Science, Federal University of Rio Grande do Sul, Bento Gonçalves Avenue # 7712, Porto Alegre, RS, Brazil article info Article history: Received 27 October 2013 Received in revised form 30 April 2014 Accepted 2 May 2014 Available online 13 August 2014 Keywords: Biocide MBO Storage Illumina HiSeq Microbial community Biodiesel PCR abstract Microbial biofilms are formed at oil/water interfaces in storage tanks containing dieselebiodiesel blends, decreasing fuel quality and increasing economic and environmental losses. Biocides may suppress the microorganisms responsible for the damage, but they are not used in all parts of the world. A B10 diesel ebiodiesel blend (oil as received with or without an inoculum derived from diesel sludge) was incubated with or without 3,3 0 -methylene bis(5-methyloxazolidine, MBO) e 100% and 50% formulations at 1000, 500, and 0 ppm e over 60 days. The biofilms formed at the oilewater interface were collected for extraction of genomic DNA followed by amplification, purification, and Illumina HiSeq sequencing of the 16S rRNA gene. The prevalent genera in the control fuel (as-received and inoculated) were similar at 28 days (Pseudomonas, Comamonas, and Burkholderia); by the 60th day, the microbial community had changed only in the as-received fuel, where the prevalent genera were Comamonas, Klebsiella, and Tol- umonas. Archea were detected in samples at 28 and 60 days. 500 ppm (as supplied) MBO 50% did not control the microbial growth and an interfacial biofilm was formed. After 28 days of incubation, taxo- nomic diversity in the as-received fuel and inoculated fuel decreased by 99.7% and 80.9%, respectively. The analysis also revealed that Firmicutes dominated the communities in the treatments with 500 ppm (as supplied) MBO 50%, followed by Proteobacteria, except in the 60 days sample from the as-received fuel, where Proteobacteria dominated, followed by Firmicutes. Inoculation increased degradation of the fuel. © 2014 Elsevier Ltd. All rights reserved. 1. Introduction Microbial contamination of fuel can occur at various points in the distribution system, but it is particularly noticeable during storage, especially if there is free water in the tank. Biodeterioration of the fuel is shown by the accumulation of biomass and degra- dation of fuel components (hydrocarbons, fatty acid esters, etc.) and can result in blockage of filters, damage to the injection system, and reduced product quality (Gaylarde et al., 1999; Bento et al., 2005; Passman and Dobranic, 2005; Bücker et al., 2011; Passman, 2013; Zimmer et al., 2013). It is accepted that problems in diesel oil are chronic, but recent changes in the composition of this fuel, the introduction of biodiesel mixtures, reduction of sulfur levels, and increase in the use of additives, have increased the biodeterioration risks (Passman, 2013). Various studies have demonstrated that diesel/biodiesel blends are more susceptible to biodegradation and biomass formation during storage (Passman and Dobranic, 2005; Bücker et al., 2011; Sørensen et al., 2011; Silva et al., 2012; Passman, 2013). The removal of bottom water and/or chemical control with biocides are the preventive treatments most frequently recom- mended (Gaylarde et al., 1999; Bento and Gaylarde, 2001; ABNT 15512, 2008; Passman, 2013). The free water formed in the tank bottom by condensation from humid air stimulates the production * Corresponding author. Tel.: þ55 51 3308 4497. E-mail address: franbucker@gmail.com (F. Bücker). Contents lists available at ScienceDirect International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod http://dx.doi.org/10.1016/j.ibiod.2014.05.030 0964-8305/© 2014 Elsevier Ltd. All rights reserved. International Biodeterioration & Biodegradation 95 (2014) 346e355