Fuel biodegradation and molecular characterization of microbial biofilms in stored diesel/biodiesel blend B10 and the effect of biocide

•Illumina high throughput sequencing showed Archaea in B10 microbial population.•Sub-effective doses of the multifunctional biocide package may increase microbial growth.•Infrared analyses showed biodiesel degradation by the microbial population in fuel both with and without biocide.

Microbial biofilms are formed at oil/water interfaces in storage tanks containing diesel–biodiesel 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–biodiesel blend (oil as received with or without an inoculum derived from diesel sludge) was incubated with or without 3,3′-methylene bis(5-methyloxazolidine, MBO) – 100% and 50% formulations at 1000, 500, and 0 ppm – over 60 days. The biofilms formed at the oil–water 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 Tolumonas. 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, taxonomic 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.