Anaerobic biodegradation of squalene: Using DGGE to monitor the isolation of denitrifying Bacteria taken from enrichment cultures.

Abstract The process of enrichment and subsequently isolation of squalene degrading denitrifying bacteria has been developed. The enrichment method used in this study targeted denitrifying bacteria, therefore an initial enrichment incubation using nitrate amendments under anaerobic conditions was performed before squalene amendment. Denaturant gradient gel electrophoresis (DGGE) analysis of polymerase chain reaction (PCR)-amplified DNA fragments prepared from extracted DNA was used to compare the composition of bacterial communities at various steps of enrichment cultures and the diversity of the 80 isolated strains obtained by classical culture methods. After 8 months of anaerobic incubation, the squalene biodegradation rate reached 80%. The community composition changed substantially during the incubation time. The enrichment cultures were dominated by 12 phylotypes, of which eight corresponded to cultivatable strains. Their identities were established by sequencing V3-V5 16SrRNA PCR fragments directly or after excision of DGGE bands and comparing the sequences with those available in GenBank. Most of the isolates were Proteobacteria of the gamma subgroup; among them, seven novel denitrifying bacteria which were capable of using squalene as the sole carbon source, were isolated and characterized.