Isolation and characterization of a bacterial consortium capable to degrade the saturated fraction of the mineral oil. Giovanna Siracusa1, Simona Di Gregorio1, Lorenzo Mariotti1, Roberto Lorenzi1 1Department of Biology, University of Pisa, Via Ghini 13, 56126 Pisa, Italy River estuaries in urbanized area are sites for several industrial activities. Their management and navigability depends on the periodical dredging of sediments. The confined disposal in dedicated area and the monitored natural attenuation of the contamination is currently the more common and strategy adopted to treat drudged sediments. The acceleration of the process and the fast safe reallocation of decontaminated sediments is actually a priority for the protection of the environment. In this context many different approaches have been adopted as bioaugmentation with specific bacterial strains able to deplete the contamination. Autochthonous microbial strains are the best candidates to eventually harbor the physiological traits of interest. A bacterial consortium is actually a better representation of the real environment (Ledin 2000). The aim of the present study was the isolation of a bacterial consortium able to use diesel oil as a sole carbon source to be eventually exploited for bio-based processes dedicated to the treatment of dredged sediments mainly contaminated by shipyard activities. The capacity of the isolated consortium has been analyzed with reference to the depletion of the saturated fraction of the mineral oil in liquid culture, containing diesel as sole carbon source. The metabolic capacity of interest has been recorded by the GC-MS analysis of the depletion of the normal and branched alkanes and of the fraction of the fatty acid methyl ester (FAME) recovered in the diesel oil here exploited and eventually deriving from the blending of diesel and biodiesel. Isolation of the consortium, depletion of diesel oil in liquid culture The bacterial consortium, able to use diesel oil as a sole carbon source, has been isolated from enrichment cultures maintained at 28°C for 35 days. The depletion of the saturated fraction of the diesel oil (1%) in BSM has been analyzed by GC-MS after 3 and 6 days of incubation. Results obtained are shown in Figure 1. A progressive depletion of the different normal alkanes (Cn-alkane, C12-C30) has been observed. Any depletion of the saturated fraction of the diesel oil has been recorded in not inoculated flasks. Figure 1: Concentration of normal alkanes Cn-alkane, C12-C30 at three different time of incubation by the isolated consortium in BSM with 1% of diesel oil. Panel A The Figure 2 shows the chromatograms of the saturated fraction of the diesel oil at the beginning of the incubation and at the successive two times of incubation, after three (panel B) and six (panel C) days of incubation. The chromatograms at the beginning of the experimentation (Figure 2, panel A) showed the major picks corresponding to the different normal alkanes (Cn-alkane, C12-C30) and the minor picks corresponding to the branched alkanes (Cb-alkane) that distilled before the n-alkane characterized by the same number of carbons. The pick corresponding to the nC18 FAME was evident as part of the profile of the saturated fraction of the diesel oil utilized. The chromatograms showed a progressive and total depletion of the different n-alkanes . The percentages of depletion of the Cn-alkane, C12-C30 by the consortium, spanning from 78.81 and 100 (Table 1). Panel B Panel C Figure 2: Chromatograms plot of the saturated fraction of diesel oil at three time incubation; Panel A: T0 of experimentation at 0 day incubation; Panel B: T3 after three days incubation; Panel C: T6 after six days incubation. Table 1: Percentages of depletion of the Cn-alkanes C12-C30 and C18 FAME for the seven morphotypes and the consortium in BSM amended with 1% diesel oil after six days of incubation. Percentages values represent mean values of percentages of depletion deriving from three flasks per morphotypes and for the bacterial consortium. Isolation and identification of the bacterial strains composing the bacterial consortium A total of nine bacterial morphotypes were recovered and screened by ARDRA analysis. The nine morphotypes were grouped in two ARDRA profiles corresponding to two operational taxonomic units (OTU). The partial sequencing of the corresponding 16S rRNA gene indicated that the morphotypes belonged to the Stenotrophomonas and to the Pseudomonas sp. (Table 2). The nine bacterial morphotypes were analyzed for their capacity to growth in BSM with 1% v/v diesel by counting the serial dilution of the CFU on LB plates inoculated with the different morphotypes in axenic culture. Results obtained indicated that only seven morphotypes out of nine were capable to utilize gasoline in BSM as the sole carbon source and they are divided into 5 groups. One group depletes alkanes between C12 and C22;one group is able to deplete C12 to C25 alkanes; one group depleting alkanes between C12-C28, a fourth group deplete C12-C30 alkanes and a fifth group is able to deplete C12-C30 alkanes with a low percentage of depletion of C28-C30 alkanes. Figure 3: ARDRA analisys of 9 morphotypes isilated from dredged sediment . Table 2: Taxonomic characterization of the nine bacterial morphotypes. Results obtained confirm the autochthonous bacterial consortium ability to degrade diesel oil. The consortium will be exploited for bioaugmentation with reference to the assumption that bioaugmentation with autochthonous bacterial strains represents a valid biotechnological instrument to deplete the contamination in environmental matrices. Bibliografia : Ledin M. Accumulation of metal by microorganisms—processes and importance for soil systems. Earth-Sci Rev 2000;51:1-31.