Bacterial strains of the genus Sphingomonas are often isolated from contaminated soils for their ability to use polycyclic aromatic hydrocarbons (PAH) as the sole source of carbon and energy. The direct detection of Sphingomonas strains in contaminated soils, either indigenous or inoculated, is, as such, of interest for bioremediation purposes. In this study, a culture-independent PCR-based detection method using specific primers targeting the Sphingomonas 16S rRNA gene combined with denaturing gradient gel electrophoresis (DGGE) was developed to assess Sphingomonas diversity in PAH-contaminated soils. PCR using the new primer pair on a set of template DNAs of different bacterial genera showed that the method was selective for bacteria belonging to the family Sphingomonadaceae. Single-band DGGE profiles were obtained for most Sphingomonas strains tested. Strains belonging to the same species had identical DGGE fingerprints, and in most cases, these fingerprints were typical for one species. Inoculated strains could be detected at a cell concentration of 10 4 CFU g of soil ؊1 . The analysis of Sphingomonas population structures of several PAHcontaminated soils by the new PCR-DGGE method revealed that soils containing the highest phenanthrene concentrations showed the lowest Sphingomonas diversity. Sequence analysis of cloned PCR products amplified from soil DNA revealed new 16S rRNA gene Sphingomonas sequences significantly different from sequences from known cultivated isolates (i.e., sequences from environmental clones grouped phylogenetically with other environmental clone sequences available on the web and that possibly originated from several potential new species). In conclusion, the newly designed Sphingomonas-specific PCR-DGGE detection technique successfully analyzed the Sphingomonas communities from polluted soils at the species level and revealed different Sphingomonas members not previously detected by culture-dependent detection techniques.

Fast-growing mycobacteria are considered essential members of the polycyclic aromatic hydrocarbons (PAH) degrading bacterial community in PAH-contaminated soils. To study the natural role and diversity of the Mycobacterium community in contaminated soils, a culture-independent fingerprinting method based on PCR combined with denaturing gradient gel electrophoresis (DGGE) was developed. New PCR primers were selected which specifically targeted the 16S rRNA genes of fast-growing mycobacteria, and single-band DGGE profiles of amplicons were obtained for most Mycobacterium strains tested. Strains belonging to the same species revealed identical DGGE fingerprints, and in most cases, but not all, these fingerprints were typical for one species, allowing partial differentiation between species in a Mycobacterium community. Mycobacterium strains inoculated in soil were detected with a detection limit of 10(6) CFU g(-1) of soil using the new primer set as such, or approximately 10(2) CFU g(-1) in a nested PCR approach combining eubacterial and the Mycobacterium specific primers. Using the PCR-DGGE method, different species could be individually recognized in a mixed Mycobacterium community. This approach was used to rapidly assess the Mycobacterium community structure of several PAH-contaminated soils of diverse origin with different overall contamination profiles, pollution concentrations and chemical-physical soil characteristics. In the non-contaminated soil, most of the recovered 16SrRNA gene sequence did not match with previous described PAH-degrading Mycobacterium strains. In most PAH-contaminated soils, mycobacteria were detected which were closely related to fast-growing species such as Mycobacterium frederiksbergense and Mycobacterium austroafricanum, species that are known to include strains with PAH-degrading capacities. Interestingly, 16S rRNA genes related to M. tusciae sequences, a Mycobacterium species so far not reported in relation to biodegradation of PAHs, were detected in all contaminated soils.

Summary Mycobacterium is often isolated from polycyclic aromatic hydrocarbon (PAH)-contaminated soil as degraders of PAHs. In model systems, Mycobacterium shows attachment to the PAH substrate source, which is considered to be a particular adaptation to low bioavailability as it results into increased substrate flux to the degraders. To examine whether PAH-degrading Mycobacterium in real PAH-contaminated soils, in analogy with model systems, are preferentially associated with PAH-enriched soil particles, the distribution of PAHs, of the PAH-mineralizing capacity and of Mycobacterium over different fractions of a soil with an aged PAH contamination was investigated. The clay fraction contained the majority of the PAHs and showed immediate pyrene- and phenanthrene-mineralizing activity upon addition of (14)C-labelled pyrene or phenanthrene. In contrast, the sand and silt fractions showed a lag time of 15-26 h for phenanthrene and 3-6 days for pyrene mineralization. The maximum pyrene and phenanthrene mineralization rates of the clay fraction expressed per gram fraction were three to six times higher than those of the sand and silt fractions. Most-probable-number (MPN)-polymerase chain reaction demonstrated that Mycobacterium represented about 10% of the eubacteria in the clay fraction, while this was only about 0.1% in the sand and silt fractions, indicating accumulation of Mycobacterium in the PAH-enriched clay fraction. The Mycobacterium community composition in the clay fraction represented all dominant Mycobacterium populations of the bulk soil and included especially species related to Mycobacterium pyrenivorans, which was also recovered as one of the dominant species in the eubacterial communities of the bulk soil and the clay fraction. Moreover, Mycobacterium could be identified among the major culturable PAH-degrading populations in both the bulk soil and the clay fraction. The results demonstrate that PAH-degrading mycobacteria are mainly associated with the PAH-enriched clay fraction of the examined PAH-contaminated soil and hence, that also in the environmental setting of a PAH-contaminated soil, Mycobacterium might experience advantages connected to substrate source attachment.

Sphingomonas is an organism of major interest for the degradation of organic contaminants in soils and other environments. A medium based on the aminoglycoside antibiotic streptomycin (Sm) was developed, which, together with the yellow pigmentation of Sphingomonas, facilitated the detection, recovery and quantification of culturable Sphingomonas from soils. All 29 previously described bacterial strains belonging to 17 different Sphingomonas species were able to grow on mineral media containing 200 microg ml(-1) streptomycin, showing that the capacity to resist high concentrations of Sm is a common characteristic within Sphingomonas. Incorporation of Sm into the mineral medium led to a significant reduction in the background microbial population and a concomitant 100 times more sensitive detection of Sphingomonas inoculated in non-sterile soil matrices. The Sm-containing medium was used to examine a variety of hydrocarbon-contaminated soils for the presence and biodiversity of Sphingomonas. Incorporation of Sm in the medium led to a significant increase in the number of yellow-pigmented colonies. Comparison of contaminated and non-contaminated soils derived from the same site revealed colonization by culturable yellow-pigmented Sm-resistant bacteria of the polluted location solely. Both yellow and non-yellow-pigmented colonies were purified from plates containing glucose and Sm, and BOX-polymerase chain reaction (PCR) was used to sort out clonally related strains. Representative strains from the major BOX-PCR clusters were identified using FAME and partial 16S rRNA gene sequencing. Forty-eight of 58 Sm-resistant isolates were identified as Sphingomonas sp. Streptomycin-resistant Sphingomonas isolates generated BOX-PCR diversity patterns that were site dependent and represented different species mainly belonging to Sphingomonas subgroups containing species formerly designated as Sphingopyxis and Sphingobium. The ability to degrade phenanthrene was only found in a minority of the Sphingomonas isolates, which all originated from soils containing high phenanthrene concentrations.

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