Biodiversity of a microbial consortium from activated sludge: molecular and confocal microscopy analysis

The biodegradation of organic pollutants in sewage treatment plants is carried out mainly by bacterial communities thriving in activated sludge. Although these ecosystems have been studied extensively from the civil engineering point of view, only few reports deal with biodiversity of resident microorganisms. To date few microbial species have already been identified. Our purpose was to contribute to the knowledge of the activated sludge biodiversity in order to understand better the dynamics of the biodegradation pathways with the aim to improve its efficiency. The experimental strategy was based on microbiological, microscopic and molecular methods, and aimed to identify bacterial species, their activities and to determine inter- and intra-specific variability. This strategy was applied to the microbial community of a sewage treatment plant collecting mainly surfactants-enriched wastes from textile industries. Samples, from different collection sites, were plated on rich or on selective media and a population of 50 random colonies from each medium, was isolated and maintained. From each isolate the DNA coding for 16S rRNA (16S rDNA) was amplified by means of polymerase chain reaction (PCR) using two universal primers (27f and 1495r) complementary to the highly conserved 5’ and 3’ ends of the coding region. The amplified 16S rDNAs were then analyzed for restriction polymorphisms (ARDRA) using the enzyme AluI, that, by generating species-specific restriction patterns, distinguishes between species. The 16S rDNA from at least one representative of each ARDRA group, was sequenced by using eight universal primers, allowing the identification at the species level. The intraspecific variability was assessed by the analysis of plasmid content and by random amplified polymorphic DNA (RAPD) fingerprinting. Fluorescently labelled, rRNA-targeted oligonucleotide probes and other fluorochrome probes were used togeter with scanning confocal laser microscopy (SCLM) to image the in situ bacterial distribution and biodiversity, and to detect activities such as: production of esopolysaccharides (ESP), important for aggregate formation in sludge, the storage of neutral lipids (i.e. hydroxialkanoates), and mixed-function oxidases for testing general catabolic activity.