POPULATION ANALYSIS IN A DENITRIFYING SAND FILTER - CONVENTIONAL AND IN-SITU IDENTIFICATION OF PARACOCCUS SPP. IN METHANOL-FED BIOFILMS

The microbial community of a denitrifying sand filter in a municipal w astewater treatment plant was examined by conventional and molecular t echniques to identify the bacteria actively involved in the removal of nitrate, In this system, denitrification is carried out as the last s tep of water treatment by biofilms growing on quartz grains with metha nol as a supplemented carbon source, The biofilms are quite irregular, having a median thickness of 13 to 20 mu m. Fatty acid analysis of 56 denitrifying isolates indicated the occurrence of Paracoccus spp, in the sand filter, 16S rRNA-targeted probes were designed for this genus and the species cluster Paracoccus denitrificans-Paracoccus versutus and tested for specificity by whole-cell hybridization, Stringency req uirements for the probes were adjusted by use of a formamide concentra tion gradient to achieve complete discrimination of even highly simila r target sequences, Whole-cell hybridization confirmed that members of the genus Paracoccus were abundant among the isolates. Twenty-seven o f the 56 isolates hybridized with the genus-specific probes, In situ h ybridization identified dense aggregates of paracocci in detached biof ilms, Probes complementary to the type strains of P, denitrificans and P. versutus did not hybridize to cells in the biofilms, suggesting th e presence of a new Paracoccus species in the sand filter, Analysis us ing confocal laser scanning microscopy detected spherical aggregates o f morphologically identical cells exhibiting a uniform fluorescence, C ell quantification was performed after thorough disruption of the biof ilms and filtration onto polycarbonate filters, An average of 3.5% of total cell counts corresponded to a Paracoccus sp,, whereas in a paral lel sand filter with no supplemented methanol, and no measurable denit rification, only very few paracocci (0.07% of cells stained with 4',6- diamidino-2-phenylindole) could be detected. Hyphomicrobium spp, const ituted approximately 2% of all cells in the denitrifying unit and coul d not be detected in the regular sand filter, This clear link between in situ abundance and denitrification suggests an active participation of paracocci and hyphomicrobia in the process, Possible selective adv antages favoring the paracocci in this habitat are discussed.