Suivi de la croissance sous forme filamenteuse et planctonique de Sphaerotilus natans par une méthode de biologie moléculaire

[Departement_IRSTEA]Ecotechnologies [TR1_IRSTEA]TED [Axe_IRSTEA]TED-EPUREInternational audienceIntroduction: Activated sludge is the most widespread biological wastewater treatment technology used over the world. However, the main problem of this process is due to the invasive proliferation of filamentous bacteria. Sphaerotilus natans is a model bacterium involved in certain cases of bulking. This bacterium is able to grow as planktonic cells or as sheathed filaments. Conventional microbiological methods are problematic under filamentous growth and PCR techniques can only quantify the total cell amount. Objectives: The aim of this work is to develop a method to quantify bulking-involved bacteria under their two growing morphologies: filamentous and planktonic, with the purpose of evaluating the factors inducting filamentation, such as nutrient availability. Materials & methods: A set of qPCR primers specifics to S. natans has been designed, to be used in pure cultures as well as in complex matrices (i.e. activated sludge). These primers are targeted against the gene sthA. qPCR has been used to quantify cells from global culture samples as well as 3 µm-pore size polycarbonate filters and filtrates, in which filaments and planktonic cells are respectively the sole cell form. The different fractions were observed in scanning electron microscopy through the filtration process to validate its efficiency. Results: The set of designed qPCR primers reveals high specificity to S. natans. It amplifies the strains ATCC 15291, 13338 and 13929 but not Leptothrix strains, which are phylogenetically close, or strain 29330, which has recently been reassigned as S. hippei. The 3 µm filtration allows effective separation of both growth forms, and filament free suspensions of planktonic cells are obtained. This method has been validated by monitoring the effect of nutrient-limited media on S. natans cultures. Its filamentous induction effect is clearly reflected by the growth kinetics obtained. Conclusion: This powerful method allows quantifying the effect of factors inducing bacterial filamentation and therefore it may shed light on bulking and other environmental processes where filamentous growth is crucial