Artificial Reefs in the Cote Bleue Marine Park: Assessment After 25 Years of Experiments and Scientific Monitoring

Abstract

Marine microbes play a very important role in biogeochemical cycles. Because of their biodiversity, the diversity of their metabolisms, and their physiological heterogeneity (live/dead, active/inactive cells), it is nec- essary to study them at the single cell level. Flow cytometry has become a technique of choice to reach this goal, providing a fast, reliable, and mul- tiparametric analysis of individual cells. However, the cost of such a technique makes flow cytometry out of reach for small institutes or laboratory units. It also requires a qualified and well-trained operator. To make this technique available to all the microbiolo- gists of the “Provence Alpes Côte d’Azur” Region, a Regional Flow Cytometry Platform (PRECYM), hosted by the Oceanology Centre of Marseille, was created by a consortium of six laboratories. Its goal is to provide researchers with the possibility to consider new approaches and to guarantee optimal technical, eco- nomic, and scientific exploitations of these instruments.An example of such a technical development is described in this chapter. The ELF97 phosphatase sub- strate has been shown to be a performing tool to assess the phosphorus status of phytoplankton populations. Recently, this technique has been successfully applied to marine heterotrophic bacteria in culture samples in which ELF-labeling was observed by microscopy. In this study, we describe a new protocol for ELF-labeling of heterotrophic bacteria that allow detection by flow cytometry. ELF-labeled samples were stored in liquid nitrogen for up to 4 months before analysis without any significant loss of total or ELF- labeled cell abundance and of relative ELF fluorescence intensity. This method enables studying ectoenzymatic alka- line phosphatase activity of heterotrophic bacteria at the single cell level in environments even with low cell abundance. It has been successfully applied in both fresh and marine samples. This approach is set to improve our understanding of the physiological response of heterotrophic bacteria to phosphorus limitation.