Periodic Reporting for period 1 - MAPAPAIMA (Measure of Alkaline Phosphatase Activity of Plankton: An Integrated Microfluidic Approach) Período documentado: 2018-10-01 hasta 2020-09-30 Resumen del contexto y de los objetivos generales del proyecto Phytoplankton are microscopic organisms located at the base of the trophic web. These autotrophic cells play a major role in the biological production of the Oceans and are also responsible of roughly 40 % of the inorganic carbon fixation on the Earth, underlying their significant role in climate control. In the last decades, evidences of climate changes accompanying increase of Ocean stratification suggest that a decrease in mixing rates of deep nutrient-rich layers with surface nutrient-depleted waters. This increase of stratification leads to the decrease in nutrient availability for phytoplankton and a nutrient limitation in some areas. As a consequence an extension of areas with low phytoplankton concentration will arise in the future and lead to a drastic modification of the carbon cycle on the Earth. In the framework of an increase of Ocean stratification, the phosphate is an interesting candidate for controlling growth of organisms both in open and coastal oceanic regions. One of the main reasons of its potential limitation of phytoplankton growth is its very low concentrations usually measured in the upper layers in spite of its high turn-over rates. Phosphate acquisition is particularly complex both in open ocean and coastal environments because phytoplankton can also use organic phosphorus matters to survive to low phosphate concentrations. The main strategy to acquire dissolved organic phosphorus is to use a set of enzymes, such as Alkaline Phosphatase. A high rate of alkaline phosphatase activity (APA) is often detected in natural samples, especially in the dissolved fraction of the sample (<0.2µm). Although the dissolved fraction can represent the entire APA of a sample, nothing is known about the species responsible for this activity, the nature, and the fate of the high APA measured in the dissolved fraction. To better understand the role of this enzyme in phosphorus uptake and survival rate of the cell, the development of a new method capable to efficiently link the measurement of APA to a phytoplankton species in natural sample is essential.Why is it important for society?By their volumes and their places on Earth, the oceans are particularly important. Indeed, they act directly on the climate, by regulating surface temperatures, provide basic resources and ecosystem services to hundreds of millions of people and play a key role in the food security of many countries (FAO 2016). The main conclusions of the climate report published by the IPCC panel experts indicated that over a period of 40 years, the oceans are overall less salty, more acidic and warmer. This dynamic does not seem to be changing and the high temperature increasing stratification of the oceans also seems to induce increasingly severe limitations in nutrients. These profoundly and durably modify communities of species and the structure of ecological niches. Therefore, to better understand the future of the oceans and carbon fixation on the Earth, it is critical to investigate how the phytoplankton can develop adaptation to survive to nutrient limiting condition. In this project, 4 different objectives are required to successfully measure the APA at the single cell level using microfluidic technology. 1) Optimize the encapsulation of plankton species in a microfluidic chip. 2) Screen droplet containing a targeted plankton species. 3) Control the incubation and 4) Perform an alkaline phosphatase assay at a single cell level. Trabajo realizado desde el comienzo del proyecto hasta el final del período abarcado por el informe y los principales resultados hasta la fecha At the beginning of this project, we defined a career plan with the supervisors in order to optimize the work flow during the postdoctoral course. Then, during this WP1, we developed the new microfluidic platform that we used during the entire fellowship. From the 7th month, we started to adapt the sorting part and trap of microfluidic chip to the plankton morphology. Among the different species tested, we decided to focus on the APA of two dinoflagellate species (Scripiella acuminata and Alexandrium minutum). During the months 10-12, we tested the viability of cells in small droplets and confirmed that a volume of 500pL is suitable for the APA assay. From the month 13th, we transported the microfluidic platform to IFREMER (Brest, France) in order to test the APA at a single cell level. The goals of this campaign were to test APA in the cultures and samples collected in the Bay of Brest. APA assays were performed on 3 Scripiella acuminata and 3 Alexandrium minutum strains revivified form a sediment core. Finally, we developed a complete microfluidic platform and chip capable to simultaneously measure the APA at the single cell level and in the dissolved fraction. The microfluidic platform included a series of image processing algorithms for the detection of target cell, sorting droplet and autonomous image analysis system. By using this analytical approach, we measured the APA in the dinoflagellate cells revivified from sediment core in order to investigate the evolution and the adaptation in the APA expression as function of time (Figure 1). Currently, a manuscript is under review in the ISME journal and another in a preparation. The microfluidic technology associated with image processing algorithm was also useful for the scientific community and used in the study of Beneyton et al., (2020). Girault, M., Siano, R., Labry, C., Latimier, M, Jauzein, C, Beneyton T, Buisson, L, Del Amo, Y., Baret J-C, Inter- and intra-species variability of alkaline phosphatase production in revived dinoflagellate revealed by single cell microfluidic analyses, The ISME journal, under review.Beneyton, T, Love, C., Girault, M., Tang, T.-Y. D., Baret, J.-C. High-Throughput Synthesis and Screening of Functional Coacervates Using Microfluidics ChemSystemsChem, 2, e2000022, (2020). Avances que van más allá del estado de la técnica e impacto potencial esperado (incluida la repercusión socioeconómica y las implicaciones sociales más amplias del proyecto hasta la fecha) The first originality of this project is based on the exploitation of the shape recognition algorithm to identify the plankton. This project included high speed recognition system open some new perspectives in the flow cytometry where, the morphology of plankton species is not only included as data but as a sorting parameter. Moreover this innovative approach associated with a library of plankton images created an easy access to the complex world of the plankton taxonomy. For instance, it has been possible to automatically recognize and isolate some toxic microplankton species (Alexandrium minutum), increasing the possibility of monitoring the seawater mass affected by harmful phytoplankton species. Miniaturization of the cultivation chamber for a single cell also opens numerous new opportunities in plankton research. Indeed, the simple possibility to automatically isolate a microplankton species in the natural sample and follow in real-time its physiological adaptation to any modification of its microenvironment should considerably help the research in the field of plankton ecology, physiology or even chronobiology. Finally, using low cost disposable chips, the system developed was able to create rapid and axenic cultures to test APA at the single cell level. This analytical method associated with the new portable microfluidic platform provide the tools needed to better characterize the high APA found in the dissolved fraction of some samples as well as directly assess the question of P-limited phytoplankton species in field. This project also contributes to create a new scientific network between the researchers studying the microfluidic and Ocean sciences. figure-1.jpg
