This study describes, for the first time, the water chemistry and microbial diversity in Dziani Dzaha, a tropical crater lake located on Mayotte Island (Comoros archipelago, Western Indian Ocean). The lake water had a high level of dissolved matter and high alkalinity (10.6–14.5 g L-1 eq. CO32-, i.e. 160–220 mM compare to around 2–2.5 in seawater), with salinity up to 52 psu, 1.5 higher than seawater. Hierarchical clustering discriminated Dziani Dzaha water from other alkaline, saline lakes, highlighting its thalassohaline nature. The phytoplankton biomass was very high, with a total chlorophyll a concentration of 524 to 875 μg chl a L-1 depending on the survey, homogeneously distributed from surface to bottom (4 m). Throughout the whole water column the photosynthetic biomass was dominated (>97% of total biovolume) by the filamentous cyanobacteria Arthrospira sp. with a straight morphotype. In situ daily photosynthetic oxygen production ranged from 17.3 to 22.2 g O2 m-2 d-1, consistent with experimental production / irradiance measurements and modeling. Heterotrophic bacterioplankton was extremely abundant, with cell densities up to 1.5 108 cells mL-1 in the whole water column. Isolation and culture of 59 Eubacteria strains revealed the prevalence of alkaliphilic and halophilic organisms together with taxa unknown to date, based on 16S rRNA gene analysis. A single cloning-sequencing approach using archaeal 16S rDNA gene primers unveiled the presence of diverse extremophilic Euryarchaeota. The water chemistry of Dziani Dzaha Lake supports the hypothesis that it was derived from seawater and strongly modified by geological conditions and microbial activities that increased the alkalinity. Dziani Dzaha has a unique consortium of cyanobacteria, phytoplankton, heterotrophic Eubacteria and Archaea, with very few unicellular protozoa, that will deserve further deep analysis to unravel its uncommon diversity. A single taxon, belonging to the genus Arthrospira, was found responsible for almost all photosynthetic primary production.

Flow cytometry offers an easy and powerful way to assess multi-parametric data in different domains, notably in the environmental sciences. Because evaluating heterotrophic prokaryotic abundance is crucial to understand an ecosystem's functioning, we propose a quick and efficient protocol for (1) cell's detachment in muddy coastal sediments followed by (2) enumeration of prokaryotes by flow cytometry compared to epifluorescence microscopy and (3) a type of storage adapted for benthic samples. First, sample preparation by incubation in a detergent mix containing sodium pyrophosphate (0.01M final concentration) and Tween 80 (0.1% final concentration) drastically increased cell detachment from sediment particles (+130.40%) compared to extraction with sodium pyrophosphate only. Cell sorting allowed to control the efficiency of the extraction as few cells were found attached to sediment particles in epifluorescence microscopy after sorting. Flow cytometry gave consistent results with strong reliability by counting 1.81 times more cells compared to epifluorescence microscopy. Thirdly, results revealed that sediment samples fixed with formaldehyde and then liquid-N2 frozen and directly stored at -80°C can be analyzed within 3months. In routine, our method of extraction and counting allowed to evaluate 83.67% of the real abundance in a sediment sample. Finally, this optimized technique was applied on sandy and muddy coastal and freshwater sediments and allowed us to prove the high efficiency of this new method. Flow cytometry is a fast, replicable and low-cost method for counting heterotrophic prokaryotes, even for sediment samples. The two-step method that we developed enables high frequency analyses (30 samples in less than 4h).

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