Characterization of the cyanobacterium Oscillatoria sp. isolated from extreme sulphureous water from Los Baños de la Hedionda (S Spain)

Los Baños de la Hedionda (LH, Málaga, S Spain) is a natural sulphureous (150200 µM sulphide) spa. Although this high sulphide levels can affect the photosynthetic process (1), there are numerous photosynthetic microorganisms inhabiting the spa (2). Among them, we isolated a strain of the cyanobacterium Oscillatoria sp., a genus well known by its tolerance to sulphide (3). The aim of this work was to isolate and to characterize this cyanobacterium, and to analyze the resistance of the isolated strain to sulphide, studied by analyzing the effect of increasing sulphide levels on photosynthetic performance and growth. On the other hand, the limit of genetic adaptation to sulphide was also explored using an evolution experiment named as rachet protocol (4). Introduction Material and methods Results and Discussion Conclusions References •Oscillatoria sp. showed stable growth despite the irradiance. •Oscillatoria sp. LD is around 1 mM, presenting the maximum growth rate on 100350 µM S (mean sulphide concentration at LH). The ID for Fv/Fm is almost 9 times higher than M. aeruginosa ID, a sulphide-sensitive strain. •Following the ratchet protocol, Oscillatoria sp. cultures above the LD have been achieved, so it can be concluded that the maximum adaptation is higher than 1.1 mM S. 1. Czyzewski, B. K., & Wang, D. N. (2012). Identification and characterization of a bacterial hydrosulphide ion channel. Nature, 483(7390), 494-497. 2. Flores‐Moya, A., Costas, E., Bañares‐España, E., García‐Villada, L., Altamirano, M., & López‐Rodas, V. (2005). Adaptation of Spirogyra insignis (Chlorophyta) to an extreme natural environment (sulphureous waters) through preselective mutations. New Phytologist, 166(2), 655-661. 3. Miller, S. R., & Bebout, B. M. (2004). Variation in sulfide tolerance of photosystem II in phylogenetically diverse cyanobacteria from sulfidic habitats. Applied and environmental microbiology, 70(2), 736-744. 4. Rouco, M., López-Rodas, V., González, R., Huertas, I. E., García-Sánchez, M. J., Flores-Moya, A., & Costas, E. (2014). The limit of the genetic adaptation to copper in freshwater phytoplankton. Oecologia, 175(4). doi: 10.1007/s00442-014-2963-1 Fig. 7 Number of days required to grow under increasing doses of S during the ratchet experiment. Four independent cultures (represented by different column patterns) were tested. Grey texture over a step of increased dose of S shows the concentration interval where initial lethal dosis was found Acknowledgments •This work has been financially supported by the Spanish Ministry of Science and Innovation through CGL2014-53682-P project. •Besides, the Predoctoral State Grant from Scientific and Technical Research and Innovation Plan, Spanish Ministry of Economy, Industry and Competitiveness I+D+i ECC/1402/2013, 2015 Fig 4 Growth rate (m) as a function of S concentration in Oscillatoria sp. Data are mean ± SD (n= 4). LD is indicated on the graph. Elena Martín-Clemente1, Ignacio José Melero-Jiménez1, Andreas Reul2, Miguel Hernández-López3, Enrique Salvo1, Elena Bañares-España1, María Jesús García-Sánchez1, Antonio Flores-Moya1 1 Departamento de Biologí3 a Vegetal, Facultad de Ciencias, Universidad de Málaga, Campus de Teatinos s/n, E-29071 Málaga, Spain; 2Departamento de Ecología y Geología, Facultad de Ciencias, Universidad de Málaga, Campus de Teatinos s/n, E-29071 Málaga, Spain; Departamento de Química Analítica, Facultad de Ciencias, Universidad de Málaga, Campus de Teatinos s/n, E-29071 Málaga, Spain Characterization of the cyanobacterium Oscillatoria sp. isolated from extreme sulphureous water from Los Baños de la Hedionda (S Spain) Isolation and maintenance: The Oscillatoria sp isolation was made by a successive dilution (SD) process (Fig. 1). The column A was filled with LH water; the rest of the wells, with 180 µL of BG11. By rows, and successively, a 20 µL aliquot from each well was transferred to the right next one. Following this method, the last well with cells of each row has a population formed by a single cell. Figure 1. Representation of the SD design. Inhibitory Dose (ID) and Lethal Dose (LD) Inhibitory dose for maximum quantum yield of PS II, on dark adapted cell cultures, (Fv/Fm) was measured using a PAM-2000 fluorimeter (Walz). Measurements of Fv/Fm were carried out after 1 hour incubation in BG 11 plus 20 mM HEPES (pH 7), under different S concentrations (0, 25, 50, 100, 150, 200, 400, 800 µM). To measure the LD, growth rate was measured after five days incubation in the same medium as indicated above at different S concentration (0, 50, 100, 200, 350, 500, 900 µM). Chla was used as a biomass estimator. In other experiments, fresh weight was used. The isolated strain was grown in a 250-mL cell culture flasks, with 100 mL of BG-11 medium buffered with 5 mM HEPES (pH 7). Sulphur (200 µM) was added to the medium every day from a Na2Saqueous NaOH master stock solution (pH ~13, 210-240 mM). At pH 7, sulfur is on the form of H2S (50%) and SH- (50%). Flasks were maintained under continuous irradiance of 50 μmol m−2 s−1, at 20°C. Ratchet protocol: the limit of the genetic adaptation to sulphide During the initial phase, four replicates of control cultures containing growth medium (BG11, 20 mM HEPES, pH 7) plus 200 µM S (mean concentration at LH), and four replicates of three treatments with increasing S concentrations were prepared (Fig. 2) Those cultures were kept seven days and then observed. Cultures showing the same concentration than control ones, were transferred to the next S level (+100 µM). Cultures not reaching a similar biomass to that found in control cultures were not transferred (red asterisks) and maintained in the same S concentration until they reached the control biomass. 0 5 10 15 20 25 30 0 50 100 150 m (mg·day-1) Irradiance (µmol ·s-1 ·m-2) 0 0,05 0,1 0,15 0,2 0,25 0,3 0,35 0,4 0 200 400 600 800 1000 m (Chla · day-1) [S] µM LD: 960 µM Fig 5 Effect of sulphide on Fv/Fm on dark-adapted cells of Oscillatoria sp strain. Data from a cyanobacterium strain isolated from a non-sulphureous waters (M. aeruginosa) are shown to compare. Data are mean ± SD (n= 4). ID are indicated on the graph. 0 0,1 0,2 0,3 0,4 0,5 0,6 0 200 400 600 800 Fv/Fm [S] µM Oscillatoria M. aeruginosa M. aeruginosa ID= 89 µM Fig 3 Growth rate (m) as a function of irradiance in Oscillatoria sp. Data are fitted to a von Bertalanffy model. Data are mean ± SD (n= 4) Oscillatoria ID= 760 µM 0 5 10 15 20 25 200→300 300→400 400→500 500→600 600→700 700→800 800→900 900→1000 1000→1100 1100→1200 No. of days Increased dose of S (uM)) 0 5 10 15 20 25 200→400 400→500 500→600 600→700 700→800 800→900 900→1000 1000→1100 1100→1200 No. of days Increased dose of S (µM) 0 5 10 15 20 25 200→500 500→600 600→700 700→800 800→900 900→1000 1000→1100 1100→1200 No. of days Increased dose of S (µM) Do you want to know more about this experiment? Get in contact with us. Fig. 2 Ratchet protocol design Most of the initial cultures are growing above the lethal dose, and even at concentrations over 1 mM. The number of days required to overcome the lethal dose is lower when the initial S concentration is minor (historical contingency). There were no differences on Oscillatoria sp. growth rate (m) on the 10-100 µmol · s-1 · m-2 irradiance range (Fig. 3). Oscillatoria sp. growth rate (m) showed a maximum on the 100-350 µM S range (Fig. 4). Inhibitory dose for Fv/Fm was much higher for Oscillatoria sp. than for a Microcystis aeruginosa strain isolated from a non-sulphureous waters.