Ann Microbiol (2014) 64:599–609 DOI 10.1007/s13213-013-0693-y ORIGINAL ARTICLE Phylogeny and ecophysiological features of prokaryotes isolated from temporary saline tidal pools Spyridon Ntougias Received: 19 March 2013 /Accepted: 8 July 2013 /Published online: 31 July 2013 # Springer-Verlag Berlin Heidelberg and the University of Milan 2013 Abstract Although hypersaline environments have been Introduction extensively examined, only a limited number of microbial community studies have been performed in saline tide pools. Halophilic and halotolerant microorganisms are distributed We have studied a temporary salt-saturated tide pool and in several terrestrial and aquatic habitats, such as the Dead isolated prokaryotes from the water. Chlorinity measure- Sea, the hypersaline lakes of Asia and Africa, the benthic ments revealed that the tide pool brine could be characterized zone, salt marshes and marine sediments (Takai and as one of the most hypersaline ecosystems on earth. Enu- Horikoshi 1999; Mutlu et al. 2008; Tsiamis et al. 2008; meration of microorganisms at different salinities showed Bodaker et al. 2010). From a biotechnological point of view, that the tide pool was dominated by moderate halophiles. halophiles can be used in environmental applications, such Based on 16S rRNA gene sequence analysis, the prokaryotic as the bioremediation of highly polluted marine and saline strains isolated were related to the bacterial genera Rhodo- terrestrial ecosystems, and in the development of new bio- vibrio, Halovibrio, Aquisalimonas, Bacillus and Staphylo- technological products, such as the use of their metabolites coccus and to the haloarchaeal species Haloferax alexan- (e.g. ectoine and β-carotene) in the cosmetic and food in- drinus. Four bacterial isolates were distantly related to their dustry (Oren 2010). closest validly described species Aquisalimonas asiatica Microbial communities in hypersaline environments have (96.5 % similarity), representing a novel phylogenetic link- been extensively studied during the last decade (Burns et al. age. Ecophysiological analysis also revealed distinct pheno- 2004; Sørensen et al. 2005; Caton et al. 2009), resulting in typic profiles for the prokaryotic strains analyzed. The her- the identification of a plethora of novel halophilic bacteria bicide 2,4-dichlorophenoxyacetate could be effectively uti- and archaea. However, the identification of halophilic diver- lized by selected strains as the sole carbon source, but phe- sity in the tidal and intertidal zone is limited. To our knowl- nolic compounds could not be utilized by any of the halo- edge, only Bolhuis and Stal (2011) have indirectly examined philic isolates examined. None of the halophilic strains were the halophilic community in coastal microbial mats located able to grow without the presence of sea salt or seawater. along a tidal gradient, reporting a massive increase in Based on these results, we conclude that moderate halophilic halobacterial sequences during the summer. Thus, saline bacteria rather than extremely halophilic archaea dominate in tidal pools are ideal environments for exploring novel halo- such a hypersaline environment. philic microbiota as to date no microbial community studies have been performed in such naturally occurring saline eco- systems. The only related work reported thus far has been an Keywords Ecophysiology . Extremophiles . Halophilic examination of fecal contamination levels in tidal pools by bacteria . Haloarchaea . Marine ecosystems . Tidal zone Genthe et al. (1995), although these authors provided no information on tide pool microbiota. Tidal or tide pools are formed after seawater entrapment in pools of the coastal zone. Several physical and biogeo- chemical parameters affect microbial life in such tidal pools * S. Ntougias ( ) (Huggett and Griffiths 1986; Kooistra et al. 1989; Netto et al. Department of Environmental Engineering, Democritus University of Thrace, Vas. Sofias 12, 67100 Xanthi, Greece 2003). The pools can be shallow or deep, large or small, with e-mail: [email protected] rocky or muddy bottoms. Large and shallow pools are more 600 Ann Microbiol (2014) 64:599–609 exposed to the surface than small and deep ones. These dilution, aliquots of 0.1 mL were placed in five different features impact nutrient availability and exposure of the isolation media. The procedure was repeated in triplicate. microorganisms to certain environmental factors. Air tem- The isolation media used were prepared as follows: (1) 0.10 perature and sunlight influence the temperature of the tide volume of seawater per volume of isolation medium pool and consequently the photosynthetic and respiration (≅ 0.35 % salt concentration), (2) 0.85 volumes of seawater rates, thereby affecting the levels of carbon dioxide and per volume of isolation medium (0.85×seawater salt oxygen in the water of the pools. The size, shape and volume concentration≅3.0 % salt concentration), (3) 0.85 volume of the tide pool also affect the concentration of dissolved of seawater per volume of isolation medium and 90 g L−1 oxygen, while increasing levels of dissolved carbon dioxide NaCl (≅12.0 % salt concentration), (4) 0.85 volume of turn the water acidic (Schulte 2007). The water evaporation seawater per volume of isolation medium and 220 g L−1 rate (as influenced by the water surface area, air temperature NaCl (≅25.0 % salt concentration) and (5) 0.85 volume of and speed) and rainfall affect the salinity of the tidal pool. seawater per volume of isolation medium, 200 g L−1 NaCl −1 The aim of our study was to identify the diversity of and 50 g L MgCl2.6H2O(≅25.0 % salt concentration— cultured aerobic heterotrophic prokaryotes isolated from addition of high amount of Mg). Peptone and yeast extract temporary rocky tide pools of high salinity and to investigate were in all media tested at concentrations of 5 and 1 g l−1 the ecophysiology of both the bacteria and archaea isolated, respectively. Media were solidified with 17 g L−1 bacterio- with special reference to their halophilicity and ability to logical agar. For both isolation and enumeration, plates were utilize certain organic substrates. incubated at 37 °C for 2 months, with the exception of cultures obtained from the isolation medium containing a high concentration of Mg, which were incubated at 50 °C. Materials and methods All other experimental tests were carried out at 37 °C, unless otherwise specified. After colony enumeration, the cultured Sample and physicochemical analysis prokaryotic population was expressed as colony forming units (CFUs) per milliliter of tide pool saline water. Saturated brine was obtained from a tide pool with a rocky bottom located at the seaside of Kardamili, South Greece. Kardamili tidal pools are formed seasonally on the top of Morphology and salt tolerance huge rocks located at the edge of the shore (latitude 36.885589, longitude 22.23187). The tidal pool under study Cell morphology and presence of flagella were examined was approximately 50×20 cm, with a depth of 10–15 cm. using a Zeiss Axiostar Plus microscope (Carl Zeis AG, Electrical conductivity (EC) and pH were determined using Oberkochen, Germany) after flagella staining (Flagella Stain CRISON CM-35 (CRISON Instruments, Alella, Spain) and Droppers; Becton Dickinson & Co., Franklin Lakes, NJ). Metrohm 632 (Metrohm, Herisau, Switzerland) conductivity Salt tolerance was investigated in media consisting of the and pH meters respectively. Dissolved oxygen in the tide nutritional base described above (i.e. 5 g L−1 peptone and pool water was measured using a CRISON oxi 45 oxymeter. 1gL−1 yeast extract) and the following saline solutions: 0.10 Ions in the brine were determined using a ICS-3000 Dual Ion v/v seawater (≅ 0.35 % salt concentration), 0.85 v/v seawater Chromatography System (Dionex Corp. Sunnyvale, CA). (≅ 3 % salt concentration), 0.85 v/v seawater and 3 % NaCl The anion/cation analysis was carried out using Dionex (≅ 6 % salt concentration), 0.85 v/v seawater and 6 % NaCl AG22/CG12A guard (4×50 mm) and analytical columns (≅ 9 % salt concentration), 0.85 v/v seawater and 9 % NaCl (4×250), 4-mm ASRS-300 (31 mA)/CSRS-300 (59 mm) (≅ 12 % salt concentration), 0.85 v/v seawater and 12 % suppressors and CR-ATC/CR-CTC trap columns. Sodium NaCl (≅ 15 % salt concentration), 0.85 v/v seawater and carbonate (4.5 mM)/sodium hydrogen carbonate (1.4 mM) 15 % NaCl (≅ 18 % salt concentration), 0.85 v/v seawater and methanesulfonic acid (20 mM) were used as eluents for and 18 % NaCl (≅ 21 % salt concentration), 0.85 v/v seawa- anions and cations at flow rate of 1.2 and 1 mL min−1, ter and 22 % NaCl (≅ 25 % salt concentration) and 0.85 v/v respectively. The sample loop size was 50 μL and a conduc- seawater and 27 % NaCl (≅ 30 % salt concentration). Tem- tivity detector was used for the determination of ions. perature and pH range for growth were tested in media consisting of the nutritional base and the appropriate saline Isolation and enumeration of prokaryotes solution. Temperatures of 5, 10, 15, 20, 25, 30, 37, 40, 45, 50, 55 and 60 °C were examined. pH within 4 and 10 (in In order to isolate and enumerate the cultured prokaryotic steps of 1 pH) was tested, and a pH check was performed population, 10 mL of saline water collected from the tide before the inoculation and after the end of the experiment. pool was mixed with 90 mL of sterile tide pool saline water, Before and after sterilization, the pH was adjusted by using a and a series of tenfold dilutions was performed. From each sterile 0.5 M HCl or 0.5 M NaOH solution. Ann Microbiol (2014) 64:599–609 601 Utilization of organic compounds as the sole carbon source Table 1 Physicochemical characteristics of the tide pool brine Brine characteristics Value To investigate substrate utilization, we prepared media con- − taining a specific organic substrate, 0.02 g L 1 yeast extract pH (1/50 diluted) 7.07 and saline solution (at the optimum salt concentration of each Electrical conductivity (EC) (1/50 diluted) (mS cm−1) 11.9 −1 isolate examined).