Ann Microbiol (2015) 65:995–1005 DOI 10.1007/s13213-014-0944-6 ORIGINAL ARTICLE Comparison of bacterial diversity from solar salterns and a simulated laboratory study Kabilan Mani & Sivaraman Chandrasekaran & Bhakti B. Salgaonkar & Srikanth Mutnuri & Judith M. Bragança Received: 8 January 2014 /Accepted: 14 July 2014 /Published online: 2 August 2014 # Springer-Verlag Berlin Heidelberg and the University of Milan 2014 Abstract Bacterial diversity in solar salterns and a simulated Introduction solar saltern under laboratory conditions was studied. The two systems were compared at the pre-salt harvesting phase and Hypersaline environments like salt lakes and solar salterns are salt harvesting phase using Denaturing Gradient Gel Electro- extreme ecosystems in which the change in salinity is the phoresis (DGGE). Bacterial composition was dominated by dictating factor which determines microbial diversity at any Gammaproteobacteria and more specifically with members of given point of time (Rodríguez-Valera et al. 1985). Prokary- Alteromonas, Vibrio, Pseudomonas, Tolumonas, otic diversity in any ecosystem is an important factor to be Marinobacter, Pseudoalteromonas and novel uncultured bac- considered because of its role in nutrient turnover, element teria. The Shannon–Weaver index (H), Simpson diversity recycling and as a potential hub for recovery of microorgan- index (D) and Equitability index (E) values showed that isms for industrially important metabolic products (Garcia- salterns can support a wide range of microbes during the Martinez et al. 1999; Lorenz and Eck 2005). Solar salterns pre-salt harvesting phase (3–4 % salinity) when compared to serve as a good model for studying the changes in biodiversity the salt harvesting phase (21–29 % salinity). Range-weighted over salinity, thereby providing us with information on ex- richness (Rr) values indicated that solar salterns are habitable tremes of life. Since diversity studies on hypersaline environ- only by a limited group of microbes as they have medium ments are gaining momentum in the past decade, the chances richness, indicated by physico-chemical characteristics. Prin- of obtaining novel isolates are relatively high when compared cipal Coordinate Analysis (PCO) of the simulated study with any other ecosystem. Novelty of isolates is not limited to showed a variation in diversity at a large scale with increase bacteria and archaea but includes eukaryotes like fungi, pro- in salinity. Solar salterns as well as the simulated tank showed tists and algae (Casamayor et al. 2013). more diversity during the pre-salt harvesting phase. Many reports have focussed on the occurrence of halophil- ic archaea in hypersaline regions (Munson et al. 1997; Ochsenreiter et al. 2002; Ahmad et al. 2008; Braganca and Keywords Solar salterns . Halophiles . Bacterial diversity . Furtado 2009;Ohetal.2010;Zafrillaetal.2010). However, DGGE . 16S rRNA information on what happens to the bacterial community in increasing salinity gradients from 2 to 30 % are limited (Henriques et al. 2006; Demergasso et al. 2008;Deshmukh K. Mani : S. Chandrasekaran : B. B. Salgaonkar : S. Mutnuri : et al. 2011; Boujelben et al. 2012). It is generally accepted that J. M. Bragança (*) diversity decreases with increase in salinity, as the case with Applied and Environmental Biotechnology Laboratory, Department any extreme ecosystem. Previous studies employing genetic of Biological Sciences, Birla Institute of Technology and Science fingerprinting techniques like clone library construction, Pilani K K Birla, Goa Campus, NH-17B Zuarinagar, Goa 403726, India DGGE, T-RFLP and RISA analysis has shown the general e-mail: [email protected] trend of decrease in diversity with increase in salinity. Fur- thermore, these studies have shown that a diverse group of Present Address: bacteria dominates salterns in low (4 %), moderate (15 %) and S. Chandrasekaran Water Pollution Group, Center of Excellence in Environmental high (32 %) salinity ponds (Casamayor et al. 2002). But trends Studies, King Abdulaziz University, Jeddah, Saudi Arabia in archaea are different with few detected in low salinity 996 Ann Microbiol (2015) 65:995–1005 ponds, and there is a gradual increase in diversity with in- Materials and methods crease in salinity. Eukaryotes, similar to bacteria, dominate low and moderate salinity ponds but gradually decrease in Sampling site, experimental set-up and physico-chemical high salinity ponds. Usually, there is a drastic variation in analysis diversity along a salinity gradient from low (4 %) to moderate salinity (15 %) and a stable community is obtained at high Sampling was carried out in Ribandar (15°25’N, 73°92’E) salinity (32 %), which is sustained until saturation (Estrada and Siridao (15°44’N, 73°86’E) salterns of Goa, India et al. 2004). Studies employing both culture-dependent and - (Fig. 1). These salterns experience two phases: namely, pre- independent techniques have shown that euryarchaeal mem- salt harvesting phase and salt harvesting phase. The first bers belonging to DHVEG-6, and Halobacteriaceae like sampling was done in January 2010, during the pre-salt har- Halorubrum sp. and Haloquadratum walsbyi are found to vesting phase and the second sampling was done in April be dominant in moderate and high salinity ponds. Low salinity 2010 during the peak salt harvesting phase. Water/brine sam- ponds are dominated by diverse bacterial groups such as ples were collected at a depth of 10 cm using sterile bottles Proteobacteria, CFB (Cytophaga–Flavobacterium– which were previously rinsed with the same sample. The Bacteroides), Actinobacteria, Firmicutes and Cyanobacteria. samples were stored at 4 °C and processed within 48 h. For With an increase in salinity, bacterial groups belonging to laboratory simulation study, 25 l of saltern inlet water from Gammaproteobacteria and CFB group members like Ribandar saltern was collected in a sterile container and trans- Halomonas and Salinibacter phylotypes become dominant. ferred to a glass tank of dimensions 0.33 × 0.23 × 0.28 m. This Organisms belonging to halotolerant and halophilic fungi like set-up was maintained under sunlight. The salinity of the Debaryomyces hansenii, Hortaea werneckii and Wallemia sampling site was measured using a conductivity meter ichthyophaga, chlorophytes like Dunaliella sp., flagellates like (Equip-tronics model EQ-682). pH of the sampling site was Halocafeteria seosinensis, ciliates like Fabrea salina,crusta- measured using a portable pH meter (Equip-tronics model ceans like Artemia sp. and novel organisms belonging to EQ-632). Salinity of the experimental set-up was measured Opisthokonta and Rhizaria taxa dominate hypersaline environ- on a daily basis. Conductivity readings were correlated to ments at various salinity gradients (Benlloch et al. 1996, 2002; salinity as described previously (Mani et al. 2012a). Anton et al. 2000, 2008; Øvreås et al. 2003;Burnsetal.2004; Estrada et al. 2004; Triado-Margarit and Casamayor 2013). Total community DNA extraction Large numbers of microbes present in the environment remain uncultured due to a lack of ambient culturable condi- Fifty ml of water sample was vacuum-filtered through tions. Therefore, biodiversity studies employing only culture- 0.22-μm pore size polycarbonate membrane. The membrane dependent techniques provide limited insight (Amann et al. was then treated with 1 ml of cell suspension buffer (0.15 M 1995). Culture-independent techniques employing 16S rRNA NaCl, 0.1 M EDTA, pH 8.0) followed by 1 ml of lysis buffer fingerprinting have provided us with much valuable and di- (0.1 M NaCl, 0.01 M EDTA, 1 % SDS, pH 8.0). To this verse information in the past two decades. Denaturing gradi- mixture, 0.5 ml of 10 % SDS was added and incubated at ent gel electrophoresis (DGGE) is a 16S rRNA fingerprinting 55 °C for 30 min. After incubation, 1.6 ml of technique is widely used for studying the structure of micro- phenol:chloroform:isoamyl alcohol (25:24:1 v/v/v) was added bial communities (Muyzer et al. 1998). and centrifuged at 12,000 rpm for 5 min. DNA was precipi- Goa is a coastal state in India having a tropical monsoon tated from the supernatant by adding 2 volumes of 95 % climate with hot and humid summers (January–May) follow- ethanol, suspended in TE (10:0.1 mM) buffer and stored at ed by lengthy monsoons (June–November). Therefore, the −20 °C for further use. DNA from the simulated glass tank salt pans operate only for a period of 3–4 months. Salterns was extracted using the same protocol on the first day, follow- in Goa experience two phases namely pre-salt harvesting ed by an interval of 15 days. The extracted DNAs were phase and salt harvesting phase. Salinity in salterns varies designated as ET0, ET15, ET30, ET45, ET60 and ET75. drastically throughout the year being around 3–4 % during the pre-salt harvesting phase and 26–29 % during the peak salt Amplification of 16S rRNA gene fragment harvesting phase (Mani et al. 2012a, b). Such salinity changes would ideally affect the distribution of microorganisms, both PCR was performed for amplifying 16S rRNA gene frag- bacteria and archaea, and their microbial succession along the ments from total genomic DNA, with primers 27F (AGAG salinity gradient (Boujelben et al. 2012). TTTGATCMTGGCTCAG) and 1492R (GGTTACCTTGTT In this study, bacterial diversity from two solar salterns ACGACTT) (Tanner et al. 1999; Sivaraman et al. 2011). during pre-salt and salt harvesting phases and a simulated Single reaction contained 10× PCR buffer, 10 mM of each solar saltern under laboratory conditions was studied using dNTPs, 2 mM MgCl2, 2 U of Taq Polymerase (Sigma), DGGE. 10 mM primers (IDT Technologies) and 1 μl of template Ann Microbiol (2015) 65:995–1005 997 Fig. 1 The location of Siridao and Ribandar solar salterns (source: Google Earth, 2010) DNA. The reaction was carried out in a thermal cycler prepared according to the manufacturer’s instructions. The (Eppendorf, Germany) with initial denaturation at 94 °C for run was performed in 1× Tris-acetate–EDTA buffer at 60 °C 5 min followed by 30 cycles of denaturation at 94 °C for 30 s, at a constant voltage of 100 V for 10 h.