Int.J.Curr.Microbiol.App.Sci (2016) 5(4): 608-614 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 5 Number 4 (2016) pp. 608-614 Journal homepage: http://www.ijcmas.com Original Research Article http://dx.doi.org/10.20546/ijcmas.2016.504.069 Studies on Microbial Diversity of a Soda Lake in India by Winogradsky Column Technique Harshil H. Bhatt1, Bhimaraj M. Sharma2 and Vivek N. Upasani3* 1Department of Biotechnology, Kadi SarvaVishwavidyalaya, Gandhinagar 382015, India 2Fisheries Officer, Commissioner of Fisheries, Bharuch, India. 3Department of Microbiology, M. G. Science Institute, Navrangpura, Ahmedabad 380009, India *Corresponding author ABSTRACT K eywo rd s Halophilic organisms are found in habitats with a high concentration of salt (mainly NaCl) such as the Great Salt Lake, Utah; Owens Lake, California; Microbial the Dead Sea, soda lakes such as Lake Magadii, Kenya and Sambhar Salt diversity, soda lake, Sambhar Lake, India; marine salterns across the globe, etc. Organisms living in such Salt Lake (SSL), habitat require high salt concentration (5-25% NaCl) and/or alkaline pH for Winogradsky growth. Algae, archaea, purple and green sulfur bacteria are the dominant Column, t-RFLP. microflora of these extreme habitat. Samples were collected from Sambhar Lake and microbial diversity was examined by cultivating halophilic Article Info organisms in Winogradsky Column. Successive changes in the column lead Accepted: to changes in diversity also. Here we report on microbial diversity based on 19 March 2016 photomicrography and t-RFLP method. This is the first report on the use of Available Online: Winogradsky column technique for enrichment and study the microbial 10 April 2016 diversity of Sambhar Salt Lake. Introduction Sergei Winogradsky, a Russian display of photoautotrophy, microbiologist developed “Winogradsky chemoautotrophy and chemoorganotrophy Column” technique a simple way to grow in aquatic ecosystem. and study the complex microbial interactions in natural samples. Each The soda lakes are wide spread in the arid organism is dependent on the other under and desert regions around the world. The given conditions for growth; inorganic salts, African Rift valley lakes, Transbaikal lakes organic matter, and light are the energy (Russia), Kulunda steppe soda lakes sources. This technique is a classical way of (Russia), Great Salt Lake (USA), Sambhar showing the diversity of microbes based on Salt Lake (India), etc. are some of the their metabolic and physiological famous lakes that have been studied for their requirements. It remains an important microbial diversity (Grant and Tindall, 608 Int.J.Curr.Microbiol.App.Sci (2016) 5(4): 608-614 1986; Grant et al., 1998; Gorlenko et al., (% w/v) NaCl and inoculated with the pink 2010; Samylina et al., 2014). The adaptation to red coloured brine of high density for of haloalkaliphilic bacteria to the enrichment of photosynthetic bacteria. The environmental pressures such as high pH, medium composition (g/L) is as follows: salt concentration, temperature, etc., in soda calcium chloride, 0.05;magnesium chloride, lakes, and their role in biogeochemical cycle 0.10; potassium dihydrogen orthophosphate, has been described by Sorokin et al. 2014 0.50; ammonium chloride, 0.80; sodium (Sorokin et al., 2014). Study on the diversity sulfate, 20.00; sodium carbonate, 20.00; of Purple sulfur bacteria (mainly genus sodium chloride, 160.00; yeast extract, 0.50; Ectothiorhodhospira) has been reported sodium succinate, 2.00; vitamin solution, from Transbaikal lakes. 1.00 ml; SLA solution, 1.00 ml. Final pH was adjusted to 8.5 with NaHCO3. Organic We have developed several Winogradsky and inorganic media were autoclaved columns to enrich and study the diversity of separately. extremely halophilic bacteria and algae present in the Sambhar Salt Lake brines and Composition of SLA solution (g/L): sodium soil. The change in color of the column, and ethylene diamine tetra acetic acid, 3.00; iron morphological forms due to the effect of sulfate, 1.10; cobalt chloride, 0.19; temperature, salt concentration, pH and light manganese chloride, 0.10; zinc chloride, intensity over a period of time was 0.07; sodium molybdate dihydrate, 0.018; monitored. Several strains of nickel chloride, 0.02; boric acid, 0.30; haloalkaliphilic archaea, anoxygenic purple copper chloride, 0.002.The same medium sulfur bacteria, green sulfur bacteria and with 5% (w/v) NaCl concentration resulted cyanobacteria were observed and few were in the enrichment of haloalkaliphilic characterized. microalgae. Other columns were inoculated with low density brine (green coloured) for This study, will be useful for others to microalgal cultivation. understand the exploration of the rich microbial diversity found in this unique The soil, brine sample (s) 200ml, chalk, and extreme environment, Whatman no. 1 paper strips were added to a 2 L glass measuring cylinder and the final Materials and Methods volume was made to 2L with the above mentioned medium. It was then incubated in Sample Collection sunlight for 3-4 months and growth was observed periodically. Soil and brine samples (various densities/salt concentrations) were collected Morphological Studies from Devyani Kyars (pans), Sambhar Lake, Rajasthan during May and November The samples collected from the site and months. Samples were stored at 4 °C. enriched in column were centrifuged; wet mount were observed microscopically and Enrichment by Winogradsky Column photomicrographs were taken using 10X, Technique 45X and 100X objectives with a Lawrence and Mayo binocular microscope attached A few cylinders were prepared using Trüper with a Nikon 6000P camera. and Imhoff medium (1978) containing 16.0 609 Int.J.Curr.Microbiol.App.Sci (2016) 5(4): 608-614 t-RFLP for Algal Diversity season, algal diversity was dominant (green brine). The brine samples collected were Terminal Restriction Fragment Length green, orange to red, or purple pink Polymorphism (t-RFLP) is a molecular depending on the salt concentration and pH. biology technique used to study microbial The various forms (oval, rod-shaped, spiral diversity using automated DNA analyser. and filamentous) of algae were observed in Genomic DNA was isolated from the the green coloured brine samples as shown biomass collected from green brine samples in Fig. 1. The filamentous and spiral forms as well as enriched microalgal culture were less in number as compared to the obtained by inoculating the same sample other forms. The size of filamentous spiral into Algae Culture Broth (ACB) forms was as long as 40-50 µm, unusual medium(Hi-media). The ITS fragment forms of square shaped cells in chain were (~800 bp) was amplified using high–fidelity also observed. The microbial cell density as PCR polymerase with 6-carboxy fluorescein well as diversity was astonishingly high in (FAM)-labelled primers. The PCR product the natural samples. Most of the algae were was subjected to restriction digestion. The prokaryotic, however eukaryotic algae fluorescently labelled t-RFs were size belonging to the genus Arthrospira (typical separated on an automated sequencer using spiral morphology with terminal oval- an internal size standard. The t-RFLP shaped akinete), and Dunaliella (Fig. 1D) electropherograms were analyzed with were also observed. GeneScan 3.7 software (Applied Biosystems). This analysis was carried out Enrichment by Winogradsky Column at Chromous Biotech Pvt. Ltd, Bangalore. Technique Results and Discussion We opted for Winogradsky column technique which supports growth of both Sampling and Microscopic Observations bacteria and algae. Use of brines of different densities (salt concentration) allowed the The Sambhar Lake is an intermittent salt development of cyanobacteria/algal flora lake, with varying physicochemical and phototrophic purple sulfur bacteria as properties depending upon the season and described by several researchers. The site. Sambhar Lake has shallow solar pans columns were set-up in May and inoculated and salinity of the pans ranges from 5.0 to with pink/red brine and soil samples. After 30% depending on the salt production stage, about a month, purple-pink colour growth Samples were collected from Devyani Kyars was initially observed at the bottom. Further, with salinity ranging from was 10-25.0 (% incubation lead to dense growth in the w/v NaCl), so that both algae and bacterial column (bloom) giving it a dark purple-pink could be enriched from the samples. The colour (Fig. 2A). Microscopic observation microbial diversity changes with season as revealed the presence of sheathed bacteria, reported in these tropical desert lakes and the curved or slightly spiral anoxygenic (Jakher et al., 1990; Kumar, 2008; Upasani purple sulfur bacteria. and Desai, 1990; Upasani, 2008) hence we have collected samples during two different We observed the presence of the anoxygenic seasons (summer and early winter). The purple sulfur bacteria in the natural as well colour of the brine indicated that during as the enriched samples, which belong to the summer season, bacterial diversity was genus Ectothiorhodhospira, reported by dominant (pink brine), and during winter 610 Int.J.Curr.Microbiol.App.Sci (2016) 5(4): 608-614 Imhoff and Trüper (1977) from Lake temperatures (10-20 °C). However, after Magadii, Kenya. One species was identified February as the temperature gradually as E. halophila (Imhoff et al., 1978). increased (max. 25-30 °C) and there was a Extremophilic anoxygenic phototrophic sudden remarkable