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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 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

(mainly NaCl) such as the Great , Utah; , California; Microbial the Dead , soda such as Lake Magadii, Kenya and Sambhar Salt diversity, , 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, , purple and green 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 .

Introduction

Sergei Winogradsky, a Russian display of photoautotrophy, microbiologist developed “Winogradsky chemoautotrophy and chemoorganotrophy Column” technique a simple way to grow in aquatic . 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 regions around the world. The given conditions for growth; inorganic salts, African , Transbaikal lakes organic matter, and light are the energy (Russia), Kulunda steppe soda lakes sources. This technique is a classical way of (Russia), (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,

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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 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 chloride, 0.05; 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 , 20.00; sodium , 20.00; of (mainly genus , 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 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 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

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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 (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 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 change with the bacterial diversity from soda lakes was development of light green colour in the reviewed by Madigen (Madigen, 2003). column (Fig. 2B). Upon incubation of more than 3-4 months, the column turned into During the first few months (November to dark green colour, and the rod-shaped January) there was not much change in the cyanobacteria became dominant form in the column that was inoculated with the algal column (Fig. 3). sample, probably due to the low

Fig.1 Photomicrographs of Microalgae from the Natural Samples

(A) (C)

( (B) (D) Fig.2 A) Enrichment of Anoxygenic Phototrophic (Purple Sulfur Bacteria), B) Enrichment of Microalgae by Winogradsky Column Technique

(A) (B) 611

Int.J.Curr.Microbiol.App.Sci (2016) 5(4): 608-614

Fig.3 Photomicrographs of Cyanobacteria from Winogradsky Column

Fig.4A t-RFLP from Algal Sample

Fig.4B t-RFLP from Enriched Sample

There were successful in the isolation and commonly found in hypersaline lakes has characterization of Euhalothece sp. from the been reported from Sambhar lake by Sharma enriched green cultures from Winogradsky et al., 2012. The presence of Akinete (thick column (Bhatt et al., 2016). The isolation of walled, resting cells) in Spirulina sp. Dunaliella sp., an eukaryotic alga (Arthrospira sp.) is the distinctive

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Int.J.Curr.Microbiol.App.Sci (2016) 5(4): 608-614 morphological feature, and its presence has The present study clearly shows that the been described in Arthrospira maxima. Winogradsky Column technique can be very Cyanobacterial biodiversity in India has useful for the enrichment of both microalgae been reviewed by Thajuddin and and bacteria to explore their diversity from Subramanian (2005); however specific hypersaline soda lakes. The microbial information on these organisms from succession was similar to that described for Sambhar Lake is not included. other aquatic environments. The use of different samples, media and cultural t-RFLP for Algal Study conditions allows the enrichment of specific group of microbes (microalgae and The use of algal specific primers ensured phototrophic purple sulfur bacteria as amplification of algal 18S rDNA, which was observed in our studies). This is the first size separated. The use of 6-carboxy report on the use of this well-known fluorescein (FAM)-labelled primers gave technique for microbiological studies from a blue colour in Genescan analysis; hence the soda lake in India. number of blue peaks in t-RFLP’s was equivalent to the number of algal species Acknowledgement present. Orange peaks are internal size standards. The results revealed the presence The authors thank the authorities at M. G. of six (6) and three (3) algal species in the Science Institute and Ahmedabad Education brine sample and enriched samples, Society (AES) for proving the laboratory respectively (Fig.4a and 4b). facilities. The grants from UGC (Minor research project no. F. No. 47-590/13, WRO We have used t-RFLP technique to know the to VN Upasani), and DBT (New Delhi) cultivable and non-cultivable species from under the “Star College Scheme” is duly the sample. From the t-RFLP data, species acknowledged. count was calculated as six (6) in the sample collected analysis, which showed the References presence of three (3) algal species in the enriched sample. This sample was 1. Adams, D.G., Duggan, P.S. 1999. inoculated in specific algal growth media Heterocyst and akinete differentiation in and incubated for 3 weeks then enriched cyanobacteria. New phytol., 144: 3–33. samplewas used for t-RFLP, it shows that 2. Bhatt, H.H., Renu, P., Upasani, V.N. three algal species were unable to grow 2016. Isolation and Characterization of under the growth conditions used in our a Halophilic Cyanobacterium laboratory. Among the three algal species Euhalothece SLVH01 from Sambhar that could be cultivated using this technique Salt Lake, India. Int. J. Curr. Microbiol. one was identified as Euhalothece sp., and App. Sci., 5(2): 215–224. two belonged to the genera Spirulina and 3. Grant, W.D., Tindall, B.J. 1986. The Dunaliella, respectively. Research work on Alkaline saline environments. In: the isolation and identification of these Herbert RA, Codd GA, editors. microorganisms for biotechnological Microbes in extreme environments. applications is being undertaken by our London: Academic Press, p. 25–54. laboratory. 4. Grant, W.D., Jones, B., Duckworth, A., Owenson, G. 1998. Microbial diversity

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of soda lakes. , 2: 191– 11. Madigen, T.M. 2003. Anoxygenic 200. phototrophic bacteria from extreme 5. Gorlenko, M.V., Buryukhaev, S.P., environments. Photosynth. Res., 76: Matyugina, E.B., Borzenko, S.V., 157–171. Namsaraev, Z.B., Bryantseva, I.A., 12. Samylina, O.S., Sapozhnikov, F.V., Boldareva, E.N., Sorokin, D.Y., Gainanova, O.Y., Ryabova, A.V., Namsaraev, B.B. 2010. Microbial Nikitin, M.A., Sorokin, D.Y. 2014. Communities of the Stratified Soda Algo-Bacterial communities of the Lake Doroninskoe (Transbaikal Kulunda Steppe (Altai Region, Russia) Region). Microbiol., 79: 410–421. Soda Lakes. Microbiol., 83: 849–860. 6. Imhoff, J.F., Trüper, H.G. 1977. 13. Sorokin, Y.D., Berben, T., Melton, Ectothiorhodospira halochloris sp. E.D., Overmars, L., Vavourakis, C.D., nov., a new extremely halophilic Muyzer, G. 2014. Microbial diversity phototrophic bacterium containing and biogeochemical cycling in soda bacteriochlorophyll b. Arch. Microbiol., Lakes. Extremophile, 18: 791–809. 130: 238–242. 14. Sharma, P., Agarwal, V., Mohan, M.K., 7. Imhoff, J.F., Hashwa, F., Trüper, H.G. Kachhwaha, S., Kothari, S.L. 2012. 1978. Isolation of extremely halophilic Isolation and Characterization of phototrophic bacteria from the alkaline Dunaliella Species from Sambhar Lake Wadi Natrun, Egypt. Arch. Hydrobiol., (India) and its Phylogenetic Position in 84: 381–388. the Genus Dunaliella Using 18S rDNA, 8. Jakher, G., Bhargava, S., Sinha, R. Natl. Acad. Sci. Lett., 35: 207–213. 1990. Comparative limnology of 15. Thajuddin, N., Subramanian, G. 2005. Sambhar and Didwana lakes (Rajasthan, Cyanobacterial biodiversity and NW India) Hydrobiologia, 197: 245– potential applications in biotechnology. 256. Curr. Sci. Sp. Ed., 89. 9. Kompantseva, E.I., Bryantseva, I.A., 16. Upasani, V.N., Desai, S.G. 1990. Komova, A.V., Namsaraev, B.B. 2007. Sambhar Salt Lake: chemical The Structure of Phototrophic composition of brines and studies on Communities of Soda Lakes of the haloalkaliphilic archeobacteria. Arch. Southeastern Transbaikal Region. Microbiol., 154: 589–593. Microbiol., 76: 211–219. 17. Upasani, V.N. 2008. Microbiological 10. Kumar, S. 2008. Conservation of studies on Sambhar Lake (Salt of Earth) Sambhar Lake- An important waterfowl Rajasthan, India. In Proceedings of habitat and a Ramsar Site in India. TAAL2007-The 12th World Lake Proceedings of TAAL2007-The 12th Conference, 448–450. World Lake Conference, 1509–1517.

How to cite this article:

Harshil H. Bhatt, Bhimaraj M. Sharma and Vivek N. Upasani. 2016. Studies on Microbial Diversity of a Soda Lake in India by Winogradsky Column Technique. Int.J.Curr.Microbiol.App.Sci.5(4): 608-614. doi: http://dx.doi.org/10.20546/ijcmas.2016.504.069

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