Haloarchaea Associated with Salt Crystals Obtained from Solar Salterns of Goa, India

FIG. 1. Haloarchaeal organisms entrapped in salt crystals obtained from solar salterns of Goa, India. Solar salt was obtained by evaporation of sea water in open pans. Orange-red colored salt revealed the occurrence of haloarchaeal organisms entrapped in the halite crystal (0.5 cm x 0.7 cm). The crystals were photographed using a Canon camera (Powershot A580, 4X zoom: 35(W) – 140 (T) mm).

FIG. 2. The scanning electron micrograph is of Haloferax, a haloarchaeal isolate. This isolate (ATCC BAA 644) was recovered from solar salterns of Ribandar, Goa, India and exhibits a cup-shaped or involuted morphology, characteristic of the genus Haloferax (1, 3).

Introduction

Haloarchaea are microorganisms that grow at salinities between 20 and 30% NaCl concentration (2, 4). Salt pans, also known as solar salterns, are manmade large open pans where evaporation of sea water leads to formation of brine and then salt. This salt is then collected as heaps on the borders of the pans. Some of these salt pans show a characteristic orange-red coloration. Closer examination of the salt at the base of the heap revealed large crystals with orange-red centers as seen in Fig. 1.

Methods

The natural solar salt crystals (Fig. 1) were collected from solar salterns of Ribandar, Goa, India, during the salt harvesting season from February to March. These solar salt crystals were incubated in 25% NaCl tryptone–yeast extract (NTYE) broth on a rotary shaker at 150 rpm for 10 days at 30°C. One milliliter of this enriched broth was spread on a 25% NTYE agar plate and incubated at 30°C for 10 days. On the eighth day of incubation tiny, round, orange-red colonies were seen; these colonies were capable of growth exclusively on medium containing 20 to 25% NaCl. An isolated colony was picked and streaked onto a slant and then identified as Haloferax based on chemotaxonomic analysis.

For electron microscopy (Fig. 2), a culture pellet of the isolate was dispersed in NaCl synthetic medium to an absorbance of 0.8 at 600 nm. 100 ml of suspension was mounted onto stubs and square cover slips, fixed with 2.0% glutaraldehyde fixative (prepared in NaCl synthetic medium), and left overnight at room temperature. The stubs and coverslips were then exposed for 10 minutes to an increasing gradient of acetone-water at concentrations of 30%, 50%, 70%, 90%, and 100% acetone for 30 minutes, air dried, and placed onto a sputter coater (SPI Module specimen holder). The position of the stage was set in such a way that the stub was approximately 50 mm from the bottom of the sputter head. After sputtering the specimen with a 10 to 15 nm film of gold, the stub was placed in the sample chamber of the electron microscope (JEOL-5800 LV SEM) and observed.

The image clearly depicts microbial ecology. Organisms living in extreme conditions are known as extremophiles and dominant among them are the archaea. This visual can be used to demonstrate extremophilic microorganisms in their natural habitat thriving in saturating salt conditions. It can also be used for the comparative study of the two domains Archaea and Bacteria.

References

1. Mojica, F. J. M., F. Charbonnier, G. Juez, F. Rodríguez Valera, and P. Forterre. 1994. Effects of salt and temperature on plasmid topology in the halophilic archaeon Haloferax volcanii. J. Bacteriol. 176:4966–4973.

2. Oren, A. 1994. The ecology of extremely halophilic archaea. FEMS Microbiol. Rev. 13:415–439.

3. Rainey, F. A., and A. Oren (ed.). 2006. Extremophiles. Academic Press, St. Louis, MO.

4. Soppa, J. 2005. From replication to cultivation: hot news from haloarchaea. Curr. Opin. Microbiol. 8:737–744.

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Author	Judith Braganca
Author - Secondary	Irene Furtado

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Halite crystal (Enlarged view)
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Halite crystal (Labeled view)
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Haloferax (Enlarged view)
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Haloferax (Labeled view)