Isolation and Cultivation of Halophilic Archaea from Solar Salterns Located in Peninsular Coast of India
K Asha, D Vinitha, S Kiran, W Manjusha, N Sukumaran, J Selvin
archaea, haloarcula quadrata, haloarcula vallismortis, halorchaea, rhodopsin-pigment, saltern
K Asha, D Vinitha, S Kiran, W Manjusha, N Sukumaran, J Selvin. Isolation and Cultivation of Halophilic Archaea from Solar Salterns Located in Peninsular Coast of India. The Internet Journal of Microbiology. 2004 Volume 1 Number 2.
Two brightly red-pigmented, motile, rod and triangular-shaped, extremely halophilic archaea were isolated from saltern crystallizer ponds located in peninsular coast of India. They grew optimally at salt concentrations between 25 and 35% and did not grow below 20% salts. Thus, these isolates are among the most halophilic organisms known within the domain Bacteria. The isolate HA3 showed optimal growth at 42°C whereas HA9 showed optimal growth at 52°C. These haloversatile microorganisms were presumed as new strains of
Though the oceans are invariably considered as largest saline body, hypersaline environments are, particularly, those containing salt concentrations in excess of seawater (3.5% total dissolved salts). Many hypersaline bodies derive from the evaporation of seawater and are called thalassic (DasSharma and Arora, 2001). The thalassic environment is obvious in the crystallizer ponds of solar salterns. Despite the prevailing extreme environment, a great diversity of microbial life has been observed in hypersaline bodies of greater than 3.5 mol/L NaCl, a point at which only a few extreme halophiles can grow (Anton et al., 1999). These extreme halophiles grow best at the highest salinities (3.4–5 mol/L NaCl), forming dense blooms, and resulting in the red colour of many salterns (Guixa-Boixereu et al., 1996). Common species of halobacteria are rod-, cocci- or disc-shaped, although triangular and even square-shaped species exist. Many are pleomorphic, especially when the ionic conditions of the media are altered, and most lyse below 1–1.5 mol/L NaCl. Halobacteria are classified as archaea (and are also called halophilic archaea or haloarchaea) and belong to the family Halobacteriacea. More than eleven genera have been reported,
Solar salterns consist of a series of shallow ponds connected in a sequence of increasingly saline brines located along the peninsular coast of India was chosen for the isolation of haloarchaea. Obviously cystallizers are almost completely dried ponds and have salinity of above 30% (Benlloch et al., 1995). Therefore, microorganisms present in the cystallizers are invariably haloarchaea. Since the haloarchaea obviously grow optimum between 25% to saturated concentration of NaCl, the possible contamination during the isolation procedure is scanty. The analysis of microbial diversity has shifted in the last two decades from cultivation-dependent approaches to 16S rRNA-based cultivation-independent approaches, which led to the discovery of many novel microbial taxa. Nevertheless, this approach also has important limitations and is often confined to naming 16S rDNA clones through sequence similarity and speculation on their ecophysiology on the grounds of this similarity. Therefore, cultivation is still the method of choice to understand fully the physiology and complex ecological interactions in which microorganisms engage. The present study was initiated to achieve isolation, characterization and cultivation of halophilic archaea from solar salterns located in the southeast coast of India.
Material and Methods
The saltern soil sample was collected from Kanyakumari coast (peninsular coast) of India. The sampling site was flourished with more than 5000 salterns, which makes a congenial environment for the growth of haloarchaea. Isolations were performed on modified DSC-97 medium (Casamino acids – 7.5 g, Yeast extract – 10.0 g, Trisodium citrate – 3.0 g, KCl – 2.0g, MgSo4.7H2O – 20.0 g, FeCl2 – 0.023 g, NaCl – 250 g, Agar-agar – 15 g, distilled water – 1000 ml, pH 7.4). After incubating at 30 °C for 4 days, 9 visible colonies, designated as HA1-9, were transferred and subcultured until pure culture was obtained. The isolates were maintained as a glycerol suspension (20%, w/v) at - 20 °C.
Morphological and biochemical characterization
The cultures for morphology were prepared by incubating the test strain on DSC-97 medium 37 °C for 3 d. The morphological properties of colonies were observed as colour, margin and elevation. Cultural characteristics were observed using starch agar media, Czapek medium, Nutrient agar, Gelatin agar, Casein agar, Simmon's citrate medium and SIM medium. All media were supplemented with 25% NaCl. Biochemical tests such as ONPG, nitrate reduction, phenylalanine deaminase, and amino acid decarboxylase were performed after MacFaddin (2000). Liquefaction of gelatin was tested using DSC-97 supplemented with gelatin (12%, w/v). Hydrolysis of urease was detected using basal medium (1 g glucose, 1 g casein-peptone, 1.98 g disodium hydroxyphosphate, 1.51 g monobasic potassium phosphate, 0.5 g magnesium sulfate, 250 g NaCl, 0.012 g phenol red, pH 9.0, 1 L distilled water). Urea was filter-sterilized and added to a final concentration of 2% (w/v). Catalase production was assayed by using 0±3% hydrogen peroxide with colonies taken from CM plates. Production of H2S was determined using Triple-sugar iron agar (Difco). Fermentation reaction was tested using phenol red basal medium supplemented with appropriate carbohydrates and saturated concentration of NaCl.
Physiological and antibiotic sensitivity tests
All tests were done at 30 °C, unless otherwise specified. In order to find out the halophilic range of isolates, they were inoculated in DSC-97 medium having varied concentration of NaCl as 0 to 35% (saturated concentration). The temperature ranges for growth were determined at varied incubation in 25,35,45,55,65,75,80 and 100°C. Rhodopsin pigments were extracted from cell pellets with methanol/acetone (1:1, v/v) and absorption spectra were recorded against the solvent in a spectrophotometer (Geneys10) (Anton et al., 2002). Both bacterial strains were subjected to
Results and Discussion
The supplementation NaCl had direct effect on the growth of halophiles. The number of colonies grown on the nutrient media was consecutively decreased with the increase of NaCl concentration. Among the 9 isolates (HA1 – HA9), HA 3 and HA9 were showed optimal growth at 35% NaCl and were grown well at saturated salt. Based on the NaCl requirement, the chosen isolates were grouped under ‘haloversatile' which require 25-35% NaCl for optimum growth. It was noteworthy, the colonies of HA3 further grow like a crystalline tree on the media whereas HA9 grows as square type crystals. The characteristic crystalline structure (accumulation of NaCl) was increased with the growth of colonies. It may be a peculiar characteristic of haloarchaea. It was presumed that the haloarchaea might increase the rate of crystallization process in the solar salterns. Halophilic microorganisms can be conveniently grouped according to NaCl requirements for growth (Ventosa et al., 1998). Larsen (1986) defined moderate halophiles as organisms growing optimally between 5 and 20% NaCl. The
The morphological, biochemical and physiological characteristics of the isolates are presented in Table 1.
The isolate HA9 had a peculiar characteristic feature as it grow as square type colonies whereas HA3 grow as discrete yellowish colonies. The colony morphology of HA9 was flat with undulated margin. HA3 was entire and raised colonies. Both the isolates were Gram negative motile rods (HA3) and square shaped (HA9). Invariably the isolates contained intra-cellular catalase and extra-cellular amylase enzymes. The isolate HA3 showed cellulose degradation, which was not reported for any of the reference strains. It was presumed that the isolate HA3 might have an active role in the biotransformation of humus in the soil of solar salterns. Both isolates produced hydrogen sulfide and indole, but nitrate reduction was restricted to HA3. The production of beta- galactosidase was observed for both isolates. Based on the morphological, biochemical physiological characteristics and comparison with characteristics of reference strains and standard reaction given in Bergy's manual of Systematic Bacteriology (Grant and Larsen, 1989), the present isolates were tentatively identified as
One of these uncultivated microorganisms, the enigmatic square archaeon first described more than 20 years ago by A. E. Walsby (1980). This intriguing microorganism was detected by conventional microscopy in brine samples collected from a salt crust forming the surface of a hypersaline pool on the Sinai Peninsula. Based on the presence of a regular hexagonal surface layer, similar to that of other halobacteria, Walsby suspected that the square prokaryotes belong to the group of the ‘Archaebacteria'. Later, this morphotype was shown to belong to the group of halophilic archaea and named as
The antibiogram indicated that
It is noteworthy that bright-red pigmentation is common in microorganisms inhabiting salt lakes and saltern ponds (Anton et al., 2002). Pigmentation was noticed in both species and showed maximum rhodopsin pigmentation in incubation under mercury lamb (1000 lux). Pigments showed absorption maxima at 482 nm and a shoulder at 506–510 nm. Members of the
In summary, the isolates HA3 and HA9 showed optimum growth at 25% to saturated concentration of NaCl. These haloversatile microorganisms were presumed as new strains.
The authors thank Dr P. Natarajan, Director, ICBM and Rev Fr Premkumar, Secretary, Malankara Catholic College for facilities and encouragement. This paper is a part of Ph.D research of KRTS.
Dr Joseph Selvin Department of Biotechnology, Malankara Catholic College, Mariagiri, Kaliakavilai - 629 153, Kanyakumari District, India Email: firstname.lastname@example.org