Arch Microbiol (1990) 154 : 589- 593 Archives of

Microbiology 9 Springer-Verlag 1990

Sambhar Salt Lake Chemical composition of the brines and studies on haloalkaliphilic archaebacteria*

Vivek Upasani** and Suresh Desai

Department of Microbiology, School of Sciences, Gujarat University. Ahmedabad - 380009, Gujarat, India

Received September 26, 1989/Accepted August 8, 1990

Abstract. The saline and alkaline brines from the Sambhar Salt Lake (SSL), both from the main lake and from the solar evaporation pans at Sambhar Salt Limited, Sambhar, Rajasthan, India, were studied with respect to their chemical composition and presence of red, ex- tremely haloalkaliphilic archaebacteria. The brines had pH values of 9.5 + 0.2 and a total salt content ranging from 7% (w/v) to more than 30% (w/v). Sodium chloride, sodium carbonate, sodium bicarbonate and sodium sul- phate were the principal salts present in these brines which lacked divalent cations (calcium and magnesium). Six strains of red, extremely haloalkaliphilic bacteria, desig- nated SSL 1 to SSL 6, were isolated. All the isolates showed obligate requirements for sodium chloride (> 15%, w/v) and high pH ( > 9.0). Magnesium ions were required in traces for maintaining morphological struc- ture and pigmentation. All these strains possessed the diether core lipids, phosphatidylglycerol (PG), phospha- tidylglycerophosphate (PGP), and bacterioruberins characteristic of halophilic archaebacteria. The strains were assigned to the newly proposed genus Natrono- bacterium.

Key words: Alkaline saline environment - Sambhar Salt Lake - Haloalkaliphilic bacteria - Archaebacteria - Natronobacterium - Natronobacterium SSL 1 (ATCC 43988) - Natronobacterium SSL 6 (ATCC 43987) - Dunaliella salina

ments. Among the former, the alkaline soda lakes of the African Rift Valley namely the Wadi Natrun, Egypt ( Imhoff et al. 1979; Soliman and Trfiper 1982) and Lake Magadi, Kenya (Tindall et al. 1980), have been studied in detail with respect to their geochemistry and microbial ecology. The isolation of red haloalkaliphilic bacteria from these environments has indicated further diversity within the archaebacterial halophiles, and has led to the creation of two new genera, Natronobacterium and Natronococcus (Tindall et al. 1984).

We report here the chemical composition of the brines collected both from the main lake as well as the solar evaporation pans (locally "kyars") of the saline and alka- line Sambhar Salt Lake (SSL), Sambhar, Rajasthan, India. The lake is situated at 26 ~ 58' N, and 75 ~ 5' E, in the middle of a closed depression in the Aravalli schists, approximately 65 km northwest of Jaipur, with its axis northwest to southeast (Aggarwal 1951). Six strains of red, extremely haloalkaliphilic bacteria were isolated from the high density brines, and characterized.

Materials and methods

Organisms

Natronobacterium gregot3,i (NCMB 2189) and N. magadii (NCMB 2190) were provided as gift by Dr. H. N. M. Ross and Dr. W. D. Grant, UK. Strains SSL 1 to SSL 6 were isolated from the SSL brines.

Highly saline and alkaline environments are rather rare compared to highly saline, neutral to acidic environ-

* Part of the paper was presented by the authors at XIV Inter- national Congress of Microbiology 7-13 September 1986, Manchester, UK ** Present address: Department of Microbiology, JJ College of Science, Nadiad-387001, Gujarat, India

Offprint requests to. S. Desai

Chemical analyses

Chemical analyses of the brines collected in July, 1984 were carried out at the laboratory of Gujarat Water and Air Pollution Board, Ahmedabad, India, by methods of Taras et al. (1980). Sodium and potassium were determined by the flame photometric method at wavelength 589nm and 776.5nm, respectively. Calcium and hardness were determined by EDTA titrimetric method. Magnesium was estimated by calculating the difference between hardness and CaCO3 content. Chloride was determined by an argentometric method. Sulphate was determined turbidometrically as BaSO4. Car-

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bonate was determined by titrimetric estimation of hardness and the alkalinity was reported as CaCO3. Bicarbonate was determined by titration against 0.02 N HzSO4 using phenolpthalein and methyl orange as indicators. Total dissolved solids were determined by a gravimetric method, using 50 ml of filtrate obtained by filtering the brine sample using Whatman filter paper No. 42. Suspended solids were determined by calculating the difference between total solids and total dissolved solids. The density (d) of the brine was measured using a heavy liquid densitometer at room temperature (35 ~ _+ 1 ~ C). pH was measured using a Systronics pH meter 324.

Enrichment and isolation

Enrichment cultures were obtained in modified Brown medium as described by Tindall et al. (1980). A brine sample (2%, w/v) was inoculated into 100 ml of the broth dispensed in 500-ml Erlenmeyer flasks and incubated on a shaker at 37~ for 7-10 days (Mullakhanbhai and Larsen 1975) Pure cultures were obtained on agar plates prepared by the addition of 2.5% (w/v) Difco agar to the medium. The isolates were maintained on slants at 5 ~ 10~ pH 9.5.

spectra were taken in a Beckman DU-40 UV-Vis double beam spectrophotometer.

Lipid analyses

Whole-organism methanolysate was prepared and the diether core llplds were extracted in hexane as described by Minnikin et al. (1975). Diether core lipids were detected using a thin-layer chroma- tographic method (Ross et al. 1981).

Total lipids were extracted from wet cells by the method of Bhgh and Dyer (1959) as modified for extremely halophilic bacteria (Kates 1972). Polar lipids were freed from non-polar components by acetone precipitation (Kates 1972). Thin-layer chromatography (TLC) of polar lipids was performed on silica gel G layers (analytical, 0.25-mm-thick; preparative 0.75-mm-thick) in the solvent system: chloroform-methanol-acetic acid-water (85: 22.5 : 10:4, by volume). Lipids were detected by the following spray reagents: (NH4)2MoO4- HC104 for phosphatides; 0.5% ~-naphthol-H2SO4 for glycolipids; H2SO4-ethanol (1 : 1, by volume) followed by charring for detection of all lipids (Kates 1972; Kushwaha et al. 1982).

Growth experiments

The strains were grown in media, pH 9.5, in which the concen- trations of NaC1, MgSO~, 7H20, and KC1 varied. Varying amount of sterile 20% (w/v) Na2CO3. 10H20 was added to produce pH values of 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 11.0 and 12.0. Incubation occurred in 50 ml of liquid medium in 250-ml Erlenmeyer flasks that were vigorously shaken at 37~ In all growth experiments, growth was followed by determining the optical density of the cell suspensions at 610 nm in a spectrophotometer (Systronics 105).

Light microscopy

A Carl-Zeiss light microscope was used with phase-contrast and dark-field optics.

Biochemical tests

Routine biochemical tests were performed using modified Brown medium as described by Holding and Collee (1971), Gonzalez and Gutierrez (1970) and Tindall et al. (1984). Chemicals used were of analytical grade.

The utilization of carbon sources including glucose, fructose, lactose, maltose, mannitol, ribose, sucrose and xylose was tested in shaken liquid medium supplemented with 1% (w/v) of the carbon source under test. Growth was measured by determining the optical density at 610 nm.

Sensitivity to chemotherapeutic agents was tested by the disc method. Discs were obtained from Pasteur Biological Laboratories, Umbergaon Road. India. Sensitivity was observed by inhibition zones around the respective disc. The agents tested were ampicillin (10 gg), bacitracin (5 units), carbenicillin (50 gg), cephaloridine (30 gg), chloramphenicol (30 p,g), cloxacillin (1 gg), colistin (10 gg), co-tnmoxazole (25 ~tg), erythromycin (15 ~tg). gentamycin (10 Ixg), kanamycin (30 gg), lincomycin (2 ~tg), novobiocin (10 gg), penicil- lin-G (10 umts), polymyxin-B (300 units), streptomycin (10 gg), sulphatriad (300 ~tg) and tetracycline (30 gg).

Carotenoid extraction

Carotenoid pigments were extracted in acetone: methanol (1:1, v/v) by the method described by Gochnauer et al. (1972). Absorption

G C mol% determination

The halobacterial DNA was prepared by the modification of the method of Marmur (1961). The lysozyme treatment step was omit- ted, as the cells lysed in hypotonic solutions. The G + C content of the DNA preparations in 0.012 M and 0.12 M phosphate buffer was determined from its melting point by the method of Marmur and Doty (1962), at Biochemical Division, National Chemical Lab- oratory, Pune, India. The wavelength scanning of the DNA solution was carried out in the range 220 to 320 nm on a Shimadzu double beam spectrophotometer model UV-210 A.

Results

Chemical composition

The brines collected f rom the Sambhar Salt Lake in July, 1984 had a total sal inity of ~ 7% (w/v) (~ 1.035, d) and the concentrat ion of salts in the brines var ied from 12 to 30% w/v. The lake dries up dur ing the hot summer season (40 ~ - 45 ~ C, atmospher ic temperature) and the salt crys- tallizes on the surface of the lake bed. The pr incipal ions contr ibut ing to the salinity of the brines, which have pH 9 .5 -10 .5 , were sulphates, carbonates, b icarbonates, chlorides, sodium and smaller amounts of potass ium (Table 1). These ions accounted for up to 99% of the total salts. However, the brines lacked the divalent cations, calcium and magnesium. The brines were rich in sus- pended solids (0 .7 -2 .8% w/v).

Ecological and microbiological observations

The brines of Sambhar Salt Lake appeared light green after the monsoons. The lake brine (1 .035-1 .042, d) flows into the various condensers and crystall izer pans (kyars) located at Jhapog, Gudha, Nawa (New), Ma in Line and Deodani . The colour of the brines var ied from yellow to green and red to pink, depending on their densities. The low density (1 .074-1 .115, d) brines at

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Table 1. Chemical composition of the brines from Sambhar Salt Lake and kyars, India collected in July, 1984 (g/liter)

Main Lake Gudha Jhapog Deodani SSL kyars kyars kyars

Na + 37.50 79 70 110.50 112.40 K + 0.50 0.30 2.15 1.58 Mg 2 + 0.00 0.00 0.00 0.00 Ca z + 0.00 0.00 0.00 0.00 C1- 21.46 84.52 139.60 142.56 SO~ 6.00 18.45 26.35 22.68 CO 2- 0.60 4.30 19.82 16.75 HCO3 1.94 4.10 8.70 6.30 Total dissolved

solids 71.27 205.16 302.00 305.60 Suspended solids 5.00 15.18 89.64 30.60 Density (d) 1.05 1.16 1.219 1.24 pH 9.0 9.5 9.5 10.5

Jhapog, Gudha and Deodani were dark green and contained dense cyanobacterial mats and abundant algal cells. The brines having density above 1.115, were brownish-red, and the red-pink colour became more in- tense from density 1.179 to saturation. The red brines (~-- 1.16, d) showed the presence of numerous red oval algal cells (~ 16 gin, diameter), which were motile with two polar flagella, and possessed vacuoles. This alga re- sembled the green alga Dunaliella salina. Besides the red- coloured alga, short and long rod-shaped halobacteria, and motile spirilla, possibly Ectothiorhodospira spp., were present in these brines. In the concentrated brines the dominating organisms were the halobacteria and to a lesser extent the spirilla. The brines were devoid of life above the rank of unicellular protozoa. The typical hy- drogen sulphide odour was distinctly present in the at- mosphere around the Deodani kyats.

Morphology of the isolates

Six strains of red haloalkaliphilic bacteria were isolated from the satterns at Sambhar Salt Ltd, Sambhar, and were designated as SSL 1 - SSL 6. Strains SSL 1 and SSL 2 were motile, long, rod-shaped, 0 .5-0.8 by 4 -10 ~m in liquid medium, and spherical 1 .0-1.5 gm (diameter) occurring singly or in clusters when grown on solid me- dium. Colonies were round, entire, slightly raised, mu- coid, opaque, pale pink, and 2 -4 mm in diameter after 7 -10 days incubation at 37~ The opaque nature of their colonies is characteristic of halobacterial cells con- taining gas vacuoles. On ageing, the colonies were stri- ated. Strains SSL 3, SSL 4, SSL 5 and SSL 6 were small motile, rods, 0 .7-0 .9 by 2 -4 gm in liquid medium, and spherical on solid medium. Colonies of these strains were round, entire, convex, transluscent (SSL 3 and SSL 4), opaque (SSL 5 and SSL 6), red-pink, and 1 -2 mm in diameter,

Growth requirements

All the six strains, SSL 1 - SSL 6, grew over the salt range 15--30% (w/v) NaC1 at 37~ Growth did not occur

below 7.5% (w/v) NaC1, and the oval forms predomi- nated at 12% (w/v) NaC1 concentration. All these strains lysed upon lowering the salt (NaC1) below 10% (w/v). The optimal NaC1 requirement of the strains SSL 1 and SSL 2 was 17.5% (w/v) at 37 ~ C, whereas the strains SSL 3 to SSL 6 grew optimally at 20% (w/v) at 37~ Strains SSL I and SSL 2 had a rapid doubling time (6 -8 h) as compared to the strains SSL 3 -SSL 6 and Natronobacterium magadii. The Mg 2 requirement of these strains was relatively low as compared to the non- alkaliphilic halobacteria. The optimal concentration was less than 0.5% (w/v) MgSO4 7H20 in the presence of optimal NaC1 concentration and growth conditions. In the absence of Mg 2+ ions, all the strains appeared oval in shape and showed poor pigmentation. Variation in the concentration of KC1 in the medium did not affect the growth of these strains.

Strains SSL 1 and SSL 2 grew over the temperature range 35 ~ 45~ whereas the strains SSL 3 -SSL 6 grew over the temperature range 30 ~ 50 ~ C. All these strains grew optimally at 40~ and pH 9.5. The pH range for optimal growth was 8.5-11.0. Growth did not occur below pH 8.0.

Biochemical characteristics

All SSL strains were gram-negative, oxidase- and catalase-positive. All these strains liquefied gelatin, hydrolyzed casein and produced H2S. Starch was hy- drolyzed by strains SSL 1 and SSL 2. Strains SSL 1 and SSL 2 hydrolyzed Tween 80; whereas strains SSL 5 and SSL 6 hydrolyzed Tween 40 and Tween 60. Strains SSL I and SSL 2 reduced nitrates to nitrites. The biochemical characteristics of these strains are given in Table 2.

The sugars glucose, lactose and mannitol stimulated the growth of SSL 1, SSL 2, SSL 5, and SSL 6. The growth of SSL 1 was also enhanced by fructose, ribose and sucrose, and that of SSL 2 by the presence of maltose and ribose. Strains SSL 3 and SSL 4 failed to show enhancement of growth in the presence of the sugars tested.

All strains were sensitive to bacitracin, co-trimoxazole (bactrim), erythromycin, sulphatriad and novobiocin. They were insensitive to other agents tested including ampicillin, penicillin, polymixin-B, streptomycin and tetracycline.

Lipid analyses

Whole-organism methanolysates of SSL strains and N. gregoryi (NCMB 2189) showed the presence of a single elongated spot of glycerol diether moiety (GDEM) having an Rf value 0.2 in preparative TLC. This spot was resolved into two distinct spots on double development as described by Ross et al. (1981).

Polar lipids of all the strains were analyzed by thin layer chromatography and total lipid patterns compared. Individual lipids were identified from the data of Kates (1972) and Kushwaha et al. (1982). All SSL strains

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Table 2. Biochemical characteristics of SSL isolates

Biochemical test SSL1 SSL2 SSL3 SSL4 SSL5 SSL6

Catalase + + + + + + Oxidase + + + + + + Tween hydrolysis

20 ng ng ng ng ng ng 40 . . . . + + 60 . . . . + + 80 + + . . . .

Gelatin hydrolysis + + + + + + Starch hydrolysis + + . . . . Casein hydrolysis + + + + + + H2S production + + + + + + Reduction of nitrate + + . . . . Reduction of nitrite . . . . . .

ng - no growth

contained phosphatidylglycerophosphate (PGP), phos- phatidylglycerol (PG) and phosphatidic acid (PA). Small amounts of two unidentified phospholipids were detected in the strains SSL i and SSL 2. Glycolipids were absent.

Absorption spectra of pigments

The absorption spectra of acetone: methanol (1:1) cell extracts of SSL strains showed maxima at 526, 494, 388 and 370 nm, and a shoulder at 470-475 nm, which is characteristic of the C5o isoprenoid pigments (bacterioruberins).

G + C tool% determination

The melting temperature range of DNA from all the SSL strains and N. gregoryi (NCMB 2189) was 85 ~ 100~ in 0.12 M phosphate buffer and 75~176 in 0.012 M phosphate buffer. The G + C content of the DNA of the SSL strains was found to be 60.2 _+ 0.4, the value representing standard deviation calculated from 3 deter- minations of Tm values.

Discussion

The Sambhar Salt Lake (SSL) is an intermittant salt lake and the largest inland salt source in India (Aggarwal 1951). The four main streams Rupnagar, Mendha, Kharain and Khandel add considerable amounts of salt, silt and organic matter from a catchment area of over 500 km 2. The Sambhar Salt Limited, Sambhar, manufac- tures salt and other by-products from the lake brines by solar evaporation process. The SSL brines are remark- ably similar in chemical composition and microbial ecol- ogy to the brines from Wadi Natrun, Egypt (Imhoff et al. 1979) and Lake Magadi, Kenya (Tindall et al. 1980). They lack the divalent cations Mg 2 + and Ca 2 +, they have a high pH (> 9.0), and they have high concentrations of carbonates and sulphates. The genesis of such saline and

alkaline environments, as well as their chemistry and ecology, have been reviewed by Grant and Tindall (1986).

Field observations revealed the absence of life ranking higher than the protozoa. The green alga, probably Dunaliella sp., and the photosynthetic purple sulphur bacteria possibly Ectothiorhodospira, occurred in blooms over a range of low brine densities (1.074-1.179, d) and served as the primary producers of the ecosystem. At higher densities (>1.179, d) the brines were highly viscous, possibly due to the lysis of algal cells; this pro- vided nutrients for the growth of chemoorganotrophic red halobacteria and spirilla. Sulphate-reducing bacteria were probably present in the sediments. Their presence was indicated by the high sulphate concentrations in the brines and by the hydrogen sulphide odour in the atmos- phere. Various algal species reported from Sambhar Salt lake brines include Anabaena sp., Arthrospira platensis, Synechococcus cedrorum and D. salina (Thivy 1966).

In the first edition of the Bergey's Manual of Systematic Bacteriology, vol. 3 (Staley et al. 1989), the red haloalkaliphilic bacteria are plaed in Section 25 Archaeobacteria, Group III Extremely halophilic archaeobacteria, Order Halobacteriales, Family Halo- bacteriaceae, genera Natronobacterium and Natrono- coccus (Grant and Larsen 1989). Recent studies have shown that the archaebacterial haloalkaliphiles are clearly distinct from the non-alkaliphilic halobacteria, and both cannot exist in the same enviroment (Tindall et al. 1984; Ross and Grant 1985). Based on the phenotypic differences, polar lipid analysis data and nucleic acid hybridization studies, two generic names: Natronobac- terium, for the rod-shaped haloalkaliphilic archaebacteria to include three species: N. pharaonis, N. gregoryi, and N. magadii; and Natronococcus, for the coccoid haloalkaliphilic archaebacteria isolated, N. occultus were proposed (Tindall et al. 1984). The members of the haloalkaliphilic genera Natronobacterium and Natrono- coccus contain phospholipids, but no glycolipids, a fea- ture common to alkaliphiles examined to date (Tindall et al. 1984; Morth and Tindall 1985; Tindall and Triiper 1986).

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The six SSL strains studied showed the following characteristics: they were gram-negative, motile rods, ex- tremely halophilic (17 .5 - 25% w/v, NaC1), and were ob- ligately alkaliphilic (pH 9.5, optimun). They had a low Mg z+ requirement (