The diversity of cultivable Halophilic bacteria inhabiting solar salt ponds of Covelong salt works, Kelambakkam, near Chennai (Southeast coast of India) was explored. Totally 11 bacteria were isolated and identified through culture, microscopic and biochemical characterization. All the isolates were able to grow optimally in 1.3M to 3.1M NaCl. Identified bacteria represent the members of genera Sediminibacillus, Oceanobacillus, Virgibacillus, Salicola, Halomonas, Marinococcus, Haloferax, Halogeomatriculum and Rhodovibrio belonging to four different families Bacillaceae, Halomonadaceae, Halobacteriaceae and Rhodospirillaceae. This study provides information on the diversity of halophilic bacteria in the salterns of Kelambakkam. Significances of these bacteria are discussed in relation to salt production in the salt works.
Keywords: Saltpan, Halobacteria, Bacillaceae, Halomonadaceae, Halobacteriaceae
Solar salterns are a unique ecosystem that supports life for hypersaline organisms. The bacteria that survive in high salinity brine are called as Halobacteria and constitute a large proposition of biota in the saline environment. Halophilic bacteria and archaea have a worldwide distribution. They have been isolated from a wide variety of habitats, including areas of high salt concentrations (Ventosa et al., 2008). Halophilic bacteria are either Gram negative or Gram positive and exhibit aerobic, facultatively anaerobic or obligatory anaerobic metabolism (Johnson et al., 2007).
They are characterized as organisms capable of growing from 8% to saturation level (35%) of sodium chloride (NaCl). Their ability to survive in such extreme environments like temperature, salinity and pH, offer a multitude of potential application in various fields (Oren et al., 1997). However, there exists several problems associated with the accuracy of halophilic bacterial systematics (Kushner, 1993). Therefore studies on bacterial diversity involve combination of phenotypic and genotypic characterization, in order to establish a stable bacterial taxonomy (Prakash et al., 2007). In this context, the present study emphasis on the diversity of cultivable/noval halobacterial isolates from the solar salterns of Kelambakkam (also called as Covelong salt works).
Materials and methods
Sampling site and sample collection
The present study was conducted at the Covelong salterns, Kelambakkam (12°45’01.28”N, 80°12’44.08”E) during March to June, 2011 (Fig. 1). Traditionally the saltern is divided into three series of ponds, namely reservoir, evaporation and crystallizer. All the three series of ponds had different shape, size, depth and width spread to facilitate salt production through solar evaporation. Salinity in these ponds varied from 10 to 40 ppt in reservoir ponds, 60 to 200 ppt in evaporation pond and 240 to 270 ppt in crystallizer ponds. For bacterial study brine samples with varying salinities were collected aseptically from the evaporation ponds at different salinity.
Fig. 1 Schematic diagram of Kelambakkam saltpan
Culture of bacteria
Each sample was divided into 50 mL aliquots and then filtered through 0.45mm and 0.22mm nitrocellulose membranes (Millipore, Inc.). The membranes were transferred onto agar plates containing Seghal and Gibbons (1960) medium (SG) and SG with 1% glucose (SGG) (without yeast extract and with 0.1% casamino acids). Inoculated plates were incubated in sealed bags at 40°C. In order to obtain pure culture by the streak plate method, representative of each sample was transferred to SG broth medium. The VKMM medium DSM372 (Manikandan et al., 2009) was used as growth medium. Artificial medium was prepared with marine agar 2216 Himedia (Zobell Marine Agar) and the salinity was increased by the addition of NaCl.
Characterization of halophiles
Morphological characteristics like shape and motility were performed according to Forbes et al. (2007). Gram staining was done using acetic acid ﬁxed samples (Oren et al., 1997). The biochemical tests such as catalase, oxidase, Methyl Red, Vogues–Proskauer, indole production, utilization of urease and citrate, nitrate and nitrite reduction and fermentation of sugar such as glucose, sucrose, maltose, lactose and mannose were tested as described by Gerhardt et al. (1998). The biochemical medium was supplemented with 10% of NaCl (w/v) and the results were observed after 72hr of incubation.
A total of 11 bacterial isolates were identified, based on the colony morphology, pigmentation, from the brine samples of Covelong salt works, Kelambakkam. The isolated bacterial isolates showed different shades of coloured colonies such as pink, red, orange, white, transparent etc.(Figs. 2a-k).
Figs. 2 a-k Colony morphology of bacterial isolates from the saltpan
(a) 2b; (b) 2ai; (c) 3a; (d) R280; (e) P180; (f) PC1; (g) PC22 (h) CN10; (i) CN12; (j) C25; (k) 2C6
Note the variations in colony colour viz. red, yellow, creamy etc.
Growth performance at different salinity
The bacterial isolates showed growth in varying NaCl concentrations. The minimum requirement of salt varied from 0-300g/L (w/v) of NaCl. The isolates such as 2b, 2ai, 3a, CN10, CN12, C25 and 2C6 showed growth in medium contain 0g/L of NaCl. On the other hand, the minimum salinity tolerance limit for pigment producing isolates like R280 and P180 was 70g of NaCl and they grew in maximum of 300g/L NaCl concentration (more than saturating concentration). In general, all the isolates exhibited optimum growth at 70 to 180g/L of NaCl with significant growth at 150g/L NaCl. These results revealed the halophilic and halotolerant nature of all isolates (Table 1).
Table 1. Growth performance of the halobacterial isolates at varying NaCl concentrations
+: Positive; -: Negative
Isolated colonies exhibited different cell shapes and pigmentation. Among the 11 isolates, 2b, 2ai, 3a, R280, PC22 and 2C6 showed rod shaped cells; P180 and C25 had cocci shaped cells; PC1 and CN12 isolates showed pleomorphic cell structure (Table 2). Certain isolates like P180 and PC1 were negative to motility test and the remaining were positive. Most of the isolates were shown positively to Gram’s staining (Table 2). Interestingly, certain bacterial isolates were showed strong pigmentation like pink (CN12), red (R280), yellow (PC1), creamy with yellow tinge and creamy with brown tinge and cream color.
Table 2. Morphological characteristics of halobacterial isolates
+: Positive; -: Negative; ND: Not determinable
All the isolates were positive for catalase test. Only two isolates, viz. 3a and C25 were negative for oxidase test. Among the 11 bacterial isolates, only R280 and P180 produced indole. Isolates such as 2ai, P180, PC22 and CN10 failed to convert pyruvate to acetoin (VP test) and PC1, PC11, PC22, CN10, CN12 and 2C6 were negative to perform mixed-acid formation (MR test). Bacterial isolates such as 2ai, CN10 and C25 were positive for Urease test and R280, P180, PC22, CN12, and 2C6 were positive for Citrate utilization test. Among the 11 isolates, 2b alone showed positive response for H2S test and the remaining were negative. Only two isolates responded negatively for nitrate reduction test and four isolates positively for nitrite reduction test. One among the 11 isolates, CN10 produced gas. Certain bacterial isolates responded positively for sugar fermentation test (Table 3).
Table 3. Biochemical characteristics of halobacterial isolates
+: Positive; -: Negative; MR: Methyl red; VP: Vouges-Proskauer; H2S: Hydrogen sulphide; Glu: Glucose; Suc: Sucrose; Mal: Maltose; Lac: Lactose; Man: Mannitol; ND: Not determinable
The bacteria isolated from brine samples of Covelong salterns were found to have highly diverse group of halotolerant and halophilic organisms with different morphological characteristics. However, morphological characteristics alone
were not enough to categorize the isolates, due to their flexibility with regard to the variations in the environmental conditions, such as temperature, NaCl concentrations, pH and medium composition (Fritze, 2002). Halobacteria can tolerate high salinity. The enzyme activity, stability, allosteric regulation, conformation and subunit association of these bacteria were affected by the salt. Halophilic bacteria were classified on the basis of their NaCl tolerance into slightly, moderately and extremely halophilic bacteria. However, this approach is practically ineffective for this purpose as the optimum and range of halophilic bacteria for tolerating NaCl is critical.
Therefore, in order to classify the halotolerant / halophilic bacterial isolates in response to NaCl the halophilic bacteria were grouped using the results of salt tolerant test proposed by Kushner (1993). The present study documents the diversity of Halobacteria in Covelong salt works, Kelambakkam using cultivation techniques. VKMM medium (Manikandan et al., 2009) with slight modification, was used to initiate the growth of wide spectrum of Halobacteria (Quesada et al., 1982; Rodriguez-Valera, 1988).
Morphological and biochemical characterization revealed that the microbial community of Covelong salt works is most closely related to the type strains of halobacterial genera Virgibacillius (Heyndrickx et al., 1998), Halobacillius (Spring et al., 1996), Halomonas (Vreeland et al., 1980), Marinococcus (Hao et al., 1984), Sediminibacillius (Carrasco et al., 2008), Oceanobacillius (Lu et al., 2002), Rhodovibrio, Salicola (Maturrano et al., 2006), Haloferax (Torreblanca et al., 1986) and Halogeomatricum (Montalvo-Rodriguez et al., 1998) for the isolates 3a, PC22, CN10, C25, 2b, 2ai, CN12, 2C6, PC1, R280 and P180 respectively. These specific isolates are reported first time in India and thus appear to be specific for the saltern studied. Among these, most of the Halobacteria were recently discovered and not much work has been carried out and thus appears to have a global distribution. Results of this study are in accordance with those of Rohban et al. (2009), who isolated bacterial strains belonging to the genera Salicola, Halomonas, Oceanobacillus, Virgibacillus, Halobacillus from Saltpan lake of Iran.
Further more the members of Haloferax (Manikandan et al., 2009), Halogeometricum, Salicola, Halobacillus and Halomonas were dominant group among microbial community in Covelong salterns, Kelambakkam. Manikandan et al. (2009) had first reported the presence of Sediminibacillus, Oceanobacillius, Virgibacillus, Marinicoccus in Indian salterns and thus appear to confirm their record of these bacteria from this saltern. Among the 11 isolates, Haloferax alexandriuns, Salicola marasensis and Halobacillus trueperi were predominant in high salinity (upto 300ppt) and temperature (Hongyu et al., 2009) isolated the halophilic bacteria from salt ponds of China at high temperature (35–40ºC).
Manikandan et al. (2009) studied the diversity of microorganisms in selected salterns of Tamilnadu, including Kelambakkam salt works. It is pertinent to mention that the microorganisms, isolated from the saltpan are different from the previous report on such environment, other than Haloferax sp. and Halogeometricum sp. (Manikandan et al., 2009). In comparison with other salterns studied Covelong salt works provide newer information, especially on the distribution of Haloferax, Halogeometricum, Sediminibacillius, Oceanobacillius, Salicola, Virgibacillius and Marinococcus etc.
Further in-depth study will provide more authentic information on their role in hypersaline (salt lake) ecosystem as well as their interaction with Artemia.
In comparison with other salterns studied, the salterns of Tamil Nadu region provided some unexpected ﬁndings including novel Haloferax and Halorubrum isolates. Our work further conﬁrms that NaCl saturated systems at different geographical locations represent a valuable source of microbial diversity as they harbour microbial communities differing in composition and structure.
Carrasco, I.J., Marquez, M. C., Xue, Y., Ma, Y., Cowan, D. A., Jones, B. E., Grant, W. D. and Ventosa, A. (2008) Sediminibacillus halophilus gen. nov., sp. nov., a moderately halophilic, Gram-positive bacterium from a hypersaline lake. Int. J. Syst. Evol. Microbiol. 58: 1961–1967.
Fritze, D. (2002) Bacillus identification- traditional approaches. In: R. Berkeley, M. Heyndrickx, N. Logan. and P. De Vos (Eds.), Applications and Systematics of Bacillus and Relatives, Oxford, Blackwell, pp. 100-122.
Forbes, B.A., Sahm, DF. and Weissfeld, A. (2007) Bailey and Scott’s diagnostic microbiology. Mosby, St Louis
Gerhardt, P.R., Murray, G.E. and Wood, W.A. (1998) Phenotypic characterization. In: P.R. Gerhardt, G.E. Murray, W.A. Wood and N.R. Krieg (Eds.), Methods for general and molecular bacteriology, American Society for Microbiology, Washington, pp. 617–791.
Lu, J., Nogi, Y. and Takami, H. (2002) Oceanobacillus iheyensis gen. nov., sp. nov. In Validation of the Publication of New Names and New Combinations Previously Effectively Published Outside the IJSEM, List no. 85. Int. J. Syst. Evol. Microbiol. 52: 685-690
Hao, M.V., Kocur, M. and Komagata, K. (1984) Marinococcus gen. nov., a new genus for motile cocci with meso-diaminopimelic acid in the cell wall; and Marinococcus albus sp. nov. and Marinococcus halophilus (Novitsky and Kushner), J. Gen. Appl. Microbiol. 30: 449–459.
Heyndrickx, M., Lebbe, L., Kersters, K. P., Vos, De., Forsyth, G. and Logan, N. A. (1998) Virgibacillus: a new genus to accommodate Bacillus pantothenticus (Proom and Knight 1950). Emended description of Virgibacillus pantothenticus. Int. J. Syst. Bacteriol. 48: 99-106.
Hongyu, W., Yang, L., Shen, L., Hu, B., Zongyun, L. and Qijiang, J. (2009) Isolation and characterization of culturable halophilic Microorganisms of salt ponds in Lianyungang, China. World J. Microbiol. Biotechnol. 25(10): 1727-1732.
Johnson, A.M., Thurlow, L.R., Zwenger, R.S. and Gillock, E.T. (2007) Partial characterization of two moderately halophilic bacteria from a Kansas Salt Marsh. The Prairie Naturalist. 39: 29-39.
Kushner, D.J. (1993) Growth and nutrition of halophilic bacteria. In: R.H. Vreeland and L.I. Hochstein (Eds.), The Biology of Halophilic Bacteria, CRC Press, Inc., Boca Raton, Fla, pp 87-89.
Litchfield, C.D., Irby, A., Kis-Papo T. and Oren, A. (2000) Comparisons of the polar lipid and pigment profiles of two solar salterns located in Newark, California, USA, and Eilat, Israel. Extremophiles, 4: 259-265.
Manikandan, M., Kannan, V. and Pasic, L. (2009) Diversity of microorganisms in solar salterns of Tamil Nadu, India. World J. Microbiol. Biotechnol. 25:1007–1017
Maturrano, L., Valens-Vadell, M., Rossello-Mora, R. and Anton, J. (2006) Salicola marasensis gen. nov., sp. nov., an extremely halophilic bacterium isolated from the Maras solar salterns in Peru. Int. J. Syst. Evol. Microbiol. 56(7): 1685-1691.
Montalvo-Rodriguez, R., Vreeland, R.H., Oren, A., Kessel, M., Betancourt, C. and Lopez-Garriga, J. (1998) Halogeometricum borinquense gen nov., sp. nov., a novel halophilic archaeon from Puerto Rico". Int. J. Syst. Bacteriol. 48(4): 1305–131
Oren, A., Ventosa A. and Grant, W.D. (1997) Proposed minimal standards for description of new taxa in the order Halobacteriales. Int. J. Syst Bacteriol. 47: 233–238.
Prakash, O., Verma, M., Sharma, P., Kumar, M., Kumari, K., Singh, A., Kumari, H., Jit, S., Gupta, S. K., Khanna, M. & Lal, R. (2007) Polyphasic approach of bacterial classification – an overview of recent advances. Indian J Microbiol. 47, 98–108.
Quesada, E., Ventosa, A., Rodriguez-Valera, F. and Ramos-Cormenzana, A. (1982) Types and properties of some bacteria isolated from hypersaline soils. J. Appl. Microbiol. 53: 155-161.
Rodriguez-Valera, F. (1988) Characteristics and microbial ecology of hypersaline environments. In: F. Rodriguez-Valera (Ed.), Halophilic Bacteria I, Boca Raton: CRC Press. pp. 3-30.
Rohban, R., Amoozegar, M.A. and Ventosa, A. (2009) Screening and isolation of halophilic bacteria producing extracellular hydrolyses from Howz Soltan Lake, Iran. Ind. J .Microbiol. Biotechnol. 36: 333-340.
Spring, S., W. Ludwig, M.C. Marquez, A. Ventosa and Schleifer, K.H. (1996) Halobacillus gen. nov., with description of Halobacillus litoralis sp. nov. and Halobacillus truperi sp. nov., and transfer of Sporosarcina halophilia to Halobacillus halophilus comb. nov. Int. J. Syst. Bacteriol. 46: 492-496.
Torreblanca, M., F. Rodriguez-Valera, G. Juez, A. Ventosa, M. Kamekura and Kates, M. (1986) Classification of non-alkaliphilic halobacteria based on numerical taxonomy and polar lipid composition, and description of Haloarcula gen. nov. and Haloferax gen. nov. Syst Appl Microbiol. 8: 89–99
Ventosa, A., Mellado, E., Sánchez-Porro, C. and Marquez, M.C. (2008) Halophilic and Halotolerant Micro-Organisms from Soils. In: P. Dion and C.S. Nautiyal (Eds.), Microbiology of Extreme Soils, Soil Biology 13, Springer-Verlag Berlin Heidelberg, pp. 87-115.
Vreeland, R.H., Litchfield, C.D., Martin, E.L. and Elliot, E. (1980) Halomonas elongata, a new genus and species of extremely salt-tolerant bacteria. Int. J. Syst. Bacteriol. 30: 485-495.
Yumoto, I., Hirota, K., Nodasaka, Y. and Nakajima, K. (2005) Oceanobacillus oncorhynchi sp. nov., a halotolerant obligate alkaliphile isolated from the skin of a rainbow trout (Oncorhynchus mykiss), and emended description of the genus Oceanobacillus. Int. J. Syst. Evol. Microbiol. 55: 1521–1524.