5. Ecology of Hypersaline Microorganisms Savita Kerkar Department of Biotechnology, Goa University, Goa Introduction 1. Slight halophiles which grow optimally at 2-5% NaCl (0.2-0.85 M). The oceans are the largest bodies of saline water with average salinities ranging from 32-35 psu. Hypersaline 2. The moderate halophiles show rapid growth at 5- environments, with salinities far more than the normal 20% NaCl (0.85-3.4 M). seawater salinities, generally originate as a result of 3. The extreme halophiles which optimally grow at evaporation of seawater. Such environments are 20-30% NaCl (3.4-5.1 M). inhabited by halophiles, the salt loving organisms. Halophiles are distributed in hypersaline environments The non-halophiles grow optimally at less than 2% all over the world, mainly in natural hypersaline brines NaCl (0.2 M). Many halophiles and halotolerant in arid, coastal and deep sea locations as well as in microorganisms can grow over a wide range of salt artificial salterns. Halophiles include prokaryotes and requirement or salt tolerance at times depending on eukaryotes which are adapted to these hypersaline environmental and nutritional factors. environments at the highest salt concentrations at or close to the solubility limit of NaCl. Characteristics of hypersaline Due to the general absence of predators, the halophilic environments communities are denser in high salt concentration zones. They withstand extreme saline conditions and Hypersaline environments are those with salt have the capacity to regulate the osmotic pressure, concentrations above that of sea water (3.3% total thereby resisting the denaturing effects of salt in their dissolved salts).Based on their origin, they can be environment. classified into thalassohaline environments with salt concentration similar to that of sea water: sodium and The hypersaline microorganisms comprise of a variety chloride being the dominating ions and the pH is near of heterotrophic and methanogenic Archaea; neutral to slightly alkaline as these environments photosynthetic lithotrophs and heterotrophic bacteria, result from evaporation of sea water. During as also photosynthetic and heterotrophic eukaryotes. evaporation, some changes may occur in the ionic Examples of most widely distributed halophilic composition out of precipitation of gypsum microorganisms include archeal Halobacterium sp., (CaSO4.2H2O) or other minerals once their solubility Cyanobacteria such as Aphanothece halophytica and has been exceeded. NaCl saturated thalassohaline the green algae, Dunaliella salina (DasSarma and brines, such as those found in saltern crystallizer Arora, 2001). ponds often display a bright red colouration as they harbor a large number of pigmented microorganisms Halophilism (Oren, 2002). A great diversity of microbial life is observed in such A common phenomenon in hypersaline environments brines of marine salinity upto 3 - 3.5 M. Hypersaline is the occurrence of gradients in salinity as a result of ponds of Mediterranean salterns (Salins-de-Giraud) the evaporation of sea water. Halophiles are in France contain waters with total salinity ranging characterized based on their requirement of salt for from 3.4 - 5.1 M (130 - 300 psu) in secondary series growth in hypersaline conditions. In contrast, and salinities increasing to 340 - 350 psu in the final halotolerant bacteria do not require NaCl for growth, series where NaCl precipitates in the form of halite although they grow in high salinity and in crystals (Caumette et al., 1994). environments devoid of high concentration of salt. Halophiles can be classified into three groups on the Athalassohaline hypersaline environments in which the basis of their response to NaCl (Ollivier et al., 1994). ionic composition differs greatly from that of seawater, are likewise populated by microorganisms. A prime example is the Dead Sea, a lake in which the 37 concentration of divalent cations (about 1.9 M Mg+2 have suggested that microbial activity occurs in some and 0.4 M Ca+2) exceeds that of monovalent cations Deep Sea hypersaline basins and viable (1.6 M Na+ and 0.14 M K+) and of which the pH is microorganisms have been recoverable from brine relatively low (around 6.0). Even such a hostile inclusions in ancient salt deposits which are over 100 environment periodically supports dense microbial million years old (Vreeland and Powers, 1999). blooms (Oren, 1988). The two largest and best-studied hypersaline lakes Adaptations to high and changing are the Great Salt Lake in the Western United States salt concentrations and the Dead Sea, in the Middle East. The Great Salt To be able to live at high salt concentrations, halophilic Lake is the larger of the two (3900 km2) but shallower (10 m) and contains salts that are close in relative and halotolerant microorganisms must maintain a proportion to seawater. The Dead Sea, on the other cytoplasm that is osmotically isotonic with the outside hand is smaller (800 km2) and deeper (340 m) medium. Two different strategies have been used to containing a very high concentration of magnesium achieve this osmotic equilibrium. The first option, used salts. Both these lakes have close to neutral pH, by the aerobic halophilic Archaea of the family although the Great Salt Lake is slightly alkaline while Halobacteriaceae and the anaerobic halophilic bacteria the Dead Sea is slightly acidic. Compared to smaller of the order Haloanaerobiales involves the hypersaline ponds, the compositions of these lakes maintenance of high intracellular ionic concentrations, remain fairly constant due to expanse of their size, where K+ is the dominant cation instead of Na+ and although recent human activities have had significant adaptation of the entire intracellular machinery to effects on the chemistry and biology of both function in the presence of high salt (Dennis and (DasSarma and Arora, 2001). Schimmin, 1997). Wherever seawater penetrates due to seepage through narrow inlets from the sea, small evaporation The second option observed in most halophilic and ponds result. Notable among these are Solar Lake, halotolerant representatives of the Bacteria, Eukarya Gavish Sabka and Ras Muhammad Pool near the Red and also in halophilic methanogenic Archaea, involves Sea coast, Guerrero Negro on the Baja California the maintenance of cytoplasm lower in salt coast, Lake Sivash near the Black Sea, Shark Bay in concentrations and the accumulation of ‘compatible’ Western Australia and Ribandar salt pans in Goa, osmotic solutes that serve to achieve osmotic India. Hypersaline evaporation ponds have also been equilibrium while not being too inhibitory to enzymatic found in Antarctica e.g. Deep Lake, Organic Lake and activity (Gallinski, 1995; Ventosa et al, 1998b). The Lake Suribati, several of which are stratified with concentrations of these osmotic solutes are regulated respect to salinity. according to the salt concentration in which the cells A number of alkaline hypersaline soda brines also are found and can be rapidly adjusted as required when exist, e.g. the Wadi Natrun lakes of Egypt, Lake the outside salinity is changed. The number of Magadii in Kenya and the Great Basin lakes of the compatible solutes synthesized de novo or by Western United States (Mono Lake, Owens Lake, accumulation from the medium keeps steadily growing. Searles Lake and Big Soda Lake), many of which may In either case intracellular sodium concentrations are go intermittently dry. Soda brines lack magnesium kept as low as possible and outward-directed sodium and calcium divalent cations because of their low solubility at alkaline pH. Such environments are very pumps in the cytoplasmic membranes are of utmost dynamic experiencing significant seasonal variations importance, both in maintaining the proper intracellular in size, salinity and temperature. In additional to natural ionic environment, and in pH regulation. Adaptation hypersaline lakes, numerous artificial solar lakes are and adaptibility of halophilic bacteria depend on the man-made for the production of sea salt. Once NaCl regulation of synthesis of such organic osmolytes as precipitates, the concentration of potassium and glycine betaine, ectoine (1,4,5,6-tetrahydro-2-methyl- magnesium chloride and sulfate brines (bitterns) that 4-pyrimidine carboxylic acid), glucosylglycerol among remain, are usually returned to the sea. others (Galinski and Louis, 1998). Hypersaline environments also occur in sub-terranean evaporate deposits e.g. the Deep Sea basins created Halocins by the evaporation and flooding of ancient seas. Deep Halocins were first isolated by Francisco Rodriguez- Sea brines are relatively stable as a result of their Valera and co-workers in 1982. Out of 40 extreme higher density as reported in the Red Sea and Gulf of halophiles screened for halocin production, seven Mexico (MacDonald et al, 1990). Preliminary studies 38 strains inhibited a broad spectrum of strains some 19 Ecological significance of halocins – 35 out of 40 strains and 2 strains inhibited 1 – 3 strains. Halocins are generally produced in the stationary phase. Production of halocins can be advantageous Halocins are bacteriocins produced by extremely since bacteriolytic halocins can lyse sensitive cells, halophilic members of the domain Archaea. Their releasing their contents and enriching the environment. bactericidal modes of action are wide ranging and Bacteriolytic and bacteriostatic halocins, reduce include inhibition of transcription, translation, DNA and competition when nutrients become abundant again. RNA nuclease