Revival of Halophiles from Recently Formed Great Salt Lake Salt Crystals
by Jennifer Day
The present boundaries of Great Salt Lake (GSL) were formed approximately 12,000 years ago. GSL is a remnant of the ancient freshwater lake, Lake Bonneville, which dissipated following the Ice Age. The resulting terminal lake collects all of the minerals flowing into the basin. This lake has the second highest saline concentration on earth (1), and it is the forth-largest terminal lake (2; 3). GSL is largely composed of sodium chloride, with a high sulfate concentration and a very low level of divalent cations (4)
Between 1955 and 1959, a railroad causeway was built which separated the lake into North Arm and South Arm; it allowed for the development of different saline concentrations in each arm. The arid climate of Utah causes substantial evaporation of the lake's water. The freshwater input to North Arm of the lake is negligible relative to South Arm, which receives 95% of the freshwater input by means of three rivers (5). Recent sampling found North Arm to have a saline concentration between 30% and 32% (5.1 and 5.5mol/L) and South Arm ranged between 15% and 16 % (2.6 and 2.7mol/L; 4).
A variety of organisms thrive in this hypersaline ecosystem. Two hundred and fifty species of birds, totaling five million individuals, stop at GSL during their migration path (3). Yeast and protozoa have been isolated from South Arm but not from North Arm (6). Twenty-five different species of algae were reported to inhabit South Arm of the lake, including types of green algae, dinoflagellates, and diatoms. Dunaliella spp. was the only species of algae reported to inhabit North Arm of GSL (7).
The real complexity of the GSL ecosystem is at the microbial foundation. Moderate halophiles, which are abundant in South Arm of GSL, are defined to grow optimally in saline concentrations between 5 and 20% (.86 and 3.4mol/L). North Arm is populated by extreme halophiles, which are defined to grow optimally in concentrations greater than 18% (3.4 mol/L; 8). The results presented in this paper will pertain only to the North Arm.
Sampling of GSL North Arm in June of 2004 showed a variety of halophilic morphotypes or cell shapes. The predominant cell morphology was cocci (45%); it was followed by squares (23%), rods (15%), triangles and pyramids (10%), and curved rods (7%). Several types of halophilic bacteriophage, or halophage, were also seen (4).
The saline concentration of North Arm GSL is near the saturation level for sodium chloride (4). Evaporation at the surface of GSL results with the molecules of salt being closer together. These molecules form small crystals of salt that float on the surface of the brine. The edges of these tiny crystals, which are surrounded by the hypersaline lake, grow by the addition of salt molecules. As the crystal becomes heavier, it floats lower and lower in the brine, which results with the pyramid-shaped crystal. Crystals eventually reach the surface of the lakebed, where they continue to grow (9).
As the crystals grow, small pockets of brine are trapped within the salt structure. As the rate of crystal growth increases, the quantity of fluid inclusions also increases. Quantities of inclusions are greatest in the center of the crystal (10). As a crystal forms, sometimes halophiles become trapped within the fluid inclusion of the halite crystals. These enclosed halophiles may remain viable in the inclusions for many years (11; 12; 13; 14; 15). The population of viable halophiles is hypothesized to decrease as resources are depleted over time (11).
Various species of halophilic Archaea (halophiles) have been revived from fluid inclusions in ancient salt crystals (12; 13; 14; 15). A new species, Halococcus salifodinae, was one novel isolate discovered in an Austrian salt mine (13). Many different species were isolated from salt crystals in two British salt mines. Based on lipid patterns, three out of nine taxonomic groups of halophiles were isolated from both of the salt mines (12).
The quantity of ancient crystals that harbor viable halophiles, however, is low. This is probably due to depletion of resources. In one study of 250 million year-old salt crystals (15), only two of the 52 crystals studied contained Archaea that survived dormancy. The effects of depleted resources can also be observed in recently formed salt crystals. Norton and Grant (11) found that rod-shaped halobacteria become spherical in shape within two or three weeks of crystal formation. This plieomorphism is typical of rod-shaped halophilic Archaea in a starved state.
This study seeks to determine the diversity of halophiles capable of surviving a short period of dormancy in recently formed hopper-shaped halite crystals extracted from GSL. The morphotypes of halophiles viable in fluid inclusions are predicted to be diverse, because they are diverse in ancient fluid inclusions. Spheres, rods, triangles, squares, and curved rods, which are the morphotypes present in GSL brine, are predicted to survive dormancy in a hopper-shaped halite crystal. The quantity of recently formed halite crystals harboring viable halophiles is predicted to be greater than the number in ancient crystals. Greater resources present allows for increased populations of microorganisms.
Few studies have been done to determine the effects of a desiccated environment on viruses, and none on bacteriophage. GSL crystals provide an excellent environment to study the effects of desiccation on halophage. Since larger halophilic Archaea are capable of being sequestered in salt crystals, the tiny halophage are expected to more easily become entrapped inside the fluid inclusions. Encephalomyocarditis virus was found to become inactivated with time in desiccate environments created through dehydration (16). Various human enteric viruses are able to survive desiccation (17). The extent of time depends on the virus, environmental factors, and the surface in which it was desiccated upon. The viruses were dried on to various objects such as aluminum, latex, paper and china to study the effect of dehydration of these viruses on various media forms. Since many viruses can withstand desiccation for long periods of time, these extreme types of bacteriophage are expected to demonstrate tolerance for limited water inside the crystal.
Materials and Methods
Crystal Selection. Hopper Shaped Halite crystals were randomly selected from the surface of the salt bed in North Arm GSL at Rozel Point. All crystals selected were submerged by approximately six to twelve inches of water. At the time of selection in mid-October, 2004, the water level of the lake was at its low point of the year.
Hopper-shaped halite crystals were separated into groups of four crystals all with similar dimensions. A total of thirty-six crystals were placed into groups of four members. Olympus America's MicroFire Camera and the software program entitled PictureFrame(TM) Version 2.1 was used to take and computer process pictures of the crystals before dissolution. Pictures of each crystal's side, top, and bottom views were taken (Fig. 1). The dimensions of the square base and amplitude of each crystal's peak was measured. The volume of the crystal was estimated from these measurements. Crystals were arbitrary categorized based on their calculated volume into small (less than .11cm3), large (greater than .38cm3), or medium (between .11cm3 and .38cm3) crystal size.
Figure 1. A picture from the side view of each crystal was taken along with a bottom view with the peak of the crystal closest to view.
Crystal Sterilization and Dissolution. All crystals were surface sterilized for one hour in 70% ethanol before dissolution. Crystals were dissolved in Modified Growth Media (MGM; 18) with different saline concentrations of 5, 12, 18, or 23%. Each member of the crystal groups discussed above was dissolved in media with one of these saline concentrations. A total of nine crystals were dissolved in each saline concentration in a Forma Scientific 420 Orbital Shaker.
Cultures were incubated in an Orbital Shaker at 80 rpm and 37ºC. These samples were subcultured four times before they were examined microscopically. As a control for contamination, pure media from each saline concentration was incubated in the shaker along with the crystal cultures.
Microscopy. Light microscopy with a Nikon 212133 was used to determine the morphotypes of halophiles present in culture after crystal dissolution. Photographs of the microbes were taken using the same program and camera as the crystal pictures. Slides of cultures were prepared and stained for light microscopy using the procedure described by Dyall-Smith (18).
Electron microscopy was used to examine cultures for the presence of phage in a total of eight media cultures at 12, 18, or 23% salinity. Media was centrifuged, and the cells were concentrated. The cell suspension was applied to a thin carbon foil for 5 minutes. Cells were stained with 20 successive drops of 2% uranyl acetate in water with a final wash with one drop of water. The samples were imaged in a Philips/FEI Tecnai 12 TEM at 60 KV and the images recorded on a 4kx4k GATAN CCD camera.
Only three of the nine crystals dissolved in media with a 5% saline concentration had growth. All three of them were white colored and the pellets had a different texture than the typical pink-colored halophiles. The 5% controls were contaminated often with a white fungal species. Every 5% stock media was made was also contaminated with a white fungus. All other halophilic growth in other saline concentrations had a pink tent to it. No contamination was ever noted at these saline concentrations. Therefore, the data for crystals dissolved in 5% media were discarded due to probable contamination.
Out of the 25 crystals dissolved in media containing the other saline concentrations, 22, or 88%, of the crystals dissolved contained viable pink-colored halophiles. The size of the crystal used or the saline composition of the media used to dissolve the crystal did not affect the viability of secluded halophiles.
Cocci, bacilli, square, triangle, and bent or curved rod morphotypes were present in all cultures containing viable halophiles as predicted (Fig. 2). Long chains were present 19 of the 24 or 79% of crystals possessing viable halophiles (Fig. 2). Some of these chains appeared to be aggregates of cocci, but electron microscopy suggested that long chained rod-shaped bacteria were present (Fig. 3). A chlorophyll producing species of algae was also noted in multiple cultures containing growth that was distinct from the Dunaliella species that inhabit the North Arm (7). No halophage were identified in cultures (Fig. 4).
Figure 2. Representative microorganisms found viable in dissolved GSL salt crystals. Starting from the left and moving up and down, the following are seen: a long chain, a bent rod, curved rod, square, rod, two pictures of green algae, cocci, and a triangle.
Figure 3. The picture on the right shows a long chain composed of aggregating cocci. The electron micrographs on the left and in the middle show long chains that do not appear to consist of aggregation microorganisms.
Figure 4. Pictures taken using electron microscopy showed that viruses (bacteriophage) are clearly present in the brine on the left and absence of viruses in a culture grown from a dissolved GSL halite crystal on the right.
As hypothesized, a greater percentage of recently formed halite crystals harbor viable halophiles than ancient crystals. This supports the major postulate that the low percentage of crystals harboring a diverse array of halophiles or microorganisms, in general, is the result of depleted resources. As time passes after initial crystal formation, the resources present to support the microbes in the inclusion diminishes. Accordingly, the quantity of microbes that the enclosed resources can support decreases with time. Eventually the resources are completely depleted resulting in the demise of all microorganisms in the inclusion.
A diverse array of halophilic microorganisms remains viable inside fluid inclusions of hopper-shaped halite crystals for short periods of time. If any growth occurred after crystal dissolution, all predicted morphotypes including cocci, bacilli, squares, bent and curved rods, and triangles were present in all cultures. This shows that representative species of all cell shapes present in North Arm GSL can be secluded into a salt crystal, and it remains viable until dissolution a short time later. These results do not say anything about the viability of different morphotypes for long periods of time secluded in hopper-shaped halite crystals. It does suggest that variation in morphotypes present in ancient salt crystals is due to the ability of the morphotype to survive seclusion in the crystal rather than its ability to actually become secluded. Another possible cause of the absence of certain morphotypes in ancient salt crystals is that the morphotype was not present in the brine at the time of crystal formation.
The species present in these diverse populations have not been determined. This work is ongoing, and we are currently performing 16S rDNA analysis and biochemical tests to classify the isolates. This study is made more difficult by the fact that very little microbiology has been accomplished on GSL. Only a handful of isolates have been characterized, and very few are from the North Arm.
Due to the difficulties occurred with contamination in five percent salt media, future studies should not utilize low concentrations similar to this. A media with a higher salinity should be used in future experiments to study seclusion in crystals from North Arm GSL. The present salinity of North Arm GSL is around 30% (4), but the highest saline percentage used was 23%. Future studies should address this same question of morphotype diversity in higher concentrations of salt. This more accurately displays what actually happens to brine in North Arm GSL with repeated crystal formation and dissolution.
Crystal size appears to not affect the viability of morphotypes present in the crystal. This finding is consistent with the current theory of hopper-shaped halite crystal formation, which suggests that the largest inclusions are formed in the beginning of the process and occupy the center of the crystal (10). These large fluid inclusions are present in all sized crystals. These large inclusions are hypothesized to contain the largest quantity of halophiles. Since the microbe population is most numerous, the probability of having a great variety of morphotypes in the inclusion is at its highest.
Long chains were seen for the first time in GSL. It is not known whether these chains consisted of one bacterium or aggregates of multiple bacteria. Microscopy data indicated that both are probably present. Future studies need to determine if aggregation is occurring. If this is the case, studies need to determine the factors that cause this excessive aggregation in microbes from a halite crystal.
This procedure showed another novel morphotype of GSL microorganisms. Bent rods were seen in all cultures with growth (Fig. 2), but they were never seen before in GSL. The identity of the rods is unknown. They may be a result of the staining method; however, they have never been seen before in the brine using the same staining method. Like the long chains of bacteria, these bent rods may be rods aggregation at ninety-degree angles to each other. They also might actually be a single bacterium that has never been seen before in GSL. Future studies need to determine the identity of these novel morphotypes.
The inability of halophage to survive crystal formation, desiccation, and crystal dissolution was not expected. It is hypothesized that the phage went into a lysogenic cycle inside the desiccate environment of the crystal instead of the predicted lytic cycle. Future work needs to be done to determine where the phage goes in this desiccate environment and why they do not reappear when the crystals are dissolved and cells cultured.
The discovery of novel halophiles in GSL halite crystals suggests that these methods are an excellent way to isolate novel organisms in GSL and other lakes with high saline concentrations. Culturing from environmental ecosystems is a difficult endeavor, and GSL is no different. In fact, only 10% of microbes are culturable when compared to direct cell counts of the brine (4). Futures studies of microbial diversity in these ecosystems should utilize these methods of crystal isolation/dissolution that may avoid the "kill factor" when sampling brine with plastic bottles.
Another implication of these methods is for determining the presence of life on the planet Mars. GSL, with its sodium chloride content with high sulfate levels are similar to the postulated ancient Martian sea. Salt-containing evaporates are present on Mars; suggesting that organisms similar to halophilic Archaea may have existed there. If there were halophiles when there was water, we know from experiments such as the ones presented in this paper that they could be alive still, sequestered in the halite. These methods should be developed as they may provide a way to search for potential halophilic organisms in the salt evaporates on the red planet when samples are returned from Mars.
The authors wish to express appreciation to Harmony Smith, Michael Accord, Rylan Larsen, and Dr. David Goldsmith for their contributions and collaborations on the project.
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