Culturable halophilic archaea at the initial and crystallization stages of salt production in a natural solar saltern of Goa, India

Haloarchaea, a group of archaea that depend on high salt concentrations, have been found to inhabit salterns that produce salt that has a wide use domestically. There are possible implications for human health, dependant on the species present, if they survive to inhabit the salt that is harvested. This study investigates the diversity of culturable haloarchaea present in the salt, during two stages of salt production: pre-harvesting, and at peak harvesting time.

Sampling of water and sediments (surface and at a depth of 10cm) was carried out. Species were isolated, purified, and identified, then the lytic effect of distilled water and low-salt water was tested. Isolation was carried out using plating techniques on two types of medium: NaCl Tryptone Yeast Extract (NTYE) and NaCl Tri-Na-citrate (NT). Initial plates were created using 100ul aliquots of water and a loop full of sediment. Enrichments were created by transferring 1ml of water samples and 1g off sediment samples onto media. The plates were incubated at 37oC for up to 5 days. Appropriate isolates were purified by selecting 10ul aliquots from these and incubating at 30oC, and repeating until orange or red colonies were observed. This colour is a noted character for identifying haloarchaeal species. There were other colonies found in both phases that were white or cream, which were removed when plated onto agar containing ampicillin. The colonies that were investigated are therefore ampicillin resistant.  The morphology of these isolates was checked using Gram staining and electron microscopy, to determine if they were in fact haloarchaea. The lytic ability of water and low-salt concentrations (3.5%) were determined of those characterised as haloarchaea by subjecting cells to their presence, followed by incubation for 24 hours. Viable colony formation was observed on medium plates. Genomic sequencing using the 16S rRNA was done to identify strains of haloarchaea. The salinity of the sampling sites were determined at the point of sampling by testing conductivity.

The species present and their responses vary depending on the sampling time, and the type of agar. Organisms present in the pre-salt collecting phase were all found to be under the species Halococcus salifodinae, to not lyse in water or low salt concentrations, and all but one to be of bright orange-red colour. There were also highly similar genetically, with 98% minimum similarity to Halococcus. Whereas, of the haloarchaea found at the peak of harvesting, all but one lysed in water and low salt concentrations, 3 gave orange colonies and 7 gave bright red. Their morphology also varied, though they were all gram negative, confirming their haloarchaeal classification.

Colonies in the pre-harvesting phase appeared at 20-30 days, suggesting there are slow growers or in lower quantities initially. Both are likely theories as the environment varies greatly, with an extreme increase in salt concentrations allowing for organisms that are more adapted to these conditions to have a competitive advantage. Also, their ability to survive at the lower concentrations suggests they do not have a high demand for salt, suggesting a slow metabolism and slow growth. This could also account for low number. The strain found in both concentrations, Halococcus salifodinae AB588757, exhibited a colour change from red-orange to orange when in high salt. This could be a reflection of lower numbers.

There were two months in between the sampling times. This is sufficient time for new bacterial species to be introduced to the saltern through natural means. It would be interesting to investigate if the species found in the pre-harvest phase are also present in the harvesting phase, since not all bacteria were represented in this study: those that did not produce visible colonies were not identified. They could be present in small quantities, therefore require a different method to that used here. The lysis test shows that this does not need to be done in the pre-harvest phase since these bacteria lyse in low salt concentrations, which the pre-harvesting phase environment consists of: 3-4% salt.

I was surprised that the species found at peak harvesting were so varied, since it makes sense for the most competitive organisms to outcompete the rest. Presumably this is not the case because of the frequency the environment changes, acting as a form of disturbance, which is said to increase diversity. The salt increase act as a disturbance, allowing a variety of species to colonise initially, before the salt concentrations become more stable, at which point the influence of competition is increased and the variation in species is reduced. This is seen by 10 species being found at peak salt and only 1 species, but several strains, are found pre-salt harvesting.

It was beneficial to use multiple agars as NT agar is capably in supporting more ‘choosey’ bacteria, hence more strains were found compared to NTYE. This of course does not enable bacteria that are not currently culturable to be identified. Another method would be to extract all the DNA from the samples, then PCR and run on a gel such as DGGE, and sequence significant bands. This would create a more representative view of the diversity present. 

The results of this study provide useful information about the diversity and change in species compositions as a result of altering the salt concentrations, with links to ecology and disturbance.

Mani et al. (2012) Culturable halophilic archaea at the initial and crystallization stages of salt production in a natural solar saltern of Goa, India, Aquatic Biosystems, 8:15

http://www.aquaticbiosystems.org/content/pdf/2046-9063-8-15.pdf