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

BACKGROUND Goa is a coastal state in India and salt making is being practiced for many years. This investigation aimed in determining the culturable haloarchaeal diversity during two different phases of salt production in a natural solar saltern of Ribandar, Goa. Water and sediment samples were collected from the saltern during pre-salt harvesting phase and salt harvesting phase. Salinity and pH of the sampling site was determined. Isolates were obtained by plating of the samples on complex and synthetic haloarchaeal media. Morphology of the isolates was determined using Gram staining and electron microscopy. Response of cells to distilled water was studied spectrophotometrically at 600nm. Molecular identification of the isolates was performed by sequencing the 16S rRNA. RESULTS Salinity of salt pans varied from 3-4% (non-salt production phase) to 30% (salt production phase) and pH varied from 7.0-8.0. Seven haloarchaeal strains were isolated from water and sediment samples during non-salt production phase and seventeen haloarchaeal strains were isolated during the salt production phase. All the strains stained uniformly Gram negative. The orange-red acetone extract of the pigments showed similar spectrophotometric profile with absorption maxima at 393, 474, 501 and 535 nm. All isolates obtained from the salt dilute phase were grouped within the genus Halococcus. This was validated using both total lipid profiling and 16S rRNA data sequencing. The isolates obtained from pre-salt harvesting phase were resistant to lysis. 16S rRNA data showed that organisms belonging to Halorubrum, Haloarcula, Haloferax and Halococcus genera were obtained during the salt concentrated phase. The isolates obtained from salt harvesting phase showed varied lysis on suspension in distilled water and /or 3.5% NaCl. CONCLUSION Salterns in Goa are transiently operated during post monsoon season from January to May. During the pre-salt harvesting phase, all the isolates obtained belonged to Halococcus sp. During the salt harvesting phase, isolates belonging to Halorubrum, Haloarcula, Haloferax and Halococcus genera were obtained. This study clearly indicates that Halococcus sp. dominates during the low salinity conditions.