Archaeal Biodiversity in Crystallizer Ponds from a Solar Saltern: Culture versus PCR

The culturable haloarchaeal diversity in a crystallizer pond from a solar saltern has been analyzed and compared with the biodiversity directly retrieved by analysis of rRNA genes amplified from the environment. Two different sets of culture conditions have been assayed: solid medium with yeast extract as carbon source and liquid media with either yeast extract or a mixture of fishmeal, Spirulina sp., and Artemia salina. Seventeen colonies grown on plates with yeast extract incubated at 30°C were analyzed by 16S rDNA partial sequencing. Sixteen were closely related to haloarchaea of the genus Halorubrum; 13 of them to Halorubrum coriense, a haloarchaeon isolated from a solar saltern pond in Australia, which had not been previously isolated from the pond analyzed in this study; and one to Haloarcula marismortui. Liquid cultures were analyzed by ribosomal internal spacer analysis (RISA) and partial sequencing of the 16SrRNA genes. A total of 18 sequences were analyzed, 15 corresponding to RISA bands obtained from cultures, and 3 from the environmental sample used as inoculum. Thirteen sequences obtained from cultures were related to several Halorubrum species, and 2 to Haloarcula. One of the clones obtained directly from the environmental sample was distantly related to a Natronobacterium, whereas two were related to SPhT, the phylotype most frequently retrieved from this environment by culture independent techniques. Our results show an extremely low diversity for the haloarchaea retrieved by cultivation even when modifications to the standard technique are introduced.