A survey of the alkalithermophilic prokaryotic diversity from the hot spring waters in Coamo Puerto Rico

Thermal springs are high temperature aquatic environments. The prokaryotic diversity in these environments has been studied world wide. However, there are few studies on the prokaryotic diversity in thermal springs in Puerto Rico. The main objective of this research was to isolate and characterize alkalithermophilic bacteria from the main thermal spring in Coamo Puerto Rico. To determine the culturable diversity of these bacteria, thermal spring water samples were filtered, plated into Thermus medium (TM) and Alkaline Yeast Extract Malt Medium (AYEMM) and incubated at 70°C. Selected isolates were characterized by standard microbiological techniques, by scanning electron microscopy, and by growing at optimal pH and different temperatures. Molecular analysis using Restriction Fragment Length Polymorphism (RFLP) patterns of a Polymerase Chain Reaction (PCR) amplicon containing the 16S rDNA region was also used to classify the isolates. Taxonomic results indicated that the isolates belonged to the Bacteria Domain. All the 185 strains recovered represented the spore forming genus Geobacillus. This genus has been reported from other hot springs and during this study it was recovered recurrently and was the most abundant in all the three sampling sites of the spring. Many of these isolates showed a wide range of growth at various temperatures and pH values. The ability of these isolates to grow at high temperatures might indicate that they represent the extremophilic diversity of the thermal springs. The Bacterial diversity was also analyzed by culture independent methods like 16S rDNA environmental clone libraries and Terminal Restriction Fragment Length Polymorphism (TRFLP). Clone libraries were very difficult to achieve due to the low diversity present in the community and chemical complexity of the sample. However, after protocol optimization some clones were obtained and in silico analysis was performed. Environmental sequences belonging to the beta-Proteobacteria were the most frequent Operational Taxonomic Units (OTUs), but some OTUs were closely related to members of the Bacteroidetes and gamma-Proteobacteria. In addition, some cyanobacterial OTUs were obtained from this environment. TRFLP patterns were also used to study structure and diversity of the 16S rDNA microbial community. The profiles obtained with different enzymes were similar among all the springs, suggesting that the community structure was very homogeneous throughout the sampling area. The combination of morphological, physiological and molecular approaches was very useful to describe the bacterial community present at the thermal spring of Coamo Puerto Rico.