Culture-dependent and culture-independent approaches to study the bacterial and archaeal diversity from Jordanian hot springs

The microbial diversity was investigated at five major hot springs in Jordan (Ashounah, Waggas, Zara, Zarqa Ma'in and Afra springs) using both microbiological culture-based and molecular culture-independent approaches. One hundred and thirty two (132) bacterial isolates were obtained and characterized morphologically. Out of 132 isolates, 125 isolates were gram positive rods, while the other seven isolates were gram positive coccobacilli. The bacterial growth at different high temperatures was determined, and revealed that 19 out of 132 isolates were able to grow at high temperatures of up to 75 degrees C. All bacterial isolates grew at pH 7.0, except one single isolate from Zarqa Ma'in spring which was able to grow at pH 3.5. Culture-independent approaches using polymerase chain reaction (PCR) was used for the amplification of specific 16S rDNA sequences of Bacteria, Archaea, Green sulfur bacteria, Green nonsulfur bacteria, Heliobacteria, and methanogenic Archaea from metagenomic DNA extracted directly from water and mat samples from each thermal spring. Nine water samples and 9 mat samples from all spring sites revealed the presence of bacteria yielding the amplicon size of (1500 bp), while 4 water samples from Zarqa Ma'in springs and 5 mat samples from Zarqa Ma'in springs and Waggas well revealed the presence of archaea with an amplicon size of (650 bp). When the primer pair targeting purple phototrophic bacteria was used, positive PCR amplification product (229 bp) appeared in 6 mat samples from Zarqa Ma'in, Afra, Zara, Ashounah and Waggas springs. Also PCR identification of bacterial isolates using primer pair specific to the 16S rDNA gene sequences of the genus Bacillus indicated that 96.97% (128 out of 132) of bacterial isolates have the size of PCR amplicon (320 bp) for the genus Bacillus.