The Naica Underground System (NUS) in Northern Mexico comprises a lead, zinc and silver producing mine and displays the largest gypsum crystals ever found in natural caves. Besides its geological, economical and touristic importance, the bacterial diversity in the NUS has not been fully explored yet. The caves are now close to the public and mining activities have been suspended for an undefined period since October 2015. Here, we surveyed for bacteria present on different mineral substrates, gypsum crystals, iron oxide crusts, and groundwater samples collected before the NUS was inaccessible using culture-dependent and culture–independent (PCR-DGGE) methods. Cluster analysis of DGGE fingerprints revealed a correlation between communities from either caves or tunnels and according to their substrate type. Both approaches revealed the presence of bacteria from the Firmicutes, Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria in gypsum crystals, oxide crusts, and groundwater, the Deinococcus-Thermus and Actinobacteria were only detected by DGGE in groundwater samples. Heterotrophic bacteria were isolated under aerobic conditions and phylogenetic analysis showed that they were affiliated with Bacillus, Brevibacillus, Paenibacillus, Schlegelella, Cupriavidus, Pseudoxanthomonas and Lysobacter. Some of these isolates were able to precipitate iron or calcium in solid media but their possible participation in biomineralization processes in the NUS has to be investigated. This study is the first reporting the isolation of microorganisms from Naica. Most of the isolates and sequences retrieved by DGGE were closely related to organisms previously detected in other extreme subsurface environments. The detected microflora is likely autochtonous but some may be allochtonous and the role of these bacteria in geobiological processes in the NUS requires further investigation.
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