There are no reports about the microbial diversity in the intertidal zone in the acidic ecosystem of RíoTinto (Huelva, Spain). In this study, we found San Juan del Puerto to be the farthest spot from the river mouth along the river in which environmental and geochemical conditions change together with the tidal cycle twice a day. Seasonal bacterial diversity has been studied during one year with maximal and minimum tidal difference and it has been compared with the sampling spot of La Rábida, which is in the mouth of the river. Acidophilic microorganisms have been found in the low tide samples like Acidocella, Acidobacterium, Acidiphilium and Ferromicrobium, genera. Iron oxidizing denitrifying Acidovorax genera has been found in different water samples in this study. Some genera form oceanic samples has been found in low tide samples, likeArcobacter, Methylophaga, Mesorhizobium, Marinobacter, Flavobacterium and Pseudomonas. Likewise, Marinomonas, Limnobacter and Rhodobacter. genera have not been found in these samples. A correspondence analysis (PCA and CCA) has been performed integrating biological diversity results with physical-chemical measurements: it shows high correlation between acidophilic microorganisms, low tide samples and heavy metal presence. High tide samples, even if more dispersed, reveals a clear oceanic influence showing a certain correlation with high conductivity, salinity, pH and chlorine concentration.
From high and low tide samples in winter and spring, and from low tide sample in autumn in San Juan del Puerto, six microalgae isolates have been obtained and named as: SJP_EBP_1, SJP_EAP_2, SJP_EBO_3, SJP_EBO_4, SJP_EBI_52, cultured in BG11 medium (pH7 and pH4) and SJP_EAI_6 in L1 medium at pH7. In order to characterize these microalgae, 18S rRNA has been sequenced and analysed. They were identified as five chlorophytes and a rhodophyte: Parachlorella sp., Trebouxiophyaceae sp., Tetradesmus obliquus, Coelastrella sp., Stichococcus bacillaris and Porphyridium sp.
Two possible biotechnological applications have been studied in this work: on one side their potential in removing metal pollution from aqueous solution and on the other their capability to synthetize silver nanoparticles.
The tolerance assay on different concentrations of cadmium, copper, iron and lead revealed that Trebouxyphiaceae sp. and Porphyridium sp. show a better tolerance to cadmium, Coelastrella sp. and Porphyridium sp. to copper, S. bacillaris to iron and Trebouxiophyaceae sp. to lead. By optical microscopy, morphometric variations due to the metal exposure have been detected as well as an extracellular mucilage in the majority of the samples that were not observed in controls. Adsorption of cadmium and lead has been observed in Parachlorella sp. and Coelastrella sp cultivated at pH7, of iron in Parachlorella sp. and T. obliquus cultivated a pH7 and in Coelastrella sp. culture at pH4 and pH7.
The chlorophytes isolated in this work showed, at both pH4 and pH7, the ability to produce extracellularly silver nanoparticles. Their antibacterial activities have been studied on Gram negative Escherichia coli ATCC10536, Klebsiella pneumoniae ATCC29665 and Gram positive Staphilococcus epidermidis ATCC6538P. The study of the kinetics of production and of the stability of the nanoparticles has been carried out depending on the phase of growth of the microalga culture used. The nanoparticles produced in stationary phase presented greater stability in all the cases. The antibacterial activity of the nanoparticles synthesized from the isolated microalgae was higher compared to S. epidermidis than to E. coli and K. pneumoniae being the nanoparticles produced from the isolates Parachlorella sp. and Trebouxiophyaceae sp. the ones that showed greater activity against the three bacteria used.