Tinto river (Huelva, Spain) is a natural acidic rock drainage (ARD) environment produced by the bio-oxidation of metallic sulfides from the Iberian Pyritic Belt. So far, and despite their ecological interest, the underlying sediments were studied only very sparsely and no complete studies were undertaken. In this thesis, an extensive survey of the Tinto River anaerobic sediment microbiota is presented combining culture independent and dependent methods following the “full-cycle rRNA approach”.
A geomicrobiological model of the different microbial cycles operating in the sediments has been developed through molecular biological methods. Culture independent methods targeting the small subunit ribosomal RNA (SSU rRNA) such as denaturing gradient gel electrophoresis (DGGE), 16S rRNA gene sequencing (cloning) and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) were used. Microorganisms involved in the iron (Acidithiobacillus ferrooxidans, Sulfobacillus spp., etc.), sulfur (Desulfurella spp., Desulfosporosinus spp., Thermodesulfobium spp., etc.), carbon (Acidiphilium spp., Bacillus spp.,Clostridium spp., Acidobacterium spp., etc.) and nitrogen (Alcaligenes spp., Pseudochrobactrum spp., etc.) cycles were identified and their distribution correlated with physicochemical parameters of the sediments. Where the pH and redox potential are closer to those of the water column (pH 2.5 and +400 mV), the most abundant organisms were identified as iron-reducing bacteria: Acidithiobacillus spp. and Acidiphilium spp., probably related to the higher iron solubility at low pH. At higher pH (4.2-6.2) and more reducing redox potential (50, -210 mV) and therefore, lower solubility of iron, members of the sulfate-reducing genera Syntrophobacter, Desulfosporosinus and Desulfurella were dominant. The design of a specific probe (DSU655) targeting sulfur-reducing organisms belonging to Desulfurella genus was conclusive to the work.
Additionally, we used targeted enrichment incubations to validate this model and prove the existence of the potential anaerobic activities detected in the acidic sediments of Tinto River. Methanogenic, sulfate-reducing, denitrifying, iron-reducing and hydrogen-producing enrichments yield positive results. Classical techniques for bacterial isolation were applied and some microorganisms were isolated such as acidophilic sulfate-reducing bacteria (two new species and a new genus, related to the Desulfosporosinus/Desulfitobacterium cluster), fermenters (a new genus, closed related to Paludibacter) and hydrogen-producers.
Finally, a biotechnological application using Tinto river sediments was tested. Bioremediation of acid rock drainage (ARD) with sulfate-reducing bacteria using domestic wastewater (DW) as a cost-effective carbon-source showed a complete cleaning of the ARD water obtaining an effluent with neutral pH and no metal content.