Heavy metals resistance in eukaryotic microorganisms isolated from sediments of an abandoned gold mine in San Luis, Argentina

BONILLA JO; DELLA VEDOVA M CECILIA; LAPADULA WALTER J; CASTRO MF; GIL, RAÚL A.; VILLEGAS LB

San Luis

Congreso; XIII Congreso Argentino de Microbiología General SAMIGE; 2018

SAMIGE

In San Luis, Argentina, the acid mine drainage of an abandoned gold mine is released into La Carolina stream. In previous studies we demonstrated that the drainage influences on the physicochemical parameters of the stream sediments and on the microbial community structure. The aim of this work was to isolate and characterize microorganisms heavy metals-tolerant from sediments of this acid mine drainage-affected environment and to investigate the ability of those isolates to remove heavy metals from the culture medium.A total of 28 sediment samples were taken from inside the mine and from La Carolina stream bed. Sediments were used for a sequential isolation process in the presence of Cu(II), Fe(II) and Cr(VI) as selection pressure, using EG* medium (g L-1): glucose 10.0; yeast extract 1.0; K2HPO4 0.5 and KH2PO4 0.5. Isolated microorganisms were then subjected to qualitative tolerance tests on solid medium. The resistant microorganisms were identified by PCR amplification with universal primers and 18S rRNA gene sequencing. The Minimum Inhibitory Concentration (MIC) was determined in liquid culture medium for each metal. Growth performance and heavy metal toxicity for the isolates were evaluated. Heavy metal concentrations were determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) in cell-free supernatants obtained during 120h and in acid digested cells, in order to determine the intracellular metal concentration.After the isolation processes, three microorganisms were isolated and identified as: Fusarium sp. M6 (NCBI Accession number KY596033); Pochonia sp. M8 (NCBI Accession number KY596046); Apiotrichum loubieri M12 (NCBI Accession number KY596699). MIC values obtained were (µg L-1): 125 (Fe), 250 (Cr) and 90 (Cu) for Fusarium sp. M6; 250 (Fe), 45 (Cr) and 90 (Cu) for Pochonia sp. M8; and 375 (Fe), 60 (Cr) and 90 (Cu) for A. loubieri M12. None of the three microorganisms showed removal capability of Fe(II) or Cr(VI) from the culture media. Consequently, the growth and removal capability of microorganisms were evaluated in the presence and absence of 35 µg L-1 Cu(II). Fusarium sp. M6 and A. loubieri M12 showed a similar behavior, since they showed a growth inhibition around 60% in the presence of the metal. Both microorganisms showed a Cu(II) removal capability between 30 and 35%. Pochonia sp. M8 was the most affected one by the heavy metal presence. These results were compared with Saccharomyces cerevisiae ATCC 32051. This strain showed MIC values of 60 µg L-1 of Cu and its growth was affected by around 70% and the metal removal capacity was around 15% showing a similar behavior with Pochonia sp. M8.The selected microorganisms were obtained from sediments with low pH values and the highest heavy metal concentrations. These extreme environments allow us to isolate microorganisms with heavy metal resistance phenotype for their subsequent metalloproteomics studies and to compare their metalloprotein profiles with S. cerevisiae