Determination of sulfur and iron oxidation bacteria by the Most Probable Number (MPN) technique

The number and the activity of acidophilic bacteria present in bioleaching processes is one of the most important parameters to understand the behaviour of a leaching process. The most frequently isolated species from these ecosystems are strains of Thiobacillus ferrooxidans, Leptospirillum ferrooxidans and Thiobacillus thiooxidans. A widely recognized difficulty in quantifying these autotrophic bacteria is problems with growth on solid medium. This work presents a modified methodology of the Most Probable Number technique (MPN) to enumerate the active bacterial populations with the capacity to oxidize Fe(II) and reduced inorganic sulfur compounds. Ln both cases, specific reagents were used to read the positive and negative tubes. The presence of ferric ion in the cultures of ferrous iron oxidizing bacteria was detected using 1% solution of potassium thiocyanate. For the sulfur oxidizing bacteria, the sulphuric acid generation was determined with 0.1% thymol blue solution. The methods were developed using pure cultures of Thiobacillus ferrooxidans growing on ferrous sulfate or in sodium tetrathionate solution; a culture of Leptospirillum ferrooxidans was also utilized to set up the technique for ferrous iron oxidizing bacteria. Later the MPN tecniques were utilized to estimate the active populations of ferrous and sulfur oxidizing bacteria present in samples of pregnant solutions of the copper mineral bioleaching operations in the north of Chile.