Enumeration and characterization of acidophilic microorganisms isolated from a pilot plant stirred-tank bioleaching operation

Abstract

Microorganisms were enumerated and isolated on selective solid media from a pilot-scale stirred-tank bioleaching operation in which a polymetallic sulfide concentrate was subjected to biologically accelerated oxidation at 45 degrees C. Four distinct prokaryotes were isolated: three bacteria (an Acidithiobacillus caldus-like organism, a thermophilic Leptospirillum sp., and a Sulfobacillus sp.) and one archaeon (a Ferroplasma-like isolate). The relative numbers of these prokaryotes changed in the three reactors sampled, and the Ferroplasma isolate became increasingly dominant as mineral oxidation progressed, eventually accounting for >99% of plate isolates in the third of three in-line reactors. The identities of the isolates were confirmed by analyses of their 16S rRNA genes, and some key physiological traits (e.g., oxidation of iron and/or sulfur and autotrophy or heterotrophy) were examined. More detailed studies were carried out with the Leptospirillum and Ferroplasma isolates. The data presented here represent the first quantitative study of the microorganisms in a metal leaching situation and confirm that mixed cultures of iron- and sulfur-oxidizing prokaryotic acidophiles catalyze the accelerated dissolution of sulfidic minerals in industrial tank bioleaching operations. The results show that indigenous acidophilic microbial populations change as mineral dissolution becomes more extensive.

FIG. 1.

Neighbor-joining tree showing the relationship of the isolates from the Mintek biooxidation plant to other iron- and sulfur-oxidizing acidophiles. The topology of this tree is supported by DNA parsimony analysis. Scale bar = 0.1 nucleotide change per 100 for the horizontal branch lengths. S., Sulfolobus; Sb., Sulfobacillus.

FIG. 2.

Plate counts for acidophiles in each of the three in-line bioreactors. The microbes were grouped and tentatively identified by using the criteria shown in Tables 2 and 3.

FIG. 3.

Effects of temperature (a) and pH (b) on the growth of Leptospirillum strain MT6 (•) and Ferroplasma strain MT17 (▴). Each data point is the mean for two analyses (variation, <10%).

FIG. 4.

Growth (▪ and □) and ferrous iron oxidation (• and ○) by Ferroplasma strain MT17 at 45°C (• and ▪) and at 50°C (○ and □). Cultures were grown under controlled conditions (at pH 1.5) in a 2-liter bioreactor. OD (660 nm), optical density at 660 nm.

FIG. 5.

Leaching of pyrite by Leptospirillum strain MT6 (○) and by Ferroplasma strain MT17 (▵) in pyrite-basal salts medium and by Ferroplasma strain MT17 in the same medium amended with 0.02% (wt/vol) yeast extract (▴). The data points are the means for triplicate cultures, and the standard deviations were generally <5%. The arrow indicates the point at which additional yeast extract (0.02%, wt/vol) was added to the yeast extract-containing Ferroplasma cultures.

FIG. 6.

Growth, as measured by optical density at 600 nm [OD (600 nm)] (○ and ▵), and oxidation of tetrathionate (• and ▴) by Ferroplasma isolates MT16 (○ and •) and MT17 (▵ and ▴).