The cells of 'Thiobacillus ferrooxidans were rapidly adsorbed on the solid surfaces of an agitated flask containing 1 % pulp density of pyrite particles. More than ca. 99 % of the inoculated cells were adsorbed. However, considerably fewer cells were adsorbed on pyrite particles than on the glass wall of the flask. Scanning electron microscope observation revealed that T. ferrooxidans cells were adsorbed aggregatively on restricted areas of the pyrite particles. The surfaces of the pyrite particles were characteristically eroded to show etched polyhedral pits, but without prominent cell adsorption of the extensively eroded surfaces during markedly enhanced leaching. When T. ferrooxidans cells were adsorbed on the solid surfaces, the iron-oxidizing activity of the bacteria was strongly inhibited, resulting in a failure to enhance pyrite oxidation. Adsorbed cells did not proliferate on the solid surfaces. When the adsorbed cells were released into an aqueous phase by the addition of the surface active agent Tween 20, the bacterial iron-oxidizing activity inhibited by adsorption was recovered and, as a result, pyrite oxidation was markedly promoted. Significant enhancement of pyrite oxidation by T, ferrooxidans was ascribed to the development and iron-oxidizing activity of the freely dispersed cells in an aqueous phase. The function of the surface active agent is to prevent tenacious adsorption of the bacterial cells to solid surfaces and organic substances examined, such as protein, nucleic acid, yeast extract, peptone, and cell free extracts, operate in the same way as the surface active agent. The enhancement of the bacterial pyrite oxidation by the intact cells of Thiobacillus thiooxidans is thought to be attributable to the organic substances excreted from T. thiooxidans cells and/or to the exchange adsorption of cells of both thiobacilli. The present results indicate that bacterial concentration 63

Fundamental investigations were made on the oxidation of pyrite and consequent dissolution of iron by pure and mixed cultures of Thiobacillus ferrooxidans and Thiobacillus thiooxidans. The release of iron from pyrite was remarkably enhanced with large inocula above ca. 109 cells per flask (or 1 % pulp density) of 2-day (active) cultures of T. ferrooxidans, but not with inoculum of 108 cells or less. Furthermore, a phenomenon was observed that the enhanced oxidation of pyrite always proceeded with the coexistence of a 108 cells or less inoculum of T. ferrooxidans and T. thiooxidans incapable of oxidizing pyrite. During the bacterial oxidation of pyrite, high iron oxidation ratios (Fe3+/total Fe) above 90% were maintained, coincident with the enhanced release of iron from pyrite. Contrarily, in the absence of T. ferrooxidans, a major portion of iron was in the ferrous form and iron release was not promoted. Thus, it was thought that T. ferrooxidans contributes to the oxidation of pyrite through the regeneration reaction of ferric iron.The bacterially assisted oxidation of insoluble sulfide minerals has been studied by many investigators using the acidophilic, chemolithotrophic, iron-oxidizing bacteria Thiobacillus ferrooxidans and related species (1-4). Two main mechanisms of bacterial attack on sulfide minerals, indirect and direct oxidation mechanisms, have been proposed (3)(4)(5)(6)(7)(8).In the indirect oxidation mechanism, sulfide minerals are oxidized chemically by ferric iron serving as an effective oxidizing agent, and the bacteria are involved in the oxidation of ferrous iron formed to the ferric state (3, 4). In the direct oxidation mechanism, sulfide minerals are oxidized biochemically by the close contacts between the bacterial cells and solid substrates, 331

The role of Thiobacillus thiooxidans was investigated in the enhancement of pyrite oxidation by the mixtures of Thiobacillus ferrooxidans and T. thiooxidans. The oxidation of pyrite by T. ferrooxidans was remarkably enhanced by the addition of dead cell suspensions and cell-free extracts of both chemolithotrophic thiobacilli and heterotrophic bacteria. The same promoting effect on the bacterial pyrite oxidation was recognized with various substances such as protein, nucleic acid, yeast extract, peptone, and also surface-active agent. These results indicate that the enhancement of bacterial pyrite oxidation by the intact cells of T. thiooxidans is attributed at least partly to the organic substances excreted from T. thiooxidans cells into the pyrite medium, and that cellular organic substances have a physico-chemical effect on a direct interaction between iron-oxidizing bacteria and solid particles.In a previous paper (1), we reported that the oxidation of pyrite and consequent dissolution of iron was markedly accelerated by adding sufficient numbers of Thiobacillus ferrooxidans of above ca. 109 cells per flask (50 ml of pyrite medium, 1 % pulp density), below which the pyrite oxidation did not proceeded. However, the oxidation of pyrite was always enhanced by the mixed addition of T. ferrooxidans and Thiobacillus thiooxidans even if the inoculum cell size of T. ferrooxidans was below the critical cell number. In this paper, to define the contribution of T. thiooxidans in the acceleration of pyrite oxidation by the mixtures of T. ferrooxidans and T. thiooxidans, we studied the effects of dead cell suspension and cell-free extracts of both acidophilic thiobacilli and other heterotrophic bacteria, and of various organic substances and surface-active agents on the release of iron from pyrite by T. ferrooxidans.

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