Among several suggested new processes for treating marginal ores in the United States, none is more intriguing than the possibility of extracting metals from low-grade materials by the action of microorganisms. One phase of a Federal Bureau of Mines investigation toward developing such a procedure was to isolate and identify specific chemosynthetic iron- and sulfur-oxidizing autotrophic bacteria native to certain domestic copper deposits where leaching is practiced. The bacteria or microorganisms considered important to this investigation are those capable of producing solutions of ferric sulfate and sulfuric acid. Solutions of this type are excellent solvents for some copper minerals. By establishing the presence of these bacteria at certain copper-leaching areas, strength would be given to a theory that microbial activity is partly responsible for the oxidation that takes place at these areas. Their presence also advances possibilities for controlled use of microbial action in metal extraction processes. Leathen has demonstrated that one of the chemosynthetic iron-oxidizing bacteria, Ferrobacillus ferrooxidans, can oxidize 200 parts per million of ferrous iron to the ferric state in 3 days, whereas atmospheric oxidation requires 2 years, Zimmerley and colleagues have been granted a patent that cites the iron-oxidizing Thiobacillus ferrooxidans as occurring generally in mine waters; the bacteria thrive in acid solutions with high concentrations of metallic ions, can acquire high tolerance to such ions by cultivation in successively more concentrated solutions; and can be used in a cyclic process for extracting metals from metallurgical minerals.
The objective of this report was to establish the presence of iron- and sulfur-oxidizing bacteria in leaching waters at several mining areas where copper leaching is practiced.
Equipment and Procedure
Sampling, Culturing, and Isolating Bacteria
Water samples were collected from five Arizona copper mines; New Cornelia at Ajo, Inspiration at Inspiration, Miami and Castle Dome at Miami, and Bagdad at Bagdad. Studies on bacteria isolation and identification were conducted on samples from various mine area locations designated as mine effluents, dump effluents precipitating launder effluents, and reservoirs.
Special precautions were taken in collecting samples to provide oxygen for aerobic bacteria and prevent contamination. Sample containers (750-milliliter plasma bottles) were thoroughly rinsed inside with three fillings of water from the sampling area. The bottles were then half-filled with sample, stoppered, and airmailed directly to the laboratory. The time of transit was 2 to 5 days. The following precautionary step was taken to assure the delivery of live bacteria to the laboratory: 1-milliliter portions of the samples being collected were inoculated in the field into capped culture tubes containing sterilized media. These were transported to the laboratory in a small portable incubator maintained at 30° C. They were 14 to 16 days in transit. As this investigation was concerned primarily with isolating and identifying chemosynthetic iron- and sulfur-oxidizing autotrophic bacteria, only a few selective media were used. Procedures for preparing the media are described in the appendix.
Air-mailed samples were examined macroscopically and microscopically as soon as they were delivered to the laboratory, and 20-millilter portions of each sample were analyzed for copper, ferrous iron, and ferric iron.
The isolation of chemosynthetic iron- and sulfur-oxidizing autotrophic bacteria was accomplished by inoculating selective media I and II with 5-milliliter portions of the air-mailed water samples; 1-milliliter portions of the portable incubator-samples were inoculated into the media containing the corresponding air-mailed sample. When growth was visually evident in the liquid medium, usually after 10 to 20 days’ incubation at 25° C., a 5-millimeter loop was dipped into the culture and streaked on an appropriate solid medium. Media I and IV were used for isolating sulfur-oxidizing bacteria, and media II and V were used for isolating the iron-oxidizing autotrophs. After incubating the plates for 7 to 20 days at 25° C, isolated colonies were transferred to fresh liquid media. When growth developed, a second streak plate was prepared. The development of morphologically-similar colonies signified the completion of the isolation step.
This investigation is concerned with the morphologically-similar bacteria; Th. thiooxidans, Th. concretivorus, Th. neopolitanus, Th. ferrooxidans, and F. ferrooxidans. The main distinguishing characteristics are that Th. thiooxidans will oxidize elemental sulfur and thiosulfate to sulfuric acid while using ammonium ions as a source of nitrogen, but will neither oxidize ferrous iron nor utilize nitrates; Th. concretivorus is similar to Th. thiooxidans, except it will use nitrates as well as ammonium ions as a nitrogen source but not nitrites; Th. neopolitanus is similar to Th. concretivorus, except it will use nitrites as well as nitrates and ammonium ions as a nitrogen source; F. ferrooxidans will oxidize ferrous iron in an acid environment (pH 3.5-2.0) to ferric iron, but will not oxidize elemental sulfur or thiosulfate; the Th. ferrooxidans will oxidize both ferrous iron to ferric iron and thiosulfate to sulfuric acid (elemental sulfur is not appreciably oxidized);
A partial identification was made by considering the energy requirements of these chemosynthetic iron- and sulfur-oxidizing autotrophic bacteria. Isolated sulfur-oxidizing microbes were tested for their ability to utilize iron by inoculating medium II (ferrous iron medium), and isolated iron-oxidizing microbes were inoculated into medium I (sulfur medium) to test for sulfur utilization. Those bacteria which oxidized both iron and sulfur were designated as Th. ferrooxidans. The bacteria which utilized only iron were identified as F. ferrooxidans.
The sulfur-oxidizing bacteria that did not oxidize iron required another step, which entailed successive transfers into media VI and VII, to determine if nitrate and/or nitrite ions could be used as a nitrogen source, further classifying the sulfur- oxidizing bacteria Into their respective species. Medium III was also inoculated to test for thiosulfate utilization.
Experimental Results and Conclusions
Macroscopic examinations of the field water samples revealed a wide range of colors (pale yellow to deep blue) commonly associated with the effluent waters of copper ore deposits containing iron sulfide minerals. All of the solutions contained precipitates except those collected from mine effluents. Microscopic examinations, using a hanging-drop technique, showed the bacteria as short, motile rods. The solutions from which chemosynthetic iron- and sulfur-oxidizing autotrophic bacteria were isolated ranged from 0.010 to 5.645 grams of iron per liter and from 0.001 to 3.865 grams of copper per liter.
The autotrophic bacteria identified were Th. concretivorus, F. ferrooxidans, and a bacterium closely resembling Th. ferrooxidans. Th. thiooxidans and Th. neopolitanus were not found in any of the samples examined. Table 1 lists the four areas and five mines under investigation and the corresponding bacteria isolated and identified from these localities. Each plus sign on the table represents a single set of isolations for a specific bacterium. The morphological and physiological characteristics of the bacteria isolated and identified are reported in table 2.
According to Bergey’s Manual Th. ferrooxidans does not appreciably use elemental sulfur. Of particular interest was the fact that the bacterium that so closely resembled Th. ferrooxidans in this investigation readily oxidized elemental sulfur, but developed only slowly in a thiosulfate medium. This minor discrepancy would tend to suggest the isolation of a new Th. ferrooxidans strain, which closely resembles the bacterium isolated by L. C. Bryner from mine waters in Utah and Mexico.
Three strains of the bacteria isolated and identified in this Report of Investigations have been placed with the American Type Culture Collection, Washington, D. C. These bacteria and their ATCC accession numbers are as follows: Th. concretivorus No. 13730, Th. ferrooxidans No. 13728, and F. ferrooxidans No. 13729.
Based on the results of this investigation, Th. concretivorus and Th. ferrooxidans appear to be common to waters associated with Arizona copper deposits, but F. ferrooxidans does not appear to be common to these areas.
Preparation of Culture Media— Medium I
One gram of precipitated sulfur was placed in each of 10 dry 250-millilitre Erlenmeyer flasks. The remaining constituents were dissolved in one liter of tap water and 100-millilitre portions carefully transferred to the flasks by allowing the solution to flow down the side, thereby floating the sulfur on top. The medium was sterilized in free flowing steam for one-half- hour on 3 consecutive days.
When the medium was to be used in the field, the following preparation was employed. The constituents were dissolved in tap water and transferred in 10-millilitre portions into capped culture tubes which were then sterilized in an autoclave for 15 minutes at 121° C. (15 pounds per square inch). Portions of precipitated sulfur weighing 0.1 gram each were transferred to capped culture tubes and sterilized in free flowing steam for one-half hour on 3 consecutive days. Before inoculating the medium in the field with the water samples, the sulfur was floated by aseptically transferring the sterile solution into the culture tube containing the sulfur.
The constituents of solution A were dissolved in a liter of distilled water, 100-milliliter portions were transferred into 250-milliliter Erlenmeyer flasks and sterilized in an autoclave for 20 minutes at 121° C. (15 pounds per square inch). Solution B was prepared separately, then sterilized by passing it through a Seitz filter. When not in use, solution B was frozen to reduce air oxidation of the ferrous sulfate. One milliliter of solution B was aseptically placed in 100 milliliters of solution A before inoculation.
When the medium was to be used in the field, the following preparation was employed. The constituents of solution A were dissolved in a liter of distilled water, then transferred in 10-milliliter portions into capped culture tubes and sterilized in an autoclave for 15 minutes at 121° C. (15 pounds per square inch). One-gram portions of FeSO4·7H20 were placed in capped culture tubes and carried into the field. Before inoculating this medium, a 10-milliliter portion of sterile distilled water was added aseptically to the culture tube containing one gram of FeSO4·7H2O and thoroughly mixed. One-tenth milliliter portions of this 10-percent ferrous sulfate solution were then aseptically transferred to the 10-milliliter portions of solution A. Tubes of this medium that were carried as controls did not become contaminated.
Solutions A, B, and C were prepared separately. Solution A was transferred in 90-milliliter portions into 250-milliliter Erlenmeyer flasks. The solutions were sterilized in an autoclave for 20 minutes at 121° C. (15 pounds per square inch). Before inoculating this medium, 5-milliliter portions of solutions B and C were aseptically transferred to the flasks containing solution A.
The constituents were dissolved in tap water, to which agar was then added and dissolved by heating the solution in a water bath. Medium IV was sterilized for one-half hour in an autoclave at 121° C. (15 pounds per square inch). The resulting medium could be stored for relatively long periods of time. When needed, the agar was dissolved by heating in a water bath, the solution aseptically transferred into sterile petri dishes, and the medium allowed to solidify.
Constituents of solution A were dissolved and then transferred in 25-milliliter portions into 125-milliliter Erlenmeyer flasks. This solution was sterilized in an autoclave for 15 minutes at 121° C. (15 pounds per square inch). Solution B was autoclaved as above, while solution C was sterilized by passing it through a Seitz filter.
Solution D was passed through an ion exchange column. The column consisted of glass tubing 2 inches in diameter and approximately 20 inches long; 500 grams of analytical grade “Amberlite” IR-120 (H) were placed in the column. The solution was passed through the column at approximately 20-30 milliliters per minute. Only the fraction of the silicic acid which had a pH reading of 2.0 was used. A rapid rise in the pH of the solution coming out of the column indicated exhaustion of the resin. The column was then rinsed with distilled water until it was free of any precipitate, and then with 1,500 milliliters of a 10-percent solution of hydrochloric acid. The resin was freed of chloride ions by back washing with distilled water.
Aseptically 1 milliliter of solution C was added to 75 milliliters of solution D, and 1 milliliter of solution B was added to 25 milliliters of solution A. The two resulting solutions were combined, thoroughly mixed, and poured into sterile petri dishes. A suitable gel usually formed in 24 hours. Control plates remained uncontaminated.
Media VI and VII
Media VI and VII were prepared in the same way as Medium I.