Copper Sulfide Precipitation by Yeasts from

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1 APPLIED MICROBIOLOGY, Jan., 1967, p Copyright 1967 American Society for Microbiology Vol. 15, No. 1 Printed in U.S.A. Copper Sulfide Precipitation by Yeasts from Acid Mine-Waters H. L. EHRLICH AND SALLY I. FOX Department of Biology, Rensselaer Polytechnic Institute, Troy, New York Received for publication 19 August 1966 ABSTRACT Two strains of Rhodotorula and one of Trichosporon precipitated dissolved copper with H2S formed by reducing elemental sulfur with glucose. Iron stimulated this activity under certain conditions. In the case of Rhodotorula strain L, iron stimulated copper precipitation aerobically at a copper concentration of 18 but not 180,g/ml. Anaerobically, the L strain required iron for precipitation of copper from a medium with 180,ug of copper per ml. Rhodotorula strain L was able to precipitate about five times as much copper anaerobically as aerobically. The precipitated copper was identified as copper sulfide, but its exact composition could not be ascertained. Iron was not precipitated by the H2S formed by any of the yeasts. Added as ferric iron, it was able to redissolve copper sulfide formed aerobically by Rhodotorula strain L from 18 but not 180 ug of copper per ml of medium. Since the yeasts were derived from acid mine-waters, their ability to precipitate copper may be of geomicrobial importance. The presence of yeasts and other fungi in acid mine-waters has been reported by workers from various parts of the world (6). The consensus has been that the life processes of these organisms are not important in influencing the transformation of mineral sulfides. Marchlewitz and Schwartz (5) reported minor effects of fungi on acid-formation by thiobacilli in acid mine-waters. According to their findings, Rhodotorula glutinis and R. rubra accelerated acid formation by Thiobacillus ferrooxidans and T. thiooxidans. Spicaria divaricula and Penicillium waksmani retarded acid-formation by T. ferrooxidans. This paper shows that, under appropriate conditions, at least some yeasts from acid mine-waters can precipitate dissolved copper with H2S which they produce from elemental sulfur. MATERIALS AND METHODS Cultures. Two Rhodotorula cultures and a Trichosporon culture were used. They were isolated from acid mine-waters at two different sites in Arizona. One of the Rhodotorula cultures is Rhodotorula glutinisgraminis. The other Rhodotorula culture is designated only as strain L, its specific identity being unknown. It consists of predominantly oval budding cells which form occasional true mycelium. The yeast forms a wrinkled colony which develops a pinkish carotenoid pigment. Most of the work in this paper utilized this strain. Media. A mineral salts solution, modified 9K medium (7), was prepared with the following ingredients (grams per liter): (NH)2SO, 6; KCI, 0.2; K2HPO, 1.0; MgSO 7H20, 1.0; and Ca(NO)2, 0.02; the ph was adjusted to.5. Of this solution, 25 ml was added to each of a series of 250-ml Erlenmeyer flasks, each of which contained 1 g of precipitated sulfur. The contents of these flasks were then sterilized by tyndallization. After sterilization, each flask received 25 ml of a sterile 20% glucose solution and 1 ml of sterile 0.2% anhydrous CUSO solution. Some flasks also received 0.96% sterile FeNH(SO)2 12H20 solution; those 15 which did not received 1 ml of sterile distilled water instead. The inoculum consisted of 1 ml of washed yeast cells prepared as given below. Uninoculated flasks received 1 ml of sterile mineral solution in place of the inoculum. Inoculum preparation. Inocula for all experiments were grown in 20 ml of iron-free 9K medium (7) containing 1% glucose at an initial ph of.5. Cells were harvested by centrifugation after 2 to of incubation at 0 C and were resuspended in glucose-free mineral solution. The final yeast suspensions contained from 105 to 107 cells per milliliter. Anaerobic incubation. To create an anaerobic environment, a vacuum desiccator was used. For fully anaerobic conditions, the internal air was replaced by "high-purity" nitrogen (Union Carbide Corp., Linde Div., New York, N.Y.). For a semianaerobic environment, the internal air pressure was lowered to approximately 0.1 atm. Chemical determinations. Copper was determined colorimetrically with 2,2'-biquinoline reagent, and 10% hydroxylamine hydrochloride was used as reduc-

2 16 EHRLICH AND FOX APPL. MICROBIOL. ing agent for cupric copper (). Total iron was determined colorimetrically with o-phenanthroline reagent, and 10% hydroxylamine hydrochloride was used as reducing agent for ferric iron (2). Ferrous iron was determined by the same method, but the hydroxylamine hydrochloride reagent was omitted. Sulfide was qualitatively detectedwithiodine-azide reagent according to Feigl (). The ph values of the media during experiments were measured with Alkacid ph Paper (Fisher Scientific Co., Pittsburgh, Pa.). RESULTS Aerobic copper precipitation. Table 1 summarizes observations made when Rhodotorula strain L was grown aerobically in sulfur-mineral medium containing 10% glucose and 0.05 g of anhydrous CuS0 per liter with and without 0.18 g of FeNH(S0)2.12H20 per liter. The data show that without added iron most of the copper disappeared from solution in 1 and remained out of solution thereafter. The data also show that in the presence of added iron copper disappeared from solution by the 6th day, but returned into solution between the 1th and 27th day. Much of the added iron disappeared rapidly from solution upon addition, presumably because of hydrolysis to ferric hydroxide. This iron reappeared in solution within 6 in inoculated flasks and remained in solution thereafter. It never reappeared in solutions of uninoculated control flasks. Redissolving of the iron is attributed to the drop in ph resulting from yeast growth. It occurred whether or not copper was present in the medium. Sampling time TABLE 1. Some of the sulfur particles in flasks in which copper was precipitated developed a blackish coating, and a dark brown-black sediment accumulated at the bottom of the flasks. Phasecontrast microscopy showed no evidence of dark precipitate inside yeast cells from flasks with precipitated copper. The changes in ph of the medium during growth, noted in Table 1, were indicative of yeast activity. Values for ph as low as 1.0 were measured in some experiments. As the last two columns in Table 1 show, the unidentified acid which caused the ph drop was formed by the yeast in glucose-mineral medium even without added sulfur, copper, or iron, although more slowly than when they were added. Copper precipitation depended upon yeast action on elemental sulfur. In the absence of elemental sulfur, the yeast grew, but no copper was precipitated. In the absence of yeast, the dissolved copper concentration, the dissolved iron concentration (after some inevitable initial precipitation), and the ph remained constant. No dark precipitate formed. Identification of the dark deposit. The dark deposit was found to be soluble in hot concentrated HCI and HN0 with an increase in soluble copper. It did not dissolve in hot concentrated H2S0, dilute HCI, or dilute HN0. These observations are consistent with known properties of copper sulfides. The possibility that the dark precipitate, resulting from yeast activity in coppercontaining media with added iron, was iron Copper precipitation by Rhodotorula strain La Concn (,jg/ml) of Cu in Concn Gag/ml) of Fe in supernatant liquid supematant liquid ph -Fe +Fe -Cu +Cu +Cu, -Fe +Cu, +Fe -Cu, +Fe -S,-Cu, v Inoculum = 2 X 106 cells. Results reported for duplicate flasks. when a particular substance is added and when it is not, respectively. Plus and minus signs indicate

3 VOL. 15, 1967 COPPER SULFIDE PRECIPITATION BY YEASTS 17 sulfide could be ruled out, because all added iron was found in solution by the time most or all of the copper was precipitated. Other observations lending support to the identification of the black precipitate as copper sulfide included the demonstration with iodineazide reagent of H2S formation by the yeast from the interaction between glucose and sulfur. In the absence of sulfur, dissolved copper was not precipitated by the yeast. Lowering the initial glucose concentration from 10 to 1 % slowed the rate of H2S formation, as indicated by slower copper precipitation. The exact composition of the sulfide is unknown, because it could not be isolated and purified for analysis. Activity of other yeasts from acid mine-water. Experiments with R. glutinis-graminis and with Trichosporon species gave results qualitatively similar to Rhodotorula strain L. However, Trichosporon species caused slower precipitation of copper than the two Rhodotorula cultures in a medium containing 10% glucose and 0.05 g of anhydrous CuS0 per liter with and without 0.18 g of FeNH(SO)2-12H20 per liter (Table 2). This slower activity was attributable in part to the growth habit of Trichosporon. It formed mycelial clumps at the surface of the medium and thus resulted in more limited contact with sulfur. Effect of increasing initial copper concentration. Since essentially all of the initially dissolved Sampling time TABLE 2. Copper precipitation by Trichosporona copper in the medium of the previously described experiments was precipitated by the yeasts, it was thought possible that larger amounts of copper might be precipitated by increasing the initial copper concentration from 18 to 180 Ag/ml of medium. However, in 2 of incubation in duplicate flasks, Rhodotorula strain L precipitated only 8 and 20,ug of copper per ml of medium in the absence of iron and 15 and 19 pig of copper per ml of medium containing 0.18 mg of ferric ammonium sulfate per ml. Therefore, increasing the copper concentration of the medium did not increase the amount of copper precipitated aerobically, whether or not iron was present. Indeed, iron did not stimulate copper precipitation in this experiment, nor did it cause extensive redissolving of copper sulfide. Effect of anaerobiosis on copper precipitation. Since the aerobic production of H2S by the yeasts from the interaction between elemental sulfur and glucose suggested that the sulfur could substitute partially for oxygen as terminal electron acceptor, it was thought possible that anaerobiosis might increase H2S production and thereby increase the extent of copper sulfide precipitation. Semianaerobic and anaerobic incubation of Rhodotorula strain L did increase the amount of copper sulfide precipitated from a medium containing 180 ug of dissolved copper per ml (Table ). It is noteworthy that the presence of iron as 0.18 g of FeNH(SO)2* 12H20 per liter was Concn (ug/ml) of Cu in Concn (,g/ml) of Fe in ph supernatant liquid supernatant liquid -Fe +Fe -Cu +Cu +Cu, -Fe +Cu, +Fe -Cu, +Fe a Inoculum = 105 cell clumps. Results reported for duplicate flasks. Plus and minus signs indicate when a particular substance is added and when it is not, respectively.

4 18 EHRLICH AND FOX APPL. MICROBIO. TABLE. Copper precipitation at lowered oxygen tension by Rhodotorula strain La Concn (tg/ml) of Cu in Concn (ug/ml) of Fe in ph Gas phase6b Sampling time supernatant liquid supernatant liquid -Fe +Fe +Cu +Cu, -Fe +Cu, +Fe Airc (0.1) (9)e (9) N2c (1) (9) (9.5) Aird (0.1) a Results reported for duplicate flasks. Plus and minus signs indicate when a particular substance is added and when it is not, respectively. b Numbers in parentheses represent atmospheres of air pressure. c Inoculum =.8 X 106 yeast cells. d Inoculum = 5 X 106 yeast cells. e Numbers in parentheses represent Fe++ concentration in micrograms per milliliter. required for this precipitation. The lack of copper precipitation in the absence of added iron in these experiments is attributable to the lack of yeast growth as reflected by the negligible ph change of the medium. Once precipitated, the copper showed no tendency to redissolve under semianaerobic or anaerobic conditions. DIscUSSION The results of these experiments show that at least some strains of Rhodotorula and Trichosporon associated with acid mine-waters have the ability to precipitate dissolved copper with H2S generated by reducing elemental sulfur with glucose. This activity was stimulated by iron under certain conditions. Anaerobically, Rhodotorula strain L was able to precipitate as much as five times the amount of copper that it precipitated aerobically, provided iron was present in the medium. Iron was not precipitated by H2S formed in these experiments. The cause of copper sulfide redissolving in aerobic experiments with Rhodotorula is best explained as reoxidation of copper sulfide by ferric iron. The results in Table 1 indicate a direct relationship between iron concentration and copper sulfide redissolving. When the iron concentration in solution had become maximal, the copper concentration in solution began to increase. Acid formation cannot have been a direct cause of copper redissolving because, in the absence of iron, the acid did not redissolve the copper sulfide formed by the yeast. Ferric iron in acid solution can oxidize copper sulfide. Hydrogen sulfide formation by reduction of elemental sulfur has been reported to be a widespread activity of fungi as well as other microorganisms (8). Copper sulfide precipitation as a result of sulfate reduction by copper-resistant Saccharomyces ellipsoideus was reported by Naiki in 1957, as cited by Ashida et al. (1), and was also studied by Ashida et al. (1). In the experiments by Ashida et al., the copper sulfide was deposited inside the cell wall of the yeasts, but not externally, as demonstrated by sections viewed by electron microscopy. Attempts by us to precipitate copper with H2S produced from sulfate by Rhodotorula have so far failed. Extracellular precipitation of copper sulfide, but not of iron sulfide, as a result of elemental sulfur reduction by yeast in an acidglucose-mineral salts medium has not been reported previously. Copper sulfide precipitation by yeasts may be important geomicrobiologically. In nature, or in field operations involving copper sulfide leaching, elemental sulfur reduction by yeast could be important in redepositing solubilized copper in reducing zones of some copper sulfide ore bodies and waste dumps. In reducing zones, where, because of a lack of oxygen, bacterial oxidation or

5 VOL. 15, 1967 COPPER SULFIDE PRECIPITATION BY YEASTS 19 autoxidation of copper sulfides cannot occur, ferric iron introduced by waters from the oxidation zone would oxidize the copper sulfides to copper sulfate and sulfur, as was shown by Sullivan (9-11); for example, CuS + 2Fe Cu- + SO + 2Fe+. Thus, if surface waters, percolating into the reducing zone of copper sulfide ore bodies, carried enough organic matter and nitrogen, yeasts in the reducing zone could reprecipitate some of the dissolved copper with H2S they produce by reducing any sulfur there with some of the organic matter. ACKNOWLEDGMENTS This investigation was supported by a grant from Duval Corp. We are grateful to D. M. Ahearn, University of Miami, Miami, Fla., for identifying one of the Rhodotorula cultures, and we thank Alice R. Ellett and J. Dockendorf for helpful technical assistance. LiTERATuRE CrrED 1. ASHIDA, J., N. HIGASHI, AND KIKUCHI An electronmicroscopic study on copper precipitation by copper-resistant yeast cells. Protoplasma 57: EHRLICH, H. L Bacterial action on orpiment. Econ. Geol. 58: EHRLICH, H. L Bacterial oxidation of arsenopyrite and enargite. Econ. Geol. 59: FEIGL, F Qualitative analysis by spot tests. Inorganic and organic application. Nordmann Publishing, Inc., New York. 5. MARCHLEWITZ, B., AND W. SCHWARTZ Untersuchungen uber die Mikroben-Assoziation saurer Grubenwaesser. Z. Allgem. Mikrobiol. 1: SILVERMAN, M. P., AND H. L. EHRLICH Microbial formation and degradation of minerals. Advan. Appl. Microbiol. 6: SILVERMAN, M. P., AND D. G. LUNDGREN Studies on the chemoautotrophic iron bacteriumferrobacillusferrooxidans. I. Animproved medium and a harvesting procedure for securing high cell yields. J. Bacteriol. 77: STARKEY, R. L Transformations of sulfur for microorganisms. Ind. Eng. Chem. 8: SULLIVAN, J. D Chemistry of leaching chalcocite. U.S. Bur. Mines Tech. Paper SULLIVAN, J. D Chemistry of leaching covellite. U.S. Bur. Mines Tech. Paper SULLIVAN, J. D Chemistry of leaching bornite. U.S. Bur. Mines Tech. Paper 86. Downloaded from on November 1, 2018 by guest