APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1994, p. 3444-3446 0099-2240/94/$04.00+0 Copyright © 1994, American Society for Microbiology
Vol. 60, No. 9
Maximum Temperature Limits for Acidophilic, Mesophilic Bacteria in Biological Leaching Systems SEPPO I. NIEMELA,l CARITA SIVELA,' TAINA LUOMA,' AND OLLI H. TUOVINEN2* Department of Applied Chemistry and Microbiology, FIN-00014 University of Helsinki, Helsinki Finland,' and Department of Microbiology, The Ohio State University, Columbus, Ohio 43210-12922 Received 24 January 1994/Accepted 8 June 1994 The maximum temperature for growth (Tmax) was determined for pure and mixed cultures of acidophilic thiobacilli. The experimental system was based on incubating the cultures in liquid media exposed to a linear temperature gradient. The Tmax values varied within the range of 36.1 to 43.60C.
Biological leaching systems involve sulfide mineral oxidations which are highly exothermic reactions, releasing excess energy as heat and causing elevated temperatures in interiors of leach piles (10, 17). Considerable heat evolution may also occur in stirred tank reactors at high pulp densities of sulfide minerals. In natural settings such as leach piles, both mesophilic and moderately thermophilic bacteria are usually present (1, 4). The optimum temperature for acidophilic iron-oxidizing mesophiles is usually reported in the range of 28 to 33°C. Maximum temperature (Tmax) limits in the range of 35 to 40°C have been reported (6) but have not been well defined for these organisms. Mesophilic iron-oxidizing acidophiles are active at temperatures as low as 2°C, but truly psychrophilic isolates have not been found (2, 3, 8). Industrial applications of biological leaching systems require that the maximum temperature limits for the bacteria are established in order to control heat evolution within the permissive temperature ranges. In heap and dump bioleaching processes the temperature limits can not be externally controlled, whereas Tmn, values are particilarly important to know for bioreactor applications of biologicai leaching processes, because intense oxidation of sulfide concentrates releases excessive heat and may lead to thermail inactivation of the inoculum. The purpose of this work was to experimentally determine the Tm~, range for mesophilic thiobacilli, with emphasis on ferrous-iron-oxidizing acidophiles. Pure cultures of Thiobacillus ferrooxidans and several mixed cultures of iron-oxidizing acidophiles were maintained with ferrous sulfate as the substrate (Table 1). The iron-oxidizing cultures were grown in a mineral salts medium [3.0 mM (NH4)2SO4, 2.3 mM K2HPO4, and 1.6 mM MgSO4 * 7H20] supplemented with 120 mM ferrous sulfate, initial pH 1.5 or 2.0. A pure culture of Thiobacillus thiooxidans was included in the study, and it was grown with 10 mM K2S406 in a mineral salts medium (7). The cultures were maintained in shake flasks (180 rpm) at 28°C. A temperature gradient incubator of a thermal bridge type (Gradiplate W10; Biodata Ltd., Helsinki, Finland), principally designed for tests with solid media (11), was employed for this study. The experimental system has been mainly used for studies of temperature relations and thermotolerance of coliform organisms (12-14). The instrument produces a linear temperature gradient across a metal base plate (98 by 300 mm) * Corresponding author. Mailing address: Department of Microbiology, The Ohio State University, 484 West 12th Ave., Columbus, OH 43210-1292. Phone: (614) 292-3379. Fax: (614) 292-8120. Electronic mail address:
[email protected].
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housed in an incubation chamber. The gradient is established with two thermostated water baths which control the upper and lower temperatures in each experiment. One water bath serves as the heat source and controls the upper temperature, which was fixed between 46 and 50°C in these experiments. The second water bath serves as the heat sink and controls the lower temperature limit, which was fixed between 30 and 35°C in this work. By varying the upper and lower temperatures within these ranges, it was possible to produce temperature gradients spanning a difference of 11 to 20°C between the upper and lower temperatures across the 98-mm length of the metal base plate in the incubation chamber. Thus, the instrument produced temperature gradients of 0.11 to 0.20°C per millimeter of the base plate, depending on the settings of the two water baths. The actual temperatures prevailing at two points on the gradient were monitored with high-class IC Temperature Transducers type AD 590 (Analog Devices, Norwood, Mass.) attached to the metal bridge. The transducers were calibrated against a certified mercury thermometer. The temperature at a desired point on the gradient was calculated from the distance measured from the cool end, by using an empirical formula derived by calibrating the instrument with a series of Grant's thermocouples (type TH) and a series 1200 Squirrel meter/logger (Grant Instruments, Cambridge, United Kingdom). Previous applications of the temperature gradient technique for coliforms utilized solid media for test cultures, yielding sharp growth patterns with well-defined Tmax values (12). In this study, initial experiments with solid media, based on either agarose or gelrite, failed to support good growth, and thus a distinct, well-defined TmaX value could not be determined. The subsequent experiments were carried out with liquid media. The bacteria were incubated either in small test tubes (outer diameter, 5.8 mm) or in microtiter wells. In both cases, therefore, temperature gradients were obtained only in discrete steps. Liquid media (1 ml) in 2-ml test tubes received 10% inocula and were capped and stacked horizontally along the isotherms of the metal base of the incubation chamber. The incubation chamber accommodated 16 test tubes in the horizontal position per experiment, yielding temperature gradients which were equivalent to 0.64 to 1.16°C per tube width (0.11 to 0.20°C per millimeter of the base plate) depending on the lower and higher temperature settings. The tubes were incubated for up to 7 days in the temperature gradient system before the cultures were scored. These results were based on the ability of the organisms to survive at elevated temperatures. Seven days contact time was considered sufficient for thermal inactivation at temperatures above the Tmax. The
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TABLE 1. Test cultures used in this study Source
Test culture and designation
T. ferrooxidans TFI-35 ......................
A-19 ...................... SG-1 ...................... Tf 583 ...................... T. thiooxidans Tt 504 ...................... Mixed culture of iron oxidizers
OF-1 ...................... OF-2 ...................... OF-3 ...................... OF-4 ...................... OF-5 ...................... OF-6 ...................... OF mixture ......................
SB/P-II ...................... SF-V2 ...................... SF-V4 ...................... SF mixture ......................
This laboratory A subculture of T. ferrooxidans AP-19 (18) S. N. Groudev, Higher Institute of Geology and Mining, Sofia, Bulgaria Subculture of T. ferrooxidans 583 from DSMa
Subculture of T thiooxidans 504 from DSMa
Enrichment culture from a bioleaching pilot plant Enrichment culture from a bioleaching pilot plant Enrichment culture from a bioleaching pilot plant Enrichment culture from a bioleaching pilot plant Enrichment culture from a bioleaching pilot plant Enrichment culture from a bioleaching pilot plant Mixture of OF-1 through OF-6 Enrichment culture from mine water samples and maintained on a sulfide ore (15) Enrichment culture from a bioleaching pilot plant Enrichment culture from a bioleaching pilot plant Mixture of enrichment cultures SF-V2 and SF-V4 from a bioleaching pilot plant
a DSM, Deutsche Versammlung von Mikroorganismen und Zellkulturen GmbH, W-3300 Braunschweig, Germany.
results were verified by transferring the test cultures to grow in shake flasks in fresh ferrous sulfate media at 28°C. For microtiter plates (96 wells), the media (10% inoculum; total volume, 300 ,ul) were incubated for up to 4 days. In general, growth and iron oxidation were complete within 2 days of incubation. Therefore, incubation for 4 days was deemed sufficient for detection of growth even at Tma,. Some plates were modified by removing a ledge from the underside in order to maximize the contact between the plate and the bottom of the chamber. Subsequent tests demonstrated that the removal of the ledge was not necessary for achieving a temperature gradient across the microtiter plates. Each well (diameter, 9 mm) represented between 0.99 and 1.80°C in the gradient, depending on the minimum and maximum temperature settings. The results were scored by reading the plates at 405 nm. Sterile blanks were included in both the test tube and microtiter plate systems. The experimentally determined Tma, values are shown in Fig. 1. For culture SB/P-TI, the test tube assay yielded a Tmax value of 42.7 + 1.5°C, and the microtiter plate system showed a value of 39.5 ± 1.0°C. The differences in the Tma, values between the two types of assay may reflect the respective methods of scoring of the positive cultures. In the test tube assay, the cultures exposed to the temperature gradient for as long as 7 days were subsequently incubated at 28°C to recover the surviving population, whereas the microtiter plate assay was based on the ability of the test cultures to grow while exposed to the temperature gradient. This difference favors a somewhat higher Tmax for the tube cultures, because the assay is based on the maximum permissive temperature for survival rather than that for growth. The relatively large standard error in the Tmax determinations is due to the assay technique with liquid media, which can be related to the temperature gradient only in discrete steps. The maximum value in this data set was 43.6°C for a mixed culture and 42.2°C for a pure culture of T. ferrooxidans. The lowest Tma values were 37.2°C for a mixed culture and 38.3°C for a pure culture of T. ferrooxidans. A Tmax value of 36.1°C, based on a single test, was determined for the DSM strain of T. thiooxidans. The results demonstrate that mesophilic thiobacilli relevant in biological leaching systems are confined to temperature ranges of
