generation was determined with 0.1% thymol blue solution. The methods were developed using pure cultures of Thiobacillusferrooxidans growing on ferrous ...
681
Determination o f sulfur and iron oxidation bacteria by the most probable n u m b e r (MPN) technique B. Escobar and I. Godoy Departamento de Ingenieria Quimica. Facultad de Ciencias Fisicas y Matemfiticas. Universidad de Chile. Beauchef 861. Cp 6511266. Santiago, Chile. Fax 56 2 6991084 The number and the activity of acidophilic bacteria present in bioleaching processes is one of the most important parameters to understand the behaviour of a leaching process. The most frequently isolated species from these ecosystems are strains of Thiobacillus ferrooxidans, Leptospirillum ferrooxidans and Thiobacillus thiooxidans. A widely recognized difficulty in quantifying these autotrophic bacteria is problems with growth on solid medium. This work presents a modified methodology of the Most Probable Number technique (MPN) to enumerate the active bacterial populations with the capacity to oxidize Fe(II) and reduced inorganic sulfur compounds. In both cases, specific reagents were used to read the positive and negative tubes. The presence of ferric ion in the cultures of ferrous iron oxidizing bacteria was detected using 1% solution of potassium thiocyanate. For the sulfur oxidizing bacteria, the sulphuric acid generation was determined with 0.1% thymol blue solution. The methods were developed using pure cultures of Thiobacillusferrooxidans growing on ferrous sulfate or in sodium tetrathionate solution; a culture of Leptospirillum ferrooxidans was also utilized to set up the technique for ferrous iron oxidizing bacteria. Later the MPN tecniques were utilized to estimate the active populations of ferrous and sulfur oxidizing bacteria present in samples of pregnant solutions of the copper mineral bioleaching operations in the north of Chile.
1. INTRODUCTION The bioleaching of copper sulfide ores is applied in Chile specially for the recovery of copper from secondary sulfide ores (1). It is now recognized that the active bacterial population in these processes is represented by acidophilic bacteria that oxidize ferrous iron and reduced inorganic sulfur compounds, being the most frequently isolated from the mesophilic environment Thiobacillus ferrooxidans, Leptospirillum ferrooxidans and Thiobacillus thiooxidans (2). However, the importance of other iron oxidizing bacteria is now being considered (3). The isolation and enumeration of these microorganisms by the classical plating techniques present serious difficulties due to the incapacity of these microorganisms to develop on the agar (4). A number of methodologies have been reported to determine the bacterial growth in the leaching process: a two layered gel of agarose (5), an overlay plate (6), a floating filter on a solution (7), and silicate plates (8). Unfortunately, some strains fail to grow or present a low efficiency of plating. Another complication is that many of the microorganims involved in
682 bioleaching processes are present not only in solutions but also adhered to the ore particles. A number of molecular PCR (2) and immunological methods (9,10) have been developed and applied to detect these acidophilic bacteria. These methodologies measure both live and dead cells , but to study the role and population dynamics in a bioleaching process it is only necessary to determine the active bacteria. Furthermore, these methodologies require specialized equipment and trained personnel. The most probable number of iron oxidizing bacteria has been frequently used to detemaine these bacteria (11,12). Lafleur et al. (13) developed a modified methodology to improve the enumeration of T. ferrooxidans which uses both a low ferrous iron concentration and a small volume of growth medium. This work aims to adapt this methodology so that it can be used with T. ferrooxidans and other ferrous iron oxidizing bacteria, and further to develop the same technique to enumerate the sulfur oxidizing bacteria in samples of pregnant leaching solutions.
2. MATERIAL AND METHODS 2.1. Microorganisms The methodologies were calibrated using Thiobacillusferrooxidans ATCC 19859 and a strain of Leptospirillum ferrooxidans grown in MC medium (0.4 (NH4)2804,0.4 MgSO4x7H20 and 0.056 K2HPO4x3H20 g/l), acidified with sulfuric acid to pH 1.6, and ferrous (II) sulphate 54 mM for T.ferrooxidans and 49 mM for Leptospirillum strain. For sulfur oxidizing bacteria, the same strain of T. ferrooxidans was used with sodium tetrathionate 10 mM as energy source, in basal medium (6.0 (NH4)2SO4, 1.0 MgSO4x 7H20, 0.02 Ca(NO3)2 ,1.0 K2HPO4x3H20 and 0.2 KC1 g/l), acidified with sulfuric acid to pH 4.0 (14). 2.2. Preparation of cell suspension for enumeration The bacteria coming from pregnant leaching solutions must be free of ions in the solution, in particular ferric iron, which could indicate the unreal presence of iron oxidation bacteria, when the MPN technique could be applied. Therefore, the bacteria must be diluted in series six or seven fold in the respective medium containing ferrous sulfate or sodium tetrathionate; only the second or third dilution can be used to the determination, according to the cell number initially in the sample (determined by direct count with a Petroff-Hauser counting chamber with a microscope). 2.3. Most Probable Number of iron oxidizing bacteria This technique employed polystyrene Nunc Immunoplates (13). The cultures were diluted in MC medium of pH 1.8 containing 3.6 mM ferrous sulfate. This low concentration of Fe(II) ion was previously determined with different strains (T. ferrooxidans, Leptospirillum ferrooxidans and wild strain isolated from the bioleaching process) as the lowest concentration to minimize its inhibiting effect on bacterial growth. The MPN was calibrated using cultures of T. ferrooxidans and L. ferrooxidans growing in Fe(II) iron and collected during exponential phase. After vortexing the bacterial suspension, 250 ~tl aliquots of the dilutions were added to the wells (5 wells per dilution) and incubated at 30~ The surrounding wells were also filled with distilled water to minimize evaporation from the cultures. The immunoplates (in duplicate) were incubated during 7,10, 14 and 20 days. At the end of these periods the MPN was determined using published tables (15); the positive tubes (wells) turned red with the addition of 50 ~tl of 1% solution of potassium thiocyanate, thus indicating the presence of Fe(llI) in the solutions.
683 The detection limit for iron oxidizing bacteria was determined using a cell culture of T.
ferrooxidans collected during exponential phase, counted with a microscope and then diluted to obtain suspensions of 104, 103 and 102 bacteria/ml. As microscopic counting at concentrations as low as 106 bacteria/ml is not possible, the number of bacteria in the diluted samples was estimated starting at the number determined for a suspension of 107 bacteria/ml and multiplying for the dilution factor in each case. Two layer agarose plates (5) were also utilized in this study. 2.4. Most Probable Number of sulphur oxidizing bacteria
The technique for sulfur oxidizing bacteria was calibrated using a culture of T. ferrooxidans growing in basal medium containing 10mM sodium tetrathionate pH 4.0. The cells were also collected in exponential phase and microscopically counted to determine the initial bacterial concentration. The immunoplates (in duplicate) were incubated at 30~ during 7, 10 and 15 days. At the end of the incubation periods the MPN was determined by adding 50 ~tl of a pH indicator (methyl orange 0.05% or thymol blue both 0.1% solution) in each well. The colour change indicated a net acid production as the result of sulfur oxidation by bacteria (pH 4.0 to pH 2.0). The detection limit for the sulfur oxidizing bacteria was also determined using an active culture of T. ferrooxidans in tetrathionate medium.
3. RESULTS AND DISCUSSION 3.1 Calibration of the Most Probable Number (MPN) of iron oxidizing bacteria
According to the results in Table 1 the MPN of iron oxidizing bacteria obtained after 7 and 14 days of growth were according to the direct counts for strains of T. ferrooxidans and L. ferrooxidans, respectively, showing that this last strain had a slower growth rate. Therefore to determine iron oxidizing bacteria in solutions coming from the bioleaching process requires about 14 days to ensure good development of the cells in the culture medium. Table 1 Direct Count and Most Probable Number (MPN) of iron oxidazing bacteria (bact/ml) Incubation Time
Direct Counts (*)
MPN Fe oxid (**)
7 (days) T. ferroox.
6 +/- 0.6 x 108
9.3 +/- 4.6 x 108
10 "
6 +/- 0.6 x 108
7.5 +/- 3 x 10 s
6 +/- 0.6 x 108
9.3 +/- 3.3 x 108
6 +/-0.6 x 108
9.3 +/- 3.3 x 108
"
14 . . . .
20 "
"
7 "
L. ferroox.
3 +/- 0.3 x 107
5.0 +/- 2.5 x 106
14 "
"
3 +/- 0.3 x 107
1.3 +/- 0.4 x 107
20 "
"
3 +/- 0.3 x 107
6.0 +/- 2.1 x 106
MPN Fe oxid: Most probable Number of Fe(II) iron oxidizing bacteria (*) Mean of 10 areas in the chamber. (**) Mean of two sets of plates.
684 Table 2 Detection limit for the iron oxidizing bacteria by the Most Probable Number (MPN). (bact/ml)
Culture 1
Direct Counts 6 +/-0.6 x 107
MPN Fe oxid *
Dilution factor x 10.2
4.3 +/- 2.2 x 105
Culture 2
"
"
x 10.3
2.3 +/- 0.9 x 104
Culture 3
"
"
x 10-4
7.5 +/- 1.9 x 103
Culture 4
"
"
x 10.5
3.5 +/- 0.9 x 102
* Mean of two sets of plates. Furthermore, the 14 days required to detect the cells is very important to permit the enzymatic system of ferrous iron oxidation to be induced (16). This technique can be applied to bacterial concentration as low as 102 bacteria per ml (Table 2).
3.2. Calibration of the Most Probable Number (MPN) of sulfur oxidizing bacteria. Table 3 presents a comparison of the results obtained using both indicators. It aims to select, the indicator that best represents the decrease of pH due to bacterial activity. The higher numbers obtained for the MPN of sulfur oxidizing bacteria determined with methyl orange after 15 days of growth is probably due to the difference in the pH range of both indicators, between 1.2 and 2.8 for thymol blue and 2.0 and 4.0 for methyl orange, therefore, the pH decrease is detected before with methyl orange. By comparing these results with the direct counts, it can be concluded that the MPN of sulfur oxidizing bacteria determined with thymol blue is more precise when using initial pH conditions (pH:4.0). An incubation time of 10 days was sufficient to obtain a similar number to the direct count method. As can be seen in Table 4, the limit of detection of sulfur oxidizing bacteria, using this MPN technique, was 500 bacteria/ml. Table 3 Direct Count and Most Probable Number (MPN) of sulfur oxidizing bacteria using methyl orange and thymol blue pH indicators (bact/ml) Direct Counts
MPN S oxid (MO)
MPN S oxid (TB)
6 (days)
1.6 +/-0.16
x 10 7
7 +/- 3.5
x 10 6
3.3 +/- 1.0
10
"
1.6 +/- 0.16
x
1.1+/- 0.3
X 10 7
2.2 +/- 0.4
X 10 7
15 "
1.6 +/-0.16
x 10 7
1.7 +/- 0.3
x 10 7
Incubation Time
10 7
(MO): Methyl Orange; (TB): Thymol Blue
6.3 +/- 2.2
x 10 7
x 10 6
685 Table 4 Detection limit for the sulfur oxidizing bacteria (bact/ml)
by the Most Probable Number (MPN).
Direct Counts 5 +/- 0.5 x 107
MPN S oxid
Dilution factor x 10-3
4.3 +/- 1.3 x 104
Culture 1 Culture 2
"
"
x 104
4.3 +/- 0.9 x 103
Culture 3
"
"
x 10.5
4.3 +/- 1.5 x 102
MPN S oxid: Most probable Number of sulfur oxidizing bacteria
3.3 Applications of the MPN of sulfur and iron oxidizing bacteria to pregnant leaching solutions The results presented in Table 5, correspond to totals (direct counts), iron and sulfur oxidizing bacteria present in samples of different bioleaching processes. These values were determined only with the intention of applying them to process samples.. It is not possible to reach conclusions concerning the process efficiency. Table 5 Comparison of cell number determined by direct count, agarose plates and Most Probable Number (MPN) of iron and sulfur oxidizing bacteria in pregnant leaching solution (bact/ml) Sample
Direct Counts
Agarose Plates
Aglom RH
2.5 +/- 0.3 x 105
4.0 +/- 0.8 x 10z
1.0+/-0.42 x 103
1.0 +/- 0.3 x 104
GS Ore A
7.5 +/- 0.8 x 105
1.6 +/- 0.3x 102
4.0 +/- 1.7 x 104
Not Deter.
MS Ore B
5.0 +/- 0.5 x 105
1.0 +/- 0.2 x 102
5.0 +/- 2.1 x 104
Not Deter.
MS Ore BC1
1.0 +/- 0.1x
Not Detected
8.0 +/- 3.4 x 102
Not Deter.
Aglom D
Not Detected
Not Detected
2.1 +/- 0.9 x 104
Not Deter.
MS Ore BC2
2.8 +/- 0.3 x 106
Not Detected
1.0 +/- 0.4 x 103
1.0 +/- 0.3 x 104
Refin
1.9 +/- 0.2 x 106
Not Detected
2.3 +/- 0.9 x 105
2.0 +/- 0.5 x 106
Aglom 64
1.1 +/- 0.1 x 106
Not Detected
2.3 +/- 0.9 x 105
1.8 +/- 0.5 x 106
Aglom 43
3.0 +/- 0.3 x 105
Not Detected
3.3 +/-1.4 x 105
6.0 +/- 1.6 x 105
Aglom 44
3.3 +/- 0.3x 106
60 +/- 12
2.3 +/- 0.9x 106
2.5 +/- 0.7 x 105
10 6
MPN Fe oxid
MPN S oxid
- Not Detected: The number of bacteria were too low to count with the microscope or didn't grow in plates. - Not Deter: The numbers of bacteria were not determined.
686 In all cases, presented in Table 5, values determined by direct microscopic counts were higher than the sum of the number of iron and sulfur oxidizing bacteria; this could be due to the (direct count) method includes heterotrophic bacteria, which can not be determined by means of this method. On the other hand, the number of bacteria determined by the MPN are underestimated (17). This is probably due to an error that can be produced by the several dilutions of the samples. Also some strains form aggregates in the solution, which may not give homogeneous solution in the respective dilutions. However, the MPN measures viable bacteria and provides diluted samples of cells that can be used later to identify the present species (17). In several samples it was not possible to determine iron oxidizing bacteria by plating because the bacteria did not grow in agarose medium; however, they were detected and determined by the MPN technique, which allows the determination of all autotrophic iron oxidizing bacteria present in the mining solution; This method can detect Leptospirillum ferrooxidans which can not grow in agarose medium. In the case of the solution called Aglom D, the direct count with a microscope was not possible because the number was too low to be determined, i.e.(104 bacteria/ml); however using the MPN technique of iron oxidizing bacteria, a population of 2.1 x 10 4 bacteria/ml was determined. When the MPN technique was applied to the sulfur oxidizing bacteria, similar numbers were determined as for the iron oxidizing bacteria (Table 5). These results confirm the observations of other ecological studies in pregnant leaching solutions and indicate that in these operations, the number of sulfur oxidizing bacteria is as high as the number of iron oxidizing bacteria (10,11). The last four cases correspond to samples that presented pHs as low as 1.4. In each case, the number of sulfur oxidizing bacteria were similar or greater than found for the iron oxidizing bacteria. Similar observations were made in samples of slurries coming from the end of continous bioleaching processes of sulphide ores, probably due to the relative increase of sulfur intermediates (10). The variability of MPN presented in Tables 1-5 are according with the variability of the statistical analysis of the most probable number technique (15). It is widely accepted that an important part of the active bacterial population in a bioleaching process may be adhered to the ore particles or the agglomerated ore particles. Thus to determine these populations, it first requires the release of the bacteria from the ore in a manner that maintains the viability of the bacteria and secondly the application of the MPN techniques. Preliminary results indicated that the hand stirred samples of agglomerated ore with MC medium at pH 1.8 released bacteria in a high percentage (more than 90%). Once the bacteria were liberated from the ore, they developed well when cultivated in Fe(II)iron and tetrathionate medium. Therefore, these bacteria may be enumerated using these tecniques, when there is not equipment available to determine consumed oxygen and carbon dioxide. The procedures presented here can be utilized for inexperienced personnel in a bioleaching plant with basic laboratory equipment. 4. CONCLUSIONS A modified technique to determine the MPN of iron oxidizing bacteria (including
T.ferrooxidans and Leptospirillum ferrooxidans) has been calibrated and applied to quantify bacteria coming from pregnant leaching solutions. The use of the MPN in bioleaching solution requires about 14 days to ensure the determination of L. ferrooxidans, since the growth rate of this strain in Fe(II) medium is slower than T. ferrooxidans species.
687 The same technique was developed, calibrated, and applied to sulfur oxidizing bacteria using a medium containing sodium tetrathionate solution at pH 4. The results can be read according to the change in pH of the solution, from 4.0 to 2.0 or lower, using a pH indicator (thymol blue), after 10 days. The detection limit of these methodologies was 102 bacteria/ml.
ACKNOWLEDGEMENTS
The authors thank the University of Chile as well as the Convenio CODELCO-Universidad de Chile.
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