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Oxidation of H2S in acid solution by Thiobacillus ferrooxidans and Thiobacillus thiooxidans
Process Biochemistry 37 (2001) 111– 114 www.elsevier.com/locate/procbio
Oxidation of H2S in acid solution by Thiobacillus ferrooxidans and Thiobacillus thiooxidans Maria E.A.G. Oprime a, Oswaldo Garcia Jr a,*, Arnaldo A. Cardoso b a
Departamento de Bioquı´mica e Tecnologia Quı´mica, Instituto de Quı´mica, UNESP, C.P. 355, 14801 -970 Araraquara, SP, Brazil b Departamento de Quı´mica Analı´tica, Instituto de Quı´mica, UNESP, C.P. 355, 14801 -970 Araraquara, SP, Brazil Received 12 May 2000; received in revised form 1 March 2001; accepted 25 March 2001
Abstract Qualitative and quantitative oxidation tests of H2S in acid solution were carried out using Thiobacillus ferrooxidans and Thiobacillus thiooxidans species. Experiments were performed using solutions of H2SO4 (pH 2.0) containing H2S in initial concentrations ranging from 5 to 100 ppm, in shake flasks at 150 rpm and 30°C. In these solution, this gas was not very stable and was quickly liberated. However, at low concentration (less than 5 ppm) it becomes stable and could only be removed from solution by oxidation. The results obtained indicated that the presence of either T. ferrooxidans or T. thiooxidans causes a significant reduction in H2S concentration (more than 99%) in relation to the sterile control. No differences in oxidation efficiency between these two species were detected. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: H2S oxidation; Thiobacillus thiooxidans; Thiobacillus ferrooxidans
1. Introduction The removal of H2S from biogas, natural or industrial gases is an important concern of environmental technology, since this gas is extremely hazardous to human health and corrosive, besides its strong unpleasant smell. It is produced during the anaerobic treatment of wastewater and during effluent treatment in some industries such as paper and sour products or in petrochemical and natural gas refineries. Conventional physical-chemical processes of H2S removal are mainly based in its oxidation by air in the presence of several catalysts, such as KMnO4, Fe3 + and active coal or by direct oxidation by agents like Cl2, H2O2 and NaClO. Processes based upon such agents are expensive due to the high cost involved in the facility installations, as well the operational cost due to the high energy demand (high pressure and temperature) [1].
* Corresponding author. Tel.: + 55-16-2016677; fax: +55-162227932. E-mail address: [email protected] (O. Garcia, Jr).
The use of microorganisms able to oxidize H2S, producing sulphate or elemental sulphur as a consequence of complete or incomplete metabolism, respectively, has been considered a potential alternative for its application on a large scale [2–7]. The most used bacteria in these investigations belong to the genus Thiobacillus, mainly T. denitrificans. These chemolithotrophic species use sulphur and its reduced forms (including H2S) as energy source for growth. Thiobacillus ferrooxidans, also oxidizes ferrous iron to ferric iron to obtain energy for its cellular processes [8]. In general, the possible advantages of a biotechnological process would be low energy demand for the process operation, reduction of the initial invested capital and reduced consumption of chemicals. Studies of H2S oxidation with T. denitrificans have been carried out at pHs near neutrality. The use of the acidophilic species T. ferrooxidans and T. thiooxidans in this pH condition, would be virtually not feasible because the optimum pH range for growth of these bacteria is 1.0 –3.5 [8]. The scope of this work was to investigate the oxidation of H2S present in acid solutions at low concentrations, using acidophilic T. ferrooxidans and T. thiooxidans.
0032-9592/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 1 ) 0 0 1 7 9 - 0
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M.E.A.G. Oprime et al. / Process Biochemistry 37 (2001) 111–114
2. Material and methods
2.1. Bacterial strains T. ferrooxidans-LR and T. thiooxidans-FG01 isolated from acid mine drainage were used in this work [9].
2.2. Culture media For growth purposes and periodic maintenance of T. ferrooxidans-LR and T. thiooxidans-FG01, media were used respectively, a slightly modified ‘T&K’ [10] and ‘9K’ with S° as energy source (10 g l − 1) [11].
2.3. Preparation of cell suspensions After growth cultures were filtered through filter paper to eliminate precipitated iron generated during growth of T. ferrooxidans or residual sulphur during growth of T. thiooxidans. Cultures were centrifuged at 3300×g for 30 min at 4°C (Sorvall-RT7, Du Pont). Precipitated cells were washed three times with 0.01 mol l − 1 sulphuric acid and re-suspended in fresh acid solution and maintained at 4°C for at most 15 days. The cellular biomass was standardized by determining the total protein by Lowry’s method as modified by Hartree [12].
2.4. H2S production H2S gas was produced by the reaction of FeS with HCl in a Kipp apparatus and collected in acid solution (0.01 mol l − 1 H2SO4).
2.5. Determination of H2S The quantitative method used to determine H2S present in the solution was that of methylene blue production by the reaction of sulphide, ferric chloride, and dimethyl-p-phenylenediamine [13].
2.6. Qualitati6e test A volume (1 ml) of cell suspension of T. ferrooxidans-LR or T. thiooxidans-FG01 (258 mg ml − 1 total protein) was inoculated in 250 ml Erlenmeyer flasks, containing 100 ml of acid solution of H2S ( 100 mg l − 1). Incubation was performed under constant shaking (150 rpm) at 30°C. Samples (1 ml) were periodically withdrawn and added to filter paper impregnated with 0.1 mol l − 1 lead acetate. A positive result of the presence of H2S in the samples was the formation of a black precipitate (PbS). Parallel to this test another was performed by replacing the cellular suspensions by a solution of Fe3 + (8.38 g l − 1) obtained through com-
plete oxidation of the ‘T&K’ medium by T. ferrooxidans-LR. Prior to the experiment this solution was filter sterilized through a membrane (0.45 mm). A flask containing only H2S solution was used as control for these tests.
2.7. Quantitati6e test In order to evaluate the effect of H2S concentration on the oxidative activity of T. ferrooxidans-LR or T. thiooxidans-FG01 strains, tests were carried out in 250 ml Erlenmeyer flasks containing 100 ml of H2S solution at initial concentrations ranging from approximately 5–100 mg l − 1. Flasks were inoculated with 1 ml cell suspension at the same protein concentration as that of the previous test, were sealed with rubber corks and incubated under constant shaking (150 rpm) at 30°C. All tests were compared with a control flask containing only H2S. Samples were periodically withdrawn for analysis of the presence of residual H2S, using the methylene blue method [13].
3. Results and discussion
3.1. Qualitati6e test In flasks containing cell suspensions of T. ferrooxidans-LR or T. thiooxidans-FG01, H2S was not detected after 120 min, indicated by the disappearance of the characteristic colour (absence of the dark precipitate on the filter paper impregnated with lead acetate). On the other hand, in the control flask, the presence of H2S could be observed throughout the test period (210 min). It was observed the rapid disappearance of H2S in the flask containing the Fe3 + solution; after approximately 4 min the characteristic dark colour of the PbS precipitate was no longer detected. In the test flask using Fe3 + solution as an oxidizing agent for H2S, a light yellow coloured precipitate was detected after the test was carried out. This precipitate was identified as elemental sulphur by XRD analysis (data not shown), following previous methodology [14]. As mentioned by some authors [6,7,15] the application of an industrial process based on Fe3 + purification of waste gas stream containing H2S, associated with bacterial recycling of Fe3 + , should taking account of the commercial potentialities of sulphur recovery in the process.
3.2. Quantitati6e test The results of kinetic evaluation of H2S oxidation by T. ferrooxidans-LR and T. thiooxidans-FG01, in concentrations ranging from 5 to 100 mg l − 1 in acid solutions, are shown in Figs. 1 and 2, respectively. H2S
M.E.A.G. Oprime et al. / Process Biochemistry 37 (2001) 111–114
113
is very unstable in acid solutions, the concentration decreasing quickly from initial concentrations to around 5 mg l − 1, as can be seen in the curves of control flasks in these figures. In order to improve the visualization of the results obtained, the curves were plotted in a semi-logarithm scale, since the differential concentration between controls and inoculated flasks was very low (5 mg l − 1). After approximately 100 min of test period, the H2S oxidation become more significant in the flasks where the bacteria, no matter which species, was present. This tendency could be observed for all initial concentrations utilized, up to a 300 min period of test. After this period, the concentrations of H2S in inoculated flasks decreased toward a minimum; below of the detection limit of the method utilized (0.02 mg l − 1), while in control flasks the quantitative determination of the gas after this period of test was always possible. The results obtained in this work showed that both acidophilic Thiobacillus species tested were capable of oxidising H2S present in acid solution efficiently at
Fig. 2. Oxidation of H2S in acid solutions containing the following initial concentrations (mg l − 1): A (6.95); B (25.71); C (32.70); D (100,0). ( ), T. thiooxidans-FG01; (), abiotic control. Curves, in semi-logarithmic scale, were obtained by the exponential decay equations from original curves.
concentrations below 5 mg l − 1, which can be considered common in industrial gases containing sulphidic gas. H2S oxidation by the Fe3 + solution, which was completed in less than 4 minutes, according to the qualitative test performed, should be considered an interesting way to remove this contaminant from industrial gases, since the oxidation of Fe2 + to Fe3 + by T. ferrooxidans can achieve high rates, depending on of the system utilized (for instance, through immobilized cells). So, Fe2 + oxidation by T. ferrooxidans to produce Fe3 + , a reactant for H2S oxidation, would be a non-limiting step in such a process. Acknowledgements
Fig. 1. Oxidation of H2S in acid solutions containing the following initial concentrations (mg l − 1): A (5.62); B (16.21); C (42.0); D (100.0). ( ), T. ferrooxidans-LR; () abiotic control. Curves, in semi-logarithmic scale, were obtained by the exponential decay equations from original curves.
We thank FAPESP (Fundac¸ a˜ o de Amparo a` Pesquisa do Estado de Sa˜ o Paulo) for the graduate student fellowship (M.E.A.G.O.) and CNPq (Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´ gico) for the research fellowship (O.G.). We also acknowledge W.A. Meneses for technical assistance.
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M.E.A.G. Oprime et al. / Process Biochemistry 37 (2001) 111–114
References [1] Sorokin DY. Use of microorganisms in protection of environments from pollution by sulfur compounds. Microbiology 1994;63:533 – 47. [2] Sublette KL, Sylvester ND. Oxidation of hydrogen sulfide by Thiobacillus denitrificans: desulfurization of natural gas. Biotechnol Bioeng 1987;29:249 –57. [3] Sublette KL, Sylvester ND. Oxidation of hydrogen sulfide by mixed cultures of Thiobacilus denitrificans and heterotrophs. Biotechnol Bioeng 1987;29:759 –61. [4] Gadre RV. Removal of hydrogen sulfide from biogas by chemoautotrophic fixed-film bioreactor. Biotechnol Bioeng 1989;34:410 – 4. [5] Sublette KL, Hesketh RP, Hasan S. Microbial oxidation of hydrogen sulfide in pilot-scale bubble column. Biotechnol Progr 1994;10:611 – 4. [6] Pagella C, Perego P, Zilli M. Biotechnological H2S gas treatment with Thiobacillus ferrooxidans. Chem Eng Technol 1996;19:79 – 88. [7] Pagella C, Silvestri P, De Faveri DM. Hydrogen sulphide removal with a biochemical process: the biological step. Chem Biochem Eng Q 1996;10:165 –74. [8] Leduc LG, Ferroni GD. The chemolithotrophic bacterium
.
Thiobacillus ferrooxidans. FEMS Microbiol Rev 1994;14:103 –20. [9] Garcia O Jr. Isolation and purification of Thiobacillus ferrooxidans and Thiobacilus thiooxidans from some coal and uranium mines of Brazil. Rev Microbiol (Sa˜ o Paulo) 1991;20:1 – 6. [10] Tuovinen OH, Kelly DP. Studies on the growth of Thiobacilus ferrooxidans. I. Use of membrane filters and ferrous iron agar to determine viable number and comparison 14CO2-fixation and iron oxidation as measures of growth. Arch Microbiol 1973;88:285 – 98. [11] Silverman MP, Lundgren DG. Studies on the chemoautotrophic iron bacterium Thiobacillus ferrooxidans. I. An improved medium and a harvesting procedure for securing high cell yields. J Bacteriol 1959;77:642 – 7. [12] Hartree EF. Determination of proteins: a modification of the Lowry method that gives a linear response. Anal Biochemist 1972;48:422 – 7. [13] Clesceri LS, Greeberg AE, Trussell RR, editors. Standard Methods for the Examination of Water and Wastewater, 17th edn., part4000. Baltimore, MD: Port City Press, 1989, pp. 191 –197. [14] Garcia O Jr, Bigham JM, Tuovinen OH. Oxidation of galena by Thiobacillus ferrooxidans and Thiobacillus thiooxidans. Can J Microbiol 1995;41:508 – 14. [15] Imaizumi T. Some industrial applications of inorganic microbial oxidation in Japan. Biotechnol Bioeng Symp Ser 1986;16:363 – 71.
Oxidation of H2S in acid solution by Thiobacillus ferrooxidans and Thiobacillus thiooxidans Maria E.A.G. Oprime a, Oswaldo Garcia Jr a,*, Arnaldo A. Cardoso b a
Departamento de Bioquı´mica e Tecnologia Quı´mica, Instituto de Quı´mica, UNESP, C.P. 355, 14801 -970 Araraquara, SP, Brazil b Departamento de Quı´mica Analı´tica, Instituto de Quı´mica, UNESP, C.P. 355, 14801 -970 Araraquara, SP, Brazil Received 12 May 2000; received in revised form 1 March 2001; accepted 25 March 2001
Abstract Qualitative and quantitative oxidation tests of H2S in acid solution were carried out using Thiobacillus ferrooxidans and Thiobacillus thiooxidans species. Experiments were performed using solutions of H2SO4 (pH 2.0) containing H2S in initial concentrations ranging from 5 to 100 ppm, in shake flasks at 150 rpm and 30°C. In these solution, this gas was not very stable and was quickly liberated. However, at low concentration (less than 5 ppm) it becomes stable and could only be removed from solution by oxidation. The results obtained indicated that the presence of either T. ferrooxidans or T. thiooxidans causes a significant reduction in H2S concentration (more than 99%) in relation to the sterile control. No differences in oxidation efficiency between these two species were detected. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: H2S oxidation; Thiobacillus thiooxidans; Thiobacillus ferrooxidans
1. Introduction The removal of H2S from biogas, natural or industrial gases is an important concern of environmental technology, since this gas is extremely hazardous to human health and corrosive, besides its strong unpleasant smell. It is produced during the anaerobic treatment of wastewater and during effluent treatment in some industries such as paper and sour products or in petrochemical and natural gas refineries. Conventional physical-chemical processes of H2S removal are mainly based in its oxidation by air in the presence of several catalysts, such as KMnO4, Fe3 + and active coal or by direct oxidation by agents like Cl2, H2O2 and NaClO. Processes based upon such agents are expensive due to the high cost involved in the facility installations, as well the operational cost due to the high energy demand (high pressure and temperature) [1].
* Corresponding author. Tel.: + 55-16-2016677; fax: +55-162227932. E-mail address: [email protected] (O. Garcia, Jr).
The use of microorganisms able to oxidize H2S, producing sulphate or elemental sulphur as a consequence of complete or incomplete metabolism, respectively, has been considered a potential alternative for its application on a large scale [2–7]. The most used bacteria in these investigations belong to the genus Thiobacillus, mainly T. denitrificans. These chemolithotrophic species use sulphur and its reduced forms (including H2S) as energy source for growth. Thiobacillus ferrooxidans, also oxidizes ferrous iron to ferric iron to obtain energy for its cellular processes [8]. In general, the possible advantages of a biotechnological process would be low energy demand for the process operation, reduction of the initial invested capital and reduced consumption of chemicals. Studies of H2S oxidation with T. denitrificans have been carried out at pHs near neutrality. The use of the acidophilic species T. ferrooxidans and T. thiooxidans in this pH condition, would be virtually not feasible because the optimum pH range for growth of these bacteria is 1.0 –3.5 [8]. The scope of this work was to investigate the oxidation of H2S present in acid solutions at low concentrations, using acidophilic T. ferrooxidans and T. thiooxidans.
0032-9592/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 1 ) 0 0 1 7 9 - 0
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M.E.A.G. Oprime et al. / Process Biochemistry 37 (2001) 111–114
2. Material and methods
2.1. Bacterial strains T. ferrooxidans-LR and T. thiooxidans-FG01 isolated from acid mine drainage were used in this work [9].
2.2. Culture media For growth purposes and periodic maintenance of T. ferrooxidans-LR and T. thiooxidans-FG01, media were used respectively, a slightly modified ‘T&K’ [10] and ‘9K’ with S° as energy source (10 g l − 1) [11].
2.3. Preparation of cell suspensions After growth cultures were filtered through filter paper to eliminate precipitated iron generated during growth of T. ferrooxidans or residual sulphur during growth of T. thiooxidans. Cultures were centrifuged at 3300×g for 30 min at 4°C (Sorvall-RT7, Du Pont). Precipitated cells were washed three times with 0.01 mol l − 1 sulphuric acid and re-suspended in fresh acid solution and maintained at 4°C for at most 15 days. The cellular biomass was standardized by determining the total protein by Lowry’s method as modified by Hartree [12].
2.4. H2S production H2S gas was produced by the reaction of FeS with HCl in a Kipp apparatus and collected in acid solution (0.01 mol l − 1 H2SO4).
2.5. Determination of H2S The quantitative method used to determine H2S present in the solution was that of methylene blue production by the reaction of sulphide, ferric chloride, and dimethyl-p-phenylenediamine [13].
2.6. Qualitati6e test A volume (1 ml) of cell suspension of T. ferrooxidans-LR or T. thiooxidans-FG01 (258 mg ml − 1 total protein) was inoculated in 250 ml Erlenmeyer flasks, containing 100 ml of acid solution of H2S ( 100 mg l − 1). Incubation was performed under constant shaking (150 rpm) at 30°C. Samples (1 ml) were periodically withdrawn and added to filter paper impregnated with 0.1 mol l − 1 lead acetate. A positive result of the presence of H2S in the samples was the formation of a black precipitate (PbS). Parallel to this test another was performed by replacing the cellular suspensions by a solution of Fe3 + (8.38 g l − 1) obtained through com-
plete oxidation of the ‘T&K’ medium by T. ferrooxidans-LR. Prior to the experiment this solution was filter sterilized through a membrane (0.45 mm). A flask containing only H2S solution was used as control for these tests.
2.7. Quantitati6e test In order to evaluate the effect of H2S concentration on the oxidative activity of T. ferrooxidans-LR or T. thiooxidans-FG01 strains, tests were carried out in 250 ml Erlenmeyer flasks containing 100 ml of H2S solution at initial concentrations ranging from approximately 5–100 mg l − 1. Flasks were inoculated with 1 ml cell suspension at the same protein concentration as that of the previous test, were sealed with rubber corks and incubated under constant shaking (150 rpm) at 30°C. All tests were compared with a control flask containing only H2S. Samples were periodically withdrawn for analysis of the presence of residual H2S, using the methylene blue method [13].
3. Results and discussion
3.1. Qualitati6e test In flasks containing cell suspensions of T. ferrooxidans-LR or T. thiooxidans-FG01, H2S was not detected after 120 min, indicated by the disappearance of the characteristic colour (absence of the dark precipitate on the filter paper impregnated with lead acetate). On the other hand, in the control flask, the presence of H2S could be observed throughout the test period (210 min). It was observed the rapid disappearance of H2S in the flask containing the Fe3 + solution; after approximately 4 min the characteristic dark colour of the PbS precipitate was no longer detected. In the test flask using Fe3 + solution as an oxidizing agent for H2S, a light yellow coloured precipitate was detected after the test was carried out. This precipitate was identified as elemental sulphur by XRD analysis (data not shown), following previous methodology [14]. As mentioned by some authors [6,7,15] the application of an industrial process based on Fe3 + purification of waste gas stream containing H2S, associated with bacterial recycling of Fe3 + , should taking account of the commercial potentialities of sulphur recovery in the process.
3.2. Quantitati6e test The results of kinetic evaluation of H2S oxidation by T. ferrooxidans-LR and T. thiooxidans-FG01, in concentrations ranging from 5 to 100 mg l − 1 in acid solutions, are shown in Figs. 1 and 2, respectively. H2S
M.E.A.G. Oprime et al. / Process Biochemistry 37 (2001) 111–114
113
is very unstable in acid solutions, the concentration decreasing quickly from initial concentrations to around 5 mg l − 1, as can be seen in the curves of control flasks in these figures. In order to improve the visualization of the results obtained, the curves were plotted in a semi-logarithm scale, since the differential concentration between controls and inoculated flasks was very low (5 mg l − 1). After approximately 100 min of test period, the H2S oxidation become more significant in the flasks where the bacteria, no matter which species, was present. This tendency could be observed for all initial concentrations utilized, up to a 300 min period of test. After this period, the concentrations of H2S in inoculated flasks decreased toward a minimum; below of the detection limit of the method utilized (0.02 mg l − 1), while in control flasks the quantitative determination of the gas after this period of test was always possible. The results obtained in this work showed that both acidophilic Thiobacillus species tested were capable of oxidising H2S present in acid solution efficiently at
Fig. 2. Oxidation of H2S in acid solutions containing the following initial concentrations (mg l − 1): A (6.95); B (25.71); C (32.70); D (100,0). ( ), T. thiooxidans-FG01; (), abiotic control. Curves, in semi-logarithmic scale, were obtained by the exponential decay equations from original curves.
concentrations below 5 mg l − 1, which can be considered common in industrial gases containing sulphidic gas. H2S oxidation by the Fe3 + solution, which was completed in less than 4 minutes, according to the qualitative test performed, should be considered an interesting way to remove this contaminant from industrial gases, since the oxidation of Fe2 + to Fe3 + by T. ferrooxidans can achieve high rates, depending on of the system utilized (for instance, through immobilized cells). So, Fe2 + oxidation by T. ferrooxidans to produce Fe3 + , a reactant for H2S oxidation, would be a non-limiting step in such a process. Acknowledgements
Fig. 1. Oxidation of H2S in acid solutions containing the following initial concentrations (mg l − 1): A (5.62); B (16.21); C (42.0); D (100.0). ( ), T. ferrooxidans-LR; () abiotic control. Curves, in semi-logarithmic scale, were obtained by the exponential decay equations from original curves.
We thank FAPESP (Fundac¸ a˜ o de Amparo a` Pesquisa do Estado de Sa˜ o Paulo) for the graduate student fellowship (M.E.A.G.O.) and CNPq (Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´ gico) for the research fellowship (O.G.). We also acknowledge W.A. Meneses for technical assistance.
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M.E.A.G. Oprime et al. / Process Biochemistry 37 (2001) 111–114
References [1] Sorokin DY. Use of microorganisms in protection of environments from pollution by sulfur compounds. Microbiology 1994;63:533 – 47. [2] Sublette KL, Sylvester ND. Oxidation of hydrogen sulfide by Thiobacillus denitrificans: desulfurization of natural gas. Biotechnol Bioeng 1987;29:249 –57. [3] Sublette KL, Sylvester ND. Oxidation of hydrogen sulfide by mixed cultures of Thiobacilus denitrificans and heterotrophs. Biotechnol Bioeng 1987;29:759 –61. [4] Gadre RV. Removal of hydrogen sulfide from biogas by chemoautotrophic fixed-film bioreactor. Biotechnol Bioeng 1989;34:410 – 4. [5] Sublette KL, Hesketh RP, Hasan S. Microbial oxidation of hydrogen sulfide in pilot-scale bubble column. Biotechnol Progr 1994;10:611 – 4. [6] Pagella C, Perego P, Zilli M. Biotechnological H2S gas treatment with Thiobacillus ferrooxidans. Chem Eng Technol 1996;19:79 – 88. [7] Pagella C, Silvestri P, De Faveri DM. Hydrogen sulphide removal with a biochemical process: the biological step. Chem Biochem Eng Q 1996;10:165 –74. [8] Leduc LG, Ferroni GD. The chemolithotrophic bacterium
.
Thiobacillus ferrooxidans. FEMS Microbiol Rev 1994;14:103 –20. [9] Garcia O Jr. Isolation and purification of Thiobacillus ferrooxidans and Thiobacilus thiooxidans from some coal and uranium mines of Brazil. Rev Microbiol (Sa˜ o Paulo) 1991;20:1 – 6. [10] Tuovinen OH, Kelly DP. Studies on the growth of Thiobacilus ferrooxidans. I. Use of membrane filters and ferrous iron agar to determine viable number and comparison 14CO2-fixation and iron oxidation as measures of growth. Arch Microbiol 1973;88:285 – 98. [11] Silverman MP, Lundgren DG. Studies on the chemoautotrophic iron bacterium Thiobacillus ferrooxidans. I. An improved medium and a harvesting procedure for securing high cell yields. J Bacteriol 1959;77:642 – 7. [12] Hartree EF. Determination of proteins: a modification of the Lowry method that gives a linear response. Anal Biochemist 1972;48:422 – 7. [13] Clesceri LS, Greeberg AE, Trussell RR, editors. Standard Methods for the Examination of Water and Wastewater, 17th edn., part4000. Baltimore, MD: Port City Press, 1989, pp. 191 –197. [14] Garcia O Jr, Bigham JM, Tuovinen OH. Oxidation of galena by Thiobacillus ferrooxidans and Thiobacillus thiooxidans. Can J Microbiol 1995;41:508 – 14. [15] Imaizumi T. Some industrial applications of inorganic microbial oxidation in Japan. Biotechnol Bioeng Symp Ser 1986;16:363 – 71.