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Treatment of tetrathionate effluents by continuous oxidation in a flooded packed-bed bioreactor
International Journal of Mineral Processing 155 (2016) 91–98
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International Journal of Mineral Processing journal homepage: www.elsevier.com/locate/ijminpro
Treatment of tetrathionate effluents by continuous oxidation in a flooded packed-bed bioreactor Nieves Iglesias ⁎, Rafael Romero, Alfonso Mazuelos, Cristina Montes-Rosua, Francisco Carranza Departamento de Ingeniería Química, University of Seville, Spain
a r t i c l e
i n f o
Article history: Received 6 June 2014 Received in revised form 2 March 2016 Accepted 18 August 2016 Available online 24 August 2016 Keywords: Bioreactor Tetrathionate Thiosalts Continuous bio-oxidation Acidithiobacillus ferrooxidans
a b s t r a c t Thiosalts are compounds commonly present in process plant liquor for the concentration of metal sulphide ores by milling and flotation. Since thiosalts are metastable species that are oxidized to sulphuric acid as an end product, they cannot be discharged nor recycled into the process. Therefore, the development of a cost-effective process for the treatment of these effluents is necessary. In this paper, the continuous bio-oxidation of tetrathionate is carried out in a flooded packed-bed reactor by an immobilized microbial consortium. Batch tests show that the initial tetrathionate concentration and pH slightly affect the tetrathionate bio-oxidation process in the ranges 3 to 5 g/L and 1.5 to 2, respectively. The specific bio-oxidation rate is 0.08 h−1. Only sulphate ions and protons have been detected as end products. For the starting up of the bioreactor, a new method of biofilm formation for tetrathionate bio-oxidation is implemented. The maximum bio-oxidation rate in continuous operation is 0.415 gh−1 (0.830 kg h−1 m−3). The biofilm was stable for the whole period studied, i.e. 35 days. These results indicate that a flooded packed-bed reactor is an interesting option from an economic point of view for the treatment of waters contaminated by thiosalts. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Thiosalts are chemicals commonly present in process plant liquor for the concentration of metal sulphide ores by milling and flotation. Alkalinity conditions and the additives employed in these plants favour the partial conversion of sulphides into thiosalts. A common practice in this kind of facility is to recycle the liquors in process. However, the accumulation of thiosalts in liquors negatively affects the efficiency of flotation; this effect renders it necessary to continuously purge the generated thiosalts from the circuit. In these effluents, tetrathionate ion is usually the most abundant thiosalt, since it remains stable in solution at temperatures below 30 °C (Druschel et al., 2003). Thiosalts are not considered toxic in themselves; however, their solutions can have an indirect impact on the environment. The incorporation of thiosalts into a receptor aquatic body can be the cause of a decrease in pH and in the dissolved oxygen concentration, and can severely affect its chemical-biological equilibrium. This is due to a natural oxidation process whose end product is sulphuric acid. This process is catalysed by metal ions in solution (Chanda et al., 1984; González-Lara et al., 2009; Senanayake, 2005a,b) and by the metabolic action of sulphur-oxidizing microorganisms (Eccleston and Kelly, 1978; Friedrich et al., 2001; Kelly and Tuovinen, 1975; Shiers et al., 2011),
⁎ Corresponding author. E-mail address: [email protected] (N. Iglesias).
http://dx.doi.org/10.1016/j.minpro.2016.08.010 0301-7516/© 2016 Elsevier B.V. All rights reserved.
commonly present in these mining environments (Dopson and Johnson, 2012; Ghosh and Dam, 2009; Hedrich et al., 2011). These are frequently Acidithiobacillus genus bacteria, of which the most abundant species is usually Acidithiobacillus ferrooxidans. Currently, the purification of effluents containing thiosalts consists of their oxidation in ponds (Kuyucak and Yaschyshyn, 2007). This is a simple and economic but very slow method, which leads to designs characterized by very large areas. One of the main inconveniences of these ponds is their low activity in cold seasons. Alternatively, several active treatments have been developed that consist of adding oxidants, such as Caro's acid (H2SO5), Fenton reagent, ozone, and hypochlorite (Kuyucak and Yaschyshyn, 2007; Lu et al., 2010).These methods are faster and more controllable but involve very high reagent costs, potentially dangerous operation, and include the incorporation of undesirable substances into the environment. The controlled biological oxidation of thiosalts is postulated as an attractive future option. This option will be developed in bioreactors designed to hold high concentrations of cells and to operate continuously. Very few reactors have been described in the literature for this purpose (Miranda-Trevino et al., 2013). Liljeqvist et al., 2011 tested a bioreactor design that included a biogenerator and a main reactor containing A. ferrivorans attached to a structured plastic bed. Despite containing a biofilm carrier, this reactor needs to be assisted by an external supply of cells throughout its operation. A bioreactor for the continuous bio-oxidation of thiosalts should meet the needs of treatment, with a size that enables the control of
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the variables (mainly temperature) and the stability of operation. In addition, its construction should be economical and simple. Flooded packed-bed bioreactors have been developed for the oxidation of ferrous ion. In flooded packed-bed bioreactors, if the energetic substrate is Fe (II), then it is possible to reach steady states and very high oxidation rates without any supply of cells (Mazuelos et al., 1999 and 2000). In these bioreactors, biomass is immobilized in a biofilm that consists of an inorganic matrix, formed by precipitated ferric compounds (mainly oxyhydroxides and jarosites), in the pores of which cells are attached. This biofilm covers the surface of particles that form the bed (Karamanev, 1991; Mazuelos et al., 2012). In these bioreactors, biofilm is very robust against changes in composition and dynamic fluid conditions. The present work aims to study the oxidation of tetrathionate in a flooded packed-bed reactor with the following objectives: 1) To achieve tetrathionate oxidation in a stable operation over time. 2) To establish a simple and quick methodology for the commissioning of bioreactors, consistent with the above objective. 3) To lay the foundation for optimization and piloting studies for the implementation of this technology.
2. Materials and methods 2.1. Batch tetrathionate bio-oxidation All cultures performed in the present work originate from a mixed culture obtained from the effluents from the Rio Tinto Mines (Huelva, Spain). This culture has been maintained for decades by successive inoculations in 9K medium (Silverman and Lundgren, 1959). The composition of 9K medium, in which the energetic substrate is Fe (II), is shown in Table 1. Phylogenetic analysis of the culture shows the three major phylotypes to be Acidithiobacillus, Leptospirillum, and Ferrimicrobium. The dominant microbial species is Acidithiobacillusferrooxidans _ATCC23270 (Mazuelos et al., 2012). The culture was pelletized in order to remove the iron. Pellets of cells were prepared by centrifugation at 12,000g for 15 min in a Sorvall SS 3 Automatic Centrifuge Du Pont Instruments. The decanted cells were washed and resuspended in tetrathionate medium, whose composition is shown in Table 1. In order to ascertain the species originated during bio-oxidation and to study the influence of pH and of tetrathionate concentration, batch assays were performed in sterilized 250 mL Erlenmeyer flasks, placed in an orbital shaker at 180 rpm and at a temperature of 31 °C. Into these flasks, 96 mL of tetrathionate culture medium, modified in terms of pH and in the concentration of Na2S4O6·2H2O (see Table 1), was added. Four initial concentrations of tetrathionate (2, 3, 3.5 and 5 g/L) and three initial pH values (1.5, 2 and 3), adjusted with dilute sulphuric acid, were studied.
To follow the evolution of cultures, pH, and concentrations of tetrathionate, the thiosulphate and sulphite were measured over time. To detect intermediate species, Ionic Chromatography was used. 2.2. Continuous tetrathionate bio-oxidation The continuous bio-oxidation of tetrathionate was performed in a flooded packed-bed reactor (Mazuelos et al., 1999) by immobilized cells. This reactor is a column of 15 cm in height and 8.4 cm in diameter. It consists of a lower chamber, (hollow cylindrical, 5 cm in height), and an upper chamber (the bed) of 10 cm in height (Fig. 1). In the lower chamber, two nozzles are placed for air and liquid medium inlet. The liquid rises through the bed and leaves the reactor via the overflow. The bed consists of siliceous sand particles between 6 and 8 mm in size. The bed porosity is 0.45. The liquid is fed by a peristaltic pump and the air by a small compressor controlled by a rotameter. Unless specified otherwise, the liquid and air flows tested were 150 mL/h and 750 mL/min, respectively. The reactor operated in a chamber with the thermostat set at 31 °C. For the starting of the bioreactor, a new method of biofilm formation for tetrathionate bio-oxidation was implemented. The method was developed in the following stages: a. Biofilm formation for the Fe(II) bio-oxidation by the Mazuelos et al. (2000) method. The medium used was 9K and the inoculum was the RT culture. b. Continuous operation feeding of 9K medium modified in pH. The pH was adjusted to 1.7 with sulphuric acid to prevent an excessive iron precipitation. c. Change of feed from 9K medium to modified tetrathionate medium. The tetrathionate medium was modified in its concentration of Na2S4O6·2H2O to 2.5 g/L and its pH to 1.7. d. Batch bio-oxidation. In this stage, the feeding pump is stopped while the reactor is maintained aerated. This serves as a test of whether attached cells accept tetrathionate as energetic substrate. e. Recirculating bio-oxidation. The reactor was fed from an external reservoir of 2.5 L containing modified tetrathionate medium. In this tank, the initial concentration of Na 2 S 4 O .6 2H 2 O was 2.5 g/L. The effluent from the bioreactor was returned to the reservoir. The initial pH in the tank was set to 2.8 to prevent an excessive fall in pH of the bioreactor, which could mean substantial dissolution of the precipitated iron that formed the matrix of the biofilm.
8.4 cm
outlet stream by overflow
siliceous
6-8 mm stone
10 cm Table 1 Chemical composition of culture media used in this study. Salt (g)
9K
Tetrathionate
(NH4)2SO4 KCl K2HPO4 MgSO4·7H2O Ca(NO3)2 CaCl2 Na2S4O6·2H2O FeSO4·7H2O H2O (mL)
3.0 0.1 0.5 0.5 0.01 300 mL (14.7%) 700
3.0 3.0 0.5 0.2 4.0 1000
Inlet solution
5 cm
Biofilm (ferric precipitates and cells)
air
Fig. 1. Flooded packed-bed bioreactor used in this study. On the right, a portion of the packed bed is schematically shown; the siliceous stone particles bear the biofilm consisting of bacteria and a matrix of ferric precipitates.
N. Iglesias et al. / International Journal of Mineral Processing 155 (2016) 91–98
93
a
b
c
Fig. 2. a) Chromatogram of culture F at time t = 0; b) Expanding the left-hand side. The peaks at 1.7 and 4 min correspond to chloride ion and dihydrogen phosphate, respectively; c) Expanding the right-hand side, the tetrathionate peak can be clearly observed.
Once the protocol of biofilm formation was concluded, the continuous operation of bioreactor was tested with two different feeds, both at pH 2.8: 1) Modified tetrathionate medium of 2.5 g/L of Na2S4O6·2H2O (1.8 g/L of tetrathionate ion). Operation time 600 h 2) Modified tetrathionate medium of 4.5 g/L Na2S4O6·2H2O (3.3 g/L of tetrathionate ion). Operation time 250 h 2.3. Analysis 2.3.1. Chemical analysis Thiosulphate and sulphite ions were analysed by iodometry; discrimination between the two ions was achieved by masking the sulphite ion with formaldehyde. (Kurtenacker and Bittner, 1924; KoH, 1990). The total concentration of thiosalts was determined by the acidimetric method with mercury chloride (Makhija and Hitchen,
1978). Tetrathionate concentration was expressed as the concentration of total thiosalts minus the concentrations of sulphite and thiosulphate. The pH was measured by a WTW-3310 pH-meter equipped with software for multi-buffer calibration and a combined glass electrode with Sentix 41 automatic temperature compensation. The ferrous iron concentration was determined with 0.3 M potassium dichromate by an automatic titrator with potentiometric control of the endpoint (Radiometer-Copenhagen). 2.3.2. Ion chromatography (IC) The 883 Basic IC plus with chemical suppression by Metrohm Suppressor Module (MSM) was used for the ion chromatographic determinations of polythionates (tetrathionate, trithionate, dithionate), sulphate, thiosulphate, and sulphite ions. The MSM was used for chemical suppression during anion analysis. This analysis consisted of 3 suppressor units in total, regenerated with 250 mM sulphuric acid or rinsed with ultra-pure water in rotation.
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a
b
c
Fig. 3. a) Chromatogram of culture F at time t = 48 h; b) Expanding the left-hand side. The peaks at 1.7 and 4 min correspond to chloride ion and dihydrogen phosphate, respectively; c) Expanding the right-hand side, the tetrathionate peak cannot be observed.
A 50 mm separation column was used. The eluent was 3.2 mM Na2CO3 with a flow rate of 0.7 mL/min. Baseline conductivity 12.3–12.6 μS/cm. Magic Net Basic was the software used for the calculation of peak areas. 3. Results and discussion 3.1. Batch tetrathionate bio-oxidation Batch tests were performed in order to establish conditions in continuous operations. A. ferrooxidans metabolizes tetrathionate ions at pH ranges between 1.33 and 4.44 (Eccleston and Kelly, 1978; Kupka et al., 2009; Tuovinen and Kelly, 1974). The overall stoichiometry of this process is given by the equation 2S4 O6 2− þ 7O2 þ 6H2 O→8SO4 2− þ 12Hþ
ðr1Þ
Although the reaction pathway for tetrathionate oxidation should yield sulphate as a final product, in order to detect intermediate species generated in tetrathionate bio-oxidation, the evolution of concentrations of tetrathionate, trithionate, dithionate, sulphate, thiosulphate, and sulphite ions were analysed by ion chromatography over time. As representative examples, chromatograms corresponding to the beginning and to the end of a culture containing 3 g/L of tetrathionate are shown in Figs. 2 and 3. Fig. 2 shows the chromatogram of the culture at the initial time. Extended chromatograms (Fig. 2b and c) clearly show the peaks of chloride, dihydrogen phosphate, sulphate, thiosulphate, and tetrathionate. Fig. 3 shows the chromatogram of the same culture at 48 h. Enlargements of this chromatogram (Fig. 3b and c) show peaks of chloride, di-hydrogen phosphate and sulphate, but not of thiosulphate nor tetrathionate. Initially, sulphate, chloride, and di-hydrogen phosphate, which come from the culture medium, and traces of thiosulphate, probably
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95
4
[tetrathionate] (g/L)
3.5 3 2.5 2 1.5 1 0.5 0 0
6
12
18
24
30
36
42
48
time (hours) A
B
C
Fig. 4. Evolution of tetrathionate concentration for batch cultures: A (initial pH 3), B (initial pH 2) and C (initial pH 1.5). Reactor: Erlenmeyer flask. Inoculum: Pellet of RTT culture. Medium: modified tetrathionate medium. Initial tetrathionate concentration 3.5 g/L. Shaking speed: 180 rpm. Temperature: 31 °C.
as a tetrathionate impurity, were detected. With the advancement of time, peaks of tetrathionate and thiosulphate disappeared and only sulphate peak remained. In no case were dithionate nor trithionate peaks observed (they should have been visible at 15.7 and 30 min, respectively). These results are in accordance with Reaction 1 in which sulphate and protons are end products. Figs. 4 and 5 present the evolution of tetrathionate concentration and the pH versus time, respectively, for tests with different initial pH values (3, 2, and 1.5) and the same initial concentration of tetrathionate (3.5 g/L). In Fig. 4, it can be observed that tetrathionate is completely consumed in less than 48 h in all cases. A clear decrease of pH was observed in Fig. 5 that corresponds with the decrease in the tetrathionate concentration (Reaction r1). The final pH in the cultures is close to 1.5 since the medium is buffered by the sulphate/bisulphate balance. Fig. 6 shows the evolution of tetrathionate concentration versus time for tests, in which the variable is the initial tetrathionate concentration (5, 3.5 and 2 g/L) and the initial pH is 3.
No presence of thiosulphate or sulphite was detected in any of the tests. In all cultures, the tetrathionate was completely consumed in 42–48 h. The lack of intermediates allows a conventional kinetic characterization based on the postulate of balanced growth in the exponential growth phase, where specific growth rate and tetrathionate biooxidation rate are constant. In an environment favourable for growth, cells regulate their metabolism and adjust the rates of various internal reactions so that a condition of balanced growth occurs (Doran, 2013). The semi-logarithmic plot of substrate consumption versus time, (Ln([S4O 26 −]o-[S4O 26 −]) versus time) is a straight line whose slope, the specific growth rate, is characteristic of each culture (inoculum, medium, and operation conditions). Table 2 shows the initial values of pH and tetrathionate concentration for each test together with the specific bio-oxidation rate calculated from the semi-logarithmic plot. The correlation coefficients of linear fit (R2) are also shown in Table 2. From a kinetic point of view, the worst case corresponds to the culture performed with the lowest concentration of tetrathionate, probably due to substrate limitation. In the remaining assays, the specific rates are very similar and present an average value of 0.079 h−1 and a standard
3.5 3 2.5
pH
2 1.5 1 0.5 0 0
6
12
18
24
30
36
42
48
time (hours) A
B
C
Fig. 5. Evolution of pH for batch cultures: A (initial pH 3), B (initial pH 2) and C (initial pH 1.5). Reactor: Erlenmeyer flask. Inoculum: Pellet of RTT culture. Medium: modified tetrathionate medium. Initial tetrathionate concentration 3.5 g/L. Shaking speed: 180 rpm. Temperature: 31 °C.
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5 4.5
[tetrathionate] (g/L)
4 3.5 3 2.5 2 1.5 1 0.5 0 0
6
12
18
24
30
36
42
48
time (hours) A
E
D
Fig. 6. Evolution of tetrathionate concentration for batch cultures: A (3.5 g/L of tetrathionate), D (5 g/L of tetrathionate) and E (2 g/L of tetrathionate). Reactor: Erlenmeyer flask. Inoculum: Pellet of RTT culture. Medium: modified tetrathionate medium. Initial pH 3. Shaking speed: 180 rpm. Temperature: 31 °C.
3.2. Continuous bio-oxidation
Table 2 Initial conditions and specific bio-oxidation rate of tetrathionate for batch cultures. Culture
pH
[Tetrathionate](g/L)
Specific bio-oxidation rate (h−1)
R2
A B C D E F
3 2 1.5 3 3 1.9
3.5 3.5 3.5 5 2 3
0.075 0.078 0.081 0.083 0.054 0.079
0.9979 0.9925 0.9984 0.9907 0.9903 0.9971
deviation (SD) of 0.003, which reveals a low impact of pH and tetrathionate concentration within the ranges tested for these variables. In accordance with the literature, the results show that A. ferrooxidans previously grown on Fe(II), can adapt its metabolic system using the tetrathionate ion as an energy substrate (Kelly and Tuovinen, 1975; Sugio et al., 1996; Ghosh and Dam, 2009).
Continuous tetrathionate bio-oxidation was tested in a flooded packed-bed bioreactor with attached cells. After biofilm formation using 9K medium and RT culture as inoculum, the bioreactor was continuously fed with 9K medium modified in pH (pH = 1.7) for 192 h. The maximum bio-oxidation of Fe(II) rate was 970 mg/h (stage b). The feed was then changed to modified tetrathionate medium (2.5 g/L, pH = 1.7). This stage lasted 43 h, longer than 12 times the mean residence time of the liquor in the reactor. In this step, dissolved iron and the cells not attached to the support were evacuated. The feeding pump was then stopped while the reactor was maintained aerated until all the tetrathionate had been oxidized. This step ended after 48 h and served as a test that the attached cells accept tetrathionate as energetic substrate. Finally, fresh feed was recycled into the reactor for 72 h, the time necessary for tetrathionate oxidation. Once the protocol of biofilm formation was concluded, the bioreactor was continuously fed with modified tetrathionate medium of
4 3.5
[tetrathionate] (g/L)
3 2.5 2 1.5 1 0.5 0 0
100
200
300
400
500
600
700
800
900
time (hours) inlet solution
outlet stream
Fig. 7. Continuous tetrathionate bio-oxidation in flooded packed-bed bioreactor. Tetrathionate ion concentration in inlet and outlet streams. Liquid flow rate 150 mL/h. Air flow rate 760 mL/min.
N. Iglesias et al. / International Journal of Mineral Processing 155 (2016) 91–98
97
tetrathionate biooxidation rate (mg/h)
450 400 350 300 250 200 150 100 50 0 0
100
200
300
400 500 time (hours)
600
700
800
900
Fig. 8. Continuous tetrathionate bio-oxidation in flooded packed-bed bioreactor. Tetrathionate bio-oxidation rate in g/h. Liquid flow rate 150 mL/h. Air flow rate 760 mL/min.
2.5 g/L of Na2S4O6·2H2O (1.8 g/L of tetrathionate ion) and pH 2.8. After 600 h of operation, the concentration of Na2S4O6·2H2O was increased to 4.5 g/L (3.3 g/L of tetrathionate ion). The results of continuous operation are shown in Figs. 7 and 8. Fig. 7 shows the concentration of tetrathionate ion in inlet and outlet bioreactor streams and Fig. 8 shows the tetrathionate bio-oxidation rate versus operation time. Note that the bioreactor is stable throughout continuous operation. Variations observed in the bio-oxidation rate in the initial hours (Fig. 8) are due to fluctuations in the flow supplied by the pump at the start of the continuous operation. The bioreactor continuously operated in a stable manner for 35 days. In terms of substrate conversion and stability, it can be stated that the protocol of biofilm formation was successful. The bioreactor oxidized 0.213 g of tetrathionate per hour when it was fed with 1.8 g/L of tetrathionate ion, and 0.390 g/h when it was fed with 3.5 g/L, which, in terms of bed volume, corresponds to productivities of 0.426 and 0.780 kg/(m3 h), respectively. The differences between tetrathionate bio-oxidation rates, obtained by increasing the concentration of this ion in feeding from 1.8 to 3.3 g/L, indicate substrate limitation; this result is similar to those obtained in batch tests discussed in Section 3.2 (Fig. 6). It has been shown that the loading protocol implemented by replacing the Fe (II) with tetrathionate once a firm biofilm is formed, achieves the objective of continuous stable bio-oxidation. This is significant in the case of industrial scaling since it would render unnecessary the creation of a large inoculum with cells adapted to tetrathionate. To the best of the authors' knowledge, the stable continuous biooxidation of tetrathionate in bioreactors with cells attached to a solid support, without the necessity of feeding cells during operation, constitutes a milestone in itself. Regarding thiosalts treatment in ponds whose residence times are in the order of weeks, the flooded packed-bed bioreactor provides a substantial improvement by reducing the operating time to the order of hours. Concerning active methods, it has been demonstrated that tetrathionate oxidation is possible in a continuous and controlled operation without the addition of any reagent, since biooxidation only needs air. 4. Conclusions and considerations It has been shown that tetrathionate bio-oxidation can be effectively performed in a flooded packed-bed reactor. The reactor has successfully operated in terms of stability and substrate conversion. The results
shown in this study offer a promising alternative for the management of effluents containing thiosalts in comparison with the methods that have hitherto been applied. Currently this study is being continued on a pilot-plant scale in order to define optimum operating conditions and to evaluate the technical feasibility of the process. A bioreactor, of 1 m in height and 0.9 m in diameter, is currently treating a flow rate of 100 L/h of mine water contaminated with tetrathionate. References Chanda, M., O'driscoll, K.F., Rempel, G.L., 1984. Catalyzed air oxidation for treatment of thiosalt effluents. Appl. Catal. 9, 291–307. Dopson, M., Johnson, D.B., 2012. Biodiversity, metabolism and applications of acidophilic sulfur-metabolizing microorganisms. Environ. Microbiol. 14 (10), 2620–2631. Doran, P.M., 2013. Bioprocess Engineering Principles. Second ed. Academic Press Elsevier, pp. 632–652. Druschel, G.K., Hamers, R.J., Banfield, J.F., 2003. Kinetics and mechanism of polythionate oxidation to sulfate at low pH by O2 and Fe3 +. Geochim. Cosmochim. Acta 67 (23), 4457–4469. Eccleston, M., Kelly, D.P., 1978. Oxidation kinetics and chemostat growth kinetics of Thiobacillus ferrooxidans on tetrathionate and thiosulfate. J. Bacteriol. 134 (3), 718–727. Friedrich, C.G., Rother, D., Bardischewsky, F., Quentmeier, A., Fischer, J., 2001. Oxidation of reduced inorganic sulfur compounds by bacteria: emergence of a common mechanism? Appl. Environ. Microbiol. 67 (7), 2873–2882. Ghosh, W., Dam, B., 2009. Biochemistry and molecular biology of lithotrophic sulfuroxidation by taxonomically and ecologically diverse bacteria and archaea. FEMS Microbiol. Rev. 33, 999–1043. González-Lara, J.M., Roca, A., Cruells, M., Patiño, F., 2009. The oxidation of thiosulfates with copper sulfate. Application to an industrial fixing bath. Hydrometallurgy 95, 8–14. Hedrich, S., Schlomann, M., Johnson, D.B., 2011. The iron-oxidizing proteobacteria. Microbiology 157, 1551–1564. Karamanev, D.G., 1991. Model of the biofilm structure of Thiobacillus ferrooxidans. J. Biotechnol. 20, 51–64. Kelly, D.P., Tuovinen, O.H., 1975. Metabolism of inorganic sulphur compounds by thiobacillus ferrooxidans and some comparative studies on thiobacillus a2 and T. neapolitanus. Plant Soil 43, 77–93. KoH, T., 1990. A review analytical chemistry of polythionates and thiosulfate. Anal. Sci. 6, 3–14. Kupka, D., Liljeqvist, M., Nurmi, P., Puhakka, J.A., Tuovinen, O.H., Dopson, M., 2009. Oxidation of elemental sulfur, tetrathionate and ferrous iron by the psychrotolerant Acidithiobacillus strain SS3. Res. Microbiol. 160, 767–774. Kurtenacker, A., Bittner, K., 1924. Iodometric analysis of a mixture of sulphide, sulphite and thiosulphate. Z. Anorg. Chem. 141, 297–303. Kuyucak, N., Yaschyshyn, D., 2007. Managing thiosalts in mill effluents: “studies conducted at the kidd metallurgical site” Paper presented at Mining and the Environment IV Conference, Sudbury, Ontario, Canada, October 19–27. Liljeqvist, M., Sundkvist, J.-E., Saleh, A., Dopson, M., 2011. Low temperature removal of inorganic sulfur compounds from mining process waters. Biotechnol. Bioeng. 108 (6), 1254–1259. Lu, Y., Gao, Q., Xu, L., Zhao, Y., Epstein, I.R., 2010. Oxygen-sulfur species distribution and kinetic analysis in the hydrogen peroxide-thiosulfate system. Inorg. Chem. 49, 6026–6034.
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Senanayake, G., 2005b. Gold leaching by thiosulphate solutions: a critical review on copper (II)–thiosulphate–oxygen interactions. Miner. Eng. 18, 995–1009. Shiers, D.W., Ralph, D.E., Watling, H.R., 2011. Batch culture of Acidithiobacillus caldus on tetrathionate. Biochem. Eng. J. 54, 185–190. Silverman, M.P., Lundgren, D.G., 1959. Studies on the chemoautotrophic iron bacterium Ferrobacillus. J. bacteriol. 78, 326–331. Sugio, T., Kanao, T., Furukawa, H., Nagasawa, T., Blake, R.C., 1996. Isolation and identification of an iron-oxidizing bacterium which can grow on tetrathionate medium and the properties of a tetrathionate-decomposing enzyme isolated from the bacterium. J. Ferment. Bioeng. 82 (3), 233–238. Tuovinen, 0.H., Kelly, D.P., 1974. Studies on the growth of Thiobacillus ferrooxidans. V. Factors affecting growth in liquid culture and development of colonies on solid media containing inorganic sulphur compounds. Arch. Microbiol. 98, 351–364.
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International Journal of Mineral Processing journal homepage: www.elsevier.com/locate/ijminpro
Treatment of tetrathionate effluents by continuous oxidation in a flooded packed-bed bioreactor Nieves Iglesias ⁎, Rafael Romero, Alfonso Mazuelos, Cristina Montes-Rosua, Francisco Carranza Departamento de Ingeniería Química, University of Seville, Spain
a r t i c l e
i n f o
Article history: Received 6 June 2014 Received in revised form 2 March 2016 Accepted 18 August 2016 Available online 24 August 2016 Keywords: Bioreactor Tetrathionate Thiosalts Continuous bio-oxidation Acidithiobacillus ferrooxidans
a b s t r a c t Thiosalts are compounds commonly present in process plant liquor for the concentration of metal sulphide ores by milling and flotation. Since thiosalts are metastable species that are oxidized to sulphuric acid as an end product, they cannot be discharged nor recycled into the process. Therefore, the development of a cost-effective process for the treatment of these effluents is necessary. In this paper, the continuous bio-oxidation of tetrathionate is carried out in a flooded packed-bed reactor by an immobilized microbial consortium. Batch tests show that the initial tetrathionate concentration and pH slightly affect the tetrathionate bio-oxidation process in the ranges 3 to 5 g/L and 1.5 to 2, respectively. The specific bio-oxidation rate is 0.08 h−1. Only sulphate ions and protons have been detected as end products. For the starting up of the bioreactor, a new method of biofilm formation for tetrathionate bio-oxidation is implemented. The maximum bio-oxidation rate in continuous operation is 0.415 gh−1 (0.830 kg h−1 m−3). The biofilm was stable for the whole period studied, i.e. 35 days. These results indicate that a flooded packed-bed reactor is an interesting option from an economic point of view for the treatment of waters contaminated by thiosalts. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Thiosalts are chemicals commonly present in process plant liquor for the concentration of metal sulphide ores by milling and flotation. Alkalinity conditions and the additives employed in these plants favour the partial conversion of sulphides into thiosalts. A common practice in this kind of facility is to recycle the liquors in process. However, the accumulation of thiosalts in liquors negatively affects the efficiency of flotation; this effect renders it necessary to continuously purge the generated thiosalts from the circuit. In these effluents, tetrathionate ion is usually the most abundant thiosalt, since it remains stable in solution at temperatures below 30 °C (Druschel et al., 2003). Thiosalts are not considered toxic in themselves; however, their solutions can have an indirect impact on the environment. The incorporation of thiosalts into a receptor aquatic body can be the cause of a decrease in pH and in the dissolved oxygen concentration, and can severely affect its chemical-biological equilibrium. This is due to a natural oxidation process whose end product is sulphuric acid. This process is catalysed by metal ions in solution (Chanda et al., 1984; González-Lara et al., 2009; Senanayake, 2005a,b) and by the metabolic action of sulphur-oxidizing microorganisms (Eccleston and Kelly, 1978; Friedrich et al., 2001; Kelly and Tuovinen, 1975; Shiers et al., 2011),
⁎ Corresponding author. E-mail address: [email protected] (N. Iglesias).
http://dx.doi.org/10.1016/j.minpro.2016.08.010 0301-7516/© 2016 Elsevier B.V. All rights reserved.
commonly present in these mining environments (Dopson and Johnson, 2012; Ghosh and Dam, 2009; Hedrich et al., 2011). These are frequently Acidithiobacillus genus bacteria, of which the most abundant species is usually Acidithiobacillus ferrooxidans. Currently, the purification of effluents containing thiosalts consists of their oxidation in ponds (Kuyucak and Yaschyshyn, 2007). This is a simple and economic but very slow method, which leads to designs characterized by very large areas. One of the main inconveniences of these ponds is their low activity in cold seasons. Alternatively, several active treatments have been developed that consist of adding oxidants, such as Caro's acid (H2SO5), Fenton reagent, ozone, and hypochlorite (Kuyucak and Yaschyshyn, 2007; Lu et al., 2010).These methods are faster and more controllable but involve very high reagent costs, potentially dangerous operation, and include the incorporation of undesirable substances into the environment. The controlled biological oxidation of thiosalts is postulated as an attractive future option. This option will be developed in bioreactors designed to hold high concentrations of cells and to operate continuously. Very few reactors have been described in the literature for this purpose (Miranda-Trevino et al., 2013). Liljeqvist et al., 2011 tested a bioreactor design that included a biogenerator and a main reactor containing A. ferrivorans attached to a structured plastic bed. Despite containing a biofilm carrier, this reactor needs to be assisted by an external supply of cells throughout its operation. A bioreactor for the continuous bio-oxidation of thiosalts should meet the needs of treatment, with a size that enables the control of
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the variables (mainly temperature) and the stability of operation. In addition, its construction should be economical and simple. Flooded packed-bed bioreactors have been developed for the oxidation of ferrous ion. In flooded packed-bed bioreactors, if the energetic substrate is Fe (II), then it is possible to reach steady states and very high oxidation rates without any supply of cells (Mazuelos et al., 1999 and 2000). In these bioreactors, biomass is immobilized in a biofilm that consists of an inorganic matrix, formed by precipitated ferric compounds (mainly oxyhydroxides and jarosites), in the pores of which cells are attached. This biofilm covers the surface of particles that form the bed (Karamanev, 1991; Mazuelos et al., 2012). In these bioreactors, biofilm is very robust against changes in composition and dynamic fluid conditions. The present work aims to study the oxidation of tetrathionate in a flooded packed-bed reactor with the following objectives: 1) To achieve tetrathionate oxidation in a stable operation over time. 2) To establish a simple and quick methodology for the commissioning of bioreactors, consistent with the above objective. 3) To lay the foundation for optimization and piloting studies for the implementation of this technology.
2. Materials and methods 2.1. Batch tetrathionate bio-oxidation All cultures performed in the present work originate from a mixed culture obtained from the effluents from the Rio Tinto Mines (Huelva, Spain). This culture has been maintained for decades by successive inoculations in 9K medium (Silverman and Lundgren, 1959). The composition of 9K medium, in which the energetic substrate is Fe (II), is shown in Table 1. Phylogenetic analysis of the culture shows the three major phylotypes to be Acidithiobacillus, Leptospirillum, and Ferrimicrobium. The dominant microbial species is Acidithiobacillusferrooxidans _ATCC23270 (Mazuelos et al., 2012). The culture was pelletized in order to remove the iron. Pellets of cells were prepared by centrifugation at 12,000g for 15 min in a Sorvall SS 3 Automatic Centrifuge Du Pont Instruments. The decanted cells were washed and resuspended in tetrathionate medium, whose composition is shown in Table 1. In order to ascertain the species originated during bio-oxidation and to study the influence of pH and of tetrathionate concentration, batch assays were performed in sterilized 250 mL Erlenmeyer flasks, placed in an orbital shaker at 180 rpm and at a temperature of 31 °C. Into these flasks, 96 mL of tetrathionate culture medium, modified in terms of pH and in the concentration of Na2S4O6·2H2O (see Table 1), was added. Four initial concentrations of tetrathionate (2, 3, 3.5 and 5 g/L) and three initial pH values (1.5, 2 and 3), adjusted with dilute sulphuric acid, were studied.
To follow the evolution of cultures, pH, and concentrations of tetrathionate, the thiosulphate and sulphite were measured over time. To detect intermediate species, Ionic Chromatography was used. 2.2. Continuous tetrathionate bio-oxidation The continuous bio-oxidation of tetrathionate was performed in a flooded packed-bed reactor (Mazuelos et al., 1999) by immobilized cells. This reactor is a column of 15 cm in height and 8.4 cm in diameter. It consists of a lower chamber, (hollow cylindrical, 5 cm in height), and an upper chamber (the bed) of 10 cm in height (Fig. 1). In the lower chamber, two nozzles are placed for air and liquid medium inlet. The liquid rises through the bed and leaves the reactor via the overflow. The bed consists of siliceous sand particles between 6 and 8 mm in size. The bed porosity is 0.45. The liquid is fed by a peristaltic pump and the air by a small compressor controlled by a rotameter. Unless specified otherwise, the liquid and air flows tested were 150 mL/h and 750 mL/min, respectively. The reactor operated in a chamber with the thermostat set at 31 °C. For the starting of the bioreactor, a new method of biofilm formation for tetrathionate bio-oxidation was implemented. The method was developed in the following stages: a. Biofilm formation for the Fe(II) bio-oxidation by the Mazuelos et al. (2000) method. The medium used was 9K and the inoculum was the RT culture. b. Continuous operation feeding of 9K medium modified in pH. The pH was adjusted to 1.7 with sulphuric acid to prevent an excessive iron precipitation. c. Change of feed from 9K medium to modified tetrathionate medium. The tetrathionate medium was modified in its concentration of Na2S4O6·2H2O to 2.5 g/L and its pH to 1.7. d. Batch bio-oxidation. In this stage, the feeding pump is stopped while the reactor is maintained aerated. This serves as a test of whether attached cells accept tetrathionate as energetic substrate. e. Recirculating bio-oxidation. The reactor was fed from an external reservoir of 2.5 L containing modified tetrathionate medium. In this tank, the initial concentration of Na 2 S 4 O .6 2H 2 O was 2.5 g/L. The effluent from the bioreactor was returned to the reservoir. The initial pH in the tank was set to 2.8 to prevent an excessive fall in pH of the bioreactor, which could mean substantial dissolution of the precipitated iron that formed the matrix of the biofilm.
8.4 cm
outlet stream by overflow
siliceous
6-8 mm stone
10 cm Table 1 Chemical composition of culture media used in this study. Salt (g)
9K
Tetrathionate
(NH4)2SO4 KCl K2HPO4 MgSO4·7H2O Ca(NO3)2 CaCl2 Na2S4O6·2H2O FeSO4·7H2O H2O (mL)
3.0 0.1 0.5 0.5 0.01 300 mL (14.7%) 700
3.0 3.0 0.5 0.2 4.0 1000
Inlet solution
5 cm
Biofilm (ferric precipitates and cells)
air
Fig. 1. Flooded packed-bed bioreactor used in this study. On the right, a portion of the packed bed is schematically shown; the siliceous stone particles bear the biofilm consisting of bacteria and a matrix of ferric precipitates.
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a
b
c
Fig. 2. a) Chromatogram of culture F at time t = 0; b) Expanding the left-hand side. The peaks at 1.7 and 4 min correspond to chloride ion and dihydrogen phosphate, respectively; c) Expanding the right-hand side, the tetrathionate peak can be clearly observed.
Once the protocol of biofilm formation was concluded, the continuous operation of bioreactor was tested with two different feeds, both at pH 2.8: 1) Modified tetrathionate medium of 2.5 g/L of Na2S4O6·2H2O (1.8 g/L of tetrathionate ion). Operation time 600 h 2) Modified tetrathionate medium of 4.5 g/L Na2S4O6·2H2O (3.3 g/L of tetrathionate ion). Operation time 250 h 2.3. Analysis 2.3.1. Chemical analysis Thiosulphate and sulphite ions were analysed by iodometry; discrimination between the two ions was achieved by masking the sulphite ion with formaldehyde. (Kurtenacker and Bittner, 1924; KoH, 1990). The total concentration of thiosalts was determined by the acidimetric method with mercury chloride (Makhija and Hitchen,
1978). Tetrathionate concentration was expressed as the concentration of total thiosalts minus the concentrations of sulphite and thiosulphate. The pH was measured by a WTW-3310 pH-meter equipped with software for multi-buffer calibration and a combined glass electrode with Sentix 41 automatic temperature compensation. The ferrous iron concentration was determined with 0.3 M potassium dichromate by an automatic titrator with potentiometric control of the endpoint (Radiometer-Copenhagen). 2.3.2. Ion chromatography (IC) The 883 Basic IC plus with chemical suppression by Metrohm Suppressor Module (MSM) was used for the ion chromatographic determinations of polythionates (tetrathionate, trithionate, dithionate), sulphate, thiosulphate, and sulphite ions. The MSM was used for chemical suppression during anion analysis. This analysis consisted of 3 suppressor units in total, regenerated with 250 mM sulphuric acid or rinsed with ultra-pure water in rotation.
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a
b
c
Fig. 3. a) Chromatogram of culture F at time t = 48 h; b) Expanding the left-hand side. The peaks at 1.7 and 4 min correspond to chloride ion and dihydrogen phosphate, respectively; c) Expanding the right-hand side, the tetrathionate peak cannot be observed.
A 50 mm separation column was used. The eluent was 3.2 mM Na2CO3 with a flow rate of 0.7 mL/min. Baseline conductivity 12.3–12.6 μS/cm. Magic Net Basic was the software used for the calculation of peak areas. 3. Results and discussion 3.1. Batch tetrathionate bio-oxidation Batch tests were performed in order to establish conditions in continuous operations. A. ferrooxidans metabolizes tetrathionate ions at pH ranges between 1.33 and 4.44 (Eccleston and Kelly, 1978; Kupka et al., 2009; Tuovinen and Kelly, 1974). The overall stoichiometry of this process is given by the equation 2S4 O6 2− þ 7O2 þ 6H2 O→8SO4 2− þ 12Hþ
ðr1Þ
Although the reaction pathway for tetrathionate oxidation should yield sulphate as a final product, in order to detect intermediate species generated in tetrathionate bio-oxidation, the evolution of concentrations of tetrathionate, trithionate, dithionate, sulphate, thiosulphate, and sulphite ions were analysed by ion chromatography over time. As representative examples, chromatograms corresponding to the beginning and to the end of a culture containing 3 g/L of tetrathionate are shown in Figs. 2 and 3. Fig. 2 shows the chromatogram of the culture at the initial time. Extended chromatograms (Fig. 2b and c) clearly show the peaks of chloride, dihydrogen phosphate, sulphate, thiosulphate, and tetrathionate. Fig. 3 shows the chromatogram of the same culture at 48 h. Enlargements of this chromatogram (Fig. 3b and c) show peaks of chloride, di-hydrogen phosphate and sulphate, but not of thiosulphate nor tetrathionate. Initially, sulphate, chloride, and di-hydrogen phosphate, which come from the culture medium, and traces of thiosulphate, probably
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4
[tetrathionate] (g/L)
3.5 3 2.5 2 1.5 1 0.5 0 0
6
12
18
24
30
36
42
48
time (hours) A
B
C
Fig. 4. Evolution of tetrathionate concentration for batch cultures: A (initial pH 3), B (initial pH 2) and C (initial pH 1.5). Reactor: Erlenmeyer flask. Inoculum: Pellet of RTT culture. Medium: modified tetrathionate medium. Initial tetrathionate concentration 3.5 g/L. Shaking speed: 180 rpm. Temperature: 31 °C.
as a tetrathionate impurity, were detected. With the advancement of time, peaks of tetrathionate and thiosulphate disappeared and only sulphate peak remained. In no case were dithionate nor trithionate peaks observed (they should have been visible at 15.7 and 30 min, respectively). These results are in accordance with Reaction 1 in which sulphate and protons are end products. Figs. 4 and 5 present the evolution of tetrathionate concentration and the pH versus time, respectively, for tests with different initial pH values (3, 2, and 1.5) and the same initial concentration of tetrathionate (3.5 g/L). In Fig. 4, it can be observed that tetrathionate is completely consumed in less than 48 h in all cases. A clear decrease of pH was observed in Fig. 5 that corresponds with the decrease in the tetrathionate concentration (Reaction r1). The final pH in the cultures is close to 1.5 since the medium is buffered by the sulphate/bisulphate balance. Fig. 6 shows the evolution of tetrathionate concentration versus time for tests, in which the variable is the initial tetrathionate concentration (5, 3.5 and 2 g/L) and the initial pH is 3.
No presence of thiosulphate or sulphite was detected in any of the tests. In all cultures, the tetrathionate was completely consumed in 42–48 h. The lack of intermediates allows a conventional kinetic characterization based on the postulate of balanced growth in the exponential growth phase, where specific growth rate and tetrathionate biooxidation rate are constant. In an environment favourable for growth, cells regulate their metabolism and adjust the rates of various internal reactions so that a condition of balanced growth occurs (Doran, 2013). The semi-logarithmic plot of substrate consumption versus time, (Ln([S4O 26 −]o-[S4O 26 −]) versus time) is a straight line whose slope, the specific growth rate, is characteristic of each culture (inoculum, medium, and operation conditions). Table 2 shows the initial values of pH and tetrathionate concentration for each test together with the specific bio-oxidation rate calculated from the semi-logarithmic plot. The correlation coefficients of linear fit (R2) are also shown in Table 2. From a kinetic point of view, the worst case corresponds to the culture performed with the lowest concentration of tetrathionate, probably due to substrate limitation. In the remaining assays, the specific rates are very similar and present an average value of 0.079 h−1 and a standard
3.5 3 2.5
pH
2 1.5 1 0.5 0 0
6
12
18
24
30
36
42
48
time (hours) A
B
C
Fig. 5. Evolution of pH for batch cultures: A (initial pH 3), B (initial pH 2) and C (initial pH 1.5). Reactor: Erlenmeyer flask. Inoculum: Pellet of RTT culture. Medium: modified tetrathionate medium. Initial tetrathionate concentration 3.5 g/L. Shaking speed: 180 rpm. Temperature: 31 °C.
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5 4.5
[tetrathionate] (g/L)
4 3.5 3 2.5 2 1.5 1 0.5 0 0
6
12
18
24
30
36
42
48
time (hours) A
E
D
Fig. 6. Evolution of tetrathionate concentration for batch cultures: A (3.5 g/L of tetrathionate), D (5 g/L of tetrathionate) and E (2 g/L of tetrathionate). Reactor: Erlenmeyer flask. Inoculum: Pellet of RTT culture. Medium: modified tetrathionate medium. Initial pH 3. Shaking speed: 180 rpm. Temperature: 31 °C.
3.2. Continuous bio-oxidation
Table 2 Initial conditions and specific bio-oxidation rate of tetrathionate for batch cultures. Culture
pH
[Tetrathionate](g/L)
Specific bio-oxidation rate (h−1)
R2
A B C D E F
3 2 1.5 3 3 1.9
3.5 3.5 3.5 5 2 3
0.075 0.078 0.081 0.083 0.054 0.079
0.9979 0.9925 0.9984 0.9907 0.9903 0.9971
deviation (SD) of 0.003, which reveals a low impact of pH and tetrathionate concentration within the ranges tested for these variables. In accordance with the literature, the results show that A. ferrooxidans previously grown on Fe(II), can adapt its metabolic system using the tetrathionate ion as an energy substrate (Kelly and Tuovinen, 1975; Sugio et al., 1996; Ghosh and Dam, 2009).
Continuous tetrathionate bio-oxidation was tested in a flooded packed-bed bioreactor with attached cells. After biofilm formation using 9K medium and RT culture as inoculum, the bioreactor was continuously fed with 9K medium modified in pH (pH = 1.7) for 192 h. The maximum bio-oxidation of Fe(II) rate was 970 mg/h (stage b). The feed was then changed to modified tetrathionate medium (2.5 g/L, pH = 1.7). This stage lasted 43 h, longer than 12 times the mean residence time of the liquor in the reactor. In this step, dissolved iron and the cells not attached to the support were evacuated. The feeding pump was then stopped while the reactor was maintained aerated until all the tetrathionate had been oxidized. This step ended after 48 h and served as a test that the attached cells accept tetrathionate as energetic substrate. Finally, fresh feed was recycled into the reactor for 72 h, the time necessary for tetrathionate oxidation. Once the protocol of biofilm formation was concluded, the bioreactor was continuously fed with modified tetrathionate medium of
4 3.5
[tetrathionate] (g/L)
3 2.5 2 1.5 1 0.5 0 0
100
200
300
400
500
600
700
800
900
time (hours) inlet solution
outlet stream
Fig. 7. Continuous tetrathionate bio-oxidation in flooded packed-bed bioreactor. Tetrathionate ion concentration in inlet and outlet streams. Liquid flow rate 150 mL/h. Air flow rate 760 mL/min.
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tetrathionate biooxidation rate (mg/h)
450 400 350 300 250 200 150 100 50 0 0
100
200
300
400 500 time (hours)
600
700
800
900
Fig. 8. Continuous tetrathionate bio-oxidation in flooded packed-bed bioreactor. Tetrathionate bio-oxidation rate in g/h. Liquid flow rate 150 mL/h. Air flow rate 760 mL/min.
2.5 g/L of Na2S4O6·2H2O (1.8 g/L of tetrathionate ion) and pH 2.8. After 600 h of operation, the concentration of Na2S4O6·2H2O was increased to 4.5 g/L (3.3 g/L of tetrathionate ion). The results of continuous operation are shown in Figs. 7 and 8. Fig. 7 shows the concentration of tetrathionate ion in inlet and outlet bioreactor streams and Fig. 8 shows the tetrathionate bio-oxidation rate versus operation time. Note that the bioreactor is stable throughout continuous operation. Variations observed in the bio-oxidation rate in the initial hours (Fig. 8) are due to fluctuations in the flow supplied by the pump at the start of the continuous operation. The bioreactor continuously operated in a stable manner for 35 days. In terms of substrate conversion and stability, it can be stated that the protocol of biofilm formation was successful. The bioreactor oxidized 0.213 g of tetrathionate per hour when it was fed with 1.8 g/L of tetrathionate ion, and 0.390 g/h when it was fed with 3.5 g/L, which, in terms of bed volume, corresponds to productivities of 0.426 and 0.780 kg/(m3 h), respectively. The differences between tetrathionate bio-oxidation rates, obtained by increasing the concentration of this ion in feeding from 1.8 to 3.3 g/L, indicate substrate limitation; this result is similar to those obtained in batch tests discussed in Section 3.2 (Fig. 6). It has been shown that the loading protocol implemented by replacing the Fe (II) with tetrathionate once a firm biofilm is formed, achieves the objective of continuous stable bio-oxidation. This is significant in the case of industrial scaling since it would render unnecessary the creation of a large inoculum with cells adapted to tetrathionate. To the best of the authors' knowledge, the stable continuous biooxidation of tetrathionate in bioreactors with cells attached to a solid support, without the necessity of feeding cells during operation, constitutes a milestone in itself. Regarding thiosalts treatment in ponds whose residence times are in the order of weeks, the flooded packed-bed bioreactor provides a substantial improvement by reducing the operating time to the order of hours. Concerning active methods, it has been demonstrated that tetrathionate oxidation is possible in a continuous and controlled operation without the addition of any reagent, since biooxidation only needs air. 4. Conclusions and considerations It has been shown that tetrathionate bio-oxidation can be effectively performed in a flooded packed-bed reactor. The reactor has successfully operated in terms of stability and substrate conversion. The results
shown in this study offer a promising alternative for the management of effluents containing thiosalts in comparison with the methods that have hitherto been applied. Currently this study is being continued on a pilot-plant scale in order to define optimum operating conditions and to evaluate the technical feasibility of the process. A bioreactor, of 1 m in height and 0.9 m in diameter, is currently treating a flow rate of 100 L/h of mine water contaminated with tetrathionate. References Chanda, M., O'driscoll, K.F., Rempel, G.L., 1984. Catalyzed air oxidation for treatment of thiosalt effluents. Appl. Catal. 9, 291–307. Dopson, M., Johnson, D.B., 2012. Biodiversity, metabolism and applications of acidophilic sulfur-metabolizing microorganisms. Environ. Microbiol. 14 (10), 2620–2631. Doran, P.M., 2013. Bioprocess Engineering Principles. Second ed. Academic Press Elsevier, pp. 632–652. Druschel, G.K., Hamers, R.J., Banfield, J.F., 2003. Kinetics and mechanism of polythionate oxidation to sulfate at low pH by O2 and Fe3 +. Geochim. Cosmochim. Acta 67 (23), 4457–4469. Eccleston, M., Kelly, D.P., 1978. Oxidation kinetics and chemostat growth kinetics of Thiobacillus ferrooxidans on tetrathionate and thiosulfate. J. Bacteriol. 134 (3), 718–727. Friedrich, C.G., Rother, D., Bardischewsky, F., Quentmeier, A., Fischer, J., 2001. Oxidation of reduced inorganic sulfur compounds by bacteria: emergence of a common mechanism? Appl. Environ. Microbiol. 67 (7), 2873–2882. Ghosh, W., Dam, B., 2009. Biochemistry and molecular biology of lithotrophic sulfuroxidation by taxonomically and ecologically diverse bacteria and archaea. FEMS Microbiol. Rev. 33, 999–1043. González-Lara, J.M., Roca, A., Cruells, M., Patiño, F., 2009. The oxidation of thiosulfates with copper sulfate. Application to an industrial fixing bath. Hydrometallurgy 95, 8–14. Hedrich, S., Schlomann, M., Johnson, D.B., 2011. The iron-oxidizing proteobacteria. Microbiology 157, 1551–1564. Karamanev, D.G., 1991. Model of the biofilm structure of Thiobacillus ferrooxidans. J. Biotechnol. 20, 51–64. Kelly, D.P., Tuovinen, O.H., 1975. Metabolism of inorganic sulphur compounds by thiobacillus ferrooxidans and some comparative studies on thiobacillus a2 and T. neapolitanus. Plant Soil 43, 77–93. KoH, T., 1990. A review analytical chemistry of polythionates and thiosulfate. Anal. Sci. 6, 3–14. Kupka, D., Liljeqvist, M., Nurmi, P., Puhakka, J.A., Tuovinen, O.H., Dopson, M., 2009. Oxidation of elemental sulfur, tetrathionate and ferrous iron by the psychrotolerant Acidithiobacillus strain SS3. Res. Microbiol. 160, 767–774. Kurtenacker, A., Bittner, K., 1924. Iodometric analysis of a mixture of sulphide, sulphite and thiosulphate. Z. Anorg. Chem. 141, 297–303. Kuyucak, N., Yaschyshyn, D., 2007. Managing thiosalts in mill effluents: “studies conducted at the kidd metallurgical site” Paper presented at Mining and the Environment IV Conference, Sudbury, Ontario, Canada, October 19–27. Liljeqvist, M., Sundkvist, J.-E., Saleh, A., Dopson, M., 2011. Low temperature removal of inorganic sulfur compounds from mining process waters. Biotechnol. Bioeng. 108 (6), 1254–1259. Lu, Y., Gao, Q., Xu, L., Zhao, Y., Epstein, I.R., 2010. Oxygen-sulfur species distribution and kinetic analysis in the hydrogen peroxide-thiosulfate system. Inorg. Chem. 49, 6026–6034.
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Senanayake, G., 2005b. Gold leaching by thiosulphate solutions: a critical review on copper (II)–thiosulphate–oxygen interactions. Miner. Eng. 18, 995–1009. Shiers, D.W., Ralph, D.E., Watling, H.R., 2011. Batch culture of Acidithiobacillus caldus on tetrathionate. Biochem. Eng. J. 54, 185–190. Silverman, M.P., Lundgren, D.G., 1959. Studies on the chemoautotrophic iron bacterium Ferrobacillus. J. bacteriol. 78, 326–331. Sugio, T., Kanao, T., Furukawa, H., Nagasawa, T., Blake, R.C., 1996. Isolation and identification of an iron-oxidizing bacterium which can grow on tetrathionate medium and the properties of a tetrathionate-decomposing enzyme isolated from the bacterium. J. Ferment. Bioeng. 82 (3), 233–238. Tuovinen, 0.H., Kelly, D.P., 1974. Studies on the growth of Thiobacillus ferrooxidans. V. Factors affecting growth in liquid culture and development of colonies on solid media containing inorganic sulphur compounds. Arch. Microbiol. 98, 351–364.