Zeta potential of silver absorbing Thiobacillus ferrooxidans

Minerals Engineering, Vol. 11, No. 2, pp. 189--194, 1998

Pergamon 0892-6875(97)00150-7

© 1998 Elsevier Science Ltd All rights rcservvd. Printed in Great Britain 0892--6875/98 $19.00+0.00

ZETA POTENTIAL OF SILVER ABSORBING THIOBA CILLUS FERROOXIDANS

R.J. WEST, G.M. STEPHENS and J.J. CILLIERS Department of Chemical Engineering, UMIST, Manchester, M60 1QD, U.K. E-mail: j.j.cilliers @umist.ac.uk (Received 2 September 1997; accepted 20 October 1997)

ABSTRACT Thiobacillus ferrooxidans was grown in shake flask cultures, using powdered sulphur as the energy source. Growth of cultures was followed by monitoring the pH of the medium. Low levels (lOppm) of soluble silver nitrate (AgN03) were introduced to some cultures, where it was absorbed by the bacteria. Silver absorption resulted in a lag time in the growth phase, but not a decrease in the subsequent growth rate, and a change in the colour of the culture from bright sulphur yellow to dark grey. Zeta potentials for both these silver-loaded bacteria and a control culture were measured using a particle microelectrophoretic technique. The zeta potentials of the control bacteria were founzt to be consistent with those recorded by other workers, with an IEP at pH 2.4. Zeta potentials of silver loaded bacteria were found to be very different, exhibiting an IEP below pH 2.0, a minimum zeta potential of -32mV at pH 7.0, and an increase in zeta potential at higher pH conditions. The zeta potential of pure silver sulphide was also measured, and fOund to have an IEP similar to that of the control culture. Between the IEP and pH 5, the zeta potential of the silver loaded bacteria is close to the sum of the potentials of the control bacteri and the pure mineral. It is postulated that the enhanced zeta potential is due to the additional surface potential of the silver sulphide precipitates. The results raise the possibility of selective recovery of silver-absorbing bacteria, and a novel silver recovery system. © 1998 Elsevier Science Ltd. All rights reserved

Keywords Bacteria; biotechnology; surface modification

INTRODUCTION T. ferrooxidans is a chemolithotrophic autotrophic bacteria, which can oxidise ferrous ions, sulphur, or reduced sulphur compounds for energy, whilst fixing CO 2 as its sole source of carbon. It is important in the bioleaching and biomining industries, due to its ability to release metals into solution, and its good resistance to many metal cations. The bacterium is Gram negative, acidophilic, mesophilic, and aerobic. A comprehensive review of the current understanding of the bacteria has been produced [1]. 189

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Zeta potentialof Thiobacillusferrooxidans

When T. ferrooxidans is using a solid energy source it can grow in the presence of what should be lethal concentrations of silver in solution [2]. Pooley [3] observed that this ability to survive was coupled to the bacteria changing colour, and he found that this was due to the bacteria absorbing the silver and nucleating it intracellularly. Pooley and Shrestha [4] found that the silver was nucleated throughout the bacteria as silver sulphide, although they were unable to determine if it was crystalline in structure. Dry weight determinations carried out during the course of their work also found that up to 20% of the dry weight of the silver-loaded-bacteria was silver sulphide. Whilst the variation of zeta potential with pH of non-silver-loaded bacteria has been previously determined [5,6], the effect that silver-loading may have on the zeta potential of the bacteria, and whether this effect may be useful, has yet to be reported. This work reports the effect of the silver, and discusses possible uses.

MATERIALS AND METHODS

T. ferrooxidans NCIMB 9490 was grown in 9K medium [7], containing 100 g 1-~ powdered sulphur as the energy source. Cultures were grown on an orbital shaker as shake flask cultures (100ml) at 200 rpm and 30°C. To avoid melting the sulphur, sterilisation of the cultures was achieved by tyndallising the entire culture at 105°C for 30 minutes on two successive days. Where required, silver was added to the cultures from a silver nitrate stock solution of 1000 ppm, at the rate of lmg per culture per day. This rate of silver addition maintained the silver concentration at well below the maximum that the bacteria can survive (approximately 70ppm) [2]. Growth was followed by measuring the pH of the medium during incubation, pH provides a ready indicator of growth of T. ferrooxidans when using sulphur as the energy source, because sulphur is oxidised to sulphuric acid during growth, thus reducing the pH [1]. Bacteria for zeta potential measurements were harvested by allowing the cultures to stand for at least 10 minutes to allow the sulphur to settle, and removing lml aliquots of the supernatant. These aliquots were then centrifuged in a microfuge at 13,000 rpm for up to 40 minutes, until a pellet had formed and the supernatant was clear. The supernatant was removed, and the pellets resuspended in distilled water. Two such pellets were suspended in 20ml of distilled water for each zeta potential sample. Zeta potentials were calculated, using the Smoluchowski equation, from electrophoretic mobilities determined on a Rank Brothers machine, using a flat cell and a constant voltage of 50V. The time taken to travel 115 }am was recorded for ten separate bacteria, with the electrical field applied in either direction, and an average velocity determined from the average of these times. The samples were suspended in an electrolyte of total ionic strength of 0.002M. pH was adjusted using 0.05M HC1 or NaOH as appropriate, and the ionic strength made up using 0.05M NaC1 solution. The zeta potential of laboratory grade Ag2S was determined as for the bacteria. Particles were wet ground before testing.

RESULTS AND DISCUSSION

Sulphur-Containing Cultures To allow the bacteria to acclimatise to sulphur as the energy source, two stock cultures were inoculated from a culture grown in a ferrous solution. The change in pH of these sulphur-containing cultures is shown in Figure 1. There was a lag phase of about 3 days, before any significant growth was observed, which was not unexpected for bacteria adapting to growth on a new energy source. Most of the growth occurred during days 3 to 6, after which growth slowed significantly. The measured growth was consistent in pH and rate

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with that previously observed [2,4]. 2.5

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Fig. 1 Growth of T. ferrooxidans on sulphur. When the medium had first been made up, the sulphur had not easily become wetted, remaining on the surface in large clumps. This remained so until growth began, when the sulphur began to change in appearance. Instead of remaining on the surface, the sulphur became wetted, and was suspended in the medium during shaking, but settled readily on standing. This yellow precipitate was found to contain sulphur and bacteria when viewed under the microscope and remained in the cultures even after extended periods of incubation (25 days). Whilst T. ferrooxidans needs to be absorbed onto the surface of the sulphur particles to grow [5], it has also been shown that soluble reduced sulphur compounds (mostly thiosulphate) can, and do, support bacterial growth in free suspension, although only after incubation for several days [8]. This was the case for these cultures, as, after allowing the cultures to stand for about 10 minutes, the supernatant became opaque, and the presence of free bacteria was confirmed by microscopic examination of the supernatant. Silver-Containing Cultures Two cultures were inoculated from the cultures grown on sulphur. Silver was added to one culture and growth progress followed by measuring pH (Figure 2). Addition of silver to cultures containing sulphur resulted in an elongated lag phase, and whilst this phenomenon has been recorded by others [2,4], the lag phase observed during these experiments was longer. The likely reason for this is that the cultures had not been acclimatised to low levels of silver beforehand, as in that work. There were clear differences between the cultures containing both silver and sulphur, and those containing only sulphur. The culture containing only sulphur grew much faster than the silver-containing culture. In the silver containing culture, there was an elongated lag phase of about 9 days (Figure 2) before significant growth was observed. After this lag phase, the silver- containing culture grew at a rate comparable to the culture containing only sulphur. This indicates that once the bacteria had adjusted to the presence of the silver, growth rates can be maintained. There were marked differences in the appearances of the cultures during their growth periods. The culture that had silver addexi was observed to change colour after several days incubation, from bright yellow to a dark grey colour, as previously observed [4]. This is due to the bacteria accumulating silver sulphide [4]. This discolouration increased over the growth period. The colour was not only in the sediment, which settled out on standing, but also in the supernatant and centrifuge pellets. Under microscopic examination, the

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silver-loaded bacteria were found to be different in appearance, being somewhat larger and more readily visible without staining. 2.5

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Fig.2 Growth of T. ferrooxidans on sulphur, in the presence and absence of silver.

Zeta Potentials

Bacteria were harvested from the supernatant of the silver containing and control cultures by centrifugation in a desk microfuge. Non-silver-loaded-bacteria required about 40 minutes in the microfuge, at 13,000 rpm, to sediment, whereas silver-loaded-bacteria could be sedimented in 20 minutes. When placed in the microfuge tubes, both bacterial suspensions were coloured; the sulphur-grown-bacteria a pale yellow and the silver-loaded-bacteria a dark grey. After centrifugation, the pellets exhibited these same characteristics. Zeta potentials for both silver-loaded-bacteria and non-silver-loaded-bacteria were determined. Samples of non-silver-loaded-bacteria were examined at three pH values. Samples of silver-loaded-bacteria were examined at ten pH values. The results for the silver containing and control cultures are shown in Figure 3. The non-silver-loaded-bacteria were found to have an IEP (PZC) at pH 2.4, and the zeta potential continued to become more negative with increasing pH, until at pH 11 it had reached almost -40mV. These zeta potentials closely correspond to those determined before for T. ferrooxidans grown on pyrite [6], although the IEP determined in that study is slightly higher. A closely corresponding IEP at pH 2.4 was found [6] when the growth took place using iron as the feedstock. Devasia et al. [5] identified the IEP of T. ferrooxidans grown on sulphur at pH 3.8, but quote only electrophoretic mobilities, thus not allowing comparison at other pH values. For the silver-loaded bacteria the trend in zeta potential with pH was quite different. The low pH required to determine the IEP could not be used in the experiment due to the high ionic concentration. The IEP was estimated to be in the pH range 1.0 - 1.5, which was considerably lower than for the control culture. At higher pH values, the surface charge became more negative, as expected. A minimum zeta potential of -32mV was found at pH 7. At lower pH values the variation in zeta potential with pH was large, and small changes in pH gave rise to large changes in the zeta potential :of the bacteria. Between pH 4 and 8 the change in zeta potential with pH was relatively small, and above pH 8 the zeta potential increased with pH. A reversal of zeta potential at high pH values has been observed previously in bacterial systems [9].

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These results show that the zeta potential of the silver-loaded-bacteria can be readily manipulated by adjusting the pH of the suspension, and especially so at pH values below 5. Since T. ferrooxidans is a strict acidophile, exhibiting optimal growth between pH 1.0 and 5.0, and not at all above pH 7.0 [1], this low pH region is of interest in the industrial application of the bacteria.

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Fig.3 Zeta potentials of T. ferrooxidans grown in the presence and absence of silver, and of pure Ag2S. For the pure AgeS, the zeta potential decreased monotonically from 21.7 mV at pH 1.3 to -49.4 mV at pH 10.3. An increase to -38.1 mV at pH 11.9 was observed. The IEP in this case is between pH 2.1 and 3.2, corresponding closely to that of the control culture. It appears that in the pH range between the IEP and a pH of approximately 5, that the zeta potential of the silver-loaded bacteria is approximately equal to the sum of the potentials of the control culture and the pure mineral. Above this pH, when the zeta potential reversal of the silver-loaded bacteria comes into effect, this relationship does not hold. Gadd and Griffiths [10] describe two methods by which bacteria overcome heavy metal toxicity; nonspecific binding of 'the metal to cell surfaces, and metabolism-dependent inlracellular uptake. An example of this mechanism is that of some yeast strains which have the ability to precipitate thallium, as thallium oxide, within their mitochondria [10]. Investigation of micrographs of silver-absorbing bacteria [4] show wide distribution of the particles formed throughout the cell. Particles are found on the cell surface, in the cellular interior and can also be observed between the outer membranes of the bacteria. This indicates that metabolism-dependent intracellular uptake is a likely mechanism. Gadd and Griffiths [10] suggest that this may have a genetic origin, either from the DNA of the microorganism, or from a plasmid. It is therefore likely that T. ferrooxidans possesses a metabolic pathway which allows it to remove silver from solution and precipitate it as silver sulphide precipitates. Since all strains of T. ferrooxidans exhibit this ability, it is likely that the required genes are contained within the genome of the bacteria, rather than that of a plasmid. The enhanced zeta potential of the silver absorbing bacteria can therefore be postulated to arise from the additional surface potential of the surface silver sulphide precipitates in addition to that of the bacteria themselves.

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Zeta potential of Thiobacillusferrooxidans

CONCLUSIONS The modification of the surface characteristics of T. ferrooxidans by growth in the presence of silver allows the zeta potential to be easily modified and altered from that of bacteria grown in the absence of silver. It has been shown that the zeta potential of the bacteria is enhanced, possibly due to the additional potential of the surface silver sulphide precipitates. This raises the possibility of applying a surface charge dependent separation system (e.g. selective flocculation or flotation), which could be the starting point for a novel silver recovery system. There is clearly much scope for the industrial use of this unusual bacterial phenomenon, but further work is necessary before it can become industrially viable and useful.

REFERENCES

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Leduc, L.G. & Ferroni, G.D., (1993) The chemolithotrophic bacterium Thiobacillus ferrooxidans.

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Norris, P.R. & Kelly, D.P., (1978) Toxic metals in leaching systems. In: Metallurgical Applications of Bacterial Leaching and Related Microbiological Phenomena, 83-102. Eds. Murr, L.E., Torma, A.E. and Brierley, J.A., London: Academic Press. Pooley, F.D., (1982) Bacteria accumulate silver during leaching of sulphide ore minerals. Nature. 296, 642. Pooley, F.D. & Shrestha, G.N., (1996) The distribution and influence of silver in pyrite bacterial leaching systems. Min. Eng., 9, 825-836. Devasia, P., Natarajan, K.A., Sathyanarayana, D.N. & Ratmananda Rao, G. (1993) Surface chemistry of Thiobacillus ferrooxidans relevant to adhesion on mineral surfaces. Appl. Env. Microbiol., 59, 4051-4055. Misra, M., Bukka, K. & Chen, S., (1996) The effect of growth medium of Thiobacillus ferrooxidans on pyrite flotation. Min. Eng., 9, 157-168. Silverman, M.P. & Lundgren, D.G., (1959) Studies on the chemoautotrophic iron bacterium Ferrobacillus ferrooxidans. I. An improved medium and a harvesting procedure for securing high cell yields. J. Bacteriol., 77, 642-647. Shrihari, Bhavaraju, S.R., Modak, J.M., Kumar, R. & Gandhi, K.S., (1993) Dissolution of sulphur particles by ThiobaciUus ferrooxidans: substrate for unattached growth. Biotech. Bioeng., 41, 612-616. Mozes, N, Leonard, A.J. & Rouxlet, P.G. (1988) On the relations between the elemental surface composition of yeasts and bacteria and their charge and hydrophobicity. Biochimica et Biophysica Acta, 945, 324-334. Gadd, G.M & Griffiths, A.J., (1978) Microorganisms and heavy metal toxicity. Microb. Ecol., 4, 303-317.

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