Abstract

Reduction of chromium (VI) by the indirect action of Thiobacillus thioparus

E.Donati; C.Oliver; G.Curutchet

Centro de Investigación y Desarrollo de Fermentaciones Industriales, CINDEFI, CONICET, Facultad de Ciencias Exactas UNLP, 47 y 115, 1900 La Plata, Argentina

^{Address to correspondence} Address to correspondence E.Donati E-mail: donati@quimica.unlp.edu.ar

ABSTRACT

The microbial reduction of chromium(VI) to chromium(III) has been one of the most widely studied forms of metal bioremediation. Recently, we have found that Thiobacillus ferrooxidans and Thiobacillus thiooxidans, growing on elemental sulphur, can indirectly promote chromium(VI) reduction by producing reducing agents such as sulphite and thiosulphate, which abiotically reduce chromium(VI). Those species of Thiobacillus are acidophilic bacteria which grow optimally at pH values lower than 4. However, most of those reducing agents are stabilised at higher pH values. Thus, the present paper reports on the ability to reduce chromium(VI) using another specie of Thiobacilli, Thiobacillus thioparus, which is able to grow at pH close to 7.0. T. thioparus cultures were carried out in a fermentation vessel containing medium and sulphur as the sole energy source and maintained at 30^oC and 400 rpm. The pH was adjusted to 6.0, 7.0 or 8.0 and maintained with the automatic addition of KOH. Our results show high chromium (VI) reduction values (close to 100% at the end of bacterial growth) at the three pH values. The results of these experiments are very promising for development of a microbiological process to be used in the detoxification of chromium(VI)-polluted effluents.

Keywords:Thiobacillus thioparus, chromium(VI) reduction, bioremediation.

INTRODUCTION

Hexavalent chromium is one of the toxic heavy metals with high mobility in soil and groundwater which can produce harmful effects on organisms including humans. Wastewaters containing chromium(VI) are generated by many industries such as chromite ore processing, electroplating, and leather-tanning processes, among others (Chuan and Liu, 1996; Lawson, 1997). As a result of unregulated application and inappropriate waste-disposal practices, chromium is introduced into the environment (mainly into soils and streams of water). As reduction of toxic chromium(VI) leads to the formation of stable and non-toxic chromium(III), this reduction may be implemented so as to achieve detoxification and therefore environmental cleanup. Chemical reduction followed by precipitation or immobilisation can be produced abiotically by different substances (Cifuentes et al., 1996; Quintana et al., 2001), but recent reports have demonstrated the feasibility of using biological reduction for the treatment of chromium(VI)-containing wastes (Rege et al., 1997; Rajwade and Paknikar, 1997; Mel Lytle et al., 1998; Salunkhe et al., 1998).

Thiobacilli are gram-negative chemoauthotrophic bacteria that can obtain energy for growth from the oxidation of a variety of inorganic sulphur compounds (Rawlings, 1997). Oxidation of these sulphur compounds, particularly that of elemental sulphur, generates a series of sulphur compounds (sulphite, thiosulphate and polythionates) with high reducing power (Steudel et al., 1987). This activity has been used in cultures of T. thiooxidans to catalyse the reduction of manganese(IV) (Porro et al., 1990), iron(III) (Donati et al., 1995) and vanadium(V) (Briand et al., 1996). Our previous results have showed that T. ferrooxidans and T. thiooxidans cultures are able to reduce chromium(VI) and that their reducing capability may be in the colloidal sulphur appearing in cultures (Sisti et al., 1998; Quintana et al., 2001). Most of those reducing agents are stabilised at high pH values (Donati et al., 2000). For this reason, another specie of Thiobacilli, Thiobacillus thioparus, which is able to grow at pH close to 7.0 using sulphur as the energy source (Rawlings, 1997), was selected to study chromium(VI) reduction at high pH values (5, 6 and 7).

MATERIALS AND METHODS

Microorganisms

A T. thioparus strain (ATCC 8158) routinely subcultured in thiosulphate medium was used. The medium contained (g/l) Na₂HPO₄ 1.2, KH₂PO₄ 1.8, MgSO₄.7H₂O 0.1, (NH₄)₂SO₄ 0.1, CaCl₂ 0.03, FeCl₃ 0.02, MnSO₄ 0.02 and Na₂S₂O₃.5H₂O 10. The medium was adjusted to pH 7.2 and sterilised by filtration through a 0.45 mm filter. After six days, the culture was filtered to eliminate residual sulphur and passed through a 0.22 mm filter to retain cells. Cells were washed with medium at pH 2.0 and suspended in medium (at pH 5.0, 6.0 or 7.0) without Na₂S₂O₃.

Chromium Reduction at Different Growth pH

T. thioparus cultures were prepared in a fermentation vessel containing 0.6 l of the medium described above inoculated at 10% v/v with T. thioparus. Thiosulphate was replaced with 12 g of analytical grade powdered sulphur as energy source. The culture pH was adjusted to 5.0, 6.0 or 7.0 and maintained at each pH value with the automatic addition of 1.25 M KOH. Cultures were incubated at 30^oC and 400 rpm. Samples of 100 ml were taken periodically and filtered through blue ribbon filter paper to eliminate sulphur particles. Then the medium was filtered through a 0.45 mm filter. Filtration membranes with both retained biomass and colloidal sulphur (strictly speaking, sulphur particles size less than 3 m m in) were used in chromium reduction experiments. Ten ml of potassium dichromate solution containing 20 mg.l^-1 chromium(VI) at pH 2.0 were filtered through the filtration membranes described above, using an accessory vacuum filter. Contact time between filter and solution was 20 minutes. After the reduction step, the medium pH 2.0 was used to flush chromium possibly adsorbed in the filter. All reduction experiments were carried out in duplicate.

Analytical Methods

Free (not attached) bacterial population was determined by using a Petroff-Hausser counting chamber in a microscope with a contrast phase attachment. Chromium(VI) was determined by the diphenylcarbazide method: 0.50 ml solution prepared with 0.025 g of diphenylcarbazide in 10 ml of acetone were added to 10 ml of sample (diluted when necessary). After for incubation 10 minutes at room temperature, absorbance at 540 nm was determined. Total chromium concentration was determined in the same way after oxidising chromium(III) with a solution of KMnO₄ boiled for 10 minutes (Urone, 1955).

RESULTS AND DISCUSSION

Figure 1 (outer graph) shows chromium(VI) reduction by sulphur and cells from T. thioparus culture grown at pH 5.0 when circulating chromium(VI) solutions with pH 2.0. The highest chromium reduction was 96% after 12 days. The inner graph shows the evolution of free bacterial population and the sulphuric acid produced in the culture where samples were taken. Sulphuric acid was estimated by the amount of KOH added to maintain the pH in the culture. This culture showed a long lag phase of bacterial growth; the highest chromium(VI) reduction (96%) was found when the culture reached the maximum free bacterial population.

Figures 2 and 3 (outer graph) show percentage of chromium(VI) reduction by colloidal sulphur and cells from cultures grown at different fixed pH values (6.0 and 7.0 respectively). The inner graph shows free bacterial population and mmoles of sulphuric acid in the cultures where the samples were taken to evaluate chromium reduction.

Cultures at pH 6.0 and 7.0 showed lag phases shorter than that at pH 5.0. After that, as a function of the free bacterial population, chromium(VI) reduction increased until it reached the maximum value (100% and 90% respectively). At pH 6.0 (probably the optimum pH value for T. thioparus growth on sulphur), cultures had the highest free bacterial populations and the highest chromium reduction values. Thus, the amount of reducing compounds (polythionates) associated with the colloidal sulphur and cells should have been at a maximum at this pH. On the other hand, no chromium(VI) reduction was detected in sterile controls at the three pH values.

Polythionates have been detected in cultures of other species of Thiobacillus such as T. thiooxidans and T. ferrooxidans. In these cultures, reduced glutathione (GSH) is required to oxidise elemental sulphur (Steudel et al., 1987):

After that, polysulphide is successively oxidised to different compounds such as thiosulphate, sulphite, other polythionates (S_n(SO₃)₂^2-) and finally sulphate. Polythionates are also formed upon acid decomposition of thiosulphate. Sulphite, thiosulphate and polythionates could be responsible for reductive reactions. According to Steudel, colloidal sulphur in cultures of T. ferrooxidans would be present as long-chain polythionates forming micelles of globules of up to a few mm. Because of this, the ability of chromium reduction by a Thiobacillus culture was evaluated using colloidal sulphur and cells. Sulphite and thiosulphate, among other reducing compounds, can be decomposed by acid-producing sulphur as the main product. Thus in cultures at pH close to 7, those reducing compounds should be stabilised and chromium(VI) reduction should be higher than that at low pH values. However, in similar experiments using acidophilic bacteria (T. ferrooxidans), we detected neither significant growth nor chromium(VI) reduction at pH higher than 4.0. On the other hand, samples from T. thiooxidans (another acidophilic bacterium) cultures grown at higher pH values showed higher chromium(VI) reduction values although the bacterial growth was not so important as at low pH values (Donati et al., 2000).

In contrast, the neutrophilic T. thioparus showed an adequate growth and high percentages of chromium(VI) reduction at pH close to 7. Although the maximum efficiency of chromium(VI) reduction at pH 6.0 was similar in T. thiooxidans, the advantages of T. thioparus were the following: a) chromium(VI) reduction was high even at the beginning of the bacterial growth (from six days), b) there was a shorter lag phase and a longer exponential phase and c) there was bacterial growth and consequent chromium(VI) reduction even at pH 7.

CONCLUSIONS

Summarising, the present paper proves the chromium(VI) reduction ability in T. thioparus cultures using sulphur as energy source. The highest chromium(VI) reduction was found at pH 6.0, which was also the optimum pH value for T. thioparus growth. Chromium(VI) reduction in these cultures was as high as that in T. thiooxidans cultures and much higher than that in T. ferrooxidans cultures at the same pH. Therefore, the use of T. thioparus cells growing immobilised on elemental sulphur at pH values close to 7 or colloidal sulphur coming from these cultures are an adequate process for use in the detoxification of chromium(VI)-polluted effluents.

ACKNOWLEDGEMENTS

Dr. Edgardo Donati and Dr. Gustavo Curutchet are research members of CONICET (Argentine National Research Council). This research was supported in part by Agencia Nacional de Promoción Científica y Tecnológica.

Received: March 5, 2002

Accepted: August 22, 2002

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