Hydrometallurgy 77 (2005) 97 – 102 www.elsevier.com/locate/hydromet Bioremediation of acid mine drainage contaminated by SRB Alena Luptakova*, Maria Kusnierova Department of Mineral Biotechnologies, Institute of Geotechnics of Slovak Academy of Sciences, Watsonova 45, 043 53 Kosice, Slovak Republic Received 27 May 2004; received in revised form 13 July 2004; accepted 9 October 2004 Abstract The aim of this work is to study the possibility of using sulphate-reducing bacteria for the heavy metals removing from acid mine drainage (AMD), which is considered to be the major environmental problem associated with mining activities. Tests were conducted to determine if the bacterial produced hydrogen sulphide could be used for the elimination of soluble heavy metals from AMD in the form of sparingly soluble sulphides. We investigated the kinetics of the copper precipitation in the form of sulphides from the model solution containing Cu2+ by sulphate-reducing bacteria on the ground of two different approaches. In the first approach one reactor was used, which provides the simultaneous running of basic processes in study method, i.e. the hydrogen sulphide bacterial production and the copper precipitation by the bacterial produced hydrogen sulphide. The second approach allowed the successive running of aforementioned processes and used two interconnected reactors. The hydrogen sulphide bacterial production was realised in the first reactor and the copper precipitation by the bacterial produced hydrogen sulphide was realised consequence in the second reactor. Under these conditions this method involves three stages such as: the hydrogen sulphide production by sulphate-reducing bacteria, the cooper precipitation by the bacterial produced hydrogen sulphide and the cooper sulphides filtration from the liquid phase. The advantage of the second approach is the faster running of the Cu2+ elimination, as well as the possibility of the selective metal precipitation in the form of sulphides. D 2005 Elsevier B.V. All rights reserved. Keywords: Bioremediation; Acid mine drainage; Sulphate-reducing bacteria; Bacterial hydrogen sulphide 1. Introduction One of the most important problems affecting mining companies around the world is the occurrence and the treatment of acid mine drainage (AMD). This waste water exhibits a harsh and extreme favourable conditions for biological activity and must be contained and treated before it can be discharged, since it * Corresponding author. E-mail address: luptakal@sake.sk (A. Luptakova). 0304-386X/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.hydromet.2004.10.019 can have severe impact on the environment. AMD generates when metal sulphide minerals, particularly the pyrite, come in contact with oxygen and water in the presence of a naturally occurring sulphur-oxidising bacteria such as Acidothiobacillus ferrooxidans that act as a biological catalyst. Its pH is very low, of about 1.5–2.0. AMD contains sulphuric acid, dissolved heavy metals, sulphates, and iron precipitates. These components of AMD have a deleterious influence on the biota of streams receiving AMD (Kontopouls, 1988). 98 A. Luptakova, M. Kusnierova / Hydrometallurgy 77 (2005) 97–102 Several methods exist for the treatment of AMD, but only few of them have been applied under commercialscale conditions. The most common treatment methods are chemical methods, e.g. the neutralization using lime or other alkaline components. This treatment results in the precipitation of sulphate anions and heavy metal cations in the form of gypsum and metal hydroxides respectively, which have been discharged. The operating costs of these processes are high, whereas sulphate and metals removal efficiencies are relatively low (Boonstra et al., 1999). In addition, all valuable metals are lost in the sludge. Microbial remediation of AMD by sulphate-reducing bacteria (SRB) is a promising alternative to hydroxide precipitation. The bacterial precipitated metal sulphides can be recovered and recycled (Boonstra et al., 1999). The treatment of AMD by SRB is based on the ability of SRB to reduce sulphates to hydrogen sulphide, which binds readily with metals to form sparingly soluble precipitates. Thereafter metals are removed from the solution in a stable form. The metabolism of SRB also generates alkalinity, which contributes towards neutralising the acidity of the AMD. The following reactions (1), (2) and (3) represent the transformation of the principal constituents of AMD by SRB attempted in this study to investigate the role that SRB plays in the precipitation of cooper from sulphate model solutions. We investigated the kinetics of the copper precipitation by sulphatereducing bacteria on the ground of two different approaches. In the first case one reactor was used for the running of basic processes of study method, i.e. the hydrogen sulphide bacterial production and the copper precipitation by the bacterial produced hydrogen sulphide. Hereunder these processes running simultaneous. The second approach allowed the successive running of aforementioned processes and used two interconnected reactors. The hydrogen sulphide bacterial production was realised in the first reactor and the copper precipitation by the bacterial produced hydrogen sulphide was realised in the second reactor. Under these conditions this method involves three stages such as: the hydrogen sulphide production by sulphate-reducing bacteria, the metals precipitation by bacterial produced hydrogen sulphide and the metal sulphides filtration from the liquid phase. 2. Materials and methods 2.1. Microorganisms 4H2 + SO24— + H+ SRB HS— + 4H2O Organic matter (C, H, O) + SO42— SRB ð1Þ HS— + HCO3— ð2Þ Me2++HSYMeS(A)+H+(Me2+—the metal cation) (3) The SRB represents a group of chemoorganotrophic and strictly anaerobic bacteria, which include representatives of the genera Desulfovibrio, Desulfomicrobium, Desulfobacter, Desulfosarcina, Desulfotomaculum , Thermodesulfobacterium , Archaeoglobus, etc. (Odom and Singleton, 1993). The closing and flooding of mines in Slovakia generate large volumes of AMD. Therefore we studied the possibility of using SRB for a bioremediation process of AMD. At this time we Applied cultures of SRB (genera Desulfovibrio and Desulfotomaculum) were isolated from a mixed culture obtained from: – the waste water collection tank used for washing machinery in a metallurgical plant with pH 7.5– 8, strong H2S odour, heavily polluted, black colour and the presence of oil (the designation SRB-VSZ), – the potable mineral water (Gajdovka spring, the locality Kosice-north, Slovakia), hygienically acceptable with pH 7.5 and slight odour of H2S (the designation SRB-GJ). The SRB was selected from the mixed cultures grown on agar plates as well as in agar shake tubes containing modified Postgate’s medium E (Postgate, 1984). The isolation was performed by the modified dilution method (Ronald, 1995). Postgate’s medium C (Postgate, 1984) was used for the preparation of active A. Luptakova, M. Kusnierova / Hydrometallurgy 77 (2005) 97–102 SRB cultures and the cultivation of SRB. This medium contained (in g/L): 0.5 K2HPO4, 1.0 NH4Cl, 1.0 Na2SO4, 0.1 CaCl2d 2H2O, 2.0 MgSO4d 7H2O, 3.5 sodium lactate, 1.0 yeast extract, 0.5 FeSO4d 7H2O, 0.1 ascorbic acid, 0.1 thioglycolic acid. The pH of the media was adjusted to 7.5 with 5 M NaOH. 2.2. Model solutions The experiments were conducted with model solutions containing Cu2+. They were prepared by dissolving of CuSO4. 5H2O of analytical grade in distilled water. The pH of solutions was adjusted to the desired value using sulphuric acid. 2.3. Analytical procedures Turbidimetric method was used to measure the concentrations of sulphate ion (APHA, 1989) using a Spectromom195 (Hungary) instrument. The absorbance of the sample was measured at a wavelength of 420 nm. The cooper concentration in the liquid samples taken from the bioreactor was determined by atomic absorption spectrophotometery (AAS) using Spectrometer AA-30 Varian (Australia) instrument. A glass pH-electrode combined with the reference Ag/AgCl electrode and a platinum redox plus Ag/ AgCl reference electrode were used to measure pH and redox potential (Eh) respectively. Digital pHmeter GPRT 144 AGL (Germany) and mV-meter Ionactivity-meter MS 20 (Czech Republic) were used. The qualitative analysis of precipitates obtained by bacterial produced hydrogen sulphide was realized by energy dispersive spectrometry (EDS) analysis using instruments that consisted of a scanning electron microscope BS 300 (Tesla, Czechoslovakia) and an X-ray micro-analyser EDAX 9100/60 (Philips, Holland). Samples of precipitates were dried and coated with gold prior to the EDS analysis. 2.4. Simultaneous running of the hydrogen sulphide bacterial production and the copper precipitation by the bacterial produced hydrogen sulphide—the application of one reactor Under these conditions the copper precipitation by the bacterial produced hydrogen sulphide was studied 99 in a discontinuous reactor in the thermostat at 30 8C and ran during a period of 10 days under anaerobic conditions. Anaerobiosis was generated by introducing an inert gas (N2) into the reactor at the beginning of the experiment, i.e. at the filling of reactor by Postgate’s medium C, the model solution with Cu2+ in the concentration of 20 mg of copper ion per litre, and the inoculum containing SRB. Residual copper concentration in the solution was measured by atomic absorption spectrophotometry in samples taken from the reactor. After 10 days formed precipitates were removed from the liquid phase by filtration through membrane filter, next dried and analysed by the qualitative EDS analysis. 2.5. Successive running of the hydrogen sulphide bacterial production and the copper precipitation by the bacterial produced hydrogen sulphide—the application of two reactors Under these conditions experiments were performed in two bioreactors with a volume 1000 mL (the first bioreactor) and 250 mL (the second bioreactor). This method contains several steps and can be divided into three stages: I. stage Biological hydrogen sulphide production—in this stage SRB using organic matter (lactate) and hydrogen (as electron donor) to convert the sulphate to hydrogen sulphide. This part was carried out under the following conditions: a discontinuous hermetically closed reactor (the first reactor), lstatically, temperature 30 8C, selective nutrient medium for D. desulfuricans (Postgate’s medium C), pH 7.5, anaerobic conditions. II. stage Cooper precipitation by the bacterial produced hydrogen sulphide—this stage followed after the indication of the sulphate reduction (blackening of the medium by production of FeS in the first bioreactor). It was carried out the continuous transfer of a gas mixture (H2, CO2 and H2S) by N2 from the first bioreactor into the second reactor, which was filled with by the model solution of copper ions. The continuous precipitation of Cu2+ was 100 A. Luptakova, M. Kusnierova / Hydrometallurgy 77 (2005) 97–102 realized by the bacterial produced H2S at pH 1.5, 2.0, 2.5, 3.0 and 3.5, respectively. III. stage Separation of copper sulphides—the filtration of precipitates from the model solution. They were dried and analysed using the qualitative EDS analysis. 3. Results and discussion 3.1. Simultaneous running of the hydrogen sulphide bacterial production and the copper precipitation by the bacterial produced hydrogen sulphide—the application of one reactor In these experiments we investigated the efficiency of copper removal from the model solution by SRB that was obtained from heavily polluted waste water (SRB-VSZ) and hygienically acceptable potable mineral water (SRB-GJ). Fig. 1 shows the results of copper precipitation at these conditions (discontinuous batch tests). They indicate high activity of both bacterial cultures of SRB. The 98–99% elimination of copper from liquid phase by SRB-VSZ and SRB-GJ was registered after 5–6 days of batch experiments. During the experiments, the formation of brown-black precipitates was observed, as well as the sensorial detection of H2S smell during the samples taking from the liquid phase in the reactor. These observations were not found in the abiotic control experiment. Fig. 2 proves the presence of SRB in the precipitates. Fig. 2. Scanning electron micrographs of precipitates containing sulphate-reducing bacteria SRB-GJ. Fig. 3 and Fig. 4 show results of EDS qualitative analysis of precipitates originated by using of SRB and in the abiotic control (i.e. in the absence of SRB). EDS spectrum (Fig. 3) confirms the origin of copper as well as iron sulphides in the presence of SRB. The iron presence was involved of the Postgate’s medium C application. Peaks of Fe, P, Ca, K and Mg were registered in the composition of precipitates from the abiotic control experiment (Fig. 4). These peaks are related with the presence of the nutrient medium representative components, and the Cu and S peaks probably suggest a low cooper precipitation. cps S Fe Concentration of Cu2+ in liquid phase (mg/L) 100 25 Cu 20 50 15 10 C 5 O Cu Au Fe 0 0 100 200 Gj Au Au 0 Time (hours) VSZ Cu Energy [keV] K Fig. 1. Precipitation of Cu2+ by sulphate-reducing bacteria. Kabiotic control, GJ-sulphate-reducing bacteria from the potable mineral water (spring Gajdovka), VSZ-sulphate-reducing bacteria from the industrial waste water (the metallurgical plant). 0 5 10 Fig. 3. EDS qualitative analysis of precipitates originated from the model solution by using one reactor (the simultaneous running of the hydrogen sulphide bacterial production and the copper precipitation by the bacterial produced hydrogen sulphide). A. Luptakova, M. Kusnierova / Hydrometallurgy 77 (2005) 97–102 Concentration of Cu2+ in liquid phase (mg/L) cps 50 Fe P Au S 10 O Mg K Cu Ca Ca Fe Cu pH 1.5 pH 2.0 pH 2.5 pH 3.0 pH 3.5 25 20 15 10 5 0 0 Au 2 4 6 8 10 Time (hours) 0 0 5 10 Energy [keV] Fig. 4. EDS qualitative analysis of precipitates originated from the model solution without SRB—the abiotic control experiment by using of one reactor. 3.2. Successive running of the hydrogen sulphide bacterial production and the copper precipitation by the bacterial produced hydrogen sulphide—the application of two reactors Table 1 shows typical changes of sulphate concentration, pH and Eh during the discontinuous cultivation of SRB in the first reactor, i.e. during the bacterial hydrogen sulphide production. The decrease of sulphate concentration and Eh, the increase of pH values, the formation of black precipitates and the sensorial detection of H2S smell were not observed in the abiotic control. It confirms that aforesaid changes were caused by bacterial metabolism of SRB. Fig. 5 presents the results of cooper precipitation (the second stage of this method). It demonstrates that copper was effectively recovered from the solution using bacterial produced H2S. An initial copper Fig. 5. Precipitation of Cu2+ by the bacterial produced hydrogen sulphide by SRB from the model solution at different pH values, by using two reactors (the successive running of the hydrogen sulphide bacterial production and the copper precipitation by the bacterial produced hydrogen sulphide). concentration 20 mg/L was decreased to less than 1 mg/L after 8 h. The most adequate pH value for cooper precipitation was 2.5. Fig. 6 indicates that Cu was precipitated in the form cooper sulphides. The composition of originated precipitates correspond with this fact. Successive running of the hydrogen sulphide bacterial production and the copper precipitation by cps Table 1 Values of pH, Eh and sulphates concentration in the first reactor at the beginning and the end of SRB-GJ cultivation and in abiotic control With SRB (GJ) Without SRB (abiotic control) pH Eh [mV] SO2 4 [mg/L] pH Eh [mV] SO2 4 [mg/L] 7.6 8.9 140 260 1.82 0.89 7.5 7.6 158 165 1.84 1.82 S 60 Cu Cu Au 40 Au Au 20 START END 101 S Cu Au Au 0 0 5 10 Energy [keV] Fig. 6. EDS qualitative analysis of precipitates originated during the precipitation of Cu from the model solution by using two reactors (the successive running of the hydrogen sulphide bacterial production and the copper precipitation by the bacterial produced hydrogen sulphide). 102 A. Luptakova, M. Kusnierova / Hydrometallurgy 77 (2005) 97–102 the bacterial produced hydrogen sulphide, i.e. the application of two reactors, allowed faster Cu2+ elimination, as well as the possibility of selective metals precipitation in the form of sulphides (Foucher et al., 2001). Acknowledgement The presented paper was prepared due to a support of grant agency VEGA for project No. 2 210622, Slovak–Czech project No. 104 and State order No. 51/ 03R 06 042. 4. Conclusions References The following conclusions can be drawn from this study: ! Sulphate-reducing activities of SRB isolated from natural and industrial sources were equivalent. ! SRB from these sources effectively removed Cu2+ from model solutions after 5–6 days, by using one batch reactor that provides the simultaneous running of basic processes of study method, i.e. the hydrogen sulphide bacterial production and the copper precipitation by the bacterial produced hydrogen sulphide. Under these conditions the created precipitates contained the mixture of copper and iron sulphides. ! The successive running of the hydrogen sulphide bacterial production and the copper precipitation by the bacterial produced hydrogen sulphide, i.e. the application of two reactors, allowed faster Cu2+ elimination (during 8 h), and probably as well the possibility of selective metals precipitation in the form of sulphides, which will depend on the solution’s pH values. APHA, 1989. Standard Methods for the Examination of Water and Wastewater, 17th ed. American Public Health Association, USA, Washington D.C. Boonstra, J., van Lier, R., Janssen, G., Dikman, H., Buisman, C.J.N., 1999. Biological treatment of acid mine drainage. In: Amils, R., Ballester, A. (Eds.), Biohydrometallurgy and the Environment Toward the Mining of the 21st Century, Part B. Elsevier, Amsterdam, pp. 559 – 567. Foucher, S., Battaglia-Brunet, F., Ignatiadis, I., Morin, D., 2001. 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