Solubilization of heavy metals from a fluvial AMD generating tailings sediment by heterotrophic microorganisms

Journal of Geochemical Exploration 92 (2007) 177 – 185 www.elsevier.com/locate/jgeoexp

Solubilization of heavy metals from a fluvial AMD generating tailings sediment by heterotrophic microorganisms Part I: Influence of pH and solid content S. Willscher ⁎, C. Pohle, J. Sitte, P. Werner TU Dresden, Faculty of Forest, Geo and Hydro Sciences, Institute of Waste and Site Management, Pratzschwitzer Str. 15, D-01796 Pirna, Germany Accepted 8 August 2006 Available online 13 November 2006

Abstract The study demonstrates that a heterotrophic mixed culture is able to mobilize considerable amounts of heavy metals (Fe, Zn, Mn, Pb, Cu, Cd, Cr) from a sulfidic AMD generating fluvial tailings material even under moderately acidic conditions as they exist at real sites. The experiments were carried out in shaking flasks by inoculation with a mixed culture of heterotrophic bacteria. The solid content and a decrease in initial pH of the suspensions were investigated as important factors of the viability of the heterotrophic microorganisms. With the mixed culture, up to 17% Pb, 27% Cd, 100% Cr, 25% Fe, 35% Cu, 79% Mn, and 28% Zn were mobilized in one batch run. Compared to the autotrophic processes, especially Pb seems to be mobilized by heterotrophic microbial action. Even under increasingly unfavorable conditions (increase in solid content, decrease in initial pH) the mixed culture demonstrated a high diversity and a good viability. As a result, it was clearly shown that heterotrophic processes also play an important role in the mobilization of metals in such acid-generating materials. Heterotrophic microorganisms improve the solubility of metals primarily released by biooxidation, and enhance the transport of these environmentally relevant metals by excretion of complexing agents. The synergistic action of both autotrophic and heterotrophic bioleaching processes generates at such sites a long-range contamination of ground- and surface-water. © 2006 Elsevier B.V. All rights reserved. Keywords: Acid mine drainage (AMD); Fluvial tailings deposit; Heterotrophic microorganisms; Complexing organic matter

1. Introduction Fluvial tailings deposits consist of mine waste and tailings materials that have been transported from a mining district and deposited by natural fluvial processes at some distance from their origin. These deposits can contain pyrite and related sulfidic metal and arsenic-

⁎ Corresponding author. Fax: +49 3501 530022. E-mail address: [email protected] (S. Willscher). 0375-6742/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.gexplo.2006.08.013

bearing minerals. Flood plains downstream from mining districts can contain abundant amounts of this material, particularly in older mining districts where mining activity occurred before the implementation and enforcement of environmental regulations (Walton-Day et al., 1999). The weathering of such materials may cause contamination of ground- and surface-waters by acidity, dissolved metals and salinity. Microbial processes play an important role in the weathering of sulfidic rocks and minerals, causing acid mine drainage (AMD) (Colmer and Hinkle, 1947; Silverman and Ehrlich, 1964; Singer

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and Stumm, 1970; Karavaiko et al., 1972; Nordstrom, 1982; Harris and Ritchie, 1983; Moses et al., 1987; Watzlaf, 1988; Ehrlich, 1990; Kleinman et al., 1991; Bosecker, 1994; Evangelou, 1995; Schippers et al., 1995; Yu, 1996; Gray, 1997; Johnson and Bridge, 1997; Kwong et al., 1997; Schüring et al., 1997; Geldenhuis and Bell, 1998; Mills et al., 1998; Booth and Bertsch, 1999; Edwards et al., 2000; Iribar et al., 2000). A previous study of fluvial tailings material focused on the potential for the chemical elution of metals, acidity and salinity from this acid generating material (Walton-Day et al., 1999). In continuing works, the material was investigated for its capacity to generate AMD by biooxidation processes with the aim to assess the long-term risks of similar mining sites exposed to geomicrobial weathering processes (Willscher et al., 2004). In those previous works it was observed that besides the well known autotrophic weathering processes also heterotrophic processes play a role even in acid generating material of this type with some organic content. Especially in percolator experiments, a formation of dissolved organic carbon (DOC) of up to 1206 mg/l was detected during the leaching experiments (Willscher et al., 2004). The enhanced solubilization of Zn, Mn, Al, and, to some extent, of Cd and Cu in the percolator experiments was explained by the occurrence of complexing organic substances in the leachates. The experimental data for Mn, Zn and Al were consistent with the observed environmental measurement data showing a high mobility of these metals in surfacewaters in the vicinity of such mining sites (Walton-Day et al., 1999; Willscher et al., 2004). The weathering behavior of heterotrophic microorganisms on rocks and minerals was subject of several research works. In some cases, heterotrophs were investigated for their capability to bioleach metals from ores, minerals (Rossi, 1978; McKenzie et al., 1987; Portier, 1991; Sukla and Panchanadikar, 1993; Steemson et al., 1997; Styriakova et al., 2003) and waste materials (Hahn et al., 1993; Willscher and Bosecker, 2003). Other studies focused on their potential use to decontaminate soils (Karavaiko and Groudev, 1985; Francis and Dodge, 1990; Rusin, 1992; Bock and Bosecker, 1996; Bosecker, 1999; Groudev et al., 1999). In most of these works, mixed cultures of heterotrophs were used to examine the weathering and leaching behavior (Wagner and Schwartz, 1967; MeunierLamy and Berthelin, 1987; McKenzie et al., 1987; Sukla and Panchanadikar, 1993; Hahn et al., 1993; Steemson et al., 1997; Styriakova et al., 2003). There, however, heterotrophic activity was examined only under optimal (neutral pH) growth conditions. The contribution of heterotrophs to the mobilization of metals under acidic

conditions was already assumed in some of the studies (Hahn et al., 1993; Johnson and Roberto, 1997; Steemson et al., 1997; Amaral Zettler et al., 2002; Willscher et al., 2004), but never investigated in particular. Hence, to investigate if heterotrophic microorganisms are able to perform a solubilizing activity at such very low pH conditions occurring in this acid-generating fluvial tailings material is the aim of the present study. Moreover, it examines the extent to which environmentally relevant metals are solubilized from this fluvial tailings material by heterotrophic processes only and without the influence of autotrophic weathering. Weathering processes at real places in nature take place with a high complexity of microbial ecosystems; therefore, a mixed culture of metal-leaching microorganisms was used in these heterotrophic leaching experiments. The influence of an increase in solid content and of a decrease in pH on the leaching activity of the mixed culture should be investigated in the experiments as important factors for the growth and activity of the microorganisms. The tailings material contained enhanced concentrations of heavy metals such as Pb and Zn. Therefore the activity of the heterotrophs at these specifically enhanced heavy metal concentrations was examined at an increase in solid content. Furthermore, the changing pH in the environment of the microorganisms was expected to be a sensitive parameter for the microbial activity. In the fluvial tailings area, microorganisms have to exist in environs with a periodically changing pH, and so this parameter was evaluated prior to conducting percolator experiments. 2. Materials and methods 2.1. Fluvial solids In addition to siliceous components, the mineralogy of the fluvial tailings deposit material included small fractions of unaltered metal sulfides such as pyrite, sphalerite and chalcopyrite, measured by XRD (WaltonDay et al., 1999; Willscher et al., 2004). Minerals such as plumbojarosite, indicative of previous weathering, were also present. Sediment cores were homogenized, digested, and analyzed for their metal content using inductively coupled plasma atomic emission spectroscopy (ICP–AES), as described in detail in Walton-Day et al. (1999). Chemical data obtained after acid digestion of the mixed sample used here correlated well with former analyses of the particular core materials (WaltonDay et al., 1999; Willscher et al., 2004). Its elemental composition was as follows: 6.77% Fe, 0.37% Al, 0.3% Zn, 0.3% Pb, 509 ppm Mn, 161 ppm Cu, 107 ppm As,

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11.4 ppm Cr and 25 ppm Cd. The total sulfur content was 3.24%. An aqueous suspension of the material reacted acidic (pH of 3.13 at 3.75% w/v). Unlike a typical metal sulfide feed, the fluvial tailings material also contained a fraction of plant derived organic material (total organic carbon (TOC) of 3.13%) in various stages of decay. The tailings area itself was largely devoid of vegetation, presumably because of the low pH and the toxicity of the near surface and the plant material was likely imported by wind and periodic flooding events. 2.2. Microorganisms A mixture of 10 different isolates of heterotrophic microorganisms with known metal leaching activities was used in the experiments. The isolates originated from heavy-metal-contaminated environmental samples. Among the isolates were strains of Microbacterium sp., Promicromonospora sp. and Pseudomonas cedrina (identification by the DSMZ/Germany). The remaining cultures are currently being identified. Presumptively, one of the cultures used was yeast. The cells of the single cultures were grown in a culture medium containing 0.5% meat peptone, 0.3% yeast extract and 1% sucrose to provide a maximum yield of each single strain. After 24 to 36 h, the cells were separated by centrifugation, washed, suspended in a mineral salt solution (Schlegel, 1992), merged to a mixed culture and used as inoculum for the leaching experiments. 2.3. Leaching experiments The leaching experiments were carried out in 500-ml Erlenmeyer flasks on a shaking table at 175 rpm and 30 °C for up to 78 days. Samples of the tailings material were sterilized before the bioleaching experiments. The leaching suspensions contained the fluvial tailings material and 4% of sucrose as carbon source in a mineral salt solution (Schlegel, 1992). During the experiments, the carbon source was replenished regularly by addition of sterilized sucrose to give a concentration of 4% (Hahn et al., 1993). Control experiments were carried out in the same way, but without inoculum and under sterile conditions. The results reported here are the average data of duplicate experiments (2% to 5% average variation). In experiments with an increase in solid content were used concentrations of 2, 4, and 6 % tailings material (m/v) in the leaching suspensions. Before inoculation, the pH of the suspensions and the controls was adjusted with sterile KOH solution. In the experiments with increase in solid content, the initial pH was adjusted

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to 8, and in the experiments with decrease in pH, the initial pH values of 8, 6.5, 5 and 4 were adjusted at a solid content of 4%. The metal content in the solutions, the pH, Eh and biomass concentrations were measured during and after the leaching experiments. 2.4. Analyses Concentrations of mobilized species of general interest in metal bioleaching (Fe, Zn, Cu) as well as some from the standpoint of overall environmental or water quality impact (Mn, Cr, Cd and Pb) were monitored. Periodic sub-samples were centrifuged at 10,000 rpm for 7 min, filtered by 0.2 μm, and diluted into 1% HNO3 prior to analyses by AAS. Spike recoveries of standards at regular intervals were used for analytical quality control. The sucrose content of the solution was measured by the anthrone method (Abidin and Maier, 1980), the biomass concentration by the Bradford method (Kleber et al., 1987). Colony counts were carried out on peptone/yeast extract agar (Schlegel, 1992). 3. Results and discussion 3.1. Leaching results at different solid contents of the fluvial tailings material The microorganisms decreased the pH in all inoculated samples to a value below 2.8 on the 2nd day of the experiment (see Fig. 1). This fast decline of pH is also due to the low buffering capacity of the tailings material caused by the long term AMD processes it was exposed to in the past 100–140 years. After the 2nd day of the experiment, the pH in the inoculated samples increased slightly (see Fig. 1), supposedly because of the adaptation phase of the microorganisms to the changed environmental conditions. The neutrophilic microorganisms were shocked by the immediate decrease in pH and the rapid increase in the concentrations of dissolved heavy metals, with some requiring up to several weeks to adapt to the dramatically changed environmental conditions. The pH in the microbial leaching solutions did not rise again above pH 3.3 during the remainder of the experiment. In contrast, the pH in the sterile control remained circumneutral (see Fig. 1). However, the pH in the inoculated samples finally decreased by nearly 1 unit (at 6% solid content), clearly indicative of an adaptation phase. At the end of the experiment, final pH values of 2.2 (2% solid content), 2.3 (4% solid content) and 2.5 (6% solid content) were determined. This low pH is sufficient for the solubilization of metals from the fluvial tailings material.

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Fig. 1. Course of the pH in the leaching solutions depending on the solid content.

The result clearly demonstrates that the decrease of the pH in the inoculated samples is caused by microbial activity. The microorganisms seemed to tolerate well the low pH and the increase of metal concentrations in the solution. These findings support the thesis that heterotrophic microbial activity is important in moderately acidic environments (down to pH 2) such as the fluvial tailings deposit. The data for the Eh and biomass measurements support the conclusions deduced from the pH data. Fig. 2 shows the Eh data. In the inoculated solutions, an initial rise in Eh

is observed caused by initial aerobic microbial growth. It is followed by a decrease of the Eh in the microbial adaptation phase, which depends on the solid content of the solutions. At 2% solid content, the Eh was only slightly depressed, whereas at 6% solid content a significant decrease of the Eh was observed (see Fig. 2). After 10 to 20 days, the Eh increased again until the end of the experiment. The biomass measurements show analogous data: an adaptation phase in the first weeks and a subsequently growth of the biomass (data not shown). The redox potential of all microbial experiments

Fig. 2. Course of the Eh in the leaching solutions depending on the solid content.

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Fig. 3. Maximum yields of solubilized heavy metals depending on the solid content: (a) in the adaptation phase; (b) at the end of experiment (78 days).

was in a range of heterotrophic aerobic reactions; the Eh data of autotrophic growth are significantly higher up to 720 mV (see Willscher et al., 2004). The measured concentrations of solubilized lead showed no significant differences between the different solid contents (data not shown). An explanation for that might be that the lead compounds in the tailings sediments (e.g. the plumbojarosite) are relatively insoluble, and so a maximum solubility product was exceeded in the experiments. The other metals (Cd, Cr, Fe, Mn, Zn) show the expected increase of the metal concentrations with increasing solid content. In contrast to these metals, Cu tends to a better solubilization at a low solid content. The main metals solubilized by the heterotrophic microorganisms were N400 mg/l iron (N7.17 mmol), 36 mg/l zinc (0.55 mmol) and 16 mg/l manganese (0.291 mmol). The highest achieved concentrations of the other metals were as follows: 9 mg/l Pb (0.043 mmol), 1.6 mg/l Cu (0.025 mmol), 0.2 mg/l Cd (1.78 × 10− 3 mmol), and 0.55 mg/l Cr (0.011 mmol). The experimentally determined concentrations of the solubilized contaminants correlated well with data from ground- and surface-water samples collected during the field study (Walton-Day et al., 1999). Consequently,

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besides autotrophic weathering processes, heterotrophic processes are able to solubilize heavy metals and cause changes in acidity from fluvial tailings materials of this type to an environmentally relevant extent. Fig. 3 shows the maximum yields of the solubilized metals dependent on the solid content in the adaptation phase and at the end of the experiment (100% yield is the maximum theoretical dissolvation of each metal). Fig. 3 also demonstrates that a substantial mobilization occurred in the adaptation phase (around 20 days, Fig. 3(a)), with the extent of solubilization increasing further over the remaining course of the experiment (Fig. 3(b)). The solubilization yield of the metals decreased with an increase of the solid content of the suspensions. This may reflect an inhibition of microbial activity at higher solid content and, therefore, a limited concentration of complex forming substances. Another reason for the decreasing mobilization yields of the metals is the mass transport limitation between solid phase and solution. The experiment demonstrates clearly that neutrophilic and acidotolerant heterotrophic microorganisms show an activity in an acid-generating material even in such a short experimental time under a moderately acidic pH and under enhanced heavy metal concentrations, all the more in geological time periods in the fluvial tailings material at the real site. 3.2. Leaching results at different initial pH values of the leaching solutions Besides the evaluation of the influence of the solid content on the solubilization of heavy metals by heterotrophs and their growth behavior, the influence of the initial pH as a sensitive parameter for the growth of neutrophilic heterotrophs was also investigated. It was expected that the growth and hence the solubilizing activity of the neutrophiles declines when the initial pH of the solution is lowered. Fig. 4 illustrates the pH behavior of the solutions. All inoculated suspensions showed an initial decrease of the pH to 2.23 up to 2.6 on the 2nd day, depending on the initial pH of the solutions (see Fig. 4). The lower the initial pH of the suspension, the lower was the pH over the course of the experiments in the first half of the experiment. After adaptation of the microorganisms (here around 40 days), the experiments at a more neutral initial pH (8, 6.5) again showed a decrease in pH by half a unit on average (see Fig. 4). In the experiments with a “low” initial pH (5 and 4), the adaptation time took obviously longer (more than 50 days), and the pH decreased more gradually (see Fig. 4). It is assumed

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Fig. 4. Course of the pH in the leaching solutions depending on the initial pH.

that the neutrophilic heterotrophs adapt better to the changing environmental conditions in the experiments with a nearly neutral initial pH. The Eh and biomass data (data not shown) correspond to the pH data of an initial growth, a following adaptation phase and then a further growth of the biomass, analogous to Fig. 2. The biomass data suggest an adaptation time in the first 20 days, and after that a growth up to the end of the experiment. At the end of the experiments, final pH data of 2.3 (initial pH 8), 2.0 (initial pH 6.5), 2.35 (initial pH 5) and 1.82 (initial pH 4) were achieved (see Fig. 4). Analogous to the solid content experiment, the solubilization behavior of the metals was different, too. The solubility of Pb did not increase at a lower pH thus reinforcing the thesis of the insolubility of the lead compounds in the tailings sediment (see Section 3.1), whereas Zn and Cu showed a slightly increased solubility at a lower initial pH. With regard to the influence of pH up to 380 mg/l Fe (6.81 mmol), 31 mg/l Zn (0.47 mmol) and 14 mg/l Mn (0.255 mmol) were solubilized. The highest concentrations achieved for the other metals were up to 6.2 mg/ l Pb (0.030 mmol), 1.68 mg/l Cu (0.026 mmol), 0.2 mg/ l Cd (1.78 × 10− 3 mmol) and 0.55 mg/l Cr (0.011 mmol). Fig. 5 shows the calculated yields of the metal solubilization in this experiment. The mobilization yields differ from the results of the previous experiment with increasing solid contents (see Section 3.1 and Fig. 3). The yields of Pb and distinctly of Cr decreased with declining pH (see Fig. 5a and b, respectively). The solubilization of Fe and Mn seems to be largely independent of the initial pH of the solution. Mn is largely soluble under slightly acidic conditions.

Iron, on the other hand, is largely insoluble under these conditions. In contrast, Zn and Cu demonstrate slightly better leaching yields when starting at low initial pH values.

Fig. 5. Maximum yields of solubilized heavy metals depending on the initial pH of the suspensions: (a) in the adaptation phase; (b) at the end of experiment (78 days).

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The reason for the different behavior is the speciation of the elements and their dissolution behavior in acidic solutions. Especially Cr and Mn seem to occur in a speciation well to mobilize, compared to the other heavy metals (see Fig. 5). A distinct decrease of the Cr concentration with declining initial pH of the solutions is supposedly caused by two reasons: (a) with the declining pH, the microorganisms are slightly inhibited and produce less solubilizing agents and (b) the complexing agents are protonated by a higher degree and form less complexes with heavy metals. The experiment demonstrates clearly, that even the neutrophilic heterotrophs can exist in a moderate acidic environment (down to pH 2) and develop their leaching activities there. The low final pH of the suspensions does not completely inhibit the growth and activity of the mixed culture. After an adaptation time of a few weeks in this case, the heterotrophs can exist and grow in an acidic environment of this type. The results of metal mobilization in the inoculated experiments are in all cases environmentally relevant. 3.3. Microbial life At the end of each experiment, colony counts (CFU) of the heterotrophs were determined (see Fig. 6a and b). The colony counts (Fig. 6) confirm the former experimental data (pH, Eh, biomass) and demonstrate a good final growth of the microorganisms at the low pH after some time of adaptation. Even in more extreme environmental conditions like an increase in solid content or a lower initial pH of the suspensions, a good final growth of the mixed culture was observed (see Fig. 6). The microorganisms seem to form a stable community in this kind of material. It is remarkable that under increasing unfavorable conditions (increase in solid content, decrease in initial pH) especially enhanced numbers of the bacteria (e.g. Microbacterium sp., Promicromonospora sp.) within the mixed community were counted (see Fig. 6). The good resistance of the used isolates at increasing concentrations of heavy metals under these conditions may serve as an explanation. It was assumed that the largely natural composition of this material supports the growth of the mixed cultures under increasingly stringent conditions. A comparable community of heterotrophs, together with iron- and sulfur-oxidizing autotrophic microorganisms, is conceivable on the fluvial tailings site. Whereas the autotrophs solubilize the sulfidic minerals by biooxidation, the heterotrophs degrade undissolved organic

Fig. 6. Colony counts (CFU) at the end of the leaching experiments: (a) influence of the solid content; (b) influence of initial pH (4% solid content).

matter and enhance the solubility of metal ions by excreting H+ and complex forming organic substances. Thus, a synergistic result regarding the solubilization and transport of environmentally relevant heavy metals is obtained. 3.4. Long-term environmental impact The fluvial tailings material contains organic material (TOC of 3.13%) (Walton-Day et al., 1999; Willscher et al., 2004), and even under biooxidation conditions (very low pH, high salinity) enhanced concentrations of DOC can be produced (1206 mg/l) (Willscher et al., 2004). This study demonstrates that these environmental conditions are a sufficient prerequisite for the metal solubilizing activity by heterotrophs. If the tailings material will remain in the fluvial area for further centuries, an entry of more organic matter (plant material, organic sediments, partially degraded material) will continue and organic carbon will accumulate, which is the carbon and energy source of heterotrophic microorganisms. Thus, besides the autotrophic weathering processes (Willscher et al., 2004), an enhancement of heterotrophic mobilization processes of heavy metals will be conceivable in the future on such a site.

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It is assumed that by action of fungi and bacteria, which degrade complex plant matter and are integrated into a heterotrophic mixed culture, sufficient DOC can be formed to solubilize and complex metal ions. In the fluvial tailing deposits, heterotrophic and autotrophic weathering and solubilization processes occur simultaneously. The complexed metals, especially Zn, Mn and Al, can be transported with the water of the creeks and rivers over a long distance, which was confirmed by measurements obtained for the surface-waters in the immediate vicinity of the fluvial tailings site (WaltonDay et al., 1999). Due to the encouraging results of these experiments, the heterotrophic microbial leaching behavior of the fluvial tailings material in percolator columns was subsequently investigated, which shall be reported in a future article. 4. Conclusions In this study it is shown that a heterotrophic mixed culture is able to mobilize considerable amounts of heavy metals (Fe, Zn, Mn, Pb, Cu, Cd, Cr) from a sulfidic AMD generating fluvial tailings material even under moderately acidic conditions as they exist at real sites. This weathering and solubilization process is proceeding as a natural process in combination with the autotrophic biooxidation of the insoluble sulfidic material in the fluvial tailings, which results in a synergistic effect on the metal mobilization. Evidently, even neutrophilic and acid tolerant heterotrophs can exist under moderately acidic conditions (down to pH 2), they are able to form viable mixed communities which may vary with and adapt to the specific environmental conditions and develop their metal remobilizing activity. The microorganisms are resistant to enhanced heavy metal concentrations at an increase in solid content. At a decrease in initial pH of the solutions, very low final pH values of the suspensions down to 1.82 (initial pH 4) were reached. Biomass measurements and CFU counts demonstrated a surprising viability of the mixed culture after some adaptation time. Especially enhanced numbers of the bacteria within the mixed community were counted under increasing unfavorable environmental conditions (increase in solid content, decrease in initial pH), but lower counts of the yeast and of fungi were observed. Besides the expansion of our general knowledge of microbial weathering processes, the perception of the participation of heterotrophic acidotolerant microbes in the weathering even of sulfidic AMD generating materials should be considered in the mitigation of environmental contaminations originating from such materials.

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