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a School of Textiles, Soongsil Univ., 1-1 Sangdo-dong, Dongjak-gu, Seoul 156-743, Korea
b Dep. of Environmental Science and Engineering, Ewha Womans Univ., 11-1 Daehyun-dong, Seodaemun-gu, Seoul 120-750, Korea
* Corresponding author (kscho{at}ewha.ac.kr)
Received for publication December 25, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Abbreviations: MW, modified Waksman medium
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Numerous methods have been proposed to remove heavy metals from sewage sludge, including chlorination, use of chelating agents, and acid treatment at high temperatures. However, these methods are generally ineffective in practical applications due to high cost, operational difficulties, and low metal leaching efficiency (Oliver and Carey, 1976; Wonzinak and Huang, 1982; Lo and Chen, 1990; Tyagi et al., 1988; Sreekrishnan and Tyagi, 1996).
An alternative way to replace chemical methods in removing heavy metals is microbial leaching with Acidithiobacillus species (Tyagi and Couillard, 1989; Tyagi et al., 1992, 1997; Jain and Tyagi, 1992; Sreekrishnan et al., 1993; Blais et al., 1993; Couillard and Mercier, 1993; Sreekrishnan et al., 1996). This method shows several advantages over chemical methods in terms of its simplicity, high yield of metal extraction, lower acid and alkali consumption, and minimum reduction in sludge nutrients such as N and P (Tyagi et al., 1988; Couillard and Mercier, 1993). In addition, microbial leaching has been shown to be 80% less expensive than acid leaching (Couillard and Mercier, 1993).
The most widely used microorganisms in metal leaching are Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans (Tyagi et al., 1988, 1991; Lo and Chen, 1990; Cho et al., 1999; Sreekrishnan and Tyagi, 1996; Sreekrishnan et al., 1996; Ryu et al., 1998). Metal leaching from the sludge occurs directly with the microorganism or indirectly with sulfuric acid formed in the presence of FeSO4 and S0. However, in microbial leaching with A. ferrooxidans and A. thiooxidans, the reaction time to leach heavy metals from the sludge to a recommended level was found to be very long (8–32 d) and the initial pH should be maintained to 2.5 to 4 (Tyagi et al., 1991, 1992; Blais et al., 1992; Jain and Tyagi, 1992; Sreekrishnan et al., 1996; Cho et al., 1999). To minimize such a problem, Tyagi et al. (1992) established mixed cultures by adding elemental sulfur during digestion; these cultures allowed heavy metal leaching without controlling the initial pH. These microorganisms were found to be the mixed cultures of neutral sulfur-oxidizing bacterium (A. thioparus) and acidophilic bacterium (A. thiooxidans).
To develop a microbial metal leaching process of sludge, a steady supply of efficient microorganisms must be ensured. Greater process efficiency can be achieved by co-inoculation of highly active isolated pure cultures to the leaching system containing indigenous microorganisms from the sludge and the mixed culture obtained by the enrichment. Once the pure culture is isolated, the leaching ability of the microorganism can be enhanced by recombination of the strain. Moreover, its application in the microbial leaching process can be optimized by understanding characteristics of the pure culture.
The objective of the present study is therefore to isolate sulfur-oxidizing pure culture A. thiooxidans, which is active in a wide range of pH values, by enrichment from the anaerobically digested sludge in the presence of elemental sulfur. We also evaluated metal leaching efficiency of the isolated culture at various sludge solids concentrations to investigate the feasibility of using this culture at high sludge solids contents. Furthermore, a relationship between the pH of the sludge solution and metal leaching efficiency was investigated.
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MATERIALS AND METHODS |
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Isolation and Identification of a Sulfur-Oxidizing Bacterium
For enrichment of sulfur-oxidizing microorganisms, a modified Waksman (MW) medium (Cho et al., 1991) of the following composition was used: 3.0 g L-1 K2HPO4, 0.1 g L-1 MgSO4·7H2O, 0.3 g L-1 CaCl2·2H2O, 0.01 g L-1 FeSO4·7H2O, and 10 g L-1 S0 as an energy source. The initial pH of the MW medium was 4.0. The dewatered sludge (5 g dry weight) was placed in a 250-mL Erlenmeyer flask containing 100 mL MW medium followed by incubation for three weeks at 30°C and 180 rpm. The pH change of the medium was monitored during the incubation. The mixed culture obtained by the enrichment was inoculated in MW medium at 10% (v/v) and the culture was again incubated at the same conditions. When the medium pH dropped to less than 2, the culture was further transferred to fresh MW medium. After 20 successive transfer cultures, the culture was finally plated in MW agar medium and the enrichment culture was isolated. This isolated culture was named MET. Identification of the isolated microorganism was performed at the Korea Research Institute of Bioscience and Biotechnology, Korean Collection for Type Cultures.
Effect of Initial pH on Sulfur Oxidation Rate of the Isolate
The pure culture of the isolated bacterium preincubated in MW medium for 3 d was inoculated at 10% (v/v) concentration in a large container containing 4 L of MW medium. The container was continuously aerated by compressed air during 8 to 10 d of incubation. The culture was then centrifuged for 3 min at 4500 x g to remove residual sulfur. The supernatant was again centrifuged for 20 min at 7600 x g and the cells were harvested. After washing the harvested cells with inorganic salt medium, the cells were further suspended within the same salt medium to obtain the concentrated cell suspension, which was used as an inoculum. The composition of the salt medium was the same as MW medium but in the absence of S0.
To investigate effects of initial pH on the sulfur oxidation rate of the isolate, the pH values of the MW medium were adjusted to 2, 3, 4, 5, 6, 7, and 8 by adding 0.2 to 2 M NaOH and HCl solutions. The concentrated cell suspension previously inoculated into the pH-adjusted MW medium was incubated at 30°C and 180 rpm. Initial optical density (OD) after inoculation was 0.05 at 660 nm. The culture broth (10 mL) was sampled every 1 or 2 d during incubation to determine pH, OD, and sulfate concentrations. When OD of the cell suspension of the isolated bacterium reached 1 at 660 nm, the actual concentration of the microorganisms corresponded to 0.47 g dry cell weight (DCW) L-1. Experiments were conducted in duplicate and the average value determined.
Effect of the Isolate Inoculation
To investigate the effect of the isolated bacterium on metal leaching, four incubation systems were prepared in 250-mL Erlenmeyer flasks containing 20 g L-1 sewage sludge, 100 mL MW medium, and 10 g S0 L-1, as follows:
System I: sterilized for 20 min at 121°C and 0.1515 MPa
System II: nonsterilized
System III: nonsterilized, inoculated with the mixed sulfur-oxidizing culture (10%, v/v)
System IV: nonsterilized, inoculated with the isolated bacterium culture (10%, v/v)
The pH of the incubation bath was measured every day to indirectly monitor growth of the microorganisms.
Effect of Sludge Concentration in Microbial Leaching of Heavy Metals by the Isolate
The isolated bacterium was cultivated in 500-mL Erlenmeyer flasks containing 200 mL MW medium and sewage sludge (50 g L-1) at 30°C and 180 rpm for 3 d. The resulting culture broth, which had been previously adapted in the sludge, was used as an inoculum to elucidate the effect of various sludge concentrations on microbial leaching of heavy metals. This culture (10%, v/v) was inoculated in 500-mL Erlenmeyer flasks containing the sewage sludge at various concentrations (20, 50, 90, and 130 g L-1 in dry weight) in 180 mL inorganic salt medium and 10 g S0 L-1. No adjustment was made for initial pH. Experiments were performed in duplicate at 30°C and 180 rpm in a shaking incubator.
Analyses of Metal and Sulfate Concentrations
Ten milliliters of sample from the flask was centrifuged for 15 min followed by filtration of the supernatant to measure concentrations of sulfate and metal in the filtrate. A finely ground dewatered sludge (0.1 g) was suspended in a Teflon vessel containing 5 mL of mixed acid solution, such as hydrofluoric acid, nitric acid, and perchloric acid (4:4:1, v/v), to determine initial concentrations of heavy metals in the sludge. The vessel was heated at 150 to 200°C to ensure a complete digestion of the sludge. Concentrations of heavy metal were measured by inductively coupled plasma spectroscopy (Plasma 40; PerkinElmer, Wellesley, MA). Sulfate concentrations in the medium were determined by ion chromatography (Waters 510 pump; Waters 432 conductivity detector; IC-Pak anion column [4.6-mm diameter x 50-mm length]; Waters, Milford, MA) at 1.2 mL min-1 and 35°C along with sodium borate–gluconate solution as a mobile phase.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Effect of Initial pH on Sulfur Oxidation Rate of Acidithiobacillus thiooxidans MET
To clarify the effect of initial pH on sulfur oxidation rate of A. thiooxidans MET, the pH values of the culture broth, dry cell weights, and sulfate concentrations were measured at all the initial pH conditions (pH 2–8). The results from pH values 2, 4, and 8 are shown in Fig. 1
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, although the reaction rate was slow in the early phase of the incubation. The pH of the medium decreased continuously and in 8 d of incubation it dropped to less than 5, thus indicating significant bacterial growth. Maximum bacterial growth was decreased after 14 d of incubation. At all other pH conditions, initial dry cell weights leveled off as the pH of the medium decreased to around 1.0 to 0.8 due to sulfur oxidation, as evidenced by increasing sulfate concentrations with incubation time. This suggested that the sulfur-oxidizing ability of MET bacterium was not adversely affected by highly acidic conditions (e.g., pH 1).
The initial sulfur oxidation rate (ISOR) of MET bacterium with time is illustrated in Fig. 2a . The ISOR for all pH treatments (pH 2–8) was determined from a ratio between concentrations of consumed energy source (S0) and incubation time during the initial 1 to 2 d of incubation. Consumed sulfur concentration was based on measurements of sulfate concentrations. Results indicated that up to pH 4, the ISOR of the MET bacterium tended to increase with increases in the initial pH of the medium. The maximum ISOR (6.1 g S L-1 d-1) was observed at pH 4. Although the ISOR decreased with increases in the pH at initial pH values greater than 4, the rates were still 3.2 and 2.2 g S L-1 d-1 at pH 6 and 7, respectively. This revealed that the MET bacterium continued to oxidize sulfur even in neutral conditions.
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Unlike most ecotypes of A. thiooxidans that are acidophilic bacteria (Tyagi et al., 1991; Jain and Tyagi, 1992), A. thiooxidans MET isolated in this study was able to oxidize sulfur not only in acidic conditions, but also in neutral conditions. Since the pH of the sewage sludge is mainly neutral, the initial pH must be adjusted to pH 2 to 4 to employ conventional A. thiooxidans in microbial leaching (Tyagi et al., 1988, 1991; Lo and Chen, 1990; Cho et al., 1999; Sreekrishnan and Tyagi, 1996; Sreekrishnan et al., 1996; Ryu et al., 1998). By contrast, A. thiooxidans MET can be directly used in a microbial leaching process without adjustment of initial sludge pH.
Effect of Acidithiobacillus thiooxidans MET Inoculation
In general, a rapid rate of acidification and low final pH of the sewage sludge are indicative of effective microbial sulfur oxidation and hence ability to leach heavy metals. Four systems comparing sterilization and inoculation treatments were analyzed for their ability to acidify sludge solutions (Fig. 3)
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Effect of Sludge Solids Concentration
Results for effects of sludge solid concentrations on pH, redox potential, and sulfate concentration are illustrated in Fig. 4 .
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The initial pH of the sludge solution inoculated with the strain MET increased with an increase in sludge solids concentration. At high sludge concentrations, such as 90 and 130 g L-1, an increase in sludge pH was observed during the first half day. However, after the initial increase, the sludge pH decreased considerably across all treatments, due to oxidation of elemental sulfur to sulfuric acid by the strain MET. The rate of acidification was much faster at lower sludge solids concentration and became slower at pH values less than 2.0. The slow acidification at high solids concentration was due to an increase in buffering capacity as described in previous studies (Sreekrishnan et al., 1993; Tyagi et al., 1997; Cho et al., 1999). The buffering effects were mainly due to the presence of basic components such as carbonate in the sludge (Brombacher et al., 1998), which hindered microbial leaching efficiency (Sreekrishnan et al., 1993; Tyagi et al., 1997; Cho et al., 1999). After 5 to 8 d of leaching, the final pH of the sludge solution decreased to 1.2, 1.3, 1.4, and 1.6 for 20, 50, 90, and 130 g L-1 sludge solids concentrations, respectively.
The sludge redox potential continuously increased with increasing reaction time and the increase in the rate of redox potential was rapid at low solids concentrations (Fig. 4b). The final redox potentials obtained for 20, 50, 90, and 130 g L-1 were 420, 42, 390, and 370 mV, respectively, suggesting a high rate of sulfur oxidation and an easy transfer of air into the sludge solution at low sludge solids concentrations. As shown in Fig. 4c, the concentration of sulfate produced by the strain MET increased with time and, regardless of sludge solids concentration, was approximately the same across treatments at any given time up to 5 d of incubation. The rate of S oxidation decreased after 5 d. Despite sulfur oxidation being similar across solids concentration treatments, pH reduction differed due to buffering by the sludge. Since the concentration of elemental sulfur in the sludge solution was 10 g L-1, the stoichiometric sulfate concentration should be 30 g L-1. Approximately 50 to 60% of sulfur was oxidized during 6 to 8 d of incubation (Fig. 4c).
Figure 5 shows concentrations of various heavy metals (Zn, Cu, Cr, Pb, Ni, and Al) solubilized by microbial leaching with A. thiooxidans MET. Regardless of type of heavy metal, the rate of leaching was faster at low sludge solids concentration than that at high solids concentration, due to a faster reduction of pH at low solids concentrations (Fig. 4a).
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The slopes (g metal per g sulfate) of the initial first-order line (before equilibrium) from the relationship between sulfate concentration and amount of leached metal shown in Fig. 7 are as follows: 2.63 for Zn, 2.43 for Cu, 3.42 for Cr, 0.85 for Pb, 1.93 for Ni, and 4.42 for Al. The greater the slope, the greater the influence of sulfate concentration on metal leaching. The influence decreased in the order of Al >> Cr > Zn > Cu > Ni >> Pb. With knowledge of such a relationship, the sole measurement of sulfate concentration in the sludge solution could permit estimation of amounts of leached metal.
Figure 8 shows the relationship between total metal content (solid metal) in the sludge and the amount of metal leached in the solution (solubilized). The latter values were selected from Fig. 6, where the amount of leached metal reached equilibrium at below pH 2. Some data points (open circles) were derived from the values cited in the reference (Sreekrishnan et al., 1993). Results revealed that the amounts of solubilized metal were proportional to solid metal content in the sludge, regardless of metal types. The slopes shown in Fig. 8 correspond to the amount of potentially leachable metal per total metal content (solid metal) within the unit volume of sludge.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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, dry cell weight; 
, sulfate.
, nonsterilized sludge; 
, nonsterilized sludge with the inoculation of A. thiooxidans MET.
, 130 g L-1.
, Cu; 
, Cr; 
, Pb.