Chemosphere 41 (2000) 283±287 Removal of heavy metals from anaerobically digested sewage sludge by isolated indigenous iron-oxidizing bacteria L. Xiang, L.C. Chan, J.W.C. Wong * Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR, People's Republic of China Abstract The removal of heavy metals (Cr, Cu, Zn, Ni and Pb) from anaerobically digested sludge from the Yuen Long wastewater treatment plant, Hong Kong, has been studied in a batch system using isolated indigenous iron-oxidizing bacteria. The inoculation of indigenous iron-oxidizing bacteria and the addition of FeSO4 accelerated the solubilization of Cr, Cu, Zn, Ni and Pb from the sludge. pH of the sludge decreased with an increase in Fe2 concentrations and reached a low pH of 2±2.5 for treatments receiving both bacterial inoculation and FeSO4 . After 16 days of bioleaching, the following heavy metal removal eciencies were obtained: Cr 55.3%, Cu 91.5%, Zn 83.3%, Ni 54.4%, and Pb 16.2%. In contrast, only 2.6% of Cr, 42.9% of Cu, 72.1% of Zn, 22.8% of Ni and 0.56% of Pb were extracted from the control without the bacterial inoculation and addition of FeSO4 . The residual heavy metal content in the leached sludge was acceptable for unrestricted use for agriculture. The experimental results con®rmed the eectiveness of using the isolated iron-oxidizing bacteria for the removal of heavy metals from sewage sludge. Ó 2000 Elsevier Science Ltd. All rights reserved. 1. Introduction Huge amounts of sewage sludge potentially contaminated with heavy metals are expected to be generated in South China due to the increasing number of wastewater treatment facilities being constructed or to be built in the next few decades (Tyagi and Couillard, 1991). Dierent disposal options including incineration, land®lling, marine discharge and land application, are commonly used in most municipalities. Land application provides an economical alternative for the ®nal disposal of sewage sludge but heavy metals in sewage sludge is always an issue restricting its general use (Davis, 1987). Reduction of heavy metals in sewage sludge by source control is relatively expensive and the sources are often dicult to identify. Therefore, removal of heavy metals prior to land application is likely to be a possible and practical means for reducing metal contents * Corresponding author. Fax: +852-2339-5995. E-mail address: [email protected] (J.W.C. Wong). in sewage sludge especially for sewage sludge derived from mixed domestic and industrial sources (Sreekrishnan et al., 1993). Several chemical methods, such as EDTA extraction and acid treatment, have been suggested for solubilization of heavy metals from sewage sludge. Addition of EDTA to sewage sludge showed high removal eciencies of Cu, Pb and Cd, but low eciencies for Fe, Ni and Cr (Jenkins et al., 1981). In acid treatment, mineral and organic acids have been commonly used to solubilize heavy metals at low pH (Hayes et al., 1980; Lo and Chen, 1990). In spite of the good extraction achieved in the acid treatment method for most metals, Cu does not show a high solubilization. The high cost, operational diculties and large consumption of chemical agents have made these chemical methods unattractive. In the last decade, research work has been performed mostly in Canada to investigate the feasibility of removing heavy metals such as Cu, Zn, Pb and Cd from sewage sludge by means of a bioleaching method using Thiobacillus ferrooxidans (an iron-oxidizing bacterium) and T. thiooxidans (a sulfur-oxidizing bacterium) 0045-6535/00/$ - see front matter Ó 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 5 - 6 5 3 5 ( 9 9 ) 0 0 4 2 2 - 1 284 L. Xiang et al. / Chemosphere 41 (2000) 283±287 (Jenkins et al., 1981; Tyagi et al., 1988; Tyagi and Couillard, 1991; Chartier and Couilard, 1997). Bioleaching processes could reduce the cost by 80% as compared to the traditional chemical methods. It was reported that the adapted indigenous iron and sulphur oxidizing bacteria eectively reduced sludge pH to 2±3. More than 80% of Cu, Zn, Mn, Cd and Ni could be removed from the sludge in 8±10 days, while the removal of Cr and Pb was about 30±40%. In addition, the pH of sewage sludge needed to be adjusted to 6 4.5 for the growth of the ATCC T. ferrooxidans strains. In view of the shortcomings, adapted sulphur oxidizing bacteria were used to leach metals from sewage sludge with the addition of mineral sulphur as the major energy source (Chartier and Couilard, 1997). However, the co-existence of T. thiooxidans and residual sulphur in the decontaminated sludge would lead to acidi®cation of soil. Therefore, the aim of the present work is to investigate the feasibility of removing heavy metals such as Cr, Cu, Zn, Ni and Pb from the sludge using the isolated indigenous iron-oxidizing bacteria to render the sludge suitable for agricultural application. 2. Materials and methods 2.1. Collection and properties of sewage sludge The anaerobically digested sewage sludge was collected from the wastewater treatment plant in Yuen Long district, Hong Kong. The pH of the sewage sludge was determined immediately after collection while solid contents were determined by oven-drying at 105°C. Subsamples were digested with di-acid digestion (conc. HNO3 + conc. HClO4 ) and heavy metals in the digestates were determined by atomic absorption spectrometry (Page et al., 1982). Table 1 gives the physicochemical properties of the sewage sludge. 2.2. Microorganisms and inoculum The indigenous iron-oxidizing bacteria were isolated from the native sewage sludge and kept at 4°C and pH 2.0 in the improved Leathen medium with 20 g/l FeSO4 á 7H2 O as the energy source (Xiang, 1992). The composition of the medium was as follows (g/l): 0.45, K2 HPO4 0.15, KCl 0.05, (NH4 )2 SO4 MgSO4 á 7H2 O 0.5, Ca(NO3 )2 á 2H2 O 0.01. The bacteria were activated by repeated inoculation (n 3) at 28°C before being used in bioleaching experiments. The inoculum was prepared by growing the isolated indigenous iron-oxidizing bacteria in 250 ml Erlenymeyer ¯asks containing 100 ml of the improved Leathen medium at pH 2.0 and 28°C with 20 g/l FeSO4 á 7H2 O. The ¯asks were agitated on a horizontal shaker at 180 rpm for 30 h, which was determined in a preliminary experiment to delineate the time period required for the highest biological activity. 2.3. Bioleaching experiments A sludge slurry of 250 ml contained in a 500 ml Erlenmeyer ¯ask was pre-acidi®ed with sulphuric acid to pH 3.0 and then agitated at 28°C, 180 rpm for 24 h to increase the initial redox potential (ORP). An inoculum of 15% volume was added to the acidi®ed sludge slurry followed by the addition of Fe2 at a concentration of 0, 0.5, 1, 1.5, 2.0, 3.0 and 4.0 g/l (in the form of FeSO4 á 7H2 O). The pH of the mixture was then readjusted to 3.0 with 5 N NaOH and the whole set up was weighed. The water loss due to evaporation was replenished with distilled water. pH and ORP values were monitored every two days and 8.0 g of sample was drawn from each ¯ask for the determination of heavy metal content in the liquid phase. All treatments were done in duplicate. 2.4. Analytical methods and statistical analyses Samples collected were centrifuged at 12,000 rpm for 15 min, and the supernatants were then ®ltered, acidi®ed to pH 2 with 16 N HNO3 and stored at 4°C prior to the determination of Cr, Cu, Zn, Ni, Pb and Fe by atomic absorption spectrometry. All data were analyzed using the SAS statistical package through an IBM personal computer. One-way ANOVA was carried out to compare the means of dierent treatments; where signi®cant F values were obtained, dierences between individual means were tested using the Least Signi®cance Dierence test (Little and Hills, 1978). Table 1 Selected physico chemical properties of Yuen Long sludge Selected characteristics Solid (%) Ash (%) Total P (g/kg dry sludge) Total N (g/kg dry sludge) pH EC (mS/cm) Heavy metals (mg/kg) 2.21 31.5 15.4 51.6 7.1 3.8 Cr Cu Zn Ni Pb Fe 663 255 2823 622 57.0 72,200 L. Xiang et al. / Chemosphere 41 (2000) 283±287 3. Results and discussion 3.1. Changes in pH and ORP with time The changes in pH and ORP with bioleaching time for sludge at a Fe2 concentration of 0±4 g/l are shown in Fig. 1(a) and (b), respectively. pH of all treatments decreased sharply to the lowest pH at day 5 and then increased slightly till the end of the leaching period except for treatments receiving <2 g/l FeSO4 and the control which showed a slight increase at day 2. Increasing the concentrations of FeSO4 caused an increase in acidi®cation as indicated by the lower pH. After 16 days of bioleaching, the pH of the control was higher than that of all other treatments. Inoculation alone caused a decrease in pH to 2.7 indicating the possible utilization of sulphide and iron in the sludge as the energy source for the growth of T. ferrooxidans. Opposite to the trend of change in pH, the ORP of all treatments decreased slightly in the ®rst two days, then increased to the highest ORP at day 6 and remained at a fairly constant value till the end of the bioleaching period. All treatments had an ORP of about 580 mV except for the control, which had a lower ORP of 497 mV. This should provide a favourable condition for the oxidation of Fe2 to Fe3 in the presence of bacteria. Fig. 1. Changes in pH (a) and ORP (b) during the bioleaching of Yuen Long sewage sludge with isolated indigenous ironoxidizing bacteria. 285 Without the presence of bacteria and FeSO4 , pH decreased only slightly and Fe2 was oxidized slowly to Fe3 . A high ORP coupled with a low pH value was considered as an indicator of the existence of a substantial population of iron-oxidizing bacteria. The increase in ORP can be attributed to the increase in the Fe3 /Fe2 ratio, which may occur naturally in the presence of air or through biological catalysis: 1 2Fe2 O2 2H ! 2Fe3 H2 O 2 E E° 2:3RT =nF log Fe3 =Fe2 where R is the gas constant, T represents temperature (°K), F the Faraday constant, and n is the number of electrons transferred in this process. The observed acidi®cation of sludge can be explained by the bio-oxidation of S° compounds to SO2ÿ 4 . The precipitation of Fe3 in the form of ferric hydroxide and jarosite also generates sulphuric acid: Fe3 3H2 O ! Fe OH3 3H 3Fe3 K 2HSOÿ 4 6H2 O ! KFe3 SO4 2 OH6 8H 3.2. Removal of heavy metals from sewage sludge Fig. 2(a) shows the in¯uence of initial Fe2 concentrations on the removal of Cr from Yuen Long sludge. Solubilization of Cr seems to be closely dependent on the presence of bacteria and FeSO4 as indicated by the low Cr solubilization of 2.6% for the control, throughout the bioleaching period. There was a lag period of 2±6 days prior to leaching of Cr from the sludge for all treatments receiving the addition of FeSO4 . The lag period decreased while the removal eciency increased with an increase in Fe2 concentrations. After 10±16 days of bioleaching, 55.3±70.6% of Cr was extracted in the presence of iron oxidizing bacteria and an initial Fe2 concentration of 4.0 g/l. Therefore, both the biological and chemical factors contributed to the solubilization of Cr. The pattern of Cu solubilization diered from that of Cr with an initial increase to the maximum at days 6±8 followed by a gradual decrease till the end of the leaching period. Inoculation of iron-oxidizing bacteria increased the extraction of Cu from 70% to >90% while the addition of FeSO4 showed little in¯uence on Cu removal. After 6±10 days of bioleaching, the maximum Cu removal ranged from 88% to 98%, depending on the amount of FeSO4 added. The decrease in Cu removal eciency with time may be due to the re-adsorption of soluble Cu by solid sludge and/or the complexation between the soluble Cu and the organic compounds in the sludge. 286 L. Xiang et al. / Chemosphere 41 (2000) 283±287 Fig. 3. Removal of Ni (a) and Pb (b) from Yuen Long sewage sludge after the inoculation of the isolated indigenous ironoxidizing bacteria and addition of FeSO4 . Fig. 2. Removal of Cr (a), Cu (b) and Zn (c) from Yuen Long sewage sludge after the inoculation of the isolated indigenous iron-oxidizing bacteria and addition of FeSO4 . The variation of Zn concentration over time for each treatment is shown in Fig. 2(c). pH appears to be the major factor responsible for the solubilization of Zn. There was only a slight increase in the solubilization of Zn following the inoculation of the isolated iron-oxidizing bacteria and the addition of FeSO4 . Zinc removal eciency ranged from 72% in the control to about 87% in the treatment receiving 4.0 g/l of initial Fe2 . The removal of Ni and Pb from sewage sludge was also investigated and the results are given in Fig. 3(a) and (b), respectively. Maximum removal was achieved for treatment receiving 4.0 g/l Fe2 at day 10 for Ni and Pb with 54% and 16% removal, respectively. Similar to Cu, further extension of the bioleaching time to 16 days signi®cantly reduced the removal eciency of Ni. This may be due to the re-adsorption of soluble Ni by the solid sludge. In spite of the extension of the leaching period to 16 days, little Pb (less than 17%) was leached into the solution phase, which may be due partly to the insoluble property of PbSO4 in the pH range 2±3. Nevertheless, the concentration of Pb in the sewage sludge form Yuen Long sewage treatment plant was below the permissible level for unrestricted use (Beavers, 1993). Dierence in metal solubilization eciencies can be explained in terms of the inorganic species predominance in sewage sludge. Heavy metals associated with the organic fraction of sludge are less available to the disposal environment than inorganic precipitates. In case of acid environments, inorganic precipitates could be solubilized rapidly. For metals, which are organically bound, a prolonged reaction time would be required (Hayes and Thesis, 1978). Table 2 shows the contents of heavy metals in the original sludge and the sludge after 16 days of bioleaching. Compared to the original sludge, the contents of heavy metals in sewage sludge decreased sharply after the inoculation of 15% (v/v) bacteria of 4.0 g/l Fe2 , which were lower than the permitted levels for restricted L. Xiang et al. / Chemosphere 41 (2000) 283±287 287 Table 2 Contents of heavy metals (mg/kg dry sludge) in sludge before and after bioleaching Before bioleaching After bioleaching Permissible levelsa a Cr Cu Zn Pb Ni 663 143 200 255 75.7 140 2823 264 300 64.5 31.6 150 62.1 6.0 60 Limits on heavy metals for unrestricted use in the State of Queensland (Beavers et al., 1993). use according to the sludge guidelines of Queensland, Australia (Little and Hills, 1978). 4. Conclusion The present study con®rmed that the isolated indigenous iron oxidizing bacteria were eective in removing Cr, Cu, Zn and Ni from the anaerobically digested sludge collected from the Yuen Long wastewater treatment plant to the solution phase. The inoculation of isolated indigenous iron-oxidizing bacteria and addition of 4 g/l of Fe2 (in the form of FeSO4 á 7H2 O) resulted in the following heavy metal solubilization eciency after 10 days of bioleaching: Cr 55%, Cu 92%, Zn 83%, Ni 54%, Pb 16%. Extending the bioleaching time to 16 days increased the removal of Cr to 71%, but reduced Cu removal eciency to 67% due to the re-adsorption of soluble Cu by the solid sludge. The removal of Cr and Cu appeared to be dependent on the microbiological activities of the sludge, especially for Cr. The relatively low removal eciency for Cr indicates that the bioleaching system should be further improved through optimizing the bioleaching parameters and also the development of more eective bioleaching microorganisms. Nevertheless, the heavy metal content of the leached sludge was within the permitted level for agricultural use. Further experiments should be performed to shorten the time required for the bioleaching of Cr in order to improve the overall removal eciency for Cr. References Beavers, P.D., 1993. Guidelines for use of biosolids products ± The Queensland scene. Australian Water and Wastewater AssociationÕs Water Journal 20 (6), 23±26. Chartier, M., Couilard, D., 1997. 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