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Auto- and heterotrophic acidophilic bacteria enhance the bioremediation efficiency of sediments contaminated by heavy metals
Chemosphere 74 (2009) 1321–1326
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Auto- and heterotrophic acidophilic bacteria enhance the bioremediation efficiency of sediments contaminated by heavy metals Francesca Beolchini a,*, Antonio Dell’Anno a, Luciano De Propris b, Stefano Ubaldini c, Federico Cerrone a, Roberto Danovaro a a
Department of Marine Sciences, Polytechnic University of Marche, Via Brecce Bianche, 60131 Ancona, Italy Istituto Centrale Ricerca Scientifica e Tecnologica Applicata al Mare – ICRAM, Rome, Italy c Istituto di Geologia Ambientale e Geoingegneria IGAG-CNR, Via Bolognola, 7, Rome, Italy b
a r t i c l e
i n f o
Article history: Received 24 July 2008 Received in revised form 21 November 2008 Accepted 21 November 2008 Available online 31 December 2008 Keywords: Contamination Bioaugmentation Metals Dredged sediments
a b s t r a c t This study deals with bioremediation treatments of dredged sediments contaminated by heavy metals based on the bioaugmentation of different bacterial strains. The efficiency of the following bacterial consortia was compared: (i) acidophilic chemoautotrophic, Fe/S-oxidising bacteria, (ii) acidophilic heterotrophic bacteria able to reduce Fe/Mn fraction, co-respiring oxygen and ferric iron and (iii) the chemoautotrophic and heterotrophic bacteria reported above, pooled together, as it was hypothesised that the two strains could cooperate through a mutual substrate supply. The effect of the bioremediation treatment based on the bioaugmentation of Fe/S-oxidising strains alone was similar to the one based only on Fe-reducing bacteria, and resulted in heavy-metal extraction yields typically ranging from 40% to 50%. The efficiency of the process based only upon autotrophic bacteria was limited by sulphur availability. However, when the treatment was based on the addition of Fe-reducing bacteria and the Fe/S oxidizing bacteria together, their growth rates and efficiency in mobilising heavy metals increased significantly, reaching extraction yields >90% for Cu, Cd, Hg and Zn. The additional advantage of the new bioaugmentation approach proposed here is that it is independent from the availability of sulphur. These results open new perspectives for the bioremediation technology for the removal of heavy metals from highly contaminated sediments. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Marine sediments represent the main repositories of anthropogenic pollutants at the global scale. This is particularly critical in the sediments accumulated in harbours and ports, where extremely high levels of several contaminants are typically reported (US EPA, 2005). These areas are also characterised by high sedimentation rates, which require periodic dredging to allow maritime transportation. As a consequence, in the last decade, a particular attention has been paid to identify the most effective treatments of decontamination of the sediment deposits, with the final objective to re-use the treated sediments either in building industries or in beaches nourishment. Among a variety of pollutants bound to the fine (silty) fraction of the harbour sediments, heavy metals are particularly dangerous for their persistency and toxicity. The concentrations of most metals in harbour sediments are usually so high that dredged sediments are included within hazardous and toxic materials (US EPA, 2005). Harbour sediments are also difficult to handle because of their muddy/pasty * Corresponding author. Tel.: +39 071 2204225; fax: +39 071 2204650. E-mail address: [email protected] (F. Beolchini). 0045-6535/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2008.11.057
characteristics and their high geochemical complexity. The most widely utilised technology for the treatment of these sediments is the separation of the coarse fraction (sands characterised by lower contamination levels), from the fine fraction (silt-clay typically highly contaminated) in order to facilitate subsequent treatments and to minimise the portion that requires disposal (Seidel et al., 2004). Current approaches for the treatment of sediments contaminated by heavy metals include the chemical extraction and/or the thermo stabilization, but their environmental impact is typically high. A promising alternative to all these technologies is based on bioremediation processes, in which microbial metabolism is utilised to change the metals’ speciation, thus enhancing their mobilisation and solubilization. Bioremediation strategies depend on the specific conditions created to stimulate bacterial metabolism; these include two main approaches: (i) bioaugmentation in which specific bacterial strains are inoculated, and (ii) biostimulation in which the metabolism of autochthonous microbial community is stimulated by inoculating specific substrates. Previous attempts made to reduce the environmental impact of the removal of heavy metals from soils utilised bio-surfactants (Mulligan et al., 2001a; Kyung et al., 2002), but little information
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is available yet for sediments bioremediation (Lors et al., 2004). The bioremediation of dredged sediments contaminated by heavy metals is generally based on bioaugmentation treatments with Acidithiobacillus spp. which are chemo-litho-autotrophic bacteria, able to oxidise sulphur (S0) and/or Fe(II) under acidic conditions, and are responsible for the solubilization of the heavy metals contained within the sulphide fraction of the sediment (Chen and Lin, 2001). Other approaches for the bioremediation of dredged sediments are based on the stimulation of the autochthonous assemblages of Fe/S oxidizing bacteria (Lors et al., 2004; Seidel et al., 2004). All bioremediation treatments of heavy metals reported so far in the literature have been focused exclusively on metals associated with sulphides (the so called ‘‘oxidisable fraction”). However several authors have suggested that the so called ‘‘reducible fraction” composed by oxides of Fe and Mn, can be very efficient in binding the heavy metals of the sedimentary matrix (Fan et al., 2005). Furthermore, while the sulphide fraction largely depends on the metabolism of the heterotrophic sulphate-reducing bacteria under anoxic conditions (Zaggia and Zonta, 1997), the Fe/Mn oxides are formed under opposite conditions (i.e. by sediment oxidation in water and/or resuspension). The aim of the present study was to test new bioremediation approaches based on bioaugmentation of different acidophilic microbial strains, in order to improve the efficiency of removal of heavy metals from highly contaminated dredged sediments. The tested treatments were based on: (i) inocula of Acidithiobacillus spp. and Leptospirillum ferrooxidans, chemoautotrophic bacteria able to oxidise both iron and sulphur, (ii) inocula of Acidiphilium cryptum strain, a chemo-heterotrophic bacterium able to reduce the Fe fraction, co-respiring oxygen and ferric iron (due to its ability to grow at low pH levels) (Küsel et al., 2002), (iii) inocula of a mixed culture of previous chemoautotrophic and chemo-heterotrophic bacteria. This latter treatment was designed hypothesising that the two microbial components can create a consortium in which the activity of A. cryptum could cooperate with chemoautotrophic bacteria (Fournier et al., 1998). 2. Materials and method
and carbonates, ca 5% by iron and manganese oxides, and less than 5% by sulphides (data not shown). 2.2. Microorganisms A mixed consortium of Acidithiobacillus thiooxidans, Acidithiobacillus ferrooxidans and L. ferrooxidans isolated in a natural environment (acid mine drainage) was provided by Professor Stoyan Groudev group, Department of Engineering and Geoecology, University of Mining and Geology ‘‘Saint Ivan Rilski”, Sofia, Bulgaria. This mixed culture is composed of chemo-litho-autotrophic bacteria, able to oxidise S0 and Fe(II) in acidic conditions. The strain was routinely cultivated in 9 K liquid medium (Cote, 1996) with iron sulphate as the energy source. The acidophilic heterotrophic A. cryptum strain was provided by German National Resource Centre for Biological Material, DSMZ No. 2390. The medium for the optimal growth conditions was prepared mixing the following solutions: 980 mL of a solution containing (in g L 1) (NH4)2SO4 2, KCl 0.1, K2HPO4 0.5, MgSO4 7H2O 0.5 and H2SO4 (pH 3); 10 mL of a solution of glucose 10%; 10 mL of a solution containing 3% yeast extract (Oxoid). All solutions were sterilized separately and afterwards mixed. Flasks were incubated at 35 °C on a rotary shaker at 150 rpm. 2.3. Experimental procedures Bioaugmentation experiments were conducted on dry sediment (2% w/v, weight of the dry sediment to volume of the medium), by inoculating different strains according to the experimental conditions. This sediment concentration was chosen in order to avoid possible autotrophic bacterial inhibition due to the high content of organic matter in the sediment. Tests were performed in 100 mL Erlenmeyer flasks, filled with 50 mL culture medium. The inoculum (5 mL) was obtained from cultures in exponential growth, with a bacterial density in the range 1.5–2 108 cells mL 1. Flasks were kept at room temperature on a rotary shaker (150 rpm). Control tests were also performed, where the sediment sample was suspended in artificial sterilised sea water. All tests were performed in triplicate.
2.1. Sediment 2.4. Experimental design Sediment samples (top 25 cm) were collected in the Ancona harbour (Central Adriatic Sea, Mediterranean Sea) by means of a modified Van Veen grab (about 14 L). Sediment samples were dried and sieved at <63 lm. Total organic matter content (ca 0.5% w/w) was determined as the difference between dry weight (60 °C, 24 h) of the sediment and weight of the residue after combustion for 2 h at 450 °C. Before combustion, sediment samples were treated with an excess of 10% HCl to remove carbonates. The X-ray diffraction analysis (Siemens D-500 diffractometer) revealed that ca 90% of the sediment was composed of silicates
Bioaugmentation experiments were performed with different acidophilic microbial strains: (i) autotrophic bacteria (A. thiooxidans, A. ferrooxidans and L. ferrooxidans), (ii) heterotrophic bacteria, and (iii) a mixed culture of both autotrophic and heterotrophic bacteria. In the first set of treatments, the mixed culture of Acidithiobacillus spp. and L. ferrooxidans was inoculated into the sediment with the addition of the 9 K medium, without iron sulphate, but in the presence of sulphur at different concentration levels: 0 (i.e. no sulphur), 0.125 and 0.5%.
Table 1 Total metals content in the sediment samples collected from the Ancona harbour and a comparison with the eco-toxicological thresholds utilised in Italy and US. Metal
Observed value (ppm)
Eco-toxicological limit Italy (Pellegrini et al., 2002)
Cu Zn Cd Hg Ni As Pb Cr
410 ± 10 500 ± 20 1.2 ± 0.1 5.4 ± 0.3 105 ± 6 21 ± 2 76 ± 4 141 ± 8
65 179 0.42 0.83 76 42 98 494
United States (US EPA, 2005) 25% Probability toxic effect
50% Probability toxic effect
50 140 0.6 0.2 24 11 48 76
157 384 2.5 0.9 80 33 161 233
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2.5. Analytical methods Heavy-metal content in the sediment was determined after acid digestion, as follows: the dried sediment (0.5 g) was transferred in Teflon boxes, added with 5 mL fluoridric acid and 1 mL of ‘‘aqua regia” (i.e. HCl:HNO3 = 3:1) incubated for 90 min at 150 °C. At the end of the incubation period, 5 mL of 10% boric acid were added and the obtained extracts were analysed by atomic absorption spectrophotometry (Varian SpectrAA 400 PLUS, atomizator GTA96, graphite furnace) for cadmium and by inductively coupled plasma-atomic emission spectrometry (Jobin Yvon JY 24) for other heavy metals. Metals distribution in different mineralogical fractions were determined by means of sequential selective extraction, according to European Union protocol (Quevauviller et al., 1997). Four different fractions are considered: (i) exchangeable fraction and carbonates, extracted utilising acetic acid 0.11 M (pH 2.8); (ii) iron and manganese oxides fraction, extracted with NH2OHHCl 0.1 M at pH 2; (iii) organic and sulphide fraction, extracted by 30% hydrogen peroxide and treated with ammonium acetate pH 2; and (iv) the residual fraction, that remains in the residual solid, is determined by difference with the total metal content. The total bacterial number in the suspension was determined by epifluorescence microscopy after staining with Acridine orange according to standard procedures for marine sediments described elsewhere (Danovaro et al., 2000).
3. Results and discussion 3.1. Heavy-metal partitioning The geochemical characterisation of the sediments is of primary importance in bioremediation technologies, as different geochemical components can influence differently the heavy-metal sequestration. For instance, the availability of sulphides, iron and manganese is crucial for several biogeochemical processes and for predicting the ability of bacteria to affect heavy-metal speciation (Lòpez-Sánchez et al., 1996; Schippers and J?rgensen, 2002). Moreover, a geochemical characterisation with a detailed analysis of heavy-metal speciation can provide important insights on the potential fate (mobilisation) of each heavy metal. In the present study, a detailed analysis of the total content of heavy metals in the harbour sediments was initially conducted. Metal and metalloid content in the sediment (ppm based on dry weight of sediment) was as follows: Cu (410 ± 10 lg g 1), Zn (500 ± 20 lg g 1), Cd (1.2 ± 0.1 lg g 1), Hg (5.4 ± 0.3 lg g 1), Ni (105 ± 6 lg g 1), As (21 ± 2 lg g 1), Pb (76 ± 4 lg g 1) and Cr (141 ± 8 lg g 1). The toxicological thresholds for marine sediment in Italy (Pellegrini et al., 1999) and in the United States (US EPA, 2004) were used to assess the contamination of the sediments utilised in the present study. These values are not adopted by law, but they can be used as safety thresholds for aquatic life, as above these value, sediments become
toxic for most biological components. All of the heavy metals encountered in the sampled sediments, with the exception of lead, arsenic and chromium, were above the Italian upper safety limit. The concentrations of some heavy metals (such as Cu) were 6 times higher than the highest accepted levels for the use and displacement of the dredged sediments. Furthermore, considering the US standards, copper, zinc and mercury were above the 50% probability toxic effect and the other metals (cadmium, nickel, arsenic, lead and chromium) were all above the 25% probability toxic effect. Therefore, the sediments utilised in the present study can be considered as highly contaminated, and their treatment for the removal of heavy metals is customary prior to any possible disposal or re-use. The metal speciation in the investigated sediment is illustrated in Fig. 1. Results presented here are in agreement with data reported in the literature (Lòpez-Sánchez et al., 1996; Pellegrini et al., 1999; Ngiam and Lim, 2001), as concerns the general trend of metal partitioning. Metals can be ordered in terms of decreasing mobility as follows: Cu, Zn, Cd, Hg, Pb, Ni, As and Cr. Furthermore, Cu was largely bound to the sulphide and the organic fraction, while Zn and Cd are bound to exchangeable and Fe/Mn fractions, Hg was mostly associated with silicates and to the exchangeable fraction, whereas Pb was bound to silicates and the sulphide fraction, and finally Ni, As and Cr were mostly bound to the residual fraction of silicates. 3.2. Bioremediation experiments In the last decade, several attempts to improve the available techniques of bioremediation for the treatment of sediments contaminated by heavy metals have been conducted (Chen and Lin, 2001; Mulligan et al., 2001b), including studies at pilot scale (Seidel et al., 2004). These studies have typically focused their attention on the ability of Fe/S oxidizing bacteria to mobilise the heavy metal, probably due to their consolidated application in bio-hydrometallurgy (Vegliò et al., 2000). At the same time, since toxic metals are distributed among different geochemical fractions (Fig. 1), it may be hypothesised that the use of Fe/S-oxidising strains in association with other micro-organisms, able to act on fractions different than sulphides, could improve metal extraction from sediments by bioleaching. In particular, Fe/Mn oxides could be identified as the complementary target fraction, due to the fact that the labile fractions are struck in an acid environment. Consequently, an essential process to improve the heavy-metal bioleaching would be to let heterotrophic bacteria respire the Fe oxides in
100 90 80
fraction,%
In the second set of treatments the bioaugmentation was conducted by inoculating the heterotrophic bacterium A. cryptum into the sediment suspended in its optimal growth medium. The latter medium was modified for the concentration of glucose, which was set at two alternative levels (i.e. 0.01 and 0.1 g L 1, corresponding to 1% and 10% of the optimal glucose concentration, respectively). Finally, in the third set of treatments the bioaugmentation was conducted by inoculating the autotrophic and the heterotrophic strains pooled together, with a volumetric ratio of autotrophic: heterotrophic set at three alternative levels: 10:1, 1:1 and 1:10. All of the bacterial strains were inoculated into a suspension at pH 2 containing the sample sediment, the 9 K medium and glucose (0.01 g L 1).
70 60 50 40 30 20 10 0 Cu
Zn
Cd
exchangeable and carbonate associated with Fe/Mn oxides
Hg
Pb
Ni
As
Cr
associated with sulphides and organics residual
Fig. 1. Metal speciation in the investigated sediments (the percentage of metal extracted in each step of the sequential extraction procedure is represented as bar diagrams; 5% standard errors are also represented). The 100% values for each metal are shown in Table 1.
F. Beolchini et al. / Chemosphere 74 (2009) 1321–1326
the sediments (Lovley et al., 2004). Moreover, this process can be done under aerobic and acidic conditions where the autotrophic bacteria (the Fe/S-oxidising strain) live. In this case a mutual cooperation between the autotrophic and the heterotrophic strains could take place, with the consequence of improving the metal
60
40 30
9
10 cells g
-1
50
20 10 0 0
3
6
9
12
15
days no S
S 0.125%
S 0.5%
control test
metal extraction yield (%)
Fig. 2. Temporal changes in bacterial abundance (cells per gram of sediment dry weight) in contaminated sediments during the bioleaching process mediated by iron and sulphur oxidising bacteria, in the presence of elemental sulphur. Reported are the effects of sulphur concentrations (sediment 2%, temperature 30 °C, stirring rate 150 rpm). The bars refer to the standard errors.
100 80 60 40 20 0 Cu
Zn no S
Cd
Hg
Pb
S 0.125%
Ni
As
S 0.5%
Cr
control
Fig. 3. Metal extraction yields at the end of bioleaching of the sediments mediated by iron and sulphur oxidising bacteria, in the presence of 0%, 0.125% 0.5% of elemental sulphur. As a comparison, extraction yields observed in control test (sediment suspended in artificial sterilised sea water) are also reported. Temporal changes in bacterial abundance are shown in Fig. 2. The bars refer to the standard errors.
a
glucose 0.01 g L-1 glucose 0.1 g L-1 control test
b
109 cells g-1
20 15 10 5 0 0
3
6
9
12
extraction yields. The results of the different bioaugmentation treatments based only on Fe/S-oxidising strains are reported in Fig. 2, which illustrates temporal changes in bacterial abundance. Heavy-metal extraction yields and metal residual concentrations at the end of the bioleaching treatment (15 d) are reported in Fig. 3. Bacterial abundance and growth rates were closely dependent upon the availability of elemental sulphur in the microcosm. Bacterial abundance was indistinguishable from the control when no sulphur was added to the medium, whereas in presence of sulphur bacterial abundance reached 5.0 1010 cells g 1 13 d after the inoculum. The differences between treatments and controls were significant already after 6 d from the inoculum at 0.5% elemental sulphur. For most metals, extraction yields increased along with the increasing sulphur supply to the growth medium. As expected, no changes in heavy-metal mobilisation were observed in the controls (i.e., sediment suspended in artificial sterilised sea water). In the treatments where low bacterial growth was observed (i.e. no sulphur addition) the metal extraction was likely due to a chemical action of the very low pH (range: 2.0–2.5) as confirmed by additional control tests performed in the same pH range without the inoculum of the autotrophic Fe/S oxidizing bacteria. Conversely, the highest performances were observed in bioaugmented sediments, in the presence of sulphur 5%. In particular the following extraction yields were observed: 78% for Ni, 57% for Hg, 46% for Cu, 36% for Zn, 35% for Cd, 18% for Pb, and 14 for As and Cr. The addition of elemental sulphur enhanced the efficiency of heavy-metal removal, and this can be explained with the need of this component as energy source for accelerating Fe/ S oxidizing bacteria metabolism (Chen and Lin, 2001) and promoting the acidic conditions, which consequently increase the mobilisation of the heavy metals (Seidel et al., 2004). Experimental tests of bioaugmentation with the only A. cryptum strain, performed at different levels of glucose concentration (0.01 and 0.1 g L 1), displayed a fast growth rate of this heterotrophic strain (specific growth rate estimated assuming a first order kinetic of 0.61 ± 0.05 d 1, Fig. 4a), higher than rates observed for the autotrophic bacteria (specific growth rate of 0.23 ± 0.03 d 1, Fig. 2). Heterotrophic bacterial abundances increased significantly after 3 d of treatment and reached a maximum of about 1.2 1010 cells g 1 after the first week. The efficiency of heavy-metal mobilisation was always <50% (Fig. 4b), and it was likely largely associated with the chemical action of the acid medium. The obtained yields were comparable to those observed for treatments based on autotrophic Fe/S-oxidising mixed culture in the absence of elemental sulphur (Fig. 3a), Cu, Zn, Cd and Hg were mobilised for a 40–50% while lower extraction yields were achieved for all of the other heavy metals. It
15
metal extraction yield (%)
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glucose 0.01 g L-1 glucose 0.1 g L-1 control test 100 80 60 40 20 0 Cu Zn Cd Hg Pb Ni
As Cr
days Fig. 4. Cell growth (a) and metal extraction yields at the end of sediments bioleaching (b) with heterotrophic bacteria, in the presence of 0.01 and 0.1 g L 1 glucose. As a comparison, extraction yields observed in control test (sediment suspended in artificial sterilised sea water) are also reported. The bars refer to the standard errors.
F. Beolchini et al. / Chemosphere 74 (2009) 1321–1326
a
b metal extraction yield (%)
10:1 1:1 1:10
30 20
9
10 cells g
-1
40
10 0 0
3
6
9
12
15
1325
10:1 1:1 1:10 control test
100 80 60 40 20 0
Cu Zn Cd Hg Pb Ni A s Cr
days Fig. 5. Cell growth (a) and metal extraction yields (b) in the microcosms inoculated with a mixed consortium of autotrophic and heterotrophic bacteria in the ratio 10:1 (circles); 1:1 (stars) and 1:10 (triangles), respectively. The bars refer to the standard errors. As a comparison, extraction yields observed in control test (sediment resuspended in artificial sterilised seawater) are also reported.
can be also noticed that the highest heavy-metal extraction yields were observed at the lowest levels of glucose and that the extraction yields did not increase with increasing glucose concentration (Fig. 4b). A possible explanation of this phenomenon is that at low glucose concentrations, the heterotrophic bacteria are stimulated to use complementary organic sources from the sediment with the consequent dissolution of the organically bound metals. The bioaugmentation treatment based on the inoculum of the autotrophic Fe/S oxidizing bacteria together with the heterotrophic acidophilic strain (i.e. A. cryptum) resulted in significantly higher extraction yields of all heavy metals investigated. The growth rates of the bacteria inoculated were significantly higher than those reported for the treatments based on either autotrophic or heterotrophic bacteria alone. In particular, after 5 d, bacterial abundances reached values of 1.4 1010 cells g 1 and 2.4 1010 cells g 1 in microcosms inoculated with autotrophic and heterotrophic bacteria in the ratio 10:1 and 1:10, respectively (Fig. 5a). Even faster bacterial growth rates were observed in the bioaugmentation treatment where the autotrophic Fe/S oxidizing bacteria and the heterotrophic bacteria were inoculated in the ratio 1:1 (highest bacterial abundance: 2.8 1010 cells g 1, achieved after 3 d of treatment). As a consequence, in the presence of the autotrophic and heterotrophic bacteria together, the efficiency of removal increased significantly for all of the heavy metals, reaching extraction yields >90% for Cu, Cd, Hg and Zn (Fig. 5b). With the except of Zn, no significant differences in heavy-metal extraction yields were observed among experimental systems containing autotrophic and heterotrophic bacteria inoculated in different proportion. Therefore all tested proportions of autotrophic and heterotrophic bacteria inoculated to the sediment are equal efficient in metal mobilisation from the different mineralogical fractions. These results suggest that the presence of a mixed consortium of heterotrophic and autotrophic bacterial strains may determine a higher heavy-metal extraction efficiency due to a coupled and synergistic metabolism. In fact in the absence of elemental sulphur the presence of A. cryptum may enhance the metal bioleaching activity of Fe/S-oxidising strain (Fournier et al., 1998). At the same time the growth of autotrophic bacteria and the release of labile organic material from their metabolism seems to be enough for the heterotrophic needs of the Fe-reducing bacteria. Overall data reported in the present work may open new perspectives for the application of bioleaching technologies to remediation purposes based on mutual interactions among bacterial strains displaying different metabolic pathways and requirements. Further investigations are needed to fully understand the potential
application of this technology, to evaluate the efficiency of this bioremediation approach to different kinds of contaminated sediments and the extent by which these treatments can be further optimised. Acknowledgements Authors are grateful to Professor Francesco Vegliò (University of L’Aquila), for his precious suggestions during the design of experimental tests. This research was financially supported by the Italian Ministry of Research (MUR). References Chen, S., Lin, J., 2001. Effect of substrate concentration on bioleaching of metal contaminated sediment. J. Hazard. Mater. B 82, 77–89. Cote, R., 1996. ATCC Bacteria and Bacteriophages. Rockville, Maryland. Danovaro, R., Marrale, D., Dell’Anno, A., Della Croce, N., Tselepides, A., Fabiano, M., 2000. Bacterial response to seasonal changes in labile organic matter composition on the continental shelf and bathyal sediments of the Cretan Sea. Prog. Oceanogr. 46, 345–366. Fan, M., Boonfueng, T., Xu, Y., Axe, L., Tyson, T.A., 2005. Modeling Pb sorption to microporous amorphous oxides as discrete particles and coatings. J. Colloid Interf. Sci. 254, 214–221. Fournier, D., Lemieux, R., Couillard, D., 1998. Essential interactions between Thiobacillus ferrooxidans and heterotrophic microorganisms during a wastewater sludge bioleaching process. Environ. Pollut. 101, 303–309. Küsel, K., Roth, U., Drake, H.L., 2002. Microbial reduction of Fe(III) in the presence of oxygen under low pH conditions. Environ. Microbiol. 4, 414–421. Kyung, J.H., Tokunaga, S., Kajiuchi, T., 2002. Evaluation of remediation process with plant derived biosurfactant for recovery of heavy metals from contaminated soils. Chemosphere 49, 379–384. Lòpez-Sánchez, J.F., Samitier, C., Rubio, R., Rauret, G., 1996. Trace metal partitioning in marine sediments and sludges deposited off the coast of Barcelona (Spain). Water Res. 30, 153–159. Lors, C., Tiffreau, C., Laboudigue, A., 2004. Effects of bacterial activities on the release of heavy metals from contaminated dredged sediments. Chemosphere 56, 619– 630. Lovley, D.R., Holmes, D.E., Nevin, K.P., 2004. Dissimilatory Fe(III) and Mn(IV) reduction. Adv. Microb. Physiol. 49, 219–286. Mulligan, C.N., Yong, C.N., Gibbs, B.F., 2001a. Heavy metal removal from sediments by biosurfactants. J. Hazard. Mater. 85, 111–125. Mulligan, C.N., Yong, R.N., Gibbs, B.F., 2001b. An evaluation of technologies for the heavy metal remediation of dredged sediments. J. Hazard. Mater. 85, 145–163. Ngiam, L.S., Lim, P.E., 2001. Speciation pattern of heavy metals in tropical estuarine anoxic and oxidized sediments by different sequential extraction schemes. Sci. Total Environ. 275, 53–61. Pellegrini, D., Ausili, A., Onorati, F., Ciuffa, G., Gabellini, M., Bigongiari, N., De Ranieri, S., 1999. Characterisation of harbour and coastal sediments: specific destination of dredged material. Aqua. Ecosyst. Health Manage. 2, 455–464. Quevauviller, P., Rauret, G., Lopez-Sanchez, J.F., Rubio, R., Ure, A., Muntau, H, 1997. The certification of the extractable contents (mass fractions) of Cd, Cr, Ni, Pb and Zn in sediment following a three-step sequential extraction procedure. Report EUR 17554 EN European Commission, Bruxelles.
1326
F. Beolchini et al. / Chemosphere 74 (2009) 1321–1326
Schippers, A., J?rgensen, B.B., 2002. Biogeochemistry of pyrite and iron sulfide oxidation in marine sediments. Geochim. Cosmochim. Acta 66, 85–92. Seidel, H., Löser, C., Zehnsdorf, A., Hoffmann, P., Schmerold, R., 2004. Bioremediation process for sediments contaminated by heavy metals: feasibility study on a pilot scale. Environ. Sci. Technol. 38, 1582–1588. US EPA, 2004. The Incidence and Severity of Sediment Contamination in Surface Waters of the United States, National Sediment Quality Survey, second ed., EPA823-R-04-007, Office of Science and Technology, Standards and Health Protection Division, Washington DC, November 2004.
US EPA, 2005. Contaminated Sediment Remediation Guidance for Hazardous Waste Sites, EPA-540-R-05-012, Office of Solid Waste and Emergency Response OSWER 9355.0-85, December 2005. Vegliò, F., Beolchini, F., Nardini, A., Toro, L., 2000. Bioleaching of a pirrhotite ore by a sulfoxidans strain: kinetic analysis. Chem. Eng. Sci. 55, 783–795. Zaggia, L., Zonta, R., 1997. Metal-sulphide formation in the contaminated anoxic sludge of the Venice canals. Appl. Geochem. 12, 527–536.
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Auto- and heterotrophic acidophilic bacteria enhance the bioremediation efficiency of sediments contaminated by heavy metals Francesca Beolchini a,*, Antonio Dell’Anno a, Luciano De Propris b, Stefano Ubaldini c, Federico Cerrone a, Roberto Danovaro a a
Department of Marine Sciences, Polytechnic University of Marche, Via Brecce Bianche, 60131 Ancona, Italy Istituto Centrale Ricerca Scientifica e Tecnologica Applicata al Mare – ICRAM, Rome, Italy c Istituto di Geologia Ambientale e Geoingegneria IGAG-CNR, Via Bolognola, 7, Rome, Italy b
a r t i c l e
i n f o
Article history: Received 24 July 2008 Received in revised form 21 November 2008 Accepted 21 November 2008 Available online 31 December 2008 Keywords: Contamination Bioaugmentation Metals Dredged sediments
a b s t r a c t This study deals with bioremediation treatments of dredged sediments contaminated by heavy metals based on the bioaugmentation of different bacterial strains. The efficiency of the following bacterial consortia was compared: (i) acidophilic chemoautotrophic, Fe/S-oxidising bacteria, (ii) acidophilic heterotrophic bacteria able to reduce Fe/Mn fraction, co-respiring oxygen and ferric iron and (iii) the chemoautotrophic and heterotrophic bacteria reported above, pooled together, as it was hypothesised that the two strains could cooperate through a mutual substrate supply. The effect of the bioremediation treatment based on the bioaugmentation of Fe/S-oxidising strains alone was similar to the one based only on Fe-reducing bacteria, and resulted in heavy-metal extraction yields typically ranging from 40% to 50%. The efficiency of the process based only upon autotrophic bacteria was limited by sulphur availability. However, when the treatment was based on the addition of Fe-reducing bacteria and the Fe/S oxidizing bacteria together, their growth rates and efficiency in mobilising heavy metals increased significantly, reaching extraction yields >90% for Cu, Cd, Hg and Zn. The additional advantage of the new bioaugmentation approach proposed here is that it is independent from the availability of sulphur. These results open new perspectives for the bioremediation technology for the removal of heavy metals from highly contaminated sediments. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Marine sediments represent the main repositories of anthropogenic pollutants at the global scale. This is particularly critical in the sediments accumulated in harbours and ports, where extremely high levels of several contaminants are typically reported (US EPA, 2005). These areas are also characterised by high sedimentation rates, which require periodic dredging to allow maritime transportation. As a consequence, in the last decade, a particular attention has been paid to identify the most effective treatments of decontamination of the sediment deposits, with the final objective to re-use the treated sediments either in building industries or in beaches nourishment. Among a variety of pollutants bound to the fine (silty) fraction of the harbour sediments, heavy metals are particularly dangerous for their persistency and toxicity. The concentrations of most metals in harbour sediments are usually so high that dredged sediments are included within hazardous and toxic materials (US EPA, 2005). Harbour sediments are also difficult to handle because of their muddy/pasty * Corresponding author. Tel.: +39 071 2204225; fax: +39 071 2204650. E-mail address: [email protected] (F. Beolchini). 0045-6535/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2008.11.057
characteristics and their high geochemical complexity. The most widely utilised technology for the treatment of these sediments is the separation of the coarse fraction (sands characterised by lower contamination levels), from the fine fraction (silt-clay typically highly contaminated) in order to facilitate subsequent treatments and to minimise the portion that requires disposal (Seidel et al., 2004). Current approaches for the treatment of sediments contaminated by heavy metals include the chemical extraction and/or the thermo stabilization, but their environmental impact is typically high. A promising alternative to all these technologies is based on bioremediation processes, in which microbial metabolism is utilised to change the metals’ speciation, thus enhancing their mobilisation and solubilization. Bioremediation strategies depend on the specific conditions created to stimulate bacterial metabolism; these include two main approaches: (i) bioaugmentation in which specific bacterial strains are inoculated, and (ii) biostimulation in which the metabolism of autochthonous microbial community is stimulated by inoculating specific substrates. Previous attempts made to reduce the environmental impact of the removal of heavy metals from soils utilised bio-surfactants (Mulligan et al., 2001a; Kyung et al., 2002), but little information
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is available yet for sediments bioremediation (Lors et al., 2004). The bioremediation of dredged sediments contaminated by heavy metals is generally based on bioaugmentation treatments with Acidithiobacillus spp. which are chemo-litho-autotrophic bacteria, able to oxidise sulphur (S0) and/or Fe(II) under acidic conditions, and are responsible for the solubilization of the heavy metals contained within the sulphide fraction of the sediment (Chen and Lin, 2001). Other approaches for the bioremediation of dredged sediments are based on the stimulation of the autochthonous assemblages of Fe/S oxidizing bacteria (Lors et al., 2004; Seidel et al., 2004). All bioremediation treatments of heavy metals reported so far in the literature have been focused exclusively on metals associated with sulphides (the so called ‘‘oxidisable fraction”). However several authors have suggested that the so called ‘‘reducible fraction” composed by oxides of Fe and Mn, can be very efficient in binding the heavy metals of the sedimentary matrix (Fan et al., 2005). Furthermore, while the sulphide fraction largely depends on the metabolism of the heterotrophic sulphate-reducing bacteria under anoxic conditions (Zaggia and Zonta, 1997), the Fe/Mn oxides are formed under opposite conditions (i.e. by sediment oxidation in water and/or resuspension). The aim of the present study was to test new bioremediation approaches based on bioaugmentation of different acidophilic microbial strains, in order to improve the efficiency of removal of heavy metals from highly contaminated dredged sediments. The tested treatments were based on: (i) inocula of Acidithiobacillus spp. and Leptospirillum ferrooxidans, chemoautotrophic bacteria able to oxidise both iron and sulphur, (ii) inocula of Acidiphilium cryptum strain, a chemo-heterotrophic bacterium able to reduce the Fe fraction, co-respiring oxygen and ferric iron (due to its ability to grow at low pH levels) (Küsel et al., 2002), (iii) inocula of a mixed culture of previous chemoautotrophic and chemo-heterotrophic bacteria. This latter treatment was designed hypothesising that the two microbial components can create a consortium in which the activity of A. cryptum could cooperate with chemoautotrophic bacteria (Fournier et al., 1998). 2. Materials and method
and carbonates, ca 5% by iron and manganese oxides, and less than 5% by sulphides (data not shown). 2.2. Microorganisms A mixed consortium of Acidithiobacillus thiooxidans, Acidithiobacillus ferrooxidans and L. ferrooxidans isolated in a natural environment (acid mine drainage) was provided by Professor Stoyan Groudev group, Department of Engineering and Geoecology, University of Mining and Geology ‘‘Saint Ivan Rilski”, Sofia, Bulgaria. This mixed culture is composed of chemo-litho-autotrophic bacteria, able to oxidise S0 and Fe(II) in acidic conditions. The strain was routinely cultivated in 9 K liquid medium (Cote, 1996) with iron sulphate as the energy source. The acidophilic heterotrophic A. cryptum strain was provided by German National Resource Centre for Biological Material, DSMZ No. 2390. The medium for the optimal growth conditions was prepared mixing the following solutions: 980 mL of a solution containing (in g L 1) (NH4)2SO4 2, KCl 0.1, K2HPO4 0.5, MgSO4 7H2O 0.5 and H2SO4 (pH 3); 10 mL of a solution of glucose 10%; 10 mL of a solution containing 3% yeast extract (Oxoid). All solutions were sterilized separately and afterwards mixed. Flasks were incubated at 35 °C on a rotary shaker at 150 rpm. 2.3. Experimental procedures Bioaugmentation experiments were conducted on dry sediment (2% w/v, weight of the dry sediment to volume of the medium), by inoculating different strains according to the experimental conditions. This sediment concentration was chosen in order to avoid possible autotrophic bacterial inhibition due to the high content of organic matter in the sediment. Tests were performed in 100 mL Erlenmeyer flasks, filled with 50 mL culture medium. The inoculum (5 mL) was obtained from cultures in exponential growth, with a bacterial density in the range 1.5–2 108 cells mL 1. Flasks were kept at room temperature on a rotary shaker (150 rpm). Control tests were also performed, where the sediment sample was suspended in artificial sterilised sea water. All tests were performed in triplicate.
2.1. Sediment 2.4. Experimental design Sediment samples (top 25 cm) were collected in the Ancona harbour (Central Adriatic Sea, Mediterranean Sea) by means of a modified Van Veen grab (about 14 L). Sediment samples were dried and sieved at <63 lm. Total organic matter content (ca 0.5% w/w) was determined as the difference between dry weight (60 °C, 24 h) of the sediment and weight of the residue after combustion for 2 h at 450 °C. Before combustion, sediment samples were treated with an excess of 10% HCl to remove carbonates. The X-ray diffraction analysis (Siemens D-500 diffractometer) revealed that ca 90% of the sediment was composed of silicates
Bioaugmentation experiments were performed with different acidophilic microbial strains: (i) autotrophic bacteria (A. thiooxidans, A. ferrooxidans and L. ferrooxidans), (ii) heterotrophic bacteria, and (iii) a mixed culture of both autotrophic and heterotrophic bacteria. In the first set of treatments, the mixed culture of Acidithiobacillus spp. and L. ferrooxidans was inoculated into the sediment with the addition of the 9 K medium, without iron sulphate, but in the presence of sulphur at different concentration levels: 0 (i.e. no sulphur), 0.125 and 0.5%.
Table 1 Total metals content in the sediment samples collected from the Ancona harbour and a comparison with the eco-toxicological thresholds utilised in Italy and US. Metal
Observed value (ppm)
Eco-toxicological limit Italy (Pellegrini et al., 2002)
Cu Zn Cd Hg Ni As Pb Cr
410 ± 10 500 ± 20 1.2 ± 0.1 5.4 ± 0.3 105 ± 6 21 ± 2 76 ± 4 141 ± 8
65 179 0.42 0.83 76 42 98 494
United States (US EPA, 2005) 25% Probability toxic effect
50% Probability toxic effect
50 140 0.6 0.2 24 11 48 76
157 384 2.5 0.9 80 33 161 233
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2.5. Analytical methods Heavy-metal content in the sediment was determined after acid digestion, as follows: the dried sediment (0.5 g) was transferred in Teflon boxes, added with 5 mL fluoridric acid and 1 mL of ‘‘aqua regia” (i.e. HCl:HNO3 = 3:1) incubated for 90 min at 150 °C. At the end of the incubation period, 5 mL of 10% boric acid were added and the obtained extracts were analysed by atomic absorption spectrophotometry (Varian SpectrAA 400 PLUS, atomizator GTA96, graphite furnace) for cadmium and by inductively coupled plasma-atomic emission spectrometry (Jobin Yvon JY 24) for other heavy metals. Metals distribution in different mineralogical fractions were determined by means of sequential selective extraction, according to European Union protocol (Quevauviller et al., 1997). Four different fractions are considered: (i) exchangeable fraction and carbonates, extracted utilising acetic acid 0.11 M (pH 2.8); (ii) iron and manganese oxides fraction, extracted with NH2OHHCl 0.1 M at pH 2; (iii) organic and sulphide fraction, extracted by 30% hydrogen peroxide and treated with ammonium acetate pH 2; and (iv) the residual fraction, that remains in the residual solid, is determined by difference with the total metal content. The total bacterial number in the suspension was determined by epifluorescence microscopy after staining with Acridine orange according to standard procedures for marine sediments described elsewhere (Danovaro et al., 2000).
3. Results and discussion 3.1. Heavy-metal partitioning The geochemical characterisation of the sediments is of primary importance in bioremediation technologies, as different geochemical components can influence differently the heavy-metal sequestration. For instance, the availability of sulphides, iron and manganese is crucial for several biogeochemical processes and for predicting the ability of bacteria to affect heavy-metal speciation (Lòpez-Sánchez et al., 1996; Schippers and J?rgensen, 2002). Moreover, a geochemical characterisation with a detailed analysis of heavy-metal speciation can provide important insights on the potential fate (mobilisation) of each heavy metal. In the present study, a detailed analysis of the total content of heavy metals in the harbour sediments was initially conducted. Metal and metalloid content in the sediment (ppm based on dry weight of sediment) was as follows: Cu (410 ± 10 lg g 1), Zn (500 ± 20 lg g 1), Cd (1.2 ± 0.1 lg g 1), Hg (5.4 ± 0.3 lg g 1), Ni (105 ± 6 lg g 1), As (21 ± 2 lg g 1), Pb (76 ± 4 lg g 1) and Cr (141 ± 8 lg g 1). The toxicological thresholds for marine sediment in Italy (Pellegrini et al., 1999) and in the United States (US EPA, 2004) were used to assess the contamination of the sediments utilised in the present study. These values are not adopted by law, but they can be used as safety thresholds for aquatic life, as above these value, sediments become
toxic for most biological components. All of the heavy metals encountered in the sampled sediments, with the exception of lead, arsenic and chromium, were above the Italian upper safety limit. The concentrations of some heavy metals (such as Cu) were 6 times higher than the highest accepted levels for the use and displacement of the dredged sediments. Furthermore, considering the US standards, copper, zinc and mercury were above the 50% probability toxic effect and the other metals (cadmium, nickel, arsenic, lead and chromium) were all above the 25% probability toxic effect. Therefore, the sediments utilised in the present study can be considered as highly contaminated, and their treatment for the removal of heavy metals is customary prior to any possible disposal or re-use. The metal speciation in the investigated sediment is illustrated in Fig. 1. Results presented here are in agreement with data reported in the literature (Lòpez-Sánchez et al., 1996; Pellegrini et al., 1999; Ngiam and Lim, 2001), as concerns the general trend of metal partitioning. Metals can be ordered in terms of decreasing mobility as follows: Cu, Zn, Cd, Hg, Pb, Ni, As and Cr. Furthermore, Cu was largely bound to the sulphide and the organic fraction, while Zn and Cd are bound to exchangeable and Fe/Mn fractions, Hg was mostly associated with silicates and to the exchangeable fraction, whereas Pb was bound to silicates and the sulphide fraction, and finally Ni, As and Cr were mostly bound to the residual fraction of silicates. 3.2. Bioremediation experiments In the last decade, several attempts to improve the available techniques of bioremediation for the treatment of sediments contaminated by heavy metals have been conducted (Chen and Lin, 2001; Mulligan et al., 2001b), including studies at pilot scale (Seidel et al., 2004). These studies have typically focused their attention on the ability of Fe/S oxidizing bacteria to mobilise the heavy metal, probably due to their consolidated application in bio-hydrometallurgy (Vegliò et al., 2000). At the same time, since toxic metals are distributed among different geochemical fractions (Fig. 1), it may be hypothesised that the use of Fe/S-oxidising strains in association with other micro-organisms, able to act on fractions different than sulphides, could improve metal extraction from sediments by bioleaching. In particular, Fe/Mn oxides could be identified as the complementary target fraction, due to the fact that the labile fractions are struck in an acid environment. Consequently, an essential process to improve the heavy-metal bioleaching would be to let heterotrophic bacteria respire the Fe oxides in
100 90 80
fraction,%
In the second set of treatments the bioaugmentation was conducted by inoculating the heterotrophic bacterium A. cryptum into the sediment suspended in its optimal growth medium. The latter medium was modified for the concentration of glucose, which was set at two alternative levels (i.e. 0.01 and 0.1 g L 1, corresponding to 1% and 10% of the optimal glucose concentration, respectively). Finally, in the third set of treatments the bioaugmentation was conducted by inoculating the autotrophic and the heterotrophic strains pooled together, with a volumetric ratio of autotrophic: heterotrophic set at three alternative levels: 10:1, 1:1 and 1:10. All of the bacterial strains were inoculated into a suspension at pH 2 containing the sample sediment, the 9 K medium and glucose (0.01 g L 1).
70 60 50 40 30 20 10 0 Cu
Zn
Cd
exchangeable and carbonate associated with Fe/Mn oxides
Hg
Pb
Ni
As
Cr
associated with sulphides and organics residual
Fig. 1. Metal speciation in the investigated sediments (the percentage of metal extracted in each step of the sequential extraction procedure is represented as bar diagrams; 5% standard errors are also represented). The 100% values for each metal are shown in Table 1.
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the sediments (Lovley et al., 2004). Moreover, this process can be done under aerobic and acidic conditions where the autotrophic bacteria (the Fe/S-oxidising strain) live. In this case a mutual cooperation between the autotrophic and the heterotrophic strains could take place, with the consequence of improving the metal
60
40 30
9
10 cells g
-1
50
20 10 0 0
3
6
9
12
15
days no S
S 0.125%
S 0.5%
control test
metal extraction yield (%)
Fig. 2. Temporal changes in bacterial abundance (cells per gram of sediment dry weight) in contaminated sediments during the bioleaching process mediated by iron and sulphur oxidising bacteria, in the presence of elemental sulphur. Reported are the effects of sulphur concentrations (sediment 2%, temperature 30 °C, stirring rate 150 rpm). The bars refer to the standard errors.
100 80 60 40 20 0 Cu
Zn no S
Cd
Hg
Pb
S 0.125%
Ni
As
S 0.5%
Cr
control
Fig. 3. Metal extraction yields at the end of bioleaching of the sediments mediated by iron and sulphur oxidising bacteria, in the presence of 0%, 0.125% 0.5% of elemental sulphur. As a comparison, extraction yields observed in control test (sediment suspended in artificial sterilised sea water) are also reported. Temporal changes in bacterial abundance are shown in Fig. 2. The bars refer to the standard errors.
a
glucose 0.01 g L-1 glucose 0.1 g L-1 control test
b
109 cells g-1
20 15 10 5 0 0
3
6
9
12
extraction yields. The results of the different bioaugmentation treatments based only on Fe/S-oxidising strains are reported in Fig. 2, which illustrates temporal changes in bacterial abundance. Heavy-metal extraction yields and metal residual concentrations at the end of the bioleaching treatment (15 d) are reported in Fig. 3. Bacterial abundance and growth rates were closely dependent upon the availability of elemental sulphur in the microcosm. Bacterial abundance was indistinguishable from the control when no sulphur was added to the medium, whereas in presence of sulphur bacterial abundance reached 5.0 1010 cells g 1 13 d after the inoculum. The differences between treatments and controls were significant already after 6 d from the inoculum at 0.5% elemental sulphur. For most metals, extraction yields increased along with the increasing sulphur supply to the growth medium. As expected, no changes in heavy-metal mobilisation were observed in the controls (i.e., sediment suspended in artificial sterilised sea water). In the treatments where low bacterial growth was observed (i.e. no sulphur addition) the metal extraction was likely due to a chemical action of the very low pH (range: 2.0–2.5) as confirmed by additional control tests performed in the same pH range without the inoculum of the autotrophic Fe/S oxidizing bacteria. Conversely, the highest performances were observed in bioaugmented sediments, in the presence of sulphur 5%. In particular the following extraction yields were observed: 78% for Ni, 57% for Hg, 46% for Cu, 36% for Zn, 35% for Cd, 18% for Pb, and 14 for As and Cr. The addition of elemental sulphur enhanced the efficiency of heavy-metal removal, and this can be explained with the need of this component as energy source for accelerating Fe/ S oxidizing bacteria metabolism (Chen and Lin, 2001) and promoting the acidic conditions, which consequently increase the mobilisation of the heavy metals (Seidel et al., 2004). Experimental tests of bioaugmentation with the only A. cryptum strain, performed at different levels of glucose concentration (0.01 and 0.1 g L 1), displayed a fast growth rate of this heterotrophic strain (specific growth rate estimated assuming a first order kinetic of 0.61 ± 0.05 d 1, Fig. 4a), higher than rates observed for the autotrophic bacteria (specific growth rate of 0.23 ± 0.03 d 1, Fig. 2). Heterotrophic bacterial abundances increased significantly after 3 d of treatment and reached a maximum of about 1.2 1010 cells g 1 after the first week. The efficiency of heavy-metal mobilisation was always <50% (Fig. 4b), and it was likely largely associated with the chemical action of the acid medium. The obtained yields were comparable to those observed for treatments based on autotrophic Fe/S-oxidising mixed culture in the absence of elemental sulphur (Fig. 3a), Cu, Zn, Cd and Hg were mobilised for a 40–50% while lower extraction yields were achieved for all of the other heavy metals. It
15
metal extraction yield (%)
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glucose 0.01 g L-1 glucose 0.1 g L-1 control test 100 80 60 40 20 0 Cu Zn Cd Hg Pb Ni
As Cr
days Fig. 4. Cell growth (a) and metal extraction yields at the end of sediments bioleaching (b) with heterotrophic bacteria, in the presence of 0.01 and 0.1 g L 1 glucose. As a comparison, extraction yields observed in control test (sediment suspended in artificial sterilised sea water) are also reported. The bars refer to the standard errors.
F. Beolchini et al. / Chemosphere 74 (2009) 1321–1326
a
b metal extraction yield (%)
10:1 1:1 1:10
30 20
9
10 cells g
-1
40
10 0 0
3
6
9
12
15
1325
10:1 1:1 1:10 control test
100 80 60 40 20 0
Cu Zn Cd Hg Pb Ni A s Cr
days Fig. 5. Cell growth (a) and metal extraction yields (b) in the microcosms inoculated with a mixed consortium of autotrophic and heterotrophic bacteria in the ratio 10:1 (circles); 1:1 (stars) and 1:10 (triangles), respectively. The bars refer to the standard errors. As a comparison, extraction yields observed in control test (sediment resuspended in artificial sterilised seawater) are also reported.
can be also noticed that the highest heavy-metal extraction yields were observed at the lowest levels of glucose and that the extraction yields did not increase with increasing glucose concentration (Fig. 4b). A possible explanation of this phenomenon is that at low glucose concentrations, the heterotrophic bacteria are stimulated to use complementary organic sources from the sediment with the consequent dissolution of the organically bound metals. The bioaugmentation treatment based on the inoculum of the autotrophic Fe/S oxidizing bacteria together with the heterotrophic acidophilic strain (i.e. A. cryptum) resulted in significantly higher extraction yields of all heavy metals investigated. The growth rates of the bacteria inoculated were significantly higher than those reported for the treatments based on either autotrophic or heterotrophic bacteria alone. In particular, after 5 d, bacterial abundances reached values of 1.4 1010 cells g 1 and 2.4 1010 cells g 1 in microcosms inoculated with autotrophic and heterotrophic bacteria in the ratio 10:1 and 1:10, respectively (Fig. 5a). Even faster bacterial growth rates were observed in the bioaugmentation treatment where the autotrophic Fe/S oxidizing bacteria and the heterotrophic bacteria were inoculated in the ratio 1:1 (highest bacterial abundance: 2.8 1010 cells g 1, achieved after 3 d of treatment). As a consequence, in the presence of the autotrophic and heterotrophic bacteria together, the efficiency of removal increased significantly for all of the heavy metals, reaching extraction yields >90% for Cu, Cd, Hg and Zn (Fig. 5b). With the except of Zn, no significant differences in heavy-metal extraction yields were observed among experimental systems containing autotrophic and heterotrophic bacteria inoculated in different proportion. Therefore all tested proportions of autotrophic and heterotrophic bacteria inoculated to the sediment are equal efficient in metal mobilisation from the different mineralogical fractions. These results suggest that the presence of a mixed consortium of heterotrophic and autotrophic bacterial strains may determine a higher heavy-metal extraction efficiency due to a coupled and synergistic metabolism. In fact in the absence of elemental sulphur the presence of A. cryptum may enhance the metal bioleaching activity of Fe/S-oxidising strain (Fournier et al., 1998). At the same time the growth of autotrophic bacteria and the release of labile organic material from their metabolism seems to be enough for the heterotrophic needs of the Fe-reducing bacteria. Overall data reported in the present work may open new perspectives for the application of bioleaching technologies to remediation purposes based on mutual interactions among bacterial strains displaying different metabolic pathways and requirements. Further investigations are needed to fully understand the potential
application of this technology, to evaluate the efficiency of this bioremediation approach to different kinds of contaminated sediments and the extent by which these treatments can be further optimised. Acknowledgements Authors are grateful to Professor Francesco Vegliò (University of L’Aquila), for his precious suggestions during the design of experimental tests. This research was financially supported by the Italian Ministry of Research (MUR). References Chen, S., Lin, J., 2001. Effect of substrate concentration on bioleaching of metal contaminated sediment. J. Hazard. Mater. B 82, 77–89. Cote, R., 1996. ATCC Bacteria and Bacteriophages. Rockville, Maryland. Danovaro, R., Marrale, D., Dell’Anno, A., Della Croce, N., Tselepides, A., Fabiano, M., 2000. Bacterial response to seasonal changes in labile organic matter composition on the continental shelf and bathyal sediments of the Cretan Sea. Prog. Oceanogr. 46, 345–366. Fan, M., Boonfueng, T., Xu, Y., Axe, L., Tyson, T.A., 2005. Modeling Pb sorption to microporous amorphous oxides as discrete particles and coatings. J. Colloid Interf. Sci. 254, 214–221. Fournier, D., Lemieux, R., Couillard, D., 1998. Essential interactions between Thiobacillus ferrooxidans and heterotrophic microorganisms during a wastewater sludge bioleaching process. Environ. Pollut. 101, 303–309. Küsel, K., Roth, U., Drake, H.L., 2002. Microbial reduction of Fe(III) in the presence of oxygen under low pH conditions. Environ. Microbiol. 4, 414–421. Kyung, J.H., Tokunaga, S., Kajiuchi, T., 2002. Evaluation of remediation process with plant derived biosurfactant for recovery of heavy metals from contaminated soils. Chemosphere 49, 379–384. Lòpez-Sánchez, J.F., Samitier, C., Rubio, R., Rauret, G., 1996. Trace metal partitioning in marine sediments and sludges deposited off the coast of Barcelona (Spain). Water Res. 30, 153–159. Lors, C., Tiffreau, C., Laboudigue, A., 2004. Effects of bacterial activities on the release of heavy metals from contaminated dredged sediments. Chemosphere 56, 619– 630. Lovley, D.R., Holmes, D.E., Nevin, K.P., 2004. Dissimilatory Fe(III) and Mn(IV) reduction. Adv. Microb. Physiol. 49, 219–286. Mulligan, C.N., Yong, C.N., Gibbs, B.F., 2001a. Heavy metal removal from sediments by biosurfactants. J. Hazard. Mater. 85, 111–125. Mulligan, C.N., Yong, R.N., Gibbs, B.F., 2001b. An evaluation of technologies for the heavy metal remediation of dredged sediments. J. Hazard. Mater. 85, 145–163. Ngiam, L.S., Lim, P.E., 2001. Speciation pattern of heavy metals in tropical estuarine anoxic and oxidized sediments by different sequential extraction schemes. Sci. Total Environ. 275, 53–61. Pellegrini, D., Ausili, A., Onorati, F., Ciuffa, G., Gabellini, M., Bigongiari, N., De Ranieri, S., 1999. Characterisation of harbour and coastal sediments: specific destination of dredged material. Aqua. Ecosyst. Health Manage. 2, 455–464. Quevauviller, P., Rauret, G., Lopez-Sanchez, J.F., Rubio, R., Ure, A., Muntau, H, 1997. The certification of the extractable contents (mass fractions) of Cd, Cr, Ni, Pb and Zn in sediment following a three-step sequential extraction procedure. Report EUR 17554 EN European Commission, Bruxelles.
1326
F. Beolchini et al. / Chemosphere 74 (2009) 1321–1326
Schippers, A., J?rgensen, B.B., 2002. Biogeochemistry of pyrite and iron sulfide oxidation in marine sediments. Geochim. Cosmochim. Acta 66, 85–92. Seidel, H., Löser, C., Zehnsdorf, A., Hoffmann, P., Schmerold, R., 2004. Bioremediation process for sediments contaminated by heavy metals: feasibility study on a pilot scale. Environ. Sci. Technol. 38, 1582–1588. US EPA, 2004. The Incidence and Severity of Sediment Contamination in Surface Waters of the United States, National Sediment Quality Survey, second ed., EPA823-R-04-007, Office of Science and Technology, Standards and Health Protection Division, Washington DC, November 2004.
US EPA, 2005. Contaminated Sediment Remediation Guidance for Hazardous Waste Sites, EPA-540-R-05-012, Office of Solid Waste and Emergency Response OSWER 9355.0-85, December 2005. Vegliò, F., Beolchini, F., Nardini, A., Toro, L., 2000. Bioleaching of a pirrhotite ore by a sulfoxidans strain: kinetic analysis. Chem. Eng. Sci. 55, 783–795. Zaggia, L., Zonta, R., 1997. Metal-sulphide formation in the contaminated anoxic sludge of the Venice canals. Appl. Geochem. 12, 527–536.