Chemosphere 74 (2009) 1321–1326

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Chemosphere

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Auto- and heterotrophic acidophilic bacteria enhance the bioremediationefficiency 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, Italyb Istituto Centrale Ricerca Scientifica e Tecnologica Applicata al Mare – ICRAM, Rome, Italyc Istituto di Geologia Ambientale e Geoingegneria IGAG-CNR, Via Bolognola, 7, Rome, Italy

a r t i c l e i n f o a b s t r a c t

Article history:Received 24 July 2008Received in revised form 21 November 2008Accepted 21 November 2008Available online 31 December 2008

Keywords:ContaminationBioaugmentationMetalsDredged sediments

0045-6535/$ - see front matter � 2008 Elsevier Ltd. Adoi:10.1016/j.chemosphere.2008.11.057

* Corresponding author. Tel.: +39 071 2204225; faxE-mail address: f.beolchini@univpm.it (F. Beolchin

This study deals with bioremediation treatments of dredged sediments contaminated by heavy metalsbased on the bioaugmentation of different bacterial strains. The efficiency of the following bacterial con-sortia was compared: (i) acidophilic chemoautotrophic, Fe/S-oxidising bacteria, (ii) acidophilic heterotro-phic bacteria able to reduce Fe/Mn fraction, co-respiring oxygen and ferric iron and (iii) thechemoautotrophic and heterotrophic bacteria reported above, pooled together, as it was hypothesisedthat the two strains could cooperate through a mutual substrate supply. The effect of the bioremediationtreatment based on the bioaugmentation of Fe/S-oxidising strains alone was similar to the one based onlyon 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 oxidizingbacteria 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 bioaugmen-tation approach proposed here is that it is independent from the availability of sulphur. These resultsopen new perspectives for the bioremediation technology for the removal of heavy metals from highlycontaminated sediments.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Marine sediments represent the main repositories of anthropo-genic pollutants at the global scale. This is particularly critical inthe sediments accumulated in harbours and ports, whereextremely high levels of several contaminants are typically re-ported (US EPA, 2005). These areas are also characterised by highsedimentation rates, which require periodic dredging to allowmaritime transportation. As a consequence, in the last decade, aparticular attention has been paid to identify the most effectivetreatments of decontamination of the sediment deposits, withthe final objective to re-use the treated sediments either in build-ing industries or in beaches nourishment. Among a variety of pol-lutants bound to the fine (silty) fraction of the harbour sediments,heavy metals are particularly dangerous for their persistency andtoxicity. The concentrations of most metals in harbour sedimentsare usually so high that dredged sediments are included withinhazardous and toxic materials (US EPA, 2005). Harbour sedimentsare also difficult to handle because of their muddy/pasty

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: +39 071 2204650.i).

characteristics and their high geochemical complexity. The mostwidely utilised technology for the treatment of these sedimentsis the separation of the coarse fraction (sands characterised by low-er contamination levels), from the fine fraction (silt-clay typicallyhighly contaminated) in order to facilitate subsequent treatmentsand to minimise the portion that requires disposal (Seidel et al.,2004).

Current approaches for the treatment of sediments contami-nated by heavy metals include the chemical extraction and/orthe thermo stabilization, but their environmental impact is typi-cally high. A promising alternative to all these technologies isbased on bioremediation processes, in which microbial metabo-lism is utilised to change the metals’ speciation, thus enhancingtheir mobilisation and solubilization. Bioremediation strategies de-pend on the specific conditions created to stimulate bacterialmetabolism; these include two main approaches: (i) bioaugmenta-tion in which specific bacterial strains are inoculated, and (ii)biostimulation in which the metabolism of autochthonous micro-bial community is stimulated by inoculating specific substrates.Previous attempts made to reduce the environmental impact ofthe removal of heavy metals from soils utilised bio-surfactants(Mulligan et al., 2001a; Kyung et al., 2002), but little information

1322 F. Beolchini et al. / Chemosphere 74 (2009) 1321–1326

is available yet for sediments bioremediation (Lors et al., 2004).The bioremediation of dredged sediments contaminated by heavymetals is generally based on bioaugmentation treatments withAcidithiobacillus 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 con-tained within the sulphide fraction of the sediment (Chen and Lin,2001). Other approaches for the bioremediation of dredged sedi-ments are based on the stimulation of the autochthonous assem-blages of Fe/S oxidizing bacteria (Lors et al., 2004; Seidel et al.,2004). All bioremediation treatments of heavy metals reported sofar in the literature have been focused exclusively on metals asso-ciated with sulphides (the so called ‘‘oxidisable fraction”). Howeverseveral authors have suggested that the so called ‘‘reducible frac-tion” composed by oxides of Fe and Mn, can be very efficient inbinding the heavy metals of the sedimentary matrix (Fan et al.,2005). Furthermore, while the sulphide fraction largely dependson the metabolism of the heterotrophic sulphate-reducing bacteriaunder anoxic conditions (Zaggia and Zonta, 1997), the Fe/Mn oxi-des are formed under opposite conditions (i.e. by sediment oxida-tion in water and/or resuspension).

The aim of the present study was to test new bioremediationapproaches based on bioaugmentation of different acidophilicmicrobial strains, in order to improve the efficiency of removal ofheavy metals from highly contaminated dredged sediments. Thetested treatments were based on: (i) inocula of Acidithiobacillusspp. and Leptospirillum ferrooxidans, chemoautotrophic bacteriaable to oxidise both iron and sulphur, (ii) inocula of Acidiphiliumcryptum strain, a chemo-heterotrophic bacterium able to reducethe Fe fraction, co-respiring oxygen and ferric iron (due to its abil-ity to grow at low pH levels) (Küsel et al., 2002), (iii) inocula of amixed culture of previous chemoautotrophic and chemo-hetero-trophic bacteria. This latter treatment was designed hypothesisingthat the two microbial components can create a consortium inwhich the activity of A. cryptum could cooperate with chemoauto-trophic bacteria (Fournier et al., 1998).

2. Materials and method

2.1. Sediment

Sediment samples (top 25 cm) were collected in the Anconaharbour (Central Adriatic Sea, Mediterranean Sea) by means of amodified Van Veen grab (about 14 L). Sediment samples were driedand 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 hat 450 �C. Before combustion, sediment samples were treated withan 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

Table 1Total metals content in the sediment samples collected from the Ancona harbour and a c

Metal Observed value (ppm) Eco-toxicological limit

Italy (Pellegrini et al., 2002)

Cu 410 ± 10 65Zn 500 ± 20 179Cd 1.2 ± 0.1 0.42Hg 5.4 ± 0.3 0.83Ni 105 ± 6 76As 21 ± 2 42Pb 76 ± 4 98Cr 141 ± 8 494

and carbonates, ca 5% by iron and manganese oxides, and less than5% by sulphides (data not shown).

2.2. Microorganisms

A mixed consortium of Acidithiobacillus thiooxidans, Acidithioba-cillus ferrooxidans and L. ferrooxidans isolated in a natural environ-ment (acid mine drainage) was provided by Professor StoyanGroudev group, Department of Engineering and Geoecology, Uni-versity of Mining and Geology ‘‘Saint Ivan Rilski”, Sofia, Bulgaria.This mixed culture is composed of chemo-litho-autotrophic bacte-ria, able to oxidise S0 and Fe(II) in acidic conditions. The strain wasroutinely cultivated in 9 K liquid medium (Cote, 1996) with ironsulphate as the energy source. The acidophilic heterotrophic A.cryptum strain was provided by German National Resource Centrefor Biological Material, DSMZ No. 2390. The medium for the opti-mal growth conditions was prepared mixing the following solu-tions: 980 mL of a solution containing (in g L�1) (NH4)2SO4 2, KCl0.1, K2HPO4 0.5, MgSO4 � 7H2O 0.5 and H2SO4 (pH 3); 10 mL of asolution of glucose 10%; 10 mL of a solution containing 3% yeastextract (Oxoid). All solutions were sterilized separately and after-wards mixed. Flasks were incubated at 35 �C on a rotary shakerat 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), byinoculating different strains according to the experimental condi-tions. This sediment concentration was chosen in order to avoidpossible autotrophic bacterial inhibition due to the high contentof organic matter in the sediment. Tests were performed in100 mL Erlenmeyer flasks, filled with 50 mL culture medium. Theinoculum (5 mL) was obtained from cultures in exponential growth,with a bacterial density in the range 1.5–2 � 108 cells mL�1. Flaskswere kept at room temperature on a rotary shaker (150 rpm).Control tests were also performed, where the sediment samplewas suspended in artificial sterilised sea water. All tests wereperformed in triplicate.

2.4. Experimental design

Bioaugmentation experiments were performed with differentacidophilic microbial strains: (i) autotrophic bacteria (A. thiooxi-dans, A. ferrooxidans and L. ferrooxidans), (ii) heterotrophic bacteria,and (iii) a mixed culture of both autotrophic and heterotrophicbacteria. In the first set of treatments, the mixed culture of Acidi-thiobacillus spp. and L. ferrooxidans was inoculated into the sedi-ment with the addition of the 9 K medium, without ironsulphate, but in the presence of sulphur at different concentrationlevels: 0 (i.e. no sulphur), 0.125 and 0.5%.

omparison with the eco-toxicological thresholds utilised in Italy and US.

United States (US EPA, 2005)

25% Probability toxic effect 50% Probability toxic effect

50 157140 3840.6 2.50.2 0.924 8011 3348 16176 233

F. Beolchini et al. / Chemosphere 74 (2009) 1321–1326 1323

In the second set of treatments the bioaugmentation was con-ducted by inoculating the heterotrophic bacterium A. cryptum intothe sediment suspended in its optimal growth medium. The lattermedium was modified for the concentration of glucose, which wasset at two alternative levels (i.e. 0.01 and 0.1 g L�1, correspondingto 1% and 10% of the optimal glucose concentration, respectively).

Finally, in the third set of treatments the bioaugmentation wasconducted by inoculating the autotrophic and the heterotrophicstrains 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 atpH 2 containing the sample sediment, the 9 K medium and glucose(0.01 g L�1).

2.5. Analytical methods

Heavy-metal content in the sediment was determined after aciddigestion, as follows: the dried sediment (0.5 g) was transferred inTeflon boxes, added with 5 mL fluoridric acid and 1 mL of ‘‘aqua re-gia” (i.e. HCl:HNO3 = 3:1) incubated for 90 min at 150 �C. At theend of the incubation period, 5 mL of 10% boric acid were addedand the obtained extracts were analysed by atomic absorptionspectrophotometry (Varian SpectrAA 400 PLUS, atomizatorGTA96, graphite furnace) for cadmium and by inductively coupledplasma-atomic emission spectrometry (Jobin Yvon JY 24) for otherheavy metals. Metals distribution in different mineralogical frac-tions were determined by means of sequential selective extraction,according to European Union protocol (Quevauviller et al., 1997).Four different fractions are considered: (i) exchangeable fractionand carbonates, extracted utilising acetic acid 0.11 M (pH 2.8);(ii) iron and manganese oxides fraction, extracted with NH2OH�HCl0.1 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, isdetermined by difference with the total metal content. The totalbacterial number in the suspension was determined by epifluores-cence microscopy after staining with Acridine orange according tostandard procedures for marine sediments described elsewhere(Danovaro et al., 2000).

0102030405060708090

100

Cu Zn Cd Hg Pb Ni As Cr

fract

ion,

%

exchangeable and carbonateassociated with Fe/Mn oxides

associated with sulphides and organicsresidual

Fig. 1. Metal speciation in the investigated sediments (the percentage of metalextracted in each step of the sequential extraction procedure is represented as bardiagrams; 5% standard errors are also represented). The 100% values for each metalare shown in Table 1.

3. Results and discussion

3.1. Heavy-metal partitioning

The geochemical characterisation of the sediments is of primaryimportance in bioremediation technologies, as different geochem-ical components can influence differently the heavy-metal seques-tration. For instance, the availability of sulphides, iron andmanganese is crucial for several biogeochemical processes andfor predicting the ability of bacteria to affect heavy-metal specia-tion (Lòpez-Sánchez et al., 1996; Schippers and J?rgensen, 2002).Moreover, a geochemical characterisation with a detailed analysisof heavy-metal speciation can provide important insights on thepotential fate (mobilisation) of each heavy metal. In the presentstudy, a detailed analysis of the total content of heavy metals inthe harbour sediments was initially conducted. Metal and metal-loid content in the sediment (ppm based on dry weight of sedi-ment) 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 tox-icological thresholds for marine sediment in Italy (Pellegrini et al.,1999) and in the United States (US EPA, 2004) were used to assessthe contamination of the sediments utilised in the present study.These values are not adopted by law, but they can be used as safetythresholds for aquatic life, as above these value, sediments become

toxic for most biological components. All of the heavy metalsencountered 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 timeshigher than the highest accepted levels for the use and displace-ment of the dredged sediments. Furthermore, considering the USstandards, copper, zinc and mercury were above the 50% probabil-ity toxic effect and the other metals (cadmium, nickel, arsenic, leadand chromium) were all above the 25% probability toxic effect.Therefore, the sediments utilised in the present study can be con-sidered as highly contaminated, and their treatment for the re-moval of heavy metals is customary prior to any possibledisposal or re-use. The metal speciation in the investigated sedi-ment is illustrated in Fig. 1. Results presented here are in agree-ment with data reported in the literature (Lòpez-Sánchez et al.,1996; Pellegrini et al., 1999; Ngiam and Lim, 2001), as concernsthe general trend of metal partitioning. Metals can be ordered interms of decreasing mobility as follows: Cu, Zn, Cd, Hg, Pb, Ni, Asand Cr. Furthermore, Cu was largely bound to the sulphide andthe organic fraction, while Zn and Cd are bound to exchangeableand Fe/Mn fractions, Hg was mostly associated with silicates andto the exchangeable fraction, whereas Pb was bound to silicatesand the sulphide fraction, and finally Ni, As and Cr were mostlybound to the residual fraction of silicates.

3.2. Bioremediation experiments

In the last decade, several attempts to improve the availabletechniques of bioremediation for the treatment of sediments con-taminated by heavy metals have been conducted (Chen and Lin,2001; Mulligan et al., 2001b), including studies at pilot scale (Sei-del et al., 2004). These studies have typically focused their atten-tion on the ability of Fe/S oxidizing bacteria to mobilise theheavy metal, probably due to their consolidated application inbio-hydrometallurgy (Vegliò et al., 2000). At the same time, sincetoxic metals are distributed among different geochemical fractions(Fig. 1), it may be hypothesised that the use of Fe/S-oxidisingstrains in association with other micro-organisms, able to act onfractions different than sulphides, could improve metal extractionfrom sediments by bioleaching. In particular, Fe/Mn oxides couldbe identified as the complementary target fraction, due to the factthat the labile fractions are struck in an acid environment. Conse-quently, an essential process to improve the heavy-metal bioleach-ing would be to let heterotrophic bacteria respire the Fe oxides in

1324 F. Beolchini et al. / Chemosphere 74 (2009) 1321–1326

the sediments (Lovley et al., 2004). Moreover, this process can bedone under aerobic and acidic conditions where the autotrophicbacteria (the Fe/S-oxidising strain) live. In this case a mutual coop-eration between the autotrophic and the heterotrophic strainscould take place, with the consequence of improving the metal

0

10

20

30

40

50

60

0 3 6 9 12 15days

109 c

ells

g-1

no S S 0.125% S 0.5% control test

Fig. 2. Temporal changes in bacterial abundance (cells per gram of sediment dryweight) in contaminated sediments during the bioleaching process mediated byiron and sulphur oxidising bacteria, in the presence of elemental sulphur. Reportedare the effects of sulphur concentrations (sediment 2%, temperature 30 �C, stirringrate 150 rpm). The bars refer to the standard errors.

0

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100

Cu Zn Cd Hg Pb Ni As Cr

met

al e

xtra

ctio

n yi

eld

(%)

no S S 0.125% S 0.5% control

Fig. 3. Metal extraction yields at the end of bioleaching of the sediments mediatedby iron and sulphur oxidising bacteria, in the presence of 0%, 0.125% 0.5% ofelemental sulphur. As a comparison, extraction yields observed in control test(sediment suspended in artificial sterilised sea water) are also reported. Temporalchanges in bacterial abundance are shown in Fig. 2. The bars refer to the standarderrors.

0

5

10

15

20

0days

109 c

ells

g-1

glucose 0.01 g L-1glucose 0.1 g L-1control test

3 6 9 12 15

a b

Fig. 4. Cell growth (a) and metal extraction yields at the end of sediments bioleachingcomparison, extraction yields observed in control test (sediment suspended in artificial

extraction yields. The results of the different bioaugmentationtreatments based only on Fe/S-oxidising strains are reported inFig. 2, which illustrates temporal changes in bacterial abundance.Heavy-metal extraction yields and metal residual concentrationsat the end of the bioleaching treatment (15 d) are reported inFig. 3. Bacterial abundance and growth rates were closely depen-dent upon the availability of elemental sulphur in the microcosm.Bacterial abundance was indistinguishable from the control whenno sulphur was added to the medium, whereas in presence of sul-phur bacterial abundance reached 5.0 � 1010 cells g�1 13 d afterthe inoculum. The differences between treatments and controlswere significant already after 6 d from the inoculum at 0.5% ele-mental sulphur. For most metals, extraction yields increased alongwith the increasing sulphur supply to the growth medium. As ex-pected, no changes in heavy-metal mobilisation were observed inthe controls (i.e., sediment suspended in artificial sterilised seawater). In the treatments where low bacterial growth was ob-served (i.e. no sulphur addition) the metal extraction was likelydue to a chemical action of the very low pH (range: 2.0–2.5) as con-firmed by additional control tests performed in the same pH rangewithout the inoculum of the autotrophic Fe/S oxidizing bacteria.Conversely, the highest performances were observed in bioaug-mented sediments, in the presence of sulphur 5%. In particularthe 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 forAs and Cr. The addition of elemental sulphur enhanced the effi-ciency of heavy-metal removal, and this can be explained withthe need of this component as energy source for accelerating Fe/S oxidizing bacteria metabolism (Chen and Lin, 2001) and promot-ing the acidic conditions, which consequently increase the mobili-sation of the heavy metals (Seidel et al., 2004).

Experimental tests of bioaugmentation with the only A. cryptumstrain, performed at different levels of glucose concentration (0.01and 0.1 g L�1), displayed a fast growth rate of this heterotrophicstrain (specific growth rate estimated assuming a first order kineticof 0.61 ± 0.05 d�1, Fig. 4a), higher than rates observed for the auto-trophic bacteria (specific growth rate of 0.23 ± 0.03 d�1, Fig. 2). Het-erotrophic bacterial abundances increased significantly after 3 d oftreatment and reached a maximum of about 1.2 � 1010 cells g�1

after the first week. The efficiency of heavy-metal mobilisationwas always <50% (Fig. 4b), and it was likely largely associated withthe chemical action of the acid medium. The obtained yields werecomparable to those observed for treatments based on autotrophicFe/S-oxidising mixed culture in the absence of elemental sulphur(Fig. 3a), Cu, Zn, Cd and Hg were mobilised for a 40–50% while lowerextraction yields were achieved for all of the other heavy metals. It

0

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Cu Zn Cd Hg Pb Nimet

al e

xtra

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n yi

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(%)

glucose 0.01 g L-1

control testglucose 0.1 g L-1

CrAs

(b) with heterotrophic bacteria, in the presence of 0.01 and 0.1 g L�1 glucose. As asterilised sea water) are also reported. The bars refer to the standard errors.

0

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0 9 12 15days

109 c

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10:11:11:10

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Cu Zn Cd Hg Pb Ni As Cr

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10:11:11:10control test

a b

3 6

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 re-suspended in artificial sterilised seawater) are also reported.

F. Beolchini et al. / Chemosphere 74 (2009) 1321–1326 1325

can be also noticed that the highest heavy-metal extraction yieldswere observed at the lowest levels of glucose and that the extrac-tion yields did not increase with increasing glucose concentration(Fig. 4b). A possible explanation of this phenomenon is that atlow glucose concentrations, the heterotrophic bacteria are stimu-lated to use complementary organic sources from the sedimentwith the consequent dissolution of the organically bound metals.

The bioaugmentation treatment based on the inoculum of theautotrophic Fe/S oxidizing bacteria together with the heterotrophicacidophilic strain (i.e. A. cryptum) resulted in significantly higherextraction yields of all heavy metals investigated. The growth ratesof the bacteria inoculated were significantly higher than those re-ported for the treatments based on either autotrophic or heterotro-phic bacteria alone. In particular, after 5 d, bacterial abundancesreached values of 1.4 � 1010 cells g�1 and 2.4 � 1010 cells g�1 inmicrocosms inoculated with autotrophic and heterotrophic bacte-ria in the ratio 10:1 and 1:10, respectively (Fig. 5a). Even faster bac-terial growth rates were observed in the bioaugmentationtreatment where the autotrophic Fe/S oxidizing bacteria and theheterotrophic bacteria were inoculated in the ratio 1:1 (highestbacterial abundance: 2.8 � 1010 cells g�1, achieved after 3 d oftreatment). As a consequence, in the presence of the autotrophicand heterotrophic bacteria together, the efficiency of removal in-creased significantly for all of the heavy metals, reaching extrac-tion yields >90% for Cu, Cd, Hg and Zn (Fig. 5b).

With the except of Zn, no significant differences in heavy-metalextraction yields were observed among experimental systems con-taining autotrophic and heterotrophic bacteria inoculated in differ-ent proportion. Therefore all tested proportions of autotrophic andheterotrophic bacteria inoculated to the sediment are equal effi-cient in metal mobilisation from the different mineralogical frac-tions. These results suggest that the presence of a mixedconsortium of heterotrophic and autotrophic bacterial strainsmay determine a higher heavy-metal extraction efficiency due toa coupled and synergistic metabolism. In fact in the absence of ele-mental sulphur the presence of A. cryptum may enhance the metalbioleaching activity of Fe/S-oxidising strain (Fournier et al., 1998).At the same time the growth of autotrophic bacteria and the re-lease of labile organic material from their metabolism seems tobe enough for the heterotrophic needs of the Fe-reducing bacteria.

Overall data reported in the present work may open new per-spectives for the application of bioleaching technologies to remedi-ation purposes based on mutual interactions among bacterialstrains 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 bio-remediation approach to different kinds of contaminated sedi-ments and the extent by which these treatments can be furtheroptimised.

Acknowledgements

Authors are grateful to Professor Francesco Vegliò (University ofL’Aquila), for his precious suggestions during the design of experi-mental tests. This research was financially supported by the ItalianMinistry of Research (MUR).

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