Biogenic Nanopalladium Based Remediation of Chlorinated

Dec 10, 2013 - Biogenic catalysts have been studied over the last 10 years in freshwater and soil environments, but neither their formation nor their ...
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Biogenic Nanopalladium Based Remediation of Chlorinated Hydrocarbons in Marine Environments Baharak Hosseinkhani,† Tom Hennebel,†,‡ Sam Van Nevel,† Stephanie Verschuere,§ Michail M. Yakimov, ∥ Simone Cappello, ∥ Mohamed Blaghen,⊥ and Nico Boon*,† †

Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, Coupure Links 653, B-9000 Gent, Belgium Department of Civil and Environmental Engineering, University of California at Berkeley, Berkeley, California 94720, United States § Department of Pathology, Ghent University, De Pintelaan 185, 9000 Ghent, Belgium ∥ Institute for Coastal Marine Environment (IAMC) CNR of Messina, Spianata San Raineri, 86-98121 Messina, Italy ⊥ Unit of Bio-Industry and Molecular Toxicology, Laboratory of Microbiology, Biotechnology, Pharmacology and Environment, Faculty of Sciences Aïn Chock, University Hassan II-Aïn Chock, Km 8 route d’El Jadida, B.P. 5366, Mâarif, Casablanca, Morocco ‡

S Supporting Information *

ABSTRACT: Biogenic catalysts have been studied over the last 10 years in freshwater and soil environments, but neither their formation nor their application has been explored in marine ecosystems. The objective of this study was to develop a biogenic nanopalladium-based remediation method for reducing chlorinated hydrocarbons from marine environments by employing indigenous marine bacteria. Thirty facultative aerobic marine strains were isolated from two contaminated sites, the Lagoon of Mar Chica, Morocco, and Priolo Gargallo Syracuse, Italy. Eight strains showed concurrent palladium precipitation and biohydrogen production. X-ray diffraction and thin section transmission electron microscopy analysis indicated the presence of metallic Pd nanoparticles of various sizes (5−20 nm) formed either in the cytoplasm, in the periplasmic space, or extracellularly. These biogenic catalysts were used to dechlorinate trichloroethylene in simulated marine environments. Complete dehalogenation of 20 mg L−1 trichloroethylene was achieved within 1 h using 50 mg L−1 biogenic nanopalladium. These biogenic nanoparticles are promising developments for future marine bioremediation applications.

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INTRODUCTION Contamination of marine systems by chlorinated hydrocarbons has become an issue of great concern given their high rate of bioaccumulation and the harmful effect on human health and aquatic ecosystems.1 Among chlorinated organic compounds, trichloroethylene (TCE) has been detected as a common groundwater, soil, and seawater contaminant.2 TCE has been shown to have carcinogenic and toxic effects on the liver and kidneys, central nervous and endocrine systems of humans.3 Recently, nanomaterial-mediated remediation of various contaminants including TCE have been evaluated as a costeffective and efficient alternative to conventional remediation methods.4 Due to their high activity, large surface to volume ratio and catalytic properties, nanomaterials have received attention as a candidate for the effective remediation of © 2013 American Chemical Society

environmental pollutants. In addition, nanoparticles can be successfully applied for in situ remediation of various contaminants.5 Specifically, the catalytic potential of palladium nanoparticles (Pd-NPs) has been reported for dechlorination of chlorinated solvents6 and the remediation of chromium.7 Microbially driven reduction and precipitation of Pd is considered to be a promising eco-friendly method to prepare catalytically active Pd nanoparticles. Previously, Pd nanocatalysts were synthesized by the bacteria Shewanella oneidensis8 and Desulfovibrio desulf uricans.9 In these studies, bio-Pd was Received: Revised: Accepted: Published: 550

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were resuspended in M9 medium to obtain an optical density at 610 nm (OD610) of 0.7 ± 0.3. Palladium(II) and formate were added to a final concentration of 50 mg L−1 and 25 mM, respectively, from anaerobic stock solutions of Na2PdCl4 (Sigma−Aldrich, Seelze, Germany) and sodium formate. The tubes were purged with repeated cycles of N2 overpressure and vacuum under-pressure. The tubes were then incubated for 24 h at 28 °C with mild shaking (120 rpm). Characterization of the NPs. For the TEM analysis of morphology and localization of bio-Pd nanoparticles on bacteria, palladized cells (biomass loaded by bio-Pd nanoparticles) were harvested by centrifugation (4920g for 10 min). The bacteria were fixed and subsequently ultrathin section TEM images were collect by a Zeiss TEM 900 transmission electron microscope (Carl Zeiss, Oberkochen, Germany) at 50 kV as previously described by Hennebel et al. 2011.15 Size distributions of NPs were determined using the software ImageJ.16 Compositions of nanoparticles were obtained using XRD. Biomass pellets containing Pd were collected by centrifugation (4420g for 10 min). Air-dried substrate on glass was then applied for characterization by X-ray diffraction. X-ray diffractograms (CuKα radiation, λ = 1.5418 Å) were collected with a D8 Discover HTS diffractometer (Bruker) using a Vantec 2000 detector with a 2K cobalt source. Intensities were recorded in transmission mode, with the following parameters: 1θ = −90°, 2θ = 20°, step time of 1200 s and step size of 28 s. Catalytic Activity of Novel Bio-Pd Catalysts in Marine Water. Batch experiments were conducted to investigate TCE dechlorination by nanoparticles. In a first set of experiments, the goal was to test the activity of the biogenic Pd NPs formed by the different species using the same amount of H2. Bio-Pd NPs were separated from the growth medium by centrifuging for 5 min at 3000g and 50 mg L−1 of bio-Pd was resuspended in sterile marine water (26 g L−1 synthetic sea salt, Instant Ocean, France). Subsequently, bio-Pd NPs were activated by supplying 100% v/v of H2 from a gas tank into the headspace of serum flasks. In a second set of experiments, the bio-Pd with the highest activity was selected and dehalogenation experiments were performed in the same serum flask used for fermentative production of H2 with Pd decorated cells and native cells. In all experiments, TCE degradation was initiated by spiking 20 mg L−1 of TCE (Sigma−Aldrich).17 The following controls were included: (1) inactive bio-Pd (no H2 added), (2) Pd-free control containing marine sterile water provided with H2, and (3) viable biomass in the absence of Pd provided with hydrogen gas. All experiments were performed in triplicate. Analytical Methods. Analysis of biogenic hydrogen gas in the headspace was performed by gas chromatography (GC; Global Analyzer Solutions, Interscience, Breda, The Netherlands) using a thermal conductivity detector (TCD). The precolumn and column were a Parabond Q (2 m × 0.32 mm) and a Molsieve 5A column (7 m × 0.32 mm), respectively. The GC was programmed as follows: column temperature = 50 °C; column pressure = 100 kPa; and detector temperature = 60 °C, with a detection limit of 1 ppm. Pd (II) concentrations were quantified in the supernatant using a Shimadzu AA-6300 atomic absorption spectrophotometer (AAS) (Kyoto, Japan). TCE degradation was monitored by measuring TCE in the headspace using GC (CP-3800, Varian, Palo Alto, CA) equipped with a flame ionization detector (FID) with the

combined with an external hydrogen donor in order to produce reactive radicals that can dissociate the chloro-carbon bond.8,10 As recently demonstrated by Chidambaram et al., the activation of bio-Pd NPs can also be achieved by biohydrogen produced during the fermentation of a carbon source.11 Applying bio-Pd formed by exogenous bacteria such as S. oneidensis and D. desulf uricans for in situ remediation of polluted marine environments has some drawbacks. First, exogenous bacteria may not survive in a contaminated marine site, nor perform metabolic activities such as the production of biohydrogen for activation of catalysts. Second, the addition of exogenous bacteria to natural ecosystems should be avoided due to their potential effects on biodiversity. To our knowledge, the formation of Pd NPs by an indigenous marine bacterial community has not been investigated yet. This study demonstrates the ability of indigenous marine bacteria to produce both biohydrogen and Pd-NPs. The size and composition of NPs were investigated by thin section transmission electron microscopy (TEM) and Xray diffraction (XRD). The catalytic feasibility of novel catalysts was evaluated by monitoring the dechlorination of TCE in a marine environment.

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EXPERIMENTAL PROCEDURES Sample Collection and Sediment Characterization. Sampling was performed in May 2011 at two marine sites: (Site A) at station SY-01, Priolo Gargallo Syracuse (37°09′31.11″N, 15°12′10.32″E) Italy; and (Site B), the Lagoon of Mar Chica (35°08′52″N, 2°50′53″W) Morocco, as previously described.12 Briefly, samples were collected at a depth of 6 m by scuba diving using acrylic cores approximately 100 cm long and with an inner diameter of 20 cm. All samples were stored at 4 °C during transport to the laboratory. Physico−chemical analyses of sediments were carried out using International Standard ISO 10390:1994. Analytic determination of heavy metals was performed according to official methods of heavy metals estimation (EPA 7060A (As), EPA 7131A (Cd), EPA 6010C (Cr, Co, Cu), EPA 7421 (Pb)). All measures were carried out using an AAS Thermo M Series (Thermo Fischer Scientific, Inc., USA). Isolation Strains from Contaminated Marine Sediments. Indigenous microorganisms were isolated from mud samples by dilution plating on the LB marine rich complete solid media containing the following per liter: 10 g tryptone, 5 g yeast extract, 75% of artificial seawater (ASW: 0.3 M NaCl, 0.01 M KCl, 0.05 M MgSO4 and 0.01 M CaCl2), 1 mL trace element solution13 and 15 g agar. Incubation of all plates was performed at room temperature in facultative anaerobic conditions using a gas-pack cultivation jar for 48h.13 Amplified rDNA Restriction Analysis. For amplification of 16SrRNA genes, the primers 63F (5′-CAGGCCTAACACATGCAAGTC −3′) and 1378R (5′-CGGTGTGTACAAGGCCCGGGAACG-3′) were used in a standard 30cycle PCR with Taq polymerase and an annealing temperature of 53 °C.14 Reduction of Pd (II) and bio-Pd(0) Formation by Marine Isolates. Pure colonies of each isolate were grown under anaerobic conditions at 28 °C for 48h in marine LB broth with mild shaking (120 rpm). Preparation of bio-Pd nanoparticles by marine-isolated bacteria was performed according to De Windt et al.8 Biomasses harvested by centrifugation (4420g for 10 min) were washed three times with 50 mL M9 medium. Washed cells 551

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following program: injection temperature = 200 °C; detector temperature = 250 °C; initial column temperature = 35 °C (hold 2 min), increase to 75 °C at a rate of 5 °C min−1; column pressure = 153 kPa (hold 2 min), increase to 176.5 kPa at a rate of 3 kPa min−1. The column used was a Factor Four TM low bleed capillary column (VF-624 ms, 30 m × 0.25 mm ID [inner diameter], DF [film thickness] = 0.25 μm, Varian). The detection limit was 100 μg L−1.

traffic transporting both crude and refined oil, is contaminated with high concentrations of heavy metals including As (91.8 mg kg−1), Cd (4.9 mg kg−1), Cr (89.4 mg kg−1), Pb (112.1 mg kg−1), and Cu (1337 mg kg−1). The second site, an area of 12 km2 linked to the Mediterranean Sea by an artificial pass, is heavily polluted with the heavy metals As (12.0 mg kg−1), Cd (2.7 mg kg−1), Cr (22.8 mg kg−1), Pb (109.3 mg kg−1), and Cu (46.8 mg kg−1). The COD content of the mud samples was 215 and 307 mg kg−1 for site A and B, respectively. Biogenic Hydrogen Production by Marine Isolates. The activation of palladium nanocatalysts for dehalogenation requires an external electron donor such as hydrogen. Isolates were therefore screened according to their ability to produce biohydrogen. Eight out of 30 facultative aerobic isolates were able to produce bio-H2 in batch experiments via fermentation of glucose (Figure 2). Further investigations focused on these strains. A strain closely related to Thalassospira species (99%, isolated from the sediment of Site A) and three strains closely related to Vibrio natriegens, Lysinibacillus sphaericus, and Pseudoalteromonas sp. species (99%, 100%, and 100%, respectively, isolated from sediments of Site B) produced the highest yields of H2, in the range of 0.29 to 2.2 mmol d −1, after 48 h of incubation in marine LB media supplemented with 20 g L−1 glucose. In contrast, S. oneidensis, used as a control and bench-mark for bio-Pd, was neither capable of surviving in this salty medium nor generating hydrogen. Palladium Precipitation Properties of Biohydrogen Producing Marine Isolates. The palladium precipitation capacity of the eight biohydrogen producing marine isolates was tested using the color shift from amber (soluble Pd (II)ions) to black (insoluble Pd (0) precipitates). Both viable and abiotic control experiments were carried out under anaerobic conditions using the eight biohydrogen producing marineisolated strains. Upon addition of formate, all biotic samples showed the formation of black precipitates. In contrast, large black aggregates were observed in cell-free controls after 24 h. In the absence of formate, the amber color of the suspension remained stable for greater than 24 h. Atomic absorption spectroscopy confirmed the sorption of Pd to the bacterial biomass in both biotic and abiotic experiments (SI Figure SI1). Approximately 98% of the added 50 mg L−1 Pd (II) was associated with bacterial cells in all hydrogen-producing marine isolates after 24 h. Pd(II) was removed up to 66% from the supernatant of formate-free control after 24h. Characterization of Bioformed Palladium Nanoparticle Using Marine Isolates. The reduction of palladium and precipitation of palladium as nanoparticles were characterized with TEM and XRD. Ornamentation, size and morphology of bioformed Pd-NPs by different types of marine isolates were investigated by TEM (Figure 3). For this purpose, the same ratio of biomass to Pd (II) (OD600 0.7 ± 0.3: 50 mg L−1) was applied for reduction of palladium. Thin section micrographs of biomass decorated with Pd-NPs, in combination with size distribution analysis (Figure 4), showed that NPs with an approximate size range of 5−10 nm decorated the cytoplasm and periplasmic space of the Gram-negative marine isolates (Thalassospira sp, Halomonas sp., and V. natriegens) (Figure 3a−c). In Gram-positive marine isolates, microbial-based PdNPs were located predominantly in the periplasmic space and on the cell wall (Figure 3d−e). In the case of Bacillus sp (OHM2), most NPs were deposited extracellularly, with a size range of 15−20 nm (Figure 3f).

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RESULTS Isolation and Identification of Marine Bacteria from Contaminated Marine Sediments. Two sites in the Mediterranean were sampled to determine a representative indigenous bacterial community for contaminated marine sediments: (Site A) Priolo Gargallo Syracuse, Italy and (Site B) the Lagoon of Mar Chica, Morocco. Thirty facultative aerobic colonies were isolated from both sites after enrichment by plating dilution series on marine media. 16S rRNA gene amplicons of the isolated strains were generated, aligned and compared with sequences from the NCBI database using the BLAST algorithm. The closest match of each of the thirty isolates was identified. Sequences of all strains were submitted to GenBank (accession numbers presented in SI Table SI1). Figure 1 presents the diversity of the indigenous bacterial

Figure 1. Bacterial diversity of the marine isolates assessed by 16 s RNA clone library of isolates from Priolo Gargallo Syracuse, Italy (Site A) and the Lagoon of Mar Chica, Morocco (Site B).

community isolated from different marine mud samples from the Lagoon of Mar Chica and Priolo Gargallo. Bacterial diversity differed significantly between these two marine mud samples. In samples from site A, 10 species were identified belonging to the orders of Oceanospirillales, Rhodospirillales, Clostridiales, and Bacillales. In samples from site B, 20 species were identified belonging to the orders of Vibrionales, Alteromonadales, and Bacillales. The majority of recovered isolates across both sites belonged to Bacillales and amounted to 40% and 50% for site A and B, respectively. Remarkably, the bacterial composition was completely different for the remaining percentages. Site A contained three species of Thalassospira and one of Halomonas, whereas site B contained mainly Vibrio species and Pseudoalteromonas species. To link the presence of these bacteria to their ability to produce H2 and Pd nanoparticles, the physicochemical characteristics of the collected sediments from both sampling sites were determined (SI Table SI2). The first site, an area characterized by heavy industrialization and extensive tanker 552

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Figure 2. Hydrogen generation rate by native and palladized cells of isolates from site A and site B in marine LB medium supplemented with 20 g L−1 glucose. Bars represent mean ± standard deviation; n = 3.

Figure 3. Transmission electron micrographs of bio-Pd (0) nanoparticles formed in the periplasmic space and cytoplasm of Thalasospira sp. (a) and Halomonas sp. (b) in the periplasmic space of V. natriegens (c), on the cell wall and in the periplasmic space of Clostridium sp (d) and L. sphaericus (e), extracellularly by Bacillus sp (OHM2) (f), in the periplasmic space and cytoplasm of Pseudoalteromonas sp. (g) and on the cell wall and extracellularly formed by Bacillus sp (Z9) (h).

times greater than native cells, with a maximum yield of 1.47 and 0.54 mmol d −1 H2 gas respectively (Figure 2). Catalytic Activity of Novel Bio-Pd Formed by Marine Isolates. Finally, the bioformed Pd-NPs of the indigenous marine isolates and the bench-mark bio-Pd formed by S. oneidensis were tested for catalyzing the dechlorination of TCE in marine water, after activation by different sources of hydrogen (external H2 from a gas tank, or bio-H2 formed by native cells or palladized cells after glucose fermentation) (Figure 5). In the case of external H2 as an electron donor for activation of the catalysts, all spiked TCE was completely dechlorinated in 60 min using 50 mg L−1 bio-Pd formed by Bacillus sp. and L. sphaericus. In the same time only 60% of TCE was removed by Pd NPs formed by Thalassospira sp. TCE dechlorination by other strains in marine water took up to 24h

X-ray diffractograms of the biogenic nanoparticles showed that metallic palladium and palladium oxide (PdO) were the main NP constituents. The crystalline diffraction peaks of 2θ = 47° and 55° in the X-ray diffractograms corresponded to Pd (0) with a family of (200) planes and PdO with a family of (103) planes, respectively (SI Figure SI2). Biogenic Hydrogen Production Using Palladized Marine Isolates. The final goal of the experimental setup was to verify that cells could still form hydrogen gas after the palladization step (Figure 2). Therefore, the bio-Pd was resuspended in sterile marine LB supplemented with glucose. The hydrogen formation rates are shown in Figure 2. Surprisingly, the Pd decorated cells of Thalassospira sp. and Clostridium sp. were capable of producing hydrogen at a rate 5 553

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Figure 4. Size distribution histogram of palladium nanoparticles formed by different marine isolates.

Figure 5. Time course of TCE dechlorination (with initial concentration 20 mg L−1) in marine environment treated with 50 mg L−1 bio-Pd synthesized using different marine isolates and S.oneidensis (catalysts)and hydrogen or biohydrogen gas as hydrogen donors. Bars represent mean ± standard deviation; n = 3.

incomplete after 90 min and the dehalogenation rate was slower compared to the samples using the same bio-Pd but external H2 (Figure 5). No TCE dechlorination was detected in control experiments consisting of (1) inactive bio-Pd (no hydrogen gas added), (2) Pd-free controls containing sterile marine water provided with H2 and (3) viable biomass in the absence of Pd (0). In the samples showing complete TCE removal, TCE was completely dechlorinated to harmless end-products (ethene and ethane), as determined using gas chromatography (data not shown).

(data not shown). TCE was completely reduced by the benchmark bio-Pd formed by S. oneidensis in marine water in 30 min. Further dechlorination experiments focused on bio-Pd produced by Bacillus sp. because of its highest catalytic activity and on Thalassospira sp., because of its high bio-H2 production after palladization of the cells (Figure 2). In the case of Bacillus sp. OHM2, the amount of bio-H2 produced by its native and palladized cells was not high enough to obtain TCE dechlorination (data not shown). Therefore, bio-H2 produced by Pseudoalteromonas sp. (2.2 mmol H2 L −1 d −1) was provided for activation of the bio-Pd. Result showed no differences in dechlorination rates between bio-Pd activated by external hydrogen or bio-H2 produced by marine, nonpalladized species. Indeed, almost complete dechlorination was obtained after 1 h. Subsequently, TCE removal was studied using bio-Pd and bio-H2 produced by Thalassospira sp. TCE dechlorination was

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DISCUSSION Identification of Marine Bacteria from Contaminated Marine Sediments. This study aimed to determine the feasibility of a nanoparticle-based remediation strategy for TCE in marine environments. Monitoring throughout the Medi554

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TCE Dehalogenation by Pd NPs with H2 Gas. Microbial reductive dechlorination of TCE in marine systems is currently the most widely applied technology for remediation of this compound. This method needs to use slow-growing anaerobic bacteria such as Dehalococcoides, and is prone to relatively slow reaction rates. While several studies have shown that a wide range of environmental contaminants, including TCE, can be successfully removed from groundwater systems by bio-Pd NPs,10 the catalytic activity of bio-Pd NPs in marine system has heretofore not been reported. In this study complete dechlorination of TCE in synthetic marine water was achieved with 50 mg L−1 of bio-Pd-NPs formed by the marine species Bacillus sp. and L. sphaericus, as well as bio-Pd-NPs formed by the fresh water bacterium S. oneidensis (Figure 5). TCE dehalogenation using bio-Pd formed by S. oneidensis showed similar rates in marine waters compared to previously determined rates in freshwater. High salt concentrations did not significantly inhibit nor improve the catalytic functioning of Pd NPs. In contrast, dehalogenation by bio-Pd formed by marine isolates occurred at a lower rate as compared to bio-Pd formed by freshwater species. Remarkably, there were large differences in TCE degradation rates using bioPd formed by marine isolates and using equal amounts of H2 as a hydrogen donor. It was clear that species which produced Pd NPs extracellularly or on cell walls dehalogenated TCE more effectively than species which only produced Pd NPs intracellularly. Specifically, the highest activity was obtained for Thalassospira sp. and L. sphaericus, which showed Pd precipitates mainly on the cell wall and for Bacillus sp., which showed Pd precipitates in the growth medium. No relationship between the size of the NPs and catalytic activity was apparent. This is probably due to the wide size distribution present in most of the bio-Pd samples (Figure 4). There was not a positive correlation between smaller size and activity: Thalassospira sp. was among the species with the smallest Pd nanoparticles (mainly in the 5−10 nm range) but its activity was lower as compared to bio-Pd formed by L. sphaericus and Bacillus sp., which had much larger nanoparticles (mainly in the 15−20 nm and 10−15 nm range, respectively). Activation of Bio-Pd by H2 Produced by Indigenous Marine Bacteria. Bio-Pd is typically activated with an external hydrogen donor for dehalogenation reactions.11,15 Fermentative generation of biogenic hydrogen by palladized bacteria is a desirable process for providing a suitable reductive agent for bio-Pd nanocatalysts.15 Due to the fast formation of a narrow size distribution of NPs on the cell wall of S. oneidensis and its high catalytic activity in marine media, this bacterium could be considered as a promising candidate for remediation of chlorinated contaminants in marine waters. However, S. oneidensis did not survive at high salt concentrations and hence was unable to generate hydrogen. In contrast, all eight species isolated from marine test sites showed hydrogen production upon addition of glucose at varying rates (Figure 2). It was also tested whether Bacillus sp. OHM2, the species with the highest catalytic activity, produced enough hydrogen to dechlorinate TCE at the same rate, but this was unsuccessful. Production of hydrogen gas by Pseudoalteromonas sp. could activate the Bacillus bio-Pd, suggesting that mixed marine microbial cultures could have different functions in the remediation strategy and that H2 does not necessarily have to come from the same species that produced the bio-Pd.

terranean has shown that these regions are heavily polluted by oil hydrocarbons, heavy metals, and halogenated compounds.18−20 In this study, sediments from the Lagoon of Mar Chica, Morocco, and Priolo Gargallo, Syracuse were considered representative of these contaminated regions. These sites are ideal since the envisioned remediation technology consists of adding small amounts of Pd to the sediments, which would not significantly increase the toxicity given their high concentrations of heavy metals (SI Table SI 1). Moreover, it was hypothesized that metal-resistant bacteria would be more abundant in these polluted samples. Thirty facultative anaerobic species were isolated from both sites and it was observed that Bacillales was the most prevalent order recovered from the two areas as determined by 16S rRNA gene sequence analysis (Figure 1). This confirmed that Bacillales is an important heavy metal-resistant order, as previously reported among strains isolated from the Mediterranean Sea.21 Eight out of the 30 bacteria were selected to determine their Pd reduction abilities and each of the six orders were represented. Surprisingly, all of the bacteria reduced Pd while nonviable controls only adsorbed the Pd partially. This finding confirmed our hypothesis that species isolated from these metal contaminated sites are ideal for our envisioned Pdbased technology. Pd Nanoparticle Formation by Marine Species. There is currently a great deal of interest in the isolation of metalresistant bacteria for bioleaching,22 metal recovery and bioremediation.23 However, little research has focused on the isolation of bacteria for the formation of NPs. Although in recent years bio-Pd NPs have been suggested as a green catalyst for removing a wide range of environmental contaminants,24 synthesis of bio-Pd NPs by heavy metal-resistant, marine bacteria has not been reported until now. The formation of bioPd NPs has been shown for more general orders such as Clostridiales and Bacillales, but was undocumented for typical marine water species such as Halomonas sp., Thalassospira sp., Pseudoalteromonas sp., and Vibrio natriegens. Formation of Pd NPs occurred solely by active biotic samples, however, Pd (II) was also adsorbed on nonactive biomass. X-ray diffractograms of the biogenic NPs demonstrated that elimination of Pd (II) from solution was partially caused by the reduction to Pd (0) and partially by the formation of PdO crystals. These results suggest that the formation of NPs starting from Pd metal ions is a widespread ability in microbial communities present in marine mud samples, and that it might be an active resistance mechanism used by marine isolates. However, the exact reaction mechanisms of metal NPs formation by metal-resistant marine bacteria remain unknown. The location and size of bio-Pd NPs were investigated by TEM (Figure 3). Our isolates formed NPs of various sizes in the periplasmic space and in the cytoplasm of the Gramnegative bacteria and in the periplasmic space, on the cell wall or in the growth medium of the Gram-positive marine bacteria. Intracellular precipitation of NPs in the case of Gram-negative bacteria may be caused by the much thinner peptidoglycan layer, which makes the transport for Pd(II) to the cytoplasm much easier. Similar observations were accounted for in freshwater species by attachment of Pd to amine or carboxy functional groups, and subsequent reduction by enzymatic activity.25 It is likely that similar mechanisms are valid for marine species. 555

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In contrast with Bacillus sp., Thalassospira sp. were able to both form Pd NPs and activate them with H2 after glucose fermentation, resulting in TCE degradation by a single organism. Interestingly, Thalassospira cells produced more H2 when palladized (Figure 2). One hypothesis to explain the increase in hydrogen production by palladized cells as compared to Pd-free cells is the improved chemical glucose oxidation and the increased dehydrogenation of organic components such as fatty acids and alcohols by Pd nanocatalysts. For example, Pd-based catalysts have recently been shown to effectively dehydrogenate formic acid.26 Another hypothesis is the participation of Pd nanoparticles in the electron transport chain or in crucial metabolic pathways.27 Beckers et al. have recently reported that the fermentative production of H2 from glucose by Clostridium butyricum was enhanced in the presence of nanometer-sized metallic particles such as Pd NPs.28 The generation of biohydrogen from glucose by palladized marine isolates supports a novel and effective procedure to provide reusable marine compatible Pd nanocatalysts. We opted to add glucose as a carbon source since the sediments we focused on in this research had very low and recalcitrant carbon contents, with COD values around several hundreds of mg kg−1. Depending on the amount and type of organics in polluted sites, a slower degrading carbon source such as polylactate could be provided instead. In this study we have assessed the application of indigenous Mediterranean marine isolates for the synthesis of palladium nanocatalysts and investigated these catalysts for remediation of chlorinated hydrocarbons in a synthetic marine environment. These novel biogenic nanoparticles open up new possibilities for future marine nanobased remediation applications. A treatment strategy for halogenated contaminants present in marine environments based on biogenic Pd nanocatalysis in full scale technology will be investigated and confirmed in future work.

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*Phone: +32 (0)9 264 59 76; fax: +32 (0)9 264 62 48; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the project grant from the EU Commission within the Program of the Seventh Framework (FP7-KBBE-2010-4): EU ULIXES project (266473) and S.V.N. is supported by the project grant no. G.0808.10N of the FWO Flanders. T.H. was supported by a postdoctoral Fellowship from the Research Foundation Flanders (FWOVlaanderen. We thank Tim Lacoere for his assistance during the molecular work and Stephen J. Andersen and Justin Jasper for critically reading the manuscript. We thank Johan Paul for XRD measurements. 556

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