Development of autochthonous microbial consortia for enhanced phytoremediation of salt-marsh sediments contaminated with cadmium

Science of the Total Environment 493 (2014) 757–765

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Development of autochthonous microbial consortia for enhanced phytoremediation of salt-marsh sediments contaminated with cadmium Catarina Teixeira a,b, C. Marisa R. Almeida a, Marta Nunes da Silva a, Adriano A. Bordalo a,b, Ana P. Mucha a,⁎ a b

Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR/CIMAR), Universidade do Porto, Rua dos Bragas, 289, 4050-123 Porto, Portugal Laboratório de Hidrobiologia e Ecologia, Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, Rua Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal

H I G H L I G H T S • • • •

Cd resistant microbial consortia were developed and applied to salt-marsh sediments. In Phragmites australis the consortia amendment promoted metal phytoextraction. The consortia addition increased Juncus maritimus phytostabilization capacity. No long term changes on the rhizosediment bacterial structure were observed.

a r t i c l e

i n f o

Article history: Received 10 February 2014 Received in revised form 10 June 2014 Accepted 11 June 2014 Available online xxxx Editor: Charlotte Poschenrieder Keywords: Autochthonous bioaugmentation Microbial consortia Metal contamination Cadmium Phytoremediation Salt-marsh plants

a b s t r a c t Microbial assisted phytoremediation is a promising, though yet poorly explored, new remediation technique. The aim of this study was to develop autochthonous microbial consortia resistant to cadmium that could enhance phytoremediation of salt-marsh sediments contaminated with this metal. The microbial consortia were selectively enriched from rhizosediments colonized by Juncus maritimus and Phragmites australis. The obtained consortia presented similar microbial abundance but a fairly different community structure, showing that the microbial community was a function of the sediment from which the consortia were enriched. The effect of the bioaugmentation with the developed consortia on cadmium uptake, and the microbial community structure associated to the different sediments were assessed using a microcosm experiment. Our results showed that the addition of the cadmium resistant microbial consortia increased J. maritimus metal phytostabilization capacity. On the other hand, in P. australis, microbial consortia amendment promoted metal phytoextraction. The addition of the consortia did not alter the bacterial structure present in the sediments at the end of the experiments. This study provides new evidences that the development of autochthonous microbial consortia for enhanced phytoremediation of salt-marsh sediments contaminated with cadmium might be a simple, efficient, and environmental friendly remediation procedure. Capsule abstract: Development of autochthonous microbial consortia resistant to cadmium that enhanced phytoremediation by salt-marsh plants, without a long term effect on sediment bacterial diversity. © 2014 Published by Elsevier B.V.

1. Introduction Metal pollution has become a major environmental problem since the beginning of the industrial revolution, as a consequence of mining, burning of fossil fuels, sewage, municipal wastes, fertilizers, and pesticides (Aafi et al., 2012). Temperate salt marshes have an important ecological role since they are not only among the most productive ecosystems on Earth (Costanza et al., 1997), but also among the most sensitive ecosystems. Salt marsh areas are important sinks for contaminants, namely metals and metalloids, that tend to accumulate in plant roots (Mishra et al., 2008). Phytoremediation takes advantage of the ⁎ Corresponding author. Tel.: +351 3401822; fax: +351 3390608. E-mail address: [email protected] (A.P. Mucha).

http://dx.doi.org/10.1016/j.scitotenv.2014.06.040 0048-9697/© 2014 Published by Elsevier B.V.

ability of plants to uptake, adsorb, and/or concentrate contaminants from the surrounding environment constituting a potentially harmless and cost effective method for the recovery of contaminated areas. Plants have developed mechanisms to tolerate metal contamination, such as synthesis of metal binding peptides, vacuolar sequestration, immobilization of metals in cell walls, exclusion through the action of plasma membrane, phytovolatilization, among others (Callahan et al., 2005; Memon and Schröder, 2008; Pilon-Smits, 2005). These mechanisms play a key role not only in protecting living organisms from the adverse effects of metals, but also in remediating metal contaminated environments (Memon and Schröder, 2008; Pilon-Smits, 2005; Valls and de Lorenzo, 2002). Phytoremediation can therefore be translated by two main strategies: (i) reduction of the metal mobility through absorption, adsorption, and/or precipitation by plant roots, hence decreasing their

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bioavailability (phytostabilization); and (ii) uptake of contaminants by plant roots and translocation to aboveground parts of the plant (phytoextraction) (Mendez and Maier, 2008; Peng et al., 2008). Nonetheless, for the phytoremediation process to be efficient, plants also rely on their interactions with microorganisms. Microbial populations that are associated to plant roots (rhizobacteria) can positively affect plants by improving growth and health, enhancing root development, or increasing plant tolerance to various environmental stresses (Glick, 2010). In fact, rhizobacteria have been reported to stimulate plant acquisition and recycling of nutrients, and control plant pathogens (e.g. Rajkumar et al., 2010; Aafi et al., 2012). Therefore, rhizobacteriaassisted phytoremediation has recently emerged as a promising field to enhance phytoremediation efficiency (Weyens et al., 2009; Zhuang et al., 2007). This “rhizoremediation” could be theoretically further enhanced by using bioaugmentation, i.e., by the addition of microorganisms resistant to the contaminant in question. However, the addition of exogenous microbial populations to the environment can be quite questionable because the effects on the natural microbial diversity are usually unknown. In addition, several studies have demonstrated that bioaugmentation carried out with exogenous microbial populations is in most cases not successful because those exotic microorganisms are not able to compete with the indigenous population, being their survival dependent on “field”/real environmental conditions (Hosokawa et al., 2009 and references therein). An alternative could be bioaugmentation with autochthonous rhizobacteria. Nevertheless, this approach is little explored in the literature (e.g., Hosokawa et al., 2009; Kidd et al., 2009), and as far as we know even inexistent in the application of phytoremediation to salt marsh sediments contaminated with metals. Therefore, further research is needed. We hypothesized that the bioaugmentation with selectively enriched resistant autochthonous bacteria could be a simple and straightforward way to enhance phytoremediation. The aim of this study was, therefore, to develop autochthonous microbial consortia resistant to cadmium that could enhance phytoremediation of salt marsh sediments contaminated with this metal. Cadmium is one of the most active elements in estuaries since it is readily mobilized during river water and seawater mixing, being one of the most toxic metals (Waeles et al., 2005). The microbial consortia were selectively enriched from sediments colonized by Juncus maritimus and by Phragmites australis, two salt-marsh plants commonly found in temperate salt marshes. The applicability of the microbial consortia and the effect on cadmium phytoremediation, as well as the microbial community structure in the sediments associated to the roots of the two plants were assessed using a microcosm experiment. To separate the effects of microorganisms from those of plants, non-vegetated sediment was also enriched and tested.

2. Methods 2.1. Sampling Sampling was performed in September 2011 in the salt marsh area of the Lima River estuary, in the north-western coast of Portugal at low tide. Rhizosediments (sediments around plant roots) colonized by J. maritimus and by P. australis, as well as non-vegetated sediment, were collected at the lower estuary (41.688° N, 8.814° W). Sediments were retrieved between 5 and 15 cm, the depth with the higher plant belowground biomass, placed in individual sterile bags, and immediately refrigerated in an ice chest for transportation. At the same location, estuarine water was also collected into plastic sterile bottles. Additionally, specimens of each plant were collected together with the sediment attached to their roots (cubes of approximately 20 × 20 × 20 cm), and placed in plastic plant pots for the microcosm experiments. Cubes of non-vegetated sediment of similar dimensions were also retrieved and placed in similar individual pots.

2.2. Sediment characterization Sediment samples were characterized in terms of water and organic matter (OM) contents, grain size distribution, microbial abundance and cadmium concentration. Sediment water and OM contents were measured as percentage of weight loss following overnight drying at 105 °C, and 4 h at 500 °C, respectively. Grain size analysis was performed by dry sieving in a mechanical shaker (CISA RP08). Initial cadmium concentration and microbial abundance were determined as described below. 2.3. Microbial consortia development The enriched cadmium-resistant autochthonous microbial consortium (AMC) was developed using a procedure freely adapted from the literature (Lorah et al., 2008), where the original sediment and water slurries were enriched in microbial species resistant to cadmium by repeated amendments with that contaminant, and by sequential dilutions (SI, Fig. 1). For each type of sediment (J. maritimus rhizosediment, P. australis rhizosediment, and non-vegetated sediment), 600 mL slurries were prepared in culture bottles, with sediment and estuarine water (1:2, v:v), 14 mg L−1 of glucose, and 96 mg L−1 of cadmium (as CdCl2, corresponding to ca. 10 times the effects range median (ERM) value (O'Connor et al., 1998) (see definition below — Section 2.6)). The slurries were incubated for 4 days at room temperature (RT), under constant agitation. The slurries were then diluted (1:1) with saline nutrient solution constituted by half-strength Hoagland solution (Hoagland and Arnon, 1950) with 10 g L−1 NaCl, to match the conditions used later in the microcosm experiments. Again, slurries were enriched with glucose (14 mg L−1) and cadmium (96 mg L−1). After a new incubation of 4 days at RT with agitation, slurries were diluted with nutrient solution and glucose (1:1), and the process repeated without cadmium addition. Sequential dilutions without cadmium were repeated 2 more times, being the total time for microbial consortia development 20 days. At each step, sub-samples were collected for total cell counts. After the final step, sub-samples were collected for further characterization. Each type of slurry was divided in two batches, being one kept as obtained, and the other autoclaved, for the subsequent use in the microcosm experiments. 2.4. Microbial community characterization To monitor microbial abundance variation during the consortium preparation, at each step of the procedure, total cell counts (TCC) were estimated in the slurries by the DAPI direct count method (Porter and Feig, 1980; Kepner and Pratt, 1994). Briefly, triplicates of 0.9 mL of slurry were fixed with 0.1 mL of formaldehyde (37%). Sub-samples of fixed slurry samples were then stained with 4′,6′diamidino-2-phenylindole (DAPI), incubated for 12 min and filtered onto black Nucleopore polycarbonate filters (0.2 μm, Whatman, UK). Membranes were set up in glass slides and cells counted at 1875 × magnification on an epifluorescence microscope (Labophot, Nikon, Japan). A minimum of 10 random microscope fields for each replicate were counted in order to accumulate at least 300 cells per filter. For initial sediment characterization, 1 g of homogenized sediment was added to 9 mL of NaCl solution (9 g L− 1), fixed with 1 mL of formaldehyde (37%), and stained with DAPI as described above. Total DNA was extracted from slurry and sediment samples using a modification of the CTAB extraction protocol as described by Barrett et al. (2006). Three to four extraction replicates were performed for each sample. Quality of extracted DNA was evaluated by visualization on 1.5% agarose gels and each DNA preparation was quantified with the Qubit fluorometer (Invitrogen). Bacterial community structure of the microbial consortia was performed by automated rRNA intergenic spacer analysis (ARISA). Extracted DNA was amplified using ITSF and ITSReub primer set (Cardinale et al., 2004), with the reverse primer

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Fig. 1. Hierarchical clustering based on Bray–Curtis similarities of ARISA fingerprints. For initial sediments: RP, RJ, and S represent P. australis rhizosediments, J. maritimus rhizosediments, and non-vegetated sediment, respectively. For the autochthonous microbial consortia: P, J, and S represent consortia enriched from P. australis rhizosediments, J. maritimus rhizosediments, and non-vegetated sediment, respectively. Lower case letters and numbers represent replicate samples.

labeled with the phosphoramidite dye 6-FAM (6-carboxyfluorescein). PCRs were performed in 25 μL volumes containing between 10 and 50 ng of DNA, 400 nM of both primers, 200 mM dNTPs, 3 × Taq PCR buffer, 2.5 U Taq DNA polymerase, 2.5 mM MgCl2 and 1 μg bovine serum albumin. The PCR mixture was held at 94 °C for 2 min, followed by 30 cycles of 94 °C for 45 s, 55 °C for 30 s, 72 °C for 2 min, and a final extension at 72 °C for 7 min. PCR products were examined on 1.5% agarose gel, and PCR replicates were combined and purified using a GFX PCR DNA purification kit (GE-Healthcare). Purified product was quantified using the Quant-it dsDNA assay kit and the Qubit fluorometer (Invitrogen), and a standardized amount of the purified PCR product was diluted 1 in 5 and mixed with 0.5 mL of ROX-labeled genotyping internal size standard (ROX 1000, Applied Biosystems). The sample fragments were run on a ABI3730 XL genetic analyzer at STABVIDA Sequencing Facilities (Lisbon, Portugal). 2.5. Microcosm experiments Plant pots with the salt marsh plants and the non-vegetated sediments were placed outdoors in two greenhouses exposed to natural light and temperature conditions. The pots were randomly arranged within and between greenhouses to guarantee that the disposition of the greenhouses did not have any impact on the experimental conditions. Plants and sediments were watered with saline nutrient solution (half-strength Hoagland solution with 10 g L−1 NaCl, to simulate the average salinity of the estuary) through an automated irrigation system, regulated to mimic natural floods (2 daily tidal cycles). Twenty days after acclimation (with tidal simulation), 6 pots of J. maritimus, 6 pots of P. australis, and 6 pots of non-vegetated sediment were spiked with a 20 mg L−1 cadmium solution (9.6 μg g−1 cadmium concentration in the sediment, ca. ERM value, the sediment quality guideline that indicates the concentration above which adverse biological effects may frequently occur (O'Connor et al., 1998)) with salinity 10. One liter of a 10 g L−1 NaCl solution without metal was added to the remaining pots. In the following day, 6 pots of each type (J. maritimus, P. australis and sediment), 3 non-spiked and 3 spiked with cadmium, were inoculated with a solution containing the enriched cadmiumresistant AMC (200 mL per pot). To the remaining 18 pots an autoclaved AMC solution (200 mL per pot) was added. As this solution still had measurable cadmium levels, non-spiked vessels presented ca. 1.0 μg g−1 cadmium concentration in the sediment, corresponding to the effects range low (ERL) value, the sediment quality guideline that indicates the concentration below which adverse biological effects rarely occur (O'Connor et al., 1998).

Altogether, 4 treatments (with 3 replicates each) were obtained: (A) (B) (C) (D)

ERL cadmium concentration without AMC (autoclaved solution), ERL cadmium concentration with AMC (non-autoclaved solution), ERM cadmium concentration without AMC (autoclaved solution), ERM cadmium concentration with AMC (non-autoclaved solution).

The pots were kept in the greenhouses with tidal simulation as mentioned above for 2 months, being afterwards dismantled. Sub-samples of plants, rhizosediment, and sediment were separated for cadmium determination and microbial community characterization. 2.6. Cadmium determinations Cadmium content was determined in sediments, rhizosediments and in the different plant tissues. Samples were digested in a highpressure microwave system and analyzed by atomic absorption spectrophotometry as described by Almeida et al. (2004). 2.7. Data analysis At least three independent replicates of each sample and determination were performed, and mean values and respective standard deviations were calculated. Before statistical analysis was carried out, data were tested for normality and homogeneity of the variances. Statistically significant differences between cadmium levels among sediment samples and plant tissues were evaluated through ANOVA tests, while differences in bacterial richness among samples were evaluated through Kruskal–Wallis tests, using STATISTICA v.10 software (StatSoft, Tulsa, USA). The bacterial richness was estimated as the total number of unique OTUs (peaks) identified within each ARISA profile, assuming that the number of peaks represented the species number (phylotype/ genotype richness). For community structure analysis, a matrix of ARISA aligned fragments and fluorescence values was created as previously described (Mucha et al., 2013). This matrix was imported to the PRIMER 6 software package (version 6.1.11) (Clarke and Gorley, 2006) to be analyzed. Data were normalized using the presence/absence of pretreatment function and samples were then analyzed using the Bray–Curtis similarity method and clustered in the complete linkage mode with the default parameters (5% significance, mean number of permutations, 1000; number of simulations, 999), for generating a dendrogram based on percent similarity. A multidimensional scaling (MDS) plot was then generated using the default parameters with a minimum stress of 0.01 to generate a configuration plot based on percent similarity. To assess the similarity of bacterial community composition among

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microcosms, an analysis of similarities (two-way crossed ANOSIM, based on Bray–Curtis similarity) was carried out. The ANOSIM is a permutation-based hypothesis statistical test, an analog of the univariate ANOVA, which tests for differences among groups of (multivariate) samples from different locations or experimental treatments; the values of the R statistic were an absolute measure of how well the groups were separated and ranged between 0 (indistinguishable) and 1 (well separated). 3. Results and discussion 3.1. Initial characterization Rhizosediments presented muddy characteristics, being dominated by the silt and clay fraction (b 63 μm) (Table 1). Silt and clay were also the dominant fraction present in non-vegetated sediment. The presence of the plants in the vegetated sediments resulted in higher water and organic matter contents, when compared to non-vegetated sediment (Table 1), which is a common feature already observed for these and other plants (Almeida et al., 2004, 2011). Cadmium levels were identical among the three types of sediments. No significant differences (p N 0.05) were found in microbial abundance between the three types of sediment (Table 1). However, ARISA profiles showed only about 30% similarity between non-vegetated and vegetated sediments (Fig. 1), indicating that plants can influence the structure of the microbial communities. Plants are known to be able of oxidizing the sediment through the movement of oxygen towards the roots (Weis and Weis, 2004), or acidifying its rhizosphere through the release of root exudates (Mucha et al., 2008), which can differ with the plant species. Therefore, the presence of vegetation influences the characteristics of sediment, which can condition the microbial communities.

over the other. Indeed, microorganisms may be capable of acclimating to metal toxicity (Liebert et al., 1991). It has been previously suggested that the presence of high metal concentrations creates selective pressure for metal-resistant microorganisms (Sandrin and Maier, 2003; Kidd et al., 2009). Changes in the microbial community structure, towards a community with more metal-tolerant or metal-resistant organisms, have been widely observed in studies investigating the effects of metals on soils (Becker et al., 2006 and references therein; Turpeinen et al., 2004). When comparing the three different consortia obtained from the different types of sediment (non-vegetated, J. maritimus rhizosediment and P. australis rhizosediment), clear differences between their ARISA fingerprints can be noted (Figs. 1 and 3), with only about 20% similarity between community structures, being the consortium obtained from J. maritimus rhizosediment the most dissimilar one. Other authors, working with organic contaminants, have shown that the microbial community was greatly affected by the soil from which the consortia were enriched (Wu et al., 2013). Regarding metals, only few studies have dealt with the enrichment of microbial consortia from soils. For example, Chai et al. (2009) stimulated an indigenous bacteria consortium with nutrient broth for the successful remediation of Cr(VI)contaminated soil. Nonetheless, bioaugmentation studies involving the enrichment and use of autochthonous microbial consortia in the remediation of metal contaminated salt-marsh sediments are lacking in the literature. On the other hand, most of the previous researches on bioaugmentation were conducted using pure cultures or mixtures of isolated microorganisms (Albarracín et al., 2010; Belimov et al., 2005). The use of microbial consortium enrichments may present a broader range of applicability perspectives in opposition to the use of culturable microorganisms currently applied in bioaugmentation strategies, which have difficulty in adapting to the environmental conditions (Hosokawa et al., 2009).

3.2. Microbial consortium development and characterization 3.3. Microcosm experiments Enriched cadmium-resistant AMC was developed by incubating sediment slurries with repeated amendments of cadmium and nutrient solution. Three different AMC were produced from the 3 types of sediment (non-vegetated, J. maritimus rhizosediment, and P. australis rhizosediment). Non-vegetated sediment was used for comparison purpose as plant can influence microbial communities (Fig. 1). Total cells counts were monitored during the consortia development (Fig. 2), and no differences were detected either between sediment types or during the 20-day period, except for un-vegetated sediment slurry where a slight decrease in TCC was noted. Nonetheless, during the development of the consortia, the microbial community structure changed drastically (Figs. 1 and 3), with only about 10% similarity between initial sediments and the microbial consortia. Moreover, at the end of the incubation the microbial consortium slurries were characterized by less OTU numbers when compared to the respective initial sediments (Table 2), showing that despite the total number of bacteria remained similar, the growth of certain bacteria was apparently favored

Table 1 Characterization of sediment samples collected from Lima estuary: grain size distribution, water (H2O) and organic matter (OM) contents, cadmium (Cd) concentration and microbial abundance (TCC) (values represent mean ± SD, n = 3).

H2O (%) OM (%) Grain size N2 mm (%) b63 μm (%) Cd (μg g−1) TCC (log cell g−1)

P. australis rhizosediment

J. maritimus rhizosediment

Non-vegetated sediment

55 ± 3 13 ± 1

52 ± 1 11 ± 1

46 ± 1 7.5 ± 0.6

0.5 84 0.15 ± 0.03 8.7 ± 0.1

1.0 70 0.15 ± 0.08 8.5 ± 0.0

0.7 58 0.27 ± 0.01 8.5 ± 0.0

Microcosm experiments were used to assess the effect of the enriched microbial consortia on the phytoremediation of cadmium contaminated sediments. The microcosm approach has been proved to be a useful tool for the evaluation of microbial mediated remediation of soil contaminants (Albarracín et al., 2010), or the assessment of the potential impact of contaminants on soil microorganisms (Ranjard et al., 2006). Microcosm studies performed under geochemical conditions similar to those of in situ field tests could be valuable to identify intrinsic mechanisms as conditions are controlled and erratic variations are minimized. Nonetheless, the extrapolation of the results to field conditions is always complex and should be supported with larger scale experiments. Two different cadmium levels were tested, lower cadmium concentration (ERL, ca. 1 μg g−1), and higher cadmium concentration treatments (ERM, ca. 10 μg g−1). At the end of the experiment period, no differences were detected between sediment types regarding bacterial richness (Table 2; H(2) = 2.97, p = 0.23, n = 36). However, differences were found between treatments (Table 2; H(2) = 9.17, p = 0.03, n = 36), with OTU number mean ranks ordered as follows: D (ERM with AMC) b B (ERL with AMC) b A (ERL without AMC) b C (ERM without AMC). Regarding the microbial community structure at the end of the experiments, it was observed that the presence of a plant was a determinant factor, as samples from the rhizosediment pots were grouped apart from those of non-vegetated sediment (Fig. 4). The MDS plot indicated that the most dissimilarity was observed between bacterial communities of non-vegetated and vegetated sediments, which were clustered apart at 40% of similarity (Fig. 4). Additionally, plant species also modulated community structure with a clear separation (at 50% of similarity) among bacterial community from the different rhizosediments. These features overpowered differences between

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9.5

761

J. maritimus rhizosediment P. australis rhizosediment Non-vegetated sediment

log cell ml-1

9.0

8.5

8.0

7.5 4

8

12

16

20

Days

Fig. 2. Variation of total cell numbers in the slurries during the development of the microbial consortia (error bar = 1 SD, n = 3).

treatments. Analysis of similarities (two-way crossed ANOSIM) confirmed that the separation between sediment groups was higher than for treatment groups (Table 3). Nonetheless, cadmium level was also a significant grouping factor (Table 3). Mucha et al. (2011) also observed that plants had an effect on microbial community structure, being different from that in non-vegetated sediments, either in the presence or in the absence of Cu. A similar effect was also reported in a study with petroleum hydrocarbons, in which the presence of plants, together with the plant species, acted as a selection factor on the bacterial

assemblage composition, overriding the effect of the different treatments tested (Ribeiro et al., 2013). It is well known that plants exert an important influence and can shape the structure and composition of microbial communities by enhancing their activity by root exudate composition and quantity (Bais et al., 2006; Koranda et al., 2011), being root exudates dependent, among other factors, on plant species (Bais et al., 2006). The fact that, after two months, there were no significant differences in the community structures between treatments with and without AMC (Table 3), is a meaningful finding, indicating that the

Fig. 3. ARISA electropherograms of ITS amplicons amplified from initial sediments and microbial consortium samples.

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Table 2 Range of peak numbers (OTUs) in the ARISA profiles of initial sediments, developed microbial consortium (AMC) slurries, and treatment pots at the end of the microcosm experiment: A — ERL Cd without AMC; B — ERL Cd with AMC; C — ERM Cd without AMC; D — ERM Cd with AMC (values represent mean ± SD, n = 3). Initial sediments

AMC

Treatment pots

P. australis rhizosediment

133 ± 13

35 ± 3

J. maritimus rhizosediment

138 ± 13

68 ± 6

A B C D A B C D A B C D

Non-vegetated sediment

120 ± 23

40 ± 3

196 204 201 131 171 143 199 159 128 151 197 143

± ± ± ± ± ± ± ± ± ± ± ±

Table 3 Results of the two-way crossed ANOSIM test for different sediments (P. australis and J. maritimus rhizosediments, and non-vegetated sediment), treatment effects, cadmium level, and AMC addition, based on ARISA results at the end of the experiment.

26 38 30 8 8 23 45 16 14 42 33 33

Statistic value (R)

Significance level (%)

Sediment type vs treatment Global test Sediment Global test Treatment

0.99 0.68

0.1 0.1

Sediment type vs Cd level Global test Sediment Global test Cd level

0.99 0.52

0.1 0.1

Sediment type vs AMC addition Global test Sediment Global test AMC addition

0.98 0.21

0.1 1.4

2014). Plant-associated microorganisms can change metal availability by altering their solubility and transport in soil, through secretion of organic substances, such as chelators and siderophores, and/or through variation in soil pH and redox potential (Kidd et al., 2009). So, the addition of AMC resistant to metal could be a suitable strategy to decrease the spreading of metals in the estuarine areas preventing metals from reaching the more coastal areas. Cadmium levels in the plant tissues showed that both plants accumulated cadmium in their tissues, being in general the higher cadmium levels found in ERM treatments (Fig. 6). However, for the same treatments, J. maritimus and P. australis showed different cadmium distributions among different tissues, showing diverse metal allocation strategies (Fig. 6). In the presence of the AMC, J. maritimus revealed an increase in cadmium concentration in belowground tissues for the higher cadmium concentration (Fig. 6), increasing metal phytostabilization in these plant structures. The ability to phytostabilize cadmium had been previously noted for these plants in situ (Almeida et al., 2006, 2011). On the other hand, for P. australis, the addition of the AMC led to a significantly (p b 0.05) higher accumulation of cadmium in the stems (Fig. 6), increasing metal translocation for both cadmium treatment levels. So, metal phytoextraction potential of this plant was enhanced. In fact, it has been previously observed that due to the high aboveground biomass of P. australis, this plant

addition of the AMC did not alter the bacterial structure in the sediments which might be important for in situ application of autochthonous bioaugmentation. Concerning metal concentrations at the end of the experiment, higher cadmium concentrations were found in sediments exposed to ERM cadmium treatments (Fig. 5). Moreover, the addition of the AMC in the treatments with higher cadmium levels (ERM) resulted in an increase of cadmium concentration in sediments (significant (p b 0.05) for P. australis rhizosediments, and for non-vegetated sediments), indicating that the respective consortia might have decreased cadmium mobility in these sediments. Curiously, the communities from these consortia (P. australis rhizosediment and non-vegetated sediment), showed higher similarity between them than with those from J. maritimus rhizosediment, as stated by the hierarchical clustering of ARISA profiles (Fig. 1). Microorganisms can interact directly with the metals to reduce their toxicity and/or change their bioavailability (Ma et al., 2011; Kidd et al., 2009), which can be the reason why a higher retention of cadmium in these sediments occurred. In fact, sediment sequential extraction carried out to assess metal availability in sediments indicated that in these sediments metal was bound to less available fractions (lower acid soluble/exchangeable fraction, Nunes da Silva et al.,

Transform: Presence/absence Resemblance: S17 Bray Curtis similarity 2D Stress: 0.08

C1J D1J

B2J

D2J D3J

C3J B2S D2S A1S

C2J

A3J A1J A2J B1J

treatment A B C D

Similarity 40 50

B3J

D3S C3S D1S C1SC2S

B1S A2S A3S

B3S C2P C3P D2P B3P D1P

A3P C1P

D3P

B1P A1P

B2P A2P

Fig. 4. Multidimensional scaling (MDS) ordination based on Bray–Curtis similarities in the presence/absence matrix obtained from ARISA fingerprints of microbial communities at the end of the experiment for the different sediments (P. australis (P), and J. maritimus (J) rhizosediments, and non-vegetated sediment (S)), and treatments (A — ERL Cd without AMC; B — ERL Cd with AMC; C — ERM Cd without AMC; D — ERM Cd with AMC). Numbers correspond to replicates. Samples enclosed by green and blue lines cluster at 40% and 50% similarity, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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6.0

763

without AMC

*

with AMC

Cd (ug g-1)

4.0

*

2.0

0.0 ERL

ERM

J. maritimus rhizosediment

ERL

ERM

ERL

P. australis rhizosediment

ERM

non-vegetated sediment

Fig. 5. Cadmium levels in J. maritimus and P. australis rhizosediments, and in non-vegetated sediment, at the end of the microcosm experiment. Significant effects of AMC addition are marked with * (p b 0.05). Error bars represent standard errors of 3 replicates. Adapted from Nunes da Silva et al. (2014).

has potential to be considered a cadmium phytoextractor (Almeida et al., 2011), a feature that can be enhanced by the addition of the AMC. These results showed that the addition of AMC resistant to

cadmium enhanced phytoremediation in both plants, although they presented different metal removal pathways. This clearly shows that plant–microbial interactions are specific and that the outcomes of

J. maritimus 300

120

Cd (ug) in Bellowground tissues

250

*

with AMC

100

200

80

150

60

100

40

* 50

20

*

0

Cd (ug) in Aboveground tissues)

without AMC

0 roots

rhizomes

stems

leaves

roots

ERL

rhizomes

stems

leaves

ERM

P. australis 160

without AMC

*

with AMC

140

250

120 200

100

* 150

80 60

100

* 50

40

*

20

0

Cd (ug) in Aboveground tissues)

Cd (ug) in Bellowground tissues

300

0 roots

rhizomes stems ERL

leaves

roots

rhizomes stems ERM

leaves

Fig. 6. Total cadmium levels (considering cadmium concentration and biomass values of each plant tissue) in J. maritimus and P. australis below ground (roots and rhizomes; left axis), and above ground tissues (stems and leaves: right axis), at the end of the microcosm experiment (adapted from Nunes da Silva et al., 2014). Significant effects of AMC addition are marked with * (p b 0.05). Error bars represent standard errors of 3 replicates.

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sediment inoculation with metal-resistant microorganisms are highly influenced by the plants to which they are associated with. Microbialinduced metal phytoextraction is thought to be accomplished through two mechanisms, increase of plant biomass and/or changes in metal availability (Kidd et al., 2009). Previous studies showed that the inoculation of metal-tolerant bacteria significantly enhanced metal uptake of the tomato plants and Sedum alfredii (He et al., 2009; Li et al., 2007). In S. alfredii, the bacterial inoculation also resulted in a better translocation of metals from root to shoot (Li et al., 2007). However, the studies regarding bacterially assisted phytoremediation are directed to soil and soil plants, whereas studies with wetland plants are to our knowledge almost inexistent. In fact, only two studies indicated that rhizobacteria could be important in metal tolerance mechanisms in wetland plants (de Souza et al., 1999; Caslake et al., 2006). No other studies were carried out regarding the inoculation of rhizospheric microorganisms into the salt marsh rhizosphere to enhance metal tolerance and uptake. Indeed, to our knowledge, the present study is the first to show that the development of autochthonous microbial consortia resistant to metals to enhanced phytoremediation may present a new technique for the decontamination of metal-polluted sediments, providing ground for further research. The different removal pathways noted for the two plants is also relevant for the design of future in situ remediation experiments.

4. Conclusions In the present work, cadmium resistant microbial consortia were developed and successfully applied to the phytoremediation in salt-marsh sediments contaminated with cadmium. The addition of the consortia increased J. maritimus metal phytostabilization capacity, and promoted metal phytoextraction potential in P. australis, while no changes on the bacterial structure present in the sediments at the end of the experiments occurred. This study provides, therefore, new evidences that the use of microbial-assisted phytoremediation, involving the use of tailormade consortia, might be a simple, efficient, and environmental friendly remediation procedure for salt-marsh sediments.

Conflict of interest I disclose any actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations within three years of beginning the submitted work that could inappropriately influence, or be perceived to influence, the work, including that related with the submission of the manuscript entitled “Development of autochthonous microbial consortia for enhanced phytoremediation of salt-marsh sediments contaminated with cadmium”.

Acknowledgments Research was partially supported by the European Regional Development Fund (ERDF) through the COMPETE — Operational Competitiveness Programme and national funds through FCT, under the projects PHYTOBIO (PTDC/MAR/099140/2008) and PEst-C/MAR/ LA0015/2013. Acknowledgements to Cristina Rocha, Hugo Ribeiro, Catarina Magalhães, Paula Salgado, Carla Silva, and Carolina Carli for the help in sampling collection, experiment assembling, vessel dismantling and sample preparation for analysis.

Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.scitotenv.2014.06.040.

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