Microbiological accumulation by the Mediterranean invasive alien species Branchiomma bairdi (Annelida, Sabellidae): Potential tool for bioremediation

Marine Pollution Bulletin 86 (2014) 325–331

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Microbiological accumulation by the Mediterranean invasive alien species Branchiomma bairdi (Annelida, Sabellidae): Potential tool for bioremediation Loredana Stabili a,b,⇑, Margherita Licciano a, Marco Lezzi a, Adriana Giangrande a a b

Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali (Di.S.Te.B.A.), Via Prov. Lecce-Monteroni, 73100 Lecce, Italy Istituto per l’Ambiente Marino Costiero – Sezione di Taranto – CNR, Via Roma 3, 74100 Taranto, Italy

a r t i c l e

i n f o

Article history: Available online 25 July 2014 Keywords: Bacterial accumulation Bioremediation Branchiomma bairdi Microbial pollution Filter feeding Polychaetes

a b s t r a c t We examined the bacterial accumulation and digestion in the alien polychaete Branchiomma bairdi. Microbiological analyses were performed on worm homogenates from ‘‘unstarved’’ and ‘‘starved’’ individuals and on seawater from the same sampling site (Ionian Sea, Italy). Densities of culturable heterotrophic bacteria (22 °C), total culturable bacteria (37 °C) and vibrios were measured on Marine Agar 2216, Plate Count Agar and TCBS Agar, respectively. Microbial pollution indicators were determined by the most probable number method. B. bairdi was able to accumulate all the six considered microbiological groups which, however, differ in their resistance to digestion. B. bairdi results more efficient than the other two co-occurring sabellids in removing bacteria suggesting that it may counteract the effects of microbial pollution playing a potential role for in situ bioremediation. Thus a potential risk, such as the invasion of an alien species, could be transformed into a benefit with high potential commercial gain and economic feasibility. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Branchiomma is a widespread genus of the family Sabellidae (Annelida) comprising a large number of species (Giangrande and Licciano, 2004), some of which have been translocated out of their natural expected distributional range (Capa et al., 2013). Currently, two non-native species of this genus have been reported in the Mediterranean: Branchiomma luctuosum Grube and Branchiomma bairdi McIntosh (Licciano and Giangrande, 2008; Giangrande et al., 2012; Arias et al., 2013). The first one was introduced from the Red sea about 25 years ago (Bianchi, 1983), whilst the introduction of B. bairdi, native of the Caribbean, is more recent. Both species can be considered invasive (Zenetos et al., 2012), especially B. bairdi which rapidly expanded in several Mediterranean localities reaching high densities in confined and degraded impacted areas, with possible effects on local communities (Arias et al., 2013). These two alien species show ecological features similar

⇑ Corresponding author at: Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali (Di.S.Te.B.A.), Università del Salento, Via Prov.le Lecce-Monteroni, 73100 Lecce, Italy. Tel.: +39 (0)832 298659; fax: +39 (0)832 298626. E-mail addresses: [email protected] (L. Stabili), margherita.licciano@ unisalento.it (M. Licciano), [email protected] (M. Lezzi), adriana.giangrande @unisalento.it (A. Giangrande). http://dx.doi.org/10.1016/j.marpolbul.2014.06.047 0025-326X/Ó 2014 Elsevier Ltd. All rights reserved.

to those of the autochthonous Sabella spallanzanii Gmelin. Although we have not enough knowledge to infer whether biotic relationships among native and alien species may affect niche dimensions, the investigation on their functional traits could help to predict their invasiveness (Sarà et al., in press). As among the functional traits one of the most important is the feeding strategy, studies on this topic may represent a contribution in understanding species interactions and functional roles. All the member of Sabellidae family are filter feeders, a guild which represent a large component of coastal marine ecosystems, both in terms of biomass and number of species (Riisgård and Larsen, 1995). Filter feeders are able to filter organic particles within a size range 0.1–50 lm (i.e., heterotrophic bacteria, heterotrophic eukaryotes, phytoplankton and detritus), processing the water column within few hours and retaining up to 80% of the suspended particles (Stabili et al., 2006b). Studies conducted on S. spallanzanii and B. luctuosum, showed their ability to accumulate and concentrate bacteria from the surrounding environment as well as their impact on bacterial community. Further laboratory experiments carried out on these species demonstrated that Vibrio alginolyticus employed as only food source, was efficiently filtered and retained (Licciano et al., 2005; 2007a,b; Stabili et al., 2006a,b; 2008a). On account of these features both the species were proposed to be utilized as bioremediators.

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The ability to remove from the surrounding water the free-living bacteria may indeed also have a large interest for seawater restoration taking into consideration that coastal zones, as the natural interface between watersheds and the oceans, are usually under intense anthropogenic pressure affecting both the ecosystem and human health through sewage waste-water discharge and disposal practice. These may lead to the introduction of pathogens causing diseases (Koop and Hutchins, 1996; Danulat et al., 2002; Canuel et al., 2009; Díaz-Asencio et al., 2009). Moreover, during the last years, the significant expansion of aquaculture activities has increased the impact on coastal ecosystems (Rana, 1998; Trovar et al., 2000) generating considerable amount of animal waste containing faecal matter, protozoans, bacteria, and viruses leading to the deterioration of water quality and disease outbreaks (Reilly and Kaferstein, 1997; Gifford, 2004; Aguado-Giménez et al., 2007). The introduction of organic matter in large amounts provides bacteria, including those potentially pathogenic to humans and marine organisms, with the complex of substrates available as a food source determining changes in their community structure and dynamics (Bedwell and Goulde, 1997). In the present paper we evaluated the performance of the alien species B. bairdi to filter and consequently remove bacteria from the water column. In order to investigate its potential for the development of strategies aimed to in situ bioremediation, the bacterial removal was than compared with that observed in the two co-occurring sabellid species. 2. Materials and methods 2.1. Sample collection and starvation

Agar 2216 (Difco, USA). The plates were incubated at 22 °C over 7 days. At the end of the incubation period the number of colony-forming units (CFU) were counted through a 10 magnifying glass. Total culturable bacteria at 37 °C (including human potential pathogens) in seawater and polychaete samples were determined by the spread plate method, using Plate Count Agar medium (Difco) (seeding 0.1 ml of each sample). After 48 h at 37 °C the growing CFU were counted. To enumerate the vibrios in seawater, 1, 5 and 10 ml were filtered on 0.45 lm pore size filters that were aseptically placed onto thiosulphate-citrate-bile-salt-agar (TCBS) plus 2% NaCl. After incubation for 48 h, the emerging colonies were counted according to the colony forming units (CFU) method. For the enumeration of vibrios in polychaetes, 0.1 ml of each worm homogenate and of appropriate decimal dilutions were plated on TCBS agar and after incubation at 30 °C for 24–48 h, the culturable vibrios were counted. For total coliforms determination, lactose broth was used as culture media in the presumptive test (incubation at 37 °C for 24–48 h). All presumptive positive (gas production) tubes were transferred to tubes containing brilliant green-lactose broth and incubated for 24–48 h at 37 °C (confirmatory test). The number of test tubes giving positive results (gas production) was recorded and used for most probable number (MPN) index calculation according to Mc Grady tables. Escherichia coli densities were determined using the miniaturized MPN, in accordance with ISO 9308-3 (incubation at 44 °C for 36–72 h). Intestinal enterococci, were calculated using the miniaturized MPN, ISO 7899-1 (incubation at 44 °C for 24–48 h). Results were expressed as MPN 100 ml 1 and MPN 100 g 1 for water and animal samples, respectively.

Adult specimens of B. bairdi were collected by SCUBA divers in the Mar Grande of Taranto (Ionian Sea, Italy) in proximity of an aquaculture plant (Fig. 1). Water samples were collected aseptically from the same sampling site with 5 l sterilized Niskin bottles and processed for enumeration of bacteria, within 4 h of sampling. Immediately upon return to the laboratory worms were manually cleaned of any tube epibionts, washed with sterile seawater and randomly divided into two sets. Specimens from one set (90 individuals) were separated in 9 groups each consisting of 10 individuals, extracted from the tubes and immediately utilized for the analysis (unstarved worms). The second set (90 individuals) was divided and placed in 9 aquaria each containing 10 individuals. Each aquarium was filled with filtered (0.22 lm pore size filters, Millipore) seawater (salinity 36‰) and kept in a temperature controlled room (T = 22 °C; photoperiod: 12:12) for 48 h before the analyses (starved worms).

The experimental design consisted of two factors: Concentration (Co), with two levels (i.e. seawater and polychaetes), fixed and Time (Ti), with two levels (i.e. before and after worm starvation), fixed and orthogonal to Co. Analysis of variance was used to assess differences in the mean abundance of the bacteriological categories between seawater and worms before (Ti0) and after (Ti1) worm starvation. Prior to analysis, the homogeneity of variance was tested using Cochran’s test. The Student–Newman–Keuls test (SNK) was used for post hoc comparisons among means (Underwood, 1997). The analysis was done using GMAV 5 computer program (University of Sidney, Australia).

2.2. Bacteriological analyses

3.1. Microbiological accumulation

The bacteriological analyses were carried out on seawater from the worm sampling site and on both starved and unstarved worms. Each group of 10 worms (9 groups of starved and 9 of unstarved worms) was processed separately for the enumeration of bacteria. Worms were homogenized for 90 s in a Waring blender after their extraction from the tubes. The homogenates (ca. 100 g) were filtered through sterile gauze and diluted with filtered (0.22 lm) seawater to obtain a 1:10 (w/v) dilution. Quantitative analyses of culturable heterotrophic bacteria (22 °C), total culturable bacteria at 37 °C, culturable vibrios, fecal and total coliforms as well as intestinal enterococci were performed in seawater and in worm homogenates. Culturable heterotrophic bacteria abundance was determined by plating 0.1 ml of undiluted seawater or worm homogenates and the respective serial dilutions in triplicate on Bacto Marine

For all the six analysed microbiological groups, ANOVA revealed that bacterial density was significantly lower in starved than in unstarved worms (P < 0.001), whilst no significant differences were found in seawater samples over time (Table 1). In the surrounding seawater mean bacterial total counts were 1.74  104 and 1.6  106 CFU ml 1 at 22 (Fig. 2A) and 37 °C (Fig. 2B) respectively. Mean vibrios density (Fig. 3) was 69 CFU ml 1, intestinal enterococci (Fig. 4A) reached a concentration of 2 MPN 100 ml 1, whilst total coliforms and Escherichia coli were absent (Fig. 4B). The concentration of bacteria at 22 and 37 °C was significantly lower in seawater than in the homogenate of unstarved B. bairdi (P < 0.001), where values respectively six orders of magnitude (1.15  1010 CFU g 1) and three orders of magnitude (1.10  109 CFU g 1) higher were reached (Fig. 2A and B). In starved worms

2.3. Statistical analysis

3. Results

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Fig. 1. Map of the Mar Grande of Taranto (Ionian Sea, Italy) showing the location of the sampling site (A); adult specimen of Branchiomma bairdi (B), scale: 1 cm.

the abundance of culturable heterotrophic bacteria was 3.3  107 CFU g 1 while the density of culturable bacteria at 37 °C including potential pathogens was five order of magnitude lower than in unstarved worms reaching a value of 5.3  104 CFU g 1. Fig. 3 reports the abundances of culturable vibrios growing at 22 and 35 °C detected in B. bairdi homogenates and the surrounding seawater. Unstarved and starved animals showed mean vibrios of 2.6 ± 0.1  107 CFU g 1 and 2.3 ± 0.1  107 CFU g 1. As shown in Table 1, vibrios were significantly more concentrated in both unstarved and starved polychaetes in comparison to the seawater (P < 0.001). In order to evaluate B. bairdi accumulation capability of the classical microbial pollution indicators, we analyzed the densities of total coliforms, Escherichia coli as well as intestinal enterococci. MPN 100 g 1 values of total coliforms as well as Escherichia coli (Fig. 4B) showed that the unstarved worms contained a bacterial concentration higher than the surrounding seawater. In the starved worms, Escherichia coli was completely absent and the concentration of total coliforms was about negligible. Regarding to intestinal enterococci concentrations (Fig. 4A), the values recorded were the same in unstarved and starved animals

(about 9 MPN 100 g 1), whilst in seawater samples 2 MPN 100 l 1 were counted. The results of B bairdi bacterial accumulation of all the considered microbiological parameters are summarized in Table 2 and compared with those obtained from the two other studied sabellids collected from the same area. As shown in the table B. bairdi shows a higher performances in bacterial accumulation.

4. Discussion This paper represents a first contribution in the understanding of feeding strategy of the alien species B. bairdi filtering on the microbial component. This species is able to accumulate all of the six analysed microbiological groups in comparison with the surrounding environment, however, a selectivity in digesting bacteria can be inferred. Significant differences were found between mean total culturable bacteria at 22 and 37 °C abundances in starved and unstarved individuals. The lower bacterial densities in starved worms suggest that within 48 h they are able to digest a conspicuous amount of the filtered bacteria. In the case of

1.0000 None

Pol Ti1 > Pol Ti2 SW Ti1 < Pol Ti1

0.9804 None

Pol Ti1 > Pol Ti2 SW Ti1 < Pol Ti1

0.9999 None

Pol Ti1 > Pol Ti2 SW Ti1 < Pol Ti1 SW Ti2 > Pol Ti2

0.9222 None

SW Ti1 < Pol Ti1 SW Ti2 < Pol Ti2

1.0000 None

Cochran’s test Transform SNK test Co (Ti) Ti (Co)

Pol Ti1 > Pol Ti2 SW Ti1 < Pol Ti1 SW Ti2 < Pol Ti2

P

Reported are SW Ti1 bacterial concentration measured at Ti1 in seawater samples; SW Ti2 bacterial concentration measured at Ti2 in seawater samples; Pol Ti1 bacterial concentration in unstarved polychaetes; Pol Ti2 bacterial concentration in starved polychaetes; ⁄⁄⁄P < 0.001.

0.5000

0 1411.2 0

F MS

0 441 0 0.3125 ⁄⁄⁄ ⁄⁄⁄ ⁄⁄⁄ 291.6 291.6 291.6 4556.25 4556.25 4556.25 15.625 ⁄⁄⁄ ⁄⁄⁄ ⁄⁄⁄ 870.12 1039.53 870.12 ⁄⁄⁄ ⁄⁄⁄ ⁄⁄⁄ ⁄⁄⁄ ⁄⁄⁄ ⁄⁄⁄ 1 1 1 32 35 Concentration = Co Time = Ti CoxTi Residual Total

2.91E + 20 2.94E + 20 2.91E + 20 4.38E + 13

6,644,227 6,723,139 6,644,227

⁄⁄⁄ ⁄⁄⁄ ⁄⁄⁄

2.38E + 16 2.24E + 16 2.38E + 16 5E + 09 4,287,692 1.13E + 09 4,287,721 2.07E + 13 5.45E + 15 2.07E + 13 4,822,504

4,767,559 4,470,043 4,769,308

16,641 19,881 16,641 19.125

P F MS

Escherichia coli

P F

Total coliforms

MS P MS

F

Total culturable bacteria (37 °C)

F MS

P

Halophilic Vibrios (22 and 35 °C)

P F MS

Heterotrophic bacteria (22 °C)

df Source of variation

Table 1 Summaries of ANOVAs testing for differences in average bacterial abundances measured at Ti1 (before starvation period) and Ti2 (after starvation) in both polychaete and seawater samples.

F=0 ⁄⁄⁄ F=0

L. Stabili et al. / Marine Pollution Bulletin 86 (2014) 325–331

Intestinal enterococci

328

Fig. 2. Mean abundance and relative standard deviations of bacterial densities in seawater and worm samples at Ti1 (unstarved) and Ti2 (starved): (A) culturable heterotrophic bacteria at 22 °C; (B) total culturable bacteria at 37 °C. (y-axes are logged).

Fig. 3. Mean abundance and relative standard deviations of vibrios densities in seawater and worm samples at Ti1 (unstarved) and Ti2 (starved). (y-axes are logged).

culturable bacteria at 37 °C the digestion might be due to the inability of non-indigenous bacteria to find a suitable environment for their proliferation and accumulation inside the worms. Heterotrophic bacteria, typically marine, could include some members which, although present in the sampling area, might result allochthonous for the normal microflora of the alien worms and thus unable to establish symbiotic relationships with this polychaete species. Not significant decrease in the Vibrio abundances was observed in starved versus unstarved worms. The high concentration of vibrios in B. bairdi also after starvation does not indicate the inability of this polychaete to digest these bacteria. Some Vibrio species, such as Vibrio alginolyticus are digested by the worms (personal observation) while others presumably find a suitable environment for their survival and growth inside B. bairdi. It is well known indeed that in aquatic environments vibrios are not only

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free-living bacteria, but are often found in and/or on marine organisms, preferring an attached lifestyle in or on animals, plants, and sediment (Thompson et al., 2004; Stabili et al., 2008b; Vezzulli et al., 2010). Vibrios, for example, are normal components of the intestinal microflora of aquatic invertebrates, including shrimp Litopenaeus vannamei (Vandenberghe et al., 1999) and blu crabs Callinectes sapidus (Huq et al., 1986). The role of an ecological reservoir for vibrios has been suggested for other marine invertebrates including mussels, crustaceans, polychaetes and cnidarians (Carli et al., 1993; Kaspar and Tamplin, 1993; Montanari et al., 1999; Cavallo and Stabili, 2002; Stabili et al., 2006a; Licciano et al., 2007a, 2007b; Covazzi Harriague et al., 2008; Vezzulli et al., 2013). We may hypothesize that bacterial proliferation processes of some Vibrio species as well digestion processes of other Vibrio species occur in the worms. However, our data are merely descriptive, and we do not know the mechanisms involved in B. bairdi bacterial degradation. More qualitative studies are needed to clarify the exact mechanisms involved, including the phagocytosis of the here considered bacterial groups by the different worm cellular types. Significantly higher values of total coliforms and Escherichia coli were found in unstarved worms compared to starved ones indicating the ability of B. bairdi to digest these bacteria. By contrast, the concentration of intestinal enterococci in starved polychaetes remained unchanged with respect to unstarved worms. The higher capability of B. bairdi to digest coliforms in comparison to intestinal enterococci could be due to their different bacterial cell walls as already hypothesized for the polychaete S. spallanzanii by Stabili et al. (2006a). It has been demonstrated that Gram positive bacteria, including intestinal enterococci, may be more resistant than Gram negative bacteria to digestion by marine invertebrates (Plante and Shriver, 1998). Coliforms are bacterial organisms inhabiting the gut of warm blooded animals. Although not necessarily harmful themselves, their presence can indicate waters recently contaminated by sewage releasing more harmful bacteria and the risk of human exposure to disease by ingestion or by contact with polluted water. The Water Framework Directive (WFD by the Commission of the European Union: EU 2000/60/EC) has the purpose to preserve, protect, and ensure the good status of all water bodies (including inland surface waters, transitional or estuarine waters, coastal waters and ground-waters) as well as to protect human health across Europe. Therefore, policies and actions are set up in order to prevent and to mitigate water scarcity and drought situations, with the priority to move towards a water-efficient and water-saving economy. In the framework of strategies aimed to improve and technologically update water treatment facilities, filter-feeder invertebrates, including the Demospongiae Spongia officinalis and Hymeniacidon perlevis, the bivalve Mytilus galloprovincialis and the sabellids S. spallanzanii and B. luctuosum have been recently proposed as bioremediators for restoring water quality based on their ability to remove pathogenic bacteria through their filtration process (Ostroumov, 1998; Milanese et al., 2003; Fu et al., 2006, 2007; Stabili et al., 2005, 2006a,b, 2008a; Licciano et al., 2007a,b; Longo et al., 2010). In this scenario, the digestion of coliforms by B. bairdi is noteworthy for the applicative relapses related to the potential employment also of this filter feeder as a bioremediator of sewage polluted seawater. The potential practical application of an invasive alien taxon as a tool to mitigate pollution, however, can lead to other issues, especially considering the European Directive Marine Strategy MSFD (descriptor 2 related to managing marine invasive species). Introduction and spread of alien species are in fact considered one of the main threats to the well-being of coastal marine ecosystems and biodiversity at different scales and extent (Bax et al., 2003; Molnar et al., 2008; Hulme et al., 2009). Although marine invasions have been well documented all over the world, they are particularly conspicuous in the

329

Fig. 4. Mean abundance and relative standard deviations of microbial pollution indicators in seawater and worm samples at Ti1 (unstarved) and Ti2 (starved): (A) intestinal enterococci (IE); (B) total coliforms (CT) and Escherichia coli (EC).

Mediterranean Sea (Zenetos et al., 2010, 2011, 2012). Alien species can displace native species, reduce community biodiversity, change species composition and abundance across habitats, modify habitat structure and produce cascading effects or trophic web shifts that could result in major negative impacts on the ecosystem. As an example, the introduction in Mediterranean Sea of the gasteropod Raphana venosa, a voracious predator of bivalve molluscs, caused a decline in local bivalve populations (Otero et al., 2013). Nevertheless, the effects of the alien species on the biodiversity and habitats cannot be generalized and along with negative effects, invasions can also have positive effects on several components of invaded ecosystems (Rodriguez, 2006). On account of these considerations, understanding the biology of non-native species is basical also for environmental managers to predict/pre-empt their arrivals as well as control or eradicate them At this regard, we are currently investigating the biology of B. bairdi (Lezzi et al., in press). B. bairdi is a Caribbean species, whose expansion to several Mediterranean localities is largely a consequence of its high capacity to colonize extremely different habitats and substrates, and the occurrence of both sexual and asexual reproductive strategies (Arias et al., 2013). However, B. bairdi appears abundant especially in confined environments and areas degraded due to anthropogenic impacts (Arias et al., 2013). The same habitats are colonized also by B. luctuosum, the other alloctonous Branchiomma species introduced in the Mediterranean at least 25 years ago, and by now considered an integrated taxon of the Mediterranean fouling community (Zenetos et al., 2010, 2011). Both these alien Branchiomma species have reached the investigated euthrophic area of the Ionian Sea (Gulf of Taranto), where B. luctuosum was observed since the year 2000 (Licciano et al., 2002) and B. bairdi was recorded from 2011 (Giangrande et al., in press). After B. luctuosum

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Table 2 Comparison of bacterial densities measured in Branchiomma bairdi with those obtained from two other sabellid species collected from the same area (Gulf of Taranto, Italy).

Sabella spallanzanii (Stabili et al., 2006) Branchiomma luctuosum (Licciano et al., 2007) Branchiomma bairdi

Culturable bacteria at 22 °C (CFU/g)

Culturable bacteria at 37 °C (CFU/g)

Culturable vibrios (CFU/g)

Total coliforms (MPN/100 g)

Fecal coliforms (MPN/100 g)

Intestinal enterococci (MPN/100 g)

1.4  109 8.8  104 1.1  1010

4.5  104 1.4  104 1.1  109

3.7  106 2.4  104 2.6  107

81 40 90

52 40 45

958 11,000 9

introduction, the autochthonous sabellid S. spallanzanii showed an initial decrease in abundance (Mastrototaro et al., 2014). At present, however, the two species seem to have reached an equilibrium sharing the same habitat with a similar abundance (Giangrande et al., in press). In this scenario the recent introduction of B. bairdi, which has reached very high densities, represents an interesting issue from an ecological perspective. Preliminary observations indicate that the 3 species do not outcompete each other (Giangrande et al., in press), thus suggesting possible differences in their functional roles and not overlapped trophic niche. However, in the study area as well as in other polluted environments, trophic sources are not a limiting factor, unlike the colonizing substrate availability. It has been demonstrated that this last concern is overcome by increasing the colonizable surface (Lezzi et al., in press). Thus, the 3 species, on account of their powerful bacterial accumulation capability, may act synergically when simultaneously employed for the development of strategies aimed to in situ bioremediation. At present, this strategy is practically applied and experienced in the study area by using panels as substrates to increase the biomass of the above mentioned species and monitoring the development of the fouling community as well as the microbial pollution decrease. In conclusion, on account of B. bairdi capability to accumulate bacteria, we suggest the use of this filter-feeder as a new tool in bioremediation. This hypothesis is particularly attractive since this species, notwithstanding its smaller size, is more efficient than the other 2 co-occurring sabellids in removing bacteria from the surrounding environment. Despite B. bairdi is an invasive species in the Mediterranean Sea and thus considered as a potential threat for the ecological well-being of the autochthonous species and ecosystem, the use of this species as bioremediator may allow to transform a potential risk into a benefit with high potential commercial gain and economic feasibility. Acknowledgement Financial support was provided by the RITMARE Flagship Project both funded by the Italian Ministry of University and Research. References Aguado-Giménez, F., Marín, A., Montoya, S., Marín-Guirao, L., Piedecausa, A., GarcíaGarcía, B., 2007. Comparison between some procedures for monitoring offshore cage culture in western Mediterranean Sea sampling methods and impact indicators insoft substrata. Aquaculture 271, 357–370. Arias, A., Giangrande, A., Gambi, M.C., Anadón, N., 2013. Biology and new records of the invasive species Branchiomma bairdi (Annelida: Sabellidae) in the Mediterranean Sea. Mediterranean Mar. Sci. 14 (1), 162–171. Bax, N., Williamson, A., Aguero, M., Gonzalez, E., Geeves, W., 2003. Marine invasive alien species: a threat to global biodiversity. Mar. Policy 27, 313–323. Bedwell, M.S., Goulde, R., 1997. Increase in specific growth rate of suspended bacteria through ponds and tanks used in intensive fish farming. Lett. Appl. Microbiol. 25, 212–214. Bianchi, C.N., 1983. Serpuloidea (Annelida, Polychaeta) delle lagune costiere laziali e campane. Annali Museo Civico di Storia Naturale di Genova 84, 231–243. Canuel, E.A., Lerberg, E.J., Dickhut, R.M., Keuhl, S.A., Bianchi, T.S., Wakeham, S.G., 2009. Changes in sediment and organic carbon accumulation in a highly

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