Bacterial accumulation by the Demospongiae Hymeniacidon perlevis: A tool for the bioremediation of polluted seawater

Marine Pollution Bulletin 60 (2010) 1182–1187

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Bacterial accumulation by the Demospongiae Hymeniacidon perlevis: A tool for the bioremediation of polluted seawater C. Longo a, G. Corriero a, M. Licciano b, L. Stabili b,c,* a

Dipartimento di Biologia Animale ed Ambientale, Università di Bari, Via Orabona, 4-70125 Bari, Italy Disteba, Università del Salento, Via Prov. le Lecce-Monteroni, 73100 Lecce, Italy c IAMC, Sezione di Taranto–CNR, Via Roma, 3-70400 Taranto, Italy b

a r t i c l e

i n f o

Keywords: Hymeniacidon perlevis Microbial pollution Total coliforms Filter-feeders Water quality Water self-purification

a b s t r a c t Sponges can filter large amounts of water, which exerts an important grazing impact on free bacteria, an important component of the diet of sponges. We examined the accumulation of bacteria in the Demospongiae (Hymeniacidon perlevis). Analyses were performed on homogenates from unstarved and starved sponges in seawater from their sampling site (the Ionian Sea). Culturable heterotrophic bacteria (22 °C), total culturable bacteria (37 °C) and vibrios densities were measured on marine agar 2216, plate count agar and TCBS agar, respectively. Total and fecal coliforms, as well as fecal streptococci, were determined by the most probable number method (MPN). H. perlevis was able to accumulate all of the six microbiological groups. Bacterial groups differed in their resistance to digestion by H. perlevis. Our data suggest that H. perlevis may accumulate, remediate and metabolize bacteria and that they may be employed as a useful bioindicator and bioremediator. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Food in the water column ranges from detrital organic carbon (DOC) to larger forms of particulate organic carbon, including live carbon (bacteria, cyanobacteria, protozoa, phytoplankton and zooplankton) and DOC (Ribes et al., 1999). The uptake of food from the water column is a major trophic pathway in marine ecosystems. Filter-feeding organisms (hereafter referred to as filter-feeders) utilize the available food in the water column and play an important role in the bentho-pelagic coupling that occurs throughout the oceans (Doering and Oviatt, 1986; Gili and Coma, 1998; Widdows et al., 1998, 2000, 2004). Filter-feeders represent a large component of coastal marine ecosystems in terms of biomass and number of species. Quantitative data on the filtering activity of various benthic filter-feeders have been acquired (Alimov, 1981; Gutelmaher, 1986; Monakov, 1998; Ostroumov, 2005; Ostroumov and Widdows, 2006; Widdows and Donkin, 1992). In these studies, the filtration rates of benthic invertebrates were traditionally measured as the rate of suspended material removed over short time-periods from confined experimental vessels under static conditions. The particles that filter-feeders capture are dependent on the mechanisms by which water flow delivers particles to the ani* Corresponding author at: Disteba, Università del Salento, Via Prov. le LecceMonteroni, 73100 Lecce, Italy. E-mail address: [email protected] (L. Stabili). 0025-326X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2010.03.035

mals’ food-gathering apparatus (Cole et al., 1992). Most of the studies on filtration rates have been carried out on molluscan populations that dwell in soft bottom habitats while few studies have looked at the filtration rates of dense filter-feeder assemblages that reside on hard substrates (Licciano et al., 2007a; Milanese et al., 2003; Pile et al., 1996; Ribes et al., 1999; Stabili et al., 2005, 2006a, 2006b, 2008). Sponges are one of the major invertebrate groups that inhabit hard bottom communities. Sponges can filter large amounts of water (0.002–0.84 ml/s cm3 of sponge tissue) through their aquiferous canal system (Osinga et al., 1999; Reiswig, 1990; Vogel, 1977). Sponges can also retain a wide range of particulate matter (0.1–50 lm) to extract edible material, including organic matter, bacteria and phytoplankton (Larsen and Riisgård, 1994; Riisgård and Larsen, 1995; Simpson, 1984). Therefore, abundant sponge populations can have an important grazing impact on the habitats they reside in. The importance of free bacteria in the diet of sponges (Van de Vyver et al., 1990) and the ability of sponges to concentrate large numbers of microorganisms suggest that sponges could be effective in reducing bacterial abundance, including microbial pollution, caused by sewage in coastal areas (Gifford et al., 2006). Excessive release of microbial pathogens, such as bacteria, viruses, and protozoans that come from human and animal waste, has become a major concern with increasing coastal urbanization and aquaculture practices because of their potential impact on coastal marine environments (Gifford et al., 2004). There are at

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least two major consequences that would result from increasing the pathogen loads from human and animal sewage into marine environments. First, pathogenic bacteria represent a significant health hazard to aquatic species and can cause recurrent occurrences of marine diseases. Marine diseases are becoming a global problem and have caused huge economic losses for the aquaculture industry (Reilly and Käferstein, 1997). Second, seafood products are severely contaminated by pathogenic bacteria and represent a bio-hazard to human health through human consumption (Reilly and Käferstein, 1997). Milanese et al. (2003) and Stabili et al. (2008) reported that the marine sponges Chondrilla nucula and Spongia officinalis can successfully remove bacteria. In harbors and in semi-enclosed basins where bacteria reach high densities, the utilization of bacteria by sponges could represent an applicative tool for bioremediation purposes. Sponges, like other invertebrates including oysters, mussels, clams, and polychaetes, have simple life histories, are resistant to toxicity and have the ability to generate an economic return following remediation activities (Gifford et al., 2005; Giangrande et al., 2005). Sponges have a renowned filtering capacity (in large communities they can filter the overlying water column in as little as 24 h; Reiswig, 1974) and high particle retention rates (Pile et al., 1996; Milanese et al., 2003; Stabili et al., 2006b). Sponges can be economically valuable through their use as bath sponges (Stabili et al., 2006b) or through the production of their novel metabolites (e.g., cytotoxin latrunculin B) for pharmaceutical uses (Hadas et al., 2005). We focus here on the marine sponge Hymeniacidon perlevis (Montagu, 1818), a common intertidal demosponge found in the Mar Piccolo of Taranto (Northern Ionian Sea). The marine sponge, found often in semienclosed or enclosed basins and in polluted seawaters such as ports and harbors (Gaino et al., submitted for publication; Corriero et al., 2004, 2007; Xue et al., 2009), is able to survive for a relatively long time when exposed to air (Gaino et al., submitted for publication). H. perlevis can reach up to 30 cm in diameter and can range in color from orange to pale green. We provide evidence that H. perlevis can filter and concentrate several groups of bacteria. We also assess whether H. perlevis contributes substantially to the removal of bacteria from the water column through accumulating bacteria and through its digestion process and thus if H. perlevis is a potential candidate for bioremediation of seawater polluted by microbial accumulation.

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2. Materials and methods 2.1. Samples collection Sponge samples were collected from the Second Inlet of the Mar Piccolo of Taranto (Northern Ionian Sea; Fig. 1). Water samples were collected from the same sampling site with 5 L sterilized Niskin bottles and they were immediately utilized for the bacteriological analysis (T0). Immediately upon return to the laboratory, the sponges were cleaned of any epibionts and were randomly divided into two sets. Specimens from one set (27 individuals) were separated into nine groups of three individuals and were immediately (T0) utilized for the analysis (‘‘unstarved” condition). The second set of specimens (27 individuals) was divided into nine aquaria (each aquaria contained three individuals). Each aquarium was filled with filtered seawater (0.22 lm pore size Millipore™ filters), acclimatized in a temperature controlled room (22 °C) and analysed after 48 h (T1; ‘‘starved” condition).

2.2. Bacteriological analyses Bacteriological analyses were performed on seawater collected at T0 from the sponge sampling site on unstarved (T0) and starved (T1) sponges. Each group of three sponges (nine groups of starved and nine groups of unstarved sponges) was processed separately for the enumeration of bacteria. Bacteriological analyses included the quantitative analyses of culturable halophilic vibrios (22 and 35 °C), culturable heterotrophic bacteria (22 °C), total culturable bacteria (37 °C), fecal and total coliforms and fecal streptococci. Enumeration of the culturable vibrios in seawater was achieved by filtering 1, 5 and 10 ml of seawater on 0.22 lm pore size filters and aseptically placing the filter disks onto thiosulphate-citratebile salt-sucrose agar (TCBS) plus 2% NaCl. Incubation was carried out at 20–25 °C and 35 °C for 2 days and the colonies of presumptive vibrios were counted according to the colony-forming unit (CFU) method. The fraction of vibrios that were potentially pathogenic to humans was estimated with the incubation temperature of 35 °C. The incubation temperature of 20–25 °C was chosen because some Vibrio spp., such as Vibrio anguillarum, do not grow well at higher temperatures (Høi et al., 1998). Mean values for three

Fig. 1. Map of the Mar Piccolo of Taranto (Ionian Sea). The arrow indicates the sampling point.

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replicate samples were determined. Each sponge sample was gently squeezed with a glass stick and the sponge pieces were homogenized for 9 s in a sterile WaringÒ blender. The homogenates (ca. 100 g) were filtered through sterile gauze and subjected to a series of dilutions (10 1, 10 2, 10 3 and 10 4). For the vibrios quantitative analysis 0.1 ml of each sponge homogenate or an appropriate decimal dilution of the homogenate (a sterilized seawater sample from the collection area was used for the dilution) was plated onto the TCBS agar. After incubating for 2 days, the culturable vibrios were counted according to the CFU method. Our results are the mean value recorded at 22 and 35 °C. Final bacterial concentrations were obtained taking into account the dilution factors. For the enumeration of heterotrophic bacteria, the number of CFU was determined by plating 100 lL of undiluted and serial dilutions of seawater, as well as sponge extract, in triplicate on Bacto marine agar 2216 (Difco). The plates were incubated at 22 °C for 7 days. All of the colonies were counted using the CFU method through a 10 magnification lens at the end of the incubation period. The total culturable bacterial densities that grew at 37 °C (indicating the fraction of bacteria potentially pathogenic to humans) were determined by the same methods previously described for the heterotrophic bacteria; however, a plate count agar (PCA) was used as the culture medium. For each microbiological parameter counted according to the CFU method, bacterial densities were expressed as CFU ml 1 for seawater and CFU g 1 (wet weight) for sponge samples. Total and fecal coliforms as well as fecal streptococci were determined by the most probable number (MPN) method using the standard five-tube method of 10-fold dilutions for seawater samples and the three-tube MPN series for sponge homogenates (APHA, 1998). For total and fecal coliform determination, lactose broth and brilliant-green-lactose broth were used as cultural media in the presumptive and confirmative tests, respectively. For fecal streptococci the presumptive test was performed using azide broth and the confirmative test was performed using ethyl violet azide broth. Results are reported as MPN 100 ml 1 or MPN 100 g 1 (wet weight) for water and sponge samples, respectively.

starved (T0) and starved (T1) sponge samples. The ANOVA was done using the GMAV 5 computer program (University of Sidney, Australia). 3. Results H. perlevis had a high capability of concentrating all six microbiological parameters considered with respect to the surrounding seawater. The densities of culturable vibrios growing at 22 and 35 °C detected in seawater at T0 and in unstarved and starved H. perlevis homogenates are reported in Fig. 2. Starved sponges had the highest densities of culturable vibrios (mean value of 3.9 ± 0.3  105 CFU g 1). Bacteria were more concentrated in unstarved sponges in comparison to the seawater (p < 0.01). In addition, the concentration of halophilic vibrios in unstarved sponges was lower than the concentration found in starved ones (p < 0.01). Culturable heterotrophic bacteria (22 °C) densities are shown in Fig. 3. The mean bacterial abundance was 2.1 ± 0.1  105 CFU ml 1 in seawater at T0, 3.5 ± 0.2  107 in unstarved sponges and 3.7 ± 0.2  107 CFU g 1 in the starved ones. Significant differences were found in bacterial concentrations between seawater and unstarved sponges at T0 (p < 0.01) as well as between starved and unstarved sponges (p < 0.01).

Seawater

1.00E+07 1.00E+06 1.00E+05 1.00E+04 1.00E+03 1.00E+02 1.00E+01 1.00E+00 T0

2.3. Statistical analysis Analysis of variance (ANOVA) was used to assess differences in the mean abundance of the bacteriological groups between the seawater and sponge samples at T0 as well as between the un-

Sponges

1.00E+08

CFU ml -1 CFU g-1

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T1

Fig. 3. Mean culturable heterotrophic bacteria abundance and relative standard deviation in seawater at T0 and sponge samples from the T0 (unstarved) and T1 (starved) conditions.

Seawater

Sponges

1.00E+06 Seawater

Sponges

1.00E+06

1.00E+05

CFU ml -1 CFU g -1

CFU ml -1 CFU g-1

1.00E+05 1.00E+04 1.00E+03

1.00E+04

1.00E+03

1.00E+02

1.00E+02

1.00E+01 1.00E+01

1.00E+00 1.00E+00

T0 TO

T1

T1

Fig. 2. Mean vibrios abundance and relative standard deviation in seawater at T0 and sponge samples from the T0 (unstarved) and T1 (starved) conditions.

Fig. 4. Mean total culturable bacteria at 37 °C abundance and relative standard deviation in seawater at T0 and sponge samples from the T0 (unstarved) and T1 (starved) conditions.

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The abundance of total culturable bacteria at 37 °C did not differ significantly in unstarved (6.5 ± 0.5  105 CFU g 1) or starved (6.4 ± 0.2  105 CFU g 1) H. perlevis samples (Fig. 4). Moreover, the mean bacterial concentrations measured in sponges at T0 were significantly higher (p < 0.01) than those measured in the surrounding seawater (9.6 ± 0.2  102 CFU ml 1). The accumulation capability of H. perlevis with respect to the classical microbial pollution indicators is reported in Fig. 5. At T0 the total coliform concentrations were 150 ± 14.1 MPN g 1 in the unstarved sponges and 9 ± 1.5 MPN ml 1 in seawater; fecal coliform concentrations were 110 ± 6.2 MPN g 1 in H. perlevis and 9 ± 1.2 MPN ml 1 in the seawater. Both the total and the fecal coliform concentrations found in the unstarved sponges were significantly higher than the concentrations found in the surrounding environment (p < 0.01). These indicators were not found in their homogenates after the sponges were starved. H. perlevis fecal streptococci concentrations (Fig. 6) were 2–3 orders of magnitude more dense in the sponge tissues than in the surrounding environment (p < 0.01). A significant decrease in bacterial density was detected in the starved sponges (420 ± 11.2 MPN g 1) in comparison to the unstarved ones (2100 ± 32.4 MPN g 1; p < 0.01).

CT seawater CF seawater

CT sponges CF sponges

180

MPN 100ml-1 MPN 100g -1

160 140 120 100 80 60 40 20 0 T0

Sponges

2500

MPN 100ml -1 MPN 100g-1

2000

1500

1000

500

0 T0

4. Discussion H. perlevis was able to accumulate all of the six microbiological groups we analysed in comparison with the surrounding environment. A similar microbiological accumulation capability has been demonstrated in other filter-feeding macroinvertebrates, including the Demospongiae S. officinalis (Stabili et al., 2008), the sabellid polychaetes Sabella spallanzanii (Stabili et al., 2006), and Branchiomma luctuosum (Licciano et al., 2007b) and the bivalve Mytilus galloprovincialis (Stabili et al., 2005). The impact that bacterial grazing has may be crucial especially in areas where H. perlevis could reach high densities and where bacteria constitute a major biomass component (e.g., coastal semi-enclosed or enclosed basins or in harbors). We found higher microbial concentrations inside the sponges than in the surrounding environment, which suggests that H. perlevis could act as a useful bioindicator; the dilution of microbial pollutants in coastal environments can make their direct measurement difficult. The European Water Framework Directive (WFD, 2000/60/EC) urges that the status of the ecological quality of ecosystems be monitored based upon biological, hydro-morphological and physical–chemical criteria and under the premise of accurate simplicity. The selection of a bioindicator depends on both its sensitivity and its cost-effectiveness (Hodson, 2002). H. perlevis could be used in the biomonitoring of marine microbial pollution because it can filter large volumes of water, accumulate the microbial pollution indicators and survive in polluted environments. Indeed, our specimens of H. perlevis were collected from the Mar Piccolo of Taranto, a semi-enclosed basin subjected to strong anthropogenic pollution. Moreover, the clearance rates for H. perlevis (27.2–59.0 ml/h g 1 in wet weight; 226.7–491.7 ml/h g 1 in dry weight; Fu et al., 2006) are higher than those reported for other species of sponges (e.g., C. nucula, 0.2–1.4 ml/h cm3 living sponge; and S. officinalis, 210 ml/h g 1 in dry weight; Milanese et al., 2003; Stabili et al., 2006b). Future studies will be conducted to confirm the hypothesis that H. perlevis may be a useful bioindicator (e.g., the demonstration that the bacterial load in sponges has a robust relationship with environmental bacterial loadings and that this relationship is maintained in time and in space). We infer a selectivity in digesting bacteria indicating the following:

T1

Fig. 5. Mean abundance and relative standard deviation of total and fecal coliforms in seawater at T0 and sponge samples from the T0 (unstarved) and T1 (starved) conditions. CT = total coliforms; CF = fecal coliforms.

Seawater

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T1

Fig. 6. Mean abundance and relative standard deviation of fecal streptococci in seawater at T0 and sponge samples from the T0 (unstarved) and T1 (starved) conditions.

– no significant differences were found between starved and unstarved H. perlevis in their ability to accumulate total culturable bacteria at 37 °C. These bacteria do not find a suitable environment inside the sponge like they find in the filter-feeding polychaete B. luctuosum (Licciano et al., 2007b); – a significant increase in the total culturable heterotrophic bacteria (22 °C) and the Vibrio abundances was observed in starved sponges versus unstarved sponges. Such bacteria, being typically marine, could include some members of the sponge microflora. Thus, H. perlevis could represent an ecological reservoir for the survival and growth of those symbiotic bacteria. Indeed, several molecular and culture-based studies indicate that the microbial community in some sponges is distinct from that found in surrounding seawater, with sponge-specific microbes being reported in some instances (Hentschel et al., 2002; Taylor et al., 2004). The role of an ecological reservoir for heterotrophic bacteria and vibrios has been suggested for other marine invertebrates including mussels, crustaceans, polychaetes and cnidarians (Carli et al., 1993; Cavallo and Stabili, 2002; Covazzi Harriague et al., 2008; Kaysner et al., 1989; Kaspar and Tamplin, 1993; Hood and Winter, 1997; Licciano et al., 2007a, 2007b; Montanari et al., 1999; Stabili et al., 2006a; Wai et al., 1999). – significantly higher values of coliforms and fecal streptococci were found in unstarved sponges than in starved sponges indicating the ability of H. perlevis to digest these bacteria. The

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higher capability of H. perlevis to digest coliforms in comparison to fecal streptococci could be due to the different bacterial cell walls of these bacteria. In fact, it has been demonstrated that Gram positive bacteria may be more resistant than Gram negative bacteria to digestion by marine invertebrates (Plante and Shriver, 1998). The selectivity of microorganism digestion has been studied in several species of sponges and the bacterial adherence properties have been postulated to play a key role in the selective retention rate (Willenz and Van de Vyver, 1984; Van de Vyver et al., 1990). Recently Fu et al. (2006) in two independent laboratory experiments demonstrated that Escherichia coli and V. anguillarum II are retained by H. perlevis. Moreover, the digestion of these bacteria was assessed by confocal microscope observation of stained E. coli and V. anguillarum II cells fed to sponge archaeocytes. We observed a significant increase in vibrios abundance in starved sponges in comparison to the unstarved ones. This does not indicate the inability of H. perlevis to digest these bacteria since Fu et al. (2006) only studied the digestion of one Vibrio species; we examined all of the culturable halophilic vibrios. Presumably some vibrio species, such as V. anguillarum II, are digested by the sponge while others find a suitable environment for their survival and growth inside the sponge. We may hypothesize that the increased abundance of vibrios in starved sponges is due to the bacterial proliferation processes of some Vibrio species prevailing over the digestion processes of other Vibrio species. As our data are merely descriptive, we cannot suggest which mechanisms H. perlevis use to degrade the examined bacteria. More qualitative studies must be carried out to determine the exact mechanisms involved, including the phagocytosis of the bacterial groups we examined by the different sponge cellular types. Based on our results about coliform digestion, another applicative purpose could be the use of H. perlevis as a biofilter of sewage polluted by microbes. Total coliforms, directly released from human and animal sewage, give a general indication of the sanitary condition of water due to the potential presence of pathogens. Sewage may contain many different human enteric pathogens, which are frequently transmitted by ingestion or by contact with polluted water. Improved and technologically updated water treatment facilities are critically needed because those currently available function poorly and are characterized by an inadequate capacity to cope with the required level of treatment. Moreover, the overall objective of the WFD (2000/60/EC) is to achieve a ‘‘good ecological quality status” for all bodies of water (including inland surface waters, transitional or estuarine waters, coastal waters and ground-waters) by 2015. The recovery of the coastal marine environments is required because coastal waters are used for multiple purposes including domestic and industrial discharge of wastewaters, recreation and aquaculture, which all increase the rates of eutrophication and microbial pollution (Mendez and Comin, 2000). Likewise, the WFD urges for the development of tools that allow for the ecological quality status to be established. Bioremediation represents an innovative approach for the recovery of impacted ecosystems with few negative impacts on their ecology (Bragg et al., 1994; Vezzulli et al., 2004). Bioremediation is usually confined to processes employing microorganisms or plants; the use of animal systems as bioremediators has not been seriously considered (Baker and Herson, 1994). Nonetheless, recent examples suggest that some animal species are effective bioremediators of heavy metals, microbial contaminants, hydrocarbons, nutrients and persistent organic pollutants, particularly in aquatic environments (Gifford et al., 2006; Ostroumov, 2002). In particular, filter-feeder invertebrates have been recently proposed as bioremediators for restoring water quality based on their ability to remove pathogenic bacteria through their filtration process

(Fu et al., 2006, 2007; Ledda et al., 2008; Licciano et al., 2007b; Milanese et al., 2003; Ostroumov, 1998; Stabili et al., 2006a, 2006b, 2008). In previous studies (Stabili et al., 2006b, 2008), we suggested using S. officinalis for bioremediation of oligotrophic environments and off-shore fish farms. H. perlevis could be more useful in removing bacteria in eutrophic environments because of its ability to accumulate bacteria and tolerate high levels of microbial pollution. In addition, H. perlevis could be used as a bioremediator of microbial pollution in impacted semi-enclosed basins in the presence of aquaculture plants. Our results agree with the conceptual framework and theory presented by Ostroumov (2005), which emphasized the vital role filter-feeders have in improving water quality and in running water self-purification. The use of H. perlevis as a bioremediator in appropriate environments is attractive because their market value lies with the extraction of secondary metabolites from their tissues for pharmaceutical uses. Future studies that sample sponges from a variety of locations with different water bacterial loads are required to confirm the hypothesis that H. perlevis may accumulate, remediate and metabolize bacteria.

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