RESEARCH PAPER New Biotechnology

Volume 27, Number 6

December 2010
Bioremediation of bacteria in aquaculture
waste using the polychaete
Sabella spallanzanii
Loredana Stabili
1,2
, Roberto Schirosi
1
, Margherita Licciano
1
, Emanuela Mola
3
and
Adriana Giangrande
1
1
Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Universita` del Salento, Italy
2
Istituto per lAmbiente Marino Costiero, Sezione di Taranto, CNR, Italy
3
Orovivo dellAdriatico, Brindisi, Italy
The excessive release of bacterial pathogens from animal waste into the aquaculture environment has
become a major concern for the aquaculture industry. The biological ltration by macroinvertebrates
contributes to water purication as a result of the bacterioplankton removal from the water. The lter-
feeder polychaete Sabella spallanzanii is known for its ability to accumulate bacteria from the marine
environment. In the present study we evaluated the survival, growth and capability of this species to
remove several bacterial groups from aquaculture waste in order to ascertain its employment as
bioremediator in a farming scenario coupled with the conversion of the wastes into polychaete protein-
rich biomass of potentially marketable value. In comparison to other technologies, the employment of
S. spallanzanii in waste treatment represents a more attractive option to reduce bacterial loads.
Introduction
Aquaculture provides over a quarter of the worlds seafood supply,
a percentage that will increase as aquaculture expands and the
worlds conventional sh catch continues to decline because of
overshing and environmental damage [1]. As currently practiced,
however, aquaculture also causes environmental damage, raising
questions about how best to meet food demands and preserving
environmental quality for the sustainable development of aqua-
culture. There is a general consensus that closed recirculating
aquaculture has the potential to reduce impact on the aquatic
environment and several trials have been conducted to develop
intensive production systems [2].
In most sh culture systems, an amount of metabolic by-pro-
ducts, residual food, faecal matter and residues of prophylactic and
therapeutic inputs, are discharged without treatment, thus leading
to the deterioration of water quality and disease outbreaks [310].
Excessive release of microbial pathogens such as bacteria, viruses
and protozoans from animal waste into the aquaculture environ-
ment has become a major concern for the aquaculture industry,
with increasing aquaculture practices [11]. There are at least two
major consequences with increasing bacterial pathogen loads in
aquaculture waters. The rst one is that pathogenic bacteria repre-
sent a signicant health hazard to the reared species causing
aquaculture diseases [12]. Many important aquaculture species
including oysters, shrimps and scallops have been infected widely.
The second consequence is that the seafood products severely
contaminated by these pathogen bacteria represent a bio-hazard
to human health as a result of human consumption [12]. Most
bacterial disease agents are part of the normal ora of the water
and can cause disease only when the reared species are stressed
owing to poor environmental conditions, inadequate diet and
poor husbandry techniques. Many, but not all, of these infections
are caused by Vibrio spp. [13,14]. Several reports consider disease
outbreaks as a signicant constraint to the development of the
aquaculture sector, with a global estimate of disease losses in the
range of about US$ 3/4 billion per year [15].
In an attempt to control bacteria, prophylactic use of antibiotics
has become a frequently used strategy [16,17]. Antibiotics, which
have been used in large quantities, are in many cases ineffective, or
result in increases in virulence of pathogens and, furthermore, are
R
e
s
e
a
r
c
h
P
a
p
e
r
Corresponding author: Stabili, L. (stabili@iamc.cnr.it), (loredana.stabili@iamc.cnr.it)
774 www.elsevier.com/locate/nbt 1871-6784/$ - see front matter 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.nbt.2010.06.018
cause for concern in promoting transfer of antibiotic resistance to
human pathogens. Immunological intervention, particularly in
the form of vaccination, offers a nancially and environmentally
sustainable means of reducing the impact of infectious disease in
aquaculture. However, the development of effective and econom-
ically viable vaccination requires a detailed knowledge base of the
immune systems of the various species being farmed [18]. There-
fore, to make the aquaculture industry more sustainable, new
strategies to control infections or minimise the impact of patho-
gen bacteria in aquaculture ecosystemare urgently needed [19,20].
Probiotic technology has been proposed for solution to these
problems [21]. That is microbial species composition in hatchery
tanks or large aquaculture ponds can be changed by adding
selected bacterial species to displace deleterious normal bacteria.
Alternatively the reduction of microbial pollution within aqua-
culture plants may be achieved by the use of living organisms.
Specic examples in the literature reveal that some invertebrates
are potential remediators of heavy metals, microbial contami-
nants, hydrocarbons, nutrients and persistent organic pollutants
[22]. In particular, lter-feeding marine macroinvertebrates lter
large volumes of waters for their food requirements and exert high
efciency in retaining small particles including bacteria [23,24].
Many of these macroinvertebrates such as oysters, mussels, clams,
polychaetes and sponges, are suitable bioremediators of organic
and microbial polluted seawater and have the ability to generate
an economic return following remediation activities [20,2427].
The present paper deals with the reduction of the bacterial load
in an aquaculture farm using the lter-feeder polychaete Sabella
spallanzanii, one of the best known and widely distributed Med-
iterranean lter-feeding polychaete. Recent eld and laboratory
studies conducted on this species showed its ability to accumulate
and concentrate bacteria from the surrounding environment [28
30]. The aim of the present study is to provide evidence for the
capability of S. spallanzanii to remove several bacterial groups from
aquaculture waste in order to employ this polychaete as a potential
candidate for bioremediation in an aquaculture farming scenario.
In addition we evaluated the survival and growing capability of
these worms in an experimental tank integrated to an aquaculture
farm to ascertain the advantage of the conversion of the wastes
into polychaete biomass that can be removed and potentially
managed as a valuable by-product.
Materials and methods
Sample collection
In November 2007 (T
0
), adult specimens of S. spallanzanii were
collected by hand using SCUBA equipment (depth range = 5
15 m) from the Gulf of Taranto (Italy) and transferred to the
aquaculture plant Orovivo dellAdriatico located in Brindisi
(Italy). Here the worms were randomly divided in two sets each
consisting of 1000 individuals. The rst set was immediately (T
0
)
utilised to evaluate the following parameters: body and tube wet
weight and dry weight, and tube length. The worms of the second
set were cleaned of any tube epibionts and inserted in a total of 40
nets eachcontaining 25 individuals. The nets were thensuspended
in an experimental recirculating aquaculture concrete tank
(treatment tank) receiving the wastes from a sh rearing tank
(Fig. 1A, B). Animals were acclimatised to these conditions for
2 months and hereafter we evaluated their survival, and growing
capability. Another experimental recirculating aquaculture con-
crete tank without polychaetes (control tank) receiving the wastes
from the same sh rearing tank, was equipped and maintained at
the same conditions of the treatment tank. Both control and
treatment tanks had a rectangular shape with a length of 16 m,
a width of 6 mfor a total surface of 96 m
2
and a capacity of 240 m
3
.
The owrate in both the tanks was 0.8 L/s. In order to evaluate the
bacterial removal by the lter-feeding polychaetes under static
water conditions at each sampling time, seawater samples were
collected 48 h after the recirculating ow system was stopped.
After the collection, seawater samples were transferred to the
laboratory on ice for enumeration of bacteria within 4 h of sam-
pling.
In the control and treatment tanks we estimated several bacter-
ial groups densities at two different times: January 2008 (T
1
) and
July 2008 (T
2
), whenthe water temperature reached the lowest and
the highest annual value respectively. Temperature and dissolved
oxygen in the tank system were measured in situ using a multi-
parametric sounding-line Ocean Seven 401, Jolzonant, Italy. At
each sampling time, seawater samples (n = 3) were aseptically
New Biotechnology

Volume 27, Number 6

December 2010 RESEARCH PAPER

FIGURE 1
(A) Experimental apparatus in the aquaculture plant showing the three
sampling points: P1 (waste in-ow from the sh rearing, i.e. initial seawater),
P2 (waste ow, i.e. treated seawater) and P3 (waste ow, i.e. untreated
seawater); (B) Sabella spallanzanii; (C) tubes of S. spallanzanii. The arrows
indicate the increasing in tube length from T
0
(November 2007) to T
2
(July
2008) in the aquaculture system.
www.elsevier.com/locate/nbt 775
R
e
s
e
a
r
c
h
P
a
p
e
r
collected using 5-L Niskin bottles at the tank P1 (waste in-ow
fromthe sh rearing, i.e. initial seawater); at the treatment tank P2
(waste ow with polychaetes, i.e. treated seawater) and at the
control tank P3 (waste ow without polychaetes, i.e. untreated
seawater) (Fig. 1A). According to Karunasagar and Otta [31], before
the beginning of the experimental procedure, physical removal of
biolms on pipes/tank surfaces was assured with a preliminary
drying to reduce the presence of pathogenic bacteria inthe system.
Bacteriological analyses
The bacteriological analyses were performed on the waste samples
collected at the three sampling points in the two sampling times.
Bacteriological analyses included the quantitative analyses of
culturable heterotrophic bacteria (22 8C), total culturable bacteria
at 37 8C, culturable halophilic vibrios at 22 and 35 8C, faecal and
total coliforms as well as faecal streptococci.
Inorder to enumerate the culturable vibrios inseawater, 1, 5 and
10 mL of seawater were ltered on 0.22 mm pore size lters; then
the lter disks were aseptically placed onto triosulfatecitrate
bilesucrosesalt agar (TCBS) plus 2% NaCl, recommended for
culturing vibrios. Incubation was carried out at 2025 8C and
35 8C for two days and the colonies of vibrios were counted
according to the colony-forming unit (CFU) method. The incuba-
tion temperature of 35 8C was chosen to estimate the fraction of
vibrios potentially pathogenic to humans. The incubation tem-
perature of 2025 8C was chosen since some Vibrio spp., such as V.
anguillarum, do not grow well at higher temperature [32]. Results
were referred as mean value recorded at 22 and 35 8C.
For enumeration of heterotrophic bacteria, the number of CFU
was determined by plating 100 mL of undiluted seawater and serial
dilutions of each water sample in triplicates on Bacto Marine Agar
2216 (Difco), recommended for culturing heterotrophic marine
bacteria. The plates were incubated at 22 8C for 7 days. At the end
of the incubation period all colonies were counted in terms of
colony-forming unit (CFU) method through a 10 magnication
lens.
Total culturable bacterial densities growing at 37 8C, indicating
the fraction of bacterial potentially pathogenic to humans, were
determined by plating 100 mL of undiluted seawater and serial
dilutions of each water sample in triplicates on Bacto Plate Count
agar (Difco) recommended for culturing this kind of bacteria. The
plates were incubated at 37 8C for 48 h.
For all the microbiological parameters counted according to the
CFU method, bacterial densities were expressed as CFU mL
1
.
Total and faecal coliforms as well as faecal streptococci were
determined by the most probable number (MPN) method using
the standard ve-tube method of 10-fold dilutions [33]. For total
and faecal coliform determination, lactose broth and brilliant-
green-lactose brothwere used as cultural media inthe presumptive
and conrmative test, respectively. For faecal streptococci the
presumptive test was performed using Azide broth and the con-
rmative one using Ethyl violet azide broth. Results were referred
as MPN/100 mL.
Estimation of growing capability and survival
An estimation of the growth capability of S. spallanzanii was
obtained by measuring the following parameters: the tube length,
the wet and dry weight of the body including the crown, the wet
and dry weight of the tube. In particular, the tube length of each
worm was estimated by a scale correct to within 0.5 mm at T
0
, T
1
and T
2
. The wet and dry weight of either the body including the
crown or the tube of each wormwere measured at the beginning of
the experimental period (T
0
) as well as at the end (T
2
). The wet
weights were measured on an analytical balance as well as the dry
weights after drying the worms at 60 8C for 48 h.
During the waste exposure the worms were checked periodically
for mortality. The criteria for death were the absence of crown
reexes, when this structure was withdrawn out of the tube and
the worms were prodded, and/or the emptiness of the tube. More-
over, at T
2
all test organisms were examined under a stereomicro-
scope in order to detect possible morphological modications.
Statistical analysis
Analysis of variance (ANOVA) was used to assess differences in the
mean abundance of the bacteriological groups among the three
sampling points in January and July. The experimental design
consisted of two factors: Time (T), xed with two levels (i.e. T
1
and
T
2
) and Sampling Points (P), xed with three levels (i.e. P1, P2 and
P3) and crossed to Time. Before analysis, the homogeneity of
variance was tested using Cochrans test and, if necessary, data
were transformed to remove heterostochasticity. The Student
NewmanKeuls test (SNK) was used for post hoc comparisons
among means.
ANOVA-1-way was used to test for differences in the mean tube
length, wet and dry weight of the body including the crown, and
wet and dry weight of the tube measured at T
0
, T
1
and T
2
.
All the analyses were done using GMAV5 computer programme
(University of Sidney, Australia).
Results
Abiotic parameters
The mean temperature in the tank systemat T
1
was 13.7 0.12 8C
and 22 0.13 8C at T
2
. The mean dissolved oxygen was
8.5 0.2 mg/L at T
1
, and 8 0.15 at T
2
.
Removal of bacteria
Culturable heterotrophic bacteria, culturable Vibrio spp., cultur-
able bacteria at 37 8C and bacterial pollution indicators were
enumerated in the waste collected from the three sampling points
in the two sampling times.
The trends of culturable heterotrophic bacterial (22 8C) abun-
dance in T
1
(January) and T
2
(July) are reported in Figure 2. In T
1
the mean bacterial density at P1 was 6.6 0.7 10
4
CFU/mL and
it decreased signicantly (p < 0.01) in the presence of worms
reaching a value of 2.1 0.18 10
4
CFU/mL at P2. By contrast,
in the absence of polychaetes bacterial abundance did not vary
signicantly at P3 in comparison to P1 (Table 1). In T
2
a similar
trend was observed indeed bacterial abundance was one order of
magnitude higher at P1 (1.2 0.06 10
6
CFU/mL) than at P2
(2.0 0.1 10
5
CFU/mL) (p < 0.01), whilst it did not signicantly
increase at P3 (9.0 0.13 10
5
CFU/mL). For each sampling
point, analysis of variance revealed signicant differences in the
bacterial abundances recorded at T
1
and T
2
(Table 1).
As regards culturable vibrios (Fig. 3) the lowest meandensities in
the waste were observed in T
1
at P1, P2 and P3. In both the
sampling periods S. spallanzanii concentrated vibrios, removing
RESEARCH PAPER New Biotechnology

Volume 27, Number 6

December 2010
776 www.elsevier.com/locate/nbt
R
e
s
e
a
r
c
h
P
a
p
e
r
themfromthe surrounding water as inferred fromthe comparison
of the bacterial abundances between P1 and P2. Indeed vibrios
concentrations, which at P1 were 6.0 0.06 10
2
CFU/mL and
1.3 0.15 10
3
CFU/mL in T
1
and T
2
respectively, dropped dras-
tically at P2 in both the times. The post hoc comparison among
means revealed that in both the sampling times vibrios were
signicantly more concentrated in the initial seawater in compar-
ison to the treated seawater (p < 0.01). By contrast, in both the
sampling periods no signicant differences in vibrios concentra-
tion were found between initial and untreated seawater (Table 1).
Figure 4 reports the densities of total culturable bacteria at 37 8C
at the three sampling points in the two sampling times. In com-
parison to P2 and as already observed for culturable heterotrophic
bacteria and vibrios, the highest densities of this bacterial group
were observed at P1 with mean values of 2.8 0.075 10
3
and 3.0 0.09 10
3
CFU/mL in T
1
and T
2
respectively. High
abundances were also recorded at P3 (2.2 0.3 10
3
and
2.6 0.07 10
3
CFU/mL at T
1
and T
2
respectively). Analysis of
variance performed on the abundance of this bacterial group,
showed a signicant T P interaction (p < 0.001). The SNK
test revealed that in both the sampling periods no signicant
differences in total culturable bacteria at 37 8Cconcentration were
found between initial and untreated seawater (Table 1).
Total and faecal coliforms concentration trends were similar
(Fig. 5A, B) and in both cases bacterial densities were higher in the
initial seawater than in the treated seawater. Particularly, at P1
total coliforms were 17 2.6 MPN/100 mL in T
1
with a signicant
increase in T
2
(33 2.6 MPN/100 mL) whereas these indicators
were not detected at P2 in both the sampling times. In the control
tank total coliforms densities were 14 3.9 and 27 7.8 MPN/
100 mL in T
1
and T
2
respectively. As regards faecal coliforms their
abundances in the initial seawater were 4 1 MPN/100 mL and
33 2.6 MPN/100 mL in T
1
and T
2
respectively. Also these indi-
cators were absent at P2 but still detectable at P3 in both the
sampling times.
Regarding to faecal streptococci concentrations (Fig. 5C), their
densities accounted to 0 MPN/100 mL at P2 in T
1
. In T
2
the values
recorded signicantly differed among P1 and P2 (p < 0.01) with
densities of 70 3.6, 2 1 and 6 2 MPN/100 mL respectively.
Faecal streptococci densities in the initial seawater did not vary
signicantly in comparison to the untreated seawater (Table 1)
where these bacteria attained values of 2 and 63 10.4 MPN/
100 mL in T
1
and T
2
respectively.
Growing capability and survival
The tube length signicantly increased (p < 0.001) from a mean
value of 10.8 2.06 cm in T
0
, to 19.6 2.65 cm in T
2
(Fig. 6).
Thus, the total mean increase of length was 8.8 1.35 cm corre-
sponding to a mean increase of 0.33 mm/day (Fig. 1C). In T
0
the
mean wet weight of the body including the crown was
0.78 0.36 g and in T
2
achieved the value of 2.1 0.6 g signi-
cantly different from the former (p < 0.001) (Fig. 7A). By dry
weight the body signicantly increased from 0.32 0.11 g (T
0
)
to 0.53 0.18 g (T
2
) (p < 0.001) (Fig. 7B). A signicant increase
was also recorded for the wet weight of the tube as well as for the
dry weight (p < 0.001). From these results we can infer that the
total mean dry weight (tube and body including the crown)
increased of 0.68 0.41 g and the total mean wet weight increased
of 2.48 0.62 g corresponding to a mean increase of total poly-
chaete biomass of 9 mg/day.
New Biotechnology

Volume 27, Number 6

December 2010 RESEARCH PAPER

FIGURE 2
Mean abundance (standard deviations) of culturable heterotrophic bacteria
at 22 8C in T
1
and T
2
at the three sampling points: at P1 (waste in-ow from
the sh rearing, i.e. initial seawater), P2 (waste ow, i.e. treated seawater) and
P3 (waste ow, i.e. untreated seawater).

FIGURE 4
Mean abundance (standard deviations) of total culturable bacteria at 37 8C
in T
1
and T
2
at the three sampling points: at P1 (waste in-ow from the sh
rearing, i.e. initial seawater), P2 (waste ow, i.e. treated seawater) and P3
(waste ow, i.e. untreated seawater).

FIGURE 3
Mean abundance (standard deviations) of culturable vibrios in T
1
and T
2
at
the three sampling points: at P1 (waste in-ow from the sh rearing, i.e. initial
seawater), P2 (waste ow, i.e. treated seawater) and P3 (waste ow, i.e.
untreated seawater).
www.elsevier.com/locate/nbt 777
R
e
s
e
a
r
c
h
P
a
p
e
r
R
E
S
E
A
R
C
H
P
A
P
E
R
N
e
w
B
i
o
t
e
c
h
n
o
l
o
g
y

V
o
l
u
m
e
2
7
,
N
u
m
b
e
r
6

D
e
c
e
m
b
e
r
2
0
1
0
TABLE 1
Summaries of ANOVAs testing for differences in average bacterial abundances measured at T
1
(January) and T
2
(July) in waste samples collected fromthe three sampling points
Source of
variation
Heterotrophic
bacteria (22 8C)
Halophilic Vibrios
(22 and 35 8C)
Total culturable
bacteria (37 8C)
CT CF SF
df MS F p MS F p MS F p MS F p MS F p MS F p
Sampling
Points = P
1 33.1736 7360.38
***
1093080 3608.51
***
0.7381 246.78
***
420.5 20.51
***
6.628 252.44
***
106.149 1059.99
***
Time = T 2 4.1832 928.15
***
1747457 5768.76
***
5.0799 1698.42
***
1065.5 51.98
***
12.5781 479.07
***
19.9711 199.43
***
T x P 2 0.2447 54.29
***
254139 838.97
***
0.4068 136.01
***
108.5 5.29
*
1.6745 63.78
***
11.8068 117.9
***
Residual 12 0.0045 302.9172 0.003 20.5 0.0263 0.1001
Total 17
Cochrans test NS NS 0.7716 NS NS 0.6813
Transform Ln(x) None Ln(x) None Ln(x + 1) Sqrt(x + 1)
SNK test
T(P) P1 T
1
< P1 T
2
P1 T
1
< P1 T
2
P2 T
1
< P2 T
2
P1 T
1
< P1 T
2
P1 T
1
< P1 T
2
P1 T
1
< P1 T
2
P2 T
1
< P2 T
2
P3 T
1
< P3 T
2
P3 T
1
< P3 T
2
P3 T
1
< P3 T
2
P3 T
1
< P3 T
2
P2 T
1
< P2 T
2
P3 T
1
< P3 T
2
P3 T
1
< P3 T
2
P(T) P1 T
1
> P2 T
1
P1 T
1
> P2 T
1
P1 T
1
> P2 T
1
P1 T
1
> P2 T
1
P1 T
1
> P2 T
1
P1 T
1
> P2 T
1
P2 T
1
< P3 T
1
P2 T
1
< P3 T
1
P2 T
1
< P3 T
1
P2 T
1
< P3 T
1
P2 T
1
< P3 T
1
P2 T
1
< P3 T
1
P1 T
1
= P3 T
1
P1 T
1
= P3 T
1
P1 T
1
= P3 T
1
P1 T
1
= P3 T
1
P1 T
1
= P3 T
1
P1 T
1
= P3 T
1
P1 T
2
> P2 T
2
P1 T
2
> P2 T
2
P1 T
2
> P2 T
2
P1 T
2
> P2 T
2
P1 T
2
> P2 T
2
P1 T
2
> P2 T
2
P2 T
2
< P3 T
2
P2 T
2
< P3 T
2
P2 T
2
< P3 T
2
P2 T
2
< P3 T
2
P2 T
2
< P3 T
2
P2 T
2
< P3 T
2
P1 T
2
= P3 T
2
P1 T
2
= P3 T
2
P1 T
2
= P3 T
2
P1 T
2
= P3 T
2
P1 T
2
= P3 T
2
P1 T
2
= P3 T
2
Reported are CT total coliforms, CF faecal coliforms, SF faecal streptococci, P1 T
1
bacterial concentration measured at T
1
in samples from waste in-ow (initial seawater), P2 T
1
bacterial concentration measured at T
1
in samples from the waste
ow with polychaetes (treated seawater), P3 T
1
bacterial concentration measured at T
1
in samples from waste ow without polychaetes (untreated seawater), P1 T
2
bacterial concentration measured at T
2
in samples from waste in-ow (initial
seawater), P2 T
2
bacterial concentration measured at T
2
in samples from the waste ow (treated seawater), P3 T
2
bacterial concentration measured at T
2
in samples from waste ow (untreated seawater);
***
p < 0.001,
*
p < 0.05, NS not
signicant.
7
7
8
w
w
w
.
e
l
s
e
v
i
e
r
.
c
o
m
/
l
o
c
a
t
e
/
n
b
t
R e s e a r c h P a p e r
At the end of the experimental period (T
2
) mortality percentage
was less then 10%. All the survived worms, examined under a
stereomicroscope did not show any morphological modications.
Discussion
Withthe explosive expansion of aquaculture farming considerable
negative environmental impacts become obvious. FAO cautioned
that such an increase will mainly be restricted by two factors,
environmental impacts and aquaculture diseases. One of the most
prominent issues is that the water-borne bacterial pathogens
represent a signicant bio-hazard for both aquaculture species
and human health [12]. To prevent the pathogen-related aqua-
culture disease and the potential contamination of aquaculture
products by pathogens, bioremediation is an attractive option
with few negative impacts on the aquaculture ecosystems. In
particular, lter-feeders invertebrates on account of their capabil-
ity to remove pathogenic bacteria, through the ltration process,
have been recently proposed as potential bioremediators. The
present study represents a signicant advance on this topic since
for the rst time a lter-feeder was actually employed in an
integrated aquaculture system as a novel bioremediation technol-
ogy to reduce the bacterial concentration and restoring water
quality. We clearly demonstrated that the lter-feeder S. spallan-
zanii is able to reduce the bacterial abundance in the waste from a
recirculating aquaculture system.
The ltering activity of the investigated species was previously
studied in several works both from open sea and laboratory con-
ditions. Lemmens et al. [34] reported that S. spallanzanii inhabiting
the bare sediment in the Southern Flats of Cockburn Sound
(Australia), has a similar ltering capacity to the macro-lter-
New Biotechnology

Volume 27, Number 6

December 2010 RESEARCH PAPER

FIGURE 5
Mean abundance (standard deviations) of total coliforms (a), faecal
coliforms (b) and faecal streptococci (c) in T
1
and T
2
at the three sampling
points: at P1 (waste in-ow from the sh rearing, i.e. initial seawater), P2
(waste ow, i.e. treated seawater) and P3 (waste ow, i.e. untreated seawater).

FIGURE 6
Mean tube length (standard deviations) of S. spallanzanii measured in T
0
and at the treatment tank (P2) in T
1
and T
2
.

FIGURE 7
Mean body and tube wet weight of S. spallanzanii (standard deviations)
measured in T
0
, and at the treatment tank in T
1
and T
2
(a) and mean body and
tube dry weight of S. spallanzanii (standard deviations) measured in T
0
, T
1
and T
2
(b).
www.elsevier.com/locate/nbt 779
R
e
s
e
a
r
c
h
P
a
p
e
r
feeder community inhabiting Posidonia sinuosa meadows. Filtering
capacity of the habitat refers to the ltering rates of the animals
combined with ecological data on the biomass of the main sus-
pension-feeders in a marine habitat. These authors estimated a
mean ltering capacity of about 12 m
3
d
1
m
2
habitat. Recent
eld and laboratory studies conducted in Mediterranean area
showed the ability of this polychaete to accumulate and concen-
trate bacteria from the surrounding environment with a higher
efciency for autochthonous bacteria in oligotrophic conditions
as well as in eutrophic ones [28]. Further laboratory experiments
carried out on this species demonstrated that bacteria, employed
as only food source, were efciently ltered and retained [29,30].
Moreover the lter activity of this polychaete can also promote the
transfer of organic matter from the water column to the sediment
as faeces and pseudofaeces and, contrary to what is observed for
mussels in the marine environment [27,35], organic matter can be
denitively removed from the system being also compacted with
the mucus during the tube building by worms.
Present experiments showed that S. spallanzanii is able to lter,
accumulate and remove fromthe waste all the considered bacterial
groups, including human potential pathogens and vibrios.
The highest values of heterotrophic bacterial abundances were
observed in T
2
in all the three sampling points. The effect of
temperature on bacterial density was already assessed by Pomeroy
and Wiebe [36]. A similar explanation can be also suggested for
vibrios density. Also concerning this bacterial group the highest
values were indeed reported in July in the initial seawater (waste
in-owing). These results are in agreement with previous ndings
indicating that several ecological parameters, including the tem-
perature, inuence the presence of different Vibrio species in the
seawater [37,38]. However, owing to its high ltering capability, S.
spallanzanii concentrated both heterotrophic bacteria and vibrios
and this was reected in the reduced bacterial abundances in their
surrounding environment in comparison to those recorded in the
waste in-owing the system. Thus, our results demonstrated that
the polychaete S. spallanzanii can effectively control the growth of
potential pathogenic bacteria in the waste. The removal of vibrios
from their surrounding waste is of particular interest taking into
account that among different pathogens in aquaculture waters,
Vibrio spp. are key pathogens owing to their extreme infectivity to
aquaculture animals and humans [14,39]. Also the control of
heterotrophic bacteria in the waste by S. spallanzanii represents
an interesting issue since the majority of bacteria causing disease
in marine sh are opportunistic pathogens that are present as part
of the normal seawater microora.
The employment of S. spallanzanii in aquaculture waste treat-
ment represents a more attractive option to reduce bacterial loads
in comparison to other technologies with high negative impacts
on the aquaculture ecosystems. In particular, antibiotics-supple-
mented feeds are commonly used in farms [26]. The massive use of
antibiotics to control infections in aquaculture has resulted in the
development of resistant strains, which have rendered antibiotic
treatments ineffective.
In addition, the results obtained on the growing capability of S.
spallanzanii in the treatment tank clearly showed an increasing in
biomass during the experimental period. In particular, we
obtained a mean increase of total polychaete biomass of 9 mg/
day in dry weight and a mean increase of 0.33 mm/day in length.
Moreover, at the end of the experimental period, a mortality
percentage less then 10% was recorded.
The use of S. spallanzanii as bioremediator in appropriate envir-
onments is attractive owing to both its effectiveness as bioreme-
diator of microbial pollution and the potential market value of its
biomass. We indeed estimated that 5.5 g (wet weight) of S. spal-
lanzanii remediate from a microbiological point of view 1 m
3
of
waste; moreover, the worm tissues can be utilised for sh con-
sumption on account of their high protein content compared to
other marine invertebrates (personal observation). Although
viable bacteria present in the polychaetes and the tubes, as well
as the levels of other bio-hazards (e.g. viruses, protozoans and
algae) were not measured to assess the marketable value of the
biomass obtained, our results are consistent with the possibility of
rearing this species in an aquaculture plant suggesting a novel
application of an animal system of bioremediation with high
potential commercial gain. Thus, we propose an aquaculture
farm model ecologically and economically self-sustaining where,
in one embodiment, it contains sh cultivation and polychaete
rearing.
Future studies should be aimed at investigating the effects of
bioaccumulation of microbial pollutants in S. spallanzanii and
determine the tolerance limits for the selected species, as well as
the detection of bio-hazards in polychaete tissues. The investiga-
tion of the capacity for these worms to utilise coliform bacteria
may also lead to new realms of bioremediation for coastal com-
munities.
Acknowledgements
Financial support was provided by the Project ACTIBIOMAR
(www.actibiomar.it <http://www.actibiomar.it>) granted by
Apulian Region.
References
1 Tidwell, J.H. and Allan, G.L. (2001) Fish as food: aquacultures contribution. EMBO
Rep. 2 (11), 958963
2 Van Rijn, J. (1996) The potential for integrated biological treatment systems in
recirculating sh culture a review. Aquaculture 139, 181201
3 Hyland, J. et al. (2005) Organic carbon content of sediments as an indicator of
stress in the marine benthos. Mar. Ecol. Progr. Ser. 295, 91103
4 Edgar, G.J. et al. (2005) Broadscale effects of marine salmonids aquaculture on
macrobenthos and the sediment environment in southeastern Tasmania. J. Exp.
Mar. Biol. Ecol. 327, 7090
5 Focardi, S. et al. (2005) Safety issues and sustainable development of European
aquaculture: new tools for environmentally sound aquaculture. Aquacult. Int. 13,
317
6 Zaccone, R. et al. (2005) Microbiological indicators for aquaculture impact in Mar
Piccolo (Taranto, Italy). Aquacult. Int. 13, 167173
7 Lee, H.W. et al. (2006) Temporal changes in the polychaete infaunal
community surrounding a Hawaiian mariculture operation. Mar. Ecol. Prog. Ser.
307, 175185
8 Yokoyama, H. et al. (2006) Quantifying aquaculture derived organic matter in the
sediment in and around a coastal sh farm using stable carbon and nitrogen
isotope ratios. Aquaculture 254, 411425
9 Mallet, A.L. et al. (2006) Impact of suspendedand off-bottomEasternoyster culture
on the benthic environment in eastern Canada. Aquaculture 255, 362373
10 Aguado-Gimenez, F. et al. (2007) Comparison between some procedures
for monitoring offshore cage culture in western Mediterranean Sea:
RESEARCH PAPER New Biotechnology

Volume 27, Number 6

December 2010
780 www.elsevier.com/locate/nbt
R
e
s
e
a
r
c
h
P
a
p
e
r
sampling methods and impact indicators in soft substrata. Aquaculture 271,
357370
11 Gifford, S. (2004) Pearl aquaculture protable environmental remediation? Sci.
Total Environ. 319, 2737
12 Reilly, A. and Kaferstein, F. (1997) Food safety hazards and the application of the
principles of the hazard analysis and critical control point (HACCP) system for
their control in aquaculture production. Aquacult. Res. 28, 735752
13 Cavallo, R.A. and Stabili, L. (2004) Culturable vibrios biodiversity in the Northern
Ionian Sea (Italian coasts). Sci. Mar. 68 (1), 2329
14 Zampini, M. et al. (2005) Vibrio cholerae persistence in aquatic environments and
colonization of intestinal cells: involvement of a common adhesion mechanism.
FEMS Microbiol. Lett. 244, 267273
15 Bondad-Reantaso, M.G. et al. (2005) Disease and health management in Asian
aquaculture. Vet. Parasitol. 132, 249272
16 Gatesoupe, F.J. (2002) Probiotic and formaldehyde treatment of Artemia
nauplii as food for larval pollack, Pollachius pollachius. Aquaculture 212, 347
360
17 Defoirdt, T. et al. (2007) The natural furanone (5Z)-4-bromo-5(bromomethylene)-
3-butyl-2(5H)-furanone disrupts quorum sensing-regulated gene expression in
Vibrio harveyi by decreasing the DNA-binding activity of the transcriptional
regulator protein LuxR. Environ. Microbiol. 9, 24862495
18 Jrgensen, J.B. et al. (2001) CpG oligodeoxynucleotides and plasmid DNA
stimulates Atlantic salmon leucocytes to produce supernatants with antiviral
activity. Dev. Comp. Immunol. 25, 313321
19 Defoirdt, T. et al. (2007) Alternatives to antibiotics to control bacterial infections:
luminescent vibriosis in aquaculture as an example. Trends Biotechnol. 25 (10),
472479
20 Milanese, M. et al. (2003) The marine sponge Chondrilla nucula Schmidt, 1862 as an
elective candidate for bioremediation in integrated aquaculture. Biomol. Eng. 20,
363368
21 Moriarty, D.J.W. (1998) Control of luminous Vibrio species in penaeid aquaculture
ponds. Aquatic zooremediation: deploying animals to remediate contaminated
aquatic environments. Aquaculture 164, 351358
22 Gifford, S. et al. (2006) Aquatic zooremediation: deploying animals to remediate
contaminated aquatic environments. Trends Biotechnol. 25 (2), 6065
23 Riisgard, H.U. and Larsen, P.S. (1995) Filter-feeding inmarine macro-invertebrates:
pump characteristics, modelling and energy cost. Biol. Rev. Camb. Philos. Soc. 70,
67106
24 Ostroumov, S. (2005) Some aspects of water ltering activity of lter feeders.
Hydrobiologia 542, 275286
25 Giangrande, A. et al. (2005) Utilization of the lter feeder Sabella spallanzanii as
bioremediator in aquaculture. Aquacult. Int. 13, 129136
26 Fu, W. et al. (2006) Potential of the marine sponge Hymeniacidon perleve as a
bioremediator of pathogenic bacteria in integrated aquaculture ecosystems.
Biotechnol. Bioeng. 93 (6), 11121122
27 Ostroumov, S. and Widdows, J. (2006) Inhibition of mussel suspension feeding by
surfactants of three classes. Hydrobiologia 556 (1), 381386
28 Stabili, L. et al. (2006) Sabella spallanzanii lter-feeding on bacterial community:
ecological implications and applications. Mar. Environ. Res. 61 (1), 7492
29 Licciano, M. et al. (2005) Clearance rates of Sabella spallanzanii and Branchiomma
luctuosum(Annelida: Polychaeta) on a pure culture of Vibrio alginolyticus. Wat. Res.
39, 43754384
30 Licciano, M. et al. (2007) Filter-feeder macroinvertebrates as key players in
culturable bacteria biodiversity control: a case of study with Sabella spallanzanii
(Polychaeta: Sabellidae). Mar. Environ. Res. 64, 504513
31 Karunasagar, I. and Otta, S.K. (1996) Biolm formation by Vibrio harveyi on
surfaces. Aquaculture 140, 241245
32 Hi, L. et al. (1998) Occurrence of Vibrio vulnicus biotypes in Danish marine
environments. Appl. Environ. Microb. 64 (1), 713
33 American Public Health Association (APHA), (1992) Standard Methods for the
Examination of Water and Wastewater (Marshall, R.T., ed.), American Public Health
Association (APHA), Washington, DC
34 Lemmens, J. et al. (1996) Filtering capacity of seagrass meadows and other habitats
of Cockburn Sound, Western Australia. Mar. Ecol. Prog. Ser. 143, 187200
35 Widdows, J. et al. (1998) Use of annular umes to determine the inuence of
current velocity and bivalves on material ux at the sedimentwater interface.
Estuar. Coast. 21, 552559
36 Pomeroy, L.R. and Wiebe, W.J. (2001) Temperature and substrates as interactive
limiting factors for marine heterotrophic bacteria. Aquat. Microb. Ecol. 23, 187204
37 Kaspar, C.W. and Tamplin, M.L. (1993) Effects of temperature and salinity on the
survival of Vibrio vulnicus in seawater and shellsh. Appl. Environ. Microbiol. 59,
24252429
38 Weichart, D. et al. (1992) Lowtemperature induced nonculturability and killing of
Vibrio vulnicus. FEMS Microbiol. Lett. 100, 205210
39 Stabili, L. et al. (2008) Epibiotic Vibrio luminous bacteria isolated from some
Hydrozoa and Bryozoa species. Microb. Ecol. 56, 625636
New Biotechnology

Volume 27, Number 6

December 2010 RESEARCH PAPER

www.elsevier.com/locate/nbt 781
R
e
s
e
a
r
c
h
P
a
p
e
r