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A dead-end filtration method to removeparticle-associated pathogens in aquaculture systems
Songzhe Fu • Jiazheng Shen • Kang Chen •
Junling Tu • Ying Liu
Received: 7 September 2011 / Accepted: 18 December 2011 / Published online: 31 January 2012� Springer Science+Business Media B.V. 2012
Abstract To reduce the incidence of bacterial diseases in recirculating aquaculture
systems, 27 marine bacterial species were introduced into Instant Ocean maintained at
25�C. Those species were enumerated before and after filtration to evaluate the efficiency
of the filtration procedure. The effects of sari filter and nylon filter on the survival of sea
bass challenged with Vibrio alginolyticus were also determined. The results of laboratory
studies indicated that the ability to remove pathogens was typically 1–3 log orders. Above
90% Vibrio sp., i.e., which were attached to particles, were removed by either 20-lm nylon
net or four layers of sari. A 9.53% mortality of sea bass was reported in pilot filtration test
using sari material as an end filter, while this percentage increased to 33.35% in control
groups. It is concluded that a simple filtration procedure that involves the use of four-layer
sari material can reduce the numbers of pathogens attached to particles in aquaculture
system. The results of this study provide the basis for pathogen reductions in full-scale
facilities.
Keywords Filtration � Pathogens � Removal rate � Recirculating aquaculture systems �Sari material
AbbreviationsRAS Recirculating aquaculture system
UV Ultraviolet
POU Point of use
LB Luria-Bertani
IO Instant Ocean
TCBS Thiosulfate-citrate-bile salts-sucrose
CFU Colony-forming units
S. Fu (&) � J. TuNanchang Center for Disease Control and Prevention, No. 833 Lijing Road, Honggutan District,Nanchang 330038, Chinae-mail: [email protected]
J. Shen � K. Chen � Y. LiuInstitute of Oceanology, The Chinese Academy of Sciences, Qingdao 266071, China
123
Aquacult Int (2012) 20:657–672DOI 10.1007/s10499-011-9494-0
HB Heterotrophic bacteria
MA Marine agar 2216
LOSWM Low-organic seawater medium
CMCC China Microbiological Culture Collection
Introduction
Aquaculture is becoming the world’s fastest growing food production sector, with an
annual increase of about 10% (FAO 1997). The outbreak of infectious bacterial diseases in
aquaculture is a major concern in the industry. Among those pathogens, Vibrio sp. are the
most important bacterial pathogens of cultured animals. They are responsible for several
diseases, and mortalities up to 100% due to vibriosis have been reported (Karunasagar
et al. 1994). Due to the rapid biofilm formation ability, the persistence of Vibrio sp. in
aquatic ecosystems often leads to seasonal outbreaks in aquaculture systems. The high
density of fish production also had led to the development of pathogen removal techniques
in RAS. In the past, the removal of those pathogens relied mostly on the use of antibiotics.
This can deliver partial successes, but the massive use of antimicrobials often led to drug
resistance and the development of potentially transferable antibiotic resistance (Cabello
2006). Concern over the potential negative effects of antibiotics used in the aquaculture has
increased considerably. The development of other pathogen removal techniques, therefore,
is critical in RAS. Currently, pathogen treatment in RAS is approached in two ways:
physical removal processes (i.e., filtration) and chemical inactivation processes (i.e., UV
radiation or ozonation). UV radiation or ozonation requires installation of an ultraviolet
sterilizer or ozonator sterilizer unit on a water line to kill the bacteria when the water flows
through the unit. The use of ultraviolet irradiation for the sterilization of bacteria has been
studied previously (Hedrick et al. 2000; Oppenheimer et al. 1997). Oye and Rimstad
(2001) also found that UV inactivated three different fish pathogenic viruses from a fish
processing plant. Unfortunately, when it is exported to full-scale practice, however, this
application suffered from various drawbacks. Some authors doubted its effectiveness
(Spotte and Adams 1981). Because of low penetrating power, it was not effective against
the bacteria attached to particles. Islam et al. (2007) found that biofilm acts as a micro-
environment that provides shelter for plankton including Vibrio cholerae in the aquatic
environment. This structure promotes resistance to environmental stresses (such as UV
radiation) present in aquatic environments (Watnick and Kolter 2000). Therefore, this
method is not effective if the water system is recontaminated in the downstream or if the
bacteria come back since the biofilms in the plumbing are not destroyed. Removal pro-
cesses via coarse filters (e.g., gravel, sand) or other membrane filters are another approach
to reduce pathogen populations and gross turbidity (Elliott et al. 2008). This strategy
gained some success and improved the overall bacterial removal efficiency and reducing
the risk of introducing UV-shielded bacteria (Wanda et al. 2007; Arndt and Wagner 2003;
Liltved and Cripps 1999). Tilleya et al. (2002) also successfully constructed wetlands as
recirculation filters in large-scale shrimp aquaculture. However, such simple treatment
systems are rarely efficient by themselves to meet the high pathogen removal standards
after a long-time operation (e.g., clogging). Besides, solid accumulation also happened in
such large-sized fixed facility, resulting from heavy loading of organic matter and the
difficulty of back flushing. It is unlikely to be suitable for the filtration of suspended solids
658 Aquacult Int (2012) 20:657–672
123
for a long-time operation. Therefore, the demand for disposable filters at the POU is
increasing.
Recent studies suggest that improving drinking water quality at the POU is beneficial in
avoiding possible waterborne diseases (Fewtrell et al. 2005; Sobsey et al. 2008). Several
POU technologies that involve water filtration have become available during the past
decade. More recently, Colwell et al. (2003) and Huq et al. (2010) found that the plankton-
associated Vibrio sp. can be removed by a filter constructed from either nylon net or sari
material. In a five-year study, such simple filtration system protected villagers from cholera
in Matlab, Bangladesh. Simple filtration via sari gained some success if properly imple-
mented and maintained (Huq et al. 1996; Lea 2008). Takashima et al. (2004) also found the
reduction in numbers of bacteria in solutions incubated with the fibers. Therefore, we also
interested in assessing whether four layers of sari materials would be effective in removing
particle-associated pathogens since the rearing water contains a large amount of feces and
unconsumed feed.
At present, it is not clear whether Vibrio sp. is unique among heterotrophic bacteria, or
merely one of many functionally similar species (most of which are not human pathogens).
If other heterotrophic bacteria operate similarly to Vibrio sp., then they should be func-
tionally similar in the attachment of particles and removed by filters. The aim of this work
was to evaluate the possibility of end-point filtration as a tool to reduce the abundance of
pathogens, thereby improving aquatic animal health.
Materials and methods
Bacterial strains
Bacterial strains used in this study were isolated from a variety of sources and are listed in
Table 1. Standard strains were purchased from CMCC. Water samples were collected at
different sites, including the costal seawater from Qingdao, Yantai and Tianjin. The
indigenous bacteria isolated from water samples were identified previously (Fu et al. 2009;
Gao et al. 2011). Bacterial strains were stored in 10% (w/v) glycerol broth at -70�C.
Laboratory filtration experiments
Isolates (Table 1) were grown in 100 ml of LB broth in 250-ml flasks and incubated at
30�C on a shaker (150 rpm). Filtration experiments were conducted in accordance with the
description of Huq et al. (1996). After washing twice in sterile IO (Aquarium Systems,
Mentor, Ohio), the cells were resuspended in 250 ml of IO and incubated at 25�C for 18 h
to ensure starvation (Roszak and Colwell 1987). Starved cells were added to sterilized
rearing water to a final concentration of 105 CFU ml-1 and then were incubated at 25�C for
18 h to allow the attachment of bacterial cells to the particles. This step (namely starvation
step) ensured that bacteria cells attached to particles would be retained on the filter. To
remove large particles, 100 ml of rearing water was filtered through 180- and 20-lm nylon
nets (Millipore Corp., Bedford, MA) and four layers of sari net obtained in local market,
respectively. After filtration, each of the content of filtrate was suspended in 100 ml
phosphate buffered saline containing sterile glass beads (0.1 mm, BioSpec Products).
Then, each filtrate was resuspended and homogenized for 2–5 min to dislodge the attached
bacteria, and the homogenates were used for direct plating. All experiments were per-
formed independently in triplicate. Treatment efficacy was measured using removal rate:
Aquacult Int (2012) 20:657–672 659
123
(Cin - Cout)/Cin, where Cin is influent pathogen concentration and Cout is effluent pathogen
concentration.
Enumeration of bacteria attached to particles
Bacteria associated with surfaces of particles were determined before and after the fil-
tration. The procedures followed the guidelines of ISO 7218:2007 ‘Microbiology of food
and animal feeding stuffs—general requirements and guidance for microbiological
examinations’ (ISO 2007). For direct plate count analyses of Vibrio sp., serially diluted
samples were spread onto TCBS agar and incubated at 37�C for 24 h. For Staphylococcusaureus, samples were subjected to 10-fold dilution prior to spreading onto Barid-Parker
agar. For other strains isolated from ocean or estuary, the abundance of isolates was
obtained by pour-plating MA (Difco Laboratories, USA) which was incubated at 30�C for
72 h. Only statistically valid plates, those that have between 25 and 250 colonies per plate,
Table 1 Bacterial strains used in this study
Bacteria Family Class or phyluma Origin
Salmonella Braenderup H9812 Enterobacteriaceae GAM CMCC
Escherichia coli ATCC29522 Enterobacteriaceae GAM CMCC
Vibrio parahaemolyticus ATCC17802 Vibrionaceae GAM CMCC
Bacillus pumilus N3-6 Bacillaceae FIR Seawater
Bacillus aquimaris R-1 Bacillaceae FIR Seawater
Alteromonas marina WB-1 Alteromonadaceae GAM Seawater
Vibrio alginolyticus FS-2 Vibrionaceae GAM Seawater
Vibrio natriegens FS-1 Vibrionaceae GAM Seawater
Vibrio hispanicus 2-9 Vibrionaceae GAM Seawater
Pseudomonas pseudoalcaligenes 5-4 Pseudomonadaceae GAM Seawater
Alcaligenes faecalis SR-2 Alcaligenaceae BETA Seawater
Marinobacter sp.F6 Alteromonadaceae GAM Seawater
Pseudoalteromonas piscicida Y-1 Pseudoalteromonadaceae GAM Seawater
Staphylococcus aureus F15 Staphylococcaceae GAM Seawater
Serratia plymuthica WT-1 Enterobacteriaceae GAM Seawater
Cyclobacterium marinum HDY-7 Cyclobacteriaceae CFB Seawater
Microbacterium paraoxydans 5-2 Microbacteriaceae ACT Seawater
Vibrio cholerae NE-1 (Serogroup O1) Vibrionaceae GAM Estuary
Vibrio choleraeNE-9 (Serogroup O139) Vibrionaceae GAM Estuary
Acinetobacter baumannii DW-1 Moraxellaceae GAM Estuary
Sphingomonas paucimobotis DY-1 Sphingomonadaceae ALF Estuary
Listeria monocytogenes NC0120 Listeriaceae FIR Estuary
Enterobacter cloacae NC1103 Enterobacteriaceae GAM Estuary
Pasteurella haemolytica NC1127 Pasteurellaceae GAM Estuary
Aeromonas sobria DT-1 Vibrionaceae GAM Estuary
Klebsiella pneumoniae NC0621 Enterobacteriaceae GAM Estuary
Fecal streptococcus NC1228 Streptococcaceae FIR Estuary
a ALF a-Proteobacteria, GAM c-Proteobacteria, BETA b-Proteobacteria, FIR Firmicutes, ACT Actinobac-teria, CFB Cytophaga-Flexibacter-Bacteroidetes
660 Aquacult Int (2012) 20:657–672
123
were considered for the determination of the viable counts. The viable counts were
determined by colony counter (WIGGENS Galaxy 230, Germany). To validate the effect
of homogenization for each species, known numbers of cells of each species were inoc-
ulated into the rearing water and 3.5% saline water, respectively, and conducted in
accordance with the description of Epstein and Rossel (1995). Bacterial counts of the
homogenates were compared with counts of unhomogenized saline water. Only relative
standard deviation in the range of ±5% is acceptable.
Biofilm-forming ability assay
Modified microtiter plate test was employed to determine the biofilm-forming ability
(Stepanovic et al. 2000). Biofilms were incubated in LOSWM and visualized by staining
with a 1-mg ml-1 aqueous solution of crystal violet as previously described (Fu et al.
2009) and then the optical density at 595 nm was determined. For reproducibility, the cell
suspension was prepared in LOSWM adjusting the turbidity to 0.25 ± 0.01 at 600 nm
(OD600) (corresponding to 107–108 CFU ml-1). Unless specifically indicated otherwise,
cells were allowed to adhere to the surface during 24 and 48 h of incubation at 30�C
without shaking. Wells containing only the culture medium (without bacteria) were used as
a negative control. Positive control was obtained by incubating the well with E. coliATCC29522. All experiments were performed in triplicate. Biofilm-forming ability of a
strain was reported as OD595. Bacteria were classified using the scheme of Stepanovic et al.
(2000).
Pilot facilities and filtration procedures
Nine identical pilot scale RAS were divided into three experimental groups. Running
parameters for biofilters and nutrient composition (dry-weight basis) of feed used in this
study are listed in Tables 2, 3. The biofilters were 0.6 9 0.4 9 0.4 m (length 9 width 9
height) and filled with a layer of 0.4 m of bamboo ball media. The fish tanks were
0.8 9 0.4 9 0.5 m (length 9 width 9 height). Each experimental group comprised three
independent RAS. Two groups were equipped with four layers of sari and 180-lm nylon
filter, respectively. Another three tanks without filter were treated as a control group. The
filters were installed at the inlet point of fish tanks as the end filters during the whole trial.
Sea bass (Dicentrarchus labrax) were obtained from a commercial sea bass hatchery in the
Shandong Province, China. The sea bass were acclimatized for 10 days before use in order
Table 2 Running parametersfor the biofilter
Parameter Value
Working volume 100 l
Temperature 14–20�C
Inlet ammonia-N 0.13–0.97 mg/l
Outlet ammonia-N 0.03–0.09 mg/l
Inlet nitrite-N 0.040–0.096 mg/l
Inlet dissolved oxygen 6–9 mg/l
pH 6.9–7.4
Salinity 28–30 g/l
Hydraulic retention time 30 min
Aquacult Int (2012) 20:657–672 661
123
to ensure adequate health. Ten percent of fish were randomly selected for standard
microbiological test. The sea bass had not been exposed to diseases and were deemed
pathogen-free by standard microbiological techniques. Potential sea bass pathogens
including Aeromonas spp., pathogenic vibrio spp., Streptococcus spp., Flexibacter col-umnaris and other parasitic protozoa were tested to guarantee their health as described by
Zorrilla et al. (2003). After the acclimation period, the sea bass were divided into nine
100-l tanks; the stocking density of the fish was 18 kg/m3. Vibrio alginolyticus FS-2 was
grown for 36 h at 28�C in LB broth. Challenge tests were performed as described by
Defoirdt et al. (2006). After incubation, the vibrios were washed twice in autoclaved
seawater and were inoculated into the outlet of biofilter at 103CFU ml-1, which was below
the LD50 value for sea bass (Kahla-Nakbi et al. 2006). After the infection, the survival of
sea bass was recorded within 24 days. Each treatment was performed in triplicate.
Statistical analysis
The results were analyzed by the Student’s t-test to determine differences (P \ 0.05)
between the tested groups (nylon, sari and control groups). The Pearson test was employed
to check for the correlation between biofilm formation in microtiter plates and removal
rate. All statistics were performed with SPSS for Windows, version 11.5 (SPSS Inc.,
Chicago, IL, USA).
Results
Laboratory filtration test
The experiments carried out in this study showed that both types of filters decreased the
counts of bacteria in water. Overall, it was found that most strains showed above 90%
removal rate after filtration by 20-lm nylon net; 1–3 log reduction was readily achievable
(Table 4). The removal rate was highest for Pseudomonas pseudoalcaligenes 5-4 (99.59%)
and lowest for Microbacterium paraoxydans 5-2(32.58%). The bacterial cells of Vibrioparahaemolyticus and other Vibrionaceae species attached themselves to the particles,
yielded about 2-log reduction. The reduction between 1.1 and 2.0 log found in our study is
in agreement with the data reported by Huq et al. (1996). Data from various species
such as Fecal streptococcus NC1228, Sphingomonas paucimobotis DY-1, Cyclobacterium
Table 3 Nutrient composition(dry-weight basis) of feed usedin this study
Composition Proximate andmineral levels%
Crude protein 48.0
Carbohydrate 5.0
Total ash 16.0
Crude fat 14.0
Crude fiber 4.0
Calcium 5.0
Phosphorus 0.5
Potassium 2.0
Sodium 4.0
662 Aquacult Int (2012) 20:657–672
123
Tab
le4
Effi
cien
cies
of
dif
fere
nt
po
resi
zeo
fn
ylo
nn
etem
plo
yed
asfi
lter
sfo
rre
arin
gw
ater
(co
nta
inin
gp
arti
cles
of
feed
tow
hic
his
ola
tes
had
atta
ched
)
Str
ain
sV
iab
leco
un
t(C
FU
ml-
1±
SD
)
Bef
ore
filt
rati
on
Aft
erfi
ltra
tio
n
20
lm(%
reduct
ion)
180
lm(%
red
uct
ion
)
Esc
her
ich
iaco
liA
TC
C2
95
22
(1.7
0±
0.2
4)
91
07
(1.4
7±
0.5
4)
91
06
(91
.36%
)(9
.80
±0
.87
)9
10
6(4
2.2
1%
)
Sa
lmo
nel
laB
raen
der
up
H9
81
2(1
.95
±0
.67)
91
07
(4.5
0±
0.7
3)
91
05
(97
.69%
)(7
.80
±0
.60
)9
10
6(6
0.0
0%
)
Ba
cill
us
pu
mil
us
N3
-6(4
.30
±0
.70)
91
07
(3.3
0±
0.6
2)
91
06
(92
.33%
)(1
.93
±1
.73
)9
10
7(5
4.9
3%
)
Ba
cill
us
aq
uim
ari
sR
-1(1
.89
±0
.49)
91
07
(1.7
7±
0.3
2)
91
06
(90
.61%
)(2
.35
±0
.11
)9
10
6(8
7.5
5%
)
Alt
ero
mo
nas
ma
rin
aW
B-1
(3.9
5±
0.9
5)
91
05
(1.3
5±
0.0
5)
91
04
(96
.58%
)(5
.50
±1
.50
)9
10
4(8
6.0
7%
)
Vib
rio
alg
ino
lyti
cus
FS
-2(4
.67
±0
.13)
91
06
(2.1
0±
0.0
8)
91
05
(95
.50%
)(2
.36
±0
.15
)9
10
6(4
9.4
6%
)
Vib
rio
na
trie
gen
sF
S-1
(2.6
3±
0.2
3)
91
06
(2.0
4±
0.0
2)
91
05
(92
.24%
)(1
.36
±1
.15
)9
10
5(4
8.2
9%
)
Pse
udoalt
erom
onas
pis
cici
da
Y-1
(7.8
0±
0.2
2)
91
06
(3.2
8±
0.7
2)
91
05
(95
.79%
)(2
.86
±0
.27
)9
10
5(6
3.3
3%
)
Alc
alig
enes
faec
ali
sS
R-2
(6.0
0±
0.0
4)
91
06
(6.5
0±
3.5
0)
91
05
(89
.17%
)(1
.23
±1
.96
)9
10
5(8
0.0
0%
)
Vib
rio
his
pa
nic
us
2-9
(1.4
0±
0.2
7)
91
08
(4.2
0±
1.1
8)
91
07
(97
.00%
)(8
.53
±0
.65
)9
10
7(3
9.0
7%
)
Ma
rin
ob
acte
rsp
.F6
(1.2
0±
0.0
2)
91
06
(4.7
3±
0.8
0)
91
04
(96
.06%
)(5
.28
±0
.15
)9
10
4(5
6.0
0%
)
Vib
rio
pa
rah
aem
oly
ticu
sA
TC
C17
80
2(2
.43
±0
.43)
91
06
(2.0
0±
0.0
8)
91
05
(91
.77%
)(1
.36
±0
.15
)9
10
5(4
4.1
0%
)
Cyc
lob
act
eriu
mm
ari
nu
mH
DY
-7(1
.46
±0
.47)
91
07
(3.6
3±
0.6
2)
91
06
(75
.14%
)(9
.87
±0
.46
)9
10
7(3
2.4
8%
)
Mic
robact
eriu
mpara
oxy
dans
5-2
(2.6
7±
0.7
5)
91
07
(1.8
0±
0.7
5)
91
07
(32
.58%
)(1
.78
±0
.67
)9
10
7(3
3.3
3%
)
Sta
ph
ylo
cocc
us
au
reus
F1
5(8
.78
±0
.53)
91
06
(2.1
3±
0.1
8)
91
06
(75
.79%
)(2
.87
±0
.22
)9
10
6(6
7.3
0%
)
Pse
ud
om
on
as
pse
udo
alc
ali
gen
es5
-4(2
.08
±0
.75)
91
08
(8.6
0±
0.7
3)
91
06
(99
.59%
)(3
.53
±0
.13
)9
10
7(8
3.3
3%
)
Ser
rati
ap
lym
uth
ica
WT
-1(1
.00
±0
.21)
91
08
(1.6
0±
0.2
8)
91
07
(98
.40%
)(3
.83
±0
.86
)9
10
7(6
1.6
8%
)
Aer
om
on
as
sob
ria
DT
-1(2
.42
±1
.32)
91
06
(1.0
7±
0.1
6)
91
05
(95
.57%
)(2
.48
±0
.45
)9
10
5(8
9.7
6%
)
V.
cho
lera
eN
E-1
(4.2
0±
1.1
0)
91
06
(2.0
4±
1.0
6)
91
05
(95
.14%
)(2
.24
±0
.50
)9
10
6(4
6.7
0%
)
V.
cho
lera
eN
E-9
(4.6
5±
2.0
8)
91
06
(2.9
1±
1.5
9)
91
05
(93
.74%
)(2
.59
±0
.55
)9
10
5(4
4.3
0%
)
Aci
net
ob
acte
rb
au
man
nii
DW
-1(9
.00
±0
.21)
91
07
(1.3
2±
0.2
0)
91
05
(95
.57%
)(1
.75
±0
.20
)9
10
5(8
0.5
6%
)
Sp
hin
go
mon
as
pa
uci
mo
bo
tis
DY
-1(1
.22
±0
.02)
91
06
(3.3
1±
0.2
0)
91
05
(72
.87%
)(4
.02
±0
.20
)9
10
5(6
5.5
7%
)
Lis
teri
am
on
ocy
tog
enes
NC
01
20
(5.2
0±
0.7
3)
91
05
(1.6
0±
0.2
4)
91
04
(96
.92%
)(1
.37
±0
.24
)9
10
5(7
3.6
5%
)
Aquacult Int (2012) 20:657–672 663
123
Tab
le4
con
tin
ued
Str
ain
sV
iab
leco
un
t(C
FU
ml-
1±
SD
)
Bef
ore
filt
rati
on
Aft
erfi
ltra
tio
n
20
lm(%
reduct
ion)
180
lm(%
red
uct
ion
)
En
tero
bac
ter
clo
aca
eN
C11
03
(2.4
5±
0.4
5)
91
07
(3.9
3±
0.2
0)
91
06
(83
.96%
)(1
.30
±0
.10
)9
10
7(4
6.9
4%
)
Kle
bsi
ella
pneu
mon
iae
NC
06
21
(2.6
0±
0.1
6)
91
07
(5.1
3±
1.8
8)
91
06
(79
.80%
)(1
.47
±0
.19
)9
10
7(4
3.5
9%
)
Fec
al
stre
pto
cocc
us
NC
12
28
(2.2
0±
0.1
7)
91
07
(1.2
0±
0.2
0)
91
07
(45
.45%
)(1
.51
±0
.90
)9
10
7(3
1.3
6%
)
Pa
steu
rell
ah
aem
oly
tica
NC
11
27
(1.6
9±
0.6
7)
91
07
(2.8
3±
0.9
7)
91
05
(98
.33%
)(8
.28
±0
.65
)9
10
6(5
1.0
0%
)
664 Aquacult Int (2012) 20:657–672
123
marinum HDY-7 and Alcaligenes faecalis SR-2 yielding less than 1-log reduction were
also obtained. For 180-lm nylon net, the removal rate was significantly lower than its
value for 20-lm nylon net (P [ 0.05). The average filtration effect of 180-lm nylon net
was between 32.48 and 89.76%, less than 1 log CFU ml-1. The removal rate was highest
for Aeromonas sobria DT-1 (89.76%) and lowest for Cyclobacterium marinum HDY-7
(32.48%).
Data from the attachment experiments showed that above 90% of the vibrios cells that
attached to particles were removed by sari filter (Table 5). About 99.87% counts of Alt-eromonas marina WB-1 in the filtrate were subtracted from filtration. The results obtained
using sari were consistent with those value obtained in filtration test with 20-lm nylon net,
showing the same extent of attachment to particles and the same degree of removal by
filtration (Table 5); there were no significant differences in removal effect by applying
20-lm nylon net and four layers of sari (P \ 0.05).
Table 5 Counts of isolates attached to the particles of feed in rearing water before and after filtrationthrough four layers of white sari material
Strains Viable count (CFU ml-1 ±SD)
Before filtration After filtration % Reduction
Escherichia coli ATCC29522 (8.30 ± 0.32) 9 107 (1.02 ± 0.32) 9 106 98.77%
Salmonella Braenderup H9812 (1.95 ± 0.20) 9 107 (2.78 ± 0.57) 9 105 98.57%
Bacillus pumilus N3-6 (2.07 ± 0.29) 9 107 (2.70 ± 0.28) 9 106 86.96%
Bacillus aquimaris R-1 (1.74 ± 0.14) 9 106 (1.02 ± 0.74) 9 105 94.25%
Alteromonas marina WB-1 (1.58 ± 0.17) 9 106 (2.00 ± 0.33) 9 103 99.87%
Vibrio alginolyticus FS-2 (1.20 ± 0.14) 9 106 (4.73 ± 0.14) 9 104 96.06%
Vibrio natriegens FS-1 (3.70 ± 0.19) 9 105 (7.50 ± 0.18) 9 103 97.97%
Pseudoalteromonas piscicida Y-1 (2.70 ± 0.15) 9 106 (1.24 ± 0.14) 9 105 95.41%
Alcaligenes faecalis SR-2 (1.27 ± 0.33) 9 106 (2.05 ± 0.33) 9 105 87.19%
Pseudomonas pseudoalcaligenes 5-4 (1.20 ± 0.02) 9 106 (4.73 ± 0.80) 9 104 96.06%
Vibrio parahaemolyticus ATCC17802 (1.60 ± 0.02) 9 106 (5.20 ± 0.30) 9 105 96.75%
Cyclobacterium marinum HDY-7 (5.40 ± 0.30) 9 106 (1.40 ± 0.71) 9 106 74.10%
Microbacterium paraoxydans 5-2 (4.07 ± 0.80) 9 106 (2.70 ± 0.88) 9 106 32.50%
Staphylococcus aureus F15 (1.60 ± 0.88) 9 107 (5.60 ± 0.32) 9 106 56.00%
Serratia plymuthica WT-1 (1.68 ± 0.80) 9 107 (5.20 ± 0.30) 9 105 96.90%
Marinobacter sp.F6 (1.27 ± 0.85) 9 107 (4.13 ± 0.66) 9 105 96.74%
Aeromonas sobria DT-1 (7.70 ± 0.18) 9 106 (3.28 ± 0.59) 9 105 95.74%
V. cholerae NE-1 (4.20 ± 1.10) 9 106 (2.24 ± 0.77) 9 105 94.67%
V. cholerae NE-9 (4.65 ± 2.08) 9 106 (4.17 ± 0.55) 9 105 91.03%
Acinetobacter baumannii DW-1 (6.00 ± 0.26) 9 107 (9.25 ± 0.15) 9 106 84.58%
Sphingomonas paucimobotis DY-1 (1.16 ± 0.20) 9 107 (1.06 ± 0.32) 9 106 90.86%
Listeria monocytogenes NC0120 (1.95 ± 0.25) 9 107 (2.10 ± 0.20) 9 106 89.23%
Enterobacter cloacae NC 1103 (2.76 ± 0.80) 9 107 (6.00 ± 0.41) 9 106 78.26%
Klebsiella pneumoniae NC0621 (1.44 ± 0.20) 9 107 (3.90 ± 0.88) 9 106 72.92%
Fecal streptococcus NC1228 (2.20 ± 0.17) 9 107 (1.02 ± 0.72) 9 107 53.64%
Pasteurella haemolytica NC1127 (1.69 ± 0.67) 9 107 (1.06 ± 0.63) 9 106 93.73%
Aquacult Int (2012) 20:657–672 665
123
Biofilm formation ability of different species
The biofilm-forming ability of different species was assayed and quantified at OD595
(Fig. 1a, b). Overall, it was found that most strains showed strong biofilm-forming ability
into the seawater medium, with the exception of Microbacterium paraoxydans 5-2,
Marinobacter sp.F6 and Cyclobacterium marinum HDY-7. It was also found, however,
that the biofilm-forming capacity was not always consistent with the filtration effects of
corresponding species (Fig. 2).The mean OD595 of isolates was relatively high for Pseu-domonas pseudoalcaligenes 5-4, Vibrio parahaemolyticus and other Vibrio sp. However,
the growth of Alcaligenes faecalis SR-2, Fecal streptococcus NC1228 and Bacillusaquimaris R-1 were relatively lower and lead us to consider them as moderate biofilm
producer. Likewise, the mean OD595 of Microbacterium paraoxydans 5-2 and Cyclobac-terium marinum HDY-7 was significantly lower (P \ 0.05) than other species tested.
These species formed weak biofilm in culture medium. It is notable that, however, those
species were unable to move. It seems that strong biofilm producer was removed more
efficiently by sari filter. The level of removal rate of sari filter did not correlate with the
level of biofilm formation in microtiter plates nor indicate removal rate would be improved
(a)
0
0.5
1
1.5
2
2.5
OD
595n
m
(b)
0
0.5
1
1.5
2
2.5
EC BP PP MP VA VH CM AM BA VN VP AF FS SA SE SB
VC VC9 PD AB SP LM CA MA PH AS KP
OD
595n
m
Fig. 1 Biofilm formation capacity of various species originated from seawater (a) and estuary (b) (blackbar) the level of biofilm buildup in 24 h; (white bar) the level of biofilm buildup in 48 h. a EC: Escherichiacoli ATCC25922; BP: Bacillus pumilus N3-6; BA: Bacillus aquimaris R-1; VA: V. alginolyticus FS-2; PP:Pseudoalteromonas piscicida Y-1; VH: V. hispanicus 2-9; VN: V.natrigens FS-1; AM: Alteromonasmarinum WB-1; SE: Serratia plymuthica WT-1; SS: Staphylococcus aureus F15; AF: Alcaligenes faecalisSR-2; VP: Vibrio parahaemolyticus ATCC17802; CM: Cyclobacterium marinum HDY-7; FS: Fecalstreptococcus NC1228; SB: Salmonella Braenderup H9812. b VC: V. cholerae NE-1; VC9: V. cholerae NE-9; PD: Pseudomonas pseudoalcaligenes 5-4; AB: Acinetobacter baumannii DW-1; SP: Sphingomonaspaucimobotis DY-1; LM: Listeria monocytogenes NC0120; CA: Enterobacter cloacae NC101103; KP:Klebsiella pneumoniae NC 0621; PH: Pasteurella haemolytica NC1127; AS: Aeromonas sobria DT-1; MA:Marinobacter sp.F6
666 Aquacult Int (2012) 20:657–672
123
due to the stimulation of biofilm buildup (R2 = 0.281, P = 0.5736). Pathogen removal
was not influenced by the level of biofilm buildup in microtiter plates. It should be
mentioned that whether or not mobility had an effect on the level of biofilm buildup
remains to be proved.
Removal of HB and Vibrio spp. by nylon and sari filters
We determined the effect of nylon or sari filters on the survival of sea bass challenged with
the pathogen V. alginolyticus FS-2. After the infection with V. alginolyticus FS-2, the final
mortality of sea bass without end filter was 33.35%, whereas mortality was 18.87% in sea
bass tanks where nylon filter was used. A 9.53% mortality of sea bass was observed in the
tanks where sari filter was used (Table 6). Statistical analysis demonstrated significant
differences (P \ 0.05) in mortality between sari filter groups and control groups, while
there were no significant differences between nylon filter groups and control groups
(P [ 0.05). And the results showed that the addition of V. alginolyticus FS-2 to waters has
no significant effect on body length, body weight and water quality. The mean final weight
was 282.3 ± 14. 4 g in the groups equipped with nylon filter, 288.0 ± 54.0 g in the sari
groups and 293.0 ± 25.3 g in the control groups.
In the tanks equipped with filters, the fluctuation in the levels of heterotrophic bacteria
(HB) was less pronounced in both sari and nylon filter groups (Fig. 3a). In both conditions,
12-day treatment with filters resulted in maintenance of the HB below the level of 106
CFU ml-1 (except for the day 7 in the tanks with nylon filters), slightly lower than the
control groups. The removal rate was peaked at 10th day in both conditions and then
drastically decreased in sari filter groups. However, compared with the control groups, the
treatments with filters did not significantly decrease the levels of HB (P [ 0.05). Similar
trends were also observed for the variation in the total number of Vibrio sp. (Fig. 3b). After
the installment of filters, the number of Vibrio sp. gradually decreased in both sari and
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0.0% 20.0% 40.0% 60.0% 80.0% 100.0%
Removal rate%O
D59
5nm
Fig. 2 Correlation betweenbiofilm formation in microtiterplates and removal rate of sarifilters. The biofilm results aregiven as OD 595 values, andremoval rate are given as %
Table 6 Effect of different filtration treatments on body length, body weight and mortality of sea bass
Treatments Body length (cm) Body weight (g) Final mortality (%)
0 day 24 days 0 day 24 days
Sari filter 23.3 ± 2.16 30.3 ± 1.15 256.4 ± 30.0 288.0 ± 54.0 9.53 ± 8.25
Nylon filter 23.6 ± 1.63 27.7 ± 0.58 263.4 ± 32.6 282.3 ± 14. 4 18.87 ± 7.91
Control 23.3 ± 1.08 27.3 ± 0.58 249.9 ± 26.9 293.0 ± 25.3 33.35 ± 8.23
Aquacult Int (2012) 20:657–672 667
123
nylon filter groups, whereas up to 105 CFU ml-1 Vibrio sp. was achieved in control
groups. A significantly lower number of Vibrio species were noted in sari groups when
compared to the control (P \ 0.05). However, the removal rate from both groups drasti-
cally decreased after the 9th day probably due to the clogging. After the 12th day, all types
of filters were replaced with new one. Then, the similar trends were observed in the next
12 days. Over the period from day 12 to day 21, the removal rate of HB in sari and nylon
filter groups increased from 91.17 to 99. 56% and raised from 52.92 to 96.76%,
(a)
3.5
4
4.5
5
5.5
6
6.5
7
7.5
8
Time(d)
Log
(10)
CFU
/ml
-20.00%
0.00%
20.00%
40.00%
60.00%
80.00%
100.00%
Rem
oval
rat
e
(b)
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
0 1 2 3 5 7 9 10 12 14 17 19 21 24
0 1 2 3 5 7 9 10 12 14 17 19 21 24
Time(d)
Log
(10)
CFU
/ml
-40.00%
-20.00%
0.00%
20.00%
40.00%
60.00%
80.00%
100.00%
Rem
oval
rat
e
Fig. 3 a Variation in the total number of heterotrophic bacteria (HB) in fish tanks and its removal rate.b Variation in the total number of Vibrio sp. in fish tanks and its removal rate. (Open triangle) Removal rateof HB (a) and Vibrio sp. (b) in nylon filter groups; (filled triangle) Removal rate of HB (a) and Vibrio sp.(b) in sari groups; (open circle) Control. (Black bar) variation in the total number of HB (a) and Vibrio sp.(b) in nylon filter groups; (white bar) variation in the total number of HB (a) and Vibrio sp. (b) in sarigroups; (yellow bar) variation in the total number of HB (a) and Vibrio sp. (b) in control groups
668 Aquacult Int (2012) 20:657–672
123
respectively. At the end of the study, bacteriological analysis found 7.4 9 102 CFU ml-1
and 3.0 9 103 CFU ml-1 Vibrio sp. in the rearing water of nylon filter groups and control
groups, whereas sari filter groups showed\102 CFU ml-1 Vibrio sp. The treatments with
sari filters increased the survival of challenged sea bass.
Discussion
The reduction in viable counts of bacterial cells in this study confirmed that attachment to
particles is an important phenomenon in aquatic systems, especially for Vibrio sp. Solid
waste removal as an important parameter should be controlled in RAS. United States EPA
also found potential health risks associated with particles in reclaimed wastewater and
determined an allowable particle limit for reclaimed water (Dietrich et al. 2009). To
remove the large-size particles, rotating microscreens are commonly used at land-based
intensive RAS. Screen mesh pore sizes of 60–200 lm are common. Such facility is prone
to many technical problems after a long-time operation, and in most situations, it requires
time-consuming washing and back flushing. Besides, the downstream biofilters also suffer
from solid accumulation and back flushing. The operations of back flushing for ‘dirty
packing media’ of biofilters often lead to the return of pathogens into the rearing tanks due
to the fact that biofilters would exhaust their filtering ability after a long-time operation. In
our study, four layers of sari cloth were used for filtration and retained smaller-sized
particles. The results obtained using 20-lm nylon net and sari material showed no variation
in the removal of bacteria attached to particles. Treatment with four layers of sari cloth
resulted in protection of the sea bass from the pathogen. Probably due to the fact that
biofilters also played a role in filtration of large-size particles, the number of HB and
number of Vibrio sp. also decreased in the control groups.
In an intensive RAS, there is high concentration of waste produced by these animals’
normal metabolic processes. Fishes exposed to this environment over time are more sus-
ceptible to bacterial infections and lost their appetite. High concentration of waste also
stimulates the growth of pathogens. The ability to maintain a pathogen-free system is a
very difficult task; however, reducing the levels of pathogens to below the infective levels
by filtration should decrease the chance of fish becoming infected. One of the key aspects
for improving the sanitation of such RAS is having the ability to ‘manage’ these pathogen
populations. In aquatic ecosystems, although prokaryotes are small in size, they play a
major role in biogeochemical processes. Michaud et al. (2006) have reported the bacterial
community structure and composition related in RAS. The findings revealed that organic
carbon plays important roles in determining the relative abundance and impact of pro-
karyotes in aquatic systems. Some microbiologists proposed that microorganisms have
different survival strategies and divided them into two groups based on growth properties:
‘zymogenous’ with rapid growth and ‘autochthonous’ with slow but continuous activity
(Thompson et al. 2005). Even though this theory of survival may now seem to be simple, it
is accepted that all microbial populations will be involved in maintaining an effective and a
stable rearing environment by releasing the chemical substances that have a bactericidal or
bacteriostatic effect on other microorganisms (Verschuere et al. 2000). Most of the
pathogens may belong to ‘zymogenous’ microbes. Natural seawater as an oligotrophic
system has low nutrient concentrations. In such systems, interactions between autotrophs
and heterotrophs are tightly coupled. The dominant heterotrophs have similar growth rates
and help maintain homeostasis with autotrophs, as well as making a balance between
‘zymogenous’ and ‘autochthonous’ (Cotner and Biddanda 2002).
Aquacult Int (2012) 20:657–672 669
123
In RAS, high concentration of fish feces and unconsumed feed promoted the growth of
‘zymogenous’ microbes. The filtration sieved out the increased ‘zymogenous’ microbes
and retained the slow growth ‘autochthonous’, most of which are not human pathogens.
Although any heterotrophic bacterial cell that is attached to small particles may pass
through the sari filters, the levels of fish pathogens were reduced below the outbreak dose.
Therefore, it is less likely to present an infectious dose, as determined by previous reports
(a minimum of 104–106 pathogen cells per ml represents an LD50 value) (Cash et al. 1974;
Kahla-Nakbi et al. 2006).
It should be mentioned, however, that any fixed filtration facility is readily clogged even
after a short-time operation. In our experiment, the clogging time for sari or nylon filter
was typically 10–12 days, which means maintenance at a regular base is very important.
The old sari or nylon filter can be disposable or reused after the disinfection by detergents
as reported by Huq et al. (1996). Based on the results of the pilot study and laboratory
filtration experiments reported here, we suggest that improving inlet water quality with end
filter would minimize the incidence of disease in the whole systems. Besides, the
installment of filters in the outlet of rearing tank can also prevent the transmission of
pathogens back from the black-flushed biofilters. Armed with this information and
employing a dead-end preventive filtration measure, we will be able to reduce the bacterial
load and prevent the disease transmission in the aquaculture systems where the environ-
mental factors (e.g., temperature) are highly variable. To the best of our knowledge, this is
the first demonstration of the filtration capacity of sari material for the removal of
pathogens in the aquaculture systems. In the near future, filters will be adopted in a full-
scale aquaculture system for pathogen reductions. Further study will investigate its long-
term ecological consequence.
Conclusion
The results of experiments reported here suggest that sari or 20-lm nylon nets, in fact, may
be a useful practice to institute to reduce the abundance of pathogens. It would be an
interesting alternative for large-sized sand or gravel filters, since sari can be disposable and
easily replaced without back flushing. Taking into consideration all of the findings
described above, we proposed that a dead-end filtration, if maintains at a regular base,
could help in controlling bacterial infections within the RAS. It could be an effective way
to, at the least, reduce the number of pathogens below the potential infectious dose.
Acknowledgment This work was supported by a grant from the National Natural Science Foundation ofChina (30972267), CAS Knowledge Innovation Project (KZCX2-EW-Q212), Public Service Sectors(Agriculture) Special Project (201003024) and Atlantic Salmon Research Fund (Y12605101I).We wouldespecially like to thank Ms. Shaoshao Liu for sampling assistance.
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