Aquacult Int (2016) 24:357–372

DOI 10.1007/s10499-015-9930-7

Ammonia and nitrite nitrogen removal in shrimp culture

by Vibrio alginolyticus VZ5 immobilized in SA beads

H. W. Shan1 • W. Y. Bao2 • S. Ma1 •

D. P. Wei1 • L. Gao3

Received: 10 October 2014 / Accepted: 14 July 2015 / Published online: 19 July 2015
Ó Springer International Publishing Switzerland 2015

Abstract The toxicity of nitrogen in the shrimp culture water has been well established.
In this study, SA beads composed of Vibrio alginolyticus VZ5, sodium alginate (SA) and
sugarcane bagasse were used for ammonia nitrogen (NH4-N) and nitrite nitrogen (NO2-N)
removal. A 50-day cultivation experiment was carried out in aquaria to evaluate the
activity of the SA beads in shrimp culture. The results indicate that SA beads have a
maximum capacity of 1.06 9 108 colony-forming units (cfu)/bead. However, the optimal
initial density of the bacteria embedded in the SA beads is 104–105 cfu/bead. The maxi-
mum NO2-N degradation rate achieved for the SA beads was 8.44 mg/L/day, and the
average NO2-N degradation per bead was 0.06 mg. The addition of a carbon source
accelerated the degradation of NH4-N and NO2-N by the SA beads. The NH4-N and NO2-N
concentrations after treatment with SA beads were below 1.55 and 1.62 mg/L, respec-
tively, at later time points, and these concentrations were significantly lower than in the
group without any treatment (P \ 0.05, df = 17). There were no significant differences in
the NH4-N and NO2-N concentrations following treatments with SA beads and water
exchange (P [ 0.05, df = 29), and the yield resulting from water treatment with SA beads
reached approximately 70 % of the yield with water exchange treatment. Moreover, the
particulate organic carbon and dissolved organic carbon concentrations in the water were
enhanced by the addition of SA beads. At later time points, some of the SA beads had
broken down, and the sugarcane bagasse from the SA beads may have served as a carbon

& S. Ma
[email protected]
H. W. Shan
[email protected]
1
The Key Laboratory of Mariculture, Ministry of Education, Ocean University of China,
Qingdao 266003, China
2
Marine Science and Technology Institute, Yangzhou University, Yangzhou 225127, China
3
Liaoning Key Laboratory of Marine Fishery Molecular Biology, Liaoning Ocean and Fishery
Science Institute, Dalian 116023, China

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source for forming bioflocs. The new approach proved effective for NH4-N and NO2-N
removal in shrimp culture.

Keyword NH4-N and NO2-N removal
Immobilization
Vibrio alginolyticus
Shrimp

culture

Introduction

The rapid development of the aquaculture industry has been accompanied by increasing
environmental impacts (Crab et al. 2007). Nutrient-enriched wastewater and bottom sed-
iments from prawn ponds discharging into adjacent coastal waters have frequently resulted
in eutrophication and oxygen depletion, with adverse consequences for marine ecology and
fisheries (Páez-Osuna et al. 1998). Nitrogen accumulation is the main problem in aqua-
culture. Many conventional techniques are used to remove nitrogen from culture water in
intensive aquaculture systems, including rotating biological contactors, trickling filters,
bead filters and fluidized sand biofilters (Crab et al. 2007). Compared with physiochemical
processing, biological treatment may be more cost-effective for nitrogen removal (Ahn
2006; Jeong et al. 2006).
The immobilization of bacteria onto beads has been found to be highly effective for
aquaculture wastewater treatment due to their high operational stability, ease of use in
continuous reactors, optimization of microbial growth and metabolic rates and high cell
densities on the immobilized beads (Yang 1997; Kim et al. 2000; Shan and Obbard 2001;
Manju et al. 2009). Highly efficient bacteria play an important role in immobilization. The
bacteria used in immobilization include nitrifying bacteria, denitrifying bacteria, effective
microorganisms (EMs) and Bacillus sp., among others (Seo et al. 2001; Hsieh et al. 2002;
Hill and Khan 2008; Lee and Cho 2010; Xiao et al. 2011). In addition to the bacteria, the
gel matrices are also important in immobilization. Many different gel matrices have been
proposed as carriers, including natural biopolymers (polysaccharides, alginate, carrageenan
and agar), and synthetic polymers (polyethylene, polyurethanes and polyethers) (Lozinsky
and Plieva 1998; Naidu et al. 2011). In addition to permeability, physical stability in water
is required for the matrices as well as a microstructure that is conducive to the growth of
bacteria on the beads.
Vibrio sp. exists as part of the normal microflora in the environment. Some studies have
reported that Vibrio alginolyticus can be used as a probiotic in aquaculture to improve
survival rates, growth and disease resistance (Austin et al. 1995; Garriques and Arevalo
1995; Gatesoupe 1997). In preliminary experiments, V. alginolyticus strain VZ5 isolated
from shrimp culture was found to be highly efficient at NH4-N and NO2-N removal (Shan
et al. 2013). Sugarcane bagasse has a microstructure that promotes the adherence and
growth of bacteria (Shan et al. 2014). Moreover, sugarcane bagasse contains many types of
cellulosic materials that can supply a carbon source for microbial decomposition (Pandey
et al. 2000). Carbon is one of the key factors affecting the absorption of nitrogen by
bacteria; the C/N ratio can affect nitrogen removal by immobilized cells (Cao et al. 2002).
In this study, SA beads composed of V. alginolyticus VZ5, sodium alginate and sug-
arcane bagasse were used for NH4-N and NO2-N removal in shrimp culture. The degra-
dation of NH4-N and NO2-N by V. alginolyticus VZ5 was investigated first. Next, variation
in the bacterial density and the beads’ capacity for NO2-N degradation as well as the

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effects of carbon source addition on the degradation of NH4-N and NO2-N were investi-
gated. Finally, a 50-day cultivation experiment was carried out to evaluate the activity of
the SA beads in shrimp culture.

Materials and methods

Vibrio alginolyticus VZ5 and culture media

The microorganism used in this study was V. alginolyticus VZ5, which was isolated from
the bottom of a shrimp pond. V. alginolyticus VZ5 was cultured in Luria–Bertani (LB)
media (composed of 10 g of peptone, 5 g of yeast powder and 30 g of sodium chloride in
1 L of distilled water) for 24 h at 30 °C and then centrifuged (4000 rpm, 5 min) to harvest
the cells.

Preparation of SA beads

The preparation method for the immobilized V. alginolyticus VZ5 via the SA–calcium
chloride method is shown in Fig. 1a. V. alginolyticus VZ5 cells were harvested by cen-
trifugation (4000 rpm, 5 min), washed twice with sterile seawater and then suspended for
immobilization. The sugarcane bagasse was prepared by screening for lengths of
0.25–0.33 mm. The screened bagasse was autoclaved at 121 °C for 20 min. SA (3 g) and
sodium chloride (3 g) were dissolved in 100 mL of deionized water, and the solution was
heated to allow the SA to dissolve. The concentrated V. alginolyticus VZ5 cells and
screened bagasse were added to the cooled SA solution at volume proportions of 1:2.5:10,
respectively. The mixture obtained was placed in a syringe and then added dropwise into a

Centrifuged Screened
3% SA solution
bacterial cells sugarcane bagasse

Mixture

Drop into 6% CaCl2 solution

Mixture

o
Keep the solution for 12 h at 4 C Hole

6% CaCl2 solution
Washing

SA beads

A B
Fig. 1 Preparation of SA beads (a) and the apparatus used to prepare the SA beads for the culture
experiment (b)

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sterile 6 % calcium chloride solution with gentle stirring to form spherical beads. The
calcium chloride solution with the spherical beads was maintained at 4 °C for 12 h to form
SA beads with entrapped bacterial cells. Because using a syringe was inefficient for the
mass production of SA beads, a stainless steel pressure surface machine was used to
prepare the SA beads used in the cultivation experiment. The preparation of the SA beads
for the cultivation experiment is outlined in Fig. 1b.

Experimental design

Degradation of NH4-N and NO2-N by Vibrio alginolyticus VZ5

The concentrated V. alginolyticus VZ5 cells were added to 100 mL of sterile seawater
(salinity 30 ppt) in a 100-mL Erlenmeyer flask. Ammonium chloride and sodium nitrite
were used as nitrogen sources to determine the utilization of NH4-N and NO2-N by V.
alginolyticus VZ5. Glucose was used as a carbon source and was added to the seawater to
adjust the C:N ratio to 10. The flasks were placed in a shaker with an agitation rate of
120 rpm for 12 h at 30 °C. OD600 values and the NH4-N and NO2-N concentrations were
determined every 3 h. Initial and final total nitrogen (TN) concentrations were also
determined. Each group had three replicates.

Bacterial density variation of the SA beads

The effect of cross-linking on the bacterial density of the SA beads was studied at various
densities of bacteria embedded in the SA beads: 6.00 9 103 colony-forming units (cfu)/
bead, 3.00 9 104 cfu/bead, 2.50 9 105 cfu/bead and 5.33 9 108 cfu/bead. The bacterial
densities of the SA beads were recorded at the start and end of the cross-linking process.
Each group had three replicates.
Various initial densities were studied during the tests using synthetic wastewater:
7.00 9 103 cfu/bead, 3.25 9 104 cfu/bead, 2.00 9 105 cfu/bead and 4.00 9 107 cfu/bead.
The synthetic wastewater was composed of 5 mg of glucose, 7.5 mg of peptone, 5 mg of
sodium nitrite and 0.3 mg of sodium dihydrogen phosphate in 100 mL sterile seawater. The
bacterial density of the SA beads was recorded every 2 days. Each group had three replicates.

NO2-N degradation by SA beads

Bacterial solution (BS), SA beads with bacteria (B–B) and SA beads with no bacteria
embedded (NB-B) were added into 100 mL of synthetic wastewater to test the NO2-N
degradation capacity of the SA beads. Untreated synthetic wastewater was used as a
control (BL). The total bacterial amount in the BS was equal to the amount in the B–B. The
NO2-N concentration in the synthetic wastewater was measured every 2 days. Each group
had three replicates.
NO2-N degradation using SA beads was studied at various bacterial densities:
2.50 9 104 cfu/bead, 7.75 9 105 cfu/bead, 5.50 9 106 cfu/bead and 8.50 9 107 cfu/bead.
Sixty SA beads were added to 100 mL of synthetic wastewater, and the mixture was
maintained in an incubator at 30 °C. The NO2-N concentration in the synthetic wastewater
was measured every day. Each group had three replicates.
Sixty SA beads (at bacterial density of 2.25 9 104 cfu/bead) were added to 100 mL of
synthetic wastewater, and the cumulative NO2-N degradation was measured. When the

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NO2-N concentration in the synthetic wastewater fell below 2 mg/L, the synthetic
wastewater was replaced with new synthetic wastewater with the same composition. The
NO2-N concentration was recorded every day.

Effect of carbon source addition on NH4-N and NO2-N degradation by SA beads

Glucose was added to the solution to test the effect of carbon source addition on the NH4-N
and NO2-N degradation by SA beads. Glucose (50 mg) was added to 100 mL of solution.
NH4-N, C ? NH4-N, NO2-N and C ? NO2-N represent the solutions generated using
ammonium chloride, glucose and ammonium chloride, sodium nitrite, and glucose and
sodium nitrite, respectively. Sixty SA beads (at bacterial density of 2.5 9 104 cfu/bead)
were added to the solution and maintained in an incubator at 30 °C. The NH4-N and NO2-N
concentrations were recorded every day. Each group had three replicates.

Activity of SA beads in shrimp culture

The cultivation experiment to evaluate the activity of the SA beads was carried out for
50 days in aquaria (50 cm 9 35 cm 9 35 cm) containing approximately 50 L of seawa-
ter. One aquarium was used as a breeding unit where 17 shrimp (Litopenaeus vannamei)
were bred to a density of 100 shrimp/m2. The body length of the shrimp was
7.65 ± 0.4 cm. The shrimp were fed a commercial feed twice daily at 8:00 and 20:00, and
2 g of feed total was provided every day. Every morning, the residual feed and feces were
removed with a siphon.
NW, WE, SW and RW represent the water treatment groups with no water exchange, water
exchange, in situ water treatment and recirculating water, respectively. There were no SA
beads added and no water exchange in the NW group. Approximately 20 % of the water in the
WE group was exchanged for fresh seawater every 10 days. The water in the SW group was
treated with SA beads in the breeding unit, and no water was exchanged during the experi-
ment. In the RW group, 20 % of the water was filtered daily using a sand filter, and the water
was stored in the aquarium (treatment unit) with SA beads before being returned to the
breeding unit. SA beads were added to the SW group at 0.16 % on the 20th day and 0.80 % on
the 30th day according to the water volume in the breeding units. The SA beads added to the
treatment units in the RW group were 0.47 % on the 20th day and 1.17 % on the 30th day,
according to the water volume in the treatment units. Each group had three replicates. The
bacterial densities immobilized in SA beads were about of 2.5 9 104 cfu/bead.
Temperature, salinity, pH and dissolved oxygen (DO) were monitored every 10 days.
The NH4-N, NO2-N and total nitrogen (TN) concentrations were determined in the
breeding unit once every 10 days. In addition, the dissolved organic carbon (DOC) and
particulate organic carbon (POC) in the NW and SW groups were measured once every
10 days. The carbon content of the sugarcane bagasse in the RW treatment unit was
measured, and parameters tracking shrimp growth performance (e.g., average body length,
yield and survival) were assessed at the end of the culture.

Sample analysis methods

OD values were determined at 600 nm using a spectrophotometer (722 visible spec-

trophotometer). The bacterial density of the SA beads was measured as follows: SA beads
were placed in 5.5 % sodium citrate solution for 10 min and mixed with a turbine mixer

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(Vortex Genie 2). After appropriate dilution, the bacteria in the diluted solution were
counted using a plate counting method on LB plates, and the bacterial densities of the SA
beads were calculated according to the dilution factor used.
Temperature, salinity, pH and DO were monitored every 10 days using a water quality
analyzer (YSI-6600). The NH4-N and NO2-N concentrations were analyzed by nessler-
ization and diazotization according to the National Specification for Marine Monitoring
(SOA 2007). Some water samples were frozen to determine the TN concentration using an
ultraviolet spectrophotometer with potassium persulfate–boric acid–sodium hydrate as the
digestive reagent (Xing et al. 2006).
Each water sample was assayed to determine the amount of POC and DOC. Any POC
was collected using pre-combusted Whatman GF/F filters, exposed to HCl fumes to purge
the inorganic carbon and then dried for further analysis. Water filtered through GF/F filters
was frozen to analyze the DOC content. The concentrations of POC and DOC were
measured using an elemental analyzer (Elementar, Vario ELIII) and an organic carbon
analyzer (Analytik Jena, 2100S), respectively.

Statistical analyses

All the treatment groups were compared in each experiment. One-way repeated-measure
ANOVA was used to analyze the data measured for dependent variables after certain time
interval during experiments. Other data comparisons were analyzed by a one-way ANOVA
followed by Tukey’s test using the Statistical Package for Social Sciences (SPSS) (19.0).
All of the data were checked for normality, and percentage data were arcsine-transformed
prior to analysis. Differences were considered significant at P \ 0.05.

Results

NH4-N and NO2-N degradation by Vibrio alginolyticus VZ5

The NH4-N concentration and OD values were affected by the time significantly. The NH4-
N concentration decreased sharply in the first 3 h (P \ 0.05, df = 5) and then remained
stable (Fig. 2a). With the decreasing NH4-N concentrations, the OD values increased for
the first 3 h (P \ 0.05, df = 5) and then decreased slightly. V. alginolyticus VZ5 degraded
the NH4-N from 9.67 to 2.41 mg/L over 12 h, a degradation rate of 75.08 %. The results
(Fig. 2b, c) demonstrated that the NO2-N concentration declined with V. alginolyticus VZ5
growth and increasing NH4-N concentration. V. alginolyticus VZ5 degraded the NO2-N
from 11.41 to 5.46 mg/L over 12 h. There was no significant difference between the TN
concentrations during the experiment (P [ 0.05, df = 5) (Fig. 2d).

Immobilization of Vibrio alginolyticus VZ5 by the SA–calcium chloride

method

The shape and internal structure of the beads prepared using the SA–calcium chloride
method are shown in Fig. 3. The diameter of the SA beads was 4.31 ± 0.26 mm. Poly-
porous particles of sugarcane bagasse embedded in SA beads could provide more area for
bacterial adhesion.

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A
12 0.5

NH4-N concentration
NH4-N
10 OD600
0.4
8

OD 600
(mg/L)
0.3
6
0.2
4
2 0.1
0 0
0 3 6 9 12
Time(hour)
B 0.4

0.3
OD 600

0.2

0.1

0.0
0 3 6 9 12
Time(hour)
C 14 2.0
NO2-N concentration

NH4-N concentration
NO2-N NH4-N
12
10 1.5
(mg/L)

(mg/L)
8
1.0
6
4 0.5
2
0 0.0
0 3 6 9 12
Time(hour)
D 25 0h
TN concentration

12 h
20
(mg/L)

15

10

0
NH4-N NO2-N
group

Fig. 2 Variations in the OD600 and NH4-N concentrations a when Vibrio alginolyticus VZ5 absorbs NH4-N.
Variations in the OD600 (b) and the concentrations of NH4-N and NO2-N c when Vibrio alginolyticus VZ5
absorbs NO2-N. Variations in the TN concentration d when Vibrio alginolyticus VZ5 absorbs NH4-N and
NO2-N. Data are represented as the mean ± standard deviation

Bacterial density variation of the SA beads

Figure 4a shows the effect of cross-linking on the bacterial density of SA beads. Bacteria
embedded in SA beads at 6.00 9 103 cfu/bead, 3.00 9 104 cfu/bead and 2.50 9 105 cfu/
bead remained stable or increased slightly, but bacteria embedded in SA beads at

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Fig. 3 Photograph of SA beads prepared by the SA–calcium chloride method (a). Scanning electron
microscopy (SEM) micrograph of the interior of the SA beads (b). The structure of sugarcane bagasse
embedded in an SA bead (c)

A 1.0E+10 6.00E+03 3.00E+04

2.50E+05 5.33E+08
Bacterial dendity (cfu/bead)

1.0E+08

1.0E+06

1.0E+04

1.0E+02

1.0E+00
0 12
Time(hour)

B 1.0E+09 7.00E+03 3.25E+04

2.00E+05 4.00E+07
Bacterial dendity (cfu/bead)

1.0E+08

1.0E+07

1.0E+06

1.0E+05

1.0E+04

1.0E+03
0 2 4 6 8
Time(day)

Fig. 4 Effect of cross-linking on bacterial density of SA beads immobilized with different initial bacterial
densities (6.00 9 103 cfu/bead, 3.00 9 104 cfu/bead, 2.50 9 105 cfu/bead, 5.33 9 108 cfu/bead) (a) and the
variation in bacterial density of SA beads immobilized with different bacterial densities (7.00 9 103 cfu/
bead, 3.25 9 104 cfu/bead, 2.00 9 105 cfu/bead, 4.00 9 107 cfu/bead) in a synthetic solution (b). Data are
represented as the mean ± standard deviation

5.33 9 108 cfu/bead decreased from 5.33 9 108 cfu/bead to 4.00 9 107 cfu/bead during
the cross-linking process.
The results (Fig. 4b) showed the variations in the bacterial density of the SA beads with
different initial bacterial densities in synthetic wastewater. Bacterial densities of 7.00 9 103

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cfu/bead, 3.25 9 104 cfu/bead and 2.00 9 105 cfu/bead increased to 8.75 9 106–5.00 9 107
cfu/bead. In contrast, bacterial density of 4.00 9 107 cfu/bead slightly increased to 1.6 9 108
cfu/bead.

NO2-N degradation by SA beads

The SA beads were highly efficient at degrading NO2-N (Fig. 5a). In 2 days, the NO2-N
concentration in B–B (SA beads used) was reduced by 87.12 % and reached zero after 4 days.
In contrast, the NO2-N concentration showed almost no changes in the other three treatments.

Fig. 5 Variation in the A BL BS

concentrations of NO2-N in the 12
NO2-N concentration (mg/L)

NB-B B-B
BL, BS, NB-N and B–B groups
(a) and for SA beads 10
immobilized with different
8
bacterial densities (2.50 9 104
cfu/bead, 7.75 9 105 cfu/bead, 6
5.50 9 106 cfu/bead, 8.50 9 107
cfu/bead) (b). The degradation 4
rates of the SA beads (c). Data
are represented as the 2
mean ± standard deviation
0
0 2 4
Time(day)

B 12
2.50E+04 7.75E+05
NO2-N concentration (mg/L)

5.50E+06 8.50E+07
10

0
0 1 2
Time(day)

C 10
Degradation rate (mg/L/d)

9
8
7
6
5
4
3
2
1
0
1 2 3 4 5 6 7 8 9
Time(day)

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Figure 5b shows the variations in NO2-N concentration for SA beads with different bacterial
densities. In the first day, the NO2-N concentration was reduced sharply, and the degradation
rate of the SA beads with bacterial densities of 2.50 9 104 cfu/bead, 17.75 9 105 cfu/bead,
5.50 9 106 cfu/bead and 8.50 9 107 cfu/bead was 46.95, 58.29, 62.28 and 36.90 %, respec-
tively. The NO2-N concentration in all four treatments was almost zero after 4 days.
Figure 5c shows the NO2-N degradation rates for the SA beads. Initially, the NO2-N
degradation rate increased, and the maximum degradation rate was 8.44 mg/L
day on the
third day. The degradation rate then gradually declined, and NO2-N degradation had nearly
ceased by the eighth day. Approximately 3.88 mg of NO2-N was degraded by the SA beads
in 8 days, and the average NO2-N degradation per bead was 0.06 mg.

Effect of carbon source addition on the degradation of NH4-N and NO2-N

by SA beads

Addition of a carbon source promoted the removal of NH4-N (Fig. 6a) and NO2-N
(Fig. 6b) (P \ 0.05, df = 29). The NH4-N concentration in the C ? NH4-N group
decreased sharply in the first 3 days (P \ 0.05, df = 29) and increased slightly during the

A 10 C+NH4-N
NH4-N
NH4-N concentration (mg/L)

0
0 1 2 3 4 5
Time(day)

B 14 C+NO2-N NO2-N 14
C+NO2-N NO2-N
12 12
NH4-N concentration (mg/L)
NO2-N concentration (mg/L)

10 10

8 8

6 6

4 4

2 2

0 0
0 1 2 3 4 5
Time(day)

Fig. 6 Effect of carbon source addition on NH4-N degradation (a) and NO2-N degradation (b). Data are
represented as the mean ± standard deviation. Note open bar and closed bar represent the concentrations of
NH4-N and NO2-N, respectively

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final period. As in the C ? NH4-N group, the NH4-N concentration in the NH4-N group
decreased in first 3 days, but the NH4-N degradation rate was only 52.27 %, which is
significantly lower than the rate observed in the C ? NH4-N group (P \ 0.05, df = 29).
The NO2-N degradation rate in the C ? NO2-N group was significantly greater than the
rate observed in the NO2-N group (P \ 0.05, df = 29). The NO2-N in the C ? NO2-N
group was completely degraded in 3 days, and it was completely degraded in the NO2-N
group in 4 days. The NO2-N concentration in the NO2-N group decreased as the NH4-N
concentration increased, but this effect was not observed in the C ? NO2-N group. In
contrast, the NH4-N concentration in the C ? NO2-N group was significantly lower than in
the NO2-N group (P \ 0.05, df = 29).

Activity of SA beads in shrimp culture

During the experiment, the temperature, salinity, pH and DO were within normal ranges,
and there were no obvious fluctuations (Table 1).
The NH4-N concentrations in all of the treatment groups started falling after 10 days
(Fig. 7a). In contrast, the NH4-N concentration in the NW group declined more slowly than
in the other groups. It is noteworthy that NH4-N concentrations in the WE, SW and RW
groups were much lower than in the NW group (P \ 0.05, df = 29). In addition, there
were no significant differences between the WE, SW and RW groups (P [ 0.05, df = 29).
Compared with the other treatments, the NO2-N concentration in the NW group was higher
(Fig. 7b) (P \ 0.05, df = 29), especially in the late stage. The NO2-N concentrations in the
SW and RW groups declined sharply from the 30th day to the 40th day. By the 50th day,
the NO2-N concentrations in the WE, SW and RW groups had fallen below 1.62 mg/L.
The trends for the TN concentrations in all of the treatments were similar (Fig. 7c),
showing an increase in the late period. Compared with the NW, SW and RW groups, the
TN concentration in the WE group was lower (P \ 0.05, df = 29).
During the first 20 days, the DOC concentrations in the SW and NW groups were
similar (Fig. 7d). After 20 days, the DOC concentration in the SW group increased and
remained higher than the DOC concentration in the NW group until the end of experiment
(P \ 0.05, df = 29). The maximum DOC concentrations in the SW and NW groups were
18.00 and 14.69 mg/L, respectively. After 30 days, the POC concentration in the SW
group was higher than in the NW group (Fig. 7e), and the POC concentrations in the SW
and NW groups at the end of experiment were 14.05 and 6.66 mg/L, respectively. The
results (Fig. 8) showed the variation in carbon content in the sugarcane bagasse in the
treatment unit in the RW group during the experiment. The carbon content in the sugarcane
bagasse decreased from 36.28 to 31.75 %, and the degradation rate reached 12.49 %.

Table 1 Variations in the temperature, salinity, pH and DO during the cultivation experiment
Temperature (°C) Salinity (%) pH DO (mg/L)

NW 24.7 ± 0.78 19.96 ± 0.41 6.72–8.14 5.31 ± 0.95

WE 24.70 ± 0.78 19.76 ± 0.27 7.13–8.17 5.44 ± 0.90
SW 24.69 ± 0.70 20.13 ± 0.30 6.87–8.09 5.35 ± 0.86
RW 24.71 ± 0.91 19.96 ± 0.23 6.88–8.12 5.48 ± 0.92
NW, WE, SW and RW represent treatment with no water exchange, water exchange, in situ treatment and
recirculating water, respectively. Data are represented as the mean ± standard deviation

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A D
NH4-N concentration (mg/L

25

DOC concentration (mg/L)

NW WE
20
)

20 SW RW NW SW

15 15

10 10

5 5

0 0
0 10 20 30 40 50 0 10 20 30 40 50
Time(day) Time(day)
B E
NO2-N concentration (mg/L)

20

POC concentration (mg/L)

30 NW WE
NW SW
SW RW
25 15
20
15 10

10
5
5
0 0
0 10 20 30 40 50 0 10 20 30 40 50
Time(day) Time(day)
C
50 NW WE
TN concentration (mg/L)

SW RW
40

30

20

10

0
0 10 20 30 40 50
Time(day)

Fig. 7 Variation in the concentrations of NH4-N (a), NO2-N (b), TN (c), POC (d) and DOC (e) during the
cultivation experiment. NW, WE, SW and RW represent treatment with no water exchange, water exchange,
in situ treatment and recirculating water, respectively. Data are represented as the mean ± standard
deviation

The best growth parameters were observed in the WE group (Table 2). Compared with
the WE group, the body length, survival rate and yield were lower in the NW group
(P \ 0.05, df = 5). There was no significant difference between the WE and SW groups in
terms of survival rate or yield (P [ 0.05, df = 5). The yield after treating the water with SA
beads reached approximately 70 % of the yield of treating the water via water exchange.

Discussion

Previous studies have demonstrated that Vibrio sp. can fix nitrogen (Flores-Mireles et al.
2007; Chimetto et al. 2008), and some strains, such as V. alginolyticus, have been used in
aquaculture as a probiotic (Gomez-Gil et al. 2002). However, in some studies, V.

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Fig. 8 Variation in carbon 40

Carbon content in sugarcane bagasse (%)

content in the sugarcane bagasse a
in the treatment units. Data are
represented as the
mean ± standard deviation.
Different letters indicate
35
significant differences between
groups (P \ 0.05) b

30

25
Initial Final

Table 2 Body length, survival rate and yield at the end of the cultivation experiment
Body length (cm) Survival rate (%) Yield (kg/m2)

NW 8.08 ± 0.08a 19.61 ± 3.40a 0.12 ± 0.02a

WE 9.03 ± 0.31b 50.98 ± 12.25b 0.40 ± 0.08c
SW 8.72 ± 0.41ab 39.22 ± 8.99ab 0.29 ± 0.06bc
RW 8.29 ± 0.10a 37.25 ± 3.40ab 0.24 ± 0.03ab
NW, WE, SW and RW represent treatment with no water exchange, water exchange, in situ treatment and
recirculating water, respectively. Data are represented as the mean ± standard deviation
Different letters indicate significant differences between groups (P \ 0.05)

alginolyticus was reported as a pathogen of shrimp (Lee et al. 1996). This may be related to
the fact that different V. alginolyticus strains have obvious genetic diversity, resulting in
virulence greatly different (George et al. 2005). The V. alginolyticus VZ5 used in our study
was identified as a non-virulence strain in previous experiments by hemolytic test, drug
sensitive test and shrimp infection test. In addition, it is safe to use V. alginolyticus VZ5 in
the food industry because the bacteria released into water could not reach the densities
threatening human health. This study presents the first evidence that V. alginolyticus could
be used in immobilization technology for ammonia and nitrite nitrogen removal in shrimp
culture.
The discoveries of aerobic denitrification bacteria provided a new biological technology
pathway of denitrification and renewed the traditional theory of biological nitrogen
removal (Pai et al. 1999). In the present study, V. alginolyticus VZ5 showed efficient NH4-
N and NO2-N removal capacity. The NO2-N concentration decreased accompanied by the
increase in NH4-N concentration indicating that V. alginolyticus VZ5 could transform
NO2-N into NH4-N in the process of denitrification. Based on the data in Fig. 2, we assume
that the NH4-N and NO2-N removal processes of V. alginolyticus VZ5 proceed as NH4-
N ? bacterial protein and NO2-N ? NH4-N ? bacterial protein, respectively.
The SA beads had a maximum capacity of 1.06 9 108 cfu/bead. The loading capacity of
the immobilized beads was limited because too many bacteria could produce a large
amount of metabolites to restrain bacteria growth and cause intense competition. The best

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initial bacterial density in the SA beads was 104–105 cfu/bead when the effect of cross-
linking on bacterial density was considered. Sugarcane bagasse embedding in SA beads
provided good substrates for bacteria growth; therefore, even a lower bacterial density was
used in the SA beads at initially, the immobilized bacteria would be able to replicate inside
the SA beads and attain a higher density later on.
Sodium alginate is a naturally occurring polysaccharide used as an excipient in the food
industry (Gombotz and Wee 2012). In BS group, the C:N ratio is low in the wastewater to
restrain the V. alginolyticus VZ5 growth, resulting in little NO2-N removal (Fig. 5a). In
contrast, SA could be used as a carbon and energy source by the bacteria, so the SA beads
showed higher efficiency for NO2-N degradation in B–B group. The activity of the SA
beads gradually decreased with the deterioration and structural change. It is noteworthy
that for applications in aquaculture systems, biodegradable materials are preferred (Manju
et al. 2009). Sodium alginate was selected as gel matrices because it could be degraded by
bacteria without forming new pollution.
The addition of a carbon source promotes NH4-N and NO2-N removal. The C:N ratio in
the medium is known to affect microbial ammonia assimilation and microbial degradation
of organic nitrogen (Zehr and Ward 2002). Denitrifying organisms require an organic
carbon source for their respiration and growth (Cao et al. 2002). Thus, carbon addition can
accelerate nitrogen degradation by bacteria (Xiao et al. 2011). Water exchange is a
common and effective method for NH4-N and NO2-N removal in aquaculture. However, it
can cause many environmental problems (Briggs and Funge-Smith 1994; Naylor et al.
1998). The present study demonstrated that NH4-N and NO2-N removal using SA beads
could achieve the same effect as water exchange, indicating that SA beads can be used for
NH4-N and NO2-N removal in shrimp culture. No mass mortality was observed during the
culture period, indicating that V. alginolyticus VZ5 was not pathogenic to the shrimp. The
yield for water treatment with SA beads reached approximately 70 % of the yield for water
treatment with water exchange.
It is also noticeable that the POC and DOC concentrations in the water were enhanced
by the addition of SA beads. The reason for this may be that in the late period of the study,
some of the SA beads had broken down, and the particles embedded in the SA beads
became suspended in the water, contributing to the increased POC concentration. The
cellulose of the sugarcane bagasse could be degraded to produce dissolved carbohydrates
(Pandey et al. 2000), and the DOC increase may be ascribed to the degradation of the
cellulose in the sugarcane bagasse. In addition, suspended particles of sugarcane bagasse
carrying large amounts of bacteria can help to maintain water quality. Ramesh et al. (1999)
reported that sugarcane bagasse is a favorable carrier for bacteria because it forms a
primary bacterial biofilm and can enhance the growth of Cyprinus carpio carp and Labeo
rohita rohu when used as a supplementary food. Thus, although some of the SA beads were
broken down in the later period of the present study, water quality might be maintained
well by sugarcane bagasse, contributing no environmental stress during shrimp culture.
Additionally, the effects of suspended particles of sugarcane bagasse from the SA beads
should be assessed in further studies. The SA beads themselves should also be optimized to
improve their physical stability.

Acknowledgments This study was supported by the National Science and Technology Supporting Plan of
the Twelfth Five-Year (2011BAD13B10, 2011BAD13B03), Ministry of Agriculture’s special funds for
scientific research on public causes (201103034). The authors are grateful to the referees for their helpful
comments on the manuscript.

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