Aquacultural Engineering 52 (2013) 7379

Contents lists available at SciVerse ScienceDirect

Aquacultural Engineering
journal homepage: www.elsevier.com/locate/aqua-online

Inorganic nitrogen dynamics in sequencing batch reactors using biooc

technology to treat aquaculture sludge
Guo-zhi Luo a,1 , Yoram Avnimelech b,1 , Yun-feng Pan a,1 , Hong-xin Tan a,,1
a
b

College of Fisheries and Life Science of Shanghai Ocean University, Shanghai 201306, China
Faculty of Agricultural Engineering, Technion, Israel Institute of Technology, Haifa 32000, Israel

a r t i c l e

i n f o

Article history:
Received 25 January 2012
Accepted 23 September 2012
Keywords:
Sequence batch reactor
External biooc reactor
Aquacultural suspended solids
Organic carbon source

a b s t r a c t
Inorganic nitrogen dynamics in a sequencing batch reactor (SBR) using biooc technology (BFT) to treat
suspended solids (SS) in a recirculating aquaculture system (RAS) stocking jade perch (Scortum barcoo)
were explored in the current study. One group was dosed with glucose (SBR-glu) as an organic carbon
source, and the other group had no added carbon source (SBR-con). The results showed that ammonium
released from aquaculture SS was assimilated by heterotrophic bacteria to form bioocs with sufcient
additions of a carbon source. Autotrophic nitrication was also observed, perhaps due to the shortcomings
of the carbon source. The 5-min oc volume index (FVI-5) was lowest on the 8th day, when the dissolved
oxygen was also at its lowest level. The crude protein content of the ocs from SBR-glu was 30.42 0.55%,
whereas that of SBR-con was 26.32 0.78% during the rst 33 days. Sharp reductions in ammonium
and nitrate in SBR-glu were found: within 6 h, the ammonium decreased from 13.22 0.98 mg N/L to
0.40 0.02 mg N/L, and within 5 h, the nitrate decreased from 72.41 1.34 mg N/L to 0.10 0.02 mg N/L.
2012 Elsevier B.V. All rights reserved.

1. Introduction
Aquaculture is predicted to increase 5-fold until 2050 (FAO,
2012). To meet this growing demand, aquaculture is shifting from
extensive cultivation systems to more intensive systems. With the
intensication of aquaculture, the focus has increasingly shifted to
its negative environmental and social impacts. Even RAS, which are
considered to provide more advantages than traditional aquaculture models, have also been reported to accumulate 1140% of the
applied feed in the form of discharged sludge (Hopkins et al., 1994).
Currently, the focus has been on capturing and reusing dissolved
waste (Neori et al., 2004); however, less attention has been given
to feces and feed residue, which are often discharged as sludge,
leaving a signicant amount of unused nutrients (Chen et al., 1996).
A potential alternative, referred as BFT, could utilize heterotrophic bacteria to convert nitrogen in aquaculture SS into
bacteria biomass (De Schryver and Verstraete, 2009), which could
potentially be used to feed sh, thereby increasing the efciency
of nitrogen. BFT, i.e. a co-culture of aquaculture species and heterotrophic bacterial biomass within the same solution (called an
in situ BFT-aquaculture system) has already been exploited in pond
aquaculture systems for tilapia (Azim and Little, 2008; Crab et al.,

Corresponding author. Tel.: +86 21 61900402; fax: +86 21 61900413.

E-mail address: drizzles@163.com (H.-x. Tan).
1
Present address: Department of Fisheries and Life Science, Shanghai Ocean University, Hucheng Ring Road 999, Shanghai 201306, China.
0144-8609/$ see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.aquaeng.2012.09.003

2009), shrimp (Asaduzzaman et al., 2008; Burford et al., 2004),

shrimp and tilapia together (Asaduzzaman et al., 2009), and catsh
and tilapia together (Brune et al., 2003). However, the in situ BFT
aquaculture system does not appear to be appropriate for species
that do not graze on biooc directly, such as carnivorous sh. As a
result, further development of mixed systems has been advocated
in which culture units are portioned with ocs to develop a hybrid
technology of BFT and RAS (Avnimelech and Teubal, 2006; Azim
and Little, 2008; De Schryver and Verstraete, 2009).
BFT is based on the assimilation of inorganic nitrogen species
(TAN, NO2 -N, and NO3 -N) by the microbial community present
within the aquaculture water (Avnimelech, 2006); it is affected
and controlled by adjusting a high C:N ratio in the system (Azim
et al., 2008). For BFT used in the biological treatment of municipal and industrial wastewaters, studies have primarily focused
on the physical properties of ocs that contribute to hindering
ocs settling and compactability (Nguyen et al., 2008). The goal
for BFT in aquacultural applications is to remove and recover the
nitrogen. Thus, water quality, nitrogen syndrome, and the nutrient composition of bioocs during the bioocculation process are
worthy of a research investigation focused on more than their physical properties. There are numerous reports regarding water quality
dynamic changes for in situ BFT-aquaculture systems, while reports
focused on the treatment of aquacultural sludge are limited. In a
BFT aquaculture system, the assimilation of heterotrophic bacteria
was stimulated because the increase in microbial biomass production is 40 times greater than the biomass production generated
from the nitrication process (8.07 g versus 0.20 g) (Ebeling et al.,

74

G.-z. Luo et al. / Aquacultural Engineering 52 (2013) 7379

2006). However, nitrication was very frequently found in in situ

BFT aquaculture systems (Avnimelech, 2012). Nitrication in a BFT
aquaculture system is adequate for water quality control but not
suitable for reusing N. In the current study, the dissolved inorganic
nitrogen dynamics of a lab-scale SBR to treat SS from a RAS stocking
Scortum barcoo were explored. The main objective of this study was
to evaluate some of the principle processes enabling the operation
of in sequencing batch reactors using biooc technology to treat
aquaculture sludge and focused on the reuse of nitrogen in aquaculture sludge. This work represents the rst part of the requisite
comprehensive research and development efforts.
2. Materials and methods
A laboratory-scale integrated RASBFT combined recycle system was established in the Center for Recirculation Aquaculture
Systems of Shanghai Ocean University. The system was comprised
of two compartments: (a) a lab-scale RAS culturing adult S. barcoo
and (b) BFT reactors constructed of polyvinylchloride drums (30 L
each), where RAS efuent treatment was performed and observed.
Water and sludge collected in the sand lter was batch fed into
the BFT reactors periodically. In the current experiment, the BFT
reactors received a one-time addition of water and waste from
RAS. It was focused on the water quality, nitrogen syndrome and
the nutrient composition of bioocs during the start-up period of
reactors.
2.1. RAS compartment features and operation
Three RAS were used in this experiment. Each RAS was equipped
with four tanks (volume of each tank was 1 m3 ), sand, and biological
lters. A moderate ow-through rate allowed the renewal of water
to the system 24 times per day at a rate of approximately 10 L/min.
The water was continuously aerated using compressed air root
blowers. The total water volume of each RAS was 10 m3 . The average stocking density of S. barcoo in each RAS was 40 2.2 kg/m3 .
The sh were fed a commercial pellet diet (moisture 3%; crude
protein 45.0%; crude lipid 8.0%; Ca 1.8%; P 1.5%; lysine 2.9%; and
methionine 1.4%; Suzhou Tong Wei Special Feed Co., Ltd., Wujiang,
China).

biooc reactors (SBR-glu) and (b) control biooc reactors without

an added carbon source (SBR-con). The dissolved organic carbon
to total ammonium nitrogen ratio (DOC:TAN) in the glucose treatment was maintained at >10, adjusted to the TAN concentration
measured. During the start-up period, a total of 46 g of glucose was
added to the SBR-glu, consisting of 6 g on May 12, 6 g on May 13,
6 g on May 14, 18 g on May 15, and 10 g on May 16.
Each reactor was supplied with 8 L of efuent from the RAS
system. The range of concentrations in the efuent were as follows: dissolved oxygen (DO): 7.28 0.45 mg/L, pH: 7.23 0.32,
DOC: 35.23 0.17 mg/L, TAN: 5.23 0.68 mg/L, nitrite (NO2 -N):
0.65 0.12 mg/L, and nitrate (NO3 -N): 15.67 2.13 mg/L. The suspended solids (total SS: 129.6145.3 g/L; pH: 6.256.67; total
COD: 182.7241.2 g/L; SCOD: 25.332.5 g/L; total Kjeldahl nitrogen (TKN): 20.124.3 g/L) from RAS were completely pulverized,
ltered through a 10-m mesh, and the ltrate added to each reactor in equal amounts as a TSS source; the initial TSS concentration
was 1000 mg/L.
The water temperature, DO, pH, TAN, NO2 -N, NO3 -N, DOC, TN,
and 5-min oc volume index (FVI-5) were determined at 09.00 h
each day during the start-up period. Biooc samples for biochemical analysis were collected from each reactor on day 33, at the end
of the start-up experiment.
2.3.2. The second experimental phase
The second experimental phase began after the 33-day startup period when the TAN, NO2 -N, and NO3 -N in reactors were
stable. The goal of this phase was to focus on the capacity of the
reactors to assimilate TAN. Glucose (15 g) and 0.5 g of NH4 Cl were
added to SBR-glu; only 0.5 g of NH4 Cl was added to SBR-con. Water
temperature, DO, pH, TAN, NO2 -N, NO3 -N, DOC, TN, and FVI-5
were determined hourly.
2.3.3. The third experimental phase
Two days later, the capacity of the reactors to remove nitrate
was tested. An additional 10 g of glucose and 0.5 g of KNO3 were
added to the SBR-glu, and only 0.5 g of KNO3 was added to SBRcon. The water temperature, DO, pH, TAN, NO2 -N, NO3 -N, DOC,
TN, and FVI-5 were measured hourly.

2.2. Lab-scale external biooc reactors

2.4. Methods of determining experimental parameters

In the current study, six SBRs were installed. Each reactor had
an internal diameter of 20 cm and a liquid-lled height of 16 cm
(equivalent to an 8-L working volume) and was maintained at
2326 C. The water in each reactor was intensively mixed with
an airstone placed at the bottom of the reactor powered by means
of a 138-W air pump (ACO-008, SenSen Co., Ltd., Zhejiang, China)
operating at a rate of approximately 20 L/min.

2.4.1. Water quality parameters

Water temperature, pH, and DO were measured using a YSI556
meter (YSI Inc., 1725 Brannum Lane, Yellow Springs, OH, USA). DOC,
TAN, NO3 -N, and NO2 -N were computed by using grab samples
ltered through a 0.45-m lter. TAN, NO2 -N, NO3 -N, total Kjeldahl nitrogen (TKN), and TSS were determined according to APHA
(2005). The DOC sample was evaluated using a Total Organic Carbon Analyzer (TOC-V.CPH, Shimadzu Seisakusho, Japan). Dissolved
inorganic nitrogen (DIN) was calculated as the sum of the concentrations of TAN, NO2 -N, and NO3 -N.

2.3. Experimental design

The current study focused on the start-up process. SBR was used;
however, wastewater and SS were added only at the beginning, and
there were no additions of water and SS during the experiment
period. Thus, the hydraulic retention time (HRT) and sludge retention time (SRT) were not calculated. The experiment was divided
into three phases.
2.3.1. The start-up phase
The rst part of the study consisted of building up the reactors
until reaching the point at which the water composition stabilized. Start-up of the reactors was conducted from May 12, 2010
to June 14, 2010 using two treatments (three replications each)
of the following: (a) glucose (carbon content 40%) added to the

2.4.2. Biochemical composition of the ocs

The ash content was determined by suspension ltered through
a glass ber lter. The lter was dried, weighed and burned in
a mufe furnace at 550 C for 4 h; the ash was then cooled and
weighed. The crude protein content was determined by the Kjeldahl
method using the Kjeldahl nitrogen determination apparatus (Kjeltec 8400, FOSS, Sweden) according to standard methods (APHA,
2005). The protein content was calculated using the assumption
that it contained 16% nitrogen and was expressed as a percentage
of the bio-oc dry matter weight (DMW) (AOAC, 1999). The FVI was
calculated from the oc volume after 5 min of sedimentation in an
Imhoff cone (1000 mL; FVI-5, %).

G.-z. Luo et al. / Aquacultural Engineering 52 (2013) 7379

A
(a)

N (mg/L)

100

60
(c)

40
20
10

15

80

(a) TAN in SBR-CON

(b) Nitrite in SBR-CON
(c) Nitrate in SBR-CON

60

(c)

100

40

(b)
5

(a)

120

(a) TAN in SBR-GLU

(b) Nitrite in SBR-GLU
(c) Nitrate in SBR-GLU

80

140

N (mg/L)

120

75

(b)

20

20

25

30

35

10

15

20

25

30

35

Fig. 1. (A) Changes in TAN (a), NO2 -N (b), and NO3 -N (c) concentrations during the start-up period of sequencing batch reactors with glucose added; (B) changes in TAN
(a), NO2 -N (b), and NO3 -N (c) concentrations during the start-up period of sequencing batch reactors for the control group.

2.5. Statistical analysis

Statistical analysis was performed using SPSS 16.0 for Windows.
Differences in water quality were considered signicant when
P < 0.05. One way analysis of variance was used on the water parameters and the oc composition. The carbon addition treatment was
the factor.
3. Results
3.1. Dissolved inorganic nitrogen syndrome during the start-up
period
The TAN, NO2 -N, and NO3 -N concentrations throughout the
start-up period are shown in Fig. 1. In both the control and
glucose-treated samples, there was an initial build-up of TAN. The
peak of TAN concentration in the control treatment was at day 8
(120 mg N/l); thereafter, TAN dropped to undetectable levels on
day 15. A similar rise in TAN was seen in SBR-glu; however, it
peaked on day 6 (110 mg N/L) and dropped to an undetectable level
on day 10. NO2 -N concentrations increased conformingly with
TAN decline, and NO3 -N concentrations increased correspondingly with a decline in NO2 N for both SBR-con and SBR-glu.
3.2. Removal of DOC and the C:N ratio during the start-up period
The DOC level in SBR-glu was signicantly higher than that of
SBR-con due to the added glucoses. Interestingly, the DOC values of SBR-glu and SBR-con were similar from day 15 through the
end of the start-up experiment, measuring 103.4 2.9 mg/L and
75.3 3.1 mg/L, respectively. The removal of the DOC in SBR-glu
was very effective because of the 46 g glucose added, which was
equivalent to 18.4 g DOC.

The DOC:TAN ratios of SBR-glu were above 15 during the 33-day

period. The DOC:TAN ratios of SBR-con were below 6 prior to day
12, and then sharply rose due to a decrease in TAN.
3.3. FVI-5 and DO during the start-up period
The trends in FVI-5 and DO of SBR-glu were similar to those
of SBR-con (Fig. 2), while the FVI-5 and DO of SBR-glu were signicantly lower than those of SBR-con; the lowest values of FVI-5
(36%) and DO of SBR-glu occurred at the same time, on day 8.
3.4. Second experimental phase: assimilation of ammonium
After the start-up period, the additions of 0.5 g of NH4 Cl and
10 g of glucose were observably eliminated within 6 h, while no
production of NO2 -N or NO3 -N was observed during these 6 h
in SBR-glu (Fig. 3A). TAN and DOC were reduced by 12.6 mg/L and
433.3 mg/L, respectively (Fig. 4). Interestingly, NO3 -N in SBR-glu
decreased along with TAN and DOC, and the reduction in NO3 N in this trial was 36.72 mg N/L (Fig. 3). The reduction in DIN was
50 mg N/L in 6 h.
The DOC of SBR-glu was signicantly higher than that of SBR-con
due to the added glucose and decreased from 622.70 25.78 mg/L
to 198.40 16.78 mg/L, whereas the DOC of SBR-con did not
change, remaining at approximately 65 mg/L (Fig. 4A). The DO
(3.0 0.8 mg/L) of SBR-glu was noticeably lower than that of SBRcon (6.0 0.45 mg/L) (Fig. 4B). FVI-5 of SBR-glu decreased within 6 h
and was observably lower than that of SBR-con (Fig. 4C). The pH of
SBR-glu was measurably higher than that of SBR-con (Fig. 4D). A
signicant difference was observed between SBR-glu and SBR-con.
There were no perceptible changes in DO, FVI-5, DIN, DOC, TAN,
NO3 -N, or NO2 -N in SBR-con (Figs. 3 and 4).

100

(b)

90

FlVI (ml)

DO (mg/L)

110

5
4

(a) DO in SBR-GLU

(b) DO in SBR-CON

(a)

(b)

80
70
60

(a) FVI-5 in SBR-GLU

50
(a)

40

(b) FVI-5 in SBR-CON

30
0

10

15

20

25

30

35

10

15

20

25

30

35

Fig. 2. (A) Changes in dissolved oxygen (DO) during the start-up period of sequencing batch reactors with glucose added (a) and that of sequencing batch reactors for the
control group (b). (B) Changes in the ocs volume index (ocs volume index)-5 during the start-up period of sequencing batch reactors with glucose added (a) and that of
sequencing batch reactors for the control group (b).

76

G.-z. Luo et al. / Aquacultural Engineering 52 (2013) 7379

45

40

60

(a) TAN in SBR-GLU

(b) Nitrite in SBR-GLU
(c) Nitrate in SBR-GLU

35

50

(c)

25

N (mg/L)

N (mg/L)

30

20
(a)

15

(c)

40

(a) TAN in SBR-CON

(b) Nitrite in SBR-CON
(c) Nitrate in SBR-CON

30
20

(a)

10
10

(b)

(b)
0

Fig. 3. (A) Changes in TAN (a), NO2 -N (b), and NO3 -N (c) concentrations during the start-up period of sequencing batch reactors with glucose added in the assimilation of
ammonium experiment. (B) Changes in total ammonia nitrogen (TAN), nitrite (NO2 -N), and nitrate (NO3 -N) concentrations of sequencing batch reactors for the control
group in the assimilation of ammonium experiment.

3.5. Third experimental phase: reduction in NO3 -N

The observed sharp reduction in NO3 -N during the 6-h of
ammonium assimilation experiment prompted the subsequent
trial. After the water composition stabilized, a further 10 g of glucose and 0.5 g of KNO3 were added to SBR-glu and 0.5 g of KNO3 was
added to SBR-con. A signicant difference was observed between
SBR-glu and SBR-con. The NO3 -N concentration in the SBR-glu
decreased from 72.41 1.34 mg N/L to 0.10 0.02 mg N/L within
5 h. DIN decreased from 74.83 10.76 mg N/L to 2.4 0.87 mg N/L
(Fig. 5A). The concentration of DOC decreased 489.8 mg/L within
5 h (Fig. 6A). The DO level and FVI-5 of SBR-glu was signicantly

3.6. Floc composition

The crude protein and ash content of ocs derived from SBRglu were signicantly higher than those of SBR-con on day 33. The
biooc quality, in terms of sh nutrition, contained 30.42 0.55%
crude protein and 28.80 1.34% ash in the present study for SBR-glu
and 26.32 0.78% crude protein and 24.21 0.98% ash for SBR-con
at 33 d.

700

8
(a) DOC in SBR-GLU

600

(b) DOC in SBR-CON

500

(a)

DO (mg/L)

DOC (mg/L)

lower than that of SBR-con. The NO3 -N, TAN, NO2 -N, DOC, pH,
FVI-5, and DO levels remained nearly constant in SBR-con within
5 h (Figs. 5 and 6).

400
300
200

(a) DO in SBR-GLU
5

(b) DO in SBR-CON

(a)

(b)

100

(b)

95

(b)
(a) FVI-5 in SBR-GLU

75

(b) FVI-5 in SBR-CON

65

pH

FVI-5-(ml)

85

55

(a)

45
35
25
0

8.8
8.6
8.4
8.2
8.0
7.8
7.6
7.4
7.2
7.0
6.8
6.6
6.4

(a) pH in SBR-GLU
(a)

(b) pH in SBR-CON

(b)

Fig. 4. (A) Changes in dissolved organic carbon (DOC) of SBR-glu (a) and SBR-con (b) in the assimilation of ammonium experiment. (B) Changes in the dissolved oxygen (DO)
of SBR-glu (a) and SBR-con (b) in the assimilation of ammonium experiment. (C) Changes in the ocs volume index (FVI)-5 of SBR-glu (a) and SBR-con (b) in the assimilation
of ammonium experiment. (D) Changes in the pH of SBR-glu (a) and SBR-con (b) in the assimilation of ammonium experiment. SBR-glu, sequencing batch reactors added
with glucose; SBR-con, sequencing batch reactor for control group.

G.-z. Luo et al. / Aquacultural Engineering 52 (2013) 7379

A
(a) TAN in SBR-GLU
(b) Nitrite in SBR-GLU
(c) Nitrate in SBR-GLU

60
50

(c)

60
50
N (mg/L)

70

N (mg/L)

70

80

(c)

40
30

(a) TAN in SBR-CON

(b) Nitrite in SBR-CON
(c) Nitrate in SBR-CON

40
30
20

20

77

(a)

10

10
(a)
0

(b)

(b)

Fig. 5. (A) Changes in TAN (a), NO2 -N (b), and NO3 -N (c) concentrations during the start-up period of sequencing batch reactors with glucose added in the reducing nitrate
experiment. (B) Changes in TAN (a), NO2 -N (b), and NO3 -N (c) concentrations during the start-up period of sequencing batch reactors for controls in the reduced nitrate
experiment.

4. Discussion

4.1. TIN syndrome during start-up of the reactor

In a new BFT system, heterotrophic and nitrifying bacterial communities and biooc development will develop normally a few days
(Avnimelech, 2012). For ex situ SBR using BFT, the start-up progress
is necessary to establish an appropriate microbial community. And,
it is essential to decide the operation efciency of reactors. The current study focused on inorganic nitrogen dynamics during start-up
period in a SBR using BFT to treat aquacultural SS. The main objective of this study was to evaluate some of the principle processes
enabling to operate a combined RASBFT combination.

The TAN in SBR during the start-up period originated from the
crude protein of the SS added during this current study. Although
the TAN concentration was as high as 117.28 1.50 mg N/L, it
decreased within 5 days to less than 2 mg N/L (from day 5 to
day 10) with the addition of glucose, while no obvious production of NO2 -N or NO3 -N was observed during this phase. It
was thought that the TAN removed between day 5 and day 10
in SBR was mainly assimilated by lamentous bacteria to form
ocs in the current study, for the following reasons. Autotrophic

(b)

600

(b) DOC in SBR-CON

500

(a)

(a) DO in SBR-GLU

(b) DO in SBR-CON

400
300
200

(a)

(b)

100

100

pH-con
(a)

8.5

90

(b)

80

FVI(ml)

(a) DOC in SBR-GLU

DO(mg/L)

DOC(mg/L)

A
700

8.0

(a) FVI-5 in SBR-GLU

70

7.5

(b) FVI-5 in SBR-CON

60
50

(a) pH in SBR-GLU
(b) pH in SBR-CON

7.0

(a)

40

(b)

6.5

30
0

Fig. 6. (A) Changes in the dissolved organic carbon (DOC) of SBR-glu (a) and SBR-con (b) in the reduced nitrate experiment. (B) Changes in the dissolved oxygen (DO) of
SBR-glu (a) and SBR-con (b) in the reduced nitrate experiment. (C) Changes in the ocs volume index (FVI)-5 of SBR-glu (a) and SBR-con (b) in the reduced nitrate experiment.
(D) Changes in the pH of SBR-glu (a) and SBR-con (b) in the reduced nitrate experiment. SBR-glu, sequencing batch reactors with glucose added; SBR-con, sequencing batch
reactors for the control group.

78

G.-z. Luo et al. / Aquacultural Engineering 52 (2013) 7379

nitrication can be completed only when BOD5 < 20 mg/L (Okabe

et al., 1996). At the beginning of the experiment, the DOC in SBRglu was 1213.20 10.23 mg/L because of the added glucose (total
of 46 g). The high organic loading was thought to be the main factor inhibiting the autotrophic nitrication of SBR-glu. The DOC of
SBR-con was approximately 100 mg/L, and there were productions
of NO2 -N and NO3 -N following the decrease in TAN. In addition,
the lowest FV-5 and DO occurred simultaneously on day 8, which
also indicated that this was the time period when the ocs were
forming rapidly.
The effects of the organic substrate in inhibiting the nitrifying
bacteria were not toxic but stimulated the heterotrophic microorganisms to grow rapidly and to compete for dissolved oxygen,
space, TAN, and micro-nutrients. The organic substrate affected
neither the growth of the nitrifying bacteria nor the progress of
nitrication (Okabe et al., 1996). The production of NO2 -N and
NO3 -N was observed after day 15, while TAN was at a low level
until the end of the start-up experiment. It may be that this period
coincided with the establishment of the autotrophic nitrication
process. The same phenomena were observed in a study by Azim
et al. (2008): high rates of nitrication, indicated by constant nitrate
accumulation (Azim et al., 2008), were observed during BFT treatments.
It is more difcult to control C:N for BFT to treat aquacultural SS
than to treat waste water because of the ammonication process
of solid organic nitrogen in SS. The lack of a carbon source, even
for a very short period, will result in ammonia nitrication and the
production of NO2 -N. Consequently, further studies of necessary
pre-treatment of the SS are needed.
4.2. Assimilation of TAN and removal of NO3 -N
Theoretically, the process of TAN assimilation in BFT is relatively
fast if sufcient carbonaceous substrate is available. In a demonstration in which sugar was added to a pond containing 7 mg N/L
TAN, the ammonium concentration decreased to approximately
1 mg N/L (Avnimelech, 1999) within 5 h. In addition, it was found
that within a few hours, virtually all of the added ammonium was
contained in the biooc (Avnimelech and Kochba, 2009), which is
consistent with the results of the current study (Fig. 4).
The nitrogen formed in a typical reaction of BFT is from
TAN (NH4 + + 1.18 C6 H12 O6 + HCO3 + 2.06 O2 C5 H7 O2 N + 6.06
H2 O + 3.07 CO2 (Ebeling et al., 2006)). Some studies also reported
that BFT can introduce inorganic nitrogen into the microbial
biomass (De Schryver et al., 2008). However, there have been
few studies on the utilization of inorganic nitrogen sources other
than ammonium, for example, NO3 -N. Our research showed
that the reduction of NO3 -N by BFT is effective. It is noteworthy
that the reduction of NO3 -N may also be due to heterotrophic
denitrication in a BFT aquaculture system. This reduction can be
determined by the change in total nitrogen or the crude protein
content in ocs. 15 N tracing can be used to evaluate the pathways of
nitrate. Because the experiment involving the removal of NO3 -N
was derived from the experiment involving the assimilation of
NH4 -N in the current study, test indicators were not complete.
In the subsequent study, we focused on the quantication of the
NO3 -N pathways in SBR-BFT treating aquacultural SS; this study
is in the submission process.
4.3. Consumption of DO and the external BFT reactor
An adequate DO level is not only essential for the metabolic
activity of cells within aerobic ocs, but it is also thought to inuence oc structure (De Schryver et al., 2008). In this experiment, the
change in FVI-5 was the same as DO (Fig. 3). This result is similar to

the reports of Wiln and Balmr (1999), indicating a trend towards

larger and more compact ocs at higher DO concentrations.
In a BFT system, intensive heterotrophic growth in the culture
water results in turbidity and may induce variations in DO levels due to increased microbial metabolism resulting from carbon
dosing. Such varying DO levels may have a negative inuence on
the aquaculture organisms of in situ BFT. The implementation of
external heterotrophic growth reactors, in which high turbidity and
large variations in DO did not inuence the species in the aquaculture pond, may offer a solution (De Schryver and Verstraete, 2009).
In this case, the external reactor expands the use of BFT in aquaculture to include the treatment of the SS collected from the cage
aquaculture and RAS in large-scale processing. The water treated
in the external reactor is redirected to the aquaculture organisms,
and the excess microbial protein in the reactor can be harvested
and used in other aquaculture units or as a replacement ingredient for sh meal and soybean protein in shrimp feed (Kuhn et al.,
2009).
4.4. C:N ratio of BFT
When bacteria are fed with an organic substrate that contains primarily carbon and little or no nitrogen (sugar, starch,
molasses, etc.), they have to take up nitrogen from their aqueous environment in order to produce the protein needed for cell
growth and multiplication (Avnimelech and Kochba, 2009). The
C:N ratio of most of the feed stocks used in semi-intensive aquaculture ponds is approximately 10:1 (w/w), but bacteria require
approximately 20 units of carbon per unit of nitrogen assimilated
(w/w) (Avnimelech, 1999). Therefore, with such a low C:N ratio in
the feed, carbon is the limiting nutrient for heterotrophic bacteria populations in aquaculture ponds. Glucose was added in this
experiment as a carbon source to optimize the process of biooc
production.
The C:N ratio has been widely used as an index of the rate at
which organic matter will decompose (Alexander, 1961). Regulating the inorganic nitrogen by manipulating the C:N ratio is a
potential control method for aquaculture systems (Avnimelech,
1999). Theoretically, C:N refers to the carbon:nitrogen ratio of
atoms. However, there are several forms of C:N in related publications, such as TOC:NO3 -N (Sauthier et al., 1998), COD:NO3 -N
(Labelle et al., 2005), COD:TKN (Klas et al., 2006), C:N (w/w)
(Azim and Little, 2008; De Schryver and Verstraete, 2009), and
BOD5 :TKN (Metcalf and Eddy, 1991). The carbon in this experiment was biodegradable, as it mainly came from the sh food.
The C:N in this experiment refers to DOC:TAN, based on the
typical action of a BFT reactor (Ebeling et al., 2006). However,
rapid reduction of NO3 -N was also observed in this experiment.
Therefore, the DOC:TAN ratio used in this experiment may not
be the most appropriate. Accordingly, the most appropriate form
of C:N ratio in BFT needs to be further optimized in a future
study.
5. Conclusions
Inorganic nitrogen dynamics in sequencing batch reactors using
biooc technology to treat aquaculture sludge was explored in
the current study. The production of NO2 -N and NO3 -N was
observed, and it indicated the presence of nitrication during the
start-up period of the reactors. A sharp decrease in TAN and NO3 N in a short time period was enhanced by the addition of an organic
carbon supplement. This work represents the rst part of a requisite
comprehensive research and development effort entailing further
research concerning the pretreatment of aquacultural SS and the
quantication of NO3 -N pathways in BBR to treat aquacultural
sludge using BFT.

G.-z. Luo et al. / Aquacultural Engineering 52 (2013) 7379

Acknowledgments
This study was funded by the National Natural Science Foundation of China (31202033), Special Fund for Agro-Scientic
Research in the Public Interest (Project 201003024), and the
Twelve Five National Science and Technology Support Program
(2012BAD25B03). We would like to thank the anonymous reviewers for their remarks and suggestions.
References
Alexander, M., 1961. Introduction to Soil Microbiology. John Wiley and Sons, New
York, 472.
AOAC, 1999. Ofcial Methods of Analysis of the Association of Ofcial Analytical
Chemists, Method Nr. 92307. Washington, DC.
APHA, 2005. Standard Methods for Examinations of Water and Wastewater, 21st ed.
APHA/AWWA/WEF, Washington, DC.
Asaduzzaman, M., Wahab, M.A., Verdegem, M.C.J., Benerjee, S., Akter, T., Hasan,
M.M., Azim, M.E., 2009. Effects of addition of tilapia Oreochromis niloticus and
substrates for periphyton developments on pond ecology and production in
C/N-controlled freshwater prawn Macrobrachium rosenbergii farming systems.
Aquaculture 287, 371380.
Asaduzzaman, M., Wahab, M.A., Verdegem, M.C.J., Huque, S., Salam, M.A., Azim,
M.E., 2008. C/N ratio control and substrate addition for periphyton development jointly enhance freshwater prawn Macrobrachium rosenbergii production
in ponds. Aquaculture 280, 117123.
Avnimelech, G., Teubal, M., 2006. Creating venture capital industries that co-evolve
with high tech: Insights from an extended industry life cycle perspective of the
Israeli experience. Research Policy 35, 14771498.
Avnimelech, Y., 1999. Carbon/nitrogen ratio as a control element in aquaculture
systems. Aquaculture 176, 227235.
Avnimelech, Y., 2006. Bio-lters: the need for an new comprehensive approach.
Aquacultural Engineering 34, 172178.
Avnimelech, Y., 2012. Biooc Technology. World Aquaculture Society, Baton Rouge,
LA, USA.
Avnimelech, Y., Kochba, M., 2009. Evaluation of nitrogen uptake and excretion by tilapia in bio oc tanks, using 15N tracing. Aquaculture 287,
163168.
Azim, M.E., Little, D.C., 2008. The biooc technology (BFT) in indoor tanks: water
quality, biooc composition, and growth and welfare of Nile tilapia (Oreochromis
niloticus). Aquaculture 283, 2935.
Azim, M.E., Little, D.C., Bron, J.E., 2008. Microbial protein production in activated
suspension tanks manipulating C:N ratio in feed and the implications for sh
culture. Bioresource Technology 99, 35903599.
Brune, D., Schwartz, G., Eversole, A., Collier, J., Schwedler, T., 2003. Intensication
of pond aquaculture and high rate photosynthetic systems. Aquacultural Engineering 28, 6586.

79

Burford, M.A., Thompson, P.J., McIntosh, R.P., Bauman, R.H., Pearson, D.C., 2004. The
contribution of occulated material to shrimp (Litopenaeus vannamei) nutrition
in a high-intensity, zero-exchange system. Aquaculture 232, 525537.
Chen, S., Ning, Z., Malone, R.F., 1996. Aquaculture sludge treatment using an anaerobic and facultative lagoon system. In: International Conference on Recirculation
Technology, Roanoke, Virginia, Virginia-Tech.
Crab, R., Kochva, M., Verstraete, W., Avnimelech, Y., 2009. Bio-ocs technology application in over-wintering of tilapia. Aquacultural Engineering 40, 105112.
De Schryver, P., Crab, R., Defoirdt, T., Boon, N., Verstraete, W., 2008. The basics of bioocs technology: the added value for aquaculture. Aquaculture 277, 125137.
De Schryver, P., Verstraete, W., 2009. Nitrogen removal from aquaculture pond water
by heterotrophic nitrogen assimilation in lab-scale sequencing batch reactors.
Bioresource Technology 100, 11621167.
Ebeling, J.M., Timmons, M.B., Bisogni, J.J., 2006. Engineering analysis of the stoichiometry of photoautotrophic, autotrophic, and heterotrophic removal of
ammonia-nitrogen in aquaculture systems. Aquaculture 257, 346358.
Food and Agriculture Organization (FAO), 2012. The State of World Fisheries
and Aquaculture 2010. FAO Fisheries and Aquaculture Department, Food
and Agriculture Organization of the United Nations, Rome (accessed 11.7.12)
http://www.fao.org/docrep/013/i1820e/i1820e00.htm
Hopkins, J.S., Sandifer, P.A., Browdy, C., 1994. Sludge management in intensive pond
culture of shrimp: effect of management regime on water quality, sludge characteristics, nitrogen extinction, and shrimp production. Aquacultural Engineering
13, 1130.
Klas, S., Mozes, N., Lahav, O., 2006. A conceptual, stoichiometry-based model for
single-sludge denitrication in recirculating aquaculture systems. Aquaculture
259, 328341.
Kuhn, D.D., Boardman, G.D., Lawrence, A.L., Marsh, L., Flick Jr., G.J., 2009. Microbial oc meal as a replacement ingredient for sh meal and soybean protein in
shrimp feed. Aquaculture 296, 5157.
Labelle, M.-A., Juteau, P., Jolicoeur, M., Villemur, R., Parent, S., Comeau, Y., 2005. Seawater denitrication in a closed mesocosm by a submerged moving bed biolm
reactor. Water Research 39, 34093417.
Metcalf, E., Eddy, H., 1991. Wastewater Engineering: Treatment, Disposal, Reuse.
McGraw Hill Inc., Boston, MA.
Neori, A., Chopin, T., Troell, M., Buschmann, A.H., Kraemer, G.P., Halling, C., Shpigel,
M., Yarish, C., 2004. Integrated aquaculture: rationale, evolution and state of the
art emphasizing seaweed bioltration in modem mariculture. Aquaculture 231,
361391.
Nguyen, T.P., Hilal, N., Hankins, N.P., Novak, J.T., 2008. Characterization of synthetic
and activated sludge and conditioning with cationic polyelectrolytes. Desalination 227, 103110.
Okabe, S., Oozawa, Y., Hirata, K., Watanabe, Y., 1996. Relationship between population dynamics of nitriers in biolms and reactor performance at various C:N
ratios. Water Research 30, 15631572.
Sauthier, N., Grasmick, A., Blancheton, J.P., 1998. Biological denitrication applied
to a marine closed aquaculture system. Water Research 32, 19321938.
Wiln, B.-M., Balmr, P., 1999. The effect of dissolved oxygen concentration on the
structure, size and size distribution of activated sludge ocs. Water Research 33,
391400.