Aquaculture Research, 2013, 110

doi:10.1111/are.12126

Dietary protein level and C/N ratio manipulation in

zero-exchange culture of Litopenaeus vannamei:
Evaluation of inorganic nitrogen control, biofloc
composition and shrimp performance
Wu-Jie Xu & Lu-Qing Pan
The Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao 266003, China
Correspondence: Lu-Qing Pan, Laboratory of Environmental Physiology of Aquatic Animal, Fisheries College, Ocean University of
China, Yushan Road 5, Qingdao 266003, China. E-mail: panlq@ouc.edu.cn

Abstract
A 30-day experiment was conducted to evaluate
inorganic nitrogen control, biofloc composition
and shrimp performance in zero-exchange culture
tanks for juvenile L. vannamei offered a 35% (P35)
or 25% (P25) crude protein feed, each feed supplemented with additional carbohydrate to increase
the C/N ratio to 20:1 (CN20) or 15:1 (CN15).
Sucrose was used as a carbohydrate to manipulate
the two C/N ratios based on the carbon and nitrogen content of both the feeds and sucrose. The
four treatments were referred to as: P35 + CN20,
P35 + CN15, P25 + CN20 and P25 + CN15. Each
treatment consisted of four replicate tanks (125 L),
each stocked with 28 shrimp (equivalent to 224
shrimp m 3). Bioflocs formed and developed based
on initial inoculation in all four treatments; and
monitored water quality parameters were maintained within acceptable ranges for shrimp culture
throughout the experiment. No significant effects
(P > 0.05) of dietary protein level, C/N ratio or
their interaction were observed on biofloc development (BFV, TSS and BFVI) and inorganic nitrogen
(TAN, NO2 -N and NO3 -N) concentrations.
At the end of the experiment, proximate analysis
of the bioflocs collected from the four treatments
showed crude protein levels of 21.3% ~ 32.1%,
crude lipid levels of 1.6% ~ 2.8% and ash levels of
43.4% ~ 61.4%. Extracellular protease and amylase activities of the bioflocs were 9.9 ~ 14.4 U g 1
TSS and 293.5 ~ 403.8 U g 1 TSS respectively.
Biofloc composition and enzyme activity were both
affected by dietary protein level (P < 0.01) and
C/N ratio (P < 0.05). Survival, per cent weight
2013 Blackwell Publishing Ltd

gain and protein efficiency ratio of shrimp were

not affected (P > 0.05) by dietary protein level, C/
N ratio or their interaction; however, the feed conversion ratios were significantly lower (P < 0.05)
in treatments with high dietary protein (P35)
compared with those in treatments with low dietary protein (P25). The results from this study
demonstrate that dietary protein level and C/N
ratio manipulation can have important implications for water quality, biofloc composition and
shrimp performance in intensive, zero-exchange
biofloc-based culture systems.

Keywords: biofloc technology, zero-exchange,

inorganic nitrogen control, biofloc composition,
shrimp growth, feed utilization
Introduction
The application of biofloc technology (BFT) in
zero-exchange shrimp culture systems has gained
popularity because it offers a practical solution to
maintain water quality and recycle feed nutrients
simultaneously (Avnimelech 2006, 2008; Crab,
Avnimelech, Defoirdt, Bossier & Verstraete 2007;
De Schryver, Crab, Defoirdt, Boon & Verstraete
2008). In such systems, inorganic nitrogen control
is based on the promotion of dense populations of
active heterotrophic microorganisms that can
assimilate the nitrogen from the water column
resulting in the production of new microbial biomass (cellular proteins) (Avnimelech 2006; Crab
et al. 2007). As these microbial communities
develop, bioflocs (microbial flocs) are formed from
heterogeneous aggregates of microorganisms and

Biofloc manipulation for shrimp culture W-J Xu & L-Q Pan

organic particles (Hargreaves 2006; De Schryver

et al. 2008). These bioflocs can be consumed as a
supplemental food source by the shrimp, creating a
nutrient recycling process within the systems and
subsequently increasing feed utilization efficiency
(Burford, Thompson, McIntosh, Bauman & Pearson
2004; Schneider, Sereti, Eding & Verreth 2005;
Hargreaves 2006; Wasielesky, Atwood, Stokes &
Browdy 2006; Xu & Pan 2012; Xu, Pan, Zhao &
Huang 2012b). More importantly, several studies
noted that the application of BFT could improve
growth performance of cultured shrimp, primarily
attributed to the promotion of bioflocs (Wasielesky
et al. 2006; Arnold, Coman, Jackson & Groves
2009; Megahed 2010; Xu & Pan 2012; Xu et al.
2012b). It was thought that the biofloc, apart from
providing a portion of nutritional demand, can also
exert a positive effect on digestive enzyme activity of
shrimp and produce some microbial extracellular
enzymes, both of which could facilitate feed digestion and utilization (Xu & Pan 2012).
In zero-exchange BFT applications, a high feed
C/N ratio (10:1 to 20:1) is recommended for the
establishment of biofloc because the growth of heterotrophic bacteria can be greatly promoted by
providing substrates with a C/N ratio of 10:1 or
greater (Avnimelech 1999; Hargreaves 2006;
Asaduzzaman, Wahab, Verdegem, Huque, Salam
& Azim 2008; Ballester, Abreu, Cavalli, Emerenciano, Abreu & Wasielesky 2010). In many cases,
the C/N ratio can be increased by reducing the
protein content of feeds and/or supplementing the
feeds with carbohydrates (Avnimelech 1999; Hari,
Kurup, Varghese, Schrama & Verdegem 2004,
2006). Proteins are the most expensive ingredients
in practical feeds; and because feed represents at
least 50% of the total cost of shrimp production
(Naylor, Goldburg, Mooney, Beveridge, Clay, Folke,
Kautsky, Lubchenco, Primavera & Williams
1998), it is more cost-effective to use relatively
low protein feeds. Several authors have reported
that reduction in dietary protein levels had no significant effect on shrimp growth when the shrimp
were cultured in the presence of bioflocs (Wasielesky et al. 2006; Ballester et al. 2010; Xu et al.
2012b). On the other hand, as the C/N ratio of
most practical feeds (25% ~ 35% crude protein) is
around 10:1, requiring the addition of an alternative carbohydrate source to further increase the
feed C/N ratio, thereby promoting the development
of bioflocs within the systems (Avnimelech 1999;
Hari et al. 2004, 2006; Asaduzzaman et al. 2008;

Aquaculture Research, 2013, 110

Asaduzzaman, Rahman, Azim, Islam, Wahab, Verdegem & Verreth 2010; Xu et al. 2012b). Furthermore, several studies suggest that manipulating a
higher feed C/N ratio could increase biofloc community volume without compromising the nutritional quality of the biofloc (Azim, Little & Bron
2008; Asaduzzaman et al. 2010).
Although BFT has been applied and developed
in intensive culture systems of several shrimp species, especially L. vannamei (Crab et al. 2007;
Ballester et al. 2010; Xu & Pan 2012; Xu et al.
2012b; Zhao, Huang, Wang, Song, Yang, Zhang
& Wang 2012), the information concerning how
biofloc can be manipulated to maximize nutritional
benefits to the cultured shrimp is limited, and
therefore the further investigation into the nutritional composition and extracellular enzymes
activity of biofloc is necessary for the continued
development of BFT. This study was conducted to
evaluate the effects of two dietary protein levels
and two C/N ratios in zero-exchange culture tanks
stocked with L. vannamei on (1) inorganic nitrogen
control, (2) primary nutritional contents and
extracellular enzyme activities of bioflocs and (3)
growth performance and feed utilization of shrimp.
Materials and methods
Tank facilities and shrimp stocking
The experiment was conducted in 16 indoor fibreglass tanks (72 cm 56 cm 40 cm) with a water
volume of 125 L each. Tanks were filled with
sand-filtered seawater (salinity 32 g L 1, pH 8.2).
Prior to stocking shrimp, each tank was inoculated
with 62.5 mL concentrated bioflocs (equivalent to
0.5 mL L 1 of tank water volume) collected from
an indoor biofloc-based shrimp culture pond. Predominant bacteria of the inoculant were characterized as Bacillus sp. (Zhao et al. 2012) and
harvested using a 10-lm mesh size nylon bag filter (Burford et al. 2004). All tanks were aerated
and mixed continuously using air stones. Water
temperature of the experimental tanks was maintained at around 26C during the culture period.
No water was exchanged during the experimental
period and dechlorinated freshwater was added to
compensate for evaporation losses. The photoperiod was maintained on a 12:12 hour light-dark
cycle (artificial luminosity of ~600 lux).
Juvenile L. vannamei were obtained from Laoshan Aquaculture Station (Qingdao, China), and
2013 Blackwell Publishing Ltd, Aquaculture Research, 110

Aquaculture Research, 2013, 110

acclimated for 10 days. Shrimp (6.95  0.22 g)

in the intermolt phase were selected and randomly
stocked into the study tanks at 28 shrimp per tank
(equivalent to 224 shrimp m 3).

Biofloc manipulation for shrimp culture W-J Xu & L-Q Pan

Table 1 Ingredient and proximate composition of the

two experimental feeds (% dry weight basis) containing
different levels of crude protein (CP) fed to L. vannamei
35% CP
feed

Experimental design and culture management

The experiment had a 2 9 2 factorial design with
two dietary protein levels (35% crude protein
[P35] vs. 25% crude protein [P25]) and two C/N
ratios (C/N of 20:1 [CN20] vs. C/N of 15:1
[CN15]). Sucrose was used as a carbohydrate to
manipulate the two C/N ratios based on the carbon and nitrogen content of both the feeds and
sucrose. The four treatments were referred to as:
P35 + CN20, P35 + CN15, P25 + CN20 and
P25 + CN15. Each treatment had four randomly
assigned replicate tanks.
The two experimental feeds (pellet size 2 mm)
were prepared according to Xu et al. (2012b). Feed
formulation, proximate composition, energy content
and C/N ratio are given in Table 1. Feeding was
done by hand to apparent satiation three times per
day (06:00, 14:00 and 22:00 hours). Daily feed
rations were reduced gradually from approximately
5% of total body weight to 3% during the experimental period, and adjusted daily according to feed tray
observations. Feed inputs were calculated, weighed
and recorded daily. Locally purchased sucrose (~95%
purity) containing 38% (w/w) carbon was preweighed and added daily with the 14:00 hours feed
(calculated to be 124%, 52%, 68% and 15% of the
daily feeds input for the P35 + CN20, P35 + CN15,
P25 + CN20 and P25 + CN15 treatments respectively), so as to promote the development of bioflocs
in the respective tanks. Sucrose was completely
mixed in a beaker with corresponding tank water
and uniformly distributed over the water surface.
The shrimp were cultured for a period of 30 days. At
the end of the experiment, shrimp were harvested
after draining off water; live shrimp were then
counted and weighed individually.
Water quality monitoring
Throughout the experimental period, water
temperature, salinity, dissolved oxygen (DO) and pH
were measured daily at 08:00 ~ 10:00 hours using
a hand-held multi-metre (YSI-6600V2, Yellow
Springs Instruments Inc., Ohio, USA). When the pH
in any tank dropped below 7.5, Na2CO3 was added
to the water to raise the pH to 7.9. Water samples
2013 Blackwell Publishing Ltd, Aquaculture Research, 110

Ingredients
Peruvian fish meal*
Soybean meal
Squid visceral meal
Wheat meal
Soybean lecithin
Fish oil
Vitamin premix
Mineral premix
Vitamin C**
Choline choride
Sodium alginate
Filler
Total
Proximate composition
Moisture
Crude protein
Crude lipid
Ash
Gross energy (kJ g 1)
C/N ratio

25% CP
feed

25
20
7
33.0
2
0.35
0.2
1
0.1
0.3
5
6.05
100

15
10
5
50.0
2
1.4
0.2
1
0.1
0.3
5
10
100

7.6
35.0
8.0
15.9
16.5
8.8

7.8
25.2
8.1
13.5
15.9
12.1

*Peruvian fish meal: crude protein 69.2% (dry weight basis).

Soy bean meal (defatted): crude protein 48.0% (dry weight
basis).
Squid visceral meal: crude protein 44.0% (dry weight basis).
Kindly provided by Qingdao Master Bio-Tech Co
Ltd (Qingdao,
Shandong,China).
**Ascorbyl phosphate (Stay C-35%, Roche).
cellulose.
Carbon/Nitrogen ratio.

(100 mL) were collected every 5 days at

14:00 hours from each tank. Half of the water sample was analysed spectrophotometrically for total
ammonia nitrogen (TAN), nitrite nitrogen (NO2 -N)
and nitrate nitrogen (NO3 -N) and the remaining
half was analysed for total suspended solids (TSS)
following standard methods (APHA 1998). Biofloc
volume (BFV) was determined weekly using Imhoff
cones, registering the volume of the biofloc in
1000 mL of the tank water after 30 min sedimentation (Avnimelech & Kochba 2009). The biofloc volume index (BFVI) is defined as the volume of biofloc
in millilitres occupied by 1.0 g of TSS after 30 min
of settling (Xu, Pan, Sun & Huang 2012a).
Biofloc collection and sampling
After 30 days, bioflocs produced in all four treatments were collected by passing tank water

Biofloc manipulation for shrimp culture W-J Xu & L-Q Pan

through a 10-lm mesh size nylon bag. The concentrated biofloc samples from each tank were
dried in an oven at 105C until constant weight
and then stored at 20C until proximate composition analysis. In addition, water samples (50 mL)
were collected from each tank, transferred to
Eppendorf tubes and then centrifuged at 2, 000 9 g
for 15 min at 4C. The supernatant was decanted
and the bottom sediment was re-suspended in
1/10 of its original volume using filtered seawater
(0.45 lm filters), and 5 mL of the resulting
re-suspended material was put into a polyethylene
bottle with an ice water bath and treated using
the Vibra cellTM ultrasound processor (VCX 130,
Sonics & Materials, Inc., Connecticut, USA) at the
frequency of 20 kHz for 2 min. The extracted solution was then centrifuged at 20, 000 9 g for
20 min and the supernatant (enzyme extract) was
used as the enzyme source for enzymatic assay.
The above method for enzyme extraction of biofloc
was modified from the method of Yu, He, Shao
and Zhu (2009).
Proximate composition and enzyme activity
analysis of bioflocs
Crude protein, crude lipid and ash content of the
biofloc samples were performed using standard
methods (AOAC1995). Protein was determined by
measuring nitrogen using the Kjeldahl method
and multiplying by 6.25; lipid by Soxhlet extraction with ether, and ash by oven incineration at
550C.
All enzymatic assays were conducted in triplicate. Protease activity was determined according
to the method of Lowry, Rosebrough and Farr
(1951) using casein as the substrate and reacting
it with Folin-phenol reagent. Amylase activity was
analysed according to the 3, 5-dinitrosalicylic acid
(DNS) colorimetric method (Pan & Wang 1997)
using soluble starch as the substrate. Enzyme
activity was determined using spectrophotometry
(SpectraMax 190, Molecular Devices Inc., California, USA); measured as the change in absorbance
and expressed as specific activity (U g 1 TSS). One
unit of enzyme activity was expressed as 1 lmol of
tyrosine or maltose released per min.
Calculations and statistics
Survival, per cent weight gain (WG), feed conversion ratio (FCR) and protein efficiency ratio (PER)

Aquaculture Research, 2013, 110

were calculated using the following equations:

Survival (%) = 100 9 (final shrimp count initial
shrimp count), WG (%) = 100 9 (final individual
weight
initial individual weight) initial individual weight, FCR = total dry weight of feed offered
total shrimp wet weight gained, PER = total
shrimp wet weight gained total dry weight of feed
protein offered. In this study, the efficiency parameters (FCR and PER) are referred to as apparent
efficiency and are of more practical than biological
significance, because actual consumption of the
feeds could not be monitored in tanks, nor could
the impact of cannibalism and consumption of biofloc be directly assessed (Tacon, Cody, Conquest,
Divakaran, Forster & Decamp 2002; Xu et al.
2012b).
Biofloc development parameters (BFV, TSS and
BFVI) and water quality parameters (TAN, NO2 -N
and NO3 -N) were compared using split-plot
ANOVA with treatments as the main factor and
time as the sub-factor (Gomez & Gomes 1984).
Subsequently, a two-way ANOVA was carried out
to determine the effect of dietary protein level, C/N
ratio and their interaction. Data obtained from biofloc (crude protein, crude lipid and ash content;
protease and amylase activity) and shrimp performance (survival, WG, FCR and PER) were analysed using two-way ANOVA to determine the effect
of dietary protein level, C/N ratio and their interaction. In the absence of interactions, one-way
ANOVA of treatments was employed and Tukeys
test at P < 0.05 level of significance was employed
as the means separation procedure. All statistical
analyses were performed using SPSS 11.5 software
(SPSS, Chicago, USA).
Results
Biofloc development and water quality
Suspended bioflocs were observed as mostly brown
in colour, ranging in size from 0.2 to 2 mm. The
biofloc development in terms of BFV and TSS over
time is shown in Fig. 1. The bioflocs were well
formed and developed in all four treatments during
the 30-day experimental period. Significant
increases (P < 0.05) in the levels of both BFV and
TSS were detected as time progressed, reaching
their highest values at the end of the experiment
(day 30). No significant effects (P > 0.05) of dietary protein level, C/N ratio or their interaction
were observed on BFV or TSS levels at most of
2013 Blackwell Publishing Ltd, Aquaculture Research, 110

Biofloc manipulation for shrimp culture W-J Xu & L-Q Pan

Aquaculture Research, 2013, 110

P35+CN20

BFV (mL L1)

25.0

P35+CN15

P25+CN20

P25+CN15

20.0
15.0
10.0
5.0
0.0

400
300
200
100
0
100

BFVI (mL g1 TSS)

Figure 1 Changes of biofloc volume (BFV), total suspended solids

(TSS) and biofloc volume index
(BFVI) in four treatment tanks
stocked with L. vannamei throughout the 30-day experimental period.
P35+CN20 = treatment with
35% CP feed and C/N ratio 20;
P35+CN15 = treatment with 35%
CP feed and C/N ratio 15,
P25+CN20 = treatment with 25%
CP feed and C/N ratio 20;
P25+CN15 = treatment with 25%
CP feed and C/N ratio 15. Values are
means (S.D.) of four replicate tanks
per sampling time in each treatment.

TSS (mg L1)

500

80
60
40
20
0

sampling times. BFIV increased significantly

(P < 0.05) in the first 15 days and then remained
relatively stable at ~65 mL g 1 TSS; and no significant effects (P > 0.05) of dietary protein level, C/N
ratio or their interaction were detected.
The results of water quality are presented in
Table 2. The pH was sometimes slightly below the
range considered to be optimal, but was corrected
when detected; and all other measured water quality parameters were maintained within acceptable
ranges for shrimp culture throughout the 30-day
experimental period (Fast & Lester 1992).
Inorganic nitrogen control
Concentrations of TAN, NO2 -N and NO3 -N were
maintained low in all four treatments during the
30-day experimental period, not exceeding
0.46 mg L 1, 0.86 mg L 1 and 8.26 mg L 1
respectively (Fig. 2). Concentrations of TAN and
NO2 -N
fluctuated
significantly
(P < 0.05)
between most sampling times, and peaked at
2013 Blackwell Publishing Ltd, Aquaculture Research, 110

10

15

20

25

30

Sampling time (day)

different sampling times between the two dietary

protein levels (P35 vs. P25). However, no significant effects (P > 0.05) of dietary protein level, C/N
ratio or their interaction were observed on TAN or
NO2 -N concentrations at most of sampling times.
Concentrations of NO3 -N increased significantly
(P < 0.05) over time, ranging from 0.21 mg L 1
at stocking to 8.26 mg L 1 at harvest; however,
no significant effects (P > 0.05) of dietary protein
level, C/N ratio or their interaction were observed.
Nutritional content and extracellular enzyme
activity of bioflocs
Primary nutritional values of the bioflocs collected
from the four treatments showed crude protein levels of 21.3% ~ 32.1%, crude lipid levels of 1.6% ~
2.8% and ash levels of 43.4% ~ 61.4% on dry
matter basis (Table 3). The crude protein, crude
lipid and ash contents of bioflocs were affected by
dietary protein level (P < 0.01) and C/N ratio
(P < 0.05), but there were no interaction effects
5

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Aquaculture Research, 2013, 110

Parameters

P35 + CN20

P35 + CN15

P25 + CN20

P25 + CN15

Temperature (C)

26.6  1.4
(24.8, 28.1)
32.3  1.1
(30.8, 33.6)
7.6  2.3
(5.1, 10.0)
7.92  0.12
(7.73, 8.07)
0.10  0.11
(0.01, 0.39)
0.43  0.36
(0.02, 1.25)
3.15  2.67
(0.18, 9.59)

26.2  1.2
(24.8, 27.8)
31.8  0.9
(30.8, 32.9)
7.6  1.8
(5.5, 9.5)
7.87  0.18
(7.43, 8.06)
0.13  0.16
(0.00, 0.51)
0.43  0.34
(0.02, 1.22)
3.19  2.72
(0.25, 8.66)

26.5  1.3
(24.7, 28.0)
32.1  1.0
(30.7, 33.4)
7.7  2.0
(5.4, 9.8)
7.93  0.12
(7.73, 8.17)
0.15  0.24
(0.00, 0.85)
0.40  0.28
(0.02, 0.94)
3.43  2.92
(0.21, 10.04)

26.2  1.3
(24.6, 27.7)
31.9  0.8
(30.7, 33.0)
7.9  1.7
(5.7, 9.9)
7.88  0.14
(7.61, 8.07)
0.15  0.22
(0.00, 0.79)
0.39  0.35
(0.03, 1.21)
3.75  3.16
(0.20, 10.97)

Salinity (g L 1)
DO (mg L 1)
pH
TAN (mg L 1)
NO2 -N (mg L 1)
NO3 -N (mg L 1)

Table 2 The overall means  S.D.

and range values (minimum, maximum) of measured water quality
parameters in four treatments
stocked with L. vannamei during the
30-day experimental period

DO: dissolved oxygen.

TAN: total ammonia nitrogen.
P35 + CN20 = treatment with 35% CP feed and C/N ratio 20; P35 + CN15 = treatment with 35% CP feed and C/N ratio 15; P25 + CN20 = treatment with 25% CP feed
and C/N ratio 20; P25 + CN15 = treatment with 25% CP feed and C/N ratio 15.

P35+CN20

TAN (mg N L 1)

0.6

P35+CN15

P25+CN20

P25+CN15

0.5
0.4
0.3
0.2
0.1
0.0

NO2-N (mg N L1)

1.2
1.0
0.8
0.6
0.4
0.2

NO3-N (mg N L1 )

0.0
12.0
10.0
8.0
6.0
4.0
2.0
0.0

10

15
20
Sampling time (day)

(P > 0.05). The crude protein contents of the bioflocs in treatments P25 + CN20 and P25 + CN15
were significantly higher (P < 0.05) than that in
treatment P35 + CN15 while the crude lipid
contents of the bioflocs in treatments P25 + CN20
6

25

30

Figure 2 Fluctuations of total

ammonia nitrogen (TAN), nitrite
nitrogen (NO2 -N) and nitrate
nitrogen (NO3 -N) concentrations
in four treatment tanks stocked
with L. vannamei during the
30-day
experimental
period.
P35+CN20 = treatment with 35%
CP feed and C/N ratio 20;
P35+CN15 = treatment with 35%
CP feed and C/N ratio 15;
P25+CN20 = treatment with 25%
CP feed and C/N ratio 20;
P25+CN15 = treatment with 25%
CP feed and C/N ratio 15. Values
are means (  S.D.) of four replicate tanks per sampling time in
each treatment.

and P25 + CN15 were significantly lower

(P < 0.05) than that in treatment P35 + CN20.
The ash content of the biofloc in treatment
P35 + CN15 was significantly higher (P < 0.05)
than those in the other three treatments.
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Biofloc manipulation for shrimp culture W-J Xu & L-Q Pan

Aquaculture Research, 2013, 110

Table 3 Effects of dietary protein level and C/N ratio on the proximate composition (% dry weight basis) and the extracellular enzyme activities (U g 1 TSS) of bioflocs in zero- exchange culture tanks stocked with L. vannamei
Treatments
Variables

P35 + CN20

Proximate composition
Crude protein
26.6
Crude lipid
2.8
Ash
48.7
Enzyme activities
Protease
14.4
Amylase
293.5

Significance (P-value)
P35 + CN15

P25 + CN20

P25 + CN15

CN

P 3 CN

 1.5ab
 0.1a
 3.2a

21.3  1.7b
2.7  0.2ab
61.4  3.2b

32.1  2.1a
2.2  0.2bc
43.4  2.8a

30.0  1.4a
1.6  0.1c
48.0  2.4a

** (0.001)
*** (0.000)
** (0.009)

* (0.048)
* (0.025)
* (0.015)

NS (0.349)
NS (0.162)
NS (0.241)

 1.1a
 21.3a

10.7  0.8b
335.5  15.7ab

11.1  0.7ab
391.4  16.2b

9.9  0.9b
403.8  17.2b

** (0.001)
** (0.009)

* (0.048)
* (0.015)

NS (0.349)
NS (0.241)

Each value represents mean  S.E. (n = 4). Mean values in the same row with different superscript letters indicate significant difference (P < 0.05).
Results from two-way ANOVA; P = Dietary protein level (35% crude protein vs. 25% crude protein); CN = C/N ratio (C/N of 20
vs. C/N of 15); P 9 CN = Interaction of different dietary protein level and C/N ratio; *P < 0.05; **P < 0.01; *** P < 0.001; NS,
not significant.
P35 + CN20 = treatment with 35% CP feed and C/N ratio 20; P35 + CN15 = treatment with 35% CP feed and C/N ratio 15;
P25 + CN20 = treatment with 25% CP feed and C/N ratio 20; P25 + CN15 = treatment with 25% CP feed and C/N ratio 15.

Extracellular enzyme activities of the bioflocs collected from the four treatments exhibited protease
activities of 9.9 ~ 14.4 U g 1 TSS and amylase
activities of 293.5 ~ 403.8 U g 1 TSS (Table 3).
Both protease activity and amylase activity of bioflocs were affected by dietary protein level
(P < 0.01) and C/N ratio (P < 0.05), but there was
no interaction effect (P > 0.05). The protease activities of the bioflocs in treatments P25 + CN15 and
P35 + CN15 were significantly lower (P < 0.05)
than that in treatment P35 + CN20 while the amylase activities of the bioflocs in treatments P25 +
CN15 and P25 + CN20 were significantly higher
(P < 0.05) than that in treatment P35 + CN20.
Growth performance and feed utilization of shrimp
The results of shrimp performance are presented in
Table 4. Survival, per cent weight gain and protein efficiency ratio were not affected (P > 0.05)
by dietary protein level, C/N ratio or their interaction. The dietary protein level significantly affected
(P < 0.05) the feed conversion ratio while the C/N
ratio had no effect on the feed conversion ratio
(P > 0.05). The feed conversion ratios in treatments with high dietary protein (P35) were significantly lower (P < 0.05) than those in treatments
with low dietary protein (P25).
Discussion
This study demonstrated that juvenile L. vannamei
could be cultured with high survival (>90.5%) at
2013 Blackwell Publishing Ltd, Aquaculture Research, 110

a stocking density of 224 shrimp m 3 without

water exchange for at least 30 days. Bioflocs were
promoted and effective control of inorganic nitrogen (TAN, NO2 -N) concentrations was achieved
through manipulating high feed C/N ratio (15 or
20) irrespective of whether the shrimp were fed
with the 35% or 25% dietary protein feed. Furthermore, neither dietary protein level (35% vs.
25%) nor C/N ratio (20 vs. 15) had any significant effect on shrimp performance in terms of survival, per cent weight gain and protein efficiency
ratio. The results also demonstrate that the dietary
protein level could be reduced from 35% to 25%
without affecting shrimp performance in a bioflocdominated culture system.
Adjusting the protein level in the feed and/or
adding carbohydrates to the culture water are two
practical management strategies for promoting the
growth of heterotrophic bacteria and subsequently
the establishment of bioflocs. In this study, bioflocs
formed and developed in the tank water of all the
four treatments, as indicated by the gradual
increases in BFV and TSS levels during the 30-day
experimental period. In addition, the BFVI in the
four treatments had similar values of ~65 mL g 1
TSS from day 15 and hence, the bioflocs had the
capacity to remain stable and suspended in the
water column under constant aerated and mixed
conditions (Xu et al. 2012a). However, no significant differences in the levels of BFV and TSS were
observed among the four treatments. This indicates that whatever dietary protein level was
applied within tested ranges (25% ~35%), suitable
7

Biofloc manipulation for shrimp culture W-J Xu & L-Q Pan

Aquaculture Research, 2013, 110

Table 4 Growth performance and feed utilization of L. vannamei in the four treatments during the 30-day experimental
period
Treatments
Variables
Initial individual weight (g)
Final individual weight (g)
Survival (%)
Percent weight gain (%)
Feed conversion ratio (%)
Protein efficiency ratio

Significance

P35 + CN20
6.98
10.70
92.6
55.0
1.48
1.93








P35 + CN15
a

0.21
0.33a
2.8a
4.8a
0.10a
0.13a

6.92
10.90
90.5
57.9
1.45
1.97








P25 + CN20
a

0.13
0.18a
3.7a
2.3a
0.09a
0.11a

6.95
10.12
95.2
46.7
1.94
2.06








P25 + CN15
a

0.17
0.35a
2.7a
4.6a
0.15b
0.18a

6.94
10.42
94.6
51.1
1.96
2.04








0.14
0.16a
2.2a
2.2a
0.12b
0.14a

CN

P 3 CN

NS
NS
NS
NS
*
NS

NS
NS
NS
NS
NS
NS

NS
NS
NS
NS
NS
NS

Each value represents mean  S.E. (n = 4). Values in the same row with different superscript letters are significantly different
(P < 0.05).
Results from two-way ANOVA; P = Dietary protein level (35% crude protein vs. 25% crude protein); CN = C/N ratio (C/N of 20
vs. C/N of 15); P 9 CN = Interaction of different dietary protein level and C/N ratio; *P < 0.05; NS, not significant.
P35 + CN20 = treatment with 35% CP feed and C/N ratio 20; P35 + CN15 = treatment with 35% CP feed and C/N ratio 15;
P25 + CN20 = treatment with 25% CP feed and C/N ratio 20; P25 + CN15 = treatment with 25% CP feed and C/N ratio 15.

supplementation of sucrose could offer enough

favourable organic substrates for the development
of bioflocs as long as C/N ratio was manipulated
above 15. In this respect, we can speculate that
the biofloc system had the capacity to buffer
against input changes in dietary protein levels and
carbohydrate additions under given conditions.
Along with the development of the bioflocs, low
levels of TAN and NO2 -N were maintained in all
the four treatments throughout the 30-day experimental period. It has been demonstrated that the
formation and development of bioflocs in the culture water are most likely linked with the direct
assimilation of dissolved organic and inorganic
matters from feed residues and shrimp excretions
by heterotrophic bacteria (Avnimelech 1999;
Schneider et al. 2005; Ebeling, Timmons & Bisogni
2006), and thereby levels of TAN and NO2 -N
can be controlled within recommended ranges for
shrimp culture (Samocha, Patnaik, Speed, Ali,
Burger, Almeida, Ayub, Harisanto, Horowitz &
Brock 2007; Arnold et al. 2009; Ballester et al.
2010; Xu et al. 2012a). In other words, organic
residues and excreted nitrogen are continually
converted into microbial biomass rather than
accumulating as toxic ammonia and nitrite in the
system. This could be reflected in the gradual
increases of BFV and TSS levels, and the low levels
of TAN and NO2 -N in this study. Meanwhile, the
moderate accumulation of NO3 -N concentration,
which occurred in all the four treatments, indicates that nitrifying bacteria were also present in
the bioflocs (Ebeling et al. 2006; Xu et al. 2012a).
In fact, both heterotrophic assimilation and autotrophic nitrification processes occurred in the tank

systems and were responsible for inorganic nitrogen control. In general, whether using high or
low protein feed, the addition of appropriate
amounts of sucrose can be effective in promoting
the development of bioflocs and simultaneously
controlling inorganic nitrogen.
Previous studies have demonstrated the beneficial effects of promoted bioflocs on shrimp nutrition
(Burford et al. 2004; Kuhn, Boardman, Craig, Flick
& McLean 2008; Xu et al. 2012b). It is plausible to
speculate that the nutritional contribution of bioflocs may be related to their nutritional contents
and extracellular enzymes (Burford et al. 2004;
Wasielesky et al. 2006; Ju, Forster, Conquest, and
Dominy 2008; Xu & Pan 2012; Xu et al. 2012b).
In this study, the bioflocs collected from the four
treatments showed certain nutritional values with
21.3% ~ 32.1% crude protein, 1.6% ~ 2.8% crude
lipid and 43.4% ~ 61.4% ash; and all of these values were affected by dietary protein level and C/N
ratio. Ju, Forster, Conquest, Dominy, Kuo, and Horgen (2008) also reported that there were differences in crude protein, crude lipid and ash
contents of the collected bioflocs from white shrimp
systems with different predominant microorganisms (e.g. chlorophytes, diatoms or bacteria) present in the microbial community. This suggested
that the microbiota that constitutes the biofloc is
likely to affect the nutritional values of the biofloc.
Unfortunately, the microbial community structures
of the bioflocs collected in this study were not
determined; however, it can be inferred that the
differences in nutritional compositions between bioflocs resulting from the different dietary protein levels and C/N ratios treatments were probably due to
2013 Blackwell Publishing Ltd, Aquaculture Research, 110

Aquaculture Research, 2013, 110

the differences in the microbial community. Not

only that, both dietary protein levels and C/N
ratios also affected the activities of extracellular
proteases and amylases. These enzymes in bioflocs
could help break down proteins, carbohydrates and
other nutritional ingredients of the feed into smaller units, thereby facilitating feed digestibility and
absorption by the shrimp (Xu & Pan 2012).
Together these results emphasize the need for further study of biofloc composition and activity under
different management practices and raise the possibility of manipulating biofloc composition and
activity to achieve a desired nutritional contribution for cultured shrimp.
In this study, the shrimp receiving either feed,
the high dietary protein (P35) or the low dietary
protein (P25), had similar growth performance and
protein efficiency ratios, which indirectly confirms
the nutritional contribution of the bioflocs. The fact
that the shrimp in treatments with low dietary protein (P25) had higher feed conversion ratios than
those in treatments with high dietary protein
(P35), may be due to shrimp consuming more low
protein feed to compensate for a protein deficiency,
despite the apparent microbial proteins available
from the bioflocs. It is possible that the shrimp did
not actually ingest much of the bioflocs under current conditions. In short-term tank experiments of
L. vannamei, Burford et al. (2004) showed that the
nitrogen retention levels of the shrimp contributed
by the natural biofloc were lower in 5 and 9 g
shrimp than that in 1 g shrimp, and statistically
higher in treatment with higher biofloc density for
the 9 g shrimps. Together these results suggest
that the contribution of biofloc to shrimp protein
nutrition probably depends on the feeding traits,
shrimp size, biofloc density and biofloc size. These
factors should be considered when attempting to
reduce the protein level in feed without compromising shrimp performance or feed utilization while
operating biofloc systems.
Acknowledgments
This study was supported by the Special Fund for
Agro-scientific Research in the Public Interest from
the Ministry of Agriculture of China (grant no.
201103034). We thank the staff at the Laboratory
of Environmental Physiology of Aquatic Animal
for their assistance in conducting the experiment.
A special thank to Timothy Morris for critically
reading the manuscript.
2013 Blackwell Publishing Ltd, Aquaculture Research, 110

Biofloc manipulation for shrimp culture W-J Xu & L-Q Pan

References
AOAC (1995) Official Methods of Analysis of the Association of Official Analytical Chemists International(16th
edn). Association of Official Analytical Chemists,
Arlington, VA.
APHA (1998) Standard Methods for the Examination of the
Water and Wastewater(22nd edn). American Public
Health Association, Washington, DC.
Arnold S.J., Coman F.E., Jackson C.J. & Groves S.A.
(2009) High-intensity, zero water-exchange production
of juvenile tiger shrimp, Penaeus monodon: An evaluation of artificial substrates and stocking density. Aquaculture 293, 4248.
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.
Asaduzzaman M., Rahman M.M., Azim M.E., Islam M.A.,
Wahab M.A., Verdegem M.C.J. & Verreth J.A.J. (2010)
Effects of C/N ratio and substrate addition on natural
food communities in freshwater prawn monoculture
ponds. Aquaculture 306, 127136.
Avnimelech Y. (1999) Carbon nitrogen ratio as a control element in aquaculture systems. Aquaculture 176,
227235.
Avnimelech Y. (2006) Bio-filters: The need for an new
comprehensive approach. Aquaculture Engineering 34,
172178.
Avnimelech Y. (2008) Sustainable land-based aquaculture- rational utilization of water, land and feed. Mediterranean Aquaculture Journal 1, 4555.
Avnimelech Y. & Kochba M. (2009) Evaluation of nitrogen uptake and excretion by tilapia in bio floc tanks,
using N-15 tracing. Aquaculture 287, 163168.
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 fish
culture. Bioresource Technology 99, 35903599.
Ballester E.L.C., Abreu P.C., Cavalli R.O., Emerenciano
M., Abreu L. & Wasielesky W. (2010) Effect of practical diets with different protein levels on the performance of Farfantepenaeus paulensis juveniles nursed in
a zero exchange suspended microbial flocs intensive
system. Aquaculture Nutrition 16, 163172.
Burford M.A., Thompson P.J., McIntosh R.P., Bauman
R.H. & Pearson D.C. (2004) The contribution of flocculated material to shrimp (Litopenaeus vannamei) nutrition in a high-intensity, zero-exchange system.
Aquaculture 232, 525537.
Crab R., Avnimelech Y., Defoirdt T., Bossier P. & Verstraete W. (2007) Nitrogen removal techniques in aquaculture for a sustainable production. Aquaculture 270,
114.
De Schryver P., Crab R., Defoirdt T., Boon N. & Verstraete W. (2008) The basics of bio-flocs technology:

Biofloc manipulation for shrimp culture W-J Xu & L-Q Pan

The added value for aquaculture. Aquaculture 277,

125137.
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.
Fast A.W. & Lester L.J. (1992) Marine Shrimp Culture:
Principles and Practices. Elsevier Science Publishers,
Amsterdam, New York.
Gomez K.A. & Gomes A.A. (1984) Statistical Procedures
for Agricultural Research)2nd edn). Wiley, New York,
680 pp.
Hargreaves J.A. (2006) Photosynthetic suspended-growth
systems in aquaculture. Aquaculture Engineering 34,
344363.
Hari B., Kurup B.M., Varghese J.T., Schrama J.W. & Verdegem M.C.J. (2004) Effects of carbohydrate addition
on production in extensive shrimp culture systems.
Aquaculture 241, 179194.
Hari B., Kurup B.M., Varghese J.T., Schrama J.W. & Verdegem M.C.J. (2006) The effect of carbohydrate addition on water quality and the nitrogen budget in
extensive shrimp culture systems. Aquaculture 252,
248263.
Ju Z.Y., Forster I., Conquest L. & Dominy W. (2008a)
Enhanced growth effects on shrimp (Litopenaeus vannamei) from inclusion of whole shrimp floc or floc fractions to a formulated diet. Aquaculture Nutrition 14,
533543.
Ju Z.Y., Forster I., Conquest L., Dominy W., Kuo W.C. &
Horgen F.D. (2008b) Determination of microbial community structures of shrimp floc cultures by biomarkers and analysis of floc amino acid profiles.
Aquaculture Research 39, 118133.
Kuhn D.D., Boardman G.D., Craig S.R., Flick G.J. & McLean
E. (2008) Use of microbial flocs generated from tilapia
effluent as a nutritional supplement for shrimp, Litopenaeus vannamei, in recirculating aquaculture systems.
Journal of the World Aquaculture Society 39, 7282.
Lowry O.H., Rosebrough N.J. & Farr A.L. (1951) Protein
measurement with the Folin-phenol reagent. Journal of
Biological Chemistry 193, 265275.
Megahed M.E. (2010) The effect of Microbial Biofloc on
water quality, survival and growth of the green tiger
shrimp (Penaeus Semisulcatus) fed with different crude
protein levels. Journal of the Arabian Aquaculture Society
5, 119142.
Naylor R., Goldburg R.J., Mooney H., Beveridge M., Clay
J., Folke C., Kautsky N., Lubchenco J., Primavera J. &

10

Aquaculture Research, 2013, 110

Williams M. (1998) Natures subsidies to shrimp and

salmon farming. Science 282, 883884.
Pan L.Q. & Wang K.X. (1997) The experimental studies
on activities of digestive enzyme in the larvae penaeus
chinensis. Journal of Fisheries of China 21, 2631.
Samocha T.M., Patnaik S., Speed M., Ali A.-M., Burger
J.M., Almeida R.V., Ayub Z., Harisanto M., Horowitz
A. & Brock D.L. (2007) Use of molasses as carbon
source in limited discharge nursery and grow-out systems for Litopenaeus vannamei. Aquaculture Engineering
36, 184191.
Schneider O., Sereti V., Eding E.H. & Verreth J.A.J.
(2005) Analysis of nutrient flows in integrated intensive aquaculture systems. Aquaculture Engineering 32,
379401.
Tacon A.G.J., Cody J.J., Conquest L.D., Divakaran S., Forster I.P. & Decamp O.E. (2002) Effect of culture system
on the nutrition and growth performance of Pacific
white shrimp Litopenaeus vannamei (Boone) fed different
diets. Aquaculture Nutrition 8, 121139.
Wasielesky W., Atwood H., Stokes A. & Browdy C.L.
(2006) Effect of natural production in a zero exchange
suspended microbial floc based super-intensive
culture system for white shrimp Litopenaeus vannamei.
Aquaculture 258, 396403.
Xu W.J. & Pan L.Q. (2012) Effects of bioflocs on growth
performance, digestive enzyme activity and body composition of juvenile Litopenaeus vannamei in zero-water
exchange tanks manipulating C/N ratio in feed. Aquaculture. doi:doi:10.1016/j.aquaculture.2012.05.022.
Xu W.J., Pan L.Q., Sun X.H. & Huang J. (2012a) Effects
of bioflocs on water quality, and survival, growth and
digestive enzyme activities of Litopenaeus vannamei
(Boone) in zero-water exchange culture tanks.
Aquaculture Research. doi:10.1111/j.1365-2109.2012.
03115.x.
Xu W.J., Pan L.Q., Zhao D.H. & Huang J. (2012b) Preliminary investigation into the contribution of bioflocs
on protein nutrition of Litopenaeus vannamei fed with
different dietary protein levels in zero-water exchange
culture tanks. Aquaculture. doi:doi:10.1016/j.aquaculture.2012.04.003.
Yu G.H., He P.J., Shao L.M. & Zhu Y.S. (2009) Enzyme
extraction by ultrasound from sludge flocs. Journal of
Environmental Science 21, 204210.
Zhao P., Huang J., Wang X.H., Song X.L., Yang C.H.,
Zhang X.G. & Wang G.C. (2012) The application of
bioflocs technology in high-intensive, zero exchange
farming systems of Marsupenaeus japonicus. Aquaculture.
doi:10.1016/j.aquaculture.2012.03.034.

2013 Blackwell Publishing Ltd, Aquaculture Research, 110