Aquaculture 356-357 (2012) 147–152

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

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
Wu-Jie Xu, Lu-Qing Pan ⁎
The Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao 266003, China

a r t i c l e i n f o a b s t r a c t

Article history: A 30-day feeding experiment was conducted to investigate the effects of promoted bioflocs on growth per-
Received 18 March 2012 formance, feed utilization, digestive enzyme activity and whole body composition of Litopenaeus vannamei
Received in revised form 12 May 2012 juveniles (average 6.95 ± 0.22 g) in zero-water exchange culture tanks. Two bioflocs treatments and one con-
Accepted 14 May 2012
trol were evaluated: Bioflocs-based tanks with two levels of C/N ratio (15, 20) by addition of carbohydrate
Available online 19 May 2012
referred to as ‘CN15’ and ‘CN20’, and clear water tanks operated with water exchange and without addition
Keywords:
of carbohydrate referred to as ‘Control’. Each group consisted of quadruplicate tanks (125 L) and each tank
Litopenaeus vannamei contained 28 shrimp (equivalent to shrimp density of 224 individuals and biomass of ~1.56 kg per cubic
Microbial floc meter of water volume). Original concentrated bioflocs were collected from an indoor bioflocs-based shrimp
Growth performance culture pond, and inoculated into all bioflocs-based tanks with the same amount (0.5 mL L − 1 bioflocs vol-
Digestive enzyme activity ume) just before stocking shrimp. Sucrose was applied as a source of carbohydrate and added separately to
C/N ratio the CN15 and CN20 treatment tanks in addition to the applied feed (35% crude protein), so as to raise the
Zero-water exchange C/N ratio of the feeds input (feed and sucrose) to 15 and 20 and subsequently promote the development
of bioflocs. The monitoring of water quality parameters showed that they all remained within recommended
levels for shrimp culture in the three groups. At the end of the experiment, survival rates of the shrimp were
above 90%, with no significant differences among the three groups (P > 0.05); and the growth (in terms of
final weight, weight gain and specific growth rate) of the shrimp in both bioflocs treatments were significant-
ly better (P b 0.05) than that obtained in the control while the feed conversion rate was significantly lower
(P b 0.05). An overall enhancement in protease and amylase activities of the shrimp in both bioflocs treat-
ments was observed, though the effect of the bioflocs on each enzyme activity performed inconsistently
among different digestive tissues: digestive gland, stomach and intestine. Proximate composition analysis
showed that the crude lipid and ash contents of the shrimp in both bioflocs treatments tended to increase.
The bioflocs collected from both bioflocs treatments showed good prime nutritional values and appropriate
extracellular enzymes activities. The crude protein and crude lipid contents ranged from 27.3% to 31.6%
and 3.7% to 4.2%, respectively; and protease and amylase activities ranged from 10.7 to 14.4 μmol min − 1 g− 1
TSS and 293.5 to 335.5 μmol min − 1 g− 1 TSS, respectively. The results from this study suggest that the pro-
moted bioflocs can improve growth performance and feed utilization of the cultured shrimp, probably
through providing a supplemental food source and enhancing feed digestion and utilization.
© 2012 Elsevier B.V. All rights reserved.

1. Introduction (Avnimelech, 2006; Crab et al., 2007). As the microbial community

The bioflocs technology is a sustainable technique used in zero-
water exchange shrimp culture systems (Avnimelech, 2008; Crab et
al., 2007; De Schryver et al., 2008). Dense and active heterotrophic
microorganisms are manipulated so as to control water quality main-
ly by the immobilization of ammonium into microbial biomass
⁎ Corresponding author at: Lab. of Environmental Physiology of Aquatic Animal,
Fisheries College, Ocean University of China, Yushan Road 5, Qingdao 266003, China.
Tel./fax: + 86 532 82032963.
E-mail address: panlq@ouc.edu.cn (L-Q. Pan).
develops, bioflocs (microbial flocs) are formed containing heteroge-
neous mixture of microorganisms and organic particles (De
Schryver et al., 2008; Hargreaves, 2006). Relatively high C/N ratio in
feed (10 to 20) was recommended for the establishment of bioflocs
in such a system (Asaduzzaman et al., 2008; Avnimelech, 1999;
Ballester et al., 2010; Hargreaves, 2006). As the C/N ratio of most of
the artificial feeds used in intensive aquaculture systems is around
10, adding carbohydrates (e.g. sugar) in addition to the regular feed
can be a practical way to increase the C/N ratio, thereby promoting
the development of bioflocs within the systems (Asaduzzaman et
al., 2008; Avnimelech, 1999; De Schryver et al., 2008). Several studies

0044-8486/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2012.05.022
148 W-J. Xu, L-Q. Pan / Aquaculture 356-357 (2012) 147–152
suggested that manipulating higher C/N ratio in feed could in-
crease bioflocs community volume without compromising the nu-
tritional quality of bioflocs (Asaduzzaman et al., 2010; Azim et al.,
2008).
Many researchers have reported that bioflocs produced within the
culture systems could enhance shrimp growth performance (Arnold
et al., 2009; Megahed, 2010; Wasielesky et al., 2006). It was deduced
that in situ bioflocs are available 24 h a day as a supplemental food
source for the cultured shrimp (Avnimelech, 1999). They can be
harvested by the shrimp, digested and may replace a significant frac-
tion of nutrition demand (Burford et al., 2004; Crab et al., 2010; Hari
et al., 2004, 2006; Wasielesky et al., 2006). Meanwhile, the consump-
tion and regeneration of bioflocs can increase feed utilization efficien-
cy by recycling feed residues and/or recovery of some fraction of
excreted nutrients (Hargreaves, 2006; Schneider et al., 2005). More
importantly, it is interesting to note that bioflocs or its attached mi-
croorganisms could exert a positive effect on the digestive enzyme
activity of shrimp (Moss et al., 2001; Xu et al., 2012a, 2012b). Presum-
ably, all of these effects may be related to bioflocs characteristics such
as nutritional content and extracellular enzyme activity. Yet so far no
study has actually measured the activities of extracellular enzymes of
bioflocs produced within the culture systems.
The white shrimp Litopenaeus vannamei (Boone) is most widely
cultured in many parts of the world. Rapid growth, good survival in
high-density culture and disease tolerance make it a good choice for
intensive and/or bio-secure closed grow-out strategies (Cuzon et al.,
2004). Over the past decade, production of L. vannamei in bioflocs-
based intensive systems with zero-water exchange has become pop-
ular and achieved sustainable (Burford et al., 2004; McIntosh, 2000;
Tacon et al., 2002; Wasielesky et al., 2006); however, much is still un-
known about the beneficial effects of bioflocs on the shrimp
performance in this type of system. This study was conducted to in-
vestigate the effects of bioflocs on growth performance, feed utiliza-
tion, digestive enzyme activity and whole body composition of
L. vannamei juveniles in zero-water exchange tanks through manipu-
lation of the feed C/N ratio. Testing also included evaluation of the nu-
tritional content and extracellular enzyme activity of the promoted
bioflocs to obtain further information about the pathways and mech-
anisms how they affect the shrimp performance.
2. Materials and methods
2.1. Experimental design and tank facilities
The experiment was carried out in indoor fiberglass tanks
(72 cm × 56 cm × 40 cm) with a water volume of 125 L each. Two bio-
flocs treatments and one control were compared: Bioflocs-based
tanks under zero-water exchange with two levels of C/N ratio (15,
20) by addition of sucrose referred to as ‘CN15’ and ‘CN20’, and
clear water tanks operated with water exchange and without addi-
tion of sucrose referred to as ‘Control’. All tanks are in the same size
and each group consisted of quadruplicate tanks.
All tanks were filled with sand-filtered seawater. Original bioflocs
characterized by Bacillus sp. as the predominant bacteria (Zhao et al.,
2012) were collected from an indoor bioflocs-based shrimp culture
pond by passing pond water through a 10-μm mesh size nylon bag
and then inoculated into all bioflocs-based tanks with the same
amount (0.5 mL L − 1 bioflocs volume) just before stocking shrimp.
All tanks were aerated and agitated continuously using air-stones
connected to an air pump. No water was exchanged in the treatment
tanks and half of the water was renewed daily in the control tanks
during the experiment period. Water heating systems were used to
keep the water temperature of all experimental tanks at around
26 °C during the culture period; and for the replacement water of
control tanks, it was also heated to the equal temperature before
renewing. Dechlorinated freshwater was added to compensate for
evaporation losses. The photoperiod was maintained on a 12:12 h
light–dark cycle (artificial luminosity of ~600 lx).
2.2. Shrimp stocking and tank management
Juvenile shrimps L. vannamei were obtained from Laoshan Aqua-
culture Station (Qingdao, China). After acclimation for 10 days, the
shrimp (6.95 ± 0.22 g) in the intermoult period were selected and
randomly stocked into 12 tanks. Each tank contained 28 shrimp
(equivalent to shrimp density of 224 individuals and biomass of
~1.56 kg per cubic meter of water volume). In addition, 16 shrimp
were randomly sampled for proximate composition analysis of the
whole body at the time of the experiment initiation. The shrimp
were cultured for a period of 30 days.
A locally formulated and prepared pellet feed containing 35%
crude protein (Xu et al., 2012b) with C/N ratio close to 9 was used.
Feeding was done by hand to apparent satiation 3 times per day at
06:00, 14:00 and 22:00 h. The daily feeding rates were slowly re-
duced from approximately 5% of total body weight to 3% during the
30-day experiment period, and adjusted daily according to feeding
trays to make sure that the feeds were fully consumed. Feed inputs
in all tanks were recorded daily.
Locally purchased sucrose (~95% purity) contained 38% (w/w)
carbon was used as a carbohydrate source for manipulating the feed
C/N ratio. The amount of sucrose added was calculated based on the
C/N ratio of the daily feeds input to the tank, considering the carbon
and nitrogen concentration in the feed and sucrose. In order to raise
the feed C/N ratio to 15 and 20, 0.68 and 1.24 kg sucrose were applied
for each kg of formulated feed in the CN15 and CN20 treatment tanks,
respectively. The pre-weighed sucrose was completely mixed in a
beaker with corresponding tank water and uniformly distributed
over the tank's surface directly after the feed application at 14:00 h
to promote the development of bioflocs.
2.3. Assessment of water quality parameters
Throughout the 30-day experimental period, water temperature,
salinity, dissolved oxygen (DO) and pH were measured daily at
08:00–10:00 h using YSI-6600V2 Multi-Parameter Water Quality
Sonde (YSI Incorporated, Yellow Springs, OH, USA). Whenever the
pH of the water in any tank dropped below 7.5, Na2CO3 was added
into the tank water to raise the pH value to 7.9 slowly. Water samples
(100 mL) were collected weekly at 14:00 h from each tank. Half of the
water sample was analyzed spectrophotometrically for total ammo-
nia nitrogen (TAN), nitrite nitrogen (NO2−–N) and nitrate nitrogen
(NO3−–N) following ‘Standard methods for the examination of
water and wastewater’ (APHA, 1998); the remaining half was
filtered under vacuum pressure through pre-dried and pre-weighed
Whatman GF/C filter paper. The filter paper containing suspended
materials was dried at 105 °C in an oven until constant weight, and
the dried sample was weighed to 0.01 mg (Azim and Little, 2008).
The weight difference was calculated and an estimate of the total
suspended solids (TSS) was obtained. Bioflocs volume (BFV) was de-
termined on site using Imhoff cones weekly, registering the volume
taken in by the bioflocs in 1000 mL of the tank water after 30 min
sedimentation (Avnimelech and Kochba, 2009).
2.4. Bioflocs collecting and shrimp sampling
After 30 days, bioflocs produced in the treatment tanks were col-
lected by passing tank water through a 10-μm mesh size nylon bag.
The concentrated bioflocs samples from each tank were dried in an
oven at 105 °C until constant weight and then stored at −20 °C
until proximate composition analysis. Additionally, water sample
(50 mL) was collected from each treatment tank, transferred to
Eppendorf tubes and then centrifuged at 2000 g for 15 min at 4 °C.
 W-J. Xu, L-Q. Pan / Aquaculture 356-357 (2012) 147–152
The supernatant was decanted and the bottom sediment was re-
suspended in 1/10 of its original volume using filtered seawater
(0.45 μm filters). The bioflocs supernatant (5 mL) was put into a poly-
ethylene bottle with an ice water bath and treated using the Vibra cell
™ ultrasound processor at the frequency of 20 kHz for 2 min. The
extracted solution was then centrifuged at 20,000 × 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 bio-
flocs was modified from the method of Yu et al. (2009).
At the end of the experiment, shrimp were harvested after
draining off water: live shrimp were counted and final body weight
(wet weight) of each individual was weighed. Six shrimp from each
tank were collected randomly for sampling of digestive tissues. The
digestive glands, stomachs and intestines of the sampled shrimp
were excised, pooled and then were weighed and homogenized
twice with distilled water (1:3 w/v) at 0 °C for 30 s each time, using
an electric blender operating at 8000 r min − 1. The homogenate was
centrifuged at 10,000 × g, 4 °C for 30 min to eliminate tissue debris
and lipids. The supernatant (enzyme extract) was dispensed into
1.5 mL Eppendorf tubes and kept at − 20 °C until enzymatic assay.
Additionally, 6 shrimp from each tank were randomly sampled for
proximate composition analysis of the whole body.
2.5. Enzyme activity and proximate composition analysis
Protease activity was assayed according to the method of Lowry et
al. (1951) using casein as the substrate and reacting it with Folin-
phenol reagent. Amylase activity was assayed according to the 3,5-
dinitrosalicylic acid colorimetric method (Pan and Wang, 1997)
using soluble starch as the substrate. For protease and amylase of
shrimp samples, enzyme activity was measured as the change in ab-
sorbance, using the SpectraMax 190 spectrophotometer (Molecular
Devices Inc., California, USA) and expressed as specific activity
(U mg − 1 protein). One unit of enzyme activity was expressed as
1 μg of tyrosine or maltose released per min. The total soluble protein
content was measured in diluted homogenates by the Bradford meth-
od (Bradford, 1976) using bovine serum albumin as a standard. For
protease and amylase of bioflocs samples, enzyme activity was mea-
sured as the change in absorbance, using the SpectraMax 190 spectro-
photometer (Molecular Devices Inc., California, USA) and expressed
as specific activity (U g − 1 TSS). One unit of enzyme activity was
expressed as 1 μmol of tyrosine or maltose released per min.
Proximate composition analysis of crude protein, crude lipid and
ash contents of the bioflocs samples and shrimp samples were per-
formed by the standard methods of AOAC (1995). Protein was deter-
mined by measuring nitrogen (N · 6.25) using the Kjeldahl method;
lipid by ether extraction using Soxhlet and ash by oven incineration
at 550 °C. Moisture of the shrimp sample was determined by oven-
drying at 105 °C for 24 h.
2.6. Calculations and statistics
Survival rate, weight gain, specific growth rate and feed conver-
sion rate were calculated using the following equations: Survival
rate (%) = 100 × (final shrimp count ∕ initial shrimp count), Weight
gain (%) = 100 × (final body weight − initial body weight) ∕ initial
body weight, Specific growth rate (% day − 1) = 100 × [Ln(final body
weight) − Ln(initial body weight)] ∕ experimental duration (days),
Feed conversion rate = total dry weight of feed offered ∕ total shrimp
wet weight gained. In the present study, the feed conversion rate is
referred to as ‘apparent’ efficiency and is of more practical than bio-
logical significance, because actual consumption of the feeds could
not be monitored in the biofocs-based tanks, nor could the impact
of cannibalism and consumption of bioflocs be directly assessed
(Tacon et al., 2002).
149
All statistical analyses were performed using SPSS 11.5 software
(SPSS, Chicago, USA). Data obtained from the experimental shrimp
were analyzed by one-way ANOVA after homogeneity of variance
test. Significant differences were considered at P b 0.05. When signifi-
cant differences were found, Duncan's multiple range test was used to
identify differences among experimental groups.
3. Results
3.1. Bioflocs development and water quality
The bioflocs development in terms of BFV and TSS over time is
shown in Fig. 1. Both BFV and TSS levels increased gradually through-
out the experiment period and the changing tendency of them over
time are basically consistent. After 30 days, average BFV and TSS
levels in both bioflocs treatments were around 21 mL L − 1 and
320 mg L − 1, respectively.
The results of water quality parameters monitored are shown in
Table 1. The measured water quality in all experimental groups
remained within recommended levels for shrimp culture throughout
the 30-day experimental period. The only exception was pH in the
tanks of both bioflocs treatments, which was sometimes slightly
below the range considered to be optimal; but when detected, was
slowly corrected.
3.2. Nutritional content and extracellular enzyme activity of bioflocs
The primary nutritional contents and extracellular enzyme activi-
ties of the bioflocs collected from the tanks water of both bioflocs
treatments are shown in Table 2. The crude protein and crude lipid
contents possessed appropriate values: 27.3% and 3.7% respectively
in the CN15 treatment and 31.6% and 4.2% respectively in the
CN20 treatment. The ash content was somewhat high: 49.4% in the
CN15 treatment and 43.7% in the CN20 treatment. Protease
activities were 10.7 μmol min − 1 g − 1 TSS in the CN15 treatment and
14.4 μmol min − 1 g − 1 TSS in the CN20 treatment. Amylase activities
were 335.5 μmol min − 1 g − 1 TSS in the CN15 treatment and
293.5 μmol min − 1 g − 1 TSS in the CN20 treatment.
Control CN15 CN20
25
20
BFV (mL L−1)
15
10
5
0
500
400
TSS (mg L−1)
300
200
100
0
0 5 10 15 20 25 30
Sampling time (day)
Fig. 1. Changes of bioflocs volume (BFV) and total suspended solids (TSS) in the control
and two bioflocs treatments with two C/N ratios (15, 20) during the 30-day experi-
mental period. Values are means (± S.D.) of four replicate tanks per sampling time in
each treatment.
150 W-J. Xu, L-Q. Pan / Aquaculture 356-357 (2012) 147–152

Table 1 Table 3
The overall means ± S.D. and range values (minimum, maximum) of water quality pa- Growth performance and feed utilization of juvenile Litopenaeus vannamei in the con-
rameters in the control and two bioflocs treatments with two C/N ratios (15, 20) during trol and two bioflocs treatments with two C/N ratios (15, 20) at the end of 30-day feed-
the 30-day experimental period based on repeated measures ANOVA. ing experiment.

Parameter Control CN15 CN20 Parameter Control CN15 CN20

a a
Temperature (°C) 26.3 ± 1.1 26.2 ± 1.2 26.6 ± 1.4 Initial individual weight (g) 6.95 ± 0.10 6.92 ± 0.13 6.98 ± 0.21a
(24.7, 27.8) (24.8, 27.8) (24.8, 28.1) Final individual weight (g) 9.77 ± 0.13a 10.90 ± 0.18b 10.70 ± 0.33b
Salinity (g L− 1) 31.5 ± 0.5 31.8 ± 0.9 32.3 ± 1.1 Weight gain (%) 41.7 ± 2.0a 57.9 ± 2.3b 55.0 ± 4.8b
(30.7, 32.4) (30.8, 32.9) (30.8, 33.6) Specific growth rate (% day− 1) 1.16 ± 0.05a 1.52 ± 0.05b 1.46 ± 0.11b
DOa (mg L− 1) 8.3 ± 1.6 7.6 ± 1.8 7.6 ± 2.3 Feed conversion rate 1.95 ± 0.07a 1.45 ± 0.09b 1.48 ± 0.10b
(6.6, 10.3) (5.5, 9.5) (5.1, 10.0) Survival rate (%) 93.9 ± 1.8a 90.5 ± 3.7a 92.6 ± 2.8a
pH 8.03 ± 0.02 7.87 ± 0.18 7.92 ± 0.12
Each value represents mean ± S.E. (n = 4). Values in the same row with different
(7.98, 8.08) (7.43, 8.06) (7.73, 8.07)
superscript letters are significantly different (P b 0.05).
TANb (mg L− 1) 0.09 ± 0.07 0.13 ± 0.16 0.10 ± 0.11
(0.03, 0.27) (0.00, 0.51) (0.01, 0.39)
NO2−–N (mg L− 1) 0.13 ± 0.07 0.43 ± 0.34 0.43 ± 0.36
(0.03, 0.25) (0.02, 1.22) (0.02, 1.25) 3.5. Body composition of shrimp
NO3−–N (mg L− 1) 0.66 ± 0.42 3.19 ± 2.72 3.15 ± 2.67
(0.17, 1.68) (0.25, 8.66) (0.18, 9.59)
Proximate composition of the shrimp whole body is shown in
a
DO: dissolved oxygen. Table 4. No significant differences (P > 0.05) in the moisture and pro-
b
TAN: total ammonia nitrogen. tein content between two bioflocs treatments and the control. The
crude lipid and ash content tended to increase in both bioflocs
treatments.

3.3. Growth performance and feed utilization of shrimp 4. Discussion

Growth performance was evaluated through final weight, weight
gain (expressed as a percent of initial body weight) and specific
growth rate. The growth of the shrimp in both bioflocs treatments
was significantly better (P b 0.05) than that obtained in the control
(Table 3). The feed conversion rate of the shrimp in both bioflocs
treatments was significantly lower (P b 0.05) than that obtained in
the control (Table 3). The survival rate of the shrimp in the three
groups was all above 90% during the 30-day experimental period
(Table 3).
3.4. Digestive enzyme activity of shrimp
As can be seen from Fig. 2, protease and amylase activities of
the shrimp in the three groups were tissues-specific. Both enzymes
activities were highest in the digestive gland, intermediate in the
stomach, and lowest in the intestine. Although not statistically sig-
nificant, enhanced protease and amylase activities in the digestive
glands of the shrimp were observed in both bioflocs treatments
(Fig. 2A–B). Protease and amylase activities in the stomachs of
the shrimp in both bioflocs treatments were significantly higher
(P b 0.05) than those in the control (Fig. 2C–D); while a decreasing
trend of protease and amylase activities in the intestines of the
shrimp in both bioflocs treatments was observed (Fig. 2E–F). A
general enhancement in protease and amylase activities of the
shrimp in both bioflocs treatments was observed, though the effect
of the bioflocs on each enzyme activity performed inconsistently
among different digestive tissues: digestive gland, stomach and
intestine.
Table 2
Proximate composition (% dry weight basis) and extracellular enzyme activity
(μmol min− 1 g− 1 TSS) of the bioflocs produced in the two bioflocs treatments with
two C/N ratios (15, 20) at the end of 30-day feeding experiment for juvenile
Litopenaeus vannamei.
Treatment Proximate composition Extracellular enzyme
activity
Crude protein Crude lipid Ash Protease
CN15 27.3 ± 3.7 3.7 ± 0.9 49.4 ± 8.2 10.7 ± 1.6
CN20 31.6 ± 4.5 4.2 ± 0.8 43.7 ± 7.1 14.4 ± 2.1
Each value represents mean ± S.D. (n = 4).
The promoted bioflocs significantly improved shrimp growth
performance and feed utilization in the present study. The growth
(in terms of final weight, weight gain and specific growth rate) of
the shrimp in both bioflocs treatments was significantly higher
than that obtained in the control, while the feed conversion rate
was significantly lower. These results are in agreement with previ-
ous findings that the growth rate and feed utilization improved in
L. vannamei (Wasielesky et al., 2006), P. monodon (Arnold et al.,
2009) and P. semisulcatus (Megahed, 2010) with bioflocs present
in the culture systems. Using L. vannamei juveniles, our latest
study also demonstrated that adding sugar as a carbohydrate
source to promote bioflocs in zero-water exchange tank systems
could improve the growth of the shrimp (Xu et al., 2012b). In con-
trast, Samocha et al. (2007) reported that addition of molasses did
not result in a significant effect on growth performance of
L. vannamei under limited water discharge.
Although previous studies have demonstrated the beneficial
effects of promoted bioflocs on shrimp production, the pathways
affecting improvement of growth rate and feed utilization are large-
ly unknown. An explanation proposed by several authors is that
bioflocs can provide a supplemental food source for the cultured
shrimp (Burford et al., 2004; Kuhn et al., 2008; Megahed, 2010).
The bioflocs, as an important and nutritional natural food within
the culture systems, which continuously available in situ can pro-
vide additional protein, lipid, mineral and vitamin for the shrimp
(Avnimelech, 1999; Izquierdo et al., 2006; Ju et al., 2008b; Moss
et al., 2006; Tacon et al., 2002; Wasielesky et al., 2006). Not sur-
prisingly, proximate analysis of the bioflocs from the current exper-
iment reveals that they contained appropriate crude protein
(27.3%–31.6%) and crude lipid (3.7%–4.2%) on dry matter basis
in shrimp nutrition terms for at least omnivorous L. vannamei
(Cuzon et al., 2004). Moreover, the bioflocs exhibited relatively
high protease and amylase activities, indicating that relevant extra-
cellular enzymes could be produced by the microorganisms
attaching to the bioflocs. As these microbial enzymes can help
break down proteins, carbohydrates and other nutritional ingredi-
ents of the feed into smaller units, the promoted bioflocs could
presumably facilitate feed digestibility and absorption. This contri-
Amylase
bution is especially important in the case that those acquired en-
335.5 ± 35.7 zymes also work effectively in the digestive tracts of the shrimp
293.5 ± 31.3 after being ingested along with the bioflocs, as exogenous enzyme
supplementation in diets performed the effective digestive function
 W-J. Xu, L-Q. Pan / Aquaculture 356-357 (2012) 147–152 151

A C 8.0
E
Digestive gland a 10.0 b

Specific activity of protease

37.0 Stomach Intestine

(µg min−1mg−1 protein)

9.5 b 7.5 a
36.5 a ab
36.0 9.0 7.0 b
a
35.5 8.5 6.5

35.0 a 8.0 6.0

34.5 7.5 5.5

5.0

3.0
B Digestive gland 3.0
D Stomach 1.5 F Intestine
Specific activity of amylase

b a
(mg min−1mg−1protein)

ab
a 1.2
2.7 2.7
a b b
a
2.4 2.4 0.9

2.1 2.1 0.6

a
1.8 1.8 0.3

1.5 1.5 0.0

Control CN15 CN20 Control CN15 CN20 Control CN15 CN20

Fig. 2. Specific activities of protease and amylase in digestive glands, stomachs and intestines of juvenile Litopenaeus vannamei in the control and two bioflocs treatments with two
C/N ratios (15, 20) at the end of 30-day feeding experiment. Means ± S.E. indicated. n = 4. Means within the same tissue with different letters are significantly different (P b 0.05).

(Lin et al., 2007). Therefore, as a supplemental food source, the bio-
flocs which can not only supplement microbial nutrition but also
contribute to digestion and utilization of the feed for the cultured
shrimp, can exert an enhancing effect on shrimp growth perfor-
mance and feed utilization. Additionally, the development and re-
generation of the bioflocs in the culture tanks can recycle residual
feeds and associated wastes, resulting in the recycling and
reutilization of feed nutrients by the shrimp and eventually improv-
ing overall feed assimilation, especially under zero-water exchange
(Avnimelech, 2006, 2008).
Another important way in which bioflocs enhance the digestive
capability of cultured shrimp is by increasing digestive enzyme activ-
ity. In penaeid shrimps, digestive enzymes, such as protease and am-
ylase, are synthesized and secreted by the digestive gland (Dall et al.,
1990). Digestion and absorption occur mainly in the stomach and in-
testine respectively (Dall et al., 1990). This is reflected by the levels of
enzyme activity in the three digestive tissues of the shrimp in the pre-
sent study. On this basis, compared to the control, an enhancement in
protease and amylase activities of digestive glands and stomachs of
the shrimp was observed in both bioflocs treatments. According to
Ju et al. (2008a), the bioflocs can provide a complete source of cellular
nutrition as well as various bioactive compounds and may contain
some as yet undiscovered factors. It can be speculated that shrimp
can adapt well to changes in diet composition in the presence of bio-
flocs (Le Moullac et al., 1996) because their digestive enzyme
Table 4
Proximate composition (% wet weight basis) of the whole body of juvenile Litopenaeus
vannamei in the control and two bioflocs treatments with two C/N ratios (15, 20) at the
end of 30-day feeding experiment.
Parameter Initial Experimental groups
Control CN15
a a
Moisture 75.87 ± 0.16 76.09 ± 0.19 75.22 ± 0.26
Crude protein 18.32 ± 0.06 17.96 ± 0.09a 18.78 ± 0.36a
Crude lipid 1.65 ± 0.02 1.80 ± 0.02a 1.91 ± 0.04ab
Ash 2.73 ± 0.03 2.65 ± 0.05a 2.82 ± 0.08ab
Each value represents mean ± S.E. (n = 4). Values in the same row with different
superscript letters are significantly different (P b 0.05).
activities change in response to different nutritional conditions.
More probably, the presence of the bioflocs could in some way stim-
ulate the production of digestive enzymes by the shrimp and/or en-
hance their activity (Moss et al., 2001; Xu et al., 2012a, 2012b). It
should be noted that there was a slight decrease in protease and am-
ylase activities in the intestines of the shrimp in both bioflocs treat-
ments. This might be unfavorable to the digestion of the ingested
feed in the intestine. As a massive number of live microorganisms
existed in the bioflocs, they could transit through the stomach into
the intestine and interfere with resident intestinal microflora balance
which plays an important role in the production or secretion of diges-
tive enzymes (Harris, 1993; Moss et al., 2000; Xu et al., 2012a). In
general, the observed increase in the digestive enzymes activities in
the digestive tissues of the shrimp in both bioflocs treatments might
have led to enhanced digestion and increased absorption of the
feed, which may in turn have contributed to the improved growth
performance and feed utilization of the shrimp. Certainly more
work is necessary to fully clarify the inconsistent effects of the bio-
flocs on the same digestive enzyme activity among different digestive
tissues: digestive gland, stomach and intestine.
The bioflocs could also influence the whole body composition of
the cultured shrimp. Although the shrimp gained limited increases
(41.7%–51.9%) in the body weight in this study, the crude lipid and
ash contents of the whole body tended to increase in both bioflocs
treatments compared to those obtained in the control. An increasing
trend of whole body lipid content has also been observed by
Izquierdo et al. (2006). In their study, L. vannamei reared in meso-
cosms systems with the bioflocs had a higher increase in the whole
body lipid content than that obtained in clear water systems, which
was attributed to the effective assimilation of several fatty acids
such as 16:1n-7 and 17:1 from the bioflocs. It is plausible to hypoth-
esize that the shrimp fed on the artificial feed and the bioflocs might
have better nutrient assimilation when compared to those fed only
CN20 the formulated feed, because of the greater amount of essential
amino acids, fatty acids (PUFA and HUFA) and other nutritional ele-
75.37 ± 0.28a
18.53 ± 0.17a ments supplied by the bioflocs (Izquierdo et al., 2006; Ju et al.,
1.96 ± 0.06b 2008b; Tacon et al., 2002). Also, the increased whole body ash con-
2.85 ± 0.05b tent of the shrimp might be explained by continuous availability of
abundant minerals and trace elements from the bioflocs as indicated
by high ash content in the present study (Tacon et al., 2002).
152 W-J. Xu, L-Q. Pan / Aquaculture 356-357 (2012) 147–152
5. Conclusions
The present study confirmed the beneficial effects of promoted
bioflocs on the shrimp performance in zero-water exchange culture
tanks through manipulating C/N ratio in feed. The bioflocs could not
only provide supplemental microbial nutrition in situ, but also pro-
duce extracellular enzymes which can facilitate feed digestion and
utilization. More importantly, the bioflocs could exert a positive effect
on digestive enzyme activity of the shrimp, which also contribute to
feed digestion and utilization. Therefore, improved growth perfor-
mance and feed utilization of the shrimp could be achieved in the
presence of the bioflocs. The data obtained from this study also sug-
gest that the bioflocs could influence shrimp body composition by in-
creasing nutrient retention in the shrimp. Further research is needed
to better understand pathways and mechanisms of bioflocs effects on
the nutrition physiology of shrimp and how the bioflocs can be ma-
nipulated to maximize shrimp production performance.
Acknowledgments
This work 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.
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