Aquaculture Reports 18 (2020) 100518

Contents lists available at ScienceDirect

Aquaculture Reports
journal homepage: www.elsevier.com/locate/aqrep

First use of biofloc technology for Penaeus monodon culture in Bangladesh:

Effects of stocking density on growth performance of shrimp, water quality
and bacterial growth
Sheikh AftabUddin a, Mohammad Abdul Momin Siddique b, c, *, Aung Sein d, Probin Kumar Dey e,
Md. Rashed-Un-Nabi e, Md. Ashraful Haque f
a
Institute of Marine Sciences, University of Chittagong, Chittagong 4331, Bangladesh
b
Department of Oceanography, Noakhali Science and Technology University, Noakhali 3814, Bangladesh
c
University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of
Hydrocenoses, Research Institute of Fish Culture and Hydrobiology, Zatisi 728/II, 389 25 Vodnany, Czech Republic
d
Harbour Aquaculture Research Center (HARC), Kalatoly, Cox’s Bazar, Bangladesh
e
Department of Fisheries, University of Chittagong, Chittagong 4331, Bangladesh
f
Bangladesh Fisheries Research Institute, Marine Fisheries and Technology Station Cox’s Bazar 4700, Bangladesh

A R T I C L E I N F O A B S T R A C T

Keywords: The bacterial population plays a crucial role in recycling the organic matter and metabolizing the toxic
Heterotrophic bacteria nitrogenous compounds under biofloc technology (BFT). The biofloc technology has been successfully imple­
Vibrio bacteria mented for several fish species, but not for shrimp culture in Bangladesh. The major objectives of this study were
Tiger shrimp
to determine the effects of stocking density on the growth performance of tiger shrimp Penaeus monodon under
biofloc technology (BFT) rearing system using commercial probiotics and to investigate the abundance of total
heterotrophic bacteria (THB) and vibrio-like bacteria (VLB) in different biofloc treatments and their water
quality parameters. The experiment was conducted with three different stocking densities in three BFT tanks (2.5
× 2.0 × 1.0 m) with triplicate and three control tanks filled in clearwater. All these tanks were built with cement
and can contain 5000 L of water. Visually healthy, disease-free P. monodon postlarvae (weighted 0.18 ± 0.02 g)
were collected from a local hatchery and stocked in three different stocking densities, i.e., 400 PL/m3 (CW), 400
PL/m3 (BFT1), 450 PL/m3 (BFT2) and 500 PL/m3 (BFT3). Shrimps were fed four times a day with commercial
feed (Biomer, 40 % crude protein) for 127 days. Significant differences (P < 0.05) in specific growth rate, food
conversion ratio, and protein efficiency ratio of shrimp were found among the treatments and the control group.
The overall final biomass in BFT1 (5.88 ± 0.12 kg m3) was significantly higher (P < 0.05) than that of other BFT
groups and the control (3.40 ± 0.09 kg m3). The present findings showed that the biofloc technology with lower
stocking density was reducing the total ammonia nitrogen, nitrite-N, and nitrate-N in water and significantly
increased THB and reduced VLB populations.

1. Introduction people are involved in Bangladesh across the entire shrimp production
value-chain (Paul and Vogl, 2012). There are about 300,000 shrimp
Global farmed shrimp production in 2016 was more than 5.7 million aquaculture farms (Feed the Future, 2016); many of them are in the
tonnes, where Bangladesh contributed about 2–3 % of the total pro­ coastal region of the country.
duction and ranked the 12th largest cultured shrimp producer in the Furthermore, most of the shrimp farms follow the extensive culture
world (FAO, 2018). Coastal shrimp farming is one of the fastest-growing practices relying on natural products and annually produce 160− 230 kg
aquaculture industries in Bangladesh. The tiger shrimp Penaeus monodon shrimp per hectare (Belton et al., 2011). Since 2012, shrimp farmers in
is one of the primary species and has been extensively cultured in the Bangladesh have been using better management practices. Recently, an
southern part of Bangladesh (AftabUddin et al., 2018). About 1.2 million intensive culture system is gaining popularity to increase the

* Corresponding author at: Department of Oceanography, Noakhali Science and Technology University, Noakhali 3814, Bangladesh.
E-mail address: [email protected] (M.A.M. Siddique).

https://doi.org/10.1016/j.aqrep.2020.100518
Received 14 June 2020; Received in revised form 26 September 2020; Accepted 16 October 2020
Available online 29 October 2020
2352-5134/© 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
S. AftabUddin et al. Aquaculture Reports 18 (2020) 100518

productivity of shrimp farms, and the average productivity has risen to mainly consists of various beneficial microbial species, but the function
3.7 ± 1.7 metric tons per ha (Rahman et al., 2019). However, disease of these beneficial microbes is still little known. On the other hand,
outbreaks remain a bottleneck problem for shrimp production all over probiotics for single or multiple known live microbial strains and their
the world, and Bangladesh has no exception. Pathogen invasion or actions on animals have well established. Therefore, probiotics are
illegal aquaculture practices resulting in disease outbreaks. Among the known as essential components in shrimp aquaculture. The biofloc
etiologic agents, viruses, and bacteria had taken as a heavy economic technology has been successfully implemented for several fish species,
toll worldwide (AftabUddin et al., 2017). but not for shrimp culture in Bangladesh. For the first time in
Beneficial microbiota-based biofloc culture is getting popular among Bangladesh, we have introduced commercial probiotic in a BFT system
the shrimp farming sector as it appears to be the most sustainable for culturing P. monodon. Optimization of stocking density of P. monodon
strategy to achieve improved water quality, disease resistance, and im­ in the BFT culture system is crucial for managing water quality in the
mune response (Zhou et al., 2009; Crab et al., 2012; Martínez-Córdova tanks and achieving higher production. Therefore, the major objectives
et al., 2015). Therefore, biofloc based nursery rearing system has pro­ of this study were (i) to determine the effects of stocking density on the
moted for better survival, healthy juvenile production, decrease nitro­ growth performance of tiger shrimp Penaeus monodon under BFT system
gen metabolites and reduce the cost of production by decreasing feed using commercial probiotics and (ii) to investigate the abundance of
conversion ratio (Emerenciano et al., 2011). Probiotic addition in bio­ total heterotrophic bacteria (THB) and vibrio-like bacteria (VLB) in
floc systems has been reported by some studies concerning the design of different biofloc treatments along with their water quality parameters.
the BFT system (Avnimelech, 2012), stocking density and growth per­
formances (Fóes et al., 2011; Anand et al., 2015; Arias-Moscoso et al.,
2018) and water quality management (Arnold et al., 2009). Biofloc

Fig. 1. The layout of the experimental setup of biofloc system.

2
S. AftabUddin et al. Aquaculture Reports 18 (2020) 100518

2. Materials and methods method of APHA (2005). Total alkalinity and total suspended solids
(TSS) of water were measured volumetrically and trapping a filter paper
2.1. Tank preparation (American Public Health Association (APHA, 2005). The volume of floc
was measured by PYREX® Brand Imhoff Cone (1000 mL), Thomas Sci­
The experiment was carried out at Harbour Aquaculture Research entific; the standardized sedimentation time was 30 min.
and Training Centre (21◦ 24′ 3.05′′ N, 91◦ 59′ 41.54′′ E), Cox’s Bazar,
Bangladesh for over 127 days. Twelve in-door cemented tanks 2.5. Growth parameters
measuring 2.5 × 2.0 × 1.0 m = 5 m3 (5000 L capacity) were washed by
5% HCL and detergent mixed water, respectively. Before that, the tanks Shrimps were harvested at the end of the experiment after draining
were painted with epoxy paint to provide a smooth interior surface. the tanks. The final weight, average daily growth rate (ADG), specific
After washing nicely, all the tanks were filled up to the marks of a beam growth rate (SGR), survival rate, final biomass production, and feed
(100 cm) with disinfected seawater and then fully discharged after 24 h conversion ratio (FCR) of shrimps were determined on fortnight basis
for rinsing purposes. Rows of PVC (1 inch) perforated L shape pipes were according to the method described earlier work (AftabUddin et al.,
laid on the tank bottom to serve as a lift aeration system (Fig. 1). A 5 hp 2017).
central blower was used for 24 h aeration throughout the experiment for
Final body weight × Initial body weight
ensuring better bioflocuation, to maintain the solids in suspension and to Average daily growth (ADG) =
Duration of experiment
ensure that dissolved oxygen remained at a saturation level. Aeration
pipe in each tank was provided with an air stone and a regulator for × 100
controlling the air pressure.
LN of final weight LN of initial weight
Specific growth rate (SGR) =
2.2. Biofloc production Duration of experiment
× 100
Before starting the trial, 0.6 g urea, 10 g soybean, 2 g rice bran, 0.5
mL molasses and 0.2 g NH4PO4 per liter of seawater, were added in a 400 Number of live shrimps
L FRP (Fiber Reinforced Plastic) tank. Two air stones were used for Survival (%) =
Initial number of total shrimps
× 100
continuous air supply for the biofloc production. The tank was covered
with black polythene to prevent the sunlight and kept for two weeks. Total feed consumed (Dry wt. basis)
Food conversion ratio (FCR) =
Floc volume (FV) was measured by using the Imhoff Cone (Merck Spe­ Weight of shrimp (g)
cialties, Mumbai, India). × 100

2.3. Experimental shrimp stocking and management Growth performances were assessed by using scoop Net (Size: 100 μ
mesh) with a standard scale and an electronic analytical balance (Precisa
The complete randomized experimental design was carried out XB 220A, Switzerland). Protein efficiency ratio (PER) was calculated
(Fig. 1) with two kinds of treatments: clearwater (CW) and biofloc according to the formulae given below:
treatments (BFT), applying three different levels of stocking density with PER = net weight gain (wet weight; g)/ protein consumed (g)
three replications per treatment. Nine experimental (BFT) culture tanks
were filled up with disinfected seawater (16 ± 1 ppt) up to the 80 cm 2.6. Total heterotrophic and Vibrio bacteria count
height (4 m3) of 100 cm. Twenty liters of biofloc were transferred to
each BFT tank by a plastic bucket. The aeration was continued, and the Count of total heterotrophic bacteria (THB) and Vibrio-like bacteria
density of the biofloc in the experimental tanks was monitored for seven (VLB) was performed once every two weeks during the experimental
days interval to make sure the biofloc density is still at an expected level. period. The water samples were collected in sterile glass bottles (250
Clearwater culture tanks also filled up with disinfected seawater and mL) at a depth of 40 cm randomly from all experimental and control
used as a control group. Visually healthy and disease-free P. monodon tanks and maintained at 4 ◦ C until further analysis. Bacteriological
postlarvae (0.18 ± 0.02 g) were collected from a local hatchery and were analysis of the water samples was performed according to the methods
stocked with four different stocking density, i.e., 400 PL/m3 (CW), 400 described by APHA (2005). Briefly, the water sample was diluted (10− 1
PL/m3 (BFT1), 450 PL/m3 (BFT2) and 500 PL/m3 (BFT3). Shrimps were to 10-3) with sterilized saline solution (2.5 % NaCl). Aliquots of 0.5 mL of
fed four times a day (07.00 a.m., 11 a.m., 07.00 pm, and 11.00 pm) with the serial dilutions were spread on Marine Zobell Agar media (HiMedia,
a commercial feed (BioMar, 40 % crude protein, France) with various Mumbai, India) for THB and Thiosulfate-citrate bile salts-sucrose (TCBS)
sizes (mm) from the beginning to the end of the experiment. Two g/m3 agar (Oxoid, UK) plates for the VLB count from the water samples. Each
of commercial probiotic (containing Bacillus licheniformis 1.8 × 108 cfu/ analysis was performed in triplicate by the spread plate method. Inoc­
g, Bacillus pumilus 2.70 × 108 cfu/g, Bacillus amyloliqufaciens 7.20 × 108 ulated plates were incubated at 35 ◦ C for 48 h. The colony was counted
cfu/g, and Bacillus subtilis 6.30 × 108 cfu/g) were distributed to each with a digital colony counter equipped with a guide plate ruled in square
tank at fortnightly intervals. The biofloc treatments were operated with centimeters and the counted colony expressed as colony forming units
minimal water exchange (10 %) every two weeks. Disinfected seawater (cfu/mL).
was added to compensate for the exchanged water.
2.7. Calculation of C: N ratio
2.4. Water quality parameters
Commercial diet (Biomer, 40 % crude protein; 200 μm~0.5 mm
Water samples were collected at fortnightly interludes from the sizes) was fed four times a day. The floc density in the culture tanks was
control and experimental tanks during morning time between 8:00 and controlled and maintained at a certain level (10 mg L− 1) in the two-week
9:00 h for 127 days. The water temperature, salinity, and pH were interval, if the floc was higher, a volume of water in the culture medium
measured directly using a digital thermometer, hand refractometer was siphoned. Contrary, when the floc density was decreased, a volume
(ATAGO, Japan), and a digital pH meter, respectively. Dissolved oxygen of floc from the BFT growing tank was taken and added into the culture
(DO) was measured by a digital DO meter (SI analytics, lab 747, tank. The molasses was used in all biofloc treatments as a carbon source.
Thailand). The total ammonia-nitrogen (TAN), nitrite (NO2-N), and ni­ The ratio of carbon and nitrogen (12:1) was maintained through the
trate (NO3-N) were analyzed spectrophotometrically following the additional carbon source and nitrogen content (feed), which facilitate

3
S. AftabUddin et al. Aquaculture Reports 18 (2020) 100518

the removal of waste (nitrogen metabolites) by a heterotrophic con­ significantly higher than those of biofloc treatments. The floc volume
version. Ten percent of water exchanges were performed every two (FV) and TSS were significantly higher in the biofloc tanks compared to
weeks by removing the sludge from culture water employing a standing the control tanks and gradually increased during the experimental
pipe covered with a net (150 μ) in all biofloc tanks, but 25 % of water period and turned into brown. The maximum floc volume in BFT3 was
was exchanged once every three days in control tanks (CW) to keep the 23.73 ± 2.58 mg L− 1 which was significantly higher than the other
concentration of ammonia and nitrite within the recommended level. treatments (P < 0.05).
The carbon-to-nitrogen ratio was measured according to Avnimelech
(2015) and Emerenciano et al. (2017). 3.2. Growth performance and survival rate

There were significant differences (P < 0.05) in average body weight

2.8. Data analysis
(ABW), specific growth rate (SGR), feed conversion ratio (FCR), and
protein efficiency ratio (PER) among the treatments (Table 2). The BFT1
All data were analyzed using STATISTICA V.13 software (StatSoft
treatment had significantly higher average daily growth (ADG), SGR,
Inc., Tulsa, OK, USA). Residuals were tested for normality (Shapiro–Wilk
FCR, and PER compared to other biofloc treatments and control (P <
test) and homogeneity of variance (plot of residuals versus predicted
0.05). The total weight gain and survival rate (81.87±1.65 %) was
values). Water quality parameters were compared by two-way repeated-
significantly higher in BFT1 (P < 0.05) than that of other biofloc treat­
measures ANOVA with treatment as the main factor and sampling date
ments and the control (Table 2). Contrary, the feed conversion ratio
as repeated measures factor. A series of one-way ANOVA was performed,
(FCR) in BFT1 was significantly lower (P < 0.05) than the control and
followed by Fisher’s LSD test to determine the differences among the
other biofloc treatments, but the protein efficiency ratio (PER) was
treatments for the growth parameters of shrimp and bacterial abun­
significantly higher (P < 0.05) than that of other treatment groups
dance. All proportional data were arcsin square-root transformed before
(Table 2). The accumulated biomass varied from 3.40 ± 0.09–5.88 ±
analyses. Data were presented as mean ± SE. Alpha was set at p < 0.05.
0.12 kg m− 3, where BFT1 treatment had the highest biomass of 5.88 ±
0.12 kg m− 3.
3. Results
3.3. Total heterotrophic and Vibrio bacteria count
3.1. Water quality parameters
The mean concentration of the total heterotrophic bacterial (THB) in
The water quality parameters of the experimental tanks are pre­
the water was significantly higher (P < 0.05) in the biofloc treatment
sented in Table 1. Water quality parameters in the experimental tanks
tanks than the control tank (Fig. 2). The mean THB count had higher in
remained within the acceptable ranges for the culture of P. monodon
number in BFT1 (6.29 ± 0.43 × 104) followed by BFT2 (5.93 ± 0.23 ×
throughout the trial. There were no significant differences (P > 0.05)
104) and BFT3 (5.79 ± 0.29 × 104) than the control group (4.36 ± 0.61
among the treatments in terms of water temperature and salinity. Dis­
× 103 cfu mL− 1). The mean concentration of Vibrio-like bacteria (VLB)
solved oxygen and pH significantly differed between control and biofloc
was significantly higher (4.8 ± 0.52 × 103 cfu mL− 1) in the control tank
treatments. The BFT1 treatment (5.02 ± 0.34 mg L− 1) had significantly
(P > 0.05) compared to BFT1 (2.15±0.41×102 cfu mL− 1), BFT2 (2.85 ±
lower DO than that of control (6.12 ± 0.41 mg L− 1) and other treat­
0.49 × 102 cfu mL− 1), and BFT3 (2.96 ± 0.25 × 102 cfu mL− 1) treat­
ments. The pH value had a significant variation (P < 0.05) in the biofloc
ments (Fig. 3). The mean concentration of THB was not significantly
treatment tanks compared to the control tank (Table 1). The mean pH
different among the biofloc treatments (P > 0.05), but an increasing
concentrations were 7.59, 7.84, 7.76, and 8.02 in the BFT1, BFT2, BFT3,
trend of THB was observed with the increase of culture period (Fig. 4).
and control groups, respectively. The total ammonia nitrogen (TAN),
The mean concentrations of VLB were significantly lower in BFT1
nitrite-N, and nitrate-N concentrations in the control group were
compared to other biofloc treatments (P < 0.05) and had a significant
difference (P < 0.05) during several sampling days (Fig. 5).
Table 1
Water quality parameters of P. monodon stocked in various densities in Biofloc
technology (BFT) and control groups over 127 days.
Biofloc treatment
Parameters Control
BFT1 BFT2 BFT3

Temperature (◦ C) 27.68 ± 27.6 ± 27.8 ± 27.75 ± Table 2

0.21a 0.13a 0.24a 0.2a
Growth parameters of P. monodon in the biofloc treatments with different
pH (no unit) 8.02 ± 7.59 ± 7.84 ± 7.76 ±
stocking density and the control (means ± SE).
0.1c 0.1a 0.1b 0.1b
DO (mg L− 1) 6.12 ± 5.02 ± 5.78 ± 5.56 ± Biofloc Treatment
0.41c 0.34a 0.24b 0.36b Parameters Control
BFT1 BFT2 BFT3
Salinity (‰) 16.94 ± 16.85 ± 16.77 ± 16.8 ±
0.2a 0.4a 0.7a 0.5a Average body weight (g) 12.95 ± 17.97 ± 15.25 ± 14.54 ±
Total ammonium nitrogen 1.15 ± 0.43 ± 0.57 ± 0.68 ± 0.03a 0.19c 0.13b 0.09b
(TAN) (mg L− 1) 0.10d 0.05a 0.12b 0.08c Average daily growth 0.10 ± 0.14 ± 0.12 ± 0.11 ±
Nitrite-N (mg L− 1) 1.61 ± 0.58 ± 0.73 ± 0.96 ± (ADG) (g/day) 0.004a 0.008b 0.007a 0.002a
0.18d 0.07a 0.13b 0.08c Specific growth rate 3.36 ± 3.62 ± 3.49 ± 3.45 ±
Nitrate-N (mg L− 1) 3.02 ± 1.89 ± 2.34 ± 2.53 ± (SGR) (%/day) 0.07a 0.02b 0.03a 0.05a
0.1c 0.17a 0.12b 0.14b Survival (%) 65.73 ± 81.87 ± 74.07 ± 68.17 ±
Alkalinity (mg CaCO3 L− 1) 166.68 ± 138.56 ± 150.56 ± 156.42 ± 1.73a 1.65c 2.43b 2.45a
8.43c 4.9a 3.8b 5.8b Feed conversion ratio 2.30 ± 1.42 ± 1.82 ± 2.16 ±
Total suspended solids 74.39 ± 128.5 ± 97.82 ± 149.5 ± (FCR) 0.25b 0.16c 0.40b 0.28a
(mg L− 1) 21.5a 14.6c 16.9b 24.3d Protein efficiency ratio 1.32 ± 1.70 ± 1.49 ± 1.38 ±
Floc volume (mg L− 1) 5.68 ± 15.94 ± 12.87 ± 23.73 ± (PER) 0.13a 0.1c 0.12b 0.23a
2.75a 2.91b 2.77b 2.58c Final biomass (kg m− 3) 3.40 ± 5.88 ± 5.08 ± 4.95 ±
0.09a 0.12c 0.16b 0.18b
The values are means (±SE, n = 30) of three replications in ten sampling dates
for the control and experimental tanks. Mean values in the same row with Mean values in the same row with different superscript differ significantly (P <
different superscript differ significantly (P < 0.05). 0.05).

4
S. AftabUddin et al. Aquaculture Reports 18 (2020) 100518

biofloc treatments than the control because of the higher oxygen de­
mand by the bacteria and other microorganisms. The lowest level of
dissolved oxygen observed in BFT1 (5.02 ± 0.34 mg L− 1) was sufficient
for good survival and growth of P. monodon (McGraw et al., 2001;
Panigrahi et al., 2020). However, the sufficient DO level in all the
treatments during the experimental period were attributed to contin­
uous aeration in the biofloc tanks. The alkalinity of water was above the
recommended levels of 130 mg L− 1 (Boyd et al., 2002), which were
provided with the physiological conditions allowing shedding and
proper formation of the exoskeleton, promoting growth and survival of
microorganisms (McGraw and Scarpa, 2003). Alkalinity influences the
process of nitrification by the development of nitrifying bacteria (Ebel­
Fig. 2. Mean (±SE) total heterotrophic bacteria in different treatment tanks.
Different letters indicated a statistically significant difference among the ing et al., 2006).
treatments (p < .05). The pH value in the biofloc treatments (ranged between 7.59–7.84)
was lower than that of the control treatment (8.02), probably due to the
respiration of heterotrophic bacteria, which increased the carbon diox­
ide concentration in the water of the biofloc treatments. The TAN con­
centrations were slightly higher (0.43 to 0.68 mg L− 1) in the present
study compared to earlier reports of the biofloc systems for P. monodon
by Thakur and Lin (2003) and Panigrahi et al. (2020). In the present
study, a low concentration of NO2-N and a high concentration of NO3-N
was observed, nitrite is derived by the oxidation of ammonia into nitrite
by autotrophic bacteria (Ebeling et al., 2006) during the nitrification
process. Due to this process, the BFT system may generate higher con­
centrations of nitrite than ammonia during the shrimp production cycle.
Silva et al. (2013) found that out of the 39 % of dissolved nitrogen
entered into the BFT system, about 0.9 % was in the form of total
ammonia, while 12 % was in the form of total nitrite after 42 days.
Fig. 3. Mean (±SE) total Vibrio-like bacteria in different treatment tanks.
Different letters indicated a statistically significant difference among the
Nitrite causes a delay in growth and reduces the survival of cultured
treatments at the end of the experiment (p < 0.05). organisms at high concentrations (Lin and Chen, 2003).
Biofloc based shrimp rearing system can improve water quality pa­
rameters by the presence of beneficial microorganisms (Schveitzer et al.,
2017; Panigrahi et al., 2018). Khanjani et al. (2017) reported significant
improvement in the nitrogen metabolites in terms of pH, ammonia, ni­
trite, and nitrate in the biofloc based nursery rearing of P. vannamei. In
this experiment, the probiotics incorporated bioflocs treatments signi­
ficantly reduced the TAN level when compared with the control. As most
of the ammonia in the culture system is taken up by heterotrophic
bacteria, the availability of NO3-N in the BFT system thus allows the
phytoplankton to grow (Middelburg and Nieuwenhuize, 2000). The TSS,
observed in this study, was within the recommended level of <500 mg
L− 1 for penaeid shrimps (Samocha et al., 2007). Several authors have
Fig. 4. The mean concentration of total heterotrophic bacteria at different
indicated that a similar trend of concentration of TSS is beneficial for the
sampling periods in the treatment tanks at the end of the experiment.
shrimp and the biofloc system stability (Schryver et al., 2008; Baloia
et al., 2013).

4.2. Growth parameters

ABW, SGR, final gain weight, and PER varied significantly between
the biofloc and control groups (Table 2). Growth and survival rates in
BFT1 were significantly higher than the other treatments, showing that
adequate stocking density with the addition of probiotic was helpful in
the growth performance and survival of P. monodon. These may be due
to the synergistic effects of improved water quality and a high hetero­
trophic bacterial density. Several previous studies have shown that
Fig. 5. The mean concentration of total Vibrio-like bacteria at different sam­ growing penaeid shrimp in the biofloc systems can improve the survival
pling periods in the treatment tanks. and growth performance of shrimp compared to growing in clear water
(Cohen et al., 2005; Mishra et al., 2008). In the present study, the
4. Discussion average daily growth of P. monodon was slightly lower than the previous
report of L. vannamei reared in similar biofloc system (Ray et al., 2011;
4.1. Water quality Schveitzer et al., 2013). However, the survival rate of P. monodon
(68.17–81.87 %) in our biofloc system was consistent with the results
Temperature, salinity, DO, and pH value of water in both control and (79.73–85.4 %) obtained by Panigrahi et al. (2020), but this survival
the biofloc groups were within the required level for the penaeid species rate is much higher compared to L. vannamei (57.8–73.8 %) reported by
(Hari et al., 2006; Zhang et al., 2006; Wasielesky et al., 2006; Panigrahi Arantes et al. (2017) under similar culture conditions.
et al., 2020). In the present study, the level of DO was lower in the At the end of the feeding period, P. monodon achieved a higher

5
S. AftabUddin et al. Aquaculture Reports 18 (2020) 100518

average body weight (17.97 ± 0.19 g) and a total output of 5.88 ± 0.12 2014; Hostins et al., 2017). The key reasons for the probiotics used in
kg m− 3 with a lower FCR (1.42 ± 0.16) in the BFT1 tanks compared to BFT are (i) helping to maintain the heterotrophic community and
the control tanks. Panigrahi et al. (2020) observed a lower FCR value competing with autotrophic microorganisms (mainly in the initial
(1.0–1.22) for P. monodon cultured in nursery based BFT compared to phases), (ii) helping to recycle organic matter, and (iii) regulating solids
the control group (1.9). Mishra et al. (2008) reported FCR values of 1.5 and TAN levels (Emerenciano et al., 2017). In this experiment, the
for P. vannamei in the BFT-based nursery, while the FCR value of the minimum water exchange (10 %) conditions could have favored the
control group was 2.49. Different microbial communities present in the beneficial bacteria proliferation; in fact, most biofloc experiments have
biofloc system provide vital nutrients for shrimp such as protein been developed without probiotics addition.
(Emerenciano et al., 2011), lipid (Wasielesky et al., 2006), amino acids
(Ju et al., 2008), and fatty acids (Ekasari et al., 2010). Therefore, it could 5. Conclusion
be postulated that the biofloc system can significantly reduce the FCR
value for the shrimp. Furthermore, the biofloc increases the availability The present study concludes that
of natural feed for shrimps, thus increasing the production performance,
and this is more important during the nursery and growing phase. Bal­ 1) P. monodon shrimps reared with a low stocking density (400 PL/m3)
lester et al. (2010) reported that the protein efficiency rate (PER) was showed better growth performance in terms of average daily growth,
higher in the zero-water exchange of the Farfantepenaeus paulensis specific growth rate, final biomass, and survival rate with a lower
nursery phase fed by different diets. About 18–29 % of the daily feed for FCR and PER.
penaeid shrimp comes from the flocculated heterotrophic culture system 2) Biofloc technology with lower stocking density significantly
(Burford et al., 2004). The addition of probiotics in the biofloc tanks may increased THB and reduced VLB populations.
develop a healthier and stable microbial community, providing benefits
to the cultivated animals (Tacon et al., 2002; Burford and Lorenzen, Data availability statement
2004).
All data analyzed during this study are included in this manuscript.
4.3. Total heterotrophic and Vibrio bacteria
CRediT authorship contribution statement
When a culture system operates with a zero or minimal exchange of
water, organic matter appears to accumulate in the water column and
Sheikh AftabUddin: Conceptualization, Project administration,
influence the growth of the heterotrophic microbial population (Martí­
Funding acquisition, Methodology, Investigation, Data curation, Writing
nez-Córdova et al., 2015). Heterotrophic bacteria degrade organic
- original draft, Writing - review & editing. Mohammad Abdul Momin
matter and use it as an energy source as well as for the creation of new
Siddique: Data curation, Formal analysis, Software, Writing - original
cells (Ebeling et al., 2006; Emerenciano et al., 2017). In this study, the
draft, Validation, Visualization, Writing - review & editing. Aung Sein:
number of total heterotrophic bacteria in the biofloc community was
Project administration, Resources, Methodology, Writing - original
significantly higher than the control. The mean concentration of THB
draft. Probin Kumar Dey: Data curation, Formal analysis, Investigation.
(6.29 ± 0.43 × 104 in BFT1; 5.93 ± 0.23 × 104 in BFT2, and 5.79 ± 0.29
Md. Rashed-Un-Nabi: Formal analysis, Resources, Writing - original
× 104 in BFT3) are very close to the previous report obtained in the
draft, Writing - review & editing. Md. Ashraful Haque: Project
BFT-based nursery ponds for P. vannamei (Panigrahi et al., 2020). The
administration, Funding acquisition, Methodology, Writing - original
presence of heterotrophic bacteria in the control tank (4.36 ± 0.61 × 103
draft.
cfu mL− 1) could be attributed to the proliferation of the endemic mi­
crobial community, and the introduction of protein-rich feeds. Devaraja
et al. (2002) found the mean concentrations of THB ranging from 1.78 × Declaration of Competing Interest
104 to 6.82 × 104 cfu mL− 1 in the bottom water of the probiotic treated
ponds where the mean density of THB in the control ponds was 2.4 × 104 The authors report no declarations of interest.
cfu mL− 1. However, the mean concentration of THB could be ranged
from 12.1–26.9 × 104 cfu mL− 1 in the in-door experiments for Acknowledgments
P. monodon culture with biofloc (Hari et al., 2006). Panigrahi et al.
(2020) fund the THB count was substantially higher in the biofloc The authors are thankful to Professor Dr. Md. Shafiqul Islam, Di­
consortium (7.8 ± 0.5 × 104) than the control groups (4.19 ± 0.5 × 104 rector, Institute of Marine Sciences and Professor Dr. Hossain Jamal,
cfu/mL), where the Vibrio count decreased substantially in the biofloc Chairman, Department of Fisheries, University of Chittagong,
consortium (7.2 ± 0.1 × 102) compared to the control group (1.94 ± 0.2 Bangladesh, for their encouragement to carry out this work. The authors
× 103 cfu mL− 1). Our present study shows a similar trend and abundance would like to thank the Authority of Harbour Aquaculture Research
of THB and Vibrio bacteria in the biofloc and the control treatments. Center (HARC), Kalatoly, Cox’s Bazar, for their enormous supports for
The dominance of THB will minimize pathogenic bacteria by the conducting this study.
action of beneficial microorganisms in the biofloc system (Panigrahi
et al., 2017). Besides, these heterotrophic bacteria can control the References
presence of ammonia and other nitrogen metabolites and convert waste
to additional food sources in the shrimp rearing system (Ebeling et al., AftabUddin, S., Siddique, M.A.M., Romkey, S.S., Shelton, W.L., 2017. Antibacterial
2006; Schveitzer et al., 2013). Colonies of Vibrio-like bacteria were function of herbal extracts on growth, survival and immunoprotection in the black
tiger shrimp Penaeus monodon. Fish Shellfish Immunol. 65, 52–58.
higher in the control system (107.1 × 103 cfu mL-1) but lower (35.5 × AftabUddin, S., Roman, W.U., Hasan, C.K., Ahmed, M., Rahman, H., Siddique, M.A.M.,
103 cfu mL− 1) in the biofloc-treated commercial farms in Mexico for 2018. First incidence of loose-shell syndrome disease in the giant tiger shrimp
L. vannamei (Arias-Moscoso et al., 2018). The introduction of carbohy­ Penaeus monodon from the brackish water ponds in Bangladesh. J. Appl. Anim. Res.
46 (1), 210–217.
drates had a positive effect on THB in the water column and sediment
American Public Health Association (APHA), 2005. Standard Methods for the
(Hari et al., 2006). The predominance of THB and a low concentration of Examination of Water and Wastewater. American Public Health Association,
Vibrio-like bacteria in the probiotic treated tanks indicating that the Washington, p. 560.
probiotic bacteria, i.e., Bacillus licheniformes, B. pumilus, Anand, P.S., Panigrahi, A., Ravichandran, P., Kumar, S., Dayal, J.S., Deo, A.D.,
Ponniah, A.G., Pillai, S.M., 2015. Growth performance of black tiger shrimp (Penaeus
B. Amyloliquifaciens and B. Subtilis has proliferated in the water and led monodon) in substrate based zero water exchange system. Indian J. Geo-Marine Sci.
to the succession and dominance of these species (Krummenauer et al., 44 (10), 1495–1503.

6
S. AftabUddin et al. Aquaculture Reports 18 (2020) 100518

Arantes, R., Schveitzer, R., Magnotti, C., Lapa, K.R., Vinatea, L., 2017. A comparison Krummenauer, D., Poersch, L., Romano, L.A., Lara, G.R., Encarnação, P.,
between water exchange and settling tank as a method for suspended solids Wasielesky Jr, W., 2014. The effect of probiotics in a Litopenaeus vannamei biofloc
management in intensive biofloc technology systems: effects on shrimp (Litopenaeus culture system infected with Vibrio parahaemolyticus. J. Appl. Aquacult. 26 (4),
vannamei) performance, water quality and water use. Aquac. Res. 48 (4), 370–379.
1478–1490. Lin, Y.C., Chen, J.C., 2003. Acute toxicity of nitrite on Litopenaeus vannamei (Boone)
Arias-Moscoso, J.L., Espinoza-Barrón, L.G., Miranda-Baeza, A., Rivas-Vega, M.E., Nieves- juveniles at different salinity levels. Aquaculture 224, 193–201.
Soto, M., 2018. Effect of commercial probiotics addition in a biofloc shrimp farm Martínez-Córdova, L.R., Emerenciano, M., Miranda-Baeza, A., Martínez-Porchas, M.,
during the nursery phase in zero water exchange. Aquac. Reports 11, 47–52. 2015. Microbial-based systems for aquaculture of fish and shrimp: an updated
Arnold, S.J., Coman, F.E., Jackson, C.J., Groves, S.A., 2009. High-intensity, zero water- review. Rev. Aquac. 7 (2), 131–148.
exchange production of juvenile tiger shrimp, Penaeus monodon: an evaluation of McGraw, W.J., Scarpa, J., 2003. Minimum environmental potassium for survival of
artificial substrates and stocking density. Aquaculture 293 (1-2), 42–48. Pacific white shrimp Litopenaeus vannamei (Boone) in freshwater. J. Shellfish Res.
Avnimelech, Y., 2015. Biofloc Technology, A Practical Guidebook, third edition. Words 22 (1), 263–268.
Aquaculture Society. McGraw, W., Teichert-Coddington, D.R., Rouse, D.B., Boyd, C.E., 2001. Higher minimum
Ballester, E.L.C., Abreu, P.C., Cavalli, R.O., Emerenciano, M., De Abreu, L., dissolved oxygen concentrations increase penaeid shrimp yields in earthen ponds.
Wasielesky Jr., W., 2010. Effect of practical diets with different protein levels on the Aquaculture 199, 311–321.
performance of Farfantepenaeus paulensis juveniles nursed in a zero exchange Middelburg, J.J., Nieuwenhuize, J., 2000. Nitrogen uptake by heterotrophic bacteria and
suspended microbial flocs intensive system. Aquac. Nutr. 16 (2), 163–172. phytoplankton in the nitrate-rich Thames estuary. Mar. Ecol. Prog. Ser. 203, 13–21.
Baloia, M., Arantes, R., Schveitzer, R., Magnotti, C., Vinatea, L., 2013. Performance of Mishra, J.K., Samocha, T.M., Patnaik, S., Speed, M., Gandy, R.L., Ali, A., 2008.
Pacific white shrimp Litopenaeus vannamei raised in biofloc systems with varying Performance of an intensive nursery system for the Pacific white shrimp, Litopenaeus
levels of light exposure. Aquacult. Eng. 52, 39–44. vannamei, under limited discharge condition. Aquacult. Eng. 38, 2–15.
Belton, B., Karim, M., Thilsted, S., Collis, W., Phillips, M., 2011. Review of Aquaculture Panigrahi, A., Saranya, C., Sundaram, M., Vinoth Kannan, S.R., Das, R.R., Satish
and Fish Consumption in Bangladesh. Studies and Reviews 2011-53. The WorldFish Kumar, R., Rajesh, P., Otta, S.K., 2018. Carbon: nitrogen (C:N) ratio level variation
Center. November 2011. influences microbial community of the system and growth as well as immunity of
Boyd, C., Thunjai, T., Boonyaratpalin, M., 2002. Dissolved salts in water for inland low shrimp (Litopenaeus vannamei) in biofloc based culture system. Fish Shellfish
salinity shrimp culture. Global Aquac. Advocate 5, 40–45. Immunol. 81, 329–337.
Burford, M.A., Lorenzen, K., 2004. Modeling nitrogen dynamics in intensive shrimp Panigrahi, A., Saranya, C., Ambiganandam, K., Sundaram, M., Sivakumar, M.R., 2020.
ponds: the role of sediment remineralization. Aquaculture 229 (1-4), 129–145. Evaluation of biofloc generation protocols to adopt high density nursery rearing of
Burford, M.A., Thompson, P.J., McIntosh, R.P., Bauman, R.H., Pearson, D.C., 2004. The Penaeus vannamei for better growth performances, protective responses and
contribution of flocculated material to shrimp (Litopenaeus vannamei) nutrition in a immunomodulation in biofloc based technology. Aquaculture 522, 735095.
high-intensity, zero-exchange system. Aquaculture 232 (1-4), 525–537. Paul, B.G., Vogl, C.R., 2012. Key performance characteristics of organic shrimp
Cohen, J., Samocha, T.M., Fox, J.M., Lawrence, A.L., 2005. Biosecured production of aquaculture in southwest Bangladesh. Sustainability 4 (5), 995–1012.
juvenile Pacific white shrimp in an intensive raceway system with limited water Rahman, M.M., Ali, H., Jaman, A., Eltholth, M., Murray, F., 2019. In: The 2nd
discharge. Aquacult. Eng. 32, 425–442. International Scientific Conference in Aquatic Animal Epidemiology (4 - 6 Nov
Crab, R., Defoirdt, T., Bossier, P., Verstraete, W., 2012. Biofloc technology in 2019). Hua Hin, Thailand p.30.
aquaculture: beneficial effects and future challenges. Aquaculture 356, 351–356. Ray, A.J., Dillon, K.S., Lotz, J.M., 2011. Water quality dynamics and shrimp Litopenaeus
Devaraja, T.N., Yusoff, F.M., Shariff, M., 2002. Changes in bacterial populations and vannamei production in intensive, mesohaline culture systems with two levels of
shrimp production in ponds treated with commercial microbial products. biofloc management. Aquacult. Eng. 45, 127–136.
Aquaculture 206 (3-4), 245–256. Samocha, T.M., Patnaik, S., Speed, M., Ali, A.M., Burger, J.M., Almeida, R.V., Ayub, Z.,
Ebeling, J.M., Timmons, M.B., Bisogni, J.J., 2006. Engineering analysis of the Harisanto, M., Horowitz, A., Brock, D.L., 2007. Use of molasses as carbon source in
stoichiometry of photoautotrophic, autotrophic, and heterotrophic removal of limited discharge nursery and grow-out systems for Litopenaeus vannamei. Aquacult.
ammonia–nitrogen in aquaculture systems. Aquaculture 257, 346–358. Eng. 36 (2), 84–191.
Ekasari, J., Crab, R., Verstraete, W., 2010. Primary nutritional content of bio-flocs Schryver, P.D., Crab, R., Defoirdt, T., Boon, N., Verstraete, W., 2008. The basics of bio-
cultured with different organic carbon sources and salinity. Hayati J. Biosci. 17 (3), flocs technology: the added value for aquaculture. Aquaculture 277, 125–137.
25–130. Schveitzer, R., Arantes, R., Costódio, P.F.S., do Espírito Santo, C.M., Arana, L.V.,
Emerenciano, M., Ballester, E.L., Cavalli, R.O., Wasielesky, W., 2011. Effect of biofloc Seiffert, W.Q., Andreatta, E.R., 2013. Effect of different biofloc levels on microbial
technology (BFT) on the early postlarval stage of pink shrimp Farfantepenaeus activity, water quality and performance of Litopenaeus vannamei in a tank system
paulensis: growth performance, floc composition and salinity stress tolerance. Aquac. operated with no water exchange. Aquacult. Eng. 56, 59–70.
Int. 19 (5), 891–901. Schveitzer, R., de Lorenzo, M.A., do Nascimento Vieira, F., Pereira, S.A., Mouriño, J.L.P.,
Emerenciano, M.G.C., Martínez-Córdova, L.R., Martínez-Porchas, M., Miranda-Baeza, A., Seiffert, W.Q., Andreatta, E.R., 2017. Nursery of young Litopenaeus vannamei
2017. Biofloc technology (BFT): a tool for Water quality management in aquaculture. postlarvae reared in biofloc and microalgae-based systems. Aquacult. Eng. 78,
In: Tutu, Hlanganani (Ed.), Water Quality. Intech, pp. 91–109. 140–145.
FAO, 2018. The State of World Fisheries and Aquaculture 2018, Meeting the Sustainable Silva, K.R., Wasielesky, W.J., Abreu, P.C., 2013. Nitrogen and phosphorus dynamics in
Development Goals. Rome. the biofloc production of the pacific white shrimp, Litopenaeus vannamei. J World
Feed the Future, 2016. Bangladesh Shrimp Hatchery Goes from ‘Zero to Hero. Accessed Aquac. Soc. 44, 30–41.
on 24 March 2020. https://www.feedthefuture.gov/article/bangladesh-shrimp-ha Tacon, A.G.J., Cody, J.J., Conquest, L.D., Divakaran, S., Forster, I.P., Decamp, O.E.,
tchery-goes-zero-hero. 2002. Effect of culture system on the nutrition and growth performance of Pacific
Fóes, G.K., Fróes, C., Krummenauer, D., Poersch, L., Wasielesky, W., 2011. Nursery of white shrimp Litopenaeus vannamei (Boone) fed different diets. Aquac. Nutri. 8,
pink shrimp Farfantepenaeus paulensis in biofloc technology culture system: survival 121–137.
and growth at different stocking densities. J. Shellfish Res. 30 (2), 367–373. Thakur, D.P., Lin, C.K., 2003. Water quality and nutrient budget in closed shrimp
Hari, B., Kurup, B.M., Varghese, J.T., Schrama, J.W., Verdegem, M.C.J., 2006. The effect (Penaeus monodon) culture systems. Aquacult. Eng. 27 (3), 159–176.
of carbohydrate addition on water quality and the nitrogen budget in extensive Wasielesky Jr, W., Atwood, H., Stokes, A., Browdy, C.L., 2006. Effect of natural
shrimp culture systems. Aquaculture 252 (2-4), 248–263. production in a zero-exchange suspended microbial floc based super-intensive
Hostins, B., Lara, G., Decamp, O., Cesar, D.E., Wasielesky Jr., W., 2017. Efficacy and culture system for white shrimp Litopenaeus vannamei. Aquaculture 258 (1-4),
variations in bacterial density in the gut of Litopenaeus vannamei reared in a BFT 396–403.
system and in clear water supplemented with a commercial probiotic mixture. Zhang, P., Zhang, X., Li, J., Huang, G., 2006. The effects of body weight, temperature,
Aquaculture 480, 58–64. salinity, pH, light intensity and feeding condition on lethal DO levels of white
Ju, Z.Y., Forster, I., Conquest, L., Dominy, W., Kuo, W.C., David Horgen, F., 2008. shrimp, Litopenaeus vannamei (Boone, 1931). Aquaculture 256, 579–587.
Determination of microbial community structures of shrimp floc cultures by Zhou, X.X., Wang, Y.B., Li, W.F., 2009. Effect of probiotic on larvae shrimp (Litopenaeus
biomarkers and analysis of floc amino acid profiles. Aquac. Res. 39 (2), 118–133. vannamei) based on water quality, survival rate and digestive enzyme activities.
Khanjani, M.H., Sajjadi, M.M., Alizadeh, M., Sourinejad, I., 2017. Nursery performance Aquaculture 287 (3-4), 349–353.
of Pacific white shrimp (Litopenaeus vannamei Boone, 1931) cultivated in a biofloc
system: the effect of adding different carbon sources. Aquac. Res. 48 (4), 1491–1501.