Aquaculture 500 (2019) 443–450

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Aquaculture
journal homepage: www.elsevier.com/locate/aquaculture

Does the biofloc size matter to the nitrification process in Biofloc T

Technology (BFT) systems?
Janaína Souza, Alessandro Cardozo, Wilson Wasielesky Jr, Paulo Cesar Abreu
⁎

Aquaculture and Marine Biotechnology Group, Institute of Oceanography, Federal University of Rio Grande – FURG, Cx P. 474., 96201-900 Rio Grande, RS, Brazil

ARTICLE INFO ABSTRACT

Keywords: In addition to environmental factors, particle size can interfere with the nitrification process because smaller
Particle size flocs are less efficient in terms of ammonia and nitrite oxidation. The objective of the present study was to
Nitrification evaluate, in three experiments, the effect of the biofloc size on the nitrification process during the production of
Filtration the white shrimp Litopenaeus vannamei in a BFT system. In all experiments, 1% of biofloc inoculum from pro-
Water Turbulence
duction tanks was used. Water samples were taken to photograph and measure the flocs. Measurements of
temperature, salinity, nitrogen compounds and total suspended solids were also carried out in the experiments.
In experiment 1, the flocs were separated by sifting the water through mesh nets of 50, 150 and 300 μm in size.
In addition, there was a control treatment with integral flocs. In experiment 2, the bioflocs were separated into
300 and 600 μm size classes and were compared with an integral control. Experiment 3 aimed to evaluate the
nitrification process in treatments with flocs smaller than 150 μm, in flocs without handling (Control), and in the
treatment where flocs were partially filtered in a 50 μm mesh for five days. This treatment resulted in a reduction
in the particle size; however, when the meshes were later removed, and the biofloc size increased again.
Different from our original hypothesis, the results of these three experiments indicate that the size of the biofloc,
as well as the amount of total suspended solids, do not affect the nitrification process in the BFT system.
However, all treatments that included manipulation through sifting showed a delay or a decrease in the ni-
trification activity. Thus, it is likely that, more than the particle size or floc abundance, the rupture of the floc
structure affects the distribution and interaction of nitrifying bacteria with reflections in the nitrification process.

1. Introduction nitrification performed by autotrophic nitrifying bacteria is established

The superintensive production of aquatic organisms using Biofloc
Technology (BFT) is characterized by low water exchange, high
stocking densities and predominantly aerobic microbiota inhabiting
heterotrophic flocs. These microorganisms, besides acting in the
maintenance of the water quality, represent an important com-
plementary food source for the produced organisms (De Schryver et al.,
2008; Avnimelech, 2009).
The removal of toxic nitrogen compounds in the BFT system is the
key for the success of this aquaculture technology. In this system, the
decrease of ammonia results from the action of heterotrophic bacteria,
which assimilate this element after the addition of an organic carbon
source (molasses or other type of sugar). However, the supplement of
molasses is a palliative process, since it is difficult to control the am-
monia levels over time due to the consumption of the heterotrophic
bacteria by the protozoa (De Schryver et al., 2008; Silva et al., 2013).
Thus, fertilization with molasses must be conducted until the
(Hargreaves, 2006). Moreover, the addition of a carbon source pro-
duces a significant amount of suspended solids that can negatively af-
fect the produced organisms.
The nitrifying autotrophic microorganisms actually conduct the
complete removal of toxic nitrogenous compounds, by oxidizing am-
monia into nitrite, which is performed by ammonia-oxidizing bacteria
(AOB) and archaean-oxidizing bacteria (AOA). Later, the nitrite oxi-
dizing bacteria (NOB) transform nitrite into nitrate, eliminating from
the system these two forms of nitrogenous compounds that present
greater toxicity to the produced organisms (Ebeling et al., 2006;
Hargreaves, 2006). However, these bacteria have slow growth in
comparison to heterotrophic microorganisms and are also more sus-
ceptible to environmental conditions.
In general, the nitrifying community is very sensitive to environ-
mental and operational factors, such as extreme pH, low concentrations
of dissolved oxygen and temperature oscillations (Koops and
Pommerening, 2001). In addition, the size and composition of the

⁎
Corresponding author.
E-mail address: docpca@furg.br (P.C. Abreu).

https://doi.org/10.1016/j.aquaculture.2018.10.051
Received 27 February 2018; Received in revised form 22 October 2018; Accepted 22 October 2018
Available online 25 October 2018
0044-8486/ © 2018 Elsevier B.V. All rights reserved.
J. Souza et al.
biofloc seems to influence the action of nitrifying bacteria (Carvalho
et al., 2006; Delatolla et al., 2009). Recently, Lara et al. (2017) studied
the effect of different types of aerators on the microbial communities of
a BFT system and verified high concentrations of ammonia and low
levels of nitrite and nitrate in treatments with propeller aerators and
vertical pumps, which produced smaller biofloc sizes. Thus, these au-
thors suggested that the reduction in particle size interfered negatively
in the nitrification process of BFT systems.
In this context, the objective of the present study was to evaluate, in
three experiments, the effect of the biofloc size on the nitrification
process during the intensive production of the white shrimp Litopenaeus
vannamei in a BFT system, considering the hypothesis that systems with
smaller particle sizes would have a less-effective nitrification process.
The results of our experiments did not corroborate the size hypothesis
but rather demonstrated that the biofloc manipulation negatively af-
fected the nitrification process. This fact has great influence on the
management of BFT systems, since bioflocs are subject to different
water turbulence levels generated by aeration that can cause the rup-
ture of flocs and, therefore, affect the nitrification process.
2. Materials and methods
2.1. Local and installations
The experiments were carried out at the Aquaculture Marine Station
(AME) – Institute of Oceanography of the Federal University of Rio
Grande, in the city of Rio Grande – RS, Southern Brazil. Nauplius of the
white shrimp Litopenaeus vannamei were acquired from the commercial
laboratory Aquatec Ltda. (RN – Brazil) and were further kept in the
laboratory at EMA until reaching the postlarvae stage.
In experiment 1, 310 L tanks with a useful volume of 200 L were
used, whereas 60 L tanks with 40 L useful volume were employed in
experiments 2 and 3. In the three experiments, the tanks were filled
with seawater one day before the beginning of the experiment and were
constantly aerated.
In experiments 1 and 2, the temperature was maintained only with
the aid of an air-conditioner, whereas in experiment 3, the temperature
was kept stable using submerged electric heaters.
A 1% of inoculum of bioflocs was used in all experiments (Table 1).
The bioflocs were originally from tanks used for the production of L.
vannamei in the BFT system in the AME. In the experiments where the
ammonia concentration exceeded 1 mg L−1, organic fertilization was
carried out with the addition of sugarcane molasses, which aimed to
maintain the C:N ratio of approximately 15:1 (Avnimelech 1999).
Throughout the experiments, the shrimp were fed twice a day with
commercial shrimp feed specific for the species, which contained 38%
crude protein (Potimar Active 38 – Guabi), and the daily feeding rate
was determined according to the methodology described by Jory et al.
(2001).
2.2. Experimental design
2.2.1. Experiment 1
The study was carried out from February 6 to 24, 2016 (19 days)
and comprised four treatments, in triplicate: (1) 50 μm treatment
Table 1
Characterization of nitrogen compounds in the 1% bioflc inoculum from the
production of white shrimp Litopenaeus vannamei from the Aquaculture Marine
Station – AME in the three experiments.
Ammonia Nitrite Nitrate
Experiment 1 (GH3) 1,5 mg/L−1 2,6 mg/L−1 9,2 mg/L−1
Experiment 2 (GH3) 0 mg/L−1 5 mg/L−1 27 mg/L−1
Experiment 3 (larviculture) 0 mg/L−1 0 mg/L−1 48 mg/L−1
444
Aquaculture 500 (2019) 443–450
(constant filtration of particles up to 50 μm); (2) 150 μm treatment
(constant filtration of particles up to 150 μm); (3) 300 μm treatment
(constant filtration of particles up to 300 μm) and (4) Control – without
particulate filtration. The shrimp were stored in their respective ex-
perimental units, with a mean initial weight of 1.22 ± 0.46 g and a
stocking density of 300 shrimps per m3, which resulted in 60 shrimps
per tank.
2.2.2. Experiment 2
The study was carried out for 45 days (March 18 to May 31 of 2016)
with larger shrimps and comprised three treatments, in triplicate: (1)
300 μm treatment (constant filtration of particles up to 300 μm); (2)
600 μm treatment (constant filtration of particles up to 600 μm) and (3)
Control – without filtration of particles. The shrimp were stored in their
respective experimental units with a mean initial weight of
4.13 ± 0.04 g and a stocking density of 300 shrimps per m3.
2.2.3. Experiment 3
The study was conducted between September 16 and October 19,
2016 (34 days). The experiment comprised three treatments, in tripli-
cate: (1) 150 μm treatment (constant filtration of particles up to
150 μm); (2) Partial Filtration treatment – with filtration of particles in
50 μm mesh during only 5 days and (3) Control – without filtration of
particles. In the partial filtration treatment, filtration with 50 μm mesh
was carried out between days 6 and 10 of the experiment. After this
period, the meshes were removed, and the treatment continued without
filtration until the end of the experiment. The shrimp, with an initial
mean weight of 1.75 ± 0.02 g, were stored in their respective experi-
mental units at a stocking density of 300 shrimps per m3.
2.3. Physical and chemical parameters
The dissolved oxygen and temperature were measured twice a day
using a multiparameter YSI Pro 20 (Yellow Springs, USA). The pH was
measured once a day using a Mettler Toledo/FEP20 digital pH-meter
(Toledo PR, Brazil). Alkalinity (mg L−1 of CaCO3) was checked every
3 days following the methodology described in APHA (1989). Correc-
tions to pH and alkalinity were performed to maintain values above 7
and 100 mg CaCO₃ L−1, respectively, by adding hydrated lime – Ca
(OH) 2.
Water samples for the ammonia and nitrite analyses were collected
daily, and for the nitrate analysis, the water samples were collected
every seven days. The concentrations of ammonia were determined
according to UNESCO (1983). The nitrite and nitrate measurements
were performed according to the methodologies described in Strickland
and Parsons (1972).
The weight of total suspended solids was determined by gravimetry
with a filtration of 20 mL aliquots of water in GF 50-A glass fiber filters,
which were previously dried and weighed. The filters were oven dried
at 60 °C for 24 h and then were weighed in a precision analytical bal-
ance (Sartorius MC1, analytic AC 210S – São Paulo, Brazil) for de-
termination of the final weight. The total suspended solid (TSS) weights
were estimated by the difference between the initial and final weights
of each filter and were divided by the filtered volume (AOAC, 1999).
2.4. Biofloc size
In all experiments, the biofloc size was maintained with the use of
filtration systems composed of nets with different mesh sizes according
to each treatment. Every two days, water samples were collected and
photographed to measure the floc sizes. Samples were observed under a
Nikon E200 microscope with a Moticam 5.0 camera with final magni-
fication of 400 ×. Measurements of the major axis of the particle were
made through the Motic Images Plus 3.0 program.
J. Souza et al. Aquaculture 500 (2019) 443–450

2.5. L. vannamei zootechnical parameters

Initial weight measurements (n = 100) were performed before the

shrimps were stored. Weekly weighing was performed for 30 shrimps,
and the amount of feed offered was adjusted according to the average
weight. At the end of each experiment, all live shrimps were weighed to
evaluate the growth and survival in different treatments. In addition,
the following parameters were determined:
Survival: (number of shrimp stocked – number of survivors) × 100;
Weight gain (g) = Average final weight (g) – Initial average weight
(g);
Final biomass (g) = final weight of all live shrimps;
Productivity (kg/m3) = (Final biomass/volume).
The apparent food conversion rate was calculated according to the
formula:
TCA = RF/Bf-Bi.
Where: TCA = apparent feed conversion rate; RF = feed given;
BF = final biomass and BI = initial biomass;
2.6. Statistical analyzes
The homoscedasticity (Levene test) and normality (Shapiro-Wilk
test) of the data were determined. When the assumptions were not met,
the data went through mathematical transformations. Afterwards, the
one-way analysis of variance (ANOVA) was applied. In the case of
significant differences, the “post hoc” test of Tukey was further applied.
The analyses were conducted with a level of significance of 95%
(α = 0.05) (Zar, 2010).
3. Results
Concentrations of ammonium, nitrite and nitrate present in the 1%
inoculum are presented in Table 1, and the results of each experiment
are presented below.
3.1. Experiment 1
Table 2 presents the mean values of salinity, temperature, oxygen,
pH, alkalinity and total suspended solids (TSS). The salinity throughout
the experiment did not differ statistically (p > 0.05) among the
treatments, which was similar to the water temperature. The con-
centrations of dissolved oxygen remained almost constant during the
Fig. 1. Mean ( ± SD) size value (μm) of the bioflocs in treatments 50 μm,
150 μm, 300 μm and Control during 19days of experiment.
experiment and did not differ among treatments (p > 0.05). The pH
values also did not show significant differences (p > 0.05) among the
treatments.
For alkalinity, significant differences (p < 0.05) were detected on
days 7, 10 and 19, when the Control showed lower concentrations than
that of the 150 and 300 μm treatments.
The concentration of total suspended solids was higher in the
Control with a maximum value of 126.6 mg L−1, whereas the minimum
value (41.6 mg L−1) occurred in the 50 μm treatment. Statistical dif-
ferences (p < 0.05) were detected in the first week between the 50 μm
treatment and the others; in weeks 2 and 3, the 150 and 300 μm
treatments had similar values (p > 0.05), while the Control was sig-
nificantly different (p < 0.05) from the 50 μm treatment.
Fig. 1 shows the size of the bioflocs during the study. There were
significant differences between the treatments throughout the experi-
ment (p < 0.05). The maximum size reached was 401.55 μm in the
treatment without filtration (Control), whereas in the treatments in
which the particles were filtered, the size remained constant according
to the sizes of the respective meshes, reaching 45, 140 and 250 μm for
the meshes of 50, 150 and 300 μm, respectively.
Regarding the nitrogen compounds, it can be observed that the
ammonia (Fig. 2A) presented large oscillations during the experiment.
To maintain concentrations of ammonia below 1 mg L−1, molasses was
added after the third day in all treatments. However, the application of
molasses was more frequent in treatments where bioflocs were filtered
(50, 150 and 300 μm). It was observed that after day 10, the con-
centrations of ammonia decreased in the Control, thus differing

Table 2
Mean ± SD of the water quality parameters of the 3 experiments in the biofloc culture system Litopenaeus vannamei.
Experiments Treatments Salinity T (°C) D.O. (mg.L−1) pH Alkalinity (mg.L−1) TSS (mg.L−1)

Experiment 1 50 μm 31,55 ± 0,35 27,28 ± 0,38 5,77 ± 0,34 8,08 ± 0,05 133,57 ± 6,55a 51,11 ± 12,28 a
(29–34,33) (26,3–28,26) (5,28–6,57) (7,96–8,15) (126,66–140) (41,66–65)
150 μm 31,33 ± 0,38 27,38 ± 0,38 5,76 ± 0,36 8,08 ± 0,04 144,04 ± 9,85b 95,88 ± 13,19 b
(29–34,11) (27,73–28,03) (5,17–6,37) (8,00–8,15) (131,66–160) (86,66–111)
300 μm 31 ± 0,38 27,38 ± 0,34 5,74 ± 0,36 8,07 ± 0,03 145,71 ± 3,83b 96,11 ± 12,05 b
(29–33) (26,93–28,36) (5,32–6,33) (8,00–8,14) (140–150) (88,33–110)
Control 30,71 ± 0,33 27,32 ± 0,36 5,81 ± 0,35 8,08 ± 0,06 127,14 ± 14,32c 107,7 ± 20,09 b
(28–33,13) (26,66–28,30) (5,28–6,55) (7,97–8,16) (100–146,66) (86,66–126)
Experiment 2 300 μm 29,33 ± 088 27,01 ± 1,61 5,78 ± 0,36 7,98 ± 0,09 199,70 ± 23,74 b 229,54 ± 166,79
(28,01–29,66) (25,23–29,16) (5,11–6,80) (7,84–8,16) (164,44–225) (23,66–470)
600 μm 29,28 ± 0,86 27,02 ± 1,65 5,77 ± 0,32 8,02 ± 0,09 208,80 ± 34,09 b 292,45 ± 177,32
(28,01–29,66) (25,26–29,36) (5,26–6,59) (7,87–8,16) (162,5–250) (32,33–458,33)
Control 29,33 ± 088 27,81 ± 1,62 5,79 ± 0,33 7,72 ± 0,28 134,16 ± 26,83 a 586,04 ± 329,35
(28,01–30,33) (25,13–29,83) (5,12–6,65) (7,03–8,15) (97,5–166,66) (55–1006,66)
Experiment 3 150 μm 31,33 ± 1,07 26,95 ± 0,51 5,35 ± 0,20 8,02 ± 0,24b 208,80 ± 34,09b 250,77 ± 112,92
(30–32,66) (26,26–27,50) (5,08–5,80) (7,53–8,33) (162,5–250) (79–390)
Partial filtration 31,38 ± 1,16 26,94 ± 0,47 5,34 ± 0,24 8,01 ± 0,25b 194,16 ± 46,61b 296,77 ± 155,87
(30–32,66) (26,36–27,76) (4,90–5,71) (7,51–8,32) (126,66–250) (72,33–515)
Control 31,44 ± 1,25 27 ± 0,52 5,29 ± 0,21 7,72 ± 0,17a 157,5 ± 50,88a 440,11 ± 280,32
(30–33,33) (26,30–27,56) (4,66–5,71) (6,92–8,31) (95–250) (72,33–856,66)

*Superscript letters indicate statistical differences.

445
J. Souza et al. Aquaculture 500 (2019) 443–450

treatment had significantly lower values, whereas the Control had the
highest values, and the 150 and 300 μm treatments showed no differ-
ence between them (p > 0.05). Survival was > 95% in all treatments.
The apparent food conversion rate was significantly higher in the 50 μm
treatment, whereas that of the other treatments was statistically similar
(p > 0.05).

3.2. Experiment 2

Fig. 2. Mean values ( ± SD) of A) Ammonia, B) Nitrite and C) Nitrate con-
centrations (mg L-1) in the treatments 50 μm, 150 μm, 300 μm and Control for
19 days of experiment.
statistically (p < .05) from the other treatments.
The nitrite (Fig. 2B) showed a significant difference (p < 0.05)
among treatments after the 13th day. Lower values were measured in
the Control, while the remaining treatments continued to oscillate until
the end of the study.
The nitrate (Fig. 2C) was higher in the Control than that of the other
treatments, but there were no significant differences (p > 0.05) be-
tween the experimental days.
Table 3 presents the results of the zootechnical performance of the
L. vannamei shrimp during the 19 days of experiments with the different
treatments. The values of final weight, weight gain and final biomass of
the treatments differed from those of each other (p < 0.05). The 50 μm
The salinity mean value was around 29, with small oscillations
occurring during the study period; however, there were no significant
differences (p > 0.05) among treatments (Table 2). Similarly, the
water temperature did not present significant differences (p > 0.05),
ranging from 25 to 29 °C. The values of dissolved oxygen did not show
significant differences among treatments (p > 0.05), which was simi-
larly to the pH values (p > 0.05). Alkalinity showed significant dif-
ferences (p < 0.05) among treatments from the 13th day until the end
of the experiment, with the Control presenting lower concentrations
than the other treatments. The decrease in alkalinity was corrected with
the addition of hydrated lime when the concentration was below
100 mg L−1.
The concentration of total suspended solids was significantly higher
(p < 0.05) in the Control than that of other treatments and reached
1000 mg L−1 (Table 2). The high solids concentration in this treatment
had to be reduced by filtration. After this procedure, the concentration
decreased but increased again afterwards. In treatments that included
filtration (300 and 600 μm), the TSS were lower.
Fig. 3 shows the variation in the size of the flocs during the ex-
periment. In the treatment without filtration (Control), the particles
reached a maximum size of 584 μm; in the treatment with a 600 μm
mesh maximum, the particle size was 518 μm, whereas the particles
remained at 250 μm throughout the experiment in the 300 μm treat-
ment. Significant differences (p < 0.05) were detected only among the
Control and 300 μm treatment.
The ammonia (Fig. 4A) showed oscillations during the experiment.
Fertilizations with molasses were performed each time the ammonia
reached 1 mg L−1. All treatments began to receive molasses on the 3rd
day, and fertilizations were most frequently performed in the 300 and
600 μm treatments. These treatments presented oscillations during the
45 days of study. It can be observed that from the 35th day, ammonia
concentrations decreased in the Control and were not detected after the
41st day, whereas the treatments with ammonia filtration began to
decrease only after day 40. Statistical differences were detected
(p < 0.05) from days 20 to 23 and 26 to 28 between the Control and
the other treatments. The maximum concentration was 4.86 mg L−1 in
the 600 μm treatment.
The nitrite (Fig. 4B) showed high concentrations in the first week in
the Control, reaching 2 mg L−1, whereas in the 300 and 600 μm treat-
ments, the concentrations remained below 1 mg L−1. After the 21st day,

Table 3
Zootechnical parameters of shrimp Litopenaeus vannamei in the 3 experiments.
Experiments Treatments Initial weight (g) Final weight (g) Weight gain (g) Final biomass (g) Weekly weight Survival (%) Apparent Food Productivity
gain (g) Conv. (Kg/m3)

Experiment 1 50 μm 1,22 ± 0,46 2,37 ± 0,89a 1,15 ± 0,89a 141,2 ± 6,84a 0,38 ± 0,03 98,88 2,03 ± 0,18a 0,70 ± 0,03
150 μm 1,22 ± 0,46 2,59 ± 0,96b 1,37 ± 0,96b 152,4 ± 4,99b 0,45 ± 0,01 97,77 1,83 ± 0,11b 0,76 ± 0,02
300 μm 1,22 ± 0,46 2,62 ± 0,86b 1,4 ± 0,86b 153,7 ± 4,28b 0,46 ± 0,02 97,77 1,84 ± 0,12b 0,76 ± 0,01
Control 1,22 ± 0,46 2,83 ± 1,16c 1,61 ± 1,16c 164 ± 2,05c 0,53 ± 0,01 97,22 1,64 ± 0,04b 0,82 ± 0,01
Experiment 2 300 μm 4,13 ± 0,04 9,10 ± 0,17b 4,97 ± 0,17b 127,33 ± 6,71b 0,71 ± 0,02 93,33b 1,96 ± 0,89b 3,18 ± 3,65b
600 μm 4,13 ± 0,04 9,97 ± 0,46a 5,84 ± 0,46a 146,16 ± 6,41c 0,83 ± 0,05 97,77b 1,76 ± 0,72a 3,65 ± 0,09c
Control 4,13 ± 0,04 10,03 ± 0,20a 5,9 ± 0,20a 112,1 ± 7,61a 0,84 ± 0,04 79,99a 1,76 ± 1,17a 2,80 ± 0,05a
Experiment 3 150 μm 1,75 ± 0,02 5,30 ± 0,32b 3,55 ± 0,32b 75,97 ± 0,17b 0,71 ± 0,06b 96 2,44 ± 0,24b 1,89 ± 0,13b
Partial 1,75 ± 0,02 5,48 ± 0,45b 3,73 ± 0,45b 80,51 ± 9,21b 0,74 ± 0,09b 98 2,26 ± 0,38b 2,01 ± 0,23b
filtration
Control 1,75 ± 0,02 6,45 ± 0,04a 4,70 ± 0,04a 96,81 ± 3,46a 0,94 ± 0,01a 98 1,72 ± 0,02a 2,42 ± 0,01a

*Superscript letters indicate statistical differences.

446
J. Souza et al.
Fig. 3. Mean ( ± SD) size value (μm) of the bioflocs in treatments 300 μm,
600 μm and Control, during 45 days of experiment.
Fig. 4. Mean values ( ± SD) of A) Ammonia, B) Nitrite and C) Nitrate con-
centrations (mg L−1) in the treatments 300 μm, 600 μm and Control for 45 days
of experiment.
nitrite concentrations in the Control remained below 1 mg L−1, whereas
the other treatments had higher concentrations until the end of the
experiment. Statistical differences (p < 0.05) were detected from day
34 and afterwards between the Control and the other treatments.
Nitrate (Fig. 4C) showed increasing concentrations in all treatments,
reaching 50 mg L−1 (maximum concentration) in the Control; in the
other treatments, the rising nitrate concentration occurred only after
the third week, when it reached the maximum values of 23 and
13 mg L−1 for the 300 and 600 μm treatments, respectively, at the end
of the experiment. Statistical differences were detected (p < 0.05)
between the Control and 300 and 600 μm treatments, which were sig-
nificant (p > 0.05).
Table 3 shows the results of the zootechnical performance of L.
vannamei shrimps during the 45 days of the experiment. The values of
final weight, weight gain and apparent food conversion were statisti-
cally similar (p > 0.05) for the Control and 600 μm treatment, whereas
the treatment of 300 μm mesh size presented significantly lower values
447
Aquaculture 500 (2019) 443–450
(p < 0.05). The final biomass and productivity presented significant
differences (p < 0.05) among all treatments. The Control had the
lowest survival, being significantly different (p < 0.05) from the other
treatments.
3.3. Experiment 3
There were no significant differences (p > 0.05) in salinity and
temperature among treatments. Oxygen showed little variation
throughout the experiment. In addition, this parameter did not present
significant differences (p > 0.05) among treatments. For pH values,
there was a significant difference (p < 0.05) between the Control and
the other two treatments. When pH reached a value lower than 7, a
correction was made with the use of hydrated lime. Alkalinity was
lower in the Control than in the other treatments. There were sig-
nificant differences (p < 0.05) during the experiment, except on days
31 and 34.
Concentrations of total suspended solids increased throughout the
study. The Control presented the highest values, reaching
856.66 mg L−1, differing statistically (p < 0.05) from 150 μm and
partial filtration (P.F.) treatments, except during the first week. The
150 μm and partial filtration treatments did not present significant
differences (p > 0.05) between each other during the experiment
(Table 2).
Fig. 5 shows the size of the bioflocs throughout the experiment. The
maximum size was 380 μm in the Control, whereas in the 150 μm
treatment, the particle size remained, on average, 140 μm. In the P.F.
treatment, the particles started the experiment with 315 μm (similar to
the Control). On day 11, the particle size reached the minimum value of
49.29 μm. After this day, when the 50 μm meshes were removed, the
particles began to increase in size, reaching 199 μm at the end of the
experiment. The mean particle size in the Control and 150 μm treat-
ments were 345 and 134 μm, respectively. Statistical differences were
observed between the Control and 150 μm treatments throughout the
study, whereas P.F. treatment was similar (p > 0.05) to the Control
between days 1 and 7 and was similar to the 150 μm treatment between
days 18 and 28.
For ammonia concentrations (Fig. 6A), significant differences were
detected (p < 0.05) only for Control and 150 μm treatment between
days 8 and 14. In the Control, ammonia stabilized at low levels after the
8th day, whereas the 150 μm and P.F. treatments had greater oscilla-
tions.
The nitrite (Fig. 6B) presented higher values in the P.F. and 150 μm
treatments, both reaching 27 mgL−1 on day 14. The Control stabilized
at low values after day 13, whereas in the other two treatments, the
nitrite was lower after day 28. Significant differences (p < 0.05) were
detected between the Control and the other treatments after the 12th
day, but no differences occurred after day 28 (p > 0.05).
The highest nitrate values (Fig. 6C) were measured in the Control.
Statistical differences (p < 0.05) were found from the second week,
with the Control presenting higher values than that of the other
Fig. 5. Mean ( ± SD) size values (μm) of the bioflocs in treatments 150 μm,
Partial Filtration (P. F.) and Control for 34 days of experiment.
J. Souza et al.
Fig. 6. Mean values ( ± SD) of (A) Ammonia, (B) Nitrite and (C) Nitrate con-
centrations (mg L−1) in treatments 150 μm, Partial Filtration P(P.F.) and
Control for 34 days of experiment.
treatments. There were significant differences (p < 0.05) between the
150 μm and P.F. treatments, which occurred only in the last week of the
experiment.
The values of final weight, weight gain, final biomass, weekly
weight gain, and productivity were significantly higher (p < 0.05) in
the Control, whereas this treatment presented the smallest apparent
feed conversion. Survival was > 95% in all treatments without a sta-
tistical difference (p > 0.05) (Table 3).
4. Discussion
In intensive shrimp production, the water quality can be compro-
mised by the accumulation of nitrogen compounds. The input of am-
monia occurs due to the excretion of the animals and the decomposition
of the organic material, with mainly feed remaining. High concentra-
tions of ammonia and nitrite can make shrimp production unfeasible
(Lin and Chen, 2003; Li et al., 2007).
The toxicity of ammonia may vary depending on the shrimp life
stage and the physicochemical characteristics of the water, but in
general, it causes gill irritation, which affects the growth and survival of
shrimp. The toxicity of nitrite, on the other hand, is related to the
transport of oxygen because this compound binds to hemocyanin and
transforms it into metahemocyanin, which is unable to transfer oxygen
to the tissues, thus reducing the amount of oxygen available for the
metabolism and causing hypoxia and, later on, death of the animals
(Boyd, 2007).
Due to the potential toxicity of nitrogen compounds in aquaculture,
water renewal was always employed with the exchange of large vo-
lumes of water. This scenario started to change with the Biofloc
Technology (BFT), where the ammonia toxicity is temporarily bypassed
with the uptake of this nitrogen compound by heterotrophic bacteria,
448
Aquaculture 500 (2019) 443–450
after the addition of an organic carbon source (Avnimelech, 1999,
2009). In general, the rapid growth of heterotrophic bacteria leads to a
decrease in ammonia, which is incorporated in the formed bacterial
protein (Avnimelech, 1999). However, this is a palliative process, since
the grazing of the newly produced bacteria by the protozoan leads to an
increase in the ammonia concentration after the excretion of this ele-
ment by flagellates and ciliates, thereby making it necessary to perform
new fertilizations until the nitrifying microorganisms become active
(Silva et al., 2013). In the end, nitrifying bacteria are responsible for the
significant and constant removal of ammonia and nitrite from aqua-
culture systems (De Schryver et al., 2008).
All experiments conducted in this study presented good conditions
for the growth of the white shrimp Litopenaeus vannamei, with adequate
values of salinity (Decamp et al., 2003); temperature (Wyban et al.,
1995); dissolved oxygen (Van Wyk and Scarpa, 1999); pH (Wasielesky
Jr et al., 2006); alkalinity (Chen et al., 2006; Furtado et al., 2011) and
TSS (Gaona et al., 2011). Thus, the main differences observed for the
zootechnical parameters were probably related to the higher ammonia
and nitrite concentrations measured in treatments where bioflocs were
manipulated. In these treatments, the ammonia and nitrite surpassed
the safety levels (Lin and Chen, 2001, 2003; Van Rijn et al., 2006),
being necessary the addition of a carbon source.
Nitrifying microorganisms, including Bacteria and Archaean, con-
sume inorganic carbon and nitrogen compounds for their growth. These
bacteria are classified as ammonia-oxidizing (AOB) and nitrite-oxi-
dizing (NOB), whereas archaean present only as ammonia-oxidizing
(AOA). The AOB and AOA obtain their energy by using nonionized
ammonia and transform it into nitrite, whereas NOB obtains energy
from the nitrite oxidation, producing nitrate (Madigan et al., 2004).
Usually, the autotrophic nitrifying bacteria presents slow growth
and low biomass in comparison to heterotrophic ones, especially in
saltwater. This occurs because the requirements of these groups are
distinct. For example, nitrifying groups are sensitive to variations in
dissolved oxygen, alkalinity, pH and temperature, which interfere with
their development and activities. In addition, nitrification can be af-
fected by high concentrations of organic material, which contribute to
the growth of heterotrophic bacteria (Figueroa and Silverstein, 1992;
Wijeyekoon et al., 2004).
It is also well reported that the biofloc size can interfere with the
nitrification process. Small particles lead to a slower nitrification in
comparison to larger particles (Carvalho et al., 2006; Vlaeminck et al.,
2010), mainly due to uneven distribution of AOB and NOB in particles
of different sizes. More recently, Lara et al. (2017) evaluated the effects
of different types of aerators on the formation of bioflocs and observed
that certain aerators cause the breaking of flocs, thus decreasing the
particle size, and also slowing down or even stopping the nitrification
process, which leads to a great accumulation of ammonia. Thus, the
authors hypothesized that a reduction in biofloc size would, in some
way, hinder the nitrification process. However, the results of the three
experiments conducted in this work did not corroborate this hypothesis,
since we could not find any direct relationship between the particle size
and nitrification.
As can be observed in the first experiment, treatments with smaller
particles (50, 150 and 300 μm) showed similar concentrations of am-
monia and nitrite but were significantly different of the Control, in
which particles were not manipulated. Likewise, in the second experi-
ment, which was performed with larger shrimp, the treatments with
300 and 600 μm bioflocs showed similar behavior and concentrations of
ammonia and nitrite (Figs. 3 and 4). Even the suspension of filtration in
the P.F. treatment of the third experiment, with a partial recovery of the
original biofloc size, did not allow an increase in the nitrification pro-
cess in the same level as in the Control. Thus, it can be concluded that
there is no direct relationship between the size of the bioflocs and ni-
trification.
After rejecting the initial hypothesis, we created an alternative hy-
pothesis that the effect on the nitrifying community would not be
J. Souza et al.
related to the size of the bioflocs, but to the amount of suspended
material, since the filtration of particles used in the different experi-
ments reduced the total suspended solids and, consequently, decreased
the substrate availability for nitrifying bacteria and impaired the action
of these microorganisms within the culture system. In this way, the
filtration used in the present study would have function as a clarifica-
tion process, with the retention of larger particles and nitrifying bac-
teria. It is known that clarification may significantly interfere with the
nitrification process (Ebeling et al., 2006).
However, the results of this study also did not confirm this alter-
native hypothesis. For example, in the first experiment, we noticed that
the treatment with particles up to 50 μm had a significantly lower solids
concentration than that of the other treatments, but the 150 and 300 μm
treatments, which had a higher amount of suspended solids showed
ammonia and nitrite levels comparable to the Control. Similarly, when
analyzing data from the second experiment, we observed that the
Control presented higher values of suspended solids but was not sig-
nificantly different from the other manipulated treatments. However,
the ammonia levels remained lower in the Control most of the time,
whereas the nitrite was statistically higher in the 300 and 600 μm
treatments than in the Control. Thus, we cannot assure that the amount
of suspended solids is a limiting factor to the nitrification process. In
addition, if this were the case, systems with structures for biofilm
fixation, which provide a greater amount of substrate for nitrifying
microorganisms, should be more efficient. However, this was not was
confirmed by Ferreira et al. (2016), who showed that the presence of
biofilm did not lead to lower levels of ammonia or nitrite compared to
systems with only bioflocs, which therefore indicated that a greater
availability of substrate does not guarantee a more efficient nitrification
process in the systems.
In conclusion, we state that neither the size of flocs, nor the amount
of particles affected the nitrification process. However, all treatments in
which the particles were manipulated through filtration presented a
lower activity of the nitrifying microorganisms. It is, thus, very likely
that the nitrification process is related to the distribution of the ni-
trifying bacteria in the particles and possibly the way these micro-
organisms interact with the environment and other bacterial groups.
Vlaeminck et al. (2010) showed that the distribution of the micro-
colonies of nitrifying bacteria in flocs occurs according to the oxygen
gradient, being a determinant factor for the higher growth and activity
of these bacteria. These authors showed that the bacteria located in the
innermost area of the floc are under hypoxic conditions, whereas the
bacteria living in the outer layer of the particles experience aerobic
conditions. In this case, AOB are located in the outermost part of the
flocs, while NOB, which are more sensitive to higher oxygen con-
centrations, are located in the innermost portion. Likewise, Carvalho
et al. (2006) investigated the distribution of nitrifying bacteria in flocs
and in granules and stated that AOB bacteria are present in the aerobic
zone, whereas NOB prevails in the inner layers (anoxic zone) of the
flocs.
Thus, another hypothesis that could explain the low efficiency of the
nitrifying bacteria in treatments in which bioflocs were manipulated
would be the fact that sifting caused the breaking of the particle
structure, with the consequent rupture of chemical gradients and the
“architecture” of the flocs regarding the distribution of the different
groups of nitrifying microorganisms, and the possible communication
among them. It is likely that, in these conditions, nitrifying bacteria of
AOB, AOA and NOB groups stopped the uptake and oxidation of ni-
trogen compounds such as ammonia and nitrite.
The third experiment was performed to test the hypothesis that the
decrease in nitrification would be caused mainly by the manipulation of
the bioflocs and the rupture of its internal structure. When we con-
ducted a limited time filtration, we expected to see a recovery in the
nitrifying capacity of the P.F. treatment. This partially occurred since
filtration with a mesh of 50 μm for only 5 days caused a less detrimental
effect on the nitrification than that observed in the 150 μm treatment,
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Aquaculture 500 (2019) 443–450
in which the particles were continuously filtered during the experi-
mental period. After stopping the filtration, the ammonia values were
lower in the P.F. than in the 150 μm treatment, however no significant
differences were observed for nitrite between these two treatments, and
both presented higher values than the Control. On the other hand, ni-
trate presented higher values in the P.F. than the 150 μm treatment,
especially in the last week.
The higher concentrations of nitrite measured in the 150 μm and
P.F. treatments may have resulted from the fact that bacteria belonging
to the NOB group have greater limitations regarding the availability of
oxygen, since it is known that these bacteria are sensitive to higher
levels of dissolved oxygen (Figueroa and Silverstein, 1992). Thus, the
breakdown of bioflocs would expose NOB to higher oxygen con-
centrations, thus limiting nitrite uptake and nitrate production, and
these would only return to their normal activities after restructuring of
the bioflocs and establishing new oxygen, ammonia gradients and ni-
trite within them.
5. Conclusion
The results presented here lead us to conclude that the rupture of
the bioflocs structure is the main cause of serious problems that were
observed in the process of nitrification. In this study, the breakdown of
the bioflocs occurred due the filtration process. However, it is likely
that high levels of water turbulence may also cause similar particle
disruption and decrease nitrification in BFT systems. It is known that
turbulence may also influence the nitrification process due to the
transfer of dissolved oxygen and nutrients to the microorganisms
(Rusten et al., 2006), but our results indicate that the disruption of the
physical and chemical structure of the bioflocs could have caused larger
oscillations of ammonia and nitrite, probably due to the rupture of the
architecture and the breakdown of oxygen and nitrogen compounds.
Biofloc Technology (BFT) is a new paradigm in aquaculture pro-
duction. However, it is clear that further studies should be directed to
generate a better comprehension of the effects of water turbulence on
the floc size and microbial composition and, by consequence, their ef-
fect on nitrifying bacteria. Such knowledge will surely improve the
efficacy of this and other aquaculture systems, resulting in a faster and
more efficient nitrification process.
Acknowledgements
We appreciate the suggestions of two anonymous Reviewers to a
previous version of the text. This study had financial support of the
project “Biotechnology network for the study of Antioxidant elements
from marine organisms (Rede SAO-Mar) (CNPq. Proc. 408921/2013-7).
W. Wasielesky Jr. and P.C. Abreu are research fellows at Conselho
Nacional de Desenvolvimento Científico e Tecnológico – CNPq of the
Brazilian Ministry of Science and Technology. We also thank the service
Periodicos of CAPES
References
American Public Health Association (APHA), 1989. Standard Methods for the
Examination of Water and Waste Water, 16th edn. American Public Health
Association, AWWA, WPFC, New York.
Association of Official Analytical Chemists (AOAC), 1999. Off. Methods Anal. Arlington 2
(35), 1–30.
Avnimelech, Y., 1999. Carbon/nitrogen ratio as a control element in aquaculture systems.
Aquaculture 176, 227–235.
Avnimelech, Y., 2009. Biofloc Technology—A Practical Guide Book, 1st edn. The World
Aquaculture Society, Baton Rouge.
Boyd, C.E., 2007. Nitrification an important process in aquaculture. Global Aquac. Adv. 5,
64–66.
Carvalho, G., Meyer, R.L., Yuan, Z., Keller, J., 2006. Differential distribution of ammonia-
and nitrite-oxidising bacteria in flocs and granules from a nitrifying/denitrifying
sequencing batch reactor. Enzym. Microb. Technol. 39, 1392–1398.
Chen, S., Ling, J., Blancheton, J.P., 2006. Nitrification kinetics of biofilm as affected by
water quality factors. Aquac. Eng. 34, 179–197.
J. Souza et al.
De Schryver, P., Crab, R., Defroidt, T., Boon, N., 2008. The basics of bio-flocs technology:
the added value for aquaculture. Aquaculture 277, 125–137.
Decamp, O.E., Cody, J., Conquest, L., Delanoy, G., Tacon, A.G.J., 2003. Effect of salinity
on natural community and production of Litopenaeus vannamei (Boone) within ex-
perimental zero-water exchange culture systems. Aquac. Res. 34, 345–355.
Delatolla, R., Tufenkj, N., Comeau, Y., Lamarre, D., Gadbois, A., Berk, D., 2009. In situ
characterization of nitrifying biofilm: minimizing biomass loss and preserving per-
spective. Water Res. 43, 1775–1787.
Ebeling, J.M., Timmons, M.B., Bisogni, J.J., 2006. Engineering analysis of the stoichio-
metry of photoautotrophic, autotrophic and heterotrophic removal of ammonia –
nitrogen in aquaculture systems. Aquaculture 257, 346–358.
Ferreira, L.M., Lara, G., Wasielesky Jr., W., Abreu, P.C., 2016. Biofilm versus biofloc: are
artificial substrates for biofilm production necessary in the BFT system. Aquac. Int.
24, 921–930.
Figueroa, L., Silverstein, J., 1992. The effect of particulate organic matter on biofilm
nitrification. Water Environ. Res. 64, 728.
Furtado, P.S., Poersch, L.H., Wasielesky Jr., W., 2011. Effect of calcium hydroxide, car-
bonate and sodium bicarbonate on water quality and zootechnical performance of
shrimp Litopenaeus vannamei reared in bioflocs technology (BFT) systems.
Aquaculture 321, 130–135.
Gaona, C.A.P., Poersch, L.H., Krummenauer, D., Foes, G.K., Wasielesky Jr., W., 2011. The
effect of solids removal on water quality, growth and survival of Litopenaeus vannamei
in a biofloc technology culture system. J. Rec. Aquac. 11, 54–73.
Hargreaves, J.A., 2006. Photosynthetic suspended-growth systems in aquaculture. Aquac.
Eng. 34, 344–363.
Jory, D.E., Cabrera, T.R., Dugger, D.M., Fegan, D., Lee, P.G., Lawrence, A.L., Jackson,
C.J., McIntosh, R.P., Castañeda, J., 2001. A global review of shrimp feed manage-
ment: status and perspectives. In: Browdy, C.L., Jory, D.E. (Eds.), The New Wave:
Proceedings of the Special Session on Sustainable Shrimp Culture. The World
Aquaculture Society, Baton Rouge, pp. 104–152.
Koops, H., Pommerening, R.A., 2001. Destribuition and icophysiology of the nitrifying
bacteria emphasizing cultured species. FEMS Microbiol. Ecol. 37, 1–9.
Lara, G., Krummenauer, D., Abreu, P.C., Poersch, L.H., Wasielesky Jr., W., 2017. The use
of different aerators on Litopenaeus vannamei biofloc culture system: effects on water
quality, shrimp growth and biofloc composition. Aquaculture 25, 147–162.
Li, E.L., Chen, Z., Zeng, X., Chen, N.Y.U., Lai, Q., Qin, J.G., 2007. Growth, body com-
position, respiration and ambient ammonia nitrogen tolerance of the juvenile white
450
Aquaculture 500 (2019) 443–450
shrimp, Litopenaeus vannamei, at different salinities. Aquaculture 265, 385–390.
Lin, Y.C., Chen, J.C., 2001. Acute toxicity of ammonia on Litopenaeus vannamei Boone
juveniles at different salinity levels. J. Exp. Mar. Biol. Ecol. 259, 109–119.
Lin, Y.C., Chen, J.C., 2003. Acute toxicity of nitrite on Litopenaeus vannamei (Boone) ju-
veniles at different salinity levels. Aquaculture 224, 193–201.
Madigan, M.T., Martinko, J.M., Parker, J., 2004. Microbial Evolution and Systematics.
Microbiology of Brock. Prentice Hall, São Paulo, pp. 302–330.
Rusten, B., Eikebrokk, B., Ulgenes, Y., Lygren, E., 2006. Design and operations of the
Kaldnes moving bed biofilm reactors. Aquac. Eng. 34, 322–331.
Silva, K.R., Wasielesky Jr., W., Abreu, P.C., 2013. Nitrogen and phosphorus dynamics in
the biofloc production of the pacific white shrimp, Litopenaeus vannamei. J. World
Aquacult. Soc. 44, 30–41.
Strickland, J.D.H., Parsons, T.R., 1972. A Practical Handbook of Seawater Analysis, 2nd
edn. Fisheries Research Board of Canada, Ottawa.
UNESCO (United Nations Educational, Scientific and Cultural Organization), 1983.
Chemical Methods for Use in Marine Environmental Monitoring. Intergovernmental
Oceanographic Commission Manual and Guides, Paris.
Van Rijn, J., Tal, Y., Schreier, H.J., 2006. Denitrification in recirculating systems: theory
and applications. Aquac. Eng. 34, 364–376.
Van Wyk, P., Scarpa, J., 1999. Water quality requirements and management. In: Van
Wyk, P., Davis-Hodgkins, M., Laramore, R., Main, K., Mountain, J., Scarpa, J. (Eds.),
Farming Marine Shrimp in Recirculating Freshwater Systems. Florida Department of
Agriculture and Consumer Services, Tallahassee 128p.
Vlaeminck, S.E., Terada, A., Smets, B.F., De Clippeleir, H., Schaubroeck, T., Bolca, S.,
Demeestere, L., Mast, J., Boon, N., Carballa, M., Verstraete, W., 2010. Aggregate size
and architecture determine microbial activity balance for one-stage partial nitritation
and anammox. Appl. Environ. Microbiol. 76, 900–909.
Wasielesky Jr., W., Atwood, H.I., Stokes, A., Browdy, C.L., 2006. Effect of natural pro-
duction in brown water super-intensive culture system for white shrimp Litopenaeus
vannamei. Aquaculture 258, 396–403.
Wijeyekoon, S., Mino, T., Satoh, H., Matsuo, T., 2004. Effects of substrate loading rate on
biofilm structure. Water Res. 38, 2479–2488.
Wyban, J., Walsh, W.A., Godin, D.M., 1995. Temperature effects on growth, feeding rate
and feed conversion of the Pacific white shrimp (Penaeus vannamei). Aquaculture
138, 267–279.
Zar, J.H., 2010. Biostatistical Analysis. Prentice Hall, Upper Saddle River.