Aquaculture 514 (2020) 734516

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

Effect of aeration intensity on the biofilm nitrification process during the T

production of the white shrimp Litopenaeus vannamei (Boone, 1931) in
Biofloc and clear water systems
Ana Paula Mariane de Moraisa, Paulo Cesar Abreub, Wilson Wasielesky Jrc,
⁎
Dariano Krummenauera,
a
Laboratory of Ecology of Microorganisms Applied to Aquaculture, Institute of Oceanography, Federal University of Rio Grande, FURG, C. P. 474, Rio Grande, RS CEP
96201-900, Brazil
b
Laboratory of Phytoplankton and Marine Microorganisms, Institute of Oceanography, Federal University of Rio Grande, FURG, C. P. 474, Rio Grande, RS CEP 96201-
900, Brazil
c
Laboratory of Mariculture, Institute of Oceanography, Federal University of Rio Grande, FURG, C. P. 474, Rio Grande, RS CEP 96201-900, Brazil

A R T I C LE I N FO A B S T R A C T

Keywords: Artificial substrates have great importance for the establishment of the biofilm, and their use in the culture
Nitrifying bacteria systems represents a complementary source of food, increase space for animals and aid in the metabolism of
Nitrogen compounds nitrogen compounds. Nitrifying bacteria present in biofilm play an important role in the maintenance of water
Ammonia quality, and several factors such as pH, temperature, salinity and dissolved oxygen can interfere in the estab-
Nitrite
lishment and efficiency of these bacterial communities. However, there is not much information in the literature
on the influence of aeration intensity on the bacterial community present in the biofilm. Thus, the objective of
this study was to determine the response of nitrifying bacteria present in the biofilm submitted to different
aeration intensities during the production of Litopenaeus vannamei (Boone, 1931) in a clear water system and also
with bioflocs. The study was composed of two experiments, where the first experiment was carried out without
shrimp and consisted of four treatments with three replicates, in 800 Liter tanks distributed in: 1) W/Air (control
- without aeration); 2) V7.5 (flow rate 7.5 L/min); 3) V33.75 (flow rate of 33.75 L/min) and V75 (flow rate of
75 L/min). All treatments use “Needlona®” as artificial substrate (Needlona® - 100% polyester fiber; 250 g/m2
weight; 1.4 mm thickness; 0.18 g/cm3 density), in the proportion of 200% of the lateral area of the tank.
Experiment two was established after the results of the previous experiment, with three treatments and three
replicates each: 1) BFT (biofloc, with flow rate of 20.00 L/min); 2) BFT + BF (biofloc and biofilm with flow rate
of 33.75 L/min) and 3) BF (biofilm with flow rate of 33.75 L/min), in which the shrimp (7.89 ± 0.24 g) were
stocked in 9 tanks (800 L) with a density of 500 shrimps m−3. In both experiments Ammonia and nitrite were
measured daily, while nitrate was analyzed weekly. The first experiment showed no difference in the ammonia
concentrations of the different treatments, whereas nitrite showed higher concentrations in the treatment
without aeration. The 33.75 L/min flow rate was chosen for experiment 2 to be compared with the aeration
normally employed in our systems (20.00 Liter/min). The nitrification process was more efficient in the treat-
ments with biofilm and bigger air flow rate, presenting smaller concentrations of ammonia and nitrite in
comparison to the BFT treatment. Similarly, treatments with biofilm and stronger flow rate showed better
zootechnical performance of the shrimp.

1. Introduction may be a limiting factor for primary production in these ecosystems,

but can also be toxic for aquatic organisms when present in higher
Nitrogen is an important nutrient for living organisms, as it is an concentrations (Vieira, 2017).
essential component for the constitution of proteins and nucleic acids. It In the nitrification process, the successive oxidation of ammonia to

Abbreviations: AOB, Ammonia-Oxidizing Bacteria; NOB, Nitrite-Oxidizing Bacteria; TAN, Total Ammonia Nitrogen; BFT, Biofloc Technology; EMA/FURG, Marine
Aquaculture Station; N-NO2−, Nitrite; CaCO3, Alkalinity; N-NO3−, Nitrate; TSS, Total Suspended Solids
⁎
Corresponding author.
E-mail address: dariano.krummenauer@furg.br (D. Krummenauer).

https://doi.org/10.1016/j.aquaculture.2019.734516
Received 8 July 2019; Received in revised form 11 September 2019; Accepted 13 September 2019
Available online 14 September 2019
0044-8486/ © 2019 Elsevier B.V. All rights reserved.
A.P.M. de Morais, et al. Aquaculture 514 (2020) 734516

nitrite and subsequently to nitrate is mainly made by auto-

chemolitotrophic microorganisms (Ebeling et al., 2006) belonging to
two groups of bacteria. The first, the ammonia-oxidizing bacteria (AOB)
are responsible for the oxidation of ammonia to nitrite. Most of these
organisms belong to the genera Nitrosomonas, Nitrosococcus, Ni-
trosospira, Nitrosolobus and Nitrosovibrio. The second group, the nitrite-
oxidizing bacteria (NOB), performs the conversion of nitrite to nitrate,
and the majority of these microorganisms belong to the genera Ni-
trobacter, Nitrococcus, Nitrospira and Nitrospina (Ebeling et al., 2006;
Madigan et al., 2016; Lara et al., 2016). Recent studies have demon-
strated that microorganisms of the Archaea domain also participate in
the nitrification process (Ward, 2013).
In the production of aquatic organisms, like fish and shrimp, higher
concentrations of nitrogen compounds as ammonia and nitrite can be-
come a problem, when they accumulate in the aquatic environment due
to the excreta of the produced organisms, decomposition of un-
consumed foods and organic waste (Timmons and Ebeling, 2010).
Therefore, the control of nitrogen elements within the breeding en-
vironment is important, since they can cause damage to the produced
shrimp and fish (Lin and Chen, 2001, 2003).
The concentration of ammonium (TAN) in the medium increases
with increasing pH and water temperature, and reduces with increasing
salinity (Boyd and Tucker, 2012) Exposure to inadequate concentra-
tions of these compounds can cause stress, triggering various physio-
logical changes, and compromising performance leading to death,
thereby impairing production (Girotto, 2010). Nitrite, on the other
hand, binds to hemocyanin, transforming it into metahemocyanin,
preventing the transport of oxygen to tissues and reducing the amount
of oxygen available for metabolism (Tahon et al., 1988). This process
can lead to hypoxia and, consequently, mortality of organisms pro-
duced (Chen et al., 1986).
An efficient aeration system is important for the oxygen supply to
produce animals, and to keep the flocs in suspension in the BFT system.
However, low concentrations of dissolved oxygen limit or suppress ni-
trification (Avnimelech, 2009; Zhu et al., 2008), since the nitrifying
bacteria, AOB and NOB present a demand for oxygen for cellular ac-
tivity, growth and reproduction. Thus, in order to carry out the ni-
trification process it is essential that these microorganisms settle in the
growing environment like bioflocs and biofilm, but also have ideal
environmental conditions to absorb and transform nitrogen com-
pounds.
The biofilm can be defined as an organic matrix adhered to any
submerged substrate, which is colonized by a microbial community
composed of bacteria, protozoa, fungi and algae (Ramesh et al., 1999).
It has been shown that biofilm is responsible for removing nitrogenous
compounds from water, especially ammonia and nitrite. Thompson
et al. (2002), evaluated the biofilm efficiency in the maintenance of
water quality through the absorption of dissolved inorganic nutrients
(ammonia and phosphate). Moreover, Ballester et al. (2003) evaluating
the influence of biofilm on Farfantepenaeus paulensis production, con-
cluded that the biofilm positively influenced shrimp growth, especially Fig. 1. Dynamic of mean ± standard deviation of a) ammonia, b) nitrite and c)
by providing an alternative food source. nitrate over time in a clear water system.
In order to increase efficiency in shrimp production, the use of ar-
tificial substrates for biofilm fixation in the Biofloc Technologic system In BFT systems, aeration must be maintained at lower rates in order to
has already been carried out by Ferreira et al. (2016). However, these keep the flocs in suspension without causing their rupture and to
authors concluded that biofilm served only as a source of com- guarantee the nitrification by the bacteria present in the biofloc (Lara
plementary food, but did not observe any difference in the metaboli- et al., 2017b; Souza et al., 2019). However, this low aeration rate may
zation of nitrogen compounds in comparison to bioflocs alone. Thus, in be a problem to nitrifying bacteria present in the biofilm. Thus, the
BFT systems the use of substrates for biofilm development would not be objective of this study is to determine the response of nitrifying bacteria
necessary to keep water quality in good standards, since the bacteria present in the artificial substrate biofilm submitted to different aeration
present in the bioflocs would be enough to keep ammonia and nitrite at intensities in the production of Litopenaeus vannamei (Boone, 1931) in
low levels and also represent an extra food source. systems with clear water and also with bioflocs.
However, it is likely that biofilm in aquaculture systems was not
analyzed in all its dimensions. For instance, there is little information in
the literature on the lower efficiency of the nitrifying bacteria in the
biofilm in BFT systems and its relationship with the oxygen limitation.

2
A.P.M. de Morais, et al. Aquaculture 514 (2020) 734516

Table 1
Mean, standard deviation (overall mean) minimum and maximum of the physical and chemical parameters over the 10-day study of different aeration intensity with
different flow rates (tree replicates).
Treatment W/AIR V7.5 V33.75 V75

Total ammonia nitrogen (mg L−1) 3.07 ± 0.49 (0.30–7.53) 2.60 ± 0.57 (0.06–7.53) 2.52 ± 1.03 (0.06–7.43) 2.65 ± 0.37 (0.06–7.67)
Nitrite (mg L−1) 0.17 ± 0.08a (0.00–0.30) 0.05 ± 0.03b (0.00–0.14) 0.08 ± 0.08b (0.00–0.49) 0.05 ± 0.03b (0.00–0.13)
Nitrate (mg L−1) 2.21 ± 0.04 (0.00–4.83) 2.85 ± 0.61 (0.00–8.67) 3.32 ± 0.96 (0.00–6.67) 3.55 ± 0.97 (0.00–6.26)
Alkalinity (mg L−1) 153 ± 27 (107–250) 149 ± 25 (103–225) 145 ± 25 (100–237) 155 ± 24 (105–232)
Dissolved oxygen (mg L−1) 6.23 ± 0.03a (6.20–6.25) 6.33 ± 0.03b (6.30–6.35) 6.34 ± 0.04b (6.35–6.38) 6.40 ± 0.02b (6.40–6.48)

Different letters on the same line represent statistical difference p < 0.05.
W/AIR: without aeration contribution; 2) V7.5: flow rate 7.5 L min-1; 3) V33.75: flow rate 33.75 L/min and 4) V75: flow rate 75 L/min in a clear water system.

Table 2 2.3. Experiment 2: biofilm and biofloc

Mean and standard deviation of molasses, hydrated lime, settling hours, water
exchange and amount of water to produce 1 kg of shrimp over the 47-day study. The experiment, performed during 47 days used the best aeration
Treatment BFT BFT+BF BF rate (33.75 L/min) determined in experiment 01. The experimental
consisted of three treatments with three triplicates, being: 1) BFT -
Total of Molasses 2507.34 ± 22.06 380.01 ± 8.50 – Biofloc with 20.00 L/min flow rate; 2) BFT+BF - Bioflocs and biofilm
(g)
with flow rate 33.75 L/min and 3) BF - clear water and biofilm with
Hydrated lime (g) 1280.07 ± 22.58 1521.59 ± 24.73 1282.46 ± 24.34
Settling hours (h) 10.00 ± 0.58 40.00 ± 1.51 – flow rate 33.75 L/min. The shrimp (7.81 ± 0.24 g) were stocked at a
Water Exchange (L) 4480.0 l ± 66.03 – – density of 500 m−3 and fed with Guabi® 1.6 mm commercial feed with
Water Use (L kg−1 719.69 ± 46.72 174.64 ± 6.93 167.65 ± 10.29 40% crude protein. The food was supplied twice a day (08,00 and
shrimp)
16,00) after samples according to the methodology of Garça de Yta
et al. (2004).
To begin the biofloc formation, organic fertilizations were carried
2. Materials and methods
out with the addition of sugar cane molasses (37% of carbon) when the
concentrations of TAN reached 1.0 mg L−1 to maintain the relation C:N
2.1. Location and facilities
15:1.
During the experiment, the dissolved oxygen, temperature and pH
The study was carried out at the Marine Aquaculture Station (EMA/
were monitored twice a day using a multiparameter (YSI PRO 20). The
FURG) - Institute of Oceanography of the Federal University of Rio
alkalinity was analyzed three times a week. When pH and alkalinity
Grande - FURG, located in the city of Rio Grande, RS, Brazil (32° 19 ′S,
values were below 7.3 and 150 mg L−1, respectively, there was the
52° 15 ′W).
addition of hydrated lime as described by Furtado et al. (2011). Salinity
In both experiments, 800 L tanks of useful volume were filled with
and total suspended solids (SST) were measured on a weekly basis,
seawater, chlorinated with 10 ppm of sodium hypochlorite, later de-
according to AOAC (2000). When SST concentrations exceeded
chlorinated with 1 ppm of ascorbic acid. Water temperature was
500 mg L−1, clarifiers (biofloc settling tank) were used in order to re-
maintained with submerged electric heaters (Hydor Theo 200W). The
move surplus solids, as recommended by Gaona et al. (2011).
aeration system was composed of a 4 HP blower and Aerotubes® mi-
Nitrogen compounds such as total ammonium nitrogen (TAN) and
croperforated hoses, to maintain constant aeration. The aeration rate of
nitrite (N-NO2) were analyzed daily, and nitrate (N-NO3) once a week
20.00 L/min is normally used in the aquaculture systems of EMA/
according to Aminot and Chaussepied (1983). Every time that the
FURG, but other rates were tested. In order to measure the airflow,
concentration of nitrite reached 26 mg L−1, the safety level for the
individual rotormeters (TRP-255-H-7 1 POL NPT-Tecnofluid®) were
salinity employed, 30% of water renewal was done (Lin and Chen,
coupled to the aeration inlet of each experimental unit and regulated in
2003).
the flow according to the treatment.
The artificial substrates used for colonization of the biofilm were
non-floating Needlona®, in a proportion of 200% of the lateral area of 2.4. Sampling of microorganisms
the tank. Needlona® used comprised of 100% polyester fiber; 250 g/m2
weight; 1.4 mm thickness, 0.18 g/cm3 density. Before the beginning of To characterize the microbial community of the second experiment,
the experiments, the substrates were kept for 30 days in a biofloc weekly samples of 18 mL of water were collected from each experi-
system. mental unit and fixed in formalin at the final concentration of 4% for
subsequent identification of the microorganisms in the Laboratory of
Phytoplankton and Marine Microorganisms of the FURG, among the
2.2. Experiment 1: biofilm in clear water treatments with and without substrate, so the CW+BF treatment was
not analyzed. According to the methodology of Utermöhl (1958). Bac-
The experiment consisted of four treatments with three replicates terial abundance analysis was performed on days 0, 17, 26 and 47 for
each, denominated: 1) W/AIR: without aeration; 2) V7.5: flow rate BFT and BFT+BF treatments.
7.5 L/min; 3) V33.75: flow rate 33.75 L/min and 4) V75: flow rate 75 L/
min in a clear water system, without shrimp. To determine the effi- 2.5. Zootechnical performance
ciency of the nitrifying bacteria, 7 mg L−1 ammonium chloride was
added representing the safety limit for the L. vannamei species at sali- In the second experiment, the zootechnical performance of the an-
nity 35 (Lin and Chen, 2001). The study lasted for 10 days. imals was determined after weekly samples of 30 animals, using a di-
Samples were collected every four hours for analysis of ammonia gital scale with an accuracy of 0.01 g.
(TAN), nitrite (N-NO2), alkalinity (CaCO3) following methodologies
UNESCO (1983), Aminot and Chaussepied (1983) e APHA (2012) re- 2.6. Statistical analysis
spectively and dissolved oxygen with Multiparameter.
Data were expressed as mean ± standard deviation. Undergo tests

3
A.P.M. de Morais, et al.
Fig. 2. Dynamic of mean ± standard deviation of a) ammonia, b) nitrite and c)
nitrate over the 47-day study of 500 m−3 Litopenaeus vannamei (7.81 ± 0.24 g)
in BFT with different aeration intensity (Biofloc with flow rate of 20.00 L/min,
BFT + BF: Bioflocos and biofilm with flow rate of 33.75 L/min and BF: biofilm
with flow rate of 33.75 L/min) with tree replicates.
of normality (Shapiro-Wilk) and homoscedasticity (Levene), with the
proof of these premises. Analysis of multiple variables was conducted
with the One-way Analysis of Variance (ANOVA) and post-hoc Tukey
test. Data that did not satisfy the assumptions for ANOVA were sub-
mitted to the non-parametric test of Kruskall-Wallis followed by a
multiple comparison test (ZAR, 2010). The level of significance was 5%
in all cases (p < 0.05).
4
Aquaculture 514 (2020) 734516
3. Results
3.1. Experiment 1 - biofilm in clear water
There was no significant difference for the ammonia and nitrate
parameters among treatments (Fig. 1a, c). The nitrite of treatments
V7.5, V33.75 and V75 showed similar values throughout the study, but
were lower than concentration measured in W/AIR, which reached
0.17 mg L−1 (Table 1, Fig. 1b).
For the alkalinity values the V33.75 treatment had the lowest mean
value, significantly different from the other treatments. The values of
dissolved oxygen were higher in the three aeration intensities tested
than in the without aeration treatment.
3.2. Experiment 2 - biofilm in clear water and biofloc
The products (molasses, hydrated lime) used to control the amount
of floc and the settling time, water exchange during the study and
amount of water to produce 1 kg of shrimp is represented in the Table 2.
The temperature, salinity, pH, nitrate (Fig. 2c) and SST did not show
significant difference (p > 0.05) between treatments during the ex-
perimental period. There was a significant difference (p < 0.05) for
ammonia, nitrite, dissolved oxygen and alkalinity. The BFT treatment
presented higher values of ammonia, dissolved oxygen and alkalinity
than BF (Fig. 2a), whereas the N-NO2 of the BFT+BF and CW+BF
treatments were significantly lower than the BFT treatment (Fig. 2b).
The dissolved oxygen levels in the BFT+BF and CW+BF treatments
were statistically higher than those of the BFT treatment. This pattern
was demonstrated throughout the experiment. The alkalinity presented
lower values in the treatments BFT+BF and CW+BF, when compared
to the BFT treatment (Table 3).
Bacterial abundance analysis was performed on days 0, 17, 26 and
47 for BFT and BFT+BF treatments due to the high concentrations of
ammonia and nitrite. The total of free bacteria did not present sig-
nificant differences (p > 0.05) among treatments. However, there
were statistical differences for the groups of analyzed bacteria.
The BFT treatment differed from the BFT+BF treatment for bacilli
and filamentous, bacteria, with higher abundances of bacteria (Fig. 3).
It is possible to observe the gradual increase of microorganisms in the
system as time passes, only on day 26 the number of organisms de-
creases and increases again as shown on day 47. The BFT+BF treat-
ment has a stability in the organisms from start to finish of the ex-
periment having an increase for filamentous and amoebae over time.
The results of the zootechnical performance of the shrimp at the end
of the experiment are presented in Table 3. There was no significant
difference between the treatments (p < 0.05) in relation to the final
weight. Survival and final biomass were significantly higher in the
biofilm treatments (BFT+BF and BF) than in the biofloc treatment
(BFT) alone (Table 4).
4. Discussion
The first experiment demonstrated the importance of the aeration in
the production system for a greater efficiency of the nitrification pro-
cess by biofilm, especially for NOB bacteria. This was evidenced by the
higher concentrations of nitrite in the treatment without aeration when
compared to the others. The lack of water movement and the con-
sequent limitation of the oxygen transfer can generate a gradient in the
concentration of this gas along the biofilm, with the presence of hypoxic
or anoxic areas in the innermost regions of the biofilm (Vlaeminck
et al., 2010). In this case, nitrite oxidizing bacteria will be less active
and this nitrogen compound will accumulate in the water.
In general, AOB are found in the outermost part of the biofilm,
whereas NOB are present in the deepest region (Gieseke et al., 2003).
Therefore, NOB is likely to be more subject to the decrease in dissolved
oxygen concentration than AOB. This is evident in this experiment,
A.P.M. de Morais, et al. Aquaculture 514 (2020) 734516

Table 3
Mean, standard deviation overall mean and minimum and maximum of the physical and chemical parameters of the water over the 47-day study.
Treatment BFT BFT+BF BF

−1 a b
Total ammonia nitrogen (mg L ) 1.73 ± 0.41 (0.00–13.20) 0.51 ± 0.11 (0.00–5.20) 0.70 ± 0.54b (0.00–6.30)
Nitrite (mg L−1) 15.36 ± 5.03a (0.00–57.00) 1.13 ± 0.56b (0.00–28.00) 1.11 ± 0.44b (0.00–3.50)
Nitrate (mg L−1) 43.91 ± 9.02 (1.70–124.00) 73.85 ± 8.25 (4.79–204.00) 52.61 ± 17.78 (4.79–124.00)
Dissolved oxygen (mg L−1) 5.02 ± 0.22a (3.75–6.15) 5.18 ± 0.16b (4.30–6.30) 5.22 ± 0.14b (3.95–6.20)
pH 7.55 ± 0.11 (7.07–8.05) 7.55 ± 0.09 (6.96–8.06) 7.58 ± 0.07 (7.17–8.12)
Temperature (°C) 29.13 ± 0.76 (24.55–32.40) 29.44 ± 0.83 (24.70–34.40) 29.02 ± 0.31 (25.00–32.05)
Salinity 30.80 ± 0.90 (26.00–33.3) 31.41 ± 1.89 (27.10–35.00) 30.14 ± 1.30 (26.00–35.00)
Total suspended solids (mg L−1) 298.19 ± 88.73 (0.00–665) 346.30 ± 57.28 (0.00–665) 332.56 ± 73.18 (0.00–575)
Alkalinity (mg L−1) 154.75 ± 15.10a (95.00–250) 135.92 ± 22.11b (55.00–185) 136.17 ± 22.14b (55–270)

Different letters on the same line represent statistical difference p < 0.05.
500 m−3 Litopenaeus vannamei (7.81 ± 0.24 g) in BFT with different aeration intensity (Biofloc with flow rate of 20.00 L/min, BFT + BF: Bioflocos and biofilm with
flow rate of 33.75 L/min and BF: biofilm with flow rate of 33.75 L/min) with tree replicates.

Table 4
Mean and standard deviation of the zootechnical performance of the L. van-
namei over the 47-day study.
Treatment BFT BFT+BF CW+BF

Initial weight 7.81 ± 0.24 7.81 ± 0.24 7.81 ± 0.24

(g)
Final weight (g) 13.50 ± 0.40 13.14 ± 0.19 13.63 ± 0.63
Survival (%) 62 ± 41.49a 87 ± 9.54b 88 ± 6.93b
Biomass final 3998.40 ± 490a 5732.02 ± 180.13b 5979.47 ± 289.47b
(m3)

Different letters on the same line represent statistical difference p < 0.05.
BFT: Biofloc without flow control, BFT + BF: Biofloc and biofilm with flow rate
33.75 L/min and CW + BF: Clear water and biofilm with flow rate 33.75 L/min.

employed in our production systems, but not as strong as 75.00 L/min,

Fig. 3. Total bacterium in treatments Bioflocos and Bioflocos+Biofilm.
since nitrite variation was more influenced by the oxygen concentration
than ammonia. Ammonia levels did not present significant differences
among treatments, indicating that AOB were not affected by the oxygen
concentration. On the other hand, the nitrification process did not seem
to be affected by the intensity of the aeration, since no differences were
observed in the three aeration intensities tested. However, for the
second experiment we have chosen the aeration rate of 33.75 L/min,
which is bigger than the aeration rates of 20.00 L/min, normally
which is leads to a high energy consumption.
The water quality parameters of the second experiment, such as
temperature, pH, salinity, nitrate and total suspended solids, remained
throughout the experiment under ideal conditions for the production of
L. vannamei (Furtado et al., 2014; Gaona et al., 2011; Van Wyk and
Scarpa, 1999), except for dissolved oxygen that presented lower values
in the BFT treatment. However, mean concentrations of dissolved
oxygen were > 5 mg L−1 required for bacteria in the nitrification pro-
cess, as well as for shrimp requirements (Timmons and Ebeling, 2010;
Van Wyk and Scarpa, 1999).
In the second experiment it is possible to observe the benefits of the
incorporation of the artificial substrate colonized with biofilm, since the
biofilm treatments presented lower ammonia and nitrite values, in-
dicating a faster and more efficient removal of nitrogen compounds
when compared to bioflocs alone. Holl et al. (2011), also consider that
the nitrifying community fixed in the substrate is capable of completely
performing the nitrification of the system, even if there is no activity of
the bacteria present in the water. On the other hand, in a similar study
Ferreira et al. (2016) observed that the biofilm had no positive effect on
the removal of the nitrogen compounds in comparison to bioflocs.
It is possible to observe a greater abundance of bacteria in the BFT
treatment compared to the BFT+BF treatment, where the BFT+BF
treatment remains more stable throughout the period, different from
the BFT that presents an increase of bacteria over time. This difference
of bacteria between the treatments is related to the formation of the
microbial community in the BFT treatment and the presence of the
substrate colonized in the BFT+BF. The different amounts of bacteria
indicate that great part of these microbes, especially the nitrifying ones,
would be attached to the biofilm and not on the flocs, or free in the
water. In our study, the lower abundances of free bacteria in the biofilm
treatment compared to the treatment with only bioflocs may have re-
sulted from a transfer of bacteria from the water column to the sub-
strate which, as observed by Oliveira et al. (2006).
There were no differences in growth among the shrimp submitted to

5
A.P.M. de Morais, et al.
different treatments, but there was a significant difference in survival
rate, with higher values in treatments with biofilm. In addition, the
inclusion of the substrate resulted in higher biomass due to higher
survivals in these treatments. This result may be a consequence of the
higher levels of nitrite in the BFT treatment, which remained about
24 days with concentrations above the safety level proposed by Lin and
Chen (2003). However, it is possible that the high concentrations of
nitrite, the stocking density and the initial size of the animals may have
negatively influenced survival in this treatment, mainly resulting in a
high standard deviation of survival data in this treatment since the
treatments that had the artificial substrate with biofilm show an in-
crease of area decreasing the relative density. Otoshi et al. (2006)
evaluated the growth and survival of juveniles of L. vannamei produced
with and without artificial substrate observing an increase in growth
and survival in substrate treatments. Similarly, Schveitzer et al. (2013)
studied the use of artificial substrates in a biofloc system with different
storage densities, concluding that the best survival was in the treat-
ments with the presence of the substrates.
5. Conclusion
Through the results obtained in this study it was possible to prove
that the nitrification process is less efficient in the absence of adequate
aeration. In addition, the use of artificial substrates to fix the biofilm
becomes a viable alternative of implantation, due to its low cost and
potential benefits to the culture environment and, consequently, of the
organisms produced.
Declaration of Competing Interest
None.
Acknowledgements
The authors are grateful for the financial support provided by the
National Council for Scientific and Technological Development
(CNPq),), Chamada Universal Process n° 409904/2018-0, Coordination
for the Improvement of Higher Level Personnel (CAPES) and FAPERGS,
Research Support Foundation of the State of Rio Grande do Sul State.
Wasielesky, W.Jr. and Abreu, P.C.A. are a research fellow of CNPq
under process number: 310652/2017-0 and 303256/2018-4, respec-
tively. Special thanks to Centro Oeste Rações S.A. (GUABI) and
AQUATEC, TREVISAN and All-Aqua Aeration for donating the experi-
mental diets and post-larvae and Aeration system respectively
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