Aquaculture 524 (2020) 735234

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

Integrated culture of white shrimp Litopenaeus vannamei and mullet Mugil T

liza on biofloc technology: Zootechnical performance, sludge generation,
and Vibrio spp. reduction
⁎
Bruno Augusto Amato Borgesa, , João Lucas Rochaa, Paulo Henrique Oliveira Pintoa,
Thiago Zacheua, Ana Clara Chedea, Caio Cesar Franca Magnottib, Vinicius Ronzani Cerqueirab,
Luis Alejandro Vinatea Aranaa
a
Laboratório de Camarões Marinhos, Departamento de Aquicultura, Universidade Federal de Santa Catarina, Rua dos Coroas, 503, CEP 88061-600, Barra da Lagoa,
Florianópolis, Santa Catarina, Brazil
b
Laboratório de Piscicultura Marinha, Departamento de Aquicultura, Universidade Federal de Santa Catarina, Rua dos Coroas, 503, CEP 88061-600, Barra da Lagoa,
Florianópolis, Santa Catarina, Brazil

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

Keywords: We grew mullet Mugil liza and white shrimp Litopenaeus vannamei in two different culture ways, a monoculture
Biofloc and an integrated culture for 41 days. They are (1) shrimp monoculture (SM), (2) mullet monoculture (MM), (3)
White shrimp integrated culture of mullet and shrimp in a same tank (SMI), and (4) integrated culture of mullet and shrimp in
Integrated culture two separated tanks (SMIS). The experimet units were composed of a matrix tank (800 L) and a recirculation
Solids
tank (90 L). After 41 days, no significant difference in shrimp final weight (1.43 ± 0.05 g) was observed among
Vibrio spp.
MM, SMI, and SMIS groups. The yield of shrimp in SM was higher than that of shrimp in SMI and SMIS. Growth
of mullet in SMI and SMIS significantly reduced the zootechnical performance of shrimp. A reduction of sludge
production in water indicated biofloc consumption by fish in SMI and SMIS. Mullet could reduce population of
Vibrio spp. in SMI and SMIS. In conclusion, integrated culture of mullet and shrimp could be placed in a same
tank, with no need to use additional tank.

1. Introduction
Over time, aquaculture has been proving to be a very promising
activity with high potential for growth. Over the last ten years, the
activity has almost doubled total production, including all species cul-
tivated worldwide, and reaching around 80 million tons in 2016 (FAO,
2018).
In the search for the growth of the activity, a new technology has
been developed and improved over the last few years that allows yield
increase in a smaller area, called biofloc technology (BFT - Biofloc
Technology), which makes it possible to produce in high densities with
minimum or zero water renewal, only to compensate for evaporation
(Avnimelech, 2015). Minimal water renewal increases biosafety, since
it reduces the chances of entry of external pathogens (Wasielesky Jr
et al., 2006). However, this system is relatively complex, especially in
relation to microbiology, nitrogen compounds, and the dynamic of the
present solids, requiring constant aeration and water movement to keep
them suspended in the water column (Avnimelech, 2015).
One of the main problems that occur in this cultivation system is the
large accumulation of solids and their possible precipitation in the tank
bottom (Wasielesky Jr et al., 2006; Hargreaves, 2013; Ray et al., 2010),
impairing the water quality and well-being of cultured organisms,
mainly due to the increased concentration of nitrogen compounds
(Thakur and Lin, 2003; Emerenciano et al., 2013). Also, the excess of
solids in the environment may cause clogging of gills of Litopenaeus
vannamei shrimp, significantly reducing their growth (Schveitzer et al.,
2013; Samocha et al., 2011; Spelta, 2016).
Currently, decanters and water renewal are the main methods used
to control and remove the solids present in biofloc production system.
Arantes et al. (2017) demonstrated that the use of decanters is sig-
nificantly better than the water renewal to remove solids in this system,
consequently improving some water quality aspects. However, decan-
ters requires excessive handling (Gaona et al., 2011; Hargreaves, 2013)
and still generates sludge rich in organic compounds that must undergo
treatment or be reused in some way. In this way, efforts have been
directed toward developing ways to minimize the generation of waste

⁎
Corresponding author.
E-mail address: bruno_roxo@hotmail.com (B.A.A. Borges).

https://doi.org/10.1016/j.aquaculture.2020.735234
Received 7 May 2019; Received in revised form 12 March 2020; Accepted 13 March 2020
Available online 14 March 2020
0044-8486/ © 2020 Elsevier B.V. All rights reserved.
B.A.A. Borges, et al.
without harming productivity. The integration of different species that
occupy different trophic levels on culture (IMTA – Integrated Multi-
Trophic Aquaculture) is already accomplished and has been proving to
be an interesting path to follow for the sustainable development of the
activity. This culture system is already used to produce white shrimp L.
vannamei integrated with Nile tilapia Oreochromis niloticus (Tendencia
et al., 2006; Poli et al., 2019), but there is still a vast field to be ex-
plored.
Thus, it comes up the possibility to control the culture solids
through the integrated cultivation with mullet Mugil liza. First, there
must be considered some important aspects that will define the success
of the system. It is essential to verify in advance if there will be con-
sumption of the solids by the mullets, also accompanying their condi-
tions of well-being and growth. In addition, it is necessary to know if
the presence of mullet in the environment generates some competition
for food, damaging the growth of shrimp. Mugil genus have potential to
integrated culture systems, since it feeds on the lowest trophic level
(detritus, microorganisms, aggregate of algae, bacteria, ciliates, rotifers,
nematodes) from the surface of substrates such as rocks or plants
(Odum, 1970; FAO, 2006–2018; Lupatsch et al., 2003; Rao and Babu,
2013; Mondal et al., 2015), whose composition is similar to biofloc, that
consists in an aggregate of algae, bacteria, ciliates, rotifers, nematodes
and detritus (Wasielesky Jr et al., 2006; Emerenciano et al., 2013;
Manan et al., 2017), reaching a concentration around to 50% crude
protein (Azim and Little, 2008; Emerenciano et al., 2013). Also, it was
adapted to the biofloc culture, tolerating solid concentrations of up to
700 mg L−1 (Rocha, 2012). Some studies reported the use of mullet as a
bioremediation species (Lupatsch et al., 2003; Katz et al., 2002), where
individuals that were kept close to intensive cultivations in cage tanks
obtained enough nutrition for growth, besides to improving sediment
characteristics. Therefore, it is believed that mullet would significantly
consume the solids present in culture with bioflocs.
Integrated culture of shrimp and mullet has shown to be successful
in productive and water quality ways. Melo et al. (2016) demonstrated
that the number of settleable solids and total suspended solids present
in a polyculture of white shrimp with parati Mugil curema was sig-
nificantly lower than the monoculture of shrimp (depending on mullet
biomass), evidencing their consumption of solids. Aghuzbeni et al.
(2017) showed that in earthen pounds the presence of mullet Mugil
cephalus on L. vannamei culture improved water quality parameters and
shrimp zootechnical performance. The Mugil genus has a high potential
for the cultivation in integrated systems; therefore, one must seek
greater knowledge in this area of study.
Also, on biofloc shrimp culture, Vibrio spp. infections are re-
sponsible for reducing production in several proportions and can infect
about 70 to 80% of individuals (Chandrakala and Priya, 2017) in all
stages of life in penaeid shrimp, leading to up to 100% mortality in
some cases (Aguirre-Gusmán, 2010). The biological interface between
Fig. 1. Side and upper view of experimental units, components and water flow.
2
Aquaculture 524 (2020) 735234
fish and aquatic environment, formed by a mucus layer, is composed of
several secretions which act like lubricant (Rosen and Comford, 1971),
mechanical protection and osmoregulation (Cameron and Endean,
1973), besides presenting potential on the prevention of parasites,
bacteria and fungi (Ebran et al., 2000). The mucus of several fish spe-
cies have been studied due to the antibiotic (Austin and Mcintosh,
1988; Magarinos et al., 1995; Ebran et al., 1999) and antifungal (Hellio
et al., 2002) properties present in the proteins of their epidermis. In this
way, it was also sought to verify if the presence of mullet reduces the
presence of Vibrio spp. in water culture.
This work evaluated the possibility of mullet M. liza and white
shrimp L. vannamei integrated culture in biofloc system, comparing two
different methods: living in the same tank and separately.
2. Materials and methods
2.1. Location and object of study
The experiment was conducted for 41 days at the Laboratory of
Marine Shrimp (LCM), Federal University of Santa Catarina – UFSC,
located in Barra da Lagoa, Florianópolis, Santa Catarina (27.57° S,
48.43° O).
The shrimp L. vannamei were obtained from Aquatec LTDA – Speed
Line lineage. The mullet M. liza were acquired from Marine Fish
Laboratory (LAPMAR) - UFSC, from a spawning of wild individuals in
the laboratory. This work was previously analyzed and approved by the
Ethics Committee on Animals Use – CEUA/UFSC (Protocol 9181090418
).
2.2. Design and experimental units
The experiment was performed in a completely randomized design
with three replicates, and composed of four treatments: shrimp mono-
culture (SM), shrimp and mullet integrated into the same tank (SMI),
shrimp and mullet integrated into separated tanks (SMIS), and mullet
monoculture (MM).
The experimental units were composed of two tanks in recircula-
tion: matrix tank and recirculation tank. Water was recirculated using a
pump (650 L h−1) and returned to matrix tank by gravity (Fig. 1).
The matrix tank consisted of circular water tanks with capacity of
800 L, with central microperforated aeration ring in order to provide
agitation and oxygenation (≥5 mg L−1) of the water column and avoid
the sedimentation of solids. Temperature was controlled (28 ± 0.5 °C)
through the thermostat and maintained in the desired range with the
use of 800 W heaters. Tanks were covered by a net and were allocated
in a greenhouse with natural photoperiod. An artificial Needlona® type
substrate composed of four rectangular plates (0.40 × 0.55 m) was
used, representing 80% of the surface area of the tank.
B.A.A. Borges, et al.
The recirculation tank consisted of circular water tanks with a ca-
pacity of 90 L, where the mullet on SMIS treatment tanks were condi-
tioned. In the other treatments, the recirculation tank had no organisms
inside. This tank has its own aeration, with four porous stones on the
bottom, to ensure that the solids do not precipitate.
2.3. Stocking and feeding
On the first day of experiment, shrimp were stocked with a density
of 2500 shrimp m−3, totaling 2000 shrimps (mean weight = 0.18 ±
0.02 g; initial biomass = 360 g) per experimental unit. The mullet
stocking (mean weight = 0.95 ± 0.09 g) was performed at the end of
the first week, with 10% of initial shrimp biomass (Hoang et al., 2018)
estimated at the end of the first week (initial biomass = 40.74 ±
0.57 g). During the seven days prior to stocking, the mullet had un-
dergone a process of acclimatization to biofloc, so that the concentra-
tion of total suspended solids in the water was gradually increased
every day, avoiding an abrupt change in the environmental character-
istics.
The shrimp were fed four times a day (08:30, 11:30, 14:00, 17:00)
with commercial shrimp feed Guabi Potimar© (40% crude protein, 10%
moisture, 7.5% ethereal extract, 4% crude fiber, mineral matter 14%).
In MM treatment, the mullet were fed with commercial feed for marine
fish Nutripisces© (crude protein 45%, moisture 13%, ethereal extract
9%, crude fiber 3.6%, mineral matter 16%) four times a day (08:30,
11:30, 14:00, 17:00). No additional feed was offered to mullet in the
SMI and SMIS treatments.
2.4. Water quality parameters
During the experiment, temperature (°C) and dissolved oxygen
(mg L−1) were measured daily, twice a day (8:30 and 14:00), with the
YSI pro20 oximeter. Twice a week were evaluated the salinity (g L−1)
(Ecosense EC300A), pH (Thermo Scientific Orion Star A211 pH), al-
kalinity (mg L−1) (APHA, 2005), total suspended solids (TSS) (mg L−1)
(APHA, 2005), total ammonia nitrogen (TAN) (mg L−1) (Grasshoff
et al., 1983), nitrite-N (NO2-N) (mg L−1) (Strickland and Parsons,
1972), and the settleable solids (SS) (mL L−1) through the Imhoff cone
method. At day 7, 25 and 39 of the experiment nitrate-N (NO3-N)
(mg L−1) (APHA, 2005) was analyzed.
To maintain the C:N ratio in 15:1 (Avnimelech, 1999), molasses
were added as a carbon organic source, assuming that the shrimp as-
similate 25% of the nitrogen present in the feed and that the rest is
excreted in the form of TAN (Crab et al., 2007). In addition, 20 g of
molasses was added to each gram of TAN present in the water when the
amount exceeded 1 mg L−1 (Avnimelech, 1999). The alkalinity was
corrected by the addition of hydrated lime (Ca(OH)2) when necessary,
so that it remained around 150 mg L−1 of CaCO3, values that did not
compromise the zootechnical performance of shrimp on cultivation
(Ebeling et al., 2006; Hargreaves, 2013; Piérri et al., 2015). When al-
kalinity was lower than 150 mg L−1, hydrated lime was added in an
amount of 10% of total shrimp biomass. Before the application, mo-
lasses and hidrated lime was diluted in the cultivation water in a
bucket. During the experiment, freshwater was added to replace the
evaporation, when necessary.
The initial water was prepared with 400 L of water from a biofloc
shrimp culture tank from the laboratory. The remaining volume was
filled with sea water, seeking an initial concentration of 300 to
400 mg L−1 of TSS for stocking. The initial water quality parameters
were: TAN = 0.75 ± 0.04 mg L−1; nitrite-N (NO2-
N) = 0.27 ± 0.04 mg L−1; TSS = 266.7 ± 28.6 mg L−1;
pH = 8.40 ± 0.08; salinity = 32.6 ± 0.15 mg L−1;
alkalinity = 152 ± 4 mg L−1.
3
Aquaculture 524 (2020) 735234
2.5. Sludge removal and quantification
To verify the amount of sludge generated between treatments, all
the sludge removed through the decanter was quantified. When the
concentration of TSS exceeded 600 mg L−1 (Schveitzer et al., 2013;
Avnimelech, 2015), the solids were removed by the decanter (60 L)
(one for each experimental unit), operating at a flow rate of 33 L h−1.
The sludge removed from decanter was quantified in a graduated
bucket and the amount of total suspended solids (TSS) present in the
sludge was quantified by the gravimetric method (APHA, 2005). The
removed solids (RS) of the system were quantified, and at the end of the
experiment it was possible to verify the sludge generation (SG) (g) in
each treatment during the cultivation, according to the equation: SG
(g) = {[(TSS final × volume) – (TSS initial × volume)]/1000} + RS.
The amount of sludge generated by the biomass produced (sludge/
biomass) was also quantified, according to the equation: sludge/bio-
mass ratio = sludge produced/total biomass.
2.6. Zootechnical performance of shrimp and mullet
To keep track of shrimp growth and adjust the amount of feed
supplied as recommended by Van Wik (1999), biometrics were per-
formed weekly.
At the end of the experiment the final mean weight (g), survival (%),
specific growth rate (SGR), apparent feed conversion rate (FCR), bio-
mass (kg) and yield (kg m−3) of shrimp were measured according to
equations: N = final biomass/mean weight; SGR (% day−1) = [(ln
final weight – ln initial weight)/time (days)] × 100; Survival (%) = (N
final/N initial) × 100; FCR = feed input/biomass gain; Yield
(kg.m−3) = final biomass/volume.
For mullet, two biometrics (initial and final) were performed to
measure weight gain (WG) (g), specific growth rate (SGR), biomass (g)
and survival (%) during cultivation, according to equations: WG
(g) = final mean weight – initial mean weight; SGR (% day−1) = [(ln
final weight – ln initial weight)/time (days)] × 100; survival (%) = (N
final/N initial) × 100.
2.7. Mullet: hepatosomatic index (HSI) and condition factor (K)
To verify the well-being conditions of the mullet, the hepatosomatic
index (HSI) and the condition factor (K) were checked at the beginning
and at the end of the experiment.
To assess HSI at beginning, fifteen mullets were selected and eu-
thanized randomly from the initial acclimation tank. In the end, five
fishes were randomly selected from each experimental unit, totaling
fifteen per treatment. In both moments, the liver of every fish was re-
moved for weighing, to apply in the formula: HSI = (body weight/liver
weight) × 100. For this procedure, the mullets were euthanized by
anesthetic deepening, by immersion in a solution with eugenol (clove
oil) 75 mg L−1. To perform the condition factor (K) of fishes, seven
individuals were randomly selected from each treatment at stocking
and harvesting for weighing and total length measurement, to apply in
the formula: K = [total weight/(total length)3] × 100. All procedures
met the Animal Ethics Committees criteria.
2.8. Vibrio spp. and total heterotrophic bacteria (THB) score
The initial and final counts of Vibrio spp., as well as the final count
of total heterotrophic bacteria (THB) were performed. Water samples
were collected from each experimental unit, homogenized, serially di-
luted (1/10) in 3% sterile saline solution (SSS) and seeded in Marine
Agar culture and TCBS for THB and Vibrio spp., respectively. Culture
media was incubated in an oven at 30 °C and after 24 h the total colony
forming units (CFU) were counted.
B.A.A. Borges, et al. Aquaculture 524 (2020) 735234

2.9. Statistical analysis
The data were analyzed through the one-way analysis of variance
(ANOVA), followed by the Tukey test when necessary (Zar, 2010). The
pre requisites of homoscedasticity and normality were verified pre-
viously, through the Levene and Shappiro-Wilk tests, respectively (Zar,
2010). When ANOVA requirements were not met, data were analyzed
by the Kruskall-Wallis test followed by confidence intervals, if neces-
sary. All tests were performed considering a significance level of
p < .05.
3. Results
statistically different (p > .05) among the SM, SMI and SMIS treat-
ments, and these were different from the MM treatment, due to the
lower mullet biomass (Table 2).
3.3. Mullet: hepatosomatic index (HSI) and condition factor (K)
For the mullet, the initial hepatosomatic index (HSI) was like the
final values obtained in MM, SMI, and SMIS. However, a difference
(p < .05) was observed between the final SMI and SMIS values
(Table 3), being SMIS the lowest value of HSI. There was no significant
difference into the initial and final condition factor (Table 3).

3.1. Water quality parameters, solids production and sludge generation

3.4. Vibrio spp. and total heterotrophic bacteria (THB) score
During the experiment, dissolved oxygen remained around
At the end of the experiment, it was found that the final amount of
5.5 mg L−1, being higher in MM (p < .05). The temperature remained
Vibrio spp. was significantly lower (p < .05) in the treatments with
near 28 °C and the salinity around 32 g L−1. The pH remained stable
mullet (Table 4). At the beginning of the experiment, the Vibrio spp.
throughout the experiment, with values around 8.0 and alkalinity
count was different among the treatments (p < .05), presenting higher
varied around of 160 mg L−1, being constantly corrected throughout
values in SM and SMI. In the end, a larger number of Vibrio spp.
the experiment by the addition of hydrated lime (Ca(OH)2). The pH was
(7.6 × 104 ± 4.71 × 104) was observed in SM, followed by SMI,
higher in MM, due to more stable values of alkalinity on this treatment
SMIS, and MM. The amount of THB at the end was not different
during the experiment. No difference was observed for total ammonia
(p > .05) between treatments (Table 4).
nitrogen concentration (p > .05), but there was a difference for N-
nitrite and N-nitrate concentration (p < .05). Significant differences
were observed in most water quality variables (Table 1), mainly related
4. Discussion
to MM treatment. During the experiment, it was not necessary to add
molasses in SM, SMI, and SMIS to control the C:N ratio, which remained
4.1. Water quality parameters
stable during the experiment. In MM, molasses were added daily to
maintain the C:N ratio in 15:1.
During the experiment, the concentration of dissolved oxygen
No statistical differences were observed for total suspended solids
(above 5.5 mg L−1) was not a limiting factor for shrimp and mullet
(TSS) concentration between SM, SMI, and SMIS, but these were sig-
growth. Temperature, pH, salinity, and alkalinity were also kept within
nificantly different from MM (p < .05). The sludge generated during
the limits suggested for the cultivation of white shrimp (Boyd, 1990;
the experiment was also different among the treatments, being statis-
Van Wik and Scarpa, 1999; Timmons et al., 2009). The salinity pre-
tically smaller in MM, followed by SMI, SMIS, and SM. For sludge/
sented higher values in MM, possibly due to the lower average tem-
biomass ratio, the value for MM was higher (Table 1).
perature in this treatment which resulted in lower evaporation of water,
and consequently lower fresh water replenishment. The pH decreases
3.2. Zootechnical performance of shrimp and mullet when alkalinity is consumed, mainly in the nitrification process and
metabolic activities of the heterotrophic bacteria (Chen et al., 2006;
Except for the yield, the performance of shrimp was statistically Ebeling et al., 2006). Nitrogen compounds also remained adequate for
similar between treatments (Table 2). For mullet, a significant differ- the growth of both species (Lin and Chen, 2001; Sampaio et al., 2002;
ence was obtained for the variables weight gain, specific growth rate Cobo et al., 2014). The formation of N-nitrite and N-nitrate was lower
and final biomass. The largest differences were observed in the SMIS in MM, possibly due to lower biomass and consequently lower feed
treatment, where the mullet only consumed biofloc as food source. The supply in this treatment.
total final biomass of the system (shrimp and mullet) was not

Table 1
Water quality parameters of shrimp monoculture (SM), shrimp and mullet integrated with different methods (SMI and SMIS) and monoculture of mullets (MM),
cultured in a biofloc system during 41 days.
MS ISM ISMS MM p

Temperature (°C) 28.10 ± 0.82ab 28.35 ± 0.71b 28.31 ± 0.72b 27.78 ± 0.83a 0.0000#
Dissolved oxigen (mg L−1) 5.58 ± 0.29b 5.58 ± 0.28b 5.58 ± 0.3b 6.06 ± 0.27a 0.0001#
pH 8.02 ± 0.17b 8.05 ± 0.15b 8.01 ± 0.16b 8.24 ± 0.04a 0.0001#
Salinity (g L−1) 31.67 ± 1.81b 32.08 ± 1.21ab 32.19 ± 1.24ab 33.19 ± 0.71a 0.0001#
Alkalinity (mg L−1) 167 ± 22.95b 158.11 ± 14.61ab 154.61 ± 16.28a 164.26 ± 14.05ab 0.0491#
TAN (mg L−1) 0.45 ± 0.43 0.39 ± 0.28 0.34 ± 0.23 0.25 ± 0.10 0.1011#
Nitrite-N (mg L−1) 1.46 ± 2.00b 1.41 ± 2.17b 1.39 ± 2.08b 0.32 ± 0.28a 0.0436#
Nitrate-N (mg L−1) 15.44 ± 11.17b 15.85 ± 11.36b 14.04 ± 11.99b 3.1 ± 1.06a 0.0021#
SS (mL L−1) 7.69 ± 6.9 5.98 ± 5.14 5.68 ± 4.43 – 0.6621#
TSS (mg L−1) 709.97 ± 255.94b 693.16 ± 215.33b 706.52 ± 252.13b 393.76 ± 71.43a 0.0000#
Sludge production (g) 1235.63 ± 130.70c 861.20 ± 201.39b 945.25 ± 48.02bc 127.20 ± 17.54a 0.0000
Sludge: biomass 0.41 ± 0.05ab 0.29 ± 0.07a 0.31 ± 0.01a 0.53 ± 0.11b 0.0163

Data presented in mean ± standard deviation. Different letters indicate statistical difference by the Tukey test (p < .05).
Initial shrimp weight = 0.18 ± 0.02 g; initial fish weight = 0.95 ± 0.03 g.
Different letters indicates statistical difference.
#
Indicates that the Kruskall-Wallis test was performed followed by confidence intervals.

4
B.A.A. Borges, et al. Aquaculture 524 (2020) 735234

Table 2
Zootechnical performance of shrimp monoculture (SM), shrimp and mullet integrated with different methods (SMI and SMIS) and mullet monoculture (MM),
cultured in a biofloc system during 41 days.
SM SMI SMIS MM p

Shrimp performance Mean final weight (g) 1.50 ± 0.03 1.37 ± 0.05 1.42 ± 0.06 – 0.0762
Survival (%) 98.74 ± 1.23 97.23 ± 3.19 98 ± 2.70 – 0.7720
Feed conversion rate 1.55 ± 0.08 1.61 ± 0.03 1.51 ± 0.08 – 0.2751
Specific growth rate (% day−1) 5.17 ± 0.06 4.96 ± 0.10 5.04 ± 0.11 – 0.0685
Final biomass (kg) 3.00 ± 0.17 2.67 ± 0.08 2.81 ± 0.03 – 0.0823
Yield (kg m−1) 3.75 ± 0.21b 3.34 ± 0.1a 3.51 ± 0.04ab – 0.0307
Mullet performance Weight gain (g) – 6.26 ± 0.77b 2.70 ± 0.17a 5.41 ± 1.58b 0.0128
Survival (%) – 95.55 ± 4.19 95.60 ± 3.81 98.46 ± 1.33 0.5230
Specific growth rate (% day−1) – 5.73 ± 0.17b 3.91 ± 0.25a 5.09 ± 0.82ab 0.0124
Final biomass (g) – 288.06 ± 24.14b 152.38 ± 8.73a 242.6 ± 32.21b 0.0011
Shrimp + mullet Total final biomass (kg) 3.00 ± 0.17b 2.96 ± 0.1b 2.96 ± 0.03b 0.24 ± 0.03a 0.0001#

Data presented in mean ± standard deviation. Different letters indicate statistical difference by the Tukey test (p < .05).
Initial shrimp weight = 0.18 ± 0.02 g; initial fish weight = 0.95 ± 0.03 g.
Different letters indicates statistical difference.
#
Indicates that the Kruskall-Wallis test was performed followed by confidence intervals.

Table 3
Hepatosomatic index (HSI) and condition factor (K) values of mullet monoculture (MM) in different integration methods (SMI and SMIS) on a biofloc culture system
during 41 days.
INITIAL MM SMI SMIS p

ab ab b a
HSI 2.12 ± 0.39 2.26 ± 0.46 2.34 ± 0.41 1,92 ± 0.34 0.0364
K initial 1.13 ± 0.08 1.12 ± 0.09 1.11 ± 0.13 0.8640
K final 1.20 ± 0.1 1.20 ± 0.06 1.23 ± 0.13 0.5448

Data presented in mean ± standard deviation. Different letters indicate statistical difference by the Tukey test (p < .05). Initial shrimp weight = 0.18 ± 0.02 g;
initial fish weight = 0.95 ± 0.03 g.
Different letters indicates statistical difference.

Table 4
Amount of Vibrio spp. at the beginning, and Vibrio spp. at the end, and total heterotrophic bacteria (THB) at the beginning and at the end in shrimp monoculture (SM),
shrimp and mullet integrated with different methods (SMI and SMIS) and mullet monoculture (MM), in a biofloc culture system during 41 days.
SM SMI SMIS MM p

Vibrio spp. initial (102. CFU mL−1) 18.00 22.00 4.00 3.00 –
Vibrio spp. final (102. CFU mL−1) 760.00 ± 471.00c 76.00 ± 50.30b 46.30 ± 29.70b 2.33 ± 1.53a 0.0224
THB final (104. CFU mL−1) 53.30 ± 58.60 76.70 ± 47.30 66.70 ± 55.10 13.30 ± 5.70 0.1051

Data presented in mean ± standard deviation. Different letters indicate statistical difference by the Kruskall-Wallis test (p < .05), followed by confidence intervals,
if necessary. Initial shrimp weight = 0.18 ± 0.02 g; initial fish weight = 0.95 ± 0.03 g.
Different letters indicates statistical difference.

4.2. Zootechnical performance of shrimp and mullet
The results showed that the presence of mullet negatively affected
the shrimp yield, possibly due to competition for space and food. The
survival obtained was higher than that found in several studies for the
different stages of shrimp growth (Xu and Pan, 2012; Schveitzer et al.,
2013; Kim et al., 2013; Correia et al., 2014), although the concentration
of solids has exceeded that recommended by Samocha et al. (2007) and
Avnimelech (2015). The zootechnical performance of shrimp was sta-
tistically similar between SMI and SMIS, and the mullet performance
was statistically higher on SMI, suggesting that there is no need for an
additional tank for the integration of both species.
The zootechnical performance of mullet was different among
treatments, except for survival. On SMIS treatment, due to the feed
restricted only to biofloc by the fish, the weight gain, the specific
growth rate (SGR), and the final biomass were smaller than MM and
SMI treatments. However, the values of SGR obtained in SMIS were
similar to those obtained in other studies with Mugil sp., in which the
mullet was fed with food with high protein level in clear water and
bioflocs (Carvalho et al., 2010; Rocha, 2012). The results showed that
mullet grew by being fed only by the biofloc present in the cultivation,
with no need of additional commercial feed, which is fundamental in
integrated systems that look to reduce the use of resources and wastes.
In SMI, although a specific diet for the mullet is not offered, it is pos-
sible that fish have fed on the shrimp feed, promoting competition for
food.
It is known that biofloc contains proteins, whose amount varies
according to their composition (De Schryver et al., 2008). It already
reported amounts between 30 and 50% of crude protein (Azim and
Little, 2008; Ballester et al., 2010), serving as supplementary food for
the cultivated organisms, and making it possible to reduce feed costs
and decrease the amount of crude protein in the diet (Avnimelech,
2007). The mullet that were fed restrictedly from the biofloc (SMIS)
obtained a specific growth rate higher than that obtained for M. ce-
phalus in bioflocs and recirculation system fed with 54% (Vinatea et al.,
2018) and 24% crude protein in bioflocs (El-Dahhar et al., 2015), and
M. liza fed with 40% crude protein in clear water (Ribeiro, 2017). In
this experiment, it was possible to remove completely the artificial
feeding of the mullet, which confirmed to be an exceptional species to
be used in integrated culture biofloc system.
4.3. Solids production and sludge generation
Due to high solids generation and excessive management of the

5
B.A.A. Borges, et al.
decanters, the total suspended solids and the settleable solids were
higher than the recommended by Samocha et al. (2007) and
Avnimelech (2015), but the values of TSS obtained in our experiment
were already reported by other authors (Schveitzer et al., 2013; Serra
et al., 2015; Krummenauer et al., 2014; Gaona et al., 2016;
Krummenauer et al., 2016; Xu et al., 2016), and in the present work it
did not affect the zootechnical performance of shrimp.
The results show that the solids present in the system were con-
sumed by mullet. The integration of mullet in shrimp cultivation proved
to be an interesting alternative for reducing solids. In MM the amount
of solids generated was lower due to the lower biomass, but this re-
sulted in higher values of sludge/biomass, also due to the constant
addition of carbon source in this treatment. Statistically, the sludge/
biomass ratio did not differ between SM, SMI, and SMIS, although it
was numerically smaller in the treatments with integrated mullet (SMI
and SMIS). In a study conducted by Poli et al. (2019), with L. vannamei
and O. niloticus integrated system on biofloc, the sludge/biomass ratio
decreases as the tilapia density increases, also showing the biofloc
consumption by the fish. The results demonstrate that the integration
with mullet M. liza is an interesting alternative for the solid reduction
generated in white shrimp L. vannamei cultivation on bioflocs, but more
studies are still necessary to reduce the negative effect of mullets in the
shrimp final yield.
4.4. Mullet: hepatosomatic index (HSI) and condition factor (K)
Besides to reduce solids on the culture, M. liza has been shown to be
adapted to the water quality conditions established for shrimp L. van-
namei in biofloc systems. On the other hand, mullet had greater weight
gain in MM and SMI. The initial and final condition factor (K) was si-
milar among the treatments, demonstrating that individuals that were
fed only with biofloc obtained welfare conditions in relation to en-
vironmental and physiological conditions (Le Cren, 1951; Nehemia
et al., 2012), similar to those consuming commercial feed with high
protein levels (40 and 45% CP).
Before the beginning of the experiment, the mullets were kept in a
recirculation system (Marine Fish Laboratory - UFSC) fed with 45% CP
and food frequency of six times a day. Thus, it can be affirmed that
environment change (clear water to biofloc) did not affect the well-
being of the fish, even with restricted feeding. The results of the con-
dition factor (K) obtained in the present work were higher than those
obtained by Wassef et al. (2002) and Ribeiro (2017) for M. cephalus and
M. liza, respectively, fed with 40% CP in the diet. On the other hand,
Hossain and Furuichi (2000) and Vinatea et al. (2018) reported higher
K values for Liza haematocheila and M. cephalus, fed with 44% and 54%
CP in the diet, respectively.
The hepatosomatic index (HSI) obtained in the MM, SMI, and SMIS
was not statistically different between the beginning and the end of the
experiment, confirming that the mullet remained in favorable condi-
tions from the environmental and physiological conditions. However,
HSI differences (p < .05) were observed between the SMI
(2.34 ± 0.41) and SMIS (1.92 ± 0.34) treatments. The liver is the
organ in which fish accumulate their energy reserves and glycogen, and
the amount of fat and glycogen in this organ directly influences their
weight, causing changes in the HSI (Hoar and Randall, 1971). When
compared to the present study, Hossain and Furuichi (2000) obtained
higher HSI for mullet L. haematocheila. On the other hand, Baker et al.
(1998) obtained similar condition factor values (1.30 ± 0.06) and
lower hepatosomatic index (1.28 ± 0.25), using the mullet Chelon
labrosus fed ad libitum with 38% CP diet.
It can be stated that Mugil liza has a high potential for integrated
cultivation on biofloc system. As bioremediation species, it showed to
be adapted to biofloc feed, obtaining similar or better parameters of
well-being than found in other researches with Mugilidae.
6
Aquaculture 524 (2020) 735234
4.5. Vibrio spp. and total heterotrophic bacteria (THB) score
In our integrated culture systems, there was a reduction in the
concentration of Vibrio spp. bacteria in the water. Hoang et al. (2018)
found similar values to SMI and SMIS for total heterotrophic bacteria
and Vibrio spp. concentrations, but in the present study, values were
significantly higher in SM when compared to integrated cultures. Even
so, this difference did not affect the performance of shrimp monoculture
(SM). Some studies present mortality for white shrimp L. vannamei at
Vibrio spp. concentrations equal to or greater than 104 CFU mL−1 (Liu
et al., 2004; Liu and Chen, 2004; Tseng and Chen, 2004; Cheng et al.,
2005; Wang and Chen, 2005). In the present work, besides the con-
centrations of Vibrio spp. (mean of 7.6 × 104 CFU mL−1), there were no
negative effects on L. vannamei production, possibly due to other factors
such as nutrition and water quality.
The results suggest that the presence of mullet integrated into the
shrimp culture showed benefits in reducing the amount of Vibrio spp.,
possibly due to the pathogen inhibitory properties present in the mucus
(Goméz et al., 2013). Tendencia et al. (2006) showed that in poly-
culture with tilapia and grouper, tiger shrimp obtained positive survival
results, also due to the antibacterial properties present in this fish
mucus. Antimicrobial molecules have already been observed in the
mucus present in fish epidermis, among them glycoproteins (Ebran
et al., 2000) enzymes (Fuochi et al., 2017), proteins (Cole et al., 1997;
Jurado et al., 2015), and antimicrobial peptides (Masso-Silva and
Diamond, 2014), mainly responsible for combating pathogens (Reverter
et al., 2018). Several studies suggest that integrated fish and shrimp
culture is an alternative to reduce the Vibrio spp. bacteria in water
(Dash et al., 2017).
The present work suggests that M. liza may have been responsible
for the reduction of Vibrio spp. during the cultivation, however, it is
recommended to do continuous work on the mullet mucus properties
and the benefits that it can bring to the production of white shrimp L.
vannamei in a biofloc system.
5. Conclusion
Integrated culture of the mullet M. liza with L. vannamei shrimp is
possible to be made simultaneously, there is no need to separate the
species in different tanks. Mullet was able to reduce the quantity of
solids in the water. Integrated culture of M. liza and L. vannamei could
reduce population of Vibrio spp. in water.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
The authors thank CAPES and CNPq for the financial support that
made this research possible. Thanks to all the personnel of the
Laboratory of Marine Shrimp - LCM, for the technical support, and to
the group of Marine Fish Laboratory - LAPMAR, for the partnership and
mullet donation. We also thank the Postgraduate Program in
Aquaculture and Fishery Resources (PPG - AQI), for all work and ded-
ication. This study was financed in part by the Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) –
Finance Code 001.
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