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Citation: Carvalho, A.; Brandão, H.;
Zemor, J.C.; Cardozo, A.P.; Vieira,
F.N.; Okamoto, M.H.; Turan, G.;
Poersch, L.H. Effect of Organic or
Inorganic Fertilization on Microbial
Flake Production in Integrated
Cultivation of Ulva lactuca with
Oreochromis niloticus and Penaeus
vannamei.Fishes 2024,9, 191.
https://doi.org/10.3390/
fishes9060191
Academic Editor: Chunsheng Liu
Received: 25 March 2024
Revised: 16 May 2024
Accepted: 21 May 2024
Published: 23 May 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
fishes
Article
Effect of Organic or Inorganic Fertilization on Microbial Flake
Production in Integrated Cultivation of Ulva lactuca with
Oreochromis niloticus and Penaeus vannamei
Andrezza Carvalho
1,
*, Hellyjúnyor Brandão
1
, Julio C. Zemor
1
, Alessandro Pereira Cardozo
1
, Felipe N. Vieira
2
,
Marcelo H. Okamoto 1, Gamze Turan 3and Luís H. Poersch 1
1Marine Aquaculture Station, Institute of Oceanography, Federal University of Rio Grande—FURG, Rua do
Hotel, no.2, Cassino, Rio Grande 96210-030, RS, Brazil; hellyjunyor@gmail.com (H.B.);
juliozemor@hotmail.com (J.C.Z.); ocalessandro@hotmail.com (A.P.C.); mar.okamoto@gmail.com (M.H.O.);
lpoersch@gmail.com (L.H.P.)
2Laboratório de Camarões Marinhos, Departamento de Aquicultura, Centro de Ciências Agrárias,
Univesidade Federal de Santa Catarina, Rua dos Coroas 503, Barra da Lagoa,
Florianópolis 88061-600, SC, Brazil; felipe.vieira@ufsc.br
3
Aquaculture Department, Fisheries Faculty, Ege University, Izmir 35100, Turkey; gamzeturan2000@gmail.com
*Correspondence: andrezzachagas@hotmail.com
Abstract: Different fertilization regimes in biofloc systems influence the predominance of distinct
bacterial populations, impacting water quality and organism performance. This study evaluates
the growth and nutrient absorption of the macroalgae Ulva lactuca when cultivated in an integrated
system with Penaeus vannamei and Oreochromis niloticus in chemoautotrophic and heterotrophic
systems. The experiment lasted 45 days and comprised two treatments, each with three replicates:
chemoautotrophic—utilizing chemical fertilizers; heterotrophic—employing inoculum from mature
biofloc shrimp cultivation, supplemented with organic fertilizers. Each treatment consisted of
three systems
, each containing a 4 m
3
tank for shrimp, 0.7 m
3
for tilapia, and 0.35 m
3
for macroalgae,
with continuous water circulation between tanks and constant aeration. Water quality analyses were
carried out during the experiment, as were the performances of the macroalgae and animals. The
data were subjected to a statistical analysis. Results revealed an increase in macroalgae biomass
and the removal of nitrate (57%) and phosphate (47%) during cultivation, with a higher specific
growth rate observed in the chemoautotrophic treatment. Nonetheless, the heterotrophic treatment
exhibited higher levels of protein in the macroalgae (18% dry matter) and phosphate removal rates
(56%), along with superior maintenance of water quality parameters. Tilapia performance varied
across treatments, with a higher final weight and weight gain recorded in the heterotrophic treatment.
The recycling of water from an ongoing biofloc cultivation with organic fertilization demonstrated
viability for macroalgae cultivation within an integrated system involving shrimp and fish.
Keywords: nitrate; phosphate; growth; biocompounds; water renewal
Key Contribution: The use of macroalgae in an integrated system with different fertilizations showed
a high rate of nitrate and phosphate removal. The use of partial harvests promoted high biomass
production with a high protein content.
1. Introduction
Numerous recent studies have explored marine macroalgae as a source of human
food, bioactive compounds, and supplements for marine organisms [
1
,
2
]. According
to Chopin [
3
], macroalgae cultivation can be deemed sustainable due to their minimal
need for feed, reduced land footprint, and bioremediation capabilities. The genus Ulva
exhibits a global distribution, with the morphology and composition adapting to the
environmental variables of the production site, rendering it a feasible and intriguing option
Fishes 2024,9, 191. https://doi.org/10.3390/fishes9060191 https://www.mdpi.com/journal/fishes
Fishes 2024,9, 191 2 of 15
for cultivation [
4
]. The cultivation of macroalgae integrated with other aquatic organisms
has gained traction in aquaculture, referred to as Integrated Multitrophic Aquaculture
(IMTA) [
5
]. Resende et al. [
6
] demonstrated the feasibility of incorporating macroalgae
for nutrient absorption and biomass production in open cultivation systems with Sea
bream Sparus aurata and European sea bass Dicentrarchus labrax. In addition to macroalgae
serving as inorganic consumers, the IMTA system also includes organic consumers to
consume the solids produced in the system. The tilapia Oreochromis niloticus, known for
its ease of handling and feeding habits, has been utilized in integrated systems, showing
positive results in solid filtration [
7
,
8
]. Due to its robustness, tilapia can also be produced
in brackish water without negative effects on its zootechnical performance [
9
]. Both species
could bring benefits when integrated into the cultivation of a main species, as in the
farming of the Pacific white shrimp Penaeus vannamei, the most cultivated shrimp in the
world, especially because it is an euryhaline [
10
], easy to manage and adapt [
11
], and
holds significant economic interest [
12
], which has been employed as a primary species in
integrated systems.
In conjunction with integrated systems, the utilization of biofloc technology has
intensified production by improving the utilization of accumulated waste in cultivation.
Studies by Brito et al. [
13
], Legarda et al. [
14
], and Morais et al. [
15
] have reported positive
results from the production of shrimp, tilapia, and macroalgae in an integrated biofloc
system. In general, biofloc technology offers enhanced biosecurity with reduced water
exchange, facilitates water quality control through microbial activity, and serves as a
supplementary food source for cultivated species [
16
]. Various fertilization approaches
promote the growth and dominance of distinct bacterial groups within the system, including
heterotrophic bacteria, chemoautotrophic bacteria, or a combination of both in mixed or
mature systems [
17
]. The bacterial groups play a crucial role in nitrogen consumption
and oxidation within the system, contributing to the maintenance of water quality for
the organisms. Heterotrophic bacteria are favored by daily carbon source fertilization,
typically at a ratio of 15 g of carbohydrate per gram of available nitrogen in the system [
18
].
Ammonia consumption by heterotrophic bacteria leads to bacterial biomass production,
increasing the total suspended solids concentration in the water, which should ideally be
maintained within the range of 100 to 350 mg L
−1
, as suggested by Gaona et al. [
19
] to
avoid adverse effects on animal performance.
Another significant bacterial group comprises chemoautotrophs, which, according
to Ebeling et al. [
18
], oxidize nitrogen within the system, converting ammonia to nitrite
and eventually to nitrate, a less toxic final product for organisms. Chemical fertilization
is utilized for system establishment, requiring approximately 30 to 45 days of chemical
fertilization prior to cultivation initiation to maintain low concentrations of ammonia and
nitrite [
17
]. Unlike heterotrophic bacteria, chemoautotrophic bacteria generate fewer solids
in the system and consume less oxygen. However, they utilize more inorganic carbon,
requiring alkalinity adjustments to maintain levels above 150 mg of CaCO
3
L
−1
[
20
].
Nitrate accumulates as the final nitrogen product in this system’s oxidation process, posing
toxicity risks to cultivated organisms at high concentrations [
21
], and, when discharged
untreated, can lead to diseases such as methemoglobinemia in humans [
22
]. Another viable
option is to utilize a biofloc inoculum from an ongoing cultivation, providing enhanced
stability in nitrogen control and greater sustainability through water reuse [
17
]. This
approach results in a mixed system containing both heterotrophic and chemoautotrophic
bacteria, aimed at regulating water quality by promoting bacterial biomass production and
nitrification [
23
]. Organic fertilization is typically employed at the onset of cultivation to
expedite the stabilization of ammonia until nitrifying bacteria become established [24].
The biofloc system is complex and subject to variations based on the fertilization
strategy utilized, which can impact water quality, production costs, and animal perfor-
mance. Brandão et al. [
23
] reported greater shrimp growth in mixed systems compared
to heterotrophic systems. Conversely, tilapia performance was negatively impacted in
chemoautotrophic systems due to low organic matter loads [
8
]. However, limited informa-
Fishes 2024,9, 191 3 of 15
tion exists regarding macroalgae performance within these systems. High concentrations
of solids produced by heterotrophic bacteria may accumulate on macroalgae, hindering
photosynthesis and affecting their performance [
25
]. Additionally, elevated nutrient con-
centrations present in the chemoautotrophic system can induce stress in macroalgae and
trigger reproductive events [26]. Choosing the right cultivation system can provide better
growing conditions and biomass production for the macroalgae. In addition to growth
and nutrient absorption, the specific physical and chemical variables inherent to each
cultivation system also influence the nutritional composition of macroalgae [
27
]. The pro-
duction of biomass with enhanced nutritional value can offer economic advantages for the
system through the generation of valuable by-products. For instance, utilizing the biomass
of macroalgae cultivated in the integrated system as a food source for shrimp and fish
could prove beneficial for aquaculture [
2
]. Therefore, the objective of this study was to
evaluate the growth performance, nutrient absorption, and bioactive compounds of the
macroalga Ulva lactuca when cultivated in an integrated system with Pacific white shrimp
Penaeus vannamei and tilapia Oreochromis niloticus using two biofloc fertilization strategies:
a chemoautotrophic system and a heterotrophic system.
2. Materials and Methods
2.1. Location and Origin of the Animals
The experiment was conducted in an agricultural greenhouse situated at the Marine
Station of Aquaculture (EMA), Institute of Oceanography, Federal University of Rio Grande
(IO-FURG), located on Cassino Beach, Rio Grande, Rio Grande do Sul. The greenhouse
was devoid of shading, and aeration within the tanks was provided by a blower through
continuous air injection via micro-perforated hoses (aerotubes).
2.2. Animal Materials
The shrimp originated from a biofloc cultivation system within a grow-out greenhouse
at EMA, with an initial weight of 7.13
±
0.18 g. Tilapia were sourced from a recircu-
lation system grow-out cultivation, starting with an initial weight of 412.33
±
72.58 g.
The macroalgae were cultivated in a greenhouse in a 1 m
3
tank containing water with
35.1 ±2.74 mg L−1of nitrate and 2.24 ±1.2 mg L−1of phosphate.
2.3. Experimental Design
The experiment, which spanned 45 days, was conducted on six experimental produc-
tion systems. Each system comprised a 4 m
3
tank for shrimp (350 shrimp m
−2
), a
0.7 m3
tank for tilapia (10 fish per m
3
), and a 0.35 m
3
tank for macroalgae cultivation (0.1 g m
3
of the useful volume of the entire system). A submerged pump circulated the system,
transferring water into the macroalgae tank, which then flowed by gravity into the shrimp
tank before returning to the tilapia tank (Figure 1). The macroalgae were contained within
the tank using a circular structure with a diameter of 0.60 m positioned near the surface,
constructed from polyethylene netting with 5 mm mesh openings.
2.4. Treatments
Two treatments were employed, each with three replicates: chemoautotrophic—a
system utilizing chemical inorganic fertilization; heterotrophic—a system supplemented
with organic fertilizer. Inoculum preparation for the chemoautotrophic system involved
maintaining water with a salinity of 20 ppt in an 8 m
3
tank. Over 35 days, daily fertilization
with sodium nitrite (Neon Comercial, São Paulo, SP, Brazil) and ammonium chloride (Neon
Comercial, São Paulo, SP, Brazil) was conducted to achieve a concentration of 1 mg L
−1
for each compound in the water. To establish bacterial populations in the system, six
pillow-like structures containing biological media were placed in the main tank and then
distributed among the replicates. The tank was continuously aerated and devoid of light,
and no heaters were utilized to simulate greenhouse cultivation conditions.
Fishes 2024,9, 191 4 of 15
Fishes2024,9,xFORPEERREVIEW4of16
Figure1.Designoftheexperimentalsystem,consistingofashrimptank,afishtank,andamacroal-
gaetank,withwaterrecirculatingbetweenthem.
2.4.Treatments
Twotreatmentswereemployed,eachwiththreereplicates:chemoautotrophic—a
systemutilizingchemicalinorganicfertilization;heterotrophic—asystemsupplemented
withorganicfertilizer.Inoculumpreparationforthechemoautotrophicsysteminvolved
maintainingwaterwithasalinityof20pptinan8m3tank.Over35days,dailyfertilization
withsodiumnitrite(NeonComercial,SãoPaulo,SP,Brazil)andammoniumchloride
(NeonComercial,SãoPaulo,SP,Brazil)wasconductedtoachieveaconcentrationof1mg
L−1foreachcompoundinthewater.Toestablishbacterialpopulationsinthesystem,six
pillow-likestructurescontainingbiologicalmediawereplacedinthemaintankandthen
distributedamongthereplicates.Thetankwascontinuouslyaeratedanddevoidoflight,
andnoheaterswereutilizedtosimulategreenhousecultivationconditions.
Oncetheammoniaandnitriteconcentrationsstabilizedandwereconvertedintoni-
trate,theexperimentstarted.Chemoautotrophictreatmentreplicateswerepreparedby
blending40%inoculumwithwaterofsalinity20,uptoausefulvolumeof5m3.Atthe
onsetoftheexperiment,thewaterparameterswereasfollows:atemperatureof26.0±0.4
°C,dissolvedoxygenof7.2±0.5mgL–1,pHof8.18±0.3,alkalinityof170.0±2.0mg
CaCO3L–1,totalammonianitrogenof0.02±0.02mgL–1,nitriteof1.5±0.2mgL–1,nitrate
of64.0±1.7mgL–1,phosphateof1.2±0.4mgL–1,andtotalsuspendedsolidsof160.0±5.8
mgL–1.
Thetanksdesignatedfortheheterotrophictreatmentwerepreparedwith40%ma-
turebioflocinoculumandseawateratasalinityof20ppt,uptoausefulvolumeof5m3.
Thebioflocinoculumwassourcedfromashrimpcultivationsystemwithausefulvolume
of237m3,adensityof184shrimpm−2,andanaverageweightof7.1±1.2g,cultivatedfor
68days.Theinitialwaterqualityparametersintheshrimpproductiontankbeforethe
experimentwereasfollows:atemperatureof25.6°C,dissolvedoxygenof5.4mgL–1,pH
of7.47,alkalinityof215.0mgCaCO3L–1,totalammoniacalnitrogenof0.20mgL–1,nitrite
of0.20mgL–1,nitrateof147.0mgL–1,phosphateof4.0mgL–1,andtotalsuspendedsolids
of700.00mgL–1.
2.5.ChemicalandPhysicalWate rParameters
Waterqualityanalyseswereconductedonsamplescollectedfromtheshrimptanks,
consideringwaterhomogenizationduetothecirculationwithinthesystems.Temperature
anddissolvedoxygenweremeasuredtwicedailyusingaPro-20model(YSIInc.,Yell ow
Springs,OH,USA),andpHwasmeasureddailywithabenchpHmeter(Seven2GoS7
Basic,MelerToledo,SãoPaulo,SP,Brazil).Salinitywasassessedtwiceaweekusinga
Pro-20model(YSIInc.,OH,USA),and,ifnecessary,freshwaterwasaddedtomaintain
salinityat20.Alkalinity(mgCaCO3L–1)wasmonitoredtwiceaweekfollowingtheAPHA
Figure 1. Design of the experimental system, consisting of a shrimp tank, a fish tank, and a macroalgae
tank, with water recirculating between them.
Once the ammonia and nitrite concentrations stabilized and were converted into
nitrate, the experiment started. Chemoautotrophic treatment replicates were prepared by
blending 40% inoculum with water of salinity 20, up to a useful volume of 5 m
3
. At the onset
of the experiment, the water parameters were as follows: a temperature of
26.0 ±0.4 ◦C
,
dissolved oxygen of 7.2
±
0.5 mg L
−1
, pH of 8.18
±
0.3, alkalinity of 170.0
±
2. 0 mg
CaCO
3
L
−1
, total ammonia nitrogen of 0.02
±
0.02 mg L
−1
, nitrite of 1.5
±
0.2 mg L
−1
,
nitrate of 64.0
±
1.7 mg L
−1
, phosphate of 1.2
±
0.4 mg L
−1
, and total suspended solids of
160.0 ±5.8 mg L−1.
The tanks designated for the heterotrophic treatment were prepared with 40% mature
biofloc inoculum and seawater at a salinity of 20 ppt, up to a useful volume of 5 m
3
. The
biofloc inoculum was sourced from a shrimp cultivation system with a useful volume of
237 m
3
, a density of 184 shrimp m
−2
, and an average weight of 7.1
±
1.2 g, cultivated for
68 days. The initial water quality parameters in the shrimp production tank before the
experiment were as follows: a temperature of 25.6
◦
C, dissolved oxygen of 5.4 mg L
−1
, pH
of 7.47, alkalinity of 215.0 mg CaCO
3
L
−1
, total ammoniacal nitrogen of 0.20 mg L
−1
, nitrite
of 0.20 mg L
−1
, nitrate of 147.0 mg L
−1
, phosphate of 4.0 mg L
−1
, and total suspended
solids of 700.00 mg L−1.
2.5. Chemical and Physical Water Parameters
Water quality analyses were conducted on samples collected from the shrimp tanks,
considering water homogenization due to the circulation within the systems. Temper-
ature and dissolved oxygen were measured twice daily using a Pro-20 model (YSI Inc.,
Yellow Springs
, OH, USA), and pH was measured daily with a bench pH meter (Seven2Go
S7 Basic, Mettler Toledo, São Paulo, SP, Brazil). Salinity was assessed twice a week using a
Pro-20 model (YSI Inc., OH, USA), and, if necessary, fresh water was added to maintain
salinity at 20. Alkalinity (mg CaCO
3
L
−1
) was monitored twice a week following the APHA
methodology [
28
], with calcium hydroxide added to both treatments when alkalinity fell
below 150 mg CaCO3L−1, as per Furtado et al. [20] recommendation.
Total ammoniacal nitrogen (mg L
−1
) and nitrite (mg L
−1
) were initially measured daily
and then twice a week after nutrient stabilization, according to UNESCO methodology [
29
].
Nitrate (mg L
−1
) and phosphate (mg L
−1
) were measured twice a week, according to the
method proposed by Aminot and Chaussepied [
30
]. Total suspended solids (mg L
−1−
TSS)
and settleable solids (ml L
−1−
SS) were quantified twice a week, using the methodology
described by Baumgarten et al. [
31
] and APHA [
28
], respectively. For the heterotrophic
system, organic carbon (molasses) was added when the total ammoniacal nitrogen exceeded
1 mg L
−1
to promote nitrogen uptake through heterotrophic bacteria growth, as proposed
by Wasielesky et al. [
32
]. In the chemoautotrophic system, inorganic carbon (calcium
hydroxide) was added when ammonia and nitrite concentrations exceeded 1 mg L
−1
and
Fishes 2024,9, 191 5 of 15
5 mg L−1
, respectively. In this treatment, alkalinity was maintained at 300 mg CaCO
3
L
−1
for optimal nitrifying bacteria performance, as recommended by Furtado [20].
2.6. Macroalgae Growth and Biochemical Analysis
Macroalgae biomass was weighed every 15 days. Before the weighing process, the
macroalgae were gently shaken inside the holding structure to eliminate any solids adhering
to the surface. Subsequently, the circular holding structure was removed from the tank
and set aside to air-dry for 10 min to remove excess water before weighing. The initial
weight of macroalgae in each replicate was 502.7
±
0.5 g. After each weighing, the extra
macroalgae biomass was removed, ensuring that the initial weight of the macroalgae was
maintained. The following formula was used to calculate the macroalgae specific growth
rate (SGR) [33]:
SGR (% d−1): 100 ×[ln (final weight (g)/initial weight (g))/(final time −initial time)] (1)
The nutrient absorption efficiency (NRR) of the macroalgae was calculated using the
following formula [33]:
NRR (%): 100 ×[(nutrient concentration at initial time (mg L−1)−nutrient concentration
at final time (mg L−1))/nutrient concentration at initial time (mg L−1)] (2)
At the conclusion of the experiment, random samples of macroalgae were collected
from each replicate. Wet samples were weighed and then subjected to drying in an
oven at 60
◦
C for 24 h after obtaining the dry weight. To determine the concentration
of chlorophyll-a, chlorophyll-b, and carotenoids, 500 mg of the dry sample was macerated
and then incubated in 5 mL of methanol in the dark for 60 min at 4
◦
C. After that, the
solution was centrifuged (12,000
×
g, 10 min), and the supernatant was used to quantify
the pigments. The wavelengths of 664 and 647
η
m were used to calculate chlorophyll a
(
Chla = 11.75 ×A664 −2.35 ×A647
), chlorophyll b (Chlb = 18.61
×
A647
−
3.91
×
A664),
and carotenoids (Car = (1000
×
A470
−
2.27
×
Chla
−
81.4 Chlb)/227), according to the
methodology of Lichtenthaler & Wellburn [34].
Protein quantification was conducted using the Bradford method. An extract was
obtained from the dried macroalgae, following the protocol of Barbarino & Lourenço [
35
],
with the addition of 1 mL of sodium hydroxide and centrifugation. The extract and TCA
(25%) were added in a ratio of 2.5:1 (v/v) to precipitate the protein and kept in an ice-cold
bath for 30 min. The solution was then centrifuged and washed with dilutions of TCA
(10 and 5%), removing the supernatant, until the protein pellet was formed. To the pellet
suspension, 0.5 mL of sodium hydroxide (0.1 N) was added, and 20
µ
L of the solution was
combined with 1 mL of the total protein kit for the final analysis procedure.
2.7. Feed Management and Performance of the Animals
Shrimp were fed twice daily with 1.6 mm feed (Guabi aqua QS 1–2 mm, Guabi
Nutrition and Animal Health S.A., Campinas, São Paulo, SP, Brazil), and weekly biometrics
were conducted to adjust feed quantities following the method proposed by Jory et al. [
36
].
The tilapia were fed twice a day with commercial feed containing 40% protein (Guabi Tech,
Guabi Nutrition and Animal Health S.A., Campinas, São Paulo, SP, Brazil) at a rate of
1% of the biomass to encourage biofloc consumption. To evaluate shrimp performance,
measurements were taken at the beginning, middle, and end of the experiment. Fish
biometrics were conducted at the beginning and end of the experiment. The animals’
performance was assessed using the following formulas:
−Final average weight (g): final biomass of live animals (g)/total number of animals;
−Weekly weight gain (g week−1): weight gain (g)/number of weeks;
−Final biomass (g): ∑final weight of all live animals (g);
−Feed conversion rate (FCR) = ∑feed offered (g)/(biomass gain (g));
−Survival (%) = (final number of animals/initial number of animals) ×100;
Fishes 2024,9, 191 6 of 15
−Yield (kg m−3): (final biomass (kg)/tank volume (m3);
−
Weight gain rate (%) = 100
×
[(final mean weight
−
initial mean weight)/initial
mean weight]
.
2.8. Statistical Analysis
The data mean (
±
standard deviation) values are presented in Tables 1–3. Data nor-
mality and homoscedasticity were assessed using the Shapiro–Wilk and Levene tests,
respectively. Upon meeting these assumptions, a Student’s t-test was employed to compare
treatment differences. In cases where the assumptions of the Student’s t-test were not met,
the non-parametric Kruskal–Wallis test was utilized. Additionally, a one-way ANOVA
followed by a Tukey post-hoc test was conducted to evaluate nitrate and phosphate con-
centrations over time in each treatment. A significance level of 5% (p
≤
0.05) was applied
to all analyses. The tests were carried out using the PAST 4.03 2020 software [37].
3. Results
3.1. Physical and Chemical Parameters
During the 45-day trial period, there were significant differences (p< 0.05) observed in
pH, alkalinity, and calcium hydroxide consumption between the treatments. The chemoau-
totrophic system exhibited the highest values, along with higher consumption of calcium
hydroxide (Table 1).
Regarding nutrient levels, the chemoautotrophic system demonstrated higher concen-
trations of ammonia and nitrite, reaching maximums of 3.1 and 20.0 mg L
−1
, respectively.
Conversely, the heterotrophic system exhibited higher concentrations of total suspended
solids and settleable solids (Table 1). Significant nitrate and phosphate removal (p< 0.05)
was observed in both treatments, although the heterotrophic treatment displayed a higher
phosphate removal rate compared to the chemoautotrophic system.
Table 1. Water quality parameters (mean
±
standard deviation) (maximum–minimum) of chemoau-
totrophic and heterotrophic biofloc systems during the 45 days of integrated cultivation of Ulva
lactuca with Oreochromis niloticus and Penaeus vannamei.
Treatments
Parameters Chemoautotrophic Heterotrophic
Temperature (◦C) 22.82 ±0.30 (27.4–15.5) 22.67 ±0.38 (27.3–14.9)
DO (mg L −1)7.06 ±0.04 (9.9–5.3) 6.96 ±0.06 (9.7–5.2)
pH 8.15 ±0.02 a(8.9–7.7) 7.89 ±0.04 b(8.1–7.5)
Salinity (g L−1)20.20 ±0.26 (22.1–19.1) 21.83 ±0.76 (23.3–20.3)
Alkalinity (mg CaCO3L−1)280.00 ±8.19 a(365.0–155.0) 189.58 ±7.02 b(230.0–150.0)
TAN (mg L−1)0.95 ±0.07 b(3.1–0.0) 0.18 ±0.08 a(1.9–0.0)
N—Nitrite (mg L−1)7.96 ±1.16 b(20.0–0.0) 0.86 ±0.60 a(5.2–0.0)
N—Nitrate (mg L−1)26.41 ±2.72 (68.0–10.0) 26.04 ±4.03 (75.0–15.0)
P—Phosphate (mg L−1)1.04 ±0.25 (2.0–0.3) 1.19 ±0.21 (2.2–0.4)
SS (ml L−1)0.39 ±0.21 a(3.0–0.0) 8.44 ±2.91 b(15.0–3.0)
TSS (mg L−1)189.22 ±26.02 a(270.0–70.0) 335.38 ±47.92 b(452.5–175.0)
Calcium hydroxide (g L−1)#0.29 ±0.02 b(0.32–0.28) 0.08 ±0.03 a(0.10–0.04)
Water exchange (m−3)&2.0 ±2.0 a0.0 ±0.0 a
Removal rate
Nitrate (%) 56.47 ±4.93 57.00 ±7.00
Phosphate (%) 47.75 ±4.75 b56.14 ±1.14 a
DO (dissolved oxygen); TAN (total ammonium nitrogen); SS (settleable solids); TSS (total suspended solids).
#Use
of calcium hydroxide during cultivation.
&
Volume of water used for renovations. Different letters in the
same line represent significant differences (p≤0.05) between treatments after Student’s t-test.
Over the weeks of cultivation, there was a reduction in the concentration of nitrate
and phosphate (Figures 2and 3). The highest nitrate concentrations were observed at
the beginning of cultivation, with a decrease from day 14 onward in both treatments
(Figure 2). Similarly, phosphate concentrations were higher during the initial week, after
Fishes 2024,9, 191 7 of 15
which they stabilized in the heterotrophic treatment. In contrast, the chemoautotrophic
treatment exhibited a decrease in phosphate concentration until the first week, followed by
stabilization and a subsequent increase in the final week (Figure 3).
Fishes2024,9,xFORPEERREVIEW7of16
TAN(mgL−1)0.95±0.07b(3.1–0.0)0.18±0.08a(1.9–0.0)
N—Nitrite(mgL−1)7.96±1.16b(20.0–0.0)0.86±0.60a(5.2–0.0)
N—Nitrate(mgL−1)26.41±2.72(68.0–10.0)26.04±4.03(75.0–15.0)
P—Phosphate(mgL−1)1.04±0.25(2.0–0.3)1.19±0.21(2.2–0.4)
SS(mlL−1)0.39±0.21a(3.0–0.0)8.44±2.91b(15.0–3.0)
TSS(mgL−1)189.22±26.02a(270.0–70.0)335.38±47.92b(452.5–175.0)
Calciumhydroxide(gL−1)#0.29±0.02b(0.32–0.28)0.08±0.03a(0.10–0.04)
Waterexchange(m−3)&2.0±2.0a0.0±0.0a
Removalrate
Nitrate(%)56.47±4.9357.00±7.00
Phosphate(%)47.75±4.75b56.14±1.14a
DO(dissolvedoxygen);TAN(totalammoniumnitrogen);SS(seleablesolids);TSS(totalsuspended
solids).#Useofcalciumhydroxideduringcultivation.&Volumeofwaterusedforrenovations.Dif-
ferentleersinthesamelinerepresentsignificantdifferences(p≤0.05)betweentreatmentsafter
Student’st-test.
Overtheweeksofcultivation,therewasareductionintheconcentrationofnitrate
andphosphate(Figures2and3).Thehighestnitrateconcentrationswereobservedatthe
beginningofcultivation,withadecreasefromday14onwardinbothtreatments(Figure
2).Similarly,phosphateconcentrationswerehigherduringtheinitialweek,afterwhich
theystabilizedintheheterotrophictreatment.Incontrast,thechemoautotrophictreat-
mentexhibitedadecreaseinphosphateconcentrationuntilthefirstweek,followedby
stabilizationandasubsequentincreaseinthefinalweek(Figure3).
Figure2.Averageweeklynitrateconcentrations(mgL−1)duringtheexperimentalperiodinthe
chemoautotrophic(chemicalfertilizationpriortostocking)andheterotrophic(useofaninoculum
fromanongoingbioflocshrimpcultivation)treatmentsinanintegratedcultivationofUlvalactuca
withOreochromisniloticusandPenaeusvannamei.Capitalleersshowdifferencesbetweenthechemo-
autotrophictreatmentsovertime.Lowercaseleersshowstatisticaldifferencesovertimeinthema-
turetreatment.
Figure 2. Average weekly nitrate concentrations (mg L
−1
) during the experimental period in the
chemoautotrophic (chemical fertilization prior to stocking) and heterotrophic (use of an inoculum
from an ongoing biofloc shrimp cultivation) treatments in an integrated cultivation of Ulva lac-
tuca with Oreochromis niloticus and Penaeus vannamei. Capital letters show differences between the
chemoautotrophic treatments over time. Lowercase letters show statistical differences over time in
the mature treatment.
Fishes2024,9,xFORPEERREVIEW8of16
Figure3.Weeklyaveragephosphateconcentrations(mgL−1)duringtheexperimentalperiodinthe
chemoautotrophic(chemicalfertilizationpriortostocking)andheterotrophic(useofaninoculum
fromanongoingbioflocshrimpcultivation)treatmentsinanintegratedcultivationofUlvalactuca
withOreochromisniloticusandPenaeusvannamei.Capitalleersshowdifferencesbetweenthechemo-
autotrophictreatmentsovertime.Lowercaseleersshowstatisticaldifferencesovertimeinthema-
turetreatment.
Totalsuspendedsolidsexhibitedsignificantdifferences(p<0.05)betweentreatments
duringmostoftheexperimentalweeks.Highconcentrationsofsolidsreaching452mgL−1
wereobservedintheheterotrophictreatment,incontrasttomaximumconcentrationsof
270mgL−1inthechemoautotrophictreatment(Figure4).
Figure4.Averageweeklyconcentrationsoftotalsuspendedsolids(mgL−1)duringtheexperimental
periodinthechemoautotrophic(chemicalfertilizationpriortostocking)andheterotrophic(useof
aninoculumfromanongoingbioflocshrimpcultivation)treatmentsinanintegratedcultivationof
UlvalactucawithOreochromisniloticusandPenaeusvannamei.Anasterisk(*)meansastatisticaldif-
ferenceonthesamedaybetweentreatments.
3.2.MacroalgaeGrowthandBiochemicalAnalysis
Therewasanincreaseinmacroalgaebiomassinbothtreatments,withhighercon-
centrationsofproteininthemacroalgaetissueintheheterotrophictreatment(p<0.05).
Figure 3. Weekly average phosphate concentrations (mg L
−1
) during the experimental period
in the chemoautotrophic (chemical fertilization prior to stocking) and heterotrophic (use of an
inoculum from an ongoing biofloc shrimp cultivation) treatments in an integrated cultivation of Ulva
lactuca with Oreochromis niloticus and Penaeus vannamei. Capital letters show differences between the
chemoautotrophic treatments over time. Lowercase letters show statistical differences over time in
the mature treatment.
Total suspended solids exhibited significant differences (p< 0.05) between treatments
during most of the experimental weeks. High concentrations of solids reaching 452 mg L
−1
were observed in the heterotrophic treatment, in contrast to maximum concentrations of
270 mg L−1in the chemoautotrophic treatment (Figure 4).
Fishes 2024,9, 191 8 of 15
Fishes2024,9,xFORPEERREVIEW8of16
Figure3.Weeklyaveragephosphateconcentrations(mgL−1)duringtheexperimentalperiodinthe
chemoautotrophic(chemicalfertilizationpriortostocking)andheterotrophic(useofaninoculum
fromanongoingbioflocshrimpcultivation)treatmentsinanintegratedcultivationofUlvalactuca
withOreochromisniloticusandPenaeusvannamei.Capitalleersshowdifferencesbetweenthechemo-
autotrophictreatmentsovertime.Lowercaseleersshowstatisticaldifferencesovertimeinthema-
turetreatment.
Totalsuspendedsolidsexhibitedsignificantdifferences(p<0.05)betweentreatments
duringmostoftheexperimentalweeks.Highconcentrationsofsolidsreaching452mgL−1
wereobservedintheheterotrophictreatment,incontrasttomaximumconcentrationsof
270mgL−1inthechemoautotrophictreatment(Figure4).
Figure4.Averageweeklyconcentrationsoftotalsuspendedsolids(mgL−1)duringtheexperimental
periodinthechemoautotrophic(chemicalfertilizationpriortostocking)andheterotrophic(useof
aninoculumfromanongoingbioflocshrimpcultivation)treatmentsinanintegratedcultivationof
UlvalactucawithOreochromisniloticusandPenaeusvannamei.Anasterisk(*)meansastatisticaldif-
ferenceonthesamedaybetweentreatments.
3.2.MacroalgaeGrowthandBiochemicalAnalysis
Therewasanincreaseinmacroalgaebiomassinbothtreatments,withhighercon-
centrationsofproteininthemacroalgaetissueintheheterotrophictreatment(p<0.05).
Figure 4. Average weekly concentrations of total suspended solids (mg L
−1
) during the experimental
period in the chemoautotrophic (chemical fertilization prior to stocking) and heterotrophic (use of an
inoculum from an ongoing biofloc shrimp cultivation) treatments in an integrated cultivation of Ulva
lactuca with Oreochromis niloticus and Penaeus vannamei. An asterisk (*) means a statistical difference
on the same day between treatments.
3.2. Macroalgae Growth and Biochemical Analysis
There was an increase in macroalgae biomass in both treatments, with higher con-
centrations of protein in the macroalgae tissue in the heterotrophic treatment (p< 0.05).
However, no significant differences (p
≥
0.05) were found in biomass gain, chlorophyll-a,
chlorophyll-b, or carotenoids between the treatments (Table 2).
Table 2. Performance and biochemistry of the macroalgae (mean
±
standard deviation) in the
chemoautotrophic and heterotrophic treatments at the end of 45 days of an integrated culture of Ulva
lactuca with Oreochromis niloticus and Penaeus vannamei.
Treatments
Chemoautotrophic Heterotrophic
Initial mean weight (g–FW) 502.76 ±0.65 502.64 ±0.44
Biomass gain (g–FW) 964.63 ±290.57 708.11 ±141.70
Protein (%) 15.13 ±0.56 b18.49 ±0.56 a
Chlorophyll-a(mg g−1)2.20 ±0.02 2.18 ±0.06
Chlorophyll-b(mg g−1)3.28 ±0.02 3.26 ±0.08
Carotenoids (mg g−1)0.06 ±0.00 0.04 ±0.02
Different letters in the same line represent significant differences (p
≤
0.05) between treatments after
Student’s t-test.
The specific growth rate indicates that the growth of macroalgae in the chemoau-
totrophic treatment remained consistent throughout the entire experiment, with no signifi-
cant difference (p
≥
0.05) observed between weighings. In contrast, for the heterotrophic
treatment, the highest growth rate was recorded on day 21, followed by a subsequent
decrease in growth rate. Notably, a significant difference in growth rate was only detected
in the last weighing among the treatments (Figure 5).
3.3. Performance of the Animals
Shrimp performance remained unaffected by the different biofloc strategies, with no
discernible differences observed between treatments. However, the performance of the fish
exhibited a significant difference (p< 0.05) between treatments, with a higher final weight,
weight gain, and weight gain rate recorded in the heterotrophic treatment compared to the
chemoautotrophic treatment (Table 3).
Fishes 2024,9, 191 9 of 15
Fishes2024,9,xFORPEERREVIEW9of16
However,nosignificantdifferences(p≥0.05)werefoundinbiomassgain,chlorophyll-a,
chlorophyll-b,orcarotenoidsbetweenthetreatments(Table2).
Tab le2.Performanceandbiochemistryofthemacroalgae(mean±standarddeviation)inthechemo-
autotrophicandheterotrophictreatmentsattheendof45daysofanintegratedcultureofUlvalac‐
tucawithOreochromisniloticusandPenaeusvannamei.
Treatments
ChemoautotrophicHeterotrophic
Initialmeanweight(g–FW)502.76±0.65502.64±0.44
Biomassgain(g–FW)964.63±290.57708.11±141.70
Protein(%)15.13±0.56b18.49±0.56a
Chlorophyll-a(mgg−1)2.20±0.02 2.18±0.06
Chlorophyll-b(mgg−1)3.28±0.023.26±0.08
Carotenoids(mgg−1)0.06±0.000.04±0.02
Differentleersinthesamelinerepresentsignificantdifferences(p≤0.05)betweentreatmentsafter
Student’st-test.
Thespecificgrowthrateindicatesthatthegrowthofmacroalgaeinthechemoauto-
trophictreatmentremainedconsistentthroughouttheentireexperiment,withnosignifi-
cantdifference(p≥0.05)observedbetweenweighings.Incontrast,fortheheterotrophic
treatment,thehighestgrowthratewasrecordedonday21,followedbyasubsequentde-
creaseingrowthrate.Notably,asignificantdifferenceingrowthratewasonlydetected
inthelastweighingamongthetreatments(Figure5).
Figure5.Macroalgaespecificgrowthrate(%day−1)inthechemoautotrophic(chemicalfertilization
priortostocking)andheterotrophic(useofaninoculumfromanongoingbioflocshrimpcultiva-
tion)treatmentsinanintegratedcultivationofUlvalactucawithOreochromisniloticusandPenaeus
vannamei.Anasterisk(*)meansastatisticaldifferenceonthesamedaybetweentreatments.Capital
leersmeandifferencesinthesametreatmentbetweensamplingdays.
3.3.PerformanceoftheAnimals
Shrimpperformanceremainedunaffectedbythedifferentbioflocstrategies,withno
discernibledifferencesobservedbetweentreatments.However,theperformanceofthe
fishexhibitedasignificantdifference(p<0.05)betweentreatments,withahigherfinal
weight,weightgain,andweightgainraterecordedintheheterotrophictreatmentcom-
paredtothechemoautotrophictreatment(Table3).
Figure 5. Macroalgae specific growth rate (% day
−1
) in the chemoautotrophic (chemical fertilization
prior to stocking) and heterotrophic (use of an inoculum from an ongoing biofloc shrimp cultivation)
treatments in an integrated cultivation of Ulva lactuca with Oreochromis niloticus and Penaeus vannamei.
An asterisk (*) means a statistical difference on the same day between treatments. Capital letters
mean differences in the same treatment between sampling days.
Table 3. Animal performance (mean
±
standard deviation) in the chemoautotrophic (chemical
fertilization prior to stocking) and heterotrophic (use of an inoculum from an ongoing biofloc
shrimp cultivation) treatments at the end of 45 days of an integrated cultivation of Ulva lactuca with
Oreochromis niloticus and Penaeus vannamei.
Treatments
Chemoautotrophic Heterotrophic
Shrimp
Final mean weight (g) 9.60 ±0.60 10.75 ±0.49
WWG (g week−1)#0.41 ±0.10 0.60 ±0.08
Final biomass (kg) 10.02 ±0.43 10.79 ±0.60
FCR ## 2.16 ±0.38 2.36 ±0.24
Survival (%) 84.71 ±4.50 84.08 ±5.22
Fish
Final mean weight (g) 447.55 ±1.05 b456.25 ±3.25 a
WWG (g week−1)#5.93 ±0.18 b7.38 ±0.55 a
Final biomass (kg) 2.89 ±0.54 2.98 ±0.25
FCR ## 1.03 ±0.10 1.18 ±0.02
Survival (%) 90.48 ±16.50 95.24 ±8.25
WGR (%) ### 8.63 ±0.25 b10.74 ±0.79 a
#
WGW (weekly weight gain);
##
FCR (food conversion rate);
###
WGR (weight gain rate). Lowercase letters mean
differences between treatments.
4. Discussion
It is widely acknowledged that the utilization of biofloc technology, compared to
conventional cultivation methods, leads to reduced water consumption and enhanced
control over water quality parameters [
16
]. The various fertilization strategies employed
in this experiment resulted in differences in water quality maintenance, organism perfor-
mance, water usage, and inputs. The chemoautotrophic system utilizes more inputs, such
as sodium hydroxide, compared to the heterotrophic system. This variance is necessary
to maintain optimal alkalinity values. In the chemoautotrophic system, the optimal func-
tioning of nitrifying bacteria occurs at values maintained at around 300 mg
CaCO3L−1
,
consequently resulting in higher pH values [
20
]. As a result, there is a more frequent
Fishes 2024,9, 191 10 of 15
application of sodium hydroxide to correct these values and stimulate the growth of nitri-
fying bacteria with higher alkalinity. Furthermore, differences in nutrient concentrations
were observed between the adopted systems, which play a crucial role in macroalgae
development. According to Messyasz et al. [
38
], most marine Ulva species thrive in envi-
ronments with high concentrations of ammonia or nitrate. Ammonia, originating from
waste feed and animal excretion, is the primary nitrogenous compound formed in the
system and can be lethal to cultivated organisms at low levels [
39
]. In both systems in this
study, an initial increase in ammonia concentration was observed due to animal stocking.
Wasielesky et al. [32]
suggest that the use of organic carbon fertilization could promote the
growth of heterotrophic bacteria in the system, which consume produced ammonia and
generate bacterial biomass. For the chemoautotrophic system, only inorganic fertilization
with calcium hydroxide was employed to encourage the growth of nitrifying bacteria with
higher alkalinity [
20
]. However, the slow establishment of nitrifying bacteria resulted in
maximum values of 3.1 mg L
−1
of ammonia in the system, which were higher than those
found in the heterotrophic system. Nevertheless, according to Lin & Chen [
39
], the values
obtained in our study were not toxic to the organisms.
The produced ammonia is oxidized into nitrite by ammonium-oxidizing bacteria and
subsequently into nitrate by nitrite-oxidizing bacteria. However, the observed increase in
nitrite levels in the chemoautotrophic treatment suggests that the nitrite-oxidizing bacteria
were not fully established in the system to facilitate this transformation. Despite the
use of artificial substrate in this experiment, it is likely that the bacterial population was
insufficient to oxidize the nitrite produced following the stocking of shrimp. The use of
artificial substrate in the system is necessary for bacterial adherence and to increase their
numbers [
40
]. According to Lin & Chen [
41
], the safe level for nitrite at a salinity of 25 is
15.2 mg L
−1
, and concentrations exceeding this limit can be lethal to shrimp. Consequently,
in our experiment, we carried out partial water exchange, and a reduction in shrimp and
fish feeding was necessary to control nitrite levels in the system, resulting in higher water
usage than in the heterotrophic system and the dilution of nutrients.
In biofloc systems, elevated concentrations of nitrate and phosphate are common
in long-term production due to low water exchange rates and high animal densities,
providing an advantageous environment for macroalgae development. Carneiro [
42
] noted
that when macroalgae inhabit eutrophicated environments, they tend to absorb significant
nutrient concentrations initially for storage, serving as a precautionary measure in case of
sudden nutrient depletion. Additionally, Hanisak et al. [
43
] suggested that a constant high
nitrogen availability in the environment does not necessarily result in increased removal,
as macroalgae nitrogen absorption capacity saturates quickly at high concentrations. This
phenomenon may have occurred in both treatments in our study, resulting in a substantial
reduction in nitrate and phosphate concentrations at the onset of cultivation. Following the
second week, nutrient stabilization occurred. It is documented that 57% of nitrogen is lost
from the water daily, with an increase over time [
44
], suggesting that the stabilization of
these nutrients in the experiment may be attributed to the continuous absorption carried out
by the macroalgae. Studies such as Massocato et al. [
45
] have demonstrated that 85% of the
nitrate from a fish cultivation was absorbed within the first five days of algae cultivation.
Phosphorus is also another compound accumulated in the system and produced daily
through waste feed [
44
]. It is an important element in photosynthesis and the transfer of
energy from macroalgae [
46
], which shows the advantage of integrating macroalgae into
closed systems. Phosphorus absorption is connected with nitrogen absorption, with an ideal
ratio of 30:1 (nitrogen:phosphorus), so that phosphorus or nitrogen are not limiting [
47
].
The higher removal rate found in the heterotrophic treatment may be linked to the pH
values. According to Rathod et al. [
48
], higher phosphate absorption occurs at pH levels
below neutrality. The maintenance of high alkalinity and pH in the chemoautotrophic
treatment may have negatively impacted phosphate absorption.
The utilization of macroalgae as a biofilter has advanced due to their excellent per-
formance in nutrient absorption, ease of management, and high biomass production [
49
].
Fishes 2024,9, 191 11 of 15
Alencar et al. [
50
] demonstrated that the macroalgae Ulva lactuca absorbed 94% of the
ammonia concentration in an integrated cultivation with shrimp. Conversely, the impact
of the organic load generated in macroalgae cultivation remains poorly understood. Due
to the intensive production of bacterial biomass, the heterotrophic system in this study
exhibited higher concentrations of total suspended solids and settleable solids. In contrast,
the chemoautotrophic system, with its use of inorganic fertilizers and water exchange,
exhibited a lower organic load, with a maximum of 270 mg L
−1
. Similar outcomes were
reported by Ferreira et al. [
17
] in their study of the two biofloc systems. Despite the ab-
sence of a significant difference in macroalgae biomass gain between the treatments, a
higher growth rate was observed toward the end of cultivation in the chemoautotrophic
treatment, potentially attributable to the lower solids content in the system compared
to the heterotrophic system. The accumulation of microbial biomass and waste in the
heterotrophic system intensified toward the end of cultivation, likely directly affecting
macroalgae growth. Carvalho et al. [
51
] demonstrated that the presence of macroalgae in
the heterotrophic system led to solid deposition due to the formation of a physical barrier,
reducing light exposure for the macroalgae and consequently impacting their performance.
Despite the lower concentration of solids in the chemoautotrophic system, they still
accumulated on the surface of the macroalgae, representing one of the challenges of biofloc
systems. Studies like Resende et al. [
6
] reported significantly higher growth rates, with a
maximum growth rate of 15.33
±
2.87% day
−1
when macroalgae were cultivated freely in
tanks with fish farm effluent, characterized by minimal solids concentrations. The results
found in our experiment are in agreement with studies by Martins et al. [
52
], who observed
a growth rate of 3.0
±
0.6% day
−1
with the macroalga Ulva ohnoi in a biofloc system. Studies
with red algae in biofloc have also been carried out, showing a maximum growth rate of
1.19
±
0.04% day
−1
[
53
], similar to those observed in our heterotrophic treatment results
in the last weeks of cultivation. However, unlike studies such as Carvalho et al. [
25
] and
Legarda et al. [
14
], which did not observe macroalgae growth in biofloc systems, our use
of partial harvests might have reduced macroalgae density in the cultivation structure
and minimized shading, resulting in improved biomass production. Biancacci et al. [
54
]
showed that the use of partial harvests in the cultivation of the macroalga Macrocystis
pyrifera promoted greater biomass gain, a lower incidence of epiphytes, and a change in the
macroalgae biochemical composition.
In addition to serving as a bioremediator, macroalgae possess economic value, as the
biomass they produce can be utilized in the pharmaceutical and food industries [
55
], thereby
fostering sustainability and profitability in production. Macroalgae serve as vital sources
of nutrients and vitamins and possess antioxidant and immunostimulant properties [
56
].
The higher protein values observed in macroalgae from the heterotrophic system may be
attributed to reduced luminosity in the system due to the gradual accumulation of solids
over the cultivation period. Ganesan et al. [
57
] showed a correlation between high pigment
concentrations in low-light and salinity environments in their study on the macroalga
Ulva fasciata, indicating potential adaptations to environmental conditions. The observed
high values of chlorophyll-a and chlorophyll-b in our study compared to those reported
by
Silva et al. [58]
may be linked to the necessity of increasing pigment concentrations in
macroalgae to maximize photosynthesis, likely due to reduced light penetration caused by
suspended particles in a biofloc system. Similar trends were noted by Fillit et al. [
59
], who
reported increased pigment concentrations during periods of low light availability.
In the integrated system, all species must have productivity in cultivation and eco-
nomic potential [
60
]. Despite the elevated nitrite concentrations in the chemoautotrophic
system, shrimp and fish performance was not affected. However, growth outcomes and sur-
vival in both treatments were lower than those reported by Ferreira et al. [
17
] in their study
on shrimp cultivation in chemoautotrophic, heterotrophic, and mature systems. This can
be attributed to temperature differences between the studies. The minimum temperature
recorded in our experiment was 14.9
◦
C, directly impacting the survival of the organisms.
Furthermore, the overall average temperature in our study (22.0
◦
C) was lower compared
Fishes 2024,9, 191 12 of 15
to studies conducted with shrimp and fish [
61
], which also influenced the growth of the
animals due to their decreased metabolism. Fish performance in terms of weight gain was
superior in the heterotrophic system compared to the chemoautotrophic system, possibly
due to the higher availability of suspended organic matter. The reduced feed supply aimed
to induce floc consumption in the system, as demonstrated by Holanda et al. [
7
], with floc
serving as a supplementary food source for organisms [
62
]. Hence, the higher concentration
of total suspended solids in the heterotrophic system might have positively influenced
fish weight gain. Similar results were reported by Poli et al. [
8
], who observed lower fish
growth in an integrated system with chemoautotrophic floc.
The use of integrated multi-trophic systems aims to balance system productivity with
sustainability, ensuring that all organisms adapt to the cultivation conditions. According to
Khanjani et al. [
63
], the utilization of integrated systems has been consistently increasing,
highlighting potential species for inclusion in the system, with crustaceans being among
the most commonly produced target species. Zimmermann et al. [
64
] discuss the future of
tilapia production, emphasizing multitrophic cultivation and biofloc technology as promis-
ing systems for maximizing production, considering greater sustainability, biosecurity, and
increased density. However, the integration of macroalgae into biofloc systems has not
yet been fully stabilized. The inclusion of macroalgae in biofloc systems has presented
challenges due to their low productivity [
14
,
15
,
25
], but their role as bioremediators in
nutrient absorption has shown promise, as demonstrated by the data presented in this
study. Furthermore, the production of macroalgae biomass with an increase in nitrogen
content in tissues, as reported by Legarda et al. [
14
] and Carvalho et al. [
25
], adds value to
the product and enhances its applicability. The incorporation of macroalgae produced in
integrated systems into fish and shrimp feed has yielded significant results, as evidenced by
Marinho et al. [65]
and Valente et al. [
66
]. Improved methods for managing the incorpora-
tion of macroalgae into biofloc systems are needed to enhance production and sustainability
in intensive production systems.
5. Conclusions
The use of macroalgae in an integrated system with organic fertilization proved to be
viable for increasing biomass production and nitrate and phosphate absorption, improving
the system’s sustainability. The use of a system with a low concentration of solids, as in
the chemoautotrophic system, promoted better growth rates for the macroalgae. However,
the use of an inoculum from a heterotrophic system intensified the removal of phosphate
and nitrate and increased the protein content of the macroalgae. A better maintenance of
water quality was found in the heterotrophic system with the use of organic fertilization,
without the need for water renewal. Finally, the heterotrophic system contributed to the
better performance of the tilapia, with an increase in weight gain and a higher average
final weight.
Author Contributions: Conceptualization, A.C., J.C.Z., A.P.C., F.N.V., M.H.O., G.T. and L.H.P.;
data curation, A.C.; formal analysis, A.C. and H.B.; funding acquisition, L.H.P.; investigation, H.B.,
J.C.Z., A.P.C., F.N.V., M.H.O. and L.H.P.; methodology, A.C., H.B., A.P.C., F.N.V. and L.H.P.; project
administration, G.T. and L.H.P.; supervision, F.N.V., G.T. and L.H.P.; validation, A.C.; visualization,
J.C.Z., A.P.C., M.H.O., G.T. and L.H.P.; writing—original draft, A.C.; writing—review and editing,
H.B., J.C.Z., A.P.C., F.N.V., M.H.O., G.T. and L.H.P. All authors have read and agreed to the published
version of the manuscript.
Funding: This research was funded by the ASTRAL Project—H2020 grant agreement 863034.
Institutional Review Board Statement: All applicable international, national, and/or institutional
guidelines for the care and use of animals were followed by the authors. The experiment was ap-
proved by the Ethics and Animal Welfare Committee of FURG (Case number 23116.005895/
2016-42
).
Data Availability Statement: Data are contained within the article.
Fishes 2024,9, 191 13 of 15
Acknowledgments: Special thanks to the Brazilian Council of Research (CNPq), the Coordination
for the Improvement of Higher Level or Education Personnel (CAPES), and the Rio Grande do Sul
State Government. Luís H. Poersch and Felipe N. Vieira received a productivity research fellowship
from CNPq.
Conflicts of Interest: The authors declare no conflicts of interest.
References
1.
Gordalina, M.; Pinheiro, H.M.; Mateus, M.; da Fonseca, M.M.R.; Cesário, M.T. Macroalgae as Protein Sources—A Review on
Protein Bioactivity, Extraction, Purification and Characterization. Appl. Sci. 2021,11, 7969. [CrossRef]
2.
Wan, A.H.L.; Davies, S.J.; Soler-Vila, A.; Fitzgerald, R.; Johnson, M.P. Macroalgae as a Sustainable Aquafeed Ingredient. Rev.
Aquac. 2019,11, 458–492. [CrossRef]
3.
Chopin, T. Marine Aquaculture in Canada: Well-Established Monocultures of Finfish and Shellfish and an Emerging Integrated
Multi-Trophic Aquaculture (IMTA) Approach Including Seaweeds, Other Invertebrates, and Microbial Communities. Fisheries
2015,40, 28–31. [CrossRef]
4.
Queirós, A.S.; Circuncisão, A.R.; Pereira, E.; Válega, M.; Abreu, M.H.; Silva, A.M.S.; Cardoso, S.M. Valuable Nutrients from Ulva
rigida: Modulation by Seasonal and Cultivation Factors. Appl. Sci. 2021,11, 6137. [CrossRef]
5.
Troell, M.; Joyce, A.; Chopin, T.; Neori, A.; Buschmann, A.H.; Fang, J.G. Ecological Engineering in Aquaculture—Potential for
Integrated Multi-Trophic Aquaculture (IMTA) in Marine Offshore Systems. Aquaculture 2009,297, 1–9. [CrossRef]
6.
Resende, L.; Flores, J.; Moreira, C.; Pacheco, D.; Baeta, A.; Garcia, A.C.; Rocha, A.C.S. Effective and Low-Maintenance IMTA
System as Effluent Treatment Unit for Promoting Sustainability in Coastal Aquaculture. Appl. Sci. 2022,12, 398. [CrossRef]
7.
Holanda, M.; Wasielesky, W.; de Lara, G.R.; Poersch, L.H. Production of Marine Shrimp Integrated with Tilapia at High Densities
and in a Biofloc System: Choosing the Best Spatial Configuration. Fishes 2022,7, 283. [CrossRef]
8.
Poli, M.A.; Legarda, E.C.; de Lorenzo, M.A.; Pinheiro, I.; Martins, M.A.; Seiffert, W.Q.; do Nascimento Vieira, F. Integrated
Multitrophic Aquaculture Applied to Shrimp Rearing in a Biofloc System. Aquaculture 2019,511, 734274. [CrossRef]
9.
de Souza, R.L.; de Lima, E.C.R.; de Melo, F.P.; Ferreira, M.G.P.; de Souza Correia, E. The Culture of Nile Tilapia at Different
Salinities Using a Biofloc System. Rev. Cienc. Agron. 2019,50, 267–275. [CrossRef]
10.
Li, E.; Chen, L.; Zeng, C.; Chen, X.; Yu, N.; Lai, Q.; Qin, J.G. Growth, Body Composition, Respiration and Ambient Ammonia
Nitrogen Tolerance of the Juvenile White Shrimp, Litopenaeus vannamei, at Different Salinities. Aquaculture 2007,265, 385–390.
[CrossRef]
11.
Natori, M.M.; Sussel, F.R.; Santos, E.D.; Previero, T.D.C.; Viegas, E.M.M.; Gameiro, A.H. Desenvolvimento Da Carcinicultura
Marinha No Brasil e No Mundo: Avanços Tecnológicos e Desafios. Informações econômicas 2011,41, 61–73.
12. FAO. State of the World Fisheries and Aquaculture—2022 (SOFIA); FAO: Rome, Italy, 2022; ISBN 9789251072257.
13.
Brito, L.O.; Arantes, R.; Magnotti, C.; Derner, R.; Pchara, F.; Olivera, A.; Vinatea, L. Water Quality and Growth of Pacific White
Shrimp Litopenaeus vannamei (Boone) in Co-Culture with Green Seaweed Ulva lactuca (Linaeus) in Intensive System. Aquac. Int.
2014,22, 497–508. [CrossRef]
14.
Legarda, E.C.; da Silva, D.; Miranda, C.S.; Pereira, P.K.M.; Martins, M.A.; Machado, C.; de Lorenzo, M.A.; Hayashi, L.; do
Nascimento Vieira, F. Sea Lettuce Integrated with Pacific White Shrimp and Mullet Cultivation in Biofloc Impact System
Performance and the Sea Lettuce Nutritional Composition. Aquaculture 2021,534, 736265. [CrossRef]
15.
de Morais, A.P.M.; Santos, I.L.; Carneiro, R.F.S.; Routledge, E.A.B.; Hayashi, L.; de Lorenzo, M.A.; do Nascimento Vieira, F.
Integrated Multitrophic Aquaculture System Applied to Shrimp, Tilapia, and Seaweed (Ulva ohnoi) Using Biofloc Technology.
Aquaculture 2023,572, 739492. [CrossRef]
16.
Crab, R.; Avnimelech, Y.; Defoirdt, T.; Bossier, P.; Verstraete, W. Nitrogen Removal Techniques in Aquaculture for a Sustainable
Production. Aquaculture 2007,270, 1–14. [CrossRef]
17.
Ferreira, G.S.; Santos, D.; Schmachtl, F.; Machado, C.; Fernandes, V.; Bögner, M.; Schleder, D.D.; Seiffert, W.Q.; Vieira, F.N.
Heterotrophic, Chemoautotrophic and Mature Approaches in Biofloc System for Pacific White Shrimp. Aquaculture 2021,
533, 736099. [CrossRef]
18.
Ebeling, J.M.; Timmons, M.B.; Bisogni, J.J. Engineering Analysis of the Stoichiometry of Photoautotrophic, Autotrophic, and
Heterotrophic Removal of Ammonia-Nitrogen in Aquaculture Systems. Aquaculture 2006,257, 346–358. [CrossRef]
19.
Gaona, C.A.P.; de Almeida, M.S.; Viau, V.; Poersch, L.H.; Wasielesky, W. Effect of Different Total Suspended Solids Levels on a
Litopenaeus vannamei (Boone, 1931) BFT Culture System during Biofloc Formation. Aquac. Res. 2017,48, 1070–1079. [CrossRef]
20.
Furtado, P.S.; Poersch, L.H.; Wasielesky, W. Effect of Calcium Hydroxide, Carbonate and Sodium Bicarbonate on Water Quality
and Zootechnical Performance of Shrimp Litopenaeus vannamei Reared in Biofloc Technology (BFT) Systems. Aquaculture 2011,
321, 130–135. [CrossRef]
21.
Furtado, P.S.; Campos, B.R.; Serra, F.P.; Klosterhoff, M.; Romano, L.A.; Wasielesky, W. Effects of Nitrate Toxicity in the Pacific
White Shrimp, Litopenaeus vannamei, Reared with Biofloc Technology (BFT). Aquac. Int. 2015,23, 315–327. [CrossRef]
22. Macedo, C.F.; Sipaúba-Tavares, L. Eutrophication and Water Quality in Pisciculture: Consequences and Recommendations. Bol.
Do Inst. De Pesca 2010,36, 149–163.
Fishes 2024,9, 191 14 of 15
23.
Brandão, H.; Xavier, Í.V.; Santana, G.K.K.; Santana, H.J.K.; Krummenauer, D.; Wasielesky, W. Heterotrophic versus Mixed BFT
System: Impacts on Water Use, Suspended Solids Production and Growth Performance of Litopenaeus vannamei.Aquac. Eng. 2021,
95, 102194. [CrossRef]
24.
Krummenauer, D.; Samocha, T.; Poersch, L.; Lara, G.; Wasielesky, W. The Reuse of Water on the Culture of Pacific White Shrimp,
Litopenaeus vannamei, in BFT System. J. World Aquac. Soc. 2014,45, 3–14. [CrossRef]
25.
Carvalho, A.; de Oliveira Costa, L.C.; Holanda, M.; Poersch, L.H.; Turan, G. Influence of Total Suspended Solids on the Growth of
the Sea Lettuce Ulva lactuca Integrated with the Pacific White Shrimp Litopenaeus vannamei in a Biofloc System. Fishes 2023,8, 163.
[CrossRef]
26.
Copertino, M.D.S.; Tormena, T.; Seeliger, U. Biofiltering Efficiency, Uptake and Assimilation Rates of Ulva clathrata (Roth) J.
Agardh (Clorophyceae) Cultivated in Shrimp Aquaculture Waste Water. J. Appl. Phycol. 2009,21, 31–45. [CrossRef]
27.
Duke, C.S.; Litaker, W.; Ramus, J. Effects of the Temperature, Nitrogen Supply and Tissue Nitrogen on Ammonium Uptake Rates
of the Chlorophyte Seaweeds Ulva Curvata and Codium Decorticatum.J. Phycol. 1989,25, 113–120. [CrossRef]
28. American Public Health Association (APHA). Standard Methods for the Examination of Water and Waste Water, 16th ed.; American
Public Health Association: Washington, DC, USA, 1989.
29.
UNESCO. Chemical Methods for Use in Marine Environmental Monitoring; Intergovernmental Oceanographic Commission: Paris,
France, 1983.
30.
Aminot, A.; Chaussepied, M. Manuel Des Analyses Chimiques En Milieu Marin; Centre National pour L’exploitation des Océans:
Paris, France, 1983.
31.
Baumgarten, M.D.G.Z.; De Barros Rocha, J.M.; Niencheski, L.F.H. Manual de Análises Em Oceanografia Química; FURG: Rio Grande,
Brazil, 1996.
32.
Wasielesky, W.; Krummenauer, D.; Lara, G.; Fóes, G. Cultivo de Camarões Em Sistema de Bioflocos: Realidades e Perspectivas.
2013,15, 30–36. Revista ABCC.
33.
Loureiro, R.R.; Reis, R.P.; Critchley, A.T. In Vitro Cultivation of Three Kappaphycus alvarezii (Rhodophyta, Areschougiaceae) Vari-
ants (Green, Red and Brown) Exposed to a Commercial Extract of the Brown Alga Ascophyllum nodosum (Fucaceae, Ochrophyta).
J. Appl. Phycol. 2010,22, 101–104. [CrossRef]
34.
Lichtenthaler, H.K.; Wellburn, A.R. Determinations of Total Carotenoids and Chlorophylls a and b of Leaf Extracts in Different
Solvents. Biochem. Soc. Trans. 1983,11, 591–592. [CrossRef]
35.
Barbarino, E.; Lourenço, S.O. An Evaluation of Methods for Extraction and Quantification of Protein from Marine Macro- and
Microalgae. J. Appl. Phycol. 2005,17, 447–460. [CrossRef]
36.
Jory, D.E.; Cabrera, T.R.; Dugger, D.M.; Fegan, D.; Lee, P.G.; Lawrence, L.; Jackson, C.J.; Mcintosh, R.P.; Castañeda, J.I.; Al, B.; et al.
A Global Review of Shrimp Feed Management: Status and Perspectives. Aquaculture 2011,318, 104–152.
37.
Hammer, Ø.; David, A.; Harper, T.; Paul, D. Ryan National Knowledge Resource Consortium -a National Gateway of S&T on-Line
Resources for CSIR and DST Laboratories. Curr. Sci. 2013,105, 1352–1357.
38.
Messyasz, B.; Rybak, A. Abiotic Factors Affecting the Development of Ulva Sp. (Ulvophyceae; Chlorophyta) in Freshwater
Ecosystems. Aquat. Ecol. 2011,45, 75–87. [CrossRef]
39.
Lin, Y.C.; Chen, J.C. Acute Toxicity of Ammonia on Litopenaeus vannamei Boone Juveniles at Different Salinity Levels. J. Exp. Mar.
Bio. Ecol. 2001,259, 109–119. [CrossRef]
40.
De Lara, G.R.; Poersch, L.H.; Wasielesky, W., Jr. The Quantity of Artificial Substrates Influences the Nitrogen Cycle in the Biofloc
Culture System of Litopenaeus vannamei.Aquac. Eng. 2021,94, 102171. [CrossRef]
41.
Lin, Y.C.; Chen, J.C. Acute Toxicity of Nitrite on Litopenaeus vannamei (Boone) Juveniles at Different Salinity Levels. Aquaculture
2003,224, 193–201. [CrossRef]
42.
Carneiro, M.A.D.A. Estudo Do Crescimento, Eficiência de Biofiltração e Cinética de Absorção de Nutrientes (N-NH, N-NO e
P-PO4
3
) Da Macroalga Gracilaria cervicornis (Turner). Master ’s Thesis, Universidade Federal do Rio Grande do Norte, Rio Grande,
Brazil, 2007.
43.
Hanisak, M.D. The Use of Gracilaria tikvahiae (Gracilariales, Rhodophyta) as a Model System to Understand the Nitrogen Nutrition
of Cultured Seaweeds. Hydrobiologia 1990,204–205, 79–87. [CrossRef]
44.
Da Silva, K.R.; Wasielesky, W.; Abreu, P.C. Nitrogen and Phosphorus Dynamics in the Biofloc Production of the Pacific White
Shrimp, Litopenaeus vannamei.J. World Aquac. Soc. 2013,44, 30–41. [CrossRef]
45.
Massocato, T.F.; Robles-Carnero, V.; Moreira, B.R.; Castro-Varela, P.; Pinheiro-Silva, L.; da Silva Oliveira, W.; Vega, J.; Avilés, A.;
Bonomi-Barufi, J.; Rörig, L.R.; et al. Growth, Biofiltration and Photosynthetic Performance of Ulva Spp. Cultivated in Fishpond
Effluents: An Outdoor Study. Front. Mar. Sci. 2022,9, 981468. [CrossRef]
46.
Tsagkamilis, P.; Danielidis, D.; Dring, M.J.; Katsaros, C. Removal of Phosphate by the Green Seaweed Ulva lactuca in a Small-Scale
Sewage Treatment Plant (Ios Island, Aegean Sea, Greece). J. Appl. Phycol. 2010,22, 331–339. [CrossRef]
47.
Zirino, A.; Elwany, H.; Facca, C.; Maicu’, F.; Neira, C.; Mendoza, G. Nitrogen to Phosphorus Ratio in the Venice (Italy) Lagoon
(2001–2010) and Its Relation to Macroalgae. Mar. Chem. 2016,180, 33–41. [CrossRef]
48.
Rathod, M.; Mody, K.; Basha, S. Efficient Removal of Phosphate from Aqueous Solutions by Red Seaweed, Kappaphycus alverezii.J.
Clean. Prod. 2014,84, 484–493. [CrossRef]
Fishes 2024,9, 191 15 of 15
49.
Chopin, T.; Buschmann, A.H.; Halling, C.; Troell, M.; Kautsky, N.; Neori, A.; Kraemer, G.P.; Zertuche-González, J.A.; Yarish, C.;
Neefus, C. Integrating Seaweeds into Marine Aquaculture Systems: A Key toward Sustainability. J. Phycol. 2001,37, 975–986.
[CrossRef]
50.
de Alencar, J.R.; Junior, P.A.H.; Celino, J.J. Cultivo de Camarão Branco Litopenaeus vannamei (Boone, 1931) Com a Macroalga Ulva
lactuca Linneaus (Chlorophyta) No Tratamento de Efluentes Em Sistema Fechado de Recirculação. Rev. Biol. e Ciências da Terra
2010,10, 117–137.
51.
Carvalho, A.; de Oliveira Costa, L.C.; Holanda, M.; Gonçalves, M.; Santos, J.; Costa, C.S.B.; Turan, G.; Poersch, L.H. Growth of the
Macroalgae Ulva lactuca Cultivated at Different Depths in a Biofloc Integrated System with Shrimp and Fish. Phycology 2023,
3, 280–293. [CrossRef]
52.
Martins, M.A.; da SILVA, V.F.; Tarapuez, P.R.; Hayashi, L.; Vieira, F.D.N. Cultivation of the Seaweed Ulva Spp. With Effluent from
a Shrimp Biofloc Rearing System: Different Species and Stocking Density. Bol. do Inst. Pesca 2020,46, 1–6. [CrossRef]
53.
Pires, C.M.; Bazzo, G.C.; Barreto, P.L.M.; do Espírito Santo, C.M.; Ventura, T.F.B.; Pedra, A.G.L.M.; Rover, T.; McGovern, M.;
Hayashi, L. Cultivation of the Red Seaweed Kappaphycus alvarezii Using Biofloc Effluent. J. Appl. Phycol. 2021,33, 1047–1058.
[CrossRef]
54.
Biancacci, C.; Visch, W.; Callahan, D.L.; Farrington, G.; Francis, D.S.; Lamb, P.; McVilly, A.; Nardelli, A.; Sanderson, J.C.;
Schwoerbel, J.; et al. Optimisation of At-Sea Culture and Harvest Conditions for Cultivated Macrocystis pyrifera: Yield, Biofouling
and Biochemical Composition of Cultured Biomass. Front. Mar. Sci. 2022,9, 951538. [CrossRef]
55.
Mabeau, S.; Fleurence, J. Seaweed in Food Products: Biochemical and Nutritional Aspects. Trends Food Sci. Technol. 1993,
4, 103–107. [CrossRef]
56.
Peñalver, R.; Lorenzo, J.M.; Ros, G.; Amarowicz, R.; Pateiro, M.; Nieto, G. Seaweeds as a Functional Ingredient for a Healthy Diet.
Mar. Drugs 2020,18, 301. [CrossRef]
57.
Ganesan, M.; Mairh, O.P.; Eswaran, K.; Subba Rao, P.V. Effect of Salinity, Light Intensity and Nitrogen Source on Growth and
Composition of Ulva fasciata Delile (Chlorophyta, Ulvales). Indian J. Mar. Sci. 1999,28, 70–73.
58.
Silva, A.F.R.; Abreu, H.; Silva, A.M.S.; Cardoso, S.M. Effect of Oven-Drying on the Recovery of Valuable Compounds from Ulva
rigida,Gracilaria Sp. and Fucus vesiculosus.Mar. Drugs 2019,17, 90. [CrossRef] [PubMed]
59.
Fillit, M. Seasonal Changes in the Photosynthetic Capacities and Pigment Content of Ulva rigida in a Mediterranean Coastal
Lagoon. Bot. Mar. 1995,38, 271–280. [CrossRef]
60.
Buck, B.H.; Troell, M.F.; Krause, G.; Angel, D.L.; Grote, B.; Chopin, T. State of the Art and Challenges for Offshore Integrated
Multi-Trophic Aquaculture (IMTA). Front. Mar. Sci. 2018,5, 165. [CrossRef]
61.
Wyban, J.; Walsh, W.A.; Godin, D.M. Temperature Effects on Growth, Feeding Rate and Feed Conversion of the Pacific White
Shrimp (Penaeus vannamei). Aquaculture 1995,138, 267–279. [CrossRef]
62.
Ahmad, I.; Babitha Rani, A.M.; Verma, A.K.; Maqsood, M. Biofloc Technology: An Emerging Avenue in Aquatic Animal
Healthcare and Nutrition. Aquac. Int. 2017,25, 1215–1226. [CrossRef]
63.
Khanjani, M.H.; Zahedi, S.; Mohammadi, A. Integrated Multitrophic Aquaculture (IMTA) as an Environmentally Friendly System
for Sustainable Aquaculture: Functionality, Species, and Application of Biofloc Technology (BFT). Environ. Sci. Pollut. Res. 2022,
29, 67513–67531. [CrossRef] [PubMed]
64.
Zimmermann, S.; Kiessling, A.; Zhang, J. The Future of Intensive Tilapia Production and the Circular Bioeconomy without
Effluents: Biofloc Technology, Recirculation Aquaculture Systems, Bio-RAS, Partitioned Aquaculture Systems and Integrated
Multitrophic Aquaculture. Rev. Aquac. 2023,15, 22–31. [CrossRef]
65.
Marinho, G.; Nunes, C.; Sousa-Pinto, I.; Pereira, R.; Rema, P.; Valente, L.M.P. The IMTA-Cultivated Chlorophyta Ulva Spp. as a
Sustainable Ingredient in Nile Tilapia (Oreochromis niloticus) Diets. J. Appl. Phycol. 2013,25, 1359–1367. [CrossRef]
66.
Valente, L.M.P.; Araújo, M.; Batista, S.; Peixoto, M.J.; Sousa-Pinto, I.; Brotas, V.; Cunha, L.M.; Rema, P. Carotenoid Deposition,
Flesh Quality and Immunological Response of Nile Tilapia Fed Increasing Levels of IMTA-Cultivated Ulva spp. J. Appl. Phycol.
2016,28, 691–701. [CrossRef]
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