Aquaculture Research, 2017, 48, 41554164 doi:10.1111/are.

13235

Effect of the addition of Chaetoceros calcitrans,

Navicula sp. and Phaeodactylum tricornutum
(diatoms) on phytoplankton composition and growth
of Litopenaeus vannamei (Boone) postlarvae reared
in a biofloc system

Yllana Ferreira Marinho1, Luis Otavio Brito2, Clarissa Vilela Figueiredo da Silva Campos2,
William Severi2, Humber Agrelli Andrade2 & Alfredo Olivera Galvez2
1
Centro de Ci^encias Humanas, Naturais, Sa
ude e Tecnologia, Universidade Federal do Maranh~
ao, Pinheiro, Maranh~
ao,
Brazil
2
Departamento de Pesca e Aquicultura, Universidade Federal Rural de Pernambuco, Recife, Pernambuco, Brazil

Correspondence: Y F Marinho, Centro de Ci^encias Humanas, Naturais, Sa

ude e Tecnologia, Universidade Federal do Maranh~
ao,
Pinheiro, Maranh~
ao 65200-000, Brazil. E-mail: yllanamar@gmail.com

decreasing cyanobacteria and improving growth of

Abstract
The aim of this study was to evaluate the effect of
the addition of Chaetoceros calcitrans, Navicula sp.
and Phaeodactylum tricornutum (diatoms) on phyto-
plankton composition and the growth of Litope-
naeus vannamei postlarvae reared in a biofloc
system. Four treatments were used: BFT (biofloc
system without feed and no addition of diatoms);
BFT-F (biofloc system with feed and no addition of
diatoms); BFT-D (biofloc system with the addition
of diatoms and no feed); and BFT-FD (biofloc sys-
tem with the addition of feed and diatoms), all in
triplicate. The shrimp (16  0.02 mg) were
stocked at 2500 postlarvae m 3 and fed a com-
mercial feed. Diatoms were added on the 1st, 5th,
10th and 15th day at a density of 5 9 104
cells mL 1 for each species. No significant differ-
ences (P > 0.05) between treatments were
observed for gross primary production, net ecosys-
tem production and water column respiration rate.
However, significant differences (P < 0.05) were
observed for nitrite, orthophosphate, alkalinity,
final weight, weight gain, yield, feed conversion
ratio (FCR), phytoplankton and cyanobacteria
composition. The BFT-FD treatment had better per-
formance parameters for final weight (270 mg),
weight gain (254 mg), yield (0.67 Kg m 3) and
FCR (0.61), indicating the benefits of the diatoms
C. calcitrans, Navicula sp. and P. tricornutum for
L. vannamei postlarvae reared in biofloc systems.
Keywords: diatoms, water quality, cyanobacteria,
growth, survival
Introduction
Shrimp farming is considered a consolidated activ-
ity in the aquaculture sector, and the Pacific white
shrimp, Litopenaeus vannamei, is recognized as the
most cultivated species in the world, with a global
production of 3.66 million metric tons in 2014
(FAO 2016). However, production in eco-friendly
and pathogen-free conditions at low economic cost
is one of the biggest challenges for this activity
(Becerra-Dorame, Martnez-Porchas, Martnez-
Cordova, Rivas-Vega, Lopez-Elias & Porchas-
Cornejo 2012).
The biofloc system is a promising option for
aquaculture because its chemical, physical and
biological interactions stimulate the formation of
microbial flocs composed of a heterotrophic and
aerobic biota of microalgae, bacteria, ciliates,
cyanobacteria and organic detritus (Martnez-
Cordova, Emerenciano, Miranda-Baeza & Martnez-
Porchas 2014). The microbiota associated with
the bioflocs assimilate nitrogenous compounds,
allowing reutilizing the culture water in many
cycles (Avnimelech 2009), and the use of shrimp

2016 John Wiley & Sons Ltd 4155

Diatoms addition on shrimp reared in a biofloc system Y F Marinho et al. Aquaculture Research, 2017, 48, 41554164
feed with lower protein levels (Xu & Pan 2014),
thus decreasing the use of fishmeal (Valle, Dantas,
Silva, Bezerra, Correira, Peixoto & Soares 2015).
Moreover, bioflocs improve the shrimps digestive
enzyme activity (Anand, Kohli, Kumara, Sun-
daray, Roy, Venkateshwarlu, Sinha & Pailan
2014) and strengthen their immunological
response (Kim, Pang, Seo, Cho, Samocha & Jang
2014), and zootechnical performance (Xu, Pan,
Zhao & Huang 2012).
In biofloc systems, a healthy microbial commu-
nity must be developed and maintained (Ray,
Lewis, Browdy & Leffler 2010). Algae provide a
nutritional complement for shrimp, making nutri-
ents available for the growth of the bacterial com-
munity, and are a source of food for zooplankton,
which in turn serves as food for the shrimp
(Marinho, Brito, Silva, Santos & Galvez 2014).
Diatoms are the preferred group of microalgae
for feeding shrimp larvae and postlarvae, because
of their high concentration of essential amino
acids and polyunsaturated fatty acids (PUFA),
specifically those in the x-3 family: eicosapen-
taenoic and docosahexaenoic, which are critical
for shrimp growth and survival (Pi~ na, Voltolina,
Nieves & Robles 2006). However, the high con-
centration of suspended organic matter, which sig-
nificantly decreases light penetration in the water
column, thus reducing photosynthesis, inhibits
diatom growth in biofloc systems (Godoy, Ode-
brecht, Ballester, Martins & Wasielesky 2012).
In spite of the importance of diatoms in exten-
sive, semi-intensive, intensive and integrated cul-
ture systems (Elezuo 2011), their role in biofloc
systems is still not well understood, especially their
impact on water quality and shrimp zootechnical
characteristics. Therefore, this study aimed to eval-
uate the effect of the addition of Chaetoceros calci-
trans, Navicula sp. and Phaeodactylum tricornutum
(diatoms) on phytoplankton composition and the
growth of Litopenaeus vannamei postlarvae reared
in a biofloc system.
Materials and methods
A 20-day indoor trial was conducted at the Sus-
tainable Mariculture Laboratory (LAMARSU) of
the Department of Fisheries and Aquaculture
(DEPAq) of the Federal Rural University at Per-
nambuco (UFRPE), Recife, Brazil. The experimental
design was completely randomized with four treat-
ments: BFT (biofloc system without the addition of
4156 2016 John Wiley & Sons Ltd, Aquaculture Research, 48, 41554164
feed or diatoms); BFT-F (biofloc system with feed
and no addition of diatoms); BFT-D (biofloc system
with the addition of diatoms and no feed); and
BFT-FD (biofloc system with the addition of feed
and diatoms), each in triplicate.
Five days prior to stocking, water from an
indoor biofloc matrix tank (temperature 27C, pH
8.0, salinity 30 g L 1, total suspended solids
107 mg L 1 and settleable solids (SS) 0.5 mL L 1)
was mixed and distributed equally to fill twelve
rectangular black-plastic tanks (the useful dimen-
sions of the tanks were 50 9 35 9 23 cm equiva-
lent to 40 L) up to ~50% of the volume, with the
remaining volume filled with sea water (tempera-
ture 27C, pH 7.8, salinity 30 g L 1). The experi-
mental units were maintained under constant
aeration using three cylindrical airstones (diameter
2.4 cm and length 2.6 cm) per tank. There was
no water exchange during the experimental per-
iod, except for the weekly addition of dechlorinated
freshwater (2% by volume) to compensate for
evaporation losses. Light intensity was maintained
at ~ 1000 lux using a fluorescent lamp with a
12-h light/12-h dark regime.
Molasses (40% organic carbon) was added once
a day as an organic carbon source to maintain the
C:N ratio at 12:1, assuming that 50% of the feed
is organic carbon, and that 1 kg of the 40% crude
protein feed with 6.25%- N has 64 g of nitrogen,
268 g of organic carbon or 670 g molasses was
needed. In the treatments without feed addition,
molasses were added on the same amount calcu-
lated for the treatments with feed addition.
Hydrated lime (Ca(OH)2) was added at 10% (by
weight) of the daily feed allotment throughout the
study for maintaining the alkalinity at about
100 mg L 1 and pH 7.5 (Furtado, Gaona, Poersch
& Wasielesky 2013).
Specific pathogen-free postlarvae (PL15,
16  0.02 mg) of L. vannamei were obtained from
a commercial laboratory (Potipor~ a, PE, Brazil) and
stocked at a density of 2500 shrimp m 3 (100
shrimp per experimental unit 1). The postlarvae
were fed five times a day (at 0800, 1100, 1300,
1500 and 1800 h), with a commercial shrimp
feed (0.4 mm to 1 mm in diameter) with 40%
crude protein and 8% lipids (Aquavita Premiun,
Guaraves, Paraba, Brazil). The daily feeding rate
was 35% body weight at the start of the experi-
ment, gradually reduced to 20% body weight at
the end of the 20-day experiment based on the
Van Wyk table (1999).
Aquaculture Research, 2017, 48, 41554164 Diatoms addition on shrimp reared in a biofloc system Y F Marinho et al.
The benthic diatoms Chaetoceros calcitrans, Nav-
icula sp. and Phaeodactylum tricornutum were
obtained from LAMARSU-DEPAq-UFRPE and cul-
tured in a Conway medium (Walne 1966) in
water with 30 g L 1 salinity, pH 8.0, temperature
25  1C and light intensity kept at ~2000 lux
using a fluorescent lamp with a 12-h light/dark
photoperiod. Diatoms were added on days 1, 5, 10
and 15 of the experiment in the BFT-D and BFT-
FD tanks, at a concentration of 15 9 104 mL 1
(5 9 104 mL 1 for each diatom species), corre-
sponding to an addition of approximately 500 mL
of microalgae of each species to the tanks, regard-
less of the waste of unconsumed diatoms.
Dissolved oxygen and temperature were moni-
tored (YSI model 55, Yellow Springs, Ohio, USA)
twice a day (at 0800 and 1600 h). Salinity (YSI
model 30, Yellow Springs, Ohio, USA); pH (YSI
model 100, Yellow Springs, Ohio, USA); total ammo-
nia nitrogen (Koroleff 1976), nitrite nitrogen (Golter-
man, Clymo & Ohnstad 1978), nitrate (Mackereth,
Heron & Talling 1978), total suspended solids
(APHA 2005), orthophosphate (APHA 2005) and
alkalinity (mg L 1 CaCO3) (Felf oldy, Szabo & Tothl
1987) were recorded on days 1, 7, 14 and 20 of cul-
tivation.
The phytoplankton and cyanobacteria concen-
trations (cells mL 1) were recorded on days 1, 7,
14 and 20 of cultivation by methods described by
Pereira-Neto, Dantas, Galvez and Brito (2008), cal-
culated according to the following formula:
C = [(nm/nq) 9 1000]/F, where C is the phyto-
plankton or cyanobacteria concentration; nm is the
number of organisms found in the quadrants anal-
ysed in the Sedgewick-Rafter chamber; nq is the
number of quadrants analysed in the chamber;
1000 is the number of quadrants in the chamber;
and F is the dilution (50) correction factor.
The methods used to estimate microbial activity
were those described by Bratvold and Browdy
(1998, 2001) and Vinatea, Galvez, Browdy,
Stokes, Venero, Haveman, Lewis, Lawson, Shuler
and Leffler (2010). Once weekly, water from the
tanks was collected at a depth of 10 cm and
placed in triplicate sets of 300 mL glass bottles
(clear or black). Initial and final oxygen concentra-
tions were measured with an YSI 55 digital oxy-
gen meter (Yellow Spring Instruments, Yellow
Springs, OH, USA). Once initial oxygen was
recorded, the bottles were sealed with glass stop-
pers held in place by plastic lids of the same colour
as the bottle. The bottles were attached to a
2016 John Wiley & Sons Ltd, Aquaculture Research, 48, 41554164
rotating table and incubated for 2 h. Gross pri-
mary production, net ecosystem production and
water column respiration rate were recorded on
days 1, 4, 8, 12, 16 and 20 of cultivation by the
classic dark and light bottle method using the fol-
lowing formulae: gross primary production (mg
O2 L h 1) = final O2 of light bottle final O2 of
dark bottle/time (h); net ecosystem production (mg
O2 L h 1) = final O2 of light bottle initial O2 of
light bottle/time (h): water column respiration rate
(mg O2 L h 1) = initial O2 of dark bottle final O2
of dark bottle/time (h) (Strickland & Parsons
1972) and transformed for mg C m- h 1 accord-
ing to Wetzel and Likens (2000).
Once a week, 10 shrimp were randomly sam-
pled from each unit and individually weighed in a
digital balance (M523 Series Milligram Balances;
BEL Engineering, Italy) and put back to the tank.
Mean weights were used to determine shrimp
growth and adjust the amount of feed and organic
carbon offered. At the end of the trial, the total
shrimp biomass and the individual shrimp weights
from each tank were recorded for determining:
biomass gain (g) = final biomass (g) initial bio-
mass (g); final average weight (g) = final biomass
(g)/number of individuals at the end; FCR = feed
supplied/biomass gain; survival (%) = (number of
individuals at the end of the evaluation period/
initial number of individuals) 9 100; and yield
(kg m 3) = final biomass (kg)/volume of the exper-
imental unit (m3).
Water quality parameters, gross primary produc-
tion, net ecosystem production and water column
respiration rate were analysed by performing
repeated ANOVA measurements. A parametric one-
way ANOVA was used to analyse shrimp performance
parameters after confirming homoscedasticity
(Cochran P < 0.05) and normality (ShapiroWilk
P < 0.05). Tukeys test (P < 0.05) was performed
to compare and rank means from the three
treatments and the control. Nonparametric Kruskal
Wallis tests were used to check significant differ-
ences between the cell counts for each algal division
in the different treatments at specific moments
during the experiment. Data analyses were per-
formed using R Statistical software Version 3.1.1. R
Core Team 2014.
Results
There were no significant differences (P > 0.05) in
the water quality parameters for dissolved oxygen,
4157
Diatoms addition on shrimp reared in a biofloc system Y F Marinho et al. Aquaculture Research, 2017, 48, 41554164

temperature, pH, salinity, total ammonia nitrogen,
nitrite nitrogen, nitrate and total suspended solids
between the four treatments (Table 1). Significant
differences (P < 0.05) were observed for
orthophosphate and alkalinity (Table 1). Nitrite
nitrogen increased until day 14 in the BFT and
BFT-D treatments, while in the other treatments
(with the addition of feed), the increase lasted only
until day 7. The concentration of orthophosphate
tended to increase along the experiment. The dif-
ference in concentration became significant
between the BFT and BFT-D treatments at the end
of the experiment. Alkalinity tended to drop during
the experiment for BFT-D and BFT-FD treatments.
The phytoplanktonic community consisted of 11
genera at the beginning of the experiment and 24
genera at the end. The most frequent genera at the
beginning were Fragilaria, Rhabdonema (Heterokonto-
phyta) and Mychonastes (Chlorophyta). At the end,
the most frequent were Navicula, Diatoma, Phaeodacty-
lum (Heterokontophyta) and Mychonastes (Chloro-
phyta) (Table 2). There were significant differences
(P < 0.05) in the cyanobacterial count (cells mL 1)
between the four treatments (Table 2).
There were no significant differences (P > 0.05)
for gross primary productivity, net primary pro-
ductivity and water column respiration rate
(Table 3). The values for gross primary productiv-
ity and water column respiration rate were posi-
tive throughout the experiment, unlike net
primary productivity, which had negative values
after day 4 (Table 3).
The shrimp survival rates were all above 91%
during the 20-day experimental period and did not
differ significantly (P > 0.05) between treatments
(Table 4). However, significant differences
(P < 0.05) were observed for final weight, weight
gain and yield for the BFT (205 mg; 189 mg;
0.46 Kg m 3) and BFT-FD (270 mg; 254 mg;
0.67 Kg m 3) treatments respectively (Table 4).
Moreover, FCR in the BFT-FD was significantly
lower (P < 0.05) than in the BFT-F (Table 4).
Discussion
The concentrations of water quality parameters
(dissolved oxygen, temperature, salinity and pH) in
the culture water were within the ranges recom-
mended for marine shrimp cultivation (Van Wyk
& Scarpa 1999).
According to Ebeling, Timmons and Bisogni
(2006), there are three ways to convert nitrogen in
aquaculture systems: photoautotrophic removal by
algae and conversion by autotrophic and hetero-
trophic bacteria. Our study used water from a
matrix biofloc tank, where high concentrations of
total ammonia-nitrogen and nitrite nitrogen were
not observed, remaining within the range recom-
mended by Schuler, Boardman, Kuhn and Flick
(2010). The low concentrations of the total ammo-
nia-nitrogen and nitrite nitrogen compounds could
be attributed to the maturity of the biofloc system
used in our study and to the establishment of het-
erotrophic and nitrifying bacteria in the system.

Table 1 Water quality parameters during the culture (20 days) of Litopenaeus vannamei postlarvae reared in biofloc
system, with and without the addition of feed and/or diatoms

Treatments

Parameters BFT BFT-F BFT-D BFT-FD

Salinity (g L 1) 27  0.04a 27  0.21a 27  0.13a 27  0.45a

Temperature (C) 26  0.07a 26  0.16a 26  1.00a 26  1.00a
Dissolved oxygen (mg L 1) 6.6  0.16a 6.5  0.06a 6.7  0.15a 6.6  0.12a
pH 8.0  0.24a 8.0  0.05a 7.9  0.17a 7.7  0.05a
Total ammonia nitrogen (mg L 1) 0.35  0.37a 0.45  0.62a 0.23  0.33a 0.28  0.37a
Nitrite nitrogen (mg L 1) 0.34  0.29a 0.40  0.25ab 0.43  0.26ab 0.38  0.25ab
Nitrate-nitrogen 0.90  1.14a 2.92  2.33a 3.04  2.24a 3.02  2.62a
Alkalinity (mg CaCO3 L 1) 163.89  26.8a 158.94  28.5a 102.72  23.5b 96.81  33.7b
Orthophosphate (mg L 1) 1.63  0.47a 1.74  0.56ab 2.13  0.67b 2.19  0.63b
Total suspended solids (mg L 1) 143.95  62.5a 162.09  65.9a 137.36  63.2a 135.45  55.5a

Results from repeated ANOVA measures and Tukeys test. Mean values in the same row with different superscripts differ significantly
(P < 0.05); the data correspond to the mean  standard deviation; BFT, biofloc system without feed and no addition of diatoms;
BFT-F, biofloc system with feed and no addition of diatoms; BFT-D, biofloc system with the addition of diatoms and no feed; BFT-FD,
biofloc system with the addition of feed and diatoms.

4158 2016 John Wiley & Sons Ltd, Aquaculture Research, 48, 41554164
Aquaculture Research, 2017, 48, 41554164 Diatoms addition on shrimp reared in a biofloc system Y F Marinho et al.

Table 2 Phytoplankton and cyanobacteria composition during the culture of Litopenaeus vannamei postlarvae reared in
biofloc, with and without addition of feed and/or diatoms

Final

Division/Genera Initial BFT BFT-F BFT-D BFT-FD

1
Dinophyta (cells mL ) 0.00 4.62 3.46 4.62 1.15
Peridinium 0.00 4.62 3.46 4.62 1.15
Heterokontophyta (cells mL 1) 2889.54 27.70 63.48 219.30 522.85
Chaetoceros 0.00 1.15 2.31 32.31 58.86
Cymbella 0.00 3.46 0.00 0.00 1.15
Diatoma 0.00 2.31 0.00 0.00 116.57
Diploneis 380.88 0.00 0.00 0.00 0.00
Fragilaria 1019.73 0.00 2.31 0.00 2.15
Navicula 0.00 13.85 32.31 151.20 286.24
Orthoseira 363.57 0.00 0.00 0.00 0.00
Phaeodactylum 0.00 5.77 9.23 58.86 109.25
Pseudonitzschia 0.00 0.00 0.00 8.08 0.00
Rhizosolenia 3.46 1.15 8.08 1.15 3.46
Rhabdonema 1121.88 0.00 0.00 0.00 0.00
Synedra 0.0 0.00 9.23 0.00 4.62
Chlorophyta (cells mL 1) 1766.22 975.30 769.85 1608.96 1364.27
Botryococcus 0.00 0.00 69.25 0.00 0.00
Chlosterium 0.003 0.00 0.00 0.00 1.15
Chlorella 0.00 0.00 1.15 3.46 5.77
Haematococcus 2.86 0.00 0.00 0.00 0.00
Mychonastes 952.22 957.99 686.75 1604.34 1338.87
Pleurotaenium 3.46 17.31 12.70 1.15 17.31
Spirogyra 0.00 0.00 0.00 0.00 1.15
Spirotaenia 2.49 0.00 0.00 0.00 0.00
Staurodesmus 0.00 0.00 0.00 0.00 1.15
Ulothrix 578.25 0.00 0.00 0.00 0.00
Euglenophyta (cells mL 1) 146.01 8.08 8.08 0.00 6.93
Trachelomonas 146.01 8.08 8.08 0.00 6.93
Cyanobacteria (cells mL 1) 3790.86a 749.08c 1674.75b 190.44e 475.53d
Anabaena 72.71 3.46 0.00 0.00 0.00
Plectonema 463.56 0.00 0.00 0.00 0.00
Merismopedia 259.70 173.13 403.97 0.00 0.00
Oscillatoria 30 644.04 230.84 86.57 23.08 0.00
Schizothrix 5294.32 311.63 789.47 124.65 475.53

Mean values in the same row with different superscripts differ significantly (P < 0.05) by KruskalWallis tests. Abbreviations as in
Table 1.

Table 3 Mean values of gross and net primary productivity and respiration during the experimental period of
Litopenaeus vannamei postlarvae reared in biofloc system, with and without addition of feed and/or diatoms

Gross primary productivity Water column respiration rate Net primary productivity
mg C m 3 h 1 mg C m 3 h 1 mg C m 3 h 1

Days BFT BFT-D BFT-F BFT-FD BFT BFT-D BFT-F BFT-FD BFT BFT-D BFT-F BFT-FD

1 80.2 107.2 96.3 98.4 71.8 98.7 83.7 91.2 20.3 25.0 18.2 22.4
4 32.8 120.3 57.2 106.7 111.2 125.6 118.7 118.7 59.9 15.6 41.6 7.8
8 56.7 59.3 75.5 47.9 170.6 293.7 276.8 230.0 85.4 185.4 155.2 143.7
12 76.5 178.1 31.5 213.5 250.0 377.5 213.7 370.6 131.7 136.4 146.8 95.3
16 92.7 293.7 74.7 256.2 313.7 400.0 273.1 461.8 168.7 39.5 153.1 128.6
20 131.2 187.5 113.0 192.7 243.1 250.6 213.1 263.7 71.3 21.3 64.6 27.1

Abbreviations as in Table 1.

2016 John Wiley & Sons Ltd, Aquaculture Research, 48, 41554164 4159
Diatoms addition on shrimp reared in a biofloc system Y F Marinho et al. Aquaculture Research, 2017, 48, 41554164
Table 4 Shrimp performance during the culture (20 days) of Litopenaeus vannamei postlarvae reared in biofloc system,
with and without addition of feed and/or diatom
Final weight
Parameters/Treatments mg
BFT 205  28.9b
BFT-D 221  25.1ab
BFT-F 256  15.2ab
BFT-FD 270  26.4a
Results from repeated ANOVA measures and Tukeys test. Mean values in the same row with different superscripts differ significantly
(P < 0.05); the data correspond to the mean  standard deviation. Abbreviations as in Table 1.
During our study, there was an accumulation of
nitrate, which was the most predominant nitrogen
compound at the end of the experiment. Higher
concentrations of nitrate than total ammonia and
nitrite were reported by Brito, Chagas, Silva,
Soares, Severi and Galvez (2016). Nitrate is the
nitrogenous compound least harmful to shrimp,
and more than 60 mg L 1 would be required to
cause any harmful effect (Van Wyk & Scarpa
1999). Furtado, Campos et al. (2015) found that
nitrate concentrations up to 177 mg L 1 at salin-
ity of 23 g L 1 would be acceptable to L. vannamei
in a biofloc system.
The conversion from ammonia to nitrite and its
subsequent conversion into nitrate is one of the
processes that reduces alkalinity in a biofloc sys-
tem. Alkalinity reduction occurs due to the con-
sumption of inorganic carbon by the heterotrophic
and nitrifying bacteria that compose the bioflocs
(Ebeling et al. 2006). Alkalinity in bioflocs may
also be lower due to total suspended solids. Ray
et al. (2010) observed that after the removal of
suspended solids, the water changed from a brown
to green colour (due to the presence of an algal
community), which caused a rise in pH and alka-
linity levels due to photosynthesis. Even with the
introduction of microalgae in the BFT-D and BFT-
FD treatments, lower alkalinity was also observed.
Ebeling et al. (2006) recommended alkalinity of
around 100 and 150 mg CaCO3 L 1 in systems
without water exchange. However, Furtado, Poer-
sch et al. (2015) found satisfactory zootechnical
performance of L. vannamei in a biofloc system
using 75 mg L 1 CaCO3.
Orthophosphate concentrations rose during the
culture period, reaching a maximum of
2.19 mg L 1. Phosphate accumulation is attribu-
ted to excretion and decomposition of waste feed
(Penaflorida 1999). This accumulation does not
4160
Weight gain Yield Survival
3
mg Kg m % FCR
189  28.9b
0.46  0.09b
91.3  8.9a
205  25.1ab 0.52  0.04ab 94.0  5.5a
240  15.2ab 0.63  0.04ab 97.6  0.5a 0.64  0.05b
254  26.4a 0.67  0.05a 98.3  2.8a 0.61  0.02a
directly affect shrimp growth but causes a prolifer-
ation of cyanobacteria (Silva, Wasielesky & Abreu
2013). In this study, this accumulation may have
affected the increase in some phytoplankton com-
munity genera as microalgae metabolize nutrients
through photosynthesis by utilizing total ammo-
nia, nitrate and phosphate for growth and building
of the cellular structure (Xin, Hong-Ying, Ke &
Ying-Xue 2010). Some evidence of this was
reported in a study conducted by Magnotti, Lopes,
Derner and Vinatea (2016), which utilized the
effluent from the culture of L. vannamei in a biofloc
system as the culture medium for the production
of biomass of Chaetoceros muelleri, Nannochloropsis
oculata and Tetraselmis chuii. Increases in the
planktonic population in biofloc systems have also
been reported in many other studies (Brito, Vina-
tea, Soares, Severi, Miranda, Silva, Coimbra &
Galvez 2014; Marinho et al. 2014).
Cyanobacteria were the most abundant taxon at
the beginning of the experiment, but the concen-
tration of some genera (Oscillatoria and Schizothrix)
decreased drastically at the end, and other genera
(e.g. Merismopedia, Anabaena and Plectonema) were
no longer observed in the BFT-D and BFT-FD. The
diatoms C. calcitrans, Navicula sp. and P. tricornu-
tum probably inhibited the growth of cyanobacte-
ria due to nutrient competition for metabolic
processes. A decreasing abundance of cyanobacte-
ria in a system without water exchange and with
the introduction of diatoms was also observed by
Marinho et al. (2014). Cyanobacteria blooms can
cause lower dissolved oxygen levels as well as the
production of toxins that cause off-flavour, which
negatively affects the quality of the shrimp (Schra-
der, Green & Perschbacher 2011). While studying
the mortality of L. vannamei in a commercial aqua-
culture farm, Zimba, Camus, Allen and Burkholder
(2006) found that dead shrimp had traces of
2016 John Wiley & Sons Ltd, Aquaculture Research, 48, 41554164
Aquaculture Research, 2017, 48, 41554164 Diatoms addition on shrimp reared in a biofloc system Y F Marinho et al.
microcystin in their hepatopancreas, which was
caused by the presence of Microcystis aeruginosa
and Anabaena sp. in the shrimp cultivation ponds.
Perez-Linares, Cadena, Rangel, Unzueta-Busta-
mante and Ochoa (2003) observed that exposing
L. vannamei postlarvae to Schizothrix calciola
affected their final weight and caused gastrointesti-
nal lesions.
In this study, the net ecosystem production had
positive values (an oxygen surplus) at the begin-
ning of the experimental time with diatoms, but
these values declined and remained negative dur-
ing the experiment. In the systems tested, with
stimulus of natural productivity, the quantity of
suspended solids increased, thus reducing light
penetration and consequently decreasing photo-
synthesis levels (Vinatea et al. 2010). Using car-
bon sources with a C:N ratio above 10:1 promotes
the succession and dominance of bacteria over
microalgae (Avnimelech 2009).
The results obtained in this study show that bio-
mass gain and final weight were higher in the
BFT-FD treatment. The diatoms C. calcitrans, Navic-
ula sp. and P. tricornutum are highly used by
shrimp larvae and postlarvae because of their high
content of PUFA from the x-3 family (EPA- eicosa-
pentaenoic and DHA - docasahexaenoic), which
are crucial for good nutrition and tolerance to
stress. P. tricornutum contains 8.3 mg of DHA and
111.5 mg of EPA for each gram of its oil (Rycke-
bosch, Bruneel, Termote-Verhalle, Goiris, Muylaert
& Foubert 2014). Navicula sp. contains 82 g of
EPA and 22 g of DHA for each kilogram of total
fatty acids (Khatoon, Banerjee, Yusoff & Shariff
2009), and C. calcitrans contains 4.48 mg of EPA
for each kilogram of biomass (Seraspe, Gabotero,
De la Pe~ na, Pahila & Amar 2014). When Seraspe
et al. (2014) analysed the effect of diets supple-
mented with C. calcitrans to control infection of
Penaeus monodon by Vibrio harveyi, they observed
that supplementation improved shrimp resistance,
as it increased the immune system capacity and
the antibacterial action against infections, attribut-
ing this to the presence of the PUFA, linoleic and
EPA.
According to Godoy et al. (2012) and Marinho
et al. (2014), the introduction of diatoms to L. van-
namei cultivated in biofloc systems led to better
shrimp zootechnical performance. Ju, Forster, Con-
quest and Dominy (2008) registered higher
growth rates in L. vannamei fed with flocs that
contained phytoplankton (246 g Kg 1), composed
2016 John Wiley & Sons Ltd, Aquaculture Research, 48, 41554164
mostly by diatoms (82%) with some chlorophytes
(7.9%), because these microorganisms have good
levels of carotenoids, chlorophylls, phytosterols,
bromophenols and vitamins, which can improve
the bioflocs proximal composition.
The feed conversion ratio (FCR) of 0.61 in the
BFT-FD indicates that diatoms are an important
food source for shrimp postlarvae. The consump-
tion of microalgae and other organisms associated
to the bioflocs reduces the FCR according to Brito,
Santos, Abreu, Ara ujo, Severi and Galvez (2015),
who also found low FCR values in biofloc systems
with the addition of diatoms. The reduction of FCR
in biofloc systems occurs due to the recycling of
nutrients that are converted into microbial protein
(Perez-Fuentes, Perez-Rostro & Hern andez-Vergara
2013), which improves the activity of digestive
enzymes (Anand et al. 2014).
Currently, the production of microalgae in com-
mercial farms for use in nursery phase is still com-
plex due to the high cost of nutrients. Some
studies demonstrate the possibility of using efflu-
ents from the biofloc system for algae production
(Magnotti et al. 2016). The inoculation of these
microorganisms in the bioflocs can improve their
nutritional quality and is also an important tool in
the current situation of global and Brazilian
shrimp farming, mainly due to white spot syn-
drome virus. Because of this problem, the water
used in commercial nursery phase has been trea-
ted with high doses of chlorine, reducing the avail-
ability of the natural feed for the shrimp, given
that many are vectors of the virus. Inoculation
with beneficial non-contaminated micro-organ-
isms, such as microalgae, is a strategy for the ini-
tial production of shrimp, increasing the
zootechnical performance and strengthening the
immune system of the cultivated animal for the
various challenges of fattening.
In summary, the results indicate that the addi-
tion of diatoms Chaetoceros calcitrans, Navicula sp.
and Phaeodactylum tricornutum every five days pro-
vides an improved natural food source for the
shrimp (postlarvae), improving shrimp growth and
helps control potentially harmful cyanobacteria.
Future research is needed to evaluate the potential
use of other diatoms species and other densities
and frequencies to understand the potential role of
diatoms in shrimp nutrition in biofloc systems.
Moreover, the biofloc composition and shrimp
digestibility should be investigated in systems with
diatoms addition.
4161
Diatoms addition on shrimp reared in a biofloc system Y F Marinho et al. Aquaculture Research, 2017, 48, 41554164

Brito L.O., Chagas A.M., Silva E.P., Soares R.B., Severi

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