Aquaculture Research, 2017, 110
13294
Effect of inoculation of the cyanobacteria Oscillatoria
sp. on tilapia biofloc culture
Anselmo Miranda-Baeza1 opez1, Jos

, Mar
a de los A Mariscal-L

1 3
Martha E Rivas-Vega , Mauricio Emerenciano , Adolfo S
anchez-Romero1 &
1
Jos

e L Esquer-M

endez
1
Universidad Estatal de Sonora (UES) Unidad Navojoa, Navojoa, Sonora, Mexico
2
Departamento de Investigaciones Cient
ficas y Tecnol

ogicas de la Universidad de Sonora (DICTUS), Universidad de
Sonora, Hermosillo, Sonora, Mexico
3
Universidade do Estado de Santa Catarina (UDESC) Centro de Educaca
Catarina, Brazil
Correspondence: A Miranda-Baeza, Universidad Estatal de Sonora (UES) Unidad Navojoa, Carretera a Huatabampo, km 5,
Navojoa, Sonora 85800, M
exico. E-mail: anselmo.miranda@ues.mx
Abstract
Cyanophytes are the most ancient photosynthetic
organisms. During its evolution, they have devel-
oped various ecophysiological adaptation strategies
to survive in extreme conditions. The environment
prevailing under biofloc cultures provides various
conditions appropriate for cyanobacterial prolifera-
tion. An outdoor experiment (7 weeks) was per-
formed with a simple random design consisting of
four inoculation levels (by triplicate) of Oscillatoria
sp. (0.0, 0.1, 0.5 and 1 mg L
1) in saltwater bio-
floc. The objective was to evaluate the effect of the
cyanobacteria inoculation on water quality and tila-
pia production parameters. The results indicated
that the amount of Oscillatoria sp. inoculated signifi-
cantly affected water quality (pH, chlorophyll a, TSS
and NO3-N) and tilapia parameters (final weight,
feed conversion ratio, specific growth rate and sur-
vival). No significant effects on dissolved oxygen,
total ammonia nitrogen (TAN) or NO2-N were
observed. We recommend identifying the cyanobac-
teria species that are able to grow in a biofloc sys-
tem and their possible adverse effects on the system.
Keywords: biofloc, cyanobacteria, nitrogenous
compounds, Oscillatoria sp., tilapia culture
Introduction
Tilapia is one of the most cultivated fish in the
world. Several features have favoured their
2017 John Wiley & Sons Ltd
doi:10.1111/are.
opez-El
as2,
e A L

~o Superior da Regi~
ao Sul, CERES, Laguna, Santa
expansion, including its good taste and fast
growth. Tilapia can be grown in ponds and cages,
supports high stocking densities, resists adverse
environmental conditions, tolerates low oxygen
concentrations and uses the primary productivity
of ponds as food source (El-Sayed 2006). In addi-
tion to the biological and operational characteris-
tics that facilitate its growth, tilapia is widely
accepted in the markets of both developed and
developing countries (FAO 2014).
One of the problems associated with tilapia
farming in arid regions is the availability of fresh-
water; therefore, its cultivation in salt water and
biofloc could be one of the most viable alterna-
tives. Biofloc technology (BFT) allows operate with
minimal water exchange and reduces the use of
pelletized food. In a traditional culture, excess of
nutrients and organic matter must be discarded to
prevent the increase in ammonia and other toxic
compounds, whereas biofloc systems take advan-
tage of these components through stimulation of
microbial biomass, which is used as a live and per-
manent source of food (Ekasari, Crab & Verstraete
2010; L
opez-El
as, Moreno-Arias, Miranda-Baeza,
Mart
nez-C
ordova, Rivas-Vega & M
arquez-R
os
2015).
In biofloc systems, a highly diverse consortium
of unicellular organisms is present including auto-
trophs, heterotrophs, prokaryotes and eukaryotes
(Mart
nez-C
ordova, Mart
nez-Porchas, Emeren-
ciano, Miranda-Baeza & Gollas-Galv
an 2016). Sev-
eral authors have reported the presence of
1
Oscillatoria sp. on biofloc tilapia culture A Miranda-Baeza et al.
cyanobacteria in biofloc cultures (Ju, Forster, Con-
quest, Dominy, Kuo & Horgen 2008; Ballester,
Abreu, Cavalli, Emerenciano, De Abreu & Wasie-
lesky 2010; Becerra-D
orame, Mart
nez-C
ordova,
L
opez-El
as & Mart
nez-Porchas 2011). However,
the impact that a high concentration of these
micro-organisms may have on water quality and
productive parameters has not been assessed. To
know this, information is important, specially
because the cyanobacteria can grow under hetero-
trophic conditions, fix N2 and generate
cyanotoxins.
The genus Oscillatoria belongs to the class Oscil-
latoriales and division Cyanobacteria and is char-
acterized by long filamentous flattened cells
without a dark green mucilaginous sheath. It is
very common in microbial mats in both marine
and freshwater environments, especially in the
presence of high organic load (Fuentemayor, Jonte,
Rosales-Loaiza & Morales 2009).
Cyanobacteria are photosynthetic micro-organ-
isms with great morphological and structural vari-
ability; throughout evolution, they have developed
various ecophysiological adaptive strategies in
response to adverse environmental conditions
(L
opez-Rodas, Maneiro & Costas 2006). As a
result, cyanobacteria can be found in places with
extreme temperature, humidity, solar radiation,
pH and nutrient concentrations.
Cyanobacteria are opportunistic micro-organ-
isms. Specific conditions, such as nutrient avail-
ability, temperature and light intensity, can
promote the explosive growth of their populations
(Carpenter & McCarthy 1975). In addition, the
ability of cyanobacteria to adapt to growth in a
wide range of radiation levels is regulated by a
complex system of thylakoid components (Mura-
kami & Yoshihiko 1991).
Heterocysts in cyanobacterial cells are almost
colourless, have a thick wall and perform the func-
tion of molecular nitrogen fixation (N2), which
can occur under anaerobic conditions (Neuer,
Papen & Bothe 1983). In addition to species with
heterocysts, other species without these cells may
be able to fix N2, including the multicellular gen-
era Oscillatoria (Carpenter & McCarthy 1975) and
Microcoleus (Malin & Pearson 1988).
Cyanobacteria are micro-organisms associated
with toxin production. Cyanotoxins are a diverse
group of toxins with complex chemical structure
and varied toxicological effects. The type of toxin
produced depends on the cyanobacterial genus. In
2 2017 John Wiley & Sons Ltd, Aquaculture Research, 110
Aquaculture Research, 2017, 110
Oscillatoria, cyclic peptides and toxic alkaloids have
been identified (Sivonen & Jones 1999).
According to De Schryver, Crab, Defoirdt, Boon
and Verstraete (2008) and Mart
nez-C
ordova et al.
(2016), biofloc systems convert excess nutrients in
the microbial biomass. These nutrients are subse-
quently consumed by the cultured animals,
increasing the water residence time (Ekasari et al.
2010), and reducing costs from water
consumption.
Avnimelech (2007) evaluated the assimilation
of biofloc by tilapia and concluded that its compo-
nents can contribute to approximately 50% of the
protein requirement for tilapia. Azim and Little
(2008) evaluated tilapia (Oreochromis niloticus) cul-
tured in biofloc and determined that fish produc-
tion was 45% increased in the experimental tanks
compared with the control (traditional culture).
However, to contribute to the qualitative nutri-
ent requirements and obtain good growth rates, it
is necessary that the biofloc be ingested, digested
and assimilated. The environment that develops
under biofloc cultures provides various conditions
appropriate for cyanobacterial proliferation. The
risk of pollution by an aggressive cyanobacterial
strain could occur during fish farming. Thus, it is
necessary to generate information about the effects
of the presence of cyanobacteria in BFT. The main
objective of this study was to evaluate the effect of
Oscillatoria sp. inoculation at four concentrations
on nitrogenous compounds, suspended solids and
the production parameters of tilapia (Oreochromis
niloticus 9 Oreochromis mossambicus) reared in
saltwater biofloc system.
Materials and methods
Isolation and maintenance of the strain
The cyanobacterial strain was obtained from the
bottom of shrimp ponds in Sonora State (north-
west Mexico), isolated by serial dilution as
described by Band (2007) and subsequently main-
tained under laboratory conditions until the exper-
iments began. Identification at the genus level was
conducted by the Institute of Marine Sciences and
Limnology of the Universidad Nacional Aut
onoma
de M
exico. The F/2 (Guillard & Ryther 1962) cul-
ture medium was used during strain maintenance.
Light was provided by white lamps, and the tem-
perature was maintained between 22 and 25C.
The strain was transferred every 15 days.
Aquaculture Research, 2017, 110
Experimental design
This experiment was a simple random design with
four treatments by triplicate. The treatments con-
sisted of inoculation with 0.0, 0.1, 0.5 and
1 mg L
1 Oscillatoria sp. corresponding to treat-
ments C0, C1, C2 and C3 respectively.
The 12 experimental units (plastic tanks with a
capacity of 70 L) were filled with 50 L of salt
water (35 &) previously sterilized with calcium
hypochlorite (3 ppt) and neutralized with sodium
thiosulfate. Each tank was stocked with 250 g of
hybrid red tilapia juveniles (Oreochromis mossambi-
cus x Oreochromis niloticus), equivalent to
5 kg m
3. The organisms (14.5  2.6 g) were
randomly selected from a pre-breeding tank. Dur-
ing the experiment, the tilapia were fed to satia-
tion with commercial diet with 35% protein
(Purina; Agribrands Purina M
exico, S.A. de C.V.
Estado de M
exico, MEX).
To supply oxygen and maintain solids in sus-
pension, constant aeration was provided by an
electric blower of 1/3 HP and air stones placed on
the bottom. The study was carried out in outdoor
conditions with a natural photoperiod (14-h light;
10-h dark) and no temperature control. No water
exchange was performed, but evaporation losses
were compensated by freshwater. The carbon/ni-
trogen (C/N) ratio was maintained at 1:15, and
the input of C and N due to food was considered
and complemented with glucose (unrefined sugar)
according to Avnimelech (2009). The growth per-
iod lasted 7 weeks. To maintain the total sus-
pended solids below to 400 mg L
1, 50% of tank
volume was filtered through 10-lm mesh on days
20 and 35.
Water variables measurement
Temperature and dissolved oxygen were mea-
sured twice daily (8:00 and 16:00 hours) using
YSI 58 digital oximeter (YSI Inc., Yellow Springs,
OH, USA). The pH was measured daily with a
Denver Instruments pH-10 digital pH meter
(Denver Instruments, Denver, CO, USA), and
salinity was measured weekly with an Aquatic
Eco-Systems refractometer (Pentair Aquatic Eco-
Systems, FL, USA). Total alkalinity was measured
on a weekly basis according to APHA (1998).
Corrections of alkalinity (as CaCO3) were per-
formed to maintain concentrations above
100 mg L
1 using bicarbonate (NaHCO3)
2017 John Wiley & Sons Ltd, Aquaculture Research, 110
Oscillatoria sp. on biofloc tilapia culture A Miranda-Baeza et al.
following the suggestion of Furtado, Poersch and
Wasielesky (2011).
The chlorophyll a was measured weekly, and
10 mL of samples was obtained and concentrated
in a refrigerated centrifuge (10 000 rpm for 5 min
and 4C). The supernatant was discarded, and
9 mL of 90% acetone was added following the
technique of Strickland and Parsons (1972). Total
suspended solids were measured as outlined in
Hach Method 8006 (Hach 2007), using a Hach
DR/2800 spectrophotometer (Hach Co. Loveland,
CO, USA). The method was previously calibrated
and corrected by the traditional gravimetric
method according to Miranda-Baeza, Voltolina and
Cordero-Esquivel (2006).
Total ammonia nitrogen (TAN), nitrite nitrogen
(NO2-N) and nitrate nitrogen (NO3-N) were mea-
sured every week. For this purpose, water samples
(250 mL) were collected and centrifuged, and the
supernatant was used to measure the respective
nutrients. The TAN was measured with the salicy-
late method (8155), NO2-N by the diazotization
with ferrous sulphate in acid medium method
(8507) and NO3-N by the cadmium reduction
method (8171) according to the procedures
described in the Hach spectrophotometer manual
(Hach 2007).
Tilapia production parameters
The specific growth rate (SGR) of the tilapia was
estimated from the equation suggested by El-Har-
oun, Goda and Chowdhury (2006)
SGR Ln final weightg

Ln initial weightg100=culture time:
The survival rate was obtained from the rela-
tionship (final number of organisms/initial num-
ber) 9 100.
Data analysis
The assumptions of normality and homogeneity of
variance were verified. Furthermore, one-way
ANOVA (Zar 1996) was performed to determine sig-
nificant differences between the basic water vari-
ables and tilapia production parameters. In cases
where the differences were significant (P < 0.05),
a posteriori multiple comparison Tukeys tests
were applied. STATISTICA for Windows 5.1, StatSoft
Inc. software was used for data processing.
3
Oscillatoria sp. on biofloc tilapia culture A Miranda-Baeza et al. Aquaculture Research, 2017, 110

Results 1000

Chlorophyll a (g L1)
The average temperature throughout the study 800

ranged between 25.3 and 25.8C in the morning 600

and between 27.9 and 28.6C in the afternoon.
No significant differences among treatments were 400
y = 914.95x2+ 1394.2x + 293.28
R = 0.924
observed (Table 1). Dissolved oxygen ranged from 200
4.29 to 4.47 mg L
1 in the morning and from
3.54 to 3.80 mg L
1 in the afternoon without sig- 0
0 0.2 0.4 0.6 0.8 1 1.2
nificant differences between treatments. The pH Oscillatoria inoculation (mg L1)
presented means between 7.70 and 7.81 in the
Figure 1 Relationship among inoculation level of
morning and 7.62 and 7.71 in the afternoon. In Oscillatoria (0.0, 0.1, 0.5 and 1 mg L
1) and chloro-
both cases, the difference between treatments was phyll a (global averages for each treatment) into the
significant (P < 0.05). The salinity varied from tilapia culture tanks.
34.9 to 35.7& without a significant difference
among treatments.
The overall average of chlorophyll a ranged Table 2 Chlorophyll a total suspended solids and
from 226 to 780 lg L
1. The minimum corre- alkalinity (mean  SD) in the different treatments
sponded to treatment C0 and the maximum to
Chlorophyll a TSS Total alkalinity
treatment C3. The ANOVA revealed significant differ-
Treatment (lg L
1) (mg L
1) (mg CaCO3 L
1)
ences (P < 0.05) between treatments (Table 3).
The averages of chlorophyll a versus the inocula- C0 226  75a 277  48a 108.2  7.2
C1 517  120b 258  57a 114.1  8.4
tion level were described by a polynomial model
C2 728  117c 288  97ab 112.4  10.2
(Fig. 1). The average total suspended solids varied C3 780  98c 337  102b 105.3  12.1
from 258 to 337 mg L
1. The minimum corre-
sponded to treatment C1 the maximum to C3, and One-way ANOVA. Different letters in the same column indicate
significant differences, P < 0.05; a < b < c.
significant differences were identified (P < 0.05)
(Table 2). The total alkalinity (as CaCO3) varied
from 105.3 to 114.1 mg L
1, with no significant Throughout the experiment, the value ranged
differences among treatments (P < 0.05) (Table 2). between 0.82 and 0.85 mg L
1 without a signifi-
The TAN was not detected during day 1. How- cant difference (P < 0.05).
ever, TAN values ranged between 3 and 5 mg L
1 The NO3-N increased from day 9, peaked at day
on day 9. Subsequently, the concentration 18 (55 and 65 mg L
1) and ended between 10
decreased significantly and was close to zero at and 20 mg L
1 (Fig. 2c). The average throughout
day 18 (Fig. 2a). The overall average for the entire the experiment ranged between 22 and
experiment ranged between 0.96 and 33 mg L
1. The lowest concentration corresponded
1.75 mg L
1 with no significant differences to treatment C3, and the highest concentration
between treatments (P < 0.05). was found with treatment C0. The difference was
The NO2-N increased from day 6 and peaked significant (P < 0.05).
around day 12. At the end of the experiment, the The initial biomass of tilapia varied from 5.0 to
concentration reached 0.01 mg L
1 (Fig. 2b). 5.1 kg m
3. At the end of the study, the average

Table 1 Temperature, dissolved oxygen and pH (mean  SD) in the different treatments

Temperature (C) Oxygen (mg L
1) pH

Tr am pm am pm am pm

C0 25.8  2.0a 27.9  1.4a 4.30  0.28a 3.80  0.26a 7.70  0.02a 7.62  0.02a
C1 25.8  2.4a 28.6  1.6a 4.47  0.28a 3.78  0.20a 7.80  0.06b 7.71  0.04b
C2 25.7  2.2a 28.1  1.7a 4.39  0.39a 3.73  0.39a 7.81  0.05b 7.71  0.03b
C3 25.3  2.3a 28.1  1.6a 4.29  0.35a 3.54  0.42a 7.74  0.04ab 7.68  0.06ab

One-way ANOVA. Different letters in the same column indicate significant differences, P < 0.05, a < b.

4 2017 John Wiley & Sons Ltd, Aquaculture Research, 110

Aquaculture Research, 2017, 110 Oscillatoria sp. on biofloc tilapia culture A Miranda-Baeza et al.

(a) 6 Discussion
C0
5 C1 Temperature and salinity were maintained within
C2 the suitable ranges reported by El-Sayed (2006)
TAN (mg L1)
4

3
C3 for growing tilapia in seawater. In biofloc systems,
dissolved oxygen (DO) is considered one of the crit-
2
ical variables due to the high respiratory rate of
1 micro-organisms. In such systems, the cultured
0 species consumes a reduced proportion of the DO
0 10 20 30 40 50
(b) (Ray, Seaborn, Leffler, Wilde, Lawson & Browdy
2.0
C0 2010; Vinatea, G
alvez, Browdy, Stokes, Venero,
C1 Haveman, Lewis, Lawson, Shuler & Leffler 2010),
1.5
NO2-N (mg L1)

C2 and the remainder is used by the micro-organisms

C3 present: the bacterial population, which consumes
1.0
oxygen continuously (Avnimelech 2009), and
0.5 microalgae, which consume oxygen during the
night or on cloudy days.
0.0 In this study, the overall mean of dissolved oxy-
0 10 20 30 40 50
(c) gen was similar between treatments. However, in
80 C0 the afternoon, the concentration was reduced in
70 C1 treatments with more Oscillatoria inoculum. In
60
NO3-N (mg L1)

50
40
30
20
10
0 ture. An increase of 3C (the difference between
0 10 20 30
Culture day
Figure 2 Average values of total ammonia nitrogen,
nitrite nitrogen and nitrate nitrogen (mg L
1) during
the experiment.
ranged from 6.0 to 8.2 kg m
3 with significant dif-
ferences (P < 0.05); the lowest yields were obtained
in the C2 and C3 treatments, and the highest were
noted in C1 and C0. The final individual weight
varied from 21.6 to 24.1 g with significant differ-
ences between treatments (P < 0.05). The lowest
weight was obtained in the C2 and C3 treatments,
whereas the highest was in C1 and C0 (Table 3).
The feed conversion ratio (FCR) varied from
1.35 to 1.85 with significant differences between
treatments (P < 0.05). The lowest averages were
obtained in C0 and C1, and the highest averages
were observed in C2 and C3. Survival ranged from
72% to 98%; the lowest corresponded to C3 and
the highest to C0, with significant differences
between treatments (P < 0.05). Specific growth
also showed significant differences (P < 0.05); the
lowest average (0.36) was obtained in C3 and the
highest (1.05) in C0 (Table 3).
C2
contrast to expectations, the morning readings
C3
exhibited higher levels of DO, which indicates that
the production of oxygen by photosynthesis was
unable to compensate the loss of DO due to
decreased solubility caused by afternoon tempera-
40 50
morning and afternoon) provoked a decreased of
1 mg L
1 DO. Therefore, the high content of
micro-organisms in the biofloc may increase the
risk of hypoxia when aeration is not efficient.
However, most tilapia species can tolerate low DO
levels; Teichert-Coddington and Green (1993)
found that reducing aeration in tilapia ponds from
30% to 10% of oxygen saturation did not affect
fish growth. In this study, the overall DO averages
were between 3.54 and 4.29 mg L
1, which is
appropriate for tilapia culture (El-Sayed 2006).
Additionally, the levels were into the interval
(3.07.5 mg L
1) reported by Azim and Little
(2008) who reared tilapia in biofloc system.
Another important variable in biofloc systems is
pH, due to its effect on the ionization of ammonia.
Higher values (pH >9) reduce this ionization and
cause a considerable proportion of NH4+ to
become NH3, which even at low concentrations is
toxic to many aquatic species (Hopkins, Hamilton,
Sandier, Browdy & Stokes 1993). Biological factors
that influence pH include photosynthesis and res-
piration, both of which have great activity in bio-
floc systems. Photosynthesis can have a noticeable
effect on the pH of water. Photosynthetic activity

2017 John Wiley & Sons Ltd, Aquaculture Research, 110 5

Oscillatoria sp. on biofloc tilapia culture A Miranda-Baeza et al. Aquaculture Research, 2017, 110

Table 3 Tilapia production parameters (mean  SD) in the different treatments

Biomass

Treatment Initial (kg m
3) Final (kg m
3) Final weight (g) FCR SGR Survival (%)

a b b a b
C0 5.0  0.03 8.22  0.48 24.0  1.7 1.35  0.12 1.05  0.14 98  0.5b
C1 5.1  0.15a 8.12  0.23b 24.1  1.0b 1.40  0.18a 0.97  0.12b 96  2.6b
C2 5.0  0.05a 6.26  0.15a 21.7  0.6a 1.83  0.14b 0.45  0.06a 82  3.6a
C3 5.1  0.05a 6.00  0.34a 21.6  0.5a 1.85  0.08b 0.36  0.13a 72  3.0a

One-way ANOVA. Different letters in the same column indicate significant differences, P < 0.05, a < b.

removes CO2 from the system and causes an
increase in the pH of the water. In contrast, respi-
ration provides CO2, and the pH decreases (Tim-
mons, Ebeling, Wheaton, Summerfelt & Vinci
2002). Lower pH levels were recorded in treat-
ment C0, whereas it was the highest in the C1C3
treatments. These results were consistent with
expectations, as C0 had the lowest photosynthesis
rate.
In aquaculture ponds, carbon dioxide, pH, alka-
linity and hardness are interrelated. The water
with moderate-to-high alkalinity and slightly basic
pH (7.08.3) has good buffering capacity; biofloc
systems lose this ability and therefore require fre-
quent restoration (Azim & Little 2008). Furtado
et al. (2011) suggest alkalinity greater than
100 mg L
1 and pH above to 7 for saltwater bio-
floc systems; in this study, alkalinity was con-
trolled; as a result, no significant differences
among treatments were determined. It was not
necessary to restore pH because its levels were
greater than 7.
Several authors have reported the presence of
cyanobacteria in biofloc cultures. In a study with
post-larval shrimp, Becerra-D
orame et al. (2011)
reported cyanobacteria concentrations of
2.1 9 104 cells mL
1 in a heterotrophic biofloc
and 3.3 9 106 in an autotrophic biofloc. These
results demonstrate the ability of cyanobacteria to
proliferate in biofloc systems. In another study,
Ballester et al. (2010) reported that filamentous
cyanobacteria reached 218 filaments mL
1 and
17 200 cells mL
1 in a biofloc system. Further-
more, Ju et al. (2008) indicated that cyanobacteria
accounted for 5.9% of the total phytoplankton
cells in a biofloc shrimp culture, whereas chloro-
phytes reached 82.3%.
The presence of cyanobacteria in BFT is
explained by their ability to adapt to adverse
conditions of light, temperature and high organic
matter load (Fuentemayor et al. 2009); in
addition, they can be grown in fully hetero-
trophic conditions (Gallon, Hasem & Chaplin
1991). This phenomenon occurred in this experi-
ment; although treatment C0 was not inoculated
with Oscillatoria, their presence became observ-
able after 10 days of culturing and increased
moderately.
In the present experiment, chlorophyll a global
means ranged from 226 to 780 lg L
1. The high-
est concentration was noted in treatment C3 and
was consistent with the range of 500 to
900 mg L
1 reported by Decamp, Conquest, Cody,
Forster and Tacon (2007) for shrimp farming with
zero water exchange. Figure 1 describes the effect
of inoculation over chlorophyll a concentration
and indicates that the Oscillatoria inoculum effect
is nonlinear, and the lower initial inoculum of
0.1 mg L
1 generated an average concentration of
517 lg L
1 of chlorophyll a, while with 1 mg L
1
was of 780 lg L
1.
Although several authors have reported the
abundance of cyanobacteria in biofloc as
cells mL
1, it is clear that errors potentially
occurred during the quantification of filamentous
forms, because these organisms are entangled in
the bioflocs. In addition, fragmentation occurs
during any separation process. Thus, in this
study, we decided to perform indirect determina-
tion through chlorophyll a. Fuentemayor et al.
(2009) conducted an Oscillatoria sp. culture
under controlled conditions and estimated that
for each mg of biomass, between 2.10 and
2.97 lg L
1 of chlorophyll a was extracted.
Because the water was sterilized, it is possible to
assume that the chlorophyll a detected in this
study came from Oscillatoria; at the end of the
culture, we obtained cyanobacterial biomass
ranging from 125 mg L
1 (C0) to 260 mg L
1
(C1, C2 and C3), corresponding to slightly
greater than 50% of TSS in the C1, C2 and C3
treatments.

6 2017 John Wiley & Sons Ltd, Aquaculture Research, 110

Aquaculture Research, 2017, 110
The presence of TSS is another major variable
in biofloc systems. Azim and Little (2008) reported
values of TSS from 560 to 597 mg L
1 in a cul-
ture of tilapia in biofloc, whereas Avnimelech
(2007) reported a range of 460 to 643 mg L
1. In
the present study, the average was between 277
and 337 mg L
1, which is lower than those
reported in the papers cited. We decided to main-
tain TSS below 400 mg L
1 by conducting partial
biofloc harvests to prevent clogging of gills and
risks due to low oxygen diffusion from the high
concentration of Oscillatoria sp. in the C1, C2 and
C3 treatments. Emerenciano, Gaxiola and Cuzon
(2012) indicate that high levels of TSS can affect
the oxygen diffusion through the gills, but we did
not found specific information to determine the
exact effect of biofloc biomass over the oxygen dif-
fusion in tilapia gills; it could be an interesting
topic for further research.
At the beginning of the experiment, the TSS
level in all treatments was 100 mg L
1 with a
rapid increase. Therefore, it was necessary to crop
biofloc on days 20 and 35 (50% of volume was fil-
tered through 10-lm mesh). The C3 treatment
recorded the highest increase (reaching 570 mg
L
1 at day 20). For treatment C0, although it did
not receive inoculum, the amount of TSS was sim-
ilar to that in C1 and C2, indicating that the rate
of formation and accumulation of SST in an auto-
trophic system may be similar to that in a mixo-
trophic system.
The different inoculation levels of Oscillatoria sp.
showed no significant differences in TAN concen-
tration. However, during the first 15 days, in the
C3 treatment, the concentration remained lower
than that in the other treatments. Some microal-
gae prefer NH4+ over other chemical forms of N.
Hii, Soo, Chuah, Mohd-Azmi and Abol-Munafi
(2011) conducted a study with Nannochloropsis sp.
In the presence of ammonium and nitrate, the
microalgae had a preference for NH4+ and also
exhibited increased growth when this compound
was the unique source of N. These phenomena are
consistent with our study because the use of NO3
was initiated when TAN was consumed (day 15)
(Fig. 2).
The maximum level of NO3-N was achieved on
day 20; afterwards, it began to decline along with
NO2-N. In the case of C1, C2 and C3, the decrease
in NO3-N can be explained because Oscillatoria
started using oxidized N compounds when the
TAN had reached low levels (below 1 mg L
1) at
2017 John Wiley & Sons Ltd, Aquaculture Research, 110
Oscillatoria sp. on biofloc tilapia culture A Miranda-Baeza et al.
day 15 of the experiment. The gap between the
TAN, NO2-N and NO3-N curves coincides with the
appearance of ammonia-oxidizing bacteria (Nitro-
somonas) and subsequently nitrite-oxidizing bacte-
ria (Nitrobacter). This process has been widely
documented in recirculating water systems (Tim-
mons et al. 2002).
In this study, the nitrate concentration showed
an increase from day 1 to day 16, with a later
decrease to the end of the experiment. The outdoor
biofloc cultures do not have light-limited condi-
tions, and it is possible to obtain high autotrophic
production. This condition and the moderate C/N
ratio (1:15) used in our study caused high
demand of nutrients; therefore, it is possible that
Oscillatoria had consumed NO3-N when TAN was
scarce (Fig. 2); Azim and Little (2008) as well as
Ekasari and Maryam (2012) reported in biofloc
tilapia cultures. It has been documented the
microalgae metabolism is capable to use different
sources of inorganic nitrogen (Li, Horsman, Wang,
Wu & Lan 2008).
The presence of microalgae may have an advan-
tage in biofloc cultures because they permit nitro-
gen compounds to re-enter into the food chain.
However, at high concentrations, microalgae could
be a risk because they can become consumers of
O2 on cloudy days. In microalgae-dominated sys-
tems, the maximum NH4+ assimilation capacity is
0.7 g m
2 day
1. In contrast, in heterotrophic
bacteria-dominated systems, the assimilation
capacity can be unlimited if an appropriate C/N
ratio is maintained (Avnimelech 2009).
The cyanobacterium Oscillatoria sp. had signifi-
cant effects on the tilapia biomass produced. Lower
production was recorded in treatments C2 and C3,
whereas similar production was noted in treat-
ments C0 and C1. The final average weight in
treatments C2 and C3 was between 21.6 and
21.7 g, whereas the final average was 24 g in
treatments C0 and C1.
The incorporation of Oscillatoria had a signifi-
cant effect on survival. Treatment C0 had 98%
survival, whereas survival was only 72% in treat-
ment C3. The genus Oscillatoria may produce toxic
cyclic peptides, including microcystins, anatoxin-a
and aplysiatoxins (Sivonen & Jones 1999).
Although the toxicity of Oscillatoria in tilapia has
not been studied, the results of this experiment
indicate that its presence caused negative effects
on the growth performance of tilapia, especially in
treatments with greater abundance (C2 and C3).
7
Oscillatoria sp. on biofloc tilapia culture A Miranda-Baeza et al.
More research is needed to evaluate the pres-
ence of cyanobacteria in biofloc systems consider-
ing that some species have the ability to use both
autotrophic and heterotrophic mechanisms to pro-
liferate (Bonaurab, Data & Loudiki 2004). These
species can assimilate organic molecules of low
molecular weight, such as amino acids, monosac-
charides, glucose and acetate (Neilson & Lewin
1974; Bonaurab et al. 2004), and they can com-
pete with heterotrophic bacteria for the carbon
substrates.
Treatments C0 and C1 registered lower feed
conversion ratios (1.35 and 1.40), which exhibited
a clear trend towards increasing along with the
concentration of Oscillatoria sp. in culture. These
results clearly indicate that the presence of the
cyanophyte negatively affected feed utilization, but
the metabolic mechanism was not studied. The
FCR values obtained in the C0 and C1 treatments
were better than the range of 1.6 to 3.5 reported
by Rivas-Vega, Gil-Romero, Miranda-Baeza, San-
doval-Muy, L
opez-El
as and Nieves-Soto (2013),
who worked with the same hybrid (Oreochromis
mossambicus x O. niloticus) cultivated in salt water
under laboratory conditions. Despite the presence
of Oscillatoria, our results were similar to De Silva,
Gunasekera and Shim (1991) who reported a FCR
of 1.10 to 2.32 (12 weeks) for the same hybrid in
a recirculating system.
The inoculum level of Oscillatoria sp. exhibited
significant effects on pH, chlorophyll a, TSS, NO3-
N and productive parameters of a tilapia hybrid. It
is advisable to identify the species of cyanobacteria
that are capable of growing in biofloc, and further
studies related to the presence, abundance and
dynamics of these populations should be con-
ducted. In addition, we recommend studies on the
effects of toxins from cyanobacteria on the species
of interest in aquaculture as well as studies related
to the interaction of cyanobacteria and hetero-
trophic bacteria in BFT.
Acknowledgments
We thank CONACyT, 206155 project, for financial
support.
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