Aquaculture 502 (2019) 391–399

Contents lists available at ScienceDirect

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

Addition of commercial probiotic in a biofloc shrimp farm of Litopenaeus T

vannamei during the nursery phase: Effect on bacterial diversity using
massive sequencing 16S rRNA
⁎
José Alberto Huerta-Rábagoa,b, Marcel Martínez-Porchasc, Anselmo Miranda-Baezaa, ,
Mario Nieves-Sotod, Martha Elisa Rivas-Vegaa, Luis Rafael Martínez-Córdovae
a
Universidad Estatal de Sonora (UES), Navojoa, Sonora 85875, Mexico
b
Maestría en Sistemas de producción biosustentables, Universidad Estatal de Sonora (UES), Navojoa, Sonora 85875, Mexico
c
Centro de Investigación en Alimentación y Desarrollo, A.C. (CIAD), Hermosillo, Sonora 83304, Mexico
d
Universidad Autónoma de Sinaloa (UAS), Mazatlán, Sinaloa 80000, Mexico
e
DICTUS, Universidad de Sonora, Hermosillo, Sonora 83000, Mexico

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

Keywords: The aim of this study was to describe the effect of the addition of commercial probiotics on the bacterial diversity
Bacterial diversity in biofloc generated in a commercial farm of whiteleg shrimp Litopenaeus vannamei. The experiment consisted of
Bacterial ecology a simple random design with three treatments by triplicate (two commercial probiotics: PA and PB, and one
Biofloc control, C). The PA was composed by a mixture (50:50) of Efinol PT and Mix Laboratory Robles; the second,
Probiotics
coded as PB was composed by the mixture (50:50) of Epicin Ponds and Epicin Hatcheries. The control treatment,
Shrimp farms
coded as C (natural endemic microbial consortium), consisted of the traditional biofloc system without probiotic
application. The samples were obtained during tree periods of the shrimp nursery (beginning, medium and
final). The abundance and diversity indexes (alpha, beta and gamma) were calculated. The shrimp productive
parameters were determined. The ponds receiving the PA mixture registered 22 phyla, being Proteobacteria
(49.99–53.66%), Planctomycetes (9.62–18%) and Bacteroidetes (11.41–29.66%) the most abundat. The ponds
receiving the PB mixture, registered 19 phyla, and Proteobacteria (49.09–60.81%), Bacteroidetes (8.18–26.56%)
and Planctomycetes (6.38–25.05%) were the most abundant. The control ponds had 19 phyla, and
Proteobacteria (43.15–73.88%), Bacteroidetes (16.29–25.56%) and Planctomycetes (5.03–9.37%) registered the
highest abundance. During the tree sampling periods, the Shannon index (alpha diversity) varied from 1.54–1.40
in PA; 1.34 to 1.41 in PB and 0.88 to 1.59 in C. The beta diversity indicated 86% of similarity among PA-C; and
90% among PB-C. At the end of the study, the gamma diversity in bioflocs depends 96% of alpha diversity and
3.70% of beta diversity. The autochthonous bacteria had the greatest influence on the diversity. The productive
parameters did not show significant differences among treatments (P < 0.05).

1. Introduction aggregates contain diverse bacteria, microalgae, fungi and detritus, as

well as flagellates, ciliates, rotifers and nematodes (Emerenciano et al.,
Aquaculture is the food industry sector with the highest growth rate 2017). In some cases to promote and maintain the biofloc, the shrimp
in the world. The whiteleg shrimp Litopenaeus vannamei is the sixth farms use commercial probiotics (Crab et al., 2012; Vargas-Albores
most cultured species (FAO, 2016). In recent decades, aquaculture and et al., 2017).
other animal production activities have used diverse substances to in- Probiotics are defined as “live microorganisms, which, administered
crease production. The most used include hormones, vitamins, anti- in adequate doses confer a health benefit to the host” (FAO, 2001). In
biotics and immunostimulants, and recently, probiotics. To reduce the aquaculture, and particularly in shrimp farming, probiotics began to be
shrimp diseases, commercial farms started to culture shrimp in biofloc used as an alternative to reduce the use of antibiotics. The micro-
technology systems (BFT). The biofloc is a term used to designate the organisms commonly used include acidolactic bacteria, bifidobacteria
formation of aggregates of particles in a colloidal dispersion. These and yeast. Several studies have reported that the use of probiotics in

⁎
Corresponding author at: Universidad Estatal de Sonora (UES), Navojoa, Sonora 85875, Mexico.
E-mail address: anselmo.miranda@ues.mx (A. Miranda-Baeza).

https://doi.org/10.1016/j.aquaculture.2018.12.055
Received 17 July 2018; Received in revised form 16 December 2018; Accepted 17 December 2018
Available online 18 December 2018
0044-8486/ © 2018 Elsevier B.V. All rights reserved.
J.A. Huerta-Rábago et al. Aquaculture 502 (2019) 391–399

shrimp and fish increases growth and survival, resulting in better di- 2.3. Culture conditions
gestion and stimulation of the immune system (Verschuere et al., 2000;
Kesarcodi-Watson et al., 2008; Vargas-Albores, et al., 2016). The whiteleg shrimp postlarvae (PL 12) were obtained from the
The natural water ecosystems contain a great diversity of bacteria, laboratory of the same company (Proveedora de larvas S.A. de C.V.).
which are essential in the recycling of organic matter and play an im- The experimental specimens (7.3 mg/ind) were stocked in ponds (70 m3
portant roles in biogeochemical cycles. volume) at density of 500 ind/m2. The ponds were covered with a high-
Despite probiotics are widely used in aquaculture, there are no density polyethylene (HDPE). The nine experimental units were placed
systematic studies to evaluate their effect on the diversity of pre-ex- in the greenhouses and the natural photoperiod was maintained. The
isting native bacteria of farms (Martínez-Porchas and Vargas-Albores, aeration was supplied by electric aerators during the experiment the
2017). During the water exchange or harvest processes, the bacteria aeration power varied from 70 to 100 Hp/Ha. Aeration grills (elabo-
contained in commercial probiotics could reach the natural environ- rated with porous tube) were placed at the bottom of the tanks to
ment. The diversity of native species is essential to maintain the sta- supply oxygen and maintain the solids in suspension.
bility of the ecosystems. Thus is a highly relevant topic for biological The culture protocol of the company consisted of two phases, the
conservation (Vargas-Albores et al., 2017). first one suggests a zero water exchange (30 d) and the second (60 d)
Until a few years ago, studies of bacterial diversity were limited to allow performing a low water exchange (3–5%/day). To avoid inter-
culture dependent techniques, and only 1–15% of bacteria were de- ference on bacterial community by the effect of the natural microbiota
tected (Streit and Schmitz 2004; Ortiz-Estrada et al., 2018). However, present on influent water, this study was focused on the zero water
current molecular techniques including high throughput sequencing of exchange phase.
metagenomics DNA or taxonomic biomarker genes provide insights into Three days previous to stocking, the ponds were filled with filtered
the structure of microbial communities, including the non-cultivable seawater (5 μm). Commercial food containing 35% of protein (Purina ®,
species. Agribrands Purina Mexico, S.A. de C.V.) was used, and the daily ration
Tracking bacterial diversity changes during the probiotic supply in varied from 20 to 8% according to the shrimp weight. The feeding
the farms allow estimating possible changes in the natural environment. frequency varied from 12 to 8 times during 24 h. In all treatments the
In biodiversity studies, one of the most used approaches was proposed carbon: nitrogen (C:N) ratio was of 12:1, to maintain this balance dif-
by Whittaker in 1960 which analyses the diversity of a community ferent amounts of molasses were daily added as carbon source.
considering three components (alpha, beta and gamma), and this ap- During the experimental period, the physicochemical characteristics
proach is still valid (Whittaker et al., 2001). The objective of this study of the culture ponds were: dissolved oxygen, 5.22–5.59 mg/L; water
was to describe the effect of the addition of commercial probiotics on temperature, 32.4–32.7 °C; salinity, 35.3–36.8‰; pH, 7.0–8.3 and set-
the bacterial diversity of the bioflocs generated in a commercial shrimp table solids, 2.53–3.12 mL/L.
farm.
2.4. Shrimp productive parameters
2. Material and methods
At the end of the study, each tank was sampled to determine
2.1. Experimental design growth, survival and feed conversion rate. The specific growth rate of
the shrimp was estimated as follows:
The experiment was developed in the facilities of the company
[ Ln final weight (g) − Ln initial weight (g)]
Proveedora de Larvas S.A. de C.V., located in Sinaloa, Mexico. The Specific growth rate =
culture time (d)
hyper-intensive farm has ponds of 70 m3 and 2000 m3 (volume). This
experiment was developed in 70 m3 ponds and consisted of a simple
random design with three treatments by triplicate (commercial pro- 2.5. Collect of bacteria samples
biotics: PA and PB, and one control, C). The PA was composed by a
50:50 mixture of Efinol PT (containing Bacillus spp., lactic acid, During weeks 1, 3 and 6 of the experiment (periods: initial, medium
Lactobacillus spp., Saccharomyces spp.) and Mix Laboratory Robles and final), water samples (200 mL) were taken from all the experi-
(containing a native microbial consortia). PB was composed by the a mental units. The samples were collected in sterile Whirl-Pak® bags of
50:50 mixture of Epicin Ponds and Epicin Hatcheries (the labels of both 250 mL capacity, which were immediately placed on ice and trans-
products declare the content of non-toxic compounds, natural microbial ported to the laboratory. Once in the laboratory, the supernatant was
cultures, and enzymes with stabilizers and growth stimulants). The removed and the precipitated biofloc was placed in 15 mL falcon tubes.
control treatment coded as C (natural endemic microbial consortium), For each treatment, a mixture (of the tree replicates) was obtained. The
consisted of the traditional biofloc system without probiotics applica- tubes with the corresponding samples were placed in an ultrafreezer
tion. (Thermo) at −80 °C until processing.

2.2. Probiotic use 2.6. DNA isolation

Two bioreactors of 1000 L (operative volume) were used to activate To isolate DNA from the collected bioflocs, the commercial DNA
and incubate the bacterial consortia. Each bioreactor received vigorous extraction kit PowerBiofilm® (MO BIO Laboratories, Solana Beech, CA,
aeration by microporous tube placed at the bottom. The water USA) was used, and DNA free of inhibitors and high molecular weight
(33–35‰) was filtered (5 μm) and sterilized with sodium hypochlorite, was obtained. For this, a biofloc mass of 0.20 mg of each sample (pre-
neutralized with sodium thiosulfate and after 12 h. The respective viously thawed) was homogenized in a Fisher Scientific ™ vortex. From
bioreactors were inoculated with probiotics in a dose of 100 g/m3, and this step and forward the procedure indicated by the manufacturer of
1 kg of molasses/m3 as carbon source, as well as a mix of micronutrients the DNA isolation kit was strictly followed.
with other compounds (not revealed by the company; it was roughly The quality of the extracted DNA was evaluated by capillary mi-
composed by trace metals, vitamins and pH stabilizers). The probiotics croelecrophoresis in a 2200 Tape Station micro-electrophoresis device
were incubated at 30 ± 3 °C during 36 h. Under these conditions the (Agilent, Palo Alto CA, USA). Briefly, 1 μL of the isolated DNA solution
viable heterotrophic bacteria reached concentrations ranging from 80 was taken and mixed with 10 μL of gDNA buffer (Agilent, USA). For
to 120 × 106 CFU/mL (determined in marine agar; plate spread at 30 °C reference, 1 μL of gDNA Ladder (Agilent, USA) was used. After the
in 24 h). mixture, 1 μL of each sample was taken and inserted into a microfluidic

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chip (gDNA ScreenTape, Agilent, USA) to measure DNA quantity and resulting sequences were analyzed and classified with Kraken 1.0.0
quality, the DNA fragments of 200 to > 60,000 bp were considered. (Illumina; basespace.illumina.com; Wood and Salzberg, 2014).
Finally, the microfluidic chip with the nucleotide samples was in- In this study we considered the phyla with abundance superior to
troduced in the 2200 Tapestation (Agilent, USA). All samples with DIN 0.001% of the total. The diversity estimators were calculated using
(DNA integrity number) ratings > 7, were considered for the prepara- multivariate techniques based on sampling patterns. To evaluate the
tion of the library. effectiveness of the sampling, the ACE, CHAO 1, JACK 1, Boostrap and
Unique estimators were used, which were determined with the software
2.7. Library preparation EstimateS 9.1.0 (Colwell, 2013). Alpha beta and gamma diversity were
calculated using the indexes of: Shannon, Pielou, Jaccard, and Schluter
For the preparation of the amplicon library, the “Library prepara- and Ricklefs, as described by Moreno (2001) and Carmona-Galindo and
tion guide for 16S gene sequencing” of Illumina was used. In short, the Carmona (2013); according the following equations:
protocol is designed to carry out the amplification, purification and Alpha diversity (Shannon-Wiener index):
sequencing of the fragment containing the V3 and V4 regions, con- S
sidering the primers reported by Klindworth et al. (2013), which were H´ = − ∑ Pi ln (Pi )
combined with the barcode of sequencing adapters and dual indicators. i=1
Adapter Forward of amplicon 16S for PCR + adapter over
where;
hang = 5′TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGG-
S = number of species (or Phyla); Pi = proportion of individuals of
GNGGCWGCAG 3′
species i; H ‘= 0 when the sample has only one species; H ‘has a
Adapter Reverse amplicon 16S for PCR + adapter Over hang = 5′
maximum value when all S species are represented by the same number
GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTA-
of individuals.
TCTAATCC 3′.
Equitability was estimated with the Pielou index:
The first amplification was carried out in 25 ul reactions, using 2×
KAPA HiFi HotStart ReadyMix (KAPA Biosystems, USA), by PCR under H´
J´ =
the following thermal cycle conditions: an initial denaturation at 95 °C H ´ Max
for 3 min; a pairing of 25 cycles of 95 °C for 10 s, 55 °C for 30 s, 72 °C for where; H'max = ln (S); H = value of the Shannon-Wiener index.
30 s, and a final extension at 72 °C for 5 min. Thereafter, the resulting To estimate the β diversity, the similarity index of Jaccard was used:
amplicons between 450 and 550 bp were subjected to a cleaning pro-
c
cess using the AMPure XP magnetic bead protocol (Beckman Coulter, Ij =
a+b−c
USA) to remove free primers, as well as primer dimers species, fol-
lowing the manufacturer's specifications. where; a is the number of species in site A; b is the number of species in
After cleaning, the indexing of samples continued, using dual in- site B; c is the number of species present in both sites. The range of this
dexes and sequencing adapters of the Nextera Index XT kit (Illumina, index varied from zero (when there are no shared species), to 1 (when
San Diego, CA, USA) and 2× KAPA HiFi HotStart Readymix (KAPA both sites share the same species).
Biosystems). A second PCR was carried out by repeating the same The gamma diversity was estimated using the Schluter and Ricklefs
thermal cycle and finishing a new cleaning process through the afore- method:
mentioned process.
γ = αaverage x β x dimmension of the sample
Finally, the resulting library was quantified and graded by electro-
phoresis (2200 Tapestation, Agilent, USA) using microfluidic chips where; average alpha diversity = average number of species in a
(D1000 Screen Tape, Agilent, USA) with a range of analysis from 35 to community; beta diversity = inverse of the specific dimension, that is
1000 bp. For this, 2 μL of the sample from the indexed library were 1/average number of communities occupied by a species; sample
taken and mixed with 2 μL of High Sensitivity D1000 buffer. Finally the size = total number of communities.
samples were inserted in Screentapes High Sensitivity D1000 and The calculation was made based on the Shannon index, using the
analyzed with the 2200 Tapestation equipment to know the con- following equation:
centration and size of purified amplicons and continue with the se-
H´beta = − ∑ Pi lnPi − ∑ qj Hj
quencing.
i j

2.8. Sequencing where;

The libraries were adjusted to a concentration of 4 nM using 10 mM

Pi = ∑ qj pij
j
Tris (pH 8.5) as a diluent. Then, the libraries were denatured with 0.2 N
NaOH. At the same time, a standard library of PhiX Control (Illumina) which represents the average frequency of species i in the set of com-
was denatured, which was used as an internal control, this allows to munities, weighted according to the importance of the communities
calculate the error rates, since it is a short genome sequence well de- (qj). In this study, the weighting was done according to the culture area,
fined. Both the denatured library and the PhiX Control were adjusted to since all treatments had the same, so the weighting was 0.333 for each
a concentration of 8 pM and were mixed (95% of the library + 5% PhiX community.
Control). Finally, the mixture was heated at 96 °C for 2 min and im-
mediately cooled on ice for 5 min. The samples were loaded into a 3. Results
MiSeq v3 Reagent Tray (Illumina) cartridge, which contained a MiSeq
v3 Flow Cell (Illumina) with a capacity of 25 million readings. The A total of 5,727,411 readings were obtained from the biofloc sam-
results were obtained after 602 cycles (2 × 301). ples; 82% were classified within a taxonomic level, whereas the rest
corresponded to unidentified readings (in some cases correspond to
2.9. Data analysis and estimation of diversity indexes uncompleted sequences). In the analysis we used the classified readings.
The ponds that received the mixture of probiotics A, registered 22
Primers, linkers and adaptors were trimmed from the sequences and phyla, and the most abundant were Proteobacteria (49.99–53.66%),
sequences shorter than 50 bp were removed. The resulting files were Planctomycetes (9.62–18%) and Bacteroidetes (11.41–29.66%) (Fig. 1).
uploaded to Basespace (Illumina) for the automatized pipeline and the The ponds that received the mixture of probiotics B, showed 19 phyla,

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100
PA
80 Others

Relative abundance (%)

Verrucomicrobia
60 Actinobacteria
Firmicutes
40 Chlamydiae
Cyanobacteria
20 Planctomycetes
Bacteroidetes
0 Proteobacteria
Week 1 Week 3 Week 6

100
PB
80
Relative abundance (%)

Others
Cyanobacteria
60
Chlamydiae
Firmicutes
40
Actinobacteria

20 Planctomycetes
Bacteroidetes
0 Proteobacteria
Week 1 Week 3 Week 6

100
C
Relative abundance (%)

80 Others
Candidatus Sacc.
60 Cyanobacteria
Chlamydiae
40 Firmicutes
Actinobacteria
20 Planctomycetes
Bacteroidetes
0 Proteobacteria
Week 1 Week 3 Week 6

Fig. 1. Bacterial profile of the main bacteria phyla in the treatments, during the sampling periods.

where Proteobacteria (49.09–60.81%), Bacteroidetes (8.18–26.56%) Table 1

and Planctomycetes (6.38–25.05%) were the most abundant. The ponds Indexes of α diversity (H), and equitability (J) of the bacteria phyla in the
not receiving inoculation of commercial probiotics (control), had 19 biofloc of each treatment, during three sampling periods.
phyla, the most abundant were Proteobacteria (43.15–73.88%), Bac- Treatment Shannon index (H) Pielou index (J)
teroidetes (16.29–25.56%) and Planctomycetes (5.03–9.37%).
At the beginning of the experiment, treatment C obtained the lowest Week 1 Week 3 Week 6 Week 1 Week 3 Week 6
alpha diversity (Shannon index H = 0.88 nits/ind); although at the end
PA 1.54 1.30 1.40 0.52 0.48 0.46
of the study increased to: H = 1.59 nits/ind. The values of the equit- PB 1.34 1.28 1.41 0.45 0.45 0.49
ability index (Pielou index, J) presented the same trend as alpha di- C 0.88 1.30 1.59 0.32 0.47 0.55
versity respect to the beginning and end of the study (Table 1).
The beta diversity was calculated using the Jaccard similarity index
(Ij), which allowed a comparison per couple of treatments, PA - C and
PB - C. The highest similarity was observed between PB - C treatments

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Table 2 4. Discussion
Beta diversity (β) of the bacteria phyla registered in
the biofloc of probiotics A (PA) y Probiotics B (PB) In all treatments the phyla with the greatest presence in the bioflocs
in respect to the control (C). were Proteobacteria (50–73%), Bacteroidetes (9–27%), Planctomycetes
Jaccard index (Ij) (10–25%), Actinobacteria (4–11%) and Firmicutes (0.5–2%). The pro-
teobacteria phylum is usually the most abundant in aquaculture en-
PA - C 0.86
vironments, considering biofloc, biofilm and recirculation systems
PB - C 0.90
(shrimp or tilapia) (Martins et al., 2013, Lee et al., 2016, Martínez-
Córdova et al., 2016).
Table 3 At the beginning of the study, the control treatment showed the
Variation of the gamma diversity index of the bacteria phyla in the bioflocs highest abundance of Proteobacteria (73%), which could be correlated
during the weeks 1, 3 and 6. with its abundance in natural environment. In the ocean, species be-
longing to this phylum constitute up to 79% of the bacterial biomass
Gamma diversity
from the bottom and 64% from the water column; while in freshwater
Week 1 Week 3 Week 6 they represent up to 40% of the bacterial diversity (Battistuzzi and
Hedges, 2009).
H′ gamma 1.32 1.32 1.52 Proteobacteria have a wide variety of metabolic pathways to obtain
%α 95.00 97.60 96.30
%β 5.00 2.40 3.70
energy, among which phototrophic, chemo-trophic and chemogano-
trophic routes are abundant (Madigan et al., 1997); therefore, they play
an important role in the nutrient cycle and in the mineralization of
with 0.90 (90% of similarity in bacterial diversity), while between PA - organic compounds (Kirchman, 2002; Kersters et al., 2003).
C the value was 0.86 (86% similarity in bacterial diversity) (Table 2). The second most dominant phyla found in this study and that
The gamma diversity index (H′ gamma; which consider the whole agreeing with the reports of Lee et al. (2016) and Porchas-Cornejo et al.
community; 3 treatments) was the same between weeks 1 and 3, (2017) was Bacteroidetes; it is common to find it colonizing macro-
however, in the week 1 it was composed of 95% diversity α, and 5% scopic particles of organic matter (Woebken et al., 2007). Additionally,
diversity β; while for week 3, the contribution of α diversity increased it is found naturally in the bottom of the sea, where it represents ap-
to 97.6% and diversity β decreased to 2.4%. By week 6, the gamma proximately 8% bacterial diversity, while on the surface it reaches 9%;
diversity index increased slightly reaching 1.52, and was composed of however, its presence increases remarkably in humid soils, reaching up
96.3% by diversity α and by 3.7% by diversity β (Table 3). to 19% (Battistuzzi and Hedges, 2009). It can be assumed that the
The predictors of bacteria richness, determined by the different biofloc has physico-chemical characteristics suitable for the prolifera-
mathematical models used, had a high coincidence with the number of tion of this phylum, allowing it to remain as the second most dominant;
phyla recorded during the sampling (sobs: observed phyla; Fig. 2). In besides that, the relative abundance of Bacteridetes increased in the PB
the PA treatment 22 phyla were observed, while the models predicted and C treatments from the first to the last sampling.
an interval from 22 to 24. According to the mathematical models Despite the great variety of phyla thriving in the natural environ-
(predictors) in this treatment the most rigorous estimator was the first ment, the particular conditions of the microenvironments favor the
order Jacknife (Jack 1). The highest precision of the estimators was presence of some of them. For example, in soils or solid surfaces nine
presented in the PB treatment, all predicting 19 phyla, the same amount abundant phyla can be found, but four of them (Proteobacteria,
as those recorded during the sequencing. In the control treatment, 19 Acidobacteria, Actinobacteria and Bacteroidetes) accumulate 90% of
phyla from the interval 19–21 expected according to the estimators the total diversity (Tsai et al., 2009). Comparing these statistics with
were collected. In this case, the two most rigorous estimators were Jack the present study, it was found that about 90% of the bacterial diversity
1 and ICE. was represented by four phyla: Proteobacteria, Bacteroidetes, Plancto-
In all treatments Uniques (phyla that only had one reading) tended mycetes and Actinobacteria.
to descend according to the sampling periods. These values were 4, 0 The analysis of the 16S rRNA gene sequences allows knowing the
and 3 for PA, PB and C respectively. bacterial diversity with high accuracy (Porteous et al., 1997,
Both the estimators and the Uniques indicate that in the PB treat- Stackebrandt and Ebers, 2006). In a similar study, Lee et al. (2016)
ment, the best sampling was performed, although in the PA and C evaluated the bacterial diversity thriving in six different points of a
treatment the estimators were very close to the real data and in both recirculation aquaculture system; the Shannon-Wiener indexes in-
cases the Uniques tended to descend. dicated that the biofilter index was four times higher compared to that
In all treatments, only five genus constituted between 46 and 78% obtained in the fish tanks. The high diversity in the biofilter could be
of the total abundance (Fig. 3). At the end of the study, the most related with the numerous biological processes influencing the con-
abundant genera that were repeated in the 3 treatments were: Rhodo- sumption of organic matter and the transformation of nitrogen com-
pirellula, Ketogulonicigenium, Ruegeria, Sulfurimonas (Fig. 3) which can pounds.
be considered as the most important in the biofloc system under the Evaluating the effectiveness of the sampling is relevant for biodi-
conditions in which the study was developed. versity studies. There are several models that allow determining the
The productive parameters did not show significant differences species richness and construct an asymptote of the species accumula-
among treatments (P < .05); by the end of the study (30 d) the shrimp tion curve through various functions (Gotelli and Colwell, 2011). These
weight (g/ind) was: 1.54 ± 0.25 (PA), 1.46 ± 0.33 (PB) and models are called diversity estimators or predictors (Colwell et al.,
1.44 ± 0.04 (C). The specific growth rate (SGR %/day): 14.17 ± 0.34 2012); the similarity among the estimated and the observed value, in-
(PA), 14.03 ± 0.48 (PB) and 14.05 ± 0.05 (C). The survival (%): dicates that the sampling is efficient and representative of the bacterial
92.0 ± 4.35 (PA), 85.0 ± 8.66 (PB) and 92.3 ± 6.54 (C). The feed community. In a study of bacterial diversity indicating fecal con-
conversion rate (FCR): 0.98 ± 0.05 (PA), 0.90 ± 0.04 (PB) and tamination in incoming tide (FLD) and outgoing tide (EBB) in a wet-
0.93 ± 0.05 (C). land, Dorsey et al. (2013) obtained approximately 51 species of bac-
teria, their sampling was evaluated using the EstimateS software
(Colwell, 2013), the estimators, that came closest to the observed va-
lues were: Bootstrap (approximately 49–50 for EBB and 50–52 for FLD)
and Jacknife (between 55 and 58 for EBB and 59–61 for FLD). While the

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J.A. Huerta-Rábago et al. Aquaculture 502 (2019) 391–399

Sobs ICE Jack 1 Bootstrap Uniques

18 25
25 25 20
23 24
23

Number of Uniques
23

Number of phyla
22 15
22
21 21
10
19 18
5 4
5
17

15 0
1 2 3 PA

Sobs ICE Jack 1 Bootstrap Uniques

21 18 20 20

20
20

Number of Uniques
19 15
Number of phyla

19
19 19
18
18 10

17
5
16 2
0
15 0
1 2 3 PB

Sobs ICE Jack 1 Bootstrap Uniques

22 20
16 21
21 20 21
Number of phyla

Number of Uniques
20 20 15
20
19 19 19
18 10
18
17 16 3 3 5
16
15 0
1 2 3
C
Sampling period
Fig. 2. Estimators of species richness and Uniques in treatments during the tree sampling periods (determined by the software EstimateS 9.1.0; Colwell, 2013).

most rigorous estimators predicted higher values: Chao 1 (79–81 for community (by treatment and by sampling). In week 1 the gamma
EBB and 114–116 for FLD) and ICE (19–21 for EBB and 160–162 for index was 1.32, of which 95% corresponded to diversity α and 5%
FLD), such differences indicated that the bodies of water had a greater diversity to β, while in weeks 3 and 6 the values were very similar to
number of species than those detected in their samplings. In the same the first sampling (1.32 and 1.52), as well as the contributions of di-
study a Jaccard similarity index was used, and it was concluded that versity α and β.
between EBB and FLD there was a 40% of shared species, considered as Evidently the replacement of phyla between the treatments (di-
a medium or moderate similarity. versity β) was almost negligible compared to the richness of the phyla
In this study, the number of phyla present in the biofloc collected in each treatment (α diversity). The results make sense when observing
during the samplings was very similar to that predicted by the esti- that 19 of the 22 phyla recorded by metagenomic analysis were shared
mators. The results of the estimators indicate that the sampling was among the treatments. The effect found with the addition of commer-
sufficient to determine with precision the richness of the phyla in the cial probiotics was contrary to expectations. Local diversity had the
three treatments. greatest contribution to the diversity of the community. This is result
Taking as a reference the bacterial diversity developed in the con- could be associated to a better adaption of the local diversity to the
trol treatment, the similarity between PA-C and PB-C was evaluated marine environment compared that contained in both probiotic mix-
using the Jaccard index. The PA-C index was 0.86, which indicates an tures.
86% match; the index between PB-C was 0.90, that is, 90% of bacterial In the RAS, biofilm or biofloc systems, the Proteobacteria and
similarity between both treatments. Bacteroidetes phyla tend to dominate (Martins et al., 2013; Monroy-
The gamma diversity index allowed determining the impact of local Dosta et al., 2013; Martínez-Córdova et al., 2016, Lee et al., 2016).
diversity (α) and the replacement of species (β) within the bacterial While biofloc systems have a large number of bacteria, few genera

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J.A. Huerta-Rábago et al. Aquaculture 502 (2019) 391–399

PA Rhodopirellula (Planctomycetes)
100
Rhodobacter (Proteobacteria)

Relative abundance (%)

Planctomyces (Planctomycetes)
Ketogulonicigenium (Proteobacteria)
50 Cyanobacterium (Cyanobacteria)
Ruegeria (Proteobacteria)
Sulfurimonas (Proteobacteria)
Croceibacter (Bacteroidetes)
0
1 3 6 other
weeks

PB Rhodopirellula (Planctomycetes)
100
Rhodobacter (Proteobacteria)
Relative abundance (%)

Planctomyces (Planctomycetes)
Ketogulonicigenium (Proteobacteria)
Ruegeria (Proteobacteria)
50
Sulfurimonas (Proteobacteria)
Croceibacter (Bacteroidetes)
Teredinibacter (Proteobacteria)
Cytophaga (Bacteroidetes)
0 Echinicola (bacteroidetes)
1 3 6
other
Weeks

Control
Rhodopirellula (Planctomycetes)
100
Rhodobacter (Proteobacteria)
Relative abundance (%)

Ketogulonicigenium (Proteobacteria)
Ruegeria (Proteobacteria)

50 Sulfurimonas (Proteobacteria)
Croceibacter (Bacteroidetes)
Vibrio
Muricauda
0 Teredinibacter
1 3 6
Other
Weeks
Fig. 3. The five more abundant bacteria genus in each treatment registered during the weeks 1, 3 and 6.

dominate. Since the sum of the 5 most abundant genera accumulated and it is known that the excess organic matter promotes its proliferation
between 46.43 and 78.29% of the total readings. (Austin et al., 1995; Fuentes and Pérez, 1998). The biofloc is char-
The genus with greater presence was Rhodopirellula, which is acterized by having high concentrations of organic matter which in
abundant in the marine environment; Žure et al. (2015) indicate that theory, can encourage its proliferation; however, it is documented that
these microorganisms apparently play an important role in the meta- this culture system favors the growth of beneficial bacteria that com-
bolism of nitrogen compounds. Carbohydrates are the main source of pete for resources (space and nutriments) that also generates complex
carbon and energy for this genus (Schlesner et al., 2004), but also play a substances inhibiting the growth of potential pathogens (Wu et al.,
significant role in carbon cycling (Glöckner et al., 2003). It can be 2012). In control treatment, the Vibrio inhibition could have occurred
considered that the biofloc developed in aquaculture systems provide considering that it was not detected appear in the five most abundant
conditions for the proliferation of these microorganisms, their abun- genera.
dance in oceanic waters is around 1.4% (Porchas-Cornejo et al., 2017). Another abundant genus that appeared in the three treatments was
The genus Vibrio was the most abundant in the control treatment Ketogulonigenium, which includes Gram-negative facultative anaerobic
(22.22%) during the first week. This genus is part of the natural biota of bacteria; these are used in the biotechnology industry for their ability to
aquatic environments. It may contain potentially pathogenic species, produce vitamin precursors, specifically 2-keto-L-gulonic acid for the

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