693 Revista MVZ Córdoba Rev. MVZ Córdoba 0122-0268 1909-0544 Universidad de Córdoba Colombia revistamvz@gmail.com 69360025014 https://doi.org/10.21897/rmvz.1648 Artículos Originales Describing the planktonic and bacterial communities associated with bocachico Prochilodus magdalenae fish culture with biofloc technology Comunidades planctónicas y bacterianas asociadas al cultivo de bocachico Prochilodus magdalenae con tecnología biofloc http://orcid.org/0000-0003-4869-223X Ayazo-Genes Julia julia.ayazog2606@hotmail.com http://orcid.org/0000-0002-1916-4262 Pertúz-Buelvas Vicente vpertuz@correo.unicordoba.edu.co http://orcid.org/0000-0002-4996-149X Jiménez-Velásquez César cesarjimenez@correo.unicordoba.edu.co http://orcid.org/0000-0001-9737-1163 Espinosa-Araujo José jaespinosaaraujo@correo.unicordoba.edu.co http://orcid.org/0000-0002-2533-1995 Atencio-García Víctor vatencio@correo.unicordoba.edu.co http://orcid.org/0000-0003-3458-4983 Prieto-Guevara Martha mprieto@correo.unicordoba.edu.co Universidad de Córdoba, Facultad de Medicina Veterinaria y Zootecnia, Departamento de Ciencias Acuícolas, Instituto de Investigación Piscícola-CINPIC, Montería, Colombia. Universidad de Córdoba Colombia Universidad de Córdoba, Facultad de Medicina Veterinaria y Zootecnia, Departamento de Ciencias Acuícolas, Instituto de Investigación Piscícola-CINPIC, Montería, Colombia. Universidad de Córdoba Colombia Universidad de Córdoba, Facultad de Medicina Veterinaria y Zootecnia, Departamento de Ciencias Acuícolas, Instituto de Investigación Piscícola-CINPIC, Montería, Colombia. Universidad de Córdoba Colombia Universidad de Córdoba, Facultad de Medicina Veterinaria y Zootecnia, Departamento de Ciencias Acuícolas, Instituto de Investigación Piscícola-CINPIC, Montería, Colombia. Universidad de Córdoba Colombia Universidad de Córdoba, Facultad de Medicina Veterinaria y Zootecnia, Departamento de Ciencias Acuícolas, Instituto de Investigación Piscícola-CINPIC, Montería, Colombia. Universidad de Córdoba Colombia Universidad de Córdoba, Facultad de Medicina Veterinaria y Zootecnia, Departamento de Ciencias Acuícolas, Instituto de Investigación Piscícola-CINPIC, Montería, Colombia. Universidad de Córdoba Colombia May-August 2019 24 2 7209 7217 02 07 2018 01 11 2018 01 05 2019 Abstract

Objective. To describe the planktonic communities and bacteria associated with the bocachico Prochilodus magdalenae fish culture with biofloc technology (BFT). Materials and methods. Bocachico fingerlings, with an average weight of 1.6±0.2 g, were stocked at three densities, i.e., 5 (T1), 10 (T2) and 20 (T3) fish/m3, with BFT in nine rectangular, 6.0 m3 concrete tanks for 120 days of culture. Identification and quantification of the microorganisms was performed every eight days in a sample of 250 ml of water per tank by analyzing aliquots on a Sedgwick-Rafter and/or in Neubauer chambers on a microscope at 10x and 40x magnification. On days 15, 45, and 90 of the fish culture, the bacterial communities were characterized by taking 2 g samples of floc and adding them to 90 ml of sterile saline solution, then subjecting them to conventional microbiological tests. Results. Five planktonic groups (microalgae, rotifers, cladocerans, copepods, and protists with ciliates predominating) with more rotifers and protists in the fish cultures at lower density (T1 and T2) were identified, and the largest amount of microorganisms oscillated between 174.9±21.4 ind/ml (T1) and 125.6±16.1 ind/ml (T2). It was possible to identify ten bacterial strains: Escherichia coli, Enterobacter sp., Klebsiella sp., Salmonella sp. (Enterobacteriaceae), Bacillus subtilis, Bacillus sp., Lactobacillus sp., Pseudomonas sp. (Vibrionaceae), Micrococcus sp., and Staphylococcus sp. (Coccus Gram+). Conclusions. The composition of plankton was similar in all treatments, with rotifers and protists being the most abundant; the bacteria showed a higher proportion of enterobacteria and heterotrophs.

Resumen

Objetivo. Describir las comunidades planctónicas y bacterianas asociadas al cultivo de bocachico Prochilodus magdalenae con tecnología biofloc (BFT). Materiales y métodos. En nueve tanques rectangulares de concreto con volumen útil de 6.0 m3, se sembraron alevinos de bocachico con peso promedio de 1.6±0.2 g, a tres densidades 5 (T1), 10 (T2) y 20 (T3) peces/m3 con BFT, durante 120 días de cultivo. La identificación y cuantificación de los microorganismos se realizó cada ocho días, en una muestra de 250 ml de agua por tanque, mediante análisis de alícuotas en cámaras Sedgwick-Rafter y/o Neubauer bajo microscopio a 10x y 40x. Los días 15, 45 y 90 del cultivo se caracterizaron las comunidades bacterianas tomando una muestra de 2 g de floc en 90 ml de solución salina estéril y sometidas a pruebas microbiológicas convencionales. Resultados. Se identificaron cinco grupos planctónicos (microalgas, rotíferos, cladóceros, copépodos y protistas con predominancia de ciliados) con mayor cantidad de rotíferos y protistas en los cultivos con menor densidad (T1 y T2); y la mayor afluencia de microorganismos osciló entre 174.9±21.4 ind/ml (T1) y 125.6±16.1 ind/ml (T2). En el grupo de bacterias fue posible identificar 10 cepas: Escherichia coli, Enterobacter sp., Klebsiella sp., Salmonella sp. (Enterobacteriaceae) Bacillus subtilis, Bacillus sp, Lactobacillus sp, Pseudomonas sp (Vibrionaceae), Micrococcus sp, Staphylococcus sp (Cocos gram+). Conclusiones. La composición del plancton fue similar en todos los tratamientos, con rotífero y protistas como los más abundantes; la mayor proporción de bacterias fueron Enterobacterias y Heterotróficas.

 Keywords Bacteria ciliates rotifers zooplankton Palabras clave Bacterias ciliados rotíferos zooplancton How to Cite Ayazo Genes, J., Pertuz Buelvas, V., Jiménez Velásquez, C., Espinosa Araujo, J., Atencio-García, V., & Prieto-Guevara, M. (2019). Describing the planktonic and bacterial communities associated with bocachico Prochilodus magdalenae fish culture with biofloc technology. Journal MVZ Cordoba, 24(2), 7209-7217. https://doi.org/10.21897/rmvz.1648 <bold>INTRODUCTION</bold>

Biofloc technology (BFT) is based on the manipulation of a microbial community under controlled conditions in a culture system, producing aquatic animals in a sustainable and biosecure way (1). This technology relies on the oxidation-reduction ratios of the nitrogen cycle and the addition of carbon-rich substrates (molasses, cassava flour, and glycerol, among others) for the sequestration and recycling of nitrogen in the form of a microbial protein (2).

These conditions stimulate the development of a beneficial microbial community, established in small flocs of organic matter re-suspended continuously in the water column by the action of permanent aeration, which recycles the ammonium nitrogen produced by the fish in the synthesis of unicellular protein. This protein can be ingested and assimilated by fish, who graze on the communities of planktonic microorganisms associated with the flocs, enabling the use of low-protein diets compared to conventional fish farming systems (3,4,5).

The transformation of particulate organic matter and other organisms in the microbial trophic chain has been proposed as a possible source of food for fish cultures produced with BFT (5). This transformation is based on the complex interactions occurring in the entire water column among the organic matter, the physical substrate, and a wide range of microorganisms. The microorganisms include microalgae, free and adhered bacteria, aggregates of organic matter, and herbivores, such as rotifers, ciliates, flagellated protozoa, and copepods (6).

As a result of these interactions, the continuous recycling of nitrogen compounds is established in the system, making available a rich, natural source of protein and lipids for the cultivated species in situ 24 hours a day (5,7,8). The assimilation of this source of food will depend mostly on the feeding habit of the species, the conditions of the system, and the populations of microorganisms that predominate. In the case of omnivorous species, such as tilapia (5,9) and shrimp, this technology is widely applicable for their cultivation (8,10,11), but such wide applicability does not exist for detritivorous species, such as bocachico Prochilodus magdalenae.

Considering the importance of natural productivity in the recycling of nutrients and maintenance of water quality, knowledge of the microbiological composition of the communities adhering to the floc is necessary in order to provide adequate management and maximize the beneficial effects for the communities involved, such as the removal of nitrogenous compounds and the feeding of farmed fishes (12). Nevertheless, little information is available about the community structure of the microorganisms present in biofloc systems, the role played by the microbial biomass, and the applicability of this technology to native fish species of commercial importance.

Therefore, since the implementation of environmentally sustainable and economically profitable intensive systems of fish production is needed, the present work characterizes the planktonic communities present in the intensive culture of bocachico in the pre-growing phase at three stocking densities using biofloc technology.

 <bold>MATERIALS AND METHODS</bold>

Localization. The study was carried out at the Fish Culture Research Institute of the University of Córdoba (CINPIC, Montería, Córdoba, Colombia). A total of 630 bocachico fingerlings with 1.6±0.5 g average weight were stocked in nine rectangular, 6.0 m3 concrete tanks at three stocking densities—5 (T1), 10 (T2), and 20 (T3) fish/m3—to characterize the planktonic communities and microbiota associated with the culture of these bocachico with BFT in the pre-growing phase during 120 days of cultivation. The culture was maintained with permanent aeration supplied by a 1.5-HP blower and polyethylene diffuser tubing, and the tanks were covered with polyester mesh to reduce the light pass (60%) and to protect the fingerlings from predators.

Initial inoculum floc. The biofloc system was established with water from the bottom of a fish farming pond containing autotrophic and heterotrophic bacteria. For the promotion and development of the latter, molasses was added as a carbon source, doing so maintained the C:N ratio at nearly to 20:1. The molasses was applied using the following equation: Molasses = NT*(20-12), where 20 is the theoretical relation of C:N (20:1) for the BFT culture management, 12 is the relation of C:N (12:1) for the food rations containing 24% crude protein (2), and TN corresponds to the total amount of nitrogen in the culture, estimated via the Kubitza equation (13), where TN = [(amount of N-NH3 + N-NH4 + + amount of N-NO2 -) * volume of water] / 1000.

After the initial inoculum stabilization (14 days), based on the nitrogen compound management and the qualitative and quantitative characterization of the microorganisms present in the inoculum, 600 L of inoculum was transferred into each experimental unit, and the volume was filled to 6.0 m3 with CINPIC distribution water.

The water quality water was evaluated during stabilization and culture periods. Dissolved oxygen (DO), pH, and temperature were measured twice a day with an oximeter (YSI, 550A, USA) and a pH meter (YSI, pH100, USA). The total ammonium, nitrites, and nitrates were measured every two days, and the total hardness and total alkalinity were assessed every eight days with a photometer (YSI, 9500, USA).

Characterization of planktonic communities. Weekly, five 50-ml floc samples were taken at five different points of each culture tank and homogenized in a 250 ml Erlenmeyer flask, then 1 ml subsamples in triplicate were fixed with 10% buffered formaldehyde for the subsequent identification and quantification of the microorganisms associated with the flocs. The samples were analyzed using a Sedgwick-Rafter and/or Neubauer cameras, an optical microscope (Carl Zeiss, Axiostar, Germany), and a positive-phase-contrast inverted microscope (Carl Zeiss, Primo Vert, Germany) with objectives between 10x and 40x, and an image analyzer (Carl Zeiss, Axiovision 4, Germany). The identification of species in the different groups of plankton was carried out with the taxonomic keys of Aladro-Lubel et al (14), among others.

The abundance of microorganisms (A) per identified item was estimated with the equation: A=((Vcf)(Ni))/(Vti)Vc, where Vcf corresponds to the final volume of the concentration, Ni is the number of counted individuals, Vti is the initial total volume, and Vc is the volume of the sample analyzed. The abundance was expressed as the number of individuals/ml (Ind/ml). The relation between the number of species and their relative abundance in time and space was analyzed weekly by treatment. Also, three ecological indices were estimated: the Shannon-Wiener index (H’) for species diversity, the Pielou index (J’) for uniformity, and the Simpson index (λ) for organism dominance.

Bacterial isolation and characterization. The bacterial community analyses were carried out according to the Monroy-Dosta et al. method (15). From the formation of the small flocs in the initial inoculum, and at 15, 45, and 90 days of culture, 2 g samples were taken and inoculated in 90 ml of sterile saline, with successive dilutions (1:10), and 0.1 ml was seeded in triplicate in Man-Rogosa Sharpe (MRS), Heart-Brain Infusion (HBI), Thiosulfate-Citrate-Bile Salts (TCBS), and Trypticase-soy (TSA) agar boxes. The plates were incubated at 27°C for 24h, and then the colony-forming units (CFU/ml) were counted; subsequently, through successive reseeding, the strains were purified. Gram staining was used to observe the cell morphology via microscopic observations (Carl Zeiss, Axiostar, Germany). Conventional microbiological tests were performed on isolated strains for identification of catalase, oxidase, fermentation oxide, mobility, and indoles. Finally, the identification of the strains was confirmed with the API20E, API20NE, APICHL, and APICHL50 tests. In addition to the water column bacterial samples, at the end of the culture, using a mesh of 100 μm, a sample of mud was taken from the bottom of each experimental tank and analyzed.

Statistical analysis. A randomized experimental design was utilized. The variables were checked for normality (Shapiro-Wilk test) and homoscedasticity (Bartlett’s test), and then analysis of variance (ANOVA) was applied, followed by Duncan’s multiple range test. In all cases, a confidence level of 95% was used. All statistical analyses were performed with SAS for Windows 9.2 (SAS Institute Inc., Cary, NC, USA).

 <bold>RESULTS</bold>

The water quality during bocachico culture in the biofloc system is shown in Table 1. Dissolved oxygen presented averages above 6.0 mg/l; the temperature ranged between 28.6±0.3°C (T1) and 28.4±0.3°C (T3); the pH remained at 8.0, on average, in all three treatments; and the hardness ranged between 101.9±67.8 mg/l CaCO3 (T2) and 90.2 ± 52.4 mg/l CaCO3 (T1). No significant difference was observed (p>0.05) for any of these variables between the treatments. The alkalinity was significantly higher (p<0.05) in the treatments with higher densities (T2=114.5±57.9 mg/l CaCO3, T3=101.9±67.8 mg/l CaCO3). The highest averages of TAN (5.1±5.3 mg/l) and NH3 (2.3±2.4 mg/l) were recorded in T3, and these averages were significantly different from those for the other treatments (p<0.05). The values of NO2- maintained an average of 0.7 mg/L in all the treatments; the average values of NO3- were significantly higher (p<0.05) in the treatments with higher densities (T2=19.2±12.3 mg/l, T3=19.2±2.2 mg/l) (p>0.05).

Parameters of water quality during the cultivation of P. magdalenae in the pre-growing phase with biofloc technology. Table 1 Parameters of water quality during the cultivation of P. magdalenae in the pre-growing phase with biofloc technology.
T1 T2 T3
Temperature (°C) 28.6 ± 0.3a 28.5 ± 0.3a 28.4 ± 0.3a
OD (mg/l) 6.9 ± 0.3a 6.9 ± 0.2a 6.8 ± 0.3a
pH 8.0 ± 0.3a 8.0 ± 0.1a 8.0 ± 0.2a
Alkalinity (mg/l CaCO3) 95.3 ± 44.5b 114.5 ± 57.9a 108.6 ± 63.2a
Hardness (mg/l CaCO3) 90.2 ± 52.4a 101.9 ± 67.8a 101.5 ± 59.0a
TAN (mg/l) 2.6±1.1c 3.3±2.1b 5.1 ± 5.3a
NH3 (mg/l) 1.2 ± 0.5b 1.5 ± 1.0b 2.3 ± 2.4a
NO2- (mg/l) 0.7 ± 0.5a 0.7 ± 0.6a 0.7 ± 0.5a
NO3- (mg/l) 16.4 ± 11.2b 19.2 ± 12.3a 19.2 ± 2.2a
Different letters in the same row indicate a significant difference (p<0.05).

Planktonic microorganisms present during the stabilization and maturation of the floc inoculum. The microorganism taxa identified during the stabilization and maturation period of the floc inoculum are listed in Table 2. The copepods were the most abundant (13.0±0.0 ind/ml), showing a significant difference in abundance (p<0.05) from the other groups. Rotifers and microalgae were the groups with the greatest diversity of species. In the rotifers, the species Lecane luna (21.0±4.4 ind/ml) and Brachionus havanaensis (17±2.6 ind/ml) were the most abundant, while, for the microalgae, Scenedesmus securiformis (5.7±2.1 ind/ml) and Pinnularia sp. (4.0±1.0 ind/ml) predominated.

Microorganisms identified during the stabilization and maturation of the floc inoculum prior to the start of the cultivation of P. magdalenae in the pre-growing phase with biofloc technology. Table 2 Microorganisms identified during the stabilization and maturation of the floc inoculum prior to the start of the cultivation of P. magdalenae in the pre-growing phase with biofloc technology.
Microorganism ind/ml Mean±SD
Rotifers
Brachionus havanaensis 17.0±2.6 8.1±6.3
Gastropus sp. 3.7±3.2
Habratrocha sp. 5.0±2.6
Rotaria sp. 7.7±1.5
Euchlanis sp. 6.3±2.1
Lecane luna 21.0±4.4
Lecane sp. 13.0±1.0
Blepharisma undulans 3.7±0.6
Mytilina saw 2.0±1.0
Colurella sp. 2.3±0.6
Philodina sp. 7.7±1.5
Microalgae
Anabaena sp. 3.7±0.6 2.6±1.1
Acinetobacter baumannii is spherical 3.7±2.1
Actinastrum sp. 3.7±0.6
Asterococcus proud 3.7±1.5
Ankistrodesmus sp. 3.7±0.6
Coelosphaerium sp. 3.0±1.0
Square Crucigenia 2.0±1.0
Diatoma mesodon 1.7±0.6
Diatoma sp. 1.3±0.6
Dactylococcopsis acicularis 1.3±0.6
Gyrosigma attenuated 2.0±1.0
Monoraphidium contortum 2.0±1.0
Monoraphidium komarkova 1.3±0.6
Square microspore 1.7±0.6
Pinnularia sp. 4.0±1.0
Rhizosolenia sp. 2.3±1.5
Scenedesmus securiformis 5.7±2.1
Staurodesmus cuspidatus 3.3±1.5
Staurastrum gracile 2.0±1.0
Staurastrum boreal 2.3±1.5
Scenedesmus rectangularis 3.0±1.0
Scenedesmus platydiscus 3.0±1.0
Scenedesmus acuminatus 2.0±1.0
Scentesmus javanaensis 1.3±0.6
Staurodesmus extension 2.0±1.0
Treubaria sp. 1.7±1.6
Ulothrix sp. 2.0±1.0
Ciliates
Strombilidium sp. 3.0±1.0 4.8±2.5
Vorticellas sp. 5.0±1.0
Stombidium sp. 3.0±1.0
Paramecium sp. 8.3±3.2
Bacteria
Streptococcus margaritaceus 2.0±0.0 2.0±0.0
Protists
Euglena 5.0±2.6 4.0±0.9
Euglypha alveolate 3.7±3.2
Nebela sp. 3.3±1.5
Cladoceros
Alona 7.7±1.5 7.7±0.0
Copepods
Ciclopoids 13.0±0.0 13.0±0.0

Characterization of planktonic microorganisms at the different densities of bocachico culture. The zooplankton groups identified during the culture period in the pre-growing phase utilizing biofloc technology are shown in Table 3. The greatest abundance of zooplankton was registered in T1 (174.9±21.4 ind/ml) and the lowest in T3 (121.3±15.9 ind/ml), with a significant difference (p<0.05). In the lower stocking density tank (T1), a greater abundance of rotifers (35.6±18.6 ind/ml), protists (69.6±28.6 ind/ml), annelids (14.3±4.2 ind/ml), amoebas (11.4±2.3 ind/ml), and cladocerans (6.6±2.2 ind/ml) was observed, with these groups showing significant differences between stocking densities (p<0.05). In T1 (18.8±10.2 ind/ml) and T3 (13.9±12.1 ind/ml), the highest abundance of ciliates was recorded without significative differences (p>0.05). The group of copepods showed no significant difference between treatments (p>0.05), ranging from 5.2±1.5 ind/ml (T3) and 6.7±1.5 ind/ml (T1).

Abundance of zooplankton in the cultivation of P. magdalenae with biofloc technology. Table 3 Abundance of zooplankton in the cultivation of P. magdalenae with biofloc technology.
Microorganism Ind/ml
T1 T2 T3
Rotifers
Blepharisma undulans 20.0 ± 5.2a 14.0 ± 3.2a
Brachionus havanaensis 14.0 ± 0.5a 5.5 ± 0.3b 7.0 ± 0.4b
Colurella sp. 24.2 ± 1.5a 7.0±2.0c 13.7 ± 8.0b
Colurella uncinate 14.0 ± 3.6b 19.0 ± 8.2a 27.0 ± 5.2a
Euchlanis sp. 42.5 ± 18.5a 32.7±20.1b 30.1 ± 20.3b
Gastropus sp. 3.7 ± 0.5a 5.5 ± 0.2a 4.3 ± 1.0a
Habrotrocha cans 52.5 ± 25.3a 47.1 ± 28.2a 42.8 ± 22.0a
Habrotrocha sp. 33.5 ± 23.5b 21.4 ± 16.3b 53.0 ± 32.1a
Keratella sp. 2.0 ± 0.2b 9.5 ± 1.0a
Lecane luna 72.2 ± 25.6a 57.8 ± 32.4b 45.2 ± 25.3b
Lecane sp. 53.2 ± 16.5a 29.3 ± 12.6b 33.7 ± 19.5b
Mytilina sp. 150.0 ± 39.5a 10.0 ± 2.0b
Mytilina saw 41.6 ± 21.0b 48.7 ± 18.3a 37.7 ± 14.3b
Philodina sp. 9.1 ± 0.5b 4.2 ± 0.3c 14.6 ± 0.4ª
Polyarthra sp. 2.0 ± 0.0a
Rotaria sp. 1.0 ± 0.0b 2.5 ± 0.0a 2.3 ± 0.5a
35.6 ± 18.6a 23.4 ± 19.6b 21.7 ± 16.8b
Ciliates
Strombilidium. 24.0 ± 12.6a 8.0 ± 1.0b 5.0 ± 2.0b
Vorticellas sp. 37.0 ± 29.4a 17.8 ± 15.3b 21.3 ± 15.4b
Stombidium sp. 11.0 ± 0.5a 4.0 ± 0.5b 13.0 ± 2.5a
Euplotes charon 2.0 ± 1.0b 12.0 ± 2.8a
Euplotes patella 1.0 ± 1.0b 5.1 ± 2.2a 2.3 ± 1.2b
Paramecio sp. 37.7 ± 22.5a 20.0 ± 16.2b 28.1 ± 14.6a
18.8 ± 10.2a 11.1 ± 8.0b 13.9 ± 12.1a
Protists
Arcella magastoma 40.0 ± 2.0a
Arcella sp. 25.5 ± 2.5b 34.0 ± 2.5a 29.3 ± 2.5b
Arcella vulgaris 60.0 ± 22.4b 79.5 ± 31.8a 71.0 ± 28.6a
Centropyxis aculeata 38.0 ± 12.8a 21.7 ± 14.6b 27.0 ± 12.2a
Euglena sp. 57.2 ± 18.4a 56.9 ± 16.4a 40.0 ± 22.6b
Euglypha acanthophora 72.3 ± 32.1a 59.5 ± 28.6b 77.5 ± 36.4a
Euglypha alveolata 106.0 ± 36.2a 81.3 ± 24.8b 84.0 ± 41.2b
Euglypha sp. 130.0 ± 45.2a 4.0±1.0c 16.0 ± 4.3b
Nebela sp. 97.2 ± 36.2a 77.5 ± 28.6b 77.6 ± 30.2b
69.6 ± 28.6a 51.8 ± 31.4b 52.8±24.1b
Anelids
Monhytera similis 18.7 ± 5.6a 7.2 ± 2.6b 7.0 ± 1.8b
Aeleosoma sp. 9.9 ± 2.1a 9.5 ± 3.2a 11.5 ± 1.5a
14.3 ± 4.2a 8.4 ± 3.4b 9.3±2.1b
Amebas
Amebas sp. 14.0 ± 2.8a
Radiant astramoeba 8.8 ± 2.4a 6.3 ± 2.1a 5.5 ± 2.8a
11.4 ± 2.3a 6.3 ± 1.6b 5.5 ± 1.5b
Cladocerans
Moina sp. 6.8 ± 1.8a 5.6 ± 2.1a 5.2 ± 1.8a
Alona sp. 6.4 ± 2.2a 3.9 ± 2.5b 3.0 ± 1.8b
6.6 ± 2.2a 4.8 ± 2.2b 4.1 ± 2.8b
Copepods
Ciclopoids 6.7 ± 1.5a 5.7 ± 2.2a 5.2 ± 1.5a
Total 174.9 ± 21.4a 125.6±16.1b 121.3 ± 15.9b
Different letters in the same row indicate a significant difference (p<0.05).

Among the rotifers, Euchlanis sp., Habrotrocha lata, Habrotrocha sp., Lecane luna, Lecane sp., Mytilina sp., and Mytilina videns were the most abundant; among the ciliates, the morphospecies that were the most abundant were Vorticellas sp. and Paramecio sp. The most abundant protists were Arcella vulgaris, Euglypha acanthophora, Euglypha alveolata, Euglypha sp., and Nebela sp.

Table 4 shows the abundance of the species and morphospecies of phytoplankton identified. The most abundant were Ankistrodesmus acicularis (31.5±8.0 cells/ml, T1) (p<0.05), Ankistrodesmus sp. (25.5±6.4 cells/ml, T3) (p<0.05), Crucigenia quadrata (50.5±2.0 cells/ml, T1 and T2) (p>0.05), Monoraphidium komarkova (50.9 ±22.3 cells/ml, T3) (p<0.05), and Scenedesmus securiformis (30.2±10.2 cells/ml, T1) (p<0.05).

Abundance of phytoplankton species (cells/ml) identified during the cultivation of P. magdalenae with biofloc technology (Mean ± SD). Table 4 Abundance of phytoplankton species (cells/ml) identified during the cultivation of P. magdalenae with biofloc technology (Mean ± SD).
Microalgae T1 T2 T3
Actinastrum sp. 1.5±0.5 b 8.0±1.0 a 1.0±1.0b
Anabaena sp. 2.0±0.3 c 4.0±0.3 b 10.7±1.2 a
Anabaena sphaerica 6.0±0.5 b 1.0±0.0 c 13.7±2.5 a
Ankistrodesmus acicularis 31.5±8.0 a 11.0±3.0 b
Ankistrodesmus sp. 18.5±8.0 a 20.4±5.0 a 25.5±6.4 a
Asterococcus superbus 2.0±0.0 a 1.0±0.0 a 1.0±0.0 a
Coelosphaerium sp. 20.9±0.5 a 17.2±1.0 a 18.6±3.5 a
Crucigenia quadrata 50.5±2.0 a 50.5±3.5 a 41.6±12.6 a
Crucigenia tetrapedia 22.2±1.0 b 10.9±2.0 c 29.5±3.5 a
Dactylococcopsis acicularis 5.0±1.0 c 18.8±2.8 a 10.8±2.5 b
Diatoma mesodon 3.2±1.0 a 1.7±1.0 b 4.4±2.0 a
Diatoma sp. 6.0±1.0 a 2.5±1.0 b 2.0±0.5 b
Gyrosigma attenuatum 9.2±2.0 a 2.5±1.8 b 8.0±3.2 a
Microspora quadrata 24.5±12.0 a 28.0±16.5 a 16.4±4.2 b
Monoraphidium contortum 17.4±5.2 a 11.1±0.5 b 19.6±2.8 a
Monoraphidium komarkova 42.1±32.1 b 20.4±12.8 c 50.9±22.3 a
Oscillatoria sp. 20.0±5.0 a
Pediastrum clathratum 4.0±1.0 a 2.0±1.0 a
Pediastrum sp. 4.0±1.0 a
Pediastrum tetras 12.0±0.5 a 6.0±2.0 b
Pinnularia sp. 1.0±0.0 b 2.0±1.0 a 3.5±2.5 a
Rhizosolenia sp. 1.5±1.5 c 14.0±2.0 a 6.5±2.2 b
Rhizosolenia sp. 2.0±1.0 a 1.0±1.0 a
Scenedesmus acuminatus 7.0±1.5 b 6.0±2.0 b 16.7±8.5 a
Scenedesmus javanaensis 22.4±0.5 b 17.8±2.2 b 24.6±0.2ª
Scenedesmus platydiscus 1.5±0.5 b 11.0±2.4 a
Scenedesmus quadricauda 33.9±0.8 a 33.1±1.0 a 30.9±0.5 a
Scenedesmus rectangularis 1.0±1.0 b 1.0±1.0 b 14.0±2.0 a
Scenedesmus securiformis 30.2±10.2 a 20.1±12.4 b 27.7±16.4 a
Staurastrum boreale 4.0±2.0 b 8.0±1.0 a 2.0±1.0 c
Staurastrum gracile 2.5±1.5 a 1.5±1.5 a 2.0±1.0 a
Staurastrum sp. 4.5±1.2 b 3.2±2.5 b 8.0±2.0 a
Staurodesmus cuspidatus 1.0±1.0 a 1.5±1.5 a 2.0±1.0 a
Staurodesmus extensus 1.5±1.5 a 1.5±1.5 a 1.5±1.0 a
Stauroneis sp. 8.3±2.5 a 9.4±3.2 a 2.4±1.2 a
Tetraedron caudatum 2.0±1.0 a
Treubaria sp. 3.0±1.5 a 4.0±2.0 a 1.0±1.0b
Ulothrix sp. 2.0±1.0 a 2.0±1.0 a 5.0±2.5 a
11.9±13.9 a 9.9±9.4 a 13.1±11.2 a
Different letters in the same row indicate a significant difference (p<0.05).

Ecological indices. The ecological indices evaluated in the planktonic communities during cultivation are described in Table 5. The Shannon-Wiener index registered the highest value in T1 (2.5-3.2 bits/ml) and T2 (2.4-3.2 bits/ml), which were significantly different that found for T3 (1.8-2.8 bits/ml) (p<0.05), except for weeks 9, 10, 14, and 16 since no significant difference (p>0.05) was found between these weeks. The Pielou uniformity index was higher in T1 (0.8-0.9) and T2 (0.8-0.7) during the first two weeks of culture; the lowest values of this index (0.6-0.7) were recorded in T1 in weeks 9, 10, and 15; no significant difference was observed for the remaining weeks among the treatments. Except for the 6th and 12th week of culture, the dominance (Simpson index) did not present a statistic difference between the treatments, ranging between 0.7 and 0.9 (p>0.05).

Ecological indices of the planktonic microorganisms associated with the cultivation of P. magdalenae with biofloc technology. Different letters in the same row indicate a significant difference (P<0.05) between treatments for each week of culture. Table 5 Ecological indices of the planktonic microorganisms associated with the cultivation of P. magdalenae with biofloc technology. Different letters in the same row indicate a significant difference (P<0.05) between treatments for each week of culture.
Culture Weeks Diversity of Shannon-Wiener (bits/ind) Uniformity of Pielou Dominance of Simpson
T1 T2 T3 T1 T2 T3 T1 T2 T3
0 3.0± 0.0 a12 2.9± 0.0 a1 2.0± 0.0 b2 0.8± 0.2ª 1 0.7± 0.2ª 1 0.6± 0.2 b2 0.8± 0.2 a1 0.8± 0.2 a1 0.8± 0.1 a1
1 3.1± 0.0 a12 3.2± 0.0 a1 2.1± 0.0 b2 0.9± 0.0 a1 0.8± 0.0 a1 0.7± 0.0 b2 0.8± 0.2 a1 0.8± 0.1 a1 0.7± 0.1 a2
2 3.1± 0.1 a12 3.0± 0.1 a1 2.1± 0.1 b2 0.9± 0.0 a1 0.8± 0.0 a1 0.6± 0.0 b2 0.8± 0.2 a1 0.9± 0.1 a1 0.8± 0.1 a1
3 3.5± 0.1 a1 2.5± 0.1 b2 2.5± 0.1 b1 0.8± 0.0 a1 0.8± 0.0 a1 0.8± 0.0 a1 0.8± 0.2 a1 0.8± 0.1 a1 0.7± 0.1 a2
4 3.2± 0.0 a12 3.1± 0.0 a1 2.2± 0.0 b2 0.7± 0.1ª 1 0.7± 0.1ª 1 0.8± 0.1ª 1 0.8± 0.2 a1 0.8± 0.2 a1 0.7± 0.1 a
5 3.2± 0.2 a12 2.8± 0.2 a1 2.2± 0.2 b2 0.8± 0.5 a1 0.8± 0.5 a1 0.8± 0.5 a1 0.9± 0.1 a1 0.9± 0.1 a1 0.8± 0.1 a1
6 2.9± 0.1 a12 2.9± 0.1 a1 1.9± 0.1 b2 0.9± 0.4 a1 0.9± 0.4 a1 0.8± 0.4 a1 0.8± 0.2 a1 0.7± 0.1 b2 0.7± 0.1 b2
7 2.8± 0.5 a2 2.7± 0.5 a2 1.8± 0.5 b2 0.8± 0.2 a1 0.8± 0.2 a1 0.8± 0.2a1 0.8± 0.1 a1 0.7± 0.1 a2 0.8± 0.1 a1
8 2.8± 0.0 a2 2.6± 0.0 a2 2.0± 0.0 b2 0.8± 0.2ª 1 0.8± 0.2ª 1 0.9± 0.2ª 1 0.9± 0.1 a1 0.9± 0.1 a1 0.7± 0.1 b2
9 2.6± 0.1 a2 2.6± 0.1 a2 2.6± 0.1 a1 0.7± 0.1 b1 0.7± 0.1 b1 0.9± 0.1 a1 0.8± 0.1 a1 0.8± 0.1 a1 0.8± 0.1 a1
10 2.8± 0.2 a2 2.8± 0.2 a1 2.8± 0.2 a1 0.6± 0.2 b2 0.8± 0.2 a1 0.9± 0.2 a1 0.8± 0.2 a1 0.7± 0.1 a2 0.8± 0.2 a1
11 3.9± 0.2 a1 2.6± 0.2 b2 1.9± 0.2 c2 0.9± 0.0 a1 0.8± 0.0 a1 0.7± 0.0 b2 0.9± 0.1 a1 0.8± 0.1 a1 0.8± 0.1 a1
12 3.1± 0.0 a12 3.0± 0.0 a1 2.1± 0.0 b2 0.8± 0.2ª 1 0.8± 0.2ª 1 0.7± 0.2ª 2 0.9± 0.1 a1 0.7± 0.1 b2 0.7± 0.1 b2
13 3.8± 0.2 a1 2.8± 0.2 b1 2.2± 0.2 c2 0.8± 0.1ª 1 0.9± 0.1ª 1 0.6± 0.1 b2 0.9± 0.1 a1 0.8± 0.1 a1 0.8± 0.1 a1
14 2.6± 0.1ª 2 2.4± 0.1ª 2 2.2± 0.1ª2 0.8± 0.2ª 1 0.9± 0.2ª 1 0.8± 0.2ª 1 0.8± 0.2 a1 0.8± 0.2 a1 0.8± 0.2 a1
15 2.6± 0.0 a2 2.2± 0.0 b3 2.2± 0.0b2 0.7± 0.1 b1 0.9± 0.1ª 1 0.9± 0.1ª 1 0.9± 0.1 a1 0.9± 0.1 a1 0.9± 0.1 a1
16 2.5± 0.0 a2 2.5± 0.0 a2 2.3± 0.0 a2 0.8± 0.2ª 1 0.9± 0.2ª 1 0.8± 0.2ª 1 0.8± 0.2 a1 0.8± 0.1 a1 0.8± 0.1 a1
Different numbers in the same column indicate a significant difference within each treatment (p<0.05).

Bacterial characterization. In the initial inoculum, eight groups were identified (Table 6) and included bacterial colonies from the family Enterobacteriaceae, such as colonies of Escherichia coli, Enterobacter sp., Klebsiella sp., and Salmonella sp., which are characterized as gram-negative bacilli and occurred in concentrations of 2.0x102 to 22.0x107 CFU/ml. A group of sporulated Gram+ bacilli, i.e., Bacillus subtili, and Bacillus sp., were identified; these bacilli are related to the species Lactobacillus sp. Bacteria functionally described as heterotrophic, sulfite-reducing, cocci, and nitrogen-fixing were also identified.

Bacterial groups isolated in the period of stabilization and maturation of the floc inoculum used in the culture of P. magdalenae with biofloc technology. Table 6. Bacterial groups isolated in the period of stabilization and maturation of the floc inoculum used in the culture of P. magdalenae with biofloc technology.
Group Species Shape Gram stain UFC/ml
Heterotrophic totals Bacillus - 8x105
Enterobacterias (Enterobacteriaceae) Escherichia coli Bacillus - 5x106
Enterobacter sp. Bacillus - 22x107
Klebsiella sp. Bacillus - 2x102
Salmonella sp. Bacillus - 3x103
Sulfite-reducing bacteria Salmonella sp. Bacillus - 3x103
Total Bacillus Bacillus subtilis Sporulated bacillus + 12x106
Bacillus sp. Sporulated Bacillus + 10x106
Total Lactobacillus Lactobacillus sp. Bacillus + 7x103
Vibrionaceae Pseudomonas sp. Bacillus - 12x103
Gram+ Coccus Micrococcus sp. Coccus + 25x104
Staphylococcus sp. Coccus + 16x104
Nitrogen fixers Coccus and Bacillus +/- 20x106

The qualitative characterization of the bacterial groups identified (group/species), relative to their presence/absence during the four months of the culture of P. magdalenae with biofloc technology are listed in Table 7. In addition to the groups present initially in the inoculum, other colonies of bacteria were also present, such as Bacillus cereus, Vibrio cholerae, and Vibrio sp. The groups of bacterial observed in the mud at the bottom of the culture were similar to those identified previously, and heterotrophic bacteria was prevalent.

Bacterial groups identified in the different treatments evaluated in the cultivation of P. magdalenae in the pre-growing phase with biofloc technology. +: presence, -: absence. Table 7. Bacterial groups identified in the different treatments evaluated in the cultivation of P. magdalenae in the pre-growing phase with biofloc technology. +: presence, -: absence.
Genus/species Culture days 15 Culture days 45 Culture days 90
T1 T2 T3 T1 T2 T3 T1 T2 T3
Nitrogen fixers - + - - + + - + +
Heterotrophs + + + + + + + + +
Lactobacillus sp. - + - + + - - + +
Bacillus cereus - + - - + - - + +
Bacillus subtilis + + + + + + + + +
Bacillus sp. + + + + + + + + +
Pseudomonas sp. + + - + - - + - -
Vibrio cholerae - + - + + - - + +
Vibrio sp. + + + + + + + + +
Escherichia coli - - + - + + - + -
Klebsiella sp. + + + + + + + + +
Salmonella sp. - - - + - + + + +
Sulfite-reducing + + + + + + + + +
Enterobacter sp. + + + + + + + + +
Micrococcus sp. + + + + + + + + +
Staphylococcus sp. + - - - - + - - -

Figure 1 shows the CFU/ml of the groups of bacteria identified at 15, 45, and 90 days of culture. In T2, the highest CFU/ml were present on day 15 (373.000 CFU/ml) and day 90 (336.000 CFU/ml), whereas the lowest dominance was recorded in T3 on day 15 (73.667 CFU/ml) and day 90 (120.000 CFU/ml), with a significant difference between these treatments (p<0.05).

Number of bacteria (CFU/ml) at 15, 45, and 90 days of culture of P. magdalenae with biofloc technology. Figure 1 Number of bacteria (CFU/ml) at 15, 45, and 90 days of culture of P. magdalenae with biofloc technology.

<bold>DISCUSSION</bold>

In the present study, the parameters of water quality (OD, temperature, and pH) did not show differences between treatments; these parameters are considered optimal for the development of culture in biofloc systems (6,8,16) and are appropriate for the management of bocachico (17); also, the alkalinity and hardness are considered adequate for the maintenance and the nitrification route of the bacteria in the system (9,4,12).

The values of TAN and NH3 were higher at the highest density evaluated (20 fish/m3), which seems to be a consequence of the greater amount of food offered, along with the higher proportion of organic matter present in this treatment. In tilapia cultures with BFT, lower values were reported: between 1.9 and 2.5 mg/l of TAN (13). This result suggests that TAN values should be stabilized by bacterial activity or by the joint action between the planktonic communities and the microbiota, where the toxic fraction of these compounds is assimilated and incorporated for efficient recycling of the nutrients. NH3 is a compound that is toxic to fish, even at very low concentrations; concentrations above 1.5 mg/l in commercial-scale cultures are considered lethal (2,8,18), with acceptable levels being below 0.5 mg/l (19). In the present study, the average levels of NH3 were similar to those reported by several authors, who evaluated the behavior and dynamics of the biofloc system in the performance of fish and shrimp cultures, mostly with values not higher than 3.0 mg/l (9,20,21).

In BFT culture, high levels of nitrogen compounds (TAN, NH3) are present; but the toxicity of these compounds will depend on the management conditions and the degree of tolerance of the cultivated species. Since, although they were well above the optimum values for the management of the system (2,20,21), these levels did not affect the system’s management and the welfare of the species in culture, these findings suggest the tolerance of the species to the registered concentrations of these nitrogenous compounds. No reports of a medium lethal dose (LC50) were found in the literature for the bocachico; therefore, it is necessary to study the tolerance of this species to these nitrogen compounds.

The nitrite values remained similar in all treatments, whereas the nitrates at the higher stocking densities (T2 and T3) were higher. This dynamic can be attributed to the management offered as a function of the analyzed density, i.e., the food ration and therefore the organic matter load were greater in T3. In general, the water quality of the culture is associated with the management conditions of the system, with some parameters being affected; in particular, the nitrogen compounds were affected by the evaluated densities. Importantly, the alimentary habit of the bocachico (iliophagous-detritivorous) (22) is a factor to be considered when considering the adaptation of this cultivation system for species with this feeding habit.

The composition of the plant communities associated with the culture of bocachico in BFT suggests a culture system rich in microorganisms of biological and nutritional importance, with nutrient flow and availability of primary food. Six planktonic groups were identified (not including bacteria) in association with the macro-aggregates of floc, such as microalgae, rotifers, ciliates, cladocerans, protists, and copepods, with a greater abundance of the latter group being observed in the stabilization period of the inoculum, whereas there was a greater abundance of rotifers, protists, and ciliates during the pre-growing phase. These results agree with the reports of other authors that have characterized the planktonic communities of macro-aggregates during the conformation and stabilization of the inoculum, as well as in the development of cultures with this technology (8,12,15).

The abundance of microorganism groups was related to the stocking density; therefore, some pressure from the species on the abundance of the microorganisms present can be inferred, depending on the density of fish evaluated and the time of stabilization of the system. This pressure was described by Ballester et al (23) and Miaca et al (24), who suggested that the elements that produce the floccules, such as the carbon source and balanced feed as well as the fish in the system, can have a direct influence on the groups of developed organisms and their abundance to the cultivation time.

In the present study, the resuspension of solids in the water column was observed. This resuspension is suggested to be a consequence of the behavior and feeding habit of the bocachico (benthic-iliophagous-detritus), a phenomenon that has been observed in fish with similar behaviors (25), i.e., causing strong agitation on the bottom, increasing the turbidity, and facilitating the flow of nutrients through the trophic chain (26). Although excessive resuspension could affect primary productivity, possibly, at 20 fish/m3 (T3), excessive pressure from grazing will be felt in the generation and reproductive cycles of the microorganisms.

In the culture with the BFT systems, the interaction and dominance of the planktonic and bacterial communities are presented as a significant contribution in the protein and energy requirements for the species of farming interest. This contribution is made through the flow of nutrients through the established trophic chains from primary producers to secondary consumers, preferably through the consumption of microalgae and bacteria through continuous grazing in macroaggregates of floc (12,27).

The protists were the group with the highest density (Table 3). Protists are an important group in the elimination of contaminants, especially nitrogen compounds, contributing to the formation of bioaggregates and flocs (28); in the depredation of bacterial populations that can become pathogenic (29); and in the formation, distribution, and composition of the bacterial community (28).

The rotifers groups constitute another of the representative groups within the trophic chains present in the culture environments with BFT (12). In the present study, rotifers were the group with the second greatest density, which oscillated in the reported range (5 to 196 ind/ml) of a similar study (12,15). Associated in the same way with the dynamics of the system and with the formation of the macroaggregates, the ciliates (Vorticellas sp. and Paramecium sp.) were also characterized in the tilapia and shrimp cultures developed in the biofloc system (15,24,27).

When considering the less abundant species, the presence of Monhytera similis is noteworthy within the group of annelids, a group considered to be of great importance within the biofloc system due to its high content of crude protein and essential fatty acids; as well as species of the amoebas, the cladocerans, and the copepods. The genera Ankistrodesmus and Scenedesmus (Chlorophyta) were predominant in the microalgae, sometimes referenced for the quantity and quality of their nutrients and for the selective consumption of certain species of ciliates and rotifers that control microalgal populations (12).

The characterization of the groups above as microorganisms is recurrent in most studies of culture with BFT (12,15,16,23,27). Therefore, the establishment of a specific, dominant, uniform and diverse planktonic community is suggested, with characteristics such as reproductive strategy, small size, short life cycle, and broad tolerance to environmental factors. These microorganisms can also become established due to system conditions, such as carbon source, initial inoculum, and, as observed in the present study, according to the behavior and nutritional habits of the cultivated species.

The diversity of the microorganisms found showed a maximum value of 3.9 bits/ind, with high dominance (>0.7) and uniformity (>0.6). When identified by group, the rotifers and microalgae were the groups with the highest taxonomic richness and abundance during cultivation. These results allow us to infer that an index of low diversity may indicate that few species exist relative to the total of some individuals characterized in BFT systems. In general, the BFT system is not a closed production system, and, when selected inoculum are used, the pattern of microorganisms and planktonic communities associated with macroaggregates of floc is predictable.

The microbiota present affect the nutrient dynamics in the system; these were characterized for Enterobacteriaceae, Bacillus, Lactobacillus, heterotrophic, sulfite-reducing, Coccus, and nitrogen-fixing bacteria, agreeing with the reports of Monroy-Dosta et al. (15). Those authors when examining the microbial community associated with the biofloc in a tilapia culture, determined that heterotrophic bacteria, such as Pseudomonas, Bacillus, Vibrios, Enterobacter, and Micrococcus, were present.

This type of bacteria promotes defined routes in the elimination of toxic nitrogen compounds in farming systems. Accordingly, Ebeling et al. (4) defined the elimination of nitrogen by photoautotrophic algae, its immobilization by heterotrophic bacteria of the microbial protein biomass, and its chemo-autotrophic oxidation in nitrate by nitrifying bacteria as the main routes within the flow of nutrients in BFT culture systems. The relative importance of each one varies according to the type and intensity of the production system. In the present study, although immobilization of the heterotrophic bacteria was encouraged, the system was dominated by nitrifying bacteria.

Microbial communities in BFT systems are presented in a variety of ways and include opportunistic bacteria and pathogens as well as neutral and beneficial bacteria. The presence of colonies of the genera Bacillus and Lactobacillus, genera with probiotic characteristics in the development of fish culture, is emphasized mainly because these genera secrete a great variety of exoenzymes and polymers that generate a hostile environment for pathogenic bacteria (30). The maintenance of the system by the established bacterial communities is notable, although routes of nitrification and recycling of nitrogen compounds exist under the minimal conditions of nutrients and the management of water quality in suitable ranges for the cultivation of the species.

The results of the present study allow us to conclude that the composition of the planktonic and bacterial communities was similar in all the treatments, with the rotifer, protists, and ciliates groups being the most abundant, all sources of food for bocachico. The present microbiota is directly related to the dynamics of the system, with a higher quantity of enterobacteria and heterotrophic bacteria.

Interests Conflict.

The authors of this article declare no conflict of interest that puts their publication at risk.

 <bold>REFERENCES</bold> 1. Vinatea L, Gálvez A, Browdy C, Stokes A, Venero J, Haveman J, Lewis B, Lawson A, Shuler A, Leffler G. Photosynthesis, water respiration and growth performance of Litopenaeus vannamei in a super-intensive raceway culture with zero water exchange: interaction of water quality variables. Aquacultural Engineering 2010; 42(1):17–24. DOI: https://doi.org/10.1016/j.aquaeng.2009.09.001 Vinatea L Gálvez A Browdy C Stokes A Venero J Haveman J Lewis B Lawson A Shuler A Leffler G Photosynthesis, water respiration and growth performance of Litopenaeus vannamei in a super-intensive raceway culture with zero water exchange: interaction of water quality variables. Aquacultural Engineering 2009 42 1 17 24 10.1016/j.aquaeng.2009.09.001 2. Avnimelech Y. Biofloc Technology - A Practical Guidebook. The World Aquaculture Society; 2009. https://books.google.com.co/books/about/Biofloc_Technology.html?id=FI-CrgEACAAJ&redir_esc=y Avnimelech Y Biofloc Technology - A Practical Guidebook. The World Aquaculture Society; 2009 https://books.google.com.co/books/about/Biofloc_Technology.html?id=FI-CrgEACAAJ&redir_esc=y 3. Wasielsky J, Atwood H, Stokes A, Browdy C. Effect of natural production in a zero exchange suspended microbial floc based super-intensive culture system for white shrimp Litopenaeus vannamei. Aquaculture 2006; 258:396–403. DOI: https://doi.org/10.1016/j.aquaculture.2006.04.030 Wasielsky J Atwood H Stokes A Browdy C Effect of natural production in a zero exchange suspended microbial floc based super-intensive culture system for white shrimp Litopenaeus vannamei. Aquaculture 2006 258 396 403 10.1016/j.aquaculture.2006.04.030 4. Ebeling J, Timmons M, Bisogni J. Review of autotrophic and heterotrophic bacterial control of ammonia-nitrogen in zero-exchange production systems: stoichiometry and experimental verification. Aquaculture 2006; 257:346-358. DOI: https://doi.org/10.1016/j.aquaculture.2006.03.019 Ebeling J Timmons M Bisogni J Review of autotrophic and heterotrophic bacterial control of ammonia-nitrogen in zero-exchange production systems: stoichiometry and experimental verification. Aquaculture 2006 257 346 358 10.1016/j.aquaculture.2006.03.019 5. Avnimelech Y. Feeding with microbial flocs by tilapia in minimal discharge bio-flocs technology ponds. Aquaculture 2007; 264:140–147. DOI: https://doi.org/10.1016/j.aquaculture.2006.11.025 Avnimelech Y Feeding with microbial flocs by tilapia in minimal discharge bio-flocs technology ponds Aquaculture 2006 264 140 147 10.1016/j.aquaculture.2006.11.025 6. Ray A, Lewis B, Browdy C, Leffler J. Suspended solids removal to improve shrimp Litopenaeus vannamei, production and an evaluation of a plant based feed in minimal-exchange, superintensive culture systems. Aquaculture 2010a; 299:89–98. DOI: https://doi.org/10.1016/j.aquaculture.2009.11.021 Ray A Lewis B Browdy C Leffler J Suspended solids removal to improve shrimp Litopenaeus vannamei, production and an evaluation of a plant based feed in minimal-exchange, superintensive culture systems Aquaculture 2009 299 89 98 10.1016/j.aquaculture.2009.11.021 7. Ekasari J, Crab R, Verstraete W. Primary nutritional content of Bio-flocs cultured with different organic carbon sources and salinity. HAYATI Journal of Biosciences. 2010; 17(3):125-130. DOI: https://doi.org/10.4308/hjb.17.3.125 Ekasari J Crab R Verstraete W Primary nutritional content of Bio-flocs cultured with different organic carbon sources and salinity. HAYATI Journal of Biosciences. 2010 17 3 125 130 10.4308/hjb.17.3.125 8. Crab R, Defoirdt T, Bossier P, Verstraete W. Biofloc technology in aquaculture: Beneficial effects and future challenges. Aquaculture 2012; (356–357):351–356. https://doi.org/10.1016/j.aquaculture.2012.04.046 Crab R Defoirdt T Bossier P Verstraete W Biofloc technology in aquaculture: Beneficial effects and future challenges. Aquaculture 2012 (356–357) 351 356 10.1016/j.aquaculture.2012.04.046 9. Azim M, Littlea D. The biofloc technology (BFT) in indoor tanks: Water quality, biofloc composition, and growth and welfare of Nile tilapia Oreochromis niloticus. Aquaculture 2008; 283(1-4):29-35. https://doi.org/10.1016/j.aquaculture.2008.06.036 Azim M Littlea D The biofloc technology (BFT) in indoor tanks: Water quality, biofloc composition, and growth and welfare of Nile tilapia Oreochromis niloticus. Aquaculture 2008 283 (1-4) 29 35 10.1016/j.aquaculture.2008.06.036 10. Kunh D, Lawrence A, Boardman G, Patnaik S, Marsh L, Flick G. Evaluation of two types of bioflocs derived from biological treatment of fish effluent as feed ingredients for Pacific white shrimp, Litopenaeus vannamei. Aquaculture 2010; 303(1-4):28-33. https://doi.org/10.1016/j.aquaculture.2010.03.001 Kunh D Lawrence A Boardman G Patnaik S Marsh L Flick G Evaluation of two types of bioflocs derived from biological treatment of fish effluent as feed ingredients for Pacific white shrimp, Litopenaeus vannamei. Aquaculture 2010 303 (1-4) 28 33 10.1016/j.aquaculture.2010.03.001 11. Suita S, Ballester E, Abreu P, Wasielesky WJr. Dextrose as carbon source in the culture of Litopenaeus vannamei (Boone, 1931) in a zero exchange system. Latin American Journal of Aquatic Research 2015; 43(3): 526-533. http://www.lajar.cl/pdf/imar/v43n3/Articulo_43_3_13.pdf Suita S Ballester E Abreu P Wasielesky WJr. Dextrose as carbon source in the culture of Litopenaeus vannamei (Boone, 1931) in a zero exchange system. Latin American Journal of Aquatic Research 2015 43 3 526 533 http://www.lajar.cl/pdf/imar/v43n3/Articulo_43_3_13.pdf 12. Ray A, Seaborn G, Leffler J, Wilde S, Lawson A, Browdy C. Characterization of microbial communities in minimal-exchange, intensive aquaculture systems and the effects of suspended solids management. Aquaculture 2010b; 310:130–138. DOI: https://doi.org/10.1016/j.aquaculture.2010.10.019 Ray A Seaborn G Leffler J Wilde S Lawson A Browdy C Characterization of microbial communities in minimal-exchange, intensive aquaculture systems and the effects of suspended solids management. Aquaculture 2010 310 130 138 10.1016/j.aquaculture.2010.10.019 13. Kubitza F. Criacão de tilapia em sistema com bioflocos sem renovacão de água. Panorama da Aqüicultura 2011; 14-23. URL Disponible en http://www.acquaimagem.com.br/docs/Pan125_Kub_bioflocos_piscicultura.pdf . Kubitza F Criacão de tilapia em sistema com bioflocos sem renovacão de água. Panorama da Aqüicultura 2011 14 23 http://www.acquaimagem.com.br/docs/Pan125_Kub_bioflocos_piscicultura.pdf 14. Aladro-Lubel M. Manual de protozoarios [Manual of protozoa]. Faculty of Sciences, Autonomous University of Mexico, Mexico 2009; p 123 Aladro-Lubel M Faculty of Sciences, Autonomous University of Mexico 2009 123 Mexico Manual de protozoarios [Manual of protozoa]. 15. Monroy-Dosta M, De Lara-Andrade R, Castro-Mejia J, Castro-Mejia G, Coelho-Emerenciano M. Composición y abundancia de comunidades microbianas asociadas al biofloc en un cultivo de tilapia. Rev Biol Mar Oceanogr. 2013; 48(3):511-520. http://dx.doi.org/10.4067/S0718-19572013000300009 Monroy-Dosta M De Lara-Andrade R Castro-Meji J Castro-Mejia G Coelho-Emerenciano M Composición y abundancia de comunidades microbianas asociadas al biofloc en un cultivo de tilapia. Rev Biol Mar Oceanogr. 2013 48 3 511 520 10.4067/S0718-19572013000300009 16. Emerenciano M, Gaxiola G, Cuzon G. Biofloc Technology (BFT): A Review for Aquaculture. Application and Animal Food Industry. INTECH 2013; 12:301-327. DOI: http://dx.doi.org/10.5772/53902 Emerenciano M Gaxiola G Cuzon G Biofloc Technology (BFT): A Review for Aquaculture. Application and Animal Food Industry. INTECH 2013 12 301 327 10.5772/53902 17. Garcia JJ, Celis LM, Villalba EL, Mendoza LC, Brú SB, Atencio VJ, Pardo SC. Evaluation of the bocachico polyculture Prochilodus magdalenae and Oreochromis niloticus tilapia using perifiton fixing surfaces. Rev Fac Med Vet Zoot. 2011; 58(2):71-83. https://revistas.unal.edu.co/index.php/remevez/article/view/25398 Garcia JJ Celis LM Villalba EL Mendoza LC Brú SB Atencio VJ Pardo SC Evaluation of the bocachico polyculture Prochilodus magdalenae and Oreochromis niloticus tilapia using perifiton fixing surfaces. Rev Fac Med Vet Zoot. 2011 58 2 71 83 https://revistas.unal.edu.co/index.php/remevez/article/view/25398 18. Hargreaves J. Biofloc Production Systems for Aquaculture. SRAC 2013; 4503:8-10. https://aquaculture.ca.uky.edu/sites/aquaculture.ca.uky.edu/files/srac_4503_biofloc_production_systems_for_aquaculture.pdf Hargreaves J Biofloc Production Systems for Aquaculture SRAC 2013 4503 8 10 https://aquaculture.ca.uky.edu/sites/aquaculture.ca.uky.edu/files/srac_4503_biofloc_production_systems_for_aquaculture.pdf 19. Pérez-Fuentes J, Hernández-Vergara M, Pérez-Rostro C, Fogel I. C:N ratio affect nitrogen removal and production of nile tilapia Oreochromis niloticus raised in a biofloc system under high density cultivation. Aquaculture 2016; 452:247-251. https://doi.org/10.1016/j.aquaculture.2015.11.010 Pérez-Fuentes J Hernández-Vergara M Pérez-Rostro C Fogel I C:N ratio affect nitrogen removal and production of nile tilapia Oreochromis niloticus raised in a biofloc system under high density cultivation. Aquaculture 2015 452 247 251. 10.1016/j.aquaculture.2015.11.010 20. Schveitzer R, Arantes R, Costódio P, Espírito-Santo C, Arana LV, Seiffert W, Andreatta E. Effect of different biofloc levels on microbial activity, water quality and performance of Litopenaeus vannamei in a tank system operated with no water exchange. Aquacultural Engineering 2013; 56:59–70. https://doi.org/10.1016/j.aquaeng.2013.04.006 Schveitzer R Arantes R Costódio P Espírito-Santo C Arana LV Seiffert W Andreatta E Effect of different biofloc levels on microbial activity, water quality and performance of Litopenaeus vannamei in a tank system operated with no water exchange. Aquacultural Engineering 2013 56 59 70 10.1016/j.aquaeng.2013.04.006 21. Lorenzo M, Souza E, Schledera D, Rezendea P, Seifferta W, Vieiraa F. Intensive hatchery performance of Pacific white shrimp in the biofloc system under three different fertilization levels. Aquacultural Engineering 2016; 72-73:40–44. https://doi.org/10.1016/j.aquaeng.2016.04.001 Lorenzo M Souza E Schledera D Rezendea P Seifferta W Vieiraa F Intensive hatchery performance of Pacific white shrimp in the biofloc system under three different fertilization levels. Aquacultural Engineering 2016 72 73 40 44 10.1016/j.aquaeng.2016.04.001 22. Marrugo-Negrete J, Navarro-Frómeta A, Ruiz-Guzman J. Total mercury concentrations in fish from Urrá reservoir (Sinú river, Colombia). Six years of monitoring. Rev MVZ Córdoba 2015; 20(3): 4754-4765. https://doi.org/10.21897/rmvz.45 Marrugo-Negrete J Navarro-Frómeta A Ruiz-Guzman J Total mercury concentrations in fish from Urrá reservoir (Sinú river, Colombia). Six years of monitoring. Rev MVZ Córdoba 2015 20 3 4754 4765. doi.org/10.21897/rmvz.45 23. Ballester E, Abreu P, Cavalli R, Emerenciano M, Abreu L, Wasielesky W. Effect of practical diets with different protein levels on the performance of Farfantepenaeus paulensis juveniles nursed in a zero exchange suspended microbial flocs intensive system. Aquaculture Nutrition 2010; 16: 163-172. https://doi.org/10.1111/j.1365-2095.2009.00648.x Ballester E Abreu P Cavalli R Emerenciano M Abreu L Wasielesky W Effect of practical diets with different protein levels on the performance of Farfantepenaeus paulensis juveniles nursed in a zero exchange suspended microbial flocs intensive system Aquaculture Nutrition 2009 16 163 172 10.1111/j.1365-2095.2009.00648.x 24. Maicá P, Borba M, Wasielesky W. Effect of low salinity on microbial floc composition and performance of Litopenaeus vannamei (Boone) juveniles reared in a zero water-exchange super-intensive system. Aquaculture Research 2012; 43:361-370. https://doi.org/10.1111/j.1365-2109.2011.02838.x Maicá P Borba M Wasielesky W Effect of low salinity on microbial floc composition and performance of Litopenaeus vannamei (Boone) juveniles reared in a zero water-exchange super-intensive system. Aquaculture Research 2011 43 361 370 10.1111/j.1365-2109.2011.02838.x 25. Della Rosa P, Ortiz J, Cáceres A, Sánchez S, Roux J. Desempeño del sábalo Prochilodus lineatus en policultivo con pacú Piaractus mesopotamicus. Lat Am J Aquat Res 2016; 44(2):336-341. http://dx.doi.org/10.3856/vol44-issue2-fulltext-14. Della Rosa P Ortiz J Cáceres A Sánchez S Roux J Desempeño del sábalo Prochilodus lineatus en policultivo con pacú Piaractus mesopotamicus. Lat Am J Aquat Res 2016 44 2 10.3856/vol44-issue2-fulltext-14. 26. Milstein A, Ahmed AF, Masud OA, Kadir A, Wahab MA. Effects of the filter feeder silver carp and the bottom feeder’s mrigal and common carp on small indigenous fish species (SIS) and pond ecology. Aquaculture 2006; 258 (1-4): 439-451. https://doi.org/10.1016/j.aquaculture.2006.04.045 Milstein A Ahmed AF Masud OA Kadir A Wahab MA Effects of the filter feeder silver carp and the bottom feeder’s mrigal and common carp on small indigenous fish species (SIS) and pond ecology. Aquaculture 2006 258 (1-4) 439 451 10.1016/j.aquaculture.2006.04.045 27. Loureiro K, Wilson W, Abreu P. Use of protozoa, rotifers and nematodes as live feed for shrimp cultured in the BFT system. Atlântica Rio Grande2012; 34(1):5-12. https://doi.org/10.5088/atl.2012.34.1.5 Loureiro K Wilson W Abreu P Use of protozoa, rotifers and nematodes as live feed for shrimp cultured in the BFT system. Atlântica Rio Grande 2012 34 1 5 12 10.5088/atl.2012.34.1.5 28. Abreu C, Ballester E, Odebrecht C, Wasielesky WJr, Cavalli R, Granéli W, Anésio A. Importance of biofilm as food source for shrimp (Farfantepenaeus paulensis) evaluated by stable isotopes. J Exp Mar Bio Ecol. 2007; 347 (1-2):88–96. https://doi.org/10.1016/j.jembe.2007.03.012 Abreu C Ballester E Odebrecht C Wasielesky WJ Cavalli R Granéli W Anésio A Importance of biofilm as food source for shrimp (Farfantepenaeus paulensis) evaluated by stable isotopes. J Exp Mar Bio Ecol. 2007 347 (1-2) 88 96 10.1016/j.jembe.2007.03.012 29. Wu L, Peng C, Peng Y, Li L, Wang S, Ma Y Effect of wastewater COD/N ratio on aerobic nitrifying sludge granulation and microbial population shift. Journal Environment Science. 2012; 24(2):234-241. https://doi.org/10.1016/S1001-0742(11)60719-5 Wu L Peng C Peng Y Li L Wang S Ma Y Effect of wastewater COD/N ratio on aerobic nitrifying sludge granulation and microbial population shift. Journal Environment Science. 2012 24 2 234 241 10.1016/S1001-0742(11)60719-5 30. Monroy D, Castro B, Fernández P, Mayorga R. Inhibition of Aeromonas hydrophila by probiotic strains isolated from the digestive tract of Pterophyllum scalare. Rev Mex Ing Quím 2010; 9(1):37-42. http://rmiq.org/iqfvp/Pdfs/Vol9%20no%201/RMIQ_Vol9_No_1_5.pdf Monroy D Castro B Fernández P Mayorga R Inhibition of Aeromonas hydrophila by probiotic strains isolated from the digestive tract of Pterophyllum scalare. Rev Mex Ing Quím 2010 9 1 37 42 http://rmiq.org/iqfvp/Pdfs/Vol9%20no%201/RMIQ_Vol9_No_1_5.pdf