Rearing of the Pacific white shrimp Litopenaeus vannamei (Boone, 1931) in BFT system with different photoperiods: Effects on the microbial community, water quality and zootechnical performance
Wellica G. Reis, Wilson Wasielesky, Paulo C. Abreu, Hellyjúnyor Brandão, Dariano Krummenauer
PII: S0044-8486(19)30295-9
DOI: https://doi.org/10.1016/j.aquaculture.2019.04.067
Reference: AQUA 634101
To appear in: aquaculture Received date: 5 February 2019 Revised date: 22 April 2019 Accepted date: 25 April 2019
Please cite this article as: W.G. Reis, W. Wasielesky, P.C. Abreu, et al., Rearing of the Pacific white shrimp Litopenaeus vannamei (Boone, 1931) in BFT system with different photoperiods: Effects on the microbial community, water quality and zootechnical performance, aquaculture,https://doi.org/10.1016/j.aquaculture.2019.04.067
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Rearing of the Pacific white shrimp Litopenaeus vannamei (Boone, 1931) in BFT system with different photoperiods: effects on the microbial community, water quality and zootechnical performance
1 Wellica G. Reis, 2Wilson Wasielesky Jr, 3Paulo C. Abreu, 2Hellyjúnyor Brandão,
1Dariano Krummenauer*
1Laboratory of Ecology of Microrganisms Applied to Aquaculture, Institute of
Oceanography, Federal University of Rio Grande - FURG, C. P. 474, Rio Grande (RS), CEP 96201-900, Brazil.
*Corresponding Author: [email protected]
2Laboratory of Carcniculture, Institute of Oceanography, Federal University of Rio Grande - FURG, C. P. 474, Rio Grande (RS), CEP 96201-900, Brazil.
3Laboratory of Phytoplankton and Marine Microrganisms, Institute of Oceanography, Federal University of Rio Grande - FURG, C. P. 474, Rio Grande (RS), CEP 96201- 900, Brazil.
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2 Abstract
In Biofloc Technology (BFT) Systems, there are changes in microbial communities during a shrimp culture cycle; usually, heterotrophic bacteria are replaced by a photoautotrophic or chemoautotrophic community, depending on the management employed. When the water is exposed to sunlight, it can abruptly change from a heterotrophic system (dominated by bacteria and protozoa) to a predominantly photoautotrophic system, with a dominance of microalgae. However, there is little information on the characterization and quantification of the microbial community in the culture of the Pacific white shrimp Litopenaeus vannamei in a BFT system with light restriction, especially the contribution of these microrganisms to the zootechnical performance of shrimp. The aim of this study was to evaluate the microbial community in a rearing environment with light restriction. The study was conducted in 12 tanks (800 L) with a storage density of 500 shrimp m-³ and an initial shrimp weight of 0.053 g. The experiment was designed with three treatments and four replicates distributed into the following groups: i) natural photoperiod - 12 h light/12 h dark (NP); ii) 24 h light (24hLI); and iii) 24 h dark (24hDA). Significant differences were found in water quality parameters (lower values of ammonia and nitrite in the 24hDA treatment), and higher values of CO2, light (Ix) and chlorophyll were observed for the NP and 24hLI treatments than for the 24hDA treatment (p<0.05). Zootechnical performance registered higher values in final weight, final biomass, weekly growth rate and survival in the NP and 24hLI treatments than in the 24hDA treatment (p<0.05). Proximal analysis of the biofloc showed higher concentrations of proteins and lipids in the NP and 24hLI
treatments than in the 24hDA treatment (p<0.05), and there was a greater abundance of bacteria (attached coccoid, free filamentous, and attached filamentous bacteria, Vibrio
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and Bacillus) in NP and 24hLI than in 24hDA (p<0.05). There was a greater abundance of photoautotrophic flagellates, heterotrophic flagellates, ciliates, dinoflagellates, rotifers and nematodes in NP and 24hLI than in 24hDA (p<0.05). The results show that light restriction favored nitrification. However, light exposition promoted an increased abundance of microrganisms, providing an additional food source and reflecting a lower rate of feed conversion, higher survival and better growth performance for L. vannamei.
Keywords
Heterotrophic; Chemoautotrophic; Luminosity; Nitrification; Super intensive
Abbreviations
DO (DissolvedOxygen), TSS (Total Suspended Solids), EE (Ethereal Extract), ENN (Non-Nitrogen Extracted), FCR (Feed Conversion Ratio)
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4 1. Introduction
The BFT (Biofloc Technology) System is a technology designed to increase productivity while improving environmental control over production, reducing or eliminating water exchange, reducing waste, minimizing rearing area, restricting the spread of diseases, and increasing biosecurity in the culture of shrimp and fish (Krummenauer et al., 2014; Avnimelech., 2015; Samocha et al., 2017). The bioflocs consist of aggregates colonized by bacteria, microalgae, protozoa, zooplankton, and nematodes as well as feces and leftover feed (Hargreaves., 2013; Lara et al., 2017).
They can also serve as a complementary food source for reared organisms, reflecting higher growth, weight gain, and survival and lower feed conversion (Wasielesky et al., 2006; Ballester et al., 2010). According to Cardona et al. (2015), bioflocs can contribute approximately 37 to 40% of natural productivity, which in turn stimulates digestive enzyme activities; this increased activity may contribute to promoting the growth of shrimp reared in biofloc.
In the BFT system, changes in microbial communities occur during a culture cycle. Usually, photoautotrophic or chemoautotrophic community replacement occurs depending on the management used. Exposure to sunlight can abruptly change the community from a heterotrophic system (mainly dominated by bacteria and protozoa) (Kirk., 2010; Hargreaves., 2013) to one with photoautotrophic characteristics dominated by microalgae. The relative importance of each photoautotrophic and heterotrophic process depends on many factors, such as daily feed rate, suspended solids
concentration, ammonia concentration, light intensity and the carbon-nitrogen ratio (C/N) (Avnimelech., 1999., 2015). Inside the biofloc, there are different communities
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that can control nitrogenous compounds in water, such as photoautotrophic,
heterotrophic and autotrophic nitrifying bacteria (Ebeling et al., 2006; Crab et al., 2012).
The heterotrophic process is based on removing ammonia (TA-N) by assimilation of this nitrogen compound into the bacterial biomass as protein. This incorporation occurs through the manipulation of the carbon and nitrogen ratio (C/N) in the system, that is, by the addition of an organic carbon source in the form of
carbohydrate (Avnimelech e Kochba, 2009). This strategy allows for acceleration of the removal of inorganic nitrogen quickly and efficiently, reducing the total ammonia concentration in the water (Ebeling et al., 2006). In the chemoautotrophic process, small amounts of bacterial biomass are produced, mainly due to the slow growth rate of nitrifying bacteria. These bacteria perform oxidation of ammonia to nitrite (ammonia- oxidizing bacteria - AOB) and later to nitrate (nitrite-oxidizing bacteria - NOB) (Ebeling et al., 2006; Crab et al., 2007). In this way, it is difficult to have full control over the bacterial communities that will form along the rearing process due to the complexity of the interactions that take place within the culture systems (Hargreaves., 2013).
In the BFT system, when exposed predominantly to sunlight, a dense bloom of photoautotrophic organisms (microalgae) develop in response to the input of light and nutrients that come from food and decomposing organic matter, such as dead
microrganisms, feces and unconsumed rations (Hargreaves., 2013; Samocha et al., 2017). When there is a primary dependence on photoautotrophic organisms in this system, the concentration of ammonia can increase in cloudy weather conditions, and a fluctuation in dissolved oxygen concentration and pH can also occur, despite extra aeration (Avnimelech., 2015).
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Light is one of the factors that can modify the abundance of microalgae consisting of cyanobacteria groups, diatoms, green algae, and dinoflagellates, among others, resulting in intense blooms of species that are often toxic. These blooms can be detrimental to water quality, affecting animal development and producing toxins that can cause physiological stress and even shrimp mortality (Alonso-Rodriguez et al., 2003; Wasielesky et al., 2012; Samocha et al., 2017).
A complex mixture of processes between algae and bacteria controls the quality of the water in BFT systems. Basically, there are two types of production with bioflocs; those that are exposed to natural light, such as ponds and outdoor tanks
(intensive rearing), are normally located in tropical or subtropical regions where there is abundance of natural light where photoautotrophic organisms predominate, resulting in the water having greenish colour (Ebeling et al., 2006; Prangnell et al., 2016). In environments in temperate regions, rearing is carried out in greenhouses or indoors (super-intensive culture) without exposure to natural light. In these greenhouse systems, the colour of the water tends to be brown, where bacterial processes control the quality of the water (Hargreaves., 2013; Samocha et al., 2017). However, this variation in water colour will depend exclusively on the microbial composition of each culture, and water colour is not an accurate indicator of system maturity status (Hargreaves., 2013).
Light is considered an abiotic factor of paramount importance for organisms living in an aquatic environment. Studies show significant differences in behaviour, growth, feed intake, maturation, and reproduction and possibly changes in the swimming activity of penaeid shrimp when exposed to different conditions of
luminosity (Gardner et al., 1998; Guerra-Santos et al., 2017). That is, luminous intensity can directly affect the animals being reared. Thus, the light intensity, when appropriate
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for shrimp in farms, provides advantages such as increased productivity and reduction of production, food and electricity costs (Coyle et al., 2011).
The importance of light on the performance of penaeids have already been described by several authors, however, the results are, sometimes, not conclusive. For instance, in a study carried out by Baloi et al. (2013), it was observed that the
zootechnical performance of L. vannamei reared in bioflocs in the absence of light showed significant differences in feed conversion and survival. On the other hand, Fleckenstein et al. (2019) investigating the supplemental effects of light on the performance of L. vannamei in intensive systems found that the presence of light can improve shrimp production, whereas Esparza-Leal et al. (2017) reported the
zootechnical performance of the same species exposed to light limitation at low salinity, also in BFT system, was not significantly affected by the light limitation.
Besides, there is little information on the characterization and quantification of the microbial community of the culture of the Pacific white shrimp L. vannamei in the BFT system with light restriction and especially on the contribution of these
microrganisms to the zootechnical performance of shrimp, so studies to elucidate this gap are of paramount importance. In this context, the aim of this study was to evaluate the microbial community in the culture of the Pacific white shrimp L. vannamei in a BFT system with light restriction (natural photoperiod, 24 hours light and 24 hours dark).
2.0 Materials and Methods 2.1 Culture conditions
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The experiment was conducted between October 16 and 25 December 2017 at the Aquaculture Marine Station - Institute of Oceanography of the Federal University of Rio Grande - FURG, located in the city of Rio Grande, Cassino - RS, Brazil (32º 19 'S, 52º 15' W). The species used in the experiment was the Pacific white shrimp
Litopenaeus vannamei. The post-larvae (Speedline Aqua) were obtained from
Aquatec® LTDA (Rio Grande do Norte). The shrimp were first kept in a pre-nursery facility at the Shrimp Production Laboratory at EMA for a period of approximately 30 days until reaching an average weight of 0.053 ± 0.010 grams. Then, animals were transferred to the experimental units for the beginning of the experiment.
2.2 Experimental design
The water used in each experimental unit was previously pumped from the Cassino beach. Prior to the experiment, the water was chlorinated using a concentration of 10 ppm and left to act for four h before being neutralized using ascorbic acid (vitamin C) in the proportion of one gram per thousand litres of water. The water was transferred to twelve rectangular cement tanks with a capacity of 1000 litres, and a useable volume of 800 litres with a bottom surface area of 1.4 m² was used. In each experimental unit, an aeration system with a microperforated hose (aerotubes) supplied by an air blower (Ibram® LTDA) of 2 CV was installed. The stocking density was 500 shrimp m-³, and the experiment simulated a direct stocking (nursery and grow-out phase) and lasted 25 days.
For the development of the experiment, three treatments were carried out with four replicates each, as follows: 1) NP - natural photoperiod with natural light during
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the day and covered with a black plastic sheet at night; 2) 24hLI (24 h light) with natural light during the day and artificial lighting during the night, using a fluorescent lamp of 80 watts; and 3) 24hDA (24 h dark) with tanks covered with black plastic for 24 h. The amount of light that reached the surface of the water (ambient light) was
measured at two different points in the tank daily at 12:00 with the aid of a Chauvin Arnoux® (C.A 810 model) luxmeter device.
2.3 Biofloc Formation
To stimulate the formation of bioflocs, the C/N ratio of the system was
maintained at 15/1, according to the methodology proposed by Avnimelech (1999) and Ebeling et al. (2006). Organic fertilization was performed using sugar cane molasses with 37.27% carbon; to maintain a C/N ratio of 15/1, 6.0 g of carbon (molasses) was added for every 1.0 g of total ammonia nitrogen (TA-N) measured in the water.
Moreover, commercial probiotics (INVE® Sanolife PRO-W) were applied once a week in a proportion of 1.0 grams per thousand litres of water to assist in the maintenance of water quality in all treatments.
2.4 Shrimp feeding and monitoring
Shrimp were fed twice daily using the commercial feed Potimar GUABI®
(40% crude protein in the nursery phase and 38% in the grow-out phase). To control feed consumption, 10% of the daily quantity of feed was offered in food trays, and the remainder was spread in the tank. The feeding rate of the nursery determined was according to the method revised by Jory et al. (2001); while in the grow-out phase, adjustments in feeding rates were conducted following the method described Yta by Garza et al. (2004).
2.5 Zootechnical performance
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The zootechnical performance of the shrimp was monitored weekly by measuring the mean weight of 30 animals per experimental unit using a digital scale with a precision of 0.01 g (Marte® scientific AS5000C). At the end of the experiment, we also evaluated other weekly zootechnical performance indices (Bagenal and Tesch, 1978; Van Wyk and Scarpa, 1999) by the following calculations:
WG = (FW - IW) / n weeks of culture
where WG = weight gain, IW = initial weight, FW = final weight;
FCR = feed offered / biomass increase where FCR = apparent feed conversion rate;
Survival: (final biomass / individual mean weight) / number of individuals stocked) x100; and Productivity: (final biomass - initial biomass) / tank volume.
2.6 Water quality parameters
The physical and chemical parameters that were monitored twice a day were temperature and dissolved oxygen (YSI® Pro 20) and pH (pH meter® FE 20 / FG2).
Salinity was checked every three days (Hach® HQ40d). Turbidity was determined once a week with a turbidimeter (Hach® 2100P, Hack Company). Water samples were collected daily to quantify total ammonia nitrogen (N-(NH3 + NH4+
)) and nitrite (N- NO2). When values exceeded the safe level, water renovations were carried out at a rate of 20%. Ammonia concentrations were determined according to the methods of
UNESCO (1983) and Strickland & Parsons (1972). The alkalinity was analysed every three days according to the methodology proposed by APHA (2012). When alkalinity reached values below 150 mg CaCO3. L-1 and/or the pH reached values below 7.6, adjustments were made following the methodology described by Furtado et al. (2011)
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and Zhang et al., (2017). The quantities of the chemical compounds required to correct pH and alkalinity were calculated according to the equations below:
K= Nf – Ni
K= where K is the level to be increased (pH or CaCO3L-1); Nf = is desired level (pH or CaCO3L-1); Ni = is current level (pH or CaCO3L-1).
Y = (W*K)/Z
Y = where Y is the chemical compound concentration to be used gL-1; W = tabulated gL-1; Z = is the capacity of W in increasing the parameter (pH or alkalinity).
Nitrate (N-NO3-
) and phosphate (P-PO4-
) concentrations were examined weekly using the methodology of Strickland and Parsons (1972). Suspended solids (ml L-1) were measured one time per week using an Imhoff cone with readings recorded after 15–20 min, following the method of Eaton et al. (1995) adapted by Avnimelech (2007). Water samples were filtered with GF50-A glass fibre filters using a vacuum pump (Prismatec®) to determine the total suspended solids concentration. The levels of total suspended solids were maintained up to 500 mg. L-1 as recommended by Gaona et al. (2011), and when values exceeded the recommended value, clarifiers were used (Ray et al., 2010; Gaona et al., 2011).
2.7 Proximate composition of biofloc
At the end of the experiment, the bioflocs were collected from tanks for analysis of the proximal composition. These samples were filtered through a 50-micron mesh. Subsequently, the samples were dried in an oven for 48 h at a temperature of 60 ºC until the displayed constant weight. The analyses were carried out at the Aquatic Organisms Nutrition Laboratory at EMA according to AOAC (2000) protocols. For ash quantification (MM), the samples were dried and then taken to a muffle furnace at
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600°C for five h. The analysis of crude protein was performed according to the Kjeldahl method, where previous digestion of the samples occurs prior to distillation and titration of the nitrogen, multiplying all the results by 6.25. The ethereal extract value was obtained using the warm extraction method by the Soxhlet extractor, using petroleum ether as solvent, for six h. For the crude fibre analysis, acidic and basic digestion of the sample was used for 30 min in each digestion, followed by burning of the residue in a muffle furnace at 500 ºC for one h, and the value of was obtained from the difference.
2.8 Microbial community assessment
For quantification of the microrganisms present in the culture water, water samples (20 mL) were collected once a week from each experimental unit. The samples were fixed in 4% formalin (final concentration) and kept in amber flasks for further counting and identification of the main microorganism groups present. For
determination of the abundance of bacteria, fixed samples were filtered on polycarbonate membrane filters (Nuclepore, 0.2 µm and 2.5 mm pore diameter) previously darkened with Irlan Black and stained with 1% acridine orange at a
concentration of 1 μg / mL (Hobbie et al., 1977). Bacteria were photographed using a camera coupled to an epifluorescence Axioplan- Zeiss microscope, with a final
magnification of 1000 X, for later counting of 30 randomly chosen fields. For protozoa, an Olympus IX51 inverted microscope with 200x final magnification was used, where aliquots of 2.1 mL of sample were placed in a sedimentation chamber and 30 random fields were counted (Utermöhl, 1958).
The microrganisms were sorted into different groups: for bacteria: free coccoid, attached coccoid, free filamentous, and attached filamentous bacteria, Vibrio and Bacillus; and for protozoa: autotrophic flagellates, heterotrophic flagellates, ciliates,
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amoeba and groups rotifers, nematodes, dinoflagellates. Counting was performed at the Laboratory of Ecology of Phytoplankton and Marine Microrganisms at FURG.
2.9 Analysis of chlorophyll a
The concentration of chlorophyll was determined once a week from 10 ml samples of culture water filtered through glass fibre GF 50a microfilters. The extraction of the photosynthetic pigment was carried out by immersing the filters in 10 mL of 90%
acetone (Merck® PA), which were packed in bottles and kept in a freezer (-12 ºC).
After a period of 24 h, the chlorophyll concentration was measured using a Turner Designs Trilogy fluorimeter. The concentration of chlorophyll a was determined by a fluorimeter (Welschmeyer, 1994).
2.10 Statistical analysis
The data were submitted to homoscedasticity of the variances and normality of the data distribution analyses. When the assumptions were not met, data were submitted to statistical transformations. Subsequently, a one-way analysis of variance - ANOVA (α = 0.05) was applied followed by a post hoc Tukey's test when significant differences were found (Zar 2010).
3.0 Results
The mean values (±SD) of the chemical and physical parameters of water during the 70-day study period are shown in (Table 1). Variables such as ammonia, nitrite, chlorophyll a, CO2 and light (Ix) were significantly greater in treatments exposed to light than in the treatment that was not (p<0.05). The other variables showed no significant differences between treatments (p> 0.05).
There was no significant difference between treatments (p> 0.05) for pH nor alkalinity, with a maximum value of 8.26 in the 24hDA treatment and a minimum of 7.6
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pH in all treatments. For alkalinity, there was a maximum of 160 mg L-1 for 24hLI and a minimum of 120 mg L-1 for 24hDA. In all treatments, corrections were made to
maintain the levels of alkalinity required for the species under study. However, in the 24hDA treatment, the corrections were performed one week before the other treatments.
Differences were observed (p <0.05) in the concentrations of total ammonia nitrogen (TA-N) among treatments, especially after the 11th day of the experiment when an increase in the concentration occurred with maximum values of 2.7 mg L-1 in the NP treatment and 2, 4 mg L-1 in the 24hLI treatment, whereas the 24hDA treatment reached 1.5 mg L-1 in the same period. After the seventh day, the ammonia concentration
decreased more rapidly in the 24 h dark treatment than in the other treatments (Figure 1A).
The highest values of nitrite were 32 mg L-1 in the 24hLI treatment, 28 mg L-1 in the NP treatment and 23 mg L-1 in the 24hDA treatment. It is worth mentioning that we worked with a safety level of nitrite of 26 mg L-1. In the NP and 24hLI treatments, a renewal of 20% of the tank volume was performed when they exceeded safety levels, whereas in the 24hDA treatment, no renewal was performed. The 24hDA treatment showed a decrease in nitrite concentrations before the other treatments, initiating the decrease on the 31st day and presenting values not detectable after the 40th day (Figure 1B).
Figure 1C shows nitrate levels throughout the experiment. There was no
significant difference between treatments (p> 0.05), with a maximum value of 97 mg L-
1 obtained in the 24hDA and 24hLI treatments and 89 mg L-1 for the NP treatment.
Chlorophyll a presented significant differences (p <0.05). The maximum value obtained
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was 250 μg L-1 for the 24hLI treatment. The dark treatment (24hDA) presented a residual chlorophyll a of 2.4 μg L-1.
Figure 2A shows free coccoid bacteria concentrations throughout the
experiment. There was no significant difference between treatments (p> 0.05), with a maximum of 2.6x106 and a minimum of 0.6x106 in the NP treatment. For attached coccoid bacteria, there was a maximum of 2.5x105 for NP and a minimum of 0.1x105 for 24hDA. There were significant differences among the treatments at 15 days (p
<0.05) (Figure 2B).
Figure 2C shows the free filamentous bacteria concentrations throughout the experiment. There was a significant difference between treatments at 43days (P< 0.05), with a maximum of 2.4x106 for 24hLI and a minimum of 0.4x106 for 24hDA. For attached filamentous bacteria, there was a maximum of 17.8x104 for NP and a minimum of 0.6x104 for NP and 24hLI. Significant differences were observed among the
treatments on days 43and 70(p <0.05) (Figure 2D).
Figure 2E shows the Vibrio concentrations throughout the experiment. There was a significant difference between treatments on days 15and 43 (P< 0.05), with a maximum of 4.2x104 for 24hLI and a minimum of 0.2x104 for 24hDA. For Bacillus, we observed no significant differences among the treatments (p> 0.05), where there was a maximum of 8.3x105 for NP and a minimum of 2.3x105 for 24hDA (Figure 2F).
Figure 2G shows photoautotrophic flagellate concentrations throughout the experiment. There was a significant difference between treatments on days 15, 43and 70 (P<0.05), with a maximum of 10x103 for NP and a minimum of 0.1x103 for
24hDA. For heterotrophic flagellates, there was a maximum of 29x102 for NP and a
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minimum of 2x102 for 24hLI. There were significant differences among the treatments on days 43and 70(p<0.05) (Figure 2H).
Figure 2I shows the ciliate concentrations throughout the experiment. There was a significant difference between treatments on days 15,43and 70 (P<0.05), with a maximum of 4.3x103 for NP and a minimum of 0.3x103 for 24hDA. For nematodes, there was a maximum of 14x102 for 24hDA and a minimum of 0.1x102 for 24hLI and NP. There were significant differences among the treatments at 70days (p<0.05) (Figure 2J).
Figure 3 presents photomicrographs of microrganisms in the three treatments at 15 and 70 days of the experiment. Treatments with light showed a higher abundance of flagellates and filamentous bacteria (Figures 3A and 3B) in comparison to those of the dark treatment (24hDA) (Figure 3E and 3F). It is also possible to identify the presence of amoeba (3C) and free coccoid bacteria (3D) as shown in the micrographs.
The results of proximal analysis of bioflocs after 70days of study are presented in Table 2.Crude protein and lipid quantification showed significant differences (p<0.05) among treatments.
The results of zootechnical performance analysis for L. vannamei shrimp on the 70th day of study are shown in Table 3.The treatments exposed to light presented higher values of final weight, final biomass, feed conversion, survival, weekly growth after one gram and growth rate than those for the 24 h dark treatment, with significant differences (p<0.05).
4.0 Discussion
4.1 Influence of light on water quality
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There were statistically significant differences for the results of ammonia, nitrite, chlorophyll a, CO2 and lux quantification in the present experiment; nonetheless, all levels were within the safe level for the raised species. Other parameters such as
temperature, dissolved oxygen, pH, nitrate, phosphate, alkalinity, total suspended solids, and turbidity did not show significant differences. All these variables are within the range found acceptable for the species under study and thus probably caused no difference in performance indices (Van Wyk and Scarpa, 1999; Gaona et al., 2011;
Furtado et al., 2011; Zhang et al., 2017).
In this study, significant differences were found in ammonia levels between treatments with light exposure (NP) and (24hLI) and that with light restriction (24hDA), but the values did not reach safety levels, and the maximum value recorded was 2.7 mgL-1. It should be noted that in the BFT system, the ammonia concentration is
controlled by adding an organic carbon source (Avnimelech. 2009). It is known that the growth rate of heterotrophic bacteria is significantly faster than that of autotrophic bacteria. Therefore, with the rapid growth of these bacteria, a fast removal of ammonia from water occurs by incorporation into bacterial biomass (Ebeling et al., 2006;
Avnimelech. 2009).
The safety levels for nitrite concentrations in L. vannamei cultures are 15.2-25.7 mg. L-1 for salinities of 25-35, respectively (Lin and Chen. 2003). In the present study, we worked with a safe level of 26 mg of NO2. L-1 and exchange of 20% of the total tank volume was carried out in the treatment in which nitrite levels exceeded the safe level.
In general, it was observed that nitrification in the 24hDA treatment was better, which can be explained by the photosensitivity of the nitrifying bacteria and by competition for nutrients.
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Nitrifying bacteria respond to light in different ways. In a study by Guerrero (1996), the authors observed photosensitivity of nitrifying bacteria when exposed to different wavelengths and colours of light. They observed inhibition of the activity of nitrifying bacteria when exposed to sunlight, but the effect of light on bacteria depends not only on the type of bacteria but also on environmental conditions. In another Vergara study (2016), they also observed light sensitivity of nitrifying bacteria, where irradiation with 500 and 1250 µmol m2 s-1 caused a reduction of 20 to 60% of the ammonia removal rates and approximately 26 to 71% of the nitrite removal rates. This photosensitivity may explain why in the dark, nitrification occurred in advance and more readily compared to in the treatments exposed to light. Light restriction provided a favourable culture environment for the growth of these bacteria, avoiding a possible inhibition of the activity of nitrifying bacteria.
In addition, there is competition for nutrients in the culture system. For each gram of the consumed ammonium, the amount of 3.13 g / g NH4+
of alkalinity for photoautotrophic bacteria, 7.05 g / g NH4+
of alkalinity for chemoautotrophic and 3.57 g / g NH4+ of alkalinity for heterotrophic carbonates is necessary (Ebeling et al., 2006).
Thus, in cases in which we have this type of bacteria in the culture, there is competition for carbonates and nitrogen compounds. This fact may explain why nitrification in the treatment with light restriction was better than in the treatments exposed to light. In 24hDA, the heterotrophic bacteria presented better growth, providing a source of nutrients available for the bacteria (carbonates and nitrogen compounds). Consequently, there is less competition with photoautotrophic organisms for nutrients. Highlighting that finding in the 24hDA treatment, more corrections were performed to maintain
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alkalinity, asthis treatment had decreased levels of alkalinity before the other treatments.
Dissolved carbon dioxide (CO2) in cultured water is the result of respiration of organisms grown along with the microbial community in the culture system (Van Wyk and Scarpa. 1999);in systems that work with high stocking densities, there is an increase of CO2.The culture tanks exposed to light showed higher survival rates and more quantification of microrganisms in the tank than those of the light-restricted treatment, generating higher concentrations of CO2.According to Furtado (2016), adequate levels are less than 5 mg L-1 CO2 during the culture of L. vannamei to reduce oxygen consumption. In the present study, there was a significant difference between NP and 24hLI and 24hDA treatments. However, the highest value found in the
experiment was 4.32 mg CO2 L-1, which did not exceed the safety values recommended by the literature.
4.2 Influence of light on the abundance of microrganisms
Variations in the culture environment, such as nutrients, natural competition of communities between different species and light can influence the primary productivity and predominance of specific microrganisms’ groups adapted to the established
environmental conditions (Irigoien and Castel. 1997; Pereira Neto et al., 2008). In this study, it was possible to verify the abundant presence of microrganisms during the culture period in different photoperiods. That is, in treatments exposed to light, there was greater abundance of bacteria such as free coccoid, attached coccoid, free
filamentous, and attached filamentous bacteria and Bacillus and protozoan groups such as autotrophic flagellates, ciliates, amoeba and group rotifers and when compared to those in treatment with total light restriction.
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In environments with abundant light, there is a tendency of microalgae to establish first, and these organisms serve as a basic food source for the development of a zooplanktonic and nutrient community; in addition to providing supplementary feed for shrimp, they also provide nutrients for the growth of bacteria in the water culture system (Ju et al., 2008). In the present study, the abundance of microrganisms
corresponded to the culture environment; that is, treatments exposed to light showed a higher abundance of photoautotrophic organisms, while light-restricted treatments had a higher concentration of heterotrophic organisms. In general, it is possible to observe that the presence of light had a positive influence on bacteria and protozoa, with significant differences (p <0.05) between treatments.
The presence of light influenced attached coccoid bacteria compared with treatment with light restriction, especially in the first two weeks of study, and
subsequently there was a significant increase over the treatment period in all treatments.
In a study carried out by (Rocha et al., 2012) on the formation of microbial flakes formed by two shrimp species (L. vannamei and Farfantepenaeus paulensis), which may be related to the abundance of biofloc-forming bacteria, mainly coccoid bacteria, there was a close relationship between the amount of aggregates and the presence of attached coccoid bacteria.
In general, the presence of light in FPN and 24hCL treatments contributed positively to the abundance of free and attached filamentous bacteria. This fact may be related to the higher availability of organic carbon dissolved in the water, especially at the beginning of the experiment when we introduced an external source of organic carbon (molasses). Anesio et al. (2003) found that free and attached bacteria act in different ways in the environment, and when there is a high availability of organic
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carbon dissolved in the water, a high abundance of free bacteria occurs; after that period, the attached bacteria predominate, leading to detrital aliment binding at higher trophic levels.
The amount of Vibrio decreased during the study, which occurred after the increase of probiotic bacteria that contain high bacilli concentrations. With this result, the inverse relationship between the Vibrio and bacilli bacteria in the culture tank became clear, proving the antagonistic effect caused by probiotic bacteria in the water.
This result corroborates findings of authors that found better control of Vibrio and an increase in Bacillus-type bacteria when using probiotics in the BFT system
(Krummenauer et al., 2014; Hostins et al., 2017). In a study conducted by Esparza-Leal et al. (2017) where L. vannamei was exposed to low-salinity and light- limiting bioflocs, the authors observed diversity in the total counts of bacteria such as Vibrio and bacilli.
However, in the present study, it was not possible to identify the species of Vibrio.
There was a higher abundance of ciliates in the system in treatments exposed to light. The ciliates function as a water quality indicator (Decamp et al., 1999). In the present study, it was possible to find a higher amount of flagellates and ciliates in the light-exposed treatments compared to that in the light-restriction treatment, which had higher concentrations of heterotrophic flagellates. Throughout the study period, there was a decrease in the concentration of flagellates and a corresponding increase in the concentration of ciliates. This fact may be related to predation of autotrophic flagellates by ciliates, occurring in the existence of trophic interactions between microrganisms in culture tanks.
Nematodes are one of the most important groups in bioflocs. In addition, the predation rate of the cultured organisms can interfere with their concentration (Ray et
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al., 2010). This low concentration may be related to the predation of nematodes by shrimp that thus decreased predation on bacteria, causing a greater increase in their concentration "Top Down" (Carperter and Kitchell, 1993). However, an increase in nematode concentration was observed in the final period of the study, mainly in the light-restricted treatment (24hDA), seeming to have no predation of nematodes by shrimp. Therefore, the presence of light positively influenced the microbial community, both in abundance and in trophic levels in the cultivation system.
4.3 Influence of light on Proximal Analysis
Microrganisms are not only known as important sources of protein but also for supplying shrimp’s lipid, mineral and vitamin requirements. In addition, they serve as a potential source of exogenous enzymes that aid in digestion (Decamp et al., 2003;
Ballester et al., 2010; Becerril-Cortés et al., 2018). The different photoperiods influenced the abundance of the microbial community, and consequently, there were alterations in the lipid and protein levels of the bioflocs with and without light
restriction but no differences in ash and fiber levels.
The percentage of lipids in bioflocs exposed to light was significantly higher than the values recorded by Wasielesky et al. (2006); Ballester et al. (2010) and Emerenciano et al. (2012) (0.49, 0.47 and 0.47%, respectively) and was similar to the values found by Silva et al. (2013) and Fugimura et al. (2015) (2.48 and 2.57%, respectively). This high lipid concentration in the present study may be related to an increased abundance of flagellates, filamentous bacteria, ciliates, coccoid bacteria and bacilli in the NP and 24hsLI treatments. The flagellates are a source of polyunsaturated fatty acids (PUFAS) and sterols (Decamp et al., 2003). A higher abundance of
microrganisms can contribute to a greater amount of that nutrient. In addition,
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nematodes, heterotrophic flagellates, filamentous cyanobacteria, ciliates and unice llular heterotrophic bacteria also have a high relationship with lipids and function as an important lipid source for shrimp (Silva et al., 2008; Rocha et al., 2012).
The levels of proteins of the bioflocs for treatments exposed to light were significantly higher than those of the treatment with light restriction. Protein values were similar to those reported by other authors (Ballester et al., 2010; Wasielesky et al., 2012; Gaona et al., 2016). The levels of proteins found in the present study may be related to the abundance of microrganisms and their protein contribution in each treatment. According to Silva et al. (2008), nematodes, coccoid bacteria and Bacillus- type bacteria are important protein sources. In the NP and (24hLI treatments, higher concentrations of coccoid and bacilli bacteria were observed than in the 24hDA treatment, so these organisms may have contributed to the protein values in the treatments. These results corroborate those of Rocha et al. (2012), who found that bioflocs with a higher level of crude protein, close to 30%, had a higher abundance of coccoid-type bacteria, while bioflocs with lower protein levels showed a lower
abundance of coccoid-type bacteria in the same period.
The levels of ash of the microbial flakes in the culture of juvenile L.vannamei shrimp in the BFT system were similar to the high ash levels found by the authors
Wasielesky et al. (2006), Silva et al. (2008), and Gaona et al. (2016) (44.85%, 45.50 and 55.51%, respectively). Tacon et al. (2002) found that flakes are rich in phosphorus, calcium, potassium, and magnesium, among others. The abundance of fibre
corroborates that observed by Silva et al. (2013), Arias Moscoso et al. (2016) and Gaona et al. (2016).
4.4 Influence of light on zootechnical performance
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The presence of light in the culture of L. vannamei positively influenced growth performance. Microbial community abundance and changes in animal behaviour associated with the presence of light may help explain these differences. Studies show that Pacific white shrimp have the ability to take advantage of natural productivity in BFT farming systems (Hargreaves, 2013; Samocha et al., 2017). Natural food serves as a supplementary source of nutrients for shrimp, reflecting zootechnica l performance in weight gain and feed conversion rates (Wasielesky et al., 2006; Becerril-cortés et al., 2018). The L. vannamei display activity in the light and dark phase; however, this activity varies according to specific behaviors. Swimming performance occurs mostly at night, and exploration in the substrate (search for food) occurs in both phases, but the animals tend to initiate food consumption during the clear phase hours (Pontes and Arruda, 2005; Pontes, 2006).
This study showed variations in survival rate with significant differences
between treatments (NP, 24HLI and 24hDA); treatments under light exposure achieved higher survival rates than those of the light-restricted treatment. These results
corroborate those of Wasielesky et al. (2012) and Baloi et al. (2013), and the results for treatments with light restriction were similar to those described by Neal et al. (2010).
Although it was not possible to identify the dinoflagellates, the low survival rate in the treatment with total light restriction could be attributed to the presence of the order Gymnodiniales since some species of Gymnodiniales have some toxicity (Smayda, 2002). Moreover, non-photosynthetic dinoflagellates absorb dissolved organic compounds and can survive in unenlightened depths, and in water and sediment, and they benefit in the summer months (Taylor et al., 2008).
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The feed conversion rates of the present study in treatments exposed to light (natural photoperiod and 24 h light) are considered low when compared to other studies with light restriction (24hDA) in biofloc systems (Wasielesky et al., 2012; Baloi et al., 2013; Esparza- Leal et al., 2017), and the results of the treatment with light restriction (24hDA) are similar to those of Neal et al. (2010) and Baloi et al. (2013). The presence of light provided greater availability of microrganisms such as bacteria, protozoa and photoautotrophic organisms in the culture system, thereby generating a biofloc with a higher concentration of protein and lipid that served as a supplemental source reflecting better FCR.The growth rate and final weight were significantly higher in treatments exposed to light (NP and 24hLI) when compared to those of the light restriction treatment. In the NP and 24hLI treatments, an abundance of microrganisms, mainly photoautotrophic organisms, contributed to the primary production of food, influencing the lipid rates in the bioflocs. The greater abundance of microrganisms can also
contribute to the protein content in the flake; however, this fact may have influenced the growth of shrimp, resulting in a greater specific weight. The performance results are different from those reported by Esparza-Leal et al. (2017) and similar to Guo et al.
(2012), Wasielesky et al. (2012) and Baloi et al. (2013).
The restriction of light provided better nitrification, avoiding photoinhibition of the nitrifying bacteria and generating a better quality of water due to the growth of heterotrophic bacteria. Thus, the nutrients were destined for these bacteria, avoiding competition with photoautotrophic organisms for nutrients. On the other hand, the presence of light resulted in a greater abundance of bacteria, protozoa and
photoautotrophic organisms, which generated higher concentrations of lipids and proteins, contributing as an additional source of food for the shrimp.
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The presence of light positively influences the abundance of microrganisms, reflecting a better performance of L. vannamei.The results suggest that photoperiod treatment (NP) is indicated for culture in the BFT system.
6.0 Acknowledgements
The authors are grateful for the financial support provided by the National Council for Scientific and Technological Development (CNPq), Coordination for the Improvement of Higher Level Personnel (CAPES) and FAPERGS, Research Support Foundation of the State of Rio Grande do Sul State. Wasielesky, W.Jr. and ABREU, P.C.A. are a research fellow of CNPq under process number: 310652/2017-0 and 303256/2018-4 respectively. Special thanks to Centro Oeste Rações S.A. (GUABI) and AQUATEC, TREVISAN and Al Aqua for donating the experimental diets and post-larvae and Aeration system respectively.
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