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Effect of aeration intensity on the biofilm nitrification process during the production of the white shrimp Litopenaeus vannamei (Boone, 1931) in Biofloc and clear water systems
Aquaculture 514 (2020) 734516
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Effect of aeration intensity on the biofilm nitrification process during the production of the white shrimp Litopenaeus vannamei (Boone, 1931) in Biofloc and clear water systems
T
Ana Paula Mariane de Moraisa, Paulo Cesar Abreub, Wilson Wasielesky Jrc, ⁎ Dariano Krummenauera, a
Laboratory of Ecology of Microorganisms Applied to Aquaculture, Institute of Oceanography, Federal University of Rio Grande, FURG, C. P. 474, Rio Grande, RS CEP 96201-900, Brazil Laboratory of Phytoplankton and Marine Microorganisms, Institute of Oceanography, Federal University of Rio Grande, FURG, C. P. 474, Rio Grande, RS CEP 96201900, Brazil c Laboratory of Mariculture, Institute of Oceanography, Federal University of Rio Grande, FURG, C. P. 474, Rio Grande, RS CEP 96201-900, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Nitrifying bacteria Nitrogen compounds Ammonia Nitrite
Artificial substrates have great importance for the establishment of the biofilm, and their use in the culture systems represents a complementary source of food, increase space for animals and aid in the metabolism of nitrogen compounds. Nitrifying bacteria present in biofilm play an important role in the maintenance of water quality, and several factors such as pH, temperature, salinity and dissolved oxygen can interfere in the establishment and efficiency of these bacterial communities. However, there is not much information in the literature on the influence of aeration intensity on the bacterial community present in the biofilm. Thus, the objective of this study was to determine the response of nitrifying bacteria present in the biofilm submitted to different aeration intensities during the production of Litopenaeus vannamei (Boone, 1931) in a clear water system and also with bioflocs. The study was composed of two experiments, where the first experiment was carried out without shrimp and consisted of four treatments with three replicates, in 800 Liter tanks distributed in: 1) W/Air (control - without aeration); 2) V7.5 (flow rate 7.5 L/min); 3) V33.75 (flow rate of 33.75 L/min) and V75 (flow rate of 75 L/min). All treatments use “Needlona®” as artificial substrate (Needlona® - 100% polyester fiber; 250 g/m2 weight; 1.4 mm thickness; 0.18 g/cm3 density), in the proportion of 200% of the lateral area of the tank. Experiment two was established after the results of the previous experiment, with three treatments and three replicates each: 1) BFT (biofloc, with flow rate of 20.00 L/min); 2) BFT + BF (biofloc and biofilm with flow rate of 33.75 L/min) and 3) BF (biofilm with flow rate of 33.75 L/min), in which the shrimp (7.89 ± 0.24 g) were stocked in 9 tanks (800 L) with a density of 500 shrimps m−3. In both experiments Ammonia and nitrite were measured daily, while nitrate was analyzed weekly. The first experiment showed no difference in the ammonia concentrations of the different treatments, whereas nitrite showed higher concentrations in the treatment without aeration. The 33.75 L/min flow rate was chosen for experiment 2 to be compared with the aeration normally employed in our systems (20.00 Liter/min). The nitrification process was more efficient in the treatments with biofilm and bigger air flow rate, presenting smaller concentrations of ammonia and nitrite in comparison to the BFT treatment. Similarly, treatments with biofilm and stronger flow rate showed better zootechnical performance of the shrimp.
1. Introduction
may be a limiting factor for primary production in these ecosystems, but can also be toxic for aquatic organisms when present in higher concentrations (Vieira, 2017). In the nitrification process, the successive oxidation of ammonia to
Nitrogen is an important nutrient for living organisms, as it is an essential component for the constitution of proteins and nucleic acids. It
Abbreviations: AOB, Ammonia-Oxidizing Bacteria; NOB, Nitrite-Oxidizing Bacteria; TAN, Total Ammonia Nitrogen; BFT, Biofloc Technology; EMA/FURG, Marine Aquaculture Station; N-NO2−, Nitrite; CaCO3, Alkalinity; N-NO3−, Nitrate; TSS, Total Suspended Solids ⁎ Corresponding author. E-mail address: [email protected] (D. Krummenauer). https://doi.org/10.1016/j.aquaculture.2019.734516 Received 8 July 2019; Received in revised form 11 September 2019; Accepted 13 September 2019 Available online 14 September 2019 0044-8486/ © 2019 Elsevier B.V. All rights reserved.
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nitrite and subsequently to nitrate is mainly made by autochemolitotrophic microorganisms (Ebeling et al., 2006) belonging to two groups of bacteria. The first, the ammonia-oxidizing bacteria (AOB) are responsible for the oxidation of ammonia to nitrite. Most of these organisms belong to the genera Nitrosomonas, Nitrosococcus, Nitrosospira, Nitrosolobus and Nitrosovibrio. The second group, the nitriteoxidizing bacteria (NOB), performs the conversion of nitrite to nitrate, and the majority of these microorganisms belong to the genera Nitrobacter, Nitrococcus, Nitrospira and Nitrospina (Ebeling et al., 2006; Madigan et al., 2016; Lara et al., 2016). Recent studies have demonstrated that microorganisms of the Archaea domain also participate in the nitrification process (Ward, 2013). In the production of aquatic organisms, like fish and shrimp, higher concentrations of nitrogen compounds as ammonia and nitrite can become a problem, when they accumulate in the aquatic environment due to the excreta of the produced organisms, decomposition of unconsumed foods and organic waste (Timmons and Ebeling, 2010). Therefore, the control of nitrogen elements within the breeding environment is important, since they can cause damage to the produced shrimp and fish (Lin and Chen, 2001, 2003). The concentration of ammonium (TAN) in the medium increases with increasing pH and water temperature, and reduces with increasing salinity (Boyd and Tucker, 2012) Exposure to inadequate concentrations of these compounds can cause stress, triggering various physiological changes, and compromising performance leading to death, thereby impairing production (Girotto, 2010). Nitrite, on the other hand, binds to hemocyanin, transforming it into metahemocyanin, preventing the transport of oxygen to tissues and reducing the amount of oxygen available for metabolism (Tahon et al., 1988). This process can lead to hypoxia and, consequently, mortality of organisms produced (Chen et al., 1986). An efficient aeration system is important for the oxygen supply to produce animals, and to keep the flocs in suspension in the BFT system. However, low concentrations of dissolved oxygen limit or suppress nitrification (Avnimelech, 2009; Zhu et al., 2008), since the nitrifying bacteria, AOB and NOB present a demand for oxygen for cellular activity, growth and reproduction. Thus, in order to carry out the nitrification process it is essential that these microorganisms settle in the growing environment like bioflocs and biofilm, but also have ideal environmental conditions to absorb and transform nitrogen compounds. The biofilm can be defined as an organic matrix adhered to any submerged substrate, which is colonized by a microbial community composed of bacteria, protozoa, fungi and algae (Ramesh et al., 1999). It has been shown that biofilm is responsible for removing nitrogenous compounds from water, especially ammonia and nitrite. Thompson et al. (2002), evaluated the biofilm efficiency in the maintenance of water quality through the absorption of dissolved inorganic nutrients (ammonia and phosphate). Moreover, Ballester et al. (2003) evaluating the influence of biofilm on Farfantepenaeus paulensis production, concluded that the biofilm positively influenced shrimp growth, especially by providing an alternative food source. In order to increase efficiency in shrimp production, the use of artificial substrates for biofilm fixation in the Biofloc Technologic system has already been carried out by Ferreira et al. (2016). However, these authors concluded that biofilm served only as a source of complementary food, but did not observe any difference in the metabolization of nitrogen compounds in comparison to bioflocs alone. Thus, in BFT systems the use of substrates for biofilm development would not be necessary to keep water quality in good standards, since the bacteria present in the bioflocs would be enough to keep ammonia and nitrite at low levels and also represent an extra food source. However, it is likely that biofilm in aquaculture systems was not analyzed in all its dimensions. For instance, there is little information in the literature on the lower efficiency of the nitrifying bacteria in the biofilm in BFT systems and its relationship with the oxygen limitation.
Fig. 1. Dynamic of mean ± standard deviation of a) ammonia, b) nitrite and c) nitrate over time in a clear water system.
In BFT systems, aeration must be maintained at lower rates in order to keep the flocs in suspension without causing their rupture and to guarantee the nitrification by the bacteria present in the biofloc (Lara et al., 2017b; Souza et al., 2019). However, this low aeration rate may be a problem to nitrifying bacteria present in the biofilm. Thus, the objective of this study is to determine the response of nitrifying bacteria present in the artificial substrate biofilm submitted to different aeration intensities in the production of Litopenaeus vannamei (Boone, 1931) in systems with clear water and also with bioflocs.
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Table 1 Mean, standard deviation (overall mean) minimum and maximum of the physical and chemical parameters over the 10-day study of different aeration intensity with different flow rates (tree replicates). Treatment
W/AIR
V7.5
V33.75
V75
Total ammonia nitrogen (mg L−1) Nitrite (mg L−1) Nitrate (mg L−1) Alkalinity (mg L−1) Dissolved oxygen (mg L−1)
3.07 ± 0.49 (0.30–7.53) 0.17 ± 0.08a (0.00–0.30) 2.21 ± 0.04 (0.00–4.83) 153 ± 27 (107–250) 6.23 ± 0.03a (6.20–6.25)
2.60 ± 0.57 (0.06–7.53) 0.05 ± 0.03b (0.00–0.14) 2.85 ± 0.61 (0.00–8.67) 149 ± 25 (103–225) 6.33 ± 0.03b (6.30–6.35)
2.52 ± 1.03 (0.06–7.43) 0.08 ± 0.08b (0.00–0.49) 3.32 ± 0.96 (0.00–6.67) 145 ± 25 (100–237) 6.34 ± 0.04b (6.35–6.38)
2.65 ± 0.37 (0.06–7.67) 0.05 ± 0.03b (0.00–0.13) 3.55 ± 0.97 (0.00–6.26) 155 ± 24 (105–232) 6.40 ± 0.02b (6.40–6.48)
Different letters on the same line represent statistical difference p < 0.05. W/AIR: without aeration contribution; 2) V7.5: flow rate 7.5 L min-1; 3) V33.75: flow rate 33.75 L/min and 4) V75: flow rate 75 L/min in a clear water system.
2.3. Experiment 2: biofilm and biofloc
Table 2 Mean and standard deviation of molasses, hydrated lime, settling hours, water exchange and amount of water to produce 1 kg of shrimp over the 47-day study. Treatment
BFT
BFT+BF
BF
Total of Molasses (g) Hydrated lime (g) Settling hours (h) Water Exchange (L) Water Use (L kg−1 shrimp)
2507.34 ± 22.06
380.01 ± 8.50
–
1280.07 ± 22.58 10.00 ± 0.58 4480.0 l ± 66.03 719.69 ± 46.72
1521.59 ± 24.73 40.00 ± 1.51 – 174.64 ± 6.93
1282.46 ± 24.34 – – 167.65 ± 10.29
The experiment, performed during 47 days used the best aeration rate (33.75 L/min) determined in experiment 01. The experimental consisted of three treatments with three triplicates, being: 1) BFT Biofloc with 20.00 L/min flow rate; 2) BFT+BF - Bioflocs and biofilm with flow rate 33.75 L/min and 3) BF - clear water and biofilm with flow rate 33.75 L/min. The shrimp (7.81 ± 0.24 g) were stocked at a density of 500 m−3 and fed with Guabi® 1.6 mm commercial feed with 40% crude protein. The food was supplied twice a day (08,00 and 16,00) after samples according to the methodology of Garça de Yta et al. (2004). To begin the biofloc formation, organic fertilizations were carried out with the addition of sugar cane molasses (37% of carbon) when the concentrations of TAN reached 1.0 mg L−1 to maintain the relation C:N 15:1. During the experiment, the dissolved oxygen, temperature and pH were monitored twice a day using a multiparameter (YSI PRO 20). The alkalinity was analyzed three times a week. When pH and alkalinity values were below 7.3 and 150 mg L−1, respectively, there was the addition of hydrated lime as described by Furtado et al. (2011). Salinity and total suspended solids (SST) were measured on a weekly basis, according to AOAC (2000). When SST concentrations exceeded 500 mg L−1, clarifiers (biofloc settling tank) were used in order to remove surplus solids, as recommended by Gaona et al. (2011). Nitrogen compounds such as total ammonium nitrogen (TAN) and nitrite (N-NO2) were analyzed daily, and nitrate (N-NO3) once a week according to Aminot and Chaussepied (1983). Every time that the concentration of nitrite reached 26 mg L−1, the safety level for the salinity employed, 30% of water renewal was done (Lin and Chen, 2003).
2. Materials and methods 2.1. Location and facilities The study was carried out at the Marine Aquaculture Station (EMA/ FURG) - Institute of Oceanography of the Federal University of Rio Grande - FURG, located in the city of Rio Grande, RS, Brazil (32° 19 ′S, 52° 15 ′W). In both experiments, 800 L tanks of useful volume were filled with seawater, chlorinated with 10 ppm of sodium hypochlorite, later dechlorinated with 1 ppm of ascorbic acid. Water temperature was maintained with submerged electric heaters (Hydor Theo 200W). The aeration system was composed of a 4 HP blower and Aerotubes® microperforated hoses, to maintain constant aeration. The aeration rate of 20.00 L/min is normally used in the aquaculture systems of EMA/ FURG, but other rates were tested. In order to measure the airflow, individual rotormeters (TRP-255-H-7 1 POL NPT-Tecnofluid®) were coupled to the aeration inlet of each experimental unit and regulated in the flow according to the treatment. The artificial substrates used for colonization of the biofilm were non-floating Needlona®, in a proportion of 200% of the lateral area of the tank. Needlona® used comprised of 100% polyester fiber; 250 g/m2 weight; 1.4 mm thickness, 0.18 g/cm3 density. Before the beginning of the experiments, the substrates were kept for 30 days in a biofloc system.
2.4. Sampling of microorganisms To characterize the microbial community of the second experiment, weekly samples of 18 mL of water were collected from each experimental unit and fixed in formalin at the final concentration of 4% for subsequent identification of the microorganisms in the Laboratory of Phytoplankton and Marine Microorganisms of the FURG, among the treatments with and without substrate, so the CW+BF treatment was not analyzed. According to the methodology of Utermöhl (1958). Bacterial abundance analysis was performed on days 0, 17, 26 and 47 for BFT and BFT+BF treatments.
2.2. Experiment 1: biofilm in clear water The experiment consisted of four treatments with three replicates each, denominated: 1) W/AIR: without aeration; 2) V7.5: flow rate 7.5 L/min; 3) V33.75: flow rate 33.75 L/min and 4) V75: flow rate 75 L/ min in a clear water system, without shrimp. To determine the efficiency of the nitrifying bacteria, 7 mg L−1 ammonium chloride was added representing the safety limit for the L. vannamei species at salinity 35 (Lin and Chen, 2001). The study lasted for 10 days. Samples were collected every four hours for analysis of ammonia (TAN), nitrite (N-NO2), alkalinity (CaCO3) following methodologies UNESCO (1983), Aminot and Chaussepied (1983) e APHA (2012) respectively and dissolved oxygen with Multiparameter.
2.5. Zootechnical performance In the second experiment, the zootechnical performance of the animals was determined after weekly samples of 30 animals, using a digital scale with an accuracy of 0.01 g. 2.6. Statistical analysis Data were expressed as mean ± standard deviation. Undergo tests 3
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3. Results 3.1. Experiment 1 - biofilm in clear water There was no significant difference for the ammonia and nitrate parameters among treatments (Fig. 1a, c). The nitrite of treatments V7.5, V33.75 and V75 showed similar values throughout the study, but were lower than concentration measured in W/AIR, which reached 0.17 mg L−1 (Table 1, Fig. 1b). For the alkalinity values the V33.75 treatment had the lowest mean value, significantly different from the other treatments. The values of dissolved oxygen were higher in the three aeration intensities tested than in the without aeration treatment. 3.2. Experiment 2 - biofilm in clear water and biofloc The products (molasses, hydrated lime) used to control the amount of floc and the settling time, water exchange during the study and amount of water to produce 1 kg of shrimp is represented in the Table 2. The temperature, salinity, pH, nitrate (Fig. 2c) and SST did not show significant difference (p > 0.05) between treatments during the experimental period. There was a significant difference (p < 0.05) for ammonia, nitrite, dissolved oxygen and alkalinity. The BFT treatment presented higher values of ammonia, dissolved oxygen and alkalinity than BF (Fig. 2a), whereas the N-NO2 of the BFT+BF and CW+BF treatments were significantly lower than the BFT treatment (Fig. 2b). The dissolved oxygen levels in the BFT+BF and CW+BF treatments were statistically higher than those of the BFT treatment. This pattern was demonstrated throughout the experiment. The alkalinity presented lower values in the treatments BFT+BF and CW+BF, when compared to the BFT treatment (Table 3). Bacterial abundance analysis was performed on days 0, 17, 26 and 47 for BFT and BFT+BF treatments due to the high concentrations of ammonia and nitrite. The total of free bacteria did not present significant differences (p > 0.05) among treatments. However, there were statistical differences for the groups of analyzed bacteria. The BFT treatment differed from the BFT+BF treatment for bacilli and filamentous, bacteria, with higher abundances of bacteria (Fig. 3). It is possible to observe the gradual increase of microorganisms in the system as time passes, only on day 26 the number of organisms decreases and increases again as shown on day 47. The BFT+BF treatment has a stability in the organisms from start to finish of the experiment having an increase for filamentous and amoebae over time. The results of the zootechnical performance of the shrimp at the end of the experiment are presented in Table 3. There was no significant difference between the treatments (p < 0.05) in relation to the final weight. Survival and final biomass were significantly higher in the biofilm treatments (BFT+BF and BF) than in the biofloc treatment (BFT) alone (Table 4). Fig. 2. Dynamic of mean ± standard deviation of a) ammonia, b) nitrite and c) nitrate over the 47-day study of 500 m−3 Litopenaeus vannamei (7.81 ± 0.24 g) in BFT with different aeration intensity (Biofloc with flow rate of 20.00 L/min, BFT + BF: Bioflocos and biofilm with flow rate of 33.75 L/min and BF: biofilm with flow rate of 33.75 L/min) with tree replicates.
4. Discussion The first experiment demonstrated the importance of the aeration in the production system for a greater efficiency of the nitrification process by biofilm, especially for NOB bacteria. This was evidenced by the higher concentrations of nitrite in the treatment without aeration when compared to the others. The lack of water movement and the consequent limitation of the oxygen transfer can generate a gradient in the concentration of this gas along the biofilm, with the presence of hypoxic or anoxic areas in the innermost regions of the biofilm (Vlaeminck et al., 2010). In this case, nitrite oxidizing bacteria will be less active and this nitrogen compound will accumulate in the water. In general, AOB are found in the outermost part of the biofilm, whereas NOB are present in the deepest region (Gieseke et al., 2003). Therefore, NOB is likely to be more subject to the decrease in dissolved oxygen concentration than AOB. This is evident in this experiment,
of normality (Shapiro-Wilk) and homoscedasticity (Levene), with the proof of these premises. Analysis of multiple variables was conducted with the One-way Analysis of Variance (ANOVA) and post-hoc Tukey test. Data that did not satisfy the assumptions for ANOVA were submitted to the non-parametric test of Kruskall-Wallis followed by a multiple comparison test (ZAR, 2010). The level of significance was 5% in all cases (p < 0.05).
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Table 3 Mean, standard deviation overall mean and minimum and maximum of the physical and chemical parameters of the water over the 47-day study. Treatment
BFT −1
Total ammonia nitrogen (mg L ) Nitrite (mg L−1) Nitrate (mg L−1) Dissolved oxygen (mg L−1) pH Temperature (°C) Salinity Total suspended solids (mg L−1) Alkalinity (mg L−1)
BFT+BF a
BF b
1.73 ± 0.41 (0.00–13.20) 15.36 ± 5.03a (0.00–57.00) 43.91 ± 9.02 (1.70–124.00) 5.02 ± 0.22a (3.75–6.15) 7.55 ± 0.11 (7.07–8.05) 29.13 ± 0.76 (24.55–32.40) 30.80 ± 0.90 (26.00–33.3) 298.19 ± 88.73 (0.00–665) 154.75 ± 15.10a (95.00–250)
0.70 ± 0.54b (0.00–6.30) 1.11 ± 0.44b (0.00–3.50) 52.61 ± 17.78 (4.79–124.00) 5.22 ± 0.14b (3.95–6.20) 7.58 ± 0.07 (7.17–8.12) 29.02 ± 0.31 (25.00–32.05) 30.14 ± 1.30 (26.00–35.00) 332.56 ± 73.18 (0.00–575) 136.17 ± 22.14b (55–270)
0.51 ± 0.11 (0.00–5.20) 1.13 ± 0.56b (0.00–28.00) 73.85 ± 8.25 (4.79–204.00) 5.18 ± 0.16b (4.30–6.30) 7.55 ± 0.09 (6.96–8.06) 29.44 ± 0.83 (24.70–34.40) 31.41 ± 1.89 (27.10–35.00) 346.30 ± 57.28 (0.00–665) 135.92 ± 22.11b (55.00–185)
Different letters on the same line represent statistical difference p < 0.05. 500 m−3 Litopenaeus vannamei (7.81 ± 0.24 g) in BFT with different aeration intensity (Biofloc with flow rate of 20.00 L/min, BFT + BF: Bioflocos and biofilm with flow rate of 33.75 L/min and BF: biofilm with flow rate of 33.75 L/min) with tree replicates. Table 4 Mean and standard deviation of the zootechnical performance of the L. vannamei over the 47-day study. Treatment
BFT
BFT+BF
CW+BF
Initial weight (g) Final weight (g) Survival (%) Biomass final (m3)
7.81 ± 0.24
7.81 ± 0.24
7.81 ± 0.24
13.50 ± 0.40 62 ± 41.49a 3998.40 ± 490a
13.14 ± 0.19 87 ± 9.54b 5732.02 ± 180.13b
13.63 ± 0.63 88 ± 6.93b 5979.47 ± 289.47b
Different letters on the same line represent statistical difference p < 0.05. BFT: Biofloc without flow control, BFT + BF: Biofloc and biofilm with flow rate 33.75 L/min and CW + BF: Clear water and biofilm with flow rate 33.75 L/min.
employed in our production systems, but not as strong as 75.00 L/min, which is leads to a high energy consumption. The water quality parameters of the second experiment, such as temperature, pH, salinity, nitrate and total suspended solids, remained throughout the experiment under ideal conditions for the production of L. vannamei (Furtado et al., 2014; Gaona et al., 2011; Van Wyk and Scarpa, 1999), except for dissolved oxygen that presented lower values in the BFT treatment. However, mean concentrations of dissolved oxygen were > 5 mg L−1 required for bacteria in the nitrification process, as well as for shrimp requirements (Timmons and Ebeling, 2010; Van Wyk and Scarpa, 1999). In the second experiment it is possible to observe the benefits of the incorporation of the artificial substrate colonized with biofilm, since the biofilm treatments presented lower ammonia and nitrite values, indicating a faster and more efficient removal of nitrogen compounds when compared to bioflocs alone. Holl et al. (2011), also consider that the nitrifying community fixed in the substrate is capable of completely performing the nitrification of the system, even if there is no activity of the bacteria present in the water. On the other hand, in a similar study Ferreira et al. (2016) observed that the biofilm had no positive effect on the removal of the nitrogen compounds in comparison to bioflocs. It is possible to observe a greater abundance of bacteria in the BFT treatment compared to the BFT+BF treatment, where the BFT+BF treatment remains more stable throughout the period, different from the BFT that presents an increase of bacteria over time. This difference of bacteria between the treatments is related to the formation of the microbial community in the BFT treatment and the presence of the substrate colonized in the BFT+BF. The different amounts of bacteria indicate that great part of these microbes, especially the nitrifying ones, would be attached to the biofilm and not on the flocs, or free in the water. In our study, the lower abundances of free bacteria in the biofilm treatment compared to the treatment with only bioflocs may have resulted from a transfer of bacteria from the water column to the substrate which, as observed by Oliveira et al. (2006). There were no differences in growth among the shrimp submitted to
Fig. 3. Total bacterium in treatments Bioflocos and Bioflocos+Biofilm.
since nitrite variation was more influenced by the oxygen concentration than ammonia. Ammonia levels did not present significant differences among treatments, indicating that AOB were not affected by the oxygen concentration. On the other hand, the nitrification process did not seem to be affected by the intensity of the aeration, since no differences were observed in the three aeration intensities tested. However, for the second experiment we have chosen the aeration rate of 33.75 L/min, which is bigger than the aeration rates of 20.00 L/min, normally 5
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different treatments, but there was a significant difference in survival rate, with higher values in treatments with biofilm. In addition, the inclusion of the substrate resulted in higher biomass due to higher survivals in these treatments. This result may be a consequence of the higher levels of nitrite in the BFT treatment, which remained about 24 days with concentrations above the safety level proposed by Lin and Chen (2003). However, it is possible that the high concentrations of nitrite, the stocking density and the initial size of the animals may have negatively influenced survival in this treatment, mainly resulting in a high standard deviation of survival data in this treatment since the treatments that had the artificial substrate with biofilm show an increase of area decreasing the relative density. Otoshi et al. (2006) evaluated the growth and survival of juveniles of L. vannamei produced with and without artificial substrate observing an increase in growth and survival in substrate treatments. Similarly, Schveitzer et al. (2013) studied the use of artificial substrates in a biofloc system with different storage densities, concluding that the best survival was in the treatments with the presence of the substrates.
Ferreira, L.M.H., Lara, G., Wasielesky, W., Abreu, P.C., 2016. Biofilm Versus Biofloc: Are Artificial Substrates for Biofilm Production Necessary in the BFT System? pp. 921–930. https://doi.org/10.1007/s10499-015-9961-0. Furtado, P.S., Poersch, L.H., Wasielesky, W., 2011. Effect of calcium hydroxide, carbonate and sodium bicarbonate on water quality and zootechnical performance of shrimp Litopenaeus vannamei reared in bio-flocs technology (BFT) systems. Aquaculture 321, 130–135. https://doi.org/10.1016/j.aquaculture.2011.08.034. Furtado, P.S., Poersch, L.H., Wasielesky, W., 2014. The effect of different alkalinity levels on Litopenaeus vannamei reared with biofloc technology (BFT). Aquac. Int. 23, 345–358. https://doi.org/10.1007/s10499-014-9819-x. Gaona, C. a P., Poersch, L.H., Krummenauer, D., Foes, G.K., 2011. The Effect of Solids Removal on Water Quality, Growth and Survival of Litopenaeus vannamei in a Biofloc Technology Culture System. vol. 12. pp. 54–73. https://doi.org/10.21061/ijra. v12i1.1354. Gieseke, A., Bjerrum, L., Wagner, M., Amann, R., 2003. Structure and activity of multiple nitrifying bacterial populations co-existing in a biofilm. Environ. Microbiol. 5, 355–369. https://doi.org/10.1046/j.1462-2920.2003.00423.x. Girotto, M.V.F., 2010. Efeitos da amônia sobre juvenis de Litopenaeus vannamei (Boone, 1931) e Litopenaeus schmitti (Burkenroad, 1936): excreção e toxicidade. Thesis Thesis in Veterinary Sciences. Universidade Federal do Paraná. Holl, C.M., Otoshi, C., Unabia, C.R., 2011. Production nitrifying biofilms critical for water quality in intensive shrimp RAS. Glob. Aquac. Advocate 38–39. Lara, G., Furtado, P.S., Hostins, B., Poersch, L., Wasielesky, W., 2016. Addition of sodium nitrite and biofilm in a Litopenaeus vannamei biofloc culture system. Lat. Am. J. Aquat. Res. 44 (4), 760–768. https://doi.org/10.3856/vol44-issue4-fulltext-11. Lara, G., Krummenauer, D., Abreu, P.C., Poersch, L.H., Wasielesky, W., 2017b. The use of different aerators on Litopenaeus vannameibiofloc culture system: effects on water quality, shrimp growth and biofloc composition. Aquac. Int. 25, 147–162. https:// doi.org/10.1007/s10499-016-0019-8. Lin, Y., Chen, J., 2001. Acute toxicity of ammonia on Litopenaeus vannamei Boone juveniles at different salinity levels. J. Exp. Mar Bio Ecol. 109–119. https://doi.org/10. 1016/S00220981(01)00227-1. Lin, Y., Chen, J., 2003. Acute toxicity of nitrite on Litopenaeus vannamei (Boone, 1931) juveniles at different salinity levels. Aquaculture 224, 193–201. https://doi.org/10. 1016/S0044-8486(03)00220-5. Madigan, Michael T., Martinko, John M., Bender, Kelly S., Buckley, Daniel H., Stahl, David A., 2016. Microbilogia de Brock. (14th ed). Oliveira, S.S., Wasielesky Jr., W., Ballester, E.L.C., Abreu, P.C., 2006. Caracterização da assembléia de bactérias nitrificantes pelo método “fluorescent in situ hybridization” (fish) no biofilme e água de larvicultura do Camarão-Rosa farfantepenaeus paulensis. Atlântica 28, 33–45. Otoshi, C., Montgomery, A.D., Matsuda, E., Moss, S.M., 2006. Effects of artificial substrate and water source on growth of juvenile Pacific white shrimp, Litopenaeus vannamei. J. World Aquacult. Soc. 37 (2). Ramesh, M.R., Shankar, K.M., Mohan, C.V., Varghese, T.J., 1999. Comparison of three plant substrates for enhancing carp growth through bacterial biofilm. Aquac. Eng. 19, 119–131. https://doi.org/10.1016/S0144-8609(98)00046-6. Schveitzer, R., Arantesa, R., Baloia, M., Costódio, P.F.S., Aranaa, V.L., Seiffert, W.Q., Andreattaa, E.R., 2013. Use of artificial substrates in the culture of Litopenaeus vannamei (Biofloc system) at different stocking densities: effects on microbial activity, water quality and production rates. Aquac. Eng. 55, 93–103. https://doi.org/10. 1016/j.aquaeng.2012.12.003. Souza, J., Cardozo, A., Wasielesky, W., Abreu, P.C., 2019. Does the biofloc size matter to the nitrification process in Biofloc Technology (BFT) systems? Aquaculture 500, 443–450. https://doi.org/10.1016/j.aquaculture.2018.10.051. Tahon, J., Hoof, D.V.A.N., Vinckier, C., Witters, R., Ley, M.D.E., Lontie, R., 1988. The reaction of nitrite with the haemocyanin of Astacus leptodactylus. Biochem. J. 249, 891–896. https://doi.org/10.1042/bj2490891. Thompson, F.L., Abreu, P.C., Wasielesky, W., 2002. Importance of biofilm for water quality and nourishment in intensive shrimp culture. Aquaculture 203, 263–278. https://doi.org/10.1016/S0044-8486(01)00642-1. Timmons, M.B., Ebeling, J.M., 2010. Recirculating Aquaculture Systems, 2nd edition. pp. 939. UNESCO, 1983. Chemical Methods for Use in Marine Environmental Monitoring. pp. 53. Utermöhl, H., 1958. Zur Vervollkommung der quantitativen Phytoplankton-Methodik. Int. Vereinigung für Theor. und Angew. Limnol. Kom. für Limnol. Methoden 9, 1–39. Van Wyk, P., Scarpa, J., 1999. Farming marine shrimp. In: Van Wyk, P. (Ed.), Farming Marine Shrimp in Recirculating Freshwater Systems. Florida Department of Agriculture and Consumer Services, Tallahassee, pp. 128–138. Vieira, R.F., 2017. Ciclo do nitrogênio em sistemas agrícolas. Embrapa, Brasília, DF, pp. 163. http://ainfo.cnptia.embrapa.br/digital/bitstream/item/175460/1/2017LV04. pdf. Vlaeminck, S.E., Terada, A., Smets, B.F., De Clippeleir, H., Schaubroeck, T., Bolea, S., Demeestere, L., Mast, J., Boon, N., Carballa, M., Verstraete, W., 2010. Aggregate size and architecture determine microbial activity balance for one-stage partial nitritation and anammox. Appl. Environ. Microbiol. 76, 900–909. https://doi.org/10.1128/ AEM.02337-09. Ward, B.B., 2013. Nitrification Encyclopedia of Ecology, Second edition. pp. 1–8. https:// doi.org/10.1016/B978-0-12-409548-9.00697-7. ZAR, J.H., 2010. Biostatistical Analysis. Prentice Hall, Upper Saddle River. Zhu, G., Peng, Y., Li, B., Guo, J., Yang, Q., Wang, S., 2008. Biological removal of nitrogen from wastewater. Rev. Environ. Contam. Toxicol. 192, 159–195. https://doi.org/10. 1007/978-0-387-71724-1_5.
5. Conclusion Through the results obtained in this study it was possible to prove that the nitrification process is less efficient in the absence of adequate aeration. In addition, the use of artificial substrates to fix the biofilm becomes a viable alternative of implantation, due to its low cost and potential benefits to the culture environment and, consequently, of the organisms produced. Declaration of Competing Interest None. Acknowledgements The authors are grateful for the financial support provided by the National Council for Scientific and Technological Development (CNPq),), Chamada Universal Process n° 409904/2018-0, 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 All-Aqua Aeration for donating the experimental diets and post-larvae and Aeration system respectively References Aminot, A., Chaussepied, M., 1983. Manuel des analisa chimiques en milieu marin. In: Edições Jouve. CNEXO, Paris (395 p). AOAC (Association of Official Analitycal Chemists), 2000. In: Cunniff, Patricia (Ed.), Official Methods of Analysis of AOAC, 16th ed. (Washington, DC). APHA/AWWA/WEF, 2012. Standard methods for the examination of water and wastewater. Stand. Methods 541 (ISBN 9780875532356). Avnimelech, Y., 2009. Biofloc Technology - a Practical Guide Book, 2nd edition. The Word Aquaculture Society, Baton Rouge, Louisiana, United States, pp. 272. Ballester, E.L.C., Wasielesky Jr., W., Cavalli, R.O., Silva Santos, M.H., Abreu, P.C., 2003. Influência do biofilme no crescimento do camarão-rosa Farfantepenaeus paulensis em sistemas de berçário. vol. 25 (2). Atlântica, Rio Grande, RS, pp. 37–42. Boyd, C.E., Tucker, C.S., 2012. Pond Aquaculture Water Quality Management, 2nd ed. pp. 700. Chen, J., Chin, C., CK, L., 1986. Effects of Ammonia and Nitrite on Larval Development of the Shrimp Penaeus monodon. Asian Fish. Soc, pp. 657–662. de Yta, A.G., Rouse, D.B., Davis, D., 2004. Influence of nursery period on the growth and survival of litopenaeus vannamei under pond production conditions. J. World Aquac. Soc. 35, 357–365. https://doi.org/10.1111/j.1749-7345.2004.tb00099.x. Ebeling, J.M., Timmons, M.B., Bisogni, J.J., 2006. Engineering analysis of the stoichiometry of photoautotrophic, autotrophic, and heterotrophic removal of ammonia-nitrogen in aquaculture systems. Aquaculture 257, 346–358. https://doi.org/10.1016/ j.aquaculture.2006.03.019.
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Effect of aeration intensity on the biofilm nitrification process during the production of the white shrimp Litopenaeus vannamei (Boone, 1931) in Biofloc and clear water systems
T
Ana Paula Mariane de Moraisa, Paulo Cesar Abreub, Wilson Wasielesky Jrc, ⁎ Dariano Krummenauera, a
Laboratory of Ecology of Microorganisms Applied to Aquaculture, Institute of Oceanography, Federal University of Rio Grande, FURG, C. P. 474, Rio Grande, RS CEP 96201-900, Brazil Laboratory of Phytoplankton and Marine Microorganisms, Institute of Oceanography, Federal University of Rio Grande, FURG, C. P. 474, Rio Grande, RS CEP 96201900, Brazil c Laboratory of Mariculture, Institute of Oceanography, Federal University of Rio Grande, FURG, C. P. 474, Rio Grande, RS CEP 96201-900, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Nitrifying bacteria Nitrogen compounds Ammonia Nitrite
Artificial substrates have great importance for the establishment of the biofilm, and their use in the culture systems represents a complementary source of food, increase space for animals and aid in the metabolism of nitrogen compounds. Nitrifying bacteria present in biofilm play an important role in the maintenance of water quality, and several factors such as pH, temperature, salinity and dissolved oxygen can interfere in the establishment and efficiency of these bacterial communities. However, there is not much information in the literature on the influence of aeration intensity on the bacterial community present in the biofilm. Thus, the objective of this study was to determine the response of nitrifying bacteria present in the biofilm submitted to different aeration intensities during the production of Litopenaeus vannamei (Boone, 1931) in a clear water system and also with bioflocs. The study was composed of two experiments, where the first experiment was carried out without shrimp and consisted of four treatments with three replicates, in 800 Liter tanks distributed in: 1) W/Air (control - without aeration); 2) V7.5 (flow rate 7.5 L/min); 3) V33.75 (flow rate of 33.75 L/min) and V75 (flow rate of 75 L/min). All treatments use “Needlona®” as artificial substrate (Needlona® - 100% polyester fiber; 250 g/m2 weight; 1.4 mm thickness; 0.18 g/cm3 density), in the proportion of 200% of the lateral area of the tank. Experiment two was established after the results of the previous experiment, with three treatments and three replicates each: 1) BFT (biofloc, with flow rate of 20.00 L/min); 2) BFT + BF (biofloc and biofilm with flow rate of 33.75 L/min) and 3) BF (biofilm with flow rate of 33.75 L/min), in which the shrimp (7.89 ± 0.24 g) were stocked in 9 tanks (800 L) with a density of 500 shrimps m−3. In both experiments Ammonia and nitrite were measured daily, while nitrate was analyzed weekly. The first experiment showed no difference in the ammonia concentrations of the different treatments, whereas nitrite showed higher concentrations in the treatment without aeration. The 33.75 L/min flow rate was chosen for experiment 2 to be compared with the aeration normally employed in our systems (20.00 Liter/min). The nitrification process was more efficient in the treatments with biofilm and bigger air flow rate, presenting smaller concentrations of ammonia and nitrite in comparison to the BFT treatment. Similarly, treatments with biofilm and stronger flow rate showed better zootechnical performance of the shrimp.
1. Introduction
may be a limiting factor for primary production in these ecosystems, but can also be toxic for aquatic organisms when present in higher concentrations (Vieira, 2017). In the nitrification process, the successive oxidation of ammonia to
Nitrogen is an important nutrient for living organisms, as it is an essential component for the constitution of proteins and nucleic acids. It
Abbreviations: AOB, Ammonia-Oxidizing Bacteria; NOB, Nitrite-Oxidizing Bacteria; TAN, Total Ammonia Nitrogen; BFT, Biofloc Technology; EMA/FURG, Marine Aquaculture Station; N-NO2−, Nitrite; CaCO3, Alkalinity; N-NO3−, Nitrate; TSS, Total Suspended Solids ⁎ Corresponding author. E-mail address: [email protected] (D. Krummenauer). https://doi.org/10.1016/j.aquaculture.2019.734516 Received 8 July 2019; Received in revised form 11 September 2019; Accepted 13 September 2019 Available online 14 September 2019 0044-8486/ © 2019 Elsevier B.V. All rights reserved.
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nitrite and subsequently to nitrate is mainly made by autochemolitotrophic microorganisms (Ebeling et al., 2006) belonging to two groups of bacteria. The first, the ammonia-oxidizing bacteria (AOB) are responsible for the oxidation of ammonia to nitrite. Most of these organisms belong to the genera Nitrosomonas, Nitrosococcus, Nitrosospira, Nitrosolobus and Nitrosovibrio. The second group, the nitriteoxidizing bacteria (NOB), performs the conversion of nitrite to nitrate, and the majority of these microorganisms belong to the genera Nitrobacter, Nitrococcus, Nitrospira and Nitrospina (Ebeling et al., 2006; Madigan et al., 2016; Lara et al., 2016). Recent studies have demonstrated that microorganisms of the Archaea domain also participate in the nitrification process (Ward, 2013). In the production of aquatic organisms, like fish and shrimp, higher concentrations of nitrogen compounds as ammonia and nitrite can become a problem, when they accumulate in the aquatic environment due to the excreta of the produced organisms, decomposition of unconsumed foods and organic waste (Timmons and Ebeling, 2010). Therefore, the control of nitrogen elements within the breeding environment is important, since they can cause damage to the produced shrimp and fish (Lin and Chen, 2001, 2003). The concentration of ammonium (TAN) in the medium increases with increasing pH and water temperature, and reduces with increasing salinity (Boyd and Tucker, 2012) Exposure to inadequate concentrations of these compounds can cause stress, triggering various physiological changes, and compromising performance leading to death, thereby impairing production (Girotto, 2010). Nitrite, on the other hand, binds to hemocyanin, transforming it into metahemocyanin, preventing the transport of oxygen to tissues and reducing the amount of oxygen available for metabolism (Tahon et al., 1988). This process can lead to hypoxia and, consequently, mortality of organisms produced (Chen et al., 1986). An efficient aeration system is important for the oxygen supply to produce animals, and to keep the flocs in suspension in the BFT system. However, low concentrations of dissolved oxygen limit or suppress nitrification (Avnimelech, 2009; Zhu et al., 2008), since the nitrifying bacteria, AOB and NOB present a demand for oxygen for cellular activity, growth and reproduction. Thus, in order to carry out the nitrification process it is essential that these microorganisms settle in the growing environment like bioflocs and biofilm, but also have ideal environmental conditions to absorb and transform nitrogen compounds. The biofilm can be defined as an organic matrix adhered to any submerged substrate, which is colonized by a microbial community composed of bacteria, protozoa, fungi and algae (Ramesh et al., 1999). It has been shown that biofilm is responsible for removing nitrogenous compounds from water, especially ammonia and nitrite. Thompson et al. (2002), evaluated the biofilm efficiency in the maintenance of water quality through the absorption of dissolved inorganic nutrients (ammonia and phosphate). Moreover, Ballester et al. (2003) evaluating the influence of biofilm on Farfantepenaeus paulensis production, concluded that the biofilm positively influenced shrimp growth, especially by providing an alternative food source. In order to increase efficiency in shrimp production, the use of artificial substrates for biofilm fixation in the Biofloc Technologic system has already been carried out by Ferreira et al. (2016). However, these authors concluded that biofilm served only as a source of complementary food, but did not observe any difference in the metabolization of nitrogen compounds in comparison to bioflocs alone. Thus, in BFT systems the use of substrates for biofilm development would not be necessary to keep water quality in good standards, since the bacteria present in the bioflocs would be enough to keep ammonia and nitrite at low levels and also represent an extra food source. However, it is likely that biofilm in aquaculture systems was not analyzed in all its dimensions. For instance, there is little information in the literature on the lower efficiency of the nitrifying bacteria in the biofilm in BFT systems and its relationship with the oxygen limitation.
Fig. 1. Dynamic of mean ± standard deviation of a) ammonia, b) nitrite and c) nitrate over time in a clear water system.
In BFT systems, aeration must be maintained at lower rates in order to keep the flocs in suspension without causing their rupture and to guarantee the nitrification by the bacteria present in the biofloc (Lara et al., 2017b; Souza et al., 2019). However, this low aeration rate may be a problem to nitrifying bacteria present in the biofilm. Thus, the objective of this study is to determine the response of nitrifying bacteria present in the artificial substrate biofilm submitted to different aeration intensities in the production of Litopenaeus vannamei (Boone, 1931) in systems with clear water and also with bioflocs.
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Table 1 Mean, standard deviation (overall mean) minimum and maximum of the physical and chemical parameters over the 10-day study of different aeration intensity with different flow rates (tree replicates). Treatment
W/AIR
V7.5
V33.75
V75
Total ammonia nitrogen (mg L−1) Nitrite (mg L−1) Nitrate (mg L−1) Alkalinity (mg L−1) Dissolved oxygen (mg L−1)
3.07 ± 0.49 (0.30–7.53) 0.17 ± 0.08a (0.00–0.30) 2.21 ± 0.04 (0.00–4.83) 153 ± 27 (107–250) 6.23 ± 0.03a (6.20–6.25)
2.60 ± 0.57 (0.06–7.53) 0.05 ± 0.03b (0.00–0.14) 2.85 ± 0.61 (0.00–8.67) 149 ± 25 (103–225) 6.33 ± 0.03b (6.30–6.35)
2.52 ± 1.03 (0.06–7.43) 0.08 ± 0.08b (0.00–0.49) 3.32 ± 0.96 (0.00–6.67) 145 ± 25 (100–237) 6.34 ± 0.04b (6.35–6.38)
2.65 ± 0.37 (0.06–7.67) 0.05 ± 0.03b (0.00–0.13) 3.55 ± 0.97 (0.00–6.26) 155 ± 24 (105–232) 6.40 ± 0.02b (6.40–6.48)
Different letters on the same line represent statistical difference p < 0.05. W/AIR: without aeration contribution; 2) V7.5: flow rate 7.5 L min-1; 3) V33.75: flow rate 33.75 L/min and 4) V75: flow rate 75 L/min in a clear water system.
2.3. Experiment 2: biofilm and biofloc
Table 2 Mean and standard deviation of molasses, hydrated lime, settling hours, water exchange and amount of water to produce 1 kg of shrimp over the 47-day study. Treatment
BFT
BFT+BF
BF
Total of Molasses (g) Hydrated lime (g) Settling hours (h) Water Exchange (L) Water Use (L kg−1 shrimp)
2507.34 ± 22.06
380.01 ± 8.50
–
1280.07 ± 22.58 10.00 ± 0.58 4480.0 l ± 66.03 719.69 ± 46.72
1521.59 ± 24.73 40.00 ± 1.51 – 174.64 ± 6.93
1282.46 ± 24.34 – – 167.65 ± 10.29
The experiment, performed during 47 days used the best aeration rate (33.75 L/min) determined in experiment 01. The experimental consisted of three treatments with three triplicates, being: 1) BFT Biofloc with 20.00 L/min flow rate; 2) BFT+BF - Bioflocs and biofilm with flow rate 33.75 L/min and 3) BF - clear water and biofilm with flow rate 33.75 L/min. The shrimp (7.81 ± 0.24 g) were stocked at a density of 500 m−3 and fed with Guabi® 1.6 mm commercial feed with 40% crude protein. The food was supplied twice a day (08,00 and 16,00) after samples according to the methodology of Garça de Yta et al. (2004). To begin the biofloc formation, organic fertilizations were carried out with the addition of sugar cane molasses (37% of carbon) when the concentrations of TAN reached 1.0 mg L−1 to maintain the relation C:N 15:1. During the experiment, the dissolved oxygen, temperature and pH were monitored twice a day using a multiparameter (YSI PRO 20). The alkalinity was analyzed three times a week. When pH and alkalinity values were below 7.3 and 150 mg L−1, respectively, there was the addition of hydrated lime as described by Furtado et al. (2011). Salinity and total suspended solids (SST) were measured on a weekly basis, according to AOAC (2000). When SST concentrations exceeded 500 mg L−1, clarifiers (biofloc settling tank) were used in order to remove surplus solids, as recommended by Gaona et al. (2011). Nitrogen compounds such as total ammonium nitrogen (TAN) and nitrite (N-NO2) were analyzed daily, and nitrate (N-NO3) once a week according to Aminot and Chaussepied (1983). Every time that the concentration of nitrite reached 26 mg L−1, the safety level for the salinity employed, 30% of water renewal was done (Lin and Chen, 2003).
2. Materials and methods 2.1. Location and facilities The study was carried out at the Marine Aquaculture Station (EMA/ FURG) - Institute of Oceanography of the Federal University of Rio Grande - FURG, located in the city of Rio Grande, RS, Brazil (32° 19 ′S, 52° 15 ′W). In both experiments, 800 L tanks of useful volume were filled with seawater, chlorinated with 10 ppm of sodium hypochlorite, later dechlorinated with 1 ppm of ascorbic acid. Water temperature was maintained with submerged electric heaters (Hydor Theo 200W). The aeration system was composed of a 4 HP blower and Aerotubes® microperforated hoses, to maintain constant aeration. The aeration rate of 20.00 L/min is normally used in the aquaculture systems of EMA/ FURG, but other rates were tested. In order to measure the airflow, individual rotormeters (TRP-255-H-7 1 POL NPT-Tecnofluid®) were coupled to the aeration inlet of each experimental unit and regulated in the flow according to the treatment. The artificial substrates used for colonization of the biofilm were non-floating Needlona®, in a proportion of 200% of the lateral area of the tank. Needlona® used comprised of 100% polyester fiber; 250 g/m2 weight; 1.4 mm thickness, 0.18 g/cm3 density. Before the beginning of the experiments, the substrates were kept for 30 days in a biofloc system.
2.4. Sampling of microorganisms To characterize the microbial community of the second experiment, weekly samples of 18 mL of water were collected from each experimental unit and fixed in formalin at the final concentration of 4% for subsequent identification of the microorganisms in the Laboratory of Phytoplankton and Marine Microorganisms of the FURG, among the treatments with and without substrate, so the CW+BF treatment was not analyzed. According to the methodology of Utermöhl (1958). Bacterial abundance analysis was performed on days 0, 17, 26 and 47 for BFT and BFT+BF treatments.
2.2. Experiment 1: biofilm in clear water The experiment consisted of four treatments with three replicates each, denominated: 1) W/AIR: without aeration; 2) V7.5: flow rate 7.5 L/min; 3) V33.75: flow rate 33.75 L/min and 4) V75: flow rate 75 L/ min in a clear water system, without shrimp. To determine the efficiency of the nitrifying bacteria, 7 mg L−1 ammonium chloride was added representing the safety limit for the L. vannamei species at salinity 35 (Lin and Chen, 2001). The study lasted for 10 days. Samples were collected every four hours for analysis of ammonia (TAN), nitrite (N-NO2), alkalinity (CaCO3) following methodologies UNESCO (1983), Aminot and Chaussepied (1983) e APHA (2012) respectively and dissolved oxygen with Multiparameter.
2.5. Zootechnical performance In the second experiment, the zootechnical performance of the animals was determined after weekly samples of 30 animals, using a digital scale with an accuracy of 0.01 g. 2.6. Statistical analysis Data were expressed as mean ± standard deviation. Undergo tests 3
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3. Results 3.1. Experiment 1 - biofilm in clear water There was no significant difference for the ammonia and nitrate parameters among treatments (Fig. 1a, c). The nitrite of treatments V7.5, V33.75 and V75 showed similar values throughout the study, but were lower than concentration measured in W/AIR, which reached 0.17 mg L−1 (Table 1, Fig. 1b). For the alkalinity values the V33.75 treatment had the lowest mean value, significantly different from the other treatments. The values of dissolved oxygen were higher in the three aeration intensities tested than in the without aeration treatment. 3.2. Experiment 2 - biofilm in clear water and biofloc The products (molasses, hydrated lime) used to control the amount of floc and the settling time, water exchange during the study and amount of water to produce 1 kg of shrimp is represented in the Table 2. The temperature, salinity, pH, nitrate (Fig. 2c) and SST did not show significant difference (p > 0.05) between treatments during the experimental period. There was a significant difference (p < 0.05) for ammonia, nitrite, dissolved oxygen and alkalinity. The BFT treatment presented higher values of ammonia, dissolved oxygen and alkalinity than BF (Fig. 2a), whereas the N-NO2 of the BFT+BF and CW+BF treatments were significantly lower than the BFT treatment (Fig. 2b). The dissolved oxygen levels in the BFT+BF and CW+BF treatments were statistically higher than those of the BFT treatment. This pattern was demonstrated throughout the experiment. The alkalinity presented lower values in the treatments BFT+BF and CW+BF, when compared to the BFT treatment (Table 3). Bacterial abundance analysis was performed on days 0, 17, 26 and 47 for BFT and BFT+BF treatments due to the high concentrations of ammonia and nitrite. The total of free bacteria did not present significant differences (p > 0.05) among treatments. However, there were statistical differences for the groups of analyzed bacteria. The BFT treatment differed from the BFT+BF treatment for bacilli and filamentous, bacteria, with higher abundances of bacteria (Fig. 3). It is possible to observe the gradual increase of microorganisms in the system as time passes, only on day 26 the number of organisms decreases and increases again as shown on day 47. The BFT+BF treatment has a stability in the organisms from start to finish of the experiment having an increase for filamentous and amoebae over time. The results of the zootechnical performance of the shrimp at the end of the experiment are presented in Table 3. There was no significant difference between the treatments (p < 0.05) in relation to the final weight. Survival and final biomass were significantly higher in the biofilm treatments (BFT+BF and BF) than in the biofloc treatment (BFT) alone (Table 4). Fig. 2. Dynamic of mean ± standard deviation of a) ammonia, b) nitrite and c) nitrate over the 47-day study of 500 m−3 Litopenaeus vannamei (7.81 ± 0.24 g) in BFT with different aeration intensity (Biofloc with flow rate of 20.00 L/min, BFT + BF: Bioflocos and biofilm with flow rate of 33.75 L/min and BF: biofilm with flow rate of 33.75 L/min) with tree replicates.
4. Discussion The first experiment demonstrated the importance of the aeration in the production system for a greater efficiency of the nitrification process by biofilm, especially for NOB bacteria. This was evidenced by the higher concentrations of nitrite in the treatment without aeration when compared to the others. The lack of water movement and the consequent limitation of the oxygen transfer can generate a gradient in the concentration of this gas along the biofilm, with the presence of hypoxic or anoxic areas in the innermost regions of the biofilm (Vlaeminck et al., 2010). In this case, nitrite oxidizing bacteria will be less active and this nitrogen compound will accumulate in the water. In general, AOB are found in the outermost part of the biofilm, whereas NOB are present in the deepest region (Gieseke et al., 2003). Therefore, NOB is likely to be more subject to the decrease in dissolved oxygen concentration than AOB. This is evident in this experiment,
of normality (Shapiro-Wilk) and homoscedasticity (Levene), with the proof of these premises. Analysis of multiple variables was conducted with the One-way Analysis of Variance (ANOVA) and post-hoc Tukey test. Data that did not satisfy the assumptions for ANOVA were submitted to the non-parametric test of Kruskall-Wallis followed by a multiple comparison test (ZAR, 2010). The level of significance was 5% in all cases (p < 0.05).
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Table 3 Mean, standard deviation overall mean and minimum and maximum of the physical and chemical parameters of the water over the 47-day study. Treatment
BFT −1
Total ammonia nitrogen (mg L ) Nitrite (mg L−1) Nitrate (mg L−1) Dissolved oxygen (mg L−1) pH Temperature (°C) Salinity Total suspended solids (mg L−1) Alkalinity (mg L−1)
BFT+BF a
BF b
1.73 ± 0.41 (0.00–13.20) 15.36 ± 5.03a (0.00–57.00) 43.91 ± 9.02 (1.70–124.00) 5.02 ± 0.22a (3.75–6.15) 7.55 ± 0.11 (7.07–8.05) 29.13 ± 0.76 (24.55–32.40) 30.80 ± 0.90 (26.00–33.3) 298.19 ± 88.73 (0.00–665) 154.75 ± 15.10a (95.00–250)
0.70 ± 0.54b (0.00–6.30) 1.11 ± 0.44b (0.00–3.50) 52.61 ± 17.78 (4.79–124.00) 5.22 ± 0.14b (3.95–6.20) 7.58 ± 0.07 (7.17–8.12) 29.02 ± 0.31 (25.00–32.05) 30.14 ± 1.30 (26.00–35.00) 332.56 ± 73.18 (0.00–575) 136.17 ± 22.14b (55–270)
0.51 ± 0.11 (0.00–5.20) 1.13 ± 0.56b (0.00–28.00) 73.85 ± 8.25 (4.79–204.00) 5.18 ± 0.16b (4.30–6.30) 7.55 ± 0.09 (6.96–8.06) 29.44 ± 0.83 (24.70–34.40) 31.41 ± 1.89 (27.10–35.00) 346.30 ± 57.28 (0.00–665) 135.92 ± 22.11b (55.00–185)
Different letters on the same line represent statistical difference p < 0.05. 500 m−3 Litopenaeus vannamei (7.81 ± 0.24 g) in BFT with different aeration intensity (Biofloc with flow rate of 20.00 L/min, BFT + BF: Bioflocos and biofilm with flow rate of 33.75 L/min and BF: biofilm with flow rate of 33.75 L/min) with tree replicates. Table 4 Mean and standard deviation of the zootechnical performance of the L. vannamei over the 47-day study. Treatment
BFT
BFT+BF
CW+BF
Initial weight (g) Final weight (g) Survival (%) Biomass final (m3)
7.81 ± 0.24
7.81 ± 0.24
7.81 ± 0.24
13.50 ± 0.40 62 ± 41.49a 3998.40 ± 490a
13.14 ± 0.19 87 ± 9.54b 5732.02 ± 180.13b
13.63 ± 0.63 88 ± 6.93b 5979.47 ± 289.47b
Different letters on the same line represent statistical difference p < 0.05. BFT: Biofloc without flow control, BFT + BF: Biofloc and biofilm with flow rate 33.75 L/min and CW + BF: Clear water and biofilm with flow rate 33.75 L/min.
employed in our production systems, but not as strong as 75.00 L/min, which is leads to a high energy consumption. The water quality parameters of the second experiment, such as temperature, pH, salinity, nitrate and total suspended solids, remained throughout the experiment under ideal conditions for the production of L. vannamei (Furtado et al., 2014; Gaona et al., 2011; Van Wyk and Scarpa, 1999), except for dissolved oxygen that presented lower values in the BFT treatment. However, mean concentrations of dissolved oxygen were > 5 mg L−1 required for bacteria in the nitrification process, as well as for shrimp requirements (Timmons and Ebeling, 2010; Van Wyk and Scarpa, 1999). In the second experiment it is possible to observe the benefits of the incorporation of the artificial substrate colonized with biofilm, since the biofilm treatments presented lower ammonia and nitrite values, indicating a faster and more efficient removal of nitrogen compounds when compared to bioflocs alone. Holl et al. (2011), also consider that the nitrifying community fixed in the substrate is capable of completely performing the nitrification of the system, even if there is no activity of the bacteria present in the water. On the other hand, in a similar study Ferreira et al. (2016) observed that the biofilm had no positive effect on the removal of the nitrogen compounds in comparison to bioflocs. It is possible to observe a greater abundance of bacteria in the BFT treatment compared to the BFT+BF treatment, where the BFT+BF treatment remains more stable throughout the period, different from the BFT that presents an increase of bacteria over time. This difference of bacteria between the treatments is related to the formation of the microbial community in the BFT treatment and the presence of the substrate colonized in the BFT+BF. The different amounts of bacteria indicate that great part of these microbes, especially the nitrifying ones, would be attached to the biofilm and not on the flocs, or free in the water. In our study, the lower abundances of free bacteria in the biofilm treatment compared to the treatment with only bioflocs may have resulted from a transfer of bacteria from the water column to the substrate which, as observed by Oliveira et al. (2006). There were no differences in growth among the shrimp submitted to
Fig. 3. Total bacterium in treatments Bioflocos and Bioflocos+Biofilm.
since nitrite variation was more influenced by the oxygen concentration than ammonia. Ammonia levels did not present significant differences among treatments, indicating that AOB were not affected by the oxygen concentration. On the other hand, the nitrification process did not seem to be affected by the intensity of the aeration, since no differences were observed in the three aeration intensities tested. However, for the second experiment we have chosen the aeration rate of 33.75 L/min, which is bigger than the aeration rates of 20.00 L/min, normally 5
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different treatments, but there was a significant difference in survival rate, with higher values in treatments with biofilm. In addition, the inclusion of the substrate resulted in higher biomass due to higher survivals in these treatments. This result may be a consequence of the higher levels of nitrite in the BFT treatment, which remained about 24 days with concentrations above the safety level proposed by Lin and Chen (2003). However, it is possible that the high concentrations of nitrite, the stocking density and the initial size of the animals may have negatively influenced survival in this treatment, mainly resulting in a high standard deviation of survival data in this treatment since the treatments that had the artificial substrate with biofilm show an increase of area decreasing the relative density. Otoshi et al. (2006) evaluated the growth and survival of juveniles of L. vannamei produced with and without artificial substrate observing an increase in growth and survival in substrate treatments. Similarly, Schveitzer et al. (2013) studied the use of artificial substrates in a biofloc system with different storage densities, concluding that the best survival was in the treatments with the presence of the substrates.
Ferreira, L.M.H., Lara, G., Wasielesky, W., Abreu, P.C., 2016. Biofilm Versus Biofloc: Are Artificial Substrates for Biofilm Production Necessary in the BFT System? pp. 921–930. https://doi.org/10.1007/s10499-015-9961-0. Furtado, P.S., Poersch, L.H., Wasielesky, W., 2011. Effect of calcium hydroxide, carbonate and sodium bicarbonate on water quality and zootechnical performance of shrimp Litopenaeus vannamei reared in bio-flocs technology (BFT) systems. Aquaculture 321, 130–135. https://doi.org/10.1016/j.aquaculture.2011.08.034. Furtado, P.S., Poersch, L.H., Wasielesky, W., 2014. The effect of different alkalinity levels on Litopenaeus vannamei reared with biofloc technology (BFT). Aquac. Int. 23, 345–358. https://doi.org/10.1007/s10499-014-9819-x. Gaona, C. a P., Poersch, L.H., Krummenauer, D., Foes, G.K., 2011. The Effect of Solids Removal on Water Quality, Growth and Survival of Litopenaeus vannamei in a Biofloc Technology Culture System. vol. 12. pp. 54–73. https://doi.org/10.21061/ijra. v12i1.1354. Gieseke, A., Bjerrum, L., Wagner, M., Amann, R., 2003. Structure and activity of multiple nitrifying bacterial populations co-existing in a biofilm. Environ. Microbiol. 5, 355–369. https://doi.org/10.1046/j.1462-2920.2003.00423.x. Girotto, M.V.F., 2010. Efeitos da amônia sobre juvenis de Litopenaeus vannamei (Boone, 1931) e Litopenaeus schmitti (Burkenroad, 1936): excreção e toxicidade. Thesis Thesis in Veterinary Sciences. Universidade Federal do Paraná. Holl, C.M., Otoshi, C., Unabia, C.R., 2011. Production nitrifying biofilms critical for water quality in intensive shrimp RAS. Glob. Aquac. Advocate 38–39. Lara, G., Furtado, P.S., Hostins, B., Poersch, L., Wasielesky, W., 2016. Addition of sodium nitrite and biofilm in a Litopenaeus vannamei biofloc culture system. Lat. Am. J. Aquat. Res. 44 (4), 760–768. https://doi.org/10.3856/vol44-issue4-fulltext-11. Lara, G., Krummenauer, D., Abreu, P.C., Poersch, L.H., Wasielesky, W., 2017b. The use of different aerators on Litopenaeus vannameibiofloc culture system: effects on water quality, shrimp growth and biofloc composition. Aquac. Int. 25, 147–162. https:// doi.org/10.1007/s10499-016-0019-8. Lin, Y., Chen, J., 2001. Acute toxicity of ammonia on Litopenaeus vannamei Boone juveniles at different salinity levels. J. Exp. Mar Bio Ecol. 109–119. https://doi.org/10. 1016/S00220981(01)00227-1. Lin, Y., Chen, J., 2003. Acute toxicity of nitrite on Litopenaeus vannamei (Boone, 1931) juveniles at different salinity levels. Aquaculture 224, 193–201. https://doi.org/10. 1016/S0044-8486(03)00220-5. Madigan, Michael T., Martinko, John M., Bender, Kelly S., Buckley, Daniel H., Stahl, David A., 2016. Microbilogia de Brock. (14th ed). Oliveira, S.S., Wasielesky Jr., W., Ballester, E.L.C., Abreu, P.C., 2006. Caracterização da assembléia de bactérias nitrificantes pelo método “fluorescent in situ hybridization” (fish) no biofilme e água de larvicultura do Camarão-Rosa farfantepenaeus paulensis. Atlântica 28, 33–45. Otoshi, C., Montgomery, A.D., Matsuda, E., Moss, S.M., 2006. Effects of artificial substrate and water source on growth of juvenile Pacific white shrimp, Litopenaeus vannamei. J. World Aquacult. Soc. 37 (2). Ramesh, M.R., Shankar, K.M., Mohan, C.V., Varghese, T.J., 1999. Comparison of three plant substrates for enhancing carp growth through bacterial biofilm. Aquac. Eng. 19, 119–131. https://doi.org/10.1016/S0144-8609(98)00046-6. Schveitzer, R., Arantesa, R., Baloia, M., Costódio, P.F.S., Aranaa, V.L., Seiffert, W.Q., Andreattaa, E.R., 2013. Use of artificial substrates in the culture of Litopenaeus vannamei (Biofloc system) at different stocking densities: effects on microbial activity, water quality and production rates. Aquac. Eng. 55, 93–103. https://doi.org/10. 1016/j.aquaeng.2012.12.003. Souza, J., Cardozo, A., Wasielesky, W., Abreu, P.C., 2019. Does the biofloc size matter to the nitrification process in Biofloc Technology (BFT) systems? Aquaculture 500, 443–450. https://doi.org/10.1016/j.aquaculture.2018.10.051. Tahon, J., Hoof, D.V.A.N., Vinckier, C., Witters, R., Ley, M.D.E., Lontie, R., 1988. The reaction of nitrite with the haemocyanin of Astacus leptodactylus. Biochem. J. 249, 891–896. https://doi.org/10.1042/bj2490891. Thompson, F.L., Abreu, P.C., Wasielesky, W., 2002. Importance of biofilm for water quality and nourishment in intensive shrimp culture. Aquaculture 203, 263–278. https://doi.org/10.1016/S0044-8486(01)00642-1. Timmons, M.B., Ebeling, J.M., 2010. Recirculating Aquaculture Systems, 2nd edition. pp. 939. UNESCO, 1983. Chemical Methods for Use in Marine Environmental Monitoring. pp. 53. Utermöhl, H., 1958. Zur Vervollkommung der quantitativen Phytoplankton-Methodik. Int. Vereinigung für Theor. und Angew. Limnol. Kom. für Limnol. Methoden 9, 1–39. Van Wyk, P., Scarpa, J., 1999. Farming marine shrimp. In: Van Wyk, P. (Ed.), Farming Marine Shrimp in Recirculating Freshwater Systems. Florida Department of Agriculture and Consumer Services, Tallahassee, pp. 128–138. Vieira, R.F., 2017. Ciclo do nitrogênio em sistemas agrícolas. Embrapa, Brasília, DF, pp. 163. http://ainfo.cnptia.embrapa.br/digital/bitstream/item/175460/1/2017LV04. pdf. Vlaeminck, S.E., Terada, A., Smets, B.F., De Clippeleir, H., Schaubroeck, T., Bolea, S., Demeestere, L., Mast, J., Boon, N., Carballa, M., Verstraete, W., 2010. Aggregate size and architecture determine microbial activity balance for one-stage partial nitritation and anammox. Appl. Environ. Microbiol. 76, 900–909. https://doi.org/10.1128/ AEM.02337-09. Ward, B.B., 2013. Nitrification Encyclopedia of Ecology, Second edition. pp. 1–8. https:// doi.org/10.1016/B978-0-12-409548-9.00697-7. ZAR, J.H., 2010. Biostatistical Analysis. Prentice Hall, Upper Saddle River. Zhu, G., Peng, Y., Li, B., Guo, J., Yang, Q., Wang, S., 2008. Biological removal of nitrogen from wastewater. Rev. Environ. Contam. Toxicol. 192, 159–195. https://doi.org/10. 1007/978-0-387-71724-1_5.
5. Conclusion Through the results obtained in this study it was possible to prove that the nitrification process is less efficient in the absence of adequate aeration. In addition, the use of artificial substrates to fix the biofilm becomes a viable alternative of implantation, due to its low cost and potential benefits to the culture environment and, consequently, of the organisms produced. Declaration of Competing Interest None. Acknowledgements The authors are grateful for the financial support provided by the National Council for Scientific and Technological Development (CNPq),), Chamada Universal Process n° 409904/2018-0, 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 All-Aqua Aeration for donating the experimental diets and post-larvae and Aeration system respectively References Aminot, A., Chaussepied, M., 1983. Manuel des analisa chimiques en milieu marin. In: Edições Jouve. CNEXO, Paris (395 p). AOAC (Association of Official Analitycal Chemists), 2000. In: Cunniff, Patricia (Ed.), Official Methods of Analysis of AOAC, 16th ed. (Washington, DC). APHA/AWWA/WEF, 2012. Standard methods for the examination of water and wastewater. Stand. Methods 541 (ISBN 9780875532356). Avnimelech, Y., 2009. Biofloc Technology - a Practical Guide Book, 2nd edition. The Word Aquaculture Society, Baton Rouge, Louisiana, United States, pp. 272. Ballester, E.L.C., Wasielesky Jr., W., Cavalli, R.O., Silva Santos, M.H., Abreu, P.C., 2003. Influência do biofilme no crescimento do camarão-rosa Farfantepenaeus paulensis em sistemas de berçário. vol. 25 (2). Atlântica, Rio Grande, RS, pp. 37–42. Boyd, C.E., Tucker, C.S., 2012. Pond Aquaculture Water Quality Management, 2nd ed. pp. 700. Chen, J., Chin, C., CK, L., 1986. Effects of Ammonia and Nitrite on Larval Development of the Shrimp Penaeus monodon. Asian Fish. Soc, pp. 657–662. de Yta, A.G., Rouse, D.B., Davis, D., 2004. Influence of nursery period on the growth and survival of litopenaeus vannamei under pond production conditions. J. World Aquac. Soc. 35, 357–365. https://doi.org/10.1111/j.1749-7345.2004.tb00099.x. Ebeling, J.M., Timmons, M.B., Bisogni, J.J., 2006. Engineering analysis of the stoichiometry of photoautotrophic, autotrophic, and heterotrophic removal of ammonia-nitrogen in aquaculture systems. Aquaculture 257, 346–358. https://doi.org/10.1016/ j.aquaculture.2006.03.019.
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