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Using poly-β-hydroxybutyric as an additional carbohydrate for biofloc in a shrimp Litopenaeus vannamei bioflocs nursery system with brackish water
Aquaculture 506 (2019) 181–187
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Short communication
Using poly-β-hydroxybutyric as an additional carbohydrate for biofloc in a shrimp Litopenaeus vannamei bioflocs nursery system with brackish water
T
⁎
Guozhi Luoa,b,c, , Zefeng Liua, Lina Shaoa, Hongxin Tana,b,c a
Shanghai Engineering Research Center of Aquaculture (Shanghai Science and Technology Committee), Shanghai Ocean University, Shanghai 201306, China Key Laboratory of Freshwater Aquatic Genetic Resources (Ministry of Agriculture of PRC), Shanghai Ocean University, Shanghai 201306, China c National Demonstration Center for Experimental Fisheries Science Education (Ministry of Education of PRC), Shanghai Ocean University, Shanghai 201306, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: C/N ratio Biofloc technology Litopenaeus vannamei Nursery Poly-β-hydroxybutyric Ammonium nitrogen
Biofloc technology involves manipulation of the C/N ratio to convert toxic nitrogenous wastes into useful microbial protein and helps improve the water quality in a closed system. Poly-β-hydroxybutyric (PHB) has been demonstrated to have positive effects on aquatic animals in an aquaculture system. A 40-day lab scale trial was carried out to investigate the potential of PHB as an additional carbohydrate for a shrimp Litopenaeus vannamei bioflocs nursery in brackish water. Three methods of carbohydrate supplementation were investigated. The first method was the addition of glucose daily at a percentage of 75% of the feed (GLU-group). The second method was the addition of PHB plus glucose; when the total ammonia nitrogen (TAN) level above 2 mg L−1 was observed, glucose was added, 6 g C to 1 g TAN (PHB + GLU-group). The third method was the addition of PHB only (PHB-group). During the 40-d trial, most of the ratios of dissolved organic carbon to total ammonium nitrogen (DOC/TAN) were maintained at 20 in the three groups. The average concentration of TAN in the PHB + GLUgroup (0.23 ± 0.19 mg L−1) was significantly lower than those of the GLU-group (0.28 ± 0.26 mg L−1) and the PHB-group (0.30 ± 0.33 mg L−1) (P < .05). The average concentration of nitrite nitrogen in the PHBgroup (0.16 ± 0.24) was significantly lower than those of the GLU-group and the PHB-group (P < .05). The concentrations of TAN and nitrite nitrogen from the PHB + GLU-group were more stable than those from the PHB-group and GLU-group. Nitrate nitrogen accumulated, as expected, in the three groups, and no significant differences were observed (P > .05). The survival rate and final weight in the PHB + GLU group were significantly higher than those of the other groups (P < .05). The food conversion ratio of the PHB group (1.52 ± 0.23) was significantly lower than those of the other groups. Taken together, our results showed that PHB is a favorable and convenient additional carbohydrate for shrimp Litopenaeus vannamei biofloc nurseries in brackish water. PHB + GLU was the best carbohydrate supplementation method.
1. Introduction Biofloc technology (BFT) in aquaculture systems refers to the rearing of aquatic animals in high stocking densities renewal with heterotrophic biota that form microbial flocs and without replenishing water (Avnimelech, 2007; Hargreaves, 2013). It has been extensively proven that biofloc in an aquaculture system provides two critical services — treating wastes from feeding and providing nutrition from floc consumption, saving the cost of an external biological filter for the normally closed aquaculture system and improving the efficiency of the nutrient utilization of the feed (Schryver et al., 2008). Heterotrophic biota uses carbohydrates to generate energy, grow and produce new cells (Avnimelech, 1999). Considering that the C/N ratio of bacterial cells is 5:1 (Rittmann and McCarty, 2001) and ⁎
accounting for the demand of respiration, the optimal C/N ratio might be higher than that of bacteria under aerobic conditions. Therefore, a C/N ratio of 15–20 is required for the assimilation of ammonia by heterotrophic bacteria (Avnimelech, 1999; Asaduzzaman et al., 2008). The C/N ratio can be increased by adjusting the C/N ratio of the fish feed or via external carbohydrates (Avnimelech, 1999; Hargreaves, 2013; Dauda et al., 2018). Some soluble carbohydrates, such as glucose, glycerol, acetate and molasses have been commonly used (Schneider et al., 2006). These materials are added several times a day or over several days during the production period (Khatoon et al., 2016). In practice, supplementation with a soluble carbohydrate is always associated with the risk of overfeeding or starvation of organic carbon, and the need for daily additions is also criticized as an extra task (Serfling, 2006). Moreover, pulsed additions of these organic materials into water
Corresponding author at: Hucheng Ring Road, 999, Shanghai Ocean University, 201306 Shanghai, China. E-mail address: [email protected] (G. Luo).
https://doi.org/10.1016/j.aquaculture.2019.03.021 Received 11 October 2018; Received in revised form 3 January 2019; Accepted 11 March 2019 Available online 12 March 2019 0044-8486/ © 2019 Elsevier B.V. All rights reserved.
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nylon bags filled with 40 g of PHB granules placed at the bottom of each tank as for the PHB-group, when TAN level above 2 mg L−1 was observed, 6 g C from glucose was added to 1 g TAN (Avnimelech, 1999), referred to as PHB + GLU group. The consumption of glucose and PHB was recorded. PHB beads ([C4H7O3]) (Ningbo Tian Co. Ltd., Zhejiang, China) were ultrasonically cleaned (0.1 kW, 40 kHz) to remove the unpolymerized small organic molecules on the surface (Zhang et al., 2016), then dried to a constant mass of ± 0.0001 g at 35 °C. The physical properties of the PHB (Ningbo Tian Co. Ltd., Zhejiang, China) were 2–3 mm diameter, 120,000 molecular weight, 1.25 kg L−1 density, 600% elongation and 175 °C melting point.
may result in a sudden lowering of the dissolved oxygen (DO) content (Schryver et al., 2008). It was found that a single addition of 0.12 g L−1 of molasses produced a dramatic reduction of the DO content from 3.2 to 1.5 mg L−1 (Pérez–Fuentes et al., 2016). Recently, biological degradation polymers (BDPs), such as poly-βhydroxybutyric (PHB), and polycaprolactone (PCL), have been investigated as additional carbohydrates and biofilm carriers for denitrification (Müller et al., 1992; Boley et al., 2000; Zhang et al., 2016; Luo et al., 2017). BDPs are subject to enzymatic attack to release dissolved organic carbon (DOC), which is helpful to save the calculation of the needed amount of additional carbohydrates (Müller et al., 1992; Boley et al., 2000; Chu and Wang, 2011). PHB has been proved to efficiently maintain the C/N ratio in a BFT-tilapia aquaculture system (Zhang et al., 2016). Litopenaeus vannamei (L. vannamei) is the most common species reared in biofloc-based systems (Lara et al., 2017. Ray and Ray, 2017). The choice of the most appropriate additional carbohydrate supplementation method is critical to the culture process. PHB is produced or accumulated by a wide range of bacteria. Positive effects of PHB on aquatic animals in an aquaculture system have been widely demonstrated (Defoirdt et al., 2007; Schryver et al., 2008; García et al., 2014; Zhang et al., 2016). The primary objective of this study was to investigate the potential of the using PHB as an external source of carbohydrates for a shrimp L. vannamei biofloc nursery in brackish water. This method can decrease the complexity of manipulating the C/N ratio in a BFT-based shrimp rearing system.
2.2. Methods for the determination of experimental parameters 2.2.1. Water quality parameters, flocs volume index in 5 min (FVI-5), crude protein content of bioflocs and components of DOC Water temperature (°C), dissolved oxygen (DO) and pH were measured daily with a WTW meter (WTW Multi 3430 SETF, Germany). Total nitrogen (TN), TSS, total ammonium nitrogen (TAN), nitrite nitrogen (NO2−-N) and nitrate nitrogen (NO3−-N) were analyzed daily according to standard methods (APHA, 2005). The dissolved organic carbon (DOC) was evaluated using a Total Organic Carbon Analyzer (Multi N/C 2100, Germany). The volume of bioflocs in 5 min (FVI-5) was measured every 10 days using an Imhoff graduated cone. On days 10 and 40, the crude protein content of the bioflocs was determined as nitrogen (N, 6.25) with the Kjeldahl method. The components of DOC were analyzed using solid-phase microextraction gas chromatography mass spectrometry.
2. Materials and methods
2.2.2. Shrimp growth parameters At the end of the experiment (40 d), all the live shrimp in each experimental unit were weighed and counted to determine growth and survival in the different treatments.
2.1. Experimental design This study was performed in 9 plastic tanks (effective volume 0.5 L, as described previously) (Zhang et al., 2016; Luo et al., 2017) filled with brackish water (salinity 15) and 300 ± 20 mg L−1 mature biofloc initially established from the suspended growth reactors. The pH in each tank was maintained at 8.0 using NaHCO3. The water temperature was maintained at approximately 30 ± 1 °C. Three compressed air stones in the center of each BFT tank were used for aeration to keep biofloc suspended and to maintain a DO above 6 mg L−1. No water was renewed during the experimental period, which lasted 40 days. Dechlorinated municipal water was added because of the evaporation and sampling. Measurement of TSS was performed once daily. When the concentrations of total suspended solids (TSS) exceeded 500 mg L−1 in the BFT tanks, some water containing flocs was removed. After being settled for half hour, the supernatant was return to the BFT tank and the bioflocs were collected and stored at −20 °C for subsequent analysis. Post-larvae of L. vannamei (0.02 ± 0.004 g L−1) were obtained from Global Biotechnology Ltd., Shenzhen, China. After being acclimated for 2 d to the experimental conditions, they were stocked in tanks (300 L−1 m−3). Beginning on the first day, the shrimp were fed Plus Post-Larval Diet (Yuequn Marine Biotechnology Ltd., Guangdong, China) with varying crumble sizes according to the size of shrimp. According to the manufacturer, this diet contained 46% protein, 8% fat, 5% fiber and 7.5% ash. Beginning on day 11, we changed to the feed containing 42% crude protein, 4% fat, 12% fiber, 18% ash, Ca 5%, P 1.0%) (Quanxing Nutrition Ltd., Jiangsu, China), and the feeding amount was based on a percentage (5%) of the assumed shrimp biomass. The photoperiod was natural, and temperature was controlled at 30 °C. Three different methods of supplying carbon sources for BFT systems were carried out with three replicates: Glucose only, edible glucose (40% w w−1 carbon, purity 99%) was added daily at a rate of 75% of the feed, the C/N ratio input was approximately 10/1 (Gao et al., 2012), referred to as GLU-group; PHB only, 4 nylon bags filled with 40 g of PHB granules placed at the bottom of each tank, no other supplementation with glucose, referred to as PHB-group; PHB plus glucose, 4
Survival ratio (SR%) = 100 × (final n / initial n); Final mean weight (g) = Final total biomass / final n; Weight gain rate (WGR, %) = 100 × (Final weight - initial weight) / initial weight； Specific growth rate (SGR % d−1) = 100 × (ln final weight-ln initial weight)/days of experiment; Feed conversion ratio (FCR) = (Feed applied -leaching)/ weight gain where n = number of shrimps. 2.3. Statistical analysis Statistical analysis was performed using the SPSS Statics 19.0 software for Windows (IBM Corporation, NY, USA). Differences in water quality parameters, the growth performance of the shrimp and the parameters involving bioflocs were analyzed using a one-way ANOVA. Difference was considered significant at a P < .05. Tukey's test was used to assess differences among the three groups. 3. Results 3.1. Water quality parameters The results of selected water quality parameters are presented in Table 1. No differences were found among the three treatments, in terms of pH, DO, or temperature (°C) during the experimental period. Furthermore, all the parameters measured were within acceptable ranges for the shrimp (L. vannamei) throughout the experiment (Van and Scarpa, 1999). Both the concentrations of TAN and NO2−-N in all groups were kept at low levels during the whole experimental period. The concentrations of TAN and NO2−-N in the PHB and GLU groups fluctuated noticeably, while those in the PHB + GLU group were more stable (Fig. 1). The average TAN concentration was significantly lower in the PHB + GLU 182
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Table 1 Water quality parameters during the 40 days shrimp nursery experiment. Data are presented as mean ± standard error. (range), and different superscript letters in a row indicate significant differences. Item
Treatments
pH Dissolved oxygen /mg L−1 Temperature /°C NO2−-N/mg L−1 Total ammonium nitrogen /mg L−1 NO3−-N/mg L−1 Total nitrogen /mg L−1 Alkalinity/mg CaCO3 L−1 Dissolved organic carbon (DOC)/ mg L−1
GLU
PHB + GLU
PHB
7.90 ± 0.16 a 7.53, 8.14 7.34 ± 0.30 a 6.91, 7.77 30.33 ± 1.50 a 28.80, 32.83 0.20 ± 0.22 a 0, 0.83 0.28 ± 0.26 a 0, 1.21 19.39 ± 13.6 a 0.33, 37.23 24.76 ± 10.97 a 10.57, 42.52 121.15 ± 25.79 a 72.65, 146.43 64.9 ± 5.5 a 12.9, 935.5
7.92 ± 0.15 a 7.56, 8.13 7.28 ± 0.28 a 6.79, 7.67 30.55 ± 1.31 a 29.17, 33.03 0.19 ± 0.12 a 0, 0.66 0.23 ± 0.19b 0.02, 0.96 20.46 ± 12.21 a 0.46, 36.47 25.57 ± 11.98 a 11.46, 43.54 121.72 ± 25.37 a 71.13, 159.67 33.7 ± 2.3 b 9.7, 182.3
7.94 ± 0.14 a 7.58, 8.13 7.37 ± 0.25a 7.02,7.73 30.29 ± 1.40 a 28.87, 32.8 0.16 ± 0.24 b 0, 1.00 0.30 ± 0.33 a 0.06, 1.26 19.59 ± 12.25 a 0.53, 35.65 23.9 ± 11.23 a 10.68, 43.74 122.25 ± 18.47 a 94.21, 150.59 23.9 ± 1.2 c 8.4, 75.0
-1
Total nitrogen concentration (mg L )
GLU PHB+GLU PHB
-1
(a)
Total ammonium nitrogen concentration (mg L )
Note: Values in the same row with different superscripts are significantly different (P < .05).
7LPHd
GLU PHB+GLU PHB
Nitrate nitrogen concentration (mg L-1 )
Nitrite nitrogen concentration (mg L-1 )
7LPHd
(c)
GLU PHB+GLU PHB
(b)
7LPHd
(d)
GLU PHB+GLU PHB
7LPHd
Fig. 1. Total nitrogen (TN) (a), total ammonium nitrogen (TAN) (b), nitrite nitrogen (NO2–N) (c) and nitrate nitrogen (NO3–N) in the shrimp culture tanks throughout this study. Data points represent treatment means. Error bars are one standard error around the mean. Different superscript letters indicate significant differences. 183
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Dissolved organic carbon concentration (mg L )
G. Luo, et al.
Consumption of carbohydrates
Treatments GLU
Glucose PHB DOC from glucose DOC from PHB
-1
Table 2 The total addition of carbohydrates (g) and organic carbon (g) in three groups.
PHB + GLU a
141.67 ± 2.00 – 56.67 ± 0.80 –
36.97 48.05 14.79 26.91
± ± ± ±
PHB b
0.19 3.18a 0.08 1.78
– 58.13 ± 4.18b – 32.55 ± 2.34
Note: Values in the same row with different superscripts are significantly different (P < .05). DOC: dissolved organic carbon. PHB: Poly-β-hydroxybutyric.
group than in the GLU group, although differences in mean values were subtle (Table 1). The average NO2−-N concentration was significantly lower in the PHB group; no significant difference in this parameter was observed between the GLU group and PHB + GLU group (Table 1). The nitrate nitrogen buildup lasted until the end of the experiment, increasing to 37.23 ± 3.01 mg L−1 in the GLU group, 36.47 ± 2.41 mg L−1 in the PHB + GLU groups, and 35.65 ± 4.73 mg L−1 in the PHB group. There were no significant differences in nitrate nitrogen concentrations among the groups.
(a)
GLU PHB+GLU PHB
DOC/TAN
(b)
3.2. The consumption of PHB and glucose, and the dynamics of the DOC/ TAN ratio The consumption of PHB and glucose during the 40-d trial for the three groups are presented in Table 2. An average mass of 141.67 ± 2.00 g of glucose with 56.67 ± 0.80 g C was added into each tank in the GLU group; to the PHB + GLU group was added 36.97 ± 0.19 g glucose with 14.79 ± 0.08 g C. Although the same initial weight of PHB was put into both the PHB + GLU and PHB groups, the consumption of PHB for each tank was 48.05 ± 3.18 g (26.91 ± 1.78 g C) in the PHB + GLU group, while in the PHB group consumption was 58.13 ± 4.18 g with 32.55 ± 2.34C, suggesting suggested that the addition of glucose decreased the degradation of PHB in this study. The C/N (w/w) input was approximately 10 in the GLU group. The average DOC concentration in the GLU group was 20.57 ± 9.06 mg L−1, significantly higher than those of the PHB + GLU and PHB groups (p < .05, Table 1). There was no significant difference in average DOC concentration between the PHB + GLU and PHB groups (Table 1). The DOC in the PHB + GLU group was more stable than that of GLU group or the PHB group (Fig. 2 a). Except for the DOC on day 36, the DOC in the PHB group was as stable as that of the PHB + GLU group. The changes in the DOC/TAN ratio are shown in Fig. 2 b. During the 40-d trial, most of the DOC/TAN ratios in the three groups were higher than 20 and fluctuated noticeably.
Time/d Fig. 2. Dynamics of the changes in the dissolved organic carbon (DOC) and DOC/TAN ratio in the reactors of the GLU group, PHB + GLU group and PHB group throughout the 40-d trial. Data points represent the treatment means. Error bars are one standard error around the mean. TAN: total ammonium nitrogen. Different superscript letters indicate significant differences.
3.4. Growth performance of the shrimp Growth performance during the experimental period was not very good for shrimp in any of the groups. At the end of the study, the survival rate of shrimp in the PHB + GLU group was 67 ± 6%, the highest among the three groups, followed by the PHB group. No significant difference in FCR was observed between the GLU group and PHB + GLU groups, both of which had significantly lower values than that of the PHB group (Table 3). The survival rate and the final weight in the PHB + GLU group were higher significantly than those of the other two groups.
3.3. Biofloc FVI-5, crude protein content, and C/N ratio 4. Discussion
The biofloc development in terms of FVI-5 min over 40 days is presented in Fig. 3. A significant increase (p < .05) in FVI-5 was found on day 20, then this parameter decreased on day 30. No continuous decrease was found between day 30 and day 40. The FVI-5 of bioflocs in the PHB + GLU group was significantly higher than that of the PHB and GLU groups. The FVI-5 of bioflocs in the PHB group was the lowest. The protein content in bioflocs in the three groups increased significantly at the end of the experiment. Protein content was similar in the GLU group and PHB + GLU group, both of which were significantly higher than that of the PHB group. No significant differences in C/N of bioflocs were observed among the three groups, ranging from 5.11 to 5.52 (Fig. 3d).
In this study, pH, DO, temperature, alkalinity and the inorganic nitrogen measured were within acceptable ranges for the shrimp (L. vannamei) throughout the 40-d trial (Van Wyk and Scarpa, 1999). TAN and NO2−-N in PHB + GLU remained more stable than in the GLU and PHB groups. The growth performance of shrimp in the PHB + GLU group was also the best among the three groups, suggesting that the carbohydrate supplementation method in the PHB + GLU group, in which PHB was submerged in the tank and in which glucose was added 6 g C to 1 g TAN when TAN level above 2 mg L−1 was observed, was the best method. PHB can be replaced with other BDPs, such as PCL or poly (butylene succinate), and glucose can also be replaced with other 184
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Fig. 3. Then day changes in the flocs volume index in 5 min (FVI-5) (a), crude protein content (b) and C/N (c) of the bioflocs in the reactors of the three groups in the 40-d trial. Error bars are one standard error around the mean. Different superscript letters indicate significant differences. Table 3 Growth performance of Litopenaeus vannamei in different treatment groups. Item
Initial weight (g) Final weight (g) Initial stocking density (shrimp m−3) Survival rate (%) Weight gain rate (%) Specific growth rate (%d−1) Feed conversion ratio (%)
Treatments GLU
PHB + GLU
PHB
0.016 ± 0.004 0.78 ± 0.11 a 300 60 ± 3 a 4756.25 ± 1273.82 9.71 ± 0.65 a 1.63 ± 0.26 a
0.016 ± 0.004 1.01 ± 0.06 b 300 67 ± 6 b 6187.50 ± 656.28 10.35 ± 0.29 b 1.63 ± 0.62 a
0.016 ± 0.004 0.87 ± 0.10 c 300 62 ± 5 c 5325.00 ± 924.91c 9.98 ± 0.39 c 1.52 ± 0.23 b
a
b
Note: Values in the same row with different superscripts are significantly different (P < .05).
4.1. PHB as additional organic carbon source for BFT
soluble organic materials, such as molasses or sucrose. More research needs to be conducted about this. However, the growth performance and survival rate of the shrimp in the three groups were not consistently ideal. The postlarvae from the same batch as this study were found to be infected with White Spot Syndrome Virus in other places (Not presented here), which is perhaps one the reasons that the survival rate was low in this study.
Manipulating the C/N ratio is the most critical for BFT in aquaculture (Avnimelech, 1999). To date, the real-time monitoring of the C/ N ratio in the water in BFT aquacultural systems could not be performed. The amount of carbon added should be varied with the nitrogen in the feed or TAN in the water, and both of two parameters constantly change as production progresses, especially the TAN level. All these considerations complicate the decision for supplying the optimal amount of soluble external carbohydrate as a carbon source. 185
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TAN ratio to describe the C/N station in the water. Assimilation of inorganic nitrogen is considered to take place when the C/N ratio is higher than 10 (Lancelot and Billen, 1985). Alteration of the C/N ratio may result in a shift from an autotrophic to a heterotrophic system (Avnimelech, 1999; Browdy and Bratvold, 2001). Maintaining a C/N ratio higher than 15–20 in a biofloc aquacultural system is thought to develop sufficient bacteria to assimilate ammonia into biomass even under intensive farming with unchanged water. However, several reports have found that when nitrate accumulated as high as hundreds mg L−1 even the C/N ratio was higher than 15 or 20 (Azim and Little, 2008; Xu et al., 2016；Zhang et al., 2016; Pérez–Fuentes et al., 2016; Luo et al., 2017; Dauda et al., 2018). In this study, the nitrate nitrogen in the PHB + GLU and PHB groups were almost twice the nitrogen in bioflocs (Fig. 4), suggesting that most unused nitrogen was converted into nitrate, not bacterial biomass; even the DOC/TAN ratio was higher than 20 (Fig. 2). The mechanism of nitrate nitrogen accumulation in this study is unclear. This aspect merits further investigation. The organic carbon in water supplies is mainly composed of humic and fulvic acids, carbohydrates, proteins, and carboxylic acids (Escobar et al., 2000). Only a fraction of DOC is assimilated and/or mineralized by a heterotrophic flora (Huck, 1990). At the end of the experiment, the constituents of DOC were further analyzed to determine if the DOC can be used (Table 4). Seven compounds were found in the GLU group. In the PHB and PHB + GLU groups, only methyl methacrylate and semi-carbazide were obtained. Methyl methacrylate was the main compound in the three groups. However, to our knowledge information related to these compounds and heterotrophic bacteria cannot be found. So, an explanation cannot be given here. The C/N ratio of bacterial cells is 5:1 (Rittmann and McCarty, 2001). The C/N ration of bioflocs in this study was equal to this value. No significant difference in C/N ratios of bioflocs were observed among the three groups or between day 10 and day 40. Few investigations about BFT include data on the C/N ratio of bioflocs. Perhaps the C/N ratio of bioflocs can be used as a single parameter to describe the characteristics of bioflocs. The paper proposed a method to control TAN and NO2−-N in biofloc culture Litopenaeus vannamei systems. In BFT aquaculture system, carbohydrates, such as Molasses, glucose, and starch) are always to be added to the water one more time in a day in order to stimulate dissolved inorganic nitrogen assimilation. Instead of the current daily additions, using biological degradable polymers (poly-β-hydroxybutyric (PHB) as carbon sources to release soluble carbon source slowly to control the system is more conveniently. This is an interesting and new approach in BFT-white prawn aquaculture. However, this paper only deals with the primary process. A lot of research needs to be carried out in the future, including the optimal ratio of carbohydrates added, stocking biomass and flocs amounts, and the cost of this treatment. Though there are still some questions needed to be handled prior to in practice, this topic is interesting.
Fig. 4. Nitrogen content in biofloc and nitrate on day 10 and day 40 in the reactors of the three groups. Error bars are one standard error around the mean. Different superscript letters indicate significant differences.
Using BDPs to maintain the C/N ratio in BFT offers no such degree of freedom. This study proved the potential for the use of PHB as a sole external carbohydrate to support a shrimp Litopenaeus vannamei nursery system. The best strategy is to use PHB as regular carbohydrate and add glucose 6 g C to 1 g TAN when TAN level above 2 mg L−1 is observed. PHB is produced or accumulated by a wide range of bacteria, and it has shown positive effects on aquatic animals in an aquacultural system (Defoirdt et al., 2007; Schryver et al., 2008; García et al., 2014). Unlike glucose, PHB must be degraded to DOC first. Biotic and abiotic factors act synergistically to decompose organic matter. Biodegradation is considered to be the predominant process (Lucas et al., 2008). Although the biodegradation of PHB has become better understood, no research reports on biodegradation under conditions such as those used in this study were found. The amount of the PHB used in this study was merely based on a rough estimate and may be not the best amount; therefore, the amount of PHB used needs to be optimized.
4.2. C/N and nitrate nitrogen accumulation In theory, “C/N” refers to the carbon/nitrogen ratio of atoms, which should be used directly by the bacteria (Avnimelech, 1999). However, in practice, both the “C” and “N” exist as many forms and the amount of the added carbon was varied with the nitrogen in the feed or TAN in the water. Both parameters constantly changed, especially the TAN level. Therefore, the supplying water soluble carbohydrates to maintain a favorable C:N ratio for heterotrophic bacteria is complicated. Using BDPs to maintain the C:N ratio in BFT offers no such degree of freedom. In addition, even if the value of C/N input into the water is very appropriate for the bacteria, it does not insure that the C/N in the water is also appropriate for the bacteria. Therefore, this study used the DOC/
Table 4 Compounds in the water in the three groups at the end of the experiment obtained from the GC/MS with the extract of DOC. Name of the compound
Methyl methacrylate Semicarbazide Allethrin 3-Methyl-6(1-methylethylidene) cyclohexene Curcumin Artemisinic acid
Molecular formula
Peak Area % of DOC in different group
C4H8O2 CH5N3O C19H26O3 C10H16 C21H20 C15H22O2
186
GLU
PHB + GLUU
PHB
86.0–87.0 1.0–2.0 1.0–2.0 1.0–2.0 1.5–2.5 7.0–8.0
88.5–89.5 10.5–11.5 – – – –
94.0–95.0 5.0–6.0 – – – –
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5. Conclusion
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Three different organic carbon sources for water quality control in a bioflocs shrimp Litopenaeus vannamei nursery system were investigated: (1) glucose added daily at a percentage of 75% of the feed, (2) PHB plus 6 g C glucose to 1 g TAN when a TAN level above 2 mg L−1 was observed and (3) only PHB. The salinity was 15. The water was not changed for 40 days. The DOC/TAN ratio in the water of all three groups was maintained up to 15. All the water parameters measured were within the acceptable ranges for the shrimp (L. vannamei) throughout the experiment. Nitrate nitrogen noticeably accumulated in the three groups, and no significant differences were observed. The survival rate and final weight in the PHB + GLU group were significantly higher than those of the other groups. The food conversion ratio of the PHB group was significantly lower than those of the other groups. Taken together, these results indicate that PHB is a favorable and convenient additional carbohydrate for a shrimp L. vannamei bioflocs nursery in brackish water. PHB + GLU was the best organic carbon supplementation method. However, this paper only deals with the primary process. A lot of research needs to be carried out in the future. Acknowledgements This study was funded by the Shanghai Science and Technology Commission Project (14320501900). References APHA, 2005. Standard Methods for the Examination of Water and Wastewater. American Public Health Association, Washington D. C. Asaduzzaman, M., Wahab, M.A., Verdegem, M.C.J., Huque, S., Salam, M.A., Azim, M., 2008. C/N ratio control and substrate addition for periphyton development jointly enhance freshwater prawn Macrobrachium rosenbergii production in ponds. Aquaculture 280, 117–123. Avnimelech, Y., 1999. Carbon/nitrogen ratio as a control element in aquaculture systems. Aquaculture 176, 227–235. Avnimelech, Y., 2007. Feeding with microbial flocs by tilapia in minimal discharge bioflocs technology ponds. Aquaculture 264, 140–147. Azim, M., Little, D.C., 2008. The biofloc technology (BFT) in indoor tanks: water quality, biofloc composition, and growth and welfare of Nile tilapia (Oreochromis niloticus). Aquaculture 283, 29–35. Boley, A., Müller, W.R., Haider, G., 2000. Biodegradable polymers as solid substrate and biofilm carrier for denitrification in recirculated aquaculture systems. Aquacult. Eng. 22, 75–85. Browdy, D., Bratvold, C.L., 2001. Effects of sand sediment and vertical surfaces (AquaMatsTM) on production, water quality, and microbial ecology in an intensive Litopenaeus vannamei culture system. Aquaculture 1–2, 81–94. Chu, L., Wang, J., 2011. Nitrogen removal using biodegradable polymers as carbon source and biofilm carriers in a moving bed biofilm reactor. Chem. Eng. J. 170, 220–225. Dauda, A.B., Romano, N., Ebrahimi, M., Ajadi, A., Chong, C.M., Karim, M., Natrah, I., Kamarudin, M.S., 2018. Influence of carbon/nitrogen ratios on biofloc production
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Short communication
Using poly-β-hydroxybutyric as an additional carbohydrate for biofloc in a shrimp Litopenaeus vannamei bioflocs nursery system with brackish water
T
⁎
Guozhi Luoa,b,c, , Zefeng Liua, Lina Shaoa, Hongxin Tana,b,c a
Shanghai Engineering Research Center of Aquaculture (Shanghai Science and Technology Committee), Shanghai Ocean University, Shanghai 201306, China Key Laboratory of Freshwater Aquatic Genetic Resources (Ministry of Agriculture of PRC), Shanghai Ocean University, Shanghai 201306, China c National Demonstration Center for Experimental Fisheries Science Education (Ministry of Education of PRC), Shanghai Ocean University, Shanghai 201306, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: C/N ratio Biofloc technology Litopenaeus vannamei Nursery Poly-β-hydroxybutyric Ammonium nitrogen
Biofloc technology involves manipulation of the C/N ratio to convert toxic nitrogenous wastes into useful microbial protein and helps improve the water quality in a closed system. Poly-β-hydroxybutyric (PHB) has been demonstrated to have positive effects on aquatic animals in an aquaculture system. A 40-day lab scale trial was carried out to investigate the potential of PHB as an additional carbohydrate for a shrimp Litopenaeus vannamei bioflocs nursery in brackish water. Three methods of carbohydrate supplementation were investigated. The first method was the addition of glucose daily at a percentage of 75% of the feed (GLU-group). The second method was the addition of PHB plus glucose; when the total ammonia nitrogen (TAN) level above 2 mg L−1 was observed, glucose was added, 6 g C to 1 g TAN (PHB + GLU-group). The third method was the addition of PHB only (PHB-group). During the 40-d trial, most of the ratios of dissolved organic carbon to total ammonium nitrogen (DOC/TAN) were maintained at 20 in the three groups. The average concentration of TAN in the PHB + GLUgroup (0.23 ± 0.19 mg L−1) was significantly lower than those of the GLU-group (0.28 ± 0.26 mg L−1) and the PHB-group (0.30 ± 0.33 mg L−1) (P < .05). The average concentration of nitrite nitrogen in the PHBgroup (0.16 ± 0.24) was significantly lower than those of the GLU-group and the PHB-group (P < .05). The concentrations of TAN and nitrite nitrogen from the PHB + GLU-group were more stable than those from the PHB-group and GLU-group. Nitrate nitrogen accumulated, as expected, in the three groups, and no significant differences were observed (P > .05). The survival rate and final weight in the PHB + GLU group were significantly higher than those of the other groups (P < .05). The food conversion ratio of the PHB group (1.52 ± 0.23) was significantly lower than those of the other groups. Taken together, our results showed that PHB is a favorable and convenient additional carbohydrate for shrimp Litopenaeus vannamei biofloc nurseries in brackish water. PHB + GLU was the best carbohydrate supplementation method.
1. Introduction Biofloc technology (BFT) in aquaculture systems refers to the rearing of aquatic animals in high stocking densities renewal with heterotrophic biota that form microbial flocs and without replenishing water (Avnimelech, 2007; Hargreaves, 2013). It has been extensively proven that biofloc in an aquaculture system provides two critical services — treating wastes from feeding and providing nutrition from floc consumption, saving the cost of an external biological filter for the normally closed aquaculture system and improving the efficiency of the nutrient utilization of the feed (Schryver et al., 2008). Heterotrophic biota uses carbohydrates to generate energy, grow and produce new cells (Avnimelech, 1999). Considering that the C/N ratio of bacterial cells is 5:1 (Rittmann and McCarty, 2001) and ⁎
accounting for the demand of respiration, the optimal C/N ratio might be higher than that of bacteria under aerobic conditions. Therefore, a C/N ratio of 15–20 is required for the assimilation of ammonia by heterotrophic bacteria (Avnimelech, 1999; Asaduzzaman et al., 2008). The C/N ratio can be increased by adjusting the C/N ratio of the fish feed or via external carbohydrates (Avnimelech, 1999; Hargreaves, 2013; Dauda et al., 2018). Some soluble carbohydrates, such as glucose, glycerol, acetate and molasses have been commonly used (Schneider et al., 2006). These materials are added several times a day or over several days during the production period (Khatoon et al., 2016). In practice, supplementation with a soluble carbohydrate is always associated with the risk of overfeeding or starvation of organic carbon, and the need for daily additions is also criticized as an extra task (Serfling, 2006). Moreover, pulsed additions of these organic materials into water
Corresponding author at: Hucheng Ring Road, 999, Shanghai Ocean University, 201306 Shanghai, China. E-mail address: [email protected] (G. Luo).
https://doi.org/10.1016/j.aquaculture.2019.03.021 Received 11 October 2018; Received in revised form 3 January 2019; Accepted 11 March 2019 Available online 12 March 2019 0044-8486/ © 2019 Elsevier B.V. All rights reserved.
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nylon bags filled with 40 g of PHB granules placed at the bottom of each tank as for the PHB-group, when TAN level above 2 mg L−1 was observed, 6 g C from glucose was added to 1 g TAN (Avnimelech, 1999), referred to as PHB + GLU group. The consumption of glucose and PHB was recorded. PHB beads ([C4H7O3]) (Ningbo Tian Co. Ltd., Zhejiang, China) were ultrasonically cleaned (0.1 kW, 40 kHz) to remove the unpolymerized small organic molecules on the surface (Zhang et al., 2016), then dried to a constant mass of ± 0.0001 g at 35 °C. The physical properties of the PHB (Ningbo Tian Co. Ltd., Zhejiang, China) were 2–3 mm diameter, 120,000 molecular weight, 1.25 kg L−1 density, 600% elongation and 175 °C melting point.
may result in a sudden lowering of the dissolved oxygen (DO) content (Schryver et al., 2008). It was found that a single addition of 0.12 g L−1 of molasses produced a dramatic reduction of the DO content from 3.2 to 1.5 mg L−1 (Pérez–Fuentes et al., 2016). Recently, biological degradation polymers (BDPs), such as poly-βhydroxybutyric (PHB), and polycaprolactone (PCL), have been investigated as additional carbohydrates and biofilm carriers for denitrification (Müller et al., 1992; Boley et al., 2000; Zhang et al., 2016; Luo et al., 2017). BDPs are subject to enzymatic attack to release dissolved organic carbon (DOC), which is helpful to save the calculation of the needed amount of additional carbohydrates (Müller et al., 1992; Boley et al., 2000; Chu and Wang, 2011). PHB has been proved to efficiently maintain the C/N ratio in a BFT-tilapia aquaculture system (Zhang et al., 2016). Litopenaeus vannamei (L. vannamei) is the most common species reared in biofloc-based systems (Lara et al., 2017. Ray and Ray, 2017). The choice of the most appropriate additional carbohydrate supplementation method is critical to the culture process. PHB is produced or accumulated by a wide range of bacteria. Positive effects of PHB on aquatic animals in an aquaculture system have been widely demonstrated (Defoirdt et al., 2007; Schryver et al., 2008; García et al., 2014; Zhang et al., 2016). The primary objective of this study was to investigate the potential of the using PHB as an external source of carbohydrates for a shrimp L. vannamei biofloc nursery in brackish water. This method can decrease the complexity of manipulating the C/N ratio in a BFT-based shrimp rearing system.
2.2. Methods for the determination of experimental parameters 2.2.1. Water quality parameters, flocs volume index in 5 min (FVI-5), crude protein content of bioflocs and components of DOC Water temperature (°C), dissolved oxygen (DO) and pH were measured daily with a WTW meter (WTW Multi 3430 SETF, Germany). Total nitrogen (TN), TSS, total ammonium nitrogen (TAN), nitrite nitrogen (NO2−-N) and nitrate nitrogen (NO3−-N) were analyzed daily according to standard methods (APHA, 2005). The dissolved organic carbon (DOC) was evaluated using a Total Organic Carbon Analyzer (Multi N/C 2100, Germany). The volume of bioflocs in 5 min (FVI-5) was measured every 10 days using an Imhoff graduated cone. On days 10 and 40, the crude protein content of the bioflocs was determined as nitrogen (N, 6.25) with the Kjeldahl method. The components of DOC were analyzed using solid-phase microextraction gas chromatography mass spectrometry.
2. Materials and methods
2.2.2. Shrimp growth parameters At the end of the experiment (40 d), all the live shrimp in each experimental unit were weighed and counted to determine growth and survival in the different treatments.
2.1. Experimental design This study was performed in 9 plastic tanks (effective volume 0.5 L, as described previously) (Zhang et al., 2016; Luo et al., 2017) filled with brackish water (salinity 15) and 300 ± 20 mg L−1 mature biofloc initially established from the suspended growth reactors. The pH in each tank was maintained at 8.0 using NaHCO3. The water temperature was maintained at approximately 30 ± 1 °C. Three compressed air stones in the center of each BFT tank were used for aeration to keep biofloc suspended and to maintain a DO above 6 mg L−1. No water was renewed during the experimental period, which lasted 40 days. Dechlorinated municipal water was added because of the evaporation and sampling. Measurement of TSS was performed once daily. When the concentrations of total suspended solids (TSS) exceeded 500 mg L−1 in the BFT tanks, some water containing flocs was removed. After being settled for half hour, the supernatant was return to the BFT tank and the bioflocs were collected and stored at −20 °C for subsequent analysis. Post-larvae of L. vannamei (0.02 ± 0.004 g L−1) were obtained from Global Biotechnology Ltd., Shenzhen, China. After being acclimated for 2 d to the experimental conditions, they were stocked in tanks (300 L−1 m−3). Beginning on the first day, the shrimp were fed Plus Post-Larval Diet (Yuequn Marine Biotechnology Ltd., Guangdong, China) with varying crumble sizes according to the size of shrimp. According to the manufacturer, this diet contained 46% protein, 8% fat, 5% fiber and 7.5% ash. Beginning on day 11, we changed to the feed containing 42% crude protein, 4% fat, 12% fiber, 18% ash, Ca 5%, P 1.0%) (Quanxing Nutrition Ltd., Jiangsu, China), and the feeding amount was based on a percentage (5%) of the assumed shrimp biomass. The photoperiod was natural, and temperature was controlled at 30 °C. Three different methods of supplying carbon sources for BFT systems were carried out with three replicates: Glucose only, edible glucose (40% w w−1 carbon, purity 99%) was added daily at a rate of 75% of the feed, the C/N ratio input was approximately 10/1 (Gao et al., 2012), referred to as GLU-group; PHB only, 4 nylon bags filled with 40 g of PHB granules placed at the bottom of each tank, no other supplementation with glucose, referred to as PHB-group; PHB plus glucose, 4
Survival ratio (SR%) = 100 × (final n / initial n); Final mean weight (g) = Final total biomass / final n; Weight gain rate (WGR, %) = 100 × (Final weight - initial weight) / initial weight； Specific growth rate (SGR % d−1) = 100 × (ln final weight-ln initial weight)/days of experiment; Feed conversion ratio (FCR) = (Feed applied -leaching)/ weight gain where n = number of shrimps. 2.3. Statistical analysis Statistical analysis was performed using the SPSS Statics 19.0 software for Windows (IBM Corporation, NY, USA). Differences in water quality parameters, the growth performance of the shrimp and the parameters involving bioflocs were analyzed using a one-way ANOVA. Difference was considered significant at a P < .05. Tukey's test was used to assess differences among the three groups. 3. Results 3.1. Water quality parameters The results of selected water quality parameters are presented in Table 1. No differences were found among the three treatments, in terms of pH, DO, or temperature (°C) during the experimental period. Furthermore, all the parameters measured were within acceptable ranges for the shrimp (L. vannamei) throughout the experiment (Van and Scarpa, 1999). Both the concentrations of TAN and NO2−-N in all groups were kept at low levels during the whole experimental period. The concentrations of TAN and NO2−-N in the PHB and GLU groups fluctuated noticeably, while those in the PHB + GLU group were more stable (Fig. 1). The average TAN concentration was significantly lower in the PHB + GLU 182
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Table 1 Water quality parameters during the 40 days shrimp nursery experiment. Data are presented as mean ± standard error. (range), and different superscript letters in a row indicate significant differences. Item
Treatments
pH Dissolved oxygen /mg L−1 Temperature /°C NO2−-N/mg L−1 Total ammonium nitrogen /mg L−1 NO3−-N/mg L−1 Total nitrogen /mg L−1 Alkalinity/mg CaCO3 L−1 Dissolved organic carbon (DOC)/ mg L−1
GLU
PHB + GLU
PHB
7.90 ± 0.16 a 7.53, 8.14 7.34 ± 0.30 a 6.91, 7.77 30.33 ± 1.50 a 28.80, 32.83 0.20 ± 0.22 a 0, 0.83 0.28 ± 0.26 a 0, 1.21 19.39 ± 13.6 a 0.33, 37.23 24.76 ± 10.97 a 10.57, 42.52 121.15 ± 25.79 a 72.65, 146.43 64.9 ± 5.5 a 12.9, 935.5
7.92 ± 0.15 a 7.56, 8.13 7.28 ± 0.28 a 6.79, 7.67 30.55 ± 1.31 a 29.17, 33.03 0.19 ± 0.12 a 0, 0.66 0.23 ± 0.19b 0.02, 0.96 20.46 ± 12.21 a 0.46, 36.47 25.57 ± 11.98 a 11.46, 43.54 121.72 ± 25.37 a 71.13, 159.67 33.7 ± 2.3 b 9.7, 182.3
7.94 ± 0.14 a 7.58, 8.13 7.37 ± 0.25a 7.02,7.73 30.29 ± 1.40 a 28.87, 32.8 0.16 ± 0.24 b 0, 1.00 0.30 ± 0.33 a 0.06, 1.26 19.59 ± 12.25 a 0.53, 35.65 23.9 ± 11.23 a 10.68, 43.74 122.25 ± 18.47 a 94.21, 150.59 23.9 ± 1.2 c 8.4, 75.0
-1
Total nitrogen concentration (mg L )
GLU PHB+GLU PHB
-1
(a)
Total ammonium nitrogen concentration (mg L )
Note: Values in the same row with different superscripts are significantly different (P < .05).
7LPHd
GLU PHB+GLU PHB
Nitrate nitrogen concentration (mg L-1 )
Nitrite nitrogen concentration (mg L-1 )
7LPHd
(c)
GLU PHB+GLU PHB
(b)
7LPHd
(d)
GLU PHB+GLU PHB
7LPHd
Fig. 1. Total nitrogen (TN) (a), total ammonium nitrogen (TAN) (b), nitrite nitrogen (NO2–N) (c) and nitrate nitrogen (NO3–N) in the shrimp culture tanks throughout this study. Data points represent treatment means. Error bars are one standard error around the mean. Different superscript letters indicate significant differences. 183
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Dissolved organic carbon concentration (mg L )
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Consumption of carbohydrates
Treatments GLU
Glucose PHB DOC from glucose DOC from PHB
-1
Table 2 The total addition of carbohydrates (g) and organic carbon (g) in three groups.
PHB + GLU a
141.67 ± 2.00 – 56.67 ± 0.80 –
36.97 48.05 14.79 26.91
± ± ± ±
PHB b
0.19 3.18a 0.08 1.78
– 58.13 ± 4.18b – 32.55 ± 2.34
Note: Values in the same row with different superscripts are significantly different (P < .05). DOC: dissolved organic carbon. PHB: Poly-β-hydroxybutyric.
group than in the GLU group, although differences in mean values were subtle (Table 1). The average NO2−-N concentration was significantly lower in the PHB group; no significant difference in this parameter was observed between the GLU group and PHB + GLU group (Table 1). The nitrate nitrogen buildup lasted until the end of the experiment, increasing to 37.23 ± 3.01 mg L−1 in the GLU group, 36.47 ± 2.41 mg L−1 in the PHB + GLU groups, and 35.65 ± 4.73 mg L−1 in the PHB group. There were no significant differences in nitrate nitrogen concentrations among the groups.
(a)
GLU PHB+GLU PHB
DOC/TAN
(b)
3.2. The consumption of PHB and glucose, and the dynamics of the DOC/ TAN ratio The consumption of PHB and glucose during the 40-d trial for the three groups are presented in Table 2. An average mass of 141.67 ± 2.00 g of glucose with 56.67 ± 0.80 g C was added into each tank in the GLU group; to the PHB + GLU group was added 36.97 ± 0.19 g glucose with 14.79 ± 0.08 g C. Although the same initial weight of PHB was put into both the PHB + GLU and PHB groups, the consumption of PHB for each tank was 48.05 ± 3.18 g (26.91 ± 1.78 g C) in the PHB + GLU group, while in the PHB group consumption was 58.13 ± 4.18 g with 32.55 ± 2.34C, suggesting suggested that the addition of glucose decreased the degradation of PHB in this study. The C/N (w/w) input was approximately 10 in the GLU group. The average DOC concentration in the GLU group was 20.57 ± 9.06 mg L−1, significantly higher than those of the PHB + GLU and PHB groups (p < .05, Table 1). There was no significant difference in average DOC concentration between the PHB + GLU and PHB groups (Table 1). The DOC in the PHB + GLU group was more stable than that of GLU group or the PHB group (Fig. 2 a). Except for the DOC on day 36, the DOC in the PHB group was as stable as that of the PHB + GLU group. The changes in the DOC/TAN ratio are shown in Fig. 2 b. During the 40-d trial, most of the DOC/TAN ratios in the three groups were higher than 20 and fluctuated noticeably.
Time/d Fig. 2. Dynamics of the changes in the dissolved organic carbon (DOC) and DOC/TAN ratio in the reactors of the GLU group, PHB + GLU group and PHB group throughout the 40-d trial. Data points represent the treatment means. Error bars are one standard error around the mean. TAN: total ammonium nitrogen. Different superscript letters indicate significant differences.
3.4. Growth performance of the shrimp Growth performance during the experimental period was not very good for shrimp in any of the groups. At the end of the study, the survival rate of shrimp in the PHB + GLU group was 67 ± 6%, the highest among the three groups, followed by the PHB group. No significant difference in FCR was observed between the GLU group and PHB + GLU groups, both of which had significantly lower values than that of the PHB group (Table 3). The survival rate and the final weight in the PHB + GLU group were higher significantly than those of the other two groups.
3.3. Biofloc FVI-5, crude protein content, and C/N ratio 4. Discussion
The biofloc development in terms of FVI-5 min over 40 days is presented in Fig. 3. A significant increase (p < .05) in FVI-5 was found on day 20, then this parameter decreased on day 30. No continuous decrease was found between day 30 and day 40. The FVI-5 of bioflocs in the PHB + GLU group was significantly higher than that of the PHB and GLU groups. The FVI-5 of bioflocs in the PHB group was the lowest. The protein content in bioflocs in the three groups increased significantly at the end of the experiment. Protein content was similar in the GLU group and PHB + GLU group, both of which were significantly higher than that of the PHB group. No significant differences in C/N of bioflocs were observed among the three groups, ranging from 5.11 to 5.52 (Fig. 3d).
In this study, pH, DO, temperature, alkalinity and the inorganic nitrogen measured were within acceptable ranges for the shrimp (L. vannamei) throughout the 40-d trial (Van Wyk and Scarpa, 1999). TAN and NO2−-N in PHB + GLU remained more stable than in the GLU and PHB groups. The growth performance of shrimp in the PHB + GLU group was also the best among the three groups, suggesting that the carbohydrate supplementation method in the PHB + GLU group, in which PHB was submerged in the tank and in which glucose was added 6 g C to 1 g TAN when TAN level above 2 mg L−1 was observed, was the best method. PHB can be replaced with other BDPs, such as PCL or poly (butylene succinate), and glucose can also be replaced with other 184
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Fig. 3. Then day changes in the flocs volume index in 5 min (FVI-5) (a), crude protein content (b) and C/N (c) of the bioflocs in the reactors of the three groups in the 40-d trial. Error bars are one standard error around the mean. Different superscript letters indicate significant differences. Table 3 Growth performance of Litopenaeus vannamei in different treatment groups. Item
Initial weight (g) Final weight (g) Initial stocking density (shrimp m−3) Survival rate (%) Weight gain rate (%) Specific growth rate (%d−1) Feed conversion ratio (%)
Treatments GLU
PHB + GLU
PHB
0.016 ± 0.004 0.78 ± 0.11 a 300 60 ± 3 a 4756.25 ± 1273.82 9.71 ± 0.65 a 1.63 ± 0.26 a
0.016 ± 0.004 1.01 ± 0.06 b 300 67 ± 6 b 6187.50 ± 656.28 10.35 ± 0.29 b 1.63 ± 0.62 a
0.016 ± 0.004 0.87 ± 0.10 c 300 62 ± 5 c 5325.00 ± 924.91c 9.98 ± 0.39 c 1.52 ± 0.23 b
a
b
Note: Values in the same row with different superscripts are significantly different (P < .05).
4.1. PHB as additional organic carbon source for BFT
soluble organic materials, such as molasses or sucrose. More research needs to be conducted about this. However, the growth performance and survival rate of the shrimp in the three groups were not consistently ideal. The postlarvae from the same batch as this study were found to be infected with White Spot Syndrome Virus in other places (Not presented here), which is perhaps one the reasons that the survival rate was low in this study.
Manipulating the C/N ratio is the most critical for BFT in aquaculture (Avnimelech, 1999). To date, the real-time monitoring of the C/ N ratio in the water in BFT aquacultural systems could not be performed. The amount of carbon added should be varied with the nitrogen in the feed or TAN in the water, and both of two parameters constantly change as production progresses, especially the TAN level. All these considerations complicate the decision for supplying the optimal amount of soluble external carbohydrate as a carbon source. 185
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TAN ratio to describe the C/N station in the water. Assimilation of inorganic nitrogen is considered to take place when the C/N ratio is higher than 10 (Lancelot and Billen, 1985). Alteration of the C/N ratio may result in a shift from an autotrophic to a heterotrophic system (Avnimelech, 1999; Browdy and Bratvold, 2001). Maintaining a C/N ratio higher than 15–20 in a biofloc aquacultural system is thought to develop sufficient bacteria to assimilate ammonia into biomass even under intensive farming with unchanged water. However, several reports have found that when nitrate accumulated as high as hundreds mg L−1 even the C/N ratio was higher than 15 or 20 (Azim and Little, 2008; Xu et al., 2016；Zhang et al., 2016; Pérez–Fuentes et al., 2016; Luo et al., 2017; Dauda et al., 2018). In this study, the nitrate nitrogen in the PHB + GLU and PHB groups were almost twice the nitrogen in bioflocs (Fig. 4), suggesting that most unused nitrogen was converted into nitrate, not bacterial biomass; even the DOC/TAN ratio was higher than 20 (Fig. 2). The mechanism of nitrate nitrogen accumulation in this study is unclear. This aspect merits further investigation. The organic carbon in water supplies is mainly composed of humic and fulvic acids, carbohydrates, proteins, and carboxylic acids (Escobar et al., 2000). Only a fraction of DOC is assimilated and/or mineralized by a heterotrophic flora (Huck, 1990). At the end of the experiment, the constituents of DOC were further analyzed to determine if the DOC can be used (Table 4). Seven compounds were found in the GLU group. In the PHB and PHB + GLU groups, only methyl methacrylate and semi-carbazide were obtained. Methyl methacrylate was the main compound in the three groups. However, to our knowledge information related to these compounds and heterotrophic bacteria cannot be found. So, an explanation cannot be given here. The C/N ratio of bacterial cells is 5:1 (Rittmann and McCarty, 2001). The C/N ration of bioflocs in this study was equal to this value. No significant difference in C/N ratios of bioflocs were observed among the three groups or between day 10 and day 40. Few investigations about BFT include data on the C/N ratio of bioflocs. Perhaps the C/N ratio of bioflocs can be used as a single parameter to describe the characteristics of bioflocs. The paper proposed a method to control TAN and NO2−-N in biofloc culture Litopenaeus vannamei systems. In BFT aquaculture system, carbohydrates, such as Molasses, glucose, and starch) are always to be added to the water one more time in a day in order to stimulate dissolved inorganic nitrogen assimilation. Instead of the current daily additions, using biological degradable polymers (poly-β-hydroxybutyric (PHB) as carbon sources to release soluble carbon source slowly to control the system is more conveniently. This is an interesting and new approach in BFT-white prawn aquaculture. However, this paper only deals with the primary process. A lot of research needs to be carried out in the future, including the optimal ratio of carbohydrates added, stocking biomass and flocs amounts, and the cost of this treatment. Though there are still some questions needed to be handled prior to in practice, this topic is interesting.
Fig. 4. Nitrogen content in biofloc and nitrate on day 10 and day 40 in the reactors of the three groups. Error bars are one standard error around the mean. Different superscript letters indicate significant differences.
Using BDPs to maintain the C/N ratio in BFT offers no such degree of freedom. This study proved the potential for the use of PHB as a sole external carbohydrate to support a shrimp Litopenaeus vannamei nursery system. The best strategy is to use PHB as regular carbohydrate and add glucose 6 g C to 1 g TAN when TAN level above 2 mg L−1 is observed. PHB is produced or accumulated by a wide range of bacteria, and it has shown positive effects on aquatic animals in an aquacultural system (Defoirdt et al., 2007; Schryver et al., 2008; García et al., 2014). Unlike glucose, PHB must be degraded to DOC first. Biotic and abiotic factors act synergistically to decompose organic matter. Biodegradation is considered to be the predominant process (Lucas et al., 2008). Although the biodegradation of PHB has become better understood, no research reports on biodegradation under conditions such as those used in this study were found. The amount of the PHB used in this study was merely based on a rough estimate and may be not the best amount; therefore, the amount of PHB used needs to be optimized.
4.2. C/N and nitrate nitrogen accumulation In theory, “C/N” refers to the carbon/nitrogen ratio of atoms, which should be used directly by the bacteria (Avnimelech, 1999). However, in practice, both the “C” and “N” exist as many forms and the amount of the added carbon was varied with the nitrogen in the feed or TAN in the water. Both parameters constantly changed, especially the TAN level. Therefore, the supplying water soluble carbohydrates to maintain a favorable C:N ratio for heterotrophic bacteria is complicated. Using BDPs to maintain the C:N ratio in BFT offers no such degree of freedom. In addition, even if the value of C/N input into the water is very appropriate for the bacteria, it does not insure that the C/N in the water is also appropriate for the bacteria. Therefore, this study used the DOC/
Table 4 Compounds in the water in the three groups at the end of the experiment obtained from the GC/MS with the extract of DOC. Name of the compound
Methyl methacrylate Semicarbazide Allethrin 3-Methyl-6(1-methylethylidene) cyclohexene Curcumin Artemisinic acid
Molecular formula
Peak Area % of DOC in different group
C4H8O2 CH5N3O C19H26O3 C10H16 C21H20 C15H22O2
186
GLU
PHB + GLUU
PHB
86.0–87.0 1.0–2.0 1.0–2.0 1.0–2.0 1.5–2.5 7.0–8.0
88.5–89.5 10.5–11.5 – – – –
94.0–95.0 5.0–6.0 – – – –
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5. Conclusion
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Three different organic carbon sources for water quality control in a bioflocs shrimp Litopenaeus vannamei nursery system were investigated: (1) glucose added daily at a percentage of 75% of the feed, (2) PHB plus 6 g C glucose to 1 g TAN when a TAN level above 2 mg L−1 was observed and (3) only PHB. The salinity was 15. The water was not changed for 40 days. The DOC/TAN ratio in the water of all three groups was maintained up to 15. All the water parameters measured were within the acceptable ranges for the shrimp (L. vannamei) throughout the experiment. Nitrate nitrogen noticeably accumulated in the three groups, and no significant differences were observed. The survival rate and final weight in the PHB + GLU group were significantly higher than those of the other groups. The food conversion ratio of the PHB group was significantly lower than those of the other groups. Taken together, these results indicate that PHB is a favorable and convenient additional carbohydrate for a shrimp L. vannamei bioflocs nursery in brackish water. PHB + GLU was the best organic carbon supplementation method. However, this paper only deals with the primary process. A lot of research needs to be carried out in the future. Acknowledgements This study was funded by the Shanghai Science and Technology Commission Project (14320501900). References APHA, 2005. Standard Methods for the Examination of Water and Wastewater. American Public Health Association, Washington D. C. Asaduzzaman, M., Wahab, M.A., Verdegem, M.C.J., Huque, S., Salam, M.A., Azim, M., 2008. C/N ratio control and substrate addition for periphyton development jointly enhance freshwater prawn Macrobrachium rosenbergii production in ponds. Aquaculture 280, 117–123. Avnimelech, Y., 1999. Carbon/nitrogen ratio as a control element in aquaculture systems. Aquaculture 176, 227–235. Avnimelech, Y., 2007. Feeding with microbial flocs by tilapia in minimal discharge bioflocs technology ponds. Aquaculture 264, 140–147. Azim, M., Little, D.C., 2008. The biofloc technology (BFT) in indoor tanks: water quality, biofloc composition, and growth and welfare of Nile tilapia (Oreochromis niloticus). Aquaculture 283, 29–35. Boley, A., Müller, W.R., Haider, G., 2000. Biodegradable polymers as solid substrate and biofilm carrier for denitrification in recirculated aquaculture systems. Aquacult. Eng. 22, 75–85. Browdy, D., Bratvold, C.L., 2001. Effects of sand sediment and vertical surfaces (AquaMatsTM) on production, water quality, and microbial ecology in an intensive Litopenaeus vannamei culture system. Aquaculture 1–2, 81–94. Chu, L., Wang, J., 2011. Nitrogen removal using biodegradable polymers as carbon source and biofilm carriers in a moving bed biofilm reactor. Chem. Eng. J. 170, 220–225. Dauda, A.B., Romano, N., Ebrahimi, M., Ajadi, A., Chong, C.M., Karim, M., Natrah, I., Kamarudin, M.S., 2018. Influence of carbon/nitrogen ratios on biofloc production
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