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The effect of different carbon sources on the nutritional composition, microbial community and structure of bioflocs
Aquaculture 465 (2016) 88–93
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The effect of different carbon sources on the nutritional composition, microbial community and structure of bioflocs YanFang Wei a,b, Shao-An Liao a, An-li Wang a,⁎ a b
Key Laboratory of Aquaculture Health and Safety, College of Life Science, South China Normal University, Guangzhou 510631, PR China Department of Environmental Science and Engineering, College of Chemical Engineering, Huaqiao University, Xiamen 361021, PR China
a r t i c l e
i n f o
Article history: Received 8 June 2016 Received in revised form 24 August 2016 Accepted 29 August 2016 Available online 31 August 2016 Keywords: Bioflocs Carbon sources Amino acid Microbial community
a b s t r a c t The objective of this study was to document how different carbon sources affect the quality of biofloc (BF). The experiment consisted of three types of biofloc systems in which biofloc was produced by daily (25 days in all) supplementation with three different carbon sources, glucose (Glu), starch (Sta) and glycerol (Gly), in each 45-L tank; the C/N ratio was 15. The highest protein content was obtained in the BF (Glu), with a value of 41.2 ± 0.8% dry weight (DW). The BF (Sta) and BF (Gly) had lower values of 31.5 ± 0.6% and 35.5 ± 1.2% dry weight (DW). A higher lipid content was observed in the BF (Sta). The essential amino acid and nonessential amino acid contents were similar in BF (Glu) and BF (Gly), but both were higher than those of BF (Sta). The Essential Amino Acid Index (EAAI) for BF (Glu), BF (Sta) and BF (Gly) was 0.99, 0.93 and 0.98, respectively; which indicated that the biofloc produced in this experiment can be considered a good-quality protein source for shrimp. High-throughput sequencing of different bioflocs revealed that three types of bioflocs were dominant in Proteobacteria and Bacteroidetes. In addition, Cyanobacteria were the dominant biofloc in BF (Sta). Microscopic examination revealed that BF (Sta) was more closely related to the formation of the floc and contained more algae. Overall, this study demonstrated that bioflocs grown on different carbon sources have different qualities and suggested that the choice of carbon source used for growing bioflocs is of prime importance. Statement of relevance: The present study indicated that different carbon sources could affect nutritional composition of biofloc, morphostructure of biofloc and the microbial community of biofloc. This result may provide a theoretical basis for the biofloc technology used in aquaculture. © 2016 Elsevier B.V. All rights reserved.
1. Introduction In recent years, biofloc technology (BFT) was introduced into aquaculture. It is an emerging environmentally friendly aquaculture production system. This technology was developed to create economic and environmental benefits via reduced water use, effluent discharges, artificial feed supply and improved biosecurity (Wasielesky et al., 2006; Avnimelech, 2007; Mishra et al., 2008). Biofloc (BF) is the core of BFT. It can provide nutrients such as “native protein” (Emerenciano et al., 2011), lipids (Wasielesky et al., 2006), amino acids (Ju et al., 2008) and fatty acids (Izquierdo et al., 2006; Ekasari et al., 2010) in the form of diverse microorganisms. Another advantage of BF has is that it is available as a food source all day and can reduce artificial feed inputs and costs (Browdy et al., 2001; Avnimelech, 2007; Samocha et al., 2007). Some studies have indicated that the different types of carbon sources can affect the composition of the biofloc. The structure and stability of a biofloc are determined by the selection of an organic carbon source to some extent (Hollender et al., 2002; Oehmen et al., 2004). ⁎ Corresponding author. E-mail address: [email protected] (A. Wang).
http://dx.doi.org/10.1016/j.aquaculture.2016.08.040 0044-8486/© 2016 Elsevier B.V. All rights reserved.
The cost of an organic carbon source also determines the use of the biological flocculation (Wilén et al., 2000). Crab et al. (2010) conducted a small–scale laboratory experiment and found that different carbon sources led to differences in the protein, lipid, carbohydrate, and fatty acid compositions of the bioflocs. Literature describing the difference in quality of BFs as a result of different carbon sources remains scarce, especially concerning the relationship between amino acid content and carbon source. A BF is mainly composed of different microorganisms, which play a key role as producers and consumers of dissolved oxygen, as nutrient recyclers, and as a food source for other organisms from higher trophic levels in aquaculture (McIntosh et al., 2000; Ray et al., 2010,; MartínezCórdova et al., 2016). Therefore, analyzing the microbial community of biofloc can help to understand the nutritional differences of different bioflocs. Different carbon sources can affect the composition of the microbial community. Knowledge of the community structure of a biofloc and its nutritional value, especially its amino acid (AA) composition, will help in the development of cost-effective shrimp feed formulations (Ju et al., 2008). Biofloc can be used as an additional feed source in aquaculture. Yet, its effect is dependent on the carbon source used (Crab et al., 2010). The
Y. Wei et al. / Aquaculture 465 (2016) 88–93
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goal of the present study was to assess the nutritional value of bioflocs grown on different organic carbon sources.
1981). The essential amino acid index (EAAI) was calculated according to Peñaflorida (1989) using the formula:
2. Materials and methods
EAAI ¼
2.1. Experimental design and conditions
with aa being the essential amino acid ratio (E/A) in the biofloc, AA being the E/A ratio in the animal body, and 1, 2, 3, …, 9 being each of the essential amino acids.
The experiment was conducted at the laboratory of life science (South China Normal University, China). The biofloc was produced in outdoor plastic tanks, containing 42 L of dechlorinated tap water and 3 L of fresh pond water to inoculate the tank with natural microorganisms such as bacteria and algae. No water exchange was performed, but evaporation losses were compensated with dechlorinated fresh water. The salinity was maintained at approximately 5 ppt. Three carbon sources were investigated: glucose, starch and glycerol. Each carbon source has three replicates and was assigned randomly. Feed pellets containing 42% protein (Haid Feed Co., Ltd. Guangdong, China) were used. The carbon source and feed were added once a day at an amount corresponding to a C/N ratio of 15. The daily quantity of carbon added was calculated according to Avnimelech (1999). The preweighed carbon source and feed were mixed in a beaker with tank water and then uniformly dispersed into the tanks. Each tank was inoculated with yeast only once at the beginning of the experiment. After inoculation, the final concentration of yeast in each tank was 1.0 × 105 cells·mL−1. The tank water was aerated and agitated continuously using air stones connected to an air pump. The diameter of the aeration tube was 6 mm, and the diameter of the air stone was 40 mm. The experiment was carried out for a period of 25 days. 2.2. Assessment of water quality parameters During the 25-day experimental period, the water temperature, dissolved oxygen (DO) and pH were measured on site every day using a YSI556 meter (YSI Incorporated 1725, Yellow Springs, OH, USA). Water samples (100 mL) were collected weekly at approximately 08:00 h and filtered through 0.45-μm GF/C filter paper under vacuum pressure. Ammonia nitrogen (NH4–N), nitrite (NO2–N) and nitrate (NO3–N) concentrations in the filtrate were analyzed according to standard methods (APHA, 1998).
p ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 9 aa1=AA1 aa2=AA2 ⋯ aa9=AA9
2.4. Bacterial metagenome sequencing and bioinformatics analysis When the experiment was finished, water samples were taken from 50 mL of each biofloc tank, and biomass was collected by centrifugation (10 min, 4000g). Total DNA was extracted from the samples using a water DNA extraction kit (E.Z.N.A.R, Omega) according to the manufacturer's protocol. MisSeq metagenome sequencing and bioinformatics was conducted by the Shenzhen HENGCHUANG Gene Technology Co Ltd. using a sequencing machine (Illumina HiSeq2500 sequencing, United States Illumina company completed). Community structure analysis examines the relative distribution of species at the phylum level. 2.5. Biofloc volume and morphostructure Floc volume was determined by sampling 1000 mL of water into a series of Imhoff cones (1000–0010, Nalgene) at 10:00 am every 3 days. The volume of the floc plug accumulating on the bottom of the cone was determined 15 min after sampling. Then, the floc plug was collected from the turn-knob at the bottom tip of the cone, and the floc morphostructure was observed with a biologic microscope (BX51, Olympus) and a fluorescence microscope (Leica, DMI3000B). 2.6. Statistical analysis Data obtained from the experiment were analyzed using SPSS 17.0 software (SPSS, Chicago, USA) for Windows. One-way ANOVA was performed on the experimental parameters. Differences were considered significant at P b 0.05. When significant differences were found, Duncan's multiple range test was used to identify differences between the experimental groups.
2.3. Proximate analysis of different bioflocs 3. Results Proximate analysis was carried out on the different types of bioflocs. Concentrated biofloc samples were collected from each tank by passing tank water through a 10-μm mesh nylon bag (Xu and Pan, 2012) after 25 days. The samples were dried in an oven at 105 °C until they reached a constant weight and then preserved in a refrigerator (− 20 °C) until proximate composition analysis. The crude protein content was calculated based on the assumption that protein contains 16% nitrogen (AOAC, 1999). For ash content, a known amount of dry sample was burnt in a muffle furnace at 550 °C for 4 h before the ash was cooled and weighed. The lipid content was determined with Soxhlet apparatus. Protein, lipid and ash contents were expressed as a percentage of the dry weight (% DW) of the bioflocs. The total carbohydrate amount was calculated according to the following formula: carbohydrate (% DW) = 100 − (crude protein (% DW) + lipid (% DW) + ash (% DW)) (Manush et al., 2005). The gross energy content of the diets was calculated using kJ·g−1 DW values of 23.0, 38.1 and 17.2 for protein, lipids and carbohydrates, respectively (Tacon, 1990). The amino acid composition of the biofloc was measured by a professional laboratory using high-performance liquid chromatography (HPLC). A selection of amino acids that are considered essential for aquatic animals in general was chosen as described by Tacon (1987) and Babarro et al. (2011). The essential amino acid ratio (E/A) for each essential amino acid (EAA) was expressed as a percentage of each amino acid of the total amount of essential amino acids measured (Arai,
3.1. Water quality The physical and chemical water quality parameters monitored throughout the experiment are presented in Table 1. There was no significant difference in temperature and pH observed between different BFs (P N 0.05). However, DO, NH4-N、NO2-N and NO3-N had significant differences (P b 0.05). The relationship between the resulting DO concentrations from the three treatments was BF (Sta) N BF (Glu) N BF (Gly). The BF (Gly) treatment had the lowest concentrations of NH4-N (0.12 ± 0.03 mg/L), NO2-N (0.02 ± 0.01 mg/L) and NO3-N (0.10 ± 0.02 mg/L). The concentrations found in the BF (Glu) treatment were Table. 1 The water quality parameters in three kinds of bioflocs treatment during the experiment. Parameter
BF (Glu)
BF (Sta)
BF (Gly)
Temperature (°C) DO (mg/L) pH NH4—N (mg/L) NO2—N (mg/L) NO3—N (mg/L)
23.3 ± 1.9a 7.5 ± 0.2ab 7.6 ± 0.2a 0.13 ± 0.01a 0.04 ± 0.02a 0.14 ± 0.01a
23.5 ± 1.6a 7.9 ± 0.1b 8.0 ± 0.3a 0.20 ± 0.03b 0.13 ± 0.02b 0.69 ± 0.01b
23.8 ± 1.3a 7.1 ± 0.3a 7.7 ± 0.2a 0.12 ± 0.03a 0.02 ± 0.01a 0.10 ± 0.02a
Each value represents mean ± S.D. Values in the same row with different superscript letters are significantly different at P b 0.05.
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similar to those of the BF (Gly) treatment. In the BF (Sta) treatment, the concentrations of NH4-N, NO2-N and NO3-N were 1.67 times, 6.5 times and 6.9 times those of the BF (Gly) treatment, respectively.
3.3. Biofloc investigation After 10 days of culture, the water in the different groups had different colors. When using glucose as a carbon source, the water was pale yellow; when using starch as a carbon source, the water was light green; when using glycerol as carbon source, the water was brown. The average volume of floc that settled in the Imhoff cones from water samples from the three replicated tanks as a function of culture time is shown in Fig. 3. Biofloc development in the water column was similar and initially slow in all three treatments. The biofloc grew rapidly after 10 days of culture. Twenty days later, the biomass was essentially unchanged. Biofloc grown with glucose had significant differences compared to bioflocs grown in glycerol and starch (P b 0.05). The morphology and structure of biological flocculations were observed with a biologic microscope with a 10 × objective (Fig. 4a) and with a fluorescence microscope with a 20× objective (Fig. 4b). The biological flocculation had irregular agglomerates. Bioflocs grown in different carbon sources had the same structure and composition; all had single-cell algae, protozoa, rotifers and bacterial communities, etc. Table. 2 Proximate analysis of three kinds of bioflocs treatment (mean ± S.D., n = 3). Composition
BF (Glu)
BF (Sta)
BF (Gly)
Crude protein (% DW) Crude lipid (% DW) Carbohydrate (% DW)⁎
41.2 ± 0.8a 6.1 ± 0.1a 37.7 ± 0.2a
31.5 ± 0.6b 8.5 ± 0.2b 47.6 ± 0.3b
35.5 ± 1.2c 4.2 ± 0.1c 45.1 ± 0.2c
Ash (% DW) Gross energy (kJ g−1 DW)⁎⁎
15.0 ± 0.1a 18.2 ± 0.3ab
12.4 ± 0.2b 18.7 ± 0.2b
15.2 ± 0.3a 17.5 ± 0.3a
Values on the same line followed by different superscript letters are significantly different (P b 0.05). DW, dry weight. ⁎ Values were calculated based on crude protein, crude lipid and ash content. ⁎⁎ Values were calculated based crude protein, crude lipid, ash and carbohydrate content.
EAA Concentration (µmol/g)
Proximate analysis of the different types of bioflocs is shown in Table 2. There was a significant difference (P b 0.05). The protein content in the BF (Sta) and BF (Gly) treatments was 31.5 ± 0.6% and 35.5 ± 1.2% of DW, respectively. Biofloc grown with glucose had a protein content that was significantly higher (41.2 ± 0.8%) than those of the other treatments. The lipid content ranged from 4.2% of DW for the BF (Gly) treatment to 8.5% of DW for the BF (Sta) treatment. The ash content was similar in both BF (Glu) and BF (Gly) treatments: 15.0 ± 0.1% and 15.2 ± 0.3% of DW, respectively. Biofloc grown on starch had an ash content that was significantly lower (12.4 ± 0.2%) than those of the other treatments. The carbohydrate contents varied between treatments. The highest calculated carbohydrate levels (47.6% on DW) were found in the BF (Sta) treatment. The lowest values were found (37.7% on DW) in the BF (Glu) treatment. The gross energy in BF (Glu), BF (Sta) and BF (Gly) was 18.2 ± 0.3 kJ g−1, 18.7 ± 0.2 kJ g−1, 17.5 ± 0.3 kJ g−1, respectively. The essential amino acid concentrations of the three types of bioflocs are shown in Fig. 1. In general, the three bioflocs seem to be rich in threonine, arginine, tyrosine, phenylalanine, isoleucine, leucine and lysine but are deficient in methionine and histidine. Furthermore, there was no cysteine in any of the bioflocs. The concentrations of nonessential amino acids in the three types of bioflocs are shown in Fig. 2. Generally, they are all rich in asparagine, glutamate, serine, glycine, alanine and proline, but deficient in tyrosine. In general, the amino acid content in BF (Sta) was less than those in BF (Glu) and BF (Gly).
100
80
60
40
20
0 BF(Glu)
BF(Sta)
BF(Gly)
Biofloc Type Fig. 1. Essential amino acid pattern (μmol/g biofloc dry weight) of different biofloc. Values are means ± standard deviation of three replications.
They varied in density and size. BF (Sta) had a higher density and smaller size; thus, it had a low floc volume. Fig. 4b shows that bioflocs grown on different carbon sources have different amounts of algae. BF (Sta) had the most, BF (Glu) had an intermediate amount, and BF (Gly) had the least. In BF (Sta), the algae cluster together, while the algae in BF (Glu) and BF (Gly) are more dispersed. 3.4. Microbial community The microbial communities at the phylum level in the three types of biofloc are shown in Fig. 5. Both bioflocs shared a large portion of Proteobacteria and Bacteroidetes. The BF (Sta) was clearly different from the BF (Glu) and BF (Gly); it also has a domain of Cyanobacteria. BF (Glu) and BF (Gly) have a more diverse microbial community than BF (Sta). 4. Discussion This study demonstrated that different carbon sources significantly affect the quality of bioflocs. The levels in BF (Sta) were significant higher than those in the other two groups. DO and pH values were lower in BF (Glu) and BF (Gly), which was likely a result of higher respiration rates due to the presence of a heterotrophic microbial community that increased the carbon dioxide concentration in a limited water exchange system (Tacon et al., 2002; Wasielesky et al., 2006). It has 120
Asparagic Glutamate Serine Glycine Tyrosine Alanine Proline
100
NEAA Concentration (µmol/g)
3.2. Proximate analysis of different bioflocs
Histidine Threonine Arginine Valine Methionine Phenilalnine Isoleucine Leucine Lysine
120
80
60
40
20
0 BF(Glu)
BF(Sta)
BF(Gly)
Biofloc Type Fig. 2. Nonessential amino acid pattern (μmol/g biofloc dry weight) of different biofloc. Values are means ± standard deviation of three replications.
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30 BF (Glu) BF (Sta) BF (Gly)
Floc volume (ml /L)
25
20
15
10
5
Fig. 5. Bacterial composition at phylum level of three biofloc types Treatments: Glu presence of biofloc grown in glucose; Sta presence of biofloc grown in starch; Gly presence of biofloc grown in glycerol. Each treatment has three parallel.
0 1
4
7
10
13
16
19
22
25
Culture day (d) Fig. 3. Dynamic changes of floc volume in different treatment throughout the experimental period. Values are means ± standard deviation of three replications in each sampling date.
been shown that there are different effects of simple versus complex carbohydrates used as the carbon source in biofloc-based systems (Avnimelech, 2012). Simple sugars result in faster ammonia removal, while more complex carbohydrates require more time for decomposition into simple sugars, thereby resulting in slower ammonia removal. This may explain the higher NH4-N level in BF (Sta) compared to BF (Glu) and BF (Gly). During the experiment, more algae were observed in BF (Sta) than in BF (Glu) and BF (Gly). Each type of biofloc grown on a different carbon source (glucose, starch and glycerol) was adequate in maintaining the water quality parameters in a normal range for shrimp growth (Van Wyk and Scarpa, 1999). Toxic ammonia and nitrite levels can lead to low survival rates or decreased growth. Therefore, BFT may make it possible to increase growth yields and survival levels at low
water replacement rates, while adding a potential additional natural food resource in the form of bioflocs (Asaduzzaman et al., 2008). Feed costs represent at least 50% of total aquaculture production costs, predominantly due to the cost of the protein component in commercial diets (Bender et al., 2004). In this experiment, except for the composition of crude protein and ash, other elements of the composition (crude lipid, carbohydrate and gross energy) are higher than the level reported by Crab et al. (2010). This could be due to differences in the production of biofloc, such as carbon source, C/N ratio and inoculum. The protein content in the diet is an important factor for the nutrition of most aquatic organisms. Most aquaculture species require protein in a range of 20–50% in their diet (Tacon, 1987). These three bioflocs have good prime nutritional values when compared to a reference commercial prawn feed analyzed by Hossain and Paul (2007). The protein content in the BF (Glu) was the highest, followed by those of BF (Gly) and BF (Sta). This could be due to the different concentration of extracellular polymeric substances (EPS), which accounts for 80–95% of the organic matter in the flocs (Wilén et al., 2008). The lipid content was low in
a
BF (Glu)
BF (Sta)
BF (Gly)
BF (Glu)
BF (Sta)
BF (Gly)
b
Fig. 4. a. Morphology of different biofloc types under light microscope. b. Morphology of different biofloc types under fluorescence microscope.
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BF (Glu) and BF (Gly), but not in BF (Sta). This is because BF (Sta) had more Cyanobacteria (Sharathchandra and Rajashekhar, 2011; Maslova et al., 2004). It has been reported that lipids are a major metabolic energy source (Roustaian et al., 2001), which implies that commercial feed is still necessary to provide adequate lipid levels in BF (Glu) and BF (Gly). Microalgae, also called ‘miniature sunlight-driven biochemical factories’ (Verma et al., 2010), are capable of producing large amounts of lipids and hydrocarbons in the presence of sunlight and carbon dioxide from flue gases. BF (Sta) had a lower ash content and higher carbohydrate content. BF (Sta) also had the highest gross energy. High ash content lowers the digestibility of other ingredients in the diet resulting in poor shrimp growth. BF (Glu) and BF (Gly) had higher ash contents, which can decrease the digestibility of the biofloc. BF (Sta) had a high concentration of carbohydrates, which could be the result of the poor solubility of starch because starch particles may still be present in the flocs. Taken together, these results show that the biofloc quality is significantly influenced by the added carbon source. There may be major differences in the way the microbes that take part in biofloc formation benefit from these different carbon sources (Rittmann and McCarty, 2001). Moreover, the impact of various carbon sources on the outgrowth of bioflocs in activated sludge systems has been documented (Bodík et al., 2009). The amino acid composition and the bioavailability of the amino acids present determine the protein quality of a diet for aquaculture. An amino acid is classified as nutritionally essential (indispensable) or non-essential (dispensable) by the nutritional requirement and the organism's ability to perform amino acid synthesis. EAAs are those that either cannot be synthesized or are inadequately synthesized by animals relative to their needs and consequently must be provided by the diet. Since amino acids are building blocks for proteins, optimal synthesis of proteins is therefore determined by the dietary amino acid profile (Mente et al., 2002). There are reports in the literature that the dietary amino acid requirement of a particular aquatic organism is strongly dependent on its body amino acid composition (Tacon, 1987). Peñaflorida (1989) recommended the calculation of the EAAI to evaluate the EAA profile in the diet relative to the EAA composition of the animal and determined that a good-quality diet had an EAAI of N0.9, a useful diet had a value between 0.7 and 0.9, and the diet was considered inadequate when this value was b0.7. The EEAI values of BF (Glu), BF (Sta) and BF (Gly) were 0.99, 0.93 and 0.98 respectively. This shows that the EAA composition of different types of biofloc could meet the requirement of the aquatic species tested. Based on this index, the biofloc produced in this study can be considered a good-quality protein source for shrimp. The biofloc can be a feed supplement, whether for specific amino acids or for total amino acid content. This also indicates that biofloc grown in glucose and glycerol had a higher EAAI than bioflocs grown in starch, which suggests that BF (Glu) and BF (Gly) were higher in quality than BF (Sta) in terms of EAAs under the experimental conditions. Ebeling et al. (2006) reported that in BFT systems, the concentration of suspended flocs is, after oxygen, the most important limiting factor for productivity. In this experiment, BF (Sta) was more dense and compact. This was because it had a higher DO concentration (see Table 1). An adequate DO level is not only essential for the metabolic activity of cells within aerobic flocs, but it is also thought to influence floc structure (De Schryver et al., 2008). Higher DO concentrations also induce more compact flocs (Wilén and Balmér, 1999). Ballester et al. (2010) showed that the microorganisms present in BFT systems play an important role in the provision of essential nutrients. It is believed that under conditions with abundant incident light, such as a shrimp pond or outdoor aquarium, phytoplankton algae are the first to grow, and these organisms provide a basic source of food for the subsequent development of a zooplankton community and nutrients for the growth of bacteria in the culture system. In Fig. 5, it can be seen that Proteobacteria and Bacteroidetes are the dominant bacteria in aquaculture and occupy a very large of proportion in all types of
biofloc. BF (Sta) was clearly different from the BF (Glu) and BF (Gly), suggesting that the microbial community was shaped by the carbon source. The majority of Proteobacteria detected in both groups were considered to be symbiotic bacteria in aquaculture (Sakami et al., 2008). Proteobacteria removes organic matter (Wagner et al., 1993; Miura et al., 2007), especially in the wastewater treatment of a biofloc. The class of Proteobacteria also plays a major role in the composition of bacteria (Wagner et al., 1995; Daims et al., 1999). This demonstrates that when a biofloc is applied to a culture system, it can effectively regulate the quality of the aquaculture water. Actinobacteria is a common probiotic, which can produce beneficial substances in specific environments (Das et al., 2008). Gerardi (2006) reported that Flavobacterium can produce glue-like extracellular polymers and has the ability to bind cells together. 5. Conclusion Biofloc rearing media provide a potential food source for shrimp reared in limited or zero water exchange systems. This culture system is environmentally friendly because it is based on limited water use and minimal effluent is released into the surrounding environment. The major aim of this study was to compare the water quality, nutritive value, morphology and microbial community of different types of biofloc. The overall results show that BF (Sta) has higher crude lipids, carbohydrates and gross energy and lower crude protein and ash compared to BF (Glu) and BF (Gly). In addition, BF (Sta) has more prokaryotic algae and is more closely aggregated. In terms of EAAI, all three bioflocs could meet the requirement of shrimp. They can be used to replace or partly replace feed oil in culture to reduce costs and pollution. Further studies are recommended to optimize the biofloc microbial biomass ratios in order to set benchmarks for shrimp culture systems. Moreover, the inhibition of pathogens should also be studied. Acknowledgments This work was supported by the major projects of science and technology and development of marine fishery in Guangdong Province, China (grant number A201401B01, A201001H02); the Project of Natural Science Foundation of Fujian Province (grant number 2014J01049); and The Guangzhou Science and Technology Program key projects (grant number 2014J4100052). We would like to thank the anonymous reviewers for their remarks and suggestions. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.aquaculture.2016.08.040. References APHA, 1998. Standard Methods for the Examination of the Water and Wastewater. 22nd ed. American Public Health Association, Washington, DC. Arai, S., 1981. A purified test diet for coho salmon, Oncorhynchus kisutch, fry. Nippon Suisan Gakkaishi 47, 547–550. Asaduzzaman, M., Wahab, M.A., Verdegem, M.C.J., Huque, S., Salam, M.A., Azim, M.E., 2008. C/N ratio control and substrate addition for periphyton development jointly enhance freshwater prawn Macrobrachium rosenbergii production in ponds. Aquaculture 280 (1), 117–123. Association of Official Analytical Chemists, 1999. Official Methods of Analysis. AOAC, Washington, DC. Avnimelech, Y., 1999. C/N 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 (1), 140–147. http://dx.doi.org/10.1016/j. aquaculture.2006.11.025. Avnimelech, Y., 2012. Biofloc Technology-a Practical Guide Book. 2nd ed. The World Aquaculture Society, Baton Rouge, United States. Babarro, J.M.F., Fernández-Reiriz, M.J., Labarta, U., Garrido, J.L., 2011. Variability of the total free amino acid (TFAA) pool in Mytilus galloprovincialis cultured on a raft system. Effect of body size. Aquac. Nutr. 17 (2), 448–458.
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The effect of different carbon sources on the nutritional composition, microbial community and structure of bioflocs YanFang Wei a,b, Shao-An Liao a, An-li Wang a,⁎ a b
Key Laboratory of Aquaculture Health and Safety, College of Life Science, South China Normal University, Guangzhou 510631, PR China Department of Environmental Science and Engineering, College of Chemical Engineering, Huaqiao University, Xiamen 361021, PR China
a r t i c l e
i n f o
Article history: Received 8 June 2016 Received in revised form 24 August 2016 Accepted 29 August 2016 Available online 31 August 2016 Keywords: Bioflocs Carbon sources Amino acid Microbial community
a b s t r a c t The objective of this study was to document how different carbon sources affect the quality of biofloc (BF). The experiment consisted of three types of biofloc systems in which biofloc was produced by daily (25 days in all) supplementation with three different carbon sources, glucose (Glu), starch (Sta) and glycerol (Gly), in each 45-L tank; the C/N ratio was 15. The highest protein content was obtained in the BF (Glu), with a value of 41.2 ± 0.8% dry weight (DW). The BF (Sta) and BF (Gly) had lower values of 31.5 ± 0.6% and 35.5 ± 1.2% dry weight (DW). A higher lipid content was observed in the BF (Sta). The essential amino acid and nonessential amino acid contents were similar in BF (Glu) and BF (Gly), but both were higher than those of BF (Sta). The Essential Amino Acid Index (EAAI) for BF (Glu), BF (Sta) and BF (Gly) was 0.99, 0.93 and 0.98, respectively; which indicated that the biofloc produced in this experiment can be considered a good-quality protein source for shrimp. High-throughput sequencing of different bioflocs revealed that three types of bioflocs were dominant in Proteobacteria and Bacteroidetes. In addition, Cyanobacteria were the dominant biofloc in BF (Sta). Microscopic examination revealed that BF (Sta) was more closely related to the formation of the floc and contained more algae. Overall, this study demonstrated that bioflocs grown on different carbon sources have different qualities and suggested that the choice of carbon source used for growing bioflocs is of prime importance. Statement of relevance: The present study indicated that different carbon sources could affect nutritional composition of biofloc, morphostructure of biofloc and the microbial community of biofloc. This result may provide a theoretical basis for the biofloc technology used in aquaculture. © 2016 Elsevier B.V. All rights reserved.
1. Introduction In recent years, biofloc technology (BFT) was introduced into aquaculture. It is an emerging environmentally friendly aquaculture production system. This technology was developed to create economic and environmental benefits via reduced water use, effluent discharges, artificial feed supply and improved biosecurity (Wasielesky et al., 2006; Avnimelech, 2007; Mishra et al., 2008). Biofloc (BF) is the core of BFT. It can provide nutrients such as “native protein” (Emerenciano et al., 2011), lipids (Wasielesky et al., 2006), amino acids (Ju et al., 2008) and fatty acids (Izquierdo et al., 2006; Ekasari et al., 2010) in the form of diverse microorganisms. Another advantage of BF has is that it is available as a food source all day and can reduce artificial feed inputs and costs (Browdy et al., 2001; Avnimelech, 2007; Samocha et al., 2007). Some studies have indicated that the different types of carbon sources can affect the composition of the biofloc. The structure and stability of a biofloc are determined by the selection of an organic carbon source to some extent (Hollender et al., 2002; Oehmen et al., 2004). ⁎ Corresponding author. E-mail address: [email protected] (A. Wang).
http://dx.doi.org/10.1016/j.aquaculture.2016.08.040 0044-8486/© 2016 Elsevier B.V. All rights reserved.
The cost of an organic carbon source also determines the use of the biological flocculation (Wilén et al., 2000). Crab et al. (2010) conducted a small–scale laboratory experiment and found that different carbon sources led to differences in the protein, lipid, carbohydrate, and fatty acid compositions of the bioflocs. Literature describing the difference in quality of BFs as a result of different carbon sources remains scarce, especially concerning the relationship between amino acid content and carbon source. A BF is mainly composed of different microorganisms, which play a key role as producers and consumers of dissolved oxygen, as nutrient recyclers, and as a food source for other organisms from higher trophic levels in aquaculture (McIntosh et al., 2000; Ray et al., 2010,; MartínezCórdova et al., 2016). Therefore, analyzing the microbial community of biofloc can help to understand the nutritional differences of different bioflocs. Different carbon sources can affect the composition of the microbial community. Knowledge of the community structure of a biofloc and its nutritional value, especially its amino acid (AA) composition, will help in the development of cost-effective shrimp feed formulations (Ju et al., 2008). Biofloc can be used as an additional feed source in aquaculture. Yet, its effect is dependent on the carbon source used (Crab et al., 2010). The
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goal of the present study was to assess the nutritional value of bioflocs grown on different organic carbon sources.
1981). The essential amino acid index (EAAI) was calculated according to Peñaflorida (1989) using the formula:
2. Materials and methods
EAAI ¼
2.1. Experimental design and conditions
with aa being the essential amino acid ratio (E/A) in the biofloc, AA being the E/A ratio in the animal body, and 1, 2, 3, …, 9 being each of the essential amino acids.
The experiment was conducted at the laboratory of life science (South China Normal University, China). The biofloc was produced in outdoor plastic tanks, containing 42 L of dechlorinated tap water and 3 L of fresh pond water to inoculate the tank with natural microorganisms such as bacteria and algae. No water exchange was performed, but evaporation losses were compensated with dechlorinated fresh water. The salinity was maintained at approximately 5 ppt. Three carbon sources were investigated: glucose, starch and glycerol. Each carbon source has three replicates and was assigned randomly. Feed pellets containing 42% protein (Haid Feed Co., Ltd. Guangdong, China) were used. The carbon source and feed were added once a day at an amount corresponding to a C/N ratio of 15. The daily quantity of carbon added was calculated according to Avnimelech (1999). The preweighed carbon source and feed were mixed in a beaker with tank water and then uniformly dispersed into the tanks. Each tank was inoculated with yeast only once at the beginning of the experiment. After inoculation, the final concentration of yeast in each tank was 1.0 × 105 cells·mL−1. The tank water was aerated and agitated continuously using air stones connected to an air pump. The diameter of the aeration tube was 6 mm, and the diameter of the air stone was 40 mm. The experiment was carried out for a period of 25 days. 2.2. Assessment of water quality parameters During the 25-day experimental period, the water temperature, dissolved oxygen (DO) and pH were measured on site every day using a YSI556 meter (YSI Incorporated 1725, Yellow Springs, OH, USA). Water samples (100 mL) were collected weekly at approximately 08:00 h and filtered through 0.45-μm GF/C filter paper under vacuum pressure. Ammonia nitrogen (NH4–N), nitrite (NO2–N) and nitrate (NO3–N) concentrations in the filtrate were analyzed according to standard methods (APHA, 1998).
p ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 9 aa1=AA1 aa2=AA2 ⋯ aa9=AA9
2.4. Bacterial metagenome sequencing and bioinformatics analysis When the experiment was finished, water samples were taken from 50 mL of each biofloc tank, and biomass was collected by centrifugation (10 min, 4000g). Total DNA was extracted from the samples using a water DNA extraction kit (E.Z.N.A.R, Omega) according to the manufacturer's protocol. MisSeq metagenome sequencing and bioinformatics was conducted by the Shenzhen HENGCHUANG Gene Technology Co Ltd. using a sequencing machine (Illumina HiSeq2500 sequencing, United States Illumina company completed). Community structure analysis examines the relative distribution of species at the phylum level. 2.5. Biofloc volume and morphostructure Floc volume was determined by sampling 1000 mL of water into a series of Imhoff cones (1000–0010, Nalgene) at 10:00 am every 3 days. The volume of the floc plug accumulating on the bottom of the cone was determined 15 min after sampling. Then, the floc plug was collected from the turn-knob at the bottom tip of the cone, and the floc morphostructure was observed with a biologic microscope (BX51, Olympus) and a fluorescence microscope (Leica, DMI3000B). 2.6. Statistical analysis Data obtained from the experiment were analyzed using SPSS 17.0 software (SPSS, Chicago, USA) for Windows. One-way ANOVA was performed on the experimental parameters. Differences were considered significant at P b 0.05. When significant differences were found, Duncan's multiple range test was used to identify differences between the experimental groups.
2.3. Proximate analysis of different bioflocs 3. Results Proximate analysis was carried out on the different types of bioflocs. Concentrated biofloc samples were collected from each tank by passing tank water through a 10-μm mesh nylon bag (Xu and Pan, 2012) after 25 days. The samples were dried in an oven at 105 °C until they reached a constant weight and then preserved in a refrigerator (− 20 °C) until proximate composition analysis. The crude protein content was calculated based on the assumption that protein contains 16% nitrogen (AOAC, 1999). For ash content, a known amount of dry sample was burnt in a muffle furnace at 550 °C for 4 h before the ash was cooled and weighed. The lipid content was determined with Soxhlet apparatus. Protein, lipid and ash contents were expressed as a percentage of the dry weight (% DW) of the bioflocs. The total carbohydrate amount was calculated according to the following formula: carbohydrate (% DW) = 100 − (crude protein (% DW) + lipid (% DW) + ash (% DW)) (Manush et al., 2005). The gross energy content of the diets was calculated using kJ·g−1 DW values of 23.0, 38.1 and 17.2 for protein, lipids and carbohydrates, respectively (Tacon, 1990). The amino acid composition of the biofloc was measured by a professional laboratory using high-performance liquid chromatography (HPLC). A selection of amino acids that are considered essential for aquatic animals in general was chosen as described by Tacon (1987) and Babarro et al. (2011). The essential amino acid ratio (E/A) for each essential amino acid (EAA) was expressed as a percentage of each amino acid of the total amount of essential amino acids measured (Arai,
3.1. Water quality The physical and chemical water quality parameters monitored throughout the experiment are presented in Table 1. There was no significant difference in temperature and pH observed between different BFs (P N 0.05). However, DO, NH4-N、NO2-N and NO3-N had significant differences (P b 0.05). The relationship between the resulting DO concentrations from the three treatments was BF (Sta) N BF (Glu) N BF (Gly). The BF (Gly) treatment had the lowest concentrations of NH4-N (0.12 ± 0.03 mg/L), NO2-N (0.02 ± 0.01 mg/L) and NO3-N (0.10 ± 0.02 mg/L). The concentrations found in the BF (Glu) treatment were Table. 1 The water quality parameters in three kinds of bioflocs treatment during the experiment. Parameter
BF (Glu)
BF (Sta)
BF (Gly)
Temperature (°C) DO (mg/L) pH NH4—N (mg/L) NO2—N (mg/L) NO3—N (mg/L)
23.3 ± 1.9a 7.5 ± 0.2ab 7.6 ± 0.2a 0.13 ± 0.01a 0.04 ± 0.02a 0.14 ± 0.01a
23.5 ± 1.6a 7.9 ± 0.1b 8.0 ± 0.3a 0.20 ± 0.03b 0.13 ± 0.02b 0.69 ± 0.01b
23.8 ± 1.3a 7.1 ± 0.3a 7.7 ± 0.2a 0.12 ± 0.03a 0.02 ± 0.01a 0.10 ± 0.02a
Each value represents mean ± S.D. Values in the same row with different superscript letters are significantly different at P b 0.05.
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similar to those of the BF (Gly) treatment. In the BF (Sta) treatment, the concentrations of NH4-N, NO2-N and NO3-N were 1.67 times, 6.5 times and 6.9 times those of the BF (Gly) treatment, respectively.
3.3. Biofloc investigation After 10 days of culture, the water in the different groups had different colors. When using glucose as a carbon source, the water was pale yellow; when using starch as a carbon source, the water was light green; when using glycerol as carbon source, the water was brown. The average volume of floc that settled in the Imhoff cones from water samples from the three replicated tanks as a function of culture time is shown in Fig. 3. Biofloc development in the water column was similar and initially slow in all three treatments. The biofloc grew rapidly after 10 days of culture. Twenty days later, the biomass was essentially unchanged. Biofloc grown with glucose had significant differences compared to bioflocs grown in glycerol and starch (P b 0.05). The morphology and structure of biological flocculations were observed with a biologic microscope with a 10 × objective (Fig. 4a) and with a fluorescence microscope with a 20× objective (Fig. 4b). The biological flocculation had irregular agglomerates. Bioflocs grown in different carbon sources had the same structure and composition; all had single-cell algae, protozoa, rotifers and bacterial communities, etc. Table. 2 Proximate analysis of three kinds of bioflocs treatment (mean ± S.D., n = 3). Composition
BF (Glu)
BF (Sta)
BF (Gly)
Crude protein (% DW) Crude lipid (% DW) Carbohydrate (% DW)⁎
41.2 ± 0.8a 6.1 ± 0.1a 37.7 ± 0.2a
31.5 ± 0.6b 8.5 ± 0.2b 47.6 ± 0.3b
35.5 ± 1.2c 4.2 ± 0.1c 45.1 ± 0.2c
Ash (% DW) Gross energy (kJ g−1 DW)⁎⁎
15.0 ± 0.1a 18.2 ± 0.3ab
12.4 ± 0.2b 18.7 ± 0.2b
15.2 ± 0.3a 17.5 ± 0.3a
Values on the same line followed by different superscript letters are significantly different (P b 0.05). DW, dry weight. ⁎ Values were calculated based on crude protein, crude lipid and ash content. ⁎⁎ Values were calculated based crude protein, crude lipid, ash and carbohydrate content.
EAA Concentration (µmol/g)
Proximate analysis of the different types of bioflocs is shown in Table 2. There was a significant difference (P b 0.05). The protein content in the BF (Sta) and BF (Gly) treatments was 31.5 ± 0.6% and 35.5 ± 1.2% of DW, respectively. Biofloc grown with glucose had a protein content that was significantly higher (41.2 ± 0.8%) than those of the other treatments. The lipid content ranged from 4.2% of DW for the BF (Gly) treatment to 8.5% of DW for the BF (Sta) treatment. The ash content was similar in both BF (Glu) and BF (Gly) treatments: 15.0 ± 0.1% and 15.2 ± 0.3% of DW, respectively. Biofloc grown on starch had an ash content that was significantly lower (12.4 ± 0.2%) than those of the other treatments. The carbohydrate contents varied between treatments. The highest calculated carbohydrate levels (47.6% on DW) were found in the BF (Sta) treatment. The lowest values were found (37.7% on DW) in the BF (Glu) treatment. The gross energy in BF (Glu), BF (Sta) and BF (Gly) was 18.2 ± 0.3 kJ g−1, 18.7 ± 0.2 kJ g−1, 17.5 ± 0.3 kJ g−1, respectively. The essential amino acid concentrations of the three types of bioflocs are shown in Fig. 1. In general, the three bioflocs seem to be rich in threonine, arginine, tyrosine, phenylalanine, isoleucine, leucine and lysine but are deficient in methionine and histidine. Furthermore, there was no cysteine in any of the bioflocs. The concentrations of nonessential amino acids in the three types of bioflocs are shown in Fig. 2. Generally, they are all rich in asparagine, glutamate, serine, glycine, alanine and proline, but deficient in tyrosine. In general, the amino acid content in BF (Sta) was less than those in BF (Glu) and BF (Gly).
100
80
60
40
20
0 BF(Glu)
BF(Sta)
BF(Gly)
Biofloc Type Fig. 1. Essential amino acid pattern (μmol/g biofloc dry weight) of different biofloc. Values are means ± standard deviation of three replications.
They varied in density and size. BF (Sta) had a higher density and smaller size; thus, it had a low floc volume. Fig. 4b shows that bioflocs grown on different carbon sources have different amounts of algae. BF (Sta) had the most, BF (Glu) had an intermediate amount, and BF (Gly) had the least. In BF (Sta), the algae cluster together, while the algae in BF (Glu) and BF (Gly) are more dispersed. 3.4. Microbial community The microbial communities at the phylum level in the three types of biofloc are shown in Fig. 5. Both bioflocs shared a large portion of Proteobacteria and Bacteroidetes. The BF (Sta) was clearly different from the BF (Glu) and BF (Gly); it also has a domain of Cyanobacteria. BF (Glu) and BF (Gly) have a more diverse microbial community than BF (Sta). 4. Discussion This study demonstrated that different carbon sources significantly affect the quality of bioflocs. The levels in BF (Sta) were significant higher than those in the other two groups. DO and pH values were lower in BF (Glu) and BF (Gly), which was likely a result of higher respiration rates due to the presence of a heterotrophic microbial community that increased the carbon dioxide concentration in a limited water exchange system (Tacon et al., 2002; Wasielesky et al., 2006). It has 120
Asparagic Glutamate Serine Glycine Tyrosine Alanine Proline
100
NEAA Concentration (µmol/g)
3.2. Proximate analysis of different bioflocs
Histidine Threonine Arginine Valine Methionine Phenilalnine Isoleucine Leucine Lysine
120
80
60
40
20
0 BF(Glu)
BF(Sta)
BF(Gly)
Biofloc Type Fig. 2. Nonessential amino acid pattern (μmol/g biofloc dry weight) of different biofloc. Values are means ± standard deviation of three replications.
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30 BF (Glu) BF (Sta) BF (Gly)
Floc volume (ml /L)
25
20
15
10
5
Fig. 5. Bacterial composition at phylum level of three biofloc types Treatments: Glu presence of biofloc grown in glucose; Sta presence of biofloc grown in starch; Gly presence of biofloc grown in glycerol. Each treatment has three parallel.
0 1
4
7
10
13
16
19
22
25
Culture day (d) Fig. 3. Dynamic changes of floc volume in different treatment throughout the experimental period. Values are means ± standard deviation of three replications in each sampling date.
been shown that there are different effects of simple versus complex carbohydrates used as the carbon source in biofloc-based systems (Avnimelech, 2012). Simple sugars result in faster ammonia removal, while more complex carbohydrates require more time for decomposition into simple sugars, thereby resulting in slower ammonia removal. This may explain the higher NH4-N level in BF (Sta) compared to BF (Glu) and BF (Gly). During the experiment, more algae were observed in BF (Sta) than in BF (Glu) and BF (Gly). Each type of biofloc grown on a different carbon source (glucose, starch and glycerol) was adequate in maintaining the water quality parameters in a normal range for shrimp growth (Van Wyk and Scarpa, 1999). Toxic ammonia and nitrite levels can lead to low survival rates or decreased growth. Therefore, BFT may make it possible to increase growth yields and survival levels at low
water replacement rates, while adding a potential additional natural food resource in the form of bioflocs (Asaduzzaman et al., 2008). Feed costs represent at least 50% of total aquaculture production costs, predominantly due to the cost of the protein component in commercial diets (Bender et al., 2004). In this experiment, except for the composition of crude protein and ash, other elements of the composition (crude lipid, carbohydrate and gross energy) are higher than the level reported by Crab et al. (2010). This could be due to differences in the production of biofloc, such as carbon source, C/N ratio and inoculum. The protein content in the diet is an important factor for the nutrition of most aquatic organisms. Most aquaculture species require protein in a range of 20–50% in their diet (Tacon, 1987). These three bioflocs have good prime nutritional values when compared to a reference commercial prawn feed analyzed by Hossain and Paul (2007). The protein content in the BF (Glu) was the highest, followed by those of BF (Gly) and BF (Sta). This could be due to the different concentration of extracellular polymeric substances (EPS), which accounts for 80–95% of the organic matter in the flocs (Wilén et al., 2008). The lipid content was low in
a
BF (Glu)
BF (Sta)
BF (Gly)
BF (Glu)
BF (Sta)
BF (Gly)
b
Fig. 4. a. Morphology of different biofloc types under light microscope. b. Morphology of different biofloc types under fluorescence microscope.
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BF (Glu) and BF (Gly), but not in BF (Sta). This is because BF (Sta) had more Cyanobacteria (Sharathchandra and Rajashekhar, 2011; Maslova et al., 2004). It has been reported that lipids are a major metabolic energy source (Roustaian et al., 2001), which implies that commercial feed is still necessary to provide adequate lipid levels in BF (Glu) and BF (Gly). Microalgae, also called ‘miniature sunlight-driven biochemical factories’ (Verma et al., 2010), are capable of producing large amounts of lipids and hydrocarbons in the presence of sunlight and carbon dioxide from flue gases. BF (Sta) had a lower ash content and higher carbohydrate content. BF (Sta) also had the highest gross energy. High ash content lowers the digestibility of other ingredients in the diet resulting in poor shrimp growth. BF (Glu) and BF (Gly) had higher ash contents, which can decrease the digestibility of the biofloc. BF (Sta) had a high concentration of carbohydrates, which could be the result of the poor solubility of starch because starch particles may still be present in the flocs. Taken together, these results show that the biofloc quality is significantly influenced by the added carbon source. There may be major differences in the way the microbes that take part in biofloc formation benefit from these different carbon sources (Rittmann and McCarty, 2001). Moreover, the impact of various carbon sources on the outgrowth of bioflocs in activated sludge systems has been documented (Bodík et al., 2009). The amino acid composition and the bioavailability of the amino acids present determine the protein quality of a diet for aquaculture. An amino acid is classified as nutritionally essential (indispensable) or non-essential (dispensable) by the nutritional requirement and the organism's ability to perform amino acid synthesis. EAAs are those that either cannot be synthesized or are inadequately synthesized by animals relative to their needs and consequently must be provided by the diet. Since amino acids are building blocks for proteins, optimal synthesis of proteins is therefore determined by the dietary amino acid profile (Mente et al., 2002). There are reports in the literature that the dietary amino acid requirement of a particular aquatic organism is strongly dependent on its body amino acid composition (Tacon, 1987). Peñaflorida (1989) recommended the calculation of the EAAI to evaluate the EAA profile in the diet relative to the EAA composition of the animal and determined that a good-quality diet had an EAAI of N0.9, a useful diet had a value between 0.7 and 0.9, and the diet was considered inadequate when this value was b0.7. The EEAI values of BF (Glu), BF (Sta) and BF (Gly) were 0.99, 0.93 and 0.98 respectively. This shows that the EAA composition of different types of biofloc could meet the requirement of the aquatic species tested. Based on this index, the biofloc produced in this study can be considered a good-quality protein source for shrimp. The biofloc can be a feed supplement, whether for specific amino acids or for total amino acid content. This also indicates that biofloc grown in glucose and glycerol had a higher EAAI than bioflocs grown in starch, which suggests that BF (Glu) and BF (Gly) were higher in quality than BF (Sta) in terms of EAAs under the experimental conditions. Ebeling et al. (2006) reported that in BFT systems, the concentration of suspended flocs is, after oxygen, the most important limiting factor for productivity. In this experiment, BF (Sta) was more dense and compact. This was because it had a higher DO concentration (see Table 1). An adequate DO level is not only essential for the metabolic activity of cells within aerobic flocs, but it is also thought to influence floc structure (De Schryver et al., 2008). Higher DO concentrations also induce more compact flocs (Wilén and Balmér, 1999). Ballester et al. (2010) showed that the microorganisms present in BFT systems play an important role in the provision of essential nutrients. It is believed that under conditions with abundant incident light, such as a shrimp pond or outdoor aquarium, phytoplankton algae are the first to grow, and these organisms provide a basic source of food for the subsequent development of a zooplankton community and nutrients for the growth of bacteria in the culture system. In Fig. 5, it can be seen that Proteobacteria and Bacteroidetes are the dominant bacteria in aquaculture and occupy a very large of proportion in all types of
biofloc. BF (Sta) was clearly different from the BF (Glu) and BF (Gly), suggesting that the microbial community was shaped by the carbon source. The majority of Proteobacteria detected in both groups were considered to be symbiotic bacteria in aquaculture (Sakami et al., 2008). Proteobacteria removes organic matter (Wagner et al., 1993; Miura et al., 2007), especially in the wastewater treatment of a biofloc. The class of Proteobacteria also plays a major role in the composition of bacteria (Wagner et al., 1995; Daims et al., 1999). This demonstrates that when a biofloc is applied to a culture system, it can effectively regulate the quality of the aquaculture water. Actinobacteria is a common probiotic, which can produce beneficial substances in specific environments (Das et al., 2008). Gerardi (2006) reported that Flavobacterium can produce glue-like extracellular polymers and has the ability to bind cells together. 5. Conclusion Biofloc rearing media provide a potential food source for shrimp reared in limited or zero water exchange systems. This culture system is environmentally friendly because it is based on limited water use and minimal effluent is released into the surrounding environment. The major aim of this study was to compare the water quality, nutritive value, morphology and microbial community of different types of biofloc. The overall results show that BF (Sta) has higher crude lipids, carbohydrates and gross energy and lower crude protein and ash compared to BF (Glu) and BF (Gly). In addition, BF (Sta) has more prokaryotic algae and is more closely aggregated. In terms of EAAI, all three bioflocs could meet the requirement of shrimp. They can be used to replace or partly replace feed oil in culture to reduce costs and pollution. Further studies are recommended to optimize the biofloc microbial biomass ratios in order to set benchmarks for shrimp culture systems. Moreover, the inhibition of pathogens should also be studied. Acknowledgments This work was supported by the major projects of science and technology and development of marine fishery in Guangdong Province, China (grant number A201401B01, A201001H02); the Project of Natural Science Foundation of Fujian Province (grant number 2014J01049); and The Guangzhou Science and Technology Program key projects (grant number 2014J4100052). We would like to thank the anonymous reviewers for their remarks and suggestions. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.aquaculture.2016.08.040. References APHA, 1998. Standard Methods for the Examination of the Water and Wastewater. 22nd ed. American Public Health Association, Washington, DC. Arai, S., 1981. A purified test diet for coho salmon, Oncorhynchus kisutch, fry. Nippon Suisan Gakkaishi 47, 547–550. Asaduzzaman, M., Wahab, M.A., Verdegem, M.C.J., Huque, S., Salam, M.A., Azim, M.E., 2008. C/N ratio control and substrate addition for periphyton development jointly enhance freshwater prawn Macrobrachium rosenbergii production in ponds. Aquaculture 280 (1), 117–123. Association of Official Analytical Chemists, 1999. Official Methods of Analysis. AOAC, Washington, DC. Avnimelech, Y., 1999. C/N 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 (1), 140–147. http://dx.doi.org/10.1016/j. aquaculture.2006.11.025. Avnimelech, Y., 2012. Biofloc Technology-a Practical Guide Book. 2nd ed. The World Aquaculture Society, Baton Rouge, United States. Babarro, J.M.F., Fernández-Reiriz, M.J., Labarta, U., Garrido, J.L., 2011. Variability of the total free amino acid (TFAA) pool in Mytilus galloprovincialis cultured on a raft system. Effect of body size. Aquac. Nutr. 17 (2), 448–458.
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