Novel Biofloc Technology (BFT) for Ammonia Assimilation and Reuse in Aquaculture In Situ

*Hai-Hong Huang*

### **Abstract**

Ammonia is one of the most harmful risks for success of fish and shrimp culture. There is no effective solution for harmlessness of ammonia in traditional aquaculture operations except exchanging water, which would bring negative effects on environment, or fixing expensive equipment. Biofloc technology (BFT) that appeared in recent years supplies a novel solution for this issue without exchanging huge water and fixing equipment. This technology could assimilate ammonia almost in real time with many other supplemental benefits. Because of the very high nutritional value for fish and shrimp, bioflocs, the by-product of BFT, could also be reused as a complemented food in situ or a gradient for feedstuff to replace expensive fishmeal or be processed to pellet diet to feed fish and shrimp directly. However, some aspects with regard to the effective use of biofloc as a food source for fish and shrimp, such as high lipid content, productivity, and palatability, need to be further researched in detail.

**Keywords:** ammonia, assimilation, reuse, biofloc technology, aquaculture

#### **1. Introduction**

The world population will exceed 9 billion people by the middle of the twentyfirst century, indicating proportionate food should have to provide. Fisheries and aquaculture are the critical important sources against this challenge of food and nutrition [1]. Between 1961 and 2016, the average annual increase in global food fish consumption (3.2%) outpaced population growth (1.6%) and exceeded that of meat from all terrestrial animals combined (2.8%). Total fish production in 2016 reached 171 million tones, of which 88% was directly utilized for human consumption. In per capita terms, food fish consumption grew from 9.0 kg in 1961 to 20.2 kg in 2015, accounting for about 17% of their average per capita intake of animal protein consumed by the global population [1].

Since the late 1980s, the fishery production has been stable without obvious increase. But aquaculture has become more and more important, which production grew faster than other major food production sectors. The contribution of aquaculture to the global production of capture fisheries and aquaculture combined has risen continuously, reaching 46.8% in 2016 and representing 53% of fish production for food uses [1].

However, the development of aquaculture has faced challenges because of lack of land and water source and degradation of environment [1]. Therefore, turning of aquaculture to intensive even high intensive model from extensive or semiextensive model is a tendency all over the world. Intensive aquaculture utilizes limited land source to culture more fish and shrimp by excessively increasing aquatic animal density with little water exchange or even zero water exchange. However, one of the most harmful risks for success of fish and shrimp in intensive aquaculture system, especially in closed intensive culture system with little water exchange, is the accumulation of ammonia. Unfortunately, there is no effective solution for harmlessness of ammonia in practical operations except exchanging water or fixing some very expensive equipment for water treatment [2].

Biofloc technology (BFT) that appeared in recent years supplies a novel solution for this issue without exchanging huge water or fixing equipment [2]. BFT could assimilate ammonia almost in real time and reuse the by-product as a natural food source in situ in aquaculture water column. In this chapter, problems referred to ammonia in aquaculture (Part 2 and Part 3), principles of ammonia removal (Part 4), main operations of BFT (Part 5), applications of using biofloc produced as a by-product of BFT in aquaculture (Part 6 and Part 7), and some highlighting issues that should be paid attention to or need to be further researched (Part 8) are introduced in brief.

#### **2. Toxicity of ammonia to fish or shrimp**

Ammonia is one of the most harmful inorganic nitrogen compounds for fish or shrimp in aquaculture (another is nitrite), whose accumulation in pond water may deteriorate water quality, reduce growth, increase oxygen consumption, alter concentrations of hemolymph protein and free amino acid levels, and even cause high mortality [3]. For example, in water with pH 8.05 and temperature 23°C, the 96 h median lethal concentration (LC50) value of ammonia on *Litopenaeus vannamei* (Pacific white-leg shrimp, the most important cultured crustacean species in the world [1]) juveniles is 35.4 mg/L at salinity of 25‰, indicating that the safety level of ammonia for rearing *L. vannamei* juveniles is only 3.55 mg/L [3]. A research conducted by the author of this chapter revealed that in a closed aquaculture system for *L. vannamei* without water exchange, the ammonia concentration accumulated up to a very high level of 10.81 mg/L, with an average of 6.35 mg/L, leading to a mortality rate as high as 70%.

There are two existent types for ammonia, ionic type (NH4 + ) and free type (NH3), both of which in general named together as total ammonia nitrogen (TAN). In fact, the toxicity of TAN is mainly from the free NH3; in water, the 96 h LC50 value to *L. vannamei* juveniles is 1.57 mg/L under conditions of pH 8.05, temperature 23°C, and salinity of 25‰, and the safety level is 0.16 mg/L [3]. Van Wyk et al. [4] even recommend a value of ≤0.03 ppm or mg/L for NH3 when farming this shrimp in recirculating freshwater systems. There is an equilibrium between the two existences of ammonia in water [5]:

$$\mathrm{NH}\_3 + \mathrm{H}^+ \leftrightharpoons \mathrm{NH}\_4^+ \tag{1}$$

In this equation,*T* means the water temperature. Based on Eq. (1), the pro-

*Novel Biofloc Technology (BFT) for Ammonia Assimilation and Reuse in Aquaculture In Situ*

25°C) could be calculated and pictured, respectively (**Figure 1**). From **Figure 1**, it is well known that there is almost no NH3 existing in water when pH is below 7.0; however, after that, the proportion of NH3 will exponentially elevate with increas-

Ammonia in aquaculture water body is mostly produced from artificial feeds fed to fish animals. Estimated about 78% of nitrogen existing in aquaculture water body comes from feedstuff [7]. Artificial formulated feed for aquaculture animals contains a very high content of protein; in general, the crude protein content in finfish feedstuff is 25–30% [8] and higher for crustacean animals, which is even up to 40– 45% for shrimp species like white-leg shrimp [4]. However, the utilization efficiency of those feeds in water is very low. When feed is added to water, only 25% of protein nitrogen in feedstuff is assimilated to body growth of aquatic animals, and the rest of about an approximate 75% proportion will lose into the water body, via directly excreting as metabolic ammonia from gill, evacuating as urea and feces by cloaca system, or dissolving as other organic nitrogen compounds [9], which are further degraded as inorganic ammonia by microorganisms with hydrolysis enzymes.

**4. The main routes for ammonia transformation in aquaculture**

nitrobacteria, and assimilation of heterotrophic bacteria [10].

of well-known photosynthesis as follows [10]:

**5**

**4.1 Route 1: photoautotrophic intake by algae or phytoplankton**

There are three routes for ammonia removal or transformation in aquaculture system: intake by photoautotrophic algae, nitrification and nitration of autotrophic

Actually, the intake route of ammonia by photoautotrophic algae is the process

<sup>+</sup> and NH3 along pH gradient under a certain temperature (such as

portions of NH4

**Figure 1.** *Percent of NH4*

ing of pH, as well as the toxicity of TAN.

*DOI: http://dx.doi.org/10.5772/intechopen.88993*

**3. Deriving of ammonia in aquaculture water body**

*<sup>+</sup> and NH3 changing with pH under 25°C.*

This equilibrium indicates that NH4 <sup>+</sup> and NH3 exist in water at the same time and their proportions are determined by the pH of the water body so that the toxicity of TAN is highly related to water pH. Actually, the relationship among water pH and the concentrations of NH4 <sup>+</sup> and NH3 could be descripted with an equation [6] as follows:

$$\frac{[\text{NH}\_3]}{[\text{NH}\_3] + [\text{NH}\_4^+]} = \frac{\mathbf{10^{pH}}}{\exp\left(\frac{6344}{273 + T}\right) + \mathbf{10^{pH}}} \tag{2}$$

*Novel Biofloc Technology (BFT) for Ammonia Assimilation and Reuse in Aquaculture In Situ DOI: http://dx.doi.org/10.5772/intechopen.88993*

**Figure 1.** *Percent of NH4 <sup>+</sup> and NH3 changing with pH under 25°C.*

aquaculture to intensive even high intensive model from extensive or semiextensive model is a tendency all over the world. Intensive aquaculture utilizes limited land source to culture more fish and shrimp by excessively increasing aquatic animal density with little water exchange or even zero water exchange. However, one of the most harmful risks for success of fish and shrimp in intensive aquaculture system, especially in closed intensive culture system with little water exchange, is the accumulation of ammonia. Unfortunately, there is no effective solution for harmlessness of ammonia in practical operations except exchanging

*Emerging Technologies, Environment and Research for Sustainable Aquaculture*

water or fixing some very expensive equipment for water treatment [2].

introduced in brief.

mortality rate as high as 70%.

two existences of ammonia in water [5]:

This equilibrium indicates that NH4

concentrations of NH4

**4**

**2. Toxicity of ammonia to fish or shrimp**

Biofloc technology (BFT) that appeared in recent years supplies a novel solution for this issue without exchanging huge water or fixing equipment [2]. BFT could assimilate ammonia almost in real time and reuse the by-product as a natural food source in situ in aquaculture water column. In this chapter, problems referred to ammonia in aquaculture (Part 2 and Part 3), principles of ammonia removal (Part 4), main operations of BFT (Part 5), applications of using biofloc produced as a by-product of BFT in aquaculture (Part 6 and Part 7), and some highlighting issues that should be paid attention to or need to be further researched (Part 8) are

Ammonia is one of the most harmful inorganic nitrogen compounds for fish or shrimp in aquaculture (another is nitrite), whose accumulation in pond water may deteriorate water quality, reduce growth, increase oxygen consumption, alter concentrations of hemolymph protein and free amino acid levels, and even cause high mortality [3]. For example, in water with pH 8.05 and temperature 23°C, the 96 h median lethal concentration (LC50) value of ammonia on *Litopenaeus vannamei* (Pacific white-leg shrimp, the most important cultured crustacean species in the world [1]) juveniles is 35.4 mg/L at salinity of 25‰, indicating that the safety level of ammonia for rearing *L. vannamei* juveniles is only 3.55 mg/L [3]. A research conducted by the author of this chapter revealed that in a closed aquaculture system for *L. vannamei* without water exchange, the ammonia concentration accumulated up to a very high level of 10.81 mg/L, with an average of 6.35 mg/L, leading to a

(NH3), both of which in general named together as total ammonia nitrogen (TAN). In fact, the toxicity of TAN is mainly from the free NH3; in water, the 96 h LC50 value to *L. vannamei* juveniles is 1.57 mg/L under conditions of pH 8.05, temperature 23°C, and salinity of 25‰, and the safety level is 0.16 mg/L [3]. Van Wyk et al. [4] even recommend a value of ≤0.03 ppm or mg/L for NH3 when farming this shrimp in recirculating freshwater systems. There is an equilibrium between the

NH3 þ Hþ⇋NH<sup>þ</sup>

their proportions are determined by the pH of the water body so that the toxicity of TAN is highly related to water pH. Actually, the relationship among water pH and the

<sup>¼</sup> <sup>10</sup>pH

exp <sup>6344</sup> 273þ*T*  +

<sup>4</sup> (1)

<sup>þ</sup> <sup>10</sup>pH (2)

<sup>+</sup> and NH3 exist in water at the same time and

<sup>+</sup> and NH3 could be descripted with an equation [6] as follows:

) and free type

There are two existent types for ammonia, ionic type (NH4

½ � NH3 ½ �þ NH3 NH<sup>þ</sup>

4

In this equation,*T* means the water temperature. Based on Eq. (1), the proportions of NH4 <sup>+</sup> and NH3 along pH gradient under a certain temperature (such as 25°C) could be calculated and pictured, respectively (**Figure 1**). From **Figure 1**, it is well known that there is almost no NH3 existing in water when pH is below 7.0; however, after that, the proportion of NH3 will exponentially elevate with increasing of pH, as well as the toxicity of TAN.

## **3. Deriving of ammonia in aquaculture water body**

Ammonia in aquaculture water body is mostly produced from artificial feeds fed to fish animals. Estimated about 78% of nitrogen existing in aquaculture water body comes from feedstuff [7]. Artificial formulated feed for aquaculture animals contains a very high content of protein; in general, the crude protein content in finfish feedstuff is 25–30% [8] and higher for crustacean animals, which is even up to 40– 45% for shrimp species like white-leg shrimp [4]. However, the utilization efficiency of those feeds in water is very low. When feed is added to water, only 25% of protein nitrogen in feedstuff is assimilated to body growth of aquatic animals, and the rest of about an approximate 75% proportion will lose into the water body, via directly excreting as metabolic ammonia from gill, evacuating as urea and feces by cloaca system, or dissolving as other organic nitrogen compounds [9], which are further degraded as inorganic ammonia by microorganisms with hydrolysis enzymes.

#### **4. The main routes for ammonia transformation in aquaculture**

There are three routes for ammonia removal or transformation in aquaculture system: intake by photoautotrophic algae, nitrification and nitration of autotrophic nitrobacteria, and assimilation of heterotrophic bacteria [10].

#### **4.1 Route 1: photoautotrophic intake by algae or phytoplankton**

Actually, the intake route of ammonia by photoautotrophic algae is the process of well-known photosynthesis as follows [10]:

$$\text{16NH}\_4^+ + \text{92CO}\_2 + \text{92H}\_2\text{O} + \text{14HCO}\_3^- + \text{HPO}\_4^{2-} \rightarrow \text{C}\_{106}\text{H}\_{269}\text{O}\_{110}\text{N}\_{16}\text{P} + \text{106O}\_2\text{O} \tag{3}$$

where C5H7O2N represents the chemical formula for microbial biomass like route 2, or Eq. (5). Compared to route 2, sufficient dissolved oxygen is needed for

*Novel Biofloc Technology (BFT) for Ammonia Assimilation and Reuse in Aquaculture In Situ*

exhausted. Differently, in Eq. (6) of route 3, carbohydrate (C6H12O6) is needed, and

**5. Novel solution for ammonia assimilation and reuse in aquaculture**

several routes referring to ammonia clearance mentioned above. However, routes 1 and 2 are all not suitable to apply in aquaculture. For route 1, intake of phytoplankton or algae might produce a large number of algae exceeding the biological capacity of water body, and those planktons will be old and die quickly and release toxins harmful to aquatic animals. In regard to route 2, it is mainly applied for effluent treatment in sewage plant, which needs inferior procedures of wastewater, and thus is not suitable in aquaculture as well. Fortunately, according to the principles of route 3 displayed in Eq. (6), a novel technology, in generic nicknamed as biofloc technology (BFT), is developed for aquaculture in recent years, to be used as effectively and environmental-friendly for transforming

Ammonia accumulation is the head issue faced in aquaculture, and there are

In accordance with Eq. (6), existing of carbohydrate will promote assimilation of ammonia, companied with synthesis of microbial biomass. However, the content of carbohydrate or C:N in aquaculture water body is lower than the need for bioreaction of Eq. (6) in general. Although the C:N of bacterial cell composition is about 5:1 [12], it needs a C:N of 15:1 for blooming growth of heterotrophic bacteria to assimilate ammonia [13, 14]. In aquaculture water body, the carbohydrate is mainly from feedstuff added in [7], whose content is usually inadequate for blooming growth of heterotrophic bacteria. For example, taking white-leg shrimp feed usually used in China into consideration, the contents of ingredients, such as crude protein, lipid, fiber, ash, and moisture, are 40, 5.0, 5.0, 15, and 12%, respectively, indicating a calculated C:N of approximate 6:1 according to the relationship

Carbon% ¼ 0*:*80 � Lipid% þ 0*:*53 � Protein% þ 0*:*42

Carbohydrate% ¼ 100 � ð Þ Protein þ Lipid þ Fiber þ ash þ moisture % (8)

Therefore, additional exogenous organic carbon source containing carbohydrate (C6H12O6) should be supplemented to prompt assimilation of ammonia by improving growth of heterotrophic bacteria, and this is one of the two principal operations

The other principal operations for BFT are aeration and treatment of byproduct. Known from Eq. (6), a huge number of dissolved oxygen is needed to assimilate ammonia by heterotrophic bacteria, and also massive bacteria biomass is

�Carbohydrate% <sup>þ</sup> <sup>0</sup>*:*<sup>42</sup> � Fiber% (7)

between contents of carbohydrate and feed ingredients [15, 16]:

produced as by-product, which needs to be treated.

� will be

the processing of bio-reaction of Eq. (6) as well, but about half of HCO3

about 40 times microbial biomass is produced.

*DOI: http://dx.doi.org/10.5772/intechopen.88993*

of ammonia.

for BFT [17].

**7**

**5.1 Principal operations of BFT**

**based on route 3: biofloc technology (BFT)**

Or when nitrate is as the nitrogen source

$$\text{16NO}\_3^- + \text{124CO}\_2 + \text{140H}\_2\text{O} + \text{HPO}\_4^{2-} \rightarrow \text{C}\_{\text{166}}\text{H}\_{2\text{63}}\text{O}\_{110}\text{N}\_{1\text{67}}\text{P} + \text{138O}\_2 + \text{18HCO}\_3^- \tag{4}$$

where C106H263O110N16P represents the stoichiometric formula for algae.

In this process, the ionic ammonia of NH4 <sup>+</sup> is the first-order utilized inorganic nitrogen for synthesis of organic materials. However, a carbon to nitrogen to phosphorus ratio (C:N:P) of about 106:16:1 is also needed, indicating that to promote ammonia assimilation, exogenous additions of inorganic carbon and phosphorus sources are needed and that in general make the growth of algae, especially bluegreen algae or cyanobacteria, to be very difficult to control and easily result in cyanobacteria blooming, a serious deterioration of water quality and a disaster for human daily life.

#### **4.2 Route 2: autotrophic oxidation by nitrobacteria**

Autotrophic nitrobacteria, the chemical autotrophic bacteria, can oxidize ammonia by using inorganic carbon sources without the need of phosphorus [10]:

$$\begin{aligned} \text{NH}\_4\text{}^+ + \text{1.83O}\_2 + \text{1.97HCO}\_3^- &\rightarrow \text{0.0244}\text{C}\_5\text{H}\_7\text{O}\_2\text{N} \\ + \text{0.976NO}\_3^- &+ \text{2.9H}\_2\text{O} + \text{1.86CO}\_2 \end{aligned} \tag{5}$$

where C5H7O2N represents the chemical formula for microbial biomass.

However, the growth rate of nitrobacteria is very low when compared to heterotrophic bacteria, which in turn leads to a low oxidized rate for ammonia. There are also no other efficient supplemental approaches to accelerate this process, which mainly relies on the natural development of nitrobacteria. Furthermore, an intermediate product of this process, nitrite or NO2 �, another toxic inorganic nitrogen compound for aquaculture animals, would be produced. Nitrite is an unstable product with high oxidized ability comparable to oxygen and thus will oxidize Fe2+ in the center of hemoglobin to Fe3+. As a result, oxygen could not combine to hemoglobin and transport to tissues, and thus animals will be asphyxiated, even though there is enough oxygen dissolved in water body [11]. Moreover, the oxidization of ammonia by nitrobacteria would cause numerous accumulation of nitrate (NO3 �), another inorganic nitrogen compound which could be easily taken by phytoplankton, indicating a potential risk of algae blooming [10, 11]. Finally, the nitrification process could affect water quality, such as exhausting carbonate alkalinity (HCO3 �) and resulting in reduction of water pH [10].

#### **4.3 Route 3: assimilation by heterotrophic bacteria**

Ammonia also could be assimilated by heterotrophic bacteria through a process different from those of photoautotrophic algae (route 1) and autotrophic nitrobacteria (route 2) [10]:

$$\mathrm{NH\_4}^+ + \mathrm{1.18C\_6H\_{12}O\_6} + \mathrm{HCO\_3}^- + \mathrm{2.06O\_2} \rightarrow \mathrm{C\_5H\_7O\_2N} + \mathrm{6.06H\_2O} + \mathrm{3.07CO\_2} \tag{6}$$

*Novel Biofloc Technology (BFT) for Ammonia Assimilation and Reuse in Aquaculture In Situ DOI: http://dx.doi.org/10.5772/intechopen.88993*

where C5H7O2N represents the chemical formula for microbial biomass like route 2, or Eq. (5). Compared to route 2, sufficient dissolved oxygen is needed for the processing of bio-reaction of Eq. (6) as well, but about half of HCO3 � will be exhausted. Differently, in Eq. (6) of route 3, carbohydrate (C6H12O6) is needed, and about 40 times microbial biomass is produced.

### **5. Novel solution for ammonia assimilation and reuse in aquaculture based on route 3: biofloc technology (BFT)**

Ammonia accumulation is the head issue faced in aquaculture, and there are several routes referring to ammonia clearance mentioned above. However, routes 1 and 2 are all not suitable to apply in aquaculture. For route 1, intake of phytoplankton or algae might produce a large number of algae exceeding the biological capacity of water body, and those planktons will be old and die quickly and release toxins harmful to aquatic animals. In regard to route 2, it is mainly applied for effluent treatment in sewage plant, which needs inferior procedures of wastewater, and thus is not suitable in aquaculture as well. Fortunately, according to the principles of route 3 displayed in Eq. (6), a novel technology, in generic nicknamed as biofloc technology (BFT), is developed for aquaculture in recent years, to be used as effectively and environmental-friendly for transforming of ammonia.

#### **5.1 Principal operations of BFT**

16NH4

16NO3

human daily life.

nitrate (NO3

NH4

**6**

alkalinity (HCO3

nitrobacteria (route 2) [10]:

<sup>þ</sup> þ 1*:*18C6H12O6 þ HCO3

<sup>þ</sup> þ 92CO2 þ 92H2O þ 14HCO3

*Emerging Technologies, Environment and Research for Sustainable Aquaculture*

Or when nitrate is as the nitrogen source

� þ 124CO2 þ 140H2O þ HPO4

In this process, the ionic ammonia of NH4

**4.2 Route 2: autotrophic oxidation by nitrobacteria**

NH4

mediate product of this process, nitrite or NO2

**4.3 Route 3: assimilation by heterotrophic bacteria**

� þ HPO4

where C106H263O110N16P represents the stoichiometric formula for algae.

Autotrophic nitrobacteria, the chemical autotrophic bacteria, can oxidize ammonia by using inorganic carbon sources without the need of phosphorus [10]:

where C5H7O2N represents the chemical formula for microbial biomass. However, the growth rate of nitrobacteria is very low when compared to heterotrophic bacteria, which in turn leads to a low oxidized rate for ammonia. There are also no other efficient supplemental approaches to accelerate this process, which mainly relies on the natural development of nitrobacteria. Furthermore, an inter-

compound for aquaculture animals, would be produced. Nitrite is an unstable product with high oxidized ability comparable to oxygen and thus will oxidize Fe2+ in the center of hemoglobin to Fe3+. As a result, oxygen could not combine to hemoglobin and transport to tissues, and thus animals will be asphyxiated, even though there is enough oxygen dissolved in water body [11]. Moreover, the oxidization of ammonia by nitrobacteria would cause numerous accumulation of

by phytoplankton, indicating a potential risk of algae blooming [10, 11]. Finally, the nitrification process could affect water quality, such as exhausting carbonate

�) and resulting in reduction of water pH [10].

Ammonia also could be assimilated by heterotrophic bacteria through a process

different from those of photoautotrophic algae (route 1) and autotrophic

� þ 2*:*9H2O þ 1*:*86CO2

�), another inorganic nitrogen compound which could be easily taken

� þ 2*:*06O2 ! C5H7O2N þ 6*:*06H2O þ 3*:*07CO2

<sup>þ</sup> þ 1*:*83O2 þ 1*:*97HCO3

þ0*:*976NO3

nitrogen for synthesis of organic materials. However, a carbon to nitrogen to phosphorus ratio (C:N:P) of about 106:16:1 is also needed, indicating that to promote ammonia assimilation, exogenous additions of inorganic carbon and phosphorus sources are needed and that in general make the growth of algae, especially bluegreen algae or cyanobacteria, to be very difficult to control and easily result in cyanobacteria blooming, a serious deterioration of water quality and a disaster for

<sup>2</sup>� ! C106H263O110N16P <sup>þ</sup> 106O2

<sup>2</sup>� ! C106H263O110N16P <sup>þ</sup> 138O2 <sup>þ</sup> 18HCO3

� ! 0*:*0244C5H7O2N

�, another toxic inorganic nitrogen

<sup>+</sup> is the first-order utilized inorganic

(3)

(4)

(5)

(6)

�

In accordance with Eq. (6), existing of carbohydrate will promote assimilation of ammonia, companied with synthesis of microbial biomass. However, the content of carbohydrate or C:N in aquaculture water body is lower than the need for bioreaction of Eq. (6) in general. Although the C:N of bacterial cell composition is about 5:1 [12], it needs a C:N of 15:1 for blooming growth of heterotrophic bacteria to assimilate ammonia [13, 14]. In aquaculture water body, the carbohydrate is mainly from feedstuff added in [7], whose content is usually inadequate for blooming growth of heterotrophic bacteria. For example, taking white-leg shrimp feed usually used in China into consideration, the contents of ingredients, such as crude protein, lipid, fiber, ash, and moisture, are 40, 5.0, 5.0, 15, and 12%, respectively, indicating a calculated C:N of approximate 6:1 according to the relationship between contents of carbohydrate and feed ingredients [15, 16]:

$$\begin{aligned} \text{Carbon\%} &= 0.80 \times \text{Lipid\%} + 0.53 \times \text{Protein\%} + 0.42\\ &\times \text{Carbonydrate\%} + 0.42 \times \text{Fiber\%} \end{aligned} \tag{7}$$

Carbohydrate% ¼ 100 � ð Þ Protein þ Lipid þ Fiber þ ash þ moisture % (8)

Therefore, additional exogenous organic carbon source containing carbohydrate (C6H12O6) should be supplemented to prompt assimilation of ammonia by improving growth of heterotrophic bacteria, and this is one of the two principal operations for BFT [17].

The other principal operations for BFT are aeration and treatment of byproduct. Known from Eq. (6), a huge number of dissolved oxygen is needed to assimilate ammonia by heterotrophic bacteria, and also massive bacteria biomass is produced as by-product, which needs to be treated.
