1. Introduction
A major requirement for meeting the current fish protein demand for human consumption is the intensification of aquaculture [
1]. Increasing the rearing density of fish increases the productivity per unit area [
2]. A limited ability to control pathogens poses a major challenge to production intensification [
3]. Sustainability in feed management is also considered to be a major component in intensifying any aquatic organism’s production [
4]. In addition, a sustainable cultural system that does not pollute the environment and utilizes limited natural resources is needed [
5]. Such an ecological aquaculture system is biofloc technology, which ensures sustainable feed management. This system provides a high yield with limited water exchange [
6]. The advantage includes maintaining a high C/N ratio; therefore, the microbial community can take up ammonium and enhance the management of health and biosecurity with a limited exchange of water [
4].
The biofloc culture system assembles various suspended organic particles with useful microorganisms involved with polymeric extracellular substance, making it a heterogeneous system [
7]. It is possible to increase the carbon ratio of the feed by adding various organic carbonaceous sources such as tapioca, glucose, corn, wheat, acetate, glycerol, and molasses, etc., to the aquaculture system or by altering the feed composition by adding additional organic carbon sources [
8]. The organic carbon source significantly influences the composition of flocs, specifically, the kind of storage polymer used and its amount [
9]. Tilapia and
Litopenaeus vannamei were successfully farmed using biofloc technology, which substantiated to be better in terms of feed efficacy and water than traditional methods [
5]. In the biofloc system, the type of carbon source used affects its management, nutritional value, microbial community, and the biofloc system organisms [
8]. Wei et al. [
10] described that adding diverse carbonaceous sources may influence the NH
4 elimination process, and simpler carbon sources (such as glucose and sucrose) may eradicate ammonia more rapidly than more complex carbon sources such as starch. Aquatic organisms have been treated with microorganisms and their cell components to enhance their immunity, growth, disease resistance, and antioxidant status [
11]. Several bioactive compounds are present in bioflocs, such as polysaccharides, chlorophyll, fat-soluble vitamins, taurine, carotenoids, and phytosterols [
12,
13]. It is widely recognized that several microorganisms and their metabolites present in biofloc are immune-stimulants, which enhance immunity and are protective against several diseases [
14]. Furthermore, bacterial species are typically linked with suspended particles in biofloc, facilitating with exogenous digestive enzymes and additional nutrients, thereby contributing to bacterial growth and survival [
5]. Many studies have been conducted by utilizing several sources of carbons and their effects on several species cultured in biofloc technology, such as tapioca and plant starch for
Pelteobagrus vachelli [
8], Longan powder for Nile tilapia [
9], and wheat bran and molasses for
Litopenaeus vannamei [
12]. Additional research is required to clarify the effects of various carbon sources on the water quality, growth, immunity, and stress response of species. The current study used tapioca flour (TF) and sugarcane molasses (SM), the best alternatives to the exhausted sources, which are readily available.
Globally, tilapia farming has increased in popularity and is the second-most farmed fish species [
4]. It has a high growth rate and is a stress-tolerant species. Moreover, it is still under investigation to assess the effects of different carbon sources on the water quality, fish growth, haematology, immunity, and antioxidant status of biofloc-reared species. Therefore, the current research work was intended to assess the effect of various carbon sources (sugarcane molasses and tapioca flour) on the water characteristics, growth, hematology, and non-specific immune and antioxidant status of juvenile
Oreochromis niloticus for the duration of 10 weeks.
4. Discussion
The composite carbon source in biofloc can enhance the nutritional value of the bacterial species in the system, which can further serve as an additional source of food for fish. Thus, it can play a significant role in the improvement of body weight. This study evaluated different carbon sources for
O. niloticus reared in a biofloc system, and the results of the present research revealed that the rearing of juvenile Nile Tilapia in a biofloc system with variant carbon sources affects the water quality characteristics, growth performance, blood profile, non-specific immune response, and antioxidant status in no water exchange. Aquatic animals depend mainly on water quality to maintain their health and limit growth [
28]. The water quality parameters (temperature, DO, pH, nitrite, nitrate, NH3, and TSS) observed in the present investigation were in the appropriate range, suitable for biofloc fish farming. The present study’s findings are according to the water parameters reported by other studies [
29,
30]. Moreover, temperature is an essential factor affecting the formation and composition of biofloc [
31] and is appropriate in the present research work. The decrease in microbial activity within the flocs led to deflocculation at lower temperatures (4 °C) than at higher temperatures (18–20 °C) [
32]. According to Krishna and Van Loosdrecht [
33], stable microbial flocs might be obtained at a temperature of (25–25 °C). In this study, the temperature range for both treatments was 27.01, slightly higher than the optimum level. The current research work also revealed a significant difference in the dissolved oxygen (DO) and pH levels in the control and treated groups. However, a lower DO level and pH were recorded in the BFT + SM group. This may have been due to higher respiration by the heterotrophic microbial community. As a result of the oxygen consumption by microbes and CO
2 emission, H
2CO
3 is produced in a limited water exchange system, reducing pH levels [
2,
10]. The DO level influences the structure of aerobic flocs due to its role in the metabolic activity of cells [
34]. According to Martins et al. [
35], filamentous bacteria were more numerous than zoogloeal bacteria at DO levels (less or equal to 1.02 mg/L). The floc volume index increases with DO levels above 3.5 mg/L [
36,
37]. The floc volume observed in the current study for the tapioca meal with biofloc was 28.94 at 6.31 mg/L DO level. The physiological function of tilapia is not adversely affected by pH 4.0–8.5 [
38]. In this study, the pH value was 8.21 (control), (7.32) BFT + SM, and (7.78) BFT + TF. The treated groups had lower levels of nitrite and ammonia combined with higher levels of nitrate, which showed a greater bacteria abundance that oxidized nitrite and ammonia compared to the control group [
39]. In addition, the control treatment involved constant water exchange, so lower levels of nitrogen compounds were expected in this treatment. According to Xu et al. [
40], a change in TSS concentrations over time can serve as an indicator of the development of biofloc in aquaculture systems, which is consistent with the findings of the current study. A lower TSS in the current study was recorded in the control group compared to the treatment groups. It has been reported that, during 14 weeks of raising
Labeo rohita fingerlings, the highest TSS level (1.32840 mg/L) was recorded in a biofloc system [
41]. Similarly, Azim and Little [
42] observed the same observations for tilapia. However, in the current research, the TSS level did not exceed 351.7 mg/L. A TSS value higher than 1000 mg/L impacts the tilapia’s health.
Additionally, biofloc microorganisms contribute to sustaining the water parameters, fecal waste, and uneaten feed metabolism, and, thus, decrease the nitrogenous compounds, particularly nitrite and NH
3 [
43]. There is, however, an association between the reduction in ammonia concentrations and the development and formation of microbes in the biofloc [
6]. According to Soliman and Abdel-Tawwab [
29], the carbon source in biofloc technology improves microbial diversity, particularly ammonia-oxidizing bacteria, which decreases the NH
3 concentration. In current study, there was no evident variation in the nitrite and nitrate levels, which indicated that heterotrophic uptake was the key ammonia removal path in the system, and this is consistent with the studies that have been explained before. The biofloc volume recorded in the current study was appropriate for tilapia production [
15].
Earlier studies have shown that, due to probiotic properties, the biofloc technology system increases fish growth performance and FCR [
4,
37,
44,
45]. Similarly, the current study results show that BFT with different carbon sources improves the fish growth performance more than the control. The BFT with TF significantly increased the fish growth and provided the best FCR. This shows that microbial floc with BFT + TF acts as a supplementary diet source that provides extra protein, vitamins, minerals, and polyunsaturated fatty acids [
9,
29,
37]. The TF contain more than 90% carbohydrates, which might show better results for microbial growth as compared to SM. In biofloc technology, the FCR should be close to one, as reported in several studies such as Khanjani et al. [
39], who observed an FCR of 0.99 while using starch as a carbon source in biofloc technology for Nile tilapia fingerlings. Similar findings were obtained by García-Ríos et al. [
46] for cultured Nile tilapia fingerlings. However, in this study, the FCR value was found to be higher. This might have been due to the short experimental duration. The biofloc system represents a suitable environment for fish growth and feed utilization without affecting the water quality or survival of the fish, which revealed the significant effects of biofloc on fish growth, as reported by previous studies findings [
12,
43,
47]. The carbon source is effective for the growth performance of reared species because it enhances the floc production, including its chemical composition and volume, and can also store different bioactive compounds (carotenoids, extracellular enzymes, polymers, and phytosterols [
43,
48]. Biofloc-reared fish such as rohu (
Labeo roita) and tilapia (
Oreochromis niloticus) have previously shown a better growth performance [
49,
50], which is according to the findings of the present study. The feed comprising different ingredients represents the major production cost in commercial aquaculture; as a result, improving the efficiency of fish nutrition is a key priority. Therefore, applying the BFT system to intensive tilapia culture can be advantageous.
Hematological parameters are essential for understanding abnormalities due to health status [
51,
52]. The findings of the current study showed that the WBCs, HCT, HB, and lymphocytes were largely affected by the carbon sources and their values were greater in BFT + TF than the other groups. The increased number of WBCs was due to lymphocytes. Fish health is closely associated with the number of leukocytes, which play a significant role in innate immunity during inflammation [
53]. According to Mansour and Esteban [
15], different carbon sources improve the number of WBCs, HB, and HCT in biofloc-cultured
O. niloticus, which agrees with the current study’s findings. According to the findings of many studies, stress, environmental conditions, carbon source type or amount, aquaculture system, microbial diversity, disease, and feeding treatment affect the blood profile of cultured fish [
4,
54,
55,
56].
It has been established that innate immunity is connected to increased levels of plasma proteins, albumin, and globulin, representing the significant proteins in plasma [
57]. In the current study, all these proteins were significantly affected by the carbon sources and were higher in BFT + TF. Besides this, the lysozyme, immunoglobulin, and ACH50 levels were significantly higher in the fish cultured in biofloc technology with tapioca flour as a carbon source. The notable increase in these parameters is consistent with the findings of Mansour and Esteban [
15]. They used wheat flour and rice bran as carbon sources for biofloc-cultured tilapia. Tapioca flour (TF) contains essential minerals such as iron and potassium. Besides this, it is also considered to be a good source of vitamin B, such as riboflavin (B2) and Niacin (B3) [
58]. Vitamin B, potassium, and iron have long been known for their roles in immunity [
59]. Therefore, an increase in the immunity of the fish reared in biofloc with tapioca flour can be linked to its nutritional value.
Moreover, Verma et al. [
60] found that tapioca flour (TF) as a carbon source significantly increased the plasma proteins, globulin, and immunoglobulin levels in
Labeo rohita reared in biofloc technology. Similar findings were revealed in the current study. The lysozyme produced by fish leukocytes causes bacterial cell wall lysis, which stimulates the complement system and facilitates the phagocytosis of many pathogens [
61]. The complement activity plays a vital role in teleost’s antibacterial defense mechanism [
62]. Researchers might investigate in future studies whether the increases in the immune parameters are associated with improved defenses against disease or stressful situations.
The antioxidant status in the present study was also greater in the fish reared in the BFT with tapioca as a carbon source compared to the BFT with sugarcane molasses and control groups. In particular, the biofloc treatment significantly increased the activity of the CAT and SOD enzymes. The increased activity of antioxidants in the case of tapioca may be linked to the antioxidants present in this plant. Several studies have suggested that all possible tapioca extracts show significantly higher antioxidant activity [
63,
64]. The present research was according to the findings of other studies [
15,
18,
45]. CAT and SOD are enzymes that prevent the oxidation of lipids. The catalytic reaction of SOD produces hydrogen peroxide from superoxide anion and is further decomposed by CAT to oxygen and water to prevent lipid peroxidation [
65]. The increased SOD and CAT activity levels in the present study may be attributed to enhanced fish well-being and decreased oxidative stress.