Effect of different carbohydrate sources on water quality and growth performance of GIFT tilapia, Oreochromis niloticus (Linnaeus, 1758) fry in biofloc system

Introduction

Aquaculture additionally called aquafarming, is the farming of fish, crustaceans, molluscs, aquatic plants, algae and other organisms. It's a food manufacturing sector that offers ample opportunities to alleviate hunger, poorness and nutrient deficiency diseases. It additionally generates economic process and ensures higher use of natural resources (FAO, 2026) [1] . There is necessity to increase aquaculture production to satisfy the increasing demand per capita and increasing global population. In this world, more than 800 million people still suffer from chronic malnutrition. By 2050, the global population is predicted to grow by another 2 billion to 9.6 billion people. A huge challenge is to safeguard these natural resources for future generations. The development of sustainable aquaculture industry is challenged by the availability of limited natural resources as well as the impact of the industry on the environment. With these limitations in mind, the development of sustainable aquaculture industry should focus on the conceptualization of systems that utilize fewer resources including water, space, energy, capital, and at the same time has lower impact on the environment. One of the strategies to improve aquaculture production and sustainability is to focus on enhancing feed nutrient utilization. This may be developed by two different approaches, i.e. (i) by increasing the feed quality and feeding strategy so that the nutrients can be efficiently delivered and finally utilized and (ii) by re-utilizing the nutrient waste through modifications within the culture system. In an aquatic system, nutrient waste can be removed by various numerous natural biogeochemical processes involving mostly microorganisms with various functions in nutrient cycles.

The nutrient waste in an aquaculture system is mostly the undigested feed and faeces. This nutrient waste can be reutilized directly by the lower tropic level organisms, which can utilize feed particles as their food source, or indirectly by the conversion of the nutrients into microbial biomass that may eventually be consumed by the cultured animal itself or other animals as their food source. Biofloc is as an environmentally friendly aquaculture technique which is mainly based on the principle of recycling waste nutrients particularly nitrogen into microbial biomass that can be used by the cultured animals or be harvested and processed into feed ingredients (Avnimelech, 2007;Kuhn et al., 2009) [6, 21] . BFT is considered as a new "blue revolution" because nutrients can be continuously recovered and reused in the medium, which benefits from minimal or zero water exchange. Biofloc plays an important role in nutrient recycling and maintaining water quality. Shrimp or fish consuming bioflocculants has shown many benefits, such as increased growth rate, reduced FCR and related feed costs. Microorganisms play a key role in BFT systems mainly heterotrophic bacteria. The main characteristics of this system are high stocking density, heavy mixing, intense aeration, low or no water exchange and high organic matter input (Schveitzer et al., 2013). Mainly these systems are developed to improve the environmental conditions, feed efficiency and immunity (Crab et al., 2012) and to conserve water. The microbiota present in the system can easily take up these nitrogenous by-products, which can serve as a substrate for operating biofloc systems and production of microbial protein cells (Mishra et al., 2008). Heterotrophic microbiota is stimulated to grow by maintaining the C/N ratio in the water by the modification of the carbohydrate content in the feed or by the addition of an external carbon source in the water (Avnimelech, 1999), so that the bacteria can assimilate the waste ammonium for new biomass production. High C:N ratio helps to convert ammonium and organic nitrogenous wastes to bacterial protein biomass (Avnimelech et al., 1994;Avnimelech, 1999;Schneider et al., 2005). Therefore, in biofloc technology, an addition of carbon sources directly to the pond water is done to promote multiplication of heterotrophic bacteria (Crab et al., 2007;Uddin et al., 2007). Hence, ammonium or ammonia can be maintained at a low and non-toxic concentration, and no water exchange is required. On addition of carbon, microorganisms in biofloc use nitrogen for maintenance and multiplication. Carbon source is an important input in the biofloc system that directly affect the growth of total heterotrophic bacteria. Several studies have been conducted to determine the impact of carbon sources such as glucose, acetate and glycerol (De Schryver et al., 2008), wheat flour (Azim & Little, 2008), tapioca starch and maize flour (Asaduzzaman et al., 2010), sucrose (Gao et al., 2012), maize flour (Liu et al., 2014) on development of biofloc and growth of the culture species. The results of these studies showed a similar impact of various carbon sources on biofloc development and growth of culture species [11, 25, 5, 7, 5, 30, 10, 34, 13, 8, 20, 19, 23, 3] . Tilapia is very suitable for biofloc systems. This filter feeding herbivore adapt to the bioflocculants suspended in water and therefore this robust, stable fish can grow and flourish in dense systems. The basic characteristics of biofloc tilapia production system, particularly as compared with the shrimp system, is very high biomass. According to the author's experience, the biomass of tilapia can reach 200-300 metric tons/ha, whereas shrimp biomass is about 20 metric tons/ha in well-managed pool (Avnimelech, 2011). Tilapia showed a good growth in biofloc systems. Tilapia in the BFT regime showed the highest body weight (BW), the longest body length (BL), the best weight gain (WG) and specific growth rate (SGR) and consumed the highest amount of feed and showed the best feed conversion quantitative relation (FCR) and protein efficiency ratio (PER) compared to the opposite over-wintering regimes and control (Soltan et al., 2015).The role of locally available different carbohydrate sources, on growth, survival and water quality improvement while rearing of GIFT tilapia fry is not known. Hence, the present study was undertaken to evaluate the effect of locally available different carbon sources on rearing of GIFT tilapia fry along with its effect on growth performance, survival and for improvement of water quality [3, 32] .

Materials and methods

Experimental fish and design

Fry of GIFT tilapia were procured from RGCA, Vijayawada, Andhra Pradesh. Experiment was carried out in 100 L circular HDPE containers, covered with net (mesh size 0.5 mm) at the wet laboratory of College of Fisheries, Shirgaon, Ratnagiri, Maharashtra, India. Experimental design was Completely Randomized Design (CRD) with a total of four treatments among them three treatments were of different carbon sources i.e., molasses (T 1 ), sugar (T 2 ) and jaggery (T 3 ) along with a control (T 0 ), each having five replicates. The fry weighing about 1.19 ± 0.2 g were stocked at 25 number in each tank. The fish were fed with commercial floating feed having 35% dietary protein. Feeding rates were fixed at 10%, 8%, and 4% of the total biomass and adjusted at 30 days intervals after sampling. Daily feed ration was split into three times (40%, 20% and 40%) and fed at 09:30, 13:30 and 17:30 h.

Biofloc Development and C/N ratio

Biofloc was developed in three 500 L capacity tanks as per the method given by Avnimelech (1999) and was maintained at a higher TSS level throughout the experiment which served as source of inoculum for the experimental tanks. At the start of the experiment, 60 L of biofloc and 40 L of freshwater were added to all the experimental tanks. A C/N ratio of approximately 15:1 (Mirzakhani et al., 2019) was maintained according to method described by De Schryver et al. (2008) [5,24,13] .

Physico-chemical water parameters

Water quality parameters such as temperature (°C), pH, dissolved oxygen (mg L -1 ), free carbon-dioxide (mg L -1 ), total alkalinity (mg L -1 ), total hardness (mg L -1 ), ammonianitrogen (NH 3 -N) (mg L -1 ), nitrite-nitrogen (NO 2 -N) (mg L - 1 ), nitrate-nitrogen (NO 3 -N) (mg L -1 ), and total suspended solids (mg L -1 ) were recorded fortnightly as per the standard methods given by Boyd (1981) and APHA (2005). Floc volume (ml L -1 ) was measured using Imhoff cone [9, 1] .

Growth parameters

To evaluate the growth and performance of the experimental animals, some parameters were considered. Length and weight of all the fishes were recorded prior to stocking. Growth and survival of all the survived fishes was recorded after every 15 days to the end of the experiment. Length of fry was measured in centimetre using a measuring scale with 0.1 mm accuracy. Gross weight of fry was taken by using electronic balance (Contech, CT-1501). The total number of survived fishes was counted during each sampling to determine the survival. Length gain (%), weight gain (%), survival (%) and specific growth rate were calculated as follows Lengthgain (%) = Final length-Initial length/Initial length x 100 (Rahman & Marimuthu, 2010) [27] Weightgain (%) = Final weight-Initial weight/Initial weight x 100 (Tolussi et al., 2010) Survival (%) = Total number of survived fishes/Initial number of fish x 100 (Tolussi et al., 2010) [7] Specific growth rate = In W2-W1/T x 100 (Tolussi et al.,

2010)

Statistical analysis

All data on length, length gain, weight, weight gain, specific growth rate and survival were analysed by One-way ANOVA. Differences were considered significant at (p<0.05). If difference was found significant, the means were compared by Duncan. The statistical analysis was performed using SPSS 16.0.

Results

Physico-chemical water parameters

The temperature, pH, dissolved oxygen, free carbon dioxide, total alkalinity, and hardness values were within the optimal range throughout the experiment. The water parameters are presented in Table 1. and Figure 1. No significant difference was found among the mean values of all the treatments for temperature, alkalinity and pH. Dissolved oxygen was found lower in T 1 (5.44 mg L -1 ) and T 2 (5.48 mg L -1 ) and no significant difference was found between these two treatments and a significant difference was found between T 3 and T 0, with the highest dissolved oxygen in T 0 (6.04 mg L -1 ). The highest free CO 2 (p<0.05) was found in T 1 (4.69 mg L -1 ) when compared to all other treatments. The lowest mean value for hardness was found in T 0 (p<0.05) when compared to other treatments. The mean value of ammonia nitrogen was highest in control (0.28 mg L -1 ) and no significant difference was found between all the treatments. Lowest mean value of nitrite nitrogen was observed in T 0 (0.18 mg L -1 ) while highest mean value was found in T 3 (0.23 mg L -1 ) and no significant difference was found among all the treatments. The mean value of nitrate nitrogen was found significantly lower (p<0.05) in T 0 . Significantly higher (p<0.05) mean value of floc volume and TSS was observed in T 1 (18.23 ml L -1 and 267.54 mg L -1 ) respectively.

Growth parameters

The effect of different carbon sources on the growth performance of GIFT tilapia fry is presented in Table 2. and Figure 2. The final length (10.60 ± 0.09 cm), weight (15.80 ± 0.10 g), length gain (%) (144.42 ± 3.47), weight gain (%) (1360.20 ± 94.09), specific growth rate (2.97 ± 0.07) and survival (%) (91.20 ± 1.50) are found to be significantly higher in T 1 followed by T 3 , T 2 and T 0 treatments. No significant difference between T 2 and T 3 were found for final length and lowest (p>0.05) mean value for final length was observed in T 0 . All the treatments showed significant difference (p<0.05) for final weight, while the lowest mean value for final weight were found in T 0 . And for length and weight gain (%) no significant differences were found between T 2 and T 3 and between T 2 and T 0 . For specific growth rate no significant (p>0.05) differences were observed between T 1 and T 3 and between T 2 and T 0 . Significantly less survival was found in T 0 and no significant difference was found between T 3 and T 2 and between T 3 and T 1 .

Discussion

Water Quality Parameters

The results of this study revealed that use of different carbon sources for rearing GIFT tilapia fry in biofloc system affects water quality, growth and survival. Water quality directly affects the well-being of fishes. Water quality parameters crucial for the growth of tilapia remained consistently within optimal physiological thresholds across all biofloc treatments, irrespective of the specific organic carbon input used. The temperature recorded throughout the experimental period was in optimal range for tilapia fry. At the start of the experiment, pH was slightly less than 7.0, it was elevated and maintained above 7.0 by adding alkaline water during water exchange and when required. The pH values were lower as compared to control in biofloc treatments (Rajkumar et al., 2016). The drop in pH was attributed to addition of acid by the activity of nitrifying bacteria in biofloc system (Hargreaves, 2013). In the present study we also observed lower pH in all the experimental tanks expect in the control. This may probably be due to the addition of carbon sources into the water, which reduced the pH and increased microbial metabolism for decomposition of organic matter in biofloc treatments. Dissolved oxygen levels in all experimental tanks were maintained near optimal range i.e., above 4.0 mg L -1 . The DO level was appropriate because there was a 24h continuous aeration. Highest free CO 2 was found in T 1 which may be because of the respiration of fish and microbes of the biofloc. The total alkalinity in the experimental tanks fluctuated throughout the experimental period. This fluctuation could be due to the acid added by the activity of the nitrifying bacteria in biofloc system (Hargreaves, 2013). In addition, heterotrophic process also consumes alkalinity in biofloc system (Ebeling et al., 2006). The decrease in alkalinity may be the cause for the decrease in pH levels (Hargreaves, 2013). The total alkalinity and total hardness levels as observed in this system were in the optimal range for tilapia fry (de Holanda Cavalcante et al., 2012). The level of ammonia-nitrogen in all experimental tanks increased and reached peak levels on day 45 in T 1 and T 2 and on day 30 in T 3 and T 0 and then decreased till the end of the experiment. The increase in ammonia-nitrogen at the beginning of the culture may be due to less microbial load and their inability to convert the existing ammonia nitrogen into microbial biomass. Increment in the ammonia-nitrogen may be also due to growing biomass, high feeding rate, feed input and excretion of ammonia from fishes. In subsequent periods, ammonia-nitrogen may be used by heterotrophic bacteria, which can reduce ammonia-nitrogen. But the ammonianitrogen was within the optimum range (Liu et al., 2018). Fluctuations in NO 2 -N and NO 3 -N content were observed throughout the experimental period. These fluctuations are due to nitrification by nitrifying bacteria and their assimilation by heterotrophic bacteria or occurrence of denitrification in the flocs settled at the bottom of tanks (Kamilya et al., 2017). The range of nitrite-N and nitrate-N observed in the present study was suitable for culture of tilapia (less than 1 mg L -1 for nitrite and 0.5-20 mg L -1 for nitrate) (Emerciano et al., 2017). The floc volume was observed to be in the favourable range (5 to 25 ml L -1 ) (Liu et al., 2018). The floc volume increased from the start of the experiment to the end of the experiment. Total suspended solids (TSS) were kept in the range of 129-352 mg L -1 in the present study. Initially TSS was low which increased with progress in culture period and then it was kept less than 400 mg L -1 (Long et al., 2015). A TSS of 200 -500 mg L -1 is sufficient for good system functionally and control ammonia without excessive water respiration (Hargreaves, 2013). So, adding carbohydrates helps grow a diverse and balanced bacterial community in the zero-water exchange system. This active bacterial population keeps the water clean and within safe limits for tilapia farming [20, 14, 12, 17] .

Growth Parameters: The maximum length was observed in molasses (T 1 ) at the end of the experiment, while the lowest was observed in control (T 0 ). Similar results were also obtained in tilapia growth by Rono et al. (2018). Molasses was found to be better among the carbohydrates used by Ekasari et al. (2014) for shrimp growth. Molasses contains simple soluble sugars (sucrose, glucose, and fructose) paired with more complex organic by-products. This specific chemical matrix ensures a steady, sustainable release of carbon, minimizing the risk of a secondary carbon spike or bacterial starvation between doses and oxygen depletion (Li et al., 2023) [29, 15, 22] . Growth performances results showed that biofloc enhances the growth performances of the tilapia juveniles in indoor condition. Azim and Little (2008) also reported the similar findings with different cultured organisms. The higher growth performance in biofloc treatments may be due to high feed intake i.e., both feed and biofloc as compared to control. The maximum length gain (%) was observed in molasses (T 1 ), while the lowest was observed in control (T 0 ). Similar results were also obtained in tilapia growth by Rono et al. (2018). The bacteria contribute to the maintenance of growth and survival of Nile tilapia by acting as a probiotic (Perez Fuentes et al., 2018). The maximum average weight and weight gain (%) was observed in T 1 while T 0 showed the lowest average weight. In the treatment of the molasses supplement system, the highest growth performance was recorded, this may be due to the high load of microorganisms and traces of molasses in the system can bind some of suspended particles and make them useful and available as source of food for cultured species (Rono et al., 2018). The maximum SGR was observed in T 1 while T 0 showed the minimum SGR. So better SGR was observed in biofloc system when compared to the control. This finding confirmed the results of different researchers who reported that the BFT system improved tilapia growth (Azim & Little, 2008;Avnimelech & Kochba, 2008). The increase in growth performance in the BFT treatment may be partly due to the maintenance of the optimum water quality and the continuous supply of nutritious floc, the richest source of many essential fatty acids (Ekasari et al., 2015;Toledo et al., 2016). Many biologically active compounds are evaluated in biofloc such as carotenoids, chlorophyll and phytosteroids which are expected to contribute better growth of cultured organisms in biofloc system [8, 29, 26, 29, 8, 4, 16] . 7.48 ± 0.29 a (7.1 -7.9) 7.54 ± 0.30 a (6.9 -7.9) 7.49 ± 0.30 a (6.9 -7.9) 7.54 ± 0.28 a (7.0 -7.9) Dissolved oxygen (mg L -1 ) 5.44 ± 0.24 a (5.1 -6.

Conclusion

This study demonstrates that implementing biofloc technology (BFT) using different carbon sources significantly enhances water quality, growth performance, and survival rates of GIFT tilapia fry compared to traditional control systems. Throughout the experimental period, vital water quality parameters including temperature, dissolved oxygen, total hardness, and total alkalinity consistently remained within the optimal physiological thresholds required for healthy tilapia cultivation. Although biofloc treatments experienced expected drops in pH and fluctuations in alkalinity due to acid production from nitrifying bacteria and microbial respiration, the parameters were successfully managed. Total suspended solids and floc volumes stayed within favorable limits. Furthermore, initial increases in ammonia-nitrogen, nitrite-N, and nitrate-N eventually stabilized as thriving heterotrophic and nitrifying bacterial communities efficiently assimilated and reduced toxic nitrogenous compounds. Among the evaluated carbon inputs, molasses (T 1 ) emerged as the most effective carbon source. Its unique chemical matrix provides simple soluble sugars alongside complex organic by-products, ensuring a stable carbon release that prevents bacterial starvation and oxygen crashes. Consequently, the molasses-supplemented system achieved the highest average weight, maximum length gain, and superior Specific Growth Rate (SGR). This enhanced performance is attributed to optimum water quality maintenance and the continuous availability of nutritious, probiotic-rich biofloc aggregates as a supplemental food source. In conclusion, utilizing carbon sources like molasses in zero-water exchange systems promotes a balanced microbial ecosystem that keeps culture water clean while maximizing tilapia growth metrics.

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