Utilizing Alternative Carbon Sources for Biofloc System for Growth and Survival of Pacific Whiteleg Shrimp (Litopenaeus vannamei)

Abstract

Selection of a suitable carbon source is crucial in the advancement of biofloc technology. This study aims to evaluate the usability and effect of leftover bread and surplus rice as carbon source for the biofloc system for growth and survival of Pacific whiteleg shrimp (Litopenaeus vannamei). Biofloc was developed 2 weeks before stocking using probiotics (0.03 g/L) with carbohydrate (0.1 g/L) source. The experimental treatments: T0 (clear water), T1 (leftover bread), T2 (surplus rice), and T3 (50% surplus rice+50% leftover bread) replicated three times. Stocked with 12 postlarvae (PL10) weighing 0.028–0.052 g each (6PL/L), parameters were regularly monitored. During a 30-day culture period, shrimp were fed twice daily with a diet containing 55% protein, and the carbon source was maintained at a C:N ratio of 15:1. One-way ANOVA of sampling data (collected every 15 days) indicated no significant differences (p > 0.05) in growth performance, survival rate, and feed conversion ratio (FCR) across treatments. Physicochemical parameters of the water are at the optimum; the leftover bread biofloc attained more ideal parameters (dissolved oxygen: 7.54, temperature: 27.30, salinity: 29.10, and pH: 7.6) due to simpler starch structure. Poor fermentation and slow degradation properties of leftover bread and surplus rice on biofloc formation were the reason why biofloc has no effect on shrimp growth and survival. Leftover bread shows promising results among the BFT treatments; for further study with greater area and period, additional parameters are encouraged.

1. Introduction

The global consumption of fish has been drastically increasing at an average annual rate of 3.1% recorded from 1961 to 2017, which doubled the annual world population growth rate (1.6%) in the same period [1]. With this documented higher demand, aquaculture firms attempt to intensify their production. However, maximizing aquaculture produces higher aggregates of organic materials, resulting in toxic effects and long-run environmental risks [2]. Enhancing productivity is the anticipated solution and simplest method addressing this heightened demand, which is also one of the focal concerns in improving aquaculture and shrimp farming [3, 4, 5, 6].

Sustainable aquaculture enhancement and responsible use of resources realizing the U.N. 2030 Agenda for Sustainable Development Goals principally: ending poverty (SDG 1), ending hunger, achieving food security and improved nutrition (SDG 2), and promoting sustained, inclusive, and sustainable economic growth (SDG 8) [7]. Furthermore, embracing the modernization of the aquaculture industry, particularly through the economic management of biofloc systems, is crucial for local development.

Shrimp farming utilizing biofloc technology exhibits gains compared to traditional fish farming, where there is zero water exchange and more negligible ecological effect – minimized contaminant on the production system. The nitrogen compounds generated from the unconsumed 20–40% incorporated feeds were recycled and converted into bacterial biomass, which can be used as supplemental feeds [3, 8].

Subsequently, the use of biofloc shrimp is a low cost, sustainable, and environment-friendly technology. The shrimp industry has been a promising venture as market potential and sustainability supporting food security [8]. Biofloc systems have been a potential aquaculture technology improving shrimp farming, and introducing this to the locale with the available raw materials can be a capable livelihood opportunity. Moreover, biofloc technology was productively utilized in nearly 20–25% of the shrimp aquaculture farms. Most Asia’s highest shrimp production industries are adapting to biofloc – Singapore, Indonesia, Myanmar, and Malaysia [9, 10]. Shrimp farming shows a promising future through the nutrient recycling principles of biofloc technology. BFT has previously been initially tested in three farms in Luzon, two in the Visayas, and two in Mindanao, employing whiteleg shrimp (L. vannamei) as cultured species [11].

This study highlights the shrimp culture practices using biofloc technology through utilizing alternative carbon sources. The BFT has been adapted worldwide, and through this research, the locale is informed on the technology and a supplementary livelihood opportunity. The result of this study will serve as an alternative carbon source on maintaining the biofloc sources reducing production cost and at the same time reduction of wastage of leftover food. This study will serve as a baseline information to those who plan on adapting biofloc technology on culturing whiteleg shrimp. The data sought can be a guide in support to the continuous improvement of Bureau of Fisheries and Aquatic Resources and other non-government agencies on their implementation of BFT as program. The result of this research is useful to the academe in need of further studies in modifying biofloc systems as well as to the people in fisheries sector visioning for the development of shrimp industry.

2. Materials and methods

2.1 Description of the study site, experimental species, treatment, and setup

This study was conducted at Balasinon, Sulop, Davao del Sur, Philippines. The location has the convenience of available materials and equipment needed and laboratory for water parameter analysis from Davao del Sur School of Fisheries, taking into consideration the status of the COVID-19 Protocols of the locale. The experiment species used in the study were 150 specific pathogen-free (SPF) Pacific whiteleg shrimp postlarvae (L. vannamei) with an approximate length of 5.0 mm, procured from available accredited hatchery from Sta. Cruz, Davao del Sur. The 5 L containers were used with 2 L water with 12 postlarvae (PL10) [12]. Figure 1 shows the experimental setup.

Figure 1.

A. Experimental setup for biofloc system for L. vannamei with constant aeration and lighting; B. Daily water parameter monitoring for pH, salinity, dissolved oxygen, temperature, and turbidity; C. Second sampling activity measuring the weight and length of the shrimp.

The experiment was done indoors using 12 cylinder containers 15 cm in diameter and 18 cm in height with 5 L capacity filled with 2 L of seawater arranged in a Complete Randomized Design (CRD). The four treatments were replicated three times: T0 (clear water system), T1 (leftover bread), T2 (surplus rice), and T3 (bread and rice) as shown in Table 1. The containers were stocked with 15 days postlarvae at a density of 6 PLs/liter and were constantly aerated throughout the study.

Treatment	Carbon content needed (g)	Carbon content g/100 g (NHS, 2016; FAO, 199)	Carbon incorporated to maintain C/N ratio (g)
T0
T1 LB*	1.06	50	2.12
T2 SR*	1.06	30	3.18
T3 LB + SR	0.53 + 0.53	50+ 30	1.06 + 1.59

Table 1.

The different treatments used in the study.

^*

LB = leftover bread, SR = surplus rice.

2.2 Development of biofloc water, carbon addition, and feeding management

The biofloc was inoculated a week prior to the procurement of the experimental species. The process flow of developing biofloc water presented in, where there are four treatments including a control treatment and three biofloc preparation tanks: T1 (leftover bread), T2 (surplus rice), and T3 (bread and rice) that were filled with approximate 7 L seawater. The proportion of the solution was 0.1 g/L carbohydrate source and 0.03 g/L probiotics to the exact amount per treatment as shown in Table 2. The pH of the prepared water was monitored after 24 hours. Application of lime at 0.05 g/L was applied to treatment 3 with bread and rice as carbon sources as the pH level dropped below 5. Aeration was maintained to the optimum dissolved oxygen requirements 4.0 mgL⁻¹ and at least 60% of saturation [4, 13]. Carbon addition was done a week before the procurement of the seeds, and star commercial feeds were utilized following the feeding rates: 3% biomass (0–20 days of culture) and 4% feeding rate (21–40 days of culture) [14, 15]. The culture species were fed twice a day (6 am and 6 pm) with 55% protein (5% lipid and 3% fiber) Figure 2 [4, 10]. The formulation of the biofloc was computed as shown in Table 2.

Treatment	Sea water (L)	Carbohydrates; (0.1 g/L)	Probiotics (0.03 g/L)	Lime* (0.05 g/L)
T0: clear water	7	—	—	—
T1: bread	7	0.7	0.21	0.35
T2: rice	7	0.7	0.21	0.35
T3: bread + rice	7	0.35 × 2	0.21	0.35

Table 2.

The formulation of different treatments used in the study.

^*

Addition done if pH decreases below.

Figure 2.

Schematic procedure in preparing and developing biofloc water [16].

2.3 Physicochemical monitoring

Reading of the temperature, pH, turbidity, and dissolved oxygen were done and carried out at 7:00–8:00 am and 4:00–6:00 pm. Measuring ammonia and observing the biofloc through microscope were done every week. The physico-chemical parameters: dissolved oxygen, pH, temperature, and salinity using a 5-in-1 multifunction water quality test probe, while turbidity was measured through identifying Secchi depth using Secchi disk (four-inch diameter). Ammonia was measured by a colorimetric ammonia kit [17].

2.4 Sampling and data gathering

Growth measurement was done every day after 15 days, and the increase in total length and weights were determined. The total length of shrimp was determined using ruler, while weight was determined using the electronic weighing scale. Sampling was conducted in the morning. Samples of five shrimps per container were taken. The shrimps were held in restraint container and then measured as soon as possible to prevent the stocks from being stressed. The total length was measured from the tip of the rostrum to the tip of the [18]. The shrimps were weighed individually. After the sampling, the shrimp will be immediately returned to the containers. The procedure was done simultaneously per individual container to minimize stress on the cultured organisms [17].

2.5 Statistical analysis

Data were statistically analyzed using a one-way ANOVA at 5% significance level to identify significant difference using Statistical Package for Social Sciences (SPSS) version 22.

3. Results

3.1 Growth performance

The use of different biofloc carbon sources in shrimp culture (L. vannamei) using biofloc systems obtained comparable results among all treatments (p > 0.05) in terms of growth parameters (weight and length increments and specific growth rate). Among the BFT treatments, the leftover bread has a higher final mean weight and length output (0.180 g ± 0.129; 3.139 cm ± 0.668). The same trend was observed on specific growth rates where all treatments were comparable as shown in Table 3.

	T0 – CW	T1 – LB	T2 – SR	T3 – LB + SR
IBW	0.040 ± 0.018	0.037 ± 0.012	0.033 ± 0.016	0.027 ± 0.013
ITL	0.593 ± 0.234	0.620 ± 0.174	0.527 ± 0.205	0.440 + 0.118
FTW^ns	0.242 ± 0.135	0.180 ± 0.129	0.166 ± 0.082	0.144 ± 0.052
FTL^ns	3.250 ± 0.625	3.139 ± 0.668	2.389 ± 0.772	2.528 ± 0.475
SGR^ns	0.68	0.48	0.44	0.39
DGR^ns	0.023	0.016	0.015	0.013

Table 3.

Growth performance of Pacific whiteleg shrimp postlarvae (Litopenaeus vannamei) grown on biofloc systems with different carbon sources for the 30 days culture period.

Mean values (±SD) in the same row are not significantly different (ns); p > 0.05. Abbreviations used: IBW = initial weight (g); ITL = initial total length (cm); FTW = final weight (g); FTL = final total weight (cm); SGR = specific growth rate (%); DGR = daily growth rate (%); CW = clear water; LB = leftover bread; SR = surplus rice.

3.2 Survival rate

The effect of different treatments on the survival of L. vannamei reared in biofloc system is presented in Table 4. The highest survival rate (58.33%) was observed in treatment 1, while the lowest was attained in treatment 2 (47.22%) on the biofloc system with surplus rice as carbon source. But statistically, the survival rate of the different treatments was not significantly different (p > 0.05).

Treatment	Number of stock	Survival^ns	Percentage (%)^Ns
0CW	36	19	52.78
ILB	36	21	58.33
IISR	36	17	47.22
III SR + LB	36	18	50

Table 4.

Survival rate of pacific whiteleg shrimp postlarvae (Litopenaeus vannamei) grown on biofloc systems with different carbon sources.

Values in the same column are not significantly different (ns) p > 0.05. Abbreviations used: CW = clear water; LB = leftover bread; SR = surplus rice.

3.3 Feed conversion ratio

Table 5 shows the final mean FCR of Pacific whiteleg shrimp postlarvae (Litopenaeus vannamei) for 30 days of culture. Results showed that the lowest feed conversion ratio in treatment 1 obtained 2.208 with leftover bread as carbon source among the treatments. Highest feed conversion ratio in treatment 2 was obtained in those Pacific whiteleg shrimp postlarvae that were grown on the biofloc system with surplus rice as carbon source, but statistically there were no significant difference, and all treatments were comparable (p > 0.05).

		Sampling period
Treatment		Initial sampling to 1st sampling^ns	1st sampling to 2nd sampling^ns	Average^ns
0	CW	0.23	0.40	0.32
I	LB	0.31	0.32	0.32
II	SR	0.28	0.44	0.36
III	SR + LB	0.26	0.44	0.35

Table 5.

Feed conversion ratio of Pacific whiteleg shrimp postlarvae (Litopenaeus vannamei) grown on biofloc systems with different carbon sources.

Values in the same column are not significantly different (ns); p > 0.05. Abbreviations used: CW = clear water; LB = leftover bread; SR = surplus rice.

3.4 Physicochemical parameters

Results showed that there was a statistically significant difference in dissolved oxygen levels on biofloc systems for shrimp using alternative carbon sources (p < 0.005). The level of dissolved oxygen in treatment 1 is significantly higher than in treatment 2 and treatment 3, and the difference is statistically significant. Figure 3 shows a declining trend of dissolved oxygen level; however, all the treatments were within the normal range for biofloc system.

Figure 3.

Mean dissolved oxygen (mgL⁻¹) levels of biofloc systems with different carbon sources for culturing Pacific whiteleg shrimp postlarvae (Litopenaeus vannamei).

The water temperature range in tanks was observed at 26.51–27.62°C for the entire duration of the experiment. Water temperature was highest during the fourth week of the experiment, with treatment 1 where carbon source with leftover bread as the highest (27.72 ± 0.72°C) and carbon source with treatment 2 where combined surplus rice and leftover bread (27.41 ± 0.65°C) as the lowest. The recorded value shows significant difference (p < 0.05), yet all treatments attained the normal range for temperature for biofloc (Figure 4). However, the consequent week shows a drop of temperature in the biofloc systems with the means (T0: 27.08 ± 0.73°C; T1: 27.62 ± 0.92°C; T2: 27.36 ± 0.78°C; T3: 27.16 ± 0.72°C).

Figure 4.

Mean temperature (°C) of biofloc systems with different carbon sources for culturing pacific whiteleg shrimp postlarvae (Litopenaeus vannamei).

The water salinity shows an inclining trend during the 30 days of experiment. However, in treatment 0, clear water system recorded fluctuating ppt levels, where in the first week, it started to decline and then rise on the fourth week as shown in Figure 5. The lowest mean salinity was 26.90 ± 0.68 ppt during the third week on treatment 0. The salinity measurements in the first week of the experiment ranged at 28.08 ± 0.59–28.56 ± 0.78 ppt and rose at the levels 29.07 ± 0.49–30.56 ± 0.48 ppt.

Figure 5.

Mean salinity (ppt) of biofloc systems with different carbon sources for culturing Pacific whiteleg shrimp postlarvae (Litopenaeus vannamei).

The level of pH on the control treatment is significantly higher among the biofloc treatments; however, the pH of leftover bread biofloc is significantly higher among other treatments of carbon sources. The level of pH level in T0 is significantly higher than in T1, T2, and T3, while T1 is significantly higher than T2 and T3. Shown in Figure 6, the recorded pH levels indicate they are within the ideal range for shrimp biofloc systems. The pH levels show a gradual acid transition with lowering of pH measurements. In first sampling, treatment 3 recorded as the most acidic with mean 7.44 ± 0.08 during day and 7.39 ± 0.42 during night, while on the second sampling, treatment 2 shows more acidity than the other treatments with mean of 7.26 ± 0.20 and 7.26 ± 0.15 at night and day, respectively.

Figure 6.

Mean pH of biofloc systems with different carbon sources for culturing Pacific whiteleg shrimp postlarvae (Litopenaeus vannamei).

Figure 7 shows the trend of turbidity on the experimental biofloc systems. There is significant difference (p < 0.05) in the turbidity of L. vannamei biofloc system with different alternative carbon sources applied. The level of turbidity in T0 significantly higher than in T2 and T3, while T1 is significantly higher than T2 and T3, and T3 is significantly higher than T2. Obviously, the control treatment is significantly less turbid than the biofloc system, while the most turbid biofloc treatment is the surplus rice and the least turbid biofloc system is the treatment with leftover bread as carbon source followed by the 50% surplus rice +50% leftover bread, and the differences are statistically significant.

Figure 7.

Mean turbidity (cm) of biofloc systems with different carbon sources for culturing Pacific whiteleg shrimp postlarvae (Litopenaeus vannamei).

The maximum ammonia measurement observed was 0.51 ± 0.0 mgL⁻¹, while the minimum observed ammonia was 0.18 ± 0.2 mgL⁻¹. The flow of pattern in ammonia level did not follow of any regular pattern as demonstrated in Figure 7. The biofloc systems as culture medium were within the safe threshold (mgL⁻¹) (Figure 8) [19].

Figure 8.

Mean ammonia (ppm) of biofloc systems with different carbon sources for culturing Pacific whiteleg shrimp postlarvae (Litopenaeus vannamei).

4. Discussion

The complex carbohydrates needed more digestion duration and less fermentation time required [20]. Leftover bread was easier to breakdown, hence easier for bacterial aggregates and manageable parameters to develop as to compare the structure of surplus rice and the combination of CR + LB [19, 21]. The inadequate fermentation of biofloc with leftover bread and surplus rice has no significant difference as carbon source as these are classified as complex starches with longer duration to be fully fermented [19, 22]. Hapsari [23] also observed that weight gain was better with fermented biofloc compared to a clear water system and unfermented biofloc, with no significant difference between the latter two.

The BFT treatments using the experimental carbon sources manifested sticky and gel-like texture due to its soluble proteins. The occurrence of floc attaching on the part of the container wall on the BFT treatments caused the decomposition process to not maximally compare to clear water systems making it unable to achieve its optimal yield [24, 25]. Monosaccharides with less protein and lipids attained the best increase in biomass in BFT farming, and this can be the reason for low value output from the more complex starches [21, 26]. Combining carbohydrates affects its digestibility base on its glycemic index and can reduce its conversion to simpler sugar, and hence, it can explain the observance of low mean weight gain on the LB + SR carbon source biofloc system [21, 27].

Fermentation was done for a short period of time, aggregates were not fully matured as mentioned by Ogello et al. [28], small quantities of bacteria cannot form substantial flocs in the culture system, and this can further explain the high yield on leftover bread as simpler starch; however, the combination of these carbohydrates can drastically affect its digestibility and reducing it, making it difficult to convert into simpler sugars [27, 29] relating this to the low growth of biofloc systems with 50% leftover bread + 50% surplus rice.

High mortality rate was evident in the experiment. This could be due to cannibalism, which is induced by the absence of hides. This is a problem for L. vannamei species in the nursery and early juvenile stages, which is understandable given their high metabolic rates and frequent molting [30]. The issue of low survival may also be linked to natural response to captivity, lack of shelter, ecdysis, increased density, lack of food, and poor water quality [31].

The parameters were maintained within the normal range; however, the significant difference in turbidity between treatments can be connected to the survival yield between the BFT systems and the clear water systems. Comparing the results, the turbidity of the BFT system using only leftover bread as a carbon source was 9.94 cm, and it had a higher survival rate (58.33%) compared to the other treatments. As explained by Kathyayani et al. [32], high water turbidity in farming of Litopenaeus vannamei is one of the environmental stressors that hinder survival of cultured species due to gills blockage as a compensatory reaction to the disruption of osmotic and ionic balance produced by high turbidity in the rearing medium.

Simpler starches, such as leftover bread, are easier to digest and convert into biomass compared to complex chains [26, 33]. In the context of this study, surplus rice facilitated experimental investigations into different carbon sources in biofloc systems, where simpler sugars resulted in slight improvements in survival rate, FCR, and water quality [34, 35, 36, 37]. The choice of carbon sources is critical to the biofloc system’s performance, as it can affect the availability, palatability, and digestibility for the cultured species [20].

It was also mentioned by Peiro-Alcantar et al. [38] that despite on the desirable water quality, survival, and growth yield of the biofloc, there were no perceived significant differences among treatments and the control. Another biofloc study by Arias-Moscoso et al. [39] revealed that while the microbial community’s dependence on floc palatability is influenced by the carbon sources used, this palatability affects the metabolization of harmful nitrogen compounds. However, the study found that probiotics have no effect on crop production.

The observed dissolved oxygen and temperature of all the recorded value during the whole duration of the study were still within the tolerable range ideal for biofloc culture system [4]. This could be caused by the simpler starch structure of bread compared to rice which is simpler to digest and attain optimal parameters [24, 29, 40]. The optimum temperature for the growth of microorganisms is around 28–32°C that was manifested on biofloc with leftover bread carbon source. Thus, more microorganisms thriving on the culture medium are consequences of the greater metabolism [19].

Low dissolved oxygen on biofloc with combined rice and bread is possible because of the digestibility characteristics of the combined carbon source [27]. This can be due to the metabolic activity of the heterotrophic and autotrophic components of the biofloc formed and more microorganism thriving on the culture medium as consequences of the greater metabolism. The leftover bread is simpler starch; then, it can be digested easier and able to facilitate a more desirable BFT system to better yield as can be seen on its survival output [24, 26].

Betanzo-Torres et al. [41], Manan et al. [42], and Ray et al. [43] pointed out that the presence of planktons consuming the available dissolved oxygen in the medium the levels was differentiated due to the difference of carbon source, whereas the simpler starch yields high in terms of its consumability, yet the D.O levels were maintained with the constant aeration as requirement for biofloc sustenance [3, 10].

The complex starch structure of rice matched to bread which is easier to convert simpler sugar [24, 29, 40]. Becerril-Cortés et al. [19] observed that the optimal temperature for the growth of microorganisms is around 28–32°C. This was also confirmed in an experiment using a biofloc system with leftover bread as the carbon source, which resulted in favorable survival rates. This can be associated with Nugroho et al.’s study [44] which states that temperature differs for colonies of various compositions, different algae species, and different groups at their ideal temperature. The biomass of community phytoplankton exposed to various temperature settings is influenced by abiotic factors. The limited water exchange might be the reason of increasing salinity levels, and this can be associated with evaporation loss though. This is controlled by adding water to conform to the setup stocking density [38, 43, 45].

Different salinity can be derived from the level of complexity of polysaccharide properties of bread and rice due to the saturation of the water evaporation which slows down, and hence the salinity increases [24, 40, 46]. The disparities which observed salinity levels of culture systems sought no significance was found in terms of survival and growth performance. The use of more complex starches caused slow degradation; hence, metabolic systems affect the parameters of the culture medium. The presence of organic matter from unfermented floc from complex starches gained more H⁺ ions making it slightly acidic than others, as to be observed on the leftover bread and compared to the control, rice, and its combination (rice and bread) [21, 24, 47]. However, recorded levels are within the normal range.

Leftover bread as carbon source gained a lesser turbid biofloc among the biofloc treatments,and this can be connected to the degradation properties of the used carbon source sought by El-husseiny et al. [47] stating that complex starch tends to have slow degradation, and hence, high total suspended solids and salinity follows. The turbidity of the biofloc treatment with leftover bread was less turbid among the other treatments depicted in Figure 9. As discerned by Kathyayani et al. [32], raised water turbidity in the intensive farming of Litopenaeus vannamei is one of the environmental stressors due to gills blockage as a compensatory reaction to the disruption of osmotic and ionic balance produced by high turbidity in the rearing medium. Biofloc with moderate turbidity and floc aggregate density are more reliable in L. vannamei growing systems – favorable element in the maintenance of efficient and stable production [40].

Figure 9.

Images of biofloc from different carbon sources on their fourth week of DOC, seen 20× magnified under the compound microscope. (A1–2. T0 – clear water system, B1–3. T1 – leftover bread, C1–3. T2 – surplus rice, D1–3. T3–50% surplus rice +50% leftover bread).

Minimal variation in ammonia observed in all treatments during the whole duration of the study. The values were all within the ideal range (1 mgL⁻¹) [4]. The current study shows that the supplementation of different carbon source in biofloc system has no effect on the ammonia levels on culturing whiteleg shrimp. The short duration of fermentation and the complex polysaccharide structure of the carbon source can be the reasons [24, 29, 47]. The reason of lesser area used can also affect the development of biofloc and the supposed progress of nitrification of ammonia for microbial aggregates [28].

5. Conclusion

The current study recommends that the use of leftover bread and surplus rice is recommended as alternative carbon sources in biofloc systems. Further investigation for leftover bread as probable carbon sources is recommended. Conducting a similar study using a higher stocking density for a longer duration of culture period and other physico-chemical parameters is recommended to validate the result of this study.

Acknowledgments

The author acknowledges the support of Institute of Fisheries and Marine Sciences of Southern Philippines Agribusiness and Marine and Aquatic School of Technology, Bureau of Fisheries and Aquatic Resources – XI, and Davao del Sur School of Fisheries.