The recirculated aquaculture system (RAS) development with nanobubble application to improve growth performance of grouper fish fry culture Pengembangan recirculated aquaculture system (RAS) dengan aplikasi

One of the aquaculture commodities with high economic value is grouper fish (Epinephelus sp.). RAS is known as one of superior and suitable aquaculture systems in juvenile fish culture. RAS installed with NBs is expected to increase the stocking density and production of hybrid brown-marbled grouper. This study aimed to analyze the system performance of grouper fish juvenile culture in high stocking density with water exchange system, RAS, and combination of RAS and NBs. This study used a factorial design with two factors, namely different stocking densities and cultivation systems. The densities were 500, 600 and 700 fish/m3, while the treatment systems were RAS without NBs, RAS installed with NBs, and control treatment with 200% water change. Each treatment was replicated three times. The total aquaria used for this study were 27 as each size was 1.5 m × 0.5 m × 0.5 m. The study results showed that the RAS installed with NBs and a stocking density of 600 fish/m2 showed the best results on fish production performance.


INTRODUCTION
Fisheries sector, including aquaculture, became one of the economic sectors that contributes to the National Gross Domestic Product in 2019 at 6.25% or Rp 62.24 trillion as higher than in 2018 at 4.83% or Rp 58.58 trillion. One of the aquaculture commodities that has high economic value and export-oriented product is grouper fish (Epinephelus sp.). This fish is exported alive to China, Singapore, Hong Kong, and Japan. The production center for grouper fish grow-out in Indonesia is distributed in several regions, namely Riau Islands, North Sumatera, Bangka Belitung, Jakarta, East Java, Bali, Maluku etc. This value chain condition above causes issues and problems of living fish transport, followed by time and place management that becomes important. Period and transport density, followed by water movement, waste accumulation, and packaging procedure influence the transported fish stress response (Shabani et al., 2016;Hong et al., 2019). The optimal time for living fish transport, mainly through air transport mode causes a repackaging and/or temporary transport, which causes an additional cost.
In grouper fish fry culture, the fish transport cost can be minimized by transporting the fish in smaller size and higher stocking density. These small fish are initially reared in juvenile culture system before entering the grow-out system using tank and floating net cage (Akbar et al., 2012;Effendi et al., 2021). Recently, a recirculation system has been developed or known as a recirculated aquaculture system (RAS) with less water consumption, high stability level that can be controlled, and the location is closed to the consumers. RAS is a culture system that recycles water through several filters and components to improve the water quality and reuse the water (Losordo et al., 2009). Nanobubbles (NBs) are air bubbles with diameter less than 200 nm that can live longer and more stable in the water as the liquid internal pressure is higher than the environment which accelerates solubility (Eriksson et al., 1999;Agarwal et al., 2011;Matsuki et al., 2012;Ebina et al., 2013). In recent years, the nanobubble technology has been applied widely in science and technology sector, such as water processing, biomedical engineering, and nanomaterials (Agarwal et al., 2011). This technology has also been used in aquaculture sector to increase dissolved oxygen (DO), namely stimulating Nile tilapia Oreochromis niloticus and vaname shrimp Penaeus vannamei growth, besides decreasing the total pathogenic bacteria and modulating the host immune system against bacterial infection (Imaizumi et al., 2018;Mahasri et al., 2018;Mauladani et al., 2018;Dien et al., 2021;Linh et al., 2021;Nghia et al., 2021;Rahmawati et al., 2021). The development of RAS installed with NBs becomes one of the solutions to improve the grouper fish fry culture productivity through increased stocking density. This study aimed to evaluate the recirculation system production performance installed with NBs in hybrid brown-marbled grouper in high stocking density condition.

Experimental design
The experimental design used in this study was a factorial design with two factors, stocking density and culture system, as each treatment was replicated three times (Table 1). The stocking densities tested were 500, 600, and 700 fish/ m 3 , while system treatments applied were RAS without NBs, RAS with NBs, and control (200% water exchange). The stocking density treatments were developed from SNI 8036.2:2014 for hybrid grouper with 2.8-3.2 cm size at 250-350 fish/m 3 in water exchange system. Control 5K 6K 7K Note: 5R = RAS without NBs at 500 fish/m 3 stocking density, 6R = RAS without NBs at 600 fish/m 3 stocking density, 7R = RAS without NBs at 700 fish/ m 3 stocking density, 5RN = RAS + NBs at 500 fish/ m 3 stocking density, 6RN = RAS + NBs at 600 fish/ m 3 stocking density, 7RN = RAS + NBs at 700 fish/m 3 stocking density, 5K = Control at 500 fish/m 3 stocking density, 6K = Control at 600 fish/m 3 stocking density, 7K = Control at 700 fish/m 3 stocking density.

Culture technique
The 27 units of aquarium with the size of 1.5 m × 0.5 m × 0.5 m were filled with seawater at 1000 L. The seawater was disinfected using 30 mg/L chlorine and neutralized by 75 mg/L Nathiosulfate, while being precipitated and aerated.
A probiotic was also used at 5 mg/L in three days before stocking the fish. For RAS without NBs treatments (5R, 6R, and 7R), aquaria were equipped with aeration and filtration systems containing green woll, zeolith, bio ring ceramic, and bioball filters (Lawson, 1995). Aquaria for RAS with NBs (5RN, 6RN, and 7RN) were equipped with nanobubble regulator.
The hybrid brown-marbled groupers Epinephelus sp., produced from the cross-breeding of female brown-marbled grouper E. fuscoguttatus and male camouflage grouper E. polyphekadion at 3.51 ± 0.05 cm (approximately on one-month age) were obtained from the breeding center in Situbondo, East Java and stocked in aquaria. Fish were stocked based on the treatments applied. The fish used were obtained after sortation and grading. The fish were acclimatized for three days in the experimental aquaria, cultured or 30 days, and fed with commercial sinking pellet diet at 1.5 mm size (48% protein content) until apparent satiation to avoid canibalism. During the culture period, water exchange was performed at 10% in RAS and NBs treatments to replace the water loss due to evaporation.

Parameters
Grouper fish length and weight was measured every 10 days during 30 days of culture. Fish samples were taken at 10% of the total fish and measured using a scale with 0.01 g accuracy, while length measurement was performed using calipers with 0.01 mm accuracy. The weight and length data were collected to calculate the fish growth performance.

Water physical-chemical parameters
Parameters tested during the experimental period contained water physical-chemical contents (pH, salinity, dissolved oxygen, ammonia, nitrite, nitrate) and growth performance (survival rate, spesific growth rate, feed conversion ratio, and productivity) ( Table 2). The temperature, pH, salinity, and DO were measured directly everyday at 07.00 GMT+7. Meanwhile, ammonia, nitrite, and nitrate were measured every 10 days by taking the water samples from each aquarium at 07.00 GMT+7.

Growth performance
Survival rate level was determined at the end of culture period using the formula (Ye et al., 2020): Whereas, SR: fish survival rate (%); Nt: total final fish; and No: total initial fish. The specific growth rate is an increased percentage of fish weight which was calculated with the formula (Wang et al., 2020): Whereas, SGR: Specific growth rate (%/day); Wt: average final fish weight on t-period (g); Wo: average initial fish weight on 0-period (g); dan t: culture period (days). The feed conversion ratio was calculated using the following formula (Goddard, 1996): Whereas, FCR: feed conversion ratio; F: Total feed consumed (kg); Wt: fish biomass on final culture period (kg); Wo: fish biomass on initial culture period; Wd: dead fish biomass during the culture period (kg). Whereas, FWD: fish weight diversity (%); S: standard deviation of fish weight on each treatment; x̅: average of fish weight on each treatment. Biomass production in the system was calculated using the following formula (Saefulhak, 2004):

Water quality
The nanobubble regulator was installed along with the recirculation system at 500, 600, and 700 fish/m 3 stocking densities (5RN, 6RN, and 7RN). This condition obtained the highest dissolved oxygen value for 30 days of culture period compared to other treatments, then declining slowly. The dissolved oxygen in other treatments tended to be stable until the end of culture period, namely 4.62-6.37 mg/L (Figure 1).
The pH measurement results during the culture period for 30 days obtained a decreased pH value tendency until the end of culture period, except in control treatment at 7.8-8.3. The lowest pH value was occurred in the 7RN treatment at 7.0 on the end of culture period. The highest pH value was occurred in all treatments, namely on the initial culture period of hybrid brown-marbled grouper at 8.2 ( Figure 2). Whereas, P: productivity (kg/m 2 ); Bt: biomass at the end of culture period (kg); and L: tank surface area (m 2 ).

Data analysis
The data obtained were tabulated for the continued statistical analysis. The production performance parameter data were analyzed using the analysis of variance (ANOVA) at 5% confidence degree. If the data were significantly different, Duncan's test was applied further. The business analysis and water quality data were analyzed descriptively using figures and tables. Data analysis was performed using Microsoft Excel 2016 and IBM SPSS Statistics 23.0 software program. The salinity measurement results during 30 days of culture period showed that all salinity values increased in all treatments based on Figure  3. The highest salinity value increase was shown by all control treatments on the 20 th day at among 31.0-30.7 g/L and on the 30 th day at 32 g/L. This figure below is the graphic of salinity level during the hybrid brown-marbled grouper culture period for 30 days (Figure 3).
The measurement results of ammonia, nitrite, and nitrate during hybrid brown-marbled grouper culture period on various applied systems and stocking densities demonstrated fluctuating values in RAS with and without nanobubble regulator. Ammonia, nitrite, and nitrate in control treatment tended to be stable and lower than other treatments. The graphic below describes the measurement results of ammonia, nitrite, and nitrate levels during 30 days of culture period (Figure 4).

Growth performance
After 30 days of hybrid brown-marbled grouper culture period in RAS with NBs obtained the best growth performance results (Table 3). The grouper fish fry reared in RAS with NBs also demonstrated a quite better productivity level (Table 4).

Discussions
Nanobubble application in recirculation system for hybrid brown-marbled grouper culture in various stocking densities will directly affect the environment or culture media. The environment designed by the system will influence fish stress and fish appetite. Moreover, the effect of recirculation system and utilization of nanobubble regulator with various stocking densities can be identified directly by producing different production performance.
Dissolved oxygen is a parameter that plays important role in water quality improvemet (Boyd, 2017), mainly on the fish culture activity. Therefore, increased dissolved oxygen becomes the main attention for grouper fish culture. The dissolved oxygen value during the experiment period tended to be stable in the culture media of hybrid brown-marbled grouper fish. However, the best result was obtained in RAS with nanobubble at different stocking density treatments.
Optimal dissolved oxygen for hybrid brown-marbled grouper growth has never been specifically studied. However, several studies mentioned that the dissolved oxygen condition of 5.2-6.2 mg/L showed a good growth for larvae until 3cm fry (Ismi, 2010;Ismi et al., 2016). Furthermore, the dissolved oxygen standard used in hybrid grouper fry culture is suggested to be >4 mg/L (SNI 8036.2:2014), while brownmarbled grouper fish is suggested to be >5 mg/L (SNI 6488.3:2011). Overall, the dissolved oxygen level in all treatments can be said to follow the standard quality, even a higher value was found in nanobubble treatment at 500 fish/m 3 (5RN) at 11.75 mg/L.
The acidity level or pH in the waters is one of the important chemical parameters in waters, mainly in grouper fish culture using recicurlation system. Imtolerable physical and chemical water  quality changes including pH value in grouper fish culture media can affect the grouper fish gills and oxygen consumption level. The lowest pH value obtained during the study was 7.0 in the 7RN treatment. Meanwhile, pH in all control treatments with various stocking densities tended to be more stable, as control treatments applied 200% water exchange per day. Water exchange and circulation systems can provide an optimal water quality for fish culture. However, it can become a bad impact on the environment if being maintained improperly and the aquaculture industry pollution has been lately become a big problem for the water environment (Wang et al., 2018). The suggested pH value for hybrid grouper is 7.5-8.5 (SNI 8036.2:2014) as the standard for one of the cross-bred fish cultures. The 5RN, 6RN, and 7RN treatments obtained a lower pH value than the suggested pH value. However, this pH value was still in an optimum limit for hybrid brown-marbled grouper based on blood glucose level, oxygen consumption level, and gill histology analyses. Moreover, it can also be proven on the production performance discussed below.
Salinity value should be noticed in marinefish culture as increased or decreased salinity affect the fish osmoregulation system. Salinity is an important abiotic factor for fish culture that can affect fish growth and physiological performance. In addition, the hybrid brown-marbled grouper has large osmotic gradient. The recirculation system used affects the evaporation level that increases the salinity level compared to the non-flow water system without water exchange. Optimal salinity for hybrid brown-marbled grouper culture needs a further study, but the data proves that the hybrid brown-marbled grouper has a high tolerance on salinity range. The salinity level applied based SNI 6488.3:2011 for brown-marbled grouper (E. fuscoguttatus) is 24.0-33.0 g/L.
Ammonia, nitrite, and nitrate levels are physical-chemical water quality parameters that become a challenge in recirculation system with high dissolved oxygen concentration. Ammonia and nitrite accumulations become the highest toxicant compounds for fish, shrimps, and other aquatic organisms (Shao et al., 2019), that will inhibit the RAS application in the aquaculture industry. The ammonia level during the experiment was fluctuating in RAS without NBs treatments. The highest ammonia level on the 10th day of culture obtained that all RAS without NBs treatments in various stocking densities reached at 1.340 mg/L (5R treatment). Decreased ammonia level in RAS without NBs treatments occurred on the 20th day of culture, except in the 6R treatment, which obtained a value of 1.007 mg/L. RAS with NBs and control treatments tended to be stable at 0.001-0.096 mg/L from the beginning to the end of the culture period. The suggested ammonia level in some grouper fish fry cultures are below 0.01 mg/L (SNI 6488.3:2011;SNI 8036.2:2014). The nitrite level during the experiment showed that the 5R, 6R, and 7R treatments had nitrite levels at 51.780, 69.720 dan 73.397 mg/L. Meanwhile, ammonia level during the culture period tended to be stable in all control treatments at 0.001-0.097 mg/L. Nitrite levels in RAS with NBs increased on the 20th day of culture and decreased entirely until the end of culture period. The nitrate level during the experiment showed that the RAS treatments obtained the highest nitrate level in 600 fish/m 3 stocking density at 265.73 mg/L. The fish specific growth rate produced in this study was among 3.49-4.25%/hari with the highest value was obtained from the nanobubble treatments. Based on the previous studies, the specific growth rate in juvenile hybrid grouper fish (E. fuscoguttatus ♀ x E. lanceolatus ♂) was around 3.13 ± 0.04%/day (Sutarmat & Yudha, 2013;Marzuki et al., 2020;He et al., 2021). The nanobubble treatments showed that the utilization of this equipment can provide a better feed conversion ratio. The best feed conversion ratio in this study was obtained from the 5RN treatment at 1.04 ± 0.01. The feed conversion ratio on the previous study in juvenile hybrid grouper fish (E. fuscoguttatus♀ x E. lanceolatus♂) was 1.04 (He et al., 2021).
Smaller length and weight diversities indicates that fish high uniformity level. The best length diversity was obtained from the 5RN treatment at 1.87 ± 0.06% and the weight diversity was obtained from the 7RN treatment at 6.50 ± 1.77%. Size variation is influenced by diets and water quality in optimal condition that will produce fish biomass with relatively uniform size (Lante et al., 2011). Based on the field observation of grouper fry agribusiness, consumers prefer choosing fry in uniform size, specifically in length size. Health conidition can be monitored thorugh physical capability in sustaining the fish survival rate (Muthmainnah, 2013).
The survival rate in this study was among 92.23 ± 1.29-100.00 ± 0.00%. The fish survival rate was among the range limit based on the previous studies, namely at 66.67-100% (Yin et al., 2018;Riduan et al., 2019;Sun et al., 2019;Zhou et al., 2019;Tan et al., 2019). The survival rate of juvenile hybrid grouper (E. fuscoguttatus ♀ x E. lanceolatus ♂) was 98.89 ± 1.92% (He et al., 2021). Survival rate is influenced by adaptability, physical conditions, age, competition, stocking density, handling, parasites, and water quality (Ismi et al., 2016). The production performance parameters in system can be seen also through its productivity level. The highest productivity level in this study was obtained from the 6RN treatment that could produce the highest grouper fish fry biomass at 2.78 ± 1.11 kg/m 2 .

CONCLUSION
The RAS with NBs application for juvenile hybrid brown-marbled grouper culture obtained a better production performance, mainly in growth performance and productivity.