## 4 Discussion
With the increase in shrimp biomass and the proliferation of microorganisms in the biofilm, the oxygen consumption rate of both the shrimp and microorganisms increased, and the DO in the aquaculture water gradually decreased from the initial value of 7.0 mg/L. The adding of liquid oxygen and the DO value above 5.0 mg/L ensures adequate shrimp growth and floc metabolism [26]. Under the highly intensive aquaculture conditions of the RAS, the pH value dropped rapidly due to the carbon dioxide produced through respiration and hydrogen ions produced by the nitrification of the biofilter [27]. After a month of RASculture, the pH value dropped from 8.2 to about 7.0. By adding a small amount of calcium oxide (CaO) or sodium bicarbonate to the system every day, the pH was maintained above 6.4. This necessary daily addition increases aquaculture costs, while maintaining a weakly acidic pH reduces the toxicity of ammonia nitrogen in the aquaculture water [28,29] and does not add costs. Pathogenic _Vibrio_ is one of the main pathogens affecting _Litopeneus vammaei_ aquaculture, and estimates of the total number of _Vibrio_ present in the culture can be used as an important indicator of the impact on the culture's health [30,31]. During the entire culture period, the total number of _Vibrio_ bacteria in the system was maintained at 300-10,000 cfu/mL, and no diseases or abnormal mortality rates were reported. The number of bacteria increased gradually in later stages, but through the use of ultraviolet lamp sterilization and increased ozone input, it was successfully kept under control. The maximum abundance of _Vibrio_ in aquaculture water was \(10^{4}\) CFU/mL, which was almost two logarithmic units lower than the \(10^{6}\) CFU/mL pathogenicity level in water [31]. This pathogen concentration is comparable to that observed in previous research by [PERSON] et al. (2018) where the total number of _Vibrio_ in a RAS culture of _Litopeneus vammaei_ was successfully controlled under low salinity conditions [9]. Ammonia nitrogen (especially free ammonia) and nitrite concentrations are also important factors that affect the health of _Litopeneus vammaei_ cultures [29,32]. In RASs, these concentrations are mainly reduced by biofilters [33]. In the initial period of recirculating aquaculture in this study, 10% of mature biomedia (sourced from a grouper RAS with a culture density of 80 kg/m\({}^{3}\)) was added to promote the fast growth of biofilm in the new biomedia. During the first 20 days of culture, the TAN value remained within 2.0 mg/L. With the increase in feed amount, it exceeded 3.95 mg/L for four consecutive days by the 41 st day. At this time, the total feed amount in the two systems reached 226.8 kg/d, which represented the maximum feeding load of the system. Studies have reported that the \"safe level\" of TAN for rearing juvenile _Litopeneus vammaei_ is 3.95 mg/L [34]. Subsequently, 1 m\({}^{3}\) of new biomedia was added to each system, and the daily feed amount was appropriately reduced to match the control of TAN in the systems. This was reduced to about 2 mg/L again through these measures. Studies recommended that the filling rate of the MBBR should not exceed 70% [35,36]. In this study, it approached this critical value, giving full play to the water treatment capacity of the MBBR. With the increase in shrimp biomass, the demand for feed increased. The daily feed amount gradually increased to 270 kg/d (on the 46 th day), and the TAN concentration on the 47 th day was 4.8 mg/L, reaching again the maximum feeding load of the system. In later stages, TAN was controlled by appropriately reducing the feed amount until the end of the experiment. Ammonia tolerance in _Litopeneus vammaei_ increases with age [37], so the safe concentration of TAN tolerated by _Litopeneus vammaei_ should be greater than 3.95 mg/l in the later period of the culture. In this study, TAN was maintained below 3.95 mg/L as much as possible.