Introduction
Biofloc technology (BFT) enables intensive aquaculture production with low water exchange, but this can lead to the accumulation of nitrogen compounds, particularly nitrate. Nitrate buildup may affect water quality, animal performance, and system sustainability, and may also pose an environmental risk if untreated water is discharged. In BFT systems, nitrate removal has traditionally been linked to denitrification under anoxic or anaerobic conditions. However, recent evidence suggests that denitrification may also occur under aerobic conditions, highlighting the functional complexity of biofloc microbial communities.
Material and methods
Two trials were carried out to evaluate aerobic and anoxic denitrification in P. vannamei production system. First, a 48 h assay was conducted to optimize nitrate removal in external bioreactors using biofloc with approximately 300 mg/L total suspended solids (TSS) and water with a high nitrate concentration. Anoxic denitrification was performed by adding molasses to adjust the C:N ratio to 3:1, and removing aeration. While, aerobic denitrification was performed under controlled oxygenated conditions (>4 mg/L) and adjusting the C:N ratio to 20:1 with a carbon source composed of a mix of di-, poly- and monosacharides (Maltose 53%, corn starch 24% and glucose 2%. Microbial populations were analyzed by qPCR to assess the functional genes associated with nitrification and denitrification under each condition. In the second trial, the denitrification strategies were applied for 4 weeks in shrimp production tanks. Once per week, between 15-20% of the tank water was subjected to a 48 h denitrification period under either anoxic or aerobic conditions and then reintroduced into the production system. In both trials, water quality was monitored through dissolved oxygen, temperature, salinity, pH, alkalinity, ammonium, nitrite, and nitrate. The pH was maintained above 8 by adding 1 M NaOH, and each denitrification period was considered complete when nitrate concentrations approached 0 mg/L.
Results and discussion
The results showed that both strategies reduced nitrate in biofloc systems, although through different dynamics. In the optimization trial, anoxic denitrification achieved the highest nitrate removal efficiency, close to 95.7%, but was accompanied by transient nitrite and ammonium accumulation and increased alkalinity after pH correction, indicating greater operational instability. In contrast, the aerobic pathway showed a more gradual and lower nitrate reduction, approximately 77.2%, but maintained greater water quality stability. The increase in TSS under this condition suggests a relevant contribution of heterotrophic assimilation, consistent with carbon supplemented biofloc systems (Ebeling et al., 2006; Avnimelech, 2009). qPCR analysis supported these differences: under anoxic conditions, the upward trend in narG, together with the low abundance of downstream denitrification genes, were consistent with transient nitrite accumulation; whereas, under aerobic conditions, the higher abundance of narG, nirS, and nirK, together with the increase in AOB and Nitrospira trend, suggests the coexistence of nitrification, heterotrophic assimilation, and denitrification in anoxic microenvironments within the flocs (Deng et al., 2020; Bhattacharya and Mazumder, 2021).
In the in vivo trials with P. vannamei, the weekly use of external reactors helped control nitrate accumulation. The anoxic pathway produced a more marked reduction of nitrate in the culture tanks compared with the control, reaching concentrations close to 0 mg/L in the reactors. However, it required a longer treatment time (96 h), to avoid risks associated with nitrite, ammonium, and low dissolved oxygen. In contrast, the aerobic pathway was more stable, but its impact on nitrate accumulation in the culture tanks was limited, likely because continuous nitrate production from feeding, excretion, and nitrification partially exceeded the treatment removal capacity. This agrees with the preliminary trial and with the higher relative abundance of nitrifying bacteria observed under aerobic conditions (Kim et al., 2022; Han et al., 2024). Additionally, both strategies were viable and did not compromise shrimp growth or survival, in agreement with previous studies using denitrified water in biofloc systems (Brand��o et al., 2024). Therefore, despite requiring stricter management, anoxic denitrification appears to be the most promising strategy for effectively controlling nitrate accumulation in biofloc systems; while the aerobic pathway provides greater operational stability, but further optimization is needed to achieve comparable results under culture conditions.
Acknowledgments
This abstract is part of the "ZeroFloc" project PID2023-149570OB-I00, funded by MICIU/AEI/10.13039/501100011033 and by the European Union NextGenerationEU/PRTR. S. Ferrando-Juan is supported by a predoctoral grant PRE2021-098367, funded by MICIU/AEI/10.13039/501100011033. J. Brol's contract was funded by the Santiago Grisol��a 2021 programme of the Generalitat Valenciana (CIGRIS/2021/109).
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