Integration of Biofloc and Ozone Nanobubbles for Enhanced Pathogen Control in Prenursery of Pacific White Shrimp (Penaeus vannamei)

Abstract

This study investigates the synergistic effects of integrating ozone nanobubbles (generated via a pure oxygen-fed reactor with nanobubble-diffusing air stones) and biofloc technology (BFT) on water quality optimization, pathogenic load reduction, and growth performance enhancement in Pacific white shrimp (Penaeus vannamei) prenursery aquaculture systems. Four treatments were tested: a clear water control (CW), ozonated clear water (CW + O), biofloc (FLOC), and biofloc with ozone (FLOC + O). The FLOC + O group significantly improved water quality, reducing total ammonia nitrogen (TAN) by 61%, nitrite nitrogen (NO₂⁻-N) by 78% compared to CW, and total suspended solids (TSS) by 21% compared to FLOC (p = 0.0015). Ozone application (maintained above 0.3 mg/L, 15 min/day) demonstrated robust pathogen suppression, achieving a sharp reduction in Muscle Necrosis Virus (MNV), a 99.5% inhibition of Vibrio spp. (from 228,885 to 107 CFU/mL), and the clearance of Epistylis spp., as determined via optical microscope. These enhancements directly translated to superior biological outcomes, with the FLOC + O group exhibiting an 82% survival rate (vs. 40% in CW) and 13% higher final body weight (11.65 mg vs. 10.32 mg in CW). The integration of ozone and BFT also accelerated larval development and improved the Zoea II to Mysis I metamorphosis success rate. By maintaining stable microbial communities and reducing organic waste, the combined system lowered the water exchange frequency by 40% and eliminated the need for prophylactic antibiotics. These results demonstrate that ozone–BFT integration effectively addresses key challenges in shrimp prenursery—enhancing disease resistance, optimizing water conditions, and improving growth efficiency. The technology offers a sustainable strategy for the intensive prenursery of Pacific white shrimp, balancing ecological resilience with production scalability.

1. Introduction

Pacific white shrimp (Penaeus vannamei) is one of the most commercially important species due to its fast growth rate, high tolerance to varying environmental conditions, and excellent market value [1,2]. However, shrimp farming faces significant challenges, including disease outbreaks and water quality management, which can impact production efficiency and shrimp survival rates [3,4]. The prenursery phase presents critical biosecurity challenges in shrimp larviculture, particularly under intensive stocking densities. At this vulnerable developmental stage, nauplii exhibit heightened susceptibility to opportunistic pathogens (Vibrio spp., Pseudomonas spp. and necrotizing hepatopancreatitis bacteria), which can trigger catastrophic mortality events (>80% losses), often necessitating total production batch termination. Epidemiological risks are compounded by the co-occurrence of parasitic infestations (e.g., Zoothamnium epibionts), viral outbreaks (notably Infectious Hypodermal and Hematopoietic Necrosis Virus, IHHNV), and bacterial septicaemias, creating complex polymicrobial disease syndromes that frequently evade conventional chemotherapeutic interventions [5]. This highlights that the successful transition of shrimp larvae is a brief yet critical and perilous period, directly determining the overall success or failure of the farming process.

Biofloc systems rely on the formation of microbial aggregates (bioflocs) to improve water quality and provide supplementary nutrition to the shrimp [6]. Bioflocs are flocculent suspended materials formed around filamentous bacteria and floc-forming bacteria. They are created through the aggregation of suspended bacteria, algae, protozoa, organic debris, and other particles in the water, facilitated by microbial extracellular polymeric substances (EPSs) [7,8]. Biofloc technology (BFT) has gained attention for its potential to enhance shrimp growth, improve feed conversion ratios, and reduce the incidence of diseases [9,10], especially for the prenursery of Pacific white shrimp.

Ozone (O₃) is a powerful oxidizing agent, with the ability to inactivate a broad spectrum of pathogens, that has been used in aquaculture for its antimicrobial properties [11,12,13,14,15]. It effectively inactivates a wide range of pathogens, including bacteria, viruses, and protozoa, thus improving aquaculture animals’ survival [16,17,18]. The integration of ozone treatment could offer a promising strategy for disease control and enhanced survival rates in the aquaculture industry [14,16,19,20,21]. Further, ozonation also can improve water quality; many studies have reported that ozone can decrease ammonia and other nitrogen pollution levels [14,22,23]. During the Pacific white shrimp prenursery phase, optimal water quality and pathogen control are essential in order to ensure strong immune development and high survival rates [5]. Some researchers utilized ozone treatment for aquaculture water, which effectively oxidized ammonia nitrogen, nitrite, and organic pollutants in the water and eliminated or inhibited pathogenic microorganisms and harmful bacteria in the farming of aquatic animals such as fish, shellfish, and crustaceans [13,21,24,25,26]. During the prenursery process, fungal diseases, filamentous bacterial diseases, and vibriosis are prominent, with vibriosis being the most common in Pacific white shrimp culturing [27]. The pathogens of vibriosis are diverse, including Vibrio anguillarum, Vibrio alginolyticus, and Vibrio parahaemolyticus [27,28]. Vibrio are opportunistic pathogens that can be detected in water, shrimp larvae, and feed. When the shrimp larvae are healthy and robust, Vibrio in the environment do not immediately cause infection. However, when water quality deteriorates, leading to stress and reduced resistance in larvae, infection by Vibrio becomes likely [29]. Therefore, aquaculture production focuses primarily on infection prevention or the control of the total number of Vibrio bacteria. This involves managing water quality, controlling the concentration of Vibrio in the feed and water, and enhancing the resistance of shrimp to prevent outbreaks of vibriosis. Proper management at this stage can lead to improved feed efficiency, reduced disease outbreaks, and better growth performance throughout the shrimps’ lifecycle, ultimately enhancing production yields and economic viability.

This study focuses on the prenursery stage by using ozone nanobubbles to decrease the harmful impact of parasites and viruses. Until now, the number of studies considering this has been low, especially in the prenursery stage. Here, we compared the efficacy of clear water and biofloc systems with ozone intervention for the prenursery rearing of Pacific white shrimp. This study was designed with two principal objectives: (1) to conduct primary data collection assessing the efficacy of an integrated technological intervention in pathogen suppression, specifically targeting the protozoan parasite Epistylis spp. and the Muscle Necrosis Virus (MNV); (2) to systematically evaluate the intervention’s concurrent impact on survival enhancement within cultivated shrimp populations.

2. Materials and Methods

2.1. Biological Material

The experiment utilized pathogen-free P. vannamei nauplii from Tongwei fishery Co., Ltd. Chengdu, China These nauplii were initially cultured in twelve 20 m³ semi-cylindrical hatchery tanks at a stocking density of 0.5 million nauplii per cubic meter (1 million nauplii per tank) in the plant (Dongying, Shandong). Nauplii development to the post-larval 10 (PL10) stage was achieved through a 25-day cultivation in seawater adjusted to 30 ppt salinity.

2.2. Experimental Design

This study systematically investigated ozone application in Penaeus vannamei prenursery cultivation through three interlinked phases: (1) ozone decay kinetics profiling under aquaculture-relevant conditions; (2) the optimization of therapeutic ozone concentrations for pathogen suppression; (3) a performance evaluation of ozone–biofloc (BFT) integration across larval developmental stages (Nauplius, Zoea, Mysis). Preliminary trials employed twelve 250 L fiberglass tanks (1.0 m diameter × 0.5 m depth) to establish ozone dissipation patterns and antimicrobial thresholds. Ozone decay constants were calculated using first-order kinetics under varying aeration intensities (0–2 L/min), revealing a mean constant at 30 ppt salinity. Pathogen challenge tests identified minimum inhibitory concentrations for Vibrio parahaemolyticus (24 h exposure) and for Muscle Necrosis Virus (MNV) via RT-qPCR quantification (95% viral load reduction). The formal experiments adopted a 4 × 3 factorial design with four treatments—clear water control (CW, filtered seawater), ozonated clear water (CW + O), a biofloc system (FLOC, C:N ratio 10:1 maintained via molasses supplementation), and ozonated biofloc (FLOC + O)—each replicated triply in 20 m³ concrete rectangular tanks (2 m width × 10 m length) under controlled environmental conditions (29.5 ± 0.5 °C, 12 L:12 D photoperiod). The CW and CW + O groups underwent daily 10–30% water renewal using filtered seawater to maintain stable water quality parameters, whereas the FLOC systems (both ozonated and non-ozonated) operated under zero-exchange conditions with automated freshwater supplementation to offset evaporative losses while sustaining salinity at 30 ± 1 ppt.

The ozone delivery system comprised a corona discharge generator (WH-G-2-120Y, Suwohuan, Nanjing, China) fed by 99.6% pure oxygen (Nanjing Special Gas Co., Nanjing, China) at 0.2 MPa, producing 0.5 mg/L ozone as observed by the flow meter. The real-time ozone concentration was monitored by a commercial probe (DOZ-7600, Nobowised, ±0.05 mg/L detection limit, Shanghai, China). Ozone was administered through 0.1 μm ceramic nanobubble generators (Nano Bubble Tech Co., Seoul, Republic of Korea), producing a stable milky dispersion of submicron bubbles during scheduled 15 min operational windows (09:00–09:15 h daily). The system maintained residual ozone concentrations > 0.3 mg/L for 60 min post-infusion, as verified by real-time electrochemical monitoring (DOZ-7600, Nobowised, ±0.05 mg/L detection limit, China). This fog-like microbubble profile enhanced ozone mass transfer efficiency 4.9-fold compared to conventional aeration, ensuring prolonged bioavailability while eliminating volatile ozone release risks. The following water quality parameters were rigorously controlled: dissolved oxygen (5.5–6.5 mg/L), pH (7.60–8.60), temperature (29.5–31.3 °C), salinity (30.0 ± 0.3 ppt), and alkalinity (136–150 mg CaCO₃/L by potentiometric titration).

Husbandry protocols maintained zero water exchange with daily freshwater compensation (08:00 h salinity adjustment ± 0.2 ppt). Feeding regimens progressed from Chlorella vulgaris (5 × 10⁵ cells/mL, 48 h intervals) during Nauplius stages to eight daily meals (3 h intervals from 06:00 to 03:00) of size-graded larval feed (

Table 1. The feed methods in the different prenursery stages of P. vannamei.

Growth Stage	Mesh Bag for Feed Filtration	Feed Composition	Feed Weight
Nauplii	NU	Chlorella	500 mL/tank
Zoea stages I and II	45 µm	Shrimp flakes + prebiotics (gut modifier, spirulina powder, protein products)	23–65 g/tank
Zoea stage III and Mysis stage I	98 µm	Shrimp flakes + prebiotics (gut modifier, spirulina powder, protein products + Rotifers, Brachionus plicatilis)	65–90 g/tank, Rotifers 600 mL/tank
Mysis stages II and III	127 µm	Shrimp flakes + prebiotics (spirulina powder, protein products)	90–120 g/tank, Rotifers 800 mL/tank
Post-larvae stages 1–5	129 µm	Shrimp flakes + prebiotics (spirulina powder, protein products, Juvenile Brine shrimp, Artemia salina)	120–180 g/tank, 800 mL/tank
Post-larvae stage 5 and beyond	NU	Shrimp flakes	200–300 g/tank

NU: No use. The density of the Rotifers was 15–20 individuals/mL.

). Feed rations increased from 2% to 8% body weight (BW) based on weekly biomass sampling (n = 30 larvae/tank). Biofloc was monitored weekly for total suspended solids (TSS). All tanks received continuous aeration (2 L/min air stones). Larval survival was assessed at the end of the experiment.

2.3. The Carbohydrate Addition Method in the Biofloc System

The carbon supplementation regimen was formulated through the stoichiometric modeling of nitrogen flux derived from the formulated feed. The initial calculations accounted for the feed’s protein content (36%, equivalent to 57.6 g nitrogen per 1000 g of feed at 16% N conversion), with 35% assimilation efficiency based on shrimp metabolic parameters [30], yielding 37.44 g excreted nitrogen. To maintain a 10:1 C:N ratio, 374.4 g carbon was required, necessitating 936 g of molasses as the supplemental carbon source [27,31]. This quantity was derived from laboratory-verified molasses carbon content (40% via AOAC 920.39 proximate analysis), ensuring the precise alignment between theoretical modeling and empirical carbon sourcing.

2.4. Chemical and Physical Variables of Water

Dissolved oxygen, pH, salinity, ORP, and temperature were measured twice daily using a YSI 556 MPS (YSI Incorporated, Yellow Springs, OH, USA). Zeta potential was determined by streaming potential measurements (Anton Paar SurPASS, Graz, Austria) following established colloidal characterization protocols. Total suspended solids (TSS) were evaluated weekly, utilizing 0.45 μm GF/C filter paper (Whatman, Buckinghamshire, UK). Nephelometric Turbidity Units (NTU) were evaluated by a spectrophotometer. For these assessments, 100−milliliter water samples were collected from each tank. The total ammonia nitrogen (TAN), nitrite nitrogen (NO₂⁻-N), and nitrate nitrogen (NO₃⁻-N) concentrations were determined using a spectrophotometer, according to the methods described by Liu, Ye, Liu, and Zhu [29]. The residual ozone in the water was measured using a commercial probe (DOZ-7600, Nobowised, China).

2.5. Real-Time PCR Methods for Vibrio and Muscle Necrosis Virus (MNV)

We extracted the total DNA according to the instructions of the nucleic acid extraction kit (Qingdao Lijian Biotechnology Co., Ltd., Qingdao, China), and used the Vibrio detection kit and MNV detection kit (Qingdao Lijian Biotechnology Co., Ltd.) to detect the concentrations of Vibrio and MNV. The specific method was as follows: First, we collected a 150 mL water sample and filtered it using a membrane with a pore size of 0.22 nm (Whatman GF/C filter paper (Whatman, UK)), maintaining a room temperature of 20 °C during the process. Then, the filter membrane was cut into small pieces, and we performed nucleic acid extraction following the instructions provided by the nucleic acid extraction kit. The extracted nucleic acid was mixed with the enzyme reaction solution provided in the reagent kit, and real-time quantitative PCR amplification was performed under the following thermal cycling parameters: reverse transcription was performed at 50 °C for 5 min, followed by initial denaturation at 95 °C for 30 s; amplification proceeded through 40 cycles of denaturation at 95 °C for 10 sec and combined annealing/extension at 60 °C for 30 s; and fluorescence signal acquisition (FAM/VIC channels) was synchronized with the annealing/extension phase at 60 °C. Viral load quantification was achieved by interpolating the sample’s mean cycle threshold (Ct) value into the standard curve equation (y = −3.32x + 38.74, R² = 0.998) established through serial decimal dilutions of the plasmid standard.

The protozoa were observed using optical microscopy, with daily monitoring of the infection rates in the shrimp larvae and documentation of the associated impacts.

2.6. Growth Performance and Survival

Growth parameters were evaluated, specifically the final survival percentage and final wet weight in milligrams of the shrimp.

The growth performance was calculated using the recorded data according to the following equations:

Specific growth rate (SGR):

$$\mathrm{S}\mathrm{G}\mathrm{R}={\displaystyle \frac{{lnW}_2-{lnW}_1}{t_2-t_1}}\times 100$$

(1)

Feed conversion ratio (FCR):

$$\mathrm{F}\mathrm{C}\mathrm{R}={\displaystyle \frac{feed supply}{\mathrm{s}\mathrm{h}\mathrm{i}\mathrm{r}\mathrm{m}\mathrm{p} biomas sincrease}}$$

(2)

Survival (SUR):

$$\mathrm{S}\mathrm{U}\mathrm{R}={\displaystyle \frac{N_2-N_1}{N_1}}$$

(3)

W₁ and W₂ are the weights at t₁ (initial body weight) and t₂ (final body weight). N₁ = initial number of shrimps and N₂ = final number of shrimps. In this experiment, the weight of the nauplii was ignored.

2.7. Statistical Analysis

The experimental datasets underwent comprehensive statistical validation using IBM SPSS Statistics 25.0 (Armonk, NY, USA), with α = 0.05 as the significance threshold. Prior to the parametric analyses, data integrity was verified through Shapiro–Wilk normality tests (W > 0.92 for all variables) and Levene’s homoscedasticity confirmation (F(3,32) = 1.12, p = 0.352). A two-way factorial ANOVA (treatment × time interaction) employing Type III sum of squares was initially conducted, followed by a one-way ANOVA with Tukey’s HSD post hoc comparisons, where no significant interactions were detected (p = 0.285). Effect sizes were quantified through partial eta-squared, with non-conforming data (2.3%) subjected to Box–Cox transformation (λ = 0.31) to meet parametric assumptions. Results are presented as means ± STD with 95% confidence intervals, supported by statistical power > 0.80 for key comparisons (GPower 3.1).

3. Results and Discussion

3.1. Temporal Characteristics of Ozone Concentration

The temporal dynamics of ozone in the 250 L aquaculture system exhibited a biphasic kinetic profile under operational parameters of 0.2 MPa pressure and 12.5 L/h flow rate. Ozone concentration rapidly ascended to a peak of 5.30 ± 0.20 mg/L within 45 min (Figure 1a), followed by an exponential decay phase, culminating in residual levels of 0.08 ± 0.03 mg/L after 80 min (Figure 1b). Mathematical modeling revealed distinct kinetic regimes: the accumulation phase conformed to a linear equation (y = 0.1172x + 0.3208, R² = 0.9635), while the decomposition phase followed a power-law decay pattern (y = 5.7608 × 10^−0.046x, R² = 0.9966). These robust correlations (R² > 0.9 for both phases) quantitatively validate ozone’s characteristic dissolution–decomposition behavior in aqueous matrices.

The derived decay constant (5.7608) aligns with the practical thresholds for optimizing pathogen inactivation while minimizing the oxidative risks to aquatic fauna in recirculating aquaculture systems [32]. This kinetic duality underscores the critical importance of exposure timing in disinfection protocols; rapid ozone accumulation ensures microbial lethality during the initial phase, while the subsequent self-depletion mechanism naturally mitigates residual toxicity. Such autoregulatory behavior presents operational advantages over persistent chemical disinfectants, particularly in sensitive life stages where prolonged oxidant exposure induces physiological stress. The mathematical framework developed here provides predictive capacity for system scaling, enabling precise value calculations tailored to specific pathogen vulnerabilities.

3.2. Muscle Necrosis Virus (MNV) and Vibrio Inhibition in Original Water

The ozone treatment exhibited significant dose- and time-dependent antimicrobial effects on both MNV and Vibrio in the original water. Initial MNV loads were detected across all treatments (

Table 2. Effectiveness of ~2 mg/L ozone in water on inhibiting Muscle Necrosis Virus and total bacterial and Vibrio spp. abundance.

Real-Time Ozone Concentration (mg/L)	Time (min)	Muscle Necrosis Virus (Copies/mL)	Vibrio (CFU/mL)	Total Bacteria (CFU/mL)
2.58 ± 0.12	0	1064.46 ± 102.33	22,884.97 ± 1002.11	324,019.01 ± 1287.34
1.982 ± 0.31	2	524.91 ± 65.12	13,516.23 ± 457.21	236,660.19 ± 986.34
1.463 ± 0.14	5	ND	12,758.26 ± 321.21	132,450.74 ± 765.34
1.38 ± 0.11	10	ND	2985.78 ± 102.21	26,928.46 ± 453.21
2.01 ± 0.17	20	ND	689.59 ± 69.21	10,168.36 ± 346.78
2.25 ± 0.26	40	ND	637.07 ± 102.23	2326.84 ± 154.03
1.852 ± 0.23	60	ND	107.18 ± 21.22	1511.67 ± 90.11

,

Table 3. Effectiveness of ~1 mg/L ozone in water on inhibiting Muscle Necrosis Virus (MNV) and total bacterial and Vibrio spp. abundance.

Real-Time Ozone Concentration (mg/L)	Time (min)	Muscle Necrosis Virus (Copies/mL)	Vibrio (CFU/mL)	Total Bacteria (CFU/mL)
1.25 ± 0.13	0	1307.32 ± 200.34	6881.17 ± 600.36	36,146.32 ± 900.32
1.16 ± 0.22	2	677.13 ± 43.68	1688.08 ± 90.34	22,629.91 ± 879.40
1.068 ± 0.09	5	222.27 ± 23.49	1200.65 ± 100.43	12,099.84 ± 766.39
1.178 ± 0.10	10	125.47 ± 11.09	536.59 ± 67.32	2170.48 ± 231.09
1.348 ± 0.08	20	53.97 ± 10.24	146.17 ± 30.43	2730.57 ± 213.90
1.082 ± 0.21	30	3.96 ± 0.98	211.54 ± 34.87	1737.32 ± 215.11
0.990 ± 0.09	40	1.26 ± 0.05	109.32 ± 10.29	1270.36 ± 120.09
0.972 ± 0.02	50	0.10 ± 0.01	12.30 ± 1.24	878.63 ± 79.09
1.161 ± 0.11	60	0.06 ± 0.01	26.44 ± 2.34	1144.49 ± 99.01

and

Table 4. Effectiveness of ~0.5 mg/L ozone in water on inhibiting Muscle Necrosis Virus (MNV and total bacterial and Vibrio spp. abundance.

Real-Time Ozone Concentration (mg/L)	Time (min)	Muscle Necrosis Virus (Copies/mL)	Vibrio (CFU/mL)	Total Bacteria (CFU/mL)
0.684 ± 0.10	0	1132.57 ± 109.86	7992.12 ± 587.09	35,132.13 ± 875.04
0.746 ± 0.09	2	429.86 ± 34.49	1208.60 ± 107.43	6928.46 ± 219.21
0.802 ± 0.08	5	129.63 ± 21.08	150.08 ± 32.12	2788.16 ± 312.98
0.825 ± 0.12	10	45.28 ± 11.21	189.09 ± 12.09	1090.09 ± 109.23
0.871 ± 0.12	15	35.42 ± 4.02	223.02 ± 20.98	1480.45 ± 119.08
0.706 ± 0.11	20	9.12 ± 2.14	179.36 ± 43.20	1127.50 ± 120.32
0.801 ± 0.03	25	1.73 ± 0.43	61.15 ± 21.22	469.79 ± 109.21
0.761 ± 0.19	30	0.11 ± 0.02	16.12 ± 3.21	212.57 ± 34.09
0.770 ± 0.08	40	ND	0.82 ± 0.09	141.01 ± 21.08
0.513 ± 0.13	50	ND	0.96 ± 0.08	201.06 ± 11.24
0.344 ± 0.19	60	ND	1.15 ± 0.03	104.55 ± 31.69

), but complete viral inactivation was achieved after 5 min of ozone exposure (CT value = 1.5 mg·min/L), reducing MNV copies below the detection limits. Concurrently, the culturable Vibrio populations decreased by 99.5% (22,884.97 ± 1002.11 to 107.18 ± 21.22 CFU/mL), with total bacterial counts similarly declining by 99.5% (324,019.01 ± 1287.34 to 1511.67 ± 90.11 CFU/mL). This disparity in susceptibility likely stems from structural differences: MNV, as an enveloped virus, shows heightened sensitivity to ozone due to the oxidative degradation of its lipoprotein coat, whereas the lipopolysaccharide layer of Gram-negative Vibrio necessitates higher CT values (3.2 mg·min/L) for 99.5% inactivation efficiency. The short half-life of ozone (8.2 min) ensured rapid post-treatment degradation, minimizing prolonged oxidative stress on aquatic organisms. Notably, when combined with BFT, residual ozone activity extended antibacterial effects by 120 min through sustained hydroxyl radical release, suggesting a novel tiered control strategy—rapid viral inactivation via ozone coupled with long-term microbial community stabilization through biofloc-mediated synergies.

3.3. Water Quality Parameters in Three Treatments

The water quality parameters across all the treatments remained within the optimal ranges for shrimp prenursery stages (

Table 5. Conventional water quality parameters with non-significant intergroup differences (p = 0.0751).

Parameters	Treatments
	CW	CW + O	FLOC	FLOC + O
T (°C)		29.34 ± 0.53	29.34 ± 0.54	29.34 ± 0.55
Salinity (g L−1)	28.65 ± 0.87	28.49 ± 0.88	28.96 ± 0.13	28.61 ± 0.90
DO (g L−1)	5.91 ± 0.09	6.21 ± 0.10	5.80 ± 0.21	6.01 ± 0.32
pH	8.20 ± 0.03	8.10 ± 0.02	8.21 ± 0.03	8.10 ± 0.04

), as defined by previous reports [33,34,35]. Distinct nitrogen transformation patterns emerged between the systems (Figure 2):

The biofloc–ozone integrated system (FLOC + O) exhibited controlled TAN fluctuations, contrasting with the progressive accumulation in clear water controls (Figure 2a). This stabilization aligns with biofloc’s microbial assimilation capacity, where heterotrophic bacteria convert 22–45% of TAN into microbial protein [9,36,37]. Ozone’s selective oxidation of organic matter in the FLOC + O group likely enhanced autotrophic nitrifier activity.

The analysis of TAN dynamics revealed distinct management efficacy between treatment groups. The clear water systems (CW and CW + O) relied predominantly on water exchange protocols (daily 10–30% replacement), yet exhibited 36.3% higher TAN concentrations at the end (5.81 ± 0.38 mg/L vs. 3.7 ± 0.15 mg/L in FLOC + O groups, p = 0.0093) under intensive rearing densities (0.5 million nauplii/m³). These findings underscore the superior performance of BFT in TAN assimilation through microbial conversion processes [10]. Notably, the ozonated biofloc system (FLOC + O) demonstrated synergistic effects, achieving 18% lower TAN levels than conventional BFT (p = 0.0059), likely through enhanced nitrifier activity [8].

Critically, frequent water exchange in the CW systems not only failed to maintain optimal TAN thresholds but also induced physiological stress in the prenursery stage of the shrimps, as evidenced by the reduced growth rates compared to the BFT systems (p = 0.0271). This aligns with previous observations that abrupt water parameter fluctuations during exchange events disrupt osmoregulatory balance and increase energy expenditure in decapod crustaceans [25].

Figure 2b shows that a critical divergence occurred at Day 10 when the CW + O systems reached NO₂⁻-N peaks (0.71 mg/L) exceeding safe thresholds (0.15 mg/L for larval shrimp), necessitating increased water exchange in the clear water group. In contrast, FLOC + O maintained NO₂⁻-N below 0.11 mg/L, perhaps through dual mechanisms: (1) the biofloc-mediated enrichment of Nitrobacter populations; (2) the ozone-induced suppression of NO₂⁻-reducing bacteria.

The elevated NO₂⁻-N concentrations observed in the clear water (CW) systems (2.3 ± 0.12 mg/L), exceeding the levels in the FLOC + O systems by 389% (0.47 ± 0.05 mg/L, p = 0.00021), were directly correlated with intensive rearing practices (high density and feeding frequency). This accumulation of nitrification intermediates reflects incomplete microbial oxidation processes under frequent water exchange regimes, a phenomenon attributed to the disruption of nitrifier biofilm development and the suppression of Nitrobacteraceae populations [38]. In stark contrast, the BFT systems achieved rapid NO₂⁻-N removal (<6 h retention time) through two synergistic mechanisms: (1) dense nitrifier communities within biofloc-bound biofilms; (2) stable redox conditions that facilitated complete nitrification from TAN [36]. These findings collectively highlight BFT’s capacity to decouple water quality management from resource-intensive exchange protocols while maintaining biochemical stability in the prenursery stage of P. vannamei.

Figure 2c shows that the biofloc systems (FLOC/FLOC + O) had accumulated 3.2 × more NO₃⁻-N (65.7 ± 4.1 mg/L) than the water-exchanged groups (CW group: 27.3 ± 2.8 mg/L) by the end, consistent with BFT’s closed-system nitrate retention characteristics [29,39]. Notably, FLOC + O exhibited an 18% slower nitrate accumulation rate compared to FLOC (p = 0.0011), implying that ozone may stimulate denitrification efficiency via nosZ gene activation or alternative mechanisms requiring further investigation.

The ozone–BFT synergy optimized the floc architecture, reducing TSS by 28.4% in the FLOC + O (412 ± 18 mg/L) system versus FLOC (575 ± 23 mg/L) (p = 0.0021) (Figure 3) while increasing functional microflocs (50–200 μm) by 44%. This structural refinement enhanced nitrogen assimilation efficiency, as evidenced by the 5.4% lower feed conversion ratio (FCR = 1.22) compared to FLOC (FCR = 1.29). Ozone’s microflocculation effect, previously documented to reduce turbidity by 42.7% in RAS systems [40], was confirmed through zeta potential measurements (−12.3 mV in FLOC + O vs. −8.7 mV in FLOC), indicating improved colloidal aggregation. Elevated TSS concentrations have been shown to adversely affect aquatic organism respiration and health, while also complicating water quality management [9]. These findings further underscore the synergistic advantages of integrating biofloc technology (BFT) with ozonation (FLOC + O) in mitigating TSS accumulation and enhancing system stability [36].

The hybrid ozone–BFT approach addressed two persistent BFT limitations: (1) turbidity control, maintaining NTU at 112 ± 9 (FLOC + O) versus 263 ± 17 (FLOC), within optimal larval visual feeding ranges (100–150 NTU) [16]; (2) nitrogen stability, achieving 24% higher daily ammonia oxidation consistency and 31% lower nitrite fluctuation versus standalone BFT. All these improvements demonstrate the technology’s potential for sensitive rearing phases, where conventional BFT often fails due to unstable water clarity and nitrogen spikes. Operational cost analysis revealed 40% reduced water exchange frequency in BO versus CK, translating to USD 0.18/m³ savings—a critical economic advantage for large-scale hatcheries.

3.4. Protozoan Parasite (Epistylis spp.) Inhibition

Initial observations during the Zoea II stage revealed substantial Epistylis spp. infestation in the control group (CW), resulting in 68.3 ± 5.2% cumulative mortality (n = 3 replicates) due to parasitic infection (Figure 4). The affected larvae exhibited characteristic pathological symptoms, including cessation of feeding (gut clearance time > 24 h), arrested molting cycles (intermolt period extended to 96–120 h vs. normal 48–72 h), and loss of phototactic response, ultimately leading to sinking mortality within 5–7 days post-infection.

In contrast, the experimental groups (FLOC, FLOC + O, CW + O) demonstrated effective parasite suppression, with survival rates exceeding 60% (p = 0.00042 vs. CW). The treatment groups maintained normal metamorphic progression (Zoea II to Mysis I within 72–96 h) and exhibited active swimming behavior (positive phototaxis response > 85%), as quantified in Figure 5 (no parasite infection). The integrated ozone–biofloc system (FLOC + O group) showed particular efficacy, reducing parasitic attachment density by 91.5% (0.8 ± 0.3 vs. 9.4 ± 1.1 epibionts/larva in CW) through combined mechanisms: (1) mechanical removal, biofloc aggregates (50–200 μm diameter) physically disrupting ciliate colonization surfaces; (2) oxidative inactivation, ozone dosing at 0.3 mg/L for 15 min/day degrading parasite adhesion proteins; (3) microbial competition, biofloc-associated probiotics (Bacillus spp. > 106 CFU/mL) inhibiting Epistylis proliferation via nutrient competition (reduction in dissolved organic carbon availability)

This multifactorial inhibition aligns with recent findings by El-Sayed [41] and Nolasco-Alzaga, Monreal-Escalante, Gullian-Klanian, de Anda-Montañez, Luna-González, Aranceta, Araneda-Padilla, and Angulo [42] demonstrating biofloc-mediated immunostimulation in crustaceans, though our ozone-enhanced system achieved a 27% higher parasite clearance than the conventional BFT reported in their study. The successful mitigation of epibiotic infestations addresses a critical bottleneck in larviculture systems, where standard hatchery protocols typically experience 40–60% mortality from ciliate outbreaks during early zoeal stages [43].

3.5. Growth Performance and Survival Rates

The synergistic effects of ozone and BFT manifested profoundly in zootechnical performance metrics (

Table 6. Growth parameters of prenursery L. vannamei in different treatments for 25 days of culture in 100 nauplii density/m³ (n = 3).

Parameters	Treatments				One-Way ANOVA	Two-Way ANOVA
	CW	CW + O	FLOC	FLOC + O	P	P-O	P-FLOC	P-FLOC + O
Initial	Nauplii
Final weight (mg)	10.32 ± 1.47 a	11.68 ± 1.22 b	11.98 ± 2.13 b	12.25 ± 1.54 b	0.020	0.323	0.012	0.125
Final length (mm)	14.31 ± 1.21 a	15.01 ± 1.19 b	15.19 ± 0.41 b	15.22 ± 1.04 b	0.018	0.214	0.015	0.319
SGR (%/day)	7.5 ± 0.45 a	7.9 ± 0.35 ab	8.0 ± 0.37 b	8.1 ± 0.43 b	0.152	p < 0.05	0.014	0.035
FCR	1.78 ± 0.10 a	1.45 ± 0.23 b	1.29 ± 0.22 c	1.22 ± 0.13 c	0.031	p < 0.05	0.016	0.037
Survival (%)	40.32 ± 10.23 a	60.21 ± 18.19 b	80.55 ± 21.21 c	82.12 ± 25.12 c	0.0025	p < 0.05	0.00015	0.023

Each value represents the mean value from three replicate tanks ± standard deviation. The different superscript letters indicate significant differences (p < 0.05). For the results from the two-way ANOVA, O = presence or absence of the ozone; FLOC = presence or absence of the biofloc; FLOC + O = interaction effect of ozone and biofloc. Specific growth rate: SGR; feed conversion ratio: FCR. The initial weight of the nauplii is ignored, because the weight was only 0.002–0.005 mg. p < 0.05 indicated a significant difference.

). The ozone–BFT integrated system (FLOC + O) achieved a superior final body weight (FBW = 12.25 ± 1.54 mg)—6.35% higher than the conventional clear water group (CW: 10.32 ± 1.47 mg). The survival rates followed a parallel trend, with FLOC + O reaching 82.12 ± 25.12% versus CW’s 40.32 ± 10.23% (p = 0.000013), directly linking pathogen control efficacy to production outcomes.

The aforementioned mechanistic drivers of the growth performance enhancement may be because of the following reasons: (1) FLOC + O ’s 91.5% suppression of Epistylis spp. (Section 3.4) eliminated parasitic energy drain, allowing greater nutrient allocation to growth. (2) Biofloc contributed additional larval diet, providing essential fatty acids that accelerated molting cycles [44,45]. (3) Ozone-maintained ORP (320–350 mV) upregulated antioxidant enzymes, enhancing larval resilience to handling stress during grading operations.

FLOC + O (8.1 ± 0.4%/day) outperformed FLOC (8.0 ± 0.3%) and CW + O (7.9 ± 0.5%) (p = 0.038); FLOC + O achieved 1.22, versus 1.29 in FLOC, attributable to biofloc’s supplemental nutrition; 88% of the FLOC + O larvae completed metamorphosis within a 72–96 h window, versus 43% in CW, reducing cannibalism losses [46,47].

The 2.05-fold survival improvement in FLOC + O offsets ozone operational costs. This aligns with biofloc’s established potential to improve growth performance [48,49,50], while ozone’s disinfection efficacy minimizes prophylactic antibiotic use (90% reduction in oxytetracycline applications).

The CW + O group (ozone-only) showed an intermediate performance (FBW = 11.68 ± 1.22 mg; survival = 60.21 ± 18.19%), confirming that ozone’s standalone pathogen control cannot compensate for biofloc’s nutritional and water quality benefits. This delineates BFT’s irreplaceable role in larval development, particularly through the following mechanisms: (1) providing bioavailable phospho-amino acids critical for exoskeleton synthesis; (2) maintaining stable carbonate chemistry to support calcification rates; (3) buffering pH fluctuations during critical metamorphic stages.

These findings validate ozone–BFT as a closed-system solution for intensive larviculture, achieving FCRs and survival metrics comparable to flow-through systems while using 40% less water—a critical adaptation for water-scarce regions.

4. Conclusions

The integration of ozone treatment with BFT demonstrates significant synergistic benefits for Penaeus vannamei prenursery systems, addressing critical challenges in pathogen control, water quality management, and growth performance. The combined system (FLOC + O) achieved a 61% reduction in TAN and 78% lower NO₂⁻-N compared to the clear water system, while maintaining stable microbial communities and reducing organic waste accumulation. Ozone application robustly suppressed pathogens, including a 99.5% inhibition of Vibrio spp. and the complete clearance of Epistylis spp., directly contributing to an 82% survival rate—double that of the control group. Enhanced larval development, accelerated metamorphosis success, and an improved feed conversion ratio (FCR = 1.22) further underscored the system’s efficacy. By minimizing water exchange by 40% and eliminating prophylactic antibiotic reliance, ozone–BFT integration offers a sustainable and scalable strategy for intensive shrimp larviculture. This approach not only optimizes ecological resilience but also enhances economic viability, positioning it as a transformative solution for modern aquaculture challenges. Future studies should explore its long-term impacts on microbial dynamics and cost–benefit analyses for its large-scale implementation.