Aquaculture is a prominent and rapidly growing industry that significantly contributes to the economic growth of many countries (Rosa et al., 2019). Tilapia fish, in particular, exhibit strong tolerance to high stocking densities, demonstrating high productivity rates and adaptability to a variety of culture conditions (Mohamed et al., 2024). The resilient mucus layer of tilapia serves as a protective barrier against damage and bacterial infections, enabling successful development and survival even in densely populated tank such as temperature, dissolved oxygen levels, pH, and waste control facilitates efficient production and provides fish farmers with a systematic approach to managing their stock (De Long et al., 2009). However, inadequate water quality has led to reduced production rates due to increased fish mortality. Low water quality negatively affects the usability of water as an aquaculture medium by depleting dissolved oxygen levels and elevating concentrations of nitrite and ammonia, both of which are toxic to fish. The release of unconsumed feed, feces, and urine from fish farms contributes to water quality decline and the occurrence of eutrophication by algae, which is detrimental to fish. High concentrations of nitrogenous compounds can harm aquatic organisms and pose a public health concern (Setiadi et al., 2018).
Photosynthetic bacteria (PSB) have shown good potential in effectively eliminating nutrients, heavy metals, and organic contaminants from various types of wastewater, contributing to sustainability (Ying et al., 2020). PSB have been widely used in fish and shellfish hatcheries and farms in China (Chen et al., 2020), as well as in wastewater treatment since the 1960s (Lu et al., 2019). PSB cells, which contain abundant active compounds, can serve as a natural source of probiotics. They have the potential to be incorporated into tilapia feed formulations as a growth supplement (Banerjee et al., 2000). Despite their high tolerance, PSB exhibit a wide range of metabolic modes that enable them to effectively eliminate nutrients, heavy metals, and organic pollutants from various water sources, thereby contributing to sustainable practices (Talaiekhozani et al., 2017).
This study focuses on the efficacy of isolated PSB compared to commercially available PSB in the industry for sustainable wastewater treatment in red hybrid tilapia (Oreochromis spp.) tank culture systems. The objective is to ensure long-term water quality for the fish and evaluate the potential of PSB to meet sustainable wastewater treatment needs.

2. Materials and Methods

2.1 Ethical approval statement

Not applicable.

2.2 Material preparation and study site

Red Hybrid Tilapia fingerlings (Oreochromis spp.) were obtained from a fish farm (Farm X) located at Jalan Samarakan, Tatau, Bintulu, Sarawak (Figure 1). Meanwhile, the PSB were isolated from previous research conducted at the Microbiology and Parasitology Laboratory (MPL) at Universiti Putra Malaysia Bintulu Sarawak Campus (UPMKB). The isolates were identified following the procedures outlined by Cho and Kim (2022). The experiment was carried out in the MPL and Wet Laboratory at UPMKB, using a polyethylene round tapered tank.

Figure 1. Maps showing the location of Farm X, marked with a red pin at coordinates 2°55'59.4"N 113°03'50.6"E, situated on Jalan Samarakan in Tatau, Bintulu, Sarawak.

2.3 Nutrient agar preparation

Nutrient agar (Merck Millipore, pH 7.0) was prepared by dissolving 28 grams of nutrient agar powder in 1 liter of distilled water. The mixture was autoclaved at 121 °C for 15 minutes and then cooled to 55 °C before being poured into petri dishes. The prepared agar was stored at 4 °C.

2.4 Serial dilution and bacterial enumeration

A serial dilution was performed under aseptic conditions. A range of 30 to 300 colonies was required for the plate count. The number of colonies on each plate was counted after 24 hours of incubation. The colony-forming units (CFU) were calculated using the following formula:

2.5 Isolation of pure culture

A single colony from the incubated culture was inoculated onto sterile nutrient agar and incubated overnight at 37 °C to obtain a pure culture. After 24 hours of incubation, the optical density (OD) at 600 nm was measured.

2.6 Procurement and cultivation of photosynthetic bacteria

A specific and carefully selected set of ingredients was incorporated into the medium for PSB culturing. The culture was then left outside, exposed to direct sunlight (30–36 ºC) for 14 days. After this period, the water began to exhibit a dark red color, indicating successful bacterial growth.
The photosynthetic bacterial (PSB) isolate used in this study belongs to the family Rhodospirillaceae, which includes purple non-sulfur bacteria (PNSB). The isolate was previously obtained and characterized by Wan Morni et al. (2023), who confirmed its identity based on colony morphology, pigmentation, Gram staining, catalase activity (3% H₂O₂), citrate utilization on Simmons Citrate Agar, and growth on Nutrient Agar and Sorbitol MacConkey Agar. The results indicated that the isolate was a Gram-negative, catalase-positive bacterium with biochemical traits consistent with purple non-sulfur photosynthetic bacteria. In the present study, the same strain was re-cultivated and integrated into the tilapia culture system for wastewater treatment assessment.

2.7 Bacterial stock and preservation

A bacterial stock for future use was prepared. A total of 1 ml of 10 ml bacterial culture was incubated overnight at 37 °C and then transferred into a 2.0 ml micro-centrifuge tube. The final mixture consisted of 50% bacteria and 50% diluted glycerol, which was stored at -20 °C.

2.8 Red hybrid Tilapia (Oreochromis spp.) preparation

The tilapia fish were acclimatized for 7 days before the experiment was conducted. The fish were fed a commercial pellet diet (Cargill (Malaysia) Sdn Bhd) at a rate of 5% of their body weight, twice daily at 0800 and 1700. They were placed in three polyethylene round tapered tanks, and the experiment was conducted indoors at room temperature under natural light conditions. Red hybrid tilapia juveniles (Oreochromis spp.) were stocked in tanks measuring 47 inches in top diameter, 36 inches in bottom diameter, and 30 inches in height, with a total volume of approximately 450 litters. Each tank was filled with 428 litters of tap water that had undergone preliminary anti-chlorine treatment to neutralize chloride ions and reduce harmful contaminants. A total of 50 fish were stocked in each tank. The tanks were maintained as a static system with no water exchange throughout the two-month culture period to simulate realistic wastewater accumulation conditions.

2.9 Integration of photosynthetic bacteria in tilapia tanks

The integration of PSB into the tank followed the procedures outlined by Ying et al. (2020), with slight modifications. Sixty millilitres of PSB were administered to the respective tanks, except for Tank 1, which served as a negative control (T1). The experiment was conducted over two months in the laboratory, during which the efficacy of different types of PSB was assessed and compared.

2.10 Assessment of wastewater treatment efficacy

Physicochemical parameters and water quality were measured every three days during the experiment, following the procedure outlined by Ying et al. (2020) with slight modifications. All readings were taken and recorded in triplicate.

2.11 Water quality parameter measurement

Total ammonia, nitrite, and nitrate levels were measured using an API freshwater test kit. Turbidity was assessed with a portable turbidimeter (Hach 2100P, United States), while pH, temperature, total dissolved solids (TDS), conductivity, and dissolved oxygen (DO) were analyzed using a portable multiparameter device (Model WQC-24, Japan).

2.12 Laboratory analysis

Water samples from each tank were taken at 15-day intervals for chemical oxygen demand (COD) and total suspended solids (TSS) analysis, following the method described by Hyman (2019). The COD was calculated using the formula,

2.13 Health assessment criteria

Every seven days, 10 fish were sampled, and the growth rate of Red Hybrid Tilapia (Oreochromis spp.) was measured. The growth parameters, including survival rate, weight gain, specific growth rate (SGR), and feed conversion ratio (FCR), were determined at the end of the experiment.

2.14 Statistical analysis

The data were analyzed using SAS 9.4 (Statistical Analysis System) for Windows. Analysis of covariance (ANCOVA) was employed to determine significant differences between groups, followed by Tukey's multiple comparisons test. The mean and standard error of the mean (SE) were reported for all findings. A significance level of P < 0.05 were considered significant.

3. Results and Discussion

3.1 Analysis of water quality parameters

Ammonia concentrations varied across treatments, with T1 recording the highest levels (8.0 ppm) on days 7, 21, 28, and 63, while the lowest reading (0.25 ppm) occurred on day 49. In T2, the highest ammonia concentration was also 8.0 ppm on day 7, with the lowest reading (0.5 ppm) on day 28. T3 showed a high concentration of 8.0 ppm on day 7, while the lowest readings (0.3 ppm) were recorded on days 28, 35, and 49, with a concentration of 0.5 ppm on day 43 (Table 1). Ammonia originates from fish faeces and unconsumed feed. Furthermore, ammonia levels should not exceed 1 mg Lˉ¹, according to guidelines from BFAR-NFFTC (2000) and the Indian Council of Agricultural Research (2006). In this study, ammonia concentrations decreased dramatically from 8.0 ppm to 1.0 ppm, which is suitable for the preservation of Red Hybrid Tilapia (Oreochromis spp.), indicating that cultured PSB positively impacts water quality (Wan Morni et al., 2023; Zhang et al., 2013).
For nitrite concentration, T1 shows a high reading of 5.0 ppm on days 14, 21, 35, 43, 49, and 56, while the lowest reading (0.0 ppm) for the negative control occurs from day 0 to day 7. In T2, nitrite concentration reaches a high of 5.0 ppm on day 35, with the lowest readings (0.0 ppm) recorded on days 0, 1, 2, and 49. T3 shows a high concentration of 5.0 ppm on days 14 and 35, with the lowest readings (0.0 ppm) on days 0 and 7, and a reading of 0.3 ppm on days 56 and 63.
Regarding nitrate concentration, T1 records high readings of 160.0 ppm from days 14 to 21 and from days 43 to 63. Days 28 and 35 show readings of 80.0 ppm, while the lowest readings (5.0 ppm) occur on days 0 and 7. In T2, nitrate concentration peaks at 160.0 ppm on days 28 and from days 43 to 63, with a reading of 80.0 ppm on day 35, and the lowest readings (5.0 ppm) on days 0 and 7. T3 reports a high concentration of 160.0 ppm on days 14, 28, and from days 43 to 63, with the lowest readings (5.0 ppm) on days 0 and 7.
The nitrite levels recorded in this study are low, ranging from 5.0 ppm to 0.3 ppm. According to Fujaya et al. (2022), nitrite levels ranging from 0 to 1 ppm are optimal for fish culture, although levels below 4 ppm are acceptable. Conversely, the nitrate levels in this study are high, reaching 160 ppm. Elevated nitrate concentrations in water can affect fertility, as nitrate is a limiting factor in primary productivity and other physico-chemical properties (Ying et al., 2020). Therefore, monitoring nitrate levels is crucial, as high concentrations can negatively impact animal growth and survival. Additionally, high nitrate levels may be associated with limited growth and decreased survivability in aquaculture (Ying et al., 2020).
The pH levels varied throughout the treatment period, from day 0 to day 63, showing the highest and lowest pH values for T1 (pH 7.5 to pH 4.6), T2 (pH 7.3 to pH 4.5), and T3 (pH 7.4 to pH 5.1). These pH values are crucial for the survivability of tilapia fish, which can thrive in a pH range of 5 to 10, with an optimal range of 6 to 9 (DeLong et al., 2009). Sudden fluctuations in pH can affect enzymatic activity and compromise the integrity of enzymatic structures. Furthermore, pH levels may also influence the growth of Phosphate Solubilizing Bacteria (PSB) (Chen et al., 2020).
The temperatures across all treatments were relatively consistent, ranging from 27.6 °C to 29.0 °C. According to Leonard and Skov (2022) and Nay and Cho (2019), the optimal temperature for tilapia is between 26 °C and 30 °C, indicating that the conditions in this study are favourable for tilapia survival. Additionally, fish metabolism can be influenced by surrounding temperature. Hülsen et al. (2016) noted that low temperatures do not significantly affect the growth rate of PSB. Thus, the advantages of a PSB wastewater treatment system operating effectively at low temperatures could enhance the implementation of PSB technology.
The TDS varied across treatments, with T1 exhibiting the highest and lowest readings, fluctuating between 0.0 ppm and 0.8 ppm from day 0 to day 63. During the same period, T2 showed a similar range from 0.0 ppm to 0.7 ppm. T3 also demonstrated a high to low range of 0.0 ppm to 0.7 ppm. The values obtained in this study (0.7 ppm to 0.2 ppm) exceed the standard value of 0.13 mg/l recommended by Khan et al. (2017) but remain within an acceptable range. High levels of TDS can lead to fish mortality due to osmotic stress (Khan et al., 2017).
Turbidity levels were recorded with high and low readings from day 0 to day 63, with T1 ranging from 640.7 NTU to 0.0 NTU, T2 from 591.0 NTU to 0.0 NTU, and T3 from 427.7 NTU to 0.0 NTU. In this study, turbidity decreased from 427.7 NTU to 324.0 NTU. High turbidity can negatively impact fish productivity by limiting light penetration (Khan et al., 2017). Additionally, dissolved suspended solids can cause filter clogging and injure fish gills. Peralta et al. (2010) suggest that the adsorptive immobilization of Phosphate Solubilizing Bacteria (PSB) holds significant potential for improving water turbidity. Therefore, these findings underscore the importance of PSB in reducing turbidity in aquaculture systems.
The concentration of conductivity varied significantly across all treatments during the experimental period. In T1, the readings ranged from 109.0 mS/m to 11.4 mS/m, while T2 ranged from 103.7 mS/m to 10.3 mS/m, and T3 ranged from 96.3 mS/m to 12.3 mS/m. Conductivity measures a solution's ability to conduct electricity and is closely associated with its salt content. Changes in salt content can lead to variations in osmotic pressure, potentially causing metabolic issues, hindered growth, and reduced offspring production in fish (Chen et al., 2020). Therefore, understanding the role of conductivity in aquaculture is essential for improving the quality of aquaculture systems.
Similarly, the concentration of dissolved oxygen (DO) also showed considerable variation across all treatments. In T1, the readings ranged from 5.8 mg/L to 0.1 mg/L, T2 from 5.41 mg/L to 0.25 mg/L, and T3 from 6.38 mg/L to 0.2 mg/L. Overall, the DO content in this study ranged from 6.38 to 0.2 mg/L, suggesting that the DO levels were within the recommended range for the survival and preservation of red tilapia (Yildiz et al., 2017). Furthermore, PSB is non-toxic and poses no threat to the aquatic ecosystem. Thus, the use of PSB could enhance the quality of aquatic ecosystems by increasing the level of dissolved oxygen in the water (Wan Morni et al., 2023).

3.2 Analysis of effect of photosynthetic bacteria to the weight and length of the red hybrid tilapia (Oreochromis spp.)

Initial measurements at Day 0 indicated no significant differences in weight and length among the groups, confirming a uniform starting point. During the early growth phase (days 7–21), no notable differences in these parameters were observed on day 7 and day 14. However, by day 21, fish in the cultured PSB tank (T3) demonstrated significantly better length growth (11.91 ± 0.51 cm) compared to the positive control (T2), although their weight remained comparable to that of the negative control (T1) (Table 2).
In the mid-phase of growth (Days 28–43), T3 consistently recorded significantly higher weight and length gains (47.69 ± 10.69 g, 12.98 ± 0.96 cm) than both T1 and T2. While T2 occasionally outperformed T1, particularly in terms of weight, the trend of T3 > T2 > T1 became increasingly evident, demonstrating the clear impact of cultured PSB on enhancing growth performance in tilapia.
By the late phase and final measurements (days 49–63), T3 achieved the highest growth outcomes, reaching 91.85 g in weight and 17.37 cm in length by day 63, significantly surpassing both control groups. T2 performed moderately well (68.18 g, 15.07 cm), significantly better than T1, which exhibited the lowest growth (54.89 g, 13.24 cm). According to Chowdhury et al. (2016), PSB contain enzymes that play a crucial role in promoting fish weight gain. Furthermore, extracellular enzymes from phototrophic purple bacteria may aid in early digestion and enhance the metabolism of fingerlings (Chowdhury et al., 2016).
Supplementation with cultured photosynthetic bacteria (PSB) significantly enhances both weight and length gain in Red Hybrid Tilapia compared to the control groups. These effects are particularly evident during the second half of the rearing period, indicating that PSB not only promotes early-stage growth but also supports sustained development. The consistent trend of T3 > T2 > T1 in both parameters confirms the efficacy of PSB as a growth-promoting additive in aquaculture systems. However, integrating PSB into aquaculture presents several challenges, including ensuring compatibility with the Red Hybrid Tilapia culture system. This requires optimizing environmental conditions such as temperature, pH, and nutrient levels to suit both organisms. The pH must be appropriate for both tilapia and PSB, with an optimum of around 7.0 for PSB and a range of 6 to 9 for tilapia (DeLong et al., 2009). Fluctuations in pH can inactivate enzymes and denature proteins, potentially inhibiting the growth and metabolism of both tilapia and PSB (Chen et al., 2020).

3.3 Analysis of Growth performance and FCR of red hybrid Tilapia (Oreochromis spp.) of all treatment’s tanks

Initial weight data shows that fish in T1 started with a significantly higher average weight (21.24 g) compared to T2 (18.23 g) and T3 (17.21 g) (Table 3). Despite starting at a lower initial weight, fish in the T3 group demonstrated the highest final weight at 91.85 g, outperforming T2 (68.18 g) and T1 (54.89 g). Correspondingly, weight gain followed the same trend, with fish in T3 recording a significant gain of 74.64 g, compared to 49.95 g in T2 and 33.65 g in T1, indicating the growth-promoting potential of cultured PSB supplementation. The SGR data also reflects this pattern, with T3 achieving the highest growth rate at 2.65%, followed by T2 at 2.08% and T1 at 1.49%, confirming that the PSB-treated group exhibited the most efficient growth rate. Importantly, survival rates across all groups remained at 100%, indicating that none of the treatments negatively impacted fish viability.
The FCR, an essential indicator of feed efficiency, was most favourable in the PSB group (T3) at 1.202, significantly lower than T2 (1.558) and T1 (2.211). A lower FCR denotes greater feed efficiency, meaning that the fish in T3 required less feed to achieve greater biomass gain. Overall, these results underscore the effectiveness of cultured PSB in significantly enhancing both growth performance and feed efficiency in Red Hybrid Tilapia. The consistent trends across parameters—where T3 > T2 > T1 in final weight, weight gain, and SGR; T3 < T2 < T1 in FCR; and no differences in survival rate. Thus, this validates the potential of PSB as a valuable bio-enhancer in aquaculture systems.

3.4 Chemical Oxygen Demand (COD) analysis

Initial COD measurements on Day 0 were relatively similar across all treatments, with values of 6.72 mg/L (T1), 6.68 mg/L (T2), and 6.84 mg/L (T3), indicating a uniform starting point. As the days progressed, COD levels increased in all groups due to the accumulation of organic matter. However, the rate and extent of COD elevation varied notably among the treatments. By day 60, T1 recorded the highest COD concentration at 67.32 mg/L, followed by T2 at 40.72 mg/L, while T3 maintained the lowest level at 34.68 mg/L. Notably, from Day 15 onwards, COD levels in the T3 group were significantly lower than in both T1 and T2, highlighting the efficiency of cultured PSB in reducing organic pollution in the system (Figure 2).
These results suggest that the metabolic activity of PSB plays a crucial role in breaking down organic compounds, thereby improving water quality. This aligns with previous findings, where PSB demonstrated effective remediation in various high-COD environments (Zhou et al., 2020; Wang et al., 2016). However, it is important to note that the effectiveness of PSB can be diminished when the effluent COD concentration is extremely high (200–1000 mg/L), as reported by Lu et al. (2019), implying that PSB functions optimally in waters with lower organic loads. In conclusion, the application of cultured PSB (T3) significantly enhances water quality by reducing COD more effectively than other treatments in this study, supporting its potential as a sustainable bio-remediation agent in aquaculture systems.

3.5 Total Suspended Solid (TSS) analysis

By the end of the study, T1 recorded the highest TSS level at 0.861 mg/L, followed by T2 at 0.667 mg/L, while T3 maintained the lowest level at 0.431 mg/L. T3 consistently demonstrated lower TSS concentrations, especially during the mid to late phases of the experiment. Statistically significant differences were observed at multiple time points, establishing the trend: T3 < T2 < T1. This suggests that the introduction of cultured PSB effectively reduced the amount of suspended particulate matter in the water.

Figure 2. Variation in Chemical Oxygen Demand (COD) concentration (mg O₂/L) over time in different treatment groups: negative control (T1), positive control (T2), and cultured PSB (T3). Error bars indicate standard deviations (SD) from triplicate samples.

The observed reduction in TSS in T3 may be attributed to microbial floc formation or the PSB’s ability to degrade organic particles, thereby enhancing their settling and removal (Figure 3). Reduced suspended solids are beneficial for fish health, as excessive TSS can clog gills and respiratory passages, causing physical damage or mortality in aquatic organisms (Talaiekhozani and Rezania, 2017). Furthermore, PSB have been reported to remove heavy metals and utilize nutrients such as ammonia, nitrite, and nitrate through photosynthesis and biomass recycling (Puyol et al., 2017), which further contributes to improved water quality. Based on this pattern, we can conclude that the use of cultured PSB (T3) significantly lowers TSS levels, thereby enhancing water clarity and supporting a healthier aquaculture environment for red hybrid tilapia.

Figure 3. Changes in Total Suspended Solids (TSS) concentration (mg/L) over time in different treatment groups: negative control (T1), positive control (T2), and cultured PSB (T3). Error bars represent standard deviations (SD) from triplicate samples. Bars with different letters at the same time point are significantly different (P < 0.05).

4. Conclusions

The current study demonstrates the significant impact of Phosphate Solubilizing Bacteria (PSB) on water quality and growth performance in Red Hybrid Tilapia (Oreochromis spp.). The introduction of PSB effectively reduced ammonia, nitrite, turbidity, and total suspended solids while maintaining optimal levels of pH, temperature, and dissolved oxygen. The PSB treatment resulted in superior fish growth, achieving the highest weight gain (91.80 g), length (17.37 cm), and feed efficiency (FCR = 1.202). Additionally, PSB effectively decreased Chemical Oxygen Demand (COD) and enhanced nutrient cycling, highlighting its potential as an eco-friendly solution for sustainable aquaculture. Despite challenges in optimizing PSB conditions, its ability to improve water quality and enhance fish health suggests promising applications in aquaculture systems. Future studies should explore the long-term stability and large-scale implementation of PSB technology.

Acknowledgements

The authors would like thanks to the Universiti Putra Malaysia Bintulu Sarawak Campus for resources and facilities support.

Funding information

Not applicable.

Data availability

Data will be made available on request.

Informed consent statement

Not applicable.

Conflict of interest

The authors declare no conflict of interest.

Authors’ contribution

Conceptualization: Suhaili Bin Mustafa and Wan Zabidii Wan Morni; Data collection: Muhammad Zaki Zulkiffle, Sophia Peter, Veronica Minnie Gumoi; Data analysis: Muhammad Zaki Zulkiffle, Sophia Peter, Veronica Minnie Gumoi, Suhaili Bin Mustafa; Figure preparation: Lirong Yu Abit; Manuscript internal review: Suhaili Bin Mustafa, Tan Toh Hii, Juriah Kamaludeen, Lirong Yu Abit. All authors also review the final manuscript before submission.