Cultivating Marine Microalgae Nannochloropsis oculata and Chaetoceros muelleri in Shrimp Farming Effluent: Growth and Nutrient Removal for Sustainable Aquaculture

ABSTRACT

Shrimp farming effluents (SFEs) are rich in nitrate and phosphate, posing significant eutrophication risks to receiving water bodies if discharged untreated, necessitating sustainable bioremediation strategies. Although microalgae‐based treatment has been explored, limited research has systematically compared the performance of different species under varying dilution ratios and initial cell densities specifically for shrimp effluent. This study assessed the cultivation performance and nutrient removal efficiency of Nannochloropsis oculata and Chaetoceros muelleri for bioremediation of SFE, focusing on nitrate and phosphate removal. Experiments evaluated nutrient dynamics and algal growth across SFE dilutions (0%–50%) and initial cell densities ( C. muelleri : 35 × 10⁴ and 70 × 10⁴ cells/mL; N. oculata : 200 × 10⁴ and 400 × 10⁴ cells/mL). Both species demonstrated effective nutrient removal, with higher removal efficiencies observed at lower initial densities. Under optimal 25% SFE conditions, N. oculata achieved 97% nitrate and 95% phosphate removal within 12 days, while C. muelleri reached 81% nitrate and 94% phosphate removal within 6 days. Specific growth rates surpassed controls, yielding maximum biomasses of 2425 × 10⁴ ( N. oculata , 12 days) and 595 × 10⁴ cells/mL ( C. muelleri , 10 days). The novelty of this study lies in the systematic optimization of operational parameters and identification of species‐specific advantages for tailored bioremediation. These findings highlight the robustness of both microalgae across SFE gradients and support their application in sustainable aquaculture wastewater treatment, nutrient recycling, and biomass valorization. Future studies should focus on pilot‐scale validation, long‐term stability assessment, and comprehensive biomass characterization for downstream applications.

Summary

• N. oculata removes 97% nitrates from SFE in 12 days at low density.

• C. muelleri achieves 81% nitrate, 94% phosphate removal in 6 days.

• Max biomass: N. oculata 2425 × 10⁴ cells/mL at 25% SFE in 12 days.

• C. muelleri peaked at 595 × 10⁴ cells/mL in 10 days, 25% SFE.

• Both species boost growth rates, aiding sustainable SFE bioremediation.

Both Nannochloropsis oculata and Chaetoceros muelleri effectively removed nitrate and phosphate from shrimp farming effluents. Under optimal 25% SFE, N. oculata (200 × 10⁴ cells/mL) removed 97% nitrate and 95% phosphate within 12 days, while C. muelleri (35 × 10⁴ cells/mL) achieved 81% nitrate and 94% phosphate removal within 6 days.

1. Introduction

Microalgae are regarded as a promising alternative source of nutrition due to their rich biochemical composition, including protein content, all essential amino acids, essential vitamins, minerals, and unsaturated fatty acids with multiple double bonds (Kumar et al. 2022). Their nutritional profile positions them as valuable contributors to both human and animal diets. In aquatic ecosystems, microalgae play a vital role during the early developmental stages of larval organisms by supplying essential nutrients required for successful metamorphosis and maturation (Chen et al. 2021). Beyond their nutritional value, microalgae have gained prominence in bioremediation applications, particularly for the treatment of various wastewater types such as urban, aquaculture, and industrial effluents (Omokaro et al. 2025). Through photosynthetic activity, microalgae efficiently convert solar energy into biomass while simultaneously assimilating nutrients like nitrogen and phosphorus from wastewater streams (Calatrava et al. 2024; Omokaro et al. 2025). This dual functionality not only reduces nutrient loads that can cause eutrophication and harmful algal blooms in aquatic environments but also produces biomass that can be exploited for biofuel production, animal feed, and other bioproducts, thereby offering considerable environmental and economic advantages.

Aquaculture production has reached unprecedented scales globally, with seafood output reported at 223.2 million tons in 2022 (FAO 2024). However, land‐based aquaculture systems demand substantial volumes of water to maintain productivity, consequently generating large amounts of effluents that contain suspended solids and pollutants such as phosphorus, carbon, and nitrogen (Ansari et al. 2019). If inadequately managed, these effluents can severely degrade receiving water bodies, leading to ecological imbalances (Thomsen et al. 2020). Numerous technologies have been employed to minimize water pollution, among which bioremediation is an emerging and promising approach. Various technological interventions have been developed to mitigate water pollution, among which bioremediation, using naturally occurring microorganisms, has emerged as an effective and cost‐efficient approach (Calatrava et al. 2024; Omokaro et al. 2025). Bioremediation reduces organic carbon compounds and, importantly, decreases concentrations of nitrogen and phosphorus prior to effluent discharge, which is critical because elevated nutrient levels negatively influence dissolved oxygen availability, nutrient balance, and the health of aquatic flora and fauna (Tsani et al. 2020). Enhancing the ecological status of water bodies by reducing nitrogen and phosphorus in wastewater remains a priority for both developed and developing nations.

Microalgae‐based bioremediation leverages their capacity to assimilate nitrogen and phosphorus from wastewater for cellular growth. These organisms absorb and convert pollutants into biomass, which can be harvested for various uses. However, challenges persist regarding the economic feasibility of microalgal cultivation due to high costs associated with continuous supplementation of expensive nutrient media such as Conway or Walne formulations (Dias et al. 2025). The yield and nutritional quality of microalgal biomass are strongly influenced by the availability and balance of macronutrients and micronutrients, necessitating carefully optimized culture media. Aquaculture effluent, including wastewater from shrimp farming, represents a viable alternative culture medium due to its nutrient richness. Utilizing such effluent not only lowers operational costs but also supports environmentally sustainable practices by improving water quality through nutrient removal (Calatrava et al. 2024; Omokaro et al. 2025). Additionally, microalgae have demonstrated the capability to remediate other contaminants, including heavy metals and pesticides found in industrial and agricultural wastewaters (Rawat et al. 2011).

Despite these advantages, research on the concurrent production of microalgal biomass and bioremediation of aquaculture wastewater remains limited. While several studies have individually explored microalgal cultivation in aquaculture effluents, a notable research gap persists regarding the systematic comparison of different microalgal species under identical operational conditions, particularly with respect to the interactive effects of effluent dilution and initial cell density on nutrient removal kinetics and biomass productivity. Furthermore, most existing investigations have focused on either single species or have not provided a comprehensive assessment of both nutrient removal efficiency and growth performance across varying effluent concentrations. The novelty of the present study lies in its comprehensive and systematic approach that directly compares two commercially and ecologically relevant species, Nannochloropsis oculata and Chaetoceros muelleri , under a matrix of SFE dilutions (0%–50%) and two distinct initial cell densities for each species. This study uniquely identifies species‐specific optimal conditions, demonstrating distinct temporal patterns in nutrient removal, where N. oculata achieves superior nitrate removal and C. muelleri exhibits faster phosphate uptake. Additionally, the investigation confirms that both species exhibit enhanced growth rates in shrimp effluent compared with conventional nutrient media, highlighting the dual benefit of wastewater treatment and high‐value biomass production. These integrated systems present complexity and potential economic constraints that may hinder scalability (Zhu et al. 2022). Optimal system performance depends on the selection of microalgal strains adept at adapting to wastewater conditions and efficiently adsorbing pollutants. The phytoremediation efficiency and biomass productivity of microalgae are further modulated by wastewater characteristics such as pollutant type and concentration, pH, salinity, and turbidity (Hussain et al. 2021). In this context, the present study aims to assess the biomass productivity and nutrient uptake efficiency of two microalgal species, N. oculata and C. muelleri , cultivated using shrimp farm effluent (SFE) as a growth medium. Furthermore, it intends to evaluate their potential utility in wastewater treatment and nutrient removal, thus emphasizing their dual role in biomass generation and environmental remediation. This research contributes to advancing sustainable aquaculture practices and integrated wastewater management strategies by harnessing the functional properties of microalgae.

2. Materials and Methods

2.1. Microalgae Strains and Culture Media

Two marine microalgae species, N. oculata and C. muelleri , were used in this study. Both strains were obtained from the Fisheries Research Station in Bandar Lengeh, Hormozgan Province, southern Iran. The cultures were grown in F/2 Guillard's medium prepared with filtered and sterilized artificial seawater adjusted to a salinity of 30 practical salinity units (psu). The artificial seawater was prepared by dissolving 30 g of synthetic sea salt (Continuum Aquatics Halcyon Marine Reef Salt; Nasimeyas Co., Iran) per 1 L of deionized water. Salinity was verified using a refractometer to ensure measurement accuracy. Each experimental culture was initiated by inoculating an appropriate volume of exponentially growing parental culture into the test medium to achieve an initial biomass concentration representative of the exponential growth phase.

2.2. Wastewater Sample

The SFE used in this study was collected from the First Shour site in Bandar Abbas, Hormozgan Province, southern Iran. The effluent originated from a 3‐month‐old shrimp pond. The mean and standard deviation (SD) for the physicochemical characteristics of the SFE are summarized in Table 1. The parameters were quantified using standardized analytical methods for the examination of water and wastewater (Lipps et al. 2022).

TABLE 1.

The physicochemical characteristics of the SFE.

Characteristic	Value
pH	8.11 ± 0.12
Nitrate (mg/L)	5.86 ± 0.14
Phosphate (mg/L)	0.98 ± 0.11
Salinity (g/L)	49.15 ± 1.88
TDS (mg/L)	85.4 ± 3.92
COD (mg/L)	9.2 ± 1.86

Among the various physicochemical parameters characterizing SFEs, nitrate and phosphate were specifically selected as the primary target nutrients in this study due to their established role as the main limiting factors driving eutrophication in coastal and marine ecosystems. These nutrients are discharged in substantial concentrations in aquaculture wastewater and are directly associated with the proliferation of harmful algal blooms, oxygen depletion, and subsequent degradation of water quality in receiving water bodies. While other parameters such as pH, salinity, total dissolved solids, and chemical oxygen demand are important for characterizing the overall quality of the effluent and ensuring suitable conditions for microalgal cultivation, the primary objective of bioremediation in this context is the reduction of inorganic nutrient loads, particularly nitrogen and phosphorus, to mitigate their environmental impact. Therefore, the present study focused on nitrate and phosphate removal as the key performance indicators for evaluating the bioremediation potential of the selected microalgal species, while the additional physicochemical parameters presented in Table 1 were monitored to provide a comprehensive baseline characterization of the SFE and to confirm that the experimental conditions remained within the optimal range for microalgal growth throughout the cultivation period.

2.3. Cultivation Procedure

As an initial step to facilitate cultivation and establish an adequate inoculum density, a two‐phase cultivation process was implemented for both N. oculata and C. muelleri . In the first phase, each species was cultured separately by inoculating 10 mL of its respective starter culture into 250‐mL Erlenmeyer flasks containing 190 mL of F/2 Guillard's medium. The cultures were maintained for 15 days. In the subsequent phase, an appropriate volume of biomass from the first phase was transferred to experimental media consisting of either F/2 Guillard's medium alone (as control) or medium supplemented with 10%, 25%, or 50% (w/v) SFE to reach the initial cell densities of 35 × 10⁺⁴ and 70 × 10⁺⁴ cells/mL for C. muelleri and the initial cell densities of 200 × 10⁺⁴ and 70 × 10⁺⁴ cells/mL for N. oculata . The selection of these specific initial cell densities was based on a series of preliminary screening experiments in which a broader range of densities (including lower and higher concentrations) was tested to determine the optimal range for both species under SFE conditions. These preliminary trials revealed that excessively low densities resulted in prolonged lag phases and insufficient nutrient uptake, while excessively high densities led to rapid nutrient depletion, self‐shading, and early growth cessation. The total culture volume was adjusted to 400 mL to achieve an initial cell density corresponding to the exponential growth phase. Cultivation continued until the stationary (constant growth) phase was reached. All experiments were conducted at a controlled temperature of 25°C ± 0.5°C, under a light intensity of 3500 lx, with a 16:8 h light:dark photoperiod. Continuous aeration was supplied from the bottom of each vessel throughout the experiment. All treatments were performed in triplicate. Table 2 summarizes the specific experimental conditions, including initial cell densities and the percentage of culture medium replaced with SFE.

TABLE 2.

Experimental conditions, including initial cell densities and the percentage of culture medium replaced with SFE.

Experimental condition	Microalgae	Initial cell density (cells/mL)	SFE percentage (%)
1	C. muelleri	35 × 10+4	0% (control)
2			10%
3			25%
4			50%
5	C. muelleri	70 × 10+4	0% (control)
6			10%
7			25%
8			50%
9	N. oculata	200 × 10+4	0% (control)
10			10%
11			25%
12			50%
13	N. oculata	400 × 10+4	0% (control)
14			10%
15			25%
16			50%

2.4. Analytical Methods

Algal growth was monitored every 24 h by counting cells with a hemocytometer. A = N1 − N02 × t1 + N1 + N2 − 2N02 × t2 − t1 + … + Nn − 1 + Nn − 2 N02 × (tn − tn − 1). Nitrate (NO₃ ⁻) and phosphate (PO₄ ³⁻) concentrations were determined following the standard methods 4500‐NO₃ ⁻ B and 4500‐P E, respectively, using a UV–visible (UV–Vis) spectrophotometer (Cecil Instruments, ce 2501, UK). These analyses followed the procedures described in Standard Methods for the Examination of Water and Wastewater (Lipps et al. 2022). The nutrient removal efficiency (%) of nitrate and phosphate by microalgae in SFE was calculated based on the initial (C _i) and final (C _f) concentrations of these nutrients during batch cultivation, according to Equation (1):

$$\text{Removal efficiency}\left(\%\right)=100\left(C_{\mathrm{i}}-C_{\mathrm{f}}\right)/C_{\mathrm{i}}$$ (1)

where C _i and C _f represent the initial and final concentrations of nitrate (mg NO₃ ⁻‐N/L) or phosphate (mg PO₄ ³⁻‐P/L), respectively. Chlorophyll a (Chl. a) content was determined following the method of Jeffrey and Humphrey (1975). Briefly, 5 mL of 90% acetone (Merck) was added to 0.01 g of harvested biomass from each test culture. The suspension was vortexed vigorously for 2 min and then incubated in the dark at 4°C for 24 h. Samples were centrifuged at 1300 g for 15 min, and the absorbance of the supernatant was measured at 664 and 630 nm using a UV–Vis spectrophotometer (Cecil Instruments, ce 2501, UK). Chl. a concentration (μg L⁻¹) was calculated using Equation (2):

$$\text{Chlorophyll}a=11.47{\mathrm{A}}_{664}-0.40{\mathrm{A}}_{630}$$ (2)

where A₆₆₄ and A₆₃₀ are the absorbance values at 664 and 630 nm, respectively. Media samples were centrifuged at 1300 g for 15 min to obtain clear supernatants, which were analyzed immediately for nitrate and phosphate concentrations to prevent sample degradation. All measurements were performed in triplicate.

2.5. Statistical Analysis

Data were assessed for normality and homogeneity using the Kolmogorov–Smirnov and Levene's tests, respectively. Differences among treatments were analyzed using one‐way analysis of variance (ANOVA), and Duncan's multiple range test was applied at a 95% confidence level to identify significant differences (p < 0.05). One‐way ANOVA was applied separately for each factor to compare treatment means within specific categories, such as comparing different SFE concentrations at a fixed initial density or comparing different initial densities at a fixed SFE concentration. This approach was chosen because the primary objective was to evaluate the individual effects of each factor on growth and nutrient removal independently, rather than to assess interactive effects among all factors simultaneously. Moreover, the experimental design consisted of independent treatment groups, and the data for each factor were analyzed separately to avoid overcomplicating the statistical model while still providing clear and interpretable results. All statistical analyses were conducted using SPSS software (Version 19.0). When variances were homogeneous, post hoc comparisons were performed to evaluate differences between treatment means. Graphs and figures were generated using Microsoft Excel and Simplot 14.0. All data are presented as mean ± SD from three replicates.

3. Results and Discussion

3.1. Effects of the SFE on C. muelleri Cell Growth Rate

Figure 1 illustrates the growth of C. muelleri at initial cell densities of 35 × 10⁺⁴ and 70 × 10⁺⁴ cells/mL in various culture media over periods of 6 and 10 days, respectively. The data indicate that this microalga can grow in SFE, but the growth rate depends on the SFE percentage. Initially, growth across all treatment groups was comparable with or even exceeded that the control; however, during the exponential growth phase, low to moderate SFE percentages (10% and 25%) exhibited superior growth rates. The highest cellular proliferation of 10,595 × 10⁺⁴ cells/mL was observed at the 25% percentage. Conversely, growth was reduced at higher (50%) and lower (10%) SFE percentages compared with the 25% treatment group. By the final days, growth in all groups plateaued, likely due to a combination of nutrient depletion, metabolic waste accumulation, and the progressive inhibitory effects of certain SFE components on algal physiology. Notably, preliminary experiments using SFE concentrations above 50% (70%, 80%, and 100%) resulted in complete cell death within 48 h, indicating the presence of toxic or growth‐inhibitory substances in the effluent that are lethal at elevated concentrations. This observation further supports the hypothesis that growth inhibition at higher SFE percentages is not solely due to nutrient limitation but rather to the accumulation of toxic compounds. These findings suggest that moderate SFE percentages (25%) represent the optimal balance between sufficient nutrient availability and minimal inhibitory effects, thereby effectively stimulating algal proliferation and nutrient removal efficiency.

FIGURE 1.

The growth of C. muelleri at initial cell densities of 35 × 10⁺⁴ and 70 × 10⁺⁴ cells/mL in various culture media over periods of 6 and 10 days, respectively.

Figure 2a presents a comparative analysis of C. muelleri cell counts on Days 6, 8, and 10 at an initial density of 70 × 10⁺⁴ cells/mL, demonstrating that SFE percentage significantly affects cell growth. On Day 6, cell counts at 25% (535 × 10⁴) and 50% (560 × 10⁴) SFE were not significantly different from the control (540 × 10⁺⁴ cells/mL). The 10% SFE percentage showed a significant reduction in growth relative to other percentages; however, the cell counts at this level (480 × 10⁺⁴ cells/mL) remained substantial. These results suggest an adaptation of the algae at higher SFE percentages after 6 days of cultivation.

FIGURE 2.

Comparative analysis of C. muelleri cell counts under different conditions (a) on Days 6, 8, and 10 at initial density of 70 × 10⁺⁴ cells/mL; (b) on Days 4, 6, and 8 at initial density of 35 × 10⁺⁴ cells/mL; and (c) the comparative effects of different SFE percentages on the growth of C. muelleri at two initial cell densities: 35 × 10⁺⁴ (after 6 days) and 70 × 10⁺⁴ cells/mL (after 10 days).

After 8 days, cell counts at 10% SFE concentration were lower compared with the control group, which may be attributed to limited nutrient availability in the culture medium. At 25% SFE percentage, cell counts (585 × 10⁺⁴ cells/mL) increased relative to the 10% treatment, and cell density was similar to the control (600 × 10⁺⁴ cells/mL), indicating no adverse effects. Both 10% and 50% exhibited significant declines.

After 10 days, the same trend persisted: 25% SFE supported optimal growth (595 × 10⁺⁴ cells/mL), while 10% and 50% suppressed cell proliferation. Overall, optimal growth over the 10‐day period was observed at moderate SFE percentages (25%) with 595 × 10⁺⁴ cells/mL.

Figure 2b compares the cell counts of C. muelleri after 4, 6, and 8 days under different conditions, with an initial density of 35 × 10⁺⁴ cells/mL. After 4 days, only the 50% treatment (285 × 10⁺⁴ cells/mL) significantly exceeded the control, indicating a positive early effect. The cell count data after 6 days revealed contrasting effects of different SFE percentages on growth. In the 10% and 50% SFE treatment groups, cell counts were lower than those recorded in the control group, suggesting that at 10% SFE, nutrient availability was insufficient to support optimal growth, while at 50% SFE, growth inhibition was likely due to the presence of inhibitory or toxic compounds in the effluent rather than nutrient limitation. However, in the 25% SFE treatment, cell counts were 440 × 10⁺⁴ cells/mL higher than other SFE percentages but remained lower than the control (520 × 10⁺⁴ cells/mL), suggesting that moderate wastewater levels support improved growth relative to other SFE treatments.

After 8 days, the growth trend of the C. muelleri was similar to that observed on Day 6, except that at 25% SFE percentage, cell proliferation increased further. The 25% treatment (410 × 10⁺⁴ cells/mL) approached control levels (450 × 10⁺⁴ cells/mL), while 10% (240 × 10⁺⁴ cells/mL) and 50% (320 × 10⁺⁴ cells/mL) confirmed inhibitory effects at both extremes.

Overall, the growth rate of C. muelleri was higher at moderate SFE percentages, indicating the existence of an optimal percentage for algal cultivation. These findings support the potential application of C. muelleri for treating low to medium percentages of organic wastewaters while simultaneously producing valuable bioproducts.

The growth pattern of C. muelleri across all treatment groups consistently followed a sigmoidal or logistic growth curve, yet the duration of the lag phase varied notably depending on both the initial inoculation density and the concentration of SFE. At the higher initial density of 70 × 10⁺⁴ cells/mL, the lag phase was uniformly less than 2 days for all treatments, indicating that a sufficiently dense inoculum facilitates rapid acclimatization and early onset of exponential division. In contrast, when the initial density was lowered to 35 × 10⁺⁴ cells/mL, a distinct divergence in lag phase duration emerged: The control group and the 25% SFE treatment both exhibited a prolonged lag period of 4 days, whereas the 10% and 50% SFE treatments displayed a markedly shorter lag of only 2 days, suggesting that the nutritional bioavailability and stimulatory efficacy of SFE are concentration dependent and do not follow a simple linear trend. Despite these initial discrepancies in lag phase, the subsequent growth trajectories of all experimental groups converged toward a similar pattern, with the exponential phase consistently initiating after 2 days of cultivation and progressing into the early stationary phase by Day 6, which indicates that once the cells overcome the adaptation barrier, the intrinsic specific growth rate is predominantly governed by the ambient physicochemical conditions rather than by the initial treatment differences. Such alterations in lag phase duration in response to varying initial cell densities and organic supplementation have been previously documented in microalgal cultivation studies, reinforcing the observation that inoculum size and medium composition interact in a complex manner to shape the early growth kinetics of marine diatoms (Nallapan et al. 2022).

These findings carry significant biotechnological implications, as optimizing the initial cell density and fine‐tuning the concentration of organic supplements like SFE can effectively reduce the lag period and thereby enhance the productivity of large‐scale C. muelleri cultures intended for aquaculture feeding or bioactive compound extraction.

Figure 2c compares C. muelleri growth at two initial densities, 35 × 10⁺⁴ (after 6 days) and 70 × 10⁺⁴ cells/mL (after 10 days), across different SFE percentages, corresponding to the periods of maximum cell proliferation. At 25% SFE, both densities showed higher growth than other percentages. Notably, at the initial density of 70 × 10⁴ cells/mL and 25% SFE after 10 days, the cell count (595 × 10⁺⁴ cells/mL) was statistically similar to the control (640 × 10⁺⁴ cells/mL). In contrast, all other SFE percentages exhibited significantly lower cell counts. Thus, the higher initial density (70 × 10⁴ cells/mL) over 10 days yielded superior performance. These findings support using wastewater as a sustainable, cost‐effective nutrient source for microalgal biomass production and environmental remediation.

3.2. Effects of the SFE on N. oculata Cell Growth Rate

Figure 3 shows N. oculata growth over 14 days at initial densities of 400 × 10⁺⁴ and 200 × 10⁺⁴ cells/mL across SFE concentrations. The results indicate that adding 10% SFE to the culture medium represents the optimal percentage for N. oculata growth, as cell densities in this treatment were equivalent to or exceeded the control on Day 10 at initial cell densities of 400 × 10⁺⁴ and 200 × 10⁺⁴ cells/mL, respectively. Notably, at 200 × 10⁺⁴ cells/mL and 10% SFE, growth surpassed the control and other treatments after 12 days.

FIGURE 3.

The changes in the cell count of N. oculata over 14‐day cultivation period, using initial cell densities of 400 × 10⁺⁴ and 200 × 10⁺⁴ cells/mL in different culture media.

Figure 4a presents of N. oculata cell counts after 4, 6, 8,10, and 12 days at an initial cell density of 400 × 10⁺⁴ cells/mL across SFE percentages. After 4 days, cell growth in 10% (930 × 10⁺⁴ cells/mL) and 50% (1305 × 10⁺⁴ cells/mL) SFE exceeded the control (785 × 10⁺⁴ cells/mL), while in 25% (645 × 10⁺⁴ cells/mL) was lower. After 6 days, all treatments surpassed the control, with 25% (1615 × 10⁺⁴ cells/mL) and 50% (1515 × 10⁺⁴ cells/mL) showing the highest counts. The overall trend demonstrated an increase in cell numbers with increasing SPE percentage.

FIGURE 4.

Comparative analysis of the cell count of N. oculata (a) after 4, 6, 8,10, and 12 days at an initial cell density of 400 × 10⁺⁴ cells/mL; (b) after 8, 10, 12, and 14 days at an initial cell density of 200 × 10⁺⁴ cells/mL in different culture media; and (c) the comparative effects of different SFE percentages on the growth of N. oculata at two initial cell densities: 200 × 10⁺⁴ (after 12 days) and 400 × 10⁺⁴ cells/mL (after 10 days).

After 8 days, cell growth at 10% (1915 × 10⁺⁴ cells/mL) and 50% (1745 × 10⁺⁴ cells/mL) was statistically similar to the control (2120 × 10⁺⁴ cells/mL), and 25% (~1700 × 10⁺⁴ cells/mL) remained substantial. Cell counts reached approximately 1700 × 10⁺⁴ cells/mL at 25% SFE percentage, reflecting that all three SPE percentages yielded notable improvements in biomass production.

After 10 days, cell counts only in the 10% treatment (2190 × 10⁺⁴ cells/mL) matched the control (2230 × 10⁺⁴ cells/mL), while in the 25% and 50%, they declined but remained viable (~1750–1850 × 10⁺⁴ cells/mL). Despite the reduction in cell growth at higher SFE percentages relative to the control, cell proliferation remained substantial, indicating that SFE supplementation significantly influences cell growth, with all three concentrations demonstrating favorable performance.

After 12 days, interesting growth patterns emerged. Ten percent SFE (1980 × 10⁺⁴ cells/mL) significantly exceeded the control (1655 × 10⁺⁴ cells/mL), whereas 50% (1800 × 10⁺⁴ cells/mL) was comparable with the control, and 25% was lower.

Overall, N. oculata exhibited a time‐ and percentage‐dependent response to SFE‐containing media over a 12‐day cultivation period starting from an initial cell density of 400 × 10⁺⁴ cells/mL. The additives influenced growth in a nuanced, nonmonotonic manner, with distinct patterns emerging at different time points. The observed trend suggests that with increasing incubation time, 10% and 50% SFE percentages could serve as suitable substitutes for standard culture media. For practical applications, aligning SFE percentage with the intended harvest window is crucial to maximize biomass production within the 10–12 days range.

Figure 4b illustrates the changes in the cell count of N. oculata after 8, 10, 12, and 14 days at an initial cell density of 200 × 10⁺⁴ cells/mL across SFE percentages. After 8 days, the control showed the highest growth. By Day 10, the control (1960 × 10⁺⁴ cells/mL) and 10% SFE (2075 × 10⁺⁴ cells/mL) performed comparably, while 25% and 50% also supported notable proliferation despite reduced growth. After 12 days, 25% SFE (2425 × 10⁺⁴ cells/mL) significantly exceeded the control (1985 × 10⁺⁴ cells/mL), whereas 10% (1870 × 10⁺⁴ cells/mL) and 50% (1750 × 10⁺⁴ cells/mL) did not differ significantly from the control.

After 14 days, 25% SFE remained optimal with maximum growth (2125 × 10⁺⁴ cells/mL), while both 10% and 50% resulted in reduced growth. Overall, 25% SFE provided the most sustained and robust cell proliferation over extended cultivation.

Over the 14‐day cultivation period at 200 × 10⁺⁴ cells/mL, N. oculata exhibited a clear percentage‐dependent response to SFE. The 25% SFE provided the most robust and sustained proliferation, particularly at Days 12 and 14, while 50% SFE generally imposed inhibitory effects, and 10% SFE offered only modest early growth comparable with the control.

The growth pattern of N. oculata across all treatment groups consistently followed a sigmoidal growth curve, yet the duration of the lag phase exhibited marked variation depending on both the initial inoculation density and the concentration of SFE applied. At the higher initial density of 400 × 10⁺⁴ cells/mL, the control group and the 25% SFE treatment both displayed a lag phase of 4 days, whereas the 10% and 50% SFE treatments showed a significantly shorter lag period of only 2 days, indicating that at specific concentrations, the organic supplementation effectively accelerated cellular acclimatization and reduced the time required for the population to enter exponential division. In contrast, when the initial density was lowered to 200 × 10⁺⁴ cells/mL, the lag phase was consistently prolonged across all treatments, with the control, the 25% SFE, and the 50% SFE groups all exhibiting a lag period of 6 days, while only the 10% SFE treatment demonstrated a comparatively shorter lag of 4 days. This observation reveals that the beneficial effect of SFE on shortening the lag phase is not only concentration dependent but also critically modulated by the initial cell density. The prolongation of the lag phase at the lower density across nearly all treatments is biologically consistent with the well‐documented phenomenon that sparse populations require extended time to initiate coordinated division, and this inherent delay can only be partially mitigated by organic enrichment, as seen in the 10% SFE treatment which still required 4 days to exit the lag phase. The differential response to SFE concentrations further suggests that the bioactive constituents of SFE exert a biphasic or nonlinear influence on cellular physiology, where an optimal concentration such as 10% provides the most effective nutritional priming, while higher concentrations like 50% may introduce inhibitory components or imbalanced nutrient ratios that diminish their growth‐promoting efficacy, particularly when the population is already challenged by low initial density. The transition into the exponential phase and subsequent progression to stationary growth followed a similar temporal framework across all treatments once the lag phase was overcome, indicating that the intrinsic maximum growth rate of N. oculata under the given culture conditions was largely governed by the physicochemical parameters of the medium rather than by the initial treatment differences. Such alterations in lag phase duration in response to varying cell densities and organic supplementation have been previously documented in microalgal cultivation studies, reinforcing the understanding that inoculum size and medium composition interact in a complex manner to shape early growth kinetics, as similarly reported by Nallapan et al. (2022) in their investigation of natural extracts as growth enhancers for microalgae. These findings collectively demonstrate that the addition of SFE does influence the lag phase of N. oculata in a manner that is both concentration and density dependent, with the most pronounced stimulatory effect observed at 10% SFE under both high and low initial densities, while the control and other concentrations either failed to shorten or even prolonged the adaptation period under suboptimal inoculum conditions.

Figure 4c presents the growth of N. oculata at two initial densities, 200 × 10⁺⁴ (after 12 days) and 400 × 10⁺⁴ cells/mL (after 10 days), corresponding to peak proliferation periods across SFE percentages. At 25% SFE, cultures initiated at 200 × 10⁺⁴ cells/mL achieved the highest cell count (2425 × 10⁺⁴ cells/mL), exceeding the control and other SFE percentages. At 10% SFE, cultures at 400 × 10⁴ cells/mL showed superior growth (2190 × 10⁺⁴ cells/mL), while those at 200 × 10⁺⁴ cells/mL and 10% SFE performed comparably with the control (1870 × 10⁺⁴ vs. 1985 × 10⁺⁴ cells/mL) after 12 days. Notably, the 200 × 10⁺⁴ cells/mL inoculum at 25% SFE after 12 days significantly outperformed the control, highlighting the optimal combination of moderate SFE and lower initial density for maximizing biomass production.

These findings support the use of wastewater as a sustainable and economical nutrient source for microalgal cultivation, offering a viable strategy for biomass production and environmental remediation.

3.3. Changes in Chl. a Production

The total Chl. a in the microalga C. muelleri after 10 days of cultivation with an initial cell density of 70 × 10⁺⁴ cells/mL indicated that the addition of SFE to the culture medium had a significant effect on Chl. a production (Figure 5a). This initial cell density was selected because it exhibited greater growth after 10 days compared with the lower initial density of 35 × 10⁺⁴ cells/mL. At SFE percentages of 10% and 50%, Chl. a production decreased, suggesting that these percentages may negatively affect the photosynthesis process. At 25% SFE, Chl. a production (6.2 mg/L) increased compared with the 10% (5.0 mg/L) and 50% (5.3 mg/L) treatments, but it remained lower than in the control group (9.1 mg/L). These results demonstrate that low and high SFE percentages reduce Chl. a production, while intermediate percentages have a less pronounced effect. Thus, optimizing SFE concentration is crucial for maintaining photosynthetic efficiency in C. muelleri during wastewater treatment applications.

FIGURE 5.

The total Chl. a content in (a) the microalga C. muelleri after 10 days of cultivation with an initial cell density of 70 × 10⁺⁴ cells/mL and (b) the N. oculata after 12 days of cultivation with an initial cell density of 200 × 10⁺⁴ cells/mL, in different SFE percentages.

Figure 5b illustrates the Chl. a content in N. oculata after 12 days of cultivation with an initial cell density of 200 × 10⁺⁴ cells/mL, indicating that the addition of SFE had a statistically significant effect on Chl. a production. The 25% SFE treatment yielded the highest Chl. a content (1.2 mg/L), exceeding both the control (0.8 mg/L) and other SFE percentages, indicating optimal conditions for pigment synthesis at this concentration. These findings demonstrate that N. oculata can sustain Chl. a production even in wastewater‐supplemented media, a trait that may be advantageous for wastewater treatment and the production of biofuels and other high‐value bioproducts.

Chl. a production was assessed as a proxy for photosynthetic activity in both microalgal species, C. muelleri and N. oculata . The results indicated that SFE percentage modulates Chl. a production in C. muelleri : At low (10%) and high (50%) SFE percentages, Chl. a declined, while at the intermediate percentage (25%), Chl. a was higher than in the other SFE treatments but still below the control (Figure 5a). This pattern suggests that intermediate SFE percentages may create more favorable conditions for photosynthesis and pigment synthesis, likely by balancing nutrient availability with potential inhibitory effects associated with extreme percentages. Because Chl. a content reflects photosynthetic performance, these findings have practical implications for wastewater treatment and the production of valuable biomass for industries such as biodiesel and pharmaceuticals.

The microalga N. oculata maintained Chl. a production in the presence of SFE, and under certain conditions, specifically at 25% wastewater percentage, Chl. a production was significantly higher than in the control group (Figure 5b). This trait may be advantageous for bioproduction processes, particularly in wastewater‐containing environments where nutrient availability is often limited, enabling the alga to sustain photosynthetic performance and effectively meet its nutritional requirements.

The relationship between Chl. a content and nutrient availability is well documented in microalgal physiology. Changes in intracellular pigment concentrations are directly linked to nutrient uptake and assimilation strategies that serve the metabolic needs of the cell rather than photosynthesis alone (Pérez‐Morales et al. 2023). Nitrogen, as a critical component of chlorophyll molecules, directly influences pigment synthesis, and studies have demonstrated that nitrate availability significantly affects Chl. a production across various microalgal species (Akter et al. 2024). In C. muelleri , Chl. a production decreased at both low (10%) and high (50%) SFE concentrations while showing improvement at the intermediate 25% treatment, suggesting that suboptimal SFE percentages induce physiological stress that impairs photosynthetic pigment synthesis. This pattern is consistent with findings that both nutrient deprivation and excessive organic loading can disrupt cellular metabolism and reduce pigment content (Pérez‐Morales et al. 2023). The increased Chl. a production observed in N. oculata at 25% SFE, exceeding even the control conditions, indicates that moderate wastewater supplementation can provide an optimal balance of nutrients that stimulates photosynthetic apparatus development, a phenomenon previously attributed to enhanced nitrogen availability facilitating greater pigment biosynthesis (Akter et al. 2024). Regarding physiological stress, high SFE concentrations likely impose oxidative stress on the cells, as salinity and organic loads in shrimp effluent can disrupt cellular homeostasis and trigger stress responses that divert energy from pigment synthesis toward protective mechanisms (Pérez‐Morales et al. 2023). The species‐specific responses observed between C. muelleri and N. oculata further confirm that metabolic efficiency and stress tolerance vary markedly among different phytoplankton groups, with some species demonstrating greater capacity to maintain photosynthetic performance under wastewater conditions.

3.4. Nitrate and Phosphate Removal by C. muelleri

Figure 6a compares nitrate removal by C. muelleri at initial densities of 35 × 10⁺⁴ and 70 × 10⁺⁴ cell/mL after 6 and 10 days, respectively. At the lower density, nitrate removal reached ~79% and remained consistent across SFE percentages, whereas significantly lower removal was observed at the higher density. A similar density‐dependent trend was noted for phosphate removal, indicating that initial cell density critically influences nutrient uptake efficiency. These findings confirm the high nitrate removal capacity of C. muelleri and support its potential as an effective biological agent for treating nitrate‐laden wastewater.

FIGURE 6.

The effect of the initial density of (a) the C. muelleri on nitrate and (b) phosphate removal at two cell densities: 35 × 10⁺⁴ (after 6 days) and 70 × 10⁺⁴ cell/mL (after 10 days) and (c) the N. oculata on nitrate and (d) phosphate removal at two cell densities: 200 × 10⁺⁴ (after 12 days) and 400 × 10⁺⁴ cell/mL (after 10 days).

Figure 6b further demonstrates the high phosphate removal capacity of C. muelleri . At the lower initial density (35 × 10⁺⁴ cells/mL), phosphate removal reached ~92% and remained consistent across all SFE percentages, significantly exceeding that observed at the higher density (70 × 10⁺⁴ cells/mL). These findings confirm the alga's effectiveness in reducing phosphate pollution, consistent with its typical nutrient uptake physiology. The resilience of C. muelleri across varying SFE concentrations highlights its potential as a sustainable and efficient biological agent for nutrient removal in wastewater treatment systems.

3.5. Nitrate and Phosphate Removal by N. oculata

Figure 6c shows the effect of the initial density of the alga N. oculata on nitrate removal at two cell densities: 200 × 10⁺⁴ (after 12 days) and 400 × 10⁺⁴ cell/mL (after 10 days). At the lower density, removal efficiency reached ~95% and remained consistent across all SFE percentages, significantly outperforming the higher density. At 400 × 10⁺⁴ cells/mL, nitrate removal increased with SFE concentration, exceeding 80% at 25% and 50% SFE. A significant difference was observed between the two initial algal cell densities (200 × 10⁺⁴ and 400 × 10⁺⁴) in the extent of nitrate removal. These findings underscore that initial cell density and treatment duration are critical factors in optimizing nitrate removal, providing valuable insights for designing N. oculata ‐based wastewater treatment systems.

Figure 6d shows the effect of the initial cell density of the microalga N. oculata on phosphate removal at two cell densities: 200 × 10⁺⁴ (after 12 days) and 400 × 10⁺⁴ cell/mL (after 10 days). The highest removal efficiency (95%) was achieved at the lower density after 12 days, with consistently superior performance across all SFE percentages compared with the higher density treatment.

The efficacy of N. oculata in removing phosphate and nitrate from wastewater, as demonstrated in this study, aligns with previous findings (Kumari et al. 2021; Nasir et al. 2023; Safari et al. 2021; Shafiq et al. 2021). Phosphate removal exceeded 95% at an initial density of 200 × 10⁺⁴ cells/mL, with no significant differences observed across SFE percentages or compared with the control (Figure 6d). At higher SFE percentages (25% and 50%), the phosphate removal percentages remained high and did not differ significantly from the control environment, which implies that wastewater, even at high percentages, can serve as an effective medium for pollution treatment. Similarly, nitrate removal reached approximately 90% after 12 days across all treatments, showing no significant variation relative to the control (Figure 6c). These results confirm that N. oculata effectively absorbs nutrients even at elevated SFE concentrations (25% and 50%), highlighting its robustness and suitability for treating nitrate‐ and phosphate‐rich industrial wastewaters.

In a comparative context, Nasir et al. (2023) reported that Chlorella sp. achieved removal of ammonia, nitrite, and orthophosphate at rates of 77.96%, 82.07%, and 75.96%, respectively. Safari et al. (2021) cultured two microalgae strains, Spirulina platensis and Chlorella vulgaris , on ammonia‐rich wastewater and observed reductions in total nitrogen by 55% and 96%, respectively. The results of Shafiq et al. (2021) demonstrated removal of ammonia, total nitrogen, and total phosphorus at rates of 95%, 87%, and 88% after 10 days, respectively. Kumari et al. (2021) showed that during the growth of the microalga Chlorella pyrenoidosa on municipal wastewater, nitrate and phosphate concentrations decreased by 99.2% and 70.1%, respectively.

3.6. Comparative Removal of Phosphate and Nitrate by C. muelleri and N. oculata

In the present study, both C. muelleri and N. oculata demonstrated high phosphate and nitrate removal efficiency in SFE at each of their respective initial cell densities. Lower initial cell densities ( C. muelleri : 35 × 10⁺⁴ vs. 70 × 10⁺⁴ cell/mL; N. oculata : 200 × 10⁺⁴ vs. 400 × 10⁺⁴ cell/mL) yielded superior removal performance. At these lower densities, nitrate and phosphate removal remained unaffected by varying SFE concentrations for both species, suggesting that initial cell density, rather than wastewater proportion, is a critical determinant of nutrient uptake efficiency. These findings confirm that both microalgae are effective biological agents for treating wastewater with variable nutrient loads.

As shown in Figure 7a, C. muelleri exhibited higher nitrate removal efficiency than N. oculata across all SFE concentrations, indicating greater robustness to varying conditions. Time‐course data, however, revealed a trade‐off: N. oculata achieved approximately 90% removal after 12 days, whereas C. muelleri reached about 80% after 6 days. Thus, C. muelleri is preferable for rapid remediation and consistent performance under variable conditions, while N. oculata is better suited for maximizing final nitrate removal over a 12‐day period. The optimal choice thus depends on operational priorities, speed and robustness versus endpoint efficiency.

FIGURE 7.

Comparative analysis of (a) the nitrate and (b) phosphate removal potential of C. muelleri and N. oculata .

Phosphate removal did not differ significantly between C. muelleri and N. oculata across all SFE percentages (Figure 7b). However, C. muelleri achieved approximately 90% removal within 6 days, whereas N. oculata required about 12 days to reach the same endpoint. Thus, while both species exhibit comparable ultimate phosphate removal capacity, C. muelleri offers a distinct kinetic advantage. Consequently, C. muelleri is preferred for applications requiring rapid phosphate reduction or short hydraulic retention times, whereas either species may be suitable for longer term processes where final removal equivalence can be fully realized.

The findings from this study demonstrate the superior efficacy of C. muelleri and N. oculata in nitrate and phosphate removal from SFE, achieving removal efficiencies of 79%–95% for nitrates and 92%–95% for phosphates across varying SFE percentages. Therefore, these microalgae demonstrate strong potential for the treatment of wastewater containing nitrate and phosphate and are particularly well suited for wastewater treatment systems operating with SFE and for industries addressing nitrate and phosphate pollution. These results surpass or align closely with prior research, including Nasir et al. (2023) (77%–83% for ammonia, nitrite, and orthophosphate), Safari et al. (2021) (55%–96% nitrogen reduction), Shafiq et al. (2021) (87%–95% for nitrogen and phosphorus), and Kumari et al. (2021) (70%–99% for nitrate and phosphate). The resilience of these microalgae to high SFE concentrations (up to 50%) underscores their potential for scalable, sustainable wastewater treatment.

The present study aligns with and contributes to the growing body of research that has successfully employed microalgae for the bioremediation of aquaculture wastewater, as demonstrated across multiple investigations which have explored various microalgal species and culture systems for nutrient removal and biomass production. Yaacob et al. (2021) and the associated studies (Nallapan et al. 2022) on virgin soil extracts from Malaysian forest reserves effectively demonstrated that natural organic amendments can serve as sustainable and cost‐effective growth promoters for microalgae including C. vulgaris , Nannochloropsis oceanica, and Chlorella sorokiniana , with the extracted nutrients supporting enhanced growth kinetics and biomass yields compared with synthetic media. Similarly, investigations into shrimp aquaculture wastewater treatment have utilized C. vulgaris in both high‐rate algae ponds and membrane photobioreactors, achieving substantial removal of COD, nitrogen species, and phosphorus while simultaneously recovering valuable biomass, thereby validating the dual‐purpose approach of wastewater treatment coupled with resource recovery (Nguyen et al. 2023). The application of Dunaliella salina in membrane bioreactor systems for Persian Gulf shrimp pond wastewater has further confirmed the feasibility of microalgae‐based treatment, with successful microalgal growth observed alongside high removal efficiencies for organic and inorganic pollutants (Shiri et al. 2023). Additionally, studies comparing macroalgae species such as Gracilaria pygmaea, Rhizoclonium riparium , and Sargassum glaucescens with the microalga N. oculata have consistently shown that microalgae exhibit superior nutrient uptake capacity, reinforcing the rationale for focusing on microalgal systems in aquaculture wastewater management (Bibak et al. 2026). Collectively, these studies have established that alternative nutrient sources, whether derived from natural soil extracts or from wastewater streams themselves, can effectively support microalgal cultivation and that microalgae possess inherent physiological capabilities for efficient nutrient removal, thereby providing a solid foundation for the present investigation which similarly employs N. oculata and C. muelleri cultivated in SFE as the growth medium. The present research therefore builds upon this established knowledge by extending the application of microalgae‐based treatment to SFE specifically, utilizing the wastewater itself as the culture medium while systematically evaluating the performance of two distinct microalgal species under varying dilution ratios and initial cell densities, and the findings consistently demonstrate effective nitrate and phosphate removal alongside enhanced growth rates, corroborating the broader scientific consensus that microalgae offer a robust and sustainable solution for aquaculture wastewater treatment and biomass valorization.

4. Conclusion

This study successfully achieved its primary objectives of assessing the cultivation performance and nutrient removal efficiency of N. oculata and C. muelleri for bioremediation of SFE across varying dilution ratios and initial cell densities. Both species demonstrated effective nutrient removal, with N. oculata achieving 97% nitrate and 95% phosphate removal under optimal 25% SFE conditions within 12 days, while C. muelleri reached 81% nitrate and 94% phosphate removal within 6 days, with higher removal efficiencies consistently observed at lower initial densities for both species. Specific growth rates surpassed controls, yielding maximum biomasses of 2425 × 10⁴ cells/mL for N. oculata and 595 × 10⁴ cells/mL for C. muelleri , while Chl. a production was modulated by SFE concentration, showing species‐specific responses with enhanced production in N. oculata at 25% SFE. The novelty of this study lies in the systematic optimization of operational parameters and the identification of species‐specific advantages for tailored bioremediation, demonstrating that C. muelleri offers faster nutrient removal kinetics suitable for shorter residence times, whereas N. oculata provides superior long‐term nutrient removal and biomass accumulation. These findings highlight the robustness of both microalgae across SFE gradients and support their application in sustainable aquaculture wastewater treatment, nutrient recycling, and biomass valorization. Future studies should focus on pilot‐scale validation, long‐term stability assessment, comprehensive biomass characterization for downstream applications, and investigation of the biochemical composition of the resulting biomass for specific industrial uses.

Author Contributions

Mohamad Shamsaei: investigation, writing – original draft. Zahra Ghasemi: conceptualization, methodology, validation, software, formal analysis, writing – review and editing, supervision, visualization. Zahra Aminikhoei: resources, project administration, writing – review and editing.

Funding

The authors have nothing to report.

Ethics Statement

The authors confirm that all experiments were performed in accordance with relevant guidelines and regulations.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

Authors can confirm that all relevant data are included in the article.