2.1. Preparation of Chicken Feathers Medium Supplemented with Moringa oleifera Leaf Extract

The chicken feathers were collected from a slaughterhouse in Douala, Cameroon. The samples were washed at 50 °C in 0.5% v/v H₂O₂ to remove residues of blood, feces, fat, offal, and sand. The washed chicken feathers were then rinsed and dried at 105 °C for 24 h. Once dried, the barbs were manually separated from the shaft and carbonized at 440 °C for 10 min in a muffle furnace to obtain ash [22]. Subsequently, 10 g of ash was dispersed in 100 mL of distilled water (1:10 w/v) and left to stand for 24 h to create a homogenate of chicken feathers. After the homogenization process, the mixture was passed through the Whatman No. 2 filter paper. The filtrate collected was used as chicken feather medium (CF).

Fresh M. oleifera leaves were harvested at the SAGRIC farm in Douala, Cameroon. The leaves were cleaned, and different quantities (20 g, 30 g, and 40 g) were mixed with 1 L of distilled water for 7 days under agitation using a homogenizer [15]. The suspension was subsequently filtered through sterile muslin cloth and Whatman No. 1 filter paper to eliminate solid particles. The filtrates obtained were diluted with an additional 1 L of distilled water to achieve final concentrations of 10, 15, and 20 g L⁻¹ Moringa oleifera leaf extracts and sterilized at 121 °C for 30 min. The obtained extracts were used as the M. oleifera leaf extract (MLE).

2.2. Microalgae and Inoculum Culture Conditions

The strain of S. platensis used in the experiments was harvested from the algal culture collection of the SAGRIC farm in Douala, Cameroon. To produce the inoculum, the S. platensis strain was cultured in Jourdan’s medium, which contained per liter: NaHCO₃ (8 g), NaCl (5 g), KNO₃ (2 g), MgSO₄ (0.16 g), (NH₄)₂HPO₄ (0.12 g), (NH₂)₂CO (0.05 g), FeSO₄ (0.02 g), and CaCl₂ (0.02 g). The culture of the inoculum was maintained at 28 ± 0.5 °C, under alkaline conditions (pH = 9.0 ± 0.4) in a greenhouse with white LED tube lamps (200 µmol photons m⁻² s⁻¹) and constant aeration supplied by a membrane pump (Laboport KNF, Freiburg, Germany).

2.3. Experimental Design

The study used a systematic methodology, wherein S. platensis was cultivated in chicken feather medium (CF). Culture media were prepared by supplementing CF medium with 0, 10, 15, or 20 g L⁻¹ of MLE for CF-MLE-0 (control), CF-MLE-10, CF-MLE-15, or CF-MLE-20, respectively. Jourdan’s medium served as the established standard medium (Table 1). The microalgae S. platensis was grown in 20 L cylindrical vessels set in a greenhouse maintained at 28 ± 0.5 °C, under white LED illumination (200 µmol photons m⁻² s⁻¹) and continuous aeration supplied by a membrane pump. A 10% inoculum (Inoculation volume/Medium volume) was used at the beginning of the cultivation experiment. The salinity and the pH were adjusted to 11.4 ± 0.5 PSU with NaCl and 9.0 ± 0.4 with 2 N NaOH, respectively. The trials were conducted in triplicate over the 27-day experimental period.

2.4. Estimation of the Eco-Friendly Chicken Feathers Medium Supplemented with Moringa oleifera Leaf Extract

The expense associated with chicken feather medium enriched with Moringa oleifera leaf extract was calculated by taking into account the concentration and cost of every substance employed in the production of 1 kg of the material. Taxes, energy consumption, and transportation costs were excluded from the analysis. The prices for all the chemicals utilized were sourced from previous research reported by Magwell et al. [23] (Table 2).

2.5. Biomass and Growth Rate Assessment

S. platensis samples were collected every 3 days to evaluate the dry biomass and cell optical density. The optical density of the cells was measured at 680 nm via a UV-VIS spectrophotometer (UV-VIS Spectrophotometer BK-S380, Shandong, China). Dry biomass determination was performed by filtering 20 mL samples through 47 mm GF/C glass fiber filters (X₁, g). The cells within the filters were rinsed twice with distilled water, oven-dried at 50 °C for 24 h, and subsequently weighed (X₂, g). The dry biomass was calculated per Equation (1):

The biomass productivity (P_x) and growth rate (μ) of S. platensis were determined from Equations (2) and (3), respectively:

X₀ and X_f represent the dry biomass at the initiation and termination of the cultivation process (t), respectively.

Upon completion of the exponential growth phase (21 days), S. platensis samples were collected to assess protein, cysteine, and pigment contents.

2.6. Biochemical Analysis

4.1. Effects of Chicken Feathers Medium Supplemented with Moringa oleifera Leaf Extract on Spirulina platensis Growth Performance

The variations in the cell density and dry biomass of S. platensis cultivated in CF medium enriched with M. oleifera leaf extract are illustrated in Figure 1A,B. An exponential increase in cell density and dry biomass was noted from day 1 to day 21. However, a significant decrease was observed from day 21 to day 27, especially in the control medium with chicken feather medium, which resulted in reduced growth from day 9 onwards. The chicken feathers medium supplemented with 15 g L⁻¹ M. oleifera leaf extract (CF-MLE-15) presented significantly (p < 0.05) higher cell density (1.68 ± 0.03) and dry biomass (1.98 ± 0.05 g L⁻¹) than standard Jourdan medium (JM), with a cell density and dry biomass of 1.58 ± 0.05 g L⁻¹ and 1.83 ± 0.04 g L⁻¹, respectively. In contrast to the other treatments, the chicken feathers medium without M. oleifera leaf extract (CF-MLE-0) showed the lowest cell density (0.50 ± 0.03) and dry biomass (0.42 ± 0.06 g L⁻¹) of S. platensis (Table 3). These results are attributed to the nutrient content, namely nitrogen, potassium, phosphorus, calcium, sodium, and magnesium, present in the leaves of Moringa oleifera and chicken feathers, which collectively enhanced cell growth and carbon metabolism during the photosynthetic activity of Spirulina platensis [22,28,29]. Therefore, CF-MLE-15 is more conducive to growth and consequently higher cell density. The findings presented a higher level of efficacy than those reported by Wamba et al. [15] for a medium containing M. oleifera leaf extract enriched with either kanwa or sodium bicarbonate. The variation observed may be related to the formulation of the medium and the duration of the cultivation.

Biomass productivity (Px) and growth rate (µ) showed an upward trend in chicken feather media with increasing concentration of M. oleifera leaf extract. It was observed that the standard Jourdan medium resulted in the highest biomass productivity (0.032 ± 0.003 g L⁻¹ d⁻¹) and growth rate (0.040 ± 0.001 d⁻¹). Nonetheless, the CF-MLE-15 medium significantly (p < 0.05) increased the biomass productivity (0.028 ± 0.002 g L⁻¹ d⁻¹) and growth rate (0.038 ± 0.001 d⁻¹) compared with those of the different chicken feathers media supplemented with M. oleifera leaf extract and the control medium (−0.011 ± 0.001 g L⁻¹ d⁻¹ and −0.057 ± 0.002 d⁻¹) (Table 3). These findings can be explained by the provision of essential nutrients from Moringa oleifera leaf and chicken feather extracts during the cultivation of Spirulina platensis. These results are congruent with the findings of Suparmaniam et al. [30], emphasizing the essential role of chicken feather nutrients in promoting microalgal growth.

4.2. Effects of Chicken Feathers Medium Supplemented with Moringa oleifera Leaf Extract on the Chlorophyll and Carotenoid Contents and Productivity of Spirulina platensis

The contents of carotenoids, chlorophyll a, and chlorophyll b in S. platensis biomass cultivated from chicken feathers supplemented with various concentrations of M. oleifera leaf extract are presented in Table 4 and Figure 2. The variations in the carotenoid and chlorophyll a and b contents were correlated with the M. oleifera leaf extract concentrations in the medium. Supplementation of the chicken feathers medium with 15 g L⁻¹ M. oleifera leaf extract (CF-MLE-15) led to a significant (p < 0.05) increase in the carotenoid (0.82 ± 0.02 mg L⁻¹), chlorophyll a (1.67 ± 0.07 mg L⁻¹), and chlorophyll b (1.20 ± 0.09 mg L⁻¹) contents. Nevertheless, the Jourdan standard medium presented significantly higher (p < 0.05) contents of carotenoids (0.90 ± 0.003 mg L⁻¹), chlorophyll a (1.72 ± 0.07 mg L⁻¹), and chlorophyll b (1.33 ± 0.12 mg L⁻¹) than the other treatments. Similarly, the productivity of chlorophyll and carotenoids of S. platensis showed a downward trend with the M. oleifera leaf extract concentration in the chicken feathers medium (Table 5). The productivity of chlorophyll and carotenoids of S. platensis was significantly higher (p < 0.05) in CF-MLE-15 with 0.16 ± 0.001 g L⁻¹ d⁻¹ and 0.11 ± 0.002 g L⁻¹ d⁻¹, respectively, than the control (0.02 ± 0.002 g L⁻¹ d⁻¹ and 0.01 ± 0.001 g L⁻¹ d⁻¹) (Table 5). The range of chlorophyll a, b, and carotenoids in the current study is consistent with that reported by Herrera et al. [31], who reported values between 0.83 and 2.66 mg L⁻¹, using Schlösser and whey media under mixotrophic conditions as a substitute for synthetic media. This increase is attributed to the potassium, calcium, and iron supplied by chicken feather and M. oleifera leaf extracts, which activated metabolic enzymes, regulated cellular processes, and maintained the structural integrity of the photosynthetic apparatus [32,33]. The ability to enrich chicken feathers medium with concentrations of 15 g L⁻¹ MLE was noteworthy, as it influenced growth, dry biomass, and photosynthetic pigments. Conversely, at higher concentrations of MLE, cellular growth and photosynthetic efficiency were significantly reduced, resulting in decreased chlorophyll and carotenoid levels. Furthermore, Saad et al. [34] reported that the pigment content of S. platensis was lower under mixotrophic conditions than autotrophic conditions.

4.3. Effects of Chicken Feathers Medium Supplemented with Moringa oleifera Leaf Extract on the Phycobiliprotein Content and Productivity of Spirulina platensis

The phycobiliprotein contents in the S. platensis biomass grown in the different media are shown in Table 4 and Figure 3. The mean phycocyanin concentrations measured in biomass grown in CF-MLE-10, CF-MLE-15, and CF-MLE-20 were 0.75 ± 0.01, 0.77 ± 0.03, and 0.75 ± 0.01 mg L⁻¹, respectively (Figure 3). Although the phycocyanin content in the JM treatment was higher than that in the CF-MLE treatment, no significant difference was observed. The mean content of phycoerythrin and allophycocyanin, measured in the biomass grown in CF-MLE-15, was 0.28 ± 0.03 and 0.19 ± 0.001 mg L⁻¹, respectively (Figure 3). Similar to the phycocyanin content, the JM treatment exhibited the highest concentrations of phycoerythrin and allophycocyanin, which were not significantly different from those observed in the CF-MLE treatments. The productivity of phycocyanin, phycoerythrin, and allophycocyanin of S. platensis in CF-MLE-15 was found to be similar to the standard medium. This result was significantly higher (p < 0.05) than the control treatment, which had a yield of 0.006 ± 0.001 g L⁻¹ d⁻¹, 0.003 ± 0.001 g L⁻¹ d⁻¹, and 0.002 ± 0.001 g L⁻¹ d⁻¹, respectively (Table 5). The results for phycocyanin content differ from those of Simo et al. [13], who received 0.14, 0.20, and 0.05 mg L⁻¹ for C-phycocyanin, allophycocyanin, and phycoerythrin, respectively, in a medium formulated with 8 g L⁻¹ oil palm empty fruit bunch medium. These differences may be attributed to the essential role of potassium and nitrogen supplied by Moringa oleifera leaf extract and chicken feather in the medium. Indeed, activating enzymes involved in phycobiliprotein synthesis affect phycobiliprotein production in metabolic responses to changes in medium nutrient availability, as reported by Baslam et al. [32].

4.4. Effects of Chicken Feathers Medium Supplemented with Moringa oleifera Leaf Extract on the Protein and Cysteine Contents of Spirulina platensis

The protein content of S. platensis biomass grown in chicken feathers medium supplemented with different concentrations of M. oleifera leaf extract is presented in Figure 4. The protein content (94.5 ± 1.52 mg L⁻¹) was higher in CF-MLE-15 than in the other treatments and the control (39.3 ± 4.21 mg L⁻¹). A high protein content (110.2 ± 6.14 mg L⁻¹) was also observed in Jourdan’s standard medium. Compared with the control, the addition of M. oleifera leaf extract to the chicken feather medium significantly increased the protein content of S. platensis biomass in all the treatment groups (p < 0.05). Therefore, mineral elements such as nitrogen, magnesium, and potassium in CF-MLE may be essential cofactors for protein production [35]. The cysteine content of S. platensis grown on CF-MLE-15 (12.5 ± 1.14 mg L⁻¹) and Jourdan standard medium (14.1 ± 0.81 mg L⁻¹) was significantly higher (p < 0.05) than that of the other treatments and the control (3.8 ± 0.27 mg L⁻¹). The cysteine content observed in this study was lower than the 0.16 g L⁻¹ reported by Magwell et al. [36] in MgSO₄-enriched medium. This difference could be explained by the composition of the culture medium in which the microalgae grew.