Introduction: The rapid growth of the global population, projected to reach 9.9 billion by 2050, has intensified the challenge of food security, prompting the exploration of alternative protein sources. Microalgae, particularly Spirulina (Arthrospira platensis), have gained attention due to their high nutritional value, rich in proteins, pigments, antioxidants, carbohydrates, and lipids, making them viable alternatives for food and feed industries. Spirulina is widely cultivated for its exceptional protein content and ease of production under diverse conditions, including high pH and mixotrophic growth, minimizing contamination risks. In Iran, the expanding sturgeon farming industry for caviar production generates substantial byproducts, such as heads and fins from farmed beluga (Huso huso), which are rich in proteins but often underutilized. These byproducts represent a sustainable resource for value-added products. Conventional Spirulina cultivation relies on Zarrouk's medium with inorganic nitrogen (sodium nitrate), which is costly and contributes to environmental pollution through unused nitrate discharge. Organic nitrogen sources, such as fish protein hydrolysates (FPH), offer a promising alternative by providing readily absorbable peptides and amino acids, enhancing growth and biochemical composition while valorizing fishery wastes. Previous studies have demonstrated the potential of FPH from fish wastes as nitrogen substitutes. A study reported a 34% increase in dry biomass and 17% in protein content when fully replacing nitrate with FPH from tuna wastes in Spirulina culture. Similarly, some investigations showed improvements in biomass and pigments in other microalgae with partial FPH substitution. However, no prior research has utilized enzymatically hydrolyzed peptides (<3 kDa) from sturgeon byproducts as an organic nitrogen source for Spirulina. The objective of this study was to evaluate the effects of partial or complete replacement of inorganic nitrogen (sodium nitrate) in Zarrouk's medium with bioactive peptides (<3 kDa) derived from enzymatic hydrolysis of farmed beluga heads and fins on the growth performance and biochemical composition of Arthrospira platensis. The hypothesis was that partial replacement (up to 40-50%) would enhance biomass, protein, and pigment contents due to better nitrogen bioavailability, while complete replacement might cause inhibitory effects from ammonia toxicity.

Materials and Methods: This study was conducted as a completely randomized design with six treatments and three replications over 11 days. Bioactive peptides (<3 kDa) were produced from farmed beluga (Huso huso) heads and fins. Byproducts were minced and subjected to enzymatic hydrolysis using Alcalase (2% w/w relative to crude protein) at pH 8.0-8.5, 56°C for 3 hours. The hydrolysate was inactivated at 90°C for 15 minutes, centrifuged (5000 g, 15 min), and the supernatant ultrafiltered (<3 kDa, 3 bar pressure) to isolate low-molecular-weight peptides, followed by freeze-drying. Proximate analysis confirmed high soluble protein (92.00 ± 5.29%) and nitrogen content (12.11 ± 0.25%). Spirulina (Arthrospira platensis) stock was obtained from the Iranian Biological Resource Center and cultivated in Zarrouk's medium at 30°C, 5000 lux light intensity (12:12 light-dark cycle), and continuous aeration in 10 L vessels. Treatments included: control (100% inorganic nitrogen), and 20%, 40%, 60%, 80%, or 100% replacement of sodium nitrate with the peptide fraction (on nitrogen-equivalent basis). Biomass was measured as dry weight (filtered, rinsed, dried at 75°C to constant weight). Pigments (chlorophyll a, b, and total carotenoids) were extracted with 96% methanol and quantified spectrophotometrically. Proximate composition included protein (Lowry-based method; Slocombe et al., 2013), lipids (Soxhlet extraction), ash (550°C incineration), and carbohydrates. Data normality was verified (Kolmogorov-Smirnov test), and differences analyzed by one-way ANOVA followed by Tukey's test (P < 0.05).

Results and Discussion: The hydrolyzed peptides exhibited high solubility (92%) and nitrogen content (12.11%), superior to raw byproducts (45% crude protein), confirming effective enzymatic breakdown into bioactive low-molecular-weight fractions. Growth monitoring revealed no differences on day 1 (lag phase), but from day 2 onward, the 40% replacement treatment consistently yielded the highest biomass, reaching 1.580 ± 0.009 g/L by day 11, significantly higher than the control (1.368 ± 0.026 g/L) (P < 0.05). The 20% treatment also showed improvements, while 60-100% replacements reduced biomass, with 100% yielding the lowest (0.914 ± 0.008 g/L). This pattern aligns with enhanced nitrogen uptake from short-chain peptides in moderate doses, but ammonia accumulation and osmotic stress at higher levels inhibiting nitrate reductase and causing toxicity. Proximate composition at day 11 showed the 40% treatment with highest protein (71.08 ± 1.41%), followed by 20% (67.08 ± 1.41%), exceeding the control (60.33 ± 1.63%) (P < 0.05). Lipids were highest in 60% (8.40 ± 0.27%), but carbohydrates and ash increased markedly in 80-100% treatments (up to 28.03% carbohydrates), indicating metabolic shift toward storage compounds under nitrogen stress. These results achieved 68% protein with full tuna FPH replacement, likely due to the purified <3 kDa fraction reducing inhibitory large peptides. Pigment contents were optimal in 40% treatment: chlorophyll a (5960 ± 80 μg/g), chlorophyll b (860 ± 50 μg/g), and total carotenoids (2310 ± 30 μg/g), significantly higher than control (3760, 120, and 1540 μg/g, respectively) (P < 0.05). Higher replacements drastically reduced pigments, attributed to impaired photosynthetic metabolism from ammonia toxicity. The 20% treatment also enhanced pigments moderately. Overall, moderate organic nitrogen substitution directed metabolism toward protein and pigment synthesis, improving nutritional quality, while excessive levels induced stress responses. Compared to inorganic-only media, partial peptide integration increased biomass by ~15% and protein by ~18%, supporting sustainable valorization of sturgeon wastes and reducing environmental impacts from mineral nitrogen.

Conclusion: This study demonstrates that bioactive peptides (<3 kDa) from enzymatic hydrolysis of farmed beluga byproducts effectively serve as partial substitutes for inorganic nitrogen in Spirulina cultivation. The optimal 40% replacement significantly enhanced biomass (1.58 g/L), protein content (71.08%), and pigments (chlorophyll a: 5960 μg/g; carotenoids: 2310 μg/g) compared to the inorganic control, while reducing carbohydrates. The 20% level also provided benefits, but higher substitutions (≥60%) impaired performance due to ammonia toxicity and metabolic imbalance. These findings highlight the potential of sturgeon processing wastes as a sustainable, cost-effective organic nitrogen source, promoting circular bioeconomy in aquaculture and microalgae industries. Applications include high-protein Spirulina supplements for human nutrition or animal feed, with added value from natural pigments for food and pharmaceutical uses. Recommendations include scaling up in photobioreactors with pH and CO₂ control for industrial yields, further characterizing peptide bioactivity (e.g., antioxidant properties), and evaluating economic feasibility. Future research should explore combined organic-inorganic feeds or other microalgal species to broaden waste valorization strategies and minimize environmental footprints from nitrogen fertilizers.