Introduction
Algae are the most important photosynthetic groups in aquatic environments, as they are carbon and nitrogen fixers and considered as primary producers (Sigee, 2005; Falkowski and Raven, 2013; Fowler et al., 2013). The growth of algae in wastewater are common phenomenon that has a crucial role in removing minerals and metabolic byproducts. The wastewater from sugar production contains high levels of carbon, nitrogen, and phosphorus, along with high BOD and COD. Molasses is a byproduct of sugar factories and is primarily used as a raw material for yeast production. It also has considerable commercial value in various fermentation processes, animal feed, and biofertilizers (Dahiya et al., 2001; Torabian and Mahjuri, 2004; Kobya and Delipinar, 2008). Molasses contains 45-50% carbohydrates, 15-20% non-aromatic organic compounds, 10-15% ash (minerals), and about 20% water (Kalyuzhnyi and Murray, 2005). Molasses wastewater includes chemicals such as propionic acid, various salts, and fermentation metabolites (Blonskaja and Zub, 2009). In addition to molasses concentration and the stated conditions, which greatly impact the growth of microscopic algae, the role of suspended solids in molasses as another influential factor in algal growth and reproduction can be investigated. The negative effects of suspended solids, particularly at high concentrations are crucial due to their reduction in light intensity. Furthermore, suspended solids can settle on algae surfaces, hindering gas exchange (e.g., oxygen and carbon dioxide), which negatively affects photosynthesis efficiency (Boyd, 2020). On the other hand, the positive effects of suspended solids include stimulating the production of certain secondary metabolites through stress induction in algal cells (Huang et al., 2024).
 This study aims to investigate the role of suspended solids and molasses dilution in the growth and biomass production of the green microalga Scenedesmus quadricauda and its potential for bioremediation. Understanding suspended solids and molasses dilution levels can contribute to the management of algal cultures in terms of optimizing light intensity, turbidity, culture system design, and the production of valuable secondary metabolites.
 Methodology
 Molasses wastewater was collected from the Eqlid Sugar Factory, located in Fars province, Iran. The factory processes sugar beets grown by 800 local farmers. The wastewater sample (10 liters) was collected before entering the treatment section of the factory in November 2023. The green microalga S. quadricauda was cultured in raw pre-treated wastewater, diluted to target concentrations of 1%, 5%, and 10%, under two conditions: with suspended solids and without suspended solids (Table 1) for 14 days. Seven experimental treatments were prepared, including BBM medium (control) and molasses wastewater with and without suspended solids at three dilution levels: 1% (10 ml/L) 5% (50 ml/L) 10% (100 ml/L). These solutions were added to 5-liter glass Erlenmeyer flasks. The initial pH of all cultures was adjusted to 6.8 using concentrated NaOH and HCl solutions. Before introducing the algal stock, all samples were autoclaved at 121°C for 15 minutes. After cooling, 5% (v/v) of the initial algal stock containing 2 × 10⁶ cells/mL was added. The cultures were incubated under appropriate light conditions provided by fluorescent lamps (60 µmol photons/m²/s) with a 12-hour light/12-hour dark photoperiod and gentle aeration. The water temperature in all treatments was kept constant at 25 ± 2°C. The experiment followed a completely randomized design with three replicates over 14 days. Daily cell counting of S. quadricauda was performed using a hemocytometer (depth: 0.1 mm, area: 0.0025 mm²), following the method proposed by Martinez et al. (2000). The specific growth rate was calculated using the formula by Omori and Ikeda (1984). To measure biomass, 100 mL of the algal culture was filtered using pre-weighed membrane filter papers (0.45 µm). The filtered samples were dried in an oven at 80°C for 4 hours. Dry Biomass Measurement and Analytical Methods After drying the algal biomass, it was placed in a desiccator to reach equilibrium with the laboratory environment. The dry weight was then measured, and the difference in weight was used to calculate the dry biomass of the algae (Omori and Ikeda, 1984). Nitrate concentrations were measured using a spectrophotometric colorimetric method at 275 nm and 220 nm, using a UV-VIS spectrophotometer (Nanombana UVISNM98 UV-VIS). Phosphate concentration was determined using spectrophotometry at 880 nm, with a JENWAY 6300 spectrophotometer (Baird et al., 2017). The five-day biochemical oxygen demand (BOD₅) was assessed by adding a specific amount of wastewater to a dilution water solution in 300 mL dark Winkler bottles. The dissolved oxygen (DO) content was measured at the start and after five days of incubation at 20°C, using an oxygen meter. The chemical oxygen demand (COD) was determined using potassium dichromate and silver sulfate reagents, with digestion in a COD reactor for 2 hours, followed by absorbance reading at 600 nm using a spectrophotometer (Baird et al., 2017). This study was conducted using a completely randomized design (CRD) with different treatments (Table 1), each with three replicates. One-way ANOVA was used to determine significant statistical differences, and Duncan’s test and Student’s t-test were performed for mean comparisons. All statistical analyses were conducted using SPSS software, and the graphs were generated using Excel.
Results
The total suspended solids (TSS) and dissolved solids (TDS) in the raw molasses wastewater were measured at 7697.2 mg/L and 2540.8 mg/L, respectively. The nitrate and phosphate concentrations were 1595.24 mg/L and 12.73 mg/L, respectively. The BOD₅ and COD values of molasses wastewater were 42,790 mg/L and 136,156 mg/L, respectively. The pH was 5.92, and the electrical conductivity (EC) was 3.97 mS/cm. The cell density of S. quadricauda in different treatments, including the control (BBM), molasses wastewater without suspended solids (1%, 5%, 10%), and molasses wastewater with suspended solids (1%, 5%, 10%), was: 1.03 × 10⁶, 1.22 × 10⁶, 1.45 × 10⁶, 7.25 × 10⁵, 1.0 × 10⁶, 9.85 × 10⁵, and 1.05 × 10⁶ cells/mL, respectively. These values correspond to days 14, 14, 14, 14, 11, 14, and 14 of the cultivation period. The highest cell density at the end of the experiment (day 14) was observed in the 1% molasses wastewater with suspended solids treatment. Overall, 1% and 5% molasses wastewater with suspended solids and 1% molasses wastewater without suspended solids showed higher cell densities than the control (BBM). The specific growth rate at the end of day 14 ranged from 0.064 – 0.104 per day, with the highest growth in the 1% molasses wastewater with suspended solids and the lowest in the 10% molasses wastewater with suspended solids. The biomass concentration ranged from 2199.1 – 6343.7 mg/L, with the highest value in the 10% molasses wastewater without suspended solids and the lowest in the 1% molasses wastewater with suspended solids. At low concentrations (1% and 5%), suspended solids had no significant effect on biomass. However, at 10% concentration, the treatment without suspended solids resulted in significantly higher biomass production (p < 0.05). The phosphate concentration decreased from 278.3 – 250.4 mg/L to 206.35 – 149.4 mg/L (Figure 2B). Nitrate and phosphate removal was significant in all treatments (p < 0.05). The highest nitrate removal (58%) and phosphate removal (46%) were observed in the 10% molasses wastewater without suspended solids (Figure 2C). The BOD₅ removal in molasses wastewater treatments ranged from 92.37 – 99.19%. The highest BOD value after cultivation was observed in the 5% molasses wastewater with suspended solids, and the lowest in the 5% molasses wastewater without suspended solids and 1% molasses wastewater with suspended solids. The highest BOD₅ removal (99.19%) was found in the 5% molasses wastewater without suspended solids, while the lowest (92.37%) occurred in the 1% molasses wastewater without suspended solids. The COD removal ranged from 94.05 – 99.46%, with the highest COD after cultivation found in the 10% molasses wastewater, and the lowest in the 1% molasses wastewater without suspended solids.
Discussion and conclusion
After the 14-day cultivation period, the treatments with 1% and 5% molasses containing suspended solids and 1% molasses without suspended solids exhibited higher cell densities compared to the control treatment, whereas other treatments had lower cell densities than the control. Similar to the present study, Farhadian et al. (2022) reported that the highest cell density of the marine microalga Tetraselmis tetrahele was observed in 1% molasses and concluded that the biomass production of T. tetrahele in 1% molasses was higher than in 0.5% diluted molasses. They also found that biomass production in molasses treatments was higher compared to other culture media. The results of this study under mixotrophic conditions showed that the highest biomass production occurred in the 10% molasses treatment without suspended solids. Biomass production showed an increasing trend with increasing molasses concentration from 1% to 10% in treatments without suspended solids. Moreover, the biomass produced in the 1% and 5% molasses treatments was almost equal and higher than in the control treatment. However, in the 10% molasses treatment with suspended solids, a noticeable reduction in biomass production was observed, which could be attributed to reduced light penetration in the culture medium. Suspended solids can limit light penetration into the water column, reducing photosynthesis and subsequently decreasing algal biomass. Suspended solids in water can pose serious challenges to aquatic ecosystems. These particles, often introduced through human activities such as agriculture and industrial pollution, can reduce water clarity, hinder the respiration of aquatic organisms, and even cause direct harm. Studies have shown that suspended solids not only affect water quality but also impact microscopic organisms in aquatic environments. These particles can cover the gills of fish and other aquatic organisms, making respiration difficult. Additionally, they can absorb sunlight, limiting photosynthesis in aquatic plants and disrupting the food chain (Bilotta and Brazier, 2008). In this study, all treatments involving S. quadricauda resulted in nitrate and phosphate uptake. The highest nitrate removal percentage (58%) was observed in the 10% molasses treatment without suspended solids, while the highest phosphate removal percentage (48%) was recorded in the 10% molasses treatment. Heydari et al. (2011) found that S. quadricauda grows well in nitrogen-rich environments, making it suitable for treating nitrogen-enriched wastewater due to its high growth rate and survival. The process of nitrate and phosphate removal by Scenedesmus microalgae has been reported in multiple studies (Oswald and Gotass, 1995; Martinez et al., 2000; Voltolina et al., 2004; Wang and Lan, 2011; Arora et al., 2021). Microalgae utilize nitrogen and phosphorus from wastewater to synthesize energy-storing molecules such as adenosine triphosphate (ATP) and adenosine diphosphate (ADP), as well as genetic material. Additionally, inorganic phosphate forms such as orthophosphate, HPO₄²⁻, and H₂PO₄⁻ are preferred by microalgal cells, which absorb them via phosphorus transporters in the plasma membrane (Ahmed et al., 2022). A comparison of nitrate and phosphate removal percentages across different concentrations (Figure 2–C) indicated that removal rates were significantly higher in molasses treatments without suspended solids than in those with suspended solids. This suggests that the presence of suspended solids may hinder nitrate and phosphate removal. Therefore, separating suspended solids from the culture medium is crucial for improving the efficiency of algal bioremediation in wastewater treatment. Biochemical Oxygen Demand (BOD) and Chemical Oxygen Demand (COD) were measured and evaluated at the beginning and end of the mixotrophic cultivation experiments.
The highest BOD removal percentage (99.19%) was observed in the 5% molasses treatment without suspended solids, while the lowest BOD removal percentage (92.37%) was recorded in the 1% molasses treatment without suspended solids. Similarly, the highest COD removal percentage (99.46%) was measured in the 10% molasses treatment without suspended solids, whereas the lowest COD removal percentage (94.05%) was found in the 1% molasses treatment without suspended solids. Wang and Lan (2011) stated that biomass production in mixotrophic culture systems is generally higher than in heterotrophic models, possibly due to greater access to carbon sources (CO₂) in mixotrophic conditions. Nagarajan et al. (2019) also reported significant reductions in BOD and COD through microalgal cultivation in wastewater.
The content is subject to carbon reduction. Carbon and organic matter uptake and consumption are also common phenomena in microalgae. Microalgae contribute to the removal of organic substances such as urea and inorganic nutrients, including nitrate and phosphate from wastewater, which helps reduce BOD and COD (Arora et al., 2021). Overall, the results indicate that the biomass obtained from the microalga S. quadricauda, cultivated under mixotrophic conditions in molasses wastewater in this study, has numerous advantages, including a short reproductive cycle, enhanced photosynthesis, higher and more efficient nutrient consumption, and effective bioremediation of wastewater (significant reduction of nitrate, phosphate, color, COD, and BOD).
Acknowledgment
This research was supported by Isfahan University and Technology, Isfahan, Iran.