The Scenedesmus sp. Niva-Chl 99 used in the experiment was obtained from the Norwegian Culture Collection of Algae, NORCCA, Oslo, Norway. A sterilized standard BBM media was used to promote the initial inoculum growth of microalgae in a Pyrex bottle (1 L), and the composition of the media was the same as mentioned by Chakraborty et al. [16] (Section S1.1). The Scenedesmus sp. culture was allowed to grow under a 14:10 h light-dark cycle, room temperature 25 ± 2 ̊C, and constant light intensity (200 µmol photons m⁻² s⁻¹) maintained through four 20-Watt LED lights (Race Technology, China). An aeration pump with a flow rate (52.5 L h⁻¹) was utilized to maintain the culture in suspension, and syringe filters made of 0.2 µm cellulose acetate (Sartorius, 1784B) were used to maintain the sterilized conditions [17].

Scenedesmus sp. biomass was harvested via centrifugation at 1500 x g for 5 min. Deionized water was used to wash the harvested algae and centrifuged a second time at 1500 x g for 5 min to avoid interference from the nutrient media before being used for experimentation. Algae pellets were then evaluated in terms of optical density at 680 nm (OD₆₈₀) spectrophotometrically (PG Model T6OU, PG Instruments, UK) and introduced into an open raceway pond to provide an initial biomass concentration of 0.2 g L^− 1 based on Eq. 3.

An open raceway pond made up of acrylic (5 mm, thickness) was used in this experiment. The pond consists of two straight flow channels (length: 80 cm, width: 14 cm, depth: 9.8 cm) and two bend flow channels (radius: 11 cm, depth: 9.8 cm) created through a center acrylic wall partition. Each bend flow channel also has two acrylic slits (5 mm, thickness) of radius 5.81 cm and 4.3 cm, separated by a channel of 1.5 cm from each other, respectively. A locally manufactured submersible pump (SHAFI) of power (8 W, DC 12V) was installed in one of the straight flow channels to promote the circulation of microalgae culture. The pump employed in this system functions by drawing the microalgal culture through an inlet at the bottom, situated at a height (1 cm) from the bottom surface of the pond, and subsequently propelling it through an outlet located along the channel parallel to the base as shown in Fig. 1C. No spargers were used for air bubbling and turbulence generated by the culture flow was the sole supplier of pure air. A DC power supply (Matrix) was used to operate the submersible pump at constant voltage (9 V) to maintain a mean flow velocity of 0.35 m s^− 1 inside the open raceway pond, ensuring turbulent mixing and no splash out. Light intensity was maintained at 200 µmol photons m^− 2 s^− 1 using four 9 W fluorescent bulbs about 24 cm apart and was measured using the quantum sensor (Apogee quantum sensor, MQ 500, USA).

The experiments were conducted in the Algal Biotechnology Lab located inside the Environmental Engineering Department (IESE), NUST (N 33° 38' 44.0592", E 72° 59' 25.242"). To explore the impacts of mixing duration on Scenedesmus sp. biomass production, experiments were conducted in three distinct raceway systems (R1, R2, and R3), each having the same configuration but different operating conditions in terms of daily mixing duration. In system R1, the submersible pump operated continuously to permit mixing for 24 h per day. In system R2, the pump was activated for 20 h and was not operational for 4 h per day, causing the culture not to mix during the interruption period. In system R3, the pump was activated for 16 h per day only, halting the culture mixing for 8 h per day. The experiment was performed for a duration of 10 days, and all experiments were performed in triplicate. The cultivation of microalgae in all systems was conducted with a 6 L working volume containing algae inoculum and standard BBM. The working volume of 6 L was opted as the optimum operating volume for the system to impart efficient mixing and to avoid overflowing. The pH and electric conductivity were measured daily using a multimeter (inoLab pH/Cond 720, Germany), and pH was adjusted initially at 6.5 ± 0.1 by adding either 8% NaOH or 2% HCl solution as required [18].

A correlation between biomass concentration and optical density was developed first to observe Scenedesmus sp. growth in an open raceway pond. For this purpose, 5, 10, 15, 20, and 25 mL samples of Scenedesmus sp. culture were collected and diluted up to 50 mL with distilled water. A UV visible spectrophotometer (PG Model T6OU, PG Instruments, UK) operated at 680 nm was used to observe the optical densities of each sample [21, 22]. The corresponding samples with the same amount of culture were placed in china dishes and oven dried at 80 ^oC for 8 h to observe the biomass concentration. The biomass concentration was calculated through the difference in the final weight of the china dish containing algae residue and the initial weight of the empty petri dish. The linear regression established between the OD₆₈₀ and biomass concentration (g_dw L^− 1) for the calculation of Scenedesmus sp. dry biomass concentration is as shown in Eq. 3.

Where C_b is the biomass concentration (g L^− 1) and a is the absorbance at OD₆₈₀.

For experimental purposes, culture sampling was done at 24-hour intervals. To avoid bias, 5 mL samples were collected each from the four sides (two straight flow channels and two bend flow channels) and combined to make a representative sample of 20 mL. The samples were subjected to spectrophotometry at a wavelength of 680 nm by using the UV visible spectrophotometer.

The specific growth rate was calculated using Eq. 4 and the doubling time was determined by using the first-order kinetics in Eq. 5 according to Liu et al. [23].

Where is specific growth rate, and C_b(i) and C_b(f) are the initial and final biomass concentration of Scenedesmus sp. at initial (t_i) and final (t_f) days, respectively.

Where t_Doubling is doubling time and is the specific growth rate of microalgae.

The maximum biomass productivity was calculated according to Saleem et al. [24] as shown in Eq. 6.

Where C_biomass(i) and C_biomass(f) are the initial and final biomass concentration of microalgae at time, initial (T_i) and final (T_f) days, respectively.

The biomass samples were collected on every alternative day and centrifuged at 1500 x g for 5 min [25]. The obtained supernatant was filtered through a 0.45 µm filter paper using a vacuum filtration assembly and analyzed for orthophosphate (PO₄³⁻), nitrate (NO³⁻), and ammonia (NH₄⁺) following the standard methods mentioned by [1]. Removal parameters were calculated as removal efficiency (%) by using Eq. 7 and nutrient removal rates (RR_x, mg L ^− 1 d ^− 1) were calculated by using Eq. 8.

Where C_in and C_eff are the initial and final concentrations of the nutrient, and t is the time duration of the experimental phase.

All experiments were performed in triplicate for the growth and nutrient measurement throughout each phase. The results obtained are presented in the form of arithmetic means ± standard deviation. The difference in results and measurement was computed and determined using analysis of variance (ANOVA) with a p-value of < 0.05 on statistical software Origin 8 (Origin Lab Corporation, USA). The Tukey method was employed to further investigate the variations among biomass production and nutrient removal.

The specific growth rate, biomass productivity and dry biomass of Scenedesmus sp. cultivated in BBM demonstrate the impact of different mixing durations in the raceway pond (Fig. 1A, B and D). Green, robust, and stable Scenedesmus sp. cultures were observed in all three experiments, indicating the productivity advantages offered through controlled environment in raceway systems. All three mixing duration systems (R1, R2 and R3) showed an initial lag phase of two days where growth was stationary. This might be because Scenedesmus sp. was not acclimatized to the gas exchange dynamics based on natural air entrapment due to turbulence generated by liquid flow, since the cultures before the experiment initiation were aerated by bubbling pure air. Hemalatha et al. [26] reported that microalgae need two days to adjust to their new environment before the biomass of algae begins to increase.

Scenedesmus sp. specific growth rate increased with an increase in the mixing duration as the system R1 achieved the highest specific growth rate compared to the R2 and R3 systems, respectively, as shown in Fig. 2(A). The highest dry biomass yield of 1.0136 g L^− 1 and biomass productivity of 0.0813 g L^− 1 d^− 1 were observed in the R1 system. The biomass productivities observed in R2 and R3 systems were 20.45% and 48.31% lower than that obtained in the R1 system and the probable reason might be the absence of Scenedesmus sp. culture mixing leading to the creation of stagnant zones [27]. The static flow of culture in the pond limits the growth of microalgae generating layers of settled algal cells in the stagnant zone, which further promotes the formation of anaerobic conditions [27, 28]. Another reason for reduced growth in R2 and R3 systems might be the development of a nutrient and oxygen gradient around the Scenedesmus sp. cells in response to the absence of mixing. The oxygen gradient develops when mixing within the algae culture becomes interrupted, which becomes problematic during the dark regime when algae switch from photosynthesis to respiration. The accumulated oxygen around the algae cell limits the proper supply of carbon dioxide (CO₂), leading to stress and biomass loss in algae culture [29]. The continuous mixing disperses this gradient by providing a continuous supply of nutrients to algae and uniform distribution and removal of oxygen in the culture. The continuous mixing ensures that the algal cells continuously change their positions, which results in even distribution of algal cells in the culture and prevents the production of stagnant dead zones in the pond [30]. The biomass outcomes obtained in this study also correspond to the literature where biomass productivity for daytime mixing (12 h d^− 1) was 41% lower compared to the continuous mixing of Nannochloropsis sp., when cultivated in an outdoor open raceway pond [1]. The Tukey HSD test was used to perform comparisons between the dry biomass of different mixing duration systems. A statistically significant difference existed for the dry biomass of Scenedesmus sp. in all three mixing duration systems.

The temperature, oxygen, and pH profiles were also monitored and are described in addition to the growth kinetics in Section S2.1. The positive growth rates for all three systems during the entire experiment suggest the suitability of environmental conditions for biomass growth as shown in Fig. 2(C). The drop-in growth rates for the last four days of the experiment might have resulted from the shielding effect of the cells, and elevated pH in systems might have converted dissolved CO₂ into the carbonate and bicarbonates, which remained unconsumed and slowed down the growth of microalgae [31]. A comparison of the current system with other studies in terms of biomass productivity and environmental conditions indicates the potential of the submersible regime for enhanced biomass output under optimal environmental conditions (e.g., aeration, CO₂ supply, and high light intensity) as shown in Table 1. No statistically significant difference was found in the specific growth rate of Scenedesmus sp. among R2 and R3 mixing durations (p > 0.05). However, the specific growth rate was significantly higher when Scenedesmus sp. was cultivated under continuous mixing duration.

The mixing efficiency experiment analysis indicated that the biomass production varied only slightly among the samples (± 0.32 mg L^− 1) taken from different heights (Section S2.2). This small variation indicates that the mixing system was highly efficient, as it ensured a uniform distribution of Scenedesmus sp. along the entire area of the pond. The liquid velocity initiated by the submersible pump in the push channel was 0.74 m s^− 1, resulting in a turbulent flow necessary for homogeneity (Fig. 1(C, D)). The flow velocity greatly reduces in the bent channels and along the straight channel due to pressure loss from clockwise movement and head loss associated with liquid frictional resistance (range, 0.25 to 0.29 m s^− 1). The mean flow velocity of 0.35 m s^− 1 maintained in submersible regime exceeds the operational flow velocities (range, 0.2 to 0.3 m s^− 1) employed for mixing of culture in traditional raceway ponds, ensuring efficient mixing and avoiding formation of dead zones [9]. The pump inlet in the pull channel draws liquid from the bottom layer and propels it into the top culture layers through an outlet that allows further agitation and mixing as indicated in Fig. 1(C).

The estimated evaporation rate was 0.405 L d^− 1 in the R1 system, which represents 6.75% of the total working volume of 6 L. The evaporation rate values were 0.337 L d^− 1 and 0.270 L d^− 1 in R2 and R3 systems, corresponding to 5.6% and 4.5% of total working volume, respectively. The higher evaporation rate in the R1 system might be due to high surface turbulence compared to the R2 and R3 systems. The consistent agitation in the culture promotes better heat transfer from the culture surface to the air, speeding up the evaporation losses [32]. The surface turbulence replaces the layer of humid air with fresh air on the surface of the open raceway pond, leading to accelerated evaporation of the culture [33]. In R2 and R3 systems, the mixing was stopped for 4 and 8 h, respectively, causing fewer disruptions to the air-liquid interface, ultimately leading to slower evaporation rates. The evaporation rate was estimated as the ratio of culture volume height to time multiplied by the area, as shown in Eq. 1, and was verified with the evaporation losses estimated from Eq. 2 proposed by Malek et al. [19] and Matanguihan et al. [20]. There was a difference of ± 20 mL d^− 1 in culture volume corresponding to 0.33% error, suggesting that the evaporation loss can be expressed as a simplified function of height for controlled raceway ponds.

A few studies have suggested intermittent mixing during cultivation as an energy saving alternative to continuous mixing [34, 10]. However, the biomass settling experiment indicated that 8 h of mixing cessation resulted in 27% settlement of biomass compared to the initial concentration of algae in the R3 system, suggesting sinking of Scenedesmus sp. under the influence of gravity. Similarly, after 4 h of stoppage, 14% of biomass was found to be sinking to the bottom of the R2 system. In the previous study, the flagella of Terselmis suecica ceased to sink to the bottom of the pond after switching off the pump for a night, as fast moving and lively cells were observed under the microscope [34]. Since the Scenedesmus sp. is nonmotile due to a lack of flagella, this is more susceptible to settling due to natural sedimentation at the bottom, when there is no mixing [35]. The stoppage of mixing in R2 and R3 systems might have led to the development of stagnant or dead zones in the raceway ponds. These zones reduce effective pond volume and contribute to biomass loss through the creation of anaerobic conditions and respiration in dark zones [36, 37]. This might be the reason for reduced biomass production in R2 and R3 systems as compared to the R1 system.

The present study entails a promising approach of utilizing submersible pumps in raceway systems to improve the design and operations of raceway ponds. The traditional raceway systems are largely dependent on the immersion depth of the paddle wheel, where an increase in depth can lead to culture volume spilling out by the paddle wheel, allowing a maximum operating velocity in the range of 0.15–0.3 m s^-1 [45]. Furthermore, the energy consumption of the paddle wheel also increases with an increase in culture volume depth as the power directly depends on the culture load [46]. The inclusion of a submersible pump in place of a paddle wheel for raceway ponds permits a wider range of operating velocities (0.10–0.45 m s^-1) without causing spill out. In the current study, it was observed that the pump impeller caused the diffuser to generate pressure and propel the culture volume turbulently, such that the flow caused the culture volume to be uniformly mixed throughout the raceway system. Additionally, a sparger connected with a compressor allows pure air bubbling in conventional open raceway ponds. In the current study, no spargers were used and the system received air through flow turbulence generated at the surface by submersible pump output thrust. The presence of two slits at each bent zone created three narrow channels that stabilized the flow and improved mixing. These curved slits enhance the mixing of microalgae in culture and prevent the formation of dead zones in areas with minimal water flow [47]. Nevertheless, further studies are required for optimization of operational conditions in submersible regime systems to maximize energy utilization and enhance biomass productivity.

The submersible pumps are also known for energy efficiency as they are placed directly in water and can operate at variable frequency drives. The net energy efficiency (η) for the submersible pump was estimated as the ratio of the theoretical power (W_h) to the actual measured energy consumption (W_m) as shown in section S2.3. The net energy efficiency corresponds to 62% for a mean velocity maintained at 0.35 m s^-1. The energy efficiency of the raceway system dropped to 11% when estimated for the maximum working volume (~ 9L), providing 0.35 m s^-1 mean velocity indicating the energy demand increases alongside increasing culture depth and mixing duration. It is estimated that paddle wheels contribute to about ~ 25% of the total power consumption for raceway cultivation systems [46]. The net energy ratio (NER), 0.049, was calculated based on the difference between the energy produced from biomass to the energy consumed for its production, as described in section S2.3. The input energy supplied by the submersible pump was 720 KJ d^-1, whereas the theoretical energy recovery for Scenedesmus sp., 19.5 MJ kg^-1, was estimated based on calorific values evaluated by [48]. The NER values in the range of 0.04–0.09 are typical of open raceway systems and differ by the category of harvesting method, microalgae secondary products, economies of scale and inclusion of solar panels [49].

In algal cultivation systems, priority should be on reducing the energy requirements as well as focusing on optimizing the mixing. Considering the experimental outcomes of the present study, an R1 based raceway system can be used to treat 42 m³ of working volume of wastewater by installing raceway systems in an area of 100 m². R1 system with 24 h mixing was considered for scale-up as the system produced the highest biomass production corresponding to the largest nutrient recovery. It is assumed that six submersible regime ponds will be operated with a 65% working volume compared to the total capacity of 6528 m³ each having an area of 13.6 m². Based on the experimental results achieved for nutrient removal and biomass production in the R1 system, the average removal for nitrate and phosphate would be 65.2 kg and 31.2 kg per year while the Scenedesmus sp. production would be 1.6 x 10³ kg per year. According to the scale, a 1.0HP submersible pump (Flow 300 L min^− 1, Power 0.75 KW, Pipe diameter 50.8 mm) can be used for each raceway pond to circulate the culture liquid with a mean velocity of about 0.35 m s^− 1. The total pump driven energy required to produce 1 kg biomass would be 24.8 MJ, resulting in energy efficient biomass production. The proposed upscaling scheme indicated that 100 m² will be able to produce 1657.6 kg per year dry biomass of Scenedesmus sp., which can be utilized for further applications including biofuels, nutraceuticals and animal feed [50], as shown in Fig. 4. However, these are theoretical values that are calculated based on the results of a lab-scale study. The actual biomass production and nutrient removal of the proposed raceway pond may differ slightly due to variations in environmental conditions and the source of wastewater used as a nutrient media for Scenedesmus sp. growth.

Fishmeal is a common protein source in fish feeds. However, its extensive use in large amounts has led to a decrease in the availability of this protein-rich resource [51, 52]. According to Patnaik et al. [53], Scenedesmus sp. biomass is rich in protein, carbohydrates, and lipids, making it a promising alternative protein source for fish feed.