Evaluation of Biomass, Lipid and Chlorophyll Production of a Microalgal Consortium Cultured in Dairy Wastewater

Abstract

Currently, microalgae cultivation requires strategies to improve yield and reduce production costs for biotechnological purposes. Dual-purpose systems are one of the most widely used strategies for these purposes, using culture media supplemented with wastewater. This study evaluated the growth of a microalgal consortium in three treatments with different proportions of dairy wastewater (WWDI) and synthetic BBM-3N medium (Bold Basal Medium), with the aim of optimizing biomass, chlorophyll, and lipid production. The treatment with 60% BBM-3N medium and 40% WWDI (Case 3) showed the best performance under experimental conditions, reaching a biomass concentration of 0.7543 g/L, a chlorophyll production of 10.6890 µg/mL, and a lipid content of 14.63%. These results exceeded those obtained in the cases with 100% BBM-3N or 100% WWDI. In addition, a techno-economic evaluation was carried out via SuperPro Designer simulation, which allowed the annual operating costs for each treatment to be estimated. Case 3 stood out as the most viable option, combining good biological performance with lower operating costs compared to the raw material, demonstrating the potential of using wastewater as a partial source of nutrients in microalgal cultures intended for the production of value-added products.

1. Introduction

Cow milk production in Mexico has increased by 9% nationwide over the past five years, with the state of Guanajuato contributing 7% of the total [1]. Most of this production is used to make various products such as cheese, yogurt, milk formulas, powdered milk, and other dairy products. This generates a considerable amount of wastewater that can and should be put to good use [2].

Wastewater from the dairy industry (WWDI) mainly includes residues from the manufacturing cycle such as spilled milk, spoiled milk, skimmed milk, and curdled milk. It also includes by-products from processing operations (whey and milk), washing contaminants, and various additives introduced during product manufacturing [3]. The most important characteristics of WWDI are organic matter, high protein content, low heavy metal content, and ease of biodegradation [4]. Due to these characteristics, discharging these effluents without prior treatment poses risks to the environment and human health [5].

The dairy industry is required to implement conventional systems for treating its wastewater (raw water), and adapting the process to the specific needs of each company leads to inefficient operations that would result in an economic burden for any industry [6]. That is why an important selection criterion for the treatment system is its ability to cope with constant fluctuations in organic load while maintaining an economically viable process [7].

On the other hand, the growing interest in research on microalgae crops and their derivatives shows their biotechnological potential for various industries, notably wastewater treatment, bioenergy production, health, human food, livestock feed, aquaculture, pigments, and the production of antioxidants, triglycerides, and polysaccharides [8].

The treatment of wastewater based on microalgae cultivation is a promising alternative, as wastewater contains the macro- and micronutrients required for microalgae biomass growth [9]. It has been found that microalgae cultivation for wastewater treatment reduces contaminant levels and promotes biomass production, and such systems have been termed dual-purpose systems [10]. The microalgae species most studied for dual-purpose systems are mainly Chlorella spp. and Scenedesmus spp. and many other native microalgae [11]. Microalgae species have been used to treat domestic, agricultural, and industrial wastewater, for example, in the dairy, paper, aquaculture, and pig farming industries [12]. The dairy and food industries in general discharge large amounts of wastewater with a high concentration of nutrients, which meet the conditions for an environment suitable for the growth of microalgae biomass [13].

It has been reported that the use of local consortia and native microalgae species are an ideal alternative for the use of wastewater components, which can contribute to the management and protection of water bodies since they are low-cost and environmentally responsible systems. Likewise, the use of wastewater for microalgae cultivation reduces the use of fresh water, reduces the cost of adding nutrients, removes compounds, and reduces the organic load [14]. However, there are few studies reporting the use of microalgal biomass native to central Mexico for the treatment of wastewater from the dairy industry.

This study examines a dual-purpose system for treating dairy wastewater and producing biomass, lipids, and chlorophyll from a consortium of microalgae native to central Mexico in order to evaluate the biotechnological potential of local inputs.

2. Materials and Methods

2.1. Collection of Biological Material and Wastewater

The microalgal consortium was collected at the Neutla Dam located in the city of Comonfort (20°42’33.5” N 100°51’48.5” W), Guanajuato, Mexico. The consortium was maintained under laboratory conditions using BBM-3N culture medium [15]. The following growth conditions were maintained: 25 °C, an initial pH of 7.0, aeration of 0.03 vvm, and a photoperiod of 16 h of light and 8 h of darkness. The morphological identification of the consortium was done by observation through a microscope and compared with the culture collection of the University of Texas (UTEX) and identified as Chlorella spp., Chlamydomonas spp., Pediastrum duplex, Scenedesmus spp., Closteriosis spp., Schroederia spp., and Spirulina spp.

The wastewater was collected from the dairy industry, GRUPO CUADRITOS®, located near the city of Celaya (20°37’56.5” N 100°47’04.8” W), Guanajuato, Mexico. The WWDI was filtered using 25 µm membranes to remove the largest suspended solids. The WWDI was characterized, yielding values of pH 7.6, chemical oxygen demand (COD) 3015.34 mg/L, phosphates (PO₄) 40.02 mg/L, nitrates (NO₃) 50.98 mg/L, ammonium (NH₄) 7.2 mg/L, dissolved oxygen (DO) 5.64 ppm, proteins 121.6 mg/L and TKN 66.57 mg/L (total Kjeldahl nitrogen).

2.2. Description of the Experimentation Stage, Production of Biomass, Lipids, and Chlorophyll

Three cases were considered for the experimental evaluation of biomass, lipid, and chlorophyll production using WWDI and BBM-3N, as described above. The amount of each component in the mixture is described in Figure 1, and each experiment was performed in duplicate. The culture type was mixotrophic, using glucose as the organic carbon source at 15 g/L in all cases. The same amount of inoculum was used, and in all cases, a total production volume of 4 L was maintained under optimal conditions of light (photoperiod, 16 h light and 8 h darkness), aeration (0.03 vvm), and constant agitation (bubbling). The 9-day growth time was based on the behavior of the microalgal consortium observed in the adaptation phase. During the experimental phase, the biomass growth kinetics [16], pH, chlorophyll content [17] (the total chlorophyll (a + b) was evaluated by measuring the absorbance of the methanol extract at 652 nm and 665 nm, with a UV/Vis spectrophotometer), nitrate consumption [18] (the NitraVerX reagent kit distributed by HACH was used (HACH Company, Loveland, CO, USA), along with the N method, Nitrate RA TNT, preloaded in the HACH DR3900 spectrophotometer—this method measures absorbance at 410 nm), and COD removal [18] (the COD HR reagent kit distributed by HACH was used(HACH Company, Loveland, CO, USA), along with the COD 1500 method preloaded in the HACH DR3900 spectrophotometer(HACH Company, Loveland, CO, USA)—this method measures absorbance at 435 nm), were determined daily. In addition, the accumulated lipid production at the end of the growth phase was estimated [19] (Bligh–Dyer method, using a mixture of chloroform–methanol 1:2 v/v as solvents, with extraction temperature of 60 °C and drying time of 15 min).

2.3. Process Simulation and Economic Projection of Biomass, Lipid and Chlorophyll Production

In order to analyze the technical and economic feasibility of the process at an industrial level, the process was simulated using SuperPro Designer v10.0 software (Intelligen, Inc., Scotch Plains, NJ, USA). The process was considered to be carried out in continuous operation mode, with 330 days of operation per year. The process includes the cultivation stage, primary harvesting of biomass through flocculation with chitosan, secondary harvesting through filtration, chlorophyll extraction with methanol as a solvent (prior mechanical maceration and exposure to cold solvent), and subsequent evaporation of the solvent to 60 °C, Figure 2 (flowsheet). On the other hand, the wet and residual biomass from the chlorophyll extraction stage is combined with a mixture of solvents, chloroform/methanol 1:2 v/v, for lipid extraction. This last stage of the process is very similar to the chlorophyll extraction stage.

For the cultivation stage, a flow of 2720 L/h is fed, including the nutrient mixture, inoculum, and glucose to the P-5/AFR-100 stoichiometric bioreactor in the proportions considered for each case analyzed. The culture maintains the operating conditions considered in the experimental phase, and a residence time of 9 days was considered. It was considered that only 1 ha of surface area needed to be covered for cultivation, and for this purpose, each bioreactor would have a volume of 300 L and would cover an area of 4.6 m² to maximize the use of sunlight and avoid dark areas. Artificial light consumption was not considered, as it was assumed that natural sunlight would be used. Under these conditions, it was determined that 2176 units would be necessary for the cultivation stage.

After biomass cultivation, the effluent stream from P-5/AFR-100 is mixed with chitosan (1 g/L in a solution of acetic acid and water 20:80 v/v) to act as a flocculant, and this stream is fed into the clarifier P-10/CL-101. At this stage, the first harvest is carried out, and the biomass that exits the bottom of the clarifier is considered to reach a concentration of 50 g/L and represent 95.71% of the biomass fed to this stage [20]. The S-116 stream that exits at the bottom of the clarifier passes to the second harvesting stage, for which filtration through a filter press, P-11/PFF-101, is considered. At this stage, the biomass is washed with fresh water to remove impurities, and the biomass is concentrated to 200 g/L, with a 90% recovery of the biomass that is fed to the secondary harvest [20]. The S-102 stream containing biomass with 80% w/w moisture is fed to the chlorophyll extraction stage, equipment P-3/SMSX-102. It was considered that the microalgae biomass has 7.03% lipids, 1.52% chlorophyll, and the rest was considered as cell debris still rich in carbohydrates and proteins (according to the results of the experimental phase of this study).

For chlorophyll extraction, the wet biomass solution is mixed with methanol (2:1 v/v ratio biomass/solvent) for a residence time of 5 min and then kept at 4 °C for 24 h (residence time) without stirring. The organic phase (chlorophyll + methanol, S-110) is transferred to an evaporator in order to separate the methanol at a temperature of 60 °C and recirculate it to the process, Figure 2 (flowsheet). The operating conditions for chlorophyll extraction were taken from Cabrera-Capetillo et al. (2023) [21]. In the chlorophyll extraction stage, there is also an outlet stream containing the residual biomass + a small amount of methanol, stream S-123; the residual biomass is still rich in lipids, carbohydrates, proteins, and other compounds. This stream is fed to the chlorophyll extraction stage, P-14/SMSX-101, together with a mixture of chloroform/methanol solvents (ratio 2:1 v/v), stream S-128. The feed to P-14/SMSX-101 maintains a 10:1 (v/v) ratio of solvent mixture (stream S-128) to Residual Biomass (stream S-123). For chlorophyll extraction, the mixture is kept in the P-14/SMSX-101 equipment for 4 h of residence time in the mixer and then 4 h of residence time in the sedimenter. In the simulation of this stage, it was considered that 100% of the lipids reported in the experimental extraction phase were extracted. There are two output streams from the extraction stage: (1) organic phase, stream S-120 (containing lipids + solvent mixture) and (2) aqueous phase, stream “Residual Biomass” (residual biomass, still rich in carbohydrates, proteins, and other compounds). After extraction, the organic phase (lipids + solvents) is sent to a solvent evaporation stage at 60 °C to finally obtain a pure lipid stream. The operating conditions for the lipid extraction stage were taken from [22].

Once the simulation for Case 1 was completed, the process was simulated for Cases 2 and 3 (Figure 3 and Figure 4). The operating conditions remained the same in all cases, but the BBM-3N and WWDI feeds were different, resulting in different concentrations of biomass, chlorophyll content, and lipids.

With the results of the simulations (material and energy balances), we proceeded to analyze the technical evaluation of the process. For this analysis, we considered only the energy consumption of the process and the behavior of this variable at each stage of the process. For the economic evaluation analysis, we considered the report on total and unit production costs produced by the simulator. The production cost is calculated directly in the simulator using the results of the material balance and including the cost of raw materials, labor, services, and complementary operating costs related to the use of the facilities. The costs used for the economic evaluation were taken as reported in Cabrera-Capetillo, et al. (2023) [21].

Continuous mode simulation was used as an industrial-scale projection scenario to leverage average productivity data obtained experimentally. The limitations of extrapolating batch data (experimental data) to continuous operation lie in the dynamics of operation, control of conditions, and the behavior of the biological system. In a real industrial case, these must be taken into account, but for the simulation, they are not considered determining factors.

The validation of kinetic parameters in continuous pilot tests is a critical step when seeking to design, scale, or control a bioprocess that will operate continuously. Although kinetic parameters can be estimated in batches, there are specific needs for which it is essential to validate them in continuous pilot tests, such as reproducibility, refining simulation models, control and optimization, and errors in design and scaling.

3. Results and Discussion

3.1. Experimental Evaluation of Biomass, Lipid, and Chlorophyll Production

For Cases 2 and 3, which contain 40% WWDI, at the end of the 9 days of cultivation and after harvesting, measurements of two parameters of the resulting water were taken in order to ensure proper disposal of the effluent. The first was nitrogen in its inorganic form (nitrates), which represented nitrogen consumption by the microalgal consortium. Initially, in the experiment for Case 2, we had a value of 20.4 mg/L, and at the end, we obtained 0 mg/L of nitrates, which represents 100% consumption of this nutrient. For Case 3, the initial estimated value was 68.9 mg/L higher due to the contribution from the addition of BBM-3N medium and the final value was 46.7 mg/L, representing 32.22% consumption. In both cases, the microalgae consumed 20 to 22 mg/L of nitrates. In this sense, this value can be taken as an indicator of the savings of this nutrient. This indicates that 35.22 mg NO₃⁻ and 61.95 mg NO₃⁻ consumed per g biomass are required for Case 3 and Case 2, respectively. The second value measured was COD, which is important for determining the reduction in organic load and thus disposing of the effluent correctly. For Case 2, initial values of 1321 mg/L were obtained in the experiment, and at the end, 196 mg/L of COD; for Case 3, the initial value was 1330 mg/L and the final value was 382 mg/L of COD, representing a decrease of 85.16% and 71.27%, respectively. In this regard, a clear reduction in key pollution indicators was recorded, which supports the use of the microalgal consortium as a bioremediation agent, in addition to its use for obtaining value-added compounds.

In line with other recent studies combining wastewater treatment with useful microalgal biomass production, Rashd et al. (2025) [23] evaluated the use of Scenedesmus parvus and Coccomyxa dispar in raw lake water, observing significant contaminant removal under real lake conditions (62% reduction in COD). Although the focus was mainly ecological, their results support the use of microalgae to enhance water resources. Similarly, Ooi et al. (2023) [24] cultivated Coccomyxa dispar and Scenedesmus parvus in palm oil industry effluents with outstanding results in biomass and lipid production (up to ~229 mg/L), as well as 81% COD removal. These findings reinforce the viability of integrating wastewater or contaminated water treatment with the production of compounds of biotechnological interest.

Figure 5 shows the behavior of microalgal biomass production during the 9 days of cultivation. The maximum values obtained were as follows: 0.7470 g/L for Case 1 (100% BBM-3N culture medium), 0.4493 g/L for Case 2 (60% fresh water + 40% WWDI), and 0.7543 g/L for Case 3 (60% BBM-3N culture medium + 40% WWDI). It should be noted that Case 3 achieved a final biomass comparable to that of the control (Case 1), despite partially replacing the culture medium (source of fresh nutrients) with WWDI. This indicates that the microalgal consortium has adapted well to the environment enriched with dairy effluent, allowing for a reduction in the use of synthetic nutrients without compromising production, which could improve the economics of the process. In contrast, Case 2 showed lower performance, reaching its maximum concentration between days 7 and 8, followed by a decrease towards the end of the culture. This trend can be attributed to a limitation in nutrient availability, given that this treatment does not contain BBM-3N culture medium, but only diluted wastewater.

This study used a native microalgal consortium, which has shown advantages over monocultures, particularly in complex media such as WWDI. For example, Qin et al. (2016) [25] reported higher productivity with consortia compared to pure cultures of Chlorella spp., obtaining biomass concentrations of 5.41 g/L versus 4.72 g/L, respectively. This superiority has been associated with metabolic complementarity between species and greater resilience to fluctuating conditions. In our study, a progressive change in the composition of the consortium was observed during cultivation. Initially, in the adaptation stage of the consortium, the population was diverse, including Chlorella spp., Chlamydomonas spp., Pediastrum duplex, Scenedesmus spp., Closteriosis spp. and Schroederia spp. However, when cultivated in dairy wastewater, the dominant species were Chlamydomonas sp., Chlorella sp. and Scenedesmus sp., while other species declined or disappeared, such as Pediastrum duplex.

This dynamic behavior in the consortium structure could be explained by selective pressure from the waste environment and possible interaction with the native microbiota of the effluent. Previous studies, such as that by Chinnasamy et al. (2010) [26], also document the influence of CO₂ and organic load on population dynamics and productivity in microalgal consortia cultivated in dairy effluents, reporting yields of up to 1.47 g/L in 9 days.

Total chlorophyll production (a + b), a key indicator of photosynthetic activity, showed differences observed between treatments. Case 2, which used only WWDI and fresh water as the culture medium, had the lowest chlorophyll concentration, with a maximum value of 2.0731 µg/mL (Figure 6). This low production can be explained by the absence of synthetic nutrients that promote photosynthesis, as well as a possible effect of the dilution of essential nutrients in the medium. In contrast, Cases 1 and 3 showed the highest concentrations: 8.6062 µg/mL and 10.6890 µg/mL, respectively. Both treatments included BBM-3N medium, either in its entirety (Case 1) or partially (Case 3). It is noteworthy that Case 3, which incorporated 40% WWDI into the mixture, achieved higher chlorophyll production than the control, suggesting a positive effect of the compounds present in WWDI on the metabolism of the microalgal consortium.

All growth curves reached their maximum chlorophyll production peak on day 7 of cultivation, which coincides with the stationary phase of biomass. This behavior suggests that chlorophyll accumulates mainly during the active growth phases and subsequently tends to decrease. From a biotechnological point of view, these results are relevant, as chlorophyll is a pigment of high commercial value in industries such as food, cosmetics, and pharmaceuticals. The higher production observed in Case 3 highlights the potential of integrating wastewater as an alternative culture medium, allowing for a reduction in the costs associated with the use of synthetic nutrients without compromising yield.

Compared to monocultures reported in the literature, the microalgal consortium evaluated showed lower chlorophyll production. Previous studies indicate that monocultures of Chlorella spp. grown under similar wastewater conditions can reach total chlorophyll concentrations between 20 and 30 µg/mL [27]. In the present study, the values obtained were below this range, even in treatments with higher nutrient input (Cases 1 and 3). This difference could be attributed to the population changes characteristic of consortia, where some species adapt and proliferate, while others decline or disappear, thus affecting the overall pigment balance [28].

In addition, the possible interaction with the effluent’s own microbiota must be considered, as it could compete with or interfere with the development of certain microalgae species [28]. Another important factor is the turbidity of the medium, generated by the solids and organic compounds present in the wastewater. This turbidity reduces light penetration, limiting the photosynthetic efficiency of the culture and, therefore, chlorophyll synthesis [29].

Lipid production varied significantly between treatments (Figure 7). Case 1, corresponding to the control with 100% BBM-3N medium, had a lipid content of 7.03 ± 0.24%. Case 2, cultivated with wastewater and fresh water, reached a value of 8.72 ± 1.97%, while Case 3, with a mixture of BBM-3N and wastewater, showed the highest content, with 14.63 ± 0.78%. These results indicate that the greatest accumulation of lipids occurs when the microalgal consortium is exposed to moderate stress conditions, as in Case 3, where nutrient availability is limited due to the partial replacement of the culture medium with WWDI. This phenomenon has been widely documented, as nutritional imbalance, particularly nitrogen limitation, stimulates lipid accumulation as an energy reserve mechanism in various microalgae species [30].

Under ideal conditions, some microalgae species can accumulate between 20% and 30% of their dry weight in lipids [31]. However, this value depends on the type of strain, culture conditions, and type of substrate. For example, studies with monocultures of Chlorella zofingiensis grown in dairy effluents have reported lipid accumulations of up to 27.7% [32], while native consortia have reached values close to 21%. Comparing this background information with the results obtained, it can be stated that the consortium used shows a moderate lipid response, particularly noteworthy in Case 3, where a balance between biomass productivity and lipid content is observed. This treatment not only allowed for significant lipid production, but also took advantage of the nutrients in the wastewater, thus reducing the need for synthetic media, which represents an advantage in economic and environmental terms. In contrast, the lower values observed in Case 1 may be due to the abundance of available nutrients, which favors cell growth but does not promote lipid accumulation. On the other hand, the greater variability in the data from Case 2 could be related to the lack of control over nutrient levels, which creates conditions of uneven stress during cultivation.

Below is a comparative summary of the maximum values obtained for biomass, total chlorophyll, and lipid percentage for each of the experimental treatments evaluated (

Table 1. Summary of the values obtained for biomass, total chlorophyll, and lipid content for the three experimental treatments. Values used for the simulations.

Case	Composition of the Medium	Biomass (g/L)	Total Chlorophyll (µg/mL)	% Lipids
1	100% BBM-3N (control)	0.7470	8.6062	7.03
2	60% Fresh water + 40% WWDI	0.4493	2.0731	8.72
3	60% BBM-3N + 40% WWDI	0.7543	10.6890	14.63

Average production values, values measured in duplicate. WWDI (wastewater dairy industry). BBM-3N (Bold Basal Medium).

). This summary provides an integrated view of the behavior of the microalgal consortium under different culture medium compositions, as well as identifying the most efficient treatment to produce compounds of biotechnological interest.

3.2. Results of the Techno-Economic Evaluation for the Production of Chlorophyll and Lipids from a Microalgal Consortium

Based on the results obtained during the experimental phase, the biomass, chlorophyll, and lipid production process were simulated on an industrial scale using SuperPro Designer v10.0 software. This simulation made it possible to evaluate the technical and economic feasibility of each of the three cases presented in this study, considering the operating conditions previously described in the methodology, as well as to detect areas for improvement in the process. Figure 2, Figure 3 and Figure 4 show the flowsheet developed in SuperPro Designer v10.0 for the simulation of the cultivation process corresponding to Cases 1, 2, and 3, respectively.

The main objective of this simulation was to estimate the efficiency of the process not only in terms of productivity but also in relation to energy consumption, use of inputs, and costs associated with each case analyzed experimentally; this analysis was from an industrial perspective, considering 1 ha as the area to be used only for the cultivation stage. In particular, the impact of partially replacing the BBM-3N culture medium with WWDI on the overall yield and production cost of chlorophyll and lipids was analyzed.

Table 2. Summary of the maximum values obtained for biomass, total chlorophyll, and lipid content at the end of cultivation for the three experimental treatments.

Case	Medium Composition	Biomass (kg/yr)	Chlorophyll (kg/yr)	Lipids (kg/yr)
1	100% BBM-3N (control)	15949.97	211.86	988.87
2	60% Fresh water + 40% WWDI	9130.33	49.17	701.34
3	60% BBM-3N + 40% WWDI	15501.28	258.23	2000

WWDI (wastewater dairy industry). BBM-3N (Bold Basal Medium).

shows the results of the estimated annual production of chlorophyll and lipids for the three cases evaluated. As shown in Table 2, the combination of BBM-3N culture medium with 40% WWDI (Case 3) produced higher levels of chlorophyll and lipid production, 258.23 kg/year of chlorophyll and 2000 kg/year of lipids, reflecting superiority over the other two cases, not only in terms of productivity but also in terms of efficient use of resources. Compared to the control (Case 1), chlorophyll increased by approximately 21% and lipids by 50%. This result highlights the beneficial effect of integrating WWDI into the culture medium on improving the production of compounds of interest. In a similar study, where dairy wastewater was used for lipid accumulation, Kumar et al. (2023) [33] reported a 27.5% increase compared to the control. This reflects positive results when mixing WWDI with BBM-3N in microalgae consortia.

Fresh water consumption is a critical indicator in the technical evaluation of sustainable processes. According to the simulation, and as shown in Figure 8, Case 1 had the highest annual freshwater consumption (21,184.73 m³/yr), due to the exclusive use of synthetic culture medium, BBM-3N, prepared with fresh water. In contrast, Cases 2 and 3, which integrate WWDI at 40%, showed reductions of 30% compared to the control (Case 1). This difference reflects the potential of using effluents as a strategy to reduce freshwater demand, which is a key aspect of process sustainability. The use of WWDI reduces the impact on freshwater consumption, which will be directly proportional to the level of substitution. This is determined by considering the production of metabolites of interest, in this case the accumulation of lipids and chlorophyll.

Energy consumption (Std Power) is similar in all cases: 17,454,436 kWh. However, steam consumption for each of them does vary, and this is related to the production of chlorophyll and lipids in each process. Case 2 has the lowest steam consumption, 273 MT (megatons), while in Cases 1 and 3 it is much higher, 364 MT and 363 MT, respectively.

In terms of economic evaluation, the results obtained in the simulator reveal differences observed between treatments (

Table 3. Total production cost, values calculated using the simulator.

Case	Labor (USD/yr)	Material (USD/yr)	Utilities (USD/yr)	Facility- Dependent (USD/yr)	Operating (USD/yr)
1	5,032,729	2,021,992	2,716,876	436,101,000	445,873,000
2	5,802,358	328,687	2,714,377	436,364,000	445,209,000
3	5,812,693	1,346,801	2,717,453	436,387,000	446,264,000

USD /yr (American Dollar per year).

). Case 3 showed the highest operating costs, 446,264,000 USD/yr (American Dollar per year), mainly attributable to the intensive use of nutrients. The operating cost of the process is mainly the sum of labor cost, material cost, utilities cost, and facility-dependent cost. Although Case 2 significantly reduced raw material costs, the operating cost is high mainly due to the facility-dependent cost, which is very similar in all cases. In this study, the complete process simulation indicated that operating costs are dominated by the cultivation stage, which accounted for 99% of the total costs. Approximately 98% of the operating costs correspond to facility-dependent expenses, which include equipment maintenance, fixed capital depreciation, and other costs such as insurance and local taxes, all calculated as a percentage of the direct fixed capital cost, which is itself derived from the estimated equipment purchase cost. The simulator’s database was used to determine equipment costs, applying the Chemical Engineering Cost Index for inflation adjustments. The cultivation stage alone represents 99% of the total equipment cost, explaining its high contribution to facility-dependent expenses. This trend is consistent with Cabrera et al. (2023) [31], who reported that operating closed photobioreactors entails high energy requirements to maintain mixing and controlled conditions, with energy consumption exceeding 170 kWh per kg of biomass, making cultivation the most impactful stage in processes involving microalgae. Similarly, Valdovinos et al. (2024) [34] reported production costs ranging from 836.9 to 1131.5 USD/kg, noting that the integration of wastewater reduces these costs by 10%, an observation which aligns with our findings in Case 3, where resource consumption and operating costs were reduced without compromising productivity. Although the evaluated process is not competitive compared to open pond systems, it offers advantages in terms of cultivation control and product quality, as highlighted in recent studies on algal biorefineries.

The cost of equipment was similar in all cases, being USD 373,284,000 for Case 1 and USD 373,520,000 and USD 373,540,000 for Cases 2 and 3, respectively. In terms of labor costs, only operators were considered to keep the process running continuously, and in all cases, this cost is similar. Regarding the cost of raw materials, in Case 1, 62.68% is due to the consumption of EDTA (chelating agent), followed by chitosan (9.41%), which is used as a flocculant, and acetic acid (6.36%), which is also used to prepare the flocculant. Case 3 shows similar results to Case 1, with 56.46% due to EDTA, 13.69% to chitosan, and 9.43% to acetic acid. In Case 2, 56% of raw material costs are also associated with chitosan and 37.92% with acetic acid. In this case, there is no impact on raw material costs due to the nitrogen source, as it is consumed directly from wastewater. Based on these results, it would be advisable to analyze whether there is another flocculant that could maintain flocculation efficiency but at a lower cost. The choice of a suitable flocculant applied to a microalgae strain or consortium is crucial, as this will impact the yield of the primary harvest and the quality of the biomass coming out of the first harvest stage. Flocculation is applied before the second harvest stage with the aim of concentrating on the biomass and reducing the impact on the energy consumption of the secondary harvest [35], which can concentrate the biomass by centrifugation or filtration. Regarding the cost associated with solvents for the extraction of compounds of interest, in Case 2, these represent almost 5% of the total cost of raw materials. Although they are not the most significant cost, it is desirable to analyze alternatives for use in order to improve extraction yield, reduce operating costs, and lessen the environmental impact of the process. In terms of extraction sustainability, recent advances offer promising alternatives to conventional solvent-based methods. For instance, ultrasound-assisted extraction using natural deep eutectic solvents (NaDES) has proven both effective and scalable for pigment recovery from Spirulina [36]. Green extraction techniques, as reviewed by Kopp (2025), demonstrate yields comparable or even superior to traditional approaches, while improving energy efficiency [37]. Comprehensive reviews, such as Fatima et al. (2023), outline a range of advanced methods (including NaDES, microwave-assisted, and ultrasound-assisted extraction) that outperform conventional methods in terms of efficiency and solvent use [38]. These greener extraction routes represent viable directions for future improvements of the evaluated process, aiming to reduce solvent toxicity, operational costs, and overall environmental impact.

Overall, the simulation results also suggest that Case 3 represents the most favorable scenario, combining high chlorophyll and lipid production yields with a significant reduction in water and nutrient use, which has a positive impact on operating costs, thus meeting the technical and economic feasibility criteria for sustainable dual-purpose processes.

4. Conclusions

The results of the exploratory study helped to select the volumetric proportions of WWDI and BBM-3N for the optimal cultivation of a consortium of native microalgae. This indicated the usefulness of these exploratory analyses for selecting not only the medium, but also the growth conditions and efficiency of native microalgae consortia for optimal biomass production in dual-purpose systems. Furthermore, it can be concluded that supplementing BBM-3N with 40% of the volume of WWDI favored the growth of the native microalgal consortium studied and the maximum removal of nutrients and organic load. Among the treatments evaluated, Case 3 (60% BBM-3N + 40% WWDI) showed the best performance in terms of cell growth, with a biomass concentration of 0.7543 g/L, chlorophyll production of 10.6890 µg/mL, and a lipid content of 14.63%.

Chlorophyll and lipid production were not affected by the use of WWDI; on the contrary, it helped reduce costs and raw materials, as the results showed similar behavior in Cases 1 and 3, which used BBM-3N culture medium. Likewise, when evaluating large-scale production with the help of the simulator, it was observed that Case 3 was the one that obtained the highest production of biomass, chlorophyll, and lipids at the lowest cost of nutrients.

Overall, the findings of this study support the viability of using wastewater in microalgae cultivation processes, with potential applications in bioenergy, natural pigment production, and effluent treatment. Future research should focus on scaling up the process, efficiently recovering the extracted compounds, and analyzing the comprehensive environmental impact of the proposed system.