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Article

Adaptation of Microalgae for the Production of Settling Flocs, Carotenoids, and Mineral Recovery from Municipal Secondary Effluents

by
Claudio Guajardo-Barbosa
1,
Tomás Guajardo-Rodríguez
1,
Ulrico Javier López-Chuken
1,
Icela Dagmar Barceló-Quintal
2,
David Cruz-Chávez
1 and
Julio César Beltrán-Rocha
3,*
1
Facultad de Ciencias Químicas, Universidad Autónoma de Nuevo León, San Nicolás de los Garza 66455, Nuevo León, Mexico
2
Departamento de Ciencias Básicas, Universidad Autónoma Metropolitana-Azcapotzalco, Ciudad de México 02200, Mexico
3
Facultad de Agronomía, Universidad Autónoma de Nuevo León, General Escobedo 66050, Nuevo León, Mexico
*
Author to whom correspondence should be addressed.
Phycology 2025, 5(4), 57; https://doi.org/10.3390/phycology5040057
Submission received: 5 September 2025 / Revised: 4 October 2025 / Accepted: 7 October 2025 / Published: 9 October 2025

Abstract

Microalgae cultivation offers a sustainable approach for nutrient recovery from municipal effluents and the production of valuable biomass, although efficient harvesting remains challenging. This study evaluated the adaptation of the microalgal consortium MC-10 in a sequential batch system through reinoculation of its flocculating fraction to enhance harvesting efficiency and mineral recovery. The consortium was initially cultivated under high ionic stress to promote cell aggregation. Laboratory preadaptation using secondary municipal effluents was then conducted, followed by an outdoor evaluation. In the initial propagation stage, flocculation efficiency reached 98%. Using municipal effluents, flocculation values of 99% were obtained, with a 149% increase in flocculating biomass under laboratory conditions, and 84% flocculation with a 125% increase in biomass production under outdoor conditions, demonstrating the consortium’s stability under environmental fluctuations and its suitability for biomass harvesting. The resulting biomass showed high potential as a biofertilizer due to its mineral content (47% dry weight, DW) and acid solubility (83%), indicating high nutrient bioavailability. Additionally, it contained a total carotenoid concentration of 451 μg/g DW, adding antioxidant value. These findings support the use of microalgae cultivation for the valorization of municipal effluents through the production of easily harvestable biomass with potential for reintegration into agricultural systems.

1. Introduction

Secondary effluents are the liquid discharges that remain after the biological stage of municipal wastewater treatment plants (MWWTPs) and still contain dissolved nutrients. These effluents represent a constant source of water and unused nutrients [1]. If these nutrients are not recovered, they can lead to the excessive enrichment of surface waters, resulting in eutrophication, a globally recognized environmental problem that degrades water quality and disrupts ecological balance by diminishing biodiversity in aquatic ecosystems [2]. Thus, there is an urgent need for strategies that simultaneously prevent eutrophication and enable nutrient valorization [3].
The treatment of secondary effluents from MWWTPs for nutrient removal (e.g., nitrogen and phosphorus) through microalgae cultivation has shown promise as a sustainable, efficient, and cost-effective strategy. In this context, microalgae not only contribute to water quality improvement but also serve as bio-factories, producing valuable resources through nutrient assimilation. Leveraging this dual role, microalgal cultivation further enables the recovery of value-added compounds from the resulting biomass, such as antioxidants, biofertilizers, and other high-value bioproducts [4,5]. However, despite these advantages, the large-scale application of microalgal systems remains limited because current harvesting methods are costly and technically challenging, remaining one of the main bottlenecks for large-scale commercialization [6,7]. large-scale recovery of microalgal biomass, whether from municipal secondary effluents or nutrient solutions, faces significant technical challenges due to low biomass concentrations in cultures (0.5–5.0 g dry weight, DW/L), small cell size (e.g., 2–20 μm in diameter), and the high colloidal stability of suspensions [8]. Therefore, systems designed for nutrient recovery coupled with microalgae production must overcome the high cost of biomass harvesting, which can account for 20–30% or more of the total production cost and remains a major barrier to commercialization [9]. Considering the need to produce biomass at large operational volumes, a microalgae harvesting technique that is both practical and economically viable is required. Conventional separation methods, such as centrifugation and filtration, have significant limitations due to their high energy demand and operational complexity [10]. Alternatively, chemical flocculants, both organic (e.g., polyacrylamide) and inorganic (e.g., Al2(SO4)3 and FeCl3), to induce cellular aggregation (i.e., flocculation) also present several limitations. These include high input costs, the need for strict pH control, and the risk of reducing biomass quality or contaminating the harvested biomass [10,11]. Therefore, there is a strong motivation to develop low-cost, efficient, and environmentally compatible alternatives for biomass harvesting [12].
In light of these limitations, naturally induced flocculation has been proposed as a viable operational alternative. This process is generally based on an increase in pH driven by photosynthetic activity, as well as on the composition of the culture medium (carbon, nitrogen, phosphorus, and micronutrients) and the neutralization of the negative charge on the cell surface by cations, which promotes the formation of cell aggregates that readily settle [11,13,14]. An example of this strategy was reported by Galán-González et al. [15], who achieved 62% flocculation efficiency in Haematococcus pluvialis. Among the most relevant findings, the study highlighted that flocculation was primarily influenced by the high ionic strength associated with elevated concentrations of dissociated nutrients and the neutralization of negative charges on the cell surface, whereas pH had no significant effect. These results suggest that effluents with high ionic loads may favor natural flocculation, although further experimental evidence is required to validate this under real wastewater conditions [16].
Another effective strategy to optimize microalgal biomass production and enhance nutrient recovery in aqueous media involves the selection of suitable strains and their preadaptation to the specific cultivation conditions. Factors such as the composition of the culture medium, pH, photoperiod, and temperature can induce physiological adaptations in microalgae, resulting in improved growth, higher substrate utilization efficiency, and increased tolerance to stress conditions [17]. For example, Li et al. [18] evaluated the adaptation of Chlorella sp. AE10 over 97 days and 31 batch culture cycles under a 10% CO2 atmosphere as a stress condition. Subsequently, both the original strain (previously maintained at 1% CO2) and the adapted strain Chlorella sp. AE10 were cultivated under a 30% CO2 concentration, with the latter reaching a productivity of 3.68 g/L, representing a 2.94-fold increase compared to the non-adapted strain. Additional examples of microalgae cultivation and adaptation under various stress conditions, including high salinity, acidic pH, high light intensity, extreme temperatures, and exposure to toxic compounds, have been compiled in the review by He et al. [19]. Nevertheless, few studies have addressed whether the preadaptation of microalgae to the ionic composition of secondary effluents can simultaneously enhance growth, nutrient assimilation, and biomass settleability. This remains an underexplored aspect in the literature, as most research has only highlighted the challenges imposed by wastewater ionic variability and salinity stress without systematically evaluating preadaptation effects [20,21,22].
Microalgae cultivation technology has the potential to recover nutrients present in wastewater by integrating them into the biomass produced during the process. This biomass can be valorized as a biofertilizer and soil conditioner, promoting nutrient reuse and strengthening the circular economy framework in agricultural systems [23]. Building upon this foundation, Beltrán-Rocha et al. [24] demonstrated that the cultivation of microalgal consortia using secondary effluents from MWWTPs produced biomass with a high mineral content, ranging from 29% to 53%. This biomass showed great potential as a biofertilizer due to its richness in minerals and organic compounds containing carbon, nitrogen, and phosphorus, such as carbohydrates, proteins, and lipids. Moreover, a distinctive feature of microalgal biomass is its antioxidant content, which can positively contribute to crop yield and quality. Antioxidants such as carotenoids, in addition to their role in seed, fruit, and flower pigmentation, also act as precursors of key phytohormones involved in plant growth regulation and signaling. These phytohormones include abscisic acid and strigolactones, which play a crucial role in plant responses to abiotic stress conditions such as water deficit, salinity, and phosphorus deficiency [25,26]. Taken together, the current evidence indicates that microalgae preadaptation and natural flocculation could represent a promising combined strategy for nutrient recovery and biomass valorization from municipal effluents. However, experimental validation remains limited [27,28].
In this context, the present study addresses this research need by evaluating the preadaptation of a microalgal consortium to high ionic loads and its ability to produce flocculating biomass in a sequential batch cultivation system using municipal secondary effluents. Specifically, we investigate how preadaptation enhances biomass harvesting efficiency under these conditions. In addition, we quantify the total mineral content and total carotenoids in the resulting biomass to assess their potential contribution as biofertilizer components.

2. Materials and Methods

2.1. Microalgal Consortium Propagation

The MC-10 microalgal consortium used in this study was non-axenic and consisted mainly of green microalgae (Chlorella sp., Scenedesmus sp.) with a minor presence of the filamentous cyanobacterium Leptolyngbya sp. The microalgal component represented the dominant fraction in terms of both relative abundance and functional activity. The consortium was originally isolated from the San Juan hydrological sub-watershed in Nuevo León State, northeastern Mexico, and represents a native microbial community adapted to local environmental conditions. It was subsequently maintained and supplied by the Environmental Sciences Laboratory of the Universidad Autónoma de Nuevo León.
The LC-Y nutrient solution [29] was selected for the propagation of the MC-10 consortium due to its high ionic strength, which reaches approximately 230 mS/m. This value, primarily attributed to the high concentration of dissociated nutrients, exceeds the typical electrical conductivity range reported for microalgae cultivation (45–90 mS/m). Such conditions may induce ionic stress, which in turn promotes the formation of cell aggregates [30]. The autoflocculation effect associated with the LC-Y solution has been previously documented by Galán-González et al. [15], who reported a flocculation rate of 62% in Haematococcus pluvialis. The composition of the LC-Y nutrient solution is shown in Table 1.
The propagation of the MC-10 consortium was carried out in bubble column reactors with a total capacity of 1 L, containing 0.7 L of the LC-Y nutrient solution. To supply atmospheric CO2 and ensure culture agitation, filtered air (0.45 μm pore size, Millex-HV filter, Merck Millipore, Burlington, MA, USA) was injected at a rate of 0.5 vvm (volume of air per volume of culture per minute). The photoperiod was set to 16 h light and 8 h dark (16L:8D), with a light intensity of 78 μmol photons m−2 s−1 (LED light source, Philips, Amsterdam, The Netherlands). To promote active growth, enhance adaptation to the LC-Y medium, and induce the formation of cell aggregates (autoflocculation), the MC-10 consortium was subcultured weekly by completely replacing the nutrient solution for a period of six months.

2.2. Determination of Flocculation in Microalgae

In the present study, the recultivation of the sedimentable fraction of the MC-10 consortium was performed. To quantify the flocculation percentage (%), measurements were taken at the end of propagation with the LC-Y nutrient solution and from each batch culture using secondary effluents. Flocculation percentage was determined as follows: 100 mL aliquots of the culture were transferred into 100 mL graduated cylinders and allowed to settle undisturbed for 15 min settling time. After this settling period, a 10 mL sample was carefully pipetted from a height corresponding to one-third of the total depth to measure the suspended microalgal biomass (g DW/L). The flocculation percentage was calculated according to the following equation [15]:
Flocculation (%) = (A/B) × 100
where A is the DW (g/L) of the suspended biomass after the specified settling period, and B is the total biomass concentration (g DW/L) determined in the same culture. This procedure was carried out without the addition of external flocculants.

2.3. Secondary Wastewater Effluent Used for Microalgal Cultivation

The direct use (i.e., without any modification) of secondary effluents from an MWWTP in northeastern Mexico was evaluated as a cultivation medium for microalgae. The secondary effluents were collected in accordance with the ISO 5667-10 [31] protocol, at the interconnection point between the municipal network and the irrigation system of the Universidad Autónoma de Nuevo León campus (coordinates: 25°43′37.8″ N, 100°18′27.0″ W, Mexico). Samples of 20 L were collected in disinfected plastic containers, transported immediately to the laboratory, and stored at 4 °C until use (within less than 4 h). The effluents were used directly without filtration or chemical modifications. The physicochemical characteristics of the secondary effluents used in this study are shown in Table 2.

2.4. Preadaptation and Cultivation of Microalgae in Secondary Municipal Effluents

This trial evaluated the adaptability of microalgae to grow in MWWTP effluent and their potential to generate easily recoverable biomass through partial purification, based on supernatant removal (i.e., non-flocculant microalgal cells suspended in the MWWTP effluent) and selective recultivation of the sedimentable fraction of the consortium (i.e., flocculant microalgae). The assay evaluated biomass production and flocculation of the MC-10 microalgal consortium over five sequential batch cultures, each with a cultivation period of two days.
The experiment was conducted using 1.5 L bubble column reactors with a working volume of 1 L, filled with MWWTP effluent. During the trial, the cultures were propagated at room temperature (27 ± 2 °C) with an aeration rate of 0.5 vvm (filtered atmospheric air through a 0.45 μm Millex-HV filter), a 16:8 light/dark photoperiod, and a light intensity of 78 μmol photons m−2 s−1. The initial inoculum concentration was adjusted to 0.05% (equivalent to 0.5 g DW/L) for each cycle. At the end of each batch cycle (2 days), a 15 min settling period (i.e., without aeration) was applied before replacing the MWWTP effluent in each photobioreactor. Biomass concentration (g DW/L) was determined by filtration and drying (Whatman GF/C filters, Cytiva, Marlborough, MA, USA), at 70 °C for 48 h, and flocculation (%) was evaluated for each 2-day batch culture. All measurements were performed in triplicate.

2.5. Flocculent Microalgal Biomass Production Using Secondary Effluents Under Outdoor Conditions

An experiment was conducted to evaluate the production of easily harvestable microalgal flocs using secondary effluents. Three sequential batch cultivation cycles were performed under outdoor conditions, each lasting five days. Inoculum concentration and biomass production during the three cycles were determined by filtration followed by gravimetric measurement of DW (Whatman GF/C filters, 70 °C for 48 h). For each cycle, the inoculum was adjusted to 0.01% (equivalent to 0.1 g DW/L) in a working volume of 25 L of secondary effluent from the MWWTP.
The MC-10 consortium was cultivated in 30 L Fast Ferment™ conical reactors (FastFerment Inc., Toronto, ON, Canada) adapted as bubble column reactors. Aeration was supplied at a rate of 0.1 vvm (equivalent to 2.5 L/min) using atmospheric air filtered through 0.45 μm pore-size membranes (Millex-HV filter). The average temperature and daily light duration during the 15-day experiment were 22.1 ± 8.8 °C and 11.6 ± 0.2 h, respectively. At the end of each batch cultivation cycle, flocculation (%) was measured in triplicate, and the suspended microalgal fraction was removed after a 15 min settling period (see Section 2.2).

2.6. Analysis of Mineral and Carotenoid Content in Microalgal Biomass

To determine the assimilated mineral content in the MC-10 consortium, the biomass obtained during propagation with the LC-Y nutrient solution, as well as from the cultivation cycles using secondary effluents (both during the preadaptation phase and the outdoor trial), was thoroughly rinsed with deionized water to remove surface-adsorbed minerals and extracellular residues, and then concentrated via three consecutive centrifugation cycles (CFG-03 centrifuge, Onof Instruments, Guangzhou, China) at 4000 rpm for 10 min each. Subsequently, the total mineral content, corresponding mainly to the intracellularly assimilated fraction, was analyzed following the AOAC 942.05 method [32].
With the aim of estimating the bioavailability of minerals in soil, the acid solubility of the biomass obtained under outdoor conditions during the three cultivation cycles was evaluated using the method described by New [33], which consists of treating the ash residue with hydrochloric acid, filtering, and quantifying the insoluble fraction after ignition in a muffle furnace (Thermo Scientific Thermolyne, Waltham, MA, USA) at 600 °C. Additionally, at the end of the third outdoor cultivation cycle, the total carotenoid content in the biomass was determined according to the method of Vo et al. [34], in which photosynthetic pigments (carotenoids) were extracted and quantified by UV-Vis spectrophotometry (Genesys 10S UV–Vis, Thermo Fisher Scientific, Waltham, MA, USA), based on solvent partitioning and absorbance measurement at 450 nm. This analysis aimed to confirm a key functional property of the microalgal biomass for its potential application as a biofertilizer, considering that carotenoids contribute to plant growth promotion and enhance tolerance to abiotic stress.

3. Results

3.1. Flocculent Biomass Production

The MC-10 consortium showed visible macroscopic floc formation during cultivation, reflecting its capacity to aggregate under both nutrient medium and effluent conditions. Figure 1 illustrates the macroscopic appearance of MC-10 flocs, which remained stable across sequential cultivation cycles. This visual evidence supports the quantitative measurements of flocculation efficiency described below.
The MC-10 microalgal consortium reached a flocculation percentage of 97.8 ± 0.9% at the end of the propagation period in the LC-Y nutrient solution (Figure 2). Meanwhile, the preliminary preadaptation assay of the MC-10 consortium in secondary effluents maintained high flocculation, starting at 92.1 ± 0.4% during the first cultivation cycle and showing an increasing trend that reached 99.4 ± 0.2% by the end of the fifth cycle (Figure 2). In the outdoor trial, favorable flocculation values were observed, increasing from 74.6 ± 0.7% (cycle 1) to 84.0 ± 0.9% (cycle 3) (Figure 2). It is noteworthy that floc recovery in MC-10 consortium cultures, whether in the LC-Y nutrient solution or in secondary effluents, required only a temporary cessation of aeration. Although the methodology established a 15 min settling period for sampling, flocs were consistently observed to recover and settle in less than 5 min, allowing their rapid collection without the addition of external flocculants. This characteristic represents a significant operational advantage, as it allows partial harvesting without the need for additional processing steps.
In addition to the selective recultivation of the flocculant fraction through sequential batch cycles to facilitate biomass harvesting, the adaptation of the microalgal consortium to secondary effluents was indirectly assessed by monitoring biomass production. At the end of the preadaptation trial in secondary effluents (batch cycle 5), biomass production by MC-10 increased to 149% relative to the initial inoculum concentration (0.5 g DW/L) (Figure 3). Subsequently, algal biomass production under outdoor cultivation conditions showed a progressive increase, rising from 28.9 ± 2.6% in cycle 1 to 124.7 ± 10.8% in cycle 3, relative to the initial inoculum concentration (0.1 g DW/L) (Figure 3).

3.2. Mineral and Carotenoid Content in Biomass

At the end of the propagation stage in the LC-Y nutrient solution and the preadaptation phase in secondary effluents, the mineral content analysis of the MC-10 consortium showed values of 47.5 ± 0.7% and 21.7 ± 0.2%, respectively (Figure 4). In contrast, mineral content in the biomass produced during the outdoor trial was 27.9 ± 0.4% in cycle 1, 39.7 ± 1.4% in cycle 2, and 46.6 ± 0.5% in cycle 3 (Figure 4). A marked variability in the mineral content of the MC-10 consortium was observed depending on cultivation conditions. The highest value was recorded during propagation in the LC-Y nutrient solution (47.5%), while an upward trend was evident during the outdoor experiment, with the mineral content in cycle 3 (46.6%) approaching levels comparable to those obtained under controlled conditions. These results suggest a gradual adaptation of the consortium to external environmental conditions, as reflected by the progressive increase in biomass mineral content.
To complement the analysis of total mineral content during the outdoor batch cultivation cycles, acid-soluble mineral content was evaluated, yielding values of 10.4 ± 0.4%, 44.8 ± 0.6%, and 82.8 ± 0.5% for cycles 1, 2, and 3, respectively (Figure 4). Notably, acid-soluble mineral content exhibited a significant increase throughout the cultivation cycles, rising from 10.4 ± 0.4% in cycle 1 to 82.8 ± 0.5% in cycle 3. This pronounced increase indicates a progressive accumulation of soluble minerals as cultivation advanced.
Additionally, at the end of the outdoor cultivation (cycle 3), total carotenoid content was determined in triplicate, yielding a value of 451.1 ± 29.3 µg/g DW. The presence of carotenoids in the biomass suggests a functional contribution from the photosynthetic microorganisms comprising the MC-10 consortium during cultivation.

4. Discussion

4.1. Flocculent Biomass Production and Microalgal Adaptation in Secondary Effluents

In laboratory-based studies involving the external addition of flocculants, high flocculation efficiencies have been reported in microalgae, ranging from 78 to 100% with inorganic flocculants and 82 to 99% with organic flocculants [35]. Notably, these values are comparable to the flocculation efficiencies observed in the present study, both under controlled laboratory conditions and in outdoor cultivation systems. Specifically, flocculation efficiencies reached 98% during the propagation phase in the LC-Y nutrient solution, 99% during the preadaptation phase with secondary effluents (MWWTP), and 84% during outdoor operation using secondary effluents (Figure 2). Although these results highlight the robust flocculating capacity of the MC-10 consortium under ionic stress conditions, the present study was not designed to investigate the physicochemical mechanisms responsible for this behavior. Nevertheless, it is well established that extracellular polymeric substances (EPS), such as polysaccharides, proteins, and other secreted metabolites, play a central role in cell–cell adhesion and aggregation in many microalgal systems [6,36,37,38]. Therefore, it is reasonable to hypothesize that EPS production and excretion by the consortium contributed to the observed flocculation performance. Detailed characterizations of the biomass, such as surface charge and aggregation tendencies, were not performed. Future research should incorporate such analyses to better elucidate the mechanisms underlying biomass aggregation.
The decrease in flocculation efficiency observed under outdoor conditions (84%), although it might initially be interpreted as a performance loss, actually demonstrates the ability of the microalgal consortium to maintain functionality in uncontrolled environments subject to fluctuations in abiotic factors such as solar radiation and temperature. This response may suggest a progressive adaptation process of the consortium, not only through physiological acclimation but also through the enrichment of microbial groups with enhanced EPS secretion capacity and self-aggregation potential [39,40]. This could require additional sequential cultivation cycles to reach maximum efficiency under outdoor conditions. In this context, it is reasonable to hypothesize that seasonal environmental changes (e.g., winter vs. summer) might influence not only the physiological behavior but also the taxonomic composition of the consortium. Although not investigated in the present study, future research is recommended to include taxonomic and molecular characterizations of the consortium at different stages and environmental conditions. Such analyses would contribute to the establishment of a functional strain repository to preserve adapted consortia with high performance under specific environmental scenarios. This resource could facilitate the reactivation and targeted use of optimized consortia depending on the season or application context, thereby enhancing the practical scalability of the system and adding long-term biological value to the microbial material developed.
The high flocculation values observed in this study highlight the remarkable flocculating capacity of the MC-10 consortium, which may be attributed to both the prior preselection of the flocculating fraction and the secretion of biopolymers favoring aggregation [41,42]. These results are consistent with those reported by Van Den Hende et al. [43], who also employed sequential batch cultures with floc reinoculation. In their sequencing batch reactors, the enrichment of a flocculation-prone fraction became evident after approximately 17–18 sequential batches (35–36 days of operation with a 2-day hydraulic retention time) [43]. In the same study, the flocculation efficiencies achieved using a filter press with a 200-μm pore size were 79%, 88%, 97%, and 99% for wastewater from the chemical, food, manure treatment, and aquaculture industries, respectively. Similar evidence of progressive enrichment has also been reported in recent studies, where flocculation acted as a preconcentration and selection step [44] and where community restructuring under sequential operation favored more flocculation-prone fractions [45]. Consistently, in the present study, preadaptation in secondary effluents (5 cycles of 2 days) and outdoor cultivation (3 cycles of 5 days) both showed a marked increase in flocculent biomass from the second to third cycle (Figure 3), further supporting the hypothesis of progressive selection of a flocculating fraction. Although those studies were conducted with microalgal–bacterial systems, the same principle of selective enrichment may be applicable to the MC-10 consortium.
The sequential cultivation of the MC-10 consortium, combined with floc reinoculation, may promote the progressive selection of the best-adapted microbial fraction, resulting in greater biomass production efficiency. This mechanism aligns with adaptive evolution principles and has been reported in other microbial consortia, including microalgal–bacterial systems, where floc-forming strains are progressively enriched over time [45,46]. The results obtained under both controlled laboratory conditions (preadaptation phase) and outdoor conditions using secondary effluents (MWWTP) showed a sustained increase in flocculent biomass with successive recultivation cycles (Figure 3).
Although the secondary effluents used as the cultivation medium represent a complex, non-sterile environment with variations in physicochemical composition, the use of cyclic batch recultivation to enhance biomass production of the MC-10 consortium is consistent with the laboratory adaptive evolution (ALE) approach. This process is characterized by the gradual adaptation of cell populations to specific conditions through cyclic transfers in batch cultures or maintenance in continuous cultures, resulting in improved biological fitness, such as increased growth rates [19]. Despite the observed increase in flocculent biomass production by the MC-10 consortium, future studies should focus on validating this strategy at larger scales or in continuous cultivation systems using municipal effluents. While this study primarily addressed functional parameters, including biomass yield, flocculation efficiency, and mineral accumulation, the importance of understanding potential temporal shifts in microbial community composition is also recognized. Future investigations should include detailed analyses of species composition dynamics alongside functional metrics to provide deeper insights into the ecological interactions and adaptive mechanisms governing the consortium.

4.2. Adaptive Responses, Mineral Composition, and Carotenoid Content in Microalgae

The mineral content in microalgae is determined by their bioaccumulation capacity, a metabolic process involving the active transport of metals in ionic form into living cells [47]. In this study, the mineral content of the MC-10 consortium demonstrated a high bioaccumulation potential, exceeding values commonly reported in microalgae, which typically range between 4 and 20% DW [48]. Concentrations of 47.5% during the propagation stage in nutrient solution, 21.7% during the preadaptation phase with secondary effluents, and 46.6% during outdoor cultivation were recorded, indicating significant variations between the different stages (Figure 4). In addition to microalgae, cyanobacteria within the consortium also contribute to mineral retention through both active and passive mechanisms. Active bioaccumulation involves ion transport and osmotic adjustment under saline stress, whereas passive biosorption relies on physicochemical interactions with EPS and surface-bound polysaccharides that can bind cations and trace metals [49,50]. Comparable adaptive strategies have also been described in cyanobacteria, which regulate intracellular ion homeostasis while secreting EPS with high affinity for divalent cations such as Ca2+, Mg2+, and heavy metals [51,52]. These dual processes provide a mechanistic explanation for the high inorganic fraction observed in the MC-10 biomass.
The bioaccumulation of inorganic matter in microalgae has been attributed to their tolerance and adaptation to high concentrations of dissolved salts, a process involving specific metabolic adjustments. These include the regulation of ion uptake and export across the cell membrane, as well as the accumulation of organic osmolytes such as proline and glycine betaine [53,54]. The distinction between active bioaccumulation and passive biosorption is important: the first requires metabolic energy for ion transport, while the second depends on surface interactions mediated by extracellular compounds. In this context, the results obtained during the initial propagation phase of the MC-10 consortium in the LC-Y nutrient solution demonstrated an adaptive response to a high ionic load (230 mS/m), manifested as mineral bioaccumulation in the biomass at 47.5%. This result supports the potential of the MC-10 consortium as a biological agent for the recovery of minerals and metallic nutrients from secondary effluents (MWWTP). These findings represent biochemical indicators of the consortium’s adaptive response, highlighting metabolic adjustments that sustain and enhance its performance under high ionic stress. This behavior was further confirmed during the final stage of the outdoor experiment, where, in cycle 3 of the sequential cultivation in secondary effluents, the consortium biomass maintained a high mineral content (46.6%), demonstrating the stability of the inorganic accumulation process under variable environmental conditions.
Microalgal biomass has demonstrated considerable potential as a biofertilizer, as it can improve soil structure, provide essential nutrients, and stimulate beneficial microbiota activity. Depending on its composition, this biomass can supply macronutrients such as N, P, K, Ca, Mg, and S, as well as micronutrients (e.g., Fe, Zn, Cu, among others) that act as cofactors in various metabolic processes. These micronutrients, present in the mineral fraction of the biomass, directly influence nutrient uptake, photosynthesis, and plant defense mechanisms [23,26]. In this context, the high mineral content (46.6%) recorded in the MC-10 consortium could enhance its effectiveness when applied as a biofertilizer, due to the combined contribution of both micronutrients and macronutrients.
In soil fertilization, it is crucial to consider that plants primarily absorb minerals as inorganic nutrients dissolved in the soil solution. An element is considered plant-available when it exists as a free ion, or can be converted into this form, within the root zone [55,56]. This is particularly important because acid solubilization can enhance the release of metallic micronutrients bound to organic matter, thereby increasing their availability [57]. In this study, the soluble mineral content determined by acid extraction of the MC-10 consortium biomass produced outdoors using secondary effluents (MWWTP) was 10.4% in cycle 1, 44.8% in cycle 2, and 82.8% in cycle 3 (Figure 4).
The soluble mineral values obtained in cycles 2 and 3 fall within the range reported by Liu [58], who, upon evaluating 12 microalgal biomass samples, found a solubility range of 38.4 to 99.5%. This suggests that minerals recovered from municipal effluents could, to a large extent, achieve a high level of acid solubility (82.8%) and, consequently, greater bioavailability when applied as a biofertilizer.
The increase in the proportion of soluble minerals depends on the characteristics of the assimilated nutrient type. For example, Zhang et al. [57] reported that, in plant biomass, the acid solubilities of P, K, Ca, Mg, and Na were higher than their water solubilities, whereas for S and Cl, solubility was not affected by solution acidity. The acid solubility of macronutrients such as P, K, Ca, and Mg in microalgal biomass recovered from municipal effluents could have practical implications for its application as a controlled-release biofertilizer. This is because the gradual release of nutrients from this type of biofertilizer would depend on factors such as pH, temperature, and microbial activity, thereby contributing to reduced leaching losses and lower pollution generation [59].
On the other hand, the organic fraction of microalgal biomass represents a valuable source of bioactive compounds with antioxidant properties, such as carotenoids, which can stimulate plant growth and enhance tolerance to oxidative stress [26,60]. In this study, the presence of carotenoids was confirmed in the biomass obtained during cycle 3 of outdoor cultivation, with a content of 451.1 μg/g DW. Although this value is considerably lower than that reported for species such as Haematococcus pluvialis, known for its high carotenoid accumulation capacity, particularly under optimized cultivation conditions, where concentrations can reach up to 28,700 μg/g DW [61], the concentration obtained represents an additional attribute supporting its potential application in agriculture. This concentration falls within the range reported for the phylum Cyanobacteria (230–2720 μg/g DW) and is slightly lower than that observed in species of the phylum Chlorophyta (530–7200 μg/g DW) [62]. Microalgal biomass obtained from cultivation in secondary municipal effluents, which represent an underutilized source of water and nutrients, could be applied in agricultural production by providing bioavailable and bioactive compounds with beneficial effects.

5. Conclusions

The selective reinoculation of the flocculating fraction of the microalgal consortium MC-10, under an initial high ionic load (230 mS/m) in a sequential batch cultivation system, achieved flocculation efficiencies of up to 99% under laboratory conditions and 84% under outdoor cultures, confirming the robustness of the consortium under variable environments. The progressive adaptation of MC-10 was evidenced by significant increases in flocculating biomass, with a 149% increase during the preadaptation phase and 125% under outdoor cultivation using municipal effluents. MC-10 also demonstrated a strong capacity for mineral bioaccumulation, with a biomass mineral content of 47% DW, substantially exceeding the typical range reported for microalgae (4–20%). Moreover, under outdoor cultivation, the acid-soluble fraction represented 83% of the minerals, indicating high nutrient bioavailability and supporting its potential as a biofertilizer. The organic fraction of the biomass also contained 451 µg/g DW of carotenoids, highlighting the bioactive value of the biomass for agricultural applications. Altogether, these findings demonstrate that MC-10 adaptation enhances biomass harvestability, nutrient recovery, and functional compound accumulation from municipal secondary effluents. This strategy proved effective for sustainable biomass generation under real-world conditions, with considerable potential for circular bioeconomy and agricultural use.

Author Contributions

Conceptualization, J.C.B.-R. and C.G.-B.; methodology, J.C.B.-R., C.G.-B., T.G.-R., U.J.L.-C., I.D.B.-Q. and D.C.-C.; investigation, J.C.B.-R., C.G.-B., T.G.-R., U.J.L.-C., I.D.B.-Q. and D.C.-C.; writing—original draft preparation, J.C.B.-R. and C.G.-B.; writing—review and editing, J.C.B.-R., C.G.-B., T.G.-R., U.J.L.-C. and I.D.B.-Q. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Macroscopic view of the MC-10 microalgal consortium.
Figure 1. Macroscopic view of the MC-10 microalgal consortium.
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Figure 2. Microalgal flocculation (%) in the LC-Y nutrient solution (initial) and during sequential cultivation cycles in secondary effluents (MWWTP). Error bars represent the SE of three analytical replicates (n = 3). The absence of error bars indicates negligible SE.
Figure 2. Microalgal flocculation (%) in the LC-Y nutrient solution (initial) and during sequential cultivation cycles in secondary effluents (MWWTP). Error bars represent the SE of three analytical replicates (n = 3). The absence of error bars indicates negligible SE.
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Figure 3. Microalgal biomass concentration during sequential cultivation cycles in MWWTP secondary effluents. Error bars represent SE, with n = 3 analytical replicates. The absence of error bars indicates negligible SE.
Figure 3. Microalgal biomass concentration during sequential cultivation cycles in MWWTP secondary effluents. Error bars represent SE, with n = 3 analytical replicates. The absence of error bars indicates negligible SE.
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Figure 4. Dynamics of total and acid-soluble mineral content in microalgal biomass. Error bars represent SE, with n = 3 analytical replicates.
Figure 4. Dynamics of total and acid-soluble mineral content in microalgal biomass. Error bars represent SE, with n = 3 analytical replicates.
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Table 1. Composition of the LC-Y nutrient solution.
Table 1. Composition of the LC-Y nutrient solution.
CompoundFinal Concentration
KH2PO41 mM
MgSO4 × 7H2O2 mM
KNO35 mM
Ca(NO3)2 × 4H2O6.25 mM
MnCl2 × 4H2O9.15 μM
FeSO4 × 7H2O20 μM
Na2EDTA (C10H14N2Na2O8)20 μM
H3BO346 μM
(NH4)6Mo7O24 × 4H2O15 nM
CuSO4 × 5H2O320 nM
ZnSO4 × 7H2O765 nM
Note: Final pH of the solution: 4.9.
Table 2. Physicochemical characteristics of secondary effluents from the MWWTP.
Table 2. Physicochemical characteristics of secondary effluents from the MWWTP.
ParametersValue
Total alkalinity (H2CO3, HCO3, CO32−)209.3 ± 2.5 mg/L as CaCO3
Nitrate (NO3)77.9 ± 0.6 mg/L
Phosphate (PO43−)11.7 ± 0.2 mg/L
pH7.6 ± 0.1
Electrical conductivity110.7 ± 0.5 mS/m
Mean ± SE (standard error).
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Guajardo-Barbosa, C.; Guajardo-Rodríguez, T.; López-Chuken, U.J.; Barceló-Quintal, I.D.; Cruz-Chávez, D.; Beltrán-Rocha, J.C. Adaptation of Microalgae for the Production of Settling Flocs, Carotenoids, and Mineral Recovery from Municipal Secondary Effluents. Phycology 2025, 5, 57. https://doi.org/10.3390/phycology5040057

AMA Style

Guajardo-Barbosa C, Guajardo-Rodríguez T, López-Chuken UJ, Barceló-Quintal ID, Cruz-Chávez D, Beltrán-Rocha JC. Adaptation of Microalgae for the Production of Settling Flocs, Carotenoids, and Mineral Recovery from Municipal Secondary Effluents. Phycology. 2025; 5(4):57. https://doi.org/10.3390/phycology5040057

Chicago/Turabian Style

Guajardo-Barbosa, Claudio, Tomás Guajardo-Rodríguez, Ulrico Javier López-Chuken, Icela Dagmar Barceló-Quintal, David Cruz-Chávez, and Julio César Beltrán-Rocha. 2025. "Adaptation of Microalgae for the Production of Settling Flocs, Carotenoids, and Mineral Recovery from Municipal Secondary Effluents" Phycology 5, no. 4: 57. https://doi.org/10.3390/phycology5040057

APA Style

Guajardo-Barbosa, C., Guajardo-Rodríguez, T., López-Chuken, U. J., Barceló-Quintal, I. D., Cruz-Chávez, D., & Beltrán-Rocha, J. C. (2025). Adaptation of Microalgae for the Production of Settling Flocs, Carotenoids, and Mineral Recovery from Municipal Secondary Effluents. Phycology, 5(4), 57. https://doi.org/10.3390/phycology5040057

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