Current Insights into Growing Microalgae for Municipal Wastewater Treatment and Biomass Generation

: Municipal wastewater (MWW) provides a promising platform for microalgae cultivation due to its rich content of essential nutrients. Recent research has showcased the multifaceted ben-eﬁts of microalgae-based wastewater treatment, from the potent depollution capabilities of these organisms to their biomass potential for ecofriendly applications. A signiﬁcant advantage lies in the ability of these systems to promote environmental sustainability without producing secondary pollutants, aligning with the circular economy model. This approach encompasses various stages, from cultivating microalgae to biomass separation and subsequent valorization. However, challenges arise when scaling these systems to industrial levels. A predominant barrier is the difﬁculty in maintaining consistent control over all the factors inﬂuencing wastewater phytoremediation. This can compromise both biomass survival and the efﬁciency of pollution removal and valorization. Notably, using native microalgal consortiums from the efﬂuent appears to be a promising strategy. These autochthonous communities often demonstrate superior adaptability and treatment capacity, emphasizing the importance of further exploring their potential to provide effective and economically viable solutions for wastewater treatment.


Introduction
As global urbanization accelerates, the imperative for efficient municipal wastewater (MWW) treatment intensifies.Traditional treatment methods, while effective, often incur high operational costs, increased energy consumption, and the generation of secondary pollutants.Utilizing microalgae for wastewater treatment is a promising alternative, offering significant advantages.It represents a more sustainable approach and microalgae-based systems also excel in nutrient removal without the extensive chemical usage typical of conventional methods [1].
This biologically driven treatment ensures the removal of harmful nutrients and sets the stage for sustainable biomass production.As Wu et al. [1] underscored, this biomass can be vital for several industries, including biofuels, animal feed, and nutraceuticals.However, the economic feasibility hinges upon the cost-effective production of microalgal biomass.As Zhou et al. [2] pointed out, a significant constraint is the high cost of essential nutrients for microalgal cultivation.
Scholars have identified MWW as a potential medium in search of solutions due to its richness in macro-and micronutrients [3,4].The nutrient balance, especially the molar ratio of carbon (C), nitrogen (N), and phosphorus (P)-often referred to as the N:P ratio, is crucial for maximizing microalgal growth.The Redfield ratio, a well-established C:N:P ratio (106:16:1), representing the elemental stoichiometry in marine phytoplankton, Resources 2023, 12, 119 2 of 28 provides a starting point.However, the optimal N:P ratio can significantly vary based on the environment and the microalgal species, warranting tailored approaches.
However, integrating microalgae cultivation with MWW treatment has been fraught with challenges.Most existing studies have been limited in scope, focusing mainly on the photoautotrophic mode of microalgae cultivation.Such studies aim for biomass production or nutrient removal, rarely both [5,6].There are evident gaps in knowledge: determining the most compatible wastewater streams, investigating various cultivation methods, understanding the distinct roles of different microalgae species in wastewater treatment, and more.Furthermore, ensuring the stability of the microalgae-bacteria consortia in varying wastewater conditions while simultaneously striving for optimal biomass yield remains daunting [7,8].
It is essential to address these knowledge gaps to propel the economic viability of microalgae cultivation and augment wastewater treatment efficiency.This paper endeavors to fulfill this need, providing an exhaustive review of the current state of the art and outlining a path forward.Our primary goal is to evaluate the compatibility of MWW for microalgae cultivation, emphasizing refining cultivation methods and enhancing nutrient removal for large-scale applications.

Municipal Wastewater Characterization
MWW is generated from everyday household and business activities, such as using the restroom, cooking, and laundry.It primarily consists of water (99.9%) but also contains organic and inorganic solids (0.01%) and microorganisms (0.01%) [9].
The composition of MWW can vary based on factors like climate, socioeconomic status, and population habits.However, in many instances, organic matter constitutes approximately 75% of the solids [10].Excessive organic matter entering natural water bodies, like lakes and rivers, disrupts the ecological balance.This influx increases the biochemical oxygen demand (BOD), promoting the rapid growth of microalgae and the proliferation of other microorganisms in a process known as eutrophication [11].
Eutrophication is characterized by the overgrowth of aquatic plants in natural waters due to elevated levels of nutrients, mainly nitrogen (N) and phosphorus (P).Often, these nutrients stem from agricultural runoff or discharges from untreated wastewater.The effects of eutrophication on water bodies are wide-ranging.Beyond threatening aquatic life and biodiversity, it complicates the treatment of water sourced from these environments.Excessive microalgae affect the water's appearance, taste, and smell.Addressing these issues during water treatment necessitates the removal of these contaminants, which, in turn, requires more chemical products, thus increasing the treatment costs [12].
High levels of organic matter and nutrients in wastewater can intensify eutrophication if not treated effectively before discharge into natural waters.Thus, the treatment of MWW is crucial, not just to combat eutrophication but also to protect the environment and curb the spread of waterborne diseases and pathogens [13].
Current MWW treatment plants utilize large-scale technologies for sewage purification.However, despite their efficacy in managing many pollutants, these conventional methods sometimes falter in removing specific contaminants like nitrogen, phosphorus, and micropollutants.This gap has spurred interest in alternative or complementary treatment methods that amplify efficacy and facilitate the recovery of essential nutrients from MWW.
A standout in this regard has been biological processes, especially those harnessing the power of microalgae.Such processes present a sustainable option and are recognized for their efficiency and potential cost benefits [14].
Microalgae have demonstrated efficiency in extracting primary nutrients like nitrogen and phosphorus from wastewater and treating a diverse set of compounds.It is pivotal to note, especially when discussing heavy metals, that microalgae do not degrade them.Instead, they adsorb and accumulate them within their cell structure.This bioaccumulation is one of the reasons why microalgae are seen as a promising tool in emerging biotechnologies for wastewater treatment [13].
Furthermore, microalgae have shown remarkable efficacy in reducing bacterial concentrations, notably total Coliforms, E. coli, Pseudomonas, and Enterococcus [15].The underlying mechanisms for this pathogen-reducing ability are multifaceted.For instance, microalgae compete with pathogens for essential nutrients like nitrogen and phosphorus.A robust microalgal population can effectively limit the resources available to pathogens, inhibiting their growth.Additionally, through photosynthesis, microalgae produce oxygen, suppressing specific anaerobic pathogens' activities.Their photosynthetic activity can also elevate the pH of the water, making conditions less hospitable for specific pathogens.Certain microalgae species release compounds with antimicrobial properties, offering a direct means to counter pathogens.
Moreover, the physical interactions between microalgae and bacteria can form algalbacterial flocs, which trap and immobilize pathogens.In systems exposed to sunlight, microalgae might indirectly heighten the exposure of pathogens to harmful UV radiation, further aiding in their inactivation.Given these multipronged approaches, it is fitting that some studies have noted the complete eradication of Coliforms and E. coli with microalgal treatment [14][15][16].Another benefit of using microalgae is their adaptability in treating MWW with various characteristics [16].For instance, treating MWW using anaerobic processes can result in a high residual organic matter content.Therefore, employing microalgae as a tertiary treatment for MWW undergoing anaerobic processing can effectively address this residual organic matter [17].
Microalgae, when grown in wastewater, can assimilate and sequester contaminants, leading to a significant reduction in pollutant concentrations.As the microalgae grow and proliferate, they form larger aggregates, which are easier to settle and separate from the treated water.This natural flocculation property of microalgae reduces the need for chemical coagulants or other separation processes, making biomass recovery more efficient and cost-effective.Moreover, the treated effluent, now cleared of a substantial portion of its contaminants, becomes suitable for various reuse applications, including irrigation, industrial processes, or groundwater recharge [18].
Microalgae treatment also permits high nutrient recovery rates, particularly nitrogen and phosphorus [19].The role of microalgae in wastewater treatment, particularly in phosphorus recovery, aligns with the principles of a circular economy.Phosphorus recovery is crucial because of its finite nature and importance in agriculture and other sectors.El Wali et al. [19] underscored the significance of a phosphorus circular economy model.Their findings suggest substantial savings in water used for agriculture and potential socioeconomic benefits, such as poverty reduction.This emphasis on phosphorus recovery becomes even more pertinent, considering the United Nations data indicates that only 56% of global MWW is treated safely [20].Given microalgae's efficiency in phosphorus removal, their role in MWW treatment could be a cornerstone of advancing toward a phosphorus circular economy.

Municipal Wastewater Treatment Plants
MWW treatment processes have evolved to meet the increasing demands for cleaner effluents and stricter environmental regulations.MWW treatment plants are meticulously designed to cater to the level of purification required for the product.As de Matos et al. [21] described, these treatments can span multiple stages, including preliminary, primary, secondary, and tertiary.
The preliminary and primary treatment stages involve the initial screening and removal of larger debris, solids, and coarse materials from the wastewater.These processes are essential for preparing the wastewater for further treatment in the secondary and tertiary stages.Within these later stages, the activated sludge process plays a critical role, primarily aimed at reducing the organic load present in the effluent [22].The choice of activated sludge as a preferred treatment mechanism arises from its widespread application and effectiveness in organic load reduction.
While several wastewater treatment processes have been established, each comes with its unique set of challenges.For instance, although effective at mitigating organic contaminants, the activated sludge method often struggles to efficiently remove nutrients, heavy metals, and specific pathogens [23].Table 1 provides a detailed analysis of the physicochemical and biological attributes of MWW after its treatment with activated sludge, highlighting the achievable removal efficiencies.
The activated sludge process typically aligns with the standards established by the European Union's Water Framework Directive [24] for effluent discharges and adheres to the World Health Organization (WHO) guidelines on wastewater treatment.However, secondary treatments, like activated sludge, may require tertiary interventions to eliminate further nutrients, pathogenic organisms, nonbiodegradable compounds, heavy metals, and remaining suspended solids.Such advanced treatments help mitigate the eutrophication potential of effluents and safeguard water resources [9].
Despite the evident advantages, tertiary treatments are not universally adopted, primarily due to cost constraints.However, their inclusion is more commonplace in regions like Europe, driven by the ambition to achieve superior water quality standards.Managing the sludge resulting from MWW treatments can account for 20 to 60% of operational costs [25].This emphasizes the growing urgency for innovative solutions that champion the principles of a circular economy.Such solutions may encompass reusing treated water, biofuel-driven energy generation, and harnessing the biomass produced [26].
Each wastewater treatment technology outlined in Table 1 comes with distinct cost implications.The activated sludge process, for instance, has higher operational costs due to its energy-intensive nature.Anaerobic digestion requires initial investments in temperature regulation and biogas collection.Trickling filters and constructed wetlands may have lower energy demands, but their efficiency can be climate-dependent, leading to costs for system optimization.Though ensuring high-quality effluent, membrane bioreactors demand higher capital and maintenance expenses due to their intricate membrane systems.
Incorporating microalgae cultivation in MWW treatment introduces a circular economy dimension to wastewater management.Resources are optimized by converting treated wastewater into valuable microalgal biomass, offering dual benefits: wastewater purification and biomass production.This approach reduces the need for fresh water in algal cultivation and provides a nutrient-rich algae medium.The resulting biomass can be valorized for various applications, from biofuels to high-value compounds, potentially offsetting treatment costs and generating revenue.[35,36] However, integrating microalgae into wastewater treatment is not without challenges.Upfront investments are needed for setting up cultivation systems.Furthermore, while algal biomass holds promise, its market value can vary based on its composition and the specific products derived from it.
Embracing this circular economy model, where wastewater becomes a resource rather than a liability, could pave the way for sustainable wastewater management.However, understanding the associated costs, benefits, and market dynamics is crucial for its successful implementation.

Microalgae
Algae refers to any organism with an undifferentiated thallus and chlorophyll, molecules in its constitution that enable the performance of oxygenic photosynthesis.They are primarily autotrophic and typically aquatic, being able to inhabit both fresh and saltwater [37].Microalgae is an umbrella term for simple, primarily microscopic plants, including free-moving plants, phytoplankton, and attached benthic microalgae [38].
Microalgae are unicellular organisms that comprise varying amounts of carbohydrates, proteins, and lipids, depending on the specific species [39].Table 2 details the chemical composition, specifically the content of proteins, carbohydrates, and lipids, of predominant microalgae species employed in MWW treatment.Understanding this composition is paramount, as it directly dictates these microorganisms' potential applications and utilization.
For instance, microalgae with high lipid content are particularly coveted for biofuel production.Species such as Scenedesmus dimorphus, which exhibits lipid percentages ranging from 16-40%, and Prymnesium parvum, with a lipid content between 22-38%, emerge as potential candidates for biodiesel production.Their substantial lipid content makes them viable for oil extraction processes that convert algal lipids into biodiesel through transesterification.On the other end of the spectrum, species with pronounced protein percentages, such as Spirulina maxima (60-71%) and Synechoccus sp.(63%), hold promise in the nutritional domain.Given their rich protein profile, these microalgae can serve as protein supplements in animal feed or even human diets.Carbohydrates play a pivotal role in bioethanol production.Microalgae species like Spirogyra sp., which boasts a carbohydrate content between 33-64%, and Porphyridium cruentum, with carbohydrates ranging from 40-57%, present themselves as potential raw materials for bioethanol extraction.
Furthermore, certain microalgae can serve multiple purposes due to their balanced proteins, lipids, and carbohydrate composition.For instance, Dunaliella salina, showcasing a harmonious blend of proteins (57%), carbohydrates (32%), and lipids (6%), might be utilized in both the nutritional sector and biofuel production, though the latter to a lesser extent.Conclusively, understanding the chemical makeup of microalgae species is crucial.It guides researchers in selecting the appropriate cultivation strategies and dictates the downstream applications in biofuels, nutrition, or other industrial sectors.Recognizing these compositional variations and their implications is essential for harnessing the full potential of microalgae in a sustainable and economically viable manner.
Algae serve as primary producers at the base of the food chain because they use photosynthesis to convert carbon dioxide, oxygen, and water into organic compounds [40].Microalgae can be classified into three groups according to their metabolism for obtaining energy and carbon sources: autotrophic, heterotrophic, and mixotrophic [41].
In the autotrophic regime, microalgae use light as an energy source and require inorganic compounds as a carbon source to carry out photosynthesis and maintain their vital functions.Microalgae can harness atmospheric carbon dioxide through photosynthesis to produce biomass, effectively capturing and storing carbon dioxide from the environment [41].In heterotrophs, microalgae grow independently of light intensity, and they need an external source of organic compounds to supply the absence of light [42].Combining these metabolisms results in the mixotrophic process; microalgae can carry out photosynthesis and assimilate organic compounds concomitantly [43].
Research indicates that microalgae biomass cultivated in MWW is rich in macronutrients and micronutrients, making it a viable option for agricultural fertilization [44].In addition to the essential nutrients commonly found in agricultural fertilizers (NPK), microalgae biomass also provides micronutrients, such as magnesium (Mg) and zinc (Zn).However, it is essential to consider the potential adsorption of heavy metals, like mercury (Hg), by microalgae during cultivation in wastewater.Proper monitoring and assessment are necessary to ensure the safety and suitability of biomass as an agricultural fertilizer [45].However, it is essential to carefully monitor the heavy metal content in such biomass to ensure its safe application and avoid potential soil contamination [46].The interaction between chemical, physical, and biological factors directly influences the promotion or inhibition of microalgae development.Some factors may be the availability of nutrients, nitrogen and phosphorus ratio (N:P), pH, temperature, light intensity, aeration, and type of cultivation system [47].Source: Data sourced from Johansen [48].

Microalgae and Their Growth Factors
Microalgae, as unicellular organisms, are uniquely equipped for wastewater treatment, capable of assimilating various nutrients and contaminants from the environment.However, many environmental and operational parameters influence their growth and efficiency.Table 3 provides a succinct overview of these factors, detailed in the sections below.

Nutrient availability
Nutrients, vitamins, and trace metals are essential for optimal microalgae growth.Nitrogen and phosphorus play crucial roles in cell synthesis and energy storage.
[ 49,50] pH level pH can influence metabolic activity, with optimal growth observed close to neutral conditions.Photosynthetic activity can lead to pH fluctuations.[51,52] Temperature Optimal temperatures are species-specific and can influence metabolic activity, photosynthesis, and cellular tolerance.[53,54] Light intensity Light intensity and photoperiod are crucial for photosynthetic activity, affecting biomass production and growth.[55,56] Aeration and mixing Proper aeration and mixing prevent sedimentation, enhance nutrient contact, and aid in removing unwanted gases.However, excessive mixing can damage cells.
[ 57,58] Type of culture Choosing between pure cultures or consortia can influence adaptability, resilience, and efficiency in wastewater treatment.Native cultures may offer advantages in adaptability and cost-effectiveness.
[ 59,60] Type of cultivation system Open systems are cost-effective but susceptible to environmental fluctuations.Closed systems (photobioreactors) offer more control but can be costlier. [61,62]

Nutrient Availability
The ideal medium for microalgae cultivation should comprise nutrients, vitamins, and certain trace metals [49].The consumption of these compounds by microalgae varies depending on the specific species and its characteristics, such as tolerance to extreme temperatures, chemical composition, rapid sedimentation, and the ability to grow mixotrophically, among others [63].The medium's lack of nutrients can limit microalgae development.Among these, the main elements essential for microalgae growth are carbon, nitrogen, phosphorus, and iron [64].
Nitrogen is a fundamental compound for forming cells, nucleic acids, and proteins [65].Its scarcity can directly affect the synthesis and accumulation of total lipids in microalgae cells.During cultivation, microalgae convert inorganic nitrogen into organic forms, making it bioavailable in the medium.It is worth noting that while true microalgae utilize the nitrogen available in the medium, certain cyanobacteria have the unique ability to fix atmospheric nitrogen, thereby supplementing their nitrogen source.The nitrogen in the effluent after activated sludge treatment is predominantly in the form of ammonium.Research indicates that microalgae preferentially consume ammonium due to its lower energy expenditure for uptake.Once ammonium is nearly exhausted, ammonia becomes the primary nitrogen source consumed, followed by nitrates [66].In systems with temperatures exceeding 30 • C and under specific conditions, such as significantly elevated pH levels, ammonia (NH 3 ) volatilization can occur, potentially leading to decreased availability [67].
Phosphorus, an essential nutrient for microalgae growth, is commonly considered a limiting factor in many natural environments and aquatic ecosystems.Its availability in wastewater can vary depending on the specific wastewater composition and treatment processes.In some cases, wastewater may contain sufficient phosphorus levels, reducing its limitation on microalgae growth.
Microalgae utilize phosphorus in various cellular processes, including synthesizing phosphate compounds, sugars, nucleic acids, ATP (adenosine triphosphate), and phosphorylated enzymes [68].Phosphorus assimilation primarily occurs in the form of inorganic compounds such as orthophosphate (PO 4 3− ) and polyphosphates.The exact phosphorus consumption required for optimal microalgae growth varies among species.When phosphorus is readily available, microalgae exhibit a luxury uptake, storing surplus phosphorus within their cells [69].This feature ensures continued growth, even when phosphorus becomes scarce in the environment, with microalgae capable of accumulating phosphorus levels 8 to 16 times the minimum required amount [50].
In secondary post-treatment effluent, nitrogen exists primarily in the form of organic nitrogen and ammonia, forming what is known as TNK (total nitrogen Kjeldahl).The pH of the medium significantly influences ammonia speciation.At a pH below 8.0, most ammonia exists in the ionized form (NH 4 + ).Temperature also affects ammonia volatilization, with higher temperatures increasing ammonia release [70].Ammonia stripping leads to the loss of ammonia through volatilization, rendering it unavailable for assimilation by microalgae cells.

pH
The pH indicates the concentration of H + ions present in the medium, which directly affects the metabolism and the availability of chemical elements for the development of microalgae.These ions can promote the precipitation of some elements and, thus, make them unavailable for consumption.Therefore, for adequate absorption of these components by microalgae, the pH value must be close to neutrality [51].
During the photosynthetic activity of microalgae, the phytoplankton present consumes the dissolved CO 2 in the medium.This consumption reduces the production of carbonic acid, increasing the pH value, which can vary throughout the day [71].The fluctuations in pH values, naturally occurring between day and night, are crucial in inhibiting pathogens in biological sewage treatments.Variations in pH can create conditions that are less favorable for the survival and growth of pathogens, thus, contributing to their inhibition [72].
Most microalgae species have better development in the pH ranges between 7.0 and 9.0, with the optimal range being 8.2 to 8.7, as it maintains the most suitable photosynthetic rate for bacterial decomposition [73].However, biomass production studies with high pH values did not inhibit microalgae growth, with pH values being 8.5 to 10 [52].

Temperature
Temperature is essential for the metabolic development of microalgae, biomass composition, and cellular components' structures.When temperatures are below the optimal level, microalgae growth becomes limited and directly affects the enzymatic processes of the cell [53].However, when temperatures rise above the optimum level, the enzymes responsible for the photosynthesis process become denatured, leading to reduced performance and potentially resulting in cell death [74].
Temperature can exert a significant influence on various parameters in the microalgal environment.For instance, it affects the solubility of CO 2 in the medium and can also impact pH values [75].Furthermore, when temperatures are within the optimal range, microalgae exhibit enhanced tolerance to high light intensities, effectively preventing photoinhibition [76].
Each microalgae species has its optimal temperature, but these values may vary between studies.Sforza et al. [77] identified that the optimal temperature for the C. protothecoides species for primary effluent treatment is 30 • C, while Binnal and Babu [78] reported an optimal temperature of 25 • C for secondary effluent treatment also using the C. protothecoides species.A study using a consortium of Chlorella sorokiniana, Monoraphidium sp., and Scenedesmus identified the ideal temperature of 35 • C for removing heavy metals [79].
Studies carried out in controlled environments do not express the actual range of temperatures that occur in outdoor conditions.Higher temperatures occur inside closed photobioreactors since no evaporative loss regulates the temperature [80].Even when there are significant variations in temperature throughout the day, microalgae activity can be maintained if the peak temperature is not constant [81].The native microalgae in the effluent are more adaptable to the environment and thus grow better, even in external cultivation conditions, than in pure cultures [54].

Light Intensity
The light intensity and the light-dark cycle (photoperiod) of exposure are determining factors for the growth of photosynthetic microalgae, as they are the energy source for the activities [55].The light intensity can optimize the microalgae cultivation process along with other factors, such as temperature and pH [82].Microalgae species react differently to light intensity; some require greater or lesser intensity for photosynthesis.Light intensity is directly related to the amount of carbon fixed by microalgae during the photosynthetic process, which influences biomass production and microalgae growth [83].
For the ideal development of microalgae, the photoperiod balance must occur since, under low lighting, there is not enough energy for development, while under excessive light, photoinhibition occurs [84].Using natural light in outdoor systems reduces growth and production costs [56].

Aeration and Mixing
In microalgal cultivation, "aeration" and "mixing" often intersect but serve distinct purposes.Aeration refers to introducing compressed air into the system, facilitating the exchange of gases, notably the supply of CO 2 for photosynthesis and removing evolved O 2 .This process ensures an optimal gaseous environment for microalgae and can be instrumental in stripping unwanted gases, such as ammonia, from the wastewater.While ammonia can be a nutrient source for microalgae, excessive concentrations can be toxic.Efficient aeration can enhance the treated effluent's quality, making it more conducive to microalgal growth [38,85].
On the other hand, mixing pertains to the agitation of the cultivation medium, ensuring the even distribution of microalgae, nutrients, and light.Adequate mixing prevents microalgal sedimentation and ensures that all cells have equitable access to light and nutrients.
In the context of a photobioreactor, both aeration and mixing are crucial.While aeration maintains the gaseous equilibrium, mixing counteracts thermal stratification and ensures uniform light exposure.
The importance of these processes is equally underscored in high-rate algal ponds (HRAP).Given their shallow depth and expansive surface area, HRAPs rely heavily on thorough mixing to prevent sedimentation and guarantee optimal light distribution across the pond.Moreover, in HRAPs, aeration plays a vital role in off-gassing unwanted volatiles and maintaining an environment conducive to microalgal proliferation.
While both aeration and mixing play integral roles in microalgal cultivation, their primary functions-gas exchange and medium agitation, respectively-should be distinctly recognized for a clearer understanding of their impact [86].

Type of Culture
The treatment of effluents using pure cultures, that is, a single species of microalgae, is usually carried out on a small scale due to the need to be careful not to contaminate the environment with other species [59].This contamination may occur due to microalgae in the MWW that will be used.Due to this, recent studies have focused on the advantages of using the intercropping, cocultivation, or polyculture of microalgae [87].
In the consortium, the different microalgae species interact synergistically with each other, thus, resulting in better metabolic control from this association.Consortia can be between microalgae or with other microorganisms; one of the most used is the microalgabacteria [88] (Figure 1).
The use of a microalgae consortium has several advantages: (i) the possibility of more robust systems due to their resistance to fluctuations in environmental conditions and invasion by other species; (ii) the greater consumption of nutrients by the combination of microorganisms and their different needs; and (iii) the increase in treatment efficiency resulting from the cooperation between various microorganisms.However, the use of a consortium faces some limitations, such as the wide variety of possible combinations of microalgae, which makes developing models difficult.In addition, it is difficult to maintain the same consortium for long periods, especially in open lagoons where there is a high risk of culture contamination [89,90].

Type of Culture
The treatment of effluents using pure cultures, that is, a single species of microalgae, is usually carried out on a small scale due to the need to be careful not to contaminate the environment with other species [59].This contamination may occur due to microalgae in the MWW that will be used.Due to this, recent studies have focused on the advantages of using the intercropping, cocultivation, or polyculture of microalgae [87].
In the consortium, the different microalgae species interact synergistically with each other, thus, resulting in better metabolic control from this association.Consortia can be between microalgae or with other microorganisms; one of the most used is the microalgabacteria [88] (Figure 1).Another critical issue for selecting the culture to be used as a tertiary effluent treatment is its origin since it can be inserted into the medium through inoculation, or a native culture already present in the effluent can be used [91].According to Galès et al. [92], when using the microalgae present in the effluent, these are more adaptable to environmental conditions and their variations.According to Baldisserotto et al. [93], the performance of the native microalgae consortium was evaluated against that in a synthetic medium.The consortium exhibited its highest performance in the original effluent, to which it was already adapted.The microalgae demonstrated superior growth and more effective nutrient removal in this environment.
Using a native consortium is a sustainable alternative since it allows the treatment of effluents without the demand for controlled conditions; there are no concerns with the contamination of the system, and mainly, the consortium is already adapted to the environment, reducing cultivation costs [60,94].

Type of Cultivation System
Microalgae can be grown in both closed and open systems (natural or artificial ponds) or closed systems (photobioreactors) [61].
Open systems are generally more economical [82] and are commonly used for largescale cultivation.However, in this type of system, microalgae remain exposed to natural conditions of lighting, temperature, evaporation, and contamination that can negatively alter the development of microalgae and can even suffer more from climate change [92,95].
Most open systems studies evaluate the effect of temperature and light intensity on microalgae cultivation [96,97].Rodríguez-Miranda et al. [98] studied temperature variations over a year in Spain using a large-scale raceway-type open system.Hernández et al. [99] studied the importance of temperature control using indirect methods in a raceway-type open system cultivation.
Closed systems, also known as photobioreactors (PBR), have advantages over open systems, as they guarantee greater biomass productivity per unit area, shorter harvest time, high surface area/volume ratio, lower risk of contamination, and greater efficiency [100].However, depending on the type of PBR, this system may be more expensive, and it is not easy to control the internal temperature and adhesion of some cultures to the walls [101].
The critical PBR systems are (i) tubular, which consists of a set of tubes with a diameter of less than 0.1 m and a length of up to 80 m.In these systems, the flow is turbulent, preventing sedimentation from occurring [102]; (ii) airlift, which consists of a vessel with two zones, the first where the liquid descends and the other where the air is injected to create a circular movement [103]; and (iii) flat panel, which consists of parallel plates with little thickness between them and can contain agitation performed by air bubbles along the length [104].
Tubular PBR systems have been associated with high microalgae biomass production due to high solar incidence and the possibility of large-scale systems [105].Studies using the airlift system usually have a few days of cultivation due to the insertion of air that accelerates the development of cultures [106,107].
In the comparative study of Yaqoubnejad et al. [108], the airlift-type system showed outstanding biomass production and nutrient removal, reaching 97% phosphorus removal after seven days of cultivation.When grown in MWW and outside a controlled culture room, contamination is expected if a pure culture is used.The microalgae consortium can increase productivity due to greater adaptability to changing conditions and more excellent resistance and efficiency in the use of resources.Due to this, external cultivations usually use microalgae consortia to present better results [62].

Municipal Wastewater Treatment Plant Effluents and Microalgae Growth
The use of microalgae in waste treatment began in the 1950s, with investigations into the ability of microalgae and bacteria to treat MWW [109].With the advancement of studies, it was realized that the combination of microalgae and bacteria growth, together with MWW treatment, could be a solution to overcome the high costs of microalgae cultivation, achieve the expected removal of nutrients from the medium, and produce high added value products such as biodiesel [110].
The use of microalgae in MWW treatment depends on the interaction between trophic chains.Heterotrophic bacteria, including anaerobic microorganisms, consume organic material and release carbon dioxide (CO 2 ), ammonium (NH 4 + ), nitrate (NO 3 − ), and phosphorus (PO 4 3− ), which are used by microalgae to generate new cells [111].Microalgae, in turn, release photosynthetic oxygen, allowing aerobic and facultative anaerobic heterotrophic bacteria to degrade organic matter and reduce the BOD of the effluent [112].
Microalgae are versatile and can remove nitrogen and phosphorus from MWW to limit values below those required by norms and the World Health Organization Guidelines for MWW discharge into water bodies [113].Microalgae cultures are capable of treating effluents with different properties, such as effluent after preliminary treatment, primary effluents after removal of suspended solids, secondary effluents after clarification obtained by the degradation of organic matter; concentrated effluents, that is, the liquid phase of the sludge from the anaerobic digester [114]; and even effluents from treatment by anaerobic systems.Depending on the effluent's origin, the treatment system's configuration will differ [113].
After treatment by aerobic systems, MWW typically exhibits a reduced organic load but maintains high nutrient concentrations.Conversely, anaerobically treated MWW may present different nutrient profiles, with specific processes possibly increasing the nutrient content of the effluent.Regardless of these distinctions, both postaerobic and postanaerobic effluents, due to their nutrient richness, are apt for microalgae treatment, acting as effective subsequent treatment options [115].Table 4 provides an overview of the nitrogen and phosphorus removal by microalgae and cyanobacteria across various stages of MWW treatment, underscoring the competence of these organisms in managing nutrient burdens from both process types.Specifically, the decrease in nitrogen levels in cyanobacteria-treated effluents can be attributed to their capacity to absorb multiple nitrogenous compounds from wastewater, supporting growth and protein synthesis.Moreover, certain strains of cyanobacteria can fix atmospheric nitrogen, which further diminishes the nitrogen levels in the effluent.The studies presented in Table 4 show that secondary treatment effluents and anaerobic system effluents have the highest nutrient removal values.In addition, several studies showed a considerable reduction in inorganic nutrient concentrations and the removal of other contaminating compounds, such as heavy metals and drugs, using microalgae [131,132].On the other hand, the conditions generated within the systems using microalgae allow considerable removal of pathogenic bacteria (total Coliforms, E. coli, Pseudomonas, and Enterococcus) during effluent cultivation and treatment [133].

Microorganisms
In work by Marchello et al. [134], the potential for microalgae growth in the effluent after anaerobic treatment and its effect on Coliforms bacteria was evaluated.The study showed increased treatment efficiency when aeration was added to the medium, showing a 4-log reduction in colony-forming units for total Coliforms and E. coli.
According to Slompo et al. [15], the use of microalgae to treat blackwater after anaerobic treatment in an up-flow anaerobic sludge blanket (UASB) reactor showed a removal log of 0.76 log for total Coliforms and 2.73 log for E. coli, representing an efficiency of 68.8% and 99.8%, respectively.
In the study conducted by Ruas et al. [135], the removal of pathogens, including Pseudomonas aeruginosa, E. coli, and Enterococcus faecalis, was assessed using a microalgaebacteria consortium.The study considered multiple factors, such as the microalgae Chlorella vulgaris, activated sludge, aeration, CO 2 supplementation, photoperiod, and the type of system (open and closed).
The findings revealed that the consortium significantly increased the removal of pathogens, exceeding 50%, and the removal efficiency reached an impressive 99.4% with the addition of aeration.Notably, the 24:0 photoperiod, characterized by a lack of solar radiation during the dark period, did not effectively remove P. aeruginosa and E. coli.This observation underscores the crucial role of solar radiation in promoting photosynthesis, which is essential for the microalgae's metabolic activity.Enhanced photosynthesis, driven by solar radiation, likely contributes to the microalgae's ability to generate oxygen and degrade organic matter, leading to improved pathogen removal in the presence of light.
Gutiérrez-Alfaro et al. [136] conducted a study to evaluate the ability to remove E. coli, Enterococcus sp., and Clostridium perfringens from different secondary processes and the behavior of these pathogens in solar disinfection.It was concluded that solar radiation significantly increased the removal of these pathogens in the effluent after secondary treatment (removal of 2.9 log of E. coli).It can thus be inferred that the application of treatment with microalgae using the appropriate photoperiod has a high potential for MWW treatment and disinfection [136].Microalgae-based MWW treatment generates valuable byproducts that can be effectively repurposed.The microalgae biomass produced during treatment holds great potential as an agricultural fertilizer, enriching and rejuvenating depleted soils [137].Additionally, after undergoing the microalgae-based treatment, the treated effluent is a water resource that can be safely reused for various nonpotable purposes [138].This includes less restrictive activities where the stringent quality standards required for drinking water are unnecessary, offering an ecofriendly and sustainable approach to wastewater management.

Removal Mechanisms of Dominant Pollutants Using Microalgae
In the realm of MWW, microalgae-based technologies have emerged as a beacon of sustainability and efficiency.One of the primary mechanisms that microalgae employ is adsorption.Due to their expansive surface area and cell walls rich in lipids, proteins, and polysaccharides, microalgae can adsorb many pollutants commonly found in MWW [63].Additionally, biosorption is a significant process wherein dead or living algal biomass passively uptakes and concentrates pollutants.The inherent functional groups, such as carboxyl, hydroxyl, and amine on microalgal cell surfaces, enhance their biosorption capacity, especially for heavy metals [139].
As microalgae proliferate in MWW, they assimilate and uptake, absorbing critical nutrients like nitrogen and phosphorus and integrating them into their biomass [140].
Another salient mechanism is biodegradation.Select strains of microalgae possess the capability to metabolize organic pollutants, converting them into benign or less harmful compounds.Certain strains can degrade intricate organic molecules in pharmaceutical residues frequently found in MWW [141].
The bioconversion ability of microalgae is another testament to their versatility.Through photosynthesis, they can process considerable amounts of carbon dioxide in wastewater, subsequently releasing oxygen-a process that mitigates greenhouse gas emissions and aids in wastewater oxygenation [142].Some calcifying microalgae strains bring about coprecipitation, precipitating pollutants such as phosphates in the form of calcium phosphate [143].
Moreover, particular strains of microalgae bolster pathogen reduction in MWW by producing compounds toxic to bacteria and other pathogens [144].Finally, the oxygenproduction capability of microalgae, a byproduct of photosynthesis, is indispensable.It enhances the dissolved oxygen levels in wastewater, promoting the activity of aerobic bacteria and facilitating the breakdown of organic pollutants [145].
In summary, when applied to MWW treatment, microalgae present a multifaceted approach, targeting various pollutants through diverse mechanisms.The optimal application hinges on the judicious selection of microalgae strains and fine-tuning the treatment conditions [146].

Reuse of Treated Municipal Wastewater
The reuse of water makes it possible to alleviate the pressure on water resources caused by the high consumption of water for the development of human activities.Reuse after treatment and biomass recovery allows substituting water sources in less restrictive demands to release better quality water for more restrictive uses such as human supply [147].
Generally, treated MWW is used for: • Irrigation of parks and gardens located in public areas, centers, sports facilities, soccer fields, golf courses, school gardens and universities, lawns, decorative trees, and shrubs along avenues and highways; For the sake of the circular economy's aims, various studies have been carried out on the reuse of MWW treated with microalgae and its potential for reuse [148].

Recovery of Microalgae Biomass
The post-treatment recovery of microalgae biomass is an integral step in wastewater treatment, offering potential applications in diverse domains.However, the inherent challenges associated with this separation make it a cost-intensive process.
Separating microalgae biomass can account for 20 to 60% of the total production cost [149].As a result, the selected separation process must be efficient, swift, and costeffective.Table 5 presents a comparative analysis of the primary techniques used for microalgae biomass separation, outlining their benefits and drawbacks.
The most widely adopted separation method on a large scale is coagulation/flocculation, followed by sedimentation.This approach destabilizes particles, facilitating the separation of the solid-liquid phases [150].Traditional coagulants, such as aluminum sulfate and ferric chloride, have been utilized in this context.However, a notable concern with these coagulants is their potential to induce biomass toxicity during the coagulation/flocculation process [151].Given the challenges with traditional coagulants, the shift towards natural coagulants, especially those based on vegetable tannins, has gained traction.These tannin-based coagulants, derived from sources like quebracho, chestnut, or black wattle, have proven effective in microalgae separation without introducing harmful metals into the process [153].
Several studies have evaluated the efficacy of Tanfloc SG, a commercial coagulant/ flocculant, in microalgae separation.For instance, Teixeira et al. [154] demonstrated that Tanfloc SG, at an optimal concentration of 100 mg•L −1 , achieved over 90% removal efficiency in various parameters within a short sedimentation time.Ruggeri et al. [155] found that 35 mg•L −1 of Tanfloc SG led to 99% turbidity removal in treating microalgae Monoraphidium contortum.
The method chosen for recovering microalgae biomass post-treatment plays a pivotal role in determining the efficiency and safety of the process.As the shift towards natural coagulants gains momentum, it offers a promising avenue for sustainable and effective biomass recovery.

Potential Applications of Algal Biomass
The rapid growth of the global population has resulted in increased energy consumption, resource depletion, and escalating pollution levels.To combat these challenges, there is a pressing need for environmentally sustainable production and consumption systems that emphasize reuse and recycling.In this context, the integration of the biorefinery concept has emerged as a beacon of sustainable development.One such promising approach is the cultivation of microalgae in wastewater, which not only aids in wastewater treatment but also produces valuable microalgal biomass with many potential applications (Figure 2).Microalgae are renowned for their rich content of bioactive compounds, including carbohydrates, proteins, lipids, and pigments, which find uses across various sectors [156].These applications span from their incorporation in animal feeds and soil biofertilization to their utility in cosmetics, pharmaceuticals, and energy production.

Biomass for Animal Feed and Soil Fertilization
Microalgae's high content of lipids, essential amino acids, and minerals positions them as a potent nutritional powerhouse, especially beneficial for mammals that cannot synthesize these components.This unique nutritional profile underscores their potential as a natural supplement in animal feeds, with particular significance for the booming aquaculture sector [157].

Biomass for Animal Feed and Soil Fertilization
Microalgae's high content of lipids, essential amino acids, and minerals positions them as a potent nutritional powerhouse, especially beneficial for mammals that cannot synthesize these components.This unique nutritional profile underscores their potential as a natural supplement in animal feeds, with particular significance for the booming aquaculture sector [157].In the realm of agriculture, microalgae biomass demonstrates efficacy as a biofertilizer.Its application enriches the soil with organic matter and boosts levels of essential plant nutrients, including nitrogen, phosphorus, calcium, potassium, iron, and manganese, among others [158].However, a caveat that warrants attention is the potential concentration of heavy metals in the harvested microalgal biomass.Elevated heavy metal concentrations can pose risks when the biomass is applied directly to the soil or used in animal feeds.It is imperative to employ pretreatment techniques or quality-control measures to ensure the safe application of this biomass.Recent research has delved into methods for reducing heavy metal concentrations in microalgal biomass, ensuring its safe use in various applications [159,160].Emphasizing these precautions underscores the holistic approach toward harnessing the potential of microalgae while ensuring environmental and health safety.

Biomass for the Production of Bioenergy
The potential use of microalgae as renewable energy has been inspired due to their rapid growth rate and high lipid content.In the last decade, microalgae have become a third-generation biofuel feedstock due to their high triglyceride.Algal polysaccharides and triacylglycerol can also be starting materials for synthesizing bioethanol and biodiesel.Biomethane from wastewater-grown microalgae biomass was reported to depend on the biomass's composition and the type of converted molecules (carbohydrates, proteins, or lipids).Towards the sustainability concept, the CO 2 generated during anaerobic digestion and the conversion of methane to electricity could be integrated with microalgae cultivation in wastewater.Generally, energy recovered from wastewater-grown microalgal biomass is through different conversion techniques like hydrothermal liquefaction, anaerobic digestion and fermentation, transesterification, and pyrolysis.The choice of an adequate technique depends strictly on the strain used and the type of energy to produce [42].
Despite several scientific initiatives, the commercialization of microalgal biofuels has not yet been achieved since it is not economically feasible because of drying and extraction costs [161].Therefore, microalgal biomass can also be used to produce other value-added compounds.

Other Valuable Compounds
Several bioactive compounds have been discovered and purified from marine microalgae.These valuable compounds, including polysaccharides, pigments, fatty acids, and proteins, demonstrate several biological activities [162].For example, the peptide fraction isolated from pepsin-hydrolyzed microalgae protein waste had antigastric cancer properties, and the Angiotensin-converting enzyme isolated from microalgae C. vulgaris had a role in the regulation of hypertension.Polyunsaturated fatty acids, including ω-3, have also been shown to have antioxidant capacities, reduce hypertension, and have immuneregulating qualities.Microalgal pigments, like astaxanthin, β-carotene, phycocyanin, lutein, and violaxanthin, have been shown to have anticoagulant, antimutagenic, antibacterial, radioprotective, anticancer, and anti-inflammatory bioactivities [163].Another emerging application of microalgal biomass is lactic acid and bioplastic production after converting carbohydrates.Glycerol could also be produced with a percentage of 10% as a byproduct during microalgal lipid biodiesel production.For cosmetic applications, different secondary metabolites and specific extracts from Tetraselmis sp. and Dunaliella sp. could be used as an alternative feedstock for producing various cosmetics, like antioxidants, UV-protectants, and antiaging, for skin care [42].

Challenges and Future Perspectives in Microalgal Municipal Wastewater Treatment
Microalgal MWW treatment, when viewed through the lens of biorefinery and valorization applications, carries a promise that could redefine how we perceive wastewater management.However, the journey from laboratory benches to large-scale industrial applications is riddled with challenges, each demanding unique solutions.
From an environmental perspective, microalgae cultivation is at the mercy of Mother Nature.Sunlight, the primary energy source for photosynthetic microalgae, varies with seasons.Similarly, temperature fluctuations can significantly affect their metabolic activities.While seemingly trivial, these environmental factors can pose significant challenges, especially when maintaining consistent growth rates.Additionally, MWW itself is not a static entity.Its composition, characterized by changing pH levels, turbidity, nutrient concentration, and hue, is anything but constant.Some wastewaters can have dark col-orations, hazardous pollutants, or highly variable nutrient concentrations-each a potential roadblock to efficient microalgal activity.
The technological challenges are manifold.One of the primary hurdles is the efficient separation of microalgal biomass from the treated MWW postphytoremediation.While this might sound straightforward, it is a complex process that can contribute significantly to production costs.Scaling up operations presents another set of challenges.Pilot studies conducted under controlled environments often yield promising results.However, with all the unpredictability they entail, translating these results to large-scale operations is no easy feat.The nonsterile nature of wastewater introduces another layer of complexity.In such environments, a plethora of microbial interactions occur.While some of these interactions are beneficial, others can harm microalgae.Exploring coculturing techniques, where microalgae are grown alongside other beneficial microorganisms like fungi, yeast, or bacteria, has shown potential.However, these cocultures introduce their complexities, needing precise controls and monitoring.The pretreatment of influents is another technological challenge.Ensuring an ideal nutrient balance and mitigating factors like turbidity are essential for optimal microalgal growth.While techniques like electrocoagulation and granular activated carbon adsorption offer solutions, they demand additional resources and optimization.
From an economic vantage point, microalgae cultivation can strain finances.Establishing a microalgae plant, especially one equipped with advanced monitoring and control mechanisms, requires significant capital investment.Operational costs, especially those associated with separating microalgal biomass and the pretreatment of influents, add to the financial burden.Another economic challenge lies in the realm of CO 2 sequestration.While environmentally beneficial, the costs associated with sequestration, especially transportrelated, can be daunting.Traditional methods, like compression for pipeline transport, are energy-intensive and, by extension, costly.However, it is not all challenges.The potential applications of microalgae present a glimmer of hope.Their biomass is versatile, finding applications in bioenergy production, extraction of high-value compounds, and biofertilizers.These applications not only offer environmental benefits but also have the potential to provide economic returns, potentially offsetting cultivation costs.
In conclusion, while challenges in microalgal MWW treatment are numerous, they are not insurmountable.The sector can achieve sustainable and economically viable solutions with continued research, technological advancements, and policy support.The key lies in addressing challenges in isolation and viewing them as parts of an interconnected web, each influencing the other.

Conclusions
Many research efforts have been directed toward developing microalgae wastewater treatment.This application has many benefits, ranging from the depolluting-detoxifying power of these versatile organisms to their biomass potential green application without generating secondary pollution.Such treatment contributes to environmental sustainability and a circular economy model but requires integrating multiple unit operations, including microalgal cultivation, biomass separation, and valorization.However, there are several limits to scaling up efficiently to an industrial scale.These limits converge in the inability to control all the factors involved in the wastewater phytoremediation process, which significantly affects the survival of the biomass and the efficiency of depollution and valorization.Therefore, the technical and economic feasibility of providing operational guidelines for microalgae-based WWT must be studied mainly using the native microalgal consortium of the effluent.In most cases, this autochthonous flora of the wastewater has shown a better capacity for adaptation, control, and treatment.

Figure 1 .Figure 1 .
Figure 1.Symbiotic communities of microalgae and bacteria for the MWW treatment.The use of a microalgae consortium has several advantages: (i) the possibility of more robust systems due to their resistance to fluctuations in environmental conditions and invasion by other species; (ii) the greater consumption of nutrients by the combination of Figure 1.Symbiotic communities of microalgae and bacteria for the MWW treatment.

Figure 2 .
Figure 2. The potential applications of the generated biomass following microalgal treatment of wastewater.

Figure 2 .
Figure 2. The potential applications of the generated biomass following microalgal treatment of wastewater.

Author
Contributions: I.D.; writing-review and editing, M.F.; visualization, R.H.; supervision, S.A.; project administration, I.D.; funding acquisition.All authors have read and agreed to the published version of the manuscript.

Table 1 .
Municipal wastewater treatment technologies, resulting water quality, and associated microalgae cultivation studies.

Table 2 .
Chemical composition of some species of microalgae.

Table 3 .
Factors affecting microalgae growth and the relevant literature findings.

Table 5 .
Advantages and disadvantages of main separation processes of microalgae.