Microalgae-Based Wastewater Treatment Processes for the Bioremediation and Valorization of Biomass: A Review

Abstract

Conventional wastewater treatment methods often rely on energy-intensive physical and chemical processes that are costly and may generate secondary pollution. These limitations have prompted the exploration of more sustainable alternatives. Among them, phycoremediation, particularly using microalgae, has emerged as a promising strategy for mitigating environmental pollution. Microalgae possess unique capabilities to sequester heavy metals, assimilate nutrients, and degrade emerging contaminants while simultaneously producing valuable biomass. The efficacy of microalgal bioremediation can be enhanced through omics-based approaches, which enable these biological agents to convert toxic compounds into non-toxic forms and improve ecosystem health. Additionally, forming microalgae–microorganism consortia can enhance process efficiency and cost-effectiveness. This review highlights multi-pronged strategies for pollutant mitigation in wastewater, focusing on environmentally and economically viable microalgal cultivation systems. It also identifies research gaps and discusses the potential for biomass valorization into economically important products.

1. Introduction

Increasing population and rapid industrialization have exerted tremendous pressure on water resources and are responsible for ever-increasing wastewater generation, causing enormous harm to the ecosystem and environment. Anthropogenic activities have significantly enhanced the municipal contaminants through industrial discharges, agricultural runoff, and improper waste disposal, leading to increased levels of pollutants in the environment. These activities introduce a wide range of contaminants, including heavy metals, pesticides, pharmaceuticals, and other contaminants into water and soil, posing risks to both ecosystems and human health [1,2]. Wastewater could be from various sources, for example, from the industries, breweries, beverages, fertilizers, tanneries, or chemical or bioprocess factories; from domestic activities (washing, kitchens, hotels); or from agriculture (poultry, piggery, dairy, nutrient runoff), including both inorganic and organic substances [3]. Currently, bioremediation through conventional strategies such as physical (filtration, screening, sedimentation, etc.), chemical (precipitation, adsorption, and ion exchange), and biological methods (activated sludge and biosorption) has been adopted, but they have various demerits such as consumption of excessive energy, resource waste, expense, and production of secondary pollution [2,4]. In brief, the rapid industrialization and expansion of human activities have resulted in the pollution of water bodies, causing serious threats to the planet’s health [4]. Waterborne diseases have been affecting the health of the human population and other forms, causing degradation of the ecosystem. Pollutants mainly originate from the excessive discharge of untreated water produced from various sources, such as agricultural, municipal, domestic, and industrial wastes [5]. These pollutants have damaging effects on the ecosystem of aquatic life, as well as on humans via the food chain [6,7,8]. The chemical pollutants in wastewater contain organic and inorganic pollutants (heavy metals, phosphorus, and trace elements). Organic impurities mainly constitute lipids, carbohydrates, and proteins [9]. Similarly, the micropollutants present in wastewater are a huge concern to environmental safety [10]. Unfortunately, many conventional treatment processes fail to remove micropollutants (pesticides, heavy metals) and contaminants (bacteria, viruses, protozoa, helminths) in wastewater [11]. Despite the presence of harmful contaminants, wastewater often provides a nutrient-rich environment that supports the growth of photosynthetic microalgae, due to its abundance of nitrogen, phosphorus, and carbon sources [8]. Moreover, they have the ability to sequester CO₂ from the environment and produce oxygen. Microalgae produce high-value biomolecules such as carotenoids, fatty acids, and proteins, which have industrial applications. These photosynthetic forms are also potential sources of bioenergy molecules [12,13,14,15], biofertilizers, and primary sources of nutrients to the lifeforms in the aquatic and semiaquatic systems [7,8,9,16,17]. Besides being easily cultivated, they exhibit high photosynthetic efficiency and growth rates [18,19].

Today, microalgae are exploited for their dual role in phycoremediation (i.e., bioremediation with the help of microalgae) and for generating high-value biomass capable of producing useful metabolites. They have been employed for bioremediation due to their capability of assimilating large amounts of carbon, nitrogen, and phosphorus for their growth and metabolite production [12]. Microalgae have the abilities of bioaccumulation, biodegradation, and adsorption. Utilizing a variety of physiological mechanisms, microalgal strains can reduce pollutants. Many important and expensive carotenoids with huge demand in the market have been produced utilizing microalgae cultivated in wastewater as a nutrient source [13]. Microalgae species, such as Porphyridium, Scenedesmus, Chlorella, Anabena, Chlamydomonas, Nannochloropsis, Synechococcus, Dunaliella, Scenedesmus, and Arthrospira/Limnospira/Spirulina, are reported for wastewater treatment, and the method has been proven to be effective and efficient [20,21,22].

Recently, there has been an upsurge in the adoption of omics approaches for value addition to micro-algal technologies for enhanced efficiencies and productivities. The omics technology associated with algae is also known as ‘algomics’, which helps in understanding the basic system of genus and species as well as sub-types, offering a huge amount of data that can be interpreted with the help of available computational tools and software [23,24]. The whole range of omics technologies, like genomics, transcriptomics, proteomics, metabolomics, and metagenomics, has been applied to study various algal processes. The combination of screening microalgae and molecular phylogeny not only helps to reveal the phenotypic but also the genotypic traits that are held responsible for microalgal growth and the removal of nutrients via omics techniques in wastewater [25]. In this review, insights into the role of microalgae and microalgae-bacteria consortia responsible for treating wastewater, along with the production of bioactive metabolites, are discussed.

2. Bioremediation Based on Microalgae

2.1. Microalgae Diversity

Microalgae are unicellular or colonial photosynthetic organisms, typically ranging in size from less than 1 µm (picoplankton) to around 100 µm, depending on the species and growth conditions [26]. These organisms are considered to be among the most ancient life forms on Earth [27]. Microalgae and Cyanobacteriophyta are a polyphyletic group that is distributed in four main classes: Cyanophyceae, Chlorophyceae, Bacillariophyceae, Chrysophyceae and Eustigmatophyceae. Microalgae are traditionally classified based on morphological characteristics such as cell structure, pigmentation, and flagella type. Advances in molecular biology have also enabled classification based on phylogenetic relationships and nutritional modes. Moreover, eukaryotic microalgae are efficient in carrying out photosynthesis to accumulate biomass, lipids, and co-products, simultaneously with wastewater treatment [28], in a most effective manner [29,30,31].

Microalgal diversity is not only reflected in their morphology and taxonomy but also in their biochemical composition. Different species of microalgae exhibit varying levels of proteins, carbohydrates, and oils, which influence their suitability for applications such as omega-3 fatty acid production and animal feed. Additionally, micronutrient profiles vary among species, contributing to their use in food supplements and functional products. With certain strains flourishing in various effluent types, microalgae have demonstrated significant promise for wastewater treatment. Species like Chlorella vulgaris, Chlorella sorokiniana, Tetradesmus obliquus, Dictyochloris, and Halochlorella rubescens (Chlorophyta) are frequently found in municipal wastewater because of their strong growth rates, high nutrient (nitrogen and phosphorus) uptake efficiency, and efficient removal of BOD and COD. Research studies revealed that the C. sorokiniana removal efficiency of total nitrogen is ~97% and phosphorus is ~99% in domestic livestock wastewater with improved CO₂ fixation [32]. On the other hand, Halochlorella rubescens showed high nitrate (~83%) and phosphate (~73%) removal efficiency from secondary municipal wastewater [33]. On the other hand, Porphyridium purpureum (Rhodophyta), Limnospira platensis, and Nostoc sp. (Cyanophyceae) grown in wastewater from the food industry revealed a removal efficiency up to 98% of COD, 94% of inorganic nitrogen, and 100% of phosphates while also recovering valuable pigments [34]. Moreover, microalgae like Micractinium, Dictyosphaerium, Scenedesmus, and Chlorella sorokiniana (Chlorophyta) are effective for agricultural and livestock wastewater because of their strong nutrient removal capabilities, mixotrophic growth capabilities, and ability to fix CO₂ [35]. Genetic engineering of microalgae could be a promising option to produce pharmaceuticals, antibodies, and other valuable products like hormones, vaccines, blood clotting factors, growth factors, anti-cancer agents, and immune regulators [36]. Among the biological approaches, microalgae are a significant class of microorganisms that can remove heavy metals from wastewater and have a wide range of environmental applications [37,38]. Researchers have identified three key mechanisms for the bioremediation of environmental contaminants: bio-adsorption, bioaccumulation, and biodegradation. These processes confirm the ability of microalgae to remove different types of environmental contaminants [38].

2.2. Bio-Adsorption

Bio-adsorption is the process by which harmful substances adhere to the cell wall or other cellular constituents (extracellular polysaccharides) of microalgal cells [39]. The contaminant’s chemical composition, such as its hydrophobicity, is also crucial to adsorption. Compared to hydrophilic substances, positively charged pollutants are more readily drawn by electrostatic interactions to the negatively charged cell surface of microalgae [40]. Ion exchange, adsorption, surface complexation, precipitation, and chelation are some of the mechanisms involved in bio-adsorption [41]. Even in dead cells, microalgae’s cell surface receptors have been shown to remain intact with contaminants [42]. It has been widely demonstrated that microalgae cells can bio-adsorb a variety of pharmacological pollutants. In one study, 7-aminocephalosporanic acid and microalgae species of Chlorella, Mychonastes, and Chamydomonas (Chlorophyta) showed 100% bio-adsorption [43]. In another study, microalgae used the bio-adsorption process to remove arsenic, mercury, cadmium, lead, boron, cobalt, iron, molybdenum, zinc, and chromium [44]. Through chelation, reducing enzymes, heavy metal immobilization, and gene control, microalgae defend themselves against the toxicity of heavy metals. The primary method for removing heavy metals from wastewater is thought to be bio-adsorption.

2.3. Bioaccumulation of Wastewater Pollutants

Bioaccumulation is the process by which contaminants move into the cell from the cell wall or cell surface. Living microalgae cells engage in bio-uptake, which involves the absorption of pollutants through three different mechanisms: active transport, passive-facilitated transport, and passive transport [40]. Due to the hydrophobic structure of the cell membrane, passive transport takes place across the cell wall from an area of high (external) contaminant quantity to an area of low (internal) contaminant amount; in contrast, polar molecules and ions cannot passively diffuse across the cell membranes. For the elimination of trimethoprim, florfenicol, carbamazepine, and sulfamethoxazole, bioaccumulation via passive diffusion has been reported [45,46,47]. While active transport is an energy-driven process, passive facilitated transport uses transporter proteins to diffuse pollutants across the cell membrane [40]. Desmodesmus subspicatus was shown to bioaccumulate 17-ά-ethinylestradiol at a rate of 23% [48], whereas Nannochloris species showed a 42% bioaccumulation rate for triclosan [45]. A study examined C. vulgaris’ capacity to bioaccumulate silver nanoparticles [49]. They discovered that C. vulgaris actively bioaccumulated glucose-coated silver nanoparticles and that the rate of accumulation increased in a dose-and time-dependent way. Without going through biotransformation, the silver nanoparticles remained in crystal form after accumulating in large vacuoles [49]. It was discovered that Spirogyra biomass, both living and dead, was effective at accumulating AsO₄⁻³ [50].

2.4. Biodegradation

By using metabolic biodegradation, contaminants can be bio-transformed into simple, non-toxic compounds [51,52]. By using pollutants as a source of carbon and electrons, metabolic degradation is the process by which microalgal biodegradation takes place [53]. Microalgae biodegradation can take place intracellularly, extracellularly, or concurrently, with the initial degradation events taking place outside of cells and the subsequent breakdown taking place inside [53]. The breakdown of contaminants by microalgae can be summed up as a two-step catalytic enzyme process. The cytochrome P450, cytochrome b5, monooxygenase, and mixed-function oxidases catalyze the following reactions in the first phase: carboxylation, hydroxylation, decarboxylation, hydrogenation, methylation, demethylation, ring cleavage, and oxidation-reduction [38,54]. It has been noted that metabolites generated during first-phase processes make contaminants more polar and hydrophilic, which facilitates their removal from the cell. In order to increase their water solubility, pollutants or their transformation products from phase I processes can be conjugated to large, polar molecules in second-phase reactions [54]. Numerous phase II enzymes have been shown to transform and detoxify a variety of pollutants, including violaxanthin de-epoxidase, alkaline and acid phosphatase, dehydratase, catalase, glutamyl-tRNA reductase, hydrolases, laccases, malate/pyruvate dehydrogenase, oxygenase, pyrophosphatase, transferase, uroporphyrinogen III carboxylase/decarboxylase, and others [55,56]. The breakdown of ciprofloxacin by Chlamydomonas sp. Tai-03 was studied [57]. The researchers discovered that the microalgal bacteria degraded the 10 antibiotics by 23–99% [58]. Moreover, numerous studies have evaluated the use of microalgal consortia to boost biomass and value-added bioproduct output while also supporting environmentally friendly wastewater treatment [59].

3. Role of Microalgae in Wastewater Treatments

Phycoremediation is the use of microalgae to remove pollutants, xenobiotics, and nutrients like phosphorus, nitrogen, sulfur, etc., along with heavy metals from contaminated environments [60,61]. To explore the capability and feasibility of employing microalgae for treating wastewater, several studies have been conducted [4,62]. The removal of nitrogen, phosphorus, and heavy metals from different effluents, as well as the heavy metals caused by pollutants, has been reported by various authors [63,64,65,66,67].

3.1. Removal of Heavy Metals by Phycoremediation: Role of Phycofilteration Mechanism

Phycofiltration (biosorption, bioaccumulation, biodegradation, assimilation, photosynthetic oxygenation, physical filtration, and flocculation) refers to the process by which microalgae remove contaminants from wastewater through a combination of biological, chemical, and physical pathways. Heavy metals in the environment are a serious concern to human beings, flora, and fauna [68]. The discharge of heavy metals from industrial, agricultural, municipal, and livestock operations poses health risks. With increasing industrialization, heavy metal ions have become a major pollutant in wastewater, increasing the burden on natural ecosystems [69]. Several microalgal species have demonstrated effective performance in the removal of heavy metals from wastewater treatment. These species can be grouped as follows: Chlorophyta: Scenedesmus, Haematococcus, Chlorella, Chlamydomonas; Coelastrum, Tetradesmus, Micractinium, Desmodesmus, Ankistrodesmus, and Auxenochlorella; Cyanobacteriophyta: Arthrospira, Limnospira, and Microcoleus; Rhodophyta: Porphyridium; Euglenophyta: Euglena; Bacillariophyceae: Diatom; and other groups: Characium, Pithophora, Nannochloropsis, Microchloropsis, and Tetraselmis. These species are widely studied for their nutrient uptake capabilities, heavy metal removal, and biomass productivity under different wastewater conditions.

In wastewater, heavy metals like chromium (Cr), zinc (Zn), copper (Cu), nickel (Ni), mercury (Hg), lead (Pb), and cadmium (Cd) are of major concern [70]. Many studies have reported that tannery effluents and saline wastewater contain a high concentration of Cr (VI) of about 155 mg/L [71]. On the other hand, many industrial wastewaters have also been reported to be high in Pb²⁺ and Cd²⁺ metal toxicity [72]. Several microalgal species have demonstrated the removal of heavy metals (Zn, Cu, Cd, Cr, Ni, Hg, As) in domestic wastewater treatment, including Porphyridium, Scenedesmus, Chlorella, Chlamydomonas, and Limnospira (formerly Spirulina) [32]. Some of the promising algal strains reported for the removal of heavy metals from wastewater streams are Chlorella vulgaris, Chlorella sorokiniana, Scenedesmus quadricauda, Coelastrum sp. (Chlorophyta), and Limnospira maxima (formerly Spirulina maxima), Limnospira platensis (formerly Spirulina platensis) (Cyanobacteriophyta) [15]. These species are widely studied for their nutrient uptake capabilities and biomass productivity under wastewater conditions. Microalgae-mediated removal of heavy metals from wastewater could be an excellent alternative to traditional methods [71].

Microalgae remove heavy metals initially through adsorption to their cell surfaces. The cell walls of microalgae contain functional groups such as carboxyl, hydroxyl, phosphate, and sulphate, which can bind metal ions via ion exchange, complexation, and electrostatic interactions [57,65]. This passive mechanism is often the first step in metal sequestration. Beyond surface adsorption, microalgae can internalize heavy metals through active transport mechanisms. Once inside the cell, metals may be compartmentalized into vacuoles or bound to intracellular ligands, reducing their toxicity. This bioaccumulation contributes to long-term sequestration and detoxification [57,65].

Researchers have reported the Cd removal capacity of Scenedesmus quadricauda to be 66% [73]. Three ponds cultivated with algae with two different metal loading rates for 7 days under 16/8 h light/darkness and 24 h light showed 98% chromium removal and 70% Zn removal at a low metal loading rate. Under a continuous light regime, the removal efficiency of Zn rose back to 80%. While numerous species demonstrate high removal efficiencies for heavy metals, C. vulgaris and S. quadricauda consistently show strong performance across multiple contaminants. However, variability in experimental conditions and wastewater types suggests the need for standardized testing protocols.

Pb, Cd, and Cu showed a removal efficiency of 36%, 33%, and 27%, respectively [74]. Furthermore, from a mixture of sewage, well water, and seawater, microalgae species C. vulgaris and Chlorella salina have been reported to remove Cu, Zn, Cr, and Ni with a high removal capacity [75]. These studies reveal that the use of microalgae in wastewater not only removed the heavy metal toxicity but also contributed to the growth and production of biomass from wastewater-grown species. Some microalgae possess enzymatic systems capable of transforming toxic metals into less harmful forms. For example, reductases may convert hexavalent chromium (Cr⁶⁺) to trivalent chromium (Cr³⁺), which is less toxic. These biochemical conversions are species-specific and influenced by environmental conditions [71]. The removal efficiency of various heavy metals, such as cadmium, lead, arsenic, cobalt, copper, boron, iron, manganese, mercury, nickel, zinc, and chromium, by algal species is presented in

Table 1. Reported removal efficiency of heavy metals by algal species.

Algal Species	Heavy Metals	Rate of Bioremediation with Time Duration	References
Cyanobacteriophyta
Anabaena sphaerica	Cd	111.1 mg/g	[76]
Anabaena sphaerica	Pd	121.95 mg/g	[76]
Phormidium ambiguum	Pd	70%	[77]
Phormidium ambiguum	Hg	97%	[78]
Chlorophyta
Chlorococcum sp.	As	239.09 µg/g	[79]
Auxenochlorella pyrenoidosa (formerly Chlorella pyrenoidosa), Tetradesmus obliquus (formerly Scenedesmus acutus)	Cd	57.14% in 3 h	[80]
Scenedesmus almeriensis	As	40.7% in 3 h	[81]
Ulva lactuca	Mn	74% in 12 h	[82]
Scenedesmus almeriensis	Br	38.6% in 10 min	[83]
Heterochlorella sp. MAS3	Cd	58% in 16 days	[84]
Chlorococcum infusionum	Cd	17% in 6 days	[85]
Oedogonium westii	Cd	5% in 7 days	[85]
Chlorella vulgaris	Cd	80% in 1 day	[86]
Oedogonium westii	Ni	32.85 mg/g in 240 min	[87]
Chlorococcum infusionum (formerly Chlorococcum humicola)	Co	44%	[88]
Ulva lactuca	Cu	86% in 12 h	[80]
Chlorococcum infusionum (formerly Chlorococcum humicola)	Fe	74.47% in 6 days	[80]
Oedogonium westii	Pd	61–96% in 7 days	[44,89]
Chlorella vulgaris	Mn	99.4% in 3 h	[90]
Ulva lactuca	Mn	74% in 12 h	[82]
Chlorophyta spp.	Zn	91.9% in 3 h	[81]
Ulva reticulata	As	8.12 mg/g/59.5% in 30 min	[91]
Phaeophyceae
Fucus vesiculosus	Cd, Ni, Pd	143.2 mg/g, 70.1 mg/g, 516.3 mg/g	[86]
Polycladia indica (formerly Cystoseira indica)	Ni, Cd	18.17 mg/g; 55.34 mg/g	[92]
Durvillaea antarctica	Cr	102.72 mg/g	[93]
Gongolaria barbata (formerly Cystoseira barbata) Ericaria crinita (Cystoseira crinita)	Cr	90.38% in 24 h 82.86% in 24 h	[77]
Rhodophyta
Neoporphyra leucosticta	Cd	75% in 2 h	[88]
Gelidium amansii	Pd	100% in 2–24 h	[94]
Hypnea valentiae	Co	10.98 mg/g	[95]

Cd—cadmium; Pd—lead; As—arsenic; Co—cobalt; Cu—copper; Br—boron; Fe—iron; Mn—manganese; Hg—mercury; Ni—nickel; Zn—zinc; Cr—chromium.

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3.2. Removal of Micropollutants

Micropollutants (MPs) are anthropogenic chemicals that originate from human activity and are found in water bodies at trace-level concentrations, typically up to the microgram per liter range. These consist of pharmaceuticals, personal care products (PPCPs), steroids, hormones, industrial chemicals, and hydrocarbons (HCs) [96]. These are of concern for human health even at low levels. Continuous discharge of MPs can result in abnormalities in the growth, development, and reproduction of sensitive species in the ecosystem [97]. For many years, researchers have been working on the removal of MPs from wastewater [9,98]. Among them is the use of microalgae species for the removal of MPs from wastewater. Microalgae can biodegrade and assimilate molecules like HCs, antibiotics, pharmaceuticals, and personal care products (PCPs) [99]. They have been employed for bioremediation due to their capability of assimilating large amounts of carbon, nitrogen, and phosphorus for their growth and metabolite production [12]. Various treatment technologies, such as activated sludge, membrane technology, biodegradation, photodegradation, volatilization, and sorption into the biomass, are some of the most relevant contaminant removal processes occurring in microalgae when grown in photobioreactors [100,101,102]. Microalgae-based wastewater treatment and photodegradation pathways are among the most promising and sustainable solutions for removing micropollutants, including pharmaceuticals, personal care products, and industrial chemicals, which can be toxic even at trace concentrations [103]. A study that investigated the effect of MPs on the environment and human health accounted for the adsorptive interactions of 30 MPs with C. vulgaris [104]. Biodegradation of diclofenac was studied using Tetradesmus obliquus (Chlorophyta), which resulted in 99% degradation of the compound from a 25 mg/L initial concentration [105], whereas photodegradation of the same MPs using C. sorokiniana resulted in 40–60% degradation [64]. Cephalosporin antibiotic 7-ACA was degraded completely by Chlamydomonas sp. and Chlorella sp., as reported in the study by [106]. Similarly, degradation of hormones such as 17-α-estradiol and 17-β-estradiol was reported by Tetradesmus dimorphus (formerly Scenedesmus dimorphus) (Chlorophyta) [107]. Ruksrithong and Phattarapattamawong [108] revealed that Tetradesmus obliquus and Chlorella vulgaris removed 99% of 17-β-estradiol. Apart from the degradation of MPs, the algal growth may also be enhanced, as observed in one case from Scenedesmus sp. Interestingly, using Scenedesmus sp. for treating textile industries wastewater, which comprises organic chemicals and dyes, showed a high utilization efficiency of 98.2% for butyrate, with a growth rate of 0.53 g/d [33]. In a study by De Godos et al. [109], Chlorella vulgaris was shown to remove 50% of tetracycline in algal ponds primarily through adsorption, attributed to positively charged molecules on its cell surface. Separately, Tetradesmus dimorphus demonstrated the ability to biodegrade up to 85% of 17-α-estradiol, as reported in [107]. The removal efficiency of various micropollutants by algae, including bacteria consortia, is presented in

Table 2. Reported removal efficiency of micropollutants by algae and algae plus bacteria.

Algal Species	Treatment System and Wastewater Type	Micropollutants	The Removal Efficiency (%)	Methods/Process	References
Tetradesmus obliquus	Lagoon water	Ibuprofen	60	Biodegradation	[110]
Chlorella vulgaris	Lagoon water	Triclosan	100	Phototransformation	[110]
Chlamydomonas oblonga (formerly Chlamydomonas mexicana)	BBM media	Ciprofloxacin	56	Biodegradation	[111]
Mixed algal–bacterial culture	Trickling filter SWW	LAS Caffeine	99–95.6	Phototransformation, biodegradation	[112]
Mixed algal–bacterial culture	Anoxic–aerobic photobioreactor DWW	Ibuprofen, Triclosan, Naproxen, Salicylic Acid, Propylparaben	94, 100, 52,98 and 100	Biodegradation, bioaccumulation, sorption,	[113]
Nannochloris sp.	Lake water	Sulfamethoxazole, Ciprofloxacin, Triclosan	40, 100, 100	photolysis, biodegradation	[114]
Auxenochlorella pyrenoidosa	Algae-activated sludge combined system BG11 media	Cefradine, Cephalexin, Ceftazidime Cefixime	89.9, 94.9, 89.7, 89.7	Photodegradation, biodegradation	[115]
Chlorella sp.	Aerated batch reactors	Caffeine	99	Volatilization	[116]
Scenedesmus sp.	Urban wastewater	Ibuprofen, Galaxolide, Tributyl Phosphate, 4-octylphenol, Tris (2-chloroethyl) Phosphate, Carbamazepine	60, 99, 99, 99, <20, <20	Biodegradation	[116]
Chlorella sp. Nitzschia acicularis (Bacillariophyceae)	2.5 L reactor Secondary wastewater	Bisphenol-A	46	Biodegradation	[117]
		Bisphenol-AF, Bisphenol-F, 2,4-dichlorophenol	80, 87,76	Bio-adsorption	[117]
Chlorella vulgaris Scenedesmus sp. Westella botryoides	HRAP Sewage wastewater	2,4-dichlorophenol, Hormones, Pharmaceuticals, Xenoestrogens	76, 7–55, 17–54, 41–53	Bio-adsorption, biodegradation, photodegradation, volatilization	[118]
Mixed algal culture	HRAP, urban wastewater	Naproxen, Caffeine, Carbamazepine, Ibuprofen, Galaxolide, Methylparaben, Triclosan, Celestolide, Atrazine, Diclofenac, Biophenol A, Caffeine	99, 89, 98, 62, 99,97,75, 95, 53, 85, 92, 85, 89.7, 84.7	Bio-adsorption, biodegradation, photodegradation, volatilization	[118]
Mixed algal culture, diatom plus bacteria, algae plus bacteria	Laundry wastewater	Cumene Hydroperoxide, LAS, Disulfoton-sulfone, Hexazinone, 4-Nitrophenol, Caffeine, Cumene Hydroperoxide, LAS, Disulfoton-sulfone, Hexazinone, 4-nitrophenol, Caffeine, Cumene hydroperoxide, LAS, Disulfoton-sulfone, Hexazinone, 4-Nitrophenol	84.7, 61.6, 53.9, 82.3, 96.6, 87.3, 81.3, 100, 100, 100, 100, 87.9, 52.1, 100, 100, 100, 70.9	-	[119]
Phaeodactylum tricornutum (Bacillariophyceae)	-	Oxytetracycline	99	Biodegradation	[120]
Monoraphidium capricornutum (formerly Selenastrum capricornutum) Chlamydomonas reinhardtii	-	17-β-estradiol, 17-α-ethinylestradiol	60–100	Biodegradation	[121]
Tetradesmus obliquus, Chlorella vulgaris, Chlorella sorokiniana	-	Diclofenac	99, 71, 67	Biodegradation	[122]
Chlorella sorokiniana	-	Paracetamol Metoprolol	99, 99	Biodegradation	[123]
Microalgal plus bacteria	-	Tritosan, Propylparaben,	85–100, 87–100,	-	[124]
Monoraphidium capricornutum	-	17-β-estradiol	42	Bio-adsorption	[125]
Chlamydomonas reinhardtii	-	17-β-estradiol	54	Bio-adsorption	[126]
Chlorella sorokiniana	-	Diclofenac, Ibuprofen, IH-benzotriazole, Xylytriazole, 5-methyl-1H-benotriazole, 5-chlorobenzotriazole	40–60, 100, 79, >42, >97, and 52	Photodegradation	[127]

HRAP, high-rate algal ponds; LAS, linear alkaline sulfonate; DWW, domestic wastewater; SWW, synthetic wastewater.

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A promising strategy for removing micropollutants using a combination of biological and physicochemical processes is revealed by the investigation of different algal species and their potential in wastewater treatment. Through biodegradation, Tetradesmus obliquus, Chlamydomonas oblonga, and Scenedesmus sp. showed moderate to high removal efficiency for medications such as ciprofloxacin and ibuprofen, with removal rates ranging from 56% to 60%. Similarly, through volatilization and bio-adsorption, Chlorella sp. (Chlorophyta) and Nitzschia acicularis (Bacillariophyceae) were successful in eliminating contaminants like caffeine and bisphenol compounds, respectively (Table 2). Superior adaptability and efficacy were demonstrated by mixed algal–bacterial cultures in a variety of wastewater types, including sewage, household, and laundry effluents. Through biodegradation, bioaccumulation, sorption, and photodegradation, these consortia were able to achieve high removal efficiencies (up to 100%) for a variety of micropollutants, such as caffeine, LAS, naproxen, salicylic acid, and different estrogens. Notably, mixed algal cultures showed up to 99% removal of acetaminophen and other common contaminants in high-rate algal ponds (HRAPs) (Table 2). With biodegradation and photodegradation efficiencies of 89.7–99%, Auxenochlorella pyrenoidosa and Chlorella sorokiniana demonstrated exceptional efficacy against antibiotics such as cephalosporins and medications like diclofenac, paracetamol, and metoprolol. Oxytetracycline was degraded by the diatom Phaeodactylum tricornutum with 99% efficiency (Table 2). Using both biodegradation and bio-adsorption processes, Monoraphidium capricornutum, Chlamydomonas reinhardtii, and algae ponds were able to remove endocrine-disrupting compounds such as 17-β-estradiol, 17-α-ethinylestradiol, and estrone with varying degrees of success. Removal efficiency ranged from 42% to 100% (Table 2).

3.3. Removal of Nitrogen and Phosphate from Wastewater

In wastewater treatment, removing excess nutrients and pollutants from wastewater is the most important goal. High nutrient concentration leads to eutrophication of marine habitats [128]. Microalgae can assimilate 2–10-ton N/ha/year in low solar irradiation (150 W/m²) conditions, upon modification of photosynthetic efficiency (1% to 5%) [129]. The common forms of inorganic nitrogen sources needed for microalgal growth are nitrite salts, nitrate salts, and ammonia. Among these, ammonium is a favorable nitrogen (N) source for microalgae as its assimilation requires comparatively lower energy expenditure [130]. Many studies have reported the utilization of available nitrogen from wastewater for the growth and biomass production of algae. For example, C. vulgaris, when grown in synthetic domestic wastewater, reduced total nitrogen by 98.69% and total phosphorus by 86.07% with a cell density of 17.94 × 10⁶ mL⁻¹ at the lab-scale level [131]. Another study by Mujtaba et al. [132] showed enhancement in nutrient removal with a decrease in activated sludge with N and P removals of 99.8% and 100% in 2 days when grown in a single reactor with co-culture of activated sludge and immobilized C. vulgaris. Phosphates in wastewater can exist in many forms, such as polyphosphates. The orthophosphate represents the bioavailable form of phosphate that can be readily assimilated. It acts as a growth-limiting factor for microalgal biomass production, and phosphorus scarcity and low P concentrations are associated with reduced cell densities [133]. C. sorokiniana was cultivated in tannery wastewater, achieving an orthophosphate removal efficiency of 81.94% and an ammonium removal efficiency of 62.04%. On the other hand, T. obliquus cultivated in urban wastewater showed 4.4 mg L⁻¹ d⁻¹ of total nitrogen removal efficiency with 1.4 g L⁻¹ of biomass production and 29.8 mg L⁻¹ of lipid productivity [134]. In another study, the removal of nitrate (84.51%) and ammonium (75.56%) was reported along with the production of lipids (150.2 mg L⁻¹ d⁻¹), carbohydrates (172.9 mg L⁻¹ d⁻¹), and protein (141.5 mg L⁻¹ d⁻¹) in C. sorokiniana-mediated treatment of aquaculture wastewater [135]. Micractinium sp. IC-76 in municipal wastewater showed 77% PO₄-P removal efficiency with a biomass productivity of 37.18 mg L⁻¹ d⁻¹ and a total lipid content of 36% [136]. In another study, the cultivation of Desmodesmus abundans yielded 16% lipid content and was found to result in 87.52% of phosphate removal [137]. It is advised to select microalgae by exposing them to combined nitrogen (N) and phosphorus (P) stress in order to achieve high lipid productivity while enabling more cost-effective and sustainable wastewater treatment [138]. Walls et al. [139] revealed nutrient removal efficiencies of 91% orthophosphates, 97% for nitrates, 95% for total ammonium phosphate within 3 days when microalgae were cultured with yeast under heterotrophic conditions. This highlights the potential of co-cultivation strategies to enhance nutrient removal performance in wastewater treatment systems.

Moreover, metalloproteins play an active role in electron transport and cell protection against reactive oxygen species [140,141]. The study conducted by Lane et al. [142] revealed that cadmium is an essential metal for some algae; it can replace zinc as a catalyst of carbon anhydrase, as found in Conticribra weissflogii (formerly Thalassiosira weissflogii) (Mediophyceae). Moreover, heavy metals can restore the essential metal ions (Zn²⁺, Fe²⁺/Fe³⁺, Ca²⁺, Mg²⁺, Se², Na⁺, K⁺) and alter the production of reactive oxygen species [143]. The enhancement of heavy metal removal efficiency can be significantly improved by the immobilization of algae biomass and the design of algae-based nanocomposites [144]. Nanocomposites play a key role in the removal of toxic compounds from wastewater. A study by Karimi-Maleh et al. [145] reported that magnetic nanocomposites can be used as adsorbents for the removal of phenazopyridine. Organic ligand-based composite materials are also used for the recognition and removal of heavy metals from contaminated water [145]. The inorganic chemicals include arsenic, cadmium, mercury, nitrate salts, phosphate salts, lead, and fluorides. These pollutants cause harm to the environment. The presence of inorganic nitrates in groundwater causes industrial pollution [146]. The excess of nitrogen and phosphate salts can lead to eutrophication [147]. The overload of these nutrients leads to oxygen depletion that destroys aquatic life. Various biological nitrification and denitrification methods can remove nitrogen from water bodies. Under aerobic conditions, bacteria like Nitrosomonas oxidize ammonia to nitrite and further oxidize nitrite to nitrate [148]. On the other hand, phosphate removal from water bodies can be conducted through chemical precipitation by using chemicals such as alum and lime to remove the excess phosphate from water. Moreover, polyphosphate-accumulating organisms can be enriched under aerobic/anaerobic conditions to take excessive phosphorus and store it as polyphosphates [133,149]. Beyond conventional technologies, nutrient removal technologies such as membrane aerated biofilm, adsorption, ion exchange, or algal/plant uptake systems can play a major role in the removal of phosphates from water bodies [150]. The contamination of copper in wastewater is too high, so it needs to be treated. The permissible limit of copper in water was >1 mg/L [151]. The removal of metals from wastewater becomes very important due to serious health concerns. For the removal of metal ions from water, modified activated charcoal and clay materials are used to remove the metal toxicity, as clay materials are known to expand and adsorb the metal ions quickly, as they have more specificity towards the water [152]. Moreover, zeolite can be used to remove Fe, Pb, Cd, and Zn due to its crystalline aluminosilicate structure, with oxygen bridges binding to Si or Al atoms [153]. Additionally, the use of nanoparticles, mainly alumina nanoparticles, can help remove the heavy metal contamination from water due to their high surface area and thermal stability [154]. There are various other adsorption techniques available for the removal of pollutants from wastewater, which include rice husk, sugarcane bagasse, electrocoagulation, and membrane-based technologies [155,156]. Moreover, microalgae utilize nutrients like phosphate and nitrogen, also removing pollutants from water bodies such as sewage wastewater, and industrial effluents. The use of eco-friendly water treatment processes and carbon dioxide sequestration using photosynthetic organisms is gaining attention worldwide for safety, efficacy, and energy efficiency [157]. Exposure to heavy metals induces oxidative stress in microalgae, triggering the production of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase. These enzymes mitigate reactive oxygen species (ROS) and protect cellular integrity. Additionally, metallothioneins and phytochelatins bind heavy metals, aiding in detoxification and stress tolerance [157].

In the current scenario, conventional and integrated approaches for eliminating metal toxicity from wastewater streams are proven to be inadequate as they are very costly and not economical [158,159]. Many physicochemical methods have been studied extensively for wastewater treatment, such as ion exchange, chemical precipitation, coagulation–flocculation, membrane filtration, oxidation with ozone or H₂O₂ adsorption, electrochemical methods, and flotation [160,161]. The selection of treatment methods mainly depends on many factors, such as the concentration, type of waste, required removal efficiency, and economic factors. The drawbacks of these methods include high energy consumption, process instability, long residence times, high carbon emissions, excessive sludge discharge, and resource wastage. A combination of different methods has been employed to reduce the huge economic barrier [162]. Removal efficiency of nitrogen and phosphorus in different wastewater streams by algal species associated with bacteria is presented in

Table 3. Microalgal strains in the treatment of wastewater from various sources.

Microalgae	Source of Wastewater (WW)	Removal Efficiency (%)	Reference
Chlorella sp.	Synthetic aquaculture	COD—15%; TN—91%; NH4—100%; TP—93%	[163]
Chlorella sp.	Domestic WW	COD—50.90%; TN—68.40%; NH4—82.40%; PO4—83.20%	[28]
Chlorella vulgaris	Sewage WW collected from the treatment plant	COD—66%; BOD—70%; TN—71%; TP—67%	[164]
C. vulgaris	Treated piggery WW	TN—49%; TP—18%	[164]
Chlorella sorokiniana	Tannery WW	NH4—62.04%; PO4—81.94%	[165]
Scenedesmus sp.	Noodle processing tank (aeration tank)	COD—71.85%	[166]
Microalgal mixture	POME WW	COD—71.6%	[167]
Microalgal mixture	Textile WW	TN—70.10%; TP—100%	[168]
Microalgal mixture	Urban WW	BOD—51%; COD—91%; TN—95.10%; TP—88.9%	[169]
Haematococcus lacustris (formerly Haematococcus pluvialis)	Primary treated sewage, primary treated piggery wastewater	Successfully removed nitrogen and phosphorus	[170]
Auxenochlorella pyrenoidosa	Soybean processing WW	COD—77%; TP—88-8%; TN—70%	[171]
Desmodesmus communis	Urban WW	100% removal of ammonia and phosphorus	[172]
Chlorella vulgaris + Bacillus licheniformis	Municipal WW	TN—88.82%; ammonium—84.98%; P—84.87%; COD—82.25%	[173]
Nannochloropsis oculata (Eustigmatophyceae)	Aquaculture WW—recirculation aquaculture system	NO2—84.38%; PO4—14.70%	[174]
Characium sp.	POME from anaerobic pond	COD—45.41%; TN—88.60%; NH3—90.35%; NH4—87%; TP—99.5%; PO4—99.10%	[175]
Pithophora sp.	Thermal WW collected from power station	BOD—88.23%; COD—87.75%; NO3—23.07%; PO4—89.37%	[176]

WW—wastewater; BOD—biological oxygen demand; COD—chemical oxygen demand; TP—total phosphorus; TN—total nitrogen; POME—palm oil mill effluent; NO₃—nitrate; NH₃—ammonia; NH₄—ammonium; NO₂—nitrite; PO₄—phosphate.

,

Table 4. Removal efficiency of nitrogen and phosphorus in different wastewater streams by algal species associated with bacteria.

Wastewater	Algae	Culture Volume and Time (h, days)	C (TOC or COD) Removal Efficiency (%)	N (TN) Removal Efficiency (%)	P (TP) Removal Efficiency (%)	References
SWW	Chlorella vulgaris and Microcystis aeruginosa	1 L, 7 d	86.55	88.95	80.28	[177]
MWW	Mixed algal–bacterial culture	14 L, 14 d	91.2–96.2	41.7–91.0	64.0–93.7	[178]
SWW	Chlorella vulgaris and activated sludge	30 L, 2.7–4 d	78–86	33–66	-	[179]
MWW	Mixed algal bacterial culture	14 L, 8 d	95–98	77–98	55–73	[180]
Municipal river water (Shanghai)	Chlorella vulgaris + Bacillus licheniformis	0.5 L, 2 d	86.6	80.3	88.9	[181]
SWW	Chlorella and Scenedesmus	2.0 L, 2–5 da	-	36–66	17.2–35.9	[182]
Various wastewaters	Chlorella variabilis + Desmodesmus sp. + Paracoccus sp.	-	77.8	71.1	69.3	[183]
DWW	Mixed algal bacterial culture	8000 L, 4 d	-	92–97	70–73	[184]

SWW—synthetic wastewater; MWW—municipal wastewater; DWW—domestic wastewater; COD—chemical oxygen demand; TOC—total organic carbon; TN—total nitrogen; TP—total phosphorus; N—ammonia nitrogen; P—phosphate

and

Table 5. Reported algal species used in wastewater treatments.

Algal Species	Wastewater and Nutrient (N and P) Removal Efficiency	Reference
Ankistrodesmus; Scenedesmus quadricauda	Olive oil mill wastewater and paper industry wastewater treatment	[185]
Acinetobacter haemolyticus, Ralstonia basilensis (bacteria)	Treatment of aromatic pollutants in the wastewater	[186]
Auxenochlorella protothecoides	Concentrated municipal wastewater treatment	[187]
Botryococcus braunii	Secondary treated sewage wastewater treatment, domestic sewage treatment, treats secondarily treated sewage in batch and continuous cultures	[188,189]
Chlorella vulgaris	Domestic sewage treatment, swine slurry treatment, dye wastewater treatment	[189,190,191]
Chlorella sorokiniana	Food and municipal wastewater treatment, cattle manure effluent treatment, wastewater treatment under aerobic dark heterotrophic conditions, reduce pollutants in palm oil mill effluent and fixes CO2	[192,193,194]
Chromochloris zofingiensis (formerly Chlorella zofingiensis)	Olive oil mill waste treatment	[195]
Parachlorella kessleri	Removal of nitrogen (8–19%) and phosphate (8–20%) from artificial medium treatment by algae	[196]
Chlorella sp.	Removal of nitrogen (76–83%) and phosphate (63–75%) from digested manure treatment; dairy wastewater treatment	[197,198]
Chlorella reinhardtii	Removal of nitrogen (42–83%) and phosphate removal (13–14%) from artificial medium treatment	[199]
Auxenochlorella pyrenoidosa	Degraded azo dye wastewater, industrial wastewater treatment, synthetic wastewater and sewage treatment	[171,200,201]
Chlorella variabilis	Dairy wastewater treatment	[202]
Chlorococcum sp.	Synthetic wastewater treatment	[203]
Chlamydomonas reinhardtii, Chlamydomonas sp.	Municipal wastewater treatment reduced the ammonia nitrogen by 83% from landfill leachate treatment	[199,204]
Comamonas sp.	Treatment of acetonitrile in the wastewater	[205]
Consortia microalga/diatoms	Carpet mill wastewater treatment, slaughterhouse wastewater treatment, dairy wastewater treatment, poultry waste treatment	[206,207,208,209]
Desmodesmus communis	Biological wastewater treatment	[210]
Diplosphaera sp.	Diluted dairy wastewater and winery wastewater treatment	[211]
Ettlia oleoabundans	Agricultural anaerobic waste effluent treatment	[212]
Gonium sp.	Textile effluent treatment	[187]
Haematococcus lacustris	Primary treated sewage and primary treated piggery wastewater	[213]
Mucidosphaerium pulchellum	Domestic wastewater treatment	[214]
Monoraphidium braunii	Organic matter waste treatment	[215]
Neochloris vigensis, Neochloris oleoabundans	Synthetic wastewater treatment, nitrate utilization up to 90–95% from cow manure waste treatment	[203,216]
Nannochloropsis sp.	Municipal wastewater treatment, tannery effluent treatment	[217]
Oscillatoria sp. (Cyanophyceae)	Removal of 100% nitrogen and 100% phosphate from municipal wastewater treatment	[218]
Oedogonium sp.	Piggery wastewater treatment	[219]
Prototheca zopfii	Degrades petroleum hydrocarbons; utilized 41.4% of crude and 10.7% of motor oil waste	[220]
Tetradesmus obliquus	Removal of nitrogen (79–100%) and phosphate (47–98%) from municipal wastewater treatment, dairy wastewater treatment, brewery effluent treatment	[202,221,222]
Scenedesmus dimorphus	Removal of 20–55% phosphate from industrial wastewater	[223]
Scenedesmus acutus	Municipal wastewater after aerobic treatment	[224]
Scenedesmus rubescens	Synthetic wastewater treatment	[203]
Arthrospira sp. (Cyanophyceae) Limnospira platensis (Cyanophyceae)	Removal of nitrogen (84–96%) and phosphate (72–87%) from piggery waste treatment, removal of nitrogen (96–100%) and phosphate (87–99%) from industrial wastewater treatment, swine wastewater treatment, olive oil mill wastewater treatment, poultry wastewater treatment, poultry litter leachate treatment	[225,226,227,228,229]
Tetraselmis indica	Domestic wastewater treatment	[230]

.

4. Algal Biomass Production Capability in Phyco-Remediation Processes

Several researchers have reported that microalgae, when supplemented with low-quality effluent water, for example, municipal, industrial, and agrochemical industrial waters, serve as feed for growth and biomass production [187]. Cultivation of microalgae in wastewater proves to be beneficial in both ways as an alternative to conventional wastewater treatment strategies and the production of biomass for industrial applications [231,232]. Reported microalgal species used in the treatment of wastewater in raceway ponds and photobioreactors for the removal of nutrients, biomass productivity, and lipids are presented in

Table 6. Reported algal species used for the removal of nitrogen and phosphate for the production of lipids and biomass during the cultivation in raceway ponds and photobioreactors.

Algal Species	Mode of Cultivation	Light (µmoles/m2/s) and Photoperiod (Light/Dark Cycle) h	Biomass Productivity (g/L/d or g/m2/d or g/L)	HRT (Day)	C (%)	N (%)	P (%)	Reference
Microchloropsis salina (formerly Nannochloropsis salina) (Eustigmatophyceae)	Flat plate	150–600, 12:12	0.2	4.7	-	-	-	[233]
Scenedesmus obtusus (formerly Scenedesmus ovalternus)	Flat plate	1300, 24:0	25.0	-	-	-	-	[234]
Sphaeropleales	Tubular PBR	Sunlight	-	2–5	85.86 (COD)	-	-	[235]
Auxenochlorella protothecoides	Tubular PBR	138, 16:8	1.96	10	78.03 (COD)	100 (TN)	100 (TP)	[236]
Chlorella sorokiniana	Air lift PBR	Sun light	-	-	86.84 (COD)	100 (TN)	100 (TP)	[237]
Mixed algal bacterial culture	Column PBR	121, 12:12	-	0.5	95.5–96.7 (COD)	60.4–70.5) TN	93.2–96.4	[238]
Chlorella sp. FC2	Bubble column PBR	1130, 12:12	0.27–0.85	6–16	-	-	-	[239]
Chlorella vulgaris	Osmotic membrane PBR	46, 24:0	2.0	1–2	-	92–99 (AN)	100 (TP)	[239]
Chlorella vulgaris	Membrane PBR	46, 24:0	-	-	-	84–97 (AN)	28–47	[240]
Nitzschia palea, Nitzschia umbonata, Nitzschia amphibia (Bacillariophyceae)	Algal flowy (Culture media: UWW)	780–1147, 12:12	34.83	-	-	-	-	[169]
Mixed algal and bacterial culture	Trickling filter Culture media: SWW	15, 12:12	-	0.3–0.5	85	15	49	[112]
Benthic polyculture	Algal turf scrubber Culture media: BG11	32 watts; 24:0	3.5	-	-	5–25	31–70	[241]
Mixed algal bacterial culture	Algal turf scrubber (Concentrate wastewater)	88, 16:8	-	10	91 (TOC)	70 (TN)	85	[242]
Mixed culture	Algal turf scrubber (culture media: secondary wastewater)	6 watts, 24:0	-	-	72 (COD)	70 (TN)	44	[243]
Scenedesmus sp., Chroococcus sp., Closterium sp., diatoms, Chlorella sp. (Chlorophyta), and Oscillatoria sp. (Cyanobacteriophyta)	Biofilm carrier (culture media: SWW)	200, 24:0	-	0.5	90 (COD)	90 (AN)	30	[244]
Oscillatoria sp. (Cyanobacte-riophyta), Navicula sp., Nitzschia sp., Cyclotella sp. (Bacillariophyceae)	Algal turf scrubber (culture media: Secondary effluent)	-	24	-	-	40 (TN)	50 (TP)	[245]

PBR—photobioreactor; COD—chemical oxygen demand; TN—total nitrogen; P—phosphate; AN—ammonia nitrogen; SWW—synthetic wastewater; UWW—urban wastewater.

and

Table 7. Cultivation of algae in wastewater streams for algal productivity and nutrients removal in raceway ponds.

Wastewater Type	Volume (m3)	Depth	Surface Area	Algal Productivity	HRT (Days)	C (%)	N (%)	P (%)	Reference
MWW	0.47	0.3	1.5	20	4.5	65 (COD)	48 (TN)	25	[246]
DWW	9.54	0.3	31.8	2	5–8	-	31–92	32–76	[247]
DWW	8	0.3	31.8	9–16.7	4–8	95 (COD)	-	-	[248]
UWW	22	0.3	31.8	30–65	6–2.5	-	-	-	[248]
PSWW	0.43	0.25	2.082	-	2, 4, 6, 8,	-	64, 81.8, 85.38, 81.81 (TP)	51.1 62.26 77.81 75.78 (TP)	[249]
SHWW	0.075	0.18	0.43	12.7	10–15	84.91 COD	70–80 (AN)	57–90 (P)	[250]
MWW	0.06	0.3	-	0.5	2	85.44 COD	92. 74 (TN)	82.85 (TP)	[251]
DWW	0.180	0.15	1.33	5	10	77 (COD)	83 (TKN)	94 (TP)	[252]
DWW	-	0.32	200	-	5	91.75 (BOD)	-	-	[253]
MWW	4375	0.35	12,500	9.7	5.5–9	-	47–79 (AN)	20–49 (P)	[254]

COD—chemical oxygen demand; TN—total nitrogen; P—phosphate; AN—ammonia nitrogen; DWW—domestic wastewater; UWW—urban wastewater; MWW—municipal wastewater; PSWW—primary settled wastewater; SHWW—slaughterhouse wastewater; TKN—total Kjeldahl nitrogen; BOD—biochemical oxygen demand.

.

The treatment of metals and acidic wastewater, carbon dioxide sequestration, and heavy metal toxicity detection in wastewater are among specialized applications [255]. Microalgae species like Chlorella, Scenedesmus, Ankistrodesmus (Chlorophyta), and Euglena species (Euglenophyta) have already been explored for the treatment of wastewater generated from industrial sources like paper, mills, and olive oil [256,257,258]. Several researchers have studied the effect on biomass production from microalgae species grown in different wastewater generated from municipal and industrial sources and produced various highly valued metabolites from the generated biomass, such as lipids, fatty acids, carotenoids, and proteins [259,260,261,262].

Chlorella vulgaris FACHB-8 cultivated in anaerobically digested municipal wastewater produced 1.62 g L⁻¹ of biomass and 26.4% of lipids [263]. Adopting Chlorella sorokiniana MB-1M12 for the treatment of shrimp culture wastewater influenced the production of biomass at 3.5 g L⁻¹ biomass along with carotenoid production (lutein content of 3.89 mg g⁻¹) [264]. When dairy wastewater was used for the growth of Scenedesmus quadricauda, it resulted in a biomass production of 0.36–0.43 g L⁻¹ [265]. Growth and production of biomass from microalgae species using wastewater treatment systems have been proven to be advantageous. This process is economical and sustainable in every aspect. Cultivating microalgae in wastewater has been a promising approach in terms of addressing the cost of production, considering nutrient supply, and the need for clean water [266]. The use of municipal wastewater can be directly supplemented to microalgae for their growth, which is an added benefit to phycoremediation and the generation of biomass [232]. The microalgae grown in abundant nutrients can be processed into bio-fertilizers for crops (rice cultivation), as reported by Dinesh and Goswami [267]. Moreover, some microalgae have been found to fix nitrogen, which reduces costs in crop production, increasing yields and sustainability. Algal biomass is also considered to be an important energy source as it is a cheap and efficient carbon dioxide fixer. It can be converted to biochar using pyrolysis to form a carbon-rich product that improves the pH of acidic soils. It becomes relevant to use dry biomass processing for a substrate with more than 80% of water after harvest, as moisture content is beneficial to the pyrolysis process to produce hydrogen-rich pygas. The moisture content also promotes the reforming reaction and cracking of aromatic compounds [268].

Due to the presence of various functional groups in the biochar of algae, it can also be used as an adsorbent for wastewater remediation [269]. Thus, phycoremediation is essential in treating wastewater effluents so that a sustainable wastewater treatment system model can be achieved with dual benefits that contributes to the circular bioeconomy.

5. Omics Approaches in Wastewater Treatment

A wide range of omics techniques, including transcriptomics, proteomics, metabolomics, metagenomics, and genomics, can now be utilized to study the different algal processes. Although many algal genomes have also been sequenced, only a small number of species have resulted in whole-genome sequences. Understanding how protein expression may be upregulated or downregulated under various conditions that impact a particular product, as well as cell survival, has been made easier by proteomics [270]. Microscopy, cell sorting, mass spectrometry, and genetic engineering are some of the conventional techniques used to investigate consortia interactions in various biotechnological applications; however, the introduction of omics tools has led to a more comprehensive and clear understanding of their relationships [271].

To move beyond descriptive analysis, this review proposes a conceptual framework for integrating omics tools into microalgal bioremediation systems. This framework emphasizes how transcriptomics, proteomics, and metabolomics can be strategically applied to optimize pollutant removal, guide species selection, and enhance stress tolerance. Omics approaches combined with microbiological and biochemical analyses provide reliable information about the impact of environmental factors on genes, transcriptomes, metabolites, protein expression, and their respective regulation. This is because co-culture strains have better genetic and metabolic capabilities than individual representative strains because of their establishment of symbiotic association. These biological mechanisms—adsorption, uptake, conversion, and stress response—are increasingly being studied through omics approaches. Transcriptomics reveals gene expression changes under metal stress, while proteomics identifies key enzymes involved in detoxification. Metabolomics helps trace metabolic shifts and the accumulation of protective compounds. Together, these tools provide a system-level understanding of microalgal bioremediation. Integrated omics approaches allow researchers to map the molecular interactions between microalgae and pollutants. Proteomic studies can identify stress-responsive proteins, while metabolomics can track the accumulation of detoxifying metabolites. These insights deepen our understanding of how microalgae adapt and mitigate environmental stressors. By combining transcriptomic and metabolomic data, researchers can pinpoint key genes and metabolic pathways involved in pollutant uptake, such as those regulating antioxidant defense, metal chelation, and nutrient assimilation. This system-level view supports the rational design of microalgal consortia and bioreactors for enhanced remediation [272,273].

Algae are becoming increasingly beneficial options for bioremediation, particularly in wastewater treatment and their development as microbial cell factories [274,275]. The phenotypic and genotypic characteristics that contribute to superior microalgal growth and nutrient removal via omics technologies in wastewater can be identified by combining microalgal screening with molecular phylogeny [276]. In addition to identifying individual genes and gene arrangements, genome analysis is the most advanced method for obtaining sequence information about microalgal populations. It also facilitates comparisons between genomes in terms of gene arrangement and sequence similarity [277]. Genomic research has advanced during the last few years from sequencing individual genomes to analyzing metagenomic (culture-independent molecular technique) information. As part of a study at the Amherstview Water Pollution Control Plant (WPCP), researchers used metagenomics sequencing to analyze the distributions and profiles of algal species in three wastewater stabilization ponds in order to determine the relative abundances of microalgae [278].

According to another metagenomic sequencing study, the bioaugmentation of Phanerochaete chrysosporium (fungi) into phenol wastewater enhanced reactor performance by changing the structure of the microbial population and reducing its toxicity [279]. The up- and downregulated proteins in the ammonia oxidation pathway of bacteria and algae in wastewater under stress have been identified using proteomic techniques [280]. In order to optimize the production of particular products, metabolomics research entails both qualitative and quantitative examination of metabolites in microalgae metabolic pathways [281]. Today, a lot of research on microalgae wastewater employs metabolomic techniques. Lastly, transcriptomics is also a vital molecular technique that helps in the research of gene expression and structure, as well as an improved understanding of genomic regulation. The use of transcriptomics tools has helped in the analysis of various cyanobacterial species and the observation of how they react to various environmental factors [269]. Omics data can be used to identify microalgal strains with superior pollutant uptake capabilities. For instance, transcriptomic profiling under heavy metal stress can reveal upregulated genes involved in metal transport or detoxification. These insights can guide the selection or genetic enhancement of strains for targeted applications. While genomic research on single microalgae utilized in wastewater treatment is still very limited, the metagenomics of microalgae has been examined with more emphasis on genes involved in nitrogen metabolism. When exogenous microalgae Chlorella were introduced to the native microbiota of a swine lagoon wastewater, a metagenomics analysis using high-throughput sequencing via the “Illumina Miseq” platform revealed maximal algal growth along with simultaneous optimal N and P removal in a CO₂ air bubbling phycoremediation mode [282].

When C. reinhardtii was cultivated in a wastewater containing nitrate and ammonia that was suitable for outdoor wastewater treatment, label-free shotgun proteomics using Multi-dimensional Protein Identification Technology (MudPIT) identified 2358 proteins with abundances of enzymes and proteins related to nitrogen metabolism and assimilation, synthesis and utilization of carbohydrates, recycling of amino acids, evidence of oxidative stress, and little in terms of lipid biosynthesis [283]. The enzymes of the enriched pathways were found to be promising targets for genetic improvement, and their subsequent metabolic dissection may contribute to a better understanding of microalgal growth and nutrient removal. This study also helped shed light on the microalgal response to nitrogen sources (ammonia/nitrate).

While proteomics and metabolomics have also been used to identify the enzymes responsible for biocatalysis and to reveal microalgal responses to pollutant-induced stresses, transcriptome analysis of Picochlorum SENEW3 revealed that half of the coding regions were differentially expressed in response to a pharmaceutical contaminant in wastewater [102]. Experiments showed that mero-cyclophane C was significantly produced at low phosphate, whereas tolytoxin, a possibly new peptide, was produced at higher phosphate levels. These kinds of metabolomic investigations will soon help in improving wastewater resource recovery techniques.

Currently, omics approaches of microalgae–bacteria consortia (co-culture) are being thoroughly researched because the efficiency of photosynthetic microalgae for wastewater treatment is primarily dependent on the associated bacteria present in the effluents [284]. Co-culture involvement in efficient nutrient removal from wastewater was studied with the use of functional genomics [279]. Therefore, research showing multi-omics data along with NGS platforms shed light on the mechanism and interaction of natural assemblages while producing unbiased results. According to a metabolomics study using ultra performance liquid chromatography (UPLC) in conjunction with quadrupole time of flight (QTOF), by improving metabolic capabilities like enhanced lipid accumulation, glucose uptake, and wastewater treatment, bacterial cofactors like thiamin pyrophosphate and 4-amino-5-hydroxymethyl-2-methylpyramidine helped the microalgal partner Auxenochlorella protothecoides in the consortia produce more biomass [285]. While bacterial genome data indicated its role in stimulating the growth of microalgae, metagenome analysis exploited algal–bacterial consortia during simultaneous biohydrogen generation and wastewater treatment, which provided crucial hints for the primary importance of the green algae partner in nitrogen and phosphorus removal [284]. The combined application of metabolomics, metagenomics, transcriptomics, and other conventional techniques, such as biochemical and microbial analysis, indicates that omics in bacterial–algal interactions would be very beneficial in comprehending and elucidating some fundamental questions regarding their association in wastewater treatment.

A combination of omics approaches has been used, such as proteomics, transcriptomics, and metabolomics, for the effective removal of metal contaminants from algae. Besides revealing the post-translational changes of proteins, proteomics analysis tells the exact location of proteins present in the cells [286]. Another study indicates that using a comparative 2-DE proteomics approach resulted in the identification of different pathways involved with respect to copper stress in Sargassum fusiforme (Phaeophyceae) [287]. On the other hand, the proteomic analysis helped in identifying the important metabolic enzymes that are downregulated during cadmium stress [288]. Metallomics is beneficial in terms of providing results at the molecular level and helps to understand the mechanisms involved in the toxicity of metals. Multi-metal systems (Cu, Cd, Pb) were applied on Chlorella sp., and results were evaluated using a combined approach of metallomics and nuclear magnetic resonance spectroscopy (NMR) to understand the possible relation between heavy metals and the fate of multi-metal systems in C. vulgaris [289,290]. Furthermore, the combined approach of transcriptomic (cDNA microarray) and metabolomic studies using particularly gas chromatography–mass spectrometry (GC–MS) and NMR spectroscopy revealed the effect of Cd exposure on C. reinhardtii. The transcriptome approach showed that genes for the defense mechanism to Cd tolerance, mainly related to oxidative stress, are upregulated. Furthermore, the metabolomics strategy led to elucidating the effect of Cd exposure on the algal metabolites involved in the glutathione pathway [291]. On the other hand, Cd uptake and effects of a carboxylate-terminated CdTe/CdS quantum dots (QD) have been evaluated in C. reinhardtii. Moreover, whole-transcriptome analysis using RNA-Seq analysis indicates that free Cd and QD had different biological effects [292]. The omics approach has been considered as an alternative to studying the adverse effects of metal toxicity better than the traditional approaches. Despite their limitations, such approaches have proven effective for both biomass treatment and the treatment of wastewater. While conventional and biological methods have shown promising results in nutrient removal, recent advancements in omics technologies offer deeper insight into the molecular mechanisms underlying these processes.

It has become the most promising tool in systems biology and in finding sustainable solutions through biotechnological interventions. Techniques involved in omics have been used to examine an organism that is involved in treating wastewater thoroughly. The techniques that are used in the omics approaches are shown in Figure 1. Metabolomics, genomics, and transcriptomics are techniques that have the advantage of rendering the processes for throughput analyses and provide the relevant information regarding the microbes involved in the process of treating, their mechanism, and the capability to produce various metabolites [293]. Advanced omics methods like transcriptomics, proteomics, metabolomics, interactomics, fluxomics, etc., are being adopted to overcome the conventional methods for the removal of metals [294]. Owing to the above-mentioned advantages, the omics approaches in algal systems are being employed in wastewater treatment to enhance the efficiency of the process. The literature showed that the genomic analysis of a unialgal strain of microalgae used in wastewater treatment is still very rare, whereas metagenomics of microalgae has been explored with a focus on genes involved in nitrogen metabolism. Metagenome analysis exploited algae–bacteria consortia during wastewater treatment that brought significant clues in nitrogen and phosphorus removal, while bacterial genome data revealed its role in growth elevation of microalgae [284]. The microalgae–bacteria consortium proliferation is more intense under the symbiotic relationship of bacteria and algae. Algae and bacterial consortia exist in the form of commensalism, mutualism, and parasitism. The study suggests that the algae are known to produce independent enzymes for vitamin B₁₂ and dependent methionine synthases, whereas, on the other hand, bacteria are able to deliver vitamin B₁₂ [295]. Moreover, microalgae are known to provide nutrients such as carbohydrates, proteins, and lipids for bacteria, whereas cell-free extract-containing metabolites, such as enzymes and hormones produced by bacteria, are vital for algal growth. The structural stability of microalgae–bacteria consortia is solely dependent on the exopolysaccharides for ecological function [296]. Numerous functional groups of lipids and proteins in the structural constituents of EPS are known to adsorb the organic pollutants [297]. Microalgae can remove organic pollutants by algal remediation, which includes biosorption and bioaccumulation. Bioaccumulation of organic pollutants functions to transfer organic compounds inside algal cells. Furthermore, bioaccumulation occurs when organic pollutants enter the cell membranes without changing the cell structure [298]. Organic pollutants may bind to the proteins, which may increase the antioxidant activity [299]. The microalgae sustainability based on wastewater treatments can be effectively obtained through algomics approaches to improve the bioremediation. Since sustainable bioremediation is based on microalgae bacterial symbiosis, further insights into omics approaches are needed to improve microbial compositions, genes, and metabolites [300].

Metagenomics is an approach that can investigate metabolic activities of microorganisms by DNA sequencing techniques [301]. Metagenomics analysis revealed that N and P were effectively removed from wastewater through phycoremediation using the algae Chlorella sp. Next-generation sequencing methods of paper wastewater treatments showed that total suspended solids led to the growth of microalgae. Metagenomic sequencing revealed that augmentation of Phanerochaete chrysosporium (Fungi) into wastewater reduced the microbial community and toxicity [279]. Kadri et al. [302] identified the purple photosynthetic bacteria in piggery wastewater treatment via 16 s rDNA of algal–bacterial consortia.

The proteomics approach is used to study the variation in proteins in the microalgae bacterial consortium [303]. Fan et al. [304] implemented transcriptomic approaches to study the effect of sulfamethoxazole on microalgae bacterial consortia in wastewater treatment. The scientist found that genes such as photosystem II, Cytochrome b6/f complex, and photosystem I. Mainly the PsbB, PsbJ, PsbE, and PsbF genes involved in photosystem II were upregulated, which are responsible for producing chlorophyll apolipoprotein. On the other hand, PsaA and PsaB genes in photosystem 1, which are involved in the generation of chlorophyll apolipoprotein, were also up-regulated. Shen et al. [305] employed proteomic approaches to study the impact of bacteria on the algae carbon utilization in wastewater and found the differential expression of 262 proteins with upregulation and downregulation of 82 and 180 proteins, respectively. Figure 1 shows that multi-omics approaches provide a vital step in supporting the application of microalgae–bacteria consortia for organic pollutant remediation in wastewater. Some of the algal strains exploited through the omics approach are shown in

Table 8. Omics approaches wastewater treatment by algal species.

Algae	Aim of the Study	Source of Wastewater (WW)	Omics Approach	References
C. vulgaris, B. licheniformis	Removal of contaminants	Municipal	16s RNA Illumina MiSeq high-throughput sequencing	[173]
Pseudanabena (Cyanophyceae), Chlamydomonas, Nitrospira, and Nitrosomonas (Bacteria)	Biomass production with nutrient recovery	Domestic	16s RNA Illumina MiSeq high-throughput sequencing	[306]
Algal-activated system	C, P, and N removal	Municipal and activated sludge	PCR and denaturing gradient gel electrophoresis	[307]
Auxenochlorella protothecoides	Biomass production and effect of bacterial consortia on WW treatment	Wastewater treatment plant	UPLC coupled with quadrupole time of light	[285]
P. chrysosporium	Fungi augmentation of WW treatment	Phenol	Metagenomic sequencing	[279]
Desmodesmus sp. MAS1 Heterochlorella sp. MAS3	Fe (40–80%) and Mn (40–60%) heavy metal removal	Heavy metal-contaminated WW	NR	[308]
C. vulgaris F1068	Ammonia uptake	Municipal	Isotope fractionation	[309]

C—carbon; P—phosphorus; N—nitrogen; Fe—iron; Mn—manganese; WW—wastewater; PCR—polymerase chain reaction; UPLC—ultra-performance liquid chromatography.

. Omics techniques, like 16S rRNA sequencing, enhance system performance by combining information from multiple biological levels (genomics, transcriptomics, proteomics, metabolomics, and lipidomics) to discover biomarkers, forecast responses in different systems, such as the human microbiome, and obtain a comprehensive understanding of biological processes. Furthermore, by connecting microbial identity to function, the combination of proteomics and 16S data helps validate the expression of particular microbial proteins. Understanding how microbes affect or react to metabolite changes is made easier by the use of 16S sequencing in the context of metabolomics, which links microbial communities to particular metabolic profiles or shifts (Figure 1).

Numerous genetic components and engineering tools, including promoters, gene vectors, selection markers, and gene editors, are now available for altering metabolic pathways in microalgae due to ongoing advancements in metabolic engineering and synthetic biology [36]. Algae can be genetically altered to improve their effectiveness in bioremediation procedures by overexpressing important enzymes involved in the breakdown of contaminants. These enzymes are essential for metabolism and the breakdown of particular pollutants. The algae can efficiently target and break down environmental pollutants by upregulating the expression of these enzymes through genetic engineering [310,311]. Model species such as Chlamydomonas reinhardtii, Phaeodactylum tricornutum, and Nannochloropsis gaditana have become focal points for functional genomics studies due to their tractable genetics and comprehensive omics resources. With the use of specific genetic nucleases tools like CRISPR-associated protein 9 (Cas9) and transcription activator-like effector nucleases (TALENs), the choice for microalgae genetic engineering has become easier [312,313]. However, because of its simplicity and versatility in comparison to TALENs, the CRISPRCas system has emerged as the go-to option for genome editing in microalgae [314,315]. To illustrate the interconnectedness of stress tolerance, energy metabolism, and detoxification at the systems level, transcriptomic and metabolomics profiling of Euglena gracilis under mercury stress showed the coordinated upregulation of antioxidant pathways (e.g., glutathione production), DNA repair mechanisms, and TCA cycle components [316]. Comparably, integrated transcriptomic and metabolomic analyses of Chlorella pyrenoidosa exposed to silver nanoparticles revealed changes in the metabolism of carbohydrates and amino acids, involving important enzymes like citrate synthase (CS), isocitrate dehydrogenase (IDH1), and malate dehydrogenase (MDH), signaling a complex reprogramming of carbon–nitrogen metabolism under nanoparticle stress [317].

When exposed to cadmium, lead, and copper, transcriptome analyses have shown a coordinated transcriptional response involving these genes, especially in Chlamydomonas reinhardtii and Tetradesmus obliquus. The accumulation of metal-binding proteins and stress-related enzymes like catalase (CAT) and superoxide dismutase (SOD) under pollutant stress is further confirmed by proteomic profiling, suggesting a tightly controlled antioxidant defense system. Furthermore, transcriptomic analysis revealed that cadmium exposure increased the expression of transporter families, glutathione-related genes, and metallothioneins in the diatom Tetratostichococcus sp. P1. With an IC₅₀ of approximately 57 µM and an impressive Cd uptake capacity (up to 1.55 mg g⁻¹ dry weight), this strain is a promising candidate for heavy metal bioremediation [318]. Another study by Khatiwada et al. [319] revealed that Euglena gracilis under heavy metal stress also showed higher levels of major facilitator superfamily (MFS) transporters, thiol-rich metal-binding proteins, and metal-transporting ATPases (P1B ATPases), which are important molecular actors that could be used to engineer improved metal uptake capabilities. This, in turn, informs genetic enhancement strategies, such as overexpressing MFS transporters, P1B ATPases, or enzymes in phytochelatin synthesis to improve performance.

Algal transcriptome research in response to various pollutant stresses is a widely used method to identify molecular changes precisely and sensitively evaluate biological risks that may improve microalgae’s capacity for bioremediation [320,321,322]

6. Bio-Refinery Approach from the Downstream Processing of Biomass to Produce Multiple Products

Microalgal metabolites are considered commercially important due to their potential health benefits [158]. Microalgae produce secondary metabolites and show various biological activities such as antioxidant, anti-cancer, anti-infective, and photo-protective activities by adopting different metabolic pathways [323]. Microalgae not only contain a high amount of protein but also contain carbohydrates and oils, which can be the main substrates for the production of omega- 3 fatty acids and as animal feeds for cattle, poultry, shellfish, and fish. In addition, micronutrients present in biomass are used as food supplements, such as oil derivatives and in desserts, dairy items, and pasta [324]. Microalgae are potential sources of naturally active molecules such as chlorophylls, polyunsaturated fatty acids, essential amino acids (e.g., valine, leucine, and isoleucine), carotenoids (e.g., astaxanthin, lutein, and β-carotene), phycobiliprotein pigments, vitamins (e.g., B₁₂), enzymes, etc. [325]. Furthermore, metabolomics examines the qualitative and quantitative estimation of metabolites present in microalgal cells with enhanced production of specified products. Thus, the optimization of metabolic pathways may lead to an increase in flux rates towards desired metabolites and increased tolerance or mitigation of contaminant compounds [75]. In recent years, genetic approaches have significantly enhanced biomass accumulation for improved metabolite production and overproduction of triacylglycerols (TAGs) [326]. A clear understanding of the metabolic pathways and development in genetic engineering is very important for the fruitful exploitation of algal biomass for the synthesis of value-added products. Various genetic approaches, such as genome sequencing, selection of markers, zinc-finger nucleases (ZFN), and clustered regularly inter-spaced short palindromic repeats (CRISPR), have been identified as useful tools in the regulation of the metabolic pathways in microalgae.

Reports suggest that microalgae C. reinhardtii can be made to produce numerous novel secondary proteins like human growth hormone, autoantigens, anti-bacterial enzymes, and cancer therapeutics in the chloroplast [327]. Furthermore, in C. reinhardtii, the amalgamation of transcriptomics and lipidomics led to the identification of major enzymes (phospholipase A2 homolog and DAG acyltransferase DGTT1) in the triacylglycerols (TAGs) synthesis pathway [328].

Moreover, the proteomics approach helps in the identification of post-translational changes, such as protein synthesis and enhanced production of TAGs under varying environmental stress factors [329]. Thus, the scientific understanding of the structural, biochemical, and biophysical characteristics of enzymes is important to further stimulate the biosynthetic pathways. In addition, the connection between genetics, protein modifications, the folding pattern of proteins, and toxic effects is important to examine in the pharmaceutical and therapeutic applications of microalgae. Moreover, molecular tools such as reverse transcription polymerase chain reactions aid in the actual assessment of uptake rates of carbohydrates and polysaccharides for maximizing cosmeceutical biosynthesis [330].

While the system proves to enhance growth and biomass production, it also treats wastewater [206]. The biomass that has been produced from microalgae grown in wastewater proves to be valuable; for example, the production of pigments [331] and fatty acids [332] has been reported in several studies from the resultant biomass.

In one such study where Chlorella vulgaris and Limnospira platensis were cultivated in tofu production wastewater, they were reported to produce 1.16 mg L⁻¹ of carotenoid content and 72.20 mg L⁻¹ of carotenoid when supplemented with 5% and 10% of the tofu wastewater, respectively [293]. Another study accounted for the production of lutein (0.76 mg g⁻¹), β-carotene (70.22 mg g⁻¹), and violaxanthin (1.40 mg g⁻¹) among the 20 important carotenoids produced by Microcoleus autumnalis (formerly Phormidium autumnale) (cyanobacteria) cultivated in wastewater generated from the slaughterhouse [294]. A microalga not only produces biomass and pigments when grown in wastewater, but the production of essential fatty acids like docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) is also reported. Another study demonstrated the production of DHA (1.7%) with 6.1 µg.mg⁻¹ of carotenoids and 2400 mg L⁻¹ of biomass production in Auxenochlorella pyrenoidosa when cultivated in olive oil mill effluent [333]. The source of wastewater supplemented with microalgae has an important role in producing high-value products, as the characteristics of wastewater determine which metal nutrients are present in abundance, which is beneficial for microalgae growth, biomass, and metabolites production, along with treating the effluents, as the higher the amount of essential nutrients available in wastewater, the more it is utilized by microalgae. In addition to technical feasibility, regulatory and safety considerations are critical for biomass applications in food and feed. Wastewater from food industries such as palm oil mill effluent (POME), molasses, cheese whey, and brewery effluent offer nutrient-rich media for cultivating microalgae. These sources, when properly treated, can support the production of functional compounds that meet food safety standards. Incorporating such substrates enhances the sustainability and economic viability of microalgal biorefineries. Figure 2 shows the bio-refinery approach (processing inputs, cultivation, extraction, and drying) for the processing of biomass into high-value compounds used in animal feed, biodiesel, and biogas applications.

To protect consumers and gain market acceptance, the use of microalgae in biorefinery systems for human and animal consumption poses serious safety and regulatory issues that need to be resolved. The European Union enforces stringent guidelines under the Novel Foods Regulation (EU) 2015/2283, which requires safety evaluations for microalgal species not commonly consumed prior to 1997. Regulatory frameworks differ by region. Comparably, the Food and Drug Administration (FDA) in the US mandates that products made from microalgae achieve Generally Recognized as Safe (GRAS) status, especially for species like Schizochytrium, Chlorella, and Spirulina.

The possible buildup of heavy metals, persistent organic pollutants, and cyanotoxins, particularly in open cultivation systems, poses a serious safety risk for both human and animal applications [334]. The European Food Safety Authority (EFSA) and the Association of American Feed Control Officials (AAFCO) are two important regulatory bodies that usually require proof of nutritional value, safety information, and the absence of contaminants in order to approve animal feed applications [335].

Large amounts of nutrient-rich wastewater are produced by the food industry and, if left untreated, can cause an environmental burden. The use of wastewater also offers a great chance to be used as an inexpensive growth medium for algal and microbial bioprocesses. In recent times, the utilization of food industry-based wastewater as alternative growth media has emerged as a sustainable and economically viable strategy for the production of high-value functional compounds, including organic acids, bioactive peptides, enzymes, pigments, vitamins, and microbial biomass with nutraceutical potential. Food industry wastewater products, including molasses, cheese whey, brewery effluent, palm oil mill effluent (POME), cassava processing wastewater (CPW), aquaculture wastewater (AqW), and dairy wastewater (DW), are excellent substrates for microbial cultivation because they contain trace elements and a lot of carbon and nitrogen are produced as byproducts. The increasing demand for dairy products has led to the growth of numerous dairy industries, which produce wastewater with a high potential for pollution [336]. The study by Bentahar et al. [337] showed that acid whey permeate was found to be the best compensation for biomass growth, enzyme production, and nutrient utilization in a Tetradesmus obliquus culture when 20% (v/v) of Bold’s basal medium (BBM) was substituted. On the other hand, slaughter wastewater was found useful for growing Porphyrium cruentium microalgae with high levels of phycoerythrin, according to Balaraman et al. [338]. Furthermore, a study by Fernando et al. [339] showed high accumulation of astaxanthin in Haematococcus lacustris and Chromochloris zofingiensis when cultivated in palm oil mill effluent (POME). Another study by Ma et al. [340] revealed the accumulation of lipids up to 28.9% in Scenedesmus sp. Research conducted by Tsotsouli et al. [341] showed that cultivating Dunaliella tertiolecta in cheese whey (CW) effluents led to a 30% increase in exopolysaccharides (EPS) production and 8 times higher biomass productivity. Moreover, the fucoxanthin yield was enhanced up to 3.64 mg/L when cultivated using a mixture of 33 mL/L CW, with specific BM and CSL [342]. Moreover, cultivation of Chlorella sorokiniana in urea-supplemented POME increased the chlorophyll content to 1.59 mg/g dry cell and the biomass (dry cell weight, DCW) to 1.07 g/L [343]. According to the report of Putri et al. [344], cultivated Spirulina in 15% POME yielded 4.67 g/L biomass, whereas Nannochloropsis oculata with 20% POME yielded 4.43 g/L biomass.

7. Challenges and Issues in Phycoremediation

The real challenge is to pre-treat the wastewater, developing a consortium of algae or an algae–microbe combination; separating the biomass, valorizing the biomass, and addressing issues regarding the biorefinery approach to separate the valuable constituents. Several groups of researchers have investigated the life cycle assessment of microalgae production to disclose its economic, energy, social, and environmental impacts and challenges faced throughout the whole process from growth to production [345]. The key challenges that are faced in the treatment of wastewater using microalgae are the presence of turbidity and total solids. The light penetration needed for the growth of microalgae is affected due to high total solids and turbidity in untreated water. High organic carbon content also limits the growth of microalgae and supports other microbial communities, which ultimately hinder the growth rate of microalgae.

It is well reported in studies that microalgae species can remove nutrients in wastewater effectively, but some species are inefficient [130]. Thus, it becomes important to select and screen the most suitable microalgae strains capable of removing nutrients from wastewater. Harvesting microalgae is also one of the challenges faced in integrated microalgae wastewater treatment, due to the small size of cells and the low biomass concentration. The separation of biomass from water accounts for 30% of the total microalgae biomass production cost [346]. Many issues are faced in developing the omics techniques that can be utilized in the upstream and downstream strategies with coupled wastewater treatment, to which researchers are finding solutions.

8. Conclusions and Future Perspectives

The treatment of wastewater using microalgae has proved to remove nutrients in an effective way and is an alternative technique that is both economical and sustainable. Wastewater supplemented with algae can produce high-value biomass and metabolites with health benefits to humans as well as animals in the form of feed, biofuels, and nutraceuticals [14]. Coupling wastewater treatment with the production of microalgae biomass results in overall cost reduction. Further investigations are needed for this approach, combining omics technology for wastewater treatment using microalgae strains with reproducibility capabilities. Due to the presence of heavy metals in the environment, more reactive oxygen species are generated, leading to cell structure damage and nutritional imbalance in the biotic components with profound metabolic disturbances. These toxic metals are known to cause up- and downregulation of chaperones, which can eventually affect protein metabolism. Besides nitrogenous compounds, sulfur-containing compounds play an important role in metal sequestration [347], wherein metallothioneins bind to heavy metals, mainly Zn, Cu, and Cd, and function as chaperones, providing essential ions to metal-dependent enzymes [348]. In order to provide effective solutions, the research should be focused more on developing genetic tools such as genomics and transcriptomics, as well as metabolomics, metallomics, and metallo-proteomics. The omics approaches are helping scientists to obtain in-depth knowledge of heavy metal stress response at next-generation sequencing platforms. Algal strains should be engineered well according to the required product and should possess important traits of producing high yields of biomass, lipids, fatty acids, carotenoids, etc. Incorporating omics approaches and bioinformatics tools with databases will play a key role in achieving economically feasible microalgae production of bio-commodities. Extensive research in the development of molecular and biochemical tools is needed, which facilitates microalgae-based treatment of wastewater as well as the production of metabolites and bioactive compounds of great interest. Future research should focus on integrating multi-omics datasets with computational modeling to predict microalgal behavior under complex pollutant mixtures. Such predictive tools will be instrumental in developing robust, scalable, and adaptive bioremediation systems.