The Potential of Microalgae in Phycoremediation

Abstract

Heavy metals (HMs) and polycyclic aromatic hydrocarbons (PAHs) can have negative impacts on the marine and freshwater biota. Utilizing microbes, particularly microalgae, which are able to extract metals and hydrocarbons from environmental matrices, the impact of these contaminants in freshwaters, seas, and wastewaters can be reduced. In fact, the contaminants can be passively absorbed and actively accumulated within the organelles of microalgae, reducing their negative impact. River streams, untreated municipal, agricultural, and industrial wastewaters, represent one of the most important issues for the anthropogenic pollution. Microalgae and bacteria can grow in polluted waters containing different metals as cadmium (Cd), lead (Pb), zinc (Zn), chromium (Cr), copper (Cu), and mercury (Hg). They are able to accumulate metal elements within biomass in a dose-dependent manner and are employed in bioremediation thanks to their growth ability in polluted sites. In the following chapter, we analyze the potential of employing microalgae in phycoremediation, their mechanisms of tolerance and resistance to heavy metals, the possibility to use consortia of microorganisms instead of single specie to cope with complex polluted matrices, the possibility to metabolic engineer microalgae to gain their function, and the employment of microalgae in both liquid and solid devices.

1. Introduction

1.1 Origin of pollution

Anthropogenic pollution is affecting marine environments worldwide, posing serious issues to the sustainability of current economic activities. Mining, intensive agriculture, metallurgical, tanning, and canning industries have led to significant contamination of terrestrial and marine ecosystems with toxic metals and organic pollutants such as petroleum hydrocarbons and other emerging contaminants.

The release of pollutants, such as polycyclic aromatic hydrocarbons (PAHs), heavy metals, pesticides, and plastic waste, has resulted in climate change, habitats’ destruction, and loss of biodiversity. Anthropogenic impacts have disrupted ecosystems, altered natural processes, and posed significant threats to human health and the well-being of the ecosystems. Among different classes of pollutants, we will address, in this chapter, two particular classes, whose impacts are crucial for the sustainability of our planet and future generations, e.g., heavy metals (HMs) and polycyclic aromatic hydrocarbons (PAHs). Heavy metals, including lead (Pb), mercury (Hg), cadmium (Cd), arsenic (As), and chromium (Cr), are primarily derived from industrial activities, such as mining, metal smelting, and manufacturing processes. These metals can enter in the ecosystems through atmospheric deposition, leaching from mining sites, and industrial wastes [1].

Additionally, agricultural practices, such as the use of fertilizers and pesticides, can contribute to heavy metal contamination in soil and water systems. Polycyclic aromatic hydrocarbons (PAHs) are a group of organic compounds that are formed during the incomplete combustion of organic materials, such as fossil fuels, wood, and tobacco. Major sources of PAHs include vehicle emissions, industrial combustion processes, and residential heating using solid fuels. Activities, such as oil spills, industrial discharges, and improper waste disposal, also contribute to PAH contamination in aquatic environments. Both HMs and PAHs are persistent pollutants that can bioaccumulate in organisms through a process called biomagnification that affects species tropic chain at different levels till arriving to human beings [2]. Effective management strategies, including pollution prevention, proper waste management, and the implementation of strict regulations, are necessary to mitigate the sources and impacts of HMs and PAHs on the environment.

Bioremediation, i.e., the use of living organisms to remove or neutralize pollutants from the environment, has emerged as a promising and sustainable approach to cope with different pollutants. Bringing novel pollution-tolerant microbes in culture and documenting their ability to degrade or uptake selected pollutants in the laboratory are likely to facilitate bioremediation research by providing novel species and information on their tolerance/resistance thresholds. Among the diverse microorganisms used for bioremediation, bacteria and microalgae have gained significant attention due to their unique capabilities to mitigate pollution while offering additional benefits. Several microbe-based approaches to promote hydrocarbons’ degradation and metals’ removal are currently under investigation.

In this chapter, we explore the mechanisms by which microorganisms and microalgae, in particular, contribute to bioremediation process.

1.2 Why microorganisms can be useful?

The use of microorganisms in the bioremediation of heavy metals (HMs) offers several advantages over traditional remediation methods. First, microorganisms possess the ability to react with heavy metals and transform them into less toxic forms through various metabolic processes. This natural ability of microorganisms can avoid the recurrence of costly chemical treatments. Second, microorganisms can thrive in different environments, including contaminated sites with extreme pH, temperature, light, and salinity conditions. Their adaptability makes them able to survive in challenging environments where chemical-physical remediation methods may be less effective or unfeasible.

Furthermore, microorganisms have the capability to bioaccumulate heavy metals within their biomass. This property makes them suitable for the removal and recovery of heavy metals from contaminated waters or soils. The metal-laden microorganisms can be harvested and further processed, allowing for the potential extraction and recycling of valuable metals. In addition, the use of microorganisms in heavy metals remediation is generally considered environmentally friendly and sustainable. Microbial processes occur naturally and do not produce harmful byproducts or contribute to secondary pollution. The use of microorganisms in remediation can also be combined with other eco-friendly techniques, such as phytoremediation, where plants and microorganisms co-work to remove contaminants.

Lastly, microorganisms can be engineered or selected for specific heavy metal tolerance or remediation capabilities, enhancing their efficiency in targeted remediation processes. Genetic engineering and microbial consortia approaches can be employed to optimize the remediation potential of microorganisms and tailor them to specific contamination scenarios. Overall, the use of microorganisms in heavy metals remediation offers a cost-effective, adaptable, sustainable, and environment-friendly approach that holds significant promise for addressing heavy metals pollution and restoring contaminated environments [3]. Below, we present the peculiar processes of photosynthetic eukaryotes, such as microalgae, which are small photosynthetic organisms either prokaryotic (cyanobacteria) or eukaryotic and which can live in all types of water and use carbon dioxide (CO2) and direct light as their only sources of energy and carbon to make organic chemicals [4]. Microalgae are able to cope with the presence of HMs and PAHs in the environments, making them promising microorganisms to be investigated for future applications in the phycoremediation field:

1. Photosynthetic activity: microalgae harness the power of photosynthesis, a process that utilizes light energy to convert carbon dioxide into organic matter, thereby reducing greenhouse gas emissions. This photosynthetic activity also plays a pivotal role in bioremediation. Microalgae absorb pollutants, such as heavy metals and organic contaminants, from their surroundings. The energy acquired through photosynthesis allows them to metabolize and break down these pollutants into less toxic forms, effectively removing them from the environment.

2. Nutrient uptake and eutrophication control: excessive nutrient runoff, particularly nitrogen and phosphorus, leads to eutrophication, a phenomenon causing harmful algal blooms (HABs) and oxygen depletion in aquatic ecosystems. Microalgae can help combat eutrophication by efficiently assimilating and incorporating these nutrients into their biomass. By doing so, they compete with harmful algal species for resources, limiting their growth and mitigating the negative impacts of eutrophication.

3. Biosorption and bioaccumulation: microalgae possess the ability to accumulate and sequester various pollutants through biosorption and bioaccumulation processes. Biosorption involves the passive binding of contaminants onto the surface of microalgal cells or extracellular polymeric substances (EPS) produced by microalgae. Through this mechanism, microalgae can remove heavy metals, organic pollutants, and even radioactive substances from the environment.

   Bioaccumulation, on the other hand, involves the active uptake and concentration of contaminants within the cellular structures of microalgae. These contaminants can be transformed, stored, or further metabolized within the cells. Microalgae, with high bioaccumulation potential, can be employed in bioremediation strategies to reduce the concentration of pollutants in contaminated areas.

4. Enzymatic degradation: microalgae possess a diverse range of enzymes that play a crucial role in the degradation and detoxification of pollutants. For example, certain microalgae produce extracellular enzymes like lipases, proteases, and peroxidases that can break down complex organic compounds, including petroleum hydrocarbons. These enzymatic activities enhance the bioremediation efficiency of microalgae by facilitating the degradation of recalcitrant pollutants into less harmful byproducts.

5. pH adjustment and oxygen production: microalgae can influence the pH of their surroundings through their metabolic activities. They can remove excess carbon dioxide from the environment during photosynthesis, leading to a rise in pH. This ability to regulate pH is particularly useful in bioremediation processes, as certain pollutants are more soluble and easily removable under specific pH conditions. Additionally, microalgae are oxygenic organisms, releasing oxygen as a byproduct of photosynthesis. The oxygen released by microalgae enhances the aerobic conditions necessary for the activity of other microorganisms involved in the bioremediation process.

1.3 Chemical versus biological remediation processes

Classical chemical remediation processes are employed to mitigate environmental contamination caused by various pollutants. These processes involve the use of chemical agents or reactions to transform or remove contaminants from soil, water, or air. Heavy metals have been removed using a variety of standard techniques, including adsorption, chemical precipitation (carbonate, hydroxide, and sulfide precipitation), chemical oxidation, chemical reduction, solvent extraction, reverse osmosis ion exchange, and electrodialysis [5]. One widely used chemical remediation technique is chemical oxidation, which involves the introduction of oxidizing agents, such as hydrogen peroxide, ozone, or persulfate, into contaminated sites. These agents react with the pollutants, breaking them down into less harmful substances. Another method is the chemical reduction, where reducing agents like zerovalent iron or sodium dithionite are utilized to convert toxic contaminants into less harmful or nontoxic forms. Additionally, chemical precipitation is used to remove heavy metals from wastewater by adding chemicals that cause the metals to form insoluble precipitates, which can then be easily separated. Chemical remediation processes offer efficient and targeted approaches for treating contaminated environments, but their implementation requires careful consideration of the specific contaminants, site conditions, and potential impacts on surrounding ecosystems. However, at concentrations between 1 and 100 mg/L, these traditional methods are ineffective for entirely removing the heavy metals. Other traditional methods have also been described, including the utilization of inorganic adsorbents such as ores, clay, metallurgical wastes, industrial waste materials, and alum, as well as organic adsorbents like plants and animal-derived waste products. However, they are neither a cheap, energy-intensive, nor an ineffective procedure, and they do not totally remove the heavy metals from the effluent [6, 7]. For the above-mentioned reasons, the phycoremediation research field is rising in the last 10 years. In Table 1 are shown the classic methods for chemical remediation.

Methods: Physical	Advantages	Disadvantages
Adsorption	Economical, absorbent, low sludge volume, flexibility in operation, applicability in wide pollutants, and fast kinetics	Adsorption chemicals are required, and adsorbent-dependent performance
Ion exchange	Potentials for metal recovery and maximized degree of regeneration and can be metal selective	Has high operation cost and sensitivity toward the particles
Membrane filtration	High efficiency, space saving, low solid waste generation, and low chemical consumption	Requires high power, restoration of the membrane is required, and high initial capital cost
Photocatalysis	Removes heavy metals and organic pollutants	Time-consuming process
Reverse osmosis	Pure effluent generation and removes most of the heavy metals	Expensive method required
Chemical methods
Chemical precipitation	No metal selectivity and simple technique	Ineffective in higher concentration, large sludge generated, high maintenance, and sludge disposal cost
Coagulation and flocculation	Cost-effective techniques, aids dewatering, bacterial inactivation capacity, and good sludge settling features	High utilization of chemical and solvent, high sludge disposal required, and cannot stand alone to remediate the heavy metals
Electrochemical treatment	Moderate metal selectivity, a low chemical required, and pure metal can be achieved	Requires high electricity supply, requirement of floc filtration, and high capital cost
Evaporation	Pure effluents generated that can be reused	Energy-intensive process, sludge disposal required, and a costly process

Table 1.

Remediation of heavy metals using various conventional methods (Monteiro et al. [8]; Cheng et al. [5]).

2. Phycoremediation

Phycoremediation, among the various phytoremediation strategies, specifically focuses on the use of macro- and microalgae for environmental remediation purposes. In this chapter, we will focus on microalgae’s capabilities to remediate environments from heavy metals pollution. Phycoremediation harnesses the natural capabilities of microalgae to remove or transform pollutants from air, water, and soil. Microalgae have a remarkable ability to uptake and accumulate contaminants, thanks to their high surface area-to-volume ratio and efficient uptake mechanisms. They can accumulate and sequester pollutants within their biomass, thus reducing their presence in the surrounding environment. Moreover, certain species of microalgae have the capability to metabolize and degrade various organic contaminants, such as hydrocarbons, pesticides, and pharmaceuticals, through their unique enzymatic machinery. This capacity makes them valuable agents for the biodegradation and detoxification of organic pollutants in aquatic and terrestrial environments. Phycoremediation is a sustainable and eco-friendly approach that operates through natural biological processes, minimizing the need for extensive infrastructure or chemical interventions. Furthermore, the biomass generated during phycoremediation processes can be harnessed for various value-added applications. Microalgal biomass can serve as a potential source of biofuels, feedstock for livestock and aquaculture, or as a raw material for the production of bioactive compounds. By harnessing the power of these microscopic organisms, phycoremediation offers a greener and more holistic solution to environmental cleanup, while also providing opportunities for resource utilization and sustainable development. In Figure 1 is explained the flowchart of microalgae phycoremediation.

Figure 1.

Flowchart of operation working for microalgae use in the bioremediation of heavy metals, HMs.

2.1 Benefits of using microalgae, a realm of biodiversity

Microalgae are highly adaptable organisms, which are capable of thriving in diverse environmental conditions, including saline, brackish, or wastewater-impacted environments. Their rapid growth rates and high biomass production further contribute to their efficacy as phycoremediation agents. They encompass a vast realm of biodiversity, comprising numerous genera and species with diverse characteristics and functionalities.

Different microalgae have been utilized to remediate wastewater all around the world for the past 30 years [9]. Microalgae Scenedesmus incrassatulus were used in an experiment by Pena-Castro and associates [10] to extract copper (II), cadmium (II), and chromium (VI). S. incrassatulus eliminated 25–78% of the heavy metals, according to the findings. Similar to Omar [11], who investigated the removal of Zn using Scenedesmus quadricauda and Scenedesmus obliquus, demonstrated that S. obliquus had a better removal efficiency than S. quadricauda. Additionally, Soeprobowati and Hariyati [12] investigated the capacity of the microalgae Chlorella sp., Spirulina sp., Chaetoceros sp., and Porphyridium sp. to remove heavy metals including Cd, Cu, and Pb. Chlorella sp. provided the maximum removal effectiveness of Pb (90%), Cd (62%), and Cu (83%) after 14 days of cultivation. However, Spirulina sp. and Chaetoceros sp. reacted with the lowest level of effectiveness, while Porphyridium sp. demonstrated good decrease of Cd (70%) and Cu (96%). These findings revealed that different microalgae species reduce heavy metals content to varying degrees, and that these species are also influenced by the types of heavy metals present in the culture medium or wastewater. By using adsorption and desorption techniques, dead microalgae cells (passive adsorbed biomass, PAB) and microalgae-based products can remove heavy metals. Daneshvar and collaborators [13] investigated the use of microalgal char in the removal of Cr (VI) from water. According to the study, microalgal biochar completely eliminated Cr (VI) from water. Cheng [14] also studied the biosorption and kinetics of Cd (II) removal using living and dead cells of Chlorella vulgaris.

The biosorption capacity of Scenedesmus sp. biomass for the removal of Cr (VI) from the solution was also studied by Pradhan and associates [15]. The findings showed that 89% of Cr (VI) was deposited in Scenedesmus sp. biomass. According to an analysis using Fourier transformation-infrared spectroscopy (FT-IR), microalgae cells contain a variety of functional groups, including amides, aldehydes, carboxylic acids, halides, and phosphates, all of which are positively charged. This allows microalgae to adsorb and aid the biosorption process. Using the Pb (II)-tolerant microalga S. obliquus, Danouche and colleagues [16] investigated the mechanism of Pb (II) reduction. In all the studies cited, the percentages of metals’ uptake capability were higher than 80%. The most part of experiments has been conducted with species belonging to Chlorophyceae and Diatoms genera.

Another source of HMs can be represented by the acid mine drainage; acid mine drainage can be directly discharged into water systems, which damages aquatic habitat and has detrimental effects on the ecosystem. However, by eliminating the hazardous heavy metals that are present in acid mine drainage, microalgae can remediate it [9]. The microalgae Chaetoceros muelleri, Tetraselmis chuii, Pavlova lutheri, and Nannochloropsis gaditana were utilized by Richards and Mullins [17] to extract heavy metals from municipal leachate and hypersaline effluent. The findings showed that 95% of the heavy metals in both wastewaters were removed by microalgae. Furthermore, a significant amount of lipids was generated by Nannochloropsis gaditana and Chaetoceros muelleri. Using a biorefinery strategy, the integration of microalgae-based heavy metal wastewater treatment with microalgal lipid production may offer a new way to keep the circular bio-economy alive. In order to investigate this integrated strategy on a laboratory scale, Yang and colleagues [18] heterotrophically cultured Chlorella minutissima UTEX 2341 in the presence of heavy metals as Cd, Cu, Mn, and Zn in order to produce lipids. The findings showed that Chlorella minutissima UTEX 2341 eliminated heavy metals by intracellular accumulation and extracellular immobilization. The presence of Cd and Cu in the media also increased the accumulation of lipid. The removal of Ni and Cu using C. vulgaris and Desmodesmus sp. was also studied by Rugnini and associates [19]. The findings showed that both microalgae were able to survive for up to 12 days when Cu and Ni were present, and Desmodesmus sp. was able to remove more than 90% of the Cu from the water samples. The capacity of Nannochloropsis oculata for the uptake of Cu (0,05, 0,1, 0,15, 0,2, and 0,25mmol/L) from the acid mine drainage was assessed by Martnez-macias and colleagues [20]. They also investigated that N. oculata species’ lipid and biomass production eliminated 92% of the Cu content, according to the findings. Among them, the most conspicuous part was removed by microalgal metabolism, while a small percentage has to be ascribed to adsorption mechanism.

Additionally, it generated biomass rich in saturated and monounsaturated fatty acids (SFAs), which was used as a fuel for the manufacture of biodiesel.

There is a significant volume of copper-rich water produced by the copper industry. The removal of Cu and Mo from wastewater by C. vulgaris had the highest removal efficiency of the two microalgae that Urrutia and associates [21] examined. Chlorella kessleri, a microalga, was also employed by Sultana and associates [22] as a biosorbent agent to remove heavy metal ions from synthetic wastewater.

The above-mentioned studies give an idea on microalgal biodiversity that extends far beyond the examples provided here. The microalgal biodiversity, in fact, is huge, with almost 64 classes (phyla) and more than 70,000 species that could represent the basin to draw for investigation of the microalgal diversity, with bioremediation aims [23]. The most recent and diversified taxon group, from an evolutionary and biogeochemical point of view, is the diatoms’ group that has been estimated to have approximately 10⁵ species [24]. Most of the diatoms’ biodiversity, for example, is hidden due to cryptic and pseudocryptic nature of species belonging to the group. For this reason, in recent years, taxonomy has been using molecular tools, in addition to morphological identification to better decipher the richness of the microalgal realm [25].

There are thousands of genera and species of microalgae, each with unique characteristics and potential applications. The study and exploration of microalgae biodiversity hold immense value in unlocking their full potential for various industries and environmental applications. Table 2 illustrates examples of microalgae successfully employed in phycoremediation.

Microalgal species	Experimental condition	Heavy metal removal efficiency	Reference
Chlorella sp.	IT: 14 days	Pb: 90%, Cd: 62%, Cu: 83%	Soeprobowati and Hariyati [12]
Chaetoceros sp.	IT: 14 days	Pb: 81%	Soeprobowati and Hariyati [12]
Porphyridium sp	IT: 14 days	Cd: 70%, Cu: 96%	Soeprobowati and Hariyati [12]
Spirulina sp.	IT: 14 days	Cd: 73%	Soeprobowati and Hariyati [12]
Chlorella vulgaris, Arthrospira platensis	pH: 5–5 5, T: 20 °C	Ni (II), Zn (II), Cd (II), Cu (II)	Piccini et al. [27]
Pseudochlorococcum typicum	T: 20.1°C, LI: 30 mE m 2 s1, D:N cycle: 16 h:8 h	Hg2+: 97%, Pb2+: 70%, Cd2+: 86%	Shanab et al. [28]
Live and dead cells of Chlorella vulgaris	T: 25°C, CT: 5–15 min	Live cells: Cd removal by live cells (95.2%) and dead cells (96.8%)	Cheng [14]
Scenedesmus quadricauda biochar	CT: 4 h, pH: 2, T: 5–35°C	Cr (VI): 100%	Daneshvar et al. [13]
Chlorella sorokiniana, Scenedesmus acuminatus	IT: 7 days, T: 25.2°C, LI: 100 μmol m2 s1, D:N cycle: 16 h:8 h	Cu (na)	Hamed [29]
Desmodesmus sp. MAS1 and Heterochlorella sp. MAS3	pH: 3 5, IT: 7 days, T: 23.1°C, LI: 60 μmol m2 s1	Cd: >58%	Abinandan et al. [30]
Scenedesmus sp. 10 mg l	pH: 1.0, T: 23°C	Cr (VI): 92.89%	Pradhan et al. [15]
Chlamydomonas reinhardtii	pH: 9.5, IT: 180 h	As: 38.6%	Saavedra et al. [31]
Chlorella vulgaris	pH: 5.5, IT: 180 h	As: 32.4%	Saavedra et al. [31]
Scenedesmus almeriensis	pH: 9.5, IT: 180 h	As: 41.7%	Saavedra et al. [31]
Chlorella vulgaris, Scenedesmus quadricauda	pH: 2, T: 25°C, IT: 240 h, pH: 6 for Cr (III) and 1 for Cr (VI), T: 25°C, IT: 120 h	Cr (VI): 43%; Cr(III): 98.3%	Sibi [32], Khoubestani et al. [33]
Chlorella sp. and Chaetoceros sp.	pH: 6, T: 25°C, IT: 180 h	Chlorella sp. and Chaetoceros sp. removed Pb 78 and 60%, respectively	Molazadeh et al. [34]
Chlorella pyrenoidosa	T 26–31°C	Ammonium nitrogen 100%, total nitrogen 57.14%, total phosphorus 75.51%, copper 73.39%, lead 72.80%, and cadmium 48.42%	Rahmani et al. [35]
Nannochloropsis oculata	LI: 2000 lux, D:N cycle 18 h:6 h, T: 25°C	Oil 66.5%, COD 54%	Ammar et al. [36]
Nannochloropsis oculata	D:N cycle 14 h:10 h, T:21°C, pH 7.5–9	Ammonium nitrogen 100%, COD 70%, iron 100%	Parsy et al. [37]
Nannochloropsis oculata	LI: 57 μmol m − 2⋅s − 1, D:N cycle 12 h:12 h, T: 21°C, pH: 7	PAHs 94%, NAP 96%, APT 95%, FLU 91%, PHE 83%, BbF 95%, DA 90%, BaP 95%, iron 96.80%	Marques et al. [38]
Galdieria sulphuraria	T: 42°C, D:N cycle 24 h, LI: 4000 lux	Total nitrogen 100%	Rahman et al. [39]

Table 2.

Modified from Grosswami et al. [26], different microalgae reported to remove heavy metals, hydrocarbons, nitrogen N, phosphorus P, and chemical oxygen demand, COD; T, temperature; IT, incubation time; LI, light intensity; DN, day-night cycle.

2.2 How do microalgae work to cope with heavy metals in the environment?

Two main processes have been detected in the study of microalgae responses to metals’ tolerance and resistance, they can be resumed in passive and active mechanisms, e.g., passive biosorption and active intake of metals through specific transporters together with antioxidant responses and redox cellular state changes. In the following sections, we will sum up the processes one by one. Ion exchange, chelation, complexation, hydroxide condensation, covalent binding, redox interaction, biomineralization, and precipitation of insoluble metal complexes are some of the molecular mechanisms by which HMs are biosorbed onto cell walls and EPS. These mechanisms involve electrostatic, van der Waals, or hydrophobic interactions between positively charged HM cations and negatively charged groups on the cell surface [40].

1. Passive biosorption is a key mechanism by which microalgae can remove heavy metals from the environment. Microalgae have a high surface area-to-volume ratio, which facilitates the binding and accumulation of heavy metals onto their cell walls, extracellular polymeric substances (EPS), and intracellular compartments. The cell wall of microalgae contains various functional groups, such as carboxyl, amino, hydroxyl, sulfhydryl, sulfate, phosphate, carbonyl, amide, imidazole, thioether, and phenol groups, which can interact with heavy metal ions through electrostatic attraction, ion exchange, complexation, and coordination reactions. The constitutive macromolecules of the cell wall, which contain a variety of negatively charged functional groups, act as the interface between the intracellular compartment and the external environment, creating a background moiety that can bind to ions from the surrounding medium in the absence of steric or conformational barriers [41]. The biosorption capacity of microalgae depends on factors, such as the metal concentration, pH, temperature, biomass concentration, and the presence of other ions in the environment. Certain species of microalgae, such as Chlorella, Spirogyra, and Scenedesmus, have demonstrated efficient biosorption capabilities for a range of heavy metals, including copper (Cu), cadmium (Cd), lead (Pb), and zinc (Zn). Biosorption offers a cost-effective and eco-friendly approach for the removal and recovery of heavy metals from contaminated water bodies. Death microalgal biomass, PAB, can be employed for the passive biosorption process.

2. Molecular processes and antioxidant responses: when exposed to heavy metals, microalgae activate a series of molecular processes and antioxidant responses to mitigate the toxic effects. Microalgae respond to HM-generated oxidative stress by managing the cell redox status and overexpressing heat shock proteins (HSPs), just like other cell types do [42]. MicroRNAs (miRNAs) are essential parts of the gene regulatory system that controls cellular HMs. By causing the complexation of excess HMs, oxidative stress defense, and signal transduction for biological control purposes, their contribution to posttranscriptional cleavage and translational inhibition of target mRNAs or methylation of target DNAs regulates responses intended to maintain cellular homeostasis [43]. Additionally, it appears that a number of genes involved in HM absorption, transport, and detoxification are activated by metal-responsive transcription factors (TFs), creating a systemic defense against HM toxicity in the cell. Microalgae consider alternative strategies, such as sexual reproduction, expression of metal-modifying enzymes, and phenotypic plasticity as a means of surviving HM toxicity [44].

   Heavy metals can induce the cells to produce reactive oxygen species (ROS), which can cause oxidative stress and damage to proteins, lipids, and DNA. More specifically, they act by impairing the photosynthetic apparatus, preventing enzyme activity, and/or stopping cellular divisions [45], or by obstructing the normal function of the thylakoid membrane and chlorophyll biosynthesis, acidifying the cytoplasm, or harming the cell membrane [46].

   Microalgae employ various antioxidant defense mechanisms to counteract the harmful effects of ROS and maintain cellular homeostasis. Some of these molecular processes include:

• Activation of antioxidant enzymes: microalgae upregulate the activity of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and ascorbate peroxidase (APX). These enzymes scavenge ROS, convert them into less harmful molecules, and maintain the redox balance within the cells.

• Accumulation of antioxidants: microalgae increase the synthesis and accumulation of antioxidant compounds such as glutathione (GSH), ascorbic acid (vitamin C), tocopherols (vitamin E), and carotenoids. These antioxidants directly neutralize ROS and protect cellular structures from oxidative damages. Increased expression of peptides that include metal-binding amino acids, such as proline, histidine, and glutamate, is another strategy used by microalgae. In order to counteract the otherwise harmful effects of HMs in the cytoplasm, these organometallic complexes are normally transported and partitioned them into vacuoles [47]. When HMs are released from their organic carrier in the acidic environment of vacuoles, they are most likely stabilized and chelated by sulfides or organic acids in vacuoles [48].

• Induction of stress-responsive genes: exposure to heavy metals triggers the activation of stress-responsive genes, including those involved in metal detoxification, chelation, and transport. For instance, metallothionein and phytochelatin genes are upregulated, leading to the synthesis of metal-binding proteins that sequester heavy metals and prevent their toxic effects.

• Activation of repair mechanisms: Microalgae activate DNA repair mechanisms, including DNA damage sensing and repair enzymes, to mitigate the genotoxic effects of heavy metals. These repair mechanisms help maintain genomic stability and prevent mutations.

Overall, the biosorption capacity of microalgae enables them to sequester heavy metals from the environment, while their molecular processes and antioxidant responses protect them from the toxic effects of heavy metal-induced oxidative stress. Understanding these mechanisms is crucial for harnessing the potential of microalgae in bioremediation strategies and developing effective strategies for environmental cleanup and sustainable resource utilization, also through the use of genetic engineered microalgae. For the active intake of metals, microalgae employ various mechanisms through membrane transporters to fulfill their nutritional requirements. The net metal flow is therefore lowered, and the expulsion of trace metal complexes may potentially impact the chemical speciation of the HMs. Several metal efflux pumps do regulate the algal membrane permeability by actively moving HMs into and out of the cell [49]. The membrane transporters facilitate the uptake of essential metals, such as iron (Fe), copper (Cu), manganese (Mn), zinc (Zn), and others. It can happen that for some of these membrane transporters, toxic metals, such as cadmium (Cd), chromium (Cr), and lead (Pb), are internalized because the transporters cannot discriminate between toxic and essential cations. The mechanism can be broadly categorized into two types: primary active transport and secondary active transport:

1. Primary active transport: primary active transport involves the direct expenditure of energy (typically adenosine triphosphate (ATP)) to drive the uptake of metals against their concentration gradient. This process is mediated by ATP-binding cassette (ABC) transporters, which are energy-dependent membrane proteins. These transporters utilize the energy derived from ATP hydrolysis to actively pump metal ions across the cell membrane.

2. Secondary active transport: secondary active transport relies on the preexisting electrochemical gradient of ions generated by primary active transporters or ion pumps. The energy stored in the electrochemical gradient is then utilized to drive the uptake of metals. Two common types of secondary active transporters involved in metal uptake are symporters and antiporters:

   • Symporters: Symporters cotransport metal ions along with other ions across the membrane in the same direction. The movement of one ion down its electrochemical gradient provides the energy required to transport the other ion against its concentration gradient. For example, the sodium-dependent phosphate symporter (NaPi) mediates the cotransport of sodium and phosphate ions, with the sodium gradient providing the energy for phosphate uptake.

   • Antiporters: Antiporters facilitate the exchange of one ion for another across the membrane. They couple the movement of one ion against its concentration gradient with the movement of another ion down its gradient. For instance, the proton-coupled divalent metal transporter (DMT) exchanges protons for divalent metal ions, allowing the uptake of metals using the proton gradient as the driving force.

   These membrane transporters play a crucial role in maintaining metal homeostasis within microalgae cells, in the most part, but not always discriminating between essential and toxic metals. The expression and activity of these transporters are regulated by the availability of metals and the specific needs of the microalgae. It’s important to note that the specific membrane transporters and their mechanisms can vary among different microalgae species and even within different growth culture stages of the same species. The understanding of these transport mechanisms is essential for exploring the bioavailability and uptake of metals in microalgae and harnessing their potential in bioremediation, nutrient supplementation, and other applications. In Figure 2 are shown the cellular mechanisms which are implied in microalgae responses to HMs.

Figure 2.

Modified from Cavalletti et al. [50], the figure illustrates the effects of high concentrations of cadmium (Cd), copper (Cu), and zinc (Zn).

2.2.1 Mechanisms against cadmium, copper, and zinc metals and species for metals remediation

Microalgae employ specific mechanisms to cope especially with a high concentration of cadmium (Cd), and essential metals such as zinc (Zn) and copper (Cu), when their concentrations exceed the normal cellular functions, and mitigate their toxic effects; obviously, the mechanisms acted against these metals can also work with other elements. Cd is a heavy metal that can cause severe damage to cellular components and disrupt biological processes in cells. Here are some specific mechanisms used by microalgae to cope with Cd toxicity:

1. Metal binding and sequestration: microalgae possess cell wall components, such as polysaccharides and proteins, which can bind and sequester cadmium ions. These functional groups present on the cell surface can form complexes with cadmium through coordination reactions, reducing its bioavailability and preventing its entry into the intracellular compartments. This sequestration mechanism helps protect the vital cellular machinery from cadmium-induced damage [51].

2. Phytochelatin synthesis: phytochelatins (PCs) are small cysteine-rich peptides that are synthesized by microalgae in response to cadmium exposure. PCs have a high affinity for heavy metals, including cadmium, and form stable complexes with them. The synthesis of PCs is catalyzed by the enzyme phytochelatin synthase (PCS), which is induced upon cadmium stress. PCs function as metal chelators, binding to cadmium ions and sequestering them in vacuoles or other intracellular compartments, reducing their toxicity.

3. Antioxidant defense system: cadmium exposure leads to the generation of reactive oxygen species (ROS) within microalgal cells, causing oxidative stress. Microalgae activate their antioxidant defense system to scavenge ROS and maintain redox homeostasis. This includes the upregulation of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and ascorbate peroxidase (APX), which detoxify ROS and protect cellular components from oxidative damage.

4. Enhanced metal efflux: microalgae can enhance the efflux of cadmium ions from their cells, reducing the intracellular cadmium concentration. This efflux is facilitated by membrane transporters such as ATP-binding cassette (ABC) transporters, metal ion transporters, or plasma membrane H + -ATPases. These transporters actively pump cadmium ions out of the cells, reducing the cellular cadmium burden and minimizing its toxic effects.

5. Metallothionein synthesis: microalgae synthesize metallothioneins (MTs) in response to Cd exposure. MTs are small, cysteine-rich proteins that have a high affinity for metal ions, including Cd. These proteins bind to copper ions and sequester them, reducing their availability and toxicity. The induction of MT synthesis is mediated by metal-responsive transcription factors, enabling microalgae to regulate Cd homeostasis [45].

   The next examples are specific mechanisms used by microalgae to cope with copper and zinc toxicity:

6. Enhanced copper efflux: microalgae enhance the efflux of excess copper ions from their cells to minimize copper toxicity. This is achieved through the activity of membrane transporters, such as ATP-binding cassette (ABC) transporters, copper-transporting P-type ATPases, or copper-effluxing P-type ATPases. These transporters actively pump copper ions out of the cells, reducing the intracellular copper concentration and preventing copper-induced damage [50].

7. Metal chelation: microalgae produce various chelating agents, such as organic acids, amino acids, and peptides, which can bind to copper ions and form complexes. These chelators, including citric acid, oxalic acid, and phytochelatins, reduce the bioavailability and toxicity of copper by sequestering it in nontoxic forms. Copper chelation helps protect cellular components from copper-induced oxidative stress and damage.

8. Zinc efflux: microalgae enhance the efflux of excess zinc ions from their cells to prevent zinc accumulation and toxicity. This is achieved through the activity of membrane transporters, such as ATP-binding cassette (ABC) transporters and zinc-effluxing P-type ATPases. These transporters actively pump zinc ions out of the cells, reducing the intracellular zinc concentration and protecting cellular components from zinc-induced damage.

9. Zinc precipitation and complexation: microalgae can precipitate excess zinc ions by forming insoluble complexes or precipitates. They produce molecules such as phosphate and sulfate, which can bind to zinc ions and form insoluble zinc phosphates or zinc sulfates. This precipitation mechanism reduces the bioavailability of zinc and limits its potential toxicity to microalgae.

Several microalgae species have demonstrated potential for heavy metals remediation, also described in Section 2.1. Some of the commonly studied species include:

• Chlorella vulgaris: this species has shown a high capacity for cadmium (Cd), copper (Cu) and zinc (Zn) biosorption and accumulation, making it suitable for metals remediation applications. For instance, transcriptomic analysis of Chlorella vulgaris revealed that intracellular carotenoids and proline contents as well as the activity of antioxidant enzymes like catalase (CAT), peroxidase (POX), polyphenol oxidase (PPO), and superoxide dismutase (SOD) had significantly increased after exposure to Cd cations. In response to metal stress, photosystem II (PSII) and CO2 assimilation appear to be blocked in microalgae; a corresponding reduction in growth rate and cell density has been seen for different Chlorella vulgaris strains during exposure to high doses of Cd [52].

• Scenedesmus obliquus: Scenedesmus obliquus has been extensively studied for its ability to tolerate and remove cadmium from contaminated aqueous solutions, showing that heat-inactivated cells removed a maximum of Cd. The highest extent of metal removal, analyzed at various pH values, was at pH 7.0. [53].

• Chlamydomonas reinhardtii: This model microalga has been utilized to investigate the molecular mechanisms involved in cadmium (Cd) and lead (Pb) tolerance and detoxification thanks to extracellular polymeric substances (EPS), intracellular starch granules, lipid droplets, and glutathione that were significantly increased under Cd and Pb treatments [54].

• Arthrospira platensis: this species, also known as Spirulina, has shown a high potential of copper (Cu) bioaccumulate thanks to its capability to produce biomass and can be used in remediation efforts [55].

• Euglena gracilis: Euglena gracilis has been studied for its ability to tolerate and remove cadmium (Cd), lead (Pb), and mercury (Hg) from contaminated waters and wastewater and its capability to hyperaccumulate these metals [56].

• Dunaliella salina: Dunaliella salina is a halophilic microalga that can tolerate elevated levels of zinc together with pigments and antioxidant molecules’ accumulation and has potential for zinc bioremediation applications [57].

• Microcystis aeruginosa: This cyanobacterial species has demonstrated the ability to tolerate and remove cadmium and zinc at high concentration from water bodies without being affected in its growth [58].

These microalgae species possess inherent mechanisms to cope with cadmium (Cd), copper (Cu), and zinc (Zn) stress, making them suitable candidates for these elements’ remediation. Their ability to sequester, chelate, and detoxify cadmium (Cd), copper (Cu), and zinc (Zn) highlights their potential in mitigating metals pollution and improving environmental quality.

2.2.2 The use of microorganism consortia in bioremediation

Microorganism consortia play a crucial role in bioremediation as they can exhibit enhanced pollutant degradation capabilities compared to individual microorganisms. Microalgae and cyanobacteria could be considered suitable for metals and hydrocarbons depletion from aquatic environments, since they are able to passively adsorb them onto their cellular wall polymers and, in living cells, they can accumulate HMs and PAHs through membrane transporters from the cytosol to organelles. Microalgae and cyanobacteria are suitable candidates for HMs and PAHs bioremediation because of their autotrophy and their ability to achieve fast growth leading to a high biomass productivity.

In fact, it has been demonstrated that some of the genes involved in heavy metals decontamination are activated in decontamination catabolism PAHs. Among microalgae, chlorophytes show the highest bioaccumulation and heavy metals removal (Cd, Cu, and Zn) capability from liquid medium making them suitable candidates for bioremediation [59].

Numerous studies have shown that when cyanobacterial biomass increases, metal concentrations, ammonium ions, and mineral phosphorus levels in the water decline. With the use of micro- and macroalgae and cyanobacteria, the efficacy of purification can reach 90–97% [60]. It has been demonstrated that cyanophytes contribute to the breakdown of hydrocarbons and have a significant role in the removal of oil-based contaminants from water [61]. Cyanobacterial biomass is regarded as one of the most successful bio-accumulators due to its widespread distribution, affordable cultivation methods, and high absorption capacity. Cyanobacteria are categorized as complex biological objects that function as biosorbents. This ability depends on the kind of microbe and the growth conditions; assemblages of microalgae and cyanobacteria might be advantageous in the restoration of polluted environments. It is influenced by the composition of their cell walls as a structure that immobilizes the metals. The consortia consist of multiple species or strains of microorganisms that work synergistically to degrade and remove pollutants from contaminated environments. There are some key aspects of using microorganism consortia for bioremediation as functional diversity that allows them to collectively target a wide range of pollutants, including organic compounds, heavy metals, and hydrocarbons. The consortium members can complement each other’s metabolic pathways, ensuring more efficient degradation and removal of various pollutants, giving synergistic interactions, where the metabolic activities of one species or strain can support or enhance the activities of the others. For example, certain species may produce growth factors, enzymes, or metabolites that stimulate the growth or activity of other consortium members [62]. This synergy promotes the overall degradation efficiency and adaptability of the consortium to diverse environmental conditions. Microorganism consortia can utilize complementary nutrient sources available in the contaminated environment, ensuring efficient utilization of available resources and preventing resource competition among the consortium members. Some species may specialize in the degradation of specific compounds or metabolize different carbon sources, thereby enhancing the overall pollutant removal efficiency. Microorganism consortia provide redundancy, meaning that if one member fails to thrive or loses activity due to unfavorable conditions or pollutant toxicity, other members can compensate for the loss; this redundancy improves the stability and resilience of the consortium, ensuring pollutant degradation even under changing environmental conditions.

Moreover, microorganism consortia can exhibit complex biodegradation pathways involving multiple enzymatic reactions and intermediate metabolites. This complexity allows for the breakdown of complex or recalcitrant pollutants that cannot be efficiently degraded by individual microorganisms. The consortium members work together to sequentially degrade the pollutants into simpler and less toxic compounds. Microorganism consortia have a higher environmental adaptability than single strains to different pH levels, temperature ranges, or salinity conditions. Examples of microorganisms’ consortia used in bioremediation include those employed in the degradation of petroleum hydrocarbons, chlorinated solvents, pesticides, and various industrial pollutants; there are several examples in the literature of microbial consortia based on bacterial species belonging to Veillonellaceae, Pseudomonadaceae, and Clostridiaceae which are able to remediate metalloid polluted environments [63] or Bacillus species can decontaminate hydrocarbon polluted sites [64]. In the last decade, attention has been placed on the capability of bacterial-microalgal consortia for the bioremediation of pesticides. For the past 50 years, nutrient removal from wastewater, agro-industrial effluent, and heavy metals pollution has been accomplished using bacterial-microalgal consortia [65]. The rate at which pesticides are accumulated or converted by bacterial-microalgal consortia has been measured in numerous pilot-scale investigations. These microbes are frequently cultivated after being extracted from an area with high pesticide contamination levels, where they are combined with other microbes to biodegrade or bioenhance organic pollutants [66]. A consortium of proteobacteria including Ralstonia, Pseudomonas, Burkholderia, Sphingomonas, and Acinetobacter was employed in conjunction with microalgae including Chlorella sp., Selenastrum sp., Phormidium sp., and Scenedesmus sp. to digest a variety of organic contaminants [67]. Polyaromatic hydrocarbons (PAHs) are primarily broken down by the cytochrome P450 monooxygenase (CYP) enzyme family, with wild-type enzymes from Bacillus and Pseudomonas species being modified to increase degradation activity [68]. The use of genetically altered bacteria and/or microalgae can provide fitness increases through upregulation of enzymatic activity, ameliorating the rate-limiting steps in metabolic pathways [69].

2.2.3 The use of microalgae free cell compared to immobilized cells

In bioremediation applications, microalgae can be utilized in two different forms: free cells or immobilized cells. Each approach offers distinct advantages and considerations, depending on the specific bioremediation requirements. Let us explore the use of microalgae free cells and immobilized cell devices in bioremediation:

1. Microalgae free cells: microalgae free cells refer to individual cells that are suspended in a liquid medium. They are widely used in bioremediation due to their ease of handling and scalability. Here are some key points regarding the use of microalgae free cells:

   • Versatility: microalgae free cells can be easily adapted to various bioremediation scenarios, including wastewater treatment, the removal of heavy metals, and the degradation of organic pollutants. They can be introduced directly into the contaminated medium, allowing for efficient contact and interaction with the pollutants.

   • Nutrient availability: microalgae free cells require a suitable nutrient supply to support their growth and metabolic activities. Adequate concentrations of carbon, nitrogen, phosphorus, and other essential elements need to be maintained to ensure optimal bioremediation performance.

   • Harvesting and reusability: harvesting microalgae free cells can be relatively straightforward by employing sedimentation, self-flocculation, centrifugation, or filtration techniques. Additionally, free cells can be harvested and reused in subsequent bioremediation processes, enhancing cost-effectiveness.

2. Immobilized cell devices: Immobilized cells refer to microalgae cells that are attached or confined to a solid support matrix or carrier material. This immobilization technique offers several advantages for bioremediation applications:

   • Enhanced stability and longevity: immobilization provides physical support and protection to microalgae cells, increasing their stability and longevity during bioremediation processes. The immobilization matrix acts as a barrier against shear forces and environmental fluctuations, ensuring the sustained activity of the microalgae.

   • Improved retention: immobilized cell devices allow for the retention of microalgae cells in a specific location or within a reactor system, providing better control over their distribution and interaction with the target contaminants. This can enhance the efficiency of pollutants’ removal and reduce the risk of cells’ washout.

   • Reusability and removability: immobilized cells can be reused in multiple cycles of bioremediation, reducing the need for continuous addition of fresh cells and can be simply removed. This can be cost-effective and environmentally friendly.

   • Scalability: immobilized cell devices are easily scalable, allowing for the design and construction of larger-scale bioremediation systems. The immobilized cells can be incorporated into fixed-bed reactors, fluidized-bed reactors, or other immobilization setups, enabling efficient pollutant treatment in larger volumes.

   The choice between microalgae free cells and immobilized cell devices depends on factors, such as the nature of the contaminants, the scale of the remediation project, and the specific requirements of the bioremediation process. Both approaches have been successfully employed in various bioremediation applications, highlighting their potential to address environmental pollution and promote sustainable remediation practices. Table 3 shows examples of biopolymer matrices already employed with immobilized microalgae.

			Species
Matrix type	Phase shape	Application	Motile	Not motile
Luffa sponge	solid	Cadmium removal		Chlorella sorokiniana
Luffa sponge	solid	Chromium III removal		Chlorella sorokiniana
Plain printing paper	45 g m–2, newsprint “Kölner Stadt-anzeiger” DuMont Schauberg	Microalgae growth for feeding in PBR	Isochrysis spp. Tetraselmis suecica	Phaeodactylum tricornutum; Nannochloropsis spp.
Polyvinylidene fluoride (PVDF)		N and P in wastewater removal		Chlorella vulgaris
Alginate	beads	Value-added products		Botyrococcus; Spp,
Several polymers not specified		N and P in wastewater removal		Chlorella vulgaris
Several polymers not specified		N and P in wastewater removal		Scenedesmus spp.
Several polymers not specified		N and P in wastewater removal	Dunaliella spp.
Blank alginate Na and Ca	beads	Nickel removal		Chlorella vulgaris
Kappa-carrageenan	beads 5 mm diameter	Zinc chromium and cadmium		Scenedesmus acutus
Polyurethane	foam	Zinc chromium and cadmium		Chlorella vulgaris
Balginate Ca and bario	beads	CdCl2 and CuSO4 x 17 days		Nannochloropsis gaditana
Aginate Ca	beads	CdCl2 and CuSO4 at 0–10–60 min and 24 h (total Cu removal) N and P wastewater treatment and metals in wastewater	Tetraselmis chui
Polysulfone and epoxy resin	beads	Cu(II), Fe(II), Ni(II), and Zn(II)		P. laminosum + microcystis
Alginate Ca	beads	Chromium		Anacystis nidulans
Zeolite 13X-Algal-Alginate Beads (ZABs)	beads	CuSO4		Chlorella vulgaris
Blank-Alginate Beads (BABs)	beads	CuSO4		Chlorella vulgaris
Chlorella-Alginate Beads (CABs)	beads	CuSO4		Chlorella vulgaris
Alginate beads	beads	N and P wastewater		Chlorella sorokiniana
Silica gel	biofilm	Thorium		Jania rubens/Saccharomyces; cerevisiae

Table 3.

Illustrated examples of different microalgae immobilized in polymeric matrices.

2.3 Gain the response: The genetic engineering of microalgae for ameliorating the bioremediation fitness

Genetic engineering of microalgae holds significant promise for improving bioremediation capabilities. By manipulating the genetic makeup of microalgae, scientists can enhance their ability to degrade pollutants, tolerate higher concentrations of toxic substances, and improve overall remediation efficiency. The molecular toolkits created for microalgal bioengineering include numerous promoters, vectors, reporter genes, and regulatory elements as well as the genome technologies that allow gene editing as the technology based on zinc-finger nucleases (ZFNs), transcription activator-like endonucleases (TALENs), clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems, and RNA interference (RNAi) [70]. Any desired DNA sequence can be targeted using these molecular techniques to produce knockout, knockdown, or insertion-deletion (indel) mutants. The new horizons of genetic modification, made possible by recent technological developments in microalgal molecular cloning, include the modification of the simultaneous expression of numerous genes and the production of marker and transgene-free knockout mutants [71]. Below, there are some key aspects of genetic engineering in microalgae for bioremediation:

1. Gene overexpression: different mechanisms can be fine-tuned in response to heavy metals tolerance and resistance, such as the overexpression of genes implied in metal transportation, chelation, biotransformation, oxidative stress response regulation, and cell surface bioengineering [72]. This allows for increased production of enzymes or proteins responsible for pollutant breakdown, thereby enhancing the microalgae’s remediation potential. CrMTP4, a metal-tolerant protein (MTP) from the family of metal transporters known as cation diffusion facilitators, was overexpressed in C. reinhardtii; this resulted in a notable improvement of both bioaccumulation efficiency and resistance to the toxicity of Cd [73].

2. Metabolic pathway engineering: microalgae can be genetically modified to optimize metabolic pathways relevant to bioremediation. A chromate reductase (ChrR) has been identified in C. vulgaris that converts Cr (VI) to Cr (III) by the oxidation of glutathione (GSH), providing a high level of resistance to Cr toxicity [74]. Arsenic biotransformation is done in a variety of microalgae strains via a number of different mechanisms, the most prevalent of which is the conversion of As (V) to As (III). Tolerance to the As element can be increased by interfering with the arsenic reduction enzymatic pathway. According to mercury resistance mediated by microalgae, Selenastrum minutum, Chlorella fusca, and Galdiera sulphuraria strains are able to biotransform Hg2+ to less harmful elemental Hg when mercuric reductase is expressed [75]. Genes encoding enzymes like cytochromes or dehalogenases can be overexpressed to increase the breakdown of organic pollutants. Phosphate transporters and/or ABCC transporters, and their overexpression, were explicitly blamed for the increased tolerance and bioaccumulation of As and Cr [76]. Other HM volatilization mechanisms in microalgae have been reported as photoreduction pathway for the biotransformation of Cr in C. vulgaris [77], intracellular and extracellular metal nanoparticle biosynthesis, and reductive interactions with functional groups of biomolecules inside and outside the cell. Modifying genes involved in nutrient uptake, detoxification pathways, or stress response mechanisms can improve the effectiveness of pollutant breakdown and metabolic activity by adding or changing genes implicated in pollutant degradation pathways.

3. Heavy metal tolerance: genetic engineering, in microalgae, can involve the introduction of genes encoding metal-binding proteins or transporters that can sequester or actively transport heavy metals, reducing their toxicity within their cells. MTs are a class of polypeptides that are either genetically encoded (classes I and II) or produced (class III) by an enzyme and are present in all living things [78]. They play a significant role in metal homeostasis and trafficking. They have a higher (15–35%) percentage of cysteine residues and a lower (15–20%) percentage of histidine residues. Also, phytochelatins (PCs) are involved in metals chelation, helping microalgae in coping with a high concentration of HMs. Glutamate-cysteine ligase catalyzes the first step in the biosynthesis of PCs, which is the creation of γ-glutamylcysteine from cysteine and glutamic acid. Following the ligation of γ-glutamylcysteine with glycine by GSH synthase, GSH is produced and functions as the primary redox buffer in eukaryotic cells, shielding them from oxidative and metal stressors. The strategy linked to phytochelatins and metallothioneins is widely used in cyanophytes as Synechococcus sp. and seven other blue-green microalgae strains that have been found to contain MT genes [79]. The ability of MTs and their host cells to bind and remediate HM has been hypothesized to depend on the length of the oligopeptide chain and the distribution of cysteine residues. Some strains of microalgae with a better tolerance for HMs do appear to produce MTs with longer chains and more cysteine residues. Through the heterologous expression of enzymes for the synthesis of long, cysteine-rich MTs from hypertolerant species in transgenic strains, it is possible way to engineer microalgae with increased HM removal capability [80].

4. Stress response enhancement: microalgae can be genetically engineered to enhance their stress response mechanisms, enabling them to better cope with pollutants and adverse environmental conditions. This may involve the introduction of stress-related genes or transcription factors that regulate the expression of stress-responsive genes, improving the microalgae’s ability to withstand and remediate contaminated environments when HMs induce the generation of superoxide radical (O₂), hydrogen peroxide (H₂O₂), hydroxyl radical (HO), and singlet oxygen (¹O₂)—collectively known as ROS [81]. In microalgae cells under HMs stress, various antioxidant enzymes’ encoding genes were increased, according to transcriptomic studies of D. salina and C. reinhardtii exposed to Cd and Pb, respectively [82]. In addition to upregulating antioxidant enzymes, Auxenochlorella protothecoides, C. vulgaris, and C. reinhardtii cells overexpressed thioredoxin (Trx), heat shock proteins (HSPs), and carotenoids in response to toxic amounts of metals [83]. Similar studies conducted under HM stress in Amphora coffeaeformis, Navicula salinicola, and D. salina revealed a significant upregulation of genes associated with antioxidant defense [84]. Superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), guaiacol peroxidase (GPX), and glutathione-S-transferase (GST) are well-known enzymatic antioxidants that actively convert superoxide radicals to hydrogen peroxide, till the final oxygen acceptor, while proline, ascorbic acid, and GSH are nonenzymatic antioxidants that may directly cope with ROS via complexation [85]. Heat shock proteins (HSPs) function as molecular chaperones to protect and repair proteins during HM stress, and are part of no-direct mechanisms used by cells [86]. HSPs play important roles in the transport, folding, unfolding, assembly, and disassembly of proteins as well as the destruction of misfolded or aggregated proteins in microalgae and other organisms. In the green microalga Tetraselmis suecica, the function of HSPs, such as HSP20, HSP70, and HSP100, in preventing protein denaturation has been demonstrated [87].

5. Bioaccumulation and biomonitoring: genetic engineering can be utilized to enhance microalgae’s ability to accumulate and bioconcentrate pollutants, enabling more efficient remediation. By introducing or modifying genes associated with pollutant uptake, transport, or storage, microalgae can accumulate higher concentrations of pollutants, facilitating their removal from the environment. Due to the need for thiols and thiol-containing molecules like GSH and PCs and their capacity to chelate metal cations, sulfur metabolism is essential for the mitigation of microalgal HM. When C. reinhardtii was exposed to Hg2+, it was found that the genes involved in sulfur metabolism—biotin biosynthesis genes—and S-transporter genes were upregulated [88]. Numerous HM transporters in microalgae and plants still need to be molecularly identified and described in terms of localization, transport characteristics, and specificity. Therefore, research in this area is essential for learning more about the routes used by HM trafficking and accumulation in microalgae. Additionally, genetic engineering can enable microalgae to serve as biomonitoring tools by introducing genes that produce detectable biomarkers in response to specific pollutants, aiding in pollution detection and monitoring efforts.

To conclude, it is impossible to disregard the importance of genetic engineering in creating and comprehending the mechanisms by which microalgae detoxify such contaminants. In order to strengthen the widespread usage of genetically modified microalgae and to close the knowledge gap in this area, more concentrated research is being acquired to address the growing environmental threat that waters pose to sustainable development. While genetic engineering offers tremendous potential for improving microalgae bioremediation capabilities, it is essential to consider the potential risks and ethical implications associated with releasing genetically modified organisms into the environment. Careful regulation, risk assessment, and monitoring are crucial to ensure the safe and responsible use of genetically engineered microalgae for bioremediation purposes.

3. Patents on use of microalgae in bioremediation

Patents related to microalgae bioremediation can cover a wide range of aspects, including:

1. Novel microalgae strains: patents may be filed to protect newly discovered or genetically engineered microalgae strains specifically designed for bioremediation applications. These strains may exhibit enhanced pollutant uptake, degradation capabilities, or stress tolerance.

2. Bioremediation methods and systems: patents can be obtained for innovative methods and systems that utilize microalgae for bioremediation purposes. These patents may cover the design and configuration of bioreactors, cultivation techniques, harvesting and biomass processing methods, and other aspects related to the efficient and effective use of microalgae in pollutant removal.

3. Genetic engineering techniques: patents may be filed to protect novel genetic engineering techniques or tools used for modifying microalgae genomes. These techniques can enable the development of microalgae strains with enhanced bioremediation capabilities, such as improved pollutant degradation pathways or increased metal uptake and tolerance.

4. Bioproducts and value-added applications: in some cases, patents may focus on the extraction and utilization of value-added products produced during microalgae bioremediation.

For example, patents may cover the extraction of biofuels, bioplastics, or bioactive compounds from microalgae biomass generated during the bioremediation process. These patents protect the intellectual property associated with the specific methods, compositions, and applications of these bioproducts. Nowadays, we still do not have patents on “Genetically Engineered Microalgae Strain for Enhanced Heavy Metal Remediation”, while we can find some examples of patentes on “Integrated Microalgae Bioremediation System for Wastewater Treatment” (patent number RU2758690C; US11577111B2) as bioderived compositions for use in environmental remediation (patent number US20150223470A1). These methods have been developed for integrated purification of complex multicomponent wastewater, containing perfluoro- and polyfluoroalkyl substance (PFAS). We can also find biotreatment methods for efficiently purifying biogas slurry with microalgae (patent number CN103663715A), algal system for improving water quality based on Spirogyra grevilleana (patent number WO2014201298A1). “Highly Efficient Microalgae Device for Bioremediation Applications” includes methods of wastewater treatment with microalgae culture supplemented with organic carbon or consortia made of bacteria-microalgae for N content decreasing (patent number WO2018053071A1; CN108623099A). Several examples of using microalgae grown in bioreactor for water decontamination can be found in the literature, e.g., USA patented the systems US20110151547A1.

4. Future perspectives of using microalgae in bioremediation

The future of using microalgae in bioremediation holds great promises and potentials. As research in this field continues to advance, several exciting future perspectives emerge:

• Enhanced genetic engineering and tailored microalgae strains: genetic engineering techniques will develop even better, allowing for precise manipulation of microalgae genomes and the development of microalgae strains with enhanced pollutant degradation capabilities, higher stress tolerance, and increased biomass production. The targeted genetic modifications will optimize metabolic pathways, improve pollutant uptake, and facilitate the production of valuable compounds during the bioremediation process. Customized microalgae strains will exhibit a higher pollutant uptake, degradation, and tolerance, making them highly effective in cleaning up diverse pollution scenarios.

• Integration of advanced technologies: the integration of advanced technologies, such as omics approaches (genomics, transcriptomics, proteomics, and metabolomics), will provide a deeper understanding of microalgae’s molecular responses to pollutants. This knowledge will enable the identification of key genes, enzymes, and metabolic pathways involved in bioremediation processes. Additionally, advanced monitoring techniques, remote sensing, and modeling tools will allow for real-time assessment and optimization of bioremediation strategies using microalgae.

• Algal bioreactors and bioprocessing: algal bioreactors will be developed and optimized for large-scale production of microalgae biomass dedicated to bioremediation applications. These systems will incorporate innovative designs, efficient nutrient recycling, and controlled environmental conditions to maximize microalgae growth and pollutant removal.

• Synergistic bioremediation approaches: future research will focus on developing synergistic approaches that combine microalgae bioremediation with other remediation strategies, such as phytoremediation, bacterial consortia, or physical remediation methods. These integrated approaches will leverage the strengths of different remediation techniques to achieve enhanced and comprehensive pollutant removal, making them more effective and sustainable.

• Field applications and commercialization: as research progresses and the efficacy of microalgae-based bioremediation becomes more established, the field applications and commercialization of microalgae-based bioremediation systems will likely increase. Industries, environmental agencies, and stakeholders will recognize the potential of microalgae as a cost-effective and eco-friendly solution for tackling pollution. The development of scalable, efficient, and economically viable microalgae bioremediation systems will drive their widespread adoption in different environmental settings.

5. Conclusions

Microalgae have demonstrated remarkable potential as bioremediation agents, owing to their diverse mechanisms and unique capabilities. Through their photosynthetic activity, microalgae can metabolize and break down HMs and PHA, effectively removing them from the environment. Their ability to efficiently uptake and assimilate nutrients helps mitigate eutrophication and control harmful algal blooms. Additionally, microalgae exhibit biosorption and bioaccumulation abilities, enabling them to sequester heavy metals, organic pollutants, and even radioactive substances. Their enzymatic degradation capabilities further contribute to the breakdown and detoxification of complex contaminants. Moreover, microalgae play a crucial role in pH adjustment and oxygen production, creating favorable conditions for other microorganisms involved in bioremediation processes, such as bacteria or fungi. Harnessing these mechanisms provides a sustainable and eco-friendly approach to address various environmental challenges. The concept of coupling environmental remediation with the generation of sustainable resources adds an economic dimension to phycoremediation, making it an attractive option for environmental restoration and resource recovery. Further research and optimization of microalgae-based bioremediation strategies hold great promise for mitigating pollution and restoring the health of our ecosystems.

Conflict of interest

The authors declare no conflict of interest.

Authors’ contribution

Both authors substantially contributed equally to the conception of the work and to the interpretation of data for the work; they equally participated in drafting and revising it.