Factors and mechanisms regulating heavy metal phycoremediation in polluted water

Rapid advances to industrialization and population increases have put aquatic ecosystems at high risk of pollution from various industrial and municipal effluents. The effluents consist of heavy metals (HM), micropollutants, nutrients, microorganisms, solids, particulates and dissolved matter. To this effect, pollutant remediation in such ecosystems is inevitable and of interest in global research. In this study, phycoremediation and its potential to bioremediate HM from polluted aqueous solutions is of focus. The factors influencing the process and the mechanisms involved are explored. The study established that available functional groups in microalgae, cell surfaces characteristics, type of microalgae species used, nutrient availability, size of biosorbent and metal concentration are some environmental factors, which influence phycoremediation success. Uptake of HM from contaminated water is regulated by mechanisms such as volatilization, bio-methylation, enzyme catalyzation, compartmentalization, extracellular polymeric substances-complexation, extracellular biosorption and intracellular bioaccumulation. To ensure high pollutant removal efficacy, improved adaptability of microalgae to HM-polluted systems and high resilience to attack by foreign agents, a number of mechanisms can be adopted. These include microalgal pretreatment with chemicals, bioengineering and biotechnological advances such a gene encoding, synthesis of transgenic proteins, gene overexpression, modification of microalgal cell surfaces with nanoparticles and the use of a consortium of microbes. This study noted that optimizing the discussed factors and mechanisms will promote field-scale application of phycoremediation in water treatment to remove HM.


Introduction
The growth in human population, urbanization and industrialization has been exponential in the 21st century globally though it is accompanied by extensive environmental pollution [1].These tendencies are associated with increased generation of solid waste, wastewater, greenhouse gas emissions, degradation of aquatic habitats, overuse of natural resources and energy.The excessive generation of waste including wastewater and its subsequent disposal and release to the environment has induced great risk on the planet's sustainability through its capacity to deteriorate the quality of land and water resources [2].The discharge of untreated effluent and solid waste to freshwater bodies for instance, is attributable to extensive pollution of such aquatic bodies, which play a great role in shaping sustainable development especially in developing countries [3].The untreated wastewater is loaded with micropollutants such as heavy metals (HM), surfactants, aromatic hydrocarbons and phenolic compounds.The foreign constituents are non-biodegradable and refractory once they buildup in the environment and cause human health risks on entering in food chains [4].Leong and Chang [5] noted that HM even in minute concentrations induce mutagenesis, carcinogenesis, hepatoxicity, neurotoxicity and teratogenesis among other toxic health effects once ingested or inhaled by living organisms.
With the known pollution effects of effluent mixing with freshwater, there is need to decontaminate polluted water off HM. Remediation of such pollutants can be done using a number of techniques including chemical precipitation, coagulation-flocculation processes, membrane filtration, flotation and ion exchange processes [6].Application of the conventional techniques is however limited due to their high operation and maintenance costs and their capacity to produce toxic sludge, which causes extensive pollution after accumulation [6,7].The conventional water treatment approaches are done in facilities, which are expensive to setup, energy-intensive, prone to mechanical failure and require technical expertise to operate and maintain [2].
To overcome the challenges associated with conventional water treatment approaches, bioremediation, which involves the employment of microorganisms (bacteria, fungi and microalgae) to transform, destroy, inactivate and remove toxic HM resulting to non-toxic substances is growing as a viable alternative [6,8].Bioremediation is preferred due to its cost efficiency if HM concentrations are low, effectiveness and its environmentally friendly nature due to production of low quantities of sludge.Phycoremediation, which is a category of bioremediation that uses microalgae to remove HM from contaminated environs is also growing as a wastewater treatment approach [9].The use of the bioremediation approach to remove HM from polluted aqueous environments is as shown in Fig. 1.Examples of microalgae used in phycoremediation of HM from aqueous solutions include the Chlorella, Botrycoccus and Spirulina species among others as detailed in Table 1.During phycoremediation, microalgae are cultivated before harvesting for use [1].Cultivation occurs via the photoautotrophic, mixotrophic and heterotrophic modes [5,8].In photoautotrophic cultivation, the microbes utilize light energy to integrate and use CO 2 , which influences the quantities of the gas in the atmosphere.Heterotrophic cultivation entails the growth of microalgae in the dark while using organic compounds, which contain carbon as the energy source.Mixotrophic cultivation involves the assimilation of organic compounds and CO 2 for microalgal growth using both photoautotrophic and chemoheterotrophic metabolic pathways.Afterwards, microalgal biomass is prepared for bioremediation use.The use of phycoremediation is favorable because of the ability of microalgae to withstand, grow and reproduce in harsh environmental conditions such as extreme salinity, extreme temperature, low levels of nutrients and high heavy metal concentrations [2].Additionally, the microorganisms are fast maturing and actively photosynthetic.They have a simple structure, high affinity for HM, a large surface area, many pollutant binding sites and can be used for phycoremediation in their living or dead biomass form [10].Such characteristics make the microbes suitable for bioremediation.Phycoremediation is also a pathway to reduced emissions of CO 2 to the atmosphere.The observation is attributable to the rapid growth of microalgae and their ability to use CO 2 from the atmosphere and solar radiation efficiently during photosynthesis [11].The microalgal biomass can be reused after phycoremediation to manufacture Fig. 1 The mechanism of heavy metal phycoremediation from polluted aqueous solutions [12] valuable products such as biofuels and fertilizers (Fig. 1), which makes the process less waste-producing and sustainable.Owing to the advantages associated with phycoremediation, it is important to optimize the process for high decontamination efficacy.For such advances to occur, it is essential to understand the working of microalgal systems and the regulators that influence successful heavy metal remediation in order to optimize their working.The aim of this review is to explore the factors that influence phycoremediation process and the mechanisms used in the uptake of HM from polluted water to understand suitable conditions of its successful field scale application.

Phycoremediation of heavy metals from polluted water
The rise of industrialization and urbanization among other anthropogenic activities has led to increased concentrations of HM in land and water resources up to alarming levels [11].The toxic metals accumulate in water and contaminate it since they cannot be reduced biologically and chemically and as such, become health and environmental hazards.In addition, HM destroy the self-purification ability of aquatic ecosystems [13].Microalgae use HM such as B, Co, Cu, Fe, Mo, Mn and Zn as trace elements in their cell metabolic and enzymatic activities unlike the case of As, Cd, Cr, Hg, Ni and Pb that are hazardous to such organisms [14].The toxic HM are found in water that has mixed with effluents from electroplating, tannery, fertilizer, pesticide and paper industries.Due to the innate hormesis effect of microalgae, they can grow and metabolize in low concentrations of such toxic elements.[2] noted that Phormidium, Spirogyra, Anabaena and Oscillatoria cyanobacteria species developed naturally in HM polluted water.
Phycoremediation works on the basis of using microalgae or microalgal biomass to remove HM from polluted environments.During the process, microalgae take up HM from polluted water using three approaches (Fig. 2).The HM can bond covalently with exo-polysaccharides from the microbes, they can exchange the toxins with ions of microalgal cell wall and cationic HM can adhere to anionic uronic acids found on the microorganisms [15].Microalgal cell wall has lipids, proteins and carbohydrates that possess anionic functional groups including the sulfhydryl (SO 3 H -), amino (NH 2 -), phosphate (PO 3 H -), hydroxyl (OH -) and carboxyl (COOH -) moieties that bind on cationic HM from polluted water [16,17].Through biosorption, dead or living microalgal biomass removes HM from polluted water through processes such as diffusion, adsorption, chelation and ion exchange [16].In bioaccumulation, HM are sequestered by living microalgae into their cytoplasm and vacuole [2,16].Some of the microalgal species used in heavy metal remediation from polluted aquatic ecosystems and their removal efficacies are as shown in Table 1.

Type of microalgae species
Multiple factors including influence phycoremediation process.These include the rate of microalgae proliferation, characteristics of the targeted wastewater, the specific strains used and their habitats; although the species used influence the process the most [17].Different microalgal species have varied removal efficacies based on their targeted HM as shown in Table 1.As such, it is important to identify the most efficient microalgal strains within a particular species to ensure effective pollutant remediation [32].Such strains should have optimal growth rates, should not be vulnerable to predation and shear and must have the ability to reproduce and survive in varied environmental conditions.The propagation and geographic proximity of the microbes is also essential in phycoremediation since its influences their ability to adapt in targeted remediation habitats and enhances the efficacy off pollutant removal [17,32].
Local strains have better survival, reproduction and bioremediation capacity compared to non-local ones although a varied geographical spread is preferable.Green microalgae such as Chlorella species are useful in water decontamination for a variety of pollutants including HM [33].In the presence of ammonia, Coelastrella sp. are stable [34].Using microalgal consortia in phycoremediation leads to individual and synergistic effects among and/or between the microorganisms used through metabolite interchanges and collaborative connections [17].Such consortiums can conversely inhibit the growth of other microbes following production of allelochemicals during photosynthesis.A case example is the inhibition of bacterial growth in a consortium of Muricauda sp., Nannochloropsis gaditana and Tetraselmis chuii attributable to the production of chlorellin [34].
To ensure microalgal species used in phycoremediation are appropriate for HM remediation from polluted water, selection and screening on strains is done before use [35].Selection involves isolation of pure cultures of a given strain using single selection, customary selection, flow cytometry and /or bioinformatics processes [35].This is followed by screening of the selected strains, which occurs in three levels.The first level uses Nile red lipophilic fluorescent dye to assess the stability of the microalgae while the second level evaluates the appropriate medium of growth to cultivate the microbes.In the third level, the strains that were successful in the previous screening stages are subjected to varied oxygen, CO 2 and pH levels to evaluate their growth suitability before scaling up their production and use.

Nutrient availability
Microalgae use nitrogen and carbon in polluted water as the source of nutrient for growth.The availability and sources of the nutrients have an effect on phycoremediation since they influence the growth of the microorganisms [36,37].For instance, Chlorella vulgaris grows better in effluent from aquaculture, which contains urea as the nitrogen source [17].Providing enough carbon to microalgae species such as Chlorella gracilis, C. vulgaris, Scenedesmus obliquus, Botryococcus braunii LB-572, Chlorella pyrenoidosa, and Chlamydomonas reinhardtii promoted their growth and multiplication [35].The pollutants found in the contaminated water control the nutrient availability, selection, choice and elimination by a particular microalgal strain.Addition of nutrient is also done for bioaugmentation and biostimulation to enhance bioremediation though the effect can also be inhibitory [6].Biostimulation and bioaugmentation promote metabolic activity of microalgae and optimizes the ratio of carbon to nitrogen and phosphorous (C:N:P).Excessive addition could however be counterproductive [38].In Chlorella vulgaris, addition of NaHCO 3 and PO 4 resulted to higher biomass production due to improved microbial growth and ultimately, better bioremediation efficacy [17].In another study, addition of nutrients in Scenedesmus almeriensis and Chlorella vulgaris reduced their ability to take up As from polluted water and inhibited the growth of the microbes [39].

Dosage and size of biosorbent
A higher amount of biosorbent (microalgal) dosage has a synergistic effect to HM pycoremediation due to increase in surface area and number of binding sites available.Therefore, using high biosorbent concentrations increases the rate of HM uptake irrespective of the microalgal strain applied for remediation [40,41].In using Chlorella colonials in Fe phycoremediation from polluted water, removal efficacy was directly proportional to dosage increases up to a maximum of 86.7% [42].An increase in biosorbent dosage from 10 to 25 g/l while the biosorbent size decreased from 104 to 44 μm resulted increased Cr biosorption using Gracilaria corticate biomass [43].Therefore, a decrease in the size of biosorbent increases the number of active binding sites and the its surface area exposing it to the sorbate (HM) and resulting to improved phycoremediation efficacy.

pH
The effective working of various functional groups found on the microalgal cell wall surface is influenced by the pH of polluted water [44].The pH of the environment where phycoremediation is occurring affects the metabolism of the microorganisms and the dissociation or association of functional moieties.As such, pH changes could be stimulatory or inhibitory to the process.In most cases, microalgae have optimal growth at neutral pH although specific strains have different optimal ranges [35].For example, Spirulina platensis has an optimal pH between 7 and 9 while S. almeriensis and S. obliquus both have an optimal pH of 8 [35].Some of the moieties are anionic under acidic conditions and hence, adsorb HM ions from polluted water.At a pH of 2, Cu ion biosorption is low while pH increases between 3 and 4 enhance adsorption of the metal ions [43].An increase in H + can inhibit biosorption capacity of microalgae by hindering the binding of metal cations to ligands on the cell wall [45].In a study by Lestari et al. [46], the bioremediation of Cd by Tetraselmis sp., Nitzschia sp. and Chaetoceros sp. was low at a pH of 4 with optimal removal efficacy being realized at a pH of 7. The observation was attributable to increased positive charges of the microalgae at low pH, which reduced metal cation uptake significantly.In alkaline pH (> 8), biosorption decreases as a result of soluble hydroxylated complexes formed by metal ions and their increased competition for active binding sites [41].At different pH levels, metal phycoremediation differs due to modification of functional groups and the formation of complexes between pollutant cations and microalgal cells [47].

Metal concentration
The uptake of HM also depends on the concentration of metal ions in polluted water.Initially, biosorption is directly proportional to the initial metal concentration but afterwards the metal sorption stabilizes and does not change with concentration increases [47].The reported trend where biosorption is directly proportional to metal concentration is attributable to accumulation of HM ions on the microalgae surface.Further metal concentration increases result to reduced biosorption as the binding sites on the microbes are all occupied with metal ions.According to Kavitha et al. [43], Cr uptake by Gracilaria corticata in polluted water decreased to 56.23% from 89.37% following an increase in the initial metal concentration from 5 mg/l to 150 mg/l.In another study, increasing Cr concentration decreased the adsorption capacity of Dunaliella salina as a result of saturation of active binding sites and reduced tolerance for the metal toxicity by the microalgae [47].The decreased Cr uptake was also attributable to reduced surface area to take up more of the metal cations in the solution.Reduced biological activity of microalgae and low bioremediation efficacy in high HM concentration was also because of limited binding sites of the microbial surface [47].

Contact time
Microalgae adsorb HM on their cell wall surface rapidly and passively in the first few minutes of contact and reach saturation within 30 min [47].A study by Sen et al. [48] estimated the time taken to reach saturation as much shorter (5 min) after the initial contact between the microalgae and HM compared to [45].For this reason, the contact time influences phycoremediation efficacy [47].With increase in contact time, biosorption of HM reduces owing to saturation of the remaining metal ions [38,40].For instance, in the first 30 min, 93.5 mg/g of Cr in contact with diatoms of Chlorella sp. and Phormidium sp sorbent and 60% of the metal was removed within the first 24 h [49].As such, biosorption occurs in two stages [43].The first stage is fast and aims at realizing an equilibrium between the biosorbent and sorbate while the second stage consists of progressive and slow adsorption followed by desorption if contact time is increased even further.

Temperature
Temperature has a direct influence on the growth of microalgae in that it regulates photosynthesis, cell division and enzymatic activities.For every 10 °C rise in temperature, the growth of microalgae doubles until optimum temperature (which is different for various species) is realized and after that, growth decreases [35].Decrease in growth is attributable to enzyme denaturation and inactivation at elevated temperature, which slows down photosynthesis.Being an exothermic process, adsorption of HM by microalgae follows the Le Chatelier's principles where adsorption and temperature changes are indirectly proportional.Initially, chemical adsorption increases with temperature increase and then begins to decrease.The initial trend is as a result of bond rupture that avails many binding sites for sorption and increased affinity of metal ions to microbial surfaces [41].Temperature changes induce a biosorption potential difference in the microalgae [40].Temperature change between 283 and 323 K increased Cr biosorption using Gracillaria corticata from 79.3 to 91.4% [43].(2023) 3:14 | https://doi.org/10.1007/s43832-023-00041-1Review 1 3

Agitation speed
In aqueous solutions such as polluted aquatic ecosystems, increased agitation increases biosorption capacity since it reduces resistance associated with mass transfer [38].A study by Mahmood et al. [50], which evaluated the microalgal remediation of Zn using Sargassum sp.biomass reported that uptake of the metal cations increased from 76.7 to 90.5% after agitation speed was raised from 50 to 200 revolutions per minute [50].

Physical and chemical composition of microalgal cell wall
During phycoremediation, HM from polluted water are adsorbed on microbial cell wall.Therefore, the physicochemical characteristics of the cell wall regulate metal ion uptake because of their multiple functional groups and active binding sites.The functional groups and binding sites change at different growth phases of the microalgae and depending on the environmental conditions [45].Some of the microalgal biomass used in HM remediation and the functional groups they possess are shown in Table 2.
The surface of microalgal cell wall acts as a barrier between the outer environment and the intracellular compartment.The presence of functional groups on the wall enables HM binding on the surface leading to the uptake of the toxins from polluted environs.The ability of functional groups to attract HM for adsorption controls removal efficacy and is influenced by physicochemical factors such as adsorbent ratio, presence of other ions, pH, and temperature [58].

Mechanisms of heavy metal uptake through phycoremediation
Phycoremediation of HM occurs via extracellular and intracellular mechanisms.The processes involved in the mechanisms are as shown in Fig. 3.The pathways in the approaches are discussed in subsequent subtopics.

Extracellular mechanisms
There are two mechanisms of extracellular uptake of HM by microalgae in polluted water [9] as shown in Fig. 3.The first involves passive biosorption where the ionic metals adhere and bind on cell surfaces of microalgae without any cell metabolism.Active biosorption, which is metabolism-based is the second mechanism.It involves the active uptake of HM into the extracellular polymeric substances (EPS) in response to stress.In passive biosorption, metal cations bind on the polyanionic cell wall of the microbes via complexation and chelation or ions are exchanged between HM ions and protons at the cell surface.During ion exchange, cations such as Ca 2+, Mg 2+ , Na + and K + found on microalgal cell surfaces are reversibly substituted with HM cations from polluted water [9].Adsorption in this context occurs via bioaccumulation, micro-precipitation, surface complexation, electrostatic interactions and through physical processes including biomineralization, redox interaction, covalent bonding and through van der Waals forces [9].The cell wall of microalgae is made up of glycoproteins such as xylans, alginic acid, mannan and glycan, which play a major role in the passive biosorption [47].Using the functional groups on the cell wall, coordinate bonds are formed with HM facilitating their uptake.Although it is affected by environmental factors, passive biosorption is rapid (5-10 min), occurs in living and dead microalgae biomass, is reversible and not influenced by metabolic inhibitors [41].
Using high molecular weight EPS such as humic substances, sugar, lipids, proteins, nucleic acids and inorganic compounds, which bind on carbohydrates and are produced by microalgae, active biosorption can occur [59].EPS can be soluble in growth media, attached, tightly or loosely bound on microbial cell wall [60].They are produced as a response to stress resulting from the presence of hazardous metals in aquatic environs.During phycoremediation, loosely bound EPS were produced by Chlamydomonas reinhardtii in response to Cd [61] and Pb [62] presence in polluted water [61][62][63].EPS not only improved the biosorption capacity of Cu ions by Chlorella sp. but they also increased tolerance to the metal and prevented its accumulation intracellularly, which disallowed any harm to the microalgal intracellular environment and maintained cellular integrity [.The EPS also have many hydrophobic moieties that are active bindings sites for HM ions [9].The binding of HM on EPS is followed by entrapment and penetration of the toxins to the cell membrane by active transport.The presence of the HM in microalgal cell membrane enhances molecular imitative mechanisms where the elements compete with thiols for binding sites.The process is slow compared to passive biosorption and depends on variations in cellular metabolism.

Intracellular mechanisms
Intracellularly, microalgal species can adjust to the presence of HM to function normally and maintain cellular integrity in the presence of such toxins.Such changes occur through modifications in the function of the cell wall and permeability of the plasma membrane, stimulation of phytochelatin synthase, formation of polyphosphates and metallothioneins (MTs) for HM, activation of the metal efflux system and through organelle compartmentalization [64].

Chelation
MTs are small peptides classified into three with the first two groups (I and II) being gene-encoded while the last class (III) comprises of polypeptides that are enzymatically synthesized such as phytochelatins (PCs) [9].Class II MTs have cysteine in their cytosol.Cysteine combines with other amino acids to form proteins, which bind on metal cations and regulate their concentration levels in microalgae intracellularly.Examples of microalgae with these proteins include Ostreococcus genera, Thalassiosira, Nannochloropsis, Symbiodinium and Chlorella aureococcus species [65].The microbes can also form other MTs in response to the presence of HM in aquatic environments to enhance biosorption.
Microalgae also produce PCs and thiol containing amino acids such as glycine (Gly), cysteine (Cys) and glutamate (Glu), which have the sulfhydryl moiety in their cysteine structure that binds to HM. Chelation by PCs begins formation of Ƴ-Glu-Cys with the help of Ƴ-glutamylcysteine synthase followed by formation of glutathione (GSH) using glutathione synthetase (GS) enzyme.Two GSH molecules bind to form 2-Gly.In this case, GSH serves as a ligand in the presence of low HM concentrations while PCs are present if concentrations are high [9].In phycoremediation of Hg, Cu, Pb and Cd from polluted water using C. sorokiniana, S. bijugatus, Stichococcus bacillaris and Chlamydomonas microalgal species, PCs that stimulated bioremediation at varying degrees were produced [9].Wang et al. [66] reported the production of GSH by Dunaliella salina after its exposure to As-polluted water.Once the PCs bind on HM, they form organometallic complexes stored in organelles of microalgae.
Microalgae accumulate orthophosphate polymers (polyps) in their vacuoles that vary based on their consistency, composition and disposition [67].Polyps are also found in the nucleus, endoplasmic reticulum, mitochondria, cell wall and cytoplasm [68].Polyps are produced in response to osmotic stress resulting from the presence of HM in polluted water.The polymers help to sequester and detoxify the trace elements.In C. reinhardtii, polyps enable metal sequestration, compartmentalization and bioaccumulation to maintain cell homeostasis intracellularly [68].

Compartmentalization of HM in cell organelles
There are various theories that attempt to explain the complexation of HM with various proteins and their subsequent sequestration to specific cell organelles such as mitochondria, chloroplasts and vacuoles.For this reason, the intracellular localization of HM by microalgae has been studied using various biophysical techniques including microscopy and spectroscopic imaging [69].In various phycoremediation studies using biophysical techniques, Pb ions were accumulated in vacuoles of Pseudochlorococcum typicum [70], Cr ions in vacuoles of Micrasterias denticulate, Cd in chloroplast of Euglena gracilis and C. reinhardtii [71] while Cu was found in pyrenoid and thylakoids of Oocystis nephrocytioides [72].

Biotransformation and HM avoidance mechanisms
Through biotransformation mechanisms, the activity, excretion and toxicity of pollutants is changed to favor deactivation and detoxification to non-harmful chemicals [73].During decontamination of HM from aqueous solutions, enzymatic and biochemical processes enhance biotransformation.Enzymatic biotransformation converts HM from being highly toxic to less harmful inorganic complexes through redox reactions catalyzed by oxidoreductase enzymes found in microalgae [9].Examples of such enzymes in microalgae include arsenate reductase, mercuric reductase and chromate reductase that target the biotransformation of As, Hg and Cr, respectively [5].Microalgal strains such as C. vulgaris convert Cr (VI) to Cr (III) using chromate reductase [74] while Galdiera sulphuraria, Selenastrum minutum and C. fusca bio-transform Hg 2+ to meta-cinnabar (β-HgS) and elemental Hg (Hg 0 ) using mercuric reductase [75].In C. reinhardtii, arsenate biotransformation in the presence of arsenate reductase was reported [76].
Biochemical mechanisms also propagate HM phycoremediation.Case examples include the reduction of Cr (VI) to Cr (III) after electrons are transferred to GSH in its reduced form [74]. Similar biochemical mechanisms have been found to detoxify As (V) to As (III) using C. salina [77] and C. aciculare [78].In both cases, the metalloid is compartmentalized in cellular fractions including debris, cell membrane, cytosol and lipids.First, As (V) is converted to As (III) before its methylation to form monomethylarsonate {MMA(V)}, a process catalyzed by S-adenosymethionine (SAM) and oxidase [9].MMA (V) is subsequently reduced to dimethylarsinate {DMA(V)} and dimethylarsinic acid {DMA(III)} before its bioconversion to arsenoribosides, arsenobetaine, arsenosugars and arsenolipids, which are less toxic forms of the metalloid [79].
In addition to biotransformation, other mechanisms can occur intracellularly and extracellularly to promote HM phycoremediation.Metal nanoparticles can be biosynthesized in or on the surface of microalgae cells [80].The nanoparticles modify HM to malleable forms by complexing them with antioxidant, pigment, carbohydrate, lipid and protein molecules, which reduce the metal cation charge to a valence of zero [81].Extracellular production of nanoparticles is simpler than intracellular because of the presence of reducing agents on the cell surface of microalgae [17].Some microalgae such as E. gracilis bio-transformed Hg to a less toxic form via biological or non-biological volatilization [82] while C. vulgaris used photoreduction to transform Cr (VI) to Cr (III) [83].

Advances and future prospects in phycoremediation
In this study, it is evident that microalgae have great potential in HM bioremediation from polluted aquatic environments.This was evident from the laboratory experiments reported (Table 1), which showed high metal removal efficacies.However, the microbes must adapt effectively at varied environments, withstand attack by foreign agents and have high metal tolerance to enhance their metal removal efficacy [16].The microalgae should have high lipid and biomass production capacity to increase the active binding sites and also adsorb high metal concentrations.These microalgal characteristics will enable the advancement of laboratory experiments to field-scale applications.To optimize the activity of microalgae in HM remediation of polluted water, it is essential to select and screen for fast and abundant growing native strains to make such treatment processes efficient.The factors influencing phycoremediation need optimization based on the specific microalgal strain since they improve selectivity and uptake of targeted HM, enhance the biotransformation, mitigation and intracellular bioaccumulation capacity of the toxins in the microbes [9].Modern advances that are physicochemical and biotechnologically motivated have been suggested to improve phycoremediation and its inherent mechanisms.Selection and screening of the toxins has been optimized using bioinformatics advances.
Physicochemical approaches such as functionalization of cell surfaces, chemical pretreatment of microbes and magnetic modification of microalgae with micro-and nano-particles (NP) have been used to advance HM phycoremediation research.The approaches aim at modifying the cell surface characteristics to enhance sorption and affinity to metal 1 3 cations resulting to their immobilization and the introduction of new available active biding sites by removing the blocking ions [9].In Neochloris alveolaris and N. minuta, biosorption of Cd, Cu, Ni, Pb and Zn cations was enhanced by addition of metal acid to functionalize its binding sites [84].Additionally, organic solvents such as toluene, acetone and alcohol as well as inorganic salts (Na 2 CO 3 and NaCl) have been used to pretreat microalgae cell surfaces to remove their blocking ions and enable uptake of metal cations from contaminated water [85].Coating C. vulgaris with magnetic iron oxide NP enhanced the removal of Cd and Pb from polluted water [86] while modifying Chlorella sp. with polyethylenimine magnetic NP enhanced the removal efficacy of Zn cations [87].Careful execution of the physicochemical modifications during HM phycoremediation is essential to prevent biomass loss and decreased selectivity of targeted metal cations and avoid cost increases during field-scale applications [9].
Through bioengineering of microbes and techniques such as cell surface display and cell surface engineering, new biosorbents have been synthesized and have enhanced metallosorption features to increase HM adsorption selectivity [88].Bioengineering is based on the over-expression of passenger proteins such as PCs and MTs on the cell surface.The passenger proteins bind on HM to enable their exportation into the cell and also promote the affinity of the toxins to the binding sites on the cell wall of microbes [89].The man-made proteins made through protein and genetic engineering are located in cell compartments of microalgae and have pre-programed properties to enable high metal affinity [9].Transgenic C. reinhardtii 2AMT-2, which expressed MT polymer on cell membrane showed high capacity to remove Hg from polluted aqueous solutions [90].Such bioengineering studies are done with other microbes (bacteria and fungi) but rare for microalgal strains, which needs advanced research for their optimized application.
Genetic engineering to enable HM phycoremediation involves gene encoding to enable biotransformation mechanisms, high affinity for metal cations to enzymes and chelators among other binding proteins and the transportation of the toxins across microalgal cell membrane [9].Through genetic manipulation, exogenous DNA is introduced to microalgae cells to make them transgenic in addition to overexpression of genes related to phycoremediation mechanisms [91].In C. reinhardtii, a metal tolerance gene (MTG) was overexpressed, which enhanced the tolerance to Cd toxicity and improved its bioaccumulation in the microalgae [92].Similarly, the internalization and bioaccumulation capacity of C. reinhardtii for Cd and Zn cations was improved by genetically modifying the microbe with AtHMA 4 transporter protein [93].C. reinhardtii complexed with HISN3 gene had high tolerance to Ni cations from polluted water unlike the microalgae that was not genetically modified [94].On the other hand, overexpression of the P5CS gene in the same microalgae (C.reinhardtii) resulted to increased tolerance for Cd cations [95].Genetic transformation of Chlorella sp. with mercuric reductase (merA) resulted to a double uptake of Hg in aqueous solutions compared to the wild type [96].Modification of C. reinhardtii with CrGNAT gene for acetyltranferase [97] and gshA synthetic gene [98] promoted Cd and Cu cation bioaccumulation, respectively.Uptake of the metal cations was enhanced by the production of PCs, Cys and GSH, which promoted intracellular phycoremediation.Incorporation of microalgal consortium in HM remediation from polluted water is also growing since it improves the resilience and stability of such systems to nutritional and environmental changes and resists pathogenic attacks [16].Overall, the mechanisms of phycoremediation require further studying to decipher the best-fit mechanisms and optimal factors for bioremediation.Such undertakings will enhance microalgae specificity to metal biosorption, adaptability to stressful conditions and pollutant bioaccumulation efficacy based on the HM characteristics of the targeted polluted aquatic ecosystems.

Conclusion
This study was aimed at exploring the factors that regulate phycoremediation and the mechanisms involved in the process.Obtained information was aimed at promoting a shift from laboratory scale to field scale application of phycoremediation.Various studies reported that microalgal species were capable of removing toxic HM from aqueous solutions at different efficacy rates and using a variety of mechanisms.The removal mechanisms applied were broadly categorized as extracellular biosorption and intracellular bioaccumulation based.The mechanisms were influenced by a number of factors including the available functional groups, characteristics of cell surfaces, type of microalgae species/ strains used, nutrient availability, size of biosorbent and metal concentration among other environmental aspects.The study found that the application of phycoremediation of HM in water treatment at field-scale is still at nascent stages.To this effect, the study suggested the need to advance research in the area to enhance the selectivity and efficiency of the process as well as make it cost efficient.Some of the suggestions made include the use of bioinformatics in selecting and screening appropriate microalgal strains, genetic engineering of microalgae to enhance their adsorptive capacity,

Review
Discover Water (2023) 3:14 microbial pretreatment and the use of consortium to enhance remediation efficacy and enable large-scale application of phycoremediation.

Table 1
Microalgal species used in heavy metal remediation

Table 2
Functional groups of various microalgal species used in HM phycoremediation