The Cell Wall of Green Microalgae and Its Role in Heavy Metal Removal

Received: 14 December 2020 Accepted: 15 March 2021 DOI: 10.1111/ppl.13405

SPECIAL ISSUE ARTICLE Physiologia Plantarum

The cell wall of green microalgae and its role in heavy metal removal

Olivia Spain | Martin Plöhn | Christiane Funk

Department of Chemistry, Umeå University, Umeå Abstract Heavy metals in industrial wastewaters are posing a serious threat to the environ- Correspondence Christiane Funk, Department of Chemistry, ment and to human health. Microalgae are increasingly being seen as potential solu- Umeå University, 901 87 Umeå, Sweden. tions to this problem as they can remove pollutants through biosorption. This Email: [email protected] process offers certain advantages over other more traditional metal removal tech- Funding information niques as it is simple, inexpensive, eco-friendly, and can be performed over a wide Energimyndigheten, Grant/Award Number: 2018-017772; NordForsk, Grant/Award range of experimental conditions. Biosorption is possible due to the unique and com- Number: 82845; Svenska Forskningsrådet plex structure of the microalgal cell wall. The variety of functional groups on the sur- Formas, Grant/Award Number: 2019-00492; Umeå Universitet; Vinnova, Grant/Award face of the cell wall (such as carboxyl or amino groups) can act as binding sites for the Number: 2017-03301 heavy metals, thus removing them from the environment. This review focuses on the

Edited by P.-E. Jensen cell wall composition and structure of the most commonly used microalgae in heavy metal removal and shows the role of their cell wall in the biosorption process. This review also aims to report the most commonly used models to predict the velocity of microalgal biosorption and the removal capacities.

1 | INTRODUCTION can be seen either as an obstacle (e.g., for extraction of valuable compounds from within the cells) or as an opportunity (e.g., for bio- Microalgae are a diverse group of unicellular and simple multicellular sorption of heavy metals or the production of carbohydrates). A photosynthetic microorganisms that are present in all existing ecosys- precise and in-depth knowledge of the cell wall properties is therefore tems on Earth (Mata et al., 2010). Due to their very limited growth required to optimize microalgal industrial processes and thus to requirements and high adaptability, they can be grown in an extensive reduce their overall costs. However, surprisingly little is known about variety of environmental conditions and do not require fertile land algal cell wall properties, probably because their composition, struc- (Benedetti et al., 2018). Microalgae only need sunlight, simple nutri- ture, and thickness depend greatly on the strain, growth phase and ents including nitrogen, sulfur, phosphorous, and carbon dioxide environmental conditions in which the algae are grown. Due to its (Pignolet et al., 2013) and can complete a full growth cycle within high costs and energy requirements, cell wall disruption is considered hours. Their inexpensive growth requirements as well as their advan- to be one of the most challenging bottlenecks in algal industrial appli- tage of being utilized simultaneously for multiple technologies cations. Gaining knowledge of the composition of algal cell walls (e.g., carbon mitigation, biofuel production, and bioremediation) have would enable the enhancement and/or design of sustainable disrup- made microalgae more and more popular for various biotechnological tion techniques and a better understanding of processes which applications (Suresh Kumar et al., 2015) (Figure 1). The algal cell wall involve the cell wall such as heavy metal removal. has important implications in these biotechnological applications, and Heavy metals are natural elements that can be found in every ecosystem on Earth. Even though they are essential for basic bio-

Olivia Spain and Martin Plöhn contributed equally to this study. chemical and physiological functions in both the animal and plant

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. © 2021 The Authors. Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.

Physiologia Plantarum. 2021;1–10. wileyonlinelibrary.com/journal/ppl 1 2 SPAIN ET AL. Physiologia Plantarum

FIGURE 1 Schematic view of the product value chain of microalgae kingdoms, some heavy metals can be toxic to living cells. In certain applications such as heavy metal removal. In this review, we mainly concentrations and environmental conditions, metal ions can damage focus on the strains that have been reported in both cell wall studies DNA, causing conformational modifications that can then lead to car- and heavy metal removal studies. cinogenesis or cell death (Tchounwou et al., 2012). Due to anthropo- Chlorella and Scenedesmus are reported to be two of the most genic activities, the quantity of heavy metals in the environment has widely used algae for heavy metal removal, due to their high bio- significantly risen over the past decades. This increase in concentra- sorption capacities (Dwivedi, 2012). The genus Chlorella includes tion has inevitably led to an increase of our exposure to the metals single-celled, spherical green microalgae of about 2–10 μm in diame- and by consequence, an increase in heavy-metal related diseases. The ter. Chlorella is currently the most cultivated microalga worldwide, metals that are currently considered to be the most problematic are mainly due to its rapid growth rate, high photosynthetic efficiency copper (Cu), cadmium (Cd), chromium (Cr), magnesium (Mn), iron (Fe), and high nutritional value (Masojídek & Torzillo, 2008). Chlorella cells zinc (Zn), mercury (Hg), and lead (Pb) as these metals are toxic even in can contain up to 70% of protein (in dry weight), making the biomass low concentrations (Javanbakht et al., 2014). Industrial wastewaters very valuable to the food industry (Liu & Hu, 2013). Chlorella vulgaris from mining, agriculture, battery manufacturing etc. are often highly is one of the most commonly reported Chlorella species for heavy polluted with heavy metals. Conventional adsorbents (e.g., activated metal removal. carbons, zeolites, clays), nanostructures (reviewed in [Burakov The genus Scenedesmus includes the species Scenedesmus, et al., 2018]), as well as biosorbents (plant material, fungi) are used to Desmodesmus, and Acutodesmus, colonial green microalgae, which fre- remove heavy metals and other contaminants from the effluents quently exist in coenobia of four or eight cells inside a mother wall. (reviewed in Ajiboye et al., 2021). Found across the world, Scenedesmus is one of the most common In this review we will show that, due to the biochemical proper- freshwater algae and is commonly farmed for applications such as oil ties of microalgal cell walls, biosorption via microalgae has the poten- production or waste-water treatment (Pignolet et al., 2013). The pres- tial to offer a new eco-friendly, efficient, and cost-effective solution ence of a very thick cell wall makes Scenedesmus species very resistant to remove heavy metals from wastewater. As it is estimated that there to digestion and predation. are more than 50 000 species of microalgae (Richmond, 2003), each This review will also mention Nannochloropsis and Neochloris spe- with different cell wall structures and compositions, this review will cies, genera of unicellular green microalgae, as their cell wall struc- focus on a selected few industrially valuable and well-reported strains tures are well reported. Both Nannochloropsis and Neochloris are of Chlorophyceae. efficient lipid producers, making them extremely interesting for biofuel production (Rashidi & Trindade, 2018; Scholz et al., 2014). Certain species of Nannochloropsis have also been shown to be very effective 2 | CURRENT KNOWLEDGE OF CELL WALL biosorbents of heavy metals (Kaparapu & Krishna Prasad, 2018; STRUCTURE AND COMPOSITION OF GREEN Sjahrul, 2013). MICROALGAE

2.1 | Most frequently used strains for heavy metal 2.2 | Cell wall architecture removal As the interface between the cell cytoplasm and the outside environ- The cell wall composition of microalgae has only been studied for a ment, the cell wall is the first barrier between the cell and heavy very limited number of strains (Table 1). These strains do not always metals. The cell wall is mainly composed of carbohydrates, proteins, coincide with those that are currently used in biotechnological and lipids, who each offer negatively charged functional groups at SPAIN TAL ET .

TABLE 1 The cell wall structure and composition of different microalgal strains

Genus Strain Cell wall structure (layers) Cell wall composition References Chlorella Chlorella vulgaris One phospholipidic inner layer Glucosamine-rigid wall (composed of galactose and rhamnose) Takeda (1991), Abo-Shady et al. (1993) 30% polysaccharides, 2.46% proteins, 15% lipids, 52.54% unknown substances Chlorella zofingiensis Inner layer and trilaminar 70% glucose and 30% mannose in its “rigid cell wall” and 65% Rodrigues and Da Silva Bon (2011) outer layer mannose, 30% glucose, plus minor amounts of rhamnose and galactose in its matrix cell wall Chlorella homosphaera 85% glucose 15% mannose in its “rigid cell wall” and 70% mannose, 20% glucose, and 10% galactose in its matrix cell wall Chlorella fusca Inner layer and trilaminar Ketocarotenoids and sporopollenin Burczyk and Hesse (1981) outer layer Scenedesmus Scenedesmus obliquus Inner layer and trilaminar 24–74% of neutral sugars, 1–24% uronic acids, 2–16% Abo-Shady et al. (1993) outer layer proteins, and 0–15% glucosamine Scenedesmus acutus Not reported Fibrallar fraction: Schiariti et al. (2004) 13% mannose and 87% glucose Non-fibrillar fraction: 23% rhamnose, 6% arabinose, 21% xylose, 50% galactose Neochloris Neochloris oleoabundans Inner layer and mono- 24.3% carbohydrates, 31.5% proteins, 22.2% lipids, and 7.8% Rashidi and Trindade (2018) electron-dense outer layer inorganic material Nannochloropsis Nannochloropsis oculata Bilayer structure 68% glucose, 4 to 8% of each rhamnose, mannose, ribose, Scholz et al. (2014) xylose, fucose, and galactose Nannochloropsis gaditana Bilayer structure 75% cellulose Scholz et al. (2014) Penium Penium margaritaceum Inner layer (consisting of Not reported Domozych et al. Domozych (2014) cellulose), an interfacing medial layer and an outer layer (HG-rich lattice) Botryococcus Botryococcus braunii Trilaminar structure with A fibrous β-1, 4- and/or β Hubert-Baudelet et al. (2017) algaenan −1, 3-glucan-containing cell wall Weiss et al. (2012) Desmodesmus Desmodesmus spp. An outer cell wall layer with Not reported Hegewald (1997)

net-like structure, lifted by Physiologia Plantarum tubes and rosettes covered or surrounded by tubes 3 4 SPAIN ET AL. Physiologia Plantarum their surface. These groups can capture and therefore remove metal While most publications currently use taxonomy as a means of ions from the surrounding environment via counter-ion interactions, classification, a recent review proposed to divide Chlorophyta into making the cell wall the main participator in heavy metal removal. three groups, based on the biochemical composition of the cell wall Thus, the knowledge of the structure and intrinsic composition of the (Baudelet et al., 2017). The first group of algae (namely Prasinophytina cell wall is extremely important when studying heavy metal removal and Chlorodendrophyceae) has cell walls that are mainly composed of via algae. 2-keto-sugar acids 3-deoxy-manno-2-octolusonicacid (Kdo), 3-deoxy- Cell walls can be categorized according to the layers composing 5-O-Methyl-manno-2-octolusonic acid (5OMeKdo), and 3-deoxylyxo- the cell wall. Three distinct conformations have been identified within 2-heptulosaric acid (Dha). The second group contains the unicellular Chlorophyta: the cell wall can either be made up of one single microfi- algae from Trebouxiophyceae and Chlorophyceae. Their cell walls are brillar layer or comprise both an inner and an outer layer. Depending principally composed of mannans, glucans, chitin-like polysaccharides on the species, the outer layer can have two different structures: a and, most importantly, an algaenan layer. This layer is the main char- mono-electron-dense layer or three sub-layers, with the latter usually acteristic of Group 2 and makes the cell wall highly resistant to many containing algaenan. Chlorella vulgaris, for example, only has an inner sorts of treatments (Rashidi & Trindade, 2018)(Rodrigues & Da Silva layer (Rashidi & Trindade, 2018). However, Chlorella zofingiensis and Bon, 2011). Group 3 is composed of green macroalgae that contain Chlorella homosphaera have both an inner and an outer layer, and xylan, mannan, glucan and sulphated and/or pyruvylated both have the trilaminar version of the outer layer (Rodrigues & Da polysaccharides. Silva Bon, 2011). The outermost layer of the Chlorella trilaminar While these current classifications are based on the type or quan- layer is composed of sporopollenin, the middle layer is principally tity of carbohydrates in the cell wall, Chlorophyta cell walls also con- mannose and chitin-like polysaccharides and the innermost layer is tain significant amounts of other components including proteins, a phospholipid bilayer (Dixon & Wilken, 2018). Scenedesmus lipids, and uronic acids. Chlorella and Scenedesmus cell walls are gener- obliquus, and many other Scenedesmus species, also have a trilaminar ally thought to be made up of 24–74% of neutral sugars, 1–24% outer layer, making the cell wall extremely resistant to strong non- uronic acids, 2–16% proteins, and 0–15% glucosamine (Abo-Shady oxidative chemical treatments and to lytic enzymes (Voigt et al., 1993). The cell wall of Chlorella fusca contains 80% of carbohy- et al., 2014). Neochloris oleabundans has a thin, but electron-dense drates (the majority being neutral sugars), 10% proteins, 0.2% ash, and mono outer layer and a thick low electron-density inner layer 13% of unknown compounds. Cell walls of N. oleabundans are com- (Rashidi & Trindade, 2018). In the case of Nannochloropsis,thecell posed of 24.3% carbohydrates, 31.5% proteins, 22.2% lipids, and wall is reported to have bilayer structure comprised of an outer 7.8% inorganic material (Rashidi & Trindade, 2018). Nannochloropsis algaenan layer, protecting an inner cellulose layer (Scholz gaditana has cell walls with a very high carbohydrate content, with et al., 2014). As we can see from these examples, the biochemical 75% of the total dry mass of the cell wall material being composed of composition of microalgal cell walls varies greatly not only between pure cellulose. The cell wall is estimated to contain only 6.2% ± 1.7% genus but even between strains, and it is not consistently similar to protein. higher plants, where carbohydrates are always and by far the main constituent of the cell wall. 2.4 | The outer layer and its role in biosorption

2.3 | General biochemical composition of algal cell The functional groups at the outer layer of the algal cell wall interact walls based on different classifications with the surrounding environment and can act as binding sites where positively charged metal ions interact with negatively charged groups Given the tremendous diversity of cell wall composition in Chlo- on the cell wall surface. The knowledge of their properties is thus rophyta, there are many different ways to categorize and compare essential when trying to understand mechanisms such as biosorption. algae based on their cell wall properties. In 1991 Takeda divided Biosorption can be defined as the rapid and reversible binding of ions 40 Chlorella strains into two categories based on the sugar composi- from aqueous solutions onto functional groups present on the surface tion of their cell wall; one group contained a glucosamine-rigid wall of biomass (Michalak et al., 2013). The process can be carried out (including C. vulgaris) and the other a glucose-mannose rigid wall under a wide range of experimental conditions and is a simple, effi- (Takeda, 1991). The matrix sugar composition of the first category cient and environmentally friendly technique, making it an interesting consisted mainly of galactose and rhamnose and that of the second process for use in heavy metal removal (Javanbakht et al., 2014). category of principally mannose. The rigid part of the cell wall usually Spectroscopic studies have shown that functional groups including represents 60–66% of the cell wall dry matter. The rest of Chlorella carboxyl, hydroxyl, sulfate, sulfhydryl (thiol), phosphate, amino, amide, cell wall is hemicellulosic. As the algae grows, the mass of the imine, thioether, phenol, carbonyl (ketone), imidazole, phosphonate, hemicellulosic section increases, whereas the mass of the rigid frac- and phosphodiester have the properties to be involved in metal bind- tion stays constant (Baudelet et al., 2017). This type of classification is ing (Figure 2) (Javanbakht et al., 2014). useful for the development of characterization techniques such as cell The carboxyl group has been reported as one of the key players wall staining for microscopy studies. in heavy metal adsorption. The carboxylic moieties can bind heavy SPAIN ET AL. 5 Physiologia Plantarum

FIGURE 2 Schematic picture of the biosorption process of metal ions to the functional groups on the microalgal cell wall (adapted from Javanbakht et al., 2014)

FIGURE 3 Influence of different factors on the removal of Cr (VI) by the microalgae Scenedesmus sp. (A) Initial pH value, (B) Contact time, (C) Initial Cr (VI) concentration, and (D) Temperature (adapted from Pradhan et al., 2019)

metals (like Cd2+) in two different ways, either replacing the H+ in the being adsorbed by different compounds of the microalgal cell wall and carboxy group (type-I) or—after longer exposure—form carboxylates its functional groups (Kotrba et al., 2011). This non-metabolic process (type-II) (Doshi et al., 2007). The impact of these groups has been con- is highly dependent on a variety of different parameters (e.g., pH, tem- firmed by a range of techniques, for example, Fourier-transform infra- perature, concentration, biosorbant dosage, or contact time), with the red spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) microalgal strain, the contact time, and the pH being the most impor- showing the interaction with heavy metals during the adsorption pro- tant aspects (see Figure 3) (Pradhan et al., 2019), whereas tempera- cess (Sheng et al., 2004). This adsorption process has been reported ture seems only to affect the kinetics but not the capacity (Sedlakova- as a biphasic mechanism. In the primary, rapid phase, metal ions are Kadukova et al., 2019). These factors are also influenced by the heavy 6 SPAIN ET AL. Physiologia Plantarum metal itself. Therefore, the Cr(VI) in Figure 3 is just one example for After integration with the boundary conditions t =0,q = 0, and at the different dependencies of heavy metal removal by microalgal spe- t = t, q = q Equation (2) is expressed as: cies. Uptake of cationic metals benefits from a higher pH, whereas the 1 1 removal of anionic ions is improved at lower pH values (Xue = + k t q −q q 2 et al., 1988). e e In the second metabolic phase, metal ions are being absorbed and accumulated within the cells. Since this involves an active The pseudo second order equation also combines the adsorption transport and the binding to proteins such as phytochelatins or on the surface within the first phase with the incorporation of the sec- metallothioneins, the process is relatively slow (Mehta & ond phase (Robati, 2013). Gaur, 2005). In contrast to the first phase, this second phase is Both equations are usually applied in parallel to describe the dependent on factors like temperature or the metabolic state itself, kinetics of biosorption. Based on the data, these equations differ as the active transport of metal ions requires energy (Perales-Vela slightly in their corresponding r2 value. Since many of these models et al., 2006). are based on empirical data, the kinetic constants will vary and need to be determined by study related curve fitting. Although being one of the most viable tools, non-linear regression often replaces linear 3 | COMMONLY USED MODELS FOR regression as it allows the usage of different error functions for the BIOSORPTION KINETICS AND ISOTHERMS best possible predictions (Foo & Hameed, 2010). The biosorption isotherms of heavy metals can also be evaluated In addition to the biological aspects of heavy metal removal, the with a big variety of models, which vary in the number of depending chemical mechanisms of biosorption are also important for a better parameters. These models can be used to describe the amount of −1 understanding of the removal process. This will allow to predict the metal ions taken up at equilibrium (qe mg g ) and the corresponding −1 velocity of the removal or the maximum amount of heavy metals that concentration of metal ions in the solution (Ce mg L ). Among those may be adsorbed. models the Langmuir- and the Freundlich-equation are commonly There are two different kinetic models, namely pseudo-first and used two-parameter isotherm models to predict the adsorption of pseudo-second order, both of which are used to describe biosorption heavy metals on algal surfaces. More than 100 years ago, Langmuir under nonequilibrium conditions. These equations include the first developed an empirical model to describe the adsorption of gases on −1 −1 −1 and second order constants k1 (h ) and k2 (g mg h ) as well as the a monolayer surface. It can be expressed as follows: amount of heavy metal adsorbed q and the maximum amount of K C heavy metal adsorbed qe at equilibrium. Due to the large number of q = q L e e max 1+K C chemical groups within the algal cell wall, there is a high number of L e possible interactions. The pseudo-first order equation by Lagergren −1 (Lagergren, 1898) covers the biosorption from the liquid phase and is where qmax describes the maximum adsorption capacity (mg g ) and −1 generally expressed as: KL represents the adsorption constant at equilibrium (L mg ) (Langmuir, 1918). The Freundlich model is used to describe adsorption ÀÁ k1t processes on a multilayer surface by the following equation: q = qe 1−e ð1Þ

1=n qe = kFCe After integration with the boundary conditions t = 0, q = 0, and at −1 t = t, q = q Equation (1) is expressed as: where kF is the relative adsorption capacity (mg g ) and n represents the heterogeneity factor (Freundlich, 1906). The SIPS model

lnðÞ qe −q = lnqe −k1t (Sips, 1948) represents one of the most used three parameter isotherm models and combines the Langmuir and the Freundlich equa- It describes the correlation between the solid capacity and the tion. In relation to experimental conditions such as pH or adsorption of liquid solid systems. Therefore, the different microalgal concentration, it reduces either to the Freundlich isotherm or predicts surfaces will affect the kinetics and its parameters. a monolayer adsorption capacity as described by Langmuir (Foo & The pseudo-second order Equation (2) was proposed by Hameed, 2010). Blanchard et al. (1984) and has been developed further (Ho & y McKay, 1999). Like the pseudo-first-order equation it highly depends ðÞKsCe q = q e max ðÞy on the solid capacity but the presence of two surface sites is essential 1+ KsCe for the theory. The presented models can be used to describe the interaction k q2t q = 2 e ð2Þ between heavy metal ions and the microalgal surface and they further 1+k q t 2 e allow to draw conclusions on the amounts of heavy metals being SPAIN ET AL. 7 Physiologia Plantarum

TABLE 2 Biosorption of different heavy metals by microalgal strainsa

−1 Metal ions Algal species pH qe (mg g ) References As (III) Chlamydomonas reinhardtii 9.5 4.6 Saavedra et al. (2018) Chlorella vulgaris 5.5 3.9 Scenedesmus almeriensis 9.5 5.0 Cd (II) Chlorella minutissima 6 303 Yang et al. (2015) Chlorella sp. 6 15.5b Shen et al. (2018) Parachlorella sp. 7 96.2 Dirbaz and Roosta (2018) Scenedesmus acutus IFRPD 1020 7 110 Inthorn et al. (2002) Cr (VI) Chlorella vulgaris 1.5 163.9c Gokhale et al. (2008) Nannochloropsis oculata 2 37.7 Kim et al. (2011) Scenedesmus quadricauda 1 46.5 Shokri Khoubestani et al. (2015) Cu (III) Chlamydomonas reinhardtii 9.5 2.4 Saavedra et al. (2018) Chlorella vulgaris 7 2.7 Scenedesmus almeriensis 9.5 1.9 Hg Chlorella vulgaris 5 17.5 Solisio et al. (2019) Chlamydomonas reinhardtii 6 106.6b Bayramoglu et al. (2006) Scenedesmus acutus IFRPD 1020 7 20 Inthorn et al. (2002) Pb (II) Chlamydomonas reinhardtii 6 380.7b Bayramoglu et al. (2006) Scenedesmus acutus IFRPD 1020 7 90 Inthorn et al. (2002) Chlorella sp. 6 10.4 Molazadeh et al. (2015) Zn (II) Chlamydomonas reinhardtii 5.5 2.7 Saavedra et al. (2018) Chlorella vulgaris 7 2.7 Scenedesmus almeriensis 5.5 2.8 aStudies were performed in controlled medium with addition of relevant heavy metal. bImmobilized. cDried biomass.

adsorbed. A large number of other isotherm models (e.g., Dubinin- functional groups of glycoproteins (Macfie & Welbourn, 2000). Further- Radushkevich, Temkin, Flory-Huggins, Redlich Peterson etc.) are more, the predicted data using the Langmuir equation fitted almost per- focusing on different aspects like the adsorption of aromatics by acti- fectly the experimental values, showing once more the high reliability of vated carbon or the structure of the adsorbent (Dubinin, 1947; this model (Bayramoglu et al., 2006). Flory, 1942; Huggins, 1942; Liu & Liu, 2008; Peterson & Cadmium is the most commonly used metal in removal studies Redlich, 1959; Temkin & Pyzhev, 1940). due to its extremely high toxicity and therefore the urgent need to eliminate this heavy metal. Chlorella sp. and Parachlorella sp. can adsorb around 90 mg of cadmium per gram of biomass, making them 3.1 | Biosorption of heavy metals by different efficient at wastewater treatment. Scenedesmus acutus IFRPD 1020 microalgal strains was shown to adsorb even more cadmium when grown under normal conditions at pH 7.0 with 12/12 h day/night cycle (Inthorn

A selection of qe values for different heavy metals, in dependence of spe- et al., 2002). Efficient biosorption of other heavy metals such as lead, cific pH values, and different microalgal strains can be found in Table 2. nickel, or copper is also commonly reported for many different algal Although the table presents only a small selection of studies, one can still strains (see Table 2). According to the studies summarized in Table 2, see that C. vulgaris, Scenedesmus sp., and Nannochloropsis sp. are three of which were performed with living algae and/or dead cells, almost all the most investigated microalgae. Chlamydomonas reinhardtii is also biosorption studies were performed under neutral pH conditions extremely potent in adsorbing heavy metals when, for example, (pH around 7.0). These environmental conditions support the survival immobilizedinoronCa2+-alginate beads (Bayramoglu et al., 2006). of the microalgal strains, even when exposed to heavy metals and at Under weak acidic conditions, this microalga can adsorb almost twice as the same time the chemical properties of the functional groups that much mercury and even higher amounts of lead than any other strain. are involved in the biosorption process are optimal. According to Macfie and coworkers, this might be related to metal ions In multiple studies, the highest amounts of adsorbed hexavalent being bound by thiol, hydroxyl or amid groups or proteins as well as by chromium were obtained under very acidic conditions at pH 1–2 8 SPAIN ET AL. Physiologia Plantarum

(Gokhale et al., 2008; Shokri Khoubestani et al., 2015) whereas the tri- the presence of diverse and well-distributed functional groups on the valent chromium was adsorbed under weak acidic conditions (pH 5), surface of their cell wall, microalgae have strong potential to remove with sorption capacities of 31–58 mg g−1, depending on the micro- heavy metals from wastewater via biosorption. However, most of the algal strain (Kim et al., 2011; Shokri Khoubestani et al., 2015). studies cited in this review were based on metal solutions rather than industrial wastewater on industrial scales. For efficient heavy metal removal in industrial applications, the combination of biosorption with 3.2 | Living vs. dead biomass for biosorption other methods such as bioconcentration or bioconversion could be a strategic procedure. Moreover, as many types of algae have not yet The biosorption process can occur in both live or dead cells. However, been evaluated for their biosorption capacities, screening more algal dead biomass is more commonly used as it presents multiple advan- species could help to select strains that have high affinities for specific tages over using living algal cells. Indeed, dead cells do not require any heavy metals. Before commercializing microalgae as biosorbents, nutrients or specific environmental conditions and can be used over a heavy metal removal by biosorption should also be performed in wide range of experimental variables (temperature, pH, etc.). Dead larger scales. This would allow the assessment of microalgae's ability biomass can easily be stored for long periods of time without loss of to adapt to a wide variation of nutrients and sunlight as well as the effectiveness (Michalak et al., 2013). In contrast to non-living biomass, evaluation of the economic feasibility of the technology. Furthermore, living algal cells can bioaccumulate heavy metals. This deeply affects additional modeling of biosorption processes is needed to better pre- their metabolism and is, in most cases, extremely toxic to the cells. dict the removal of heavy metals by microalgae. Furthermore, as enzymatic activity is preserved in living cells, enzymes Overall, the removal of heavy metals by microalgal biomass offers may alter the pollutant via biotransformation or biodegradation, which an eco-friendly and sustainable process, which could be improved by could make the recovery of the pollutant more difficult (Torres, 2020). further knowledge of the cell wall composition and of the basic mech- Other commonly reported advantages of using dead biomass over liv- anisms of biosorption. ing cells include the possibility to regenerate and reuse the biomass, the easy immobilization of the dead cells and the easier mathematical ACKNOWLEDGMENTS modeling of the kinetics (Chu & Phang, 2019). Finally, and most The authors would like to thank the Swedish Energy Agency (Grant importantly, dead microalgal cells have been reported to adsorb heavy no. 2018-017772, project: 48007-1), Vinnova (2017-03301), the metals to greater extent than living cells (Aksu & Kutsal, 1990). NordForsk NCoE program “NordAqua” (Project no. 82845), FORMAS Sibi (2014) showed that the biosorption rate of As(III) by different (2019-00492) and Umeå University as well as the Industrial Doctoral kinds of microalgae was significantly higher when using dead biomass School at Umeå University for financial support. than when using living cells (Sibi, 2014). Open access funding enabled and organized by Projekt DEAL.

DATA AVAILABILITY STATEMENT 3.3 | Advantages and disadvantages of using Data sharing is not applicable to this article as no new data were cre- biosorption ated or analyzed in this study.

Biosorption is just one of many methods used for heavy metal ORCID removal. It offers several advantages in comparison to more tradi- Christiane Funk https://orcid.org/0000-0002-7897-4038 tional wastewater treatment techniques like chemical precipitation or coagulation-flocculation. As the biomass is cheap and easy to produce, REFERENCES the overall cost of the treatment is very low. The algae can remove Abo-Shady, A.M., Mohamed, Y.A. & Lasheen, T. (1993) Chemical composi- several types of heavy metals at a time and both the biomass and the tion of the cell wall in some green algae species. Biol. Plant, 35, 629– 632. https://doi.org/10.1007/BF02928041. metals can be recovered at the end of the treatment. Furthermore, Ajiboye, T.O., Oyewo, O.A. & Onwudiwe, D.C. (2021) Simultaneous biosorption does not require the use of harsh chemicals, and is thus removal of organics and heavy metals from industrial wastewater: a sustainable and environmentally friendly. However, the use of bio- review. Chemosphere, 262, 128379. https://doi.org/10.1016/j. sorption for heavy metal removal also has some disadvantages such chemosphere.2020.128379. Aksu, Z. & Kutsal, T. (1990) A comparative study for biosorption character- as the rapid saturation of the active sites on the surface of the bio- istics of heavy metal ions with C. vulgaris. Environmental Technology, mass and the reversible sorption of the metals to the binding sites 11, 979–987. https://doi.org/10.1080/09593339009384950. (Dodbiba et al., 2015). Baudelet, P.H., Ricochon, G., Linder, M. & Muniglia, L. (2017) A new insight into cell walls of Chlorophyta. Algal Research, 25, 333–371. https:// doi.org/10.1016/j.algal.2017.04.008. Bayramoglu, G., Tuzun, I., Celik, G., Yilmaz, M. & Arica, M.Y. (2006) Bio- 4 | CONCLUSIONS sorption of mercury(II), cadmium(II) and lead(II) ions from aqueous sys- tem by microalgae Chlamydomonas reinhardtii immobilized in alginate The architecture and composition of Chlorophyta cell walls is very beads. International Journal of Mineral Processing, 81, 35–43. https:// complex yet very important for biotechnological applications. Due to doi.org/10.1016/j.minpro.2006.06.002. SPAIN ET AL. 9 Physiologia Plantarum

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