Magnetically modified microalgae and their applications.

http://informahealthcare.com/bty ISSN: 0738-8551 (print), 1549-7801 (electronic) Crit Rev Biotechnol, Early Online: 1–11 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/07388551.2015.1064085

REVIEW ARTICLE

Magnetically modified microalgae and their applications Ivo Safarik1,2, Gita Prochazkova3, Kristyna Pospiskova2, and Tomas Branyik3

Critical Reviews in Biotechnology Downloaded from informahealthcare.com by University of Otago on 07/11/15 For personal use only.

1

Department of Nanobiotechnology, Institute of Nanobiology and Structural Biology of GCRC, Ceske Budejovice, Czech Republic, 2Regional Centre of Advanced Technologies and Materials, Palacky University, Olomouc, Czech Republic, and 3Department of Biotechnology, University of Chemistry and Technology Prague, Prague, Czech Republic Abstract

Keywords

The majority of algal cells can interact with a wide range of nano- and microparticles. Upon interaction the modified cells usually maintain their viability and the presence of foreign material on their surfaces or in protoplasm can provide additional functionalities. Magnetic modification and labeling of microalgal biomass ensures a wide spectrum of biotechnological, bioanalytical and environmental applications. Different aspects of microalgal cell magnetic modification are covered in the review, followed by successful applications of magnetic algae. Modified cells can be employed during their harvesting and removal, applied in toxicity microscreening devices and also as efficient adsorbents of different types of xenobiotics.

Harvesting algal cells, magnetic labeling, magnetic modification, magnetic separation, microalgae

Introduction Prokaryotic cyanobacteria (blue-green algae) and eukaryotic microalgae are promising sources for biomass and primary/ secondary metabolite production with considerable use in the food, feed, aquaculture, pharmaceutical, cosmetic or fuel industries. Commercial large-scale production systems date back to the 1960s for Chlorella and 1970s for Arthrospira (Spirulina), reaching from 1980 worldwide distribution (Spolaore et al., 2006). Microalgae are also an essential part in many environmental applications where they can act as biofertilizers/soil N2-fixators (Pulz & Gross, 2004), biocatalysts in bioaccumulation and biodegradation of organic pollutants (Semple et al., 1999), materials for wastewater treatment (Abdel-Raouf et al., 2012; Markou & Nerantzis, 2013) or parts of biosensors for detection of toxic compounds (Haigh-Florez et al., 2014). From an industry perspective the attractiveness of these organisms is not only based on their rich metabolic reactions leading to numerous valuable products, but they are also easy to cultivate, fast growing and able to adapt to various environmental conditions. Process costs can be reduced as microalgae are able to use wastewater, sunlight or flue gas as nutrient and energy sources. Additionally, they reach higher

Address for correspondence: Ivo Safarik, Department of Nanobiotechnology, Institute of Nanobiology and Structural Biology of GCRC, Na Sadkach 7, 370 05 Ceske Budejovice, Czech Republic and Regional Centre of Advanced Technologies and Materials, Palacky University, Slechtitelu 27, 783 71 Olomouc, Czech Republic. Tel: +420 387775608. Fax: +420 385310249. E-mail: [email protected] or URL: http://www.nh.cas.cz/people/safarik/

History Received 19 November 2014 Revised 2 May 2015 Accepted 14 May 2015 Published online 7 July 2015

productivities compared to conventional forestry, agricultural crops and other aquatic plants, thus much less land area is required. Unlike agricultural plants, microalgal metabolite/ biomass production can be all year-round, independent of weather conditions with cultivation sites built upon nonarable land or wasteland (Mata et al., 2010; Xu et al., 2009). Nevertheless, studies also question the sustainability and costefficiency of various microalgal biotechnological processes, especially concerning the production of biofuels, and several bottlenecks are discussed which still need to be resolved (Lam & Lee, 2012; Lardon et al., 2009; Singh & Olsen, 2011; Slade & Bauen, 2013). To further improve desirable production properties of selected microalgal species, different genetic techniques are being exploited and tested in order to obtain recombinant strains. Both cyanobacteria and eukaryotic microalgae would be very promising potential cell factories, but the development of transformation systems has yet to be optimized in many cases. Additionally, the risks of genetically modified organism release needs to be assessed for full scale production (Wijffels et al., 2013). It must not be forgotten that in addition to the biotechnologically attractive species there are also those species that display negative effects, for example, unpleasant tastes and odors (Van Durme et al., 2013) or biofouling. In recent years, the occurrence of harmful algal blooms has increased due to eutrophication. As cyanobacteria are often present, they can accumulate in the food chain leading to animal poisoning and risks to human health (Mata et al., 2010; Mundt et al., 2001). Thus, research is also focused on removing such species from the environment by environmentally attractive means.

2

I. Safarik et al.

Crit Rev Biotechnol, Early Online: 1–11


Figure 1. Transmission electron microscopy of dried C. vulgaris cell [(a) bar line is 1 mm] and magnetic fluid modified Chlorella cell [(b, c), bar lines are 2 mm and 200 nm, resp.]. Reproduced, with permission, from Safarikova et al. (2008).

Novel contributions to tackle the above-mentioned problematic areas involve the faster and easier manipulation of microalgal species via magnetic modification with subsequent application of an external magnetic field. Generally, the majority of prokaryotic and eukaryotic cells (including algal cells) is able to interact with a wide range of nano- and microparticles, such as gold, silver, palladium or silica ones, carbon nanotubes. The modified cells usually maintain their viability, but the presence of foreign material on their surfaces, in the protoplasm or in intracellular organelles can provide additional functionalities. Cell modification with magnetic nano- and microparticles is particularly important and magnetically modified cells have been successfully used in many applications (Safarik et al., 2014). This review article focuses on different procedures of magnetic modification of microalgal cells and on the most important examples of their applications by addressing various biotechnological important areas (harvesting, removing harmful algal species, etc.).

Magnetic labeling of microalgal cells The absolute majority of cells (including microalgal cells) exhibit diamagnetic behavior. The magnetic modification of originally diamagnetic algal cells can be usually performed by the attachment of magnetic nano- or microparticles onto the cell surface. The basic procedures used for the magnetic modification of algal cells involve non-specific attachment of magnetic nanoparticles (MNPs) (e.g. by magnetic fluid treatment) (Safarikova et al., 2008) or magnetic microparticles (Prochazkova et al., 2013b) onto the cell surface, polymer-modified iron oxides particles binding (Fakhrullin et al., 2010; Toh et al., 2014c), covalent immobilization of magnetic particles on algae cell surface or vice versa (Venu et al., 2013), specific interactions with immunomagnetic nano- and microparticles (Aguilera et al., 1996; Aguilera et al., 2002; Safarik & Safarikova, 1999) or entrapment (together with magnetic particles) into biocompatible polymers (Eroglu et al., 2013). Magnetic properties of the modifiers are mainly due to the presence of nano- or microparticles of magnetite (Fe3O4) or maghemite (g-Fe2O3) or their mixtures. In some cases, ferrite particles (Gao et al., 2009) can also be used. In most cases, the viability and phenotype alternation of modified cells is not negatively affected by the attached magnetic particles. It should be taken into account that in specific cases the surface bound particles can be internalized by the treated cells and will appear in

the protoplasm. The common characteristics of all magnetically modified cells are their specific interactions with an external magnetic field. The individual modification procedures will be described in more details below. Non-covalent interaction of target cells with magnetic nano- and microparticles Different types of magnetic microparticles, as well as ionically and sterically stabilized MNPs, have been used for magnetic cells modification. Magnetic modification of Chlorella vulgaris cells can be performed using an appropriate magnetic fluid. First, dried Chlorella cells were thoroughly washed several times with 0.1 M acetic acid to remove a substantial portion of soluble macromolecules which would otherwise cause the magnetic fluid to spontaneously precipitate. Subsequent addition of perchloric acid stabilized magnetic fluid led to the deposition of magnetic iron oxides nanoparticles onto the cells surface and formation of magnetically responsive algal cells (Figure 1) (Safarikova et al., 2008). The same magnetic fluid has been successfully used for magnetic modification of selected diatoms or chrysomonads (Diadesmis gallica, Mallomonas kalinae); in this case magnetic modification can be performed in methanol (Kratosova et al., 2013). Colloidal particles of hydrous iron(III) oxide were also applied to magnetize algal cell surfaces followed by cell separation using a high gradient magnet. The percentage of algal cell recovery depended on the added amount of iron oxide particles. It has reached almost 100% when a very small amount of particles was added to the algal suspension. These results demonstrate the feasibility of algal cell magnetic separation with no addition of a polymer stabilizer (Takeda et al., 2000). Alternatively, magnetite particles in combination with aluminum sulfate were used for the magnetization and separation of Anabaena and Aphanizomenon cells (Bitton et al., 1975), while magnetite combined with ferric chloride was used for magnetization and separation of Scenedesmus obliquus cells (Yadidia et al., 1977). An extremely simple procedure for the magnetic modification of algal cells has been developed recently which is based on the use of microwave synthesized magnetic iron oxides nano- and microparticles. Two very inexpensive starting chemicals are used (ferrous sulfate heptahydrate and sodium or potassium hydroxide); after their mixing and formation of mixed iron hydroxides the suspension underwent


DOI: 10.3109/07388551.2015.1064085

Magnetically modified microalgae and their applications

3

Figure 2. TEM images of the thin sections of (a) bare and (b, c) (poly)allylamine hydrochloride-stabilized MNPs coated C. pyrenoidosa cells. Reproduced, with permission, from Fakhrullin et al. (2010).

microwave treatment (a regular kitchen microwave oven can be used successfully) and nano- and microparticles of magnetic iron oxides formed (Pospiskova et al., 2013; Zheng et al., 2010). Mixing of magnetic particles with algal cell (C. vulgaris) suspensions caused cell flocculation and magnetically responsive cell aggregates (usually ca. 100–300 mm in diameter) were formed (Prochazkova et al., 2013b). Diatom particles have been magnetically labeled with human serum albumin (HSA)-coated iron oxides nanoparticles, prepared from oleic acid/oleylamine-coated nanoparticles. Subsequently, the nanoparticles were surface-exchanged with dopamine and added to an aqueous solution of HSA, where the protein molecules were adsorbed onto the particle surface to grant them good aqueous stability. Diatoms were contacted with the modified particles in PBS at room temperature for 2 h. Multiple amine groups on the surface of the MNPs ensured their partial positive charge, making them appropriate to interact with negatively charged cell surfaces (Todd et al., 2014). Chitosan (CS) can also be successfully used for surface modification of magnetic iron oxide nanoparticles (IONPs). Simple mixing promoted the linkage between Chlorella sp. and modified nanoparticles. Subsequent magnetophoretic separation of magnetized Chlorella cells was carried under low gradient magnetic separation (LGMS) with a NdFeB permanent magnet in an inhomogeneous magnetic field with magnetic field gradient (rB)580 T/m. It was also shown that MNPs can enter the cells. The internalization of nanoparticles may be through a passive uptake or adhesive interaction (Toh et al., 2014b; Toh et al., 2014c). A very useful procedure for coating algal cells by magnetite nanoparticles via electrostatic interactions (EI) has been described recently. (Poly)allylamine hydrochloride stabilized positively charged MNPs (average diameter around 15 nm) were used for the magnetization of living Chlorella pyrenoidosa cells. The single-step magnetization procedure is very simple and consisted of the dropwise introduction of the aqueous suspension of algal cells into the nanoparticle solution followed by intensive shaking for 10 min.

TEM images (Figure 2) demonstrated the uniform layer of MNPs on the cell walls with the thickness around 90 ± 20 nm (Fakhrullin et al., 2010). Another similar cationic polyelectrolyte, namely poly(diallyldimethylammonium chloride) (PDDA) promoted effective attachment of IONPs onto microalgal cells (Chlorella sp. or naturally occurring microalgae) through electrostatic attraction (Lim et al., 2012; Toh et al., 2014b; Toh et al., 2012). A magnetic material based on montmorillonite, a commonly used clay, was manufactured by the supporting of Cu(II)/Fe(III) oxides on pillared montmorillonite prepared from Na-montmorillonite using aluminum polycation. This magnetic material was used for modification and subsequent magnetic removal of the cyanobacteria Microcystis aeruginosa (Gao et al., 2009). Specific interactions with immunomagnetic nano- and microparticles Immunomagnetic detection and modification of cells is based on the use of magnetic nano- or microparticles with immobilized monoclonal or polyclonal specific antibody enabling their selective attachment to target cells when added to a cell suspension. After incubation, target cells with attached magnetic particles are isolated with the help of an appropriate magnetic separator. In the direct method, the antibodies against target surface epitopes are attached to the magnetic particles, which are then added to the cells containing sample. In the indirect method, the cell suspension is incubated with free primary antibodies which bind to the target cells. Then, magnetic particles with immobilized secondary antibodies, protein A or protein G are added, enabling the beads to bind rapidly and firmly to the primary antibodies on the target cells. Alternatively, primary antibodies can be biotinylated and magnetic particles with immobilized streptavidin are used for capturing the target cells (Safarik et al., 2014; Safarik & Safarikova 1999). Recently, the cells of toxic dinoflagellate Alexandrium fundyense have been isolated from natural seawater plankton samples using Dynabeads with immobilized monoclonal

4

I. Safarik et al.


antibody against the surface antigens. Both direct and indirect procedures were tested as well as three types of modified magnetic beads (MBs) (streptavidin, and two secondary antibodies: sheep anti-mouse and goat anti-mouse). Optimal indirect bead attachment protocol enabled separation of 90% of the labeled A. fundyense cells in unialgal cultures (with non-specific binding from 5% to 10%). Simpler ‘‘direct’’ technique (with recovery 80% and non-specific binding ca. 2%) enabled pre-coating of beads with the specific antibody in bulk before use, which shortened the procedure and eliminated target cell losses (Aguilera et al., 1996; Aguilera et al., 2002). Alternatively, purified polyclonal antibodies against Heterosigma akashiwo were immobilized to carboxylated MBs Seradyn (Huang et al., 2012). Entrapment of cells into magnetic biocompatible gels Prokaryotic and eukaryotic cells can be entrapped in natural or biocompatible synthetic carriers (gels) and the magnetically responsive gels are formed by the addition of appropriate magnetic materials (Safarik et al., 2014). Immobilization of algal cells into different non-magnetic matrices has been described many times (Mallick, 2002). However, only exceptional examples of algal cell entrapment into magnetic gels can be found in the literature such as the entrapment of microalgal cells (C. vulgaris) within a nontoxic polyvinylpyrrolidone (PVP) polymer matrix containing superparamagnetic magnetite nanoparticles using continuous flow vortex fluidic device. High entrapment efficiency (up to 95%) was obtained. Entrapped cells can be separated from the PVP matrix using mild-sonication (Eroglu et al., 2013). Other procedures for algal cells magnetization An exceptional modification procedure was used for magnetization of cyanobacterium Synechocystis sp. PCC 6803. This photosynthetic microorganism was immobilized on aminefunctionalized MBs (Dynabead M-270 Amine) by 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC)–N-hydroxysulfosuccinimide (NHS) coupling chemistry. The carboxylic groups present on the Synechocystis cell wall were activated with EDC and then subjected to NHS to produce aminereactive NHS esters enabling interaction with aminefunctionalized MBs to produce covalent amide bonds (Venu et al., 2013).

Magnetotactic algae The first magnetotactic alga, found in brackish mud and water samples coming from a coastal mangrove swamp near Fortaleza, Brazil, was tentatively identified as Anisonema platysomum; cells contained numerous, well-organized chains of bullet-shaped magnetite crystals ranging from 80 to 180 nm long by 40–50 nm wide (Torres de Araujo et al., 1986). The arrangement of magnetosomes appears to be so precisely structured in this alga that most probably this organism biomineralizes and arranges endogenous magnetite crystals in a highly controlled fashion within the cell, where intracellular structural filaments play a significant role in the synthesis of the magnetosome chain, as has been shown for magnetotactic bacteria (Posfai et al., 2013).


Application of magnetically responsive microalgal cells Already in the 1970s, magnetic modification of microalgae has been applied for removing harmful algae from lakes and ponds (Bitton et al., 1975). Since that time, the application potential of magnetically responsive algae increased substantially, as discussed in the next section of this review. Harvesting of microalgal biomass Methods for dewatering microalgal cultures, together with their advantages and disadvantages, have been extensively reviewed in numerous studies (Harun et al., 2010; Kim et al., 2013; Shelef et al., 1984). Typically, the harvesting of microalgae includes one or more solid–liquid separation steps and methods such as centrifugation, gravity filtration, various forms of flocculation, sedimentation and flotation (Chen et al., 2011; Christenson & Sims, 2011; Sukenik & Shelef, 1984; Uduman et al., 2010; Vandamme et al., 2013). Biomass recovery represents one of the critical bottlenecks of largescale process feasibility as it claims 20–30% of total biomass production costs (Mata et al., 2010). Difficult cell separation is caused by the low sedimentation velocity of microalgae, which is given by their small cell size (typically 5–10 mm), colloidal character with repulsive negative surface charges and low biomass concentrations in culture broths (50.5 kg/m3 dry biomass in some commercial production systems), so large volumes need to be processed in order to concentrate the cells (Molina Grima et al., 2003; Shelef et al., 1984). For cost efficient downstream processing it is essential to significantly reduce the volume of the microalgal suspension after cultivation not by a single, but by a two-step process with concentration factors of 100–200 in the primary and 2–10 in the secondary procedure (Shelef et al., 1984; Uduman et al., 2010). Before applying energy-consuming physical cell separation processes that require expensive equipment (e.g. centrifugation, filtration), it is beneficial to pre-concentrate the microalgal culture using a less expensive technique such as flocculation (Gonza´lez-Ferna´ndez & Ballesteros 2013; Granados et al., 2012; Vandamme et al., 2012a). It must be emphasized that the choice of the harvesting method is strongly case dependent. Among other factors such as final product price, the selection of a suitable harvesting method can be made according to the desired level of moisture in the product. Sludge obtained by gravity sedimentation contains generally a higher amount of moisture than biomass recovered by centrifugation. This could substantially influence the economics of product recovery if the subsequent downstream processing step would be an energy demanding (e.g. thermal drying) process (Mata et al., 2010). Magnetic nano- and microparticles have potential for harvesting agents. The attractiveness of bioseparation processes applying such agents is caused by the non-destructive nature of magnetic field, use of simple devices and particles biocompatibility, easy manipulation and regeneration (Cerff et al., 2012; Lim et al., 2012; Prochazkova et al., 2013b; Safarikova et al., 2008; Safarik et al., 2012; Safarik & Safarikova, 2009; Yavuz et al., 2009). Magnetic harvesting of microalgae by application of an external magnetic field after successful adsorption of a magnetic agent onto microalgal


DOI: 10.3109/07388551.2015.1064085

cells can also be viewed as a single-step process, since flocculation and separation occur simultaneously (Vandamme et al., 2013). Magnetic agents applied to harvest various microalgal species can be in the form of uncoated magnetic iron oxides nano- and microparticles (Hu et al., 2013; Lee et al., 2014; Prochazkova et al., 2013b; Xu et al., 2011) or as functional nanocomposites that consist of, e.g. a magnetic core coated with silica. This coating can additionally carry specific functional groups such as polyethylenimine (PEI) or cationic polyelectrolytes such as CS, PDDA and cationic polyacrylamide (CPAM) (Cerff et al., 2012; Hu et al., 2014; Lee et al., 2013; Lim et al., 2012; Toh et al., 2014b; Toh et al., 2014c; Prochazkova et al., 2013a; Wang et al., 2014). Subsequent sections will be devoted to a more detailed consideration of individual agents, harvesting efficiencies under various environmental conditions and mechanisms involved. Physicochemical aspects of magnetic harvesting In general microorganisms are prone to solid surface adhesion to achieve a more efficient state and so minimize the free interfacial energy. In an aqueous environment it must be considered that a whole range of physicochemical surface interactions such as non-covalent Lifshitz van der Waals forces (LW), EI and acid–base interactions (AB) occurs during the adhesion of agal cells to magnetic particles (Bos et al., 1999). Additionally, as surfaces carry different surface charges, electric double layers are formed. These phenomena lead to attraction and/or repulsion between surfaces upon contact. The surrounding environment’s ionic strength strongly influences the thickness of the electric double layer, thus it determines the decay of EI with distance (van Oss, 2003). As EI are prominent under low ionic strength conditions, i.e. with values bellow 0.1 M (Bilanovic et al., 2009), they most likely have a more important function during magnetic algal cell separation from freshwater than from marine culture media. During the study of a given freshwater microalgal magnetic harvesting system, it could be of great use to first characterize the behavior of the interacting surfaces in a defined way under model conditions (e.g. 10 mM KCl, pH 2–12) as there is no presence of complex interfacial phenomena. These can occur in culture media with interfering ions such as Ca2+ and Mg2+ that precipitate under alkaline conditions and subsequently bind to the cell surface (Vandamme et al., 2012a). Zeta potential (surface charge) measurements of the microalgal cells and magnetic particles surfaces have shown various surface charges depending on the surrounding environment. Microalgal cells maintain predominantly a negative surface charge due to the carboxyl, phosphate or hydroxyl groups over a wide pH range (Hadjoudja et al., 2010; Henderson et al., 2008; Lim et al., 2012; Zhang et al., 2012), so potential harvesting agents have to be charged positively. A pH-sensitive surface charge of magnetic particles is ensured by surface hydroxyl species that cover the metal oxide. Thus, below the isoelectric point the metal oxide’s surface is positively charged as it gains protons and vice versa being negative as it loses protons above its isoelectric point (Parks, 1965). Thus, a potentially simple


5

strategy of microalgal harvesting by pH manipulation would consist of the following steps: (i) flocculation of microalgae with magnetic particles by electrostatic attraction, (ii) magnetic separation of magnetized cells and (iii) recovery of the magnetic particles from the flocs by electrostatic repulsion (Lee et al., 2014). Subsequent studies described in the following sections will show that such an approach is significantly dependent upon the type of magnetic agent applied, the microalgal species and environmental conditions. Prochazkova et al. (2013b) have demonstrated the harvesting of C. vulgaris with iron oxide magnetic microparticles (IOMMs) prepared from Fe(II) precursor and microwave treatment. IOMMs carried a positive surface charge under low pH values having an isoelectric point (pI) equal to 6.2, with a typical ion exchange nature of (naked) iron oxide particles as has been confirmed also in other studies (Gregory et al., 1988; Hencl et al., 1995; Sun et al., 1998; Xu et al., 2011). At pH values below the magnetic particle’s pI, adhesion of IOMMs to microalgal cells was successful, accompanied by intensive EI and subsequent high separation efficiencies after subjecting the mixture to an external magnetic field. The most significant difference between zeta potentials of the interacting cells and IOMMs (67 mV) occurred under model environmental conditions (10 mM KCl at pH 4), resulting in the most intensive EI and highest separation efficiencies (95% at pH 4 at doses of 800 mg IOMMs/g microalgal biomass). Thus, high separation efficiencies can be achieved under acidic pH because the differences between the zeta potential values of the interacting particles are very pronounced. Nevertheless, it must be emphasized that IOMMs–microalgae attachment can also take place under basic conditions even as the surfaces of both cells and IOMMs are negatively charged (Prochazkova et al., 2013b). This specific phenomenon of attachment can be mediated at molecular level via positively charged domains and local attractive EI despite overall repulsion (Bos et al., 1999). Apart from EI, several other non-covalent forces may occur during microalgal cell adhesion to magnetic particles. Physicochemical approaches, such as the XDLVO (extended Derjaguin–Landau–Verwey–Overbeek) theory, can show the interplay of all non-covalent LW, EI and AB forces, and as such they are considered to be very helpful tools in predicting (un)favorable conditions for microbial cell adhesion (Hwang et al., 2012; Kurec & Branyik 2011; Sirmerova et al., 2013). Illustration of modeling using the XDLVO theory, i.e. the dependency of the total Gibbs energy of interaction (DGTOT) and its components AB, LW and EI upon the distance between a microalgal cell and a model spherical particle is shown in Figure 3. According to the trend, it is possible to predict whether microbial adhesion will be under the given conditions favorable (DGTOT50) or not (DGTOT40) (Prochazkova, 2015). Described approaches have been successfully introduced also in the case of physicochemical characterization of microalgal cell attachment/detachment to MBs under model conditions (Prochazkova et al., 2013a). Two types of defined MBs that carried ion exchange functional groups (DEAE – diethylaminoethyl and PEI) contacted microalgae (C. vulgaris) under various model conditions. Zeta potential and contact angle measurements were applied to characterize the interacting surfaces.

6

I. Safarik et al.


The importance of non-covalent interactions is reported in many studies also for harvesting microalgae directly in culture media, as will be shown in detail in the subsequent chapter. Additionally, it must be emphasized that non-covalent interactions can be accompanied by other forces especially when polyelectrolytes are included into magnetic particles, such as polymer bridging, hydrophobic interactions, the nano-effect or hydrogen bonding (Cheng et al., 2011; Li et al., 2009; Toh et al., 2014b).


Magnetic harvesting in culture media

Figure 3. Illustration of modeling using the XDLVO theory, i.e. the dependency of the total Gibbs energy of interaction (DGTOT) and its components (AB, LW and EI) upon the distance between a microalgal cell and a model spherical particle of the same size: cell diameter ¼ 6.9 mm, Hamaker constant ¼ 0.9 kT, zeta potential of the cells ¼ 27 mV, zeta potential of model particle ¼ 10 mV, ionic strength of solution ¼ 10 mmol/L (Prochazkova, 2015).

Beads carrying strong anion exchange groups (PEI) displayed a high isoelectric point (pI ¼ 9.0), while those bearing the weak ionex group (DEAE) had a pI equal to 6.3. Nevertheless, both MBs displayed positive surface charges over a wide range of pH values. Together with the cell/MB sizes the obtained data served for predicting (un)favorable conditions for cell harvesting and bead detachment using the XDLVO approach. Subsequently, test tube experiments were performed to verify the predictions. Optimal harvesting efficiencies (490%) were found for DEAE and PEI MBs, where the most effective separations were achieved in model environment (pH 4) at doses of 200 mg DEAE MBs and 100 mg PEI MBs/g of microalgal biomass, respectively. Efficient detachment was achieved only for DEAE MBs (490%) upon changing environmental conditions to basic where repulsive EI interactions occur. Previous predictions by the XDLVO model were in most cases in accordance with these findings and showed the importance of cell–magnetic particle surface complementarity for successful bead attachment and vice versa in the case of bead detachment. A discrepancy was found between the XDLVO prediction and the poor PEI MBs– microalgal surface detachment. Probably an additional interaction occurred (e.g. covalent bonds) between the microalgal surface and PEI MBs, that the XDLVO model is not able to characterize as it is based on non-covalent interacting forces. Thus, this work emphasizes the crucial influence of ionex character of the magnetic agent together with influence of the environment’s pH value upon microalgal adhesion/detachment (Prochazkova et al., 2013a). XDLVO analysis has also been recently applied to study the attachment of bare and PDDA IONPs onto microalgal cells, Chlorella sp. and Nannochloropsis sp., in both freshwater and seawater. Obtained results showed the prominent role of EI in freshwater conditions. LW and AB interactions played more dominant roles in high ionic strength media (100 mM NaCl), such as seawater. For all cases up to 99% separation efficiencies were achieved (Toh et al., 2014a).

Upon changing the conditions from model solutions to culture media, the whole system of harvesting microalgae has to be considered from several new perspectives such as the type of microalgal species, interference of various components present in the medium (e.g. ions or algogenic organic matter) leading to a higher dosage of magnetic agent, toxicity of agent applied, regeneration of the magnetic agent and reuse of culture media after magnetic cell separation. Each of these aspects will be addressed in more detail showing the simplicity, reliability, velocity, efficiency and wide applicability of various magnetic agents. The effect of ion interference and culture medium composition on magnetic harvesting has been shown in the case of IOMMs. The most positive prominent effect of composition on separation efficiencies in freshwater culture medium (pH 6.5) was shown in the case of removing the main source of phosphorus (KH2PO4), compared to other tested situations where main macro- and microelements were excluded from the culture medium. Separation efficiencies close to 100% were achieved at a dose of 3000 mg of IOMMs/g of microalgal biomass, which was significantly higher when compared to separations under optimal model conditions (800 mg/g for pH 4). The phenomenon of culture media ion interference has been shown also in the case of zeta potential measurements, where IOMMs displayed a positive surface charge only upon omitting phosphorous ions from the medium (Prochazkova et al., 2013b). Another study applying naked magnetic particles was conducted by Xu et al. (2011). Chlorella ellipsoidea and Botryococcus braunii were harvested from modified Chu 13 medium. Separation efficiencies nearing 90% were obtained for C. ellipsoidea at doses of 380 mg/g at pH values 4–9. For B. braunii separation efficiencies nearing 100% were achieved under low pH values (4–7) with doses equal to 20 mg/g. Thus, dependency upon microalgal species can be observed additionally to the effect of culture medium composition. Size of the microalgal cells can play a potentially significant role in magnetic agent dosage as has been displayed in the case of other flocculants, i.e. cationic starch (Vandamme et al., 2010). Composition of the algal cell wall, especially amount of polysaccharides, has also been shown to affect cell flocculation with CS (Cheng et al., 2011). Additionally, in the case of other flocculants such as alum or CS, the presence of algogenic organic matters (AOM) has been shown to increase flocculant dosage upon cell harvesting in culture medium (Shelef et al., 1984;

Unmodified magnetic particles.


DOI: 10.3109/07388551.2015.1064085

Vandamme et al., 2012b). As magnetic harvesting displays many similar features to common flocculation techniques, these observations should be taken into consideration upon optimizing magnetic microalgal harvesting procedures. In the above-named studies, the recovery of naked magnetic particles after microalgal magnetic harvesting was performed using diluted acids to solubilize the particles as the attachment of the particles to the algal cells was very strong. Subsequently, clean microalgal cells could be recovered by filtration (Prochazkova et al., 2013b; Xu et al., 2011). Marine Nannochloropsis sp. has also been successfully harvested with naked magnetic Fe3O4 nanoparticles (Hu et al., 2013). Dependency on growth phase was shown and after 18 days of cultivation the microalgae were harvested with the highest efficiency of 95% at doses of 99 mg particles/g dry microalgal biomass. Improved cell separations were observed under higher biomass concentrations, apparently due to increased collision between the cells and magnetic particles. After 18 days’ cultivation, the harvesting efficiency declined which was probably because of cell concentration decrease with simultaneous increase of AOM that originated from algal cell autolysis. Thus, released AOM could then compete with the microalgal cells for the available MNP active sites. The authors also showed the importance of surface charges; the algal cells were negatively charged at all tested pH values, while Fe3O4 nanoparticles had an isoelectric point at pH 7. Results showed that EI forces were one of the adsorption mechanisms for pH values 4–6. Nevertheless, the study also mentions the nano-effect (nano-size adsorption behavior) at all pH values due to the high specific surface of the nanoparticles. Furthermore, higher temperatures led to an increase in nanoparticles adsorption to microalgal cells. After magnetic separation reused culture medium proved to be effective for biomass production (Hu et al., 2013). Naked Fe3O4 magnetic particles were also applied to harvest oleaginous microalga Chlorella sp. KR-1 (Lee et al., 2014). Electrostatic attraction between microalgal cells and particles was observed with subsequent recovery of the magnetic agent from the harvested flocs by electrostatic repulsion below and above the agent’s isoelectric points (pI 5–6). Magnetic particles detachment could be achieved by submerging the microalgae–Fe3O4 particle flocs into a solution maintained above the agent’s isolelectric point and by using physical methods such as vortexing (recovery efficiency was 90% after 30 s). Thus, electrical repulsion induced by pH shift was alone not sufficient to recover algal cells as the interacting magnetic particle–cell forces were too strong. Lee et al., (Lee et al., 2014) described that microalge has to be completely covered by the Fe3O4 particles, leading to tight agglomerations. In this study, the repeated reuse of the particles has also been tested, i.e. achieving for 10 recycles 90% harvest and recovery efficiencies. Adjustment of appropriate pH value for harvesting is suggested by the simple means of intensive bubbling of CO2, followed by the stopping of CO2 supply upon the recovery step. Furthermore, neither the use of the Fe3O4 magnetic particles nor changing of pH values had any negative effect on microalgal cell growth (Lee et al., 2014).


7

In the case of studies with MBs, doses of 3000 mg/g of hydrophilic silica-coated magnetic particles MagSilica 50–85 were needed to separate 90% of C. vulgaris cells from IGV-medium at pH 8. For Chlamydomonas reinhardtii 90% of cells were separated from TAP-medium (pH 8) at doses close to 800 mg/g. The high pH values needed to achieve such high separation efficiencies may nevertheless have a negative effect on cell viability (Cerff et al., 2012). Additionally, under alkaline conditions in culture medium autoflocculation caused by Mg2+ or Ca2+ precipitation has to be considered to promote the formation of microalgal flocs (Vandamme et al., 2012a). Fe3O4 nanoparticles functionally coated with PEI successfully separated 97% of C. ellipsoidea cells within 2 min. A rise in temperature led to an increase in harvesting efficiency. Adsorption isotherm data fit the Langmuir model, suggesting that the nanoparticles adsorbed in a monolayer. The reusability of the culture medium after magnetic separation was evaluated for repeated microalgal growth in order to achieve improved water-usage in the microalgal cultivation and harvesting process. Following magnetic separation, the liquid medium was successfully reused for microalgal cultivation over 10 batch cycles without any negative effect on the final algal biomass production (Hu et al., 2014). CS–Fe3O4 nanoparticles composites displayed a microalgae harvesting efficiency of over 99% without the need of changing the pH of culture medium (Lee et al., 2013). The authors emphasize the importance of the pH value stability of media (pH 6–7) as it has a significant impact on the culture media’s reusability after magnetic harvesting as the composites will not have an inhibitory effect on algal cells when compared to, e.g. the negative effects of alum (chemical flocculant) on microalgal growth. When the composite with a higher ratio of CS to MNPs was used, higher harvesting efficiency was obtained with the same dosage. In the case of CS/MNPs ratio being 0.54, a 99% harvesting efficiency was obtained by a dosage of 1400 mg/g dry biomass. However, only 95% of microalgae was harvested with 3400 mg/g of another CS/MNPs composite (ratio 0.13) because CS provides adsorption sites for microalgae and, therefore, the composite with a higher CS/ MNPs ratio has more adsorption sites. Lee et al., (Lee et al., 2013) also tested and proved the composites biocompatibility by plating harvested flocs on agar plates. After 3 days of incubation, microalgal growth was evident as visible colonies on the agar surface. In addition, microalgae were successfully cultured by using microalgae-attached CS/MNPs as an inoculum. Subsequent cell growth displayed a typical pattern of microalgal growth. The effect of naked IONPs and composites, i.e. IONPs functionalized with CS, upon contacting microalgal cells has been studied (Toh et al., 2014b). From the cross-section TEM images of cells, naked IONPs had a tendency to internalize Chlorella sp. cells due to a passive uptake or adhesive interaction. The final biofuel quality was not affected as the algal oil has been produced during cultivation, prior to magnetic cell harvesting. When harvesting efficiencies were compared, naked IONPs achieved much lower values (6.2%) than surface functionalized-IONPs (SF-IONPs) (95%). In this MBs and composites.


8

I. Safarik et al.

study, the critical importance of the binding agent, CS, has been proven to enable successful SF-IONPs–microalgal cell adhesion followed by fast separation. Thus, CS plays important roles as: (i) stabilizing agent to ensure the colloidal stability of SF-IONPs prior to attachment, (ii) binding agent to promote the adhesion of SF-IONPs onto cells surface, and also, (iii) bridging agent to enhance flocculation of SF-IONPs-attached-cells in order to achieve rapid magnetophoretic separation under low magnetic field gradient (Toh et al., 2014b). SF-IONPs with PDDA were applied to harvest 99% of Chlorella sp. from culture media (Lim et al., 2012). This study proved that higher separation efficiencies were achieved by the composite compared to separating microalgal cells firstly by PDDA with subsequent addition of MNPs. Additionally, Lim et al. (2012) emphasized the role of magnetic particles shape, introducing rod-like particles as better candidates for magnetophoretic separation in comparison to spherical IONPs. The effect of cationic polyelectrolyte coating of IONPs has been studied by Toh et al. (2014b) with Chlorella sp. as model microorganism. PDDA and CS were tested resulting in cell separation efficiency of up to 98% for the case of PDDA and 99% for the case of CS in 6 min when 3 107 cells/mL Chlorella sp. were exposed to 300 mg/L SF-IONPs. The different polyelectrolytes did not induce significant effects on cell separation efficiency as long as the particle attachment occurred. However, the PDDA is more preferable as a binder for all types of microalgae media than the CS because it is not pH dependent. SF-IONPs coated with PDDA guarantee the cell separation performance for all pH range of cell medium, with 98.2% at pH 8.84. However, the CS performance will be affected by the cell medium pH, with only 22.9% biomass recovery at pH 9.25 (Toh et al., 2014c). Another successful magnetic composite for harvesting microalgae is composed of iron oxide and CPAM. A harvesting efficiency of more than 95% was reached within 10 min using 171.4 mg/g for C. ellipsoidea and 13.9 mg/g for B. braunii. Bridging was the primary flocculation mechanism followed by EI between the microalgal cells and the magnetic flocculant under low pH values (Wang et al., 2014). Removal of microalgal biomass For decades, eutrophication of surface water and algal blooms in lakes, ponds and drinking water reservoirs occurs worldwide because of water contamination by industrial effluents, agricultural activities and domestic wastewater discharge. Thus, toxic algae can grow excessively resulting in water quality deterioration. Additionally, drinking water resources are threatened as several algal species can penetrate filters and release toxins. Another problem occurs during the disinfection process of drinking water production as algal presence can lead to disinfection by-products. Other water quality problems affected by algae must be emphasized as well, i.e. taste, turbidity and odor. The methods used for algae removal include centrifugation, microstraining, coagulation, in-pond chemical precipitation, filtration, flotation and ion exchange (Bitton et al., 1975; Gao et al., 2009).


Magnetic separation has also been successfully applied for algae removal from environmental sources starting 1970s. Algae removal by high gradient magnetic filtration was tested by several research groups (Bitton et al., 1975; Yadidia et al., 1977). The methods were based on suspending magnetic particles (usually magnetite) in the solution. These magnetic particles were coagulated with the algae and the suspension was then passed through a magnetic filter. Algae removal efficiencies from water samples of five Florida lakes were between 55% and 94% using alum as a flocculant and a commercial magnetic filter (Bitton et al., 1975). In another study, algae removal efficiencies were higher than 90% using 5–13 ppm ferric chloride as a primary flocculant and 500–1200 ppm magnetite as a magnetic seed for both laboratory prepared and pond-grown algae suspensions formed mainly of S. obliquus (Yadidia et al., 1977). In addition to application of regular naked magnetite particles, binding of magnetic particles to the algae cell surface has been promoted using appropriate biopolymers or synthetic polymers. The formation of magnetic algae cell flocs after addition of MNPs has been improved and their removal rate increased when CS solution was added to the system. Relatively low CS concentration (less than 10 mg/L) and MNPs concentration (0.05%) was sufficient to achieve the highest removal rate (Lee et al., 2012). Alternatively, magnetite particles were surface modified with CS and subsequently used for M. aeruginosa (harvested from surface water when algal blooms occurred) magnetic modification, resulting in 99% of algal cells removal (Liu et al., 2009). Also, surface modified rod-like IONPS have been employed. In this case, the nanoparticles (20 nm diameter, 300 nm length) were modified with PDDA. The particles were used to bind algae cells present in the fishpond water, namely Scenedesmus sp., Spirulina sp., Chlorella sp., Tetraedron sp., Haematococcus sp. and Dictyosphaerium sp. Based on the magneto-shape anisotropy of rod-liked IONPs, overall separation efficiency of microalgae cells up to 90% has been achieved in less than 3 min. Both high gradient magnetic separation with 5B41000 T/m and LGMS with 5B580 T/m could be successfully used for algae removal (Toh et al., 2012). Magnetically modified inorganic materials, such as magnetic montmorillonite (Gao et al., 2009) and magnetic acidmodified fly ash (Liu et al., 2013), have been also tested for algal bloom removal by the coagulation-magnetic separation method. More than 99% of algal cells were removed within 5 min following the addition of fly ash derived magnetic coagulant at optimal loadings (200 mg/L). This method suggests the possible transformation of a waste material into an efficient coagulant/adsorbent, thus significantly reducing the production cost (Liu et al., 2013). However, fly ash may contain trace concentrations of heavy metals, polycyclic aromatic hydrocarbons as well as other substances detrimental to health (Borm, 1997). Magnetically modified microalgal cells in biotechnology and other biosciences Algae cells can be used as a part of whole-cell biosensors, immobilized on transducers by various types of immobilization techniques, for example, chemical cross-linking, physical


DOI: 10.3109/07388551.2015.1064085

adsorption or incorporation into a suitable matrix. Some drawbacks of these methods, for example, connected with the decrease in viability of the cells, can be overcome using magnetic immobilization processes. This method consists of temporary immobilization of magnetic cells on biosensor surface by an external magnetic field. Magnetic cells are held and manipulated on the electrode by small magnets positioned under the electrode (Naumenko et al., 2014). Recently, a whole-cell amperometric herbicide biosensor based on magnetically functionalized microalgae and screenprinted electrodes has been described (Zamaleeva et al., 2011). Microalgae cells (C. pyrenoidosa) were coated with biocompatible MNPs stabilized by poly(allylamine hydrochloride) (average diameter 15 nm). Suspension of cells was magnetized after mixing with a suspension of magnetic particles during vortexing. Magnetically modified cells were used for construction of amperometric biosensor with screenprinted electrodes. Screen-printed electrode was connected with supporting tetrafluoroethylene plate with inserted NdFeB cylindrical magnet (2 mm in diameter) in the cavity below the working electrode area to produce the strong magnetic field in the vicinity of the electrode. The magnetically modified cells were mixed with the electron mediator (K3Fe(CN)6) and electrolyte (Na2SO4) and the drop of this suspension was placed onto the working area of the electrode. The magnetized cells were immediately assembled above the magnet in the area of magnetic field. This biosensor was applied for the detection of triazine herbicides, inhibitors of photosynthetic activity. The biosensor was able to detect atrazine (from 0.9 to 74 mM) and propazine (from 0.6 to 120 mM) (the limits of detection 0.7 and 0.4 mM, respectively) (Zamaleeva et al., 2011). Magnetically modified diatoms have been suggested as a perspective drug carrier for potential in vivo applications. The cytotoxicity of this material was low, due to the fact that silica and protein-coated MNPs are low-toxic. This diatom-based material exhibited slow biodegradability in body fluids (Todd et al., 2014). Magnetically modified microalgal cells as xenobiotics adsorbents Nonmagnetic immobilized algae cells have often been used for the removal of various toxic compounds (Mallick, 2002). Magnetically modified C. vulgaris cells were applied as a new cost effective magnetic adsorbent for the removal of six watersoluble organic dyes. Dried cells were magnetically modified using water-based magnetic fluid stabilized with perchloric acid (individual MNPs or their aggregates were accumulated on the outer cell surface). Data from a dye adsorption process were fitted to Langmuir isotherm and the maximum adsorption capacities ranged between 24.2 and 257.9 mg of dye/g of dried magnetically modified cells for Saturn blue LBRR and aniline blue, respectively. Increasing the pH value can positively affect the adsorption capacities of some dyes (e.g. crystal violet and safranin O) (Safarikova et al., 2008).

Conclusion and outlook A combination of magnetic properties, represented by magnetic particles/materials, with the metabolic potential of


9

photosynthetic microalgae, promising cell factories, can result in many ways interesting magnetic composites. Given the fact that magnetic modification and labeling of microalgae can be carried out in a targeted way, it permits enhancement of the desired characteristics of the new composites. By selecting the suitable materials and applying the appropriate conditions, it is possible to achieve applications of magnetically modified microalgae in many fields of biotechnology, environmental technology and bioanalytics. One of the bottlenecks of using microalgae for the production of low added value products (food, feed, biofuels) is the harvesting of cells from diluted culture medium. Therefore, magnetic separation can be especially useful for harvesting microalgal biomass prior to downstream processing as well as for the removal of algal bloom from natural water reservoirs. However, both these applications of magnetic separation can be successful only on condition that the magnetic flocculant is either very cheap and/or regenerable. For some particular microalgal strains and conditions such magnetic flocculants have already been described, however, there is no single magnetic flocculant suitable for all microalgae and culture conditions. The flocculant selection and process optimization is a matter of knowledge and experiment. Similar criteria can be applied also for the utilization of magnetically modified microalgae as adsorbents of xenobiotics. The performance of various magnetic particles including unmodified and coated magnetic reagents on microalgae recovery has been summarized in tabular form in the recent paper (Wang et al., 2015). Magnetic properties can also play an important role in biosciences when using magnetically modified algae as parts of biosensors or in immunomagnetic separation procedures. These applications in particular require high specificity and reliability, thus the price of magnetic immobilization or magnetic particles is not of primary relevance. Since microalgae, marine in particular, are a biotechnologically very promising group of organisms, the development of new detection and characterization methods utilizing magnetic particles can be expected. The future research in this area and subsequent technology will depend on the existence of low-cost, easy-to-prepare, biocompatible, environmentally friendly and reusable magnetic nano- or microparticles, available both in the native and modified forms, together with availability of appropriate efficient, safe, easy-to-operate and not expensive magnetic separators. The magnetic separation of microalgal cells should become an integral part of the downstream processes leading to the production of the desired products.

Declaration of interest The authors report no declarations of interest. This research was supported by the Grant Agency of the Czech Republic (Projects Nos. 13-13709S and P503/12/1424) and by the projects LO1305 and LD13021 (Ministry of Education, Youth and Sports of the Czech Republic).

References Abdel-Raouf N, Al-Homaidan AA, Ibraheem IB. (2012). Microalgae and wastewater treatment. Saudi J Biol Sci, 19, 257–75.


10

I. Safarik et al.

Aguilera A, Gonzalez-Gil S, Keafer BA, Anderson DM. (1996). Immunomagnetic separation of cells of the toxic dinoflagellate Alexandrium fundyense from natural plankton samples. Mar Ecol Prog Ser, 143, 255–69. Aguilera A, Keafer BA, Rau GH, Anderson DM. (2002). Immunomagnetic isolation of live and preserved Alexandrium fundyense cells: species-specific physiological, chemical, and isotopic analyses. Mar Ecol Prog Ser, 237, 65–78. Bilanovic D, Andargatchew A, Kroeger T, Shelef G. (2009). Freshwater and marine microalgae sequestering of CO2 at different C and N concentrations – response surface methodology analysis. Energ Convers Manage, 50, 262–7. Bitton G, Fox JL, Strickland HG. (1975). Removal of algae from Florida lakes by magnetic filtration. Appl Microbiol, 30, 905–8. Borm PJA. (1997). Toxicity and occupational health hazards of coal fly ash (CFA). A review of data and comparison to coal mine dust. Ann Occupat Hyg, 41, 659–76. Bos R, van der Mei HC, Busscher HJ. (1999). Physico-chemistry of initial microbial adhesive interactions – its mechanisms and methods for study. FEMS Microbiol Rev, 23, 179–230. Cerff M, Morweiser M, Dillschneider R, et al. (2012). Harvesting fresh water and marine algae by magnetic separation: screening of separation parameters and high gradient magnetic filtration. Bioresour Technol, 118, 289–95. Chen CY, Yeh KL, Aisyah R, et al. (2011). Cultivation, photobioreactor design and harvesting of microalgae for biodiesel production: a critical review. Bioresour Technol, 102, 71–81. Cheng YS, Zheng Y, Labavitch JM, VanderGheynst JS. (2011). The impact of cell wall carbohydrate composition on the chitosan flocculation of Chlorella. Process Biochem, 46, 1927–33. Christenson L, Sims R. (2011). Production and harvesting of microalgae for wastewater treatment, biofuels, and bioproducts. Biotechnol Adv, 29, 686–702. Eroglu E, D’Alonzo NJ, Smith SM, Raston CL. (2013). Vortex fluidic entrapment of functional microalgal cells in a magnetic polymer matrix. Nanoscale, 5, 2627–31. Fakhrullin RF, Shlykova LV, Zamaleeva AI, et al. (2010). Interfacing living unicellular algae cells with biocompatible polyelectrolytestabilised magnetic nanoparticles. Macromol Biosci, 10, 1257–64. Gao ZW, Peng XJ, Zhang HM, et al. (2009). Montmorillonite–Cu(II)/ Fe(III) oxides magnetic material for removal of cyanobacterial Microcystis aeruginosa and its regeneration. Desalination, 247, 337–45. Gonza´lez-Ferna´ndez C, Ballesteros M. (2013). Microalgae autoflocculation: an alternative to high-energy consuming harvesting methods. J Appl Phycol, 25, 991–9. Granados MR, Acien FG, Gomez C, et al. (2012). Evaluation of flocculants for the recovery of freshwater microalgae. Bioresour Technol, 118, 102–10. Gregory R, Maloney RJ, Stockley M. (1988). Water treatment using magnetite: a study of a Sirofloc pilot plant. Water Environ J, 2, 532–44. Hadjoudja S, Deluchat V, Baudu M. (2010). Cell surface characterisation of Microcystis aeruginosa and Chlorella vulgaris. J Colloid Interf Sci, 342, 293–9. Haigh-Florez D, de la Hera C, Costas E, Orellana G. (2014). Microalgae dual-head biosensors for selective detection of herbicides with fiber-optic luminescent O2 transduction. Biosens Bioelectron, 54, 484–91. Harun R, Singh M, Forde GM, Danquah MK. (2010). Bioprocess engineering of microalgae to produce a variety of consumer products. Renew Sust Energ Rev, 14, 1037–47. Hencl V, Mucha P, Orlikova A, Leskova D. (1995). Utilization of ferrites for water treatment. Water Res, 29, 383–5. Henderson R, Parsons SA, Jefferson B. (2008). The impact of algal properties and pre-oxidation on solid–liquid separation of algae. Water Res, 42, 1827–45. Hu YR, Guo C, Wang F, et al. (2014). Improvement of microalgae harvesting by magnetic nanocomposites coated with polyethylenimine. Chem Eng J, 242, 341–7. Hu YR, Wang F, Wang SK, et al. (2013). Efficient harvesting of marine microalgae Nannochloropsis maritima using magnetic nanoparticles. Bioresour Technol, 138, 387–90. Huang J, Wen RB, Bao ZM, et al. (2012). Applications of immunomagnetic bead and immunofluorescent flow cytometric techniques for


the quantitative detection of HAB microalgae. Chin J Oceanol Limn, 30, 433–9. Hwang G, Ahn IS, Mhin BJ, Kim JY. (2012). Adhesion of nano-sized particles to the surface of bacteria: mechanistic study with the extended DLVO theory. Colloid Surf B, 97, 138–44. Kim J, Yoo G, Lee H, et al. (2013). Methods of downstream processing for the production of biodiesel from microalgae. Biotechnol Adv, 31, 862–76. Kratosova G, Schrofel A, Safarik I, et al. (2013). Bionanocomposite, the process of its production and application. Czech Patent, 304046. Kurec M, Branyik T. (2011). The role of physicochemical interactions and FLO genes expression in the immobilization of industrially important yeasts by adhesion. Colloid Surf B, 84, 491–7. Lam MK, Lee KT. (2012). Microalgae biofuels: a critical review of issues, problems and the way forward. Biotechnol Adv, 30, 673–90. Lardon L, Helias A, Sialve B, et al. (2009). Life-cycle assessment of biodiesel production from microalgae. Environ Sci Technol, 43, 6475–81. Lee H, Suh H, Chang T. (2012). Rapid removal of green algae by the magnetic method. Environ Eng Res, 17, 151–6. Lee K, Lee SY, Na JG, et al. (2013). Magnetophoretic harvesting of oleaginous Chlorella sp. by using biocompatible chitosan/magnetic nanoparticle composites. Bioresour Technol, 149, 575–8. Lee K, Lee SY, Praveenkumar R, et al. (2014). Repeated use of stable magnetic flocculant for efficient harvest of oleaginous Chlorella sp. Bioresour Technol, 167, 284–90. Li YG, Gao HS, Li WL, et al. (2009). In situ magnetic separation and immobilization of dibenzothiophene-desulfurizing bacteria. Bioresour Technol, 100, 5092–6. Lim JK, Chieh DCJ, Jalak SA, et al. (2012). Rapid magnetophoretic separation of microalgae. Small, 8, 1683–92. Liu D, Li FT, Zhang BR. (2009). Removal of algal blooms in freshwater using magnetic polymer. Water Sci Technol, 59, 1085–91. Liu D, Wang P, Wei GR, et al. (2013). Removal of algal blooms from freshwater by the coagulation-magnetic separation method. Environ Sci Pollut Res, 20, 60–5. Mallick N. (2002). Biotechnological potential of immobilized algae for wastewater N, P and metal removal: a review. Biometals, 15, 377–90. Markou G, Nerantzis E. (2013). Microalgae for high-value compounds and biofuels production: a review with focus on cultivation under stress conditions. Biotechnol Adv, 31, 1532–42. Mata TM, Martins AA, Caetano NS. (2010). Microalgae for biodiesel production and other applications: a review. Renew Sust Energ Rev, 14, 217–32. Molina Grima E, Belarbi EH, Acien Fernandez FG, et al. (2003). Recovery of microalgal biomass and metabolites: process options and economics. Biotechnol Adv, 20, 491–515. Mundt S, Kreitlow S, Nowotny A, Effmert U. (2001). Biochemical and pharmacological investigations of selected cyanobacteria. Int J Hyg Envir Health, 203, 327–34. Naumenko EA, Dzamukova MR, Fakhrullin RF. (2014). Magnetically functionalized cells: fabrication, characterization, and biomedical applications. In: Katz E, ed. Implantable bioelectronics. 1st ed. Weinheim: Wiley-VCH, 7–26. Parks GA. (1965). The isoelectric points of solid oxides, solid hydroxides, and aqueous hydroxo complex systems. Chem Rev, 65, 177–98. Posfai M, Lefevre CT, Trubitsyn D, et al. (2013). Phylogenetic significance of composition and crystal morphology of magnetosome minerals. Front Microbiol, 4, Article 344. Pospiskova K, Prochazkova G, Safarik I. (2013). One-step magnetic modification of yeast cells by microwave-synthesized iron oxide microparticles. Lett Appl Microbiol, 56, 456–61. Prochazkova G, Podolova N, Safarik I, et al. (2013a). Physicochemical approach to freshwater microalgae harvesting with magnetic particles. Colloid Surf B Biointerfaces, 112, 213–18. Prochazkova G, Safarik I, Branyik T. (2013b). Harvesting microalgae with microwave synthesized magnetic microparticles. Biores Technol, 130, 472–7. Prochazkova G. (2015). Microalgal surface interactions and their biotechnological applications. Prague: University of Chemistry and Technology. Pulz O, Gross W. (2004). Valuable products from biotechnology of microalgae. Appl Microbiol Biotechnol, 65, 635–48.


DOI: 10.3109/07388551.2015.1064085

Safarik I, Maderova Z, Pospiskova K, et al. (2014). Magnetic decoration and labeling of prokaryotic and eukaryotic cells. In: Fakhrullin RF, Choi I, Lvov YM, eds. Cell surface engineering: fabrication of functional nanoshells. RSC Smart Materials No. 9 Cambridge: RSC, 185–215. Safarik I, Pospiskova K, Horska K, Safarikova M. (2012). Potential of magnetically responsive (nano)biocomposites. Soft Matter, 8, 5407–13. Safarik I, Safarikova M. (1999). Use of magnetic techniques for the isolation of cells. J Chromatogr B, 722, 33–53. Safarik I, Safarikova M. (2009). Magnetic nano- and microparticles in biotechnology. Chem Papers, 63, 497–505. Safarikova M, Pona BMR, Mosiniewicz-Szablewska E, et al. (2008). Dye adsorption on magnetically modified Chlorella vulgaris cells. Fresenius Environ Bull, 17, 486–92. Semple KT, Cain RB, Schmidt S. (1999). Biodegradation of aromatic compounds by microalgae. FEMS Microbiol Lett, 170, 291–300. Shelef G, Sukenik A, Green M. (1984). Microalgae harvesting and processing: a literature review. Haifa: Technion Research and Development Foundation Ltd. Singh A, Olsen SI. (2011). A critical review of biochemical conversion, sustainability and life cycle assessment of algal biofuels. Appl Energ, 88, 3548–55. Sirmerova M, Prochazkova G, Siristova L, et al. (2013). Adhesion of Chlorella vulgaris to solid surfaces, as mediated by physicochemical interactions. J Appl Phycol, 25, 1687–95. Slade R, Bauen A. (2013). Micro-algae cultivation for biofuels: cost, energy balance, environmental impacts and future prospects. Biomass Bioenerg, 53, 29–38. Spolaore P, Joannis-Cassan C, Duran E, Isambert A. (2006). Commercial applications of microalgae. J Biosci Bioeng, 101, 87–96. Sukenik A, Shelef G. (1984). Algal autoflocculation – verification and proposed mechanism. Biotechnol Bioeng, 26, 142–7. Sun ZX, Su FW, Forsling W, Samskog PO. (1998). Surface characteristics of magnetite in aqueous suspension. J Colloid Interface Sci, 197, 151–9. Takeda S, Furuyoshi T, Tari I, et al. (2000). Separation of algae with magnetic iron(III) oxide particles using superconducting high gradient magnetic field. Nippon Kagaku Kaishi, 9, 661–3. Todd T, Zhen ZP, Tang W, et al. (2014). Iron oxide nanoparticle encapsulated diatoms for magnetic delivery of small molecules to tumors. Nanoscale, 6, 2073–6. Toh PY, Ng BW, Abdul LA, et al. (2014a). The role of particle-to-cell interactions in dictating the nanoparticle aided magnetophoretic separation of microalgal cell. Nanoscale, 6, 12838–48. Toh PY, Ng BW, Ahmad AL, et al. (2014b). Magnetophoretic separation of Chlorella sp.: role of cationic polymer binder. Process Saf Environ Protect, 92, 515–21. Toh PY, Ng BW, Chong CH, et al. (2014c). Magnetophoretic separation of microalgae: the role of nanoparticles and polymer binder in harvesting biofuel. RSC Adv, 4, 4114–21. Toh PY, Yeap SP, Kong LP, et al. (2012). Magnetophoretic removal of microalgae from fishpond water: feasibility of high gradient and low gradient magnetic separation. Chem Eng J, 211, 22–30.


11

Torres de Araujo FF, Pires MA, Frankel RB, Bicudo CEM. (1986). Magnetite and magnetotaxis in algae. Biophys J, 50, 375–8. Uduman N, Qi Y, Danquah MK, Forde GM, Hoadley A. (2010). Dewatering of microalgal cultures: a major bottleneck to algae-based fuels. J Renew Sustain Ener, 2, Article No. 012701. Van Durme J, Goiris K, De Winne A, et al. (2013). Evaluation of the volatile composition and sensory properties of five species of microalgae. J Agric Food Chem, 61, 10881–90. van Oss CJ. (2003). Long-range and short-range mechanisms of hydrophobic attraction and hydrophilic repulsion in specific and aspecific interactions. J Mol Recognit, 16, 177–90. Vandamme D, Foubert I, Fraeye I, et al. (2012a). Flocculation of Chlorella vulgaris induced by high pH: role of magnesium and calcium and practical implications. Bioresour Technol, 105, 114–19. Vandamme D, Foubert I, Fraeye I, Muylaert K. (2012b). Influence of organic matter generated by Chlorella vulgaris on five different modes of flocculation. Bioresour Technol, 124, 508–11. Vandamme D, Foubert I, Meesschaert B, Muylaert K. (2010). Flocculation of microalgae using cationic starch. J Appl Phycol, 22, 525–30. Vandamme D, Foubert I, Muylaert K. (2013). Flocculation as a low-cost method for harvesting microalgae for bulk biomass production. Trends Biotechnol, 31, 233–9. Venu R, Lim B, Hu XH, et al. (2013). On-chip manipulation and trapping of microorganisms using a patterned magnetic pathway. Microfluidics Nanofluidics, 14, 277–85. Wang SK, Stiles AR, Guo C, Liu CZ. (2015). Harvesting microalgae by magnetic separation: a review. Algal Res, 9, 178–85. Wang SK, Wang F, Hu YR, et al. (2014). Magnetic flocculant for high efficiency harvesting of microalgal cells. ACS Appl Mater Interfaces, 6, 109–15. Wijffels RH, Kruse O, Hellingwerf KJ. (2013). Potential of industrial biotechnology with cyanobacteria and eukaryotic microalgae. Curr Opin Biotechnol, 24, 405–13. Xu L, Guo C, Wang F, et al. (2011). A simple and rapid harvesting method for microalgae by in situ magnetic separation. Bioresour Technol, 102, 10047–51. Xu L, Weathers PJ, Xiong XR, Liu CZ. (2009). Microalgal bioreactors: challenges and opportunities. Eng Life Sci, 9, 178–89. Yadidia R, Abeliovich A, Belfort G. (1977). Algae removal by high gradient magnetic filtration. Environ Sci Technol, 11, 913–16. Yavuz CT, Prakash A, Mayo JT, Colvin VL. (2009). Magnetic separations: from steel plants to biotechnology. Chem Eng Sci, 64, 2510–21. Zamaleeva AI, Sharipova IR, Shamagsumova RV, et al. (2011). A wholecell amperometric herbicide biosensor based on magnetically functionalised microalgae and screen-printed electrodes. Anal Methods, 3, 509–13. Zhang XZ, Amendola P, Hewson JC, et al. (2012). Influence of growth phase on harvesting of Chlorella zofingiensis by dissolved air flotation. Bioresour Technol, 116, 477–84. Zheng BZ, Zhang MH, Xiao D, et al. (2010). Fast microwave synthesis of Fe3O4 and Fe3O4/Ag magnetic nanoparticles using Fe2+ as precursor. Inorg Mater, 46, 1106–11.

carbon nanotube composites and their catalytic applications.

Magnetically responsive yeast cells: methods of preparation and applications.

Editorial: Advances in Microalgae Biology and Sustainable Applications.

Inorganic Hybrid Nanofibers.

Transgene Expression in Microalgae-From Tools to Applications.

A Holistic Approach to Managing Microalgae for Biofuel Applications.

Characterization of modified tapioca starch solutions and their sprays for high temperature coating applications.

Extracellular Metabolites from Industrial Microalgae and Their Biotechnological Potential.

Nanostructured Biomaterials and Their Applications.

Model animals and their applications.

Magnetic Nanomaterials and Their Applications.

Removal of crystal violet from water by magnetically modified activated carbon and nanomagnetic iron oxide.

Genetically modified cells: applications for intracerebral grafting.

Modelling of Microalgae Culture Systems with Applications to Control and Optimization.

Isolated and Dynamical Horizons and Their Applications.

Seasonal isolation of microalgae from municipal wastewater for remediation and biofuel applications.

Advances in Microalgae-Derived Phytosterols for Functional Food and Pharmaceutical Applications.

Synthesis of base-modified 2'-deoxyribonucleoside triphosphates and their use in enzymatic synthesis of modified DNA for applications in bioanalysis and chemical biology.

MESENCHYMAL STROMAL CELLS AND THEIR ORTHOPAEDIC APPLICATIONS.

Gold-oxoborate nanocomposites and their biomedical applications.

General Bahr-Esseen inequalities and their applications.

Archaeal lipids and their biotechnological applications.

Marine lectins and their medicinal applications.

Photorefraction: two methods and their clinical applications.