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Mitigation of Fe0 nanoparticles toxicity to Trichosporon cutaneum by humic substances
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Mitigation of Fe0 nanoparticles toxicity to Trichosporon cutaneum by humic substances Karolı´na Pa´drova´1, Olga Mat’a´tkova´1, Michaela Sˇikova´1, Tibor Fu¨zik2, Jan Masa´k1, Alena Cˇejkova´1 and Vladimı´r Jirku˚1 1 2
University of Chemistry and Technology, Prague, Department of Biotechnology, Technicka´ 5, 166 28 Prague, Czech Republic University of Chemistry and Technology, Prague, Department of Biochemistry and Microbiology, Technicka´ 5, 166 28 Prague, Czech Republic
Zero-valent iron nanoparticles (nZVI) are a relatively new option for the treatment of contaminated soil and groundwater. However, because of their apparent toxicity, nZVI in high concentrations are known to interfere with many autochthonous microorganisms and, thus, impact their participation in the remediation process. The effect of two commercially available nZVI products, Nanofer 25 (nonstabilized) and Nanofer 25S (stabilized), was examined. Considerable toxicity to the soil yeast Trichosporon cutaneum was observed. Two chemically different humic substances (HSs) were studied as a possible protection agent that mitigates nZVI toxicity: oxidized oxyhumolite X6 and humic acid X3A. The effect of addition of HSs was studied in different phases of the experiment to establish the effect on cells and nZVI. SEM and TEM images revealed an ability of both types of nZVI and HSs to adsorb on surface of the cells. Changes in cell surface properties were also observed by zeta potential measurements. Our results indicate that HSs can act as an electrosteric barrier, which hinders mutual interaction between nZVI and treated cell. Thus, the application of HS seems to be a promising solution to mitigating the toxic action of nZVI.
Introduction The occurrence of nanomaterials in many industrial technologies and medical applications has increased over the past decade. Various types and forms of nanomaterials are used in production of textiles, cosmetics and in food industry (e.g. food packaging) [1,2]. Materials consisting of nanoparticles with dimensions less than 100 nm have significantly different physico-chemical properties compared to their conventional counterparts. Such changes in particle size are usually reflected in an increased reactivity, which may have, depending on the specific situation, both positive and negative effects on biological activities [3]. Nanoscale zero-valent iron particles (nZVI) represent a frequently used agent in environmental remediation technologies [4–8]. The nZVI possess high reduction activity and they react Corresponding author: Mat’a´tkova´, O. ([email protected]) www.elsevier.com/locate/nbt
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with a wide range of (toxic) substances such as halogenated organic compounds, inorganic anions and heavy metals [9]. In these chemical reactions, nZVI serve as electron donors and are oxidized to forms commonly occurring in the environment (Fe2+, Fe3+) [7]. The overall impact of this material on living organisms in the environment has not been fully elucidated in all aspects. In this context, several works have been published that highlight the toxic effects of nZVI not only to microorganisms. Diao and Yao [10] investigated nZVI toxicity against the bacteria Bacillus subtilis, Pseudomonas fluorescens and Aspegillus versicolor. The toxic effect of nZVI was shown in both bacterial strains, however, the Gram-negative bacterium P. fluorescens was less resistant than Gram-positive B. subtilis. A possible explanation for this might be in the relatively rigid cell wall of Gram-positive bacteria, with lipoteichoic acid serving as a chelating agent. A. versicolor displayed the highest http://dx.doi.org/10.1016/j.nbt.2015.09.007 1871-6784/ß 2015 Elsevier B.V. All rights reserved.
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resistance toward nZVI, no tested concentration (0.1–10 g/l) was found lethal. Li et al. [11] described high nZVI toxicity against Escherichia coli. They compared the influence of aerobic and anaerobic conditions and found that the absence of oxygen led to higher nZVI toxicity. NZVI toxicity was also demonstrated in studies on human bronchial epithelial cells [12]. Toxicity of nZVI strongly depends on a concentration and treated microorganism. Keller et al. [13] tested the effect of different nZVI concentrations on fresh water and marine microorganisms. They showed that for each microorganism there is a threshold between non-toxic and toxic concentration. Furthermore, Kadar et al. [14] reported that trace concentrations (mg/l) of nZVI lead to higher growth of studied microalgae strains. Because of their reactivity, nZVI may be dangerous to living organisms, they are even more toxic than other iron-based nanoparticles (maghemite, magnetite). The stability of iron particles increases and reactivity decreases with their increasing oxidative state. Because of their reactivity, nZVI are therefore the most dangerous to living organisms [15]. The relatively low standard reduction potential (E8H(Fe2+/Fe0) = 0.447 V) enables the reduction of many compounds, not only pollutants but also functional groups of biomolecules [16]. At a cellular level, the high redox activity of nZVI gives rise to reactive oxidative species (ROS), such as hydroxyl radical, superoxide radical and hydrogen peroxide, which cause oxidative stress [17]. Although the mechanism of nZVI toxicity has not been fully determined, recent studies support the combined effect of biophysical nanoparticle/cell interactions and oxidative stress [18], not excluding a physical contact of nZVI and cell surface. The nZVI interaction with cell walls results in membrane disruption and loss of membrane selective permeability, which facilitates nanoparticle penetration into cytosol. The nZVI can be oxidized by both extra- and intracellular oxygen (Eqn 1) to form ferrous ion [19]. 2Fe0 þ 2H2 O þ O2 ! 2Fe2þ þ 4OH
(1)
Ferrous ion can enter the Fenton reaction (Eqn 2) generating from hydrogen peroxide highly reactive hydroxyl radicals, which are responsible for the damage of biological macromolecules [20]. Fe2þ þ H2 O2 ! Fe3þ þ OH þ OH
(2)
Ferrous ion could also be oxidized by hydroxyl radicals to give ferric ion, which can react with superoxide radicals generating again ferrous ion – the so-called redox cycling (Eqn 3 and 4) [21]. Fe2þ þ OH ! Fe3þ þ OH
(3)
Fe3þ þ O2 ! Fe2þ þ O2
(4)
Reaction of ferric ion with hydrogen peroxide could produce superoxide radical (Eqn 5) [21]. Fe3þ þ H2 O2 ! Fe2þ þ OOH þ Hþ ! Fe2þ þ 2Hþ O2
(5)
The above chain reactions lead to an increase of ROS level in the cytosol. Although microorganisms possess antioxidant defence mechanisms, they are unable to neutralize ROS to such an extent. The resulting oxidative stress causes protein oxidation, lipid peroxidation and DNA damage, with ensuing apoptosis and cell
death. The nZVI are also oxidized extracellularly to form ferrous ions, which can enter the cell either by active transport or penetrate through damaged cell membrane. In cytosol these ions can interact with hydrogen peroxide in Fenton reaction and thus contribute to increasing the ROS level in the cell [19]. In the event of its application in environmental remediation processes the demonstrated cytotoxicity of nZVI can result in serious damage to the soil microflora. Effective decontamination of the environment is often realized by a combination of physicochemical processes and processes utilizing the biodegradation activity of microorganisms. The toxic properties of nZVI might significantly reduce this ability of microorganisms [22]. Therefore, there is a high demand to develop methods that would reduce the toxicity of nZVI to microorganisms, while maintaining the redox activity exploitable in the degradation of environmental pollutants. For example, Phenrat et al. [23] tested surface modification of nZVI to reduce their toxicity. Modified nanoparticles did not generate oxidative stress and proved less toxic to nZVI-exposed rodent microglia and neurons. Some works deal with the application of humic substances (HSs) as protective substances against nZVI toxicity. HSs are the main component of natural organic matter, one of the most abundant materials in the world. Natural organic matter is part of soils, coal, peat or surface and groundwater [24]. HSs form polydispersions, with a nonstoichiometric elemental composition and a molecular structure that is very complicated, irregular and heterogeneous [25]. The skeleton of the molecule is usually formed by alkyl and aromatic units and contains a large number of diverse chemical functional groups (hydroxyls, phenolic moieties, carboxylic acids, quinones) and is determined predominantly by the properties and composition of the natural organic matter from which they are isolated. Individual types of HS raw materials have undergone various geochemical transformation of the original organic matter and therefore have varied resulting properties and composition. Properties and structure of the HS also affect preparation procedures, which are used for their isolation [22]. HS can be considered as an environmental modulator mitigating the harmful consequences of stress factors [25]. Their protective characteristics are known from the nature, where they shield microorganisms and higher plants from diverse stress conditions such as UV irradiation, infections or pollution [26,27] Furthermore, HS enhance nutrient supply or catalysis of the biochemical reactions in biota [25]. Their protective characteristics have been described by Oris et al. [28], who have studied photo-induced toxicity of anthracene to fathead minnows (Pimephales promelas) and daphnia (Daphnia magna) and reported significantly reduction of acute photo-induced toxicity in both organisms by dissolved HSs. Humic acids (HAs) are important group of oxidized HSs. They are obtained for example from oxyhumolites (oxidized young brown coal) by extraction into alkaline aqueous solutions [29]. Their potential protective activity effectiveness relates to their properties, which mainly depend on their composition determined by origin, locality, isolation and purification procedure [30]. Effectiveness of HAs in mitigation of nZVI toxicity was reported by Li et al. [18] and Chen et al. [22]. The adsorbent properties of oxyhumolites toward basic and acid dyes was described by Janosˇ et al. [31]. One of the possible theories on how HAs protect cells is www.elsevier.com/locate/nbt
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based on their ability to readily adsorb to cellular and nanoparticle surfaces thus decreasing the nanoparticle–cell interactions and related nZVI toxicity. Li et al. [18] published data showing that this effect can be caused by electrosteric repulsion, which is elicited by the negative charge of HSs molecules and their ability to reduce the zeta potential [22]. Chen et al. [22] also documented that HA have no impact on nZVI reactivity and nanostructures conglomeration, therefore their application should not unduly influence nZVI applicability in bioremediation technologies, such as mobility and reducing capabilities. Moreover, antioxidant properties of HAs have been described. Aeschbacher et al. [32] have pointed to the redox activity of HAs, which play a key role in nature and may act as redox buffers. HAs accept electrons from anaerobic microbial respiration or donate them to oxygen in aerobic conditions. HAs also secure electron transfer from microorganism to a poorly accessible substrate in biogeochemical redox reactions or from abiotic reductants to organic pollutants. These antioxidant capabilities are attributed mainly to phenolic moieties, which include mono- and polyhydroxylated benzene units. Phenolic moieties represent major electron donating groups in HA molecule. By quenching oxidants they may protect other functional groups in HS molecule and thus support its stability [33]. Therefore, HAs possess several properties that could as a whole effectively protect microbial cells against nZVI. Nevertheless, the mechanism by which HAs protect cells against nanotoxicity has not been fully clarified, however, each macromolecular structure, coating additively the outer part of the cell wall, must act as a physiologically active component, offering functions related to its chemical structure, properties and the total amount of concrete HAs bound to a specific cellular component. Positive protective effect of HAs was also proved in the case of other kinds of nanoparticles, for example TiO2 [34] or silver nanoparticles [35]. The objective of this research was to investigate the toxic effect of commercially available nZVI, Nanofer 25 and its stabilized form Nanofer 25S against the yeast Trichosporon cutaneum by examining cell viability and morphology. Various applications of HSs, isolated from oxyhumolites extracted from the North-Bohemian Coal Basin in the Czech Republic, were used to mitigate the toxic effects of the nanoparticles.
Material and methods Chemicals Nanofer 25 and Nanofer 25S (Nanoiron Ltd., Czech Republic) are two commercial products of nZVI with different reactivity. Nanofer 25 is composed of particles without any surface treatment and is characterized by a high reactivity. The surface of Nanofer 25S particles is covered (stabilized) with a Na-biodegradable acrylic copolymer which results in reduced reactivity and lower tendency to agglomeration and sedimentation. Both products are distributed as a water dispersion of nanoparticles with mean particle size 50 nm and specific surface area >25 m2 g1. Purity of iron in solid phase is about 85% [14]. HSs X6 (oxidized oxyhumolite) and X3A (HA) were isolated from oxidized brown coal that occurs only in the area of north and northwest Bohemia [36]. HSs were obtained from the Research Institute of Inorganic Chemistry, Czech Republic. The isolation and characterization procedures are described in Kurkova´ et al. 146
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[30]. HA X3A is characterized by high organic part content and lower H/C ratio in comparison to oxidized oxyhumolite X6.
Yeast cultivation Yeast T. cutaneum (strain CCY 30.5.10) was used as a model microorganism. The cell populations were cultivated in 250 ml Erlenmeyer flasks at 308C on a shaker at 100 rpm. A two-step preculture was used to prepare inoculum for experimental cell populations. The first step was carried out in nutrient broth medium for 12 hours. The cells were harvested by centrifugation at 9000 g for 10 min and washed with basic mineral medium. The second step was carried out in 100 ml of basic mineral medium (g l1): (NH4)2SO4 4.0; KH2PO4 1.7; Na2HPO412H2O 1.51; MgSO47H2O 20.0; CaCl2 20.0; FeSO47H2O 1.0; MnSO4H2O 1.0 with 10 g l1 glucose as carbon source. The resulting cell population was used for inoculation (initial biomass optical density OD400 nm = 0.2) of 100 ml basic salt media (other conditions are the same as above) and 48 hours old biomass was used in the experiments to monitor nZVI toxicity.
Exposure of T. cutaneum biomass to nZVI and HSs The experiments were performed in 10 ml of 0.15 M phosphate buffer (pH 7.2) (PBS) in five parallels. The initial biomass optical density corresponded to OD400 nm = 0.2 (13 106 ml1 cells). The effect of a range of nZVI and HS concentrations applicable in remediation processes (1–10 g l1; 0.01–1 g l1, respectively) was preliminary evaluated (data not shown) and the nZVI concentrations 1 g l1 and 5 g l1 and HS concentration 0.3 g l1 were chosen for further analysis based on the nZVI performance observed in previous experiments with wider concentration scale. Three different arrangements of nZVI and HSs addition were studied. 1. Firstly, HS at concentration 0.3 g l1 was added together with nZVI at the beginning of experiments. 2. Secondly, cells were incubated with HS (0.3 g l1) for 15-min, washed two times with PBS and further exposed to both nZVI concentrations. 3. The third experiment was carried out with nZVI modified by HS. The nZVI were first left to interact with HS (0.3 g l1) for 15 min, then centrifuged (9000 g, 10 min, 108C), washed with PBS and used for the experiments as above. Controls were prepared without nZVI or HS.
Cell viability determination Influence of nZVI and HSs on cell viability was determined by the plate method. Briefly, samples of cell suspension taken after 0, 5, 10, 15 and 30 min of exposure were plated on nutrient agar plates, incubated at 308C for 48 hours, and the colony forming units (CFU) were counted.
Study of cell morphology Effect of both nZVI concentrations on T. cutaneum cell morphology was examined by light microscopy with phase contrast using Nikon Eclipse 400 (Nikon, Japan) equipped with Canon 1100D digital camera (Canon, Japan). Effect of nZVI on T. cutaneum morphology after exposure to nZVI was compared with the effect of hydrogen peroxide (1 g l1) in the same conditions. Natural shape of T. cutaneum cells under physiological conditions is usually ovoid and/or elongated. In the presence of nZVI the yeast cell
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shape was observed to be circular. Quantification of this effect was accomplished using image analysis (NIS elements 3.0, Laboratory Imaging, Czech Republic) with circularity as the measured parameter, where the circularity of circle is considered 100%. Cell morphology was examined after 30 min of each exposure experiment and the circularity was compared with that of control population. Circularity was determined as a median value of 100 cells per sample.
Surface charges of Nanofer 25 (1 g l1), Nanofer 25S (1 g l1) and T. cutaneum cells before and after HS treatment were measured at neutral pH. Incubation with HS (0.3 g l1) was carried out in the same manner. Zeta potential analyser Zetasizer Nano ZS (Malvern Instruments, UK) was used.
Scanning and transmission electron microscopy Samples for both microscopies were prepared as follows: (1) cell suspension was examined after a 30-min exposure to nZVI (1 g l1); (2) cell suspension was examined after a 15-min exposure to HS X6 (0.3 g l1) and (3) cell suspension was examined after 15min interaction with HS X6 and a subsequent 30-min exposure to nZVI suspension (1 g l1). Control was prepared without nZVI or HS. Subsequently, the samples for scanning electron microscope (SEM) were re-suspended in 5 ml of fixative solution (3% glutaraldehyde in 0.1 mol l1 phosphate buffer, pH 7.2) and left for 18 hours at 48C. The samples were immersed in liquid nitrogen for 1 min and freeze-dried. Cells were placed onto slides and coated with gold and palladium. The resulting specimens were studied by scanning electron microscope Hitachi S 4700 (Hitachi, Japan) operating at 15 kV. Samples for TEM were deposited on carbon-coated copper grids. After a few minutes of incubation, the grid was washed two times on top of a drop of deionized water and the excess liquid was blotted away. No additional staining was performed. Images were acquired by JEOL JEM-1010 transmission electron microscope (Jeol, Japan) operating at 80 kV. Images were captured by MegaView III CCD camera (Olympus, Japan) and by analySIS 2.0 software package (Olympus, Japan).
Results Toxicity of nZVI to T. cutaneum T. cutaneum was exposed to the two types of nZVI at concentrations 1 g l1 and 5 g l1. Toxicity of these materials, caused primarily by the ability to produce large quantities of reactive oxygen species, was monitored as a change in cell viability in dependence on the length of exposure. Figure 1 demonstrates that the effect is most intense during the first 5 min after application of the nanoparticles. The dynamics of the toxic effect subsequently decreases and after 30 min the number of viable cells is stable. The surface stabilization of Nanofer 25S (see Material and methods) influenced its biological activity, which resulted in lower toxicity compared to Nanofer 25. After a 30-min exposure to Nanofer 25S at both tested concentrations 40% of initial cell count retained reproductive activity, while with 1 g l1 Nanofer 25 it was 25% and with 5 g l1 it was 18%. The physical contact of nZVI with cell population of T. cutaneum has also resulted in significant changes of cell shape (morphology).
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Zeta potential
FIGURE 1
Growth of T. cutaneum exposed to nZVI particles: () control, (&) Nanofer 25 1 g l1, (~) Nanofer 25 5 g l1, (&) Nanofer 25S 1 g l1, and (~) Nanofer 25S 5 g l1.
Depiction of the statistical interpretation of observed effect is shown in Fig. 3. When described by the circularity parameter, the non-stressed elongate shape of majority of cells in the control population falls into a circularity range of 60–89% as represented by the error bars (10th and 90th percentiles). The boundaries of the box charts represent the 25th and 75th percentiles (control population circularity 66–84%). The high range of the values of the circularity parameter is caused by the very high variability of T. cutaneum cells shapes under normal conditions. This can be seen in light microscopy images in Supporting information material (see S1). The median (center line) for control population is 75% circularity. By contrast is the almost completely circular shape (majority of cell with circularity 97–99%) of cells after the exposure to Nanofer 25 (5 g l1).
T. cutaneum protection by HSs against the effects of nZVI Nanofer 25 and Nanofer 25S A mitigation of nZVI toxicity by HS X6 (oxidized oxyhumolite) and HA X3A was evaluated in terms of cell viability (CFU) and compared to control population and population exposed only to nZVI. In this work, three experiment set-ups were employed to ascertain the HSs influence: simultaneous effect, cell preincubation and nZVI preincubation. The following procedures were performed: 1. Simultaneous effect – T. cutaneum cells were exposed concurrently to nZVI and HSs; 2. Cell preincubation – T. cutaneum cells were initially incubated with the HSs (concentration 0.3 g l1) and the treated cells were exposed to nZVI; 3. nZVI preincubation – nanoparticles were incubated with HSs and this mixture was then added to cell population. Cell viability was monitored (Fig. 2) and these results served to evaluate the ability of HS to mitigate the toxic effect of both tested nZVI forms. When 1 g l1 Nanofer 25 was used (Fig. 2a), the X6 was the most effective in mitigation of nZVI toxicity in both the simultaneous effect and cell preincubation modes (see Material and methods for experiment set-ups). More than 84% and 88% of treated cells, www.elsevier.com/locate/nbt
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FIGURE 2
Mitigation of nZVI toxicity to T. cutaneum by humic substances X6 and X3 after 30-min exposure (nZVI concentration: white bars 1 g l1; gray bars 5 g l1). (a) Nanofer 25; (b) Nanofer 25S.
respectively, preserved their viability. In addition, the application of Nanofer 25 and X6 simultaneously showed the highest potential for protection against oxidative stress caused by 5 g l1 Nanofer 25. However, in all experiment set-ups the overall effect of protection was lower against the higher Nanofer 25 concentration. The protective effect of HS was also proved by the determination of cell morphology (Fig. 3). When HSs were added to Nanofer 25, the protection by HS was revealed as the diversity of cell shapes returned the more oblong shape was retained. Although the cell shape was more circular than the control population (median of circularity 86%), the overall characteristic and the variability of cell shapes indicated a lower stress. This effect was observed in all conditions studied and described above in viability tests for Nanofer 25. The circularity was in the range 70–90% (25th and 75th percentiles) for both HS and all experiment set-ups (simultaneous
effect, cell preincubation and nZVI preincubation) – in Fig. 3 are shown only the data for simultaneous effect with HS X6. The coated Nanofer 25S particles were tested in an identical manner as Nanofer 25 (Fig. 2b). They exhibited lower toxicity to T. cutaneum cells (39% viability in both concentrations), but the HSs offered less protection. The cell morphology corresponds with the observed viability. In Fig. 3, the influence of 5 g l1 Nanofer 25S and the effect of simultaneous exposure with HS X6 are shown. The exposed cells in both cases maintained their oblong shape (circularity 65–95%, as shown in 10th and 90th percentiles), the effect was similar for both HS and all experiment set-ups. To compare the impact of nZVI on cell morphology with the influence of ROS alone, cells were exposed to hydrogen peroxide (1 g l1). The exposure time and hydrogen peroxide concentration have been chosen to reflect the influence and ROS presence as caused by nZVI. After 30 min, no morphological changes were observed (data not shown).
Characterization of nZVI and T. cutaneum surface under studied conditions The zeta potential of both nanoparticles and T. cutaneum cells was determined to evaluate the surface properties that influence the mutual interactions between nanoparticles and cells. In addition, an effect of HSs on the zeta potential value was examined (see Table 1). The zeta potential of the Nanofer 25 was 3.1 mV. The TABLE 1
Effect of humic substances, Nanofer 25 (1 g l1) treatment and three experimental set-ups on zeta potential (mV) of Trichosporon cutaneum Control
Nanofer 25 Nanofer 25 + HS T. cutaneum T. cutaneum + Nanofer 25 T. cutaneum + HS
FIGURE 3
The effect of Nanofer 25 (5 g l1) and Nanofer 25S (5 g l1) and protection by HS against nZVI on T. cutaneum cell morphology expressed as cell circularity. The whiskers (error bars) represent 10th and 90th percentiles, the box charts boundaries represent the 25th and 75th percentiles and the center line depicts the median. 148
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nZVI + HS + T. cutaneum
Simultaneous effect Cell preincubation Nanofer 25 preincubation
X6 X3A
3.1 18.1 25.0 30.0 35.0 35.7 28.2
X6 X3A X6 X3A X6 X3A
31.7 36.1 32.6 34.2 40.1 39.4
X6 X3A
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zeta potential of Nanofer 25S was significantly higher (35 mV) and further interaction with both HSs did not bring about any significant change of the zeta potential. This points to a lower ability of HSs to mitigate the action of Nanofer 25S compared to Nanofer 25 – see Fig. 2. The value of surface charge can probably influence the extent of nanoparticle toxicity. The zeta potential of T. cutaneum was 30 mV. The considerable difference between the zeta potential of T. cutaneum and Nanofer 25 supports a higher toxicity of this material compared to Nanofer 25S, which was observed in viability tests. A decrease of T. cutaneum zeta potential to 35 mV was observed as a consequence of Nanofer 25 adsorption on cell surface. The interaction of both studied HSs with Nanofer 25 or T. cutaneum resulted in a change of surface charges. The surface charge of Nanofer 25 decreased after interaction with both X6 (18.1 mV) and X3A (25 mV). However, the reaction of T. cutaneum cells was different, as the application of X6 decreased the surface charge (35.7 mV) only moderately. X3A had an opposite effect and the surface charge was increased to 18.1 mV. The zeta potential of T. cutaneum under all three experimental set-ups changes according to the treatment conditions and the results correspond to those obtained by viability tests. The least change of T. cutaneum zeta potential occurred under protection with X6 and under simultaneous effect and cell preincubation conditions (31.7 mV, resp. 32.6 mV). By contrast, Nanofer 25 preincubation experiment resulted in a significant increase of cell surface charge.
TEM and SEM of T. cutaneum surface under studied conditions Electron microscopy was used to examine the T. cutaneum cell surface before and after exposure to Nanofer 25 (1 g l1) and/or oxyhumolite X6 (0.3 g l1). A crucial step of the nZVI mechanism of action is mutual interaction between nZVI and the exposed cells. The interaction of nZVI and cell surface is apparent from detailed images of both SEM (Fig. 4c) and TEM (Fig. 5c). Both methods show the ability of HS to adsorb on cell surface (Figs 4b and 5b). After incubation with X6, the cell surface was covered by an electron dense film (Fig. 5b) which presumably indicates HS adsorption. TEM images of the control population (Fig. 5a) display cells with morphology revealing the influence of nanoparticles and HS: ovoid cell shape and the cell cytosol and organelles are clearly recognizable as opposed to exposed cells. TEM analysis also proved that adhesion of nZVI to the cell surface occurs despite the HS absorption onto cells (Fig. 5d). However, cell lysis as a response to nZVI action is distinguishable only after exposure to nZVI without X6 protection (Figs 4c and 5c).
Discussion The nZVI technology attracts considerable attention mainly due to their unique properties, such as high reactivity and low standard reduction potential, which allow their use in remediation processes. However, these properties also influence biological activities and are associated with high toxicity against exposed microorganisms [37]. NZVI can considerably disturb autochthonal microbial populations and hinder their participation in environmental remediation. Our results showed that commercially available nZVI Nanofer 25 and Nanofer 25S have high toxicity against the yeast
FIGURE 4
SEM of Trichosporon cutaneum surface (a) after 15-min incubation in the presence of HS X6 0.3 g l1 (b) and 30-min exposure to Nanofer 25 1 g l1 (c).
T. cutaneum, an aerobic soil microorganism with an ability to utilize a wide range of organic pollutants [38]. Although the mechanism of nZVI action still needs to be clarified, Keenan et al. [12] have reported the ability of nZVI to induce ROS production. Because of their high reactivity, nZVI are oxidized very quickly to ferrous and ferric ion [39]. Oxidation can take place both extracellularly and in the cytosol [19]. Ferrous ion introduces a transient state and its oxidation to ferric ion leads to ROS production (Eqn 1–4). Adeleye et al. [40] studied long-term environmental fate of nZVI oxidation in aqueous media, including www.elsevier.com/locate/nbt
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Research Paper FIGURE 5
TEM of Trichosporon cutaneum (a) after 15-min incubation in the presence of oxyhumolite X6 0.3 g l1 (b), 30-min treatment with Nanofer 25 1 g l1 (c) and treatment of oxyhumolite X6 pre-incubated cells with Nanofer 25 1 g l1 (d).
groundwater samples. The nZVI oxidation to ferrous iron occurred from the very start of the experiment. A possible link between oxidative stress induced by nZVI and the change of cell morphology was not proven. The presence of ROS caused by application of hydrogen peroxide did not change cell shape (data not shown). Since the presence of the HSs, which have the ability to produce an additive layer on the surface of cells [41] significantly suppressed changes in cell morphology, it is offered as a possible explanation of these changes response to the direct physical contact of nZVI with the cell surface. The change in cell shape from elongated to spherical can be seen as an effort to capture the smallest possible cell surface area and thus minimize contact with nZVI. A similar change of shape observed by Xie et al. [42]. Application of ZnO nanoparticles caused a morphological change of Campylobacter jejuni, which underwent a change of cell shape from spiral to coccoid. However, it was also observed the opposite effect when E. coli cells became more filamentous in the presence of nanoparticles CdO [43]. The importance of autochthonal microflora in environmental regeneration is indisputable. Therefore, recent research has focused on protection of microorganisms without the loss of nZVI activity. The protective effect of HSs is known from nature [44]. HSs have properties, such as the ability to bind to surfaces [18] or antioxidant properties [32], which make these substances useful for the protection of microorganisms. The use of HSs in mitigation 150
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of nanoparticle toxicity to microorganisms has been described in several reports [18,22,35]. Chen et al. [22] focused on the influence of HA on the toxicity and reactivity of nZVI. They found that HA not only significantly mitigated nZVI toxicity but also did not affect nZVI reactivity with trichloroethylene. Our study addressed the comparison of protection abilities of two chemically different HSs, oxidized oxyhumolite and HA. Both HSs proved the ability to mitigate the impact of nZVI. In all experiments a slightly better protective character was shown by HA (Fig. 2). Nevertheless, a significant mitigation of nZVI toxicity was observed mainly with respect to 1 g l1 Nanofer 25 (Fig. 2a). The results of morphological experiments corresponded with those obtained in viability tests. The most effective experiment set-up was cell preincubation with HA X6 (1 g l1 Nanofer 25). Treated cells showed a significantly lower change of morphology and higher viability. The biological activity of nZVI strongly depends on their specific form. Keller et al. [13] reported that different modifications of nZVI can profoundly influence their toxicity. In our research the stabilization of Nanofer 25S displayed lower toxicity to T. cutaneum compared to non-stabilized Nanofer 25. The stabilization or surface modification also influences interactions of nanoparticles with HSs. Dong and Lo [45] showed that different surface modification changed the mode of the HA–nZVI interaction and resulted in different degree of nanoparticles aglomeration. Therefore, the
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A positive effect of HSs on nZVI properties including mobility [46] and stability of nanoparticles in environment [22] has been reported. Our results suggest that application of HSs together with nanoparticles into the treated area enhances the viability of treated microorganisms.
Conclusion The toxicity of nZVI to yeast cells was proven to be affected by HSs, probably due to a hindrance of a direct contact between nZVI and exposed cells caused by electrostatic repulsion. Because of the chemical diversity of HSs the mitigation of nZVI toxicity also varies with the type of HS used. Therefore, the protective ability of each HS needs to be investigated to achieve the best result in specific circumstances. The advantage of HSs employment as a protectant is their natural origin and occurrence in environment. In this study, two different HSs and three set-ups of HSs application were compared. For the soil phenol-degrading yeast T. cutaneum the simultaneous effect of HA and cell preincubation with HA were the most effective in cell protection. The application of HSs in treating contaminated environments using nanoparticles could benefit by combining bioremediation and abiotic nanoparticle remediation processes resulting in better effectivity and lower impact on environment.
Conflict of interest No conflict of interest is declared.
Acknowledgements Financial support from specific university research (MSMT No. 20/ 2015) and by project of Ministry of Industry and Trade of the Czech Republic; Identification code: FR-TI3/564.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.nbt.2015.09. 007.
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presence of stabilizing agents (Na-biodegradable acrylic copolymer) on the surface of Nanofer 25S nanoparticles caused such an difference in surface properties between Nanofer 25 and Nanofer 25S that probably lead to lower ability of both studied HSs to mitigate the toxic impact of Nanofer 25S. A correlation between cell viability and morphology was also observed. Cells under Nanofer 25S treatment did not undergo significant change of the morphology in comparison with Nanofer 25 at both studied concentrations (corresponding with the lower toxicity of Nanofer 25S in comparison with Nanofer 25). Moreover, circularity of cells protected by HSs was higher because of a diminished protective character of HSs against Nanofer 25S action. By contrast, both HS showed high ability to mitigate the effect of Nanofer 25 on cell circularity. In their presence, the cells retained morphological characteristics resemblant to control population. The mutual interactions between nanoparticles and exposed cells seem to play a crucial role in the mechanism of nZVI action. Zeta potential characterizes the surface charge and its value influences potential interactions not only between nanoparticles themselves but also between nanoparticles and exposed cells. After adsorption of HS on both cells and nZVI, the negative charge of HS molecules caused a decrease of zeta potential of both types of nZVI and the cells [22]. These premises were confirmed also in our work by determination of zeta potential of nanoparticles, HSs and exposed cells. The results are summarized in Table 1. HSs probably behaved as polyelectrolytes with high molecular weight and thus caused electrosteric repulsion between nanoparticles and cells [18]. TEM microscopy (Fig. 5d) showed that adsorption of nanoparticles on cell surface occurred despite the presence of an HS coating. Thus, electrosteric repulsion alone seemed to be the principal mechanism in cell protection. However, adsorption of both HSs and nZVI on cell surface and simultaneous improvement of cell viability indicated restriction of physical interaction between cell surface and nZVI [22].
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Mitigation of Fe0 nanoparticles toxicity to Trichosporon cutaneum by humic substances Karolı´na Pa´drova´1, Olga Mat’a´tkova´1, Michaela Sˇikova´1, Tibor Fu¨zik2, Jan Masa´k1, Alena Cˇejkova´1 and Vladimı´r Jirku˚1 1 2
University of Chemistry and Technology, Prague, Department of Biotechnology, Technicka´ 5, 166 28 Prague, Czech Republic University of Chemistry and Technology, Prague, Department of Biochemistry and Microbiology, Technicka´ 5, 166 28 Prague, Czech Republic
Zero-valent iron nanoparticles (nZVI) are a relatively new option for the treatment of contaminated soil and groundwater. However, because of their apparent toxicity, nZVI in high concentrations are known to interfere with many autochthonous microorganisms and, thus, impact their participation in the remediation process. The effect of two commercially available nZVI products, Nanofer 25 (nonstabilized) and Nanofer 25S (stabilized), was examined. Considerable toxicity to the soil yeast Trichosporon cutaneum was observed. Two chemically different humic substances (HSs) were studied as a possible protection agent that mitigates nZVI toxicity: oxidized oxyhumolite X6 and humic acid X3A. The effect of addition of HSs was studied in different phases of the experiment to establish the effect on cells and nZVI. SEM and TEM images revealed an ability of both types of nZVI and HSs to adsorb on surface of the cells. Changes in cell surface properties were also observed by zeta potential measurements. Our results indicate that HSs can act as an electrosteric barrier, which hinders mutual interaction between nZVI and treated cell. Thus, the application of HS seems to be a promising solution to mitigating the toxic action of nZVI.
Introduction The occurrence of nanomaterials in many industrial technologies and medical applications has increased over the past decade. Various types and forms of nanomaterials are used in production of textiles, cosmetics and in food industry (e.g. food packaging) [1,2]. Materials consisting of nanoparticles with dimensions less than 100 nm have significantly different physico-chemical properties compared to their conventional counterparts. Such changes in particle size are usually reflected in an increased reactivity, which may have, depending on the specific situation, both positive and negative effects on biological activities [3]. Nanoscale zero-valent iron particles (nZVI) represent a frequently used agent in environmental remediation technologies [4–8]. The nZVI possess high reduction activity and they react Corresponding author: Mat’a´tkova´, O. ([email protected]) www.elsevier.com/locate/nbt
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with a wide range of (toxic) substances such as halogenated organic compounds, inorganic anions and heavy metals [9]. In these chemical reactions, nZVI serve as electron donors and are oxidized to forms commonly occurring in the environment (Fe2+, Fe3+) [7]. The overall impact of this material on living organisms in the environment has not been fully elucidated in all aspects. In this context, several works have been published that highlight the toxic effects of nZVI not only to microorganisms. Diao and Yao [10] investigated nZVI toxicity against the bacteria Bacillus subtilis, Pseudomonas fluorescens and Aspegillus versicolor. The toxic effect of nZVI was shown in both bacterial strains, however, the Gram-negative bacterium P. fluorescens was less resistant than Gram-positive B. subtilis. A possible explanation for this might be in the relatively rigid cell wall of Gram-positive bacteria, with lipoteichoic acid serving as a chelating agent. A. versicolor displayed the highest http://dx.doi.org/10.1016/j.nbt.2015.09.007 1871-6784/ß 2015 Elsevier B.V. All rights reserved.
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resistance toward nZVI, no tested concentration (0.1–10 g/l) was found lethal. Li et al. [11] described high nZVI toxicity against Escherichia coli. They compared the influence of aerobic and anaerobic conditions and found that the absence of oxygen led to higher nZVI toxicity. NZVI toxicity was also demonstrated in studies on human bronchial epithelial cells [12]. Toxicity of nZVI strongly depends on a concentration and treated microorganism. Keller et al. [13] tested the effect of different nZVI concentrations on fresh water and marine microorganisms. They showed that for each microorganism there is a threshold between non-toxic and toxic concentration. Furthermore, Kadar et al. [14] reported that trace concentrations (mg/l) of nZVI lead to higher growth of studied microalgae strains. Because of their reactivity, nZVI may be dangerous to living organisms, they are even more toxic than other iron-based nanoparticles (maghemite, magnetite). The stability of iron particles increases and reactivity decreases with their increasing oxidative state. Because of their reactivity, nZVI are therefore the most dangerous to living organisms [15]. The relatively low standard reduction potential (E8H(Fe2+/Fe0) = 0.447 V) enables the reduction of many compounds, not only pollutants but also functional groups of biomolecules [16]. At a cellular level, the high redox activity of nZVI gives rise to reactive oxidative species (ROS), such as hydroxyl radical, superoxide radical and hydrogen peroxide, which cause oxidative stress [17]. Although the mechanism of nZVI toxicity has not been fully determined, recent studies support the combined effect of biophysical nanoparticle/cell interactions and oxidative stress [18], not excluding a physical contact of nZVI and cell surface. The nZVI interaction with cell walls results in membrane disruption and loss of membrane selective permeability, which facilitates nanoparticle penetration into cytosol. The nZVI can be oxidized by both extra- and intracellular oxygen (Eqn 1) to form ferrous ion [19]. 2Fe0 þ 2H2 O þ O2 ! 2Fe2þ þ 4OH
(1)
Ferrous ion can enter the Fenton reaction (Eqn 2) generating from hydrogen peroxide highly reactive hydroxyl radicals, which are responsible for the damage of biological macromolecules [20]. Fe2þ þ H2 O2 ! Fe3þ þ OH þ OH
(2)
Ferrous ion could also be oxidized by hydroxyl radicals to give ferric ion, which can react with superoxide radicals generating again ferrous ion – the so-called redox cycling (Eqn 3 and 4) [21]. Fe2þ þ OH ! Fe3þ þ OH
(3)
Fe3þ þ O2 ! Fe2þ þ O2
(4)
Reaction of ferric ion with hydrogen peroxide could produce superoxide radical (Eqn 5) [21]. Fe3þ þ H2 O2 ! Fe2þ þ OOH þ Hþ ! Fe2þ þ 2Hþ O2
(5)
The above chain reactions lead to an increase of ROS level in the cytosol. Although microorganisms possess antioxidant defence mechanisms, they are unable to neutralize ROS to such an extent. The resulting oxidative stress causes protein oxidation, lipid peroxidation and DNA damage, with ensuing apoptosis and cell
death. The nZVI are also oxidized extracellularly to form ferrous ions, which can enter the cell either by active transport or penetrate through damaged cell membrane. In cytosol these ions can interact with hydrogen peroxide in Fenton reaction and thus contribute to increasing the ROS level in the cell [19]. In the event of its application in environmental remediation processes the demonstrated cytotoxicity of nZVI can result in serious damage to the soil microflora. Effective decontamination of the environment is often realized by a combination of physicochemical processes and processes utilizing the biodegradation activity of microorganisms. The toxic properties of nZVI might significantly reduce this ability of microorganisms [22]. Therefore, there is a high demand to develop methods that would reduce the toxicity of nZVI to microorganisms, while maintaining the redox activity exploitable in the degradation of environmental pollutants. For example, Phenrat et al. [23] tested surface modification of nZVI to reduce their toxicity. Modified nanoparticles did not generate oxidative stress and proved less toxic to nZVI-exposed rodent microglia and neurons. Some works deal with the application of humic substances (HSs) as protective substances against nZVI toxicity. HSs are the main component of natural organic matter, one of the most abundant materials in the world. Natural organic matter is part of soils, coal, peat or surface and groundwater [24]. HSs form polydispersions, with a nonstoichiometric elemental composition and a molecular structure that is very complicated, irregular and heterogeneous [25]. The skeleton of the molecule is usually formed by alkyl and aromatic units and contains a large number of diverse chemical functional groups (hydroxyls, phenolic moieties, carboxylic acids, quinones) and is determined predominantly by the properties and composition of the natural organic matter from which they are isolated. Individual types of HS raw materials have undergone various geochemical transformation of the original organic matter and therefore have varied resulting properties and composition. Properties and structure of the HS also affect preparation procedures, which are used for their isolation [22]. HS can be considered as an environmental modulator mitigating the harmful consequences of stress factors [25]. Their protective characteristics are known from the nature, where they shield microorganisms and higher plants from diverse stress conditions such as UV irradiation, infections or pollution [26,27] Furthermore, HS enhance nutrient supply or catalysis of the biochemical reactions in biota [25]. Their protective characteristics have been described by Oris et al. [28], who have studied photo-induced toxicity of anthracene to fathead minnows (Pimephales promelas) and daphnia (Daphnia magna) and reported significantly reduction of acute photo-induced toxicity in both organisms by dissolved HSs. Humic acids (HAs) are important group of oxidized HSs. They are obtained for example from oxyhumolites (oxidized young brown coal) by extraction into alkaline aqueous solutions [29]. Their potential protective activity effectiveness relates to their properties, which mainly depend on their composition determined by origin, locality, isolation and purification procedure [30]. Effectiveness of HAs in mitigation of nZVI toxicity was reported by Li et al. [18] and Chen et al. [22]. The adsorbent properties of oxyhumolites toward basic and acid dyes was described by Janosˇ et al. [31]. One of the possible theories on how HAs protect cells is www.elsevier.com/locate/nbt
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based on their ability to readily adsorb to cellular and nanoparticle surfaces thus decreasing the nanoparticle–cell interactions and related nZVI toxicity. Li et al. [18] published data showing that this effect can be caused by electrosteric repulsion, which is elicited by the negative charge of HSs molecules and their ability to reduce the zeta potential [22]. Chen et al. [22] also documented that HA have no impact on nZVI reactivity and nanostructures conglomeration, therefore their application should not unduly influence nZVI applicability in bioremediation technologies, such as mobility and reducing capabilities. Moreover, antioxidant properties of HAs have been described. Aeschbacher et al. [32] have pointed to the redox activity of HAs, which play a key role in nature and may act as redox buffers. HAs accept electrons from anaerobic microbial respiration or donate them to oxygen in aerobic conditions. HAs also secure electron transfer from microorganism to a poorly accessible substrate in biogeochemical redox reactions or from abiotic reductants to organic pollutants. These antioxidant capabilities are attributed mainly to phenolic moieties, which include mono- and polyhydroxylated benzene units. Phenolic moieties represent major electron donating groups in HA molecule. By quenching oxidants they may protect other functional groups in HS molecule and thus support its stability [33]. Therefore, HAs possess several properties that could as a whole effectively protect microbial cells against nZVI. Nevertheless, the mechanism by which HAs protect cells against nanotoxicity has not been fully clarified, however, each macromolecular structure, coating additively the outer part of the cell wall, must act as a physiologically active component, offering functions related to its chemical structure, properties and the total amount of concrete HAs bound to a specific cellular component. Positive protective effect of HAs was also proved in the case of other kinds of nanoparticles, for example TiO2 [34] or silver nanoparticles [35]. The objective of this research was to investigate the toxic effect of commercially available nZVI, Nanofer 25 and its stabilized form Nanofer 25S against the yeast Trichosporon cutaneum by examining cell viability and morphology. Various applications of HSs, isolated from oxyhumolites extracted from the North-Bohemian Coal Basin in the Czech Republic, were used to mitigate the toxic effects of the nanoparticles.
Material and methods Chemicals Nanofer 25 and Nanofer 25S (Nanoiron Ltd., Czech Republic) are two commercial products of nZVI with different reactivity. Nanofer 25 is composed of particles without any surface treatment and is characterized by a high reactivity. The surface of Nanofer 25S particles is covered (stabilized) with a Na-biodegradable acrylic copolymer which results in reduced reactivity and lower tendency to agglomeration and sedimentation. Both products are distributed as a water dispersion of nanoparticles with mean particle size 50 nm and specific surface area >25 m2 g1. Purity of iron in solid phase is about 85% [14]. HSs X6 (oxidized oxyhumolite) and X3A (HA) were isolated from oxidized brown coal that occurs only in the area of north and northwest Bohemia [36]. HSs were obtained from the Research Institute of Inorganic Chemistry, Czech Republic. The isolation and characterization procedures are described in Kurkova´ et al. 146
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[30]. HA X3A is characterized by high organic part content and lower H/C ratio in comparison to oxidized oxyhumolite X6.
Yeast cultivation Yeast T. cutaneum (strain CCY 30.5.10) was used as a model microorganism. The cell populations were cultivated in 250 ml Erlenmeyer flasks at 308C on a shaker at 100 rpm. A two-step preculture was used to prepare inoculum for experimental cell populations. The first step was carried out in nutrient broth medium for 12 hours. The cells were harvested by centrifugation at 9000 g for 10 min and washed with basic mineral medium. The second step was carried out in 100 ml of basic mineral medium (g l1): (NH4)2SO4 4.0; KH2PO4 1.7; Na2HPO412H2O 1.51; MgSO47H2O 20.0; CaCl2 20.0; FeSO47H2O 1.0; MnSO4H2O 1.0 with 10 g l1 glucose as carbon source. The resulting cell population was used for inoculation (initial biomass optical density OD400 nm = 0.2) of 100 ml basic salt media (other conditions are the same as above) and 48 hours old biomass was used in the experiments to monitor nZVI toxicity.
Exposure of T. cutaneum biomass to nZVI and HSs The experiments were performed in 10 ml of 0.15 M phosphate buffer (pH 7.2) (PBS) in five parallels. The initial biomass optical density corresponded to OD400 nm = 0.2 (13 106 ml1 cells). The effect of a range of nZVI and HS concentrations applicable in remediation processes (1–10 g l1; 0.01–1 g l1, respectively) was preliminary evaluated (data not shown) and the nZVI concentrations 1 g l1 and 5 g l1 and HS concentration 0.3 g l1 were chosen for further analysis based on the nZVI performance observed in previous experiments with wider concentration scale. Three different arrangements of nZVI and HSs addition were studied. 1. Firstly, HS at concentration 0.3 g l1 was added together with nZVI at the beginning of experiments. 2. Secondly, cells were incubated with HS (0.3 g l1) for 15-min, washed two times with PBS and further exposed to both nZVI concentrations. 3. The third experiment was carried out with nZVI modified by HS. The nZVI were first left to interact with HS (0.3 g l1) for 15 min, then centrifuged (9000 g, 10 min, 108C), washed with PBS and used for the experiments as above. Controls were prepared without nZVI or HS.
Cell viability determination Influence of nZVI and HSs on cell viability was determined by the plate method. Briefly, samples of cell suspension taken after 0, 5, 10, 15 and 30 min of exposure were plated on nutrient agar plates, incubated at 308C for 48 hours, and the colony forming units (CFU) were counted.
Study of cell morphology Effect of both nZVI concentrations on T. cutaneum cell morphology was examined by light microscopy with phase contrast using Nikon Eclipse 400 (Nikon, Japan) equipped with Canon 1100D digital camera (Canon, Japan). Effect of nZVI on T. cutaneum morphology after exposure to nZVI was compared with the effect of hydrogen peroxide (1 g l1) in the same conditions. Natural shape of T. cutaneum cells under physiological conditions is usually ovoid and/or elongated. In the presence of nZVI the yeast cell
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shape was observed to be circular. Quantification of this effect was accomplished using image analysis (NIS elements 3.0, Laboratory Imaging, Czech Republic) with circularity as the measured parameter, where the circularity of circle is considered 100%. Cell morphology was examined after 30 min of each exposure experiment and the circularity was compared with that of control population. Circularity was determined as a median value of 100 cells per sample.
Surface charges of Nanofer 25 (1 g l1), Nanofer 25S (1 g l1) and T. cutaneum cells before and after HS treatment were measured at neutral pH. Incubation with HS (0.3 g l1) was carried out in the same manner. Zeta potential analyser Zetasizer Nano ZS (Malvern Instruments, UK) was used.
Scanning and transmission electron microscopy Samples for both microscopies were prepared as follows: (1) cell suspension was examined after a 30-min exposure to nZVI (1 g l1); (2) cell suspension was examined after a 15-min exposure to HS X6 (0.3 g l1) and (3) cell suspension was examined after 15min interaction with HS X6 and a subsequent 30-min exposure to nZVI suspension (1 g l1). Control was prepared without nZVI or HS. Subsequently, the samples for scanning electron microscope (SEM) were re-suspended in 5 ml of fixative solution (3% glutaraldehyde in 0.1 mol l1 phosphate buffer, pH 7.2) and left for 18 hours at 48C. The samples were immersed in liquid nitrogen for 1 min and freeze-dried. Cells were placed onto slides and coated with gold and palladium. The resulting specimens were studied by scanning electron microscope Hitachi S 4700 (Hitachi, Japan) operating at 15 kV. Samples for TEM were deposited on carbon-coated copper grids. After a few minutes of incubation, the grid was washed two times on top of a drop of deionized water and the excess liquid was blotted away. No additional staining was performed. Images were acquired by JEOL JEM-1010 transmission electron microscope (Jeol, Japan) operating at 80 kV. Images were captured by MegaView III CCD camera (Olympus, Japan) and by analySIS 2.0 software package (Olympus, Japan).
Results Toxicity of nZVI to T. cutaneum T. cutaneum was exposed to the two types of nZVI at concentrations 1 g l1 and 5 g l1. Toxicity of these materials, caused primarily by the ability to produce large quantities of reactive oxygen species, was monitored as a change in cell viability in dependence on the length of exposure. Figure 1 demonstrates that the effect is most intense during the first 5 min after application of the nanoparticles. The dynamics of the toxic effect subsequently decreases and after 30 min the number of viable cells is stable. The surface stabilization of Nanofer 25S (see Material and methods) influenced its biological activity, which resulted in lower toxicity compared to Nanofer 25. After a 30-min exposure to Nanofer 25S at both tested concentrations 40% of initial cell count retained reproductive activity, while with 1 g l1 Nanofer 25 it was 25% and with 5 g l1 it was 18%. The physical contact of nZVI with cell population of T. cutaneum has also resulted in significant changes of cell shape (morphology).
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FIGURE 1
Growth of T. cutaneum exposed to nZVI particles: () control, (&) Nanofer 25 1 g l1, (~) Nanofer 25 5 g l1, (&) Nanofer 25S 1 g l1, and (~) Nanofer 25S 5 g l1.
Depiction of the statistical interpretation of observed effect is shown in Fig. 3. When described by the circularity parameter, the non-stressed elongate shape of majority of cells in the control population falls into a circularity range of 60–89% as represented by the error bars (10th and 90th percentiles). The boundaries of the box charts represent the 25th and 75th percentiles (control population circularity 66–84%). The high range of the values of the circularity parameter is caused by the very high variability of T. cutaneum cells shapes under normal conditions. This can be seen in light microscopy images in Supporting information material (see S1). The median (center line) for control population is 75% circularity. By contrast is the almost completely circular shape (majority of cell with circularity 97–99%) of cells after the exposure to Nanofer 25 (5 g l1).
T. cutaneum protection by HSs against the effects of nZVI Nanofer 25 and Nanofer 25S A mitigation of nZVI toxicity by HS X6 (oxidized oxyhumolite) and HA X3A was evaluated in terms of cell viability (CFU) and compared to control population and population exposed only to nZVI. In this work, three experiment set-ups were employed to ascertain the HSs influence: simultaneous effect, cell preincubation and nZVI preincubation. The following procedures were performed: 1. Simultaneous effect – T. cutaneum cells were exposed concurrently to nZVI and HSs; 2. Cell preincubation – T. cutaneum cells were initially incubated with the HSs (concentration 0.3 g l1) and the treated cells were exposed to nZVI; 3. nZVI preincubation – nanoparticles were incubated with HSs and this mixture was then added to cell population. Cell viability was monitored (Fig. 2) and these results served to evaluate the ability of HS to mitigate the toxic effect of both tested nZVI forms. When 1 g l1 Nanofer 25 was used (Fig. 2a), the X6 was the most effective in mitigation of nZVI toxicity in both the simultaneous effect and cell preincubation modes (see Material and methods for experiment set-ups). More than 84% and 88% of treated cells, www.elsevier.com/locate/nbt
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FIGURE 2
Mitigation of nZVI toxicity to T. cutaneum by humic substances X6 and X3 after 30-min exposure (nZVI concentration: white bars 1 g l1; gray bars 5 g l1). (a) Nanofer 25; (b) Nanofer 25S.
respectively, preserved their viability. In addition, the application of Nanofer 25 and X6 simultaneously showed the highest potential for protection against oxidative stress caused by 5 g l1 Nanofer 25. However, in all experiment set-ups the overall effect of protection was lower against the higher Nanofer 25 concentration. The protective effect of HS was also proved by the determination of cell morphology (Fig. 3). When HSs were added to Nanofer 25, the protection by HS was revealed as the diversity of cell shapes returned the more oblong shape was retained. Although the cell shape was more circular than the control population (median of circularity 86%), the overall characteristic and the variability of cell shapes indicated a lower stress. This effect was observed in all conditions studied and described above in viability tests for Nanofer 25. The circularity was in the range 70–90% (25th and 75th percentiles) for both HS and all experiment set-ups (simultaneous
effect, cell preincubation and nZVI preincubation) – in Fig. 3 are shown only the data for simultaneous effect with HS X6. The coated Nanofer 25S particles were tested in an identical manner as Nanofer 25 (Fig. 2b). They exhibited lower toxicity to T. cutaneum cells (39% viability in both concentrations), but the HSs offered less protection. The cell morphology corresponds with the observed viability. In Fig. 3, the influence of 5 g l1 Nanofer 25S and the effect of simultaneous exposure with HS X6 are shown. The exposed cells in both cases maintained their oblong shape (circularity 65–95%, as shown in 10th and 90th percentiles), the effect was similar for both HS and all experiment set-ups. To compare the impact of nZVI on cell morphology with the influence of ROS alone, cells were exposed to hydrogen peroxide (1 g l1). The exposure time and hydrogen peroxide concentration have been chosen to reflect the influence and ROS presence as caused by nZVI. After 30 min, no morphological changes were observed (data not shown).
Characterization of nZVI and T. cutaneum surface under studied conditions The zeta potential of both nanoparticles and T. cutaneum cells was determined to evaluate the surface properties that influence the mutual interactions between nanoparticles and cells. In addition, an effect of HSs on the zeta potential value was examined (see Table 1). The zeta potential of the Nanofer 25 was 3.1 mV. The TABLE 1
Effect of humic substances, Nanofer 25 (1 g l1) treatment and three experimental set-ups on zeta potential (mV) of Trichosporon cutaneum Control
Nanofer 25 Nanofer 25 + HS T. cutaneum T. cutaneum + Nanofer 25 T. cutaneum + HS
FIGURE 3
The effect of Nanofer 25 (5 g l1) and Nanofer 25S (5 g l1) and protection by HS against nZVI on T. cutaneum cell morphology expressed as cell circularity. The whiskers (error bars) represent 10th and 90th percentiles, the box charts boundaries represent the 25th and 75th percentiles and the center line depicts the median. 148
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nZVI + HS + T. cutaneum
Simultaneous effect Cell preincubation Nanofer 25 preincubation
X6 X3A
3.1 18.1 25.0 30.0 35.0 35.7 28.2
X6 X3A X6 X3A X6 X3A
31.7 36.1 32.6 34.2 40.1 39.4
X6 X3A
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zeta potential of Nanofer 25S was significantly higher (35 mV) and further interaction with both HSs did not bring about any significant change of the zeta potential. This points to a lower ability of HSs to mitigate the action of Nanofer 25S compared to Nanofer 25 – see Fig. 2. The value of surface charge can probably influence the extent of nanoparticle toxicity. The zeta potential of T. cutaneum was 30 mV. The considerable difference between the zeta potential of T. cutaneum and Nanofer 25 supports a higher toxicity of this material compared to Nanofer 25S, which was observed in viability tests. A decrease of T. cutaneum zeta potential to 35 mV was observed as a consequence of Nanofer 25 adsorption on cell surface. The interaction of both studied HSs with Nanofer 25 or T. cutaneum resulted in a change of surface charges. The surface charge of Nanofer 25 decreased after interaction with both X6 (18.1 mV) and X3A (25 mV). However, the reaction of T. cutaneum cells was different, as the application of X6 decreased the surface charge (35.7 mV) only moderately. X3A had an opposite effect and the surface charge was increased to 18.1 mV. The zeta potential of T. cutaneum under all three experimental set-ups changes according to the treatment conditions and the results correspond to those obtained by viability tests. The least change of T. cutaneum zeta potential occurred under protection with X6 and under simultaneous effect and cell preincubation conditions (31.7 mV, resp. 32.6 mV). By contrast, Nanofer 25 preincubation experiment resulted in a significant increase of cell surface charge.
TEM and SEM of T. cutaneum surface under studied conditions Electron microscopy was used to examine the T. cutaneum cell surface before and after exposure to Nanofer 25 (1 g l1) and/or oxyhumolite X6 (0.3 g l1). A crucial step of the nZVI mechanism of action is mutual interaction between nZVI and the exposed cells. The interaction of nZVI and cell surface is apparent from detailed images of both SEM (Fig. 4c) and TEM (Fig. 5c). Both methods show the ability of HS to adsorb on cell surface (Figs 4b and 5b). After incubation with X6, the cell surface was covered by an electron dense film (Fig. 5b) which presumably indicates HS adsorption. TEM images of the control population (Fig. 5a) display cells with morphology revealing the influence of nanoparticles and HS: ovoid cell shape and the cell cytosol and organelles are clearly recognizable as opposed to exposed cells. TEM analysis also proved that adhesion of nZVI to the cell surface occurs despite the HS absorption onto cells (Fig. 5d). However, cell lysis as a response to nZVI action is distinguishable only after exposure to nZVI without X6 protection (Figs 4c and 5c).
Discussion The nZVI technology attracts considerable attention mainly due to their unique properties, such as high reactivity and low standard reduction potential, which allow their use in remediation processes. However, these properties also influence biological activities and are associated with high toxicity against exposed microorganisms [37]. NZVI can considerably disturb autochthonal microbial populations and hinder their participation in environmental remediation. Our results showed that commercially available nZVI Nanofer 25 and Nanofer 25S have high toxicity against the yeast
FIGURE 4
SEM of Trichosporon cutaneum surface (a) after 15-min incubation in the presence of HS X6 0.3 g l1 (b) and 30-min exposure to Nanofer 25 1 g l1 (c).
T. cutaneum, an aerobic soil microorganism with an ability to utilize a wide range of organic pollutants [38]. Although the mechanism of nZVI action still needs to be clarified, Keenan et al. [12] have reported the ability of nZVI to induce ROS production. Because of their high reactivity, nZVI are oxidized very quickly to ferrous and ferric ion [39]. Oxidation can take place both extracellularly and in the cytosol [19]. Ferrous ion introduces a transient state and its oxidation to ferric ion leads to ROS production (Eqn 1–4). Adeleye et al. [40] studied long-term environmental fate of nZVI oxidation in aqueous media, including www.elsevier.com/locate/nbt
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TEM of Trichosporon cutaneum (a) after 15-min incubation in the presence of oxyhumolite X6 0.3 g l1 (b), 30-min treatment with Nanofer 25 1 g l1 (c) and treatment of oxyhumolite X6 pre-incubated cells with Nanofer 25 1 g l1 (d).
groundwater samples. The nZVI oxidation to ferrous iron occurred from the very start of the experiment. A possible link between oxidative stress induced by nZVI and the change of cell morphology was not proven. The presence of ROS caused by application of hydrogen peroxide did not change cell shape (data not shown). Since the presence of the HSs, which have the ability to produce an additive layer on the surface of cells [41] significantly suppressed changes in cell morphology, it is offered as a possible explanation of these changes response to the direct physical contact of nZVI with the cell surface. The change in cell shape from elongated to spherical can be seen as an effort to capture the smallest possible cell surface area and thus minimize contact with nZVI. A similar change of shape observed by Xie et al. [42]. Application of ZnO nanoparticles caused a morphological change of Campylobacter jejuni, which underwent a change of cell shape from spiral to coccoid. However, it was also observed the opposite effect when E. coli cells became more filamentous in the presence of nanoparticles CdO [43]. The importance of autochthonal microflora in environmental regeneration is indisputable. Therefore, recent research has focused on protection of microorganisms without the loss of nZVI activity. The protective effect of HSs is known from nature [44]. HSs have properties, such as the ability to bind to surfaces [18] or antioxidant properties [32], which make these substances useful for the protection of microorganisms. The use of HSs in mitigation 150
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of nanoparticle toxicity to microorganisms has been described in several reports [18,22,35]. Chen et al. [22] focused on the influence of HA on the toxicity and reactivity of nZVI. They found that HA not only significantly mitigated nZVI toxicity but also did not affect nZVI reactivity with trichloroethylene. Our study addressed the comparison of protection abilities of two chemically different HSs, oxidized oxyhumolite and HA. Both HSs proved the ability to mitigate the impact of nZVI. In all experiments a slightly better protective character was shown by HA (Fig. 2). Nevertheless, a significant mitigation of nZVI toxicity was observed mainly with respect to 1 g l1 Nanofer 25 (Fig. 2a). The results of morphological experiments corresponded with those obtained in viability tests. The most effective experiment set-up was cell preincubation with HA X6 (1 g l1 Nanofer 25). Treated cells showed a significantly lower change of morphology and higher viability. The biological activity of nZVI strongly depends on their specific form. Keller et al. [13] reported that different modifications of nZVI can profoundly influence their toxicity. In our research the stabilization of Nanofer 25S displayed lower toxicity to T. cutaneum compared to non-stabilized Nanofer 25. The stabilization or surface modification also influences interactions of nanoparticles with HSs. Dong and Lo [45] showed that different surface modification changed the mode of the HA–nZVI interaction and resulted in different degree of nanoparticles aglomeration. Therefore, the
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A positive effect of HSs on nZVI properties including mobility [46] and stability of nanoparticles in environment [22] has been reported. Our results suggest that application of HSs together with nanoparticles into the treated area enhances the viability of treated microorganisms.
Conclusion The toxicity of nZVI to yeast cells was proven to be affected by HSs, probably due to a hindrance of a direct contact between nZVI and exposed cells caused by electrostatic repulsion. Because of the chemical diversity of HSs the mitigation of nZVI toxicity also varies with the type of HS used. Therefore, the protective ability of each HS needs to be investigated to achieve the best result in specific circumstances. The advantage of HSs employment as a protectant is their natural origin and occurrence in environment. In this study, two different HSs and three set-ups of HSs application were compared. For the soil phenol-degrading yeast T. cutaneum the simultaneous effect of HA and cell preincubation with HA were the most effective in cell protection. The application of HSs in treating contaminated environments using nanoparticles could benefit by combining bioremediation and abiotic nanoparticle remediation processes resulting in better effectivity and lower impact on environment.
Conflict of interest No conflict of interest is declared.
Acknowledgements Financial support from specific university research (MSMT No. 20/ 2015) and by project of Ministry of Industry and Trade of the Czech Republic; Identification code: FR-TI3/564.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.nbt.2015.09. 007.
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presence of stabilizing agents (Na-biodegradable acrylic copolymer) on the surface of Nanofer 25S nanoparticles caused such an difference in surface properties between Nanofer 25 and Nanofer 25S that probably lead to lower ability of both studied HSs to mitigate the toxic impact of Nanofer 25S. A correlation between cell viability and morphology was also observed. Cells under Nanofer 25S treatment did not undergo significant change of the morphology in comparison with Nanofer 25 at both studied concentrations (corresponding with the lower toxicity of Nanofer 25S in comparison with Nanofer 25). Moreover, circularity of cells protected by HSs was higher because of a diminished protective character of HSs against Nanofer 25S action. By contrast, both HS showed high ability to mitigate the effect of Nanofer 25 on cell circularity. In their presence, the cells retained morphological characteristics resemblant to control population. The mutual interactions between nanoparticles and exposed cells seem to play a crucial role in the mechanism of nZVI action. Zeta potential characterizes the surface charge and its value influences potential interactions not only between nanoparticles themselves but also between nanoparticles and exposed cells. After adsorption of HS on both cells and nZVI, the negative charge of HS molecules caused a decrease of zeta potential of both types of nZVI and the cells [22]. These premises were confirmed also in our work by determination of zeta potential of nanoparticles, HSs and exposed cells. The results are summarized in Table 1. HSs probably behaved as polyelectrolytes with high molecular weight and thus caused electrosteric repulsion between nanoparticles and cells [18]. TEM microscopy (Fig. 5d) showed that adsorption of nanoparticles on cell surface occurred despite the presence of an HS coating. Thus, electrosteric repulsion alone seemed to be the principal mechanism in cell protection. However, adsorption of both HSs and nZVI on cell surface and simultaneous improvement of cell viability indicated restriction of physical interaction between cell surface and nZVI [22].
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