Article pubs.acs.org/est
Membrane Alterations in Pseudomonas putida F1 Exposed to Nanoscale Zerovalent Iron: Effects of Short-Term and Repetitive nZVI Exposure Panaya Kotchaplai,†,§ Eakalak Khan,‡ and Alisa S. Vangnai*,§,∥ †
International Program in Hazardous Substance and Environmental Management, Graduate School, §Center of Excellence on Hazardous Substance Management (HSM), and ∥Biocatalyst and Environmental Biotechnology Research Unit, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand ‡ Department of Civil and Environmental Engineering, North Dakota State University, Dept. #2470, P.O. Box 6050, Fargo, North Dakota 58108, United States S Supporting Information *
ABSTRACT: In this study, we report the effect of the commercial nanoscale zerovalent iron (nZVI) on environmental bacteria, emphasizing the importance of nZVI-bacterial membrane interaction on nZVI toxicity as well as the adaptability of bacteria to nZVI. Exposure of Pseudomonas putida F1 to 0.1, 1.0, and 5.0 g/L of nZVI caused the reduction in colony forming units (CFUs) substantially for almost 3 orders of magnitude. However, a rebound in the cell number was observed after the prolonged exposure except for 5.0 g/L nZVI at which bacterial viability was completely inhibited. Upon exposure, nZVI accumulated on and penetrated into the bacterial cell membrane. Cell membrane composition analysis revealed the conversion of the cis to trans isomer of unsaturated fatty acid upon short-term nZVI exposure, resulting in a more rigid membrane counteracting the membrane-fluidizing effect of nZVI. Several cycles of repetitive exposure of cells to 0.1 g/L nZVI induced a persistent phenotype of P. putida F1 as indicated by smaller colony morphology, a more rigid membrane, and higher tolerance to nZVI. A low interaction between nZVI particles and the surface of the nZVI-persistent phenotypic cells reduced the nZVI-induced membrane damage. This study unveils the significance of nZVI-membrane interaction on toxicity of nZVI toward bacteria.
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INTRODUCTION Nanoscale zerovalent iron (nZVI) is one of the most extensively used nanoparticles for environmental remediation due to its capability of transforming/degrading a wide range of toxic contaminants by reduction, oxidation and/or removing them by sorption. For in situ application, large amounts of iron nanoparticles are directly applied in contaminated soil and groundwater for the treatment of organic contaminants as well as inorganic anions and metals.1,2 Despite the advantages of nZVI for environmental remediation, it has been reported to cause adverse effects on aquatic organisms, terrestrial organisms, and mammalian cells,3 as well as microorganisms.4 Most studies on microbial susceptibility to nZVI exposure were performed in batch4,5 and reported that nZVI toxicity to bacterial cells could range from the alteration of bacterial activities6−8 to the reduction in cell viability9−12 depending on exposure dosage, time, and environmental conditions. Bacterial nZVI toxicity is largely contributed by two effects: cell membrane disruption and oxidative stress.4 Initially, a direct contact and attachment of iron nanoparticles onto and inside a bacterial cytoplasmic membrane results in cell membrane © XXXX American Chemical Society
disruption and interference of membrane-bound protein functionality.4,13−16 Once internalized, Fe0 in the nZVI particle can directly transfer electrons to the oxygen molecule generating hydrogen peroxide.12 The oxidized forms of Fe0, Fe(II), and Fe(III) species then further interact with hydrogen peroxide, generating more reactive oxygen species (ROS), which attack bacterial biomolecules, causing cellular oxidative stress.17 Nevertheless, as most bacterial cells are generally able to adapt to environmental stresses, they could resist and adapt to nZVI-induced oxidative stress by up-regulation of specific enzymes in an oxidative stress response and iron homeostasis.12,18,19 On the other hand, although the cell membrane is known as a cellular protective layer and a front line of defense, of which the composition could be altered in response to environmental stresses for cell survival,20,21 nZVI-mediated Received: Revised: Accepted: Published: A
February 9, 2017 May 12, 2017 June 23, 2017 June 24, 2017 DOI: 10.1021/acs.est.7b00736 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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Environmental Science & Technology
was adjusted to an initial cell concentration of 108 CFU/mL before being exposed to 0.1, 1.0, and 5.0 g/L of nZVI under shaking conditions (150 rpm), 30 °C for 1 h, and then prolonged for 6 h. For cell sampling, the suspension samples were taken while shaking to ensure the dispersion of cells from nZVI-induced agglomerates, serially diluted, and the diluted samples were then vortexed before spreading on the plates. Cell viability defined as cells with the ability to reproduce38 was monitored as the colony forming unit using the plate count technique and expressed as a logarithm value of the cell number after nZVI exposure (Cn) over the initial cell number (C0). Cellular adenosine triphosphate (ATP) content, which represents cell vitality or the physiological state of cells,38 was determined (SI, S3) occasionally for comparison. A control experiment was conducted in the same manner but without nZVI addition (referred to as untreated cells). The effects of 0.1 g/L of Fe(II), Fe(III), and 3 mg/L poly(acrylic acid) (equivalent to 3% of surfactant coating material in Nanofer 25S) were also tested in the same manner. Cell Repetitive Exposure to nZVI. Cell suspension was prepared as described above. For the test, nZVI at the environmentally relevant concentration of 0.1 g/L39 was selected. For each repetitive nZVI exposure cycle, the treated cell suspension from the previous cycle was transferred using 1% inoculum to fresh M9G. Then, once nZVI was reapplied, cells were exposed to nZVI for 24 h. The nZVI redosing cycle was conducted ten times. The obtained cells were then tested for their sensitivity to nZVI by exposing cells to 0.1, 1.0, and 5.0 g/L of nZVI at 150 rpm and 30 °C for 1 h prior to colony counting. Cells with distinct colony size obtained from the 10th nZVI exposure cycle were then retreated with nZVI at 0.1 g/L and examined as indicated following the methods described below. Cell Morphology and Membrane Analysis Using Electron Microscopy. Samples were prepared as described in the SI (S3−S4). Scanning electron microscopy (SEM) with EDX and transmission electron microscopy (TEM) were used to observe interactions between nZVI and the bacterial cell membrane. Bacterial Cell Membrane Analysis. Fatty acids, the cis and trans configuration of unsaturated fatty acids, and membrane fluidity from the bacterial cells treated under the indicated condition were examined as described in the SI (S4− S5). Determination of bacterial surface properties was conducted with an initial cell concentration of 108 CFU/mL. The electrophoretic mobility was measured in 5−200 mM NaCl solutions and analyzed as described in the SI (S2; nZVI characterization section). Statistical Analysis. Experimental data were statistically analyzed using two-way ANOVA followed by Tukey’s multiple comparisons or Student t test with the Holm-Sidak method (GraphPad Prism version 6.00 for Windows, GraphPad Software, La Jolla, California, USA, www.graphpad.com). A p < 0.05 was considered statistically significant. The standard deviation of the data was calculated and presented as error bars.
membrane modification upon nZVI exposure has not yet been studied. Despite the remarkable reactivity toward pollutants, nZVI is ultimately oxidized. The reactive lifespan of nZVI typically lasts within days to a few-week time frame depending on the particle characteristics and surrounding environmental conditions. This nZVI aging results in a rapid loss of Fe0 content and decreases its reactivity with the target contaminant.22,23 As a consequence, in addition to a one-time direct injection technique, the nZVI redosing process using a multiple-time injection of nZVI or recirculation of nZVI-amended groundwater with an addition of fresh nZVI24,25 has been increasingly practiced in order to maintain and enhance the pollutant treatment efficacy.22,25−27 Regarding toxicity of the nanoparticle redosing process, there has been only one report so far on the effect of sequential exposure of nanoparticles, including nZVI, on the wastewater bacterial community.28 Although the impact of nZVI on the microbial community was not found due to rapid nZVI aggregation reducing overall reactivity and toxicity,28 it cannot be ruled out that bacterial cells may also have a responsive mechanism in order to adapt to the sequential nZVI exposure. In general, repetitive exposure of bacterial cells to stress may strengthen them through the adaptation, while the cumulative effect may weaken them.29−31 In this point of view, susceptibility and responsive mechanism(s) of bacterial cells with nZVI repetitive exposure may be distinct from that of a single exposure and thus were investigated in this study. Since the bacterial membrane is a dynamic layer of fatty acids and phospholipids of which the composition is quantitatively and/ or qualitatively modified in response to stresses, nZVI-mediated membrane modification upon repetitive exposure was focused. In addition, while a toxicity study using a mixed culture could represent the complex and ecologically relevant environment, studying with pure bacterial culture is more suitable and eligible for deciphering the toxicity mechanism.32 In addition, although nZVI particles are widely applied to the subsurface environment, the effectiveness of nZVI application under aerobic conditions has been proposed and demonstrated.33−36 The oxidation of Fe0 under oxic conditions results in the initiation of Fenton chemistry and resultant production of ROS, which are capable of degrading various contaminants. Accordingly, P. putida F1, the environmental bacterium with an ability to degrade toxic pollutants including toluene and trichloroethylene,37 was selected as a model strain to investigate the nZVI effect on bacterial cells under aerobic conditions.
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MATERIALS AND METHODS nZVI Source and Characterization. Nanofer 25S, a poly(acrylic acid)-modified nZVI, was obtained from NANOIRON s.r.o., Czech Republic. nZVI characterizations are provided in the Supporting Information (SI) (S2). Bacteria, Media, and Cultivation Conditions. P. putida F1 was kindly provided by Professor J. Kato from Hiroshima University (Japan). Bacterial cells were cultivated at 30 °C with shaking conditions (150 rpm) in M9 minimal medium (pH 7.0) supplemented with 0.4% glucose (w/v) (thus further referred to as M9G). Medium components are provided in the SI (S2). nZVI Toxicity Assessment. P. putida F1 cells from the mid log phase were harvested by centrifugation at 5,000 rpm for 15 min and washed twice with 0.85% NaCl before being resuspended in M9G medium. The bacterial cell suspension
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RESULTS nZVI Characteristics. The toxicity of nanoparticles depends on their physicochemical properties including size,40 surface properties, and the reactivity of the particles,41 which are influenced by the environmental matrix. For instance, while the primary size of nZVI is in a nanometer range, nZVI particles can react and/or interact with environmental B
DOI: 10.1021/acs.est.7b00736 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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Environmental Science & Technology constituents such as Ca2+, resulting in rapid aggregation in natural water (i.e., groundwater and seawater) and a formation of microsized aggregates during in situ applications.42,43 The nanoparticle aggregates were reported to cause lower toxicity than the discrete nanoparticles due to their lower reactive surface area for interaction.44 In this study, in order to diminish the complexity from the organic matter in the biological medium, M9G medium was selected. Based on the dynamic light scattering, Nanofer 25S in M9G medium gave a z-average hydrodynamic size of 5,064 ± 308 nm and a polydispersity index of 0.52 ± 0.05, indicating the broad-size aggregation of iron nanoparticles in M9G medium. The number-averaged and volume-averaged size distributions of nZVI are shown in the SI, Figure S1. To estimate the reactive period of Nanofer 25S suspended in M9G medium under shaking and aerobic conditions, the Fe0 and Fe(II) contents representing nZVI reactive forms were determined over time. At initial time, the main iron species of 0.1 g/L Nanofer 25S was Fe(II) with 52.4 ± 1.4% of the total iron, whereas Fe0 and Fe(III) contents were 29.3 ± 3.0% and 18.3 ± 1.6%, respectively (Figure 1A). After 30 min under the
4% (200 mg/L) after 6 h of shaking in aerobic conditions for 1.0 and 5.0 g/L, respectively (data not shown). Bacterial Viability Rebound after Prolongation of nZVI Exposure. The effect of nZVI on bacterial viability is generally dose-dependent.4 In this study, within the first hour of nZVI exposure at 0.1 and 1.0 g/L, the number of CFUs of P. putida F1 decreased by 2.6 and 3.3 orders of magnitude, respectively (Figure 1 B). On the other hand, the CFUs of cells exposed to 5.0 g/L of nZVI decreased by only 2.2 orders of magnitude within the first hour of exposure probably due to aggregation of nZVI at high concentration reducing its immediate reactivity and toxicity to bacterial cells,19 but cells eventually were eradicated after prolonged exposure (Figure 1 B; SI, Table S1). The results were in agreement to the ATP assay of cells exposed to nZVI (SI, Figure S2). To differentiate the effect of nZVI from that of other iron forms or the coating material, bacterial CFUs were determined with Fe(II), Fe(III), or poly(acrylic acid) exposure. None of those chemicals substantially reduce bacterial viability (SI, Figure S3), suggesting that the reduction in cell viability was mainly caused by the iron nanoparticles. Interestingly, after prolongation of cell exposure to nZVI for 3−6 h, a rebound in a number of CFUs was clearly observed (Figure 1 B). There were two possibilities for this phenomenon. First, after nZVI (0.1 g/L) underwent full oxidation/aging within the first 0.5 h (Figure 1 A), the remaining, uninjured or sublethally injured, bacterial population could resume growth in the less unfavorable condition. Second, there was a subpopulation of cells that could respond or adapt to survive under the stress45 caused by nZVI exposure and eventually could resume growth. Further investigations on viability and characteristics of cells with prolonged and repetitive nZVI exposure were conducted to clarify this, and the result suggested the latter case (as described below). Repetitive nZVI Exposure Induced the Emergence of Bacterial Phenotypic Variant. The test of cells with prolonged and repetitive nZVI exposure was conducted not only to elucidate the response mechanism of bacterial cells to repetitive nZVI exposure but also to imply what potentially happens to indigenous environmental bacteria that encounter the repetitive exposure to nZVI through nZVI redosing process or recirculation of nZVI practice. An altered phenotype of P. putida F1 cells, as characterized as small colony morphology, was detected when cells were repetitively exposed to nZVI for three cycles (Figure 2 A). The proportion of the small-colony phenotypic variant (referred to as the persistent phenotypic cells hereafter) increased with the increasing number of nZVI redosing cycles and became more than 75% and >90% of the total cell population after cells were repetitively exposed to nZVI for the fifth and 10th cycles, respectively (Figure 2 B). Subsequently, the persistent phenotypic cells from the 10threpetitively nZVI exposure were determined for their susceptibility to nZVI. The result clearly showed that the persistent phenotypic cells exhibited significantly less susceptibility (i.e., more tolerance) to nZVI toxicity (adjusted p = 0.0001 and 0.0003 for cells exposed to 0.1 and 5.0 g/L, respectively; adjusted p < 0.0001 for cells exposed to 1.0 g/L nZVI) compared to that of the normal cells (Figure 2 C). A further test was subsequently conducted to examine whether the small-colony phenotypic variant generated by multiple nZVI redosing was either a temporary or permanent cell characteristic. The persistent phenotypic cells from the 10th cycle nZVI exposure were recultivated in nZVI-free M9G
Figure 1. Iron content of nZVI under the test conditions and the impact of nZVI on P. putida F1 cell survival. (A) Percentage of the iron content of 0.1 g/L nZVI suspended in M9G medium maintained under shaking aerobic conditions for 6 h. The inset emphasizes the content of iron species within the first hour of oxygen exposure. (B) Time-dependent survival of P. putida F1 cells after nZVI exposure. Cells from the mid log phase with an initial concentration of 108 CFU/mL were exposed to nZVI at (■) 0.1 g/L, (▲) 1.0 g/L, and (●) 5.0 g/L. The control was nZVI-untreated cells (○). The data were means ± SD of at least three independent experiments.
indicated condition, Fe0 was fully oxidized to a nondetectable level, while Fe(II) content was decreased to 4.4 ± 0.48%, and Fe(III) became the major iron species contributing to 95.6 ± 0.48%. This indicated that the reactive period of 0.1 g/L Nanofer 25S under the test conditions in this study was considerably short, compared to the generally reported 2-h lifetime of nZVI under other aerobic conditions.13 The remaining Fe0 content was approximately 2% (20 mg/L) and C
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deal with and the first line of defense against an environmental disturbance that may affect the physiological function of the cell. Upon the cell stress challenge, the membrane flexibility and adaptation ability predominantly determine the survival of the cell.21 As our results showed that P. putida F1 survived and exhibited the transient phenotypic variant upon nZVI exposure, it was of our interest to further explore cell membrane adaptation/alteration in response to nZVI exposure. Thus, morphology and membrane of the bacterial cells presented in different conditions (with or without nZVI exposure) were initially visualized by TEM (Figure 3) and SEM (SI, Figure S5). The normal cells of P. putida F1 grown in M9G medium are rod-shaped (Figure 3 A, B; SI, Figure S5) with a smooth intact membrane (Figure 3 C). Upon a single dose application and 1h exposure of nZVI at 0.1 g/L to cells, nZVI particles extensively accumulated onto bacterial cells outer surface (Figure 3 D-F), while some penetrated inside and localized adjacent to the cell membrane (as described by the SEM-EDX result below), which may cause cell deformation and damage (Figure 3 F). Interestingly, once the nZVI exposure was prolonged to 6 h, the aggregation of nZVI on the bacterial cell surface was obviously less intense (Figure 3 G-I). The electron micrograph analysis of the nZVI-persistent phenotypic cells indicated there was no major difference in cell size between the normal and the nZVI-persistent phenotypic cells (SI, Figure S5 B and F, respectively); thus the small colony size of the persistent phenotypic cells may be due to their differences in colony forming rate. In addition, the persistent phenotypic cell surface was smooth (SI, Figure S5 H) with apparently less intense aggregation and penetration of nZVI (Figure 3 J-O). Further analysis using SEM-EDX was conducted to elaborate this phenomenon. The Fe content of the normal cells treated with nZVI (SI, Figure S5 D) was significantly higher than that of the untreated normal cells (SI, Figure S5 B) suggesting the penetration and accumulation of nZVI into the nZVI-treated normal cells. The analysis of the persistent phenotype without nZVI exposure showed that cells have inherent high Fe content (SI, Figure S5 F) at 2.73% by wt because these cells were repeatedly exposed to nZVI, and thus it was likely that Fe may be incorporated into cell structure. Then, when the Fe content of the nZVI-treated persistent phenotype was determined (SI, Figure S5 H), it was interestingly found that the Fe content of cells was similar to that of the untreated persistent phenotype (2.59% by wt). This result initially suggested that there was membrane alteration of the persistent phenotype contributing to cell higher tolerance to nZVI. Accordingly, a comprehensive examination was further studied. Decreasing Membrane Fluidity after Cell Repetitive Exposure to nZVI. Bacterial cells critically respond to environmental stressors that compromise their membrane integrity with a homeoviscous adaptation mechanism in which cell membrane lipid composition, and thus its fluidity, is regulatory altered to be a proper state for cell survival.46 The P. putida F1 cell membrane was obviously perturbed by nZVI. The distinctive cellular outer surface and membrane appearance of the persistent phenotypic cells treated with nZVI suggested that the membrane adaptation process occurred. Thus, membrane characteristics of the nZVI-persistent phenotypic cells were investigated in comparison to that of the normal cells to gain an insight on the nZVI-mediated bacterial membrane modification in response to nZVI exposure. The cell membrane fluidity state of the normal cells and the persistent phenotypic cells with and without nZVI exposure
Figure 2. P. putida F1 colony morphology and nZVI susceptibility after the repetitive nZVI exposure. (A) Colony morphology of the small-colony phenotypic variant (referred to as the persistent phenotypic cells) of P. putida F1 after 3 times repeatedly exposed to 0.1 g/L of nZVI. (B) Proportion of the normal cells and the persistent phenotypic cells with the increasing number of nZVI redosing cycle. (C) nZVI susceptibility of the normal cells and the persistent phenotypic cells generated from the 10th-repetitively nZVI exposure tested with various nZVI concentrations. CFUs were determined after 1 h of nZVI exposure, and the result was expressed as a logarithm value of CFUs (Cn) over the initial CFUs (C0). Italic letters (with and without an asterisk) indicate a significant difference (p < 0.05) from the normal, untreated cells within the same group, according to the two-way ANOVA followed by Tukey’s multiple comparisons.
medium for five cycles to determine the dynamics of normal phenotype recovery (SI, Table S2). Cells from the fifth recovery cycle were then treated with nZVI (0.1 g/L). The colony morphology and nZVI susceptibility characteristics of the persistent phenotypic cells were reverted to be as similar as those of the normal cells (SI, Figure S4). This result suggested that repetitive exposure to nZVI induced the emergence of the transient phenotypic variant capable of surviving or persisting in the presence of a lethal dose of nZVI or repetitive exposure conditions. After the nZVI stressor was removed, these persistent cells may resume the characteristics of the normal cells, as shown in this case as the susceptibility to nZVI. Visualization of Bacterial Membrane-nZVI Interaction. Acting as a barrier between cellular internal and external environments, the bacterial cell membrane is an initial target to D
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Figure 3. TEM micrographs of P. putida F1 cells: (A−C) the normal mid log phase cells without nZVI exposure; (D−F) the normal cells after 0.1 g/ L nZVI exposure for 1 h and (G−I) for 6 h; (J−L) the nZVI-persistent phenotypic cells after 0.1 g/L nZVI exposure for 1 h and (M−O) for 6 h. Scale bar differences are noted.
was assessed using DPH, a fluorescent probe capable of intercalating into the lipid bilayer of the membrane and being sensitive to lipid reorientation, and was expressed as the fluorescent anisotropy, which is inversely proportional to membrane fluidity. When the normal cells were exposed to nZVI, the intercalation of DPH into the bacterial cell membrane layer was disturbed, and the fluorescent anisotropy value increased twice as much indicating that the bacterial membrane becomes less fluid (i.e., more rigid) (Figure 4). Prior to nZVI exposure, the fluorescent anisotropy value of the persistent phenotypic cells generated from repetitive nZVI exposure was significantly (p = 0.001) higher than that of the normal cells, suggesting a more rigid membrane (Figure 4). As the nZVI-treated persistent phenotypic cells also possessed a substantially higher fluorescent anisotropy value (Figure 4 A), the result thus suggested that cells with either single nZVI exposure or repetitive nZVI exposure were likely to increase membrane rigidity against nZVI-mediated membrane damage. Cis/Trans Isomerization Is a Short-Term Response to nZVI Exposure. It was found that P. putida F1 adapted to
nZVI exposure by increasing its membrane rigidity to maintain cell survival probably by reducing the degree of interaction between cells and the particles resulting in the increase of cell tolerance to nZVI and the rebound growth. Bacterial membrane fluidity change is considered one of the adaptive responses of bacteria against unfavorable environmental factors that can occur by (i) alteration of degree of saturation of fatty acid,47 (ii) conversion of cis to trans isomer of unsaturated fatty acid, or (iii) synthesis of branched or cyclopropane fatty acids.20,48 A further investigation was conducted to gain insight into the unexplored bacterial membrane adaptation mechanism of nZVI. Fatty acid analysis of the normal cells showed that the dominant fatty acid components of P. putida F1 grown in M9G medium were C16:0, C16:1, and C18:1 (SI, Table S3) as previously reported by Fang et al.47 When the normal cells were exposed to nZVI once for 1 h, cells had an immediate response with 1.2 times increase of the degree of saturation of the fatty acids (Figure 4 B; SI Table S3), while the conversion of cisunsaturated fatty acids to trans-unsaturated fatty acids was markedly elevated by 5.4 times (Figure 4 C; SI Table S3). On E
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Figure 4. Modification in the membrane of P. putida F1 cells after exposure to 0.1 g/L of nZVI for 1 h. (A) Membrane fluidity expressed as fluorescent anisotropy ± SD. The higher fluorescent anisotropy indicates less membrane fluidity (i.e., more membrane rigidity). (B) Degree of membrane fatty acid saturation, which is the ratio between unsaturated fatty acids and saturated fatty acids. (C) The ratio between trans and cis isomers of unsaturated fatty acid (i.e., C16:1 and C18:1). (D) Membrane Viscosity Index (MVI) value. Italic letters (with and without an asterisk) indicate a significant difference (Student t test with the Holm-Sidak method, p < 0.05) from the normal without nZVI exposure. An asterisk indicates insignificant difference between with and without nZVI exposure in the same group.
the other hand, further analysis indicated that the persistent phenotypic cells generated by multiple nZVI redosing acquired a relatively higher value of the saturated-to-unsaturated fatty acid ratio than that of the normal cells even prior to nZVI exposure. Upon nZVI exposure, the ratio was substantially upshifted by 1.4 times, while the conversion of cis-unsaturated fatty acids to trans-unsaturated fatty acids was considerably low. From these results, the MVI values were then deduced from the integration of the degree of saturation and fatty acid configuration to confer membrane fluidity49 (Figure 4 D; SI Table S3). It was shown that both normal and persistent phenotypic cells responded similarly to nZVI exposure by increasing their membrane rigidity (i.e., with higher MVI values), although it was apparent that they relied on different membrane adaptation mechanisms. Bacterial Surface Properties As Determined by Soft Particle Theory. The interaction between nZVI and bacterial cell is governed by various physicochemical parameters,50 including electrostatic interaction.51 Bacterial surface properties were evaluated based on Ohshima’s soft particle theory. The persistent phenotypic cells exhibited a higher fixed charge density and lower electrophoretic softness than that of the normal cells (SI, Table S4). Previous reports showed that modification/removal of bacterial outer surface components, for example, lipopolysaccharides (LPS) or extracellular polymeric substance (EPS), could alter bacterial surface properties.52−54 In such a case, bacterial cells with truncated 4 mm LPS exhibited the higher charge density and lower electrophoretic softness than that of their highly truncated 1 mm LPS phenotype.52 In this study, the outer surface potential of the persistent phenotypic cells at −1.12 mV was substantially more negative than that at −0.77 mV of the normal cells. This result infers some degree of higher electrostatic repulsion between nZVI and surface of the persistent phenotypic cells (SI, Figure S6) and may imply the modification in outer surface
components of the persistent phenotypic cells, which resulted in less interaction between nZVI and cells.
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DISCUSSION
It is generally reported that nZVI particles cause bacterial cell damage; however, this work reveals a bacterial modification/ adaptation mechanism in response to nZVI exposure, resulting in higher tolerance and growth recovery of bacterial cells under the prolonged nZVI exposure. Although impacts of nZVI to bacterial cells were previously reported, cell susceptibility and responses were varied depending on bacterial strain, stage of growth, and exposure conditions. Xiu et al. (2010) reported that the metabolic activity of a dechlorinating-microbial consortium was inhibited upon the exposure to 1 g/L of nZVI but was resumed in the nZVI-free system.55 Studies of the bacterial pure cultures by Chaithawiwat et al. (2016) revealed that Gram-negative bacterial cells, i.e. E. coli and P. putida, in lag and stationary phases showed higher resistance than those in exponential and decline phases when exposed to 1 g/L of nZVI.16 On the other hand, a report by Sacca et al. (2014) showed that the viability of P. stutzeri was adversely affected by Nanofer 25S at 1−5 g/L but was less impacted at 10 g/L because nZVI particles were aggregated in a nonshaking test condition, thus reducing their reactivity and limiting nZVIbacterial cell contact.19 In addition, bacterial cell activity could also be influenced by particle dosing. While the dosing of nZVI, nano titanium dioxide, or nano cerium dioxide in sequencing batch reactors at increasing concentration showed an insignificant effect on nitrification and bacterial community composition in activated sludge,28 the repetitive exposure to titanium dioxide nanoparticles and carbon nanotubes reduced the activity of soil microorganisms.31,56 These discrepant effects of nanoparticles as well as nZVI to bacterial cells accentuate the importance of their ecotoxicological assessment because the F
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proteins),77,78 while the negative charge of Gram positive bacteria is mainly attributed to the peptidoglycan and teichoic acid in the outer surface.78 These bacterial surface components play an important role in the interaction between bacterial surface and environmental matrix including soil and sand particles. The cationic metal ion, for example, copper, lead, and iron, primarily binds to the phosphate headgroup of phospholipid and the LPS of bacteria.79,80 The altered surface components exhibit varied affinities for metal binding due to differences in their surface charge.79 While the adhesion between bacteria and surface is mainly attributed to a longrange electrostatic interaction, other aspects such as hydrophobic interaction or polymer bridging cannot be excluded.81,82 In addition, the increase of extracellular proteins and EPS production has been reported in bacterial cells adapted to the exposure to increasing concentrations of silver nanoparticles and zinc oxide nanoparticles.83 The increasing amount of surface components contributed to the higher negative charge83 and may provide more binding sites for the released metal ion, reducing nanoparticles dispersion, and direct contact with bacterial cells (steric repulsion).83−85 Accordingly, in this study, it cannot be ruled out that the repetitive exposure of cells to nZVI may induce the modification of bacterial cell surface components, thus reducing the interaction between the bacterial cells and nZVI. In addition, even though the persistent phenotype obtained from the repetitive nZVI exposure can revert to its parental characteristics after several sequential cultivations in the absence of nZVI, the emergence of this phenotype should not be neglected since the mechanism underlying this nZVI persistence as well as the characteristics of the emergent phenotype is not thoroughly understood.
transformation/aging of nanoparticles and bacterial adaptation likely occur.57−59 Several nanotoxicological studies showed that bacterial cells are able to cope with stresses generated by nZVI at various levels. Molecular analysis of biomarker gene expression as well as transcriptional and protein expression of Bacillus cereus, P. stuzeri, and Klebsiella oxytoca exposed to nZVI revealed the overexpressing genes and proteins involved in tricarboxylic acid cycle modulation, cellular detoxification metabolism, and oxidative-stress responses to counteract nZVI-induced damages.19,60−62 At a cellular level, bacterial cell wall adaptations to a single nZVI exposure were also reported.19 In this study, the prolonged and repetitive nZVI exposure generated stress to P. putida F1 resulting in cell adaptation and persistent cellular phenotypic change to small colony morphology. Phenotypic variation has been known as one of the adaptive responses of bacterial cells to environmental stresses.63 Long-term exposure to cold stress induced the emergence of the reversible small colony phenotype (SCV) of Staphylococcal cultures with distinct structure and amino acid composition of the cell wall.64 Repetitive exposure to triclosan has been reported to increase the SCV phenotype showing higher persistence to triclosan and altered susceptibility to other stresses.65 Stochastically epigenetic changes or altered metabolic balance leading to different physiological states of cells (phenotypic heterogeneity) may grant them the chance to survive under stress conditions.64,66,67 The long-term or repetitive exposure to stress may be a selection pressure for the persistent phenotype. Direct contact between nanoparticles and the bacterial cell membrane is known as a primary cause for cell growth inactivation and cytotoxicity. Therefore, membrane adaptation is an initial bacterial responsive mechanism to nanoparticles59,68 as well as other environmental stresses including organic solvent,69−71 heavy metal,72 and pH.73 Upon initial exposure of P. putida F1 to nZVI, although cell growth was inactivated, cells adopted the cis−trans isomerization as an urgent stress response mechanism to severely nZVI-induced membrane damage.48,74,75 This process is an energy-independent, posttranslation modification process of the membrane fatty acyl chain known only in certain bacterial strains, Pseudomonas and Vibrio,74 making them capable of adapting to and enduring under growth-suppressed conditions.74,76 On the contrary, the prolonged and/or repetitive nZVI exposure allowed cells to evolve. In this case, the persistent phenotypic cells responded by acquiring homeoviscous adaptation of the cell membrane. By increasing the degree of saturation, the membrane of persistent phenotypic cells was more rigid, which contributed to higher nZVI tolerance. This phenomenon is in agreement with the report of bacterial adaptive responses to carbon nanotubes in which membrane fluidity was increased by modifying their fatty acid composition. In that study, Gramnegative bacteria elevated the level of saturated fatty acids, while Gram-positive bacteria increased the proportion of branched-chain fatty acids to counteract the toxic effect of carbon nanotubes.59 Several factors have been reported to affect the nZVI-bacteria interaction including the difference in bacterial surface characteristics.4,11,50 Less nZVI toxicity on Bacillus subtilis is attributed to its more negative surface charge, compared to E. coli.14 Gram-negative bacteria exhibit the negative surface charge due to the presence of the carboxylic acids and phosphate esters (phosphoryl group) in their cell surface components (i.e., phospholipids, lipoproteins, LPS, and
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.7b00736. Details of methodology: 1) nZVI characterization, 2) M9G composition, 3) ATP assay, 4) sample preparations for SEM, SEM-EDX, and TEM analysis, 5) bacterial cell membrane analysis, 6) fatty acid extraction and derivatization, and 7) membrane fluidity analysis using fluorescent anisotropy measurement; Figure S1, numberand volume-averaged size distributions of nZVI; Figure S2, ATP content and colony forming units of P. putida F1 cells after exposure to nZVI; Figure S3, effect of other iron forms or the coating material on P. putida F1 cell viability; Figure S4, susceptibility to nZVI of the nZVIpersistent phenotypic cells after recultivation in nZVIfree M9G medium; Figure S5, SEM and SEM-EDX analysis of P. putida F1 cells with and without nZVI exposure; Table S1, bacterial toxicity of nZVI; Table S2, dynamics of bacterial recovery after nZVI removal; Table S3, fatty acid profiles of P. putida F1 and persistent phenotype cells; Table S4, surface characteristics of nZVI and bacterial cells based on Ohshima’s soft particle theory (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Phone: +662 218 5430. Fax: +662 218 5418. E-mail: alisa.v@ chula.ac.th. Corresponding author address: Department of G
DOI: 10.1021/acs.est.7b00736 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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Environmental Science & Technology
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Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand. ORCID
Eakalak Khan: 0000-0002-6729-2170 Alisa S. Vangnai: 0000-0001-9485-5672 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors would like to thank Wanwara Thuptimdang for MatLab processing in Ohshima’s soft particle analysis. This research was financially supported by the Thailand Research Fund (IRG 5780008) and the 90th Anniversary of Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund). The stipend for the first author was supported by the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program (Grant No. PHD/0359/2551).
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