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The rapid development of nanotechnology and its integration into everyday applications can threaten the environment. While green methods for the degradation of organic contaminants are well established, the recovery of inorganic crystalline contaminants is of major concern due to their low sensitivity to biotransformation and lack of understanding of material surface interactions with biological ones. Here, we use a Nb-based inorganic 2D MXenes model combined with a simple shape parameter analysis method to trace the bioremediation mechanism of 2D ceramic nanomaterials by the green microalgae Raphidocelis subcapitata. We found that microalgae degrade Nb-based MXenes due to surface-related physico-chemical interactions. Initially, single-layer and multilayer MXene nanoflakes were attached to the surface of microalgae, which somewhat reduced the growth of algae. However, upon prolonged interaction with the surface, microalgae oxidized MXene nanoflakes and further decomposed them into NbO and Nb2O5. Because these oxides are non-toxic to microalgae cells, they consume Nb oxide nanoparticles by an absorption mechanism that further restores the microalgae after 72 hours of water treatment. The effects of nutrients associated with absorption are also reflected in the increase in cell volume, their smooth shape and change in growth rate. Based on these findings, we conclude that the short and long term presence of Nb-based MXenes in freshwater ecosystems may cause only minor environmental impacts. It is noteworthy that, using two-dimensional nanomaterials as model systems, we demonstrate the possibility of tracking shape transformation even in fine-grained materials. Overall, this study answers an important fundamental question about surface interaction-related processes driving the bioremediation mechanism of 2D nanomaterials and provides a basis for further short-term and long-term studies of the environmental impact of inorganic crystalline nanomaterials.
Nanomaterials have generated a lot of interest since their discovery, and various nanotechnologies have recently entered a modernization phase1. Unfortunately, the integration of nanomaterials into everyday applications can lead to accidental releases due to improper disposal, careless handling, or inadequate safety infrastructure. Therefore, it is reasonable to assume that nanomaterials, including two-dimensional (2D) nanomaterials, can be released into the natural environment, the behavior and biological activity of which are not yet fully understood. Therefore, it is not surprising that ecotoxicity concerns have focused on the ability of 2D nanomaterials to leach into aquatic systems2,3,4,5,6. In these ecosystems, some 2D nanomaterials can interact with various organisms at different trophic levels, including microalgae.
Microalgae are primitive organisms found naturally in freshwater and marine ecosystems that produce a variety of chemical products through photosynthesis7. As such, they are critical to aquatic ecosystems8,9,10,11,12 but are also sensitive, inexpensive and widely used indicators of ecotoxicity13,14. Since microalgae cells multiply rapidly and quickly respond to the presence of various compounds, they are promising for the development of environmentally friendly methods for treating water contaminated with organic substances15,16.
Algae cells can remove inorganic ions from water through biosorption and accumulation17,18. Some algal species such as Chlorella, Anabaena invar, Westiellopsis prolifica, Stigeoclonium tenue and Synechococcus sp. It has been found to carry and even nourish toxic metal ions such as Fe2+, Cu2+, Zn2+ and Mn2+19. Other studies have shown that Cu2+, Cd2+, Ni2+, Zn2+ or Pb2+ ions limit the growth of Scenedesmus by altering cell morphology and destroying their chloroplasts20,21.
Green methods for the decomposition of organic pollutants and the removal of heavy metal ions have attracted the attention of scientists and engineers around the world. This is mainly due to the fact that these contaminants are easily processed in the liquid phase. However, inorganic crystalline pollutants are characterized by low water solubility and low susceptibility to various biotransformations, which causes great difficulties in remediation, and little progress has been made in this area22,23,24,25,26. Thus, the search for environmentally friendly solutions for the repair of nanomaterials remains a complex and unexplored area. Due to the high degree of uncertainty regarding the biotransformation effects of 2D nanomaterials, there is no easy way to find out the possible pathways of their degradation during reduction.
In this study, we used green microalgae as an active aqueous bioremediation agent for inorganic ceramic materials, combined with in situ monitoring of the degradation process of MXene as a representative of inorganic ceramic materials. The term “MXene” reflects the stoichiometry of the Mn+1XnTx material, where M is an early transition metal, X is carbon and/or nitrogen, Tx is a surface terminator (e.g., -OH, -F, -Cl), and n = 1, 2, 3 or 427.28. Since the discovery of MXenes by Naguib et al. Sensorics, cancer therapy and membrane filtration 27,29,30. In addition, MXenes can be considered as model 2D systems due to their excellent colloidal stability and possible biological interactions31,32,33,34,35,36.
Therefore, the methodology developed in this article and our research hypotheses are shown in Figure 1. According to this hypothesis, microalgae degrade Nb-based MXenes into non-toxic compounds due to surface-related physico-chemical interactions, which allows further recovery of the algae. To test this hypothesis, two members of the family of early niobium-based transition metal carbides and/or nitrides (MXenes), namely Nb2CTx and Nb4C3TX, were selected.
Research methodology and evidence-based hypotheses for MXene recovery by green microalgae Raphidocelis subcapitata. Please note that this is just a schematic representation of evidence-based assumptions. The lake environment differs in the nutrient medium used and the conditions (eg, diurnal cycle and limitations in available essential nutrients). Created with BioRender.com.
Therefore, by using MXene as a model system, we have opened the door to the study of various biological effects that cannot be observed with other conventional nanomaterials. In particular, we demonstrate the possibility of bioremediation of two-dimensional nanomaterials, such as niobium-based MXenes, by microalgae Raphidocelis subcapitata. Microalgae are able to degrade Nb-MXenes into the non-toxic oxides NbO and Nb2O5, which also provide nutrients through the niobium uptake mechanism. Overall, this study answers an important fundamental question about the processes associated with surface physicochemical interactions that govern the mechanisms of bioremediation of two-dimensional nanomaterials. In addition, we are developing a simple shape-parameter-based method for tracking subtle changes in the shape of 2D nanomaterials. This inspires further short-term and long-term research into the various environmental impacts of inorganic crystalline nanomaterials. Thus, our study increases the understanding of the interaction between the material surface and biological material. We are also providing the basis for expanded short-term and long-term studies of their possible impacts on freshwater ecosystems, which can now be easily verified.
MXenes represent an interesting class of materials with unique and attractive physical and chemical properties and therefore many potential applications. These properties are largely dependent on their stoichiometry and surface chemistry. Therefore, in our study, we investigated two types of Nb-based hierarchical single-layer (SL) MXenes, Nb2CTx and Nb4C3TX, since different biological effects of these nanomaterials could be observed. MXenes are produced from their starting materials by top-down selective etching of atomically thin MAX-phase A-layers. The MAX phase is a ternary ceramic composed of “bonded” blocks of transition metal carbides and thin layers of “A” elements such as Al, Si, and Sn with MnAXn-1 stoichiometry. The morphology of the initial MAX phase was observed by scanning electron microscopy (SEM) and was consistent with previous studies (See Supplementary Information, SI, Figure S1). Multilayer (ML) Nb-MXene was obtained after removing the Al layer with 48% HF (hydrofluoric acid). The morphology of ML-Nb2CTx and ML-Nb4C3TX was examined by scanning electron microscopy (SEM) (Figures S1c and S1d respectively) and a typical layered MXene morphology was observed, similar to two-dimensional nanoflakes passing through elongated pore-like slits. Both Nb-MXenes have much in common with MXene phases previously synthesized by acid etching27,38. After confirming the structure of MXene, we layered it by intercalation of tetrabutylammonium hydroxide (TBAOH) followed by washing and sonication, after which we obtained single-layer or low-layer (SL) 2D Nb-MXene nanoflakes.
We used high resolution transmission electron microscopy (HRTEM) and X-ray diffraction (XRD) to test the efficiency of etching and further peeling. The HRTEM results processed using the Inverse Fast Fourier Transform (IFFT) and Fast Fourier Transform (FFT) are shown in Fig. 2. Nb-MXene nanoflakes were oriented edge up to check the structure of the atomic layer and measure the interplanar distances. HRTEM images of MXene Nb2CTx and Nb4C3TX nanoflakes revealed their atomically thin layered nature (see Fig. 2a1, a2), as previously reported by Naguib et al.27 and Jastrzębska et al.38. For two adjacent Nb2CTx and Nb4C3Tx monolayers, we determined interlayer distances of 0.74 and 1.54 nm, respectively (Figs. 2b1,b2), which also agrees with our previous results38. This was further confirmed by the inverse fast Fourier transform (Fig. 2c1, c2) and the fast Fourier transform (Fig. 2d1, d2) showing the distance between the Nb2CTx and Nb4C3Tx monolayers. The image shows an alternation of light and dark bands corresponding to niobium and carbon atoms, which confirms the layered nature of the studied MXenes. It is important to note that the energy dispersive X-ray spectroscopy (EDX) spectra obtained for Nb2CTx and Nb4C3Tx (Figures S2a and S2b) showed no remnant of the original MAX phase, since no Al peak was detected.
Characterization of SL Nb2CTx and Nb4C3Tx MXene nanoflakes, including (a) high resolution electron microscopy (HRTEM) side-view 2D nanoflake imaging and corresponding, (b) intensity mode, (c) inverse fast Fourier transform (IFFT), (d) fast Fourier transform (FFT), (e) Nb-MXenes X-ray patterns. For SL 2D Nb2CTx, the numbers are expressed as (a1, b1, c1, d1, e1). For SL 2D Nb4C3Tx, the numbers are expressed as (a2, b2, c2, d2, e1).
X-ray diffraction measurements of SL Nb2CTx and Nb4C3Tx MXenes are shown in Figs. 2e1 and e2, respectively. Peaks (002) at 4.31 and 4.32 correspond to the previously described layered MXenes Nb2CTx and Nb4C3TX38,39,40,41 respectively. The XRD results also indicate the presence of some residual ML structures and MAX phases, but mostly XRD patterns associated with SL Nb4C3Tx (Fig. 2e2). The presence of smaller particles of the MAX phase may explain the stronger MAX peak compared to the randomly stacked Nb4C3Tx layers.
Further research has focused on green microalgae belonging to the species R. subcapitata. We chose microalgae because they are important producers involved in major food webs42. They are also one of the best indicators of toxicity due to the ability to remove toxic substances that are carried to higher levels of the food chain43. In addition, research on R. subcapitata may shed light on the incidental toxicity of SL Nb-MXenes to common freshwater microorganisms. To illustrate this, the researchers hypothesized that each microbe has a different sensitivity to toxic compounds present in the environment. For most organisms, low concentrations of substances do not affect their growth, while concentrations above a certain limit can inhibit them or even cause death. Therefore, for our studies of the surface interaction between microalgae and MXenes and the associated recovery, we decided to test the harmless and toxic concentrations of Nb-MXenes. To do this, we tested concentrations of 0 (as a reference), 0.01, 0.1 and 10 mg l-1 MXene and additionally infected microalgae with very high concentrations of MXene (100 mg l-1 MXene), which can be extreme and lethal. . for any biological environment.
The effects of SL Nb-MXenes on microalgae are shown in Figure 3, expressed as the percentage of growth promotion (+) or inhibition (-) measured for 0 mg l-1 samples. For comparison, the Nb-MAX phase and ML Nb-MXenes were also tested and the results are shown in SI (see Fig. S3). The results obtained confirmed that SL Nb-MXenes is almost completely devoid of toxicity in the range of low concentrations from 0.01 to 10 mg/l, as shown in Fig. 3a,b. In the case of Nb2CTx, we observed no more than 5% ecotoxicity in the specified range.
Stimulation (+) or inhibition (-) of microalgae growth in the presence of SL (a) Nb2CTx and (b) Nb4C3TX MXene. 24, 48 and 72 hours of MXene-microalgae interaction were analyzed. Significant data (t-test, p < 0.05) were marked with an asterisk (*). Significant data (t-test, p < 0.05) were marked with an asterisk (*). Значимые данные (t-критерий, p < 0,05) отмечены звездочкой (*). Significant data (t-test, p < 0.05) are marked with an asterisk (*).重要数据（t 检验，p < 0.05）用星号(*) 标记。重要数据（t 检验，p < 0.05）用星号(*) 标记。 Важные данные (t-test, p < 0,05) отмечены звездочкой (*). Important data (t-test, p < 0.05) are marked with an asterisk (*). Red arrows indicate the abolition of inhibitory stimulation.
On the other hand, low concentrations of Nb4C3TX turned out to be slightly more toxic, but not higher than 7%. As expected, we observed that MXenes had higher toxicity and microalgae growth inhibition at 100mg L-1. Interestingly, none of the materials showed the same trend and time dependence of atoxic/toxic effects compared to the MAX or ML samples (see SI for details). While for the MAX phase (see Fig. S3) toxicity reached approximately 15–25% and increased with time, the reverse trend was observed for SL Nb2CTx and Nb4C3TX MXene. The inhibition of microalgae growth decreased over time. It reached approximately 17% after 24 hours and dropped to less than 5% after 72 hours (Fig. 3a, b, respectively).
More importantly, for SL Nb4C3TX, microalgae growth inhibition reached about 27% after 24 hours, but after 72 hours it decreased to about 1%. Therefore, we labeled the observed effect as inverse inhibition of stimulation, and the effect was stronger for SL Nb4C3TX MXene. The stimulation of microalgae growth was noted earlier with Nb4C3TX (interaction at 10 mg L-1 for 24 h) compared with SL Nb2CTx MXene. The inhibition-stimulation reversal effect was also well shown in the biomass doubling rate curve (see Fig. S4 for details). So far, only the ecotoxicity of Ti3C2TX MXene has been studied in different ways. It is not toxic to zebrafish embryos44 but moderately ecotoxic to the microalgae Desmodesmus quadricauda and Sorghum saccharatum plants45. Other examples of specific effects include higher toxicity to cancer cell lines than to normal cell lines46,47. It could be assumed that the test conditions would influence the changes in microalgae growth observed in the presence of Nb-MXenes. For example, a pH of about 8 in the chloroplast stroma is optimal for efficient operation of the RuBisCO enzyme. Therefore, pH changes negatively affect the rate of photosynthesis48,49. However, we did not observe significant changes in pH during the experiment (see SI, Fig. S5 for details). In general, cultures of microalgae with Nb-MXenes slightly reduced the pH of the solution over time. However, this decrease was similar to a change in the pH of a pure medium. In addition, the range of variations found was similar to that measured for a pure culture of microalgae (control sample). Thus, we conclude that photosynthesis is not affected by changes in pH over time.
In addition, the synthesized MXenes have surface endings (denoted as Tx). These are mainly functional groups -O, -F and -OH. However, surface chemistry is directly related to the method of synthesis. These groups are known to be randomly distributed over the surface, making it difficult to predict their effect on the properties of MXene50. It can be argued that Tx could be the catalytic force for the oxidation of niobium by light. Surface functional groups indeed provide multiple anchoring sites for their underlying photocatalysts to form heterojunctions51. However, the growth medium composition did not provide an effective photocatalyst (detailed medium composition can be found in SI Table S6). In addition, any surface modification is also very important, as the biological activity of MXenes can be altered due to layer post-processing, oxidation, chemical surface modification of organic and inorganic compounds52,53,54,55,56 or surface charge engineering38. Therefore, to test whether niobium oxide has anything to do with material instability in the medium, we conducted studies of the zeta (ζ) potential in microalgae growth medium and deionized water (for comparison). Our results show that SL Nb-MXenes are fairly stable (see SI Fig. S6 for MAX and ML results). The zeta potential of SL MXenes is about -10 mV. In the case of SR Nb2CTx, the value of ζ is somewhat more negative than that of Nb4C3Tx. Such a change in the ζ value may indicate that the surface of negatively charged MXene nanoflakes absorbs positively charged ions from the culture medium. Temporal measurements of the zeta potential and conductivity of Nb-MXenes in culture medium (see Figures S7 and S8 in SI for more details) seem to support our hypothesis.
However, both Nb-MXene SLs showed minimal changes from zero. This clearly demonstrates their stability in the microalgae growth medium. In addition, we assessed whether the presence of our green microalgae would affect the stability of Nb-MXenes in the medium. The results of the zeta potential and conductivity of MXenes after interaction with microalgae in nutrient media and culture over time can be found in SI (Figures S9 and S10). Interestingly, we noticed that the presence of microalgae seemed to stabilize the dispersion of both MXenes. In the case of Nb2CTx SL, the zeta potential even slightly decreased over time to more negative values (-15.8 versus -19.1 mV after 72 h of incubation). The zeta potential of SL Nb4C3TX slightly increased, but after 72 h it still showed higher stability than nanoflakes without the presence of microalgae (-18.1 vs. -9.1 mV).
We also found lower conductivity of Nb-MXene solutions incubated in the presence of microalgae, indicating a lower amount of ions in the nutrient medium. Notably, the instability of MXenes in water is mainly due to surface oxidation57. Therefore, we suspect that green microalgae somehow cleared the oxides formed on the surface of Nb-MXene and even prevented their occurrence (oxidation of MXene). This can be seen by studying the types of substances absorbed by microalgae.
While our ecotoxicological studies indicated that microalgae were able to overcome the toxicity of Nb-MXenes over time and the unusual inhibition of stimulated growth, the aim of our study was to investigate possible mechanisms of action. When organisms such as algae are exposed to compounds or materials unfamiliar to their ecosystems, they may react in a variety of ways58,59. In the absence of toxic metal oxides, microalgae can feed themselves, allowing them to grow continuously60. After ingestion of toxic substances, defense mechanisms may be activated, such as changing shape or form. The possibility of absorption must also be considered58,59. Notably, any sign of a defense mechanism is a clear indicator of the toxicity of the test compound. Therefore, in our further work, we investigated the potential surface interaction between SL Nb-MXene nanoflakes and microalgae by SEM and the possible absorption of Nb-based MXene by X-ray fluorescence spectroscopy (XRF). Note that SEM and XRF analyzes were only performed at the highest concentration of MXene to address activity toxicity issues.
The SEM results are shown in Fig.4. Untreated microalgae cells (see Fig. 4a, reference sample) clearly showed typical R. subcapitata morphology and croissant-like cell shape. Cells appear flattened and somewhat disorganized. Some microalgae cells overlapped and entangled with each other, but this was probably caused by the sample preparation process. In general, pure microalgae cells had a smooth surface and did not show any morphological changes.
SEM images showing surface interaction between green microalgae and MXene nanosheets after 72 hours of interaction at extreme concentration (100 mg L-1). (a) Untreated green microalgae after interaction with SL (b) Nb2CTx and (c) Nb4C3TX MXenes. Note that the Nb-MXene nanoflakes are marked with red arrows. For comparison, photographs from an optical microscope are also added.
In contrast, microalgae cells adsorbed by SL Nb-MXene nanoflakes were damaged (see Fig. 4b, c, red arrows). In the case of Nb2CTx MXene (Fig. 4b), microalgae tend to grow with attached two-dimensional nanoscales, which can change their morphology. Notably, we also observed these changes under light microscopy (see SI Figure S11 for details). This morphological transition has a plausible basis in the physiology of microalgae and their ability to defend themselves by changing cell morphology, such as increasing cell volume61. Therefore, it is important to check the number of microalgae cells that are actually in contact with Nb-MXenes. SEM studies showed that approximately 52% of microalgae cells were exposed to Nb-MXenes, while 48% of these microalgae cells avoided contact. For SL Nb4C3Tx MXene, microalgae try to avoid contact with MXene, thereby localizing and growing from two-dimensional nanoscales (Fig. 4c). However, we did not observe the penetration of nanoscales into microalgae cells and their damage.
Self-preservation is also a time-dependent response behavior to the blockage of photosynthesis due to the adsorption of particles on the cell surface and the so-called shading (shading) effect62. It is clear that each object (for example, Nb-MXene nanoflakes) that is between the microalgae and the light source limits the amount of light absorbed by the chloroplasts. However, we have no doubt that this has a significant impact on the results obtained. As shown by our microscopic observations, the 2D nanoflakes were not completely wrapped or adhered to the surface of the microalgae, even when the microalgae cells were in contact with Nb-MXenes. Instead, nanoflakes turned out to be oriented to microalgae cells without covering their surface. Such a set of nanoflakes/microalgae cannot significantly limit the amount of light absorbed by microalgae cells. Moreover, some studies have even demonstrated an improvement in light absorption by photosynthetic organisms in the presence of two-dimensional nanomaterials63,64,65,66.
Since SEM images could not directly confirm the uptake of niobium by microalgae cells, our further study turned to X-ray fluorescence (XRF) and X-ray photoelectron spectroscopy (XPS) analysis to clarify this issue. Therefore, we compared the intensity of the Nb peaks of reference microalgae samples that did not interact with MXenes, MXene nanoflakes detached from the surface of microalgae cells, and microalgal cells after removal of attached MXenes. It is worth noting that if there is no Nb uptake, the Nb value obtained by the microalgae cells should be zero after removal of the attached nanoscales. Therefore, if Nb uptake occurs, both XRF and XPS results should show a clear Nb peak.
In the case of XRF spectra, microalgae samples showed Nb peaks for SL Nb2CTx and Nb4C3Tx MXene after interaction with SL Nb2CTx and Nb4C3Tx MXene (see Fig. 5a, also note that the results for MAX and ML MXenes are shown in SI, Figs S12–C17 ). Interestingly, the intensity of the Nb peak is the same in both cases (red bars in Fig. 5a). This indicated that the algae could not absorb more Nb, and the maximum capacity for Nb accumulation was achieved in the cells, although two times more Nb4C3Tx MXene was attached to the microalgae cells (blue bars in Fig. 5a). Notably, the ability of microalgae to absorb metals depends on the concentration of metal oxides in the environment67,68. Shamshada et al.67 found that the absorptive capacity of freshwater algae decreases with increasing pH. Raize et al.68 noted that the ability of seaweed to absorb metals was about 25% higher for Pb2+ than for Ni2+.
(a) XRF results of basal Nb uptake by green microalgae cells incubated at an extreme concentration of SL Nb-MXenes (100 mg L-1) for 72 hours. The results show the presence of α in pure microalgae cells (control sample, gray columns), 2D nanoflakes isolated from surface microalgae cells (blue columns), and microalgae cells after separation of 2D nanoflakes from the surface (red columns). The amount of elemental Nb, ( b) percentage of chemical composition of microalgae organic components (C=O and CHx/C–O) and Nb oxides present in microalgae cells after incubation with SL Nb-MXenes, (c–e) Fitting of the compositional peak of XPS SL Nb2CTx spectra and (f h) SL Nb4C3Tx MXene internalized by microalgae cells.
Therefore, we expected that Nb could be absorbed by algal cells in the form of oxides. To test this, we performed XPS studies on MXenes Nb2CTx and Nb4C3TX and algae cells. The results of the interaction of microalgae with Nb-MXenes and MXenes isolated from algae cells are shown in Figs. 5b. As expected, we detected Nb 3d peaks in the microalgae samples after removal of MXene from the surface of the microalgae. The quantitative determination of C=O, CHx/CO, and Nb oxides was calculated based on the Nb 3d, O 1s, and C 1s spectra obtained with Nb2CTx SL (Fig. 5c–e) and Nb4C3Tx SL (Fig. 5c–e). ) obtained from incubated microalgae. Figure 5f–h) MXenes. Table S1-3 shows the details of the peak parameters and overall chemistry resulting from the fit. It is noteworthy that the Nb 3d regions of Nb2CTx SL and Nb4C3Tx SL (Fig. 5c, f) correspond to one Nb2O5 component. Here, we found no MXene-related peaks in the spectra, indicating that microalgae cells only absorb the oxide form of Nb. In addition, we approximated the C 1 s spectrum with the C–C, CHx/C–O, C=O, and –COOH components. We assigned the CHx/C–O and C=O peaks to the organic contribution of microalgae cells. These organic components account for 36% and 41% of the C 1s peaks in Nb2CTx SL and Nb4C3TX SL, respectively. We then fitted the O 1s spectra of SL Nb2CTx and SL Nb4C3TX with Nb2O5, organic components of microalgae (CHx/CO), and surface adsorbed water.
Finally, the XPS results clearly indicated the form of Nb, not just its presence. According to the position of the Nb 3d signal and the results of the deconvolution, we confirm that Nb is absorbed only in the form of oxides and not ions or MXene itself. In addition, XPS results showed that microalgae cells have a greater ability to uptake Nb oxides from SL Nb2CTx compared to SL Nb4C3TX MXene.
While our Nb uptake results are impressive and allow us to identify MXene degradation, there is no method available to track associated morphological changes in 2D nanoflakes. Therefore, we also decided to develop a suitable method that can directly respond to any changes occurring in 2D Nb-MXene nanoflakes and microalgae cells. It is important to note that we assume that if the interacting species undergo any transformation, decomposition or defragmentation, this should quickly manifest itself as changes in shape parameters, such as the diameter of the equivalent circular area, roundness, Feret width, or Feret length. Since these parameters are suitable for describing elongated particles or two-dimensional nanoflakes, their tracking by dynamic particle shape analysis will give us valuable information about the morphological transformation of SL Nb-MXene nanoflakes during reduction.
The results obtained are shown in Figure 6. For comparison, we also tested the original MAX phase and ML-MXenes (see SI Figures S18 and S19). Dynamic analysis of particle shape showed that all shape parameters of two Nb-MXene SLs changed significantly after interaction with microalgae. As shown by the equivalent circular area diameter parameter (Fig. 6a, b), the reduced peak intensity of the fraction of large nanoflakes indicates that they tend to decay into smaller fragments. On fig. 6c, d shows a decrease in the peaks associated with the transverse size of the flakes (elongation of the nanoflakes), indicating the transformation of 2D nanoflakes into a more particle-like shape. Figure 6e-h showing the width and length of the Feret, respectively. Feret width and length are complementary parameters and should therefore be considered together. After incubation of 2D Nb-MXene nanoflakes in the presence of microalgae, their Feret correlation peaks shifted and their intensity decreased. Based on these results in combination with morphology, XRF and XPS, we concluded that the observed changes are strongly related to oxidation as oxidized MXenes become more wrinkled and break down into fragments and spherical oxide particles69,70.
Analysis of MXene transformation after interaction with green microalgae. Dynamic particle shape analysis takes into account such parameters as (a, b) diameter of the equivalent circular area, (c, d) roundness, (e, f) Feret width and (g, h) Feret length. To this end, two reference microalgae samples were analyzed together with primary SL Nb2CTx and SL Nb4C3Tx MXenes, SL Nb2CTx and SL Nb4C3Tx MXenes, degraded microalgae, and treated microalgae SL Nb2CTx and SL Nb4C3Tx MXenes. The red arrows show the transitions of the shape parameters of the studied two-dimensional nanoflakes.
Since shape parameter analysis is very reliable, it can also reveal morphological changes in microalgae cells. Therefore, we analyzed the equivalent circular area diameter, roundness, and Feret width/length of pure microalgae cells and cells after interaction with 2D Nb nanoflakes. On fig. 6a–h show changes in the shape parameters of algae cells, as evidenced by a decrease in peak intensity and a shift of maxima towards higher values. In particular, cell roundness parameters showed a decrease in elongated cells and an increase in spherical cells (Fig. 6a, b). In addition, Feret cell width increased by several micrometers after interaction with SL Nb2CTx MXene (Fig. 6e) compared to SL Nb4C3TX MXene (Fig. 6f). We suspect that this may be due to the strong uptake of Nb oxides by microalgae upon interaction with Nb2CTx SR. Less rigid attachment of Nb flakes to their surface can result in cell growth with minimal shading effect.
Our observations of changes in the parameters of the shape and size of microalgae complement other studies. Green microalgae can change their morphology in response to environmental stress by changing cell size, shape or metabolism61. For example, changing the size of cells facilitates the absorption of nutrients71. Smaller algae cells show lower nutrient uptake and impaired growth rate. Conversely, larger cells tend to consume more nutrients, which are then deposited intracellularly72,73. Machado and Soares found that the fungicide triclosan can increase cell size. They also found profound changes in the shape of the algae74. In addition, Yin et al.9 also revealed morphological changes in algae after exposure to reduced graphene oxide nanocomposites. Therefore, it is clear that the altered size/shape parameters of the microalgae are caused by the presence of MXene. Since this change in size and shape is indicative of changes in nutrient uptake, we believe that analysis of size and shape parameters over time can demonstrate uptake of niobium oxide by microalgae in the presence of Nb-MXenes.
Moreover, MXenes can be oxidized in the presence of algae. Dalai et al.75 observed that the morphology of green algae exposed to nano-TiO2 and Al2O376 was not uniform. Although our observations are similar to the present study, it is only relevant to the study of the effects of bioremediation in terms of MXene degradation products in the presence of 2D nanoflakes and not nanoparticles. Since MXenes can degrade into metal oxides,31,32,77,78 it is reasonable to assume that our Nb nanoflakes can also form Nb oxides after interacting with microalgae cells.
In order to explain the reduction of 2D-Nb nanoflakes through a decomposition mechanism based on the oxidation process, we conducted studies using high-resolution transmission electron microscopy (HRTEM) (Fig. 7a,b) and X-ray photoelectron spectroscopy (XPS) (Fig. 7). 7c-i and tables S4-5). Both approaches are suitable for studying the oxidation of 2D materials and complement each other. HRTEM is able to analyze the degradation of two-dimensional layered structures and the subsequent appearance of metal oxide nanoparticles, while XPS is sensitive to surface bonds. For this purpose, we tested 2D Nb-MXene nanoflakes extracted from microalgae cell dispersions, that is, their shape after interaction with microalgae cells (see Fig. 7).
HRTEM images showing the morphology of oxidized (a) SL Nb2CTx and (b) SL Nb4C3Tx MXenes, XPS analysis results showing (c) the composition of oxide products after reduction, (d–f) peak matching of components of the XPS spectra of SL Nb2CTx and (g– i) Nb4C3Tx SL repaired with green microalgae.
HRTEM studies confirmed the oxidation of two types of Nb-MXene nanoflakes. Although the nanoflakes retained their two-dimensional morphology to some extent, oxidation resulted in the appearance of many nanoparticles covering the surface of the MXene nanoflakes (see Fig. 7a,b). XPS analysis of c Nb 3d and O 1s signals indicated that Nb oxides were formed in both cases. As shown in Figure 7c, 2D MXene Nb2CTx and Nb4C3TX have Nb 3d signals indicating the presence of NbO and Nb2O5 oxides, while O 1s signals indicate the number of O–Nb bonds associated with functionalization of the 2D nanoflake surface. We noticed that the Nb oxide contribution is dominant compared to Nb-C and Nb3+-O.
On fig. Figures 7g–i show the XPS spectra of Nb 3d, C 1s, and O 1s SL Nb2CTx (see Figs. 7d–f) and SL Nb4C3TX MXene isolated from microalgae cells. Details of Nb-MXenes peak parameters are provided in Tables S4–5, respectively. We first analyzed the composition of Nb 3d. In contrast to Nb absorbed by microalgae cells, in MXene isolated from microalgae cells, apart from Nb2O5, other components were found. In the Nb2CTx SL, we observed the contribution of Nb3+-O in the amount of 15%, while the rest of the Nb 3d spectrum was dominated by Nb2O5 (85%). In addition, the SL Nb4C3TX sample contains Nb-C (9%) and Nb2O5 (91%) components. Here Nb-C comes from two inner atomic layers of metal carbide in Nb4C3Tx SR. We then map the C 1s spectra to four different components, as we did in the internalized samples. As expected, the C 1s spectrum is dominated by graphitic carbon, followed by contributions from organic particles (CHx/CO and C=O) from microalgae cells. In addition, in the O 1s spectrum, we observed the contribution of organic forms of microalgae cells, niobium oxide, and adsorbed water.
In addition, we investigated whether Nb-MXenes cleavage is associated with the presence of reactive oxygen species (ROS) in the nutrient medium and/or microalgae cells. To this end, we assessed the levels of singlet oxygen (1O2) in the culture medium and intracellular glutathione, a thiol that acts as an antioxidant in microalgae. The results are shown in SI (Figures S20 and S21). Cultures with SL Nb2CTx and Nb4C3TX MXenes were characterized by a reduced amount of 1O2 (see Figure S20). In the case of SL Nb2CTx, MXene 1O2 is reduced to about 83%. For microalgae cultures using SL, Nb4C3TX 1O2 decreased even more, to 73%. Interestingly, changes in 1O2 showed the same trend as the previously observed inhibitory-stimulatory effect (see Fig. 3). It can be argued that incubation in bright light can alter photooxidation. However, the results of the control analysis showed almost constant levels of 1O2 during the experiment (Fig. S22). In the case of intracellular ROS levels, we also observed the same downward trend (see Figure S21). Initially, the levels of ROS in microalgae cells cultured in the presence of Nb2CTx and Nb4C3Tx SLs exceeded the levels found in pure cultures of microalgae. Eventually, however, it appeared that the microalgae adapted to the presence of both Nb-MXenes, as ROS levels decreased to 85% and 91% of the levels measured in pure cultures of microalgae inoculated with SL Nb2CTx and Nb4C3TX, respectively. This may indicate that microalgae feel more comfortable over time in the presence of Nb-MXene than in nutrient medium alone.
Microalgae are a diverse group of photosynthetic organisms. During photosynthesis, they convert atmospheric carbon dioxide (CO2) into organic carbon. The products of photosynthesis are glucose and oxygen79. We suspect that the oxygen thus formed plays a critical role in the oxidation of Nb-MXenes. One possible explanation for this is that the differential aeration parameter is formed at low and high partial pressures of oxygen outside and inside the Nb-MXene nanoflakes. This means that wherever there are areas of different partial pressures of oxygen, the area with the lowest level will form the anode 80, 81, 82. Here, the microalgae contribute to the creation of differentially aerated cells on the surface of the MXene flakes, which produce oxygen due to their photosynthetic properties. As a result, biocorrosion products (in this case, niobium oxides) are formed. Another aspect is that microalgae can produce organic acids that are released into the water83,84. Therefore, an aggressive environment is formed, thereby changing the Nb-MXenes. In addition, microalgae can change the pH of the environment to alkaline due to the absorption of carbon dioxide, which can also cause corrosion79.
More importantly, the dark/light photoperiod used in our study is critical to understanding the results obtained. This aspect is described in detail in Djemai-Zoghlache et al. 85 They deliberately used a 12/12 hour photoperiod to demonstrate biocorrosion associated with biofouling by the red microalgae Porphyridium purpureum. They show that the photoperiod is associated with the evolution of the potential without biocorrosion, manifesting itself as pseudoperiodic oscillations around 24:00. These observations were confirmed by Dowling et al. 86 They demonstrated photosynthetic biofilms of cyanobacteria Anabaena. Dissolved oxygen is formed under the action of light, which is associated with a change or fluctuations in the free biocorrosion potential. The importance of the photoperiod is emphasized by the fact that the free potential for biocorrosion increases in the light phase and decreases in the dark phase. This is due to the oxygen produced by photosynthetic microalgae, which influences the cathodic reaction through the partial pressure generated near the electrodes87.
In addition, Fourier transform infrared spectroscopy (FTIR) was performed to find out if any changes occurred in the chemical composition of microalgae cells after interaction with Nb-MXenes. These obtained results are complex and we present them in SI (Figures S23-S25, including the results of the MAX stage and ML MXenes). In short, the obtained reference spectra of microalgae provide us with important information about the chemical characteristics of these organisms. These most probable vibrations are located at frequencies of 1060 cm-1 (C-O), 1540 cm-1, 1640 cm-1 (C=C), 1730 cm-1 (C=O), 2850 cm-1, 2920 cm-1. one. 1 1 (C–H) and 3280 cm–1 (O–H). For SL Nb-MXenes, we found a CH-bond stretching signature that is consistent with our previous study38. However, we observed that some additional peaks associated with C=C and CH bonds disappeared. This indicates that the chemical composition of microalgae may undergo minor changes due to interaction with SL Nb-MXenes.
When considering possible changes in the biochemistry of microalgae, the accumulation of inorganic oxides, such as niobium oxide, needs to be reconsidered59. It is involved in the uptake of metals by the cell surface, their transport into the cytoplasm, their association with intracellular carboxyl groups, and their accumulation in microalgae polyphosphosomes20,88,89,90. In addition, the relationship between microalgae and metals is maintained by functional groups of cells. For this reason, absorption also depends on microalgae surface chemistry, which is quite complex9,91. In general, as expected, the chemical composition of green microalgae changed slightly due to the absorption of Nb oxide.
Interestingly, the observed initial inhibition of microalgae was reversible over time. As we observed, the microalgae overcame the initial environmental change and eventually returned to normal growth rates and even increased. Studies of the zeta potential show high stability when introduced into nutrient media. Thus, the surface interaction between microalgae cells and Nb-MXene nanoflakes was maintained throughout the reduction experiments. In our further analysis, we summarize the main mechanisms of action underlying this remarkable behavior of microalgae.
SEM observations have shown that microalgae tend to attach to Nb-MXenes. Using dynamic image analysis, we confirm that this effect leads to the transformation of two-dimensional Nb-MXene nanoflakes into more spherical particles, thereby demonstrating that the decomposition of nanoflakes is associated with their oxidation. To test our hypothesis, we conducted a series of material and biochemical studies. After testing, the nanoflakes gradually oxidized and decomposed into NbO and Nb2O5 products, which did not pose a threat to green microalgae. Using FTIR observation, we found no significant changes in the chemical composition of microalgae incubated in the presence of 2D Nb-MXene nanoflakes. Taking into account the possibility of absorption of niobium oxide by microalgae, we performed an X-ray fluorescence analysis. These results clearly show that the studied microalgae feed on niobium oxides (NbO and Nb2O5), which are non-toxic to the studied microalgae.