Efficient Removal of Cadmium Using Edible Fungus and Its Quantitative Fluorimetric Estimation Using (Z)-2-(4H-1,2,4-Triazol-4-yl)iminomethylphenol.

Microbes accumulate heavy metals after adsorption or absorption. This study exhibited that Trametes versicolor can tolerate up to 5 mg/g concentration of cadmium. Change in fungus morphology due to cadmium along with its absorption were analyzed using SEM, XRD, and EDAX. Cadmium absorption usually increased with time, and it was determined quantitatively by a fluorimetry technique using a synthesized imine fluorophore as a specific probe and compared with results obtained from atomic absorption spectroscopy (AAS). The intensity of the cadmium-specific XRD peak gradually increased up to the seventh day, and the absorption by the organisms reduced the concentration of cadmium even from the effluent of the plating industry. After the seventh day, Trametes versicolor absorbed almost 0.300 mg/g concentration of cadmium as visualized under high content screening from the fluorescence appearance of hyphae. Hence it can be concluded that Trametes versicolor may play a key role in reducing cadmium from a contaminated environment.

Introduction

Industrial development is one of the major outcomes of science and technology development; on the other hand, these kinds of advancements are creating some serious threats not only to us but to all the living world.[1,2] Among these, endocrine disrupting effects[3−5] by heavy metals sometimes mimic estrogen and alter its activity.[6] Some evidence has shown that cadmium can cause endocrine disruption and early maturation.[7] There are several types of methods to remove heavy metals mechanically,[8−14] but the main problem is either these techniques are expensive or, to some extent, not sufficiently effective. Generally, microbes can accumulate heavy metals[15−17] on their surface and absorb them inside their body using channel or carrier proteins. Generally, fungi are more effective in removal of heavy metals than bacteria because of their higher biomass.[18,19]

Trametes versicolor is a kind of white rot fungus commonly found on wood.[20] Being a laccase producing fungus, it has various economic benefits, apart from having a huge beneficial role in nature.[21]Trametes versicolor can secrete laccase; because of its oxidoreductive nature,[22] laccase can oxidize various types of toxic chemical compounds into nontoxic ones, which is why Trametes versicolor is an important contributor in bioremediation research. Apart from the reported information, there are so many untold and untouched sides, which can really give some new methods in the field of environmental pollution. Usually, Trametes versicolor has a high growth rate, and due to its huge biomass, it can absorb a large amount of heavy metals from contaminated sites.[21]Trametes versicolor can sequester heavy metals after chelation and either accumulate them inside their body or eject them using carrier or channel proteins.[15,17] Cadmium pollution has become a very serious environmental problem.[23] Using Trametes versicolor, we can respond to cadmium pollution using their large removal ability.[21]

There are so many high cost and sophisticated methods available for cadmium metal ion quantification such as inductively coupled atomic plasma mass spectroscopy (ICP-MS)[24] inductively coupled plasma emission spectroscopy (ICP-AES),[25] atomic absorption spectroscopy (AAS), and anode stripping voltammetry.[26] Recently, fluorescence spectral analysis has been used as a powerful analytical tool because of its operational simplicity, cost effectiveness, high sensitivity, and selectivity.[27] For Cd2+ ion estimation, only a few fluorescence based sensors had reported recently with different mechanisms.[28,29] Among these, only a very few reports showed a good discrimination for Cd2+ ions from Zn2+ ions.[30] Very recently, our group has reported a simple fluorescent based chemosensor for the quantification of Cd2+ ions using a very simple salicyaldimine based fluorophore [(Z)-2-(4H-1,2,4-triazol-4-yl)iminomethylphenol]. It shows high sensitivity at the femtomolar level and selectivity toward Cd2+ ions in the presence of Zn2+ ions at physiological pH based on intramolecular charge transfer (ICT) mechanism. Moreover, its fluorescence intensity is enhanced in a linear fashion with variation of the concentration of Cd2+ ions from 5 fM to 1 mM along with a limit of detection of 1 fM. So, it could be potentially used for the quantification of Cd2+ ions even if present in femtomolar concentrations. In this work, we have used the same fluorescent probe (Scheme S1, Supporting Information) based on our reported procedure[31] and the same characterization spectra are given in Supporting Information, Figures S2–S4.

Most papers directly describe cadmium removal after introducing cadmium stress in laboratory conditions. But this effort tried to analyze not only overall removal but concentration on absorption. Other than the ability of significant cadmium removal activity in laboratory conditions, this study demonstrated cadmium removal ability of Trametes versicolor from a cadmium contaminated environmental sample. The synthesized probe, which has been designed specifically for cadmium, exhibited better identification of cadmium removal under live cell imaging of treated Trametes versicolor using high content screening, as well as correlated with increasing emission spectra over time. Compared to previous reports [(76.17%), (88%), (85%)],[32−34] this study demonstrated much higher cadmium removal (90.3%,). It can be concluded that Trametes versicolor can play a significant role in cadmium removal from contaminated sites and also that the novel cadmium specific probe can visualize as well as sense the presence of cadmium after removal using Trametes versicolor.

Experimental Procedures

Materials and Methods

Analytical grade solvents and double distilled water were used in all steps. Salicyaldehyde, 4-amino-1,2,4-triazole, glacial acetic acid, HPLC grade ethanol, HNO3, HClO4, KBr, and resazurin were purchased from Sigma-Aldrich. Glucose, NH4NO3, Na2HPO4, KH2PO4, MgSO4, CuSO4 5H2O, CaCl2, FeSO4, ZnSO4, Na2MoO4, MnSO4, H3BO3, and acetate salts of different metal ions were purchased from Merck. Yeast extract, potato dextrose broth, and DMEM media was purchased from Hi-Media. AGS and MCF-12 cell lines were purchased from NCCS, Pune, and Trametes versicolor was isolated from Pondicherry. 1H NMR and 13C NMR were measured using BrukerAV-400 spectrometer. Mass spectra were measured on a Thermo fleet LC-MS spectrometer. Measurements were made with a Eutek pH-Tutor.

Growth Condition of Trametes versicolor

Trametes versicolor, a white rot fungus, was isolated from the soil sample of Pondicherry. During isolation, the fungus has been grown in potato dextrose broth (PDB) and maintained at 28 °C with shaking at 150 rpm (rotation per minute) for 7 days. During all experiments the fungus was grown in Kirk and Ferrell media containing a particular concentration of cadmium salt and maintained similar growth conditions mentioned above.[21]

Determination of Minimal Inhibitory Concentration (MIC) of Cadmium for Trametes versicolor

Isolated Trametes versicolor(21) grown in separate conical flasks in the presence of various concentrations of cadmium (0.100 to 6 mg/g) at 28 °C with shaking at 150 rpm for 15 days to determine minimal inhibitory concentration. During this experiment, the cadmium source was cadmium acetate. After 15 days, the MIC[35] was calculated from the generated biomass in the presence of different concentrations of cadmium. The concentration at which no Trametes versicolor growth was observed is considered as MIC value of cadmium. The whole set of experiments was performed in triplicate. Biomass production after 15 days of incubation in the presence of different concentrations of cadmium was statistically analyzed using one-way ANOVA to check whether the statistical significant difference in biomass production present.

Preparation of Samples from Trametes versicolor for Cadmium Analysis

Trametes versicolor was inoculated in 50 mL of media[21] with 1 mg/g concentration of cadmium (Cd) in seven different conical flasks at 28 °C with shaking at 150 rpm for 7 days. Generally, the growth of Trametes versicolor will be observed after the second day. Hence, cadmium-treated biomass was collected every day from the third day from specifically time labeled conical flask, centrifuged, dried, and lyophilized. After lyophilization, dried biomass was kept at normal room temperature in dust free conditions. Before analysis using AAS (PerkinElmer’s PinAAcle 500 was used), 2 mg of lyophilized treated biomass was dissolved in 8 mL of concentrated nitric acid along with 2 mL of perchloric acid and kept in a hot air oven at 20 °C overnight until the solution became clear.

Detection of Cadmium Removal by Trametes versicolor Using Various Techniques

Fluorimetric Method

All the UV studies were done with JASCO 550, and fluorescence spectra titrations were carried out with a CARY Eclipse Fluorescence spectrophotometer. To 2 mL of synthesized probe at pH 7.0, various concentrations of cadmium samples were added. The resulting fluorescent changes were recorded at 433 nm with the excitation wavelength of 332 nm. The same protocol was followed for industrial samples also. The corresponding color change and UV emission data are given in Supporting Information (Figures S6 and S7).

Atomic Absorption Spectroscopy (AAS)

The clear biomass solution was used for atomic absorption microscopy analysis, and it was measured at 283 nm range.[21,36] During this experiment, all the test samples were diluted 70 times, a standard curve was prepared using 1, 3, 5, 7, 9, 11, and 13 ppm cadmium samples, and concentration of unknown samples was determined by extrapolating the absorbance value obtained from third to seventh day samples; after multiplying by 70, the actual value of removed cadmium was determined. This experiment was performed in triplicate; increasing biomass with time was analyzed statistically to determine significant difference using one-way ANOVA. (PerkinElmer’s PinAAcle 500 were used) The extrapolation data for complete cadmium removal is given in Supporting Information (Figure S8).

Fourier Transform Infrared (FT-IR) and X-ray Diffraction (XRD)

When cadmium is adsorbed on the surface of the organism, some functional groups play an important role during adsorption. Functional groups involved in the interaction with cadmium can be interpreted from the FT-IR data.[21] The above lyophilized biomass of the cadmium-treated fungus was examined through FT-IR (FT-IR-8400S SHIMADZU) using KBr pellet method. The nature of Trametes versicolor before and after treatment with cadmium was examined by XRD (PANalytical X’Pert-Pro MPD PW3040/60 XRD)), and the XRD peak was analyzed using JCPDS database.

Cyclic Voltammetry (CV)

The uptake of cadmium was again checked with cyclic voltammetry technique. After treatment with Trametes versicolor for 15 days of incubation, the heavy metal sample was filtered through 0.22 μm syringe filter, and the uptake was checked for the filtrate sample and control using cyclic voltameter (CHI1205, BIOLOGIC) .

Visualization of Cadmium up Take by High Content Screening (HCS) Using Synthesized Probe

The fluorescence probe was added to the freshly grown fungal biomass (in the presence of cadmium), and after half an hour of incubation at room temperature, the biomass was spread on a glass slide using a sterilized needle and forceps. Synthesized probe usually gives fluorescence when it binds with cadmium after incubation of fungus. The probe can interact with absorbed or adsorbed cadmium, and that fluorescence image was visualized using high content screening (HCS). During visualization under HCS, excitation and emission spectra at 332 and 436 nm were used. Treated fungus from day 3 through 7 was ruptured using ultrasonication (methodology explained previously); after centrifugation, supernatant was passed through a 0.22 μm syringe filter, and 10 μL of probe was added to 1.5 mL of intracellular materials before measuring emission spectra at 436 nm. The experiment was performed three times in triplicate. The mean value of the absorbed cadmium quantity was statistically analyzed using one-way ANOVA.

Detection Using Electron Microscopy of Surface Morphology of Fungus after Cadmium Adsorption

After the seventh day of incubation, fungal biomass was collected after centrifugation at 5000 rpm for 15 min. Fungal biomass was separated, spread on an ITO (indium tin oxide) glass plate, and visualized under scanning electron microscopy (VEGA3, TESCAN). During imaging under SEM, some specific spots on the fungal hyphae were examined using EDAX (AMETEK) to confirm the deposition of cadmium on the fungal surface.

Cytotoxicity Assay of the Synthesized Probe

To check the cytotoxic effect of the synthesized probe, AGS and MCF-12F cell lines were used, and resazurin assay was performed in 96 well plates (5000 cells/well) using DMEM (F12/HAM) culture media. After 24 h, both cell lines were treated with various concentrations of the synthesized probe (50–2000 μg/mL)[37,38] and incubated for 48 h. Resazurin (1 μM) was added in all probe treated AGS and MCF-12F cells, and after 4 h of incubation, the resorufin formation was measured fluorimetrically at 540 nm excitation and 590 nm emission.

Detection Using Synthesized Probe of Cadmium Removal by Trametes versicolor from Cadmium Contaminated Environmental Sample

A cadmium contaminated water sample was collected from an electroplating industry site in Tamil Nadu. In individual conical flasks (labeled for collection days), 50 mL of cadmium-contaminated water sample and uncontaminated water control, along with yeast extract (5 g/L) and glucose (2 g/L), were inoculated with Trametes versicolor and incubated for up to 7 days at 28 °C in shaking incubator at 150 rpm. On specified days, fungal biomass was collected after visible growth in cadmium-contaminated water and treated with ultrasonication, and the supernatant was collected after centrifugation at 5000 rpm for 20 min. A portion of 1.5 mL of the supernatant obtained from each conical flask on a specific day was mixed with 10 μL of the synthesized probe, and emission spectra were taken at 436 nm along with specific control. Simultaneously, the remaining supernatant was lyophilized and prepared using the same protocol for AAS sample preparation. The amount of cadmium absorbed in each day was quantified three times in triplicate, and statistical analysis was performed using one-way ANOVA. Using this experiment, cadmium absorbed by Trametes versicolor at each time point from cadmium-contaminated water was both qualitatively and quantitatively determined.

Results and Discussion

Determination of MIC

Minimal inhibitory concentration (MIC) was determined on the basis of fungal growth in terms of increasing dry biomass after 15 days of incubation. MIC of cadmium for Trametes versicolor has demonstrated (Figure ) significant tolerance. Cadmium MIC was 6 mg/g. The cadmium tolerance by Trametes versicolor was much higher than the usual concentration of cadmium present in cadmium-polluted areas, and this MIC value is much greater compared with a previous report (4 mg/g).[39] Increasing concentration of cadmium gradually decreased the growth of Trametes versicolor as measured by dry biomass with a statistically significant difference (p < 0.001). This MIC value indicated that Trametes versicolor may play a vital role in the removal of higher concentrations of cadmium from the cadmium-polluted areas.

Figure 1

Detection of minimal inhibitory concentration (MIC) in the presence of cadmium.

Detection Using the Synthesized Probe of Cadmium Up Take by Trametes versicolor

Visualization under HCS clearly showed the thread-like appearance of fungal hyphae (Figure a) along with fluorescence due to up take of cadmium (either adsorption or absorption). When fungus was incubated with probes for a short time, then probes bound cadmium present on the surface, and probe was absorbed and combined with absorbed cadmium; as a result, fluorescence intensity was increased and helped to visualize thread-like hyphae with its fluorescent appearance. The control sample (fungus without cadmium) did not show any fluorescence after addition of probes under HCS.

Figure 2

Visualization of cadmium up taking using cadmium specific probe. (a) Fluorescence imaging in the presence of control fungus and probe. (b) Fluorescent appearance of control fungus/probe/cadmium. (c) Emission spectra for absorbed cadmium samples on different days.

The quantity of cadmium uptake was confirmed by emission spectra (Figure c) of samples from different days, and the results clearly showed that the seventh-day sample showed maximum fluorescence enhancement followed by samples from sixth, fifth, fourth, and third days, and the concentrations were 0.365, 0.300, 0.215, 0.162, and 0.110 mg/g, respectively. This result clearly revealed that cadmium absorption increased with increased exposure time; as a result, the seventh-day sample exhibited the highest absorption. These values were correlated with the mean value of cadmium uptake analyzed using AAS; the results showed that 0.350, 0.290, 0.220, 0.170, and 0.120 mg/g cadmium was absorbed on the seventh, sixth, fifth, fourth, and third day, respectively, with statistically significant difference (p < 0.001). The data obtained from both methods correlating each other indicated that uptake of cadmium increased with increasing exposure time (Table )

Table 1

Comparison of Cadmium Quantification under Laboratory Conditions

day	fluorimetric quantification (mg/g)	AAS quantification (mg/g)
3	0.110	0.120
4	0.162	0.170
5	0.215	0.220
6	0.300	0.290
7	0.365	0.350

Detection Using Probes of Cadmium Absorption by Trametes versicolor from Cadmium Contaminated Water sample

Contaminated effluent sample analysis clearly reveals that like the previous result, sample from the seventh day of incubation showed maximum fluorescence intensity followed by those from the sixth, fifth, fourth, and third days (Figure ) because more cadmium was absorbed with increasing time. Concentrations of cadmium are 0.330, 0.290, 0.210, 0.195, and 0.140 mg/g, respectively. The binding of probe with cadmium was previously reported as 1:1, which was confirmed via Job’s plot and ESI-mass and 1H NMR spectroscopy (Figure S5a–c). Simultaneously these results quantitatively analyzed from the mean value obtained from AAS analysis (Figure S8) revealed that 0.300, 0.270, 0.200, 0.175, and 0.150 mg/g cadmium was absorbed after 7, 6, 5, 4, and 3 days incubation, respectively, with statistically significant difference (p < 0.001) (Table ).

Figure 3

Fluorescence intensity of environmental sample at different exposure day.

Table 2

Comparison of Cadmium Quantification Using an Environmental Contaminated Sample

day	fluorimetric quantification (mg/g)	AAS quantification (mg/g)
3	0.140	0.150
4	0.195	0.175
5	0.210	0.200
6	0.290	0.270
7	0.330	0.300

Functional Group Involvement in Metal Conjugation

The interaction between the control fungus and cadmium ions were confirmed by the FT-IR analysis, and the corresponding spectra are shown in Figure a. Initially the control exhibited a maximum stretching frequency at 3353 cm–1, which is due to the presence of free hydroxyl groups, which may be present in the fungus. Additionally peaks at 2928, 1644, 1064, and 1378 cm–1 may be attributed to alkyl −CH, amide −C=O and −C=C– stretching, and −CH3 bending frequencies. From the third day onward cadmium exposed sample exhibits hydroxyl broadening along with peak shift from 3353 to 3421 cm–1, and only a broadening along with decrease in intensity was observed for the remaining peaks. Most remarkably this broadening and peak shift was further enhanced at the seventh day from 3421 to 3434 cm–1. From these stretching variations, we could clearly conclude that the cadmium ions can coordinate with the −OH and amide −C=O and −C=C– functional groups of the fungus due to the soft acid–soft base mechanism between cadmium ions and these functional groups.

Figure 4

(a) Detection of the involvement of functional groups during adsorption. (b) Cadmium accumulation variation detected using XRD by exposure day.

This mode of interaction was further confirmed with the help of X-ray diffraction study. Initially for control there was a broad peak observed at the 2θ value of 25.41 (Figure b-1), which indicates the amorphous nature of the fungus. But for the third day and seventh day, a sharp peak appeared and increased with time of exposure at the 2θ value of 44.51 (JCPDS file no. 10-0454)[40] (Figure b-2,3). This phenomenon signifies that with the increasing exposure time, accumulation of cadmium also increased, and cadmium encapsulates the fungus surface. As a result, the amorphous nature decreases and the crystallinity increases owing to the crystalline cadmium nanoparticle accumulation.

Conformation of Cadmium Uptake by Cyclic Voltammetry Technique

The concentration of cadmium before and after treatment with Trametes versicolor was analyzed, and a redox couple appeared corresponding to cadmium with the favorable reduction current of 65 μA and oxidation current of 35 μA in the control sample. But after treatment, reduction and oxidation current were reduced to one-third of the original value (Figure ). The results again confirm the efficient uptake of cadmium.

Figure 5

Detection of Cadmium Removal using cyclic voltammetry.

Cytotoxicity Assay of the Synthesized Probe

Resazurin assay clearly indicated that the highest concentration (2000 μg/mL) of probes did not show any cytotoxicity against both AGS and MCF-12F cell line (Figure ).

Figure 6

Cytotoxicity analysis of synthesized probe: cytotoxicity assay of probe in the presence of (a) AGS and (b) MCF-12F.

Visualization of Cadmium Up Take Using Electron Microscopy

EDAX along with SEM imaging clearly revealed the presence of cadmium in the focused hyphae region, and a specific cadmium peak also appeared after EDAX analysis from that specifically focused region, in other side control sample (fungus without any cadmium treatment) did not show any peak corresponding cadmium (Figure ). SEM images reveal a rod shape with well-defined cell wall for Trametes versicolor cultured in control solution. However, SEM images of Trametes versicolor cultured in solution containing cadmium look similar, but cell walls are not well-defined.

Figure 7

EDAX and SEM images of (a) control fungus without cadmium treatment and (b) fungus treated with 1000 ppm concentration of cadmium.

Hence, to confirm the status of cadmium in the fungi, we have done the above analysis using SEM, EDAX, XRD, and IR spectral analysis.

From the results obtained from EDAX, it is confirmed that the cadmium is present in the fungal mass. The SEM and XRD results clearly show that the cadmium is present in the form of nanoparticles. In the SEM, we could not focus in the nanometer range because the fungi were not stable during close focusing. But one or two small spots of cadmium nanoparticles were present, and when the point analysis was carried out on the small spots in the EDAX analysis, intense peaks corresponding to cadmium were observed in the EDAX graph.

As per the earlier reports, more microorganisms and plants can take up metal contaminates from the soil and water and reduce the metal ions to metal nanoparticles. For example, recently it was reported that alfalfa plants growing in a AuCl4 rich environment can take up Au metal ions and reduced them into nanoparticles. The absorption of Au metal by the plants was confirmed by X-ray absorption studies (XAS), and transmission electron microscopy (TEM).[41] Similarly, a well-known example is magnetostatic bacteria, which can synthesize magnetic nanoparticles.[42] The metal-contaminated waters were treated using live , and the bacteria produced silver–carbon composite materials. Also, the surface trapping of nanoparticles by fungus has been reported.[43]

Conclusion

This study indicated that Trametes versicolor has a huge potential ability to resist higher concentrations of cadmium. MIC result showed that Trametes versicolor can resist 5 mg/g cadmium, and it can adhere as well as absorb a huge amount of cadmium from the environment. In laboratory conditions, we have seen that Trametes versicolor can absorb 0.100 mg/g cadmium after the second day of exposure and 0.350 mg/g on the seventh day. This study clearly revealed that due to absorption of this amount of cadmium, it can play a significant role in removal or heavy metals like cadmium from contaminated sites. The synthesized probe was very specific for cadmium and used for quantitative determination of cadmium by fluorescent technique. The data obtained from the fluorescent technique was compared with data obtained from AAS study. XRD, SEM, and CV studies were also carried out to support the cadmium uptake. The FT-IR study explains the mechanism of interaction between fungus and cadmium. In summary, we can conclude that white rot fungus, Trametes versicolor, can reduce the toxic load of cadmium from industrial effluent as well as in laboratory conditions efficiently compared to earlier reports, and the removal can be significant visualized and quantitatively determined using our probe. As it is an edible fungus, the removal of cadmium using this fungus can be considered as a green, simple, and efficient route.