Structural and Nanomechanical Properties of Termitomyces clypeatus

Jan 15, 2009 - Structural and Nanomechanical Properties of Termitomyces clypeatus Cell Wall and Its Interaction with Chromium(VI). Sujoy K. Das, Akhil...
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J. Phys. Chem. B 2009, 113, 1485–1492

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Structural and Nanomechanical Properties of Termitomyces clypeatus Cell Wall and Its Interaction with Chromium(VI) Sujoy K. Das,† Akhil R. Das,‡ and Arun K. Guha*,† Department of Biological Chemistry and Polymer Science Unit, Indian Association for the CultiVation of Science, JadaVpur, Kolkata 700 032, India ReceiVed: October 03, 2008; ReVised Manuscript ReceiVed: NoVember 26, 2008

Alterations of cell surface properties accompanying the complex life cycle of Termitomyces clypeatus have been monitored using atomic force microscopy (AFM). A new hyphae/mycelium is developed on cell division, and the cell wall of the mycelium undergoes a process of internal reorganization (or maturation) followed by morphological and chemical alterations. The changes of the surface ultrastructures during the growth process are correlated to the corresponding changes in relative viscoelasticity and rigidity of the cell wall by employing force spectroscopy. The cell wall rigidity and elasticity are found to be 0.34 ( 0.02 N/m and 27.5 ( 2.1 MPa, respectively, at the early logarithmic phase, on maturation increase to reach 0.81 ( 0.08 N/m and 92.5 ( 12 MPa, respectively, at the stationary phase, and thereafter decrease to 0.62 ( 0.06 N/m and 61.6 ( 6.6 MPa at the death phase. The alterations of the ultrastructural and nanomechanical properties of the cell surface as functions of growth phases affect the interaction involving chromium and T. clypeatus. Introduction The surface and interface of the microbial cells play crucial roles in numerous reactions and interactions in diverse fields of science and technology ranging from biological to environmental disciplines.1-7 The complex fungal surfaces consist of different polysaccharides (mainly β-1,3-D-glucans, mannan, chitin, and chitosan), a few glyocoproteins, and lipids in the cell wall,8-10 and their organization is manifested in the surface properties. The supramolecular moieties of the cell wall impart strength to the cells, control their shape, offer protection against mechanical damage, and regulate intracellular communication processes and their interactions with the environment.10-13 As the cell wall directly interacts with the extracellular environment, investigations on such surfaces are likely to yield vital information toward understanding relevant processes such as adhesion, surface recognition, biomineralization, metal-microbe interactions, etc.14,15 However, a lack of appropriate methodologies has arrested so far the desired progress in understanding the detailed physicochemical properties of microbial cell surfaces at the subcellular level.16 In recent years atomic force microscopy (AFM) has emerged as a powerful tool for investigating a number of characteristic properties associated with the microbial cells and biomolecules.17-21 Studies on microbial cells using AFM permit fundamental insights into the long-range interactions and mechanical properties of cell surfaces resulting from interaction forces involving AFM probes and such surfaces.20-25 In recent years the process of biosorption or bioaccumulation of hazardous metal ions on microbial cells has attracted considerable attention.6,26-28 However, information is not available regarding the physicochemical characteristics of the cell surfaces and also of the interface of microbial cells and aqueous solution. Lately microscopic investigations have reported morphological alterations of cells as a result of metal * Corresponding author. Telephone: +91 33 2473 4971/5904, ext 502. Fax: +91 33 2473 2805. E-mail: [email protected]. † Department of Biological Chemistry. ‡ Polymer Science Unit.

ion binding,29-32 but without providing any information on the intrinsic characteristics of the cell wall. The present investigation deals with an attempt to study the cell wall maturation during the growth process of the fungal strain Termitomyces clypeatus and the accompanying characteristic alterations following adsorption of chromium. We believe that the present AFM investigation monitors for the first time the dynamics of cell wall maturation and the transformations occurring in the cell wall due to the metal ion binding. Experimental Section Chemicals. Dehydrated microbiological media and ingredients were procured from Himedia, India. All other reagents were of analytical grade and were purchased from Merck (Germany) and Sigma (USA). Metal Solution and Analysis. A stock solution of chromium (100 mg/L) was prepared by dissolving potassium dichromate (K2Cr2O7) in double-distilled water and diluted to the desired concentration. The concentration of chromium was measured by an atomic absorption spectrometer (Varian Spectra AA 55). Methods. Preparation of Mycelia. Termitomyces clypeatus used in this study was kindly supplied by Dr. S. Sengupta, Indian Institute of Chemical Biology, Kolkata, India, and grown in a complex medium (see Supporting Information). Growth Rate of T. clypeatus. The complex medium (75 mL) was inoculated with 100 µL of mycelial suspension and then incubated at 30 °C for 240 h. At the end of incubation, mycelia were harvested from the fermented broth by filtration, washed with deionized water, and dried by lyophilization. The amount of biomass produced was then recorded. Chromium Adsorption on T. clypeatus Mycelia. The mycelia of T. clypeatus were harvested from different growth phases, washed with deionized water, dried by soaking in blotting paper, and used for adsorption of chromium. The moisture content of the biomass was determined by drying at 65 °C to constant weight.Theadsorptionexperimentswereconductedincitrate-phosphate buffer (50 mM, pH 3.0, being optimum) with 0.25 g of blotted dried T. clypeatus mycelia and 25 mL of K2Cr2O7 solution

10.1021/jp808760f CCC: $40.75  2009 American Chemical Society Published on Web 01/15/2009

1486 J. Phys. Chem. B, Vol. 113, No. 5, 2009 containing 100 mg/L chromium and incubated at 30 °C for 48 h with constant shaking (130 rpm) unless otherwise stated. On completion of the incubation process, the mycelia were separated by centrifugation (10 000 rpm for 10 min) and the chromium concentration in the supernatant was measured as described above, while the concentration of the adsorbed metal ions was calculated using the mass balance equation.33 The kinetics of chromium adsorption on T. clypeatus mycelia harvested from the stationary phase was followed at regular intervals up to 48 h as mentioned above, with the other experimental conditions remaining the same. Immobilization of Fungal Mycelia for Atomic Force Microscopic (AFM) Imaging. Fungal mycelia were harvested from the growth medium and washed with citrate-phosphate buffer (50 mM, pH 6.0). The samples for AFM analysis were then prepared following the methods of Dorobantu et al.34 by incubating the mycelial suspension with an ultrasonically cleaned glass coverslip for 60 min, followed by repeated washing with ultrapure Millipore water (18.2 MΩ) to remove loosely attached mycelia. The coverslip was then mounted for AFM study and kept hydrated by soaking in the same citrate-phosphate buffer during analysis. All the images were recorded under identical conditions. Atomic Force Microscopy. Atomic force microscopic (AFM) images of T. clypeatus mycelia were recorded in tapping mode at ambient conditions using a multimode AFM (Veeco Metrology, Autoprobe CP-II, Model No. AP0100) with silicon probes (for details, see the Supporting Information). Force-distance curves were recorded in contact mode to measure the cell wall elasticity and rigidity. A phosphorus (n) doped Si cantilever with a spring constant 0.6 N m-1 was used for force measurement. The spring constant of the cantilever was calibrated from the resonance frequency of the cantilever.35 In each experiment the force curve was recorded on a bare coverslip glass serving as a reference. Approximately 50 force curves were recorded in every experiment. The elasticity of the fungal cell wall was measured from different positions of five to six fungal hyphae grown in five independent experiments. For each experiment 50 force-distance curves were recorded at several locations of different cells harvested from each phase. A single cantilever and tip were used for all force spectroscopy measurements during the recording of relative changes in cell wall elasticity. Results and Discussion Cell Surface Ultrastructure. The alterations of the cell surface chemical compositions of a number of microbial strains following the growth process have been reported.36-38 An electron microscopic investigation reveals that the cell wall morphology changes with the growth process as well as with environmental conditions.39 However, no detailed information on the cell wall mechanical properties during the maturation process has been reported. In order to probe the surface properties of T. clypeatus under native conditions, fungal mycelia were immobilized (without using any drying or fixation) on the glass coverslip and AFM images were recorded in tapping mode to minimize possible damage. Figure 1 shows the growth of T. clypeatus in relation to biomass production as a function of time. It is observed that the growth of T. clypeatus enters an exponential phase after an initial lag of 20 h, maintains this state up to 90 h, and finally enters the death phase (after 120 h). AFM images (Figure 2) of the contoured region of the cell surface of T. clypeatus mycelia harvested from different growth phases of the organism exhibit significant alterations of the surface morphology with the progress of the growth process.

Das et al.

Figure 1. Growth curve of T. clypeatus and effect of incubation period on adsorption of chromium by T. clypeatus mycelia. Data represent an average of five independent experiments (SD shown by error bar.

All measurements have been recorded under identical conditions, and the images are representative of different cells, each imaged at the same growth stage. AFM imaging was recorded initially by scanning a 20 µm × 20 µm area that contained several fungal mycelia (see Supporting Information, Figure S1). The image size was gradually reduced to isolate a single mycelium and finally obtain high resolution images (1 µm × 1 µm). The images were recorded from a randomly selected position on a single mycelium. The mycelium was scanned in both forward and backward directions before imaging to ensure that tip artifacts, such as hysteresis, were not altering the images. A high resolution micrograph (Figure 2A) reveals that the young fungal mycelia harvested from the early logarithmic phase (30 h) have relatively smooth surfaces (sectional analysis, Figure 2M) without any detectable structural characteristics on this young hyphae; however, some protuberant structures emerge on the cell wall (Figure 2B) when the organism enters the midlogarithmic phase (70 h). The appearance of some randomly distributed nanopore-like features on the cell surface (Figure 2B) are found to develop with the progress of the growth process (80-100 h, Figure 2C,D) until the organism enters the stationary phase (120 h, Figure 2D), when the surface becomes more textured. Sectional analysis of this mature mycelial surface clearly demonstrates that the nanopores are homogeneously distributed throughout the cell wall (Figure 2N) irrespective of the scan angles, scan rates, and scan ranges during the experiments. The structural features observed on the mycelia harvested from the logarithmic and stationary phases collapse at the death phase (>130 h, Figure 2E,F). The possibility of distortion of the cell wall during scanning with the AFM tip was assessed by repeated scanning over the selected area, but no difference between the last and first scans in each set could be detected. The reproducible imaging of the surface features could be obtained irrespective of scan speed, angle, and force. We believe that the images reflect an intrinsic structure of the cell surface and reveal a high sensitivity to fine surface details.40 The phase image provides a fairly detailed view of the surface heterogeneity regarding the organization of the cell surface components and nanoscale roughness of the surface.40 The phase image (Figure 2G) of the mycelia harvested from the early logarithmic phase also corroborates the topographic findings. The phase contrast among different components of the cell surface is similar, suggesting its homogeneous distribution. The cell surfaces are found to be smooth when harvested from the midlogarithmic and early stationary phases (Figure 2H,I). However, the phase images exhibit more characteristic features than the topographic ones. The high distribution of pore-like

Properties of T. clypeatus Cell Wall

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Figure 2. Topographic (A-F) and phase contrast (G-L) AFM images of pristine mycelia, harvested from different growth phases, taken under tapping mode in air. Mycelia harvested from (A) early logarithmic phase, (B, C) logarithmic phase, (D) stationary phase, and (E, F) death phase. (M) Sectional analysis of (A), and (N) sectional analysis of (D). Scan area 1 µm × 1 µm.

structures on the cell surface are more clearly visible in the phase images (Figure 2I,J) compared to the topographic ones (Figure 2C,D). Phase images show considerable differences (dark and light regions) in the cell wall components. Enhanced contrast in the phase images is attributed to the viscoelastic differences among the different macromolecular components of the cell which presumably reflect the presence of underlying different cell wall polysaccharides (such as chitin, chitosan, glucan, and mannan) and proteins.41 We found that the cell surface heterogeneity of mycelia harvested from the midlogarithmic phase or stationary phase is greatly enhanced at the onset of the death phase (Figure 2K,L). A high degree of heterogeneity in the phase image of the death phase cells might be due to the autolysis of the cellular macromolecular components.42 Previously it was reported on the basis of cytological analysis that entry of a fungal cell into the stationary phase causes growth

arrest followed by a process of autolysis whereby mycelium actively secretes lytic enzymes that result in the ultimate macromolecular breakdown of the mycelia.42-44 The AFM images demonstrate that at the onset of the death phase (>130 h) the nanopores became larger (Figure 2E,F,K,L) due to autolytic activity and merged into one another, thus creating longer holes on the contour of the cell wall. The sectional analysis exhibits that the surface roughness (Rrms) value of the mycelia harvested from early logarithmic phase is 2.86 ( 0.2 nm, which increases to 6.86 ( 1.2 nm as the organism enters the midlogarithmic phase. At the stationary phase the Rrms value becomes 12.58 ( 2.5 nm. Interestingly, at the onset of the death phase, the surface textures collapse due to lysis of the cell and this increases the Rrms value to 19.2 ( 3.5 nm. The AFM images thus demonstrate alteration of the surface morphology on cell wall maturation.

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Figure 3. Force-distance curve of glass coverslip (A) and T. clypeatus mycelia harvested from early logarithmic (B), stationary (C), and death phases (D). Changes in cell wall rigidity (E) and Young’s modulus (elasticity) (F) of pristine T. clypeatus mycelia harvested from different growth phases and after adsorption of chromium. Mycelia harvested from (1) early logarithmic phase, (2, 3) logarithmic phase, (4) early stationary phase, (5) stationary phase, and (6) death phase. Data represent an average of five independent experiments (SD shown by error bar.

The changes of the cell wall morphology during maturation are likely to be reflective of the physicochemical properties due to variation of their macromolecular compositions.9-12 The nanomechanical properties such as cell wall elasticity or stiffness (Young’s modulus, E) and relative rigidity (Kcell) were obtained employing force spectroscopy of the fungal mycelia immobilized on the glass coverslip.17-24 A single cantilever tip was used in all the force measurements as we measured the relative changes of the cell wall elasticity during the growth process. Force curves were measured on the glass coverslip before and after measurement on the mycelia to confirm the absence of AFM tip contamination leading to nonspecific adhesion between the tip and the cell surface. Force curves were recorded from random locations (dark as well as light regions in the phase image) on the cells. The rigidity and Young’s modulus of the cell wall were then calculated from the mean values derived from those curves. The extension half of the force cycle reflects the advance of the tip toward the cell, the initial cell contact, and the deflection of the tip as it is pressed into the surface. The shape and slope of the curve depend on the surface characteristics.

The extension curve for the glass coverslip (Figure 3A) shows two regimes: a flat horizontal approach and a steep linear deflection. The transition between these two regions is sharp. However, the extension curve on the T. clypeatus mycelia (Figure 3B-D) has a less steep linear deflection than on the coverslip and is separated into three regimes. In between the horizontal approach and the linear deflection, a nonlinear transition is observed. The nonlinear region, sometimes referred to as the “repulsive” region,45 of the extension curve is caused by repulsive electrostatic and van der Waals forces between the cell surface and the AFM tip, but much of the deflection at larger deflection must be due “steric” factors, i.e., the interaction between tip and various soft polymeric biomolecules of the cell wall.45-47 A similar phenomenon is also observed by Volle et al.47 in the case of E. coli. The extension curve of the mycelia harvested from different growth phases also exhibits three regimens, but variation in the nonlinear transition is observed with the growth process. Early logarithmic phase cells (Figure 3B) show larger nonlinear deflection, and with progress of the cell wall matura-

Properties of T. clypeatus Cell Wall

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Figure 4. Topographic (A-F) and phase (G-L) contrast AFM images of chromium adsorbed biomass harvested from different growth phases, taken under tapping mode. Mycelia harvested from (A) early logarithmic phase, (B, C) logarithmic phase, (D) stationary phase, and (E, F) death phase. Scan area 1 µm × 1 µm.

tion this portion decreases and the stationary phase cells (Figure 3C) demonstrate very little deflection. As expected, the death phase cells (Figure 3D) exhibit larger deflection than the stationary phase cells. The force and distance components of the nonlinear regime between the approach to the cell wall and the linear deflection were also measured. In the early logarithmic phase cells, this large nonlinear region encompasses an average distance of 150 ( 15.5 nm and a force of 3 N. The nonlinear region decreases, in both distance and force, with the growth process (Supporting Information, Table S1), and the stationary phase cells experience the smallest values. Thus, the differences observed in the force and distance components of the nonlinear region indicate that tip-cell surface interaction varies with the wall composition. Lysis of the cell wall at the death phase weakens the integrity of the cell wall to mechanical stress and thus experiences the long-distance components in the nonlinear region. The slope of the linear portion of each extension was used to determine the stiffness of the cell wall. The relative rigidity or spring constant (Kcell) of the cell wall was determined from

the slope of the linear portion (last portion of the approach curve or first portion of the retraction cycle) of the force distance curve using the formula (eq 1)48,49

Kcell ) -

KCS KC + S

(1)

where KC is the cantilever spring constant and S is the negative slope. The obtained spring constant associated with the cell wall was used for the subsequent determination of cell wall elasticity (Young’s modulus, E) using finite element modeling (eq 2) as described by Zhao et al.50

E ) 0.80

KF R h h

1.5

()

(2)

where R is the radius of the mycelia; E and h are the elastic modulus and thickness of the cell wall. The values of h and R

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Figure 5. AFM images (A) of T. clypeatus mycelia harvested from the stationary phase of growth during adsorption of chromium at different times. Cross-sectional analysis (B, C) of images from the marked portion. Scan area 1 µm × 1 µm.

were determined from transmission electron micrographs (see Supporting Information, Figure S2 and Table S2). The cell wall rigidity and elasticity were obtained from the mean values derived from multiple force curves recorded from random locations (dark as well as light regions in the phase image) on the cells, and a slight variation (standard deviation ∼3%) was observed between the individual force-distance curves. In view of the close agreement of the estimated values, the obtained rigidity and elasticity are considered reasonable. The cell wall rigidity and elasticity of the T. clypeatus mycelia harvested from the early logarithmic phase are found to be 0.34 ( 0.02 N/m and 27.5 ( 2.1 MPa, respectively. The relative rigidity of the reference glass coverslip is found to be 0.995 ( 0.05 N/m. The effects of maturation on the cell wall mechanical properties are presented in Figure 3E,F. It is observed that the growth process of the fungal cell wall exerted a conspicuous effect on local stiffness and rigidity. The relative rigidity (Figure 3E) and elasticity (Figure 3F) of the pristine fungal mycelia increase with the growth process, attain maximum value at the stationary phase (0.81 ( 0.08 N/m and 92.5 ( 12.4 MPa), and thereafter decrease (0.62 ( 0.06 N/m and 61.6 ( 6.6 MPa) at the death phase; i.e., the stationary phase cells are indented less by the same applied force compared to the cells obtained from the other growth phases. In other words, the stationary phase (matured) cells are less viscous (more rigid) compared to the rest of the cells. The measured values of the cell wall rigidity and elasticity of the mycelia harvested from different growth phases are

significantly different (p < 0.1) from that of the other. The T. clypeatus cell wall has higher rigidity and elasticity compared to the reported values for bacterial and mammalian cells,13,51 but lower than that of Aspergillus nidulans reported by Zhao et al.50 and Ma et al.52 The lower elasticity of T. clypeatus compared to that of A. nidulans may be due to the variation of cell wall constituents (β-1,3-D-glucans, mannans, chitin, chitosan, glycoproteins, and lipids).53,54 The variation of the measured stiffness and rigidity of the mycelia harvested from different growth phases indicates alterations of the nanomechanical properties with the maturation of the cell. During the growth process a new wall material is developed and rearranged, which reflects the alterations of the cell wall rigidity and elasticity. The cell wall rigidity and viscoelasticity change with the growth process, thereby demonstrating the dynamics of the cell wall maturation. Thus, the results show a direct correlation of the alterations of the morphological and nanomechanical properties as a consequence of the cell wall maturation. The present study describes for the first time the dynamics of the cell wall maturation process on the basis of the force-distance curve along with the AFM images. Binding of Chromium(VI) to Fungal Cell Wall. Figure 1 shows that the adsorption capacity depends on the harvesting time of the mycelia and the process increases during the entire exponential growth phase of the organism, attaining maximum value at the stationary phase, and starts decreasing after entering the death phase. Enhanced adsorption of chromium exhibited

Properties of T. clypeatus Cell Wall by mycelia harvested at the stationary phase indicates that the binding of the metal ions to T. clypeatus mycelia is facilitated by the development of binding sites with the progress of the growth process.34-36 At the onset of the death phase the adsorption capacity and the binding sites decrease due to cell lysis. The cell surface ultrastructure is considered to have significant implications on the binding of chromate ions to the cell surface. We have already demonstrated (Figures 2 and 3) that the cell surface ultrastructural properties alter with the cell wall maturation and this variation of the cell wall properties during growth phase is manifested in the adsorption (Figure 3E,F) phenomenon. AFM images (Figure 4) along with the surface height profiles reveal structural alterations of the cell surface topography on chromate ion binding. The cell surface becomes covered by a densely packed layer of the metal ions (confirmed by EDXA analysis, see Supporting Information, Figure S3B) following the binding of chromium on T. clypeatus mycelia harvested from stationary phase. The surface morphology transformed to a layered structure (Figure 4D), and the nanopores present in the control mycelia disappeared (Figure 2D). The cell surface is depicted by a lighter color in the phase images, which may be due to the increased cell surface hardness.16 The surface convexes appeared relatively compact in the phase image, indicating cell surface coverage with layers of different compositions. Thus, surface heterogeneity developed as a result of chromate ion binding. The average surface roughness (Rrms), rigidity (Kcell), and elasticity (E) of the cell wall on chromium adsorption increased significantly (p < 0.1) to 32.6 ( 5.8 nm, 0.96 ( 0.07 N/m, and 175.6 ( 15.6 MPa, respectively, corresponding to the values of the stationary phase cells of 12.58 ( 2.2 nm, 0.81 ( 0.08 N/m, and 92.5 ( 12.4 MPa. The mycelia collected from the early logarithmic phase (Figure 4A) hardly exhibited any surface morphology alteration on metal ion adsorption. The adsorbed materials were localized only in some areas of the cell surface of the midlogarithmic phase (Figure 4B,C) or death phase (Figure 4E,F). The average roughness, rigidity, and elasticity are 14.61 ( 4.2 nm, 0.65 ( 0.06 N/m, and 70.5 ( 10.5 MPa, respectively, in the former species, while in the latter the corresponding values are 27.3 ( 6.2 nm, 0.68 ( 0.09 N/m, and 90.5 ( 8.5 MPa. Thus, the ultrastructural transformations of the T. clypeatus cell surface due to the binding of chromate ions suggest that the surface properties play important roles in the adsorption process. Kinetics of Chromium(VI) Adsorption on the Mycelial Surface. The kinetics of chromate ions binding to T. clypeatus was followed in order to monitor the inner details of the adsorption process along with the associated changes in ultrastructural properties. We found that the chromium adsorption increased with time and reached equilibrium after a 40 h incubation period (Figure 6A). Figure 5A shows the representative topographic images of the fungal mycelia on chromate ion adsorption as a function of the incubation period. The images depict that the surface morphology of the metal-adsorbed mycelia is conspicuously different from that of the pristine ones (Figure 2D). In the initial stage of metal ion adsorption (Figure 5A) the topographic image of the surface appears to be smooth compared with that of the pristine one (Figure 2D), and this may be due to the covering of the nanopores. On increasing the incubation period to 2 h, the surface is observed to be covered by small domain-like structures. The surface coverage increases with time (15 h), and cross-sectional analysis depicts uniform denser surface patterns (Figure 5B) of chromium throughout the cell wall having a surface roughness of 18.5 ( 2.4 nm. Drastic changes of the surface morphology are noted

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Figure 6. Adsorption kinetics of chromium (A) on T. clypeatus mycelia. Changes of the cell wall elasticity (B) of T. clypeatus on binding of chromium at different incubation period. Data represent an average of five independent experiments (SD shown by error bar.

on increasing the adsorption period (beyond 30 h), and at equilibrium a layered structure formation (Figure 5C) with increased roughness values (28.5-32.6 nm) is observed. The effect of metal ion binding on the elasticity of the surface undergoing alteration with the progress of the adsorption process as a function of time is presented in Figure 6B. The cell wall elasticity changes as a function of time during the adsorption process. This is consistent with the increase of the surface coverage with chromate ions, as already observed. The elasticity of the cell wall increases due to the metal ion adsorption resulting in increased hardness of the surface. We believe that the present investigation reports for the first time detailed ultrastructural alterations of the fungal cell surface properties and the topographical images during the metal ion adsorption process utilizing the AFM technique. Conclusions Cell wall maturation of T. clypeatus mycelium and its interaction with chromium have been monitored on the basis of the alterations of the ultrastructural and nanomechanical properties. The cell wall rigidity (Kcell) and elasticity (E) increase from 0.34 ( 0.02 to 0.81 ( 0.08 N/m and from 27.5 ( 2.1 to 92.5 ( 12.4 MPa, respectively, until the organism enters the stationary growth phase and the values decrease at the onset of the death phase. The adsorption characteristics of chromium depend on the nanomechanical properties of the cell wall, and maximum adsorption is observed at the stationary phase. The cell wall rigidity and elasticity increase significantly to 0.96 ( 0.07 N/m and 175.6 ( 15.6 MPa, respectively, on chromate ion binding compared to those at the stationary phase. The kinetic results of the chromium adsorption process on T. clypeatus speak in favor of a sequential transformation of the associated surface properties.

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