Thermal Effects on Surface Structures and Properties of Bacillus

Jun 6, 2013 - Department of Engineering Physics, Air Force Institute of Technology, 2950 Hobson Way, Wright-Patterson AFB Ohio 45433-7765,...
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Thermal Effects on Surface Structures and Properties of Bacillus anthracis Spores on Nanometer Scales Alex G. Li,* Yun Xing, and Larry W. Burggraf Department of Engineering Physics, Air Force Institute of Technology, 2950 Hobson Way, Wright-Patterson AFB Ohio 45433-7765, United States S Supporting Information *

ABSTRACT: Bacterial spores, one of the hardiest forms of life known, can survive severe environmental stresses such as high temperature. Using thermal atomic force microscopy (AFM), we show that the surface structures and properties of Bacillus anthracis spores when exposed to elevated temperatures undergo substantial changes on nanometer scales. Thermal-blister-like nanostructures, which grow in size with increasing temperature, are formed on the spore surface when it is heated by a thermal tip. Although thermal damage to the spore surface is persistent upon cooling heat-treated spores to room temperature, thermal effects on surface properties of the spores are complex. The thermally induced nanostructures show a lower surface−tip adhesion and a higher modulus than the surrounding spore surface. The overall trend is for the adhesion to decrease with increasing temperature. However, the adhesion of heat-treated spores may be smaller than, equal to, or larger than that of untreated spores, depending upon the degree of surface damage induced by heat. Although the overall spore dimensions show few changes during and after heat treatment, the size of the spore substructures decreases significantly. In addition, we demonstrate a nanoscratch AFM method for imaging the subsurface structures of spores.



INTRODUCTION Bacterial spores such as Bacillus anthracis (Ba) are highly resistant to inactivation by heat and other environmental stresses.1 Significant progress has been made by many pioneers2−16 in elucidating the fascinating mystery regarding how a spore protects molecules such as DNA, RNA, and enzymes essential to its survival. This research not only informs scientific curiosity about dormant life but also has practical applications because of the role of spores in bioterrorism weapons, food spoilage, and food-borne disease. Because heating in a moist environment is still one of the most effective methods of inactivating spores,17 it is important to relate thermal effects to initial biochemical damage that leads to the thermal inactivation of spores. Bacillus spores are formed with a concentric multilayer architecture in which the central core is surrounded by distinct successive layers: an inner membrane, a germ cell wall, a cortex, and a coat assembly.18,19 For Bacillus anthracis spores, there is an additional layer, called the exosporium, that loosely encases the spore. The spore core that contains DNA, RNA, and most © 2013 American Chemical Society

enzymes is largely composed of dipicolinic acid (DPA). The cortex of spores consists of peptidoglycan with alternating Nacetyl-glucosamine and N-acetyl-muramic acid residues.14 The coat assembly of spores is a multilayer structure consisting of the basement layer, inner coat, outer coat, and outermost crust.18,19 McKenney et al.20 recently mapped the genetic dependencies for the coat assembly by the fusion of coat proteins to green fluorescent protein. They suggested that the inner coat layer contains safA-dependent morphogenetic proteins; the outer coat and the outermost spore surface contain cotE-dependent morphogenetic proteins. The exosporium of Bacillus anthracis spores, which contains about 20 different proteins, is composed of a hexagonal crystal-like basal and hairy-nap outer layer. The filaments of the nap are composed of BclA, a glycoprotein containing distinct NReceived: November 30, 2012 Revised: June 6, 2013 Published: June 6, 2013 8343

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examination confirmed the spore purity (>95%), spores were harvested from the agar plates using a sterile cell scraper and transferred to a microcentrifuge tube, where the spores were washed at least three times with sterile DI water. Afterwards, the spores were incubated on a heating block for 30 min at 65 °C to inactivate vegetative growth and washed again with DI water before storage or measurement. For the AFM imaging of individual spores, a 10 μL drop of a diluted spore suspension containing about 105 spores was placed on freshly cleaved mica or an alcohol-cleaned silicon wafer. The spore sample was then air dried in a BSL-2 Biosafety cabinet for at least 12 h before being mounted onto an iron disk, which is magnetically attached to the sample stage of the atomic force microscope. For the AFM imaging of monolayer film of spores, a clean spore water suspension with a spore concentration for monolayer coverage was dropped onto an alcohol-cleaned silicon wafer substrate. The sporecovered substrate was then left to air dry for at least 12 h before the measurement. For AFM imaging of the substructures or internal spore components, the spores were broken down using a standard autoclave liquid cycle and a nanoscratch AFM method that we have developed. AFM Imaging of the Surface of Bacillus anthracis Spores. The AFM instrument that we used to image the surface structures and properties of spores was a Nanoscope Multimode atomic force microscope with peak force imaging capability from Bruker Corporation. For thermal mechanical measurement, the atomic force microscope was operated in intermittent contact mode in which the cantilever tip is oscillated at a small amplitude (10−100 nm) and a low modulation frequency (0.5−2 kHz). The force of contact was measured for each modulation cycle and used to control the feedback electronics. The load applied to the AFM tip was maintained as a small preset value (0.3 to 30 nN). The AFM instrument was equipped with a piezoelectric scanner (AS-130VLR) capable of imaging 125 μm × 125 μm in the lateral x- and y-axis directions and 5 μm in the vertical z-axis direction. During a typical peak force imaging process, the instrument acquires and analyzes the individual force curves from each cycle of contact. Surface properties such as adhesion, modulus, dissipation, and deformation are simultaneously measured and processed using commercial Multimode 8 nanoscope analysis software. The modulation frequency is significantly lower than the cantilever resonance frequency, so there is sufficient bandwidth for fast data sampling and for analysis in real time. The direct force measurement at high speed allows the force−distance data to be analyzed directly without ambiguity. For high-temperature AFM measurement, two thermal cantilever tips with different spring constants were utilized. The spring constant of the thermal tips was determined using the thermal tune method.39 The detailed specifications for the stiff thermal cantilever tip (AN2200) are as follows: tip height (h) = 3−6 μm, tip radius < 30 nm, and the spring constant decreased slightly from 2.9 to 2.3 N/m with increasing temperature from 25 to 350 °C. The cantilever length, thickness, and resonance frequency are 200 μm, 2 μm, and 55−80 kHz, respectively. Detailed specifications for the soft thermal cantilever tip (AN2-300) are as follows: tip height (h) = 3−6 μm, tip radius < 30 nm, and the spring constant decreased slightly from 0.4 to 0.3 N/m with increasing temperature from 25 to 350 °C. The cantilever length, thickness, and resonance frequency are 300 μm, 2 μm, and 15−30 kHz, respectively. For the nanoscratch AFM measurement, a calibrated diamond tip with a spring constant of 186 N/m was used. The detailed specifications for the diamond tip (DNISP) are as follows: tip height (h) = 50 μm, tip radius = 40 nm, resonance frequency = 50 kHz, length = 350 μm, and width = 100 μm. All AFM tips were rinsed with high-purity methanol and acetone before use. In addition, the thermal tip was briefly heated to about 400 °C for 10 s to remove possible contamination before each measurement. For thermal tip temperature calibration, the AFM was operated in contact mode. The temperature calibration curve was produced by the quadratic fitting of the melting points of poly(ethylene terephthalate) (PET, Tm = 235 °C), highdensity polyethylene (HDPE, Tm = 116 °C), and polycaprolactone (PCL, Tm = 55 °C) to their corresponding heating voltages at which the crystalline polymers start to melt, similar to our previous calibration,40 as shown in Figure 1b.

terminal (NTD) and C-terminal (CTD) domains separated by an extended collagen-like central region.21−24 The essential molecules of spores such as DNA, RNA, and enzymes are protected by multiple mechanisms involving α/βtype small, acid-soluble proteins (SASP),5−10 the mineral dipicolinic acid (DPA) matrix,11−13 and spore structures.14−16 The membrane and cross-linked protein coat of spores can limit the entry of some harmful chemicals such as hydrolytic enzymes into the spore; the cortex, a highly hydrated crosslinked peptidoglycan matrix, can maintain, in addition to structural integrity and mechanical strength, the dehydrated state of the core. Although it is believed that the layered architecture of spores plays an important contributing role in protecting the DNA, RNA, and enzymes from a variety of environmental stresses, little research has been done previously to connect thermal effects on surface structures and properties to the heat resistance of spores. Heat resistance, which is usually measured by the time duration of a constanttemperature exposure required for a decimal reduction in colony-forming units of viable spores, is known to be correlated with protoplast dehydration.25−28 The dehydration of spores may enhance heat resistance by stabilizing DNA against hydrolytic reactions and preventing the irreversible denaturation and aggregation of proteins.17,29−31 In a dry environment, the heat resistance of the spores can increase by 1000-fold in comparison to that in a wet-heat environment.28 It is generally believed that the high heat resistance of dry spores is due to low water activity in the core. However, it is intriguing how a moderate change in the core water content could produce over a 1000-fold difference in the heat resistance because the core water is well regulated between 25 and 50% of the wet weight, depending upon the state of dehydration of the spores.25−27 Atomic force microscopy (AFM) methods are routinely utilized to image various spores at room temperatures.32−38 Chada et al.32 showed the Bacillus spore surface is covered with nanometer-sized circular bumps. For Bacillus subtilis and Bacillus anthracis spores, there are ridge structures largely oriented with the long axis of the spore. Plomp et al.33,34 imaged surface structures of spores under different conditions using the AFM. Without the exosporium, the surface of Bacillus thuringiensis shows crystalline hexagonal honeycomb structures; the surface of Bacillus cereus spores shows small randomly oriented domains. Our previous AFM research36 showed that four closely related species of Bacillus spores can be distinguished by surface morphology analysis. In this work, we used thermal atomic force microscopy at elevated temperatures to measure real-time thermal effects on the topographic morphology and nanomechanical properties of Bacillus anthracis spores, including the adhesion force, elastic modulus, deformation, and energy dissipation. We used a hydrophilic silicon AFM tip to detect interfacial adhesion, which is sensitive to surface water, the water produced from chemical reactions in heated spores, and changes in surface polarity. We observed striking changes, which resemble phase transitions, in the physical properties of heated spores on the nanometer scale. We modeled thermal transport in the spore using simplified finite element analysis (FEA). In addition, we demonstrated a nanoscratch method for imaging subsurface structures of spores.



MATERIALS AND METHODS

Sample Preparation. Bacillus anthracis spores (Sterne strain) were grown on nutrient agar for about 7 days. Once the microscopy 8344

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Figure 1. Finite element analysis modeling and nanomechanical characterization of spores using thermal AFM. (a) Temperature changes on the surface and in the center of the spore in first four cycles of thermal modulations. (b) Thermal tip indentation of the Bacillus anthracis spore (Ba), poly(ethylene terephthalate) (PET), high-density polyethylene (HDPE), and polycaprolactone (PCL) at various temperatures. (c) Histogram of the elastic modulus of air-dried spores on freshly cleaved mica. (d) Peak-force error image of the spore covered by a thin layer, which we have tentatively identified as the exosporium, as shown by the dotted line. (e) Peak-force error image of the spore with straight interfaces, as shown by the dotted polygon. (f) Peak-force error image of the spores with straight interfaces forming sharp corners, as shown by the arrows. The peak force error image was generated using the sum signal of the preset peak force and the force error signal at each point of the tip−surface contact. The small differences (or errors) between the preset force (e.g., set point) and the measured force signal are sensitive to rapid changes in surface morphology and properties. The adhesion force was determined from the pull-off position in the force−distance curve on each point of contact and can be produced by attractive forces such as van der Waals, electrostatics, and the capillary meniscus of a thin liquid film on the sample surface. The topographic morphology of the surface may affect the interfacial adhesion because the adhesion typically increases with increasing tip size and area of contact. For silicon-based cantilever tips, the tip surface is usually covered with hydroxyl groups as a result of surface reactions with water in air. The adhesion force between the hydrophilic tip and sample surface is likely dominated by hydrogen bonds in water and other polar molecules of the spore surface. Young’s modulus of the sample was obtained by fitting the unloading portion of the force− distance curve using the Derjaguin−Muller−Toporov (DMT) model,41 as given by

F − Fadhesion =

two components. One is elastic deformation from which the elastic modulus is measured; the other is plastic deformation from which the energy dissipation is determined. Because the nominal radius of the cantilever tips was used in this work, the measured elastic modulus was qualitative. Energy dissipation was given by the force times the tip velocity integrated over each cycle of the tip oscillation. For the elastic surface, the dissipation is zero when the loading and unloading curves coincide. The energy dissipation is dominated by plastic deformation on the sample surface. We estimated the radius of the thermal tip and the diamond tip by varying the tip radius in the DMT model to match the measured modulus of known materials such as freshly cleaved mica, graphite, and polystyrene polymers. AFM Image Analysis. Because a spore surface is not homogeneous, the property value measured at each pixel in the AFM image may vary within a range and the surface properties may not be an ideal Gaussian distribution.36 We described the surface properties using the 50 percentile value and the deviation of the property distribution. The 50% value is defined by the percentage of the surface above the reference plane (e.g., the lowest surface), and the deviation is defined by the difference in the 95 and 5% values in the surface distribution of the height, adhesion, modulus, or dissipation. For example, the 50% height is the height at which half the points in the measured area are above and half are below the reference plane. Surface Heating at Nanometer Contact. The localized heating at the nanometer contact of the spore surface is achieved by scanning a heated AFM tip in intermittent contact with the surface. The temperature of the thermal tip is controlled through joule heating in a U-shaped resistor formed in the silicon cantilever beam by a doping process using the nano-TA controller (Anasys Instruments). For the temperature ramp AFM measurement, the AFM tip was heated at a constant rate of about 1 °C/s from room temperature to up to 350 °C and cooled to room temperature at a high rate of about 1000 °C/s. For constant-temperature AFM measurements, the AFM tip was

4 E* R(d − d0)3 3

where F is the load, Fadhsion is the adhesion force, E* is the reduced modulus, R is the radius of the tip, and (d − d0) is the deformation on the surface. The reduced modulus is defined by −1 ⎛1 − ν2 1 − νtip2 ⎞ s ⎜ ⎟ E* = ⎜ + Etip ⎟⎠ ⎝ Es

where ν is Poisson’s ratio, Es is Young’s modulus of the sample, and Etip is Young’s modulus of the tip. The deformation is approximately 85% of the maximum distance at which the tip penetrates the surface at a load equal to the peak force. The deformation usually consists of 8345

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Figure 2. Thermal damage to the surface of air-dried spores deposited in a monolayer. Peak-force error images a and b were recorded on a single spore using a stiff AN2-200 thermal tip at 20 and 325 °C, and images c and d were recorded on a small cluster of spores using the soft AN2-300 thermal tip at 20 and 325 °C, respectively. The loads on the thermal cantilever tips were about 9 and 3 nN, respectively, and the scan frequency was 1 Hz. quickly heated to the preset temperature at a rate of about 1000 °C/s. The heating duration varies, depending upon the modulation frequency and the scanning rate of the thermal tip. For example, at a modulation frequency of 2 kHz, the heating time at the tip−surface contact is 5 × 10−4 s. Our finite element analysis modeling substantiates that the temperature distribution in the spore supported by a silicon substrate can reach its steady state within a time of less than 5 × 10−4 s, as shown in Figure 1a. The maximum surface temperature at the tip−spore interface is significantly higher than that in the center of the spore. The details of the finite element analysis modeling are given in the following section. Finite Element Analysis of Thermal Transport in the Spore. We performed the finite element analysis of thermal transport in the thermal tip−spore−substrate system using commercial code (COMSOL Multiphysics). The temperature distribution in the spore was obtained using a simplified thermal transport model in which the temperature at the tip−spore surface was modulated between 30 and 477 °C at 2 kHz and the spore−silicon surface was maintained at a constant temperature of 27 °C. The heat losses from the surface of the cantilever, tip, spore, and substrate were modeled by the radiation boundary conditions that assume that the flux across the surface is proportional to the temperature difference between the surface and the surrounding air. The side surfaces of the substrate system were thermally insulated. The spore, remaining in contact with the tip and the substrate at all times, was modeled as a prolate ellipsoid of 0.75 μm in the short-axis direction (b and c) and 1.0 μm in the long-axis direction (a). The physical properties of the spore described in this modeling are as follows: thermal conductivity, 0.3 W/m·K; specific heat, 2.5 kJ/kg/K; density, 1.2 kg/m3; and heat-transfer coefficient, 50 W/m2/K. Nanoscratch Method for Subsurface Imaging of Spores. The nanoscratch method we have developed to image subsurface spore materials consists of two steps. The first step is gently to scratch the spore surface to create identifiable surface markings and remove the top layer of the spore surface by indentation with a stiff silicon or

diamond AFM tip. The normal load required to produce sufficient indentations varied from tens of nanonewtons (nN) to a few micronewtons (μN), depending upon the surface stiffness of the spores. The second step is to locate the scratched spores using fiducial marks (large clusters of spores or dust) in the substrate and scratch marks on the spore surface and image the spores with a soft, sharp AFM tip. The freshly exposed spore surface can be distinguished from the native surface by the scratch marks.



RESULTS AND DISCUSSION Finite Element Analysis (FEA) Modeling of Heat Transport in the Spore. Our simplified FEA model shows that when the temperature at the tip−spore interface (referred to as surface temperature) is modulated between 30 and 477 °C at 2 kHz the temperature in the center of the spore (referred to as the center temperature) changes periodically at the modulation frequency between 28 and 137 °C (Figure 1a). The difference in the two peak temperatures is about 340 °C. The tip heating appears to be highly localized at the top surface of the spore. The spore temperature reaches the steady-state distribution within 0.5 ms in the first cycle. There is a small phase lag between these two temperatures, equivalent to a time difference of about 15 μs. It takes approximately about 30 μs for heat to propagate through the spore from the top surface to the substrate. We compared surface resistance to tip indentation of the spore with that of three different polymers. The tip deflection was measured at a constant load of 20 nN in contact mode using the AN2-300 thermal tip. When the tip temperature was increased from 25 to 400 °C at a rate of 1 °C/s, the surfaces of polycaprolactone (PCL), high-density polyethylene (HDPE), and poly(ethylene terephthalate) (PET) showed large deformations at the corresponding melting points of 55, 116, 8346

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and 235 °C, respectively, as indicated by the arrows in Figure 1b. In contrast, the spore surface showed no significant deformation until 342 °C. The deflection curve for the spore surface was obtained by averaging 12 temperature ramps at different locations in the monolayer of spores. It is obvious that the surface structure of spores is mechanically stable at high temperature. Figure 1c shows the histogram of elastic modulus measured using a diamond AFM tip for a cluster of spores deposited on a freshly cleaved mica surface. When the modulus of the mica surface was calibrated to be 50 GPa by adjusting the tip radius, the peak modulus of the spore was 4.2 GPa. We note that our measured modulus was lower than that, 13.6 GPa, as reported by Sahin et al.42 We believe that the inconsistency may be largely due to the different viscoelasticities of spore surfaces because the modulus of viscoelastic materials generally increases with increasing frequency. We found, by examining more than 100 AFM images containing more than 5000 spores, that the spores we prepared were likely covered by the exosporium, as highlighted by the dotted line in Figure 1d. Our assessment of the surface conditions of the spores we prepared prior to the heat treatment was based on the following information. First, the structural features we observed in the AFM images (Figure 1c and Figures S1−S3 in the Supporting Information) resemble the exosporium. Second, we noted that the adhesion between the spores in the monolayer produces straight interfaces and polygons surrounding the spores, as shown in Figure 1e and f, which are similar to those produced by interfacial adhesion between biological cells.43 It is known that the formation of straight interfaces and polygons around the cells is primarily due to the dominant effect of surface tension on thin, compliant cell walls, or other thin-layer structures. Because the spore’s coat, which is tightly bound to the underlying structure, may not be sufficiently mechanically compliant to behave like the cell wall as a result of strong elastic interactions with the underlying structure,42 the cell-like adhesion characteristic we observed in the monolayer spores is likely due to the presence of a soft, flexible thin layer, which we tentatively identify as the exosporium. Third, the study of the exosporium of Bacillus spores by several researchers34,44 indicated that the spores, harvested after normal washing and centrifugation, are largely covered by the exosporium. To remove the exosporium, mechanical forces such as ultrasonication or press pressures are required. The method we used to prepare our spore samples did not involve the use of sonication or other harsh mechanical treatment. Therefore, we expect that the exosporium is loosely attached to the spores in our samples. Our AFM measurements showed that the thickness of the thin surface layer varied between 12 and 15 nm. It should be noted that the structural identifications in the AFM images are not definitely identified without confirmations using other rigorous methods. Thermal Effect on Surface Structures of a Monolayer of Air-Dried Bacillus Anthracis Spores. Thermal damage to the spore surface can be superficial or deep wounds that substantially alter the underlying spore structures. Figure 2a,b shows the AFM peak force error images, recorded at room temperature before and after the spore was heat treated with the AN2-200 thermal tip at 325 °C for 8 min in air. Thermal blisterlike nanostructures of about 10 nm in diameter were initially formed at about 85 °C on the spore surface. The size of the surface aggregates grew to about 150 nm in diameter with increasing temperature to 325 °C. The thermal blisters were persistent and largely located on the surface layer.

We noted that thermal damage to the spore surface can vary significantly. Figure 2c,d shows the AFM peak-force error images recorded using the AN2-300 thermal tip on a different spore sample that was heat treated under the same temperature and time. A large number of nanostructures of about 100 nm in diameter were produced on the spore surface. The thermal damage appeared to extend to the subsurface of the spores because the surface roughness at some locations exceeded the thickness of the cover layer of about 12 nm. The adhesion force on the nanostructures decreased by 14%, and the elastic modulus increased by a factor of 2 in comparison to that of the surrounding surface area without the thermal blisters. Thermal Effect on Surface Properties of a Monolayer of Air-Dried Bacillus anthracis Spores. Changes in surface mechanical properties, including adhesion, modulus, deformation, and dissipation, of the monolayer spores measured using the AN2-200 thermal tip at elevated temperatures are shown in Figure 3. The adhesion was nearly constant at about 18 nN

Figure 3. Thermal effect on surface properties and the corresponding deviations of air-dried Bacillus anthracis spores. The data was obtained by averaging over an image area of 8 μm × 8 μm in the monolayer containing approximately 50 spores in each image. (a) Adhesion force, (b) elastic modulus, (c) energy dissipation, and (d) deformation (elastic plus plastic component) under a fixed load of 30 nN at various temperatures. The tip temperature was ramped up at 50, 75, 100, 225, 275, and 325 °C, respectively, before it was quickly cooled to 20 °C, as shown at the reduced scale of 1/10 of the original temperature (T) in Celsius in panel a.

between 50 and 75 °C. With increasing temperature from 75 to 100 °C, the adhesion decreased slightly by about 1 nN before it returned to the original value of 18 nN at 225 °C. When the temperature was ramped up to 325 °C, the adhesion showed a large decrease of about 4 nN. There was no significant change in the adhesion while the temperature was maintained at 325 °C for an extended period of time except for a small fluctuation that was probably due to thermal damage to the exosporium and exposure of the newly created surface of the underlying spore materials. As soon as the spores were cooled to room temperature, the adhesion increased to 22 nN in about 20 min. The adhesion force of heat-treated spores was larger than that of untreated spores (Figure 3a). The rapid changes in the surface properties of spores within a narrow range of temperature and time resemble phase transitions. The 8347

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Figure 4. Thermal effect on interfacial adhesion between the spore surface and the AFM tip in a nitrogen-purged environment (a, b) and in air (c, d). The data was obtained by averaging over an image area of 8 μm × 8 μm in the monolayer, containing approximately 50 spores in each image, under a fixed load of 15 nN at elevated temperatures. The tip temperature was continuously ramped from 20 to 350 °C and cooled to room temperature at 20 °C.

temperature was maintained at 325 °C for a prolonged period of time. The deformation exhibited a large drop from 3.2 to 0.46 nm after the thermal tip was cooled to room temperature. The surfaces of heated spores showed a smaller deformation than that of unheated spores, indicating that the spores became stiffer after the heat treatment at high temperature. The deviation in the deformation remained constant except for the time period of high-temperature heating. The energy dissipation, a measure of plasticity in the spore surface, increased gradually with increasing temperature from 20 to 325 °C. The dissipation showed small further increases for the extended period of heating at 325 °C. Upon the sample returning to room temperature, the dissipation decreased sharply. In contrast to the dissipation, the deviation in dissipation decreased gradually with the initial increase in temperature and the prolonged time of heating at high temperature. The dissipation deviation showed a large jump when the tip was cooled to room temperature, indicating that the spore surface becomes more dissipative after the hightemperature heat treatment. To understand whether the increased adhesion that we observed on heated spores was related to the uptake of water from air or was due to structural changes of the spore surface, we measured the adhesion force on the same spore sample in nitrogen gas and in air using the same thermal tip (AN2-300) and the same AFM control parameters, including control gains, scan length, scan frequency, and load, for consistent

heterogeneity in the surface properties of spores, as measured by the deviation in the adhesion distribution, gradually decreased with increasing temperature, indicating that the spore surface became more hydrophobic and homogeneous at elevated temperatures. However, the spore surface became less homogeneous again when cooled to room temperature, as indicated by the large increase in the deviation. The elastic modulus decreased slightly from a nominal 960 to 895 MPa with increasing temperature to 225 °C. There was a large drop of 57% in the modulus at 275 °C, occurring at a lower temperature than the large drop observed in the adhesion. When the temperature of the thermal tip was maintained at 325 °C for an extended period of time, the modulus increased from 383 to 639 MPa during the initial heating and decreased to 511 MPa with further increases in the heating time. After the spores were cooled back to room temperature, the modulus returned to approximately the initial value prior to the heat treatment. The deviation in the elastic modulus increased significantly during the initial heating from 50 to 225 °C, above which the deviation decreased quickly with increasing temperature. Upon cooling to room temperature, the modulus deviation returned to the value of unheated spores. The deformation, which is proportional to the sum of the elastic and plastic displacement of the thermal tip on the surface, increased gradually from 0.85 to 1.3 nm with increasing temperature from 50 to 225 °C. There was a large jump of 3.2 nm in the deformation at 275 °C. The deformation showed no significant changes when the tip 8348

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Figure 5. AFM adhesion images showing thermal damage to the surface layer of the spores at high temperatures. (a) Adhesion at room temperature of 20 °C. (b) Real-time image of the adhesion at 150 °C. (c) Real-time image of the adhesion at 350 °C. (d) Adhesion after quick cooling from 350 to 20 °C in air.

Figure 6. Nanoscratch AFM method for imaging subsurface structures of spores. (a) Height image of a partially covered spore. (b) Modulus image of the plastically deformed surface of the spore. (c) Deformation image of the partially covered spore. (d) Height profiles across the steps between the newly exposed and covered surfaces of the spore.

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Figure 7. AFM images of the autoclaved Bacillus anthracis spores. The height (a) and force error (d) images were recorded using the AN2-200 thermal tip at room temperature. The height (b, c), and peak force error images (e, f) were recorded after the autoclaved spores were heated by the thermal AFM tip at 150 °C. The tentative assignments of the spore component structures, including the exosporium, coat, cortex, and core, are indicated by the black, blue, red, and white arrows, respectively. The ridge structure of the coat is highlighted by the dotted arrow in panel f.

on the adhesion of the spores measured in air was similar to that measured in nitrogen gas. We infer that observed changes in the adhesion were independent of water uptake in air. The effect of heat on the adhesion force of the spores can be visualized in AFM adhesion images in Figure 5a−d. In general, the adhesion decreased uniformly with increasing temperature (Figure 5a,b). The fluctuations in the adhesion were due to uneven thermal damage to the surface layer mostly around the edges of spores (Figure 5c), leading to fluctuations in the adhesion. Some spores showed weaker adhesion, and others showed stronger adhesion after the heat treatment (Figure 5d) compared to their original adhesion values. We propose that when the surface layer is dehydrated and denatured without disintegration, the spore surface may have a low adhesion force. However, when the surface layer is broken down by heat, the newly created spore surface may have a high adhesion force. Nanoscratch Method for Imaging Subsurface Materials of Spores. We developed a nanoscratch AFM method for imaging the subsurface structures of Bacillus anthracis spores, as illustrated in Figure 6. The surface layer was removed by deforming and scratching the surface with a stiff diamond AFM tip. When an appropriate load was applied, small nanoparticles were visible in the plastically deformed surface, as indicated by the arrow in Figure 6b. The observed nanoparticles appeared to be located beneath the surface layer. We measured the thickness of the surface layer, which we have tentatively identified as the exosporium, at three different steps between the covered and the uncovered spore surfaces, as indicated by the dotted arrows (1−3) in Figure 6c. The average step height

comparison. The adhesion force was averaged over an image area of 8 μm × 8 μm for every 8 min time interval, which was equivalent to a 25 °C temperature interval at different locations that were randomly selected in the monolayer of spores. It is seen in Figure 4 that the adhesion force measured at room temperature in a nitrogen-purged environment for nine different locations varied between 5 and 11 nN. When the temperature was increased from 20 to 350 °C at a constant rate of 1 °C/s, the general trend was for the adhesion to decrease with increasing temperature with occasional small fluctuations mostly between 100 and 170 °C. As the spores were quickly cooled from 350 to 20 °C at a rate of 1000 °C/s, the adhesion increased to different values, ranging from 5 to 11 nN, in about 20 min. Although the average value of the adhesion before and after the heat treatment was nearly the same, the adhesion for each individual temperature ramp varied remarkably, as seen in Figure 4a,b. For example, the percentage decrease in the adhesion was relatively consistent from sample to sample, reduced by 60% at the minima, during the heating period. However, the percentage change of the adhesion was scattered over a wide range from −40 to +60% during the cooling period. We show for clarity in Figure 4c only 5 representative measurements out of more than 20 adhesion measurements in air. The adhesion decreased with increasing temperature from 20 to 350 °C and increased after the spores were cooled to room temperature. The average value of the adhesion before and after the heat treatment differed by less than 1 nN. The adhesion for each individual temperature ramp showed small variations from sample to sample. The observed thermal effect 8350

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Figure 8. Thermal effect on the substructures of an autoclaved Bacillus anthracis spore coat at elevated temperatures: (a) height and (c) peak-force error image at 20 °C and (b) height and (d) peak-force error image at 275 °C.

The structural identifications of the exosporium, coat, cortex, and core of the spores are indicated by the black, blue, red, and white arrows, respectively. The number next to the arrow shows the height of the corresponding component. The exosporium and coat of the spores are distinct in their shape and thickness. The thickness of the exosporium we measured for six different samples varied from 10 to 15 nm. The thickness of the coat measured for more than 10 different autoclaved spores varied from 20 to 125 nm. The thickness of the cortex, which surrounds the core of about 500 nm in diameter, was about 200−300 nm. We noted that the core, which is expected to be fully hydrated during the autoclaving process, was susceptible to heat assault, melting away when the tip temperature was increased to 150 °C, as indicated in Figure 7b,e. The enlarged views of the coat (Figure 7c,f) show that the ridge structures in the coat were formed by small rods of 30 nm diameter and 160 nm length. These rods were arranged perpendicular to the ridge, which mostly runs along the long axis of the ellipsoid coat. Thermal AFM images of an autoclaved spore coat with ribbon structures are shown in Figure 8. When the coat was heated to elevated temperatures, the overall size of the spore structures showed few changes, but the dimensions of the substructures decreased significantly. For example, the central ridge of 160 nm in width broke into several smaller segments of about 40 nm in width. The overall height of the coat was nearly constant. The width of the ribbons decreased from 60 to 17 nm, and the height of the ribbons showed a smaller decrease from 8 to 5 nm. The ridge structures appear to be formed by folding the ribbon in a zigzag accordion fashion. The width of the ridges is determined by the folding distance or pitch. The folded ribbon forms a thick cable, which is then coiled into

was about 12 nm, consistent with our other measurements (10−15 nm) and the thickness reported in the literature.45,46 We noted that the closely packed spherical nanoparticles were about 40 nm in diameter and were arranged into a highly ordered, layered, concentric structure. The modulus of the nanoparticles was about one-fourth of the value for the original surface layer. The adhesion of the nanoparticles (about 10 nN) was larger than that for the original surface layer of the spores (about 7 nN). It is known from thin-sectioning images of transmission electron microscopy (TEM)2,3,47,48 that the outer coat of Bacillus spores is composed of multilayer fine structures, which are different from the closely packed spherical nanoparticles that we have observed. However, the nanoparticles we showed in Figure 6 resemble the nanostructures reported by Hirota et al.49 Using TEM and scanning transmission electron microscopy (STEM), they showed that the outer coat of a Bacillus cereus spore was covered by unidentified nanometer particles (denoted by SX). We speculate that these nanoparticles may be related to the unidentified interspace materials that populate the area between the coat and the exosporium of the spores. Thermal Effects on Component Structures of Bacillus anthracis Spores. To study the thermal effect on component structures of spores such as the coat and cortex, we broke down the spores using a standard autoclave liquid cycle. The autoclaved spores largely lost their core materials, which is consistent with the SEM observation of bacterial endospores by Perkins et al.50 Figure 7 shows the AFM images of a small cluster of broken spores. We tentatively identify the exosporium, coat, and cortex components of the spores on the basis of the known information about their size, shape, and morphology. The assignment of the core material is speculative. 8351

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Figure 9. AFM images of the thermal effect on the ridge structures of two different spore coats. (a) Height image at 20 °C. (b) Height image at 150 °C. (c) Peak-force error image at 20 °C. (d) Peak-force error image at 325 °C.

changes on nanometer scales at high temperatures. Thermal blisterlike, persistent nanostructures are formed on the spore surface when it is heated with a thermal tip. The nanostructures show a lower adhesion and higher modulus than the surrounding spore surface. The general trend is for the adhesion to decrease with increasing temperature. Upon cooling to room temperature, the heat-treated spores can show a smaller, equal, or larger adhesion force than can the unheated spores, depending upon the degree of thermal damage to the spore surface. Although the overall dimensions of the spore show few changes, the size of the substructures decreases remarkably at high temperatures. We have demonstrated a nanoscratch AFM method for imaging the subsurface structures of spores.

concentric, circular, or elliptical rings to produce the coat structure. With increasing temperature, the length of the cable shows no change, but the width of the cable decreases dramatically. It is worth mentioning that this zigzag folding architecture of linear chains allows the spore to expand or contract with a simple push or pull in the axial direction, depending on the environmental conditions. More details of the effect of heat on the substructures of the autoclaved spores are shown in Figure 9. The nanorods, building blocks of the ridge structures in the coat, broke into two to three smaller spherical particles at about 150 °C. The spherical particles shrank and deformed at a higher temperature of 275 °C. When the temperature was increased to 325 °C, most of the spherical particles were damaged. We observed a similar thermal effect on other autoclaved spores, as shown in Figure 9c,d. The observed thermal effect on the coat structure is qualitatively reproducible. It is clear that one of the most striking effects of heat on the coat structure is to reduce and degrade the substructures of the spores. The overall structure and shape of the coat appear to be relatively strong and stable to moderate heat assault. The substructures are relatively weak and susceptible to heat assault. Therefore, the observed thermal effects on the surface structures and properties of spores are expected to have a major impact on their lethality and survivability.



ASSOCIATED CONTENT

S Supporting Information *

AFM images of Bacillus anthracis spores. This material is available free of charge via the Internet at http://pubs.acs.org.



AUTHOR INFORMATION

Corresponding Author

*E-mail: alex.li@afit.edu.



Author Contributions

CONCLUSIONS We performed a real-time characterization of thermal effects on the surface structures and nanomechanical properties of Bacillus anthracis spores at elevated temperatures using thermal atomic force microscopy. Our study has shown that the surface structures and properties of the spores undergo remarkable

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest. 8352

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ACKNOWLEDGMENTS The views expressed in this report are those of the authors and do not reflect the official position of the United States Air Force, Department of Defense, or U.S. Government. This research was partially sponsored by the Defense Threat Reduction Agency (DTRA) in a program managed by Dr. Su Peiris.



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