The Physics of Fungi Michael Buck

1. The Physics of Fungi Michael Buck Student Collaborators Carolyne Gikunju Julia Butler Liming Zhao Faculty Mentors Dr. David Schaefer Dr. Steven Lev Dr. Gail Gasparich Dr. Mark Marten

2. Outline • What is Nanotechnology? • 2. Studies of Fungi • Adhesive Properties • - AFM Removal Measurements • - Flow Cell Experiments • Mechanical Properties • 3. Conclusions

3. What is Nanotechnology? Cat ~ 0.3 m 100 m 1 meter 10-1 m Head of Pin 1-2 mm 0.1 m (100 mm) The Macroworld Bee ~ 15 mm 10-2 m 0.01 m (10 mm) 10-3 m 1 millimeter Human Hair ~ 50 m 10-4 m 0.1 mm (100 m) 10-5 m 0.01 mm (10 m) The Microworld Red and White Blood Cells ~ 2-5 m 10-6 m Microelectromechanical Systems (MEMS) 10 – 100 m 1 micrometer Visible Spectrum 10-7 m 0.1 m (100 nm) 10-8 m 0.01 m (10 nm) Indium Arsenide Quantum Dot ~ 10 nm The Nanoworld 10-9 m 1 nanometer DNA ~ 2 nm wide 10-10 m 0.1 nm Atoms of Materials ~ 0.1 – 0.5 nm

4. Scanning Probe Microscopy Derivative Microscopes Kelvin Probe Capacitance Magnetic Force Friction Force Electrochemical I. Scanning Tunneling Microscope II Atomic Force Microscope General Operation of SPMs

5. Position Sensitive Detector Computer Control (Feedback) Cantilever ATOMIC FORCE MICROSCOPE DESIGN Diode Laser Sample AFM Image

6. Anatomy of a Force Curve Approach Withdrawal Approaching surface Cantilever at neutral deflection Field forces pull cantilever down Adhesion pulls tip downward Cantilever Deflection Tip breaks free at force of removal Cantilever at neutral deflection Piezo Position Cantilever at maximum deflection Withdrawal from surface piezo up piezo down

7. AFM Studies of Fungi Purpose:to build a better understanding of morphological development, physiology and physical properties of fungi. - Study of the interaction of fungi with substrates - Measurement of the mechanical properties of fungi Why do we care about Fungi? • medically (pathogenic fungi account for greater than 40% of all deaths in US hospital-acquired infections, • agriculturally (fungal pathogens account for millions of dollars in crop damage annually, while other species can be used as ecological biocontrol agents or “green pesticides”) • industrially (filamentous fungi are used to produce both pharmaceutical and specialty chemicals worth billions of dollars annually).

8. Crop Damage Inhibitor Fungicide Our Approach Enzyme Attachment • Mount fungi onto an AFM cantilever • Bring it into contact with a surface. • Use the AFM to determine the removal force. • Repeat this for varying contact times. Surface Forces Germination Mapping of Germination Process C Pull – Off Force ( a.u. ) B A Contact Time ( a.u ) Adhesion Measurements of Fungal Spores 2 Ways to combat fungal crop damage

9. Particle Attachment Techniques to Tipless Cantilevers CCD Microscope Light Sources 3-axis Translator Piezo Micromanipulator Slide with Glue and Spores First Glue, then Spore Mounted Particle

10. Results of Pull – Off Forces for Spores Dry: (106 ± 27.4) nN Wet: (152 ± 50.9) nN Sonicated: (300 ± 136) nN Surface morphology of Aspergillis nidulans spores. Shown are dry (A), wet (B), and sonicated (C) surface conditions. The inset of the dry spore shows a close-up of the structure of the protein layer. Bar length is 0.2μm. A B C A typical force curve set

11. Measurement of Shear Forces – Flow Cell

12. Flow Cell Apparatus

13. Purpose:to build a better understanding of morphological development, physiology and physical properties of fungi. - Measurement of the mechanical properties of fungi - Study of the interaction of fungi with substrates Why do we care about Fungi? • medically (pathogenic fungi account for greater than 40% of all deaths in US hospital-acquired infections, • agriculturally (fungal pathogens account for millions of dollars in crop damage annually, while other species can be used as ecological biocontrol agents or “green pesticides”) • industrially (filamentous fungi are used to produce both pharmaceutical and specialty chemicals worth billions of dollars annually). AFM Studies of Fungi

14. Why Study the Mechanical Properties of Fungi? Fungi are used in the bioprocessing industry to produce industrial enzymes (sales approaching $1 billion annually). Enzymes are specialty chemicals, and are used in products ranging from laundry detergent to baked goods. Agitator Oxygen The high viscosity of the broth reduces the amount of oxygen that can be supplied, and reduces the enzyme production. Collaborators at UMBC have developed an techniques to improve enzyme output (75%). they believe their method produces weak places in the fungi, allowing it to break apart during the agitation. This reduces the viscosity. Highly Viscous Broth Fermentor Enzymes AFM is being used to test this hypothesis.

15. Why Study the Mechanical Properties of Fungi Chitin Fibrils a-Glucan and Protein Outside Cell b-Glucan Cell Wall Periplasmic Space Membrane Cytoplasmic Space Inside Cell The cell wall is a composite with exceptional strength Therefore, we would like to use the AFM to study the strength of fungal cell walls.

16. Elastic Modulous Spore Treatment Force Indentation Method Contact Stiffness Method “Dry” 5.1 ±1.9 Gpa n = 50 5.0 ±1.9 Gpa n = 50 “Sonicated” 10.5 ± 4.3 Gpa n = 45 10.1 ± 3.6 Gpa n = 45 Results of Indentation Measurements Similar results are obtained on both filamentous fungi and fungal spores. Measurements under Dry Conditions Measurements under liquid

17. Conclusions • The AFM has been successfully used to measure mechanical • adhesion properties of fungi. • Flow cell experiments have begun, incorporating an ICPMS as • a novel detection apparatus. Future Plans • AFM experiment will be expanded to study effects of environmental • conditions, substrate types, and other fungal spore types. • A custom built force measurement apparatus is nearing completion • to allow us greater control over experimental conditions. • Flow cell experiments will be expanded to study other biological • systems