Virus Inactivation By Biogenic Silver

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  • 1. 14th International Biotechnology Symposium and Exhibition 2010 Rimini, Italy, September 15 th 2010 Bart De Gusseme L. Sintubin, L. Baert, T. Hennebel, M. Uyttendaele, W. Verstraete and N. Boon Laboratory of Microbial Ecology & Technology, Ghent University, Belgium
    • Virus Inactivation
    • by
    • Biogenic Silver
  • 2. Bart De Gusseme Virus inactivation by biogenic silver IBS 2010, RImini, September 15th, 2010
  • 3.
    • Viruses are important causative agents for waterborne diseases (WHO, 2003; Emerging issues in water and infectious disease )
    • More and more unknown viral diseases are discovered by novel molecular techniques
    • Endemic in developing countries (e.g. Hepatitis)
    • Acute outbreaks in
    • industrial countries (e.g. Norovirus)
    • (Boccia et al., 2002; Emerg. Infect. Dis., 8, 563-568)
    Introduction: waterborne diseases
  • 4.
    • Conventional drinking water treatment:
      • Coagulation/Flocculation
        • Physical removal
        • Virucidal activity of Al and Fe coagulants
      • Disinfection
        • Chlorine
          • Formation of byproducts
          • Aggregation: pretreatment necessary
        • Ozone: effective but formation of byproducts
        • UV: not effective against Adenovirus
        • (Krasner et al., 2006; ES&T, 40, 7175-7185 ;
        • Shannon et al., 2008; Nature Reviews, 452, 301-310)
    Conventional water disinfection Need for alternative techniques for drinking water disinfection Bart De Gusseme Virus inactivation by biogenic silver IBS 2010, RImini, September 15th, 2010
  • 5. Nanoscale silver as disinfectant
    • Recent interest in nanotechnology for water disinfection (Li et al., 2008; Wat. Res., 42, 4591-4602)
    • Silver containing nanoparticles have a broad antimicrobial spectrum
    • Chemically produced silver nanoparticles against
      • HIV-1
      • (Elechiguerra et al., 2005; J. Nanobiotech., 3, 6)
      • Monkeypox
      • (Rogers et al., 2008; Nanoscale Res. Lett., 3, 129-133)
    Bart De Gusseme Virus inactivation by biogenic silver IBS 2010, RImini, September 15th, 2010
  • 6. Biogenic silver nanoparticles 0,5 µm 1,5 µm Biogenic metalic silver nanoparticles (bio-Ag 0 ) Reduction of Ag + on bacterial cell surface of dead Lactobacillus sp pH = 12 Ag + Ag + Ag + Ag + Ag + Ag +
  • 7. Biogenic silver nanoparticles
    • Lactobacillus fermentum
      • Safe bacterium (probiotic)
      • Production of EPS: adsorption of metal ions
      • Presence of reducing sugars
    • Advantages of biogenic production of silver nanoparticles
      • Green synthesis
      • Narrow size distribution: 11.2 ± 0.9 nm
        • (Sintubin et al., 2009; AMB, 84, 741-749; LabMET)
      • Bacterial cell surface keeps the nanoparticles well dispersed and prevents them from aggregation
        • (Hennebel et al., 2009; Trends Biotechnol., 27, 90-98; LabMET)
    Bart De Gusseme Virus inactivation by biogenic silver IBS 2010, RImini, September 15th, 2010 Bio-Ag 0 = nanoscale Ag 0 on a microscale bacterial carrier
  • 8. Model organisms
    • Bacteriophage UZ1 of Enterobacter aerogenes
      • DNA-phages of enteric bacteria as a model for enteric viruses (Verthé et al., 2004; AMB, 65, 465-472; LabMET)
      • Resistant to normal chlorination
      • Detection: plaque assays
    • Murine NoroVirus-1 (mice)
      • Closely related to Human NoroVirus (RNA)
      • Detection: real-time RT-PCR & cell culture (Baert et al., 2008; AEM, 74, 543-546)
    Bart De Gusseme Virus inactivation by biogenic silver IBS 2010, RImini, September 15th, 2010
  • 9. Disinfection assay on UZ1 (Contaminated drinking water, C 0 = 6.0 x 10 6 pfu mL -1 ) Bart De Gusseme Virus inactivation by biogenic silver IBS 2010, RImini, September 15th, 2010 > 4.2 log inactivation after 3 h with addition of Bio-Ag 0 No sorption to biomass, slightly faster than Ag + LOD
  • 10. Disinfection assay on MNV-1 (C 0 = 1.1 x 10 6 pfu mL -1 , 8.9 x 10 8 gc mL -1 ) Bart De Gusseme Virus inactivation by biogenic silver IBS 2010, RImini, September 15th, 2010 > 4.7 log inactivation after 30 min with addition of Bio-Ag 0 No removal detected with real-time RT-PCR : RNA intact LOD non-infectious RNA still intact
  • 11. Inactivation mechanism (MNV-1)
    • After addition of Bio-Ag 0
      • virus is not infective anymore (see plaque assays)
      • yet, free RNA is present (see real-time RT-PCR)
    • Similar disinfection assay with 5.4 mg Bio-Ag 0 L -1 and MNV-1
      • Addition of RNase A, prior to RNA isolation
      • free RNA is sensitive for RNase A activity
      • Result: 2.5 log decrease after 24 h
    Bart De Gusseme Virus inactivation by biogenic silver IBS 2010, RImini, September 15th, 2010 Most probable inactivation mechanism: Interaction of Ag 0 with (thiol groups of the) protein capsid, preventing the virus from docking onto his host cell
  • 12. Challenge
    • Growing concern about the loss of nanoparticles in the environment
      • Cost
      • Potential impacts of nanomaterials on human health and ecosystems
    • (Benn and Westerhoff, 2008; ES&T, 42, 4133-4139;
    • European Parliament, Cosmetics resolution 76/768/EEC, March 24, 2009)
    Bart De Gusseme Virus inactivation by biogenic silver IBS 2010, RImini, September 15th, 2010
    • Need for efficient anchoring of nanoparticles
    • Great advantage of bacterial carrier matrix
  • 13. NanoCeram ® filter
    • Electropositive cartridge filter (Argonide, Stanford, FL)
      • microglass fiber matrix (0.6 μm pore size)
      • 35 wt% alumina (AlOOH) nanofibers : positively charged
    • Coated with Bio-Ag 0 (31 mg Ag 0 m -2 filter area)
      • Bacterial carrier negatively charged (van Hullebusch et al., 2003; Rev. Env. Sci. Biotech., 2, 9-33)
    • Continuous disinfection assay on UZ1
      • Q = 30 L h -1
      • HRT = 2 min
      • NanoCeram with
      • and without Bio-Ag 0
    Bart De Gusseme Virus inactivation by biogenic silver IBS 2010, RImini, September 15th, 2010
  • 14. Continuous virus removal Bart De Gusseme Virus inactivation by biogenic silver IBS 2010, RImini, September 15th, 2010 Only 1.5 log decrease of UZ1 after 10 HRTs without Bio-Ag 0 > 3.5 log decrease with addition of Bio-Ag 0
  • 15. Conclusions
    • Biogenic silver
      • Green synthesis of silver nanoparticles
      • Microscale bacterial carrier binds nanoparticles and keeps them dispersed
    • Virus inactivation demonstrated on two model organisms
      • >4.2 log removal of model for enteric viruses after 3 h (5.4 mg L -1 )
      • >4.7 log decline of model for human norovirus after 30 min (5.4 mg L -1 )
      • Most probably related to damaging of viral protein capsid
    • Coating of Bio-Ag 0 onto electropositive cartridge filter
      • 31 mg Bio-Ag 0 m -2 filter area enhances virus inactivation (+ 2 log)
      • Advantage of bacterial carrier for anchoring of silver nanoparticles
    Bart De Gusseme Virus inactivation by biogenic silver IBS 2010, RImini, September 15th, 2010
  • 16. Future perspectives
    • Household disinfection of drinking water
    Bart De Gusseme Virus inactivation by biogenic silver IBS 2010, RImini, September 15th, 2010 Bio-Ag 0 Bio- Ag 0
  • 17. Future perspectives
    • Disinfection of process water for water reuse
      • e.g. food industry, aquaculture, hospitals,… : Specific niches!
      • Immobilization in polymeric membranes for safe use of silver nanoparticles:
      • (De Gusseme et al., 2010; Water Research, submitted ; LabMET)
    Bart De Gusseme Virus inactivation by biogenic silver IBS 2010, RImini, September 15th, 2010
  • 18. Acknowledgements Bart De Gusseme (Aspirant), Tom Hennebel (project no. 7741-02) and Leen Baert (Postdoctoral fellow) are supported by the Fund of Scientific Research (FWO)-Flanders. Liesje Sintubin is supported by a project grant (no. 71333) of the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT). We gratefully thank Lieve Herman and Els Van Coillie of the Institute for Agricultural and Fisheries Research, Technology and Food Unit (Belgium) for the ability to use their laboratory facilities. Bart De Gusseme Virus inactivation by biogenic silver IBS 2010, RImini, September 15th, 2010
  • 19. B. De Gusseme, L. Sintubin, L. Baert, E. Thibo, T. Hennebel, G. Vermeulen, M. Uyttendaele, W. Verstraete & N. Boon
    • Biogenic Silver for Disinfection of Water Contaminated with Viruses.
    • Applied and Environmental Microbiology , 76(4), 1082-1087.
    [email_address] LabMET.UGent.be