1.14th International Biotechnology Symposium and Exhibition 2010 Rimini, Italy, September 15 th2010 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 GussemeVirus inactivation by biogenic silverIBS 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 GussemeVirus inactivation by biogenic silverIBS 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 broadantimicrobial 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 GussemeVirus inactivation by biogenic silverIBS 2010, RImini, September 15th, 2010 6. Biogenic silver nanoparticles 0,5 m 1,5 m Biogenic metalic silver nanoparticles (bio-Ag 0 ) Reduction of Ag +on bacterial cell surface of deadLactobacillussp 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 nanoparticleswell dispersed and prevents them from aggregation

• • (Hennebel et al., 2009; Trends Biotechnol., 27, 90-98; LabMET)

Bart De GussemeVirus inactivation by biogenic silverIBS 2010, RImini, September 15th, 2010 Bio-Ag 0= nanoscale Ag 0on a microscale bacterial carrier 8. Model organisms

Bacteriophage UZ1 ofEnterobacter aerogenes

• DNA-phages of enteric bacteria as a model forenteric viruses(Verth 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 GussemeVirus inactivation by biogenic silverIBS 2010, RImini, September 15th, 2010 9. Disinfection assay on UZ1 (Contaminated drinking water, C 0= 6.0 x 10 6pfu mL -1 ) Bart De GussemeVirus inactivation by biogenic silverIBS 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 6pfu mL -1 , 8.9 x 10 8gc mL -1 ) Bart De GussemeVirus inactivation by biogenic silverIBS 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 0L -1and 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 GussemeVirus inactivation by biogenic silverIBS 2010, RImini, September 15th, 2010 Most probable inactivation mechanism: Interaction of Ag 0with (thiol groups of the) protein capsid, preventing the virus from docking onto his host cell 12. Challenge

Growing concern about the loss of nanoparticlesin 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 GussemeVirus inactivation by biogenic silverIBS 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 m pore size)

• 35 wt% alumina (AlOOH) nanofibers : positively charged

Coated with Bio-Ag 0(31 mg Ag 0m -2filter 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 GussemeVirus inactivation by biogenic silverIBS 2010, RImini, September 15th, 2010 14. Continuous virus removal Bart De GussemeVirus inactivation by biogenic silverIBS 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 ofBio-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 0onto electropositive cartridge filter

• 31 mg Bio-Ag 0m -2filter area enhances virus inactivation (+ 2 log)

• Advantage of bacterial carrier for anchoring of silver nanoparticles

Bart De GussemeVirus inactivation by biogenic silverIBS 2010, RImini, September 15th, 2010 16. Future perspectives

Household disinfection of drinking water

Bart De GussemeVirus inactivation by biogenic silverIBS 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 GussemeVirus inactivation by biogenic silverIBS 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 theFund 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 GussemeVirus inactivation by biogenic silverIBS 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 Contaminatedwith Viruses.

Applied and Environmental Microbiology ,76(4), 1082-1087.

[email_address] LabMET.UGent.be