Fabrication and characterization of anti-microbial and biofouling resistant nanofibers with silver nanoparticles and immobilized enzymes for application in water filtration

Du Plessis, Danielle Marguerite (2011-03)

Thesis (MSc (Biochemistry))--University of Stellenbosch, 2011.

Thesis

ENGLISH ABSTRACT: Due to a global lack of access to potable water, a problem particularly affecting people in developing countries and the poor, improvement on existing water purification methods are necessary to provide more cost effective, accessible and efficient methods of water purification. In drinking water systems, biofilms are a potential source of contamination, which can affect the biological stability and hygienic safety of water. In industrial water systems, biofilms can cause corrosion, resistance in flow systems and a decrease in efficiency of membranes. Nanotechnology has been identified as a technology to utilize in water purification problem solving. Alternatives to the use of chemical biocides and antibiotics need to be investigated therefore; the focus of this study was the fabrication and characterization of polymer nanofibers containing silver nanoparticles as biocide and anti-biofouling nanofibers with hydrolytic enzymes immobilized on the surface. The aim of this study was to synthesize and compare poly (vinyl alcohol) (PVA) nanofibers and poly (acrylonitrile) (PAN) nanofibers with silver nanoparticles to determine which type of fiber will be the most appropriate for application in water sanitation. The two types of fibers were to be compared based on morphology, silver nanoparticle content, physical distribution of silver nanoparticles, levels of silver leaching from the fibers in water, which could imply toxicity, and most importantly, anti-microbial efficacy. Back scattering electron images revealed that silver nanoparticles in PVA nanofibers were more evenly dispersed than in PAN nanofibers, but that PAN nanofibers had higher silver nanoparticle content. This was confirmed by energy dispersive X-ray (EDX) analysis. Both PVA and PAN nanofibers containing silver nanoparticles had excellent anti-microbial activity, with PVA nanofibers killing between 91% and 99% of bacteria in a contaminated water sample and PAN nanofibers killed 100%. When investigated by SEM, the biocidal effect of PAN nanofibers containing silver nanoparticles can be observed as morphological changes in the cell walls. Neither PVA nor PAN nanofibers leached silver into water. PVA is a non-toxic and biodegradable synthetic polymer, and PVA-silver nanofibers have excellent anti-microbial activity, making it applicable in water sanitation in an environmental conscious milieu. PAN nanofibers are more conductive to the formation of silver nanoparticles, have higher silver nanoparticle content, allowing the complete sanitation of pathogenically contaminated water samples. PAN nanofibers also have better longevity and strength in water, making it ideal for water filtration and sanitation in higher throughput systems. Furthermore, immobilized enzymes are being investigated as possible alternatives to inefficient conventional methods of controlling and removing biofilms from filtration systems. This study demonstrates the covalent immobilization of two industrial proteases and an amylase enzyme onto polymer nanofibers widely used in filtration membranes. Confirmed by FTIR, these nanofibers were successfully activated by amidination, allowing the covalent immobilization of respectively two serine proteases and an α-amylase onto the fibers. When inspected visually, fibers largely retained their original morphology after activation and enzyme immobilization. Immobilized enzymes were, however visible as aggregated particles on the nanofiber surfaces. The large surface area to volume ratio provided by the nanofibers as immobilization surface, allowed sufficient amounts of enzymes to be immobilized onto the fibers so that all enzymes retained above 80% of the specific activity of the free enzymes. For each of the immobilized enzymes, just below 30% of initial activity was retained after 10 repeated cycles of use. Fibers with immobilized enzymes on their surface did not support the growth of biofilms, as opposed to plain nanofibers, which did support the growth of biofilms. When considering the combined advantages of this effective immobilization process, the robustness of the enzymes used in this study, and their effectiveness against biofilms in their immobilized state, a valuable addition has been made to technology available for the control of biofilm formation on filtration membranes, and could potentially be employed to control biofilm formation in water filtration systems. A combination of anti-microbial and anti-biofouling nanofibers into a single nanofiltration product may prove to be highly applicable in water sanitation systems.

AFRIKAANSE OPSOMMING: As gevolg van 'n wêreldwye gebrek aan toegang tot drinkbare water, 'n probleem wat veral mense in ontwikkelende lande en armes raak, is dit van belang dat bestaande metodes van watersuiwering verbeter word om voorsiening te maak vir meer koste-effektiewe, toeganklike en doeltreffende metodes van watersuiwering. In drinkwater stelsels is biofilms 'n potensiële bron van besoedeling, wat die biologiese stabiliteit en die higiëniese veiligheid van water beïnvloed. In industriële waterstelsels kan biofilms tot die verwering van pyplyne lei, weerstand in die stroomstelsels veroorsaak en 'n afname in die doeltreffendheid van membrane veroorsaak. Nanotegnologie is geïdentifiseer as 'n tegnologie wat aangewend kan word in watersuiwerings probleemoplossing. Alternatiewe vir die gebruik van chemiese antimikrobiese middels moet dus ondersoek word. Hierdie studie fokus dus op die vervaardiging en karakterisering van polimeer nanovesels met silwer nanopartikels wat ingesluit is as antimikrobiese middel en anti-biofilm vesels met hidrolitiese ensieme geïmmobiliseer op die oppervlak. Die doel van hierdie studie was om poli (viniel alkohol) (PVA) nanovesels en poli (akrielonitriel) (PAN) nanovesels te sintetiseer waarby silwer nanopartikels ingesluit is, en te bepaal watter tipe vesel die mees geskikte sal wees vir die gebruik in water sanitasie. Die twee tipes vesels is met mekaar vergelyk gebaseer op morfologie, silwer nanopartikel inhoud, fisiese verspreiding van silwer nanopartikels, vlakke van silwer uitloging vanuit die vesels in water, wat toksisiteit tot gevolg kan hê, en die belangrikste, antimikrobiese effektiwiteit. Terug verstrooiing elektron beelde het aan die lig gebring dat die silwer nanopartikels in PVA nanovesels meer eweredig versprei was as in PAN nanovesels, maar dat PAN nanovesels 'n hoër silwer nanopartikel inhoud gehad het. Dit is bevestig deur “energy dispersive X-ray” (EDX) analise. Beide PVA en PAN nanovesels met silwer nanopartikels het uitstekende antimikrobiese aktiwiteit getoon, met PVA vesels wat tussen 91% en 99% bakterieë in besoedelde water monsters kon doodmaak en PAN vesels wat 100% bakterieë kon uitwis. Wanneer vesels ondersoek is met ʼn skandeer elektronmikroskoop (SEM), kon die antimikrobiese effek van PAN vesels met silwer nanopartikels as morfologiese veranderinge in die selwande waargeneem word. Nie PVA of PAN nanovesels loog silwer uit in water nie. PVA is 'n nie-toksiese en bioafbreekbare sintetiese polimeer, en PVA-silwer nanovesels het uitstekende antimikrobiese aktiwiteit, wat dit van toepassing maak op water sanitasie in ʼn omgewings bewuste milieu. PAN vesels is meer gunstig tot die vorming van silwer nanopartikels, en het 'n hoër silwer nanopartikel inhoud, dus word patogeen besoedelde water volledig gesteriliseer. PAN vesels het ook 'n beter langslewendheid en weerstandige sterkte in water, wat dit ideaal vir water filtrasie en sanitasie in hoër deursettings stelsels maak. Geïmmobiliseerde ensieme word ook ondersoek as moontlike alternatiewe tot ondoeltreffende konvensionele metodes van beheer en die verwydering van biofilms uit water stelsels. Hierdie studie toon die kovalente immobilisasie van twee industriële proteases en 'n amilase ensiem op polimeer vesels wat gebruik word in filtrasie membrane. Bevestig deur FTIR, is PAN vesels suksesvol geaktiveer deur amidinasie, sodat die kovalente immobilisasie van onderskeidelik twee serien proteases en 'n α-amilase op die vesels moontlik is. Met visuele ondersoek kan gesien word die vesels behou grootliks hul oorspronklike morfologie na aktivering en ensiem immobilisasie. Geïmmobiliseerde ensieme is egter sigbaar as saamgevoegde deeltjies op die nanovesel oppervlaktes. Die groot oppervlakarea: volume-ratio van die vesels wat dien as immobilisasie oppervlak, laat toe dat voldoende hoeveelhede van ensieme geïmmobiliseer word sodat alle ensieme meer as 80% van die spesifieke aktiwiteit van die vrye ensieme behou. Vir elk van die geïmmobiliseer ensieme, is net minder as 30% van die aanvanklike aktiwiteit behou na 10 siklusse van hergebruik. Vesels met geïmmobiliseerde ensieme op hul oppervlaktes het nie die groei van biofilms ondersteun nie, in teenstelling met gewone vesels, sonder ensieme, wat die groei van biofilms ondersteun. As die gesamentlike voordele van hierdie doeltreffende immobilisasie proses, die robuustheid van die ensieme en hulle doeltreffendheid teen biofilms in hul geïmmobiliseerde toestand in ag geneem word, is ʼn waardevolle toevoeging gemaak tot tegnologie wat beskikbaar is vir die beheer van biofilm vorming op filtrasie membrane, en dit kan potensieel gebruik word om biofilm vorming filter stelsels te beheer. Die kombinasie van anti-mikrobiese en anti-biofilm vesels in ʼn enkele nanofiltrasie produk moet nagestreef word, omdat dit hoogs van toepassing sal wees in water sterilisasie stelsels.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/6580
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