Removal of indoor bacteria by using the chitosan titania nano metals composite materials

THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY NGAN THI THU UYEN REMOVAL OF INDOOR BACTERIA BY USING THE CHITOSAN-TITANIA-NANO-METALS COMPOSITE MATERIALS BACHELOR THESIS Study Mode : Full-time Major : Environmental Science And Management Faculty : International Programs Office Batch : 2013 – 2017 Thai Nguyen, September 2017 Thai Nguyen University of Agriculture and Forestry Degree Program Bachelor of Environmental Science and Management Student name NGAN THI THU UYEN Student ID DTN1353140024 Removal of indoor bacteria by using the chitosan-titania- Thesis title nano-metals composite materials Assoc Prof Kuo-Pin Yu Supervisor(s) Ph.D Nguyen Huu Tho Abstract: Recently, antimicrobial efficacy of nano-metals has been extensively investigated Nano-structured materials are attracting a great deal of attention because of their potential for achieving specific processes and selectivity, especially in biological and pharmaceutical applications Nanotechnology has become one of the most practical technologies, because of unique physical and chemical properties of nanomaterials This study has developed chitosan-TiO2-nano-metals composite materials and apply these composite materials on the removal of indoor air pollutants bacteria The nano-metal composite (Cs, TiO2, and Ag) were successfully prepared in the form of AgNPs/Cs-TiO2 micro-beads by mixing of chitosan and TiO2 powder treated with the cross-linking agent to form the chitosan-TiO2 composite materials, while silver nanoparticles supported by chitosan/TiO2 composites (AgNPs/CS-TiO2) The antibacterial experiments of Escherichia coli ( Gram negative) and Staphylococcus epidermidis (Gram-positive) were conducted on surfaces agar plates in the darkness into incubated at 37ºC through two methods: zone of inhibition assay (ZOI) and inactivation test The critical concentration of Ag to inhibit the germination and growth of Escherichia coli and Staphylococcus epidermidis of wt% nano-Ag catalyst were 4.32(µg) E coli and 4.73(µg) S.epidermidis, respectively lower than silver nitrate (AgNO3) (E coli was 44.27(µg/ml), and S.epidermidis was 70.94(µg/ml), respectively) from ZOI assay The time profiles of the survival ratio of E coli and S.epidermidisr educe on the surface of catalysts and quartz chip, testing in the darkness in the different time In addition, the initial survival fraction (SFi) of Escherichia coli and Staphylococcus epidermidis were (SfiE.coli= 0.4662; SfiS.epidermidis= 0.11079 ) on the surface of catalysts and quartz chip in the darkness after 8h Based on this study, nanoCs/TiO2 and -Ag has the efficient antibacterial effect and can be used as an antibacterial agent for the indoor environment Keywords Chitosan, TiO2, AgNPs, nanomaterials, nanoparticles, Escherichia coli, Staphylococcus epidermidi Number of pages: 42 Date of submission: 10 th Oct, 2017 ACKNOWLEDGEMENT Firstly, I would like to say thanks to the cooperation between Thai Nguyen University of Agriculture and Forestry and National Yang-Ming University for providing me an amazing opportunity to internship in Taiwan It brings me great pleasure to work and submit my thesis for graduation I would like to express my sincere gratitude and deep regards to my supervisor, Associate Professor Kuo-pin Yu who guided me wholeheartedly when I implemented this research and for the supervision, encouragement, advice, and guidance in writing this thesis I sincerely thanks to Ph.D.Nguyen Huu Tho for her advice, assistance, sharing experiences before and after I went to Taiwan, helping me to understand and complete proposal and thesis Special thanks to Ms.Wan-Tien Shen, and Ms.Yen-Chi Chen and other staffs in Laboratory of National Yang-Ming University and for helping and providing me necessary equipment as well as knowledge for analysis of bacteria samples, without them, this research could not be accomplished on time Finally, I also thank my family and dear friends for their love and moral support throughout my study Thai Nguyen, August 2017 NGAN THI THU UYEN i TABLE OF CONTENTS LIST OF FIGURES LIST OF TABLES LIST OF ABBREVIATIONS PART I INTRODUCTION 1.1 Research rationale 1.2 Research’s objectives .7 1.3 Research questions 1.4 Limitations PART II LITERATURE REVIEW 2.1 Bacteria .8 2.1.1 Definition of Escherichia coli and Staphylococcus epidermidis 2.1.2 Effecting of Escherichia coli and Staphylococcus epidermidis to indoor environments 11 2.2 Overview of chitosan (Cs)-titanium dioxide (TiO2) -Silver (Ag) 12 2.2.1 Chitosan structures and properties .12 2.2.2 Titanium oxidation structures and properties .13 2.2.3 The characteristics of Silver (Ag) 14 2.2.4 Nanomaterials .15 2.2.5 Nanoparticles (NPs) 16 ii 2.2.6 Silver nanoparticles (AgNPs) 18 PART III MATERIALS AND METHODS .20 3.1 Materials 20 3.2 Instruments .21 3.3 Preparation of silver nanoparticles supported chitosan/TiO2 composites (AgNPs/CS-TiO2) 22 3.4 Photochemical reaction for chitosan/TiO2 composites (AgNPs/CS-TiO2) 23 3.5 Zone of inhibition Assay (ZOI ) .24 3.6 Inactivation test 25 PART IV RESULT AND DISCUSSION 28 4.1 Antimicrobial activity of the AgNPs/Cs-TiO2 micro-beads ( 2wt% Ag) 28 4.2 Inactivation Experiment 31 PART V CONCLUSION 36 REFERENCES 37 iii LIST OF FIGURES Figure 2.1: Escherichia coli .9 Figure 2.2: Staphylococcus epidermidis 11 Figure 2.3: Structure of chitin and chitosan 13 Figure 2.4: Crystal structures of rutile, anatase and brookite titanium dioxide 14 Figure 2.5: Antimicrobial mechanism of nano silver 19 Figure 3.1: Some instruments used for this study 21 Figure 3.2: The process of AgNPs composites material .22 Figure 3.3: Photochemical process of AgNPs composites material 24 Figure 3.4: The process of zone of inhibition assay (ZOI) 25 Figure 4.1: Zones of inhibition of Cs and Cs/TiO2 between E coli and 29 Figure 4.2: The zone of inhibition is a function of Ag concentration 30 Figure 4.3: The critical concentration of AgNO3 (μg/ml) and 2wt% Ag beads (μg) between E.coli and S epidermids 31 Figure 4.4: The time profiles of the survival ratio of E.coliand S.epidermidis on the surface of catalysts and quartz chip in the darkness (Dark) 34 LIST OF TABLES Table 4.1: Zone of inhibition of AgNO3, Cs, Cs/TiO2 and 2wt % Ag beads against bacterial in four testing conditions 28 Table 4.2: The initial survival fraction (Sfi) of E coli and S.epidermidis on catalysts under various conditions 33 LIST OF ABBREVIATIONS ROS Reactive oxygen species SSA Specific surface area NPs Nanoparticles AgNPs Silver nanoparticles TSA Trypical soy agar LB Lysogeny broth ZOI Zone of inhibition PART I INTRODUCTION 1.1 Research rationale In recent years, indoor air quality has become a significant public health concern According to the Environmental Protection Agency has reported that indoor air can be to times more polluted than outdoor air It has been reported that a single person inhale’s an average of approximately 10 m3 of air every day (Dacarro et al., 2003)., and spend 80–95% of our lives indoors Human health effects from indoor exposures are suspected to because by the presence of substances, including allergens such as dust mite, cockroach, pet dander, tobacco smoke, volatile organic compounds, and lead A more recent concern has been the realization that many homes have substantial amounts of bacteria (Portnoy et al., 2005) Bacteria can proliferate in the home, school and office environment cause problems include structural damage to the building, aesthetic problems because mold physically destroys the building materials on which it grows The found that Escherichia coli and Staphylococcus epidermidis are the most common bacteria in indoor air, which are the confirmed or presumed causative agents of several infectious diseases, and their components are linked to the development and exacerbation of chronic respiratory illness including asthma, shortness of breath and other symptoms (DeninaHospodsky et al.,2012) Because of Shiga toxin-producing strains of E coli, such as verocytotoxin E coli (VTEC) or enterohemorrhagic E coli (EHEC) are easily infectious It normally starts when the shiga toxin-producing strains of E coli are ingested via contaminated foodstuffs and can lead to a halt in protein synthesis in the affected cells of the body (Große et al., 2014) Besides, the natural environment of PART IV RESULT AND DISCUSSION 4.1 Antimicrobial activity of the AgNPs/Cs-TiO2 micro-beads ( 2wt% Ag) The antimicrobial activity of the AgNPs/Cs-TiO2 micro-beads was evaluated by using the ZOI assay, and the results are summarized in table 4.1 Table 4.1: Zone of inhibition of AgNO3, Cs, Cs/TiO2 and 2wt % Ag beads against bacterial in four testing conditions Name E.coli S epidermidis 44.27 70.94 CS _ _ CS/TiO2 _ _ 4.32 4.73 AgNO3 ( µg/ml) wt% Ag beads (µg) According to the results of ZOI ( table 4.1) and AgNO3 result (Fig 4.2), the antibacterial activities of AgNO3 against E.coli ( Gram-negative) more efficient than against S.epidermidis (Gram-positive) Due to the different cell wall structure between Gram- Negative and Gram-positive bacteria Gram-negative E coli has a lipopolysaccharide The layer at the exterior and a thin layer of peptidoglycan next to the lipopolysaccharide layer LPS is not as rigid as peptidogly can and contains negative charges, which facilitate the attraction of Ag+ ion (Salton & Kim, 1996) In contrast, the gram-positive bacterium, S epidermidis contains multiple peptidoglycan layers Peptidoglycan has a complex structure The linear polysaccharide chains cross-link via short peptides to form a 3-dimensional rigid structure, and peptidoglycan often contains teichoic acids and lipoteichoic acids, which have strong negative charges The 28 lipoteichoic acids may contribute to the sequestration of free Ag+ ions and make it difficult for Ag+ ions to penetrate the rigid cell wall (Egger et al., 2009) This research also tested the antibacterial effectiveness of the Cs and Cs/TiO2, the antimicrobial activity of Cs and Cs/TiO2 have low inhibition to against E.coli and S epidermidis in 18 to 24 hours, and have a little bit of inhibition zone was observed, as show in fig.4.1 (a), (b) Zones of inhibition of Cs (c), (d) Zone of inhibition of Cs /TiO2 Figure 4.1: Zones of inhibition of Cs and Cs/TiO2 between E coli and S epidermidis Accordingly, AgNPs/Cs- TiO2 micro-beads had the highest antimicrobial activities when the Ag wt% values were 2wt% The antibacterial activities of wt% Ag beads were higher than that of 0.5 and 1wt% Ag beads In this study, the results show 2wt% Ag concentration The antibacterial activities of 2wt% Ag are 4.32 E.coli and 4.73 S.epidermidis respectively, and it was lower than the silver nitrate (E.coli 29 was 44.27 and S.epidermidiswas 70.94, respectively) The Cc values of the AgNPs or silver nitrate were obtained from ZOI assays When the concentrations are above the Cc, they completely inhibit microbial growth Thus, a lower Cc indicates better antimicrobial performance,as shown in fig Figure 4.2: The zone of inhibition is a function of Ag concentration Critical concentration is estimated from the y-intercept of the semilog plot of the experimental data (AgNO3 and 2wt% Ag beads ) (a), (b)Inhibition zone of AgNO3 against E.coli and S epidermids ; (c),(d) Inhibition zone of 2wt % Ag beads against E.coli and S epidermids 30 Figure 4.3: The critical concentration of AgNO3 (μg/ml) and 2wt% Ag beads (μg) between E.coli and S epidermids 4.2 Inactivation Experiment The initial survival fraction (SFi) shown in table 4.2 reflects the effectiveness of nano-metal composites on the inactivation of E.coli and S.epidermidis colonies in darkness Both the SFi and SRs (8h) in the cases of 2wt% Ag(0.2g) were larger than those in the cases of Cs-TiO2 (0.2g) between E.coli and S.epidermidis, indicating that the antifungal effectiveness of 2wt% Ag(0.2g) was higher In this study, E.coli and S.epidermidis colonies were inactivated completely by 2wt% Ag(0.2g) at the beginning of the experiment (SR(0) = 1), and were entirely inactivated at 8h (SR(8h)E.coli= 0.06667 ; SR(8h)S.epidermidis = 0.16981) and (SFiE.coli= 0.4662 ; SFiS.epidermidis= 0.11079 ) after 8h This result demonstrated the considerable antifungal effectiveness of the AgNPs/Cs-TiO2 micro-beads (wt% Ag(0.2g)) on the inactivation of E.coli and S.epidermidis colonies in darkness To estimate inactivation curves in experiment used the semi-logarithmic plot to describe the survival rate The equation followed the kinetic parameters and was 31 analyzed using first-order reaction Chick-Watson model (Maier et al., 2009) As shown in Table 4.2, after staying on the surface of bare quartz chip, AgNPs/Cs-TiO2 micro-beads (2wt% Ag(0.2g)) in the darkness for h, E.coli and S.epidermidis were still viable and its k constant as approaches zero However, when increasing the loading level of Ag+ and thermal treated would enhance the antibacterial effectiveness, the inactivation experiment of E.coli and S.epidermidis at different condition had the similar tendency The inactivation rate constants (k) at the beginning time was lower the tail (end time) The AgNPs/Cs-TiO2 micro-beads supported by 2wt% Ag(0.2g), the inactivation rate constants (k) were 0.33851 E.coli and 0.88653 S.epidermidis for 8h , respectively In addition, D-values were calculated at the linear portion of inactivation curves, assuming the logarithmic number of spores is a linear function of treatment time The D-value also shown in table 4.2 reflects the time that the catalysts could kill 90% of E.coli and S.epidermidis The lowest D-values at 2wt% Ag(0.2g) were 3.3858-hour E.coli and 2.42302-hour S.epidermidis, respectively Delgado (2012) selected two heat-resistant molds; the results showed the D-values for spores of P variotii and N fischeri with months of age at 85oC and 90oC were 3.9 and 4.5 min, respectively (Delgado et al., 2012) 32 Table 4.2: The initial survival fraction (Sfi) of E coli and S.epidermidis on catalysts under various conditions a) E coli D-value S.D K (hour) SR(0) 1 SR(1) 0.83333 0.7868 0.18232 4.00457 SR(2) 0.63333 0.21822 0.22838 3.77933 SR(4) 0.35 0.98561 0.26246 3.64026 SR(6) 0.16667 0.7868 0.29863 3.51115 SR(8) 0.06667 0.43644 0.33851 3.3858 Sfi 0.4662 b) S.epidermidis D-value S.D K (hour) SR(0) 1 SR(1) 0.98113 0.35264 0.01905 6.26337 SR(2) 0.72453 0.5348 0.16112 4.12821 SR(4) 0.58868 0.38629 0.13247 4.324 SR(6) 0.33962 0.29853 0.17999 4.01746 SR(8) 0.16981 0.27252 0.88653 2.42302 Sfi 0.11079 33 As shown in table 4.4 After staying on the surface of bare quartz chip, the AgNPs/CsTiO2 Micro-Beads (2wt% Ag(0.2g)) 2wt% in the darkness for 8h, E.coli and S.epidermidis were viable with low survival ratios (SRs) In addition, the strong cell walls of the S.epidermidis may mitigate the antibacterial efficacy of Ag Consequently, the inactivation rate of the E.coli on the bare quartz chips was higher than S.epidermidis on the surface of the AgNPs/CsTiO2 micro-beads (2wt% Ag(0.2g)) Figure 4.4: The time profiles of the survival ratio of E.coliand S.epidermidis on the surface of catalysts and quartz chip in the darkness (Dark) There was a significant decrease in the survival ratio of E.coli and S.epidermidis on the surface of catalysts and quartz chip, testing in the darkness at different periods of time 34 In the case of 2wt% Ag(0.2g), the survival ratio of E coli and S.epidermidis were testing under the same condition (SR is at the starting point) The survival ratio of E.coli and S.epidermidis on the surface of catalysts and quartz chip in the darkness are 0.35 and 0.58868 after 4h exposure After exposure 8h in the experiment, SR of E coli and S.epidermidis decreased to 0.06667 and 0.16981, respectively The result shown 2wt% Ag(0.2g) has been successfully inactivated bacteria on the surface of catalysts and quartz chip in the darkness because of silver ions (Ag+) are highly reactive with proteins, as they bind with the thiol groups (-SH) and cause structural changes in the bacterial cell wall and nuclear membrane, leading to DNA misrepresentation and cell death (Sotiriou & Pratsinis, 2010) Moreover, Ag+ can also inhibit bacterial replication by interacting with DNA and RNA, resulting in their denaturation ( Park et al., 2009) 35 PART V CONCLUSION In summary, the antibacteriall efficacy of silver nanoparticles (AgNPs) supported chitosan/TiO2 composites (AgNPs/CS-TiO2) in the differ ent ratio at various conditions Well-mixtured nano-Cs, -TiO2, and -Ag were successfully created AgNPs/Cs-TiO2 micro-beads showed broad-spectrum and stabilized antibacterial properties against E.coli and S.epidermidis Moreover, AgNPs/Cs- TiO2 micro-beads was 2wt% Ag exhibited more potent antimicrobial activity than that of 0.5wt% Ag beads AgNPs/Cs- TiO2 and 1wt% Ag beads AgNPs/Cs- TiO2 in this research In the ZOI assay, AgNPs/Cs-TiO2 micro-beans (2wt% Ag(µg)) beans against E.coli and S epidermids exhibited more potent antimicrobial activity than that of AgNO3(µg/ml) plates were incubated at 37ºC for 18-20 hours The optimal antimicrobial efficiency occurred when the dispersion of AgNPs/Cs-TiO2 micro-beads (2wt % Ag) were between 4.32 E.coli and 4.73 S.epidermidis (µg), respectively The nano-Cs,-TiO2, and -Ag loaded AgNPs / Cs-TiO2micro-beads catalysts can reduce the survival ratio of E.coli and S.epidermidis in inactivation assay The loading level of Ag is important, and the antibacterial property of AgNPs / Cs-TiO2 microbeans (2wt% Ag(0.2g)) in the darkness is obtained in higher loading level The antifungal effectiveness of AgNPs / Cs-TiO2 micro-beans (2wt% Ag(0.2g)) catalyst is better than others condition in this experiment In conclusion, AgNPs/Cs-TiO2 micro-beads (2wt% Ag) exhibit broad-spectrumbiocidal activity toward E.coli and S.epidermidisin my study Furthermore, the successful combination of nano-Cs /-TiO2 and AgNPs created the unique morphology, which is strong oxidative properties, low cost, non-toxicity, chemical and thermal 36 stability Hence, these materials have potential applications for indoor environments as the removal of indoor air pollutants bacteria REFERENCES Berger, T J., Spadaro, J A., Chapin, S E., & Becker, R O (1976) Electrically generated silver ions: quantitative effects on bacterial and mammalian cells Antimicrobial Agents and Chemotherapy, 9(2), 357–358 Brown, T., and D S (1976) The effects of silver nitrate on the growth and ultrastructure of the yeast Cryptococcus albidus Microbios Lett., 3, 155–162 Chatzimitakos, T G., & Stalikas, C D (2016) Qualitative Alterations of Bacterial Metabolome after Exposure to Metal Nanoparticles with Bactericidal Properties: A Comprehensive Workflow Based on 1H NMR, UHPLC-HRMS, and Metabolic Databases Journal of Proteome Research, 15(9), 3322–3330 Choi, O., Deng, K K., Kim, N.-J., Ross, L., Surampalli, R Y., Hu, Z., … Hu, Z (2008) The inhibitory effects of silver nanoparticles, silver ions, and silver chloride colloids on microbial growth Water Research, 42(12), 3066–3074 Dachraoui, F., Ouanes, I., & Abroug, F (2009) Enhanced antimicrobial efficacy of thermal-reduced silver nanoparticles supported by titanium dioxide Colloids and Surfaces B : Biointerfaces, 9(1), 6–11 Dacarro, C Picco, A M., Grisoli R & Redolfi M (2003) Determination of aerial contamination in scholastic sports environment J Appl Microbiol,95,904-912 De Vos, P., Garrity, G M., Jones, D., Krieg, N R., Ludwig, W., Rainey, F A., … Whitman, W B (2009) Bergey’s manual of systematic bacteriology Volume Three The Firmicutes Bergey’s Manual of Systematic Bacteriology (Vol 3) 37 Delgado, D A., De Souza Sant’Ana, A., Granato, D., & Rodriguez de Massaguer, P (2012) Inactivation of Neosartorya fischeri and Paecilomyces variotii on paperboard packaging material by hydrogen peroxide and heat Food Control, 23(1), 165–170 Edmundson, M., Thanh, N T., & Song, B (2013) Nanoparticles based stem cell tracking in regenerative medicine Theranostics, 3(8), 573–82 Egger, S., Lehmann, R P., Height, M J., Loessner, M J., & Schuppler, M (2009) Antimicrobial properties of a novel silver-silica nanocomposite material Applied and Environmental Microbiology, 75(9), 2973–2976 Elefanty, A G., Blelloch, R., Passegué, E., Wernig, M., & Mummery, C L (2010) On the streets of San Francisco: Highlights from the ISSCR annual meeting 2010 In Cell Stem Cell , 7(2), 443–450 Fey and Olson (2010) Staphylococcus epidermis Life sciences web site Retrieved from: http://healthcare.bioquell.com /microbiology/staphylococcus-epider Fotadar, U., Zaveloff, P., & Terracio, L (2005) Growth of Escherichia coli at elevated temperatures Journal of Basic Microbiology, 45(5), 403–404 Ghosh, M., Bandyopadhyay, M., & Mukherjee, A (2010) Genotoxicity of titanium dioxide (TiO2) nanoparticles at two trophic levels: Plant and human lymphocytes Chemosphere, 81(10), 1253–1262 Große, C Schleuder, G Schmole, C Nies, D.H.(2014) Survival of Escherichia coli cells on solid copper surfaces is increases by glutathione Appl Environ Microbiol 2014 Sep 02842-14 Haghi, M., Hekmatafshar, M., Janipour, M B., Seyyed, S., Faraz, M., Sayyadifar, F., & Ghaedi, M (2012) Antibacterial effect of TiO2 nanoparticles on pathogenic 38 strain of e coli International Journal of Advanced Biotechnology and Research, 3(3), 621–624 Ishii, S., & Sadowsky, M J (2008) Escherichia coli in the Environment: Implications for Water Quality and Human Health Microbes and Environments / JSME, 23(2), Kaper, J B., Nataro, J P., & Mobley, H L (2004) Pathogenic Escherichia coli Nature Reviews Microbiology, 2(2), 123–140 King, Z A., Feist, A M., Desplats, C., Beyly, A., Cuiné, S., Bernard, L., … Driessen, A J M (2013) The respiratory chains of Escherichia coli Industrial Biotechnology, 6(3), 748–766 Kroll, A., Dierker, C., Rommel, C., Hahn, D., Wohlleben, W., Schulze-Isfort, C., … Schnekenburger, J (2011) Cytotoxicity screening of 23 engineered nanomaterials using a test matrix of ten cell lines and three different assays Particle and Fibre Toxicology, 8(1), Kumar Dutta, P., Dutta, J., & Tripathi, V S (2004) Chitin and chitosan: Chemistry, properties and applications Journal of Scientific & Industrial Research, 63, 20– 31 Lee, B U., Yun, S H., Ji, J H., & Bae, G N (2008) Inactivation of S epidermidis, B subtilis, and E coli bacteria bioaerosols deposited on a filter utilizing airborne silver nanoparticles Journal of Microbiology and Biotechnology, 18(1), 176–182 Mack, D., Davies, A P., Harris, L G., Rohde, H., Horstkotte, M A., & Knobloch, J K M (2007) Microbial interactions in Staphylococcus epidermidis biofilms Analytical and Bioanalytical Chemistry Meadow, J F., Altrichter, A E., Kembel, S W., Kline, J., Mhuireach, G., Moriyama, 39 M., … Bohannan, B.J.M (2014) Indoor airborne bacterial communities are influenced by ventilation, occupancy, and outdoor air source Indoor Air, 24(1), 41–48 Morones, J R., Elechiguerra, J L., Camacho, A., Holt, K., Kouri, J B., Ramírez, J T., & Yacaman, M J (2005) The bactericidal effect of silver nanoparticles Nanotechnology, 16(10), 2346–2353 Muzzarelli, R A A (2010) Chitins and chitosans as immunoadjuvants and nonallergenic drug carriers Marine Drugs Nilsson, M., Frykberg, L., Flock, J I., Pei, L., Lindberg, M., & Guss, B (1998) A fibrinogen-binding protein of Staphylococcus epidermidis Infection and Immunity, 66(6), 2666–73 Ozerin, A N., Zelenetskii, A N., Akopova, T A., Pavlova-Verevkina, O B., Ozerina, L A., Surin, N M., & Kechek’yan, A S (2006) Nanocomposites based on modified chitosan and titanium oxide Polymer Science Series A, 48(6), 638–643 Park, H.-J., Kim, J Y., Kim, J., Lee, J.-H., Hahn, J.-S., Gu, M B., & Yoon, J (2009) Silver-ion-mediated reactive oxygen species generation affecting bactericidal activity Water Research, 43(4), 1027–32 Park, H J., Kim, J Y., Kim, J., Lee, J H., Hahn, J S., Gu, M B., & Yoon, J (2009) Silver-ion-mediated reactive oxygen species generation affecting bactericidal activity Water Research, 43(4), 1027–1032 Pelaez, M., Nolan, N T., Pillai, S C., Seery, M K., Falaras, P., Kontos, A G., … Dionysiou, D D (2012) A review on the visible light active titanium dioxide photocatalysts for environmental applications Applied Catalysis B: 40 Environmental h Pelgrift, R Y., & Friedman, A J (2013) Nanotechnology as a therapeutic tool to combat microbial resistance Advanced Drug Delivery Reviews Portnoy, J M., Kwak, K., Dowling, P., VanOsdol, T., & Barnes, C (2005) Health effects of indoor fungi Annals of Allergy, Asthma & Immunology : Official Publication of the American College of Allergy, Asthma, & Immunology, 94(3), 313-9-22, 390 h Ramalingam, S., Muralidharan, V S., & Subramania, A (2009) Electrodeposition and characterization of Cu-TiO2 nanocomposite coatings Journal of Solid State Electrochemistry, 13(11), 1777–1783 Richards, R M., Odelola, H A., & Anderson, B (1984) Effect of silver on whole cells and spheroplasts of a silver resistant Pseudomonas aeruginosa Microbios, 39(157–158), 151–157 Salton, M R J., & Kim, K.-S (1996) Medical Microbiology University of Texas Medical Branch at Galveston Shannon (Producer) (2012) PaveMaintenance Pave Maintenance web site Retrieved from:https://pavemaintenance.wikispaces.com/TiO2+Photocatalys+-+Shannon Shi, L., Zhao, Y., Zhang, X., Su, H., & Tan, T (2008) Antibacterial and anti-mildew behavior of chitosan/nano-TiO2 composite emulsion Korean Journal of Chemical Engineering, 25(6), 1434–1438 Smitha Mundasad.(2011) E coli: Are the bacteria friend or foe? BBC News web site Retrieved from: https://www.bbc.com/news/health-13639241 Sotiriou, G A., & Pratsinis, S E (2010) Antibacterial activity of nanosilver ions and 41 particles Environmental Science and Technology, 44(14), 5649–5654 Sungkaworn, T., Triampo, W., Nalakarn, P., Triampo, D., Tang, I M., Lenbury, Y., & Picha, P (2007) The Effects of TiO2 Nanoparticles on Tumor Cell Colonies : Fractal Dimension and Morphological Properties International Journal of Biomedical Sciences, 2(1), 67–74 Wang, L., Hu, C., & Shao, L (2017) The antimicrobial activity of nanoparticles: Present situation and prospects for the future International Journal of Nanomedicine Widerström, M., Wiström, J., Sjöstedt, A., & Monsen, T (2012) Coagulase-negative staphylococci: Update on the molecular epidemiology and clinical presentation, with a focus on Staphylococcus epidermidis and Staphylococcus saprophyticus European Journal of Clinical Microbiology and Infectious Diseases Xiu, Z., Zhang, Q., Puppala, H L., Colvin, V L., & Alvarez, P J J (2012) Negligible particle-specific antibacterial activity of silver nanoparticles Nano Letters Younes, I., & Rinaudo, M (2015) Chitin and chitosan preparation from marine sources Structure, properties and applications Marine Drugs Yu, K P., Huang, Y T., & Yang, S C (2013) The antifungal efficacy of nano-metals supported TiO2 and ozone on the resistant Aspergillus niger spore Journal of Hazardous Materials, 261, 155–162 Zhang, Y., & Zhang, M (2004) Cell growth and function on calcium phosphate reinforced chitosan scaffolds Journal of Materials Science: Materials in Medicine, 15(3), 255–260 42