3. Origin of the research problem 4. Review of Research and development in the Subject 4.1 National Status Endotoxin content of PM10 and its association with inflammatory activity, air pollutants and different meteorological conditions were investigated by Mahapatra et al. (2018). The mean concentration of endotoxin was found to be in the range of 0.29-0.53 EU/m3. Though positive correlation between endotoxin concentration and presence of pollutants were found in present study but inflammatory activity exhibited negative correlation with endotoxin content associated with PM10. Air quality of temple in city of Mumbai was examined by Mehta et al., (2015) in terms of microbiological load. Significant microbial concentration was found due to improper ventilation. Gangamma. (2014) characterized the airborne bacteria in ambient environment of Mumbai city. In addition, elucidation on the role of bacterial endotoxin in PM induced inflammatory responses in vivo has also been investigated by them. Total 28 species belonging to 17 genra were identified and amongst them, significant domination of Gram positive and spore forming group of bacteria were found in Mumbai city. Joshi and Srivastava, (2013) identified the microbes and fungi in indoor air of rural residential houses. Sequence obtained from PCR amplification was analyzed and confirmed the presence of bacterial species such as Arthrobacter SP. FB24 and Bacillus Cereus and fungal spores like Aspergillums cravats. Abundance and characterization of culturable bioaerosol at Delhi, India were investigated by Kumar et al. (2013). The average concentration of airborne bacteria in Delhi varied from 0.43 – 3.35×107 CFU/m3 which is relatively higher than the range reported from temperate regions. Their findings suggested the strong correlation between meteorological parameters (temperature, humidity) and abundance of microbial load at the sampling site. Shape and size controlled antimicrobial AgNPs were synthesized using Aloe-vera plant extract by Logaranjan et al. (2016). Shape, size and structural properties of synthesized nanoparticles were investigated by different characterization techniques like SEM, FTIR, XRD. Significant antimicrobial activity of AGNPs were found against both gram positive and gram negative bacteria. A simple one-pot green synthesis of stable AgNPs usingA.indicaleaf extract at room temperature was reported by Ahmed et al. (2016). The synthesized silver nanoparticles showed efficient antimicrobial activities against both E. coli and S. aureus. According to their findings only 15min were required for the conversion of silver ions into silver nanoparticles at room temperature, without the involvement of any hazardous chemical. Parwar et al. (2016) developed Sericin/PVA/Clay Nanofiber mats by Electrospinning method for antimicrobial air filtration mask. Along with structural, mechanical and antimicrobial activity, PM absorbing capacity of these synthesized mats were also examined and their results showed a promising capacity of this mat as an air filtration mask. 4.2 International Status Airborne particles in indoor environment in terms of biological components were assessed by Mirhoseini et al. (2016). Average concentration of airborne bacteria and fungi were found to be 163 and 151 CFU/m3 respectively. In this study, predominant bacteria were found to be staphylococcus SP. and Arthrobacter SP. Seo et al., (2015) investigated the concentrations of (1, 3)-b-Glucan in submicron fungal fragments, airborne mold and bacteria, and PM10 in both indoors and outdoors environments of schools. Their findings confirmed the comparable amounts of submicron fungal fragments in school. Metagenomic methods were employed by Cao et al. (2014) to analyze the microbial composition of Beijings PM pollutants. Their findings suggested that the majority of inhalable microorganisms were soil associated and nonpathogenic to human. Size segregated PM (UFP, FP and coarse) particles were collected from the peat fire by Kim et al. (2014) and analyzed them for the chemical constituents and endotoxins content. Highest endotoxins content was found in the coarse particles which elicited the greatest pro-inflammatory responses. Lin et al. (2014) synthesized PVA nanofiber mat doped with aligned AgNPs using green manner. According to their findings the prepared nanofiber mat is suitable for broad range of application starting from antimicrobial agent to water and air purification techniques. Controllable AgNPs were developed by Chandra Sekhar et al. (2016) using extracts of Limonia acidissima tree. Various phyto-chemical compounds (saponins, phenolic compounds and amines) present in natural plant extract act as a reductant and produced AgNPs easily. Kinetic study in formation of AgNPs was verified in this study by UV-Vis technique. Antibacterial activity of produced AgNPs was investigated against Escherichia Coli and Bacillus Subtilis and their results suggested the excellent antimicrobial activity of AgNPs against both of them. Min et al. (2018) has been reported efficient eco-friendly multifunctional silk-protein nano-fiber air purification filter which is effective for reducing air pollution and thus capable of protecting the environment. Their results confirmed the excellent filtration efficiency (90) of prepared air filter which exceeded even the performance of the high efficiency particulate air (HEPA) filters. Chlcone penetrated antibacterial air filter (CPF) for inactivation of bioaerosol was developed by Huang et al (2018). Their results suggested that the survival of bioaerosol depends on the concentration of CPF. This study also suggested the inactivation of captured bacteria in presence of Chlcone and in addition, higher antiseptic effect of the penetrated was found for gram negative bacteria as compared to gram positive bacteria. Successful formation of Chitosan (CS)/ PVA nano-fiber and their application in air filtration technologies were done by Shalihah et al. (2017). Morphology and structural properties of the developed fibers were examined by SEM, FTIR and XRD respectively. Synthesized fibers exhibited significant filtration efficiency and post filtration various properties of the fibers such as morphology and porosity were also investigated. Kang et al. (2016) developed a high throughput fabrication process for antimicrobial filter with eminent antimicrobial efficiency using electro-spraying and nebulization method. Their results suggested a directly proportional relationship between the amount of antimicrobial substances coated on the filter and their filtration performance. Chen et al. (2016) made AgNPs/NSP modified air filter and assessed the filtration efficiency and antimicrobial efficacy of the synthesized filter. Antimicrobial effects of the prepared air filters were evaluated against the bioaerosol including Escherichia Coli and Candida famata in testing chambers under different relative humidity conditions. AgNP/NSP filter exhibited 91 and 95 efficacy against C. famata and E.coli. Their findings suggested that this modified air filter can be applied to air cleaning purpose and would be very effective in removing bioaerosol and thus would be capable of protecting human health in indoor environment. Chitosan nano fiber electro spun with Polyethylene oxide (PEO) and AgNO3 was successfully developed by Annur et al. (2015). FTIR and XPS both techniques were used to characterize the surface of the prepared nano fiber. Antibacterial activity of this nano fiber was also evaluated by measuring zone of inhibition against E. coli. Hwang et al. (2015) fabricated antimicrobial air filter from Natural Euscapnis Japonic nano-particles. Evaluations of antimicrobial activity, cytotoxicity of coated filters were performed in this study. In addition, filtration efficiency of synthesized filters was also checked. According to their results the hybrid fibers exhibited tremendous antibacterial activity against both gram positive (99) and gram negative (97) bacteria. Excellent Cooper and their research group (2013) introduced antibacterial filter made up of Chitosan- Poly-caprolactone utilizing the natural antibacterial propertied of CS. They have found that incorporation of 25 CS into nano fibers membrane reduced S. aurous bacterial colonization. They have also demonstrated the ability of fibers to remove particles and act as a pre-filter. Electro spun of PVA CS oligosaccharide with Ag nanoparticles was done by Li et al., (2013) to produce fiber mat.. Bio compatibility (antibacterial activity) of nano fiber was evaluated and showed significant inhibition in growth of E. Coli and Staphylococcus. 5. Significance of the study Microorganisms associated with airborne particles is a significant toxic component responsible for serious public health concern and analysis of these PM bound microbes will provide interesting data, not only for the evaluation of its impact on human health and environment, but also for the identification of specific emission sources. Therefore, characterization of airborne microbes and its toxicity evaluation is of paramount importance. At the same time, alleviation of these microbes is also necessitated in order to address the health concerns. Mitigating the environmental contaminants with the help of nano-based approaches has been developed already but most of the researches are based on aquatic environment. However, researches on alleviation of PM bound microbes by the green synthesized nanofiber air filter are still untouched. So methodical research is necessitated in order to understand the role nano-fiber air filter in capturing the PM bound microbes. This proposed work will provide a sort of base, for evaluating the quality of air and health risk associated with it and will thus be helpful for the society, policy makers and environmentalist for proper planning and mitigation measures. 6. Objectives Indoor particulate pollution is acknowledged as an important public health concern accountable for broad range of adverse health effects and these adverse health effects associated with PM are attributed to biological constitutes (micro-organisms) present in it. Therefore, detail evaluation of the toxicological profiles of PM bound microbes (their concentration, trends, and toxicity assessment) is foremost important to measure the control strategies for regional as well as global air pollution. At the same time inactivation of these airborne microbes by natural polymer based air filter is also necessitated for protection of human health in indoor environment. Thus, in the lieu of this, the objectives of present study are 1) Characterization and toxicological evaluation of indoor PM in terms of its microbial components 2) Green synthesis and characterization of AgNPs from natural plant extract 3) Preparation, characterization and antimicrobial activity of CS-PVA-AgNPs nano-fibers 4) Determination of the filtration efficiency of the prepared nano-fibers 7. Methodology 7.1. Sample collection and extraction 7.2. Characterization and identification of PM bound microbes and its toxicity assessment 1 ml of PM extract will be transferred from the suspension on duplicate plates containing selective culture medium (Nutrient agar, Eosine Methylene Blue Agar and Blood Agar for bacteria culture and Potato Dextrose Agar and Malt Extract Agar for fungal culture) directly which further will be incubated at 37C for 72 h under aerobic conditions. Finally, airborne concentrations of aerobic microorganisms will be determined by calculating colony forming units (CFU). The colonies counted on each sample type will be used to identify the constituent species (genus). Counted colonies will be first categorized based on their morphological and color characteristics. Most abundant colonies will be transferred to new plates for purification and identification by a PCR procedure at a certified laboratory. Toxicological profile of microbes will be determined in terms of endotoxins measurement by Limulus Amebocyte Lysate (LAL) method. PM extracted sample will be mixed with LAL reagent and incubated at 37C for 15 minutes followed by addition of substrate solution. Presence of endotoxins will be confirmed by developing yellow colour and then mixture will be measured spectrophotometrically at 405-410 nm. Finally concentration of endotoxins will be calculated from the standard curve. 7.3. Green Synthesis of AgNPs AgNPs will be synthesized from natural plant extract and reduction of Ag ions to Ag0 nanoparticles will be done in a medium of plant extract in which no extra reducing agent will be further used. Different phytochemical compounds present in plant extract will act as a reductant to produce AgNPs. Fresh and healthy leaves of natural plants will be collected locally and rinsed thoroughly with tap water followed by doubled distilled water. Afterwards, plant leaves will be boiled in 100 ml distilled water for 20 minutes and further cooled, centrifuged and filtered. Post preparation of leaf extract, 10 ml of 0.3M AgNO3 will be added to 10 ml of extract and will be incubated in dark overnight. Complete reduction of Ag to Ag0 will be confirmed by colour change from colourless to brownish yellow. Finally, the colloidal mixture will be filtered and oven dried. 7.4. Preparation and characterization of CS-PVA-AgNPs Nanofiber by Electrospinning 7.5. Antibacterial activity of prepared nano-fiber Antibacterial activity of prepared will be measured against both gram positive and gram negative bacteria by disc-diffusion method. Disc shape sample of nanofiber with 11 cm2 will be will be sterilized first with UV for 2 hr at 37C and subsequently placed on 24 hr old nutrient culture of PM induced bacteria and fungi. Finally the relative antibacterial effect will be found by measuring the diameter of the clear zone of inhibition formed around the disc as compared to the control. 7.6. Air permeability measurement Filtration efficiency of synthesized Nanofiber mat can be measured in terms of air permeability of the fabrics. The air permeability values of CS-PVA-AgNPs nano-fiber mats will be measured using air permeability testing instrument. The measurement will be carried out according to the ASTM D737-04 standard test method for air permeability of textile fabrics. Y, dXiJ(x(I_TS1EZBmU/xYy5g/GMGeD3Vqq8K)fw9
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