Submitted in fulfilment of the requirements for the Master of Technology Degree: Environmental Health, Durban University of Technology, 2012. / Waterborne diseases represent substantial global burden of disease and children under the age of five are more susceptible to these diseases compared to adults. The aim of this study was to determine the microbiological quality of Nyabarongo River water used for domestic purposes, women’s knowledge, attitudes and practices (KAP) on water usage and waterborne diseases and its link to the diarrhoea outbreaks experienced in two rural communities: Rusheshe and Ayabaraya of Masaka in Rwanda. A total of 35water samples were collected from Nyabarongo River and from study households which used slow sand filtration (SSF) or Sûr’Eau as treatment methods and analyzed for total coliform and faecal coliform indicators. For household samples turbidity was also analyzed. Retrospective records from Masaka Health Centre were reviewed to determine the prevalence of waterborne diseases from the study areas during 2010. Further, a structured questionnaire was administered to 324 women residents of the study areas to elicit information on their KAP on water handling and waterborne diseases. SPSS Predictive Analytic Software (PASW) Statistics version 18.0 (IBM, Somers, NY) and STATA Release (Version 11.0, College Station, Texas USA) were used for data analysis. Results showed that the mean values of total and faecal coliforms of river and household water samples were above the WHO and Rwandan recommended guidelines. The mean values of total coliform and faecal coliform were significantly lower (p ≤ 0.05) in both filtered and Sûr’Eau treated water than in river water. No statistical differences of means were observed for both total coliform and faecal coliform counts between samples taken from filtered and Sûr’Eau treated water containers (p=0.80 (TC) and p = 0.56 (FC). However, turbidity values were significantly lower in filtered water using SSF than in Sûr’Eau treated water samples (p =0.002). Out of 2814 records form Masaka Health Centre during 2010, 160 cases were identified as having diarrhoeal diseases. Furthermore, respondents who used Nyabarongo River as source of water were more likely to have symptoms of diarrhoea (OR =5.35; CI: 2.12 - 14.46; p <0.05). The frequency of diarrhoea were significantly higher among people who did not wash hands before food preparation (p = 002) and after using a toilet (p = 0.007) than among those who did. There was a statistically significant association of level of education levels and drinking water treatment practices at the household level (p < 0.05). Respondents with primary school education only and those with high school education were more likely to wash their hands after using a toilet (OR= 5.24, CI 1.42-19.38, p =0.01 and OR = 7.15, CI = 1.79 -28.62, p=0.01, respectively) than those who did not attend school. No significant associations were identified between educational levels and washing hands before food preparation. The findings of this study points to the facts that water sourced from Nyabarongo River is unsafe for human consumption even after prescribed treatment, such as the use of SSF and Sûr’Eau, and could increase the prevalence of waterborne diseases and therefore calls for urgent provision of potable water. Women in the study areas had limited knowledge regarding water storage practices for prevention of household water contamination and this; underscore the need for more water handling practices and hygiene education in rural communities. / Durban University of Technology.

The spread of antibiotic-resistant microorganisms in the environment is recognized widely as an important public health issue, with concerns about future ability to treat infectious diseases. The main risk to public health is that the resistance genes are transferred from environmental bacteria to human pathogens. Safe water is one of the most important needs in public health in the twenty first century. The major health threat posed by drinking unsafe water is the transmission of infectious diseases, which are the leading causes of mortality and morbidity for children under the age of 5 and it is estimated to cause 1.5 million deaths annually in developing countries. In addition to the wide spread cases of water-borne diseases resulting from the contamination of water sources, concerns have been raised when these diseases fail to be cured due to development of resistance to most prescribed antibiotics by the contaminating microorganisms. It is now a well-established fact that E.coli is a significant cause of diarrheal illnesses both in infants and adults in many parts of the world. Data on clinical isolates is plenty while less attention has been given to environmental isolates of these enteric pathogens. Samples from the environment such as water may serve as probable reservoirs of these pathogens; this is compounded by the entry of functional compounds of antibiotics into waterways, through humans and animals that have ingested antibiotics. This is because antibiotics are not completely metabolized and may enter waterways through the waste products of these humans or animals.Studies on antimicrobial resistance is important in order to detect changes in patterns of resistance, implement control measures on the use of antimicrobial agents, and to prevent the spread of multidrug-resistant strains of bacteria. It also provides surveillance data for antibiotic resistances, necessary to define or update guidelines for empirical treatment, as well as a guide for appropriate antibiotic supplies. Study objectives: The objectives of this research were: (i) to determine the total and faecal coliform status of drinking water sources, as an indication of quality; (ii) to determine the bacteriological profile of bacteria flora in the drinking water sources; (iii) to determine prevalence and susceptibility profiles of antibiotic resistant water-borne E.coli; (iv) to investigate the virulence genes associated with multiple antibiotic resistant E. coli isolates; (v) to compare three laboratory based techniques: PCR, API 20E, and Culture based methods used for detection of E.coli and (vi) to determine the association between multiple antibiotic resistance and radiation sensitivity (D10). © University of South Africa 2014 VII Methodology: Four hundred and sixty four (464) water samples were collected for assessment between June 2011 and May 2012. The samples were collected from 57 sampling sites, from six different water sources including: boreholes (10), a canal (1), dams (15), hand-dug wells (15), a river (1), and streams (15). Total coliforms, faecal coliforms, and E. coli analysis were done by the MPN method. Bacteria isolation and identification were done using API 20E, conventional methods, and a PCR based DNA STRIP technology that allows simultaneous detection of virulence genes and confirmation of E. coli isolates. Antibiotic susceptibility testing was also conducted using the Kirby-bauer method. Radiation sensitivity was done using a cobalt 60 source. Results: The results obtained indicated that all the water sources were of poor quality in terms of microbial distributions with total coliform and faecal coliform counts ranging between 0 to 2.4x103 MPN/100ml. E. coli counts ranged between 10 to 7.9x101MPN/100ml. Disease risk assessment of the various water sources indicated that dam water sources presented a high disease risk, while borehole water sources had a low disease risk. A total of five hundred and twenty bacterial isolates (520) were obtained during the period of study. Three hundred and five (305) isolates representing 58.65% of the total were obtained during the dry season, as against (205) representing 41.35% in the rainy season. The most commonly occurring bacteria in the water samples was Klebsiella spp constituting 20%. The next most occurring organism was E. coli (18.7%). This was followed by Pseudomonas aeruginosa (15.61%), Enterobacter spp. (15.4%), Proteus vulgaris (13.1%), and Enterococcus faecalis (9.2%). The least isolated bacteria were Vibrio cholerae (1.2%) and Shigella spp. (1.2%). The prevalence of multi drug resistance E. coli was 49.48 %. E. coli isolates showed a high sistance patterns to the tested antibiotics. They were most resistant to penicillin (32.99%), cefuroxime (28.87%), erythromycin (23.71%), and tetracycline (21.45%). In contrast, they were susceptible to nitrofurantoin (93.8%), cefotaxime and amikacin (91.75%), gentamicin (90.7%), nalidixic acid (89.65%), ciprofloxacin (74.2%), chloramphenicol (69.07%), pipemidic acid (65.97%) and cefuroxime (52.58%). Sixty-three percent (63%) of the multidrug resistant E. coli strains recorded a multiple antibiotic resistance (MAR) index value of >0.2. Six (6%) percent of he multiple antibiotic resistant were eae virulence genes producing however, none of the E. coli isolates produced the stx1 and stx2 virulent gene. The analytical profile index (API) recorded specificity and sensitivity of 99.7% and 98.50 % respectively for the detection of E. coli. The © University of South Africa 2014 VIII culture/ biochemical based methods for detection of E coli recorded specificity of 81.82% and a sensitivity of 96.91%. There was no association (P> 0.05) between radiation sensitivity (D10) and antibiotic resistances. Conclusion: The study has confirmed that majority of the water sources used for drinking and domestic purposes in the study area are highly contaminated with high levels of faecal coliforms above the recommended standards. There were also resence of bacteria of public health importance in the water sources. Both animals and humans could be sources of faecal bacteria contamination of the drinking water sources. The study confirmed a high prevalence of multiple antibiotic resistances in E. coli isolates. The eae virulence gene was associated with some of the multiple resistant E. coli isolates. The study also concludes that API 20E has a high specificity and sensitivity close to that of the PCR. Lastly, There is no association between multiple antibiotic resistant indexes and radiation sensitivity (D10) of antibiotic resistant E. coli. / School of Environmental Sciences / D. Phil. (Environmental Science)

Department of Ecology and Resource Management / PhDENV / Groundwater has traditionally been perceived to be low in chemical species toxicity and microbiologically 'pure'. However, depending on the geological chemistry, formations and anthropogenic activities creating the frequent occurrence of microbiological contamination and excess toxic chemical constituents, the high quality of groundwater as a drinking water source can easily be compromised rendering it unsafe, thus, leading to severe waterborne epidemics. The rapid increase in fluoride and microbial contamination of groundwater have become a global problem to human health. Fluoride in its acceptable concentration in drinking water (< 1.5 mg/L); is known to be beneficial for human growth and development but becomes detrimental at higher concentrations (> 1.5 mg/L) leading to the prevalence of dental and crippling skeletal fluorosis. On the other hand, consumption of microbiologically contaminated water has led to many types of diseases including diarrhea, cholera, typhoid, dysentery and other serious illnesses often leading to millions of deaths annually worldwide. South Africa had experienced water-borne diseases epidemic in the recent past due to failing water treatment facilities in many parts of the country including rural areas. Fluorosis, diarrhea, and cholera are among the chronic health hazards affecting a large population in South Africa. Continuous outbreaks of water-related diseases have been at an unimaginable high level with a reported increase in death rate. The inefficiency of conventional water treatment plants to remove fluoride and disinfect these pathogens from the contaminated domestic and rural community has led to the development of many techniques. These include membrane filtration, ion-exchange, coagulation-precipitation, adsorption among others of which adsorption process proves to be a more significant technology for fluoride removal. Equally, the emergence of nanomaterials has also proved to be the natural answer to solve problems associated with microbes in water since these are absolute barriers to pathogens whose size exceeds most sorbent pore sizes. Also, materials from natural biopolymers or biomass can be utilized at an affordable cost as effective sorbent material for toxic chemical ions and pathogens removal from contaminated water. Consequently, extensive research works have been channeled into the development of more advanced low cost sustainable functionalized sorbent materials and technologies with multifunctional properties for effective water purification. The present study focused on the development of a functionalized chitosan-cellulose hybrid nanocomposite decorated with metal-metal oxides nanoparticles for simultaneous fluoride and microbial removal from groundwater. This was to increase the selectivity and disruption of such pollutants for effective groundwater purification technology. The thesis is presented in nine chapters: (1) General introduction, problem statement, and motivation, research objectives, hypothesis and delimitations of the research are briefly discussed, (2) This chapter gives the literature review of occurrence and sources of fluoride, various fluoride removal techniques; sources, control measures and prevention of microbial pollution in groundwater; the importance of biosynthesis of nanomaterials as emerging novel water treatment adsorbents, the strength of Point-Of-Use as a means of water treatment, water treatment adsorbents synthesis and types of adsorbents with emphasis on hydroxyapatites and biopolymeric based sorbent materials, (3) Optimization of microwave-assisted synthesis of silver nanoparticle by Citrus paradisi peel extracts and its application against pathogenic water strain, (4) Biosynthesis of ultrasonically modified Ag-MgO nanocomposite and Its potential for antimicrobial activity, (5) Green synthesis of Ag/MgO nanoparticle modified nanohydroxyapatite and its potential for defluoridation and pathogen removal in groundwater (6) Green Synthesis of AgMgOnHaP nanoparticles supported on Chitosan matrix: defluoridation and antibacterial effects in groundwater, (7) Biosynthesis of nanofibrous cellulose decorated Ag-MgO-nanohydoxyapatite composite for fluoride and bacterial removal in groundwater, (8) Defluoridation and removal of pathogens from groundwater by hybrid vi cross-linked biopolymeric matrix impregnated Ag-MgOnHaP nanocomposite (9) Conclusions and Recommendations. It is important to point out that Chapters 3 to 8 contains a collection of the research deliverables produced in forms of paper publications and manuscripts and are summarized in a systemic order of experimental protocol. This first output (Chapter 3) of this study evaluated the optimization of a time-dependent microwave-assisted biosynthesis of silver nanoparticles using aqueous peel extracts of Citrus paradisi (Grapefruit red) as a reducing, stabilizing and capping agent with emphasis on its antibacterial property. Optical, structural and morphological properties of the synthesized Citrus paradisi peel extract silver nanoparticle (CPAgNp) were characterized using UV-visible spectrophotometer, transmission electron microscope (TEM), scanning electron microscope (SEM), energy dispersive spectroscopy (EDS), Brunauer–Emmett–Teller (BET) and X-ray diffractometer (XRD). The antimicrobial activity was evaluated using the well- and disc-diffusion as well as microdilution methods. Characteristic surface plasmon resonance (SPR) wavelength in the range of 420-440 nm at an optimized intensity growth rate typical of silver nanoparticles was obtained. Microwave irradiation accelerates the reaction medium within seconds of nucleation compared to conventional heating methods of synthesis. The influence of the reaction mixtures affected the SPR patterns on the different nucleation, stability and nanoparticle growth. The mixing ratio of 2:3 (C. paradisi peel extracts: 1 mM AgNO3) was chosen as the optimum reaction mixing ratio relative to the bio-reduction intensity of SPR process contributing to the particle size growth of CPAgNps. The presence, interaction and shifting of the functional groups in the FT-IR spectra of biosynthesized CPAgNps indicated that bioactive compounds present in C. paradisi peel extract were responsible for the bio-reduction of the silver ion to silver nanoparticles. The electron micrographs of the synthesized CPAgNps showed a face-centered cubic (FCC) unit phase structure, spherically-shaped nanoparticles size of 14.84 ± 5 nm with a BET pore diameter of 14.31 nm. The use of biological material allowed the control of the size and stability of the nanoparticle but was obtained in low quantity. The Citrus paradisi peel extract mediated AgNp were found to possess a broad-spectrum antimicrobial activity against water-borne pathogenic microbes in the order: Escherichia coli > Staphylococcus aureus > Klebsiella pneumonia. In Chapter 4, a synergistic bi-layered Ag-MgO nanocomposite from Ag and MgO precursor salts using a natural source from the waste product (citrus fruits outer cover) as a reducing and capping agent was successfully synthesized by a simple rapid, integrated bio-mediated microwave and ultrasonic methods. This was carried out to investigate the interfacial interaction and the encapsulated growth rate behind their combination in obtaining an enhanced antibacterial activity against common water fecal pathogen (Escherichia coli). The growth sequence, structural and morphology interface as well as the composition of the nanocomposite were examined and evaluated by the different characterization techniques. The respective potential application as an antimicrobial agent was evaluated and compared against Escherichia coli. The bio-mediated core-shell Ag-MgO nanocomposite showed characteristic synergetic UV-visible absorption bands at 290 nm for MgO nanoparticle and at around 440 nm for Ag nanoparticle, which moved to a lower wavelength of 380 nm in the composite. The shifting to a lower wavelength confirmed the reduction in the particle size as influenced by the growth rate optical property of biomolecular capped Ag-MgO nanocomposite from the phytochemical constituents in the peel extract of the Citrus paradisi. FTIR analysis further elaborated the role of the organic moieties in the Citrus paradisi extracts acting as the capping and stabilizing agent in the formation of the core-shell Ag-MgO nanocomposite. SEM analysis revealed an agglomeration of layered clustered particles, which was poly-dispersed while XRD showed the cubical crystal lattice network phase structure of the Ag-MgO nanocomposite. The TEM micrograph vii showed a structurally uniform and spherical biosynthesized Ag-MgO nanocomposite with a diameter of about 20–100 nm with an average particle size of 11.92 nm. The bi-layered Ag-MgO nanocomposite exhibited a higher level broad-spectrum of antibacterial potential on E. coli with 22 mm zone of inhibition and MIC of 20 (μg/mL) in comparison with the Ag (9 mm; 40 μg/mL) and MgO (9 mm; 80 μg/mL) nanoparticles. The leaching and toxicity level of the time-dependent releases of metal ions indicates that the effluents contain a lower concentration of Ag and Mg ions as compared to World Health Organization permissible limit of < 100 ppb (Ag). The biosynthesized Ag-MgO nanocomposite exhibited an enhanced antibacterial activity synergistic effect against E. coli than Ag and MgO nanoparticles, thus, proving to be a potential disinfect material against common pathogens in water treatment. Chapter 5 presented the biosynthesis, characterization, and assessment of simultaneous fluoride and pathogen removal potential in aqueous solutions of a multi-layered Ag-MgO/nanohydroxyapatite (Ag-MgOnHaP) composite. The successful incorporation of Ag-MgO into nanohydroxyapatite (Ag-MgOnHaP) sorbent via an in-situ solution-gelation (sol-gel) method was ascertained from UV-visible absorption spectrum bands at 290 and 440-378 nm typical of MgO and Ag nanoparticles combination in Ag-MgOnHaP composite. FTIR analysis showed the main surface functional groups involved to be –OH, C=N, carbonate and phosphate species on the backbone of Mg-O-Mg vibrational mode. The hydroxyl and amine groups indicated the interaction of a variety of metabolites components present in citrus peel extract as bio-reductive compounds associated with the Ag-MgO and also in fluoride ion exchange. SEM, TEM images and XRD analysis showed a well-dispersed discretely embedded layered-spherical Ag-MgOnHaP nanocomposite without any form of agglomeration after ultrasound exposure ranging in size from 20 to 100 nm with an average mean particle size diameter of 16.44 nm. The high purity of the synthesized Ag-MgOnHaP nanocomposite was confirmed by the presence Ag, Mg and O impregnated on the nanohydroxyapatite template from EDS spectrum analysis. Batch sorption studies using the nanocomposite under different experimental parameters were conducted and optimized. Equilibrium fluoride adsorption capacity of 2.146 mg/g at 298 K was recorded with more than 90% fluoride removal at optimized conditions of 60 min, 10 mg/L initial F- concentration, 0.3 g/L dosage, and pH 6 at 250 rpm. pHpzc of Ag-MgOnHaP nanocomposite was established to be 8. The equilibrium data were best fitted to the Freundlich isotherm model and followed the pseudo-second-order kinetics model at room temperature. The presence of competing anions such as Cl−, NO3−, does not have an impact on percentage fluoride uptake efficiency, but SO42− and CO32− reduce the F- removal efficiency. Moreover, as the concentration of the co-anions increased, fluoride adsorption uptake decreases. The biosynthesized nanohydroxyapatite incorporated Ag/MgO nanoparticle adsorbent (Ag-MgOnHaP) showed strong antibacterial activity against Escherichia coli and Klebsiella pneumonia when compared to hydroxyapatite alone. The presence and interaction between the Ag, MgO nanoparticles with the respective bacterial genomes was suggested to have accounted for this bioactivity. The synthesized Ag-MgOnHaP sorbent was found to portray a better sorption capacity compared to other adsorbents of similar composition in the literature and could be successfully regenerated with 0.01 M NaOH with fluoride removal of 74.24% at the 4th cycle of re-use. The impregnation of metal-metal oxide nanoparticles on sustainable natural biopolymers from waste products was presented in Chapters 6, 7 and 8. The use of these sustainable natural biopolymers (chitosan and cellulose) was targeted with more emphasis on surface functionalization, improved structural diversity and improved specific surface area with the sole aim of increasing the adsorptive capacity of fluoride ions as well as antimicrobial properties. The selected polymers were chosen because of their biodegradability, viii non-toxicity, renewability, selectivity and abundance in nature, which makes them promising starting materials for the purpose of sustainable water treatment. Chapter 6 presents the successful sol-gel biosynthesis, characterization, potential application for fluoride and pathogens removal from aqueous solution using Ag-MgOnHaP embedded on a chitosan polymer backbone (AgMgOnHaP@CSn) sorbent material. The overall formation of the AgMgOnHaP@CSn nanocomposite from different surface functionalization precursors and phases were supported by the various characterization methods such as UV–vis spectroscopy, SEM-EDS, FTIR, TEM, and Brunauer–Emmett–Teller (BET) techniques. Batch fluoride sorption experiments were conducted to assess fluoride uptake efficiency through optimization of several operational parameters such as contact time, adsorbent dosage, initial pH and co-competing anions. The antimicrobial activity of the synthesized AgMgOnHaP@CSn nanocomposites was also determined. The presence and bio-reduction processes of both Ag and MgO chemical species due to the interaction and coordination of bonds within the bioactive functional species of the polymer matrix was confirmed by the emergence of a sharp peak appearing at around 290 nm to a broad plateau plasmon absorbance above 440 nm on the AgMgOnHaP@CSn nanocomposite. FTIR analysis further supported the presence of the main bioactive functional species to be –OH, –NH2 CO32−, PO43-, Mg–O-Mg amongst other groups on the material surface. SEM and TEM displayed homogeneously dispersed particles within the aggregated biopolymeric composite with a diameter ranging between 5-30 μm. Pore sizes were observed to be in the micro-mesoporous range with an average size of about 35.36 nm and a pore diameter of 33.67 nm. The optimized conditions were as follows: 30 mins contact time, a dose of 0.25 g/50 mL, adsorbate concentration of 10 mg/L F-, initial pH 7 while adsorption capacity decreases with increase in temperature. AgMgOnHaP@CSn composite has a pHpzc value of ≈ 10.6 and the maximum sorption capacity was established to be 6.86 mg/g for 100 mg/L F- concentration at 303 K. The effect of co-existing anions was observed to be of the following order: Cl- < NO3- < SO42- << CO32-. The fluoride sorption experimental data was well described by Langmuir adsorption isotherm while the sorption reaction mechanisms were diffusion-controlled and followed the pseudo-second-order sorption model. F- sorption process could best be described as a combination of ligand exchange, electrostatic attraction, and improved structural surface modification. The antimicrobial susceptibility analysis through the zone of inhibition (mean and standard deviation) showed the potency to pathogens of the following order: Staphylococcus aureus > Escherichia coli. Chapter 7 gives an insight into the development of cellulose nanofibrous matrix (isolated from saw-dust) decorated with Ag-MgO-nanohydroxyapatite (CNF-AgMgOnHaP) and its application in fluoride and pathogen removal from contaminated water. The synthesized CNF-AgMgOnHaP, unlike the cellulose nanofiber, showed characteristic absorption bands in UV–vis spectroscopy between 270-290 nm typical of MgO together with a broad band around 420 nm associated with the characteristic of silver nanoparticles. FTIR spectrometry suggested the presence of nanohydroxyapatite (nHaP) and MgO species impregnation within the CNF matrix. SEM, TEM, XRD, and EDS analysis showed a well-established structural and morphological modifications between cellulose nanofiber alone, biosynthesized CNF-AgMgOnHaP and fluoride sorbed CNF-AgMgOnHaP nanocomposite. A granulated aggregation of micro-mesoporous particles with an improved BET surface area of 160.17 m²/g was developed. Optimum fluoride sorption capacity was 8.71 mg/g for 100 mg/L F- solution at 303 K. F- sorption capacities decreased as the operating temperatures increases. Optimum F- removal of 93 % was achieved at optimum conditions established: pH 5, solid/liquid ratio of 0.25 g/ 50 mL, 10 mg/L F-, contact time 10 min, temperature 25 ± 3 °C and shaking speed of 250 rpm. Percent F- removal decreased with increasing initial adsorbate concentration. The pHpzc value of the CNF-AgMgOnHaP occurred at ≈ 4.7. Co-existing ions were observed to have an effect on the adsorption of F- in the following order: NO3- < Cl- < SO42- <<CO32-. Equilibrium fluoride sorption onto the CNF-AgMgOnHaP was best described by non-linear Freundlich isotherm model across all the operating temperatures. The linear Dubinin-Radushkevvich (D-R) model for F- sorption energies were in the 3.54 – 4.08 kJ/mol across all operating temperature. This suggested the physical adsorption mechanism processes were involved in the F- uptake by the CNF-AgMgOnHaP sorbent. The overall kinetic results indicated that the mechanisms not only depend on the pseudo-second-order process but were also governed by mass transfer of the adsorbate molecules across the CNF-AgMgOnHaP surface. The thermodynamic parameters revealed that the sorption process of F- onto CNF-AgMgOnHaP was endothermic and spontaneous at the sorbent/solution interface. The regeneration-reuse study showed that the synthesized adsorbent can be reused for a maximum of 5 adsorption-desorption cycles using Na2CO3 and NaOH as regenerants. Overall surface chemistry by XPS, FTIR, EDS as well as sorption isotherm and kinetic models analysis suggested that both physical and chemical adsorption processes were involved in the fluoride uptake by CNF-AgMgOnHaP nanocomposite. The observed zone of inhibition demonstrated that CNF-AgMgOnHaP adsorbent possesses antibacterial activity against all the bacterial strains in the following order: E. Coli > S. aureus > K. pneumonia. The antibacterial potency increased with increasing sorbent concentration. In chapter 8, Defluoridation and antimicrobial activity of synthesized cross-linked cellulose-chitosan impregnated with the developed nanomaterial (AgMgOnHap) are presented. The before and after fluoride sorption by the synthesized CECS@nHapAgMgO nanocomposites were characterized using several physical and chemical techniques which include, BET, SEM-EDS, TEM, XPS, XRD, and FTIR. The overall batch fluoride sorption processes and adsorption capacity through optimization of different experimental sorption parameters, sorption isotherms, and kinetic mechanisms as well as antibacterial potency were studied and reported. SEM and TEM analysis showed densely irregular multiple-layered structures, homogeneous deposition of the AgMgOnHaP on the polymeric matrices. Equilibrium fluoride sorption capacity on CECS@nHapAgMgO sorbents showed an increased affinity of 26.11 mg/g for 150 mg/L F- solution at 313 K.at optimized conditions of 40 min contact time, dosage of 0.3 g and pH of 5. The pH point of zero charge was found to be 7.27. The reaction pathway model sequence of fitness follows the order Pseudo first order < Elovich < Pseudo-second order kinetic model while intra-particle diffusion model and mass transfer of fluoride molecules from the external surface onto the improved pores of the adsorbent were found to be involved in the rate-controlling step. Although both non-linear Langmuir and Freundlich isotherms showed appropriate trends in the F- sorption process, the adsorption isotherm data were better fitted to the non-linear Freundlich isotherms models, suggesting stronger heterogeneous adsorption onto the active binding sites of the CECS@nHapAgMgO surface. The fluoride sorption was observed to be a favorable process across the operating temperatures. Temkin heat of sorption (BT) and the mean free adsorption energy (E) of the D-R isotherm model was within the range of 0.68-3.39 J/mol and 1.58 -7.45 kJ/mol respectively. The fluoride sorption process was observed to be temperature-dependent; while adsorption capacities (Qm) and Temkin heat of sorption (BT) increased with increasing temperature, D-R Mean free sorption energy (E) decreased at higher temperatures. The thermodynamic analysis demonstrated that fluoride sorption on the CECS@nHapAgMgO surface was exothermic, feasible and spontaneously inclined with a decrease in the degree of randomness at the sorbate-sorbent interface. The influence of co-existing anions on fluoride removal exhibited the following trend Cl−< NO3− <SO42- << CO32- <<HCO3−. The practical and economic viability, potential for regeneration showed its reusability up to 3 cycles with water and Na2CO3 as regenerants. The potential ability of CECS@nHapAgMgO to disinfect both gram- positive and gram-negative water bacterial was confirmed by the zone of inhibition and Minimum Inhibitory Concentration (MIC) measurements. The observed values showed the inhibitory efficiency in the following order: S. aureus > E. Coli > K. pneumonia where the MIC values of 20 μg/mL were recorded for S. aureus and E. Coli respectively and 10 μg/mL for K. pneumonia. Lastly, the applicability of the sorbents was tested with a field water sample collected from a high fluoride borehole water from a local village (Lephalale Municipality of Limpopo province, South Africa). The before and after analysis showed the excellent potential of CECS@nHapAgMgO sorbent in removing fluoride. In conclusion, the successful surface functionalization synthesis of these improved surface area hybrid nano-sorbents supported by the different morphological techniques was found to be effective in creating more surface-active binding sites for fluoride adsorption and disinfection of waterborne pathogens from aqueous solution. The originality of this developed sorbent lies firstly, in the ability to simultaneously remove both chemical and biological water pollutants; secondly, the use of biodegradable, eco-friendly and non-toxic abundance wastes raw materials to develop a water purification material and in solving waste management issues was a key factor towards environmental sustainability. Above all the developed materials were established to possess superior fluoride adsorption capacity when compared to other reported sorbent materials. Lastly, the project findings /innovation will contribute to Sustainable Development Goals (SDG) 3 and 6, aimed at improving clean water supply and health of the communities and the world at large. However, the following recommendations were made following the findings from this study: 1) In order to increase the surface area to volume ratio, greater selectivity, porosity, and mechanical stability of the polymers as well as size-exclusion mechanism without a large energy penalty of the microbes and fluoride ion for effective water treatment, a more effective and an enhanced multifunctional, multi-layer nanofibrous hybrid sorbent through electrospinning techniques should be considered for future work, 2) More studies on the mode of actions and morphological changes in the pathogens leading to the cell death through the influence of the nanocomposites should be further explored, 3) Application of this advanced technology vis-à-vis other biomaterials to generate filter membrane towards efficient microbial removal and deflouridation is a great challenge worth looking at, 4) Lastly, materials developed in the present study should be modeled, tested and fabricated at the point of use for fluoride and pathogen removal at household level. / NRF

MSc (Microbiology) / Department of Microbiology / Background: The occurrence of microorganisms and endocrine disrupting chemicals (EDCs) in water poses a serious concern due to their effects on humans, animals and environment. In recent years, EDCs have been increasingly reported in rivers that receive large amounts of wastewater effluents. Of all the EDCs, natural and synthetic hormones are among those that are recognized for their potential to mimic or interfere with normal hormonal functions of humans and animals. The present study aimed at assessing the occurrence of these hormones in relation to the molecular diversity of Aeromonas and evaluating the resistance of Aeromonas to antibiotics as well as to assess anti-bacterial activity of two selected traditional medicinal plants. Methods: Wastewater, water and fish samples were collected from various sources (rivers, wastewater treatment plants, taps, and dams) for the detection of hormones and isolation of Aeromonas species. The analysis of hormones from various organs of the fish and from water samples was conducted, after extraction using enzymelinked immunosorbent assays (ELISA). Different types of hormones including Estriol, Estradiol, Ethinylesradiol and Testosterone were detected, and their concentrations determined. Aeromonas spp were isolated rom the samples using microbiological methods and Conventional PCR was used for genotyping as well as for detection of the beta-lactamase genes. Kirby-bauer method was used to determine the susceptibility profiles of Aeromonas to different antibiotics. Microdilution assay was used to determine the Anti-bacterial activity of the plant (Annoniceae and Zornia milneana) extracts against Aeromonas species. Results: A total of 144 samples were collected from 23 different locations in two countries: South Africa and Zambia. These included wastewater and treated wastewater, River water, fish and tap water. 17α-ethinylestradiol (EE2) was detected in most of the samples (92.7%) with concentrations varying from 0.59 ng/ml to 65 ng/ml. The hormones were also detected from drinking water, with testosterone detected at high concentrations of up to 140 ng/ml in tap water. Most sewage treatment plants were not able to remove the EE2 from the wastewater as the concentration of this hormone in the final effluent was almost always higher than that in the influent. These homones were also detected in drinking water at high concentrations of up to 53.49 ng/ml in the tap water for EE2 and 1777 ng/ml for E2. The overall detection of Aeromonas species in the samples was 84.5%. A. caviae was the most prevalent species accounting for 73.6%, followed by A. veronii with 64.6%. The bacteria were completely resistant to cefuroxime accounting for 100% resistance. Aeromonas isolates also showed high resistance to trimethroprim (88.7% for A. hydrophila), cefazolin (highest 97.8% for A. cavie), and ceftazidime (83.9% for A. sobria). TEM was the most prevalent beta-lactamase gene with detection rate of 87%. All isolates lacked the presence of the CTX-M3 gene. Also, wastewater had the highest prevalence of A. veronni and A. caviae accounting for 87.5% and 82.5% respectively. Multiple antibiotic resistance was also observed with the Aeromonas isolates being resistant to up to 11 antibiotics. High prevalence of 77.1% of Aeromonas hydrophila was observed in the presence of ethinylestradiol (EE2). Aeromonas veronii and Aeromonas caviae were the most predominant species in the presence of total estriol, A. veronii had a prevalence of 57.1% and A. caviae had a prevalence of 52.8%. Aeromonas hydrophila and Aeromonas caviae had the lower prevalence in the presence of hormones with the percentages of 26.1% and 27.8% respectively. The methanol extracts of both Zornia milneana and Annona species showed good activity against the Aeromonas spp with the lowest MIC of 0.078 mg/ml. Ethyl acetate extracts were the least effective. Conclusion: This study has shown high occurrence of steroid hormones in all types of environmental samples tested. These included tap water, river water, wastewater and fish both in Zambia and South Africa. Therefore, steroid hormones constitute and important health problem in the Southern African Sub-Region. The incapacity of the wastewater treatment plants to remove EE2 is an important problem that needs to be tackled immediately. The prevalence of Aeromonas species is very high in our environmental water as well as in drinking water, with the highest prevalence observed in fish and wastewater. It was also revealed that there is relationship between steroid hormones and Aeromonas species, with the hormones supporting the growth of Aeromonas species. The presence of beta-lactamase genes which causes Aeromonas to be resistant to antibiotics was also noted. Methanol extracts of Zornia milneana and Annona spp were the most effective against Aeromonas spp and could serve as primary sources for the isolation of lead compounds. / NRF

This study focuses on the isolation and characterization of bacteria from lignocellulosic biomass obtained from the sediments of the Tyume River in Alice, Eastern Cape and to determine those bacterial isolates with good potential for modification and decomposition of lignocellulosic biomass for industrial application. Several bacterial isolates were recovered and screened for ability to degrade various lignocellulosic materials. Nine of the isolates were positive for lignocellulolytic activity. Four isolates were cellulase positive and six were xylanase positive. Moreover, one isolate (SB1) was positive for both xylanase and cellulase activities and showed the best hydrolysis zone on solid media. This isolate was then chosen as the best and identified molecularly. The 16S rDNA sequence analysis indicated that SB1 was a Bacillus cereus species. Factors affecting the cellulose and xylanase enzyme production by the organisms were studied. The organisms produced the enzymes maximally at earlier hours of incubation (12-30 hr) and optimally at acidic pH (3-5) and at moderate temperatures (35-45ºC). SB1 appears to hold promise in the decomposition of lignocellulosic wastes.

MSc (Microbiology) / Department of Microbiology / See the attached abstract below