Theses and dissertations (Applied Sciences)
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Item Assessment of biomarkers for normalization of SARS-CoV-2 concentrations in wastewater(2023-09) Osman, Aaliyah; Sheena, Kumari; Amoah, Isaac Dennis; Bux, FaizalDuring the COVID-19 pandemic, the measurement of SARS-CoV-2 RNA levels in wastewater quickly emerged as an additional tool for monitoring and to provide an early warning system. This led to development of several regional, national and international projects aimed at applying this approach. The main principle is based on the detection of the viral signature in untreated wastewater to provide an indication of infection levels within connected populations. However, the concentration of the viral signature in wastewater can be impacted by dilution factors or population changes in the sewer shed, leading to misinterpretation of measurement results. Therefore, there is the need for normalization of wastewater to ensure accurate representation of infection numbers. The aim of this study was to evaluate different viral and bacterial markers in wastewater for their efficiency in normalizing SARS-CoV-2 WBE data, which will enhance the accuracy when interpreting the SARS-CoV-2 RNA concentrations in wastewater. Weekly sampling was conducted from two wastewater treatment plants (WWTP A and WWTP B) within the eThekwini district over a period of three months (July-October 2022). Three biomarkers (crAssphage, Bacteroides (HF 183), and Pepper Mild Motile Virus) where chosen for this study to ascertain the most suitable for WBE data normalization. Biomarker and SARS CoV-2 concentrations in the wastewater samples were determined using the droplet digital PCR (ddPCR). Physicochemical characteristics of the wastewater samples were also determined to identify the potential impact of these characteristics on the concentration of SARS-CoV-2 and the biomarkers. To determine the most suitable biomarker, correlation analysis and the Adaptive neuro fuzzy inference system (ANFIS) model was used. Average concentrations of SARS-CoV-2 in the sampled WWTPs ranged from 0.28 copies/µL to 9.57 copies/µL. Among the three biomarkers studied, crAssphage recorded the highest concentration compared to PMMoV and Bacteroides HF183 in both the WWTPs. CrAssphage recorded the highest concentration of 7943 (±7.07) copies/µL for WWTP A and 8006 (±4.24) copies/µL for WWTP B. The Bacteroides HF183 highest concentrations were 10116 (±120.91) copies/µL for WWTP A and 2474 (±117.37) copies/µL for WWTP B. PMMoV had concentrations of 46 (±4.24) copies/µL for WWTP A and 84,1 (±5.48) copies/µL for WWTP B. PMMoV concentrations were observed to be the highest at Week 1. CrAssphage showed a greater association during the trend analysis with SARS-CoV-2 (0.499) than the other two biomarkers for WWTP A, (HF 183 and SARS-CoV-2 (-0.191) and PMMoV and SARS-CoV 2 (-0.562)). Among the physicochemical factors studied, electrical conductivity and temperature had a significant correlation with SARS-CoV-2 and the crAssphage biomarker for both WWTPs. Using the ANFIS model, it was shown that the levels of the measured biomarker concentrations in wastewater had a significant association with chemical oxygen demand (COD), dissolved oxygen (DO), and volatile solids (VS). These results indicate a possible impact of these parameters on the concentration of these biomarkers in the wastewater. Furthermore, the viral RNA quantities of SARS-CoV-2 in wastewater were demonstrated to be influenced by other parameters such as electrical conductivity, pH and temperature. This indicates a difference in the physicochemical parameters that influence both biomarkers and SARS-CoV-2. However, when all physicochemical parameters, biomarkers and SARS-CoV-2 were combined, it was determined that the best biomarker was crAssphage, with potential impact from COD and the VS. The results of this study highlight the significance of including wastewater characteristic in WBE studies for reliable and accurate results. As shown in this study, crAssphage can serve ix as a biomarker for efficient WBE for COVID-19 surveillance. In addition, it has been demonstrated that the detection and quantification of targets of concern, including SARS-CoV 2, may be enhanced when combined with wastewater characteristics, which may enhance the monitoring of COVID-19 infections.Item Removal of selected heavy metals from wastewater using modified agricultural waste(2023-09) Msimango, Maureen Nomaxhosa; Qwabe, L. Q.; Ntola, P.This study explores the potential of utilizing inexpensive adsorbent materials derived from agricultural waste to eliminate Zn (II), Ni (II) and Cd (II) from water-based solutions. Sugarcane bagasse was chemically modified to extract cellulose and further functionalize extracted cellulose to prepare carboxymethyl cellulose which were used as biosorbents. The biosorbents were characterized using XRD, FTIR (ATR), and SEM for confirmation of physical and chemical properties and surface morphology of the adsorbents. In batch experiments, the effect of various parameters such initial concentration (10-300 mg/L), pH (2- 8), adsorbent mass (0.1-1.7 g), and contact time (5-150 min). Adsorption was poor for all metals below pH 4 and reached maximum removal efficiency at pH 6. The increase in initial concentration favoured the increase in removal efficiency but reaches a maximum beyond 100 mg/L. The increase in biosorbent mass shows favours increase in removal efficiency for Ni (II) and Cd (II) but a decrease was observed for Zn(II). The removal efficiency increased with contact time and reached equilibrium at 60 minutes for all metals and biosorbents. The maximum adsorption capacities of Zn(II), on SCB, SCBC, and CMC were 12.3, 20.9 , and 33.5 mg/g respectively. Ni (II) adsorption capacities on SCB, SCBC, and CMC, were 41.9, 25.4, and 125.7 mg/g respectively. The maximum capacities of Cd(II) on SCB, SCBC, and CMC, were 11.3, 20.8, and 21.6 mg/g respectively. The performance of CMC superseded SCB and SCBC. Kinetic experiments showed that the adsorption process followed pseudo second order whereas the equilibrium studies showed that the adsorption process followed the Langmuir adsorption isothermItem Impact of chemical oxygen demand to nitrogen ratio on anammox bacterial growth in an up-flow anaerobic sludge blanket reactor(2023) Msimango, Sandile Simiso; Kumari, Sheena; Nasr, Mahmoud; Bux, FaizalThe anaerobic oxidation of ammonium (ANAMMOX) process has been suggested as an economical and innovative means of removing nitrogen from wastewater. Nevertheless, very few studies have evaluated the effect of the chemical oxygen demand (COD) to nitrogen (N) ratio on bacterial communities in an ANAMMOX-mediated system. Heterotrophic bacteria can readily outcompete the slow-growing ANAMMOX bacteria in the presence of organic carbon. This study examined the effect of the organic carbon to nitrogen (C/N) ratio on the performance of ANAMMOX in an upflow sludge blanket reactor using synthetic wastewater as the feedstock. Two UASB reactors (UASB-A and UASB-B) were seeded with biomass from a labscale ANAMMOX reactor and operated for a period of 593 days. Both reactors were operated using similar operational conditions during the enrichment phase (0-400 days). Thereafter, the addition of organic carbon in the medium altered the C/N ratio of one of the reactors (UASBB). During this period, UASB-A served as a control reactor. A CN ratio of 1.0, 1.5, and 2.0 was achieved in the UASB B reactor by increasing the organic carbon concentration every 60 days. The reactors were analyzed at three-day intervals per week for nitrogen and COD removal efficiency. The quantitative PCR method was used to detect the dominant N-removing organisms within both reactors at different phases. In addition, cDNA quantification or reverse transcriptase qPCR (RT-qPCR) was also conducted to determine the dominant and active nitrifying communities. The results indicated that when the C/N ratio is 1.0, almost complete removal of NH4 + -N is observed (92%), and nitrogen removal efficiency (NRE) is approximately 82%. The ratios of ΔNO2 - /ΔNH4 + and ΔNO3 - /ΔNH4 + ratios during this phase (C/N=1) fluctuated from >1.25 to <1.6 and from >0.35 to <0.45 <0.11 to >1.6, respectively, which was within the range of the expected ANAMMOX stoichiometric ratio. In addition, when the C/N ratio was increased from 1 to 1.5, NRE rose from 82 to 88%. However, a decrease of NRE to 83% was observed when the C/N ratio was further increased to 2. The quantitative PCR results showed an increase in total bacteria from 1.4 × 106 copies/µL to 2.3× 106 copies/µL, and 2.4× 106 copies/µL as the ratio of C/N increased from 1.0 to 1.5 and thereafter to 2, respectively. ANAMMOX bacteria showed an increase from 16 × 103 copies/µL to 6.5× 10 4 copies/µL, and 2.06 × 105 copies/µL when the C/N ratio was increased from 1 to 1.5, and 2, respectively. The cDNA analysis further showed an increase of ANAMMOX bacteria transcript abundance from 4.6 × 104 copies/µL to 2.52× 106 copies/µL with an increase in C/N ratio to 1.5. Subsequently, a decrease in ANAMMOX bacteria transcript abundance to 1.09 × 106 copies/µL was observed when the C/N ratio was further increased to 2. The expression of the hzo gene encoding for hydrazine dehydrogenase (HDH), which catalyses the oxidization of the unique ANAMMOX intermediate hydrazine to N2 was 169 folds of expression, which was very high at C/N=1, but showed a decrease to 39 folds expression at C/N=1.5. Almost complete inhibition of hzo gene was observed when the C/N ratio was further increased to 2. Based on chemical analysis, it was further confirmed that the decrease of both ANAMMOX and AOB abundance at a higher C/N ratio caused an increase in effluent NH4 + -N concentrations. In conclusion, the study has shown that a higher C/N ratio could significantly affect the overall nitrogen removal rate and the activity of the diverse microbial populations, more specifically the ANAMMOX bacterial activity.Item The factors affecting bacterial colonisation on microplastics and the impact of tertiary treatment of wastewater on the attached bacteria and microplastics(2023-05) Rajcoomar, Saieshna; Bux, Faizal; Kumari, Sheena; Amoah, Isaac DennisMicroplastics (MPs) in aquatic environments have become an environmental concern globally. In addition to the direct impact of these plastics on aquatic organisms, their surfaces could serve as a unique habitat for various microbial communities through the formation of biofilms. Various factors could play a role in microbial attachment and biofilm formation in wastewater. This study aimed to assess potential factors that lead to biofilm formation on different types of MPs in wastewater and determine the impact of UV and chlorine treatment on these biofilms. In a laboratory scale experiment, MPs (low density polyethylene (LDPE), high density polyethylene (HDPE), and polypropylene (PP) were exposed to untreated wastewater under various conditions of temperature (20°C, 25°C and 35°C), light and dark conditions, as well as aerobic and anaerobic conditions for a period of five weeks. The formation of biofilms on MPs was quantified using optical density (OD660) measurements. The highest biofilm formation was observed in week 3, with an OD of 1.77. Thereafter, a decline in OD was observed, reaching an OD of 1.1 by week 5. This change in biofilm concentration over the week corresponded to changes in nutrient (nitrite, nitrate and ammonia) concentration in the media. A positive correlation was observed between the changes in biofilm concentration and nitrite (r = 0.824) and ammonia (r = 0.1) levels in the media. Meanwhile, a negative correlation observed for nitrate concentration (r=-0.673). Factors such as dark conditions, 25 C, and aerobic conditions presented the highest median biofilm formation with an OD value of 1.6, 1.7 and 1.6, respectively. It was also observed that polyethylene had higher biofilm concentrations compared to the polypropylene. Furthermore, rough MPs had higher biofilm formation than smooth MPs, with median ODs of 1.7 and 1.6 respectively. The microbial communities in the biofilms and wastewater medium were characterised by 16S rRNA amplicon sequencing. The results revealed that the alpha diversity (richness, evenness, and diversity) was lower in wastewater compared to the biofilms. It was observed that PP supported the most diverse bacterial community ( H’= 2.51138 and Simpson index= 11.096), while HDPE supported the least diverse bacterial community (H’= 0.88779 and Simpson index= 1.5324). Beta diversity using the Jaccard distance index revealed that the most similar communities were observed among biofilms from the three types of MPs while the most dissimilar communities were observed between the biofilm and wastewater medium communities. The most dominant phyla in both the biofilms and wastewater medium during the five weeks were Proteobacteria, Bacteroidetes and Planctomycetes. The bacterial communities, however, varied for each type of plastic and the wastewater medium. It was observed that Methylotenera, Hydrogenophaga, and Rhodanobacter was the most abundant genera in biofilms whereas C39(45.25%) and Luteimonas(18.96%) were the abundant genera in the wastewater medium. Methylotenera mobilis was the most common species among the three types of MPs. In addition, pathogenic species such as Mycobacterium arupense and Methylobacterium adhaesivum were detected in abundance on LDPE and PP. To assess the impact of UV treatment and chlorination on the attached biofilms, the microplastics with attached biofilm were exposed to UV-C and Chlorine (5 mg/L) treatment for 60 minutes. The biofilms were inactivated (100%) after 30 mins of UV treatment, whereas 10 min was sufficient to achieve 100% inactivation of biofilm by chlorine treatment. In conclusion, the research presented in this study has made substantial contributions to our understanding of the role that environmental factors play in the formation of biofilm on MP surfaces.Item An integrated approach for biofuel and fertilizer production using microalgae grown in wastewater(2022-09) Musetsho, Pfano; Bux, Faizal; Renuka, Nirmala; Guldhe, AbhishekMicroalgae are recognized as potential candidates for resource recovery from wastewater and are projected for biorefinery models. Therefore, this study was undertaken to evaluate the potential of poultry litter and municipal wastewater as nutrient and water sources, for the cultivation of Acutodesmus obliquus for lipids production for biodiesel application. The efficacy of lipid extracted biomass (LEA) as fertilizer for mung bean crops was also assessed in microcosm. A. obliquus cultivation in acid pre-treated poultry litter extract (PPLE) showed maximum biomass production of 1.90 g L-1 , which was 74.67% and 12.61% higher than the raw poultry litter extract (RPPE) and BG11 respectively. Higher NO3-N, NH3-N, and PO4-P removal of 79.51%, 81.82%, and 80.52% respectively were observed in PPLE as compared to RPLE treatment. The highest biomass (140.36 mg L-1 d -1 ), lipids (38.49 mg L-1 d -1 ), and carbohydrates (49.55 mg L-1 d -1 ) productivities were observed in the PPLE medium. The application of LEA as a fertilizer for mung bean crops showed improvement in plant growth and soil microbial activity. A maximum increase in organic carbon (59.5%) and dehydrogenase activity (130.8%) was observed in LEA amended soil which was significantly higher than chemical fertilizer (CF) control in 30 days. Whilst plant fresh weight and leaf chlorophyll in the LEA amended soil was comparable to whole algal biomass (WA) and CF control. The findings of the present study could be a basis for sustainable biorefinery for the valorization of wastewater for the production of microalgae-derived biofuel and byproducts for agricultural applications.Item Profiling of key nitrogen converting organisms in wastewater treatment plants with diffused aeration(2022-09) Kumalo, Puseletso Constance; Bux, Faizal; Pillai, Sheena Kumari Kuttan; Awolusi, Oluyemi OlatunjiMaintaining stable nitrification rates in biological nutrient removal (BNR) systems is difficult due to the slow growth rates of nitrifying bacteria and their sensitivity to environmental and operational conditions. Dissolved oxygen (DO) concentration in the aeration tank significantly affects nitrification and nitrifying bacterial growth. Currently, diffused aeration systems are gaining popularity over conventional surface aeration systems due to their advantages like process stability, better control, and lower cost of operation. However, studies regarding the impact of this aeration type on the selection of functional microbial communities in wastewater treatment plants are still lacking. This study focused on investigating the community structure and activity of key nitrogen converting organisms within two different municipal full-scale wastewater treatment plants (WWTP A and WWTP B) operated with fine bubble diffused aeration. WWTP A was relatively a large plant with a flow rate of 71 ML/day and consisted of three parallel BNR systems (reactor 1, 2, and 3), operated using a similar mode whereas WWTP B was relatively a small plant (0.5 ML/day) with a single BNR system. Composite sludge samples from aeration tanks, as well as influent and effluent water samples, were collected monthly from August 2019 to February 2020 and from June 2020 to August 2020. The nutrient removal performance of the plant was estimated from the influent and effluent chemical analysis. Floc structure analysis and sludge volume index were calculated to assess the settling characteristics. In addition, nitrifying bacterial population dynamics and their activities were assessed using quantitative real-time and reverse transcriptase PCR, respectively in relation to selected plant operational (DO, temperature, substrate concentration) conditions. The average ammonia removal at WWTP A was 95±5.6% which correlated with DO concentration in the aeration tank and the nitrification rate of the plant, whereas the WWTP B recorded 98±02% average ammonia removal efficiency with a more stable DO level in this plant. The sludge volume index (SVI) values were below 150mL/g in both plants, indicating good sludge settling under fine bubble diffused aeration. However, the floc structure varied across the reactors during the study period and ranged from small to medium, open to compact, and irregular with occasional filaments branching mainly in WWTP A. The microbial analysis of sludge samples showed that ammonia oxidising bacteria (AOB) 16S rRNA gene abundance was high in all the three reactors in WWTP A as compared with nitrite oxidising bacteria (NOB). In WWTP B, the average 16S rRNA gene copies for NOB were observed to be higher than AOB. In addition, in WWTP A, a negative correlation was found between the AOB 16S rRNA population and DO concentration in reactor 1 (r = -0.40), while a positive correlation was found in reactor 3 (r = 0.47) with no clear correlation in reactor 2 as well as in WWTP B. In both plants, Nitrobacter spp. was the dominant NOB, while the relative abundance of Nitrospira spp. was generally consistent throughout the study. The nxrB copy number was observed to be higher than that of nxrA (encoding for Nitrobacter spp.). The highest amoA copy number was observed when the temperatures were high (22 ⁰C -26.1 ⁰C), implying that increasing temperatures possibly benefited AOB growth. In terms of functional gene expression, a rapid decrease in expression levels of amoA was observed in both plants while the expression levels of nxrB were observed to increase rapidly as the temperature increased. In contrast, expression levels of the nxrA were relatively more consistent throughout the study period in both plants. At WWTP A, there was a positive correlation between AOB expression (amoA) and DO concentration in all reactors (reactor 1: r = 0.49; reactor 2: r = 0.78 and reactor 3: r = 0.32; p = 0.05). However, no clear correlation was found between NOB expression (nxrA and nxrB) and DO concentration. At WWTP B, a negative correlation was observed between nxrA expression levels and DO concentration (r = - 0.34, p = 0.05). However, DO concentration showed no clear correlation with amoA and nxrB expression levels. The phylogenetic analysis of nxrB populations in both the plants also revealed similarities that are closely related to uncultured Nitrospira spp., nitrite oxidoreductase subunit B, which has been implicated in complete nitrification (COMAMMOX). These observations indicate a need for more research effort using next-generation sequencing to identify and quantify novel nitrifying bacterial including COMAMMOX and ANAMMOX in WWTPs that were previously unachievable using conventional molecular techniques. In conclusion, this study revealed that the fine bubble diffused aeration operated at relatively high DO concentration was able to effectively remove ammonia in both plants resulting in stable and high nitrification rates even at different seasons and loading rates. It also promoted compact flocs with good settleability as well as facilitated optimal and diverse functional nitrifying bacterial community structure and activity.