Theses and dissertations (Applied Sciences)
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Item Efficacy and mechanisms of antiretroviral drugs removal by algaefrom wastewater treatment plants(2024-05) Reddy, Karen; Bux, Faizal; Kuttan Pillai, Sheena Kumari; Renuka, Nirmal; Moodley, BrendaThe presence, risks, and fate of pharmaceutical pollutants in the environment have raised concerns worldwide. South Africa, with the largest population consuming antiretroviral (ARV) drugs in Africa, faces challenges in efficiently removing these compounds from water bodies. This study's primary focus was to investigate the efficiency and mechanisms of nevirapine (NVP) removal by algae isolated from wastewater treatment processes. It included the isolation and screening of algal strains from wastewater treatment plants for their potential to remove ARV drugs, optimizing culture conditions to enhance removal efficiency, determining the potential mechanisms employed by selected algal strains for NVP remediation, and assessing the associated metabolic responses of algal cells to NVP using gene expression and metabolomics analyses. Eleven green indigenous fresh water microalgal isolates were screened from wastewater treatment plants (WWTPs) in KwaZulu-Natal, resulting in the selection of two strains, Coelastrella tenuitheca and Tetradesmus obliquus, based on their growth rates, biomass productivity and toxicity tolerance. In the ecotoxicity study, the calculated IC50 values of NVP (0–100 mg L−1) on selected algal strains after 96 h of exposure were 23.45 mg L−1 (C. tenuitheca) and 18.20 mg L−1 (T. obliquus), which far exceeds the concentration of NVP found in wastewater. Hence, T. obliquus and C. tenuitheca was selected for further NVP remediation studies using different cultivation conditions. A concentration range of 0-4000 ng L-1 of NVP was tested to assess the potential for NVP removal by both microalgae (autotrophic cultivation). Lower concentrations of NVP (up to 200 ng L−1) have shown to have a positive impact on microalgae growth. Specifically, in T. obliquus, the highest dry cell weight of 941.27 mg L−1 was obtained when exposed to a NVP concentration of 50 ng L−1. Both microalgae showed varying removal efficiencies (19.53–74.56%) when exposed to different NVP concentrations. During the late log phase on day 8, T. obliquus achieved the highest NVP removal efficiency, removing 74.56% of the NVP, while C. tenuitheca achieved a removal rate of 48% at an NVP concentration of 50 ng L−1. The photosynthetic efficiency (Fv/Fm and rETR) of both microalgal species was found to be unaffected by environmental concentrations of NVP (up to 4000 ng L−1) during the mid-log phase of growth. Furthermore, the scanning electron microscopy (SEM) analysis demonstrated that both algal species produced distinct ridges on their cell surfaces after NVP uptake. Additional evaluations were conducted on the microalga, T. obliquus, for the removal of NVP at 4000 ng L-1, as well as their cellular response (expression of antioxidant enzymes and metabolomics) and biomass production under different cultivation modes (autotrophic, heterotrophic, and mixotrophic). The highest NVP removal efficiency was observed under mixotrophic (80.13%) growth on day 8, whilst heterotrophic and autotrophic cultivation modes removed 70.30% and 64.40%, respectively. Mass balance calculations showed that the primary removal mechanism was identified as biodegradation, with a relatively low contribution from bioadsorption (2.39-3.36%) and bioaccumulation (0.55- 0.87%). Fourier-transform infrared (FTIR) spectroscopy results of harvested microalgal cells displayed bands in the region of 950-1000 cm-1, indicating the presence of aromatic C-H rings found in NVP. Additionally, 6 possible biotransformation products of NVP were identified by untargeted liquid chromatography-time of flight mass spectrometry. Additionally, under autotrophic conditions, the gene expression analysis revealed heightened activities of superoxide dismutase (sod1), glutathione peroxidase (gpx1) and catalase (cat2) in T. obliquus. The upregulation of antioxidant genes enhances the organism's ability to defend against oxidative stress induced by NVP. The expression levels of antioxidant genes were significantly reduced during heterotrophic and mixotrophic growth, suggesting microalgae can overcome oxidative stress with glucose supplementation. To further investigate the cellular level response of microalgal cells to NVP, metabolomic analysis was carried to out to identify and quantify key algal metabolites during mixotrophic cultivation. The increase in activity of the fatty acid biosynthesis pathway and carbohydrate synthesis was observed by T. obliquus in the presence of NVP under mixotrophic growth conditions. The findings from this study emphasize the significant potential of microalgae in the field of ARV drug remediation.Item A combined metagenomics and metatranscriptomics approach to assess the occurrence and reduction of pathogenic bacteria in municipal wastewater treatment plants(2023-05) Conco, Thobela; Bux, Faizal; Kumari, Sheena; Stenström, Thor-Axel; Ismail, ArshadThe emergence and spread of pathogens, antibiotic resistant bacteria (ARB) and antibiotic resistant genes (ARG) through insufficiently treated effluents from wastewater treatment plants (WWTP) pose a risk to human health and the environment. The present study focused on assessing the occurrence, prevalence and fate of dominant pathogenic bacteria, ARGs and mobile genetic element (MGE) in different WWTPs in Durban, Kwa-Zulu Natal, South Africa. The samples were taken from three wastewater treatment plants with different configurations, including trickling filter (TF), biological nutrient removal (BNR), and conventional activated sludge processes (CAS). Total genomic DNA and RNA were extracted from the samples for metagenomic and tracriptomic analysis. A total of 23 pathogenic bacterial genera, including enteric and emerging opportunistic pathogens, were detected in the samples. Acinetobacter spp. and Aeromonas spp. were the predominant pathogens in influent metagenomes, while Escherichia coli and Acinetobacter spp. dominated influent transcripts. Based on Shannon-Wiener indices, the diversity of bacteria increased from influents to final effluents in two treatment plants. ARGsthat confer resistance to aminoglycosides, beta-lactamases, tetracycline and sulfonamides were abundant in both influent and effluent samples. Results further exposed that MGE-ARG associations were the main drivers of ARG persistence to final effluents. This included 5 plasmids: R338-R151 (sulI), pRH-1238 (strB), pPM91 (aadA), pRH-1238 (aadA4-5), pRH-1238 (sulII); two class 1 integrons (aadA and arr) and 1 transposon Tn4351 (tetX). In transcripts, the MGE-ARG associations showed two plasmids: pRH-1238 (aadA) and pPM91 (aadA) and one hybrid plasmid R338-R151 (sulI). The study investigated the potential impact of operational parameters (dissolved oxygen (DO), total suspended solids (TSS), pH and temperature) on selected bacterial pathogens (Aeromonas spp, Acinetobacter spp., Pseudomonas aeruginosa and Klebsiella pneumoniae) and their fates at different stages of the three WWTPs. Principal component analysis (PCA) showed that temperature, DO, and pH were the most relevant factors influencing pathogen abundance. Among the studied pathogens, Acinetobacter spp. was the most prevalent in the influent samples, followed by Aeromonas spp. As for the aeration samples, Aeromonas spp. was dominant in WWTP1 (CAS configuration) and WWTP2 (BNR configuration), while Acinetobacter spp dominated in WWTP3 (BNR configuration). Acinetobacter spp., Aeromonas spp., and Pseudomonas aeruginosa were the dominant ones in secondary effluents, with their dominance varying across the sampling period. In the final treated effluent, Acinetobacterspp., Aeromonas spp., and P. aeruginosa were dominant, with their dominance varying from sample to sample. Additionally, free living amoebas (FLA) were also investigated for their contribution to the propagation and persistence of pathogens in secondary and final effluents. Using the conventional isolation technique, FLAs were isolated from different samples. The internalized bacteria and ARGs were further identified using metagenomic analysis. Metagenomic profiles identified nine species belonging to Acanthamoeba and two species belonging to Entamoeba. A. castellini was the most prevalent dominant species detected in effluent and final effluent samples of all three WWTPs. P. aeruginosa, S. maltophilia, A. spanius, C. testosteroni, and E. cloacae were the most dominant bacterial endosymbionts detected. Among these, S. maltophilia and P. stutzeri were detected in FLAs isolated from the final treated effluents indicating their prevalence in the chlorinated effluents. The presence of ARGs within FLAs were also ascertained. Genes conferring resistance to aminoglycosides (aadA); trimethoprim (dfrA15 and dfrA5); sulfonamides (Sul1 and SulII), macrolides (msrA, mphC); rifamycin (Arr); quinolones (qnrE1) and tetracyclines (TetA and TetG). SulI, dfrA5, AadA, dfrA15, SulII, TetA, TetG and qnrE1 were among the resistance genes that persisted into final effluents. The results of this study have contributed significantly to our current understanding of pathogens, particularly the dominant pathogens and the role of FLAs in the dispersal of pathogens and ARGs into the environment via WWTPs. The study also indicatesthat the conventionally treated effluents may still contain human pathogens, ARGs, and MGEs, which may contribute to the propagation of emerging pathogens and antibiotic resistance in the receiving environment.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.