Faculty of 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 Evaluation of biohydrogen production potential of sugarcane bagasse using activated sludge in a dark fermentation process(2016) Reddy, Karen; Bux, Faizal; Kuttun Pillai, Sheena Kumari; Gupta, Sanjay KumarAnaerobic dark fermentation is an efficient biological process to produce hydrogen from waste material. In South Africa, this technology has not been explored adequately to extract energy from biological wastes. Within the KwaZulu Natal region of South Africa, the sugar industry is a prominent venture that produces mass quantities of sugarcane bagasse amongst other waste products. This by-product can be an ideal source of substrate for biohydrogen generation. In this study, sugarcane bagasse was used as the main substrate for biohydrogen production by anaerobic fermentation using sewage sludge as the inoculum. Different pre-treatment methods were employed to maximize the release of fermentable sugars from the lignocellulosic biomass. Among the different pre-treatment methods employed, the maximum sugar yield (294.4 mg/g) was achieved with 0.25% H2SO4 for 60 minutes at 121°C. Prior to inoculation, the sewage sludge was also subjected to thermal pre-treatment to eliminate methanogens. Thermal pre-treatment of inoculum sludge for 30 min was effective in eliminating methanogens. Fluorescence in situ hybridization was used to positively identify the hydrogen producing bacteria present before and after treatment. The pre-treated substrate and inoculum was integrated into a dark fermentation process to further optimize the effect of pH, substrate to biomass, iron and magnetite nanoparticles on hydrogen production. The maximum hydrogen production (1.2 mol/mol glucose) was achieved at a pH range of 5-6, a substrate to biomass ratio of 3.5, and iron and magnetite nanoparticle concentration of 200 mg/L. Microbial analysis using quantitative polymerase chain reaction has confirmed the dominance of Clostridium spp. in the reactor. The highest hydrogenase gene activity (number of copies of hydrogenase gene expression/ng DNA) was recorded in the reactor supplemented with magnetite nanoparticles with lowest being in the raw sludge. There was a direct positive correlation between the hydrogenase gene copy number and the hydrogen yield obtained at different reactor conditions. Scanning electron microscopy was a useful to visually analyse the interaction of microorganisms with activated sludge. This study highlights the significance of anaerobic microorganisms from waste sludge being able to utilize agricultural waste material to produce biohydrogen which could be further scaled up for continuous hydrogen production. In addition, statistical tools used to predict the possible sugar (Design of experiments) and hydrogen yields (Gompertz model) produced would be helpful in saving time during full-scale operation of biohydrogen producing reactors.