Efficacy and mechanisms of antiretroviral drugs removal by algaefrom wastewater treatment plants
Authors:   , Reddy Karen 
Published: September 2024

The 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.