School of Life Sciences

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Isolation and characterization of bacteriophages from wastewater as potential biocontrol agents for Escherichia coli.
(2022) Ntuli, Nontando Percevierance.; Schmidt, Stefan.
Host-specific lytic bacteriophages have regained momentum as an alternative treatment option to control and eliminate pathogenic bacteria. This study aimed to isolate, characterize and evaluate the potential application of Escherichia coli phages as a biocontrol agent in wastewater. In this study, four lytic Escherichia coli phages were isolated from wastewater for biocontrol purposes, using the double-layer method with E. coli (ATCC-25922) as a host. The phage morphology was characterized using transmission electron microscopy, with further parameters such as host range, phage stability at different temperatures, and pH values analyzed additionally. The genome of two selected phages (NPS and NPM) was sequenced, and the capacity of the phage isolate NPM to eliminate E. coli from artificial wastewater was evaluated and compared to conventional chlorination. All the four phage isolates showed typical T4 phage appearance with isometric capsids and contractile tails of different sizes, matching the family Myoviridae in the order Caudovirales. They exhibited a narrow host range limited to E. coli isolates, with two exceptions: phage NPS and NPM additionally lysed Salmonella Typhimurium (ATCC-14028). The four phage isolates were even able to lyse MDR (multidrug-resistant) E. coli isolates, such as the strain FP29. The four phages had burst sizes ranging from 70-115 per host cell and a latency period of 10- 20 minutes. All the four bacteriophages were stable at pH 5-9 but completely inactivated at pH 12. Exposure to 60°C for 10 minutes reduced phage titers by 1.5- log, while exposure to 80°C for 10 minutes completely inactivated all four phage isolates. The two genomes (NPS and NPM) were 99% identical and had similar sizes (169 536 bp), but phage NPS differed from phage NPM in view of its host range and plaque morphology. Another difference observed at the genome level was a shift of coding sequences between phage NPS and NPM. Phage isolate NPM achieved a 3.5- log reduction of E. coli cells present in artificial wastewater at an MOI of 0.1 in 120 minutes. A 90-minute chlorine treatment achieved a log reduction in the same range, highlighting that phages have the potential as environmentally friendly biocontrol agents in wastewater treatment.
Monitoring the efficacy of a lowland instream barrier on the lower uThukela River and the importance of river connectivity.
(2023) Van Zyl, Bradley Bruce.; Downs, Colleen Thelma.
Freshwater is the key to life on earth, with rivers being the pathway that allows it to run from mountains to the ocean, performing many important functions along its way. Humans rely on the many ecosystem services that freshwater provides, such as water quantity provision for domestic, industrial, and agricultural processes, food supply, water purification, and recreation and tourism, to name a few. Water-scarce countries, such as South Africa, are particularly vulnerable to water shortage issues and require many water supply solutions, like dams, weirs, and transfer schemes, to harvest the available rainfall. The uThukela River in KwaZulu-Natal is not only an important resource within its catchment but also to external catchments through inter-basin water transfer schemes, including that of the Thukela-Vaal transfer, which feeds the economically important hub of South Africa, the Gauteng Province. The lower uThukela River is a highly stressed system, with synergistic effects from multiple stressors relating to water quality, water quantity, habitat alterations, and wildlife disturbance affecting it. Additionally, the Lower Thukela Bulk Water Supply Scheme (LTBWSS) weir was recently constructed in its lower reaches, approximately 20 km upstream of the uThukela Mouth to the Indian Ocean, to provide bulk water to surrounding local municipalities. Due to the rich diversity of fish species in the region, particularly those with marine, estuarine, and freshwater migratory patterns, a fishway was incorporated into the design of the weir to facilitate fish movements over the weir. This study evaluated the impact that the LTBWSS weir has on fish community structures in the region and additionally assessed the efficacy of the fishway in allowing fish movements through it. Various sites were selected upstream, downstream, and on the tributary Mandeni ii Stream to assess environmental variables driving fish community structures, with passive and active sampling methods used to assess the fishway's functionality. Fish collection occurred at three sites upstream of the LTBWSS weir, four sites downstream of it on the uThukela River, and two sites on the Mandeni Stream. Abiotic variables relating to water quality, velocity, depth, and habitat were collected along with fish to determine which environmental variables were driving the fish communities at these sites. Multivariate analyses indicated that available substrate and cover, the average depth, and temperature were drivers of the fish communities in the study. Upstream sites showed lower species richness compared with downstream sites, with fish communities largely made of freshwater species and few euryhaline species. Additionally, individual species showed different responses to different environmental variables. Furthermore, since the construction of the LTBWSS, the loss of previously highly abundant cichlid species has occurred in the region. This is likely because of the synergistic effects of stressors created by it, such as the disruption of fine sediment transport, water abstraction, and pollution. Passive assessment of the fishway's efficacy in catering for migratory species used PIT telemetry. Budget constraints only allowed the installation of a single PIT antenna at the upstream entrance of the fishway, which was able to assess the upstream migration of fish from downstream. The results found that only eight individuals representing three species managed to navigate the fishway during the study successfully. This included Oreochromis mossambicus, Labeo molybdinus, and Clarias gariepinus. Active sampling involved electrofishing three key locations in the fishway on a monthly basis. Results showed that small-size classes of fish largely dominated the fishway and that the downstream entrance had the highest abundances and species richness. Further research on the role of the fishway in maintaining river connectivity is recommended. iii The outcomes of this study showed the importance that water resource managers have in maintaining the resource for humans and the environment. Knowing individual species' responses to environmental variables allows their populations to be better managed. Additionally, the outcomes of this study showed the importance of river connectivity past a barrier and highlighted the need for effective fish passage solutions in South Africa. It emphasised the need to better understand the migratory requirements of local fish to build better fish passage structures. Major stressors to be addressed include the impacts caused by barriers relating to flow releases, migration blocks, and habitat alteration upstream and downstream of them. Furthermore, the proper management of fish passage structures is essential to their functionality, which includes regular monitoring of the fishway for issues such as debris blockages and swiftly finding solutions to them to ensure that no undue delays or stress may occur for migratory fish.
The evaluation of the health status of Clarias gariepinus from the Inanda and Nagle dams in KwaZulu-Natal, South Africa.
(2021) Mdluli, Siphosakhe.; Lebepe, Jefferey.; Vosloo, Dalene.
The uMgeni River is one of the most polluted freshwater ecosystems in KwaZulu-Natal. This river is home to about 48 fish species. The study aims to determine the effects of water quality on the health of Clarias gariepinus from the Nagle and Inanda dams. Water variables were measured in situ using a YSI meter. Water and sediment samples were collected at three sites in each dam. Fish were collected and euthanized by severing the spinal cord. Different fish biometrics were measured. Fish tissues were preserved based on the analysis to be carried out. Neutral to alkaline water pH was recorded at both dams. Although the Inanda Dam exhibited higher total nitrogen concentration, both dams were mesotrophic, whereas the phosphate concentration at the Inanda Dam was eutrophic. Generally, the Nagle Dam showed good quality water compared to the Inanda Dam. The Inanda Dam fish population showed a relatively higher prevalence of alterations in the gills and liver than fish from the Nagle Dam. The degree of alterations showed some variability within each population, however, there was no significant difference (p>0.05) between the two populations. Both populations exhibited organ indexes less than 20, hence, moderate alterations. Fish ovaries and testis showed slight alterations at both dams. Acetylcholinesterase (AChE) brain activity recorded no significant difference in between the dams (p>0.05). Male populations recorded no significant difference in vitellogenin (VTG) induction between the dams (p>0.05). Despite, AChE activity and VTG induction showing no significant difference between the two populations, there was a great variability within each population. The lowest AChE activity as well as the highest VTG level were observed in the Inanda Dam fish populations. Both histopathologic and biochemical biomarkers are signs of increased pollution effect at the Inanda Dam. Nevertheless, it is evident that the uMgeni River system which is supposed to be providing sanctuary to aquatic biota is becoming deteriorated. These findings provide baseline data or a point of reference for future studies as it is the first of its kind in this river system.
The use of zebrafish to assess water quality and remediation efforts.
(2023) Zondi, Thandolwethu Beauty.; Hewer, Raymond.
Although wastewater effluents continue to be significant polluters of aquatic ecosystems in developing countries with limited water resources, little is known about the ecotoxicity induced by these effluents on fish throughout their early life stages. Several wastewater treatment plants (WWTPs) in South Africa (SA) do not adequately meet the minimal wastewater treatment requirements established by the country's Department of Water and Sanitation (DWS). Moreover, contaminants of emerging concern (CECs) originating from synthetic or natural sources, are widely distributed in aquatic environments of SA. This includes a broad range of natural and chemical compounds, such as aspirin (44243 ng/L), Fluoroquinolones (27100 ng/L), Atenolol (25900 ng/L), Nalidixic acid (25234 ng/L) and Ciprofloxacin (20514 ng/L). In addition to chemical compounds, endocrine disrupting chemicals, pharmaceuticals and personal care products are also distributed in the water systems. In the process of wastewater treatment, agents such as flocculants, coagulants, chemical precipitants (e.g., calcium hydroxide or sodium hydroxide) and chlorine disinfectants are utilized in wastewater treatment settings. However, research to understand the adverse effects that can be caused by these agents on aquatic organisms is still ongoing in SA. In order to bridge this knowledge gap, advanced techniques could be employed to help reveal adverse effects of wastewater as well as any shortcomings of current water remediation techniques. Using an appropriate aquatic model organism with highly conserved physiological pathways present in higher vertebrates (including humans), a rich behavioural repertoire, and occurrence in a variety of habitats would be a novel approach. To this effect, this study employed zebrafish with the aim to monitor six distinct wastewater samples from various regions of SA and to assess the effectiveness of currently used water remediation techniques such as chlorination. Two wastewater effluents, namely, Southern Works Final Effluents (SWFE) and Jacob’s Incoming (JB) alerted potential toxicity during chemical characterization with suboptimal pH (SWFE = 9.02 ± 0.16 and JB = 5.65 ± 0.02) and total alkalinity of zero (0 mg/L) detected for both effluents. The lethal toxicity of these effluents was seen by the elevation of mortality rate up to 77 ± 2.89 % and 100 ± 0.00 %, respectively for SWFE and JB at 40 %, with corresponding LC50 values of 17.77 % and 16.46 %. The zebrafish jaw and face, heart, brain, fins, notochord, somite and tail were significantly deformed (p < 0.05) post-exposure to these effluents, as revealed by morphological scores upon the analysis of the zebrafish’s body structure. Moreover, there was a delay in development due to the aforementioned effluents, unsuccessful hatching, craniofacial abnormalities, pericardial and yolk sac oedema, notochord abnormality somite defects and spinal cord curvature. In addition, locomotor activity of zebrafish was inhibited following observation of distance travelled, frozen moments, acceleration rates, swimming trajectories and exploration rate. Surprisingly, safety of these wastewaters was restored by chemical precipitation revealing non-lethal pH ranges of 6.02 - 8.02 and 6.65 - 7.65 for SWFE and JB, reducing the mortality rate to non-significant levels (p > 0.05) compared to the control. Also, sodium bicarbonate (NaHCO3) at 120 mg/L was found effective at supplementing the wastewater total alkalinity. In contrast, Amanzimtoti water before and after chlorination (TB and TA), Incoming Badulla (IB) and Chatsworth Incoming (CI) exhibited no consistent lethality effects on zebrafish and induced no apparent stress as demonstrated by insignificant expression (p > 0.05) of the stress protein: heat shock protein 70 (HSP70). However, the insignificant mortality v rate (p > 0.05) in the water tested before (TB) and after (TA) chlorination appeared to be the same (~25 %) indicating that chlorination is not enough at completely remediating wastewater. Our study is a pioneer in evaluating the ecotoxicological impact of wastewater effluents from localized regions of a developing country like South Africa in relation to the adjustment of water quality parameters for the neutralization of contaminants. To better understand emerging contaminants released as effluents in SA's water bodies and their interactions with aquatic organisms at the adult stage, more studies needs to be developed.

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