Faculty of Applied Sciences

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Assessment of biomarkers for normalization of SARS-CoV-2 concentrations in wastewater
(2023-09) Osman, Aaliyah; Sheena, Kumari; Amoah, Isaac Dennis; Bux, Faizal
During 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.
The efficiency of lytic enzymes for Ascaris eggs inactivation
(2024-05) Zikalala, Thabiso; Stenström, Thor Axel; Amoah, Isaac Dennis; Buckley, Christopher Andrew
Faecal sludge (FS) contains organic matter that can enhance soil quality if pathogenic organisms, such as Ascaris lumbricoides, are reduced to safe levels. A. lumbricoides is a highly resistant nematode used to assess the efficiency of FS and wastewater treatment. The standard for reusing FS is to reduce Ascaris spp eggs to <1 egg/g for helminth reduction. However, in many developing countries, untreated FS is used as a soil enhancer without following proper guidelines. There are a number of FS treatment technologies in use, such as compositing, drying and the use of calcium carbonate. These techniques are able to reduce the pathogen concentration, especially Ascaris spp to accepted standard prior to application. Other alternative FS treatment technologies are under assessment and development. The objective of this study was to evaluate the use of lytic enzymes to inactivate Ascaris spp eggs by targeting the egg shell of the parasite. Ascaris spp egg shells consist of protein, chitin, and a lipid layer. Therefore, it may be possible to disintegrate Ascaris spp by targeting these layers of the egg shell with enzymes such as protease, chitinase, and lysozyme, which are commonly produced by indigenous soil bacteria. Ascaris spp eggs were detected in all FS samples collected from urine dehydration diversion toilets (UDDTs) in Durban, with an average concentration of 976-1118 helminth eggs/gram of FS. However, the viability of recovered eggs from FS was low and eggs were in different stages of development. This would negatively affect the experiments and produce inconsistent data. Therefore, for the lytic enzyme inactivation experiments, commercially bought eggs were used instead of the helminth eggs recovered from the UDDTs FS. Exposure of the eggs to commercial lytic enzymes was done following three different approaches. Firstly, Ascaris spp eggs were exposed to each of the enzymes individually, a second approach was employed where the eggs were consecutively exposed to the eggs with a rinse in between, and lastly, the eggs were exposed to a mixture of all three enzymes at once. Viability of the Ascaris spp eggs was determined via incubation, followed by microscopic examination of the eggs for visible motile larvae. For the single enzyme exposure, chitinase was the most detrimental enzyme resulting in a reduction of viability by 34% at room temperature. Exposure of the Ascaris spp eggs using the enzymes in series, achieved an egg viability reduction of up to 90 % at 37 °C, after 5 days exposure. Exposing the eggs to the mixed enzymes gave a reduced egg viability of 75 % at 37 °C, after 5 days exposure. Furthermore, the detection of microorganisms in the FS capable of producing lytic enzymes used was also confirmed. Enzymes were produced using selective media that resulted in the production of enzymes where the concentration (mg/L) and specific activity (U/mg) was determined to 0,68 mg/mL (0,08 U/mg) of protease from nutrient broth enriched with 2.5% milk, 3,17 mg/mL (0,006 U/mg) of chitinase produced from 1% colloidal chitin, and 7132 mg/mL (2600 U/mg) lysozyme from nutrient broth enriched with 1% Micrococcus lysodeikticus culture. The lytic enzymes showed to have an antagonistic effect on the Ascaris spp eggs. This therefore serves as a proof of concept that lytic enzymes produced by microorganisms found in FS could potentially be used for the inactivation of Ascaris spp eggs. However, further work is required focusing on enhancing the enzyme production, testing of the inactivation potential of these enzymes in the presence of solids and other materials present in FS, and finally the technique for field application of such technology.
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 Dennis
Microplastics (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.