Theses and dissertations (Applied Sciences)

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The accumulation of heavy metals by aquatic plants
(2003) Maharaj, Saroja; Moodley, Kandasamy Govindsamy; Southway-Ajulu, F. A.; Baijnath, Himansu
The pollution of water bodies by heavy metals is a serious threat to humanity. The technique known as phytoremediation is used to clean up these polluted water bodies. The accumulation of heavy metals by aquatic plants is a safer, . cheaper and friendlier manner of cleaning the environment. The aquatic plants -studied in this project are A.sessilis, P.stratiotes, R.steudelii and T.capensis. The accumulation of heavy metals in aquatic plants growing in waste water treatment ponds was investigated. The water, sludge and plants were collected from five maturation ponds at the Northern Waste Water Treatment Works, Sea Cow Lake, Durban. The samples were analysed for Zn, Mn, Cr, Ni, Pb and Cu using ICP-MS. In general it was found that the concentrations of the targeted metals were much lower in the water (0.002 to 0.109 mg/I) compared to sediment/sludge (44 to 1543mg/kg dry wt) and plants (0.4 to 2246 mg/kg dry wt). These results show that water released into the river from the final maturation pond has metal concentrations well below the maximum limits set by international environmental control bodies. It also shows that sediments act as good sinks for metals and that plants do uptake metals to a significant extent. Of the four plants investigated it was found that }t.sessi[ir (leaves, roots and stems) and }A.sessilis (roots and stems) are relatively good collectors of Mn and Cu respectively. These findings are described in the thesis. The concentration of heavy metals in the stems, leaves and roots of the three plants were compared to ascertain if there were differences in the ability of the plant at different parts of the plant to bioaccumulate the six heavy metals studied.
Analysis of selected organic pollutants in water using various concentration techniques
(2014-08-08) Ramphal, Sayjil Rohith; Moodley, Kandasamy Govindsamy; Chetty, Deenadayalan Kisten
Among persistent organic pollutants, chlorobenzenes are some of the most frequently encountered compounds in aqueous systems. These compounds can enter the environment via natural and anthropogenic sources, and are ubiquitous due to their extensive use over the past several decades. Several chlorobenzene compounds, once in the environment, can biologically accumulate, and are reputed to be carcinogens and extremely hazardous to health. Several chlorobenzenes are listed as priority pollutants by the United States Environmental Protection Agency. Excessive exposure to these compounds affects the central nervous system, irritates skin and upper respiratory tract, hardens skin and leads to haematological disorders including anaemia. In spite of these harmful effects, chlorobenzenes are still used widely as process solvents and raw materials in the manufacture of pesticides, chlorinated phenols, lubricants, disinfectants, pigments and dyes. In the light of the above, it is imperative to monitor the levels of chlorinated benzenes in all types of surface waters, using low-cost but sensitive methods of preconcentration and detection. In this study, a simple and relatively cheap preconcentration method using direct immersion solid phase microextraction (DI-SPME) followed by gas chromatography equipped with a flame ionisation detector (GC-FID) was developed for the analysis of 7 chlorinated benzenes in dam water. Experimental parameters affecting the extraction efficiency of the selected chlorobenzenes, such as fibre type, sample size, rate of agitation, salting-out effect and extraction time, were optimised and applied to the Grootdraai Dam water samples. The optimised method comprises the use of a 100 µm polydimethylsiloxane (PDMS) fibre coating; 5 ml sample size; 700 revolutions per minute rate of agitation and an extraction time of 30 minutes. The calibration curves were linear with correlation coefficients ranging from 0.9957–0.9995 for a concentration range of 1–100 ng/ml. The respective limits of detection and quantification for each analyte was as follows: 1,3-dichlorobenzene, 0.02 and 0.2 ng/ml; 1,4-dichlorobenzene, 0.04 and 0.4 ng/ml; 1,2-dichlorobenzene, 0.02 and 0.2 ng/ml; 1,2,4-trichlorobenzene, 0.3 and 2.7 ng/ml; 1,2,4,5-tetrachlorobenzene, 0.09 and 0.9 ng/ml; 1,2,3,4-tetrachlorobenzene, 0.07 and 0.7 ng/ml; pentachlorobenzene, 0.07 and 0.7 ng/ml. Recoveries ranged from 83.6–107.2% with relative standard deviation of less than 9%, indicating that the method has good precision, is reliable and free of matrix interferences. Water samples collected from the Grootdraai Dam were analysed using the optimised conditions to assess the potential of the method for trace level screening and quantification of chlorobenzenes. The method proved to be efficient, as 1,3 dichlorobenzene, 1,4-dichlorobenzene and pentachlorobenzene were detected at concentrations of 0.4 ng/ml, 1.7 ng/ml and 1.4 ng/ml, respectively.
Antioxidant and anticancer properties of bioactive peptides from Lablab purpureus
(2023-05) Sipahli, Shivon; Mellem, John Jason
Cancer can be described as a non-communicable disease that develops from defective cells in the human body and grows uncontrollably. Globally in 2020, statistics revealed that the disease had affected approximately 19.3 million people. With about 51% of these cases resulting in death. Cancer treatments usually comprise surgery, chemotherapy, radiotherapy, or a combination of the three. Traditional therapies such as chemotherapy and radiotherapy drugs are effective at shrinking tumours. However, a key disadvantage is that these drugs are unable to distinguish between cancerous and healthy cells. Subsequently, the human body experiences many adverse side effects such as hair loss, vomiting, lowered immunity, and a general deterioration of health. Drug resistance and rejection are also major disadvantages of these traditional therapies. Alternative therapies are required to mitigate these drawbacks. The vital factor to consider for alternative treatments should be to selectively target cancer cells thereby alleviating the unwanted side effects. Compounds derived from non-toxic edible plants have shown to have bioactive potential. These plants are regarded as non-toxic to the human body therefore they would be able to target the tumour cells alone. Plant compounds also provide additional protection such as their antioxidant abilities and apoptotic potential. Evidence suggests that bioactive peptides derived from legumes can act as both anticancer agents and strong antioxidants. This study investigated the bioactive potential of peptides derived from Lablab purpureus. This investigation began by assessing the antioxidant capacity (2,2-diphenyl-1-picrylhydrazyl-hydrate (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic (ABTS), superoxide radical scavenging and Ferric Reducing Antioxidant Power (FRAP) assays) and antiproliferative abilities (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)) of L. purpureus protein isolate and hydrolysates (alcalase, trypsin and pepsin). The hydrolysate and fractions of interest were selected based on the MTT assay with the pepsin hydrolysate selected for further apoptotic studies (caspase-3 and -7, and annexin V-PI). Thereafter, the pepsin hydrolysate was fractionated by ultrafiltration (molecular weight cut-off: <1, 3, 5, 10, >10 kDa). The 3 kDa fraction was further fractionated by RP-HPLC. Five peaks appeared on the chromatogram, however, fraction 2 was selected, for apoptotic investigations (caspase -3 and -9, p53 and annexin V-PI). Antioxidant studies are a good measure of the isolate or hydrolysate's ability to perform as a bioactive compound. The 50% inhibitory concentration (IC50) observed for the respective antioxidant studies showed the radical scavenging ability of the isolate and hydrolysates to be 1.81-4.47 mg/mL (DPPH), 1.73-2.42 mg/mL (ABTS), 1.36-4.4 mg/mL (superoxide radical scavenging) and 19.20-21.94 mg/mL (FRAP). Anticancer activity was substantiated by the peptides' ability to induce apoptosis. The pepsin hydrolysate was selected using the MTT assay (IC50 values of A549, 119.6; MCF7, 9.80 and HEK293, 13.86 µg/mL). Pepsin hydrolysate inhibited cancerous cells (A549 and MCF-7) while causing minimal damage to healthy cells (HEK293). Thereafter apoptotic markers, caspase 3/7 and annexin V-PI were quantified. Visualisation of cells in different stages of apoptosis was investigated by Annexin V-PI staining quantified by flow cytometry. During early apoptosis; A549, 42%; MCF-7, 17%; HEK293, 34%. Caspase 3/7 assay verified that the pepsin hydrolysate caused an increase in apoptotic activity. Caspase-3 and -9 activity of cells, determined by ELISA showed that Fraction 2 treated cancer cells (A549 - 0.067 ng/mL, 21.966 ng/mL, and MCF-7 - 0.137 ng/mL, 0.205 ng/mL respectively) had a greater caspase concentration over camptothecin (A549 - 0.029 ng/mL, 20.486 ng/mL and MCF-7 - 0.051 ng/mL, 0.112 ng/mL respectively). Tumour suppressor protein, p53, acts as a protective mechanism by initiating apoptosis in ‘suspicious’ cells. The A549 cell line showed the greatest p53 expression compared to MCF-7 and HEK293. Increased p53 can regulate signalling pathways leading to targeted apoptosis. Finally, annexin V-PI confirmed that Fraction 2 did induce apoptosis in the cells (cells in early apoptosis, A549, 85%; MCF-7, 90%; HEK293, 94%). Results from this study have shown that peptides derived from L. purpureus (specifically fraction 2) have potential anticancer abilities which may be attributed to their antioxidant and apoptotic abilities.
Application of lipid extracted algae in feed and energy production
(2019) Ansari, Faiz Ahmad; Bux, Faizal; Gupta, Sanjay Kumar; Guldhe, Abhishek
Microalgae are well considered to be promising feedstocks for biodiesel production. Microalgae can be grown under different types of cultivation conditions and their biomass has tremendous potential to be used as biofuel feedstock and for other applications such as feed, food, cosmetics, pharmaceutical etc. Despite the many benefits and the significant development in the field of microalgal biodiesel production, there are several challenges including high cultivation cost and developing efficient downstream processing methods. The biomass production cost is high, which significantly hinders the use of microalgae as a feedstock. Most of the available literature is focused on upstream, single strain and single product strategy, where mainly algal lipids are used for biofuel production. Hence, for improving the sustainability of the algal biofuel production processes and related process economics, a multiple applications approach using integrated biorefinery and exploiting microalgae for environmental benefits is required. To explore the microalgal biorefinery concept it is vital to understand the various cultivation conditions and applications of biomass in different sectors. There are various strategies, which have potential to make algal biofuel technologies more economically feasible and environmentally sustainable. Use of alternative culture media, improving the biomass production and the efficiency of downstream processing (drying, cell disruption, lipid extraction etc.) algal biofuel technology economical. Utilizing lipid-extracted algae (LEA) for energy and aqua feed application will maximize overall economic return and will leave minimal residues as by-product. The major focus of this thesis was to utilize LEA as substrate for biomethane production and protein source in aquaculture feed. However, effect of preceding steps such as microalgae cultivation, biomass drying and cell disruption on major metabolites extraction was also studied. Microalgae were cultivated in different medium (domestic wastewater and BG11) and their biomass yields and biochemical composition (lipid, protein and carbohydrate) were compared. Different drying and cell disruption techniques were employed for lipid extraction and their effect on lipid, protein and carbohydrate yields were evaluated. The yield of major metabolites on whole cell and LEA were also compared. Suitable solvent systems were selected for optimum lipid extraction from wet and dry biomass with minimal toxic effect on LEA metabolites so that LEA can be further used for biomethane and aquaculture feed production. The choice of microalgae at large scale depends upon the number of factors such as their adaptability to large-scale cultivation, biomass production, major metabolites content, robustness towards the open system cultivation and contamination. In this study, S. obliquus and C. sorokiniana were cultivated in wastewater and BG11 medium at laboratory scale. Both strains are indigenous to KwaZulu-Natal. C. sorokiniana showed lower biomass and major metabolites (lipid, protein and carbohydrate) production at large scale compared to S. obliquus. Considering better adaptability to open cultivation, high biomass and metabolites yields, S. obliquus strain was selected for the LEA application study. Microalgae species, C. sorokiniana and S. obliquus were cultivated on BG11 and using different ratios of raw domestic wastewater and post-chlorinated wastewater as nutrient media. The cultivation of S. obliquus and C. sorokiniana showed biomass yield of 1.2-3.5 and 0.78-1.8 g L-1 in BG11 medium, respectively. While biomass yield observed in wastewater was 0.59-1.59 g L-1 for S. obliquus and 0.67-1.45 g L-1 for C. sorokiniana. The higher biomass yield in BG11 medium attributed to the higher nutrient contents in this medium compared to wastewater. The lipid contents for S. obliquus and C. sorokiniana were 20 and 16.5% dry cell weight (DCW), respectively when grown using BG11 medium. While increases in lipid contents of 26.25 and 29.4% DCW were found for S. obliquus and C. sorokiniana, respectively when cultivated using wastewater. Similarly, carbohydrate contents for S. obliquus and C. sorokiniana were 18 and 17% DCW, respectively for BG11 medium. Increased in carbohydrate contents of 25% for S. obliquus, 28.4% DCW for C. sorokiniana were observed for wastewater. Microalgae tend to accumulate more lipids and/or carbohydrates under nutrient stress condition. The nitrogen and phosphorus contents in wastewater are lower than BG11 medium, which were responsible for stressed condition for microalgae. With limited nutrients in wastewater compared to BG11 medium, growth of microalgae is also lower which resulted in lower protein content. Protein content for S. obliquus and C. sorokiniana in BG11 medium were 37.83-48.8 and 25-35.3% DCW, respectively. The protein contents for S. obliquus and C. sorokiniana in wastewater medium were 16.4-27.29 and 15.8-27.3% DCW, respectively. The biochemical composition depends upon the nutrient composition of the medium and cultivation conditions. The two selected microalgae have shown potential for nutrient removal while cultivated in wastewater. The removal efficiency by S. obliquus was found to be 76.13% for COD, 98.54% for nitrogen and 97.99% for phosphate. Microalgae C. sorokiniana cultivation in wastewater removed 69.38% COD, 86.93% nitrogen and 68.24% phosphates. Increased lipid accumulation in the cells was also recorded in stressed conditions due to low nutrient availability from wastewater. After harvesting of microalgae from culture media, the water content in thick algal slurry (>85% DCW) lowers the products recovery. To overcome this challenge drying and cell disruption are required to enhance the efficiency of lipid extraction. Where drying and cell disruption increase the viability of biomass for lipid extraction process. Three biomass-drying techniques viz. sun, oven and freeze-drying and four-cell disruption techniques viz. microwave, sonication, osmotic shock and autoclave disruption were studied for their effect on recovery of major metabolites from S. obliquus. Microalgae metabolites recovery from whole cell and LEA were analysed and compared. The results showed that after lipid extraction, LEA still contained comparable protein to whole algae biomass however, the carbohydrate concentration was reduced. Oven drying exhibited the highest recovery of all the major metabolites followed by freeze-drying; sun drying however, showed lower yields. Despite lower metabolites recovery sun-drying technique is preferable at large scale due to its easy application and cost-effective nature. The main drawback of sun drying technique is weather dependence and required longer period to dry. The microwave and autoclave microalgal cell disruption improved the lipid yield but loss of other compounds was observed. In osmotic shock treatment, due to poor cell disruption efficiency low lipid were obtained and comparably lower protein loss was noticed during lipid extraction. Lipid extraction is crucial step for microalgae biodiesel production. Solvent-assisted lipid extraction is widely used technique for lipid recovery from dry or wet algae biomass. In a biorefinery approach, it is vital to choose appropriate solvents for the optimum lipid extraction whilst having minimal effect on the remaining metabolites (protein and carbohydrates) in LEA. LEA could be used for energy generation or aquaculture feed applications. Six commonly used organic solvents/ solvent systems were used for lipid extraction from wet and dry biomass. The results showed that the lipid extraction efficiency depends strongly on types of biomass as well as solvent systems selected. Lipid extraction from wet algal biomass could reduce the processing steps and save energy incurred in drying. However, the water present in wet algal slurry acts as a barrier, which results in lower lipid yield compared to the dry biomass. The results revealed that among all six-selected solvents, chloroform: ethanol (1:1 v/v) was most effective if wet biomass used specifically for lipid purpose only. To explore the biorefinery concept, isopropanol/hexane composition is the most suitable solvent system because it is less toxic and resulted in high protein (20.07% DCW) and carbohydrate (22.87%) yields in LEA. For dry algal biomass, chloroform: methanol (2:1 v/v) is an appropriate solvent system if biomass used especially for lipid (19.25%) extraction. If LEA to be used for energy and/or aquaculture feed application, DCM: methanol was found to be a suitable solvent system, which gave 32.79% protein and 26.92% carbohydrate yield. Comparatively hexane has lower lipid recovery but shown higher protein and carbohydrate yield in LEA. Due to less toxic, easy to scale up and inexpensive, hexane is preferable as a solvent for lipid extraction if LEA is to be further utilized at large scale for energy or feed application. Anaerobic digestion (AD) of organic residues is well-researched technology for biomethane production. Whole microalgae and LEA has promising potential for biomethane production. The anaerobic sludge used as inoculum for microalgal biomass digestion. Biomethane production from whole algae and products extracted algae highly depends on sludge to algae biomass ratio for higher methane production. The extraction of metabolites also changes the biochemical composition of residual biomass, which can affect the biomethane production. It is vital to understand the effect of various product-extracted algae and as well as pre-treated algae on the biochemical methane potential. In order to compare biomethane potential, four types of biomass were selected namely sun dried powder algae (SDPA), mild heat-treated algae (MHTA), LEA (using hexane as lipid extracting solvent) and protein-extracted algae (PEA). The average methane (CH4) production rate was ~ 2.5 times higher for protein and lipid extracted algae than for whole algae SDPA and MHTA whilst the cumulative CH4 production was higher for pre-treated algae. Highest cumulative CH4 production (318.7mL CH4 g-1 VS) was found for MHTA followed by SDPA (307.4mL CH4 g-1 VS). The CH4/CO2 ratios of 1.5 and 0.7 were observed for MHTA and LEA, respectively. Outcome of this objective revealed that pre-treatment process disrupts the microalgae cell walls, exposing intracellular material and increasing the surface area. The product-extracted algae changes the elemental composition, which decreases the cumulative gas yield CH4/CO2 ratio. Presence of high nitrogen in the form of protein produces ammonia (NH3) which inhibits the methane production. Therefore, it is imperative to use PEA biomass to improve the methane production yield than the whole cell biomass. Due to escalating price and unstable supply of fish meal (FM), alternative protein sources are used in aqua feed, however these sources do not meet to the requirement. The use of less expensive protein source in aquaculture feed as alternative to FM is required. Microalgae are primary producers in the food chain as well as a natural food for fish. Microalgal biomass is comprised of proteins, lipids, carbohydrates, pigments and many other bioactive compounds. The microalgal proteins have an appropriate balance of all essential amino acids, while lipids are rich in polyunsaturated fatty acids (omega-3 fatty acids, EPA, DHA). Whole algae contain all required ingredients while LEA also contain protein, carbohydrates, vitamins, bioactive compounds even though most of the lipid soluble nutrients have been removed. Thus, microalgae have promising potential to be used in aquaculture feed. Aquaculture production continues to increase globally, to meet the aquaculture feed demand algae supplemented aquaculture feed will play an important role in providing good quality fish. In this study, approximately 200 kg of microalgal biomass was harvested for the feed application. Due to lower toxicity, ease of availability and ease of recovery from mixture, hexane was used as a lipid extracting solvent at pilot scale to generate LEA. The 44 weeks (from juvenile to finisher stage) feeding trials were conducted to evaluate the effect of whole and LEA supplementation of S. obliquus strain on growth performance, disease tolerance, feed utilization, physiological activity, and fillet biochemical composition of Nile tilapia (Oreochromis niloticus). In the first trial, fish were fed with an algae free diet (control) and four experimental diets (2.5, 5, 7.5 and 10 wt%) as protein source of dried S. obliquus. The study showed that microalgae could be used as a protein supplement in the Tilapia feed for enhancement of morphological characteristics and nutritional value. The 7.5% and 10% supplementation of whole algal biomass in tilapia feed showed significant improvement in weight and length of the fish compared to the control. The daily body weight gain was 0.25 g higher in experimental groups than the control. The hepatosomatic index percentage was also higher in fish feed when 7.5% whole algae was used in fish feed as a protein source. The results also showed that 7.5% and 10% have better specific growth rate (1.57 and 1.5%), daily body weight gain (1.1 and 0.86 g), overall body weight gain (427.16 and 331.48 g), protein assimilation (43.96 and 40.46%) higher than the control diet fed fish. The survival rate of fish were 100% at every inclusion level. In second trial (44 weeks), two supplementations (7.5 and 10 wt%) of LEA as protein source were used in Nile tilapia diets. Results showed 7.5% and 10% LEA supplemented feed shown better growth performance than control. The protein content were 42.2%, 41.3% and 36.1% in tilapia fed with 7.5%, 10% LEA and control feed, respectively. The body weight gain, tilapia fed with 7.5% LEA shown 357 g while 10% LEA and control have 331.78 g, and 330.08 gm, respectively. The application of whole and LEA of S. obliquus in tilapia feed, shown appropriate supplementation level for tilapia feed at demonstration scale. This thesis presents advances in knowledge in the field of microalgae biorefinery research for pilot scale operations. This research work has covered various aspects such as effect of drying, cell disruption and lipid extraction on whole and LEA metabolites yield. The extraction of lipid from wet and dry microalgal biomass using various solvent systems provides a new insight for the selection of appropriate solvent systems, which can be used for the large-scale lipid extraction. The study on LEA for biomethane production enhances the understanding about the effects of different pre-treatments and product extractions on biomethane production. The results revealed that the supplementation of whole cell and LEA using S. obliquus for tilapia feed is safe therefore, can be used as an alternative protein source. The findings of this study have both academic and industrial value.
Assessment of a biological nutrient removal process for the remediation of edible oil effluent
(2002-05-10) Mkhize, Sandile Psychology; Bux, Faizal
Eutrophication is a natural process that is greatly aggravated by the action of man in the natural environment. Deterioration of South Africa's natural water resources results directly or indirectly from the discharge of industrial effluent rich in nutrient nitrogen and phosphorus. The South African edible oil refmeries generally discharge poor quality effluent which impacts negatively on the water resources and wastewater treatment installations. The main aim of this study was to assess the capacity of a laboratory scale effluent treatment process that will produce final effluent of acceptable quality with regards to organic load and phosphate concentration prior to its discharge into the municipal sewerage system. The study was conducted in three stages: wastewater characterization, treatability studies, and laboratory scale treatment investigations. After analysing various effluent parameters, treatability studies were conducted using an aerobic-anaerobic sequencing batch reactor with a total hydraulic retention time of 24 hours. The results showed an average of 75 % reduction of COD and more than 90 % removal of fats, oils and grease (FOG). Based on the results of effluent characterisation and treatability studies, a laboratory scale activated sludge effluent treatment process was designed and operated with two bioreactors (aerobic and anaerobic) in series. The system was operated for a period of one-month resulting in 70 % removal of COD and 4% reduction in phosphate (P04-P). After some structural and operational changes from the original design configuration, the system was the operated continuously for the duration of the study period. An optimum COD removal of 75 % and 107 mgll P04-P reduction was achieved during the last operational phase of the system. More than 95 % reduction in fats, oils and grease (FOG) had been achieved in both semi-continuously and continuously operated systems.b.7
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.
Assessment of heavy metals and pathogens removal from municipal wastewater using a constructed rhizofiltration system
(2018) Odinga, Christine Akinyi; Swalaha, Feroz Mahomed; Bux, Faizal; Otieno, Fredrick Alfred O.
Wastewater discharged from municipal treatment plants contain a mixture of organic contaminants, trace metals, enteric pathogens, viruses, and inorganic materials. The presence of such pollutants in wastewater poses a huge challenge to the choice and applications of the preferred treatment method. Conventional treatment methods are inefficient in the removal of some environmentally toxic pollutants and pathogens. This study evaluated the effectiveness of a constructed rhizofiltration system in the removal of heavy metals and enteric pathogens from municipal wastewater. The study was conducted at an eThekwini municipal wastewater treatment plant in Kingsburgh - Durban in the province of KwaZulu-Natal. The pilot-scale rhizofiltration unit included three different layers of substrates consisting of medium stones, coarse gravel and fine sand. The system had one section planted with Phragmites australis and Kyllinga nemoralis while the other section was unplanted and acted as the reference section. Influent and effluent, plant tissue and sediment from the rhizofilter were sampled bi-monthly for a period of two years and assessed for the presence and removal of selected enteric pathogens, trace heavy metals and changes in physicochemical and biological parameters using standard methods. Antibacterial potential of the two experimental plants was determined by the agar-well diffusion method using plant root exudates exposed to selected pathogenic bacteria. Observation of details of plant morphology, distribution and assessment of the metals attachment onto the various plant tissues was determined using images from scanning electron microscopy (SEM). The Langmuir model was used to assess the heavy metal adsorption of the plants. There was an increase in pH from 6.95 pH units to 7.55 pH units in the planted and 6.72 to 7.23 pH units in the reference sections. There was an average reduction in biochemical oxygen demand (BOD) by 79% and chemical oxygen demand (COD) by 75%. Suspended solids were reduced by 86% in the planted section and 59.8% in the reference section, Electrical conductivity was reduced by 7.7% in the planted section and 0.83% in the reference section, Total Dissolved Solids was reduced by 11.5% in the planted section and 3.5% in the reference section, temperature was reduced by 11.9% in the planted section and 1.2% in the reference section, while dissolved oxygen was raised by 10% in the planted section and 5% in the reference section. Turbidity was reduced by 9.7 NTU in the planted section and 9.1 NTU in the reference section, while alkalinity was reduced by 46.3% in planted and 45.5% on reference sections of the rhizofilter. There was a significant reduction in organic loading in the system which was statistically significant (phosphorous, p = 0.029; ammonia, p = 0.03).These average reductions and increases were observed after the system was fully established. The results indicate a comparatively better removal efficiency in the planted than the reference sections of the system. Considering the entire rhizofilter, heavy metals were accumulated at varying percentages of 96.69% on planted and 48.98% in reference sections for cadmium. Chromium was 81% and 24%, Copper was 23.4% and 1.1%, Nickel was 72% and 46.5, Lead was 63% and 31%, while Zinc was 76% and 84% in the planted and reference section of the rhizofilter respectively. The planted section had a much higher removal efficiency as compared to the reference section of the rhizofilter. The macrophytes were found to display some metals binding potential according to observations from SEM and EDX analysis. Significant amounts of Cu deposits were recorded on the roots of K. nemoralis at 0.31wt% with a peak at 0.6cps/eV than on P. australis which was at 0.31wt% with a peak at 0.6cps/eV. Further, higher deposits of Ni at 0.01 wt% with peak at 0.5 cps/eV and 0.0 wt% with peak at 0.2 cps/eV, Pb at 0.22 wt% with peak at 0.2 cps/eV and 0.21wt% with peak at 0.2 cps/eV were recorded on the roots of K. nemoralis and P. australis respectively. Kyllinga nemoralis was found to have greater metals adsorptive capabilities than P. australis. The planted and reference sections had varied removal capacities of between 45% and 98% for the various pathogens detected in the influent wastewater. For example, the concentration of coliphage was reduced by 94.6% in the planted section and 93.6% in the reference section, Candida spp. removal was 64.7% in the planted section and 62.5% in the reference section. Escherichia coli was reduced by 65%- 85% while Salmonella spp. was removed by 94% in the planted section compared to 78% in the reference section. Ascaris lumbricoides was reduced by 77% in the planted section and 53% in the reference section. Accordingly, higher pathogens reduction was achieved in the planted section as compared to the reference section of the rhizofilter. Root exudates from Kyllinga nemoralis were found to display a wider zone of growth inhibition at 9.97±0.19 mm compared to P. australis which had a zone of 8.63 ± 0.22 mm when exposed to cultured colonies of Escherichia coli.
Assessment of the anaerobic baffled reactor for treatment of vegetable oil effluent
(2001) Frost, Lee-Anne; Tivchev, G. N.
The vegetable oil industry produces effluent containing quantities of fat, oil, sodium, phosphates as well as other pollutants. Oils and greases tend to clog sewers and pumps, thus creating difficulties within the municipal wastewater treatment works. Physico-chemical treatment methods, such as (Dissolved Air Flotation) OAF, gravity separation and the use of coagulants have been attempted providing a considerable reduction in organic loading; however, discharge standards are still not met. Thus, biological treatment methods are being sought after. Aerobic treatment has been attempted however, shock loads cause problems while running such a process. The objective of this study was to assess the efficiency of anaerobic digestion to degrade Vegetable Oil Effluent (VOE) as well as the efficiency of the Anaerobic Baffled Reactor (ABR). Anaerobic digestion involves the breakdown of organic matter by the action of microorganisms in the absence of oxygen, producing methane-rich biogas. The VOE was characterized, providing significant information on its chemical composition. It was found that the effluent had high sulphate content as well as a high COD content. High sulpahte content of wastewaters have known to promote growth of Sulphate Reducing Bacteria (SRB), which utilize the same energy source as Methane Producing Bacteria (MPB) and therefore compete for the same energy source. Sulphate and lipid reduction pretreatment experiments were carried out, using barium chloride and gravitational separation respectively. The results obtained, showed that the use of barium chloride to reduce sulphate content in VOE was successful, with significant sulphate reduction. The lipid reduction experiments however, did not show any significant lipid reduction. Batch tests were conducted in serum bottles to assess the extent of biodegradation of the VOE in its raw state as well as with reduced sulpahte content. Methanogenic toxicity tests on the raw and pretreated VOE provided a range of toxicity results. These assays are relatively simple and inexpensive. Gas production was monitored to determine the rate and extent of biodegradation. The efficiency of digestion was assessed by COD reduction. Results indicated potential inhibition of the methanogenic bacteria responsible for methane production by the
Bioaugmentation with Bacillus spp. for bioremediation of synthetic wastewater using a fluidized-bed reactor
(2020-03-25) Roets, Yrielle; Bux, Faizal; Lalloo, Rajesh; Kumar, Sheena
South Africa’s freshwater resources, including rivers, man-made lakes and groundwater are under severe threat due to an ever-expanding population and economy, which is depleting these resources. The increase in population has a direct correlation with the increase in wastewater generated. The remaining fresh water resources need to be preserved therefore recycling of wastewater, to replenish our water supplies and preserve the environment, is a solution to the problem. For a developing country, it is important to use treatment methods that are cost effective and do not exert a negative impact on the environment, such as biological wastewater treatment options. One of the systems commonly used in biological wastewater treatment is the fluidized-bed bioreactor (FBBR) due to its advantages such as higher biomass concentration and a higher mass transfer thus resulting in a higher rate of biodegradation. This study focused on evaluating the efficacy of augmenting with Bacillus spp. to enhance the bioremediation of wastewater using a FBBR. Bacillus spp. used in this study were isolated from a municipal wastewater treatment plant (10 isolates) and the remaining three isolates were selected from the CSIR Bacillus database. The isolates (13 in total) were screened for 1) their ability grow in wastewater, 2) ability to reduce high concentrations of COD, ammonium, nitrates and phosphates in flask studies containing synthetic wastewater (SWW) and 3) ability to produce common enzymes such as amylase, cellulase, lipase and protease. Isolates showed varying bioremediation potential for different compounds analysed. Isolate B006 showed the highest phosphate removal rate (3.290 mg.L-1.h-1) where as D005 showed the highest growth rate (0.955 h-1), COD reduction rate (55 mg.L-1.h-1) and cellulase activity (5.485 mm) among all the isolates. Isolate D014 presented the highest ammonium removal rate (12.43 mg.L-1.h-1), amylase (5.00 mm) and protease (10.00 mm) activity whilst B001 displayed the highest nitrate removal rate (9.4 mg.L-1.h-1). The results for the individual assays were assessed and weighted in a matrix and the isolates that scored above 50% were selected for consortium studies. Four Bacillus spp. that scored above 50% in the scoring matrix were then evaluated for their ability to co-exist as a consortium. The consortium studies were then compared with results obtained for individual isolates. The selected Bacillus isolates were identified and assessed for their safety to the environment and to the end user. Identification was conducted using 16s rDNA sequencing and results showed that B006 identified as B. cereus, D005 as B. cereus and D014 as B. subtilis. Isolates, B006 and D005 were further assessed for enterotoxin production and the presence of anthrax virulent plasmids pX01 and pX02. After conducting the biosafety assays, the isolates were rendered safe for use. The isolates were then cryopreserved as spores in 25% glycerol and stored at -80 °C. The impact of the cryopreservation method and the storage conditions on the viability of the isolates was assessed after six months of storage and it was established that the isolates were still viable and that the method was adequate. The bioremediation potential of the consortium was further evaluated using a 17 L Pilot scale fluidised-bed bioreactor. The reactors were fed at three different flow rates of 1.5 L.h-1, 2 L.h-1 and 3 L.h-1 over steady state conditions (~3months). The results showed that the FBBR augmented with the selected Bacillus isolates, resulted in improved nutrient (COD, ammonium and phosphates) removal efficiencies compared to the non-bioaugmented control. The highest ammonium removal (62.8%) was observed at a flow rate of 1.5 L.h-1 (11.30 h retention time), whereby there was an overall 29.8% improvement in ammonia removal in comparison to the non-augmented control. Similarly, an overall improvement in phosphate (14.73%) was observed at a flow rate of 2 L.h-1 (8.48 h retention time) with 50% removal efficiency. The highest COD removal was observed at a flow rate of 1.5 L.h-1 (11.30 h retention time) whereby 74.5% COD was reduced with a 32.6% improvement when compared to the non-bioaugmented control. Our work has demonstrated the potential application of Bacillus as bioaugmentation agents to enhance wastewater treatment efficiency as a potential solution to water challenges in developing countries. This technology could also be utilized for addressing the challenges of a wider range of different effluents.
Biodiesel production from microalgae by enzymatic transesterification
(2015) Guldhe, Abhishek; Bux, Faizal; Permaul, Kugen; Mutanda, Taurai
Main focus of this study is to investigate the enzymatic-conversion of microalgal lipids to biodiesel. However, preceding steps before conversion such as drying of microalgal biomass and extraction of lipids were also studied. Downstream processing of microalgae has several challenges and there is very little literature available in this area. S. obliquus was grown in the pilot scale open pond cultivation system for biomass production. Different techniques were studied for biomass drying and extraction of lipids from harvested microalgal biomass. Effect of these drying and extraction techniques on lipid yield and quality was assessed. Energy consumption and economic evaluation was also studied. Enzymatic conversion of microalgal lipids by extracellular and whole cell lipase application was investigated. For both applications, free and immobilized lipases from different sources were screened and selected based on biodiesel conversion. Process parameters were optimized using chosen extracellular and whole cell lipases; also step-wise methanol addition was studied to improve the biodiesel conversion. Immobilized lipase was studied for its reuse. Final biodiesel was characterized for its fuel properties and compared with the specifications given by international standards. Enzymatic conversion of microalgal lipids was compared with the conventional homogeneous acid-catalyzed conversion. Enzymatic conversion and chemical conversion were techno-economically investigated based on process cost, energy consumption and processing steps. Freeze drying was the most efficient technique, however at large scale economical sun drying could also be selected as possible drying step. Microwave assisted lipid extraction performed better compared to sonication technique. Immobilized P. fluorescens lipase in extracellular application and A. niger lipase in whole cell application showed superior biodiesel conversion. The extracellular immobilized P. fluorescens lipase showed better biodiesel conversion and yields than the immobilized A. niger whole cell lipase. Both the enzyme catalysts showed lower biodiesel conversion compared to conventional chemical catalyst and higher processing cost. However, techno-economic analysis showed that, the reuse potential of immobilized lipases can significantly improve the economics. Fewer purification steps, less wastewater generation and minimal energy input are the benefits of enzymatic route of biodiesel conversion. Microalgae as a feedstock and lipase as a catalyst for conversion makes overall biodiesel production process environmentally-friendly. Data from this study has academic as well as industrial significance. Conclusions from this study form the basis for greener and sustainable scaling-up of microalgal biodiesel production process.
Biological phosphorus removal from edible oil effluent by anaerobic- aerobic sequencing batch reactor
(2004) Manganyi, Abel Jwili; Bux, Faizal
The objective of this study was to evaluate the characteristics and treatability of process wastewater from an edible oil refining industry, which discharge its effluent into a sewer system. The main objective was to assess a laboratory scale treatment process that would produce effluent having a regulatory acceptable phosphate concentration (below 20 mgIL) prior to discharge into municipal sewer system. A single stage laboratory-scale anaerobic-aerobic sequencing batch reactor (BPR-SBR) with a total volume adjustable up to 10L was designed for biological phosphorus removal. The BPR-SBR was run at 10 days sludge age, 8 hours hydraulic retention time and organic load of ~ 0.38 kg COD/kg MLSS.d for 158 days to evaluate its performance for bio-P removal efficiency. The BPR-SBR system showed a consistent P removal efficiency of up to 78.40 %, 80.15 % COD and 72.43 % FOG reduction. The laboratory scale study has demonstrated that the SBR technology is suitable for treating wastewater from edible oil producing industry.
Carbapenem resistance profiling of selected bacterial species belonging to the Enterobacteriaceae family in various water matrices
(2021) Dludla, Winile Nokwazi; Stenström, T. A.; Pillai, S. K. K.; Adegoke, Anthony Ayodeji
Carbapenems are broad-spectrum β-Lactams exhibiting bactericidal activity by binding to penicillin-binding proteins (PBPs). These antibiotics are the last resort drugs prescribed for severe infections when other administered ones have failed to produce enough responses in patients. Bacteria belonging to the Enterobacteriaceae family, including Escherichia coli and Klebsiella pneumoniae were identified as “priority pathogens” by the World Health Organization (WHO) in 2017 due to their resistance against carbapenems and have been ranked as “Priority 1: Critical” for research. Among the various types of carbapenem-resistant Enterobacteriaceae (CRE), carbapenemase-producing CRE (CP-CRE) have drawn the most attention since they can contribute to the overall challenge of antimicrobial resistance. Wastewater treatment plants (WWTPs) have been considered a potential hub for antibiotic resistance gene exchange and further release into the environment. Therefore, antimicrobial resistance surveillance using sewage isolates has been presented as a way to study the occurrence and spread of specific clonal groups or sequence types within a community or population. Thus, this study aimed to investigate the occurrence of carbapenem-resistant K. pneumoniae and E. coli and their virulence genes in selected WWTPs treating municipal wastewater in Durban, South Africa, and to assess the efficiency of these WWTPs in removing these microorganisms. The samples were taken from two WWTPS (WWTP I and WWTP II) monthly from six sampling points (influent, aeration/biofilter, pre-chlorination, post-chlorination, upstream and downstream from the WWTP’s discharge points) from January to September 2018. Primary isolation and enumeration were carried out on CHROMagarTM ECC and Klebsiella ChromoSelect Selective Agar Base, for E. coli and K. pneumoniae, respectively. A total of 120 carbapenem-resistant E. coli (CR E. coli) and 100 carbapenem-resistant K. pneumoniae (CR K. pneumoniae) were randomly selected, further identified using biochemical tests and confirmed using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF), polymerase chain reaction (PCR) and 16S rRNA sequencing. The confirmed isolates were then subjected to antimicrobial susceptibility testing (AST) using eight antibiotics (third-generation cephalosporins and carbapenems) to determine their resistance profile. Extended-spectrum beta-lactamases (SHV, CTX-M, TEM, and OXA-1) and carbapenemase genes (NDM-1, OXA-48, IMP and VIM) and virulence genes such as stx 1 and 2, rfbE, hly, eae, and fliC were targeted using PCR. While carbapenem-resistant E. coli was detected in both WWTPs, the carbapenem-resistant K. pneumoniae was only detected in WWTP II. The total E. coli count in the influent ranged from 6.8 to 7.1±0.06 log10 CFU/100 mL (WWTP I) and 6.9 to 7.2±0.08 log10 CFU/100 mL (WWTP II) with a carbapenem resistance percentage of 0.01% and 0.07%, respectively. A complete reduction of CR E. coli was observed in both WWTPs surveyed and was not detected from the receiving water bodies. The total K. pneumoniae in the influent of WWTP II ranged between 7.1 to 7.3±0.1 log10 CFU/100 mL. For carbapenem-resistant K. pneumoniae, the highest percentage was detected in the aeration tank (3.03%), followed by the pre-chlorinated effluent (1.33%), with the influent having the least (0.34%). However, carbapenem-resistant K. pneumoniae was not detected from the post-chlorinated effluent and the receiving water bodies, indicating a complete removal. The third-generation cephalosporin resistance profile showed high resistance against cefixime in both E. coli and K. pneumoniae isolates. Among these, the influent of WWPT II showed the highest percentage of resistant E. coli against cefixime (92%) compared to WWTP I E. coli (62%) and WWTP II K. pneumoniae (81%). In addition, the resistance profile of carbapenems showed that most of the isolates were resistant to ertapenem in both WWTPs. Of the 120 E. coli isolates, 60 in WWTP I and II showed 100% and 95% resistance against ertapenem, respectively. Additionally, K. pneumoniae showed 100% resistance against ertapenem. The predominant ESBL genes detected in the E. coli isolates were TEM and SHV, showing a 100% carriage in both WWTPs. The least detected ESBL gene was OXA-1, at 98% and 52% carriage in WWTP I and II, respectively. The occurrence of the ESBL genes in K. pneumoniae isolates was slightly different between the sampling points. In the influent, the dominant genes were TEM and CTX-M at 75% and 62%, respectively. In the aeration and pre-chlorinated effluent, the dominant genes were TEM and SHV with 100% carriage. The least detected gene was the CTX-M at 13% in the influent, with no detection at the other sampling points. Both E. coli and K. pneumoniae isolates showed NDM-1 and OXA-48 to be the predominant genes identified of the carbapenemase-producing genes investigated. In the influent of WWTP I and II, the E. coli isolates that harboured NDM-1 were 100% and 82%, respectively. However, the modified Hodge test did not correlate with the detection of CRE since the test was positive for only 34 isolates (15%), while 168 isolates carried the carbapenemase genes based on PCR test. Out of the six virulence genes tested in CR E. coli, three (hly, rfbE, and eae) were detected. The predominant gene in both WWTPs was hly, with the highest percentage in WWTP II (88%) compared to WWTP I (33%). The least detected gene was eae, only detected in WWTP II at 8%. For K. pneumoniae, out of the three virulence genes (wabG, urea, and rmpA) tested, wabG was the only gene detected. This gene was detected at all the sampling points, with the highest percentage being in the pre-chlorinated effluent (50%), followed by the influent (11%) and the least in the aeration (10%). The investigated WWTPs showed carbapenem-resistant E. coli and K. pneumoniae isolates in their influent samples and the various stages of the treatment except in the post chlorinated effluent indicating their efficient removal during the disinfection process. Future research to determine the presence of viable but not culturable (VBNC) carbapenem-resistant Enterobacteriaceae in these treated effluent and recipient water bodies, and the use of advanced molecular methods capable of identifying these bacteria at lower concentrations is hereby recommended as these water bodies are routinely used for agricultural, industrial, and household purposes by the local communities.
Characterisation of the microbial communities present in an anaerobic baffled reactor utilising molecular techniques
(2005) Lalbahadur, Tharnija; Bux, Faizal
The provision of safe and sanitary water is a constitutional right and above all, a necessity of life. As a result of the rapid urbanisation and the past policies of apartheid, a large population of South Africa dwell in informal settlements, where there is very little hope of development, as the government does not possess the resources that are necessary for a full-scale sanitation programme. Therefore, on-site treatments have been considered to provide sanitation in these dense peri-urban areas. The anaerobic baffled reactor (ABR) is one such sanitation system. This reactor utilises the phenomenon of anaerobic digestion to degrade substrates. One of the major disadvantages of any anaerobic treatment processes is the extreme sensitivity of the bacterial communities, thus inducing slow recovery rates following toxic shocks. Therefore, an understanding of these microbial consortia is essential to effectively control, operate and optimise the anaerobic reactor. Fluorescence in situ hybridization, 4’,6-diamidino-2-phenylindole (DAPI) staining and DNA sequencing techniques were applied to determine the microbial consortium, as well as their reactions to daily operating conditions. With an understanding of these populations and their responses to perturbations within the system, it is possible to construct an anaerobic system that is successful in its treatment of domestic wastewater. In situ hybridizations were conducted for three operating periods, each characterised by specific flow rates. Results showed Eubacterial population dominance over the Archaeal population throughout both of the operating periods investigated. However, these cells cumulatively consisted of 50% of the total biomass fraction, as determined by DAPI staining. Group-probes utilised revealed a high concentration of fermentative acidogenic bacteria, which lead to a decrease in the pH values. It was noted that the ABR did not separate the acidogenic and methanogenic phases, as expected. Therefore, the decrease in pH further inhibited the proliferation of Archaeal acetoclastic methanogens, which were not present in the second operating period. DNA sequencing results revealed the occurrence of the hydrogenotrophic Methanobacterium and Methanococcus genera and confirmed the presence of Methanosarcina. Sequencing of the bacterial DNA confirmed the presence of the low G+ C Gram Positives (Streptococcus), the high G+C Gram Positives (Propionibacterium) and the sulfate reducing bacteria (Desulfovibrio vulgaris). However, justifications were highly subjective due to a lack of supportive analytical data, such as acetate, volatile fatty acids and methane concentrations. Despite this, findings served to add valuable information, providing details on the specific microbial groups associated with ABR treatment processes.
Characterization and modification of Bambara groundnut globulin fractions for the enhancement of functional properties
(2023-05) Alabi, Opeyemi Olaitan; Amonsou, Eric Oscar
There is a growing interest in the utilization of leguminous grain proteins for food and industrial applications. Bambara groundnut is a xerophyte pulse grain and a potential source of protein that can replace soybean protein, a trusted and widely used food ingredient in the food industry. However, the use of Bambara groundnut proteins including the subunits (legumin and vicilin) is limited in food applications. The understanding of the composition of Bambara groundnut proteins at the subunit level is vital to unlocking their potential and facilitating utilization. In this study, Bambara groundnut globulin is characterized in terms of the structures, composition, and physicochemical properties at the subunit level, and then modified using atmospheric plasma and enzymatic hydrolysis. Bambara globulin consisted of about 70% vicilin, whilst legumin protein was found in relatively low quantity. Gel electrophoresis revealed three major protein bands in globulin similar to vicilin with predominant β-sheet structures. The presence of a disulfide bond was also revealed in legumin. Bambara globulin showed major vicilin (7S, Mw: 120 kDa) and minor legumin (11S, Mw: 410 kDa) components. Fluorescence and hydrophobicity data suggested a folded structure for the legumin fraction dominated by the helical secondary structure compared to the vicilin fraction. Bambara proteins contain an appreciable amount of methionine that is even higher than the FAO/WHO recommended value. Bambara vicilin had the highest amount of negatively and positively charged amino acids compared to globulin and legumin. This coincides with its high solubility profile (approximately 82% at pH 3.5). The least gelation concentration (LGC) significantly increased in the order of globulin (8%) < legumin (18%) < vicilin (20%) at pH 7. Bambara groundnut proteins formed weakly structured gels as indicated by the frequencydependent behaviours of both the storage (Gʹ) and loss (Gʺ) moduli with a difference of lesser than 1 log cycle. The highest Gʹ of vicilin gel indicated more firmness of the gel compared to the gel formed by globulin and legumin. The sol-gel transition temperatures increased in the order of globulin (40℃) < legumin (50℃) < vicilin (80℃). The Gʹ and Gʺ of globulin showed relatively low dependency on heating time beyond the gel point compared to legumin and vicilin subfractions, suggesting a more rapid establishment of its gel network during gelation. Vicilin gel consisted of a microporous structure with a small lath sheet-like structure compared to globulin and legumin. Emulsifying stability of the proteins significantly differed (p < 0.05) at pH 7. The foaming capacity of the vicilin fraction was significantly (p < 0.05) higher than that of the storage protein at pH 3, 7, and 9. Atmospheric cold plasma-activated water (PAW) and enzymatic modification of Bambara groundnut globulin were further assessed. The cold plasma treatment resulted in the loss of the helical structure of Bambara globulin. The plasma treatment increased the hydrophobicity of Bambara globulin indicating an unfolded structure that was also reflected in the observed redshift in fluorescence intensity. No major changes were observed in gel electrophoresis, protein surface charge, and solubility profiles, except for about a 20% reduction in the glutamic acid content of the amino acid profile. Bambara globulin had reduced emulsifying capacity after treatment with PAW. However, foaming capacities were significantly better and stable at up to 15 mg protein/mL. Hydrolysates produced from Bambara groundnut globulin and vicilin, respectively using a combination of pepsin and pancreatin were investigated for ACE and renin inhibitory activities. The hydrophobic amino acid residues in both globulin and vicilin hydrolysates are high, improving the entry of their peptides into target organs via hydrophobic associations. Surface hydrophobicity increased significantly (p<0.05) with an increase in peptide size from <1 to <3 kDa with that of vicilin hydrolysate and membrane fractions having the highest values. The low molecular weight peptide (<1 kDa) membrane fractions from globulin at 1 mg/mL exhibited significantly higher (p<0.05) invitro ACE inhibitory activities compared to vicilin hydrolysate and its fractions. However, higher molecular peptide fraction (<3 kDa) favoured renin inhibitory activity at the same concentration. Vicilin is the major protein fraction of Bambara groundnut globulin. Bambara groundnut globulin was stabilized by disulfide linkages from the legumin, a minor fraction of the storage protein. Bambara globulin and its subfractions formed a weakly structured gel with the dominance of an elastic structure. The dominancy of the β-sheet structure in vicilin protein and the high crosslink density of the vicilin gel could be related to the firmness of the vicilin gel. The variations in the gel points of Bambara globulin and the subfractions were linked to the differences in their amino acid and subunit composition, the thermal unfolding properties of the protein fractions, and the presence of disulfide linkages. Modification of Bambara groundnut globulin using cold plasma-activated water treatment and enzymatic hydrolysis, respectively increased the hydrophobicity of the protein and influenced the emulsifying and foaming properties and the invitro angiotensin-converting enzyme (ACE) and renin inhibitory activities. Therefore, Bambara groundnut globulin could be a potential functional ingredient in the food system. The low molecular weight peptide (<1 kDa and <3 kDa) membrane fractions from globulin have the potential to serve as functional bioactive peptides against hypertension.
Cloning, expression, characterization and application of cyanase from a thermophilic fungus Thermomyces lanuginosus SSBP
(2018) Ranjan, Bibhuti; Singh, Suren; Pillai, Santhosh Kumar Kuttan; Permaul, Kugen
Rapid industrialization and proliferative development of chemical and mining industries have resulted in increased global pollution and environment deterioration, due to the release of numerous toxic substances. This has extreme relevance in the South African context due to the high amount of cyanide used by local mines in comparison to that utilized globally. This has created the need for the development of novel approaches viz., using microbial enzymes for its remediation because of lower process times, lower energy requirements, and their cost-effective, nontoxic and eco-friendly characteristics. From previous work in our lab, the whole genome sequencing and secretome analysis of the industrially-important fungus Thermomyces lanuginosus SSBP revealed the presence of a cyanate hydratase gene and enzyme, respectively. Cyanate hydratase detoxifies cyanate in a bicarbonate-dependent reaction to produce ammonia and carbon dioxide. The cyanate hydratase gene (Tl-Cyn) from this fungus was therefore cloned, overexpressed, purified, characterized and its potential in cyanate detoxification has also been evaluated. The recombinant cyanate hydratase (rTl-Cyn) showed high catalytic efficiency, suggesting that it could be used for bioremediation applications. Though, cyanate hydratase catalyzes the decomposition of cyanate, the requirement of bicarbonate is a major drawback for its effective utilization in large-scale applications. Hence, a novel strategy was developed to limit the bicarbonate requirement in cyanate remediation, by the combinatorial use of two recombinant enzymes viz., cyanate hydratase (rTl-Cyn) and carbonic anhydrase (rTl-CA) from T. lanuginosus. This integrative approach resulted in the complete degradation of cyanate using 80% less bicarbonate, compared to the cyanate hydratase alone. In addition, co-immobilization of these recombinant enzymes onto magnetic nanoparticles and evaluation of their potential in bio-remediation of cyanurated wastes together with their reusability resulted in more than 80% of cyanate detoxification in wastewater samples after 10 cycles. Another novel strategy was also developed for the simultaneous removal of heavy metals and cyanate from synthetic wastewater samples, by immobilizing the rTl-Cyn on magnetic multi- walled carbon nanotubes (m-MWCNT-rTl-Cyn). The m-MWCNT-rTl-Cyn simultaneously reduced the concentration of chromium (Cr), iron (Fe), lead (Pb) and copper (Cu) by 39.31, 35.53, 34.48 and 29.63%, respectively, as well as the concentration of cyanate by ≥85%. The crystal structure of Tl-Cyn in complex with inhibitors malonate or formate at 2.2 Å resolution was solved for the first time to elucidate the molecular mechanism of cyanate hydratase action. This structure enabled the creation of a mutant enzyme with ~1.3-fold enhanced catalytic activity as compared to the wild-type Tl-Cyn. In addition, the active site region of Tl-Cyn was found to be highly conserved among fungal cyanases. Information from the 3D structure could enabled the creation of novel fungal cyanases, which may have potential for biotechnological applications, biotransformation and bioremediation.
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, Arshad
The 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.
A comparative microbiological assessment of river basin sites to elucidate fecal impact and the corresponding risks
(2017) Sithebe, Ayanda; Stenström, Thor-Axel; Singh, Gulshan; Bux, Faizal
The study aims to assess and compare the concentration of microbial contaminants, their sources and distribution in surface water and sediment, and to determine the impact of seasonal variations and corresponding risks of faecal contamination using conventional and molecular methods. Historical data analysis was conducted using E. coli values from the eThekwini Water and Sanitation (EWS) department for 66 months (2009-2014). E. coli and Enterococci were analysed in surface water and sediment samples using the mFC/ spread plate and Colilert-18 (IDEXX) methods. The impact of seasonal variations was assessed using E. coli and Enterococci data collected during rainfall and no rainfall events, using an auto-sampler and sediment trap in parallel. Conventional standard membrane filtration methods using mFC agar, Slanetz & Bartley/ Bile Esculin and Brilliance E. coli selective agar were compared to the enzymatic Colilert-18 and Enterolert (IDEXX) test methods along the Isipingo and Palmiet Rivers. In addition, comparison of the analytical performance of droplet digital PCR (ddPCR) and qPCR for the detection of Salmonella targeting ttr gene in river sediment samples collected from the four sites of the Palmiet River in Durban, South Africa was done. In order to assess the public health risk associated with exposure of men, women and children to microbial pathogens in polluted surface water during recreational activities, the QMRA tool was employed in relation to the risk exposure to pathogenic E. coli, Campylobacter, Salmonella and Shigella. Also, the risk associated with crop irrigation (on farmers) as well as the consumption of crops irrigated with surface water from the Isipingo river was determined. Analysis of the historical data gave a baseline of the two rivers of interest, thus helps understand the current situation of the rivers enabling researchers to pick up potential gaps. In this study after the analysis of the historical data it was evident that at the Palmiet river, microbial analysis must be conducted around the QRI settlements which is a major pollution source. Also, from this study it was found that sampling points situated close to wastewater treatment plants, pump stations or informal settlements were of major concern, thus were considered for the study. It was found that sediment exhibited higher microbial concentrations than surface water, which was observed in both rivers. Also, rainfall had a significant impact on microbial variability. Higher microbial concentrations (indicator organisms) were observed in surface water after a heavy rainfall as appose to when there was no rainfall. This was due to contamination that is washed off into the river and sediment resuspension. Methodology comparison revealed that Colilert-18 and Brilliance E. coli were more selective compared to mFC agar. Brilliance E. coli /Coliform agar was comparable with Colilert-18 IDEXX, which was also observed with Slanetz & Bartley and Enterolert IDEXX. However, when mFC agar was compared with Colilert-18 IDEXX, significant difference was observed. In comparison of two Molecular methods, ddPCR were found to be fully amenable for the quantification of Salmonella and offer robust, accurate, high-throughput, affordable and more sensitive quantitation than qPCR in complex environmental samples like sediments. Quantitative Microbial Risk Assessment (QMRA) relating to recreational and occupational exposure showed that children were at the highest risk of getting infected. Also, it was observed that the probability of infection upon exposure to surface water from the Isipingo and Palmiet rivers was significantly high, hence exceeded the WHO guidelines values. Risk assessment on crops revealed that pathogenic bacteria may pose a risk to the consumer, however, a 9-log reduction may be achieved according to the WHO multi-barrier approach which involves proper washing and proper cooking of the crop before ingestion. Overall the sampling points that had the highest pollution level and constantly exceeded the WHO and DWAF guidelines at the Isipingo river were the points situated and named “Next to the WWTP”, and “Downstream of QRI” at the Palmiet River.
Comparison of lignin yield from sugarcane bagasse pellets using liquid hot water and ionic liquid pretreatment methods
(2019) Gnana, Gueh Charles; Deenadayalu, Nirmala
In this research work, lignin yield from sugarcane bagasse pellets (SBP) was investigated after treatment of sugar cane bagasse with liquid hot water (LHW) and enzymatic hydrolysis followed by ionic liquids (ILs) and only ionic liquids pretreatment methods. In the LHW and ionic liquid methods, the SBP were first treated with LHW at 200 °C, for 30 minutes in a suitable reactor, for removal of hemicellulose. The complex cellulignin residue was treated separately with either of two ionic liquids namely: 1-ethyl-3- methylimidazolium acetate ([Emim][OAc]) or 1-butyl-3- methylimidazolium hydrogen sulphate ([Bmim][HSO4]), using microwave digestion at varying time intervals. Theionicliquidmethodinvolvedthepretreatmentofsugarcanebagasse pelletswith either 1-ethyl-3-methylimidazolium acetate or 1-butyl-3-methylimidazolium hydrogen sulphate followed by microwave digestion at varying time intervals. Ultraviolet (UV) spectroscopy at a wavelength of 280 nm was used as a tool for quantification of lignin. The different functional groups of the extracted lignin were confirmed using attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. Thermogravimetric analysis (TGA) provided information on thermal characteristics of the extracted lignin. In addition to material characterization, mixed factorial ANOVA was performed to compare the extracted lignin yield using the LHW and IL and the ionic liquid pretreatment methods. High performance liquid chromatography (HPLC) was used to identify the C5 sugars in the hydrolysate after LHW pretreatment. X-ray diffraction (XRD) was used to identify cellulose peaks of cellulignin and SBP and ILs treated samples. The results indicated that the lignin yield from sugarcane bagasse pellets after liquid hot water treatment and enzymatic hydrolysis was 37.8 % (m/v). The highest percentage yield of lignin extracted from the complex cellulignin (LHW and IL) was found to be 68.00 % (m/v) and 32.04 % (m/v) for [Emim][OAc] and [Bmim][HSO4], respectively for the optimized reaction time of 10 minutes. However, 67.25 % (m/v) and 48.94 % (m/v) of the extracted lignin were obtained for the pretreated SBP with [Emim][OAc] and [Bmim][HSO4], respectively for a reaction time of 20 minutes. This comparative study revealed that, there is no significant difference between the yield of lignin extracted from the complex cellulignin (68.00%) and sugarcane bagasse pellets (67.25 %).The sugarcane bagasse pellets is the preferred method since it doesn’t require high energy input.
Design and optimization of an adsorption method for the removal of textile azo dyes from aqueous solutions using Plantago lanceolata
(2021) Kaunda, Thabisile Penelope; Gengan, Robert Moonsamy; Singh, T.
Water is an essential commodity for human survival; however, this resource is predicted to be scarce within the 21st Century due to pollution and industrialization. Textile industries are among the many polluters of water, and hence methods to remediate textile waste-water need attention. In this study, a common garden weed Plantago Lanceolata was used for the preparation of novel activated carbon materials as an adsorbent for the degradation of textile azo dyes Reactive Blue 222 (RB), Reactive Red 195 (RR), and Reactive Yellow 145 (RY). The activated carbon was modified with four different chemical activators to produce phosphoric acid-based activated carbon (H3PQ4-AC), sulfuric acid-based activated carbon (H2SO4-AC), potassium hydroxide-based activated carbon (KOH-AC), and sodium hydroxide-based activated carbon (NaOH-AC). These materials were characterized by Fourier- transfer infrared spectroscopy (FTIR), scanning electron microscope with energy dispersive Xray (SEM/EDX), high resolution transmitting electron microscope (HRTEM), and a thermogravimetric analyzer with differential scanning calorimeter (TGA/DSC). The initial concentration of the adsorbate, adsorbent dosage concentration, contact time, temperature, and pH were optimized. The four materials adsorption capacity was studied, and H3PQ4-AC produced the best results of adsorption capacity 98,98% - 100%, with optimum agitation time of 70 minutes, the optimum dosage of 0.8 g/60 ml of adsorbent, and pH of 6. The experimental data were fitted using Langmuir (type 1- 4), Freundlich, Temkin, and Dubinin-Radushkevich isotherms. The data from this study best fitted the Langmuir isotherm type 1: RB (qm -15.58 mg g 1 ), RR (qm - 11.24 mg g 1 ) and RY (qm - 11.24 mg g 1 ). Furthermore, the reaction rate followed the pseudosecond-order kinetic model while the intraparticle diffusion model had no impact. Its thermodynamic parameters indicated the reaction as spontaneous and exothermic. Furthermore, a nanocomposite was prepared from H3PQ4-AC and iron oxide to produce an iron oxide/activated carbon nanocomposite. FTIR, SEM/EDX, HRTEM, and TGA/DSC fully characterized this novel material. The iron oxide/H3PQ4-AC nanocomposite produced slightly better results compared to H3PO4-AC: RB (99.60% - 100%), RR (99.59 - 100%) and RY (99.48% - 100%). The experimental data fitted Langmuir isotherm type 1, and the reaction followed the pseudo-second-order kinetic reaction model.
Detection and evaluation of the fate of estrogen endocrine disrupting chemicals in wastewater treatment
(2014) Surujlal-Naicker, Swastika; Bux, Faizal
All over the world concerns have been raised over the possible adverse effects that may occur when exposed to chemicals that have the potential to interfere and affect the endocrine system. The concern is directed at both humans and wildlife. There is still a lack of public awareness regarding Endocrine Disrupting Chemicals (EDCs) and the harmful effects on humans and wildlife. It has only been within the last decade that South Africa began the actual task for proper management and control for water and wastewater quality. There are many ways to detect these EDCs all of which are very laborious and most of the cases these EDCs are either in the pico or nano gram per litre range, too minute for many methods to detect effectively; so therefore the research project aimed to use rapid and sensitive techniques to determine the quickest means to detect the very low concentrations of theses EDCs. Two techniques were researched, i.e., Enzyme Linked immunoassays (ELISAs) and Radio-immunoassays (RIAs). The research study thus assessed the solid phase extraction (SPE) technique for total recovery of hormones; the ELISA and RIA techniques for rapid detection of natural (estrone (E1), estradiol (E2) and estriol (E3) and synthetic ethinylestradiol (EE2) by validating the precision and reproducibility . These techniques were then applied to determine hormone EDC removal first at laboratory scale investigations and then applied to full scale wastewater treatment plants (WWTP) with different configurations in order to deduce removal efficiency of each type of plant. The next phase assessed the toxicity of individual and combined estrogen standards as well as the toxicity in the WWTPs and classify and to determine if there was a correlation between hormone concentration and toxicity in final effluents. The assessment of the SPE and the immunoassay procedures (ELISA AND RIA) using standards and controls found that both these assays can be utilised to quantify hormone estrogens in wastewater. The small sample volume required reduced the labour time and application of the procedure made it cost effective and reliable techniques. The intra-assay and inter-assay validation procedures as well as the standard recoveries confirmed reproducibility and precision of the immunoassays. The % CV were <10% for both the intra-assay and inter-assay validations. The laboratory scale investigations included the operation of a modified Ludzak-Ettinger (MLE) process which enabled control and manipulation over the operational parameters in order to establish how certain parameters influenced the removal of hormone EDCs. One such parameter that was manipulated was the sludge retention time (SRT). The MLE tests showed that the SRTs definitely have an effect on the removal of hormones from the influent as well as the overall performance of sewage treatment. The 10 day SRT proved that longer SRTs will definitely aid in the removal of hormones and possibly other EDCs in raw sewage. During the 10 day SRT the influent hormone concentrations (E1: 59.11 ng/L, E2: 61.40 ng/L) were almost double than the influent hormone concentrations (E1: 26.46 ng/L, E2: 27.60 ng/L) during the 5 day SRT, which impacted on the removal efficiency. The 5 day SRT had an overall average E2 and E1 removal of 78.11% and 81.71% respectively while the 10 day SRT had average E2 and E1 removal of 91.24 % and 80.56% respectively. The 24 hour batch test provided evidence of the reversible metabolism of the E2 hormone. This was seen by the rapid decrease of E2 and the rapid increase of E1 in less than 3 hours, which proved that E2 can be metabolized in to E1. An average reduction of 94.44% of E2 was seen after 5 hours and after 10 hours was no longer detected. After 13 hours E1 could no longer be detected. This finding also provided clarity as to the lower percentage removal of E1 during the 10 day SRT of the MLE process. The Vibrio fischeri biotox method implemented was the most economic and easiest way to conduct the toxicity tests. The validation of the test used a 52.9 mg/L K2Cr2O7 standard which provided a Cr (VI) concentration of 18.7 mg/L in the final test suspension which is the theoretical effective concentration causing 50% inhibition (EC50). This specific concentration of the Cr (VI) exhibited an EC50 at 20.08 mg/L. The toxicity investigations of the individual and mixed hormone standards revealed that at the 10 ng/L concentration the individual E2 standard had the highest percentage inhibition (%INH) of 45.99% after the 30 minute contact time (T30), and when this standard was further diluted to 5 and 1 ng/L also showed higher % INH (26.04 and 23.66 %INH, respectively) than the individual EE2 standard (21.92 %INH) at 10 ng/L. . According to the toxicity classification system and after interpretation of the data, all the hormone standards were classified as Class II as they all exhibited slight acute toxicity. The 10 ng/L E2 standard had Toxicity Units (TU) of 0.8 which was close to the Class III level; however when it was in a mixture with E1 and E3, the TU was much lower (0.6 TU). The synthetic EE2 hormone also showed slight acute toxicity and had the lowest TU of 0.4. The application of the above mentioned techniques to full scale WWTPs with different configurations showed different removal efficiencies. The WWTPs ranged from the most primary consisting of just oxidation ponds to biological trickling filters, to biological nutrient removal (BNR) to conventional activated sludge (AS) plants. Removal rates ranged from 29% to 96% for E2, 0% to 89% for E1 and 0% to 100% for EE2. The overall ranking of the WWTPs from the most efficient to least efficient in terms of hormone removal were as follows: Plant E (91%) = Plant D (before UF) (91%) > Plant B (east side) (88%) > Plant B (west side) (77%) > Plant C (east side) (71%) > Plant D (after UF) (57%) > Plant A (56%) > Plant C (west side) (12%). Using the Vibrio fischeri method to evaluate the reduction of toxicity in WWTPs C, D and E proved effective. It was seen immediately after secondary biological treatment in the clarifier effluent the toxicity was reduced. Plants C, D and E had reduced the toxicities by 100, 80 and 97 % immediately after secondary biological treatment, while after the addition of the Chlorine disinfectant in the final stage of treatment the toxicity increased having %INH of 99.9, 15.7 and 99.9 respectively. In conclusion the SPE can be used as an extraction procedure for hormones in wastewater and the immunoassays can be used as rapid techniques for quantification of hormone EDCs in wastewater. The ELISA technique proved to be the slightly superior to the RIA in terms of facilities required. The laboratory scale procedures proved that some hormones can be oxidised to other hormones and therefore longer sludge retention times may be required to improve the removal. The study of the different WWTPs configuration showed that plant configuration and operational parameters impact the removal of hormone EDCs. The composition of the influent received by the plant also has an effect on the removal, i.e., whether it’s industrial, domestic or a mixture of both. Results concluded that plants which have either mixing and/or aeration with activated sludge and longer SRTs of more than 10 days have a higher rate of hormone removal than those plants with shorter SRTs and that the activated sludge processes were capable of reducing the toxicity of the influent. Overall results indicated that hormone EDCs are indeed being discharged with the effluents from WWTPs in South Africa. However whether the concentrations left in the final effluents will still have an adverse effect on the aquatic life is a question that still remains unanswered. The aquatic ecosystems are inevitably being polluted with these EDCs and their breakdown products.