Theses and dissertations (Applied Sciences)

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Anti-HIV activity of selected South African medicinal plants
(2013-09-17) Hurinanthan, Vashka; Odhav, Bharti; Parboosing, Raveen
South Africa has the largest number of people infected with HIV/AIDS. It also has more than 30 000 species of plants and many of these have a long tradition of medicinal use. It is highly likely that the treatment for HIV will come from this traditional knowledge. The need for effective preventative and therapeutic agents for HIV remains an urgent global priority. The aim of this study was to screen selected South African medicinal plants for anti-HIV activity and to identify and characterise an active compound from a plant that can be used for HIV treatment. The aqueous and methanolic extracts of the roots, leaves, flowers and stems of thirty eight plant species (108 extracts) were screened for anti-HIV activity. The plants which had anti-HIV activity were further screened for anti-reverse transcriptase activity. Thirty-two extracts exhibited varying degrees of anti-HIV activity. Cleome monophylla, Dichrostachys cinerea and Leonotis leonurus aqueous leaf extracts had anti-HIV-1 reverse transcriptase activity. The aqueous extracts of D. cinerea showed the best anti-HIV activity with a Selectivity Index of 43.5 and significant anti-HIV-1 reverse transcriptase activity. Crude phytochemical screening of D. cinerea showed that it had tannins, saponins, flavonoids and alkaloids but did not contain any phlobatannins, terpenoids, steroids or phenols. D. cinerea displayed a high degree of free radical scavenging activity with an IC50 of 25 μg/ml, therefore the anti-HIV activity could be attributed to the flavonoids present in the plant. Bio-guided fractionation was used to isolate and purify the active compound from the D. cinerea extract. Compounds were isolated by thin layer chromatography and were tested for anti-HIV-1 and anti-reverse transcriptase activity. From these results the active compound was identified, and purified using preparative TLC. The active compound was characterised by High Performance Liquid Chromatography, Ultraviolet-visible spectrophotometry, and Ultra Performance liquid chromatography coupled to MS/MS. Structural elucidation was performed using Nuclear Magnetic Resonance. From these results, it was deduced that the compound isolated from D. cinerea was a catechin. In this study we show that the catechins present in D. cinerea are responsible for the anti- HIV-I activity and inhibits the reverse transcriptase activity which is a key factor in the progression of HIV. Potentially, these results can be used to develop a new drug for the treatment of HIV or as a cost effective therapeutic agent in treating HIV-infected individuals with oxidative stress.
Anti-inflammatory, anti-oxidant and wound-healing properties of selected South Africa medicinal plants
(2017) Mzindle, Nonkululeko Betty; Odhav, Bharti
South Africa has a wide range of medicinal plants that are used traditionally by black Zulu South Africans for the treatment of a range of illnesses, including inflammatory ailments; disease conditions caused by oxidative stress and wound healing. It has been indicated that bioactive compounds isolated from plants contribute to their anti-inflammatory, antioxidant and wound healing properties; hence, herbal remedies have been widely used traditionally in many countries in the management and treatment of wounds. Inflammation is the main condition that relates to a variety of diseases affecting most of the world’s population. It is the body’s immune response to infection and injury and is induced by the release of pro-inflammatory mediator’s —prostaglandins and leukotrienes—following wound occurrence. Wounds result in disruption of living tissue caused by oxidative stress. Anti-inflammatory agents, antioxidants, and antimicrobials play an important role in the wound healing process and they prevent aggravated wound conditions.Controlling inflammation during wound repair is important to minimize any additional complications that may result; hence, chemical agents such as non-steroidal anti-inflammatory drugs (NSAIDS), synthetic antioxidantsand steroids are frequently used. These drugs block the enzymes that are responsible for prostaglandin synthesis in inflammation, react with free radicals thereby interfering with oxidation process as a result affect one or more phases of wound healing. The use of these drugs, however, has been limited as they can cause detrimental side effects when used over long periods of time.There is, consequently, a need to find alternative natural therapeutic drugs. Studies on medicinal plants confirmed that herbal drugs exhibit fewer side effects in comparison with chemical agents and are more cost-effective.Thus the aim of this study was to investigate South African medicinal plants, for anti-inflammatory, antioxidant and wound healing properties. Dissolved extracts of thirty-eight medicinal plants were evaluated for theiranti-inflammatory activity using the 5-lipoxygenase assay as well as free radical scavenging activity using the 1; 1-diphenyl-2-picrylhydrazyl (DPPH) assay.Their safety was evaluated using brine shrimp lethality assay. Proliferation and viability of fibroblast cells was determined by the3-(4, 5-dimethylthiazolyl)-2, 5-diphenyltetrazolium bromide(MTT) assay furthermore a scratch wound assay was used to study the properties of wound healing in vitro and to confirm the anti-inflammatory activities of the dissolved extracts. Migration rate was evaluated quantitatively by an image analyzer. Methanol was chosen for extraction because it completely dissolves extracts. Anova was used for statistical analysis. Almost all aqueous extracts were found to be effective in inhibiting lipoxygenase enzyme when compared to nordihydroguaiaretic acid (NDGA). Aqueous extracts exhibited remarkably high percentage inhibition of lipoxygenase with most above 100% when compared to methanolic extracts. Amaranthus dubius and Portulaca oleracea were found to have good biological activities in the inhibition of 5-lipoxygenase enzymes when compared to the other plants. However, Galinsoga parviflora and Syzygium cordatumwere least effective in inhibiting enzyme activity with percentages as low as -2% and 34% respectively. Percentage inhibitions for methanolic extracts were lower than that of aqueous extracts. Amaranthus spinosus had the highest percentage inhibition among all the methanolic extracts andGalinsoga parviflorahad the lowest. The methanolic plant extracts were found to be more effective in scavenging DPPH free radicals than the corresponding aqueous extracts. All the methanolic extracts exhibited free radical scavenging ability in the range of 60%–104%. Asystasia gangetica, Ficus sur, Heteropyxis natalensis, Hibiscus sabdariffa, Pelargonium sp. showed notably higher scavenging abilities, ranging from 101%–104% compared to Rutin. Methanolic extracts of Heteropyxis natalensis and Hibiscus sabdariffa exhibited scavenging ability even at the lowest concentration of 10μg/ml. Furthermore, aqueous extracts displayed remarkably lower activities than methanolic extracts with thirty-one extracts having a scavenging capacity ranging from 22%—59%. None of the extracts were found to be detrimental to brine shrimp. Almost all the extracts were shown to stimulate the growth of fibroblast cells except the methanolic extract of Solanum nodiflorum, which was shown to be killing the cells at high concentrations with a percentage viability of 46%.As the concentration decreased, however, the viability of cells with this extract increased to 143%. An increase in the number of fibroblast cells was observed in the scratched area of the treated cells and a significant migration rate was also noted with some of the extracts. Aqueous extracts of Sonchus oleraceus (86%), Justicia flava (85%) and Dichrostachys cinerea (85%) and methanolic extracts of Senna occidentalis and Hibiscus sabdariffa were found to have the highest migration rate compared to untreated cells that served as a control. No cell migration was observed with methanolic extract of Solanum nodiflorum.Instead, the extract was found to be toxic to the cells. Some of the plants evaluated in this study have been studied for either anti-inflammatory, antioxidantand wound healing properties in vivo, however, no work has been conducted to demonstrate a correlation between anti-inflammatory, antioxidant and wound healing properties of plant species in vitro. The current study was, therefore, conducted to review medicinal herbs considered as anti-inflammatory, antioxidants and wound healing agents as well as collecting evidence for their effectiveness and pharmacological mechanisms in modern science. In the plant species investigated Amaranthus dubius, Asystasia gangetica, Bidens pilosa, Buddleja saligna, Carpobrotus dimidiatus, Chenopodium album, Dichrostachys cinerea, Emex australis, Ficus sur, Guilleminea densa, Hibiscus sabdariffa, Physalis viscose, Syzygium cordatum, Taraxacum officinale and Tulbaghia violacea demonstrated good anti-inflammatory and wound healing properties.In conclusion the results from this study demonstrated promising anti-inflammatory and antioxidantactivities as well as wound healing properties,furthermoreit was aslo shown that the plant extracts were not toxic to the cells hencethis suggested that the plants investigated, can be used as substitutes or to formulate wound healing agents that are safe to use in primary healthcare.
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.
Bioactive compounds from South African plants against Mycobacterium tuberculosis
(2016) Singh, Alveera; Odhav, Bharti; Coovadia, Yacoob
Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB) has infected approximately one-third of the world population, with 9.6 million TB cases in 2014. The emergence of multi-drug resistant (MDR) and extensively-drug resistant (XDR) strains of MTB has further complicated the problem of TB control. It is now imperative that novel antimycobacterial compounds are discovered in order to treat infections and reduce the duration of current TB therapy courses. For centuries, medicinal plants have been used globally worldwide for the treatment and prevention of various ailments. This occurs particularly in developing countries where infectious diseases are endemic and modern health facilities and services are inadequate. In recent years, the use and search for plant drug derivatives have been fast-tracked. Ethnopharmacologists, botanists, microbiologists, and natural product chemists are trying to discover phytochemicals which could be developed for the treatment of infectious diseases, especially TB. Plants are rich in a wide variety of secondary metabolites, such as tannins, terpenoids, alkaloids, and flavonoids, which have been found in vitro to have antimycobacterial activity. In the search for new lead compounds, nine medicinal plant species, Buddleja saligna, Capparis tomentosa, Carpobrotus dimidiatus, Dichrostachys cinerea, Ekerbergia capensis, Ficus Sur, Gunnera perpensa, Leonotis leonurus and Tetradenia riparia were collected in Kwa-Zulu Natal (KZN) following report of their therapeutic use in traditional medicine to treat symptoms and infections related to TB. They were tested in vitro for their activity against Mycobacterium smegmatis, Mycobacterium tuberculosis H37Rv (ATCC 25177) and three well-characterized clinical isolates of MDR-TB and XDR-TB using the agar incorporation method. The minimum inhibitory concentration of the active plant extracts was determined using the broth microdilution method. Our findings show that five of the nine plants screened have antimycobacterial activity with concentrations ranging from 125 µg/ml to 1000 µg/ml. The aqueous extracts of G. perpensa and T. riparia; and the methanolic extracts of B. saligna, C. tomentosa, and C. dimidiatus possessed significant activity against M. smegmatis, M. tuberculosis H37Rv (ATCC 25177) and the three well-characterized clinical isolates of MDR-TB and XDR-TB. The cytotoxic effect of the active plant extracts was evaluated against the mouse BALB/C monocyte-macrophage (J774.2) and peripheral blood mononuclear cells (PBMCs). The toxic effects of the active plant extracts were evaluated using the brine shrimp lethality assay. Except for a high concentration of G. perpensa none of the other plants which possessed antimycobacterial activity showed any toxic or cytotoxic activity. The active plant extracts were thereafter assessed to determine if they had any effect on the survival or death of mycobacterial species, M. smegmatis, bound within the macrophage (J774.2) cell line at a concentration of 100 µg/ml. B. saligna had inactivated most of the phagocytosed bacilli after 24 hours of treatment therefore, it has a bactericidal effect on the mycobacteria located within the mouse macrophage. A phytochemical investigation of the leaves of B. saligna led to the isolation of two isomeric pentacyclic triterpene compounds namely Oleanolic Acid (OA) and Ursolic Acid (UA) using thin layer chromatography followed by silica gel column chromatography. The structures of these compounds were fully characterized by detailed NMR investigations, which included 1H and 13C NMR. Ursolic acid was isolated from this plant for the first time. Two-dimensional (2D) and three-dimensional (3D) quantitative structure-activity relationship (QSAR) studies were carried out to provide insight on the interaction of the compounds with the enzyme. Molecular docking studies predicted the free binding energy of the triterpenes inside the steroid binding pocket of Mycobacterium tuberculosis fadA5 thiolase compared to a reported inhibitor. Thus, their ability to inhibit the growth of Mycobacterium tuberculosis was predicted and was confirmed to possess significant antimycobacterial activity when tested against M. smegmatis, M. tuberculosis H37Rv (ATCC 25177), clinical isolates of MDR-TB and XDR-TB using the Microplate Alamar Blue Plate (MABA) assay. The present study has scientifically validated the traditional use of medicinal plant B. saligna.
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.
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.
Characterization and application of bambara groundnut starch-lipid complexes
(2017) Oyeyinka, Samson Adeoye; Amonsou, Eric Oscar; Singh, Suren
Bambara groundnut (Vigna subterranea) is an indigenous underutilised leguminous crop to Africa. It is a good source of protein and carbohydrate including starch. Bambara groundnut is a traditional crop grown mainly for subsistence in Southern Africa. Bambara groundnut has the advantage of being drought tolerant and can thrive in hot temperatures and poor soil conditions. Therefore, it has great potential as an alternative crop to soya bean and peanuts for cultivation and utilisation. Bambara groundnut starch can potentially be used for various industrial applications. However, native starches are not suitable for most industrial applications, hence the need for modification. Bambara groundnut starch has been previously modified using physical and chemical modification methods. Natural alternatives such as the use of lipids are being sought to modify starches due to the associated risk with chemically modified starch. In this research, Bambara groundnut starch was modified with lipids to improve functional properties, utilisation and application. Specifically, the physicochemical properties of native Bambara groundnut starch obtained from five Bambara groundnut genotypes and three landraces (maroon, brown and cream) were determined. Bambara groundnut starch was modified with lipids (palmitic acid, stearic acid, oleic acid, linoleic acid and lysophosphatidylcholine) and the physicochemical properties of the modified starch were investigated. Further, the influence of high-pressure homogenization on complexation of Bambara groundnut starch with lipids was assessed in comparison with maize and potato starches. Lastly, an application of modified Bambara groundnut starch in biofilm production was also studied. Bambara groundnut landraces generally showed higher amylose contents (approx. 33%) than the genotypes (approx. 28%). Differences were observed in the crystalline patterns of these starches. Bambara groundnut genotypes exhibited the C-type-crystallinity, while the landraces showed the unusual A-type pattern. In terms of functionality, landrace starches showed better swelling than the genotypes. Subsequent studies on modification used maroon Bambara groundnut starch since the amylose content was higher than other landraces and there was a consistent supply of the grains during the period of the study. Generally, Bambara groundnut starch showed higher complexing ability with all the lipids than maize and potato reference samples. These differences in complexing ability among the starches could be due to the variation in amylose contents (Bambara groundnut starch: 31.5%, maize: 22.5% and potato: 24.6%). Fatty acids complexed better with Bambara groundnut starch than lysophosphatidylcholine, which could be due to the structural differences in comparison with the lysophosphatidylcholine molecule. The number of fatty acid in the glycerol backbone and the additional steric hindrance of the polar phosphatidic acid group in the lysophosphatidylcholine may have reduced its complexing ability. Among the fatty acids, palmitic acid complexed better than stearic and the unsaturated fatty acids, possibly due to its short chain length compared to other fatty acids. Bambara groundnut starch showed reduced peak and setback viscosities in the presence of stearic acid, linoleic acid and lysophosphatidylcholine, suggesting the formation of V-amylose complex. Bambara groundnut starch pasted with lipids displayed reduced gelling ability compared to their unmodified counterparts. XRD studies of freeze-dried paste revealed peaks at 2Ѳ = 7.4, 12.9 and 19.9o confirming the formation V-amylose complexes in Bambara groundnut starch. Modification of Bambara groundnut starch with lipids resulted in reduced digestibility. High-pressure homogenization significantly increased the complexing ability of Bambara groundnut starch with lipids. Homogenized Bambara groundnut starch-lipid complexes generally exhibited higher complex index than their unhomogenized counterparts. The higher complexing ability could be attributed to the effect of high-pressure which may have enhanced greater dispersion of lipids in the starch-water system. X-ray diffraction studies also revealed the formation of higher complexes as shown by high intensities at peaks (2Ѳ= 7.4, 12.9 and 19.9o) corresponding to V-amylose complexes. Bambara groundnut starch-lipid complexes displayed significantly higher melting temperatures (95.74-103.82oC) compared to native uncomplexed starch (77.32oC). Homogenized Bambara groundnut starch complexes were non-gelling while the unhomogenized types produced weak gels, with G′ ˃ G″ in the range of 0.1- 10 Hz. Complexation of Bambara groundnut starch with lipids using high-pressure homogenization may be employed in the production of modified starch with non-gelling properties and higher thermal stability suitable for certain industrial application, such as fat replacers in mayonnaise, frozen foods and desserts for a better mouth feel. The physicochemical and mechanical properties of biofilm prepared from Bambara groundnut starch modified with stearic acid at varying concentrations of 0, 2, 4, 6, 7 or 10% were further studied. By SEM, Bambara groundnut starch films containing stearic acid (˃ 2%) showed a progressively rough surface compared to those with 2% stearic acid and the control. The addition of 2% stearic acid to Bambara groundnut starch film reduced water vapour permeability by approximately 17%. However, mechanical properties of starch films were generally negatively affected by stearic acid. Bambara groundnut starch film may be modified with 2% stearic acid for improved water vapour permeability and thermal stability with minimal effect on tensile strength.
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.
Composition and functional bioactive properties of bambara groundnut protein and hydrolysates
(2016) Arise, Abimbola Kemisola; Amonsou, Eric Oscar; Ijabadeniyi, Oluwatosin Ademola
Bambara groundnut (Vigna substerranea) is an indigenous legume of African origin which is currently experiencing a low level utilisation. It is tolerant to drought and can grow under poor soil conditions in which other lucrative crops such as groundnut cannot grow. Bambara is a good source of protein comparable to that of cowpea and slightly lower than soya bean. In order to assess the potential use of bambara protein as a functional ingredient in food systems and as an important ingredient for the formulation of therapeutic product, the knowledge of its protein composition, structure and functionality becomes important. The main goal of this thesis was to determine the composition and bioactive properties of bambara protein and its hydrolysates. Specifically, a comparative study was carried out on the protein content, yield and functional properties of protein concentrates prepared from three different bambara landraces using different extraction methods (Salt solubilisation and Acid precipitation). There was no significant difference in protein content, yield and functional properties of the landraces. However, the method of extraction had an influence on their physicochemical and functional properties. Acid precipitation produced bambara protein concentrates with high protein content and yield (79% and 52% respectively) when compared to salt solubilisation (protein content - 57% and yield - 25%). Protein concentrates prepared through salt solubilisation method exhibited better functional properties in terms of water absorption capacity, oil absorption capacity, foaming capacity, foaming stability and emulsion activities when compared to concentrates obtained through acid precipitation. Furthermore, the composition of bambara proteins produced through isoelectric precipitation was determined. SDS PAGE revealed four major bands; a broad band at 55 kDa which was analysed to be vicilin, two medium bands at 62 kDa and 80 kDa and a high molecular weight (HMW) protein at 141 kDa. Further investigation of bambara protein revealed vicilin (55 kDa) with two sub units as the major protein in bambara and this was also confirmed by the proteomic map. The proteomic map revealed acidic amino acids as the major protein of bambara which is characteristic of vicilin, the map also showed that there were differences in the number of spots across the landraces with 77 spots matching each other. Circular dichroism spectroscopy exhibited reductions in α-helix, and β-pleated sheet conformations as pH varies. In addition, the tertiary structures as observed from the near-UV CD spectra were also influenced by shifts in pH conditions. Differential scanning calorimetry thermograms showed two endothermic peaks at around 67 and 81oC respectively. These can be attributed to thermal denaturation of vicilin and the HMW protein. Subsequent studies used isolates from red bambara since the composition of the landraces were similar. Bambara protein isolate was subjected to enzymatic hydrolysis using three proteases (alcalase, pepsin and trypsin) to produce various bambara protein hydrolysates (BPHs). BPHs were investigated for antioxidant and antihypertensive activities. The in vitro structural and functional characteristics of bambara protein and its enzymatic protein hydrolysate revealed that bambara groundnut possessed antioxidant properties against a variety of physiologically relevant free radicals. High surface hydrophobicity and the molecular size of the peptide seem to be important for scavenging of hydroxyl radicals, ferric reducing power and metal chelation. BPHs and peptide fractions were able to scavenge DPPH radicals with greater affinity for smaller size. Less than 1 and 1-3 kDa pepsin fraction was able to scavenge DPPH radical more than glutathione, BPHs and its fractions scavenge ABTS•+ three folds than the isolate. Scavenging of superoxide radicals was generally weak except for 5-10 kDa peptide fractions. All BPHs inhibited linolenic acid oxidation with greater affinity for the lower molecular size peptide. BPHs showed potential antihypertensive properties because of the in vitro inhibition of activities of angiotensin converting enzyme (ACE) and renin inhibition. The molecular size had significant effect on the ACE inhibitory properties with low molecular weight peptide (<1 kDa) fractions exhibiting significantly higher (p<0.05) inhibitory activities. However, enzyme type had synergistic effects on renin inhibition with alcalase hydrolysate showing highest inhibition at 59% when compared to other hydrolysates and their membrane fractions. The fractions with <1 and 1-3 kDa peptides showed a higher potential as antihypertensive and antioxidant peptides. Based on this study, incorporation of bambara protein isolate as an ingredient may be useful for the manufacture of high quality food products. Likewise, the bambara protein hydrolysates, especially the <1 kDa and 1-3 kDa fraction represent a potential source of bioactive peptides in formulating functional foods and nutraceuticals.
The consumption, product compliance and manufacturer insights of plant-based foods in KwaZulu-Natal
(2024-09) Govender, Camilla; Naicker, Ashika; Makanjana, Onwaba
Background: The term plant-based diet focuses predominantly on the consumption of food from plants. A plant-based diet consists mainly of wholegrains, fruits, vegetables, legumes, nuts, seeds and may also include more processed food products made from these ingredients. Food is the single most powerful lever to optimise human health and environmental sustainability. A large body of work has emerged on the environmental impact of various diets. As per the findings of the Environmental Assessment and Reference Tool (EAT)-Lancet Commission, many studies have concluded that a diet rich in plant-based foods (PBFs) has improved health and also environmental benefits. This transition has led to a noteworthy increase in the demand for PBFs globally, and South Africa is no exception. Aim: This study aimed to determine the consumption, product compliance and manufacturer insights of plant-based foods in KwaZulu-Natal (KZN). Methods: This study used a mixed methods approach, namely, qualitative and quantitative research methodology. The first objective of this study was to determine the consumption of PBFs and identify barriers and facilitators influencing the adoption of plant-based diets through a consumer survey. The study's second objective was to document the scope of plant-based products in terms of availability through in-store and online observations that were available in KZN. All products were analysed to determine the scientific and regulatory compliance of plant-based products by analysing food labels according to the Foodstuffs, Cosmetic and Disinfectant Act (Act 54, 1972), R146 Regulation relating to the labelling and advertising of foodstuffs. The qualitative aspect of the study delved into the product development practices, trends and challenges of plant-based manufacturers in South Africa through key informant interviews. The adequacy of the diet among consumers (n=100) who consumed plant-based foods was assessed through two 24-hour food recalls and analysed for dietary adequacy. Results: Three hundred and eighty consumers participated in the online survey, of which 67.7% (n=259) of participants were women and 31.3% (n=120). Regarding the frequency of consumption of PBFs, 47% (n=180) of participants consumed PBFs between 2 to 3 times a week, 27.7% (n=106) of participants consumed PBFs daily, 17.2% (n=66) of participants consumed PBFs once a week, and 4.2% (n=16) of participants consumed PBFs once a month. A significant 63.4% (n=243) of participants indicated that they spend at most R60.00 on plantbased products, p<0.001. Using the binomial test to assess if any response option was selected significantly more than others, a significant number of participants (80% n=307) p<0.001, reported that they consumed PBFs for health reasons, followed by 37% (n=142) who reported that they consumed PBFs due to religious reasons. A significant 64% (n=245) and 58.5% (n=224) of participants reported that the most common format of plant-based meat alternatives that they consumed were burgers and sausages, respectively (p<0.001). The findings of this study also revealed that there are many challenges and barriers that are associated with the adoption of PBFs. This study found that 58.5% of participants (n=224) reported that PBFs were expensive (p<0.001), and 40.5% of participants (n=155) indicated that PBFs were limited and lacked variety. A comprehensive database of plant-based products (n=431) in the South African market was documented through in-store and online observations. Eighty-nine percent (n=386) of the products documented were manufactured in South Africa.. Regarding South Africa's labelling regulation, 95% (n=411) of the plant-based products complied with legislation. Key informant interviews were conducted with manufacturers to understand the market trends and constraints within the South African plant-based food sector. Key themes emerging from the key informant interviews included consumer-centric PBFs, consumer-driven plant-based trends, growth in the plant-based market, dynamic and evolving plant-based product development, challenges in PBF manufacture and marketability, insufficient government support and market-related price of PBFs. Furthermore, the adequacy of plant-based diets was analysed using the 24-hour food recall (n=92). The majority of nutrients fell below the EAR for both males and females. Both groups exhibited dietary inadequacies in specific micronutrients: vitamin D, folate, vitamin B12, and iron. Dietary inadequacy of specific micronutrients was noted for vitamin D, Folate, vitamin B12 and iron in both groups. However, there was notable carbohydrate excess intake across both groups and genders, surpassing the recommended estimate for energy. Conclusion: This study comprehensively examined the consumption patterns, product compliance, and manufacturer insights of PBFs in KZN. The findings reveal a growing interest in flexitarian diets and frequent consumption of PBFs, driven primarily by health motivations but hindered by cost and limited variety. Retail availability of PBFs in KZN is extensive, offering consumers a wide range of choices in both fresh and frozen categories, reflecting the robust state of the plant-based industry in KZN and it can be reasonably generalised to the whole country Regulatory compliance among PBFs is generally high, although small-scale manufacturers occasionally fall short of labelling standards. Manufacturer insights indicate a consumer-driven demand for PBFs that closely mimic meat products, necessitating innovation and regulatory support to stabilise this emerging market. Nutritional assessments of plant-based diets highlight a critical need for dietary supplementation to address deficiencies in essential vitamins and minerals such as B12, iron, and zinc. The study underscores the importance of nutrition education to guide consumers towards healthier food choices within plant-based diets. In conclusion, while the plant-based food sector in KZN is expanding and meeting many consumer needs, there are significant opportunities for improvement in product variety, affordability, regulatory compliance, and nutritional adequacy. Addressing these challenges through coordinated efforts among stakeholders can further enhance the adoption and benefits of plant-based diets. The conclusions drawn from this study can be reasonably generalised to metropolitan areas of the whole country.
Cyanobacteria-microalgae consortia as bio- inoculants for enhancing soil fertility and plant growth
(2024-05) Jose, Shisy; Bux, Faizal; Kumari, Sheena; Renuka, Nirmal; Ratha, Sachitra Kumar
Modern agriculture that heavily utilizes synthetic fertilizers has raised significant environmental concerns worldwide. Microalgae-based bio-inoculants have become a more viable and eco- friendly option to reduce the reliance on chemical fertilizers for improving soil fertility and plant growth in agronomic practices. Indigenous microalgal species can colonize and thrive well under local conditions, making them well-suited for use as bio-inoculants. Additionally, it appears that using microalgal consortia of green microalgae and nitrogen (N)-fixing cyanobacteria could be beneficial because the green microalgae can supply carbon (C), the cyanobacteria can fix C and N in the soil. They both can produce agriculturally beneficial metabolites and improve soil nutrient availability. This synergistic relationship could enhance the overall effectiveness of bio- inoculants and promote sustainable agriculture practices. In this study, thirteen microalgal strains were isolated from agricultural fields of Durban, KwaZulu Natal, South Africa. Two N-fixing cyanobacteria (Nostoc sp. and Calothrix sp.) and two green microalgae (Desmodesmus armatus and Chlorella sp.) strains were selected and analyzed for metabolites of agricultural significance for the development of suitable microalgal consortia under N-deficient conditions. The amount of indole acetic acid (IAA) in biomass extracts from cyanobacteria (Calothrix sp. (2.54 ng g−1) and Nostoc sp. (1.52 ng g−1)) was significantly higher than in extracts from green microalgae (Chlorella sp. (0.32 ng g−1) and Desmodesmus armatus (0.20 ng g−1)). A completely randomized design was used to develop and evaluate eight microalgal consortia on a N-deficient medium with the selected microalgal strains. A significant improvement in biomass productivity, indole acetic acid production, nutrients viz C, N, phosphorus (P), potassium (K), calcium (Ca), copper (Cu), iron (Fe), and manganese (Mn) was observed in the selected consortium compared to the individual isolates. The microalgal consortium was further analyzed for biostimulant properties using seed germination assay in chili seeds. A significant increase in seedling length and leaf number was observed in seeds treated with biomass extracts of consortium compared to the control. The pot culture study also supported the effect of microalgal bio-inoculant on soil fertility, chili plant growth and native soil microbiomes. Soil enzyme activity increased significantly (p < 0.05) with microalgal treatments, with soil dehydrogenase activity (DHA), organic carbon (OC), soil chlorophyll (Chl), total polysaccharides (TP) and nutrients such as C, N, P, K and Mn being more enriched at 100 % and 50 % treatment applications in comparison to control (Cnt). Growth responses in terms of shoot and root (fresh and dry weight), root length and leaf number were significantly high in 50 % microalgal treatments (Al(50 %)+CF(50 %)) when compared to Cnt. With different soil nutrient parameters and microbiome (bacterial and fungal) indicators, we could successfully predict higher soil fertility and plant growth responses to microalgal inoculations. Results using 16SrRNA and ITS amplicon sequencing suggested that microalgal bio-inoculation improved the diversity and composition of native soil microbiome, leading to an increase in soil fertility, plant growth and yield. Further, shotgun metagenomics has confirmed a higher enzymatic activity involved in C, N, and P metabolisms in 50 % and 100 % microalgal treatment compared to the control. Potential shifts in microbial taxa and functional genes indicated that microalgal bio-inoculant was a major driver of microbial metabolic change. The findings from the study suggested that microalgal bio-inoculation improved the diversity and composition of native soil microbiomes, leading to an increase in soil fertility, plant growth, and yield in chili plants.
Cytotoxicity and gene expression of selected apoptotic markers in the human laryngeal carcinoma cell line (HEp-2) by Bulbine spp. fractions
(2013-07-30) Singh, Rishan; Reddy, Lalini; Permaul, Kugen
Apoptosis, or programmed cell death, is a process which is pivotal in eliminating damaged, infected, or unwanted cells from the body. It has been studied in numerous types of cell lines ranging from normal to infected cell lines, and there have been a wide range of studies on apoptosis in laryngeal cancer because this type of cancer has become one of the most common types of head or neck cancer due to the high incidence of alcohol consumption, tobacco smoking and chewing of betel quid amongst populations. Laryngeal cancer is usually treated with radiotherapy or is surgically removed, but due to the loss of the function of the larynx after surgery, it has been suggested that alternative strategies or ways of treating laryngeal cancer are required. This has prompted the use of, and research in the field of, plant medicine to combat laryngeal cancer. Plant medicine has been used for centuries by the Chinese, Indian and Arabian population in Uhani, Ayurveda and Siddha as a form of replacing conventional medicine with complementary and alternative medicine, these include many plants from the family Asphodelaceae, which have become marketable commodities owing to their medicinal values and traditional uses. Amongst these plants, the genus Bulbine has been used as a form of natural medicine in rural Africa and they are also exploited for their aloe vera properties as well as their possession of phytochemical compounds such as isoflavanoids, nor-lignans, naphthalene derivatives, anthracene and poly prenylated flavonoids. There has been a compelling amount of literature on the traditional uses of the Bulbine spp. because these are linked to the Bulbine spp. having secondary metabolites such as pyroles, chromones, coumarins, bianthraceane, benzene as well as alkaloids. However, for Bulbine natalensis and B. frutescens, the plants of interest in this study, the location of anticancer compounds in them are the only amounts of information available. It has been reported, traditionally, that B. natalensis possesses the anticancer potential in the roots, while the anticancer potential for B. frutescens is in the leaves. However, this requires scientific clarification. Therefore, this study was conducted to assess programmed cell death or apoptosis by analysing the responses of the human laryngeal carcinoma cell line (HEp-2) to crude aqueous and organic (50% and 100% ethanol) fractions of B. natalensis and B. frutescens. In order to have achieved this, the HEp-2 cell line was exposed to the above mentioned fractions at three different final concentrations (20, 2 and 1μg/ml) and assessed for cytotoxicity using the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay as an indicator of cell death after fraction utilisation (3 days) for 5 and 8 days. The differences in the potency of the Bubline spp. fractions were confirmed using the non-parametric ANOVA test. Thereafter, selected fractions were screened for apoptotic potential using reverse transcriptase-polymerase chain reaction (RT-PCR) to determine the expression of bax and caspase-3 biomarkers, which are the biomarkers that participate in mitochondrial, endoplasmic reticulum and receptor-ligand mechanism of apoptosis. The fractions of B. frutescens were selected relative to those of B. natalensis for the RT-PCR procedure (read section 3.4.1. for the selection procedure) and links between the cytotoxicity and gene expression results were analysed. It was found that the B. natalensis fractions had a much greater cytotoxic effect on the HEp-2 cell line compared to fractions of B. frutescens by the fifth day of the MTT assay. On the eight day of incubation, there was an increase in HEp-2 cell line proliferation by the fractions of both plant species administered. The fractions selected for bax and caspase-3 gene expression analysis for B. natalensis were the: 20 μg/ml root and corm aqueous fractions, 20 μg/ml leaf and corm 100% ethanol fractions, 20 μ g/ml corm 50% ethanol fraction, 2 μg/ml root aqueous fraction, 2 μg/ml leaf 100% ethanol fraction and the corm 1 μg/ml aqueous and 50% ethanol fractions. The fractions that were compared to B. natalensis were the 20 μg/ml root and leaf aqueous and 100% ethanol fractions respectively, the 2 μg/ml root aqueous fraction and the 2 μg/ml leaf 100% ethanol fraction. It was found from RT-PCR analysis that all of the B. natalensis fractions tested induced expression of caspase-3, which indicated that those fractions were capable of inducing apoptosis in laryngeal carcinoma in vitro, since caspase-3 is the molecular indicator of apoptosis. The aqueous B. frutescens root fraction, did not induce expression of caspase-3 gene, although it caused expression of bax. This implied that the root aqueous B. frutescens fraction, may be involved in some other form of cell death, other than apoptosis. It was also found that there was variability in the response of the HEp-2 cell line to the Bulbine spp. fractions because of the variation in bax expression among fractions of different concentration. It was difficult, from this study, to classify fractions into categories for their mechanism of action, because not all of the fractions that caused the expression of capase-3, induced bax gene expression. Hence, proper conclusions were unable to be made, more so, because all the mechanisms of apoptosis mentioned, involve bax gene activation in order to proceed to completion. Therefore for those Bulbine spp. fractions to which the HEp-2 cell line exhibited a variable response to, it was postulated that cell death or apoptosis occurred through some other unknown mechanism. Overall, the cytotoxicity result didn’t correlate to the gene expression results because fractions that promoted HEp-2 cell line growth by day five, expressed apoptotic markers, which highlighted the sensitivity and accuracy of the cells-to-cDNATM II kit for detecting a few possibly apoptosed cells. This was confirmed by the fact that the HEp-2 cell line used in the MTT cytotoxicity assay and gene expression study had the same passage number and were viable, the latter being achieved because the MTT assay only measures the cytotoxicity of compounds once they have been taken up by viable cells – measuring mitochondrial activities expressed as absorbances. Therefore, the deduction that HEp-2 cell death may be due to bax/caspase-3 expression was valid because the mRNA was isolated from viable HEp-2 cells that had been killed by Bulbine spp. fractions of different polarity. Furthermore, the lack of correlation between the cytotoxicity and gene expression results indicated the amount of HEp-2 cell line proliferation by the fraction out-competes those that died, thereby producing a negative cytotoxicity result. There was a relationship between the traditional information about the anticancer potential for B. natalensis and B. frutescens. For example, the aqueous root fractions of B. natalensis were found to be non-toxic to the HEp-2 cell line, but did express caspase-3, which indicated the possibility of apoptosis. Similarly, the 100% ethanol leaf B. frutescens fractions were non-toxic to the HEp-2 cell line, but were able to induce apoptosis as well. This emphasised that the MTT cytotoxicity assay should be compared with other methods of measuring cytotoxicity when performing studies like this, because although literature has emphasised many advantages of using the MTT cytotoxicity assay in apoptotic studies, this study proved otherwise. When identical HEp-2 cells were treated with the same extract, only some cells were killed (apoptosis) whereas others proliferated. This was because although the cells were identical phenotypically, they were all probably at different phases of the cell cycle resulting in the HEp-2 cells responding variably to the same fraction at different concentrations. It was also found that the responses were concentration independent. For example, the 1 μg/ml B. natalensis corm fraction exhibited the highest toxicity of the three concentrations administered. The lowest cytotoxicity was achieved for the 20 μg/ml fraction – showing a proliferative effect on the HEp-2 cell line. Similarly, the 2 μg/ml aqueous B. natalensis leaf fraction induced the highest cytotoxicity level in the HEp-2 cell line followed by the 1 μg/ml and then the 20 μg/ml fractions. Apart from the genetic variation in identical HEp-2 cells; this indicated that the HEp-2 cell line was selective to particular fractions of the Bulbine spp. for utilisation. Concentration independence and HEp-2 cell preferential selection has been reported in many other studies involving plant fractions/extracts and natural products. This study demonstrated that although all the tested B. natalensis fractions were capable of inducing HEp-2 cell death possibly via. apoptosis (caspase-3 induction), a lack of any link between apoptosis and the cytotoxicity results (hence the 20 μg/ml corm fraction had a negative cytotoxicity but expressed both apoptotic markers), indicated the need for phytochemical screening of both Bulbine spp. in future, to determine the compounds that are responsible for the cytotoxicity and gene expression result outcomes of both Bulbine spp. fractions. Furthermore, procaspase genes also have to be analysed since genes are expressed to form procaspases, which then form active caspases. Although normal cells also express caspase-3 genes during apoptosis, this study focused exclusively on the effect of Bulbine natalensis and B. frutescens fractions (selected relative to the cytotoxicity results of B. natalensis) on the HEp-2 cell line (read cell culture and cytotoxicity discussion for selection of HEp-2 cell line). The validity of this study is confirmed by similar experimental designs that assayed the cytotoxicity of plant-derived or natural compounds on cancer cell lines only, and the detection of apoptosis through caspase- 3 induction and other unrelated methods. This is the first study to report the induction of apoptosis in cancer cell lines by Bulbine spp. fractions using cytotoxicity and the expression of bax and caspase-3 apoptotic markers. It provides insight into the interaction between the HEp-2 cell line and the aqueous and organic fractions of B. natalensis and B. frutescens by analyzing links between cytotoxicity and bax and caspase-3 gene expression; which could probably contribute to drug design with selected Bulbine spp. fractions. Further investigations are required in future, to confirm the possible drug targets of the studied Bulbine spp. fractions in an attempt of assaying their therapeutic importance.
Design of a worksite intervention to lower cardiometabollc risk In South Africa
(2023-05) Singh, Shivneta; Naicker, Ashika; Grobbelaar, Hendrina Helena
Background: Non-communicable diseases (NCDs) continue to increase globally, with an unduly larger impact in low to middle income countries (LMICs). NCDs are the main cause of death worldwide. There is strong evidence that lifestyle changes, such as weight loss, increased physical activity, and improved diet quality can help avert or slow down type 2 diabetes and reduce cardiometabolic risk factors, for example, high blood glucose, plasma lipids, and blood pressure. Regardless of the verification of research data, supporting the use of lifestyle interventions to prevent diabetes (improve glucose tolerance and lower high blood pressure), and implementing interventions in real-life settings has been proven to be difficult. Aim: The purpose of the study was to guide the development of an acceptable, appropriate, and feasible worksite intervention targeting the food environment and behavioural intervention to reduce cardiometabolic risk at a worksite in South Africa (SA). Methodology: In this cross-sectional study both qualitative and quantitative methods were used. The capacity of the built environment was explored through structured observations of the food and physical environment to offer healthy food and promote physical activity. Semi-structured in-depth interviews (IDIs) were conducted with worksite managers and canteen managers to assess the appropriateness, acceptability and feasibility of changes at worksites and explore the perceptions, provisions, facilitators and barriers to healthy eating at the worksite environment. Purposive snowballing sampling was used to recruit worksite managers and canteen managers for the IDIs. Focus Group Discussions (FGDs) were conducted amongst employees to explore the perceptions, provisions, drivers of and barriers to healthy eating at the worksite. An Organisational Readiness to Implement Change (ORIC) questionnaire was administered face to face at the worksite canteens and online through emails to employees aged between 18 to 65 years to determine the worksite readiness to implement changes at the worksite. Canteen staff were approached to rate possible intervention components for the purpose of tailoring the interventions and identifying the best way to deliver the intervention. The interventions were rated by canteen managers or canteen staff on a scale from one to five with regards to the feasibility of implementing different components of the intervention: with one (1) being impossible to implement and five (5) being easy to implement. A scoping review was carried out to gather data from empirical findings on the categorisation of healthy foods through Front of Pack (FOP) labelling schemes and was reported using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) extension for Scoping Reviews (PRISMA-ScR) guidelines. Key findings from the formative work were used to select suitable food and physical environment interventions for the worksite. Worksite canteen intervention training was conducted to train canteen managers for the implementation of the food environmental intervention using the training manual that was developed by the researcher. Results: The study was conducted at six Unilever SA worksites, three in KwaZulu-Natal (KZN) and three in Gauteng province. The Unilever worksites comprised of five factories and one Head Office, namely, Unilever Maydon Wharf (Personal Care and Home Care), Unilever Indonsa (Savoury dry food plant - Knorr, Robertson’s, Rajah), Unilever Khanyisa (Home Care), Boksburg (Home care) and Lordsview (Ice cream -Ola) and La Lucia Head Office (Head Office). The study was also conducted at Retailability Head Office, a fashion apparel brand, which borders on the Unilever Head Office, making a total of seven worksites. The findings of the structured observations for the physical environment indicated that walking tracks were demarcated within the worksites; however, there were no signage prompts to encourage walking. Stairwells were clean, well lit, easily visible and accessible at each building; however, the health benefits of using the stairs were not displayed. The food environment was observed at six canteens including La Lucia Head Office and Retailability, Unilever Maydon Wharf, Unilever Indonsa, Unilever Khanyisa and Unilever Boksburg. There were two canteens at La Lucia Head Office: the main canteen and a coffee shop. There was no canteen at Unilever Lordsview, but there was a dining area with packaged food sold at the vending machine. Regarding the observation of the food environment, the worksite canteens offered five subsidised contract dishes among other unsubsidised dishes of which the healthy meal was the most expensive in a four-week menu cycle. Pre-made pre-packaged food options were sold at the canteens and displayed near to the point of sale. Ten semi-structured IDIs were conducted with worksite managers, to assess the appropriateness, acceptability and feasibility of changes at worksites and to explore the perceptions, provisions and facilitators of and the barriers to healthy eating at the worksite environment. Several themes emerged from the IDIs with worksite managers with regards to participation in a lifestyle intervention namely: availability, worksite resources and barriers to participation. Four semi-structured IDIs were conducted with canteen managers to assess the appropriateness, acceptability and feasibility of changes at worksites and barriers to healthy eating at the worksite environment. Numerous themes emerged from the IDIs with canteen managers including the enablement of a healthy food environment, information dissemination, employee preference and canteen enablers. Five FGDs were conducted, each group comprising of 4-6 employees, until data saturation was reached to understand the appropriateness, acceptability and feasibility of a range of possible changes at the worksite. Many themes emerged from the FGDs such as nutrient-dense foods, nutrient-poor foods and energy-dense beverages as well as the food environment and healthy longevity. Worksite readiness to implement change was determined through the ORIC questionnaire. The ORIC statements that Unilever employees agreed with most scored 4.51 on the scale of “I believe this change will benefit our worksite”, followed by 4.49 on the scale of “I am committed to implementing this change” and 4.41 on the scale of “I want to implement this change”. The lowest scores (disagreement) were 3.35 for “I have the equipment we need to implement this change”, 3.37 for “I have the resources we need to implement this change” and 3.46 for “I have the expertise to implement this change”. All statements were significantly different from ‘3’, on average. All mean values were >3 which indicates there was a significant agreement with all the statements. Factor analysis with promax rotation was applied to the 31 statements. A Kaiser-Meyer-Olkin Measure of Sampling Adequacy (KMO) of 0.958 and a significant Bartlett’s test indicated that the data was adequate for successful and reliable extraction. The alpha value for factor 1 and 2 was >.7, therefore indicating reliability. Composite variables were formed by calculating the average of the agreement scores for all items included in a variable. It was observed that there was a significant agreement for commitment (COM) and implementation (IMP) (with agreement that they desired the change and were committed to it being significantly higher than their agreement that they could implement the change). Results from a paired ttest showed agreement that they desired the change and were committed to it and were significantly higher than agreement that they could implement the change, t (299) = 15.229, p<.001. Further analysis to determine differences across worksites was conducted. Analysis from ANOVA showed that there was a significant difference in COM (commitment) across worksites, F (3, 296) = 5.375, p=.001. Post hoc analysis using Tukey’s test indicated that agreement to commitment was significantly higher at Maydon Wharf than at La Lucia Head Office (p=.043) and at Khanyisa (p=.021); and at Indonsa than at La Lucia Head Office (p=.031) and at Khanyisa (p=.021). Furthermore, there was a significant difference in IMP (implementation) across worksites, F (3, 296) = 9.960, p<.001. The ORIC statements that Retailability employees agreed with the most scored 4.58 on the scale: “I am committed to implementing this change”, followed by 4.53 on the scale of “I believe this change will make things better”, 4.50 on the scale of “I feel that implementing this change is a good idea” and 4.50 on the scale of “I feel this change is compatible with our values”. The lowest scores (indicating disagreement) were 3.11 for “I have the equipment we need to implement this change”, 3.24 for “I have the resources we need to implement this change” and 3.46 for “I have the expertise to implement this change.” All statements were significantly different from ‘3’, on average. All mean values were >3 which indicated there was significant agreement to all the statements. Factor analysis with promax rotation was applied to these 31 items. Two factors were extracted which accounted for 72.19% of the variance in the data. A Kaiser-MeyerOlkin Measure of Sampling Adequacy (KMO) of .784 and a significant Bartlett’s test indicated that the data was adequate for successful and reliable extraction. The alpha value for factor 1 and 2 was >.7, therefore indicating reliability. It was observed that there was a significant agreement for commitment (COM) and implementation (IMP) (with agreement that they desired the change and were committed to it being significantly higher than their agreement that they could implement the change). Results from a paired ttest showed that agreement that they desired the change and were committed to it was significantly higher than agreement that they could implement the change, t (37) = 6.727, p<.001. The intervention rating scale was completed by 12 canteen managers and canteen staff using a scale from one to five for the feasibility of implementing different components of the intervention. Interventions that were easy to implement were reported by 100% (n=12) of participants for increasing fruit and vegetable choices, 92% (n=11) for the provision of free water, followed by 75% (n=9) for the addition of a salad bar and ready-to- eat healthy meals. The one-sample t-test was done to determine whether the average ‘implementability’ score differed significantly from the central score of ‘3’. The easiest interventions to implement were reported by 83% (n=10) of participants being the strategic positioning of healthier alternatives to make healthy items more accessible with 75% (n=9) recommending traffic light labelling and healthy option stations. The most difficult to implement was reported by 25% (n=3) of participants for the display of kilojoules of a product translated into the number of minutes to perform a certain physical activity. A scoping review was conducted on FOP labelling schemes to inform the selection of a FOP labelling scheme best suited for canteen foods at worksites in SA. Several articles (n = 2513) were identified and screened after excluding duplicates (n = 2474). Overall, 1347 articles were excluded from the study because their abstracts and titles did not match the qualifying criteria. A total of six articles was used in the qualitative analysis after a full-text review of the remaining articles. It was concluded that grading foods into categories of healthfulness through evidence of key nutritional dimensions is a practical tool to inform food environmental interventions that may assist in public health promotion by influencing consumer choice in workplace canteens and beyond. The results from the observations, IDIs, FGDs, ORIC questionnaire, canteen rating intervention scale and scoping review were used to guide the development of the physical and canteen intervention and canteen staff were capacitated with training to implement the food environment intervention. A list of interventions detailing the tools to be used and responsibilities for executing the six weeks’ intervention with two weeks’ maintenance classes for phase three of the study, titled the South African Pioneer Worksite Multicomponent Lifestyle Intervention Study (WMLIS) was developed by the research team. Conclusion: Interventions are a useful tool that can be used in worksite settings to improve employees' overall occupational well-being. Interventions targeting the food environment and behavioural intervention can promote healthy eating behaviours and reduce cardiometabolic risk. It is important to take into account organisational complexity and the built environment when designing an intervention. In this study, formative research methods were used to engage worksite stakeholders to develop an intervention plan that is both theoretically and practically grounded to foster institutionalisation of the intervention. The findings of this study was used to contextualise and guide the development of acceptable, appropriate and feasible worksite food environment and behavioural intervention to reduce cardiometabolic risk among South African employees. The methods used allows for drawing of general conclusions for the implementation of lifestyle and food environment changes at worksites in SA. It is recommended that the study should be scaled up to other worksites to determine the comparativeness of this study to the response of other worksites regarding the acceptability, appropriateness and feasibility of worksite interventions.
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.
Detection and quantification of nitrifying bacteria from South African biological nutrient removal plants
(2013-07-30) Ramdhani, Nishani; Bux, Faizal; Pillai, Sheena Kumari Kuttan
Nitrification is a crucial step in biological nutrient removal (BNR) processes, mostly carried out by a group of nitrifying bacteria which includes ammonia-oxidising bacteria (AOB) and nitrite-oxidising bacteria (NOB). Nitrification failure has proven to be a common operational problem in full-scale wastewater treatment plants (WWTP) since nitrifying bacteria are very sensitive to sudden changes in environmental or plant operating conditions. The current investigation was carried out to advance our understanding of the distribution of nitrifying bacterial populations and their performance at three different BNR plants in KwaZulu-Natal, South Africa. The latest molecular techniques such as fluorescent in situ hybridisation (FISH)-confocal scanning laser microscopy (CSLM), polymerase chain reaction (PCR) and real-time quantitative PCR (Q-PCR) were applied to detect and quantify nitrifying bacteria. When using FISH to target the nitrifying population, it necessitated optimising pre-treatment protocols of the samples to improve accuracy during quantification. Sonication was found to be the superior method of dispersion based on the least disruption of nitrifier cell integrity, irrespective of the sludge type. The effect of plant configurations and wastewater characteristics on the distribution of the nitrifying bacterial population and subsequently on the nitrification performance was evaluated using FISH and PCR. FISH results revealed the dominance of Nitrosomonas (AOB), Nitrobacter (NOB) and Nitrospira (NOB) for all BNR plants. The 16S rRNA analysis of PCR products using genus-specific primers, revealed the presence of more than one species of the same group at these plants. Nitrosomonas spp. including Nitrosomonas halophila, Nitrosomonas eutropha, Nitrosomonas europaea, Nitrosomonas aestuarii and an unidentified Nitrosomonas spp. were found to dominate among the AOB and Nitrobacter vulgaris, Nitrobacter alkalicus, Nitrobacter hamburgensis and an unidentified Nitrobacter spp. were the dominant species for NOB. Among these species, Nitrosomonas aestuarii, Nitrosomonas europaea, Nitrobacter hamburgensis were detected only from the industrial wastewater samples. The efficiency of two commonly used techniques viz., FISH and Q-PCR for the detection of nitrifiers from WWTP were also studied and compared, specifically targeting Nitrobacter sp. Even though there were slight variations in the quantification results, changes in the Nitrobacter community at these plants were consistent for both FISH and Q-PCR results. Both techniques have their own limitations and advantages. This study has helped to add to the platform of understanding the distribution and activity of nitrifying bacteria by correlating population dynamics with the operational parameters at full-scale level. The observations made in this study will assist researchers and engineers to minimise future nitrification failure at full-scale BNR plants. This study also confirmed the highly complex activities of wastewater treatment processes, which is dependant on a number of factors. Specific AOB or NOB predominant in wastewater rather suggests that the wastewater type and characteristics may contribute to significantly different microbial environments. Among the AOB, Nitrosomonas dominated at all BNR plants throughout the study period and for NOB both Nitrobacter and Nitrospira were found in significant numbers but their dominance varied across the plants. These dissimilar, distinct distribution patterns could be attributed to their environment which in turn impacted on the nitrification performance of the system. It was also noted that the co-existence of more than one group of these communities at the same plant could help the plant escape complete functional failures such as nitrification, due to sudden changes in temperature and substrate concentrations, as this function can be performed by different groups. Although it would have been meritorious to conduct a nitrogen balance in this study, this was not possible since the research focused on full-scale systems.
Determining the efficiency of the anammox process for the treatment of high- ammonia influent wastewater
(2017-08) Gokal, Jashan; Bux, Faizal; Stenström, Thor-Axel; Kumari, Sheena K.
Domestic wastewater contains a high nutrient load, primarily in the form of Carbon (C), Nitrogen (N), and Phosphorous (P) compounds. If left untreated, these nutrients can cause eutrophication in receiving environments. Biological wastewater treatment utilizes a suspension of microorganisms that metabolize this excess nutrient load. Nitrogen removal in these systems are due to the synergistic processes of nitrification and denitrification, each of which requires its own set of operating parameters and controlling microbial groups. An alternative N-removal pathway termed the anammox process allows for total N-removal in a single step under anoxic conditions. This process, mediated by the anammox bacterial group, requires no organic carbon, produces negligible greenhouse gases and requires almost 50 % less energy than the conventional process, making it a promising new technology for efficient and cost-effective N-removal. In this study, a sequencing batch reactor (SBR) was established for the autotrophic removal of N-rich wastewater through an anammox-centric bacterial consortia. The key microbial members of this consortia were characterized and quantified over time using molecular methods and next generation sequencing to determine if the operational conditions had any effect on the seed inoculum population composition. Additionally, local South African wastewater treatment plants were screened for the presence of anammox bacteria through 16S rRNA amplification and enrichment in different reactor types. A 3 L bench scale SBR was inoculated with active biomass (~ 5 % (v/v)) sourced from a parent anammox enrichment reactor, and maintained at a temperature of 35 °C ± 1 °C. The reactor was fed with a synthetic wastewater medium containing no organic C, minimal dissolved oxygen (< 0.5 mg/L), and N in the form of ammonium and nitrite in the ratio of 1:1.3. The reactor was operated for a period of 366 days and the effluent ammonium, nitrite and nitrate were measured during this period. The hydraulic retention time was controlled at 4.55 days from Day 1 to Day 250, and thereafter shortened to 1.52 days from Day 251 to Day 360 due to an increased nitrogen removal rate (NRR). During Phase I of operation (Day 1 to Day 150), the reactor performance gradually increased up to an NRR of ~160 mg N/day. During Phase II (Day 151 to Day 250), the overall reactor performance decreased with the NRR decreasing to ~90 mg N/day, while Phase III (Day 251 to Day 366) displayed a gradual recovery of NRR back to the reactor optimum of ~160 mg N/day. The accumulation of nitrate in the effluent during the latter parts of Phase II and Phase III, coupled with oxygen ingress (~2.1 mg/L) in the same period, indicated that it was not the anammox pathway that was dominating N-removal within the reactor, but more likely the second half of the nitrification pathway mediated by the nitrite oxidizing bacteria (NOB). This was further confirmed through molecular analysis, which indicated that the bacterial population had shifted significantly over the course of reactor operation. Quantitative PCR methods displayed a decrease in all the key N-removing population groups from Day 1 to Day 140, and a marginal increase in anammox and aerobic ammonia oxidizing bacteria from Day 140 – Day 260. From Day 300 onwards, NOB had started dominating the system, simultaneously suppressing the growth of other N-removing bacterial groups. Despite this, the NRR peaked during this period, indicating an alternative mechanism for ammonia removal within the reactor system. A total population analysis using NGS was also performed, which corroborated the QPCR results and displayed a population shift away from anammox bacteria towards predominantly NOB and members of the phylum Chloroflexi. The proliferation of aerobic NOB and Chloroflexi, and the suppression of anammox bacteria, indicated that DO ingress was indeed the primary cause of the population shift within the reactor. Despite this population shift, N-removal within the reactor remained high. New pathways have recently emerged which implicate these two groups as potential N oxidizers, with specific NOB groups showing the ability for oxidation of ammonia through the comammox process, and members of the Phylum Chloroflexi being capable of nitrite reduction. This could imply that an alternate pathway was responsible for the majority of N-removal within the system, in addition to the anammox and conventional nitrification pathways. Additionally, in an attempt to detect a local anammox reservoir, eleven wastewater systems from around South Africa were screened for the presence of anammox bacteria. Through direct and nested PCR-based screening, anammox bacteria was not detectable in any of the activated sludge samples tested. Based on the operating conditions of the source wastewater systems, a subset of three sludge samples were selected for further enrichment. After 60-110 days of enrichment in multiple reactor configurations, only one reactor sample tested positive for the presence of anammox bacteria. Although this result indicates that anammox bacteria might not be ubiquitous within every biological wastewater system, it is more likely that anammox bacteria might only be present at undetectable levels, and that an extended enrichment prior to screening is necessary for a true representation of anammox bacterial prevalence in an environmental sample.
Development and optimization of remedial measures to control filamentous bacteria in a full-scale biological nutrient removal plant
(2014) Deepnarain, Nashia; Bux, Faizal
Wastewater treatment plants (WWTPs) frequently experience bulking and foaming episodes, which present operational challenges by affecting sludge settling due to the excessive proliferation of filamentous bacteria. Various control strategies have been implemented over the years to minimize filamentous growth, however, filamentous bulking still remains an unresolved problem in many WWTPs worldwide. The current study focused on developing and optimizing remedial measures viz., specific and non-specific methods to reduce problematic filamentous bacteria in a full-scale WWTP. Specific methods demonstrated the influence of plant operational parameters viz. chemical oxygen demand, influent N-NH4+, food to microorganism ratio, dissolved oxygen, temperature and pH on the abundance of filamentous bacteria. A cumulative logit model was used to determine the significant relationships between the individual filamentous bacteria at present and the prevailing plant operational parameters. Using the above statistical approach, significant observations and predictions were made with respect to the individual filamentous growth under certain operational parameters. With further validation, this model could be successfully applied to other full-scale WWTPs identifying specific parameters that could contribute to filamentous bulking, thus providing a useful guide for regulating specific filamentous growth. Non-specific control methods such as chlorine, ultraviolet irradiation and ozone treatment were investigated on filamentous bacteria using a live/dead staining technique. To achieve at least 50% reduction of filamentous bacteria, a chlorine dose of 10 mg Cl2/L was required, all filaments were killed at a dose of 22 mg Cl2/L. In addition, an effective UV and ozone dose of 4418.91 μw seconds/cm2 and ±20 mg O3/L respectively, was required to kill 50% of the filamentous bacterial population. Among the three non-specific methods, ozone treatment seemed to be an effective method in controlling the filamentous population with a low negative impact to the surrounding environment. This study serves as a useful guide on the problems and control of filamentous bulking in activated sludge plants.
Development of a bioprocess technology for the production of Vibrio midae, a probiotic for use in abalone aquaculture
(2019) Moonsamy, Ghaneshree; Singh, Suren; Lalloo, Rajesh
The abalone industry of South Africa is under severe pressure due to illegal harvesting and poaching of this seafood delicacy. These abalones are harvested excessively; as a result, these animals do not have a chance to replace themselves in their habitats, ensuing in a drastic decrease in natural stocks of abalone. Abalone, has an extremely slow growth rate, and takes approximately four years to reach a size that is market acceptable, therefore, it was imperative to investigate methods to boost the overall growth rate and immunity of the animal. The University of Cape Town (UCT) began research, which resulted in the isolation of two microorganisms, a yeast isolate Debaryomyces hansenii and a bacterial isolate Vibrio midae, from the gut of the abalone and characterised them for their probiotic abilities. This work resulted in an internationally competitive concept technology that was patented. The next stage of research was to develop a suitable bioprocess to enable commercial production. Numerous steps were taken to develop an efficient production process for V. midae, one of the isolates found by UCT. The initial stages of research resulted in the development of a stable and validated cell bank which allowed the development of a robust inoculum stage. This was followed by optimization of temperature and pH which resulted in improved probiotic production at a temperature of 30oC and a pH of 6.5. Once these critical growth parameters were established further media optimization studies were performed. The two key nutrient supplements investigated were corn steep liquor (CSL) and High Test Molasses (HTM) due to their suitability, availability and affordability. The optimization of CSL (6.4 g.l-1) and HTM (24 g.l-1) concentrations in the growth medium resulted in a 180% increase in cell concentration, a 5716-fold increase in cell productivity and a 97.2% decrease in the material cost of production when compared to the base case technology. Furthermore, a stable market ready liquid probiotic product, containing viable but not culturable (VBNC) state of Vibrio midae cells, was developed during the downstream processing aspect of the study. Finally, the validation of this production technology at full manufacturing scale was demonstrated which further enhances the attractiveness and commercial feasibility of this probiotic production process.