Masters Degrees (Microbiology)

Permanent URI for this collectionhttps://hdl.handle.net/10413/8006

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Development of innovative pretreatments for simultaneous saccharification and citric acid production from banana pseudostem: bioprocess optimization and kinetic assessment.
(2022) Laltha, Milesh.; Gueguim Kana, Evariste Bosco.
Abstract in PDF.
Investigating the role of small RNAS in transcriptome regulation of genetically diverse clinical strains of mycobacterium tuberculosis.
(2021) Govender, Divenita.; Mvubu, Nontobeko Eunice.
Tuberculosis (TB), caused by the human adapted members of the Mycobacterium tuberculosis complex (MTBC), is a threat to global health. Understanding the regulatory network of the MTBC members may reveal novel vaccine candidates and drug targets. The small RNAs (sRNAs) have only recently been investigated for their role in Mycobacterium tuberculosis (M. tb) transcriptome regulation with none being explored in clinical strains or within the MTBC lineages. The present study aimed to investigate the regulatory role of sRNAs on the M. tb transcriptome in a lineage-specific manner, with emphasis on the clinical strains most prevalent in South Africa. In silico whole genome sequence alignment of strains belonging to the eight MTBC lineages was performed to identify sRNAs containing lineage-specific mutations and their respective potential targets. To elucidate transcriptome regulation in clinical strains of M. tb belonging to the Beijing and F15/LAM4/KZN lineages, mRNA and sRNA sequencing were performed followed by Hisat-Ballgown Bioinformatics analysis to identify novel sRNAs and their respective targets. The sRNAs discovered from sRNA sequencing were confirmed through real time qPCR. The in silico data revealed several sRNAs that may play a role in transcriptome regulation at a lineage-specific level, such as those involved in macrophage entry, lipid biosynthesis pathway, adaptation mechanisms during antibiotic exposure, and environmental stress. They may also be able to disrupt genes that are detrimental and restore functions to those that are beneficial. The mutated and consensus sRNAs were identified to target the same function, but one pathway may be more efficient than the other. Novel sRNAs were discovered from sRNA sequencing of the Beijing and F15/LAM4/KZN clinical strains, with their predicted targets absent from the mRNA sequencing results, indicating these sRNAs may elicit an inhibitory function. Real time-PCR analysis revealed significant fold change differences between the clinical strains belonging to the Beijing, F15/LAM4/KZN, F11 and Unique families suggesting an underlying regulation of these transcripts at a family level. This data could explain the underlying phenotypic differences observed within the MTBC and understanding of the regulatory function of these sRNAs, may identify novel alternative strategies in the fight against M. tb.
Assessing the antimicrobial, anti-quorum sensing and anti-biofilm potential of bacteria isolated from pocillopora and acropora corals.
(2021) Buswana, Olona.; Chenia, Hafizah Yousuf.; Pearton, David.
Abstract available in PDF.
Isolation and characterization of bacteriophages from wastewater as potential biocontrol agents for Escherichia coli.
(2022) Ntuli, Nontando Percevierance.; Schmidt, Stefan.
Host-specific lytic bacteriophages have regained momentum as an alternative treatment option to control and eliminate pathogenic bacteria. This study aimed to isolate, characterize and evaluate the potential application of Escherichia coli phages as a biocontrol agent in wastewater. In this study, four lytic Escherichia coli phages were isolated from wastewater for biocontrol purposes, using the double-layer method with E. coli (ATCC-25922) as a host. The phage morphology was characterized using transmission electron microscopy, with further parameters such as host range, phage stability at different temperatures, and pH values analyzed additionally. The genome of two selected phages (NPS and NPM) was sequenced, and the capacity of the phage isolate NPM to eliminate E. coli from artificial wastewater was evaluated and compared to conventional chlorination. All the four phage isolates showed typical T4 phage appearance with isometric capsids and contractile tails of different sizes, matching the family Myoviridae in the order Caudovirales. They exhibited a narrow host range limited to E. coli isolates, with two exceptions: phage NPS and NPM additionally lysed Salmonella Typhimurium (ATCC-14028). The four phage isolates were even able to lyse MDR (multidrug-resistant) E. coli isolates, such as the strain FP29. The four phages had burst sizes ranging from 70-115 per host cell and a latency period of 10- 20 minutes. All the four bacteriophages were stable at pH 5-9 but completely inactivated at pH 12. Exposure to 60°C for 10 minutes reduced phage titers by 1.5- log, while exposure to 80°C for 10 minutes completely inactivated all four phage isolates. The two genomes (NPS and NPM) were 99% identical and had similar sizes (169 536 bp), but phage NPS differed from phage NPM in view of its host range and plaque morphology. Another difference observed at the genome level was a shift of coding sequences between phage NPS and NPM. Phage isolate NPM achieved a 3.5- log reduction of E. coli cells present in artificial wastewater at an MOI of 0.1 in 120 minutes. A 90-minute chlorine treatment achieved a log reduction in the same range, highlighting that phages have the potential as environmentally friendly biocontrol agents in wastewater treatment.
Optimization of antifungal production by Bacillus species in the presence of nanoparticle supplementation and preliminary scale-up.
(2023) Nzimande, Sikhulile Nosimo.; Gueguim Kana, Evariste Bosco.; Ramchuran, Santosh Omrajah.; Sanusi, Adeyemi Isaac.
The intensive agricultural practices used to meet global crop production demands have resulted in the rigorous use of chemical pesticides. These ultimately compromise crop production as well as the environment. In order to alleviate this, cheaper and environmentally friendly, biocontrol agents have been considered as an alternative to chemical pesticides. Biosurfactants are a promising alternative to chemical pesticides due to their higher biodegradability, lower toxicity, and environmental friendliness. Amongst the many bacterial and fungal biosurfactant producers, biosurfactants from Bacillus species show promise as biocontrol agents. These biosurfactants are known for their wide biotechnological use in agricultural, industrial, and medicinal fields. However, large scale production is still faced with challenges such as low yields and high production cost thus raising the need for modelling, optimization, catalytic and scale up investigations. Hence, a study was undertaken with the aim of enhancing biosurfactant production through process modelling and optimization with subsequent assessment of the scale up potential of the optimized process. A Response Surface Methodology (RSM) using box Behnken design was used to investigate the optimal process conditions for improved biosurfactant production from B. subtilis BS20. The investigated process parameters included glucose concentration (10 – 30 g/L), incubation temperature (25 – 45℃) and incubation time (24 – 96 h). The developed model gave a high coefficient of determination (R2 ) = 0.86, p-value of 0.0279 and F-value of 4.62 for the modelled biosurfactant production. Optimized process conditions of 11.5 g/L glucose concentration, 24 h incubation time and 41o C for incubation temperature were obtained and produced a maximal antifungal activity of 68 mm. Moreover, supplementary inclusion of seven (7) different nanoparticles as a biocatalyst in the cultivation of B. subtilis BS20 was carried out using the optimal process condition to further improve antifungal (biosurfactant) production. The inclusion of nanoparticles favored increased biomass yield, but biosurfactant with high antifungal activity was not obtained. Moreover, when it comes to commercializing new bioprocess and bioproduct developments, bio – process scale-up in the biotechnology industry is an essential stage. This study therefore evaluated the scale up of biosurfactant production based on constant power consumption, Reynold number and impeller tip speed. The stirrer speed (n), impeller diameter (di), number of impellers (N), power number (Np), broth density (⍴), working volume and geometric factor (fc) were correlated with impeller tip speed (Vtip), Reynolds number (𝑅𝑅𝑅𝑅) and power consumption rate (P/V) to obtain the most suitable criterion for biosurfactant production in a 10 L bioreactor. Implementing constant Vtip value from the 1 L scale: 93 rpm, Reynold number (Re) 5.9E - 04, Power (P) 0.32 W, Power to Volume ratio (P/VL) 160 W/m3 , circulation time (tc) 5.2 s and shear stress (γ) 15.5 S-1, at 41 °C, gave the highest antifungal activity of 65 mm zone of inhibition in the 10 L scale bioreactor. The antifungal activity obtained for constant Vtip were comparable to those obtained at 1L bioreactors (57 mm), this showed that the bioprocess dynamics for achieving high antifungal activity are available, further paving the way for feasible commercialization strategies. This study has elucidated the optimum process conditions for B. subtilis BS20 metabolism for improved biosurfactant production resulting in significant antifungal activity. Furthermore, findings showed that the inclusion of nanoparticles biocatalyst to the process enhanced biomass yields. Process scale up provided preliminary data for large scale production of biosurfactant production from B. subtilis BS20.
The production of monoclonal antibodies against esat6 of mycobacterium tuberculosis.
(2016) Pillay, Nethi.; Pillay, Balakrishna.
Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis (TB), was responsible for 9.6 million cases and 1.5 million deaths globally. Therefore, early TB diagnosis remains a high priority to mitigate the consequences of poor health outcomes and continuous disease transmission. The strategy in detecting the TB antigen instead of antibodies elicited against TB is preferred as immunosuppression in patients co-infected with HIV, makes antibody detection unfavourable. Monoclonal antibodies (MAbs) to TB-specific markers are therefore an attractive option for use in ELISAs and lateral flow tests that effectively meet the criteria for a rapid antigen detection test. Consequently, the objective for the present study was to produce TB-specific MAbs that would aid in the development and optimization of an ELISA for the rapid detection of TB. In order to meet these objectives this study focused on the production and characterization of MAbs that specifically target the early secretory antigenic target 6 (ESAT6) protein from M. tuberculosis. PCR was performed on M. tuberculosis H37Ra DNA, using primers specific to the esat6 gene. PCR products were inserted into pGEM-T vector followed by ligation into the expression vector pGEX6P-1 for transformation into Escherichia coli (E. coli) strain XL-1 Blue. ESAT6 GST protein was expressed and purified by glutathione sepharose affinity chromatography. Thereafter, recombinant ESAT6 GST protein was used to immunise 10 Balb/C mice and stable hybridoma cell lines were generated. The specificity of three monoclonal antibodies were confirmed and identified as anti-ESAT6 DE2-1, anti- ESAT6 KE10-1 and KE10-2. Hybridomas showing cross-reactivity to non-specific antigens, were excluded from the study. Ouchterloney Double-Diffusion was employed and the three MAbs were subtyped as an IgG and two IgM’s respectively. The multi-epitopic nature of ESAT6 is a desirable characteristic in diagnostic assay development. This characteristic was demonstrated by ELISA using anti-ESAT6 DE2-1 as a coating MAb and anti-ESAT6 KE10-1, conjugated to horse radish peroxidase, as the detection antibody. The generation of anti-ESAT6 MAbs in this study, have demonstrated their potential for use in the development of a rapid TB diagnostic test. This is critical in TB management, treatment of the disease, reducing TB transmission and incidence. Future work must therefore be aimed at the development of a diagnostic test, for use at the point-of-care, which would complement the TB diagnostic algorithm.
Optimization of biomass production and lipid formation from Chlorococcum sp. cultivation on dairy and paper-pulp wastewater.
(2021) Ngerem, Emmanuel Chidi.; Gueguim Kana, Evariste Bosco.; Olaniran, Ademola Olufolahan.
Abstract available in PDF.
Design, implementation and assessment of a novel bioreactor for dark fermentative biohydrogen production.
(2020) Khan, Mariam Bibi Hassan.; Gueguim Kana, Evariste Bosco.
The majority of the world’s energy consumption and electricity generation is derived from fossil fuel sources. Their consumption has a negative environmental impact, thus the need for renewable energies. Hydrogen being a high energy zero carbon fuel source presents a profound appeal. Hydrogen may be produced biologically via various methods, this work involves dark fermentative hydrogen production (DFHP). A review of literature on the physicochemical parameters affecting fermentative hydrogen bioprocess was conducted. Bioreactor design was identified as a fundamental component that regulates the overall process outcome and was therefore analysed at length. The review highlighted that existing reactor configurations are unable to sustain a comprehensive criteria of efficient DFHP. A consolidation of biomass retention and non-invasive agitation were distinguished as crucial. The need for a novel reactor configuration possessing these attributes was consequently accentuated. This study focuses on the design, implementation and assessment of novel bioreactor configuration for DFHP. The vessel was formed from a 2L glass and fitted with ports. Three 3D-printed permeable cartridges enclosed immobilized microbial cells and functioned as baffles. The localization and motion of the cartridges promoted improved exposure between microbial cells and substrate. Agitation was accomplished by rocking the vessel at 180°. All the control set points were adjustable, presenting the option of evaluating diverse control regimes. The implemented reactor showed a 35% increase in the peak hydrogen fraction and a 58% reduction in lag time compared to the control shake flask reactor. These findings showed that the novel reactor configuration, by means of the cartridge structure supporting the immobilized cells, enhanced the biohydrogen production process. Subsequently, a preliminary scale up of the cartridge concept was implemented and incorporated into a continuous stirred tank reactor (CSTR). The cartridge (46x40x300mm) consisted of perforated hollow rectangular tubes, joined to form a single amalgamation. This unit was used as substitute for the standard impellers of the CSTR and aligned at 120° laterally to the agitating shaft. The modified reactor prepared with Immobilized cells in cartridge (ICC) was comparatively assessed with the standard CSTR operated with suspended cells in reactor (SCR) and immobilized cells in reactor (ICR). ICC reduced fermentation time by 52 and 65% compared to SCR and ICR respectively. Gompertz model coefficients indicated a 98 and 37% increase in the maximum hydrogen production rate (Rm) using the ICC compared to the SCR and ICR fermentations respectively. ICC also showed better pH buffering capacity and complete glucose degradation. These findings further demonstrated that the scale up reactor configuration with the cartridge structure improved biohydrogen productivity, yield and process economics. The novel configuration reduced process time, improved Hydrogen yield and ensured complete substrate degradation. Furthermore, the structural integrity of immobilized cells was maintained. These findings demonstrated that the novel bioreactor design improved biohydrogen production and showed potential for further DFHP research and development.
Isolation and characterization of diaryl ester catabolizing soil fungi.
(2019) Moonsamy, Volante.; Schmidt, Stefan.
Aromatic hydrocarbons are major organic pollutants that can persist in the environment. However, many fungi and yeasts can utilize these compounds as carbon and energy sources under aerobic conditions. Salol and benzyl salicylate are diaryl ester biocides exhibiting endocrine-disrupting properties. Using mineral salts medium containing salol and benzyl salicylate as sole carbon and energy source, aerobic enrichment cultures were established by inoculation with soil samples collected from a local animal farm and Bisley Nature Reserve Pietermaritzburg, KwaZulu-Natal. A salol utilizing fungal isolate and a benzyl salicylate utilizing yeast isolate were selected after enrichment. The fungal and yeast isolate were provisionally assigned to the genus Fusarium and Trichosporon, based on phenotypic characteristics and the sequence analysis of the ITS1-5.8S rRNA-ITS2 region. Growth kinetics of the fungus were assessed by measuring the dry weight of the biomass over time in batch cultures; the growth of the yeast was assessed via OD600 determinations and verified via microscopic cell counts. Appropriate abiotic controls showed that the concentration of tested aromatic pollutants remained stable over time while no biomass formed in biotic controls without added carbon source. Salol and benzyl salicylate utilization was verified by measuring the Chemical Oxygen Demand (COD) and UV-Vis spectra over time. COD measurements and UV spectroscopy indicated that up to 10 mM salol was catabolized completely by Fusarium sp. strain VM1 within 10 days while Trichosporon sp. strain VM2 catabolized 10 mM benzyl salicylate almost quantitatively. Specific enzyme activity determinations showed that both esterase and catechol-1,2-dioxygenase were induced by growth on salol and benzyl salicylate, indicating that the catabolism of diaryl esters is initiated by hydrolysis of the ester-linkage and the monoaromatic hydrolysis products were further metabolized via catechol and the ortho-pathway. These results indicate that members of the genus Fusarium and Trichosporon present in South African soils have the potential to eliminate diaryl esters and simple monoaromatic pollutants.
Process development for biomass and lipid production from a local chlorella isolate using a miniature parallel raceway pond reactor.
(2020) Gumbi, Zanenhlanhla Gugulethu.; Gueguim Kana, Evariste Bosco.
Abstract available in pdf.
Molecula characterization of chlamdia trachomatis isolates using sequence variation in the major outer membrane protein gene (OMP1) and evaluation of their susceptibility profile.
(2019) Khanyile, Thobile Nokwazi.; Mlisana, Koleka Patience.; Singh, Ravesh.
Chlamydia trachomatis infections are the most common bacterial sexually transmitted infections (STIs) in humans, worldwide. Due to asymptomatic nature of C. trachomatis, the need for sensitive, reliable and affordable laboratory methods for diagnosis is critical. The aim of this study was to ascertain if the genetic profiles of different C. trachomatis isolates associate with antibiotic resistance.Two hundred and sixty-five Eswab™ clinical samples were screened for Ct using Anyplex™ II STI-7 Detection. We have applied High Resolution Melting Analysis (HRMA) for the genotyping of the Ct and applied it specifically to the 14 sexually transmitted infection-related genotypes: AC, D-K and L1-L3. Based on the genotype of the OMP1 (Outer Membrane Protein) gene C. trachomatis is grouped into different serovars, which present in different clinical manifestations; with type A, B, Ba, and C causing trachoma, D-K cause urogenital infections and LI, LII & LIII associated with lymphogranuloma venereum (LGV). We confirmed the presence of the OMP1 gene with the conventional PCR. HRMA was performed to identify the C. trachomatis serovars on a Quantstudio 5 real – time PCR instrument and CDC control strains were included in the analysis. HRM analysis was done on the High-Resolution Melt Softwarev3.1. We identified the following serovars A, B, C, D, E, F, G, I, J, L3 and our prevalence for the above serovars were as follows 3.2%, 6.4%, 3.2%, 9.7%, 16.1%, 29%, 9.7%, 12.9%, 3.2% and 6.4%, respectively. None of the serovars: H, K, L1, L2 were observed. A TaqMan real time PCR assay was also performed to measure the bacterial concentration of each C. trachomatis positive sample to elucidate if there is any association with the serovar type. D-K serovars had higher bacterial load compared to A-C and L3 serovars, (p =0.0045). We also performed sanger sequencing on ribosomal proteins (L4 and L22) to determine the presence of mutations that have been previously associated with drug resistance. The ribosomal protein L4 had mutations located in 7 different positions, significant mutations associated with macrolides resistance were observed at amino acid number 109 and 151. Ribosomal protein L22 had 21 samples with mutation at amino acid number 24, that has not been associated with resistance before. Based on our study and previous studies, it is clear that macrolide resistance in C.trachomatis is multifactorial besides changes in the amino acids.
Investigation of multiple concurrent Human papillomavirus infections, oncogenicity, and STI co-infection as risk factors for Human immunodeficiency virus infection.
(2017) Jewanraj, Janine.; Liebenberg, Lenine Julie.; Archary, Derseree.
Background: Human papillomavirus (HPV) is one of the most common sexually transmitted infections (STIs) globally and a necessary factor for cervical cancer development. While HPV infection has been associated with increased Human immunodeficiency virus (HIV) risk, the underlying mechanisms remain unclear. Since STIs upregulate cytokine production and immune cell recruitment, and reduce epithelial barrier integrity, this study investigated whether the immune responses associated with HPV infection contribute to a genital immune environment conducive to an increased risk of HIV infection. Methods: This study included a baseline assessment of 167 HIV negative women participating in the CAPRISA 008 trial. The Roche Linear Array was used to detect the presence of 37 HPV genotypes in cervicovaginal lavage (CVL) pellets. The concentrations of 48 cytokines and 9 matrix metalloproteinases (MMPs) were assessed in matching CVL supernatants by multiplex ELISA. The frequencies of activated or proliferating T cells, NK cells, and of HIV target cells were assessed on cervical cytobrush-derived specimens by flow cytometry. Multiplex PCR was conducted to determine infection with common discharge-associated STIs. Results: The study demonstrated a 50.8% HPV prevalence. HPV infection was associated with younger age, older male partners, not living with a regular partner, and higher parity. HPV infection was also associated with greater levels of IL-5, IL-6 and G-CSF, an association otherwise masked by the inflammatory nature of other STI. Concomitant HPV/STI infection resulted in reduced concentrations of IL-6 and IL-1RA relative to HPV-STI+ women. In multivariate analyses controlling for other STI and nugent score, HPV-infected women had increased concentrations of SDF-1α (β = 0.148 pg/ml). Women with HR-HPV had higher concentrations of MCP-1 (β = 0.127 pg/ml) and IL-13 (β = 0.117 pg/ml), and greater frequencies of lymphocytes (β = 1.987 pg/ml) relative to those infected with LR-HPV. Having multiple HPV infections was associated with reduced concentrations of IL-5 (β = -0.170 pg/ml). Conclusion: While discharge-related STIs are inflammatory, a more subtle immune profile was associated with HPV infection that did not overtly relate to an increased potential for HIV risk. However, this study demonstrated an association between HR-HPV and biomarkers of inflammation, suggesting the need for longitudinal investigation to confirm a biological mechanism for the relationship between persistent HR-HPV infection and HIV acquisition.
Synthesis, detection and quantification of inulooligosaccharides and fructooligosaccharides by extracellular and intracellular inulinase and fructosyltransferase enzymes isolated from coprophilous fungi.
(2018) Ojwach, Jeff David Okinda.; Mukaratirwa, Samson.; Mutanda, Taurai.
Exploration of fungal biodiversity capable of producing fructosyltransferase and inulinase enzymes in significant amounts is crucial for the production of oligofructans. Indigenous coprophilous fungi are predominantly sustainable bioresources, harbouring novel enzymes with potential industrial and biotechnological applications. Fructosyltransferase (Ftase) and inulinase are gaining considerable attention due to their capability to synthesise biofunctional nutraceuticals with low calories and health benefits when ingested in recommended dosages. Hence, due to several health benefits associated with prebiotics, bioprospecting for coprophilous fungi as unique bioresources of fructosyltransferase and inulinase was imperative. The present study therefore focused on the collection of herbivore dung from various terrestrial habitats in KwaZulu-Natal Province, South Africa whereby sixty-one (61) indigenous coprophilous fungal strains were isolated after repeated purification to monoculture. The axenic fungal strains were identified using morpho-taxonomic keys and molecular identification by 18S rDNA sequencing where Neocosmospora spp, Trichoderma spp., Aspergillus spp and Fusarium spp. were dominant. The fungal strains were subsequently assessed for their ability to produce extracellular and intracellular Ftase and inulinase enzymes. During the preliminary screening, the culture filtrate was examined for transfructosylating and hydrolytic activity using 2,3,5-triphenyl tetrazolium chloride (TTC) as a chromogenic marker and Lugol’s iodine solution, respectively. Zones of hydrolysis on 30 fungal isolates were observed on the TTC assay plates in diameters ranging from 15 mm to 30 mm, representing high extracellular Ftase activity. The formation of clear zones following addition of iodine solution on inulin rich media indicated the presence of inulinolytic activity. Secondary screening involved DNS assays of eight (8) isolates that secreted high concentrations of Ftase while six (6) different fungal strains showed <50 % inulinase: invertase ratio. The final screening step was tertiary screening where products of biocatalysis were qualitatively detected by thin layer chromatography to visualize saccharide spots of fructooligosaccharides and inulooligosaccharides. HPLC analysis of Ftase and inulinase reaction products revealed and further confirmed that coprophilous fungi harbour fructosyltransferase and inulinase enzymes. The crude extracellular fructosyltransferase enzyme was partially purified by 9.3-fold with a yield of 7.3 % and a specific activity of 2465.5 U mg-1 after a three-step procedure involving (NH4)2SO4 fractionation, dialysis and ion exchange chromatography. The apparent molecular weight of this Ftase was estimated by SDS-PAGE to be approximately 70 kDa. Zymogram analysis under non-reducing conditions showed the enzyme migrating as a polydisperse aggregate yielding broad band of approximately 100 kDa. The enzyme further exhibited an enhanced activity at a broad pH range of 4.0 – 8.0 and optimal activity at a temperature range of 40 °C – 80 °C, while the enzyme was stable at pH 8.0 and between 40 °C – 60 °C, respectively. Under these conditions, the enzyme remained stable retaining 95 % residual activity after incubation for 6 h. The presence of metal ions such as Hg2+ and Ag2+ inhibited Ftase activity while, Ca2+, Mg2+ and K+ at 1 mM increased the enzyme activity, with stabilization observed with Na+, Zn2+ and Cu2+. With sucrose as the substrate, the enzyme kinetics fitted the Michaelis-Menten model. The Km, Vmax and kcat values were 2.076 mM, 4.717 μmole min-1, and 4.7 min-1, respectively with a catalytic efficiency of 2.265 μmole min-1. In vitro antioxidant potential of FOS by 1,1 - diphenyl-2-picryl hydroxyl (DPPH) assay, ferric reducing antioxidant power (FRAP) assay and nitric oxide (NO) radical inhibition yielded IC50 of 6.71 μg/ml, 1.76 μg/ml and IC25 of 0.27 μg/ml, respectively. Free radical scavenging and inhibition activities showed a concentration-dependent antioxidant activity with no significant differences with oligosaccharide standards (p < 0.01). However, vitamin C was significant in FRAP and NO assays. These results clearly demonstrated that an indigenous coprophilous fungus is a potential new reservoir of salient biotechnological enzymes that can be exploited for the production of prebiotics for subsequent biotechnological applications.
The endosymbiotic bacteria of the South African bird cherry-oat aphid, rhopalosiphum padi.
(2018) Nhlapho, Bongiwe Ntombizodwa.; Lin, Johnson.; Sydenham, Scott Lloyd.
Cereal crops play a major role in human diet as staple foods, especially in developing countries. These crops are a part of a few edible crops that are widely cultivated globally. However, the production of these crops is constantly being put under strain by abiotic and biotic stresses in the environment, such as aphids. Aphids are the most important insects of cereal crops, not only causing damage through feeding. Aphids such as the bird cherry-oat aphid also transmit harmful plant viruses. They feed using piercing and sucking mouthparts that they insert into the plant while excreting saliva, which suppresses the plant’s defence mechanism. The plant sap these insects feed on is rich in sugars and deficient in essential nutrients required for their optimal growth and reproduction processes. These insects live in symbiotic associations with endosymbiotic bacteria, which synthesise the deficient nutrients for the aphids. These bacteria also produce a chaperon protein that has been hypothesised to be involved in protecting the viruses they transmit from degradation. In South Africa, there is limited information about the endosymbiotic bacteria of the bird cherry-oat aphid. This aphid is estimated to cause substantial cereal crop yield losses through feeding, mostly through transmitting barley yellow dwarf viruses. Persistence of these pests may lead to a reduced harvest of these crops, which might result in a drastic rise in hunger and poverty and serious economic consequences. This study aimed to investigate the association between the bird cherry-oat aphid and its endosymbiotic bacteria, with the hope that the findings will give further understanding on how to manage this pest. Molecular biology techniques were employed to identify the endosymbiotic bacteria of the bird cherry-oat aphid using 16S rDNA. Once identified, the effects of two antibiotics on the survival and reproduction of bird cherry-oat aphids were compared using a flask method which was found to be superior in rearing aphids compared to an artificial diet. This study also assessed the ability of bird cherry-oat aphids to acquire Hamiltonella defensa secondary endosymbiont from infested rose grain aphid through a shared food source. The results obtained in this study show that the obligate primary endosymbiont, Buchnera aphidicola, was found across all the screened samples, while sporadic occurrence was observed for the secondary endosymbionts. In addition, this study also showed that in the absence of their primary endosymbiotic bacteria, bird cherry-oat aphids could not reproduce and though aphid death was not immediate, most of the aphids had died by the end of the experiment. Lastly, this study showed that secondary endosymbionts can be passed between aphids through a shared food source. The field provides crops with a vast number of microbes, which can be interchangeable between plants and aphids. However, the most essential microbe, B. aphidicola, uses the aphid as a host and controlling this endosymbiont might lead to a potential control measure for the bird cherry-oat aphid.
Investigating the quorum-sensing inhibitory and potential HIV activity of indigenous South African sponge-associated bacterial extracts.
(2018) Jacobs, Carrie Shelouise.; Chenia, Hafizah Yousuf.
With bacterial antimicrobial resistance on the rise, alternative therapeutic agents are being sought. One such alternative is the use of anti-virulence strategies such as quorum-sensing inhibition (QSI), which has been proposed as a viable treatment option for pathogenic microorganisms such as multi-drug resistant Pseudomonas aeruginosa. Ninety-eight bacterial isolates from seven South African sponges harvested off the coast of KwaZulu-Natal underwent primary QSI screening using C. violaceum biosensor sandwich assays. Fifteen isolates presenting putative QSI activity were selected for further study, five of which were identified as Bacillus spp. while ten were identified as Actinomycete spp., following 16S rRNA PCR and sequencing. Crude extracts were obtained from these 30 isolates following shake-flask fermentations with two production media, mannitol and medium 5294, and ethyl acetate extractions. Extracts were then screened against QS-mediated P. aeruginosa virulence factor production, viz. pyocyanin, pyoverdine, elastase, protease, rhamnolipid, initial and mature biofilm production as well as swimming and swarming motilities. Extracts were additionally screened for putative anti-HIV reverse transcriptase (RT) activity due to the lack of current research examining the anti-HIV potential of sponge-associated bacteria. Actinomycete-derived mannitol extracts yielded the most significant inhibition for most of the P. aeruginosa virulence factors tested, while for the Bacillus spp. isolates medium 5294 extracts resulted in more significant inhibitions, indicating that the selection of production media may need to be carefully considered. This was not the case for the anti-HIV RT activity, with mannitol and medium 5294 extracts yielding similar activity. Broad spectrum QSI activity was observed with a number of extracts, including those from Streptomyces spp. SP4-AB2 and SP3-AB22, and Bacillus weihenstephanensis SP5-AB7, each of which was able to significantly (p < 0.05) inhibit virulence factors mediated by each of the three P. aeruginosa quorum sensing systems, with inhibitions as high as 93% being observed. It was further observed that 13 of the 30 extracts yielded RT-inhibition >90%; higher than that of the positive control nevirapine. Due to the high percentage of QSI and HIV-RT inhibition observed, sponge-associated bacterial extracts should indeed be screened further for their QSI and anti-HIV potential, especially those belonging to the genus Bacillus and the family Actinomycetes. Sponge-associated bacteria, therefore, possess the QSI and anti-HIV RT potential necessary to serve as the solution to the current resistance crisis and should be further studied in this regard.
Screening for biosurfactant production amongst aerobic endospore forming bacteria isolated from Mfabeni peatland sediment core.
(2019) Adu, Folasade Abimbola.; Hunter, Charles Haig.
Abbstract available in pdf.
Screening, purification and characterisation of anti-pseudomonas aeruginosa compounds produced by endophytic fungi from Kigelia Africana.
(2018) Mamokoena, Kuali.; Govinden, Roshini.; Moodley, R.
The emergence of new diseases and drug resistant pathogens coupled with the side effects presented by conventional or synthetic drug use calls for the discovery of new antibiotics and chemotherapeutics. Pseudomonas aeruginosa is a multifaceted Gram-negative opportunistic pathogen which is responsible for ten percent of all hospital infections. This, therefore, directed the search for novel bioactive molecules with new targets from previously understudied sources. Plants have bioactive compounds that have been used for traditional healthcare for thousands of years. In the interest of plant preservation, the focus has shifted to include the plant microbiome; interestingly, not only were the plants themselves producing the bioactive metabolites but also their associated microbiome. The focus of this study was to screen and determine the optimum time to produce anti-Pseudomonas aeruginosa metabolites, to purify the compounds of interest and characterise them. Forty-five endophytic fungi were grown in solid substrate fermentation on rice to produce extracts of varying ages (one week to four weeks). Thin layer chromatography (TLC) coupled with bio-autography revealed that the anti-P. aeruginosa compound was produced after three weeks. Average zones of inhibition of 40.33, 20.33 and 22 mm were obtained using the Kirby-Bauer disc diffusion assay. All rows A, B, C and D show very strong activity as the MICs of the extracts were 156.5 μg/mL, 39.06 μg/mL, 78.73 μg/mL and 19.53 μg/mL for T1, T2, T3 and T4, respectively. Thin layer chromatography was conducted, and optimum separation was observed using hexane: ethyl acetate (60:40, v/v). Fractionation was carried out using a silica gel column with six different ratios of a hexane: ethyl acetate solvent system. The first round of purification resulted in twenty-seven fractions with five fractions having similar TLC profiles. These fractions were combined and subjected iii to a second round of purification that gave three fractions. One fraction was observed to have good anti-P. aeruginosa activity and acceptable purity levels after nuclear magnetic resonance spectroscopy. Compound one, a dilactone (3a,10b-dimethyl-1,2,3,3a,5a,7,10b,10c-octahydro-5,8-dioxa-acephenanthrylene-4,9-dione, molecular formula C16H18O4) was isolated as a white solid from the extract of the fungus Neofusicoccum luteum. This compound was previously isolated from the fungus Oidiodedron griseum. The relative configuration of the compound was confirmed by X-ray crystallography. Although the isolated compound is not novel, its ability to inhibit the growth of P. aeruginosa is new. This suggests that known compounds need to be screened across a wide range of pathogens and organisms to determine potential activity.
Prevalence, antibiogram and molecular characterization of methicillin-resistant staphylococcus aureus recovered from treated wastewater effluent and receiving surface water.
(2018) Kerisha, Ramessar.; Olaniran, Ademola Olufolahan.
Inadequately treated wastewater effluent serves as a reservoir of potentially pathogenic bacteria and contributes to the spread of these organisms in the environment, including Staphylococcus aureus (S. aureus), a faecal bacterium known to cause pneumonia, septicaemia and skin infections in humans. The presence of S. aureus in water has become problematic as it has been shown to exhibit resistance towards β-lactam antibiotics commonly used to treat infections, including methicillin, leading to the emergence of methicillin-resistant S. aureus (MRSA). The current study aimed to determine the prevalence of MRSA and mecA (known to induce methicillin resistance) in the influent, treated effluent and receiving surface rivers of two wastewater treatment plants (WWTPs) in Durban. The study also evaluated the antibiogram and virulence gene profiles of MRSA isolates recovered from the treated effluent and receiving surface water using the Kirby-Bauer disc diffusion and PCR assays. Genetic fingerprinting was carried out to determine the phylogenetic relationship between isolates with selected antibiogram profiles. The prevalence of MRSA in WWTP1 ranged from 11.45-85.63% (influent), 16.28-39.36% (before chlorination), 2.16-5.07% (after chlorination), 1.06-7.24% (downstream) and 4.95-14.09% (upstream). In WWTP2, the prevalence of MRSA for the influent ranged from 48.25-86.18%, before chlorination; 23.73-93.75%, after chlorination; 4.28-48.82%, downstream; 1.74-19.31% and upstream; 5.90-28.78%. Correlation studies of selected physico-chemical parameters to the prevalence of MRSA was carried out. The real-time PCR assay showed a reduction in the concentration of mecA from the influent to the treated effluent in both WWTPs. The highest resistance was observed towards lincomycin (100%), followed by oxacillin (98.75%), cefoxitin and penicillin (97.50%) and ampicillin (96.25%). Additionally, 72.50%, 66.25%, 52.50%, 40% and 33.75% of the isolates showed resistance against cefozolin, azithromycin, amoxicillin/clavulanic acid, erythromycin and vancomycin, respectively. The following antibiotic resistance genes were detected in resistant isolates: aac(6ꞌ)/aph(2ꞌꞌ) in 56.25%, ermC in 62.50%, msrA in 22.50% and blaZ and tetK in 70%. The virulence genes hla and sea were detected in 57.50% of the isolates, hld in 1.25% and the lukS P/V gene was not detected. Thirteen pulsotypes v (designated A-M) was generated for selected isolates using pulse field gel electrophoresis, correlating them to their respective antibiograms. The study revealed a lower prevalence of MRSA and concentration of mecA in the treated effluent as compared to the influent of both WWTPs. It also revealed that these multi-drug resistant strains, isolated from the treated effluent and receiving surface waters, are potentially pathogenic and could contribute to the spread of disease in the environment. Hence, the need for more stringent monitoring and evaluation of treatment performance of the WWTPs.
The potential of waste sorghum (sorghum bicolor) leaves for bioethanol process development using Saccharomyces cerevisiae BY4743.
(2017) Rorke, Daneal Carmine Solange.; Gueguim Kana, Evariste Bosco.
The limitations of first generation biofuels have prompted the quest for alternative energy sources. Approximately 60 million tonnes of sorghum are generated each year, with 90% being lignocellulosic waste, which is an ideal feedstock for biofuel production. The recalcitrance of lignocellulose often demands harsh pre-treatment conditions and results in the generation of fermentation inhibitors, negatively impacting process yields and economics. In this study, an artificially intelligent model to predict the profile of reducing sugars and all major volatile compounds from microwave assisted chemical pre-treatment of waste sorghum leaves (SL) was developed and validated. The pre-treated substrate was assessed for bioethanol production using Saccharomyces cerevisiae. Monod and modified Gompertz models were generated and the kinetic coefficients were compared with previous studies on different substrates. To develop the Artificial Neural Network (ANN) model, a total of 58 pre-treatment process conditions with varying parameters were experimentally assessed for reducing sugar (RS) and volatile compound production. The pre-treatment input variables consisted of acid concentration, alkali concentration, microwave duration, microwave intensity and solid-to-liquid ratio (S:L). Response Surface Methodology (RSM) was used to optimise RS production from microwave assisted acid pre-treatment of sorghum leaves, giving a coefficient of determination (R2 ) of 0.76, resulting in an optimal yield of 2.74 g RS/g SL. A multilayer perceptron ANN model was used, with a topology of 5-13-13-21. The model was trained using the backpropagation algorithm to minimise the net error value on validation. The model was validated on experimental data and R2 values of up to 0.93 were obtained. The developed model was used to predict the profile of inhibitory compounds under various pre-treatment conditions. Some of these inhibitory compounds were: acetic acid (0-186.26 ng/g SL), furfural (0-240.80 ng/g SL), 5-hydroxy methyl furfural (HMF) (0-19.20 ng/g SL) and phenol (0-7.76 ng/g SL). The developed ANN model was further subjected to knowledge extraction. Findings revealed that furfural and phenol generation during substrate pre-treatment exhibited high sensitivity to acid- and alkali concentration and S:L ratio, while phenol production showed high sensitivity to microwave duration and intensity. Furfural generation during pre-treatment of waste SL was majorly dependent on acid concentration and fit a dosage-response relationship model with a 2.5% HCl threshold. VI The pre-treated sorghum leaves were enzymatically hydrolysed and subsequently assessed for yeast growth and bioethanol production using Saccharomyces cerevisiae BY4743. Kinetic modelling was carried out using the Monod and the modified Gompertz models. Fermentations were carried out with varied initial substrate concentrations (12.5-30.0 g/L). The Monod model fitted well to the experimental data, exhibiting an R2 of 0.95. The model coefficients of maximum specific growth rate (μmax) and Monod constant (Ks) were 0.176 h-1 and 10.11 g/L respectively. Bioethanol production data fitted the modified Gompertz model with an R2 of 0.98. A bioethanol production lag time of 6.31 hours, maximum ethanol production rate of 0.52 g/L/h and a maximum potential bioethanol concentration of 17.15 g/L were obtained. These findings demonstrated that waste SL, commonly considered as post-harvest waste, contain sufficient fermentable sugar which can be recovered from appropriate HCl-based pre-treatment, for use as a low cost energy source for biofuel production. The extracted knowledge from the developed ANN model revealed significant non-linearities between the pre-treatment input conditions and generation of volatile compounds from waste SL. This predictive tool reduces analytical costs in bioprocess development through virtual analytical instrumentation. Monod and modified Gompertz coefficients demonstrated the potential of utilising sorghum leaves for bioethanol production, by providing data for early stage knowledge of the production efficiency of bioethanol production from waste SL. The generated kinetic knowledge of S. cerevisiae growth on waste SL and bioethanol formation in this study is of high importance for process optimisation and scale up towards the commercialisation of this fuel.
Bacterial degradation of 2,4-dichlorophenol : catabolic genes detection and enzyme characterization.
(2017) Setlhare, Boitumelo.; Mokoena, Mduduzi Paulos.; Olaniran, Ademola Olufolahan.
Abstract available in PDF file.

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