Biochemistry and Microbiology

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    Public participation as a mechanism to enhance service delivery in Mtubatuba Local Municipality
    (University of Zululand, 2024) Madondo, James Nkosinathi
    The purpose of this study is to investigate anxiety associated with the use of technology among teachers in Lesotho. The sample consists of 100 high school teachers from various schools located in the Maseru district. To meet the objectives, a questionnaire was designed, which collected data on teachers’ anxieties towards technology in relation to demographic variables. The findings of the study suggest that the educators do differ in their anxiety levels in relation to technology, and that there is no correlation between age and anxiety. The relationship between the variable of gender and anxiety is revealed. Furthermore, teaching experience is found to have a significant effect on anxiety, while nationality is found to have no effect on anxiety.
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    In vitro antioxidant and antibacterial activity of Crude extracts of some freshwater cyanobacteria
    (University of Zululand, 2023) Ikhane, Olufemi Akayagboke Albert; Opoku, A.R Osunsanmi, F.O. and Mosa, R.A
    Commonly available antibiotics are increasingly becoming ineffective due to the astronomic rise of antimicrobial resistance (AMR). As a result, there is a need for the discovery of new antibiotics and antioxidants from natural products. Cyanobacteria possess a myriad of significant secondary metabolites among which potential antibacterial and antioxidant could be found. The aim of this study was to exploit the potential metabolite reservoir of cyanobacteria towards the development of novel antimicrobial compounds. Freshwater cyanobacteria, Cylindrospermum alatosporum NR125682.1 and Loriellopsis cavenicola NR117881.1, utilized in this study were isolated from Vulindlela area, KwaZulu-Natal, SA. They were propagated on BG-11 media, identified, and characterized through 16S rRNA sequencing. The cyanobacteria were sequentially extracted with hexane, dichloromethane (DCM) and ethanol. The extracts were screened for their antioxidant capacity using DPPH, ABTS, OH* radicals and metal chelating potential. The in silico molecular docking of the major constituents of the extracts against β-lactamase was also evaluated. The in vitro antibacterial potential of the extracts was evaluated using the broth microdilution method against some selected gram-positive and gram-negative clinical bacterial isolates. The effect of the extracts on the bacterial membranes was evaluated using the lactate dehydrogenase assay. The efflux pump inhibitory potential was investigated by measuring the percentage cytoplasmic accumulation of rhodamine. Beta-lactamase inhibitory potential was investigated along with synergistic potential when combined with erythromycin. The ability of the extracts to effect DNA damage was also evaluated. The crude extracts were moderate antioxidants, scavenging free radicals with IC50 range of 6-10 μg/ml and metal chelating efficiency IC50 values ranging from 44-72 μg/ml. Despite the encouraging (-6.6, -6.3 kJmol-1) binding affinity of some of the phytochemicals in the extracts following molecular docking against beta-lactamase, the in vitro inhibition of beta-lactamase indicates that all six extracts were poor inhibitors of the enzyme with a high IC50 value of 5.6 mg/ml. The ethanol crude extract of both isolates was the most efficient with a minimum MIC value of 0.7 mg/ml against the tested resistant bacteria. The ethanol extract of Loriellopsis cavenicola NR117881.1 effectively inhibited efflux pump activity, with up to 60% rhodamine accumulation in the bacteria tested. The extracts effected cell membrane damage on the tested bacteria. The extract also exhibited synergism when combined with erythromycin against some of the tested bacteria. Only the DCM extract of Loriellopsis cavenicola NR117881.1 effected DNA damage. The various bioactivity exhibited by the extracts suggest potential for the development of antibacterial and antioxidant active agents with multiple beneficial effects.
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    Anti-aging potential of some freshwater microalgae extracts
    (University of Zululand, 2023) Sithole, Siphesihle Zamajobe.; Opoku, A.R. Mosa, R.A. & Osunsanmi, F.O.
    The aging of the skin is a progressive, multi- faceted phenomenon that alters skin tissue and is impacted by both intrinsic and external causes. Age and genetics contribute to intrinsic aging, whereas ultraviolet radiation (UVR) contributes to extrinsic aging. Intrinsic aging is a predetermined process that produces fine wrinkles, whereas long-term UVR exposure (classified as photo-aging) causes patchy hyperpigmentation, dilated blood vessels, epidermal hyper proliferation, and sunburn. Existing skincare products for anti-aging are known to include hazardous substances associated with an elevated skin cancer risk. As a result, there is a need to look for natural alternative cosmetic items. Thus, this study aimed to assess the anti- aging potential of freshwater microalgal extracts. Freshwater samples collected from the University of Zululand environment were enriched with BG-11 and cultured under UV illumination. Out of the ten (10) pure colonies isolated from the culture and characterised through 16s RNA, Cylindrospermum alatosporum (NR125682.1) and Loriellopsis cavernicola (NR117881.1) were cultivated for biomass. Dried biomass was sequentially extracted with solvents with varying polarities namely hexane, dichloromethane (DCM) and ethanol. The phytochemical screening of the extract was conducted using GC-MS and FTIR. The antioxidant activities of the microalgal extracts were estimated by various in vitro assays including the scavenging of 1.1-Diphenyl-2-picryl hydrazil (DPPH), 2.2- Azinobis 3-ethylbenzothiazoline-6-sulfonate (ABTS), hydroxyl (·OH) radicals and metal ion chelating, In vitro experiments were conducted to explore the inhibition of aging enzymes, namely collagenase, elastase, hyaluronidase, and tyrosinase. UVB and UVA photoprotective activity of the microalgal extracts was also investigated. Antibacterial susceptibility testing was conducted on commensals, Propionibacterium acnes and Staphylococcus aureus. The results obtained indicate that the extracts contained phenols and sulfhydryl groupcontaining compounds that could be responsible for certain biological activities of importance to this study. The extracts were average scavengers of synthetic radicals (IC50 values in the range from 6.5 – 48.6 μg/ml) but were potent metal ion chelators (44.3- 75.7 μg/ml). All extracts inhibited (in vitro) tyrosinase (≈ 47.87%), hyaluronidase (≈ 89.51%), and collagenase (≈ 76.67%) activities, but were poor inhibitors of elastase (≈16.43%). UVA and UVB photoprotective activity were exhibited by the extracts with L cavernicola exhibiting better UVB photoprotective activity (SPF 14.67 – 78.96). besides, this is observed for antimicrobial activity against strains of Staphylococcus aureus (ATCC 25923 and APO25177.1) and Propionibacterium acnes Kwik-stikTM. In conclusion, C alatosporum (NR125682.1) and L cavernicola (NR117881.1) possess the potential to be exploited as anti-aging agents.
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    Green synthesis and characterisation of metal-based nanoparticles using Kombucha tea SCOBY yeast-based bioflocculant and their application in wastewater treatment
    (University of Zululand, 2023) Tsilo, Phakamani Hopewell; Basson, A. K. Pullabhotla, Rajasekhar V. S. R. Dlamini, N. G. and Ntombela, Z. G.
    Flocculation is a mechanical purification used in wastewater treatment, involving the precipitation of larger flocs to eliminate microbial cells and suspended particles. Microorganisms naturally secrete bioflocculants during growth, offering safer, environmentally friendly alternative to chemical flocculants. This study focuses on a bioflocculant from a yeast strain in Kombucha tea SCOBY, highlighting its production, characterization, and application. Additionally, the bioflocculant explored as a stabilizing agent in the green synthesis of copper, silver, and iron nanoparticles, with potential applications in wastewater treatment. The approach prioritizes safety, lack of secondary pollution, and biodegradability in environmental contexts. In the production of the bioflocculant, the optimal medium composition and culture condition for the bioflocculant-producing yeast, previously isolated from Kombucha tea SCOBY from GreenHeart Organics, Durban, KwaZulu Natal, South Africa, were determined through process optimization. Solvent extraction and purification techniques were employed to produce the bioflocculant. This bioflocculant was then used in synthesizing copper, iron, and silver nanoparticles. Characterization involved various analytical techniques such as FT-IR, X-ray diffraction, SEM-EDX, UV-Vis spectroscopy, TEM, and TGA. The flocculating activity of both the bioflocculant and the bio-synthesized nanoparticles was investigated, with subsequent optimization of dosage size, temperature, pH, metal ions, and shaking speed. Additionally, the study assessed the antimicrobial activity, cytotoxicity, and dye removal capabilities of the bioflocculant and bio-synthesized copper, iron, and silver nanoparticles. Spectrophotometric evaluation was conducted on the removal potential of these components for parameters including BOD, COD, total nitrogen, phosphorus, and sulphate, using wastewater samples from Tendele coal mine and Vulindlela wastewater treatment plants in South Africa. Through the analysis of the internal transcribed spacer (ITS) region sequences was used for fungi identification. The fungus was identified as Pichia kudriavzevii MH545928.1. Under optimal conditions, including a 1% (v/v) inoculum size, glucose and peptone as nutrient sources, a temperature of 35 ⁰C, pH 7, and a shaking speed of 140 rpm for 60 h, the fungus produced a bioflocculant with a remarkable flocculating activity of 99.1% and the yield of 2.836 g/L. Scanning electron microscopy (SEM) analysis unveiled a cumulus-like structure, while Fourier transform infrared spectroscopy (FT-IR) indicated the presence of hydroxyl, carboxyl, amine, and thiocyanate functional groups which is essential in the flocculation process. X-ray diffraction (XRD) analysis revealed the bioflocculant exhibited large particles with distinct diffraction peaks at 10⁰ and 40⁰, suggesting its crystalline nature. The results obtained suggest that Pichia kudriavzevii MH545928.1 holds promising potential for industrial applications as a producer of bioflocculants. The produced bioflocculant demonstrated removal efficiencies of 73% for BOD, 49% for COD, and 47% for phosphate (P). The obtained bioflocculant, with a concentration of 2.836 g/L, exhibited composition comprising carbohydrates (69%), protein (11%), and uronic acid (16%). The elemental composition analysis of the bioflocculant revealed the presence of the following elements: carbon (C) at 16.92%wt, nitrogen (N) at 1.03%wt, oxygen (O) at 43.76%wt, sodium (Na) at 0.18%wt, magnesium (Mg) at 0.40%wt, aluminium (Al) at 0.80%wt, phosphorus (P) at 14.44%wt, sulfur (S) at 1.48%wt, chlorine (Cl) at 0.31%wt, potassium (K) at 0.34%wt, and calcium (Ca) at 20.35%wt. In coal mine wastewater, the bioflocculant demonstrated removal efficiencies of 43% for chemical oxygen demand (COD), 64% for biochemical oxygen demand (BOD), 73% for phosphate (P), and 50% for nitrogen (N). Comparing the bioflocculant to traditional flocculants, it was comparable in terms of dye removal with over 72% removal efficiency across all tested dyes. The biosafety profile of the bioflocculant showed survival effect of over 80% HEK cell lines with 25 μg/μL concentration. It was noted that on both Gram-positive and Gram-negative microorganisms, the bioflocculant did not show to be effective. The viability of colour change from blue to light blue in the formation signifies the success in the synthesis of CuNPs. The peaks observed in the characteristics of the bioflocculant-treated CuNPs were at 3482 cm-1 (-OH), 3261 cm-1, 1640 cm-1, 1059 cm-1, 580 cm-1, and 519 cm-1 (Cu-O). These peaks indicate the presence of functional groups including hydroxyl, amine, and copper oxide bonds. The UV-Vis analysis of the as-synthesized CuNPs demonstrated the presence of surface plasmon resonance (SPR) within the absorbance range of 500 - 600 nm, with peak maxima observed at 535 nm and 560 nm. The XRD pattern exhibited prominent planes such as (200) and (220) at 2θ values of 40° and 51°, respectively. Furthermore, the particle size of the CuNPs, determined using the Debye-Scherrer equation, was found to be 30 nm. Transmission electron microscopy analysis confirmed the presence of spherical shaped CuNPs with an average size of 20 nm. EDX analysis showed the presence of copper (Cu) in the as-synthesized CuNPs, indicating successful biosynthesis as it was not detected in the bioflocculant used during synthesis. The biosynthesized CuNPs were thermal stable retaining above 70% flocculating activity when subjected to TG analysis. The optimal dosage for the biosynthesized CuNPs was determined to be 0.2 mg/mL, resulting in a flocculating activity of 93%. Through the optimization of various factors such as metal ions, pH, temperature, and shaking speed, flocculating activities of 93% (Ca), 94%, 93% (at 70°C), and 94% were achieved, respectively. CuNPs were effective on dye removal with over 58% removal efficiency. They were also effective on inhibiting both Gram-positive and Gram-negative microorganisms, while biosafety showed they could retain over 58% cell viability at a concentration of 200 μg/μL. CuNPs promise a potential application in an industrial wastewater treatment and dye removal and shows a good biosafety effect. Successful biosynthesis of FeNPs was confirmed by the presence of various functional groups observed in the FT-IR spectra, including hydroxyl, halogen (C-Br), carbonyl, alkanes (C-H), and Fe-O functional groups. TEM analysis demonstrated that the iron nanoparticles generated exhibited dimensions ranging from 2.6 to 6.2 nm. The UV-vis spectra displayed a peak at approximately 210, 265, and 330 nm for the newly fabricated FeNPs, providing confirmation of their formation. The EDX spectrum of the biosynthesized FeNPs indicated the presence of iron nanoparticles at a weight percentage of 0.82 %wt in addition to the elements present in the bioflocculant used in their fabrication. The FeNPs synthesized were observed to be hexagonal in shape and exhibited agglomeration, with dimensions ranging from 18 to 50 nm revealed through SEM analysis. The thermal stability of FeNPs observed through TG analysis, revealed that their thermal stable and have retained more than 60% of their initial weight at elevated temperatures. The optimal dosage for achieving the highest flocculating activity was determined to be 0.6 mg/mL for the nanoparticles. The FeNPs exhibited remarkable performance by retaining over 70% of their flocculating activity even at a temperature of 100 ⁰C. Notably, the biosynthesized FeNPs displayed the highest flocculating activity of 97% under specific conditions, including a shaking speed of 180 rpm, Fe3+ ions as the cation, and a pH level of 6. The biosynthesized Fe nanoparticles exhibited concentration-dependent cytotoxicity on HEK 293 cell lines, with the highest concentration (100 μg/μL) resulting in 34% cell survival. These nanoparticles also demonstrated potent antimicrobial properties against various Gram-positive and Gram-negative bacteria. Furthermore, they displayed high efficiency in removing dyes, with a minimum removal rate of 65% and a maximum removal rate of 93% for safranine. The Fe nanoparticles also showed remarkable effectiveness in removing different pollutants from wastewater including COD and BOD. In comparison to conventional flocculants and the bioflocculant, the biosynthesized Fe nanoparticles showed significant potential in reducing both chemical oxygen demand (COD) and biological oxygen demand (BOD) in treated wastewater samples. Making the better optimization for water treatment to replace the in use conventional flocculants. In the biosynthesis of silver NPs using a bioflocculant the SEM images unveiled the presence of smooth, close to spherical particles with an average size of 7 to 12 nm. EDX analysis revealed the presence of silver as an element, constituting 61.93% of the weight, alongside other elements found in the bioflocculant which confirm the synthesis of AgNPs from a bioflocculant. FT-IR spectra analysis indicated the existence of various functional groups, including carboxyl, polyphenols, aromatics, anhydrate, and aliphatic primary amine, amine, and halo compounds. The presence of amine groups suggested the successful synthesis of AgNPs, as they facilitated the reduction of Ag+ to Ag0. TEM imaging revealed spherical nanoparticles with sizes ranging from 7 to 12 nm. TG analysis demonstrated that AgNPs exhibited greater thermal stability, retaining over 85% of their mass at high temperatures, while the bioflocculant used for their fabrication retained 60% of its weight under similar conditions. UV-vis spectra exhibited a Surface Plasmon Resonance (SPR) band around 450 nm for the Ag nanoparticles, and X-ray diffraction analysis indicated a crystallite size of 19 nm obtained through Scherrer equation. Utilizing the bioflocculant as a capping and stabilizing agent in the biosynthesis of AgNPs, offers an ecologically safe technique that yields synthesis rates comparable to chemical methods but with faster results. The optimization process revealed that the AgNPs exhibited optimal flocculation efficiency at a dosage of 0.2 mg/mL, with the flocculating activity of 94%. The biosynthesized nanoparticles demonstrated a preference for a shaking speed of 140 rpm, resulting in a flocculating activity of 94%. Furthermore, when a pH of 7 was employed in the presence of Ca2+ as the metal ion, the optimum flocculating activity of 98% was obtained. The biosynthesized AgNPs using a bioflocculant are thermal stable as they retained more than 85% flocculating activity at 100 ⁰C. The concentration-dependent cytotoxic effects of the biosynthesized AgNPs on HEK 293 cell lines were observed and showed over 68% cell viability at a concentration of 25 μg/μL and 42% cell viability at 100 μg/μL. The AgNPs also demonstrated strong antimicrobial efficacy against various Gram-positive and Gram-negative bacteria. Additionally, the biosynthesized AgNPs exhibited efficient removal of dyes, with the removal efficiency over 75% and with a maximum removal efficiency of 96% observed for methylene blue. Furthermore, these nanoparticles proved highly effective in removing a diverse range of pollutants from wastewater, surpassing traditional flocculants in removing both BOD (92% removal efficiency) and COD (86% removal efficiency) among other pollutants. In conclusion, the Cu, Fe, and Ag nanoparticles synthesized using the bioflocculant exhibit promising potential as a substitute for traditional flocculants in wastewater treatment and dye removal due to their biocompatibility and environmentally friendly properties.
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    Genome data mining, annotation, and phylogenetic analysis of cytochrome P450 monooxygenases in the fungal class Pezizomycetes
    (University of Zululand, 2022) Nsele, Nomfundo Ntombizinhle
    Cytochrome P450 monooxygenases (CYPs/P450s) are heme proteins that play a role in organisms’ primary and secondary metabolism. P450s play an important role in organism adaptation since lifestyle influences P450 composition in their genome. This phenomenon is well-documented in bacteria but less so in fungi. This study observed this phenomenon where diverse P450 complements were identified in saprophytic and ectomycorrhizal Pezizomycetes. Genome-wide data mining, annotation, and phylogenetic analysis of P450s in 19 Pezizomycetes revealed 668 P450s that can be grouped into 153 P450 families and 245 P450 subfamilies. Only four P450 families, namely, CYP51, CYP61, CYP5093, and CYP6001, are conserved across 19 Pezizomycetes, indicating their important role in these species. A total of 5 saprophyte Pezizomycetes have 103 P450 families, whereas 14 ectomycorrhizal Pezizomycetes have 89 P450 families. Only 39 P450 families were common, and 50 and 64 P450 families, respectively, were unique to ectomycorrhizal and saprophytic Pezizomycetes. These findings suggest that the switch from a saprophytic to an ectomycorrhizal lifestyle led to both the development of diverse P450 families as well as the loss of P450s, which led to the lowest P450 family diversity, despite the emergence of novel P450 families in ectomycorrhizal Pezizomycetes.
University of Zululand