Browsing by Author "Okudoh, Vincent Ifeanyi."
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Item Anaerobic digestion of energy crop (cassava)(2019) Sawyerr, Nathaniel Olugbenga.; Trois, Cristina.; Workneh, Tilahun Seyoum.; Okudoh, Vincent Ifeanyi.Global energy demand is on the rise due to continuous increases in population, economic growth, and energy usage. Methane production through anaerobic digestion of organic materials provides a resourceful carrier of renewable energy, as methane can be used instead of fossil fuels for both heat and power generation and also as vehicle fuel, thus cutting down the emissions of greenhouse gases and hence contribution in the slowing down climate change. Several studies have been done on biogas, but in South Africa, these are biased towards industrial wastewater. Therefore, there is need to explore other alternatives for biogas generation. Furthermore, the sustainability of anaerobic digestion processes depends on the availability and the identification of the optimal substrate. The use of cassava in South Africa provides a great potential for the production of bioenergy especially biogas, due to its suitable chemical composition. Cassava codigested with other feedstock could be an alternative substrate for various communities for the production of biogas in South Africa. Since cassava is yet to be listed as a staple food crop in South Arica, its peels and other by-products from its processing can be suitable for renewable energy production for small medium enterprises (SMEs). This study’s overall objective was that of establishing the suitability of cassava tubers as an alternative source of biomass feedstock for biogas production in South Africa. The specific objectives of the study were: 1) Comparing the yield and rate of biogas production of cassava peels inoculated with cattle manure using a batch digester under anaerobic digestion conditions addressed in chapter four and five of the thesis; 2) Investigate the biogas yield and rate of different co-digestion ratios of cassava with vegetable and fruit waste using batch digestion under anaerobic digestion conditions presented in chapter six; 3) Optimize the production of biogas through process optimization by maintaining the optimum temperature during fermentation and compare inexperiments subjected to different treatment or treatment combinations and, 4) While chapter seven addresses the objective of using the experimental results to design an upscale system using baseline data information from experiment. Several feedstocks (i.e. cassava tuber, cassava peels, vegetable and fruit waste and cattle manure) were identified and analysed using the American Standard Methods for examination of Water and Wastewater (ASTM). Cassava was selected as it has several advantages compared to other crops, including the ability to grow on degraded land and where soil fertility is low. It also has the highest yield of carbohydrate per hectare (4.742 kg/carb) apart from sugarcane and sugar beet, which makes it suitable for bioenergy (biogas) generation. In the first instance, a batch experiment of were cassava peels were digested anaerobically with and without cattle manure to determine whether cassava peels (CP) in combination with cattle manure (CM) at different ratios shows better biogas yield. The following ratio combinations of mixture were used 100:0, 0:100, 80:20 and 20:80 (CM:CP). A theoretical methane production was conducted using elemental composition and the results were compared with the experimental ones. The test of biogas yield was conducted using an anaerobic digester of 600 ml at mesophilic (35 ± 1 °C) temperature. In the second experiment a 50 litres anaerobic digester was used to investigate the biogas yield of peeled cassava tuber compared to unpeeled cassava tuber that yield biogas of 635.23 L/kg VS and 460.41 L/kg VS respectively. This was based on the finding of the first experiments of biogas yield from cassava peels. The biogas yield with and without inoculum was measured and the biogas yield were modelled using two different models namely modified Gompertz and cone model. Finally, in parallel with the previous batch experiments another set of batch experiments were carried out under anaerobic conditions at mesophilic (35 ± 1 °C) temperature in a 600 ml digester, this experiments was conducted by co-digesting cassava (CB) with vegetable and fruit waste (CB:VF) at different ratios (100:0, 60:40, 40:60 and 50:50). The cumulative biogas yield were modelled for kinetics using modified Gompertz model. Based on the results obtained from the experimental study cassava co-digested with vegetable and fruits at a ratio of 40:60 which was found to produce the maximum yield, a mathematical design (upscale system) was designed. This designed biogas plant could be located in several communities especially those close to the landfills to reduce the cost of transportation from source. The study’s results revealed that: • co-digestion influenced biogas production and methane yield. The final cumulative methane yields by the co-digestion of CM and CP at the CM:CP mixing ratios of 80:20 and 20:80 were 738.76 mL and 838.70 mL respectively. The corresponding average daily methane yields were 18.42 mL/day and 20.97 mL/day. This indicates that CP enhanced the production of methane in the co-digestion process with the 20:80 CM:CP ratio. • the feedstock of peeled cassava with inoculum, produced 28.75% more biogas yield when compared to peeled cassava without inoculum. This results highlights the important of inoculum in the anaerobic digester. • peeling the cassava tuber increase the biogas yield by 38% compared to the unpeeled tuber • cassava biomass co-digested with vegetable and fruit waste increased the methane yield compared to the mono-digestion with the highest methane production was achieved from the co-digestion of cassava biomass with vegetable & fruit waste at 40:60 ratio (CB: VF) Although several challenges hampering the smooth implementation of biogas generation in South Africa, this study concludes that cassava (peeled and unpeeled) co-digested with fruit and vegetables waste has potential to generate biogas thereby presenting a substantial opportunity to promote bioenergy production from cassava considering in many rural areas the needs for fuel and electricity are not satisfied fully. Finally, cassava anaerobic digestion facility at different scales could enhance additional benefits like the integration of nutrients and residual carbon into the land as fertilizer.Item Isolation and characterization of antibiotic(s) produced by bacteria from KwaZulu-Natal soils.(2010) Okudoh, Vincent Ifeanyi.; Wallis, Frederick Michael.This work reports the continued search for new antibiotics in the relatively under investigated region of KwaZulu-Natal, South Africa. A soil bacterium designated strain N8 with antibacterial activity against both Gram-positive and Gram-negative bacteria was isolated from a poultry farm in Pietermaritzburg, South Africa. The organism was one of approximately 2600 strains isolated from various habitats in the KwaZulu-Natal midlands, South Africa during an actinomycete screening programme. The highest number of antimicrobially-active isolates came from a forest soil site whereas the lowest number was present in a riparian soil. Morphological, physiological and cultural characteristics indicated that strain N8 belonged in the genus Intrasporangium. In the literature, members of this actinomycete genus have not been associated previously with antibiotic production. Studies on the influence of different nutritional compounds on antibiotic production showed that the highest antibacterial activities were obtained when glycerol at 1% (w/v) was used as sole carbon source in the presence of mineral trace elements. Using solvent extraction and various chromatographic techniques, the antibiotic produced by strain N8 was recovered from the fermentation broth. The use of a three-solvent system, petroleum ether: acetone: ethyl acetate enhanced the separation of the antibiotic complex in broth. Bioassay results established that the antibacterial agent was in the ethyl acetate fraction (EAF) and chromatographic methods were used in its purification. The chromatographic methods used were: flash column chromatography (FCC), thin-layer chromatography (TLC), and Harrison research chromatotron (HRC). Further purification was carried out by reverse phase high performance liquid chromatography (HPLC). Most of the inactive, coloured material was removed from the antibiotic extract by FCC, while TLC chromatograms run using a range of the most polar to the least polar solvent systems [SS1 (most polar) – SS5 (least polar)] showed best separation of EAF with SS2. TLC chromatograms using SS2 usually showed 3 bands. Bioautograms of SS2-separated EAF revealed that the antibiotic activity was located in the region with an Rf value of 0.56 – 0.64. The Harrison research chromatotron technique also gave good separation of the EAF sample. Preparative HPLC was used as the final purification step for most of the EAF samples. Although, a number of peaks were observed during isocratic-HPLC (IHPLC) runs, they were not as clearly separated as those obtained with gradient-HPLC (GHPLC). Three major peaks PI, PII and PIII with elution times of 3.56 min, 4.53 min and 23.06 min respectively were revealed under GHPLC runs with decreasing concentrations (100% – 50%) of methanol in water. Methanol concentrations between 50% and 70% in water were considered the optimum GHPLC mobile phases. Since these chromatographic methods were all time consuming, required large volumes of solvents, and resulted in low yields of the antibiotic, an alternative procedure producing better results was sought. This led to the development of a procedure combining a three-solvent extraction system with a pH precipitation process which efficiently recovered the antibiotic in solid/crystal form. Using this procedure, sufficient quantities of the antibiotic were recovered from the fermentation broth to permit a degree of structural elucidation. Two types of crystals (brown and pink-yellow in colour) were obtained and their chemical natures established by means of 1H- and GCOSY- nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LC-MS). On further LC-MS analysis, the brown crystals appeared to be a protein and since it did not show inhibitory activity against any of the test organisms, no further studies were carried out on it. The pink-yellow crystals when suspended in a minimal volume of methanol showed inhibitory activity against S. marcescens confirming that the antibiotic activity resided therein. The LC-MS spectrum of these crystals showed a prominent/base peak at 304.2724 [mass to charge ratio (m/z) in positive mode]. The elemental composition of this compound suggests a molecular formula close to C16H36N2O3 with a molar mass of 304.4686 g/mol. No existing name could be assigned to it from the database of known natural compounds. Hence, the possibility that it is a novel antimicrobial compound cannot be excluded. Characterisation of the antimicrobial substance using GC-MS revealed that it contained at least seven components (A – G). These components were then subjected to mass spectrum analysis and their retention indices compared to computer database listings of known compounds. Components A and B were regarded as representing one compound (possibly isomers) since they have the same molecular weight and formula. Their different retention indices strongly suggest they are indeed isomers. Thus a total of six different compounds were detected in the extract by GC-MS and the molecular formulae assigned to them include: C6H10O (A and B); C6H12O2 (C); C9H14O (D); C8H7N (E); C21H44 (F); and C12H14N2O (G). Since only low probability matches were obtained for A – F and as the sample could not be recovered from the analyser, they were not studied further. The closest match (71% probability) with substances listed in the computer database of natural compounds was for compound G (C12H14N2O) which was thus provisionally identified as N-acetyltryptamine. A structurally related compound known as melatonin is attributed with the ability to inhibit tumour growth in vivo and in vitro. Attempts were made to assign a chemical structure to the antibiotic produced by strain N8 using all the data available. The indications are that it is a tryptamine, the chemical structure of which is postulated to be: In order to monitor the antimicrobial activity of the antibiotic produced by strain N8, bioassays were conducted after all major steps during the isolation and characterization processes. The antimicrobial activity of the pink-yellow crystals was confirmed on the test organisms used during the primary screening phase, namely, Escherichia coli, Pseudomonas fluorescens, Serratia marcescens, Staphylococcus aureus, Enterococcus faecalis and Xanthomonas campestris pv. campestris, and the yeast Candida utilis, indicating that the crude substance had maintained its inhibitory activity against Gram-positive and Gram-negative bacteria, and the yeast tested. The study was extended to include investigations into the use of combinations of the GHPLC separated peaks of the antibiotic (PI, PII and PIII) to improve the efficacy of growth inhibition of the test pathogens for possible use in chemotherapy. Data from these studies showed that PI inhibited the growth of E. coli and X. campestris pv. campestris while PII and PIII inhibited the growth of the latter organism and also that of S. marcescens. Individually, the peaks showed no growth inhibition on Pseudomonas fluorescens but the combination PI+PII+PIII was antimicrobially effective. In all cases, the use of combinations was significantly more effective than the use of any single component alone. For example, the combination of GHPLC PI and PII had a greater growth inhibitory effect (synergic action) against Serratia marcescens than did either alone; the inhibition-zone diameter being double (30mm) that caused by the single peaks (15mm) against S. marcescens. Likewise mixing PI and PIII resulted in a much improved action against X. campestris pv. campestris. These findings may meet the current call by many scientists that all infectious diseases should be treated with a combination of two antibiotics with different mechanisms of action in order to counter the serious problem of emerging bacterial resistance. Since the antibiotic isolated during this study showed activity against both mammalian and plant pathogenic bacteria it is hoped that this work will encourage further investigation in this field in South Africa. The results obtained should impact on the pharmaceutical industry as well as agriculture and will, hopefully, help curb both plant and human infectious diseases in our African communities. This study also confirmed that KwaZulu-Natal soils do harbour rare actinomycetes that produce novel antimicrobial compounds.Item Isolation and identification of antibiotic producing microorganisms from natural habitats in the KwaZulu-Natal midlands.(2001) Okudoh, Vincent Ifeanyi.; Wallis, Frederick Michael.The search for new antibiotics continues in a rather overlooked hunting ground. In the course of screening for new antibiotic-producing microorganisms, seventy-nine isolates showing antimicrobial activity were isolated from soil samples from various habitats in the KwaZulu-Natal midlands, South Africa. Existing methods of screening for antibiotic producers together with some novel procedures were reviewed. Both modified agar-streak and agar-plug methods were used in the primary screens. The use of selective isolation media, with or without antibiotic incorporation and/or heat pretreatment, enhanced the development of certain actinomycete colonies on the isolation plates. Winogradsky's nitrite medium (Winogradsky, 1949), M3 agar (Rowbotham and Cross, 1977), and Kosmachev's medium (Kosmachev, 1960), were found to be selective for actinomycetes. Statistical analysis showed highly significant interactions between isolates, assay media and the test organisms. The diameters of inhibition zones were found to be larger on Iso-sensitest agar (ISTA)[Oxoid, England] than in nutrient agar plates. Of the 79 isolates that showed antimicrobial activity, 44 isolates were selected for confirmatory screening. Of these, 13 were selected for secondary screening. Criteria for selection were based on significant inhibition of at least two test organisms and/or the inhibition of the specifically targeted organisms, Pseudomonas and Xanthomonas species. Following secondary screening eight isolates were considered for further investigation. The isolates were tentatively identified . on the basis of morphological features, using both light microscopy and scanning electron microscopy(SEM); their ability to utilize various carbon sources; and selected physiological and staining tests. Suspected actinomycetes were further characterized on the basis of selected chemical properties using thin layer chromatography (TLC) and high pressure liquid chromatography (HPLC) techniques. High pressure liquid chromatography analysis (Beckman 6300 analyzer) detected the presence of diaminopimelic acid (DAP) in whole-cell hydrolysates of six of the isolates while TLC analysis confirmed the type ofDAP present. The isolates N2, N12, N16, N19 and N35 were tentatively identified as Thermomonospora, Saccharopolyspora, Nocardiodes, Corynebacterium and Promicromonospora, respectively. Isolate N30 was identified as belonging to the coryneform group ofbacteria, possibly an Arthrobacter species. Isolate, N8, tentatively identified as Actinosynnema, was unique among the isolates tested as it showed good antimicrobial activity against all the Gram- positive and Gram-negative bacteria, and yeasts used as test organisms in the present investigation.