Microbiology

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Microbial biotransformation of kimberlite ores.
(2008) Ramcharan, Karishma.
Microbial leaching plays a significant role in the natural weathering of silicate containing ores such as diamond-bearing kimberlite. Harnessing microbial leaching processes to pre-treat mined kimberlite ores has been proposed as a means of improving diamond recovery efficiencies. The biomineralization of kimberlite is rarely studied. Therefore, this study investigated the feasibility of exploiting both chemolithotrophic and heterotrophic leaching processes to accelerate the weathering of kimberlite. Preliminary investigations using mixed chemolithotrophic leaching cultures were performed on four finely ground kimberlite samples (<100μm) sourced from different mines in South Africa and Canada. Mixed chemolithotrophic cultures were grown in shake flasks containing kimberlite and inorganic basal media supplemented either with iron (Fe2+, 15g/l) or elemental sulfur (10g/l) as energy sources. Weathering due to dissolution was monitored by Inductive Coupled Plasma (ICP) analyses of Si, Fe, K, Mg and Ca in the leach solutions at known pH. Structural alterations of kimberlite after specified treatment times were analyzed by X-ray Powder Diffraction (XRD). The results of the preliminary investigation showed that weathering can be accelerated in the presence of microbial leaching agents but the degree of susceptibility and mineralogical transformation varied between different kimberlite types with different mineralogical characteristics. In general, the results showed that the kimberlite sample from Victor Mine was most prone to weathering while the sample from Gahcho Kue was the most resistant. It was therefore deduced that kimberlite with swelling clays as their major mineral component weathered relatively more easily when compared to kimberlite that consisted of serpentine and phlogopite as their major minerals. Gypsum precipitates were also distinguished indicating that a partial alteration in the kimberlite mineralogical structure occurred. Both energy sources positively influenced the dissolution process, with sulfur producing superior results. This was attributed to the generation of sulfuric acid which promotes cation dissolution and mineral weathering. Success in the preliminary investigations led to further experimental testing performed to determine the effect of particle size and varying energy source concentrations on the biotransformation of kimberlite. It was observed that although weathering rates of the larger kimberlite particles (>2mm<5mm) were lower than that of the finer particles, slight changes in their mineralogical structures represented by the XRD analyses were seen. Optimisation studies of energy source concentration concluded that although the highest concentration of elemental sulfur (20% w/w) and ferrous iron (35% w/w) produced the most pronounced changes for each energy source tested, the leaching efficiency at these concentrations were not drastically greater than the leaching efficiency of the lower concentrations, as expected. Following the success of batch culture shake flasks weathering tests, the effect of continuous chemolithotrophic cultures on the biotransformation of larger kimberlite particles (>5mm<6.7mm) was investigated. A continuous plug-flow bioleach column was used to model the behaviour of chemolithotrophic consortia in a dump- or heap leaching system. Two sequential columns were setup, in which the first consisted of kimberlite mixed with sulfur and the second purely kimberlite. Inorganic growth medium was pumped to the first column at a fixed dilution rate of 0.25h-1 and the leachate from the first column dripped into the second. After an 8 week investigation period, the ICP and XRD data showed that weathering did occur. However, the pH results showed that the leaching process is governed by the amount of acid produced by the growth-rate independent chemolithotrophic consortia. Data from pH analyses also showed that the leaching bacteria reached ‘steady state’ conditions from day 45 onwards. The pH also remained higher in the second column than in the first column highlighting the alkaline nature of the kimberlite ores and its ability to act as a buffering agent and resist weathering. This important factor, as well as further optimisation studies in process operating conditions and efficiency, needs to be considered when establishing heap-leaching technology for these kimberlite ores. In the preliminary heterotrophic investigation, Aspergillus niger was used to produce organic metabolites to enhance kimberlite mineralization. The results demonstrated that the organic acid metabolites generated caused partial solubilization of the kimberlite minerals. However, it was deduced that for more significant changes to be observed higher amounts of organic acids need to be produced and maintained. The results obtained in this study also showed that the type of kimberlite presents a different susceptibility to the dissolution process and the presence of the fungal cells may improve the leaching efficiency. The results in this study provided an optimistic base for the use of microbial leaching processes in accelerating the weathering of kimberlite. These findings may also serve to supply data to formulate recommendations for further and future column microbial leach tests as well as validation and simulation purposes.
Application of bacterial bioflocculants for wastewater and river water treatment.
(2008) Buthelezi, Simphiwe P.; Pillay, Balakrishna.
Dyes are often recalcitrant organic molecules that produce a colour change and contribute to the organic load and toxicity of textile industrial wastewater. Untreated effluent from such sources is harmful to aquatic life in the rivers and lakes due to reduced light penetration and the presence of highly toxic metal complex dyes. The use of alum as flocculant/coagulant in wastewater treatment is not encouraged as it induces Alzheimer’s disease in humans and results in the production of large amounts of sludge. Therefore, the development of safe and biodegradable flocculating agents that will minimize environmental and health risks may be considered as an important issue in wastewater treatment. Bioflocculants are extracellular polymers synthesized by living cells. In this study, bacterial bioflocculants were assessed for their ability to remove dyes from textile wastewater as well as reducing the microbial load in untreated river water. The bacteria were isolated from a wastewater treatment plant and identified using standard biochemical tests as well as the analysis of their 16S rDNA gene sequences. Six bacterial isolates were identified viz. Staphylococcus aureus, Pseudomonas plecoglossicida, Pseudomonas pseudoalcaligenes, Exiguobacterium acetylicum, Bacillus subtilis, and Klebsiella terrigena. The flocculating activities of the bioflocculants produced by these isolates were characterized. The effect of temperature, pH, cations and bioflocculant concentration on the removal of dyes, kaolin clay and microbial load was also determined. The amount of bioflocculants produced by the bacterial isolates ranged between 5 and 27.66 g/l. According to the findings of the present study, bacterial bioflocculants were composed of carbohydrates, proteins, uronic acid, and hexosamine in varying quantities. The bioflocculants were effective to varying degrees in removing the dyes in aqueous solution, in particular whale dye, medi-blue, fawn dye and mixed dyes, with a decolourization efficiency ranging between 20-99.9%. Decolourization efficiency was influenced by the bioflocculant concentration, pH, temperature, and cations. The bacterial bioflocculants were also capable of reducing both the kaolin clay and the microbial load from river water. The flocculating activity ranged between 2.395–3.709 OD-1 while up to 70.84% of kaolin clay and 99% of the microbial load from the river water was removed. The efficiency of kaolin clay flocculation increased with higher concentration of bacterial bioflocculants. The optimum pH for the flocculating activity was observed between 6 and 9. The best flocculating activity was observed at 28oC. Divalent cations such as Mg2+ and Mn2+ improved the flocculation while salts such as K2HPO4, CH2COONa, and Na2CO3 did not. The findings of this study strongly suggest that microbial bioflocculants could provide a promising alternative to replace or supplement the physical and chemical treatment processes of river water and textile industry effluent.
Characterisation of chlorinated-hydrocarbon-degrading genes of bacteria.
(2009) Govender, Algasan.
1,2-dichloroethane (DCA) is one of the most widely used and produced chemicals of the modern world. It is used as a metal degreaser, solvent, chemical intermediate as well as a fuel additive. This carcinogen is toxic to both terrestrial and aquatic ecosystems and accidental spills and poor handling has resulted in contamination of the environment. Thus far several bacteria in the Northern hemisphere have been identified that are capable of utilizing this compound as a sole carbon and energy source. This report focuses on the isolation and characterization of bacterial isolates from the Southern hemisphere that are capable of degrading DCA as well as the global distribution of the DCA catabolic route. Samples obtained from waste water treatment plants were batch cultured in minimal medium containing DCA and repeatedly sub-cultured every five days over a 25 day period. A halogen release assay was performed in order to determine whether individual isolates possessed dehalogenase activity. Confirmation of DCA utilization by bacterial isolates positive for dehalogenase activity was done by sub-culturing back into minimal medium containing DCA. Enzyme activities were confirmed with cell free extracts using all of the intermediates in the proposed DCA degradative pathway and compared to a known DCA degrading microorganism. Biochemical tests and 16SrDNA sequencing indicated that all the South African isolates belonged to the genus Ancylobacter and were different from each other. Based on enzyme activities, it was found that the South African isolates may possess a similar degradative route as other DCA degrading microorganisms. Primers based on genes involved in DCA degradation were synthesized and PCR analysis was performed. It was found that all isolates possessed an identical hydrolytic dehalogenase gene whereas the other genes in the pathway could not be PCR amplified. Southern hybridization using probes based on known genes indicated that some of the isolates had homologous genes. Pulsed field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) analysis indicated that the five South African isolates of Ancylobacter aquaticus are distinguishable from each other. This study is the first report indicating that microbes from different geographical locations use similar metabolic routes for DCA degradation. The first gene of the pathway (dhlA) has undergone global distribution which may be due to widespread environmental contamination.
Beta-lactamase mediated resistance in Salmonella spp. at a tertiary hospital in KwaZulu-Natal.
(2008) Govinden, Usha.; Essack, Sabiha Yusuf.; Sturm, Adriaan Willem.; Moodley, Prashini.
Extended spectrum (3-lactamases (ESBLs) were characterized in Salmonella spp. isolates from a pediatric ward of a hospital in Durban. Forty one Salmonella spp. were subjected to serotyping, antibiotic susceptibility testing, E-Tests for ESBL detection, iso-electric focusing, polymerase chain reaction for detection of genes and sequencing. Isolates were screened for the presence of WaTEM, WaSHV, WaCTX-M, WaOXA , WaCMY, WaDHA and WaACC genes. The most common serotype was Salmonella Typhimurium. Isolates were multi-drug resistant with 100% susceptibility only to meropenem and ciprofloxacin. Tazobactam was the most effective inhibitor. Forty-one percent of the isolates were resistant to ceftriaxone, thus limiting therapeutic options for Salmonella infections.TEM-1 was the most predominant (3-lactamase found in 51% of isolates while SHV-12 found in 39 % was the most common ESBL. TEM-63 was evident in 29 %, TEM-116 in 10 % and TEM-131 was found in one isolate. The high ceftazidime MICs of isolates expressing only TEM-63 were indicative of R164S substitution which widens the binding cavity to accommodate the bulky side chains of oxyiminoaminothiazolyl cephalosporins. The identification of TEM-131 which differs from TEM-63 by 1 amino acid reiterates the evolutionary potential of the TEM-type plactamase. Other ESBLs identified included SHV-2, CTX-M-3, CTX-M-15 and CTX-M-37. CMY-2 and the OXA-1 p-lactamase were also detected. This is the first report of TEM-116, CTX-M-3, -15 and -37 in Salmonella spp. in South Africa. All isolates with nalidixic acid MICs > 48 ug/ml had the mutation D87N, or D87G in the QRDR of the gyrA gene. This study showed that Salmonella spp. may be multi-drug resistant with the propensity to harbour p-lactamases in unique combinations. The diversity of ESBLs and the co-expression of quinolone resistance suggests that their incidence in salmonellae needs to be monitored.
Optimizing biocontrol of purple nutsedge (Cyperus rotundus).
(2006) Brooks, Michael John.; Laing, Mark Delmege.
Cyperus rotundus L. CYPRO (purple nutsedge) and Cyperus esculentus L. CYPES (yellow nutsedge) are problematic weeds on every continent. At present there is no comprehensive means of controling these weeds.. The primary means of control is herbicides, although the weeds are becoming more resistant. Bioherbicide control of purple and yellow nutsedge is an important avenue of research, with much of the focus being to increase the virulence of current fungal pathogens of C. rotundus and C. esculentus. The primary aim of this study was to increase the virulence of a fungal pathogen of C. rotundus and C. esculentus, with the objective of creating a viable bioherbicide. A possible means of increasing the virulence of a pathogen would be to increase the amount of amino acid produced by the fungus. This was proposed as a means of increasing the virulence of Dactylaria higginsii (Luttrell) M. B. Ellis. Overproduction of amino acids such as valine and leucine result in the feedback-inhibition of acetolactate synthase (ALS), an enzyme which is a target for many herbicides currently on the market. By applying various amino acids to tubers of purple nutsedge and comparing the results with a reputable herbicide, glyphosate, it was possible to determine the success of the amino acid applications. Only glutamine treatment at 600 mg.r1 resulted in significantly less (P
Humic acid pretreatment for enhancing microbial removal of metals from a synthetic 'wastewater'.
(2004) Desta, Tsegazeab Goje.; Wallis, Frederick Michael.
The presence of heavy metal ions in waste streams is one of the most pervasive environmental issues of present times. A rotating biological contactor (RBC) was used to investigate the potential capacity of microbial biofilms in remediation of the metal ion species from a mixed metal contaminated effluent solution containing Cr+3 , Pb+2 and Cu+2 , each at a concentration of 200 mg r1 • In the first part of this study the effectiveness of various support materials for the development of microbial biofilms capable of removing heavy metals from a synthetic effluent was investigated. EDX analysis showed that none of the support matrices investigated, viz. gravel, polyester batting and sand, adsorbed metal ions on their surfaces; hence, metal adsorption was due purely to microbial activities. The biofilms attached more firmly and uniformly to polyester batting than to gravel and sand. The characteristics of polyester batting which made it a superior support matrix were its surface roughness and porous hydrophilic nature, which provided a larger surface area for the adhesion of microorganisms and attraction of nutrients during the biofilm development process. The selective accumulation of metal ion specIes by various microbial populations grown as biofilm using polyester batting as support matrix in separate compartments of a single-stage RBC bioreactor was examined. Lead ions were readily accumulated by almost all the microbial biofilms tested. Fungus-dominated biofilms selectively accumulated chromium ions whereas biofilms comprising mainly bacteria more readily accumulated copper ions from the mixed metal contaminated effluent solution. However, where interactions between the bacterial and fungal components were encouraged the mechanical stability of the biofilms was enhanced so that large amounts of all three metal ion species were removed by this biofilm. The combined effect of a series of bench-scale columns containing liquid humic acid and a three stage RBC bioreactor on the removal of metal ion species from a mixed metal contaminated effluent was investigated. After seven days of treatment the combined system had removed approximately 99% of the Cr+3, 98% of the Pb+2 and 90% of the Cu+2 ions from the mixed metal contaminated synthetic effluent. Complexation of the metal ions with humic acid was the predominant factor accounting for approximately 68-86% Cr+3 , 70-86% Pb+2 and 53-73% Cu+2 removal levels within the columns. A large proportion of the remaining Cr+3 and Pb+2, but not of the Cu+2, was removed in compartment 1 of the RBC. This suggested that the presence of the former two metals in solution might have reduced the removal of the Cu+2 ions from the system. The removal of substantially large amounts of the competing ions chromium and lead during the initial stages of the treatment process meant that copper was successfully taken up in the second and third RBC compartments. Hence, the economy of the treatment process was improved as larger quantities of the metal ions were removed in a shorter period of time than was possible when using the individual treatments (humic acid-metal complexation and biofilm adsorption) separately. More than 75%,92% and 86% of the adsorbed Cr+3 , Pb+2 and Cu+2 ions, respectively, were recovered from the three RBC bioreactor compartments following repeated washing of the biofilms with 0.1 M HCI. This relatively easy desorption suggested that the metal ions were simply adsorbed onto the surfaces of the biofilm cells rather than being taken into the cytoplasm of the cells.
The effect of water treatment residues on soil microbial and related chemical properties.
(2003) Pecku, Shantel.; Hunter, Charles Haig.; Hughes, Jeffrey Colin.
Water treatment residue (WTR), a by-product of the water treatment process, consists primarily of precipitated hydroxides of the coagulants used in the water treatment process, along with sand, silt, clay, humic compounds, and dissolved organic matter. It is usually disposed of by landfill, a technology with numerous problems that include dwindling landfill capacity, extensive dewatering requirements for the WTRs, high costs of transportation, and potential liability for landfill clean-up. Therefore, land disposal (or land treatment) presents a popular alternative disposal method based on the principle that the physical, chemical, and microbial properties of the soil can be used to assimilate applied waste without inducing any negative effects on soil quality. The objective of this study was to investigate the effects of land disposal of the WTR generated by Umgeni Water, a local water treatment authority, on soil quality. These effects were investigated using depth samples from soil profiles of Westleigh and Hutton soil forms at field trials located at Ukulinga Research Farm, near Pietermartizburg and Brookdale Farm, Howick, KwaZulu-Natal, South Africa, respectively. Four rates of WTR (0, 80, 320, and 1280Mg ha-1 incorporated into the soil) were investigated at both trials, in addition to mulched treatments at rates of 320 and 1280Mg ha-1 at Brookdale only. Sampling of plots was carried out in September 2001 and May 2002, and all treatments were investigated under fallow and grassed cultivation. Laboratory measurements used to assess soil quality included pH, electrical conductivity (EC), organic carbon (QC), and microbial activity using f1uorescein diacetate (FDA) hydrolysis. At both trials in September 2001 WTR-amended plots displayed higher pH in the 0-200mm soil in comparison to the controls, whereas by May 2002 pH had returned to the condition of the controls. Addition of WTR at Ukulinga resulted in higher QC in September 2001, but in May 2002 this was similar to the controls. However, at Brookdale QC was unaffected by WTR. At Ukulinga and Brookdale the effect of WTR on EC was variable, and microbial activity in the soil profile was unaffected by WTR addition. Observations at Ukulinga and Brookdale reflected long term changes (3 and 5 years, respectively) to soil quality following WTR addition. To examine the initial changes in soil quality a laboratory experiment was set up using the field trial soils. Research objectives were also extended to include WTRs from Rand Water (Johannesburg), Midvaal Water Company (Stilfontein), Amatola Water (East London), and two samples from the Faure Water Treatment Plant (near Cape Town). The second Faure sample (Faure2 ) was collected when blue green algal problems were experienced at the plant. The measurements used to investigate these short term effects on soil quality were soil pH, EC, and microbial activity as indicated by respiration rate. Each of the WTRs added to the Hutton and Westleigh soils increased soil pH by varying increments, and the higher the WTR application rate, the higher was the pH recorded. With the exception of the Rand and Umgeni WTRs that clearly increased soil EC, the effect of the otherWTRs on EC was variable. The Faure1 and Amatola WTRs appeared to have no effect on microbial activity, whereas the Umgeni, Rand, Midvaal, and Faure2 WTRs stimulated microbial activity by Day 2 following the addition of WTR, but this had declined by Day 14. As for pH, higher microbial activity was recorded at higher WTR application rates. Changes in microbial community structure of the Hutton soil only, following the addition of WTR were examined using denaturing gradient gel electrophoresis (DGGE) analysis. Community profiles of the different WTRs proved to be markedly different. However, WTR-amended soil retained banding patterns consistent with the control soil indicating that dominant populations in the Hutton soil had been retained. The field trials indicated that long term effects of land disposal of WTR were not detrimental to the measured indicators of soil quality namely, pH, EC, QC, and microbial activity. The laboratory assessments of the short term response of the Hutton and Westleigh soil forms to WTR addition suggested that the tested variables were altered by WTR, but not significantly changed to the detriment of soil quality. Microbial community analysis indicated that the community structure of the Hutton soil was not significantly altered by WTR amendments. Present findings provide no evidence to suggest that land disposal of WTR is detrimental to soil quality. It is therefore regarded as a feasible disposal option although there are some aspects that should be investigated further. These include investigations into rhizosphere/microbial interactions and the feasibility of growing cash crops.
Characterization of selected Bacillus isolates exhibiting broad spectrum antifungal activity.
(2004) Tewelde, Teklehaimanot Weldeslasie.; Hunter, Charles Haig.; Beukes, Mervyn.
The genus Bacillus is comprised of Gram-positive, rod-shaped, spore-forming bacteria which are well known for their ability to produce a diverse array of antimicrobial compounds. Ofparticular interest is the ability of certain strains to produce antifungal compounds. Such organisms have the potential for application in agriculture where they can be used as biocontrol agents against selected plant pathogenic fungi. A study was undertaken to further characterize selected Bacillus isolates that exhibit broad spectrum antifungal activity. Dual culture bioassays were used to screen seven selected Bacillus isolates for activity against four plant pathogenic fungi in vitro. All isolates were able to inhibit the pathogens to varying degrees. Two isolates, R29 and B81, were selected for further testing and characterization. Further bioassays were performed on five complex nutrient media which were adjusted to pH S.S and 7, and both incubated at 2SoC and 30°C" respectively. It was found that pH and media composition showed significant influences on the antifungal activities of the isolates tested, but that a SoC temperature difference in incubation temperature did not. Tryptone soy agar was found to give rise to the largest inhibition zones. Both isolates were tentatively identified using standard biochemical and morphological tests. Based on its phenotypic characteristics, R29 was identified as a strain of B. subtilis. B81 proved to be more difficult to assign to a specific group or species of Bacillus, though B. subtilis and B. licheniformis were considered to be the nearest candidates. Genomic DNA was extracted from both isolates and a portion of each of their 16s rDNA genes were amplified and sequenced for homology testing against the GeneBank database. Homology testing confirmed that both isolates were members of the genus Bacillus and most probably strains of B. subtilis. The DNA fragment used for sequencing proved to be too small to give conclusive identification of the isolates. Isolate R29 was selected for further characterization of its antifungal compound/so Growth curve studies using a defined synthetic medium showed that antifungal activity arose during the stationary phase and appeared to be closely linked to sporulation. The antifungal component of cell free culture supematant was extracted using various methods including thin layer chromatography, acid precipitation, hydrophobic interaction chromatography and methanol extractions. High performance liquid chromatography (HPLC) analysis of extracts from acid precipitation and hydrophobic interaction chromatography revealed two active peaks indicating that at least two antifungal compounds were produced. Methanol extracted samples produced the cleanest sample extract but only revealed one active peak from the HPLC fraction . Nuclear magnetic resonance analysis of purified samples indicated that the antifungal compound/s have aromatic complex and peptide structures. The extracted antifungal compounds were Protease K resistant and found to be thermostable at temperatures ranging 80-121oC, and, were active at pH ranges of 3-13. The antifungal compounds were found to exhibit similar properties to known antifungallipopeptides i.e. iturin A and fengycin A and B. Further characterization and identification of the active compounds is recommended usmg methods such as liquid chromatography mass spectrometer and matrix-assisted laser desorption ionisation time-of- flight. The results presented in this dissertation provide a basis from which antifungal compounds produced by strains ofBacillus can be further characterized.
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.
P15 trypanosome microtubule associated protein : structure/function analysis and vaccine development for the prevention of African sleeping sickness.
(2001) Rasooly, Reuven.; Balaban, Naomi.
Trypanosomes are hemoflagellated protozoan parasites causing chagas disease in South America, Leishmaniasis throughout the world, and African sleeping sickness in humans and nagana in animals in Africa. About 55 million people and 25 million cattle have been estimated to be at risk of contracting African sleeping sickness or nagana respectively. Once injected into the blood stream via the bite of a tsetse fly, the parasite evades the host's immune response by repeatedly changing its surface antigens, thus making the development of a vaccine seem impossible. Furthermore, chemotherapy existing today can be toxic, suggesting that novel methods to prevent diseases caused by trypanosomes are essential. All parasites of the Trypanosomatidae family contain unique microtubular structures called the subpellicular microtubules. Microtubules are made of tubulin and of microtubule associated proteins (MAPs). Unlike other microtubules, the subpellicular microtubules are crosslinked to one another and to the plasma membrane. The unique structure of the subpellicular microtubules has been attributed to unique trypanosome subpellicular MAPs which stabilize the microtubule polymers and crosslink them to one another. Three unique types of subpellicular MAPs have been identified: MARP, which is a high molecular mass MAP that stabilizes microtubules, p52 that is a 52kDa MAP which crosslinks microtubules, and pI5, which is a I5kDa protein which bundles microtubules. Because trypanosome MAPs have been shown to be unique to these parasites, these molecules could serve as useful target sites for therapy. In this study pI5 was cloned and sequenced and shown to contain highly organized, nearly identical tandem repeats with a periodicity of 10 amino acids, rich in positively charged and in hydrophobic amino acids. It was shown that pI5 can also bind phospholipids, suggesting that it may not only bundle the microtubule polymer through its positively charged amino acids but may also crosslink the microtubules to the plasma membrane through its hydrophobic regions, thus contributing to the stable structure of the subpellicular microtubules. To test for the efficiency of pI5 as a vaccine candidate, the recombinant pI5 was cloned into an adenovirus, which was used as a vaccine delivery system for pI5. Mice were vaccinated with the native purified pI5, with the expressed recombinant pI5 and with the adenovirus containing the recombinant pI5 gene (Ad-pI5). The results indicated that pI5 protected 100% of the animals vaccinated with the recombinant molecule (8/8), and 87% of the animals vaccinated with the native protein (7/S), while none of the control animals were protected. Animals that were vaccinated with the Ad-pI5 were protected but so were the control animals vaccinated with an adenovirus containing the lacZ gene. We have shown that vaccination with the adenovirus is associated with an elevated CDS+ T cell response which is known to be trypanostatic (S6), suggesting that animals vaccinated with Ad-pIS may have been protected not only by the specific anti-plS response but also by non specific immunity that was induced by the adenovirus itself. The source of the native and recombinant pI5 was from a different strain of T. brucei that was used for challenge. Since the subpellicular microtubules are common to all members of the Trypanosomatidae family, pI5 may ultimately serve as a common target for therapy to all types of diseases caused by trypanosomes.
Bioremediation of creosote-contaminated soil by microbial intervention..
(2002) Atagana, Harrison Ifeanyichukwu.; Haynes, Richard John.
No abstract available.
Cost-benefit analysis of the environmental impacts of Darvill Wastewater Works, Pietermaritzburg, KwaZulu-Natal.
(2002) Sikhakhane, Sindisiwe S.; Ahmed, Fethi B.; Darroch, Mark Andrew Gower.
Darvill Wastewater Works (DWWW) receives and treats both domestic and industrial wastewater from the city of Pietermaritzburg, in KwaZulu-Natal. Sludge from the wastewater treatment is sprayed onto surrounding lands, causing odour and fly problems. The plant also discharges treated effluent into the Msunduzi River, compromising water quality. This study uses several economic valuation techniques to estimate the value of the benefits of improving air and water quality to overcome these problems caused by DWWW. The benefits. are then compared with the costs of upgrading DWWW to see whether or not upgrading DWWW to improve air and water quality would be worthwhile. The Contingent Valuation Method (CVM) was used to elicit people's willingness to pay (WTP) for improvements in air quality due to the elimination of odours and flies caused by sludge deposited by DWWW. The WTP estimates reflect individual's preferences for improvements in air quality. The stated WTP amounts were positively related to household income, but negatively related to the age and gender of the respondent and the number of dependants in the household. The mean monthly WTP for the surveyed households is higher for those that are closer to the pollution source (R23.00 and R29.00 for Zones land 2) and less for those further away (RI4.00 for Zone 3). Sobantu residential area had the lowest mean monthly WTP (R18.00), followed by Lincoln Meade (R27.00) and Hayfields (R54.00). This is expected, as Sobantu has relatively high levels of unemployment and lower household incomes. Strategic, hypothetical and free rider bias may have led to the unexpected signs of some estimated regression coefficients in linear regression models used to estimate WTP. The mean WTP was estimated as R307.20 per annum per household, and when this is aggregated over the total population in the residential areas impacted by odours and flies (37192 households), the benefits of eliminating odours and flies are estimated as R11 425 382.00 per annum. A hedonic price method was used to quantify the decline in property values as a result of odours and flies caused by sludge deposited by DWWW. Properties experienced a R6650.08 decline in selling price if the distance from them to DWWW is decreased by one kilometre. Properties that are closer to DWWW were worth RI5 953.90 less than those further away from DWWW. Aggregating these values over all estimated impacted households in the study, gives an estimated benefit of improving air quality of R28 480 518.00 per annum. The impact of water pollution was quantified by estimating the revenue (R3 744 975.00) that would be lost by Pietermaritzburg if the Duzi Canoe Marathon were to be cancelled due to incidences of diarrheoa reported during the race. A cost of illness procedure was adopted to quantify the effect of water pollution on the health of communities that use the Msunduzi River as a source of potable water supply. A value of R1 243 372.50 was estimated as the annual cost of water-related illnesses in these rural areas. This value represents the costs of the river pollution to those communities. Both of these exercises indicated that improving water quality of the Msunduzi River would be beneficial to society. The effect of nutrient enrichment of the Msunduzi River was quantified by estimating the cost of removing water hyacinth from the Inanda Dam, treatment cost at Wiggins water treatment works and the value of recreation at Mahlabathini Park (Inanda Dam). The annual cost of removing water hyacinth was estimated from the direct costs of chemicals and labour as R47 202.15. The increased treatment costs at Wiggins attributable to DWWW were estimated as R1 104 999.20 and R956 924.15 per annum for removal of algae, and tastes and odours, respectively. The value of R706.90 per annum was estimated as the consumer surplus accruing to recreationists, and, therefore, the value of recreation at Mahlabathini Park to an individual. These annual benefits, when aggregated over the total study population (296 590) were over two hundred million rands (R209 659 470.00). The estimated total benefits (R256 662 840.00) of eliminating odours and flies and effluent problems were compared to the actual costs of two alternative methods of upgrading DWWW using cost-benefit analysis. These alternatives were co-disposal option (R170 473 320) and a land disposal option (R168 809377). Benefit-cost ratios of 1.51 and 1.52 suggest that from society's standpoint, it would be beneficial to upgrade the plant in order to eliminate its adverse environmental impacts. The study results have important implications for policy makers, both the DWWW management and the Pietermaritzburg-TLC municipality. At present DWWW is operating beyond its design capacity, and this problem, together with the poor status of Pietermaritzburg's reticulation system, causes overflow of untreated or compromised final effluent into the Msunduzi River during rainy seasons. These problems also impact on the efficient operation of the plant as the sludge is not properly digested before being sprayed onto surrounding land. Thus to prevent further environmental degradation, a fundamental basis of the National Environmental Management Act, DWWW would need to address these issues. Upgrading DWWW would be a short-term solution if the problems with the storm-runoff into the plant is not addressed.
Bioremediation of chemically contaminated soil : extraction/analysis methodology development.
(2002) Khan, Fatima.; Wallis, Frederick Michael.
The efficacies of soil extraction methods, namely, Soxhlet, sonication, agitation, alkaline digestion and the ethyl acetate micro-method, for monitoring soil bioremediation were evaluated using three soil types, Swartland, Rensburg and Hutton, encompassing the mineralogical range prevalent in Kwa Zulu Natal. Phenol, atrazine and the BTEX component of petrol were the molecules used in this study and were extracted under different spiking concentrations, after prolonged ageing times up to 21 days and after changing the composition of the spiking solution. It was concluded that extraction methods must be validated for the specific conditions under which they would be used, taking into consideration, soil type, spiking solutions, moisture content, weathering times and the analyte(s) in question. A preliminary appraisal of atrazine degradation in a Hutton soil was then made under the conditions of sterilized, fertilized/non-fertilized and non-sterilized, fertilized/nonfertilized soils. The predominant pathway of atrazine degradation was deemed to be chemically/abiotically mediated due to the soil pH and the presence of iron and aluminium oxides as well as the high levels of manganese in the soil. The results obtained prompted further study into atrazinecatabolism using soil-slurry reactors, under the conditions of carbon-limitation, nitrogen limitation, carbon/nitrogen non-limitation and carbon/nitrogen limitation. A comparison was made between inoculated and non-inoculated bioreactors. The ability of the indigenous microbial population to return the Hutton soil to its original pristine state was confirmed. The expense of inoculation and culture maintenance could be avoided since carbon and nitrogen supplementation would be as equally effective as inoculation.
Molecular and physiological characterization of thiosulphate-oxidizing microbial associations prior to use in hydrogen sulphide biofiltration.
(2000) Laughlin, Jamie B. A.; Senior, Eric.
Interacting microbial associations capable of utilizing thiosulphate as an energy source were enriched/isolated from activated sludge, landfill site [mal covering soil and soil from an acid mine water drainage site. The isolates were designated Lf-I, Ws-2 and Am-3, respectively. Although hydrogen sulphide was the target molecule for gas biofiltration, thiosulphate, which is a key oxidized intermediate, was used in this study due to the difficulty of working with a toxic gas. Together with thiosulphate oxidation, the microbial associations were assessed for their abilities to oxidize dissolved sulphide to elemental sulphur. Physiological analyses (temperature, pH and substrate concentration optimization) were made with closed and open cultures while morphological characterization and species compositional changes were monitored by light and scanning electron microscopy (SEM). To investigate further functional and structural responses to physiological changes, denaturing-gradient gel electrophoresis (DGGE) separation of PCR-amplified 16S DNA gene fragments and Biolog GN microtitre plates were used. The associations were found to be active metabolically between 0 and 35°C, 15 and 50°C, and 15 and 45°C, with optimum temperatures of 25, 40 and 35°C for Lf-l, Ws-2 and Am-3, respectively. The optimum pH range for microbial association Lf-l was between 3 and 4. The maximum specific growth rates of associations Lf-l , Ws-2 and Am-3 were 0.08, 0.06 and 0.03 h~l , respectively. Components of all three Gram negative rod-dominated associations were motile and displayed anaerobiosis. During open culture cultivation the species complement of Lf-l , as determined by morphological analysis, changed. The same association oxidized sulphide (40 ppm) to sulphur although Ws-2 and Am-3 did not have this capacity. Biolog GN plates detected pH-effected species compositional changes in Lf-l and these were confirmed by DGGE. The same technique showed that enrichment had occurred in the Biolog GN wells. Species composition changes also resulted in response to different pH values (2 to 9), temperatures (5 to 40°C) and dilution rates (0.003 to 0.09 h-1 ), but activity changes were not always accompanied by population profile changes.
The development of an in vitro system to assess the effect of arbuscular mycorrhizal fungi on cereal crops in KwaZulu-Natal, South Africa.
(2010) Govender, Avrashka.; Jamal-Ally, Sumaiya Faizal.
Cereal crops such as maize and sorghum are economically important in South Africa (SA) as a staple food diet. In order to meet the needs of South Africa’s growing population, higher yields in crop production need to be attained. However, the two major stress factors that affect yield production and require primary attention are nutrient deficiencies and pest infestations. Research is now being focused on certain endophytes that have become a valuable tool for agriculture as they protect crops against the above-mentioned stresses. The endophyte focused on in this study was Arbuscular Mycorrhizal fungi (AMF). This research was aimed at developing an in vitro culture system for SA cereal crops to enable interaction studies of endophytes. This dissertation is divided into two parts; the first part focused on the development of an in vitro culture system, the assessment of sorghum plant growth and exudate production in the presence of the Glomus intraradices strain. The results indicated that sorghum produces the required root exudates in the second stage of growth. Using high pressure liquid chromatography with mass spectrometry (HPLC/MS), it was noted that sorghum produced phytochemicals as chemoattractants for the respective endophytes. However, it was documented that when the plant underwent certain stresses they produced exudates, which acted as phytotoxic compounds that destroyed symbiotic organisms around sorghum rhizophere. The second part focused on optimization of the surface sterilization of maize seeds. The results indicated that maize contained unidentified endophytes, which negatively affected plant development. Surface sterilization of maize seeds was accomplished. The successful in vitro development can be used for future use to study plant development. Understanding plant development and interaction with symbiotic endophytes would not only be of great benefit but would also make it easier to create a biocontrol agent in vitro, which would bring about high crop yields at cost-effective prices and would be less labour intensive.
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.
Molecular characterisation and detection of xanthomonas albilineans, the sugarcane leaf scald pathogen.
(1994) Permaul, Kugenthiren.; Pillay, Balakrishna.; Pillay, D.
No abstract available.
Application of image analysis in microecophysiology research : methodology development.
(1998) Dudley, B. T.; Wallis, Frederick Michael.
Rehabilitation of landfill sites is important for successful land utilization. Revegetation is one key element of the process since it can overcome aesthetic problems. The inimical challenges of landfill leachate and gas are largely responsible for the difficulties associated with the revegetation of completed sites. Many components of landfill leachate can be catabolized by microbial associations thereby reducing their impacts on the environment. The importance of research on interactions between pollutants, microorganisms and soil is its applicability in environmental risk assessment and impact studies of organic pollutants which enter the soil either accidentally or intentionally. The application of image analysis with microscopy techniques to landfill soil-pollution interactions provides a means to study surface microbiology directly and to investigate microbial cells under highly controlled conditions. This research focused on the development of a method to study the real time processes of attachment, establishment, growth and division of microbial cells/associations in site covering soils. Image analysis provides a powerful tool for differential quantification of microbial number, identification of morphotypes and their respective responses to microenvironment changes. This minimal disturbance technique of examining visually complex images utilizes the spatial distributions and metabolic sensitivities of microbial species. It was, therefore, used to examine hexanoic acid catabolizing species, both free-living and in a biofilm, with respect to obviating the threat of hexanoic acid to reclamation strategies. The three sources of inoculum (soil cover, soil from the landfill base liner and municipal refuse) were compared for their ability to provide associations which catabolized the substrate rapidly. During the enrichment programme the inocula were challenged with different concentrations of hexanoic acid, a common landfill intermediate. From the rates at which the substrate was catabolized conclusions were drawn on which concentration of hexanoate facilitated the fastest enrichment. The results of initial batch culture enrichments confirmed that the soil used contained microbial associations capable of catabolizing hexanoic acid at concentrations < 50mM, a key leachate component. Exposing the landfill top soil microorganisms to a progressive increase in hexanoic acid concentration ensured that catabolic populations developed which, in situ, should reduce the phytotoxic threat to plants subsequently grown on the landfill cover. The analysis of surface colonization was simplified by examining the initial growth on newly-exposed surfaces. The microbial associations generated complex images which were visually difficult to quantify. Nevertheless, the dimensional and morphological exclusions which were incorporated in the image analysis software permitted the quantification of selected components of the associations although morphology alone was inadequate to confirm identification. The effects of increasing the dilution rate and substrate concentration on the growth of surface-attached associations in Continuous Culture Microscopy Units (CCMUs) were examined. Of the five dilution rates examined the most extensive biofilm development (9.88 jum2) during the selected time period (72h) resulted at a dilution rate of 0.5h' (at 10mM hexanoic acid). The highest growth (608 microorganisms.field"1) was recorded in the presence of 50mM hexanoic acid (D = 0.5h"1). To ensure that the different morphotypes of the associations were able to multiply under the defined conditions a detailed investigation of the component morphotypes was made. Numerically, after 60h of open culture cultivation in the presence of 50mM hexanoic acid, rods were the predominant bacterial morphotypes (43.74 field'1) in the biofilms. Both rods and cocci were distributed throughout the CCMUs whereas the less numerous fungal hyphae (0.25 field'1) were concentrated near the effluent port. The specific growth rates of the surface-attached associations and the component morphotypes were determined by area (//m2) colonized and number of microorganisms.field"' and compared to aerobic planktonic landfill associations. From area determinations ( > 0.16 h'1) and the number of microorganisms.field"1 10mM hexanoic acid was found to support the highest specific growth rate ( > 0.05 h"1) of the surfaceattached association isolated from municipal refuse. With optical density determinations, the highest specific growth rate (0.01 h'1) was recorded with 25mM hexanoic acid. The surface-attached microbial associations component species determinations by area and number showed that the hyphae had the highest specific growth rate ( > 0.11 h"1). The surface-attached microbial association specific growth rate determinations from the discriminated phase (0.023 h'1), area colonized (0.023 h"1) and number of microorganisms (0.027 h"1) calculated from the results of the component species rather than the association should give more accurate results. The specific growth rate obtained differed depending on the method of determination. Any one of these may be the "correct" answer under the cultivation conditions. Depending on the state (thickness) of the association (free-living, monolayer or thick biofilm) the different monitoring methods may be employed to determine the growth. As a consequence of the results of this study, the kinetics of microbial colonization of surfaces in situ may be subjected to the same degree of mathematical analysis as the kinetics of homogeneous cultures. This type of analysis is needed if quantitative studies of microbial growth are to be extended to surfaces in various natural and artificial environments.
Installation, commissioning and preliminary microbiological and operational investigations of full-scale septic tank digestion of sewage.
(1997) Taylor, Michelle Anne.; Senior, Eric.
This study investigated the commissioning and maintenance of a Pennells two-tank bioreactor system with specific reference to its application in rural areas of KwaZulu- Natal, South Africa to treat sewage and generate biogas. The septic tank configuration was installed in a community which lacked electricity and domestic waste disposal. An artificial wetland was constructed at the outlet of the system to facilitate further treatment. Inefficient operation and maintenance of the system occurred due to various social/community-related problems which are typical of a field- and community-based project of this nature in a rural region of a Third World African country. These problems affected both maintenance and digester performance. The Pennells system was characterized by incomplete anaerobiosis which limited methanogenesis. Despite this, and attendant problems of low temperatures and elevated pH values, COD removal resulted. Laboratory-scale batch cultures, in conjunction with fluorescence and scanning electron microscopy, were used to identify a suitable anaerobic digester sludge for inoculation purposes. Perturbation experiments with locally used detergents and toxic compounds demonstrated the inimical effects of these agents. In contrast, low concentrations of penicillin and tetracycline promoted methanogenesis. Further analysis with light, fluorescence and scanning electron microscopy identified the acidogens as the predominant bacterial species, whilst fluorescence microscopy confirmed the absence of methanogens in the bioreactor.
Exploitation of indigenous fungi in low-cost ex situ attenuation of oil- contaminated soil.
(1997) McGugan, Brandon Ross.; Senior, Eric.
The central aim of this study was to determine if indigenous fungi of an oil-contaminated soil could be effectively used in a low-cost bioremediation of the soil. Since some of the contaminant had been present at the site for over two decades, the indigenous microbial species had been subjected to specific selection pressures for a protracted period, thus facilitating key enzymatic capabilities for hydrocarbon degradation. Analysis of the pertinent influential parameters of soil bioremediation indicated that an ex situ technique, utilising the catabolic activities of the indigenous soil fungi, was a feasible low-cost option. Fungi were isolated from the contaminated soil through a variety of techniques. The abilities of these isolates to degrade the contaminant oil and a range of representative hydrocarbon molecules was evaluated by a systematic screening programme. Sixty-two isolates were initially examined for their growth potential on hydrocarbon-supplemented agar. A bioassay, utilising hydrocarbon-impregnated filter paper discs, was then used to examine the abilities of 17 selected isolates to catabolise three representative hydrocarbon molecules (hexadecane, phenanthrene and pristane) in different concentrations. In the same bioassay, the influence of a co-metabolite (glucose) on growth potential was also examined. Eight fungal species: Trichophyton sp.; Mucor sp.; Penicillium sp.; Graphium sp.; Acremoniwn sp.; Chaetomium sp.; Chrysosporium sp.; and an unidentified basidiomycete were then selected. Liquid batch cultures with a hydrocarbon mixture of hexadecane, phenanthrene, pristane and naphthalene facilitated quantitative analysis (HPLC) of the hydrocarbon catabolic abilities of the selected isolates. Ex situ bioremediation was evaluated at laboratory-scale by both bioaugmentation and biostimulation in soil microcosm trials. During the course of the study, total petroleum hydrocarbon (TPH) concentration (U.S. EPA Method 418.1) was used as a simple and inexpensive parameter to monitor hydrocarbon disappearance in response to soil treatments. Soil microbial activities were estimated by use of a fluorescein diacetate hydrolysis bioassay. This was found to be a reliable and sensitive method to measure the activity of respiring heterotrophs as compared with the unreliable data provided by plate counts. In the bioaugmentation trial, the eight selected isolates were individually used to inoculate (30% v/v) the contaminated soil. The highest rate of biodegradation (50.5% > than the non-sterile control) was effected by an Acremonium species after 50 days incubation (25°C). The second highest rate of biodegradation (47% > than the non-sterile control) was achieved with a soil treatment of sterile barley/beer waste only. Comparable rates of hydrocarbon degradation were achieved in simple biostimulation trials. Thus, due to its lower cost, biostimulation was the preferred remediation strategy and was selected for further laboratory investigation. Common agricultural or industrial lignocellulosic wastes such as: wood chips; straw; manure; beer brewery waste; mushroom compost; and spent mushroom substrate were used as soil treatments, either alone or in combination. The effect of the addition of a standard agricultural fertiliser was also examined. The highest level of biodegradation (54.4% > the non-sterile control) was recorded in a microcosm supplemented (40% v/v) with chicken manure. Finally, an ex situ bioremediation technique was examined in a pilot-scale field trial. Wood chips and chicken manure were co-composted with the contaminated soil in a low-cost, low-maintenance bioremediation system know as passive thermal bio venting. Extensive monitoring of the thermal environment within the biopile was made as an indirect measure of microbial activity. These data were then used to optimise the composting process. Three-dimensional graphical representations of the internal temperatures, in time and space, were constructed. From these graphs, it was determined that an inner core region of approximately 500 cm3 provided a realistic simulation of conditions within a full-scale biopile. During this trial a TPH reduction of 68% was achieved in 130 days. The findings of this research suggested that the utilisation of fungal catabolism is applicable to soils contaminated with a wide range of hydrocarbon contaminants. Passive thermal bioventing offers a bioremediation strategy which is highly suitable for South African conditions in terms of its low level of technological sophistication, low maintenance design and, most importantly, its relatively low cost.

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