Dissertations / Theses on the topic 'Environmental aspects of Microbial biotechnology'

Thesis (MSc)--University of Stellenbosch, 2003.
ENGLISH ABSTRACT: Biofouling of water systems is a problem extensively experienced in industry. Although this subject is the focus of many studies, the ability of microorganisms to survive exposure to biocides is still poorly understood. This study aimed to assess the biocidal effect of ozone on planktonic cells and biofilm communities, to evaluate different ozone generation techniques, and to follow population shifts within the biofilm community. Specific objectives included determining the effect of different ozone concentrations, the effect of different exposure times, and an assessment of microbial responses after exposure to sub-lethal ozone concentrations. Typically, 300 ml of an ovemight bacterial culture was exposed to ozone that was generated by anodic oxidation (0.3% wt or 18- 20% wt, respectively) or silent electric discharge (3.5% wt 03). The ozone was purged into the culture for 5-, 7-, 10- and 15 min., respectively. Enumeration of cells following ~10 min. exposure to 18-20% wt ozone showed a significant reduction in viable cell numbers. In contrast, when exposed to the two lower 03 concentrations, there was little change in the viable cell numbers even after prolonged exposure (30- and 60 min.). To evaluate biofilms, ozone was bubbled into the irrigation that was pumped through replicate flow cell channels. Response to ozone exposure was evaluated after staining the biofilms with the Baclight Viability probe, observation with fluorescence microscopy, and image analysis. The higher ozone concentration (18-20% wt 03) more effectively disrupted the biofilm structure of denser biofilms than the lower concentration, especially after 90 min. exposure. When compared to the controls, the 90 min. exposure resulted in a notable reduction in viable cells from 69% to 38% and a corresponding increase in nonviable cells from 29% to 62%. The lower concentration ozone (3.5% wt 03) was effective against the less dense, thinner biofilms evaluated, but not effective against the thicker biofilm. An analysis of the differences between continuous culture biofilms and batch culture biofilms showed that the biofilms in the batch system were less rigid. To evaluate microbial response to biocides, techniques such as Biolog whole-community metabolic profiles and terminal restriction fragment length polymorphisms (T-RFLP) were used. Biolog analysis of planktonic cells revealed changes following exposure to sub-lethal biocide concentrations, however carbon utilisation profiles resembled that of the controls after 24-48 hours. For biofilm communities, no carbon utilization differences could be detected under these conditions. There was, however differences in T-RFLP patterns between treated and untreated biofilm communities.
AFRIKAANSE OPSOMMING: Biobevuiling van watersisteme is 'n probleem wat algemeen in industriëe ervaar word. Alhoewel hierdie onderwerp die fokus van vele studies is, word die vermoëns van mikroorganismes om blootstelling aan biosiede te weerstaan steeds swak verstaan. Die doel van hierdie studie was om die biosidiese effek van osoon op planktoniese selle en biofilm gemeenskappe waar te neem, om die verskillende osoon generasie tegnieke te evalueer, asook om verskuiwings in die samestelling van die biofilm gemeenskap waar te neem. Spesifieke doelwitte sluit in die bepaling van die effek van verskillende osoon konsentrasies, die blootstellingtye, en 'n waarneming van mikrobiese reaksies na blootstelling aan sub-dodings osoon konsentrasies. Drie honderd ml van 'n oornag bakteriese kultuur was aan osoon, wat deur anodiese oksidasie (0.3% wt of 18% - 20% wt) of geluidlose elektriese ontlading (3.5% wt), gegenereer is, blootgestel. Tye van blootstelling was 5-, 7-, 10-, of 15 min., onderskeidelik. Bepaling van selgetalle na :2:10 min. blootstelling aan 18 - 20% wt osoon, het 'n betekenisvolle verlaging in die getal lewensvatbare mikrobeselle getoon. In teenstelling hiermee, het blootstelling aan twee laer osoon konsentrasies min verskil in die lewensvatbare selgetalle, selfs na verlengde blootstellingstye (30- en 60 min.), getoon. Om biofilms te evalueer is osoon in die medium geborrel wat deur replikaat vloeisel kanale gepomp is. Na osoon blootstelling, was die vloeisel onderwerp aan beeld analise deur gebruik te maak van die Baclight lewensvatbare peiler en fluoressensie mikroskopie. Die hoër osoon konsentrasie (18 - 20% wt 03) het die struktuur van dikker biofilms meer effektiefuiteengeskeur as die laer konsentrasie, veral na 90 min. blootstelling. In vergelyking met die onderskeie kontroles, het die getalle van lewensvatbare selle na 90 min. blootstelling drasties verlaag vanaf 69% tot 38% en 'n ooreenstemmende toename in die nie-lewensvatbare selgetalle vanaf 29% tot 62%. Die laer osoon konsentrasie (3.5% wt 03) was meer effektief teenoor die minder digte en dunner biofilms wat ge-evalueer was, maar nie so effektief teenoor die dikker biofilms nie. 'n Analise van die verskille tussen kontinue-kultuur biofilms en lotkultuur biofilms het getoon dat die lot-kultuur biofilms minder rigied is. Vir die evaluering van mikrobiese reaksies na biosied blootstelling, is tegnieke soos Biolog gemeenskap metaboliese profiele en eind-restriksie-fragment-lengte polimorfisme (TRFLP) gebruik. Biolog analise van planktoniese selle het verskille getoon na blootstelling aan sub-dodelike biosied konsentrasies. Koolstof benutting het wel na 24 - 48 ure met dit van die kontrole ooreengestem. Vir biofilm gemeenskappe was daar geen noemenswaardige verskille in koolstof benutting nie. Daar was wel verskille in T-RFLP patrone tussen die onbehandelde en biosied-behandelde biofilm gemeenskappe.

Since the 1900’s it has been known that microorganisms are capable of generating electrical power through extracellular electron transfer by converting the energy found organic compounds (Potter, 1911). Microbial fuel cells (MFCs) has garnered more attention recently, and have shown promise in several applications, including wastewater treatment (Yakar et al., 2018), bioremediation (Rosenbaum & Franks, 2014), biosensors (ElMekawy et al., 2018) desalination (Zhang et al., 2018) and as an alternative renewable energy source in remote areas (Castro et al., 2014). In MFCs catalytic reactions of microorganisms oxidize an electron donor through extracellular electron transfer to the anode, under anaerobic conditions, with the cathode exposed to an electron acceptor, facilitating an electrical current (Zhuwei, Haoran & Tingyue, 2007; Lovley, 2006). For energy production in remote areas a low cost and easily accessible feed stock is required for the MFCs. Sweet sorghum is a drought tolerant feedstock with high biomass and sugar yields, good water-use efficiency, established production systems and the potential for genetic improvements. Because of these advantages sweet sorghum stalks were proposed as an attractive feedstock (Rooney et al., 2010; Matsakas & Christakopoulos, 2013). Dried sweet sorghum stalks were, therefore, tested as a raw material for power generation in a MFC, with anaerobic sludge from a biogas plant as inoculum (Sjöblom et al., 2017a). Using sorghum stalks the maximum voltage obtained was 546±10 mV, the maximum power and current density of 131±8 mW/m2 and 543±29 mA/m2 respectively and the coulombic efficiency was 2.2±0.5%. The Ohmic resistances were dominant, at an internal resistance of 182±17 Ω, calculated from polarization data. Furthermore, hydrolysis of the dried sorghum stalks did not improve the performance of the MFC but slightly increased the total energy per gram of substrate. During the MFC operation, the sugars were quickly fermented to formate, acetate, butyrate, lactate and propionate with acetate and butyrate being the key acids during electricity generation. Efficient electron transfer between the microorganisms and the electrodes is an essential aspect of bio-electrochemical systems such as microbial fuel cells. In order to design more efficient reactors and to modify microorganisms, for enhanced electricity production, understanding the mechanisms and dynamics of the electron transport chain is important. It has been found that outer membrane C-type cytochromes (OMCs) (including omcS and omcZ discussed in this study) play a key role in the electron transport chain of Geobacter sulfurreducens, a well-known, biofilm forming, electro-active microorganism  (Millo et al., 2011; Lovley, 2008). It was found that Raman microscopy is capable of providing biochemical information, i.e., the redox state of c-type cytochromes (cyt-C) without damaging the microbial biofilm, allowing for in-situ observation. Raman microscopy was used to observe the oxidation state of OMCs in a suspended culture, as well as in a biofilm of an MFC. First, the oxidation state of the OMCs of suspended cultures from three G. sulfurreducens strains (PCA, KN400 and ΔpilA) was analyzed. It was found that the oxidation state can also be used as an indicator of the metabolic state of the cells, and it was confirmed that PilA, a structural pilin protein essential for long range electron transfer, is not required for external electron transfer. Furthermore, we designed a continuous, anaerobic MFC enabling in-situ Raman measurements of G. sulfurreducens biofilms during electricity generation, while poised using a potentiostat, in order to monitor and characterize the biofilm. Two strains were used, a wild strain, PCA, and a mutant, ΔOmcS. The cytochrome redox state, observed through the Raman spectra, could be altered by applying different poise voltages to the electrodes. This change was indirectly proportional to the modulation of current transferred from the cytochromes to the electrode. This change in Raman peak area was reproducible and reversible, indicating that the system could be used, in-situ, to analyze the oxidation state of proteins responsible for the electron transfer process and the kinetics thereof.

Master of Science
Department of Civil Engineering
Prathap Parameswaran
The ever-increasing demand for water, food, and energy and the simultaneous diminishment of our planets’ ecosystems wrought by humans have prompted a more sustainable approach to engineering the built environment. Wastewater treatment systems stand at the interface that connects the built and natural environment where potential solutions for resource and environmental issues exist. Wastewater treatment technologies can address issues involving water, food, energy, and environmental regulation when resources are properly captured from the wastewater while it’s being treated. This way of thought allows wastewater to be perceived as a source of valuable products rather than an obligate waste stream. For this reason, anaerobic wastewater treatment is progressively being considered because of its ability to improve energy and resource recovery, while reducing costs and environmental impacts associated with conventional domestic wastewater treatment. More specifically, anaerobic membrane bioreactors (AnMBRs) hold promise to effectively treat wastewater at low temperatures with low energy and nutrient requirements, low sludge production, while having the benefit of generating methane-rich biogas suitable as an energy source and the potential to capture nutrients used to fertilize cropland. But, at low temperatures the microbial communities that control anaerobic digestion (AD) face biochemical obstacles. Elucidating the microbial community dynamics within AnMBRs with respect to seasonal temperatures will give insight on how to efficiently operate AnMBRs with the goal of energy-neutral wastewater treatment. DNA based tools such as advanced high-throughput sequencing was coupled with AnMBR process data to explicate the mechanism of methane production in the suspended biomass of an AnMBR from a mesophilic startup leading into psychrophilic conditions, and then returning to mesophilic temperatures.

This is an exploration of the potential for a citizen science project, with the goal to get the general public involved in microbial soil biodiversity around Uppsala, Sweden. Biodiversity serves an important role in how an ecosystem performs and functions. A large part of Earth's biodiversity exists below ground in soil, where microorganisms interact with plants. It would be beneficial to analyse the abundance and spread of some microorganisms in order to gain a better understanding of soil biodiversity. We suggest that one species family to study could be Phytophthora. Phytophthora is a genus of oomycetes that often are pathogenic, causing disease in various trees and other plants. It is unknown exactly how widespread the genus is today, making it extra interesting for the proposed study. For the general public to be able to do this a device needs to be developed that is easy to use and preferably could be used directly in the field. An isothermal amplification method is suitable for identifying the microorganism under these conditions. Many isothermal amplification methods are expensive, perhaps too expensive for a citizen science study, but have great potential for easy field testing. We propose a device utilizing RPA and lateral flow strips. RPA - Recombinase Polymerase Amplification is a method for amplification that might be suitable since it is simple, sensitive, and has a short run time. It is however expensive, which is an issue, but isothermal amplifications are expensive across the board. Lateral flow strips can be used to visualize the results. They utilize antibodies to detect the previously amplified amplicons, and give a positive or negative test answer that would be understandable to even untrained study participants. One of the biggest obstacles identified in this project concerns amplifying DNA from a soil sample, because an extraction step is necessary. The methods we have identified for extraction are not performable in the field, since they require centrifugation. In the proposition for a device a possible work-around for this is proposed, but since it has yet to be tested it is not yet known whether it will work or not.

The development of coal biotechnology, using hard coal as a substrate, has been impeded by its low reactivity in biological processes. As a result, the more successful application studies have focused on lignitic soft coals. However, new studies have reported using biologically or geologically oxidized hard coal as a functional substrate option for bioprocess applications on a large scale. This study undertook a preliminary investigation into the feasibility of environmental applications of coal biotechnology using oxidized hard coal substrates in both anaerobic and aerobic processes with carbon dioxide, sulfate and oxygen as terminal electron acceptors. A preliminary characterization of the oxidized hard coal substrates was undertaken to determine and predict their viability and behavior as electron donors and carbon sources for environmental bioprocess applications of direct interest to the coal mining industry. Both biologically and geologically oxidized coal substrates showed loss of up to 17% and 52% carbon respectively and incorporation of oxygen ranging from 0.9 – 24%. The latter substrate showed greater loss of carbon and increased oxygenation. The biologically and geologically oxidized hard coal substrates were shown to partition readily into 23% and 32% organic humic acid, a 0.1% fulvic acid fraction and 65% and 59% inorganic and humin fractions respectively. These organic components were shown to be potentially available for biological consumption. In the unmodified hard coal substrate, partitioning was not observed and it did not perform as a functional substrate for any of the bioprocesses investigated. Where carbon dioxide was used as a terminal electron acceptor, methane production ranging from 9 – 26 mg CH4.g substrate-1 was demonstrated from both oxidized coal substrates. Geologically oxidized coal produced 30% more methane than biologically oxidized coal. Methane yields from the geologically oxidized coal in the presence and absence of a co-substrate were 5 – 13-fold higher than previous studies that used hard coal for methanogenesis. Based on these results, and that the development and optimization of the biological oxidation process is currently ongoing, further applications investigated in this study were undertaken using geologically oxidized coal. It was shown using pyrolysis gas chromatography mass spectrometry that the methanogenic system was dependent on the presence of an effective co-substrate supporting the breakdown of the complex organic structures within the oxidized hard coal substrate. Also that the accumulation of aromatic intermediate breakdown compounds predominantly including toluene, furfural, styrene and 2-methoxy vinyl phenol appeared to become inhibitory to both methanogenic and sulfidogenic reactions. This was shown to be a more likely cause of reactor failure rather than substrate exhaustion over time. Evidence of a reductive degradation pathway of the complex organic structures within the oxidized hard coal substrates was shown through the production, accumulation and utilization of volatile fatty acids including acetic, formic, propionic, butyric and valeric acids. Comparative analysis of the volatile fatty acids produced in this system showed that geologically oxidized coal produced 20% more of the volatile fatty acids profiled and double the total concentration compared to the biologically oxidized coal. The use of geologically oxidized hard coal as a functional substrate for biological sulfate reduction was demonstrated in the neutralization of a simulated acid mine drainage wastewater in both batch and continuous process operations. Results showed an increase in pH from pH 4.0 to ~ pH 8.0 with sulfide production rates of ~ 86 mgL-1.day-1 in the batch reactions, while the pH increased to pH 9.0 and sulfide production rates of up to 450 mgL-1.day-1 were measured in the continuous process studies using sand and coal up-flow packed bed reactors. Again, the requirement for an effective co-substrate was demonstrated with lactate shown to function as a true co-substrate in this system. However, a low cost alternative to lactate would need to emerge if the process was to function in large-scale commercial environmental treatment applications. In this regard, the aerobic growth and production of Neosartorya fischeri biomass (0.64 g.biomass.g SOC-1) was demonstrated using oxidized hard coal and glutamate as a co-substrate. Both can be produced from wastes generated on coal mines, with the fungal biomass generated in potentially large volumes. Preliminary demonstration of the use of the fungal biomass as a carbon and electron donor source for biological sulfate reduction was shown and thus that this could serve as an effective substrate for anaerobic environmental treatment processes. Based on these findings, an Integrated Coal Bioprocess model was proposed using oxidized hard coal as a substrate for environmental remediation applications on coal mines. In this approach, potential applications included methane recovery from waste coal, use of waste coal in the treatment of acid mine drainage waste waters and the recovery and use of humic acids in the rehabilitation of open cast mining soils. This study provided a first report demonstrating the use of biologically and geologically oxidized hard coals as bioprocess substrates in environmental bioremediation applications. It also provided an indication that follow-up bioengineering studies to investigate scaled-up applications of these findings would be warranted.

A polyphasic approach was used to examine the impact of contamination on soil microbial community structure. Two systems were examined using a combined biochemical and molecular biological approach. Petroleum hydrocarbon contaminated soils from two Northern Canadian sites, representing long-term contamination, were examined using Biolog GN plates and PCR-denaturing gradient gel electrophoresis (DGGE) analysis of total community 16S rDNA. Results obtained using both methods demonstrated a positive correlation between samples that was based on the geographical origin of the samples, not on contamination level. In the second system, non-contaminated soil was contaminated with the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) to monitor the effect of short- to medium-term contamination. Changes in the soil microbial community were examined using PCR-DGGE of total community 16S rDNA combined with RDX mineralization and chemical analysis of intermediates. The non-contaminated loam soil had an inherent RDX degradative capability and contamination of soil columns with 1000 mg RDX/kg soil did not significantly change the 16S rDNA bacterial community profile. The bacterial diversity remained high as estimated by the number of bands present in the DGGE and by NQ-78704 statistical rarefaction analysis of 16S rDNA clone RFLPs. The same soil, used in 10% soil slurries (w/v), demonstrated two apparently different RDX degradation mechanisms based on mineralization and chemical analysis. The differences were based on aerobic versus anaerobic conditions and the presence/absence of Na3 citrate. PCR-DGGE performed on 16S rDNA from aerobic slurries amended with Na3-citrate detected the stimulation of 3 operational taxonomic units, identified as Stenotrophomonas sp., Sphingomonas sp. and a member of the Alcaligenaceae. The results from the two systems examined (short- to medium-term and long-term contamination) demonstrated the utility of a polyphasic approach in the examina

A study of the microbial diversity in sediments of the Great Berg River estuary is carried out using modern molecular phylogenetic methods. The aim of this study was to determine the effect of (pollution by) the effluents of the fish industry on the composition of the microbial community in the sediments. The diversity in microbial groups of sediment samples that received wastewater from the local fishing industry was investigated by a PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) approach and compared to an unaffected site.

In an increasingly energy-hungry world, our capacity to meet the heightened energy demands of the future has become a pressing matter. The most urgent of these concerns are tied to the accessibility of petroleum. Various experts have proselytized both the imminent arrival of peak oil production rates and the ensuing decline of those rates thereafter. And to that end, the development of novel and advanced oil exploration methodologies has become almost as important as finding the sources of oil themselves. The soils above petroleum reservoirs play host to various communities of alkane- oxidizing bacteria that can utilize the natural gas emitted by the reservoirs as a source of carbon and energy. While methane can originate from non-petroleum sources, the only natural sources of propane and butane are oil and gas fields. The increased presence of propane and butane-oxidizing bacteria in a given soil sample is used by oil prospectors as an accurate indicator of a proximal petroleum reservoirs. For over a century, cell counts and hydrocarbon metabolic rates have been the metrics used to determine the presence of hydrocarbon-oxidizing microbes. These methods require weeks to complete. Here, we have developed a set of DNA primers for a much more rapid detection of hydrocarbon-oxidizing microbes through PCR amplification - for the chief purpose of petroleum exploration. Each primer’s design is based on a nucleotide sequence alignment of seven prmA and bmoX genes from seven organisms, which encode the large hydroxylase subunit of propane monooxygenase and the alpha hydroxylase subunit of butane monooxygenase respectively. These monooxygenases are the enzymes responsible for the initiation of propane and butane catabolism. Optimization of PCR with this primer set was accomplished using DNA extracted from known butane and propane oxidizers as positive controls, and methane and toluene oxidizers as negative controls. PCR products recovered from cultures of butane-oxidizing and propane-oxidizing bacteria, and soil samples, were sequenced. Phylogenetic trees were constructed from the sequencing data to confirm the accuracy of amplification. We demonstrate the use of PCR and agarose gel electrophoresis to detect hydrocarbon-oxidizing bacteria in culture and in complex microbial soil communities. Detection limits were elucidated through two different experiments. Potential avenues of advancements include narrowing specificity by selectively removing primer degeneracies, the use of additional positive and negative controls and the adaptation of the primers to a qPCR TaqMan assay.

D-amino acids (D-AAs) are the α-carbon enantiomers of L-amino acids (L- AAs), the building blocks of proteins in known organisms. It was largely believed that D-AAs are unnatural and must be toxic to most organisms, as they would compete with the L-counterparts for protein synthesis. Recently, new methods have been developed that allow scientists to chromatographically separate the two AA stereoisomers. Since that time, it has been discovered that D-AAs are vital molecules and they have been detected in many organisms. The work of this dissertation focuses on their place in bacterial metabolism. This specific area was selected due to the abundance of D-AAs in bacteria-rich environments and the knowledge of their part in several processes, such as peptidoglycan synthesis, biofilm disassembly, and sporulation. We focused on the bacterium Pseudomonas putida KT2440 which inhabits the densely populated plant rhizosphere. Due to its versatility and cosmopolitan character, this bacterium has provided an excellent system to study D-AA metabolism. In the first chapter, we have developed a new approach to identify specific genes encoding enzymes acting on D-AAs, collectively known as amino acid racemases. Using this novel method, we identified three amino acid racemases encoded by the genome of P. putida KT2440. All of the enzymes were subsequently cloned and purified to homogeneity, followed by a complete biochemical characterization. The aim of the second chapter was to understand the specific role of the peculiar broad-spectrum amino acid racemase Alr identified in chapter one. After constructing a markerless deletion of the cognate gene, we conducted a variety of phenotypic assays that led to a model for a novel catabolic pathway that involves D-ornithine as an intermediate. The work in chapter three identifies for the first time numerous rhizosphere-dwelling bacteria capable of catabolizing D-AAs. Overall, the work in this dissertation contributes a novel understanding of D-AA catabolism in bacteria and aims to stimulate future efforts in this research area.

This study was done to determine the effectiveness of a commercially available bioaugmentation product, BiOWiSHTM-Aqua FOG, for remediating petroleum-contaminated sandy soil. Biodegradation enhancement by BiOWiSHTM-Aqua FOG was evaluated in laboratory microcosms by directly measuring total petroleum hydrocarbon (TPH) and indirectly using respirometry. Attempts were made to enrich hydrocarbon-degrading bacteria in BiOWiSHTM-Aqua FOG, and the resulting enrichment cultures were screened using respirometry as well. Potential hydrocarbon-degrading bacteria in BiOWiSHTM-Aqua FOG were isolated. Experiments were performed at bench-scale using microcosm bottles containing sand contaminated with either motor oil or No. 2 diesel fuel. The microcosms were incubated at 25oC under aerobic conditions. TPH measurements of soil in the microcosms at 0, 25 and 56 days indicated that the addition of 500-ppm BiOWiSHTM-Aqua FOG improved biodegradation of the motor oil-contaminated soil by 45%. However, BiOWiSHTM-Aqua FOG did not have a measurable effect on biodegradation in the diesel-contaminated soil. In the respirometry experiments, BiOWiSHTM-Aqua FOG and two hydrocarbon-enriched BiOWiSHTM-Aqua FOG cultures were evaluated indirectly by the measurement of microbial carbon dioxide production and oxygen uptake using a MicroOxymaxTM respirometer. The respirometry experiments showed that in the six-day period following motor oil-contamination of soil, the addition of BiOWiSHTM-Aqua FOG substantially improves biodegradation rates. The added organisms in the product out-performed the indigenous organisms in the 5-6 days following contamination of the soil. The CO2 production observed in the BiOWiSHTM microcosms contaminated with motor oil was much greater than CO2 production without motor oil, which confirms that the observed metabolism can be attributed to motor oil biodegradation rather than metabolism of other organic material in the soil. Enriched consortia consistently generated far less CO2 than microcosms with the 500 ppm BiOWiSHTM-Aqua FOG. Stoichiometric calculations suggested that BiOWiSHTM-Aqua FOG removed approximately 1400 ppm TPH (14%) from the soil in 6.5 days, while an enrichment culture of BiOWiSHTM-Aqua FOG only reduced TPH levels by 459 ppm (5%). This result suggests that increased biodegradation rate in bioaugmented soil is aided by biodiversity in the augmenting inoculum. A potential hydrocarbon-degrading candidate organism was isolated from the product and cultured on Bushnell-Haas agar and plate-count agar (PCA). While at least two distinct colony types were successfully grown on media with motor oil, these same colonies appeared on Bushnell-Haas agar with no apparent carbon source, and survived repeated transfers onto this same medium. Therefore, their status as hydrocarbon-degraders is inconclusive. More thorough enrichment work could be pursued, especially using soil samples collected from petroleum-contaminated sites.

The objective of this thesis was to investigate BiOWiSHTM-Aqua, a commercial dry solid formulation containing a consortium of bacteria and yeast, as a biopesticide for treatment of Botrytis cinerea, a gray mold that affects strawberries. BiOWiSHTM-Aqua was compared with another commercial product specifically used as a fungicide and bacteriocide, Serenade® Garden Disease Control Spray (concentrated Bacillus subtilis strain QST 713). Both laboratory tests as well as in vivo lab tests were conducted. BiOWiSHTM-Aqua results varied widely from plate to plate, regardless of experimental conditions. In some of these plates, inhibition zones were observed around colonies from BiOWiSHTM-Aqua, indicating efficacy. The organism responsible for the inhibition zones of B. cinerea growth was isolated from BiOWiSHTM-Aqua, and 16s rRNA analysis identified this culture as a strain of B. subtilis. This strain was designated as B. subtilis ssp. KLB. The B. subtilis KLB concentration required to completely inhibit B. cinerea was 9.1x104 CFU/mL when B. subtilis KLB was inoculated 48 hours before B. cinerea, 1.3x105 CFU/mL at 24 hours, and 3.2x106 CFU/mL when both were inoculated at the same time. Various preliminary experiments using B. subtilis KLB were also conducted to investigate its economic feasibility, to characterize the organism, and to test its post-harvest in vivo viability. B. subtilis KLB cell concentration was 1.6x109 CFU/mL in a bioreactor with LB at the end of the log growth phase. B. subtilis KLB achieved cell concentrations as high as 5x109 CFU/mL in shake flasks with food-grade tapioca as a carbon source. Inoculation of B. subtilis KLB on post-harvest strawberries did not have an effect on Botrytis infection rates compared to the negative control. These various experiments were the first step in research to potentially produce B. subtilis KLB on a commercial scale.

Thesis (MTech (Environmental Health))--Cape Peninsula University of Technology, 2017.
Surface water is used as a source of water supply in many countries, including South Africa. One of the sources of surface water pollution is leachate and surface runoff from landfills. In agricultural soils, the landfill runoff and leachate deteriorate the quality and affect the fertility of soil. The entry of metals and microorganisms from landfill leachate to adjacent environments is through surface runoff due to rainfall. Adverse effects on human- and environmental health triggers a need to monitor and control contaminants in the environment. The aims of the study are to determine the effect of landfill runoff and leachate on agricultural soil and river water (Veldwachters River) running adjacent to the Devon Valley landfill site and to identify potential metal-tolerant organisms in environmental samples collected in Stellenbosch, Western Cape, South Africa. Samples (agricultural soil, river water and sediments) were collected once a month for a period of six months from the study area for analysis. Physicochemical parameters that are known to have major effects on environmental samples were assessed and the concentrations of various metals (Al, Pb, Cr, Mn, Mo, Co, Ni, Cu, Zn, Fe, Cd and V) were also determined by means of inductively coupled plasma mass spectrometry (ICP-MS). Soil texture analysis was tested in order to monitor the metal distribution in soils under the influence of environmental factors.

Thesis (MTech (Horticulture))--Cape Peninsula University of Technology, 2016.
Waste management in winery and distillery industries faces numerous disposal challenges as large volumes of both liquid and solid waste by-products are generated yearly during cellar practices. Composting has been suggested a feasible option to beneficiate solid organic waste. This incentivized the quest for efficient composting protocols to be put in place. The objective of this study was to experiment with different composting strategies for spent winery solid waste. Compost materials consisting of chopped pruning grape stalks, skins, seed and spent wine filter material consisting of a mixture of organic and inorganic expend ingredients were mixed in compost heaps. The filter material component varied (in percentage) among five treatments: T1 (40%) lined, T2 (20%) lined, T3 (0%) lined, T4 (40%) grinded material, lined and T5 (40%) unlined. Composting was allowed to proceed in open air over 12 months, from autumn to summer. Indicators such as temperature, moisture, enzyme activities, microbial counts, pH, and C/N ratio, were recorded. Generally, season (df =3, 16, P < 0.05) had significant effects (df =1, 3, P < 0.05) on heap temperature and moisture in all treatments. Similarly, microorganisms (actinobacteria and heterotrophs) varied significantly in all treatments in response to seasonal change (df = 3, 16; P < 0.05). Enzyme activities fluctuated in accordance with seasonal factors and compost maturity stages, with phosphatases, esterases, amino-peptidases, proteases and glycosyl-hydrolases being most prominent. Compared to treatments T2 and T3, compost treatments with higher percentage waste filter materials (T1, T4 and T5) had higher N (16100-21300 mg/kg), P (1500-2300 mg/kg), K (19800-28200 mg/kg), neutral pH, and lower C/N ratios (13:1-10:1), which were also comparable with commercially produced composts. Filter materials therefore, appears to be a vital ingredient for composting of winery solid waste.

Thesis (MScAgric)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: The quality of wine is largely dependent on environmental conditions and recent studies have therefore focused on agricultural practices in terms of water, soil and biodiversity conservation. The industry aims to create sustainable practice and to protect the natural resources available. Sources of potential grape contamination include: vehicle pollution, pesticides, bushfires and wood preservatives used for trellising systems. The latter have come to the attention of the South African wine industry (e.g. creosote and Copper Chromium Arsenate (CCA) products) as they may have consequences for the environment and wine quality. Creosote is a known pollutant of soil and ground water and the volatile fraction has been monitored in air. Plants may also accumulate polycyclic aromatic hydrocarbons (PAHs), which constitute up to 85% of the mass of creosote, and of which some have been proven carcinogenic. Because of the health risks associated with it, creosote has therefore been restricted for use in most applications in Europe, and also in the United States, Canada and Australia. This study focussed on the sensorial and chemical analyses of environmental and wine samples taken from the area around the creosote stockyard to determine accumulation of creosote-derived compounds. Environmental samples were collected and analysed at different distances from the affected area, over two vintages. Wines were made from grapes grown in vineyard blocks adjacent to the stockyard, to determine the effect of distance and skin contact during alcoholic fermentation treatments on wine taint. A sensory panel was trained for descriptive analysis to determine the intensity of the taint. Analytical methods were developed for the analysis of volatile organic compounds (VOCs) by gas chromatography mass spectrometry (GC-MS) and polycyclic aromatic hydrocarbons by high performance liquid chromatography with diode array detection (HPLC-DAD). The sensory results obtained showed white and rosé wines were perceived as clean, whilst the red wines were associated with burnt rubber/tar taint. The perceived taint decreased as distance from the stockyard increased. Wines made from the Cabernet Sauvignon blocks adjacent to the stockyard also showed an increase of taint with the increase of skin contact. Chemical data obtained showed that the taint consisted of a complex mixture of compounds, each with its own pattern of retention within the vineyard and wine. Only m-cresol was found above odour threshold, and only in red wines. The synergistic effect of some compounds may lead to an increase in the perceived taint. Berries and leaves had higher concentrations of volatile compounds than wines. Leaf contamination varied and followed the general trend in literature where the plants with high lipid content and exposed leaf area were the most contaminated. There may be other compounds present in creosote emissions, aside from those investigated here, with sensory attributes related to the taint found in wines. These compounds are styrene, indene, benzene, toluene, isoquinoline and quinoline and should be included in further investigations. From the PAH analysis of environmental samples and wines, it is evident that the samples closest to the stockyard were affected the most. The contamination varied with the depth of the soil: some of the heavier compounds were found in the samples taken from the deeper levels, whilst nearly all other target compounds were present in the top layer of soil. The concentrations found in the environmental samples were lower than reported in literature. Wines had few PAHs present, but at much higher concentrations than is allowed by EU legislation. From this study it is evident that the stockyard had negative effects on the surrounding environment in terms of sensory and chemical contamination. Recommendations include area rehabilitation by means of bioremediation to protect resources and ensure sustainable and safe production of crops. Industrial emissions should also be regulated and restricted in agricultural areas. Furthermore the use of creosote should be revised, and prohibited for agricultural use.
AFRIKAANSE OPSOMMING: Die kwaliteit van wyn is grootliks afhanklik van die omgewingstoestande. Daarom fokus verskeie onlangse studies op landbou aktiwiteite en die invloed daarvan op die omgewing in terme van water, grond en biodiversiteit bewaring. The wyn industrie se doelwitte sluit volhoubare praktyke in, wat die natuurlike hulpbronne bewaar en beskerm. Druiwe kan deur middel van die volgende bronne besoedel word: brandstof uitlaatgasse, insekdoders, veldbrande, gifstowwe wat gebruik word vir houtperservering wat dan verder aangewend word vir opleistelsels. Houtperservering (Koper chroom arsenaat (CCA) en kreosoot) het veral in die laaste paar jaar onder aandag gekom in die wynbedryf van Suid-Afrika as gevolg van die invloed daarvan op die omgewing en die uiteindelike wynkwaliteit. Kreosoot is ‘n gekende gifstof wat verantwoordelik is vir grond en grondwater besoedeling en wat gemonitor word in die atmosfeer. Plante akkummuleer ook poli-sikliese aromatiese hidro-koolstowwe (PAHs), wat to 85% uitmaak van die massa van kreosoot. PAHs is karsinogenies en dus is daar baie navorsing op die molekules reeds gedoen. Die gesondheidsrisikos gepaardgaande met kreosoot het gelei tot die streng geregulasies tans ingestel in Europa, die Verenigde State, Kanada en Australië. Hierdie studie het gefokus op die sensoriese en chemiese analises van omgewings- en wyn-monsters geneem van die omliggende area van die kreosoot palewerf om die akkumulasie van kreosoot-afgeleide-verbindings. Omgewingsmonsters was versamel en geanaliseer om verskillende afstande vanaf die bron van besoedeling (palewerf) te ondersoek oor ‘n twee jaar periode. Wyne is gemaak van die druiwe wat afkomstig is van die blok aangeplant langs die palewerf. Die wyne is ondersoek in terme van afstand vanaf die kreosoot bron asook oenologiese invloede, dopkontak gedurende alkoholiese fermentasie, op die kontaminasie beskryf in wyn. Die wyne is ook oor ‘n twee jaar periode voorberei en sluit die 2011 en 2012 seisoen in. ‘n Sensoriese paneel is opgelei om die beskrywende analises op die wyn uit te voer met die doel om die intensiteit van kontaminasie te identifiseer. Analitiese metodes is ook ontwikkel vir die analise van vlugtige organiese verbindings(VOCs) met gas chromatografie-massa spektrometrie (GCMS) en poli-sikliese aromaties hidro-koolstowwe (PAHs) met hoë druk vloeistof chromatografie. Die sensoriese resultate bekom het wit en rosé wyne as skoon laat blyk, terwyl rooi wyne meer geassosieer was met die gebrande rubber/ teer afgeur beskrywing. Die waargeneemde afgeur het afgeneem soos wat die afstand vanaf die palewerf toegeneem het. Wyne gemaak van die Cabernet Sauvignon blokke langsaan die palewerf het ook ‘n toename in die afgeur gehad met ‘n toename in dopkontak. Chemiese data bekom beeld uit dat die afgeur uit ‘n komplekse mengsel van verbindings bestaan, elk met sy eie patroon van verspreiding en verbinding in die wingerd as ook in die wyn. Net m-kresol was gevind bo die reuk drumpel, dit het ook net in rooi wyne voorgekom. Die sinergistiese effek van die verbindings mag egter bydra tot die waargeneemde afgeur. Druiwekorrels en blare het hoër konsentrasies van die vlugtige verbindings gehad as wat gemeet is in die wyne. Blaar kontaminasie het ook baie gewissel en het ooreengestem met die algemene tendens wat in literatuur beskryf is, naamlik dat plante met ‘n hoër lipid inhoud en grootter blaar oppervlak die meeste gekontamineer word. Daar mag egter nog baie ander verbindings bydra tot die waargeneemde afgeur gevind in die wyn. Spesifieke verbindings wat wel ‘n rol kan speel in kontaminasie en wat voorkom in die vlugtige gedeelte van kreosoot is styreen, indeen, benzeen, tolueen, isoquinoleen die vlugtige verbindings van kreosoot. Die verbindings moet ingesluit word vir verdere studies wat gedoen word op die kreosoot geassosieerde afgeur. Die PAHs analise op die omgewingsmonsters en wyne het gelei tot die bevestiging dat die naasliggende omgewing die meeste geaffekteer is. Die kontaminasie wissel in terme van die diepte in die grond wat die gifstowwe voorkom: die swaarder molekulêre verbindings is tot in die dieper vlakke waargeneem terwyl al die gemete verbindings in die boonste lae teenwoordig was. Die vlakke wat waargeneem is in dié studie is egter laer as wat voorheen in literatuur gevind is in ‘n kreosoot geaffekteerde omgewing. Wyn het PAHs teenwoordig gehad, alhoewel slegs twee verbindings gemeet is, het dit in hoër vlakke voorgekom as wat sekere Europese regulasies as toelaatbaar spesifiseer. Vanaf die studie resultate blyk dit, dat die palewerf se negatiewe invloed op die omliggende omgewing beide meetbaar was in sensories en chemiese kontaminasie. Voorstelle sluit onder andere die rehabilitasie van die omliggende omgewing deur middel van bioremediasie in. Om sodoende die natuurlike hulpbronne in die area te bewaar asook om volhoubare en veilige verbouing van gewasse te verseker. Industriële besoedeling en afval moet ook gereguleer word en beperk word in landbou areas. Verder moet die gebruik van kreosoot heroorweeg word en strenger regulasies moet in plek gestel word om aan internastionale standaarde te voldoen.
Financial support from Winetech, Trip and UFS

The influence of nitrogen (N) and phosphorus (P) limitation on pulp and paper mill activated sludge (AS) floc properties was studied using a bioreactor fed with synthetic Kraft mill effluent. The bioreactor and synthetic effluent were designed and shown to perform like the real mill system providing the AS, establishing the in vivo relevance of the results. Limitation of either N or P produced inadequate effluent biotreatment, shown by poor BOD5 and suspended solids removal, and by decreased biomass health, performance, and floc settling. Greatly enhanced poly-beta-hydroxybutyrate (PHB) (but not carbohydrate or extracellular polymeric substances [EPS]) synthesis was the common response of the floc microbial community to N limitation over many days. In contrast, P-limitation increased total carbohydrate and EPS, but not PHB.
N limitation, but not P limitation, caused the net floc surface charge to be much more negative, while P-limitation, but not N-limitation, increased the floc bound water content and surface hydrophobicity. Thus, in real pulp and paper mill AS systems, careful manipulation of N or P additions may be useful to optimize the key process of charged polymer-assisted AS dewatering.

Thesis (Master of Engineering in Chemical Engineering)--Cape Peninsula University of Technology, 2018.
Acid rock drainage (ARD) is one of the most severe environmental challenges currently faced by the mining industry worldwide. ARD is formed from the oxidation of sulphide-bearing minerals, particularly pyrite, in the presence of water and oxygen. ARD generation is accelerated by the presence of naturally occurring iron and sulphur-oxidizing micro-organisms, which regenerate leaching agents that facilitate sulphide mineral oxidation. ARD pollution is characterized by a high concentration of metals and sulphates in solution, low pH and a high salt content (salinity) in the environment, contaminating soil and groundwater. In South Africa, ARD is a major challenge in the gold and coal mining industries, where millions of tons of sulphide waste rock and overburden are generated and discarded. Characterization of these waste materials is required to develop an appropriate disposal strategy to minimise the risk of pollution and the generation of ARD. Potential ARD generation prediction from waste rock depends on the precise characterization of ARD potential using Biokinetic tests. Commonly used ARD prediction methods are static and long-term kinetic tests. Static tests provide data for a worst-case scenario focussing on strong acid chemical leaching potential to give an overall acid forming potential of a sample. Such kinetic tests provide data illustrating the rate of the net acid generation capacity of mine waste. However, these tests are capital intensive and time-consuming and fail to provide adequate information on the effect of micro-organisms on the overall net acid generation capacity of mine waste. The Biokinetic test reported herein and developed at the University of Cape Town, focusses on addressing a worst case scenario provided by static tests in a cost-effective manner and reduced time frames provided for by conventional kinetic tests. This test primarily provides relative rates of ARD generation in the presence of micro-organisms within 90 days. However, the Biokinetic test is at the developmental stage and thus far, has not been consistently used for different waste ores to determine a standardised approach. Therefore, the aim of this study was to investigate the effects of microbial consortia and to develop a standardisation approach for the test for ARD formation potential using gold-bearing and copper-bearing waste rock. Additionally, to refine the Semi-continuous Biokinetic test simulation, a flow-through system where there is minimal seepage in the waste deposit, was also developed. The sulphur content of the gold and copper-bearing samples used in this study was between 2.3 and 3.15%, respectively. These waste rock samples were found to be potentially acid- forming. In the Biokinetic test, finely milled waste rock samples were slurrified, inoculated with consortia and cultured under standard bioleaching conditions. Leaching and acidification rates were monitored.

This dissertation comprises a series of five separate papers, arranged as chapters, linked thematically and also in their conclusions. The thematic connection between the chapters is that, in each, I investigate some aspect, either historical or contemporary, of how moral limits have been, or might be, applied to the human manipulation of nature through technology. More specifically, I explore how the concept of naturalness has been, and still is, employed in ethical arguments that seek to place limits upon or defend the use of various technologies. In each chapter, I argue that arguments which appeal to nature or naturalness as a normative concept make proper sense only when understood from the perspective of virtue ethics. The conclusions of each chapter are connected, and connected to the conclusions of the dissertation as a whole: firstly, that what I call 'arguments from nature', as they are used in debates about the moral limitations on the use of technology, are defensible only from within a virtue ethics framework; secondly, that such arguments have an important, although limited, role in such debates; and, finally, that virtue ethics more broadly can inform debates about the ethics of technology and the environment. In the first two chapters, by comparing contemporary debates over the ethics of technological manipulation of nature with historical debates over the proper relationship between art and nature, I demonstrate that virtue ethics have played, and still do play, a significant role in our ethical understanding of our relationship with the non-human world. I argue that the ethical issues that arise from our relationship with the non-human world, in response to advances in technology and to problems with the environment, indicate the need for an understanding of ethics that goes further than the mere consideration of rights and utility. In chapters three and four, I argue that virtue ethical theory provides the most promising understanding of the argument from nature as it is applied in attempts to place limits on the human manipulation of nature. In the final chapter, I explore what a modern environmental or technological virtue or vice might be. I explain and defend the environmental and technological virtue of 'living in place' and, in doing so, bring together and validate the claims made in previous chapters that the appeal to human nature does have a role as a normative guide for our ethical evaluations of how we should live and, more generally, that virtue ethical theory can be of guiding and foundational significance in an overarching ethics of the environment and technology.

Thesis (MSc)--Stellenbosch University, 2003.
Full text to be digitised and attached to bibliographic record.
ENGLISH ABSTRACT: Traditionally, wine has been produced by the spontaneous fermentation of grape juice by yeast that originate from the grapes and winery equipment. Research has shown that the population composition and dynamics of these yeasts and other microorganisms are very complex. Kloeckera and its anamorph, Hanseniaspora, dominate the yeast population found on the surfaces of grapes, although prevailing Saccharomyces cerevisiae strains complete the fermentation process. The yeast S. cerevisiae is an important factor contributing to the quality of wines and, therefore, the improvement of wine yeasts receives considerable attention worldwide. Apart from classical yeast breeding studies, genetic engineering and recombinant DNA techniques are increasingly being used in strain development research programmes. These techniques might enable the wine yeasts to produce heterologous enzymes that degrade polysaccharides, convert malic acid to lactic acid, increase glycerol production, release roam and flavour compounds, secrete antimicrobial peptides, etc. The release of recombinant yeast strains (genetically modified organisms, GMOs) is subject to statutory approval. Therefore, it is important to answer several questions prior to the use of such genetically improved yeast in the commercial production of wine. For example, will recombinant yeast strains be able to multiply and spread in nature, and will this GMO be able to out-compete the natural microflora because of its newly acquired genetic traits. Since existing commercial wine yeasts are used in the abovementioned strain development research, it is essential to determine already at this early stage to what extent these wine yeast strains survive and spread in nature and to what extent they influence the fermentations of the following vintages. This study is divided into two sections. The aim of the first section is to sample a representative number of yeast strains from various vineyards in different climatological areas, mainly in the Western Cape, South Africa. These yeast strains were identified mainly by electrophoretic karyotyping (contour-clamped homogenous electric field electrophoresis; CHEF). The second part of the study summarises the results obtained when Fourier transform infrared (FT-NIR) spectroscopy was used to differentiate commercial wine yeast strains. Sets of data, containing the spectra of the mostly used commercial wine yeast strains, were constructed and used as a reference library. The spectra of the isolated yeast strains were then compared to the reference dataset with specific FT-NIR computer software using mathematical calculations. In conclusion, the two methods used in conjunction with one another proved that the commercial wine yeast strains do not easily disperse from the cellar into the vineyard. The commercial wine yeast strains are also more likely to be found near the cellar and the places where the grape skins are dumped. Therefore, should a recombinant yeast strain be used in winemaking, it would not be dispersed into the vineyard. It therefore appears that the commercial use of genetically improved yeast does not pose a high risk in terms of dominance of the indigenous microbial population in the environment
AFRIKAANSE OPSOMMING: Wyn is tradisioneel gemaak deur die natuurlike gisting van druiwesap deur giste wat op die druiwe en keldertoerusting voorkom. Navorsing het getoon dat die samestelling en dinamika van die gispopulasie en ander mikro-organismes baie kompleks is. Kloeckera en sy anamorf, Hanseniaspora, domineer die inheemse gispopulasie op druiwedoppe, terwyl Saccharomyces cerevisiae in baie klein getalle op die druiwedoppe voorkom, maar later die fermentasie oorheers en uiteindelik voltooi. Die gis S. cerevisiae speel 'n baie belangrike rol in die kwaliteit van wyn en daarom geniet die verbetering van wyngiste wêreldwyd besondere aandag. Benewens die klassieke gistelingstudies, word genetiese manipuleringstegnieke toenemnd in navorsingsprojekte gebruik wat daarop gefokus is om wyngisrasse te verbeter. Hierdie tegnieke mag die giste in staat stelom heteroloë ensieme te produseer wat polisakkariedes afbreek, appelmelksuur afbreek, gliserolproduksie verhoog, smaak- en geurkomponente vrystel, antimikrobiese peptiede afskei, ens. Voordat sulke geneties gemanipuleerde giste het egter in kommersiële wynproduksie gebruik sal kan word, is daar heelwat wetlike vereistes waaraan voldoen sal moet word en vrae wat vooraf beantwoord sal moet word. Byvoorbeeld, sal die rekombinante giste in staat wees om vinniger te vermeerder as gevolg van die nuwe genetiese eienskappe en sodoende die natuurlike populasies onderdruk? Omdat kommersiële wyngiste in bogenoemde gisverbeteringprogramme gebruik word, is dit noodsaaklik om nou reeds die verspreiding van die kommersiële giste te monitor en te bepaal hoe geredelik hulle in die natuur kan versprei en oorleef, en hoe hulle wynfermentasies van die daaropvolgende jare beïnvloed. Die studie is in twee gedeeltes verdeel. Die doel van die eerste gedeelte was om 'n verteenwoordigende aantal gisrasse uit die wingerde van 'n aantal wynplase in verskillende klimaatstreke te isoleer, spesifiek in die Wes-Kaap, Suid-Afrika. Die gisrasse was grotendeels deur elektroforetiese kariotipering (kontoer-geklampte homogene elektriese veld; CHEF) geïdentifiseer. Die tweede deel van die navorsing was gefokus op die onderskeiding tussen die mees gebruikte kommersiële wyngiste met 'Fourier-Transform Near Infrared' (FTNIR) spektroskopie. Eerstens is 'n stel data, bestaande uit die spektrum data oor die kommersiële wyngiste opgestel om as 'n verwysingsbiblioteek te dien. Tweedens is die spektrum van data oor die geïsoleerde giste onder presies dieselfde toestande met die verwysingsbiblioteek vergelyk. Dié tegniek maak dit moontlik om tussen die kommersiële wyngiste te onderskei. As die twee metodes saam gebruik word vir identifikasie, kan die afleiding gemaak word dat kommersiële wyngiste nie maklik vanaf die kelder na die wingerd versprei nie. Die kommersiële wyngiste is ook meestal naby die kelder en die dopstortingsterreine gevind. Sou 'n rekombinante gisras dus gebruik word om wyn te maak, sal dit nie maklik versprei nie. Die kommersiële gebruik van geneties gemanipuleerde wyngiste behoort dus nie In groot omgewingsrisiko in te hou nie.

[Truncated abstract] Recovery of soil nutrients, microbial populations and carbon (C) and nitrogen (N) cycling processes are critical to the success of rehabilitation following major ecosystem disturbance. Bauxite mining represents a major ecosystem disturbance to the jarrah (Eucalyptus marginata) forest in the south-west of Western Australia. Mining has created a mosaic of mined areas in various stages of succession surrounded by non-mined forest areas. Initial site preparations within rehabilitation areas such as contour ripping alter soil structure (creation of mound and furrows) and over time also influence the distribution of vegetation and litter. Current performance criteria developed by industry, government and other stakeholders have determined that before post-bauxite mined areas of jarrah forest can be integrated back into normal forest management practises they should be functional and demonstrate resilience to normal forest disturbances such as fire. Furthermore, resilience should be of a manner comparable to non-mined analogue forest sites. Currently little is known of the resilience of microbial communities and C and N cycling in rehabilitation sites to normal forest disturbances such as prescription burning. As such, before rehabilitated jarrah forests can be successfully integrated into broad scale forest management regimes, a more thorough knowledge of the potential impacts of burning practises on the soil microbial community and C and N cycling processes in these systems is required. ... While there are similar rates of C and N cycling the underlying microbial community structure was distinctly different; implying a high degree of functional redundancy with respect to C and N cycling. Differences in the C and N cycling and structure of the microbial communities were likely to be due to differences in soil environmental conditions (i.e. soil alkalinity/acidity, soil moisture) and C substrate availability which influence the physiological status of the microbial community and in turn are related to successional age of the forests. Results also suggest that the measurement of CLPP can be a useful approach for assessment of changes in the functional ability of microbial communities. However, the interpretation of how well these rehabilitation forests have recovered heterotrophic abilities was greatly affected by the methodological approach used (e.g. MicroRespTM or Degens and Harris, 1997). Importantly, results from Chapter 4 and 5 suggested that the effects of a moderate prescription fire on C and N processes, CLPP and microbial community structure of 18 year old rehabilitation forests are likely to be short-lived (< 2 years). Furthermore, the effects of the moderate spring prescription fire were not large enough to decouple C and N cycling processes over the short-term (< 1 years) which suggests that by 18 years of age rehabilitation forests demonstrate comparable functional resilience to a moderate prescription burn.

Fundamental processes underpinning the biotransformation of coal by fungal biocatalysts have been intensively investigated, however, limited large-scale industrial applications using such systems have been reported. The un-anticipated sporadic growth of Cynodon dactylon on the surface of un-rehabilitated discard coal dumps has been noted and this was found to be coupled with the breakdown of coal into a humic soil-like material in the top 1.5 metres of the dumps. Extensive fungal growth was observed to be associated with the Cynodon dactylon root system and examination of plant roots indicated the presence of mycorrhizal fungi. Analysis of the Cynodon dactylon plant roots around which coal biotransformation was occurring confirmed the presence of arbuscular mycorrhizal colonisation with the species Glomus clarum, Paraglomus occultum, Gigaspora gigantea and Glomus mosseae identified to be associated with the plants. Further molecular characterisation of non-mycorrhizal rhizospheric fungi showed the presence of fungal species with coal-degrading capabilities that most likely played a role in the coal biotransformation observed. The discard coal dump environment was simulated in pot and column studies and coal biotransformation was reproduced, with this process enhanced by the addition of mycorrhizal and non-mycorrhizal rhizospheric fungal inocula to the environment. Mycorrhizal and non-mycorrhizal species in the inoculum were re-isolated from the simulated environment fulfilling a number of Koch’s postulates and indicating a causal role in the biotransformation of coal. An inversion of conventional mycorrhizal colonisation was demonstrated in this system with reduction in extraradicular presence and an increase in intracellular colonisation compared to soil controls. A descriptive model was formulated suggesting a two-part fungal system involving organic carbon and nutrient exchange between the plant, mycorrhizal fungi and non-mycorrhizal coal-degrading rhizospheric fungi ultimately resulting in the biotransformation of coal. The biotransformation observed was comparable to reports of “rock-eating fungi”. Results suggest that the biological degradation of coal in situ with the production of a soil-like substrate could provide a feasible method of discard coal dump rehabilitation as well as provide a humic-rich substrate that can be utilised in further industrial applications.

"June, 2008".
Thesis (PhD)--Macquarie University, Division of Environmental & Life Sciences, Dept. of Chemistry & Biomolecular Sciences, 2009.
Bibliography: p. 227-259.
Literature review -- Materials and methods -- Sampling sites and sampling material -- Enrichment cultures and molecular analyses -- Optical and binding characterisation of the QDs -- Applications of the QDs -- Concluding remarks.
White island, the most active volcano in New Zealand, is a poorly studied environment that represents an ideal site for the investigation of acidophilic thermophiles. The microorganisms present on here are continually exposed to extreme environmental conditions as they are surrounded by steamy sulphurous fumaroles and acidic streams. The sediment temperature ranges from 38°C to 104°C whilst maintaining pH values below 3. A survey of the volcanic hydrothermal system of White Island was undertaken in order to gain insights onto the microbial diversity using culture-dependant techniques and molecular and phylogenetic analyses. A novel liquid medium based on "soil-extract" was designed which supported growth of bacterial and archaeal mixed cultures. Molecular analyses revealed that the dominant culturable bacterial species belong to the Bacteroidetes, Firmicutes and α-Proteobacteria groups. Several previously uncultured archaeal species were also present in the mixed cultures. The knowledge gained from these studies was intended to help in the development of a novel microbial detection technique suitable for community analysis. -- Conventional molecular techniques used to study microbial biodiversity in environmental samples are both time-consuming and expensive. A novel bead-based assay employing Quantum dots (QDs) was considered to have many advantages over standard molecular techniques. These include high detection speeds, sensitivity, specificity, flexibility and the capability for multiplexed analysis. QDs are inorganic semiconductor nanoparticles made up of crystals about the size of proteins. It has been claimed that the physical and chemical properties of the QDs have significant advantages compared to organic dyes, including brighter fluorescence and resistance to photo-bleaching. Their optical properties facilitate the simultaneous imaging of multiple colours due to their flexible excitation and narrow band emission. Functionalised QDs are able to bind to different biological targets such as DNA, allowing high-throughput analysis for rapid detection and quantification of genes and cells. -- The optical and physical characteristics of the QDs as well their interaction with biomolecules are shown to be suitable for the development of a novel bead-based technique able to target the key microbial species and identify them by flow cytometric measurements (FCM). The broad absorption and narrow emission spectra of the QDs, as well as their fluorescence intensity and specify to target biomolecules, was compared to other organic fluorophores. The potential advantages and limitations of QDs as a fluorophores for biological applications are discussed. -- The data acquired during this study provides a broad overview of the microbial diversity and ecology of the volcanically-active hydrothermal systems of White Island and constitutes the baseline for the development of a novel bead-based technique based on QDs.
Mode of access: World Wide Web.
xvii, 259 p. ill. (some col.)

Thesis (M. S.)--Civil and Environmental Engineering, Georgia Institute of Technology, 2009.
Committee Chair: Dr. Frank E. Löffler; Committee Member: Dr. Joseph B. Hughes; Committee Member: Dr. Kurt D. Pennell; Committee Member: Dr. Lawrence J. Shimkets; Committee Member: Dr. Robert A. Sanford; Committee Member: Dr. Thomas DiChristina.

The aims of the project were twofold. The initial objective of the study, based on previous results, was to develop an economically viable methodology for immobilizing yeast cells for the treatment of heavy metal-laden waste water. The non-viable yeast cross-linked by 13% (w/v) formaldehyde/1N HNO₃ exhibited satisfactory mechanical strength and rigidity in a continuous-flow column operation. No apparent disruption of the biomass after repeated use was observed. The cost of immobilizing 1kg dry yeast pellets was estimated at less than US$I. Zn uptake capacity of FA-cross-linked pellets, on batch trials, remained similar to that of raw yeast, reflecting that the immobilizing procedure did not hinder its metal removing capacity. In column studies, cation metals were effectively removed by the yeast pellets from aqueous solution at natural pHs, and then recovered completely by washing the pellets in situ with O.1M HCl. The recovered metals were concentrated in such small volumes that recycling or precipitation of them was facilitated. The metal uptake capacity of the regenerated biomass remained constant in comparison with cycle 1, indicating that reuse of the yeast would be possible. In the case of Cr⁶⁺, a gradual breakthrough curve of Cr in the column profile was noted, with a simultaneous reduction of Cr⁶⁺ to Cr³⁺. However, Cr⁶⁺ in the effluent can be markedly minimised either by accumulation onto the biomass or reduction to its trivalent form. Desorption of bound Cr⁶⁺ with either alkali or salt could not accomplish the regeneration of the biomass. A combination of reduction and desorption with FA/HNO₃ appeared promising in regeneration of the saturated biomass at 4°C. The metal sorption capacities of the yeast pellets, on a batch or a fixed-bed system are relatively lower than that of documented sorbents. Apparently more of the yeast pellets would be required for treating a certain volume of waste effluent, than with other sorbents. Therefore Azolla filiculoides was examined as a suitable sorbent for this purpose. This constitutes the second part of the project. Azolla filiculoides, a naturally-abundant water fern, was screened for its metal sorption and recovering capacities, mechanical stability, flow-permeability and reusability. The azolla biomass appeared to have fulfilled the required mechanical criteria during the repeated sorption-desorption column operations. It is water-insoluble and appears flexible under pressure when rinsed with water. These characters are of crucial importance in a continuous-flow system since a column can be operated at high flow rates without apparent compact of the biomass and pressure loss. Therefore, immobilization of the biomass can be avoided. The sorption isotherm data, obtained from batch removal of Cr⁶⁺, showed that the sorption process was effective, endothermic and highly pH dependent. Considerable amounts of Cr⁶⁺ were accumulated at the optimum pHs of 2-2.5. Column sorption of Cr⁶⁺ at a low flow rate and pH of 2.5 showed optimum performance with a total Cr uptake of 50.4mg/g at 60% saturation of the biomass. Removal of Cr⁶⁺ from an electroplating effluent using an azolla column was deemed reasonably satisfactory, although the uptake declined slightly. Desorption of bound Cr⁶⁺ with various desorbents was incomplete, which resulted in a low regeneration efficiency of about 50%. However, removal and recovery of Cr³⁺ using the azolla column was than that of Cr⁶⁺. Desorption of Cr³⁺ from the spent biomass column was accomplished with the recovery of 80% using O.5N H₂SO₄, The regeneration efficiencies for Cr³⁺ removal were up to 90% and demonstrated that the biomass is reusable. Cation metal uptake capacities of azolla, obtained either from batch or column experiments, are reasonably high in comparison with other sorbents. The uptake of Ni or Zn ions from solution is pH dependent showing the optimum pH of around 6 to 6.5, under the current experimental conditions. The sorption kinetics for cation metals was rapid with about 80% of the bound Ni ions being taken up in the first 10 min. The character of rapid binding is extremely important in a column sorption process, especially on a large scale since it favours an optimum uptake of metals at high flow rates. The Ni or Zn uptakes in column sorption were not markedly affected when the flow rates were increased from 80mllh up to 800ml/h for the 5g biomass used. The cation heavy metals removed from waste effluents were recovered in a concentrated solution of small volume. The desorption of bound Ni and Zn ions from the saturated biomass was accomplished with either O.2N HCl or H₂SO₄ that resulted in recoveries of more than 95%. The metals recovered, in the case of Ni and Zn, are identical to that of plating agents ego nickel sulphate or chloride, so that recycling of the metals is possible. An effluent-free, closed loop of Ni or Zn treatment system was proposed, whereby the Ni or Zn ions can be recycled to the plating bath whilst the purified water is fed back to the rinse tanks. Ca and Mg ions, commonly present in the electroplating effluents, appeared to affect sorption of heavy metals by azolla when metal concentrations were relatively low, presumedly through its competitive binding for the shared sites on surfaces of azolla. The data obtained from column sorption of Ni and Zn follows the BDST model well, enabling the application of the model to predicting design parameters for scale-up of the biosorption column system. It is interesting that the values of metal uptake, expressed in molar quantities, obtained on respective single-metal solutions and the multiple metal system, are similar, implying that the mechanisms involved in the sorption of all metal cations are similar and that the binding sites on surfaces of azolla are probably shared by all cation metals. The surface of the biomass provides sites for metal binding estimated in the range of 0.45-0.57mmol/g, based on the current experiments. The biomass has a surface area of 429 m²/g and water retention of 14.3 ml/g. The functional groups on the surface of azolla were partially identified using chemical modification and metal binding comparison. Among the functional groups examined, carboxyl groups, provided by amino acids and polysaccharides, appeared to play an important role in metal cation binding. The infrared spectra of the samples support this conclusion.

Dissertation (PhD(Agric)) -- Stellenbosch University, 2005.
ENGLISH ABSTRACT: Due to increased consumer demand for products labelled by origin, and the requirement that these labels are a guarantee of both quality and product character, there is an increasing global focus on delimitation of denominations of origin. The integrity of denominations of origin and their defensibility can be ensured through the use of terroirs as a basis for delimitation. The aims of this study were to establish the dominant environmental criteria that affect the viticultural behaviour and wine character of two important cultivars (Cabernet Sauvignon and Sauvignon blanc) in the Stellenbosch Wine of Origin District, to use an appropriate methodology to identify viticultural terroirs in this district based on these criteria and with the use of a geographic information system, and finally to use these viticultural terroirs to identify denominations of origin within the same area. A terroir can be defined as a grouping of homogenous environmental units, or natural terroir units, based on the typicality of the products obtained. Identification and characterisation of terroirs depends on knowledge of environmental parameters, the functioning of the grapevine and the characteristics of the final product. Field studies, resulting in point data, are necessary to investigate the functioning of the grapevine but in order for this information to be of use within zoning studies it must be placed in a spatial context. As a first phase in data acquisition, the Stellenbosch Wine of Origin District was characterised and natural terroir units were identified using existing digital data and a geographic information system. A natural terroir unit (NTU) can be defined as a unit of land that is characterised by relatively homogenous topography, climate, geological substrate and soil. A total of 1389 NTUs were identified in the Stellenbosch Wine of Origin District (84 537 ha). The identified NTUs were homogenous with respect to terrain morphological unit, altitude, aspect and soil type. Each of the identified units was further described with respect to the extent of the expected sea breeze effect and, for certain of the soil types, the associated parent material. As a second phase of data acquisition, a network of plots of Sauvignon blanc and Cabernet Sauvignon were delimited in commercial vineyards in proximity to weather stations and their viticultural and oenological response monitored for a period of seven years. Regression tree analyses were performed on the complete data set and the relative importance of the environmental and management related variables determined for each dependent variable. Excepting for scion clone, which had a high relative importance for bunch mass of Sauvignon blanc and yield to pruning mass index of Cabernet Sauvignon, no other non-environmental variable included in the analyses appeared to have a strong effect on grapevine performance and wine character. The performance of Cabernet Sauvignon was affected by the potassium content of the subsoil and the climate of the season. The performance of Sauvignon blanc appeared to be related to soil texture, wind exposure and temperature, both during the green berry growth stage and the month prior to ripening. From the results presented, it appears that environmental parameters have an overriding effect on the performance of both Cabernet Sauvignon and Sauvignon blanc, but that these two cultivars react differently to environmental stimuli. A knowledge-driven model used the rules generated in the regression tree analyses to directly classify natural terroir units with respect to expected response of Cabernet Sauvignon and Sauvignon blanc in the Stellenbosch Wine of Origin District. The natural terroir units were thus grouped into terroir units that were homogenous with respect to predicted viticultural and oenological response for each cultivar. The use of representative sites to determine the response of the grapevine to its environment is time consuming and costly and limits terroir studies to research related investigations. Vineyard managers were therefore surveyed with respect to the functioning of established Sauvignon blanc vineyards in the Stellenbosch Wine of Origin District in an attempt to obtain the necessary data. Comparison of the data generated with these questionnaires to measured data in commercial vineyards suggested that the vineyard managers were able to characterise the performance of vineyards with respect to vigour, signs of drought stress and yield. Each vineyard was mapped and the responses were linked to modelled environmental variables. Classification and regression trees were used to construct decision trees, which could be applied to environmental data in a geographic information system to determine viticultural terroirs for production of Sauvignon blanc. These terroirs, although fewer, were comparable to those generated using field data. Data gathered during terroir studies, and the identified viticultural terroirs for Cabernet Sauvignon and Sauvignon blanc, were used to revisit the boundaries of the Stellenbosch Wine of Origin District and the Simonsberg-Stellenbosch ward. Modifications were proposed based on expected wine characteristics. Boundaries for two new wards in the Helderberg basin were proposed. It was also possible to identify vineyards within a ward for the production of terroir specific wines.
AFRIKAANSE OPSOMMING: Toenemende verbruikersaanvraag vereis produkte waarvan die etikette nie net die oorsprong aandui nie, maar ook kan dien as ‘n waarborg vir die produk se kwaliteit en kenmerkendheid. Hierdie tendens verklaar die toenemende wereldwye fokus op afgebakende areas van oorsprong. Die integriteit van die afgebakende areas van oorsprong sowel as hul verdedigbaarheid kan gewaarborg word deur terroirs as basis vir afbakening te gebruik. Die doelstellings van die studie was om die oorheersende omgewingseienskappe wat wingerdprestasie en wynkarakter van twee belangrike cultivars, naamlik Cabernet Sauvignon en Sauvignon blanc, in die Stellenbosch Wyn van Oorsprongsdistrik bemvloed, te bepaal; tweedens om ‘n toepaslike metodologie te gebruik om terroirs in die die distrik te bepaal wat gebaseer is op die geidentifiseerde omgewingseienskappe met die gebruik van ‘n geografiese inligtingstelsel; en, ten slotte, om hierdie terroirs vir wingerbou te gebruik om afgebakende areas van oorsprong in dieselfde omgewing te identifiseer. ‘n Terroir kan gedefinieer word as ‘n samestelling van homogene omgewingseenhede, of natuurlike terroir-eenhede (NTE), wat gebaseer word op die kenmerkende eienskappe van die produkte wat daaruit verkry word. Identifisering en karakterisering van terroirs sal afhang van kennis van die omgewingsparameters, die funksionering van die wingerdstok en die eienskappe van die finale produk. Veldstudies waaruit puntdata verkry word, is noodsaaklik om die funksionering van die wingerdstok te ondersoek. Dit is egter noodsaaklik om eers hierdie inligting in ruimtelike konteks te plaas alvorens die inligting vir soneringstudies gebruik kan word. As 'n eerste fase van datagenerering, was die Stellenbosch Wyn van Oorsprongsdistrik gekarakteriseer en NTE’s geidentifiseer deur gebruik te maak van bestaande digitale data en ‘n geografiese inligtingstelsel. ‘n NTE kan gedefinieer word as ‘n landseenheid wat gekarakteriseer word deur ‘n relatiewe homogene topografie, klimaat, geologiese substraat en grondtipe. ‘n Totaal van 1389 NTE’s is geidentifiseer in die Stellenbosch Wyn van Oorsprongsdistrik (84 537 ha). Die geidentifiseerde NTE’s was homogeen met betrekking tot die terrein morfologiese eenheid, hoogte bo seespieSI, hellingsaspek en grondtipe. Elk van die geidentifiseerde eenhede was verder beskryf volgens die omvang van die seewindinvloed en, vir toepaslike grond tipes, die geassosieerde moedermateriaal. As ‘n tweede fase van datagenerering is ‘n netwerk van persele van Cabernet Sauvignon en Sauvignon blanc afgebaken binne bestaande kommersiele wingerde in die nabyheid van weerstasies. Hul wingerd- en wynkundige respons is vir ‘n periode van sewe jaar gemonitor. Regressieboomanalises is gebruik om die volledige stel data te analiseer en om die relatiewe belang van omgewings- en bestuurspraktykverbonde veranderlikes te bepaal. Die bostokkloon (wat 'n hoe relatiewe belang vir die trosmassa van Sauvignon blanc en die oes- tot snoeimassa verhouding van Cabernet Sauvignon het) is die enigste van die nie-omgewingsparameter wat ‘n sterk invloed op wingerdprestasie of wynkarakter blyk te he. Die prestasie van Cabernet Sauvignon is beTnvloed deur die kaliuminhoud van die ondergrond sowel as die seisoensklimaat. By Sauvignon blanc het dit voorgekom of die prestasie verband hou met grondtekstuur, windblootstelling en temperatuur tydens die groen fase van korrelgroei sowel as die maand voor rypwording. Alhoewel dit blyk uit die resultate dat omgewingsparameters 'n oorheersende invloed op die prestasie van beide Cabernet Sauvignon en Sauvignon blanc uitoefen, reageer die twee cultivars verskillend op omgewingsprikkels. ‘n Kennisgedrewe model waarvan die riglyne uit resultate van die regressieboomanalise saamgestel is, word gebruik om NTE direk te klassifiseer ten opsigte van die verwagte respons van Cabernet Sauvignon en Sauvignon blanc in die Stellenbosch Wyn van Oorsprongsdistrik. Die NTE is dus gegroepeer om terroir eenhede te vorm wat homogeen was ten opsigte van die verwagte wingerd- en wynkundige respons vir elke cultivar. Die gebruik van verwysingspersele om die respons van die wingerdstok teenoor sy onmiddelike omgewing te bepaal, is tydrowend en duur en beperk sodoende terroir studies tot navorsing. Gevolglik is ‘n opname onder wingerdbestuurders gemaak om inligting oor die prestasie van Sauvignon blanc in die Stellenbosch Wyn van Oorsprongsdistrik in te win en sodoende die nodige data te verkry. Na vergelyking van die ingewinde data, wat uit die opname verkry is, met gemete data vanaf kommersiele wingerde, kon afgelei word dat wingerdbestuurders by magte is om die prestasie van wingerde ten opsigte van groeikrag, tekens van droogtestres en opbrengs te karakteriseer. Elke wingerd is gekarteer en die respons is gekoppel aan die gemodeleerde omgewingsparamters. Klassifikasie en regressiebome is gebruik om besluitnemingsmodelle saam te stel wat toegepas kon word op omgewingsdata in ‘n geografiese inligtingstelsel om terroirs vir die produksie van Sauvignon blanc te bepaal. Hierdie terroirs, alhoewel minder, was vergelykbaar met die wat gegenereer was met behulp van veldstudies. Data verkry met terroirstudies, sowel as gei'dentifiseerde terroir-eenhede, was gebruik om die grense van die Stellenbosch Wyn van Oorsrongsdistrik en die Simonsberg-Stellenbosch wyk te herbepaal. Voorgestelde modifiserings was gebaseer op verwagte wyneienskappe. Grense vir twee nuwe wyke in die Helderbergkom is voorgestel. Dit was ook moontlik om wingerde binne ‘n wyk te identifiseer vir die produksie van terroir-spesifieke wyne.

Black band disease (BBD) of corals is a temperature dependent, highly virulent, polymicrobial disease affecting reef-building corals globally. The microbial consortium of BBD is primarily comprised of functional physiological groups that include photosynthetic cyanobacteria, sulfate reducers, sulfide oxidizers and a vast repertoire of heterotrophic bacteria. Quorum sensing (QS), the cell-density dependent communication phenomenon in bacteria, is known to induce expression of genes for a variety of virulence factors in diseases worldwide. Microbes capable of QS release signals such as acyl homoserine lactones (AHLs) and autoinducer-2 (AI-2), which coordinate microbial interaction. The focus of the present study was to investigate the presence and potential role of QS in BBD pathogenicity, utilizing culture dependent and independent methodologies. Isolates across coral health states including BBD, were screened for production of QS signals, and AHL and AI-2 production capabilities were analyzed via LC-MS/MS. The effect of temperature on AHLs was also examined. Additionally, antimicrobial production capabilities of isolates were tested. BBD metagenomes were utilized to screen for sequences related to QS, antimicrobial synthesis, and antimicrobial resistance genes. BBD isolates represented a significantly higher proportion of isolates capable of producing QS signals in comparison to healthy coral isolates. Several AHLs produced by coral derived bacterial cultures were identified, and three AHLs, specifically 3OHC4, 3OHC5 and 3OHC6, showed a significant increase in production at an elevated temperature of 30 °C, which correlates with increased BBD incidence on reefs with increasing water temperature. Most of the BBD cultured isolates were identified as vibrios. Several sequences related to QS, antimicrobial synthesis and resistance genes were detected in the BBD metagenomes. Based on the findings of this study, a model for potential microbial interactions amongst BBD heterotrophs, centered around QS, is proposed. Taken together, the findings from this study provide a clearer understanding of the potential role of QS in BBD, and serve as the basis for further studies aimed at elucidating the pathogenesis of an intricate coral disease.

Användandet av deponier är en av de vanligaste metoderna för avfallshantering globalt. Trots insatser som gjordes för att förbjuda hushållsavfall i deponier under millennieskiftet, deponier skapade innan restriktionerna är fortfarande en risk för miljön. Under 2014 öppnade SÖRAB en kontinuerlig biologisk reningsanläggning (KBR-anläggning) på Löt Avfallsanläggning för att hantera lakvatten från en gammal deponi som under en tid fylldes med hushållsavfall. Sedan dess har SÖRAB arbetat med att förbättra KBR-anläggningen. Målet med denna studie är att utforma en driftstrategi för KBR-anläggningen för att förbättra kvävereningen vid låga temperaturer. Ett antal laborativa försök genomfördes, såsom den mikrobiella konsortiets livsduglighet i lakvattnet och tillväxten i både rumstemperatur och vid 4°C, bioaugmentation genom att berika den mikrobiella cellkulturen som redan finns i lakvattnet och hur detta förbättrar kvävereningen i jämförelse med tillsatser av den kommersiella bakterieblandningen ClearBlu Environmental och andra externa kolkällor. Resultaten från dessa laborativa försök påvisade komplett nitrifikation i både rumstemperatur och 4°C i berikat lakvatten från KBR-anläggningens L2A bassäng efter fem dagar. Försöket visade även på syresatt denitrifikation. Dessutom påvisades komplett denitrifikation inom fem dagar, vid rumstemperatur i lakvatten från anläggningens L2B bassäng. Under efterföljande pilotförsök påvisades möjligheten till upplivandet av den biologiska kvävereningen genom berikningen av den mikrobiella cellkulturen i lakvattnet. I ett pilotförsök då lakvatten från L2B bassängen berikades, komplett denitrifikation skedde under en anaerob fas på 16 dagar samt nitrifikation och aerob denitrifikation under ett påföljande 17 dagar lång aerob fas. Ett annat pilotförsök då lakvatten från L2A bassängen berikades påvisade både aerob och anaerob nitrifikation, då ammoniumrening skedde i både den syresatta och syrefria fasen. Tillsatsen av nutrient broth (näringsbuljong) kan påverka KBR-anläggningen, vilket kväver vidare studier. Resultatet från detta projekt tydligt påvisar att kvävereningen i KBR-anläggningen kan förbättras genom att berika den redan närvarande mikrobiella kulturen.
Landfilling has been one of the most popular methods of handling waste globally. Despite the efforts made to stop the disposal of household waste during the turn of the millennia, the landfills formed before these restrictions are still at risk for causing harm to the environment. In 2014, SÖRAB opened a continuous-flow biological treatment (KBR) facility in Löt to treat the leachate produced in one of their older landfills, once filled with household waste. Since then, SÖRAB has been working on improving the treatment facility. The aim of this the study is to find a suitable process to enhance the nitrogen removal at low temperature. Several laboratory scale experiments were performed, such as viability of microbial consortia in the leachate and growth at room temperature and at 4°C, testing bioaugmentation by enriching the microbial cell culture in the leachate and their efficiency in removing nitrogen, compared to the commercial cell culture ClearBlu Environmental and carbon source addition. The results displayed complete nitrification at both room temperature and 4°C in bioaugmented, enriched leachate originating from the L2A basin of the KBR facility, after five days. These trials also suggested the occurrence of aerated denitrification. Complete denitrification within five days was seen at room temperature in bioaugmented, enriched leachate from the L2B basin of the same facility. The ensuing pilot scale trials proved the possibility to revive the biological nitrogen removal by microbial cell culture enrichment. In one pilot in which leachate from the L2B basin was enriched, complete denitrification in the anaerobic phase consisting of 16 days occurred, along with some nitrification and aerated denitrification in the 17 day long aerated phase that followed. Another pilot scale trail in which leachate from the L2A basin was enriched, both aerobic and anaerobic nitrification occurred, as ammonium removal occurred in both the aerated and unaerated phases. The addition of nutrient broth might influence the KBR system which needs further study. The results from this project clearly demonstrate that nitrogen removal in the KBR facility could be enhanced using a culture naturally present in the facility.

Microalgae can be grown on municipal wastewater media to both treat the wastewater and produce feedstock for algae biofuel production. However the reliability of treatment must be demonstrated, as well as high areal algae productivity on recycled wastewater media and efficient sedimentation harvesting. This processes was studied at pilot scale in the present research. A pilot facility was operated with nine CO2-supplemented raceway ponds, each with a 33-m2 surface area and a 0.3-m depth, continuously from March 6, 2013 through September 24, 2014. The ponds were operated as three sets of triplicates with two sets continuously fed primary-clarified municipal wastewater at either a 2-day or 3-day hydraulic residence time (HRT), and one set fed the clarified effluent of the 3-day pond set. This second pond-in-series was operated with a 3-day HRT. Areal biomass productivity is reported as gross and net, the former based only on biomass in the pond effluents and the latter subtracting the volatile suspended solids in the influent from those in the effluent. An estimate was also made of autotrophic biomass productivity, as differentiated from heterotrophic growth. Over a year, net productivity averaged 83 metric tons per hectare per year (MT/ha-yr) for the 2-day HRT ponds, 52 MT/ha-yr for the 3-day HRT ponds, and 44 MT/ha-yr for the 3-day HRT ponds receiving clarified effluent of the first set of 3-day HRT ponds (i.e., recycled water). The lower net productivity of the pond receiving water recycling was attributed to two factors. First, the relatively high influent suspended solids concentrations were subtracted from the effluent suspended solids concentrations before net productivity was calculated. Second, the recycled water contained less soluble organic matter than the primary-clarified wastewater leading to less heterotrophic biomass production. The accumulation of inhibitory allelochemicals is a possible third cause of lower productivity , but no specific information was collected on allelopathy. Algae were harvested from pond effluent by sedimentation, with harvest efficiency most affected by the extent of natural bioflocculation occurring in the ponds. Some forms of bioflocculation are thought to be mediated by bacteria, which often make-up a substantial fraction of the settled flocs. Pond samples settled in 1-L Imhoff cones averaged/L total suspended solids after 24 hours of settling; but all ponds fell short of meeting an averaged/L total suspended solids after a 2 hour interval which would be ideally achieved for wastewater effluent. No relationship was seen between settling performance and the bacterial content of flocs. Soluble carbonaceous biochemical oxygen demand (scBOD5) removal by the raceway ponds was sufficient to meet wastewater treatment requirements year around. Influent scBOD5 concentrations averaged 83 mg/L, and the effluent averaged 5.1 mg/L and 4.2 mg/L for the 2-day and 3-day HRT pond sets, respectively. The variable with the greatest influence on productivity in all pond sets, and settling performance in the recycled water pond set, was season (i.e., co-correlated variables of solar insolation and pond temperature). Neither productivity nor settling appeared to be related to prominent algae genera or prevalence of grazers. The high net productivity achieved with a growth medium of primary clarifier effluent and the generally high settleability of algal-bacterial flocs indicate a good potential for algae wastewater treatment and biofuel production. However, the settling of algae grown on recycled water needs improvement to achieve the full potential of wastewater-grown algae biofuel production.

[Truncated abstract] Acidic mine lakes are formed as a result of the oxidation and dissolution of metal sulfide minerals and are primarily characterized by low pH values of 2 – 4. Many strategies for the bioremediation of acidic mine lakes depend on the alkalinity generation capabilities of microbial ferric and/or sulfate reducing bacteria. However nearly all mine lakes are oligotrophic, with very low concentrations of available organic carbon and nutrients; all required for healthy microbial growth. There is also an unusual class of mine lakes characterized by low concentrations of organic carbon and also very low concentrations of dissolved iron and sulfate. Our ability to promote microbial activity in these systems is especially challenging. This study focuses on one of these systems, Lake Kepwari, a coal mine lake in Western Australia. Numerical modeling of remediation strategies is an efficient way of testing scenarios prior to expensive in-field trials. However such modeling relies on good descriptions of microbial processes, including kinetic parameterizations of ferric and sulfate reduction. There has been little research to date on the study of kinetic parameterizations of the chemical and biological alkalinity generation in acidic mine lakes. The objectives of this thesis were to investigate the viability of microbial ferric and sulfate reduction in an ultraoligotrophic, acidic mine lake, to assess the impact of these microbial processes on water quality and to parameterize the Dual Monod kinetics of neutralization under dual limitation conditions. Molecular analyses including most probable number, DNA extraction, polymerase chain reaction, polymerase chain reaction – denaturing gradient gel electrophoresis were used to examine the microbial communities in the lake sediments. ... The Monod maximum specific microbial growth rates with respect to dissolved organic carbon and ferric, and as determined in batch experiments, were 0.07 ± 0.01 and 0.048 ± 0.02 day-1, respectively, and their corresponding Monod half saturation constants and were 14.37 and 5.6 mmol L-1. The Monod maximum consumption rates under ferric and OC limitation were also estimated. The Monod maximum specific microbial growth rates with respect to dissolved organic carbon and sulfate, , and were 0.05 ± 0.01, 0.08 ± 0.01 and 0.07 ± 0.02 day-1, respectively, and their corresponding Monod half saturation constants, and were 75.5, 131.8 and 10.2 mmol L-1. The Monod maximum consumption rates under sulfate and OC limitation were also estimated. The results of this study suggest that strategies for the remediation of ultraoligotrophic, acidic mine lakes may rely on microbial ferric and sulfate reduction, however additions of both organic carbon and sulfate/ferric are essential. These results can be immediately applied to mesocosm studies in outdoor enclosures and to the management of acidic mine lakes. Furthermore, this thesis has provided a new, valuable understanding on the Dual Monod kinetic parameterizations of neutralization for an ultraoligotrophic, acidic mine lake environment. These parameterizations are essential for the lake ecological models that will be used to investigate remediation scenarios for acidic mine lakes.

The contamination of the environment, accidental or intentional, in particular with chemical toxins such as industrial chemicals and chemical warfare agents has increased public fear. There is a critical requirement for the continuous detection of toxins present at very low levels in the environment. Indeed, some ultra-sensitive analytical techniques already exist, for example chromatography and mass spectroscopy, which are approved by the US Environmental Protection Agency for the detection of toxins. However, these techniques are limited to the detection of known toxins. Cellular expression of genomic and proteomic biomarkers in response to toxins allows monitoring of known as well as unknown toxins using Polymerase Chain Reaction and Enzyme Linked Immunosensor Assays. However, these molecular assays allow only the endpoint (extracellular) detection and use labels such as fluorometric, colorimetric and radioactive, which increase chances of uncertainty in detection. Additionally, they are time, labor and cost intensive. These technical limitations are unfavorable towards the development of a biosensor technology for continuous detection of toxins. Federal agencies including the Departments of Homeland Security, Agriculture, Defense and others have urged the development of a detect-to-protect class of advanced biosensors, which enable environmental surveillance of toxins in resource-limited settings. In this study a Surface-Enhanced Raman Spectroscopy (SERS) immunosensor, aka a SERS-linked immunosensor assay (SLISA), has been developed. Colloidal silver nanoparticles (Ag NPs) were used to design a flexible SERS immunosensor. The SLISA proof-of-concept biosensor was validated by the measurement of a dose dependent expression of RAD54 and HSP70 proteins in response to H2O2 and UV. A prototype microchip, best suited for SERS acquisition, was fabricated using an on-chip SLISA to detect RAD54 expression in response to H2O2. A dose-response relationship between H2O2 and RAD54 is established and correlated with EPA databases, which are established for human health risk assessment in the events of chemical exposure. SLISA outperformed ELISA by allowing RISE (rapid, inexpensive, simple and effective) detection of proteins within 2 hours and 3 steps. It did not require any label and provided qualitative information on antigen-antibody binding. SLISA can easily be translated to a portable assay using a handheld Raman spectrometer and it can be used in resource-limited settings. Additionally, this is the first report to deliver Ag NPs using TATHA2, a fusogenic peptide with cell permeability and endosomal rupture release properties, for rapid and high levels of Ag NPs uptake into yeast without significant toxicity, prerequisites for the development of the first intracellular SERS immunosensor.

The genetic information found in each species of organism is unique, and can be used as a tool to differentiate at the molecular level. This has caused rapid genotyping methods to become the cornerstone of a new area of research dependent on reading the genome as a form of identification. One of these specific identification methods, known as pyroprinting, relies on the small variation of DNA sequences within the same species to develop a unique, reproducible fingerprint. By simultaneously pyrosequencing multiple polymorphic loci within the ribosomal operons known as the intergenic transcribed spacers, a reproducible output is obtained, known as a pyroprint, which can be used like a fingerprint to identify that organism. This section of the genome not only differs between species but also between isolated bacteria within that species, allowing for the differentiation of species subtypes, referred to as strains. While this is a viable method for generating reproducible fingerprints from individual strains it may be possible to obtain identical fingerprints from non-identical organisms. The following report uses direct sequence comparison and in silico pyrosequencing of E. coli isolates housed in the Center for Applications in Biotechnology at California Polytechnic State University, San Luis Obispo that have matching pyroprints to show that it is possible to receive near identical pyroprints from non-identical sequences of intergenic transcribed spacers. Although the exact likelihood and cause of this false positive result remains undetermined due to limitations in the sequencing method, its existence questions the accuracy of using pyroprints of the ITS regions as a method of strain classification.

Matthew R. Borglin This thesis demonstrates efficacy of Octenyl Succinic Anhydride (OSA), as a biofilm sanitizer. Biofilms allow bacteria to adhere to solid surfaces with the use of excreted polymeric compounds. For example, surfaces found in food production or processing facilities such as the interior of a raw milk holding tank, are some of the most susceptible to biofilm contamination. When present, biofilms can cause a variety of negative effects, which include; reduction of product shelf life, corrosion, and outbreaks of foodborne illnesses. The close association of biofilms with the majority of foodborne illness cases led the US Environmental Protection Agency (EPA) to create a new category of sanitizer specifically designed for treatment of mature biofilms. The efficacy of sanitizers in this new regulatory category is determined by the EPA protocols MB-19 and MB-20. The EPA’s protocols outline methods for cultivating, treating, and measuring effects on Pseudomonas aeruginosa biofilms in a continuous flow stir bar bioreactor. Biofilm modification by OSA was verified by the presence of octenyl esters on OSA treated biofilms with single point Raman spectrophotometry. OSA modified biofilm’s antimicrobial properties were first investigated with crystal violet staining in 96-well microtiter plates with inconclusive results. However, effective antimicrobial properties where apparent when using the CDC Biofilm Reactor. OSA treatments consistently returned a 6-log CFU/coupon reduction in biomass compared to controls. Inhibition of planktonic and/or biofilm regrowth was demonstrated using the 96-well plate methodology. This thesis demonstrated the effectiveness of OSA chemical esterification reaction as a biofilm treatment. In doing so, this work suggests a new approach for biofilm remediation by chemically modifying the structural components of biofilm.

Municipal treated wastewater entering rivers contain biologically active pharmaceuticals capable of inducing effects in aquatic life. Phytoremediation of three of these pharmaceuticals and an herbicide was investigated using Sandbar willow (Salix exigua) and Arabidopsis thaliana. Both plants were effective at removing compounds from solution, with removal of 86% of the synthetic estrogen, 17α-ethynylestradiol, 65% of the anti-hypertensive, diltiazem, 60% of the anti-convulsant, diazepam (Valium®), and 51% of the herbicide atrazine, in 24 hours. Distribution of compounds within roots and shoots, in soluble and bound forms, differed among compounds. Uptake and distribution of pharmaceuticals within the study plants confirmed pharmaceutical behaviour can be predicted based on a physiochemical property, their octanol-water partitioning coefficients. An effective method for detection of 17α-ethynylestradiol within surface water using solid phase extraction and gas chromatography-mass spectrometry was developed. Previously unreported breakdown of 17α-ethynylestradiol into another common estrogen, estrone, during preparative steps and gas chromatography was resolved.
xv, 216 leaves ; 29 cm.

My dissertation research examined the effect of the cultivation of insect-resistant Bacillus thuringiensis (Bt) maize on the soil environment with a goal of understanding how to obtain a balance between technological advancement and maintenance of a healthy soil ecosystem. Although Bt plants may help to reduce pesticide use, conferring benefits to farm workers and the environment, there are still unresolved questions about how the cultivation of Bt plants affects soil organisms. For this dissertation project, I used 14 different genotypes of Bt maize and non-Bt maize (Zea mays) to investigate the effects of transgenic Bt plants on the colonization ability, abundance, and diversity of symbiotic arbuscular mycorrhizal fungi (AMF) in the soil ecosystem over time. My greenhouse studies demonstrated that Bt maize plants exhibited reduced AMF colonization across multiple Bt genotypes and that effects were most pronounced when fertilizer levels were limited and spore density was high. In addition, I found that although differences in AMF colonization between Bt and non-Bt maize were difficult to detect in the field, spore density was reduced in Bt field plots after just one growing season. When I tested the effect of plot history on AMF and plant growth, I found that Bt and non-Bt maize plants had higher leaf chlorophyll content when grown in plots previously cultivated with the same maize line as the previous year, indicative of a positive feedback effect. I also examined potential mechanisms contributing to the reduced AMF colonization observed in Bt maize in greenhouse studies and determined that follow-up experiments should continue to investigate differences in root apoplastic invertase activity and root permeability in Bt and non-Bt maize. Future investigations would also benefit from examining potential differences in root exudate profiles and volatile organic compounds between Bt and non-Bt cultivars. Taken together, my dissertation results suggest that, while difficult to detect in the field, reductions in AMF colonization in Bt maize roots may be ecologically significant as they could lead to a decrease in the abundance of AMF propagules in the soil over time, potentially impacting soil structure and function in areas where Bt crop cultivation is high.

Breast cancer mortality is mainly due to distant recurrence of the disease arising from dormant tumor cells established by cancer therapies. Patients who initially respond to cancer therapies often succumb to distant recurrence of the disease. It is not clear why people with the same type of breast cancer respond to treatments differently; some escape from dormancy and relapse earlier than others. In addition, some tumor clones respond to immunotherapy while others do not. We investigated how autophagy plays a role in accelerating or delaying recurrence of neu overexpressing mouse mammary carcinoma (MMC) following adriamycin (ADR) treatment, and in affecting response to immunotherapy. We explored two strategies: 1) transient blockade of autophagy with chloroquine (CQ), which blocks fusion of autophagosomes and lysosomes during ADR treatment, and 2) permanent inhibition of autophagy by a stable knockdown of ATG5 (ATG5KD), which inhibits the formation of autophagosomes in MMC during and after ADR treatment. We found that while CQ prolonged tumor dormancy, but that stable knockdown of autophagy resulted in early escape from dormancy and recurrence. Interestingly, ATG5KD MMC contained an increased frequency of ADR-induced polyploid-like cells and rendered MMC resistant to immunotherapy. On the other hand, a transient blockade of autophagy did not affect the sensitivity of MMC to immunotherapy. Our observations suggest that while chemotherapy-induced autophagy may facilitate tumor relapse, cell-intrinsic autophagy delays tumor relapse, in part, by inhibiting the formation of polyploid-like tumor dormancy. Although immunotherapy of breast cancer by means of anti-HER2 antibodies prolongs survival of breast cancer patients, disease recurrence remains a major challenge. On the other hand administration of human vaccines against infectious disease in a preventive setting or during latency/dormancy has been successful in offering a cure. Here, we sought to use adoptive immunotherapy (AIT) at the time of tumor dormancy in order to prevent progression of breast cancer. We used a low dose immunogenic chemotherapy by means of 5-FU, Adriamycin, and Cyclophosphamide (FAC) in order to stabilize tumor progression prior to AIT using autologous tumor-reactive lymphocytes. Low dose FAC established local tumor dormancy, inhibited distant tumor dormancy occurring long before distant metastasis, and induced predominate a Ki67- quiescent type of tumor dormancy, which is less susceptible to tumor immunoediting. Dormant tumor cells expressed the cell survival pathways, including the endothelin receptor/ligand (ETRA, ETRB and ET-1) and PD-L1, thereby protecting them from elimination by AIT. In addition, tumor-reactive CD8+ T cells also produced ET-1 as a survival ligand for ETRA positive tumor cells. A combination of AIT with the blockade of tumor cell survival pathways resulted in a significant improvement of AIT against tumor dormancy. We also showed that the inhibition Bcl-xL downstream of the tumor cell survival pathways is specifically effective against dormant tumor cells, suggesting a combination of AIT with small molecules inhibitors of Bcl-xL. Altogether, we showed that distant tumor dormancy is established long before distant recurrence of breast cancer, and that the expression of several tumor cell survival pathways in dormant cells protects them from immunotherapy. Our results suggest that immunotherapeutic targeting of tumor dormancy combined with the blockade of a common downstream cell survival pathway could prevent tumor progression and recurrence of the disease.

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.
Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2004.

AN ANALYSIS OF GLOBAL GENE EXPRESSION RESULTING FROM EXPOSURE TO ENERGETIC MATERIALS A Dissertation Presented for the Doctor of Philosophy Degree University of Tennessee, Knoxville VERNON LASHAWN MCINTOSH JR. August 2010 Dedication This dissertation is dedicated to my family. My mother and father Debra and Vernon McIntosh instilled in me the respect for academic excellence and the drive maximize my potential. Early on, my younger brother Kyle started showing signs of a shared interest in biology thus my desire to be a positive role model for him kept me motivated. Last but certainly not least, my loving wife and best friend Nichole has been there to offer love and support throughout my entire undergraduate and graduate degrees. It’s difficult to imagine making it this far without her (and that’s not just because she paid the bills). Abstract Characteristic transcriptional biomarkers have been identified for microbial cultures exposed to 2, 4, 6-trinitrotoluene (TNT), 2, 6-dinitrotoluene (DNT), or triacetone-triperoxide (TATP). This study describes the generation of expression profiles for exposure to each compound, the functional significance of each response, and the identification of the characteristic alterations in gene expression associated with exposure to each compound. Expression profiles were generated from a total of three different candidate organisms: Escherichia coli, Saccharomyces cerevisiae, and Pseudomonas putida. Common to all three organisms, TNT exposure resulted in increased expression of genes involved in toxin resistance and drug efflux systems. The S.cerevisiae and E.coli expression profiles were both characterized by increased expression of genes involved in iron-sulfur cluster assembly, sulfur containing amino acids, sulfate transport and assimilation and the metabolism of nitrogen compounds. Only E.coli and Saccharomyces were used to generate DNT induced expression profiles; both profiles exhibited high degrees of similarity with each organism’s respective TNT profiles. This was especially true of the E.coli profile where 25 of the 30 alterations were also observed after exposure to TNT. A computational discriminant functional analysis was performed to identify characteristic biomarkers for each exposure. For each compound a set of transcriptional biomarkers (10 or less) was developed. An additional set of biomarkers was developed encompassing both TNT and DNT exposure. These sets of genes serve as a transcriptional fingerprint for exposure to each respective compound. The sensitivity and specificity of each transcriptional fingerprint is sufficient to correctly identify exposure to energetic materials against a background of non-energetic compound exposures. This study makes several novel contributions to the greater body of scientific knowledge: • This is the first documented study of the interactions of TATP in any biological system. • This is the first comprehensive gene expression study of the TNT response by P. putida, E.coli or E.coli. • This is the first application of computational class prediction in the development of biomarkers for exposure to energetic materials

Fermentation is a process by which primary food products are modified biochemically by the action of microorganisms and/or their enzymes. Several societies have, over the years, intentionally carried it out to enhance the taste, aroma, shelf-life, texture, nutritional value and other properties of food. It is used in many parts (lithe world. However, there are regional differences in use and these depend on the availability of raw materials, consumption habits. and other socio-cultural factors. This study was aimed at (comparatively) assessing, local commercial and homemade amahewu with respect to nutritional value, hygiene and other health benefits to the commirn ity. Methods employed were Thin Layer Chromatography (TLC) (mycotoxins), High Perliffmance Liquid Chromatography (HPLC) (mycotoxins, sugars and amino acids), Dumas (proteins), SOxhlet (lipids) and intubation technique (metabolisable energy) to analyse maize meal and amahewu samples from various regions. The regions sampled included mal3heleni (South Coast) and kwaNgcolosi (North Coast) villages. Commercial amahewu was analysed with kind permission from Clover SA. Species from the following genera were isolated and identified from amahewu samples: Lactobacillus, Saccharonivccs, Lcuconostoc, Lactococcus, Panioca, Entcrobacter and kleb•iella. Saccharotnyces was detected in commercial samples only. Gram-negative strains were identified in most of manheleni village samples. No traceable amounts of aflatoxin BI (AFB1), fumonisin B 1 (FBI) and zearalenone (ZEA) were found in Clover SA samples. AFB I was detected in 40% of both maize meal and amahewu samples from maBheleni (range 0.55 — 0.84ng/g and 8.3x10 5 — 9.1x10-5ng/g respectively). From the same village, 100% of the maize meal and 80% of the amahewu samples were contaminated with FBI (range 4.1 47.2ng/g and 1.4 ---- 6.9ng/g respectively). ZEA was detected in all maize meal samples (range 0.9 — 4.3ng/g). None of the amahewu samples contained detectable levels of ZEA. All maize meal and amahewu samples from kwaNgcolosi were contaminated with AF13 1 (range 8.3 — 30.I ng/g and 0.04 - 0.102ng/g respectively). FB I was detected in 75% of both maize meal and amahewu samples from the same village (range 0.5 — 4.1ng/g and 0.04 0.56ng/g respectively). ZEA was also found in all maize meal samples and 75% of amahewu samples (range 3.7 — 16.4ng/g and 0.03 -- 0.06ng/g respectively). MaBheleni, Clover SA and kwaNgcolosi maize meal and amahewu samples contained vitamins B1, 13 2 and B6 with a range of 0.31+0.21 - 4.48±0.81 B 1 ; 0.15±0.14 - 1.67±0.33 B2 and 0.05±0.07 - 0.77±1.45 lig/g B6. Fat levels ranged from 0.28±0.40 to 4.54±0.05 percentage by weight. The levels of proteins varied from 4.02±0.02 to 8.40±0.04 percentage by weight. Starch concentrations ranged from 31.51.5.28 to 75.911.92g/100g. Maize meal samples contained glucose and maltose, while glucose, fructose, sucrose, maltose, M-triose, DP 4 and 5 and DP >15 were detected in amahewu. Apparent and true metabolisable energy for homemade and commercial Freeze-dried amahewu was 13.194 and 13.696MJ/kg (AME N ); and 13.605 and 14.106M.Ekv ( 1 MEN ), respectively. This study has shown that lactic acid maize fermentation reduce' the levels of AF13 1 , FB I and ZEA toxins in maize meal, inhibits the growth of most Gram-negative bacteria, and in some instances, fermentation did improve the nutritional value. Metabolisable energy analysis represents an important tool to assess whether or not compounds ingested are converted to sources of energy in the body and utilised. Amahewu fermentation yielded beneficial products (probiotics: reduced mycotoxins levels and reduced starch). In conclusion, natural lactic acid maize fermentation to produce amahewu will do more good than harm to the consumer, therefore, people need to be advised on how to safely store their maize and also to be encouraged to consume their stored maize in fermented form.
Thesis (M.Med.Sc.)-University of Natal, Durban, 2003.