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Thesis (Ph. D.)--University of Wisconsin--Madison, 1966. / Typescript. Vita. Includes facsimile of one published journal article and texts for two articles to be submitted to journals, all by the author and M.L. Jackson. Description based on print version record. Includes bibliographical references (leaves 116-128).

Thesis (MScEng (Process Engineering))--University of Stellenbosch, 2009. / The abundance of alkane by-products formed in South Africa presents a feedstock opportunity for the production of a wide range of commercially important products, such as long-chain dioic acids and alcohols. These compounds are formed as intermediates through the biological conversion of alkanes, a route which is particularly attractive when compared with chemical conversion due to its operation under milder process conditions. Furthermore, advances in genetic manipulation, which enable the accumulation of a range of metabolic intermediates, make the biological route remarkably flexible. From the literature review Yarrowia lipolytica was identified as a promising organism for use in studying alkane bioconversion because of its ability to produce large quantities of fatty acids when grown on n-paraffins as a sole carbon source. The bioconversion of alkanes will not only depend on the genetic modification but also on the process conditions to maximise growth and bioconversion. The overall objective of this project was therefore to investigate the potential of Y. lipolytica for alkane bioconversion by defining the conditions that maximise both cell growth and bioconversion. The Y. lipolytica strains supplied (TVN348, TVN493 and WT), however, were not yet modified to the extent that accumulation of metabolic intermediates was possible. Use was therefore made of a model system in which the alkane substrate was substituted with an even chain alkylbenzene. Since Y. lipolytica is unable to metabolise the benzene ring, the alkylbenzene is converted to the metabolic intermediate, phenyl acetic acid (PAA), and the potential for bioconversion assessed through measuring the accumulation of PAA. The specific objectives of the project were therefore 1) to define and quantify the parameters for the establishment of an effective model system in shake flasks with respect to trace elements, buffering, added nitrogen, oxygen supply, glucose concentration, alkylbenzene substrate and inducer requirements 2) to use the defined model system to identify the most promising strain of Y. lipolytica TVN348, TVN493 and WT 3) to use the defined model system and selected strain for evaluation of the influence of time of substrate addition and glucose concentration on cell growth and bioconversion of Y. lipolytica under controlled conditions in an instrumented bioreactor Furthermore, since poor reproducibility in cell growth and bioconversion had been prevalent in previous studies, it was also aimed to identify and statistically quantify the reproducibility between duplicate or triplicate samples in each experiment and between sets of different experiments with respect to PAA formation and cell concentrations. Studies were conducted in shake flask cultures to define and quantify the parameters for the model system. The parameters assessed included trace elements, buffering, nitrogen concentration, oxygen supply, glucose concentration, alkylbenzene substrate type and possible inducer requirements. Trace elements, phosphate buffering and added nitrogen did not significantly affect the cell growth of Y. lipolytica TVN348. The cell concentration of Y. lipolytica TVN348 and TVN493 was increased by 65% and 43% respectively for an increase in oxygen supply by decreasing the working volume from 150ml to 50ml, while the cell concentration of Y. lipolytica WT was increased by 41% when oxygen supply was increased by switching from non-baffled to baffled flasks in 50ml cultures. Bioconversion was also increased for an increase in oxygen supply: 2.4mM to 29.0mM PAA (Y. lipolytica TVN348) and 1.2mM to 21.7mM PAA (Y. lipolytica TVN493) for a decrease in working volume; 10.5mM to 46.6mM PAA (Y. lipolytica WT) when switching from non-baffled to baffled flasks. These results indicated that adequate oxygen supply is crucial to both growth and bioconversion, and that further study should be conducted in 50ml working volumes. Cell concentrations obtained in 1.6% (wt/v) and 3.2% (wt/v) glucose cultures (3.95x108cells/ml and 4.03x108cells/ml respectively) indicated that cell growth was neither enhanced nor inhibited by 3.2% (wt/v) glucose. Of the range of substrates examined (propylbenzene, butylbenzene, sec-butylbenzene, hexylbenzene, ethyltoluene and tert-butyltoluene for Y. lipolytica TVN348 and TVN493; octylbenzene and decylbenzene for Y. lipolytica WT), hexylbenzene was regarded as the best substrate for bioconversion (14.7mM and 14.1mM PAA for TVN348 and TVN493 respectively; 42.6mM PAA for WT). Lastly, the absence of a requirement for an additional inducer such as ethanol or oleic acid was confirmed when PAA was formed from hexylbenzene in the culture containing additional glucose (25.0mM). This suggested that when using hexylbenzene as substrate, bioconversion was induced provided sufficient glucose was available for cell maintenance. Results from duplicate or triplicate flasks in each individual shake flask experiment were reproducible and conclusions were based solely on results which showed 95% confidence intervals. However, reproducibility problems were experienced with results between different sets of experiments carried out under the same conditions. The model system was therefore defined by: 1) no addition of trace elements, additional buffering or added nitrogen, 2) cultures grown in 50ml volumes to supply an adequate amount of oxygen crucial for growth and bioconversion, 3) 3.2% (wt/v) glucose and 4) addition of 1% (v/v) hexylbenzene at 24h with no inducer requirements. Use of the model system in shake flask cultures to identify the most promising of the three strains of Y. lipolytica supplied demonstrated that there was no significant difference in cell growth or bioconversion between these strains. Y. lipolytica WT (which has no genetic modifications) was therefore used for further investigation until an appropriate strain could be substituted when it became available. The growth and bioconversion of Y. lipolytica WT was further investigated under controlled conditions in a bioreactor. The influence of time of substrate addition (11h, 24h, 48h) and glucose concentration (3.2% and 6.4% (wt/v)) on growth and bioconversion was examined. When hexylbenzene was added at 48h, cell growth was increased (8.90x108cells/ml) when compared to two of the triplicate cultures with hexylbenzene addition at 24h (4.74x108cells/ml and 3.92x108cells/ml) and the culture with hexylbenzene addition at 11h (2.82x108cells/ml). The third of the triplicate cultures with hexylbenzene addition at 24h, on the other hand, exhibited the strongest growth (2.23x109cells/ml). The poor reproducibility between the triplicate cultures with hexylbenzene addition as 24h made it difficult to determine whether hexylbenzene addition at 24h or 48h maximised cell growth. Furthermore, the cell growth was not significantly improved when the glucose concentration was increased from 3.2% (wt/v) to 6.4% (wt/v) (7.47x108cells/ml for 6.4% glucose culture), however it was also not inhibited. The highest amount of specific PAA formed by Y. lipolytica WT was found when hexylbenzene was added at 11h (7.4x10-11mmol PAA/cell), however the highest accumulated PAA was produced in the culture that exhibited the strongest growth with hexylbenzene addition at 24h (41.4mM). This suggested that the bioconversion of hexylbenzene was maximised when it was added during the active growth phase. It is therefore recommended to conduct fed-batch experiments in future to maintain the active growth phase. Accumulated PAA was increased in 6.4% (wt/v) glucose culture (15.2mM PAA) when compared with two of the 3.2% (wt/v) glucose cultures (5.4mM and 4.3mM PAA). These results indicated that the increased glucose concentration did not inhibit the bioconversion. Furthermore, PAA was formed when 5% (wt/v) residual glucose was observed in the culture, suggesting that the bioconversion of hexylbenzene was not inhibited at glucose concentrations as high as 5.0% (wt/v). If future work were to be conducted in bioreactor culture where glucose is added in fed-batch operation, glucose concentrations in cultures of up to 5% (wt/v) could be considered for initial studies. During bioconversion by Y. lipolytica, the PAA measured after hexylbenzene exhaustion did not, however, correspond to 100% conversion. Further, poor reproducibility was found in the bioreactor cultures. The disappearance of hexylbenzene without a corresponding accumulation of PAA and poor reproducibility was investigated by determining whether PAA was further degraded or alternatively, whether other metabolic intermediates were being formed and accumulated from the hexylbenzene. However, substitution of the hexylbenzene with PAA as substrate confirmed that PAA could not be metabolised. Further, NMR analyses of both the aqueous and organic phases of the culture did not identify any additional metabolic intermediates. It is recommended that additional analyses be conducted on the aqueous and organic phases to further assess the possible accumulation of intermediates. The development of the model system in shake flask cultures demonstrated the importance of adequate oxygen supply for both cell growth and bioconversion. It was also shown that no inducer was needed because hexylbenzene acted as its own substrate inducer. Furthermore, comparison of Y. lipolytica strains TVN348, TVN493 and WT under the defined conditions of the model system revealed that the genetically modified strains (TVN348, TVN493) did not exhibit enhanced bioconversion. Bioreactor cultures using the model system under controlled conditions further showed that bioconversion was not inhibited at a 5% (wt/v) residual glucose concentration and suggested that bioconversion was maximised when hexylbenzene was added during active growth phase. This informs on future work, suggesting fed-batch operation in order to extend the active growth phase, where glucose concentrations in the bioreactor of up to 5% (wt/v) can be considered.

The aim of this study was to understand the contribution of Linear Alkylbenzene Sulfonates (LAS) to the decline of Mediterranean coastal forest exposed to marine aerosols. LAS are a group of synthetic anionic surfactants widely used in the composition of household or industrial detergents and agrochemicals. This study was part of a large project (RISICO) aiming the assessment of the environmental impact of the LAS at multiple levels: biodegradation in the coastal waters, sorption – desorption processes in the sediments, toxicity to the aquatic life and toxicity to the coastal forest (by aerosolisation of the sea water). Previous studies pointed out this group of surfactants as the main cause of the coastal forest decline. However, the quantification of this surfactant in the environmental samples (mainly sea water and foliar deposition) was done using non-specific analytical methods as methylene blue active substances (MBAS), leading to overestimation of the environmental concentrations of LAS. The work hypothesis was that at actual environmental concentrations, the LAS does not play a key role in the foliar uptake of the sea salt deposited on the coastal vegetation by the marine aerosols, therefore the LAS may not be the main cause of the coastal forest decline. The research involved both greenhouse experiments and field measurements. The experimental work was conducted on young Mediterranean trees (Laurus nobilis L., Quercus ilex L. and Pinus halepensis P. Mill.) and investigated the synergistic toxic effects of exposure to simulated marine aerosol contaminated with surfactants. An array of endpoints was used including photosynthetic activity, relative water content, foliar deposition and uptake of salt and LAS, and pigments analysis. The results of those experiments revealed that LAS itself did not have phytotoxic effects. Nevertheless, the surfactant was shown to enhance the foliar uptake of the salt in the tested species, especially in Pinus halepensis, confirming the conclusions of previous studies regarding the sensitivity of this species to polluted marine aerosols. The field work was conducted in San Rossore National Park (Italy) and Porquerolles Island (France) and was focused on evaluating the health status of the Mediterranean forest (Quercus ilex L., Pinus halepensis Mill. and Pinus pinaster Aiton.) and also on quantification of LAS in coastal aerosols using highly specific analytical methods, like the mass spectrometry (MS). The frequencies and extent of injuries in the coastal trees were found to be correlated to the salt but not with the LAS content of the leaves. The concentrations of LAS in the Pinus and Quercus leaves were comparable in the two studied sites but the concentrations of salt were extremely high in San Rossore, suggesting that other factors may determine the excessive salt foliar uptake. The parallel MS and MBAS carried out in the same set of aerosol samples revealed that MBAS measurements were not relevant for LAS concentrations in the marine aerosols. Projecting the experimental results to the real LAS and salt exposure of the coastal forest, we concluded that LAS may play a marginal role in coastal vegetation decline.

The aim of this study was to understand the contribution of Linear Alkylbenzene Sulfonates (LAS) to the decline of Mediterranean coastal forest exposed to marine aerosols. LAS are a group of synthetic anionic surfactants widely used in the composition of household or industrial detergents and agrochemicals. This study was part of a large project (RISICO) aiming the assessment of the environmental impact of the LAS at multiple levels: biodegradation in the coastal waters, sorption – desorption processes in the sediments, toxicity to the aquatic life and toxicity to the coastal forest (by aerosolisation of the sea water). <p>Previous studies pointed out this group of surfactants as the main cause of the coastal forest decline. However, the quantification of this surfactant in the environmental samples (mainly sea water and foliar deposition) was done using non-specific analytical methods as methylene blue active substances (MBAS), leading to overestimation of the environmental concentrations of LAS. <p>The work hypothesis was that at actual environmental concentrations, the LAS does not play a key role in the foliar uptake of the sea salt deposited on the coastal vegetation by the marine aerosols, therefore the LAS may not be the main cause of the coastal forest decline. <p>The research involved both greenhouse experiments and field measurements. The experimental work was conducted on young Mediterranean trees (Laurus nobilis L. Quercus ilex L. and Pinus halepensis P. Mill.) and investigated the synergistic toxic effects of exposure to simulated marine aerosol contaminated with surfactants. An array of endpoints was used including photosynthetic activity, relative water content, foliar deposition and uptake of salt and LAS, and pigments analysis. The results of those experiments revealed that LAS itself did not have phytotoxic effects. Nevertheless, the surfactant was shown to enhance the foliar uptake of the salt in the tested species, especially in Pinus halepensis, confirming the conclusions of previous studies regarding the sensitivity of this species to polluted marine aerosols. <p>The field work was conducted in San Rossore National Park (Italy) and Porquerolles Island (France) and was focused on evaluating the health status of the Mediterranean forest (Quercus ilex L. Pinus halepensis Mill. and Pinus pinaster Aiton.) and also on quantification of LAS in coastal aerosols using highly specific analytical methods, like the mass spectrometry (MS). The frequencies and extent of injuries in the coastal trees were found to be correlated to the salt but not with the LAS content of the leaves. The concentrations of LAS in the Pinus and Quercus leaves were comparable in the two studied sites but the concentrations of salt were extremely high in San Rossore, suggesting that other factors may determine the excessive salt foliar uptake. The parallel MS and MBAS carried out in the same set of aerosol samples revealed that MBAS measurements were not relevant for LAS concentrations in the marine aerosols. Projecting the experimental results to the real LAS and salt exposure of the coastal forest, we concluded that LAS may play a marginal role in coastal vegetation decline. <p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

In many countries around the world, municipal sewage and industrial wastewaters are typically treated, or sometimes only partially treated, prior to their discharge into surface waters. A major anionic surfactant, Linear Alkyl benzene Sulfonate (LAS), and a degraded product of a non-ionic surfactant, Nonylphenol (NP), are frequently found in municipal wastewaters. When wastewater containing such surfactants and their degraded products is used for irrigation, it can have an effect on the sorption/desorption and movement of pesticides in soils. Therefore, a lysimeter study was conducted, in summer 2004, to assess the effect of LAS and NP on the movement of agricultural herbicides through a sandy loam soil. The degradation of the herbicides was studied in lysimeters over a ninety-day period. Irrigation water with a concentration of 12 mg L -1 of LAS and NP was used to assess their effect on the leaching of atrazine, metolachlor, and metribuzin. Moreover, a laboratory sorption experiment was undertaken to estimate the partition coefficients (kd) of the three herbicides with water containing the same concentrations of LAS and NP. Irrigation water containing low concentrations of surfactants (LAS and NP) did not increase leaching of the three herbicides. Therefore, these results would reduce the concerns regarding pesticide leaching through sandy soil brought on by LAS and NP in wastewaters for irrigation, which is becoming more important due to increasing water scarcity in the dry climate regions of the world. / Beside the lysimeter study, mathematical models can be used effectively and economically in a very short period of time for simulating herbicide concentrations into soil. PESTFADE, a one dimensional transient flow model, was used, in this study, to simulate the fate of the three herbicides in sandy soils. Another model, Artificial Neural Network (ANN), was also used over mathematical modeling due to its faster execution period and less input parameter requirements, for predicting the concentrations of the three herbicides in a sandy loam soil. The predicted concentrations, from both models were compared with the experimental results from the lysimeter study. Although slight overestimations and underestimations were observed, both models simulated herbicide concentrations in the soil profile satisfactorily.

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