Bioremediation of aromatic pollutants using the ligninolytic enzymes of the white rot fungi has been thoroughly researched and has been shown to have considerable potential for industrial application. However, little success in scale-up and industrialisation of this technology has been attained due to problems associated with the continuous production of the pollutant-degrading enzymes using conventional bioreactor systems. The low productivities reported result from the incompatibility of conventional submerged culture reactor techniques with the physiological requirements of these fungi which have evolved on a solid-air interface, viz. wood. The enzymes are also produced only during the stationary phase of growth and can therefore be regarded as secondary metabolites. This study reports the conceptualisation, characterisation and evaluation of a novel bioreactor system as a solution to the continuous production of idiophasic pollutant degrading enzymes by the white rot fungus Phanerochaete chlysosporium. The reactor concept evolved from observation of these fungi in their native state, i. e. the metabolism of lignocellulosic material and involves the immobilisation of the organism onto a capillary ultrafiltration membrane. Nutrient gradients established across the biofilm, an inherent characteristic of fixed bed perfusion reactors, are exploited to provide both nutrient rich and nutrient poor zones across the biofilm. This allows growth or primary metabolism in the nutrient rich zone, pushing older biomass into the nutrient poor zone where secondary metabolism is induced by nutrient starvation. In effect, this represents a transformation of the events of a batch culture from a temporal to a spatial domain, allowing continuous production of secondary metabolites over time. Direct contact of the outer part of the biofilm with an air stream simulated the solid-air interface of the native state of the fungus. In order to facilitate the practical application of the membrane gradostat reactor (MGR) concept, conventional capillary membranes and membrane bioreactor modules were first evaluated. These were found to be unsuitable for application of the MGR concept. However, critical analysis of the shortcomings of the conventional systems resulted in the formulation of a set of design criteria for the development of a suitable membrane and module. These design criteria were satisfied by the development of a novel capillary membrane for membrane bioreactors, as well as a transverse flow membrane module, which is a novel approach in membrane bioreactor configuration. For the physiological characterisation of the MGR concept, a single fibre bioreactor unit was designed, which allowed destructive sampling of the biofilm for analysis. Using this system, it was shown that distinct morphological zones could be observed radially across the mature biofilm obtained through MGR operation. That these morphotypes do represent the temporal events of a typical batch culture in a spatial domain was confirmed by following the morphological changes occurring during batch culture of the immobilised fungus where the onset of primary and secondary metabolic conditions were manipulated through control of the nutrient supply. The different morphotypes were correlated to distinct growth phases by comparison of the morphology to the secretion of known enzymatic markers for secondary metabolism, viz. succinate dehydrogenase and cytochrome C oxidoreductase. Detailed structure-function analysis of the biofilm using transmission electron microscopy and adapted enzyme cytochemical staining techniques showed that the biofilm appeared to operate as a co-ordinated unit, with primary and secondary metabolism apparently linked in one thallus through nutrient translocation. This study provided new insights into the physiology of P. chrysosp,o rium and a detailed descriptive model was formulated which correlates well to existing models of wood degradation by the white rot fungi (WRF). Evaluation of the process on a laboratory scale using a novel transverse flow membrane bioreactor showed that a volumetric productivity of 1916 U.L.⁻¹day⁻¹ for manganese peroxidase, one of the pollutant degrading enzymes, could be attained, corresponding to a final concentration of 2 361 U.L.⁻¹ This may be compared to the best reported system (Moreira el at. 1997), where a volumetric productivity of 202 U.L.⁻¹day⁻¹was achieved with a final concentration of 250 U.L.⁻¹ However, MGR productivity is yet to be subjected to rigorous optimisation studies. The process could be operated continuously for 60 days. However, peak productivity could not be maintained for long periods. This was found to be due to physical phenomena relating to the fluid dynamics of the system which caused fluid flow maldistribution, which would have to be resolved through engineering analysis. In evaluation of the MGR concept for aromatic pollutant removal, in this case ρ- cresol, from growth medium, good performance was also achieved. The VmaxKm calculated by linear regression for the MGR was 0.8 (R² = 0.93), which compared favourably to that reported by Lewandowski et al. (1990), who obtained a Vmax/Km of 0.34 for a packed bed reactor treating chlorophenol. It was concluded that the MGR showed suitable potential to warrant further development, and that the descriptive characterisation of the biofilm physiology provided a sufficient basis for process analysis once engineering aspects ofthe system could be resolved.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:rhodes/vital:4032 |
| Date | January 1999 |
| Creators | Leukes, W |
| Publisher | Rhodes University, Faculty of Science, Biochemistry, Microbiology and Biotechnology |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis, Doctoral, PhD |
| Format | 218 leaves, pdf |
| Rights | Leukes, W |
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