Ho Wing Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 110-123). / Abstracts in English and Chinese. / Chapter 1 --- Introduction / Chapter 1.1 --- Bioconversion --- p.1 / Chapter 1.2 --- Functional foods & quality improvement in fermentation Edible mushroom --- p.2 / Chapter 1.3 --- Substrates --- p.4 / Chapter 1.4 --- Edible mushroom --- p.6 / Chapter 1.5 --- Nutritional value of food and feed --- p.9 / Chapter 1.6 --- Protein digestibility --- p.16 / Chapter 1.7 --- Problem caused by fungal contamination --- p.17 / Chapter 1.8 --- Antioxidant --- p.18 / Chapter 1.9 --- Research objectives --- p.20 / Tables and figures --- p.21 / Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.32 / Chapter 2.2 --- Sample preparation --- p.33 / Chapter 2.3 --- Fungal growth measurement --- p.34 / Chapter 2.4 --- Proximate compositions --- p.34 / Chapter 2.4.1 --- Moisture determination --- p.34 / Chapter 2.4.2 --- Ash determination --- p.35 / Chapter 2.4.3 --- Crude lipid determination --- p.35 / Chapter 2.4.4 --- Dietary fiber determination --- p.36 / Chapter 2.4.5 --- Crude protein determination --- p.38 / Chapter 2.4.6 --- Carbohydrate determination --- p.38 / Chapter 2.4.7 --- Glucose determination --- p.39 / Chapter 2.4.8 --- Chitin determination --- p.40 / Chapter 2.4.9 --- Phytic acid determination --- p.41 / Chapter 2.5 --- In vitro protein digestibility --- p.42 / Chapter 2.6 --- Aflatoxin determination --- p.43 / Chapter 2.7 --- Antioxidant ability --- p.45 / Chapter 2.7.1 --- Ferric reducing antioxidant powder (FRAP) assay --- p.45 / Chapter 2.7.2 --- Trolox equivalent antioxidant capacity (TEAC) assay --- p.46 / Chapter 2.8 --- Statistical analysis --- p.47 / Table --- p.48 / Chapter 3 --- Results / Chapter 3.1 --- Mycelia growth --- p.49 / Chapter 3.1.1 --- Growth diameter --- p.49 / Chapter 3.1.2 --- Chitin content --- p.50 / Chapter 3.2 --- Weigh loss in sample preparation --- p.51 / Chapter 3.3 --- Proximate composition --- p.52 / Chapter 3.3.1 --- Moisture --- p.52 / Chapter 3.3.2 --- Ash --- p.52 / Chapter 3.3.3 --- Crude lipid --- p.53 / Chapter 3.3.4 --- Dietary fiber --- p.54 / Chapter 3.3.5 --- Crude protein --- p.55 / Chapter 3.3.6 --- Carbohydrate content --- p.56 / Chapter 3.3.7 --- Glucose content --- p.56 / Chapter 3.3.8 --- Phytic acid --- p.56 / Chapter 3.4 --- In vitro protein digestibility (IVPD) --- p.57 / Chapter 3.5 --- Aflatoxin --- p.53 / Chapter 3.6 --- Antioxidant ability --- p.58 / Chapter 3.6.1 --- Ferric reducing antioxidant powder (FRAP) assay --- p.58 / Chapter 3.6.2 --- Trolox equivalent antioxidant capacity (TEAC) assay --- p.60 / Tables and figures --- p.62 / Chapter 4 --- Dissusions / Chapter 4.1 --- Mycelia growth --- p.89 / Chapter 4.2 --- Weigh loss in sample preparation --- p.90 / Chapter 4.3 --- Proximate composition --- p.90 / Chapter 4.3.1 --- Moisture --- p.90 / Chapter 4.3.2 --- Ash --- p.91 / Chapter 4.3.3 --- Crude lipid --- p.92 / Chapter 4.3.4 --- Dietary fiber --- p.93 / Chapter 4.3.5 --- Crude protein --- p.96 / Chapter 4.3.6 --- Glucose concentration --- p.98 / Chapter 4.3.7 --- Phytic acid --- p.99 / Chapter 4.4 --- In vitro protein digestibility (IVPD) --- p.101 / Chapter 4.5 --- Aflatoxin --- p.102 / Chapter 4.6 --- Antioxidant activity --- p.103 / Chapter 4.7 --- Bioconversion ability --- p.105 / Chapter 4.8 --- Best substrate --- p.105 / Chapter 4.9 --- Functional foods --- p.106 / Chapter 4.10 --- Limitation of the methodology and future development --- p.107 / Table --- p.108 / Chapter 5 --- Conclusion --- p.109 / References

Many microorganisms, and in particular fungi, are able to grow over a wide pH range. Thus, these microorganisms must possess some regulatory mechanism or system that senses the environmental pH signal and ensures that gene expression of certain molecules is tailored to the pH of the environment (Penalva and Arst, 2002). In Aspergillus species and several other fungi, pH regulation is mediated by seven genes viz. palA, palB, palC, palF, palH, palI and the global pH regulatory gene, pacC (MacAbe et al, 1996; Negrete-Urtasun, 1999; Denison, 2000). The activated form of the PacC protein activates genes that are required at alkaline pH, e.g. genes coding for alkaline phosphatases, and represses certain genes that are functional at acidic pH, e.g. genes encoding acid phosphatases (Negrete-Urtasun, 1999). PacC (and its homologues) also positively regulates genes involved in penicillin biosynthesis, e.g. the isopenicillin N synthase gene, ipnA, in A. nidulans (Penalva and Arst, 2002). It has also been hypothesised that pacC may negatively regulate aflatoxin biosynthesis, a carcinogenic secondary metabolite in several species of Aspergillus (Keller et al, 1997). To elucidate the role of pacC a novel method of post-transcriptional gene silencing known as RNA interference was utilized. This method involved the cloning of a partial pacC gene fragment first in the forward and then the reverse orientations in a fungal expression cassette to create an RNA interference (RNAi) vector. The unique structure of this vector would allow the cloned fragments to be expressed and the resulting RNA to immediately form a double stranded stem-loop structure or short hairpin RNA (shRNA; McDonald et al, 2005). The formation of this shRNA, in turn, would be responsible for activating the endogenous RNA degradation complexes that would lead to mRNA degradation and subsequent gene silencing (Liu et al, 2003; Kadotoni et al, 2003; McDonald et al, 2005). The results presented here have shown that confirmed pacC RNAi mutants produced aflatoxins irrespective of environmental pH (i.e. the mutants produce aflatoxins under acidic and alkaline conditions). Thus, pacC is essential for pH regulation of aflatoxin production in A. flavus. There are numerous other biological (e.g. presence of oxylipins, lipooxygenases) and non-biological factors (pH, carbon source etc.) which affect maize colonisation and aflatoxin production by A. flavus (Burrow et al, 1996; Wilson et al, 2001; Calvo et al; 2002; Tsitsigiannis et al, 2006). However, all the genetic mechanisms involved have as yet not been identified. It has been shown by Caracuel et al (2003) that pacC acts as a negative virulence regulator in plants and these workers have hypothesised that PacC prevents expression of genes that are important for infection and virulence of the pathogen. Therefore the physiological effects that pacC silencing had on the growth, conidiation and pathogenicity of A. flavus mutants were also investigated. The results of this study showed that pacC does not play a significant role in primary growth and development but does affect conidial production. SEM results showed that mutants have many “open ended” phialides and poorly developed conidiophores. This would suggest that pacC activation of conidial production genes is also required. Furthermore, pacC RNAi silencing severely impaired the ability of the A. flavus mutants to infect and cause damage on maize. The results obtained here are similar to that of pacC null mutants in A. nidulans, C. albicans and F. oxysporum which also exhibited low pathogenicity (Davis et al, 2000; Fonzi, W.A, 2002; Caracuel et al, 2003; Bignell et al, 2005 and Cornet et al, 2005). This study indicates that pathogenicity of A. flavus on maize is directly related to the structural integrity of conidia, which in turn is greatly influenced by PacC. This gene is a global transcriptional regulator and may either repress or activate one or many genes in each of the above pathways (Penalva and Arst, 2002). Studies on the genetic mechanisms of pacC regulation on these pathways are needed to elucidate the mechanisms of activation or repression of these genes.

A three part study was carried out to examine rheological, colloidal and floe microstructural aspects of industrial brewing yeast strains. Following a review of the literature, the rheological properties of four yeast strains (two flocculent ale and lager types and their non-flocculent variants) were examined. In related colloidal studies, orthokinetic flocculation rates of these strains as well as their cell surface charge were determined. Floc microstructure was characterized using both light and scanning electron microscopy. In a summary chapter, the cell floc model (a modification of Hunter's elastic floc model) was used to the explain the rheological and colloidal behaviour of brewing yeast suspensions. Flow behaviour studies of the commercial yeast suspensions suspended in a calcium-containing sodium acetate buffer revealed that yeast flocculent characteristics had an important influence on their suspension flow behaviour. As cell concentrations increased, suspension flow properties become increasingly non-Newtonian and could be described by the Casson model at low rates of shear and the Bingham model at shear rates above 100 s⁻¹. The cell floc model was proposed to explain the Bingham flow behaviour of these csuspensions. The Bingham yield stress in these suspensions was believed to be a function of the orthokinetic capture coefficient, cell volume and the energy to break up doublet cells. Increasing temperature tended to lower the Bingham yield stress in lager strains and increase the yield stress in ale strains. A semi-empirical explanation for the viscosity increase of deflocculated cell suspensions and the estimation of pseudo-capture coefficients was presented. Furthermore, studies of the flow behaviour of yeast strains suspended in decarbonated ale and lager beer revealed that: 1) suspensions of flocculent strains show higher yield stress values than their non-flocculent variants, 2) ale strain suspensions tended to have higher yield values than the lager strains and 3) yeast dispersed in beer had higher yield stress values than when suspended in buffered calcium suspensions. This last observation was believed to reflect the influence of ethanol on the cell binding process which has important implications for future measurements of yeast flocculation. Colloidal studies revealed for the first time, that the orthokinetic rate of flocculation of brewing yeast cells could be modelled by a first order equation, as predicted by fundamental colloid theory. While subject to considerable variation, measured rate constants led to the calculation of orthokinetic capture coefficients. Yeast cell zeta potential values generally agreed with literature data but could not be employed in the DLVO model of colloid flocculation to explain measured orthokinetic capture coefficient values. Examination of the cell zeta potential data indicated that the data had non-normal distributions. SEM examination of the four industrial yeast strains suggested that a number of distinct structures mediated cell-to-cell interaction and that intra-strain differences occurred. These findings, along with the observation of non-normal surface charge distributions, indicated that these industrially pure strains had undergone substantial variation. Treatment of the flocculent cells with pronase tended to reduce cell-to-cell contacts. In the summary chapter the cell floe model was employed to describe the rheological behaviour of the yeast suspensions. Estimation of the force needed to separate doublet yeast cells were made using critical shear rate data (i.e., the point at which Bingham flow begins). This estimate was similar to that reported for single antibody bonds and may be due to the presence of lectin-like structures on the yeast cell wall. / Land and Food Systems, Faculty of / Graduate

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Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Protein secretion and intracellular transport are highly regulated processes and involve the interplay of a multitude of proteins. A unique collection of thermosensitive secretory mutants allowed scientists to demonstrate that the secretory pathway of the yeast Saccharomyces cerevisiae is very similar to that of the higher eukaryotes. All proteins commence their journey in the endoplasmic reticulum, where they undergo amino-linked core glycosyl modification. After passage through the Golgi apparatus, where the remodelling of the glycosyl chains is completed, proteins are transported to their final destinations, which are either the cell surface, periplasmic space or the vacuole. Proteins destined for secretion are usually synthesised with a transient amino-terminal secretion leader of varying length and hydrophobicity, which plays a crucial role in the targeting and translocation of their protein cargo. Considerable effort has been made to elucidate the molecular mechanisms involved in these processes, especially due to their relevance in a rapidly expanding biotech industry. The advantages of S. cerevisiae as a host for the expression of recombinant proteins are well documented. Unfortunately, S. cerevisiae is also subject to a number of drawbacks, with a relative low product yield being one of the major disadvantages. Bearing this in mind, different secretion leaders were compared with the aim of improving the secretion of the LKA 1 and LKA2 a-amylase enzymes from the S. cerevisiae secretion system. The yeast Lipomyces kononenkoae is well known for its ability to degrade raw starch and an improved secretion of its amylase enzymes from S. cerevisiae paves the way for a potential one-step starch utilisation process. Three sets of constructs were prepared containing the LKA 1 and LKA2 genes separately under secretory direction of either their native secretion leader, the S. cerevisiae mating pheromone a-factor (MFa1) secretion leader, or the MFa1 secretion leader containing a synthetic C-terminal spacer peptide (EEGEPK). The inclusion of a spacer peptide in the latter set of constructs ensured improved Kex2p proteolytic processing of the leader/protein fusion. Strains expressing the amylase genes under their native secretion leaders resulted in the highest saccharolytic activity in the culture medium. In contrast to this, strains utilising the synthetic secretion leader produced the highest fermentation yield, but had a lower than expected extracellular activity. We hypothesise that the native amylase leaders may function as intramolecular chaperones in the folding and processing of their passenger proteins, thereby increasing processing efficiency and concomitant enzyme activity. / AFRIKAANSE OPSOMMING: Proteïensekresie en intrasellulêre transport is hoogs gereguleerde prosesse en betrek die onderlinge wisselwerking van 'n verskeidenheid proteïene. 'n Unieke versameling van temperatuur-sensitiewe sekresiemutante het wetenskaplikes in staat gestelom die ooreenkoms tussen die sekresiepad van die gis Saccharomyces cerevisiae en dié van komplekser eukariote aan te toon. Alle proteïene begin hul reis in die endoplasmiese retikulum, waartydens hulle ook amino-gekoppelde kernglikosielveranderings ondergaan. Nadat die proteïene deur die Golgi-apparaat beweeg het, waar die laaste veranderings aan die glikosielkettings plaasvind, word hulle na hul finale bestemmings, waaronder die seloppervlak, die periplasmiese ruimte of die vakuool, vervoer. Proteïene wat vir sekresie bestem is, word gewoonlik met 'n tydelike, amino-eindpuntsekresiesein, wat 'n kritiese rol in die teiken en translokasie van hul proteïenvrag speel, gesintetiseer. Heelwat pogings is in hierdie studie aangewend om die molekulêre meganismes betrokke by hierdie prosesse te ontrafel, veral as gevolg van hul toepaslikheid in 'n vinnig groeiende biotegnologiebedryf. Die voordele van S. cerevisiae as 'n gasheer vir die uitdruk van rekombinante proteïene is alombekend. S. cerevisiae het egter ook verskeie nadele, waaronder die relatiewe lae produkopbrengs die belangrikste is. Teen hierdie agtergrond, is verskillende sekresieseine met mekaar vergelyk met die doelom die sekresie van die LKA 1 en LKA2 a-amilasegene vanuit die S. cerevisiae-uitdrukkingsisteem te verbeter. Die gis Lipomyces kononenkoae is bekend vir sy vermoeë om rou stysel af te breek en 'n verbeterde sekresie van sy amilasegene vanuit S. cerevisiae baan die weg vir 'n moontlike een-stap styselgebruiksproses. Drie stelle konstrukte is gemaak wat die LKA 1- en LKA2- gene onafhanklik onder sekresiebeheer van onderskeidelik hul inheemse sekresiesein, die S. cerevisiae paringsferomoonsekresiesein (MFa1) of die MFa1-sekresiesein met 'n sintetiese koppelingspeptied aan die C-eindpunt (EEGEPK), plaas. Die insluiting van 'n koppelingspeptied in die laasgenoemde stel konstrukte verseker verbeterde Kex2p proteolitiese prosessering van die sein/proteïenfusie. Rasse wat die amilasegene onder beheer van hul inheemse sekresieseine uitdruk, het die beste saccharolitiese aktiwiteit in die kultuurmedia getoon. In teenstelling hiermee, het rasse wat van die sintetiese sekresiesein gebruik maak, die beste fermentasie-opbrengs getoon, maar met 'n laer as verwagte ekstrasellulêre aktiwiteit. Ons vermoed dat die inheemse amilaseseine as intramolekulêre begeleiers optree in die vou en prosessering van hul proteïenpassasiers, wat lei tot verbeterde prosessering en ensiemaktiwiteit.

The depletion of petroleum and other platform chemical resources are a global concern; therefore alternative substrates must be identified to replace these current sources. Thus allowing research in fungal biotechnology to prosper, as filamentous fungi can utilize second-generation feedstocks or agricultural waste to produce these petroleum derived platform chemicals. This research focuses on the ability of filamentous fungi to use different second-generation feedstocks such as wheat bran and sugar cane bagasse to generate platform chemicals of interest, namely being itaconic acid (IA) and other organic acids of interest, such as citric acid. This study focused on the metabolite producing capabilities of Aspergillus terreus, initially in a shake flask fermentation environment and then in an Airlift Bioreactor environment utilizing hydrolyzed wheat bran and sugar cane bagasse as a substrate source to produce metabolites of interest. The initial shake flask fermentation experiment involved inoculation and incubating A. terreus in hydrolyzed wheat bran with additional minerals at 30°C for 5 days at a pH range of between 3-4. The result yielded itaconic acid and citric acid concentrations of 1.01g/l and 6.23g/l at their peaks, respectively. The airlift bioreactor was run for 16 days with a constant pH range between 3-4, at a temperature of 30°C with a dissolved oxygen level of 20g/l. The result of the study yielded a high itaconic acid and citric acid concentration peaking at 59.4 g/l and 59.2 g/l, respectively.

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The aim of the project was to determine whether a by-product of industrial fermentations, Saccharomyces cerevisiae, could be utilized to bioaccumulate heavy metal cations and to partially define the mechanism of accumulation. S. cerevisiae cells were found to be capable of accumulating Cu²⁺in a manner that was proportional to the external Cu²⁺ concentration and inversely proportional to the concentration of biomass. The accumulation process was only minimally affected by temperature variations between 5 and 40°C or high ambient concentrations of sodium chloride. The accumulation process was however considerably affected by variations in pH, bioaccumulation being most efficient at pH 5 - 9 but becoming rapidly less so at either extreme of pH. Selection for copper resistant or tolerant yeast diminished the yeast's capacity for Cu²⁺ accumulation. For this and other reasons the development of heavy metal tolerance in yeasts was deemed to be generally counterproductive to heavy metal bioaccumulation. The yeast biomass was also capable of accumulating other heavy metal cations such as c0²⁺ or Cd²⁺. The yeast biomass could be harvested after bioaccumulation by tangential filtration methods, or alternatively could be packed into hollow fibre microfilter membrane cartridges and used as a fixed-bed bioaccumulator. By immobilizing the yeast in polyacrylamide gel and packing this material into columns, cu²⁺, C0²⁺ or Cd²⁺ could be removed from influent aqueous solutions yielding effluents with no detectable heavy metal, until breakthrough point was reached. This capacity was hypothesized to be a function of numerous "theoretical plates of equilibrium" within the column. The immobilized biomass could be eluted with EDTA and recycled for further bioaccumulation processes with minor loss of bioaccumulation capacity. Yeast cells were fractionated to permit identification of the major cell fractions and molecular components responsible for metal binding. Isolation of the yeast cell walls permitted investigation of their role in heavy metal accumulation. Although the amino groups of chitosan and proteins, the carboxyl groups of proteins, and the phosphate groups of phosphomannans were found to be efficient groups for the accumulation of copper, the less effective hydroxyl groups of the carbohydrate polymers (glucans and mannans) had a similar overall capacity for copper accumulation owing to their predominance in the yeast cell wall. The outer (protein-mannan) layer of the yeast cell wall was found to be a better Cu²⁺ chelator than the inner (chitinglucan) layer. It appeared that the physical condition of the cell wall may be more important than the individual macromolecular components of the cell wall in metal accumulation. It was apparent that the cell wall was the major, if not the sole contributor to heavy metal accumulation at low ambient heavy metal concentrations. At higher ambient metal concentrations the cytosol and vacuole become involved in bioaccumulation. Copper and other metals caused rapid loss of 70% of the intracellular potassium, implying permeation of the plasma membrane. This was followed by a slower "leakage" of magnesium from the vacuole which paralleled Cu²⁺ accumulation, suggesting that it may represent some form of ion-exchange. An intracellular copper chelating agent of approximately 2 kDalton molecular mass was isolated from copper tolerant yeast. This chelator was not a metallothionein and bound relatively low molar equivalents of copper compared to those reported for metallothionein. Treatment of the biomass with hot alkali yielded two biosorbents, one soluble (which could be used as a heavy metal flocculent), and an insoluble biosorbent which could be formed into a granular product to be used in fixed-bed biosorption columns. The granular biosorbent could accumulate a wide range of heavy metal cations in a semispecific manner and could be stored in a dehydrated form indefinitely, and rehydrated when required. Bioaccumulation by live algae was investigated as an alternative to yeast based processes. Various strains of algae, of which Scenedesmus and Selenastrum were the most effective, were found to be capable of accumulating heavy metals such as Cu²⁺, Pb²⁺ and Cr³⁺.